key: cord- - oca mrm authors: shen, wen-jun; cui, wenjuan; chen, danze; zhang, jieming; xu, jianzhen title: rpirls: quantitative predictions of rna interacting with any protein of known sequence date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: oca mrm rna-protein interactions (rpis) have critical roles in numerous fundamental biological processes, such as post-transcriptional gene regulation, viral assembly, cellular defence and protein synthesis. as the number of available rna-protein binding experimental data has increased rapidly due to high-throughput sequencing methods, it is now possible to measure and understand rna-protein interactions by computational methods. in this study, we integrate a sequence-based derived kernel with regularized least squares to perform prediction. the derived kernel exploits the contextual information around an amino acid or a nucleic acid as well as the repetitive conserved motif information. we propose a novel machine learning method, called rpirls to predict the interaction between any rna and protein of known sequences. for the rpirls classifier, each protein sequence comprises up to diverse amino acids but for the rpirls- g classifier, each protein sequence is represented by using -letter reduced alphabets based on their physiochemical properties. we evaluated both methods on a number of benchmark data sets and compared their performances with two newly developed and state-of-the-art methods, rpi-pred and ipminer. on the non-redundant benchmark test sets extracted from the pridb, the rpirls method outperformed rpi-pred and ipminer in terms of accuracy, specificity and sensitivity. further, rpirls achieved an accuracy of % on the prediction of lncrna-protein interactions. the proposed method can also be extended to construct rna-protein interaction networks. the rpirls web server is freely available at http://bmc.med.stu.edu.cn/rpirls. the interactions of proteins with other proteins, peptides, dnas and rnas govern most the essential molecular function. rna-protein interactions (rpis) have a critical influence on post-transcriptional gene regulation [ ] [ ] [ ] , viral assembly [ ] [ ] [ ] , cellular defence [ ] , protein synthesis [ , ] and various other fundamental biological processes [ , ] . a significant portion of transcripts is long non-coding rnas (lncrnas) which are not translated into proteins and are longer than nucleotides [ ] . lncrnas normally function with their interacting proteins [ ] . for instance, the lncrna hotair regulated the hoxd locus in trans by interacting with pcg proteins [ ] ; several lncrnas were shown to be able to interact with auf , a protein linked to aging and cancer [ ] ; lncrnas binding to jarid protein were essential for the recruitment of prc to the chromatin [ ] ; lncrna gas inhibited hepatitis c virus replication by decoying hcv ns protein [ ] . hence, the study of rpis is essential for understanding their functions. compared to those of protein-protein interactions and dna-protein interactions, current knowledge regarding rna-protein interactions, especially lncrna-protein interactions is still limited. in this study, we propose a novel machine learning method, which we call rna-protein interaction prediction based on regularized least squares (rpirls), to quantitatively predict the potential rna-protein interactions. the experimental determination of rpis remains expensive and time-consuming [ ] [ ] [ ] , but fortunately, the accumulated rpi experimental data facilitate the development of computational models for rpi prediction [ ] [ ] [ ] . in , pancaldi and b .. ahler [ ] introduced a computational approach for rbp (rna binding protein)-mrna interaction prediction. they employed support vector machines (svms) and random forests (rfs) based on more than physical and functional features of rpis, including gene ontology, chromosomal position, gene and protein physical properties, protein localization, experimental translation, mrna properties, predicted protein structure, utr properties and genetic interactions. bellucci et al. [ ] proposed a method called catrapid for the prediction of protein lncrna interaction. they evaluated the interaction propensities of protein-rna based on their physicochemical properties, including secondary structure, hydrogen bonding and van der waals. muppirala et al. [ ] developed a method called rpiseq, which predicted rpis solely based on primary sequences. the rpiseq method still employed svms and rfs but exploited different features. they represented each sequence of proteins and rnas as the normalized frequencies of the corresponding -mer and -mer, respectively. in , based on the same feature vectors presented in muppirala et al., wang et al. [ ] first reduced the dimensionality of feature vectors, and then performed the rpis prediction by using naive bayes classifier which assumed the independence of attributes and by using extended naive bayes classifier which considered the correlation between attributes. lu et al. [ ] integrated the information on the secondary structure, hydrogen bonding propensities and van der waals of lncrnas and proteins with fisher's linear discriminant model. in , suresh et al. [ ] developed a method called rpi-pred to predict rpis by considering the high-order d protein and rna structure information. in , pan et al. [ ] proposed a new method named ipminer that integrated deep learning with stacked ensembling to improve the prediction performance of ncrna-protein interactions. in this paper, we classified rna-protein pairs as interacting or non-interacting by integrating derived kernel with regularized least squares (rls) [ ] . the motivation is to relate the sequence information of proteins and rnas to their biological functions, i.e., interactions. our method attempted to extract discriminant subsequence features from amino acid sequences and nucleotide sequences. the derived kernel measures the similarity between two biological sequences by capturing nucleic acid or amino acid compositions and repetitive sequence patterns. we used regularized least squares in learning as the computations performed by rls algorithms can be expressed using just inner products, hence allowing efficient implementation of kernel-based learning, in addition the rls algorithms often perform comparable to the best batch classifiers [ ] . since the dimensionality of feature space increases exponentially with the template size, for computational sake, we set upper limit for template size. on the other hand, we categorized amino acids into several groups based on their physiochemical properties [ ] [ ] [ ] , the reduced alphabet representation of the protein sequence allows larger template size and also decreases the dimensionality of feature space. we considered the derived kernel with two-layer architecture, hence there were two template sets, denoted as t p and t r needed to be constructed for protein and rna, respectively. here we considered all possible substrings of the same length making up a template set. the template set t p for amino acid sequences was composed of substrings with k continuous amino acids, while the template set t r for nucleic acid sequences was composed of substrings with l continuous nucleic acids. in order to extract discriminant subsequence features from amino acid sequences and nucleotide sequences, we explored the effect on rpi prediction over a range of choices for the template sizes of protein and rna. the training set rpi was used to determine these parameters. in our case, we used different template sizes of protein and rna chosen from set { , , . . . , } ∪ { , , . . . , } for rpirls and { , , . . . , } ∪ { , , . . . , } for rpirls- g. with different combination of protein template size and rna template size, the combined kernelk dk was integrated with rls to predict rna-protein interactions. the ten-fold stratified cross-validation has been verified to be the best algorithm for model selection on a large scale experiments [ ] , therefore on the data set rpi , we tuned the parameter λ by ten-fold stratified cross-validation with the optional parameter set {λ = e n , n = − , · · · , }. the data set rpi was divided into ten mutually exclusive folds and the mean response of each fold was approximately equal. in each test we merged parts of the samples as the training set and left the other part as the test set. the parameter λ was chosen by leave-one-out cross-validation on the training set. for rpirls, in all the ten sets, λ = e − uniformly achieved the best performance in the training data. tables and showed the performance of the proposed method in terms of auc and accuracy with different combination of parameters k and l, respectively. the experiment results showed that when the protein template size k = and the rna template size l = , the model performs best with auc score of . and accuracy of . . the other measurements of specificity (sp) and sensitivity (se) were . and . , respectively. while for rpirls- g, λ = e − uniformly performed best in all the ten sets. table showed that the method achieved the best prediction accuracy of . when the protein template size k = and the rna template size l = . the other measurements (auc, sp and se) were observed as . , . and . , respectively. the computational results showed that the rpirls classifier outperformed the rpirls- g classifier in terms of various performance measurements, indicating that the diversity of amino acids at a sequence is important for the prediction of rpis. the performance of predicting rpis was evaluated by using -fold stratified cross-validation on the rpi data set. different combinations of parameters k and l were evaluated. remark on the symbols of template sizes: k stands for template size of amino acid sequences; l stands for template size of nucleic acid sequences. the best auc in the table is marked in bold. remark on the symbols of template sizes: k stands for template size of amino acid sequences; l stands for template size of nucleic acid sequences. the best accuracy in the table is marked in bold. in order to evaluate the reliability and robustness of rpirls and rpirls- g, we compared them with other two state-of-the-art methods rpi-pred and ipminer. the rpi and rpi data sets after removing overlapping rpis with the training data were evaluated. both the rpirls and rpirls- g classifiers were trained on the rpi data set, and tested on the rpi and rpi data sets, respectively. as shown in tables and , rpirls outperformed the rpirls- g, rpi-pred and ipminer methods on both data sets. for the rpi data set as shown in table , the performance of the rpirls method was . , . , . and . for predictive accuracy, auc, specificity and sensitivity, respectively. while the predictive accuracy of the rpi-pred and ipminer methods were just . and . , respectively which were much lower than rpirls's. the remaining measurements (specificity and sensitivity) were observed as . and . , respectively for rpirls, and . and . , respectively for ipminer. the rpirls method outperformed rpi-pred and ipminer in terms of accuracy, specificity and sensitivity on the rpi data set. table . predictive performance of, rpirls- g in terms of the accuracy on the, rpi training data set over varying template sizes. the performance of predicting rpis was evaluated by using -fold stratified cross-validation on the rpi data set. different combinations of parameters k and l were evaluated. remark on the symbols of template sizes: k stands for template size of amino acid sequences; l stands for template size of nucleic acid sequences. the best accuracy in the table is marked in bold. similar results were observed on the rpi data set in table . the specificity of the rpi-pred and ipminer methods was just . and . , respectively, indicating there was a positive bias in their predictions of performance. a low specificity increases the labor, cost, and time needed to perform the required experimental tests, but our rpirls method achieved both reasonable specificity and sensitivity. furthermore, we evaluated the rpirls classifier on large-scale rna-protein pairs in the currently available rpintdb data base. the rpirls method correctly predicted out of rpis, reaching the predictive accuracy of %. to explore the effectiveness of the proposed method on predicting ncrna-protein interactions, a large-scale ncrna-protein interaction data set (we called nrpi ) was retrieved from the npinter data base [ ] . we trained rpirls and rpirls- g on the rpi data set, and tested it on the nrpi . table showed the prediction results compared with the rpi-pred classifier on the nrpi data set. the ipminer method showed a significantly positive bias on predicting ncrna-protein interactions, thus here we didn't include it into the comparison. the predictive accuracy for different organisms were separately computed. for the six organisms, our method rpirls performed best for the homo sapiens and saccharomyces cerevisiae, rpi-pred performed best for drosophila melanogaster, escherichia coli and mus musculus, and both methods obtained the same predictive accuracy for the caenorhabditis elegans. rpirls outperformed rpirls- g over all six organisms. for ncrna-protein pairs, the rpirls method achieved an accuracy of % compared to % for rpirls- g and % for the rpi-pred method. we further tested rpirls and rpirls- g on the lnrpi data set which was a subset of the nrpi data set and consisted of only lncrna-protein interactions (lncrpis). our model achieved an overall accuracy of % compared to % for rpirls- g and % for the rpi-pred classifier as shown in table . the predictive performance of rpirls was improved in out organisms compared with its performance on the nrpi data set. the results indicated the effectiveness of our method to predict lncrna-protein interactions only by using primary sequences of proteins and rnas. predicting lncrna-protein interaction networks is useful to explore the molecular mechanisms that are regulated by lncrnas [ , ] . in this experiment, we evaluated the performance of rpirls on building lncrpi networks and further compared its performance with rpi-pred. on the basis of the data in npinter, we analyzed the results of four organisms, i.e., caenorhabditis elegans, drosophila melanogaster, escherichia coli and saccharomyces cerevisiae, consisting of , , and lncrpis, respectively. for caenorhabditis elegans, the rpirls method correctly identified all lncrpis (blue edges) while the rpi-pred method correctly identified out of . as shown in figure , rpi-pred made incorrect prediction for the pair of n -g ebf (red edges). in figure , rpi-pred correctly predicted all lncrpis, whereas rpirls missed out of lncrpis for drosophila melanogaster. these out of incorrect predictions which were observed between two proteins p and q vss (yellow rectangle) and three signal recognition particle (srp) rnas n , n and n (green ellipse), formed the srp rna-protein interactions. for escherichia coli, the rpirls classifier made much more errors than the rpi-pred method, with predictive accuracies of % vs. %. the performance of rpirls for escherichia coli was much poorer than that for the other five organisms. in order to analyze why rpirls had relative poor performance on escherichia coli, we estimated the amino acid composition of escherichia coli compared with that of the other five organisms. as shown in figure , we found that escherichia coli had relative higher observing frequencies of alanine and valine as well as much lower content of serine compared with that of the other five organisms. the amino acid composition bias in escherichia coli probably leaded to poor results. as shown in figure , among incorrect predicted pairs, rpis corresponded to protein hubs, e.g., p a h , p afz , p ag , p ce , p ce , p and p , each of which appearing as a yellow rectangle node was shown to interact with six transfer-messenger rnas (tmrnas), e.g., n , n , n , n , n and n (green ellipse). for saccharomyces cerevisiae, as showed in figure , among incorrect predicted pairs, rpis were involved in protein hubs (p , q and q ), in which each protein interacted with small nuclear rnas (snrnas: n , n , n and n ), and other rpis corresponded to protein hubs (p , p , p , q , q , q and q ), each of which interacted with three small nucleolar rnas (snornas: n , n and n ). the rpirls classifier correctly identified out of rpis, achieving a high accuracy of %, compared of % (correctly predicted out of pairs) for rpi-pred. in this work, we illustrated the effectiveness and reliability of rpirls in predicting rpis for eukaryotic organisms in networks which comprised a variety protein hubs and rna hubs. mammalian cells contain more than different proteins interacting with rna [ ] . normally, any individual rna can interact with multiple proteins [ , ] . conversely, most proteins are capable of interacting with multiple rnas [ ] . given the number of rnas and rna-binding proteins, the number of possible rpis is enormous. high-throughput sequencing methods have accumulated huge amount of rna-protein binding experimental data and opened new possibilities to measure and understand rna-protein interactions by computational methods. most of the previous computational works on rpis focus on the prediction of rna-binding proteins or rna-binding residues in a protein sequence [ , [ ] [ ] [ ] . to our knowledge, very limited works have been developed to predict the specific associations between rnas and proteins, which play a critical role in post-transcriptional gene regulatory networks. complex networks of rpis mediate post-transcriptional gene regulation and therefore prediction of rpis helps us to gain insight into regulatory networks [ , ] . the work presented here provided a computational method, called rpirls, to classify rna-protein pairs as interacting or non-interacting by integrating a sequence-based derived kernel with regularized least squares. the derived kernel exploited the contextual information around an amino acid or a nucleic acid as well as the repetitive conserved motif information. our results demonstrated that only the sequence structures of rnas and proteins provide sufficient information to accurately predict rna-protein interactions, especially long non-coding rna-protein interactions. specifically, the rpirls classifier considered each protein sequence comprising up to diverse amino acids, while the rpirls- g classifier encoded each protein sequence by using the -letter reduced alphabets according to amino acid physiochemical properties. the computational results showed that the rpirls classifier was superior to the rpirls- g classifier in reliability and effectiveness, indicating that the diversity of amino acids at a sequence has critical impact on the function of rna-binding proteins. on two non-redundant benchmark data sets extracted from the pridb, the rpirls method outperformed rpi-pred and ipminer in terms of accuracy, specificity and sensitivity. compared with rpi-pred and ipminer, the rpirls method obtained a reasonable sensitivity at a lower false positive rate. further, rpirls achieved an accuracy of % compared to % for rpi-pred on the prediction of lncrna-protein interactions. the rpirls method can be extended to construct rna-protein interaction networks and therefore helps us to gain insights into post-transcriptional gene regulation. the reason for the good performance of the proposed method may be due to several factors. firstly, the use of similarity scores is a significant conceptual change in protein/rna evaluation, quantifying the overall similarity between proteins, rnas and their interactions. combining kernels by tensor product for the set of rna-protein pairs allowing to share information across the rna-binding proteins considerably improved the prediction, especially in the case of rnas with few known binding proteins. secondly, we have found that contiguous k-mer frequencies alone captured rich statistical information on the repetitive conserved motif of rna-protein pairs and the diversity of amino acids at a sequence has also contributed to an observed improvement contrast to rpi-pred which just applied -letter frequency for both protein and rna. finally, a kernel works as a measure of similarity and supports the application of powerful machine learning algorithms such as regularized least squares which we used in this paper. the rls mehod enables us to efficiently search for an optimized parameter λ at essentially no additional cost [ ] . further, our model was trained on a large data set which contained rna-protein pairs, and yielded more robust results. in contrast, ipminer had much more model parameters to fit as combining deep learning with stacked ensembling, however, was trained on a small data set of just rna-protein pairs, and thus showed a significantly positive bias on predicting ncrna-protein interactions. the main disadvantage of the proposed method is that the method is purely data-driven, in the sense that it relies solely on information derived from amino acid sequences and nucleic acid sequences, and thus does not consider higher structural information of protein and rna. while this may be seen as an advantage, since it can predict any rna-protein pair of known sequences. the increase of the number of protein-rna complexes in protein data bank [ ] has opened possibilities for researchers to develop secondary data bases and to gain valuable insight into the structure and function of these complexes. the protein-rna interface data base (pridb) v . [ ] identifies interfacial residues in rna-protein complexes and also calculates atomic distances between interfacial residues. the rb and rb are two precalculated data sets in the pridb, which respectively consist of and rna-binding protein chains. after combining the rb and rb data sets, we obtained a total of experimentally validated non-redundant rna-protein pairs, which had at least two atoms respectively coming from rna and protein with distance no more than Å. next, we removed redundant rna-protein pairs, which are the same protein chains interacting with the same rna chains. further, we removed those rna-protein pairs with amino acid sequence length < or nucleic acid sequence length < . finally, we obtained a positive sample set which consisted of experimentally validated rna-protein pairs. so far there are no definite negative samples of rna-protein interactions that are available. to construct a balanced negative sample set ("rna-protein non-interacting pairs"), we made it by randomly permute the proteins in the positive sample set but kept the rna fixed. we repeated the permutation process until no negative pairs existed in the positive sample set. as a result, the training set, called rpi , was composed of rna-protein interacting pairs and rna-protein non-interacting pairs. several data sets were employed to evaluate the performance of the proposed methods. our rpirls method was first evaluated using two popular non-redundant data sets of rpis studied in [ ] . the rpi data set consisted of experimentally validated rna-protein pairs extracted from the pridb data base. while the rpi data set eliminated all rpi pairs with ribosomal proteins or ribosomal rnas from the rpi data set. to avoid overlapping between training and testing data sets, those rpis overlapping the training data were removed, leaving the rpi data set of rpis and the rpi data set of rpis. their corresponding negative pairs were generated by following the same steps as developing the training negative pairs. next, we tested the performance of the rpirls method on a large scale data set extracted from the rna-protein interaction data base (rpintdb) (http://pridb.gdcb.iastate.edu/rpiseq/download.php). this data set consisted of , experimentally validated rpis from several sources, including the rpidb, npinter data base and high-throughput experiments published in literature. the fourth data set were extracted from the npinter data base which we called nrpi . the nrpi data set consisted of , experimentally validated ncrna-protein pairs from six model organisms, i.e., caenorhabditis elegans, drosophila melanogaster, escherichia coli, homo sapiens, mus musculus and saccharomyces cerevisiae. we constructed the fifth data set called lnrpi by extracting only lncrna-protein pairs from the npinter data base. this data set contains , experimentally validated lncrna-protein pairs. in this paper, we proposed two classifiers for predicting rpis based on different representations of protein sequences. for the rpirls classifier, each amino acid sequence comprised up to different amino acids. while for the rpirls- g classifier, we adopted the same amino acid classification approach as [ , ] . the derived kernel was proposed by smale et al. [ ] on images inspired by neuroscience of visual cortex. in what follows, we briefly described the construction of derived kernel on sequences. suppose a is a finite set called the alphabet. in the work here a is the set of amino acids (for rpirls), alphabets (for, rpirls- g) or nucleic acids. let a = a and define a k+ = a k × a recursively for any k ∈ n. we say s is a string if s ∈ ∪ ∞ k= a k , and s = (s , . . . , s k ) is a k-mer (e.g., a sequence of length k) if s ∈ a k for some k ∈ n with s i ∈ a . the process of computing the derived kernel mainly includes three steps as below: step . set an initial kernelk at the first layer. here the initial kernel is defined as: where x, y ∈ a k . x = {x , . . . , x k } and y = {y , . . . , y k } are substrings of the same length k. x = y if and only if x i = y i for i = , . . . , k. step . let f = ( f , . . . , f n ), denote by | f | the length of f , so here | f | = n. then define the second layer neural response of f at t : where t is the template set at the first layer, here we consider all possible substrings of length k making up the template set t , so here t = a k . h is the transformation set at the first layer. step . compute the second layer derived kernel by normalizing the inner product of two neural responses: where ·, · l (t ) denotes the l inner product with respect to the uniform measure |t | ∑ t∈t δ t , where |t | is the cardinality of the template set t and δ t is the dirac measure; with correlation normalization: . this process can continue if appropriate higher level templates are defined. at each layer (local) derived kernels are built by recursively pooling over previously defined local kernels. here, for the -layer derived kernel, pooling is accomplished by taking an average over a set of transformations which calculating the frequency of a template that occurs in a sequence. in this paper we deal with inner product kernels k which satisfies the mercer condition, are known to be an instance of reproducing kernels. next with correlation normalization,k is also a reproducing kernel andk(x, x) = for all x ∈ x. the kernel function is symmetric (i.e., k( f , g) = k(g, f )), and non-negative (i.e., k( f , f ) ≥ ), therefore it can be interpreted as a measure of similarity. we first apply the kernel to the set r which contains nucleic acid sequences, and denote it byk r , and then apply the kernel to the set of amino acid sequences p, denote it byk p , and lastly combine two kernels in a natural way by tensor product for the set of rna-protein pairs . the reproducing kernel for two rna-protein pairs (r, p), (r , p ) ∈ r × p is defined by: k dk ((r, p), (r , p )) =k r (r, r )k p (p, p ). since bothk r (r, r ) andk p (p, p ) are positive definite kernels,k dk ((r, p), (r , p )) is obviously a positive definite kernel too [ ] . after combining the kernel with other kernel-based supervised learning algorithm, we can predict rpis to any rna-protein pairs with known primary sequences. the rls algorithm is one of the most widely used models for regression. let k be a kernel on a finite set x. write h k to denote the inner product space of functions on x defined by k. supposez = {(x i , y i )} m i= is a sample set (called the training set) with x i ∈ x and y i ∈ r for each i. the rls can be written as follows: we integrated rls with the combined kernel k =k dk , hence the main construction is to computē herein, we aim to develop a novel method to distinguish rna-protein interaction pairs from non-rna-protein interaction pairs. therefore, for the binary classification case with y i ∈ {− , } for each i, iff ≤ , the predicted class is − ( denotes non-interaction), otherwise it is (denotes interaction). one important step of rls is to find a "good" value of the regularization parameter λ > in equation ( ). they were selected from an optional set Λ by leave-one-out cross-validation [ ] on the training data. we never used testing data for parameter selection which is under the risk of over-fitting. the sensitivity (se) and specificity (sp) are used to measure the ability of identifying positive and negative instances, respectively. they are defined by the accuracy which is used to measure the prediction quality, is defined by accuracy = tp + tn tp + tn + fp + fn . the auc (area under the receiver operating characteristic curve) is further employed to measure the predictive performance, which is for perfect prediction and . for random prediction. rna regulons: coordination of post-transcriptional events rpicool: a tool for in silico rna-protein interaction detection using random forest a census of human rna-binding proteins sequence-specific interaction of r coat protein with its ribonucleic acid binding site rna-rna and rna-rotein interactions in coronavirus replication and transcription diverse roles of host rna binding proteins in rna virus replication rna-protein interactions in human health and disease the three-dimensional structure of the ribosome 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squares to pattern classification simulations of the dynamics at an rna-protein interface prediction of rna-binding proteins from primary sequence by a support vector machine approach prediction of rna binding sites in proteins from amino acid sequence a study of cross-validation and bootstrap for accuracy estimation and model selection the noncoding rnas and protein related biomacromolecules interaction database molecular mechanisms of long noncoding rnas function of lncrnas and approaches to lncrna-protein interactions principles and properties of eukaryotic mrnps transcriptome-wide analysis of protein-rna interactions using high-throughput sequencing a neural network method for identification of rna-interacting residues in protein a server for the computational prediction of rna-binding residues in protein sequences prediction of protein-rna binding sites by a random forest method with combined features dissecting the expression dynamics of rna-binding proteins in posttranscriptional regulatory networks deciphering the role of rna-binding proteins in the post-transcriptional control of gene expression the protein data bank pridb: a protein-rna interface database introduction to the peptide binding problem of computational immunology: new results. found generalized cross-validation as a method for choosing a good ridge parameter this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license author contributions: w.s. and j.x. conceived and designed the experiments; w.s., w.c. and j.z. performed the experiments; w.s. and w.c. analyzed the data; w.s. and d.c. contributed web tools; w.s. and j.x. wrote the paper. the authors declare no conflict of interest. key: cord- -thygu at authors: lanfranco, maria fe; mocchetti, italo; burns, mark p.; villapol, sonia title: glial- and neuronal-specific expression of ccl mrna in the rat brain date: - - journal: front neuroanat doi: . /fnana. . sha: doc_id: cord_uid: thygu at chemokine (c-c motif) ligand (ccl ) belongs to a group of chemokines that play a role in the peripheral immune system, mostly as chemoattractant molecules, and mediate tactile allodynia. in the central nervous system (cns), ccl and its receptors have multiple functions, including promoting neuroinflammation, insulin signaling, neuromodulator of synaptic activity and neuroprotection against a variety of neurotoxins. evidence has also suggested that this chemokine may regulate opioid response. the multifunctional profile of ccl might correlate with its ability to bind different chemokine receptors, as well as with its unique cellular expression. in this work, we have used fluorescence in situ hybridization combined with immunohistochemistry to examine the expression profile of ccl mrna in the adult rat brain and provide evidence of its cellular localization. we have observed that the highest expression of ccl mrna occurs in all major fiber tracts, including the corpus callosum, anterior commissure, and cerebral peduncle. in these tracts, ccl mrna was localized in oligodendrocytes, astrocytes and microglia. astrocytic and microglial expression was also evident in several brain areas including the cerebral cortex, caudate/putamen, hippocampus, and thalamus. furthermore, using a specific neuronal marker, we observed ccl mrna expression in discrete layers of the cortex and hippocampus. interestingly, in the midbrain, ccl mrna co-localized with tyrosine hydroxylase (th) positive cells of the ventral tegmental area, suggesting that ccl might be expressed by a subset of dopaminergic neurons of the mesolimbic system. the expression of ccl mrna and protein, together with its receptors, in selected brain cell populations proposes that this chemokine could be involved in neuronal/glial communication. chemokines belong to the cytokine family of peptides that induce the maturation and trafficking of leukocytes and are considered to be essential for the inflammatory responses of the immune system. some chemokines have been linked to pro-inflammatory events, associated with chronic pain (white et al., ) or brain diseases such as human immunodeficiency virus (hiv)-associated dementia (schmidtmayerova et al., ; conant et al., ) , multiple sclerosis (hvas et al., ; mcmanus et al., ; rentzos et al., ) , stroke (siniscalchi et al., ) or traumatic brain injury . of particular interest is the chemokine (c-c motif) ligand (ccl ), formerly known as regulated on activation normal t-cell expressed and secreted (rantes). ccl is a small protein of amino acids that activate mononuclear phagocytes and induce their migration across the blood brain barrier to the site of inflammation (ubogu et al., ) . the role of ccl in inflammation has also been inferred by an association between increased ccl protein expression and the degree of inflammation in a variety of disorders and pathologies, including neuropathic pain (bhangoo et al., ) , asthma, atherosclerosis and arthritis among others (marques et al., ) . however, in the brain, ccl function goes beyond the one attributed to a classic pro-inflammatory chemokine. in fact, ccl is capable of inducing proliferation of oli-neu, an oligodendrocyte precursor-like cell line (kadi et al., ) suggesting a role in myelination. moreover, ccl promotes the migration of dorsal root ganglia cells in vitro (bolin et al., ) , and regulates the differentiation of astrocytes (bakhiet et al., ) suggesting that ccl may act as a neurotrophic factor. in addition, ex vivo activation of ccl receptor (ccr ) by ccl increases glucose transporter type membrane translocation in the hypothalamus (chou et al., ) , suggesting a role of ccl in glucose uptake and metabolism. these examples highlight the potential role of ccl as a modulator of cellular metabolism and brain architecture. lastly, ccl exerts neuroprotective activity against various neurotoxins including glutamate (bruno et al., ) , β-amyloid (ignatov et al., ) , and the hiv proteins gp (campbell et al., ) and tat (rozzi et al., ) . the different effects of ccl could be due to the ability of this chemokine to bind to multiple receptors. the selective and differential expression of ccl and its receptors, ccr (avdoshina et al., ) , ccr (he et al., ) and ccr (tran et al., ) in the rodent brain supports the role of ccl as a potential modulator of brain homeostasis. nevertheless, the functional role of ccl in the brain could be more in line with the suggested properties of some chemokines to act as a third neurotransmitter system (adler et al., ) helping neuronal communication (rostene et al., ) , perhaps through modulation of the release of glutamate from nerve endings (musante et al., ) . ccl is constitutively expressed in the adult central nervous system (cns; campbell et al., ) . nevertheless, the type of cells that express ccl has been so far inferred by in vitro studies. for instance, astrocytes appear to express ccl at high levels (avdoshina et al., ) ; nevertheless, oligodendrocytes (balabanov et al., ) , microglia (avdoshina et al., ) and neurons , all release ccl . in this study, we have analyzed ccl mrna expression patterns in the rat brain using in situ hybridization combined with immunohistochemistry to detect specific cell types (grabinski et al., ; lanfranco et al., ) . this method showed adequate sensitivity and specificity to detect mrna transcripts of the ccl gene. our results show that ccl mrna follows a cellular and anatomical distribution, which is highly regionalized and restricted to certain parts of the brain. we provide evidence for the first time that ccl mrnas can be expressed in dopamineproducing neurons. two month-old c bl/ j (wild-type, wt) and ccl knock-out (ko, b . p -ccl tm ) male mice ( - g) were purchased from the jackson laboratory (bar harbor, me, usa). ccl ko mice have been validated by northern blot analysis of total rna isolated from lps-stimulated peritoneal exudate cells (makino et al., ) . three-month-old male sprague-dawley (sd) rats ( - g) were purchased from charles river laboratory (germantown, md, usa). animals were housed under standard conditions with food and water ad libitum and maintained on a -h light/dark cycle. animals were anesthetized with a mixture of ketamine/xylazine ( mg/kg and mg/kg, i.p.) and intracardially perfused with ice-cold phosphate buffered saline (pbs) followed by perfusion with % paraformaldehyde (pfa). after perfusion, whole brains were quickly removed and post-fixed in % pfa overnight and transferred sequentially into a %, % and % sucrose solution. post-fixed brains were used for rna in situ hybridization and immunohistochemistry studies. all studies were carried out following the guide for the care and use of laboratory animals as adopted and promulgated by the u.s. national institutes of health and approved by georgetown university animal care and use committee. a sliding microtome (microm hm , thermo fisher scientific, tustin, ca, usa) was used to cut the brains in coronal orientation. the brain sections ( µm thickness) were cryoprotected in an antifreeze solution ( % glycerol + % ethylene glycol + . m pbs) for storage at − • c. free-floating brain sections were washed three times in pbs before mounting in gold seal tm ultrastick tm adhesion microscope slides thermo fisher scientific) . tissue was allowed to dry at room temperature (rt) and then stored at − • c until use. rnascope in situ hybridization assay was performed according to manufacturer's instructions (advance cell diagnostics (acd), hayward, ca, usa). in short, mounted tissue sections were serially dehydrated in %, %, %, % and % ethanol for min each. in between all pretreatment steps, tissue sections were briefly washed with ultra-pure water. incubation periods were performed on the hybez tm hybridization system (acd). the pretreat solution (hydrogen peroxide reagent) was applied for min at rt and then the tissue sections were boiled in pretreat solution (target retrieval reagent) for min. mounted slices were treated with pretreat solution (protease reagent) for min at • c. custom rat ccl rnascope probe was designed and purchased from acd. ccl probe targets the region - (accession number: nm_ . ) of the ccl sequence with pairs of zz-target probes. in addition, the negative (cat. no. , acd) and positive (cat. no. , acd) control probes were applied and let hybridized for h at • c. the amplification steps were performed according to manufacturer's directions. in between every amplification step, sections were washed with × wash buffer. detection was performed using a mixture ratio of red-a to red-b solution of : . the sections were incubated for min at rt and rinsed with ultra-pure water. following in situ hybridization, the sections were processed for immunohistochemistry. briefly, following the blocking step with % normal goat serum (ngs) in pbs for h at rt, post-hybridized slides were incubated with an antibody against glial fibrillary acidic protein (gfap, : , emd millipore, temecula, ca, usa), ionized calcium binding adaptor molecule- (iba- , : , wako chemical usa, richmond, va, usa), homeobox protein transcription factor nkx . ( : , emd millipore), neuronal nuclear antigen (neun, : , emd millipore) or tyrosine hydroxylase (th, : , emd millipore) in the presence of % ngs in pbs overnight at • c. brain slices treated with neun antibody were incubated for h at • c. subsequent to three washes with pbs, the slides were incubated with corresponding alexa fluor secondary antibodies ( : ; molecular probes , thermo fisher scientific) for h at rt. brain sections were rinsed with pbs three times and incubated for min in pbs with dapi solution ( : , , sigma-aldrich, st. louis, mo, usa) for counterstained nuclei. anatomical structures were analyzed in coronal sections and mapped according to paxinos and watson atlas (paxinos and watson, ) . fluorescent signals of ccl mrna hybridization and immunohistochemistry for different cell types were imaged with a ×, ×, × and × objective lens on a leica sp confocal microscope (leica microsystems inc., buffalo grove, il, usa) and an axioplan microscope (zeiss, thorwood, ny, usa) with a photometrics camera. all microscope and camera settings were identical for all images. the color label to far red (excitation nm, emission nm) was assigned for mrna hybridization signal. the color label to green was assigned for the antigen of interest (excitation nm, emission nm). the number of ccl mrna positive/cell type marker, and the total number of ccl mrna positive cells were quantified in five (corpus callosum and cortex) and three (hippocampus and vta) microscopic fields per brain section ( x, . mm ), using the imagej software (national institute of health. bethesda, md, usa), as previously described . figures for rnascope and ihc are representative of sections obtained from three animals. data for figure , expressed as the mean ± sem (sections obtained from five animals), were analyzed using one-way analysis of variance (anova) with bonferroni's multiple comparison post hoc test using graphpad prism software v. . (graphpad). a p-value < . was considered statistically significant. several antibodies against ccl exhibit non-specific binding to an antigen as determined by positive reactions in brain sections from wt and ccl ko mice (supplementary figure s ) . thus, we used in situ hybridization to map the expression of ccl mrna. rat and mouse ccl mrna share high sequence similarity (> %). therefore, we first validated the specificity of the ccl probe for rnascope by comparing ccl mrna signal in brain sections from wt and ccl ko mice. in the cortex ( figure a ) and corpus callosum ( figure d ) of wt mice, the ccl probe produced a strong fluorescent signal, characterized by red dots co-localizing with dapi positive cells (insets in figures a,d) , suggesting a nuclear localization. moreover, we observed that only a subpopulation of cells expresses ccl mrna, evidenced by the lack of red puncta in several dapi-stained nuclei (figures a,d) . no figure | validation of ccl mrna probe using ccl knock-out (ko) mice. in situ hybridization was performed with an rnascope probe targeting ccl mrna. representative images for ccl mrna (red dots) and nuclei counterstained with dapi (blue) in the cortex (a-c) and corpus callosum (d-f) of wild-type (wt) (a,d) and ko (b,e) mice. the sections were also analyzed for peptidylprolyl isomerase b mrna (c,f) as positive control probe. insets: high magnification images depicted by the white arrows. images showed that the fluorescent in situ hybridization by the rnascope probe produces puncta (red dots) that represents a single mrna transcript. scale bar µm for (a-f) and µm for inset images. hybridization was observed in ccl ko mice (figures b,e) , supporting the specificity of the ccl mrna probe used for rnascope . additional controls for potential artifacts were performed. sections were stained with a positive control probe that targeted the peptidylprolyl isomerase b gene. this gene is ubiquitously expressed throughout the mice tissue (kouadjo et al., ) . in a parallel experiment, sections were stained with a probe that targeted the bacillus subtilis dihydrodipicolinate reductase gene. this was used as a negative control because this bacterial gene is not expressed in mice. while peptidylprolyl isomerase b probe detected an mrna species throughout the brain (figures c,f) , the negative control probe generated no fluorescence (data not shown) suggesting that the technique and conditions used do not create artifacts. rnascope was then used to map the expression of ccl mrna throughout the adult rat brain. figures a-c show a schematic representation of three representative coronal figure | distribution of ccl mrna positive cells in the rat brain. (a-c) schematic drawing of three coronal sections of the rat brain indicating where ccl mrna puncta (red dots) were detected. (a -a ) representative sections through the corpus callosum (cc) and cingulate cortex (cg), external capsule (ec) and caudate/putamen (cpu), and anterior commissure (ac). (b ) representative section through the cerebral cortex denoting ccl mrna punta in different cortical layers (ii-iv). (b ) representative section through the thalamus; ic, internal capsule; po, posterior thalamic nucleus; vpm, ventroposterior medial nucleus; vpl, ventroposterior lateral nucleus; (b ,b ) representative sections through the hippocampal formation; ldvl, laterodorsal tegmental nucleus; slu, stratum lucidum of the hippocampus; gr, gracile fasciculus; ml, medial lemniscus; cx, parietal cortex; dg, dentate gyrus; (ca - ), cornu ammonis - . (c ) representative section from the midbrain; scp, superior cerebellar peduncle; snc, substantia nigra compacta; mzmg, marginal zone of the medial geniculate nucleus; sc, superior colliculus; dpme, deep mesencephalic nucleus; pag, periaqueductal gray; dmpag, dorsomedial pag; if, interfascicular nucleus; vta, ventral tegmental area. scale bar µm for (a -c ). brain sections ( . mm, − . mm and − . mm from bregma) where ccl mrna was detected. puncta were observed throughout the brain, although some regions contained more puncta than others (table ) . for instance, the corpus callosum (cc, figure a ) and the anterior commissure (ac, figure a ) contained more puncta than the caudate/putamen (cpu, figure a ). in the cerebral cortex, more hybridization was observed in layers iii/iv than ii and vi ( figure b ) . in the thalamus, the ventroposterior medial (vpm) nucleus exhibited more ccl mrna hybridization than the ventroposterior lateral (vpl) nucleus ( figure b ) . within the hippocampus, ccl mrna was more abundant in the fornix than in the hippocampal formation (figures b ,b ) . in the midbrain, areas with ccl mrna signal included the cerebral peduncles (cp, figure c ), and the ventral tegmental area (vta, figure c ). semi-quantitative analyses revealed that ccl mrna is expressed in several brain regions, in agreement with previous studies , but that there is an overall higher expression of ccl mrna in areas containing fiber tracts. these include the corpus callosum, anterior commissure, external capsule, fimbria, optic tract and cerebral peduncle ( table ) . moreover, we observed ccl mrna positive signal in several brain areas, including the cerebral cortex, striatum, hippocampus, thalamus and midbrain ( table ) . within these brain structures, ccl mrna was detected only in selected areas. for instance, the piriform cortex was negative ( table ) . in the striatum, the dorsomedial portion exhibited more ccl mrna signal than the ventromedial one ( table ) . taken together, our data suggest that different cell type may express ccl mrna. our semi-quantitative analysis indicates that ccl mrna is most abundant in the white matter that contains fiber tracts ( table ) which includes several subtypes of glial cells. to reveal which cells express ccl mrna, we examined the corpus callosum utilizing rnascope in situ hybridization combined with immunohistochemistry for specific glial cell markers. these include gfap, an astrocytic marker, iba- , a microglia/macrophage-specific marker, and nkx . , a marker to identify oligodendrocyte progenitor cells. we found that ccl mrna in the corpus callosum is predominantly inside nuclei of cells, evidenced by the co-localization of dapi (blue) with the ccl mrna hybridization signal, that were in gfap (figures a - ) , iba- (figures b - ) and nkx . (figures c - ) positive cells. noteworthy, nkx . positive cells were aligned in parallel rows (figure c ), which is a typical property of adult oligodendrocyte progenitor cells. quantitative analyses of ccl mrna in the corpus callosum revealed that . %, . % and . % of gfap, iba- and nkx . positive cells, respectively, expressed ccl mrna while no ccl mrna was found in neurons ( figure a) . these results validate the notion that the corpus callosum contains a high percentage of oligodendrocytes expressing ccl mrna. analysis of the somatosensory cortex with the ccl probe revealed that layers iii-v contained ccl mrna, although most of the puncta were in layers iii and iv ( figure b ) . to characterize which cells express ccl mrna in these layers, serial sections were examined by rnascope in situ hybridization combined with immunohistochemistry, using glial specific markers as described above. the neuron-specific nuclear protein marker, neun, was used to detect neurons. we observed that the fluorescent puncta of ccl mrnas in layers iii and iv were co-localized with neun (figures a - ) , iba- (figures b - ) , as well as gfap (figures c - ) positive cells. more positive puncta were observed in glial cells (figures b ,c ) than neurons (figure a ) , suggesting that cortical neurons express ccl mrna in low abundance. although we did not quantify ccl protein levels due to the lack of specificity of the antibodies, this result supports previous in vitro data that neurons produce and release ccl protein at a lower level than glial cells (avdoshina et al., ) . in layer v, ccl mrna was also observed in nkx . positive cells (figures d - ) , overall suggesting that in the cerebral cortex glia cell as well neurons express ccl mrna. to confirm this finding, we carried out a quantitative analysis of ccl mrna puncta, . % of neurons, . % of astrocytes, % of microglia and . % oligodendrocytes were also positive for ccl mrna (figure b ). analysis of the hippocampal formation revealed that ccl mrna is particularly abundant in the fimbria (figure b ) , which is a prominent white matter tract along the edge of the hippocampus, supporting the notion that ccl mrna is expressed in fiber tracts. however, red puncta were also observed in sections throughout the hippocampus mainly in the cornu ammonis (ca) and dentate gyrus (dg) regions ( figure b ). the ca contains pyramidal neurons whereas the polymorphic layer of the dg consists of sparsely distributed polymorphic cells. rnascope in situ hybridization combined with immunohistochemistry revealed that in the ca region, in addition to neurons (figures a - ) , microglia (figures b - ) and oligodendrocytes (figures e - ) expressed ccl mrna. in neurons, the red puncta that correspond to ccl mrna were found outside and inside the nuclei (figure a ) , suggesting that some of this mrna is readily available for translation. in the polymorphic cells layer of the dg, microglia (figures c - ) and astrocytes (figures d - ) were positive for ccl mrna. overall, in the hippocampus, ccl mrna was found in . % of neurons and in ∼ % in glial cells ( figure c ). ccl mrna was also scattered in the midbrain (figure c ) . the majority of puncta were observed in the cerebral peduncle ( figure c) , a major descending fiber tract. however, puncta were also observed within the vta region. rnascope in situ hybridization followed by immunohistochemistry revealed that in this region, % of neurons ( figure d ) express ccl mrna (figures a - ) . microglia (figures d, b - ) , astrocytes (figures d - ) , and few oligodendrocytes (figures d, c - ) also express ccl mrna in . %, . %, and . % respectively. intriguingly, only a subpopulation of neun positive cells expressed ccl mrna ( figure a ) . to determine the type of neurons that express ccl mrna, sections were stained with an antibody against th, the rate limiting step enzyme for the synthesis of dopamine. some but not all th-positive cells exhibited ccl mrna puncta (figures e - ) , suggesting that ccl mrna is synthesized in a small subset of dopamine neurons. interestingly, these neurons, unlike those in the cerebral cortex, exhibited several puncta, mostly perinuclearly ( figure e ) , suggesting high expression of this mrna in these neurons. our results reveal a unique expression profile of ccl mrna throughout the adult rodent brain. we showed that the cellular anatomical distribution of this chemokine is highly regionalized and restricted to certain parts of the brain. most notably, we found that ccl mrna hybridization signal is the highest in white matter, such as the anterior commissure, corpus callosum and the optic tract, which are rich in glia cells and myelinated axons. however, a constitutive expression of ccl mrna was also present in some subset of neuronal cell in the cerebral cortex and hippocampus, and especially in dopamine-producing neurons of the vta. altogether, our data characterize a constitutively anatomical and cellular expression of ccl mrna in the rat brain. most importantly, we observed ccl mrna in various cells even without injury or inflammation. our work joins a growing number of studies that have shown that chemokines can be constitutively expressed throughout the brain in a region specific manner. for example, cx cl , the first chemokine shown to be expressed in neurons, is constitutively expressed in human (raport et al., ) and rat brain (nishiyori et al., ) . cxcl , also known as stromal derived factor- , is expressed in different cell types throughout the rat brain, including astrocytes, microglia, and neurons (banisadr et al., ) . similarly, ccl is constitutively expressed in neurons and astrocytes, but not in oligodendrocytes or microglia (banisadr et al., ) . our data shows that under physiological conditions ccl mrna is constitutively expressed in all glial cells (microglia, astrocytes and oligodendrocytes) as well as in a subset of neurons. although in this work we only measured mrna, the fact that ccl proteins are detected in the adult brain supports the notion that ccl , like other chemokines (banisadr et al., ) , might have a role as potential modulators of brain function. under physiological conditions, ccl is expressed in low abundance in the brain tissue, posing an experimental challenge to detect it with conventional immunostaining technology. in addition, levels of a given peptide do not necessarily reflect the site of synthesis. an alternative approach is measuring mrna by rnascope technology, which in addition to the sensitivity for the detection of low abundance mrna molecules (sørdal et al., ) . in our study, we were able to observe low number of puncta within cells, as well as an agglomerate of puncta. the highest hybridization signal was mostly observed in glial cells. moreover, in several sections, we have seen puncta in dapi positive cells, suggesting a nuclear localization. however, ccl mrna was also localized outside the nucleus. thus, it appears that from the nucleus, ccl mrna might move to the cytoplasm, where the translation into ccl protein occurs. this event could be followed by ccl release. although measuring ccl release in vivo is technically challenging, this notion is supported by previous data showing that brain cells in vitro release ccl proteins (avdoshina et al., ) . a novel discovery presented here is the expression of ccl mrna in oligodendrocytes. the significance of this result is still under investigation. ccl promotes the proliferation of an oligodendrocyte precursor-like cell line (kadi et al., ) . thus, it is possible that oligodendrocyte precursors or other glial cells might be regulating their own proliferation via the release of ccl . oligodendrocyte most well known role is to figure | distribution of ccl mrna in the hippocampus. images are representative coronal sections showing the distribution of ccl mrna (red dots) in the dentate gyrus (dg) and ca regions of the hippocampus. the sections were stained for neun (a - ), iba- (b - ,c - ), gfap (d - ), or nkx . (e - ). dapi (blue) was used as a counterstaining. panels (a -e ) are higher magnifications of areas pointed by arrows. scale bar = µm for (a -e ), and µm for high magnification images (a -e ). preserve myelin; however, oligodendrocytes also maintain axonal integrity, support axonal metabolism and aid neuronal survival (peferoen et al., ) . changes in oligodendrocytes number and dynamics are increasingly recognized as important components in the pathogenesis of neurodegenerative disorders. in taiep rats, an animal model for multiple sclerosis, ccl and its receptor ccr is down-regulated in comparison to sprague-dawley rats (soto-rodriguez et al., ) . such reduction has been shown to limit the infiltration of pro-inflammatory macrophages and therefore to prevent demyelination (glass et al., ) . on the other hand, ccl has neuroprotective properties. for example, ccl has been shown to exert neuroprotective activity against various neurotoxins including glutamate (bruno et al., ) , β-amyloid (ignatov et al., ) , and the viral proteins gp (campbell et al., ) and tat (rozzi et al., ) . the neuroprotective activity of ccl could be due to its ability to increase neurotrophic factors, such as brain-derived neurotrophic factor and epidermal growth factor (tokami et al., ) . thus, it is conceivable to suggest that oligodendrocyte-derived ccl could act as a regulator of neuronal survival. our studies showed that some neuronal populations in selected brain areas exhibit ccl mrna. ccl is not the only chemokine that is expressed in neurons. for example, ccl is constitutively expressed in neurons, and co-localizes with various neurotransmitters and neuropeptides (banisadr et al., ) . also, cxcl , cxcl and cx cl are expressed in neurons in several brain regions (nishiyori et al., ; banisadr et al., ) . in addition, neurons, at least in culture, are capable of releasing ccl upon depolarization or activation of n-methyl-d-aspartate receptors , suggesting that neurons, under the proper stimulation, are capable of releasing ccl , not dissimilar to that observed for neurotransmitters. here, we show that neun positive cells in the cerebral cortex and in the hippocampus are also ccl mrna positive; together with previous findings that cortical neurons release ccl (avdoshina et al., ) , our data suggest that ccl mrna can be translated into proteins in neurons. moreover, few th-positive cells in the vta expressed ccl mrna, indicating that ccl may be expressed by a subset of dopaminergic neurons. why ccl mrna is detected only a subset of dopamine neurons is at present unclear. the vta also contains gabaergic neurons (dobi et al., ) . thus, the co-localization of ccl mrna in th-negative but neun positive cells in the vta suggests that ccl mrna may be synthetized in gabaergic neurons. more experiments are needed to support this suggestion. our data show that ccl mrna is contained in a subset of dopaminergic neurons. the significance of this finding remains speculative at present. dopamine neurons of the vta belong to the dopaminergic mesolimbic system, which comprises several interconnected brain regions, including the nucleus accumbens and the prefrontal cortex. activation of this system is crucial for the reward and addictive properties of opioids (wise and rompre, ; pontieri et al., ) . we have previously shown that ccl protein levels are increased in morphine dependent rats in brain areas involved in opioid reward . ccl has also been shown to interact with opioid receptors causing heterologous desensitization (szabo et al., ) . thus, ccl , when produced in dopaminergic fibers innervating the nucleus accumbens or prefrontal cortex could be contributing to regulation of dependence to opioids as previously suggested . ccl might act as a neuromodulator to enhance/inhibit the synaptic transmission underlying opioid reward. although still speculative, this suggestion is supported by data showing that ccr , the receptor for ccl , is expressed in selected neuronal populations of the frontal cortex and striatum (avdoshina et al., ) . moreover, ccl increases the release of glutamate (musante et al., ; di prisco et al., ) whose activity could induce various forms of synaptic plasticity and cellular adaptations that are responsible for opioid tolerance and addiction (manzoni and williams, ; stuber et al., stuber et al., , . in conclusion, we were able to detect ccl mrna by in situ hybridization in adult rat brain in several cells, including a subset of neurons. together with previous results showing that ccl and its receptor ccr are constitutively produced in the adult rodent brain (tran et al., ; avdoshina et al., ; campbell et al., ) , our data suggest a role for ccl in the cns other than a homeostatic chemokine that attracts and activates mononuclear phagocytes at sites of inflammation. mfl, im and sv designed the experiments and wrote the manuscript. mfl and sv performed the in situ hybridization and immunofluorescence experiments and analyzed the data. im, sv and mpb revised the manuscript. all authors reviewed the results and approved the final version of the manuscript. this work was supported by the dean of biomedical research (toulmin pilot project) provided by the georgetown university medical center (im), nih grants r ns (sv), r ns (mpb) and r ns (im) from the national institute for neurological disorders and stroke. viewing chemokines as a third major system of communication in the brain neurotrophins modulate the expression of chemokine receptors in the brain morphine induces the release of ccl from astrocytes: potential neuroprotective mechanism against the hiv protein gp rantes promotes growth and survival of human first-trimester forebrain astrocytes interferon-γ-oligodendrocyte interactions in the regulation of experimental autoimmune encephalomyelitis the chemokine brak/cxcl regulates synaptic transmission in the adult mouse dentate gyrus stem cell niche highly regionalized neuronal 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accumbens facilitates reward seeking heterologous desensitization of opioid receptors by chemokines inhibits chemotaxis and enhances the perception of pain rantes has a potential to play a neuroprotective role in an autocrine/paracrine manner after ischemic stroke chemokine receptor expression by neural progenitor cells in neurogenic regions of mouse brain determinants of ccl -driven mononuclear cell migration across the blood-brain barrier. implications for therapeutically modulating neuroinflammation sexual dimorphism in the inflammatory response to traumatic brain injury chemokines: integrators of pain and inflammation brain dopamine and reward the authors would like to thank dr. valeria avdoshina for comments on the manuscript and kierra s. jenkins for technical support. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fnana. . /full#supplementary-material figure s | examples of brain sections from wild-type (wt) and ccl knock-out (ko) mice stained for ccl using different commercially available antibodies. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © lanfranco, moccheti, burns and villapol. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -u apzw authors: michael, edwin; sharma, swarnali; smith, morgan e.; touloupou, panayiota; giardina, federica; prada, joaquin m.; stolk, wilma a.; hollingsworth, deirdre; de vlas, sake j. title: quantifying the value of surveillance data for improving model predictions of lymphatic filariasis elimination date: - - journal: plos negl trop dis doi: . /journal.pntd. sha: doc_id: cord_uid: u apzw background: mathematical models are increasingly being used to evaluate strategies aiming to achieve the control or elimination of parasitic diseases. recently, owing to growing realization that process-oriented models are useful for ecological forecasts only if the biological processes are well defined, attention has focused on data assimilation as a means to improve the predictive performance of these models. methodology and principal findings: we report on the development of an analytical framework to quantify the relative values of various longitudinal infection surveillance data collected in field sites undergoing mass drug administrations (mdas) for calibrating three lymphatic filariasis (lf) models (epifil, lymfasim, and transfil), and for improving their predictions of the required durations of drug interventions to achieve parasite elimination in endemic populations. the relative information contribution of site-specific data collected at the time points proposed by the who monitoring framework was evaluated using model-data updating procedures, and via calculations of the shannon information index and weighted variances from the probability distributions of the estimated timelines to parasite extinction made by each model. results show that data-informed models provided more precise forecasts of elimination timelines in each site compared to model-only simulations. data streams that included year post-mda microfilariae (mf) survey data, however, reduced each model’s uncertainty most compared to data streams containing only baseline and/or post-mda or longer-term mf survey data irrespective of mda coverage, suggesting that data up to this monitoring point may be optimal for informing the present lf models. we show that the improvements observed in the predictive performance of the best data-informed models may be a function of temporal changes in inter-parameter interactions. such best data-informed models may also produce more accurate predictions of the durations of drug interventions required to achieve parasite elimination. significance: knowledge of relative information contributions of model only versus data-informed models is valuable for improving the usefulness of lf model predictions in management decision making, learning system dynamics, and for supporting the design of parasite monitoring programmes. the present results further pinpoint the crucial need for longitudinal infection surveillance data for enhancing the precision and accuracy of model predictions of the intervention durations required to achieve parasite elimination in an endemic location. we report on the development of an analytical framework to quantify the relative values of various longitudinal infection surveillance data collected in field sites undergoing mass drug administrations (mdas) for calibrating three lymphatic filariasis (lf) models (epifil, lym-fasim, and transfil), and for improving their predictions of the required durations of drug interventions to achieve parasite elimination in endemic populations. the relative information contribution of site-specific data collected at the time points proposed by the who monitoring framework was evaluated using model-data updating procedures, and via calculations of the shannon information index and weighted variances from the probability distributions of the estimated timelines to parasite extinction made by each model. results show that data-informed models provided more precise forecasts of elimination timelines in each site compared to model-only simulations. data streams that included year post-mda microfilariae (mf) survey data, however, reduced each model's uncertainty most compared to data streams containing only baseline and/or post-mda or longer-term mf survey data irrespective of mda coverage, suggesting that data up to this monitoring point may be optimal for informing the present lf models. we show that the improvements observed in the predictive performance of the best data-informed models may be a function of temporal changes in inter-parameter interactions. such best data-informed models may also produce plos mathematical models of parasite transmission, via their capacity for producing dynamical forecasts or predictions of the likely future states of an infection system, offer an important tool for guiding the development and evaluation of strategies aiming to control or eliminate infectious diseases [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the power of these numerical simulation tools is based uniquely on their ability to appropriately incorporate the underlying nonlinear and multivariate processes of pathogen transmission in order to facilitate plausible predictions outside the range of conditions at which these processes are either directly observed or quantified [ ] [ ] [ ] [ ] . the value of these tools for guiding policy and management decisions by providing comparative predictions of the outcomes of various strategies for achieving the control or elimination of the major neglected tropical diseases (ntds) has been highlighted in a series of recent publications [ , , ] , demonstrating the crucial role these quantitative tools are beginning to play in advancing policy options for these diseases. while these developments underscore the utility of transmission models for supporting policy development in parasite control, a growing realization is that these models can be useful for this purpose only if the biological processes are well defined and demographic and environmental stochasticity are either well-characterized or unimportant for meeting the goal of the policy modelling exercise [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this is because the realized predictability of any model for a system depends on the initial conditions, parameterizations and process equations that are utilized in its simulation such that model outcomes are strongly sensitive to the choice of values used for these variables [ ] . any misspecification of these system attributes will lead to failure in accurately forecasting the future behaviour of a system, with predictions of actual future states becoming highly uncertain even when the exact representation of the underlying deterministic process is well established but precise specification of initial conditions or forcing and/or parameter values is difficult to achieve [ , ] . this problem becomes even more intractable when theoretical models depend on parameter estimates taken from other studies [ , , ] . both these challenges, viz. sensitivity to forcing conditions and use of parameter estimates from settings that are different from the dynamical environment in which a model will be used for simulation, imply that strong limits will be imposed on the realized predictability of any given model for an application [ , , ] . as we have shown recently, if such uncertainties are ignored, the ability of parasite transmission models to form the scientific basis for management decisions can be severely undermined, especially when predictions are required over long time frames and across heterogeneous geographic locations [ , , ] . these inherent difficulties with using an idealized model for producing predictions to guide management have led to consideration of data-driven modelling procedures that allow the use of information contained within observations to improve specification and hence the predictive performance of process-based models [ , , , [ ] [ ] [ ] . such approaches, termed model-data fusion or data assimilation methods, act by combining models with various data streams (including observations made at different spatial or temporal scales) in a statistically rigorous way to inform initial conditions, constrain model parameters and system states, and quantify model errors. the result is the discovery of models that can more adequately capture the prevailing system dynamics in a site, an outcome which in turn has been shown to result in the making of significantly improved predictions for management decision making [ , , , ] . initially used in geophysics and weather forecasting, these methods are also beginning to be applied in ecological modelling, including more recently in the case of infectious disease modelling [ , ] . in the latter case, the approach has shown that it can reliably constrain a disease transmission model during simulation to yield results that approximate epidemiological reality as closely as possible, and as a consequence improve the accuracy of forecasts of the response of a pathogen system exposed to various control efforts [ - , , - ] . more recently, attention has also focused on the notion that a model essentially represents a conditional proposition, i.e. that running a model in a predictive mode presupposes that the driving forces of the system will remain within the bounds of the model conceptualization or specification [ ] . if these driving forces were to change, then it follows that even a model well-calibrated to a given historical dataset will fail. new developments in longitudinal data assimilation can mitigate this problem of potential time variation of parameters via the recursive adjustment of the model by assimilation of data obtained through time [ , , ] . apart from allowing assessment of whether stasis bias may occur in model predictions, such sequential model calibration with time-varying data can also be useful for quantifying the utility of the next measurement in maximizing the information gained from all measurements together [ ] . carrying out such longitudinal model-data analysis has thus the potential for providing information to improve the efficiency and cost-effectiveness of data monitoring campaigns [ , [ ] [ ] [ ] , along with facilitating more reliable model forecasts. a key question, however, is evaluating which longitudinal data streams provide the most information to improve model performance [ ] . indeed, it is possible that from a modelling perspective using more data may not always lead to a better-constrained model [ ] . this suggests that addressing this question is not only relevant to model developers, who need observational data to improve, constrain, and test models, but also for disease managers working on the design of disease surveillance plans. at a more philosophical level, we contend that these questions have implications for how current longitudinal monitoring data from parasite control programmes can best be exploited both scientifically and in management [ ] . specifically, we suggest that these surveillance data need to be analysed using models in a manner that allows the extraction of maximal information about the monitored dynamical systems so that this can be used to better guide both the collection of such data as well as the provision of more precise estimates of the system state for use in making state-dependent decisions [ , [ ] [ ] [ ] . currently, parasite control programmes use infection monitoring data largely from sentinel sites primarily to determine if an often arbitrarily set target is met [ ] . little consideration is given to whether these data could also be used to learn about the underlying transmission dynamics of the parasitic system, or how such learning can be effectively used by management to make better decisions regarding the interventions required in a setting to meet stated goals [ , ] . here, we develop an analytical framework to investigate the value of using longitudinal lf infection data for improving predictions of the durations of drug interventions required for achieving lf elimination by coupling data collected during mass drug interventions (mdas) carried out in three example field sites to three existing state-of-the-art lymphatic filariasis (lf) models [ , , , [ ] [ ] [ ] [ ] [ ] [ ] . to be managerially relevant to current who-specified lf intervention surveillance efforts, we evaluated the usefulness of infection data collected in these sites at the time points proposed by the who monitoring framework in carrying out the present assessment [ ] . this was specifically performed by ranking these different infection surveillance data streams according to the incremental information gain that each stream provided for reducing the prediction uncertainty of each model. longitudinal pre-and post-infection and mda data from representative sites located in each of the three major regions endemic for lf (africa, india, and papua new guinea (png)) were assembled from the published literature for use in constraining the lf models employed in this study. the three sites (kirare, tanzania, alagramam, india, and peneng, png) were selected on the basis that each represents the average endemic transmission conditions (average level of infection, transmitting mosquito genus) of each of these three major extant lf regions, while providing details on the required model inputs and data for conducting this study. these data inputs encompassed information on the annual biting rate (abr) and dominant mosquito genus, as well as mda intervention details, including the relevant drug regimen, time and population coverage of mda, and times and results of the conducted microfilaria (mf) prevalence surveys (table ) . note each site also provided these infection and mda data at the time points pertinent to the existing who guidelines for conducting lf monitoring surveys during a mda programme [ ] , which additionally, as pointed out above, allowed the assessment of the value of such infection data both for supporting effective model calibration and for producing more reliable intervention forecasts. the three existing lf models employed for this study included epifil, a deterministic monte carlo population-based model, and lymfasim and transfil, which are both stochastic, individual-based models. all three models simulate lf transmission in a population by accounting for key biological and intervention processes such as impacts of vector density, the life cycle of the parasite, age-dependent exposure, density-dependent transmission processes, infection aggregation, and the effects of drug treatments as well as vector control [ , , - , , , ] . although the three models structurally follow a basic coupled immigration-death model formulation, they differ in implementation (e.g. from individual to population-based), the total number of parameters included, and the way biological and intervention processes are mathematically incorporated and parameterized. the three models have been compared in recent work [ , ] , with full details of the implementation and simulation procedures for each individual model also described [ , , , , , , , , ] . individual model parameters and fitting procedures specific to this work are given in detail in s supplementary information. we used longitudinal data assimilation methods to sequentially calibrate the three lf models with the investigated surveillance data such that parameter estimates and model predictions reflect not only the information contained in the baseline but also follow-up data points. the available mf prevalence data from each site were arranged into four different temporal data streams to imitate the current who guidelines regarding the time points for conducting monitoring surveys during an mda programme. this protocol proposes that infection data be collected in sentinel sites before the first round of mda to establish baseline conditions, no sooner than months following the third round of mda, and no sooner than months following the fifth mda to assess whether transmission has been interrupted (defined as reduction of mf prevalence to below % in a population) [ , ] . thus, the four data streams considered for investigating the value of information gained from each survey were respectively: scenario -baseline mf prevalence data only, scenario -baseline and post-mda mf prevalence data, scenario -baseline, post-mda , and post-mda mf prevalence data, and scenario -baseline and post-mda mf prevalence data. in addition to these four data streams, a fifth model-only scenario (scenario ) was also considered where no site-specific data was introduced. in this case, simulations of interventions were performed using only model-specific parameter and abr priors estimated for each region. the first step for all models during the data assimilation exercises reported here was to initially simulate the baseline infection conditions in each site using a large number of samples ( , for epifil and transfil, and , - , for lymfasim) randomly selected from the parameter priors deployed by each model. the number of parameters which were left free to be fitted to these data by each model range from (lymfasim and transfil) to (epifil). the abr, a key transmission parameter in all three models, was also left as a free parameter whose distribution was influenced by the observed abr (table ) and/or by fits to previous region-specific datasets (see s supplementary information for model-specific implementations). the subsequent steps used to incorporate longitudinal infection data into the model calibration procedure varied among the models, but in all cases the goodness-of-fit of the model outputs for the site-specific mf prevalence data was assessed using the chi-square metric (α = . ) [ ] . epifil used a sequential model updating procedure to iteratively modify the parameters with the introduction of each subsequent follow up data point through time [ ] . this process uses parameter estimates from model fits to previous data as priors for the simulation of the next data which are successively updated with the introduction of each new observation, thus providing a flexible framework by which to constrain a model using newly available data. fig summarizes the iterative algorithm used for conducting this sequential model-data assimilation exercise [ ] . lymfasim and transfil, by contrast, included all the data in each investigated stream together for selecting the best-fitting models for each time series-i.e. model selection for each data series was based on using all relevant observations simultaneously in the fitting process [ , , ] . although a limitation of this batch estimation approach is that the posterior probability of each model is fixed for the whole simulation period, unlike the case in sequential data assimilation where a restricted set of parameters is exposed to each observation (as a result of parameter constraining by data used in the previous time step)which thereby yields models that give better predictions for different portions of the underlying temporal process-here we use both methods to include and assess the impact that this implementation difference may have on the results presented below. for all models, the final updated parameter estimates from each data stream were used to simulate the impact of observed mda rounds and for predicting the impact of continued mda to estimate how many years were required to achieve % mf prevalence. interventions were modelled by using the updated parameter vectors or models selected from each scenario for simulating the impact of the reported as well as hypothetical future mda rounds on the number of years required to reduce the observed baseline lf prevalence in each site to below the who transmission threshold of % mf prevalence [ ] . when simulating these interventions, the observed mda times, regimens, and coverages followed in each site were used (table ) , while mda was assumed to target all residents aged years and above. for making mf prevalence forecasts beyond the observations made in each site, mda simulations were extended for a total of annual rounds in each site at an assumed coverage of %. while the drug-induced mf kill rate and the duration of adult worm sterilization were fixed among the models (table ) , the worm kill rate was left as a free parameter to be estimated from post-intervention data to account for the uncertainty in this drug efficacy parameter [ , , ] . the number of years of mda required to achieve the threshold of % mf prevalence was calculated from model forecasts of changes in mf prevalence due to mda for each modeldata fusion scenario. the predictions from each model regarding timelines to achieve % mf for each fitting scenario were used to determine the information gained from each data stream compared to the in all scenarios, the initial epifil models were initialized with parameter priors and a chi-square fitting criterion was applied to select those models which represent the baseline mf prevalence data sufficiently well (α = . ). the accepted models were then used to simulate the impact of interventions on mf prevalence. the chi-square fitting criterion was sequentially applied to refine the selection of models according to the post-mda mf prevalence data included in the fitting scenario. the fitted parameters from selection of acceptable models at each data point were used to predict timelines to achieve % mf prevalence. the scenarios noted in the blue boxes indicate the final relevant updating step before using the fitted parameters to predict timelines to achieve % mf in that data fitting scenario. information attributable to the model itself [ , , ] . the relative information gained from a particular data stream was calculated as i d = h m -h md where h measures the entropy or uncertainty associated with a random variable, h m denotes predictions from the model-only scenario (scenario ) which essentially represents the impact of prior knowledge of the system, and h md signifies predictions from each of the four model-data scenarios (i.e. scenarios [ ] [ ] [ ] [ ] . the values of i d for each data scenario or stream were compared in a site to infer which survey data are most useful for reducing model uncertainty. the shannon information index was used to measure entropy, h, as follows: is the discrete probability density function (pdf) of the number of years of mda predicted by each fitted model to reach % mf, and is estimated from a histogram of the respective model predictions for m bins (of equal width in the range between the minimum and maximum values of the pdfs) [ , ] . to statistically compare two entropy values, a permutation test using the differential shannon entropy (dse) was performed [ ] . dse is defined as |h -h | where h was calculated from the distribution of timelines to achieve % mf for a given scenario, y , and h was calculated from the distribution of timelines to achieve % mf for a different scenario, y . the list of elements in y and y were combined into a single list of size y + y and the list was permuted , times. dse was then recalculated each time by calculating a new h from the first y elements and a new h from the last y elements from each permutation, from which p-values may be quantified as the proportion of all recalculated dses that were greater than the original dse. model predictions of the mean and variance in timelines to lf elimination were weighted according to the frequencies by which predictions occurred in a group of simulations. in general, if d , d ,. . .,d n are data points (model predictions in the present case) that occur in an ensemble of simulations with different weights or frequencies w ,w ,. . .,w n , then the weighted mean, here, n is the number of data points and n is the number of non-zero weights. in this study, the weighted variance of the distributions of predicted timelines to achieve % mf prevalence was calculated to provide a measure of the precision of model predictions in addition to the entropy measure, h. a similar weighting scheme was also used to pool the timeline predictions of all three models. here, predictions made by each of the three models for each data scenario were weighted as above, and a composite weighted % percentile interval for the pooled predictions was calculated for each data stream. this was done by first computing the weighted percentiles for the combined model simulations from which the pooled . th and . th percentile values were quantified. the matlab function, wprctile, was used to carry out this calculation. the extent by which parameter constraints are achieved through the coupling of models with data was evaluated to determine if improvements in such constraints by the use of additional data may lead to reduced model prediction uncertainty [ ] . parameter constraint was calculated as the ratio of the mean standard deviation of all fitted parameter distributions to the mean standard deviation of all prior parameter distributions. a ratio of less than one indicates the fitted parameter space is more constrained than the prior parameter space [ ] . this assessment was carried out using the epifil model only. in addition, pairwise parameter correlations were also evaluated to assess whether the sign, magnitude, and significance of these correlations changed by scenario to determine if using additional data might alter these interactions to better constrain a model. for this assessment, spearman's correlation coefficients and p-values testing the hypothesis of no correlation against the alternative of correlation were calculated, and the exercise was run using the estimated parameters from the epifil model. epifil was used to conduct a sensitivity analysis investigating whether the trend in relative information gained by coupling the model with longitudinal data was dependent on the interventions simulated. the same series of simulations (for three lf endemic sites and five fitting scenarios) were completed with the extended mda coverage beyond the observations given in table set here at % instead of % to represent an optimal control strategy. as before, the timelines to reach % mf prevalence in each fitting scenario were calculated and used to determine which data stream provided the model with the greatest gain of information. the results were compared to the original series of simulations to assess whether the trends are robust to changes in the intervention coverages simulated. epifil was also used to perform another sensitivity analysis expanding the number of data streams to investigate if the who monitoring scheme is adequate for informing the making of reliable model-based predictions of timelines for achieving lf elimination. to perform this sensitivity analysis, pre-and post-mda data from villupuram district, india that provide extended data points (viz. scenario - as previously defined, plus scenario -baseline, post-mda , post-mda , and post-mda mf prevalence data, and scenario -baseline, post-mda , post-mda , post-mda , and post-mda mf prevalence) were assembled from the published literature [ , ] . the timelines to reach % mf prevalence and the entropy for each of these additional scenarios were calculated to determine whether additional data streams over those recommended by who are required for achieving more reliable model constraints, which among these data might be considered as compulsory, and which might be optional for supporting predictions of elimination. differences in predicted medians, weighted variances and entropy values between data scenarios, models and sites were statistically evaluated using kruskall-wallis tests for equal medians, f-tests for equality of variance, and dse permutation tests, respectively. p-values for assessing significance for all pairwise tests were obtained using the benjamini-hochberg procedure for controlling the false discovery rate, i.e. for protecting against the likelihood of obtaining false positive results when carrying out multiple testing [ ] . here, our goal was twofold. first, to determine if data are required to improve the predictability of intervention forecasts by the present lf models in comparison with the use of theoretical models only, and second, to evaluate the benefit of using different longitudinal streams of mf survey data for calibrating the three models in order to determine which data stream was most informative for reducing the uncertainty in model predictions in a site. table summarises the key results from our investigation of these questions: these are the number of accepted best-fitting models for each data stream or scenario in the three study sites (table ) , the predicted median and range ( . th - . th percentiles) in years to achieve the mf threshold of % mf prevalence, the weighted variance and entropy values based on these predictions, and the relative information gained (in terms of reduced prediction uncertainty) by the use of longitudinal data for constraining the projections of each of the three lf models investigated. even though the number of selected best-fit models based on the chi-square criterion (see methods) differed for each site and model, these results indicate unequivocally that models constrained by data provided significantly more precise intervention predictions compared to model-only predictions ( table ). note that this was also irrespective of the two types of longitudinal data assimilation procedures (sequential vs. simultaneous) used by the different models in this study. thus, for all models and sites, model-only predictions made in the absence of data (scenario ) showed the highest prediction uncertainty, highlighting the need for data to improve the predictive performance of the present models. the relative information gained by using each data stream in comparison to model-only predictions further support this finding, with the best gains in reducing model prediction uncertainty provided by those data constraining scenarios that gave the lowest weighted variance and entropy values; as much as % to % reductions in prediction variance were achieved by these scenarios in comparison to modelonly predictions between the three models ( table ). the results also show, however, that data streams including post-mda mf survey data (scenarios and ) reduced model uncertainty (based on both the variance and entropy measures) most compared to data streams containing only baseline and/or post-mda mf survey data (scenarios and ) ( table ) . although there were differences between the three models (due to implementation differences either in how the models are run (monte carlo deterministic vs. individual-based) or in relation to how the present data were assimilated (see above)), overall, scenario , which includes baseline, post-mda , and post-mda data, was shown to most often reduce model uncertainty the greatest. additionally, there was no statistical difference between the performances of scenarios and in those cases where scenario resulted in the greatest gain of information (table ) . it is also noticeable that the best constraining data stream for each combination of site and model also produced as expected the lowest range in predictions of the numbers of years of annual mda required to achieve the % mf prevalence in each site, with the widest ranges estimated for model-only predictions (scenario ) and the shorter data streams (scenario ). in general, this constriction in predictions also led to lower estimates of the median times to achieve lf elimination, although this varied between models and sites ( table ) . the change in the distributions of predicted timelines to lf elimination without and with model constraining by the different longitudinal data streams is illustrated in fig for the kirare site (see s supplementary information for results obtained for the other two study villages investigated here). the results illustrate that both the location and length of the tail of the prediction distributions can change as models are constrained with increasing lengths of longitudinal data, with inclusion of post-mda mf survey data consistently producing a narrower or sharper range of predictions compared to when this survey point is excluded. fig compares the uncertainty in predictions of timelines to achieve elimination made by each of the three models without (scenario ) and via their constraining by the data streams providing the lowest prediction entropy for each of the models per site. note that variations in scenario predictions among the three models directly reflect the different model structures, parameterizations, and the presence (or absence) of stochastic elements. the boxplots in the figure, however, show that for all three sites and models, calibration of each model by data the lowest entropy scenario for each site is bolded and shaded grey. additional scenarios shaded grey are not significantly different from the lowest entropy scenario. data assimilation in filarial model predictions greatly reduces the uncertainty in predictions of the years of annual mda required to eliminate lf compared to model-only predictions, with the data streams producing the lowest entropy for simulations in each site significantly improving the precision of these predictions ( table ). this gain in precision, and thus the information gained using these data streams, is, as expected, greater for the stochastic lymfasim and transfil models compared to the deterministic epifil model. note also that even though the ranges in predictions of the annual mda years required to eliminate lf by the data streams providing the lowest prediction entropy differed statistically between the three models, the values overlapped markedly (e.g. for kirare the ranges are - , - , - for epifil, lymfasim and transfil data assimilation in filarial model predictions respectively), suggesting the occurrence of a similar constraining of predictive behaviour among the three models. to investigate this potential for a differential model effect, we further pooled the predictions from all three models for all the data scenarios and evaluated the value of each investigated data stream for improving their combined predictive ability. the weighted % percentile intervals from the pooled predictions were used for carrying out this assessment. the results are depicted in fig and indicate that, as for the individual model predictions, uncertainty in the collective predictions by the three lf models for the required number of years to eliminate lf using annual mda in each site may be reduced by model calibration to data, with the longitudinal mf prevalence data collected during the later monitoring periods (scenarios and ) contributing most to improving the multi-model predictions for each site. the boxplots show that by calibrating the models to data streams, more precise predictions are able to be made regarding timelines to achieve % mf prevalence across all models and sites. the results of pairwise f-tests for variance, performed to compare the weighted variance in timelines to achieve % mf prevalence between model-only simulations (scenario ) and the lowest entropy simulations (best scenario) (see table ), show that the predictions for the best scenarios are significantly different from the predictions for the model-only simulations. significance was determined using the benjamini-hochberg procedure for controlling the false discovery rate (q = . ). for epifil, lymfasim and transfil, the best scenarios are scenarios , , and for kirare, scenarios , , and for alagramam, and scenarios , , and for peneng, respectively. we attempted to investigate if model uncertainty in predictions by the use of longitudinal data was a direct function of parameter constraining by the addition of data. given the similarity in outcomes of each model, we remark on the results from the fits of the technically easier to run epifil model to evaluate this possibility here. the assessment of the parameter space constraint achieved through the inclusion of data was made by determining if the fitted parameter distributions for the model became reduced in comparison with priors as data streams were added to the system [ ] . the exercise showed that the size of the estimated parameter distributions reduced with addition of data, with even scenario data producing reductions for kirare and peneng (fig ) . in the case of alagramam, however, there was very little, if any, constraint in the fitted parameter space compared to the prior parameter space. this result, together with the fact that even using all the data in kirare and peneng produced up to only between . to % reductions in fitted parameter distributions when compared to the priors, indicate that the observed model prediction uncertainty in this study may be due to other complex factors connected with model parameterization. table provides the results of an analysis of pairwise parameter correlations of the selected best-fitting models for data scenario compared to those selected by the data stream that gave the best reduction in epifil prediction uncertainty for alagramam (scenario ). these results show that while the parameter space was not being constrained with the addition of more data, the pattern of parameter correlations changed in a complex manner between the two constraining data sets. for example, although the number of significantly correlated parameters did not differ, the magnitude and direction of parameter correlations were shown to change between the two data scenarios ( table ) . the corresponding results for kirare and peneng are shown in s supplementary information , and indicate that a broadly similar pattern of changes in parameter associations also occurred as a result of model calibration to the sequential data measured from those sites. this suggests that this outcome may constitute a general phenomenon at least with regards to the sequential constraining of epifil using longitudinal mf prevalence data. an intriguing finding (from all three data settings) is that the most sensitive parameters in this regard, i.e. with respect to altered strengths in pairwise parameter correlations, may be those representing the relationship of various components of host immunity with different transmission processes, including with adult worm mortality, rates of production and survival of mf, larval development rates in the mosquito vector and infection aggregation (table ) . this suggests that, as more constraining data are added, changes in the multidimensional parameter relationships related to host immunity could contribute to the sequential reductions in the lf model predictive uncertainty observed in this study. the lf elimination timeline predictions used above were based on modelling the impacts of annual mda given the reported coverages in each site followed by an assumed standard coverage for making longer term predictions (see methods). this raises the question as to whether the differences detected in the case of the best constraining data stream between the present study sites and between models ( table ) could be a function of the simulated mda coverages in each site. to investigate this possibility, we used epifil to model the outcome of changing the assumed mda coverage in each site on the corresponding entropy and information gain trends in elimination predictions made from the models calibrated to each of the site-specific data scenarios/streams investigated here. the results of increasing the assumed coverage of mda to % for each site are shown in fig and indicate that the choice of mda coverage in this study are unlikely to have significantly influenced the conclusion made above that the best performing data streams for reducing model uncertainty for predicting lf elimination pertains to data scenarios and . however, while the model-predicted timelines to achieve the % mf prevalence threshold using the observed mda coverage followed by % mda coverage showed that the data stream which most reduced uncertainty did not change from the impact of using the observed mda coverage followed by % mda coverage modelled for kirare and peneng (table , fig ) , this was not the case for alagramam, where data from scenario with a % coverage resulted in the greatest reduction in entropy compared to the original results using % coverage which indicated that scenario data performed best (table , fig ) . notably, though, the entropy values of predictions using the data scenario and constraints were not statistically different for this site (p-value < . ) (fig ) . epifil was also used to expand the number of calibration scenarios using a dataset with longer term post-mda data from villupuram district, india. this dataset contained two addition data streams: scenario which included baseline, post-mda , post-mda , and post-mda mf data, and scenario , which included baseline, post-mda , post-mda , post-mda , and post-mda mf data. scenario thus contained the most post-mda data and was demonstrated to be the most effective for reducing model uncertainty, but this effect was not statistically significantly different from the reductions produced by assimilating data contained in table . spearman parameter correlations for scenarios (lower left triangle) and (upper right triangle) for alagramam, india. data assimilation in filarial model predictions scenarios and ( table ). the inclusion of more data than are considered in scenario therefore did not result in any significant additional reduction in model uncertainty. epifil was used to evaluate the accuracy of the data-driven predictions of the timelines required to meet the goal of lf elimination based on breaching the who-set target of % mf for all sites, either scenario or had the lowest entropies, and scenario was not significantly different from scenario for kirare and alagramam. these results were not statistically different from the results given % coverage (see table ), suggesting that the data stream associated with the lowest entropy is robust to changes in the interventions simulated. scenarios where the weighted variance or entropy were not significantly different from the lowest entropy scenario are noted with the abbreviation ns. significance was determined using the benjamini-hochberg procedure for controlling the false discovery rate (q = . ). https://doi.org/ . /journal.pntd. .g table . predictions of timelines to achieve % mf in villupuram district, india, considering extended post-mda data. reporting those scenarios which are statistically significantly different from each other by numbers ( - ) as superscripts. for example, the weighted variance for scenario has the superscript numbers ( ) ( ) ( ) ( ) ( ) ( ) to indicate that the weighted variance for scenario is significantly different from the weighted variance for scenarios - . significance was determined using the benjamini-hochberg procedure for controlling the false discovery rate (q = . ) in all pairwise statistical tests. + information gained by each data stream (scenario - ) are presented in comparison to the information contained in the model-only simulation (scenario ) https://doi.org/ . /journal.pntd. .t data assimilation in filarial model predictions prevalence. this analysis was performed by using the longitudinal pre and post-infection and mda data reported for the nigerian site, dokan tofa, where elimination was achieved according to who recommended criteria after seven rounds of mda (table ). the data from this site comprised information on the abr and dominant mosquito genus, as well as details of the mda intervention carried out, including the relevant drug regimen applied, time and population coverage of mda, and outcomes from the mf prevalence surveys conducted at baseline and at multiple time points during mda [ ] . the results of model predictions of the timelines to reach below % mf prevalence as a result of sequential fitting to the mf prevalence data from this site pertaining to scenarios - (as defined above) are shown in table . note that in the post mda , and surveys, as no lf positive individuals were detected among the sample populations, we used a one-sided % clopper-pearson interval to determine the expected upper one-sided % confidence limits for these sequentially observed zero infection values data assimilation in filarial model predictions using the "rule of three" approximation after k empty samples formula [ ] . the results show that model constraining by scenario , which includes baseline and post-mda data, and scenario , which includes baseline, post-mda , and post-mda data, resulted in both the least entropy values and the shortest predicted times, i.e., from as low as to as high as years, required for achieving lf elimination in this site ( table ). the data in table show that the first instance the calculated one-sided upper % confidence limit in this setting fell below % mf prevalence also occurred post mda (i.e after years of mda). this is a significant result, and indicates that apart from being able to reduce prediction uncertainty, the best data-constrained models are also able to more accurately predict the maximal time ( years) by which lf elimination occurred in this site. our major goal in this study was to compare the reliability of forecasts of timelines required for achieving parasite elimination made by generic lf models versus models constrained by sequential mf prevalence surveillance data obtained from field sites undergoing mda. a secondary aim was to evaluate the relative value of data obtained at each of the sampling time points proposed by the who for monitoring the effects of lf interventions in informing these model predictions. this assessment allowed us to investigate the role of these data for learning system dynamics and measure their value for guiding the design of surveillance programmes in order to support better predictions of the outcomes of applied interventions. fundamentally, however, this work addresses the question of how best to use predictive parasite transmission models for guiding management decision making, i.e. whether this should be based on the use of ideal models which incorporate generalized parameter values or on models with parameters informed by local data [ ] . if we find that data-informed models can reduce prediction uncertainty significantly compared to the use of theoretical models unconstrained by data, then it is clear that to be useful for management decision making we require the application of model-data assimilation frameworks that can effectively incorporate information from appropriate data into models for producing reliable intervention projections. antithetically, such a finding implies that using unconstrained ideal models in these circumstances will provide only approximate predictions characterized by a degree of uncertainty that might be too large to be useful for reliable decision making [ , , ] . here, we have used three state-of-the-art lf models calibrated to longitudinal human mf prevalence data obtained from three representative lf study sites to carry out a systematic analysis of these questions in parasite intervention modelling (see also walker et al [ ] for a recent study highlighting the importance of using longitudinal sentinel site data for improving the prediction performances of the closely-related onchocerciasis models). further, by iteratively testing the reduction in the uncertainty of the projections of timelines required to achieve lf elimination in a site made by the models matching each observed data point, we have also quantified the relative values of temporal data streams, including assessing optimal record lengths, for informing the current lf models. our results provide important insights as to how best to use process models for understanding and generating predictions of parasite dynamics. they also highlight how site-specific longitudinal surveillance data coupled with models can be useful for providing information about system dynamics and hence for improving predictions of relevance to management decision-making. the first result of major importance from our work is that models informed by data can significantly reduce predictive uncertainty and hence improve performance of the present lf models for guiding policy and management decision-making. our results show that these improvements in predictive precision were consistent between the three models and across all three of our study sites, and can be very substantive with up to as much as % to % reductions in prediction variance obtained by the best data-constrained models in a site compared to the use of model-only predictions ( table ). the practical policy implications of this finding can also be gleaned from appraising the actual numerical ranges in the predictions made by each individual model for each of the modelling scenarios investigated here. in the case of epi-fil, the best data-informed model (scenario in peneng) gave an elimination prediction range of - years, while the corresponding model-only predictions for this site indicated a need for between - years of annual mda (table ). these gains in information from using data to inform model parameters and hence predictions were even larger for the two stochastic models investigated here, viz. lymfasim and transfil, where ranges as wide as - years predicted by model-only scenarios were reduced to - years for the best data-informed models in the case of lymfasim for kirare village, and from as broad as - years to - years respectively in the case of transfil for peneng (table ). these results unequivocally indicate that if parasite transmission models are used unconstrained by data, i.e. based on general parameter values uninformed by local data, it would lead to the making of predictions that would be marked by uncertainties that are likely to be far too large to be meaningful for practical policy making. if managers are risk averse, this outcome will also mean their need to plan interventions for substantially much longer than necessary, with major implications for the ultimate cost of the programme. note also that although statistically significant changes in the median years of mda required to achieve lf elimination were observed for the best datainformed models for all the three lf model types in each site, these were relatively small compared to the large reductions seen in each model's predictive uncertainly (table , fig ) . this result highlights that the major gains from constraining the present models by data lies in improving their predictive certainty rather than in advancing their average behaviour. however, our preliminary analysis of model predictive accuracy suggests that the best data-constrained models may also be able to generate more accurate predictions of the impact of control ( table ), indicating that, apart from simply reducing predictive uncertainty, such models could additionally have improved capability for producing more reliable predictions of the outcomes of interventions carried out in a setting. the iterative testing of the reduction in forecast uncertainty using mf surveillance data measured at time points proposed by the who (to support assessment of whether the threshold of % mf prevalence has been reached before implementation units can move to post-treatment surveillance [ ]) has provided further insights into the relative value of these data for improving the predictive performance of each of the present lf models. our critical finding here is that parameter uncertainty in all three lf models was similarly reduced by the assimilation of a few additional longitudinal data records (table ). in particular, we show that data streams comprising baseline + post-mda + post-mda (scenario ) and those comprising baseline + post-mda data (scenario ) best reduced parameter-based uncertainty in model projections of the impact of mdas carried out in each study site irrespective of the models used. although preliminary, a potential key finding is that the use of longer-term data additional to the data measured at the who proposed monitoring time points did not lead to a significant further reduction in parameter uncertainty (table ) . also, the finding that the who data scenarios and were adequate for constraining the present lf models appears not to be an artefact of variations in the mda coverages observed between the three study sites (fig ) . these results suggest that up to years of post-mda mf prevalence data are sufficient to constrain model predictions of the impact of lf interventions at a time scale that can go up to as high as to years depending on the site and model, and that precision may not improve any further if more new data are added ( table , table ). given that the who post-mda lf infection monitoring protocol was developed for the purpose solely focussed on supporting the meeting of set targets (e.g. the % mf prevalence threshold) and not on a priori hypotheses regarding how surveillance data could be used also to understand the evolution and hence prediction of the dynamical parasitic system in response to management action, our results are entirely fortuitous with respect to the value of the current lf monitoring data for learning about the lf system and its extinction dynamics in different settings [ ] . they do, nonetheless, hint at the value that coupling models to data may offer to inform general theory for guiding the collection and use of monitoring data in parasite surveillance programmes in a manner that could help extract maximal information about the underlying parasite system of interest. our assessment of whether the incremental increase in model predictive performance observed as a result of assimilating longitudinal data may be due to parameter constraining by the addition of data has shed intriguing new light on the impact that qualitative changes in dynamical system behaviour may have on parameter estimates and structure, and hence on the nature of the future projections of system change we can make from models. our major finding in this regard is that even though the parameter space itself may not be overly constrained by the best data stream (scenario in this case for alagramam village), the magnitude and direction of parameter correlations, particularly those representing the relationship of different components of host immunity with various transmission processes, changed markedly between the shorter (scenario ) and seemingly optimal data streams (scenario ). this qualitative change in system behaviour induced by alteration in parameter interactions in response to perturbations has been shown to represent a characteristic feature of complex adaptive ecological systems, particularly when these systems approach a critical boundary [ ] [ ] [ ] . this underscores yet another important reason to incorporate parameter information from data for generating sound system forecasts [ ] . the finding that additional data beyond years post-mda did not appear to significantly improve model predictive performance in this regard suggests that pronounced change in lf parameter interactions in response to mda interventions may occur generally around this time point for this parasitic disease, and that once in this parameter regime further change appears to be unlikely. this is an interesting finding, which not only indicates that coupling models to at least years post-mda will allow detection of the boundaries delimiting the primary lf parameter regions with different qualitative behaviour, but also that the current who monitoring protocol might be sufficient to allow this discovery of system change. although our principal focus in this study was in investigating the value of longitudinal data for informing the predictive performance of the current lf models, the results presented here have also underscored the existence of significant spatial heterogeneity in the dynamics of parasite extinction between the present sites ( table , fig ) . in line with our previous findings, this observed conditional dependency of systems dynamics on local transmission conditions means that timelines or durations of interventions required to break lf transmission (as depicted in table ) will also vary from site to site even under similar control conditions [ ] [ ] [ ] ] . as we indicated before, this outcome implies that we vitally require the application of models to detailed spatio-temporal infection surveillance data, such as that exemplified by the data collected by countries in sentinel sites as part of their who-directed monitoring and evaluation activities, if we are to use the present models to make more reliable intervention predictions to drive policy and management decisions (particularly with respect to the durations of interventions required, need for switching to more intensified or new mda regimens, and need for enhanced supplementary vector control) in a given endemic setting [ ] . as we have previously pointed out, the development of such spatially adaptive intervention plans will require the development and use of spatially-explicit data assimilation modelling platforms that can couple geostatistical interpolation of model inputs (eg. abr and/or sentinel site mf/ antigen prevalence data) with discovery of localized models from such data in order to produce the required regional or national intervention forecasts [ ] . the estimated parameter and prediction uncertainties presented here are clearly dependent on the model-data fusion methodology and its implementation, and the cost function used to discover the appropriate models for a data stream [ ] . while we have attempted to evaluate differences in individual model structures, their computer implementation, and the data assimilation procedures followed (e.g. sequential vs. simultaneous data assimilation), via comparing the collective predictions of the three models versus the predictions provided by each model singly, and show that these factors are unlikely to play a major role in influencing the current results, we indicate that future work must address these issues adequately to improve the initial methods we have employed in this work. currently, we are examining the development of sequential bayesian-based multi-model ensemble approaches that will allow better integration of each model's behaviour as well as better calculation of each model's transient parameter space at each time a new observation becomes available [ ] . this work also involves the development of a method to fuse information from several indicators of infection (e.g. mf, antigenemia, antibody responses [ ] ) together to achieve a more robust constraining of the present models. as different types of data can act as mutual constraints on a model, we also expect that such multiindicator model-data fusion methods will additionally address the problem of equifinality, which is known to complicate the parameterization of complex dynamical models [ , ] . of course, the ultimate test of the results reported here, viz. that lf models constrained by coupling to year post-mda data can provide the best predictions of timelines for meeting the % mf prevalence threshold in a site, is by carrying out the direct validation of our results against independent observations (as demonstrated by the preliminary validation study carried out here using the dokan tofa data (tables and )). we expect that data useful for performing these studies at scale may be available at the sentinel site level in the countries carrying out the current who-led monitoring programme. the present results indicate that access to such data, and to post-treatment surveillance data which are beginning to be assembled by many countries, is now a major need if the present lf models are to provide maximal information about parasite system responses to management and thus generate better predictions of system states for use in policy making and in judging management effectiveness in different spatiotemporal settings [ , ] 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probability of virus shedding. methods: employing a statistical model that is considered to capture the data-generating process of the outbreak and virus surveillance, maximum likelihood estimation of the asymptomatic ratio was implemented. results: assuming that all norovirus outbreaks (n = ) were the result of random sampling from an identical distribution and ignoring genogroup and genotype specificities, the asymptomatic ratio was estimated at . % ( % confidence interval [ci], . – . ). although not significant, separate estimation of the asymptomatic ratio of the gii. genotype appeared to be greater than other genotypes and was estimated at . % ( % ci, . – . ). conclusion: the present study offered the first explicit empirical estimates of the asymptomatic ratio of norovirus infection in natural infection settings. the estimate of about % was consistent with those derived from volunteer challenge studies. practical difficulty in controlling gii. outbreaks was supported by the data, considering that a large estimate of the asymptomatic ratio was obtained for the gii. genotype. noroviruses are an evolutionarily diverse group of singlestranded positive-sense rna viruses without the envelope, belonging to the caliciviridae family, and are responsible for a substantial part of acute viral gastroenteritis in humans. , norovirus outbreaks have happened both in developing and industrialized nations. once infected, symptoms are characterized by non-bloody diarrhea, vomiting, nausea, and abdominal cramps, and there has been no specific treatment to accelerate the cure of patients infected with the virus. vaccines have yet to be fully developed and brought into practice, and the presence of various genotypes prevents infected human from acquiring sufficient specific immunity. published studies have reported that around % of norovirus infection remains asymptomatic, while such asymptomatically infected individuals are known to excrete substantial volume of viruses. , vomit from infectious individuals contains over copies=gram of noroviruses, , and infectious vomitus is known to be sometimes aerosolized, , making it difficult to prevent secondary transmissions. the virus spreads through fecal-oral routes, such as via contaminated water, food, and people's contaminated hands. , published studies indicate that infections associated with environmental contaminants are mainly caused by contaminated water or foods (especially oysters), accounting for seasonal outbreaks that are frequently observed in winter. given these features, workers who are directly engaged in cooking and handling of foods, such as cooks, food servers, and food factory workers, are considered as one of the key subjects for prevention of secondary transmissions. [ ] [ ] [ ] one of the notable virological features of noroviruses is genotype variation, which is divided into five genogroups and many genotypes in each genogroup. as of , the most prevalent genotype in humans is gii. , , which accounts for as many as % of all reported norovirus infections with virus isolation. some pieces of evidence indicate that this genotype evolves rapidly, escaping from selection pressure. , in japan, the majority of outbreaks are caused by genogroup ii, and a large proportion is caused by gii. . gii. -associated epidemics in japan were first observed in , and the genotype has been continuously observed every year since then. to decipher the most effective preventive measures against this virus, it is vital to quantitatively clarify the natural history characteristics, including asymptomatic ratio, the risk of infection per exposure, and the probability of virus shedding. nevertheless, explicit estimates are very scarce, except for rigorous challenge studies that helped quantify the natural history , ; moreover, it is unclear if the natural history of experimentally infected individuals are similar to those based on natural infection. a small number of mathematical modelling studies estimated a part of the abovementioned values, - but explicit model-based estimates of the asymptomatic ratio have yet to be offered. the present study aims to estimate the asymptomatic ratio of norovirus infection, reanalyzing foodborne outbreak data with laboratory testing in japan, along with other parameters, including virus shedding frequency and the risk of infection. in addition to estimating the asymptomatic ratio for all noroviruses, the present study also compares the estimate across different genogroups and genotypes. we reanalyzed a published dataset that tested both symptomatic and asymptomatic individuals during norovirus outbreaks at food catering settings in japan from november , to december , (n = outbreaks). in that study, a total of , involved individuals, including food-handlers, other workers, and potentially exposed customers, were examined for the presence of symptoms (ie defined as either non-bloody diarrhea or vomiting) and also virus shedding from their feces, conducting a survey shortly after each outbreak (within week). asymptomatic individuals were defined as persons without diarrhea and vomiting, and the asymptomatic ratio was defined as the proportion of asymptomatically infected individuals among the total of infected individuals. through stool specimen tests using highly sensitive real-time reverse transcription polymerase chain reaction (rt-pcr), which has the highest detection limit among all existing genome testing methods the virus and its genogroup=genotype were identified. due to its highly sensitive nature, the real-time rt-pcr was shown to detect positive norovirus specimens if > copies were included in a well. since asymptomatic healthy individuals as well as symptomatic cases were tested, the results in each outbreak are classified into four categories, ie, (i) symptomatic and virus positive, (ii) symptomatic but virus negative, (iii) asymptomatic and virus positive, and (iv) asymptomatic and virus negative. in addition to the number of people in each category for each outbreak, genotyping results were obtained. figure summarizes the survey results, and the coded data are given in etable . it should be noted that the number of asymptomatic and virusnegative individuals includes both uninfected individuals and infected cases without virus shedding. the abovementioned study has been very rigorous, in that all individuals involved in each food-borne outbreak were fully surveyed, which has been extremely rare and not routinely attainable. in addition, such a study must employ the highly sensitive real-time rt-pcr, a condition that was also satisfied by the - study. moreover, all events were observed in the same year in japan within food handling facilities of comparable sizes. here, we describe a statistical model with which the asymptomatic ratio and other parameters were jointly estimated using the abovementioned foodborne outbreak data. first, the data generating process is schematically illustrated in figure . three pieces of information are considered: the infection process, the illness onset, and virus shedding results. we assume that all situations and population were similar across outbreaks and shared identical parameters. let p be the risk (or the probability) of norovirus infection given an exposure at food handling center, and similarly, let s be the asymptomatic ratio of norovirus infection, representing the probability that infected individuals escape from symptomatic illness. moreover, let q be the probability of virus detection from an infected individual. while these parameters are allocated, all these processes are not directly observed in the original survey. the epidemiological information we have an access includes (i) the total number of people involved (including healthy individuals) in each outbreak, (ii) the number of symptomatic cases, and (iii) the numbers of symptomatic virus shedding cases and asymptomatic virus shedding cases. from these pieces of information, we estimate p, q and s jointly. let i be an identity of an outbreak (ie, i ranges from to ). all three parameters were dealt with as deterministic parameters; we did not consider demographic stochasticity in the data generating process. since observed processes are the single and independent success=failure process, the resulting process is a bernoulli trial, and thus, we assume that each observed event was a result of binomial sampling (because every observed step was binary and mutually independent). the probability that n i cases experience symptomatic illness among a total of n i symptomatic and m i asymptomatic individuals is described by similarly, the probability that y i cases are asymptomatic but positive among a total of m i asymptomatic individuals (that include uninfected individuals) is p ðy i ; m i ; p; q; sÞ the probability that x i cases shed the virus among a total of n i symptomatic cases is it should be noted that q was assumed as independent of symptoms because the rt-pcr testing employed was highly sensitive and the cut-off value must have allowed q to be comparable between symptomatic and asymptomatic infected individuals. , sensitivity analysis was conducted to address potential difference of q between symptomatic and asymptomatic infection (see below). it should also be noted that the parameter p should have ideally reflected the demographic stochasticity of the data generating process, but p is a mixture of food-borne and human-to-human transmissions and we do not have a sufficient dataset to characterize the distribution. thus, the abovementioned process in principle adopted a deterministic modeling approach and measured the uncertainty of p based on sampling error alone. a maximum likelihood method was employed to estimate parameter values, p, q and s. these parameters were estimated assuming several different interpretation scenarios of empirical miura f, et al. data. first, a single set of p, q, and s was estimated, assuming that each outbreak was a random sampling result from the abovementioned mechanisms ( )-( ). second, parameters were assumed to be different between genogroup and , so there were six unknown parameters. third, parameters were assumed to be different by genotype. when estimating parameters by genotype, it should be noted that the available number of outbreaks differed by genotype (eg, seven outbreaks for gii. ), and we excluded outbreaks in which multiple genotypes were identified. to identify any improved fit despite increased number of parameters, akaike information criterion (aic) was computed for each scenario. using ( )- ( ), we derive the total likelihood l for each single interpretation scenario, computed as where l ð p; s; n; mÞ ¼ y i n i þ m i n i ð pð À sÞÞ ni ð À pð À sÞÞ mi ; l ð p; q; s; m; yÞ x i q xi ð À qÞ ðniÀxiÞ : maximum likelihood estimates of p, q, and s were obtained by minimizing the negative logarithm of ( ), and the % confidence intervals (ci) were computed using the profile likelihood. to analyze the sensitivity of parameter estimates to differential frequency of virus shedding between symptomatic and asymptomatic infections, we used the following α, ie, where q is the probability of virus shedding from an asymptomatic case and q is the probability from a symptomatic case. we set the baseline value of α as . , which indicates that asymptomatic and symptomatic cases have the same probability of virus shedding (due to a highly sensitive pcr testing method , ), which is supported by empirical observation. , however, as part of sensitivity analysis, the value of α was changed from . to . , anticipating that virus shedding frequency from asymptomatic cases might potentially be smaller than that from symptomatic cases. calculating the aic for each interpretation scenario, the aic of the genogroup-combined model (ie, a model with three parameters only) yielded the value of , . , while that of the genogroupseparated model (ie, a model with six parameters) was , . . the smaller value of aic for a genogroup-separated model indicated that the model with genogroup information was better fitted to outbreak data, so there might potentially be a difference between gi and gii. figure compared observed and predicted numbers of people by symptom and pcr testing result, visually demonstrating the satisfactory description of observed patterns. a χ goodness-of-fit test revealed no significant deviations between predicted and observed numbers for both genogroup-combined and genogroup-separated models (p > . for both models with degrees of freedom at and , respectively). varying the value of α from . to . , we examined the sensitivity of model parameters to differential virus shedding frequency between symptomatic and asymptomatic infections (figure ). when α gets smaller, both the risk of infection ( p) and the asymptomatic ratio (s) were estimated to be greater. the probability of virus shedding among symptomatic cases remained stable ( figure c ). of the total of outbreaks, there were two outbreaks involving large number of subjects (n = , for two outbreaks; . % of the total), which could lead us to feel that the overall results may be highly influenced by those two outbreaks. removing the two outbreaks and analyzing the remaining altogether, parameters p, s, and q were estimated at . % ( % ci, . - . %), . % ( % ci, . - . %) and . % ( % ci, . - . %), respectively. the risk of infection (p) was significantly greater than the estimate in which all outbreaks were analyzed together, but two other parameters, including asymptomatic ratio (s) and the risk of virus shedding (q), were not significantly deviated. the present study reanalyzed foodborne norovirus outbreaks in japan from - that involved laboratory testing of all individuals including healthy people. to statistically infer asymptomatic ratio in joint with the risk of infection and the probability of virus shedding, a statistical model was developed to describe the data generating process. assuming that all outbreaks occurred due to repeated random samplings, the risk of infection (p) and the asymptomatic ratio (s) were estimated to be . % and . %, respectively. the probability of virus shedding (q) was estimated at . %. possible differences by genogroup were implicated by penalized likelihoods. the risk of infection was small for gii. , while the asymptomatic ratio was estimated to be high for this common genotype. an important contribution of the present study to the literature of norovirus is that the asymptomatic ratio was estimated to be about %, which is consistent with existing literature based on volunteer challenge studies. , , not based on human volunteer challenge, our study has successfully estimated the asymptomatic ratio from naturally occurred outbreaks, as has also been achieved for other infectious diseases. in particular, the asymptomatic ratio of common genotype gii. was estimated to be high, which is in line with the conventional understanding that the control of gii. is likely more difficult than that of other genotypes. in addition to original findings of similar virus shedding frequency inducing asymptomatic infections among food handlers. not only spreading the virus via food handlers, but the greater asymptomatic ratio allows infected individuals to be freely mobile and have more opportunities to pass the virus to others. when we assumed that the actual frequency of virus shedding from asymptomatic individuals is smaller than symptomatic cases, the asymptomatic ratio was estimated to be greater than the baseline. in other words, the estimated value around % might be even greater than our result. there was no statistically significant difference between gi and gii, while the aic value was indicative that there might be a difference between gi and gii. this was caused by limited sample size, and slight non-significant differences (eg, greater risk of infection for gi than gii and smaller asymptomatic ratio for gi than gii) were identified, implying that gi might be more controllable than gii. to our knowledge, the present study is the first to epidemiologically endorse different patterns of outbreaks by genogroups in natural settings, though differences have been indicated in experimental studies. , , while a greater asymptomatic ratio for gii. than others was consistent with our existing understanding, the smaller infection risk with gii. than others calls for careful interpretation. due to the frequent circulation of gii. , a portion of exposed individuals might have been immune in advance of outbreaks (that were not widespread before but may have remained unrecognized); moreover, evolutionary changes in gii. (eg, antigenic evolution) might have later varied the virus characteristics, including the infectiousness of gii. . evolutionary epidemiological study of gii. to be adapted to human populations is a subject of our ongoing study. two limitations must be discussed. first, the survey data contained epidemiologically limited information. that is, to define symptomatic cases, diarrhea or vomiting was employed, but they were qualitative definitions that did not explicitly involve the frequency, such as the number of defecations=vomiting per day. such qualitative definition is favored for clearly defining what a symptomatic case is, and an important advantage of the present study is that an identical research group has conducted the entire survey and minimized the measurement of error that could potentially arise from researchers. nevertheless, when it comes to additionally measuring the severity of illness, which is out of the scope of the present study, it might have been unavoidable to include human perception error. such misclassification could have potentially affected the asymptomatic ratio (eg, due to broad definition of asymptomatic persons). moreover, the exact timing of stool sampling was unavailable; however, it is likely that all sampling took place in a matter of week from the onset of outbreaks, during which the virus shedding level of both symptomatic and asymptomatic cases should be substantial. second, the model involved a few simplifications. the simplified points include the ignorance of the dependent happening in the risk of infection. since the majority of outbreaks were considered as purely foodborne, a single parameter p was allocated, but if human-to-human transmission was involved, it might have been better to account for that information, which was not attainable in the present study using the same dataset. another simplification was the use of binomial sampling process for each likelihood. as a consequence, the uncertainty bound of p in the present study should be regarded as an underestimate. one of the drawbacks has been seen in significantly greater p that we obtained when two big outbreaks were removed from analysis; in other words, the estimate might not have been significantly different if the uncertainty also reflected demographic stochasticity. despite the abovementioned limitations, our study has successfully demonstrated that the asymptomatic ratio can be estimated from outbreak data in which all involved individuals undertook laboratory testing and also that the asymptomatic ratio was about % in natural outbreak settings. practical difficulty in controlling gii. outbreaks was supported by its large estimate of the asymptomatic ratio. collecting similar datasets from further outbreaks, we will be able to gain further insights into the natural history of norovirus infection, including genotype-specific differences. understanding the natural history better, similar explicit estimates based on natural infection in future could help clarify better control strategies of norovirus outbreaks, even by genotype. noroviruses: a comprehensive review norovirus disease in the united states a systematic review and metaanalysis of the global seasonality of norovirus norovirus vaccine against experimental human norwalk virus illness viral shapeshifting: norovirus evasion of the human immune system risk factors for symptomatic and asymptomatic norovirus infection in the community norwalk virus shedding after experimental human infection diagnosing norovirus-associated infectious intestinal disease using viral load detection and quantification of airborne norovirus during outbreaks in healthcare facilities effects of cleaning and disinfection in reducing the spread of norovirus contamination via environmental surfaces the epidemiology of published norovirus outbreaks: a review of risk factors associated with attack rate and genogroup norovirus outbreaks: a systematic review of commonly implicated transmission routes and vehicles increase in viral gastroenteritis outbreaks in europe and epidemic spread of new norovirus variant foodborne norovirus outbreak: the role of an asymptomatic food handler norovirus cross-contamination during food handling and interruption of virus transfer by hand antisepsis: experiments with feline calicivirus as a surrogate epidemiology of foodborne norovirus outbreaks systematic literature review of role of noroviruses in sporadic gastroenteritis mechanisms of gii. norovirus persistence in human populations structural basis for the recognition of blood group trisaccharides by norovirus norovirus gii. strain antigenic variation advances in norovirus biology coexistence of multiple genotypes, including newly identified genotypes, in outbreaks of gastroenteritis due to norovirus in japan norovirus surveillance group of japan. divergent evolution of norovirus gii= by genome recombination from norovirus surveillance group of japan. identification of monomorphic and divergent haplotypes in the - norovirus gii= epidemic population by genomewide tracing of evolutionary history shedding of norovirus in symptomatic and asymptomatic infections heterogeneity in norovirus shedding duration affects community risk mathematical model for the control of nosocomial norovirus duration of immunity to norovirus gastroenteritis fitting outbreak models to data from many small norovirus outbreaks norovirus infections in symptomatic and asymptomatic food handlers in japan broadly reactive and highly sensitive assay for norwalk-like viruses based on real-time quantitative reverse transcription-pcr identification of the asymptomatic ratio mers-cov scenario and modeling working group. estimating the severity and subclinical burden of middle east respiratory syndrome coronavirus infection in the kingdom of saudi arabia supporting online information accompanies the paper on the journal website.financial support: fm acknowledges the graduate program for social ict global creative leaders (gcl), the university of tokyo for financially supporting his study at hokkaido university. hn received funding support from the japan agency for medical research and development, jsps kakenhi grant numbers kt , k , and h , the japan science and technology agency (jst) crest program (jpmjcr ) and ristex program for science of science, technology and innovation policy. rm acknowledges the jsps kakenhi grant number h . the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.conflicts of interest: none declared. ethical standards: not applicable, as our study used published and anonymized data. key: cord- - r hg ld authors: pawliw, rebecca; farrow, rebecca; sekuloski, silvana; jennings, helen; healer, julie; phuong, thuan; sathe, pri; pasay, cielo; evans, krystal; cowman, alan f.; schofield, louis; chen, nanhua; mccarthy, james; trenholme, katharine title: a bioreactor system for the manufacture of a genetically modified plasmodium falciparum blood stage malaria cell bank for use in a clinical trial date: - - journal: malar j doi: . /s - - -x sha: doc_id: cord_uid: r hg ld background: although the use of induced blood stage malaria infection has proven to be a valuable tool for testing the efficacy of vaccines and drugs against plasmodium falciparum, a limiting factor has been the availability of good manufacturing practice (gmp)—compliant defined p. falciparum strains for in vivo use. the aim of this study was to develop a cost-effective method for the large-scale production of p. falciparum cell banks suitable for use in clinical trials. methods: genetically-attenuated parasites (gap) were produced by targeted deletion of the gene encoding the knob associated histidine rich protein (kahrp) from p. falciparum strain d . a gap master cell bank (mcb) was manufactured by culturing parasites in an fda approved single use, closed system sterile plastic bioreactor. all components used to manufacture the mcb were screened to comply with standards appropriate for in vivo use. the cryopreserved mcb was subjected to extensive testing to ensure gmp compliance for a phase investigational product. results: two hundred vials of the gap mcb were successfully manufactured. at harvest, the gap mcb had a parasitaemia of . %, with % of parasites at ring stage. testing confirmed that all release criteria were met (sterility, absence of viral contaminants and endotoxins, parasite viability following cryopreservation, identity and anti-malarial drug sensitivity of parasites). conclusion: large-scale in vitro culture of p. falciparum parasites using a wave bioreactor can be achieved under gmp-compliant conditions. this provides a cost-effective methodology for the production of malaria parasites suitable for administration in clinical trials. the use of controlled human malaria infection (chmi) for testing the efficacy of vaccines and drugs against plasmodium falciparum is now well established. induced blood stage malaria (ibsm) infection was pioneered in the s [ ] [ ] [ ] , and was subsequently refined [ ] [ ] [ ] [ ] . a limiting factor in the ibsm system has been the restricted number of defined p. falciparum strains available. this is in part due to difficulties obtaining suitable material for ibsm studies. until recently it has been necessary to rely on access to deliberately infected volunteers or malariainfected travellers returning from overseas. in both cases, ethical approval is required to collect, store and use this material and there are concerns about contaminating infectious agents. a process for culturing sufficient volumes of p. falciparum in tissue culture flasks for in vitro production of good manufacturing practice (gmp) grade blood stage malaria cell banks was recently described [ ] . this provides a culture-based alternative to sourcing parasites from individuals who have either experimental or natural infection (travellers returning from malaria endemic areas). this allows more flexibility in that new banks can be created as required, and not only when a suitable donor becomes available. it also allows for tight control over all components used in the cell bank manufacture and ensures that cell banks are renewable resources. however, bulk preparation of p. falciparum cultures is both time consuming and technically demanding; yields can be low and parasite quality can be variable when grown in static culture. multiply-infected erythrocytes (containing two or more parasites) are also observed more frequently than in suspension cultures [ ] . the presence of erythrocytes containing multiple parasites is undesirable for ibsm studies where the parasite dose should be carefully controlled. dalton et al. have developed a method for production of large scale suspension cultures of asexual and sexual blood stage p. falciparum using a wave bioreactor (wave bioreactor ™ / eht system) [ , ] . growth of p. falciparum in the wave bioreactor system is superior to static flask cultures because parasites retain synchrony over at least cycles of invasion. additionally, the development of multiply infected erythrocytes is reduced; this was shown to be both consistent and reproducible [ , ] . disposable plastic bioreactors also allow for careful control and monitoring of the cellular environment, and have been widely used for the culture of plant, animal and microbial cells, and for the production of proteins, antibodies and other biologics conforming to gmp standards [ ] . here, the manufacture and release of a cell bank that complies with the gmp requirements for a phase investigational product is described. the cell bank was manufactured using a wave bioreactor and consists of erythrocytes infected with genetically modified p. falciparum parasites. this cost-effective methodology using appropriate gmp compliant conditions allows for the production of sufficient quantities of material for ibsm studies and whole blood-stage vaccine trials, with sufficient material available for quality control and ongoing stability testing. the manufacture of the master cell bank was conducted in accordance with the united states food and drug administration (fda) guidance for industry: cgmp for phase investigational drugs. genetically attenuated parasites (gap) were produced by targeted deletion of the gene encoding the knobassociated histidine-rich protein (kahrp) from p. falciparum strain d , in a similar manner as previously described for this and other genes [ , ] . briefly, deletion of kahrp was achieved by a positive-negative selection strategy [ ] whereby the entire kahrp gene was replaced through double cross-over homologous recombination with a cassette encoding human dihydrofolate reductase (dhfr), a selectable marker that renders transfectants resistant to wr (jacobus pharmaceuticals, nj). the loxp-dhfr-loxp-pcc plasmid was created by insertion of a loxp-hdhfr-loxp cassette into the pcc plasmid backbone [ ] , as previously described [ ] . the kahrp-ko plasmid was created by cloning kahrp ′ and ′ sequences into this plasmid. plasmid dna was transfected into p. falciparum d by electroporation. following wr positive selection, parasites were grown without wr to deselect those containing the wr-containing plasmid, thereafter wr was reapplied to select those with integration of the dhfr cassette into the genome. these were then placed under negative selection with -fluorocytosine ( -fc; sigma) to select for homologous recombination. -fc resistant parasites were genotyped by pcr. kahrp-ko parasites were cloned by limiting dilution in -well flat bottom plates and the resulting clonal line, d -kahrp-ko, was characterized by whole genome sequencing (accession number prjeb ). the p. falciparum d parasites used for transfection with the kahrp knockout vector were created from the d parental strain described previously [ ] . this strain was used for the original ibsm studies [ ] and has been extensively used for subsequent studies. the gap seed bank was produced by thawing the contents of one vial of the gap pre-seed bank following a standard protocol [ ] and expanding the parasites in culture (rpmi media supplemented with % heat-treated pooled human serum and % human erythrocytes) using standard methods [ ] . at each passage, fresh complete media and erythrocytes were added and the culture was expanded to achieve the required volume for cell banking. when the culture contained a minimum of % ring-stage parasites at - % parasitaemia, the seed bank was cryopreserved using glycerolyte (fenwal inc.) in a : ratio, aliquoted into ml cryovials, and stored in vapour phase liquid nitrogen. the final gap seed bank was transferred to a therapeutic goods administration (tga) licensed gmp facility (q-gen; qimr berghofer medical research institute [qimrb]) for further expansion and production of the gap master cell bank. the d kahrp-ko gap master cell bank (gap mcb) was manufactured in a iso class biological safety cabinet within an iso class clean room using tissue culture flasks and a bioreactor (ge wave ™ system). the gap mcb dossier was reviewed by the united states food and drug administration (fda) under the investigational new drug (ind) program. a summary of the process is shown in fig. and described in detail below. a vial of the d kahrp-ko gap seed bank was thawed at °c with the stepwise addition of and . % sodium chloride following a standard protocol [ ] . the resulting red blood cell (rbc) pellet was washed in . % sodium chloride and resuspended in ml of complete rpmi-hepes media (life technologies) containing µl of % haematocrit (hct) washed rbcs, and the cell suspension was transferred to a t cm flask. parasites were cultured in flasks under standard malaria culture conditions [ ] in an atmosphere of % carbon dioxide, % oxygen and % nitrogen at °c and media was changed every h. parasitaemia was monitored by examination of giemsa-stained thin blood films. additional media and fresh rbcs were added as required to maintain the culture at a parasitaemia of between . and %, and with a - % hct. the parasite culture was expanded to a volume of approximately ml and the parasites were synchronized by a single treatment with % sorbitol [ ] prior to transfer to a l cellbag of the wave ™ bioreactor. the ready to process wave ™ system (ge healthcare) was used for this study and configuration of the equipment is shown in fig. . the system consists of a pre-sterile single use cultivation container (cellbag) made of fda approved plastic. each cellbag has builtin inlet and outlet air filters, and ports that allow the addition of culture medium and extraction of samples (fig. a) . the cellbag is placed on a temperature-controlled rocking platform (fig. b) ; gas transfer and mixing of the cell culture is facilitated by movement of the rocker unit which induces a wave motion in the culture. temperature, rocking speed, rocking angle and atmospheric conditions (fig. c ) are monitored and controlled by the cellbag control unit (cbcu). the system is controlled from a pc running unicorn software version . . or later. the synchronous parasite culture was transferred into a l cellbag and maintained under conditions controlled electronically by the bioreactor system (fig. c ). parasitaemia was monitored by examination of giemsastained thin blood films. the culture medium was changed daily, with washed rbcs added as required to maintain a parasitaemia of . - . % and a - % hct. when the culture volume reached ml it was transferred to a l cellbag and the parasite culture was expanded to a final volume of approximately l in line with the manufacturers recommended culture volumes for and l cellbags. to facilitate media change and transfer between cellbags the cell culture was removed from the in use cellbag under sterile conditions, centrifuged in ml conical bottles (corning) and the supernatant removed. the required volume of fresh media was pumped into the destination cell bag under the control of the cbc unit and the required volume of inoculum added under gravity flow. the culture was monitored to ensure synchronicity and was maintained such that the % of ring stage parasites (as a % of the total parasites) present in the culture did not fall below %. when the required volume was reached, the culture was maintained for one further invasion cycle before parasites were harvested for banking at a parasitaemia target of > % and a minimum of % ring stage parasites. progression through the final multiplication cycle was monitored by examination of giemsa-stained thin blood films every - h. the cell culture supernatant was tested for microbial contamination prior to culture in the wave ™ bioreactor system and prior to harvesting. at harvest the entire cell culture was removed under sterile conditions and centrifuged in ml conical bottles to pellet the rbcs prior to freezing. rpmi-hepes media (life technologies) was supplemented with % heat inactivated (hi) pooled human serum and hypoxanthine (ht) supplement ( . mm sodium hypoxanthine and . mm thymidine). complete media was used immediately or dispensed in aliquots and stored at a temperature of between and °c for a maximum of days. fig. wave bioreactor configuration. a each cellbag has built-in inlet and outlet air filters, and ports that allow the addition of culture medium and extraction of samples. b the ready to process wave ™ system (ge healthcare) was used for this study. c temperature, rocking speed, rocking angle and atmospheric conditions are monitored and controlled by the cellbag control unit (cbcu) the serum used was supplied, screened and tested by key biologics (memphis usa), an fda registered blood collection and processing establishment. whole blood units were collected from donors who were screened and found acceptable for donation of transfusable blood components based on all applicable fda requirements. infectious disease testing as required for qualification of blood components for transfusion to humans was carried out and was required to be non-reactive or negative. serum from individual donors was tested (using fda approved test kits) for human immunodeficiency virus (hiv)-i and hiv-ii, human t-lymphotropic virus (htlv) types i and ii, hepatitis b virus (hbv), hepatitis c virus (hcv), trypanasoma cruzi and syphilis (by serology), and underwent nucleic acid testing for hbv, hcv, hiv, and west nile virus (wnv). serum was then pooled from the multiple donors, frozen, stored and shipped under controlled conditions to qimrb. on arrival the serum was thawed, heat inactivated at °c, filtered and stored at − °c in ml aliquots until required. the pooled serum was additionally tested for epstein barr virus, parvovirus b and herpes virus type by pcr at pathology queensland. endotoxin testing and sterility testing was performed at q-gen. leukodepleted packed rbcs (group o rh (d) negative) used for in vitro cultivation of p. falciparum parasites were obtained from the australian red cross blood service (blood service). all supplied rbcs were screened according to blood service protocols to eliminate individuals with risk of exposure to transmissible spongiform encephalopathy and blood borne agents. the blood was collected and leukodepleted at the blood service and the donor's blood sample was tested and certified nonreactive for hiv, hbv, hcv, htlv and syphilis using diagnostic kits approved by the tga. a blood sample was additionally tested by pathology queensland for infectious diseases that are not included in mandatory testing for blood and blood products including anti-flavivirus igm antibodies, trypanasoma cruzi igg antibodies, barmah forest virus igm and igg antibodies, and ross river virus igm and igg antibodies. samples were also screened (by pcr testing) for epstein barr virus (ebv), cytomegalovirus (cmv), parvovirus b , human herpes virus type and type (hhv- and hhv- ), and wnv. on receipt at qimrb the cells were transferred from the blood service pack to a sterile container and a ml aliquot was removed for sterility testing. aliquots of rbcs were washed three times in rpmi and resuspended in complete rpmi to give a hct of %. blood was washed as required in aliquots not exceeding ml and stored at - °c for up to days after washing. percentage parasitaemia and parasite life cycle stage were assessed by microscopic examination of thin blood films and when cultures achieved > % parasitaemia with at least % ring-stage parasites the cell bank was cryopreserved using glycerolyte in a : ratio (i.e. two volumes of glycerolyte were added to each volume of prbc) as previously described [ ] . the cell bank was dispensed in ml aliquots in cryovials, cryopreserved using a controlled rate freezer as previously described [ ] and stored in vapour phase liquid nitrogen at - °c. a total of vials were produced; two vials were allocated as retention samples. table . parasite viability post-freezing was determined by measurement of cloning efficiency via a limiting dilution assay [ , ] with positivity determined by production of p. falciparum histidine-rich protein ii (hrp-ii). in order to determine post-freeze viability, a vial of the gap mcb was thawed following a standard protocol [ ] and a sample was removed for rbc counting using a haemocytometer. the number of parasitized/infected rbcs (prbcs) was determined via microscopic examination of a giemsa-stained thin blood smear. dilutions of prbcs were prepared in complete media and dispensed in μl aliquots in -well flat bottom plates at % hct at theoretical concentrations of , , , , . , . , . and . parasites/well. thirty-two replicates of each concentration were plated out and non-parasitized rbcs were included as a negative control. plates were incubated in an atmosphere of % o , % co and % n at °c for days and media was replenished on day . on day , supernatant was removed from each well and processed immediately or frozen for future testing. the supernatant was tested for p. falciparum hrpii using the sd malaria antigen pf elisa kit, according to the manufacturer's instructions. briefly, supernatants from each well were transferred to the corresponding well of an uncoated microplate and incubated for min following the addition of µl . a µl sample was then transferred from each well of the uncoated microplate to the corresponding well of the pre-coated assay microplate and incubated. plates were washed and µl of tetramethylbenzidine substrate added to each well. after a min incubation, the reaction was stopped by the addition of µl of ml/l ( n) hydrochloric acid. the absorbance was read at nm with reference wavelength at nm. the mean absorbance values (optical density [od]) for the positive and negative controls were calculated in order to ensure the validity of the assay. the cut off value for parasite positivity was calculated using the absorbance values of the two wells containing the rbc negative control. the cut off value was the mean plus standard deviations and this value was applied to the test samples to calculate the number of positive and negative wells. limiting dilution analysis was performed to determine the frequency of viable parasites as previously described [ ] . anti-malarial drug sensitivity testing was carried out on the gap mcb parasites using the standard [ h]-hypoxanthine uptake inhibition assay [ ] to ensure that the drug resistance phenotype of the parasites had not changed during culture. the in vitro sensitivity of the gap mcb parasites to nine anti-malarial drugs was assessed (chloroquine, dihydroartemisinin, piperaquine, lumefantrine, amodiaquine, atovaquone, pyronaridine, mefloquine, quinine). the parental d line (chloroquine sensitive) and w (chloroquine resistant) were included as controls. the assays were performed at the army malaria institute, queensland, australia. briefly, aliquots of parasites from the gap mcb were grown in vitro and synchronized to give % ring stage parasites. the parasite culture was dispensed into a -well plate containing twofold dilutions of each antimalarial drug for testing. [ h]-hypoxanthine was added and the amount incorporated into parasites was measured after a -h incubation. the ic values for each drug were determined by nonlinear regression analysis. this calculation was performed using graphpad prism software (graphpad prism version . ). drug threshold values were expressed as mean ic ( % confidence interval) in nmol/l. the results were compared to published threshold values for drug resistance [ ] . the genotype of the gap mcb was determined by pcr using primers to confirm kahrp gene deletion and hdhfr integration. in vitro culture samples were taken at the beginning of the process (day ) and at harvest (day ) and placed in qiagen lysis buffer. genomic dna was extracted from paired samples using a qiagen dnamp dna blood mini kit and gdna from the parent strain d was used as a control. standard pcr was set up using pairs of primers ( to further validate the successful genetic attenuation of p. falciparum d and verify hdhfr integration, a real time pcr assay was set up using primers designed at various regions of the p. falciparum d gap (fig. ) . primer sequences are listed in table . to determine limit of detection of possible contamination with wildtype p. falciparum d , two single copy genes (pfmdr- and pfldh) were amplified and used as reference for copy number estimation. a standard curve containing five ten-fold serial dilutions of wildtype p. falciparum d was prepared, starting from . ng/μl to amplify kahrp excised region and mdr and ldh genes. triplicate pcr reactions were set up using the roche fast start essential dna green master mix (cat. no. ), μm of each primer ( table ) and μl of each gdna sample in a μl reaction volume. quantitative pcr was performed using a roche light cycler with the following cycling conditions: °c for min; in vitro assays for the detection of viral contaminants and inapparent viruses was also performed. a cell bank consisting of erythrocytes infected with genetically-modified p. falciparum parasites was successfully manufactured using a wave bioreactor. two hundred vials of the gap mcb were produced that met the requirements for use in phase i clinical trials. gap parasites maintained an asexual blood stage cycle of approximately h during the in vitro culture period in the bioreactor, and showed a normal maturation pattern. the percent parasitaemia and percent ring stage parasites of the culture over time is illustrated in fig. . parasitaemia was maintained between . and % by the addition of rbcs during the expansion phase (day - ). during the banking phase (day - ) the parasitaemia was maintained below %. at harvest the culture had a parasitaemia of . %, with % of parasites at ring stage (high percentage of ring stage parasites is illustrated in fig. ) . thus, the pre-defined acceptable criteria of > % parasitized rbc and ≥ % ring stage parasites (table ) were met. pcr assays confirmed kahrp gene deletion and hdhfr integration in parasite samples collected at the initiation of culture (day ) and at harvest (day ) (fig. ) demonstrating that the genotype of the parasites was maintained during the culture and banking process. amplification of the kahrp excised region on serially diluted wildtype p. falciparum d and comparing ct values obtained from serially diluted gap showed undetectable contamination of the gap starting from . ng/ μl dna concentration up to as low as . × − ng/ μl. this is equivalent to a single copy or less using single copy gene references pfmdr- and pfldh. the in vitro sensitivity of the gap mcb parasites to a panel of anti-malarial drugs is shown in table . the mean ic ( % confidence interval) results for each drug are presented in table along with the corresponding results for the control p. falciparum strains d and w . gap master cell bank parasites demonstrated sensitivity to eight of the anti-malarial drugs tested (chloroquine, artemisinin, piperaquine, lumefantrine, amodiaquine, atovaquone, pyronaridine, quinine) and resistance to mefloquine. parasite viability was tested using samples taken approximately h (time ) after freezing and at months postproduction. the gap mcb was determined to contain % viable parasites at time and % viable parasites at months post-production. these values conform to the pre-defined acceptable criteria of > % viable parasites. the variability between the two time points is within normally observed limits for this assay. our aim was to produce a gmp compliant cell bank consisting of kahrp deficient p. falciparum parasites using the wave bioreactor system. previous studies have shown that the wave bioreactor system is suitable for the production of large volumes of high quality p. falciparum cultures [ ] . we have taken this method further to demonstrate that the wave bioreactor can be used to culture parasites for use in phase clinical trials. the use of a wave bioreactor system has a number of significant advantages over traditional culture of p. falciparum in flasks. foremost is that it allows close control of culture conditions that can be monitored and maintained electronically by the wave bioreactor system; the cellbags used are single use closed systems approved by the fda. significant savings in time and labour were also achieved; ml of culture was transferred to a cellbag on day , expanded to ml on day , and a culture volume of l was achieved on day with harvest/ cryopreservation on day . during this rapid expansion phase the labour required to maintain the culture (monitor parasitaemia, change media, add fresh rbcs) was less than would be required if growing in multiple tissue culture flasks. in addition, the use of multiple flasks can result in cultures spending more time in less than the continual wave motion of the wave bioreactor ensures gentle movement of cells in culture resulting in a high proportion of single infections [ ] . this allows for accurate calculation of parasite numbers which is essential for defined dose delivery in clinical trials. results confirmed that wave bioreactor cultures contained high quality ring stage parasites with a low number of multiply infected red cells (< . %). additionally, the effective cryopreservation of the mcb was demonstrated by the fact that parasite viability was over % after months. the ability to use research grade starting material, including genetically-modified parasites, to produce a gmp grade product offers significant opportunities. it allows for genetically-modified parasites to be used as vaccine candidates and for the number of p. falciparum strains available for ibsm studies to be expanded. the availability of multiple antigenically and geographically diverse p. falciparum strains for ibsm studies is essential drug threshold values are expressed as mean ic ( % confidence interval) in nmol/l the assay was repeated twice and reproducible results were obtained a third assay was performed for lumefantrine as ic values were not detectable in the first two assays to allow testing of new anti-malarials against multiple strains before field testing begins. the methodology described here is highly cost effective, with each of the vials produced containing sufficient material for doses at an approximate cost of usd . /dose. although the method requires use of a specialist item of equipment (wave bioreactor system), the fact that these units are becoming widely used for a range of applications (including tissue engineering, cellular and gene therapies, the production of therapeutic proteins and human vaccines) means that they are frequently available within research facilities or hospitals. the use of sterile, single use closed system cell bags means that a wave bioreactor system can be shared between groups without risk of cross contamination. measurement of plasmodium falciparum growth rates in vivo: a test of malaria vaccines effect of vaccination with recombinant asexual-stage malaria antigens on initial growth rates of plasmodium falciparum in non-immune volunteers immunity to malaria after administration of ultra-low doses of red cells infected with plasmodium falciparum a phase trial of msp -c , a blood-stage malaria vaccine containing isoforms of msp formulated with montanide(r) isa a phase ii pilot trial to evaluate safety and efficacy of ferroquine against early plasmodium falciparum in an induced blood-stage malaria infection study a pilot randomised trial of induced blood-stage plasmodium falciparum infections in healthy volunteers for testing efficacy of new antimalarial drugs blood-stage challenge for malaria vaccine efficacy trials: a pilot study with discussion of safety and potential value development of cultured plasmodium falciparum blood-stage malaria cell banks for early phase in vivo clinical trial assessment of anti-malaria drugs and vaccines large-scale growth of the plasmodium falciparum malaria parasite in a wave bioreactor the development of sexual stage malaria gametocytes in a wave bioreactor bag bioreactor based on wave-induced motion: characteristics and applications targeted gene disruption shows that knobs enable malaria-infected red cells to cytoadhere under physiological shear stress preerythrocytic, live-attenuated plasmodium falciparum vaccine candidates by design negative selection using yeast cytosine deaminase/uracil phosphoribosyl transferase in plasmodium falciparum for targeted gene deletion by double crossover recombination exported proteins required for virulence and rigidity of plasmodium falciparum-infected human erythrocytes gene deletion from plasmodium falciparum using flp and cre recombinases: implications for applied site-specific recombination convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over m website views per year • at bmc genetic analysis of the human malaria parasite plasmodium falciparum human malaria parasites in continuous culture synchronization of plasmodium falciparum erythrocytic stages in culture an improved method for undertaking limiting dilution assays for in vitro cloning of plasmodium falciparum parasites elda: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays estimation of plasmodium falciparum drug susceptibility by the h-hypoxanthine uptake inhibition assay. worldwide antimalarial resistance network (wwarn) field application of in vitro assays sensitivity of human malaria parasites antimalarial drugs. geneva: world health organization we would like to thank the australian red cross blood service for providing human erythrocytes. this methodology may represent a cost effective strategy by allowing extensive testing in a controlled environment (phase trials) before deciding to move to field studies (phase trials) which can be very expensive due to logistic and administrative challenges associated with carrying out clinical trials in a distant research site. the limiting factor for this methodology is that it is currently only useful for p. falciparum as other human malaria species are not easily grown in vitro. the large scale in vitro culture of p. falciparum parasites using a wave bioreactor can be achieved under gmp compliant conditions. this provides a cost-effective methodology for the production of malaria parasites suitable for administration in clinical trials. the authors declare they have no competing interests. all data generated or analysed during this study are included in this published article. not applicable. not applicable. the study was funded by nhmrc development grant app and nhmrc fellowship support app (ls) and nhmrc fellowship support app (jm). springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - owcqt d authors: iketani, sho; shean, ryan c.; ferren, marion; makhsous, negar; aquino, dolly b.; des georges, amedee; rima, bert; mathieu, cyrille; porotto, matteo; moscona, anne; greninger, alexander l. title: viral entry properties required for fitness in humans are lost through rapid genomic change during viral isolation date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: owcqt d human parainfluenza viruses cause a large burden of human respiratory illness. while much research relies upon viruses grown in cultured immortalized cells, human parainfluenza virus (hpiv- ) evolves in culture. cultured viruses differ in their properties compared to clinical strains. we present a genome-wide survey of hpiv- adaptations to culture using metagenomic next-generation sequencing of matched pairs of clinical samples and primary culture isolates (zero passage virus). nonsynonymous changes arose during primary viral isolation, almost entirely in the genes encoding the two surface glycoproteins—the receptor binding protein hemagglutinin-neuraminidase (hn) or the fusion protein (f). we recovered genomes from hpiv- primary culture isolates and hpiv- strains directly from clinical samples. hn mutations arising during primary viral isolation resulted in substitutions at hn’s dimerization/f-interaction site, a site critical for activation of viral fusion. alterations in hn dimer interface residues known to favor infection in culture occurred within days (h and n ). a novel cluster of residues at a different face of the hn dimer interface emerged (p and r ) and imply a role in hpiv- -mediated fusion. functional characterization of these culture-associated hn mutations in a clinical isolate background revealed acquisition of the fusogenic phenotype associated with cultured hpiv- ; the hn-f complex showed enhanced fusion and decreased receptor-cleaving activity. these results utilize a method for identifying genome-wide changes associated with brief adaptation to culture to highlight the notion that even brief exposure to immortalized cells may affect key viral properties and underscore the balance of features of the hn-f complex required for fitness by circulating viruses. altered to suit cultured cells and could no longer be considered clinical strains. there has been a relative dearth of sequence information available for hpiv compared to other respiratory viruses ( ) , and more importantly a lack of information about strains prior to passaging in the laboratory and thereby being subjected to selective pressure. to address the question of which features associated with fitness in humans may be lost in routine viral culture, we sought to understand the genomic changes associated with brief exposure of hpiv- from clinical samples to culture during the isolation process. we used metagenomic next-generation sequencing (mngs) of field strains of hpiv- isolated from humans to analyze the diversity and commonalities of circulating hpiv- strains at the genomic level, and we simultaneously sequenced paired specimens that were subjected to culture for viral isolation. the sequencing strategy we used to sequence clinical strains directly without passage in immortalized cells does not rely on designing sequence-specific primers that may amplify only a subset of samples (in case of mutations) ( ) . deep genomic sequencing of nine sets of paired clinical samples (primary nasal swabs in viral transport medium) and culture isolates (culture harvest from zero passage virus) led to discovery of a number of hn mutations associated with rapid evolution in culture. to assess the frequency of mutations identified earlier, we also performed deep sequencing of hpiv- clinical samples and culture isolates from the university of washington virology laboratory, allowing us to confirm that the alterations associated with brief exposure to culture for viral isolation were almost entirely found in the sequences of culture isolates and found commonly within populations of viruses in those isolates. functional characterization of the effects of several of the specific hn gene mutations found in the cultured isolates in a clinical isolate (ci) background-in expressed hn-f complexes or recombinant viruses bearing the mutated genes-reveals that the culture-associated mutations all increase fusion promotion by the hn-f complex. these results support the notion that clinical strains differ from cultured viruses in the balance of fusion properties. the locations of the residues that differ after brief culture of clinical viruses point to specific domains that are relevant to the control of viral entry in vivo. the sequencing data, when correlated to functional data, help define the flexibility and constraints of the hpiv- fusion complex. these data provide the first characterization of culture-associated hn mutations in the background of hpiv- clinical strains directly from patient samples. we show that, even during transient exposure to culture ("zero passage" virus), the act of isolating a virus is associated with genomic evolution that may significantly affect viral protein function and not reflect in vivo dynamics. mutation and mixed allele frequency comparison between clinical samples and their matched cultured specimens. to compare hpiv- sequenced directly from patients with hpiv- sequenced after brief growth in culture, nine hpiv- -positive (hpiv- ϩ) nasal swabs were sequenced metagenomically, and their paired viral isolates were sequenced after initial isolation in rhesus monkey kidney (rhmk) cells. we investigated the variant alleles with a minor allele frequency of Ͼ % that arose in culture. the nine swabs included one clinical sample that was independently cultured twice, such that ten viral isolates were sequenced. clinical respiratory samples that tested positive for hpiv- by quantitative reverse transcription-pcr (qrt-pcr) with cycle thresholds under were either sequenced directly by metagenomic nextgeneration sequencing (mngs) without exposure to culture or grown on rhmk cells and viral isolates were harvested and sequenced. the choice of rhmk cells for this study was based on the fact that these are the standard cells used for viral isolation by many or most diagnostic microbiology laboratories and would allow us to answer the question of whether this standard process leads to alteration in the viruses. seven of the nine cultured isolates were harvested before day of culture, and no viruses were passaged beyond initial isolation. sequencing reads for the cultured viruses were analyzed for mutational changes relative to the hpiv- consensus genome obtained from metagenomic sequencing of the original clinical sample (without expo-sure to any cell culture), and both assemblies were analyzed for variants with Ͼ % minor allele frequency (maf) ( ) . a total of nonsynonymous mutational changes with maf change of Ͼ % in response to exposure to cell culture were found across the nine sets of hpiv- viral isolates, with of the in the hn protein (p Ͻ . by fisher's exact test) (fig. ) . of the ten mutations found outside the hn protein, four were found in the p protein, four in the l protein, and two in the f protein. of the two l variants that increased in allele frequency in cultured specimens, one (g d) was not found in a separate viral isolation performed on the same clinical sample, while the other (p l) was found at a high allele frequency in the original clinical sample (fig. d to g). evolution of the polymerase complex will be explored separately, as the focus of this current paper is on the entry complex. during viral isolation from sample sc , we noted the appearance of the previously described hn h q mutation at % maf after only days in culture (fig. g) . hn h q has previously been isolated in laboratory-adapted hpiv- strains and is possible cpe day -cpe, had+ sc l n h f m p n r c % r , , , , , , , , , , , , , , mutational changes and allele frequencies in matched clinical samples and cultures. blue genomes represent clinical samples, and the green genomes below the blue genomes represent the same sample after it was inoculated into culture and harvested several days later. the number of days cultured, hemagglutinin adsorption assay results (had), and observed cytopathic effect (cpe) are shown for each viral isolate. virus from sample sc in panel g was isolated twice. coverage is averaged across the entire genome and depicted underneath each genome. allele frequencies are plotted to scale above the genome organization. mutational changes are described relative to the amino acid in the consensus genome of the original patient sample. variants indicated in italics were found exclusively on separate reads and are considered unlinked. iketani et al. ® associated with enhanced fusion promotion as a result of hn's f-triggering function ( , ( ) ( ) ( ) . two independent viral isolations in rhmk cells of this clinical sample resulted in selection for the hn h q variant allele. during viral isolation in rhmk cells from sample sc , an hn n d mutation emerged from % to % allele frequency in only days, the greatest change in allele frequency during the brief exposure to culture (fig. e ). previous comparison of direct clinical isolates with laboratory-adapted strains showed that every clinical strain bore asparagine (n) at position , while our laboratory strains bore aspartic acid (d) at position ( ). the hns derived from clinical isolates (cis) showed from -to -fold-higher neuraminidase activity than hns derived from the lab-adapted strains, and the substitution of the aspartic acid at this position in the laboratory strains led to an approximately -fold-lower receptor cleavage in the laboratory-adapted strains, suggesting that the asparagine at position is essential for the high propensity for receptor cleavage in field strains. the rapid emergence of this adaptation to culture by reducing receptor cleavage is remarkable. during viral isolation from both samples sc and sc , an hn l s mutation, adjacent to residues and mentioned above, arose to % and % maf ( fig. a to i). during isolation of sample sc , the same site contained an l f mutation that was unlinked to the h q mutation that also arose during that isolation (i.e., was found exclusively on separate reads). the sc isolation also resulted in selection of novel mutations hn s n and hn t i, comprising a cluster from positions to with significant alterations in the cultured viruses (fig. h) . while the region of hn corresponding to the cluster at positions to above was known to be of interest for entry in vivo (based on features of h and n ), during independent viral isolations from sample sc , a set of mutations emerged in hn at residues previously unrecognized to be relevant to fitness: p and r (hn p l and hn r k). these mutations were not linked to each other. the p l mutation described above was also recovered during viral isolations of sc and sc ( fig. c to h). adjacent to these residues, another previously undescribed hn mutation l f was selected at a high allele frequency ( %) during isolation of sc and at a low level during isolation of sc ( % maf in k and % maf in cul ). as discussed further below, these three residues describe a region of interest based on our previous structural analysis of the hn dimer interface ( ) . phylogeny and hn mutation history in original samples. to dissect the influence exerted by background hn sequence of the clinical virus on subsequent culture adaptation, we generated a phylogenetic tree based on amino acid substitution distance in the hn protein of our clinical samples (fig. ) . none of the phylogenydetermining mutations among direct clinical sample strains were located in hn's receptor binding site ii or second dimer interface domain that emerged during rapid culture adaptation. independent viral isolates from sample sc ( k and cul ) produced p l, r k, and h q. the hn mutations l f, p l, s n, and l s also arose among separate lineages. large-scale whole-genome sequencing of hpiv- direct-and culture-exposed strains shows that culture-associated mutations are rarely found in clinical strains. to place the alleles that arose in the paired clinical-culture specimens above in the broader context of hpiv- sequence information, we used mngs to obtain whole genomes from an additional hpiv- strains, of which were primary viral isolates (passage zero virus; brief exposure to culture only during viral isolation) and the remaining were directly from patient samples. all specimens were collected in or (see table s in the supplemental material). recombination analysis using rdp ( , ) revealed no recombination among these strains similarly to other nonsegmented negative-strand rna viruses ( , ) . phylodynamic analysis of the concatenated coding regions of the hpiv- sequenced strains revealed a mean mutation rate of . eϪ substitutions/site/year ( % confidence interval, . eϪ to . eϪ ). analysis of concatenated coding regions from all hpiv- sequences with complete coding sequences (cds) in the genbank nt database as of september revealed a similar mean mutation rate of . eϪ substitutions/site/year ( % confidence interval, . eϪ to . eϪ ) similar to those of other paramyxoviruses ( ) . phylogenetic analysis of the newly sequenced hpiv- coding sequences revealed multiple lineages of hpiv- present in - clinical samples and clustering by time of collection as the dominant feature rather than by sequence obtained from viral isolate versus clinical sample (fig. ) . however, examination of the deep sequencing data for of the viral isolates revealed mutations with Ͼ % maf in residues and and residues to selected during the paired sample-isolate analysis above. while the original clinical samples for these isolates are not available, none of the clinical samples sequenced metagenomically contained variant residues at these loci. of the strains with variant residues, only viral isolates and laboratoryadapted strains contained variant residues with Ͼ % maf that would be expected to be reflected in consensus sequence available publicly. in addition to the newly derived hpiv- genome sequences, we also examined all complete and partial consensus hpiv- hn sequences available in the genbank nt database as of august -a total of , sequences-in a search for the culture-associated mutations isolated here. of the culture-associated mutations in the hn protein described above, p l ( ), s n ( ), l f ( ), l s ( ), n d ( ) , and t i ( ) appeared exclusively in sequences from cultured isolates ( , ( ) ( ) ( ) ( ) ( ) ( ) . none of the consensus sequences in ncbi with a p l mutation had a mutation in the to residue range (consensus sequence, -hkslnt- ). the h q mutation appeared previously in sequences, with the vast majority of these ( / ) occurring in culture isolates; note that as above, even sequences listed as clinical may have experienced brief exposure to culture conditions. the l f mutation has not been reported in any consensus hpiv- hn sequence in ncbi. three instances of the r k alteration were found in ncbi genbank ( , ) . one of these was recovered using rt-pcr directly from clinical specimens during an hpiv- outbreak in a children's hospital cancer ward in barcelona, spain, in summer ( ) . the remaining two r k mutations reported were from a likely culture-adapted college of american pathologists proficiency testing sample (strain f , genbank accession no. ky ) and a primary culture isolate from japan (ddbj accession no. ab ) ( ) . hpiv- culture adaptations localize to the dimer interface of hn. we mapped all the residue changes in the hn protein that arose during adaptation to brief culture onto the hpiv- hn protein crystal structure wef (fig. ). all alterations localized to the dimer interface. we previously described critical residues at the hn dimer interface for hn's roles in receptor engagement and activation of f at h -which we identified as the secondary sialic acid binding site-and in receptor cleavage at n , shown at the top of the hn globular head dimer interface in the diagram (fig. ) . the diagram highlights the fact that n likely makes contact with the nac modification of n , and glycosylation could therefore have a significant effect on binding. we have shown that alterations that enhanced dimer contacts were associated with culture adaptation, while host fitness was correlated with reduced dimer contacts at these residues ( ) . alterations in residues and have been associated with other culture conditions parainfluenza virus fitness for host tissues ® as well ( - ), supporting the notion that this site is intimately involved in the mechanism whereby hn activates f in the natural host. the mutations p l and r k have not been described before and appear in a cluster at the opposing side of the dimer interface. the rapid emergence of alterations here during adaptation of a patient's hpiv- to culture suggests a role in viral entry in the natural host. of note, p does not make specific contact with the opposing monomer, but a mutation would change the conformation of the loop significantly, thereby altering the contacts that the loop can have. hn mutations in site ii and dimer interface modulate fusion promotion. to understand the selection forces that affect hpiv- fitness, we next analyzed the biological properties conferred by the culture-associated mutations in hn. here we characterized the influence of each specific residue on the function of the hn-f fusion complex. to investigate whether the identified mutations altered hn's fusion promotion ability we had previously observed in clinical isolates of hpiv- , we introduced seven of these rapidly emerging mutations into the background of a well-characterized clinical isolate hn, clinical isolate (ci- ) ( , ). we paired these mutated hn proteins with a lab-adapted f to ensure that we could observe adequate levels of fusion to compare function, since clinical isolate hn and f pairs result in nearly unobservable levels of fusion ( ) . fusion promotion by each of these mutant hn-f pairs was quantified with a ␤-galactosidase complementation assay and compared to the wildtype clinical isolate hn (ci wt) paired with the lab-adapted f (fig. ) . we included an influenza a virus hemagglutinin (ha) and f pair as a positive control for fusion, as it had been previously shown that hn has a stabilizing property that prevents f triggering; that is, an increase in fusion relative to the ci wt would be expected and was seen ( ) . the fusion exhibited by all mutant hn-f pairs was considerably higher than that of the ci wt hn-f pair, ranging from a -fold increase (hn s n) to a -fold increase (hn l f). neuraminidase activity is reduced by hn's adaptations to culture except for h q. after observing that fusion promotion was increased by all hn mutants, we examined several interacting properties of hn (see introduction) that are involved in this process. we assessed the neuraminidase (receptor cleavage) activity and also quantified the release of receptor-bearing erythrocytes (rbcs) from hn-expressing cells as a measure of the balance between avidity and receptor-cleaving properties. neuraminidase activity was measured through the cleavage of =-( -methylumbelliferyl)-␣-d-n-acetylneuraminic acid, sodium salt hydrate ( -munana) by hn (fig. a) . as previously described, almost all the culture-adapted hn mutants displayed lower neuraminidase activity than ci wt, ranging from roughly % (l s) to % (r k) relative activity ( ). the h q mutant had increased neuraminidase activity, with roughly % activity relative to ci wt. we have previously shown that this mutation at h on hn confers both higher receptor avidity and intrinsically enhanced triggering of f and thereby augments fusogenicity, an effect that overrides the increase in receptor cleavage (see the release assay below) ( ) . release of bound receptor-bearing rbcs by cell surface hns reflects the balance of neuraminidase activity, which would release the bonds tethering the cells, and avidity of the hns, which retain the bound cells (fig. b) . we quantified the amount of rbcs released at several time points when incubated at ph . and °c. differences between the hns was most readily observed at the earliest time point of min, when ci wt had the most release (~ %), whereas the mutant hns all had less release, reflecting either their higher receptor avidity, lower neuraminidase activity, or both. we observed two separate groups of mutants in their capability to release rbcs. a more rapidly releasing group-p l, r k, and s n-had roughly % release at min and complete release (~ %) after min. in contrast, the more slowly releasing group, consisting of h q, l f, l s, and n d, had to % release at min and to % release after min. as noted above, the higher receptor avidity of hn can override the higher-than-wt neuraminidase activity. (fig. b, yellow squares) , producing no detected progeny compared to a titer of to pfu/ml for each of the viruses bearing a single adaptive hn mutation. the growth in cv- cells reveals that while the clinical virus is severely handicapped in monolayer culture, the mutations that emerged during the rhmk culture isolation process confer growth in these monolayer cultures. while the wt ci shows negligible fitness in monolayer culture compared to the adapted viruses on day , by day infectious virus is released into the supernatant fluid of the virus to cell culture. at day however, adaptive mutations have appeared (table ) and correlate with increased production of infectious particles (fig. b, yellow squares) . in hae tissue, the wt ci- has a growth advantage over all the mutated viruses, showing that the individual culture-adaptive hn mutations modestly impair infection of hae (fig. b, orange circles) . at day after hae infection, wt ci releases . ϫ infectious particles into the hae apical surface, which is between and times more particles than released by the mutant viruses. as expected based on our previous studies ( ), growth in hae did not result in emergence of mutations (table ) . mechanisms that affect respiratory virus-cell interplay in the context of humans are finely tuned to the environment of the host ( , ) . to understand natural viral infection and the features that govern infectivity and transmissibility in humans, we have endeavored to analyze field strains in airway tissues. we have shown for human parainfluenza virus type that conclusions drawn from laboratory strains can be misleading with regard to the receptor interaction and viral fusion properties that govern entry and fitness in vivo ( , ) . analysis of clinical strains suggests that the hn-f fusion pairs of circulating hpiv- viruses maintain a balance of properties that result in an inverse correlation between fusion in cultured cells and growth in vivo; the fusion we have previously used whole-genome sequencing of field strains of hpiv- isolated from humans and grown only in airway cultures to analyze the diversity and the commonalities of circulating strains of hpiv- ( ). the sequences were used to define functional and structural properties of proteins of circulating strains in order to identify the genetic basis for properties that confer fitness in the field. while laboratoryadapted and ci airway-grown strains shared the basic properties of the hn-f fusion complex-receptor binding, fusion triggering, receptor cleavage-the balance between these properties at each step is shared across all cis we studied and different from all laboratory strains. the ci fusion pairs all possessed lower fusogenicity and decreased f activation compared to those of laboratory strains. here we carried out deep genomic screens of paired hpiv- clinical samples; one portion of each sample was sequenced directly from the patient with no culture step, while the other portion was exposed to brief culture for viral isolation in immortalized rhesus monkey kidney (rhmk) cells, a standard cell line used in clinical laboratories for virus identification. we hypothesized that even during such brief periods of culture, mutations arising in the hn-f fusion complex could be informative about features essential for growth in humans that would be stringently selected against in culture. hpiv- evolution upon exposure to culture was extraordinarily rapid and almost entirely limited to the hn protein. bioinformatic analyses of existing hn sequences revealed that hn mutations that arose during brief exposure to culture are almost uniformly found in consensus sequences from hpiv- that are either adapted to culture or exposed to culture-and not found in hpiv- sequenced directly from clinical samples. deep sequencing of newly derived hpiv- isolates and clinical samples revealed that more than one-third of hpiv- isolates had subpopulations of hn dimer interface variants that were not reflected in consensus sequences and were not present in hpiv- sequences obtained directly from clinical samples. the sequences were used to define functional and structural properties that differ from laboratory-selected circulating strains in order to identify the genetic basis for properties that may confer fitness in the field. the hn mutations that we identified in response to exposure to culture included both novel and previously observed and characterized adaptations to immortalized cells. remarkably, all the mutations map to the dimer interface and receptor binding site ii of hn. these results point to the critical role of this region in the hpiv- entry mechanism and for fitness in vivo, as we have deduced from smaller-scale and mechanistic studies ( - , ). the h residue at receptor binding site ii at the dimer interface of hpiv- hn influences hn's dimerization and its activation of f ( , ). we have shown that the presence of the glutamine at position at the dimer interface of the hpiv- hn increases hn oligomerization and thereby modulates f triggering, with this hn more actively triggering f ( , ). when the virus bearing this mutated hn was subjected to growth in human airway, it rapidly acquired a compensatory mutation, q r, which reduced fusion triggering and reduced hn dimerization. structural analysis of footprints of the dimer interfaces showed that when hn h q is present in receptor binding site ii, more of the interface residues are in contact with the opposing monomer face. however, in the hn with the q r compensatory mutation that permits growth in the airway, the dimer interface footprint shows decreased points of contact. the dimer interface footprint pointed to several residues and domains whose contact with the opposing monomer may be critical for the key functions governed by hn's receptor binding site ii. the emergence of the h q mutation after brief culture highlights both the flexibility of hn's site ii to adapt to new conditions and also the exquisite constraint of the virus's fusion complex with respect to alterations in the environment. adjacent to the h residue in the dimer interface, but not making contact in the dimer interface footprint mentioned above, is n , a residue we have previously shown to be highly conserved in all clinical strains. this residue was previously found to be an aspartic acid in laboratory-adapted strains, associated with a dramatic decrease in neuraminidase activity consistent with the overall high-receptor-contact/highfusion laboratory-adapted phenotype. in the present study, the n d mutation emerged during the very brief exposure to culture, highlighting the advantage of lowering neuraminidase activity to reduce receptor cleavage for culture conditions versus the importance of neuraminidase for human infection. within the same cluster, s was altered during culture adaptation; this is a residue that contacts the opposing monomer in the dimer interface cluster ( , ) . the precise role of this residue in the functions of the dimer region will be explored. strikingly, several mutations emerged in residues at the opposite side of the hn dimer interface from the cluster at positions to : residues , , and . the points of contact of n and r with the opposing hn monomer in the dimer interface footprint had been noted to correlate with fusion promotion and airway adaptation; however, the function of these residues had been unexplored ( ) . the selection for l f, p l, and r k in the absence of mutations in the domain from positions to suggests that the newly identified important region in the hn dimer interface may act independently of receptor binding site ii, and the functional relationships between these two sites will be explored in future studies. numerous deep sequenced viral isolates also demonstrated p l and r k hn variants in the absence of mutations in the domain from positions to . the finding that this second dimer interface domain undergoes rapid selection in the background of authentic clinical strains suggests that it may be relevant to fitness in the airway. to investigate the biological significance of these adaptations, we generated singly mutated hn molecules in the background of our well-characterized hpiv- clinical virus hn, ci- ( ), and generated recombinant viruses bearing the hn mutations on the genetic background of ci- . brief exposure to culture, through this diversity of strategies, resulted in increased fusion promotion for all the hn variants tested: h q, s n, l f, l s, n d, p l, and r k. while perhaps not unexpected based on our previous hypotheses, the absolute consistency of this finding-that through a range of specific mutations and specific functional and structural differences, the virus arrives at increased fusion-is remarkable. each individual hn mutation conferred fitness for recombinant virus infection of immortalized monolayer culture cells and for spread of virus through the culture, compared to the parent wt ci. to explore the components of hn's properties that contribute to the enhanced fusion promotion and infection properties for all the emerging hn mutants, we assessed neuraminidase activity and hn's release of receptor-bearing target cells, a measure of the balance between receptor avidity and cleavage ( ) . we have shown that clinical viruses, compared to laboratory strains, have a combination of high neuraminidase, low avidity, low f triggering, resulting in lower fusogenicity. the hn mutants that emerged here all except for hn h q had decreased neuraminidase activity. it is of interest that mutations at site ii affect neuraminidase enzymatic activity, since this site is not known to possess enzymatic activity, which resides in the bifunctional active site at t /d ( , ( ) ( ) ( ) . one mechanistic conjecture that will be explored is that alterations or interactions at one hpiv- hn site (site i or ii) modulate the function of the other site, as we have shown for another paramyxovirus, newcastle disease virus, where receptor interaction at hn site i leads to the activation of site ii ( , ) . these data may suggest an unexplored cross talk between site i and site ii that could alter neuraminidase activity allosterically. all the mutants-including hn h q-showed reduced release of rbcs compared to the clinical strain. the hn h q mutant's decreased release of rbcs in the face of increased neuraminidase activity relative to the ci wt is in agreement with our previous finding that this mutation confers increased receptor avidity ( ) that balances the receptor-cleaving activity for an overall retention of contact. interestingly, the h q mutation in the lab-adapted hn background does not have higher neuraminidase activity ( ), supporting the notion that the phenotype conferred by these sites may be modified in the context of the genetic background. collectively, the pattern is of enhanced receptor engagement and an increase in fusogenicity that confers fitness in vitro. the genetically engineered viruses bearing the individual alterations in hn show that each is fit for growth in monolayer culture compared to the wt parent and less fit for growth in hae. the mutations emerging during the brief viral isolation process thus led directly to fitness for growth in monolayer culture. interestingly, there is no single mutation or minor allele required for successful growth in culture, although all samples in this study did produce at least one nonsynonymous minor allele on either the f or hn proteins. the minor alleles were extremely biased toward mutations in the hn or f protein, with eight of ten cultures presenting minor alleles on the hn protein and four presenting a minor allele on the f protein, supporting the critical nature of the hn-f fusion complex for host adaptation ( ). intriguingly, single mutations in hn did not confer fatal growth disadvantage in the airway model, despite conferring fitness in monolayer culture. it will be of interest to determine whether an accumulation of several fusion-skewing mutations is necessary for an absolute inability to grow in airway and whether a more sensitive experimental system (i.e., infection in vivo) would be responsive to single alterations or only to a greater dimer interface disturbance. the genome sequences presented here provide a significant resource for future hpiv- genomic epidemiology and biochemical characterization, increasing the number of publicly available genomes by more than % ( , ) . we obtained full genome sequence coverage from as few as an estimated , starting copies ( ) and found that relatively uniform whole viral genome coverage can be achieved with , paired-end reads, suggesting that many samples could be sequenced in a single miseq run. whole-genome viral sequencing from clinical samples should be both technically and economically feasible, permitting investigators to avoid the use of laboratoryadapted strains of virus in immortalized cultured cell lines. the deep genome-wide screens of paired clinical samples and viral isolates allow for simple, rapid interrogation of viral evolution in the context of multiple clinical strain backgrounds. the methods pursued here allowed for discovery of novel hn residues of interest that heretofore had not been identified based on consensus sequence. deep sequencing after rapid selection pressure may allow for discovery of a greater number of novel mutations that may be removed from the population by drift or competition with other variant subpopulations after longer selection periods yet provide clues to function. other techniques that can more comprehensively profile single mutations, such as deep mutational scanning, are often limited by their use of singular backgrounds, often in the context of culture-adapted strains, and focus on singular proteins. our results are consistent with an "uncertainty principle" for virology: the act of attempting to study a clinical viral sample through commonly performed laboratory procedures, such as viral culture in cell lines, affects the authenticity of the viral biology even before the first passage. that viruses evolve uniquely in culture has been shown for hpiv- ( ) ; however, the extraordinarily rapid nature of evolution in culture, even without passage, is notable. most of the adaptive genotypes could be found as minor alleles only by deep sequencing of clinical samples and viral isolates. with the increased provision of deep coverage viral genomes obtained directly from clinical samples due to diagnostic mngs, experiments similar to the one outlined here will be critical for potentially pathogenic viruses in order to assess whether previous viral characterization studies apply to newly sequenced clinical samples ( , ) . for hpiv- , our results are consistent with the notion that use of authentic culture models that better reflect hpiv- growth in vivo is critical if we are to understand and characterize pathogenesis ( ) . the specific mutations that emerged in the cultured samples, while disturbing from the perspective of the "uncertainty" described above, provide a wealth of information about hn domains that may be critical for fitness in humans. in our focus on the correlation of emerging hn mutations to function, we did not characterize all combinations of hn mutations, and for functional studies, we paired hns with a lab-adapted more fusogenic f protein in order to readily discern differences in hn properties; ongoing studies will explore the potential for epistatic interactions in hpiv- evolution. of particular interest is the cluster of residues around p and r that were uncovered as critical in this study. the interactions of these residues with the opposing monomer, and these points of contact of this domain within the interface, were suggested based on structural data to be relevant for fusion promotion ( ) . future studies will address the function of this hn dimer interface domain in hpiv- fusion and cell entry. sample description. the term "clinical sample" is used to describe primary nasal swabs in viral transport media, while "viral isolate" or "culture isolate" is used to describe culture harvest from zero passage virus (i.e., brief exposure to culture only during viral isolation). for paired clinical sample/culture isolate sequencing, l of nasal swab viral transport medium from nine patients from to with an hpiv- cycle threshold (c t ) of Ͻ was inoculated onto primary rhesus monkey kidney (rhmk) cells (quidel diagnostics) and incubated on a roller drum at °c. the tubes were assessed for cytopathic effect daily and confirmed via hemadsorption with % guinea pig red blood cells (rbcs). positive cultures were harvested and stored at Ϫ °c. deidentified bronchial alveolar lavage (bal) fluid and nasal swab samples from patients from used for prior clinical testing were stored at Ϫ °c and subjected to mngs. in addition, culture harvests for which viral isolation was performed for prior clinical testing of bal fluid samples, nasal swabs, and sputum samples from to at the university of washington virology laboratory were also stored at Ϫ °c and subjected to mngs. culture harvests from to were performed using the same protocol detailed above for the paired sequencing experiment. use of excess deidentified clinical specimens was approved by the local university of washington institutional review board (irb) (protocol study ). sample metadata, including genbank accession numbers for consensus hpiv- genomes, are available in table s in the supplemental material and associated with ncbi bioproject . mngs and amplicon sequencing. mngs was performed as previously described ( ) . rna was extracted from l of thawed culture harvests and clinical samples using the zymo viral rna kit (zymo research) and treated with turbo dnase i (thermo fisher) for min at °c ( ) . doublestranded cdna was made using random hexamers, superscript iii reverse transcriptase (thermo fisher), and sequenase v . (thermo fisher) and cleaned using zymo- dna clean and concentrator (zymo research). mngs libraries were produced by adding one-third volume of nexteraxt (illumina) and finished with cycles of dual-indexed pcr amplification and cleaned using . ϫ ampure beads (beckman coulter) ( ) . libraries were sequenced using one -bp and two -bp runs on an illumina miseq system. libraries from the matched viral isolates were prepared twice in independent library preparations and sequencing runs to control for artifacts of library preparation. amplicon sequencing was performed to investigate linkage between mutations recovered during mngs. reverse transcription-pcr (rt-pcr) was performed using cycles of the default parameters (melting temperature [t m ], °c) in the one-step rt-pcr kit (qiagen) with pcr primers hpiv - f (f stands for forward) ( =-tacagatagggataataactgtaaa- =) and hpiv - r (r stands for reverse) ( =-gctttgctcctaagttttttatatt- =). amplicons were purified using . ϫ ampure beads, and dualindexed truseq libraries were prepared using the kapa hyperprep kit with eight cycles of pcr amplification ( ) . sequence data alignment and analysis. sequencing reads were adapter and quality trimmed using cutadapt and then aligned to the hpiv- reference sequence (nc_ ) with geneious v . . . consensus genomes were extracted for original patient samples, and culture sample sequencing reads were aligned to the sample consensus. consensus genomes were visualized and mutations and minor alleles with coverage of Ͼ ϫ and minor allele frequency (maf) of Ͼ % were called using geneious . . . the known locus of rna editing in the phosphoprotein gene was not included among the variant nonsynonymous alleles, nor were adenosine deaminase-like changes present on the same strand in p virus (affecting minor variants at r g and i v in ci- in table ). phylodynamic analysis. for phylodynamic analysis, coding nucleotide sequences from the newly generated hpiv- genomes sequenced were extracted, concatenated, and aligned using genious v . . and mafft ( ) . the resulting alignment was used to generate a bayesian skyline tree with an uncorrelated relaxed clock and hasegawa, kishino, and yano (hky) substitution model with gamma plus invariant site heterogeneity model (four gamma categories) and three codon partitioning in beast v . . with a markov chain monte carlo (mcmc) length of , , iterations ( ) . tree generation trace information and evolutionary clock rates were gathered and analyzed using tracer v . . ( ). the resulting trees were annotated and compiled using treeannotator v . . and then annotated and scaled in figtree v . . . the same phylodynamic protocol was also performed on all complete hpiv- genomes present in ncbi nt as of august . recombination detection was performed using rdp 's rdp, chimaera, maxchi, genecov, siscan, and seq algorithms with default settings ( ) . to determine whether culture-associated hn amino acid changes were present in our paired sample sequencing, we downloaded all partial and complete hpiv- hn sequences from ncbi nt as of august . metadata on whether specific sequences originated from clinical sample or culture material were gathered from the ncbi genbank sequence record and associated manuscripts in pubmed. however, it is not always possible to determine whether there was brief exposure to culture conditions prior to sequencing. to show how the background of hn protein sequence could influence the amino acid mutations selected during culture, a majority consensus amino acid sequence of hn protein from each putative clinical sample was extracted and aligned using mafft. a neighbor-joining tree was created using the genious tree building algorithm, jukes-cantor distance method, and , replicates. functional characterization of hpiv- hn. the hn mutants examined in this study were generated through site-directed mutagenesis of the previously constructed pcaggs mammalian expression vector with the ci- hn gene ( ) . plasmids were sanger sequence confirmed prior to usage. transfections with plasmids were performed in hek t cells using lipofectamine in accordance with the manufacturer's instructions. cell surface expression of each hn was measured through standard immunofluorescence assay procedures using monoclonal antibodies against hpiv- hn and used for normalization as appropriate. two monoclonal anti-hn antibodies used here (vt- g -b and vt- f -b ) were custom-made by aldevron using dna immunization. briefly, a cdna encoding hpiv- hn was cloned into expression plasmids (pegfp) ( ) . groups of laboratory rats (wistar) were immunized by intradermal application of dna-coated gold particles using a handheld device for particle bombardment (gene gun). serum samples were collected after a series of immunizations and tested in flow cytometry on hek cells transiently transfected with the above expression plasmids. antibody-producing cells were isolated and fused with mouse myeloma cells (ag ) according to standard procedures. measurement of fusion promotion. the ability of individual hns to promote fusion was measured through a ␤-galactosidase complementation assay as previously described ( , ) . hns were paired with the f protein from a lab-adapted strain ( ), transiently transfected in hek t cells, and then cell fusion was quantified. lab-adapted f partner molecules were used as previously described ( , ) , since fusion is minimal when ci hn and f are expressed in pairs, and does not allow for quantitative comparisons. measurement of neuraminidase activity. neuraminidase activity of hns were measured as previously described ( , ) . hek t cells were transiently transfected with individual hns, collected through detachment with mm edta, washed with dulbecco phosphate-buffered saline (dpbs) supplemented with g/liter d-glucose and mg/liter sodium pyruvate (gibco), then resuspended in co -independent medium (ph . ) (gibco). cells were then added to =-( -methylumbelliferyl)-␣-d-n-acetylneuraminic acid, sodium salt hydrate ( -munana) at a : ratio for a final concentration of . mm. fluorescence was measured every min for h at °c with a microplate reader (tecan). measurement of rbc release. the ability of hns to release bound rbcs was measured as described previously ( , ) . rbcs were bound to cells for min at °c and washed to remove unbound rbcs. co -independent medium (ph . ) was then added to the cells, and the cells were incubated at °c for the appropriate times before being collected and replaced for the next time point. after min, rbcs that remained bound to the cells were lysed with ack (ammonium-chloride-potassium) lysing buffer (gibco) and quantified by measurement of absorbance at nm. immortalized cell culture. cv- (african green monkey kidney) and t (human kidney epithelial) cells were grown in dulbecco's modified eagle's medium (dmem) (cellgro; mediatech) supplemented with % fetal bovine serum (fbs) and antibiotics at °c in % co . recombinant virus production and analysis. rhpiv clinical isolate (ci- )-egfp is a recombinant virus generated with a plasmid coding for the majority consensus sequence of hpiv- ci- ( , , ) obtained by deep sequencing and containing an enhanced green fluorescent protein (egfp) expression cassette between p and m viral genes. all mutants were derived from this unique sequence. recombinant viruses were generated by reverse genetics in the rhpiv -ci- -egfp-f k background using a modification of methods previously described ( ) . briefly, t cells were seeded in six-well plates. the following day, the cells were transfected using profection mammalian transfection system, calcium phosphate kit (promega) according to the manufacturer's recommendations with g of pflc plasmid encoding the full-length viral genome, ng of pcaggs-hpiv p, ng of pcaggs-hpiv l, ng of pcaggs-hpiv n, ng of pcaggs-t polymerase, ng of pcaggs-hpiv lab-adapted hn, and ng of pcaggs-hpiv lab-adapted f ( ). the following day, supernatant fluids were replaced with dmem supplemented with % fbs. two days after transfection, the cells were mechanically detached by pipetting up and down and overlaid onto cv- cells in a -cm culture dish. cultured cells were monitored by fluorescence microscopy until % of the cells were infected. the medium was replaced with dmem without fbs, and cells were incubated for up to days at °c in % co . supernatant fluid was collected, centrifuged at ϫ g for min at °c, aliquoted, and kept at Ϫ °c until use. all recombinant viruses were sequenced at this stage using the same mngs methods detailed above on l of supernatant fluid to confirm the correct ci backbone and hn mutation sequences before use in experiments. viral propagation in human airway epithelia. the hae epiairway air- system (mattek corporation) consists of normal human-derived tracheo/bronchial epithelial cells that have been cultured to form a pseudostratified, highly differentiated mucociliary epithelium closely resembling that of epithelial tissue in vivo. upon receipt from the manufacturer, human airway epithelium (hae) cultures were transferred to six-well plates (containing ml medium per well) with the apical surface remaining exposed to air and incubated at °c in % co . hae cultures were infected with , pfu of recombinant clinical isolate egfp ( ) (referred to as ci- -egfp-f k ) at the apical surface for min at °c. the wild type (wt) ci- -egfp-f k or ci- -egfp-f k harboring single mutations in hn (i.e., p l, r k, h q, s n, or l f) was used. at min, the medium containing the inoculum was removed, and cultures were placed at °c and fed each day with ml medium via the basolateral surface. viruses were harvested by adding l of ϫ phosphate-buffered saline (pbs) with calcium and magnesium chloride (mattek corporation) per well to the hae cultures' apical surface and equilibrating for min at °c. the suspension was then collected, and viral titers were determined as previously described ( ) . this viral collection was performed sequentially with the same wells of cells on each indicated day postinfection. viral propagation in cv- . cv- cultures were seeded in -well plates. seventy percent confluent cells were infected with , pfu of recombinant ci- -egfp-f k (wt or harboring single mutations in hn [i.e., p l, r k, h q, s n, l f]) at the apical surface for min at °c. after min, the medium containing the inoculum was replaced with ml of dmem supplemented with % fbs. the supernatant fluid was then collected, aliquoted, and replaced with the same volume of dmem supplemented with % fbs, and viral titers were determined as previously described ( ) . this viral collection was performed sequentially with the same wells of cells on each indicated day postinfection. supplemental material for this article may be found at https://doi.org/ . /mbio . - . table s , pdf file, . mb. global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis control of an outbreak of human parainfluenza virus in hematopoietic stem cell transplant recipients epidemiology and clinical presentation of the four human parainfluenza virus types features of circulating parainfluenza virus required for growth in human airway circulating clinical strains of human parainfluenza virus reveal viral entry requirements for in vivo infection interaction between the hemagglutinin-neuraminidase and fusion glycoproteins of human parainfluenza virus type iii regulates viral growth in 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virus hemagglutinin-neuraminidase receptor binding: effect of receptor avidity and steric hindrance at the inhibitor binding sites paramyxovirus receptor-binding molecules: engagement of one site on the hemagglutinin-neuraminidase protein modulates activity at the second site second sialic acid binding site in newcastle disease virus hemagglutinin-neuraminidase: implications for fusion database resources of the national center for biotechnology information clinical metagenomic identification of balamuthia mandrillaris encephalitis and assembly of the draft genome: the continuing case for reference genome sequencing rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis rapid metagenomic next-generation sequencing during an investigation of hospital-acquired human parainfluenza virus infections a metagenomic analysis of pandemic influenza a ( h n ) infection in patients from north america rule-out outbreak: -hour metagenomic next-generation sequencing for characterizing respiratory virus source for infection prevention myeloablation-associated deletion of orf in a human coronavirus e infection mafft: a novel method for rapid multiple sequence alignment based on fast fourier transform bayesian phylogenetics with beauti and the beast . tracer v mechanism of interference mediated by human parainfluenza virus type infection infection of ciliated cells by human parainfluenza virus type in an in vitro model of human airway epithelium human parainfluenza virus infection of the airway epithelium: the viral hemagglutinin-neuraminidase regulates fusion protein activation and modulates infectivity we thank the patients from western washington state and staff of the university of washington virology laboratory who contributed to the project. this work was supported by nih/national institute of allergy and infectious diseases (niaid) r ai and r ai to a.m. r.c.s. received support from the mary gates endowment for students. key: cord- - qi aibx authors: van de groep, kirsten; nierkens, stefan; cremer, olaf l.; peelen, linda m.; klein klouwenberg, peter m. c.; schultz, marcus j.; hack, c. erik; van der poll, tom; bonten, marc j. m.; ong, david s. y. title: effect of cytomegalovirus reactivation on the time course of systemic host response biomarkers in previously immunocompetent critically ill patients with sepsis: a matched cohort study date: - - journal: crit care doi: . /s - - - sha: doc_id: cord_uid: qi aibx background: cytomegalovirus (cmv) reactivation in previously immunocompetent critically ill patients is associated with increased mortality, which has been hypothesized to result from virus-induced immunomodulation. therefore, we studied the effects of cmv reactivation on the temporal course of host response biomarkers in patients with sepsis. methods: in this matched cohort study, each sepsis patient developing cmv reactivation between day and (cmv+) was compared with one cmv seropositive patient without reactivation (cmvs+) and one cmv seronegative patient (cmvs−). cmv serostatus and plasma loads were determined by enzyme-linked immunoassays and real-time polymerase chain reaction, respectively. systemic interleukin- (il- ), il- , il- , interferon-gamma–induced protein- (ip- ), neutrophilic elastase, il- receptor antagonist (ra), and il- were measured at five time points by multiplex immunoassay. the effects of cmv reactivation on sequential concentrations of these biomarkers were assessed in multivariable mixed models. results: among cmv+ patients, could be matched to cmvs+ or cmvs− controls or both. the two baseline characteristics and host response biomarker levels at viremia onset were similar between groups. cmv+ patients had increased ip- on day after viremia onset (symmetric percentage difference + % versus − % when compared with cmvs+ and + % versus + % when compared with cmvs−) and decreased il- ra (− % versus % and − % versus + %, respectively). however, multivariable analyses did not show an independent association between cmv reactivation and time trends of il- , ip- , il- , or il- ra. conclusion: cmv reactivation was not independently associated with changes in the temporal trends of host response biomarkers in comparison with non-reactivating patients. therefore, these markers should not be used as surrogate clinical endpoints for interventional studies evaluating anti-cmv therapy. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. cytomegalovirus (cmv) reactivation is observed in - % of intensive care unit (icu) patients without known prior immune deficiency [ ] [ ] [ ] and is associated with increased morbidity and mortality [ ] [ ] [ ] . in a previous study, we estimated that the population-attributable fraction of icu mortality due to cmv reactivation was % in patients with acute respiratory distress syndrome (ards) [ ] . in a subsequent study among patients with septic shock, we found an effect of cmv reactivation on icu mortality only in patients with concurrent epstein-barr virus reactivation [ ] . although multiple studies point toward a causal relationship, definitive proof that cmv reactivation worsens clinical outcome is lacking, as most data are also compatible with a scenario in which cmv reactivation is merely a marker of immune suppression in this patient group. based on previous studies in icu patients, there is a clear pathophysiological link between inflammation and immune suppression on the one hand and the subsequent risk of cmv reactivation on the other [ ] [ ] [ ] [ ] [ ] . markers reflecting impaired functioning of natural killer cells and cytotoxic t cells were predictive of cmv reactivation [ , ] . furthermore, bacterial sepsis and corticosteroids have been identified as clinical risk factors for cmv reactivation [ , , ] . however, less is known about the reverse association and thus the effects of cmv reactivation on the immune system. direct cytotoxic effects of cmv on organs have been observed primarily in immunocompromised hosts [ ] but also in previously immunocompetent patients in the icu [ ] . moreover, indirect immune-modulating effects are assumed to play a role in the pathogenicity of cmv [ , [ ] [ ] [ ] . in vitro analysis revealed multiple mechanisms encoded within the genome of cmv that may contribute to a non-specific inhibition of both cellular and humoral immunity [ ] . observational clinical studies yielded conflicting results comparing levels of multiple inflammatory markers in patients with and without cmv reactivation [ , , ] . however, these studies analyzed biomarker responses only immediately upon icu admission and thus could not assess potential immunological effects due to the onset of cmv reactivation. nevertheless, cytokine levels were used as a primary (surrogate) endpoint in a recent placebo-controlled randomized control trial in which prophylactic antiviral treatment with ganciclovir failed to reduce interleukin- (il- ) levels [ ] . hence, definite proof of immune-modulating effects induced by cmv remains to be demonstrated. naturally, such an effect can be demonstrated only after onset of cmv reactivation. therefore, this longitudinal study aimed to investigate whether the temporal course of seven host response biomarkers, including both pro-and anti-inflammatory cytokines, in previously immunocompetent icu patients with sepsis differs between patients with and without cmv reactivation. this matched cohort study was performed among patients who had been included in two previous studies conducted within the molecular diagnosis and risk stratification of sepsis (mars) cohort [ , ] . for this study, we included sepsis patients who presented with either concomitant ards or septic shock to the mixed icus of two university medical centers in the netherlands between january and june and had remained in the icu beyond day . exclusion criteria were cmv seronegative patients with cmv viremia (thus a primary infection) during their icu stay and known immunodeficiency or anti-viral treatment in the week before icu admission. the institutional review boards of both study centers approved an opt-out method of informed consent (protocol number - c). from this parent cohort, we selected patients with an onset of cmv reactivation between day and in the icu. these patients with viremia were matched to two control groups consisting of patients without viremia on any day of icu admission. first, we matched patients with reactivation in a : ratio to cmv seropositive patients without reactivation (further referred to as "primary comparison"). second, we matched patients with reactivation in a : ratio to cmv seronegative patients without cmv viremia (further referred to as "secondary comparison"). this secondary comparison was intended mainly to confirm results of the primary comparison; the rationale was that any finding suggestive for an effect of cmv reactivation should also become apparent when compared with seronegative patients who are not at risk for cmv reactivation. matching criteria to reduce confounding were length of stay until reactivation (determines t = ), sequential organ failure assessment (sofa) score at t = (± points), age (± years), sex, and high-dose corticosteroid use during days prior to t = (that is, more than mg hydrocortisone or equivalent). patients were also matched on hospital and calendar day of icu admission (± days) in order to reduce possible influences of variation in sample workup and biobank storage duration [ ] . the optimal matching result was retrieved by selecting the largest sample size after random iterations of the matching procedure. leftover plasma, obtained daily as part of routine patient care, was stored at − °c and used to determine cmv serostatus at icu admission. subsequently, cmv load in blood was measured weekly, and for intermediary days, on which quantitative polymerase chain reaction was not performed, we estimated viral loads by log-linear imputation (see electronic supplementary materials of [ ] ). cmv viremia was defined as at least international units (iu) per milliliter. this cutoff value was similar to the ones used in previous studies [ , ] . results of cmv viral load measurements in plasma performed for this study were not made available to the treating physicians, and none of the included patients received anti-cmv treatment. to map the immune response, we measured a panel of host response biomarkers in samples derived from five time points: day of viremia onset (t = ), days prior (t = − ), and after viremia onset at day , , and (sample availability depended on length of stay in the icu). a multiplex luminex immunoassay was performed by using edta plasma and included the following proteins: il- , il- , il- , tumor necrosis factor-alpha (tnf-α), tnf-related apoptosis-inducing ligand (trail), interferon-gamma (ifn-γ), ifn-γ-induced protein- (ip- ), neutrophilic elastase, granzyme-b, il- receptor antagonist (ra), and il- . based on the results of a pilot run using samples obtained from ards patients without sepsis at icu admission (whom were not included in this study), we excluded ifn-γ, tnf-α, trail, and granzyme-b from the final panel because the levels of these biomarkers were below the lower limit of detection in more than % of the samples. of note, in this pilot run, cmv reactivation was not associated with detectability of the four excluded biomarkers. measurements of biomarkers were performed by using an in-house developed and validated multiplex immunoassay (iso certified) based on luminex technology (xmap, luminex, austin, tx, usa). the assay was performed as described previously [ ] . in short, thawed edta plasma samples ( μl) were diluted : in high performance elisa (hpe) buffer (sanquin, the netherlands) and centrifuged through filtration columns to remove debris. then non-specific heterophilic immunoglobulins were pre-absorbed from all samples with heteroblock (omega biologicals, bozeman, mt, usa). next, samples were incubated with antibody-conjugated magplex microspheres for -h at room temperature with continuous shaking and this was followed by -h incubation with biotinylated antibodies and -min incubation with phycoerythrin-conjugated streptavidin diluted in hpe buffer. acquisition was performed with the flexmap d system (bio-rad laboratories, hercules, ca, usa) in combination with xponent software version . (luminex). data were analyzed by -parametric curve fitting using bio-plex manager software, version . . (bio-rad laboratories). univariable analyses were performed to compare patients and disease characteristics for matched groups with and without cmv reactivation using chi-squared, wilcoxon rank sum, or fischer exact tests as appropriate. measured host response markers were natural log-transformed concentrations in picograms per milliliter for all analyses. symmetric percentage differences were calculated for each patient at the different time points. this delta percentage reflects the relative change from the measurement days prior to cmv reactivation until the follow-up measurement [ ] . we performed additional multivariable analyses by using generalized linear mixed models to assess the effect of cmv reactivation on the time course of each individual biomarker. in the mixed model analyses, we assessed whether baseline biomarker levels were comparable between matched groups (that is, coefficient for cmv reactivation) as well as the effect of cmv on the course of the biomarker levels over time (that is, coefficient for interaction term between time and cmv reactivation). a priori we chose to model the established immune markers il- and il- . based on the observed divergence in the symmetric percentage differences over time between groups, we conducted the multivariable analyses also for the pro-inflammatory chemokine ip- and the anti-inflammatory cytokine il- ra. since not all cmv reactivation patients were included in both comparisons, we performed separate mixed model analyses for the primary and secondary comparisons. thus, in total, eight models were built (for each of the four biomarkers in each of the two comparisons). for each model, sofa score at t = (that is, the day of reactivation) and age were included as confounders since we used a range (instead of an exact value) as matching criteria for these co-variables. for the fixed part of th emodels a polynomial term for time was evaluted (that is, quadratic time effect). furthermore, a random intercept and a rondom slowe were evaluted for each model. restricted maximum likelihood estimation (reml) was used to generate unbiased variance estimates for the final models [ ] . different ways to model the time course for each host response marker were compared by using the likelihood ratio test and akaike's information criterion. to take multiple testing into account and reduce the risk of spurious findings, we performed all statistical testing against a p value of . and used a confidence interval of %. bonferroni adjustment was deemed inappropriate and too conservative as the different measurements performed over time within a single patient and hence the tests were highly correlated with each other. analyses were performed by using either sas enterprise guide . (sas institute, cary, nc, usa) or r version . . (r foundation for statistical computing, ; used packages "lme ", "lmetest"). forty-five ( %) of eligible patients with cmv reactivation during icu day - could be included after matching (fig. ) . twenty-eight patients were matched to a seropositive patient as well as a seronegative, nine to only seropositive, and eight to only seronegative, respectively. this resulted in a study population of unique patients, divided into a primary comparison (that is, with cmv reactivation matched to cmv seropositive without reactivation) and a secondary comparison (that is, with cmv reactivation matched to cmv seronegative). patient and disease characteristics at icu admission were comparable between matched groups and are presented in table . in the patients with cmv reactivation, median peak level of cmv dna load was iu/ml (interquartile range (iqr) - ). median length of stay in the icu until reactivation was days (iqr - ), which was influenced by the used inclusion criterion (that is, viremia onset between day and in the icu). of the unique sepsis patients included, ( %) presented to the icu with septic shock and ( %) patients had ards during the first week of icu admission. in the primary comparison, the median icu length of stay was days (iqr - ) for patients with cmv reactivation versus days (iqr [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] for subjects without reactivation (p = . ). this was days (iqr - ) versus days (iqr [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in the secondary comparison (p = . ), respectively. hospital mortality was % for patients with cmv reactivation and % for the matched patients without reactivation in the primary comparison (p = . ). in the secondary comparison, this was % versus % (p = . ), respectively. baseline levels of measured host response markers were comparable between patients with and without reactivation, both at t = − (that is, days prior to viremia onset) and at t = (that is, day of reactivation onset) ( table ). in general, this remained the case for each marker up to days after cmv reactivation; the exceptions were median il- levels (which were significantly higher on day ) and median il- levels (which were significantly lower on day ) in patients with cmv reactivation compared with controls (additional file : table s ). however, these differences were not consistent across both primary and secondary comparison. time trends of various markers within patients were described by symmetric percentage differences relative to their levels days prior to cmv viremia onset (fig. for primary comparison, additional file : figure s for secondary comparison). for ip- and il- ra, differences in time trends were observed between patients with and without reactivation in both comparisons. patients with cmv reactivation had a more pronounced increase of ip- (median percentage difference of % versus − %) and decrease of il- ra (median percentage difference of − % versus %) on day after viremia onset compared with cmv seropositive patients without reactivation. for the secondary comparison, with cmv seronegative patients, similar differences in trends were observed for ip- (+ % versus + %) and il- ra (− % versus + %), respectively. of importance, sample size decreased over time because of death or icu discharge with a minimum of per patient group after days (additional file table s ). in the multivariable mixed model analyses, cmv reactivation did not significantly affect the baseline levels of il- , ip- , il- , and il- ra (table ) . a significant decrease over time was observed in all patients for il- in both the primary and secondary comparison and for il- in the primary comparison only, respectively. however, cmv reactivation did not significantly affect the time trend of any of the four analyzed biomarkers. we performed an explorative study to compare time trends of host response biomarkers in patients with reactivation that were matched to non-reactivating control patients who were either seropositive or seronegative for cmv. although we initially observed differential trends of il- ra and ip- in the crude analysis, these differences did not remain in the linear mixed model analysis the hypothesis of an immune-modulating effect of cmv is based on the observation of increased mortality and morbidity in patients with viremia without organ manifestation of cmv disease [ , ] . proposed mechanisms of such indirect pathogenicity are autoantibody production, enhanced inflammation, vascular damage, and cmv-induced immunosuppression [ ] . based on this hypothesis and an observed association between plasma markers and mortality in patients with ards [ ] , il- was used as a surrogate endpoint in a recent randomized controlled trial that evaluated the safety of preventive antiviral treatment in icu patients [ ] . our finding that cmv reactivation is not associated with modified il- dynamics questions the suitability of il- as an endpoint in clinical trials evaluating preventive therapy for cmv reactivation in icu patients. furthermore, time trends of other immunological biomarkers were not robustly affected by cmv reactivation. our study has several strengths. first, to our knowledge, this is the first study with serial measurements of the immune response following (instead of prior to) cmv reactivation. second, our study design included two matched control groups. because of the used matched cohort design, we could include only out of patients with cmv reactivation but this loss was compensated by the ability to include controls that were more comparable to those patients. sepsis patients in the icu are known to be very heterogeneous [ , ] ; thus, the matching reduced in theory both confounding and unwanted variation by extraneous factors. third, by using mixed model analyses, we accounted for correlation of measurements performed within one patient by the use of random effects, which increased the statistical power to identify differences between patient groups. moreover, this type of analysis takes into account the considerable loss to follow-up of patients and allowed us to estimate an average trend over time based on available data. our study also has some limitations. first, this was an explorative study evaluating multiple host response biomarkers. we chose a lower p value threshold of significance in order to decrease the risk of spurious findings due to multiple testing, but false-negative findings remain an accessory risk to keep in mind also when considering our study sample size. unfortunately, a formal sample size calculation for this kind of statistical analysis was not possible. nevertheless, we postulate that possible immunomodulating effects of cmv reactivation il- . ( . - . ) . ( . - . ) . . ( . - . ) . ( . - . ) . ip- . ( . - . ) . ( . - . ) . seem at most to be rather limited in these patients because no large differences in biomarker levels between matched groups were observed. second, we analyzed host response biomarkers as standalone markers, which is probably a simplification of the complex immune response. however, large sample sizes are required to perform more advanced network analyses, and the integration of time series in such analyses, to our knowledge, has not been conducted before. we also measured only the plasma concentrations. since cmv pneumonitis could be an important mediator of the pathological effect of cmv reactivation in critically ill patients, bronchoalveolar lavage samples may be additionally informative but were not available [ , ] . finally, we did not evaluate all potentially relevant biomarkers for cmv reactivation; thus, future studies are needed before an immunomodulating effect of cmv can be ruled out with certainty as an important pathological mechanism in previously immunocompetent icu patients. this study could not demonstrate an independent immunomodulating effect of cmv reactivation in patients with sepsis. this finding does not lend support for the use of immunological markers as surrogate endpoints for clinical outcome in interventional studies of prophylactic or pre-emptive cmv therapy in icu patients. additional file : table s . absolute levels of host response markers during follow-up by cytomegalovirus (cmv) reactivation status. figure s . ethics approval and consent to participate all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional or national research committee (or both) and with the helsinki declaration and its later amendments or comparable ethical standards. for this study, an opt-out informed consent method was approved. not applicable. cytomegalovirus reactivation in a general, nonimmunosuppressed intensive care unit population: incidence, risk factors, 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cytomegalovirus (cmv)-specific t-cell immunity for the assessment of the risk of active cmv infection in non-immunosuppressed surgical and trauma intensive care unit patients human cytomegalovirus infections in nonimmunosuppressed critically ill patients cytomegalovirus reactivation in icu patients human cytomegalovirus: clinical aspects, immune regulation, and emerging treatments a contributive result of open-lung biopsy improves survival in acute respiratory distress syndrome patients cmv in critically ill patients: pathogen or bystander cytomegalovirus-induced immunopathology and its clinical consequences reactivation of multiple viruses in patients with sepsis the 'indirect' effects of cytomegalovirus infection virological and immunological features of active cytomegalovirus infection in nonimmunosuppressed patients in a surgical and trauma intensive care unit effect of ganciclovir on il- levels among cytomegalovirus-seropositive adults with critical illness: a randomized clinical trial cytokine assays: an assessment of the preparation and treatment of blood and tissue samples effect of anticoagulants on circulating immune related proteins in healthy subjects statistics notes: percentage differences, symmetry, and natural logarithms using the general linear mixed model to analyse unbalanced repeated measures and longitudinal data lower tidal volume ventilation and plasma cytokine markers of inflammation in patients with acute lung injury why have clinical trials in sepsis failed? unexplained mortality differences between septic shock trials: a systematic analysis of population characteristics and control-group mortality rates immunological insights into the pathogenesis of active cmv infection in non-immunosuppressed critically ill patients we thank the department of medical microbiology and the multiplex core facility of the laboratory for translational immunology for their logistical support and performance of measurements, the participating icus and research nurses of the two medical centers for their help in data acquisition, and all members of the molecular diagnosis and risk stratification of sepsis all authors declare that they have no conflicts of interest related to the subject matter. key: cord- - gjepq h authors: lee, jin-ho; cho, hyeon-yeol; choi, hye kyu; lee, ji-young; choi, jeong-woo title: application of gold nanoparticle to plasmonic biosensors date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: gjepq h gold nanoparticles (gnps) have been widely utilized to develop various biosensors for molecular diagnosis, as they can be easily functionalized and exhibit unique optical properties explained by plasmonic effects. these unique optical properties of gnps allow the expression of an intense color under light that can be tuned by altering their size, shape, composition, and coupling with other plasmonic nanoparticles. additionally, they can also enhance other optical signals, such as fluorescence and raman scattering, making them suitable for biosensor development. in this review, we provide a detailed discussion of the currently developed biosensors based on the aforementioned unique optical features of gnps. mainly, we focus on four different plasmonic biosensing methods, including localized surface plasmon resonance (lspr), surface-enhanced raman spectroscopy (sers), fluorescence enhancement, and quenching caused by plasmon and colorimetry changes based on the coupling of gnps. we believe that the topics discussed here are useful and able to provide a guideline in the development of novel gnp-based biosensors in the future. recent advances in nanotechnology have revealed new possibilities for the development of biosensors with improved performance based on the unique properties of nanoparticles [ ] . for instance, the high surface area-to-volume ratios of nanomaterials facilitate the performance of bio/chemical analyte capturing and sensing by providing more active surface regions. more specifically, magnetic nanoparticles can be employed for the enrichment of targets analytes, and polymeric or plasmonic metal nanoparticles can be introduced for signal amplification [ ] [ ] [ ] . among these nanoparticles, plasmonic nanoparticles have gained particular interest from various scientists and engineers because of their unique physical and chemical properties. they have been widely applied in the biomedical fields for diagnostics, imaging, and therapeutics [ , , ] . in particular, gold nanoparticles (gnps) have drawn enormous interest, owing to their easy preparation, biocompatibility, inertness, and intense colors [ , ] . additionally, the ease of surface modification with a wide range of biomolecules, such as oligonucleotides and proteins, through the formation of stable bonds with mercapto or amino groups to create specific/selective binding, allows for the development of new biosensor platforms with enhanced capabilities in the detection of various biological/chemical analytes [ , , ] . one of the most prominent and useful features of gnps for biosensor applications is their unique and tunable optical properties. in particular, localized surface plasmon resonance (lspr) can be modulated by different sizes, shapes, and surface coupling of the nanoparticles [ ] [ ] [ ] [ ] . upon irradiation with light, the free electrons in the gnp are excited, simultaneously inducing collective coherent non-propagating oscillations of surface plasmons, which is known as lspr [ ] . gold surface plasmons exhibit unique features underlying their strong absorption and scattering of light, making gnps fascinating candidates for biological sensing and imaging probes. briefly, the localization of the electromagnetic field results in the amplification of spr signals and surface-enhanced raman scattering (sers) [ , ] . fluorescent signals can be also quenched or enhanced due to the energy transfer between the gnp and fluorophore [ ] . additionally, a high molar extinction coefficient and significant color changes can be observed by aggregating gnps, resulting in the development of naked-eye-based biosensors [ ] . furthermore, many studies have already revealed that gnps are relatively nontoxic when compared to other nanoparticles, such as metal oxides or carbon-based materials in general [ , ] . this qualification of gnps has led to the development of biosensors for in vitro/in vivo diagnosis and imaging of molecular interactions on a cellular level as well. in this paper, we review and discuss recently developed plasmonic biosensors combined with the unique optical properties of gnps. we provide an overview of the unique optical properties of gnps utilized for the development of biosensors and then highlight newly developed gnp-based plasmonic biosensors. each section individually focuses on one of the following: lspr, sers, plasmon-enhanced fluorescence and quenching, and colorimetry changes based on the coupling of plasmonic gnps. one of the most well-established unique optical characteristics of gnps that is widely utilized for the development of biosensors is their localized surface plasmon resonance (lspr) phenomenon [ , ] . when noble metals, such as gold, are exposed to light, a resonant interaction between electron-charged oscillations near the surface of the metal and the electromagnetic field of the light creates propagating surface plasmons. this unique physical property changes enormously when the size of metal becomes nanoscale relative to the bulk material, resulting in a confined and non-propagating localized surface plasmon around the nanoparticle with a specific frequency known as the lspr [ , ] . in the particular case of gnps, the lspr yields exceptionally high absorption coefficients and scattering properties within the visible to near-infrared (nir) wavelength range [ , ] . these spectral characteristics of gnps are independent of size, shape, and the local dielectric environment, therefore, the refractive index changes induced by an adsorbate on a gnp can be used to monitor molecular binding events [ , ] . recently, several studies on the development of biosensors based on lspr have been reported. lee et al. have proposed a simple electrochemical deposition technique to fabricate gnps on a transparent indium tin oxide substrate for the detection of human immunodeficiency virus (hiv) based on the lspr mechanism ( figure a ) [ ] . the binding of biomolecules on the gnp surface was quantitatively analyzed through absorbance changes, which are caused by the change in refractive index on the gnp surface due to specific binding occurring through immunoreactions without any labeling materials. comparably, van duyne and his group used gnps as a labeling material to improve the signal of an lspr-based immunoassay. anti-biotin antibodies were labeled with nm gnps and introduced to a biotin-functionalized silver nanoprism array. as a result, a . nm wavelength shift was induced, while unlabeled anti-biotin resulted in only an nm redshift. ( figure b ) [ ] . as an alternative approach, instead of using array systems, single gnp spectroscopy is also utilized for biosensing by using dark-field microscopy techniques. compared to array systems, single-nanoparticle-based lspr biosensors have several advantages, such as higher sensitivity and smaller sample volume requirements. alivisatos and his coworkers have used spectral changes based on the dimerization of gnps to measure dna length and track hybridization kinetics. the distance was determined on the basis of plasmonic coupling between two gnps modified at two ends of a dna strand ( figure c ) [ ] . two gnps were separately conjugated to each single-stranded dna (ssdna) end by gold-thiol interactions on one end, and streptavidin-biotin interactions on the other end. the distance between the gnps was adjusted by controlling the length of the ssdna and by changing the ionic strength of the buffer. the plasmon resonance (lspr) maximum shifted to the red region at high salt concentrations ( . m nacl), indicating the decreased distance between the two gnps due to the reduced electrostatic repulsion of the particles at high ionic strength environments. conversely, low salt concentrations ( . m nacl) increased electrostatic repulsions and led to a blue shift of the lspr maximum. along with these results, the hybridization of complementary dna also resulted in a significant blue shift, which is expected considering that a structural property of double-stranded dna (dsdna) is a stiffness greater than ssdna, therefore allowing it to push apart two gnps. lee et al. also demonstrated that a combination of plasmonic dimer probes can be used to detect and image mrna splice variants in live cells [ ] . the gnps were functionalized with oligonucleotides as a probe, and the hybridization of these probes with specific mrna sequences led to the formation of nanoparticle dimers that exhibited distinct spectral shifts due to their plasmonic coupling. with this approach, they showed the spatial and temporal distribution of three selected splice variants of the breast cancer susceptibility gene (brca ) and also monitored single-copy resolutions by measuring the hybridization dynamics of nanoplasmonic gnp dimers ( figure d ). was determined on the basis of plasmonic coupling between two gnps modified at two ends of a dna strand ( figure c ) [ ] . two gnps were separately conjugated to each single-stranded dna (ssdna) end by gold-thiol interactions on one end, and streptavidin-biotin interactions on the other end. the distance between the gnps was adjusted by controlling the length of the ssdna and by changing the ionic strength of the buffer. the plasmon resonance (lspr) maximum shifted to the red region at high salt concentrations ( . m nacl), indicating the decreased distance between the two gnps due to the reduced electrostatic repulsion of the particles at high ionic strength environments. conversely, low salt concentrations ( . m nacl) increased electrostatic repulsions and led to a blue shift of the lspr maximum. along with these results, the hybridization of complementary dna also resulted in a significant blue shift, which is expected considering that a structural property of double-stranded dna (dsdna) is a stiffness greater than ssdna, therefore allowing it to push apart two gnps. lee et al. also demonstrated that a combination of plasmonic dimer probes can be used to detect and image mrna splice variants in live cells [ ] . the gnps were functionalized with oligonucleotides as a probe, and the hybridization of these probes with specific mrna sequences led to the formation of nanoparticle dimers that exhibited distinct spectral shifts due to their plasmonic coupling. with this approach, they showed the spatial and temporal distribution of three selected splice variants of the breast cancer susceptibility gene (brca ) and also monitored single-copy resolutions by measuring the hybridization dynamics of nanoplasmonic gnp dimers ( figure d ). raman spectroscopy is a spectroscopic technique based on the inelastic scattering of photons from the targeting molecules in the sample which reflects the specific chemical bond information. even though raman spectroscopy has a great potential to be applied as a biosensor by taking advantage of its specificity, the weak signal intensity is its main drawback. the lspr phenomenon of plasmonic gnps has been utilized to enhance the light scattering signal for biosensing applications. lspr leads to resonant absorption or scattering of the incident light, thereby effectively coupling the incident light energy into the metal nanoparticles, including gnps. this results in a - -order magnitude enhancement of the local electromagnetic (em) field intensity at the nanoparticle surface, which is the key to the huge enhancement in sers [ , ] . moreover, recent studies of sers on metal nanoparticles demonstrated the importance of having local field "hotspots" due to surface roughness [ , ] , nanogaps between aggregated plasmonic nps [ , ] , or np-metal surfaces [ ] which can induce higher em enhancements, and the sers contribution to such hotspots often dominates the observed response [ ] . to generate homogeneous "hotspots" using gnps, researchers have fabricated gnp arrays [ , ] . liu and coworkers utilized a simple droplet evaporation process to assemble highly ordered octahedral gnp arrays with nanoscale interparticle gaps ( figure a ) [ ] . to form highly ordered nanogaps, which have a role as a "hotspot" for sers, gold octahedra with an edge length of about nm were used as building blocks for d and d superlattices. however, the preformed gnp arrays cannot selectively place the target sample at the "hotspot" specifically to obtain the highly enhanced signal, therefore, different approaches have been reported for locating the target molecule at the hotspot. several studies have recently reported the development of a sers-based biosensor that acts as a "direct biosensor" by detecting specific signals from the target biomolecules, such as neurotransmitters [ ] , nucleosides [ ] , and microrna (mirna) [ ] . tu and his coworkers developed a sers-based serotonin analysis method by using gnps in the presence of various closely related precursors and metabolites ( figure b ) [ ] . due to their structural similarity, serotonin and tryptophan-a precursor of serotonin-show very similar sers spectra when mixed with gnps. to distinguish two amino acids, sers measurements were conducted under a range of different ph conditions covering the pk value. particularly, the sers signal intensity of serotonin was significantly higher than tryptophan at ph . and ph . conditions. this is likely a result of gnps aggregating with the positively charged ampholytes, forming d complexes. similarly, feng and his coworkers analyzed the differences of nucleosides between nasopharyngeal and esophageal cancers collected from the urine by sers after incubating with gnps [ ] . by taking advantage of the chemical-specific spectral information in raman spectroscopy, the combination of distinct raman frequencies from different chemicals is able to be utilized as a barcoding system, as well as an "indirect biosensor" [ ] . this raman barcoding system provides the potential to overcome a "multiplexing ceiling" from existing optical materials. cho and his coworkers developed a raman barcoding-based biosensor with gnps to diagnose and analyze circulating cancer stem cells (ccscs) in the presence of circulating tumor cells (ctcs) ( figure c ) [ ] . for this approach, five different combinations of raman-active nanoprobes (rans) were prepared, each consisting of a gnp (as a raman signal enhancer), aromatic thiol (as a raman reporter), antibodies, and thiolated dna (as a capturing linker). when the ccscs and the ctcs with spiked with five families of rans in the blood, the subtype-specific sers signals decorated the cell and were analyzed once the cell was captured by the microfluidic device. another way to develop an "indirect biosensor" is by combining gnps' sers and catalytic properties to provide a particularly promising platform for the real-time probing of gnp-catalyzed reactions and for developing sensitive bioassays. hu and her coworkers synthesized a nanomaterial which embedded gnps and glucose oxidases in the metal-organic framework to generate hydroperoxide. the generated hydroperoxide induced the activation of a co-delivered precursor (leucomalachite green, lmg) into the active form (malachite green, mg) ( figure d ) [ ] . the assembled oxidase oxidized the substrate (i.e., glucose or lactate), and the hydroperoxide was produced based on the amount of the substrate. the generated hydroperoxide continued to oxidize lmg to the raman-active mg via the gnps' catalysis, as measured by sers. after optimizing the in vitro sensing performance, it was further employed to monitor the change of glucose and lactate in living brains following the ischemia/reperfusion model. even though sers-based biosensors using gnps have shown great performance for the detection and analysis of target molecules, some improvements are needed to overcome current limitations, including signal reproducibility and sensitivity. green, mg) ( figure d ) [ ] . the assembled oxidase oxidized the substrate (i.e., glucose or lactate), and the hydroperoxide was produced based on the amount of the substrate. the generated hydroperoxide continued to oxidize lmg to the raman-active mg via the gnps' catalysis, as measured by sers. after optimizing the in vitro sensing performance, it was further employed to monitor the change of glucose and lactate in living brains following the ischemia/reperfusion model. even though sers-based biosensors using gnps have shown great performance for the detection and analysis of target molecules, some improvements are needed to overcome current limitations, including signal reproducibility and sensitivity. plasmonic gnps have another interesting optical phenomenon with localized fluorophores, which is highly useful for the development of biosensors [ ] . in general, gnp can lead to both fluorophore quenching and enhancement that can be explained by a well-established förster (or fluorescence) resonance energy transfer (fret) mechanism [ , ] . when the gnp and the fluorophore are within a few nanometers from each other, the non-radiative local field of one material (donor) can excite the other one (acceptor), which then translates into either the quenching or plasmonic gnps have another interesting optical phenomenon with localized fluorophores, which is highly useful for the development of biosensors [ ] . in general, gnp can lead to both fluorophore quenching and enhancement that can be explained by a well-established förster (or fluorescence) resonance energy transfer (fret) mechanism [ , ] . when the gnp and the fluorophore are within a few nanometers from each other, the non-radiative local field of one material (donor) can excite the other one (acceptor), which then translates into either the quenching or enhancement of the fluorescent signal. more specifically, the quenching happens when fret occurs from the fluorophore to gnp; alternatively, the enhancement of fluorophore, which is typically referred to as plasmon-enhanced fluorescence (pef), can happen, depending on the electromagnetic field generated from the plasmonic metal surface [ ] . particularly, it is well known that these effects can be balanced by the spatial variation (distance) between the gnp and the fluorophore [ , ] . for example, fluorophore quenching can be observed at a short distance (< nm) and enhanced fluorescence can be observed at an adjusted distance (~ nm). it should be noted that gnps can also enhance the radiative rate of the fluorophore through the purcell effect at longer distances ( ~ nm); however, since the resulting purcell effect is negligible between plasmonic metals and fluorophores, it will not be discussed herein [ ] . oh and his coworkers have developed a fluorescent biosensor based on a site-specific enzymatic cleavage reaction to detect a prostate-specific antigen (psa) by utilizing the gnps as energy acceptors (quencher) ( figure a ) [ ] . a small peptide sequence with a fluorophore (fluorescein isothiocyanate, fitc) on one end was conjugated to the gnp through the functional amine group on the other end. initially, fluorescence associated with the fluorophore was quenched due to the fret between the fluorophores and the gnps; however, when the psa recognized and cleaved the specific sequence of the peptides conjugated to the gnp, a recovery of fluorescence signal was observed due to the splitting of the fitcs from the gnp. in a similar way, degliangeli et al. functionalized gnps with fluorophore-labeled dna-probe strands to detect mirna ( figure b ) [ ] . the presence of target mirna led to the hybridization of dna-rna strands, and, following enzyme hydrolysis, fluorescence signal recovery was observed by splitting fluorophores from gnps. enhancement of the fluorescent signal. more specifically, the quenching happens when fret occurs from the fluorophore to gnp; alternatively, the enhancement of fluorophore, which is typically referred to as plasmon-enhanced fluorescence (pef), can happen, depending on the electromagnetic field generated from the plasmonic metal surface [ ] . particularly, it is well known that these effects can be balanced by the spatial variation (distance) between the gnp and the fluorophore [ , ] . for example, fluorophore quenching can be observed at a short distance (< nm) and enhanced fluorescence can be observed at an adjusted distance (~ nm). it should be noted that gnps can also enhance the radiative rate of the fluorophore through the purcell effect at longer distances ( ~ nm); however, since the resulting purcell effect is negligible between plasmonic metals and fluorophores, it will not be discussed herein [ ] . oh and his coworkers have developed a fluorescent biosensor based on a site-specific enzymatic cleavage reaction to detect a prostate-specific antigen (psa) by utilizing the gnps as energy acceptors (quencher) ( figure a ) [ ] . a small peptide sequence with a fluorophore (fluorescein isothiocyanate, fitc) on one end was conjugated to the gnp through the functional amine group on the other end. initially, fluorescence associated with the fluorophore was quenched due to the fret between the fluorophores and the gnps; however, when the psa recognized and cleaved the specific sequence of the peptides conjugated to the gnp, a recovery of fluorescence signal was observed due to the splitting of the fitcs from the gnp. in a similar way, degliangeli et al. functionalized gnps with fluorophore-labeled dna-probe strands to detect mirna ( figure b ) [ ] . the presence of target mirna led to the hybridization of dna-rna strands, and, following enzyme hydrolysis, fluorescence signal recovery was observed by splitting fluorophores from gnps. as an alternative direction for developing fluorescent based biosensors, bracamonte and his group have developed highly fluorescent nanoparticles by utilizing gnps as the core, silica as the shell (as a spacer), and rhodamine b (rhb) as a fluorophore for bacteria imaging and sensing ( figure c ) [ ] . the plasmon resonance maximum of the core-shell nanoparticles was able to be red shifted due to interactions with the gnp core based on the thickness of the silica shell. the pef effect from the gnp core was also evaluated by applying sodium cyanide as a gold leeching material. in addition, bright and clear bacteria images were obtained by laser fluorescence microscopy after labeling them with the developed core/shell pef nanoparticles. teixeira et al. also utilized gnps as cores and covered them with an polyelectrolyte shell to promote enhancements for a phthalocyanine fluorescent dye [ ] . similar to these approaches, various studies were performed to verify the effect of spatial variation by controlling spacing within shell thickness. for example, montaño-priede et al. demonstrated that the maximum fluorescence enhancement of quantum dots (qds, cdse) was observed when a silica shell between a gnp core and qds was approximately nm; however, both the reduction and the increase of shell thickness diminished the enhancement effect of the fluorescence either by energy transfer from the qds to gnp or a larger separation between the cdse qds and gnp core, respectively ( figure d ) [ ] . similar to the aforementioned unique optical properties of gnps, the color of the gnp solution is also affected by the lspr phenomenon upon the aggregation and dispersion of the gnps [ ] . the aggregation of gnps can induce the coupling of the plasmon modes when the interparticle distances are less than . times their diameters [ ] . this electromagnetic coupling of gnps induced a red shift and broadening of the plasmon resonance peak, associated with the longitudinal resonance [ ] . the aggregation or dispersion of the gnps is influenced by changes to the external environment, including ph [ ] , chemicals [ ] , metal ions [ ] , and biomolecules (dna [ ] [ ] [ ] , mirna [ ] , peptide [ ] , and proteins [ , ] ). depending on the interaction between targets and gnps, gnps can have roles as effective nucleation sites for fast-catalyzing the target aggregation. choi and his coworkers utilized gnps as a nucleation core and colorimetric optical reporters for tracking amyloid-beta (aβ) aggregation ( figure a ) [ ] . the aβ monomers were electrostatically attached to the surface of the gnps at ph ~ , since aβ has a positive charge while gnps have a negatively charged surface. the aggregation speed and size of the aggregated composites are directly related to the concentration of aβ in the sample, and it can be monitored by a color change, from red to purple or blue. this colorimetric biosensing platform was also used to confirm the inhibition properties of anti-aβ antibodies and human serum albumin on the aβ oligomerization. similarly, lou and her coworkers found that digested dna has superior abilities over intact dna for binding to unmodified gnp solution at high salt concentration [ ] . however, these non-crosslinking aggregation-based colorimetric biosensors show several limitations in signal stability, reproducibility, and target specificity because of the nonselective interaction between the target and the gnps and lack of a crosslinking method to overcome the interparticle repulsive forces. to induce target-specific gnp aggregation, gnps have been functionalized with target-recognizing molecules, such as single-stranded dna (ssdna) [ , ] , antibodies [ ] , and aptamers [ ] [ ] [ ] [ ] . after mirkin and his coworkers reported a biosensor in which can analyze a single mismatch in the hybridized dna by using the ssdna-conjugated gnps [ ] , dna and rna samples have been analyzed with an interparticle crosslinking aggregation-based colorimetry method [ ] . lee and his coworkers designed gnp networks interconnected with duplex dna with strategically placed hg + -complexed t-t mismatches ( figure b ) [ ] . the cysteine analyte can bind mercuric ion and remove it from the aggregate, thereby lowering the dna dissociation temperature. in this way, the presence of cysteine can be detected in a specific manner. similarly, aptamer-modified gnp-based colorimetric biosensors have been developed to detect proteins [ ] , peptides [ ] , and bacteria [ ] . zhu and his coworkers showed the different degrees of gnp aggregation profiles when comparing the gnp solution containing aptamer only with the gnp containing an aβ-aptamer with sodium ions ( figure c ) [ ] . the aβ-aptamer complexes were stabilized by the gnps in the presence of sodium ions in the colloidal solution. in the case of colorimetric biosensor-based protein detection, gnps are also applicable for the detection of enzymatic reactions [ , ] . de la rica and his coworkers developed a plasmonic enzyme-linked immunosorbent assay (elisa) as a colorimetric biosensor ( figure d ) [ ] . in the presence of the analyte, the enzyme catalase consumes hydrogen peroxide, and low concentrations of hydrogen peroxide favor the formation of aggregated, spherical nanoparticles that give rise to a blue solution. modified gnp-based colorimetric biosensors have been developed to detect proteins [ ] , peptides [ ] , and bacteria [ ] . zhu and his coworkers showed the different degrees of gnp aggregation profiles when comparing the gnp solution containing aptamer only with the gnp containing an aβaptamer with sodium ions ( figure c ) [ ] . the aβ-aptamer complexes were stabilized by the gnps in the presence of sodium ions in the colloidal solution. in the case of colorimetric biosensor-based protein detection, gnps are also applicable for the detection of enzymatic reactions [ , ] . de la rica and his coworkers developed a plasmonic enzyme-linked immunosorbent assay (elisa) as a colorimetric biosensor ( figure d ) [ ] . in the presence of the analyte, the enzyme catalase consumes hydrogen peroxide, and low concentrations of hydrogen peroxide favor the formation of aggregated, spherical nanoparticles that give rise to a blue solution. recent advances in both nanofabrication/nanochemistry and detection techniques have led to multiple approaches for the research and development of plasmonic biosensors. the unique plasmonic properties of gnps provide enormous potential for biosensing research when they are combined with a material, having such advantages as easy preparation, biocompatibility, inertness, and intense light emissions. a large variety of designs of gnp-based systems have been reported to improve their plasmonic properties in the specific optical systems for the targeted applications. for example, ( ) immobilizing or patterning gnps on the surface of a metal substrate for an lspr-based biosensor; ( ) controlling the interparticle gap distance of gnps to generate "hotspots" for the sers-based biosensor; ( ) controlling the distance between a fluorophore and a gnp for enhancing or quenching the fluorescent signal; and ( ) controlling the dispersion stability in a solution for construction of a colorimetric biosensor ( table ) . the performance of gnp-based plasmonic biosensors shows clear advantages when compared to other optical biosensors; however, a lack of signal reproducibility, standardization, and the necessity of confirmation with conventional methods forms a hurdle for the plasmonic biosensors to be fully implemented in practice when answering significant biomedical questions. the major concern for plasmonic biosensors is their signal reproducibility, because randomly conjugated analytes or randomly aggregated gnps can generate highly enhanced signals that do not match actual conditions. homogeneously nanopatterned substrates can help induce lspr, generate the "hotspot" uniformly, and guide the analyte into the "hotspot" specifically. moreover, analyzing the sample by combining different sensing techniques can provide new and precise information for specific analytes. for example, the chemical structure of an analyte can be changed by oxidation/reduction, therefore, spectroelectrochemical biosensors [ ] -combining plasmonic signal detection and electrochemical detection techniques-can be one of the ways to break through current limitations (i.e., difficulty distinguishing similarly structured chemicals) of plasmonic biosensors. recently, kim and his coworkers reported a potential spectroelectrochemical signal detection platform-the large-scale homogeneous nanoelectrode array-which shows a homogeneous sers signal [ ] . although the instability of the biological materials (e.g., dna, enzyme, aptamer, etc.) is still one of the major limiting factors for the development and commercialization of the biosensors, many studies have been performed to overcome this issue through the development of chemical-based biomolecules. for example, dna-binding hairpin pyrrole-imidazole (py-im) polyamide-a class of heterocyclic amino acid oligomers-was conjugated on the gnp for dna detection instead of the dna-binding protein [ ] . peptide nucleic acid (pna) is also used to improve stability by substituting nucleic acid (dna/rna) or aptamers [ ] . in addition, the proper immobilization of enzymes tends to improve stability. some immobilization techniques can diminish the activity of the enzyme due to random orientation or denaturation of the enzyme; however, proper immobilization approaches are found to be effective at maintaining enzyme activity, as well as biosensor stability [ , ] . consequently, in combination with these newly developed methods, we believe with the further improvement of sensitivity, selectivity, and reproducibility of gnp-based plasmonic biosensors, plasmonic biosensors will find more impactful applications 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biomarker by au nanoparticle-decorated bi se nanosheets fabrication of new single cell chip to monitor intracellular and extracellular redox state based on spectroelectrochemical method large-scale nanoelectrode arrays to monitor the dopaminergic differentiation of human neural stem cells sequence-specific fluorescence detection of dna by polyamide-thiazole orange conjugates multiplex paper-based colorimetric dna sensor using pyrrolidinyl peptide nucleic acid-induced agnps aggregation for detecting mers-cov, mtb, and hpv oligonucleotides recent advances in electrochemical glucose biosensors: a review immobilization strategies to develop enzymatic biosensors label-free detection and molecular profiling of exosomes with a nano-plasmonic sensor ultrasensitive surface-enhanced raman scattering detection in common fluids plasmonic substrates for multiplexed protein microarrays with femtomolar sensitivity and broad dynamic range key: cord- -yiqdsf z authors: schlub, timothy e; buchmann, jan p; holmes, edward c title: a simple method to detect candidate overlapping genes in viruses using single genome sequences date: - - journal: mol biol evol doi: . /molbev/msy sha: doc_id: cord_uid: yiqdsf z overlapping genes in viruses maximize the coding capacity of their genomes and allow the generation of new genes without major increases in genome size. despite their importance, the evolution and function of overlapping genes are often not well understood, in part due to difficulties in their detection. in addition, most bioinformatic approaches for the detection of overlapping genes require the comparison of multiple genome sequences that may not be available in metagenomic surveys of virus biodiversity. we introduce a simple new method for identifying candidate functional overlapping genes using single virus genome sequences. our method uses randomization tests to estimate the expected length of open reading frames and then identifies overlapping open reading frames that significantly exceed this length and are thus predicted to be functional. we applied this method to reference rna virus genomes and find that it has both high sensitivity and low false discovery for genes that overlap by at least nucleotides. notably, this analysis provided evidence for previously undiscovered functional overlapping genes, some of which are coded in the antisense direction suggesting there are limitations in our current understanding of rna virus replication. gene overlap occurs when two or more genes share the same region of a nucleotide sequence in a genome. this occurs frequently in viruses, especially those with rna genomes, but has also been observed in bacteria and in eukaryotes including humans (smith et al. ; keese and gibbs ; veeramachaneni et al. ; nakayama et al. ). the high prevalence of gene overlap in viruses has been attributed to two complementary theories: gene "compression" and gene "novelty." compression theory argues that the size of viral genomes is constrained by factors such as high mutation rates and the small capsid structure housing the genetic material. this constrained genome size subsequently exerts selection pressure on genes to overlap to maximize genetic potential (belshaw et al. ; chirico et al. ) . gene novelty theory asserts that the constrained nature of viral genomes, combined with their limited noncoding regions, makes the generation of new genes difficult without major changes in genomic structure or input from the host genome. mutations in current genes that generate a new open reading frame (orf) then allow the generation of new genes within an established older gene in a process called "overprinting" (keese and gibbs ; sabath et al. ; brandes and linial ) . these theories are not mutually exclusive and both processes may be operating in virus genomes. overlapping genes may also function as a mechanism for regulating gene expression and reduce the probability of mutation fixation in overlapping areas as the resident genes may have competing selection pressures (krakauer ; dreher and miller ) . due to these evolutionary constraints, overlapping genes frequently encode proteins with accessory functions that play important roles in pathogenicity or spread (rancurel et al. ). overlapping genes were first detected following the discovery that the cumulative length of protein sequences in bacteriophage u exceeded the length of the genome (barrell et al. ). today, the detection of overlapping genes still largely relies on laboratory methods that isolate, sequence, and align individual proteins to reference genomes (fellner et al. ) . these and other potential laboratory methods such as ribosome profiling (irigoyen et al. ) are costly and time intensive, making large scale screening and identification of overlapping genes expensive. necessarily, these factors have led to the development of bioinformatics and theoretical methods for the analysis of overlapping genes that rely on genome sequence analyses alone. for example, synonymous sites that exhibit a reduced nucleotide substitution rate are indicative of functional overlapping proteins; because these substitutions affect two proteins they are usually expected to be deleterious and hence are observed at a reduced rate (firth and brown , ; jagger et al. ) . a number of other properties of overlapping genes have been article ß the author(s) . published by oxford university press on behalf of the society for molecular biology and evolution. this is an open access article distributed under the terms of the creative commons attribution non-commercial license (http://creativecommons.org/licenses/by-nc/ . /), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. for commercial re-use, please contact journals.permissions@oup.com open access used as effective bioinformatics makers, such as synonymous codon dissimilarity between newly generated overlapping genes and the remainder of the genome (pavesi et al. (pavesi et al. , , and the restriction that particular codon sequence orders place on alternative reading frames (lebre and gascuel ) . for example, the reverse complementary nucleotide sequence for two adjacent tyrosines (tat/c and tat/c) will be a/gta and a/gta, which always creates a stop codon (either a taa or tag after a reading frame shift of nucleotide). although these properties help in the development of bioinformatics techniques to discover unknown overlapping genes, they are restricted by their requirement for multiple genomic sequences or by their poor sensitivity. with the rapid rise of metagenomics to discover new viruses (bekal et al. ; ballinger et al. ; shi et al. shi et al. , , efficient and sensitive approaches of identifying overlapping genes that require genome sequence information alone will be essential. herein, we present a new statistical method for detecting overlapping genes in different reading frames that relies on only a single nucleotide sequence of a gene or genome. we apply this method to a large scale computational screening of all available (linear) rna virus genomes. the method estimates the theoretical expected length of orfs before a stop codon is reached in all reading frames within an established gene. if an orf exists of much greater length than predicted by this expected length, then we surmise that there has been selection against the accumulation of stop codons that shorten the putative orf. we conclude that this constitutes evidence that the orf in question provides functional benefit to the virus. despite its simplicity, we show that this method is a powerful way to detect functional overlapping genes that can be readily applied to large scale computational screening of all known viruses and to viruses newly discovered through metagenomics. our rationale is that overlapping orfs with functional benefit will result in negative selection against nucleotide substitutions that introduce stop codons within that gene. accordingly, the length of orfs (as measured by the distance between bookend stop codons) is likely to be larger when they are functional than what would be expected by random chance alone where stop codons could be introduced without penalty. hence, the defining characteristic of our method for the detection of overlapping genes is identifying orfs larger than expected by chance alone (fig. a ). we developed three tests for estimating the distribution of expected orf lengths. briefly, in the first test, we estimate the expected length of orfs by permuting codon positions in the original reading frame and then measuring orf lengths in other reading frames. this process is repeated to generate an expected distribution of orf lengths (codon permutation test, fig. b ). in the second test, instead of permuting codon positions, the codon order is unchanged and nucleotide substitutions that would introduce synonymous mutations in the original reading frame are randomly generated (synonymous mutation test, fig. c ), before measuring orf lengths in the other reading frames. in the third test, referred to as the combined test, the p values for both the codon permutation test and the synonymous mutation test must fall below some cut-off value. to demonstrate the applicability of this method, we first considered andean potato latent virus that contains a known overlapping gene. andean potato latent virus is a positivesense single-stranded rna virus (family tymoviridae) in which the i _gp gene, that encodes the putative the expected orf length based on codon composition is calculated. orfs longer than expected by random chance are identified. the expected orf length is estimated by one of three tests. for the codon permutation test (b) the codon sequence on the original frame is permuted and orf lengths on alternative reading frames measured for each permutation. for the synonymous mutation test (c), codons that preserve the original amino acid sequence are randomly generated and the length of orfs on alternative reading frames subsequently measured (note that codon replacement is not restricted to the example mutations shown in the figure, all of which occur in the third nucleotide positions, and that codon replacement with the original codon is also possible). the third test requires both the codon permutation test and the synonymous mutation test p values to be below some cut-off value. simple method to detect candidate overlapping genes . doi: . /molbev/msy mbe movement protein, is overlapping. i _gp is codons long, is located on frame þ , and is largely contained within the larger ( codon) i _gp gene that encodes the enzymes necessary for virus replication (methyltransferase, endopeptidase, helicase, and polymerase) ( fig. a ). we calculated the distribution of expected orf lengths on frames þ and þ using the codon permutation test. the distributions of these lengths are shown with the shading in figure b and c. the actual orf lengths on frames þ and þ on the unpermuted i _gp gene are represented by black dots on top of the theoretical distribution. for frame þ , we observe orfs, all of which have lengths within expected ranges (p ¼ . ). a different picture is observed in frame þ. although there are orfs, of which have lengths within the expected range, there is a single orf whose length far exceeds the expected distribution of lengths (p < . ); this is correctly identified as i _gp . the synonymous mutation test produces similar results in this example. to explore the possibility of using this method to screen for candidate overlapping orfs, we calculated both the sensitivity and false discovery rate of the codon permutation test, the synonymous mutation test, and a combined test that requires an orf to be larger than expected by both the codon permutation and synonymous mutation test. as there are too few coding regions within a single genome to estimate the sensitivity and false discovery rate with sufficient precision, we estimated the population sensitivity and false positive rate across a subset of viruses (linear rna viruses) known to contain many overlapping genes (see materials and methods section). accordingly, whole genome sequences were downloaded from reference linear rna viruses available on genbank; this produced a total of coding regions that were used to estimate the sensitivity and false discovery rate of each test. the codon permutation, synonymous mutation and combined test all rely on detecting overlapping orfs that are larger than expected by random chance. consequently, the sensitivity of these tests will depend on how much of a gene is overlapping (denoted as overlap length). the sensitivity and false discovery rate will also be dependent on the p value cutoffs used to determine if an orf is larger than expected by random chance, with higher p values providing higher sensitivity at a cost of greater false discovery. to understand these dependencies, receiver operator characteristic (roc) curves were generated across a range of p values, and across a range of overlapping gene lengths for all three tests ( fig. ). the number of true overlapping orf's used in this sensitivity set ranged from for overlapping orf lengths greater than nucleotides, to for overlapping orf lengths greater than nucleotides. we find that the three test (codon permutation, synonymous mutation, and combined) have similar sensitivities for p value cut-offs between . and . , with the synonymous mutation generally having the highest sensitivity, followed by the codon permutation test and then the combined test ( fig. , table ). however, for all three tests we also find that sensitivities are generally insufficient when the overlapping length is below nucleotides in length (< codons), but improve considerably as the overlapping length increases above nucleotides. importantly, the three tests show the expected distribution of orf lengths in frame þ (shaded area) calculated by permutation test, and the actual orf lengths on frame þ (black dots). the known frameshifted gene, i _gp , was clearly identified using the permutation test as its length was much larger than that expected by chance alone (p < . ). schlub et al. . doi: . /molbev/msy mbe considerable differences in false discovery rates, with the synonymous mutation tests showing the highest (worst) rates and the combined test with the lowest (best) rates. as the highest and lowest sensitivity tests (synonymous mutation and combined, respectively) are also the tests with the corresponding highest and lowest false discovery rates, we used the standard measure of a diagnostic tool-the area under the curve-to compare which test gave the best sensitivity and false discovery rate combinations. the area under the curve here will lie between and , with a value of . indicating a screening tool of no benefit, and a value of indicating a perfect screening tool with no error. accordingly, we find that the combined test consistently has the best sensitivity and false discovery rate combinations across all minimum overlap lengths, with an area under the curve increasing from . to . as the minimum overlap length increases from to nucleotides (table ). this demonstrates that the combined test is a successful screening tool with both high sensitivity and relatively low false discovery. synplot is a commonly used bioinformatic approach to identify overlapping genes by detecting reduced variability at synonymous sites (firth ) . although powerful, this method is necessarily constrained by the requirement for multiple sequences of sufficient diversity to robustly detect overlapping genes. in contrast, our method requires only a single sequence, and can therefore be applied in many situations where synplot would be inviable. quantitative comparisons of sensitivity and false discovery between the methods are difficult, as the factors associated with sensitivity in synplot (sequence diversity, recombination, and window size) are not present in our method. therefore, to make this comparison informative, we apply our method to the synplot validation data set (table from firth ) and report the results in table . this validation consists of gene overlaps with a minimum overlap length of nucleotides. we find that using a p value cut of value of . , the codon permutation method, synonymous mutation method, and combined approach detects , , and of the gene overlaps, respectively. these results are in agreement with our previous sensitivity estimates. for example, figure shows that the combined approach will have $ % sensitivity for overlaps of at least nucleotides when a p value cut-off of . is used. we next screened for previously undiscovered overlapping genes by using the combined test and a p value cut-off of . . this cut-off was chosen as only . % of any discoveries are estimated to be a false positive (table ) . we find evidence for undocumented functional overlapping orfs within all reference genomes of linear rna viruses. of these orfs, two had been previously described in synplot 's rna screening in (firth mbe not annotated within genbank, they were not necessarily undiscovered, as some existed within the ncbi protein databases. to remove these already discovered or hypothesized overlapping orfs, we performed a protein blast search of the undocumented overlapping orfs and found that nine had previously been discovered but were not annotated within the reference genome, thereby leaving newly discovered functional overlapping orfs from our method (table , supplementary materials s and s , supplementary material online). of these newly discovered orfs, we would expect approximately three to be false discoveries. to test if we can detect homologs of the newly discovered overlaps in other species, we aligned their protein sequence against the ncbi nt database using tblastn (supplementary material s , supplementary material online). we filtered the results to only include alignments with a similarity of at least % and where the alignment was at least % the length of the orf (material table . sensitivity, false discovery, and area under the curve for each test across a range of p value cut-offs and overlapping lengths. the discovered orfs ranged from to codons in length, with a median and interquartile range of . ( - . ) codons; were transcribed in the same direction (sense, frames þ and þ ) as the original gene with coded in the opposite direction on complementary nucleotides (antisense frames Àc , Àc , and Àc , supplementary material s , supplementary material online). in addition, of the orfs were located completely within their reference coding region, eight lay on the boundary and four encompassed the entire coding region, suggesting that the reference coding region may lie completely within the larger discovered orf. of these discovered orfs, a number are of particular interest and discussed in more detail below. the largest detected orf was codons long and located within nhumirim virus, a positive-sense flavivirus recently isolated from mosquitoes in brazil (pauvolid-correa et al. ) . unexpectedly, this orf is coded on a reverse complementary reading frame (Àc ), which means that unlike the other proteins in this virus, transcription must occur from a negative-sense rna template. this finding invites further investigation of the potential mechanisms by which transcription of reverse complementary reading frames might occur in positive-sense rna viruses. in addition, the th codon in this orf is a methionine (a common start codon) suggesting that a large component of the codons may be transcribed. a codon long orf was detected within the phosphoprotein p coding gene of bovine respirovirus virus (singlestranded negative-sense rna virus, family paramyxoviridae). this þ reading frame orf was particularly interesting because although its protein alignment didn't match any bovine respirovirus virus proteins, it did align with v proteins and rna editing derivatives within the phosphoprotein p gene of (galinski et al. ; wells and malur ) . these derivatives may play an important role in virus replication (durbin et al. ) , virulence (huang et al. ) , and/or the disruption of interferon expression (roth et al. ) , and the discovery is in agreement with that claims all three reading frames in the p gene of bovine respirovirus are expressed (pelet et al. ). the method also detected two new orfs of length and codons in phosphoprotein in another paramyxovirus, tioman virus. although one of these orfs was in the same sense (þ reading frame) the other was in the reverse complementary frame (Àc ). the three smallest orfs detected were in ligustrum necrotic ringspot virus, a positive-sense virus from the betaflexiviridae, okahandja mammarenavirus, a negative-sense virus from the arenaviridae, and simbu orthobunyavirus, a vector-borne negative-sense virus from the peribunyaviridae. these orfs had lengths , , and codons, and were in frames þ , þ , and Àc , respectively. interestingly, these were three of the four detected orfs that completely encompass their relatively short reference genes, suggesting that the reference gene may be entirely located within the orfs discovered by this method. we present a simple new method that uses a single genome sequence to detect candidates for overlapping genes. the method assumes that functional orfs are longer than expected by random chance as they experience selective pressure against mutations that introduce stop codons. we quantify this by using three ways to estimate the null distribution for orfs lengths within each reading frame of a gene, and use the null to identify those orfs significantly longer than predicted by random chance. this approach has a number of advantages over current bioinformatics methods to detect overlapping genes. in addition to being simple and quick, it only requires a single genome sequence. this is in contrast to other bioinformatic methods that require multiple sequences to estimate and compare nucleotide or codon diversity. this feature allows the method to be applied much more broadly in both metagenomics projects where genomes of new viruses are frequently only present in a single copy (bekal et al. ; ballinger et al. ; shi et al. ) , and also in screening scenarios such as demonstrated herein. the method is best suited to refine regions of the genome that contain candidates for functional overlapping genes, after which these regions can be further tested for functionality with more resource intensive laboratory methods such as protein isolation, ribosomal profiling (michel et al. ; ingolia ) , and studying the effects of introduced knock out mutations (chung et al. ) . a second important feature of our method is the relatively high sensitivity to detect overlapping genes, whilst maintaining acceptable false discovery rates. this is best achieved by using the combined test where newly detected orfs must be larger than expected by both the codon permutation and synonymous mutation tests. the combined test is advantageous as true positives are readily detected by both tests, so the constraint of requiring both tests to detect the orf does not impact the sensitivity. however, the combined test does substantially reduce the false positives rate, as false positives detected by one test are frequently excluded by the other. there is also scope to further reduce false discovery by modifying our method, or by imposing post analysis constraints, for example by calculating orf lengths from start codon to stop codon rather than between two stop codons. this was not considered for the screening results here due to variation in alternative start codons among viruses, but would be an important optimization in more targeted screening. one caveat to this method (and other bioinformatics approaches) is that sensitivity depends on the size of overlap, with smaller regions of overlap being more difficult to detect. unlike other methods, however, we explicitly calculated the sensitivity for many lengths of overlap and find that a length of at least nucleotides ( codons) is required before the method becomes effective. as this length increases to nucleotides ( codons), the method becomes a very powerful diagnostic tool as measured by an area under the curve equal to . . the estimate of this method's sensitivity and false discovery rates for an overlapping gene detection method is a strength, as although sensitivity can be calculated for other methods, false discovery estimation is often neglected and rarely reported due to a lack of negative controls. when it is reported, it is usually based on estimates of type error rates of p values, rather than comparison to a negative control as we have done in here. to demonstrate the utility of our method's effectiveness for overlapping gene screening, we individually analyzed all reference linear rna genomes available on genbank. this provided evidence for undocumented overlapping orfs of which we expect only to be false positives, although all should be verified experimentally. one notable orf identified here is the exceptionally long ( codons) antisense orf in nhumirim virus, a single-stranded positive-sense rna virus from the family flaviviridae, which suggests that this virus may employ a novel method of transcription and clearly merits further investigation. we also identified several undiscovered orfs in the phosphoprotein p within the paramyxoviridae family, a region known to frequently contain overlapping genes in other reading frames due to rna editing. within bovine respirovirus virus , the orf codon sequence discovered here aligned with many v proteins of other members of the paramyxoviridae. as the v protein in these viruses also overlaps with the phosphoprotein p protein, this suggests that the v protein also exists in bovine respirovirus virus . in other paramyxoviridae, notably tioman virus, we also identified antisense orfs in the phosphoprotein. the detection of antisense orfs is notable. antisense overlaps have been shown to exist in a number viruses that use dna as a replication intermediate including those in the herpesviridae (ward et al. ) , rep/orf in porcine schlub et al. . doi: . /molbev/msy mbe circovirus (he et al. ) and hbz/p and hbz/p in human t lymphotropic virus (arnold et al. ) . they are also suspected to occur in many more viruses with dna intermediaries, including a long suspected antisense protein (asp) in hiv- (torresilla et al. ; cassan et al. ). in addition, they have been infrequently suggested to occur in rna viruses that do not use dna intermediates, such as a more than amino acid (a) overlapping antisense hypothetical protein in rice black streaked dwarf virus (dsrna) (zhang et al. ), a aa overlapping antisense hypothetical protein in lymphocytic choriomeningitis mammarenavirus (-ssrna) (salvato et al. ) , and a possible aa overlapping antisense orf called "neg " in human influenza a virus (clifford et al. ; sabath et al. ) . our method can be used to investigate these further. for example, in the case of neg we find that a codon orf on in the neg reading frame (Àc ) is highly statistically unlikely by both the codon permutation and synonymous mutation methods, providing further evidence for a functional benefit of this orf. interestingly, however, a further frameshift of nucleotide (frame Àc ) would make orfs of such lengths much more likely (p ¼ . and . for codon permutation and synonymous mutation methods respectively), demonstrating the importance of the expected orf lengths on every individual reading frame, rather than just the sense direction. furthermore, when applying our method to hiv- , we find that the possible antisense orf (asp) is not substantially longer than expected by chance alone (p ¼ . and p ¼ . for codon permutation and synonymous mutation methods, respectively) in that reading frame. our results do indicate that antisense orfs are present at levels higher than currently expected. this does not necessarily mean that a transcribed protein is functional, although its presence may be indicative of some functional benefit, such as regulating expression by diverting ribosomes (pelechano and steinmetz ; beltran and garcia de herreros ) . importantly, our method's high detection of antisense orfs are in contrast to other bioinformatic screening methods which have been shown to have poor sensitivity to antisense orfs by computer simulations. this is because synonymous mutations in frame þ impact the reverse complementary frame (specifically frame Àc ) much less than other reading frames (mir and schober ) . although this feature would impact the sensitivity of our synonymous mutation test, as it would for all current methods, the codon permutation test will not impacted by this, and could be used in isolation when specifically screening for antisense orfs. overlapping genes play an important role in viral evolution (simon-loriere et al. ) , and are particularly prevalent in rna viruses with small genomes. however, the study of overlapping genes is limited by detection methods that either have high laboratory costs, or require enough sequences to make reliable substitution rate comparisons. our simple, but powerful, permutation and synonymous mutation method requires only a single genome sequence and is computationally quick to run. these properties make it an ideal choice for identifying candidate orfs in screening situations such as metagenomics viral discovery projects, or applied to large genome databases such as we have done here. whole genome sequences were downloaded for all viruses available from the ncbi ftp site ftp://ftp.ncbi.nlm.nih.gov/ genomes/viruses. of the viral genomes, were rna viruses with linear genomes, and these were selected for analysis. all coding regions (annotated with a cds in genbank) were analyzed, excluding regions annotated with a "join" indicating some form of midsequence frameshift in the established gene such as a ribosomal slippage (leaving coding regions analyzed). the following notation is used to identify the different reading frames (supplementary material s , supplementary material online): þ is the original reading frame; þ or þ is a frameshift of or nucleotides, respectively ( to transcription); À , À , or À a frameshift of , or nucleotides, respectively, after the coding sequence has been reversed (i.e. to transcription); þc , þc or þc is a frameshift of , or nucleotides, respectively, on the complement of the coding sequence ( to transcription); and Àc , Àc or -c is a frameshift of , or nucleotides, respectively, on the complement and reversed coding sequence ( to transcription). þ , þ , þ , þc , þc , þc are considered the only viable reading frames as transcription on these frames occur in the to direction. for each coding region and for each viable alternative reading frame (þ ,þ ,Àc , Àc , Àc ) we performed the following analysis (summarized in fig. a ). first, the length of orfs between the stop codons "tga," "tag," and "taa" on that specific alternative reading frame was calculated. then, , new coding sequences were created by either randomly permuting the codons in frame þ (codon permutation test; fig. b ), or for each amino acid in reading frame þ , randomly choosing a replacement codon (for which the original codon is a possible candidate) that encodes the same amino acid (synonymous mutation test; fig. c ). for each of these , new coding sequences, the length of orfs between stop codons in that alternative reading frame was calculated again. the lengths of orfs over all , randomly generated coding sequences were pooled to calculate a theoretical distribution of the length of orfs on that specific alternative reading frame. for each orf length l in the original unpermuted coding sequence, the probability of observing a length as large or larger by random chance alone is calculated using this theoretical distribution as follows: where c is the empirical cumulative distribution function of the theoretical distribution of lengths calculated by permuting codons in the original coding sequence: that is, c(l) is the simple method to detect candidate overlapping genes . doi: . /molbev/msy mbe probability of sampling an orf length less than l on that specific reading frame. the value À c(l) has an interpretation similar to that of a p value testing whether or not the length l is sampled from the theoretical distribution of lengths calculated earlier. to correct this "p value" for the total number of alternative reading frame orfs, s, in the original coding sequence the following equation is used: this adjustment is analogous to a bonferroni adjustment of p values, here correcting for the number of orfs within a reading frame. small p values for an orf are interpreted as evidence that the orf in question is larger than expected by random chance alone and therefore provides evidence that there has been negative selection against mutations that introduce stop codons in this orf. from this, we can also infer that the orf is of functional benefit to the virus. the third test, denoted as the "combined test," requires that the p value for both the codon permutation test and the synonymous mutation test be below some cut-off value. the method was only applied to orfs on alternative reading frames that exist totally or partially within the parent orf. when orfs on alternative reading frames extended beyond the parent orf boundaries, its length was truncated to the length contained with the parent orf. this analysis was performed using r (version . . ; r_core_team ) and required the packages seqinr the sensitivity (true positive rate) is measured as the proportion of known overlapping genes within the downloaded reference genomes that are detected using our method. an orf identified with our method was considered a true positive if it was located on the same reading frame and overlapped with a gene already annotated in the reference genome. as the sensitivity of our method will be dependent on the extent of overlap, we calculated the sensitivity for detecting previously annotated overlapping genes where the minimum nucleotide length of overlap is , , , , , , and nucleotides. the false discovery rate calculation is more complex as the absence of an annotated overlapping gene does not exclude its biological presence so that distinguishing between false positives and new discoveries is not possible without extensive laboratory work. to overcome this, we conservatively estimated the false discovery rate of our tests by using the nonviable - reading frames (À , À , À , þc , þc , and þc , supplementary material s , supplementary material online) as a negative control. that is, as detected orfs on these frames cannot be transcribed into proteins and are therefore false positives, they serve as an estimate to what proportion of detected orfs on viable reading frames are similarly not functional. a search for sequences homologous to the newly discovered overlapping orfs was performed by aligning their protein sequences to the ncbi nt database using tblastn (tblastn . . þ). the e-value threshold was set to . while all other settings were set to their default values. the results were stored in a sqlite database ( . . ). for homologous sequence detection the alignments were filtered to include only alignments with similarity ! % and length ! % of the corresponding orf sequence. from each filtered alignment, the ncbi accession for the query (orf) and subject were extracted and the corresponding taxid and lineage obtained using ncbi entrez. a python tool taxmax.py was developed (https://gitlab.com/janpb/taxmax.git) to compare the ncbi lineage from each orf and its aligned sequence. the similarity between two lineages is described as a score between and . a score of indicates no similarity between lineages while a score of indicates both sequences have the same ncbi lineage. for each alignment the alignments positions were compared to check for orthologous positions. supplementary data are available at molecular biology and evolution online. enhancement of infectivity and persistence in vivo by hbz, a natural antisense coded protein of htlv- discovery and evolution of bunyavirids in arctic phantom midges and ancient bunyavirid-like sequences in insect genomes overlapping genes in bacteriophage phix discovery and initial analysis of novel viral genomes in the soybean cyst nematode the evolution of genome compression and genomic novelty in rna viruses antisense non-coding rnas and regulation of gene transcription gene overlapping and size constraints in the viral world concomitant emergence of the antisense protein gene of hiv- and of the pandemic why genes overlap in viruses an overlapping essential gene in the potyviridae evidence for a novel gene associated with human influenza a viruses translational control in positive strand rna plant viruses mutations in the c, d, and v open reading frames of human parainfluenza virus type attenuate replication in rodents and primates evidence for the recent origin of a bacterial protein-coding, overlapping orphan gene by evolutionary overprinting mapping overlapping functional elements embedded 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nhumirim virus, a novel flavivirus isolated from mosquitoes from the pantanal on the informational content of overlapping genes in prokaryotic and eukaryotic viruses viral proteins originated de novo by overprinting can be identified by codon usage: application to the "gene nursery" of deltaretroviruses gene regulation by antisense transcription the p gene of bovine parainfluenza virus expresses all three reading frames from a single mrna editing site r: a language and environment for statistical computing. version . . . vienna, austria: r foundation for statistical computing overlapping genes produce proteins with unusual sequence properties and offer insight into de novo protein creation deletion of the d domain of the human parainfluenza virus type (hpiv ) pd protein results in decreased viral rna synthesis and beta interferon (ifn-beta) expression is there a twelfth protein-coding gene in the genome of influenza a? a selection-based approach to the detection of overlapping genes in closely related sequences evolution of viral proteins originated de novo by overprinting the primary structure of the lymphocytic choriomeningitis virus l gene encodes a putative rna polymerase redefining the invertebrate rna virosphere meta-transcriptomics and the evolutionary biology of rna viruses the effect of gene overlapping on the rate of rna virus evolution dna sequence at the c termini of the overlapping genes a and b in bacteriophage phi x reviving an old hiv- gene: the hiv- antisense protein mammalian overlapping genes: the comparative perspective a novel herpes simplex virus gene, ul . , maps antisense to the ul gene and encodes a protein which colocalizes in nuclear structures with capsid proteins expression of human parainfluenza virus type pd protein and intracellular localization in virus infected cells package 'ggplot ' for r. elegant graphics for data analysis molecular characterisation of segments to of rice black-streaked dwarf virus from china provides the complete genome simple method to detect candidate overlapping genes this work was supported by an arc australian laureate fellowship awarded to ech (fl ). the authors acknowledge the university of sydney hpc service at the university of sydney for providing high performance computing resources that have contributed to the research results reported within this article. we thank an anonymous reviewer for suggesting the synonymous mutation method. key: cord- -jv o authors: marcos-villar, laura; díaz-colunga, juan; sandoval, juan; zamarreño, noelia; landeras-bueno, sara; esteller, manel; falcón, ana; nieto, amelia title: epigenetic control of influenza virus: role of h k methylation in interferon-induced antiviral response date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: jv o influenza virus stablishes a network of virus-host functional interactions, which depends on chromatin dynamic and therefore on epigenetic modifications. using an unbiased search, we analyzed the epigenetic changes at dna methylation and post-translational histone modification levels induced by the infection. dna methylation was unaltered, while we found a general decrease on histone acetylation, which correlates with transcriptional inactivation and may cooperate with the impairment of cellular transcription that causes influenza virus infection. a particular increase in h k methylation was observed and the use of an inhibitor of the specific h k methylase, dot l enzyme, or its silencing, increased influenza virus replication. the antiviral response was reduced in conditions of dot l downregulation, since decreased nuclear translocation of nf-kb complex, and ifn-β, mx and isg expression was detected. the data suggested a control of antiviral signaling by methylation of h k and consequently, influenza virus replication was unaffected in ifn pathway-compromised, dot l-inhibited cells. h k methylation also controlled replication of another potent interferon-inducing virus such as vesicular stomatitis virus, but did not modify amplification of respiratory syncytial virus that poorly induces interferon signaling. epigenetic methylation of h k might have an important role in controlling interferon-induced signaling against viral pathogens. chromatin is a dynamic structure that adapts itself to alter the spatial arrangement of genetic information and thus meet the changing demands of cell functions; it has a key role in the control of gene expression. epigenetic modifications of chromatin regulate heritable and reversible gene expression without altering the dna sequence, and can also modify chromatin accessibility. dna methylation and post-translational histone acetylation and methylation are major mechanisms responsible for epigenetic regulation of gene expression . dna methylation typically represses gene transcription when takes place in the promoters of regulated genes . histone acetylation opens the condensed chromatin structure by reducing dna affinity for histones; this enables the dna to uncoil from nucleosomes so that transcription factors and rna polymerase can access the dna and increase transcription . histone methylation can increase or decrease gene transcription, depending on which amino acids in the histones are modified and how many methyl groups are added to specific residues . influenza virus is an rna virus whose genome consists of eight single-stranded rna segments with negative polarity. the virus uses a very uncommon transcription mechanism. the heterotrimeric viral polymerase synthesizes capped, polyadenylated viral mrnas through an initiation process; it uses short-capped oligonucleotides as primers, which are scavenged by a viral endonuclease from newly synthesized host rna polymerase ii transcripts , . the viral transcription strategy thus requires continuous cellular transcription for viral mrna infected cells. to identify epigenetic changes induced by influenza virus infection in the cell transcription machinery, we first analyzed dna methylation levels. human a lung epithelial cells were mock-infected or infected at high multiplicity of infection (m.o.i.; pfu/ml) with a hypervirulent a/pr / / (pr hv) strain [ ] [ ] [ ] [ ] (fig. a) . at h post infection (hpi), dna was isolated and methylation profiles evaluated in triplicate using the k infinium dna methylation beadchip. correlation analysis of the , valid probes showed an extremely strong relationship between samples (pearson correlation coefficient from r = . to r = . ; fig. b ). to identify specific differentially methylated candidates, we performed a parametric analysis to compare average beta values from infected with mock-infected cells, selecting those with differences in methylation levels > (− . >delta> . ) and a standard deviation value < % (desvest< . ). we found no significant variations, even after analysis of selected cpg sites from infection-associated genes (not shown), which indicated that global methylation of dna does not change during influenza virus infection in this system. histone fractions were purified from the same samples used for the study of dna methylation and mass spectrometry (ms) analysis were performed. we used unbiased analysis with the nanolc-ms platform to identify and quantify influenza virus-induced epigenetic changes in histone post-translational modifications (ptm). three biological replicates were analyzed and quantitative analyses completed (see methods). we considered a mascot score threshold of as high-confidence peptide identifications, and all peptides methylated/acetylated on lysine (above or below this threshold) were validated manually to assure correct tandem mass spectra matches. only ptms that were present in at least two replicas are presented (fig. c) and it has to be pointed out that all modified and unmodified peptides were identified reliably more than once. results in infected cells showed a reduction in lysine acetylation of histone and . this decrease would impair host cell expression, in accordance with the role of acetylation in opening the condensed chromatin and activating transcription . non-methylated h k and non-acetylated h k levels increased moderately during infection; since h k methylation and h k acetylation are hallmarks of transcription elongation , , the increased levels of these unmodified residues would also contribute to transcriptional inactivation of host cells. histones have a large proportion of basic residues. this property impedes detection of specific residues, probably due to the presence of another lysine(s) that trypsin proteolysis would render as peptides too small to be identified reliably by ms-based techniques. lysines not detected by ms include h k and h k , whose ptm are recognized as histone marks that control cell transcription. h k trimethylation is a common modification in active chromatin , whereas h k methylation is linked to transcriptional repression , . decreased h k me levels have been observed during influenza virus infection ; here we evaluated possible changes in methylated h k and in acetylated h k , as the latter is a conventional histone marker of active chromatin . total extracts of a mock-infected or pr hv-infected cells at high m.o.i. were collected at various times post-infection and levels of acetylated h k and dimethylated h k were tested by western blot. the results indicated that at late times after infection, influenza virus triggers a decrease in h k ac and an increase in h k me (fig. d ), which could contribute to a general decrease in host cell transcription activity. in contrast to this general effect on histone modifications, which seems to impair transcription, we detected a clear increase in methylation of lysine of histone , especially monomethylation (fig. c ). this result was unanticipated, since studies in saccharomyces cerevisiae and in humans, h k methylation levels correlated clearly with transcriptional activity . as methylation of this lysine gave the highest score in ms analysis, we evaluated this increase by two additional methods. we purified histones from cells mock-or pr hv-infected at high m.o.i.; at hpi, h k me levels were evaluated by western blot analysis (fig. e ) and a quantitative colorimetric method based on anti-h k me antibody detection (epigentek) (fig. f ). both techniques verified the h k methylation increase observed in proteomic analysis. these results indicate that epigenetic modifications induced by influenza virus infection mainly target the histone component of host cell chromatin, with h k residue methylation the most frequently modified. that methylation of infected cell dna is unaffected indicates, that in these conditions, influenza virus does not permanently injure the host cell, but the epigenetic modifications may be transitory. methyltransferases modify different lysines, only one known methyltransferase, dot l, mono-, di-and trimethylates h k ; the inhibitor epz- (epz) specifically inhibits dot l. to confirm this ability to inhibit h k methylation in our system, we incubated a cells with the inhibitor and used western blot to analyze h k me accumulation at various times after epz addition. treatment with or μm epz substantially decreased h k methylation at - h post-epz addition, without affecting h k me levels (fig. s a ). the effect of dot l inhibition on cell viability was analyzed by mtt assay (see methods and ); at these doses, epz did not notably affect cell viability, even at h after treatment (fig. s b) . to determine whether h k methylation alters influenza virus infection, we inhibited dot l and analyzed its effect on production of infectious particles. a cells were plated alone or with dot l inhibitor ( h), then infected at low m.o.i. with the pr hv strain, with another laboratory-passaged influenza strain, a/wsn/ (wsn) ( fig. a ) or with one of two natural pandemic isolates, a/california/ / (cal ) and a/ california/ / (cal ) (fig. b ) and analyzed production of infectious particles in each condition. dot l inhibition caused an increase in viral replication, higher in cells infected with the natural isolates, which suggests a general role of h k methylation in control of the influenza virus life cycle. to confirm the effect of dot l activity on influenza virus replication, we tested the effect of dot l knock-down on production of infective particles. for rnai-mediated dot l silencing experiments, we used lentiviruses expressing shrna specific for dot l (shdot l , shdot l ) or a control that expresses irrelevant shrna (shtm) , . treatment of a cells with the dot l inhibitor or expression of the dot l-shrnas reduced h k me levels compared with shtm, measured by western blot analysis (fig. a ) and quantitative colorimetric method (fig. b ). dot l silencers decreased h k methylation in infected cells in a lesser extent than epz treatment. to analyze the relevance of dot l in virus replication, we transduced a cells with lentiviruses expressing the dot l silencers or control shrna ( days), followed by infection with the pr hv ( role of h k methylation in antiviral responses. the above results suggested that modification of lysine of histone may mediate the antiviral response; its demethylation could decrease the host response against the pathogen allowing higher production of infectious particles. in agreement with this proposal we did not find significant reduction of viral titer in cells infected at high m.o.i where ifn signaling does not play a major role, in the presence of the dot l inhibitor (data not shown). these data suggested that h k methylation would affect host response in conditions close to natural infections that take place in the epithelium of the respiratory tract at low multiplicity of infection and trigger an efficient ifn induction. nf-κb is a protein complex that controls dna transcription, cytokine production and cell survival. nf-κb is involved in cell responses to stimuli such as viral antigens and has a key role in regulating the innate immune response to infection . nf-κb has been shown to be activated upon accumulation of influenza virus rna . in response to specific stimuli, nf-κb translocates from cytoplasm to the nucleus, where it binds promoters of regulated genes, activates transcription of the interferon pathway , and stimulates isg transcription. type i interferon, which includes the ifnα and β subtypes, is induced during influenza virus infection and has an essential role in host defense against the virus by activating expression of a large number of isg . to analyze the effect of dot l methylase in nf-κb activation and ifn signaling, we evaluated nf-κb subcellular distribution in cells treated with tumor necrosis factor (tnf)-α ( ng/ml, min), or infected with influenza virus ( pfu/ml, hpi), alone or with dot l inhibitor ( h). after tnf-α binding to the receptor, the inhibitory protein iκbα, which normally binds nf-κb and inhibits its translocation, is phosphorylated; this is followed by ubiquitination and degradation, releasing nf-κb, which is then translocated to the nucleus . nf-κb distribution was monitored using antibodies to the p subunit of the nf-κb complex and anti-lamin a/c antibodies to identify the nuclear envelope; influenza np detection was used to monitor infection. we analyzed > cells to quantitate nuclear and cytosolic nf-κb distribution (fig. ) . tnf-α addition produced a significant increase in nuclear import of nf-κb, and dot l inhibitor treatment significantly decreased its nuclear translocation (fig. a) . influenza virus infection similarly induced nf-κb nuclear import, and inhibition of h k methylation reduced its nuclear translocation (fig. b) . subcellular distribution of nf-κb was additionally examined in cells previously transfected with lentiviruses that express the specific dot l silencers or the control ( days). using the conditions described above, the corresponding cells were treated with tnf-α, or infected with pr hv influenza virus. in agreement with the results obtained using the dot l inhibitor, the dot l silencers decreased the nuclear translocation of nf-κb both, in tnf-α treated cells (fig. a ) or in influenza virus infected cells (fig. b) , whereas the expression of the control silencer did not change the subcellular distribution of nf-κb. together these results indicate that methylation of h k modulates the pathway involved in the nuclear translocation of nk-κb. as dot l inhibition impaired nf-κb activation, we examined its effect on steps that follow its nuclear translocation, that is, ifn and isg rna production. a cells were left untreated (control), treated with u/ml ifnαβ (ifn) or were infected with pr hv strain ( pfu/cell) (pr hv), alone or with dot l inhibitor ( h). after h or h of ifn treatment or virus infection, rna was extracted and used for qpcr detection. at h, we found a weak increase on ifnβ, ifn-stimulated gene (isg ) and interferon-induced protein mx (mx ) rna levels after ifnαβ addition or influenza virus infection, and dot l inhibitor treatment did not significantly decreased their accumulation (fig. b,c) . at h, we observed an increase in ifnβ, isg and mx rna levels and a reduction of these rnas when cells were pretreated with the h k methylase inhibitor (fig. b,c) . the significant reduction in nf-κb nuclear translocation and decreased ifnβ, isg and mx rna expression after inhibition of dot l both, support a role for h k methylation in modulating the antiviral response. given the role of h k methylation in the control of ifn signaling, we analyzed the effect of dot l inhibitor on influenza virus replication in cells with normal or deficient ifn responses. we used pr hv and cal strains at low m.o.i. to infect mdck, mdck v or mdck npro cells, untreated or treated with dot l inhibitor. mdck v cells express v protein, which targets stat and for degradation , and mdck npro cells express npro protein, which targets irf- for polyubiquitination and subsequent degradation ; ifn, but not isg, is thus induced in mdck v cells, and neither ifn nor isg are induced in mdck npro cells. we analyzed viral titers at different hpi and observed that the increased infectious particle production in mdck cells treated with the inhibitor and infected with pr hv or cal (fig. a and b) was reduced or lost in 'ifn-compromised' cells treated with dot l inhibitor. the effect of influenza virus infection on h k methylation in mdck cells with normal and deficient response to ifn was checked by colorimetric assays. influenza virus infection increased h k methylation levels in normal and interferon deficient cells, likewise in a cells (fig. s a) . since h k methylation does not affect influenza virus replication in cells with impaired ifn signaling, we analyzed the effect of dot l inhibitor in subsequent stages of viral infection. we evaluated ifnβ, isg and mx rna amounts after influenza virus infection ( pfu/cell) in ifn-competent or -deficient cells, untreated or treated with dot l inhibitor. influenza virus infection increased ifnβ, isg and mx production in mdck control cells, and inhibitor treatment reduced their accumulation (fig. c, top) . infection of mdck v cells increased ifnβ production, which was reduced by dot l inhibitor, whereas accumulation of isg was not observed, in accordance with the need for stat signaling for their induction (fig. c, center) . infection of mdck npro cells did not increase ifnβ, isg or mx rnas, independently of the presence of dot l inhibitor (fig. c, bottom) , which coincided with the inability of these cells to produce ifn and isg. in addition, total extracts of mdck, mdck v and mdck npro cells infected with pr hv at m.o.i for h were used for western blot analysis to detect irf- , stat , mx and isg to confirm the differential expression of these proteins in normal or ifn-deficient cells (fig. s b) . these results indicate that the interferon antiviral response to influenza virus infection is at least partially regulated by h k methylation via canonical type i ifn signaling mediated by the stat pathway at the level of ifn activation and response. these results indicated the ability of dot l methylase to control interferon signaling pathways. to evaluate whether h k methylase has a broader role in the control of virus multiplication, we tested the effect of dot l inhibitor and the lentiviruses expressing the dot l silencers on the replication of two distinct rna viruses, respiratory syncytial virus (rsv) and vesicular stomatitis virus (vsv). like influenza virus, rsv is a respiratory virus, and is the most common cause of viral lower respiratory tract infections in infants and children . although ifnβ appears to restrict viral replication in lung epithelial cells, it is generally agreed that rsv is relatively resistant to ifnαβ, effects, and ifn has not been detected in natural infection . vsv can infect insects, cattle, horses and pigs and is a potent ifn inducer both in cell culture and animal models . human epithelial a cells, untreated or treated with dot l inhibitor ( h) (fig. a,c) , or transduced with lentiviruses expressing the dot l silencers or control shrna ( days) (fig. b,d) , were infected at low m.o.i. with rsv ( pfu/ml) (fig. a,b) or vsv ( − pfu/ml) (fig. c,d) ; at various times post-infection, cell supernatants were obtained for viral titration (see methods). no changes were observed in rsv replication in the presence of the inhibitor or the dot l silencers, whereas infectious particle production clearly increased in vsv-infected cells treated with dot l inhibitor or when dot l was down-regulated through the expression of the specific silencers. we also evaluated the expression of viral and isg proteins (isg and mxa) during infection with rsv (fig. b, right) , and vsv (fig. d, right) . the corresponding viral proteins were detected and we observed a lack of ifn-stimulated gene induction in rsv-infected cells, while infection with vsv efficiently induced ifn response in our system. these results support a role for h k methylation in the control of ifn signaling, and a potential general dot l methylase function in regulating pathogen infection controlled by the ifn pathway. viruses are obligate intracellular parasites that completely subordinate host cell metabolism. although influenza virus does not integrate into the genome of the infected cell, the virus efficiently switches off expression of host cell genes , a result of a complex interplay of virus-induced activities closely coordinated to reduce the host response to eliminate the viral infection. consequently, during infection there is a network of viral-and host-induced modifications of cellular gene expression with a close dependence of chromatin-based functions and therefore of chromatin dynamic. accordingly, specific interactions between chromatin remodelers of the chd family and influenza virus proteins have been described such as the association of chd with the non-structural protein ns or chd and chd with the viral polymerase complex , , . in spite of this association, little effort has been made to identify epigenetic changes in chromatin induced by the infection. few alterations in dna methylation have been reported during influenza virus infection [ ] [ ] [ ] . among them changes in promoter methylation levels of some proinflammatory cytokines and interleukine genes when using the high virulence h n strain have been described. infection of human respiratory epithelial cells with pr hv strain did not show variations on dna methylation (fig. b) , however it is possible that changes in promoter methylation of specific genes and their subsequent inactivation, take place in response to particular virulent influenza strains and/or in different systems. previous studies showed that influenza virus infection modulates ptm of several isg. chromatin immunoprecipitation assays and antibodies that recognize canonical marks of transcription activation such as h k me or of inactivation such as h k me showed addition or removal of these ptm in several isg; modifications depended on strain virulence when h n virus was compared with the h n pandemic strain . this study emphasized the importance of epigenetic control in the antiviral response elicited by influenza virus infection; nonetheless, they focused on the search for specific canonical transcription activation or repression marks of histones that correlate with up-or down-transcriptional regulation of the corresponding genes and do not provide an overview of all possible changes to chromatin in response to the viral infection. for a broad overview of chromatin modifications in influenza virus-infected cells, we used an unbiased search for global changes in chromatin epigenetics at the dna and histone levels. we observed a general decrease on histone acetylation in h and h lysine residues after infection, as well as increased levels of unmodified h k , h k and dimethylated h k (fig. c and d) . in addition, previous studies showed reduction of h k me ; one of the hallmark of active chromatin . histone acetylation has a key role opening the condensed chromatin, resulting in charge neutralization and a more relaxed, open, and transcriptionally active chromatin structure . decreased acetylated histones, trimethylated h k and increased non-methylated h k and dimethylated h k , all associate with transcriptional inactivation [ ] [ ] [ ] [ ] . these epigenetic changes would impair host cell expression, in accordance with the transcription inactivation of the host cell that occurs during infection and constitutes one of the major mechanisms triggered by the virus to inactivate host transcription machinery and consequently to decrease the antiviral response. unexpectedly, changes in methylation of lysine of histone were the most prominent. lysine is located within the globular domain of histone h and is mono-, di-, and trimethylated by dot l, which modifies this lysine exclusively . h k methylation is increased in actively transcribing genes and appears to have a role in cell cycle regulation and dna damage response . dot l has been studied particularly in the modulation of mixed-lineage leukemia (mll)-related leukemogenesis, as mll fusion target gene expression depends specifically on the functional dot l gene ; indeed, inhibition of dot l activity is currently in clinical trials. the role of h k methylation in viral infection, is poorly characterized. infection of human primary fibroblasts with human cytomegalovirus (hcmv), produces a marked increase in h k me levels and downregulation of dot l expression results in a decreased viral growth . the genome of hcmv is a double-stranded dna molecule that is maintained as episome during infection. the viral dna lacks histones when encapsidated in the virion however, upon infection the viral genome is transported to the nucleus, where it becomes associated with host cell histones. it has been speculated that dot l directly mediates replication of the hcv genome, in agreement with the chromatinization of its dna genome . the function of h k methylation modulating influenza virus replication, suggest a very different mechanism, since influenza virus is an rna virus that does not integrate in the host chromatin and its genome lacks histones. in physiological conditions where antiviral signaling is induced, decreased h k methylation clearly enhanced viral replication (figs and ) . moreover, nuclear translocation of nf-κb (figs and ) and accumulation of ifnβ and isgs (isg and mx ) decreased in influenza virus-infected cells with dot l downregulated (fig. ) . influenza virus and vesicular stomatitis virus induce a strong antiviral immune response characterized by robust production of antiviral type i interferons. during infection, double-stranded rna molecules are produced, which are recognized by the rig-i helicase . after activation, rig-i recruits various tnf receptor-associated factors, which trigger phosphorylation and activation of the iκκ complex, leading to iκbα phosphorylation and degradation to allow nfκb nuclear translocation . nf-κb and ifn regulatory factor direct expression of type i ifn, which promote transcription of a variety of genes that further limit viral replication , . we observed a marked increase on replication of influenza virus and vsv, both potent inducers of ifn, in cells with low levels of methylated h k (figs , and ). this effect is lost when cells deficient on ifn signaling are infected with influenza virus (fig. ) . together the data indicate that demethylation of h k would decrease antiviral ifn signaling and therefore, viruses may develop different strategies attempting to control epigenetic modification of h k that elicit the anti-pathogen response. methylation of lysine of histone might have a general role in controlling the host response of pathogens that are interferon inducers. epz was purchased from novagen, human tumor necrosis factor α from sigma-aldrich, and recombinant interferon αβ (pbl - universal type i ifn) was provided by s. guerra. lentiviral particle production and cell transduction. lentiviral particles were produced in hek t cells by cotransfection of plasmids pspax and pmd .g with each of the plko-based shrna vectors, as described , . supernatants were collected to h post-transfection, filtered through a . μm filter, and used to transduce the corresponding cells. as the lentiviral vectors confer puromycin resistance, the minimum amount of supernatant necessary to confer % resistance to puromycin ( μg/ml) was used. silencing was tested by western blotting or colorimetric assays, normally at days post-transduction. western blot was performed as described . to detect influenza virus proteins, the following antibodies were used: for pa, monoclonal antibodies (mab) and ( : ; ; for pb , mab ( : ; , and for pb , rabbit polyclonal antibody ( : ; ; for β-tubulin, mouse anti-β-tubulin mab ( : ; sigma). to analyze histones, we used h k ac ab ( : ; abcam) and h k me ( : ; active motif). polyclonal antibodies to h k me d e , h k me c d , h # and h d h ( : ) were from cell signaling. for vsv detection, a monoclonal antibody mix against g, n and m viral proteins provided by m. esteban was used. for rsv detection, a monoclonal antibody against np protein provided by j.a. melero was used. colorimetric determination of h k me . the iquik global di-methyl histone h k quantification kit (colorimetric) from epigentek was used for elisa-like measurement of total h k me amounts, following the supplier's protocol. proteomic analysis of histone modifications. enzymatic digestion and itraq- plex labeling. the enriched histone fraction ( μg) for each condition, prepared following the epigentek protocol, was precipitated by the methanol/chloroform method, reconstituted in m urea/ m thiourea/ mm teab buffer (triethylammonium bicarbonate, ph . ), reduced with mm tris( -carboxyethyl) phosphine (tcep, sciex), and alkylated with mm methyl methanethiosulfonate (mmts, pierce); this was followed by trypsin (sigma-aldrich) proteolysis at a : enzyme:protein ratio. the tryptic peptides were labeled using the itraq- plex isobaric mass tagging kit (sciex) according to manufacturer's instructions (mock, tag- ; pr lv, tag- , pr hv, tag- ). samples were pooled, dried and desalted on a sep-pak c cartridge (waters). peptide fractions were subjected to lc-ms/ms analysis using a nano liquid chromatography system (eksigent technologies nanolc ultra d plus) coupled to a high speed triple tof mass spectrometer (sciex) with a nanoelectrospray ion source. samples were injected on a c pepmap trap column ( μm, μm i.d. x cm; thermo scientific) at μl/min, in . % formic acid in water, and the trap column was switched on-line to a c nanoacquity beh analytical column ( . μm, Å, μm i.d. x cm, waters), equilibrated in mobile phase a ( . % formic acid in water), and peptide was eluted in a min linear gradient from - % b ( . % formic acid in acetonitrile) at nl/min. the mass spectrometer was operated in data-dependent acquisition mode. for tof scans, accumulation time was set to ms, and up to precursor ions were monitored per cycle. data analysis. ms and ms/ms data obtained were converted to mgf files, which were also searched against a homo sapiens protein database containing protein-coding genes (including reversed entries to calculate false discovery rate; fdr) using the mascot server v. . (matrix science). search parameters were set as follows: enzyme, trypsin; allowed missed cleavages, ; fixed modifications, beta-methylthiolation of cysteine and itraq- plex (n-term and k); variable modifications, oxidation of methionine, acetylation (k and n-term) and methylation and dimethylation (k). peptide mass tolerance was set to ± ppm for precursors and . da for fragment masses. the confidence interval for protein identification was set to ≥ % (p < . ) and only peptides with an individual ion score above the % fdr threshold were considered correctly identified. dna methylation. isolated dna was processed according to the manufacturer's protocols for illumina infinium assay, as described . data filtering. from the initial , cpg, we excluded all probes with detection p-values > . ( cpg removed). we eliminated cpg containing single-nucleotide polymorphisms (snp) located within bp of the target cpg (snp ) and internal controls (ch and rs), resulting in cpg removed. a total of , valid probes were included in the final analysis. methylation score was represented as β-values, average for each probe was calculated and β-value differences were used for analysis. confocal immunofluorescence microscopy. cells were fixed in % paraformaldehyde ( min, room temperature) and stored in pbs. for immunofluorescence, cells were permeabilized ( min) in pbs containing % triton x- and incubated with primary antibodies diluted in pbs/ % bsa (w/v) as follows: rat anti-np ( : ;, anti-p ab ( : ; abcam) and anti-lamin a/c ( )( : ; sc- , santa cruz) to label nuclear envelope. confocal microscopy was performed with a leica tcs sp laser scanning system. images of × pixels and an eight-bit grayscale depth were acquired sequentially every . - . μm using las af version . . software (leica) and analyzed using las af and metamorph premier version . . image analysis software (molecular devices). p nuclear translocation was quantified by counting at least cells/condition, and the ratio of relative p intensity in nucleus and cytoplasm of each cell was calculated. qrt-pcr analysis. for rna extraction, cell pellets were resuspended in ml trizol reagent (invitrogen) and rna was purified as recommended by the manufacturer. rna was digested with rnase-free dnase ( u/ mg; h, °c), extracted with phenol-chloroform-isoamyl alcohol and ethanol-precipitated. for reverse transcription, we used the high-capacity cdna rt kit (applied biosystems). pcr were performed in -well pcr plates using sybr green pcr master mix (applied biosystems). pcr were carried out in a prism sequence detection system (applied biosystems). the cycle threshold (ct) was determined with analytical software (sds; applied biosystems). serial dilutions of cdna were used to ensure amplification. chromatin remodeling, histone modifications, and dna methylation-how does it all fit together? cancer epigenomics: dna methylomes and histone-modification maps histone acetylation and chromatin remodeling histone modifications for human epigenome analysis the cap-snatching endonuclease of influenza virus polymerase resides in the pa subunit influenza virus transcription and replication influenza mrna translation revisited: is the eif e cap-binding factor required for viral mrna translation? pathogenic influenza viruses and coronaviruses utilize similar and contrasting approaches to control interferon-stimulated gene responses changes in methylation of genomic dna from chicken immune organs in response to h n influenza virus infection infection with influenza a viruses causes changes in promoter dna methylation of inflammatory genes interleukin- expression was regulated by epigenetic mechanisms in response to influenza virus infection or dsrna treatment influenza a virus-induced caspase- cleaves the histone deacetylase in infected epithelial cells replication fitness determines high virulence of influenza a virus in mice carrying functional mx resistance gene attenuated strains of influenza a viruses do not induce degradation of rna polymerase ii specific residues of pb and pa influenza virus polymerase subunits confer the ability for rna polymerase ii degradation and virus pathogenicity in mice influenza virus and chromatin: role of the chd chromatin remodeler in the virus life cycle histone acetylation: a switch between repressive and permissive chromatin chromatin modifications by methylation and ubiquitination: implications in the regulation of gene expression hat , a golgi apparatus-anchored b-type histone acetyltransferase, acetylates free histone h and facilitates chromatin assembly profile of histone lysine methylation across transcribed mammalian chromatin pr-set -dependent methylation of histone h lys functions in repression of gene expression and is essential for mitosis histone acetylation regulates chromatin accessibility: role of h k in inter-nucleosome interaction the many faces of histone h k methylation the diverse functions of dot and h k methylation a rapid colorimetric assay of fungal viability with the tetrazolium salt mtt human staufen protein interacts with influenza virus ribonucleoproteins and is required for efficient virus multiplication the role of nuclear factor kappab in the interferon response influenza virus-induced nf-kappab-dependent gene expression is mediated by overexpression of viral proteins and involves oxidative radicals and activation of ikappab kinase the nf-kappab regulatory network signaling to nf-kappab x-ray crystal structure of an ikappabbeta x nf-kappab p homodimer complex in vitro and in vivo specificity of ubiquitination and degradation of stat and stat by the v proteins of the paramyxoviruses simian virus and human parainfluenza virus type the npro product of bovine viral diarrhea virus inhibits dna binding by interferon regulatory factor and targets it for proteasomal degradation activity and regulation of alpha interferon in respiratory syncytial virus and human metapneumovirus experimental infections ability of the matrix protein of vesicular stomatitis virus to suppress beta interferon gene expression is genetically correlated with the inhibition of host rna and protein synthesis polypeptide synthesis in influenza-virus infected cells chd facilitates vrnp nuclear export by interacting with nes of influenza a virus ns pa subunit from influenza virus polymerase complex interacts with a cellular protein with homology to a family of transcriptional activators chd chromatin remodeler is a negative modulator of influenza virus replication that relocates to inactive chromatin upon infection influenza virus infection causes specific degradation of the largest subunit of cellular rna polymerase ii dot l/kmt recruitment and h k methylation are ubiquitously coupled with gene transcription in mammalian cells mll-rearranged leukemia is dependent on aberrant h k methylation by dot l quantitative proteomic discovery of dynamic epigenome changes that control human cytomegalovirus (hcmv) infection temporal dynamics of cytomegalovirus chromatin assembly in productively infected human cells activation and regulation of pathogen sensor rig-i mavs recruits multiple ubiquitin e ligases to activate antiviral signaling cascades. elife , e emerging role of ubiquitination in antiviral rig-i signaling a third-generation lentivirus vector with a conditional packaging system lentivirus-delivered stable gene silencing by rnai in primary cells monoclonal antibodies against the influenza virus pb and np polypeptides interfere with the initiation step of viral mrna synthesis in vitro distinct regions of influenza virus pb polymerase subunit recognize vrna and crna templates molecular cloning. a laboratory manual genetic trans-complementation establishes a new model for influenza virus rna transcription and replication we are indebted to j. ortin, p. gastaminza and u. garaigorta for critique of the manuscript. the technical assistance of s. ciordia and r. navajas from the proteomics facility (cnb-csic, proteored isciii) is greatly appreciated. we thank c. mark for editorial assistance. lmv was supported by ciber de enfermedades respiratorias. this work was funded by the spanish ministry of economy and competitivness, plan nacional de investigacion científica, desarrollo e innovacion tecnologica (bfu - -r, (aei/feder)) and ciber de enfermedades respiratorias. js is a miguel servet researcher at isciii (ms / ). supplementary information accompanies this paper at https://doi.org/ . /s - - - .competing interests: the authors declare that they have no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - ajfb m authors: liang, zhenli; pan, hongbo; wang, xijing; zhu, yinchuan; dang, yiwu; fan, xiaohui; gao, lingxi; zhang, zengfeng title: histopathological features and viral antigen distribution in the lung of fatal patients with enterovirus infection date: - - journal: virol sin doi: . /s - - -y sha: doc_id: cord_uid: ajfb m nan enterovirus (ev ) infection causes hand-foot-andmouth disease (hfmd) in infants and children. patients with hfmd usually have good prognosis; however, in some extreme cases the infection can be accompanied by central nervous system diseases, eventually leading to cardiorespiratory failure, and even death. currently, ev has become one of the most important pathogens infecting central nervous system, after poliovirus. previous studies have shown that lung injury in patients with hfmd is associated with neurogenic pulmonary edema (npe) after ev infection of the brainstem, rather than with direct viral invasion (jiang et al. ). this might be because ev was not isolated and inflammatory cell infiltration rarely occurred in lung and lung injured tissues (kao et al. ; jiang et al. ) . ev can be transmitted through respiratory tract and cause upper respiratory tract infection, such as herpangina; nevertheless, the translocation route from the upper respiratory tract to the lung tissue or through viremia to the lung still remains unclear. viral receptors have an important role in mediating the specific binding of viruses to the target cells. the distribution of viral receptors is usually closely related to the host range and tissue tropism of the viruses, and is considered as one of the key factors for viral pathogenicity. to further explore the causes of lung injury by ev , we first examined the distribution of ev receptor in normal human lung tissues, and then investigated the target cells of ev infection, thus providing the molecular basis for the lung infected ev . consequently, the pathological changes in the lung tissues of fatal ev infection were perceived, and the distribution of ev antigen and various inflammatory cells in the lung tissues were detected. to investigate whether ev could infect the lung tissues of lower respiratory tract, the distribution of ev receptor, psgl- (human p-selectin glycoprotein ligand- ) and scarb (human scavenger receptor class b member ) (nishimura et al. ; yamayoshi et al. ) were examined in the lung tissue specimens collected form patients with tumor or non-inflammatory diseases. patients including four cases of pneumonic pseudotumor, two cases of pulmonary cyst, cases of pulmonary fibroma and two cases of pulmonary myxoma. we selected normal lung tissue in the specimens of pneumonectomy in these patients. general, the distribution of those receptors is the same as health people. the tissues were fixed by % formalin, dehydrated by gradient ethanol, soaked and embedded with wax. paraffin sections were sectioned continuously at a thickness of lm. tissue sections were dewaxed into water, and endogenous peroxidase activity was blocked. primary antibodies of psgl- ( : ) and scarb ( : ) (products from vector company, california, usa) were added, and incubated overnight in a refrigerator at °c. samples were then incubated with horseradish peroxidase labeled (mouse/rabbit) igg polymers (products from vector company, california, usa) at room temperature, developed by dab, re-dyed by hematoxylin and observed under microscope. no histopathological changes and infective lesions were found in the selected tissues. results showed that scarb was positively distributed in the intrapulmonary bronchial epithelial cells, bronchiolar epithelial cells, alveolar cells and macrophages ( fig. a- c ). psgl- was positively zhenli liang and hongbo pan have contributed equally to this work. the online version of this article (https://doi.org/ . /s - - -y) contains supplementary material, which is available to authorized users. distributed in a scattered pattern in the intrapulmonary bronchial epithelial cells, bronchiolar epithelial cells and macrophages; while no expression was found in the alveolar cells ( fig. d- f ). these results were consistent with jiao et al. ( ) . furthermore, ev receptors scarb and psgl- were expressed in the lung tissues; scarb was more widely distributed than psgl- . these data suggested that ev could infect lung tissues, with respiratory tract as one of the portals of invasion. clinically, ev infection could cause fever, and hfmd/herpangina, and it can be accompanied by central nervous system diseases such as brain stem encephalitis or lung injury. previous studies have shown that lung injury lesions are characterized by pulmonary congestion, different degrees of neurogenic pulmonary edema and pulmonary hemorrhage, with no diffuse lung injury (chang et al. ; kao et al. ; jiang et al. ) . neurogenic pulmonary edema is often caused by ev infection in the brain stem. it was speculated that ev may first destroy the specific regulatory function of the brain stem, causing dysfunction of autonomic nervous system. this subsequently resulted in the excess fluid accumulation in the lung interstitium and/or alveoli, forming interstitial and/or alveolar pulmonary edema syndrome (chang et al. ; kao et al. ; jiang et al. ) . moreover, it is clinically characterized by acute dyspnea and progressive hypoxemia, which is similar to acute respiratory distress syndrome (ards) (jiang et al. ) . the pathological changes of ards include dark red coloring of the lung tissue and hyaline membrane formation and organization. in this study, the bronchial and pulmonary tissues from autopsy cases with ev infection were obtained from the department of pathology at the first affiliated hospital of guangxi medical university. patients included in this study were between and years old, and there were males and females. ev isolation and nucleic acid detection in the cerebrospinal fluid, brain, heart and lung tissues of cases were all positive (table , supplementary figure s ). he staining was used to observe the pathological changes. immunohistochemistry was performed to detect the ev antigen and kinds of inflammatory cells. antigen retrieval was performed under high temperature and high pressure after dewaxed tissue sections into water and blocking the endogenous peroxidase activity. samples were then incubated with the following primary antibodies: ev ( : , , to label ev antigen, provided by the national infectious disease diagnostic reagent and vaccine engineering technology research center, xiamen university, china), cd (to label t cells), cd (to label b cells), cd (to label nk cells), cd (to label macrophages) (all purchased from sigma, new york, usa) at °c overnight, following incubation with horseradish peroxidase labeled (mouse/rabbit) igg polymers (sigma products, new york, usa) at room temperature was applied, developed by dab, and observed under microscope. the protein edema in the alveolar space with fibrin exudation was observed. some alveoli were filled with mononuclear cells, alveolar macrophages, exfoliated epithelial cells and cell debris in out of autopsy cases with ev infection (fig. g) . also, fibrin like exudates and hyaline membrane formation in some alveolar cavities with moderately light pink stained exudation in the alveolar septum and space, fibrinous exudation and pulmonary hyaline membrane were observed. the alveolar septum was widened, and the capillaries in the septum were highly dilated and congested (fig. h) . these results confirmed the above speculation of neurogenic pulmonary edema. the mortality of neurogenic pulmonary edema remained high, thus remaining an important b fig. detection of ev in fatal patients' lung and the histopathology changes. a-f expression and distribution of ev receptor scarb and psgl- in the normal lung tissues (immunohistochemistry). scarb was positively expressed in the bronchial epithelial cells (black arrow) (a), bronchiolar epithelial cells (black arrow) (b), alveolar cells (black arrow) and macrophages (red arrow) (c); psgl- produced a scattered pattern and was positively expressed in the bronchial epithelial cells (black arrow) (d), bronchiolar epithelial cells (black arrow) and macrophages (red arrow) (e), while no expression was found in the alveolar cells (f). g-l pathological changes of lung tissues in fatal children with ev infection (he staining). protein edema in the alveolar space with fibrin exudation was observed inside, and some alveoli were filled with mononuclear cells, alveolar macrophages, exfoliated epithelial cells and cell debris (g); the alveolar septum was widened, the capillaries in the septum were highly dilated and congested, with infiltration of inflammatory cells (h); intrapulmonary bronchitis and bronchiolitis, diffuse or focal infiltration of inflammatory cells in the wall and surrounding tissues (i, j); compensatory emphysema phenomenon of ruptured alveolar wall and fusion of pulmonary alveoli (k); the lymph nodes near the hilar bronchus indicated a reactive hyperplasia, germinal center enlargement, paracortical zone atrophy, and lymphocyte depletion (l) (black arrow). m-t distribution of ev antigen and inflammatory cells in the lung tissues of fatal children with ev infection (immunohistochemistry). cd ? t lymphocytes (m) and cd ? b lymphocytes (n) were diffusely distributed in the wall of bronchus and bronchioles and surrounding tissues; cd ? nk cells (o) and cd ? macrophages (p) were scattered in the widened alveolar septum and alveolar spaces; virus antigen was positive in the intrapulmonary bronchial epithelial cells (q), bronchiolar epithelial cells (r), alveolar cells (s) and macrophages (t) (black arrow). table the ev nucleic acid detection result of cerebrospinal fluid, brain, heart and lung tissues in cases (positive cases/total cases). cerebrospinal fluid brain heart lung nucleic acid / / / / complication and main cause of death in children with ev viral infection. interestingly, bronchitis and bronchiolitis, diffuse or focal infiltration of inflammatory cells in the wall and surrounding tissues, such as lymphocytes and mononuclear macrophages around the bronchial wall (fig. i, j) , widened alveolar septum with infiltration of inflammatory cells, and some mononuclear cells in the lumen or/and alveolar space filled, alveolar macrophages and exfoliated epithelial cells (fig. g, h ) were all found in / dead infants with ev infection. these results were consistent with the pathological changes of viral pneumonia. also, the phenomenon of compensatory emphysema of ruptured alveolar wall, fusion of pulmonary alveoli (fig. k) , and the lymph nodes near the hilar bronchus demonstrated reactive hyperplasia, germinal center enlargement, paracortical zone atrophy, and lymphocyte depletion (fig. l) . furthermore, the distribution of immune cells in the infected lung tissue was detected. t cells (fig. m) and b cells (fig. n) were diffusely distributed in the bronchus and bronchioles and surrounding tissues; nk cells ( fig. o ) were distributed in a scattered manner in the widened alveolar septum and alveolar spaces; and macrophages ( fig. p) were mainly located in the alveolar space. these data suggest that the above immune cells may have an important role in the local immune response of the lung tissue in critically ill patients. literature reported that ev infection could cause respiratory diseases, such as pharyngitis, laryngitis, bronchitis and pneumonia (gilbert et al. ; merovitz et al. ; tsai et al. ) . although ev was not isolated from the autopsy of lung specimens, ev rna remained positive (vallet et al. ). to further confirm whether ev could infect the lung tissues; immunohistochemical method was used to detect ev virus antigen. briefly, our data showed that the viral antigen was positive in the bronchial (fig. q ) and bronchiolar epithelial cells (fig. r ) and positively scattered in the alveolar cells (fig. s) and macrophages (fig. t ) in out of cases. furthermore, the distribution of viral antigen in the lung tissues was consistent with that of ev receptor, indicating that the lower respiratory tract of the lung also remained the target tissue of ev infection. therefore, the above results suggested that lung injury was caused by direct injury and/or immunopathologic injury of respiratory tract epithelium after viral infection. lung tissues of ev infected patients with severe hfmd showed interstitial pneumonia and positive viral antigens in the pulmonary epithelial cells and macrophages. this in turn suggested that, lung injury, in addition to the neurogenic cause, might also be caused by ev infection that in turn invades the upper respiratory tract, spreads to the lower respiratory tract, and then infects the lung tissues. therefore, severe ev infection is considered as a systemic virally infective disease. in addition to the involvement of the central nervous system, the respiratory system and lung tissues were also considered as the main target organs of the virus. in critically ill children, neurogenic damage and/or virus that directly infect the lung causes pulmonary edema and interstitial pneumonia, leading to respiratory and circulatory failures and even death. clinical features and risk factors of pulmonary oedema after enterovirus- -related hand, foot and mouth disease outbreak of enterovirus in victoria, australia, with a high incidence of neurogenic involvement autopsy findings in children with hand, foot, and mouth disease distribution of ev receptors scarb and psgl- in human tissues mechanism of fulminant pulmonary edema caused by enterovirus enterovirus infections at a canadian center human p-selectin glycoprotein ligand- is a functional receptor for enterovirus respiratory virus infections among pediatric inpatients and outpatients in taiwan from to fatal case of enterovirus infection scavenger receptor b is a cellular receptor for enterovirus acknowledgements this study was supported by the guangxi natural science foundation grant ( gxnsfaa ). conflict of interest the authors declare that they have no conflict of interest.animal and human rights statement the study was approved by the medical ethics committee of guangxi medical university. written informed consents were obtained from all the patients, or their guardians and families prior to the study. key: cord- -fie ns authors: white, michael; freistaedter, andrew; jones, gwendolyn j. b.; zervos, emmanuel; roper, rachel l. title: development of improved therapeutic mesothelin-based vaccines for pancreatic cancer date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: fie ns pancreatic cancer is the (th) leading cause of cancer deaths, and there are no effective treatments. we developed a poxvirus platform vaccine with improved immunogenicity and inserted the mesothelin gene to create an anti-mesothelin cancer vaccine. mesothelin expression is mostly restricted to tumors in adult mammals and thus may be a good target for cancer treatment. we show here that the modified vaccinia virus ankara (mva) virus expressing mesothelin and the enhanced mva virus missing the immunosuppressive a gene and expressing mesothelin were both safe in mice and were able to induce ifn-gamma secreting t cells in response to mesothelin expressing tumor cells. in addition, the mva virus has oncolytic properties in vitro as it can replicate in and kill panc pancreatic adenocarcinoma cell line tumor cells, even though it is unable to replicate in most mammalian cells. deletion of the a gene in mva improved t cell responses as expected. however, we were unable to demonstrate inhibition of panc tumor growth in immunocompetent mice with pre-vaccination of mice, boosts, or even intratumoral injections of the recombinant viruses. vaccine efficacy may be limited by shedding of mesothelin from tumor cells thus creating a protective screen from the immune system. pancreatic cancer is the th leading cause of cancer deaths in the united states [ ] and has the lowest -year survival rate ( %) for solid tumors [ ] largely owing to the fact that radiation, surgery, and current chemotherapy options are ineffective. new treatment strategies are essential. human mesothelin is normally expressed only in mesothelial cells of the pleura, peritoneum, and pericardium. however, mesothelin is overexpressed in a high percentage of ovarian cancers, pancreatic cancers, non-small lung cancers, and mesotheliomas, making it a potential target for anti-cancer treatments [ ] [ ] [ ] [ ] [ ] . human and mouse mesothelin share sequence similarity, expression patterns, and biochemical characteristics, [ ] , and the homeostatic function of mesothelin in mammals is unknown: the gene can be deleted without apparent effect in mice plos c bl mice and thus can be grown in syngeneic mice to allow for study of an anti-tumor immune response in an immunocompetent mouse model. this has a great advantage over tumor studies performed with xeno-or allografts in immunocompromised mice. to determine whether we can improve vaccine immunogenicity and efficacy, we test the parental normal mvameso virus vaccine against the mva mesothelin expressing virus that has had the immunosuppressive a gene removed (mvamesoa del). all animal experiments were conducted with the approval of the east carolina university animal care and use committee (k ) in an aaalac accredited animal facility. isoflurane anesthesia was used for tumor injections and for euthanasia. the c bl/ chemically-induced pancreatic adenocarcinoma cell line, panc , was a kind gift from dr. keping xie (md anderson cancer center, houston, tx). panc cells were grown in rpmi, with % fetal bovine serum, mm glutamine, u/ml of penicillin and streptomycin. mesothelin over expressing panc cell lines are described in zervos, et al [ ] . cells were incubated at ˚c with % co . vaccinia virus, mva, and the mva a deletion mutant viruses were previously described [ , ] . baby hamster kidney (bhk) cells were employed for growth of mva and were cultured using dulbecco's modified eagle medium (dmem), supplemented with % fetal bovine serum, mm glutamine, u/ml of penicillin and streptomycin. in order to create a therapeutic anti-mesothelin vaccine, we inserted mouse mesothelin into the mva viral genome. we used the normal wild type mva or the mva in which the a virulence gene had been deleted [ ] to create the mvameso virus and the mvamesoa del recombinant viruses respectively. in both cases, mesothelin was cloned into a vector (plw , a gift from l. wyatt, national institutes of health) designed for insertion into an area of the mva genome where genes had been naturally deleted during passage of the virus [ ] . recombinant viruses expressing the gfp marker protein were selected, purified, and analyzed to confirm the genotype by raid prep pcr analysis [ ] . the presence of the mesothelin gene was confirmed using mesothelin-specific primers (ggctggctatggctgtaagac and agcagtgt tggatgagtccatcgt) with an annealing temperature of ˚c. in order to assess the presence of the a gene, recombinant viruses were analyzed using a specific primers (tgacta taacagatacatt and cagattatctgaaggattgat) at an annealing temperature of ˚c. to confirm the insertion sites of the mesothelin, we used primers in the mva flanking regions, (cataagtataaagtccgactattgttc and gataaagttgcatcatcacctat). the pcr amplicons were run on an . % agarose gel. peptides representing murine mesothelin coding sequence, gvygfqvseadvralgglac and cppgkepykvdedlifyqn, were synthesized and conjugated to klh carrier proteins via the terminal cysteine residues (underlined, genemed synthesis), and used for immunization of two rabbits [ ] . sera were confirmed for reactivity with the immunizing peptides by elisa. bhk cells ( x cells/well) were uninfected or infected with the mva viruses at a multiplicity of infection (moi) of plaque forming units (pfu, infectious virus particles) of virus per cell for h in % co atmosphere. cells were lysed with disruption buffer and heat inactivated at ˚c for five minutes. dna was sheered using a -gauge needle and samples were loaded onto a pre-cast - % gradient gel (thermo scientific) for sds-page and transferred overnight. membrane was blocked for minutes at room temperature with % fat free milk in tbs, and incubated with rabbit anti-mesothelin antibody ( : , ) for h at room temperature, washed, and then incubated in anti-rabbit igg (fc) ap conjugate (promega), : at room temperature for one h, washed and developed with western blue stabilized substrate (promega). hek transected cells, previously described [ ] , were used as control. for analysis of released mesothelin, supernatants were collected from wells containing the normal or mesothelin over-expressing panc cells [ ] , uninfected bhk cells or cells infected with mva viruses. the cell supernatants were run on an sds-page gel, transferred to a pvdf membrane and probed with a rabbit anti-mesothelin antibody and an alkaline phosphatase conjugated ˚antibody as described above. female c bl/ mice (charles river, - weeks of age) were used for experiments. all mice were housed in the east carolina university department of comparative medicine aaalac accredited animal facility and kept in conventional conditions with full access to food and water throughout the study. all procedures were approved by the institutional animal care and use committee and in accordance with recommendations for proper care and use of laboratory animals. in order to assess the safety of injecting mva virus expressing mesothelin, we injected mice i.m. (n = ) with pbs, normal wild type mva, mvameso, or mvamesoa del ( . x pfu/mouse i.m. in ul, and boosted month later with . x pfu/mouse) and monitored mouse health daily by body score condition and weight up to days, similar to previous work [ ] . numbers of gamma interferon (ifn-gamma)-secreting spleen cells were enumerated similarly to methods described previously [ , ] . ninety-six-well plates (immulon h b; thermo electron) were coated overnight with purified rat anti-mouse ifn-gamma ( mg/ml; pharmingen) at ˚c. plates were washed with blocking buffer before adding a titration of murine splenocytes in rpmi medium. splenocytes were harvested from pbs, mva, mvameso, or mvame-soa del infected mice month after vaccination. the in vitro stimulation of splenocytes was achieved either by the addition of panc cells, wild type vaccinia virus (moi = ), or lewis lung cells, which do not express mouse mesothelin [ ] , followed by incubation for h at ˚c. plates were then washed (pbs with . % tween , . % sodium azide) and incubated with biotinylated rat anti-mouse ifn-gamma ( . mg/ml; pharmingen) for h at ˚c. plates were washed and incubated with streptavidin-ap for h at ˚c. plates were developed with an agarose-bcip ( -bromo- -chloro- -indolylphosphate)-amp mixture, and spots were counted by using a dissection microscope. to assess viral replication over time [ , ] , , cells were plated in a -well plate. bhk cells were used as a positive control since mva is known to replicate in them. bhk or panc cells were infected with mva at an moi of pfu/cell for h, then inocula were removed and the cells were washed once. supernatants and cells were harvested separately at six time points: , , , , , and h post-infection. these samples were frozen and thawed three times before being titered in bhk cells in -well plates [ ] . panc cells ( , cells/well) were plated in triplicate in a -well plate. mva was added to the wells at a multiplicity of infection (moi) of . and . pfu/cell. after h, ul -( , -dimethylthiazol- -yl)- -( carboxymethoxyphenyl)- -( -sulphophenyl)- h-tetrazolium, inner salt/phenazine methosulphate (mts/pms) ( . mg/ml mts, promega and . mg/ml pms, sigma) was added to each well, and the absorbance was read at nm [ ] . media only was used to define the background control level. we used the syngeneic heterotopic tumor model employing the murine pancreatic adenocarcinoma panc cell line [ ] . female c bl/ mice (charles river, - weeks of age, n = - per group) were injected s.c. in the right flank with panc cells ( . x ), one injection per mouse, under isoflurane ( %) inhalation anesthesia [ ] . tumor size was measured three times per week by a digital caliper, and volume was calculated as [(smallest diameter ) x largest diameter]/ . mice were humanely euthanized if they reach endpoints of body condition score, tumor size, ulceration, or impairment. for analysis of safety and immunogenicity, mice were injected with . x pfu/mouse i.m. in ul, and boosted month later with . x pfu/ mouse. for treatment of tumor bearing mice, mice were injected s.c. in the right flank with panc cells ( . x ), and then injected i.m. contralaterally with . x pfu/mouse ul times, on days , , after tumor cell injections. for pre-vaccination of mice, mice were injected i.m. with . x pfu/mouse, and then boosted with . x pfu/mouse, and challenged with panc tumor cells as above. for viral injections into tumors, tumors were allowed to grow, and viral injections ( . x pfu/mouse in ul) into tumors began when tumors were mm . experiments were repeated at least three times, and representative data are shown. a twotailed student's t test was used to compare groups. p values < . were considered significant. while tumor associated antigens, such as mesothelin, often do not induce a robust immune response, evidence suggests it is possible to generate an anti-tumor response by presenting the tumor antigen in an immunogenic context, such as by expressing the protein in a viral infection [ , ] . in order to create a putative therapeutic anti-mesothelin vaccine, we inserted the mouse mesothelin gene into the poxvirus mva genome under a viral promoter so that mesothelin would be expressed in any cells infected with the recombinant virus. we constructed both a normal wild type mva virus expressing mesothelin (mvameso) and a mesothelin-expressing mva virus in which the viral a immunosuppressive virulence gene had been deleted (mva-mesoa del) [ ] . the purified viruses were analyzed by pcr to confirm the presence of the mesothelin gene in the expected loci in the genomes and the presence or absence of the a gene. next, we wanted to confirm the expression of mesothelin protein from the recombinant viral genomes in infected cells. as shown in fig , mesothelin protein was detected as a broad band~ kda. mesothelin is glycosylated, so differences in glycosylation can be seen as different mw bands, and glycosylated forms may vary in different cell types [ ] . mesothelin protein was strongly expressed in both mva mesothelin virus-infected bhk cells, with monomers and apparent dimers (~ kda), but not in cells infected with mva parental wild type virus or uninfected bhk cells (fig ) . this indicated that the recombinant mesothelin expressing viruses were able to express significant quantities of mesothelin protein in infected cells. cells were lysed and samples were loaded onto a pre-cast - % gradient gel. rabbit anti-mesothelin antibody and anti-rabbit igg (fc) ap conjugate (promega) detected mesothelin protein (arrow). hek transected cells, previously described [ ] , were used as control. https://doi.org/ . /journal.pone. .g as a control, mesothelin was detected by western blot in hek cells transfected with a mesothelin expressing plasmid with a strong promoter [ ] , but not in hek cells transfected with the control plasmid vector. mesothelin could also be detected in the panc murine pancreatic adenocarcinoma cell line as we have shown previously by western blot and flow cytometry [ ] . while mesothelin is a tumor associated antigen that can be detected in panc cells grown in vitro or as a tumor grown in the mouse, it is not a highly abundant protein [ ] . multiple forms and sizes of cleaved mesothelin proteins have been reported to be released from cells expressing it [ , ] . we have previously shown that forms of the mesothelin protein are found in supernatants of panc cells, a carboxy terminal region released by furin cleavage and one derived from the amino terminus [ ] . we assessed what forms of mesothelin proteins are present in the supernatants of bhk cells infected with the mva viruses (wild type mva, mvameso, and mvamesoa del) and for comparison cultured panc cells overexpressing mesothelin [ ] . as seen in fig , several protein bands that react with rabbit anti mesothelin antisera were detected by western blot, with large quantities of released mesothelin apparent in the cells infected with the recombinant viruses encoding mesothelin. in order to assess the safety of injecting mva viruses expressing mesothelin, we injected mice i.m. (n = ) with pbs, wild type mva, mvameso, and mvamesoa del. mice were boosted month later. we monitored mouse health by body score condition daily and weight for days as shown in fig . there were no significant differences between the groups. we observed no adverse effects after the vaccination. this suggests that no significant anti-mesothelin autoimmune disease was induced in the mice. to determine whether the viruses expressing mesothelin protein were able to induce an immune response in mice, we first attempted to measure anti-mesothelin antibody in vaccinated mouse sera. we attempted to measure reactivity to panc cells, which express mesothelin on the surface by flow cytometry as we have done previously [ ] , however we got very low responses, possibly due to released mesothelin (fig ) binding the anti mesothelin antibodies in vivo. we then measured the frequency of anti-mesothelin ifn-gamma secreting t lymphocytes by elispot assay. mice vaccinated with pbs or mva had very few (< ) splenocytes that secreted ifn-gamma in response to panc cells (fig ) . mvameso vaccinated mice had an average of ifn-gamma secreting t cells per splenocytes, and mvamesoa del had an average of , significantly more (p< . ) than mva and pbs treated mice. in comparison, there were very few spots ( - ) in response to stimulation with lewis lung cells that do not express mouse mesothelin, and mice vaccinated with mva, mvameso and mvamesoa del viruses all had good responses to restimulation with vaccinia virus ( , , and spots respectively). these results indicate that while all mice responded to the virus infection, and the mesothelin expressing viruses induced an immune response in mice that reactivated in response to mesothelin expressing panc cells. additionally, vaccination with the mvame-soa del virus induced % more responding t lymphocytes compared to the mvameso virus, suggesting that removal of the a induced a stronger immune response, but the difference did not reach statistical significance. some oncolytic poxviruses have been shown to specifically target and replicate in tumor tissues in vivo [ ] because the tumor or microenvironment supports viral replication. we therefore wanted to assess if mva virus was able to infect and replicate in panc cells by a one-step viral growth curve [ ] . mva is unable to replicate in almost all mammalian cells: baby hamster kidney (bhk) cells are one known exception in which mva can replicate. bhk and panc cells were infected at an moi of pfu/cell so that each cell would be infected, and the supernatants and cells were harvested separately and titered for virus quantity over time. as shown in fig , over h the number of infectious mva virions (plaque forming units, pfu) increased in bhk cells more than logs. mva replication in panc cells also increased by more than logs, suggesting that the mva viruses might be effective oncolytic viruses in vivo since they can infect and replicate selectively in the panc tumor cells, while unable to replicate in normal mammalian tissues. viruses released into the supernatants also increased during mva infection of both cell lines. these results were promising, because they indicated that the virus could potentially amplify in situ and spread to cells that were not initially infected at the time of virus inoculation. since mva was able to replicate in panc cells, we wanted to assess whether mva was able to kill panc cells in vitro. panc cells were uninfected or infected with an moi of . or pfu/cell and compared to media with no cells. the infection was allowed to proceed for h, and mts reagent was added to measure cell metabolism as a measure of viability. to test the hypothesis that administration of mesothelin expressing viruses could induce an immune response and reduce mesothelin expressing tumor growth, we established tumors in mice [ ] and then treated with the mva mesothelin viruses. to test if the removal of the immunosuppressive a gene improved this therapeutic vaccine efficacy, we assessed the mvameso virus compared to the mvamesoa del virus. tumors were established by s.c. injection of tumors in one flank of the mice. at , , and days post tumor injection, mice , and survival (b) was not improved. we have previously described the construction of stable mesothelin over expressing panc cells and shown that tumors formed by these cells were significantly smaller than the tumors expressing wild type levels of mesothelin [ ] . we also tested whether the mesothelin expressing viruses might have greater efficacy against panc cells that expressed higher levels of the panc target. however, there was also no efficacy against the panc tumor cells expressing increased levels of panc protein, and the tumors continued to grow rapidly. since the panc tumors grow rapidly, we tested whether inducing an immune response to mesothelin prior to tumor induction would be protective against tumor growth. mice were injected i.m. with pbs, wild type mva, mvameso, or mvamesoa del, and boosted weeks later. two weeks after the boost, tumor cells were injected s.c. contralaterally in the flank. as shown in fig a, the prior vaccination with mvameso or mvamesoa del did not significantly decrease tumor growth or increase survival times. next, we attempted direct intratumoral injections since mva is able to replicate in and kill panc cells in vitro. we injected tumor cells s.c. and when tumors reached mm , we injected pbs, wild type mva, mvameso, or mvamesoa del directly into tumors times, days apart, however, we were unable to detect a benefit (fig b) . we also attempted to treat stable mesothelin overexpressing panc [ ] tumors by intratumoral injection, however there was no apparent effect, as tumors continued to grow and mice had to be euthanized. we have shown that murine mesothelin can be expressed from the mva virus genome and detected as a~ kda monomer protein on sds-page immunoblot, and a~ kda presumed dimer. mesothelin is released from cells and can be detected in the supernatants of cultured cells [ ] and in much higher quantities in cells infected with mva viruses that expresses mesothelin under a viral promoter. injection of the mesothelin expressing viruses into animals caused no apparent ill effect to at least months after vaccination, suggesting that mvameso vaccine is safe and does not initiate a deleterious autoimmune response. the mvameso vaccine induced a strong response to the mesothelin expressing panc tumor cell line, only slightly smaller than the amount of anti-vaccinia virus t lymphocytes induced by injection of vaccinia virus [ ] . these results indicate that the mesothelin protein was expressed in an immunogenic form by the virus in the infected mice. furthermore, mva virus (with and without mesothelin expression) was able to replicate in and kill panc tumor cells grown in vitro, suggesting that it would be an effective oncolytic virus in vivo. our previous results suggested that mesothelin might be a good target for control by an immune response [ ] , as mesothelin overexpression reliably caused a decrease in a heterotopic tumor growth in an immunocompetent syngeneic mouse model while in vitro proliferation was not inhibited [ ] . together, these results suggested that mvameso might be an effective treatment for pancc tumors, and that the mvamesoa del might be more effective given its improved immunogenic properties [ ] . however, we were unable to demonstrate any efficacy of the mesothelin mva vaccines prophylactically or therapeutically, even with multiple boosts or when injected directly into the tumors. one possibility is that the panc tumor cell line is simply too aggressive for control in an animal model where the tumor microenvironment may act to protect and promote tumor cell growth [ ] . while we had hoped that intratumoral injection would allow the virus to replicate in, spread, and kill tumor cells, we did not see a potent oncolytic effect in animals. perhaps while the mva virus can kill panc tumor cells in vitro, the anti-viral immune response in vivo limits the replication and spread of the virus. an ineffective anti-tumor immune response may also be caused by the shedding of mesothelin by the tumor in vivo (decoy mesothelin) blocking the immune effector antibodies and t cells. mesothelin is known to be detectable in supernatants of panc cells [ ] and in sera from patients with mesothelin expressing tumors [ , , ] . this shed soluble mesothelin may be taken up by other cells and bind anti-mesothelin antibodies rendering them ineffective. these problems may be worsened by administration of these mesothelin-expressing viruses because they also release soluble mesothelin at high levels. another possibility is that the shed mesothelin induces a state of anergy/tolerance in the animal, but our data show that mesothelin specific t lymphocytes are generated in these animals even though there is soluble circulating mesothelin shed from the mesothelin virus infected cells. perhaps new developments with cart cells [ ] may be able to more effectively facilitate t cell targeting and killing of cancerous cells. our data showed that both mvameso and mvamesoa del vaccines were able to generate a t lymphocyte response that was activated by the panc cells, and the mvamesoa del induced a stronger response, however it may be possible that the panc cells have developed mutations that protect them from effective cytotoxic t lymphocyte killing, even if t lymphocytes are appropriately expanded and targeted to recognize them. tumors that develop in humans and animals have already been highly selected in vivo to avoid the immune responses that control cancers. further improvements in these oncolytic therapeutic vaccines may be made by including cytokines such as il- and gmcsf, which have been reported to improve oncolytic potential of vaccinia virus and ultimately host survival [ , ] . in addition, depletion of t regulatory cells may counteract homeostatic or cancer cell driven immunosuppression and enhance the development of mesothelin-specific cd t cells capable of killing tumor cells [ , ] . although some results suggest that immune responses can be counter-productive: the anti mesothelin immune response may induce autoantibodies to the target antigen as well as other proteins by epitope spreading, and these antigen-spreading autoantibodies may cause increased damage to the animal. it was previously shown that antigen spreading was associated with a trend toward decreased survival in patients with prostate cancer treated with a recombinant viral vaccine [ ] . finally, given the difficulties with targeting an antigen like mesothelin that is shed, it may be more effective to target an antigen that is not shed. recent results targeting a different pancreatic tumor associated antigen, the oncofetal fucose-rich glycovariants of the pathological bile salt-dependent lipase, suggest it may be a superior tumor target antigen [ ] . united states cancer statistics: - incidence and mortality web-based report cancer statistics mesothelin is overexpressed in the vast majority of ductal adenocarcinomas of the pancreas: identification of a new pancreatic cancer marker by serial analysis of gene expression (sage) localization of mesothelin in epithelial ovarian cancer mesothelin is a malignant factor and therapeutic vaccine target for pancreatic cancer mesothelin expression in human lung cancer murine mesothelin: characterization, expression, and analysis of growth and tumorigenic effects in a murine model of pancreatic cancer mesothelin is not required for normal mouse development or reproduction sirna-mediated erc gene silencing suppresses tumor growth in tsc mutant renal carcinoma model mesothelin immunotherapy for cancer: ready for prime time? control of human mesothelin-expressing tumors by dna vaccines analysis of cloned fvs from a phage display library indicates that dna immunization can mimic antibody response generated by cell immunizations control of large, established tumor xenografts with genetically retargeted human t cells containing cd and cd domains identification of novel human ctl epitopes and their agonist epitopes of mesothelin mesothelin-specific cd (+) t cell responses provide evidence of in vivo cross-priming by antigen-presenting cells in vaccinated pancreatic cancer patients mesothelin-targeted immunotherapies for malignant pleural mesothelioma phase i study of ss p, a recombinant anti-mesothelin immunotoxin given as a bolus i.v. infusion to patients with mesothelin-expressing mesothelioma, ovarian, and pancreatic cancers new high affinity monoclonal antibodies recognize non-overlapping epitopes on mesothelin for monitoring and treating mesothelioma replicating poxviruses for human cancer therapy efficacy of vaccination with recombinant vaccinia and fowlpox vectors expressing ny-eso- antigen in ovarian cancer and melanoma patients raccoonpoxvirus safety in immunocompromised and pregnant mouse models the evolution of poxvirus vaccines serum antibodies to blood group a predict survival on prostvac-vf the poxvirus vectors mva and nyvac as gene delivery systems for vaccination against infectious diseases and cancer phase i trial of recombinant modified vaccinia ankara encoding epstein-barr viral tumor antigens in nasopharyngeal carcinoma patients genome sequence and comparative virulence of raccoonpox virus: the first north american poxvirus sequence vaccination with alvac and aidsvax to prevent hiv- infection in thailand identification of poxvirus cd + t cell determinants to enable rational design and characterization of smallpox vaccines the poxvirus a protein is an immunoregulator poxvirus safety analysis in the pregnant mouse model, vaccinia and raccoonpox viruses deletion of the a gene from modified vaccinia virus ankara increases immunogenicity and isotype switching clinical development of modified vaccinia virus ankara vaccines modified vaccinia virus ankara-based vaccine vectors induce apoptosis in dendritic cells draining from the skin via both the extrinsic and intrinsic caspase pathways, preventing efficient antigen presentation assessment of the protective effect of imvamune and acam vaccines against aerosolized monkeypox virus in cynomolgus macaques clonal vaccinia virus grown in cell culture as a new smallpox vaccine epithelial immunization induces polyfunctional cd + t cells and optimal mousepox protection immunogenicity of a highly attenuated mva smallpox vaccine and protection against monkeypox shared modes of protection against poxvirus infection by attenuated and conventional smallpox vaccine viruses the evolving role of immunotherapy in prostate cancer prostvac-vf: a vector-based vaccine targeting psa in prostate cancer vaccinia virus a r inhibits mhc class ii antigen presentation vaccinia virus decreases mhc class ii antigen presentation, t cell priming, and peptide association with mhc class ii antigen presentation assays to investigate uncharacterized immunoregulatory genes severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice rapid preparation of vaccinia virus dna template for analysis and cloning by pcr clin cancer res. a viral vaccine encoding prostate-specific antigen induces antigen spreading to a common set of self-proteins in prostate cancer patients characterization of the vaccinia virus a r protein and its role in virulence establishment of the enzymelinked immunosorbent assay system to detect the amino terminal secretory form of rat erc/mesothelin establishment of a novel specific elisa system for rat n-and c-erc/mesothelin. rat erc/mesothelin in the body fluids of mice bearing mesothelioma gmcsf-armed vaccinia virus induces an antitumor immune response effective depletion of regulatory t cells allows the recruitment of mesothelin-specific cd t cells to the antitumor immune response against a mesothelinexpressing mouse pancreatic adenocarcinoma an iscom vaccine combined with a tlr agonist breaks immune evasion mediated by regulatory t cells in an orthotopic model of pancreatic carcinoma a pancreatic tumorspecific biomarker characterized in humans and mice as an immunogenic onco-glycoprotein is efficient in dendritic cell vaccination the authors wish to acknowledge the expert technical help of melinda carver. key: cord- - e xrd y authors: watanabe, tokiko; iwatsuki-horimoto, kiyoko; kiso, maki; nakajima, noriko; takahashi, kenta; jose da silva lopes, tiago; ito, mutsumi; fukuyama, satoshi; hasegawa, hideki; kawaoka, yoshihiro title: experimental infection of cynomolgus macaques with highly pathogenic h n influenza virus through the aerosol route date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: e xrd y several animal models are used to study influenza viruses. intranasal inoculation of animals with a liquid inoculum is one of the main methods used to experimentally infect animals with influenza virus; however, this method does not reflect the natural infection with influenza virus by contact or aerosol route. aerosol inhalation methods have been established with several influenza viruses for mouse and ferret models, but few studies have evaluated inoculation routes in a nonhuman primates (nhp) model. here, we performed the experimental infection of nhps with a highly pathogenic h n influenza virus via the aerosol route and demonstrated that aerosol infection had no effect on clinical outcome, but caused broader infection throughout all of the lobes of the lung compared with a non-aerosolized approach. aerosol infection therefore represents an option for inoculation of nhps in future studies. influenza viruses cause annual epidemics and periodic pandemics. we have experienced three pandemics in the th century (i.e., spanish flu in - , asian influenza in , and hong kong influenza in ) and one in the st century (i.e., pandemic influenza a (h n ) in ). these influenza pandemics have a huge impact on public health and the global economy . additionally, recent sporadic human infections with h n and h n avian influenza viruses have raised the pandemic threat of these viruses [ ] [ ] [ ] [ ] . the continued circulation of h n viruses in birds provides opportunities for them to infect humans. indeed, human cases of h n infections have been reported in several countries, with a total of confirmed cases and fatalities as of oct , which is a case fatality rate of approximately % (http://www.who.int/influenza/human_animal_interface/ _ _ _tableh n .pdf?ua= ). therefore, concern over the pandemic potential of h n viruses is clearly justified. influenza virus can be transmitted from person to person through several transmission modes, including direct contact, indirect contact with a contaminated object (fomite), droplet transmission (droplets of > µm in diameter), and droplet nuclei transmission, also referred as airborne or aerosol transmission (droplet nuclei of < µm in diameter, which can remain suspended in the air for prolonged periods). experimental infection of animal models with influenza viruses is an essential component of the characterization of influenza viruses. mice, ferrets, and nonhuman primates (nhps) are commonly used to examine the pathogenicity, replicative ability, and transmissibility of influenza viruses, although each model has its advantages and limitations. nhps have been frequently used as a model to characterize the virulence of various influenza viruses, such as highly pathogenic h n , and the and pandemic viruses, because of their close genetic relationship to humans [ ] [ ] [ ] . division of virology, department of microbiology and immunology, institute of medical science, university of tokyo, tokyo, - , japan. department of pathology, national institute of infectious diseases, shinjuku-ku, tokyo, - , japan. intranasal and/or intratracheal inoculation with a liquid inoculum of influenza viruses has been the primary method used to infect nhps; however, a liquid inoculum does not reflect the natural infection. aerosol inhalation methods for inoculation of mice and ferrets have been established with several influenza viruses, including h n viruses [ ] [ ] [ ] [ ] [ ] . in the ferret model, these studies demonstrated that the inoculation of animals with highly pathogenic avian influenza h n virus via the aerosol route led to higher nasal wash virus titers, earlier onset of clinical signs, and/or a broader spectrum of disease compared with infection via intranasal inoculation despite no difference in lethality [ ] [ ] [ ] . in a murine model, belser et al. recently established aerosol infection with highly pathogenic h n influenza virus in the nhp model; however, they have not compared the outcomes in the nhps infected via the aerosol route with those in nhps infected via the conventional method of liquid virus inoculation. therefore, in this study, we compared the virus distribution, pathogenicity, and disease outcome in nhps infected via the aerosol route with these parameters in nhps infected through multiple routes (e.g., the intranasal and intratracheal routes). establishment of an h n virus infection system in nonhuman primates through an aerosol route by using a commercial nebulizer. a wide range of commercial nebulizers are available for clinical and research use. in this study, we used the ultrasonic nebulizer ne-u from omron healthcare co, ltd, japan. this was chosen because it is easy to use and could reduce the treatment time needed compared with other nebulizer devices , thereby making the procedure less stressful for the animals. the particle size is also critical for aerosol inhalation because aerosol transmission (also referred to as droplet nuclei transmission) occurs when small particles (< µm) are inhaled. a previous study demonstrated that the ne-u nebulizer generates a large fine particle fraction of aerosol mass < µm (the mean percentage of the fine particle fraction was %) . we used -to -year-old male cynomolgus macaques that weighed - kg each. four animals were inoculated with ml of a pfu/ml solution of the highly pathogenic h n avian influenza virus a/vietnam/ ut / strain (vn ) through the aerosol route by using the ultrasonic nebulizer ne-u . these animals were defined as 'the aerosol method group' . as a control, ml of the virus solution was administered through multiple routes by the conventional method, that is, by the intranasal ( . ml each to the left and right nostril), intraoral ( . ml each to the left and right tonsil), intraocular ( . ml per eye), and intratracheal ( . ml using a tracheal catheter) routes (defined as 'the conventional method group'). the virus dose of × pfu of influenza virus was chosen for inoculation because a similar virus dose has been widely used in previous studies to infect nhps to ensure the greatest likelihood of virus infection and replication in the inoculated animals , , [ ] [ ] [ ] . upon virus infection, none of the animals exhibited noticeable clinical signs (i.e., no appreciable body weight loss or fever was observed in any of the infected animals) except for a slight decrease in appetite among all of the infected animals. to examine virus shedding, nasal swab samples were collected from the infected animals on days and post-infection for virus titration. on day post-infection, vn virus was recovered from four animals in the conventional method group, whereas viruses were detected from three of four animals in the aerosol method group (table and fig. ). the mean virus titers were not significantly different between the conventional and the aerosol method groups [ . and . log (pfu/ml), respectively] (fig. ). on day post-infection, vn virus was recovered from nasal swabs of two and three animals in the conventional and aerosol method groups, respectively, and the mean virus titers were comparable between the two groups. to compare the distribution of h n virus in animals infected via the conventional method with that in animals infected via the aerosol method, animals from each group were euthanized on day post-infection and organ samples were collected for virus titration. relatively high virus titers were detected in the tonsils of all infected animals in both groups ( table ) . viruses were recovered from the nasal mucosa, trachea, and bronchus of some of the infected animals in both groups (table ). additionally, virus was recovered from the mediastinal lymph nodes of two and one of animal id virus titers (log pfu/ml) of animals infected with vn virus via the: aerosol method # - # - # - # - # - # - # - # - table . virus titers in nasal swabs from cynomolgus macaques infected with the h n virus via the conventional or aerosol method a . a cynomolgus macaques were inoculated with ml of a pfu/ml solution of a/vietnam/ut / virus (vn ) by the conventional method (through a combination of the intratracheal, intranasal, ocular, and oral routes) or via the aerosol method by using a nebulizer. b -, virus not detected (detection limit: . log pfu/ml). virus titers in respiratory swabs from infected cynomolgus macaques. cynomolgus macaques were inoculated with ml of a pfu/ml solution of the highly pathogenic h n avian influenza virus a/vietnam/ ut / strain (vn ) through the aerosol route by using the ultrasonic nebulizer ne-u (defined as "the aerosol method group"). as a control, ml of the virus solution was administered through multiple routes, which is the conventional method; by intranasal ( . ml each to the left and right nostril), intraoral ( . ml each to the left and right tonsil), intraocular ( . ml per eye), and intratracheal ( . ml using a tracheal catheter) routes (defined as "the conventional method group"). nasal swab samples were collected on days and postinfection for virus titration. red and blue horizontal bars show the mean titers for the aerosol and conventional method groups, respectively. virus titers (log pfu/g) of animals infected with vn virus via the: four animals in the conventional and aerosol method groups, respectively. also, virus was detected in the duodenum and brain of one of four animals in the conventional and aerosol method groups, respectively (table ) . interestingly, the virus distribution in the lower respiratory tract differed between the conventional and aerosol method groups. vn virus replicated efficiently in all of the lung lobes of the infected animals in the aerosol method group, whereas no virus was recovered from some of the lung lobes of some animals in the conventional method group ( table ). the mean virus titers in right upper, right middle, left upper, and left middle lobes of the lungs of the infected animals in the aerosol method group were significantly higher than those of the infected animals in the conventional method group [the mean virus titers in the aerosol method group were . , . , . , and . log (pfu/g), whereas those in the conventional method group were . , . , . , and . log (pfu/g), respectively] (fig. ) . in contrast, vn replicated well in the right-and left-lower lung lobes of the infected animals in the conventional method group [the virus mean titers were . and . log (pfu/g), respectively]. presumably, the introduction of virus as a liquid via the intratracheal and intranasal routes led to a large influx of virus solution into the right-and left-lower lobes. we assume that this also occurs in ferrets because we often observe similar macroscopic pathological changes in the lower lung lobes of infected ferrets (unpublished data). taken together, these findings demonstrate that a productive h n virus infection in nhps can be induced via the aerosol route by using the commercial ultrasonic nebulizer ne-u , and inoculation of nhps with aerosolized h n virus can result in the development of virus infection across a large area of the lower respiratory tract and the distribution of virus in all of the lung lobes of the infected animals, whereas inoculation via the conventional method can result in some lung lobes not being exposed to or infected with virus. histopathologic changes in the respiratory tract of animals infected with h n virus via the conventional and aerosol methods. next, we compared histopathological changes in the infected animals in the aerosol method group with those in the conventional method group. for this histopathologic analysis, we focused on two animals for each group in which virus was recovered from a variety of organs (i.e., # - and # - from the conventional method group, and # - and # - from the aerosol method group). nasal cavity, trachea, lung, epiglottis, heart, liver, spleen, kidney, small intestine, large intestine, and brain (i.e., olfactory bulb, cerebrum, cerebellum, and brainstem) were examined using hematoxylin-and-eosin-stained sections obtained from the selected animals. we also examined the distribution of influenza virus antigen by immunohistochemistry. we found no obvious changes and no viral antigens in most of the tissues tested, except for the tracheal and lung tissues. in the trachea, we observed mild inflammatory cell infiltration but no virus-antigen-positive cells. pulmonary edema and inflammatory cell infiltration into the alveolar spaces were observed in all of the infected figure . replication of h n virus (vn ) in the lung lobes of infected monkeys. cynomolgus macaques were inoculated with ml of a pfu/ml solution of vn through the aerosol route or conventional multisite routes (i.e., intranasal, intraoral, intraocular, and intratracheal routes) by using the aerosol or conventional method, respectively. four animals from each group were euthanized on day post-infection for virus titration. red and blue horizontal bars show the mean titers for the aerosol and conventional method groups, respectively. in the aerosol method group, the virus titers in some of the lung lobes were significantly higher than those in the conventional method group on day post-infection (p < . in the right-upper, right-middle, and left-middle lobes, and p < . in the left-upper lobe). detection limit = . log pfu/ml/tissue sample. scientific reports | ( ) : | doi: . /s - - - animals tested. the degree of inflammation, which had spread from around the bronchi, was different for each part of the lungs of each animal. the total pathologic scores appear to be higher in the animals infected by the conventional method than those by the aerosol method, although no statistical analysis was conducted because of the small number of animals used ( table ). the parts of the lung section with a pathologic score of are shown in fig. . neutrophils and mononuclear cell infiltration with fibrinous exudates were observed in the interstitial areas and alveolar spaces. edema and desquamation of alveolar epithelial cells into the alveolar spaces were also observed. these observations are identical to those reported in human patients who died as a result of h n infection [ ] [ ] [ ] [ ] . the conventional method group showed more severe inflammation in the lungs compared with the aerosol method group (table ) . all animals showed virus-antigen-positive signals in the bronchial and alveolar epithelium (table ). the use of animal models is essential for studying the biological properties of influenza viruses such as pathogenicity, replicative ability, and disease outcome. in most cases, intranasal administration with a liquid containing the virus is used to infect animals with influenza virus; however, a liquid inoculum does not reflect the natural influenza virus infection. recently the aerosol inhalation method has been tested in several mammalian models, including mice, ferrets, and nhps due to its resemblance to human exposure to influenza virus. here, we inoculated nhps with a highly pathogenic h n influenza virus via the aerosol route and showed that aerosol infection did not affect clinical outcome, but did cause more widespread infection throughout the lower respiratory tract compared with the conventional approach. the uniform infection in all of the lung lobes and the bronchial epithelium afforded by the aerosol infection system offers an advantage in some experimental settings. for example, when researchers take lung tissues for multiple purposes (e.g., virus titration, histopathology, and various omics analyses), they need to take samples from different parts of the lungs. therefore, infection via the aerosol route would minimize experimental variability. recently, wonderlich et al. demonstrated that aerosol infection of nhps with a highly pathogenic h n avian influenza virus caused fulminant pneumonia that rapidly progress to acute respiratory distress syndrome, and some of the infected animals died or were humanely sacrificed due to respiratory failure. the same phenomenon has also been observed in the ferret model [ ] [ ] [ ] ; however, findings by wonderlich et al. are not consistent with our findings here. the differences in disease outcomes between their study and ours may be caused by the different conditions used for the aerosol infection; that is, they used a head-only exposure chamber (into which the nhp's head was inserted), which allowed the animals to inhale the aerosolized h n virus with an exposure duration ranging from to min. in contrast, we used a standard inhalation mask, which is not a tight-fitting mask and allows the animals to breathe the aerosol mist through the nose and mouth spontaneously; the exposure duration was approximately min, potentially leading to virus infection with a lower dose compared to wonderlich's study. in addition, other parameters, such as origin, gender, and age of the macaques, as well as the conditions for virus stock generation (i.e., egg-grown virus vs. mdck-grown virus (our study), which could table . pathologic scores and detection of influenza virus antigen in animals infected with h n virus via the conventional or aerosol method a . a cynomolgus macaques were inoculated with ml of a pfu/ ml solution of vn virus by using the conventional method (through a combination of intratracheal, intranasal, ocular, and oral routes) or via the aerosol method by using a nebulizer. b pathologic severity scores for infected animals. to represent comprehensive histological changes, respiratory tissue samples were evaluated by scoring pathologic changes. pathologic scores were determined for each lung lobe in summary, here we established a system for aerosol infection of nhps with highly pathogenic h n influenza virus. we found that experimental infection with aerosolized h n virus led to a wider distribution of virus in the lower respiratory tract compared with liquid virus inoculation via the conventional method. however, no difference in clinical outcome was observed between the two inoculation methods. a highly pathogenic h n avian influenza virus, a/vietnam/ut / (h n ; vn ), was isolated in our laboratory from the same human specimen from which a/vietnam/ / (h n ; vn ) was isolated. there are no amino acid differences between vn and vn although there are two nucleotide differences, one in the na gene at position and the other in the m gene at position (in both cases, vn has adenine at these positions and vn has guanine). all experiments with vn virus was performed in enhanced biosafety level (bsl ) containment laboratories at the university of tokyo and kyoto university, japan, which are approved for such use by the ministry of agriculture, forestry, and fisheries, japan. cells. madin-darby canine kidney (mdck) cells were maintained in eagle's minimal essential medium (mem) containing % newborn calf serum. the genetic origin of the mdck cells was confirmed by dna fingerprinting (using random amplified polymorphic dna) conducted by takara bio japan. mdck cells were used for plaque assays to titrate viruses. experimental infection of nonhuman primates. two-to three-year-old male cynomolgus macaques, weighing . - . kg and serologically negative by neutralization assays for currently circulating human influenza viruses, were obtained from cambodia (obtained from japan laboratory animals, inc., tokyo, japan). all experiments with macaques were performed in accordance with the regulation on animal experimentation guidelines at kyoto university and were approved by the committee for experimental use of nonhuman primates in the institute for virus research, kyoto university. for the aerosol inoculation (aerosol method group), animals were anesthetized with ketamine via intramuscular injection and, by using the ultrasonic nebulizer ne-u (omron healthcare co, ltd, japan), ml of pfu/ figure . pathological findings in the lungs of infected monkeys. shown are representative pathological findings in the lungs of monkeys infected with vn by using the conventional (a,c; right-lower lobe of animal # - , whose pathologic score and score of detection of antigen-positive cells were and +, respectively) or aerosol (b,d; right-middle lobe of animal # - , whose pathologic score and score of detection of antigen-positive cells were and +, respectively) method at day post-infection with hematoxylin-eosin (he) staining (a,b) and immunohistochemistry for influenza viral antigen (np) (c,d). the animal id numbers correspond to those shown in tables , , and . scale bar is μm. scientific reports | ( ) : | doi: . /s - - - ml of virus was aerosolized and administered to each animal via an inhalation mask, which is not a tight-fitting mask and allows the individual to breathe the aerosol mist through the nose and mouth spontaneously. for virus inoculation through multiple routes, that is, the conventional method (the conventional method group), animals were anesthetized with ketamine via intramuscular injection and inoculated with a suspension containing a total of × pfu of virus through a combination of the intratracheal ( . ml), intranasal ( . ml per nostril), ocular ( . ml per eye) and oral ( . ml each to the left and right tonsil) routes. body temperature was monitored at , , , and days post-infection by use of a rectal thermometer. at the indicated timepoints post-infection, two or four macaques per group were euthanized for virologic and pathologic examinations. the virus titers in various organs and nasal swabs were determined by using plaque assays in mdck cells. pathologic examination. animal tissues were fixed in % phosphate-buffered formalin for pathologic examination. they were then processed for paraffin embedding and cut into -µm-thick serial sections. one section from each tissue sample was subjected to standard hematoxylin-and-eosin staining, while another was processed for immunohistochemistry with a mouse monoclonal antibody for type a influenza virus np antigen that reacts comparably with all of the test viruses. this antibody was produced in our laboratory and used for immunohistochemistry at : dilution. specific antigen-antibody reactions were visualized by use of , ′-diaminobenzidine tetrahydrochloride staining and a dako envision system (dako co. ltd., tokyo, japan). for statistical analyses, we used r (www.r-project.org) and lme to fit a linear mixed effects model to the virus titer data. different models were fitted for each biological question, namely, (i) the differences in virus titers between the groups over time (table ) , and (ii) infection of different organs (table ) , and infection of different lung parts (table and fig. ). in the first case, we used as fixed effects the different groups (i.e., the aerosol or conventional infection methods), and the time of the measurement. in the second case, we used as fixed effects the different groups (i.e., the aerosol or conventional infection methods), and the different organs in which the virus titers were measured. in both models, as random effects, we had intercepts for the different animals in the study. finally, we used the lsmeans package , to compare the groups for each model separately, and the p-values were adjusted using holm's method. p-values were considered significant if they were less than . . modified organisms of the university of tokyo and kyoto university, and, when required, by the competent minister of japan. all experiments were approved by the respective committees at the university of tokyo and kyoto university. all experiments with h n viruses were performed in enhanced biosafety level laboratories at the university of tokyo (tokyo, japan), and kyoto university (kyoto, japan), which are approved for such use by the ministry of agriculture, forestry and fisheries, japan. staff working in enhanced bsl- and bsl- ag wear disposable overalls and powered air-purifying respirators. the enhanced bsl- facilities at the university of tokyo and kyoto university include controlled access, effluent decontamination, negative air-pressure, double-door autoclaves, hepa-filtered supply and exhaust air, and airtight dampers on ductwork connected to the animal cage isolators and biosafety cabinets. the structure is pressure-decay tested regularly. all personnel complete biosafety and bsl- training before participating in bsl- -level experiments. human infection with a novel avian-origin influenza a (h n ) virus epidemiology of human infections with avian influenza a(h n ) virus in china a. h n influenza-continuing evolution and spread pandemic influenza as a current threat early and sustained innate immune response defines pathology and death in nonhuman primates infected by highly pathogenic influenza virus pathogenesis of influenza a (h n ) virus infection in a primate model aberrant innate immune response in lethal infection of macaques with the influenza virus ferrets develop fatal influenza after inhaling small particle aerosols of highly pathogenic avian influenza virus a/vietnam/ / (h n ) influenza virus aerosol exposure and analytical system for ferrets comparison of the levels of infectious virus in respirable aerosols exhaled by ferrets infected with influenza viruses exhibiting diverse transmissibility phenotypes comparison of traditional intranasal and aerosol inhalation inoculation of mice with influenza a viruses experimental transmission of influenza virus infection in mice. i. the period of transmissibility influenza a virus challenge models in cynomolgus macaques using the authentic inhaled aerosol and intra-nasal routes of infection widespread virus replication in alveoli drives acute respiratory distress syndrome in aerosolized h n influenza infection of macaques identification and validation of nebulized aerosol devices for sputum induction lethal influenza virus infection in macaques is associated with early dysregulation of inflammatory related genes massive mobilization of dendritic cells during influenza a virus subtype h n infection of nonhuman primates experimental infection of macaques with a wild water bird-derived highly pathogenic avian influenza virus (h n ) molecular biology, and pathogenesis of avian influenza a (h n ) infection in humans the comparative pathology of severe acute respiratory syndrome and avian influenza a subtype h n -a review apoptosis and pathogenesis of avian influenza a (h n ) virus in humans h n infection of the respiratory tract and beyond: a molecular pathology study fitting linear mixed-effects models using lme least-squares means: the r package lsmeans we thank susan watson for scientific editing. we also thank naomi fujimoto, mikiko tanaka, kazue goto, yuko sato, hiromi sakawaki, and tomoyuki miura for technical assistance. competing interests: y.k. has received grant support from chugai pharmaceuticals, daiichi sankyo pharmaceutical, toyama chemical, and ttsumura co., ltd; royalties from medimmune; and is a co-founder of flugen.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -a f l iu authors: dou, dan; revol, rebecca; Östbye, henrik; wang, hao; daniels, robert title: influenza a virus cell entry, replication, virion assembly and movement date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: a f l iu influenza viruses replicate within the nucleus of the host cell. this uncommon rna virus trait provides influenza with the advantage of access to the nuclear machinery during replication. however, it also increases the complexity of the intracellular trafficking that is required for the viral components to establish a productive infection. the segmentation of the influenza genome makes these additional trafficking requirements especially challenging, as each viral rna (vrna) gene segment must navigate the network of cellular membrane barriers during the processes of entry and assembly. to accomplish this goal, influenza a viruses (iavs) utilize a combination of viral and cellular mechanisms to coordinate the transport of their proteins and the eight vrna gene segments in and out of the cell. the aim of this review is to present the current mechanistic understanding for how iavs facilitate cell entry, replication, virion assembly, and intercellular movement, in an effort to highlight some of the unanswered questions regarding the coordination of the iav infection process. influenza viruses belong to the orthomyxoviridae family and are classified as either type a, b, c, or the recently identified type d ( , ) . influenza a viruses (iavs) and type b viruses (ibvs) contain , negative-sense, single-stranded viral rna (vrna) gene segments ( figure a ) ( , ) , which encode transcripts for essential viral proteins, as well as several strain-dependent accessory proteins ( figure b) . in comparison, influenza type c and d viruses only possess seven vrna gene segments, as the hemagglutinin-esterase fusion protein vrna replaces the hemagglutinin (ha or h) and the neuraminidase (na or n) vrnas ( , ) . iavs will be the main focus of this review since they are the primary agents responsible for influenza pandemics, and a major contributor to the annual influenza epidemics in the human population ( ) . the natural reservoir for iavs is wild aquatic birds, but they commonly infect other species, including humans, and have even been isolated from penguins in antarctica ( ) ( ) ( ) ( ) . the ability to adapt to multiple species is a major reason why iavs are more diverse than ibvs, which are essentially exclusive to humans. despite the host-range differences, many similarities do exist between these two viruses. both possess a host-derived lipid membrane, referred to as an envelope, which is decorated on the surface with the viral membrane proteins ha, na, and to a lesser extent the matrix (m ) protein ( figure c ) ( ) ( ) ( ) . the envelope is supported underneath by the matrix (m ) protein, and inside, the eight vrnas are found as individual viral ribonucleoprotein (vrnp) complexes ( figure c , bottom). each vrnp is comprised of a vrna that is wrapped around numerous red circles represent the ′ m pppg cap, black lines denote the - nucleotide, host-derived primers that are obtained by the cap-snatching mechanism of the viral polymerase. a(n) corresponds to the ′ poly-a tail produced by reiterative stuttering of the viral polymerase. the smaller mrnas (empty boxes) represent transcripts that encode nonessential accessory proteins found in many strains, whereas those that are less prevalent (pb -s , m , and ns ) are not illustrated ( ) ( ) ( ) ( ) ( ) ( ) . (c) diagram of an influenza a or b virus. the viral membrane proteins ha, na, and m are shown, along with the eight viral ribonucleoproteins (vrnps), and the matrix protein m that supports the viral envelope. to highlight the vrnp components, the illustration beneath the virus is not to scale. a single vrna gene segment is shown wrapped around multiple nucleoprotein (np) copies with the conserved promoter regions in the ′ and ′ utrs forming a helical hairpin, which is bound by a single heterotrimeric viral rna-dependent rna polymerase (pb , pb , and pa). (d) top view of an influenza virus cross-section showing the vrnp " + " configuration. vrnps are depicted with black circles as it is not known if the positioning of a particular vrnp is conserved or interchangeable. frontiers in immunology | www.frontiersin.org july | volume | article copies of the viral nucleoprotein (np) and bound by a single copy of the heterotrimeric viral polymerase, consisting of pb , pb , and pa ( ) ( ) ( ) . the polymerase binds the vrnas at a helical hairpin that results from the base pairing between the conserved semi-complimentary ′ and ′ ends ( ) ( ) ( ) . morphologically, iavs can either form spheres with a diameter of ~ nm or filaments that can reach up to µm in length [reviewed in ref. ( ) ]. however, upon passaging in eggs, or mdck cells, the filamentous form is generally lost ( , ) . several studies have attributed the morphology change to m , presumably through its function in supporting the envelope ( ) ( ) ( ) . regardless of the virion shape, ha is the most abundant viral envelope protein, followed by na, and m ( ) . recent work has shown that the viral envelope also contains host membrane proteins ( , ) . these proteins are likely recruited based on the lipid composition at the plasma membrane budding site, which can differ between cell types ( , ) . through possible interactions with each other and m , the eight vrnps typicallly form a + configuration inside the virus ( figure d ) ( , ) . the + configuration may have a mechanistic function, as it is also conserved in type c and d viruses that only possess vrnas ( ) . further supporting the mechanistic concept, it was recently shown that iavs can package cellular ribosomal rna (as a vrnp) when one of the vrnas is made unavailable ( ) , possibly explaining how type c and d viruses acquire their "eighth" vrna. the classification of iavs into subtypes is based on the genetic and antigenic properties of the surface antigens ha and na, which mediate viral entry and release, respectively ( , ) . to date, ha (h - ) and na subtypes (n - ) have been found in iavs isolated from aquatic birds ( ) . two additional subtypes for ha (h and h ) and na (n and n ) have recently been identified in bats ( , ) , but in contrast to the ha and na subtypes from the more traditional avian iavs, these do not appear to recognize sialic acid (sa) ( ) ( ) ( ) . despite the numerous possible subtype combinations, only three have consistently persisted in the human population, causing the following pandemics in the process: and (h n ), (h n ), and (h n ) ( ) . currently, only the h n and h n subtypes, as well as the two antigenically distinct ibv lineages (victoria and yamagata), are endemic in the human population ( ) , which is why many iav vaccines include two representative iav and ibv strains ( ) . a significant challenge in battling iavs is the constant evolution of the surface antigens (ha and na) in response to pressure from the host immune system, which is referred to as antigenic drift and antigenic shift. antigenic drift is most evident in circulating seasonal iavs, where substitutions by the polymerase that cause mutations in the surface antigen epitopes have continuously been selected to enable reinfection of the same host ( ) . antigenic shift is responsible for the development of the iav pandemics, and it relies on the less the terminal sialic acid residues are displayed with an α- , linkage, as well as an α- , linkage, to illustrate the "linear" and "bent" presentations. (b) illustration of iav cell entry. (i) iavs initiate cell entry by using the ha receptor-binding domain (located in the ha region) to associate with sialylated glycoconjugates on a host "receptor." binding to the "receptor" triggers endocytosis. (ii) the virus then traffics to the endosome where the lower ph facilitates a conformational change in ha, exposing the fusion peptide (located in the ha region) for insertion into the endosomal membrane. (iii) the ha pre-hairpin conformation begins to collapse, forming a six-helix bundle that promotes hemifusion of the viral envelop with the endosomal membrane. at some point, the m channel opens to release the viral ribonucleoproteins (vrnps) from m by acidifying the viral interior. (iv) ha further collapses into a trimer of hairpins to promote the formation of the fusion pore, which (v) releases the vrnps into the cytosol. (vi) the exposed nuclear localization signals (nls) on the vrnps are recognized by the adaptor protein importin-α, leading to the recruitment of importin-β that (vii) facilitates the transport through the nuclear pore complex (npc) and into the nucleus. frontiers in immunology | www.frontiersin.org july | volume | article frequent process of reassortment, which involves the exchange of vrnas between two iavs during co-infection of a cell ( , , ) . while reassortment can happen between two related iavs, antigenic shift occurs when the reassortment process yields a new iav subtype. iavs are also under constant negative selection due to the functional requirements of the viral proteins, and the constraints of the limited genome. several roles have been reported for most of the iav proteins. these include the function of ha in receptor binding, as well as membrane fusion, and viral release by the sialidase activity of na. to perform these functions, the proteins need to correctly fold, oligomerize, and as for the genome itself, they have to be properly trafficked and packaged into new virions. thus mutations that benefit one property may hinder another. the goal of this review is to highlight these functional requirements by providing a summary of the mechanisms iavs have evolved to facilitate cell entry, replication, virion assembly and movement, with particular attention to how iavs coordinate the infection process. iavs initiate the infection process by using the ha molecules on the viral envelope. upon reaching a potential host cell, the ha receptor-binding site attaches the virus to surface glycoconjugates that contain terminal sa residues (figure a ) ( , , ) . iavs then scan the cell surface for the proper sialylated "receptor" by using the sialidase function of na to remove local sas and liberate nonproductive ha associations ( ) . currently, the "receptor's" identity remains unknown, but it is generally thought that has from avian iavs have higher specificity for receptors with α- , -linked sas that have a "linear" presentation ( , ) , whereas has from human iavs prefer an α- , linkage, which results in a more "bent" presentation ( figure a ) ( , ) . while these preferences correlate with sa linkages in the respective hosts ( ) , several studies have shown that matching ha receptor binding preferences with the sa linkages in a particular host is not essential for infection, but is more critical for transmission ( ) ( ) ( ) ( ) . this implies that the iav "receptor" either displays significant cell tropism in the airways or that iavs can potentially use more than one receptor. despite the unknown identity of the receptor, it is clear that ha-mediated binding to the receptor triggers endocytosis of the virion ( figure b , step i). the endocytosis can either occur in a clathrin-dependent manner, involving dynamin and the adaptor protein epsin- ( - ), or by macropinocytosis ( , , ) . once inside the cell, the virus is trafficked to the endosome, where the low ph activates the m ion channel ( , , ) , and causes a large conformational change in ha that exposes the fusion peptide ( figure b , step ii) ( ) ( ) ( ) . opening of the m ion channel acidifies the inside of the viral particle, releasing the packaged vrnps from m ( figure b , step iii), which enables the transfer of the vrnps to the host cytoplasm following ha-mediated fusion ( , fusion of the viral-endosomal membranes by ha occurs through multiple steps [reviewed in refs. ( , ) , and requires cleavage of ha by host cell proteases into two subunits, ha and ha ( , , ) ]. the cleavage (see ha proteolytic activation at the golgi or plasma membrane) is required to enable the exposure of the fusion peptide on the n-terminus of the ha upon the ph change in the endosome ( ). once exposed, the fusion peptide inserts into the endosomal membrane, while the c-terminal transmembrane domain (tmd) anchors ha in the viral membrane, creating a pre-hairpin conformation (see figure b , step ii "box"). the ha trimers then fold back on themselves creating a hairpin that begins to position the two membranes in close proximity to each other (see figure b , step iii "box"). the hairpin bundles then further collapse into a six-helix bundle, and in doing so, the two membranes come closer together enabling the formation of the lipid stalk, and the subsequent fusion of the two membranes ( figure b , step iv). to date, not all of these stages have been observed with ha and some have been inferred based on observations of related fusogens from other viruses. in contrast to the early steps in iav entry, vrnp trafficking to the nucleus following the fusion event is highly dependent on the host cell machinery and transport pathways [reviewed in ref. ( ) ]. supported by numerous studies, the current model is that the newly released cytoplasmic vrnps use the importin-αimportin-β nuclear import pathway to gain entry to the host cell nucleoplasm ( figure b , steps vi and vii) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . to initially engage this pathway, it is thought that the vrnps use the surface exposed nuclear localization sequences from the numerous np molecules to recruit the adapter protein importin-α ( ) ( ) ( ) . upon binding to the vrnp, importin-α is recognized by the importin-β transport receptor, which directs the vrnp to the nuclear pore complex, where it is transported into the nucleoplasm. recent improvements in imaging and rna labeling techniques have made it possible to monitor the entire entry process in single cells ( , , ( ) ( ) ( ) . the cumulative results from these studies show that iavs can deliver their vrnps from the cell surface to polyadenylation "reiterative stuttering" the nucleus in approximately h, with entry and fusion occurring rather quickly (~ min), and nuclear import requiring the bulk of the time ( ) . a striking observation from these studies is the efficiency with which the eight vrnas reach the nucleus, indicating how effectively vrnps recruit the host nuclear import factors. supporting this observation, it was shown that np adaptation to the importin-α isoforms of a particular species is crucial for productive iav infections ( ) . while the bulk of the vrnp trafficking work has been carried out using various immortalized cell lines, the potential species related differences, and the essential role of vrnp trafficking in reassortment, emphasize the need for further methodology development to examine the details of iav entry in primary cells and tissue explants. inside the nucleus, the heterotrimeric viral rna-dependent rna polymerase carries out the transcription and replication of the vrnas [reviewed in refs. ( , ) ]. the replication of the influenza genome involves two steps: transcription of complimentary rna (crna), followed by transcription of new vrna copies using the crnas as templates. the crnas are produced by an unprimed process that relies on the correct complementation of free rntps (generally gtp and atp) with the ′ end of the vrna template ( figure a ) ( , ) . the nucleotide complementation locks the vrna template into the polymerase active site within the pb subunit and results in the formation of an a-g dinucleotide from which the crna is elongated ( ) . upon exiting the polymerase, the crna associates with newly synthesized np molecules and a single copy of the viral polymerase to assemble into a crnp ( ) . currently, it is thought that the newly produced viral polymerases, which are incorporated into the crnps, generate multiple vrna copies in a manner similar to crna transcription ( figure b) . however, there is one distinction related to the difference in the positioning of the longer ′ end of the positivesense crna. due to the increased length, the crna is positioned in the polymerase such that the rntp annealing and dinucleotide formation is likely to occur at the nucleotides located and bases from the crna ′ end (figure b , pathway i) ( , ( ) ( ) ( ) . the dinucleotide primer then has to dissociate and reanneal to the nucleotides at the ′ end prior to elongation ( figure b) . alternatively, the crna ′ end could reposition within the polymerase due to rntp bin ding, resulting in the generation of full-length vrna transcripts directly ( figure b , pathway ii). the transient nature of the rntp annealing and dinucleotide formation makes it technically challenging to exclude either possibility. the remaining task of assembling a vrnp is analogous to crnp formation. viral mrna transcription from the vrna templates is primed, making it significantly more efficient than crna and vrna transcription ( ) . the viral polymerase obtains the primers through a mechanism termed cap snatching ( ) , which is aided by the association with the cellular rna polymerase ii c-terminal domain (figure ) ( ) ( ) ( ) . for cap snatching, the viral polymerase uses the pb subunit to bind to ′ caps of nascent host transcripts ( ) and the pa subunit endonuclease domain to cleave - nucleotides downstream of the ′ cap ( ) ( ) ( ) . the pb cap-binding domain then rotates to position the newly acquired capped primer into the pb catalytic center where it is extended using the vrna as a template ( ) . finally, each transcript is polyadenylated through a reiterative stuttering′ process, which occurs when the polymerase encounters the short poly-u sequence at the vrna ′ end (figure "box") ( , ) . this process likely involves multiple cycles of dissociation, repositioning, and reannealing of the mrna to this template region of the vrna to achieve polyadenylation. during the course of infection, mrna synthesis occurs before crna and vrna transcription, and mrna transcription is much more abundant because the use of primers significantly increases the initiation efficiency ( ) . the initial mrnas are transcribed by the vrnp-associated polymerases and exported from the nucleus for translation by cytoplasmic ribosomes ( ) . however, the m and ns transcripts also possess donor and acceptor splice sites that match well with those in human transcripts ( ) . these sites recruit the cell spliceosome, which produces the spliced transcripts that encode for the m and ns proteins, respectively ( ) ( ) ( ) ( ) ( ) . the ns transcript has been reported to maintain a similar ratio of non-spliced and spliced transcripts throughout infection ( ) , whereas the ratio of the spliced m transcripts (encoding m ) have been shown to increase during infection ( ) . these observations imply that ns and ns are always equally expressed, while m expression is more biased toward the later stages of infection. however, it is likely that the splicing efficiency of the ns and m transcripts differs between iav strains ( , ) . iav protein synthesis is entirely dependent on the translation machinery of the host cell. following nuclear export [reviewed in ref. ( ) ], the translation of the viral mrnas is divided between cytosolic ribosomes (for pb , pb , pa, np, ns , ns , and m ) and endoplasmic reticulum (er)-associated ribosomes for the membrane proteins ha, na, and m (figure , steps i and ii). nuclear localization sequences on the newly synthesized np proteins and polymerase subunits (pb , pb , and pa) target these proteins into the nucleus by recruiting the importin-α-importin-β pathway that is utilized for vrnp nuclear import (figure , step iii). the np and pb proteins are imported individually, whereas the pb and pa proteins are imported as a heterodimer ( , ) . in the nucleus, these newly synthesized proteins assist in viral mrna transcription and vrna replication. np monomers bind to nucleotide stretches with a partial g bias in vrnas, and presumably crnas, to assemble vrnps and crnps through a process that may be regulated by the np phosphorylation (figure , steps v and vii) ( ) ( ) ( ) . the heterotrimeric polymerase assembles and binds to the newly formed crnps to transcribe vrnas ( figure , step vi) that upon formation into vrnps can generate additional viral mrna (figure , step viii) , or crna transcripts (figure , step ix) ( , ) . the viral rna-binding protein ns is synthesized early and also imported into the nucleus, where it can act as an inhibitor of interferon signaling [reviewed in ref. ( ) ]. in addition, ns may contribute to viral mrna export from the nucleus by linking the viral transcripts to the cellular nuclear export components tap/nxf , p , rae , e b-ap , and the nucleoporin nup ( ) . ns (alternatively known as the nuclear export protein) and m are imported into the nucleus as well. multiple studies have implicated these two proteins in the nuclear export of vrnps ( , , ( ) ( ) ( ) ( ) . while the mechanism remains unclear, current data support a model where m acts as an adaptor protein linking ns to vrnps (figure , step x) ( , ) . through established interactions with crm , ns is then able to target the vrnp complex to the crm nuclear export pathway for transport to the cytoplasm ( ), where m potentially prevents the re-import of vrnps by blocking access to the np nuclear localization sequences (figure , step xi) ( ) . within the cytoplasm the vrnps are trafficked toward the plasma membrane for viral assembly by rab . rab facilitates the interaction by associating with the viral polymerase pb subunit ( ) , potentially providing a quality control mechanism that ensures new virions incorporate vrnps carrying a polymerase. earlier studies proposed that vrnps specifically associate with rab on recycling endosomes, which use microtubules for transport toward the cell surface ( figure , step xiia) ( ) ( ) ( ) ). an alternative model has recently been proposed where infection causes tubulation of the er membrane network and the vrnps bind to rab molecules that have localized to this network for trafficking toward the plasma membrane ( figure , step xiib) ( ) . currently, it is not known how vrnps are transferred to the plasma membrane in either model, or how iavs incorporate all eight of the different vrnps in a " + " configuration. while several studies have indicated that specific vrnp associations likely contribute to the packaging of the eight vrnps ( , , ) , the underlying mechanisms remain to be established. the iav membrane proteins, which are ultimately destined for the viral envelope, are synthesized by ribosomes associated with the er membrane. similar to cellular secretory proteins, ribosome-nascent chain complexes containing na, ha, or m are co-translationally directed to the er by interactions of their hydrophobic targeting sequences with the signal recognition particle (srp) (figure , step ii) ( ) ( ) ( ) ( ) . the cleavable signal sequence on ha facilitates the interaction with srp, whereas na and m use their respective tmd as an er targeting sequence. once bound, srp targets the ribosome-nascent chain complexes to the srp receptor in the er membrane (figure , step iii) , which transfers the ribosome to a sec protein-conducting channel known as the translocon ( - ) . linked to the dependence on srp, mutations that alter the targeting sequence hydrophobicity of cellular secretory proteins have been shown to decrease their er targeting and subsequent synthesis ( , ) . although this aspect has not been examined for the iav membrane proteins, there is evidence that the hydrophobicity of their er-targeting sequences change ( , ) , which suggests iavs potentially use this mechanism to titrate na and ha expression. the translocon enables passage of the elongating na, ha, and m polypeptides into the er lumen and facilitates the membrane partitioning of their respective tmd segments through a lateral gate ( , ) . to activate the membrane integration, the tmd segments have to be of the appropriate length and hydrophobicity ( , ) . in human h n and h n viruses, these criteria are conserved in the tmds of ha and m , but not in the tmd of na, as it has become progressively less hydrophobic in the h n viruses ( ) . the uncharacteristic hydrophobicity loss was shown to be possible because of the na tmd being positioned at the n-terminus ( ) . the positioning (~ amino acids from the c-terminus), combined with the slow rate of ribosomal translation (~ amino acids per second), likely provides these nontypical tmds with significant time to properly orientate and facilitate membrane insertion during the co-translational translocation process. during translocation, the n-terminus of ha and m is directly translocated into the er lumen, whereas na inverts, positioning the c-terminus in the er lumen ( , ) . in addition, ha and na receive multiple n-linked glycans. the glycans are transferred by the oligosaccharyltransferase to asn-x-ser/thr sequences, and vary in number as well as positioning based on the strain, or subtype ( ) . one function of the glycans is to increase the folding efficiency of na and ha by recruiting the lectin chaperones (calnexin and calreticulin) and the associated oxidoreductase erp , which aids in disulfide bond formation ( , ( ) ( ) ( ) . this is especially crucial for the ha and na proteins that possess a significant number of intramolecular disulfide bonds (e.g., six in has, eight in n , and nine in n ) ( ) ( ) ( ) . by contrast, m possesses two intermolecular disulfide bonds in its tetrameric conformation ( ) . depending on the subtype, na tetramers also possess or more intermolecular disulfide bonds. oligomerization of ha involves the trimerization of independently folded monomers, whereas na tetramerization has been proposed to result from the pairing of two co-translationally formed dimers, which assemble through a process involving the n-terminal tmd of na ( , ) . in line with this model, it has been shown that the tmd is essential for proper na folding, and that the decreasing hydrophobicity in the n tmds functions to frontiers in immunology | www.frontiersin.org july | volume | article support the folding and oligomerization of the enzymatic head domain ( , ) . iavs easily achieve the protein concentrationdependent requirement for oligomerization due to the abundance of ha and na that is synthesized during an infection. however, these high synthesis levels at the er can also be deleterious by activating the er-stress response. indeed, several studies have shown that iav replication does activate the er-stress induced unfolded protein response ( , ) , but this response is also mitigated by the inhibition of the eif α-kinase and stress granule formation through the functions of other viral proteins ( ) . despite everything that is known about the synthesis and assembly of the iav membrane proteins, several aspects have yet to be addressed. these include obtaining atomic structures of fulllength ha and na in a membrane, something that should become easier to address with the advances in cryo-electron microscopy structure determination. identifying if the na protein removes sa residues directly from substrates within the golgi, as this could decrease the effectivity of nonmembrane permeable na inhibitors. it is also unclear how iavs regulate the timing and expression levels of the viral proteins as viral mrna transcription shows little temporal variation ( , ) . while it is likely that m is regulated in part by splicing ( , ) , this does not apply to ha and na. recent work has linked na and ha regulation to the nucleotide composition of the ′coding regions for their er-targeting sequences, which dramatically differ from the profile of corresponding regions in human secretory protein mrnas ( , ) . an obvious candidate for post-transcriptional regulation is the viral rna-binding protein ns . indeed, many studies have shown that ns can increase translation of particular mrnas, possibly by enhancing the translation initiation rate through the recruitment of eif- g to the ′region of viral mrnas ( , ( ) ( ) ( ) ( ) ( ) . however, a clear mechanistic picture for influenza protein regulation is lacking. ha traffics from the er as a fusion incompetent precursor termed ha . to gain its fusion function, ha must be cleaved into the subunits ha and ha ( , , ) . the cleavage occurs in either a monobasic, or a multibasic, cleavage site ( ) . multibasic sites are commonly found in highly pathogenic avian iavs and are cleaved by furin, a calcium-dependent serine endoprotease that is located within the trans-golgi network ( ) . furin is also ubiquitously expressed ( ) , which is one of the major reasons why avian iavs with a multibasic cleavage site are generally more pathogenic. by contrast, human (and low pathogenic avian) iavs encode for has with a monobasic cleavage site, which have been shown to be processed by different proteases in human respiratory epithelial cells. these include the transmembrane protease serine s- member (tmprss ), human airway trypsin-like protease (hat), and possibly tmprss ( , ) . hat localizes at the plasma membrane where it can either cleave newly synthesized ha or the ha found in cell-associated virions ( , ) . similar to furin, tmprss resides in the trans-golgi network, where it cleaves ha en route to the plasma membrane. the m ion channel is thought to prevent the premature activation of ha following cleavage by equilibrating the slightly acidic ph of the golgi ( , ) . distinct from furin, tmprss expression has been found to be more restricted to the upper and lower respiratory tract, whereas hat was mainly shown to be expressed in the upper respiratory tract ( ) . these cell tropisms suggest that lower respiratory infections are likely mediated by tmprss , and could be one of the primary reasons human iavs are confined to the epithelial layer of the respiratory tract. compared with the bulk lipid profile of the plasma membrane, iav envelopes are enriched in cholesterol and sphingolipids ( ) , indicating that they bud from distinct apical plasma membrane regions often referred to as "rafts" ( ) . however, infectious iavs must possess mechanisms to target the eight vrnps, m , ha, na, and m to these sites in the membrane ( , ) . ha is believed to localize to these distinct regions based on fatty acid modifications of the c-terminal cysteine that occur in the golgi ( ) ( ) ( ) ( ) , whereas na enrichment has previously been attributed to a property in the c-terminus of the tmd ( ) . in contrast, m has been shown to accumulate at the boundaries of these budding domains ( ) , and the cytosolic protein m has been proposed to localize to the budding region by associating with the short cytoplasmic tails of ha and na ( ) . however, it is equally plausible that na and ha create membrane domains with a unique lipid profile that have a high affinity for m . finally, the vrnps, delivered to the cell periphery by rab , are thought to localize to the budding site by binding to m ( , ) . in addition to orchestrating the assembly of the correct viral components at the apical budding site, iavs also have to remodel the membrane to induce bud formation, and ultimately scission of the viral envelope from the plasma membrane. to promote bud formation, the virus must first induce significant curvature in the membrane and then constrict the two opposing membranes of the viral envelope to help to facilitate membrane scission. curvature can be induced by (i) protein or "molecular" crowding on one leaflet of a bilayer, (ii) association of curved or "bending" proteins with the bilayer, (iii) biased accumulation of cone shaped lipids in one leaflet of the bilayer, or (iv) the cytoskeleton ( ) . based on cumulative data regarding budding, iavs appear to induce membrane curvature through a combination of these mechanisms. indicative of using molecular crowding and bending proteins, several studies have demonstrated that ha and na expression is sufficient to induce budding, and that the efficiency and shape uniformity benefit from the presence of m ( ) ( ) ( ) ( ) . these results indicate that the abundance of ha and na on one side of the membrane can contribute to curvature. it also is intriguing to speculate that the asymmetric ( ) shape of na plays a role in this process as it is often seen clustering in the viral membrane ( , ) . by contrast, m appears to be analogous to a membrane-bending protein as it recruited to the cytosolic side of the membrane budding site, oligomerizes upon reaching the membrane, and these oligomers have been modeled to form curved structures ( ) ( ) ( ) . based on these properties, it is plausible that m significantly influences the membrane curvature at the budding site, potentially explaining its role in discerning whether iavs form spheres or filaments ( , ) . the ion channel m localizes to the budding site boundary and has also been shown to contribute to iav scission by functioning as a membrane-bending protein ( , ) . the membrane-bending property of m is localized in an amphiphilic α-helix that can incorporate the amino acid side chains from its hydrophobic face into a leaflet of the bilayer. with this domain positioned in the cytosol, the intercalation results in negative membrane curvature, which has been proposed to facilitate viral bud neck formation and scission, presumably by decreasing the distance between the two opposing membranes of the viral envelope ( ) . while much of the framework concerning iav budding has been established, it has been difficult to identify the details of the budding process, in part due to the mobility and heterogeneity of the plasma membrane. the lack of strong phenotypes from domains proposed to contribute to budding could also imply that iavs have built redundancy into the budding process ( ) ( ) ( ) . the possibility of redundancy is certainly plausible, as iavs contain the necessary components to allow for a combination of lipid recruitment, molecular crowding, and a membrane-bending protein. iav cell release and movement once the newly assembled iavs bud, their release is highly dependent on the sialidase activity of na. na is a homotetramer, and each subunit is comprised of a short n-terminal cytoplasmic tail (six amino acids), followed by a tmd, a length variable stalk, and a globular enzymatic head domain ( ) . the globular head domain forms a -bladed propeller structure, where each blade is comprised of four antiparallel β-sheets that are stabilized by disulfide bonds ( , , ) . the catalytic tyr residue is found in a highly conserved active site that forms a deep pocket in the center of each monomer ( ) . all of the residues necessary for catalysis exist within each monomer ( ) , which has made it difficult to reconcile why na evolved to function as a tetramer ( , , ) . structures of the enzymatic head domain indicate that na tetramers bind up to five calcium ions and calcium has been shown to contribute to na activity ( , , ) . however, it remains unclear why influenza na has evolved to position a calcium ion at the tetrameric interface. na facilitates viral release by catalyzing the hydrolysis of the glycosidic linkage that attaches sa to underlying sugar molecules ( ) ( ) ( ) . by removing local sa residues, na prevents ha binding at the cell surface, which facilitates the release of the virus during budding (figure a and step vi) ( , ). na has also been shown to promote the separation of iavs by removing sa residues from the n-linked glycans located on the ha and na molecules in the viral envelope ( figure b and step vii) ( ) . in contrast to ha, nas from human iavs show a general preference for α , -linked sa with variable abilities to cleave α , -linked sa residues ( , , ) . however, a thorough analysis of na sa preference is lacking. more recent studies have found that some strains possess nas that are inefficient enzymes, but still capable of sa binding, raising the question of whether a poor na enzyme could contribute to, or replace, the ha receptor-binding function ( , ) . the movement of iavs from cell to cell in the respiratory epithelium is significantly different from that in immortalized cell lines grown in liquid culture due to the presence of different cell types and a mucus layer. the mucus layer provides a protective barrier for the epithelium and is rich in heavily glycosylated mucins that can interact with iavs and limit cell binding ( , ) . studies measuring viral movement through mucus and respiratory epithelial cells have shown that na-mediated cleavage of sas from mucins enhances iav movement through the mucus layer and infectivity ( figure c and step viii) ( , , ) . recent work showed that this function may also apply to transmission, as iavs that possess low na activity, and are inhibited by mucus, are deficient in aerosol and contact transmission ( ) . iavs are constantly exposed to negative and positive selection pressure, which shapes how the virus evolves. the functional requirements of each iav protein, such as enzyme catalysis, substrate binding, oligomerization, and domains that perform essential interactions with host proteins all combine to create substantial negative selection pressure that often manifests in the form of sequence conservation. negative pressure can also come from functions within the vrna sequences. these include promoters and "packaging signals, " but are also likely to involve aspects such as the formation of structural elements, or possibly mediating vrnp interactions that generate the + assembly in viral particles. in addition, the exposure of iavs to the immune response and constantly changing environments such as host, temperature, ph, cell type, and antivirals result in positive selection pressure. experimentally, addressing each type of selection has its caveats, but clearly a holistic picture of both iav and host functions are required to begin predictions of evolutionary constraints on the virus. most studies on the influenza evolutionary process focus primarily on antigenic drift and antigenic shift. however, all the viral transcribed rnas are subject to replication errors by the viral polymerase, which are estimated at per , - , nucleotides ( ) ( ) ( ) . consequently, both the viruses and the viral proteins are likely to exist as large heterogeneous populations during an infection. as many iav proteins are homo-oligomers this can potentially generate heterogeneity within individual protein complexes that could have functional advantages. by applying single particle and single cell analysis, these types of aspects are beginning to be investigated ( ) . another interesting approach is deep mutational scanning, which has been used to examine the site-specific amino acid tolerance of iav proteins in general, and in the context of different selection pressure ( ) ( ) ( ) ( ) . currently, the best characterized protein in iavs is ha, which has two primary functions, (i) to initiate binding to the host cell and (ii) to deliver the vrnps to the host cell cytosol by fusing the viral and endosomal membranes. these functions are efficiently divided between the two domains of ha (ha and ha ), created by proteolysis. the receptor-binding site responsible for entry is located in the considerably larger ha subunit that is known to be immunodominant, explaining the high sequence variability in this region ( ) . by contrast, the smaller ha subunit, containing the fusion peptide that is necessary to deliver the viral genome to the host cell, shows considerably higher sequence conservation. this organization is logical from the viral perspective as the large ha subunit likely blocks antibody recognition of ha . the viral downside is the need to escape antibodies that inhibit the receptor-binding pocket without losing specificity and the binding function. based on this knowledge, several exciting new strategies are being developed to elicit the production of antibodies that target the more conserved region of ha ( ) ( ) ( ) . the hope is that these strategies will generate broadly neutralizing antibodies that recognize multiple ha subtypes from iavs and the distinct lineages in ibvs, providing longer lasting immunity and alleviating the threat of potential pandemics. a similar approach using na would likely provide additional benefits. however, our knowledge of na lags behind ha. currently, it is still not known why na has evolved to function as a tetramer, which is relevant because this property presumably restricts the potential antigenic drift (mutations) it can accommodate and still function. iav replication and spreading frontiers in immunology | www.frontiersin.org july | volume | article a relatively overlooked feature in the replication process is the contributions of host rna-binding proteins (rbps). human cells are predicted to encode over , rbps, of which are predicted to interact with mrnas ( ) . as a rna virus, it is highly likely that iavs have evolved to utilize this enormous network of rbps, which is supported by observations that some rbps inhibit iav replication, whereas others contribute ( ) ( ) ( ) . it should also be considered that changes in rbps have been associated with various cancers, which could possibly influence the susceptibility to influenza infections ( , ) . with the growing interest in rna biology, this aspect of iav 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single-molecule sensitivity fish analysis influenza a virus assembly intermediates fuse in the cytoplasm analysis of iav replication and co-infection dynamics by a versatile rna viral genome labeling method differential use of importin-alpha isoforms governs cell tropism and host adaptation of influenza virus the rna polymerase of influenza a virus: mechanisms of viral transcription and replication structural insights into rna synthesis by the influenza virus transcription-replication machine interaction of the influenza a virus nucleocapsid protein with the viral rna polymerase potentiates unprimed viral rna replication isolation and characterization of the positive-sense replicative intermediate of a negative-strand rna virus single-molecule fret reveals the pre-initiation and initiation conformations of influenza virus promoter rna different de novo initiation strategies are used by influenza virus rna polymerase on its crna and viral rna promoters during viral rna replication genetic trans-complementation establishes a new model for influenza virus rna transcription and replication internal initiation of influenza virus replication of viral rna and complementary rna in vitro structural insight into cap-snatching and rna synthesis by influenza polymerase a unique cap(m gpppxm)-dependent influenza virion endonuclease cleaves capped rnas to generate the primers that initiate viral rna transcription association of the influenza a virus rnadependent rna polymerase with cellular rna polymerase ii rna-free and ribonucleoproteinassociated influenza virus polymerases directly bind the serine- -phosphorylated carboxyl-terminal domain of host rna polymerase ii structural basis of an essential interaction between influenza polymerase and pol ii ctd the structural basis for cap binding by influenza virus polymerase subunit pb the cap-snatching endonuclease of influenza virus polymerase resides in the pa subunit crystal structure of an avian influenza polymerase pa(n) reveals an endonuclease active site the rna exosome syncs iav-rnapii transcription to promote viral ribogenesis and infectivity polyadenylation sites for influenza virus mrna direct evidence that the poly(a) tail of influenza a virus mrna is synthesized by reiterative copying of a u track in the virion rna template an in vitro fluorescence based study of initiation of rna synthesis by influenza b polymerase influenza viruses and mrna splicing: doing more with less sequence of interrupted and uninterrupted mrnas and cloned dna coding for the two overlapping nonstructural proteins of influenza virus mapping of the two overlapping genes for polypeptides ns and ns on rna segment of influenza virus genome sequences of mrnas derived from genome rna segment of influenza virus: colinear and interrupted mrnas code for overlapping proteins spliced and unspliced rnas encoded by virion rna segment of influenza virus influenza virus mrna trafficking through host nuclear speckles differences in the control of virus mrna splicing during permissive or abortive infection with influenza a (fowl plague) virus frontiers in immunology | www.frontiersin.org july regulated m mrna splicing in influenza virus-infected cells the accumulation of influenza a virus segment spliced mrnas is regulated by the ns protein inefficient splicing of segment and mrnas is an inherent property of influenza virus a/brevig mission/ / (h n ) that causes elevated expression of ns protein biogenesis, assembly, and export of viral messenger ribonucleoproteins in the influenza a virus infected cell nuclear import and assembly of influenza a virus rna polymerase studied in live cells by fluorescence cross-correlation spectroscopy genomewide analysis of influenza viral rna and nucleoprotein association nucleotide resolution mapping of influenza a virus nucleoprotein-rna interactions reveals rna features required for replication phosphorylation at the homotypic interface regulates nucleoprotein oligomerization and assembly of the influenza virus replication machinery the ns protein: a multitasking virulence factor influenza virus targets the mrna export machinery and the nuclear pore complex the influenza virus nep (ns protein) mediates the nuclear export of viral ribonucleoproteins a nuclear export signal in the matrix protein of influenza a virus is required for efficient virus replication influenza a virus ns protein mediates vrnp nuclear export through nes-independent interaction with hcrm a second crm -dependent nuclear export signal in the influenza a virus ns protein contributes to the nuclear export of viral ribonucleoproteins crucial role of the influenza virus ns (nep) c-terminal domain in m binding and nuclear export of vrnp crystal structure of the m protein-binding domain of the influenza a virus nuclear export protein (nep/ns ) a rab -and microtubule-dependent mechanism for cytoplasmic transport of influenza a virus viral rna rab a is essential for transport of the influenza virus genome to the plasma membrane apical transport of influenza a virus ribonucleoprotein requires rab -positive recycling endosome influenza virus genome reaches the plasma membrane via a modified endoplasmic reticulum and rab -dependent vesicles interaction of the influenza virus nucleoprotein with the cellular crm -mediated nuclear export pathway a functional sequence-specific interaction between influenza a virus genomic rna segments n-linked glycans direct the cotranslational folding pathway of influenza hemagglutinin nh -terminal hydrophobic region of influenza virus neuraminidase provides the signal function in translocation type ii transmembrane domain hydrophobicity dictates the cotranslational dependence for inversion integration of a small integral membrane protein, m , of influenza virus into the endoplasmic reticulum: analysis of the internal signal-anchor domain of a protein with an ectoplasmic nh terminus a yeast mutant defective at an early stage in import of secretory protein precursors into the endoplasmic reticulum protein translocation across the endoplasmic reticulum. ii. isolation and characterization of the signal recognition particle receptor inefficient srp interaction with a nascent chain triggers a mrna quality control pathway substratespecific translocational attenuation during er stress defines a pre-emptive quality control pathway solving the membrane protein folding problem x-ray structure of a protein-conducting channel molecular code for transmembrane-helix recognition by the sec translocon polar residues and their positional context dictate the transmembrane domain interactions of influenza a neuraminidases protein translocation across the rough endoplasmic reticulum the cotranslational maturation program for the type ii membrane glycoprotein influenza neuraminidase n-linked carbohydrates act as lumenal maturation and quality control protein tags the number and location of glycans on influenza hemagglutinin determine folding and association with calnexin and calreticulin structure of the haemagglutinin membrane glycoprotein of influenza virus at a resolution structure of the influenza virus glycoprotein antigen neuraminidase at . a resolution the pandemic h n neuraminidase n lacks the -cavity in its active site influenza virus m integral membrane protein is a homotetramer stabilized by formation of disulfide bonds steps in maturation of influenza a virus neuraminidase assembly of subtype influenza neuraminidase is driven by both the transmembrane and head domains the influenza virus neuraminidase protein transmembrane and head domains have coevolved influenza a viral replication is blocked by inhibition of the inositolrequiring enzyme (ire ) stress pathway influenza induces endoplasmic reticulum stress, caspase- -dependent apoptosis, and c-jun n-terminal kinase-mediated transforming growth factor-beta release in lung epithelial cells influenza a virus host shutoff disables antiviral stress-induced translation arrest real-time rt-qpcr assay for the analysis of human influenza a virus transcription and replication dynamics strand-specific real-time rt-pcr for distinguishing influenza vrna, crna, and mrna translational regulation of viral secretory proteins by the ' coding regions and a viral rna-binding protein the signal sequence coding region promotes nuclear export of mrna the ns protein from influenza virus stimulates translation initiation by enhancing ribosome recruitment to mrnas major contribution of the rna-binding domain of ns in the pathogenicity and replication potential of an avian h n influenza virus in chickens eukaryotic translation initiation factor gi is a cellular target for ns protein, a translational activator of influenza virus influenza virus ns protein stimulates translation of the m protein influenza virus ns protein enhances the rate of translation initiation of viral mrnas influenza viruses cause hemolysis and fusion of cells interaction of influenza virus hemagglutinin with target membrane lipids is a key step in virus-induced hemolysis and fusion at ph . influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease fur gene expression as a discriminating marker for small cell and nonsmall cell lung carcinomas proteolytic activation of influenza viruses by serine proteases tmprss and hat from human airway epithelium proteolytic activation of the influenza virus hemagglutinin cleavage of influenza a virus hemagglutinin in human respiratory epithelium is cell associated and sensitive to exogenous antiproteases cleavage of influenza virus hemagglutinin by airway proteases tmprss and hat differs in subcellular localization and susceptibility to protease inhibitors amantadine selection of a mutant influenza virus containing an acid-stable hemagglutinin glycoprotein: evidence for virus-specific regulation of the ph of glycoprotein transport vesicles influenza virus m protein ion channel activity stabilizes the native form of fowl plague virus hemagglutinin during intracellular transport influenza and sars-coronavirus activating proteases tmprss and hat are expressed at multiple sites in human respiratory and gastrointestinal tracts lipid rafts as a membrane-organizing principle influenza virus assembly and budding association of influenza virus proteins with membrane rafts mutations at palmitylation sites of the influenza virus hemagglutinin affect virus formation acylation-mediated membrane anchoring of avian influenza virus hemagglutinin is essential for fusion pore formation and virus infectivity s acylation of the hemagglutinin of influenza viruses: mass spectrometry reveals site-specific attachment of stearic acid to a transmembrane cysteine influenza virus hemagglutinin concentrates in lipid raft microdomains for efficient viral fusion role of transmembrane domain and cytoplasmic tail amino acid sequences of influenza a virus neuraminidase in raft association and virus budding influenza virus m protein mediates escrt-independent membrane scission influenza virus assembly: effect of influenza virus glycoproteins on the membrane association of m protein influenza virus assembly and lipid raft microdomains: a role for the cytoplasmic tails of the spike glycoproteins identification of the domains of the influenza a virus m matrix protein required for np binding, oligomerization and incorporation into virions membrane curvature in cell biology: an integration of molecular mechanisms influenza virus hemagglutinin and neuraminidase, but not the matrix protein, are required for assembly and budding of plasmid-derived virus-like particles formation of virus-like particles from human cell lines exclusively expressing influenza neuraminidase budding capability of the influenza virus neuraminidase can be modulated by tetherin structural analysis of the roles of influenza a virus membrane-associated proteins in assembly and morphology the crystal structure of the influenza matrix protein m at neutral ph: m -m protein interfaces can rotate in the oligomeric structures of m influenza virus matrix protein m preserves its conformation with ph, changing multimerization state at the priming stage due to electrostatics influenza a matrix protein m multimerizes upon binding to lipid membranes the m matrix protein controls the filamentous phenotype of influenza a virus viral membrane scission the influenza virus hemagglutinin cytoplasmic tail is not essential for virus assembly or infectivity the cytoplasmic tail of the neuraminidase protein of influenza a virus does not play an important role in the packaging of this protein into viral envelopes mutations in the membrane-proximal region of the influenza a virus m protein cytoplasmic tail have modest effects on virus replication influenza neuraminidase influenza virus neuraminidase: structure, antibodies, and inhibitors the . a resolution crystal structure of influenza b neuraminidase and its complex with sialic acid mechanism-based covalent neuraminidase inhibitors with broad-spectrum influenza antiviral activity structure of the catalytic and antigenic sites in influenza virus neuraminidase conversion of a class ii integral membrane protein into a soluble and efficiently secreted protein: multiple intracellular and extracellular oligomeric and conformational forms a (n ) neuraminidase of the x- influenza virus recombinant: determination of molecular size and subunit composition of the active unit influenza virus sialidase: effect of calcium on steady-state kinetic parameters mucin as substrate of enzyme action by viruses of the mumps influenza group mucins and mucoids in relation to influenza virus action; the destruction of francis inhibitor activity in a purified mucoid by virus action neuraminidase: the specific enzyme of influenza virus and vibrio cholerae preparation and properties of antibody directed specifically against the neuraminidase of influenza virus inhibition of influenza virus replication in tissue culture by -deoxy- , -dehydro-n-trifluoroacetylneuraminic acid (fana): mechanism of action characterization of temperature sensitive influenza virus mutants defective in neuraminidase mismatched hemagglutinin and neuraminidase specificities in recent human h n influenza viruses oligosaccharide specificity of influenza h n virus neuraminidases neuraminidase receptor binding variants of human influenza a(h n ) viruses resulting from substitution of aspartic acid in the catalytic site: a role in virus attachment? influenza virus neuraminidases with reduced enzymatic activity that avidly bind sialic acid receptors influenza a penetrates host mucus by cleaving sialic acids with neuraminidase mucoproteins in relation to virus action a beneficiary role for neuraminidase in influenza virus penetration through the respiratory mucus neuraminidase is important for the initiation of influenza virus infection in human airway epithelium pandemic swine h n influenza viruses with almost undetectable neuraminidase activity are not transmitted via aerosols in ferrets and are inhibited by human mucus but not swine mucus comparative mutational analyses of influenza a viruses rates of spontaneous mutation among rna viruses rapid evolution of rna viruses extreme heterogeneity of influenza virus infection in single cells deep mutational scanning identifies sites in influenza nucleoprotein that affect viral inhibition by mxa the inherent mutational tolerance and antigenic evolvability of influenza hemagglutinin accurate measurement of the effects of all amino-acid mutations on influenza hemagglutinin how single mutations affect viral escape from broad and narrow antibodies to h influenza hemagglutinin immunogen design for hiv- and influenza is it possible to develop a "universal" influenza virus vaccine? toward a universal influenza virus vaccine: potential target antigens and critical aspects for vaccine development hemagglutinin-stem nanoparticles generate heterosubtypic influenza protection structure and function analysis of an antibody recognizing all influenza a subtypes a census of human rna-binding proteins cellular rna binding proteins ns -bp and hnrnp k regulate influenza a virus rna splicing cellular ddx rna helicase inhibits influenza a virus replication but is counteracted by the viral ns protein multiple nuclear-replicating viruses require the stress-induced protein zc h a for efficient growth dissecting the expression landscape of rna-binding proteins in human cancers rna-binding proteins in cancer: old players and new actors inhibitors of influenza a virus polymerase the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord- - x euj authors: nickol, michaela e.; kindrachuk, jason title: a year of terror and a century of reflection: perspectives on the great influenza pandemic of – date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: x euj background: in the spring of , the “war to end all wars”, which would ultimately claim more than million lives, had entered into its final year and would change the global political and economic landscape forever. at the same time, a new global threat was emerging and would become one of the most devastating global health crises in recorded history. main text: the h n pandemic virus spread across europe, north america, and asia over a -month period resulting in an estimated million infections and – million deaths worldwide, of which ~ % of these occurred within the fall of (emerg infect dis : - , , bull hist med : - , ). however, the molecular factors that contributed to the emergence of, and subsequent public health catastrophe associated with, the pandemic virus remained largely unknown until , when the characterization of the reconstructed pandemic virus was announced heralding a new era of advanced molecular investigations (science : - , ). in the century following the emergence of the pandemic virus we have landed on the moon, developed the electronic computer (and a global internet), and have eradicated smallpox. in contrast, we have a largely remedial knowledge and understanding of one of the greatest scourges in recorded history. conclusion: here, we reflect on the influenza pandemic, including its emergence and subsequent rapid global spread. in addition, we discuss the pathophysiology associated with the virus and its predilection for the young and healthy, the rise of influenza therapeutic research following the pandemic, and, finally, our level of preparedness for future pandemics. influenza viruses have posed a continual threat to global public health since at least as early as the middle ages, resulting in an estimated - million cases of severe illness and , - , deaths annually worldwide, according to a recent estimate [ ] . regional influenza epidemics occur on an annual basis, resulting in millions of illnesses and hospitalizations despite intensive vaccination and awareness programs [ , ] . moreover, influenza pandemics arise sporadically due to the introduction of an antigenically-distinct influenza a virus within a population, which can result in devastating effects on global public health and healthcare networks. the emergence of influenza subtype h n in , which ultimately resulted in an estimated - million deaths worldwide, would forever change the course of human history and will be discussed in detail in the following sections [ ] [ ] [ ] . the aims of this short review are to discuss: i) the emergence and spread of the virus; ii) the unique severity of disease in young, healthy individuals; and iii) the subsequent influence of the pandemic on influenza virus therapeutic and future preparedness. it is postulated that % of the worldwide population is infected by an influenza virus each year, resulting in a total economic burden of $ . billion usd [ , ] . as a testament to the significant toll posed by influenza on public health and healthcare systems, the us centers for disease control and prevention (cdc) estimated that from to , influenza infections resulted in . - . million illnesses and , - , hospitalizations annually in the us alone [ ] . it has been suggested that children are likely the primary transmitters of influenza [ ] . lethal influenza infections are primarily associated with high risk populations, including infants (< year), the elderly (> years), and individuals with pre-existing comorbidities, including chronic respiratory abnormalities, cardiac disease, immunodeficiency, and pregnancy [ , ] . mortality in children and young adults is generally low [ ] . symptoms manifest as a sudden high fever, headache, pharyngitis, cough, myalgia, nausea, vomiting, and fatigue, which generally resolve within days in healthy adults [ , ] . severe and/or lethal disease is typically associated with viral pneumonia or secondary bacterial infections in the lower respiratory tract [ ] . to be considered a pandemic, an influenza virus must: i) spread globally from a distinct location with high rates of infectivity resulting in increased mortality; and ii) the hemagglutinin (ha) cannot be related to influenza strains circulating prior to the outbreak nor have resulted from mutation [ , ] . it should also be appreciated that prior to the first isolation of a human influenza virus in , the cause of influenza outbreaks and pandemics could only be inferred based on physiological symptoms of disease, along with the speed and breadth at which illness was spread [ ] . as early as bc, hippocrates, the father of modern medicine, described the first known account of an influenza-like illness in his sixth "book of epidemics" [ , ] . here, he recounted an annual recurring upper respiratory tract infection characterized by pharyngitis, coryza, and myalgia which peaked around the winter solstice [ ] . this seasonal epidemic occurred in perinthus, a northern port town located in what is now turkey, and is referred to as the "cough of perinthus" [ ] . it has been suggested that potential pandemics may have occurred in and ; however, it is unanimously agreed that the first documented influenza pandemic occurred in , resulting in high morbidity [ , ] . the virus originated in asia, before spreading to africa, and then simultaneously spreading from both continents to europe. it reportedly spread across the entire european continent within months, before eventually reaching the americas [ , ] . two pandemics were recorded in the eighteenth century. the first began in russia in , eventually moving across the entirety of europe within months and, ultimately, across the known world over the following years [ ] [ ] [ ] [ ] . the second pandemic began in china in , before spreading to russia and, subsequently, across all of europe. interestingly, this second pandemic had a high proclivity for young adults [ ] . two major pandemics also occurred throughout the nineteenth century. the first began in in china, with subsequent spread to southeast asia, russia, europe, and north america and had a low overall mortality rate [ , , , ] . a second pandemic emerged in russia in and spread rapidly to europe and north america, circumnavigating the globe in just months [ , ] . the virus, suggested to be of subtype h n , reappeared at least more times in successive years resulting in an estimated million global fatalities [ , , , ] . four influenza pandemics have occurred over the past century ( fig. ) . the - spanish flu pandemic, subtype h n , resulted in an estimated - million deaths worldwide and will be discussed in detail in the following sections. the - asian flu pandemic, subtype h n , originated in china in february and spread throughout asia and then globally by the summer. case fatality rates were approximately . % with - million deaths worldwide [ , [ ] [ ] [ ] [ ] . just a decade later, the - hong kong flu pandemic, subtype h n , emerged in china in july and spread throughout europe, north america, and australia by early [ ] . although mortality rates were low, the pandemic would ultimately claim between , and million lives [ ] . in april , the - swine flu pandemic, subtype h n , began with nearly simultaneous outbreaks in mexico and the us, before spreading globally over the next weeks. while the - pandemic had a low associated case fatality rate, resulting in , deaths worldwide, it had devastating effects on global economies and healthcare networks [ , ] . conventionally, influenza pandemics result in the extinction of previously circulating virus strains; however, this view was complicated by events in . although h n was replaced by h n as the circulating strain following the - asian flu pandemic, a descendant of the virus "re-emerged" suspiciously in , likely as a result of a man-made event, and established itself as a co-circulating strain, along with the reassortant h n virus (following the - hong kong flu pandemic) [ , ] . the suspicious "re-emergence" of a descendant of the virus in has been postulated to have been the result of a man-made event. this hypothesis has gained traction, as both the ha and na of the re-emerged virus show incredible similarity to a reference virus, and it is unlikely that this strain was maintained in an animal reservoir for almost two decades without having undergone detectable mutation [ ] . in , a triple reassortment (made up of avian, swine, and human influenza genes) pandemic h n jumped from pigs to humans, resulting in the co-circulation of three influenza strains [ ] . the first wave of the pandemic one hundred years following its emergence, the origin of the pandemic influenza virus remains shrouded in mystery. the pandemic began early in the final year of the first world war. whereas prior pandemics had spread largely along trade routes, the global context of the war enabled greater viral spread facilitated by the mass mobilisation of military personnel and civilians [ , ] . this was further augmented by the poor health and sanitation conditions found within trenches along the frontlines of the war, facilitating disease transmission [ ] . public knowledge regarding the severity of the pandemic was hindered, as many news agencies were barred from writing about the global health threat, instead reporting solely on morale boosting subjects [ ] . however, as spain was a neutral party in the war, newspapers were able to report on the devastating effects that the pandemic virus was exhibiting in spain. thus, it was generally perceived that this devastating illness originated in spain, resulting in the pandemic being incorrectly labeled as "the spanish flu" [ ] . a century following its emergence, there remains a relative paucity of knowledge regarding the ancestry and regional origin of the virus. sequence analysis suggests that the virus was derived from an avian-like influenza virus and that all eight gene segments likely evolved in parallel [ , ] . analyses of influenza virus genome sequences also suggest that the initial entry of the precursor virus into human circulation began in and did not appear to have jumped directly from an avian source [ , , ] . however, improved understanding regarding the emergence of the virus, as well as factors (biological, social, environmental) that contributed to viral transmission and pathogenesis, have been vital to the development of current epidemic and pandemic influenza outbreak response efforts. descendants of the pandemic influenza virus strain have been the cause of almost every seasonal influenza a infection worldwide over the past century [ ] . additionally, each of the pandemics occurring in , , and were caused by descendants of the pandemic ( , , and ). circulation of h n was reinitiated in and has therefore been added to this timeline. grey arrows designate the circulating or co-circulating strains during the interpandemic periods influenza virus strain, earning the viral strain the nickname "the mother of all pandemics" [ ] . investigations concerning the origins of the first wave of the pandemic, beginning in march , have primarily focused on the us and china, though recently it has been suggested that the origin may have been an outbreak of a respiratory disease misidentified as pneumonic plague in china [ , , ] . humphries suggests that the dissemination of labourers from china to assist allied war efforts during this outbreak resulted in the inadvertent spread of the virus to europe [ ] . from to , the route of travel to europe for the labourers included checkpoints in singapore, durban, cape town, north africa, and canada. additional reports of the first wave of the virus in the spring of suggest that the pandemic originated with chinese workers at camp funston, kansas, where the workers began suffering from to day fevers, gastrointestinal symptoms, and general weakness [ , ] . within weeks soldiers had been hospitalized, and thousands more had received out-patient treatment [ ] . the illness was able to spread to other military camps within the us, before traversing the atlantic ocean via soldiers supporting allied operations in europe. the us army reported that from march-may , . % of us soldiers were hospitalized due to this unidentified respiratory illness [ ] . while illness rates were high during this initial wave, mortality rates were largely similar to seasonal outbreaks of influenza. spain reported that the mortality rates for pneumonia and influenza was only . % [ ] . although there was some acceptance that this new illness was indeed influenza, this was not generally accepted [ ] . radusin reported that although the physiological symptoms were similar to influenza, the illness was too mild and short-lasting with minimal complications for it to be influenza [ ] . infections began to subside in many regions by the early summer [ ] . the generally accepted lines of spread of the first and second waves of the virus are provided in fig. . in mid-august of , reports suggesting a second wave of this severe illness began to surface [ ] . in some regions, primarily northern europe, the period between the end of the first wave and the beginning of the second wave was incredibly short, making the two waves almost indistinguishable [ , ] . this second wave, occurring from september-november , was responsible for the majority of illnesses and fatalities associated with the pandemic. although the origins of the first wave [ ] continue to be debated, the origin of the second wave is generally agreed to be the harbour town of plymouth in southern england, which allowed the pandemic influenza virus strain to easily spread to the rest of the world [ ] . ships from plymouth were dispatched to freetown, sierra leone in august , which allowed the virus to spread across the african continent [ ] . new zealand soldiers, who stopped in freetown on their way to and from the war front in europe, facilitated transfer of the pandemic virus to new zealand [ ] . from plymouth, the virus also spread to boston, from which it was able to disseminate across the rest of north america resulting in > million fatalities over the ensuing four months [ , ] . this second wave spread globally throughout the fall of with illness seen first amongst military personnel and, subsequently, within the general population [ , ] . the second wave of the pandemic differed from the first in that much higher morbidity and mortality rates were reported, with the majority of all fatalities associated with the pandemic occurring during this wave [ ] . ultimately, the pandemic would result in an estimated million infections worldwide (~ / of the world's population at the time) and a case fatality rate > . %, more than times higher than any other pandemic [ , ] . as a testament to the severity of this second wave, during the fall of , the first - pages of spanish newspapers were filled with obituaries of those who had succumbed to the pandemic virus [ ] . further, reports from philadelphia, pennsylvania stated that across hospitals in the city, every hospital bed was occupied by patients with influenza [ ] . the pandemic was especially problematic in highly isolated communities where many individuals had limited contact with prior influenza strains, thus lacking any pre-existing immunity. for example, some inuit settlements reported case mortality rates as high as %, while certain communities in africa were completely decimated [ ] . interestingly, individuals who had been infected throughout the first wave seemed to be protected against this secondary wave, and recent analyses have suggested that these individuals had up to % protection throughout the fall wave [ , ] . a third and final wave of the pandemic appeared in most of the world in the early months of [ , , ] . this final wave generally overlapped the first wave in terms of regional distribution; however, it seemed to spare areas where the second wave had been especially severe. overall, morbidity rates were lower throughout this final influenza wave; however, mortality rates are believed to have been just as severe as the second wave [ , ] . three successive annual winter post-pandemic recurrences occurred following the third wave of the pandemic with continually decreasing mortality rates, in particular within those - years of age [ ] . classically, fatal influenza infections are primarily associated with the very young (< years) and the elderly (> years) resulting in a characteristic "u"-shaped mortality curve (fig. ) . interestingly, however, the - h n influenza pandemic mortality curve exhibits a "w"-shape due to excess mortality in young adults - years of age due to influenza-related illness. it has been postulated that the increased disease severity in young adults was likely associated with immune status due to the lack of pre-existing immunity in this population [ ] . further, more than % of fatal infections occurred in those < years of age and nearly % of all influenza-related deaths during the pandemic were in those aged - years [ ] . influenza and pneumonia fatality rates in those aged - years were more than times higher than in previous years and absolute risk of influenza-related death was higher in those < years of age than those > years old [ ] . it is still not fully understood why this occurred, but it is possible that an antigenically similar influenza strain circulated prior to , providing a level of protection against the novel h n pandemic strain to those born prior to [ ] . additionally, archaeserological and epidemiological evidence have shown that an h subtype influenza virus may have been responsible for the influenza pandemic, which circulated until the emergence of the pandemic virus, leaving those individuals who had not been exposed to an h subtype virus highly susceptible to the pandemic virus [ ] . it has also been suggested that the generation of an excessive inflammatory response ("cytokine storm") in healthy, young adults infected with the virus may have contributed to the excess mortality seen within this age group [ ] . recent in vivo studies with the virus have shown a marked upregulation of inflammatory cytokines, along with the suppression of important antiviral immune responses [ , ] . in addition, other influenza strains, such as fatal h n infections in humans, have also been associated with the deleterious consequences of an excessive inflammatory response [ ] . ultimately, the case fatality rate was so severe in young adults during the - pandemic that the average life expectancy rate in the us dropped by~ years [ ] . physiological symptoms of the pandemic virus generally lasted for days and were described as feeling cold, shivering, high fever, weakness, nausea, loss of appetite, pharyngitis, cough, and bloodshot eyes [ ] . in some patients, a short "rebound" to normal health would occur that was followed by an aggressive recrudescence of disease and, ultimately, death [ ] . similar to the pandemic, the majority of fatal infections resulted from respiratory complications. however, it has also been demonstrated that excess influenza fatalities during the - pandemic were associated with an acute aggressive bronchopneumonia (including epithelial and vascular necrosis, hemorrhage, edema, and bacterial-associated variant pathology within the lungs) and a severe acute respiratory distress-like syndrome associated with severe facial cyanosis [ ] . autopsies performed on preserved lung tissues in the modern era have revealed acute pulmonary hemorrhage and secondary bacterial infections associated with pulmonary lesions in nearly all the fatal cases examined [ , , ] . streptococcus pneumoniae was present in many cases; however, staphylococcus aureus, haemophilus influenzae, and streptococcus pyogenes also appeared to complicate fatal cases [ , ] . neutrophilic pulmonary infiltration was seen in cases of pneumococcal pneumonia, while cases of staphylococcal pneumonia were marked by multiple microabscesses infiltrated by neutrophils [ ] . however, alveolar cell damage was seen in each case along with pulmonary repair and remodelling [ ] . tissues from each of the fatal cases examined had similar pathologic presentation, independent of which pandemic wave they were associated with. despite the difference in mortality rates, each wave showed similar cellular tropism, infecting both type i and type ii pneumocytes, as well as the bronchiolar respiratory epithelium [ ] . a multitude of scientific and technological advances have occurred over the past century, allowing for a greater understanding of the dynamic relationship between the host and influenza viruses during infection. these advances, along with access to autopsy samples and the reconstitution of the pandemic virus, have facilitated a greater understanding of how the pandemic virus differs from other seasonal and pandemic influenza virus strains. moreover, technological advancements following the - influenza pandemic virus have facilitated the development of preventative measures, including vaccines and antivirals, to limit widespread illness due to influenza infections. the determination of the genomic sequence of the pandemic virus, and the subsequent reconstruction of the virus, has provided us with the opportunity to decipher the viral-and host-specific properties that contributed to the severity of the - pandemic. it has been demonstrated that in contrast to other influenza viruses, the pandemic virus is highly virulent and pathogenic in multiple animal species without prior adaptation [ , ] . while obvious knowledge gaps remain, in particular with respect to the origin of the virus and the molecular mechanisms (host and/or viral) underlying differential pathogenesis as compared to other influenza viruses, there have been considerable advances in our understanding of the pandemic virus. [ , ] . means with standard deviations are presented for the prepandemic mortality curve. adapted from taubenberger and morens [ ] since the isolation of the first human influenza virus in , researchers have worked to develop an effective influenza vaccine [ ] . current influenza vaccines are reformulated seasonally and provide protection against circulating influenza a and b viruses [ ] . the world health organization conducts worldwide surveillance studies throughout the year on currently circulating influenza strains, and thus recommends which strains should be included in each influenza vaccine [ ] . while the seasonal influenza vaccine is approximately % effective, this protection is dependent on the characteristics of the individual being vaccinated, including age and overall health, as well as the match between the strains included in the vaccine formulation and currently circulating strains [ ] . individuals who have been vaccinated are generally protected from illness and provide a measure of protection for those who are not able to be vaccinated due to their age or other health issues through herd immunity [ ] . there has also been increasing interest in the development of "universal" influenza vaccines designed to provide protection against a wide range of antigenically-distinct influenza viruses, including those currently in circulation and those that may emerge in the future [ ] . these will not be discussed in detail as recent reviews have provided excellent discussions of this topic [ ] [ ] [ ] [ ] [ ] [ ] [ ] . two major classes of antivirals have emerged for therapeutic treatment of severe influenza virus infections. adamantane antivirals target the matrix- (m ) surface protein, while neuraminidase (na) inhibitors target the na viral surface protein. adamantane compounds were the first licensed influenza antivirals and block the m ion channel protein from properly functioning, thus effectively blocking membrane fusion [ , ] . unfortunately, adamantane antivirals are only able to target influenza a viruses limiting their application for influenza b virus infections [ ] . further, more than % of influenza a viruses are resistant to this class of drugs due to the high mutation rate of the virus [ , ] . thus, the use of na inhibitors is recommended [ ] . na inhibitors block the na surface protein and prevent the release of progeny virus and infection of additional cells [ ] . while resistance to na inhibitors has been observed in some influenza virus strains, they are still highly effective in the majority of patients [ ] . studies have shown that both adamantane antivirals and na inhibitors provide protection against the virus [ ] . although outside the auspice of this commentary, it should be mentioned that advances in mechanical ventilation modalities, including non-invasive positive pressure ventilation, from the s onwards, have provided an additional support mechanism for treatment of severely ill patients [ ] . the routine clinical use of antibiotics in the early twentieth century also heralded a new era for combating influenza viruses. as a testament to this, excess influenza mortality declined significantly from to onwards [ ] [ ] [ ] . however, the widespread general administration of antibiotics has resulted in an escalating public health crisis due to multi-drug resistance. this has impacted the treatment of severe influenza infections, as methicillin-resistant s. aureus (mrsa) is the most frequently isolated bacteria from patients with severe influenza-bacterial co-infections in the us [ , ] and complicated up to % of fatalities during the pandemic [ ] [ ] [ ] [ ] . influenza preparedness and lessons for the future although it has now been a century since the start of the spanish flu pandemic, lessons from this global health catastrophe continue to inform modern-day pandemic preparedness. investigations of the pandemic, including those with the reconstructed virus, have allowed researchers, as well as the global public, to understand the mechanisms that underlie pandemic emergence and escalation to public health crisis. it also allows researchers to predict the potential public health risks which may be caused by new pandemic viruses. for example, sequencing of the pandemic virus revealed similarities in the h protein of the pandemic virus, allowing researchers to predict that a lack of protection, and thus a high mortality rate may be seen in healthy, young adults throughout the h n pandemic [ ] . thus, when vaccines were limited during the early stages of the pandemic, young adults were prioritized over the elderly, who demonstrated some degree of protection to this influenza strain, resulting in a lower mortality rate in young, healthy adults [ ] . the average age for laboratory-confirmed fatalities during the pandemic was years in the us, supporting this vaccine prioritization initiative [ ] . additionally, the awareness of the complications caused by secondary bacterial co-infections from the pandemic ensured that the medical community was aware of this threat throughout the pandemic, likely resulting in a reduced mortality rate due to severe influenza infections with complications [ ] . however, the pandemic, albeit milder than previous pandemics in terms of overall mortality, resulted in significant strains on global healthcare networks and economies [ ] . in canada, direct healthcare costs (including hospitalizations, outpatient visits, and therapeutics) related to the pandemic have been estimated at $ billion cad, with $ million cad related directly to hospital care [ ] . a computational modeling study by smith and colleagues suggested that direct costs related to illness would be between . - . % of gdp in the uk for pandemics ranging from low to extreme [ ] . further, the - severe acute respiratory syndrome outbreak resulted in~$ billion total gdp loss in toronto alone [ ] . this highlights the importance of pandemic preparedness beyond a healthcare-centric approach to one that also includes downstream economic effects. the - pandemic resulted in incredible improvements to public health as well as scientific advances. however, our current understanding of influenza viruses, and their ability to cause illness in humans is still in its infancy in many aspects, and further underlines our inherent need for continued influenza research. the identification of key molecular determinants involved in the pathophysiology of severe influenza infections will also assist drug discovery and development strategies, including insights on appropriate timing for administration of antivirals and/or antibiotics. the development of efficacious broader-spectrum or "universal" influenza vaccines is also of incredible importance. the emergence of novel highly pathogenic avian influenza (hpai) viruses, including h and h subtypes, are of particular concern due to their pandemic potential. circulating hpai viruses are of potential concern to global public health [ ] . asian lineage avian influenza a (h n ), which circulates in fowl, is rarely found in humans but has resulted in life-threatening cases when able to establish stable lineages [ ] and h n has resulted in sporadic human infections in china resulting in > infections with an estimated % case fatality rate since [ ] . because hpai viruses can arise from previously known low-pathogenicity viruses with only minor mutations, it is important to be vigilant concerning these potential pandemic viruses [ , ] . in spite of the public health advancements in the years following the - pandemic, including widespread access in the developed world to an efficacious influenza vaccine, influenza viruses remain a global public health threat. this pas year, there were > , reported influenza infections, influenza-associated hospitalizations, and deaths across canada [ ] . further, during the - influenza season, vaccination rates in those - years of age was only and % in those ≥ , 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authors' contributions men and jk conceived of the ideas presented herein and made substantial contributions to the drafting and revising of the manuscript. both authors read and approved the final manuscript.ethics approval and consent to participate not applicable. not applicable. the authors have declared that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -wtestt i authors: jung, eunok; de los reyes v, aurelio a.; pumares, kurt jan a.; kim, yangjin title: strategies in regulating glioblastoma signaling pathways and anti-invasion therapy date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: wtestt i glioblastoma multiforme is one of the most invasive type of glial tumors, which rapidly grows and commonly spreads into nearby brain tissue. it is a devastating brain cancer that often results in death within approximately to months after diagnosis. in this work, optimal control theory was applied to regulate intracellular signaling pathways of mir- –ampk–mtor–cell cycle dynamics via glucose and drug intravenous administration infusions. glucose level is controlled to activate mir- in the up-stream pathway of the model. a potential drug blocking the inhibitory pathway of mtor by ampk complex is incorporated to explore regulation of the down-stream pathway to the cell cycle. both mir- and mtor levels are up-regulated inducing cell proliferation and reducing invasion in the neighboring tissues. concomitant and alternating glucose and drug infusions are explored under various circumstances to predict best clinical outcomes with least administration costs. glioblastoma multiforme (gbm) is the most common and the most aggressive type of brain cancer. the median length of survival time is approximately to months following diagnosis. gbm is characterized by anaplasia, nuclear atypia, cellular pleomorphism, mitotic activity, and more importantly, alternating phases of rapid proliferation and aggressive invasion into the surrounding brain tissue. this leads to an inevitably critical recurrence even after the surgical resection of the main tumor mass [ , ] . the mainstay of treatment for gbm is surgery, followed by radiotherapy and chemotherapy. despite advances in these approaches, glioma cells can still invade the neighboring tissues beyond detection leading to tumor recurrence. high probability of main treatment failure also encourages researchers to investigate the use of innovative treatments when the first line of therapy has failed, in order to improve clinical outcomes [ ] . in the tumor microenvironment (tme), glioma cells encounter many challenges including hypoxia, acidity, and limited nutrient availability. to maintain rapid growth, tumor cells need to adapt to these biochemical changes and modify their metabolic activity by increasing glycolysis even in the presence of oxygen. this process is called the warburg effect which requires consuming considerable amounts of glucose [ ] . the tricarboxylic acid cycle, or krebs cycle, plays an important role in the breakdown of organic fuel molecules and the survival in non-a a a a a hypoxic normal differentiated cells. these molecules include glucose, fatty acids, and some amino acids. while differentiated cells favor this type of metabolism, which is very efficient in terms of atp production, tumor cells adopt the inefficient aerobic glycolysis producing relatively large amounts of waste product in the form of lactic acid [ ] . this may provide cancer cells the advantage of not having to depend on oxygen as an energy source especially in a hostile tumor microenvironment, thus leading to longer survival [ ] . inhibition of glycolysis may also prevent drug resistance thus a better understanding of this metabolic pathway may lead to better treatment options and clinical outcomes [ ] . developing strategies of metabolic adaptation, angiogenesis, and migration is critical for cancer cells in order to survive metabolic stress and ensure enough nutrient supply as tumor mass accumulates where glucose supply may fluctuate due to heterogeneous biochemical and biophysical conditions [ ] . therefore, adequate cellular responses to glucose withdrawal are critical for cancer cell survival. cancer cells then activate the -adenosine monophosphate activated protein kinase (ampk) pathway under metabolic stress. it is the master cellular sensor of energy availability which enhances glucose uptake and conserve energy, thus avoiding cell death [ ] . micrornas, also abbreviated as mirna, are approximately nucleotide single-stranded non-coding ribonucleic acids (rnas) that are known to regulate gene expression [ ] . dysregulation of microrna expression has been linked to oncogenic and tumor suppressor activities in several types of cancer, including gbm [ , ] , where altered mirna expression contributes to tumorigenesis [ , ] . godlewski et al. [ ] identified an interesting mechanism of glioma cell migration and proliferation wherein a particular microrna, mir- , and its counterpart, ampk complex (cab / lkb /strad/ampk), determine whether the cell favors growth at the expense of invasion or conversely. moreover, they also identified a potential feedback loop between lkb and mir- allowing for a sustained and robust response to glucose withdrawal [ ] . it was found out that (i) under high (normal) glucose conditions, up-regulation of mir- leads to the down-regulation of ampk complex, which then leads to elevated proliferation and decreased migration of glioma cells and (ii) glucose withdrawal induces down-regulation of mir- and up-regulation of ampk, which promotes cell migration with reduced proliferation. the mathematical models developed by kim et al. [ , , ] describe the effects of the mir- -ampk core control system on cell proliferation and migration in glioblastoma. it explains the response of mir- to high and low glucose levels as well as the mutual antagonism between mir- and ampk complex concentrations. kim et al. [ ] then extended the model to include the dynamics of mammalian target of rapamycin (mtor), which is a protein kinase that links with other proteins as well as to the cell cycle dynamics, to form the mir- -ampk-mtor core control system. this mutual antagonism between mir- and ampk, which was predicted in these mathematical models [ , [ ] [ ] [ ] , was confirmed by recent experiments. for example, ansari et al. [ ] recently found that (i) the mir- transcription in gbm cells is induced by unrestricted activity of its transcription factor oct (official gene symbol pou f ) in the presence of abundant glucose, resulting in ampk inhibition through direct targeting of cab in the lkb complex; and (ii) the mir- level is inhibited through the phosphorylation and inactivation of oct at s by ampk in response to glucose depletion-induced metabolic stress, leading to a reciprocal negative feedback loop between mir- and ampk. in this case, suppression of mir- in turn leads to sustained ampk activities and a robust response to glucose withdrawal in gbm cells. the multiscale mathematical models [ , [ ] [ ] [ ] also predict the growth-invasion cycling patterns of glioma cells in response to fluctuating glucose uptake in the tumor microenvironment. the core control system predicts bistability and hysteresis bifurcation when delayed down-regulation of mir- activities along certain molecular pathways would induce glioma cells to stay longer in the proliferative phase despite relatively low glucose concentrations, making this mechanism a therapeutic target. the cell cycle represents an integrated series of events that regulates complex processes including cell proliferation, cell division and dna replication, regulated by a complex hierarchy of genetic and metabolic networks which involves several transition states of varied lengths and checkpoints [ ] . the stages of the cell cycle are as follows: (i) synthesis phase (s), a period where dna replication occurs; (ii) gap phase (g ), during which proteins required for mitosis are produced; (iii) mitosis phase (m), a period where chromatin condensation, nuclear envelope breakdown (nebd), chromatid separation, and cytokinesis happens; (iv) gap phase (g ), in which genes necessary for dna replication are activated and the protein agents of s phase progression are accumulated; and (v) resting phase (g ), a state in which cells can exit the cell cycle and enter a phase of quiescence or relative inactivity [ ] . the progression of mammalian cell cycle is tightly regulated by coordinated activation of cyclin-dependent kinases (cdks) family [ ] . the cdks are positively regulated by cyclins and negatively by cdk inhibitors (cdkis) such as the proteins p , p , p and p . in cancer cells, cyclins are over-expressed while cdkis are under-expressed which results in the dysregulation of the cell cycle, and promoting uncontrolled cell growth [ ] . tyson and novak [ , ] identified that the transition between two stable steady states, g and s-g -m cell-cycle phases, are described using the kinetic relations of the model that is controlled by changes in cell mass. a standard gbm treatment is surgery followed by chemotherapy and radiotherapy. however, even under best circumstances, the mean survival of this disease is about a year. poor outcomes of standard care treatments are due to the topographically diffuse nature of the disease [ ] . by the time of diagnosis, typical gbm cells may have widely spread throughout the brain tissue [ ] [ ] [ ] , increasing the potential of recurrence. thus, annihilation of distant tumor satellites is implausible despite surgically removing all the essential tumor seen on enhanced mri scan [ ] . knowing the exact margins of a tumor mass in real patients is indeed a daunting task. in this study, it is assumed that a major tumour mass has been surgically removed and that the infiltrative tumor cells are near the surgical site. the objective is to prevent the glioma cells from further diffusing into the surrounding brain tissue. localization approach will be utilized, that is, glioma cell invasion will be blocked keeping them in a proliferative phase while also attempting to limit excessive growth before a second surgery [ ] . our analytical tool is primarily based on the framework of optimal control theory, which has been successfully used to make informed decisions involving biological models such as optimal treatment strategies in human immunodeficiency virus (hiv) models [ ] [ ] [ ] [ ] , tuberculosis [ ] [ ] [ ] , and cardiopulmonary resuscitation (cpr) techniques [ , ] . optimal control theory is also applied to the mir- -ampk core control system to determine the intravenous glucose and/or drug infusion protocols with least possible cost under various circumstances in de los reyes, et al. [ ] . recently, kim et al. [ ] developed an intracellular signaling pathway model that extends the mir- -ampk-mtor core control system including the cell cycle dynamics. in this work, a potential control problem is formulated in order to maintain high levels of mir- and mtor (low levels of ampk) inducing cell proliferation prohibiting cell motility and invasion to the neighboring tissues. with glucose levels as a key regulator of mir- activity which also activates mtor in the downstream signaling pathway, glucose intravenous infusion is considered to up-regulate mir- and mtor concentrations above certain threshold values. in addition, a drug suppressing the inhibitory effect of mtor by the ampk complex is incorporated. this drug can be administered concomitantly or alternately with glucose as a secondary intravenous infusion. the controls are then given by dose rates of glucose and drug intravenous administrations regulating upstream and downstream signaling pathway, respectively. solution of the optimal control problem aims to determine infusion protocols with minimal glucose and drug amount, and least administration costs. hence, glucose and drug levels are regulated to prevent rapid tumour growth, hyperglycemia, and further drug complications. the results propose plausible glucose and drug intravenous infusion controls which indicate the time of administration, frequency, number of administrations, and dosages of glucose and drug per infusion. in the current study, we will first present the mir- -ampk-mtor-cell cycle intracellular signalling pathway developed by kim et al. [ ] . a drug module is incorporated to up-regulate mtor activities inducing cell proliferation. then an optimal control problem is formulated with the goal of activating mir- and mtor levels through glucose and drug intravenous infusions. two different infusion protocols will be explored, namely, concomitant and alternating glucose and drug intravenous administrations. optimal solutions for two strategies are presented and results on frequency, dosage per infusion, total glucose and drug amount, and relative cost incurred in the administrations are compared. the conclusion section discusses and summarizes the optimal control results, and provides outlook for future research directions. in this section, we present the basic components of intracellular signalling pathway of tumor cell containing the core control mir- -ampk-mtor and cell cycle pathway developed in kim et al. [ ] , incorporating a drug which blocks the inhibitory pathway of mtor by ampk complex. the core control model identifies a key mechanism which determines the molecular switches between the proliferative and migratory phases in response to fluctuating glucose and drug levels. the simplified signaling pathways consists of five key determinants of the intracellular structure, namely, glucose level g, mir- level m, ampk complex activity a, mtor concentration r, and drug level d. the intracellular cell cycle dynamics are developed by tyson and novak [ , ] including only the essential interactions for its regulation and control. the model captures the kinetics of chemical processes within the cell such as production, destruction, and different molecule interactions. the transition between two main steady states, g and s-g -m phases, of the cell cycle are described using the kinetic relations of the model that is controlled by changes in cell mass. . also included are the effects of the changes in oxygen dynamics at the macroscopic level through the activation and inactivation of hif- α. this results in changes in cell cycle length. in addition, the cell cycle inhibitory effect of p or p genes of hif- α is incorporated. kim et al. [ ] proposed to link both the mir- -ampk-mtor control system and the cell cycle dynamics to provide a mechanism driving the cell cycle to undergo the quiescent stage g -phase depending on the concentration level of mtor. fig a depicts the detailed schematic diagram of the intracellular signalling networks including mir- , ampk complex, mtor, and key players in the cell cycle module (cycb, cdh , p cdct, p cdca, plk ). kinetic interpretation of arrows and hammerheads represent induction and inhibition in the signaling network, respectively. the dimensionless diagram of the core control mir- , ampk complex, and mtor linking to the cell cycle is depicted in fig b. it should be noted that u g and u d are the sources of glucose and drug with decay rates μ g and μ d , respectively, which can be controlled exogenously. s and s are signaling sources to ampk complex and mtor, respectively, while α, β and γ are inhibition strengths, and ϕ denotes decay. it has been shown that high (normal) glucose concentration yields over expression of mir- levels (down-regulation of ampk complex and up-regulation of mtor) leading to elevated cell proliferation and reduced migration, while low glucose levels leads to down-regulation of mir- activities (up-regulation of ampk complex and down-regulation of mtor) reducing cell proliferation and inducing migration into the surrounding brain tissue [ , , , ] . the effect of various glucose levels in the regulation of the core control is depicted in fig . kim et al. [ ] developed a core control system of glioma cell migration and proliferation by using a regulatory network of key molecules (mir- (m), ampk (a)) as follows: as observed in experiments [ , , ], the regulatory system in eq ( ) includes a mutually antagonistic loop between mir- and ampk complex in response to high and low glucose levels (g). this genetic toggle switch induces a monostable and bistable system, characterizing proliferation and critical cell infiltration of gbm cells in brain [ ] . in the follow-up studies [ , [ ] [ ] [ ] including mtor (r) and cell cycle modules, this mutual antagonism and bistable system played a critical role in developing anti-invasion strategies. a mutually antagonistic feedback loop has been well-studied for its bistable properties by use of mathematical models ([ - ] and other references in [ ] ). for instance, in a study on a genetic toggle switch in escherichia coli [ ], a mutually inhibitory loop between repressor and repressor was in this study, we consider a drug d suppressing the inhibitory effect of mtor by ampk complex where the inhibition strength is given by z(d) = e −d . when d is large, γe −d is small which makes dr/dt to be large. thus, the presence of drug up-regulates mtor activity that could eventually lead to cell proliferation. fig illustrates the mtor bifurcation curve. observe that increasing drug concentration shifts the hysteresis curve upwards keeping the same bistability window. hence, with higher drug levels for the same glucose concentrations, mtor is activated prompting elevated cell growth. in the mir- -ampk-mtor core control system developed by kim and colleagues [ , [ ] [ ] [ ] ] , the bistability regime of main variables emerges in response to glucose levels. as it was shown in [ ], the existence and size of the bistability window (|w b |) depend on other essential parameters and may disappear under perturbations of parameters. as it will be shown later (figs and ), some of key parameters are sensitive in creation or destroying the bistability while other parameters are not. after achieving the equilibrium, continuation of the curve is computed by varying the glucose concentration g and bifurcation points are detected labeled lp and cp for limit and cusp points, respectively. fig a illustrates the hysteresis diagram (g, r)−curves for different s values. in order to obtain the cusp point (cp), fold continuation is computed starting at a limit point where two parameters g and s are activated resulting to codim bifurcations. both parameters (g, s ) are varied along the curve where each point is a limit point for the equilibrium curve at the corresponding value of s . this is depicted in fig b. the cusp point gives the threshold values th m , th a , and th r . a matlab software matcont was used for numerical continuation and bifurcation study of continuous and discrete dynamical systems [ ] . the governing model equations for the dimensionless intracellular signaling dynamics are then described by the following ordinary differential equations d½plk � dt ¼ k ½mass s �½cycb�ð À ½plk �Þ À k ½plk �; strategies in regulating glioblastoma signaling pathways and anti-invasion therapy the cell cycle dynamics and the regulatory core control system are linked by a variable called pseudo-mass ([mass s ]) given by the oxygen dynamics through hif- α is described as and the growth rate μ is expressed as wherem is the probability density function with uniform distribution between − and . this growth rate formulation introduces cell cycle heterogeneity of length between and hrs to account for the natural variability between cell growth rates and to have a non-synchronous population [ ] . the model parameters in system ( ) are listed in tables and . in the current model formulation, mtor (r) is activated/inactivated in the same way as mir- (m). in addition, r links the core control system and the cell cycle dynamics via the pseudo-mass ([mass s ]) which influences the cell mass [mass] and the intracellular proteins (refer to eq ( )). therefore, when glucose supply is high, r is up-regulated (proliferative phase; up-regulated m, down-regulated a) and [mass s ] � [mass] yielding a typical cell cycle (see fig ) . on the other hand, when glucose supply is low, r is down-regulated (migratory phase; down-regulated m; up-regulated a) and [mass s ] exceeds [mass] influencing the cells to enter into resting phase g [ ] . let us assume that glucose (g) and drug (d) can be regulated through intravenous infusions and can be periodically administered. for illustrative purposes, consider h infusions every h with maximum dosage of unit, and refer this as regular infusion. we then have the intracellular dynamics under regular infusion can be seen in fig . since glucose and drug levels oscillate, it follows that m and r also periodically fluctuates around the threshold (th m � . , th r � . ) as can be seen in fig a. note that when m and r crosses the threshold value from above, respectively, peak in pseudo-mass is generated. it can thus be inferred that these peaks indicate cell migration since m and r levels are below their respective threshold values. strategies in regulating glioblastoma signaling pathways and anti-invasion therapy as shown in fig b, a trajectory of core control concentrations in mtor-mir- -ampk space switches between proliferation and migration region. a closer look at the intracellular cell cycle proteins, mass, and mass s dynamics are illustrated in fig c. note that regular infusion significantly perturb the cell cycle dynamics stimulating several cell divisions (see mass profiles) and cell migration (see mass s ). indeed, fluctuating glucose levels in the microenvironment leads to the dichotomy of grow and go dynamics of glioblastoma cells yielding bigger tumor mass as reported in [ ] , even in the presence of (fluctuating) drug concentrations to keep the cells in proliferation phase. in the current investigation, we aim to regulate the amount of glucose and drug infusions to up-regulate mir- and mtor above its threshold values inducing cell proliferation strategies in regulating glioblastoma signaling pathways and anti-invasion therapy avoiding migration to neighboring tissues. the modeling approach utilizes optimal control theory to identify infusion administration protocols. let u g (t) and u d (t) be the controls of the system representing dose rates of glucose and drug intravenous administrations, respectively. two different administration protocols are examined, namely, ( ) concomitant, and ( ) alternating glucose and drug infusions, with the following objective functionals respectively. here, m(t) and r(t) denote the level of mir- and mtor concentrations, respectively. parameters b and b are weight factors measuring the relative cost based on maximizing mir- m(t) and mtor r(t), and administering glucose and drug intravenous infusions over a specified time interval, respectively. the control costs are modeled by the linear combination of quadratic terms u g ðtÞ and u d ðtÞ. our objective is to find optimal infusion regimen for glucose and drug administrations, denoted by u � g ðtÞ and u � d ðtÞ, such that the objective functionals are satisfied, that is, where the bounds for the controls represent the limits on dose rates for glucose and drug administrations. assuming that mir- , ampk, and mtor responses are regulated by glucose levels and influenced by drug levels, our control strategies deal with finding optimal control regimens for both glucose and drug intravenous infusions. we also note that the existence of optimal controls is guaranteed by standard results in control theory [ ] . in this maximization problem, the necessary convexity of the integrand in the objective functional holds. therefore, we can proceed with applying pontryagin's maximum principle [ ] . an iterative method is used for solving the optimality system which is a two-point boundary value problem having initial conditions for the state variables and terminal conditions for the adjoints. numerical simulations are obtained using a fourth-order iterative runge-kutta method. given the initial conditions and guess for the controls, state equations are solved using the forward scheme while the corresponding adjoint equations are solved using the backward scheme with the transversality conditions. the controls are updated by using a convex combination of the previous controls and the value from the characterizations. this is commonly referred as the forward-backward sweep method (fbsm) which is shown to be convergent [ ] . further details on the optimal control under study can be found in the s appendix. ( )) was performed in order to determine which parameters have the most/least influence on the reference output (main variables). all the core control parameters were considered to assess their corresponding influence on the mir- , ampk, and mtor activity. it was inferred that mir- activity will be enhanced by increasing glucose signal (g) and autocatalytic production rates (ℓ , ℓ ) of mir- . these parameters were negatively correlated with ampk activity due to the mutual antagonistic mechanism between mir- and ampk complex. in addition, ampk activity will be up-regulated by an increase in signaling source s and autocatalytic rates (ℓ , ℓ ) of the ampk complex in the model. it has been also noted that increasing s and the inhibition strength of mtor by ampk (γ) down-regulates mtor level. in a similar fashion, s is strongly positively correlated with mtor levels but little correlated with g, ℓ , ℓ , ℓ , ℓ , α, β, � , � at time . in this work sensitivity analysis is carried out to determine which model parameters have consequential effect in achieving or inhibiting bistability in the (g, r)−curve. as in [ ], a method from [ ] is adapted where a range for each parameter is selected and divided into n intervals of uniform length. for each parameter of interest, a partial rank the prcc values range between - and with the sign determining whether a change in the parameter value will promote (+) or suppress (−) bistability. the following algorithm is performed: . assign a probability distribution d½m j;min ; m j;max � to each parameter μ j and let n be the number of samples to be selected. divide the interval [μ j,min , μ j,max ] into n equiprobable subintervals, and draw an independent sample from each subinterval. . assemble the lhs matrix l, wherein each row of l represents a unique combination of parameters sampled without replacement. . for each row of the lhs matrix l, solve for mtor r in terms of glucose g and check for bistability window w b . if bistability exists assign to output variable w b in matrix y, else assign − . . rank-transform the matrices l and y to obtain l r and y r . by rank-transform, we mean to replace the value by its rank when the data are sorted from lowest to highest, e.g. the smallest value is assigned a rank . tied values are assigned an average rank. strategies in regulating glioblastoma signaling pathways and anti-invasion therapy . fix a parameter μ j , which is encoded in the j th column in the matrix l r . form the following linear regression models using the data matrices l r and y r for μ j and y, respectively: compute ðm j Àm j Þ and ðy ÀŷÞ, the residuals in the input parameter and the output after removing the linear effects of the other input parameters. . obtain the prcc of μ j using prcc m j ;y ¼ covðm j Àm j ; y ÀŷÞ ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi varðm j Àm j Þvarðy ÀŷÞ . repeat steps and for the remaining parameters. it illustrates that a change in the mir- autocatalytic production rate (ℓ ) or inhibition strength of ampk complex by mir- (β) enhances bistability. on the contrary, the hill-type coefficient ℓ in the regulation of ampk is responsible for losing bistability. it should be noted that due to the model's structure, ℓ up-regulates mir- which in turn increases mtor and suppress ampk activity. the parameter ℓ promotes ampk complex and down-regulates mir- and mtor. however, other parameters ℓ , α, ℓ , s , ℓ , ℓ , γ, s are little sensitive in emergence or destruction of the bistability. the bistability of mir- -ampk-mtor core control system depends on the geometric structure of its nullclines. in particular, bistability arises when m-and a-nullclines (i.e., dm dt ¼ and da dt ¼ ) intersect at three distinct points, producing one unstable and two stable steady states. the nullclines intersect three times due to their sigmoidicity influenced by catalytic rates ℓ and ℓ . these rates must be proportionate for a given glucose g level. otherwise, the nullclines will intersect only once. this bistability condition has been shown for a genetic toggle switch in e. coli [ ] . fig a- c depict the m-and a-nullclines under different g levels with various ℓ and ℓ values. the bifurcation curves for ℓ , ℓ and region of bistability for specific g levels are depicted in fig d. under given circumstances, ℓ and ℓ showed significant sensitivities in the bistability of the system. on the contrary, both ℓ and ℓ affect the r-nullcline only. it is illustrated in fig e that for several ℓ and ℓ values, r-and a-nullclines intersect at only one point, leading to a single steady state. in effect, m-and a-nullclines will also intersect once, producing monostability. hence, ℓ and ℓ show insignificant consequence in achieving bistability (see fig ) . in the following numerical experiments, it is assumed that initially glioma cells are in growth phase (a probable occurrence of post primary tumour surgery) with m > th m , a < th a , and r > th r . it is also considered that glucose and drug can be administered exogenously as intravenous infusions. further, the weight parameters b = and b = are used as default values unless specified. in this strategy, glucose and drug infusions are administered simultaneously, in particular, both controls u g (t) and u d (t) are infused at the same. thus, this is referred to as concomitant control. here, it is assumed that the drug in consideration which blocks the inhibitory pathway of mtor by ampk complex had negligible side effects and had inconsequential chemical reactions with glucose. in order to determine an efficient strategy of concomitant infusion protocol, glucose and drug are administered concurrently at initial time t = for hours using the numerical scheme fbsm. infusion spontaneously increases glucose and drug concentrations as depicted in fig (a) and (b) . consequently, mir- and mtor levels are up-regulated while ampk complex is down-regulated as shown in fig c. a closer look at the control curves shows that both u g (t) and u d (t) decrease from < t i < suggesting that both glucose and drug dose rates should be decreased from time t i . this leads to the decrease of glucose and drug concentrations due to consumption and decay. accordingly, mir- and mtor levels decrease while ampk complex increases. before mir- crosses the threshold value th m , fbsm is again applied for the next hours. this suggests a time for the next concomitant administrations in which mir- profiles are monitored subsequently. the procedures in tracking mir- profiles and applying fbsm for hours are repeated over a specified time duration. thus, the number of concomitant administrations are determined. it is important to note that keeping mir- level above its threshold value consequently confine ampk and mtor levels, below and above its corresponding threshold values, respectively (see fig c) . suppose that concurrent glucose and drug administrations is not plausible due to unwanted chemical reactions. we propose another control strategy that administers glucose u g (t) and drug u d (t) infusions alternately. at initial time t = , glucose infusion is obtained by solving the optimal control problem using the numerical scheme fbsm for hours. next, drug infusion is attained for the next hours in a similar manner. hence, the controls u g (t) and u d (t) are applied one after the other. subsequently, mir- levels are then tracked and before it crosses the threshold value, glucose infusion u g (t) and drug infusion u d (t) are again administered alternately in a similar fashion above, over the specified duration of administration. thus, the time for the next alternating infusion is determined by tracking mir- levels. this strategy is referred simply as alternating control. this infusion protocol is shown in fig (a) and (b). note that glucose infusion increases mir- levels and drug infusion activates mtor activities keeping ampk down-regulated as depicted in fig c. the intracellular dynamics under alternating control is illustrated in fig . as shown in fig a, both concomitant and alternating control strategies are able to sustain elevated mir- and mtor levels above their threshold values and ampk levels below its threshold value. it was also shown that mtor-mir- -ampk trajectory is restrained in the proliferation region prohibiting cell migration. further, number of cell divisions is reduced with slightly higher mass concentration before division compared to regular infusions but lower compared to constant (intermediate) high glucose concentrations. in this section, we compare the cost efficiency of the proposed strategies in terms of frequency of administration, dose per infusion, total glucose and drug amount, relative cost per infusion, and total cost incurred in the administrations. for the following results, the time for simulation duration is considered to be hours ( days). recall that parameters b and b are the weight factors associated in our objective functional which represent the measure of costs involved in the administration of glucose u g (t) strategies in regulating glioblastoma signaling pathways and anti-invasion therapy and drug u d (t) infusions, respectively, which also includes dosage, type, brand, medical fee for administration, etc. fig a shows that as the cost of glucose administration becomes expensive (increasing b values) with fixed drug administration cost (b = . ), frequency of concomitant and alternating infusion increases. however, it should be observed that as frequency of administration increases, the optimal glucose dose per infusion decreases with drug dose per infusion increases, see fig b. increasing drug dosage compensates the decreasing glucose dosage in order to keep mtor activities up-regulated leading to cell proliferation. on the contrary, if drug administration cost increases (increasing b values) with fixed glucose administration cost (b = . ), frequency of administration remains almost constant. this is depicted in fig c. the glucose dose per infusion is almost the same but the drug dose per infusion decreases with increasing b as illustrated in fig d. further, note that frequency of concomitant infusion control is always lower than that of alternating infusion control. in addition, drug (glucose) dose per infusion of concomitant control is always more (slightly less) than that of alternating control. for increasing glucose administration cost (increasing b ) with fixed drug administration cost (b = . ), total amount of glucose used for both control strategies generally decrease (except for high b > . values), as depicted in fig a, while total amount of drug increases, see fig b. on the other hand, when drug administration becomes expensive (increasing b strategies in regulating glioblastoma signaling pathways and anti-invasion therapy values) with fixed glucose administration cost (b = . ), the total amount of glucose dosage is almost constant (just slightly increasing for concomitant control) as shown in fig c, but the total drug amount is decreasing as can be seen in fig d. as illustrated in fig , concomitant control use less total amount of glucose than that of alternating control. contrarily, concomitant administration consumes more total drug amount as compared to that of alternating control (except possibly for high glucose administration cost, b > . , see fig b) . figs and reflect the relative cost per infusion and total administration cost of glucose and drug infusions incurred under concomitant and alternating controls. with fixed drug administration cost (b = . ), relative glucose cost per infusion and total administration cost increases as b increases. note that alternating control cost for glucose is always higher than concomitant infusions, see fig (a) and (b) . the relative and total administration cost for concomitant infusion slightly increase as compared to alternating control as b increases. again, alternating control incur more cost for higher glucose administration cost as illustrated in fig (c) and (d) . on the other hand, when b = . and drug administration cost (b ) increases, the relative glucose cost per infusion and total administration cost is almost constant. this can be seen in fig (a) and (b) . again, relative total cost for alternating control is higher than that of concomitant control. further, as b increases, relative drug cost per infusion and total administration cost decreases (fig (c) and (d) ), since total drug dosage also decreases as shown in fig d. in this case, it can be seen that drug administration cost for alternating control is generally lower than that of concomitant control. observe that in fig , both concomitant (blue) and alternating (orange) control trajectories in glucose-mtor-drug space are restricted in a smaller region avoiding aggressive invasion, rapid proliferation, and unwanted drug complications. both strategies achieved the goal of keeping mtor (mir- ) up-regulated inducing cell proliferation and thus avoiding aggressive cell migration. it is important to note that under these proposed optimal control infusions, glucose and drug levels are regulated to prevent excessive cell division and tumor growth. as a consequence, these strategies suggest safer infusion administration preventing hyperglycemia for diabetic patients and risk of drug complications. table provides the average frequency, dosage and relative cost of concomitant and alternating control strategies where b = b = . and simulation time is h ( d). the periodic switching behavior of glioblastoma cells between proliferation and invasion phases is highly influenced by fluctuating glucose levels [ , ] . in response to high glucose supply, mir- and mtor are up-regulated and ampk complex is down-regulated inducing cell growth. on the contrary, low glucose level up-regulates ampk complex, down-regulating mir- and mtor, promoting cell migration [ ] . the mutual antagonistic mechanism strategies in regulating glioblastoma signaling pathways and anti-invasion therapy between mir- (mtor) and ampk complex and the cell's strategic metabolic adaptation support the survival of cancer cells even in a nutrient-deprived microenvironment [ , ] . in addition to rapid proliferation of glioblastoma cells, aggressive invasion to the surrounding tissue is a major cause of treatment failure. despite advances in medical imaging technology such as mri and pet, glioblastoma cells can spread beyond detection leading to tumor recurrence within to cm of the resection cavity even after surgical removal of a malignant glioblastoma [ ] . these glioma cells are capable to deform cell membrane and nucleus for cell infiltration through a narrow gap between normal glial cells in brain tissue by upregulation of myosin ii along with actin bundles [ , ] . while exact migratory patterns are still poorly understood, these invasive glioma cells prefer white matter and blood vessels [ , ] with a wide range of speeds in the range of - μm/h [ ] [ ] [ ] [ ] showing sometimes saltatory patterns in the migration direction in brain [ ] . prediction of tumour invasion directions in nearby tissue may help define exact boundaries of focal treatments (surgery or radiosurgery), preventing future growth and recurrence [ ] . for example, medical doctors could determine specific locations for radiation target volumes, not just using a rough estimate of - cm in all directions as a guidance as commonly done today [ ] . assuming that migratory cells are localized near the surgery site [ ] , one possible approach is to keep the cells in its proliferative phase preventing them from invading brain tissue in a combination with transport of therapeutic drugs near blood vessels [ ] . as a result, the tumor strategies in regulating glioblastoma signaling pathways and anti-invasion therapy mass will be visible for a succeeding surgery while killing proliferative tumor cells at the blood sites. in this study, we considered the intracellular dynamics of the mir- -ampk-mtor-cell cycle signaling pathway model developed recently by kim et al. [ ] . incorporated in the model is a drug component which blocks the inhibitory pathway of mtor by ampk complex. this drug targets up-regulation of mtor activities enhancing cell proliferation. the focus of the current work is to regulate up-stream signaling pathway via glucose infusion activating strategies in regulating glioblastoma signaling pathways and anti-invasion therapy mir- , and control the down-stream pathway to cell cycle via drug infusion enhancing mtor activities. optimal control problem is formulated with the goal of keeping high levels of mir- and mtor to induce cell growth and reduce invasion to the surrounding tissues. in the framework of optimal control theory, two administration strategies are explored to achieve the goal with minimal cost incurred in glucose and drug administrations. the control strategies investigated in this study are ( ) concomitant infusion control and, and ( ) alternating glucose and drug infusion control. both strategies are able to switch on the proliferative mode of glioblastoma cells and turn off its migratory mode. cell cycle is regulated with fewer mass divisions restricting rapid growth. numerical results show that concomitant control had fewer infusions, lesser glucose dosage and cheaper administration cost. however, when glucose and drug poses unwanted chemical reactions during concurrent administration, alternating control would be beneficial with lower drug amount usage. the mathematical models of the mir- -ampk-mtor core control [ , [ ] [ ] [ ] ] are based on a 'go-or-grow' hypothesis and supporting experiments in gbm cells [ , , ] . however, the range of bistable behavior indicates a 'go-and-grow' program which has been proposed for other cancers too [ ] . in the glioma cases, 'go-or-grow' hypothesis has been long suggested [ , , [ ] [ ] [ ] [ ] in addition to mir- -induced 'go-or-grow' mechanism [ , , ] . glioma consists of a bulky, proliferative core in the center and highly invasive individual cells in the outer rim [ , , , ] and sequential transition between proliferative and invasive phenotypes characterizes tumor progression and may lead to faster growth [ ] . at least at the microscopic level, these migration/proliferation phenotypes appear to be mutually exclusive characteristics at different time frames [ ] , as suggested by in vivo imaging data of glioma cells migrating in a saltatory fashion [ ] . glioma cells was shown to pause for a short period of time (�an hour) for cell division before the daughter cells begin to move again [ ] . gal et al. [ ] further experimentally observed that this phenotypic switch can happen under different extracellular environment: (i) proliferative type in soft agar via activation of myc signaling pathways in response to hepatocyte growth factor (hgf); and (ii) infiltrative type in matrigel through ras signaling pathways in response to the same hgf. recently, dhruv et al. [ ] also affirmed the role of activation of key molecules in dichotomy between proliferation and invasion: c-myc and nfκb in the proliferative core and radially dispersed, invasive region of gbm tumors, respectively. glial cells can also interact with gbm tumor cells for phenotypic switch of gbm cells between cell migration and active proliferation [ ] . although these experiment and mathematical modeling support the 'go-or-grow' hypothesis, it is not certain if alternative mechanism such as 'go-andgrow' occurs in such a heterogenous tme in real patients [ ] . mansury et al. [ ] for instance illustrated that individual glioma cell in a mixed group of proliferative and invasive phenotypes can depend on genotype of counter part and tumor microenvironment. in a similar conceptual studies on epithelial-to-mesenchymal transition (emt) and mesenchymalto-epithelial transition (met) [ ] can give a hint on the complexities of this complex regulation of those two phenotypes and glioma in tme might not posses the simple 'go-or-grow' dogma. increasing number of evidences now suggest that the 'go-or-grow' model has similar molecular basis with emt [ ] . for instance, hgf and tgfβ are major regulators of emt, and these also provide strong stimuli of gbm invasion [ , ] and, upregulation of met and cd activities, as well as an activated nfκb signaling pathway, was reported in both mesenchymal gbm and metastatic cancer [ ] [ ] [ ] [ ] . all these experimental observations suggest that metastatic epithelial cancers and mesenchymal gbm drive common mechanisms that regulate the phenotypic transition between invasion and proliferation, suggesting the possibility of the 'go-and-grow' mechanism. the emt paradigm in gbm has not been extensively studied as relevant to the progression of the disease due to different origin and rare metastasis of glioma [ , ] . further studies and experimentation need to be done for better understanding of the fundamental principles. in this paper, we did not take into account key microenvironmental factors such as endogenous immune dynamics including nk cells [ ] and m /m macrophages [ ] , other major signaling networks [ , ] such as e f and myc [ , ] , angiogenesis [ , ] , biophysical interaction between tumor cells and blood vessels [ ] , ecm remodeling for therapy [ , [ ] [ ] [ ] , or growth factors [ , ] such as epidermal growth factors [ , , ] , fibroblast growth factors [ ] , transforming growth factor-β [ , ] , and csf- [ , , ] , that may play critical roles in proliferation, progress, aggressive invasion of gliomas and development of anticancer strategies [ ] . for example, endogenous nk cells may interfere oncolytic virus combination therapy in gbm while exogenous nk cells increase anti-tumor efficacy [ ] , illustrating the complex nature of tumor microenvironment. recently, mtor was considered to be a master regulator of cell growth and recognized as a good therapeutic target for therapies in glioblastoma [ ] . a recent study found that withaferin a and temozolomide can induce apoptosis and reduce drug resistance by g /m cell cycle arrest through intrinsic and extrinsic apoptotic signaling pathways [ ] . more detailed analysis and experiments on akt/mtor/pi k are necessary. interestingly, radiation was shown to indirectly promote the export of lactate into the extracellular space and inhibition of ampk/nfκb signaling pathways were involved in radiation-induced invasion of cancer cells [ ] . on the other hand, m microglia/macrophages induce matrix remodeling and glioma cell invasion [ , [ ] [ ] [ ] . since pi k signaling was shown to contribute to m -polarization of these tumor associated macrophages (tams) [ ] and pi k binding to csf r was shown to enhance spreading of macrophages, thus promoting glioma cell invasion [ ] . therefore, better understanding of these pi k-mtor-csf r signaling networks in macrophages would lead to development of blocking aggressive infiltration of cancer cells. signaling pathways of apoptosis and necroptosis are important parts of oncolytic virus (ovs) therapy [ , ] . tumor extracellular matrix (ecm) plays a significant role in regulation of glioma invasion in brain tissue as well as ov spread [ ] . for example, cspgs, one of major tumor ecm in brain can characterize the invasive and non-invasive gliomas in a complex tme where microglia and astrocytes mechanically interact with cspgs and tumor cells [ , [ ] [ ] [ ] . hybrid models [ , [ ] [ ] [ ] and its associated optimal control strategies can be adapted to take into account this important intracellular signaling as well as cell population dynamics and tissue dynamics. better understanding of various roles of these components in tumor microenvironment may provide better anti-invasion strategies of glioma cells. however, our mathematical model in this work is a first step toward further experimental/ clinical investigation and more optimal anti-invasion strategies of gbm by incorporating these microenvironmental components. we will address these issues in future work. supporting information s appendix. optimal control problem. formulation of the optimal control problem for concomitant and alternating glucose and drug infusion. 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bortezomib-induced unfolded protein response increases oncolytic hsv- replication resulting in synergistic antitumor effects bortezomib treatment sensitizes oncolytic hsv- treated tumors to nk cell immunotherapy choindroitinase abc i-mediated enhancement of oncolytic virus spread and anti-tumor efficacy: a mathematical model chondroitin sulfate proteoglycans potently inhibit invasion and serve as a central organizer of the brain tumor microenvironment contributions of chondroitin sulfate proteoglycans to neurodevelopment, injury, and cancer glycosaminoglycans and glioma invasion a hybrid model for tumor spheroid growth in vitro i: theoretical development and early results multiscale models of cell and tissue dynamics the role of the microenvironment in tumor growth and invasion this work is resulted from the konkuk university research support program (e.j.). this paper is also supported by the korea national research foundation (nrf) grant funded by the korean government (mest): nrf- r a b (e.j.) and the basic science research key: cord- -snruk j authors: schmidt, julius j.; lueck, catherina; ziesing, stefan; stoll, matthias; haller, hermann; gottlieb, jens; eder, matthias; welte, tobias; hoeper, marius m.; scherag, andré; david, sascha title: clinical course, treatment and outcome of pneumocystis pneumonia in immunocompromised adults: a retrospective analysis over years date: - - journal: crit care doi: . /s - - - sha: doc_id: cord_uid: snruk j background: despite modern intensive care with standardized strategies against acute respiratory distress syndrome (ards), pneumocystis pneumonia (pcp) remains a life-threatening disease with a high mortality rate. here, we analyzed a large mixed cohort of immunocompromised patients with pcp, with regard to clinical course and treatment, and aimed at identifying predictors of outcome. methods: this was a single-center retrospective analysis in a tertiary care institution across years. diagnosis of pcp required typical clinical features and microbiological confirmation of pneumocystis jirovecii. epidemiological, clinical, laboratory and outcome data were collected from patient records. results: a total of , specimens from patients were sent for microbiological assessment ( with clinical suspicion of pneumocystis pneumonia). pcp was confirmed in patients, about half of them hiv positive ( %). the remaining subjects were either solid organ transplant recipients ( . %) or suffered from malignancy ( . %) or autoimmune diseases ( . %). of note, % of patients with pcp were not receiving chemoprophylaxis. overall in-hospital mortality was . %, increasing to % if icu admission was required. multivariable regression identified lactate dehydrogenase (ldh) as predictor of in-hospital mortality (adjusted or . ( % ci . – . ), p < . ). mortality in ldh quartiles increased from % to %, and a cutoff value of u/l predicted mortality with sensitivity and specificity of %. with regard to treatment, % of patients received trimethoprim-sulfamethoxazole at doses that were lower than recommended, and these patients had a higher mortality risk (hr . ( % ci . – . ), p = . ). conclusions: pcp remains a life-threatening disease among immunocompromised patients. about half of patients with pcp do not have hiv infection. initial ldh values might serve as a stratifying tool to identify those patients at high risk of death among patients with hiv and without hiv infection. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. pneumocystis pneumonia (pcp) is a severe disease with high morbidity and mortality, which almost exclusively affects immunocompromised patients. pcp has long been known for its high prevalence among human immunodeficiency virus (hiv)-positive patients [ ] . since the implementation of combination antiretroviral therapies (cart) and chemoprophylaxis its incidence has been continuously decreasing [ ] . nowadays, most patients with hiv-associated pcp are treatment-naïve with very low cd cell counts; some of these patients do not know that they are hiv positive until they attend hospital [ ] . on the other hand, pcp is also frequently diagnosed in non-hiv-positive patients as immunosuppressive regimens are being increasingly used in a wide range of patient populations. consistently, the incidence of pcp in non-hiv-positive patients is increasing [ ] . pneumocystis infections have first been described in preterm infants following world war ii [ ] and in patients with malignancies in the late s [ ] , more than a decade before the hiv epidemic emerged. defects in cell-mediated immunity and use of glucocorticoids are among the strongest risk factors for the development of pcp [ , ] . the epidemiology of pcp has been described in several retrospective studies [ , ] . a study from the mayo clinic in non-hiv-positive patients found that most frequent underlying diseases were hematological malignancies ( %), organ transplantation ( %), autoimmune disease ( %), solid tumors ( %) and other reasons [ ] . although, most of these reports were published more than a decade ago and might not reflect the current epidemiological situation, a report from france was published a few years ago [ ] . roux et al. reported overall mortality of . %, which was significantly higher in non-hiv-positive patients ( %) than in hiv-positive patients ( %) [ ] . a few parameters such as age, prior episode of pcp, low cd cell count, lactate dehydrogenase (ldh), and coinfections have been reported to predict unfavorable outcome in patients with hiv infection [ ] [ ] [ ] . reports on outcome predictors in non-hiv-positive patients are sparse. based on the high burden of pcp and the likelihood of unfavorable outcome particularly in non-hiv-positive patients, chemoprophylaxis with trimethoprim-sulfame thoxazole (tmp-smx) is recommended in high-risk populations [ ] . tmp-smx is also the treatment of choice for pcp. adjunctive corticosteroid therapy is recommended in hiv-positive patients with severe respiratory failure [ , ] . however, while beneficial outcomes of higher dosage of corticosteroids in hiv-positive patients during pcp are reported [ ] , the outcome of using corticosteroids in non-hiv-positive patients is not clear [ ] . we here report comprehensive epidemiological, clinical, laboratory, therapeutic and outcome data on cases of pcp, including a high percentage of non-hiv-positive patients, in a tertiary care center over the last years. additionally, we aimed at identifying predictors of outcome. we performed a retrospective, single-center cross-sectional analysis of all patients with a positive finding of pneumocystis jirovecii on direct immunofluorescence testing or detection by diff-quick staining in the bronchial washing fluid or broncho-alveolar lavage (bal) fluid, from january to june . our hospital is a university tertiary care center with approximately beds. written informed consent was waived by the ethics committee due to the anonymized retrospective nature of the analysis. all bronchial washing fluids or bal samples from patients clinically suspected to have p. jirovecii pneumonia were evaluated within the study period. for every patient, clinical data on demographic characteristics, underlying disease, status of immune competence, treatment regimens of immunosuppression, pcp therapy regimen and mortality, were gathered in the study database. date of diagnosis was defined as the date of microbiological confirmation. p. jirovecii direct immunofluorescence was performed using the monofluo kit p. carinii (biorad laboratories, years to ) or the detect if pneumocystis carinii kit (axis-shield diagnostics ltd., year ). diff quick staining was performed using the stain sets provided by dade behring or siemens ag, respectively. standard descriptive statistics were used to summarize the data (e.g. continuous: mean ± standard deviation/ count: absolute and relative frequencies/time-to-event: kaplan-meier estimator). to identify predictors of in-hospital mortality or survival we applied logistic and cox proportional hazards models. consequently, both odds ratio (or) and hazard ratio (hr) estimates were reported. to obtain multivariable adjusted estimates, all main effects with univariate p values less than or equal to . were investigated simultaneously. in addition, confidence intervals (ci) were calculated with coverage of %. finally, we also created receiver operating characteristics (roc) curves for in-hospital death, which were summarized by area under the curve (auc) estimates. roc analyses were based on an increasingly complex (leave one out) logistic regression model with in-hospital mortality as the predicted outcome and the following predictors, which were all entered linearly: ldh alone, ldh + age, ldh + age + body mass index (bmi), and for ldh + age + bmi + estimated glomerular filtration rate (egfr). all reported p values are nominal and two-sided. in this explorative study, we applied a significance level of α = . (two-sided). all statistical analyses were done using graphpad prism . (la jolla, ca, usa) or r . . . during the observation period, , bal or bronchial washing fluid samples were investigated. a clinical suspicion of p. jirovecii infection was raised in of these specimens of which (i.e. . %) were microbiologically confirmed by immunofluorescence as positive for pcp. there were ultimately patients enrolled into the study ( were excluded due to incomplete data sets) (fig. ). in the overall population, patients were female ( . %) and the average age was ± years. there were patients ( %) with hiv infections; ( . %) had undergone solid organ transplant and ( . %) had undergone chemotherapy due to hematologic or oncologic malignancies: patients ( . %) received immunosuppressive therapy for rheumatoid autoimmune diseases (is/rd) and patients ( . %) had other underlying diseases (miscellaneous (misc)), e. g. common variable immune-deficiency (cvid) ( table ) . five patients could be classified into multiple categories. after case reevaluation, patients were classified to the most recent active disease prior to the pcp event. the average cumulative incidence of pcp in our institution was ± cases per year, with a peak in the years - (additional file : figure s a ). of the patients with pcp, . % were admitted to the intensive care unit (icu), with . % in need of mechanical ventilation, . % in need of renal replacement therapy (rrt), and . % in need of extracorporeal membrane oxygenation (ecmo) support (fig. a) . the overall in-hospital mortality was . %. the mortality was % in patients requiring icu treatment, . % in patients requiring rrt and . % in patients requiring ecmo support, and only . % of patients died on regular (non-icu) wards (fig. b) . mortality during the observation time from to slightly fluctuated but did not trend toward an improvement in more recent years (additional file : figure s b ). the underlying disease was associated with outcome ( table ). the lowest mortality was observed in hiv-infected patients ( . %); the respective mortality rates in the non-hiv group were . % in solid organ transplant recipients, . % in patients with rheumatic diseases and . % in patients with hematologic -oncologic diseases, respectively (additional file : figure s c ). figure c , d summarizes icu admission and icu mortality with regard to the etiology of the patients' immunosuppressive disease. of note, only patients ( %) were receiving chemoprophylaxis with tmp-smx at the time when pcp was diagnosed ( table ) . univariate regression analysis revealed that age, bmi, gfr and initial ldh were associated with death from pcp. both logistic and cox multivariable regression analyses identified ldh as a predictor of unfavorable outcome in pcp (table , adjusted or . ( % ci . - . ) per u/l, p < . , adjusted hr . ( % ci . - . ) per u/l, p < . ). the initial ldh levels were significantly higher in later non-survivors ( ± vs. ± iu/l, p < . , fig. a ) and in patients that required admission to the icu ( ± vs. ± iu/l, p < . , fig. b ). stratification of patients into ldh quartiles showed increasing (in-hospital) mortality rates among those. mortality ranged between % in the lowest and % in the highest ldh quartile (fig. c) . a roc curve for in-hospital death had an estimated auc of . ( % ci . - . ) (p < . for ldh alone, additional file : figure s ). potential alternative cutoff values and their impact on usually reported statistics are summarized in additional file : table s for descriptive purposes. we used a cutoff of iu/l for further analysis. patients with initial ldh below iu/l had lower mortality than the overall population ( . vs. . %) and lower mortality than those with initial ldh > iu/l ( . vs. . %, p < . , fig. d, e) . the addition of other clinical quantitative variables to ldh in the auc model such as age, bmi and egfr did not improve the predictive value of ldh alone (additional file : figure s , additional file : table s ) in the overall cohort. however, when icu patients were analyzed separately, we observed that the addition of age as a variable increased the predictive performance of ldh alone from an auc of . ( % ci . - . ) to an auc of . ( % ci . - . ) (p = . ). of note, we did not detect any etiology subgroup that showed better performance in the roc analysis (additional file : figure s ), nor did we detect temporal trends when applying the ldh prediction models in three strata ( ) ( ) ( ) ( ) ( ) ( ) - , - ) (additional file : figure s ). fig. influence of initial lactate dehydrogenase (ldh) as an outcome predictor. the scatter plot shows initial ldh levels at admission in later survivors (alive) and non-survivors (dead) ( ± vs. ± iu/l, p < . ) (a) and in non-icu versus icu patients ( ± vs. ± iu/l, p < . ) (b). c bar graph showing overall mortality (right y-axis) stratified for initial ldh quartiles (left y-axis) (q , < ; q , - ; q , - ; q , > iu/l). d mortality in all patients with pneumocystis pneumonia with ldh levels ≤ and > iu/l (cutoff value was derived from roc curve of all patients, with sensitivity and specificity of %.) the gray area highlights the overall mortality of . % without ldh risk stratification. e kaplan-meier analysis of survival in patients stratified for ldh ≤ and > iu/l during days (p log-rank test < . ) there were / patients ( . %) initially treated with the gold standard, tmp-smx. the remaining patients had alternative regimens with chemotherapeutics from the second line, mostly due to side effects and intolerance of tmp/smx (e.g. atovaquone and/or pentamidine, fig. ) . among the patients treated with tmp-smx, ( %) were dosed in the recommended range above mg/kg bodyweight (bw) per day, while patients ( %) received a dose lower than mg/kg bw (additional file : figure s a ). of note, these patients had a higher mortality risk (hr . ( % ci . - . ), p = . ). solid organ transplant (sot) recipients and patients with rheumatoid diseases were more likely to receive lower dosages (additional file : figure s b ). on the other side, we observed that under-dosed patients also had significantly lower egfr ( . ± ml/min vs. ± ml/min, p < . ). interestingly, the absolute dose of tmp-smx was not different in survivors ( . ± . mg/kg) and non-survivors ( . ± . mg/kg, p = . ). still, kaplan-meier survival curves revealed a significant difference when patients were grouped into those treated with < mg/kg and those treated with ≥ mg/kg tmp-smx (additional file : figure s c , p log-rang test = . ). however, tmp-smx dose did not independently predict outcome in the multivariable regression analyses. here we present the single-center experience with regard to epidemiology, treatment and outcome of pcp in a tertiary care center over a period of years. a substantial group of our pcp cases (about %) were not related to hiv infections and these patients had a worse clinical course with higher icu admission and mortality rates than patients with hiv-associated pcp. three major non-hiv-positive groups were identified: ( ) solid organ transplant recipients, ( ) patients with malignancies and ( ) patients with rheumatic diseases. pcp occurred almost exclusively in patients who did not receive chemoprophylaxis with tmp-smx. overall, about one quarter of all patients suffering from pcp did not survive the disease. in those who required intensive care (approximately % of all patients), the in-hospital mortality increased up to %. based on the -in principle -reversible nature of pcp, almost no patients were denied intensive care in the event of acute respiratory failure, as the in-hospital mortality rate in non-icu patients was . %. moreover, we found that the extent of initial ldh elevation was a predictor of in-hospital mortality, and that in univariate comparisons, in-hospital mortality was higher in patients whose tmp-smx dose was below mg/kg bw. non-hiv-positive populations at risk of pcp have been described before. the distribution across these risk groups varies widely (sot - %, hematologic diseases - %, rheumatic diseases - %), which is likely due to the clinical emphasis in predominantly monocentric studies [ , [ ] [ ] [ ] . several centers have reported a decrease in the percentage of hiv-infected patients among all cases of pcp in the era of cart [ , ] . we did not confirm this decrease, nor did fillatre et al. [ ] . our data showed that the clinical course and outcome of pcp differed between hiv-infected and non-hiv-infected patients. in general, hiv-infected patients require fewer icu admissions and have better overall survival than non-hiv-infected patients, which is in line with previous reports [ , ] . the pcp mortality rate in hiv-positive patients was in the range of - % [ , , ] compared to - % in hiv-negative patients [ ] [ ] [ ] . the better outcome of hiv-positive patients might be the consequence of ( ) lower age (− . years in our cohort), ( ) fewer co-morbidities, ( ) the reversible nature of the immune defect upon anti-viral treatment strategies and hypothetically ( ) greater awareness of pcp in hiv-positive individuals, where it is the most common aids-defining disease. in mixed cohorts, icu admission and mechanical ventilation occurred in - % and - % of patients, respectively [ , , ] . both events are frequently reported as negative predictors of survival. nevertheless, newer data on icu mortality in patients with pcp are scarce. only one recent study reported relatively high icu mortality in hiv-negative patients of % compared to % in hiv-positive patients [ ] . mortality in patients with invasive mechanical ventilation was % and %, respectively, in these two groups [ ] . compared to other reports [ , ] , we had a relatively high icu admission rate of % in hiv-positive patients, which can in part be explained by the selective transfer of more patients with more progressive disease to our university hospital as a tertiary center. the literature is inconclusive on outcome predictors, but on multivariable analysis, a large prospective study from france demonstrated a significant survival benefit in younger patients [ ] . although observed in hiv-positive patients before [ ] , here we report for the first time that in a mixed population of hiv-positive and non-hiv-positive patients the initial levels of serum ldh at hospital admission are not only useful in the diagnostic evaluation [ ] but may also be an independent predictor of survival. this might potentially help the clinician in early identification of the sickest patients at high risk of unfavorable outcome. with regard to the specificity/sensitivity, the ideal ldh cutoff value to predict outcome has yet to be defined. with regard to the actual treatment of pcp we found that a reduction in the recommended tmp-smx dose below mg/kg bw (for whatever reason) might be associated with higher mortality ( . vs. . %). given that this observation was not confirmed in the multivariable analysis, relevant confounders must be considered. our study has several limitations. this was a retrospective observational study based on the medical records of patients with pcp from a single institution. consequently, causal claims cannot be made. the observation that under-dosing with tmp-smx is associated with higher mortality might be confounded by a higher percentage of patients with renal impairment. more robust evidence from well-designed studies is needed to make firm conclusions or even to make implications about changes in standard pcp management. in summary, pcp is a rare but potentially fatal disease in immunocompromised patients with diseases of different etiology. about % of cases were non-hiv-associated. initial ldh levels -if validated by others -might be a useful predictor of in-hospital mortality, and tmp-smx treatment doses in patients at high risk of death (e.g. icu admission + ldh > u/l) should probably not be reduced below mg/kg bw. additional file : figure s . cumulative incidence of (a) and inhospital mortality in (b) pneumocystis pneumonia (pcp) at hannover medical school from to . figure s . receiver operating characteristic (roc) curves for in-hospital mortality applied to the total sample. figure s . roc curves for in-hospital mortality applied to subgroups regarding underlying etiology of immunosuppression and for the icu cohort. figure s . roc curves for in-hospital mortality with the ldh prediction model applied in three strata (years - , - , - ) . figure s . association between trimethoprimsulfamethoxazole (tmp-smx) dose and mortality (docx kb) additional file : table s . suggested, alternative data-derived ldh cutoff values and their impact on usually reported statistics (with % confidence interval in parenthesis) related to the prediction of in-hospital mortality in patients with pcp. table s . estimated aucs (with % confidence interval) of the ldh-related logistic regression models for the prediction of in-hospital mortality in patients with pcp. table s . additional descriptive information (with % confidence interval in parenthesis) on patient characteristics and their potential value as predictors of in-hospital mortality in patients with pcp. table s . pneumonia in the acquired immune deficiency syndrome opportunistic infections in patients with and patients without acquired immunodeficiency syndrome severity and outcomes of 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factors of pneumocystis pneumonia in solid organ recipients in the era of the common use of posttransplantation prophylaxis a multicenter randomized double-blind placebo-controlled trial of adjunctive corticosteroids in the treatment of pneumocystis carinii pneumonia complicating the acquired immune deficiency syndrome acute respiratory failure secondary to pneumocystis carinii pneumonia in the acquired immunodeficiency syndrome. a potential role for systemic corticosteroids adjunctive corticosteroids for pneumocystis jiroveci pneumonia in patients with hiv infection. cochrane hiv/aids group use of adjunctive corticosteroids in severe adult non-hiv pneumocystis carinii pneumonia incidence of pneumocystis jiroveci pneumonia among groups at risk in hiv-negative patients pneumocystis pneumonia suspected cases in non-hiv and hiv patients infectious disease ward admission positively influences p. jiroveci pneumonia (pjp) outcome: a retrospective analysis of hiv-positive and hiv-negative immunocompromised patients population-based analysis of invasive fungal infections pneumocystis pneumonia in hiv-infected and immunocompromised non-hiv infected patients: a retrospective study of two centers in china outcomes and prognostic factors of non-hiv patients with pneumocystis jirovecii pneumonia and pulmonary cmv co-infection: a retrospective cohort study predisposing factors, clinical characteristics and outcome of pneumonocystis jirovecii pneumonia in hiv-negative patients analysis of underlying diseases and prognosis factors associated with pneumocystis carinii pneumonia in immunocompromised hiv-negative patients intensive care of patients with hiv infection: utilization, critical illnesses, and outcomes. pulmonary complications of hiv infection study group plasma il- /il- ratio and il- , ldh, and hbdh level predict the severity and the risk of death in aids patients with pneumocystis pneumonia utility of lactate dehydrogenase vs radiographic severity in the differential diagnosis of pneumocystis carinii pneumonia no external funding was received to conduct this study. however, dr david's laboratory is supported by a grant from the german research foundation (da / - ). the datasets used and analyzed during the current study are available from the corresponding author on reasonable request. carl-neuberg-strasse , hannover, germany. authors' contributions jjs, cl, sd and sz collected the data. as performed the statistical analysis. experts in particular field were involved for specific data interpretation (ms for hiv; hh, jg, tb, mmh for transplantation; me for malignancies). jjs, cl, mmh, as and sd wrote the manuscript and all authors read, commented on and approved it.ethics approval and consent to participate written informed consent was waived by the local ethics committee at hannover medical school due to the anonymized retrospective nature of the analysis. not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -vj t hn authors: joffe, michael; wagner, simon d.; tang, julian w. title: case report: a fatal case of disseminated adenovirus infection in a non-transplant adult haematology patient date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: vj t hn background: we report a fatal case of disseminated adenovirus infection in a non-transplant haematology adult patient with chronic lymphocytic leukaemia who had completed combination chemoimmunotherapy a few months before developing respiratory symptoms. in such non-transplant patients, monitoring for adenovirus in the blood is not routine. however, with adenoviruses, when there is a more peripheral (i.e. non-blood) site of infection such as the chest, serial adenovirus monitoring in blood for the duration of that illness may be warranted. case presentation: this case started with an initial bacterial chest infection that responded to treatment, followed by an adenovirus pneumonitis that disseminated to his blood a week later with levels of up to million adenovirus dna copies/ml. despite prompt treatment with cidofovir, his respiratory function continued to deteriorate over the next two weeks and he was moved to intensive care. intravenous immunoglobulin and ribavirin were subsequently added to his treatment. however, he died soon after this with a final adenovirus load of million copies/ml in his blood. conclusions: we recommend that even in non-transplant haematology patients, where such patients present with an acute respiratory adenovirus infection, teams should consider checking the blood for adenovirus to check for signs of disseminated infection. the earlier this can be tested, the earlier treatment can be initiated (if adenovirus positive), which may produce more successful clinical outcomes. human adenoviruses are non-enveloped, doublestranded dna viruses. they exist as seven species (a-g) with greater than types identified so far that can affect different organs and cause a wide spectrum of disease in both immunocompetent [ ] [ ] [ ] [ ] and immunocompromised [ , [ ] [ ] [ ] [ ] individuals, including pneumonitis, hepatitis, gastroenteritis and conjunctivitis [ ] . although adenoviruses are important respiratory pathogens in haematology patients, adenovirus surveillance in blood is not normally performed in non-transplant adult patients. we report here a case of adenovirus pneumonitis which led to a fatal disseminated adenovirus infection in an adult patient with chronic lymphocytic leukaemia (cll) on chemotherapy. the patient was a -year old man with cll, who presented with persistent cough and coryzal symptoms in early . he had a past medical history of epilepsy and rheumatic fever. he had never smoked. cll was first diagnosed in late . the leukaemia progressed and he underwent six cycles of chemotherapy with the fcr combination (fludarabine mg/m , cyclophosphamide mg/m and rituximab mg/m ) over the next months completing the treatment in june . bone marrow and clinical findings showed a complete response to treatment. after his chemoimmunotherapy he continued on acyclovir ( mg bd) and co-trimoxazole ( mg bd, times a week) prophylaxis, both of which he was still taking at the time of presentation. this was in response to a t-cell lymphopenia that persisted up to his hospital admission in july (cd + t helper cells . × [ ]/l). in april he presented with a -week history of a non-productive cough and rhinorrhoea. there were no clinical or laboratory features of cll at that time but serum immunoglobulins were suppressed (igg . g/l (normal range - g/l) and cd + t-cells were low at . × [ ]/l (normal range . to . × [ ]/l). although there were transient improvements, productive cough persisted despite multiple courses of antibiotics (azithromycin mg od, co-amoxiclav mg tds, doxycycline mg od) and a course of oral prednisolone ( mg od). a ct scan of his sinuses showed right maxillary sinus change consistent with chronic sinusitis, and he was subsequently referred to ear nose and throat specialists for further management. however, the radiological sinus changes were not felt to be significant and no specific treatment was initiated. the patient's symptoms worsened and in the june a ct scan of the chest showed centrilobular nodular changes, right-sided patchy consolidation with surrounding ground glass opacity with halo sign. this raised the possibility of atypical infection including fungal pathogens and the patient was subsequently prescribed voriconazole mg bd. he also underwent a bronchoscopy later the same month, from which a broncho-alveolar lavage (bal) sample cultured haemophilus influenzae, but was negative for other pathogens including fungi. no viral screen was carried out on this sample. in response to this, he was given a prolonged course of co-amoxiclav ( mg tds). he showed subsequent clinical improvement on this, together with the voriconazole. one month later (july ), however, he was readmitted with a marked deterioration in the productive cough and shortness of breath on exertion. bilateral crepitations were heard on examination, which was consistent with a chest x-ray showing bilateral, patchy consolidation. c-reactive protein was mg/l. he was started on intravenous (iv) tazocin ( . g tds) and clarithromycin ( mg bd). although there were signs of improvement over the course of the week his symptoms persisted. a repeat ct scan on this readmission showed ground glass changes, tree-in-bud appearance, and nodular changes all of which had progressed from the previous imaging. he underwent another bronchoscopy examination, and was started on a treatment dose of cotrimoxazole ( mg/kg, daily in divided doses) and caspofungin ( mg od). in addition his serum immunoglobulins, which had remained low since his chemotherapy demonstrated pan-hypogammaglobulinaemia, and intravenous immunoglobulin (ivig) . g/kg was administered. this second bal was positive for adenovirus dna by pcr testing, using an in-house respiratory multiplex pcr screening assay, as described elsewhere [ ] . a beta-d-glucan test on the bal was also positive at pg/ml, having been negative in the peripheral blood. fungal, bacterial, pneumocystis and tuberculosis screens were all negative. despite this, a left mid-zone consolidation persisted on chest imaging. at this point he was diagnosed with adenovirus pneumonitis. one week later the patient remained symptomatic with persistent fevers, and peripheral blood was sent for adenovirus pcr, with a result of . million copies/ml. the qualitative and quantitative adenovirus pcr testing on this blood sample were performed at a commercial laboratory (micropathology ltd., coventry, uk). four days later this adenovirus level had risen to . million copies/ml (adenovirus type c , based on viral sequencing and analysis performed by micropathology ltd., coventry, uk). based on these results iv cidofovir ( mg/kg weekly) treatment was given. four days later the blood adenovirus dna levels had increased to million copies/ml. over the course of the following two weeks the patient deteriorated in terms of respiratory function, requiring transfer to intensive care for ventilatory support. his liver function also deteriorated during this time (bilirubin μmol, alkaline phosphatase iu/l. despite additional measures including further dosing with ivig ( . g/kg), and the addition of ribavirin (iv mg/kg), he died as a result of disseminated adenovirus infection and multi-organ failure. the last adenovirus dna level, three days before death was million copies/ml. no post-mortem investigations were performed. thus, the disseminated adenovirus infection was deemed to be the cause of the patient's multi-organ failure and death on the basis of the high levels of viremia, which coincided with the patient's rapid deterioration, as described in other cases [ ] [ ] [ ] [ ] . there are several well-known risk factors for severe adenovirus infections, including: allogeneic stem cell (or solid-organ) transplantation, particularly with t-cell depletion; treatment with anti-cd monoclonal antibody (alemtuzumab or campath) or anti-thymocyte globulin (atg); severe immunosuppression used to treat graft-versus-host disease; and any other cause of severe lymphopaenia that reduces the ability of the host's cell-mediated immunity to defend against adenovirus infection [ ] . this patient's chemotherapy regimen included fludarabine which has severe lymphopaenia as a recognised adverse effect, and which has been present in treatment regimens where various other viral reactivations have occurred, including hepatitis b [ ] [ ] [ ] , bk virus [ ] , herpes simplex and epstein-barr viruses [ ] , cytomegalovirus [ ] , as well as adenovirus [ ] . yet in this case, it was noted that throughout this period during which he acquired and was infected with adenovirus, his total lymphocyte count remained within or even above the normal range of - × [ ]/l, though their specific functionality was not tested. although fatal adenovirus infection has been reported in non-transplant paediatric and adult patients on chemotherapy [ ] [ ] [ ] [ ] , there is still no consensus on how to deal with an isolated adenovirus positive result in a peripheral (non-blood) sample type on a routine basismany such patients also have asymptomatic adenovirus infections. from a virological viewpoint, since all systemic antiviral drugs are only virostatic and not virucidal, earlier adenovirus pcr blood testing allowing earlier treatment to prevent further increases in viral load, may improve clinical outcomes [ ] . however, due to the severe nephrotoxic nature of the mainstay treatment, intravenous cidofovir, most transplant teams are reluctant to prescribe this drug empirically, unless a definitive upward trend is seen in the adenovirus blood levels. this is often the cause of delays in commencing therapy with this drug for disseminated adenovirus infection. thus, in such immunocompromised patients where a peripheral site (e.g. a non-blood sample, such as a respiratory or stool sample) has had an adenovirus pcr positive result, we would recommend that serial (once or twice weekly) monitoring for adenovirus pcr testing be performed for the duration of that specific adv illness, to check for possible disseminated adenovirus infection, earlier. such a test in this context is inexpensive and would allow earlier detection of disseminated adenovirus infection. this in turn then allows treatment to be commenced earlier, which could have a significant, positive clinical impact. finally in such immunocompromised patients, all pathogens: bacteria, fungi and viruses, should be screened for in the initial investigation of an acute infective episode, to allow prompt intervention as required. adenovirus infections in immunocompetent and immunocompromised patients epidemic of adenovirus-induced respiratory illness among us military recruits: epidemiologic and immunologic risk factors in healthy, young adults large epidemic of adenovirus type infection among military trainees: epidemiological, clinical, and laboratory studies outbreak of severe respiratory disease associated with emergent human adenovirus serotype at a us air force training facility in fatal adenovirus hepatitis during standard chemotherapy for childhood acute lymphoblastic leukemia fatal adenovirus hepatitis during maintenance therapy for childhood acute lymphoblastic leukemia disseminated adenovirus infection in cancer patients presenting with focal pulmonary consolidation fatal adenoviral and enteroviral infections and an epstein-barr virus positive large b-cell lymphoma after alemtuzumab treatment in a patient with refractory sézary syndrome resource impact of managing suspected middle east respiratory syndrome patients in a uk teaching hospital hepatitis b virus reactivation after fludarabine-based regimens for indolent non-hodgkin's lymphomas: high prevalence of acquired viral genomic mutations hbv reactivation after fludarabine chemotherapy identified on investigation of suspected transfusion-transmitted hepatitis b virus hepatitis b reactivation in a hbsag-negative, hbcab-positive patient receiving fludarabine for the treatment of chronic lymphocytic leukaemia symptomatic bk virus reactivation following fludarabine, cyclophosphamide and rituximab chemotherapy for chronic lymphocytic leukemia/small lymphocytic lymphoma herpes simplex and epstein-barr virus lymphadenitis in a patient with chronic lymphocytic leukemia treated with fludarabine cytomegalovirus oesophagitis following treatment with fludarabine for refractory lymphoplasmacytic lymphoma fulminant hepatitis due to human adenovirus no funding was required for the writing of this case report availability of data and materials any data (suitably anonymised to maintain patient confidentiality) is available for readers to review if a suitable written request to the corresponding author is made. authors' contributions mj, swparticipated in the clinical care of the patient on the ward. jwtadvised and supervised the laboratory testing, interpretation and reporting. mjwrote the first draft of the paper, which was edited for style and accuracy by jwt and sw. all authors critically reviewed the manuscript for publication. all authors have read and approved the final version of this manuscript.ethics approval and consent to participate not applicable consent for publication written consent to publish this case report and any accompanying images was obtained from the patient's next of kin. jwt is one of the virology section editors for the journal. the other authors declare that they have no competing interests.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -abs rvjk authors: liu, ming; kong, jian-qiang title: the enzymatic biosynthesis of acylated steroidal glycosides and their cytotoxic activity date: - - journal: acta pharm sin b doi: . /j.apsb. . . sha: doc_id: cord_uid: abs rvjk herein we describe the discovery and functional characterization of a steroidal glycosyltransferase (sgt) from ornithogalum saundersiae and a steroidal glycoside acyltransferase (sga) from escherichia coli and their application in the biosynthesis of acylated steroidal glycosides (asgs). initially, an sgt gene, designated as ossgt , was isolated from o. saundersiae. ossgt -containing cell free extract was then used as the biocatalyst to react with structurally diverse drug-like compounds. the recombinant ossgt was shown to be active against both β- and β-hydroxyl steroids. unexpectedly, in an effort to identify ossgt , we found the bacteria laca gene in lac operon actually encoded an sga, specifically catalyzing the acetylations of sugar moieties of steroid β-glucosides. finally, a novel enzymatic two-step synthesis of two asgs, acetylated testosterone- -o-β-glucosides (at- β-gs) and acetylated estradiol- -o-β-glucosides (ae- β-gs), from the abundantly available free steroids using ossgt and ecsga as the biocatalysts was developed. the two-step process is characterized by ecsga -catalyzed regioselective acylations of all hydroxyl groups on the sugar unit of unprotected steroidal glycosides (sgs) in the late stage, thereby significantly streamlining the synthetic route towards asgs and thus forming four monoacylates. the improved cytotoxic activities of ′-acetylated testosterone -o-β-glucoside towards seven human tumor cell lines were thus observable. steroidal glycosides (sgs) are characterized by a steroidal skeleton glycosidically linked to sugar moieties, which can be further acylated with aliphatic and aromatic acids thus forming complex acylated steroidal glycosides (asgs) . the resulting steroidal glycoside esters (sges) exhibit a wide variety of biological activities, like cholesterol-lowering effect , anti-diabetic properties , anti-complementary activity , immunoregulatory functions , and anti-cancer actions [ ] [ ] [ ] , which made asgs promising compounds with pharmaceutical potential. numerous methods, including direct extraction , chemical synthesis , and biosynthesis , have been developed to synthesize these acylated steroidal glycosides. direct extraction from varied organisms is one of the main methods to obtain asgs [ ] [ ] [ ] . however, the content of asgs was usually low in natural sources [ ] [ ] [ ] . moreover, the extraction routes were highly time-consuming and required laborious purification procedures [ ] [ ] [ ] , resulting in poor yields and/or low purity of the final products. the production of asgs was also achieved by chemical synthesis previously [ ] [ ] [ ] [ ] [ ] . however, these efforts often encounter a fundamental challenge, namely, regioselective acylation of single hydroxyl group of unprotected sgs in the late stage of the chemical synthesis of asgs. sgs generally possess multiple hydroxyl groups with similar reactivity. regioselective acylation of a particular one of multiple hydroxyl groups generally requires multi-step protection/deprotection procedures, which makes the synthetic pathway of these sges costly, wasteful, long and timeconsuming, and results in low yield in the end. the biosynthesis of asgs from free steroids based on enzymatic catalysis was deemed to reduce the number of protection/ deprotection steps due to the high selectivity of enzymes. theoretically, the biosynthesis of asgs includes two steps. in the first reaction, the sugar moiety from nucleotide-activated glycosyl donors was attached to steroids at different positions, most commonly at the c- hydroxyl group (oh), under the action of nucleotide dependent sgts , . the glycosylation of a hydroxyl group at the c- position of steroids was well characterized and a few of steroidal β-glucosyltransferases were isolated from diverse species , . however, the reports of sgts specific for positions other than c- of steroids are limited. the sugar moieties of the resultant sgs can further be acylated by sgas to form asgs in the next step , . compared to sgts, surprisingly little is known about sgas. up to date, no sga genes has yet been cloned, which in turn limit the enzyme-mediated biosynthesis of asgs. hence, the successful gene isolation and functional characterization of sgas have become a prominent challenge for bioproduction of asgs. herein, the functional characterizations of a plant-derived sgt with activity against both β-and β-hydroxyl steroids, and a bacterial sga, as well as their application in the biosynthesis of asgs are reported. initially, a steroidal glycosyltransferase ossgt was isolated from medicinal plant o. saundersiae and showed activities for β-and βhydroxyl steroids. unexpectedly, in an effort to identify the function of ossgt , we characterized laca protein (designated as essga ) from e. coli as a sga, catalyzing testosterone- -oβ-glucoside (t- β-g) and estradiol- -o-β-glucoside (e- β-g) to form corresponding acylates. further, under the synergistic actions of ossgt and essga , the biosynthetic preparation of two acylated steroidal glycosides (asgs), namely acetylated t- β-gs (at- β-gs) and e- β-gs (ae- β-gs), was first achieved, thereby yielding four monoacetylated steroidal glucosides, namely -o, -o, -o and -o-acylates (scheme ). the cytotoxic activities of these monoacylates were evaluated against seven human tumor cell lines (hct , bel , mgc , capan , nci-h , nci-h and a ) and -acetylated testosterone -o-β-glucoside was observed to display improved cytotoxic activity against these seven cell lines (scheme ). the species o. saundersiae is a monocotyledonous plant rich in steroidal glycosides, suggesting that it may contain sgts responsible for the glycosylation of steroidal aglycons [ ] [ ] [ ] . o. saundersiae is thus selected as the candidate plant for sgts isolation. the transcriptome of o. saundersiae was thus sequenced with the aim of isolating genes encoding sgts. a total of , , raw reads were generated after the transcriptome sequencing of o. saundersiae. after removal of dirty reads with adapters, unknown or low quality bases, a total of , , clean reads were retained. these clean reads were combined by assembling soft trinity to form longer unigenes. finally, an rna-seq database containing , unigenes with mean length of bp was obtained. next, these unigenes were aligned to publicly available protein databases for functional annotations, retrieving unigenes displaying the highest sequence similarity with sgts. unigene with bp in length was thus retrieved from the unigene database for its high similarity with sgts ( supplementary information fig. s ). moreover, orf finder result showed that this unigene contained a complete open reading frame (orf) of bp, starting at nucleotide with an atg start codon and ending at position with a tga stop codon. the unigene contained bp of -utr (untranslated region) and bp of -utr. therefore, unigene was selected for further investigation. to verify the identity of unigene , a nested pcr assay was therefore carried out to amplify the cdna corresponding to the orf of unigene using gene-specific primers (supplementary information table s ). an expected band with approximately . kb was obtained, as observed in agarose gel electrophoresis ( supplementary information fig. s a ). the amplicon was then inserted into peasy tm -blunt plasmid (supplementary information table s ) to form a recombinant vector for sequencing. results indicated that the amplified product was % identity with that of unigene , confirming unigene was a bona fide gene in o. saundersiae genome. the bp orf encoded a polypeptide of amino acids (aa) with a predicted molecular mass of . kda and pi of . . blast analysis of the deduced protein revealed its predominant homology with sterol β-glucosyltransferase from elaeis guineensis (xp_ . , %), musa acuminata subsp. malaccensis (xp_ . , %) and anthurium amnicola (jat . , %). the cdna was therefore designated as ossgt and submitted to genbank library with an accession number of mf . the sequence analyses of ossgt were first assessed with the aim to direct its expression and functional verification. no putative trans-membrane domain was observed in ossgt based on the prediction results by tmhmm (http://www.cbs.dtu.dk/services/ tmhmm/), suggesting ossgt is a cytoplasmic sgt and may be expressed heterologously in e. coli in a soluble form. multiple alignment of ossgt and other plant sgts indicated that the middle and c-terminal parts of these sgts were more conservative than the n-terminal region ( supplementary information fig. s ), consistent with previous notion . moreover, two conservative motifs, namely a putative steroid-binding domain (psbd) and a plant secondary product glycosyltranferase box (pspg), were observed in ossgt ( supplementary information fig. s ). the region named psbd located in the middle part of ossgt and was thought to be involved in the binding of steroidal substrates . pspg box is about aa in length and close to the carboxy-terminus. this box is a characteristic "signature sequence" of udp glycosyltransferase and deduced to be responsible for the binding of the udp moiety of the nucleotide sugar . the presence of psbd and pspg boxes suggests that ossgt may be involved in secondary metabolism, catalyzing the transfer of udp-sugars to steroidal substrates thereby forming steroidal glycosides. the phylogenetic tree based on deduced amino acid sequences of ossgt and other sgt was generated by mega . . as scheme an enzymatic two-step synthesis of at- β-g ( b- e) and ae- β-g ( b- e) from the free steroids testosterone ( ) and estradiol ( ) . firstly, two sgs, t- β-g ( a) and e- β-g ( a), were prepared from their corresponding steroidal substrates testosterone ( ) and estradiol ( ) in the presence of a steroidal glycosyltransferase ossgt from o. saundersiae. the resulting t- β-g ( a) was further regioselectively acetylated under the action of an acyltransferase ecsga from e. coli, thereby yielding four monoacetylated steroidal glucosides ( b- e) with the yield ratio of : : : . likewise, e- β-g ( a) was acetylated by ecsga to form monoacetylated products b- e in a ratio of : : : . shown in supplementary information fig. s , all selected sgts were clusted into four clades, mon, di, ba and fun clades. the four clades included sgts from monocots, dicots, bacteria and fungi, respectively. ossgt belonged to mon clade, suggesting that ossgt was most similar to sgts from monocots. ossgt was then inserted into pet- a(þ) to yield a recombinant pet a-ossgt (supplementary information table s ) , which was transformed into transetta(de ) (transgen, beijing, china) for heterologous expression. sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) indicated that most of the expressed ossgt protein was present in the form of insoluble inclusion body, which was regarded to be devoid of bioactivity. it was well known that chaperone proteins were able to assist protein folding and thus increase production of active protein . therefore, a chaperone plasmid pgro (takara biotechnology co., ltd., dalian, china) was applied to be co-expressed with pet aossgt in bl (de ) (transgen, beijing, china), facilitating the soluble expression of ossgt . as shown in supplementary information table s , the plasmid pgro contains two genes encoding chaperone proteins groes and groel. under the synergistic action of chaperones groes and groel, an intense band with an apparent molecular mass of kda was present in the crude extract of bl (de )[pet aossgt þpgro ], but not in the crude proteins of the control strain bl (de )[pet- a (þ)þpgro ] ( supplementary information fig. s b ). the immunoblot analysis with an anti-polyhistidine tag antibody showed a bound band, but the control extract did not cross-react with the antibody ( supplementary information fig. s c ). these data collectively indicated that ossgt was successfully expressed in e. coli in a soluble form (supplementary information figs. s b and c) in accord with the predicted result of soluble expression of ossgt . to identify the activity of ossgt , the ossgt -containing crude protein was used as the biocatalyst for glycosylation reactions. each member of the acceptor library ( - , fig. b and supplementary information fig. s ) was first assessed as sugar table s ). of the substrates, only steroids were observed to be glucosylated by ossgt , forming corresponding monoglucosides (figs. - and supplementary information figs. s - ) . the ten steroids included seven β-hydroxysteroids ( - , supplementary information figs. s - ), two β-hydroxylsteroids ( - , figs. and ) and one β, β-dihydroxysteroid ( , supplementary information fig. s ). the reaction activities of ossgt towards the ten substrates indicated that ossgt was an sgt showing activities towards both β-and β-hydroxysteroids, consistent with the predicted result by bioinformatics analyses (supplementary information figs. s and s ). in fact, the reports of sgts with glycosylation activity against steroids at positions other than c- were limited and only three sgts from yeast were verified to exhibit selectivity towards both β-and β-hydroxylsteroids . ossgt was therefore viewed as the first plant sgt with selectivity towards both β-and β-hydroxylsteroids (figs. and and supplementary information figs. s - ) . however, the glycosylation activity of ossgt towards βhydroxyl group would be lost if additional hydroxyl group at β-( β-oh-testosterone, ), β-( β-oh-testosterone, ), β-( β-oh-testosterone, ), or α-position ( α-oh-testosterone, ) , even a methyl group at c -position (methyltestosterone and its derivatives, - ) was attached to testosterone ( ), generating not any glycosylated products. moreover, ossgt has no activity towards other compounds, including steroids without β-and β-hydroxyl groups ( - and - ) , flavonoids ( - ), alkaloids ( ) ( ) ( ) ( ) ( ) ( ) ( ) , triterpenoids ( - ), phenolic acids ( - ) and coumarins ( - ) as shown in supplementary information fig. s . among reactive β-and β-hydroxylsteroids, dehydroepiandrosterone ( ) had a maximum conversion approaching %, followed by diosgenin ( ) with % conversion and the other compounds having conversion below % (fig. a) . to produce sufficient glucosylated products for structural characterization, scale-up of ossgt -mediated reactions to preparative scale ( ml) was conducted. the resultant glucosides ( ) glycosylation. hplc chromatogram of reaction product of estradiol ( ) incubated with ossgt protein (a) or without ossgt (b). uv spectra of and enzymatic product a are shown in upper panels. the hplc conditions are available in supplementary information table s . were prepared by hplc and subjected to nmr analysis for structural elucidation. to determine the glycosylation sites of a- a, a and a, the c nmr analyses of the corresponding aglycons - , and were also performed (supplementary information figs. s - and table s ). the c nmr glycosylation shifts (Δδ, δ glucoside -δ aglycon ) of these glycosides were thus examined to ascertain the glycosylation position (table , and supplementary information tables s - ). the steroidal glycosides were observed to have significant glycosylation shift Δδs for c- (glycosides - ) or c- position (compounds - ), showing their -or -glycosides. for a and a, the location of glucose group was determined to be at c- based on their hmbc correlations between h- and c- (supplementary information figs. s and ). the β-anomeric configuration of the d-glucose unit in these ten glucosides ( a- a) was determined from the large anomeric proton-coupling constants of h- (j ¼ . hz) ( table and supplementary information tables s - ). the structures of these glucosides were thus assigned to βglucosides ( a- a and a) or β-glucosides ( a and a) of steroids based on h nmr ( a- a) and c nmr ( a- a) signals, hsqc ( a- a, a- a, a- a), hmbc ( a- a, a) and dept ( a, a) spectra (table , supplementary information figs. s - and tables s - ). these data collectively showed that ossgt was an inverting-type glycosyltransferase. in the preparation of t- β-g ( a), when the concentrated reaction mixture was separated by reversed phase highperformance liquid chromatography (rp-hplc), we accidentally discovered that in addition to the major peak representing t- β-g ( a), a minor peak ( b) was also present in the hplc profile ( supplementary information fig. s a ). the minor product with a t r of . min was also subjected to lc-ms analysis. surprisingly, the [m þ h] þ value of the minor product was assigned to . , more than that of monoglycosylated testosterone ( supplementary information fig. s b ). this finding hints that the minor product may be an acetylated testosterone glucoside. to characterize the exact structure of b, the minor product was prepared in bulk for nmr experiment (supplementary information figs. s - ). details of h and c nmr spectra were tabulated in table . the minor product was thus identified as -acetylated testosterone -o-β-glucoside ( -at- β-g, b). to test if the acetylated product b was from glucoside a, the purified glucoside a was used as the substrate to incubate with crude extract of e. coli expressing pet- a(þ) or pet ossgt , respectively. in both conditions, we observed the presence of b ( supplementary information fig. s ). on the contrary, no acetylated product b was detected in the e. coli lysate without the addition of substrate a ( supplementary information fig. s ). we therefore inferred that testosterone ( ) was first glycosylated at the β-hydroxyl group by ossgt to form t- β-g ( a), which was then selectively acetylated at c- of sugar moiety to yield the -at- β-g ( b) by a soluble bacterial acetyltransferase ( supplementary information fig. s ) . likewise, two metabolites, e- β-g ( a) and -acetylated estradiol -o-β-glucoside ( -ae- β-g, b), were detected in the concentrated ossgt -catalyzed reaction mixture of estradiol ( ) as shown in supplementary information figs. s - , tables s and s . these data collectively revealed that e. coli cell contained at least one sgas specific for the acetylation of steroidal β-glycosides. moreover, the sugar donor promiscuity of ossgt -catalyzed glycosylation reactions was also investigated. β-sitosterol ( ) and testosterone ( ) were chosen as the sugar acceptors to react with varied sugar donors listed in the supplementary experimental section, respectively. results demonstrated that both β-sitosterol ( ) and testosterone ( ) had no reactive activity towards other udp-activated nucleotides except udp-glc under the action of ossgt , indicating ossgt was specific for udp-glc. to characterize the genes encoding sgas, the first task was to analyze the genome sequence of bl (de ), which was public in ncbi database (accession no. cp . ). this bacterial strain contains at least putative acetyltransferase genes, in which laca, maa and wech, were predicted to encode o-acetyltransferase (supplementary information table s ). as shown in supplementary information fig. s , further sequence analyses revealed wech protein was a membrane-bound protein with a total of membrane-spanning helixes, inconsistent with the above results, in which the candidate acetyltransferase was determined to be a soluble protein in bacterial lysate. laca and maa proteins were predicted to have no transmembrane helixes, suggesting their soluble form in bacterial. thus, the remaining two genes, laca and maa, were further investigated. first, the entire orfs of the two genes were isolated from bacterial genome using gene-specific primers (supplementary information fig. s a and table s ). the orfs of laca and maa genes were and bp encoding polypeptides of and aa, respectively. the predicted molecular weights of the two proteins were . and . kda. the two genes were then inserted into pet- a(þ) to generate two recombinant vectors, which were introduced into bl (de ) for heterologous expression. after isopropyl-β-d-thiogalactoside (iptg) induction, the accumulation of approximately or kda was observed in the lysate of bacterial strain harboring pet a-laca or pet a-maa (supplementary information fig. s b ). moreover, the presence of bacterially-expressed his-laca or his-maa fusion protein in the bacterial lysate was verified by western-blot with anti-his antibody ( supplementary information fig. s c ). the expressed laca or maa protein was then purified to near homogeneity by affinity chromatography ( supplementary information fig. s b ). the purified his-laca or his-maa fusion protein was used as the biocatalyst to react with t- β-g ( a) and acetyl-coa. the reactions were monitored by hplc-uv/ms analysis using the method e (supplementary information table s ). as shown in fig. , -at- β-g ( b) was detected in laca-catalyzed bioconversion of t- β-g ( a), attesting laca encoded a sga (fig. , upper panel). on the contrary, there were not any new products in maa-mediated reaction. laca was thus designated as ecsga (e. coli steroidal glucoside acetyltransferase) for convenience hereinafter and submitted to genbank with an accession number of mf . it is generally accepted that hydrolases and acyltransferases are two classes of enzymes responsible for acylation reactions of sgs . the enzymatic acylations reported now are largely performed by hydrolases like lipases . on the other hand, not any genes encoding sgas are isolated up to date , . ecsga is therefore regarded as the first steroidal glycoside acyltransferase catalyzing the attachment of acyl groups into the hydroxyl groups of steroidal β-glycosides, to our knowledge. also, ecsga was observed to catalyze another steroidal βglycoside, e- β-g ( a), to form corresponding acylate ( b, fig. , upper panel) . on the other hand, the other glucosides listed in supplementary information fig. s could not be acetylated by ecsga , testifying ecsga was specific to steroidal β-glycosides. moreover, the acyl donor promiscuity of ecsga was investigated. t- β-g ( a) or e- β-g ( a) was used as the acyl acceptor to react with different acyl donors (acetyl-coa, succinyl-coa, arachidonoyl-coa, palmitoyl-coa and acetoacetyl-coa) under the action of the purified ecsga . results manifested that neither t- β-g ( a) nor e- β-g ( a) could react with these acyl donors except acetyl-coa, indicating that ecsga had strict donor selectivity. after careful check of ecsga -catalyzed reaction mixture in hplc profile, we have found several other minor peaks adjacent to the major product b (fig. , upper panel) . these minor peaks are so close that we could not distinguish. therefore, an efficient hplc method, namely method i (supplementary information table s ), was developed to separate these peaks. as shown in fig. (lower panel) , besides the major product b (t r ¼ . min), we observed three other minor peaks at t r ¼ . , . table s ). lower panel, hplc profile of acetylated products of a separated by chromatogramic method i (supplementary information table s ). and . min, respectively. the lc-ms measurement of these minor peaks showed that all of them have an [m þ na] þ value of . , thus suggesting their monoacetylated testosterone glucosides ( supplementary information fig. s ) . likewise, e- β-g ( a, t r ¼ . min) was observed to form four acetylated glucosides using purified ecsga as the biocatalyst (fig. ) . besides the well-characterized -ae- β-g ( b, t r ¼ . min), the other three products were determined to be monoacetylated estradiol glucosides based on their ms data ( supplementary information fig. s ). it was assumed that ecsga could introduce an acyl group into different hydroxyl groups of steroidal β-glycosides, generating varied monoacetylated products (figs. and ). to obtain sufficient amount of monoacetylated testosterone glucosides for structural characterization and further cytotoxicity assay, an enzymatic two-step process for at- β-gs ( b- e) was developed (scheme ). firstly, the whole cell biotransformation for the formation of at- β-gs ( b- e) was exploited due to its simple catalyst preparation. when testosterone ( ) was incubated with the engineered strain bl (de )[pet a-ossgt þpgro ], not any new products were detected. on the other hand, when t- β-g ( a) was added into the same whole-cell system, -at- β-g ( b) was present in the reaction mixture ( supplementary information fig. s ). these data indicated that testosterone ( ) could not be transported into the cell while the glycosylation of testosterone ( ) significantly improved the intercellular transport. thus, the formation of at- β-gs ( b- e) from testosterone ( ) using the single whole-cell biocatalyst is infeasible. a two-step process is therefore established to address this limitation. specifically, ossgt -catalyzed reaction was performed in the membrane-free crude cell extract of bl (de )[pet a-ossgt þpgro ], while ecsga -mediated acetylation was conducted in the whole-cell system of bl (de )[pet a-essga ]. the optimal ph and temperature of ossgt -catalyzed reaction using the cell-free extract of bl (de )[pet a-ossgt þp-gro ] as the biocatalyst were first determined to be alkaline ph value of and c, respectively (supplementary information fig. s ). next, the μl screening scale of ossgt -catalyzed glycosylation reaction was scaled to ml scale, in which mg testosterone ( ) were glycosylated to form mg t- β-g ( a) under optimized conditions (scheme ). the resultant t- β-g ( a) was subsequently used as the substrate applied in the scale-up of the whole-cell system of bl (de )[pet a-essga ] ( ml) under optimized ph . and c. the resulting reaction mixture was subjected to high-performance liquid chromatography-solid phase extraction-nmr spectroscopy (hplc-spe-nmr) measurement. comparison of the h and c nmr spectra of c- e with those of b suggested that compounds c- e had the same framework as b and the structural difference might be the position of the acetyl group. the location of acetyl group was determined to be at c- based on the hmbc correlations between h- (δ . ) and c- " (δ . ) as shown in supplementary information fig. s . thus, compound c was assigned as -at- β-g. the isolated glucose proton at δ . (h- ) of compound d exhibited long-range correlations with carbonyl carbons at δ . ( supplementary information fig. s ). moreover, h- (δ . ) of compound e showed long-range table s ). lower panel, hplc profile of acetylated products of a separated by chromatogramic method (supplementary information table s ). correlations with c- " (δ . ), as revealed by the hmbc spectrum ( supplementary information fig. s ). these data supported that the structure of d and e was elucidated as -at- β-g and -at- β-g, respectively. hence, the three trace products at t r ¼ . , . and . min were thus assigned to be -( d), -( e) and -at- β-g ( c) based on their respective nmr data (tables - and supplementary information figs. s - ) . these data indicate that ecsga can effectively introduce the acetyl group into the primary hydroxyl group and each secondary hydroxyl group of t- β-g ( a), yielding four monoacylates without the formation of diacylates ( fig. and scheme ). because the primary c( )-oh was the most reactive of the four hydroxyl groups in t- β-g ( a), acetylation of t- β-g ( a) took place preferentially at the c ( )-oh, giving -o-acylate predominantly in % yield ( fig. and scheme ). also, ecsga can regioselectively acetylate each secondary hydroxyl of t- β-g ( a) in the presence of the primary hydroxyl group, giving -( c), -( d) and -at- β-g ( e) in %, % and % yield, respectively. these data revealed the reactivity trend of hydroxyls is -oh -oh -oh -oh. likewise, the formation of four monoacylates was also present in ecsga -catalyzed acylation of e- β-g ( a, t r ¼ . min, fig. ). in addition to the well-characterized major product b, there are three trace products c- e. because of their trace amount, we did not further enrich these monoacetylated estradiol glucosides for nmr analysis. however, according to the catalytic behavior of ecsga towards t- β-g ( a), it was easy to infer that these products were most likely -( c, t r ¼ . min), -( d, t r ¼ . min) and -ae- β-g ( e, t r ¼ . min, fig. ). the order of reactivity of the hydroxyls was determined as -oh -oh -oh -oh with a yield ratio of : : : (fig. ) . regioselective acylation of one of the multiple hydroxyl groups in sgs is the major obstacle to the synthesis of sges and direct methods for site-selective acylation of unprotected sgs have rarely been documented. in this contribution, we successfully achieved the regioselective acylation of fully unprotected sgs using ecsga as the biocatalyst, thereby leading to an extremely short-step synthesis of asgs. acetylated steroidal glucosides, namely b, c, d and e, together with b were tested for their in vitro cytotoxicity against seven human cancer cell lines including hct , bel , mgc , capan , nci-h , nci-h and a . the results indicated that -at- β-g ( d) exhibited a wide spectrum of cytotoxic activities against the tested cell lines (table ). -at- β-g ( b) displayed much less cytotoxicity than -at- β-g ( d) but showed a mild cytotoxicity against human non-small cell lung carcinoma cell line nci-h with ic values of . μmol/l ( table ) . on the contrary, the control t- β-g ( a) did not display significant cytotoxicity towards these tested cell lines (ic . μmol/l). these evidences revealed that the acyl groups of sges are of importance to their cytotoxicity and direct regioselective acylation of sgs is thus believed as a powerful tool for the discovery of drug candidates. acylated steroidal glycosides have attracted our attentions primarily due to their biological and pharmacological significances , , , . there are two enzymes, namely sgts and sgas, responsible for the biosynthesis of asgs. to synthesize asgs, the primary premise is to obtain glycosyltransferases capable of catalyzing the formation of sgs from the abundantly available free steroids. o. saundersiae is thus selected as the candidate plant for sgts isolation. o. saundersiae is a monocotyledonous plant rich in acylated steroidal glycosides, suggesting that it may contain sgts and sgas responsible for the biosynthesis of asgs . thus, the transcriptome of o. saundersiae was sequenced with the aim to facilitate the genes discovery. ossgt was then isolated from o. saundersiae based on the rna-seq data. subsequently, ossgt -containing cell-free extract was used as the biocatalyst for glycosylations of structurally diverse drug-like scaffolds. the use of cell-free extract offers a number of advantages. unlike the ambitious purification procedures, the preparation of cell-free extract was simple and timesaving. moreover, compared the purified enzymes, the recombinant proteins used in crude extract-based system were more stable. steroidal glycosides are one of the main sources of innovative drugs . sgt-catalyzed glycodiversification of steroids could expand the molecular diversification, thereby facilitating the discovery of pharmacological leads. thus, the search of sgts with catalytic promiscuity may provide potent biocatalysts for glycodiversification. therefore, a library containing structural diverse drug-like molecules was utilized to react with the recombinant ossgt with the aim to explore the substrate flexibility of ossgt . in vitro enzymatic analyses revealed that ossgt was active against various steroids, including physterols ( - , supplementary information figs. s - ) , steroid hormones ( , - , figs. and , and supplementary information figs. s , s and s ), steroidal sapogenin ( , supplementary information fig. s ) and cardiac aglycon ( , supplementary information fig. s ) , exhibiting a wider substrate range than that of previously identified sgts from plant , . to investigate the regioselectivity of ossgt , diversified steroids were selected as the sugar acceptors for ossgt -catalyzed glycosylations. as illustrated in figs. - and supplementary information s - , ossgt specifically attacked the hydroxyl groups at c- and c- positions, but no activities towards hydroxyl groups at c- ( , ), c- ( ), c- ( ), c- ( ) , c- ( ), c- ( ) , c- ( , and ) and c- ( and ) . when steroids having two potentially reactive hydroxyl groups, like α-hydroxypregnenolone ( ) or androstenediol ( ), were used as the substrate for ossgt -assisted glycosylation, only glycosides with a glycosyl substituent in c- position were detected in the reaction mixture, suggesting ossgt exhibited prominent regioselectivity towards the -oh of both substrates ( supplementary information figs. s and s ) . also, ossgt the stereoselectivity of ossgt was also assessed in this study. estradiol ( ) and α-estradiol ( ) differ for the configuration of the hydroxyl group at c- position. when each of the two compounds was used to react with ossgt , only β-configurated glycosides were generated (fig. ) . likewise, ossgt showed βselective glycosylation towards the hydroxyl group at c- position. cumulatively, these evidences revealed that ossgt catalyzed glycosylations were conducted in a region-and stereoselective fashion. one of the most striking findings of this study is the characterization of bacterial laca protein as a steroidal glycoside acyltransferase. it is well known that laca is one of three structural genes (lacz, lacy and laca) in lac operon , . the function of lacz and lacy is well-characterized , . lacz encodes a βgalactosidase, catalyzing the cleavage of lactose into glucose and galactose. lacy encodes a lactose permease responsible for lactose uptake , . the third structural protein encoded by laca gene in lac operon was initially inferred to be an acetyltransferase. the exact action of this protein, however, remains in doubt until now. in this investigation, in an effort to identify the function of ossgt , we unexpectedly characterized laca protein from e. coli as a sga. in vitro enzymatic analyses revealed that laca protein could specifically catalyze the attachment of acyl groups into the hydroxyl groups of sugar moieties of steroidal β- glycosides (figs. and ) . although we have no evidences for the role of laca protein in vivo, these findings in the present work may provide a starting point for identifying the exact activity of laca protein in lactose metabolism. the bottleneck in enzymatic synthesis of asgs is the lack of well-characterized sgas. the successful characterization of laca made it to be the first sga and laca protein was thus designated as ecsga . a novel enzymatic two-step synthesis of at- β-gs and ae- β-gs from the abundantly available free steroids under the sequential actions of a steroidal glycosyltransferase ossgt from o. saundersiae and ecsga was achieved. the two-step process is characterized by acyltransferase-catalyzed regioselective acylations of all hydroxyl groups of unprotected sgs in the late stage, thereby significantly streamlining the synthetic route towards asgs and thus forming four monoacylates. regioselective acylation could expand molecular diversity, thereby facilitating the discovery of pharmaceutical leads. in this investigation, ecsga -catalyzed acetylation of two steroid βglucosides (t- β-g and e- β-g) leaded to the production of eight new monoacylates. furthermore, the cytotoxic activities of these monoacylates were tested and -at- β-g was observed to display improved activities towards seven human tumor cell lines, suggesting this compound had promisingly pharmacological potential. this study therefore reports for the first time a novel synthetic process for the green preparation of acylated steroidal glycosides with medicinal interest. a steroidal glycosyltransferase ossgt from o. saundersiae was identified to be the first plant sgt with selectivity towards both β-and β-hydroxylsteroids. one of the most striking findings of this study is the characterization of bacterial laca protein as a steroidal glycoside acyltransferase, catalyzing the attachment of acyl groups into the hydroxyl groups of steroidal β-glycosides. a novel enzymatic two-step synthesis of at- β-gs and ae- β-gs from the abundantly available free steroids under the sequential actions of ossgt and ecsga was achieved. the two-step process is characterized by acyltransferase-catalyzed regioselective acylations of all hydroxyl groups of unprotected sgs in the late stage, thereby significantly streamlining the synthetic route towards asgs and thus forming four monoacylates. moreover, the cytotoxic activities of these monoacylates were tested and -at- β-g was observed to display improved activities towards seven human tumor cell lines. this study therefore reports for the first time a novel synthetic process for the green preparation of acylated steroidal glycosides with medicinal interest. in this contribution, four compound libraries, namely sugar acceptor, sugar donor, acyl acceptor and acyl donor libraries, were provided for enzyme-mediated reactions. the compounds listed in fig. and supplementary information fig. s include diverse structures like steroids , flavonoids ( ) ( ) ( ) ( ) ( ) ( ) ( ) , alkaloids ( ) ( ) ( ) ( ) ( ) ( ) ( ) , triterpenoids ( - ), phenolic acids ( - ) and coumarins ( - ) are used as the sugar acceptors for ossgt -catalyzed glycosylation reactions ( fig. and supplementary information fig. s ). the sugar donors consist of seven udp-activated nucleotides, among which, udp-dglucose (udp-glc), udp-d-galactose (udp-gal), udp-d-glucuronic acid (udp-glca) and udp-n-acetylglucosamine (udp-glcnac) were obtained from sigma-aldrich co., llc. (st. louis, mo, usa). udp-d-xylose (udp-xyl), udp-l-arabinose (udp-ara) and udp-d-galacturonic acid (udp-gala) was synthesized by enzymemediated reactions in our laboratory [ ] [ ] [ ] . the acyl acceptor library is made up of steroidal glucosides ( a- a) and other glucosides ( - ) listed in supplementary information fig. s . the acyl donor library includes acetyl-coa, succinyl-coa, arachidonoyl-coa, palmitoyl-coa and acetoacetyl-coa, all of which were purchased from sigma-aldrich co., llc. the other chemicals were either reagent or analytic grade when available. the resulting raw reads from sequence library of o. saundersiae was firstly filtered to obtain clean reads, discarding dirty reads with adaptors, unknown or low quality bases. these clean reads were subsequently combined to form longer unigenes by assembling program trinity. these unigenes obtained by de novo assembly cannot be extended on either end. next, these unigenes were aligned by blast x algorithm to protein databases, such as nr, swiss-prot, kegg and cog (e-valueo . ) for functional annotation. the unigenes displaying similarity to sgts were retrieved for further orf analysis. in a word, those unigenes with a complete orf and displaying high similarity to sgts were selected as the candidate for further investigation. to verify the authenticity of the candidate unigene, cdna isolation was performed using gene-specific primers by a nested pcr assay as previously described (supplementary information table s ) [ ] [ ] [ ] [ ] . the obtained amplicon was inserted into peasy tm -blunt plasmid (transgen co., ltd., beijing, china) and then transformed into e. coli trans -t competent cells for recombinant plasmid selection (supplementary information table s ). the resultant recombinant plasmid was isolated and subjected to nucleotide sequencing. the obtained cdna was thus designated as ossgt for convenience. the bioinformatics analyses of ossgt , like prediction of physiochemical properties, multiple sequence alignment and phylogenetic analysis, were performed as detailed in our previous reports [ ] [ ] [ ] [ ] . ossgt was amplified using gene-specific primers (supplementary information table s ) and the resulting pcr product was ligated into ecori and hind iii sites within the pet- a(þ) vector (novagen, madison, usa) using seamless assembly cloning kit (clonesmarter technologies inc., houston, tx, usa) as described previously . the generated construct pet a-ossgt was transformed into e. coli strain transetta (de ) for expression as described previously . also, to improve heterologous expression of ossgt , pet a-ossgt was cotransformed into e. coli bl (de ) strain with a chaperone plasmid pgro (takara biotechnology co., ltd., dalian, china) as introduced by yin et al. the expression of ossgt was induced by iptg at a final concentration of . mmol/l. the expressed ossgt was checked by sds-page and western-blot analyses as described by guo et al. next, the bl (de ) [pet a-ossgt þpgro ] suspension cells were disrupted in a high-pressure homogeniser (apv- , albertslund, denmark) operated at bar. disrupted cells were centrifuged at , rpm for min to discard the pellet. the resultant supernatant, namely the membrane-free crude extract, was used as the biocatalyst for steroidal glycosylation. after verification of heterologous expression of ossgt , the crude extract containing the recombinant ossgt was applied as the biocatalyst to react with various sugar acceptors and donors ( fig. and supplementary information fig. s ). the total reaction mixture was μl contained mmol/l phosphate buffer (ph . ), a sugar acceptor ( mmol/l), a sugar donor ( mmol/l) and μl crude ossgt proteins. the reaction mixture was incubated at c for h. the formation of glycosylated products was table the cytotoxic activities of monoacylates against human tumor cell lines. unambiguously determined by a combination of hplc-uv, hplc-ms and nmr as described previously , . the determination conditions for hplc-uv were summarized in supplementary information table s . the genome dna was extracted from e. coli strain bl (de ) using bacteriagen dna kit (cwbio co., ltd., beijing, china) according to the supplier recommendation. the resulting genome dna was then used as the template of pcr amplification to isolate these candidate sga genes using gene-specific primers (supplementary information table s ). the amplified pcr products were inserted into peasy tm -blunt plasmid to generate recombinant vectors for sequencing verification. next, these oacetyltransferase-encoding genes were heterologously expressed in bl (de ) as described above. sds-page and western-blot of these recombinant proteins were conducted as that of ossgt (see above). the recombinant o-acetyltransferase proteins were subjected to purification with ni-nta agarose columns according to the manufacture's protocol. purified protein concentrations were determined using bradford protein assay (bio-rad, hercules, ca, usa). the enzymatic activities of ecsgas were determined in μl citrate buffer solution (ph . ) containing an acyl acceptor ( mmol/l) listed in supplementary information fig. s , an acyl donor ( mmol/l) summarized in chemicals section and the purified protein ( . μg). the reactions were incubated at c for h. then μl methanol was added to terminate the reaction. the reaction mixture was monitored by hplc-uv (supplementary information table s ) and the structure of the generated product was determined by a combination of hplc-ms and nmr as reported by liu et al. . . biotransformation of testosterone and testosterone- -oβ-glucoside using the whole cell system after iptg induction, the engineered bl (de )[pet a-ossgt þpgro ] cells were harvested by centrifugation at , rpm for min and then resuspended in m medium with cell density of od value of . . the substrate testosterone ( ) or testosterone- -o-β-glucoside (t- -o-β-g, a) with the final concentration of . mmol/l was added into the m medium and continued to incubate at c overnight. the formation of products was monitored by hplc analysis as mentioned above. the effects of ph and temperature on ossgt -and ecsga catalyzed reactions were investigated. the crude extract of bl (de )[pet a-ossgt þpgro ] and the purified ecsga protein were applied as the biocatalyst in their respective reactions. the effects of ph on both reactions were determined at varied buffers including citric acid/sodium citrate buffer ( . mol/l, ph . - . ), na hpo /nah po ( . mol/l, ph . - . ), na hpo / naoh buffer ( . mol/l, ph . - ). the influences of temperature were explored in a range of to c with intervals of c (ossgt -mediated glycosylation) or c (ecsga -catalyzed acylation) in the standard reaction mixture as described above. scale-up of ossgt -and ecsga -catalyzed reactions was performed to obtain sufficient at- β-gs for structural characterization and further cytotoxicity assay. initially, the μl ossgt catalyzed reaction was directly scaled to ml, in which mg testosterone ( ) were added into and then incubated with crude cell extract at optimal ph and temperature for h. the resultant reaction mixture was applied to preparative hplc to isolate pure t- β-g, which was then used as the substrate in ml ecsga -catalyzed reaction for at- β-gs production. structure characterization of at- β-gs was performed using hplc-spe-nmr technique as described by liu et al. except some modifications on chromatographic conditions. hplc separation was carried out on an ymc-pack ph column ( μm, nm, mm  . mm) with an isocratic elution of % water-trifluoroacetic acid (a, . %: . %, v/v) and % methanol (b) at a flow rate of ml/min. seven human cancer cell lines, hct- (human colon cancer cell line), bel (human hepatocellular carcinoma cell line), mgc (human gastric carcinoma cell line), capan (human pancreatic cancer cell line), nci-h , nci-h and a (human lung cancer cell lines) were used in the cytotoxicity assay. the viability of the cells after treated with various chemicals was evaluated using the mtt ( -( , -dimethylthiazol- -yl)- , -diphenyl tetrazolium bromide) assay performed as previously reported , . the inhibitory effects of these tested compounds on the proliferation of cancer cells were reflected by their respective ic ( % inhibitory concentration). steryl glycosides and acylated steryl glycosides in plant foods reflect unique sterol patterns rice bran extract containing acylated steryl glucoside fraction decreases elevated blood ldl cholesterol level in obese japanese men structural analysis of novel bioactive acylated steryl glucosides in pre-germinated brown rice bran a potent anticomplementary acylated sterol glucoside from orostachys japonicus therapeutic potential of cholesteryl o-acyl alpha-glucoside found in helicobacter pylori immunological functions of steryl glycosides cholestane glycosides with potent cytostatic activities on various tumor cells from ornithogalum saundersiae bulbs a new rearranged cholestane glycoside from ornithogalum saundersiae bulbs exhibiting potent cytostatic activities on leukemia hl- and molt- cells acylated cholestane glycosides from the bulbs of ornithogalum saundersiae synthesis of a cholestane glycoside osw- with potent cytostatic activity first total synthesis of an exceptionally potent antitumor saponin, osw- improved enzyme-mediated synthesis and supramolecular selfassembly of naturally occurring conjugates of β-sitosterol simple method for high purity acylated steryl glucosides synthesis regioselective diversification of a cardiac glycoside, lanatoside c, by organocatalysis sterol glycosyltransferases-the enzymes that modify sterols the functions of steryl glycosides come to those who wait: recent advances in plants, fungi, bacteria and animals sterol β-glucosyltransferase biocatalysts with a range of selectivities, including selectivity for testosterone molecular cloning and biochemical characterization of a recombinant sterol -o-glucosyltransferase from gymnema sylvestre r.br. catalyzing biosynthesis of steryl glucosides cloning and functional expression of ugt genes encoding sterol glucosyltransferases from saccharomyces cerevisiae, candida albicans, pichia pastoris, and dictyostelium discoideum glycosyltransferases in plant natural product synthesis: characterization of a supergene family chaperone coexpression plasmids: differential and synergistic roles of dnak-dnaj-grpe and groel-groes in assisting folding of an allergen of japanese cedar pollen, cryj , in escherichia coli regioselective enzymatic acylation of complex natural products: expanding molecular diversity structure, bioactivity, and chemical synthesis of osw- and other steroidal glycosides in the genus ornithogalum functional diversification of two ugt enzymes required for steryl glucoside synthesis in arabidopsis lac operon induction in escherichia coli: systematic comparison of iptg and tmg induction and influence of the transacetylase laca the lac operon galactoside acetyltransferase cdna isolation and functional characterization of udp-d-glucuronic acid -epimerase family from ornithogalum caudatum transcriptome-guided discovery and functional characterization of two udp-sugar -epimerase families involved in the biosynthesis of anti-tumor polysaccharides in ornithogalum caudatum transcriptome-guided gene isolation and functional characterization of udp-xylose synthase and udp-d-apiose/udp-dxylose synthase families from ornithogalum caudatum ait transcriptome-wide identification of sucrose synthase genes in ornithogalum caudatum transcriptomeenabled discovery and functional characterization of enzymes related to ( s)-pinocembrin biosynthesis from ornithogalum caudatum and their application for metabolic engineering functional analyses of ocrhs and ocuer involved in udp-l-rhamnose biosynthesis in ornithogalum caudatum interactions among sars-cov accessory proteins revealed by bimolecular fluorescence complementation assay cdna isolation and functional characterization of squalene synthase gene from ornithogalum caudatum probing steroidal substrate specificity of cytochrome p bm variants steroids hydroxylation catalyzed by the monooxygenase mutant - from bacillus megaterium bm metabolic engineering of escherichia coli for -butanol production cytotoxic cholestane glycosides from the bulbs of ornithogalum saundersiae supplementary data associated with this article can be found in the online version at doi: . /j.apsb. . . . key: cord- -iryb v z authors: kao, kuo-chin; chang, ko-wei; chan, ming-cheng; liang, shinn-jye; chien, ying-chun; hu, han-chung; chiu, li-chung; chen, wei-chih; fang, wen-feng; chen, yu-mu; sheu, chau-chyun; tsai, ming-ju; perng, wann-cherng; peng, chung-kan; wu, chieh-liang; wang, hao-chien; yang, kuang-yao title: predictors of survival in patients with influenza pneumonia-related severe acute respiratory distress syndrome treated with prone positioning date: - - journal: ann intensive care doi: . /s - - - sha: doc_id: cord_uid: iryb v z background: patients with influenza complicated with pneumonia are at high risk of rapid progression to acute respiratory distress syndrome (ards). prone positioning with longer duration and lung-protective strategies might reduce the mortality level in ards. the aim of this study is to investigate the survival predictors of prone positioning in patients with ards caused by influenza pneumonia. methods: this retrospective study was conducted by eight tertiary referral centers in taiwan. from january to march in , all of the patients in intensive care units with virology-proven influenza pneumonia were collected, while all of those patients with ards and receiving prone positioning were enrolled. demographic data, laboratory examinations, management records, ventilator settings and clinical outcomes were collected for analysis. results: during the study period, patients with severe influenza pneumonia were screened and patients met the diagnosis of ards. totally, patients receiving prone positioning were included for analysis. the -day survivors had lower acute physiology and chronic health evaluation (apache) ii score, pneumonia severity index (psi), creatinine level and lower rate of receiving renal replacement therapy than non-survivors ( . ± . vs. . ± . , p = . ; . ± . vs. . ± . , p = . ; . ± . mg/dl vs. . ± . mg/dl, p = . ; and % vs. %, p < . ). multivariate cox regression analysis identified psi (hazard ratio . , % confidence interval . – . ; p < . ), renal replacement therapy (hazard ratio . , % confidence interval . – . ; p < . ), and increase in dynamic driving pressure (hazard ratio . , % confidence interval . – . ; p = . ) which were independent predictors associated with -day mortality. conclusions: in the present study, in evaluating the effect of prone positioning in patients with influenza pneumonia-related ards, pneumonia severity index, renal replacement therapy and increase in dynamic driving pressure were associated with -day mortality in patients with influenza pneumonia-related ards receiving prone positioning. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. severe complicated influenza including pneumonia, myocarditis and neurologic complications are still a burden on intensive care units (icu) nowadays, especially viral or secondary bacteria pneumonia-induced acute respiratory distress syndrome (ards) [ , ] . during the winter season in , there was an outbreak of influenza in taiwan. totally, subjects were admitted to icus due to severe complicated influenza pneumonia according to the data from the centers for disease control of taiwan [ ] . patients with influenza pneumonia needing mechanical ventilation were at high risk of rapid progression to ards. for the pandemic h n virus infection, - % of patients admitted to icus had complications with ards [ , ] . there are several therapeutic options for refractory hypoxemia in patients with severe ards [ , ] , but only a few options have been confirmed with clinical validity by previous studies, including higher positive endexpiratory pressure (peep) [ , ] , lower tidal volume [ ] , neuromuscular blocking agents [ ] and prone positioning [ ] . prone positioning was first suggested in [ ] ; however, the clinical benefit of prone positioning in patients with ards was not confirmed until when the proseva study showed decreased -day and -day mortality and increased ventilator-free days only when it was started early and there were sufficiently long sessions [ ] . further, meta-analysis by cochrane database also revealed that prone positioning would reduce the mortality rate when used with lung-protective strategies and longer duration in patients with severe ards [ , ] . few studies have explored the effect of prone positioning focused on influenza pneumonia-related ards patients. xu et al. [ ] studied h n influenza patients with prone positioning, and decrease in carbon dioxide retention was noted, but no clinical outcome was mentioned. moreover, what factors that can predict the efficacy of prone positioning in severe ards are not entirely clear [ ] . the aim of this study is to investigate the survival predictors of prone positioning in patients with severe ards caused by influenza pneumonia. this multicenter retrospective cohort study was conducted by the taiwan severe influenza research consortium (tsirc), which included eight tertiary referral centers (four hospitals in northern taiwan, two hospitals in central taiwan and two hospitals in southern taiwan). over a period of months from january to march in , all patients with the virology-proven influenza infection who were admitted to icus due to severe complicated influenza in these eight hospitals were collected and their data were analyzed. all patients diagnosed as severe ards according to berlin definition and also receiving prone positioning were collected for investigation [ ] . the berlin definition of ards was defined by acute onset within week, bilateral lungs opacities, no evidence of cardiac failure-related hydrostatic edema by echocardiography, and pao /fio ratio < mm hg with positive end-expiratory pressure (peep) ≥ cm h o. the demographic and laboratory data, treatment record, mechanical ventilation settings, and clinical outcomes were analyzed from the electronic medical records with a standardized case report form in each hospital. university hospital rind, tri-service general hospital - - - ). the need for informed consent was waived, and patients' data were anonymized and de-identified prior to analysis. influenza infection was confirmed by one of the following tests revealing as positive including the rapid antigen test, nucleic acid reverse transcriptase polymerase chain reaction (rt-pcr), viral culture sampling from nasopharynx swab, throat swab, sputum or bronchoalveolar lavage and positive serum antibody serologic test (antibody titers increased more than times from acute to convalescent stages). the usual practice in the units was that patients be ventilated with lung-protective strategy by low tidal volume - ml/kg of predict body weight plus low positive endexpiratory pressure (peep)-oxygen fraction in air (fio ) table for pressure-controlled or volume-controlled ventilation [ ] . ventilation was monitored by arterial blood gas measurements, with ventilator settings changed as needed. pulse oximetry (spo ) was used to monitor oxygenation, and ventilatory settings were adjusted to maintain spo > % or pao > mm hg and to avoid raising the plateau pressure > cm h o. the method of prone positioning complied with the proseva study [ ] . doses of neuromuscular blocking agent with intravenous cisatracurium and sedatives with intravenous midazolam were adjusted to maintain synchrony between the ventilator and the patient's breathing, as well as hemodynamics. the criteria for stopping prone positioning were any of the following: improvement in oxygenation (defined as a pao /fio ratio ≥ mm hg, with a peep of ≤ cm h o and an fio ≤ . ), a decrease in the pao /fio ratio ≥ % or complications happening during prone positioning such as spo ≤ % or pao /fio ratio ≤ mm hg, severe cardiac arrhythmia, systolic blood pressure ≤ mm hg and any other life-threatening condition for which the intensivist decided to stop the prone positioning. the laboratory data including baseline characteristics, underlying disease, complete blood count, differential count and biochemistry data were obtained when the patient was admitted to the icu. the mechanical ventilator settings were recorded such as peak inspiratory pressure, peep, artery blood gas, partial pressure of oxygen in arterial blood (pao ), pao /fio ratio, tidal volume, dynamic driving pressure and dynamic compliance of the respiratory system before and day after the first prone positioning. the above physiological data were recorded before prone positioning on the supine position and day after first prone positioning on the prone position. the dynamic driving pressure and dynamic compliance were computed as peak pressure minus peep and tidal volume divided by peak pressure minus peep. the severity scores including pneumonia severity index (psi) [ ] , acute physiology and chronic health evaluation ii (apache ii) score [ ] , curb- (confusion, urea > mmol/l, respiratory rate ≥ /min, blood pressure [systolic < mm hg or diastolic ≤ mm hg] and age ≥ years) pneumonia severity score [ ] and sequential organ failure assessment (sofa) score [ ] were collected on the icu admission day. statistical analyses and database management were performed using spss version . . (spss inc., chicago, il). the data were presented as number (percentages) for nominal variables, and as mean ± standard deviation for continuous variables. the chi square test was used to compare the nominal variables, and the student's t test was used to compare the continuous variables. cox proportional hazard models were used with covariates significantly different between survivors and non-survivors at the threshold of . and mortality at day as the dependent variable. calibration was assessed using hosmer-lemeshow goodness-of-fit test (c statistic, goodness of fit was defined as a p value > . ), and discrimination was assessed by the area under the receiver operating curves. even though peak airway pressure, dynamic driving pressure, and compliance are mathematically coupled, we planned to formally test the collinearity within them and, if verified, to use a specific cox model for each. we also included those collinear variables two-by-two into three additional cox regression models [ ] , besides the other covariates. one model pertained to peak airway pressure and dynamic driving pressure, one to peak airway pressure and compliance, and one to dynamic driving pressure and compliance. if both variables in the couple lacked significance, the conclusion could be that the same information was carried by each component of the couple. if one of the variables in the couple remained significantly correlated with survival, this variable would be more informative than the other in the couple. univariate and multivariate cox proportional hazard regression models were used to estimate the hazard ratio (hr). in this study, we used the two-tailed test, and the definition of significance was p value < . . in total, patients with virology-proven severe influenza pneumonia were admitted to icus and screened during the study period. there were patients with influenza a (including h n in patients and h n in patients), patients with influenza b, and patients with undetermined influenza type. of these patients, patients ( %) met the diagnosis of severe influenza pneumonia-related ards. the rates of mild, moderate and severe ards were % ( / ), % ( / ) and % ( / ), respectively. of these patients with ards, patients ( %) receiving prone positioning were included for analysis (fig. ) . the rate of receiving prone positioning was % ( / ) in mild, % ( / ) in moderate and % ( / ) in severe ards, respectively (p = . ). the characteristics of the subjects according to the -day survivors and non-survivors are summarized in table . the mean age was . ± . years, and patients ( %) were male. the duration of prone positioning of survivors and non-survivors was not significantly different ( . ± . days vs. . ± . days, p = . ). the survivors had lower apache ii score, psi, creatinine level and lower rate of receiving renal replacement therapy than did non-survivors ( . ± . vs. . ± . , p = . ; . ± . vs. . ± . , p = . ; . ± . mg/dl vs. . ± . mg/dl, p = . ; and % vs. %, p < . ). regarding the oxygenation, the mean pao /fio ratio of these patients before prone positioning was . ± . mm hg. before prone positioning, there were no significant differences in the pao /fio ratio, paco , tidal volume, peep, peak airway pressure, dynamic driving pressure and dynamic compliance between surviving and non-surviving patients. the data regarding the gas exchange and lung mechanics were recorded before prone positioning and after -day prone positioning (table ) . for the -day survivors, there were no significant differences in these parameters compared with -day non-survivors except for peak airway pressure. after prone positioning, the -day survivors had decreased peak airway pressure (− . ± . cm h o) and the -day non-survivors had increased peak airway pressure ( . ± . cm h o). compared with -day non-survivors, the peak airway pressure and dynamic driving pressure were both decreased in -day survivors (− . ± . univariate analysis was used to identify variables that have prognostic value for -day mortality, and multivariate cox regression analysis was used to identify variables that did have significant predictive value (table ) . pneumonia severity index (hazard ratio . , % confidence interval . - . ; p < . ), renal replacement therapy (hazard ratio . , % confidence interval . - . ; p < . ) and increased dynamic driving pressure (hazard ratio . , % confidence interval . - . ; p = . ) were identified as significant and independent predictors associated with -day mortality. as the collinearity between Δ dynamic driving pressure, Δ peak airway pressure and Δ dynamic compliance was statistically significant, a cox model was constructed for each of these variables. after multiple adjustments of coupled variables, three additional cox models were performed (additional file ). when Δ dynamic driving pressure and Δ peak airway pressure were analyzed two-by-two, Δ dynamic driving pressure remained significant but Δ peak airway pressure did not (model in additional file ). when Δ dynamic driving pressure and Δ dynamic compliance were analyzed two-by-two, Δ dynamic driving pressure remained significant but Δ dynamic compliance did not (model in additional file ). when Δ peak airway pressure and Δ dynamic compliance were analyzed two-by-two, both did not reveal significant (model in additional file ). receiver operating curves analysis and c statistic of variables of predictors revealed . in psi ( % confidence interval, . - . , p = . ), . in renal replacement therapy ( % confidence interval, . - . , p = . ) and . ( % confidence interval, . - . , p = . ) in delta dynamic driving pressure (fig. ). the aim of this multicenter retrospective study was to evaluate the effect of prone positioning focusing on patients with influenza pneumonia-related ards. after multivariate cox regression analysis, psi, renal replacement therapy and increased dynamic driving pressure were associated with -day mortality in patients with influenza pneumonia-related ards receiving prone positioning. most of the studies evaluating the effect of prone positioning were in ards patients with heterogeneous risk factors [ , ] . for specific conditions such as burns, prone positioning has been demonstrated to safely implement and improve oxygenation (in burn patients with severe ards) in a burn intensive care unit [ ] . the present study was more homogenous and specific to patients with ards caused by influenza pneumonia. systematic review and meta-analysis studies in prone positioning have revealed decreased mortality in patients with severe acute hypoxemic respiratory failure, but not in less severe hypoxemia. survival benefits were noted using a range of pao /fio ratio thresholds up to approximately mm hg [ ] or less than mm hg [ ] . in the present study, the pao /fio ratio was . ± . mm hg before prone positioning. however, the pao /fio ratio was not significantly different between -day survivors and -day non-survivors ( . ± . mm hg vs. . ± . mm hg, p = . ). in terms of the response of prone positioning to ards, the different entities of the risk factor possibly produce different outcomes. in addition to severity of hypoxemia, further clinical trials would assist in clarifying the survival benefits of prone positioning in the specific risk factors. some studies have shown that acute kidney injury (aki) was common and an independent risk factor for mortality in patients with influenza a [ ] [ ] [ ] [ ] . in patients with severe ards caused by h n influenza pneumonia, a recent study also revealed aki was common and demonstrated significantly increased mortality [ ] . the % mortality rate among the patients requiring renal replacement therapy was significantly higher than the % mortality rate among the patients not requiring renal replacement therapy. the present study in patients receiving prone positioning caused by influenza pneumonia-related ards demonstrated that the requirement for renal replacement therapy had nearly times the mortality rate (hazard ratio . ) than patients not requiring renal replacement therapy. in order to reduce the mortality in patients with severe ards caused by h n influenza pneumonia, it is important to prevent development ards acute respiratory distress syndrome, bmi body mass index, apache ii acute physical and chronic health evaluation, sofa sequential organ function assessment, psi pneumonia severity index, curb- curb- for pneumonia severity, wbc white blood cell count, paco atrial pressure of carbon dioxide in arterial blood, pao atrial pressure of oxygen in arterial blood, fio oxygen fraction in air, pbw predict body weight, peep positive end-expiratory pressure all values are expressed as the number of patients (percentage) or mean ± sd *p < . : survivors versus non-survivors of aki and need for renal replacement therapy by avoiding nephrotoxic agents and supplying sufficient renal perfusion and oxygenation. amato and colleagues analyzed randomized controlled trials in ards patients and demonstrated that driving pressure was the strongest predictor of mortality [ ] . a secondary analysis of data from ards patients enrolled in two independent randomized controlled trials revealed that when ventilating patients with low tidal volume, driving pressure was a risk factor for death in ards patients, as was plateau pressure or compliance of respiratory system [ ] . airway driving pressure was significantly related to lung stress and could detect lung over-stress with acceptable accuracy (r = . p < . and r = . p < . at and cm h o of peep) in ards patients [ ] . furthermore, the apronet study on prone positioning of ards patients found that prone positioning was associated with low complication rates, significant increase in oxygenation, and a significant decrease in driving pressure [ ( - cm h o) to [ ] [ ] [ ] [ ] [ ] [ ] [ ] cm h o, p = . ] [ ] . our previous study for severe ards patients with ecmo revealed that higher dynamic driving pressure [hazard ratio . ( . - . ), p = . ] during the first days of ecmo was one of the factors independently associated with icu mortality [ ] . the present study in influenza pneumonia-related ards patients receiving prone positioning also found that increased dynamic driving pressure (hazard ratio . , % confidence interval . - . ; p = . ) was identified as ards acute respiratory distress syndrome, Δ change between before and after prone positioning day, pao partial pressure of oxygen in arterial blood, paco atrial pressure of carbon dioxide in arterial blood, one of the independent predictors associated with -day mortality. it was suggested that ventilatory support with lung-protective strategy with low tidal volume and optimal peep level be applied, and these be then adjusted according to the driving pressure, ideally less than cm h o, although this limit should be addressed in future studies [ ] . despite some studies associating driving pressure with physiological and clinical outcomes, it is necessary to evaluate the driving pressure as a primary end point during ventilatory setting in ards patients in the near future. the lung safe study showed that the use of prone positioning actually depended on the severity of hypoxemia, from % in mild to . % in moderate and to . % in severe ards [ ] . a prospective international prevalence study (the apronet study, ards prone position network) found that the rates of prone positioning were up to . %, . % and . % in mild, moderate and severe ards [ ] . in our study, the rates of prone positioning were %, % and % in mild, moderate and severe ards, respectively. the substantially different rates in the use of the prone positioning may reflect the management bias of prone positioning in patients with ards between the different studies. furthermore, among our eight involved hospitals, the rate of prone positioning varied from % ( / ) to % ( / ) and the bias even existed between different hospitals in the same study. it is important to be homogenous on the indication and management in the selected prone position as one of the standard interventions in severe ards. this study has some limitations. firstly, since this study is retrospective, some patients or data might be missing. secondly, the primary end point of this study was -day mortality, and the value of computed power was . . this was a retrospective study, and patients with severe ards receiving prone positioning were analyzed. although more patients were needed to increase the power of this study, the limitation was from the nature of retrospective study within a -month period. thirdly, prone positioning is not a routine intervention in the management of ards and has no standard procedure such as how many hours a day, how to perform it or how to protect the patients. in this study, even though every patient had prone positioning for more than h a day, the exact duration showed little difference between each hospital. fourthly, the change in physiological measurements pertains to a difference between supine and prone position, and hence, the impact of chest wall is not taken into account. finally, in this study, we focused on influenza-related ards patients, and whether the result can be extrapolated to all patients with ards is unknown, requiring further investigation. to confirm the benefit of prone positioning in ards especially in influenza this study was designed to evaluate the effect of prone positioning in influenza pneumonia-related ards patients. after multivariate cox regression analysis, it was found that psi, renal replacement therapy and increased dynamic driving pressure were associated with -day mortality in patients with influenza pneumoniarelated ards receiving prone positioning. h n : viral pneumonia as a cause of acute respiratory distress syndrome pathogenesis of influenzainduced acute respiratory distress syndrome taiwan national infectious disease statistics system. taiwan centers for disease control critical care services and h n influenza in australia and new zealand critically ill patients with influenza a(h n ) infection in canada the berlin definition of ards: an expanded rationale, justification, and supplementary material european society of intensive care medicine, and society of critical care medicine an official american thoracic society/european society of intensive care medicine/society of critical care medicine clinical practice guideline: mechanical ventilation in adult patients with acute respiratory distress syndrome positive end-expiratory pressure setting in adults with acute lung injury and acute respiratory distress syndrome: a randomized controlled trial ventilation strategy using low tidal volumes, recruitment maneuvers, and high positive end-expiratory pressure for acute lung injury and acute respiratory distress syndrome: a randomized controlled trial ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome neuromuscular blockers in early acute respiratory distress syndrome prone positioning in severe acute respiratory distress syndrome conference on the scientific basis of respiratory therapy. pulmonary physiotherapy in the pediatric age group. comments of a devil's advocate prone position for acute respiratory failure in adults effect of prone positioning during mechanical ventilation on mortality among patients with acute respiratory distress syndrome: a systematic review and meta-analysis a multicenter retrospective review of prone position ventilation (ppv) in treatment of severe human h n avian flu treatment of ards with prone positioning acute respiratory distress syndrome: the berlin definition a prediction rule to identify lowrisk patients with community-acquired pneumonia apache ii: a severity of disease classification system defining community acquired pneumonia severity on presentation to hospital: an international derivation and validation study the sofa (sepsis-related organ failure assessment) score to describe organ dysfunction/failure. on behalf of the working group on sepsis-related problems of the european society of intensive care medicine effect of driving pressure on mortality in ards patients during lung protective mechanical ventilation in two randomized controlled trials prone positioning improves oxygenation in adult burn patients with severe acute respiratory distress syndrome prone ventilation reduces mortality in patients with acute respiratory failure and severe hypoxemia: systematic review and meta-analysis prone position for acute respiratory distress syndrome: a systematic review and meta-analysis writing committee of the who consultation on clinical aspects of pandemic (h n ) influenza influenza a infection and acute kidney injury: incidence, risk factors, and complications acute kidney injury among critically ill patients with pandemic h n influenza a in canada: cohort study acute kidney injury in criticallyill adult patients with seasonal influenza infection outcomes of acute kidney injury in patients with severe ards due to influenza a(h n ) pdm virus driving pressure and survival in the acute respiratory distress syndrome airway driving pressure and lung stress in ards patients a prospective international observational prevalence study on prone positioning of ards patients: the apronet (ards prone position network) study dynamic driving pressure associated with mortality in acute respiratory distress syndrome with extracorporeal membrane oxygenation driving pressure: a marker of severity, a safety limit, or a goal for mechanical ventilation? epidemiology, patterns of care, and mortality for patients with acute respiratory distress syndrome in intensive care units in countries we thank professor meng-chih lin, the president of the taiwan society of pulmonary and critical care medicine, who organized and coached the tsirc team. on behalf of all authors, the corresponding author states that there is no conflict of interest. not applicable. not applicable. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -llstohys authors: you, shu-han; chen, szu-chieh; liao, chung-min title: health-seeking behavior and transmission dynamics in the control of influenza infection among different age groups date: - - journal: infect drug resist doi: . /idr.s sha: doc_id: cord_uid: llstohys background: it has been found that health-seeking behavior has a certain impact on influenza infection. however, behaviors with/without risk perception on the control of influenza transmission among age groups have not been well quantified. objectives: the purpose of this study was to assess to what extent, under scenarios of with/without control and preventive/protective behaviors, the age-specific network-driven risk perception influences influenza infection. materials and methods: a behavior-influenza model was used to estimate the spread rate of age-specific risk perception in response to an influenza outbreak. a network-based information model was used to assess the effect of network-driven risk perception information transmission on influenza infection. a probabilistic risk model was used to assess the infection risk effect of risk perception with a health behavior change. results: the age-specific overlapping percentage was estimated to be %– %, %– %, and %– % for child, teenage and adult, and elderly age groups, respectively. individuals perceive the preventive behavior to improve risk perception information transmission among teenage and adult and elderly age groups, but not in the child age group. the population with perceived health behaviors could not effectively decrease the percentage of infection risk in the child age group, whereas for the elderly age group, the percentage of decrease in infection risk was more significant, with a . th percentile estimate of %. conclusion: the present integrated behavior-infection model can help health authorities in communicating health messages for an intertwined belief network in which health-seeking behavior plays a key role in controlling influenza infection. it has been found that health-seeking behavior has a certain impact on influenza infection. therefore, to facilitate public health decisions about intervention and management in controlling the spread of infectious diseases, it is crucial to assess to what extent, under scenarios of with/without control and preventive/protective behaviors, the age-specific network-driven risk perception influences influenza infection. to control respiratory infectious diseases, the development of vaccination, contact tracing, isolation, and the promotion of protective behaviors are the important measures. indeed, the effectiveness of control measures fundamentally depends greatly on human beliefs on public infection awareness and risk perception for driving the change in self-behavior. infection and drug resistance : submit your manuscript | www.dovepress.com you et al risk perception can be referred to as an awareness or belief about the potential hazard and/or harm, which plays an important role in shaping health-related behaviors to reducing susceptibility and infectivity. generally, risk perception is affected by social factors such as media release by health authorities, observation or interaction with relation-specific groups, past experiences of similar hazards, habits, and culture. these factors result in variation in risk perception among individuals. epidemiological studies have found that variances in risk perception can be observed by examining the behavioral responses among different age groups. steelfisher et al indicated that % of the adult population said that they did not intend to acquire the h n vaccine for themselves. in addition, perception of vaccine safety and personal vulnerability were the major reasons for vaccine acceptance. allison et al indicated that children could use accurate protective behavior; for example, they could use hand gel for preventing influenza. on the other hand, childhood vaccination is more likely to depend on parental decision making. moreover, researchers have suggested assessment of the risk perception and behavior across different age groups. , a social network could be an important social structure in which people could exchange information about risk-related events to spur the health behavior change. scherer and cho suggested that individual perceptions could be affected by self-perception in the social network. researchers have explored the interactions between epidemic spreading and risk perception in the network. , however, the influence of risk perception on the risk of infectious disease is controversial, because the perceptual capacity of individuals may both create and reduce the disease risks. therefore, the behavior-disease dynamics in the social network structure may result in amplification or attenuation of the disease outbreak. most epidemic modeling techniques have used a simple epidemic model such as the susceptible-infected-recovered (sir) model for describing a homogeneous disease network. moreover, the effects the network with human responses to disease spreading were studied extensively and attracted substantial attention. funk et al used sirbased perceptual-influenza model for examining the effects of risk perception on behavioral change and susceptibility reduction. they also indicated that the effects within a disease network can induce health behavioral changes in the population. in turn, the influence of risk perception could result in a feedback signal to alter the progress of the disease dynamics. , recently, information theoretical approaches have been applied to infer the relations in diseases or social networks. , zhao et al developed a model to quantify the effects of a dynamic network, indicating that the behavior responses correspond to the entropy derived from different information content of the dynamic social network. greenbaum et al proposed an information theoretic model to assess pandemic risk. they indicated that mutual information was a key determinant in minimizing risk of the pandemic threats. we have previously incorporated the information-theoretic framework into a behavior-influenza (bi) transmission dynamic system to understand the effect of individual behavioral change on influenza epidemics. , here we assess that if, how, and to what extent, under different scenarios of with/without control and preventive/protective behaviors, the age-specific network-driven risk perception influences influenza infection. in this study, we analyzed the emergency admission rates from the weekly ili visits, which were obtained from taiwan centers for disease control (tcdc) by sentinel primary care physicians. the ili cases were detected through the real-time outbreak and taiwan national infectious disease statistics system. the definition of an ili case must meet three criteria: ) fever (ear temperature ≥ . °c) and respiratory tract symptoms (including rhinorrhea, nasal congestion, sneezing, sore throat, cough, and dyspnea), ) one of the symptoms of muscle ache, headache, and extreme fatigue; and ) exclusion of simple running nose, tonsillitis, and bronchitis. data on emergency admission rates for six influenza seasons in the period from week of to week of were adopted to test how health-seeking behavior influences the influenza infection dynamics. influenza season was defined from july (week ) to june (week ) of the following year in taiwan. the ili-related emergency admission rates were detected by using the icd- -cm codes for influenza and pneumonia ( - ). we also estimated the age-specific admission infection fraction (if) for each age group, including child ( - years), teenage and adult ( - years) , and elderly ( + years), for different human behaviors or influenza risk perceptions. we multiplied the annual mid-year population estimates health-seeking behavior in the control of influenza infection , and then divided the result by the number of ili visits to estimate if, which is given as mid-year population incidence rate where i is the different age groups (child, teenage and adult, and elderly) and j is the yearly based time period in the period of - . the concept of the bi model developed in our previous studies , mainly incorporated the sir-based perception model into an information-theoretic framework, which was used to simulate the information flow of risk perception in response to an influenza outbreak. briefly, the bi model uses six compartments to represent the disease states of susceptible, infected, and recovered by dividing the population into a with/without perception structure. the description of input parameters for the bi model is given in table . basic reproduction number (r ) can be used to quantify disease infection severity, defined as the average number of secondary cases produced successfully by an infected individual in a totally susceptible population. therefore, based on the bi model, we can also estimate r with the perception state (r a ) and without perception state (r d ). it can be described as input source information with perception s a = r a = a / λ , where a is the rate of perception spread and l is the rate of perception loss. on the other hand, input source information can be described without perception is the infection rate describing contact between infected and susceptible populations and g is the recovery rate from infected to recovered populations. we assumed that r can be treated as the basic reproduction number resulting from individuals with risk perception information (r ,rpi ) for each age group in the period of - . thus, r ,rpi can be estimated as: . furthermore, to better characterize perception spread rate for different age groups during each year (a ij ) on the bi transmission dynamics, we adopted a ij from an epidemic equilibrium structure. the equilibrium information flow of risk perception from population without perception r d where r a e is the basic reproduction number at equilibrium with information flow of risk perception from population without perception, s i is the reduced infectivity factor from infected with perception to susceptible without perception, w is the rate of infected becoming with perception, a is the perception spread rate, and g w/o is the recovery rate of infected without perception. moreover, we assumed that people may make the decision to change behavior based on r ,rpi in the previous year. based on equation , a can then be rewritten as; where r ij ,pri, and r i j ,pri, + ( ) are the basic reproduction numbers with/without risk perception, respectively, for each age group in the period of - . to assess the effect of network-driven risk perception information transmission on influenza infection, we applied an information theoretic model referred to as the multiple access channel (mac) that is used to capture a signal r transmitting through multiple channels to the responses i , i , …, i n . we considered the network-driven risk perception information model (nm) with information bottleneck (ib). the maximum mutual risk perception information (mi max ) resulting from the nm can be estimated as; where n e is the effective information from contact numbers of individuals, s r is the variance of the r signal distribution, s ib→i is the variance introduced in each access channel through the ib to response i, and the s r →ib is the variance introduced to the ib. the ratio s s s is the signal-to-noise ratio. on the other hand, the nm model with a negative feedback was considered to explore the effect of perceived different health behaviors on reducing susceptibility. here, we used the correlation coefficient (r) and the overlapping percentage (i o ) to associate r and i from the published data (table s ) to calculate s s s in equation . we estimated the r based on the relationship between viral titer-based i and viral tier-based r corresponding to with/ without perceived different health behaviors. briefly, we selected published papers (table s ) where health behaviors treated with vaccinations and antiviral drugs for different subtypes of influenza were included. two protective behaviors (i.e., perceived of carrying the disease to vaccine and antiviral taking) were adopted in a state of greater alert. the value of r can be used to associate the amount of observed variability that is attributable to the overall biological variability and experimental noise. on the other hand, i o describes the age-specific overlapping percentage between the infected population with/ without perception adjusted by the fractions of initial infected population with perceptual state over those without perceptual state. here, we used three perceptual scenarios to assess our model with the initial infected population ratios of < , = , and > . the i o can then be estimated as algebraic manipulation of probability density functions (pdfs) of s a and s d as; thus, followed by the information-theoretic theorem with known values of r, i o , and s r , the s s can be computed as therefore, the nm model with a negative feedback in equation can be rewritten as; health-seeking behavior in the control of influenza infection where i represents the individuals perceived with/without health behaviors. we further incorporated the estimated probability distributions of the model parameter with age-specific initial population sizes in the period of - (table ) and a into the bi model, to estimate the age-specific overlapping percentages. to parameterize the reduced susceptibility factor with regard to adopting preventive behaviors, including using masks, avoiding visiting crowded places, and hand washing (s s,pre ), and protective behaviors of vaccination (s s,pro ), we applied a standard logistic regression-based equation for mathematically expressing the components of the healthbehavior model (hbm). the hbm-based health behavior with the standard logistic regression-based equation has been applied to estimate with/without preventive/protective health behaviors in respiratory infectious diseases such as severe acute respiratory syndrome (sars), influenza, - and other diseases. [ ] [ ] [ ] the estimates are equivalent to the decisions of rational individuals with influenza knowledge. here, s s can be expressed in terms of odds ratios (ors) depending on health behaviors perceived to be associated with each hbm variable. where s s is the probability of the hbm-based health behaviors (such as preventive behavior, s s,pre , and protective behavior, s s,pro ) and x is a binary variable with a value of indicating a "high" state and a value of indicating a "low" state. or is a calibration factor when all hbm variables are in a "low" state. s s represents that an individual engages in a particular behavior, and it could be calculated from equation . s s ≥ . indicates that an individual engages in a specific health behavior. r perception-based probabilistic risk assessment to develop a probabilistic risk model, a dose-response model describing the relationship between transmission potential quantified by signal r and the total proportion of the infected population (i) has to be constructed. in a previous study, we have successfully employed the joint probability distribution to assess the risk profile. it can be expressed mathematically as; where r(i) is the cumulative distribution function describing the probabilistic infection risk in a susceptible population at specific r signal, p(r ) is the probability distribution of r signal (the prior probability), and p(i|r ) is the conditional response distribution describing the dose-response relationship between i and r . the exceedance risk profile can be obtained by -r(i). in view of equation , we can relate p(i, r ) to r(i) in equation . thus, the mutual information in these interdependences between belief of risk perception and infection risk can then be expressed as a mechanism of interpersonal influence described in equation . in table , the numbers of ili visits were . × ± × (mean ± standard deviation [sd]), . × ± . × , and . × ± . × per month for child, teenage and adult, and elderly age groups, respectively. figure shows the ili-related emergency admission rates and if among the three age groups, child ( - years), teenage - ) , the ili-related emergency admission rates were estimated to be . ± . (mean ± sd), . ± . , and . ± . per , population for child, teenage and adult, and elderly age groups, respectively. overall, the ili-related emergency admission rate was the highest in the child age group ( . - . per , population, minimum-maximum), whereas the lowest one was observed in the teenage and adult age group ( . - . per , population; figure a ). on the other hand, the highest ili-related emergency admission if was in the child age group ( . ± . %), followed by teenage and adult ( . ± . %) and elderly ( . ± . %) age groups ( figure b ). to model the bi model, the age-specific perception spread rate (a) has to be determined (equation ). we first calculated age-specific r , pri based on the age-specific ili-related admission if. our results indicated that lognormal (ln) distribution with a geometric mean (gm) and a geometric standard deviation (gsd), ln (gm, gsd), was the most suitable fitted model for r , pri distributions of ln ( . , . ), ln ( . , . ), and ln ( . , . ) for child, teenage and adult, and elderly, age groups, respectively (table ). figure demonstrates age-specific overlapping percentage (i o ) between infected population with/without perception adjusted by fractions of initial infected population with perceptual state over those without perceptual state. we used three different scenarios of initial infected population fraction: i + /i -< (figure a-c) , i + /i -= ( figure d-f) , and i + /i -> ( figure g-i) . we showed that i + /i -> results in the lowest estimates of i o in child and elderly age groups ( figure g and i), whereas for teenage and adult age groups, the estimate was the highest in the case of i + /i -= ( figure e ). generally, i o estimates range from % to %, % to %, and % to % for child, teenage and adult, and elderly age groups, respectively ( figure ). thus, we used i o based on justified initial infected population fraction to further examine the mi max among each age group. health-seeking behavior in the control of influenza infection generally, the individual with risk perceptual status is more likely to have the communicable belief among the population. in the case of effective information from contact numbers of individuals (n e = ; figure a ), when i + /i -< and i + /i -= , the mi max was < bit. on the other hand, when i + /i -> , as the perceptual information increased in the population, mi max was > bit, indicating that the network-based information reflected cooperativity. our results showed that mi max -n e profile featured a smooth shape ( figure b ). in the elderly group, as the strength of n e increased, mi max -n e profile experienced a nearly smooth curve. on the other hand, in the child and elderly groups, when n e was > , mi max was > bit. our results indicated that mi max ranged from . to . bits, . to . bits, and . to . bits for child, teenage and adult, and elderly age groups, respectively, given n e ranging from to ( figure b ). to explore the impact of n e -varying perceived health behavior information on mi max , we estimated correlation coefficient (r) based on the relationship between viral titerbased i and viral tier-based r corresponding to, with and without, perceived different health behaviors. the resulting estimates were r w = . and r w/o = . ( figure s ). we further used equation to calculate mi max based on overlapping percentage (i o ) and r affected by n e . the results indicated that mi max ranged from . to . bits, . to . bits, and . to . bits for without control, and preventive, and protective behaviors in the child age group, respectively ( figure a ). for the teenage and adult age group, mi max ranged from . to . bits, . to . bits, and . to . bits for without control, and preventive, and protective behaviors, respectively ( figure b ). our results showed that individuals perceived the health behaviors to increase mi max among child, and teenage and adult, age groups ( figure a and b, respectively). our results also revealed that individuals perceived the preventive behavior to improve risk perception figure b) , and the elderly age group ( figure c ), but not in the child age group. our results indicated that there was % probability for exceeding the infected fraction of population (i); . , . , and . for child ( figure a ), teenage and adult ( figure b ), and elderly ( figure c ) age groups, respectively, in the condition of without perceived health behaviors. however, there was a % probability for reducing the infected fraction of population within the ranges of . - . for preventive behavior and . - . for protective behavior ( figure ). the age-specific ∆mi with respect to with/without health behaviors was estimated based on equations and . we found that, for instance, without any control information released, the median percentages of decrease in infection risk with ∆mis = and for elderly were ( . - ) and ( . - ), respectively, whereas the child age group had the lowest estimates of ( . - ) and ( . - ), respectively ( figure a ). on the other hand, ∆mi estimates at incremental mi changes with perceived health behaviors were %- %, %- %, and %- % for child, teenage and adult, and elderly age groups, respectively ( figure b and c). the population with perceived health behaviors could not effectively decrease the percentage of infection risk in the child group, whereas for the elderly age group, the percentage of infection risk decreased more significantly with a . th percentile estimate of % ( figure b and c). our results indicated that children may be preferable to adopt the protective behaviors. allison et al indicated that the use of hand gel for hygiene was a feasible strategy in elementary schools to prevent influenza spread. our results implicate that children could use the accurate knowledge about the protective behavior to prevent influenza infection effectively. health-seeking behavior in the control of influenza infection our results found that the perceived protective behaviors enhanced the mi max in adults, whereas the perceived vaccination behavior might not. a meta-analysis of eligible studies also confirmed that raising risk perception from low to high would have a potential effect on vaccination behavior of adults. we suggest that future studies should detect the differences among the health behaviors in adults. schneeberg et al indicated that the vaccination rate for seasonal influenza was consistently low among the elderly population in canada. walter et al indicated that the elderly population failed to obtain information about vaccine perception from the internet directly. it was also found that face mask wearing was easily performed by older adults in hong kong. elderly people also appeared to be more active in conducting preventive measures. in this article, we incorporated the probability-based hbm with regard to specific health behaviors into an sirbased epidemiological model. the hbm was used to examine individual's perceptual dimensions such as perceived susceptibility, severity, benefits, and barriers. however, the hbm has led to somewhat controversial issues in exploring the health behaviors such as for vaccination programs. , the hbm presents a rational point of view that assumes the perceiver to be uninfluenced by the emotion, on describing the human you et al response to an epidemic. , our study, however, establishes a more robust mechanistic framework on modeling the influence of network-driven risk perception on influenza infection. to our knowledge, we have conducted the first step on exploring the effects of risk perception in a population, on the spread of epidemics. we believe that our present methodology provides an innovative approach that integrates an epidemiology model with the information theory. we examined three scenarios for describing different agespecific populations to overcome susceptibility risk due to less accurate knowledge of influenza. we found that noise effect, which reflects as overlapping percentages about the uncertainty of accurate knowledge of influenza, can reduce risk perception information transfer on the network through the epidemic transmission. for example, previous studies found that participants had misconceptions between seasonal vaccine and pandemic strain. , the effect of overlapping response may have resulted from the public health campaigns. for example, people were recommended to acquire the seasonal vaccine in pandemics. this may lead to a feedback mechanism between behavior change and disease dynamics. future work should carefully consider the effects of this noise on specific age groups. moreover, each intervention should be carefully investigated to the extent possible during an epidemic. this study has several limitations. the estimation of information flow of risk perception in age groups depended on the ili data. indeed, the human response to influenza varied with time and hence is not possible to detect in realtime situations. moreover, perceptual states in specific age groups may be affected by the severity levels of disease, the amount of accurate information about influenza, and other health-related leaflets. therefore, we suggest that the health authorities could reinforce health monitoring by using information technology and then linking it to the real-time epidemiological surveillance systems. a further limitation of our study is that we did not consider the influential factors on risk perception in an epidemic model. hence, future research should explicitly consider a number of additional influential factors on risk perception within an epidemic modeling, including disease prevalence, network effects, and government and media health messages. the findings of our study have an implication for public health. risk communication might be more effective if health authorities focus on a variety of information communication channels for conveying health behavior messages. moreover, our findings concerning perception of different health behaviors show substantial differences among age groups. we found that perceived protective behaviors (e.g., covering mouth, coughing hand washing) could reduce the infection risk for all age groups. this suggests that such crucial information for control measures would allow for targeting resources to designing and implementing the education plans concerning perception of healthy behaviors that are least perceived in the health behaviors. we developed an integrated mathematical model by incorporating the epidemiological transmission dynamics, the information flow of human responses, and an information theoretic model to assess the effects of network-driven risk perception on influenza infection risk. the simulated human responses with perceived health behaviors could decrease the risk of infection among different age groups. we demonstrated that the risk perception among populations changed with the effective information varying with the contact numbers of individual. we conclude that the present integrated bi model can help public health authorities on communicating health messages for an intertwined belief network in which health-seeking behavior plays a key role in controlling influenza infection. dynamic modeling of vaccinating behavior as a function 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human influenza: randomized, controlled trials for prophylaxis and treatment double-blind evaluation of oral ribavirin (virazole) in experimental influenza a virus infection in volunteers dose response of a/alaska/ / (h n ) cold-adapted reassortant vaccine virus in adult volunteers: role of local antibody in resistance to infection with vaccine virus efficacy and safety of low dosage amantadine hydrochloride as prophylaxis for influenza a cold recombinant influenza b/texas/ / vaccine virus (crb ): attenuation, immunogenicity, and efficacy against homotypic challenge evaluation of the infectivity, immunogenicity, and efficacy of live cold-adapted influenza b/ann arbor/ / reassortant virus vaccine in adult volunteers effects of the neuraminidase inhibitor zanamivir on otologic manifestations of experimental human influenza oral oseltamivir in human experimental influenza b infection the authors acknowledge the financial support of the ministry of science and technology, republic of china, under grant most - -e- - -my . all authors contributed toward data analysis, drafting, and critically revising the paper and agree to be accountable for all aspects of the work. the authors report no conflicts of interest in this work. infection and drug resistance is an international, peer-reviewed openaccess journal that focuses on the optimal treatment of infection (bacterial, fungal and viral) and the development and institution of preventive strategies to minimize the development and spread of resistance. the journal is specifically concerned with the epidemiology of antibiotic resistance and the mechanisms of resistance development and diffusion in both hospitals and the community. the manuscript management system is completely online and includes a very quick and fair peerreview system, which is all easy to use. visit http://www.dovepress.com/ testimonials.php to read real quotes from published authors. health-seeking behavior in the control of influenza infection key: cord- - sm pgt authors: timurkan, mehmet ozkan; aydin, hakan; sait, ahmet title: identification and molecular characterisation of bovine parainfluenza virus- and bovine respiratory syncytial virus - first report from turkey date: - - journal: j vet res doi: . /jvetres- - sha: doc_id: cord_uid: sm pgt introduction: bovine parainfluenza virus- (bpiv ) and bovine respiratory syncytial virus (brsv) are the cause of respiratory disease in cattle worldwide. with other pathogens, they cause bovine respiratory disease complex (brdc) in ruminants. the aim of the study was the detection and molecular characterisation of bpiv and brsv from nasal swabs and lung samples of cows in and around the erzurum region of eastern turkey. material and methods: in total, samples were collected. of animals used in the study were males and females. the age of the animals was between months and years, mean . years. most males were in the fattening period and being raised in open sheds; females were in the lactating period and kept in free stall barns. all samples were tested for the presence of viral genes using rt-pcr. gene-specific primers in a molecular method (rt-pcr) identified brsv (fusion gene) and bpiv (matrix gene) strains at the genus level. results: rna from brsv and bpiv was detected in two ( . %) and three ( . %) samples, respectively, one of each of which was sequenced and the sequences were aligned with reference virus strains. phylogenetic analyses clustered the strains in genotype c/bpiv and subgroup iii/brsv. conclusion: the results indicate that brsv and bpiv contribute to bovine respiratory disease cases in turkey. this is the first report on their detection and molecular characterisation in ruminants in turkey. bovine respiratory disease complex (brdc) is a major problem for cattle breeders worldwide, causing serious economic losses. brdc is associated with infection by certain viruses, bacteria, and parasites ( ) . in addition to these infectious agents, stress factors such as transport, gestation, and poor management conditions play an important role in the onset of the disease ( ) . bovine herpes virus (bhv- ), bovine respiratory syncytial virus (brsv), and bovine parainfluenza virus- (bpiv ) are the most common viral agents of the respiratory system. some opportunistic agents (mannheimia haemolytica, pasteurella multocida, haemophilus somnus, and mycoplasma spp.) contribute to the appearance of clinical signs and thus increase mortality and cause losses in the herds ( ) . suppressed immunity also has an important role in the prognosis. diseases such as bovine leucosis and bovine viral diarrhoea suppress immunity and lead to more animal loss by worsening clinical symptoms. bpiv (the new name of which is bovine respirovirus ) is an rna virus assigned to the paramyxoviridae family under the respirovirus genus. brsv (the new name of which is bovine orthopneumovirus) is in the pneumoviridae family under the orthopneumovirus genus. to date, three genotypes of bpiv have been described. these genotypes, termed a, b, and c, were differentiated based on phylogenetic analysis. genotype a strains have been isolated in north america, china, and japan. genotype b was originally found in australia. isolations of genotype c were in china, south korea, and japan. in addition, all three genotypes have been reported in argentina ( ) . initially brsv subgroups were identified (a, b, and ab or intermediary) based on monoclonal antibody and polyclonal sera analyses against f and g proteins ( ) . additionally, valarcher et al. ( ) proposed that six genetic subgroups may be found in brsv strains, when f, g, and nucleoprotein sequences are phylogenetically analysed by maximum-likelihood algorithms. therefore, six subgroups were detected in brsv. these subgroups termed i (the subgroup b prior to the recommendation of valarcher et al. ( ) ), iii (subgroup a), and ii, iv, v, and vi (subgroup ab) were differentiated based on phylogenetic analysis. subgroup i consists of european strains (uk and switzerland). subgroup iii includes viruses exclusively from the usa. subgroup ii aggregates strains from the netherlands, belgium, france, denmark, sweden, and japan. subgroup iv is of european and usa strains while subgroups v and vi are found only in french and belgian isolates ( , ) . subgroup vii was detected in later years ( ) and some strains are known which are still not classified (these are regarded as untyped) ( ) . bpiv and brsv can cause mild symptoms or subclinical disease when present alone. however, when there is a co-infection, they may cause bronchopneumonia, severe cough, high fever, and nasal discharge and contribute to a more serious clinical course of infection ( ) . regardless of the infecting agent in brdc, clinical symptoms may be similar and the process of detecting the underlying primal agent may be hindered due to mixed bacterial infections. this situation makes viral diagnosis difficult and decreases the specificity and sensitivity of the molecular methods (when compared to immunofluorescence antibody tests) ( ) . data on virological detection of these agents in turkey is limited ( , ), but there are more studies on seroprevalence of these viruses among cattle herds. the studies reported lowest and highest seropositivity of % ( ) and . % ( ) for bpiv and % ( ) and % ( ) for brsv. serological studies on brsv and bpiv were previously conducted in different geographic regions of our country. in these studies the following percentage values for brsv and bpiv prevalence were determined respectively: alpay et al. ( , ) . the aim of this study was the detection and molecular characterisation of bpiv and brsv strains retrieved from nasal swabs and lung samples of cows in the eastern region of turkey. the determination of brsv and bpiv types and associated co-infections for respiratory system infections was conducted. sampling. nasal swabs and lung tissue samples were gathered from cattle from the erzurum district and its neighbouring provinces. table shows the origin and quantity of the collected samples. a total of animals were used in the study ( males and females). the herd consisted of cross-breeds of brown swiss, holstein, and simmental. the age of the animals ranged between months and years and their mean age was . years. the males were in the fattening period and kept in open sheds; the lactating females were housed in free stall barns. samples were acquired from animals with nasal discharge and severe coughing. in total samples were collected including lung tissue samples and nasal swabs. although sampling was performed throughout the year, the winter and autumn months were generally preferred as the sampling period (table ) . extraction of the viral nucleic acid. nasal swabs were vortexed in ml of pbs and centrifuged at , rpm for min. supernatant was collected and µl was used for extraction. a total of mg of each tissue sample was homogenised in µl pbs using a tissue lyser ii homogenizer (qiagen, germany) and centrifuged at , rpm at room temperature, and then µl of the supernatant was used for extraction. a gf- viral nucleic acid kit (vivantis, malaysia) was used for nucleic acid extraction according to the manufacturer's recommendations. all extracts were kept at − °c before testing. reverse transcription (rt). viral rna was subjected to reverse transcription using a first strand cdna synthesis kit (thermo fisher scientific, germany) according to the manufacturer's instructions. pcr. samples were tested by pcr using genespecific primer pairs. primers specific for the geneencoding fusion region for brsv and matrix gene for bpiv were used. primer sequences, their related gene regions, and the size of estimated amplicons are shown in table . taq dna polymerase (thermo fisher scientific, germany) was used for the pcr which was run under previously described conditions ( , ) . sequencing. after pcr, the samples with the clearest virus-specific pcr bands were subjected to sequencing. samples were sequenced (with the sanger sequencing method, bi-directionally with forward and reverse primers) by the pendik veterinary control institute, istanbul, turkey. obtained sequences were submitted to genbank as bovine respirovirus isolate bpiv /tr/erz/ under accession number ky and bovine orthopneumovirus isolate brsv/tr/erz/ under accession number ky , and then were analysed phylogenetically. phylogenetic analysis. the raw sequence data and reference virus sequences obtained from genbank were aligned using bioedit version . . . software ( ) . the aligned data were subjected to phylogenetic analysis using mega version ( ) to give bootstrap analyses ( , replicates) by the neighbour-joining method. tamura's two-parameter model was used and bootstrap values are shown in the phylogenetic tree. polymerase chain reaction (pcr). among samples, two were brsv-and three bpiv -positive. four of the positive samples were swabs, and one was a lung sample. the bpiv -positive samples originated from erzurum-aşkale province (swab), erzurum-aziziye province (swab), and erzurum slaughterhouse ( nd period, apr. ) (lung sample). brsv-positive samples originated from erzurum-aziziye province (swab) and bayburt-centre (swab). all animals identified as positive were young cattle. phylogenetic analysis. nucleotide sequences of the viruses identified in the study were closely related to asian and european strains (figs and ) . the phylogenetic analysis of the fusion (f) protein of brsv showed that the identified brsv strain belonged to subgroup iii (subgroup a) and was in the same cluster as reference viruses (ku strain usii/s /usa and fj strain /brazil). the matrix gene sequence of the bpiv strain sequenced from a bpiv -positive sample was classified into a cluster of genotype c. there are various diseases affecting young and old cattle worldwide. these diseases can impair the respiratory, digestive, and genital systems ( , , , ) . however, it is viral diseases associated with the respiratory system of cattle which are regarded as one of the main problems in cattle breeding, affecting both adults and young animals. this holds especially true when mortality rates increase in case of mixed infections. in this study, brsv and bpiv virus strains were detected in pool of lung tissue and nasal swab samples collected from cattle from erzurum and neighbouring provinces. bpiv and brsv were detected by other researchers with serological and virological methods in cases of lower respiratory tract infections in cattle in turkey ( , , ) . however, these studies were not performed by molecular methods and did not include genetic characterisation as we have done. although it was proved that in cases of lower respiratory tract infections lungs could contain a high number of virus particles ( ), we could detect only bpiv , even though samples were collected in late winter. this may be because the animals were in a convalescent phase of infection or a majority of tissue samples suspected of being from pneumonic animals did not contain virus particles. enveloped viruses, paramyxoviruses in particular, are generally susceptible when exposed to the outside environment. pirtle and beran ( ) reported that when diluted to . ccid /ml and used to contaminate different kinds of media, the viability time was . h on rubber gloves, min on skin, and a maximum of h on an indoor surface. this is why samples must be admitted to the laboratory in a short time and tested in the shortest time possible. when low positivity rates are considered, both in this study and worldwide, there is always the possibility of false negativity because of virus sensitivity. pcrs specific for the gene-encoding fusion region for brsv and the matrix gene for bpiv were used. the fusion gene of brsv is a region that determines antigenic variations ( ) , and the matrix gene for bpiv is responsible for viral assembly, pathogenesis, and antigenicity of strains ( , ) . phylogenetic analysis of brsv based on the f protein sequence classified the isolates into seven different subgroups. the topology of the phylogeny was retained when an analysis of the n and g protein gene sequences was conducted ( ) . in our study, the determined strain was found to belong to subgroup iii. knowing the genotypes helps to determine the typespecific vaccine selection in the geographic region. valarcher et al. ( ) identified vaccine failure among animals infected with brsv groups v and vi, indicating that commercial vaccines act poorly against infections caused by such viral groups. therefore, selection of vaccines is important and should be specific to the type of strains present in the region. for example in the s in europe, vaccines including rispoval rs (strain rb- , subgroup ii), bayovac (strain lehmkuhl , subgroup iii), and vacores (strain / , subgroup ii) were used. if these vaccines were currently in use, bayovac could be suitable for our country. however, in vivo trials would be required for other vaccines. therefore, genotyping studies of viruses are necessary for molecular epidemiology and vaccine studies. it is well established that respiratory tract infections are mostly mixed. these types of infections may contain viruses, bacteria, and other agents or may be caused by virus-virus or bacteria-bacteria interaction ( , ) . we investigated the samples with single or mixed (brsv + bpiv ) infections and no other mixed infections were detected. therefore, when viruses are the sole cause of infection, it is highly likely that they will cause subclinical infections; this may explain the low rate of detection in this study. aetiology in respiratory infection is quite important ( , ) . a fast and reliable method must be selected to identify the underlying agent. genotyping studies may provide information about strains that cause serious clinical onsets related to vaccine strains and preventive status. phylogenetic analysis of the brsv strain was performed (fig. ) . analysis of the partial sequence of brsv shows that our strains were more closely related to american and brazilian (usa: ku ; and brazil: fj ) strains instead of european ones. although the strains identified in the study are expected to be close to the european strains (because of the geographical location of turkey), the increase in meat and live animal imports into our country over the last years may be a factor in this situation, because our country imports meat from usa, canada, brazil, and uruguay. many infectious diseases may be spread among cities, countries, and continents as a result of animal or animal product transportation ( , ) . in our study, brsv sequences showed high genetic similarity with the american and brazilian strains. this genetic similarity among brsv strains suggests that it could have come from brazil or america, as a result of live animal exports. infection with bpiv is common in cattle worldwide. the virus is divided into three genotypes: a, b, and c ( , , , ) . genotype a is most common in america, genotype b in australia and genotype c is seen mostly in asia ( , , ) (fig. ) . our samples clustered in genotype c in the phylogenetic tree. this is the first study to report bpiv of this type (genotype c) in turkey. this also may be regarded as the first report in europe since no published data is available as far as we are aware. in conclusion, this paper is the first report of brsv and bpiv infections in cattle in the east anatolian region of turkey and specifically erzurum and its neighbouring provinces by molecular methods. there is a need for additional epidemiological studies to better understand the prevalence and control of the infections. regional and local infections should be investigated and properly addressed through random sampling. for implementing control and eradication studies at a national level, full genome sequence data should be provided in further studies. the authors declare that there is no conflict of interests regarding the publication of this article. a study on investigations of occurrence of some virus infection in buffaloes in turkey virological and serological studies on the role of pi- virus, brsv, bvdv, and bhv- on respiratory infections of cattle. i. the detection of etiological agents by direct immunofluorescence technique sığırlarda viral nedenli solunum sistemi enfeksiyonlarinin seroepidemiyolojisi. ankara Üniv vet fak derg distribution of g (vp ) and p (vp ) genotypes of group a bovine rotaviruses from turkish calves with diarrhea bir ada ekosistemindeki sığır, koyun ve keçilerde bazı viral enfeksiyonların serolojik olarak araştırılması. ankara Üniv vet fak derg detection of respiratory viral antigens in cattle lung tissues by direct elisa antibody prevalence against respiratory viruses in naturally infected cattle in central anatolia study on the etiologic role of bovine respiratory syncytial virus in pneumonia of dairy calves genetic characterization of bovine respiratory syncytial virus strains isolated in italy: evidence for the circulation of new divergent clades detection of an untyped strain of bovine respiratory syncytial virus in a dairy herd doğu ve güneydoğu anadolu bölgesinde süt sığırlarında parainfluenza virus- , bovine herpes virus- ve respiratory syncytial virus enfeksiyonlarının seroepidemiyolojisi. yyÜ vet fak derg potential risk of regional disease spread in west africa through cross-border cattle trade seroprevalence of viral upper respiratory infections in dairy cattle. kafkas Üniv vet fak derg bovine respiratory disease research laboratory test descriptions for bovine respiratory disease diagnosis and their strengths and weaknesses: gold standards for diagnosis, do they exist? the animal-human interface and infectious disease in industrial food animal production: rethinking biosecurity and biocontainment bioedit: a user-friendly biological sequence alignment editor and analysis program for windows / /nt effect of stress on viral-bacterial synergy in bovine respiratory disease; novel mechanisms to regulate inflammation identification of two distinct bovine parainfluenza virus type genotypes complete nucleotide sequence of the matrix gene of human parainfluenza type virus and expression of the m protein in bacteria sequence characterization of the matrix protein genes of parainfluenza virus types a and b isolation and characterization of bovine parainfluenza virus type from water buffaloes (bubalus bubalis) in argentina identification and genome characterization of genotype b and genotype c of bovine parainfluenza type viruses isolated in the united states molecular characterization of a korean bovine parainfluenza virus type isolate molecular characteristics of bovine virus diarrhoea virus isolates from turkey: approaches for an eradication programme analysis of the bovine respiratory syncytial virus fusion protein (f) using monoclonal antibodies virus survival in the environment identification of three antigen epitopes on the nucleocapsid protein of the genotype c of bovine parainfluenza virus type epidemiology, molecular epidemiology, and evolution of bovine respiratory syncytial virus bovine respiratory disease in feedlot cattle: environmental, genetic, and economic factors phylogenetic relationships of brazilian bovine respiratory syncytial virus isolates and molecular homology modeling of attachment glycoprotein mega : molecular evolutionary genetics analysis version . one-step multiplex real time rt-pcr for the detection of bovine respiratory syncytial virus, bovine herpesvirus , and bovine parainfluenza virus phylogenetic analysis of a partial l gene from bovine papillomavirus type isolated from naturally occurring papilloma cases in the northwestern region of turkey the detection and molecular characterization of bovine respiratory coronavirus infection by rt-pcr in erzurum evolution of bovine respiratory syncytial virus development of nested pcr assays for detection of bovine respiratory syncytial virus in clinical samples development of a real time reverse transcriptase polymerase chain reaction for the detection of bovine respiratory syncytial virus in clinical samples and its comparison with immunohistochemistry and immunofluorescence antibody testing development and evaluation of two truncated recombinant np antigen-based indirect elisas for detection of bovine parainfluenza virus type antibodies in cattle serological evaluation of viral infections in bovine respiratory tract seroprevalence of bovine respiratory viruses in north-western turkey. trop anim health prod isolation and genetic characterization of bovine parainfluenza virus type from cattle in china key: cord- -kbpvt on authors: atherstone, c.; galiwango, r. g.; grace, d.; alonso, s.; dhand, n. k.; ward, m. p.; mor, s. m. title: analysis of pig trading networks and practices in uganda date: - - journal: trop anim health prod doi: . /s - - - sha: doc_id: cord_uid: kbpvt on east africa is undergoing rapid expansion of pig rearing, driven by increasing pork consumption. introduction and expansion of pig production systems in this biodiverse landscape may create new risks, including zoonotic pathogen transmission. historically, biosecurity measures have primarily been focused at farm level, ignoring the important function pig traders fulfill between farmers and consumers. this study interviewed pig traders operating at uganda’s only registered pork abattoir to describe their characteristics, business practices, biosecurity practices, and pig health management and reporting practices. all the traders were male, and nearly all ( . %) relied on pig trading as their primary source of income. most of the pigs brought for processing at the slaughterhouse were purchased from smallholder farms ( . %). in addition, there was a significant difference in the high price paid per kilogram at farm gate by region (p = . ). high prices paid at farm gate were associated with holiday periods (p < . ), harvest season (p < . ), and drought (p < . ). traders preferred buying live pigs from male farmers ( . %) because they were considered the final decision makers and owned the pigs being sold. all pig traders were aware of clinical signs indicating a pig was sick. this study has provided baseline information on pig trader practices in uganda. improvements in local pork slaughterhouses and markets will benefit not only pig traders in accessing consistent customers but also individual pig farmers by increasing their market access. finally, given their role as a link between farmers and consumers, traders would benefit from targeted inclusion in disease control and prevention strategies. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. the domestic pig population in uganda, currently estimated at . million, plays an essential economic and social role in a country, in which % of households derive some or all of their livelihoods directly from livestock (uganda bureau of statistics ). pig keeping has grown in popularity as a livelihood activity due to their high reproduction rates, rapid weight gain, potential to provide quick financial returns, and rising demand for pork. in the past years, pork consumption has increased more than -fold, from an estimated annual per capita consumption of . kg in to . kg in . pork currently accounts for more than a third of the annual per capita meat consumption (food and agriculture organization (fao) ) . further, total pork consumption is projected to increase by % between and in uganda due to human population growth (food and agriculture organization ) . the increase in pork consumption, while exceptionally rapid in uganda, is not unique in the region. the democratic republic of congo's total consumption of pork is expected to increase by %, tanzania by %, and kenya by % between and (food and agriculture organization ) . introduction and expansion of pig production systems in these biodiverse landscapes may create new risks, including pathogen transfer from pigs to humans (ocaido et al. ; wilson ; food and agriculture organization (fao) ; atherstone et al. ; hamill et al. ; food and electronic supplementary material the online version of this article (https://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users. agriculture organization ). of particular public health interest is the role of pigs in the zoonotic transmission of emerging pathogens to people (atherstone et al. ; vergara-alert et al. ; middleton and westbury ; ma et al. ; kobinger et al. ; conlan et al. ; mccormack and allworth ; abubakar et al. ; marsh et al. ) . as pig traders form an important link between pig farms and pork customers, research informing their knowledge, attitudes, and practices is essential. african swine fever (asf) is considered the major infectious disease constraint to pig production in africa (penrith et al. ) , and as such, research to date has focused on this infection. penrith and vosloo reported that outbreaks of asf in new areas of africa have almost all been associated with movement of domestic pigs and pig products (penrith and vosloo ). in uganda, several studies to characterize practices associated with the occurrence and spread of asf identified the collection of pigs and pig products from farms (kabuuka et al. ) , distribution of infected pork by traders , pig movements due to restocking and trade (kalenzi atuhaire et al. ; nantima et al. ) , free range movement of pigs on farms (nantima et al. ) and trade of live pigs and pig products (tejler and teijler ) as risk factors. these studies focused on pig farmers and their perception of trading and pig movements. a limited number of studies have targeted pig traders, but again, these were restricted to knowledge and practices related to asf transmission and control (dione et al. ; chenais et al. ; muhangi et al. ) . despite the link between disease spread and pig movement, little is known about broader trading practices, motivations for buying, and patterns of purchases and sales of pigs in uganda. given pig traders' important role in supplying pork for a rapidly expanding consumer base and linking farmers with consistent markets, a better understanding of their practices and motivations around purchasing, transportation, and pig health management is needed. this would assist in developing policies that specifically support traders and the important functions they serve while identifying suitable interventions to ensure a safe, reliable pork supply. therefore, the objectives of this study were to ( ) describe pig trader characteristics, trading practices, biosecurity practices, pig health management, and reporting practices and ( ) map source locations of pigs purchased to supply pork through the major abattoir in uganda. wambizzi cooperative society limited is located in nalukolongo, southwestern kampala, uganda's capital city. wambizzi was selected as it is the only registered pig abattoir in uganda and has many pig traders supplying live pigs to meet the urban demand for pork. as a registered slaughterhouse, carcasses processed at wambizzi are visually inspected by kampala city council authority (kcaa) meat inspectors and stamped bfit for human consumption.^pork processed at the abattoir is sold in the greater kampala area to pubs, pork joints, hotels, butchers, supermarkets, and private organizations (non-governmental organizations, missions, and private individuals). the slaughterhouse has a capacity of pigs/ day, but the supply of pigs fluctuates substantially throughout the year (roesel et al. ) . according to slaughterhouse records, an average of , kg of pork was processed monthly from april to may . using the estimated annual per capita consumption of . kg of pork (food and agriculture organization (fao) ) and the kampala population of . million (uganda bureau of statistics ), wambizzi produced roughly . % of the pork consumed in kampala during this time frame. pig slaughtering also occurs in backyards and slaughter slabs to supply informal, roadside pork joints and butcheries. pig traders privately operating from wambizzi use the abattoir facility to slaughter their pigs and have their pork inspected and stamped to meet requirements in the formal marketplace. traders buy live pigs from farms/markets and aggregate them into groups for transport to the abattoir. during this interim period (farm gate to abattoir), pigs are under the ownership and care of traders. once processed, traders sell the pork to their own customers in the sales building adjacent to the evisceration and inspection building. while membership with the abattoir is not required, traders pay a fee per pig ( ugx, i.e., . usd in ) to use the abattoir facilities (roesel et al. ). pig traders were interviewed between october and october , during periods corresponding with national holidays when the sale and consumption of pork increases (roesel et al. ; ouma et al. ) . there is no formal register of pig traders operating at wambizzi. in preparation for this research, a member of the research team with prior experience working with pig traders informally questioned traders on site over several days to develop a more recent estimate of the number of traders operating from wambizzi. based on this, the total number of traders operating at wambizzi was estimated at . thus, we aimed to interview traders over the course of the study. a variety of methods were used to recruit participants, including direct approach and active-snowballing. traders arriving at the abattoir were approached by a local member of the research team and initially asked about their interest in learning about the study. if the trader expressed interest, information on the scope and purpose of the research was provided orally. traders who indicated that they were willing to participate in the research study were asked to give written consent to be interviewed. if the trader was unable to give written consent due to physical impairment or illiteracy, their thumbprint was provided in place of signature. additional traders were identified by asking participants who had completed the interview for the name and contact information of other pig traders operating from wambizzi. furthermore, we observed trader brands on pigs at slaughter (e.g., number or letter carved on the animal at the time of purchase) and asked participants who had completed the interview if they could identify the trader who supplied the pig. the enumerator then contacted these newly identified pig traders to invite their participation in the research study. a local enumerator with previous experience working with pig traders in uganda was recruited and trained for data collection. a structured questionnaire was adapted from previous research conducted with pig traders by the international livestock research institute (ilri) in uganda under the smallholder pig value chains development project (cgiar livestock and fish research program ) . the questionnaire captured information on pig trader characteristics, live pig buying practices, transportation practices, and pig health management. traders also reported the sub-counties where they had purchased live pigs over the months prior to the interview date. the questionnaire was developed in english and translated into the local language (luganda). the questionnaire comprised primarily closed-ended questions to keep the interview to a maximum of min. openended questions regarding buying practices and clinical signs observed in pigs were included, with answers recorded exactly as the interviewee stated. the full questionnaire is included in the supplementary materials. the slaughter process started at am each morning and preceded the selling of pork from am to am. the enumerator was on site by am each morning to identify pig traders previously not interviewed. however, to ensure that the interview did not conflict with pig trader's business, most interviews took place between am and noon each day. data from the questionnaires was entered into epi info . (centers for disease control, atlanta, ga, usa). following data cleaning, data was exported to spss . (ibm corp., armonk, ny, usa) for analysis. standard descriptive analysis was performed for categorical and quantitative variables describing pig trader characteristics, live pig purchasing practices, transportation practices, and pig health management. population pyramid style graphs were prepared to compare pork demand and pork farm gate prices by months. source locations (reported to the sub-county level) were entered into microsoft excel and checked for spelling accuracy. the subcounties were then joined to the centroid of each sub-county polygon in the global administrative unit layers for uganda (food and agriculture organization, rome, italy) using arcgis . (environmental systems research institute, redlands, ca, usa). the number of pig traders operating in each sub-county was mapped using graduated symbols. two binary outcome variables of interest were explored further, namely high price paid at farm gate over the last year ( / ) and low price paid at farm gate over the last year ( / ). reasons given for prices paid were recoded into binary variables (yes/no) and used as explanatory variables. univariable binomial logistic regression analyses were conducted to evaluate the associations of the binary explanatory variables with both the outcome variables. explanatory variables with a p value < . were included in two multivariate regression models for high or low price paid to evaluate associations after adjusting for other variables in the model. in addition, non-parametric analyses were conducted for four quantitative variables (high price paid per kilogram, low price paid per kilogram, number of live pigs bought during high demand weeks, and number of live pigs bought during low demand weeks) to identify significant differences by operating region (kruskal-wallis test), number of districts (mann-whitney u test), and number of regions (mann-whitney u test) traders purchased live pigs from. operating region was identified based on the location pig traders reported purchasing live pigs in. because price and number of pigs purchased were not identified by individual districts and many pig traders operated in multiple regions, operating region was binned into three categories: central only, eastern only, and all other regions (including western, northern, and responses that covered multiple regions). number of regions and number of districts a pig trader operated in were recoded into two responses: above median and below median. independent variables with significant differences (p < . ) were subject to post-hoc pairwise comparisons to identify which specific responses(s) were significantly different from each other. a total of interviews were conducted with pig traders operating from wambizzi between october and october . no traders declined participation in the study. pig trader characteristics are shown in table . all pig traders interviewed were male and ranged in age from to years (median years; first quartile (q ) = , third quartile (q ) = ). the median number of years working as a pig trader was years (range months- years; q = , q = . ). a large proportion ( . %; / ) of participants had not completed primary school. most pig traders were engaged in trading as their primary source of income ( . %; / ) and described their business operation as fixed ( . %; / ), meaning that they had established locations for buying live pigs and selling pork. proximity to pork customers was the primary reason for having a fixed business operation ( . %; / ) with most traders supplying pigs solely to wambizzi ( . %; / ). when asked about other pig traders operating in their areas, almost all the traders had competition for live pigs in their buying areas ( . %; / ). almost two thirds of the pig traders were not members of a trading group or cooperative ( . %; / ). buying and transportation practices are shown in table . most traders sourced their pigs directly from smallholder farms ( . %; / ). only one trader reported purchasing pigs at a livestock market. when pig traders were asked about whom they prefer to purchase pigs from at the farm, . % ( / ) preferred buying from men rather than from women. reasons offered by traders who preferred to purchase from men included that men were the following: the decision makers on the farm, faster decision makers, and owned the pigs being sold. further analysis to understand this gender preference was not possible because the number of responses for women were all less than . lorries (trucks) were the most common type of vehicle used to transport pigs to the abattoir ( . %; / ). the majority of traders rented the vehicles they used to transport pigs ( . %; / ). vehicles were cleaned after each use ( . %; / ) using both water and laundry washing powder ( . %; / ). none of the pig traders reported using bleach or any other type of disinfectant to clean their vehicles. the pig waste (feces, urine, bedding) left in the vehicle after transporting the pigs was most commonly heaped at wambizzi for crop farmers to collect and use for compost in their gardens ( %; / ). june and december were frequently identified as months with high customer demand for pork, whereas february and september were associated with low customer demand (fig. ) . during months when demand for pork was low and high, respectively, traders bought a median of . pigs (range - pigs/week; q = ; q = ) and . pigs (range - pigs/week; q = ; q = ) per week. figure shows the months traders associated with paying high or low farm gate prices to purchase pigs. the median reported high prices at farm gate was ugx/kg (range − ugx/kg; ugx = usd as of january ). when low prices were paid at farm gate, the median was ugx/kg (range - ugx/kg). reasons for paying high and low prices at farm gate are shown in table . in multivariate logistic regression, holiday period, crop/coffee harvesting season, and drought were significantly associated with high price paid, whereas drought, school fees due time, and sick pigs were significantly associated with low price paid. figure shows the source locations of pigs purchased on the day of interview and preceding months. pig traders reported buying live pigs in one to eight districts (median ) across one to three regions (median ) in uganda. thirty-six percent of traders purchased live pigs only in the central region (n = ) and % of traders purchased live pigs only in the eastern region (n = ). farm gate prices by operating region as well as the number of districts/regions that a pig trader operates in are outlined in table . pig traders operating in one region paid significantly higher prices per kilogram at farm gate than traders operating in two to three regions (p = . ). the number of live pigs purchased per week by operating region and number of districts/regions that a trader operates in is shown in table . during months when demand for pork was low, the region(s) a pig trader operated in to purchase live pigs was significantly associated with the number of pigs purchased (p = . ). pig traders operating in only central region purchased a significantly higher number of pigs during low demand months than traders operating in only eastern region (p < . ). traders operating in only the eastern region purchased a significantly lower number of pigs during low demand months than traders operating in all other regions (p = . ). all the pig traders reported recognizing clinical signs indicating a pig was sick. when asked to list these signs, the most commonly stated signs were dropping of ears ( %; / ), reddening of ears ( . %; / ), straightening of the tail ( . %; / ), and weakness or difficulty standing ( . %; / ). traders typically did not report pigs considered to be sick to anyone ( . %; / ). if there was reporting, the trader informed a meat inspector on site at wambizzi ( %; / ) or a veterinary officer ( %; / ). if sick pigs were observed while under the traders' care, . % of the traders did nothing to care for the sick pigs ( / ). if action was taken, the sick pig was slaughtered at wambizzi and the meat sold ( . %; / ). this is the first study to describe ugandan pig trader characteristics and business practices around live pig buying, transportation, and health management. the prices paid to farmers for their pigs were associated with the number of regions and districts a pig trader operates in. in addition, pig traders reported paying higher prices during holiday periods and harvest season (crops/coffee). traders preferred buying live pigs from male farmers because they considered them the final decision makers and owned the pigs being sold. finally, we found that all pig traders checked for clinical signs in pigs that indicated the animal was sick. pig traders who operate in only one region paid, on average, higher prices per kilogram to farmers for their pigs. considering that such traders are likely to travel shorter distances to source their pigs, compared to those traders that work in more regions, this may suggest that the distance travel outcome variables with less than five responses were not including in the univariable analysis b p value = . . explanatory variable removed from multivariable logistic regression model fig. source locations of live pigs on the day of interview and preceding months, as reported by pig traders interviewed at wambizzi cooperative society slaughterhouse, kampala, uganda, to source pigs has an impact on the price paid for the pigs, with traders traveling less offering higher prices to farmers. however, another possibility may be that traders who operate in multiple regions are large-scale traders, buying pigs in bulk, and therefore paying lower prices. nevertheless, given the importance of pigs as an asset within smallholder farming households, it is advantageous when pig farmers can secure more income from the sale of their pigs. given that almost half the traders in this study operated in four or more districts in uganda, pigs are traveling large distances from farm to slaughterhouse. reducing the distance pigs' travel for processing is both an animal welfare and a disease mitigating practice, especially for limiting the dissemination of asf (tejler ) . thus, there is a need for locally regulated slaughter facilities and/or improved transport infrastructure throughout the country to reduce the distance traveled from farm to slaughterhouse. centralized slaughter facilities would also help address the lack of consistent market access, a commonly cited constraint among pig farmers (ouma et al. n.d.; wabacha et al. ; kagira et al. ; muhanguzi et al. a ). moreover, traders in this study reported that the primary reason for maintaining a fixed business operation was proximity to pork customers. local slaughter facilities would provide a centralized location for traders to access customers. this study also found that holiday periods, harvest season, and drought were the most commonly cited reasons trader paid high prices at farm gate. other studies have noted that smallholder farmers keep pigs as a source of cash in times of need (dione et al. ; deka et al. ; gichohi et al. ) . when pig traders need to source live pigs around harvest season, they pay more for these pigs because farmers have recently sold their crops and, therefore, do not need the additional cash generated from the sale of a pig. the situation is a little different around the holidays. an increase in pig sales and pork consumption during festive seasons is well documented (roesel et al. ; dione et al. ; adams et al. ; kambashi et al. ) . it is possible that traders have a harder time sourcing the number of pigs they need at the holiday period or that farmers know of the increased holiday demand and raise their prices. the higher prices paid for pigs around holidays are inducements for farmers to time their pig rearing activities to take advantage of the economic benefit of having pigs ready for sale according to holiday periods. free ranging, tethering, and feeding of crop residues and grasses to pigs are common practices among smallholder pig farmers in east africa chenais et al. ; tejler ; kagira et al. ; dione et al. ; muhanguzi et al. b; nantima et al. ) . in all these production systems, drought would reduce the amount and quality of feed available for pigs and, therefore, would reduce the number of pigs suitable for sale. it is also possible that farmers intentionally chose not to rear grower and fattener pigs during known seasons of drought. it will be important to address live pig supply issues, whether at holiday periods, drought, or from other causes, to ensure a consistent pork supply so that consumers are able to access the quality and quantity of pork they demand. at the farm level, this research found that pig traders have a strong preference to buy live pigs from male pig farmers. a study in kenya found that the decision to keep pigs was made by men (simiyu and foeken ) . because of the large financial investment, technical knowledge required to raise pigs and role as sole decision makers in their home, men maintained control of pigs and leveraged this control for any financial decisions made over the animals (simiyu and foeken ) . these subtle cultural values around livestock ownership and household financial decision making come into play in accessing markets for livestock and livestock products. women tend to face more challenges than men in accessing and benefiting from markets, especially more formal markets (kristjanson et al. ) . furthermore, given women's traditional responsibility for household food security, their level of control over decisions about whether to sell or consume the family's animal products, as well as over how to use any income obtained from the sale of animal foods, could greatly determine the nutritional well-being of household members (kristjanson et al. ) . while there is clearly a need for support of women at the household level in accessing markets for their livestock, this research shows that there is also a need to work with pig traders to enable female pig farmers to access consistent markets for their pigs. in this study, all the traders interviewed observed clinical signs they described as indicating a pig was sick. the frequently observed clinical signs such as reddening of the ears, dropping of ears, and weakness or affected movements are consistent with clinical signs of asf (chenais et al. ; tejler ; dione et al. ) . despite recognition of these signs as indicators of sickness, traders failed to report these suspected cases to the proper authorities. similar findings have been reported in previous research in uganda chenais et al. ; muhangi et al. ; nantima et al. ) and are consistent with studies conducted in indonesia (leslie et al. ) . given that traders play an essential role in transporting pigs, there is a need to develop policies and strategies to integrate pig traders into disease reporting and disease mitigating strategies without fear of recrimination or detriment to their business. in addition, given the pressures pig traders are under to meet quality standards of pork customers, pig traders would benefit from training on disease mitigating strategies including safe and hygienic slaughter practices, perhaps through an industry association or group. this would also address the gap between traders admitting that they are responsible for conducting their business in support of disease prevention but do not perceive themselves as key actors in the control of disease (dione et al. ). there are several limitations to this study. first, responses to the questionnaire are subject to recall bias. this is especially true of answers around the number and location of pigs purchased over the months prior to the interview. however, the number of pigs for which traders provided a source location (n = ) was considerably less than the number of pigs processed at the slaughterhouse (n = , for july -june ) (roesel et al. ). it appears that when the pig traders were unsure of actual numbers, they underreported, or only reported the location they sourced the pigs without any accompanying number of pigs purchased. self-reported locations are likely to be reliable as the traders used communitybased scouts to identify pigs for sale. we purposely interviewed pig traders during periods when demand for pork was historically high and theorized that this would mean that more pig traders would be bringing pigs in for processing. however, it is possible that there are pig traders that only operate sporadically and would have been missed in this study. we worked with the pig traders at wambizzi to identify other pig traders to interview. we also cataloged the brands on each pig being processed, as each trader has a unique symbol to identify their pigs once they have been processed. every effort was made to identify all potential research participants. previous research, undertaken with the management at wambizzi, stated that there were pig traders regularly operating from the premises (roesel et al. ) . given this, our study team managed to identify three times the number of pig traders. further analysis beyond descriptive analyses was hindered by the low number of responses for certain variables. for example, we were unable to analyze why pig traders prefer buying live pigs from men rather than women. given the priority of gender empowerment in uganda and the significance of livestock in alleviating poverty for women and children, it is important to identify ways to support women in accessing markets for their pigs. more detailed interviews and focus groups may shed additional light on trader buying decisions. this study has provided baseline information on pig trader practices in uganda. the prices paid at farm gate for pigs are affected by the number of regions and districts a trader operates in to procure pigs. given the animal welfare and disease transmission implications of pigs traveling over multiple districts and regions from farm to slaughterhouse, consideration should be given to establishment of local pork slaughterhouses and markets and improvements to transport infrastructure. furthermore, pig traders prefer buying live pigs from male farmers. for women to overcome the challenges of accessing formal livestock markets, there is a need for additional research to identify how women can access pork markets in uganda, particularly if pig traders are involved. finally, this research shows that pig traders are observing sick pigs but fail to report these sick pigs. historically, disease control interventions have been focused 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gulu district outbreaks of african swine fever in domestic pigs in gulu district the national livestock census report livestock susceptibility to infection with middle east respiratory syndrome coronavirus characterisation of smallholder pig production in kikuyu division, central kenya zoonoses and antimicrobial resistance in kenya and malawi acknowledgements the authors would like to thank the management of wambizzi cooperative society who facilitated this research and the pig traders operating at wambizzi for their involvement in this study. this research benefited from the technical input and tools developed by ilri uganda colleagues ben lukuyu, emily ouma, michel dione, and kristina roesel.funding funding for this study was provided by the cgiar research program on agriculture for nutrition and health, led by the international food policy research institute. statement of animal rights this human study has been approved by the appropriate ethics committees and has therefore been performed in accordance with the ethical standards laid down in the declaration of helsinki and its later amendments. the authors declare that there is no conflict of interest. key: cord- -c jdp jv authors: fu, yangxi; tang, zhengzhen; ye, zhixu; mo, shi; tian, xingui; ni, ke; ren, luo; liu, enmei; zang, na title: human adenovirus type infection causes a more severe disease than type date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: c jdp jv background: human adenovirus type (hadv- ) and (hadv- ) cause significant morbidity and develop severe complications and long-term pulmonary sequelae in children. however, epidemiologic reports have suggested that nearly all highly severe or fatal adenoviral diseases in children are associated with hadv- rather than hadv- . here, we conduct in-depth investigations to confirm and extend these findings through a comprehensive series of assays in vitro and in vivo as well as clinical correlates. methods: a total of nasopharyngeal aspirate (npa) samples were collected from hospitalized children with acute respiratory infections in children’s hospital of chongqing medical university from june to may . among samples that tested positive for hadvs, clinical data of cases of hadv- ( ) and hadv- ( ) infections were analyzed. all hadv-positive samples were classified by sequencing the hexon and fiber genes, and compared with clinical data and virological assays. we also performed in vitro assays of virus quantification, viral growth kinetics, competitive fitness, cytotoxicity and c a assay of the two strains. mouse adenovirus model was used to evaluate acute inflammatory responses. results: clinical characteristics revealed that hadv- infection caused more severe pneumonia, toxic encephalopathy, respiratory failure, longer mean hospitalization, significantly lower white blood cell (wbc) and platelet counts, compared to those of hadv- . in cell culture, hadv- replicated at a higher level than hadv- , and viral fitness showed significant differences as well. hadv- also exhibited higher c a production and cytotoxic effects, and hadv- -infected mice showed aggravated pathology and higher pulmonary virus loads, compared to hadv- -infected mice. macrophages in balf remained markedly high during infection, with concomitant increase in pro-inflammatory cytokines (tnf-α, il- β, ifn-γ, and il- ), compared hadv- infection. conclusions: these results document that hadv- replicates more robustly than hadv- , and promotes an exacerbated cytokine response, causing a more severe airway inflammation. the findings merit further mechanistic studies that offer the pediatricians an informed decision to proceed with early diagnosis and treatment of hadv- infection. human adenovirus is well-recognized as an important pathogen of respiratory tract infection in childhood [ ] [ ] [ ] . more than hadv serotypes are further subdivided into seven species (a-g) [ , ] ; however, species b (hadv- , , and ), c (hadv- , , and ), and e (hadv- ) are the ones most associated with respiratory infections [ ] . epidemiological studies have reported an approximately - % hadv-positive rate among acute respiratory tract infections in children [ , ] . adenovirus infections may in fact result in high morbidity and mortality in children, and fatality rates for untreated severe hadv pneumonia or disseminated disease may exceed % [ ] . severe adenovirus infection in children can be complicated with pleural effusions [ ] , acute respiratory distress syndrome (ards) [ ] , respiratory failure [ ] , myocarditis [ ] , and central nervous system dysfunction [ ] , leading to either mechanical ventilation or extracorporeal life support, even death. unfortunately, effective adenoviral vaccine for children and specific antiviral drugs against human adenoviruses are currently not available. hadv- and hadv- , belonging to subgenus b , cause infections that are usually mild and self-limiting in immunocompetent individuals; however, severe, and even life-threatening infections and outbreaks, associated with both type and in children, are increasingly reported [ , , ] . epidemiology of the disease suggests that hadv- and hadv- are the major types responsible for lower respiratory diseases in children less than years old worldwide [ , , , ] . in beijing, china, hadv- , and hadv- were the most common serotypes causing pneumonia from to [ ] . in chongqing, china, . % of the hadv- -infected patients were diagnosed as having severe pneumonia, significantly higher than the . % of the hadv- -infected patients from to [ ] . in taiwan, hadv- and hadv- , causing complication with respiratory failure, accounted for % of infected children; seven cases due to hadv- were among ten deaths reported in a single year, between and [ ] . in korea, both hadv- and hadv- cause epidemics of severe lower respiratory tract infections in young children. overall, and %, of the children infected with hadv- and hadv- , respectively, required mechanical ventilation [ ] . certain adenovirus serotypes are associated with particular clinical features and severe illness; however, no study has offered persuasive evidence for in the different degrees of disease severity caused by hadv- and hadv- infection because it still limits on the basis of clinical observation. in the present study, therefore, we undertook a comprehensive analysis of the comparative clinical features of hadv- and hadv- infection, as well as a serial of experiments, were performed to better understand the association between severity of the disease and the serotypes of hadvs. participants, demographic data, clinical data analysis patients ranging in age from month to years and requiring inpatient treatment due to acute respiratory tract infections (arti) at the department of respiratory medicine, children's hospital of chongqing medical university between june, and may, , were enrolled in this study. parents/guardians provided demographic data and medical history during an interview. severe disease was defined as respiratory failure confirmed by an abnormal blood gas analysis result (an oxygen saturation level of approximately % or less) or toxic encephalopathy. a total of nasopharyngeal aspirates (npas) were collected on the day of hospital admission after obtaining permission from the patients or their guardians. imaging and laboratory data on admission and during hospitalization were collected. white blood cell (wbc) count > , / μl was defined as leukocytosis, whereas that < /μl was defined as leukopenia. the study procedure was approved by the ethics committee of the children's hospital of chongqing medical university. informed consent was obtained from parent or guardian of all participants. hadv isolation, identification, and molecular typing npas from hadv-positive patients were inoculated into hep- cell monolayers and cytopathic effect (cpe) was monitored for days. viral dna and rna were extracted from μl aliquots of the npa samples by a qiaampminelute virus spin kit (qiagen, hilden, germany). hadv-positive samples were used to amplify the hexon hypervariable regions (hvrs) - and fiber genes for subsequent sequencing [ , ] . the hadvs strains were molecularly typed by pcr amplification and sequencing of all seven hypervariable regions in the hexon gene, described previously [ ] . the hadv- (cq ) and hadv- strains (cq ) used in this study were originally isolated from npas of children infected with hadv. the virus was purified using a standard cesium chloride gradient centrifugation procedure, as described [ ] . the concentration of viral particles (vps) was determined by spectrophotometry according to the a value. the virus concentration was calculated as vps/ml = a *dilution* . three cell lines (a , hbe, and hek- ) at % confluency in -well plates were infected with either hadv- or hadv- at a multiplicity of infection (moi) of vp/cell. viruses were cultured as previously described [ , ] . the culture supernatants and cells were sampled at h post-infection. viral loads were quantified by quantitative pcr (qpcr). briefly, μl reaction mixtures were prepared by adding μl of sample nucleic acid extract to μl of iq supermix (bio-rad, hercules, ca), primers and probes. the sequences of primers and probes for qpcr and the amplification conditions have been detailed [ , ] . the real-time hadv primer sequences were as follows: hadv- forward, ′-ggga gacaatattactaaagaaggtttgc- ′, hadv- reverse, '-caacttgaggctctggctgata- ′. the sequence of the hadv- probe was '-cactac"t"-gaaggagaagaaaagcccatttatgcc, which was labeled with -carboxyfluorescein (fam) and internally quenched at "t" with black hole quencher- on the ′end and phosphate on the ′-end. the forward primer sequences of hadv- were '-gaggagccag atat tgatatggaatt- ′, the reverse primer sequences of hadv- were: '-aattgacattttccgtgtaaagc a- ′, and probe, '-aagctgctgacgctttttcgcc tga- ′. the hadv- probe was labeled with -carboxy fluorescein (fam) on the ′-end and ′-terminally quenched with black hole quencher- . the thermal cycling condition was as follows: one cycle of min at °c; cycles of s at °c; and min at °c. a cells at % confluency in -well plates were infected with either hadv- or hadv- (moi = vps/ cell). after h adsorption with rocking every min, the inoculum was removed, ml of fresh dmem-f with % fbs was added to each well, and the plates were incubated at °c in % co . the culture media were harvested at , , , , , , , , and h post-infection. tcid was used to titer the virus at each time point. a cells were infected with a mixture hadv- and hadv- at a ratio of : and moi of vps/cell in standard -well cell culture plates, and incubated as described under 'viral growth kinetics'. media supernatants and cells were harvested at , , , , , , , h post-infection. virus copies were quantified at each indicated time point by qpcr described earlier [ , ] . a cells ( . × ) were seeded in each well of -well flat bottom plates and incubated at °c in % co overnight. thereafter, the media were removed, and cells were infected with hadv- or hadv- (low moi = vps/cell, or high moi = vps/cell). infected and uninfected cell viability were assayed at , , , , and h post-infection and the absorbance was determined as described [ , ] . cell viability was expressed as the ratio, and the mean (mean ± sem) of replicates of two strains was presented. all samples were collected as described previously, with minor modifications [ ] . purified hadvs ( . g) in pbs were mixed with a : dilution of normal human serum (nhs) in an assay volume of μl, and incubated for , and min at °c. the samples were further diluted : and tested using an opteia human c a elisa kit (bd biosciences), according to the standard procedure provided by the manufacturer. female balb/c mice, - -week old, were purchased from the animal laboratory of chongqing medical university and housed in a dedicated pathogen-free facility at chongqing medical university. the use of all experimental animals strictly met the ethical requirements of the animal committee of chongqing medical university (license numbers: syxk(yu) - ). the mice under anesthesia were infected intranasally (i.n.) with × vps/ml of hadv- or hadv- in μl. mock-infected mice were instilled with μl of phosphate-buffered saline (pbs) under the same conditions. mice were processed after , , and days of infection. for studies of viral load, total viral nucleic acid was directly extracted from the right lung lobe, using a qiaamp mini-viral dna extraction kit (qiagen, hilden, germany) and following the manufacturer's recommendations. quantification of hadv- and hadv- copies were performed as described earlier [ , ] . after , , and days of infection ( mice in each time point in every group), the animals were euthanatized by intraperitoneal injection of pentobarbital ( mg/kg). the left pulmonary hilum was closed by clamping, in order to collect balf from the right side. the specific steps were as described previously [ ] . briefly, bronchoalveolar lavage was performed using . ml of ice-cold sterile pbs for a total of times. balf was centrifuged at rpm for min in cold ( °c). the supernatant was aliquoted and stored at °c for subsequent cytokine measurements. the precipitate was resuspended in ml of pbs, and the total number of cells was counted under a microscope. the cells were centrifuged, smeared onto slides, fixed, and stained with diff quik (baxter healthcare corp, deerfleld, miami, fl) to analyze and classify white blood cells. cell counting was performed using a microscope. a total of random cells was counted from each slide and included macrophages, lymphocytes, and neutrophils. mice were sacrificed at , , and d after infection for pathological analyses. the left lung lobe was collected, fixed in % paraformaldehyde for h, dehydrated, and then embedded in paraffin. lung tissue blocks were cut into μm thick sections that were then stained with hematoxylin and eosin to evaluate lung tissue pathology associated with hadv- and hadv- infection. the levels of interferon-γ (ifn-γ), tumor necrosis factor-α (tnf-α), interleukin- β (il- β), and interleukin- (il- ) in balf were all measured using commercial murine elisa reagent kits (neobioscience, shenzhen, china) according to the manufacturer's instructions. descriptive statistics were performed for all variables; the continuous variables were summarized as means and standard deviations (sd) or as medians and ranges, and the categorical variables were summarized as frequencies and proportions. the graph pad prism . software was used for data analysis. statistical significance was assessed by one-or two-way analysis of variance (anova). one-way anova was used to analyze significant differences between three or more groups. two-way anova was used to analyze significant differences between two variables. if an overall test was significant, the tukey's test was utilized for specific comparisons between individual groups. differences were considered significant at p < . . more severe disease in patients infected with hadv- compared to hadv- among the npas, specimens were identified as positive for adenovirus, of which hadv- ( ) and hadv- ( ) were the most common types. as shown in table , age and gender distribution, underlying disease, and coinfection were comparable between hadv- and hadv- . co-infection with other respiratory viruses were observed in . and . % of patients infected with hadv- and hadv- , respectively. the most common clinical manifestations of hadv- and hadv- infections, such as cough, wheezing, croup, dry rales, moist rales and fever, including the peak of febrile body temperature (> °c), were not significantly different. in contrast, pneumonia was the most common diagnoses of the patients. among the hadv- -infected patients, . % were diagnosed as having severe pneumonia, significantly higher than that of the hadv- -infected patients ( . % vs . %, p = . ). when the median duration of hospitalization was analyzed, we found that the hadv- inpatients had a longer mean duration of hospitalization, . ± . days (p = . ). several severe outcomes, toxic encephalopathy, and respiratory failure were significantly higher in patients with hadv- infection compared with those with hadv- (p < . ), but no statistically significant differences were found in icu admission, pleural effusion, suspicious bo and intracranial infection. notably, one child, infected by hadv- , exhibited complications of acute respiratory distress syndrome (ards). laboratory findings for the hadv- -positive inpatients were also significantly different from those infected by hadv- (table ) . specifically, the hadv- -positive inpatients had lower white blood cell count ( . ± . vs. . ± . × cells/l; p = . ), platelet count ( . ± . vs. . ± . × cells/l; p = . ). in contrast, hemoglobin and c-reactive protein levels, and the percentages of lymphocytes, neutrophils and positive sputum culture were found to be statistically similar. in the chest radiographs (table ) , alveolar infiltration, consolidation and pleural effusion were more frequently observed with the hadv- patients compared to hadv- patients but they did not differ significantly. increased lung texture and interstitial inflammation showed a similar proportion. two patients infected with type , and four patients infected with type , had atelectasis. notably, two patients infected with adenovirus type had complications with pneumothorax. since type-specific adenovirus infection is known to cause different tissue tropisms and clinical manifestations as indicated before, viral loads and fitness of hadv- and hadv- were evaluated in several human epithelial cells to determine if there were differences. as shown (fig. a) , at h post-infection, hadv- replicated to a higher number of viral copies than hadv- in all three cell lines tested, namely, a , hek- and hbe cells. hadv- also exhibited stronger growth than hadv- in a cells at , and h post-infection (fig. b) . in comparing the competitive fitness between hadv- and hadv- , however, no significant difference was observed. lastly, hadv- loads were significantly higher than those of hadv- at , , and h post-infection (p < . ) (fig. c) . cytotoxicity assay may serve as a measure of cellular distress induced by viral infection. as shown (fig. ) , at a low moi of vps/cell, viability of hadv- -infected cells was slightly higher than that of the hadv- -infected cells, but there was no statistically significant difference. in contrast, at high moi of vps/cell, hadv- induced a remarkably greater decrease in cell viability at both and h post-infection ( . and . %, respectively; p < . ) (fig. ) . to compare complement activations by hadv- and hadv- , the anaphylatoxin c a, a split fragment of c (the step where classical, lectin, and alternative pathways of complement activation merge), was measured in vitro. as shown (fig. ), hadv incubation with serum, compared with nhs incubation alone, resulted in a marked increase of c a at , and mins. importantly, elevation of c a by hadv- , compared with hadv- , was statistically higher at mins (p < . ). to better understand the differences in adenovirus-induced airway inflammation, we established a mouse respiratory infection model for human adenovirus. in this model, assays of viral load in the lung tissues revealed that virus copies of hadv- -infected groups were higher than those of hadv- -infected groups at , and days of infection, particularly on day , when virus copies in the hadv- group were . -fold higher than the hadv- group (p < . ) (fig. a) . pulmonary inflammation in mice was also observed following hadv- and hadv- infection at each time point and found to gradually increase from day to day , decreasing on day . compared with hadv- -infected mice, lung tissue damage of hadv- -infected mice was also higher on all days of infection (fig. b) . consistent with the morphological changes, the total number of cells in balf was higher, compared with those in the control group on all days (p < . ) (fig. c-e) ; importantly, the hadv- group showed a significantly greater increase in these cells, in comparison with the hadv- group (p < . ) (fig. d-e) . specifically, abundant macrophages and smaller number of lymphocytes, neutrophils infiltrated into the balf at all days post-infection. whereas the neutrophils registered a slight rise, only on day , followed by dramatic decrease, the lymphocyte and macrophage numbers remained persistently elevated through days and . in addition, macrophages in balf of the hadv- group were significantly higher than the hadv- group after and days of note: data are shown as medians (interquartile range) or number (%) abbreviations: wbc white blood cell, crp c-reactive protein " a ": a significant difference between two groups infection ( fig. d-e) , while the lymphocytes exhibited significant difference only on day (p < . ) (fig. d) . neutrophils in balf of the hadv- group was not significantly different compared with that of the hadv- group at all time points (fig. c-e) . together, these results showed that hadv- infection induced a relatively more severe acute airway inflammation. the levels of inflammatory mediators associated with airway inflammation, including il- β, tnf-α, ifn-γ, and il- , were also clearly elevated in balf at , , and days post hadv- and hadv- infection. nevertheless, the levels of il- β, il- , and tnf-α in the hadv- group were higher than the hadv- group post , , and days of infection (p < . ) (fig. f-i ). hadv- and hadv- have been prevalent throughout the world for decades, and both serotypes frequently play an important role in the etiology of lower respiratory infection in children. in the present study, coinfection with hadv- and hadv- with other respiratory viruses accounted for half of the pediatric cases, but coinfection per se did not result in a difference in clinical manifestation or severe clinical outcomes. these results and the severe respiratory diseases and complications, which we identified with hadv- infection in hospitalized children, were all consistent with previous reports [ , , , ] . remarkably, we also found a higher rate ( . %) of toxic encephalopathy associated with hadv- infection. previous studies in japan showed that mild encephalitis/encephalopathy with a reversible splenial lesion (mers) could be triggered by adenovirus infection [ , ] , which was proposed to be caused by neurotoxins, likely acting as an antigen that induced a strong immune response, leading to a series of symptoms. recently, silkworm chrysalis ingestion was shown to induce toxic encephalopathy [ ] ; however, our results constitute the first reported case of toxic encephalopathy associated with adenoviral infection. earlier studies reported that several viruses, including adenoviruses, influenza a virus, parainfluenza virus, and japanese encephalitis virus, can use the olfactory nerve as a shortcut into the central nervous system, causing infection of nerve cells and induction of immune response [ ] ; however, the exact mechanisms of such neurotropic infections remain to be elucidated. in the current study, we have compared and correlated the biological characteristics of hadv- and hadv- with their properties ex vivo and in vitro. first of all, the results of cellular infectivity (fig. a) indicated that a , hbe, and hek- are susceptible cell lines for the growth of adenovirus ex vivo. furthermore, a difference of viral loads recorded in a and hbe cell lines between adenovirus and indicated differential inflammatory reactions were produced from infection of host respiratory epithelial cells by hadv stimulated the innate immune system that might partially explain the role of hadv- infection in the pathogenesis of a more severe pneumonia. this is likely because the airway epithelial cells not only serve as a passive barrier to infectious particles but also actively participate in the innate immune response to foreign antigens. the immune response of epithelia to infection and antigen exposure involves the release of chemokines and cytokines into the submucosa, which initiates an inflammatory reaction [ ] . the results of co-infection of the two viruses indicated that distinctive fitness is type-specific and not interaction. the result of cell viability also provided useful information that was important for understanding type-specific viral virulence (fig. ) . taken together, we conclude that the differences observed in the replication, fitness, and virulence of the two adenoviruses are dependent on virus type. the complement system plays important roles in innate immunity and is vital in the protection against invading pathogens. the complement cascade can be initiated through three main pathways: classical, lectin, and alternative. the classical pathway is mainly antibodydependent. it is activated by interaction of c q (a subunit of the first complement protein c ) with the fc portion of the igm or igg immune complex, although activation can also be achieved in an antibody-independent manner by some membrane components of viruses, bacteria and fungi. the lectin pathway is first activated when the mannose-binding lectin, a structural homologue of c q, binds to carbohydrate moieties on the surface of pathogens, including yeast, bacteria and viruses. the alternative pathway is antibody-independent; it is spontaneously activated on biological surfaces, as well as in plasma and other body fluids, when the level of c hydrolysis remains consistently low. this spontaneous cleavage readily initiates amplification of the activation cascades [ ] . although every pathway is triggered independently, all of the complement cascades culminate in the central cleavage of c and the generation of its active fragments c a and c b, which can bind covalently to viral components to aid in opsonization and phagocytosis. furthermore, complement activation also mediates the inflammatory reaction via the generation of anaphylatoxins (c a and c a) and recruitment of inflammatory cells to the site of infection [ , ] . therefore, activation of the complement system leads to inflammation, opsonization, and membrane perturbation. previous studies by johnson et al. and others [ , [ ] [ ] [ ] have shown that the complement is strongly activated by a wide range of negative-strand rna viruses, including parainfluenza virus (piv ), nipah virus (niv), mumps virus ; c-e the number of total cells and the differential counting of cells in balf. f-i concentration of cytokine il- β, tnf-α, and il- in balf; n = ; *,**,*** indicate p < . , . , and . , respectively, compared with the mock group; #, ##, ### indicate p < . , . , and . , respectively, compared with hadv- group implicate the plasma level of c a as a marker of the early innate response to adenovirus [ ] . in previous studies, our group successfully established a hadv- pneumonia model in the laboratory mouse, and studies in this in vivo model suggested that hmgb is a mediator of hadv- -induced pulmonary inflammation [ ] . our current study has addressed whether different types of human adenovirus infection result in distinct inflammatory reactions in vivo. in this study, we clearly observed robust virus growth, and also documented significantly higher viral load in hadv- infected mice at days post-infection. these results are different from those reported by kajon et al. in which only low levels of hadv- and hadv- replication were seen at the early stage of infection in mice [ ] . we speculate that the apparent discrepancy is due to the effect of different experimental animal models and detection methods used. adenovirus infection is characterized pathologically by a time-dependent progression in the type of inflammatory cells present. the initial inflammation is a neutrophilic interstitial infiltration with neutrophilic alveolitis. subsequently, monocytes become evident and, finally, predominantly lymphocytic infiltrates appear. during the acute stage of hadv- and hadv- infection, we also found that the total number and classes of cells in balf increased, primarily accounted for by macrophages. neutrophils only transiently increased at days post-infection. a mixture of mononuclear cells (macrophages and lymphocytes) exclusively became evident at and days postinfection. macrophages were most prominent than neutrophils and lymphocytes at all time points. these results are fully consistent with several studies, including those of kajon et al., which showed that hadv- and hadv- -induced pneumonia mainly produces neutrophil infiltration at and days post-infection, and affects macrophages [ ] . apart from phagocytosing and killing the pathogens, macrophages also secrete chemokines to recruit cells to the sites of infection. yoon et al. found that adenovirus type produced a more robust il- and il- response than adenovirus in human bronchial epithelial cells [ , ] . diaz et al. compared cytokine responses between type and type , and documented significantly higher production of interferon-γ from type [ ] . similarly, subsequent production of il- β, induced by macrophages, also reached the highest levels after , , and days of hadv- and hadv- infection. asgari et al. have reported that engagement of the c a receptor triggers il- β processing and release via caspase- activation [ ] . the results are consistent with the immune response of the epithelia to adenovirus infection and antigen exposure, which involves the release of chemokines and cytokines into the submucosa and initiates an inflammatory reaction. lastly, complement activation mediates the inflammatory reaction via anaphylatoxins (c a and c a) and recruitment of inflammatory cells to the site of infection. the inflammation then leads to the recruitment of phagocytes that help clear the invaders. collectively, we conclude that these innate immune responses play an important role in the initial defense against adenovirus infection. although our results implicate a novel type-specific relationship between adenoviral infection, it has potential limitations. the data presented herein only interrogated virological differences; however, adenovirus infection is a very complex process that involves not only the structural proteins of the virus, such as fiber, hexon, and penton, but also includes host and environmental factors. nonetheless, our data confirm that severe adenovirus infection was associated with hadv- rather than hadv- . the significance of the study lies in that the pediatrician should be aware of the importance of early diagnosis and treatment of hadv- infection in children in clinical practice. the findings should also stimulate further studies of mechanisms of the different pathogenicity among human adenovirus serotypes. the severity of adenoviral infection, as studied in chongqing, china, may be correlated to human adenovirus type (hadv- ) instead of type (hadv- ). overall, strain of the hadv- type caused a more severe pneumonia and an exacerbated cytokine response, which also paralleled their more robust replication in cell culture, as compared to hadv- . while the exact mechanism of the type-specific pathogenicity merits further investigation, these findings may eventually contribute to better control and treatment of adenoviral infection. serum inflammatory markers in patients with adenovirus respiratory infection adenovirus serotype and infection with acute respiratory failure in children in taiwan epidemical features of hadv- and hadv- in pediatric pneumonia in chongqing human mastadenovirus type : a novel, multiple recombinant species d mastadenovirus isolated from diarrhoeal faeces of a haematopoietic stem cell transplantation recipient computational analysis of a species d human adenovirus provides evidence of a novel virus human adenovirus associated with severe 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reveals high sequence variability phylogenetic analysis of the main neutralization and hemagglutination determinants of all human adenovirus prototypes as a basis for molecular classification and taxonomy identification and typing of respiratory adenoviruses in guangzhou, southern china using a rapid and simple method a simple and efficient method for purification of infectious recombinant adenovirus a comparison of viral fitness and virulence between emergent adenovirus p and prototype adenovirus p strains in vitro characterization of human adenovirus type in comparison with its parental adenoviruses, types and pring-akerblom p. rapid and quantitative detection of human adenovirus dna by real-time pcr quantitative real-time pcr assay panel for detection and type-specific identification of epidemic respiratory human adenoviruses a novel factor i activity in nipah virus inhibits human complement pathways through cleavage of c b mmp- -mediated by sarm-trif signaling pathway contributes to ifngamma-independent airway inflammation and ahr post rsv infection in nude mice epidemiology of acute encephalopathy in japan, with emphasis on the association of viruses and syndromes transient hemiparesis and hemianesthesia in an atypical case of adult-onset clinically mild encephalitis/ encephalopathy with a reversible splenial lesion associated with adenovirus infection the effect of hemoperfusion on patients with toxic encephalopathy induced by silkworm chrysalis ingestion the olfactory nerve: a shortcut for influenza and other viral diseases into the central nervous system cytokine induction by respiratory syncytial virus and adenovirus in bronchial epithelial cells complement: a unique innate immune sensor for danger signals complement. first of two parts second of two parts virion-associated complement regulator cd is more potent than cd in mediating resistance of mumps virus and vesicular stomatitis virus to neutralization incorporation of host complement regulatory proteins into newcastle disease virus enhances complement evasion differential mechanisms of complementmediated neutralization of the closely related paramyxoviruses simian virus and mumps virus adenovirus activates complement by distinctly different mechanisms in vitro and in vivo: indirect complement activation by virions in vivo hmgb mediates hadv- infection-induced pulmonary inflammation in mice acute inflammatory response and remodeling of airway epithelium after subspecies b human adenovirus infection of the mouse lower respiratory tract differential effects of respiratory syncytial virus and adenovirus on mononuclear cell cytokine responses c a modulates il- beta secretion in human monocytes by regulating atp efflux and subsequent nlrp inflammasome activation we thank professor rong zhou, dr. xingui tian, and colleagues (state key lab of respiratory disease, guangzhou medical university, china) for kind assistance in various areas of the study. we would like to acknowledge the assistance of the patients and their caregivers involved in the study, the staff in the department of respiratory medicine and the department of intensive care unit of children's hospital of chongqing medical university help for nasopharyngeal aspirates collection. this study was supported by the national natural science foundation of china for young scholars ( ). the funders had no role in the study design, data collection, analysis and interpretation of data and writing the manuscript. the datasets used and analyzed during the current study are included within the article and its tables. additional data may be available from the corresponding author on reasonable request.authors' contributions yxf performed the experiments, analyzed and/or interpreted the data, and wrote the manuscript. zzt, zxy, sm helped to perform the experiments. xgt contributed to the design of the paper and revised the manuscript. kn contributed to the design of the study. lr assisted revision of the manuscript. n z contributed to the design of the study and assisted in the analysis and/or interpretation of data, revised the manuscript. em l contributed to conception, collected clinical information, and revision of the manuscript. all authors read and approved the final manuscript. the study procedure was approved by the ethics committee of the children's hospital of chongqing medical university, chongqing, china. written informed consent was obtained from parent or guardian of all participants. not applicable. the authors declare that they have no competing interests. key: cord- -nquw qga authors: kuchipudi, suresh v.; nissly, ruth h. title: novel flu viruses in bats and cattle: “pushing the envelope” of influenza infection date: - - journal: vet sci doi: . /vetsci sha: doc_id: cord_uid: nquw qga influenza viruses are among the major infectious disease threats of animal and human health. this review examines the recent discovery of novel influenza viruses in bats and cattle, the evolving complexity of influenza virus host range including the ability to cross species barriers and geographic boundaries, and implications to animal and human health. influenza viruses are known to constantly evolve and cross species barriers. the genetic diversity of influenza viruses is ever increasing with more novel influenza subtypes being discovered periodically. the purpose of this review is to provide an up-to-date overview of ecology and evolution of influenza viruses including the novel influenza viruses in bats and cattle. in addition, we discussed the growing complexity of influenza virus-host interactions and highlighted the key research questions that need to be answered for a better understanding of the emergence of pandemic influenza viruses. influenza is among the major infectious disease problems affecting animal and human health globally. several human influenza pandemics have been recorded since ad [ ] , with the most significant of those being the "spanish flu" of , often referred to as the "mother of all pandemics" [ ] . spanish flu pandemic is believed to have affected approximately - percent of the world's population and caused more than - million human deaths globally [ ] . influenza infections in humans occur either as epidemic (seasonal or interpandemic) influenza caused by influenza a and b viruses, or as sporadic pandemic influenza caused by influenza a viruses [ ] . study of influenza pandemics has been of great interest to epidemiologists. influenza epidemics and pandemics have been repeatedly occurring for centuries, but to date the ability to predict a pandemic has not been achieved [ ] . influenza viruses belong to the family orthomyxoviridae (from the greek orthos, meaning "standard, correct", and myxa, meaning "mucus"). members of orthomyxoviridae are characterized by virions that are either spherical or pleomorphic measuring - nm in diameter. genomes are comprised of single-stranded negative-sense rna that is arranged in either eight, seven or six segments depending on the genus. classification of the historically described a, b and c influenza viruses based on antigenic differences in nucleocapsid (np) and matrix (m ) proteins. influenza a viruses (iavs) are further classified into subtypes based on the antigenic properties of the external glycoproteins namely iavs are known to infect humans and a wide variety of animals including pigs, horses, minks, seals, whales and birds [ ] . avian influenza viruses (aivs) continue to spread, evolve and cause significant economic losses to poultry industries globally. unlike low pathogenic avian influenza viruses (lpaivs) that cause mild clinical signs in domestic poultry, infection of gallinaceous poultry with highly pathogenic avian influenza viruses (hpaivs) results in severe fatal disease often causing up to % mortality within two-three days [ ] . aivs have also been implicated in the generation of influenza viruses that have caused all human influenza pandemics. in particular, human infection with certain contemporary eurasian lineage hpai h n viruses frequently caused severe disease with a case fatality rate of up to % [ ] . ability to infect a wide range hosts is a key contributing factor to the complex and seemingly expanding genetic diversity of iavs. it is now well established that iavs infect domestic pets such as dogs and cats, adding to the list of host species that could potentially expose humans to influenza viruses. an h n equine influenza virus in and an avian virus-like h n strain around or were transmitted to dogs, and these canine influenza viruses have been circulating in the u.s. dog population ever since [ ] . after the first report of pandemic h n influenza a virus (ph n ) infection of cats in italy in [ ] , more than cats became infected with influenza subtype h n in animal shelters in new york, ny, usa during - [ ] . while transmission of these animal viruses to humans has not yet been documented, the close contact of many humans with pet dogs and cats presents an increased risk of opportunity for such a host jump. transmission of iavs between animals and humans is bidirectional such that humans are infected by animal influenza viruses and human influenza viruses have been shown to transmit to animals. in particular, studies established that domestic pets are susceptible to infection by human influenza viruses. for example, dogs experimentally inoculated with a human seasonal h n or pandemic (pdm) h n ( ) showed nasal shedding and seroconversion. however, dogs inoculated with influenza b virus did not exhibit virus shedding or seroconversion highlighting that interspecies transmissibility is a key feature of iavs and not ibvs [ ] . aquatic birds such as ducks, gulls, and shorebirds have historically been identified as the primary reservoir of all the known iav subtypes [ ] . recently, northwest atlantic gray seals were suggested as an endemically infected wild reservoir population for diverse influenza viruses [ ] . arguably, influenza virus host range could be much broader than currently known, with additional reservoirs that are yet to be revealed. prior to , antigenically different ha (h -h ) and nine different na (n -n ) types had been described, all of which were found in the aquatic bird reservoir. however, the number of ha and na types have now expanded after the identification of two novel influenza-like viruses in fruit bats. the first was in a frugivorous yellow-shouldered bat (sturnira lilium) in guatemala [ ] . soon after, a second virus was found in the flat-faced fruit bat (artibeus planirostris) in peru [ ] . the newly identified bat flu viruses are genetically distinct from all previously known iavs and hence are designated as novel subtypes namely h n and h n [ ] . serological surveys showed wide spread prevalence of h /h -specific antibodies among bat populations in central and south america [ ] . finding novel bat-influenza viruses is not surprising as bats represent % of all known mammalian species [ ] and have been known to be natural reservoirs of many deadly zoonotic rna viruses including ebola virus [ ] [ ] [ ] . it is believed that bats have the capacity to harbor more influenza virus genetic diversity than all the other mammalian and avian species combined [ ] . notably, little yellow-shouldered bats in central america have been proposed as a potential sylvatic mammalian reservoir of influenza [ ] . discovery of novel bat iavs raises numerous questions including the host range evolution of iavs and the role of bats in evolution of iavs. the potential for the newly discovered bat iavs to reassort with previously known iav types is being explored and providing novel information about the iav life cycle. bat flu viruses have been reconstructed by reverse genetics using synthetic dna which has allowed for more functional understanding of these viruses [ ] . multiple experimental studies have shown that reassortment of bat iavs and conventional iavs does not occur under experimental conditions [ ] [ ] [ ] . this is partly explained by functional differences between bat iavs and conventional iavs. iav nonstructural protein ns is a multifunctional protein that plays a crucial role in evading host immune responses and serves as a key virulence factor [ , ] . unlike the ns of conventional iavs, bat ns fails to bind to the host p β, a regulatory subunit of the cellular metabolism-regulating enzyme phosphoinositide -kinase (pi k) [ ] . in addition, the surface glycoprotein ha and na genes of bat iavs only show low nucleotide sequence identity with those of the conventional iavs [ , ] . notably, it has been found that the bat iav hemagglutinin (ha) and neuraminidase (na) proteins lack the receptor binding activity that are characteristic of the conventional iav [ , ] . ha of bat iavs does not bind to the classical avian (saα , -gal) or human (saα , -gal) iav receptors highlighting the possibility of unique entry mechanisms that are yet to be identified [ ] . consequently, it was found that bat iavs initiate infection at the basolateral membrane of cells unlike conventional iavs which preferential initiate infection on the apical surface of cells [ ] . these findings highlight that there are major evolutionary constraints to bat iavs to effectively reassert with conventional iavs and/or infect other species including humans. while the zoonotic potential of bat iavs is not yet fully established and continues to be a focus of ongoing research, bats have been found to be susceptible to infection by conventional iavs. a recent study found serological evidence of iav h subtype infection in % of frugivorous bats tested in africa [ ] . a key source of iav genetic diversity could come from the replication of iavs in a non-native host species that initiate evolution of new virus variants [ ] . the receptors for previously known iavs are sialic acids (sa) on host cells [ ] . consequently, the expression of these receptors on host cells is a key determinant of the ability of iavs to infect a host [ ] . aivs preferentially bind to sa receptors that are linked to galactose by an α , linkage (saα , -gal), while human and classical swine influenza viruses show preference to α , linked sas (saα , -gal). it is widely believed that hosts that co-express both saα , -gal and saα , -gal receptors could support reassortment of iavs and hence play a major role in the evolution of iavs [ , ] . we have demonstrated abundant co-expression of both saα , -gal and saα , -gal receptors that are compatible with avian and human iav binding in respiratory and digestive tracts of little brown bats, the most widespread bat species in north america [ ] . the potential for bats to support infection by multiple conventional iav types and thus serve as a source of novel genetic variants remains to be fully explored. although influenza viruses infect humans and a wide range of animals and birds, cattle were never considered to be susceptible to influenza virus infection. however, a novel influenza virus has recently been identified in several animals including swine, cattle, sheep, and goats. the virus was first isolated as an influenza c-like virus from pigs with respiratory illness in oklahoma, usa, in [ , ] . the virus was subsequently classified as influenza d virus (idv), and the virus has now been reported from many countries including united states, france [ ] , italy [ ] , china [ ] , japan [ ] ireland [ ] and countries in north and west africa [ ] . the international committee on taxonomy of viruses (ictv), which is responsible for developing, refining, and maintaining a universal virus taxonomy, has recently released revised classification of orthomyxoviridae [ ] . in earlier classification, orthomyxoviruses belonged to any one of the five genera: influenzavirus a, influenzavirus b, influenzavirus c, thogotovirus and isavirus [ ] . however, the most recent classification of orthomyxoviridae includes eight genera and nine species (table ) . genus alphainfluenzavirus comprises the species influenza a virus, betainfluenzavirus comprises the species influenza b virus, gammainfluenzavirus comprises the species influenza c virus, and delatinfluenzavirus comprises the species idv [ ] . like other influenza viruses, idvs possess a negative-sense single-stranded rna genome. however unlike iavs that contain segments, idvs comprise seven genomic segments that are predicted to encode nine proteins, including glycoprotein hemagglutinin-esterase fusion (he), polymerases pb , pb , and p , nucleoprotein, matrix protein (m and cm ), and nonstructural proteins (ns and nep) [ ] . idvs use -o-acetylated sialic acid as their cellular receptor on host cells such as icvs [ ] . it is well established based on several epidemiological studies that cattle are the primary reservoirs of idvs [ , ] . in addition, idvs have also been isolated from a range of animals including pigs, sheep, goat, horses and camelids. while the precise role of idvs in clinical disease in animals is not yet fully investigated, their role in causing respiratory infections in cattle has been implied. two recent studies carried out metagenomic characterizations of the virome associated with bovine respiratory disease in feedlot cattle and found correlation of idv presence with bovine respiratory disease (brd) clinical signs, raising exciting new prospects for understanding and combatting this complicated disease [ , ] . brd complex is one of the major diseases affecting the cattle industry in the usa and around the world. productivity losses due to brd are estimated to be $ . per calf with an annual economic impact of more than one billion dollars to the u.s. cattle industry [ ] . brd is associated by multiple pathogens and accounts for approximately - % of the morbidity in the usa [ ] and . - . % of the morbidity in mexican feedlot cattle. brd results in the use of widespread therapeutics and antibiotics in feedlots, which increasingly raises public health concerns of promoting antibiotic resistance [ , ] . pathophysiology of brd involves complex interactions between host, pathogen, environment and management factors. in feedlot cattle, brd is initiated by viral infection followed by stress due to travel which is typically followed by a secondary infection by resident bacteria [ ] . viral infection can cause increased susceptibility to secondary bacterial infections by either immunosuppression or by damaging the epithelium of upper airways and injuring lung parenchyma which facilitates the migration of bacterial pathogens and colonization of the lower respiratory tract. depending on several factors, the clinical outcome of brd can be variable; however, higher morbidity and mortality are observed in the event of mixed viral and bacterial infections [ ] . many viral pathogens have been implicated in brd, which include bovine viral diarrhea virus, bovine herpesvirus , bovine respiratory syncytial virus and bovine parainfluenza . experimental studies in calves with idv showed damage that results in the induction of inflammation in the trachea. idv could be a significant player in brd and could facilitate coinfections with other bovine pathogens [ ] . in addition to the animal health implications of idvs, a recent study found idv antibodies in out of persons that had contact with cattle and only out of that did not have any exposure to cattle [ ] . the results of this study raised the possibility that idv could be relevant from a public health stand point and that it could pose a zoonotic risk to cattle-exposed workers. with much still to be characterized about the new idv species, exploring its impact on human an animal health through epidemiological studies will be vital to understanding spread of this virus. emerging and novel zoonotic infections often result from pathogens jumping from their original host into novel host species [ ] . the host range evolution of mammalian viruses typically involves more closely related hosts [ , ] . in particular, rna viruses with broad host range are more likely to jump between distantly related species [ , , ] . a key determinant in the host range evolution of a virus is the mechanism of viral entry used by the viruses. analysis of human viruses revealed that the viruses that use receptors that are highly conserved in their amino acid sequence across species have the broadest host range [ ] . the ability of viruses to bind to an alternative receptor is sometimes key in species jumping. aivs need to change to preferentially bind to saα- , -gal receptor to efficiently transmit between humans. in experimental studies, it was shown that the shift from saα- , -gal to saα- , -gal binding requires four mutations for a ha of hpai h n viruses [ ] . however, several newly emerged h n aivs in egypt have been found to have acquired the human receptor saα- , -gal binding ability during their emergence in birds [ ] . influenza a, b, c and d viruses have varying susceptible host range ( figure ) . notably, the newly discovered idvs have the widest host range after iavs. further, humans and pigs are susceptible to infection by all four types of influenza viruses. with the extensive host range that continues to grow and the zoonotic potential, influenza viruses remain a major challenge to epidemiologists. owing to their tremendous potential to affect animal and human health, there is a need to carry out in-depth and comprehensive studies to unravel the ecological complexity of influenza virus host range evolution. in particular, we feel that researchers should focus on answering the key questions, "what is the role of bats in the ecology and evolution of iavs?", "are idvs involved in the epidemic influenza infections in people?", and "are birds susceptible to infection by idvs?" vet. sci. , , x for peer review of need to carry out in-depth and comprehensive 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influenza virus sublineages during their emergence in birds in egypt acknowledgments: authors acknowledge the support of penn state animal diagnostic laboratory for providing resources and access needed for the preparation of this review. the authors declare no conflict of interest.acknowledgments: authors acknowledge the support of penn state animal diagnostic laboratory for providing resources and access needed for the preparation of this review. the authors declare no conflict of interest. key: cord- -abs tc r authors: chong, ka chun; hu, pei; lau, steven; jia, katherine min; liang, wenjia; wang, maggie haitian; zee, benny chung ying; sun, riyang; zheng, huizhen title: monitoring the age-specificity of measles transmissions during - in southern china date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: abs tc r background: despite several immunization efforts, china saw a resurgence of measles in . monitoring of transmissions of individuals from different age groups could offer information that would be valuable for planning adequate disease control strategies. we compared the age-specific effective reproductive numbers (r) of measles during – in guangdong, china. methods: we estimated the age-specific r values for age groups: – months, – months, months to years, – years, – years, – years, and ≥ years adapting the contact matrix of china. the daily numbers of laboratory and clinically confirmed cases reported to the center for disease control and prevention of guangdong were used. results: the peak r values of the entire population were above unity from to , indicating the persistence of measles in the population. in general, children aged – years and adults aged – years had larger values of r when comparing with other age groups after . while the peaks of r values for children aged – years dropped steadily after , the peaks of r values for adults aged – years kept at a high range every year. conclusions: although the provincial supplementary immunization activities (sias) conducted in and were able to reduce the transmissions from to , larger values of r for children aged – years were observed after , indicating that the benefits of the sias were short-lived. in addition, the transmissions from adults aged between and years increased over time. disease control strategies should target children and adult groups that carry high potential for measles transmission. increased over time. disease control strategies should target children and adult groups that carry high potential for measles transmission. measles is a highly contagious acute viral disease. throughout the world, and most countries have set goals for its elimination. in , the national expanded program on immunization (epi) in china started to implement a standard schedule for the routine administration of one dose of measles-containing vaccine (mcv ) among children between and months of age. subsequently, the mean annual measles incidence decreased from per , in - to per , in - [ ] . in , a two-dose routine measles immunization program was implemented for children aged between months and years. the age schedule for the second dose of mcv (mcv ) was shifted to - months in . during - , the number of measles cases showed a remarkable decrease but remained around . per , on average [ ] . in , the government of china set a goal to eliminate measles by , for which purpose a series of programs was implemented, including strengthening routine immunization surveillance, supplementary immunization activities (sias), and casebased surveillance [ ] . an sia is defined as the administration of a supplementary dose of a vaccine to a specific age population in a certain area during a short period, regardless of the recipients' previous vaccination histories. sias enhance routine immunization programs, including catch-up campaigns, follow-up campaigns, and outbreak-response immunization. the estimated coverage rate of routine immunization with mcv increased from . % in to . % in , whereas the estimated coverage rate for mcv was < % before and . % in [ ] . in september , china conducted a synchronized, nationwide sia that targeted children aged months to years, covering million children with a reported coverage rate of . % [ ] [ ] [ ] . although the annual measles incidence had dropped to . per , in , it resurged to more than . per , in [ ] . despite the implementation of two-dose routine vaccines since , frequent outbreaks have occurred over the past years [ ] [ ] [ ] . guangdong, a highly populated province with million population in , is located in the southernmost part of mainland china (fig ) . the incidence of measles in guangdong dropped to a remarkable low in ( . / , ) after a series of sias targeting children aged months to years during and . however, guangdong had the highest number of reported cases in china in and , with incidences of . and . per , , respectively [ ] , even though the reported coverage of mcvs was kept above % every year after . to this end, the guangdong government implemented some mop-up vaccination programs after targeting children aged months to years (fig ) . monitoring the effectiveness of the measles control policy is done by surveillance. in china, measles is a category b infectious disease which indicates it is highly contagious and must be reported to the surveillance system within hours after confirming the laboratory samples [ ] . since , china has a direct network reporting system and automatic warning information system for infectious diseases. the system focusses on the number of reported cases, but does not evaluate the transmissibility of measles. the effective reproduction number, r, is a key epidemiologic variable that summarizes the transmissibility of infectious diseases. it is defined as the expected average number of secondary cases produced by an infectious individual in a population in which not all the individuals are susceptible [ ] . when r is larger than , an infectious individual is expected to infect more than one secondary case. when r is less than , an infectious individual tends to infect less than one secondary case, and the incidence will decrease. nevertheless, some infectious diseases have been shown to be strongly age-specific, for example, measles. age-specific r, defined as an average total number of secondary cases from all age groups generated by a single case with respect to his age group was recommended to study the differences in transmission potential taking account of social mixing [ ] [ ] [ ] [ ] [ ] [ ] . although a usual interpretation of age-specific r for gauging the control measures required to eliminate an infection is inappropriate [ , ] , age-specific rs provide valuable information on the underlying heterogeneous transmission between and within different groups of individuals. for example, glass et al. estimated the rs of pandemic influenza a(h n ) for children and adults and identified children had a higher transmission then adult cases. in china, the demography of measles infections has changed over time. while infants aged between months and months and young adults aged to years were the primary population of measles infections, children aged to months and adults aged - years became the primary sources after the national sia. apparently, the age specificity in measles transmissions could be affected by vaccination policies. chong et al. [ ] showed that even though substantial decreases in the numbers of cases were observed after mass vaccination campaign, measles could still persist in a population given a high value of r. the majority of the relevant epidemiological studies conducted in china have been based on reported cases and have aimed to describe the incidence and characteristics of population distribution [ , ] . however, the age specificity in measles transmissions had hardly been studied. in the present study, we compared the age-specific r of measles infections between different age groups by using laboratory and clinically confirmed data collected from to . daily notifications of measles cases from january , to december , were collected from the national infectious disease monitoring information system (nidmis), as complied by the center for disease control and prevention (cdc) of guangdong province. for some of the cases with typical clinical symptoms, case notifications were sent to the person in charge of reporting by outpatient or resident doctors, and the cases were then recorded as "clinically confirmed". blood samples from these cases were sent to cdc clinical laboratories for confirmation, if laboratory capacity allowed. other cases with atypical clinical symptoms were recorded as "suspected cases," and the blood samples of these cases were subsequently transferred to a diagnostic laboratory to obtain a confirmed diagnosis. the test results were returned to the patients' doctors. if the test results were positive, the cases were relabeled as "laboratory-confirmed cases," and the person in charge of reporting was notified. if the results were negative, the cases were relabeled according to the specific disease that had been detected before handing over to the reporting personnel. for the (clinically confirmed or suspected) cases without laboratory confirmation, epidemiological investigations were conducted to determine whether the patients' infections had any linkage to other confirmed cases within - days before the onset of any symptom. the epidemiological investigations were performed through direct contacts in the relevant village, community, or school, or through direct contacts for mass gathering events. the clinically confirmed and laboratory-confirmed cases were both regarded as cases, and reporting personnel were required to report such cases to the nidmis within hours. we divided the population into age groups according to the age of onset: - months (pre-vaccination age), - months (received the first dose of the measles containing vaccine, mcv- ), months to years (received the second dose of the measles containing vaccine, mcv- ), - years (primary and secondary school students), - years (high school and college students/young adults), - years (mature adults), and ! years (aged adults). this study was reviewed and approved by the medical ethics committee of the guangdong cdc. the application of the data in this study has been authorized by the guangdong cdc. all data were fully anonymized prior to access by any of the authors and does not involve patients' privacy prior collection. informed consents were exempt from the ethics committee in accordance to the cdc policy of continuing public health investigations of notifiable infectious diseases, in which the patient names, addresses, medical histories with infectious diseases, and their family information will not be disclosed to the public by guangdong cdc in any form. the data are available without restrictions (the link will be provided after the acceptance of the paper). the method for estimating the age-specific effective reproduction numbers suggested by white et al. [ ] , which is a modification of the wallinga and teunis approach [ ] , was adopted. let p d denote the probability of a serial interval of length d, (d = , ,. . .,d where d be the maximum serial interval length), n t;g i denote the frequency of symptom onset in age group g i (i = , ,. . .,g where g is the total number of age groups) on day t (t = , ,. . .,t where t is the length of the study period), r g i !g j denote the contact rate between two individuals from age group g i and age group g j (j = , ,. . .,g). the effective reproduction number of age group g i on day t, r t;g i , can be calculated by summing the expected number of individuals in each age group from t+ to t+d infected by an individual in age group g i whose symptom onset was on day t: denote the relative probability that an individual in group g j on day t+d was infected by an individual in group g i on day t. in this study, there were age groups (i.e., g = ) and the maximum serial interval length d was set at . p d was generated from a gamma distribution with a mean of days and standard deviation of days [ ] . r g i !g j was estimated by using the contact matrix of china, projected by the bayesian hierarchical model in prem et al [ ] . the estimation formulas were implemented in microsoft excel. we extended the probabilistic method described in white et al. to generate the statistical uncertainty [ ] . a parametric bootstrapping approach was employed to generate , realizations of fr t;g i g. in each iteration, we generated a new dataset by first simulating the total number of individuals in group g j (j = , , . . ., ) infected by those in group g i (i = , , . . ., ) with symptom onset of day t (t = , , . . ., t- ) from a poisson distribution with mean = n t;g i  x minðd;tÀ tÞ d¼ n tþd;g j  pði t;g i ! i tþd;g j Þ, which can be interpreted as the estimated total number of individuals in group g j infected by all the individuals in group g i with symptom onset on day t, where n t;g i and pði t;g i ! i tþd;g j Þ were directly obtained and calculated from the original dataset respectively. the simulated number was then distributed within the serial interval t + and t + according to a gamma distribution with a mean of days and standard deviation of days. the above procedure was repeated for all i,j, and t. the resulting data were used to calculate a realization of fr t;g i g and further averaged by months. the % credible intervals (ci) of the monthly estimates were obtained from the . th and . th percentiles over the , realizations. apart from china's contact matrix, contact matrixes from other countries were employed to test the sensitivity of our results [ ] . we also tried evaluating the estimates using days and days as the mean and standard deviation of the gamma distribution of the serial interval [ ] . the r values estimated for children aged - years were low across the study period in general, even though the values also increased since , indicating that primary and secondary school students had a limited contribution to measles transmissions. similarly, the results for the adults aged ! years were extremely low across the study period, indicating that these persons were unlikely to infect more than a case on average. the for adults aged between and years, a clear seasonal pattern of r values was observed after , which showed a similar trend to that observed in children. in and , the estimated annual peaks of r were . ( % ci: . to . ) and . ( % ci: . to . ) respectively. given that the r peak of the entire population was significantly above unity in , adults aged - years had the largest contribution to measles transmissions. s fig shows the sensitivity of the results to the use of contact matrices from other countries [ ] . in general, the major findings were robust with the variation of contact patterns, for example, children aged - years still had a large contribution in measles transmissions after . nevertheless, due to a difference in contact frequency, larger estimates were observed for children aged - months and - months when using the contact matrix of germany. moreover, while using poland's contact matrix drew a lower estimates for children aged - we also investigated using a different set of parameters for the distribution of the serial interval, and found that the results were generally consistent with the main analysis (s fig). the r values of children groups were slightly increased, whereas the r values of adult groups were slightly decreased. monitoring the age specificity of measles transmissions could provide information that would be valuable to officials who seek to develop adequate disease control strategies. for example, it could help to select appropriate age groups for supplementary vaccination. in this study, we compared the age-specific r of measles infections between different age groups, using laboratory and clinically confirmed data from to for guangdong province. according to the results, measles transmissions varied across most age groups before and after and the large values of r from the entire population indicated a persistence of measles in the population from to . in general, children aged - years and adults aged - years had higher contributions in measles transmissions when comparing with other age groups after . after , while the peaks of r values for children aged - years dropped steadily by years, the peaks of r values for adults aged - years remained unchanged and kept at a high range every year, demonstrating the highest contributions in measles transmissions. the findings suggest that disease control strategies should target children and adult groups that carry a high potential for measles transmission. as has been previously noted, we found that children aged - years had r values that increased after , even though sias targeted this population in and . the increasing r values could have resulted from low mcv coverage in this cohort, for which the official reported coverage was usually over-estimated [ , ] . an in-house survey of a similar cohort of children aged - months showed that mcv and mcv coverage rates were only % and %, respectively [ ] , results that were inconsistent with the generally reported figure of > % in china [ ] . the geographic heterogeneity of vaccine coverage in china could be another explanation [ , ] . a chinese study indicated that the measles antibody levels of children aged - years old were significantly lower for residents of rural areas than for residents of urban areas [ ] . the primary reasons why rural children had missed their mcvs were because they were living far from the clinics and because they were unable to access vaccination information [ ] . the incomplete immunization records of rural children also made it more difficult for public health officials to track them in order to administer the vaccine. we observed elevated transmissions in infants aged - months, which may primarily be attributed to the design of the immunization system, which regarded them as too young to be vaccinated by either routine immunization or sias. a longitudinal study of maternal measles antibody titers in infants in guangzhou (the provincial capital of guangdong province) showed that titers among infants decreased rapidly after months of age, and were generally undetectable at months of age [ ] . several other studies reported similar results [ , ] . hence, there was a remarkable immunity gap among children under months old. some studies showed that only around . % to . % of infants are seropositive for measles at months of age [ , ] . nevertheless, even though infants aged - months were identified as a high transmissibility group, reducing the minimum age for receiving mcv- to months is controversial. we also found that the transmissions from children aged - years were comparatively low, which was expected given that given that they were the main target of previous sias. moreover, many primary schools implemented screening of children's vaccination certificates and administered supplementary doses of the measles vaccine to fill immunity gaps before the annual entrance [ ] . the values of age-specific r for adults aged between and years kept at a high range from to and it could be attributed to the lower efficacy of measles vaccines, the low vaccination coverages during s and earlier, and the reduced chance of natural infections. persons aged - years at the time of the present study were thought to be the first recipients of the vaccination after the approval of routine immunization. liquid vaccines were used for immunization at that time. they had a lower effective dosage and may have resulted in the lower level or shorter protective duration of antibodies among the population. in addition, a functional cold chain, transportation, and communication system for the measles vaccine had not been established at that time; hence, the quality and efficacy of the vaccines could not be guaranteed. secondly, several parents knew nothing about the measles vaccine and underestimated the severity of measles, thereby resulting in a low vaccination rate and a high rate of unsure inoculation history. thirdly, secondary vaccine failure (i.e. measles onset after vaccination and successful seroconversion) due to waning immunity might have occurred among vaccinated adults. although our study could not identify secondary vaccine failure from other cases as serological evidence of previous successful vaccination were lacked, it has been concluded by who that waning immunity has not played a major role in the transmission of measles compared to the absence of initial immunity [ ] . the proportion of cases attributable to secondary vaccine failure varied greatly across outbreaks [ ] . in a cohort study (n = ) in zhuji county of zhejiang province, around - % of those given with single doses of domestic vaccines would become sero-negative (measured by haemagglutination-inhibition tests) after years, yet clinical measles cases rarely happened among them who had humoral immunity waned as they were still protected by secondary immune response [ , ] . finally, the subsequent sias did not cover these persons; thus, the immunity gaps among people aged to years increased. on the other hand, the transmissions from individuals aged ! years were the lowest among the age groups studied, even though there was almost no vaccination history in this group. we believe that the majority of these individuals acquired antibodies through natural infection, owing to the highly contagious nature of measles when they were young. moreover, many studies have shown that seropositivity after natural infection persists longer and generates a stronger response than the immunity acquired from vaccination [ ] [ ] [ ] . given the increasing values of r for the entire population observed after , some mopup vaccination campaigns in and appear to have had limited effectiveness, even though they aimed to control measles transmission. one reason for this is that rural families usually have a lower level of education and do not fully understand information regarding mop-up campaigns, which results in a lack of initiative to get the vaccine. moreover, some of the susceptibles were migrants, and officials reported difficulties tracking their vaccination histories. although door-to-door notifications, text messages, and telephone notifications have been used to inform migrant families to join mop-up campaigns [ ] , it is often difficult to contact these families because of changes to their addresses or phone numbers. from a policy-making perspective, these results imply that for a successful measles control campaign, the public health department should carry out control measures for appropriate age groups. for children between and months old, it is necessary to take measures to improve vaccination coverage, including providing more publicity to improve parents' awareness about vaccination against measles, creating integrated multichannel notifications to inform parents of the vaccination, and strengthening the supervision of kindergartens. for adjustments to the immunization strategy, adult-specific vaccination programs should be considered to fill the immunity gaps among adults, especially for those aged between and years. one of the major limitations in this study is the quality of notification data. indeed, a proportion (~ %) of the notification data in the early phase study ( ) ( ) ( ) was only clinically confirmed which may lead to some misdiagnosis as well as an underestimate of the age-specific rs. nevertheless, more than % of cases were laboratory confirmed after . particularly, the reliance on clinically confirmation was more in rural hospitals in which doctors may lack sufficient knowledge on measles diagnosis. the positive predictive value of a clinical definition would also be changed over time as measles has become rarer by time. to minimize the chance of misdiagnosis, the clinically confirmed cases were not only identified by clinical symptoms, but were also investigated with any potential epidemiological association with other laboratory-confirmed cases. moreover, the completeness of the data could be affected by underreporting as some of the parents might have regarded measles as a kind of skin disease or might have confused it with other diseases that involve skin rashes. apart from that, age-specific rs are common to be used as a metric to identify appropriate age groups most responsible for transmission as for a target of interventions [ , , ] . however, when determining the effort required to eliminate an infection, the interpretation of an age-specific r is different from that of an overall r in a heterogeneous population as using the original threshold of unity could lead to an underestimation of target population for interventions [ , ] . alternatively, roberts & heesterbeek [ ] suggested a type-reproduction number which can single out particular subgroup rather than averaging over all subgroups. further works such as generalizability and statistical inference [ ] on the alternative measures worth being investigated. in summary, we compared the age specificity in measles transmissions from to in guangdong province. although the provincial sias conducted in and were able to reduce the transmission rates from to , larger effective reproductive numbers for children aged - years were observed after , which indicates that the benefits of the sias were short-lived. in addition, the transmissions from adults aged between and years increased over time. based on the findings of the present study, we believe that disease control measures should strategically target those groups that carry a high potential for measles transmissions. monitoring progress towards the elimination of measles in china: an analysis of measles surveillance data beijing: world health organization china representative office world health organization. measles and rubella surveillance data investigation of a measles outbreak in china to identify gaps in vaccination coverage, routes of transmission, and interventions endemic and imported measles virus-associated outbreaks among adults new measles virus genotype associated with outbreak on the road to measles-free: where are we now? law of the people's republic of china on prevention and treatment of infectious diseases. database of laws and regulations definition and estimation of an actual reproduction number describing past infectious disease transmission: application to hiv epidemics among homosexual men in denmark determining the dynamics of influenza transmission by age estimating age-specific reproductive numbers-a comparison of methods. statistical methods in medical research estimating reproduction numbers for adults and children from case data pandemic influenza h n : reconciling serosurvey data with estimates of the reproduction number likelihood-based estimation of continuous-time epidemic models from time-series data: application to measles transmission in london estimation of the reproductive number and the serial interval in early phase of the influenza a/h n pandemic in the usa. influenza and other respiratory viruses the type-reproduction number t in models for infectious disease control. mathematical biosciences a new method for estimating the effort required to control an infectious disease interpreting the transmissibility of measles in two different post periods of supplementary immunization activities in hubei epidemiological analysis of measles in china between changes of epidemiological characteristics of measles in beijing before and after supplementary immunization campaigns of measles vaccine in different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures projecting social contact matrices in countries using contact surveys and demographic data social contacts and mixing patterns relevant to the spread of infectious diseases the correlation between infectivity and incubation period of measles, estimated from households with two cases measles vaccine coverage estimates in an outbreak three years after the nation-wide campaign in china: implications for measles elimination investigation of a case of measles outbreak in a kindergarten in hubei province immunization: measles, st dose (mcv ) immunization coverage estimates by country comparison of measles antibody levels and trends of children aged - years between and in jiujiang area, jiangxi province analysis on the immunization of measles and its influencing factors among freshmen in elementary school and kindergarten in shenzhen city analysis of the influencing factors of measles vaccine leakage dynamic maternal measles antibody level in infants: a longitudinal study fetal, and neonatal outcomes associated with measles during pregnancy: namibia early waning of maternal measles antibodies in era of measles elimination: longitudinal study pre-vaccination evolution of antibodies among infants , and months of age: a longitudinal analysis of measles, enterovirus and coxsackievirus . vaccine measles antibodies in mother-infant dyads in tianjin notice on the implementation plan for checking the vaccination certificate before children's kindergarten and primary school enrollment in guangdong province world health organization. measles vaccines: who position paper identification of primary and secondary measles vaccine failures by measurement of immunoglobulin g avidity in measles cases during the são paulo epidemic duration of immunity following immunization with live measles vaccine: years of observation in zhejiang province decreasing seroprevalence of measles antibodies after vaccination-possible gap in measles protection in adults in the czech republic immunoglobulin response in serum and secretions after immunization with live and inactivated poliovaccine and natural infection the influence of children's immunization leak replant measures on improving immunization vaccination rate we thank guangdong cdc for providing the data for analysis. we thank the two anonymous reviewers whose comments helped improve and clarify this manuscript. key: cord- -ykisq nz authors: kallel, hatem; matheus, séverine; mayence, claire; houcke, stéphanie; mathien, cyrille; lavergne, anne; hommel, didier title: capillary leak-syndrome triggered by maripa virus in french guiana: case report and implication for pathogenesis date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: ykisq nz background: we report hereby a severe case of hantavirus pulmonary syndrome” (hps) induced by maripa virus in french guiana and describe the mechanism of severity of the human disease. case presentation: a -year- old patient started presenting a prodromic period with fever, dyspnea, cough and head ache. this clinical presentation was followed by a rapid respiratory, hemodynamic and renal failure leading to admission in the icu. biological exams revealed an increased haematocrit level with a paradoxical low protein level. echocardiographic and hemodynamic monitoring showed a normal left ventricular function with low filling pressures, an elevated extravascular lung water index and pulmonary vascular permeability index. these findings were compatible with a capillary leak-syndrome (cls). conclusions: the severity of hps caused by the virus maripa in french guiana can be explained by the tropism of hantavirus for the microvascular endothelial cell leading to a cls. hantavirus generally stands as a rodent-borne virus infection. rodent shed the virus in their droppings, saliva and urine. human infection occurs after breathing air contaminated by the virus. this can result on a severe respiratory syndrome named 'hantavirus pulmonary syndrome' (hps) which can be associated to cardiac failure leading to 'hanta virus cardio-pulmonary syndrome' (hcps) [ ] . in french guiana, cases of hps due to a hantavirus named maripa virus were diagnosed between and [ , ] . we report hereby the th human case of hps diagnosed in french guiana and describe the mechanism of severity of the human disease. our -year-old patient with a history of tobacco, alcohol, and illicit-drug consumption was admitted to the icu for fever ( °c), tachycardia ( beat/min), hemodynamic shock (blood arterial pressure was / mmhg), acute respiratory distress syndrome (ards; pao /fio ratio was ) with signs of intra-alveolar haemorrhage and, acute renal failure. symptoms including headache, fever, cough and dyspnea leading to respiratory failure started days before admission. the treatment consisted of crystalloids and norepinephrine infusion as well as mechanical ventilation support and continuous renal replacement therapy (rrt). initial laboratory testing showed renal impairment (urea nitrogen at . mmol/l, serum creatinine at μmol/l), a rise in inflammatory parameters (leucocytes count at . g/l and c-reactive protein at mg/l), an increased haematocrit level ( . %), thrombocytopenia ( g/l), a low protein level ( g/l) and cellular dysoxia (lactates dosage at . mmol/l). all other biological tests including the dosage of hepatic, muscular and cardiac enzymes were normal on admission. chest x-ray showed bilateral alveolar infiltrates and bilateral pleural effusion. transthoracic echocardiography showed normal and homogeneous left ventricular contractility with low filling pressures, with an aortic velocity time integral (vti) of cm (normal: - cm), a normal right heart function with tricuspid annular plane systolic excursion (tapse) at mm (normal: - mm), and a small pericardial effusion. hemodynamic monitoring using transpulmonary thermodilution (picco system; pulsion medical systems se, feldkirchen, germany) showed a cardiac output (co) of . l/min (normal: - l/min), a global end diastolic volume index (gedvi) of ml/m (normal: - ml/m ), an extravascular lung water index (evlwi) of . ml/kg (normal: - ml/kg), and a pulmonary vascular permeability index (pvpi) of . (normal: . - . ). chest computed tomography showed vessels enlargement, peribronchial cuffing, bilateral kerley lines, alveolar edema, and abundant right pleural effusion needing pleural drainage. the protein level in the pleural fluid was g/l and the microbiological culture was sterile. the viral investigations (igm and rt-pcr in the blood) confirmed an acute infection by hantavirus. the complete rna coding sequence of the s rna segment (genbank accession no. mg ) was also generated and compared with those of the other previous hantavirus cases from french guiana using a bayesian approach. this s rna sequences showed a nucleotide identity of . to . % with the five other previously described sequences of the maripa virus belonging to the rio mamoré clade. phylogenetic relationships were inferred from alignment with nt of the s segment [ ] . laboratory testing concerning other infectious agents (bacteria, fungal or parasite) were negative. overall, the patient's management included mechanical ventilation, norepinephrine, fluid infusion, sedation (midazolam and sufentanil), curarisation (cisatracrium), broad spectrum antibiotics, corticosteroids, and renal replacement therapy. concerning the outcome, the patient recovered gradually with a concomitant rise in serum protein level and a decrease in haematocrit concentration (fig. ) . he was weaned from mechanical ventilation at day , norepinephrine was stopped at day and rrt at day . he fully recovered and left hospital on the th day. the patient was examined in the outpatient clinic three weeks after discharge and his clinical examination was normal. we report here a human case of acute maripa virus related pulmonary syndrome managed in the icu of french guiana with a clear evidence of associated capillary leak syndrome responsible for the severity of the disease. in case of hantavirus infection, initial symptoms include fever, myalgia and headache followed by gastrointestinal symptoms such as abdominal pain, vomiting and diarrhea [ ] . this stage lasts around to days before the onset of respiratory failure, hypotension and cardiovascular shock. thrombocytopenia with haemorrhagic symptoms and renal injury are also frequently reported [ ] . in our case, symptoms recorded at admission were compatible with acute infection by hantavirus and required admission to the icu. the mechanism which may explain the severity of the disease is the tropism of hantavirus for the microvascular endothelial cell [ ] . this tropism causes microvascular hyperpermeability with fluid and proteins leakage leading to hypovolemia and to a non cardiogenic pulmonary oedema. biologically, we observe an increased haematocrit level due to hemoconcentration, and a paradoxical reduced serum fig. evolution of the serum protein and hematocrit levels during the first days (norepinephrine was stopped at day ) proteins level secondary to the transfer of proteins from the vessel to the interstitial space. under normal conditions, the endothelium plays the role of a selective permeable barrier to regulate plasma fluid exchange, as well as molecules and cells trafficking. disruption of cell junctions, with combination of cell retraction process, lead to the loss of the vascular endothelium barrier function. in such conditions, fluids and proteins infusion are ineffective because of the immediate leakage to the interstitial space with a worsening of the respiratory failure without any efficacy on the hemodynamic state. this mechanism is similar to that reported by clarkson in and is explained by a plasma leakage [ ] which was also described in arbovirus infections where the diagnosis was based on thoraco-abdominal sonography and scanography [ ] [ ] [ ] . in our patient, hemodynamic investigations using echocardiography and the picco system showed hypovolemia with low filling pressures and without any ventricular dysfunction. an elevated amount of extravascular lung water as well as an increased vascular permeability were also observed. this result is confirmed by the chest ct scan findings, showing a large amount of water in the alveoli, in the perivascular and in the pleural space. the pleural effusion was exudative and contained a high quantity of protein which can be explained by a protein leakage rather than by an inflammatory origin. the pathogenesis of capillary leakage remains undefined. some evidence suggest that hantavirus disease pathogenesis is immunologically mediated by cytotoxic t lymphocytes and other immune cells in target organs producing inflammatory cytokines. overall, three hypotheses have been reported to explain the mechanism of increased capillary permeability involved in hantavirus infection: a) the attack of infected endothelial cells by virus-specific cytotoxic t lymphocytes (ctls), b) tnf-α production by infected monocyte/macrophages and finally c) the direct effect of the virus on the endothelial cell functions [ , ] . bradykinin, a potent inflammatory and vasoactive nonapeptide generated by kallikrein at the sites of tissue injury is supposed to be the key mediator of the vascular leakage resulting from hantavirus infection. it acts by disrupting inter-endothelial junctions and causes changes in vascular tone. two patients with severe capillary leak syndrome caused by a puumala hantavirus infection were successfully treated with a bradykinin receptor antagonist [ , ] . experimental data demonstrating the plasma kallikrein-kinin system activation during hantavirus infection were also reported [ ] . in the same way, the intensity of the inflammatory syndrome was correlated to the importance of the capillary leakage and to the level of thrombocytopenia [ ] . despite abundant literature on hantavirus, few reports have focused on the aetiology of shock in severe hantavirus infected patients. many studies assume that the shock associated to hantavirus pulmonary syndrome is cardiogenic and hantavirus induces a typical myocarditis. these data were based on the examination of postmortem tissue from human hps cases [ ] . however, in our case, myocardial dysfunction was neither observed during echocardiography and nor during picco investigation. any inotropic agent support has been needed. in addition, troponine levels were normal despite severe shock. consequently, we think that maripa virus is more responsible for hps rather than hcps. such a finding is important as it raises the question about the effectiveness of extracorporal membrane oxygenation (ecmo) in patients presenting maripa virus infection with severe shock. we conclude that hps secondary to maripa virus infection in french guiana can cause severe damages leading to multi organ failure. the severity of the disease may be explained by a dysregulated inflammatory and immune reaction causing a severe capillary leakage without cardiac involvement. physicians should be aware of hps occurring in french guiana and any immediate management in the icu should be considered. none. clinical data will not be made available according to the french cnil recommandations (commission nationale informatique et libertés) that require specific authorizations to transfer data from one center to another. however, data from the medical chart of the patient can be obtained contacting the corresponding author since, the patient has given consent to share his medical informations. authors are cited in the same order that they are cited in the title page. ethics approval and consent to participate the patient has given consent to participate. hantavirus pulmonary syndrome hantavirus pulmonary syndrome caused by maripa virus in french guiana hantavirus pulmonary syndrome, french guiana maripa virus rna load and antibody response in hantavirus pulmonary syndrome immunopathogenesis of hantavirus pulmonary syndrome and hemorrhagic fever with renal syndrome: do cd + t cells trigger capillary leakage in viral hemorrhagic fevers? cyclical edema and shock due to increased capillary permeability capillary leakage in travelers with dengue infection : implications for pathogenesis zika virus in the americas: a review for clinicians zika and chikungunya: emerging arboviruses in the new world increased permeability of human endothelial cell line ea.hy induced by hantavirus-specific cytotoxic t lymphocytes a severe case of puumala hantavirus infection successfully treated with bradykinin receptor antagonist icatibant severe puumala virus infection in a patient with a lymphoproliferative disease treated with icatibant endothelial cell permeability during hantavirus infection involves factor xiidependent increased activation of the kallikrein-kinin system thrombocytopenia associates with the severity of inflammation and variables reflecting capillary leakage in puumala hantavirus infection, an analysis of finnish patients mechanisms of shock in hantavirus pulmonary syndrome written informed consent was obtained from the patient for publication of this case report and accompanying images. a copy of the written consent is available for review by the editor of this journal. the authors declare that they have no competing interest. key: cord- -upogtny authors: viboud, cécile; lessler, justin title: the influenza pandemic: looking back, looking forward date: - - journal: am j epidemiol doi: . /aje/kwy sha: doc_id: cord_uid: upogtny in commemoration of the centennial of the influenza pandemic, the american journal of epidemiology has convened a collection of articles that further illuminate the epidemiology of that pandemic and consider whether we would be more prepared if an equally deadly influenza virus were to emerge again. in the present commentary, we place these articles in the context of a growing body of work on the archeo-epidemiology of past pandemics, the socioeconomic and geographic drivers of influenza mortality and natality impact, and renewed interest in immune imprinting mechanisms and the development of novel influenza vaccines. we also highlight persisting mysteries in the origins and severity of the pandemic and the need to preserve rapidly decaying information that may provide treasure troves for future generations. initially submitted august , ; accepted for publication september , . in commemoration of the centennial of the influenza pandemic, the american journal of epidemiology has convened a collection of articles that further illuminate the epidemiology of that pandemic and consider whether we would be more prepared if an equally deadly influenza virus were to emerge again. in the present commentary, we place these articles in the context of a growing body of work on the archeo-epidemiology of past pandemics, the socioeconomic and geographic drivers of influenza mortality and natality impact, and renewed interest in immune imprinting mechanisms and the development of novel influenza vaccines. we also highlight persisting mysteries in the origins and severity of the pandemic and the need to preserve rapidly decaying information that may provide treasure troves for future generations. age patterns; history of epidemiology; influenza; mortality; pandemic; prior immunity one hundred years after the fact, the influenza pandemic remains one of the most important epidemics of the modern medical era; it was significant for its impact on both human health and the development of epidemiology and other medical sciences. still, as we mark its centennial, it is sobering to realize how little we understand about the origins and lethality of this unusual outbreak despite decades of intense multidisciplinary research. although it would be years before it was possible to fully characterize the virus responsible for the pandemic ( ), contemporaneous medical authorities put commendable effort into reporting detailed epidemiologic data on the progression of the pandemic that ranged from individual-level clinical records to aggregated city-level vital statistics ( , ) . in addition to quantitative epidemiologic data, there exist many anecdotal reports from clinicians, particularly those who served military populations, that have been mined to provide modern audiences a comprehensive account of the pandemic ( ). yet, many important questions remain about the evolutionary origins of the pandemic virus; the contribution of world war i and troop displacements to pandemic emergence and progression; the unique age profile of pandemic deaths, with its signature of high mortality rate among healthy young adults; the consequences of such a large mortality event on natality; and the heterogeneity of the pandemic experience around the world. such mysteries have captured the attention of the lay public and scientific community alike. in commemoration of the centennial of the pandemic, the american journal of epidemiology has convened a collection of articles that further illuminate the epidemiology of that pandemic and consider whether we would be more prepared if an equally deadly influenza virus were to emerge today. five of the articles touch on the origins of the pandemic virus, addressing the role of swine as mixing vessels in this and other pandemic events ( ), the age-specific mortality patterns of the pandemic ( ) ( ) ( ) , and prior population immunity ( ) . others include reports on geographic and social heterogeneities in the pandemic experience in which the authors describe the spatial diffusion of the pandemic in india and portugal ( , ) , the socioeconomic predictors of high mortality risk in sweden and globally ( , ) , and the consequences of the pandemic on us natality rates ( , ) . finally, commentaries address preparedness for future influenza pandemics ( , ) . the influenza virus is remarkable for its ability to infect a variety of animal species, from bats to birds to mammals. although successful cross-species transmission events may be rare, they play a key role in the genesis of new pandemic strains. nelson and worobey ( ) discussed different lines of evidence informing the origins of the virus, including the genetic make-up of the virus and other pandemic strains, the characteristics of influenza receptors across different influenza hosts, and the frequency of cross-species transmission events. they concluded that the pandemic virus must have emerged in mammals just before , most likely from the avian reservoir, with onward transmission from humans to swine. more broadly, a re-analysis of virologic data from the and pandemics, together with a modern understanding of the swinehuman interface, suggested a twist on the long-standing concept of swine as a "mixing vessel" for influenza virus. the authors proposed that swine should be viewed as a repository of historic human viruses rather than a conduit for reassortment of genetic material between avian and human viruses. van wijhe et al. ( ) returned to the question of the origins of the virus by exploring the epidemiologic imprint of the virus on danish mortality records, echoing recent work on immune imprinting ( ) ( ) ( ) . they identified several age breakpoints in pandemic mortality that were suggestive of the cycling of different influenza strains between the mid- th century and the pandemic. most notably, they argued for co-circulation of subtypes of influenza virus (carrying type i and ii hemagglutinin surface antigens) between and . as a result, persons born between and (aged - years during the pandemic) may have been primed by either hemagglutinin type, potentially explaining the intriguing age profile of pandemic mortality in adults. cilek et al. ( ) used a similar epidemiologic approach to explore the pandemic mortality patterns in madrid, spain. madrid is particularly interesting because a lethal pandemic wave was reported in the city in june , the earliest such event recorded. similar to other regions of the world, madrid experienced a signature pandemic pattern of higher mortality rates among young adults. however, seniors in madrid suffered equally high rates of excess influenza mortality. this is unlike the experience of the rest of europe and north america, where seniors were reportedly spared, presumably because of antigenic recycling (i.e., exposure to a related strain in childhood that conferred partial protection) ( , ) . this is an intriguing finding, and it will be important for future work to reconcile the well-accepted idea that a -like virus may have circulated in europe and north america in the second half of the th century, with the notion that madrid would have escaped this virus. to understand the unique epidemiology of the virus, it can be useful to document the experience of remote populations, in which prior immunity to influenza would be expected to be low because of less frequent circulation of the virus. rice ( ) built on a rich literature in this area to document mortality patterns in new zealand between and . he found that the s were a decade associated with high rate of influenza mortality in new zealand, despite the low global connectivity of this island in the era before air travel. he also noted that influenza mortality in was highest among young adults, with a more pronounced intensity in males than in females. these patterns are broadly consistent with the those among young adults in europe and the americas, pointing to the near universality of increased influenza mortality risk in this age group in . chuah et al. ( ) used seroepidemiology and structural equation modeling to answer the inverse question: how did early-life exposure to the pandemic virus impact how people responded to the pandemic, which was caused by an antigenically similar virus? they found evidence for immunologic priming from the virus in the oldest people they studied (individuals years of age or older) that impacted both baseline titers and vaccine response in . this work adds to a growing body of evidence that early-life exposures can have profound effects on immune response and mortality patterns decades after they occur ( ) . information about global connectivity in the th century is tenuous, and influenza records before are scarce. epidemiologic reconstructions of "modern" pandemics of the type presented here ( - ) provide indirect information on the exposures of populations that are now long gone, generating valuable hypotheses about influenza circulation patterns and disease dynamics well into the th century. such reconstructions offer precious insights into what influenza may have looked like years ago in a very different world and how long-term changes in human demography and mobility may affect disease dynamics ( ) . active research topics in the field of archeo-epidemiology include the search for predictors of influenza mortality, such as socioeconomic indicators or geography, and the drivers of influenza spatial diffusion. in articles in the present issue, the authors concentrated on the spatial diffusion of influenza, focusing on british india and portugal, countries that have been poorly studied in the context of the pandemic ( , ) . both studies revealed a highly heterogeneous spread of the pandemic and geographic variation in pandemic mortality impact, albeit at different spatial scales. although portugal as a whole was severely hit by the pandemic compared with other european countries, some provinces nearly fully escaped ( ). analysis of district-level mortality records in india revealed a northeastward wave of infection from september to november that was associated with climate and population density ( ) . diffusion was driven by long-distance jumps via the railroad network, superimposed on local diffusion between neighboring provinces. further, the authors found moderate heterogeneity in the mortality experiences of different indian provinces. spreeuwenberg et al. ( ) also made use of recently unearthed data from india to revisit the global mortality impact of the pandemic. india is a particularly important country for global burden estimation because it was the one most severely hit by the pandemic, with annual pandemic excess mortality rates that were -fold higher than those in denmark for instance ( ) . in the new study, the authors placed the burden of the pandemic at a much lower number than did previous work ( ) , in part by using more detailed data to better adjust for high background mortality unrelated to flu. the results of the portuguese study by nunes et al. ( ) echoed these conclusions-that careful analyses of more detailed data tend to decrease estimates of pandemic burden. the risk factors responsible for increased mortality and morbidity from influenza remain elusive, whether at the population level (e.g., effect of population density or weather on transmission) or the individual level (socioeconomic status, comorbid conditions, etc.). this is still an active area of contemporary influenza research, with direct applications to design targeted intervention strategies. in the present issue, bengtsson et al. ( ) explored the role of social class on pandemic mortality by linking individual death records with historical census data on occupation in a powerful study that captured the entire swedish population. the authors found that low-skilled or unskilled adults had higher death rates than did more skilled workers during the pandemic period relative to prepandemic years, whereas farmers (especially men) fared particularly well. social differences tended to be smaller in women, and there was no clear gradient between social class and mortality. the authors hypothesized that these social differences were linked to differential crowding in the workplace (hence an effect on transmission) rather than differences in income or nutrition. this is a topical issue because the effects of socioeconomic status and baseline health on influenza mortality are still debated today ( ) . researchers have long thought that the pandemic could have affected birth rates ( ) because of the large impact of this event on young adult mortality, the increased risk of severe flu outcomes during pregnancy, and a possible association between influenza infection and miscarriage. two papers in this issue address the topic of natality ( , ) . key questions here include the trimester of pregnancy during which the risk of death is highest for the mother and/or the unborn child and the impact of influenza on (increased) stillbirths and (decreased) live births. the duration of the pandemic effect on natality is also important because it informs the biological mechanism at play. if influenza impacts the probability of conception or fetal deaths, one would expect a temporary natality drop in the aftermath of the pandemic, followed by a rebound in births a few months later. in contrast, a high mortality rate among young women of childbearing age due to influenza infection would result in a long-lasting natality trough. dahal et al. ( ) explored these questions using individual birth and death certificates from arizona, where there was a drop in natality - months after pandemic mortality peaked. this was a temporary depletion, consistent with a detrimental effect of influenza early in pregnancy. in a larger study of populationlevel vital statistics in us states, chandra et al. ( ) found a % drop - months after peak influenza mortality, which they ascribe to a drop in conception during the period of intense pandemic activity. they also found a natality drop in the months after peak mortality, which they linked to excess preterm births and stillbirths due to influenza infections in the last trimester. interestingly, these patterns were also found in the aftermath of the influenza pandemic wave, albeit with a less pronounced effect. one reason we still look back at the pandemic years later is because doing so will make us better able to prepare for the future. the last articles of this collection are focused on preparedness for future pandemic threats ( , ) , building on the lessons learned in and later pandemics, and on new tools to protect populations, including the very active (but still elusive) topic of universal influenza vaccines. jester et al. painted an optimistic picture of progress made in influenza surveillance domestically and internationally, antiviral treatments, and robustness in the infrastructure for vaccine production ( ) . epstein reviewed the progress of the development of a broadly cross-protective flu vaccine, focused on conserved parts of the influenza virus, such as the matrix protein, nucleoprotein, the hemagglutinin stem, and various cocktail combinations ( ) . these vaccines offer promising broad protective effects against new influenza antigenic variants and could potentially be used in pandemic situations. however, some of the candidate vaccine formulations permit limited viral replication and may foster the emergence of escape mutants fit enough to cause disease. these features could have adverse epidemiologic consequences, and these risks need to be projected and monitored carefully. the pandemic is traditionally considered a worst-case scenario for pandemic preparedness, but there were many other pandemics before about which we know very little regarding mortality impact, circulating strains, or prior immunity. in fact, the pandemic has only recently drawn attention among epidemiologists ( , ) . most european and north american countries began formal collection of vital statistics in the mid-to-late th century, so that any pandemic predating can only be explored using church or cemetery records (or indirectly through reconstruction of modern pandemics). digitization of historic records is time consuming, data lack standardization, and information is generally limited to small populations. even with regard to the pandemic, crucial questions may never be answered, including which specific virus (or even subtype) circulated before and further back into the th century and what the population immunity profile was before the pandemic. the search for archival influenza specimens predating has remained elusive, and to our knowledge no archived sera exist from this period. in the absence of further virologic evidence, our understanding of the origins of the pandemic is limited to a handful of influenza virus sequences collected during may to november and to the epidemiologic signature of the virus in different populations. as nelson and worobey noted ( ), more work can be done in this area, particularly to explore the uracil content of post- viruses in different hosts, reconstruct their evolutionary trajectories, and better characterize host receptors and barriers to cross-species jumps. further, as rice ( ) and dahal et al. ( ) noted, a systematic analysis of the age mortality profiles of the pandemic in a sample of remote and wellconnected locations would be most useful, together with modeling of plausible biological hypotheses and immune histories most consistent with the data. databases, which are often crowdsourced or maintained by state health departments ( ) . further analyses of such data could shed light on the mortality profile of the pandemic in understudied locations and would also allow identification of family linkage and host genetic risk factors, which could be tested among descendants. many other library archives exist, although paper-based records rapidly decay and need to be digitized as quickly as possible. the detailed, sometimes freeform, notes typically kept by the scientists at the time mean that careful examination of these archives can sometimes yield surprising fruit. one such resource is the work of wade hampton frost, who was the first chair of the department of epidemiology at johns hopkins university and a critical figure in the fight against the pandemic. modern reanalysis of dr. frost's detailed work ( ) has already yielded abundant insights, and we included digital copies of his papers on the pandemic from the chesney archive in the web appendix (available at https://academic. oup.com/aje). the pandemic is remarkable for the large amount of extremely detailed epidemiologic data collected by public health officials ( , ), in part because it was an era that valued epidemiology, at a time when analytical approaches and knowledge of infectious agents were limited. these exquisitely detailed records have been particularly useful in the attempt to understand the pandemic retrospectively. as a thought experiment, we can imagine ourselves in : we may ask how scientists would look back at the large amount of data we archive on a daily basis in . on the one hand, much if not all of the modern data are digital, meaning that they do not run the risk of being destroyed by fire or floods and they can be more accessible to a wide audience, spurred by the open access movement. however, digital data can also be corrupted (intentionally or unintentionally) and disappear. much from the floppy-disk era has already been permanently lost, and it is unclear if modern cloud-based archives would survive a major disruption (whether technological or civil). further, even today, there is a systematic dearth of epidemiologic and molecular data from low-and middle-income settings (including data on the pandemic ( )). we are just beginning to scratch the surface of the intricate relationship between the influenza virus and the complex immune history of a host who has had repeated influenza exposures ( , ( ) ( ) ( ) ) . it is unclear whether we will be able to fully understand these interactions in the foreseeable future; in the meantime, population birth cohorts carrying important influenza immune histories disappear. we echo earlier calls for a time-stamped global repository of human sera and pathogen specimens, ideally together with epidemiologic information (biobanks) for current and future use ( ) . we also applaud the push by the us national institute of allergy and infectious diseases to fund international influenza birth cohort studies and help untangle the complex mechanisms of influenza immunity ( ) . if again confronted with a deadly flu pandemic, we would be in a better place than we were in because of the availability of drugs, vaccines, and antibiotics and the general improvements in health and nutrition. there are high hopes for the development of universal vaccines, but we need to keep in mind that influenza is a rapidly evolving virus that has a large and diverse animal reservoir and presumably many tricks in store. we can only anticipate another hundred years of very active, and always surprising, influenza research. structure of the uncleaved human h hemagglutinin from the extinct influenza virus preliminary statistics of 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natality decline and miscarriages associated with the influenza pandemic: the scandinavian and united states experiences transmissibility and geographic spread of the influenza pandemic age-specific excess mortality patterns and transmissibility during the - influenza pandemic in madrid, spain evidence for antigenic seniority in influenza a (h n ) antibody responses in southern china use of serological surveys to generate key insights into the changing global landscape of infectious disease the ghost of influenza past and the hunt for a universal vaccine this article does not necessarily represent the views of the national institutes of health or the us government.conflict of interest: none declared. key: cord- -vpzzsdld authors: thompson, kelly b.; krispinsky, luke t.; stark, ryan j. title: late immune consequences of combat trauma: a review of trauma-related immune dysfunction and potential therapies date: - - journal: mil med res doi: . /s - - - sha: doc_id: cord_uid: vpzzsdld with improvements in personnel and vehicular body armor, robust casualty evacuation capabilities, and damage control resuscitation strategies, more combat casualties are surviving to reach higher levels of care throughout the casualty evacuation system. as such, medical centers are becoming more accustomed to managing the deleterious late consequences of combat trauma related to the dysregulation of the immune system. in this review, we aim to highlight these late consequences and identify areas for future research and therapeutic strategies. trauma leads to the dysregulation of both the innate and adaptive immune responses, which places the injured at risk for several late consequences, including delayed wound healing, late onset sepsis and infection, multi-organ dysfunction syndrome, and acute respiratory distress syndrome, which are significant for their association with the increased morbidity and mortality of wounded personnel. the mechanisms by which these consequences develop are complex but include an imbalance of the immune system leading to robust inflammatory responses, triggered by the presence of damage-associated molecules and other immune-modifying agents following trauma. treatment strategies to improve outcomes have been difficult to develop as the immunophenotype of injured personnel following trauma is variable, fluid and difficult to determine. as more information regarding the triggers that lead to immune dysfunction following trauma is elucidated, it may be possible to identify the immunophenotype of injured personnel and provide targeted treatments to reduce the late consequences of trauma, which are known to lead to significant morbidity and mortality. in modern global conflicts, asymmetric warfare has led to a number of injuries in combat personnel that differ from prior conflicts. since the start of operation iraqi freedom (oif) in , the use of improvised explosive devices (ieds) and ambushes with rocket propelled grenades has led to an increase in the number of personnel wounded or killed by explosions and fewer casualties resulting from gunshot wounds compared to prior conflicts [ ] . during the vietnam conflict, % of injuries were in the head or neck region, and . % were in the thoracic area. oif witnessed a significant shift in the injury pattern, with more than % of injuries occurring in the head or neck region and only . % occurring in the thoracic region, a trend that continues in current conflicts due to improvements in personnel and vehicular body armor. armor use has also led to a decrease in the number of deaths as a result of gunshot wounds, which were down to . % [ , ] . in data examining the timing and causes of death for patients surviving transport to a forward deployed combat surgical hospital in iraq from to , head injury and truncal and/or extremity hemorrhage were the cause of death in % of all patients. most deaths occurred in the acute phase of care, with less than % occurring more than days after admission. of unexpectant deaths with higher preventability scores, hemorrhage was the leading cause of death ( %), followed by multi-organ dysfunction syndrome (mods) ( %), hypoxia ( %), and brain injury ( %). these patients had lower mean injury severity scores (iss) and were less likely to have severe head injuries; however, they had biochemical evidence of severe injuries as evidenced by significant acidosis, coagulopathy, and hypotension on presentation and the majority required massive transfusion (> units of red blood cells in h). the survival of combat-related injuries is in part due to the need for post-injury surgical care, where amputations accounted for % of all wounds, and % of all casualties suffered spinal cord injury [ ] . though there have been significant improvements in combat casualty care, with a focus on preventing deaths from hemorrhage, the relatively high incidence of surgical care and post-injury mods contributes to the burden of morbidity and mortality in those who survive the initial trauma [ ] . the associated morbidity and mortality is multifactorial, among which dysregulation of the immune system is an important contributing factor. immune dysregulation leads to an increased risk for late onset sepsis and infection, acute respiratory distress syndrome, and delayed wound healing in addition to late mods [ , ] . an important factor in the development of mods and immune dysregulation in the setting of trauma is the need for post-injury surgical intervention. given the increased incidence of explosive injuries in modern combat that lead to multiple injury patterns, it is useful to utilize explosive injuries as a model for understanding the complex nature of trauma-induced mods. primary blast injuries caused by the overpressure wave passing through the body lead to damage at the air-fluid interfaces of the tympanic membrane, the lungs, and intestine, and may lead to hollow viscus rupture and internal hemorrhage that can be difficult to identify upon initial triage [ ] . secondary blast injuries occur as a consequence of high-energy projectiles being released from the explosive, leading to extensive tissue injury or loss and wound contamination. tertiary blast injuries result from blunt trauma sustained when the casualty is blown back from the explosion into other objects. quaternary blast injuries involve injuries sustained due to the chemical nature of the blast, including burns and inhalation injuries, as well as when other objects are blown onto the casualty, leading to crush injuries and perhaps further penetrating injuries. injuries from explosives therefore present with numerous complex wounds that are likely to be contaminated and are associated with extensive tissue damage or loss and compounded by microvascular trauma as well. based on the nato triage doctrine, following initial stabilization and basic hemorrhage control by combat medics (role care), the injured are moved to role facilities at forward operating bases (fobs) for the provision of damage control resuscitation (dcr), which includes further hemorrhage control, decontamination or debridement of wounds, and limb or life-preserving surgeries, including exploratory laparotomy with temporary wound closure and the application of negative pressure wound therapy [ ] . administration of blood products and fluids is continued in a balanced manner to restore circulating volume and organ perfusion while allowing for "permissive hypotension". in recent conflicts, approximately to % of military casualties undergo massive transfusion, receiving more than units of blood in the first h after injury. such large volume transfusion has been associated with immune suppression, coagulopathy, acidosis, organ dysfunction and hypothermia [ , ] . these effects are further compounded by uncontrolled pain associated with the injuries of the wounded, which has been shown to induce an inflammatory state leading to hypercoagulation, an increased metabolic demand of tissues, and impaired immune function [ , ] . patients are then moved to a role forward-deployed level trauma-equivalent hospital where dcr is continued. the injured undergo further operations of their wounds and are managed in a critical care facility with the aim of restoring physiological function and limiting the effects of prolonged operative times, massive transfusion, and multiple complex injuries. it is here that the late effects of trauma may begin to develop. once stabilized enough for transport, typically within days after injury, patients are evacuated to role facilities for more definitive or specialized care and rehabilitation [ , , ] . within the first few weeks after injury, patients may undergo multiple surgeries, including initial surgeries for decontamination and hemorrhage control, followed by further debridement, grafting, amputations, primary closures and reconstructions. despite timely and appropriate medical and surgical care, the consequences of immune dysfunction after the initial trauma may persist and lead to further complications for survivors. the initial concepts of immune dysregulation and dysfunction came from a consensus meeting in describing the whole-body response to an infectious or injurious stimulus, which came to be known as the systemic inflammatory response syndrome (sirs) [ ] . these concepts later evolved to incorporate the response of counter-regulatory mechanisms designed to dampen the initial pro-inflammatory signal, termed the compensatory anti-inflammatory response syndrome (cars) [ ] . the temporal association of sirs and cars was initially conceptualized to happen in the sequence of sirs and then cars, but this belief has been challenged by a model demonstrating more overlap between the two responses [ ] . in addition, our more recent understanding of the complex integrated pro-and anti-inflammatory responses to injury has also led to the acknowledgement of a protracted form of immune dysregulation, termed persistent inflammation-immunosuppression and catabolism syndrome (pics) (fig. ) [ ] . while the clinical and temporal evolutions of sirs, cars and now pics have undergone revisions as our understanding of their associated immune phenotypes has evolved, the underlying concepts of pro-and anti-inflammatory responses have remained similar since they were first postulated. following the initial trauma, a host of immune mediators are released by various cells and tissues within the body to activate the immune system and promote a pro-inflammatory state through the expansion and recruitment of various cell lines with the goal of preventing or combating infection and eliminating dead or dying tissue. this pro-inflammatory state is carefully balanced with a compensatory anti-inflammatory response to limit further tissue damage, preserve organ function, and ultimately quiet the pro-inflammatory state and return the body to homeostasis. in severe trauma, there may be an exaggerated pro-inflammatory state, which leads to further injury and rapid multiple organ failure. this may be combined with or followed by an exaggerated and prolonged compensatory anti-inflammatory response, which is associated with immunosuppression through lymphocyte dysfunction and apoptosis, down-regulation of monocyte human leukocyte antigen (hla) receptors, monocyte deactivation, and unbalanced production of cytokines and anti-inflammatory mediators. these effects place injured patients at risk for late complications, secondary to susceptibility to infection and inability to clear infections [ ] . recent studies have suggested that damage-associated molecular patterns (damps) are key to the initiation and continuation of both sirs and cars and may play a critical role in both the "one-hit" and "two-hit" models for the development of mods as well as the subsequent development of pics [ ] . under these conditions, endogenous molecules, such as cytokines (tumor necrosis factor, interleukin- beta) or alarmins (interleukin- alpha, high mobility group box , s ), are released from activated or injured cells to promote a host response, and their presence has been linked to outcomes after trauma [ , ] . more specifically, cytokines are released when pattern recognition receptors, the typical receptors to which damps bind, are activated on immune cells, while alarmins, constitutively active molecules produced by somatic cells, are released when cells undergo necrosis or apoptosis [ ] . the release of alarmins, such as high mobility group box (hmgb ), has fig. temporal association of immune dysfunction syndromes. after an initial combat-related injury, there is the development of a hyper-inflammatory response, termed the systemic inflammatory response syndrome (sirs), and an immune suppressing response, termed the compensatory antiinflammatory response syndrome (cars). these two responses happen within minutes to days, occurring nearly simultaneously, and it is during these initial inflammatory phases that death from early multi-organ dysfunction syndrome (mods) may occur. as both the pro-inflammatory and antiinflammatory responses resolve, there is a period of resolution, typically within days to weeks, that allows for the return to homeostasis and survival after the injury. however, in a percentage of injured patients, the pro-inflammatory and/or anti-inflammatory responses never resolve, leading to a period of chronic critical illness termed persistent inflammatory-immunosuppressive and catabolic syndrome (pics). this occurs in patients who have been critically ill for longer than days with significant lymphopenia and chronic inflammation. pics may persist for months and lead to the risk of developing later mods and secondary infections with subsequent morbidity and late mortality been demonstrated to occur as soon as min after injury. this rapid release in response to trauma is in contrast to the delayed release demonstrated in the setting of severe infections [ ] [ ] [ ] . while the production and release of these molecules is intended to recruit cells to the site of injury and contain its effects, they also alter the response to later infectious or injurious challenges, termed immunotolerance [ ] . this tolerant phenotype was first described in trauma patients in the mid- s, where monocytes isolated from injured patients had a reduced cytokine response to ex vivo stimulation of endotoxins [ ] . though significant debate still exists regarding the mechanisms and effects of immunotolerance following injury or infection, population-based studies have demonstrated a correlation between the presence of endotoxin tolerance and the development of organ dysfunction [ , ] . one of the more important cytokines associated with an immunotolerant phenotype is interleukin- (il- ). this was first shown in il- knockout mice that demonstrated an impaired tolerant phenotype to repeated endotoxin challenge [ ] . persistently elevated il- levels in the plasma have also been correlated with a worse outcome in patients with sepsis and have been associated with the development of secondary complications after burn injury and trauma [ ] [ ] [ ] . more specific to combat-related injuries, higher il- levels have been shown in those who develop mods, as well as in non-survivors compared to survivors [ ] . similar to il- , elevated levels of transforming growth factor β (tgf-β), another anti-inflammatory cytokine, have been shown to correlate with the severity of injury and the development of secondary infections [ ] . comparatively, for those who survive the initial injury, an excessive predominance of pro-inflammatory markers compared to anti-inflammatory markers has been associated with poor wound healing, suggesting a temporal imbalance in immune function recovery and specific trauma-related outcomes [ ] . immune dysfunction after injury has been shown to impact both the innate immune system, which is able to immediately respond without reprogramming or differentiation, and the adaptive immune system, which requires secondary activation and programing via cell-cell contact [ ] . one classical feature of immune dysfunction after systemic inflammation is a reduced expression of human leukocyte antigen dr (hla-dr) on peripheral blood mononuclear cells, which are innate immune cells. this reduced hla-dr expression is associated with impaired antigen presentation [ ] . as early as the s, it was recognized that major trauma results in decreased expression of hla-dr on monocytes and was linked to an increased risk for infection during the recovery period, leading to late morbidity and mortality [ ] . these findings have been confirmed in multiple subsequent studies, which have suggested that both a more robust initial inflammatory reaction along with the inability to recover hla-dr expression predispose and prognosticate trauma patients to subsequent development of sepsis [ , ] . in addition, reduced hla-dr expression has been observed within h after surgery and can be restored through the application of granulocyte-macrophage colony stimulating factor (gm-csf) and interferon-gamma (ifn-γ) [ ] . continued suppression of monocyte hla-dr expression has also been correlated with a worse outcome in patients with sepsis [ ] . though monocytes and the tissue variant of monocytes, known as macrophages, have been the stereotypical innate immune cells to demonstrate immune dysfunction after trauma, other innate immune cells have been shown to have impaired activity, including neutrophils, dendritic cells and natural killer cells [ ] . the immunosuppressive phenotype displayed by these innate immune cells typically involves decreased phagocytosis, decreased cytokine production, decreased cytotoxic function and an overall susceptibility for apoptosis [ ] . the inactivation of monocytes after surgery, trauma, and infections further propagates immune dysfunction through alterations in t lymphocyte function. lymphopenia itself is known to occur after severe injury, and a lack of lymphocyte recovery is known to impact survival [ ] . beyond changes in lymphocyte number, circulating effector t lymphocytes also change from a pro-inflammatory th phenotype to an anti-inflammatory th phenotype [ ] . this change in phenotype is partly due to suppression by regulatory t cells, which are important mediators of il- and tgf-β production. the impairment of effector helper t lymphocytes after trauma also results in a reduction of interferon gamma (ifn-γ) production by th polarized cells [ ] . ifnγ serves a key function in stimulating increased antigen presentation and anti-pathogen activities of innate immunity cells [ ] . after major surgery, while the number of effector t cells decreases, the number of regulatory t cells remains relatively unchanged [ ] . these regulatory t cells express the receptor programmed death (pd- ), which can act as a negative regulator on other immune cells, particularly antigen-presenting cells expressing the programmed death ligand (pd-l ) [ ] . a high expression of pd- on t lymphocytes has been correlated with the severity of illness after major trauma [ ] . beyond t lymphocytes, b lymphocytes are also affected, resulting in impaired antibody production as well as apoptosis [ ] . a summary of the initial combat injury and the subsequent major pro-and anti-inflammatory responses is shown in fig. . while the proposed mechanisms of immune dysfunction mentioned here are not exhaustive and likely involve a complex and dynamic host of responses to curtail the integrated inflammatory response, it is increasingly clear that trauma and the necessary associated surgeries alter the immune system. in those injured personnel who develop more aberrant phenotypes of immune dysfunction, there is a higher risk of developing late complications of the initial injury. despite the early and aggressive medical management of patients as they are moved through various levels of care, alterations in immune function following trauma can place patients at risk for late complications of trauma. in addition, the inability to have sufficient resolution from sirs or cars can lead to the development of pics. the consequences of these altered immune phenotypes can lead to impaired wound healing, late onset sepsis, mods, and acute respiratory distress syndrome (ards) [ , ] . in a study from investigating the incidence of wound infections from wounded personnel arriving at a role facility week after injury from combat operations in afghanistan and iraq, approximately % of the wounds biopsied were infected or critically contaminated as defined by wound tissue biopsy cultures with greater than × cfu/g of biopsied tissue at admission. of infected wounds, gram-negative bacteria predominated, with acinetobacter baumannii being the most common pathogen throughout the study period. this finding was consistent with other reports of predominance in orthopedic wounds and osteomyelitis [ ] . in combat wounds treated at a referral facility within week of injury, nine wounds ( %) in five patients ( %) demonstrated impaired healing, including five delayed wound closures in three patients and four wound dehiscences in two patients, despite appropriate surgical debridement. delays in wound closure were made due to concerns about infection (n = ) or severe systemic illness (n = ). delayed wound healing was found to be associated with increased serum concentrations of multiple inflammatory mediators, including il- , il- , and matrix metalloproteinase- (mmp- ). increased effluent concentrations of il- , il- , and macrophage inflammatory protein alpha (mip α) were also predictive of critical contamination of wounds prior to closure. each of these bio-markers was also independently associated with wound outcome. many of these patients were critically ill on admission with a mean (±sd) iss of ± and a mean acute physiology and chronic health evaluation (apache) ii score of ± on admission. the critical interplay between systemic and local inflammation and wound bacterial burden likely contributes to wound outcome. the balance of chemokines, cytokines, fig. interactions of the innate and adaptive immune systems in response to trauma. immediately following injury, damaged tissues release damage-associated molecular patterns (damps) and in response, residing innate immune cells release pro-inflammatory cytokines. these signals help recruit other innate immune cells to the site of injury in an attempt to contain the deleterious effects of the injury. however, in severe injuries, the immune response goes beyond the local site of injury and leads to systemic inflammation. to reduce the impact of systemic inflammation, the adaptive immune system, primarily through the suppression of regulatory t cells (t reg ), releases anti-inflammatory cytokines and other signals that impede the immune system as it tries to continue the pro-inflammatory response. this manifests as apoptosis of innate immune cells and decreased antigen presentation (hla-dr on monocytes), as well as apoptosis and the anergy of helper t cells causing leukopenia. in the maladaptive state, preponderance of this anti-inflammatory, immune suppressing phenotype leads to the consequences of cars and pics. the general effect of a chronic inflammatory state on immune systems in response to injury is listed below their respective cell types. for a general review of the immune system and inflammation, the reader is referred to a review by spiering [ ] and matrix metalloproteinases that are necessary for appropriate wound healing may be altered by the presence of bacteria at the wound site. furthermore, this balance may be altered by a dysregulated immune response secondary to injury, with a higher risk of immunosuppression-associated infection or failure to clear the bacterial burden and chronic local inflammation at the tissue bed [ ] . although there is a high incidence of blast injuries sustained by combat personnel, only a small percentage ( to %) suffer burn injuries [ ] . despite this, infected burns are a difficult subset of wounds to manage as wound infection leading to sepsis is the most common cause of mortality in burn patients after burn injury and an important component to delayed wound healing. furthermore, due to the disruption of the protective epithelial layer of skin, burn patients are at risk for invasive bacterial and fungal infections. clinicians must have a high index of suspicion for infection as thermal injury-induced hyperpyrexia, immune suppression, and systemic inflammatory response syndrome may alter the typical presenting features of infection and make infection difficult to control [ ] . pseudomonas aeruginosa, klebsiella pneumoniae, escherichia coli and staphylococcus aureus are independent predictors of mortality, with s. aureus being a major cause of septicemia in burn patients [ ] . furthermore, the gram-negative p. aeruginosa, e. coli, and k. pneumoniae are also associated with failure to heal infected burn wounds [ ] . additionally, skin grafting is a common surgical procedure for the management of burns; however, given the presence of co-existing injuries, amputations, and the critical illness of patients, suitable donor sites are difficult to obtain and harvest, potentially leading to delayed healing and an increased risk of infection [ ] . sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection [ ] . sepsis is a major cause of morbidity and mortality after trauma, as alterations in immune function following trauma contribute to an increased susceptibility and impaired ability to combat infection through the modification of both the innate and adaptive immune functions. furthermore, the development of mods is often associated with infection and is the most common cause of late death in trauma patients who survive past the first to h of resuscitation [ ] . following major trauma, sirs is initiated by the activation of the innate immune response. this is often soon followed by cars, which is controlled by the adaptive immune system and was previously thought to occur approximately to days after trauma [ ] . however, more recent research has demonstrated that sirs and cars may occur at the same time with the robustness of each response dependent on a variable milieu of cytokines and other mediators [ , ] . massive trauma can lead to an accelerated and substantial inflammatory response and severe sirs, independent of infection, leading to a "one-hit" initiation of mods [ , ] . patients with less severe trauma may develop late mods due to new surgical stress, general anesthesia, transfusion of blood products, infection, or ischemia/reperfusion injury triggering the reactivation of the inflammatory response in a "two-hit" model of mods [ , ] . a key inciting event in the development of mods and sepsis may be the transfusion of blood products. studies have demonstrated that the transfusion of red blood cells to an already primed immune system leads to a significant increase in il- and tnf-α production by monocytes, which can have deleterious effects following injury or infection [ ] . these effects are likely a result of damps, residual white blood cells, and other soluble and insoluble mediators within the donor blood that contribute to a cascade of transfusion-related immunomodulation (trim), although the exact mechanisms remain difficult to elucidate. regardless, the transfusion of red blood cells has been associated with worsening organ dysfunction, increased rates of infection, and increased mortality [ , , ] . cars typically occurs in conjunction with late onset mods, as immunosuppression increases the potential for hospital acquired infections via immune-inflammatory dysregulation in which the balance of pro-inflammatory and anti-inflammatory mediators is disrupted [ ] . furthermore, it has been postulated that cytokines produced during this period of immune dysregulation may actually favor or promote the growth of bacteria [ ] . according to the trauma register of the german society of traumatology, more than % of civilian trauma patients with multiple injuries develop septic complications, with % of the patients developing multiple organ failure [ ] . among combat personnel admitted to a role facility during operation iraqi freedom, of ( . %) developed infections, with % of cases having wound infections followed by % with bacteremia and % with pneumonia. infection was more likely with those patients having had surgery prior to admission, higher iss, and injuries qualified as blast, abdominal, soft tissue, ≥ injury locations, or loss of limb. s. aureus, e. coli, p. aeruginosa and a. baumannii were the dominant causative organisms of infection, with many demonstrating multidrug resistance [ ] . sepsis and other nosocomial infections increase the risk of late onset mods, which carries a significant mortality burden. in another study of combat-related trauma patients with and without sepsis, of casualties with severe trauma who developed sepsis, developed mods and died. of the matched casualties with severe trauma and no evidence of sepsis, developed mods and died, demonstrating a . -fold higher mortality when trauma is complicated by sepsis [ ] . in combat-related burn patients, the presence of k. pneumoniae bacteremia was independently associated with an increased risk of mortality and increased ventilator days [ ] . according to the american burn association national burn repository, the primary cause of death in burn patients with sepsis is multi-organ failure ( . %), followed by pulmonary failure/sepsis ( . %) and burn wound sepsis ( %), with a higher total body surface area involvement associated with an increased risk of sepsis development and mortality [ ] . acute respiratory distress syndrome (ards) is the most frequent manifestation of mods following trauma, with to % of injured patients ultimately developing the syndrome. trauma patients who develop ards along with mods have mortality rates as high as to %; however, the attributable mortality to ards alone in this population has been difficult to delineate given the severity of co-existing injuries. additionally, ards causes significant morbidity in the trauma population, demonstrating increased rates of complications, longer hospital and icu lengths of stay, and increased hospital costs [ ] . ards has been shown to have varying patterns of onset within trauma cohorts with distinct risk factors for each pattern. in a study utilizing latent class analysis examining the timing of onset of ards in patients with trauma, major phenotypes were identified: early onset ards (occurring < h after trauma) and late onset ards (occurring > h after trauma). early onset ards was associated with an increased severity of thoracic trauma score, more severe early hypotension, and increased red blood cell transfusion during the initial resuscitation, suggesting that early onset ards may be characterized by higher iss and severe hemorrhagic shock necessitating the transfusion of blood products, which is consistent with a "one hit" model of mods and immune dysfunction. late onset ards was hypothesized to be associated with progressive mods and nosocomial infections consistent with the "two-hit" model of mods, in which dysfunction of the innate and adaptive immune systems plays a role in inappropriate immunosuppression, leading to an increased risk of nosocomial infections. despite the two phenotypes, there was no significant difference in mortality between early-and late-onset ards [ ] . in one study from , of mechanically ventilated us combat casualties from operation iraqi freedom/enduring freedom, ards was identified in . % and was associated with higher military-specific iss as well as hypotension and tachycardia at initial presentation. ards was also an independent risk factor for death (or . ) [ ] . additionally, large volumes of plasma and crystalloid infusion have been identified as independent risk factors for the development of ards in combat personnel [ ] . in a study examining the incidence and mortality of ards in combat-related burn patients, . % of mechanically ventilated burn patients developed ards with an overall mortality of . %. however, mortality increased in accordance with ards severity, with severe ards demonstrating . % mortality and a -fold increased odds of death. predictors for the development of moderate or severe ards were inhalation injury, higher iss, pneumonia, and the transfusion of fresh frozen plasma (ffp). [ ] . a recent study has demonstrated that the presence of mitochondrial dna (mtdna) damps from blood products is associated with the development of ards with ffp and platelets having the highest amounts of mtdna fragments prior to transfusion. following transfusion, patient serum concentrations of mtdna fragments increased linearly, with the serum quantity at h after transfusion being a predictor for the occurrence of ards ( . vs . ) [ ] . recently, with the advances provided by critical care medicine, more patients are surviving beyond the wellestablished sirs, cars, and early mods phenotypes and developing a chronic critical illness. this chronic critical illness is characterized by ongoing protein catabolism and a combination of inflammation and immunosuppression termed persistent inflammation-immunosuppression and catabolic syndrome (pics), which serves as a prolonged form of mods with late-term mortality [ ] . pics was characterized by gentile and moore et al. [ ] in as icu stay > days, c-reactive protein ≥ μg/dl, total lymphocyte count < . × /μl of blood, weight loss > % during hospitalization or body mass index < , creatinine height index < %, albumin level < . g/dl, prealbumin level < mg/dl, and retinol binding protein level < μg/dl. pics patients suffer from increased long-term mortality and have increased morbidity associated with manageable organ dysfunctions, poor wound healing, recurrent nosocomial infections, delirium, psychosocial stress, and prolonged rehabilitation needs with a decreased likelihood of returning to pre-insult functional status. recent research has demonstrated that sirs and cars may occur and proceed concurrently for prolonged periods of time leading to pics and that in addition to the mechanisms previously discussed, myeloid-derived suppressor cells (mdscs) may also play a critical role in the development of pics by augmenting both the immunosuppressed and pro-inflammatory state [ ] . following severe trauma or infection, granulocytes rapidly demarginate from the bone marrow and lymphocytes undergo massive apoptosis, creating space for hematopoietic progenitor production in an 'emergency myelopoiesis-granulopoiesis' [ ] . production in these disease states is shifted towards myelopoietic precursors, including mdscs, with the degree of expansion and persistence of mdscs being proportional to the severity of the inflammatory insult. mdscs are both pro-inflammatory and immunosuppressive through their interaction with t-cells and the production of various cytokines. though the precise incidence and evolution of pics after combat injury has not been studied, injured combat personnel may suffer from a milder form of pics as identified by chronic manageable organ dysfunction [ ] . stewart et al. [ ] demonstrated that of the combat injured personnel admitted to an icu, the iss at admission was consistently associated with an increased risk of development of hypertension, coronary artery disease, diabetes mellitus, and chronic kidney disease and at a higher rate than would be expected when compared to military controls. the development of these chronic diseases is likely, at least in part, driven by a chronic inflammatory response initiated by the initial injury and subsequent medical care, as a number of pro-inflammatory cytokines have been implicated in the development of hypertension, diabetes mellitus, coronary artery disease, and chronic kidney disease [ ] . despite the overwhelming presence of both systemic pro-inflammatory and compensatory anti-inflammatory responses after injury, treatment to curb the exaggerated phenotypes remains elusive. the reasons for the absence of targeted therapy are numerous; however, the crux of the issue lies in appropriately identifying the dynamic immunophenotype of a patient after injury. while the pro-inflammatory state happens immediately after injury, work from the late s showed that tolerance to endotoxin challenge could happen as soon as min after traumatic injury [ ] . while this may be an appropriate response to dampen the initial pro-inflammatory cascade, persistence of an anti-inflammatory phenotype after day of illness has been associated with higher mortality [ ] . thus, it seems reasonable to prevent or attempt to reverse the anti-inflammatory phenotype before the development of immune dysfunction. several therapies have been utilized, though the results have been mixed. granulocyte-macrophage colony stimulating factor and granulocyte colony stimulating factor gm-csf and granulocyte colony stimulating factor (g-csf) have been suggested as therapies to reverse the effects of immunosuppression. in a randomized, double-blinded trial of patients who had suffered traumatic brain injury or cerebral hemorrhage, the early application of g-csf ( μg/day) was associated with a reduced incidence of bacteremia, though not on other nosocomial infections or mortality [ ] . in another randomized control trial of patients with sepsis-induced immunosuppression, defined as reduced monocyte human leukocyte antigen-dr (mhla-dr) expression, patients received either a placebo or gm-csf ( μg/ kg/day) [ ] . those in the gm-csf group had a reduced duration of mechanical ventilation and an improved ex vivo monocyte cytokine response to bacterial endotoxin. though data on the use of g-csf during conflict is limited, it has been utilized to treat the myelosuppressive effects of mustard gas during the persian gulf war, suggesting that it could be offered in forward operating areas to aid in recovery [ ] . however, these results are tempered by a meta-analysis of both g-csf and gm-csf, demonstrating that while there was a quicker reversal of sepsis in patients who received therapy, there was no improvement in -day survival [ ] . ifn-γ is a cytokine important for the regulation of t cell function. early animal studies, such as one looking at infection mortality after hemorrhagic shock, showed that ifn-γ prophylaxis could reverse the immunosuppressive phenotype after injury [ ] . a later randomized, multicenter trial tested this hypothesis in severely injured patients through the preventive application of daily subcutaneous injections of ifn-γ ( μg) for days. while early mortality was not affected, mortality from infection was reduced in the ifn-γ treatment group after days [ ] . however, a later study in burn-injured patients receiving ifn-γ prophylaxis for days showed no difference in infection rates compared to placebo controls [ ] . though the application of ifn-γ after combat-related injuries has not been tested, issues could possibly arise from late complications related to the treatment, with a specific focus on wound healing, as animal studies have suggested that systemic ifn-γ treatment can impair wound healing [ ] . conversely, data showing that in dehisced wounds from combat-related injuries, ifn-γ expression is suppressed compared to wounds that heal appropriately, suggesting that either high or low levels of ifn-γ can alter the inflammatory response related to proper wound healing [ ] . the use of pooled intravenous immunoglobulin (ivig) has been proposed as an immunomodulator for some time. the concept behind its use is multifactorial, including receptor blockade, antigen binding, and opsonization. over the past several decades, numerous studies have been conducted examining the utility of either polyclonal or antigen-specific monoclonal ivig in the treatment of sepsis. in aggregate, systemic reviews and meta-analyses have led to no definitive conclusion about the efficacy of ivig in septic patients [ ] . however, within the more specific post-surgical population, the use of ivig has improved sepsis-mediated icu outcomes, especially when combined with appropriate antibiotic therapy [ , ] . in addition, one study examined the prophylactic application of ivig therapy in trauma patients. this randomized study tested the use of polyclonal ivig compared to albumin given in escalating doses ( to mg/kg/day) on hospital days , , and after admission for trauma. these patients also received penicillin prophylaxis on hospital days through . while there were no deaths related to infections in either group, the group that received ivig had a lower rate of nosocomial pneumonia and non-catheter infections [ ] . though the application of ivig after combat-related injuries to prevent immunologically induced organ dysfunction has not been tested, ivig has been used in deployed settings as a treatment for autoimmune diseases, suggesting the feasibility of such prophylactic use in combat areas [ ] . the feasibility of ivig use in deployed settings is further enhanced through the development of lyophilized ivig, which has a similar efficacy, yet a longer shelf life, that could be maintained within forward operating areas [ ] . interleukin- and transforming growth factor β despite the association of il- and tgf-β with an immunosuppressive phenotype, the application of il- antagonism to correct immunosuppression after trauma or injury has not been fully tested. animal models have suggested that the use of il- or tgf-β blocking antibodies can improve survival in polymicrobial sepsis [ ] . in addition, the combination receptor antagonism of il- and tgf-β has led to improved control of parasitic vectors similar to those observed in veterans who served in middle eastern conflicts, suggesting an additional potential benefit of il- and tgf-β antagonism to improve immune function [ , ] . currently, data supporting the clinical use of anti-il- antibodies are limited. its use has only been tested in a single pilot study looking at il- antagonism in patients with systemic lupus [ ] . this is in contrast to tgf-β blockade, which has received significant interest within cancer immunology, with several small molecule inhibitors and antibodies in development [ ] . the successful application of such therapeutics to combat-injured personnel to reverse immunosuppression remains unknown. while the application of the previously mentioned therapies has the largest amount of clinical evidence surrounding their use as immunomodulators in the post-injured or infected, other therapies are currently under investigation. one such therapy is interleukin- (il- ). this endogenous anti-apoptotic cytokine has a main function in supporting the proliferation and survival of effector t cells [ ] . preclinical studies have supported the use of recombinant il- as an immunostimulant to improve survival in animal models of sepsis [ , ] . this has led to a recent trial of human recombinant il- therapy to patients who had evidence of lymphopenia and persistent vasoactive medication requirements in the setting of sepsis [ ] . though the trial was underpowered to detect clinical differences, recovery in t cell counts and function was noted in the il- group, and this effect persisted for several weeks after the completion of therapy, suggesting that a limited early application may have longer-lasting effects. thymosin α is a peptide derived from thymic epithelial cells that has both immunostimulating and immunotolerizing effects on antigen presenting cells and t cells. its use in humans as an immunomodulator dates back to the s when it was used as a therapy to treat immunodeficiency in athymic patients [ ] . the immunomodulatory effects eventually led to its development as a commercially available therapy, called thymalfasin, which was tested as an adjuvant therapy in hepatitis and cancer [ , ] . its properties further led to the investigation of thymosin α as an adjuvant in sepsis. a recent systematic review of clinical trials demonstrated that thymosin α offered daily during sepsis showed benefits with regard to improved t cell counts, reduced cytokinemia, and a reduction in mortality risk ratio to . [ ] . there have been no studies examining the effectiveness of thymosin α in forward operating areas but given that it is supplied as a lyophilized powder that can be injected subcutaneously, its application in such areas would be testable. programmed death- and programmed death ligand- ameliorating t cell and macrophage dysfunction after injury has also been examined by targeting the programmed death- (pd- ) and programmed death ligand- (pd-l ) axis. using animal models of sepsis, the application of pd- or pd-l antibodies around sepsis initiation was associated with reduced leukopenia and improved survival [ ] [ ] [ ] . in humans, treatment of blood with anti-pd- or anti-pd-l antibodies from patients with sepsis or surgically mediated t cell suppression demonstrated decreased t cell apoptosis and increased ifn-γ production [ , ] . clinical trials of antibodies targeting pd- have been further employed in a variety of cancers as well as human immunodeficiency virus infection [ , ] . extrapolation of these efforts into treating patients with immunosuppression after sepsis led to a phase clinical trial using an anti-pd- antibody (#nct ); however, the trial was terminated in . though the pre-clinical data for modulating the pd- /pd-l axis is promising, further data are needed to determine its potential role in reversing the immunosuppressed phenotype after combat-related injuries. the asymmetric warfare of modern conflicts has led to an increased number of wounded combat personnel injured by blast injuries due to the increased utilization of improvised and rocket propelled explosive devices. patients who survive the initial trauma of injury and resuscitation are at risk for several late consequences of their injuries. among these consequences, delayed wound healing, late onset sepsis and infection, multi-organ dysfunction syndrome, acute respiratory distress syndrome, and persistent inflammation-immunosuppression and catabolic syndrome are significant in their association with the increased morbidity and mortality of wounded personnel. these late consequences of trauma have been shown to be associated with a dysregulated immune system that leads to an immunosuppressed state with variable immunophenotypes. promising research into determining the immune profiles of trauma patients to help personalize and target therapies may provide a potential avenue in preventing late complications and directing treatment [ , , ] . recent epigenetic work by scicluna et al. 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antibody bms- in hiv- infected participants on suppressive antiretroviral therapy metalloproteinase expression is associated with traumatic wound failure biomarkers for patients with trauma associated acute respiratory distress syndrome classification of patients with sepsis according to blood genomic endotype: a prospective cohort study immunosuppression in sepsis: a novel understanding of the disorder and a new therapeutic approach blood pressure and heart rate from the arterial blood pressure waveform can reliably estimate cardiac output in a conscious sheep model of multiple hemorrhages and resuscitation using computer machine learning approaches funding rjs was supported by national institutes of health grants, k -gm .availability of data and materials not applicable. authors' contributions kbt, ltk and rjs all contributed to writing the manuscript, with kbt creating the initial draft. all authors read and approved the final manuscript.ethics approval and consent to participate not applicable. not applicable. the views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the air force, navy, the department of defense or the u.s. government (kbt, ltk). key: cord- -q zmids authors: saberian, peyman; kolivand, pir-hossein; hasani-sharamin, parisa; dadashi, fatemeh; farhoud, amir reza title: iranian emergency medical service response in disaster; report of three earthquakes date: - - journal: adv j emerg med doi: . /ajem.v i . sha: doc_id: cord_uid: q zmids introduction: the earthquake is one of the most natural catastrophic crises that can cause a lot of casualties. considering an earthquake-prone country, iran is ranked as one of the world's most dangerous countries objective: in this article, we describe the actions taken by emergency medical service (ems) after the earthquake in kermanshah, varzaghan, and bam and compared the strengths and weaknesses of the emergency response program and the limitations and challenges of this system in dealing with these major crises. method: this study is a cross-sectional study that compares some of the information and findings related to three earthquakes that occurred in iran, including bam, varzaghan and sarpol-e-zahab earthquakes. the data reported in the present article is descriptive and is based on various independent sources such as national emergency operation center, local emergency operations center (eoc), the ems of the country, the world health organization, the united nations, the statistics website, the forensic data website, the international institute of seismology and earthquake engineering, conferences and personal interviews. to ensure the credibility of the information, the authors reported data that had been verified by two or more sources. results: the characteristics of the geographic area of the earthquakes has been described. post-earthquake response activities were described in details in subheadings including rapid warning and response, surge capacity plan, rapid response teams, emergency medical teams, increasing the capacity of health facilities, increasing transfer capacity, and handling, transportation and distribution of injuries. conclusion: in the recent earthquake, had been occurred in sarpol-e-zahab, the health response of the country was largely satisfactory. the existence of structures such as eoc at various levels, the unified incident command system, emergency operations plan, and medical care monitoring center are among the most important reasons for satisfactory performance. over the past years, natural disasters have killed people, injured million people and affected more than billion people worldwide ( ) . the earthquake is one of the most natural catastrophic crises that can cause a lot of casualties ( ) . considering an earthquake-prone country, iran is ranked as one of the world's most dangerous countries. the occurrence of earthquakes with an intensity of more than magnitudes has been recorded in iran since ( , ) . in the past, iran has experienced several earthquakes in different areas, causing severe economic damage and heavy casualties, and the record number of people killed in the earthquakes in the last century has been over , , which the actual number is likely to be more than that ( ) ( ) ( ) . after an earthquake, various components of the health system and treatment centers are encountered with a large number of patients ( ) . emergency medical service (ems) is an important component of the health care system in this situation ( ) . the main goal of ems in responding to a crisis is coordination and management of the health system services, as well as the continuation of medical services through triage, life-saving activities on the scene, rapid diagnosis and treatment, or the transfer of the injured to emergency departments ( ) . it has been proven that timely emergency services will improve the outcome of patients, especially the time-sensitive ones ( ) . although there is a structured approach to the response of the ems system to crises, recent studies indicate that more activities are still needed to improve ( , ) . one of the last earthquakes recorded in iran occurred on november , , near the town of sarpol-e-zahab, kermanshah province. iranian seismological center reported this earthquake . magnitudes and a depth of . kilometers. the earthquake had three foreshocks: . , . , and . magnitudes one hour before the main earthquake and hundreds of aftershocks. this big event was felt across a large area in all directions with a maximum intensity in the eight focal areas of the earthquake. according to the statistics, people lost their lives and thousands of structures suffered serious damage, and the two major cities of ezgeleh ( km north of the earthquake center) and sarpol-e-zahab ( km south-east of the earthquake center) collapsed completely. despite the great challenges that existed in responding to this major crisis, the responses of local and national organizations to address this crisis were significant, and the ems as the first responder in the health system was responsible for coordinating and managing health services in this incident. earlier, other earthquakes like the one occurred in bam of magnitude . in , as well as in varzaghan of magnitude . in , resulted in significant casualties. the experience of large events can have a positive impact on the awareness of organizations and direct their policies to the conduct of incident management activities ( ) . in this article, we describe the actions taken by ems after the earthquake in kermanshah, varzaghan, and bam and compared the strengths and weaknesses of the emergency response program and the limitations and challenges of this system in dealing with these major crises. this study is a cross-sectional study that compares some of the information and findings related to three earthquakes that occurred in iran, including bam, varzaghan and sarpol-e-zahab earthquakes. the reason for choosing these three crises in the first place was the resemblance of earthquakes' strength and intensity, secondly, geotechnical, texture and demographic similarities, and thirdly, the time intervals of earthquakes, which made it possible to study the performance of the ems in three different timeframes. the methodology of this study and the text of this article was reviewed and approved by the board of ems of the country. the data reported in the present article is descriptive and is based on various independent sources such as national emergency operation center (neoc), local emergency operations center (leoc), the ems of the country, the world health organization (who), the united nations (un), the statistics website, the forensic data website, the international institute of seismology and earthquake engineering, conferences and personal interviews. to ensure the credibility of the information, the authors reported data that had been verified by two or more sources. the following criteria have been used for comparison of earthquakes: the first criterion is the comparison of seismology, geotechnical and texture data; the second criterion is the magnitude of the human damage caused by earthquakes; the third criterion: the notification process and rapid warning system of the medical emergency services system; the fourth criterion: the coordination and pre-hospital emergency system's surge capacity program; the fifth criterion: distribution system and transfer of patients and injured. the earthquake occurred in a residential area with a history of past earthquakes. the study area is located in zagros seismotectonic state. zagros is considered to be the most active and earthquakeprone region in iran. according to reports in kermanshah province, % of buildings are made of masonry materials, i.e., without a building structure. about % of urban buildings ( , units) and % of buildings in rural areas ( , units) of kermanshah province have been made of masonry materials. in kermanshah province, more than one million and a hundred thousand people reside in housing units without a skeleton ( ) .  bam buildings in the region were made of masonry, brick, steel, and concrete. the brick and mortar buildings, which have desert architectural features, were completely destroyed and collapsed in more than % of the cases. most of the residential structures in the rural areas and the cities of varzaghan, ahar, and herris were mortar and mud or masonry material buildings. the largest casualty in this earthquake was caused by the destruction of mud, mortar or brick rural buildings that simplest technical criteria had not been considered, and non-structural parts such as walls and false ceilings had severe damage. geological features of the earthquake, the facilities of the area and damage to humans are summarized in table . response activities after the earthquake were focusing on the priorities of the response were immediately performed. a joint effort was made by government officials and international communities in the first week after the tragedy. information is presented separately in table .  rapid warning and response due to the severity of the earthquake which was felt by most of the officials in kermanshah, relative preparation was made at local authorities from first minutes, and immediately after the earthquake, the leoc was activated. since there was no damage to the ambulances of the region, ems forces of the area immediately began to provide services to the injured. notification to the neoc was performed according to preplanned protocols for warning levels and activation levels. during the first hour after the incident, the first meeting of the emergency committee was held at the neoc site. local and central hospitals, rapid response teams at local, regional and national levels were called. the disconnection of communication and telecommunication networks in the affected areas has caused difficulties in calling, estimating damages, correct locating and estimating the required resources. the reports of the affected areas were collected from the health information management unit which was developed at neoc. all local, regional and capital hospitals were on standby. one of the major strengths of the incident was its concurrence with the arbaeen ceremony. fortunately, these forces had been stationed on the western border of the country to manage mass gatherings from months ago that resulted in a more comprehensive, efficient and faster response. in total, ambulances, ambulance buses, two mobile communication vehicles were sent from the mehran border to the earthquake zone, which began serving the wounded from the beginning hours. in the varzaghan earthquake, the leoc was immediately activated and the call for forces was made minutes after the earthquake. coordination was conducted to deploy forces from the province, road stations and neighboring provinces, and backup ems teams were sent from the road stations, city and the province, as well as adjacent provinces of west azerbaijan, ardebil and zanjan. the provincial teams were presented at the damaged site at : . unfortunately, in the bam earthquake, the eoc structure was not established at the local and national levels. also, due to the severity of the destruction of the area, the first relief teams in the affected region were announced at : ( : after the earthquake). there was no comprehensive warning and response system, which was one of the reasons for the lack of coordination in response plans.  surge capacity plan due to the extent of the kermanshah earthquake, the care needs of patients and injured were more than available resources; therefore, the main focus of the medical emergency system was on increasing care capacity. starting from the very early hours after the incident, the surge capacity in three axes of man resources, equipment and structure was started. based on reports and evidence available, hours after the receipt of earthquake news, medical teams were called from most of the province's hospitals. but the damage to some health centers poses serious challenges to treatment operations. according to the information of ems capacity assessment system and reports on the extent of damage, the amount of need for force, equipment, and ambulance was estimated by the committee of the emergency situation of health system crisis and immediately the resources were called and deployed to the region. also, all units located on the western border for the arbaeen ceremony were settled in the region immediately after the early hours of the incident.  rapid response teams following the earthquake in kermanshah, rapid response teams (rrts) of the ems system were sent to the site to assess and respond to the medical needs of the injured.  emergency medical teams after the earthquake, disaster medical assistance teams (dmats) were immediately recalled by the neoc and served to the injured of the incident. considering the damage to the health centers, there was no possibility to provide quick services to the injured in these centers, and the transfer of these injured to hospitals in kermanshah province or neighboring provinces could create serious challenges for these injured; the rapid launching of field hospitals led to the transfer of the injured with critical priority to these centers and provided essential treatment and medical services to these patients and injured near the incident site before they were transferred to a permanent hospital. performing various surgeries such as hand transplantation is one of the important functions of these hospitals. in this incident, considering the severity of the incident in the sarpol-e-zahab area and the complete destruction of the only regional hospital, the first -bed field hospital was set up in less than hours of the incident in the area, followed by field hospitals in west islamabad and salas babajani also launched. interagency coordination and management carried out by the neoc was another strength of the incident, as early as the aftermath of the incident, the islamic revolutionary guard corps (irgc) and the army arrived at the scene. the establishment of three field hospitals by the military forces in the region provided a great deal of assistance in managing injured and casualties. another effective action to increase the capacity of health facilities was the discharge of nonemergency patients, which was done by alert to all medical centers in kermanshah and neighboring provinces. there are no accurate statistics on the extent of surge capacity of the medical centers. the next effective action was converting the nonmedical centers into the temporary medical centers, as well as the conversion of singlespecialty hospitals to multi-specialty hospitals; in the earthquake of varzaghan, the martyr madani and taleghani single specialty hospitals were converted into general hospitals through the establishment of specialist expeditions from neighboring provinces. upon the completion of the debris removal and the prediction of the second wave of injured, gyms no. and of the medical university were equipped with emergency facilities and turned to the health centers to be used in case of encountering a large number of patients. also, by equipping all the capacities available in imam reza, shohada, sina and razi hospitals, the capacity of these hospitals increased by beds.  increasing transfer capacity immediately after assessing the situation, ambulances from kermanshah province and ambulances settled on the western border of the country for arbayeen, helicopters and ambulance buses were deployed to the site. in some of the affected centers where treatment facilities were severely damaged, the ems bus ambulances were utilized as the outpatient and temporary treatment sites. according to coordination by the neoc, with the commander of the army and the commander of irgc, four aircraft, and helicopters were provided to ems for the transfer of the injured. a total of flight missions resulted in the transfer of injured.  handling, transportation and distribution of injuries after the occurrence of the earthquake and the presence of ems forces, the triage of the injured and basic and advanced life support was carried out by ems forces according to the predefined protocols and the transfer and distribution of the injured to the health centers was begun. calls from the scene to the referral hospitals were often difficult due to communication problems, as well as lack of awareness of the capacity of the hospitals, that requires a direct and specific contact, so the capacity of the local, regional and tehran hospitals using the medical care monitoring center (mcmc) was determined and the patients were distributed by the coordination of mcmc with referral hospitals. of the kermanshah earthquake missions, . % ( ) were transferred to provincial hospitals and . % ( persons) to hamedan province and . % ( people) to tehran and alborz in hours. . % ( people) were transferred via air medical service and . % ( people) via land medical service. the ems of the country created a triage point with medical teams at the airport. patients with higher priority of treatment were immediately triaged at the delivery point of the airport and soon after referred to referral hospitals in accordance with defined protocols and prior notification. comparing the performance of the ems system in three recent earthquakes in rapid response and warning highlighted the importance of the existence of the eoc structure at local, regional and national levels. in bam earthquake, the lack of these structures and infrastructure communication at levels resulted in more than hours of delay in the presence of the first relief teams in the region. the coordination between the leoc and the neoc lead to a quicker and more coherent response in the varzagan and sarpol-e zahab earthquakes. also, the preparation and deployment of emergency teams on the western border of the country for the ceremony of arbaeen led to the timely presence of these teams in the sarpol-e-zahab earthquake. to increase the capacity of the emergency medical and therapeutic system, the experiences recorded in earthquakes indicate lack of a comprehensive surge capacity in bam, which could be a reason to increase the number of mortality and morbidity following the incident. comparing the program of surge capacity in varzaghan and sarpol-e-zahab earthquakes, the focus of the medical emergency system in the varzaghan earthquake was on using its provincial capacities through converting non-therapeutic centers to the therapeutic ones, the discharge of non-emergent patients and the conversion of single-specialty hospitals to general ones. in order to increase ems capacity, about one-third of the province's capacity has been deployed to the earthquake-affected area. the strengths of this program are reducing the need for the transfer of the injured to other provinces and the costeffectiveness of this program. on the other hand, in the recorded experiences of an earthquake, it is recommended that the treatment of the injured be carried out near the site of the incident so that the patient's relationship with family members and relatives is not discontinued. the negative aspect of the program was that the use of provincial capacity would disrupt the provision of the relief process and routine services and could lead to the collapse of health systems in the region. in the sarpol-e-zhahab earthquake, coordination and support of other organizations such as islamic revolutionary guard corps (irgc) and the army led to an increase in the capacity of the health systems, on the other hand, the presence of relief and medical units at the western border and the recall of these forces on the scene immediately after the occurrence of the earthquake led to decrease of need to surge capacity of the province which in turn caused organizing the provision of medical services to the injured without triage and the patients who went to local health facilities on their own cars. nor did there be a disruption in routine processes. the weakness of this program is being costly and moving the patient away from the place of his/her residence which requires a registry system for the distribution of the injured and strong follow-up systems. existence of the comprehensive mcmc system for managing and coordinating the distribution of patients resulted in a faster and more regular distribution of patients in provincial hospitals and hospitals of other provinces and in addition facilitated the registration of patients' information and follow up. the uniform incident command system (ics) was developed through the ems of the country for the integrated coordination and management of health services so that the management of facilities and resources of cooperator organizations, support, and even military forces was carried out by this system. this resulted in the transfer of a significant number of injured persons within a limited time frame, which certainly affected the mortality and morbidity of traumatized patients that were timesensitive. one of the limitations of this plan was the lack of an integrated national structure for recording information and lessons learned from events and crises, resulted in some information being inaccessible. it seems that, in the recent earthquake, the health response of the country was largely satisfactory. the existence of structures such as eoc at various levels, the unified incident command system, emergency operations plan (eop), and mcmc are among the most important reasons for satisfactory performance. however, there is still a need to strengthen integrated systems and interorganizational management. the lessons learned from these earthquakes can provide motivation for enhancing preparedness for the future and response mechanisms. public health guidance for community-level preparedness and response to severe acute respiratory syndrome (sars) orthopaedic injury analysis in the yushu, china earthquake list of earthquakes in iran annual disaster statistical review : the numbers and trends. centre for research on the epidemiology of disasters (cred) prehospital management of earthquake casualties buried under rubble lessons learnt from the past and preparedness for the future: how a developing country copes with major incidents facilitators and obstacles in pre-hospital medical response to earthquakes: a qualitative study commentary: emergency medical services: just the beginning of an effective system a national evaluation of the effect of trauma-center care on mortality systematic reporting to improve the emergency medical response to major incidents: a pilot study the initial health-system response to the earthquake in health systems effectiveness and efficiency for disasters and conflicts earthquake kermanshah province we gratefully thank the ems personnel that were involved in management of mentioned disasters. we also thank pre-hospital emergency research center, for their support and collaboration in conducting and publishing the current study. all the authors met the standards of authorship based on the recommendations of the international committee of medical journal editors. none declared. this study was conducted with a grant from tehran ems center. key: cord- -nnoni qb authors: huang, wan-ping; cho, che-pei; chang, kung-yao title: mrna-mediated duplexes play dual roles in the regulation of bidirectional ribosomal frameshifting date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: nnoni qb in contrast to − programmed ribosomal frameshifting (prf) stimulation by an rna pseudoknot downstream of frameshifting sites, a refolding upstream rna hairpin juxtaposing the frameshifting sites attenuates − prf in human cells and stimulates + frameshifting in yeast. this eukaryotic functional mimicry of the internal shine-dalgarno (sd) sequence-mediated duplex was confirmed directly in the s translation system, indicating that both frameshifting regulation activities of upstream hairpin are conserved between s and s ribosomes. unexpectedly, a downstream pseudoknot also possessed two opposing hungry codon-mediated frameshifting regulation activities: attenuation of + frameshifting and stimulation of a non-canonical − frameshifting within the + frameshift-prone cuuuga frameshifting site in the absence of release factor (rf ) in vitro. however, the − frameshifting activity of the downstream pseudoknot is not coupled with its + frameshifting attenuation ability. similarly, the + frameshifting activity of the upstream hairpin is not required for its − frameshifting attenuation function thus, each of the mrna duplexes flanking the two ends of a ribosomal mrna-binding channel possesses two functions in bi-directional ribosomal frameshifting regulation: frameshifting stimulation and counteracting the frameshifting activity of each other. genetic codes and potential secondary structures defined by local rna sequences constitute the two layers of information embedded within the primary mrna sequences of an open reading-frame (orf). the secondary structures in mrna are unwound by an intrinsic duplex-unwinding activity of ribosome during translation [ , ] , allowing exposure of the buried genetic codes for decoding in the ribosomal a-site, while refolding co-translationally after leaving the ribosome. as an mrna is translated by multiple ribosomes, the unwinding and refolding cycles of secondary structures repeat for an active mrna under translation. however, specific mrna structures could resist ribosomal unwinding and trigger backward slippage (toward the '-direction of mrna) of the ribosome by one nucleotide within a slippery sequence to stimulate translational reading-frame switch toward the − frame. an in-frame xxxyyyz slippery sequence as well as an optimally placed ( - nucleotides) downstream stimulator pseudoknot [ ] or hairpin [ ] is required for efficient − programmed ribosomal frameshifting (prf) with x and y each representing an identical nucleotide. by positioning a-and p-site trnas over the slippery sequence while keeping watson-crick base-pairing identities of the codon-anticodon interactions at the first two positions of each codon unchanged in -and − frames, this configuration helps increase probability to slip the ribosome one nucleotide in the ' direction [ , ] . by contrast, specific mrna signals can program a ribosome to slip forward (toward the '-direction) by a single nucleotide for + frameshifting [ ] [ ] [ ] . the well-documented + frameshifting in the ty retrotransposon in saccharomyces cerevisiae involves a hepta-nucleotide sequence (cuuaggc), and is caused by ribosome pausing at the agg codon due to low expression levels of agg decoding trna while being decoded within ribosomal a-site [ ] . differences in cis-element requirement between + and − frameshifting suggest that distinct mechanisms are responsible for these two frameshifting pathways, although disruption of codon-anticodon interactions between peptidyl-trna and mrna during translocation step of translation elongation has been proposed to play an initiation role for frameshifting [ ] . both + and − prfs have been characterized in prokaryotic gene expression regulation [ ] [ ] [ ] . + prf is adopted by the prfb gene of e. coli. to encode one of the translation termination proteins, release factor (rf ), in a regulatory feedback loop. when the uga recognizing rf is insufficient in a cell, a translating ribosome pauses at the stop codon within the cuuuga sequence located in the n-terminal coding region of prfb. to escape from this trap, the paused ribosome slips one nucleotide forward to continue translation in + frame to complete the synthesis of full-length rf . this results in supply of mature rf to terminate translation at the frameshifting site uga codon to prevent synthesis of more rf . by contrast, the dnax gene of e. coli. produces the γ subunit of dna polymerase iii by − prf via an aaaaaag slippery sequence (a g) and a downstream rna hairpin. [ ] . single-molecule fluorescence analysis tracking dye-labeled ribosome movement along mrna suggested that the downstream dnax hairpin impeded translocation while stimulating − frameshifting at aag codon during the incoming accommodation step [ ] . however, a kinetic analysis of a downstream − prf pseudoknot in an in vitro s translation system argued that the frameshifting event occurred in the post-translocation step after peptide bond formation between the aminoacyl-trna and peptidyl-trna that sit at the xxy and yyz codons, respectively [ ] . nevertheless, these works help establishing s ribosome as an ideal platform for mechanical analysis of frameshifting regulation. in addition to specific downstream structures, an internal shine-dalgarno sequence (sd) to nucleotides upstream of the slippery site was shown to act as a − prf stimulator [ ] . furthermore, e-site juxtaposing internal sd upstream of the cuuuga + frameshifting site can promote + frameshifting [ ] , whereas deletion analysis indicated that e-site juxtaposing upstream internal sd can downregulate − prf stimulated by a downstream dnax hairpin [ ] . although internal sd anti-sd interaction has been suggested to be an elongation pausing element for s ribosome [ ] , mechanisms underlining the opposing effects in + and − frameshifting regulation by e-site juxtaposing upstream internal sd remain unclear. recently, it was found that a potential rna hairpin upstream of xxxyyyz − frameshifting site can down-regulate mammalian − prf as well as stimulate agg codon mediated + frameshifting in saccharomyces cerevisiae [ , ] . the frameshifting regulation activities were shown to be positively correlated with the predicted stability of the upstream hairpins. given the unfolding/refolding cycles of a translating mrna, it suggests the involvement of a co-translationally refolded upstream hairpin in frameshifting regulation when the ribosome moves into a downstream frameshifting site. these findings of unexpected frameshifting regulation by upstream structures are in contrast to the well-known role of downstream rna structures in stimulation of eukaryotic − prf [ , ] and implicate a link between the mechanisms of + and − frameshifting regulation by mrna structures flanking frameshifting sites. however, differences of sequence compositions between the + and − frameshifting sites hamper further analysis. intriguingly, a trans-formed duplex mediated by an antisense dna and the sequence upstream of frameshifting site can replace the cis-acting hairpin to attenuate − prf [ ] . in addition to suggesting that formation of an upstream duplex juxtaposing frameshifting sequences is the determinant for frameshifting regulation, this result is reminiscent of the duplex formed between the internal sd sequence and s rrna in prokaryotic frameshifting regulation [ , ] although the components forming the two duplexes are different. therefore, a direct comparison of frameshifting regulation activities between upstream hairpins and internal sd mediated duplexes in s ribosome could shed light to help resolving the roles of mrna structures flanking the ribosomal mrna-binding channel in frameshifting regulation. in this study, the frameshifting regulation activities of upstream refolding hairpins were examined using the well-characterized − frameshift-prone a g and the + frameshift-prone cuuuga frameshifting sites as the model systems. in addition to showing that a stable upstream hairpin is a functional mimicry of upstream internal sd in both + and − frameshifting regulations, we discovered a downstream structure-mediated non-canonical − frameshifting under the + frameshift-prone cuuuga frameshifting site in the absence of rf . importantly, by using this frameshifting site to minimize the effects of sequence composition differences in p-site codon-anticodon interactions, we are able to demonstrate that a downstream structure can stimulate − frameshifting and attenuate + frameshifting, which are opposite to the frameshifting regulation activities of the upstream refolding hairpin. these findings indicate that stable structures flanking the two ends of mrna-binding channel within an elongating ribosome play opposite roles in regulation of bidirectional reading-frame switch. in addition to providing a framework for further mechanism analysis, these results can be used to explain non-canonical frameshifting events observed in disease-related genes containing long expanded cag trinucleotide repeat expansions. cell-free in vitro translation lysates of e. coli. have been used to reproduce cellular translation mediated processes, including ribosomal frameshifting for mechanism analysis [ , ] . to examine the frameshifting regulation activity of upstream refolding hairpins in s ribosome, a glutathione-s-transferase (gst) and renilla luciferase fusion protein coding construct (gst-rluc) was designed to serve as the in vitro frameshifting reporter. the gst open reading frame (orf) was fixed in frame, while the downstream rluc orf was fused out of frame such that it could only be accurately translated upon frameshifting through insertion of translational frameshifting signals between gst and rluc orfs ( figure s a ). premature stop codons for out of frame translation were also inserted in the n-terminal region of rluc orf ( figure s a ). they were used to prevent radioactivity-based frameshifting efficiency calculation bias caused by ribosome drop-off effect [ ] during translation of long frameshifted polypeptides. in vitro − and + prf activities using different amounts of reporter mrna as translation templates in cell-free lysates of e. coli. (figures s b,c and s a) were examined to determine the optimal amount of mrna ( ng) required for frameshifting assay. the − prf efficiencies induced by a downstream dnax hairpin in the presence of upstream internal sd mediated duplexes with different spacings toward the frameshifting site ( figure s b ,d) were similar to those reported in vivo [ ] , indicating that the experimental platform can faithfully reproduce this − prf model system. to compare upstream hairpins with sd anti-sd duplexes in frameshifting regulation, we used the a g slippery sequence with a modified pseudoknot (mpk) derived from the infectious bronchitis virus (ibv) a/ b gene [ , ] , and the cuuuga frameshifting site of rf for − and + prf analysis, respectively ( figure ) . a cgc sequence was used as the e-site codon for the cuuuga frameshifting site to avoid e-site invasion [ ] that could mask the + frameshifting stimulation activities of upstream duplexes. the effects of upstream internal sd-mediated duplexes and hairpins ( figure a ) on − and + prf efficiencies were analyzed. an rna hairpin of a gc base-pair rich base-pairs stem capped by ugcg loop sequence (h ) with a predicted stable hairpin conformation was used for comparison ( figure a ). we found that the sd-asd duplex attenuated activity of − prf stimulated by mpk the most ( figure b ,c), and was also the most potent stimulator of + prf for the cuuuga frameshifting site in the absence of rf ( figure d ,e). by contrast, rna hairpins regulated + and − frameshifting efficiencies to a lesser extent. however, the stable upstream hairpin, h reduced − prf to a similar extent as that of internal sd and possessed about % of the + frameshifting activity that can be stimulated by the internal sd ( figure d ,e). by contrast, a destabilization mutant of h with reduced stability through watson-crick base-pairing disruption and bulge insertion (h ) possessed much lower − prf attenuation and + prf stimulation activities compared with those of h . these results are consistent with the positive correlation between frameshifting regulation activity and hairpin stability observed before [ ] . furthermore, the upstream hairpin-dependent + frameshifting occurred only in the absence of rf ( figure s b ). together with previous analysis in eukaryotic systems [ ] , these results indicate that stable upstream refolding hairpins are functional elements capable of regulating frameshifting in both s and s ribosomes. . stimulation of + frameshifting as well as attenuation of - frameshifting by internal sdmediated duplex mimicries in an in vitro s translation system. (a) sequences, predicted secondary structures and free energy of distinct upstream hairpins evaluated. mfold [ ] was used for mrna secondary structure and free energy prediction. effects of upstream duplex variants on - and + figure . stimulation of + frameshifting as well as attenuation of − frameshifting by internal sd-mediated duplex mimicries in an in vitro s translation system. (a) sequences, predicted secondary structures and free energy of distinct upstream hairpins evaluated. mfold [ ] was used for mrna secondary structure and free energy prediction. effects of upstream duplex variants on − and + frameshifting were evaluated using respective a g and cuuuga frameshifting sites inserted in pet gst-rluc- reporters. due to the exclusion of rf in the reaction, a uaa or uag stop codon was used as the frame stop codon (in purple color), while uaa was used as the out of frame premature stop codons in the n-terminal region of rluc. mutations to disrupt the stem of h were typed in rose-red color. (b) radioactivity-based − prf assay by sds-page analysis of s methionine-labeled in vitro translation products, with calculated − prf efficiencies shown, for different upstream duplex constructs in (a). the values displayed are means ± sd of three independent experiments. (c) relative − prf activity of frameshifting activities in (b) compared to sd∆ with asterisks indicating p value < . using student's t-test. (d) radioactivity-based + prf assay by sds-page analysis of s methionine-labeled in vitro translation products, with calculated + frameshifting efficiencies shown, for different upstream duplex constructs in (a). the values displayed are means ± sd of three independent experiments. (e) relative + prf activity of (d) compared to sd∆ with asterisks indicating p value < . using student's t-test. the ability to stimulate + prf and attenuate − prf by a stable upstream hairpin implies a link between + frameshifting stimulation and − frameshifting attenuation. it also raises the question of whether a downstream − prf stimulator also plays an opposite role in − and + frameshifting regulation. this means that a downstream − prf stimulator may serve as a + prf attenuator. to test this hypothesis, mpk was placed downstream of the + frameshift-prone cuuuga sequence ( figure a ) to examine its effect on + frameshifting in the absence of rf . a mutant of mpk with stem disrupted (mt) was used as the negative control for measuring intact + frameshifting activity. interestingly, + frameshifting activity decreased when the downstream structure was replaced from mt to mpk ( figure b ,c). this result is consistent with + frameshifting attenuation by a downstream mpk. thus, the mpk downstream of mrna-binding channel of an elongating ribosome plays reverse regulatory roles in + and − frameshifting regulations. unexpectedly, an extra translation product appeared when the cuuuga sequence was followed by an mpk or a hairpin in the absence of rf ( figure b mt and sl are the variants of mpk with mutations (in colors) to destabilize the stem and stem , respectively. please note that there are three uaa stop codons (with different colors) to terminate translation in three different frames in the absence of rf in these constructs using the pet gst-rluc- reporter ( figure s a ) as the backbone. the − and + frame uaa stop codons are located in the n-terminal region of rluc orf with the − frame stop codon being moved to downstream of + frame stop codon in the "shift" construct. (b) the + frameshifting attenuation assays by sds-page analysis of s methionine-labeled in vitro translation products for different mpk variant constructs in (a), with + prf and potential − prf products annotated. calculated − and + prf efficiency values displayed are means ± sd of three independent experiments. (c) relative + prf activity of construct with downstream wt mpk compared to mt with asterisks indicating p values < . using student's t-test. unexpectedly, an extra translation product appeared when the cuuuga sequence was followed by an mpk or a hairpin in the absence of rf ( figure b lanes and ) , whereas it disappeared when rf was added (lane ) or the downstream mpk was replaced with an mt (lane ). sequence analysis implicated that the extra translation product could arise from a − frameshifting event although there was no xxxyyyz consensus slippery sequence of − prf. consistently, mutating the potential − frame stop codon moved the extra translation product to a higher region of the gel, corresponding to translation termination at the next − frame stop codon ("shift" in figure b ). together, these results suggest that a non-canonical − frameshifting may have occurred in addition to + frameshifting. finally, faint extra bands with low mobility in the gel appeared upon longer image exposure and disappeared upon treatment of the translation reaction with rnase before gel-loading ( figure s ). given that the translated protein products were s-methionine labelled, the senstivity to rnase treatment suggested that the low mobility bands belonged to colvant linkage adducts between proteins and rnas, and most probably a peptidyl-trna intermediate during translation in the absence of rf . to confirm the identity of the extra translation product, a flag-tagged construct (flag-cuuuga-mpk) ( figure s a ) was designed to help enrich the recovery of potential − frame products for mass spectrometry analysis. the nucleotide sequence encoding the flag amino acid sequence was inserted upstream of the corresponding frameshifting site in the frame of gst-rluc fusion orf so that translation products, including shifted and non-shifted ones all contain the flag fragment. after translation, these flag-tagged translation products were immuno-precipitated by anti-flag antibody, purified by protein g conjugated bead, and analyzed by sds-page ( figure s b-e) . comparison of the frameshifting products of the construct before and after immunoprecipitation with those of the flag-less frameshifting reporter indicated that frameshifting efficiency remained similar between the two constructs ( figure and figure s d ). attempts of lc-ms/ms analysis by thrombin treatment to remove gst from the − frameshifted c-terminal peptide were not successful (data not shown). instead, in-gel trypsin digestion was performed to generate peptide fragments for lc-ms/ms characterization. analysis of liquid chromatography tandem ms (lc-ms/ms) data of peptide fragments derived from the unusual translation product identified a amino acid tryptic peptide fragment (llitgvsvr) and this was further validated using a chemically synthesized peptide standard ( figure a,b) . this result supports a frameshifting event occurring at the cuuuga sequence with a uga to uug shift (encoding the second leucine after formation of the first leucine by decoding the frame cuu), followed by continued translation extension in the − frame ( figure c and figure s f ). as the first u of the e-site uau codon juxtaposing the cuuuga + frameshifting site in wildtype rf was reported to involve e-site invasion through base-pairing with anti-sd in s rrna [ ] , we performed e-site sequence mutagenesis to explore the mechanism of non-canonical - frameshifting. interestingly, replacing e-site sequence from cgc to cgu dramatically diminished the - frameshifting product with the restoration of + frame product (compare lanes and in figure as the first u of the e-site uau codon juxtaposing the cuuuga + frameshifting site in wild-type rf was reported to involve e-site invasion through base-pairing with anti-sd in s rrna [ ] , we performed e-site sequence mutagenesis to explore the mechanism of non-canonical − frameshifting. interestingly, replacing e-site sequence from cgc to cgu dramatically diminished the − frameshifting product with the restoration of + frame product (compare lanes and in figure a ), whereas replacement from cgc to ugc did not lead to a diminished − frameshifting product (compare lanes and in figure a ). further mutating ugc to uac to reduce codon-anticodon stability did not alter the distribution of frameshifting patterns (lanes and in figure a ), whereas mutating ugc to ugg almost abolished the − frameshifting product (lane in figure a ). thus, neither stability of e-site codon-anticodon pairing nor e site invasion is the main cause of the dramatic switch in frameshifting pathways. although the diminished − frameshifting can be explained by − prf efficiency modulation by e-site sequence composition [ , ] , we noticed that significant − frameshifting seems to require the third position of the e-site codon to be a c nucleotide, which also occurs at the first position of the p-site codon. furthermore, dramatic − frameshifting reduction by converting cgc cuu to cgu cuu can be reversed by further mutating the p-site sequence from cuu to ucu (lanes - in figure a ), resulting in an identical u bridging the e-and psites. together, these data indicate that an identical nucleotide in the two positions bridging frame eand p-sites is enough to facilitate − frameshifting with a downstream structure in the absence of rf , and is consistent with the repairing of frame peptidyl-trna to an identical nucleotide in the first position of the − frame codon to pave the way for − frame switching. a), whereas replacement from cgc to ugc did not lead to a diminished - frameshifting product (compare lanes and in figure a ). further mutating ugc to uac to reduce codon-anticodon stability did not alter the distribution of frameshifting patterns (lanes and in figure a ), whereas mutating ugc to ugg almost abolished the - frameshifting product (lane in figure a ). thus, neither stability of e-site codon-anticodon pairing nor e site invasion is the main cause of the dramatic switch in frameshifting pathways. although the diminished - frameshifting can be explained by - prf efficiency modulation by e-site sequence composition [ , ] , we noticed that significant - frameshifting seems to require the third position of the e-site codon to be a c nucleotide, which also occurs at the first position of the p-site codon. furthermore, dramatic - frameshifting reduction by converting cgc cuu to cgu cuu can be reversed by further mutating the p-site sequence from cuu to ucu (lanes - in figure a ), resulting in an identical u bridging the e-and p-sites. together, these data indicate that an identical nucleotide in the two positions bridging frame e-and p-sites is enough to facilitate - frameshifting with a downstream structure in the absence of rf , and is consistent with the repairing of frame peptidyl-trna to an identical nucleotide in the first position of the - frame codon to pave the way for - frame switching. mpk has been suggested to stimulate - prf in a uuuaaag slippery site during posttranslocation after peptide bond formation while frame uua and aag codons occupy ribosomal p-and a-sites, respectively [ ] . given that the cuu codon in the cuuuga frameshifting site represents the last coding amino acid in the frame, positioning the cuu codon into the a-site for formation of the final peptide bond results in a spacing of nucleotides in length toward downstream mpk ( figure a) . however, the absence of rf being required for frameshifting to occur indicates that frameshifting occurs after the uga codon enters the ribosomal a-site, resulting in a spacing of nucleotides in length toward mpk. we reasoned that if the first model can explain observed - frameshifting, deleting the spacer length by nucleotides should still be - frameshifting competent ( figure a ). by contrast, the second model predicted such a deletion mutant should be frameshifting incompetent because spacing toward the pseudoknot becomes non-optimal ( nucleotides in length). the results showed that the resulted construct sp lost - frameshifting activity ( figure b ), indicating figure . an identical nucleotide bridging e-and p-sites facilitates non-canonical − frameshifting. sds-page analysis of in vitro radioactivity based frameshifting assays of constructs with e-site or p-site mutagenesis. calculated − and + prf efficiencies displayed are means ± sd of three independent experiments. mpk has been suggested to stimulate − prf in a uuuaaag slippery site during post-translocation after peptide bond formation while frame uua and aag codons occupy ribosomal p-and a-sites, respectively [ ] . given that the cuu codon in the cuuuga frameshifting site represents the last coding amino acid in the frame, positioning the cuu codon into the a-site for formation of the final peptide bond results in a spacing of nucleotides in length toward downstream mpk ( figure a) . however, the absence of rf being required for frameshifting to occur indicates that frameshifting occurs after the uga codon enters the ribosomal a-site, resulting in a spacing of nucleotides in length toward mpk. we reasoned that if the first model can explain observed − frameshifting, deleting the spacer length by nucleotides should still be − frameshifting competent ( figure a ). by contrast, the second model predicted such a deletion mutant should be frameshifting incompetent because spacing toward the pseudoknot becomes non-optimal ( nucleotides in length). the results showed that the resulted construct sp lost − frameshifting activity ( figure b ), indicating that mpk stimulates − frameshifting with a five-nucleotides spacing downstream of the ribosomal a-site sitting uga stop codon. combined with − frameshifting product characterization and e-site sequence mutagenesis results, they suggest that the non-canonical − frameshifting occurs via a single slippage of peptidyl-trna in ' direction through two possible pathways with uga stop codon sitting at the ribosomal a-site ( figure c ). in pathway a, the -frame peptidyl-trna moves from cuu to − frame ccu, allowing the − frame uug codon for decoding in the a-site. by contrast, an incoming uug decoding charged trna could sample the available reading-frames in the empty a-site and is paved by the two identical nucleotides bridging e-and p-sites to accept the movement of peptidyl-trna toward the − frame, and resulting in − frame decoding in the a-site. that mpk stimulates - frameshifting with a five-nucleotides spacing downstream of the ribosomal a-site sitting uga stop codon. combined with - frameshifting product characterization and e-site sequence mutagenesis results, they suggest that the non-canonical - frameshifting occurs via a single slippage of peptidyl-trna in ' direction through two possible pathways with uga stop codon sitting at the ribosomal a-site ( figure c ). in pathway a, the -frame peptidyl-trna moves from cuu to - frame ccu, allowing the - frame uug codon for decoding in the a-site. by contrast, an incoming uug decoding charged trna could sample the available reading-frames in the empty asite and is paved by the two identical nucleotides bridging e-and p-sites to accept the movement of peptidyl-trna toward the - frame, and resulting in - frame decoding in the a-site. the proposed mechanisms put the uga stop codon in the a-site, resulting in a spacer of nts to mpk while the cuu codon is sitting in the p-site based on the data from (b) as well as the requirement of rf deficiency for the non-canonical − frameshifting. this positioning allows the downstream mpk to stimulate − frameshifting through two alternative pathways with differences in the sequential order of peptidyl-trna movement from -frame to − frame. in pathway a, the -frame peptidyl-trna moves from cuu to − frame ccu and thus positions the uug codon in the a-site. in pathway b, an incoming charged trna samples the available reading-frames while peptidyl-trna is sitting in the -frame p-site and is paved by two identical nucleotides bridging e-and p-sites that favors − frame accommodation of peptidyl-trna to facilitate − frameshifting. that non-canonical − frameshifting occurred within a + frameshift-prone cuuuga site with the presence of a downstream pseudoknot raises the possibility that reduced + frameshifting shown in figure b is not caused by a direct counteracting downstream structure (an attenuator), but rather is the result of multiple frameshifting pathways competing. however, + frameshifting efficiency was reduced significantly by the downstream mpk (from . % to . %) in a uaucuuuga construct that favors + frameshifting but disfavors − frameshifting (figure a,b) . therefore, the main cause of the observed + frameshifting reductions is not − frameshifting pathway competition, indicating direct counteraction to + frameshifting by the downstream mpk. to see if this property is also applicable to − frameshifting attenuation by an upstream hairpin, we constructed an h hairpin upstream of the a g − frameshifting site. we did not detect + frameshifting activity (lane in figure c ) although h did attenuate − prf efficiently (lane in figure c ). taken together, these results indicate that the existence of a stable mrna hairpin at either end of the mrna-binding channel of an elongation ribosome counteracts frameshifting stimulated by its counterpart at the other end of the channel. proposed mechanisms put the uga stop codon in the a-site, resulting in a spacer of nts to mpk while the cuu codon is sitting in the p-site based on the data from (b) as well as the requirement of rf deficiency for the non-canonical - frameshifting. this positioning allows the downstream mpk to stimulate - frameshifting through two alternative pathways with differences in the sequential order of peptidyl-trna movement from -frame to - frame. in pathway a, the -frame peptidyl-trna moves from cuu to - frame ccu and thus positions the uug codon in the a-site. in pathway b, an incoming charged trna samples the available reading-frames while peptidyl-trna is sitting in the -frame p-site and is paved by two identical nucleotides bridging e-and p-sites that favors - frame accommodation of peptidyl-trna to facilitate - frameshifting. that non-canonical - frameshifting occurred within a + frameshift-prone cuuuga site with the presence of a downstream pseudoknot raises the possibility that reduced + frameshifting shown in figure b is not caused by a direct counteracting downstream structure (an attenuator), but rather is the result of multiple frameshifting pathways competing. however, + frameshifting efficiency was reduced significantly by the downstream mpk (from . % to . %) in a uaucuuuga construct that favors + frameshifting but disfavors - frameshifting (figures a and b) . therefore, the main cause of the observed + frameshifting reductions is not - frameshifting pathway competition, indicating direct counteraction to + frameshifting by the downstream mpk. to see if this property is also applicable to - frameshifting attenuation by an upstream hairpin, we constructed an h hairpin upstream of the a g - frameshifting site. we did not detect + frameshifting activity (lane in figure c ) although h did attenuate - prf efficiently (lane in figure c ). taken together, these results indicate that the existence of a stable mrna hairpin at either end of the mrna-binding channel of an elongation ribosome counteracts frameshifting stimulated by its counterpart at the other end of the channel. to further explore the role of flanking structures in bidirectional frameshifting regulation, destabilizing mutations to destroy watson-crick base-pairing for stability reduction were introduced at the upper stem of upstream h hairpin ( g a) and the stem of downstream mpk pseudoknot (g c) ( figure a ) to examine the effects on + and − frameshifting in the absence of rf . the cuuuga sequence was weakly frameshifting-prone without the presence of a stable flanking structure (with the destabilized g a and g c mutants) (lane in figure b ), while + frameshifting activity increased in the presence of a stable upstream hairpin (h -wt) (lane ). by contrast, a downstream mpk strongly stimulated − frameshifting (lane ). similar to that of figure s , an extra faint rnase-sensitive band with low mobility appeared but disappeared upon rnase treatment ( figure s ). the co-existence of this rnase-sensitive band with the − frameshifting product and its disappearance in the presence of rf implicated that it is related to − frameshifting and its identity awaits further characterization in the future. finally, both + and − frameshifting products were significantly reduced in the presence of both the stable upstream h hairpin and the downstream mpk (lane ). this result is consistent with mutual attenuation of + and − frameshifting by stable downstream and upstream structures, respectively. frameshifted translation products have been reported in research on protein overexpression in e. coli. [ ] . such hungry codon-mediated frameshifting has also been proposed to be responsible for observed − frameshifting of huntingtin in huntington's disease due to the extensive use of glutamine cag codon [ ] . in this case, appropriate a-site codon occupation could be delayed due to low concentrations of cognate charged trna, leading to ribosome pausing. this is similar to an a-site sitting stop codon without its cognate release factor rf as described above. to see if the flanking structure effects are also applicable in hungry codon-mediated frameshifting regulation, we used a cysteine codon (ugu) to replace the uga stop codon in the ribosomal a-site ( figure a ). the cysteine concentration in the in vitro translation system was then reduced to mimic the hungry codon condition. because gst-rluc reporter possesses cysteine amino acid residues upstream of the cuuuga site, sequential dilutions of cysteine concentration were performed to optimize experimental conditions to ensure detectable levels of translation products while keeping the cysteine concentration as low as possible ( figure s ). by generating ugu (cys) as a hungry codon at the ribosomal a-site, we observed + and − frameshifting stimulation by upstream h hairpin and downstream mpk, respectively (lanes and in figure c ). by contrast, destabilizing both structures by watson-crick base-pairing disruption ( g a of h and g c of mpk) almost abolished − frameshifting activity and strongly reduced + frameshifting activity (lane ), while co-existence of stable flanking structures led to the compromise of both frameshifting pathways (lane ). thus, delay or interference in ribosomal a-site occupancy represents a parameter for translational reading-frame switch regulation that can be further tuned bi-directionally via stable mrna duplexes flanking the elongation ribosome ( figure d ). the + stimulation and − attenuation activities of a co-translationally refolding rna hairpin in s translation system suggest that it is a functional mimicry of an internal sd-mediated duplex that forms between mrna and rrna. therefore, their frameshifting regulation properties may share similar mechanisms. extensive mutagenesis of e-site codon composition has demonstrated that e-site codon-anticodon stability reversely correlates with + frameshifting efficiency in cuuuga frameshifting sites [ , ] and was proposed to be related to the e-site trna dissociation. consistently, biochemical experiments on rf + frameshifting have shown that extending the internal sd-mediated duplex by base-pairing with e-site sequences can enhance + frameshifting efficiency and accelerate the dissociation of e-site trna [ ] . alternatively, the involvement of peptidyl-trna dissociation has also been proposed based on toe-printing analysis [ ] . by contrast, the mpk − prf stimulator downstream of a non-xxxyyyz sequence was shown to delay e-site trna dissociation [ ] . thus, it is tempting to explain the reverse role in frameshifting regulation by the structures flanking different frameshifting sites through the mediation of opposite effects in the dissociation of e-site trna. however, our experimental approach was not capable of kinetic measurement for e-site trna dissociation although limited mutagenesis analysis of e-site sequence composition in figure suggests that stability of codon-anticodon interaction is not the main cause for switch in frameshifting pathways. alternatively, upstream duplexes juxtaposing the e-site could simply act as a roadblock to hinder the backward movement of ribosome to attenuate − prf. nevertheless, our finding of co-existence of + and − frameshifting under the same sequence background represents an ideal framework for studying frameshifting pathway regulation by e-site trna dissociation kinetics as well as flanking structures via single-molecule approaches in the future. kinetic modeling and experimental analysis have suggested that − prf can occur in either accommodation or two sequential post-translocation steps along the x xxy yyz slippery site [ ] . however, the non-canonical − frameshifting observed in sequence ccuuuga does not fit into the xxxyyyz consensus for − prf slippery sequence. in a recent model of uuuaaag-mediated − prf, mpk was proposed to prolong elongation factor g (ef-g) release during post-translocation stage to interfere with the swiveling of the s subunit head and the coupled translocation [ ] [ ] [ ] [ ] , allowing the ribosome to sample alternative reading-frames [ ] . by contrast, single-molecule fluorescence analysis of the dnax − prf motif argued that the accommodation step during delayed ef-g release determines reading-frame selection [ ] . as a downstream mpk and the absence of rf are both required for the non-canonical − frameshifting in cuuuga site, together with the results in figure b , they support a model of uga codon occupying the a-site followed by a -nucleotide spaced downstream mpk upon frameshifting ( figure c) . since ef-g has probably dissociated from the ribosome after the translocation step that positions the uga codon into the a-site, it seems probable that reverse of s head swiveling is completed [ ] . however, this raises questions about the role of the downstream hairpin or mpk in this process. alternatively, downstream structures could still delay the translocation process to allow prolonged reading-frame sampling by the ribosome [ , , ] . during this process the frame and − frame codons in the a-site can both compete for their cognate ligands with the − frame pathway prevailing in the absence of rf . furthermore, slippage of peptidyl-trna by one nucleotide in the − frame is facilitated by an identical nucleotide bridging the frame e-and p-sites as revealed in figure , eventually leading to repositioning of the − frame codons in the p-and a-sites. however, our data are incapable of distinguishing the order between − frame slippage of peptidyl-trna and − frame a-site trna accommodation (pathways a and b in figure c ). within these contexts, the finding that the duplex structures flanking the two sides of ribosomal mrna-binding channel play reverse roles in frameshifting regulation complements current proposed − prf stimulation models of downstream structures for a more complete picture of the dynamic interplay between ribosome and mrna in frameshifting regulation. this study has demonstrated that − prf attenuation by a co-translationally refolding upstream hairpin is conserved between s and s ribosomes. it further suggests that frameshifting attenuation activity is an intrinsic property of duplex-mediate structures that flank the mrna-binding channel of an elongating ribosome, and could be masked by the composition of the sequence occupied by the ribosome. furthermore, the tendency of an a-site empty ribosome to shift its reading-frame in either direction depending on the existence of flanking structures could provide a working model for unusual frameshifting events mediated by hungry codons in both s and s ribosomes. unusual − frameshifting without an xxxyyyz shifty site has been reported for the cag trinucleotide expansion region of the mjd- transcript in spinocerebellar ataxias as well as huntingtin in huntington's disease [ , ] . experimental evidence from cag repeat expansion in huntingtin suggested that the consumption of glutamine-charged trna by the expanded repeats makes the glutamine-decoding cag a hungry codon and eventually makes it frameshift-prone in the presence of cag-mediated hairpin structures [ ] . however, repeated cag sequences lack an identical nucleotide to bridge the frame e-and p-sites, and is consistent with the weak − frameshifting efficiency [ ] . additionally, long repeats are capable of generating both upstream and downstream cag-mediated hairpins to flank the ribosome to further compromise frameshifting efficiency via frameshifting attenuation effects. by contrast, + frameshifting has also been reported for cag repeat in huntingtin with the involvement of a weak uucc + shifty site upstream of the expanded cag repeats [ ] . as expanded cag repeats can form a downstream stable hairpin while an upstream cag mediated hairpin refolds, attenuation of + frameshifting by a downstream hairpin could thus mask this mechanism. a pet gst-rluc reporter was designed as the backbone for cloning of different frameshifting signals to examine the frameshifting activities using radioactivity-based frameshifting assay in vitro. to construct pet gst-rluc, renilla luciferase gene (rluc) was amplified using p luc vector [ ] as the template, and then cloned into ecori/noti sites of pgex- t- (ge healthcare), generating a gst-rluc fusion protein gene (gst-rluc). the gene of gst-rluc was then amplified and inserted into nhei/noti sites of pet- a (+) (merck co. inc.) to form pet gst-rluc. to facilitate non-shifted and shifted translation products separation by electrophoresis, translation termination sites were designed into each reading-frame with optimal spacing among these stop codons. the stop codon in the frame was provided by the individual frameshifting elements under examination. unless specified, the out of frame premature stop codons were designed into the n-terminal region of rluc orf of the pet gst-rluc reporter. due to the existence of multiple + and − frame stop codons in the n-terminal region of rluc orf, further deletions and site-directed point mutations were performed to generate pet gst-rluc- for tracking single frameshifting event (− or + ) and double frameshifting events (− and + ). the nucleotide sequences of n-terminal regions of rluc orf in pet gst-rluc- are shown below with the two boldly typed taa corresponding to premature out of frame stop codons. -ataacttcgaaagtttatgatccagaacaaagga aacggaaactggtccgcagtggtgggccagatgtacacaaataa- . (notice: in single frameshifting events, the second boldly typed taa was used as the termination site of − or + frameshifting ( figure ). in double frameshifting events (figures and - ) , the first boldly typed taa was used as − frame stop codon, and the second boldly typed taa was used as + frame stop codon.) the pet gst-flag-rluc reporter was used to express flag-tagged translation products for immunoprecipitation. to generate gst-flag-rluc, the pet gst-rluc- reporter was used as the template. dna oligo nucleotides r ( -cttgtcgtcatcgtctttgtaatcatccgattttggaggatggtc- ) and r ( -cggggatccacgcggaaccagcttgtcgtcatcgtctttgt- ) were designed as reverse primers to contain nucleotides coding part of flag-tag (underlined) in the and regions, respectively. the -part of r was designed to be complementary to the c-terminal end nucleotide sequences of gst while the -part of r was extended with bamhi recognition sequences. by contrast, primer f-t ( -gcgaaattaatacgactcactataggg- ) was designed as the forward primer to anneal with the sequence upstream of gst. incubation of pet gst-rluc- with r and f-t primers for cycles of pcr amplification and followed by additional cycles of pcr with the addition of f-t and r primers generated gst-flag with a flanked nhei/bamhi cutting sites. the amplified gst-flag containing cdna was restriction enzymes treated and cloned into nhei/bamhi sites of the pet.gst-rluc- reporter to replace the gst fragment to generate the gst-flag-rluc reporter construct. unless specified, all frameshifting signals under examination were cloned into the pet gst-rluc- reporter backbone for radioactivity-based frameshifting assay. the frameshifting elements containing different sequences of upstream and downstream structures flanking a slippery sequence were constructed by pcr-based ligation approach [ ] . in brief, the elements were assembled by performing pcr using two to three pieces of chemically synthesized dna oligo-nucleotides (from genomics biosci & tech corp at taiwan) of partially overlapping sequences. forward and reverse primers used for cdnas amplification contain appropriately designed overlapping regions as well as bamhi and ecori restriction sites. the amplified cdnas were then cloned into the bamhi/ecori sites of appropritate reporters. following standard cloning procedures, the resultant recombinant vectors were transfromed into the dh α strain of escherichia coli and were selected by ampicillin. site-directed mutagenesis in this study was constructed using the quick-change mutagenesis kit from stratagene in accordance with the manufacturer's instructions. all nuelcotide sequences of cloned and mutated frameshifitng elements were confirmed by dna-sequencing analysis. for in vitro frameshifting assay, nascent polypeptides were generated using the reconstituted s translation system (purexpress ∆rf kit, neb) in accordance with manufacturer's instructions. unless specified, ng mrnas purified after transcription by t rna polymerase (thermo fisher scientific) was used as the templates in each . µl reaction, with rf excluded if required. to create cysteine deficiency condition for assaying hungry codon-mediated frameshifting, two different amino-acids mixture sets were added into the translation mixture (purexpress ∆ aa, trna kit, neb) separately. in each . ul reaction, . µl of . mm amino-acids mixture (neb) and . µl of mm minus-cysteine amino-acids mixture (promega) were used. for each assay, . µl of µci/µl s labeled methionine (nen) was added into the reaction, and incubated at °c for h. samples were then resolved by % sodium dodecylsulphate-polyacrylamide gel electrophoresis (sds-page), and exposed to a phosphorimager screen for quantification after drying of the gel. in ribonuclease-treated assay, specific amounts of ribonuclease a (thermo scientific, am ) were added into each reaction in the end. after incubation at room temperature for mins, the reactions were quenched with × sds-gel loading buffer, and analyzed by sds-page. frameshifting efficiencies were calculated, by dividing the counts of the shifted products by the sum of the counts for both shifted and non-shifted products, with calibration of the methionine content in each protein product. a µl cell-free translation reaction containing ng -flag-cuuuga-mpk reporter plasmid was incubated at • c for h. the reaction was then mixed with phosphate buffered saline (pbs) buffer ( mm nacl, . mm kcl, mm na hpo , . mm kh po , ph . ) containing : dilution of anti-flag m (sigma) in a final volume of µl, and rotated overnight at • c. the sample was then incubated with µl protein g magnetic sepharose xtra (ge healthcare) at • c for h with gentle shaking. following incubation, the beads were washed three times with µl pbs buffer, and boiled in µl × sds-gel loading buffer at • c for minutes to elute flag-tagged polypeptides. the elution fractions were resolved by % sds-page, and then stained with coomassie brilliant blue r- (panreac). after the gel was destained with destaining buffer ( % methanol, % glacial acetate acid), the target band was sliced for mass spectrometry (ms) analysis. the gel slice containing translation products excised from the resolved gels was cut into pieces for in-gel trypsin digestion. briefly, the gel pieces were first washed with mm triethylammonium bicarbonate buffer (teabc) / % (v/v) acetonitrile (acn). the washed samples were then reduced with mm dtt at • c for h and alkylated with mm iodoacetamide (iam) at room temperature for another h in the dark. trypsin buffer (protein:trypsin = : , w/w) was added to cover the gel pieces, and incubated at • c for more than h. tryptic peptides were extracted by incubating with % formic acid (fa)/ % acetonitrile (acn) for min, and vacuum dried. the sample was dissolved in µl . % (v/v) formic acid (fa) and µl used in lc-ms/ms analysis. the synthetic peptide standard was purchased from kelowna international scientific inc. (taiwan). lc-ms/ms was performed using an orbitrap fusion lumos tribrid mass spectrometer (thermo fisher scientific) equipped with a nanospray flex ion source. the trypsin-treated sample was loaded onto an ultimate nano lc system (c acclaim pepmap nanolc column, cm × µm i.d., µm, Å) connected to a mass spectrometer. a segmented gradient of min from % to % solvent b at a flow rate of nl/min and a column temperature of • c was used for separation with solvent a containing . % formic acid in water and solvent b containing % acetonitrile with . % formic acid. mass spectrometry analysis was performed in data-dependent mode with full-ms (externally calibrated to a mass accuracy of < ppm, and a resolution of , at m/z = ) followed by hcd-ms/ms of the most intense ions in s. high-energy collision activated dissociation (hcd)-ms/ms (resolution of , ) was used to fragment multiple charged ions within a . da isolation window at a normalized collision energy of ev. agc target at e and e was set for ms and ms/ms analysis, respectively, with previously selected ions dynamically excluded for s. max. injection time was ms. a customized database was used to search for the existence of translation peptides by matching the m/z value to the obtained ms spectra. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , figure s : t radioactivity-based frameshifting analysis of a − prf model system using gst-rluc- reporter by in vitro translation in cell-free lysates of e. coli., figure s : the + frameshifting stimulated by duplex structures upstream of the cuuuga frameshifting site are abolished in the presence of rf ., figure s : potential rna-protein adducts disappear upon rnase a treatment., figure s : recovery of a potential − frame translation product for mass spectrometry analysis., figure s : rnase a treatment removes a low mobility band without affecting the three major translation products., figure s : creating a cysteine hungry codon by reducing cysteine amino acid concentration in the in vitro translation system. mrna helicase activity of the ribosome the ribosome uses two active mechanisms to unwind messenger rna during translation an rna pseudoknot and an optimal heptameric shift site are required for highly efficient ribosomal frameshifting on a retroviral messenger rna stem-loop structures can effectively substitute for an rna pseudoknot in - ribosomal frameshifting an 'integrated model' of programmed ribosomal frameshifting the -a solution: how mrna pseudoknots promote efficient programmed − ribosomal frameshifting programmed translational frameshifting reprogrammed genetic decoding in cellular gene expression ribosomal frameshifting and transcriptional slippage: from genetic steganography and cryptography to adventitious use ribosomal frameshifting in the yeast retrotransposon ty: trnas induce slippage on a nucleotide minimal site p-site trna is a crucial initiator of ribosomal frameshifting rrna-mrna base pairing stimulates a programmed − ribosomal frameshift dynamic pathways of − translational frameshifting programmed − frameshifting by kinetic partitioning during impeded translocation reading frame switch caused by base-pair formation between the ' end of s rrna and the mrna during elongation of protein synthesis in escherichia coli the anti-shine-dalgarno sequence drives translational pausing and codon choice in bacteria an atypical rna pseudoknot stimulator and an upstream attenuation signal for − ribosomal frameshifting of sars coronavirus regulation of programmed ribosomal frameshifting by co-translational refolding rna hairpins a general strategy to inhibiting viral − frameshifting based on upstream attenuation duplex formation translation elongation factor ef-p alleviates ribosome stalling at polyproline stretches programmed ribosomal frameshifting generates a copper transporter and a copper chaperone from the same gene a dual-luciferase reporter system for studying recoding signals characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an rna pseudoknot maintaining the ribosomal reading frame: the influence of the e site during translational regulation of release factor mfold web server for nucleic acid folding and hybridization prediction comparative mutational analysis of cis-acting rna signals for translational frameshifting in hiv- and htlv- the three transfer rnas occupying the a, p and e sites on the ribosome are involved in viral programmed − ribosomal frameshift ribosome can resume the translation in both + or − frames after encountering an aga cluster in escherichia coli depletion of cognate charged transfer rna causes translational frameshifting within the expanded cag stretch in huntingtin analysis of effects of trna:message stability on frameshift frequency at the escherichia coli rf programmed frameshift site genetic analysis of the e site during rf programmed frameshifting short spacing between the shine-dalgarno sequence and p codon destabilizes codon-anticodon pairing in the p site to promote + programmed frameshifting dynamics of translation by single ribosomes through mrna secondary structures the many paths to frameshifting: kinetic modelling and analysis of the effects of different elongation steps on programmed − ribosomal frameshifting the process of mrna-trna translocation head swivel on the ribosome facilitates translocation by means of intra-subunit trna hybrid sites crystal structures of ef-g-ribosome complexes trapped in intermediate states of translocation the ribosome moves: rna mechanics and translocation a frameshifting stimulatory stem loop destabilizes the hybrid state and impedes ribosomal translocation ribosomal frameshifting on mjd- transcripts with long cag tracts mutant cag repeats of huntingtin transcript fold into hairpins, form nuclear foci and are targets for rna interference an expanded cag repeat in huntingtin causes + frameshifting gene synthesis, high-level expression, and mutagenesis of thiobacillus ferrooxidans rusticyanin: his is a ligand to the blue copper center the authors thank daniel flynn for english editing and chien-hung yu for reading the manuscript and comments. the lc-ms/ms analyses were supported by the instrumentation center and mass spectrometry core facility at national taiwan university. the authors declare no conflict of interest. key: cord- -jcvrqeew authors: gelain, maria elena; bonsembiante, federico title: acute phase proteins in marine mammals: state of art, perspectives and challenges date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: jcvrqeew the term “acute phase response” (apr) is referred to a nonspecific and complex reaction of an organism that occurs shortly after any tissue damage, such as infection, trauma, neoplasia, inflammation, and stress. the apr can be identified and monitored with some laboratory tests, such as the concentration of several plasma proteins, the acute phase proteins (apps). the apps are components of the non-specific innate immune response, and their plasma concentration is proportional to the severity and/or the extent of tissue damage. the evaluation of health status of marine mammals is difficult because the classical clinical signs of illness used for human and domestic animals are difficult to recognize and understand. for this reason, in the past years, several efforts were done to identify laboratory markers of disease in these animals. the apps have demonstrated their role as early markers of inflammation in veterinary medicine, thus several apps were tested in marine mammals, such as c-reactive protein (crp), serum amyloid-a (saa), and haptoglobin (hp). however, the difficulty to extrapolate the knowledge about apps in one species to another, the lack of specie-specific reagents, the absence of data about negative apps have hampered their extent use in marine mammals. herein, the state of art of apps in marine mammals is reviewed, with particular attention to pre-analytical and analytical factors that should be taken into account in validation and interpretation of apps assays. moreover, the current application, potential utility and the future developments of apps in marine mammals is highlighted and discussed. the mammalian immune system includes innate or nonspecific immunity as well as adaptive or specific immunity. the responses of these two different pathways are distinct, but highly interconnected. the first reaction of an organism to different pathological conditions is an innate, non-specific response ( ), a more conserved response during evolution which aim is the immediate reaction against pathological stimuli ( ) . after the initial recognition of pathogens or tissue damages by the tissue-resident macrophages, which express the pattern recognition receptors (prrs), a variety of different inflammatory mediators are produced by leukocytes, endothelial cells, tissue cells or are derived from plasma proteins. these mediators include different chemokine, cytokine, vasoactive amines and products of the arachidonic acid: their primary effect is to elicit inflammatory response locally and to recruit leukocytes and plasma proteins in the site of injury ( ) . glycoprotein (agp) ( ) , in ruminants is haptoglobin (hp) ( ) ; in horse is serum amyloid a (saa) ( ) and in pigs are hp, saa, and major acute phase protein (map) ( ) . recently, in veterinary medicine, studies on the role of apps as markers of infectious, inflammatory and neoplastic diseases have proliferated ( ) and at least different plasma proteins have been identified as apps ( ) . their use as marker of homeostasis perturbation provides some advantages compared with traditional parameters like the white blood cell (wbc) counts. compared to wbc count, the diagnostic sensitivity of apps is higher and the change in concentration is faster ( ) . moreover, their stability in serum/plasma is high, so it is possible to measure apps in frozen samples ( ) . one limitation of the apps is the poor diagnostic specificity, for this reason they cannot be used as primary diagnostic test for a specific disease, but they were successfully used to detect subclinical diseases and to monitor clinical evolution and to assess the response to treatment ( ) . additionally, the combined measurement of several apps provides more information than the evaluation of a single protein: in the "app value index, " proposed by gruys et al. ( ) , sensitivity and specificity are improved by combining response of both positive and negative major and minor apps. marine mammals are a group of around mammalian species which depend on water environment for most of their needs. they include orders: carnivora (pinnipeds, otters, and polar bear), cetacea (dolphins, whale, and porpoise) and sirenia (manatee and dugogons). marine mammals are differently adapted to life in water, with some species, which are fully aquatic (cetaceans and sirenia), and others that spend part of their life on land (pinniped and polar bears) ( ) . from an immunological point of view, aquatic adaptation caused few differences in distribution and function of immune system between marine and terrestrial mammals ( ) . however, nowadays the marine mammal' immune system is deeply exposed to environmental pollution because they are a long-lived animals placed at the top of food chain, thus they are exposed to a progressive bioaccumulation of fat-soluble pollutants, such as pcbs, which affect both innate and adaptive immune function ( ) . for these reasons, increasing knowledge in cellular and humoral immune response is continuously required to understand their immune system and in particular its relationship with infectious pathologies and the environmental pollution ( ) . furthermore, marine mammals live also in controlled environment like aquaria, rehabilitation facilities and research center where health assessment is fundamental to evaluate the correct management of animals and to monitor the response to therapy during rehabilitation. from this perspective, the availability of new markers to asses immune functions is fundamental both for medical care and research purpose ( ) . innate immune response represent the first line of response against pathological stimuli, it's very fast and it's based primarily on effector cells (e.g., mast cells, macrophages, neutrophils) and antimicrobial substances (e.g., complement, reactive oxygen, and nitrogen species). on the other hand, adaptive immune system is antigen-specific, it takes more days to be effective and it's based on different t-cells response and on b-lymphocytes which are responsible of humoral response mediated by the different subclass of immunoglobulin (igg, igm, iga) ( ) . several assays were proposed to evaluate both immune response in marine mammals, generally based on isolated leucocytes with the aim to evaluate the leukocytes response against in vitro stimuli ( ) . to assess the response pattern of cetaceans' cellular innate immune system, the phagocytosis and the generation of reactive oxygen species of polymorphonuclear leukocytes were investigated. in particular, in vitro ingestion of latex beads and hydrogen peroxide production have been evaluated in beluga whales (delphinapterus leucas) and in bottlenose dolphins (tursiops truncatus) ( , ) whereas phagocytosis and respiratory burst assay, using whole blood from bottlenose dolphins, were used to assess the antimicrobial activity ( ) . in addition, the investigation of apr by analyzing the cytokine expression gives important information on the functionality of lymphoid cells. the production of specie-specific antibodies allows the development of immunological assays for the quantification of cytokine expression useful to investigate the inflammatory response in whales and dolphins. the coding regions of il- , il- β, il- , and tnf-α gene of the beluga whale have been sequenced, and a cytokine-specific rabbit antisera have been produced ( ) ( ) ( ) . in harbor porpoise (phocoena phocoena), the quantification of mrna of il- β, il- , il- , il- , il- , and tnf-α have been performed by rt-pcr ( ) , and the increase of il- was seen in harbor porpoises suffering from long lasting infectious ( ) . also in bottlenose dolphins, pacific white-sided dolphins (lagenorhynchus obliquidens), and beluga whales, il- , il- , il- , il- , il- , il- , tnf-α, tgf-β, and interferon (ifn)-γ quantification was performed using rt-pcr ( ) . an il- receptor expression assay and an il- elisa were developed in bottlenose dolphins and killer whale (orcinus orca), respectively ( , ) . similarly, both innate and the cell-mediated response were studied in pinnipeds. to better understand the innate response, phagocytic activity of isolated peripheral blood leukocytes was evaluated in harbor seal (phoca vitulina), gray seal (halichoerus grypus), and harp seal (phoca groenlandica) pups, in harbor seal female during lactation and in harbor seal pups admitted to rescue center ( , ) . the authors found an age-related variation in both pups and adults: phagocytosis increased with age in gray and harbor seal pups, while in female harbor seals decreased from sub-adult to adulthood. at the same time, pups after rehabilitation showed a decreased phagocytic activity, probably due to the decreased stimulation of innate response after therapy. also cytokine response was evaluate in harbor seal. pro-inflammatory cytokine mrna (il- β, il- , il- , and il- ) in pups in a rehabilitation center were higher at admission whilst il- was higher before the release ( ), demonstrating the recovery from inflammation. recently, a multiplex canine cytokine assay was validate in harbor, gray and harp seal to measure proteins levels in cell culture supernatant of peripheral blood mononuclear cells (pbmc) ( ) . however, all these techniques are not generally applicable in a clinical setting in which the primary goal is a sensitive diagnostic tool with a rapid turnaround, even if give us important information on factors affecting cetaceans' immune system. for this reason, in the past years, several efforts were made to identify laboratory markers of disease in these animals. first parameters tested were wbc and erythrocyte sedimentation rate ( ) . however, even if they are inexpensive and rapid, they lack specificity and sensitivity. moreover, changes in wbc occur after several hours after inflammatory stimuli. thus, efforts were directed to identify inflammation at earlier stage ( ) . to examine the humoral response, species-specific antibodies against igg were produced and used to evaluate serum igg levels in killer whale by radial immunodiffusion assay ( ) and by competitive elisa in bottlenose dolphins ( , ) . the determination of igg baseline values in free-ranging and in managed dolphins revealed higher levels of immunoglobulin in the first group with several values over the accurate range of the assay, probably due to the higher parasitic load in free-ranging dolphins ( ) . serum total protein, albumin, globulin and albumin:globulin ratio (a:g) are undoubtedly among the most measured markers in basic health assessment in domestic animals as well as in marine mammals. serum protein electrophoresis is also broadly applied in veterinary medicine and it has the advantage to produce an accurate measurement of albumin and the visualization of globulin fractions ( ) . the interpretation of total proteins values and electrophoretic pattern of serum proteins is receiving increased attention also in marine mammals in which a typical pathologic pattern could be identified in inflammatory diseases ( ) . reference intervals for these markers are available for free-ranging bottlenose dolphins ( ) and, compared to these, recently data on managed dolphins showed slightly lower values of tp, α-globulins, and γ-globulins and higher albumin and albumin/globulins ratio ( ) . it's interesting to note that hp, α -antitrypsin, α antichymotripsin, and α -macroglobulin migrate in the α-globulins fraction, while the igg and crp migrate in the γ-globulins fraction. albumin acts as a negative acute phase protein since the synthesis of this protein is decreased during an inflammation ( ) . thus, the lower concentration of positive apps associated to a higher concentration of albumin and the consequent higher albumin/globulins ratio could reflect lower antigenic stimuli in managed population compared to the free-ranging populations ( ) . serum total protein analysis were used to assess health status in several cetaceans species such as pantropical spotted dolphins (stenella attenuata) ( ), beluga ( ) , minke whales (balaenoptera acutorostrata) ( ) and killer whales ( ) as well as in other marine mammals, like harbor seals (phoca vitulina) ( ) and walruses (odobenus rosmarus) ( ) . in all these species, serum total protein analysis was demonstrated to be one of the most used and commonly accepted marker of inflammation. however, specific apps have demonstrated their superior role as early markers of inflammation, so based on the results obtained in humans and companion animals, several positive apps were tested in marine mammals ( table ) . published works had the primary aims to evaluate the feasibility of the assays to measure the apps, to validate the antibody-based assay and to determine the ris. in bottlenose dolphins three apps (crp, saa, and hp) were tested, even if not always complete validation studies were performed ( , ) . for these apps, the authors established the ris in free-ranging and managed dolphins using automated assays ( ) and they found significantly lower saa and higher hp levels in free ranging animals. the only clinical significance of these alteration was a higher ability to detect chronic inflammation for hp. regarding hp, segawa and colleagues validated commercially available hp-elisa and hp-hemoglobin binding assay in bottlenose dolphins with ''acceptable" intra-and inter-assay imprecision (cv: . % healthy dolphins and . % inflamed dolphins; cv: . % healthy dolphins and . % inflamed dolphins) and demonstrated that hp levels in the serum increase under inflammatory conditions ( ) . positive apps were tested also in florida manatees (trichechus manatus latirostris) to define the more accurate marker of inflammation. five different apps were tested: agp, crp, hp, fibrinogen, and saa. saa showed the highest diagnostic sensitivity and specificity ( % for both sensitivity and specificity) in the detection of inflammatory diseases, the diagnostic specificity of hp and fibrinogen were and %, respectively, while their diagnostic sensitivity were and %, respectively, ( ) . when used in stranded manatee suffering from cold stress and trauma, saa showed % of sensitivity and % of specificity in detecting diseased animals ( ) . by contrast, the abs used for the determination of agp and crp did not cross-react in this species ( ) . in harbor seal, an ab anti-crp and a competitive immunoassay was produced ( ), but hp is probably the app most used in pinnipeds. a multispecies assay based on hemoglobin binding capacity was used to demonstrate as hp is a sensitive marker of the health vs. disease status in harbor seal ( ) . in seal pups admitted in a rescue center. hp, total protein, igg and globulin values correlated positively, but hp levels increased during the hospitalization, probably reflecting age-related changes ( ) . hp is considered a health marker also in steller sea lions (eumetopias jubatus): significantly higher levels of hp were found in declining population compared to more stable ones ( ) . however, also genetic differences between distant and isolated population of wild animals could be the causes of this difference, not only a pathological condition. if some data on marine mammals positive apps are available in literature, quite surprising are the lack of data available on negative apps. for these reasons, the possibility to evaluate the usefulness of an "app value index" is still far from being applied. the availability of sensitive markers of inflammation both for free-ranging and managed marine mammals is nowadays considered fundamental to evaluate the health status and, in rehabilitation setting, to monitor the response to therapy and to define the prognosis. as serum markers, the apps have several advantages: they have longer stability compared to other blood component such as wbc; they can be performed on frozen serum, thus the samples can be shipped to references laboratories; some assays can be automated to obtain results in an excellent turnaround time. however, is important to consider that the knowledge about apps in one species cannot be readily generalized to another species, in which healthy levels, response to inflammation or infection, and prognostic significance may be different ( ) . moreover, the evolution of marine mammals and their adaptation throughout the millennia to an aquatic environment had led to a different physiology and metabolism compared to terrestrial mammals. thus, the understanding of the genetic, phenotypical and biochemical properties of marine mammals apps are essential prior to using them as a new biomarker. an example of how the biochemical properties influence the analytic method is paroxonase- (pon ), a hdl-bound esterase which protects against organophosphate compounds, acts as negative app and as oxidative stress marker. pon is usually assessed by enzymatic method and, based on the different pon functions, several substrates have been identified to evaluate serum pon activities. nevertheless, both in humans as in some terrestrial mammals, pon gene polymorphisms highly influence the enzymatic activity toward different substrate: the single-nucleotide polymorphisms (snps) leu met and gln arg increase the paraoxonase activity ( ) in humans and different pon genotypes influence activities toward paraoxon and phenyl-acetate in rabbit ( ) . also in cows, some snps in the promotor region of pon gene are associated with serum pon activity ( ) . recently, a phylogenetic study on convergent functional losses across marine mammals, has identified a pon functional loss in marine mammals, probably related to their different lipid metabolism and fatty acid oxidation due to adaptation to the marine environment and a high concentration of ω- fatty acids on their diet. as a consequence, in several marine mammals species paroxonase activity is very low, while enzymatic activity against other pon substrates is still present, such as arylesterase activity ( ) . for these reasons, the use of classical enzymatic assays is hampered in these animals and further studies are needed to elucidate the role of pon as possible negative app, oxidative stress marker and the consequences of its inability to detoxify organophosphates compounds. from an analytical point of view, another challenge in the evaluation of apps in marine mammals is the need of speciesspecific assays, especially for the immunological assay, such as elisa or immunoturbidimetry. this means the development of a de-novo method, often a time-consuming and expensive approach, or the validation of a commercial available assay used in other species ( ) . the latter approach is surely the most used in veterinary medicine, in which some human assays were validated for dogs, cats and horses ( ) . however, even if some apps appeared highly conserved among species, an accurate validation of antibody cross reactivity is needed as well as species specific standards and control material ( ) . among positive apps, saa is the most used across different species: it appeared as the most conserved app in mammals even if some difference in circulating isoforms were reported ( ) and it's considered a major app in all the mammals in which it was investigated ( ) . some commercial saa assays showed good results also in marine mammals, such as bottlenose dolphin, manatee and striped dolphin (stenella ceoreloualba) ( , , ) and its use as diagnostic and prognostic marker appears nowadays the most promising. to obtain accurate data, all the pre-analytical factors that could influence the results should be taken into consideration. the effect of storage, temperature and different anticoagulant had to be evaluated in a correct validation process as well as the interference of hemolysis and lipemia, as done in other species ( ) . the application of a novel biomarker required a full evaluation of all the analytical performances and the clinical value. this process is usually divided in steps: the assessment of analytical features (precision, accuracy, detection limits), the overlap performance (the ability to detect difference between healthy and diseased animals), the assessment of diagnostic capacity (sensitivity, specificity, accuracy, positive, and negative predictive values) and, at the end, the evaluation of the outcome of the new methods (which is the advantage of the test and its influence in the patient management) ( ) . in veterinary medicine, the validation studies do not always follow all these steps, mainly due to the lack of resources or technical limitation ( , ) . also in marine mammals, the majority of studies had performed only some steps ( , , , , ) . this is mainly due to the limitation in species-specific reagents, the number of samples from animal with known health status and, last but not least, the capability to generate appropriate reference intervals, hampered the possibility to perform complete validation studies. population-based reference intervals derived from an appropriate group of reference individuals are usually required for diagnostic purpose ( ) . however, a number of biological factors have to be taken in consideration to select the appropriate reference population. surely, age, sex and pregnancy could be used for partitioning ( , ) , but in marine mammals greater attention should be given to the difference between wild and managed animals. serum protein electrophoresis values obtained in managed bottlenose dolphins showed lower total proteins and higher albumin levels compared to reference intervals derived from free-ranging ( ) while wild-caught manatees, apparently clinically healthy, had saa level above reference limit ( ) . these data could indicate a trend to an inflammatory status or the presence of subclinical inflammation in free-ranging animals which are more exposed to immunological stimuli. in any case, this highlights the need to define appropriate reference intervals for animals living in different environment to have an accurate toll for the evaluation of clinical condition. compared to human and companion animals, the use of apps in marine mammals is just getting started. the increasing need of knowledge on immune system and its response against infectious diseases or chemical pollutants and the request of more sensitive inflammation markers have increased the effort of researchers to study the apr and apps. even if apps are considered a sensitive, but non-specific marker of inflammation, some studies revealed that, in some infectious diseases, apps showed a specific behavior and biochemical features. one example is the modification of the glycan moiety of agp in feline infectious peritonitis, fiv and felv, influencing the host-pathogens interaction and the immune response ( ) ( ) ( ) . currently, some of the greatest threats for wild marine mammals is pathogens, like morbillivirus, herpesvirus, brucella ceti, and toxoplasma gondii ( ): the evaluation of apr and apps patterns during these infectious diseases could lead to the identification of a distinctive response of the immune system and increase the understanding of hostpathogen interaction. secondly, for managed or rescued animals, the forthcoming needs are the increase of automated assays, the standardization of procedures across laboratories and the discovery of new markers, for example negative apps, to generate an app index also in marine mammals. these new tools will certainly increase the diagnostic and prognostic skills for health assessment and, especially for stranded animals, the development of new "health status" markers will provide valuable resources in evaluating the response to treatment and rehabilitation prior to the release into the wild. mg and fb analyzed the literature review, designed, and wrote the review. 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harbour, grey and harp seals cytokine and acute phase protein expression in blood samples of harbour seal pups validation of a commercial canine assay kit to measure pinniped cytokines clinical pathology crc handbook of marine mammal medicine measurement of serum immunoglobulin concentration in killer whales and sea otters by radial immunodiffusion development and validation of monoclonal and polyclonal antibodies for the detection of immunoglobulin g of bottlenose dolphins (tursiops truncatus) baseline circulating immunoglobulin g levels in managed collection and freeranging bottlenose dolphins (tursiops truncatus) acute phase response in animals: a review hematologic and serum biochemical reference intervals for freeranging common bottlenose dolphins (tursiops truncatus) and variation in the distributions of clinicopathologic values related to geographic sampling site detection of hereditary bisalbuminemia in bottlenose dolphins (tursiops truncatus fundamentals of veterinary clinical pathology hematological, serum, and plasma chemical constituents in pantropical spotted dolphins (stenella attenuata) following chase, encirclement, and tagging serum chemistry of freeranging white whales (delphinapterus leucas) in svalbard serum chemistry of the minke whale from the northeastern atlantic hematological and serum biochemical analytes reflect physiological challenges during gestation and lactation in killer whales (orcinus orca) hematology and serum chemistry in stranded and wild-caught harbor seals in central california: reference intervals, predictors of survival, and parameters affecting blood variables serum chemistry reference values in free-ranging north atlantic male walruses (odobenus rosmarus rosmarus) from the svalbard archipelago acute phase protein quantitation in serum samples from healthy atlantic bottlenose dolphins (tursiops truncatus) evaluation of immune and stress status in harbour porpoises (phocoena phocoena): can hormones and mrna expression levels serve as indicators to assess stress? comparison of methods used to diagnose generalized inflammatory disease in manatees (trichechus manatus latirostris) assessement of serum amyloid a levels in the rehabilitation setting in the florida manatee (trichechus manatus latirostris) plasma haptoglobin levels in threatened alaskan pinniped populations characterization of haptoglobin in the blood plasma of harbor seals (phoca vitulina) fibrinogen concentrations in captive bottlenose dolphins during pregnancy characterization of the circulating serum amyloid a in bottlenose dolphins molecular characterization and validation of commercially available methods for haptoglobin measurement in bottlenose dolphin harbor seal (phoca vitulina) c-reactive protein (c-rp): purification, characterization of specific monoclonal antibodies and development of an immuno-assay to measure serum c-rp concentrations acute phase protein haptoglobin in blood plasma samples of harbour seals (phoca vitulina) of the wadden sea and of the isle helgoland assay validation and diagnostic applications of major acute-phase protein testing in companion animals human paraoxonase- (pon ): gene structure and expression, promiscuous activities and multiple physiological roles rabbits possess a serum paraoxonase polymorphism similar to the human q r characterization of single nucleotide polymorphisms in the promoter region of the bovine paraoxonase (pon ) gene affecting serum enzyme activity in dairy cows ancient convergent losses of paraoxonase yield potential risks for modern marine mammals clinico-pathological findings in a striped dolphin (stenella coeruleoalba) affected by rhabdomyolysis and myoglobinuric nephrosis effects of age and sex on clinicopathologic reference ranges in a healthy managed atlantic bottlenose dolphin population association between faecal shedding of feline coronavirus and serum alpha -acid glycoprotein sialylation hyposialylated α -acid glycoprotein inhibits phagocytosis of feline neutrophils glycan moiety modifications of feline alpha -acid glycoprotein in retrovirus (fiv, felv) affected cats emerging infectious diseases in cetaceans worldwide and the possible role of environmental stressors clinico-pathological investigation of serum proteins in odontocetes the literature review presented in the manuscript is partially included in ph.d. thesis of bonsembiante ( ), supported by a ph.d. grant from the university of padova. key: cord- -el wyu authors: zhang, qingxiu; zhu, wen; xu, fei; dai, xuejiao; shi, ligen; cai, wei; mu, hongfeng; hitchens, t. kevin; foley, lesley m.; liu, xiangrong; yu, fang; chen, jie; shi, yejie; leak, rehana k.; gao, yanqin; chen, jun; hu, xiaoming title: the interleukin- /pparγ signaling axis promotes oligodendrocyte differentiation and remyelination after brain injury date: - - journal: plos biol doi: . /journal.pbio. sha: doc_id: cord_uid: el wyu the repair of white matter damage is of paramount importance for functional recovery after brain injuries. here, we report that interleukin- (il- ) promotes oligodendrocyte regeneration and remyelination. il- receptor expression was detected in a variety of glial cells after ischemic brain injury, including oligodendrocyte lineage cells. il- deficiency in knockout mice resulted in greater deterioration of white matter over d after stroke. consistent with these findings, intranasal delivery of il- nanoparticles after stroke improved white matter integrity and attenuated long-term sensorimotor and cognitive deficits in wild-type mice, as revealed by histological immunostaining, electron microscopy, diffusion tensor imaging, and electrophysiology. the selective effect of il- on remyelination was verified in an ex vivo organotypic model of demyelination. by leveraging primary oligodendrocyte progenitor cells (opcs), microglia-depleted mice, and conditional opc-specific peroxisome proliferator-activated receptor gamma (pparγ) knockout mice, we discovered a direct salutary effect of il- on oligodendrocyte differentiation that was mediated by the pparγ axis. our findings reveal a new regenerative role of il- in the central nervous system (cns), which lies beyond its known immunoregulatory functions on microglia/macrophages or peripheral lymphocytes. therefore, intranasal il- delivery may represent a novel therapeutic strategy to improve white matter integrity in stroke and other brain injuries. introduction matter components. however, the potential effects of il- on white matter integrity after ischemic stroke remain elusive. in the current study, we discovered that il- is essential for white matter repair after stroke. intranasal delivery of il- nanoparticles after stroke robustly promoted oligodendrogenesis, enhanced white matter integrity, and improved long-term functional recovery. complementary studies in cell cultures and animals revealed that il- -mediated oligodendrogenesis was achievable even in the absence of microglia/macrophages in vitro or after microglia/macrophage significant depletion in vivo. furthermore, il- directly promoted opc differentiation into mature oligodendrocytes in a peroxisome proliferator-activated receptor gamma (pparγ)-dependent manner. these collective findings suggest that activation of the il- / pparγ signaling cascade may represent a novel recovery-enhancing strategy to promote longterm outcomes after ischemic stroke. to explore the potential cellular targets for il- , we used flow cytometry to assess expression of il- rα on various types of cells in the ischemic brain. consistent with previous reports [ , ] , il- rα was expressed on cd − cd b − gfap + astrocytes, cd − cd b − neun + neurons, cd intermediate cd b + microglia, cd high cd b + macrophages, and other infiltrated immune cells (cd + cd b − ) d after -min mcao (fig a and b) . notably, il- rα was also expressed in oligodendrocyte lineage cells (fig a- d ). different markers, including a b , o , and o /galactocerebroside (galc), were used to identify oligodendrocytes at different differentiation stages. il- rα was detected in cd − cd b − o + cells (fig a- d ), which were identified as preoligodendrocytes and premyelinating oligodendrocytes, and cd − cd b − o + cells (fig a and b) , which were identified as premyelinating oligodendrocytes or mature oligodendrocytes [ ] . the percentages of il- rα + o + cells were higher in the ipsilateral ischemic hemisphere (fig c and d) , suggesting a potential direct effect of il- on white matter cells. imagestream analyses confirmed that il- rα was expressed on both a b + opcs and o + preoligodendrocytes/premyelinating oligodendrocytes, as well as cd b + microglia/macrophages in the ischemic hemisphere ( fig e and s a-s c fig) . in light of these results, we evaluated whether il- influenced white matter integrity after ischemic stroke. wt and il- ko mice were subjected to transient focal ischemia induced by -min mcao. dual staining for myelin basic protein (mbp, a major myelin protein) and smi (a marker of demyelinated axons) was performed to assess the lesion in white matter (fig f- h ). the overall immunofluorescence intensity of mbp staining decreased in the infarct border along three white matter-rich brain regions (the external capsule [ec] , the cortex, and the striatum) (see "total" values in panel of the right of fig f) at d after mcao, indicating a significant loss of myelin protein after stroke. additionally, the overall immunofluorescence of smi increased in these three regions, resulting in a significant increase in the smi /mbp ratio in wt mice after stroke (fig g) . il- -deficient mice displayed exacerbation of the loss in mbp ( fig f) and a further increase in the smi /mbp ratio compared to wt mice (fig g) , suggesting that il- is important in maintaining axonal myelination or promoting axonal remyelination after stroke. stroke impacts both gray and white matter. in order to confirm the effect of il- in white matter, we explored the remyelination capacity of il- in a model of lysophosphatidylcholine/lysolecithin (lpc)-mediated demyelination using ex vivo organotypic cerebellar slice cultures. this toxicant-based experimental model of demyelination, although not attempting to fully mimic a disease with complex etiology and pathogenesis, has proven extremely useful for understanding the biology of remyelination [ ] . slices collected from wt and il- ko mice exhibited comparable extents of demyelination, as revealed by the loss of mbp staining along neurofilamentheavy (nfh + ) axonal fibers, at d after lpc (fig a) . partial remyelination was achieved by d following lpc in wt brain slices, but il- deficiency significantly delayed remyelination (fig a and b) , as shown by lower mbp staining intensity ( fig c) and less mbp + axonal coverage ( fig d) . il- supplementation significantly enhanced remyelination in wt brain slices and rescued remyelination capacities in il- ko brain slices (fig a, c and d ). il- liposome nanoparticles were prepared to facilitate the penetration of il- into the ischemic brain. the mean particle size of il- protein-loaded liposome was . ± . nm with a and the ratio of smi to mbp immunofluorescence intensity (g) in ipsilesional ec, cortex, striatum, and the total for all three areas quantified. n = - /group. data are normalized to the intensities of contralateral hemispheres. � p < . , �� p < . , ��� p < . . one-way anova and bonferroni post hoc. (h) representative images for mbp (green) and smi (red) double immunostaining in the peri-infarct areas in brains from sham, wt mcao mice, and il- ko mcao mice d after mcao. scale bar: μm. data associated with this figure can be found in the supplemental data file (s data). cns, central nervous system; ec, external capsule; fsc-w, forward scatter width; gfap, glial fibrillary acidic protein; il- r, interleukin- receptor α; ko, knockout; mbp, myelin basic protein; mcao, middle cerebral artery occlusion; mfi, mean fluorescence intensity; opc, oligodendrocyte progenitor cell; ssc-a, side scatter area; wt, wild-type. lpc ( μg/ml) was added in wt or il- ko organotypic cultures for h to induce demyelination. il- ( ng/ml) or vehicle was added after lpc removal. culture slices were fixed d and d after lpc treatment. control cultures were maintained in normal culture media for d. (a) mbp (green) and nfh (red) staining was performed to label myelin and axonal fibers, respectively. scale bar: μm. (b) mbp and nfh double staining and imaris d reconstruction. arrows indicate myelination of individual axonal fibers that are magnified in the images to the right. scale bar: μm. (c) myelin integrity was measured as the intensity of mbp immunostaining. (d) the myelination index was calculated as the % mbp + myelin coverage along the entire nfh + fibers. n = - slices/group. � p < . , �� p < . , ��� p < . . one-way anova and bonferroni post hoc. data associated with this figure can be found in the supplemental data file (s data). il- , interleukin- ; ko, knockout; lpc, lysophosphatidylcholine/lysolecithin; mbp, myelin basic protein; nfh, neurofilament heavy; wt, wild-type. polydispersity index of approximately . ± . and a zeta potential of . ± . mv, confirming high stability of the nanodispersion. the il- nanoparticles were applied through the nasal route ( μg/kg body weight), which has been increasingly utilized for cns drug delivery because of its capacity to bypass the blood-brain barrier and avoid first-pass metabolism [ ] . intranasal administration of il- nanoparticles effectively increased il- protein levels in the corpus callosum (cc) and ec, cerebral cortex, and striatum h after il- application (fig a- c ). il- remained elevated in cortex and striatum until h after delivery (fig b and c ). c bl/ j mice were subjected to -min mcao and randomly assigned to receive il- nanoparticle ( μg/kg body weight) or control nanoparticle (vehicle) treatment. nanomaterials were intranasally delivered at h after mcao and repeated at - d, d, d, and d after mcao (fig d) . magnetic resonance imaging (mri) on these animals was performed serially at . t on days , , , and after mcao. t -weighted images showed no significant difference in the brain lesion volume of il- -versus vehicle-treated mice (fig e and f) , indicating comparable gross tissue damage across treatment groups. histological staining of the neuronal marker neun further demonstrated comparable tissue loss d after stroke in il- -and vehicle-treated mice (fig e and g) . serial whole-brain in vivo diffusion tensor imaging (dti) scans were collected d, d, and d after mcao. the same animals were then killed at d after stroke, and the fixed brains were subjected to ex vivo dti scanning (fig a and b , s a and s b fig) . longitudinal observation of fractional anisotropy (fa) maps and calculation of the ratio of fa(ipsilateral)/ fa(contralateral) demonstrated progressive white matter injury (ratio < ) with a slow recovery in the ec of vehicle-treated stroke mice at - d after stroke, which was significantly improved by day following stroke in il- -treated mice (fig c) . this trend was observed until day in the ex vivo fa values (fig c) . further analysis of the d dti images revealed that il- treatment enhanced white matter integrity, as revealed by elevated fa values in the white matter-enriched ec and internal capsule (ic) (fig d, e and g ). in addition, il- treatment significantly reduced radial diffusivity (rd, λ ? ) in the ec ( fig f) and ic (fig h) , which likely indicates improved myelin integrity [ ] . no significant differences between il- and vehicle treatment groups were observed in axial diffusivity (ad, λ || ), a measurement for axonal integrity (s c and s d fig) . to further explore the biological basis of these dti results, we compared the dti results with immunohistochemical staining of mbp performed in the same animals. there were remarkable similarities between the fa maps and mbp images (fig d, i and j). il- treatment significantly increased the mbp + area in the ec (fig d and i ). there was a significant positive correlation between the mbp + areas and the fa values in the ec (fig j) . the quantification of mbp staining confirmed a significant increase in mbp intensity in the ischemic brains of il- -treated mice compared to vehicle-treated mice (fig k) . to determine whether il- can improve axonal myelination after stroke, myelin thickness was measured in the ec using transmission electron microscopy (tem) (fig a) . frequency histograms revealed much greater reduction of axon diameters in the mcao + vehicle group compared to the mcao + il- and sham groups, suggesting higher axonal degeneration in stroke mice without il- treatment (fig b) . scatter plots of the g-ratio (the ratio of the inner axonal diameter to the total outer diameter of myelinated fiber) as a function of axon diameter in sham mice or stroke mice treated with vehicle or il- revealed significant differences in myelin thickness (average g-ratio: sham = . [n = axons from animals]; mcao + vehicle = . [n = axons from animals]; mcao + il- = . [n = axons from animals]) ( fig c) . significant increases in the g-ratio were observed in both low-and high-caliber axons at d after mcao, indicating a reduction in myelin thickness ( fig d) . il- post-treatment significantly reduced the g-ratio of the middle-sized (diameter = - nm) and large (diameter > nm) axonal fibers, suggesting that il- may maintain axonal myelination or promote axonal remyelination after stroke. the g-ratio in small axonal fibers (diameter < nm) displayed a trend toward reduction with il- treatment ( fig d) . saltatory conduction of action potentials along myelinated axons relies on normal organization of the nodes of ranvier (nor), which are rich in sodium and potassium ion channels. double immunofluorescent analyses of nav . (a nodal sodium channel protein) and contactin-associated protein (caspr, a paranodal cell adhesion protein) revealed the nor in the ec (fig e) . typical nor are characterized by punctate nav . staining at nodes between the paranodal staining of caspr. the total numbers of normal nor decreased significantly after stroke in the ipsilateral ec compared to the contralateral ec (fig f) , and this was accompanied by a decrease in the caspr + paranode length (fig g) . il- treatment significantly increased the number of nor and the paranode length in the ischemic ec (fig f and g) , suggesting an improvement in nor structure. the paranodal gap between pairs of caspr staining remained unchanged between groups ( fig h) . as il- enhanced the structural integrity of white matter after mcao, we further evaluated white matter function by measuring the transmission of action potentials in the cc and ec (fig i) . evoked compound action potentials (caps) display an early peak representing fast-conducting myelinated axons (n , fig j) [ ] . the amplitude of the n segment was significantly reduced d after mcao (fig k) , indicating impaired conduction through myelinated axons. il- -treated mice exhibited less reduction in the n amplitude after mcao, suggesting myelin preservation or improved remyelination of axons compared to the vehicle-treated subjects. sensorimotor and cognitive functions were assessed by investigators blinded to group assignments. as shown in fig a and b , il- -treated and vehicle-treated stroke mice showed comparable reductions in latencies to contact and remove adhesive tape from the paw at d after stroke, suggesting similar initial deficits in dexterity across groups. however, at later time points starting from - d after stroke, il- treatment reduced the time spent touching and removing tapes in stroke mice, culminating in improved performance in the adhesive removal test (fig a and b ). long-term sensorimotor performance in the rotarod test after mcao was also improved in il- -treated mice ( fig c) . furthermore, il- -treated mice exhibited long-term improvements in cognitive function in the morris water maze test, as manifested by a reduction in the latency to find the hidden platform (improved spatial learning, fig d and e ) and increased time spent in the target quadrant when the platform was removed (improved memory, fig d and f ). there was no difference in swimming speed among different treatment groups, indicating similar gross motor skills across groups ( fig g) . next, we measured the potential correlation between white mater integrity and behavioral performance after stroke in vehicle or il- -treated mice. the results revealed significant negative correlations between mbp intensity and the latency to touch ( fig h) and remove ( fig i) the adhesive tape at d after stroke, confirming an association between histological observations of white matter integrity and long-term functional recovery of behavioral endpoints. successful differentiation of opcs into myelinating oligodendrocytes is important for remyelination [ ] . thus, we compared the regeneration of myelin-producing oligodendrocytes in mcao mice with or without il- treatment. -bromo- -deoxyuridine (brdu) was injected at - d after mcao to label newly generated cells. mature oligodendrocytes were recognized by immunostaining with anti-adenomatous polyposis coli (apc) (or cc ) antibodies [ ] . compared to vehicle-treated mice, a significantly higher ratio of brdu + apc + newly generated oligodendrocytes was observed in il- -treated mice d after mcao (fig a and b ). the total numbers of brdu + cells and the total numbers of apc + cells were also significantly increased in il- -treated mice compared to vehicle-treated mice (fig c and d ). it has been reported that higher concentrations of the apc antibody may detect some populations of astrocytes in certain pathological conditions [ ] . however, no apc signal was detected in glial fibrillary acidic protein (gfap) + astrocytes in the ischemic brain in our preclinical model of stroke (s fig) . il- is known to promote microglia m polarization, which in turn enhances opc differentiation and remyelination [ ] . we observed in our model that il- treatment robustly promoted microglia/macrophage polarization toward an anti-inflammatory phenotype (cd + iba + ) at and d after stroke (s fig). microglia depletion through genetic targeting or pharmacological treatment is a widely used approach to evaluate the contribution of this population to various biological processes [ ] . therefore, we chose plx , an inhibitor of colony-stimulating factor receptor, to deplete microglia/macrophages and identify their role in il- -afforded oligodendrogenesis ( fig e) [ ] . consistent with previous reports [ ] , plx achieved approximately % depletion efficacy within a short period. dietary intake of plx for d dramatically reduced the number of microglia in the normal brain significant depletion of microglia/macrophages did not change the number of brdu + apc + cells or total brdu + cells compared to microglia/macrophage-competent stroke mice (fig f- h ). il- treatment increased the numbers of brdu + apc + cells or total brdu + cells after stroke in mice (h) quantification of the number of brdu + cells in the peri-infarct areas. n = - /group. � p < . , ��� p < . . one-way anova followed by bonferroni post hoc. data associated with this figure can be found in the supplemental data file (s data). apc, adenomatous polyposis coli; brdu, -bromo- -deoxyuridine; ctx, cortex; ec, external capsule; il- , interleukin- ; mcao, middle cerebral artery occlusion; str, striatum. fed with the control diet. il- treatment exhibited a trend (p = . ) toward lower capacities to increase the number of brdu + apc + newly generated oligodendrocytes in microgliadepleted mice versus microglia-competent mice, supporting a potential role of microglia in il- -afforded oligodendrogenesis. importantly, il- treatment still resulted in a significant enhancement of brdu + apc + oligodendrocyte regeneration in microglia/macrophagedepleted stroke mice (fig f and g ). considering the expression of il- r on oligodendrocyte linage cells, these results indicate that il- may exert microglia/macrophage-independent effects on oligodendrocyte regeneration after demyelination. consistent with a previous study in a model of multiple sclerosis [ ] , we found that repeated intranasal il- administration did not change t-lymphocyte subpopulations or b lymphocytes in the blood (s a-s c fig consistent with a direct effect of il- on oligodendrocytes, in vitro studies showed that il- at a concentration range of - ng/ml directly stimulated the differentiation of neuron-glia antigen (ng ) + opcs into mbp + mature oligodendrocytes without the aid of microglia ( fig a and b ). il- at concentrations of or ng/ml induced opc differentiation that was comparable to the effects of a t cocktail typically used to induce opc differentiation in vitro. the effect of il- on opc differentiation was indeed il- r-dependent, because specific il- rα neutralizing antibodies (il- rabs) blocked the il- -induced increase in mrna expression of the mature oligodendrocyte marker mbp (fig c) . il- at a concentration range of - ng/ml had no effect on opc survival (s a and s b microarray analyses were performed to further elucidate the mechanisms underlying the promotion of oligodendrogenesis by il- . opcs were treated with pbs or il- ( ng/ml) for d. total rna extraction from cell pellets was used for the affymetrix clariom s rat array. principal component analysis (pca) showed clustering of samples in a treatment-dependent manner ( fig d) . in total, differentially expressed genes (degs) were identified (false discovery rate [fdr] < . ; and fold change > ), out of which were up-regulated and were down-regulated in il- -treated opcs relative to pbs-treated opcs (fig e, s and s tables). the gene ontology (go) enrichment analysis was then performed using the metascape analysis [ ] (http://metascape.org). the top enriched go terms in three categories (biological processes, cellular components, and molecular functions) were identified in the upregulated degs by il- treatment (s a fig). a network plot further captured the relationships between the main regulated biological processes, which were related to cell adhesion and motility, cytokine production, cell responses to external stimuli, cellular component organization, and cell death (fig f) . the enriched go terms among the down-regulated degs in il- -treated opcs represent biological processes such as developmental growth, small molecule biosynthetic process, vessel development, and responses to nutrients and peptides (s b fig). these results indicated changes in cell mobilization, growth, differentiation, and defense in il- -treated opcs. a kyoto encyclopedia of genes and genomes (kegg) pathway analysis g) . the ppar transcription factor assay confirmed that pparγ activity, but not pparα or pparδ activities, was up-regulated in opcs upon il- treatment ( fig h) ; these effects were abolished when il- rα was blocked with a neutralizing antibody ( fig i) . furthermore, immunostaining (fig j and k ) and western blotting (fig l- n, and s fig) demonstrated that the pparγ inhibitor gw blocked il- -induced expression of mature oligodendrocyte markers (mbp or myelin oligodendrocyte glycoprotein [mog]), suggesting that pparγ activation is essential for the opc differentiation induced by il- . finally, opc-specific pparγ ko (pparγ-opc ko) mice were used to confirm the importance of pparγ signaling in il- -enhanced oligodendrogenesis in an in vivo stroke model ( fig a) . to confirm the ko of pparγ specifically in opcs, pdgfrα + cd − opcs, glast + cd − astrocytes, cd + immune cells, and other cd − glast − pdgfrα − cns cells were sorted from the brains collected from pparγ-opc ko mice at d after -hydroxytamoxifen injections. the dna extracts were subjected to pcr using primers flanking the loxp site and primers detecting the cre cdna. the loxp band was abolished in opcs because of the targeted deletion after cre-induced recombination, while remaining intact in astrocytes, immune cells, and other cns cells sorted from the same brain ( fig b) . the expression of cre was detected in the opcs of the pparγ-opc ko mice ( fig b) . deletion of pparγ specifically in opcs exerted no impact on il- r expression on o + oligodendrocytes (s fig). vehicle or il- treatment in pparγ-opc ko mice resulted in similar neuronal tissue loss (fig c and d ) and white matter lesion (fig c, e and f) d after stroke. the lack of pparγ expression in opcs did not significantly impair oligodendrocyte replacement after stroke, as shown by comparable numbers of brdu + apc + cells in the ischemic brain from pparγ-opc ko mice and control mice at d after mcao (fig g and h) . however, the absence of pparγ in opcs reduced the capacity of il- to increase the number of newly generated brdu + apc + oligodendrocytes (fig g and h ). consistent with a reduced capacity to promote oligodendrogenesis, il- failed to improve long-term sensorimotor (fig i and j ) or cognitive (fig k- m ) deficits after stroke in the absence of pparγ. il- /pparγ signaling improves white matter repair after stroke the ischemic brain, which is attributed, at least partly, to its release from injured neurons or from infiltrated immune cells [ ] . cerebral il- levels then return to baseline within wk after stroke [ , ] . in patients, serum levels of il- are known to change following an acute stroke [ ] . furthermore, il- gene polymorphisms are related to susceptibility to ischemic stroke and subsequent functional outcomes [ , ] . finally, a recent study reported the expression of il- r surrounding axonal filaments in postmortem brain tissue from patients with multiple sclerosis [ ] . although these clinical and experimental data are correlative, the present series of in vivo and in vitro studies firmly establish a causal link between il- repletion and white matter integrity in preclinical stroke models. deficiency of endogenous il- an improvement in white matter integrity might be achieved in two ways: preservation of preexisting white matter structures or enhancement of white matter repair. our data favor the latter as the predominant mechanism of action for il- . first, longitudinal dti revealed similar reductions in fa values shortly after stroke in vehicle or il- -treated mice, indicating comparable degrees of initial white matter injury during the early/acute phase. a significant improvement in white matter microstructure was only observed in il- -treated animals in later stages of recovery ( and d after stroke). these results strongly suggest a delayed effect of il- on white matter repair, rather than preservation shortly after injury. second, electron microscopy (em) and histological studies revealed structural signs of remyelination in the ischemic brain, including thinner myelin segments than before stroke, and reduced paranode length with caspr/nav . staining [ ] . each of these structural alterations were significantly improved by il- treatment, indicating superior remyelination. third, il- treatment increased the number of new, mature oligodendrocytes (apc + brdu + ) in the ischemic brain d after stroke. such improvements in oligodendrogenesis are critical for successful remyelination. finally, the lpc model of demyelination-remyelination unequivocally confirmed the white matter-specific effects of il- in remyelination. as expected, the il- -mediated reconstitution of myelin structure culminated in functional improvements in neural conduction and behavioral performance, despite no changes in the degree of gray matter loss. although il- is important for white matter repair after brain injury, the il- ko mice do not display deficits in myelin coverage in organotypic brain slice cultures under physiological conditions. this observation is consistent with the view that different mechanisms control myelin sheath formation during normal development and remyelination [ ] . although our t scanning did not show significant reductions in brain lesion size under conditions of il- repletion, there was a trend toward lesion volume reduction at - d after stroke. as the il- treatment was initiated h after stroke, it remains possible that il- may provide some degree of early protection [ ] . it is also possible that some neun + neurons remaining within the gray matter of mice without il- might not be physiologically functional, although they are still histologically present, perhaps because their communication via white matter tracts is significantly impeded. a previous study using mixed glial cultures demonstrated that the addition of il- promotes the differentiation of opcs into mbp-expressing oligodendrocytes in vitro, an effect mainly attributed to il- -mediated anti-inflammatory effects [ ] . an additional conclusion emerging from our studies is that il- enhances white matter repair at least partially independent of its well-established anti-inflammatory effects on microglia/macrophages. in il- -treated stroke mice, we observed an anti-inflammatory phenotypic change in microglia/ macrophages, which is known to promote remyelination after cns injury [ ] . however, dramatic depletion of microglia/macrophages in the ischemic brain did not abolish the capacity of il- to enhance oligodendrocyte replacement, supporting the involvement of microglia/ macrophage-independent mechanisms. in vitro studies then confirmed that il- can also directly target white matter components and promotes opc differentiation into mature oligodendrocytes in the absence of microglia/macrophages. previous fate mapping studies revealed that opcs are responsible for generating new oligodendrocytes during remyelination [ , ] . however, the differentiation of opcs is frequently impaired in lesioned brains, mainly because of nonpermissive environmental cues [ ] [ ] [ ] . we found that il- treatment may overcome these obstacles for opc differentiation in the ischemic brain, both directly by promoting opc differentiation and indirectly through immunomodulation. notably, endogenous oligodendrocyte differentiation has been observed until as late as mo after cns injuries [ ] , and this prolonged effort may provide a sufficiently wide therapeutic window that allows delayed il- treatment to restore white matter and encourage long-term stroke recovery. in the present study, we report a previously undefined mechanism of il- , namely that it directly induces opc differentiation by activating pparγ signaling. pparγ is a master transcription factor known to promote opc differentiation and maturation though manifold mechanisms involving enhanced myelin lipid synthesis, reduced oxidative stress, and promotion of mitochondrial function [ , ] . our previous work has shown that rosiglitazone, a pparγ agonist, enhanced opc proliferation and increased the number of newly generated mature oligodendrocytes after mcao [ ] . in addition, pparγ may interact with histone deacetylases to modulate cell differentiation [ ] . we demonstrated here that pparγ inhibition abolished the effect of il- on opc differentiation in vitro and that opc-specific ko of pparγ in vivo reduced il- -enhanced oligodendrocyte replacement in the ischemic brain. these collective results indicate that il- -induced oligodendrocyte differentiation/maturation relies at least partially on the activation of pparγ activity in opcs. precisely how il- /il- r engages pparγ activity is not yet known, but several studies suggest that il- may activate pparγ in macrophages and other cell types through signal transducer and activator of transcription (stat ) signaling [ , ] . whether this pathway is also operational in opcs lies beyond the scope of the present study but warrants further investigation. aside from the cell-specific effects of il- on opcs, we note that il- r is widely distributed in many types of cns cells as well as infiltrated immune cells. several lines of evidence from our study support the capacity of il- to target multiple types of cells in addition to oligodendrocytes. first, we did not observe significant differences in the density of brdu + apc + cells between wt and pparγ-opc ko mice following mcao, suggesting that endogenous il- is able to promote white matter integrity by targeting other types of cells. second, although pparγ inhibition eliminated the effect of il- on opc differentiation in cultures, the absence of pparγ in opcs reduced, but did not abolish, the capacity of il- to increase the number of newly generated oligodendrocytes in vivo. these results suggest that pparγ expression in opcs is an important but not exclusive mechanism for il- -induced oligodendrogenesis in preclinical stroke. indeed, we observed that il- treatment robustly promoted microglia/macrophage polarization toward an anti-inflammatory m phenotype, as mentioned above. il- -stimulated microglia have been shown to release il- induced (il i ), which in turn augments remyelination [ ] . thus, direct and indirect effects on microglia/macrophages may contribute to the white matter repair afforded by il- . it has also been reported that the effects of il- on adaptive immune responses contribute partially to recovery from neurotrauma [ ] . in our hands, however, repeated intranasal il- administration did not change t-lymphocyte subpopulations or b lymphocytes in blood and did not alter the composition of infiltrated lymphocytes. these data dispute the involvement of adaptive immune responses in il- -afforded neuroprotection in our stroke model. aside from its roles in immunoregulation, il- can also directly target other types of cns cells. for example, a recent study reported that the activation of il- r signaling in neurons may increase axonal outgrowth and ameliorate axonal pathology in a model of multiple sclerosis [ ] . in this context, it is important to note that we did not observe improvements in the dti measurement for axonal integrity (ad, λ || ). in other disease models, multiple factors including inflammation, cellular infiltration, and gliosis may contribute to ad at chronic stages of disease. therefore, the measurement of λ || appears to lack sufficient resolution to distinguish axonal damage/repair versus confounding cellular responses over the course of progressive demyelination [ ] . on the other hand, our em data demonstrated an increase in axon diameter in il- -treated mice compared to vehicle-treated controls, supporting a beneficial role of il- in maintaining axonal structure. these structural data were supported by our electrophysiological studies of axon conductance. preservation of axonal structure and electrical function might provide an important feedback signal for opc differentiation and guide remyelination [ , ] , preventing wallerian degeneration of neurons after ischemia and enhancing white matter repair. in addition to these mechanisms, astrocytes are known to produce trophic factors in response to il- stimulation, including brain-derived neurotrophic factor (bdnf) and nerve growth factor (ngf), both of which are also critical for brain function and brain repair [ ] . a series of in vitro studies assessed the effective concentrations of il- in various types of cns cells. for example, il- regulates microglia activity at concentrations of - ng/ml [ , ] . il- inhibits inflammatory responses and boosts trophic factor release in astrocytes at - ng/ml [ ] . il- protects neurons against excitotoxicity and stimulates axonal outgrowth in vitro at ng/ml [ ] . in the present study, il- directly stimulated opc differentiation within a concentration range of - ng/ml. these collective studies reveal similar effective il- concentrations (in nanoscales) in glial and neuronal cells of the cns. indeed, it is precisely the multitargeting capacities of low-dose il- that may render it an excellent therapy for cns injuries characterized by a wide array of pathological mechanisms. the ultimate implementation of preclinical research into clinical treatments requires extensive information on safety, optimal dose, route, and schedule of administration, among other variables. in the present study, il- stimulated opc differentiation in cultures at a concentration range of - ng/ml, and the optimal concentration was - ng/ml. although there is neither a mathematical nor empirical method to accurately convert in vitro concentrations to in vivo intranasal dosage, these in vitro data support the capacity of il- to directly target opcs at low concentrations. previous preclinical studies have similarly documented therapeutic effects of low doses of il- in ischemic stroke and other neurological diseases. for example, subcutaneous il- application at μg/kg/d [ ] or intracerebroventricular application of il- at ng/d [ ] significantly improved long-term outcomes after stroke. vogelaar and colleagues reported that intrathecal or intranasal ( μg/d) il- treatment ameliorated clinical signs and reversed disease progression in a model of multiple sclerosis [ ] . those studies, however, did not measure il- concentrations in the brain after treatment. here, we delivered il- intranasally at a dose of μg/kg/d (about . μg/d based on . kg average body weight), which significantly elevated il- levels in key parts of the brain. this dose is similar to the effective intranasal dose for vogelaar's study and improved long-term functional recovery in our stroke model. repeated dosing regimens or controlled continuous release was employed for all in vivo il- treatment because of its rapid clearance and inactivation. the use of liposome-entrapped peptides has been reported to protect the cytokine and enhance its tissue delivery [ , ] . consistent with those findings, we demonstrated that il- liposome nanoparticle delivery elevated il- levels in the brain for at least h. further characterization of the optimal regimen of il- treatment for stroke would accelerate its clinical translation. despite growing awareness of the importance of white matter restoration in poststroke rehabilitation, there is no effective therapy to preserve white matter function or to improve its repair in humans. our study has uncovered a novel role of il- in poststroke white matter recovery-a role that lies beyond its well-known immunoregulatory functions. furthermore, we have shown that il- promotes oligodendrogenesis and white matter repair in a pparγdependent manner. therefore, it seems reasonable to continue to test il- as a therapeutic strategy for functional recovery after ischemic stroke. in particular, the intranasal route enables cns il- delivery, which is expected to significantly reduce the risk of peripheral side effects and expedite the eventual translation of il- treatment for stroke victims. all animal procedures were approved by the university of pittsburgh institutional animal care and use committee (approval number: ), performed in accordance with the national institutes of health guide for the care and use of laboratory animals, and reported in accordance with the animal research: reporting in vivo experiments (arrive) guidelines. all efforts were made to minimize animal suffering and the number of animals used. all animals were housed in a temperature-and humidity-controlled facility with a -h light/dark cycle. food and water were available ad libitum. transient cerebral ischemia was induced by intraluminal occlusion of the left middle cerebral artery (mca) for min, as described previously [ ] . sham-operated animals underwent the same anesthesia and exposure of arteries without mcao. briefly, mice were anesthetized with % isoflurane in a % o / . % n o mixture until they were unresponsive in the tail-pinch test. mice were then fitted with a nose cone blowing . % isoflurane in a % o / . % n o mixture under spontaneous breathing for anesthesia maintenance. an - monofilament with a silicon-coated tip was introduced into the common carotid artery, advanced to the origin of the mca, and left in place to limit mca blood flow for min. rectal temperature was controlled at . ± . ˚c using a temperature-regulated heating pad during surgery. regional cerebral blood flow was measured in all stroke animals using laser doppler flowmetry. animals that did not display at least % reduction in regional cerebral blood flow during mcao compared to pre-ischemia levels were excluded from further experimentation. surgeries and quantification were performed by investigators blinded to animal genotypes and experimental grouping. animals were randomly assigned to sham or cerebral ischemia groups and received randomized treatments using a lottery drawing box. a grand total of mice ( sham-operated and ischemic mice) were used in this study, including mice that were excluded from further assessments, either because of death after ischemia or failure of ischemia induction. the mortality during mcao surgery was . % in wt male mice ( / ), . % in il- ko male mice ( / ), . % in microglia/macrophage-depleted mice ( / ), and . % in pparγ-opc ko mice ( / ). there was no significant difference in mortality rate across groups. lecithin ( mg) and cholesterol ( mg) were dissolved in ml of a chloroform-methanol mixed solution ( : , volume:volume). a lipid layer was formed after evaporating the mixed solvent under vacuum distillation. recombinant il- protein ( mg, peprotech, rocky hill, nj, usa) was dissolved in ml of water and then added to the lipid layer for hydration for about min. the mixture was homogenized by ultrasonic probes for min in an ice bath and then filtered through a . -μm membrane. the free il- protein was removed by ultrafiltration with centrifugal filter units ( -kda cutoff). the particle size and zeta potential of il- protein-loaded liposomes were determined by dynamic light scattering (dls). the concentration of il- protein was determined by the bicinchoninic protein determination kit (thermo fisher scientific, pittsburgh, pa, usa). animals were randomly assigned to receive vehicle or il- ( μg/kg body weight, intranasal) nanoparticle treatment starting h after mcao and repeated at - d, d, d, and d postsurgery. briefly, under isoflurane anesthesia, five -μl drops (total μl) of il- nanoparticles were applied alternately into each nostril with a -min interval between drops. control animals received an equal volume of vehicle control using the same anesthesia and delivery regimen. in order to label proliferating cells, animals were intraperitoneally injected with the thymidine analogue brdu ( mg/kg, b , sigma, st. louis, mo, usa) twice a day (with an interval of at least h) for consecutive days, beginning at d after mcao. behavioral tests were performed by an individual blinded to experimental groups. sensorimotor functions were measured - d after mcao. the rotarod test. briefly, mice were forced to run on a rotating drum (iitc life science, woodland hills, ca, usa) with speeds starting at rpm and accelerating to rpm within s. three consecutive trials were conducted for each mouse with an interval of min. the time at which a mouse fell off the drum was recorded as the latency to fall. data were expressed as mean values from three trials. the adhesive removal test. the adhesive removal test was performed to assess tactile responses and sensorimotor asymmetries. two × -mm adhesive tapes were applied to lesioned forepaws. tactile responses were measured by recording the time to initially contact the ipsilateral paws, as well as the time to remove the adhesive tape, with a maximum observation period of s. morris water maze test. cognitive function was analyzed with the morris water maze test - d after mcao, as described previously [ ] . in the learning test, a square platform ( × cm ) was submerged cm beneath the water surface in a circular pool (diameter = cm) of opaque water. mice were placed into the pool from one of the four locations and allowed to locate the hidden platform for s. each mouse was trained on trials (with randomly assigned starting positions) per day to locate the platform for three consecutive days before mcao. at the end of each trial, the mouse was placed on the platform or allowed to stay on the platform for s with prominent spatial cues displayed around the room. the time spent to reach the platform (learning phase) was recorded. three trials were performed on each day. the memory test was performed on day . the platform was removed, and a single -s probe trial was conducted. time spent in the goal quadrant where the platform was previously located was recorded. animals were euthanized and perfused with saline followed by % pfa (sigma-aldrich; st. louis, mo, usa) in pbs. brains were cryoprotected in % sucrose in pbs. coronal brain sections ( μm) were sliced on a freezing microtome, and six equally spaced coronal brain sections encompassing the mca territory were stained with a rabbit anti-neun antibody (abn , : ; millipore, burlington, ma, usa) or rabbit anti-mbp antibody (mbp ab , : , abcam, cambridge, ma, usa). loss of neun + or mbp + tissue was analyzed by a blinded observer using nih image j software. the infarct volume with edema correction was calculated as the volume of the contralateral hemisphere minus the noninfarcted volume of the ipsilateral hemisphere. coronal brain sections were subjected to immunofluorescence staining. briefly, floating brain sections were blocked with % donkey serum in . % triton x- in pbs (pbst) for h at room temperature, followed by overnight incubation with primary antibodies at ˚c. after three washes in . % pbst, sections were incubated with the appropriate secondary antibodies for h at room temperature. the process was repeated once for double staining. image analyses were performed on one or two randomly selected microscopic fields in the peri-infarct areas of the ec, two in the cortex, and two in the striatum of each section; two sections covering the infarct area were assessed for each mouse brain. the recorded images were loaded onto image j (nih) and were manually quantified by observers blinded to grouping. positively stained cells were electronically labeled with the software to avoid duplicated counting. the infarct area was identified as the region in which the majority of dapi-stained nuclei were shrunken. the border of the infarct was determined by the loss of neun staining and the accumulation of iba + microglia/macrophage or brdu + apc + oligodendrocytes on adjacent sections. we defined the peri-infarct area as the tissue that covers a radial distance of - μm from the border of the infarct. animals were euthanized and perfused with cold saline. brains were dissected, and the ipsilateral (left) and contralateral (right) hemispheres were collected. brains were dissociated into a single-cell suspension using a gentlemacs dissociator (miltenyl biotec) following the manufacturer's instructions. the suspension was passed through a -μm cell strainer (fisher scientific, pittsburgh, pa, usa) and resuspended in % percoll (ge health). single-cell suspensions were separated from myelin and debris by centrifugation ( g, min, ˚c) on a %- % percoll gradient. cells at the interface were collected and washed with hank's balanced salt solution (hbss; sigma-aldrich, st. louis, mo, usa) containing % fetal bovine serum (sigma-aldrich, st. louis, mo, usa) and mm edta (sigma-aldrich, st. louis, mo, usa) before staining. blood cells were prepared as described previously [ ] . single-cell samples were first incubated with antibodies to surface antigens for min on ice at ˚c in the dark. after two washes, cells were fixed and permeabilized with an intracellular staining kit (thermo fisher ebioscience, pittsburgh, pa, usa) according to the manufacturer's protocol. caps were measured in the ec. mice were decapitated after co euthanasia, and brains were removed. coronal slices ( μm thick) were prepared − . mm from bregma and transferred to an incubation chamber containing pregassed ( % o / % co ) artificial cerebrospinal fluid (acsf; mmol/l nacl, . mmol/l kcl, mmol/l na h po , . mmol/l cacl , mmol/l nahco , . mmol/l mgcl , mol/l glucose [ph . ]) for min at ˚c and then for h at room temperature ( ˚c). brain slices were then perfused with acsf at a constant rate ( - ml/min) at ˚c. for recording, a bipolar stimulating electrode (intertip distance, μm) was placed across the cc at about . mm lateral to the midline. a glass extracellular recording pipette ( - mo tip resistance when filled with acsf) was placed in the ec, . , . , . , and mm lateral from the stimulating electrode. the recording at . mm from the stimulating electrode was reported. the cap signal was digitized (digidata b plus humsilencer; molecular devices, san jose, ca, usa), amplified (× k), and recorded by the axoclamp b amplifier (molecular devices, san jose, ca, usa). the signal was then analyzed by pclamp software (molecular devices, san jose, ca, usa). the input-output curves were generated by increasing the stimulating intensity by . -ma intervals from baseline, up to ma. the amplitude of the n component of the cap (representing myelinated fibers) was calculated as the variance from the first peak to the first trough. tem was used to measure myelin thickness in the cc/ec area. mice were perfused with icecold saline, followed by % pfa and . % glutaraldehyde in . mol/l pbs buffer. the cc/ec tissue near the site of ischemia was microdissected into -mm blocks. these specimens were immersion-fixed for h in % glutaraldehyde. tissue was then rinsed in pbs and fixed in % osmium tetroxide in . m pbs for min. samples were dehydrated in increasing concentrations of acetone and embedded in araldite resin. ultrathin -nm sections were cut on a leica uct ultramicrotome with a diamond knife (diatome, wetzlar, germany), stained with uranyl acetate and lead citrate, and viewed using a jem tem (jeol, akishima, japan). two sections from each animal were analyzed. three to images ( μm each) were acquired in randomly selected areas within the cc/ec from each section at a magnification of , × and analyzed with image j by an investigator blinded to experimental groups. at least random axons per animal were analyzed by tracing the axonal circumference and the whole fiber circumference in a blinded fashion. g-ratios were calculated as the ratio of the inner axonal diameter to the total outer diameter (axonal diameter + total myelin sheath thickness). for in vivo mri, mice were initially anesthetized with % isoflurane and then positioned on an animal cradle with a stereotaxic head holder. anesthesia was maintained between % and . % isoflurane throughout the experiment. respiration and temperature were monitored continuously, and temperature was maintained by a warm animal heating system (sa instruments, stony brook, ny, usa). mri was performed on a . t/ -cm aviii hd spectrometer (bruker biospin, billerica, ma, usa) equipped with a -cm high-performance gradient set and using an -mm quadrature rf transmit volume coil and a -channel receive surface rf coil and paravision . . . a t -weighted rare sequence was used to visualize the infarct, with the following setup: repetition time (tr)/echo time (te) = , / ms, field of view (fov) = × mm, acquisition matrix = × , slices with a slice thickness of . mm, averages, and a rare factor = . the dti data were collected using an echo planar imaging (epi)-dti imaging sequence with the same geometry as the t -weighted scan, using the following setup: tr/te = , / ms, fov = × mm, acquisition matrix = × , slices with a slice thickness of . mm, averages, segments, a images and noncolinear diffusion images, Δ/δ = / ms, and a b-value = , s/mm . perfusion-fixed brains were left in the skull and imaged ex vivo using a bruker av hd . t/ -mm vertical-bore microimaging system equipped with a micro . gradient set capable of , mt/m, a -mm quadrature rf resonator, and paravision . . (bruker biospin, billerica, ma, usa). dti data were collected using a multislice spin-echo sequence with a images and noncolinear diffusion images with the following setup: te/tr / , ms, averages, × matrix, × -mm fov, slices, . mm slice thickness, bvalue = , s/mm , and Δ/δ = . / . ms. this setup has been established for the acquisition of high-resolution dti scanning in small animals [ , , ] . dti data were analyzed with dsi studio software (http://dsi-studio.labsolver.org/). rois were drawn manually in a blinded manner encompassing the ec and ic in the ipsilesional and contralesional hemispheres to determine fa, ad, and rd. dec, fa, and rd maps were generated by dsi studio software. in order to verify cre-induced deletion of flanked pparγ exons, a pair of primers (forward: -tgtaatggaagggcaaaagg; reverse: -tggcttccagtgcataagtt) that flank the downstream loxp site were used to perform pcr with genomic dna. program for pcr was ˚c for min; ( ˚c for s; ˚c for s; ˚c for s) × , and then ˚c for min. a pair of primers that detect the cre cdna (forward: -gcggtctggca gtaaaaactatc; reverse: -gtgaaacagcattgctgtcactt) were used as a positive control to confirm the absence of pcr band in opc was not due to failed pcr protocol itself. microarray experiment was performed in the genomics research core in the university of pittsburgh. total rna was prepared from il- -or pbs-treated opcs using trizol (qiagen) according to manufacturer's instruction. preparation of cdna was performed using the wt plus reagent kit (thermo fisher) according to the manufacturer's instructions. reverse transcription was performed using ng total rna and dntp-t random primers. second-strand synthesis and in vitro transcription created amplified crna. cdna from a second reverse transcription reaction was fragmented and end-labeled with biotin for hybridization to the affymetrix clariom s rat array. following overnight ( h) hybridization at ˚c with rotational mixing at rpm, arrays were processed on a genechip fluidics station using manufacturer-specified protocols. to remove unbound sample, arrays were first washed with nonstringent wash buffer a. the genechips were then stained for min in stain cocktail . buffer a was again used to wash off excess stain. signal amplification was achieved by -min incubation with stain cocktail followed by a second -min incubation with stain cocktail . the chip was washed with high-stringency buffer b and filled with array holding buffer before being removed from the fluidics station and scanned using the genearray scanner. first-level image analysis was performed using affymetrix expression console. four biologic replicates were performed for each experimental condition. data resulted in , detected probes that were used for subsequent analysis using transcriptome analysis console (affymetrix) and metascape analysis [ ] (http://metascape.org). the go network plot is visualized using cytoscape, in which terms with a similarity > . are connected by edges. each node represents an enriched term and is colored by its cluster id. primary opc cultures were isolated with the differential attachment method. briefly, brains of mixed-sex sprague dawley rat pups (postnatal day - ) were triturated after removing the meninges and blood vessels. the brain tissues were dissociated with trypsin ( . %) at c for min. after washing with ice-cold dmem, the cells were passed through a -μm filter and plated onto poly-d-lysine-coated t-flasks filled with culture media (dmem/f containing % heat-inactivated fetal bovine serum, mm l-glutamine, mm sodium pyruvate, μm nonessential amino acids, u/ml penicillin, and μg/ml streptomycin) [ ] . cells were grown to confluence ( ) ( ) ( ) ) in a humidified incubator at ˚c and with % co . microglia were removed by shaking the mixed glia-containing flasks for h at rpm. after removing microglia, flasks were subjected to shaking at rpm overnight to separate opcs from the astrocyte layer. opcs were maintained for - d in a serum-free basal defined medium (bdm: dmem, . % bovine serum albumin, μg/ ml human apo-transferrin, μg/ml insulin, nm sodium selenite, nm d-biotin, nm hydrocortisone) containing ng/ml pdgf and ng/ml bfgf. for oligodendrocyte induction, opcs were stimulated with t and cntf ( ng/ml). the medium was exchanged every d. protein was isolated from in vitro cultures. western blots were performed using the standard sds-polyacrylamide gel electrophoresis (page) method. the primary antibodies used in this study include rabbit anti-mbp (cell signaling technology, , : , danvers, ma, usa), mouse mog (santa cruz, sc- , : , santa cruz, ca, usa), and mouse anti-β-actin (a , : , ; sigma-aldrich, st. louis, mo, usa). in brief, polyvinylidene difluoride (pvdf) membranes were blocked in % nonfat milk for h at room temperature and then incubated at ˚c overnight with primary antibodies. next, the membrane was incubated with secondary antibodies for h at room temperature ( : , , li-cor biosciences, lincoln, ne, usa), and scanned with li-cor odyssey infrared imaging system - u (li-cor biotechnology, lincoln, ne, usa). imagej software was used for gel analyses. nuclear extracts were prepared from opcs using ne-pe nuclear and cytoplasmic extraction reagent kit (thermo fisher, , pittsburgh, pa, usa). ppar activity was assessed using the ppar (α, δ, γ) transcription factor assay kit (abcam, ab , toronto, on, canada) or the pparγ transcription factor assay kit (cayman chemical, , ann arbor, mi, usa) following the manufacturers' instructions. the cerebellum and attached hindbrain were isolated from p c bl/ j mouse pups, sectioned sagittally at μm on a mcilwain tissue chopper, and plated onto millipore-millicel-cm mesh inserts (thermo fisher scientific, pittsburgh, pa, usa) in -well culture plates with media containing % basal medium eagle (sigma, st. louis, mo, usa, b ), % heatinactivated horse serum (gibco - ), % earle's balanced salt solution (gibco - ), . mg/ml glucose (sigma g ), % penicillin-streptomycin, and % glutamax. media were changed every - d. to induce demyelination, cultures were treated with lpc (sigma, . mg/ml) for h. slices were washed in media for min and treated for or d with pbs or il- ( ng/ml, il- was added to fresh media every other day). slices were fixed in % pfa for h. rabbit anti-mbp antibodies (santa cruz, santa cruz, ca, usa, sc- ; : ) and mouse anti-nf-h antibodies (abcam, cambridge, ma, usa, ab , : , ) were applied for h at ˚c. fluorescence-conjugated secondary antibodies were applied overnight at ˚c. slices were mounted onto glass slides and coverslipped with fluoromount-g. confocal microscopy (fluoview fv ; olympus, tokyo, japan) was used to capture images. mbp staining intensity was analyzed with imaris software (bitplane ag). morphology reconstructions were carried out with the imaris filamenttracer module. quantitative assessment of myelination was performed in a blinded manner using the imaris colocalization module. thresholds for colocalization of red (nfh + axon) and green (mbp + myelin) channels were calculated automatically and maintained across different images from the same batch of staining. a colocalization channel was created and channel statistics were calculated automatically. the myelination index was calculated as the percent of colocalized areas in the total nfh + red pixels above threshold. for each condition, at least slices collected from three independent experiments were analyzed. two to three fields were imaged from each slice. sample sizes for animal studies were determined by power analyses based on pilot studies or the literature. results are presented as mean ± standard error of the mean (sem). graphpad prism software (version . . , la jolla, ca, usa) was used for statistical analyses. the student's t test was used for comparison of two groups for continuous variables with normal distributions. the mann-whitney u rank sum test was used for continuous variables with nonnormal 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astrocyte activation and induces ngf secretion microglia/macrophage polarization dynamics reveal novel mechanism of injury expansion after focal cerebral ischemia cytokines in liposomes: preliminary studies with il- , il- , il- , gm-csf and interferon-gamma effect of il- and il- liposomal formulations on the induction of immune response to bovine herpesvirus type- glycoprotein d adoptive regulatory t-cell therapy protects against cerebral ischemia selective role of na(+) /h(+) exchanger in cx cr (+) microglial activation, white matter demyelination, and post-stroke function recovery tissue plasminogen activator promotes white matter integrity and functional recovery in a murine model of traumatic brain injury key: cord- -eqgc v y authors: may, win lai; kyaw, myat phone; blacksell, stuart d.; pukrittayakamee, sasithon; chotivanich, kesinee; hanboonkunupakarn, borimas; thein, khin nyo; lim, chae seung; thaipadungpanit, janjira; althaus, thomas; jittamala, podjanee title: impact of glucose- -phosphate dehydrogenase deficiency on dengue infection in myanmar children date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: eqgc v y glucose- -phosphate dehydrogenase (g pd) deficiency may affect the clinical presentation of dengue due to the altered redox state in immune cells. we aimed to determine the association between g pd deficiency and severity of dengue infection in paediatric patients in myanmar. a cross-sectional study was conducted among paediatric patients aged – years with dengue in yankin children hospital, myanmar. one hundred and ninety-six patients positive for dengue infection, as determined via pcr or elisa, were enrolled. dengue severity was determined according to the who classification guidelines. spectrophotometric assays determined g pd levels. the adjusted median g pd value of males in the study population was used to define various cut-off points according to the who classification guidelines. g pd genotyping for mahidol, kaiping and mediterranean mutations was performed for out of samples by real-time multiplex pcr. of ( . %) patients had severe dengue. the prevalence of g pd phenotype deficiency (< % activity) in paediatric patients was . % ( / ), specifically, . % ( / ) in males and . % ( / ) in females. severe deficiency (< % activity) accounted for . % ( / ) of our cohort, occurring . % ( / ) in males and . % ( / ) in females. among samples genotyped, the g pd gene mutations were detected in . % ( / ) of patients, with . % ( / ) in males and . % ( / ) in females. the g pd mahidol mutation was . % ( / ) while the g pd kaiping mutation was . % ( / ). severe dengue was not associated with g pd enzyme deficiency or presence of the g pd gene mutation. thus, no association between g pd deficiency and dengue severity could be detected. trial registration: the study was registered following the who international clinical trials registry platform (who-ictrp) on thai clinical trials registry (tctr) website, registration number # tctr introduction glucose- -phosphate dehydrogenase (g pd) is an enzyme presenting in the cytoplasm of human cells that participates in the pentose phosphate pathway and supplies reducing energy by maintaining levels of the co-enzyme nicotinamide adenine dinucleotide phosphate (nadph) [ ] . g pd deficiency is an x-linked inheritance. g pd gene consists of exons with approximately kb and is situated on the distal long arm of x chromosome (xq ). about mutations of g pd gene have been reported resulting in protein variants with different levels of enzyme activity. nadph is essential for both oxidant and antioxidant systems of cells. in the antioxidant pathway, nadph maintains the reduced form of glutathione to protect cells from oxidative damage [ ] . this reduction in cells causes accumulation of redox oxidative species (ros) and leads to senescence [ ] and haemolysis in red blood cell [ ] . on the other hand, nadph is also involved in the oxidant pathway to produce ros. although the overproduction of these ros may adversely affect cell function, cells of immune system also need these reactive species to kill invading organisms. phagocytes of the immune system need these reactive species to kill invading pathogens as part of the innate immune system. in cases of severe g pd deficiency, the lack of oxidative metabolism can cause a reduction in oxygen-dependent phagocytosis as observed in chronic granulomatous disease [ ] and allows for viral replication [ ] [ ] [ ] [ ] . glucose- -phosphate dehydrogenase (g pd) deficiency affects more than million people all over the world [ ] and prevalence is approximately % in africa and ranges from . to . % in southeast asia [ ] . in myanmar, the prevalence is . % and . % in adult males and females respectively [ ] , while it is . % and . % in healthy children males and females respectively [ ] . to note, . % of males and . % of females possess the g pd mutation [ ] with the g pd mahidol variant occurring in . % of children in myanmar [ ] . dengue virus infection is one of the leading causes of morbidity and mortality in children living in tropical and sub-tropical regions [ ] . according to who, approximately million people worldwide experience a dengue infection annually [ ] . in southeast asia, dengue leads to over , deaths annually [ ] . in myanmar, all four dengue serotypes are known to co-circulate, and children are at the highest risk for infection [ ] . the reported case fatality rate was per , dengue cases in [ ] . most dengue patients present with undifferentiated febrile illness that some may progress to life threatening disease [ ] . how patients' genetic background affects the development of severe infection has become an area of interest. g pd deficiency is one of the reported genetic variants associated with infections [ , ] . in vitro studies reported that monocytes from g pd-deficient individuals had increased susceptibility to dengue virus serotype infections along with higher viral replication [ , ] . whether higher replication of dengue virus in g pddeficient individuals increases the likelihood of disease severity remains unknown. herein, we investigated the association between g pd deficiency and severity of dengue infection in paediatric patients in myanmar. a cross-sectional prospective study was conducted in yankin children hospital, yangon from august to august . paediatric patients (age range: - years) with a fever for - days and clinically suspected of having dengue fever were screened. written informed consent and verbal assent were taken from caregivers and children, respectively. three millilitres of blood were taken, and sera were tested for the ns antigen as well as for igm and igg antibodies using the sd bioline dengue duo rapid diagnostic test (rdt) (standard diagnostics, korea). patients with a positive ns antigen and/or dengue igm result were enrolled. patients who had a diagnosis other than dengue, such as co-infections and immunocompromised status (e.g., steroid use, chemotherapy, and/or positive hiv status) were excluded. patients were followed - weeks after admission for convalescent serum sample collection. complete blood count (cbc), reticulocyte count (rc) and g pd activity were measured within hours after collection of acute sera. g pd activity was assessed via spectrophotometry (randox laboratory, uk). the adjusted median value of g pd activity for males in our cohort was used to define various cut-off points [ ] . dengue severity was classified according to the who classification guidelines [ ] . both acute and convalescent samples were stored at - ˚c prior to further testing. the g pd activity, cbc and rc were re-checked at least one month after the first sample collection for patients at risk of being misdiagnosed for g pd deficiency based on analyses of the first blood sample collected, such as patients with rc values > %, those who suffered or recovered from acute haemolytic anaemia, and those who had a blood transfusion before the first sample collection. the study protocol was approved by the ethics dengue, zika and chikungunya virus detection using a reverse transcriptase pcr assay. rna was isolated from acute patient serum samples ( μl) using the qiaamp viral rna mini kit (qiagen, germany). rna extractions were performed according to the manufacturer's protocol. rna was stored at - ˚c until further use. one-step reverse transcriptase (rt) pcr was performed to detect dengue, zika and chikungunya viruses in a single assay using zdc multiples rt-pcr assay (bio-rad, usa). assays were performed according to the manufacturer's protocol, with an additional rnase p primer mix as an internal extraction control. for each rt-pcr assay, a positive control, provided from the kit; negative control (healthy donor sera); and no template control were included. all rna samples were performed in duplicate. a positive pcr was defined as when one or both duplicates had a signal above a fixed threshold of for all the respective targets: fam for zika virus, hex for chikungunya virus, and texas red for dengue virus. g pd activity. sample was stored at - ˚c, then transported to department of medical research (dmr) laboratory in yangon myanmar and processed within hours. spectrophotometry was conducted using humalyser spectrophotometer (medsource ozone biomedicals, delhi, india) and kits from randox (cat no pd , randox laboratories ltd., crumlin, uk). the procedure provided in the manual with the kit was followed. normal and deficient g pd controls (cat no pd for deficient and cat no pd for normal, randox laboratories ltd., crumlin, uk) were used. the results were considered valid if the measured activities of the controls were within the reference range. enzyme activity was determined by the spectrophotometer set at ˚c to measure the rate of absorbance at nm under ultraviolet light. enzyme activity was calculated and adjusted for the hb concentration value from complete blood count (cbc) recorded at the time of sample collection. the adjusted median value of g pd activity for males in our cohort was used to define various cutoff points [ ] . g pd genotyping by real-time pcr. dna samples were extracted from μl of whole blood using the qiaamp dna blood mini kit (qiagen, germany) according to the manufacturer's recommendation. three g pd variants (g pd mahidol c. g>a; g pd kaiping c. g>a; and g pd mediterranean c. c>t) previously reported in g pd-deficient individuals in myanmar were detected by real-time pcr with the bio-rad cfx real-time system and c thermal cycler (bio-rad, usa). genotyping pcr, with melting curve analysis using dual-labelled, self-quenched probes, was performed using μl reactions, containing . μl of iq multiplex power mix, . μl each of forward and reverse primers, . μl each of snp and wt probes, μl sterile distilled water, and μl genomic dna. the amplification conditions were minutes at ˚c for iq multiplex power mix activity, followed by cycles of seconds at ˚c for denaturation, and min at ˚c for annealing and extension for g pd genotyping. the samples were run together with the plasmid control for each targeted mutation; three separate pcr reactions were run in every sample for the three different mutations. fluorescence and ct values using the cfx manager software (bio-rad, usa) were used to determine genotypes. each sample's result was verified by examining the pcr curve generated to eliminate false-positive results due to aberrant light emission (s table) . acute dengue infection. laboratory confirmed acute dengue infection was defined by a positive qrt-pcr, ns antigen (ag) elisa, or igm elisa result. primary or secondary dengue infections were defined according to a negative or positive igg elisa result in the febrile phase (� days), respectively [ ] . dengue severity. the severity of dengue infection was classified according to the who guideline [ ] . dengue patients were classified as non-severe or severe based on clinical and laboratory parameters. patients with non-severe dengue were classified into two groups based on the absence or presence of warning signs. non-severe dengue without warning signs was defined if the patient had fever with of these following criteria: nausea/vomiting, rash, aches and pains, tourniquet test positive, leucopoenia without presentation of any warning signs. warning signs included abdominal pain or tenderness, persistent vomiting, clinical fluid accumulation, mucosal bleed, lethargy/restlessness, liver enlargement > cm and increase in haematocrit (� %) concurrent with rapid decrease in platelet count (< × per μl). severe dengue was defined as: dengue shock syndrome (dss), fluid accumulation with respiratory distress, and/or severe bleeding that necessitated blood transfusion or involved an organ [ ] . dss was diagnosed by the presence of hypotension or narrow pulse pressure (< mmhg) [ ] . g pd deficiency. the who classification is based on the g pd activity expressed as a percentage of the median g pd value of a normal male population. class i-v are defined as: class i < % (associated with chronic non-spherocytic haemolytic anaemia), class ii -< %, class iii -< %, class iv - %, and class v > %. in this study, g pd deficiency was classified as severe, moderate, or normal if enzyme activity was < %, - %, or > %, respectively [ ] . the adjusted median enzyme activity in males was determined as % activity of the study population [ ] . only patients with laboratory confirmed acute dengue infection were included in the statistical analysis. data were evaluated using descriptive statistics, medians (interquartile ranges) for continuous variables and frequencies and percentages for categorical variables. chi-squared test or fisher's exact test (for categorical variables) and mann-whitney u test (for continuous variables) were used for comparative analyses between severe and non-severe dengue infections and between patients with or without g pd deficiency. associations between g pd status and dengue severity were evaluated using the binary logistic regression test with % confidence interval. data were analysed using ibm spss statistics (ibm, armonk, ny, usa) data manager software package. in total, patients with clinically suspected dengue infection were screened by rdt. in our study, ( . %) patients were excluded ( non-dengue and with past dengue infection), and patients were enrolled in the study (defined as positive ns ag and/or igm result) as indicated on fig . out of enrolled patients, were excluded ( did not have glucose- -phosphate dehydrogenase deficiency on dengue infection in children sufficient blood volume, had a negative dengue result, and lacked quantitative g pd results). in total, patients with laboratory confirmed acute dengue infection and with quantitative g pd results were included in the analysis (fig ) . among the patients with a confirmed acute dengue infection, ( . %) were classified as severe dengue while ( . %) were classified as non-severe dengue. among the patients with non-severe dengue, ( . %) presented with warning signs and ( . %) were without. all patients with severe dengue presented with dengue shock syndrome (dss). among these dss patients, one patient also had fluid overload with respiratory distress, and one patient had severe bleeding that necessitated blood transfusion. ten out of fifty-one ( . %) of children with severe dengue were admitted to the icu, and required a blood transfusion. patients with severe dengue were significantly younger (p = . ) and were hospitalised for a longer time (p < . ) compared to non-severe dengue patients. the majority of patients were classified as secondary dengue ( . %, / ), and only . % ( / ) were classified as primary dengue. severe dengue was significantly higher in secondary dengue patients compared with the non-severe group (p = . ). nausea and vomiting were more frequent in patients with severe dengue (p = . ). however, a positive tourniquet test was less frequent in children with severe dengue (p = . ). other clinical warning signs, including tachycardia and hypotension, were significantly detected in patients with severe dengue. (p < . ). patients with severe dengue also had significantly higher haematocrit, higher leukocyte counts, and lower platelet counts than nonsevere dengue patients (table ) . the median g pd activity of dengue-confirmed patients was . u/g hb ( . - . ). the adjusted male median value of g pd activity was determined by excluding male samples with � % activity from the derived value [ ] . the adjusted male median was . u/g hb ( . - . ) ( table ). based on this value, g pd activity < . u/g hb was defined as class i, activity between . - . u/g hb was class ii, . - . u/g hb was class iii, . - . u/g hb was class iv, and > . u/g hb was class v for this population ( table ) . the prevalence of g pd phenotypic deficiency, based on these cut-off reference values revealed that / ( . %), including ( . %) males and ( . %) females were in class ii (severe g pd deficiency) and / ( . %), including ( . %) males and ( . %) females were in class iii (moderate g pd deficiency) ( table ) . real-time pcr for three known single nucleotide polymorphisms (snps) in the g pd gene in myanmar, mahidol, kaiping and mediterranean, was performed in of dengue-confirmed samples ( males and females). among them, / ( . %) had recognised snps, ( / ( . %) were males while / ( . %) were females). twenty-four patients had the mahidol mutation ( %), and one female ( %) had the kaiping mutation. the mediterranean genotype was not detected. samples from of / ( . %) ( / males, / females) patients with g pd deficient phenotypes were included in this genotypic study. among them, only / ( . %) had mutations, while / ( . %) ( male and female) did not have mutation. among the samples from patients without g pd phenotype deficient, / ( %) samples ( male and female) had mutations (table ) . among the patients with a g pd mutation, / ( . %) were classified as who class ii and among them / ( . %) were heterozygous male and one homozygous female. there was one male with the mahidol mutation in who class iv. this patient had a reticulocyte count < % while testing for g pd activity. females with genotypic mutations have varying degrees of the g pd deficient phenotype being classified as who class ii to who class iv (fig ) . severe dengue was not associated with a g pd deficiency phenotype nor genotype variants whether we used a cut off of < % (i.e. only including hemizygous males and homozygous females) or a cut off of < %, corresponding to classes i to iii of the who classification (table ) . severe dengue was diagnosed in / ( . %) children with < % enzyme activity and in / ( %) children with g pd levels > % (p = . ). in addition, / ( %) study participants had a g pd snp, and / ( %) did not have a detectable snp and severe dengue (p = . ). herein, we investigated if there is an association between dengue fever and g pd deficiency in children in myanmar. the majority of children in this study, . %, had secondary dengue infection possibly due to the four dengue serotypes circulating in this endemic area, which leads to subsequent infections [ ] . a quarter of the children in the present study had a severe dengue fever, all of whom presented with dss. the frequency of secondary dengue infection was significantly higher in the severe dengue than in the non-severe dengue group, which is consistent with a meta-analysis of studies in asia that described the association between dss and secondary dengue infection [ ] . no association between severity of dengue infection and g pd enzyme deficiency or g pd mutation was detected. the result of this study is consistent with two earlier studies conducted in thailand [ , ] . tanphaichitr and colleagues did not demonstrate a correlation between g pd deficiency and dengue severity in male paediatric patients (age range: - years) with dengue haemorrhagic fever age. in this study, out of patients had g pd deficiency [ ] . seridhoranakul et al. also did not demonstrate a significant relationship between g pd deficiency and dhf in a study with dhf patients, in which patients were g pddeficient, and controls, in which patients were g pd-deficient [ ] . tanphaichitr et al. reported a higher prevalence of g pd deficiency in male dhf patients, which was . % table . prevalence of g pd among confirmed dengue paediatric patients according to who classification. compared to % in bangkok, and suggested that g pd-deficient males may suffer more from dhf [ ] . the prevalence of g pd deficiency in paediatric patients with dengue infection in the present study was lower than the prevalence of g pd deficiency in children visiting the emergency department of yankin children hospital, ages: month to years old, who were screened for dengue via rdt ( . % vs. . %, respectively). in this study, g pd deficiency glucose- -phosphate dehydrogenase deficiency on dengue infection in children was diagnosed if enzyme activity was < % of the adjusted median value, which was slightly lower than the adjusted median value in our study ( . u/g hb vs. . u/g hb) [ ] . when comparing the results of these two studies, conducted in the same setting, g pd-deficient children may not be more susceptible to dengue infection compared to children with normal g pd activity. two in vitro studies demonstrated that human monocytes from g pd-deficient individuals had higher replication of dengue virus serotype [ , ] , and these studies suggested that the likelihood of severe dengue was likely to increase in g pd-deficient individuals. the findings from our study did not support this hypothesis. however, viral load was not assessed in our study, thus, the association of viral load or viral replication to clinical outcomes was beyond the scope of this study. the different dengue serotype also affects clinical outcomes. although dengue serotypes were not assessed in this study, previous studies described that the prevalent serotype among patients with dhf in myanmar in recent years was dengue serotype [ , ] . additionally, all four serotypes participated in the outbreak [ ] . the association between g pd deficiency, viral load and different dengue serotypes warrant future studies. more than different mutations in the g pd gene have been identified, and these variants have different impacts on enzyme activity [ ], which might result in different clinical manifestations. one study reported that patients with the g pd mediterranean mutation showed a more severe clinical course during bacterial infection compared to patients with wild type g pd [ ] . most patients with g pd mutation possessed the mahidol variant, which is consistent with previous studies in myanmar [ , ] . genotypic mutation was not detected in male and female patients with phenotypic deficiency and may have been due to the limited number of genotypes that we sought; previous research in this region has identified other uncommon g pd mutations for instance g pd union, coimbra and canton [ , , ] . bancone et al reported this molecular heterogeneity among this ethnicity along thai-myanmar border [ ] . six patients ( male and females) who had genotypic mutation had normal g pd activities; all had reticulocyte count < %, excluding a false negative phenotype due to haemolysis. g pd mahidol is considered a moderately severe variant with varying g pd activities which classifies it as who class ii/iii; almost all of male patients with mahidol variant had enzyme activities < % (i.e. were class ii). this finding is consistent with results of another study from the thai-myanmar border [ ] . a significant association between g pd mutation, in predominantly the mahidol variant, and dengue infection was not found. whether g pd genotypes other than mahidol could affect dengue severity cannot be excluded based on our current study. thus, the effect of other g pd gene mutations on dengue infection is unknown, and complementary studies should be performed in different populations. in this study, the demographic, clinical and haematological parameters of patients with severe and non-severe dengue were compared, and we found that young age is a risk factor significantly associated with severe dengue infection. data collected from to in myanmar also reported that the highest case fatality rate was seen in the infantile age group [ ] . the clinical parameters of warning signs were more frequent in severe dengue patients, which was in agreement with previous studies; hepatomegaly was a risk factor of dss or severe dengue infection [ ] [ ] [ ] [ ] [ ] [ ] , abdominal pain was a prognostic factor for severe dengue [ , ] , and lethargy was the best clinical sign to identify patients who may progress to severe dengue [ ] . although there were no patients with persistent vomiting, those who had a history of vomiting were frequently more present in the severe dengue patient group. other studies reported that persistent vomiting was one of the factors associated with dss or high mortality [ , ] . in the who classification for dengue, high haematocrit � % from baseline, concurrent with a rapidly decreasing platelet count, is listed as a warning sign [ ] . in this study, patients with severe dengue had significantly higher haematocrit and lower platelet count than those with non-severe dengue. the findings in this study also reinforced the applicability of the warning signs outlined in the who guidelines to detect severe dengue infection in children in myanmar. the strength of this study was its prospective design. all acute dengue cases were confirmed by real-time pcr for the virus and elisa assays, and g pd deficiency was confirmed by a gold standard quantitative spectrophotometric assay. although the cut-off level of g pd activity did not represent all children in myanmar, it did reflect the g pd activity of children with dengue infection seen at the yankin children hospital. there were limitations in our study. for example, dengue viral load and serotypes were not analysed; therefore, we were not able to detect the relationship among viral load, serotypes, dengue severity and g pd deficiency. additionally, g pd mutations were not assessed in all the participants, and the impact of rare g pd variants previously reported in myanmar population could not be analysed, which may have impacted our statistical analysis. the parameters, e.g., chest x-rays, liver function tests and ultrasounds of chest and abdomen that may affect dengue severity classification in terms of major organ involvement or evidence of plasma leakage were not collected, and these factors may have affected our study's outcomes. we did not find an association between g pd deficiency and dengue severity. most of our patients had g pd mahidol, the most common variant in myanmar. this study proposes activity cut-off points of enzyme activity according to the who classification, as well as the prevalence of g pd deficient and the predominant genetic mutation among paediatric patients. this study reconfirms the usefulness of the who dengue classification in paediatric dengue patients in myanmar. more research is needed to explore other factors related to dengue severity such as dengue virus viral load, dengue virus den serotypes and genotypes. supporting information s table. forward, reverse primers, snps and wt probes used in the study. 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syndrome in children. paediatr indonesia predictive factors of dengue shock syndrome at the children hospital no clinical and laboratory signs associated to serious dengue disease in hospitalized children we would like to acknowledge ms. seungyeon kwak and mr. byeon mun sub from korea university guro hospital and ampai tanganuchitcharnchai from moru for their assistance. we also thank ms. atthanee jeeyapant for her data analysis expertise and ms. gaye proctor from moru for english language editing. we are also grateful to dr. kyaw zin thant, director general, department of medical research, for his help and support throughout the study. key: cord- -n rgqyv authors: schuh, amy j.; amman, brian r.; sealy, tara s.; flietstra, timothy d.; guito, jonathan c.; nichol, stuart t.; towner, jonathan s. title: comparative analysis of serologic cross-reactivity using convalescent sera from filovirus-experimentally infected fruit bats date: - - journal: sci rep doi: . /s - - -z sha: doc_id: cord_uid: n rgqyv with the exception of reston and bombali viruses, the marburgviruses and ebolaviruses (family filoviridae) cause outbreaks of viral hemorrhagic fever in sub-saharan africa. the egyptian rousette bat (erb) is a natural reservoir host for the marburgviruses and evidence suggests that bats are also natural reservoirs for the ebolaviruses. although the search for the natural reservoirs of the ebolaviruses has largely involved serosurveillance of the bat population, there are no validated serological assays to screen bat sera for ebolavirus-specific igg antibodies. here, we generate filovirus-specific antisera by prime-boost immunization of groups of captive erbs with all seven known culturable filoviruses. after validating a system of filovirus-specific indirect elisas utilizing infectious-based virus antigens for detection of virus-specific igg antibodies from bat sera, we assess the level of serological cross-reactivity between the virus-specific antisera and heterologous filovirus antigens. this data is then used to generate a filovirus antibody fingerprint that can predict which of the filovirus species in the system is most antigenically similar to the species responsible for past infection. our filovirus igg indirect elisa system will be a critical tool for identifying bat species with high ebolavirus seroprevalence rates to target for longitudinal studies aimed at establishing natural reservoir host-ebolavirus relationships. unlike the spillover events leading to marburgvirus disease, those for ebolavirus disease have been difficult to associate with a specific environment, such as caves. furthermore, the evidence linking ebolavirus spillover to a particular species of bat is largely circumstantial. for example, the and index cases of sudan virus disease both worked in a cotton factory in sudan, where a retrospective investigation eight to nine months after the initial outbreak identified a large roof colony of trevor's free-tailed bats (mops trevori) directly over the working area of the index case . the putative index case of the ebola virus disease outbreak in the kasaï-occidental province of the drc was said to have purchased bats for consumption following a reported annual migration of hammer-headed bats (hypsignathus monstrosus) and franquet's epauletted fruit bats (epomops franquetti) . lastly, the presumed index case of the ebola virus disease outbreak that started in guinea was reported to have played in a tree hollow, where dna traces of mops condylurus were later identified . the majority of research directed towards the identification of the natural reservoir hosts of the ebolaviruses has consisted of cross-sectional surveillance of the sub-saharan african and asian bat population for evidence of active and past ebolavirus infection. the predominance of cross-sectional studies can be attributed to the large number of bat species residing within the geographic range of ebolavirus circulation that require investigation, as well as the challenges associated with obtaining diagnostic specimens from a mammal that is difficult to capture due to its elusive nocturnal nature and ability to fly. collectively, ebola virus rna has been detected in three bat species captured in the republic of the congo and gabon (hypsignathus monstrosus, epomops franquetti and myonycteris torquata) and reston virus rna has been reportedly detected in four bat species captured in the philippines (chaerephon plicatus, cynopterus brachyotis, miniopterus australis and miniopterus schreibersii) . yet, infectious virus was not isolated from any of these species , , indicating that they are either dead-end virus hosts, had cleared virus infection prior to sampling, or that current filovirus isolation techniques lack the sensitivity to recover infectious virus from specimens with low viral loads. serological reactivity of bat sera with ebolavirus antigens using indirect elisas, indirect fluorescent tests, bead-based multiplex assays and/or western blots has been reported in bats representing at least species throughout sub-saharan africa and asia [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, interpretation of this data has been exceedingly difficult due to multiple reasons that include: ( ) the absence of a panel of positive and negative bat sera for initial serological assay validation and continuing quality control, ( ) the use of various uncharacterized recombinant filovirus antigens, and ( ) the application of different statistical methods for establishing cutoff values for seropositivity. nonetheless, ebolavirus serosurveillance offers several distinct advantages compared to surveillance for active virus infection including a longer diagnostic window (i.e., length of time a diagnostic test can detect evidence of infection), no knowledge of virus-tissue tropism and no requirement for destructive sampling. although erbs are natural reservoir hosts of the marburgviruses only, they develop a robust virus-specific igg antibody response following experimental inoculation with the ebolaviruses , . in this study, we generate high quality, virus-specific antisera by prime-boost immunization of captive erbs with marburg virus, ravn virus, ebola virus, bundibugyo virus, taï forest virus, sudan virus or reston virus. these virus-specific antisera, along with a set of filovirus-naïve bat sera, are then used to validate a series of indirect elisas that utilize non-recombinant, infectious-based filovirus antigens for the detection of virus-specific igg antibodies from bats. we then assess the level of serological cross-reactivity between each virus-specific antiserum and viral antigen and use this information to generate a filovirus antibody fingerprint that is able to predict which of the six filovirus species in our system is most antigenically similar to the species responsible for past infection. filovirus-specific antisera generated from erbs. the bats used to generate the filovirus-specific antisera in this study originated from an erb breeding colony . a total of erbs were divided into seven groups (table ) (table ) , prepared in . ml of sterile dulbecco's modified eagle's medium (thermo fisher scientific inc., waltham, ma, usa). after eight weeks, the bat groups were subcutaneously challenged with log tcid of homologous virus. the bats were euthanized two to four weeks thereafter by cardiac exsanguination under isoflurane anesthesia, followed by an overdose of isoflurane. whole blood collected into serum separator tubes (fisher scientific, grand island, ny, usa) at euthanasia was allowed to clot and then centrifuged at , g for min. the sera were transferred into polypropylene cryoelite wheaton vials (dwk life sciences, millville, nj, usa), and then temporarily stored under liquid nitrogen vapors in the biosafety level four laboratory (bsl- ) until they were double bagged, passed through a dunk tank containing % micro-chem plus (national chemical www.nature.com/scientificreports www.nature.com/scientificreports/ laboratories incorporated, inc., philadelphia, pa, usa) and transported to the gamma-cell irradiator. the doublebagged serum aliquots were sandwiched between layers of dry ice and then exposed to × rads of gamma-cell irradiation using a cobalt- source. filovirus-naïve sera collected from erbs. sera obtained from filovirus-naïve bats from the breeding colony and negative control bats from previous experimental studies , [ ] [ ] [ ] were used as filovirus-naïve sera. these sera were collected and processed in the same manner as outlined above. husbandry. all bats were group-housed according to filovirus inoculum in a climate controlled bsl- animal area, with a h day/ h night cycle. bats received a quantity of fresh fruit, supplemented with protein/vitamin powder, equal to their body mass daily and water ad libitum. (table ) and uninfected control antigen lysate for the filovirus igg indirect elisas were prepared as previously described [ ] [ ] [ ] . briefly, roller bottles of vero e cells were inoculated with virus and the cultures were harvested when ≥ % of cells exhibited evidence of infection by immunofluorescence assay. filovirus antigen lysates were prepared from the cultures by detergent basic buffer extraction of infected cells. uninfected control antigen lysate was generated in the absence of virus, but otherwise prepared in the exact same manner. filovirus igg indirect elisas. each filovirus-specific antiserum (n = ) and filovirus-naïve serum (n = ) was tested for reactivity against each filovirus antigen lysate (n = ) and uninfected control antigen lysate (n = ; supplementary fig. s ). wells of -well elisa plates were coated ( µl) with the dilution of filovirus antigen lysate (diluent: pbs containing % thimerosal) that was found to result in optimal reactivity with sera pooled from each of the filovirus-infected bat groups ( : for the ebola, bundibugyo, taï forest and reston virus lysates, and : for the marburg, ravn and sudan virus lysates) and corresponding wells were coated with an equivalent dilution of uninfected control lysate ( : or : ). after incubation overnight at °c, the plates were washed with pbs containing . % tween- (pbs-t) and µl of serum diluent (pbs containing % skim milk and . % tween- ) was added to each well of the plate. after min, µl of a : dilution of gamma-irradiated bat serum pre-diluted in masterplate diluent (pbs containing % skim milk powder, . % tween- and % thimerosal) was added to the first well of the plate and four-fold serial dilutions were www.nature.com/scientificreports www.nature.com/scientificreports/ performed. final bat serum concentrations were : , : , : and : . following a hr incubation at °c, the plates were washed with pbs-t and µl of a : , dilution of goat anti-bat igg conjugated to horseradish peroxidase (bethyl laboratories, montgomery, tx, usa, cat#: a - p, lot#: a - p- ) in serum diluent was added to the plates. the manufacturer notes that this antibody reacts specifically with bat igg and with light chains common to other immunoglobulins. after incubation for hr at °c, the plates were washed with pbs-t, µl of the two-component abts peroxidase system (kpl, gaithersburg, md, usa) was added, and the plates were allowed to incubate for min at °c. the plates were then read on a microplate spectrophotometer set at nm. the optical density (od) values of each four-fold serial dilution were visually inspected to ensure linearity. to negate non-specific background reactivity, adjusted od values were calculated by subtracting the ods at each four-fold dilution of wells coated with uninfected control antigen lysate from ods at corresponding wells coated with filovirus antigen lysate. the adjusted sum od value was determined by summing the adjusted od values at each four-fold serial dilution. the average adjusted sum od of duplicate runs was reported. notably, performing each indirect elisa (seven filovirus antigen lysates and two dilutions of uninfected control antigen lysate) in duplicate required only µl of serum. influence of sex and age on the reactivity of antisera with homologous antigen. the breeding colony was founded from ugandan wild-caught, filovirus-naïve adult (≥ yr) erbs . for founder bats, age in years was determined by subtracting the date of capture from the date of euthanasia and then adding one year. for bats born in captivity, age in years was determined by subtracting the date of birth from the date of euthanasia. the bats were then classified into three age categories: < yr, ≥ yr-≤ yrs and > yrs. a two-way anova was performed to determine if serological reactivity of bat sera with homologous antigen was significantly influenced (p < . ) by sex, age category, or the interaction between sex and age category (spss statistics , ibm software, armonk, ny, usa). for each filovirus-specific igg indirect elisa, quadratic discriminant analysis was used to determine a cutoff value for seropositivity by considering: ( ) the reactivity of the filovirus-specific antisera group with homologous antigen, ( ) the reactivity of the filovirus-naïve sera group with that same antigen, and ( ) the prior probability of the filovirus-specific antisera group (vba for microsoft access , microsoft office professional plus , redmond, wa, usa). the prior probability of each filovirus-specific antisera group was set at . . this value represents the overall population seroprevalence of marburgvirus in its natural reservoir host, the erb . we assume that the seroprevalence of the ebolaviruses in their respective natural reservoir bat hosts approximates this value. however, the prior probability has relatively little influence on determining the magnitude of the cutoff value for seropositivity. the cutoff value for each filovirus-specific indirect elisa represents the value where the posterior probabilities for the reactivity of the filovirus-specific antisera with homologous antigen and the reactivity of filovirus-naïve sera with this same antigen are equal. sensitivity and specificity of the indirect elisas. for each indirect elisa at its specified seropositivity cutoff value, the filovirus-specific antiserum and filovirus-naïve serum samples were classified as true positive, false positive, false negative or true negative. the sensitivity of each assay was then calculated by dividing the number of individuals with a positive test result by the number of individuals with a history of past infection (experimentally inoculated with that particular virus), and the specificity of each assay was calculated by dividing the number of individuals without a history of past infection (filovirus-naïve group) by the number of individuals with a negative test result. level of serological cross-reactivity. the level of serological cross-reactivity between each group of filovirus-specific antisera and each heterologous virus antigen was calculated by dividing the number of antisera positive for reactivity with a virus antigen by the total number of antisera tested against that antigen (e.g., number of marburg virus antisera positive for reactivity with ravn virus antigen/number of marburg virus antisera tested for reactivity against ravn antigen), and was expressed as a percentage. after the nucleotide sequences of the filovirus isolates used to inoculate the bats in this study were retrieved from genbank, the coding regions were translated into amino acids and aligned using the muscle algorithm (geneious . . , biomatters limited, auckland, new zealand). percent identity values were then calculated from pairwise amino acid comparisons generated from the virus protein alignment. a pearson's product-moment correlation was performed to assess the relationship between percent serological cross-reactivity and percent amino acid identity (spss statistics , ibm software, armonk, ny, usa). antibody fingerprint analysis. quadratic discriminant analysis was used to predict which one of the seven filoviruses in the system was most antigenically similar to the filovirus responsible for past infection by considering the relative reactivity of each filovirus-specific antiserum and filovirus-naïve serum with each filovirus antigen, as well as their covariance (visual basic for microsoft access). first, prior probabilities were calculated for the eight classes used in the analysis (seven filovirus antigen classes and one negative class). second, classification was performed by calculating posterior probabilities for the inclusion of an antisera/sera sample in each class and then assigning the sample to the class with the highest probability. evaluating the performance and discriminatory ability of the filovirus igg indirect elisa system. to evaluate the ability of our system comprising seven filovirus-specific indirect elisas to predict the filovirus species most antigenically similar to the species responsible for past infection, we tested seven marburg virus convalescent serum or whole blood samples collected from experimentally inoculated erbs. five of these samples www.nature.com/scientificreports www.nature.com/scientificreports/ were collected four weeks post primary marburg virus inoculation , , while two of the samples were collected at and weeks post primary inoculation following a "natural" boost (i.e., marburg virus-specific antibody levels waned in these bats and then increased following contact with infectious cagemates) . serological reactivity with homologous filovirus antigen is not influenced by age or sex. a total of erbs were used in this study that was performed over a two-year time span. the total number of bats dedicated to this study, as well as the size and composition of each group was dependent on the reproductive capacity of the erb breeding colony, as well as the characteristics and number of bats needed for other experimental studies. the bats were divided into seven groups, with group sizes ranging from - individuals (table ) . two groups of bats had approximately equal sex ratios (ravn and taï forest), while two groups were comprised of mostly female individuals (bundibugyo and sudan), and the remaining five groups were comprised of all male (reston) or all female (marburg and ebola) individuals. two groups of bats were comprised solely of individuals < yr of age (bundibugyo and taï forest), while the majority of individuals in three groups were ≥ -≤ yrs of age (marburg, ebola and reston) and the majority of individuals in two groups were > yrs of age (ravn and sudan). despite the observed variability in group composition, a two-way anova revealed that serological reactivity with homologous filovirus antigen was not significantly (p > . ) influenced by sex (f( , ) = . ), age (f( , ) = . ), or the interaction between sex and age (f( , ) = . ). filovirus-specific indirect elisas are highly sensitive and specific. with the exception of the igg antibody indirect elisa using marburg virus antigen ( % sensitivity and % specificity; fig. a and table ), all of the assays exhibited % sensitivity and specificity ( fig. and table ). the mean group adjusted sum od values ranged from . (± . sd) for the igg antibody indirect elisa using marburg virus antigen to . (± . sd) for the igg antibody indirect elisa using ebola virus antigen. strong positive correlation between filovirus serological cross-reactivity and filovirus protein amino acid identity. figure and table show the level of serological cross-reactivity between each group of bat filovirus-specific antisera and the six heterologous filovirus antigens. the consistent magnitudes of serological reactivity of the individual bat antisera across all six heterologous filovirus antigens highlights the robust performance of our filovirus igg indirect elisa system. strong serological cross-reactivity was observed between marburg virus antisera and ravn virus antigen ( %), and ravn virus antisera and marburg virus antigen ( %) (fig. a,b) . (fig. e-g) . very limited cross-reactivity was observed between marburgvirus antisera and ebolavirus antigen ( %: majority of antisera-antigen combinations; %: marburg virus antisera versus taï forest virus antigen, ravn virus antisera versus bundibugyo and taï forest virus antigens; %: ravn virus antisera versus reston virus antigen), and ebolavirus antisera tested against marburgvirus antigen ( . %: sudan virus antisera versus marburg virus antigen; . % sudan virus antisera versus ravn virus antigen) (fig. ) . a pearson's product-moment correlation revealed that there was a statistically significant, strong positive correlation between percent filovirus serological cross-reactivity and percent filovirus amino acid identity (r = . , n = , p < . ). filovirus serological cross-reactivity dataset used to develop an antibody fingerprint classification system. we used the adjusted sum od data (each serum tested against each antigen) generated from bats intentionally infected with each of the seven known culturable filoviruses to develop a classification system (fingerprint) to predict which filovirus elicited the antibody response. supplementary table s shows posterior probability support values for classification of each filovirus-specific antiserum or filovirus-naïve serum sample into eight pre-defined classes (seven filovirus antigen classes and one negative class), with probabilities of each row in the table summing to . . each sample was assigned to the class with the highest posterior probability (i.e., highest degree of certainty). overall, all of the samples ( / ) were correctly classified, with posterior probabilities in support of the true class (i.e., filovirus used to prime-boost each bat) ranging from . to . . this indicates that this system, using seven independent filovirus igg indirect elisas, has the ability to establish an antibody fingerprint from filovirus convalescent bat sera that can be used to predict which of the filovirus species in the system is the most antigenically similar to the species responsible for past infection (see supplementary note for an example of how the antibody fingerprinting was performed in this study). to further assess the performance and predictive ability of our filovirus igg indirect elisa system, we tested five marburg virus convalescent sera or whole blood samples collected four weeks post primary marburg virus experimental inoculation and two whole blood samples collected at and weeks post primary experimental inoculation following a "natural" boost (i.e., marburg virus-specific antibody levels waned and then increased following contact with infectious cagemates). as expected, marburgvirus-specific igg antibody levels were markedly lower in these seven convalescent serum and whole blood samples compared to marburgvirus-specific igg antibody levels in the serum samples from the prime-boosted bats in this study (fig. a) . however, all samples collected after primary marburg virus inoculation or following a "natural" boost were classified as marburgvirus igg antibody positive. the antibody fingerprint analysis predicted that three of these bats were previously infected with marburg virus (sample ids , and with marburg posterior probability values of . , . , and . , respectively) and four were previously infected with ravn virus (sample ids , , and with ravn posterior probability values of . , . , . and . , respectively; fig. b ). www.nature.com/scientificreports www.nature.com/scientificreports/ in this study, we generated filovirus-specific antisera from prime-boosted erbs and collected filovirus-naïve sera to validate and characterize an indirect elisa system that utilizes non-recombinant, infectious-based filovirus antigens for the detection of virus-specific igg antibodies from bats. initial validation of the system revealed that the individual filovirus-specific igg indirect elisas exhibited high sensitivity ( - %) and specificity ( %). similar to previous studies using human sera, we observed limited to no serological igg cross-reactivity between the filovirus genera ( - %) [ ] [ ] [ ] , varying levels of serological igg cross-reactivity between the ebolavirus species table for statistics on the reactivity of each filovirus antigen lysate with homologous antisera and filovirus-naïve sera, as well as for the sensitivity and specificity of each of the filovirus igg indirect elisa. www.nature.com/scientificreports www.nature.com/scientificreports/ ( - %) , and strong serological igg cross-reactivity within the species marburg marburgvirus ( %). the low level of serological igg cross-reactivity between some of the ebolavirus species underscores the necessity of performing all assays within the system to avoid false negative results. although significant levels of serological igg cross-reactivity were observed between the prime-boost filovirus-specific antisera and some of the filovirus antigens, when the overall covariance of the seven-individual indirect elisas in the system were considered, we were able to predict the filovirus species responsible for past infection % of the time using as little as μl of sera (each serum was tested against each antigen in duplicate). further evaluation of the performance and predictive ability of our system using seven marburg virus convalescent serum or whole blood samples collected after primary experimental infection with marburg virus (or primary experimental infection with marburg virus plus a "natural" boost) confirmed that the system was able to predict the filovirus species most likely responsible for past infection. as expected, the filovirus igg indirect elisa system was not able to correctly predict whether past infection was due to marburg virus or ravn virus % of the time. this finding supports the current classification of marburg and ravn viruses into a single virus species, marburg marburgvirus . the robustness and discriminatory power of our system results from its underlying components and various control points. we employed infectious-based filovirus antigens, rather than recombinant virus protein antigens, as a strategy to increase both the sensitivity (i.e., ability of the filovirus igg indirect elisa system to correctly identify those with past exposure to a filovirus as filovirus igg antibody positive) and specificity (i.e., ability of the filovirus igg indirect elisa system to correctly identify those with no past exposure to a filovirus as filovirus igg antibody negative) of the system. although recombinant filovirus antigens can be generated in large quantities and do not require a bsl- laboratory for production, the use of single recombinant virus proteins for antibody detection can lead to false negative results if an individual's antibody repertoire is not directed against the particular recombinant virus protein that was employed , [ ] [ ] [ ] [ ] [ ] [ ] . alternatively, false positive serological results can occur when recombinant virus antigens unknowingly share similar epitopes with other virus antigens for which the study population possesses antibodies against , . false positive results can also arise from failing to account for non-specific serological reactivity resulting from the presence of non-virus contaminants in an antigen preparation. here, we negated non-specific serological reactivity to vero e cell components by subtracting the reactivity of wells coated with uninfected antigen lysate from the reactivity of corresponding wells coated with filovirus antigen lysate. while the majority of previously published ebolavirus serosurveys of bats used recombinant filovirus antigens generated in bacterial expression systems, not all of the studies implemented procedures to negate non-specific reactivity between residual bacterial epitopes in the antigen preparation and antibacterial antibodies in bat sera , . like the majority of published ebolavirus serosurveys of bats, we performed serial serum dilutions to assess for linearity and specificity , , , [ ] [ ] [ ] [ ] . most importantly, we included pooled filovirus-specific bat antisera as a positive control and pooled filovirus-naïve bat sera as a negative control in every run to ensure that our filovirus igg indirect elisa system continually performed as expected. using filovirus-specific antisera collected from bats that were prime-boosted at the same time not only allowed us to thoroughly examine the level of serological cross-reactivity between the virus-specific antisera and heterologous filovirus antigen, but also provided the opportunity to use this knowledge to evaluate previously published ebolavirus serosurveys of bats [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . four out of the previously published ebolavirus serosurveys of bats used ebola virus antigen only to detect ebolavirus igg antibodies , [ ] [ ] [ ] and another four of these serosurveys used ebola and reston antigens only to detect ebolavirus igg antibodies , [ ] [ ] [ ] . based on the low serological cross-reactivity between ebola virus antigen and sudan virus antisera ( . %) and reston virus antigen and sudan virus antisera ( . %) observed in our study, we predict that the serosurveys using ebola virus antigen or ebola and reston virus antigens only would have missed the opportunity to detect bats previously infected with sudan virus. likewise, due to the high level of serological cross reactivity between ebolavirus-specific antisera and heterologous ebolavirus antigen that we observed in our study, it is possible that the serological reactivity of bat sera with ebola or reston virus antigens in the previous serosurveys was due to past infection with one of the other known ebolaviruses or an undescribed ebolavirus. although we successfully used quadratic discriminant analysis to predict which of the filovirus species in the system was most antigenically similar to the species responsible for past infection, only nucleotide sequence data can be used to ascertain infection with a particular filovirus. furthermore, incrimination of an ebolavirus-natural reservoir host relationship requires consistent detection of both active (rna and virus isolation from tissues/bodily fluids suggestive of a transmission mechanism) and past infection with a particular ebolavirus through space and time. www.nature.com/scientificreports www.nature.com/scientificreports/ while our filovirus igg indirect elisa system enables the robust detection of virus-specific igg antibodies from bats and was able to predict the filovirus species most likely responsible for past infection, it does have some limitations. first, this system was validated using virus-specific antisera from prime-boosted bats and sera from table for statistics on the cross-reactivity between each filovirus-specific bat antisera and heterologous filovirus antigen. www.nature.com/scientificreports www.nature.com/scientificreports/ filovirus-naïve bats. although all seven of the marburg virus convalescent whole blood and serum samples collected from erbs after primary experimental marburg virus inoculation or following a "natural" boost were marburgvirus igg antibody positive, these samples had markedly lower marburgvirus-specific igg antibody levels compared to samples collected from the bats in this study that were prime-boosted with one of seven filoviruses. this suggests that the sensitivity of the individual filovirus igg indirect elisas may be lower than what was reported here ( - %) using filovirus-specific antisera from prime-boosted bats and filovirus naïve bat sera. however, statistical determination of seropositivity cutoff values for the indirect elisas were largely driven by the variance surrounding the mean reactivity of the filovirus-naïve sera with each of the virus antigens. it is likely that circulation and long-term maintenance of the ebolaviruses in their natural reservoir hosts leads to multiple virus exposures throughout the lifetime of an individual, resulting in boosting of virus-specific igg antibody levels. "natural" boosting of virus-specific igg antibody levels in erbs that had been previously infected with marburg virus was observed during a recent experimental transmission study shortly after documenting virus infection and seroconversion in naïve contact bats . others have reported periodic boosting of rabies virus neutralizing antibody titers in a wild colony of big brown bats (eptesicus fuscus) . second, we used a goat anti-bat igg conjugated to horseradish peroxidase as the secondary antibody in our system. this antibody was generated using www.nature.com/scientificreports www.nature.com/scientificreports/ sera from erbs, as well as sera from nine other bat species comprising four chiropteran families (manufacturer product datasheet). while this antibody has been confirmed to react with sera from these chiropteran species and has been used with success to detect igg antibodies in sera from > bat species comprising seven chiropteran families [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , it is possible that its reactivity with igg antibodies from divergent bat species might diminish. if this is the case, the concentration of secondary antibody may need to be optimized prior to testing a new species of bat or an alternative secondary antibody may need to be used. third, our filovirus igg indirect elisa system includes seven virus antigens that represent all of the reported culturable filoviruses. given the recent discoveries of filovirus rna from divergent bat families in geographically distant locations ( ), we expect that there are a number of undiscovered ebolaviruses that continue to circulate in nature. however, we believe that the genetic diversity of ebolavirus antigens (up to . %) included in our system will allow for the detection of past infection with a novel ebolavirus, and prediction of the ebolavirus species that is most antigenically similar to the species responsible for past infection. we intend to use our filovirus igg indirect elisa system to screen archived and incoming specimens from cross-sectional serosurveys of the bat population of sub-saharan africa and asia for evidence of past filovirus infection. if other specimen types exist for bat species identified as filovirus seropositive, they will be tested for evidence of active infection using virus-specific qrt-pcr, pan-filovirus rt-pcr and/or virus isolation techniques. bat species positive for evidence of active filovirus infection or species exhibiting a seroprevalence equivalent to or larger than that of marburgvirus in erbs (~ % total population seroprevalence) will be targeted for longitudinal studies aimed at collecting a wide range of specimens for evidence of active and past ebolavirus infection. while most of the virus-specific antisera generated in this study will be pooled to use as positive controls in our indirect filovirus elisa system, some will be reserved for investigations aimed at determining the mechanisms by which bats clear and control filovirus infections through virus neutralization and cross-neutralization assays, as well as epitope mapping studies. any remaining filovirus-specific bat antisera may be used to develop a pan-ebolavirus indirect elisa that utilizes a mixture of non-recombinant, infectious based antigens from culturable ebolaviruses, as well as validate the performance of indirect elisas that use recombinant filovirus proteins to detect filovirus antibodies. ecology of filoviruses taxonomy of the order mononegavirales: second update epidemiology of ebola (subtype reston) virus in the philippines the discovery of bombali virus adds further support for bats as hosts of ebolaviruses field aspects of the marburg virus outbreak: . primate supply marburg-virus disease in kenya report of a who/international study team. ebola haemorrhagic fever in sudan report of an international commission. ebola haemorrhagic fever in zaire human infection due to ebola virus, subtype cote d'ivoire: clinical and biologic presentation 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worldwide carry hepatitis e virus-related viruses that form a putative novel genus within the family. hepeviridae hibernating little brown myotis (myotis lucifugus) show variable immunological responses to white-nose syndrome isolation and characterization of a novel alphaherpesvirus in fruit bats a novel rhabdovirus isolated from the straw-colored fruit bat eidolon helvum, with signs of antibodies in swine and humans evidence of hantavirus infection among bats in brazil identification of secreted and membrane-bound bat immunoglobulin using a microchiropteran-specific mouse monoclonal antibody antibodies against henipa-like viruses in brazilian bats. vector borne zoonotic dis discovery of an ebolavirus-like filovirus in europe re-emergence of lloviu virus in miniopterus schreibersii bats characterization of a filovirus (měnglà virus) from rousettus bats in china the authors declare that all data supporting the findings of this study are available within the article or from the corresponding author upon request. we thank the diagnostics team from the viral special pathogens branch at the centers for disease control and prevention (cdc) for preparing the filovirus antigen lysates used in this study. we would also like to thank peter eworonsky, lester slough and eddie jackson from the comparative medicine branch at the cdc for providing care to the bats during this study. this study was partially funded by dtra grant hdtra- - - , subaward s- - . the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention. supplementary information accompanies this paper at https://doi.org/ . /s - - -z. publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - v qvhg authors: johansson, michael a.; reich, nicholas g.; meyers, lauren ancel; lipsitch, marc title: preprints: an underutilized mechanism to accelerate outbreak science date: - - journal: plos med doi: . /journal.pmed. sha: doc_id: cord_uid: v qvhg in an essay, michael johansson and colleagues advocate the posting of research studies addressing infectious disease outbreaks as preprints. • despite the advantages of preprints and the endorsement of journals and funders in the context of outbreaks, less than % of ebola and zika journal articles were posted as preprints prior to publication in journals. • with broader adoption by scientists, journals, and funding agencies, preprints can complement peer-reviewed publication and ensure the early, open, and transparent dissemination of science relevant to the prevention and control of disease outbreaks. emerging public health threats, such as infectious disease outbreaks, require swift and evidence-based responses informed by science. however, the communication of scientific research is notoriously slow [ ] . when rapid dissemination of new information has the chance to prevent or control epidemics affecting hundreds or thousands of people, we must fast-track this process. preprints (manuscripts posted publicly prior to peer review) have been endorsed as a solution to this challenge, yet adoption remains very low and needs to be improved. on february , , more than of the world's largest and most prestigious public health journals and funding agencies issued a landmark statement on the importance of preprints and data sharing in public health emergencies such as the ebola and zika epidemics [ ] . journal signatories pledged to ( ) make related scientific content freely accessible, and ( ) allow data and preprint manuscripts to be shared prior to publication. here, we focus on preprints, which may include both new data and new analyses and offer an opportunity to speed and democratize further scientific analyses and the availability of evidence to inform outbreak responses. a a a a a the statement coincided with a proliferation of zika research in response to the epidemic in the americas (fig a) . between november and august , we identified a total of preprint manuscripts with zika in the title or abstract in recognized public preprint repositories, including general repositories: biorxiv ( , http://www.biorxiv.org/), arxiv ( , arxiv.org), f research ( , with other that was originally posted in biorxiv, f research.com), peerj preprints ( , peerj.com/preprints/), and the world health organization zika open repository ( , www.who.int/bulletin/online_first/zika_open/), which was established explicitly for the zika epidemic (s dataset). these likely represent the majority of zika preprints, though others may have been posted in ad hoc, lesser-known, or laboratory-or university-specific webpages or repositories. over a similar time period in the ebola outbreak (may to january ), there were ebola preprints ( fig b) . there were many more publications in peer-reviewed journals over these time periods: , and , publications indexed in pubmed (http://www.ncbi.nlm.nih.gov/pubmed, s dataset) with ebola or zika, respectively, in the title or abstract and of type "journal article" ( and additional publications were classified as letters, reviews, etc., respectively). this increase in publications ( %) cannot explain the increase in preprints alone ( %). over the same time period, there was a general increase in preprints for health sciences [ ] ; for example, biorxiv submissions with the word "outbreak" rose approximately -fold between ( ) and ( ). thus, the increase in preprints related to the zika epidemic likely reflects this general trend, some increase in research, and possibly the influence of the statement on data sharing. to assess changes in preprint posting during the epidemics, we examined the subset of all preprints that could be matched with an eventual publication. for ebola, we matched ( %) of the preprints to pubmed-indexed journal articles, and for zika, ( %). four additional zika preprints were matched to "review" ( ) or "comparative study" ( ) publications in pubmed. unmatched preprints may be preprints that were never submitted for peerreviewed publication (e.g., opinions or preliminary work that was abandoned), were never accepted for publication, or were still in peer review at the end of the selected time periods. preprints were increasingly used to disseminate new data during the zika outbreak. among the subset of preprints matched to journal articles described above, the proportion of preprints including original data increased substantially from % for ebola to % for zika (difference: %, % confidence interval [ci] % to %, -sample test of proportions). while a minority of preprints contained new data, the majority of preprints in both outbreaks included novel analyses, % for ebola and % for zika (estimated difference: %, % ci − % to %). the remainder were comprised of opinions, proposals for new lines of study, or research indirectly related to ebola or zika. the increase in data sharing between epidemics appears to represent a shift away from waiting for peer review and towards rapidly reported, open science. this shift should enable other researchers to build upon those findings and recognize the high value of data sharing (academic and otherwise), a common challenge in the midst of outbreaks. preprint posting also led to earlier access to those data and analyses. excluding manuscripts in f research, which uses the preprint posting date as the publication date, the median delay between preprint posting and publication was approximately days for both outbreaks, and less than % of the manuscripts were published within days of posting. notably, this delay, which may include submission to multiple journals, is quite similar to normal publication timelines [ ] . it is unclear to what extent journals are able to accelerate publication in outbreaks, but it is clear that every time there is an editorial or peer review decision, rejection, or revision there are delays, and that preprint posting precludes delays in broad access to the information. the successes of preprints in the ebola and zika epidemics belie a more complex story about preprint use during these outbreaks. while the number of preprints increased, data sharing was more common, and scientific findings were available earlier, adoption remained extremely low. publications with preprints represented a small minority of all pubmedindexed journal articles, at approximately . %. the proportion was slightly higher for zika compared to ebola ( . % versus . %), likely indicating a small increase between outbreaks (estimated difference: . %, % ci − . % to . %). among signatory publishers, who represented approximately % and % of the publications for ebola and zika, respectively, this proportion was approximately . % ( % ci . % to . %) higher than for nonsignatory publishers ( . % versus . %) (fig c and d ). this suggests that these publishers may have policies that are supportive of preprints irrespective of the statement [ ] . although preprint adoption in both outbreaks was very low, important advances were clear. first, the relatively higher preprint usage for signatory publishers both before and after the statement indicates that policy supportive of preprints can encourage their use. second, the number of preprints posted increased between the outbreaks, likely reflecting changing attitudes towards preprints in the life sciences and changing policies, including the data sharing statement [ ] [ ] [ ] . third, preprints generally contained new analyses and increasingly shared novel data. fourth, with preprints available months before peer-reviewed publications, preprint posting can accelerate the sharing of research. preprints also bring new challenges to outbreak responses [ ] . by definition, preprints are not peer reviewed prior to posting. while preprint posting is common practice in fields such as physics and statistics, it is a new concept to many scientists in public health and even more so to public health officials, the press, and the public, all of whom may be seeking the latest information during epidemics. until preprints are broadly recognized as pre-peer review manuscripts, they may be misinterpreted as peer-reviewed research. on the other hand, peer review faces its own challenges of subjectivity, bias, transparency, and speed [ , , ] . peer review is an integral component of scientific communication, but it does not intrinsically guarantee the quality of science. moreover, peer review is particularly challenging during major outbreaks when the most qualified reviewers are also immersed in urgent research. preprint posting may help mediate this process, providing an opportunity for broad and immediate community input that is not subject to the limits of traditional peer review [ ] [ ] [ ] . assuring ethical review, participant confidentiality, recognition of preprints as pre-peer review manuscripts, and finding mechanisms to enable transparent, open feedback will be essential to limiting possible negative impacts and maximizing the benefits of preprints for outbreak responses. immediate, open access to research prior to peer review raises the possibility of misinterpretation and the misuse of science in critical decision making when lives are at stake, but it also permits early and open criticism, discussion, and consideration of findings that may save lives. further adoption of preprints also requires changes in how funders, scientists, and publishers value scientific contributions. all stakeholders agree that the best science should be brought to bear against outbreaks, yet they are also keenly aware of the importance of peer review and the scientific accolades that come with publishing novel, impactful research in prestigious journals. in the context of outbreaks, the goal of impacting the epidemic by immediately sharing important scientific findings conflicts with career goals tied to the slower, traditional, peer review-centered publication process. the low adoption of preprints during the ebola and zika epidemics is a symptom of uncertainty about preprints and this conflict of incentives. the signatory funders and publishers clearly endorsed preprints, but the majority of scientists did not realize that they could or should post preprints or thought that posting a preprint would jeopardize publication opportunities. we advocate steps to improve the adoption of preprints and speed the dissemination of science in the context of outbreaks. first, scientists should promote preprints by posting them and choosing to submit manuscripts to journals that accept preprints; many journals have policies that explicitly allow preprint posting (tools for identifying these journals are provided by jisc: http://www.sherpa.ac.uk/romeo/search.php and wikipedia: https://en.wikipedia.org/ wiki/list_of_academic_journals_by_preprint_policy). second, all publishers should endorse preprint posting for research related to outbreaks. this would send a clear signal to all scientists that preprints are integral to scientific communication and help nonscientists identify preprints as a distinct form of communication compared to peer-reviewed publications [ , [ ] [ ] [ ] . publishers should actively encourage or require preprint posting at the time of submission, driving adoption directly as faculty of has for f research. third, preprint repositories and the scientific community should ensure that preprints contain appropriate content (e.g., maintaining ethics and privacy) and are readily identifiable as preprints, and that mechanisms exist to facilitate community input prior to or concurrent with peer review. these considerations can help reduce the risks of preprints and maximize their benefits. fourth, universities and funders should recognize preprints together with peer-reviewed publications and citations as an important part of an investigator's track record, especially for any scientist involved in outbreak responses. the scientific community should not ask why preprints are posted during outbreaks, we should ask why they are not posted and make early posting the standard rather than the exception. preprints offer numerous challenges and opportunities for science in general but represent a particularly important opportunity to accelerate the dissemination of science in the midst of infectious disease outbreaks, when early actions are critical and evidence is scarce [ , ] . despite this need and the statement on preprints and data sharing, less than % of ebola and zika journal articles were posted as preprints prior to publication in journals. this low adoption reflects an intrinsic and established, yet unnecessary, prioritization of the traditional publication process over the dissemination of science. further progress is essential to ensure that science can be rapidly and broadly disseminated in the context of outbreaks. it is incumbent upon scientists, publishers, and funders alike to recognize the value of preprints and embrace them as a critical component of outbreak science. supporting information s dataset. data on preprints included in the analysis. "preprint_date" is the initial preprint submission date from the respective repository. "pmid" is the pubmed identification number the preprint was matched to (if matched). "journal" and "pub_type" are from the pubmed entry. "publisher" indicates the publisher if the publisher signed the data sharing statement. "new_data" and "new_analysis" indicate whether the authors judged the manuscript to contain new data or analysis, respectively ( = true). (csv) s dataset. data on pubmed journal articles included in the analysis. "pub_date" is the journal publication date as indicated by pubmed or the entry creation date if a specific journal publication date was not supplied. "pmid" and "journal" are the pubmed identification number and journal indicated by pubmed for the article. "publisher" indicates the publisher if the publisher signed the data sharing statement. (csv) does it take too long to publish research? nature sharing data during zika and other global health emergencies the preprint dilemma time for a prepublication culture in clinical research? accelerating scientific publication in biology ten simple rules to consider regarding preprint submission peer review: a flawed process at the heart of science and journals estimating the reproducibility of psychological science improving the evidence base for decision making during a pandemic: the example of influenza a/h n middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility key: cord- - ereip x authors: yoon, sun-woo; wong, sook-san; zhu, huachen; chen, rirong; li, long; zhang, yu; guan, yi; webby, richard j title: dysregulated t-helper type (th ):th cytokine profile and poor immune response in pregnant ferrets infected with pandemic influenza a(h n ) virus date: - - journal: j infect dis doi: . /infdis/jix sha: doc_id: cord_uid: ereip x pregnancy has been associated with severe influenza, an association highlighted during the pandemic of influenza a(h n ) virus (a[h n ]pdm ) infection. to assess the underlying mechanism, we infected pregnant and non-pregnant ferrets with a(h n ) pdm virus. a(h n )pdm -infected pregnant ferrets also had higher levels of inflammatory cytokines in their pulmonary tracts. systemically, total cd (+) t cell counts and a(h n )pdm -specific b-cell responses in blood were significantly lower in pregnant ferrets. this model predicts that the poorer outcome for pregnant women during the a(h n )pdm pandemic was due to an elevated level of viral replication and to a cytokine imbalance that led to a less effective immune response. pregnancy has been associated with severe influenza, an association highlighted during the pandemic of influenza a(h n ) virus (a[h n ]pdm ) infection. to assess the underlying mechanism, we infected pregnant and non-pregnant ferrets with a(h n ) pdm virus. a(h n )pdm -infected pregnant ferrets also had higher levels of inflammatory cytokines in their pulmonary tracts. systemically, total cd + t cell counts and a(h n )pdm -specific b-cell responses in blood were significantly lower in pregnant ferrets. this model predicts that the poorer outcome for pregnant women during the a(h n )pdm pandemic was due to an elevated level of viral replication and to a cytokine imbalance that led to a less effective immune response. keywords. pregnancy; pandemic influenza a virus; immune response. pregnant women are at risk of increased mortality and morbidity due to influenza virus infection [ ] . during the pandemic of influenza a(h n ) virus (a[h n ]pdm ) infection, pregnancy was associated with an increased risk of hospitalization [ ] , disease susceptibility [ ] , and influenza-related death in the united states. in particular, women in their third trimester were especially vulnerable. several studies have shown that pregnant women also had increased mortality and hospitalization rates during previous influenza pandemics: the [ ] . in these cases, higher risk was not restricted to infection during the third trimester, and virus exposure during early gestation was also associated with adverse outcome for the fetus [ , ] . pregnancy is considered to be a state of immune suppression, with cytokine profiles biased toward a t-helper type (th )-type response [ ] . peripheral blood mononuclear cells (pbmcs) isolated from pregnant women responded less robustly to innate immune stimulation than did those isolated from nonpregnant women. although pregnant women are known to be a high-risk group for influenza virus infections, little is known about the underlying mechanisms. while some studies have assessed the impact of pregnancy during influenza virus infection in mice, studies in more-relevant animal models are warranted. we therefore developed a pregnant ferret model and analyzed the pathologic and immunologic changes following infection with a(h n )pdm . all experiments were approved by the shantou university medical college (reference number: sumc - ) in compliance with their policies for animal ethics and welfare and use of animals in research, the guidelines of the world health organization, and the international council for laboratory animal science. eighteen-to -month-old ferrets (mustela putorius furo) were obtained from wuxi sangosho co. ltd. (jiangsu province, china). in this study, we used pregnant ferrets ( infected with a[h n ]pdm and uninfected) and nonpregnant ferrets ( infected with a[h n ]pdm and uninfected). all ferrets were serologically negative for a(h n ) pdm and seasonal influenza a(h n ) virus as determined by hemagglutination inhibition assays. on day after mating, ferrets were infected intranasally with μl of % tissue culture infective doses (tcid )/ml of the a(h n )pdm virus a/california/ / (ca ), each diluted in phosphate-buffered saline (ph . ), or received μl of phosphate-buffered saline alone, after sedation with isoflurane. all inoculated animals were monitored daily. for viral titration, nasal washes from each ferret were collected on days , , , , , , and after inoculation, and organs were collected on days after inoculation ( or ferrets per group). all virus titers were expressed as tcid in madin-darby canine kidney cells. for histopathologic analysis, tissue specimens were processed for hematoxylin and eosin staining and stained with a mouse anti-nucleoprotein (np) monoclonal antibody for analysis of pbmc responses, cells were collected on days after inoculation and isolated by density centrifugation by using histopaque- medium (sigma, ca) according to the manufacturer's instructions. for quantification of peripheral cd + cells, pbmcs were stained with allophycocyanin-labeled cd antibody (ebioscience, ca) and data acquired on a bd facsaria ii (bd biosciences, nj). all samples were analyzed by flowjo software (tree star, or). to measure the influenza virus-specific b-cell response, -well multiscreen ha plates (millipore, ma) were coated with µg of recombinant hemagglutinin (ha) from a(h n )pdm (sino biological, china), and cells were plated and incubated at °c for hours. after binding, cells were stained with alkaline phosphatase-conjugated goat anti-ferret immunoglobulin g antibody (southern biotechnology, al) and incubated overnight at °c. spots were visualized by colorimetric analysis, using the substrate -bromo- -chloro- -indolyl phosphate/nitroblue tetrazolium (bcip/nbt, kpl, md), and positive spots were counted by using a ks enzyme-linked immunospot (elispot) system (carl zeiss). cytokine and chemokine expression levels were measured using quantitative real-time polymerase chain reaction analysis (roche, ca). to determine whether pregnancy in ferrets results in enhanced influenza severity, as seen in humans, we examined the virologic and pathologic changes following ca infection. owing to the significant weight gain in pregnant ferrets, weight loss, which is typically used as a clinical marker of disease in ferrets, was not a feasible measure for this study. upon infection, we observed that there were no significant differences in the temperature changes between pregnant and nonpregnant groups (p = . ; data not shown). although our study was not designed to specifically measure this outcome, we noticed differences in the number of newborns between uninfected and infected pregnant ferrets by the end of the gestational period. two newborns were produced by the noninfected pregnant ferrets, but none were produced by the infected pregnant ferrets. to determine the pattern of virus shedding in pregnant and nonpregnant animals, we collected nasal wash samples every other day. viruses were detected in the nasal wash specimens from all inoculated ferrets, with significantly higher titers in pregnant ferrets (p < . ) that were maintained for longer periods ( figure a ). by day , all animals fully recovered, and virus was cleared in all infected ferrets. to evaluate the histopathologic changes upon ca infection, tissue samples were collected days after inoculation from pregnant and nonpregnant ferrets. viruses were detected in turbinate, tracheal, and lung tissues from all animals, with significantly higher titers (p < . ) seen in the pregnant group (mean titers ranged from . to . log tcid /ml; figure b ). viral np staining confirmed virus replication in lungs (figure dc and dd) and tracheas (figure dg and dh) of inoculated ferrets. no evidence of viral infection was observed in nonpulmonary tissues ( figure b) , including the transplacental tissues, suggesting a general lack of systemic spread of viruses in ferrets and no evidence of intrauterine infection. pathologically, a difference in lung damage was observed between pregnant and nonpregnant groups. this was characterized by more-pronounced alveolar damage, widespread edema in the lungs (figure cc and cd), and severe degeneration of epithelial cell walls in trachea (figure cg and ch) of the pregnant animals. the numbers of viral np-positive cells were also higher in pulmonary tissues of pregnant ferrets (figure dd and dh) than in those of nonpregnant ferrets (figure dc and dg) . no evidence of np positivity or tissue damage was observed in the control mock-infected ferrets (figure ca, cb, ce , and cf and figure da, db, de, and df) . overall, these results showed that during pregnancy, both virus replication and subsequent tissue damage are elevated following influenza virus infection during pregnancy. influenza virus infection in pregnant women is associated with increased levels of respiratory tract inflammatory cytokines and chemokines [ ] . to determine whether a similar phenotype is present in pregnant ferrets, we measured the expression levels of inflammatory cytokines and chemokines by quantitative real-time polymerase chain reaction in the trachea (representative of the upper respiratory tract), lungs (representative of the lower respiratory tract), and bronchoalveolar lavage to evaluate responses in resident or infiltrating immune cells lining the airways [ ] of pregnant and nonpregnant animals following infection. in general, although not exclusively, when differences in the levels of cytokines and chemokines were noted, levels were higher in pregnant animals, consistent with observations in humans. in lung tissues, levels of interferon α (ifn-α) and interleukin (il- ) were significantly higher in pregnant animals; levels of interleukin (il- ) and interleukin p (il- p ) were higher in nonpregnant animals. in tracheal tissues, messenger rna (mrna) levels of tumor necrosis factor α, ifn-γ, and interleukin (il- ) were higher in pregnant animals. in bronchoalveolar lavage, pregnant animals had higher mrna levels for ifn-α, ifn-γ, il- , and il- p . in pbmcs, nonpregnant animals had higher levels of il- and il- p (figure a) . in pregnant mice, influenza virus infection has been associated with alterations in the immune response [ , ] . correspondingly, we assessed the numbers of cd + t lymphocytes and influenza virus-specific b cells in total pbmcs days after inoculation in our infected ferrets. in a(h n )pdm -infected pregnant ferrets, the percentage of cd + t lymphocytes among pbmcs was significantly lower than in nonpregnant ferrets (p < . ; figure b ). elispot measurements of a(h n ) pdm -specific b cells also showed reduced numbers in the pregnant ferrets (p < . ; figure c ). together, these results suggest that both innate and adaptive immune responses are impaired in pregnant ferrets during influenza virus infection. although pregnancy is associated with increased susceptibility to pathogens owing to immunologic suppression, it is not entirely clear how this manifests as increased severity after influenza virus infection. while informative studies have been conducted in mice, the nature of influenza virus-mediated disease in this host is distinctly different than what is seen in humans; we thus turned to the ferret model. the pregnant ferret model has been used since the s to study the teratogenic potential of influenza viruses [ ] [ ] [ ] . these studies showed that fetal infection and pathology were only possible through virus inoculation directly into the amniotic space or maternal bloodstream, leading the authors to suggest that the teratogenic risk to the fetus due to natural influenza virus infection was low. however, these studies did not focus on the virologic or immunologic outcome of infection in the pregnant ferret itself. thus, our study aimed to address this knowledge gap. in our study, we infected the ferrets on day after mating, which, in the ferret's gestational cycle, loosely corresponds to early stages of human pregnancy. the cytokine profile in the infected ferrets was consistent with that observed in infected humans, notably the elevation levels of ifn-α, il- , and ifn-γ [ ] . this suggests that the immune response in infected ferrets to some extent mimics the response in humans [ ] . when we compared the cytokine mrna levels between pregnant and nonpregnant ferrets, however, there were striking differences in responses of several key cytokines in the trachea, lung, and bronchoalveolar lavage. stronger proinflammatory responses were observed in the trachea (ie, upper respiratory tract) of pregnant ferrets, which also had extended and higher levels of virus shedding as compared to nonpregnant ferrets. interestingly, levels of il- and tnf-α in the upper respiratory tract of influenza virus-infected humans were correlated with virus shedding [ ] . in contrast, mrna levels of the antiinflammatory cytokine il- were increased in the lungs of nonpregnant ferrets. this suggests that nonpregnant ferrets may have more capacity to regulate excessive inflammatory responses and tissue repair in the lungs, compared with pregnant ferrets, as was previously shown in the murine model [ ] . however, as was shown in same study, cytokine levels may change over time, and because of limitations of the ferret model, our results may only represent a snapshot of these changes. severe cases of influenza in pregnant women were also associated with decreased levels of immunoglobulin in sera [ ] and dysregulated t lymphocytes [ ] . consistent with these observations, we found that the numbers of a(h n )pdm -specific b cells and cd + t cells in peripheral blood in pregnant ferrets were lower than those in nonpregnant ferrets. pbmcs from pregnant ferrets produced less il- and il- p than those from nonpregnant ferrets after a(h n )pdm infection, which could account for the decreased number of influenza virus-specific b cells and cd + t cells. unlike the murine model, one major limitation of our ferret model was the lack of reagents for detailed immunologic analyses. for example, the cytokine responses could only be profiled at the mrna level and not at the protein level. at the time of the experiments, no suitable assay for the assessment of ferret-specific cd + t-cell function and specificity was available. for the same reason, we were also unable to assess the cellular innate responses or the cd + t-cell responses. these are also critical immune regulators that can modulate disease severity during influenza virus infection. our findings here will have to be interpreted with these caveats in mind. in our combined virologic and immunologic analyses, we showed that a(h n )pdm infection during pregnancy induces more-severe disease as a consequence of elevated viral loads and innate responses, combined with a diminished adaptive response. these factors likely contributed to the increased morbidity and mortality rates observed during the influenza pandemic in pregnant women. novel influenza a(h n ) pregnancy working group. h n influenza virus infection during pregnancy in the usa influenza and congenital anomalies: a systematic review and meta-analysis california pandemic (h n ) working group. severe h n influenza in pregnant and postpartum women in california influenza in pregnancy: the case for prevention the th :th dichotomy of pregnancy and preterm labour bronchoalveolar lavage constituents in healthy individuals, idiopathic pulmonary fibrosis, and selected comparison groups. the bal cooperative group steering committee wild type and mutant pandemic influenza a (h n ) viruses cause more severe disease and higher mortality in pregnant balb/c mice fatal outcome of pandemic h n influenza virus infection is associated with immunopathology and impaired lung repair, not enhanced viral burden, in pregnant mice the effects of maternal influenzal viraemia in late gestation on the conceptus of the pregnant ferret association of foetal wastage with influenza infection during ferret pregnancy the pregnant ferret as a model for studying the congenital effects of influenza virus infection in utero: infection of foetal tissues in organ culture and in vivo local and systemic cytokine responses during experimental human influenza a virus infection. relation to symptom formation and host defense local innate immune responses and influenza virus transmission and virulence in ferrets association between severe pandemic influenza a (h n ) virus infection and immunoglobulin g( ) subclass deficiency regulatory t cells mediate maternal tolerance to the fetus key: cord- -k puoq authors: azadeh, natalya; sakata, kenneth k.; saeed, ali; mullon, john j.; grys, thomas e.; limper, andrew h.; binnicker, matthew j. title: comparison of respiratory pathogen detection in upper versus lower respiratory tract samples using the biofire filmarray respiratory panel in the immunocompromised host date: - - journal: can respir j doi: . / / sha: doc_id: cord_uid: k puoq background: the filmarray respiratory panel (farp) (biofire diagnostics, inc.) is a multiplex, polymerase chain reaction (pcr) technique that can detect respiratory viruses and bacterial targets in a single reaction. immunocompromised hosts (ich) with respiratory illnesses often undergo bronchoscopy with bronchoalveolar lavage (bal). this prospective study aimed to evaluate the yield and concordance of np and bal farp testing when performed on the same patient concurrently. methods: from february to december , patients ( ich and non-ich) were enrolled. np swabs and bal samples were sent for farp testing. results: the yield of the bal farp among ich and non-ich was % ( / ) and % ( / ), respectively. the yield of positive np swabs in ich was % ( / ) versus % ( / ) in non-ich. the majority of patients ( %; / ) had concordant results between np and bal specimens. of the ich patients who had a positive bal farp, the majority ( %) had the same pathogen detected from the np swab. conclusion: the farp may be useful in the ich. given the high concordance, in patients whom a pathogen is identified on the np farp, a farp performed on bal will likely yield the same result. however, if the np farp is negative, performing the test on a bal sample may have an incremental yield. e filmarray ® respiratory panel (farp) (biofire diagnostics, salt lake city, ut) is a multiplex pcr assay that can rapidly (∼ min) detect common respiratory viruses and bacterial targets in a single reaction [ ] . published studies have shown that multiplex pcr panels are more rapid and sensitive compared to routine culture and antigen detection methods [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . although multiplex panels are rapid and accurate, the high cost to the laboratory and patient may limit their use in resource-limited settings. for the majority of immunocompetent patients with respiratory symptoms, diagnostic testing may not be required. if testing is completed, it is usually done on an easily accessible specimen type (e.g., throat swab or nasopharyngeal [ ] (np) swab) and is generally limited to targeted pathogens (e.g., influenza virus). however, in patients who are immunocompromised or critically ill, a rapid, syndromic panel may be of benefit. furthermore, patients with persistent or progressive symptoms may require a more extensive diagnostic evaluation, including bronchoscopy with collection of bronchoalveolar lavage (bal) fluid. e utility of farp testing using np swabs and bal specimens collected from immunocompromised hosts (ich) has been investigated [ , , ] . our group completed a previous, retrospective study analyzing the farp in patients who underwent bronchoscopy with bal for evaluation of a respiratory illness. e results of the multiplex panel on bal were compared to those of testing on a np swab that had been collected within days of the bronchoscopy. is study demonstrated high concordance ( %) between the farp completed on np swabs and bal specimens [ ] . however, the prior study was limited by the fact that the np swab and bal fluid were collected at different time points, and the data were analyzed mainly to compare overall concordance of the farp between the two specimen types. in order to extend on these prior observations, we sought to prospectively evaluate the yield and concordance of the farp using np swabs and bal fluid collected concurrently from ich and non-ich comparison group. is study was approved by the mayo clinic institutional review board. between february and december , patients ( ich and non-ich) who underwent a bronchoscopy with bal as part of their routine clinical care were enrolled in this study. ages ranged between and years (mean years), and % were male. an immunocompromised status was defined as patients who were ( ) recipients of systemic steroids (for ≥ days), disease-modifying agents (e.g., inhibitors of tumor necrosis factor), chemotherapy, or calcineurin inhibitors, ( ) hiv positive, ( ) solid organ or stem cell transplant recipients, and/or ( ) positive for a lymphoproliferative or myelodysplastic syndrome (table ) . ich patients (e.g., lung transplant recipients) who were undergoing routine graft rejection surveillance transbronchial biopsies and bal were also included in our analysis. ey would also serve as an ich control group to determine whether the farp tested positive in asymptomatic ich patients. twenty-five patients who did not meet the criteria listed above were also included as part of a non-ich comparison group. ese were patients undergoing bronchoscopy with bal to rule out infection but were not considered immunocompromised. once enrolled, the patients' electronic medical records were reviewed and the following information was obtained: demographics (age and sex), presence and reason of immunosuppression, microbiology testing data from the bal specimen (e.g., cultures and pcr), specifics of the respiratory illness (e.g., cough and wheezing), and other clinical or radiographic features. when the results of other microbiology studies (e.g., routine culture) were reviewed, only those results that led to an impact on patient management and were considered to be true pathogens were analyzed. e protocol for collection and testing of specimens was approved by the institutional review board at our center. patients provided informed consent and subsequently were sedated prior to bronchoscopy. following sedation, a wire-shafted, bbl cultur-eswab (becton dickinson, franklin lakes, nj) was used to sample the posterior nasopharynx per routine protocol and subsequently placed in ml of m viral transport media (remel, lenexa, ks). next, a bronchoscopy with bal was performed, in which ml aliquots of normal saline were instilled into a subsegmental bronchus corresponding to an area of radiographic abnormality on ct. forty ml of bal fluid was recovered by immediate return suction. a - ml aliquot of the bal specimen and the np swab were transported to the laboratory, stored at °c, and tested within hours. testing. in addition to farp testing, which was performed solely for study purposes, the following microbiology studies were completed on the bal sample as part of each patient's routine clinical care: gram stain, fungal smear, acid-fast smear, routine cultures (bacterial, fungal, viral, and mycobacterial), real-time pcr for influenza, respiratory syncytial virus (rsv), pneumocystis jirovecii (pjp), adenovirus, and legionella spp. e np swab was collected solely for study purposes, and no other microbiology studies were conducted on this source. testing. np swabs in viral transport media were tested by the filmarray respiratory panel according to the manufacturer's instructions. in addition, . ml of raw bal fluid was loaded into a filmarray v . pouch and tested on the filmarray . instrument (biofire) following the same protocol used for np swabs. all statistical analysis was done using comparison of proportions online chi-squared calculator (https://www.medcalc.org/calc/comparison_of_ proportions.php). among patients enrolled in this study, the multiplex panel was positive for at least one target in ( %) np specimens and ( %) bal samples. e majority ( %; (tables and ). twenty-seven ( %) of ich patients had at least one pathogen detected in the np swab. of these, / ( %) had the same pathogen(s) detected from the paired bal specimen. e remaining ( %) patients had a negative result in the paired bal sample. e farp was positive for at least one pathogen in ( %) bal specimens collected from ich patients, and of these, / ( %) had the same pathogen detected in the paired np swab sample. e remaining ( %) patients had a negative result in the np specimen (table ). in comparison with the farp, the overall yield of other microbiology studies (e.g., routine bacterial, fungal and viral culture, and individual real-time pcr studies) on the bal specimens was % ( / ) ( table ). in the ich group, at least one microbiology study (not including farp) was positive in % ( / ) of the bal samples, compared to % ( / ) of bal specimens being positive by the farp. among ich patients who had either a positive np or bal farp, ( %) had additional yield from routine (non farp) microbiology studies. among ich patients who had negative farp results from both the np swab and bal, ( %) had an organism identified by at least one of the routine microbiology tests (table ). in non-ich patients, other microbiology studies were positive for at least one pathogen in / ( %) bal specimens. e majority ( %; / ) of these patients had negative filmarray results in both np and bal samples (table ). in the ich group, the most common indication for bronchoscopy with bal in our study was a radiographic infiltrate(s) on chest imaging (n � ), followed by a diagnosis of pneumonia (n � ), cough (n � ), sepsis (n � ), e results of this study suggest that the farp may be useful in the evaluation of ich with suspicion of lower respiratory tract infection, as the yield of this test was higher in ich ( %) compared to non-ich patients ( %) (p � . ) ( tables and ). twenty-seven ich patients ( %) had a positive np farp, with / ( %) having the same pathogen(s) detected from the paired bal sample. e remaining ( %) patients had a negative farp result in bal fluid (table ) . is suggests that if a pathogen is detected from the np swab by the farp, performing the multiplex assay on bal fluid is unlikely to provide additional yield for pathogens included on the farp. among the ich group, / ( %) had a pathogen detected from bal fluid by the farp. e majority ( %; / ) of these patients had the same pathogen(s) identified from the np specimen, while ( %) had a negative farp result on the np swab (table ) . we conclude that if the farp is negative on a np swab, there may be additional value of testing bal fluid by the multiplex assay. it should be noted that regardless of the farp results on an np swab, proceeding with bronchoscopy/bal may still be indicated in ich patients with suspected infection since the farp does not test for certain pathogens (e.g., aspergillus spp., pneumocystis, nocardia spp., and mycobacteria spp.) that can be important causes of disease in the immunosuppressed population. in support of this, in our cohort, those with a positive farp on either np or bal samples had an additional pathogen(s) detected by routine microbiology studies in % of cases. furthermore, among those patients with negative farp results on both np and bal specimens, there was an additional yield from routine microbiology studies on the bal fluid in . % of cases (table ). e yield of routine microbiology testing (e.g., cultures and individual real-time pcr assays) in the ich cohort was %, compared to the farp being positive in % of np swabs and % of bal specimens. ese results suggest that the farp assay may serve as a rapid alternative to routine testing for those targets that are included on the multiplex panel. furthermore, farp testing may play an important role in patient management decisions, including infection control and antimicrobial stewardship [ ] . if a bacterial pathogen is detected, broad-spectrum coverage could be modified to offer more specific antimicrobial therapy. in contrast, if a viral pathogen is detected and bacterial cultures remain negative, it may be prudent to discontinue antibiotics and initiate targeted antiviral therapy [ ] . bronchoscopy with bal is routinely ordered in ich patients at our institution. our study showed that standard microbiology studies on bal fluid had a yield of % in this cohort, which is comparable to the yield of the farp ( %). we believe that the comparable yield of farp in this population provides additional support for this noninvasive test as a reasonable adjunct to bal testing in the ich. many ich patients with respiratory illness are placed on empiric, broad-spectrum antimicrobial therapy before the bal is collected, which can confound the yield of bal cultures. multiplex molecular testing, however, may not be affected by the prior administration of antibiotics. we propose that when used in conjunction with routine microbiology studies, farp testing on bal fluid may have a significant impact on the diagnosis and management of ich patients with respiratory illnesses. conversely, farp appears to be less useful in non-ich patients. in our study, the yield of routine microbiology studies in non-ich patients was higher than that in ich patients ( % versus %, resp.). however, the farp showed a lower yield when compared to routine cultures in the non-ich cohort ( % versus %, resp.). e performance of the farp observed in our study is comparable with other studies in the literature. our group previously performed a retrospective review of farp testing on np and bal samples collected within one week of each other. is study reported the farp to be positive in . % ( / ) of np swabs versus . % ( / ) of bal specimens [ ] . e higher yield from bal is likely due to the fact that only patients with a radiographic and/or clinical indication for bal were included. hammond et al. [ ] also assessed the performance of the farp using np swabs (n � ) and bal specimens (n � ) collected from ich patients (n � ). importantly, this study did not compare np swabs and bal fluid that were collected concurrently. e results suggested that the farp had an overall yield of % and identified more respiratory pathogens compared to conventional methods (direct fluorescent antibody (dfa), enzyme immunoassays, and viral culture) [ ] . soccal et al. [ ] assessed the detection of respiratory viruses by individual real-time pcr assays using np and bal samples collected concurrently from lung transplant patients. ey observed an overall viral positivity rate of . % in np samples and . % in bal samples. ey also demonstrated that lung transplant patients had a much slower lung function recovery if they were found to have evidence of rejection in addition to viral pathogen detection, providing important prognostic information [ ] . e lower yield of pcr in bal specimens could be explained by the inclusion of any lung transplant patient that underwent bronchoscopy, without necessarily having respiratory symptoms, pulmonary infiltrates, or decline in airflow. our data supports the notion that those without clinical or radiographic indications for bal have a lower rate of pathogen detection. is study has several limitations that should be discussed. first, the number of np (n � ) and bal (n � ) samples testing positive by the farp was relatively low, so this limits the conclusions that can be made regarding specific pathogens tested. second, due to the heterogeneous radiographic and clinical findings in these patients, it was difficult to identify specific clinical or radiologic features that would prompt the performance of farp testing of bal fluid versus a np swab alone. despite these limitations, this is the first study to compare the performance of the farp using bal fluid and np swabs collected concurrently from ich patients and a non-ich comparison group. when the farp is performed in conjunction with routine microbiology tests in the ich, it can provide important diagnostic information, especially in patients with clinical manifestations of respiratory illness. our data suggest that, in this cohort, farp testing should be initially performed on a np swab. if positive, there is limited additional yield of testing the farp on bal fluid. however, if the results of the multiplex assay are negative on the np swab, testing a bal specimen may provide additional information, especially in patients with lower respiratory tract disease. it is necessary to highlight that the farp does not cover all causes of respiratory infection, and therefore, routine microbiology testing (e.g., bacterial, mycobacterial, and fungal cultures) will continue to be essential. in conclusion, respiratory infections that are generally well tolerated in the immunocompetent host can be serious and life-threatening in the ich. e early detection, diagnosis, and appropriate management of these infections can have a substantial impact on clinical care and outcomes. erefore, multiplex testing may play an important role in the management of ich presenting with respiratory symptoms. all filmarray respiratory panel reagents were purchased using collaborative research grant funds supplied by the department of laboratory medicine and pathology at mayo clinic. e authors claim no conflicts of interest related to the products used in this study. evaluation of the filmarray ® respiratory panel for clinical use in a large children's hospital respiratory virus detection in immunocompromised patients with filmarray canadian respiratory journal respiratory panel compared to conventional methods rapid multiplex pcr assay to identify respiratory viral pathogens: moving forward diagnosing the common cold comparison of the filmarray respiratory panel and prodesse real-time pcr assays for detection of respiratory pathogens comparison of the idaho technology fil-marray system to real-time pcr for detection of respiratory pathogens in children comparison of two multiplex methods for detection of respiratory viruses: filmarray rp and xtag rvp upper and lower respiratory tract viral infections and acute graft rejection in lung transplant recipients evaluation of the biofire filmarray respiratory panel and the genmark esensor respiratory viral panel on lower respiratory tract specimens randomized phase iii trial of docetaxel versus vinorelbine or ifosfamide in patients with advanced non-small-cell lung cancer previously treated with platinum-containing chemotherapy regimens. e tax non-small cell lung cancer study group filmarray respiratory panel assay: comparison of nasopharyngeal swabs and bronchoalveolar lavage samples surveillance of upper respiratory infections using a new multiplex pcr assay compared to conventional methods during the influenza season in taiwan key: cord- -qrt zmz authors: miyakawa, kei; matsunaga, satoko; yamaoka, yutaro; dairaku, mina; fukano, kento; kimura, hirokazu; chimuro, tomoyuki; nishitsuji, hironori; watashi, koichi; shimotohno, kunitada; wakita, takaji; ryo, akihide title: development of a cell-based assay to identify hepatitis b virus entry inhibitors targeting the sodium taurocholate cotransporting polypeptide date: - - journal: oncotarget doi: . /oncotarget. sha: doc_id: cord_uid: qrt zmz sodium taurocholate cotransporting polypeptide (ntcp) is a major entry receptor of hepatitis b virus (hbv) and one of the most attractive targets for anti-hbv drugs. we developed a cell-mediated drug screening method to monitor ntcp expression on the cell surface by generating a hepg cell line with tetracycline-inducible expression of ntcp and a monoclonal antibody that specifically detects cell-surface ntcp. using this system, we screened a small molecule library for compounds that protected against hbv infection by targeting ntcp. we found that glabridin, a licorice-derived isoflavane, could suppress viral infection by inducing caveolar endocytosis of cell-surface ntcp with an ic( ) of ~ μm. we also found that glabridin could attenuate the inhibitory effect of taurocholate on type i interferon signaling by depleting the level of cell-surface ntcp. these results demonstrate that our screening system could be a powerful tool for discovering drugs targeting hbv entry. hepatitis b virus (hbv) is the causative agent of chronic hepatitis b (chb), which can lead to liver cirrhosis and hepatocellular carcinoma. the world health organization reported that over million people worldwide have chronic hbv [ ] . despite the effectiveness of the hbv vaccine, worldwide prevalence of the disease remains high, and chb is a major global health problem. current therapeutic regimens for chb include pegylated interferon (ifn) and nucleoside/ nucleotide analogues. both treatments aim to prevent progression of the disease to liver failure, cirrhosis, and hepatocellular carcinoma. however, these treatments have limited effectiveness for hbv clearance [ ] . in fact, pegylated ifn maintains viral suppression only in approximately % of patients [ ] . nucleoside and nucleotide analogues inhibit hbv replication by targeting viral dna polymerase, but long-term treatment is required to achieve clinical benefits. for example, a -month course of lamivudine achieves clearance of hepatitis b e antigen (hbeag) in approximately % of patients with chb [ ] . moreover, long-term treatment can be associated with a higher risk of side effects and emergence of drug resistant viruses, resulting in treatment failure and disease progression. therefore, it is vital to research paper www.oncotarget.com develop new types of antiviral drugs for hepatitis b treatment. in the hbv life cycle, the hepatitis b surface antigen (hbsag) initially attaches to heparan sulfate proteoglycans on the host cell surface [ ] . this attachment seems to be relatively low affinity and reversible, but is essential for the subsequent more specific interaction between hbs and the hepatocyte-specific bile acid transporter sodium taurocholate cotransporting polypeptide (ntcp) [ ] . although various membrane proteins have been reported to be hbv entry receptors, accumulating evidence now suggests that ntcp is an essential receptor for hbv infection [ ] . discovery of this receptor has dramatically increased our understanding of the molecular basis of hbv entry. viral entry is currently one of the most important targets in the search for new drugs to treat viral infections and identification of ntcp has kindled interest in exploring compounds that inhibit hbv entry. ntcp is exclusively expressed at the basolateral membrane of hepatocytes [ ] [ ] [ ] , and is involved in reuptake of conjugated bile acids from the bloodstream into hepatocytes [ ] . ntcp is one of the factors that highly restrict host tropism of hbv to hepatocytes. the specific interaction of the pres domain of hbsag with ntcp triggers hbv attachment and initiates entry into hepatocytes. the synthetic peptide drug myrcludex b exhibits significant anti-hbv activity by competitively inhibiting this interaction, both in vitro and in vivo, and is currently undergoing a phase ii clinical trial for chronically hbv-infected patients [ , ] . physiological substrates of ntcp such as taurocholic and glycocholic acids can also inhibit hbv infection, suggesting the competitive binding of bile acids and pres to ntcp [ ] . interestingly, pres -ntcp binding could trigger type i ifn signaling, whereas bile acid treatment does not [ ] . bile acid intake via ntcp was found to inhibit the ifn pathway in the physiologically relevant range of concentrations [ ] . these observations support the possibility that ntcp also takes part in the ifn-mediated innate antiviral response. because high expression often causes cell cycle inhibition, there are very few conventional hepatocellular carcinoma cell lines with ectopic expression of high levels of ntcp. moreover, there are no commercially available monoclonal antibodies (mabs) specifically recognizing cell-surface ntcp due to the difficulty of producing full-length ntcp protein with native structure. in the current study, we generated hbv-susceptible hepg cells with a tetracycline-inducible ntcp gene (intcp cells) without affecting cell growth. we also generated a high quality mab (clone a ) to efficiently detect cell-surface ntcp. using these tools, we identified the compound glabridin, which significantly inhibits hbv infection through the downregulation of ntcp from the cell surface. endogenous ntcp expression is rarely detectable in hepatoma cell lines such as hepg and huh , and these cells are not susceptible to hbv infection. therefore, we generated the intcp cell line, a hepg based cell line harboring a tetracycline-inducible human ntcp gene ( figure a) . treatment of the intcp cell line with the tetracycline analogue doxycycline (dox) caused expression of ntcp in a dox-dependent manner, and ntcp expression was -to -fold higher than endogenous expression in differentiated heparg cells and primary hepatocytes, as revealed by western blot and quantitative reverse transcription-pcr ( figure b- d) . to determine whether dox-induced ntcp protein localized to the plasma membrane, we performed a pres peptide binding assay. we found that the pres peptide detected dox-treated intcp cells but not untreated cells, indicating the presence of ntcp on the cell surface ( figure e ). consistent with a previous report [ ] , a western blot of ntcp showed major bands of - kd that were shifted to a single band of - kd after treatment with peptide n-glycosidase (pngase), implying that ntcp was modified by n-glycosylation (supplementary figure a) . although ntcp expression has been reported to affect cell proliferation [ ] , intcp cells showed no differences in cell cycle progression or cell expansion with or without dox treatment ( figure f, g) . we subsequently examined the susceptibility of intcp cells to hbv infection. at an moi of geq/cell, intcp cells showed high susceptibility to hbv infection (~ % infected) without dmso treatment ( figure h , supplementary figure b ). this infection was significantly inhibited by pres peptide treatment ( figure h ), which indicates that hbv infection was ntcp-mediated. taken together, these results show that dox-induced ntcp proteins are exposed on the cell surface and functionally interact with pres . although the above results suggest that ntcp proteins localize on the cell surface, this could not be directly demonstrated due to the lack of suitable antibodies for flow cytometry or immunofluorescence microscopy analysis. therefore, we generated a monoclonal antibody (mab) for this purpose. because recombinant ntcp protein tends to form insoluble aggregates, we utilized the wheat germ cell-free system, which has been shown to have advantages for the production of membrane proteins [ , ] . the synthesized ntcp proteins were purified and used to immunize mice, and more than hybridoma clones were established (figure a ). using a www.oncotarget.com flow cytometer-based screening assay with dox-treated and untreated intcp cells, we identified a hybridoma clone producing anti-ntcp mab, clone a ( figure b ). the a mab could recognize endogenous ntcp in differentiated heparg cells (supplementary figure ) . we performed immunofluorescence microscopy using the a mab in various cell lines and cell-surface ntcp was clearly observed ( figure c , d). because the three-dimensional organoid culture recapitulates cell-cell interactions and recent studies have indicated some advantages of hepatoma organoids in hepatitis virus infection [ ] , we investigated the localization of ntcp in hepatoma organoids. we embedded intcp cells in hydrogel before performing immunofluorescence microscopy with the a mab to visualize ntcp. ntcp localization was widespread in the membrane of internal cells as well as on the surface of the organoid ( figure e ). epitope mapping using recombinant ntcp mutants revealed that the a mab recognizes amino acids - of ntcp ( figure f ). to test whether the a antibody can inhibit hbv infection, we pretreated intcp cells and primary human hepatocytes with a mab and subsequently infected cells with wild type hbv and hbv encoding a luciferase reporter gene (hbv-nl) [ ] . the a mab failed to inhibit hbv infection ( figure g , h), suggesting that the interaction between a mab and ntcp neither blocks hbv-host cell interaction nor causes downregulation of ntcp from the cell surface. because the a mab binds ntcp but does not interfere with its localization and function, it can be used to screen for antiviral compounds that modulate the level of cell-surface ntcp. briefly, intcp cells were treated with different low-molecular-weight chemical compounds from a library derived from natural plant extracts for hours and we subsequently quantified cell viability and the amount of cell-surface ntcp using the a mab ( figure a ). we identified two compounds, geraldol and glabridin, that could decrease the amount of cell-surface ntcp without observable cytotoxicity ( figure b ). flow cytometry analysis using the a mab demonstrated that both geraldol and glabridin decrease the levels of cell-surface ntcp in a dose-dependent fashion ( figure c ), but further analysis revealed that geraldol negatively regulates the tetracycline-responsive promoter activity ( figure d ). microscale thermophoresis analysis of the biomolecular interaction [ ] revealed that glabridin could weakly but directly interact with ntcp ( figure e ). therefore, we focused on glabridin for further functional analyses. flow cytometry analysis showed that glabridin downregulated the amount of cell-surface ntcp in hepg -hntcp-c [ ] and heparg cells with ic values of µm and µm, respectively ( figure a ). notably, treatment with µm glabridin had no effect on the level of ntcp mrna (supplementary figure a) , suggesting that glabridin affects ntcp protein rather than ntcp gene expression. consistently, treatment with glabridin rapidly (within three hours) modulated the membrane localization of ntcp, but not that of the transferrin receptor ( figure b ). similar results were obtained with cell-surface biotinylation analysis in which only cell-surface proteins were purified and detected by western blotting (supplementary figure b ). immunofluorescence microscopy using the a mab showed that in glabridin-treated cells, ntcp mainly accumulated in the cytoplasm ( figure c ). to test the role of endocytosis in ntcp localization, we treated cells concurrently with glabridin and inhibitors of clathrinmediated or caveolar endocytosis. we found that genistein, a specific inhibitor of caveolar endocytosis, completely disrupted ntcp internalization ( figure d ), suggesting that glabridin causes the internalization of ntcp through caveolar endocytosis. because internalized membrane proteins are typically trafficked to degradation or recycling pathways, we next performed a cycloheximide chase assay. interestingly, ntcp protein levels were relatively stable in control cells, but the half-life of ntcp was significantly shorter in glabridin-treated cells ( figure e ). this suggests that glabridin reduces the amount of ntcp on the cell surface by promoting its endocytosis and subsequent intracellular degradation. previous reports have indicated that glabridin induces apoptosis in certain cancer cells [ ] , but we observed no negative effect on cell viability under our experimental conditions ( figure f ). we next assessed the impact of glabridin on hbv infection in hepatocytes. time-of-addition experiments demonstrated that µm glabridin inhibited hbv infection when added early in infection but became less effective when added later ( figure a ), suggesting that it acts at an early stage of the viral life cycle. indeed, when glabridin was added to hbv-producing cells, it did not affect the amounts of core-associated viral dna and/or rna (supplementary figure ) . consistent with these observations, intcp cells treated with glabridin three hours prior to infection showed a significant reduction in the percentage of infected cells and the amount of hbsag secretion ( figure b , c). we performed a parallel analysis with differentiated heparg cells and confirmed that glabridin blocked hbv infection and downregulated endogenous ntcp with an ic value of µm ( figure d ). these results suggest that glabridin inhibits hbv infection by removing ntcp from the cell surface. ntcp is a transporter for bile acid uptake. we investigated the effect of glabridin on ntcp-mediated www.oncotarget.com anti-ntcp mab generation. using a wheat germ cell-free protein production system, large amounts of ntcp were synthesized with high solubility. following mouse immunization, we established over hybridoma clones. (b) selection of a clones producing anti-ntcp mab. culture supernatants of hybridoma clones were used as primary antibodies for flow cytometry analysis of dox-treated (red line) or -untreated intcp cells (blue line). (c-e) immunofluorescence staining of cell-surface ntcp by a mab on intcp cells (c), hepg -hntcp-c cells (d), and intcp-derived spheroid (e). scale bars: µm. (f) epitope mapping of a mab. recombinant wild-type or partially truncated ntcp proteins tagged with his were generated using wheat germ extracts and subjected to western blotting using anti-his or a antibodies. the predicted epitope of a mab is shown in pink. (g, h) a mab fails to inhibit hbv infection. intcp cells (g) and primary human hepatocytes (h) were infected with hbv or its reporter virus (hbv-nl) respectively, in the presence of a mab. anti-hbs mab (clone a , which recognizes the pres domain) was used as a control. viral infectivity was determined by intracellular hbcag staining (g) or nanoluc activity (h) of infected cells. uptake of taurocholate (tca) in a sodium-containing condition and found that glabridin reduced tca uptake in a dose-and time-dependent manner ( figure a ). a previous study has shown that ntcp-mediated bile acid transport affects the expression of ifn stimulatory genes (isgs) in primary human hepatocytes [ ] , so we investigated whether glabridin counteracts this function of bile acid. primary human hepatocytes were treated with type i ifn and tca in the presence or absence of glabridin. treatment with ifn upregulated various isg proteins including mx and bst , which are known to inhibit hbv replication [ ] [ ] [ ] [ ] , while concurrent treatment with tca reduced this effect ( figure b ), in accordance with previous observations [ ] . interestingly, the addition of µm glabridin partially cancelled the effect of tca in isg expression ( figure b ). these findings suggest that glabridin enhances the innate immune response by suppressing bile acid uptake in hepatocytes through the downregulation of cell-surface ntcp. in this study, we generated intcp cells, which have high ntcp expression and high susceptibility to hbv infection, and also developed a monoclonal antibody (mab) that recognizes cell-surface ntcp. using these tools, we identified glabridin as a compound that inhibits hbv infection by downregulating levels of its entry receptor, ntcp. although primary hepatocytes express ntcp at low levels for the uptake of bile acids, endogenous ntcp in hepatocellular carcinoma cell lines is not sufficient to achieve successful infection with hbv in vitro. hepatocellular carcinoma cell lines stably expressing ntcp have been created, and exhibited increased susceptibility to hbv infection [ ] , but it has been shown that continuous ntcp expression can induce cell cycle arrest at the g /g phase [ ] , indicating that ectopic ntcp expression may be unfavorable for the long-term culture. to overcome these issues, we created the intcp cell line, featuring inducible ntcp expression, using the third-generation tetracyclineinducible gene expression system in hepg cells. upon transient treatment with tetracycline derivatives, this cell line exhibits high expression of cell-surface ntcp and becomes highly susceptible to hbv infection without any observable cell cycle arrest. this unique feature of our newly developed cell line makes it a useful tool for further biological analysis of ntcp in hepatic cells. we created a new mab recognizing cell-surface ntcp by using a wheat germ cell-free protein production system to synthesize full-length recombinant ntcp protein, which was then used to immunize mice. generally, the quality of an antibody is determined largely by the antigen used for immunization. preparation of immunogens derived from highly insoluble membrane proteins is challenging, and ntcp tends to be insoluble in conventional cell expression systems due to its multiple transmembrane domains. there are several methods for preparing antigens derived from insoluble proteins, including use of synthetic peptides containing the predicted immunogenic epitope of extracellular domains [ ] , but synthetic peptides are structurally linear and do not recapitulate the native structural features of membrane proteins. by contrast, the wheat germ cell-free protein production system utilizes a eukaryotic translation system to synthesize structurally intact and biologically active proteins similar to those expressed in mammalian cells [ , ] . this approach enabled us to create a mab capable of detecting a spatial antigen within the region of ntcp exposed on the cell surface. our work demonstrates the advantage of the cell-free system in the production of proteins with multiple transmembrane domains [ , ] . the three-dimensional structure of ntcp protein has not been well characterized and the number of transmembrane domains is controversial at present. several groups have predicted that ntcp has - transmembrane domains and that the c-terminus of ntcp is located in the cytoplasm [ , ] . we found that our a mab recognizes amino acids - of ntcp on intact cells without membrane permeabilization, implying that this epitope is possibly exposed to the extracellular space. more precise structural biological studies should be carried out to elucidate the topology of the ntcp protein. immunofluorescence microscopy experiments using our a antibody revealed that ntcp localized on the plasma membrane, frequently at cell-cell contact sites. this localization was more obvious in a hepatocyte organoid culture system, suggesting that the localization of ntcp may depend on cell polarity. because the threedimensional culture system recapitulates cell polarity, it is useful for analyzing the function of ntcp in sodium taurocholate transport as well as hbv infection. recent studies demonstrated that the three-dimensional culture system is suitable for analyzing polarized hbv transmission [ , ] . it is not well established whether ntcp plays a role in viral transmission in polarized cells, and our newly developed a mab may be useful for pursuing this intriguing question. several compounds have been shown to target ntcp. recent studies demonstrated that antagonists of retinoic acid receptor ro - [ ] or the cytokine interleukin- [ ] could reduce ntcp expression. a previous report also indicated that the green tea extract epigallocatechin- -gallate (egcg) also inhibited hbv entry into primary human hepatocytes [ ] . interestingly, egcg induced endocytosis of ntcp from the plasma membrane followed by protein degradation. in the current study, we found that glabridin, a compound from licorice extract, also causes ntcp receptor downregulation. although the precise mechanisms are unclear, our findings intcp cells were treated with glabridin ( , , and µm) for three or hours. after incubation with [ h]-taurocholate (tca) for minutes, cells were washed and intracellular radioactivity was quantified. cyclosporin a (csa, µm) was used as a positive control in this assay. * p < . , ** p < . , two-tailed unpaired t-test. (b) glabridin counteracts bile acids to promote innate immune signaling. primary human hepatocytes were sequentially treated with ifn-β ( u/ml), tca, and glabridin ( µm), as shown in left panel. the expression of representative isgs, including mx and bst , was quantified using qpcr. ns, not significant; ** p < . , two-tailed unpaired t-test. www.oncotarget.com suggest that glabridin directly binds ntcp and causes its internalization by caveolar endocytosis. by estimating protein turnover using a cycloheximide chase assay, we were able to infer that glabridin promotes the intracellular degradation of ntcp. interestingly, glabridin is orally bioavailable and is known to rapidly accumulate in the liver [ ] [ ] [ ] . since ic value of glabridin for inhibiting of hbv entry is relatively high (~ µm), this compound might have little translational benefit in hbv research. however, the data obtained from our study demonstrates the potential scope of our current methodology in drug discovery for hbv therapeutics. it is possible that the internalization of ntcp could inhibit its transporting activity thereby causing unfavorable effects on hepatocytes. however, people with i t or s f mutations in the ntcp gene show decreased surface expression and transporting activity of ntcp, but there have been no reports of serious diseases resulting from these mutations to date [ , ] . the frequent occurrence of physiological downregulation of ntcp is also notable. this downregulation is typically controlled by transcription factors, such as hnf α, under cholestasis conditions [ ] . furthermore, recent papers have shown that ntcp is the main transporter for conjugated bile acids into the liver, but other auxiliary transporters, such as oatp, may compensate when ntcp is absent [ , ] . although these reports hint that transient downregulation of ntcp might not induce immediate adverse effects on the liver, the effect should be further investigated in in vivo models to determine whether ntcp inhibition is a reasonable anti-hbv drug target. in conclusion, we identified as glabridin as a natural compound capable of inhibiting hbv infection by impairing viral entry into host cells, with an additional effect of enhancing the antiviral immune response. furthermore, our data demonstrate that our newly developed cell line and antibody will serve as powerful tools for drug discovery targeting hbv entry and for exploring molecular mechanisms underlying hbv spread. to generate intcp cells, a hepg tet-on advanced cell (clontech) parental cell line was transduced with a retroviral vector encoding the ntcp gene fused to a tetracycline-responsive element, then was selected with puromycin ( µg/ml), and cultured with dmem (wako) supplemented with % fbs. unless otherwise indicated, intcp cells were treated with doxycycline (sigma-aldrich) for hours before experiments. the intcp spheroid was made using the -d life dextran-cd hydrogel sg kit (cellendes) and cultured for seven days on a chamber slide (thermo fisher scientific). primary human hepatocytes (pxb-cells) were purchased from phoenixbio. heparg cells were purchased from biopredic international and differentiated according to the manufacturer's instructions. the chemical compound library from natural plant extracts was obtained from tokiwa phytochemical. nocodazole, cyclosporin a, genistein, and pitstop were purchased from sigma-aldrich. glabridin, staurosporine, and ifn-β were obtained from wako. a protemist xe robotic protein synthesizer (cellfree sciences) was used for the generation of full length ntcp and its truncated derivatives as previously described [ , ] . immunization of balb/c mice with recombinant ntcp and generation of hybridoma cells producing anti-ntcp antibody were performed as previously described [ ] . purification of antibodies in the culture supernatant of the hybridoma clones was performed by centrifugation at , rpm for minutes and elution with acrosep hyper df columns (pall). samples were then concentrated using amicon ultra filters (merck millipore). immunoglobulin characterization was carried out using the isostrip mouse monoclonal antibody isotyping kit (roche). cells were fixed with % paraformaldehyde, blocked with % normal goat serum (thermo fisher scientific), and stained with either anti-ntcp mab (clone a ) or anti-hbcag polyclonal antibody (dako) and alexa fluor-conjugated secondary antibodies (thermo fisher scientific). alexa fluor -conjugated phalloidin (thermo fisher scientific) was used for f-actin staining. for intracellular staining, cells were permeabilized with . % triton x- before blocking. for the pres -peptide binding assay, intcp cells pretreated with µg/ml dox for hours were incubated with nm fitc-conjugated pres peptide (the first amino acid residues of small hbs domain fused with n-terminal myristoyl group and c-terminal fitc) at ° c for two hours. cells were then washed with pbs and fixed with % paraformaldehyde. microscopic imaging was performed with an fv -d confocal laser scanning microscope (olympus) or bz- fluorescence microscope (keyence). cells were detached with mm edta in pbs and fixed with % formaldehyde before incubation with either anti-ntcp ( a ) or anti-transferrin receptor (genetex) antibodies at ° c. cells were then stained with pe-conjugated secondary antibody and analyzed using a facscanto ii instrument (bd biosciences). for the drug screen, cells were treated with candidate compounds www.oncotarget.com ( µm) hours before analysis. data were analyzed with flowjo software (treestar). wild-type hbv was derived from the supernatants of hepg . . cells, which were stably transfected with a complete hbv genome. hbv reporter viruses (hbv-nl) were produced by transient transfection of hepg cells with puc . -hbv/nl and puc-hbv-d, as previously described [ ] . the collected supernatants were filtered through a . -μm filter (merck millipore), and concentrated approximately times using a peg virus precipitation kit (biovision). cells were infected with wild-type hbv at a concentration of , genome equivalents per cell in the presence of % peg for hours. alternatively, cells in a -well plate were inoculated with µl of hbv-nl in the presence of % peg for hours. hbv-infected cells were cultured in fresh medium for an additional - days and their infectivity was determined by intracellular hbcag staining or extracellular hbsag quantification, as previously described [ ] . the infectivity of hbv-nl was quantified using the nano-glo luciferase system (promega), according to the manufacturer's instructions. samples in sds loading buffer were loaded onto % polyacrylamide gels, electrophoresed, and blotted onto pvdf membranes (merck millipore), as previously described [ ] . membranes were probed with primary antibodies and horseradish peroxidase-conjugated secondary antibodies (ge healthcare). for protein degradation analysis, cycloheximide ( µg/ml) was added - hours before harvesting cells. the primary antibodies used were as follows: anti-ntcp, anti-vinculin, anti-α-tubulin (sigma-aldrich) and anti-his (genetex). detected proteins were visualized using a fluorchem digital imaging system (alpha innotech). band analysis was performed with imagej software (national institutes of health). messenger rna extraction and subsequent cdna synthesis was performed using trizol reagent (thermo fisher scientific) and revertra ace (toyobo), respectively, according to each manufacturer's instructions. gene expression was then analyzed by qpcr using sybr premix ex taq ii (takara) and a cfx real-time pcr detection system (bio-rad). the primer pairs used were ′-ggacttcgagcaagagatgg- ′ and ′-agcactgtgttggcgtacag- ′ for actb; ′-atggaggcccacaacg cgtctgccc- ′ and ′-cagaaggtggagcaggtggtcatcac- ′ for ntcp; ′-ggctgtttaccagactccgaca- ′ and ′-cacaaa gcctggcagctctcta- ′ for mx ; and ′-tctcctgcaaca agagctgacc- ′ and '-tctctgcatccagggaagccat- ' for bst . recombinant ntcp-gfp and gfp proteins were incubated with different concentrations of compounds in mm potassium phosphate buffer (ph . ) containing mm nacl, . % bsa, and . % ddm (n-dodecylβ-d-maltopyranoside) for one hour at room temperature. samples were loaded on monolith nt. standard treated capillaries (nano temper technologies) and analyzed with a monolith nt. blue red microscale thermophoresis instrument. cell cycle analysis was performed with a tali image-based cytometer (thermo fisher scientific). cell viability was measured with cell counting kit- (dojindo) or celltiter-glo assay (promega) according to the manufacturer's instructions. cells were treated with [ h]-taurocholic acid (tca) in a sodium-free or sodium-containing buffer at ºc for minutes to allow tca uptake into cells. cells were washed and intracellular radioactivity was measured using a liquid scintillation counter. all graphs represent means and standard deviations. the statistical significance of differences between two groups was tested using a two-tailed unpaired t test with prism software (graphpad). km designed and performed research, analyzed data, and wrote the manuscript; sm, yy, md, and kf performed research and analyzed data; hk and tc analyzed data; hn, kw, ks, and tw contributed reagents and analyzed data; and ar designed and supervised the research, analyzed the data, and wrote the manuscript. global epidemiology of hepatitis b virus infection: new estimates of age-specific hbsag seroprevalence and endemicity virologic monitoring of hepatitis b virus therapy in clinical trials and practice: recommendations for a standardized approach pegylated interferon alfa- b alone or in combination with lamivudine for hbeag-positive chronic hepatitis b: a randomised trial nucleoside analogues for chronic hepatitis b: antiviral efficacy and viral resistance entry of hepatitis b and hepatitis d virus into hepatocytes: basic insights and clinical implications sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis b 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www.oncotarget.com we thank naohito nozaki for antibody production and haruka sato, kyoko ohnishi, and sho nonoyama for their technical assistance. the authors declare that they have no competing interests. this work was supported in part by an amed grant-in-aid for the program on the innovative development and the application of new drugs for hepatitis b (jp fk to ar and jp fk to km), the creation of innovation centers for advanced interdisciplinary research areas program (to ar), and by a gsk japan research grant (to km). key: cord- - ylifqo authors: kelly, j. daniel; worden, lee; wannier, s. rae; hoff, nicole a.; mukadi, patrick; sinai, cyrus; ackley, sarah; chen, xianyun; gao, daozhou; selo, bernice; mossoko, mathais; okitolonda-wemakoy, emile; richardson, eugene t.; rutherford, george w.; lietman, thomas m.; muyembe-tamfum, jean jacques; rimoin, anne w.; porco, travis c. title: projections of ebola outbreak size and duration with and without vaccine use in Équateur, democratic republic of congo, as of may , date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: ylifqo as of may , , suspected, probable and confirmed cases of ebola virus disease (evd) had been reported in Équateur province, democratic republic of congo. we used reported case counts and time series from prior outbreaks to estimate the total outbreak size and duration with and without vaccine use. we modeled ebola virus transmission using a stochastic branching process model that included reproduction numbers from past ebola outbreaks and a particle filtering method to generate a probabilistic projection of the outbreak size and duration conditioned on its reported trajectory to date; modeled using high ( %), low ( %), and zero ( %) estimates of vaccination coverage (after deployment). additionally, we used the time series for prior ebola outbreaks from to to parameterize the thiel-sen regression model predicting the outbreak size from the number of observed cases from april to may . we used these techniques on probable and confirmed case counts with and without inclusion of suspected cases. probabilistic projections were scored against the actual outbreak size of evd cases, using a log-likelihood score. with the stochastic model, using high, low, and zero estimates of vaccination coverage, the median outbreak sizes for probable and confirmed cases were cases ( % prediction interval [pi]: , ), cases ( % pi: , ), and cases ( % pi: , ), respectively. with the thiel-sen regression model, the median outbreak size was estimated to be . probable and confirmed cases ( % pi: . , . ). among our three mathematical models, the stochastic model with suspected cases and high vaccine coverage predicted total outbreak sizes closest to the true outcome. relatively simple mathematical models updated in real time may inform outbreak response teams with projections of total outbreak size and duration. a a a a a on may , , the world health organization (who) announced the occurrence of an outbreak of ebola virus disease (evd) in the democratic republic of congo (drc). [ ] from april through may , suspected evd cases were reported in iboko and bikoro, Équateur province. on may , blood samples from five hospitalized patients had been sent to kinshasa for ebola-pcr testing, and two were confirmed pcr-positive. [ ] on may , vaccination of healthcare workers started. [ ] by may , the ring vaccination campaign was being rolled out as contacts and contacts of contacts were being actively monitored. six suspected, probable and confirmed evd cases had been reported, and ( %) of probable and confirmed evd cases had died. [ ] this outbreak had several features that were worrisome for widespread transmission. cases were reported over a -kilometer distance, including four confirmed cases in thẽ , , -inhabitant provincial capital of equateur, mbandaka, which is situated on the congo river and bordering congo-brazzaville. [ ] moreover, travel to kinshasa is frequent from mbandaka. given these risk factors, early epidemic growth profiles, [ ] and evidence of unreported infection from previous outbreaks, [ , ] the risk of a substantially larger outbreak could not be ignored. the factors causing epidemic growth to peak have been debated. delayed detection of evd outbreaks and resulting widespread distributions of evd have significantly contributed to epidemic growth. [ ] in addition to traditional burial practices, ebola treatment units with low quality care and/or high mortality rates have discouraged ebola suspects from presenting to care and contribute to community-based transmission. [ ] [ ] [ ] fragile, overwhelmed public health surveillance systems have also contributed to higher rates of unreported cases, who endanger urban communities, [ ] which potentially have had higher transmission rates than rural communities. [ ] change to subcritical transmission (reproduction number below ) tends to occur when ebola response organizations deploy control, prevention and care measures, [ , ] communities adopt more protective behaviors, [ , ] and/or transmission decreases in a social network. [ , ] scientific advances with rapid diagnostics and vaccines from the west africa outbreak were deployed in the april-july evd outbreak in drc and had the potential to limit ebola virus transmission. [ ] [ ] [ ] we used reported case counts during evd outbreak in drc and/or time series from prior outbreaks to estimate the total outbreak size and duration with and without the use of vaccines. these projections were intended to help organizations anticipate and allocate sufficient resources for the duration of the april-july evd outbreak. the following methods were used to generate projections: a stochastic branching process model, [ ] statistical regression based on prior outbreaks, and gott's law. [ ] on may , evd cases ( suspected, probable and confirmed) were reported in three locations (iboko, bikoro, and mbandaka) (fig ) . based on evd situation reports from drc, we assumed the ring vaccination program started the week of may , so we used may as the time point that vaccines were implemented in the model at high and low coverage levels. data on suspected, probable, and confirmed case counts were available from who situation reports in may and used as the basis for analysis. the published situation reports reflected data available up until may , , , , and , respectively. during the outbreak, the ebola response team tested suspected cases for evd and depending on positive or negative results, cases were classified as confirmed or not a case. the final outbreak case count was probable and confirmed cases (no suspected cases). [ ] due to this reporting process, probable and confirmed cases were used to parameterize the stochastic branching process model, regression models, and gott's law. we added suspected cases to create an additional projection, using stochastic branching process model. we modeled ebola virus transmission using a stochastic branching process model, parameterized by transmission rates estimated from the dynamics of prior evd outbreaks, and conditioned on agreement with reported case counts from the evd outbreak to date. we incorporated high and low estimates of vaccination coverage into this model. then we generated a set of probabilistic projections of the size and duration of simulated outbreaks in the current setting. to estimate the reproduction number r as a function of the number of days from the beginning of the outbreak, we included reported cases by date from thirteen prior outbreaks and excluded the first historical outbreak reported in those countries (e.g., outbreak in yambuko, drc) (s table) . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] as there is a difference in the ebola response system as well as community sensitization to evd following a country's first outbreak, we employed this inclusion criterion to reflect the ebola response system in drc during what is now its ninth outbreak. the wallinga-teunis technique was used to estimate r for each case and therefore for each reporting date in the outbreak. [ ] the serial interval was defined as the interval between disease onset in an index case and disease onset in a person infected by that index case. the serial interval distribution used for this estimation was a gamma distribution with a mean of . days and a standard deviation of days, with intervals rounded to the nearest whole number of days, consistent with the understanding that the serial interval of evd cases ranges from to days with mean to days. [ , [ ] [ ] [ ] given that serial interval distribution, which we can denote as a probability w (t) of a t-day interval between given primary and secondary cases, wallinga-teunis estimation works by defining a relative likelihood p ij for each possible source j of a given case i: and then deriving from that an estimated reproduction number rj for each case: after using this technique to derive estimated reproduction numbers for each case in an outbreak, we use these estimated r values and cases' onset dates d to estimate an initial reproduction number r and quenching rate τ for each past outbreak by fitting an exponentially quenched curve: to each outbreak's r and d values (s fig) . transmission was modeled using a stochastic branching process model in which the number of secondary cases si caused by any given primary case i was drawn from a negative binomial distribution whose mean is the reproduction number r: [ , ] si � nbðr; kÞ; where r is reproduction number as a function of day, k is a dispersion parameter, and nb() denotes the negative binomial distribution. all transmission events were assumed to be independent. the serial interval between date of detection of each primary case and that of each of its secondary cases is assumed gamma distributed with mean . days and standard deviation days, rounded to the nearest whole number of days, as above. the pair of parameters r and τ estimated for the different past outbreaks used, and dispersion parameter k, were used in all possible combinations (with r and τ taken as a unit) to simulate outbreaks. this model generated randomly varying simulated outbreaks with a range of case counts per day. after the ministry of health and who conducted epidemiological investigations about the beginning of the evd outbreak in Équateur province, they concluded that the outbreak began on april , , with a single case. [ ] the simulation process occurs as follows: proposed epidemic trajectories are generated in an initial step based on the above branching process, and these are then subsequently filtered by discarding all but those whose cumulative case counts match the known counts of the april-july evd outbreak on known dates. the filtration accepts epidemics within a range of cases more or less than each recorded value. this one-step particle filtering technique produced an ensemble of model outbreaks, filtered on agreement with the recorded trajectory of the outbreak to date. this filtered ensemble is then used to generate projections of the eventual outcome of the outbreak. [ ] to model vaccination coverage with respect to total transmission (unreported and reported), we multiplied the estimate of vaccine effectiveness by low and high estimates of reported cases. in a ring vaccination study at the end of the west africa outbreak, the overall estimated rvsv-vectored vaccine efficacy was % and vaccine effectiveness was . % in protecting all contacts and contacts of contacts from evd in the randomized clusters, including unvaccinated cluster members. [ ] estimates of vaccine effectiveness were used in our stochastic model. the ring vaccination study found the vaccine to be effective against cases with onset dates days or more from the date of vaccine administration, so we modeled the vaccination program as a proportionate reduction in the number of new cases with onsets days or more after the program start date. then, past estimates of the proportion of unreported cases were used to estimate the proportion of exposed individuals not covered by the vaccination process. based on a sierra leonean study from the - outbreak, [ ] we estimated that the percentage of reported cases in drc would rise over time from a low of % to a high of %. given these low and high estimates of reported cases and the estimate of vaccine effectiveness ( . %), a low estimate of vaccination program coverage was % ( % multiplied by . %) and a high estimate of vaccination program coverage was % ( % multiplied by . %). the course of the outbreak with and without the vaccination program was modeled based on approximate dates available from situation reports. [ ] for simulation based on probable and confirmed cases, from , , simulated outbreaks, , were retained after filtering on approximate agreement with drc case counts. for simulation based on probable, confirmed, and suspected cases, from , , simulated outbreaks, , were retained after filtering. the simulated outbreaks that were retained after filtering were continued until they generated no further cases. this ensemble was used to derive a distribution of outbreak sizes and durations. mean and median values and % prediction intervals were calculated using the . and . percentiles of simulated outbreak size and duration. these analyses were conducted using r . . (r foundation for statistical computing, vienna, austria). for contrast with the stochastic model above, a simple regression forecast was conducted based solely on outbreaks of size or greater. time series for all such prior outbreaks were obtained, including seven prior ones from drc, dating back to (s table; the beginning of each outbreak was not reliably characterized; therefore, all time-series were aligned on the day they reached cases. in the april-july outbreak, we observed cases over the period from april to may (day to day ). may corresponded to day since reaching reported cases. for the prior outbreaks, linear interpolation was used to obtain the number of cases on day (after reaching cases). to reduce the influence of outliers and high leverage points, and to improve linearity, we calculated the pseudologarithm transform f(x) = arcsinh(x/ ), asymptotically logarithmic but well-behaved at . we used nonparametric theil-sen regression (r-package mblm) followed by calculation of the resulting prediction interval for a new observation. [ , ] finally, we reported the median and % central coverage intervals for the prediction distribution conditional on the value being no smaller than the observed value on day . sensitivity analysis was conducted using ordinary least squares regression. these analyses were conducted using r . . (r foundation for statistical computing, vienna, austria). gott' s law was used to estimate the outbreak size using data through may and may . we included a projection using data through may because we hypothesized that this method performs better when the first situation report is posted than at later in the outbreak period. [ ] then we included a projection with the regression models using data through may for comparison. with gott's law, we assume we have no special knowledge of our position on the epidemic curve. if we assume a non-informative uniform prior for the portion of the epidemic that still remains, the resulting probability distribution for the remaining number of cases y is: the median outbreak size was estimated, along with the two-sided % prediction interval. each of the above models assigned a probability to any possible value of the total outbreak size. the final outbreak size was probable and confirmed cases, so we identified the probability of this equivalent number ( ) from each model, as of may . probabilistic projections were scored using a log-likelihood (ignorance) score. [ ] as of may , , there were suspected, probable and confirmed evd cases. bikoko had ten confirmed cases, probable cases, and one suspected case. iboko had confirmed cases, two probable cases, and one suspected case. mbandaka had four confirmed cases and one suspected case (fig ) . with the stochastic model, we projected outbreak size and duration of probable and confirmed cases. in the absence of any vaccination program, the projected median outbreak size was . cases (mean . ; % prediction interval: . , . ). median duration of projected outbreaks was . days (mean . ; % prediction interval: . , . ). using a lower estimate of % vaccination coverage, the median outbreak size was . cases (mean . ; % prediction interval: . , . ) and median duration was . days (mean . ; % prediction interval: . , . ). using a higher estimate of % vaccination coverage, the median size was . evd cases (mean . ; % prediction interval: . , . ), and the median duration was . days (mean . ; % prediction interval: . , . ). these projections with the stochastic model were repeated to estimate suspected, probable and confirmed cases (table and table with the regression based on past outbreaks, the median outbreak size was estimated to be . probable and confirmed cases ( % prediction interval: . , . ), while use of ordinary least squares produced a median size of . probable and confirmed cases ( % prediction interval: . , . ). outbreak projections were also reported using data through may in table . gott's law suggests that given probable and confirmed cases, the median estimate of outbreak size was . cases ( % ci: . , . ). using the probable and confirmed cases as of may , the median estimate of outbreak size was . cases ( % ci: . , . ). of the mathematical models employed, the stochastic model that included suspected cases and high vaccination coverage had the best probabilistic score (log likelihood of - . ). likelihood scores of each model can be found in table . when we were conducting our projections in late may, this outbreak still had the potential to become the largest outbreak in drc since . vaccine use, regardless of coverage levels, was projected to prevent more than half of the total outbreak size. vaccines, however, were only part of concurrent prevention, control, and care strategies. [ , [ ] [ ] [ ] we also found that the stochastic model with vaccine use projected that rare, large outbreaks (tail of the distribution of the model without vaccinations) were prevented, suggesting that repeat epidemics such as the - west african outbreak may have been highly unlikely once vaccines were rolled out. multiple models were used to estimate total outbreak size. this study exemplified how mathematical models, including simple regressions, can be useful for advising real-time decision making because they provided rapid projections and similar estimates of r as compared to complex models, [ ] even though real-time modeling projections historically overestimated outbreak size and duration. [ , ] our projections that included suspected cases did not suggest that vaccines had as much of an impact as our model using only probable and confirmed cases. the trends associated with suspected cases were subject to several factors, including operational choices of response teams and maturity of the outbreak. nevertheless, suspected case counts may at times provide a better glimpse into the near future of an outbreak than the confirmed and probable case counts. in our case, use of the time series of confirmed, probable, and suspect cases yielded a forecast closer to the final outbreak size. however, as model projections can be highly sensitive to inclusion of suspected cases and use of exact case counts, particularly the last few counts in the available data, conclusions must be taken with caution. thus far, there had been a strong local and international response, and deployment of vaccines and rapid diagnostic tests (rdt) occurred early in response efforts. [ ] rdts were being used to screen ebola suspects while the vaccines are being administered to high-risk groups for evd, including healthcare workers, contacts, and contacts of contacts. to further limit epidemic growth from unreported cases, particularly those who have non-specific symptoms but table . distribution of projected outbreak size from stochastic branching process model. mean, median and % prediction interval of outbreak size, by proportion of vaccine coverage, using probable and confirmed cases with and without suspected cases. probable table . distribution of projected outbreak duration from stochastic branching process model. mean, median and % prediction interval of outbreak duration, by proportion of vaccine coverage, using probable and confirmed cases with and without suspected cases. there are limitations to our projections. projection distributions were right-skewed, with long tails (and we therefore reported the median instead of the mean). while there have been observed evd outbreaks with a case count greater than ten cases, we were unable to include all prior outbreaks in our estimates due to data availability. [ , ] note that the simple regression projection is based entirely on past outbreaks of evd (measured and reported in different ways), and cannot account for the improved control measures and vaccination in the way that a mechanistic model does. we included, however, as much real-time information into our estimates as possible, but situations such as the introduction of evd into a large urban population and implementation of rdts and vaccines are new to drc. we did not include vaccination of healthcare workers in the stochastic model. our estimates of vaccination effectiveness and reported cases were obtained from west africa because these estimates were not available for the evd outbreak in Équateur. these modeling assumptions may not have been consistent with estimates in drc and should be carefully considered prior to use in other evd outbreaks. a strength of our approach was the use of multiple methods to estimate the outbreak size, although we note that gott's law has not been validated for outbreak projections in other evd outbreaks. additional limitations of the models were that they did not include parameters to address spatial spread, urban settings, conflict zone, or other factors that may have influenced the accuracy of the predictions, particularly in the - evd outbreak in northeastern drc (ongoing in january ). while it can also be useful and achievable to use models of these kinds to make short-term forecasts for evaluation of model performance and to inform outbreak response, [ ] the present study was limited to projections of final outbreak size and duration. among our three mathematical models, the model that performed the best (stochastic model with suspected cases and high vaccine coverage) predicted total outbreak sizes close to the true outcome. when evd cases were introduced into mbandaka, there was concern that the total outbreak size could exceed most prior evd outbreaks in drc. indeed, our projections were consistent with this concern because models without vaccine coverage projected higher total outbreak sizes. in our stochastic model projections, vaccine use reduced mean total outbreak size by more than half, regardless of coverage levels (p< . , welch's t-test). as vaccine coverage was scaled up, an influx of support was warranted to support and bolster the evolving rapid response; however, continued efforts to strengthen the health system are equally as warranted so that we can respond to future outbreaks before they become epidemics. relatively simple mathematical models updated in real time may inform outbreak response teams with projections of total outbreak size and duration. supporting information s table. list of prior ebola outbreaks from to by time period, country, confirmed/probable reported and time series case count, outbreak inclusion into the regression and stochastic models. world health organization regional office for africa. health topics: ebola virus disease world health organization. emergencies preparedness, response excitement over use of ebola vaccine in outbreak tempered by real-world challenges mathematical models to characterize early epidemic growth: a review minimally symptomatic infection in an ebola 'hotspot': a cross-sectional serosurvey anatomy of a hotspot: chain and seroepidemiology of ebola virus transmission after ebola in west africa-unpredictable risks, preventable epidemics the ebola suspect's dilemma biosocial approaches to the - ebola pandemic ebola virus disease in west africa-clinical manifestations and management unreported cases in the - ebola epidemic: spatiotemporal variation, and implications for estimating transmission heterogeneity in district-level transmission of ebola virus disease during the - epidemic in west africa the impact of control strategies and behavioural changes on the elimination of ebola from lofa county dynamics and control of ebola virus transmission in montserrado, liberia: a mathematical modelling analysis risk communication and ebola-specific knowledge and behavior during - outbreak a theory-based socioecological model of communication and behavior for the containment of the ebola epidemic in liberia modeling household and community transmission of ebola virus disease: epidemic growth, spatial dynamics and insights for epidemic control ebola control: effect of asymptomatic infection and acquired immunity rapid antigen test for ebola. for use under emergency use authorization (eua) only efficacy and effectiveness of an rvsv-vectored vaccine in preventing ebola virus disease: final results from the guinea ring vaccination, open-label, cluster-randomised trial (ebola Ç a suffit!) ebola control: rapid diagnostic testing on the probability of the extinction of families implications of the copernican principle for our future prospects high-resolution global maps of st-century forest cover change report of a who/international study team ebola virus disease in southern sudan: hospital dissemination and intrafamilial spread ebola hemorrhagic fever outbreaks in gabon, - : epidemiologic and health control issues the reemergence of ebola hemorrhagic fever, democratic republic of the congo prise en charge des malades et des défunts lors de l'épidémie de fièvre hé morragique due au virus ebola d'octobre à décembre a limited outbreak of ebola haemorrhagic fever in etoumbi, republic of congo ebola virus disease in the democratic republic of congo filovirus outbreak detection and surveillance: lessons from bundibugyo different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures ebola virus disease in west africa-the first months of the epidemic and forward projections a systematic review of early modelling studies of ebola virus disease in west africa transmission dynamics and control of ebola virus disease (evd): a review comparing methods for estimating r from the size distribution of subcritical transmission chains superspreading and the effect of individual variation on disease emergence ebola virus disease in the democratic republic of the congo outbreak(s) of ebola haemorrhagic fever, congo and gabon potential for large outbreaks of ebola virus disease world health organization. weekly epidemiological record. abonnement annuel university of bergen dissertation for phd ebola virus disease outbreak in nigeria: transmission dynamics and rapid control ebola (ebola virus disease): history of ebola virus disease: - ebola outbreak in west africa: case counts estimates of the regression coefficient based on kendall's tau a rank-invariant method of linear and polynomial regression analysis. i, ii, iii. nederl akad wetensch, proc scoring probabilistic forecasts: the importance of being proper beyond vaccines: improving survival rates in the drc ebola outbreak food insecurity as a risk factor for outcomes related to ebola virus disease in kono district, sierra leone: a cross-sectional study the symbolic violence of 'outbreak': a mixed methods, quasiexperimental impact evaluation of social protection on ebola survivor wellbeing estimating the future number of cases in the ebola epidemic -liberia and sierra leone models overestimate ebola cases centers for disease control and prevention. outbreaks chronology: ebola hemorrhagic fever real-time projections of epidemic transmission and estimation of vaccination impact during an ebola virus disease outbreak in the eastern region of the democratic republic of congo we thank the ebola responders for their efforts in the evd outbreak in Équateur, democratic republic of congo. key: cord- - dg i i authors: xiong, qingqing; lee, gha young; ding, jianxun; li, wenliang; shi, jinjun title: biomedical applications of mrna nanomedicine date: - - journal: nano res doi: . /s - - - sha: doc_id: cord_uid: dg i i as an attractive alternative to plasmid dna, messenger rna (mrna) has recently emerged as a promising class of nucleic acid therapeutics for biomedical applications. advances in addressing the inherent shortcomings of mrna and in the development of nanoparticle-based delivery systems have prompted the development and clinical translation of mrna-based medicines. in this review, we discuss the chemical modification strategies of mrna to improve its stability, minimize immune responses, and enhance translational efficacy. we also highlight recent progress in nanoparticle-based mrna delivery. considerable attention is given to the increasingly widespread applications of mrna nanomedicine in the biomedical fields of vaccination, protein-replacement therapy, gene editing, and cellular reprogramming and engineering. messenger rna (mrna) is a transient carrier of genetic information from genes to ribosomes for protein synthesis. during the first decade after its discovery in [ ] , most studies focused on understanding the structure, function, and metabolic activity of mrna in eukaryotic cells. in the s, the translation of protein from isolated mrna was first achieved in living cells [ ] . the concept of using nucleic acid as a drug was not proposed until when wolff et al. demonstrated that direct injection of in vitro transcribed (ivt) mrna resulted in the expression of the encoded protein in mouse muscle [ ] . since then, mrna has been explored as a new class of nucleic acid therapeutics for the treatment of various diseases, such as cancer and genetic diseases. as a promising alternative to conventional plasmid nano res. , ( ) : - dna (pdna) therapy, mrna-based therapy possesses multiple unique features. first, since mrna is translated in the cytoplasm, it does not need to enter the nucleus to exert its bioactivity. second, unlike pdna, mrna has a negligible chance of integrating into the host genome, which may avoid the risk of insertion mutagenesis [ , ] . third, mrna has more consistent and predictable protein expression kinetics than the random onset expression kinetics of dna [ ] [ ] [ ] . fourth, mrna is only transiently active, which may be beneficial for applications where transient protein expression is required [ ] . finally, in vitro synthesis of mrna is relatively convenient and robust, which further enhances the prospect of mrna being used in therapeutics. despite these unique features of mrna, its clinical application has been limited due to its poor stability, propensity for immunostimulation, and the lack of an efficient delivery system. as a single-stranded polynucleotide, mrna is easily degraded by the highly active nucleases during production in vitro and delivery in vivo. moreover, the negatively charged, large mrna molecules have difficulty in directly crossing the cellular membrane. such challenges have greatly hindered the clinical translation of mrna therapeutics. recently, however, along with the increasing knowledge of the relationship between mrna structure and its translation efficacy, a variety of chemical modification methods have been developed to improve mrna stability and reduce its immunostimulation [ ] [ ] [ ] , thus potentiating its therapeutic utility. in parallel with these developments, nanotechnology has emerged as a promising method allowing these nucleic acids to withstand multiple extracellular and intracellular barriers (e.g., protecting them from enzymatic degradation, improving cytosolic transportation, and reducing renal filtration) [ ] [ ] [ ] . the rise of mrna nanomedicines is rapidly advancing their applications in a wide range of biomedical fields, such as vaccination, protein-replacement therapy, gene editing, and cell reprogramming and engineering. in this review, we discuss the chemical modification methods of mrna, summarize the nanoparticle platforms for mrna delivery, and provide an overview of their diverse biomedical applications. typically, mrna is synthesized by an in vitro transcription method in a cell-free system. this process involves a linearized dna template containing a phage promoter sequence (t , t , or sp ) and target gene sequence [ ] (fig. ) . since ivt mrna is not as biologically viable due to its instability and immunogenicity, it was originally considered to be disadvantageous compared to other therapeutic agents. in the past thirty years, substantial efforts have been devoted to developing effective modification strategies to optimize ivt mrna synthesis [ ] . here, we discuss these possible strategies for mrna modification that contribute to increased resistance to nuclease degradation and reduced recognition by the immune system. similar to the mature mrna in eukaryotic cells, ivt mrna contains the following five structural elements ( fig. ): ′ cap, ′ untranslated region ( ′ utr), proteinencoding open reading frame (orf), ′ utr, and ′ poly(a) tail [ ] . modification of these elements during mrna synthesis in vitro can improve mrna stability and lead to an efficient and stable expression of target proteins. natural eukaryotic mrna molecules have a cap structure of -methylguanosine (m g) linked to the nano res. , ( ) : - ′-end of pre-mrna via a ′- ′ triphosphate bridge. a functional ′ cap structure is a prerequisite for the efficient translation of mrna, since its binding to eukaryotic translation initiation factor e (eif e) promotes the initiation of translation [ ] , whereas its binding to mrna decapping enzymes regulates mrna decay [ ] . ivt mrna can be capped either by performing post-transcriptional modification using recombinant vaccinia virus-derived capping enzymes [ ] , or by incorporating synthetic cap analogs into the in vitro transcription reaction [ ] . however, the enzymatic capping method is only limited to the standard cap structure (m gpppg) because of enzyme specificity. by contrast, co-transcription with the cap analogs allows for more cap structures and is a simple process. it is a commonly used method of synthetic mrna preparation. however, this method also has limitations. first, due to the competition between cap analogs and native guanosine triphosphate (gtp), some of the mrna remains uncapped, which severely affects its translation efficiency [ ] . second, since cap analogs can be attached to mrna in two orientations, half of the capped mrnas are reverse-oriented and are not recognized by the cap-binding protein [ ] . to avoid the reverse cap orientation, anti-reverse cap analogs (arcas) were developed. in an arca, the ′-hydroxyl (oh) of the native mrna cap is replaced by a methoxy group (och ) [ , ] . in addition to reducing reverse orientation, a new generation of arcas has been developed to reduce the binding between the mrna cap region and the decapping enzymes, further improving the mrna translation efficiency and stability. specifically, phosphorothioate-modified cap [ ] , boranophosphatemodified cap [ , ] , phoshoroselenoate-modified cap [ ] , methylenebisphosphonate-modified cap [ ] and , -dithiodiphosphate-modified cap [ ] , dihalogenmethylenebisphosphonate-modified cap [ ] are other examples of ′-cap modifications that display resistance to decapping enzymes. polyadenylation is an essential step in the production of functional mrna in eukaryotic cells. the poly(a) tail plays an important role not only in facilitating nuclear export and translation initiation, but also in protecting the mrna from nuclease degradation through the interaction with poly(a)-binding protein (pabp) [ ] . generally, ivt mrna is tailed either by a polyadenylation reaction catalyzed by recombinant poly(a) polymerase [ ] [ ] [ ] , or by direct transcription from the poly(t) sequence encoded in the dna template [ ] . although recombinant poly(a) polymerase derived from escherichia coli is a facile method for poly(a) tail addition, the ivt mrnas prepared by this method are a mixture of rnas with different poly(a) lengths. by contrast, ivt mrna generated from poly(t)-containing dna template has a defined poly(a) length and enables reproducible batch control. thus, this approach is more preferable in practical applications. the length of poly(a) acts as an indicator of mrna stability [ ] and also contributes to mrna translational regulation [ ] . several studies have demonstrated that relatively longer poly(a) length is advantageous for mrna translation. for example, the protein expression in hela cells transfected with mrna increased as the poly(a) tail length increased from to residues [ ] . optimized mrna containing adenosines showed maximum protein expression in dendritic cells (dcs) [ ] . in another study, ivt mrna with a poly(a) length of nucleotides allowed for efficient protein expression [ ] . a linear plasmid vector system, pevl, was recently explored as the template for generating ivt mrna with poly(a) tails of up to approximately bases and with a defined length [ ] . besides the tail extension, incorporation of adenosine analogs, such as '-deoxyadenosine or -azaadenosine, provided better mrna protection from '-exonuclease degradation, which could also be used to increase protein expression [ ] . utrs are the non-coding regions located at ′ upstream and ′ downstream of orfs, which do not directly contribute to the protein sequences. however, they play a critical role in the regulation of mrna stability and protein translation via the interaction with rnabinding proteins (rbps) [ , ] . ′ utr is an important element for ribosome recruitment and start codon choice [ ] , and it may have a crucial impact on translation [ ] . ′ utr determines protein expression nano res. , ( ): - levels through the regulation of mrna stability and translation mediated by au-rich elements, and also facilitates local translation through the control of mrna localization [ ] . the sequence, secondary structure, and length of utrs influence the translation process. therefore, incorporating ′ and ′ utrs with regulatory sequences is another strategy to further improve the stability and translation of mrna. for instance, the presence of the internal ribosomal entry site in the ′ utr enables effective mrna translation even when the cell level of eif e is low [ ] . mrnas containing n -methyladenosine (m a) in their ′ utr can be translated in a cap-independent manner [ ] . the induction of the optimized kozak sequence is an effective strategy to prevent fault initiation [ ] . while the ′ utr mainly influences the initiation of the translation process, the ′ utr plays a critical role in stabilizing mrna [ ] . the ′ utr of α and β-globin mrnas are the best-characterized ′ utr sequences that can enhance the stability and translation efficiency of mrna [ ] . the length of ′ utr influences the localization of membrane proteins [ ] . it was reported that the long 'utr of cd enables efficient protein expression on the cell surface, whereas the short ′ utr primarily localizes the cd protein to the endoplasmic reticulum. an orf is the coding region that provides genetic information for protein expression. the base composition may have an impact on translational activity and stability of mrna [ , ] . for example, reduction of the frequency of uu and ua dinucleotides results in increased mrna stability and protein expression [ ] . besides this, replacing rare codons with frequently used synonymous codons is a common strategy to improve protein expression [ , ] . codon optimization in mrnas has been successfully applied in the past decade [ , ] , although its accuracy in human therapeutics is still questioned [ ] . further studies are required to verify the outcome of codon adaptation for mrna therapeutics. ivt mrna has an inherent immunostimulatory effect, as it can be recognized as exogenous rna by pattern recognition receptors (prrs) of the innate immune system, including toll-like receptors (tlrs) [ , ] and cytoplasmic retinoic-acid-inducible gene i (rig-i)like receptors (rlrs) [ ] . activation of these prrs induces a downstream cascade of the immune response, resulting in the expression of proinflammatory cytokines and type i interferons (ifns). ivt mrna-induced immune activation is considered beneficial for vaccination, because it may provide an adjuvant activity to facilitate dendritic cell maturation as well as t cell activation [ , ] . for non-immunotherapy applications, however, this immunostimulatory activity of ivt mrna is a major disadvantage since it can seriously reduce the translation efficiency of mrna [ ] . therefore, modulation of mrna immunostimulation could provide promising opportunities to further improve the therapeutic effect of ivt mrna. the immunostimulatory activity of ivt mrna can be reduced by incorporation of chemically modified nucleosides. pseudouridine (ψ), a naturally occurring modified nucleoside, is one of the most common modifications used in ivt mrna preparation. ψmodified mrnas reportedly diminish the activation of prrs, leading to a low immune response and high protein expression [ , ] . in addition, they reduced the activation of ′- ′-oligoadenylate synthetase and show increased resistance to rnase l-mediated cleavage [ ] . meanwhile, incorporation of chemically modified nucleotides into mrnas may potentially increase the translation efficacy of the mrnas. in a recent study, ψ was employed as a suitable chemical modification for mrna encoding ascpf protein, a type-v crispr-cas effector endonuclease from acidaminococcus sp. [ ] . the combination of modified crrna (cr ' f, containing five ′-fluoro ribose at the ′ terminus) and fully ψ-modified ascpf mrna increased the gene-cutting efficiency by over -fold compared to the control group with unmodified nucleosides. besides ψ, other modified nucleosides commonly utilized in mrna modifications (fig. ) . they include n -methyladenosine (m a), n -methyladenosine (m a), -thiouridine (s u), -methyluridine (m u), -methoxyuridine (mo u), n -methylpseudouridine (m ψ), -methylcytidine (m c), -hydroxymethylcytidine (hm c), -methoxycytidine (mo c), and nano res. , ( ): - -methylguanosine (m g) [ , ] . a recent study identified m ψ, mo u, and ψ as the top three favorable nucleosides for mrna modification, which significantly augmented protein expression [ ] . in addition, combining ψ and m c in modified mrna can produce higher protein expression compared to a single modification [ , ] . in another study, the combination of -thiouridine (s u) and m c in modified mrna considerably reduced activation of prrs and extended the protein expression to four weeks [ ] . m ψ modification alone or in combination with m c was superior to ψ or m c/ψ modification in its ability to reduce innate immune response and increase protein expression [ ] . although many studies on chemically modified nucleosides have contributed to the development of mrna modifications, the best modification remains unclear. moreover, it should be noted that while most chemically modified nucleosides can reduce mrna immunostimulation, translation efficiency of a modified mrna can sometimes be decreased with respect to that of an unmodified mrna [ ] . considering future in vivo studies and clinical applications, we need to screen the optimal modifications depending on the practical applications. due to its physicochemical properties that include large size and highly negative charge, chemically modified mrnas still face multiple extracellular and intracellular barriers. to overcome these barriers, a suitable delivery vehicle may be needed to facilitate the cell entry and endosomal escape of mrna, protect it against enzymatic degradation, and prolong its circulation life when used for systemic delivery. inspired by the experience from gene therapy and rna interference (rnai) therapy, viral and non-viral vectors have been explored as an mrna delivery tool for in vitro and in vivo applications [ , ] . however, viral vectors are associated with several inherent limitations, such as the risk due to their immunogenicity [ ] , limited loading efficiency, and difficulty of large-scale production [ , ] . alternatively, non-viral vectors, particularly those based on biocompatible nanotechnologies, are preferable for nucleic acid delivery due to their diverse properties. moreover, non-viral vectors are easier to synthesize and modify. although a number of non-viral vectors have been investigated for pdna and rnai delivery, these vectors have not always been effective for mrna delivery due to their different structures. here, we highlight the non-viral nanoplatforms for mrna delivery, including lipid nanoparticles, polymer nanoparticles, polypeptide nanoparticles, hybrid nanoparticles, and gold nanoparticle-dna conjugates (fig. ). lipid nanoparticles (lnps) represent a class of non-viral vectors formulated with synthetic or naturally derived lipids containing hydrophilic heads and hydrophobic tails. cationic lipids are often used to complex the anionic nucleic acid via electrostatic interaction. the utilization of cationic lipids for mrna transfection dates back almost years. then, n-[ -( , -dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (dotma) was demonstrated to condense and deliver luciferase-encoding mrna into various cell lines [ ] . subsequently the dotma derivative , -dioleoyl- -trimethylammonium-propane (dotap) was extensively studied for mrna delivery and better transfection efficiency was demonstrated [ , ] . in a recent study, the zwitterionic lipid , -dioleoyl-snglycero- -phosphoethanolamine (dope) was used in combination with dotap to facilitate endosomal escape, which enhanced gene expression [ ] . a number of lipid-based transfection reagents, such as lipofectamine tm , are commercially available and have been widely used for mrna transfection in vitro [ ] . however, these reagents are relatively toxic and have poor pharmacokinetics, limiting their in vivo applications. similar to lnp formulations used in small interfering rna (sirna) delivery, other components can be incorporated to optimize lnps for mrna delivery, which may significantly improve their transfection efficiency. typically, optimized lnps are composed of four components ( fig. (a) ): (a) a cationic or ionizable amino lipid to complex the nucleic acid and enhance endosomal escape; (b) a helper phospholipid to support the bilayer structure and facilitate cell uptake; (c) cholesterol to enhance the stability and promote membrane fusion; and (d) a polyethylene glycol (peg) conjugated lipid to reduce aggregation, avoid reticuloendothelial clearance, and decrease non-specific uptake [ ] . however, mrna has a structure distinct from sirna and may differ in its packing affinity with nanoparticles. therefore, it is critical to optimize the parameters for formulating lnps specifically for mrna delivery. to this end, a generalized strategy was developed to optimize lnp formulations for mrna delivery in vivo using design of experiment (doe) methodologies including definitive screening and fractional factorial designs [ ] . erythropoietin mrna-loaded c - lnps optimized for mrna delivery were screened by varying their formulation parameters (composition ratios or phospholipid types). this optimization resulted in a -fold higher potency than the previously used formulations for sirna delivery. new ionizable lipid or lipid-like materials have been also developed to improve mrna delivery. a class of n ,n ,n -tris( -aminoethyl) benzene- , , tricarboxamide (tt), which contains a phenyl ring, three amide linkers, and three amino lipid chains, was utilized to formulate lnps for mrna delivery in vivo. among them, tt was identified as the lead material for the most optimized formulation, improving the efficacy of luciferase-mrna delivery in vitro by over -fold with significantly reduced experimental workload [ ] . more recently, this formulation was employed to encapsulate both mrna and gadoliniumbased contrast agents [ ] . these dual-functional lnps showed comparable or slightly higher delivery efficiency for mrna than the original tt lnps. in another study, a series of bioinspired alkenyl amino alcohol ionizable lipids were synthesized to formulate lnp together with cholesterol, dope, and c -peg- for human erythropoietin coded mrna delivery [ ] . to successfully engineer the next generation of highly potent lnps, the intracellular delivery mechanisms of these lnp-encapsulated mrna should be understood. a recent study identified the late endosome/ lysosome formation as an essential process for nano res. , ( ): - functional mrna delivery [ ] . in addition to providing the structural backbone of the lnps, lipids can serve as signaling molecules that regulate endosome biogenesis and degradation [ ] . thus, incorporating bioactive lipids enriched with endo/lysosomal compartment into lnps can boost intracellular delivery of lnp-encapsulated mrna and protein expression [ ] . lnps are currently the most popular non-viral delivery system in clinical trials for genetic drugs [ , ] . in fact, alnylam has just announced positive data from the phase iii apollo trial (nct ) of patisiran (an rnai therapeutic formulated using lnp technology) for the treatment of transthyretin (ttr)-mediated amyloidosis. this represents the first rnai therapy that has been success in a phase iii clinical trial. it is expected to be approved by the food and drug administration this year. the success of this lnp formulation in sirna delivery has bolstered confidence in the potential of the approach for similar mrna delivery applications. along with the recent development of novel lipids or lipid-like materials, lnps remain promising candidates for the clinical translation of mrna nanomedicines. similar to cationic lipids, cationic polymers have also been commonly used to complex with mrna and generate polyplex nanoparticles through electrostatic interactions ( fig. (b) ). polymers are easy to synthesize and possess a high degree of chemical flexibility, which makes them attractive for nucleic acid therapy. these cationic polymers often consist of amine groups that are protonated in the acidic environment of endosomes (approximate ph . ), allowing the polyplexes to achieve endosomal escape due to the "proton-sponge" effect [ , ] . polyethyleneimine (pei) has a high density of amine groups. pei is the most studied cationic polymer and is the gold standard control in gene transfection [ ] . however, the potential toxicity associated with its high molecular weight and lack of degradability has historically prevented its broader application. to counteract this, researchers have attempted to develop a modified low molecular weight pei for mrna delivery. in one study, kda pei conjugated to cyclodextrin was used as a safe carrier for mrna encoding human immunodeficiency virus (hiv) gp . strong systemic and mucosal anti-hiv immune responses as well as production of cytokines were demonstrated [ ] . in another study, . kda pei with its primary amines modified by different aromatic domains was evaluated. only the salicylamide-modified pei was a reliable carrier for mrna delivery in hela and u cells [ ] . as an alternative to pei, poly (β-amino esters) (pbaes) exhibit several advantages, such as easy synthesis, relatively low cytotoxicity, and good degradability, and have been demonstrated as an effective delivery system for nucleic acids [ , ] . however, serum instability may be one of the drawbacks of pbaes for systemic administration. to address this problem, pbaes with alkyl side chains were developed for non-viral gene delivery. nanoparticles formed from these pbae terpolymers exhibited significantly increased stability in physiological conditions [ ] . based on these findings, polymerlipid nanoparticles based on the interaction of pbae terpolymer and peg-modified lipid were developed and used for the intravenous delivery of functional mrna to the lungs of mice [ ] . rapid developments in polymer chemistry have included controlled radical polymerization methodologies, which have been employed to construct a variety of cationic, multi-functional polymers with well-defined architectures and low material heterogeneity for gene delivery applications [ ] . among them, poly(dimethylaminoethyl methacrylate) (p(dmaema)) is one of the most intensively investigated gene vectors. however, whereas p(dmaema) bound strongly to pdna, its observed binding to mrna was weak [ ] . incorporation of peg to the side chains of p(dmaema) increased its ability to complex mrna and improved the transfection efficiency. another study used reversible additionfragmentation chain transfer (raft) polymerization to design a series of multifunctional triblock copolymers for intracellular mrna delivery [ ] . these materials are composed of a cationic dmaema segment to condense mrna, a hydrophilic peg methyl ether methacrylate (pegma) segment to improve stability and biocompatibility, and a copolymer of diethylaminoethyl methacrylate (deaema) and butyl nano res. , ( ): - methacrylate (bma) to facilitate cytosolic entry. the optimal architecture was that with the pegma block in the center of the polymer chain, which produced the greatest stability and highest transfection efficiencies. recently, the concept of reductive decationizable cationic block copolymers was introduced in mrna delivery. by combining raft copolymerization with post-polymerization modification, a cationic block copolymer bearing disulfide-linked primary amines was synthesized. this polymer could effectively condense mrna and subsequently release it in a reductive cytoplasmic environment [ ] . the dendrimer is another type of non-viral gene carrier that is being investigated. recently, a dendrimerbased nanoparticle system was developed to deliver antigen-encoding replicon mrna in mice to generate protective immunity against various lethal pathogen challenges, including h n influenza, toxoplasma gondii, and ebola virus [ ] . besides the synthetic polymers discussed above, some naturally occurring polysaccharides, such as chitosan, can also be utilized for nucleic acid delivery [ ] . with chitosan/ hyaluronic acid nanoparticles as the carrier material, mrna could successfully be delivered into the cd -expressing hct- cells [ ] . the mrna binding strength and the internalization rate of the nanoparticles were associated with chitosan molecular weight and the degree of deacetylation. although polymer nanoparticles are not as clinically advanced as lnps and some have only been studied in vitro by analyzing reporter protein expressions, their potential in mrna delivery is undeniable. further research is needed to explore their in vivo applications. moreover, further development and optimization of polymers with improved biocompatibility and transfection efficacy will facilitate the use of polymer nanoparticles for mrna delivery. protamines are a family of small peptides. they readily condense nucleic acids via electrostatic interactions. protamines were one of the first cationic materials explored for rna delivery, in [ ] . an earlier study reported that mrna could be protected from rnase degradation after condensation by protamine, and described that these protamine/mrna complexes can act as danger signals that activate several human blood cells in a toll-like dependent manner [ ] . another example of protamine/mrna-based therapy is the rnactive® technology, an mrna vaccine from curevac. the vaccine is composed of a mixture of naked mrna for antigen expression and protamine/ mrna complexes for immune stimulation [ ] . several clinical trials have evaluated rnactive® vaccines, one of which contains self-adjuvanted mrna encoding four antigens associated with prostate cancer (psa, psca, psma, and steap ) [ ] . when intradermally administered, the vaccine was well tolerated and immunogenic towards patients with advanced castration-resistant prostate cancer. poly(lysine), which bears amine groups on its side chains, has become widely used in pdna and sirna delivery since it was first investigated as a carrier material for nucleic acid. its application was recently extended to tumor-targeted mrna delivery [ ] . in this study, mrna was condensed by a mixture of crgd-peg-polylysine (plys) (thiol) and poly(nisopropylacrylamide) (pnipam)-plys (thiol), forming a stable nanoformulation with a core consisting of plys and mrna cross-linked by redox-responsive disulfide linkage. the nanoformulation protected mrna from degrading in harsh biological environments and improved tumor accumulation and gene expression in vivo. apart from the aforementioned traditional cationic peptides, much research has also been devoted to developing polypeptides with structures that can improve the efficiency of mrna delivery. one noteworthy example is a synthetic mrna delivery system based on a dendronized polypeptide (denpol) architecture that reportedly can efficiently deliver mrna to various cells. the denpol system described in one study contained a backbone of l-lysinedicysteine polymer with multiple lysine dendrons grown on the surface. the resulting system combined the advantages of the conformational flexibility of a linear polymer, the beneficial multivalent interactions of a dendrimer, and the reduced responsivity of the disulfide linkages in the polymer backbone [ ] . in a separate study, a range of polypeptides was synthesized by n-carboxyanhydride polymerization of l-benzyl nano res. , ( ): - aspartate, followed by an exhaustive amination of the ester groups to manufacture various cationic side chains for mrna co-encapsulation as well as a recombinant form of pbap. this co-delivery strategy produced a -fold increase in mrna expression in vitro [ ] . except for the protamine/mrna nanoparticles now undergoing clinical trials, most studies using polypeptide nanoparticles for mrna delivery are still in their infancy. more studies are required to facilitate the use of this kind of material in mrna delivery. in addition to the mrna delivery nanoplatforms based on a particular material, it is also important to highlight the hybrid nanoparticles combining various components, such as lipid, polymer, peptide, and even inorganic nanomaterials. as such, hybrid nanoparticles could integrate multiple beneficial features from their individual components, and may provide more functionality and flexibility to achieve efficient mrna transfection. among these hybrid nanoparticles, lipid-polymer hybrid nanoparticles (lpns) are an archetypal example of an emerging generation of therapeutic delivery vehicles [ ] [ ] [ ] . lpns have a classic core-shell structure comprised of polymer cores and lipid/lipid-peg shells. they exhibit complementary advantages of both polymeric and lipid nanoparticles. our group has developed various kinds of lpns for systemic rnai and cancer therapy [ , ] . similar platforms are being developed for the delivery of tumor suppressor encoding mrna for cancer therapy [ ] . generally, two strategies have been used to design lpns for mrna delivery. one involves the condensation of mrna with a cationic polymer or peptide into polyplexes, followed by envelopment with a "lipid" shell ( fig. (c) ). in this approach, a protamine/ mrna complex core and dotap/cholesterol lipid bilayers were inserted into dspe-peg to generate liposome/protamine/rna (lpr) nanoparticles. the lpr nanoparticles successfully delivered modified mrna encoding herpes simplex virus -thymidine kinase to xenograft-bearing nude mice to produce superior tumor growth inhibition compared to the equivalent pdna treatment by the same formulation [ ] . in a recent study, biodegradable hybrid nanoparticles composed of pbae/mrna core and peglipid were developed for the systemic delivery of luciferase-encoding mrna to the lungs [ ] . here, peg-lipid contributed to improved in vivo serum stability of the hybrid nanoparticles. another strategy for loading mrna in hybrid nanoparticles is to absorb them onto the lipid surface layer through electrostatic interactions (fig. (d) ). one study used hybrid nanoparticles containing a ph-responsive pbae core to promote endosomal escape and a phospholipid shell to minimize the toxicity of the polycationic core [ ] . negatively charged mrna absorbed onto the surface of the cationic hybrid nanoparticles displayed efficient transfection in vitro and in vivo. organic-inorganic hybrid nanoparticles are another type of hybrid nanoparticles that have been widely investigated as a means of drug delivery [ ] . carbonate apatite developed by akaike and colleagues is a ph-sensitive inorganic crystal that has a strong affinity for charged molecules. in their early studies using carbonate appetite as luciferase mrna carriers, the researchers did not observe luciferase expression. however, applying inorganic carbonate apatite onto cationic dotap/mrna complexes (fig. (e) ) substantially increased the luciferase expression in both mitotic and nonmitotic cells as compared to dotap/ mrna complexes [ ] . the high transfection potency of the carbonate apatite/dotpa/mrna hybrid nanoparticles was mainly attributed to the enhanced cellular contact and internalization facilitated by the apparently higher gravitational force and the positive charge of the absorbed inorganic particles [ ] . the transfection potency could be further improved by complexing fibronectin, an extracellular matrix (ecm) protein, into the hybrid nanosystem [ ] . different organic materials including polymers, lipids, dendrimers, peptides attached to diverse inorganic nanoparticles like gold, mesoporous silica, magnetic iron oxide, carbon nanotubes, and quantum dots have been widely used for efficient drug delivery and imaging [ ] . with continuing research in this area, the repertoire of organic-inorganic hybrid nanoparticles for mrna delivery will be expanded. nano res. , ( ): - gold nanoparticles (aunps) functionalized with thiol-terminated dna (aunp-dna conjugates) (fig. (f) ) are another nanoparticle system that has been demonstrated to be an efficient and universal nanocarrier for drug and gene delivery due to their high cellular uptake [ ] [ ] [ ] . aunps present another potential platform for cellular mrna delivery with their conjugated dna, which can be functional by having a sequence complementary to the mrna of interest. the use of aunp-dna conjugates to deliver bcl- -associated x (bax) mrna to xenograft tumors in mice allowed for the effective expression of bax protein, which inhibited tumor growth by inducing apoptosis [ ] . with the rational design of a dna oligomer, aunp-dna conjugates could also modulate the access and recycling time of ribosomes during mrna translation, enhancing the in vitro translation efficiency of mrna [ , ] . the progress of mrna technology along with the development of nanotechnology has enabled the utilization of mrna for a wide range of therapeutic applications. in the following section, we highlight the application of mrna nanomedicine in preventing or treating various diseases. the four major biomedical applications of mrna nanomedicine include: ) nanovaccines derived from antigen-encoded mrna for the activation of the immune system; ) proteinreplacement therapy for the treatment of genetic disorder diseases and cancer due to the mutation or loss of protein expression; ) gene-editing achieved by the co-delivery of cas -encoded mrna and grna; and ) cell programming and engineering through the introduction of mrna encoding for transcript factors or other functional molecules. some selected examples of mrna nanomedicine for biomedical applications are summarized in table . over the past decades, nucleic acid-based vaccines have emerged as attractive alternatives to conventional vaccine strategies. with the major technological innovation and research investment in exploring mrna as an immunotherapeutic tool, the field of mrna-based vaccines is developing rapidly, and some are currently in clinical trials [ , ] . since mrna is a non-infectious, non-integrating therapeutic agent with excellent translation efficiency, mrna-based vaccines are safer and more efficacious than live, attenuated virus and dna-based vaccines. vaccination in vivo can be achieved using nanoparticle-based systems as the mrna carrier. vaccination with an mrna-based nanovaccine is a multifaceted and multistep process. first, the nanoparticle system should be engineered to efficiently deliver antigen-encoding mrna into antigen presenting cells (apcs). second, mrna is translated into antigenic protein in the cytosol of apcs, and consequently processed into peptide epitopes with the aid of proteasomes to bind with the major histocompatibility complex (mhc) class. finally, the mhc-peptides are transferred to the cell surface, where they present the peptide epitopes to either cd + t cell or cd + t cells, resulting in corresponding immune responses (fig. ) . remarkable progress has been made to date in developing mrna-based nanovaccines for both infectious diseases and cancer. the earliest attempt to use mrna-based nanovaccine was reported in . anionic liposomes were utilized as a carrier for mrna that encoded influenza virus nucleoprotein. virus-specific cytotoxic t cell responses were induced when the nanovaccine was administrated intravenously or subcutaneously [ ] . since then, more mrna nanovaccines have been developed to induce protection from several viral pathogens via antigen-specific antibody and cellular immune responses. one of the commonly used mrna vaccines is the self-amplifying mrna (sam) platform based on an alphavirus genome [ ] , which contains an rna replication machinery gene with the structural protein genes replaced by those for protein antigens. after immunization, the antigen-encoding rna is capable of replication and amplification in the cytoplasm of the transfected cells, thereby generating a large amount of antigen even with a low vaccine dose. an early study demonstrated that a dose as low as . μg of sam vaccine encoding respiratory syncytial virus fusion glycoprotein (rsv-f) in an lnp delivery system produced effective cellular and humoral immune responses in mice, and μg elicited potent immune responses and conferred protection from further rsv infections in cotton rats [ ] . immunization with sam vaccines encoding influenza virus hemagglutinin (ha) in a cationic nanoemulsion protected mice from influenza virus challenges [ , ] . in other studies, sam vaccines encoding influenza antigens were successfully delivered to dcs by chitosan-nanoparticles and pei-based polyplexes, which were also reported to successfully induce humoral and cellular immune responses in mice [ , ] . besides influenza antigens, protection against several other viruses by sam-based nanovaccines has been demonstrated in various species. for example, a modified dendrimer nanoparticle system was developed to deliver multiple mrna replicons to generate protective immunity against a broad spectrum of lethal pathogens including ebola, h n influenza, and toxoplasma gondii [ ] . in another study, mrna encoding zika virus premembrane (prm) and envelope (e) proteins were encapsulated into the same nanoparticle system as a vaccine candidate against zika virus. the approach successfully elicited antigen-specific antibody and cd + t cell response in mice [ ] . the other strategy is to use non-replicating mrna for vaccination. rnactive® vaccines, developed by the curevac company, are a powerful technology for non-replicating mrna vaccination by combining naked mrna for expression of an antigen and protamine/mrna complexes that stimulate the immune system. the rnactive® technology induces balanced humoral and cellular immune responses in several animal models. the first report of the protective antibody response achieved by rnactive® vaccines was published in , in which direct intradermal injection of a mixture of naked mrna encoding various influenza infection virus and a protamine/ nano res. , ( ): - mrna complexes adjuvant resulted in protective immunity against influenza infection in multiple animal models including mouse, ferret, and pig [ ] . another study demonstrated that rnactive® vaccine encoding non-replicating rabies virus glycoprotein was capable of inducing potent neutralizing antibodies in mice and pigs and protecting mice from lethal intracerebral challenges [ ] . a first-in-human phase i clinical trial (nct ) is currently underway to evaluate the safety and immunogenicity of the rnactive® rabies vaccine [ ] . non-replicating mrna-based nanovaccines can also be directly administrated to induce an immune response in vivo. one example is hiv- gag encoding mrna complexed with dotap/dope, pei or polyethyleneimine stearic acid copolymer in mice, which reportedly elicited antigen-specific cd + and cd + t cell responses [ , ] . cyclodextrin-pei k conjugate-complexed mrna encoding hiv- gp also led to strong mucosal and systemic immune responses [ ] . lnp-formulated, modified mrna encoding ha protein of h n or h n virus generated rapid and robust immune responses in mice, ferrets, non-human primates, and humans [ ] . encouraged by these results, a phase i trial (nct ) is evaluating the safety and immunogenicity of h n vaccines in humans. in addition, a zika virus vaccine has been engineered by encapsulating modified mrna encoding zika virus prm-e into lnp. in one study, two doses of the vaccine resulted in enhanced antibody production that protected against zika virus infection [ ] . maternal vaccination with these vaccines elicited protection against placental damage and fetal demise in mice [ ] . a combined phase i/ii trial testing this zika virus vaccine is now ongoing (nct ). cancer immunotherapy aims to exploit the benefit of the activated immune system that can recognize and kill cancer cells through humoral and cellular immune responses. the most widely used application of mrna vaccines in cancer immunotherapy is to transfect mrna encoding tumor-associated antigens (taas) into patient-derived dcs in vitro and then re-administer these activated dcs to the patients. dc-loaded mrna vaccines against cancer are the most widely investigated nanovaccine variety in clinical trials [ ] . although this intervention shows good efficacy in eliciting a tumor-reducing immune response in cancer patients, it involves a complex manipulation process that may complicate its practical application regarding production costs, doses used, and intrinsic phenotypic variability. as an alternative, the direct injection of mrna-based cancer nanovaccines is a simpler way to improve the vaccination effects in vivo. the nanoparticle platform protects mrna from degradation by nuclease in body fluids and facilitates its uptake by apcs. in an mrna nanovaccine was formulated by encapsulating gp into hemagglutinating virus of japan (hvj)liposome. immunization by the direct injection of the nanovaccine into the spleen of mice elicited both anti-gp antibody and ctl responses against b melanoma [ ] . other delivery routes, such as intradermal, subcutaneous, and intranasal injection, have also been used to administer mrna-based nanovaccines in cancer therapy. due to the prevalence of apcs in the skin, intradermal injection is a favorable route for mrna cancer vaccination. in a trial reported in (nct ), one of seven patients with metastatic melanoma displayed a complete clinical response after being treated by the intradermal administration of protamine-stabilized mrna coding for six melanoma-associated antigens [ ] . intradermal injection of the rnactive® vaccine cv , which encodes four prostate specific antigens, to patients with castrate-resistant prostate cancer produced a safe but unexpectedly high level of cellular immunogenicity [ ] . subcutaneous administration also has been explored, in which the mrna nanovaccines are administered to the apcs that are prevalent in lymph nodes through the lymphatic system. the subcutaneous regions between the skin and skeletal muscles can be easily accessed, making it a convenient vaccination route. lnp-encapsulated mrna coding for the tumorassociated antigens glycoprotein (gp ) and tyrosinase-related protein (trp ) was used for melanoma vaccination. subcutaneous immunization of the vaccine with a single dose delayed tumor growth and prolonged overall survival in a b f melanoma nano res. , ( ): - mouse model [ ] . by intranasal administration, mrna-based nanovaccines can be directly delivered into immune cells within lymphoid tissues located in the nasal cavity, while avoiding the systematic barrier. since the intranasal immunization route is needle-free and non-invasive, it enables repeated administrations and leads to favorable patient compliance. in one study, nasal vaccination was demonstrated as an effective administrated route for cancer vaccination with ovalbumin (ova) encoding mrna nanoparticles [ ] . furthermore, systemic delivery of mrna can be achieved by administering mrna-based nanovaccines intravenously. nanotechnology engineering of mrna enables its stability in the bloodstream and ensures its delivery to dcs of the target organs, with the aim of inducing a subsequent anticancer immune response. in one study, mice were intravenously vaccinated with mart- melanoma antigen-encoding mrna in mannosylated and histidylated lipopolyplexes. the strategy resulted in the efficient delivery of the mrna to splenic dcs and led to a significant inhibition of b f melanoma growth [ ] . in another study, rna-lipoplexes (rna-lpx) were developed by complexing liposomes with mrna encoding four tumor antigens. the rna-lpx were intravenously injected to lymphoid dcs in mice. strong effector and memory t-cell responses were successfully induced, which mediated potent interferon-gamma-dependent rejection of progressive tumors [ ] . an ongoing phase i doseescalation trial (nct ) is testing rna-lpx that encode shared tumor antigens in patients with advanced malignant melanoma. combination of mrna-based immunotherapy with other therapeutic approaches may exert a synergistic effect in cancer therapy. for instance, combining tumor-specific rnactive® mrna vaccine with local radiation therapy produced a strong synergistic antitumor effect with the efficient eradication of large established e.g -ova tumors and lewis lung cancer tumors [ ] . combination immunotherapy of glycosylated type transmembrane mucin mrna nanovaccine and anti-cytotoxic t-lymphocyte-associated protein monoclonal antibody significantly enhanced the anti-tumor immune response for the treatment of non-immunogenic triple-negative negative breast cancer [ ] . more recently, the same nanoparticle system was employed to co-deliver mrna encoding trp and sirna silencing programmed death-ligand . the combinational therapeutic approach downregulated the expression of programmed death-ligand in the tumor antigen-presenting dcs and significantly promoted t cell activation and proliferation, resulting in an enhanced immune response against established melanoma [ ] . the field of mrna nanovaccines is experiencing a very exciting phase with several clinical studies in progress. however, while preclinical studies showed encouraging results for the potential of mrna nanovaccines in animal models, a recent clinical trial reported that vaccination with the cv rnactive® rabies vaccine in humans resulted in only modest immune responses [ ] . further research is needed to understand the different immune responses and mechanisms between animal species and humans. another challenge of mrna nanovaccines is the limited nanoplatforms for mrna delivery. although the rnactive ® platform and lnps have shown some potential for clinical use, the bottleneck has not yet been overcome for most formulations. detailed knowledge of the mechanisms of mrna delivery and antigen presentation of mrna nanovaccines is needed to screen suitable formulations and doses for optimized immune responses. addressing these issues will help to achieve the true potential of mrna nanovaccines in clinical translation. abnormal protein expression is a frequent cause of many diseases. the ability to normalize protein expression in vivo has a great potential in treating these diseases. rnai technology has been widely used to treat diseases characterized by over-expression of specific proteins [ ] . various sirna-based nanomedicines are now being assessed in clinical trials [ ] . alternatively, mrna-based nanomedicine is a new approach to diseases characterized by protein loss or malfunction, using ivt-mrna as the source of therapeutic proteins. the use of modified nucleotides and improved purification methods in ivt mrna preparation reduces the immunogenicity of ivt-mrna, which is critical for its application in protein-nano res. , ( ): - replacement therapy. as shown in fig. , after mrnaloaded nanoparticles are internalized into the cell, the mrna needs to be released and then recognized by the translation machinery in the cellular cytosol to finally bind with the ribosomal complex. the ribosomal complex scans the mrna sequence in the ′ to ′ direction and begins translation when it recognizes the start codon (initiation). the amino acids are added to the elongating peptide and the translation process continues until the ribosome recognizes the stop codon (termination). the newly synthesized peptide is released from the complex for post-translational modifications, yielding the new protein for replacement therapy. several diseases have been studied in which the in vivo protein production resulted following the systemic delivery of mrna nanoformulation. inducible hsp is the most protective of the heat shock proteins against subsequent hypoxia or ischemia. in one study, cationic lipids were used to deliver mrna encoding hsp to the central nervous system by intrathecal injection. lipid/mrna complexes were able to protect the mrna from degradation in human cerebrospinal fluid in vitro and the expression of reporter protein was successfully detected in coronal sections throughout the rat brain [ ] . this was one of the earlier attempts at using mrna-based nanoparticles for proteinreplacement therapy. cancer is characterized by multiple genetic disorders in the cancer cell genome, which drive cancer pathogenesis and development. correction of the cancer cell genetic disorders by, for instance, upregulation of tumor suppressor genes could be a promising approach for cancer therapy [ ] . bax is a pro-apoptotic molecule that functions as a tumor suppressor in various cancers. the use of cationic liposomes to transfer bax mrna has resulted in a stronger anti-tumor effect against malignant melanoma in vitro and in vivo compared to the pdna-based therapy in the same formulation. enhanced expression of bax protein was observed and caspase- activity increased significantly in hmg cells following transfection with liposome-bax mrna complexes [ ] . anti-angiogenesis therapy is another important strategy for inhibiting tumor growth, which has been intensively investigated in cancer treatments [ ] . in a recent study, soluble fms-like tyrosine kinase (sflt- ), an anti-angiogenic protein, was efficiently expressed in a pancreatic tumor-bearing mouse model after intravenous injection of mrna encoding sflt- encapsulated in a nanomicelle system stabilized by cholesterol. using this mrna-based nanomedicine for systemic anti-angiogenic therapy, a remarkable anti-cancer effect was observed for the first time in an intractable pancreatic cancer model [ ] . another promising application of mrna-based nanoparticles is to restore or augment metabolic enzymes that are associated with metabolic diseases. a codon-modified mrna encoding human methylmalonyl-coa mutase (hmut) was encapsulated in a biodegradable lnp system as a remedy for methylmalonic acidemia [ ] . intravenous administration of the therapeutic into two murine models of methylmalonic acidemia resulted in robust hepatic mut expression and improved growth and survival of mice. in a recent study, a hybrid mrna delivery system comprising a lipid nanoparticle for mrna protection and a polymer micelle for hepatocytes targeting was used to deliver human ornithine transcarbamylase mrna in a murine model of ornithine transcarbamylase deficiency, which led to pronounced nano res. , ( ): - synthesis of the desired protein in the liver and prolonged survival [ ] . apart from deriving the intracellular protein from the delivered mrna in the cytosol, the translated protein could instead be secreted into the extracellular space to systemically treat the protein deficiency. in two dependent studies, intravenous administration of nanoparticle-formulated mrna encoding human erythropoietin in mice, non-human primates, and even pigs resulted in elevated human erythropoietin protein levels in the serum [ , ] . hemophilia b is a disease caused by a single defective protein factor ix that normally produced by the liver. lnps were used to deliver human fix (hfix) mrna to the liver to treat hemophilia b in a fix knockout mouse model. the hfix mrna was effectively translated into functional fix protein by hepatocytes and secreted into the circulation where it alleviated the clotting defect of the mice [ ] . several other studies have also described the use of mrna nanoparticles for protein replacement therapies in various therapeutic areas including neurological disorders [ ] , fulminant hepatitis [ ] , bone defects [ , ] , and liver and lung fibrosis [ ] . considering the diversity of proteins that may be potential candidates for replacement therapy, together with the proven feasibility of nanotechnology for systemic mrna delivery, mrna nanomedicines are a promising candidate for therapeutics targeting any kind of functional protein deficiency. however, it must be noted that these endeavors are still at preclinical stages of their development. one of the major hurdles may be the lack of understanding of the exact pharmacokinetic and pharmacodynamic properties of these nanoparticle systems in humans. moreover, while there has been much success in vitro, the challenges of delivering a therapeutically active dose of mrna in vivo to the hard-to-access cells or tissues have not been thoroughly addressed. in the past several decades, genome editing has emerged as a powerful tool for therapy of genetic diseases since it can precisely delete, replace, and insert a dna sequence at a specific site in the genome [ ] . traditional nuclease-based gene targeting technologies, such as zinc finger nucleases and transcription activator-like effector nucleases, have been applied to generate a variety of animal and cellular models [ ] . however, these nucleases recognize the target gene sequence by protein-dna interactions, and thus require a customized protein for each target, which is a complex and time-consuming process. more recently, clustered regularly interspaced short palindromic repeats-crispr-associated protein (crispr-cas) technology, which was originally identified as a prokaryotic adaptive immune system that protects bacteria against foreign dna invasions [ ] , has provided a more precise platform for the gene editing of mammalian cells [ , ] . the most widely used crispr-mediated gene editing technology is the crispr-cas system derived from streptococcus pyogenes [ ] . the cas nuclease can be directed to cleave the target gene at a precise location using a single guide rna (sgrna) that is complimentary to the target dna sequence [ ] . the power of crispr-cas lies in the fact that cas nuclease can target any site in the genome simply by modifying the sequence of sgrna. moreover, it is also possible to simultaneously edit multiple independent genes using multiple sgrnas [ ] . effective gene editing by the crispr-cas system requires delivery of a functional cas -sgrna ribonucleoprotein complex to the cell nucleus. delivery of a plasmid dna encoding both the cas protein and sgrna is a simple and convenient format for the crispr process [ ] . however, this approach risks nonspecific editing and off-target effects due to the extended presence of cas in the cell [ ] and gene editing can be delayed by natural transcription and translation mechanisms. direct delivery of the ribonucleoprotein complex seems to be the most straightforward alternative that may potentially minimize the off-target cleavage [ ] . however, this approach also suffers the challenge of delivering the large cas protein across the two cellular barriers of the cell membrane and nuclear membrane. thus, co-delivery of sgrna and cas mrna provides an alternative method of crispr gene editing with the following benefits [ ] . mrna delivery leads to transient protein expression, which may be favorable nano res. , ( ): - in limiting the off-target editing. in addition, this approach avoids the challenge of crossing the nuclear membrane, as mrna does not require nuclear entry to exert its effect. in one study, the gene editing efficiencies of these three delivery formats (pdna, cas mrna/sgrna, and direct rnp delivery) were compared. cas mrna/sgrna delivery was superior to that of pdna delivery in all cell lines tested, likely due to the quicker onset of gene editing by the relatively stable cas protein directly translated from the mrna [ ] . cas mrna has been widely utilized to generate gene-modified animal models by disrupting or inserting dna sequences ex vivo in embryonic cells (zebrafish, mouse, rabbit, and monkey) with high efficiency [ ] [ ] [ ] , as well as to generate modified chimeric antigen receptor t cells for enhanced cancer immunotherapy [ ] . nevertheless, the systemic delivery of cas mrna by nanoparticles for in vivo applications was only reported two years ago. optimal in vivo genome editing is determined mainly by the delivery routes of cas mrna and sgrna. these two components can be either encapsulated into two separate systems or co-administrated in a single dose (fig. ). an earlier study used lnp-mediated delivery of cas mrna in combination with adenoassociated viruses carrying a sgrna and a repair template to generate fumarylacetoacetate hydrolase (fah)-positive hepatocytes by correcting the causative fah-splicing mutation gene mutation in a mouse model of human hereditary tyrosinemia [ ] . in another study, cas mrna and a chemically modified sgrna targeting pcsk were separately encapsulated in lnps. simultaneous administration of these two formulations in a single dose enabled the nearly complete editing of the target gene in hepatocytes in vivo [ ] . despite its success, cas mrna must first be translated into the cas protein. thus, the timing of delivery is another concern. a recent study demonstrated that the expression of cas protein was robust hours after the injection of the nanoparticle-formulated cas mrna and rapidly decreased at hours. thus, delaying the delivery of the sgrna to hours following mrna injection may enhance the editing efficiency [ ] . since mrna and sgrna are both single-stranded rna molecules, they are also readily co-delivered in the same nanoparticles (fig. ). this strategy guarantees that these two components are delivered to the same individual cells, and has achieved greater editing efficiency [ ] . an lnp-based delivery system that co-formulated cas mrna and grna into a single particle for simultaneous delivery resulted in a significant editing of the mouse transthyretin gene in the liver, with > % reduction in serum protein levels that persisted for at least months [ ] . the development of crispr-cas has revolutionized the genome editing field. while similar to the conventional mrna-based gene therapy, the delivery of cas mrna together with sgrna still faces many challenges that need to addressed. the rapid development of the crispr technology may provide a solution. for instance, the cpf nuclease has been identified as an alternative to cas [ ] . discovery and implementation of safe and efficient delivery systems or materials are critical for the successful application of the crispr technology in clinical settings. nano res. , ( ): - cellular reprogramming is the process of differentiated cells back into pluripotent cells. this has great potential in studies of normal development, constructing patientspecific disease models, and generating autologous tissues for cell-based therapies that can repair damages from injuries or illnesses. in , yamanaka and colleagues first demonstrated that adult somatic cells can be reprogrammed into induced pluripotent stem cells (ipscs) by transfection with pdna encoding four transcription factors (oct , sox , klf , and c-myc; also known as yamanaka factors) using retroviral vectors [ , ] . the retroviral delivery strategy carries the risk of genomic integration, which may endow the derived ipscs with tumorigenic characteristics due to the consistent expression of these oncogenic transcription factors. therefore, considerable efforts have focused on developing a safe and effective means of directing the fate of ipscs. since ivt mrna is transient and does not enter the nucleus, it eliminates the risk of insertional mutagenesis and tumorigenesis. in addition, mrna can be directly translated into transcription factors within the cytoplasm, which enables higher in vitro transfection efficiency than pdna. as shown in fig. , transcription factors are expressed from mrna in the target cell, enter the cell nucleus, and bind to the enhancer or promoter sequences of genomic dna to regulate specific gene expressions. these advantages of mrna make it a powerful tool to modulate cell phenotype and function, suggesting great promise for regenerative medicine [ ] . the pioneering use of ivt mrna to mediate reprogramming of somatic cells was reported in . ivt mrna encoding four transcription factors (oct , sox , lin , and nanog) was transfected using lipofectamine to reprogram human foreskin fibroblasts into ipscs [ ] . in the same year, cocktails of mrna encoding yamanaka factors were also used to generate ipscs, followed by transfection with myod mrna to induce further differentiation into myogenic cells [ ] . subsequently, other researchers attempted to improve the mrnabased reprogramming technology by optimizing the transcript factor cocktails [ ] or employing graphene oxide-polyethylenimine as the mrna delivery system [ ] . besides reprogramming somatic cells into ipscs, somatic cells can transdifferentiate and directly transform into the desired functional cell types through the transfection of mrna encoding specific transcription factors. for example, mrnas encoding three cardiac reprogramming factors (gata , mef c, and tbx ) were formulated into a c-lipo delivery system composed of a heart-targeting peptide and lipofectamine. cardiac fibroblasts were partially reprogrammed towards a cardiomyocyte-like state via daily transfection of this mrna nanoformulation [ ] . a similar strategy has been applied to reprogram other types of cells. pancreatic exocrine cells and human pancreatic duct-derived cells could be transdifferentiated into insulin-secreting β cells by transfecting with mrna encoding pancreatic transcription factors [ , ] . human fibroblasts could directly be reprogrammed into hepatocyte-like cells by synthetically modified mrna encoding hnf a plus any two of foxa , foxa , or hnf a in the presence of an optimized hepatic growth medium [ ] . another application of ivt-mrna is to engineer mesenchymal stem cells (mscs), which are attractive candidates for cell-based therapy to treat multiple diseases. mrna transfection has been successfully used to engineer mscs with enhanced homing properties. the engineered mscs simultaneously expressed a combination of homing molecules (p-selectin glycoprotein ligand- and sialyl-lewisx and immunosuppressive cytokine interleukin- ) [ ] . in another study, the delivery of arca '-cap analog modified mrna encoding integrin α subunit in mscs resulted the strategy may enhance cell adhesion and msc migrations [ ] . ivt mrna has great potential in cellular reprogramming and engineering, which will promote the development of regenerative medicine. considerable progress has been made; however, a variety of limitations remain to be overcome. due to the transient protein expression of ivt mrna, repeated daily transfections are required for the successful transformation of functional cells, which may be timeconsuming. one potential strategy to address this issue is to develop biomaterials or nanoparticles for sustained mrna release and protein expression. moreover, most of the reported studies were limited to in vitro cellular reprogramming using commercial transfection agents, with only a few studies having investigated their further applications in vivo. more efficient mrna nanoplatforms are needed to prolong mrna-mediated protein expression and facilitate their translation into practical application. mrna-based nanomedicine holds great potential in gene-based therapies. in the past decades, substantial advances have been made in the chemical modification of mrna, addressing the key challenges associated with its instability and immunostimulation. another major issue for the in vivo application of mrna is the efficient delivery of mrna to the desired cells while reducing the systemic exposure. advances in nanotechnologies and biomaterials for mrna delivery have improved the stability of mrna in a physiological environment and have realized an efficient strategy for the desired delivery of mrna. nevertheless, the application of mrna still faces a variety of challenges. first, the systemic administration of mrna formulations sometimes results in poor accumulation in the target tissue and/or cell, or insufficient penetration in the lesion tissue, which lead to the inefficient expression of desired protein. more efforts are required to further optimize the materials and formulations used for mrna delivery that would enable the efficient protein expression in hard-to-access tissue and/or cell. second, since the chemically modified mrna is not identical to the natural mrna in eukaryotic cells, the immunogenicity of the protein derived from ivt mrna may be another concern that needs to be considered. through rational and individualized design, the synthetic mrna should be closer to the natural mrna while not affecting the therapeutic efficacy. in addition, the high cost of synthesized mrna also limits its clinical application [ ] . continuing efforts are needed to develop more efficient synthesis methods of mrna to reduce the production cost. this review has presented four main categories of the recent applications of mrna nanomedicine: vaccination, protein-replacement therapy, gene-editing, and cellular reprogramming and engineering. it is worth noting that mrna-based nanovaccines have progressed rapidly for various infectious diseases and cancer, and some have progressed to clinical trials. however, other applications are still in preclinical stages. given that the advances continue and interest is sustained in this field, we can expect to see more mrna nanomedicines to enter clinical studies in the foreseeable future. an unstable intermediate carrying information from genes to ribosomes for protein synthesis use of frog eggs and oocytes for the study of messenger rna and its translation in living cells direct gene transfer into mouse muscle in vivo mrna vaccines-a new era in vaccinology a new developing class of gene delivery: messenger rna-based therapeutics current prospects for mrna gene delivery multi-level kinetic model of mrna delivery via transfection of lipoplexes single-cell mrna transfection studies: delivery, kinetics and statistics by numbers delivering the right message: challenges and opportunities in lipid nanoparticles-mediated modified mrna therapeutics-an innate 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end-dependent reverse ' caps in rnas made in vitro by phage rna polymerases novel "anti-reverse" cap analogs with superior translational properties synthesis of anti-reverse cap analogs (arcas) and their applications in mrna translation and stability phosphorothioate cap analogs increase stability and translational efficiency of rna vaccines in immature dendritic cells and induce superior immune responses in vivo the first examples of mrna cap analogs bearing boranophosphate modification synthesis, properties, and biological activity of boranophosphate analogs of the mrna cap: versatile tools for manipulation of therapeutically relevant cap-dependent processes translation, stability, and resistance to decapping of mrnas containing caps substituted in the triphosphate chain with bh , se, and nh synthetic dinucleotide mrna cap analogs with tetraphosphate cap analogs modified with , -dithiodiphosphate moiety protect mrna from decapping and enhance its translational potential mrna cap analogues substituted in the tetraphosphate chain with cx : identification of o-to-ccl as the first bridging modification that confers resistance to decapping without impairing translation multifunctional deadenylase complexes diversify mrna control effective induction of anti-melanoma immunity following genetic vaccination with synthetic mrna coding for the fusion protein egfp.trp tailing and '-end labeling of rna with yeast poly(a) polymerase and various nucleotides poly(a) tail shortening by a mammalian poly(a)-specific '-exoribonuclease modification of antigenencoding rna increases stability, translational efficacy, and t-cell stimulatory capacity of dendritic cells control of poly(a) tail length translational control by changes in poly(a) tail length: recycling mrnas mrna with a < -nt poly(a) tail imparted by the poly(a)-limiting element is translated as efficiently in vivo as long poly(a) mrna mrna transfection of dendritic cells: synergistic effect of arca mrna capping with poly(a) chains in cis and in trans for a high protein expression level pevl: a linear plasmid for generating mrna ivt templates with extended encoded poly(a) sequences synthetic messenger rna as a tool for gene therapy untranslated regions of mrnas molecular mechanisms of translational control translational control by '-untranslated regions of eukaryotic mrnas functional ' utr mrna structures in eukaryotic translation regulation and how to find them regulation by '-untranslated regions cellular ires-mediated translation ' utr m a promotes cap-independent translation at least six nucleotides preceding the aug initiator codon enhance translation in mammalian cells the role of the ' untranslated region in post-transcriptional regulation of protein expression in mammalian cells half-lives of beta and gamma globin messenger rnas and of protein synthetic capacity in cultured human reticulocytes alternative ' utrs act as scaffolds to regulate membrane protein localization synonymous but not the same: the causes and consequences of codon bias speeding with control: codon usage, trnas, and ribosomes uu/ua dinucleotide frequency reduction in coding regions results in increased mrna stability and protein expression codon bias and heterologous protein expression coding-sequence determinants of gene expression in escherichia coli modified tumour antigen-encoding mrna facilitates the analysis of naturally occurring and vaccine-induced cd and cd t cells in cancer patients design of an optimized wilms' tumor (wt ) mrna construct for enhanced wt expression and improved immunogenicity in vitro and in vivo a critical analysis of codon optimization in human therapeutics species-specific recognition of single-stranded rna via toll-like receptor and antiviral immunity via rig-i-mediated recognition of rna bearing '-diphosphates challenges and advances towards the rational design of mrna vaccines a development that may evolve into a revolution in medicine: mrna as the basis for novel, nucleotide-based vaccines and drugs designing and incorporating a real world data approach to international drug development and use: what the uk offers incorporation of pseudouridine into mrna yields superior nonimmunogenic vector with increased translational capacity and biological stability incorporation of pseudouridine into mrna enhances translation by diminishing pkr activation nucleoside modifications in rna limit activation of '- '-oligoadenylate synthetase and increase resistance to cleavage by rnase l engineering crispr-cpf crrnas and mrnas to maximize genome editing efficiency emergence of synthetic mrna: in vitro synthesis of mrna and its applications in regenerative medicine effects of chemically modified messenger rna on protein expression modified mrna directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction mammalian synthetic circuits with rna binding proteins for rna-only delivery expression of therapeutic proteins after delivery of chemically modified mrna in mice n -methylpseudouridineincorporated mrna outperforms pseudouridine-incorporated mrna by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice screening of mrna chemical modification to maximize protein expression with reduced immunogenicity engineering adenoassociated viruses for clinical gene therapy non-viral vectors for genebased therapy immune responses to gene therapy vectors: influence on vector function and effector mechanisms progress and problems with the use of viral vectors for gene therapy viral vectors: from virology to transgene expression cationic liposome-mediated rna transfection high performance mrna transfection through carbonate apatite-cationic liposome conjugates systemic activation of antigen-presenting cells via rna-loaded nanoparticles optimization of lipid nanoparticle formulations for mrna delivery in vivo with fractional factorial and definitive screening designs reprogramming human fibroblasts to pluripotency using modified mrna delivery materials for sirna therapeutics optimization of lipid nanoparticle formulations for mrna delivery in vivo with fractional factorial and definitive screening designs an orthogonal array optimization of lipid-like nanoparticles for mrna delivery in vivo dual-functional lipid-like nanoparticles for delivery of mrna and mri contrast agents bioinspired alkenyl amino alcohol ionizable lipid materials for highly potent in vivo mrna delivery boosting intracellular delivery of lipid nanoparticleencapsulated mrna chapter -studying lipids involved in the endosomal pathway lipid nanoparticles for targeted sirna delivery-going from bench to bedside lipid nanoparticle systems for enabling gene therapies polycation gene delivery systems: escape from endosomes to cytosol dual-responsive polyplexes with enhanced disassembly and endosomal escape for efficient delivery of sirna a welldefined coil-comb polycationic brush with "star polymers" as side chains for gene delivery enhanced intranasal delivery of mrna vaccine by overcoming the nasal epithelial barrier via intra-and paracellular pathways self-aggregating . kda polyethylenimines with dissolution switch at endosomal acidic ph are delivery carriers for plasmid dna, mrna, sirna and exon-skipping oligonucleotides poly-β amino ester-containing microparticles enhance the activity of nonviral genetic vaccines dual-responsive nanoparticles based on oxidized pullulan and a disulfide-containing poly(β-amino ester) for efficient delivery of genes and chemotherapeutic agents targeting hepatoma degradable terpolymers with alkyl side chains demonstrate enhanced gene delivery potency and nanoparticle stability polymer-lipid nanoparticles for systemic delivery of mrna to the lungs polymer vectors via controlled/ living radical polymerization for gene delivery pegylation improves nanoparticle formation and transfection efficiency of messenger rna multifunctional triblock copolymers for intracellular messenger rna delivery reductive decationizable block copolymers for stimuliresponsive mrna delivery dendrimer-rna nanoparticles generate protective immunity against lethal ebola, h n influenza, and toxoplasma gondii challenges with a single dose chitosans for delivery of nucleic acids chitosan/hyaluronic acid nanoparticles: rational design revisited for rna delivery protamine enhancement of rna uptake by cultured chick cells toll-like receptor-dependent activation of several human blood cell types by protaminecondensed mrna rnactive® technology: generation and testing of stable and immunogenic mrna vaccines self-adjuvanted mrna vaccination in advanced prostate cancer patients: a first-in-man phase i/iia study a targeted and stable polymeric nanoformulation enhances systemic delivery of mrna to tumors biodegradable dendronized polymers for efficient mrna delivery differentially charged hollow core/shell lipid-polymer-lipid hybrid nanoparticles for small interfering rna delivery ultra-small lipid-polymer hybrid nanoparticles for tumorpenetrating drug delivery cell membrane-derived nanomaterials for biomedical applications multifunctional envelope-type sirna delivery nanoparticle platform for prostate cancer therapy tumor microenvironment-responsive multistaged nanoplatform for systemic rnai and cancer therapy restoration of tumor suppression in vivo by systemic delivery of chemically-modified pten mrna nanoparticles systemic delivery of modified mrna encoding herpes simplex virus thymidine kinase for targeted cancer gene therapy in vitro and in vivo mrna delivery using lipid-enveloped phresponsive polymer nanoparticles selfassembled inorganic/organic hybrid nanoparticles with multi-functionalized surfaces for active targeting drug delivery drastic effect of nanoapatite particles on liposome-mediated mrna delivery to mammalian cells akaike, t. mrna delivery through fibronectin associated liposome-apatite particles: a new approach for enhanced mrna transfection to mammalian cell recent progress in delivery of therapeutic and imaging agents utilizing organicinorganic hybrid nanoparticles multifunctional dna-gold nanoparticles for targeted doxorubicin delivery delivery of shrna using gold nanoparticle-dna oligonucleotide conjugates as a universal carrier terminal pegylated dna-gold nanoparticle conjugates offering high resistance to nuclease degradation and efficient intracellular delivery of dna binding agents inhibition of xenograft tumor growth by gold nanoparticle-dna oligonucleotide conjugates-assisted delivery of bax mrna enhancement of in vitro translation by gold nanoparticle-dna conjugates exploiting the protein corona from cell lysate on dna functionalized gold nanoparticles for enhanced mrna translation induction of virus-specific cytotoxic t lymphocytes in vivo by liposome-entrapped mrna an alphavirus replicon particle chimera derived from venezuelan equine encephalitis and sindbis viruses is a potent gene-based vaccine delivery vector nonviral delivery of self-amplifying rna vaccines proc. natl. acad. sci rapidly produced sam®vaccine against h n influenza is immunogenic in mice induction of broad-based immunity and protective efficacy by self-amplifying mrna vaccines encoding influenza virus hemagglutinin self-replicating replicon-rna delivery to dendritic cells by chitosan-nanoparticles for translation in vitro and in vivo polyethylenimine-based polyplex delivery of self-replicating rna vaccines an rna nanoparticle vaccine against zika virus elicits antibody and cd + t cell responses in a mouse model protective efficacy of in vitro synthesized, specific mrna vaccines against influenza a virus infection an mrna vaccine encoding rabies virus glycoprotein induces protection against lethal infection in mice and correlates of protection in adult and newborn pigs safety and immunogenicity of a mrna rabies vaccine in healthy adults: an open-label, non-randomised, prospective, first-in-human phase clinical trial type i ifn counteracts the induction of antigen-specific immune responses by lipidbased delivery of mrna vaccines induction of hiv- gag specific immune responses by cationic micelles mediated delivery of gag mrna preclinical and clinical demonstration of immunogenicity by mrna vaccines against h n and h n influenza viruses modified mrna vaccines protect against zika virus infection vaccine mediated protection against zika virus-induced congenital disease rna melanoma vaccine: induction of antitumor immunity by human glycoprotein mrna immunization direct injection of protamine-protected mrna: results of a phase / vaccination trial in metastatic melanoma patients final analysis of a phase i/iia study with cv , an intradermally administered prostate cancer immunotherapy based on self-adjuvanted mrna lipid nanoparticle assisted mrna delivery for potent cancer immunotherapy intranasal mrna nanoparticle vaccination induces prophylactic and therapeutic anti-tumor immunity enhancement of dendritic cells transfection in vivo and of vaccination against b f melanoma with mannosylated histidylated lipopolyplexes loaded with tumor antigen messenger rna systemic rna delivery to dendritic cells exploits antiviral defence for cancer immunotherapy mrna-based vaccines synergize with radiation therapy to eradicate established tumors combination immunotherapy of muc mrna nano-vaccine and ctla- blockade effectively inhibits growth of triple negative breast cancer mrna vaccine with antigen-specific checkpoint blockade induces an enhanced immune response against established melanoma sojourn from discovery to delivery challenges and clinics clinical experiences with systemically administered sirna-based therapeutics in cancer stability of mrna/cationic lipid lipoplexes in human and rat cerebrospinal fluid: methods and evidence for nonviral mrna gene delivery to the central nervous system bax mrna therapy using cationic liposomes for human malignant melanoma combined effects on tumor growth and metastasis by anti-estrogenic and antiangiogenic therapies in mmtv-neu mice systemic delivery of messenger rna for the treatment of pancreatic cancer using polyplex nanomicelles with a cholesterol moiety systemic messenger rna therapy as a treatment for methylmalonic acidemia targeted mrna therapy for ornithine transcarbamylase deficiency hopx and the cardiomyocyte parentage therapeutic efficacy in a hemophilia b model using a biosynthetic mrna liver depot system systemic delivery of factor ix messenger rna for protein replacement therapy treatment of neurological disorders by introducing mrna in vivo using polyplex nanomicelles messenger rna-based therapeutics for the treatment of apoptosis-associated diseases transcript-activated collagen matrix as sustained mrna delivery system for bone regeneration chemically modified rna induces osteogenesis of stem cells and human tissue explants as well as accelerates bone healing in rats translation of angiotensin-converting enzyme upon liver-and lungtargeted delivery of optimized chemically modified mrna therapeutic genome editing: prospects and challenges genome editing with engineered zinc finger nucleases small crispr rnas guide antiviral defense in prokaryotes the crispr craze multiplex genome engineering using crispr/cas systems the crispr/cas bacterial immune system cleaves bacteriophage and plasmid dna the new frontier of genome engineering with crispr-cas one-step generation of mice carrying mutations in multiple genes by crispr/ cas-mediated genome engineering development and applications of crispr-cas for genome engineering guide-seq enables genomewide profiling of off-target cleavage by crispr-cas nucleases direct cytosolic delivery of crispr/cas -ribonucleoprotein for efficient gene editing high-frequency off-target mutagenesis induced by crispr-cas nucleases in human cells rapid and highly efficient mammalian cell engineering via cas protein transfection generation of gene-modified mice via cas /rna-mediated gene targeting effective gene targeting in rabbits using rna-guided cas nucleases generation of gene-modified cynomolgus monkey via cas /rna-mediated gene targeting in one-cell embryos targeting a car to the trac locus with crispr/cas enhances tumour rejection therapeutic genome editing by combined viral and non-viral delivery of crispr system components in vivo structure-guided chemical modification of guide rna enables potent non-viral in vivo genome editing a non-viral crispr/cas delivery system for therapeutically targeting hbv dna and pcsk in vivo non-viral crispr/cas gene editing in vitro and in vivo enabled by synthetic nanoparticle co-delivery of cas mrna and sgrna a single administration of crispr/ cas lipid lanoparticles achieves robust and persistent in vivo genome editing cpf is a single rna-guided endonuclease of a class crispr-cas system induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors induction of pluripotent stem cells from adult human fibroblasts by defined factors reprogramming of human fibroblasts to pluripotent stem cells using mrna of four transcription factors highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mrna feederfree derivation of human induced pluripotent stem cells with messenger efficient mrna delivery with graphene oxide-polyethylenimine for generation of footprint-free human induced pluripotent stem cells peptide-enhanced mrna transfection in cultured mouse cardiac fibroblasts and direct reprogramming towards cardiomyocyte-like cells reprogramming of pancreatic exocrine cells ar j into insulin-producing cells using mrnas for pdx , ngn , and mafa transcription factors v-maf musculoaponeurotic fibrosarcoma oncogene homolog a synthetic modified mrna drives reprogramming of human pancreatic duct-derived cells into insulin-secreting cells direct reprogramming of human fibroblasts to hepatocyte-like cells by synthetic modified mrnas mrna-engineered mesenchymal stem cells for targeted delivery of interleukin- to sites of inflammation translation, but not transfection limits clinically relevant, exogenous mrna based induction of alpha- integrin expression on human mesenchymal stem cells key: cord- -vu b a authors: panahi, heidar ali; bolhassani, azam; javadi, gholamreza; noormohammadi, zahra title: a comprehensive in silico analysis for identification of therapeutic epitopes in hpv , , and oncoproteins date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: vu b a human papillomaviruses (hpvs) are a group of circular double-stranded dna viruses, showing severe tropism to mucosal tissues. a subset of hpvs, especially hpv and , are the primary etiological cause for several epithelial cell malignancies, causing about . % of all cancers worldwide. due to the high prevalence and mortality, hpv-associated cancers have remained as a significant health problem in human society, making an urgent need to develop an effective therapeutic vaccine against them. achieving this goal is primarily dependent on the identification of efficient tumor-associated epitopes, inducing a robust cell-mediated immune response. previous information has shown that e , e , and e early proteins are responsible for the induction and maintenance of hpv-associated cancers. therefore, the prediction of major histocompatibility complex (mhc) class i t cell epitopes of hpv , , and oncoproteins was targeted in this study. for this purpose, a two-step plan was designed to identify the most probable cd + t cell epitopes. in the first step, mhc-i and ii binding, mhc-i processing, mhc-i population coverage and mhc-i immunogenicity prediction analyses, and in the second step, mhc-i and ii protein-peptide docking, epitope conservation, and cross-reactivity with host antigens’ analyses were carried out successively by different tools. finally, we introduced five probable cd + t cell epitopes for each oncoprotein of the hpv genotypes ( epitopes in total), which obtained better scores by an integrated approach. these predicted epitopes are valuable candidates for in vitro or in vivo therapeutic vaccine studies against the hpv-associated cancers. additionally, this two-step plan that each step includes several analyses to find appropriate epitopes provides a rational basis for dna- or peptide-based vaccine development. hpvs are a large branch of the papillomaviridae family, grouped in different genera (alpha-, nu-/mu-, beta-and gamma-papillomaviruses), with more than genotypes [ ] [ ] [ ] [ ] . the classification of papillomaviruses (pvs) has been based on l gene sequence. they are clinically divided into two groups: low-risk hpvs, like hpv and , which cause benign lesions (warts and benign papillomas), and high-risk hpvs (hrhpvs), like hpv and , which are a a a a a response. however, the addition of cd + t cell epitopes can significantly augment its strength and duration [ , , ] . cd + ctls commonly recognize intracellular-originated peptides presented by mhc-i molecules. they accommodate peptides with - residues; the ideal length is residues. while cd + helper t lymphocytes (htls) commonly recognize extracellular-originated peptides presented by mhc-ii molecules. they accommodate peptides with - residues or even more; the ideal length is [ ] [ ] [ ] [ ] [ ] . the strength of the interaction between a t cell receptor and a peptide-mhc complex (pmhc), depends on the presented peptide and the mhc structure [ , ] . the binding of a peptide to mhc-i molecule is the most selective stage in the way of peptide presentation [ ] . bioinformatics tools can predict the potential immunogenic epitopes from thousands of epitopes in a short time [ ] . generally, the algorithms of these tools range from ones programmed to determine peptide-mhc molecule binding data to those based on structural similarity, molecular modeling, and molecular docking [ ] . peptides that bind to a specific mhc molecule have sequence similarity. therefore, peptide sequence patterns have been used to predict their binding to mhc molecules [ ] . in recent years, the accuracy of these methods has increased strikingly, and more than % of natural epitopes have been recognized at a high specificity of % [ ] . this improvement in performance was achieved by the expanding experimental binding data, available in the immune epitope database (iedb) and analysis resource (http://www.iedb.org/), and by the improvement of machine-learning algorithms [ ] . regarding the fundamental importance of epitope prediction in vaccine development, we investigated the best potential cd + t cell epitopes from the e , e , and e oncoproteins of four prevalent hrhpv genotypes ( , , and ) in the world and iran [ ] , as shown in a two-step plan was designed to identify the most probable cd + t cell epitopes (fig ) . for the first step, mhc-i and ii binding, mhc-i processing, mhc-i population coverage and mhc-i immunogenicity prediction analyses, and for the second step, mhc-i and ii protein-peptide docking, epitope conservation, and cross-reactivity with host antigens analyses were considered. the second step analyses were performed only for the selected peptides in the first step. in jan , in order of priority, the refseq, reviewed or unreviewed sequences of hrhpv oncoproteins (e , e , and e ) were retrieved from the national center for biotechnology information database (ncbi) (http://www.ncbi.nlm.nih.gov/) and uniprotkb/swiss-prot database (http://www.uniprot.org/). the isoform sequences of hpv , , , and oncoproteins were retrieved from hpv t cell antigen database (http://cvc.dfci.harvard.edu/hpv/ html/search.php). all the sequences are accessible in supporting information (s file). binding of epitopes to mhc-i molecules is an essential step for antigen presentation to ctls. herein, it was predicted by four online servers, as illustrated in table . the hla supertypes and frequently occurring hla-i alleles provided by the servers were included in the analysis. however, when an allele (e.g., hla-b � : ) was not provided, but its allele group (i.e., hla-b � ) was available, we used the allele group instead of the allele. the used human and mouse alleles, or allele groups are provided in supporting information (s table) . currently, eight prediction methods are available in the iedb mhc-i binding prediction tool, i.e., iedb recommended [ ] , consensus [ ] , netmhcpan [ , ] , artificial neural network (ann) [ , ] , smm with a peptide-mhc binding energy covariance matrix (smmpmbec) [ ] , stabilized matrix method (smm) [ ] , comblib_sidney [ ] , pickpocket [ ] , netmhccons [ ] and netmhcstabpan [ ] . the iedb-recommended and consensus are not independent methods; they use ann, smm and comblib_sidney methods to generate a representative index for each predicted pmhc; the median of percentile ranks (prs) or binding scores obtained from the used methods is reported as a representative pr or consensus score in the iedb-recommended or consensus method respectively. the pr is calculated by comparing the half maximal inhibitory concentration (ic ) of subjected peptide against a group of random peptides from swiss-prot database. the ic value, expressed as nanomolar, shows binding affinity. the lower ic or pr means higher binding affinity. as a rough guideline, peptides with ic values < nm are considered as high affinity, - nm intermediate affinity and more than - nm low affinity. no known t cell epitope has got an ic value > nm to date [ ] . in this study, iedb recommended method was used. the outputs for each pmhc in this method consisted of a median pr, a method-specific ic , and a method-specific pr. predictions were made against frequently occurring human mhc-i alleles (including hla supertypes) and mhc-i mouse alleles. epitope length was set on , , , and mer. peptides with median pr < . are applied for the analysis. netmhcpan mhc-i binding prediction. netmhcpan server predicts binding of peptides to the known mhc molecules using anns method. it is trained on a combination of naturally eluted ligands ( human and mouse mhc-i alleles) and binding affinity data ( mhc molecules from human, mouse, cattle, primates, and swine). besides, the user can perform a prediction against any custom mhc-i molecule by uploading its full-length sequence [ ] . in this study, predictions were performed for , , , and mer peptides against frequently occurring human mhc-i alleles and mhc-i mouse alleles. pr thresholds for strong and weak binders were set on . and . , respectively. peptides with pr < . were applied for the analysis. rankpep mhc-i binding prediction. rankpep predicts binder peptides of a given protein sequence or sequence alignments to mhc-i and ii molecules. the algorithm of rankpep based on the comparison of sequence similarities, using position-specific scoring matrices (pssms) method. it employs profiles of a group of aligned peptides recognized to bind to a specific mhc molecule and creates a consensus sequence by determining the most common residue for each position. then, it allocates an optimal score to the consensus sequence, compares the score of the subjected peptide with the optimal score, and gives the peptide a percentile optimal value for comparison. finally, it highlights strong binders in red [ , ] . herein, the prediction was made against frequently occurring hla-i and h -i alleles. the server did not provide all common lengths of epitopes for all the mhc alleles. thus, the used alleles and their provided epitope lengths are shown together, as given in supporting information (s table) . syfpeithi mhc-i binding prediction. syfpeithi (http://www.syfpeithi.de/ -home. htm/) is a database of over published and verified peptide sequences of human, mouse, and other organisms, known as natural binders of mhc-i and ii molecules. when syf-peithi analyzes a peptide for binding prediction against a specific mhc-i allele, its scoring system evaluates every residue of the query and gives it an arbitrary value between and , according to whether it is an anchor, auxiliary anchor, or preferred residue. it allocates the value to those residues which slightly preferred in that particular position, to the ideal anchor residues, and - to - to those residues which exhibit an adverse effect on the binding ability. the sum of these values is the score of the peptide. the maximal score could vary between different mhc alleles [ , ] . herein, the prediction was made against frequently occurring hla-i alleles and h -i alleles. epitope length was set on , , , and mer. every predicted pmhc which got a score less than % of the reference sequence score was excluded from the analysis. the allele-specific reference sequence was selected from rankpep's consensus sequence [ ] , or our syfpeithi predicted epitopes, whichever got the highest score in syfpeihi server. the reference sequences, their sources, and their scores are given in supporting information (s table) . recognition of high immunogenic cd + t cell epitopes was the primary aim of this study. therefore, all predictions were primarily made against epitopes with - residue length. however, it was valuable to determine that which mer mhc-i epitope is the core peptide of the mhc-ii epitope(s) too. the core peptide lies on the mhc-ii molecule grooves, and play the central role in constructing pmhc. with this strategy, the short minimal predicted epitopes could be used in designing of synthetic long peptides (slps), resulting in peptide loading to both mhc-i and ii molecules. iedb mhc-ii binding prediction. in this study, the mhc-ii binding prediction was made by iedb mhc-ii binding predictor (http://tools.iedb.org/mhcii/) [ , , ] . iedb possess seven prediction methods for mhc-ii binding prediction: iedb-recommended, consensus [ ] , netmhciipan [ ] , nn-align [ ] , smm-align [ ] , combinatorial libraries [ ] and sturniolo's method [ ] . herein, the iedb-recommended method was used, and all peptides with pr< . were selected for the analysis. the prediction was made against human alleles (iedb reference set) and three mouse alleles, given in supporting information (s table) . the server has fundamentally set the epitope length on mer. each iedb-recommended method participated in the prediction process offered a core peptide ( mer) for each predicted epitope ( mer). we associated the mer mhc-ii core peptides with the mer mhc-i predicted epitopes to determine that which mhc-i epitope is the core peptide of the mhc-ii epitope(s) too. mhc-i t cell epitope processing predictions of e , e , and e oncoproteins are made by the iedb combined predictor (http://tools.iedb.org/processing/). this tool combines predictors of three main steps of mhc-i antigen presentation pathway (proteasomal processing, transporter associated with antigen processing (tap) transport, and mhc-i binding) and calculates a total processing score for each predicted epitope. it allows the user to choose a method from ann, smm, smmpmbec, comblib_sidney , netmhcpan, netmhccons and pickpocket methods for the binding prediction. in the current update ( ), the iedb team has changed the choice of the recommended prediction method for the processing tool to be netmhcpan . rather than a consensus, since the processing tools requiring an ic value, which the consensus method does not provide. furthermore, netmhcpan . has provided all mhc alleles and has performed the predictions very well in recent comparisons [ ] . there are two types of proteasomes, the housekeeping types which are expressed instinctively, and immuno types which are provoked by ifn-γ secretion. the immunoproteasomes are believed to improve the efficiency of antigen presentation [ , ] . in this study, the immunoproteasome option was selected. the program outputs for every predicted epitope consisted of proteasome score, tap score, mhc score, processing score (proteasome + tap score), total score (proteasome + tap + mhc score), and mhc-i ic . the tap scoring system calculates a-log (ic ) value for the binding of a peptide (or n-terminal of its precursors) to the tap molecules. the higher tap score, the higher transport rate. [ , , ] . herein, the analysis was made against the human and mouse mhc-i alleles used later in the iedb binding prediction, with the iedb-recommended method and other default settings of the program. epitopes with ic < nm for hla-i alleles and < nm for h -i alleles were included in the analysis. several factors could clarify the difference between epitope and non-epitope peptides; an essential factor is epitope immunogenicity, i.e., it could be recognized by t cells. some amino acids, particularly those with large and aromatic side chains (especially tryptophan, phenylalanine, and isoleucine), are associated with immunogenicity. moreover, the positions p - of a peptide are more critical for immunogenicity [ ] . in this study, the mhc-i immunogenicity of all predicted epitopes was determined by the iedb web server (http://tools.iedb.org/immunogenicity/) [ ] . this tool uses the properties of amino acids and their locations to predict the immunogenicity of a pmhc. the default option was selected to specify which positions of the query peptide to be masked from the analysis, because it masked positions which are also suggested for the most frequent human mhc-i allele, hla-a � : . iedb population coverage prediction tool (http://tools.iedb.org/population/) [ ] is used to predict the hla-i population coverage of all - mer predicted epitopes in the first step. this tool can accept a target population by two query levels: ) area-country-ethnicity and ) ethnicity alone. it can integrate allele frequency information retrieved from the allele frequency net database (afnd) (http://www.allelefrequencies.net/default.asp) [ ] . iedb also accepts custom populations with allele frequencies defined by users. since, hla-i and hla-ii t cell epitopes elicit immune responses from two different t cell populations (ctl and htl, respectively), the server provided three different population coverage modes: ) hla-i lonely, ) hla-ii lonely, and ) hla-i and hla-ii together. herein, the mhc-i promiscuous predicted epitopes and their binding hla-i alleles (ic < nm or pr< . ) were entered as inputs for the analysis against the world population. the primary aim of molecular docking is the prediction of the binding site of a ligand at a protein receptor surface, and then docking and modeling the ligand into the recognized site. in this study, the binding ability of the first step selected peptides to human and mouse mhc molecules, was analyzed by cabs-dock (http://biocomp.chem.uw.edu.pl/cabsdock/) server. the server uses a multistage procedure that involves multiple programs, with the cα-cβ-side chain (cabs) model at its heart. the detailed information about these stages is given in supporting information (s file) [ , ] . also, fig shows the pipeline of cabs-dock protocol [ ] . cabs-dock gets the d structure of the receptor and the sequence of the peptide as obligatory inputs. furthermore, there are some non-obligatory inputs as recommendations which could improve outputs. in this study, duplicate dockings for each peptide ( dockings in total) were done against the most significant human/mouse mhc-i and ii molecules which had at least one well-structured protein data bank (pdb) file in the rcsb protein data bank (https://www.rcsb.org/), as shown in table . these pdb files are in the complex with their peptidic ligand and some x-ray crystallography solution molecules (heteroatoms). thus, these excess molecules, as well as redundant mhc molecules were removed before executing docking process. since, the binding site of epitopes on the mhc molecules was well-known previously, the unlikely regions to bind masked before the analysis. cabs-dock returns ten representative models (medoids) as the best-simulated models and ranks them by cluster density (cd). cluster density is equal to the number of elements in a cluster divided by their average ligand root mean square deviation (rmsd). the higher cd value implies greater accuracy. ligand rmsd value shows the differentiation measure between cluster elements. as a guideline; rmsd < . Å means high accuracy; rmsd � . and � . Å means medium accuracy and rmsd > . Å means low accuracy [ ] . herein, the rmsd and cd of the best-simulated models were selected for the analysis. the best model, which has the highest cd value, is not necessarily the top-ranked model, because, in some cases, peptides were not attached to their binding site properly. thus, these malformed models were excluded from the analysis. it is important to note that, due to the different frequency of mhc alleles in human populations, the equal cd value of different mhc alleles, don't have equal value regarding population coverage. thus, to involve the effect of population coverage, the cd value of every model was multiplied by its allele population coverage (divided by hundreds for more facility) to obtain a weighted index. then, the sum of all hla-i or ii weighted indexes of each peptide was calculated to get a total docking score (tds), used as a score to compare the candidate peptides. it is the first time that the tds has been formulated and used for this purpose. this formula is also applicable to the similar docking scores obtained from other servers. the use of highly conserved epitopes in a vaccine formulation reduces the risk of tumor immune escape and provides broader protection against different virus strains or genotypes. thus, the conserved areas are preferred to use in therapeutic vaccines, if they are appropriate epitopes. herein, the epitope conservancy analyses for the first step selected peptides were done in three levels: . inter-isoform conservancy: the percent of conservancy between all isoforms of each e , e , or e oncoprotein. . inter-type conservancy: the percent of conservancy between hpv and (alpha-papillomavirus ), as well as between hpv and (alpha-papillomavirus ). . inter-hrhpv conservancy: complete ( %) conservancy between all hrhpvs (hpv , , , , , , , , , , , and ). the selected peptides in the first step were analyzed to find inter-strains and inter-types conservancy percentages by iedb tool, conservation across antigens, (http://tools.iedb.org/ conservancy/). the inter-hrhpvs conservancy analysis was done by the iedb and expasy clustalw servers (https://embnet.vital-it.ch/software/clustalw.html). cross-reactivity with host antigens can cause adverse immune responses. therefore, the selected peptides in the first step were checked for similarities with the mouse and human proteomes by the ncbi blastp tool (https://blast.ncbi.nlm.nih.gov/blast.cgi). regarding the studies, different peptides usually get different scores/ranks in different analyses. this inconsistency indicates that these results needed to be analyzed with an integrated approach. indeed, integrated approach is more practical and efficient in such conditions in comparison with analysis by analysis filtering approach, in which those epitopes are chosen for the next analysis that have gotten an acceptable score in the previous analysis. herein, the integrated approach was applied in both steps of epitope selection. since the ultimate goal of the discovery of therapeutic epitopes is to use them in human vaccines, only the scores/ranks of human alleles were used to rank epitopes in some studies. however, investigators usually test therapeutic vaccines on mouse species in preclinical trials, thus in the current study, the binding status of the predicted epitopes to mouse mhc-i alleles was also studied by several binding predictors and molecular docking, as well. as stated above, ctl-mediated responses play a crucial role in killing the malignant cells. besides, the binding of epitopes to mhc-i molecules is the most selective step for antigen presentation to ctls. therefore, in the first step, the selection was made primarily by the comparison of obtained mhc-i binding, processing and immunogenicity scores/ranks, and population coverage percentages. however, the mhc-ii binding ranks were actually of secondary importance to the selection process as an added advantage. additionally, the population coverage has a dual application. first, it determines the coverage of a given peptide in the target population. second, it is the best index for summarizing and evaluating of the hla-i binding predictions too, since it is calculated from the results of hla-i binding prediction analyses. in the first step, ten peptides (tables - ) from each hpv genotype oncoprotein ( peptides in total), which got better results in the first step analyses were selected for the second step analyses, including protein-peptide molecular docking, epitope conservation, and crossreactivity with host antigens. the individual detailed results of the mhc-i and ii binding (s file), mhc-i immunogenicity (s file) and mhc-i population coverage (s file) predictions, as well as, mhc-i and ii molecular docking (s file) and epitope conservation (s file) analyses are given in supporting information, as excel files. indeed, cabs-dock returns ten representative medoids as the best-simulated models and ranks them by cluster density (cd). cluster density is derived from two factors (the number of elements in a cluster and their average ligand rmsd) that is an advantage for this server. in the second step, five peptides out of ten selected peptides in the first step (tables - ) , which got better results in all analyses of both steps, were selected as the final-predicted epitopes. none of the final predicted epitopes showed more than % sequence similarity with mouse and human proteomes. high prevalence and mortality of oncogenic infectious pathogens such as hpv and helicobacter pylori have caused serious problems for humans. currently, people who are infected with hrhpvs but show normal cytology or precancerous lesions do not have any treatment option, causing the disease progress toward invasive carcinoma in some cases. unfortunately, no fda-approved immunotherapy exists for pre-existing hpv infections or their related cancers to date. immunotherapy of hpv-associated cancers by dna or peptide-based vaccines, depends on the recognition of highly immunogenic epitopes, inducing robust and specific immune responses, particularly cell-mediated responses against the malignant cells. the primary aim of this study was the prediction of cd + t cell epitope from the e , e and e oncoproteins, using a comprehensive two-step selection plan. these proteins chose because they play a pivotal role in the cell transformation, immune evasion, and maintenance of malignancy, as well as, their permanent expression (e and e ) by the malignant cells [ ] [ ] [ ] . expression of e oncoprotein occurs in the early phase of hpv infection. evidence indicates that e play a prominent role in the genesis of hpv-associated cancers, but is not essential for cancer progression [ ] , since when hpv genome integrates into the host genome, it usually results in the disruption of e , e , and e genes. therefore, targeting e protein provides an opportunity for treatment of hpv infections and preventing the precancerous lesions from the progression to established carcinomas [ , ] . some genotypes of hrhpvs are more involved in the genesis of epithelial tissue malignancies [ ] . thus, in this study, hrhpv , , and were targeted due to their high prevalence in the hpv-associated cancers, especially cervical carcinoma. there are several limitations for epitope prediction: ) the major drawback of peptidebased vaccines is low immunogenicity [ , ] . many studies have focused on enhancing immunogenicity using immune stimulating agents or adjuvants to avoid this problem. another solution is the use of agonist epitopes [ ] . epitope immunogenicity is a crucial factor in vaccine development. however, many of known natural epitopes when are analyzed in silico by iedb mhc-i immunogenicity predictor, do not obtain a high score. therefore, in this study, epitope selection was based on the integrated approach, in which one analysis does not play an important role alone. ) there are certain drawbacks associated with the function of each method invented for the mhc-peptide binding prediction [ ] . for this reason, several predictors and a molecular docking program were used to augment the prediction accuracy. ) some web tools have been developed for mhc-ii epitope prediction. since mhc-ii groove can bind to peptides with variable lengths, and different peptides have the different number of residues between their n-terminus and first anchor [ ], the exact assignment of mhc-ii core peptide would be a difficult problem which reduces the success rate of these prediction tools. therefore, most mhc-ii prediction tools did not usually make epitope predictions as accurately noted for mhc-i molecules [ , ] . in cancer immunotherapy, the ctl-mediated responses play the central role in eradication of malignant cells, and the binding of epitopes to mhc-i molecules is an essential step for antigen presentation to ctls. thus, in this study, predicted epitopes were primarily selected by their mhc-i binding and processing scores. however, the mhc-ii binding scores were actually of secondary importance to the epitope selection process as an extra advantage. additionally, there are several other essential determinants which significantly affect the outcomes, such as antigen processing, immunogenicity, population coverage, conservancy and cross-reactivity with host antigens. vaccine development requires a comprehensive approach to cover all these effectual elements, covered in this study. the primary aim of molecular docking is the recognition of binding site of a ligand at a protein receptor surface, and docking and modeling the ligand into this recognized site. in this study, cabs-dock server was used for molecular docking analyses. cabs-dock has several main advantages: ) the method does not require any data about the peptide structure and its binding site. ) during docking process, peptide conformation is entirely flexible. ) it is possible to apply dynamic conformational changes in the receptor structure and ) to exclude some receptor regions from the docking search, leading to the more efficient search in the vicinity of the binding site at a sensible time. [ , ] . in comparison with protein-ligand (small molecules) docking, protein-peptide docking analysis is more problematic, since significant conformational changes occur during the process. as a general rule, how much the length of the query peptide to be longer, there are more torsions and conformational flexibilities. additionally, in comparison to protein-protein interactions, protein-peptide dockings are more transient, and their binding affinities are notably weaker [ ] . these factors make structural predictions of long peptides very challenging. therefore, in this study, mer peptides were preferred for selection compared to other possible lengths. they are also preferred by all mhc-i molecules as epitope and by mhc-ii molecules as the core peptide of epitopes. moreover, expansion of or mer ctl epitopes to longer peptides may create a practical alternative, containing both cd + htl and cd + ctl epitopes; especially, when cd + htl epitopes, covering ctl epitopes, are not recognized [ ] . [ , , [ ] [ ] [ ] [ ] [ ] [ ] . however, the prediction of t cell epitopes inducing strong responses has remained a big challenge. for therapeutic hpv vaccines, many candidates have been designed to trigger the activation of ctls or htls, mostly by targeting two major hpv oncoproteins, e or e [ ] , and in a few studies, e oncoprotein [ , ] . as well as, several clinical trials have been launched for immunotherapy of hpv-associated cancers [ ], although, they have not been so immunogenic, to induce a sufficient cellular immunity and eradicate malignant cells completely. some studies have suggested that the use of e and e slps, containing both cd + htl and cd + ctl epitopes, led to more potency and durability of cd + t cell reactivity in vivo, in comparison with the minimal ctl epitopes [ , ] . in , as pioneers in hpv epitope studies, feltkamp et al. recognized the hpv -e sequence rahynivtf as an mhc-i epitope that can provoke ctl-mediated responses and eradicates established hpv l -induced tumor cells in mice [ , ] . this sequence is the first hpv -e predicted epitope in our study as well. in , kumar et al. studied hpv -e oncoprotein to predict the candidate t-cell and bcell epitopes [ ] . they have screened potent epitopes for mhc-i molecules according to pr and the immunogenicity score, using iedb mhc-i binding and immunogenicity predictors. they found a mer potent epitope, safrcfivyiifvy, having the lowest pr and the highest immunogenicity score, i.e., . and . , respectively. notably, our second hpv -e predicted epitope, safrcfivy, is the n-terminal part of safrcfivyiifvy, and our first predicted epitope, flihtharf, is the c-terminal part of the third epitope of their study, vyiplflihtharf. in , tsang et al. scanned the hpv -e and e oncoproteins for the match peptides with the consensus motif of hla-a binding peptides [ ] . the bimas algorithm [ ] was employed to rank probable binding peptides according to the predicted one-half-time dissociation of pmhcs. three potential ctl predicted epitopes of the e protein (klpqlctel, kiseyr-hyc, and qqynkplcdl) and three of the e protein (ymldlqpet, tlheymldl, and rtledllmgt) were selected. they showed the immunogenicity of these peptides was enhanced when their agonist epitopes were used. the klpqlctel and tlheymldl sequences are the seventh and the fifth predicted epitopes of hpv -e and hpv -e in our study, respectively. experimental evidences about hrhpv-derived epitopes in literatures are mostly limited to e and e oncoproteins of hpv and . among our first-step predicted epitopes: fllcfcvll and yiifvyipl from the e -derived epitopes [ ] , fafrdlcivy [ ] , cyslygttl [ ] , vydfafrdl [ , ] , kfyskisey [ ] , klpqlctel [ ] [ ] [ ] , iseyrhycy [ ] , eyrhycysl [ ] , klpdlctel [ , [ ] [ ] [ ] , fafkdlfvv [ , ] and klpdlctel [ , [ ] [ ] [ ] from the e -derived epitopes, rahynivtf [ ] , ledllmgtl [ ] , tlheymldl [ , [ ] [ ] [ ] , llmgtlgiv [ , , , ] , qaepdrahy [ ] , gtlgivcpi [ , ] , fqqlflntl [ ] and tlqdivlhl [ ] from the e -drived epitopes were reported as t-cell epitopes experimentally. besides, ivyrdgnpy, cyslygttl, klpqlctel and iseyrhycy from the e -derived epitopes, and rahynivtf and gtlgivcpi from the e -derived epitopes were also reported as hla ligands [ ] . others are novel epitopes that they also require experimental studies for validation. as far as we know, this is the first time that in a laborious in silico study for epitope prediction, e , e and e oncoproteins of hrhpv , , and have been investigated altogether. moreover, in previous studies, usually only one predictor tool was used for making epitope prediction, or if several tools were used, no integrated approach was employed to make the conclusion. we believed that our predicted epitopes are valuable candidates for further in vitro and in vivo therapeutic vaccine studies. additionally, the introduction of the ten epitopes for each hpv genotype oncoprotein in the first step of the study shows which region of each oncoprotein is rich of the epitope, and thus, is more suitable for use in the design of slps. notably, the previous in vivo studies have been conducted using slps of hrhpv-e and/or-e oncoproteins, in particular hpv oncoproteins [ , [ ] [ ] [ ] [ ] [ ] . furthermore, the two-step plan of this in silico study, which each step includes several analyses to find proper epitopes by an integrated approach, would provide a basis for rational epitope prediction. however, it could be more efficient by adding other useful analyses. further studies are recommended on the peptide binding assays, the design of polyepitope constructions including e , e and e epitopes, the expansion of the minimal ctl epitopes to longer peptides (slps), the use of various adjuvants, involvement of delivery routes, mouse immunization with the designed constructs, evaluation of immune responses such as cytokines, antibodies, ctls and tumor growth for finding the best construct for clinical trials. it is important that 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against cancer experience with synthetic vaccines for cancer and persistent virus infections in nonhuman primates and patients mechanisms of peptide vaccination in mouse models: tolerance, immunity, and hyperreactivity therapeutic vaccination against human papilloma virus induced malignancies immunologic treatments for precancerous lesions and uterine cervical cancer therapeutic cancer vaccines the authors sincerely thank dr. ali namvar and miss elnaz agi for their valuable guidance and comments during preparation of the paper. key: cord- -ion n b authors: de silva senapathi, upasama; abdul-cader, mohamed sarjoon; amarasinghe, aruna; van marle, guido; czub, markus; gomis, susantha; abdul-careem, mohamed faizal title: the in ovo delivery of cpg oligonucleotides protects against infectious bronchitis with the recruitment of immune cells into the respiratory tract of chickens date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: ion n b the in ovo delivery of cytosine-guanosine (cpg) oligodeoxynucleotides (odns) protects chickens against many bacterial and viral infections, by activating the toll-like receptor (tlr) signaling pathway. although the delivery of cpg odns in ovo at embryo day (ed) has been shown to reduce infectious bronchitis virus (ibv) loads in embryonic chicken lungs pre-hatch, whether in ovo delivered cpg odns are capable of protecting chickens against a post-hatch challenge is unknown. thus, our objectives were to determine the protective effect of the in ovo delivery of cpg odns at ed against ibv infection encountered post-hatch and, then, to investigate the mechanisms of protection. we found significantly higher survival rates and reduced ibv infection in the chickens following the pre-treatment of the ed eggs with cpg odns. at days post infection (dpi), we found an increased recruitment of macrophages, cluster of differentiation (cd) α+ and cd + t lymphocytes, and an up-regulation of interferon (ifn)-γ mrna in the respiratory tract of the chickens. overall, it may be inferred that cpg odns, when delivered in ovo, provide protection against ibv infection induced morbidity and mortality with an enhanced immune response. infectious bronchitis (ib) is mainly an acute and severe disease of the respiratory system of chickens [ ] . the causative agent, infectious bronchitis virus (ibv), belongs to the family coronaviridae [ ] . there is increasing evidence of ibv infection being reported in birds other than chickens [ , ] . although ibv induced changes are observed primarily in the mucosal surfaces of the respiratory tract, the virus is also known to cause pathology in the female reproductive tract and kidneys, with a varying degree of severity dependent upon the type of strain that infects and replicates in the aforementioned tissues [ ] [ ] [ ] . ever since the first record of ib in the early s [ ] , periodic ib outbreaks associated with the isolation of heterogeneous strains of ibv have been reported globally [ , ] . major losses to the broiler meat industry are due to carcass condemnation at processing, a poor feed conversion ratio resulting in poor weight gain, and mortality. ibv is considered a highly infectious agent with near % morbidity, and with mortality reaching - % [ , ] . in breeder and layer flocks, the major losses are due to reduced egg production during and after infection with ibv. the egg drop during ibv infection has been estimated to be between - % [ ] . furthermore, the downgrading of eggs because of a poor internal egg quality and egg shell quality also account for considerable production losses [ ] . the standard preventive measures, such as strict quarantine and biosecurity measures [ ] , do not seem to sufficiently control the disease. thus far, the most efficient method for controlling ibv is by vaccination [ ] . the immunization of chickens against ibv is mainly by live attenuated and killed vaccines [ ] . however, the emergence of variant ibv strains/serotypes arising from vaccinated flocks, among other factors, has led to vaccine failure and ib outbreaks. thus, the development of novel approaches as an alternative or adjunct in order to control the current measures against ibv is becoming increasingly important. toll-like receptor (tlr)s are a family of germ line encoded pattern recognition receptors (prrs) expressed on the surface or within the endosomal compartments of cells [ ] . these receptors are crucial for recognizing whole or segments of microbial pathogens, and they initiate key host immune defenses against inciting agents. among the tlrs, tlr (in mammals)/tlr (in birds) are the only receptors capable of distinguishing bacterial, parasitic, and viral dna containing cytosine-guanosine (cpg) motifs [ ] . several studies have demonstrated the immunostimulatory and therapeutic success of cpg oligodeoxynucleotides (odns) application in various host-pathogen interaction models [ ] [ ] [ ] . the protection provided by cpg odns against lethal challenges of extracellular bacteria, such as escherichia coli [ ] and salmonella typhimurium [ ] , and viruses, such as low pathogenic avian influenza virus [ ] and infectious laryngotracheitis virus (iltv) [ , ] , in chickens have been well documented. cpg odns are known to induce an array of cytokines; chemokines; and effecter molecules, such as interferon (ifn) α, β and γ, interleukin (il)- β, il- , il- , il- , tumor necrosis factor (tnf)-α, and nitric oxide (no) [ ] [ ] [ ] . these effecter molecules are believed to play a pivotal role in protecting the host against intra and extra cellular pathogens. whilst activating a variety of immune cells, it plays an integral role in bridging the innate immune system with the adaptive immune system directing immune responses toward t helper (th) response [ ] . a study that pre-treated chicken embryos with class b cpg odns at embryo day (ed) in ovo, and then challenged them with ibv ark strain the day after (ed ) , showed an increased up-regulation of ifn-γ, il- β, il- , il- , and oligoadenylate synthetase (oas) a in the embryonic spleen [ ] . additionally, the authors saw a significant reduction in the ibv nuclear (n) gene mrna expression in various embryonic tissues pre-treated with cpg odns, compared with the control, highlighting the value of cpg odn treatment in ibv control. however, they did not demonstrate whether the in ovo cpg odn delivery is effective against the ibv challenge encountered post-hatch. in this study, we determined whether the cpg odns delivered in ovo could provide protection against a post-hatch ibv challenge. furthermore, we looked into several cytokines and immune cells that may be activated with such protection. we found that in ovo delivery was protective against ibv challenge post hatch, suggesting a potential lasting protective effect of cpg odns towards ibv infection, which could be exploited for developing control measures. the specific pathogen free (spf) eggs from white leghorn layer hens were obtained from the canadian food inspection agency (cfia), ottawa, and were incubated according to the manufacturer's instructions in digital egg incubators (kingsuromax and rcom maru deluxe max, autoelex co., ltd., gimhae, gyeongnam, korea), located at the health research innovation centre (hric) , university of calgary. all of the animal care protocols as well as the use of live chickens, embryos, and spf eggs in our experiments, have been reviewed and approved by the health science animal care committee (hsacc, ac - , november ). at ed , the incubated eggs were candled in order to select viable eggs for further incubation, and the hatched birds were transported and housed in high containment poultry isolators at the prion/virology animal facility, hric, university of calgary, with access to ad libitum food, water, and necessary veterinary care. the ibv massachusetts (m) strain was purchased from the american type culture collection (atcc, manassas, va, usa) and was used in all of the experiments. nine day old spf viable eggs were used to propagate m strain of ibv, and the allantoic fluid was harvested at dpi by careful aspiration. the end point dilution assay was employed to assess the viral titers using ed spf eggs, and was expressed as a % embryo infectious dose (eid ) [ ] . the synthetic cpg odns, class b cpg motifs recognized by chicken tlr . the control odns were diluted in pbs to the same concentration ( µg in µl per egg), and were delivered via the same route (n = ). the in ovo tlr ligand delivery was carried out as described previously [ ] . on day post-hatch, the birds in both groups were infected with ibv m strain intra-trachealy, at a dose rate of . × eid per bird, and were monitored for days post-infection (dpi) for disease progression and outcome. the humane end point of the birds was determined based on the clinical score of each bird (ruffled feathers and huddling together = , droopy wings = , depression = , mild increase in respiratory rate = , increased respiratory rate with constant beak opening = , severe increased respiratory rate marked by gasping = , and body weight loss = ). the clinical score of was considered the humane endpoint. the eggs were then incubated for days until hatching. on the day of hatching, a subset of cpg odn-treated chickens (n = ) were infected with ibv m strain intra-trachealy at a dose rate of . × eid per bird, while maintaining the rest of the birds in that treatment group as uninfected controls (n = ). similarly, a subset of the control odn-treated chickens was infected with ibv m strain with the same dose (n = ), with the remaining birds being the control odn treated birds (n = ), and all the pbs treated birds (n = ) were kept as controls. the birds were weighed, wing tagged and after infection, were placed in separate isolators until the subsets of the animals were euthanized at (n = - per group) and (n = - per group) dpi. the clinical signs were observed and recorded daily as described, and the oro-pharyngeal and cloacal swab samples obtained using puritan ® unitranz-rt ® media transport systems (vwr, edmonton, ab, canada) at and dpi, and the ibv genome load were quantified following the rna extraction. simultaneously, the lung tissue was collected at and dpi in rna save ® (biological industries, froggabio, toronto on, canada), in order to determine the viral genome loads in lungs. to evaluate the ibv n antigen in the tracheal mucosal epithelium, the tracheal tissues from the dpi birds were collected and preserved in an optimum cutting temperature (oct) compound (tissue-tek ® , sakura finetek usa inc, torrance, ca, usa), and were snap frozen in dry ice until use in immunofluorescent assay. to observe the histopathology, the tracheal tissues of the dpi birds were fixed in % neutral buffered formalin (vwr international, west chester, pa, usa) and sent to the histopathology diagnostic services unit at the university of calgary, faculty of veterinary medicine, for hematoxylin and eosin (h and e) staining. additionally, the trachea and lung tissues were collected in an oct compound (tissue-tek ® , sakura finetek usa inc, torrance, ca, usa), snap frozen, and subjected to immunofluorescent assay so as to quantify the key innate and adaptive immune cells. another portion of the tissues were collected iusing rna save ® (biological industries, froggabio, toronto on, canada) for the cytokine mrna expression analysis. the animal numbers represent the total number of animals in two independent experiments. the total rna from the lungs collected at and dpi was extracted using a trizol reagent (invitrogen, canada inc., burlington, on, canada), according to the manufacturer's guidelines. for the rna extraction of oro-pharyngeal and cloacal swabs, the e.z.n.a. ® viral rna kit (omega bio-tek inc., norcross, ga, usa) protocol was adopted as per manufacturer's guidelines. the concentration of extracted rna was measured using nanodrop spectrophotometer (thermoscientific, wilmington, de, usa), with the absorbance at a / nm wavelength. two µg of total rna from the tissue samples and ng of total rna from the swab samples were used to synthesize the cdna with the use of the high capacity cdna reverse transcription kit (invitrogen life technologies, carlsbad, ca, usa), as per manufacturer's guidelines. a rt-pcr assay was carried out using fast sybr ® green master mix (invitrogen, burlington, on, canada) in order to quantify the ibv n gene and cytokine mrna expressions. rt-pcr assays were conducted in a well un-skirted, low profile pcr plate (vwr, edmonton, ab, canada), where the final reaction volume of the qpcr was maintained at µl. each qpcr run consisted of samples of interest, a positive control/s (gene specific plasmid), negative reverse transcriptase (nrt) control (cdna construct without the multiscribe reverse transcriptase enzyme), and negative template (ntc) control. all of the cdna samples originating from the tissues, along with the plasmid dilution series used to generate the standard curves, were run in triplicate. the target genes were quantified in relation to the β actin housekeeping gene. the target gene and the housekeeping gene for each sample was run on the same plate. five picomolar (pm) of different gene specific primers (forward and reverse primers) were used in each reaction (supplementary table s ). the change in the mrna expression of the cytokines was assessed using the pfaffl method [ ] . the optimum parameters used in the thermal cycler (cfx -c ) (bio-rad laboratories, mississauga, on, canada) were • c for seconds (s) of pre-incubation, • c for s, and • c for s for amplification cycles. a melting curve analysis was performed between • c and • c, with a . • c raise in temperature every s. the acquisition of fluorescent signals was performed at • c for s. for the cluster of differentiation (cd) α+ cell and macrophage (kul +) of the lung and trachea, µm thick sections were cut from the oct preserved tissues and were fixed using cold acetone for minutes (min). the tissues were then blocked by adding % goat serum diluted in a trizma buffered saline (tbs) buffer (trizma base: . g; nacl: g in l of distilled water; ph . ) at room temperature for min. after tipping off the excess blocking buffer, as the primary antibodies, the mouse monoclonal antibody specific for chicken macrophages/monocytes, kul + (southern biotech, birmingham, alabama, usa), cd α (ct- , southern biotech, birmingham, alabama, usa), was used in a : dilution in a % goat serum for min. the secondary antibody, goat anti-mouse igg (h+l) conjugated with dylight ® (red fluorescence) (bethyl laboratories inc., montgomery, tx, usa) was then used in a : in % goat serum for hour (h), followed by adding vectashield ® mounting medium with , -diamidine- -phenylindole dihydrochloride (dapi, vector laboratories inc., burlingame, ca, usa) (blue fluorescence), placing cover slips and edges sealed with lacquer as the final step. for the cd + t cell staining, before blocking the tissues with % goat serum, sections were blocked with avidin followed by biotin (vector laboratories, inc., burlingame, ca, usa), each with min incubation periods, in between washing with tbs-t for min twice and with pbs for min once. after blocking with % goat serum for min, a primary antibody, cd (ct- , southern biotech, birmingham, alabama, usa) was added in a : dilution in % goat serum for min. next, biotinylated goat anti-mouse igg (h+l) (southern biotech, birmingham, alabama, usa) was used as a secondary antibody in a : dilution in a % blocking buffer, and was incubated for min. then, dylight ® (green fluorescence) streptavidin in a : dilution was added for min, followed by a final step of mounting the slides with a vectashield ® mounting medium with dapi (vector laboratories inc., burlingame, ca, usa). all of the incubations were performed in a humidifying chamber at room temperature. each incubation with an antibody was followed by washing the slides in a tbs-t buffer for min twice and in pbs for min once. for the quantification of the tissue kul +, cd + cells, and cd α+ cells, five areas with maximum positive fluorescent signals of kul +, cd + cells, and cd α+ cells per tissue section were captured at x magnification, along with the corresponding nuclear stained (dapi) areas. the images were then subjected to fluorescent intensity quantification using image j software (national institute of health, bethesda, md, usa). the fluorescent intensities for the dylight ® (kul +, cd α+ cells) and dylight ® (cd + cells) positive signals were expressed relative to the total area (as estimated by nuclear staining with dapi), and were given as a percentage. a log-rank test was used to identify the differences in survival percentage. the kruskal-wallis test followed by the mann-whitney u test were used to identify the group differences in the clinical score data for each time point. the differences among the two groups were identified using othe student's t test. one-way analysis of variance (anova) followed by the students-newman-keuls test were used to identify the group differences in all of the other experiments. the grubbs' outlier test was performed in order to identify the outliers before the data was analyzed. the data in the graphs are shown in the original scale of the measurements. however, because of the non-normality and inability to satisfy the model assumptions of data belonging to the cell counts and cytokine mrna expression, a natural log transformation was applied to these data sets prior to analysis. model statistics were performed using graphpad prism software , la jolla, ca, usa. a normality test, generation of histograms, box plots, and q-q plots were performed in r statistical software, r studio version . . , boston, ma, usa. * = significant at p ≤ . , ** = significant at p ≤ . *** = significant at p ≤ . . we observed a significant increase in the survival rate of the cpg odn-treated chickens (p < . ) when compared with the control odn-treated chickens, as seen in figure a . also, the clinical signs in the cpg odn-treated ibv-infected group were significantly milder compared with the control odn-treated and ibv-infected group at dpi (p < . , figure b) . , and the rest were kept as in ovo cpg pre-treated uninfected controls (n = ). similarly, a subset of birds in the in ovo control odn-treated birds was infected with ibv (n = ), and the remaining birds were kept as in ovo control odn-treated uninfected controls (n = ). the in ovo pbs treated birds were kept as uninfected controls (n = ). a subset of birds from each group was sacrificed at dpi (n = - per group), and the remaining birds were sacrificed at dpi (n = - ) in order to obtain lung tissue. (c) ibv genome loads in oro-pharyngeal swabs at and dpi, (d) ibv genome loads in cloacal swabs at and dpi, and (e) ibv genome loads in and dpi lung. (f-g) the quantitative data and representative figures from the immunofluorescent assay of the trachea for ibv n antigen is presented. scale bar = µm (h) representative images of histological observations of trachea are given. control odns -ibv: severe epithelial metaplasia with severe cellular infiltration, germinal center formation is seen (a), superficial epithelial layer has become squamous with complete loss of cilia (b) and mucus glands not detected. cpg odns-ibv: pseudostratified simple columnar epithelium and intact ciliated epithelia (arrow) is evident where some have become rounded, and a few mucus secreting glands have been distorted and elongated (arrow head). cpg odns-control, control odns-control, and pbs-control: no lesions, normal pseudostratified ciliated columnar epithelium (c) with mucus secreting glands. log-rank test was used to identify the differences in the survival rate, and the kruskal-wallis test followed by the mann-whitney u test were used to identify the differences in the clinical scores at selected time points. the student's t test was performed to identify group differences in the oropharyngeal and cloacal genome loads, and one-way analysis of variance (anova) followed by the students-newman-keuls post hoc test was used to identify the differences in the lung ibv genome loads and ibv n antigen amount in the trachea. the differences were considered significant at * = significant at p ≤ . , ** = significant at p ≤ . *** = significant at p ≤ . . c-h: the animal numbers and results represent the pooled data of the two independent experiments. in order to assess the ibv genome loads in the lungs, and to determine the degree of virus shedding through the feco-oral route, the ibv n gene was quantified at and dpi in the birds that were pre-treated with in ovo cpg odns, control odns, and pbs. we observed a significant reduction in the viral genome loads in the oro-pharyngeal swabs collected and dpi (p < . ; figure c ), but did not observe a difference in the ibv genome loads in the cloacal swabs ( and dpi) between the treatment groups (p > . ; figure d ). however, significantly lower levels of lung viral genome load in the in ovo cpg odn pre-treated ibv infected group compared to in ovo control odn pre-treated ibv-infected group were observed at dpi (p < . ; figure e ). at dpi, although the control odn pretreated ibv infected lung had a significantly higher ibv genome load when compared with the uninfected controls (p < . ; figure e) , the difference of the ibv genome load in the lungs between two ibv infected groups was not significant (p > . ; figure e ). we observed a significant reduction of the ibv-n antigen in the tracheal mucosal epithelium of the in ovo cpg odn pre-treated-ibv infected birds compared with the in ovo control odn pre-treated-ibv infected birds (p < . ; figure f-g) . this was also seen in the histology of the trachea for the degree of tracheal damage following ibv infection. the mucosal epithelium of the in ovo control odn treated-ibv infected group showed severe metaplasia with severe mononuclear cell infiltration, where the superficial epithelial layer had been replaced by squamous cells. a complete erosion/loss of the entire mucosae was evident in several areas of the trachea. also, mucus secreting glands were absent from the remaining mucosae. in contrast, in the in ovo cpg odn pre-treated-ibv infected birds, the epithelium was a pseudostratified simple columnar epithelium mostly with intact cilia on the surface. mucus glands were present with some distortion and elongation (figure h ). in general, the in ovo cpg odn pre-treated ibv infected group recorded higher macrophage and cd + and cd α+ t numbers in the trachea compared with the uninfected and control odn pre-treated groups, although not all of the specific comparisons reached statistical significance (figure a-c) . similarly, the in ovo cpg odn pre-treated ibv infected group recorded higher macrophage and cd + and cd α+ t numbers in lungs compared to uninfected and control odn pre-treated groups, although not all of the specific comparisons reached statistical significance (figure d-e) . in the trachea and lungs, the macrophage and cd α+ t recruitment patterns, respectively, indicated that the in ovo delivered cpg odns are capable of increasing the recruitment of these cells in both the ibv infected and uninfected chickens (figure a the quantitative data following immunofluorescent assays done for the trachea (a) macrophages, (b) cluster of differentiation (cd) + t cells, and (c) cd α+ t cells are given. the quantitative data following the immunofluorescent assays done for lung (d) macrophages, (e) cd + t cells, and (f) cd α+ t cells are given. one-way anova followed by the students-newman-keuls post hoc test were used to identify the group differences. the differences were considered significant at * = significant at p ≤ . , ** = significant at p ≤ . *** = significant at p ≤ . . the results represent the pooled data of two independent experiments. considering that we observed a significant reduction in the ibv induced morbidity and mortality of in ovo cpg odn pre-treated birds correlating with varying degrees of increased macrophages, cd +, and cd α+ t cells in the tracheal and lung tissues, we needed to further elucidate the mechanisms by which these immune cells were efficiently recruited. several cytokine mrna expression levels in the lungs were analyzed at dpi, and our data showed a significant increase in the up-regulation of only the ifn-γ mrna expression in the in ovo cpg odn pre-treated lungs compared with the in ovo control odn pre-treated lungs, in both the ibv infected (p < . ) and uninfected (p < . ) groups (figure a-c) . in ovo delivery of poultry vaccines has been performed routinely for decades by the poultry industry [ ] . in ovo delivery targets the deposition of cpg odns in the amniotic cavity. subsequently, the ingestion of cpg odns containing amniotic fluid by the developing embryo distributes cpg odns in the respiratory and gastrointestinal tracts, leading to immune cell recruitment in these two body systems [ ] . we have shown in this study that cpg odns when delivered in ovo are capable of protecting young chickens against a post-hatch ibv infection induced ib. in ovo cpg odn-treated birds displayed reduced ibv viral loads in the lungs and a decreased ibv replication and pathology in the trachea, which is associated with high survival rates and low morbidity. we found that the macrophages in the trachea and cd + and cd α+ t cells in the lungs play important roles in this process, as increases in these cells were observed in the in ovo cpg odns treated group. lastly, we saw an up-regulation of ifn-γ mrna in the in ovo cpg odn pre-treated lungs, suggesting the critical role of this cytokine in the in ovo cpg odn-induced clearance of ibv infection. the host survival after day post-hatch ibv infection was seen as significant in the presence of cpg odn administration in ovo compared with the controls, and a similar protective effect of in ovo delivered cpg odns has been recorded against the post-hatch iltv infection [ , ] , and e. coli and salmonella thypimurium septicemia [ , ] . in the current study, the protection-mediated by the in ovo administered cpg odns was associated with significantly lower ibv replication in the trachea and ibv genome loads in the lungs at and dpi. consequently, the ibv genome loads in the oro-pharyngeal swabs were significantly reduced at and dpi. however, we did not observe a significant reduction in the ibv genome loads in the cloacal swabs at and dpi, because of the high variability of the ibv genome loads within the control odn pre-treated group. it is difficult to explain why we observed a discrepancy in the ibv genome loads between the oro-pharyngeal and cloacal swabs, as the in ovo delivered cpg odns have been shown to recruite immune cells into the gastrointestinal mucosa [ ] . our data confirm that, when delivered in ovo, the cpg odns are able to recruit macrophages into the trachea at dpi (four days of age), when compared to the in ovo delivered control odns in both the ibv infected and uninfected groups. previously, we saw that the in ovo delivered cpg odns increased the macrophages in the trachea at one day of age [ , ] . this macrophage recruitment to the trachea is associated with a lower ibv replication in the tissue, and it is possible that the cpg odn-mediated increase of the macrophages seen in the trachea in this study, played a central role in limiting the viral replication by three possible mechanisms. first, these cells may have efficiently and rapidly phagocytized the virus-infected cells and aided in virus elimination. second, they may have alerted the adaptive immune system to the invasion via active antigen presentation to the t cells. third, it may have contributed to the t and b cell activation and proliferation through the release of cytokines [ , ] . our observation of the increased recruitment of cd + and cd α+ t cells in the lungs following in ovo cpg odns delivery indicated that cpg odns could act as a mitogen, as has been shown previously [ ] . this cpg odn-mediated increased cd + and cd α+ t cell recruitment also could be due to the increased survival of these t cells in the lungs [ ] . interestingly, we saw an expansion of the cd α+ t cell population, but not the cd + t cell population in the in ovo cpg odn pre-treated-ibv infected lungs. although a portion of this increase of cd α+ cell recruitment could be potentially attributable to the ibv specific cd + t cells, we did not determine whether these cd α+ t cells are in deed ibv specific. we are at a loss as to why we did not see a similar cd + and cd α+ t cell response in the trachea, but it is possible that the in ovo delivered cpg odn-mediated cd α+ t cell recruitment is tissue specific [ ] . it is also important to note that our data is limited to dpi, and we do not know whether the in ovo delivered cpg odn-mediated cd + and cd α+ t cell recruitments in the trachea are occurring in other time points. of the examined immune mediators, ifn-γ, a dominant product of the t helper (th) type cells, was upregulated in the cpg odn pre-treated ibv infected and uninfected groups, when compared with the control odn pre-treated ibv infected and uninfected groups. the source of the lung ifn-γ mrna could be the cd + t cell lymphocytes and cd α+ cytotoxic lymphocytes [ , ] , and we observed an increased recruitment of the cd + and cd α+ t cells in the lungs in our experiment. two other immune mediators that were induced by the cpg odns and originated from the innate immune cells, such as the macrophages in the lungs, are the chemoattractant, il- β, and the no production inducer, inos [ ] . in the current study, we did not observe that the cpg odns or ibv induced the mrna expression of il- β or inos. this discrepancy in the cpg odn-mediated lack of il- β and inos expression can be explained by the difference in the time points observed. thapa et al. [ ] observed an increase il- β mrna expression in the lungs pre-hatch, and we observed a lack of il- β mrna expression post-hatch following in ovo cpg odns delivery. the significance of the observations described in our study are two-fold. first, we found that the in ovo administration of cpg odns is capable of limiting ibv replication in the lungs and trachea, leading to an increased survival and reduced morbidity in the early post-hatch birds. second, in our study, the early recruitment and maintenance of key immune cells, such as cd α+ and cd + t cells and macrophages, and the up-regulated ifn-γ mrna, exhibited not only an initiation of the early innate response, but also an effective and early adaptive host response mediated by the cpg odns, which would facilitate protection against the ibv infections encountered in birds in their immediate post-hatch life. further experiments elucidating the mechanisms of the cpg odn-mediated adoptive response, such as cell-and antibody-mediated immune responses in chickens and the duration of protection provided by this ligand against ibv, would be greatly beneficial in order to better understand the protective effects of cpg odns, and may aid in the development of more effective ibv control measures. to conclude, we show that the cpg odn-mediated protective response against post-hatch encountered ibv infection is associated with the up-regulation of ifn-γ mrna expression (in the lungs) and the enhanced recruitment of macrophages (in trachea) and cd + and cd α+ t cells (in the lungs). our findings, although preliminary, may provide a basis for developing novel control strategies in the long term against ibv infection in chickens. infectious bronchitis virus types: incidence in the united states induction of innate immune response following infectious bronchitis corona virus infection in the respiratory tract of chickens pathogenicity of australian strains of avian infectious bronchitis virus isolation of avian infectious 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that suppresses infectious bronchitis virus replication in chicken embryos cpg-odns induced changes in cytokine/chemokines genes expression associated with suppression of infectious bronchitis virus replication in chicken lungs bacterial dna-induced nk cell ifn-γ production is dependent on macrophage secretion of il- interactions between bacterial cpg-dna and tlr bridge innate and adaptive immunity a simple method of estimating fifty per cent endpoints the use of real-time quantitative pcr for the analysis of cytokine mrna levels hatchery vaccination against poultry viral diseases: potential mechanisms and limitations immunostimulatory cpg-modified plasmid dna enhances il- , tnf-α, and no production by bovine macrophages interleukin- -induced promotion of t-cell differentiation in mice immunized with killed listeria monocytogenes creating space: an antigen-independent, cpg-induced peripheral expansion of naive and memory t lymphocytes in a full t-cell compartment distinct effects of t-bet in th lineage commitment and ifn-γ production in cd and cd t cells il- up-regulates il- receptor expression on t cells, th cells, and b cells: synergism with il- for ifn-γ production this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we would like to acknowledge the staff of the prion/virology animal facility at foothill campus, university of calgary, for the experimental animal management. the authors declare no conflicts of interest. key: cord- -d d n qa authors: gu, daqian; ao, xiang; yang, yu; chen, zhuo; xu, xiang title: soluble immune checkpoints in cancer: production, function and biological significance date: - - journal: j immunother cancer doi: . /s - - - sha: doc_id: cord_uid: d d n qa immune checkpoints play important roles in immune regulation, and blocking immune checkpoints on the cell membrane is a promising strategy in the treatment of cancer. based on this, monoclonal antibodies are having much rapid development, such as those against ctla- (cytotoxic t lymphocyte antigen ) and pd- (programmed cell death protein ).but the cost of preparation of monoclonal antibodies is too high and the therapeutic effect is still under restrictions. recently, a series of soluble immune checkpoints have been found such as sctla- (soluble ctla- ) and spd- (soluble pd- ). they are functional parts of membrane immune checkpoints produced in different ways and can be secreted by immune cells. moreover, these soluble checkpoints can diffuse in the serum. much evidence has demonstrated that these soluble checkpoints are involved in positive or negative immune regulation and that changes in their plasma levels affect the development, prognosis and treatment of cancer. since they are endogenous molecules, they will not induce immunological rejection in human beings, which might make up for the deficiencies of monoclonal antibodies and enhance the utility value of these molecules. therefore, there is an increasing need for investigating novel soluble checkpoints and their functions, and it is promising to develop relevant therapies in the future. in this review, we describe the production mechanisms and functions of various soluble immune checkpoint receptors and ligands and discuss their biological significance in regard to biomarkers, potential candidate drugs, therapeutic targets, and other topics. immune checkpoints are molecules that can increase or decrease the signals of the immune system, and they are considered to be critical factors in treating infections, cancers and autoimmune diseases. currently, immune checkpoint therapy is seen as a pillar of cancer therapy [ ] . among the different checkpoint therapies, those involving pd- and ctla- may be the most effective. ctla- is considered to be the first functional immune checkpoint, as it stops t cells in lymph nodes at the initial stage of naive t-cell activation, while the pd- pathway suppresses activated t cells at the later stages of an immune response, typically in peripheral tissues [ ] . in clinical trials, the anti-ctla- antibody and the anti-pd- antibody have shown tremendous promise against a wide spectrum of solid and hematological malignancies, significantly improving os (overall survival) in newly diagnosed and heavily pretreated patients alike [ ] . however, the influences of soluble receptors and ligands on immune regulation and cancer treatment have been less well studied. soluble receptors and ligands, which are part of a family including full-length receptors and ligands, are produced by mrna expression or by the cleavage of membrane-bound proteins and are found free in the plasma. these entities may play important roles in immune regulation via interactions between soluble receptors and full-length ligands or between soluble ligands and full-length receptors. for example, alternative splice variants of the human pd- and ctla- genes have been identified, and spd- can interfere with pd-l / (programmed cell death ligand- / , also known as b -h / ):full-length pd- interactions, thereby blocking the negative signal imparted by the transmembrane form of pd- [ , ] . several studies have documented many types of soluble receptors and ligands that can be detected in the plasma in cancer, and the plasma levels are related to the severity of cancer. since previous studies suggested that soluble receptors and ligands should be considered therapeutic targets in cancer, we introduce some common therapeutic targets. we also review the production of these soluble receptors and ligands and discuss related clinical findings. we not only consider the significance of these receptors and ligands with regards to the prognosis and treatment in cancer but also consider their mechanisms of action. finally, we conclude the use of immunotherapy based on these soluble molecules. immune checkpoints can stimulate or inhibit signals in immune cells and regulate their functions; thus, the checkpoints play important roles in the maintenance of immune homeostasis [ ] . for example, t cells need two signals for activation: binding of the tcr (t-cell receptor) and the mhc (major histocompatibility complex) and an interaction between costimulatory molecules [ ] . on the contrast, pd-l expressed by some tumors works as a coinhibitory ligand with pd- to prevent t-cell activity [ ] . in the immune system, checkpoints can be divided into two groups: stimulatory molecules such as tcr/mhc and inhibitory molecules such as ctla- /cd or cd and pd- /pd-l . increasing numbers of novel receptors and ligands have recently been found in the immune system. some take part in costimulatory interactions, such as cd l/cd and ox l/cd [ ] , while others, such as hvem/btla and mhcii/lag [ ] , are involved in inhibitory interactions. apart from these, other receptors have also become renowned for their unique functions. for example, gal- /tim- can induce the inhibition of th cell responses [ ] . in fig. , we summarize the various ligand-receptor interactions fig. various ligand-receptor interactions between t cells and apcs (generalized to include all karyocytes). tim- , lag , pd- , pd-l , btla, and ctla- are coinhibitory molecules present on the surface of t cells. ox , tcr, cd , and cd can transfer stimulatory signals into t cells, and cd can receive stimulatory signals from cd l on t cells of immune checkpoints between t cells and apcs (antigen-presenting cells). immune checkpoints can transfer signals between different immune cells, changing their activities and regulating the secretion of cytokines in response to the microenvironment. for example, when the tcr on th cells combines with mhcii on apcs, the th cells are activated and can secrete il- and ifn-γ (interferon-γ) to enhance the antitumor effects [ ] . pd- and ctla- are excellent examples of immune checkpoints. unlike other members of the cd family, pd- can only transduce signals when crosslinked with bcr or tcr. various studies have confirmed that pd-l and pd-l are expressed in cancer cells, t cells, macrophages (mostly m macrophages), myeloid dcs (dendritic cells), myeloid suppressor cells, stromal fibroblasts, and endothelial cells, suggesting that pd- / pd-l /pd-l can influence many cell types. the pd- / pd-l pathway in the tme (tumor microenvironment) can inhibit the activation of effector t cells and promote the generation of treg cells; this shows that pd- -induced immune suppression may cause cancer cells to escape immune surveillance [ ] . the blockade of this pathway in many therapeutic strategies can promote antitumor effects. ctla- is expressed in t cells, b cells, nk cells, nkt cells, and dcs. ctla- is homologous to cd , but it has an approximately -fold higher affinity for both cd and cd than cd . similar to pd- , ctla- inhibits t cell activation by binding to its ligand [ ] . moreover, ctla- also inhibits il- production and influences naive cd + t cell differentiation. both antibody blockade of ctla- and genetic deletion of ctla- induce the generation of th cells and improve th cell differentiation. in addition, ctla- can control not only t cell effector functions but also b cell responses by regulating the functions of t follicular helper cells and t follicular regulatory cells [ ] . though the critical role of ctla- in controlling t cell activation and tolerance is well known, how ctla- exerts its inhibitory effects remains unclear. with the development of immunotherapy for cancer and other diseases, the demand for identifying immune checkpoints is growing. the fda has approved a series of antibodies targeting these checkpoints. in addition, some novel strategies based on the principle of immune checkpoints have been developed. for example, a combination of synergistic immune checkpoint blockade and targeted therapy is used to treat metastatic melanoma [ ] . because of the need for the endogenous molecule used for therapy, the formation mechanisms and functions of soluble receptors and ligands produced by human body, such as spd- , are being explored [ ] .due to the rapid development of immune checkpoint therapy, it will likely become the most effective way to fight cancer, although this therapy still has some limitations, such as a lack of power in the tme [ ] . in addition to the receptors and ligands of immune checkpoints on cell membrane, a series of soluble immune checkpoints have also been analyzed, and their plasma levels have been measured. these checkpoints play an important role in immune regulation, are involved in the development and prognosis of cancer, and are considered to be potential biomarkers and therapeutic targets. a summary of the information gathered on the soluble immune checkpoints is shown in table . the spd- was reported to be a monomeric protein [ ] . christian nielsen et al. found that spd- is generated from mrna expression. four alternatively spliced pd- mrna transcripts-pd- △ex , pd- △ex , pd- △ex , , and pd- △ex , , -were described apart from the full-length pd- . these variants are generated by splicing out exon ; exon ; exons and ; and exons , , and , respectively. in contrast to the other transcripts, which do not have obvious biological functions, pd- △ex is the soluble isoform of pd- and increases following the activation of pbmcs [ ] . one study on spd- found that its existence in tumor tissue promotes tumor-specific immunity, and in immunocompetent mice, a striking degree of immune cell infiltration was observed on local tumor, that was thought to be related to prolonged survival [ ] . in addition, in a study on nsclc (non-small cell lung cancer), elevated spd- was found in % of patients receiving erlotinib and these patients experienced prolonged progression-free and os [ ] . notably, in a cohort of hbv patients, higher spd- level appears to be associated with an increased risk of hcc (hepatocellular carcinoma) [ ] . spd- can inhibit all three pd-l/pd- interactions: pd-l /cd , pd-l /pd- , and pdl /pd- [ ] . osama et al. found that expressed spd- blocks pd-l /pd- interactions, which explains the inhibition of tumor growth after local gene transfer of spd- in tumor inoculation sites [ ] . researchers have also used adenovirus to transduce the thymidine kinase gene and spd- into tumors, which causes tumor regression by upregulating the activation of cd + t cells [ ] . moreover, in research on cancer treatments using a combination of the hsp vaccine and spd- , it has been found that spd- can not only block pd-l but also reduce the expression of il- gene, a negative regulatory gene [ ] . fibronectin ch has been demonstrated to increase the activity of macrophages, and in vivo studies have demonstrated that a spd- -ch recombinant peptide increases the cytolytic activity of both macrophages and cytotoxic t lymphocytes, especially towards pd-l -positive tumor cells. this effect is due to the increased production of inducible nitric oxide synthase, tnf-α (tumor necrosis factor-α), and ifn-γ [ ] . in addition, the combination of - bbl and spd- decreases the expression of il- and tgf-β in treated mice, thus inducing the expression of il- and ifn-γ and the accumulation of cd + t cells in the tme. furthermore, one research team constructed a recombinant eukaryotic expression plasmid encoding spd- to investigate the effects of a blockade of spd- /pd-l interaction, the antitumor response of t cells to spd- and the local therapeutic effect of spd- on mouse hepatocarcinoma. after coculturing spd- with tumor cells (h cell line) and spleen lymphocytes, the group demonstrated a dual effect of spd- : an enhancement of the immune response through interaction with immune cells such as dcs and a blockade of pd-l on tumor cells [ ] . above all, we can speculate that spd- can interact with pd-l and prevent pd- from combining with pd-l ; in other words, spd- competes with pd- in vivo [ ] . however, harmjan kuipers, et al. reported a different phenomenon. they cocultured dcs and t cells with spd- and observed an inhibition of t cell proliferation and il- production. they speculated that reverse signaling may take place when spd- binds to pd-l on dcs (see fig. ) [ ] . thus far, some treatments using spd- have been tested in mice, but the use of this treatment has not been reported in humans. however, mouse models can provide insight to improve future antitumor treatments for humans. it has been reported that when the hsp vaccine and spd- -which are used to treat cancer and to block pd-l , respectively-are combined, mice experience a significantly prolonged survival time compared to mice treated with hsp or spd- separately [ ] . in china, researchers used naked plasmids to deliver spd- -ch into tumor inoculation sites and for example, spd- has been reported to be a monomer, and it can be produced by pbmcs through mrna expression. moreover, spd- can combine with pd-l and pd-l , thus blocking pd-l/pd- interactions and somehow activating cd + t cells. some soluble receptors can be produced by the cleavage of membranebound proteins or by both the cleavage of membrane-bound proteins and mrna expression found that spd- -ch stimulates more effective antitumor activity than spd- , ch or spd- /ch , which shows that the recombinant protein spd- -ch can be used as a therapeutic strategy after the surgical removal of tumors [ ] . moreover, after researchers administered spd- dna with the human papilloma virus- e dna vaccine to mice, e -specific cd + t cell responses were significantly enhanced, resulting in potent antitumor effects against e -expressing tumors and causing a significant increase in the survival rate up to %; this suggests a role for spd- dna as a genetic adjuvant and for prophylactic antitumor treatment [ ] . in mice with h hepatocarcinoma, naked plasmid of - bbl and spd- were injected for local gene transfer; ultimately, this treatment completely eradicated tumors from mice with small numbers of preexisting tumor cells and eradicated tumors from % of individuals with larger numbers of preexisting tumor cells [ ] . production spd-l can be produced and released by both tumor cells and activated mature dcs, while immature dcs, macrophages, monocytes and t cells are refractory to releasing spd-l [ ] . spd-l is detectable in human serum, and its concentration increases with age; furthermore, it has been reported that increased mmpi (matrix metalloproteinase inhibitor) reduces the production of spd-l in pd-l transfected cells, suggesting that spd-l may be produced by the proteolytic cleavage of membrane-bound proteins [ ] . pd-l is encoded by the cd gene which comprises seven exons on chromosome in mice and on chromosome in humans. however, evidence that spd-l can be produced by alternative splicing has not been found [ ] . in a french multicenter randomized phase iii clinical trial, researchers found that the levels of spd-l in the plasma of patients with dlbcl (diffuse large b-cell lymphoma) were much higher than healthy humans. these patients were treated with high-dose chemotherapy and rituximab. then, patients with elevated spd-l experienced poorer prognosis, with a -year os rate of % versus % in healthy individuals. when patients were in cr (complete remission), their spd-l levels returned to normal [ ] . in a clinical study about malignant melanoma, early changes in spd-l levels after checkpoint blockade treatment did not correspond with benefit. however, rise in spd-l after months of treatment correlated with partial responses in ipilimumabtreated patients. and rise in spd-l after pembrolizumab treatment was also associated with partial responses, and high pre-treatment levels were associated with disease progression [ ] . for nivolumab-treated patients with nsclc, lower basal plasma levels of spd-l were associated with better clinical benefit, but the changes during treatment were still vague [ ] . in another study on patients with hbv-related hcc, circulating pd-l expression was closely related to intratumoral pd-l expression and pd- /pd-l expression was associated with tumor size, blood vessel invasion and bclc (barcelona clinic liver cancer) stage. moreover, patients with higher expression of circulating pd-l and pd- had shorter os and tumor-free survival times than those with lower expression. these results show that patients with higher spd- and spd-l levels have a worse prognosis [ ] . in a study on nktcl (natural killer/t-cell lymphoma), patients with a high concentration of serum spd-l (≥ . ng/ml) or with a high percentage of pd-l expression in tumor specimens (≥ %) responded poorly to treatment and exhibited markedly worse survival than patients with lower concentrations or lower percentages of expression. furthermore, a high concentration of serum spd-l and a high percentage of pd-l expression in tumor specimens can be independent adverse prognostic factors in patients with stage i~ii nktcl [ ] . similar to the study above, studies by both wang and huang's teams found that the overall response rate to treatment was higher in low spd-l patients than in high spd-l patients with mm (multiple myeloma), indicating a poorer prognosis in patients with higher levels of spd-l (> . ng/ml) [ , ] . in patients with oral squamous cell carcinoma, the increased expression of spd-l has also been found to be associated with poor prognosis [ ] . similarly, in hl (hodgkin lymphoma), spd-l levels are positively correlated with clinical stage [ ] . however, the phenomenon in advanced gastric cancer seemed to be contradictory to the above researches, in which adenocarcinoma patients with higher pd-l expression had much better prognosis and less lymph node metastasis than low-expression patients [ ] . a study on the role of spd-l found that ifn-γ secretion by cll (chronic lymphocytic leukemia) t lymphocytes decreases significantly in the presence of spd-l . conversely, treatment with an anti-pd-l antibody leads to a significant increase in ifn-γ secretion by cll t lymphocytes [ ] , and coincubation of cd + or cd + t cells with spd-l -producing cells and mdc-derived spd-l induces t cells to undergo apoptosis [ ] . as spd-l spreads throughout the body via the blood and lymphatic circulation, it exerts a widespread inhibitory effect by interacting with cell surface receptors such as membrane-bound pd- [ ] . two novel human pd-l splice variants have been identified. in the major variant, exon is cut off, and the protein product lacks the igc-like domain and is shorter in the extracellular region. although the other variant is also generated by cutting off exon , the acceptor site for that variant is bp downstream of the canonical acceptor site. this second variant also has a frameshift such that its protein product lacks the transmembrane domain and is secreted in a soluble form, which is thought to be spd-l . these findings suggest that spd-l expression may be controlled by posttranscriptional regulation through alternative splicing [ ] . production although the prominent source of sctla- is treg cells, the sctla- transcripts have also been detected in both monocytes and immature dcs [ ] . magistrelli et al. identified an additional splice variant named ctla- deltm that lacks both the transmembrane and intracellular domains. the splice variant, derived from the deletion of exon (which encodes the transmembrane domain and the cytoplasmic tail of ctla- ), is thought to be translated into sctla- . furthermore, ctla- deltm can be produced as a soluble monomer [ ] . there have been only a few reports on sctla- levels in the serum of patients with cancer. in a study, for ipilimumab-treated patients with melanoma, those who could respond to the treatment had higher serum levels of sctla- (mean = pg/ml) and experienced longer os [ ] . in addition, recent analyses of primary melanoma cell lines have demonstrated that the cells can secrete detectable levels of sctla- , supporting the relevance of this molecule in cancer. and in b-all (b-cell acute lymphoblastic leukemia) patients, the correlation between sctla- and neoplastic b cells was apparently negative [ ] . furthermore, it has been shown that sctla- is expressed by malignant b cells, at least in pediatric all patients, and the release of sctla- from acute lymphoblastic leukemia cells may constitute a strategy for immune-surveillance escape [ ] . analysis of human t cells in vitro has shown that sctla- secretion can increase during immune responses and has potent inhibitory properties, as isoform-specific blockade of sctla- significantly increases ag(antigen)-driven proliferation and cytokine (ifn-γ, il- ) secretion [ ] . similar to full-length ctla- , sctla- can bind to b costimulatory ligands on apcs to prevent b from combining with the costimulatory receptor cd in t cells, thus inhibiting t-cell responses. furthermore, sctla- can neutralize the anti-ctla- -mab in vivo. indeed, the inhibition of sctla- with anti-sctla- -mab induces significant increases in antigen-specific immune responses both in vitro and in vivo. in human peripheral blood mononuclear cell responses, the selective blockade of sctla- activates the proliferation of cd + and cd + t cells and promotes increased cytokine secretion, most notably the secretion of ifn-γ, which in turn enhances antitumor effects [ ] . as is the case for spd- , the affinity of sctla- /cd has not been reported. however, ctla- binds to cd and cd with kd values of . μm and . μm respectively, which are approximately -fold lower than the kd values for the binding of cd to cd and cd ( μm and μm, respectively) [ ] . thus far, there have been few studies on the implications of sctla- in cancer treatment. with regards to anti-ctla- -mabs, it is interesting that selective blockade of sctla- can not only enhance antigen-specific cd + and cd + t-cell responses but also exert functional antitumor activity without requiring an interaction with full length ctla- in a murine model of melanoma [ ] . scd production cd is a costimulatory factor mainly expressed on the surface of activated monocytes, b cells and dcs. kakoulidou et al. found that a spliced form, scd , is expressed in unstimulated monocytes and b cells. scd lacks the transmembrane domain and can bind to recombinant cd -ig, cd -ig and activated t cells [ ] . scd is thought to be a homodimer based on an analysis of its structure [ ] . in one clinical study, the scd levels in the majority of patients with aml (acute myeloid leukemia) ( / ) and mm ( / ) were normal. however, significantly elevated levels were detected in cll and mcl (mantle cell lymphoma) patients. furthermore, increased scd levels in cll patients were significantly associated with poor prognosis and were accompanied by low platelet and hemoglobin levels with elevated wbc counts and the expression of cd [ ] . kakoulidou et al. reported that recombinant scd has immunomodulatory effects, as shown by its inhibition of the mixed lymphocyte reaction and t-cell proliferation; they speculated that the preferential binding of scd to cd is responsible for the inhibitory reaction [ ] . in contrast, wei et al. posited that soluble b -igg can bind to ctla- on activated t cells with a high affinity, blocking the negative signals triggered by scd -which is different from the response triggered by membranebound cd [ ] . furthermore, sturmhoefel et al. found that soluble b -igg can induce t-cell proliferation in therapy for established tumors [ ] . more concrete mechanisms have since been studied. for example, one study found that a soluble form of cd , cd -fc (in which the extracellular domains of human or mouse cd are fused to the fc domain of igg ), increased the production of ifn-γ by pd- + activated t cells more effectively than antibodies to pd- or pd-l , possibly by neutralizing pd-l or costimulating with cd [ ] . suzanne et al. and samuel et al. achieved similar results, finding that cd -fc could sustain ifn-γ production by both human and murine pd- + activated t cells in the presence of pd-l + human or mouse tumor cells, respectively. they also found that cd -fc simultaneously inhibited pd-l /pd- -mediated immune suppression [ , ] . in a preclinical study, the cd -fc was used in combination with treg cell depletion, which dramatically controlled the colon tumor size and enhanced antitumor activity. furthermore, the mice in the study exhibited immunologic memory since they can reject subsequent implants in rechallenge experiments [ ] . in another study, the cd -fc fusion protein gene was delivered to tumor cells in vivo in the context of an oncolytic replication-competent herpes simplex virus [ ] . however, zhou et al. described a nonviral intramuscular gene transfer method to deliver this therapeutic protein, after which muscle tissue can exert immune costimulatory effects for cancer therapy by producing the protein in large quantities. this gene transfer method has also been used as an adjuvant therapy for dna vaccination [ ] . combination therapy has also been considered. for example, yasushi et al. combined il- , il- , and scd with oncolytic herpes simplex virus- vectors in a treatment and showed strong antitumor activity [ ] . scd production scd is produced by resting monocytes in human beings. jeannin et al. demonstrated that the scd detected in human serum can be generated by the translation of the cd △tm mrna, which is characterized by the deletion of the transmembrane domain. and scd is formed as a monomer [ ] . hock et al. reported that the plasma of a proportion of examined leukemia patients contained elevated levels of scd , but the scd levels were not directly related to crp (c-reactive protein)levels, suggesting that increases in scd are not solely related to a broad inflammatory response. furthermore, no relationship between scd levels and prognosis was found [ ] . in another study, levels of scd were elevated (> . ng/ml) relative to normal donors in % of patients with aml and in % of patients with mds (myelodysplastic syndrome). in addition, compared to aml patients with normal scd levels, patients with aml who had elevated scd levels experienced significantly lower cr rates and poorer survival. however, the correlation between scd levels and cr rates or survival rates in patients with mds was not found [ ] . in patients from the uk medical research council myeloma vith trial, hock et al. reported that serum levels of scd were significantly elevated. they also found that elevated scd levels were associated with significantly shorter survival (median = vs. months) and event-free survival times (median = vs. months) in abcm + p patients (patients receiving adriamycin, carmustine, cyclophosphamide, and melphalan with prednisolone), which suggested that scd may be an important prognosis marker in at least some myeloma treatment groups [ ] . there have been few reports on the function of scd in serum. juan et al. found that co-delivery of scd downregulated the immune response to a dna vaccine, suggesting that scd may bind to ctla- to transfer a negative signal to t lymphocytes [ ] . production zhang et al. demonstrated that sb -h is released by monocytes, dcs, activated t cells, and various mb -h + (membrane b -h + ) cells but not by mb -h − carcinoma cells. after the addition of mmpi, the release of sb -h from cells is blocked, indicating that the release of sb -h from b -h on the cell surface is mediated by a matrix metalloproteinase [ ] . moreover, chen et al. found that sb -h is also generated by alternative splicing of mrna [ ] . in one study conducted by a single center, the expression of sb -h and spd-l in the csf (cerebrospinal fluid) of the patients with glioma was higher than the patients with a moderate traumatic brain injury. furthermore, the expression of b -h and pd-l in csf and tumor tissues was related to the glioma grade [ ] . in clear cell renal cell carcinoma, both the serum level of sb -h and sil- r (soluble il- r) are significantly correlated with the clinical stage, and the level of sb -h shows a positive correlation with sil- r [ ] . in a study, sb -h concentrations were significantly higher in patients with eshcc (early-stage hepatocellular carcinoma) than in cirrhotic patients ( . ± . ng/ml vs. . ± . ng/ml). furthermore, high levels of sb -h were correlated with poor clinical outcomes [ ] . chen et al. measured the expression of sb -h in nsclc-derived mpes (malignant pleural effusions) and found that the median value of sb -h in mpes was higher than that in npes (nonneoplastic pleural effusions). moreover, the levels of mpe-derived sb -h were correlated with smoking status, primary tumor size (t factor), regional lymph node dissemination (n factor) and distant metastasis (m factor) in nsclc patients, suggesting that increased sb -h in mpes is correlated with the tnm stage of nsclc [ ] . sb -h can bind to the b -h r (b -h receptor) on activated t cells, showing that sb -h is functional [ ] . in chen et al.'s study, t cell proliferation was significantly inhibited in the presence of sb -h compared to the control group, and sb -h significantly reduced the levels of both il- and inf-γ in the culture supernatants compared to the levels in the control group, suggesting that sb -h can negatively regulate t cell responses [ ] . sun et al. found that sb -h can induce macrophages to increase the expression of mmr (macrophage mannose receptor) and il- and decrease the expression of hla(human leukocyte antigen)-dr and il- β in vitro, which can switch the macrophage phenotype from m to m [ ] . xie et al. observed that sb -h was highly expressed in mb -h + pancreatic carcinoma cells. additionally, sb -h promoted il- and vegf expression by first increasing tlr expression and then activating nf-κb signaling, which facilitated the formation of nascent blood vessels to help the cancer cells invade and metastasize [ ] . scd production similar to murine scd , human scd is generated by alternative mrna splicing [ ] . one study found that scd can be generated by pbmcs; notably, the expression of scd in lymphocytes requires strong activation, and the levels of scd negatively correlate with lymphocyte proliferation and positively correlate with the degree of activation-induced cell death caused by mitogen overstimulation [ ] . according to a small single-center study, patients with colon cancer have significantly higher plasma levels of scd than patients with rectal cancer ( ± pg/ml vs. ± pg/ml). interestingly, the levels of scd and scd l are significantly correlated, indicating that divergent mechanisms might be involved in the pathogenesis of colorectal cancer [ ] . enhanced levels of scd can be detected in the sera of patients with leukemia and lymphoma, and high scd levels are strongly associated with cll. however, why scd is present in only a proportion of patients and whether scd levels correlate with other parameters-such as disease stage, disease progression or therapeutic success-remain unclear [ ] . labiano et al. induced tumor cells to generate scd with hypoxia and demonstrated that tumor-secreted scd prevents the costimulation of t lymphocytes by preventing the interaction of cd l with the transmembrane forms of cd expressed on t lymphocytes [ ] . in one study, breast cancer cells were treated with scd in combination with saha (suberoylanilide hydroxamic acid), and the synergistic cytotoxic effect was enhanced, suggesting that a combination of saha and scd could be a potential cancer therapy [ ] . the natural soluble forms of receptors and ligands are important components of immune regulation, although their definitive mechanisms of action have not been determined. in this review, we selected spd- , spd-l , spd-l , sctla- , scd , scd , sb -h and scd for analysis. all of these molecules may play important roles in cancer. many studies regarding these entities are ongoing, and the relevance of soluble receptors and ligands to various diseases is becoming increasingly apparent. as soluble molecules, their serum and tissue such studies supply information for the formulation of therapeutic strategies. the type of analyses refers to the way of analysis of the associations between levels and prognosis or outcomes. + means higher than controls; − means lower than controls; \ in serum/plasma levels means no significant difference between patients and controls, \ in types of analyses and prognosis/outcomes means no data found levels can be easily detected. these molecules may also be critical factors for evaluating the severity and prognosis of cancer and many other diseases since most patients experience changes in their levels (see table ); in addition, some soluble molecules have been reported to be predictive markers for the benefit of target therapy (see table ). in immunotherapy, immunogenicity of checkpoint inhibitors is still a severe problem, and the detection of anti-drug antibodies is still equated as a main way to measure immunogenicity [ ] . according to the characteristics of soluble receptors, it is probable for them to neutralize the effect of monoclonal antibodies. furthermore, whether they are included or paly important role in the hypersensitivity reactions during therapy is also unknown, since the interaction and changes of levels of these molecules are complicated. thus, it is hopeful but there is a long way to find applicable soluble molecules to predict immunogenicity. moreover, their exact functions are still unclear. thus far, studies have developed methods to assess some of these proteins, such as spd- and sctla- . thus, we can use these technologies for further research. in addition to detecting the proteins, some researchers have successfully mediated their serum levels to regulate the human immune system, suggesting that such manipulations can potentially be used in cancer treatment. based on limited experimental and clinical findings, these soluble receptors and ligands can be novel therapeutic targets. although it has been established that the concentrations of soluble receptors can influence the activation of apcs and t cells, the specific relevance of these factors is still unknown; nevertheless, we can use antibodies such as anti-pd- -mab and anti-ctla- -mab to block these targets and neutralize their various functions in the progression of diseases. however, it may be necessary-yet difficult-to find more specific antibodies to precisely mediate these targets since current antibodies cannot distinguish between full-length receptors and soluble receptors. although it will be some time before the precise regulatory roles of these soluble receptors and ligands are illuminated, it is imperative that they are considered in the formation of strategies for immunotherapy. such studies supply information for the formulation of diagnostic tools. uncertain means the levels may be as same as control or be higher or lower than control in the study; not mentioned means no data found in the study the future of immune checkpoint therapy ctla- and pd- pathways: similarities, differences, and implications of their inhibition advances in cancer immunotherapy in solid tumors alternative splice variants of the human pd- gene a native soluble form of ctla- immune checkpoint inhibitors for cancer treatment new checkpoints in cancer immunotherapy anti-cytotoxic t-lymphocyte antigen- antibody: the first in an emerging class of immunomodulatory antibodies for cancer treatment the blockade of immune checkpoints in cancer immunotherapy novel antibodies targeting immune regulatory checkpoints for cancer therapy a role for the tim- /gal- /bat pathway in determining the clinical phenotype of multiple sclerosis interleukin- is required for cancer eradication mediated by tumor-specific th cells the pd :pd-l / pathway from discovery to clinical implementation targeting cytotoxic t-lymphocyte antigen- (ctla- ): a novel strategy for the treatment of melanoma and other malignancies coinhibitory pathways in the b -cd ligand-receptor family combining forces: the promise and peril of synergistic immune checkpoint blockade and targeted therapy in metastatic melanoma in vivo blockade of the programmed cell death- pathway using soluble recombinant pd- -fc enhances cd + and cd + t cell responses but has limited clinical benefit enhancing t cell therapy by overcoming the immunosuppressive tumor microenvironment structural and functional analysis of the costimulatory receptor programmed death- reconstructed adeno-associated virus with the extracellular domain of murine pd- induces antitumor immunity increase in soluble pd- is associated with prolonged survival in patients with advanced egfr-mutated non-small cell lung cancer treated with erlotinib circulating programmed death- as a marker for sustained high hepatitis b viral load and risk of hepatocellular carcinoma enhancement of vaccineinduced primary and memory cd (+) t-cell responses by soluble pd- adenovirus expressing both thymidine kinase and soluble pd enhances antitumor immunity by strengthening cd t-cell response hsp vaccine in combination with gene therapy with plasmid dna encoding spd- overcomes immune resistance and suppresses the progression of pulmonary metastatic melanoma regulating immunity and inhibiting tumor growth by the recombinant peptide spd- -ch investigation on the effects of soluble programmed death- (spd- ) enhancing anti-tumor immune response blockade of b -h with spd- improves immunity against murine hepatocarcinoma contribution of the pd- ligands/pd- signaling pathway to dendritic cellmediated cd + t cell activation soluble pd- facilitates - bbl-triggered antitumor immunity against murine h hepatocarcinoma in vivo soluble b -h : differences in production between dendritic cells and t cells development of a sandwich elisa for evaluating soluble pd-l (cd ) in human sera of different ages as well as supernatants of pd-l + cell lines identification of a novel splice variant of human pd-l mrna encoding an isoform-lacking igv-like domain high level of soluble programmed cell death ligand in blood impacts overall survival in aggressive diffuse large b-cell lymphoma: results from a french multicenter clinical trial soluble pd-l as a biomarker in malignant melanoma treated with checkpoint blockade soluble programmed cell death ligand as a novel biomarker for nivolumab therapy for non-small-cell lung cancer upregulation of circulating pd-l /pd- is associated with poor post-cryoablation prognosis in patients with hbv-related hepatocellular carcinoma pd-l is upregulated by ebv-driven lmp through nf-kappab pathway and correlates with poor prognosis in natural killer/t-cell lymphoma serum levels of soluble programmed death ligand predict treatment response and progression free survival in multiple myeloma soluble pd-l : a biomarker to predict progression of autologous transplantation in patients with multiple myeloma levels of programmed death- and programmed death ligand- in the peripheral blood of patients with oral squamous cell carcinoma and its clinical implications high serum level of soluble programmed death ligand is associated with a poor prognosis in hodgkin lymphoma level of circulating pd-l expression in patients with advanced gastric cancer and its clinical implications role of soluble programmed death- (spd- ) and spd-ligand in patients with cystic echinococcosis cloning and identification of two novel splice variants of human pd-l the soluble isoform of ctla- as a regulator of t-cell responses a soluble form of ctla- generated by alternative splicing is expressed by nonstimulated human t cells clinical benefit from ipilimumab therapy in melanoma patients may be associated with serum ctla levels the engagement of ctla- on primary melanoma cell lines induces antibodydependent cellular cytotoxicity and tnf-alpha production a soluble form of ctla- is present in paediatric patients with acute lymphoblastic leukaemia and correlates with cd d+ expression targeting the alternatively spliced soluble isoform of ctla- : prospects for immunotherapy? a molecular perspective of ctla- function human soluble cd is generated by alternative splicing, and recombinant soluble cd binds to cd and cd influencing t-cell activation structure and dimerization of a soluble form of b - identification of a circulating soluble form of cd : levels in patients with hematological malignancies in vitro co-stimulation of anti-tumor activity by soluble b molecules potent activity of soluble b -igg fusion proteins in therapy of established tumors and as vaccine adjuvant a soluble form of cd enhances antitumor immunity by neutralizing programmed death ligand- and simultaneously providing costimulation novel strategies for inhibiting pd- pathway-mediated immune suppression while simultaneously delivering activating signals to tumor-reactive t cells soluble cd restores t cell activation and overcomes tumor cell programmed death ligand -mediated immune suppression combination b -fc fusion protein treatment and treg cell depletion therapy in situ expression of soluble b - in the context of oncolytic herpes simplex virus induces potent antitumor immunity intramuscular gene transfer of soluble b . /igg( ) fusion cdna induces potent antitumor immunity as an adjuvant for dna vaccination triple combination of oncolytic herpes simplex virus- vectors armed with interleukin- , interleukin- , or soluble b - results in enhanced antitumor efficacy soluble cd is a costimulatory molecule for human t lymphocytes human plasma contains a soluble form of cd which is present at elevated levels in some leukaemia patients the clinical significance of soluble cd levels in patients with acute myeloid leukemia and myelodysplastic syndrome circulating levels and clinical significance of soluble cd in myeloma patients codelivery of dna coding for the soluble form of cd results in the down-regulation of the immune response to dna vaccines soluble cd (b -h ) is released from monocytes, dendritic cells and activated t cells and is detectable in normal human serum characterization of a soluble b -h (sb -h ) spliced from the intron and analysis of sb -h in the sera of patients with hepatocellular carcinoma b -h and b -h expression in cerebral spinal fluid and tumor tissue correlates with the malignancy grade of glioma patients clinical significance of serum soluble t cell regulatory molecules in clear cell renal cell carcinoma early detection of hepatocellular carcinoma in patients with hepatocirrhosis by soluble b -h upregulation of soluble b -h in nsclc-derived malignant pleural effusion: a potential diagnostic biomarker correlated with nsclc staging clinical significance of the induction of macrophage differentiation by the costimulatory molecule b -h in human non-small cell lung cancer soluble b -h promotes the invasion and metastasis of pancreatic carcinoma cells through the tlr /nf-kappab pathway cd , implications in immunity and potential for therapy expression of soluble cd correlates with activationinduced cell death of lymphocytes expression of cd and cd ligand in colorectal cancer patients levels of soluble cd are enhanced in sera of leukemia and lymphoma patients and are strongly associated with chronic lymphocytic leukemia hypoxia-induced soluble cd in malignant cells blocks cd l-costimulation as an immune escape mechanism saha/ vorinostat induces the expression of the cd receptor/ligand system and enhances apoptosis mediated by soluble cd receptor in a human breast cancer cell line immunogenicity to biotherapeutics -the role of antidrug immune complexes aberrant regulation of synovial t cell activation by soluble costimulatory molecules in rheumatoid arthritis expression of programmed death- (pd- ) on cd + and cd + t cells in rheumatoid arthritis soluble programmed death (pd- ) is decreased in patients with immune thrombocytopenia (itp): potential involvement of pd- pathway in itp immunopathogenesis aberrant production of soluble inducible t-cell co-stimulator (sicos) and soluble programmed cell death protein (spd- ) in patients with chronic hepatitis c soluble pd- is associated with aberrant regulation of t cells activation in aplastic anemia predictive value of soluble programmed death- for severe sepsis and septic shock during the first week in an intensive care unit. shock soluble programmed cell death receptor- (spd- ): a potential biomarker with anti-inflammatory properties in human and experimental acute respiratory distress syndrome (ards) inverse correlation of soluble programmed cell death- ligand- (spd-l ) with eosinophil count and clinical severity in allergic rhinitis patients soluble programmed death- ligand (spd-l ) is significantly reduced in the serum of type diabetes patients increased pd- on cd (+ )cd (−) t cell and soluble pd- ligand- in patients with t dm: association with atherosclerotic macrovascular diseases contribution of soluble forms of programmed death and programmed death ligand to disease severity and progression in systemic sclerosis increased production of circulating soluble co-stimulatory molecules ctla- , cd and cd in patients with rheumatoid arthritis aberrant production of soluble co-stimulatory molecules ctla- and cd in patients with chronic hepatitis b the soluble ctla- splice variant protects from type diabetes and potentiates regulatory t-cell function increased production of soluble ctla- in patients with spondylarthropathies correlates with disease activity plasma concentrations of soluble ctla- , cd , cd and cd costimulatory molecules reflect disease severity of acute asthma in children levels of the soluble forms of cd , cd , and cd are elevated in the synovial fluid of rheumatoid arthritis patients aberrant production of soluble costimulatory molecules ctla- , cd , cd and cd in patients with systemic lupus erythematosus soluble cd protein in serum samples of patients with asthma b -h augments the inflammatory response and is associated with human sepsis detection and quantitation of soluble b -h in expressed prostatic secretions: a novel marker in patients with chronic prostatitis enhancement of membrane b -h costimulatory molecule but reduction of its soluble form in multiple sclerosis potential role of soluble b -h in liver immunopathogenesis during chronic hbv infection reduced sb -h expression in the peripheral blood of systemic lupus erythematosus patients admission levels of soluble cd are increased in patients with acute pancreatitis and are associated with subsequent complications clinical implications of elevated serum soluble cd levels in patients with acute coronary syndrome heightened intrathecal release of soluble cd in patients with multiple sclerosis serum concentrations of soluble - bb and - bb ligand correlated with the disease severity in rheumatoid arthritis increased soluble cd levels and cd + t-cell-associated expression of cd in acute atherothrombotic stroke not applicable. availability of data and materials not applicable. authors' contributions dg and xaliterature search and review, writing, graphical design, and editing; yy and zcliterature search and review; xx -conception and design and editing. all authors read and approved the final manuscript.ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -ctass hz authors: bull, james j.; nuismer, scott l.; antia, rustom title: recombinant vector vaccine evolution date: - - journal: plos comput biol doi: . /journal.pcbi. sha: doc_id: cord_uid: ctass hz replicating recombinant vector vaccines consist of a fully competent viral vector backbone engineered to express an antigen from a foreign transgene. from the perspective of viral replication, the transgene is not only dispensable but may even be detrimental. thus vaccine revertants that delete or inactivate the transgene may evolve to dominate the vaccine virus population both during the process of manufacture of the vaccine as well as during the course of host infection. a particular concern is that this vaccine evolution could reduce its antigenicity—the immunity elicited to the transgene. we use mathematical and computational models to study vaccine evolution and immunity. these models include evolution arising during the process of manufacture, the dynamics of vaccine and revertant growth, plus innate and adaptive immunity elicited during the course of infection. although the selective basis of vaccine evolution is easy to comprehend, the immunological consequences are not. one complication is that the opportunity for vaccine evolution is limited by the short period of within-host growth before the viral population is cleared. even less obvious, revertant growth may only weakly interfere with vaccine growth in the host and thus have a limited effect on immunity to vaccine. overall, we find that within-host vaccine evolution can sometimes compromise vaccine immunity, but only when the extent of evolution during vaccine manufacture is severe, and this evolution can be easily avoided or mitigated. recombinant vector vaccines are live replicating viruses that are engineered to carry extra genes derived from a pathogen-and these extra genes produce proteins against which we want to generate immunity. these vaccine genomes may evolve to lose the extra genes during the process of manufacture of the vaccine or during replication within an individual, and there is a concern that this evolution might severely limit the vaccine's efficacy. the dynamics of this process are studied here with mathematical models. the potential for vaccine evolution within the host is somewhat limited by the short-term growth of the vaccine population before it is suppressed by the immune response. we find that evolution is a problem only when the process of manufacture results in the majority of the vaccine virus being revertant. we show that increasing the vaccine inoculum size or reducing a a a a a live vaccines replicate within the host. as true of any reproducing population, these withinhost vaccine populations may evolve. for live vaccines that do not transmit, any within-host evolution is a dead end and might thus seem to be irrelevant to vaccine function. but if the process is fast enough, or the vaccine population replicates long enough, the vaccine population may evolve to a state where it is ineffective or virulent-either change would be bad. the two main types of live viral vaccines are attenuated and recombinant-vectored. most live virus vaccines in use today are attenuated, their reduced virulence typically achieved by adapting the wild-type virus to a new environment (e.g. replication in a novel cell line or low temperature), with a consequent reduced replication rate in humans. the use of attenuated vaccines is too risky for pathogens such as hiv, and a safer alternative is to develop a live, recombinant vector vaccine where one or a few pathogen genes with immunogenic activity (proteins that elicit protective immunity) are expressed from a benign virus vector. the expected consequences of within-host evolution differ between these two types of vaccines ( table ) . evolution of an attenuated vaccine is likely to be a reversion toward the wildtype state, the rate of this process depending heavily on vaccine design and the duration of vaccine virus replication in the host (reviewed in [ ] ). to a first approximation, reversion toward the wild-type state should lead to the vaccination more closely resembling natural infection [ ] , such as higher virus densities, side-effects and disease, and possibly an increased immune response. within-host evolution of an attenuated vaccine might also predispose the virus to better transmission-also reflecting the wild-type state-but this outcome is not assured: viral adaptation to different tissues within the host may hamper growth in and dissemination from tissues important in transmission (e.g., [ ] ). the expected consequences for evolution of a recombinant-vectored vaccine are fundamentally different [ ] . in most cases, the antigen against which immunity is sought comes from a foreign transgene inserted into a competent viral vector without replacing any vector genes. vectors in development include adenovirus, vsv (vesicular stomatitis virus) and cmv (cytomegalovirus). the vector genome carries out all viral amplification and transmission functions, and the transgene does not contribute to any process benefiting vector reproduction. from an evolutionary perspective, the transgene is both dispensable and potentially costly: selection may favor loss of the transgene and thus loss of vaccine's ability to elicit immunity against the antigen encoded by the transgene. this evolution therefore generates something akin to infection by the wild-type vector. as vectors are typically chosen to be avirulent for immune competent hosts, vaccine evolution will result in no more than a harmless infection that does not generate immunity to the antigen encoded by the transgene. considerable attention has recently been given to the evolution of attenuated vaccines and designs that retard their evolution. evolutionary stability of attenuated vaccines seems attainable by engineering designs, including the introduction of hundreds of silent codon changes, genome rearrangements, and some types of deletions (comparisons and reviews are provided by [ , , ] ). far less thought has gone into the consequences of evolution for recombinant vector vaccines or of strategies to minimize this evolution. although recombinant vector vaccines are not yet in widespread use, many are under development [ , ] , and their success may rest on understanding within-host evolution. here we explore how the combination of evolution during the process of vaccine manufacture and during its within-host dynamics following vaccination could affect the immune responses elicited by a recombinant vector vaccine and reduce its efficacy-the specific interaction between evolution and immunity. we consider viral vaccines and focus on vaccines that cause shortduration (acute) infections. the ideas we discuss also apply to live vaccines of bacteria and other pathogens. our overall message is that while vaccine evolution may occur, it is either unlikely to be a problem (i.e., compromise the generation of immunity), or it is easily mitigated. when vaccine evolution does limit the adaptive immune response, we identify ways of escaping such outcomes. our analysis rests on mathematical models, but most results can be explained intuitively (perhaps only in hindsight), with the main results illustrated graphically; many analyses are relegated to supporting information. our analysis assumes that vaccines replicate within the host untill cleared by host immunity; we exclude vaccines that reproduce for just a single infection cycle (e.g., modified vaccinia virus ankara), as they have no significant opportunity for evolution. our models are numerical analyses of ordinary differential equations. the equations are given in supporting information (s appendix). the were numerically evaluated and graphed in r (s file, a markdown file), sometimes also evaluated in mathematica (s file). the key question is whether evolution of the vaccine virus (henceforth just 'vaccine') meaningfully affects immunity to the antigen encoded by the foreign transgene (henceforth just 'antigen'). the potential for vaccine evolution is easy to understand. through mutation, any large vaccine population will contain mutants that inactivate or delete the foreign transgene, and those revertants will then grow amidst the vaccine. vaccine inferiority may accrue in two different ways: the transgenic insert and its expression may intrinsically impair vaccine growth, and adaptive immunity to the foreign antigen may impair the vaccine's growth but not the revertant's during an infection. it is easy to appreciate how and why the vaccine may be inferior to the revertant, and this can result in an increase in frequency of the revertant. however, the relationship between this evolution and the extent of immunity to the vaccine antigen is more complex. we thus explain some of the factors that affect how this evolution translates into a reduction in immunity to the antigen, and why in some circumstances, substantial evolution can result in little change in immunity to the antigen, while in different situations it can result in a substantial reduction. two realms of vaccine evolution. vaccine evolution can be inimical to immunization by limiting vaccine antigen levels in the host. as noted above, one realm in which evolution may occur is within the host, starting with the inoculum and ensuing until vaccine clearance. a second realm of evolution affecting antigen levels is that occuring during manufacturingduring growth of the virus to prepare the stock used for inoculation. both realms are part of a continuum, any evolution during manufacturing advancing evolution within the host (fig ) . the inoculum sets the starting point for within-host evolution, and indeed, an inoculum that is mostly revertant will limit vaccine efficacy even if no further evolution occurs within the host. from population genetics principles [ ] , even if the inoculum has a seemingly low frequency of revertant, the evolution that has occurred prior to inoculation may greatly accelerate within-host evolution (fig ) . paradoxically, when considerable evolution occurs in manufacture, such that the inoculum is primarily revertant, there is little opportunity for further evolution within the host-the damage is already done. the effect of any 'pre-host' evolution on the host immune response is potentially as important as the effect of within-host evolution. an important difference between the two realms is that pre-host evolution may be more easily mitigated than is within-host evolution. that is, controlling pre-host evolution may be a feasible way to limit within-host evolution and to limit the loss of immunity from vaccine evolution. since pre-host and within-host evolution represent different realms on a continuum, we adopt a language that attempts to distinguish them and avoid confusion. we use the following: • 'pre-host evolution' refers to evolution during manufacture that affects inoculum composition • 'within-host evolution' refers to evolution that occurs within the host after inoculation • 'evolution' refers to either or both of the above. it is easily appreciated that the specifics of evolution in the two realms may differ-vaccine growth and fitness in an in vitro environment (pre-host) will often differ from that within the host. regardless, however, any pre-host evolution will advance subsequent within-host evolution, unless the revertant is selected in opposite directions pre-host and within-host. there may even be a common molecular basis to vaccine inferiority in both the pre-host and withinhost environments that will render the two processes somewhat similar (see below). a short duration of infection limits within-host evolution. any fitness advantage of revertant means that its frequency-its abundance relative to the vaccine-will increase during active viral growth (fig ) . however, when the infection caused by the vaccine is acute, as we consider here, the magnitude of possible within-host evolution is limited by the short duration of viral growth before clearance. if the inoculum is largely free of revertant, even a moderate fitness cost of the vaccine may have little effect on vaccine evolution, such that vaccine fitness effects and evolution can be ignored. evolution versus immunity. surprisingly, vaccine evolution per se need not reduce the immune response, even when its magnitude is large. if overgrowth by revertant does not interfere with vaccine growth, then vaccine growth and antigen production are not affected (fig ) . evolution affects antigen production only to the extent that revertant superiority suppresses vaccine growth and thereby suppresses antigen production. numbers versus frequencies. models of evolution often address relative frequencies, on a scale of to . immunity develops in response to vaccine density. in addressing immunity, it is thus necessary for the model to track densities, whereas any evolution is more easily described with frequencies (the two approaches can be compared between figs and ). the challenges are thus to understand (i) when and how much vaccine evolution occurs; (ii) whether and to what extent evolution affects the abundance of vaccine virus in the host over time; and (iii) how changes in vaccine abundance affect the generation of adaptive immunity against the antigen. the arguments presented above are qualitative and only superficially identify the scope of the problem. quantitative understanding ultimately rests on analysis of mathematical models. however, as the models have many interacting processes-minimally innate immunity, adaptive immunity and intrinsic growth differences between vaccine versus revertant-we first verbally explain the biology underlying the processes that go into those models. intrinsic fitness differences. intrinsic fitness effects are considered here to be those that stem from the intracellular processes of viral gene expression and assembly, independent of host immune responses. intrinsic fitness differences between the vaccine and the revertant (wild-type vector) are plausible because the transgene is non-essential and has no evolutionary history with the vector genome. thus, the insertion may be disruptive, and the resulting antigen expression may interfere with vector functions. intrinsic fitness effects are expected to affect evolution during vaccine manufacture as well as within-host evolution, but it has largely been investigated in vitro, as would apply to manufacture and the pre-host phase. indeed, intrinsic fitness differences may be the sole or at least the most important bases of vaccine inferiority. because recombinant viruses have often been observed to evolve loss or down-regulation of engineered inserts, they are now commonly observed during in vitro growth for their 'genetic stability' (e.g., [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ). some recombinant viral genomes are stable over short term transfers in culture, others not, indicating that intrinsic fitness effects of the engineering are not universal. thus, the possibility of vaccine inferiority should not be ignored, and furthermore, even when a vaccine appears to be stable over a few transfers, the short term population where the revertant has a % fitness advantage and starts at a frequency of − . the curve shows the well-known population genetic principle that, while the favored type (revertant) is rare, its absolute frequency changes very little. but the frequency eventually reaches a level at which evolution is rapid. the yellow box represents a possible period of pre-host evolution, the green box representing the period of within-host evolution. the periods of within-host evolution are drawn to be the same length in right and left panels, as if the vaccine has the same within-host duration in both cases. the arrow represents a possible point at which manufacture would end and an inoculum be created, thus defining the boundary between pre-host and within-host evolution. the left panel depicts a short period of vaccine manufacture, the right a longer period of vaccine manufacture and one in which more pre-host evolution has occurred. it is thus easy to see the potential importance of pre-host vaccine evolution on within-host evolution, for even when the revertant is not a large component of the inoculum, it can be poised for rapid evolution within the host (right panel). the curve obeys p t ¼ p w t p w t þ À p , in which p t represents the revertant frequency in generation t and w the fitness of revertant relative to vaccine. the curve is drawn for a common evolutionary process across pre-host and within-host evolution, but evolution in the within-host phase will typically experience different parameters than evolution in the pre-host phase. retention of antigen expression may mask an underlying long term instability. thus most observations of stability merely set limits on the possible magnitudes of inferiority. yet even if vaccine selective 'neutrality' turns out to be fleeting, merely a mistaken impression from shortterm observations, we will find that the phenomenon of short-term stability mirrors a solution to minimize vaccine evolution within the host-the solution of limiting vaccine growth. the revertant virus has the superior growth rate, but in the absence of interference between the two, vaccine growth is unimpeded and immunity is triggered. https://doi.org/ . /journal.pcbi. .g three mechanisms of vaccine-revertant interference. fig presented a hypothetical case in which evolutionary superiority of revertant did not suppress vaccine growth, hence evolution had little effect on antigen production. that process was one in which there was no interference between vaccine and revertant growth. evolution does become important to antigen levels if vaccine and revertant interfere so that vaccine growth is depressed by the revertant, or if the duration of infection by the vaccine strain is reduced. in either case the revertant will then suppress antigen levels. again, the problem is complicated by the limited duration of the infection: reduced antigen production due to vaccine evolution depends not only on interference between the two genomes but also on overall growth and the extent to which it affects the level of immunity to vaccine and vector. a mechanism that forces interference between vaccine and revertant can also limit the total amount of viral growth, thereby limiting evolution. evolution of vaccine versus revertant thus depends on details, in particular, the specific mechanism by which revertant interferes with vaccine growth. we describe three different mechanisms that have been proposed that may be relevant to vaccine competing with revertant: innate immunity, resource limitation, and adaptive immunity to the vector backbone shared by the vaccine and revertant virus. (these are not the only possible mechanisms of within-host parasite competition [ ] , but they capture the relevant immune processes.) for many vaccines, each mechanism will impede revertant and vaccine equally as a collective population, thus ensuring interference. it was initially believed, implicitly if not explicitly, that the adaptive immune response played the dominant role in the control of viruses and other infections. in the 's, janeway and medzhitov identified shared pathways for the control of pathogens between vertebrates and drosophila, even though drosophila lacks an adaptive immune response (reviewed in [ ] ). this led to a resurgence of interest in the role of innate immunity in the initial control of infections. later modeling studies of influenza infections suggested yet another mechanism, that the initial control of these infections could be largely described by simple resource limitation models, of the type used in ecology for population growth [ , ] . the realization that all three different processes might suppress viral infection led to more careful examination of the roles of different factors in the early control of acute infections [ ] [ ] [ ] [ ] . the relative role of each mechanism in clearing infections is the basis of ongoing discussion, but it is widely accepted that the roles differ among infections by different viruses and that each mechanism is potentially important for some viruses. • innate immunity. there are two broad arms of immunity for suppressing vaccine growth within the host, the innate and the adaptive immune responses. innate immunity is triggered by conserved molecules associated with pathogens (pathogen associated molecular patterns, [ ] ). conserved structures of pathogens targeted by innate immunity include dsrna, frequently accompanying viral replication, plus lipopolysaccharides and endotoxins of bacteria [ ] . because innate immunity involves the activation of a standing population of immune cells such as macrophages and dendritic cells, or triggering of the complement pathway, it can be elicited much more rapidly than the adaptive response; the latter requires many rounds of clonal expansion of rare antigen-specific cells to generate a population large enough to control the infection [ ] . furthermore, recent studies have shown that the innate response is required for the initial stimulation of the adaptive response [ ] . thus, innate immunity has a major role in early control of the viral population. innate immunity can control both vector and vaccine, and it is not likely to discriminate between two genomes that differ by a single, non-essential gene (the transgene). • resource limitation another mechanism by which revertant levels can suppress vaccine levels is resource limitation. both the vaccine and revertant virus use the same resource (susceptible host cells). resource limitation can control the infection if the virus depletes this resource, whereby the rate of virus output falls below its intrinsic death rate [ ] . like innate immunity, resource limitation is expected to affect vaccine and revertant similarly. resource limitation has been considered an important mechanism for competing malarial strains within the host [ , ] . • adaptive immunity adaptive immunity can be induced by the revertant and the vaccine virus. adaptive immune responses to antigens expressed by the revertant will presumably affect the vaccine and revertant equally-because the vaccine encodes a complete vector genome, and the revertant is also a complete vector/virus. as with the preceding pair of mechanisms, adaptive immunity elicited by the revertant will also depress the abundance of the vaccine virus. adaptive immunity to the vaccine antigen will be considered shortly. all three interference mechanisms will potentially operate in any vaccinated host. with all three operating, one mechanism may take precedence over the others, simply because it is activated earlier or enforces a lower limit on viral density than the others. however, there are different stages or degrees of vaccine suppression, so an early mechanism may act to control the infection without clearing it, and another mechanism may act later to clear. because of the delay in developing an adaptive response, viral suppression by adaptive immunity typically occurs later than effects of innate immunity or resource limitation and so might seem to be unimportant in vaccine evolution. yet adaptive immunity may be important in clearing the vaccine following control by other mechanisms, in which case it could have an important role in vaccine evolution. adaptive immunity to the vaccine antigen may also contribute to vaccine inferiorityand feed back to inhibit itself. the preceding paragraphs omitted adaptive immunity to the antigen. by its very nature, adaptive immunity suppresses vaccine growth. but adaptive immunity to the antigen is specific to the vaccine and is thus another reason-besides intrinsic fitness effects-that the vaccine may have lower fitness than revertant. the evolutionary consequences should be the same for both types of inferiority, reducing the long term generation of antigen levels within the host, but adaptive immunity would be irrelevant to vaccine evolution during manufacturing and during early growth within the host. an interesting twist is that adaptive immunity to the antigen might eventually feed back negatively on itself to limit its own growth-immunity against a virus is intrinsically inhibitory, so adaptive immunity against the vaccine will limit vaccine growth and thus limit antigen build-up that would fuel further immunity. one question is whether this self-inhibition is worsened with vaccine evolution. the effect is biologically complicated because adaptive immunity to the antigen does not necessarily translate into selection against the vaccine. selection against the vaccine per se operates only when adaptive immunity specifically targets the vaccine genome over the revertant genome, and this selection need not occur-either because adaptive immunity is so delayed that it is never manifest during vaccine growth, or because the antigen is physically decoupled from its genome when attacked by the adaptive response. without imposing selection on the vaccine, antigen-directed immunity will not affect vaccine evolution. we now employ quantitative models to evaluate the intuitive ideas presented above. given the high dimensionality of the problem, we are especially interested in how well intuition works and whether generalities are observed across large regions of parameter space. a flow diagram of the elements and interactions within the host reveals the complexity of the model (fig ) and facilitates understanding the dynamical equations. v and w are the respective vaccine and revertant densities, with intrinsic growth and death rates governed by four parameters (not illustrated). the model also includes variables for resources (r), innate immunity (z), adaptive immunity to vector (y), and adaptive immunity to antigen (x) that are both influenced by and influence v and w. in the following sections, we explore the dynamics of these interactions with simulations and present results graphically (the results presented do not allow resource limitation to influence dynamics; trials where resource limitation matters were conducted but are not shown). equations and parameter values are provided in s appendix. resource limitation and innate immunity yield qualitatively similar results, so trials with resource limitation are not illustrated in the main text. the equations apply only to within-host processes; any pre-host evolution is subsumed into inoculum composition. the models assist us by forcing us to specify assumptions for how the viruses and immunity interact, and by allowing us to rigorously explore outcomes in different scenarios. however, there is uncertainty in the model structure, many parameter values are unknown, and different viruses will behave somewhat differently. consequently, we focus on broad generalities that arise from many simulations and illustrate these for a few specific cases, reserving supporting information files for further details. the presentation below briefly discusses the dynamics of individual trials for illustration but then moves to contour plots that reveal differences in outcomes as the key parameters are changed. the model used here incorporates the structure of earlier models that described immune responses [ ] [ ] [ ] ; parameter values used here were chosen as described in some of these earlier studies. evolution can matter. in the trials used for illustration, we allow innate immunity to control the infection and adaptive immunity to cause final clearance. such a scenario might correspond to the dynamics of listeria or influenza infections of mice [ ] , or the early dynamics of siv infections [ ] . to get a sense of the full dynamics in the model, we show the time course of dynamics for the different variables (fig ) under conditions of no evolution (top left), just pre-host evolution-revertant abundant in the inoculum but with no fitness advantage (top right), mostly within-host evolution (bottom left), and both (bottom right). the effect of different types of evolution is seen from a comparison of the panels. our chief interest is in final immunity to vaccine, the blue curves. the most visible effect of evolution on immunity to the antigen is evident in the lower right panel, which combines pre-host evolution with a fitness advantage of the revertant. however, the log scale diminishes the visual impact of substantial evolution in other cases. when the revertant is half the inoculum but has no fitness advantage, the immune response is diminished by nearly -fold (top right). overall, the impression is that one must at least suppress either pre-host or within-host evolution to avoid a large loss in immunity (lower right versus the others). illustrations of dynamics from individual trials convey many details. however, without a specific empirical basis for the parameter values chosen, the details have little assured relevance. we therefore provide contour plots that allow easy comparison of many different trials in which parameters of specific interest are varied (fig ) . these graphs show the cumulative vaccine load (left panel) and final level of immunity to vaccine antigen (right) as a function of initial revertant frequencies and selective advantage of the revertant (c). a strong correspondence exists between vaccine load and the level of immunity generated, as is observed empirically following infection [ ] . subsequent figures therefore illustrate the level of immunity. the initial composition of the inoculum matters somewhat more to the adaptive response than does the intrinsic cost of the vaccine (as evident by the contours being closer to vertical rather than horizontal), but this pattern rests heavily on the parameter ranges chosen. indeed, unrealistically large values of initial revertant levels (w( )) are illustrated to offer contrast, as the outcomes are otherwise moderately insensitive to vaccine composition in these graphs. the good news is that, when the inoculum is mostly vaccine and revertant fitness is not high, evolution has little effect on viral load or final level of immunity (i.e., the lower left of each panel has a broad area of one color). this occurs because of the short duration of infection. over longer periods of vaccine growth, the selective advantage of the revertant would undoubtedly play an increasing role in evolution. the large number of parameters ( ) limits the degree to which we can conduct comprehensive sensitivity tests, so the trials are confined to variations in those parameters of greatest interest. vaccine evolution driven by adaptive immunity. we focus on infections of short duration-that are cleared and do not rebound once suppressed. factors that limit the duration of infection include resource limitation, and innate and adaptive immunity. for the most part these factors act equally against vaccine and revertant virus. only one factor, adaptive immunity to the vaccine antigen (x), acts specifically on the vaccine virus and not the revertant. intuition suggests that this adaptive immunity to the antigen can potentially suppress the vaccine's growth and give an advantage to the revertant. as with intrinsic fitness costs, this selection might feed back to limit vaccine growth and thus limit the development of further immunity to antigen by allowing revertant to grow and interfere with vaccine. this section considers whether these arguments are supported by the model. any real vaccine that elicits immunity against the antigen may also experience an intrinsic fitness cost. the effect of immunity on evolution would then be confounded with the effect of intrinsic fitness effects on evolution, making it difficult to isolate one from the other. the models do not face this problem, however. they can be parameterized so that the only possible selection against the vaccine comes from immunity (by setting c = ). vaccine populations can also be freed of revertant by omitting revertant from the inoculum and setting the mutation rate to . thus, we can measure the effect of adaptive immunity on vaccine growth from trials that lack revertant and then compare those results with trials that include revertant. the revertant is included at half the inoculum (representing pre-host evolution); it has no intrinsic fitness advantage over vaccine. immunity (to vaccine) is reduced to just over a third of the level with no evolution. (lower left): the revertant is a small fraction of the inoculum ( . ) but it has a % fitness advantage over vaccine. the level of immunity is % that with no evolution. (lower right): the revertant is half the inoculum and has a % fitness advantage over vaccine. the level of immunity is now less than . % that with no evolution-a depression of almost orders of magnitude. the trials are parameterized so that virus is controlled by innate immunity with final clearance due to adaptive immunity; the mutation rate is in all cases. equations, initial conditions and parameter values not shown here are given in s appendix; r code is included in s file. there are several background points to note about the model structure. first, adaptive immunity specific to vaccine (x) develops at a rate proportional to the vaccine abundance (v) and parameters s (rate of clonal expansion of adaptive immunity) and ϕ x (antigen concentration yielding half the maximum growth rate of adaptive immunity x). in contrast, the impairment specific to vaccine is due to the level of immunity (x) and the vaccine impairment parameter k x . thus, immunity can develop while imposing little or no impairment, i.e., when k x ! . second, adaptive immunity to the vector (y) develops according to its own growth rate parameter (ϕ y ) in response to vaccine plus revertant abundance (v + w), and it impairs both vaccine and revertant growth equally by impairment parameter k y . when revertant is present, it increases the level of immunity to vector backbone/revertant but does not directly affect immunity specific to the vaccine. this immunity will result in faster clearance of both revertant and vaccine, and this results in decreased immunity to the antigen; this is the 'interference' that causes a problem from vaccine evolution. trials were run that contrasted revertant absence versus revertant introduced at % of the inoculum-no evolution versus primairly pre-host evolution, respectively (fig ) . absence of the revertant is the baseline against which the effect of evolution can be compared. the horizontal axis varies k x , the parameter for impairment/killing specific to vaccine, and the vertical axis varies k y , impairment to vector, which affects vaccine and revertant equally. in both panels, increasing impairment against vaccine leads to lower levels of immunity to the vaccinethis is the self-limiting effect of adaptive immunity, which exists even in the absence of evolution. as expected, impairment of immunity to vaccine by immunity to vector is also found. a large effect of inoculum composition on vaccine immunogenicity is evident by comparing the left panel (no mutation, no evolution) and right panel (chiefly prehost evolution): introduction of revertant reduces the level of immunity against vaccine up to -fold. for the -host evolution) . the final vaccine load and immunity against the vaccine antigen depends heavily on two parameters, the inoculum composition (plotted on the x-axis as initial abundance of the revertant virus, w( )) and the growth advantage of the revertant within the host (c, plotted on the y-axis). the heat maps show how, as the composition shifts toward revertant or as vector superiority increases (as we move to the right or up), there is a reduction in the viral load of the vaccine (defined as r v dt, left panel) and in the magnitude of immunity to the vaccine antigen (x, right panel). the initial amount of vaccine virus is always v( ) = (i.e. logv( ) = ). note that the graphs span high frequencies of revertant in the inoculum that should be easily avoided (log w( ) = , i.e. w( ) = v( ))-if the researcher is alert to the possibility. we include such extremes merely to show that the outcome is relatively insensitive to small changes in vaccine composition. equations, initial conditions and parameter values not shown here are given in s appendix; r code is included in s file. https://doi.org/ . /journal.pcbi. .g within-host vaccine evolution right panel, the revertant is / the inoculum and has no intrinsic advantage over vaccine; total inoculum size is unchanged. all reduction of immunity against vaccine is thus due to revertant in the inoculum and any within-host evolution from the selective effect that stems from immunity against vaccine. of the two parameters, k y has a much larger effect than does k x : compared to the left panel, the right panel, with revertant present, has vaccine-specific immunity suppressed more than an order of magnitude along the k y axis, much less so on the k x axis. we attribute this effect of k y to interference by the revertant: the revertant elicits high levels of immunity (y) that indiscriminately also suppress vaccine, thereby suppressing vaccine-specific immunity x. the magnitude of interference depends not only on revertant abundance but also on the values of k x , k y , and the innate immune response (k z ), so interference can appear more or less important in other trials using the same revertant abundance. a question motivating this analysis was one step deeper in the complexity of these effects: does the self-limiting effect of adaptive immunity worsen when revertant is present? this question can be answered by comparing the self-inhibitory effect between left and right panels as k x is increased. by inspection of colors along the horizontal axes, it is seen that the self-inhibitory effect is actually somewhat reduced by the revertant. the revertant lowers the overall response to vaccine, but when correcting for that difference, the effect of increasing k x is slightly weaker in the right panel than in the left. we attribute this weakening of self-limitation as due to vaccine levels being increasingly controlled by immunity against revertant. in sum, therefore, immunity to the vaccine (x) is reduced by itself (depending on the immunity parameter, k x ) and by revertant. the two effects do not interact to make the problem worse than from their separate effects. the results above suggest that vaccine evolution is only likely to compromise immunity if there is substantial pre-host or within-host evolution and if this evolution depresses vaccine effect of evolution on the suppression of immunity by impairment parameters. the final level of immunity to the vaccine antigen (x) depends heavily on the inhibitory parameters k x and k y -which respectively describe how strongly immunity to the vaccine (x) and revertant (y) suppress the viral populations. the left plot considers the absence of revertant, hence no evolution. the right panel introduces revertant at / virus in the host. as the short duration of infection limits within-host evolution, one means of achieving vaccine efficacy is to control the inoculum. two ways of controlling the inoculum are to control its composition and to control its size. pre-host evolution can be reversed by purifying the inoculum after the fact or by taking care to start with a pure isolate and limiting growth (e.g., fig ) . the benefit of suppressing revertant frequency in the inoculum is evident in fig : the magnitude of immunity to the vaccine increases by orders of magnitude as the initial frequency of the revertant is decreased. the effect is strongest at low inoculum levels, pointing to the other solution-increase inoculum size. intuition also suggests that the deleterious effects of evolution can be reduced by increasing the inoculum size, provided the composition does not change: to achieve a threshold antigen level, a large inoculum requires less growth than a small one. less growth reduces the potential for evolution-in the extreme, a large enough inoculum requires no vaccine growth, as with killed vaccines. these conjectures are supported by fig : when the revertant frequency in the inoculum is high, increasing the inoculum size appreciably increases the magnitude of immunity; a much reduced benefit is seen when revertant frequency is low, likely because there is less evolutionary interference from the revertant. these results suggest parallel benefits from reducing the frequency of the revertant in the inoculum and increasing the dose. consideration of the gains from each could help choose an economically feasible strategy, since both purifying the inoculum and increasing its dose are likely to incur financial costs. whether and how well controlling the inoculum will work in practice will depend on details. solutions may be quantitative rather than absolute. intuition is useful for guidance but needs to be confirmed by formal analyses, guided by data from the specific implementation. any live viral vaccine may evolve within the host. the potential for attenuated viruses to revert to wild-type virulence is well appreciated [ , ] , even if it presents a problem for relatively few vaccines (e.g., attenuated polio, [ ] ). there is also a potential for live, recombinant vector vaccines to evolve-our focus in this paper-with the main concern being loss or reduced expression of the transgenic insert [ , ] . if such a vaccine were to evolve fast enough or long enough that it lost the insert, vaccine efficacy might well suffer. we find that evolution during manufacture (pre-host evolution) can play a more important role than within-host evolution in reducing vaccine efficacy, and furthermore that it may be the more easily mitigated. we developed and analyzed models to explore ways in which vaccine evolution could lead to a reduction in vaccine efficacy. an intrinsic fitness advantage of the revertant virus, expected because transgene expression is likely to have metabolic and other costs, will lead to vaccine being gradually overgrown by revertant. this is only likely to cause a reduction in the immunity to the vaccine antigen if it leads to a reduction in the absolute amount (as opposed to merely a reduction in relative frequency) of the vaccine virus. there are in fact several mechanisms by which an ascending revertant population may suppress vaccine: revertant can reduce the amount of the vaccine virus in the host if the revertant uses resources required for virus replication or if the vaccine virus is cleared by the innate or adaptive responses elicited by the revertant. the clear and positive message from our study is that vaccine evolution, if it proves to be a problem for immunization, should be easily mitigated by manipulating the vaccine inoculum. critical to understanding and addressing this problem is recognizing that the vaccine may evolve both within the host and also during manufacture, whereby the inoculum already carries modest to high levels of revertant. the composition of the inoculum can have a large effect on within-host evolution and immunity. by limiting the amount of revertant in the inoculum, and also by boosting the inoculum level, it should usually be possible to limit the amount of within-host vaccine evolution and ensure that immunization is effective. we emphasize, however, that this solution will typically not work for transmissible vaccines and vaccines that establish long term infections within the host. furthermore, using a large inoculum may seem to defeat the purpose of using a live vaccine. there may be cases in which vaccine evolution is so rapid that controlling the inoculum is not sufficient. the solution in this case is to develop or engineer the vaccine with less of a disadvantage. the timing and tissues of antigen expression, location of the transgene in the vector genome, and the size of the transgene may all influence intrinsic fitness effects [ , , , , ] . directed evolution approaches might also improve vaccine efficacy: one simple approach in reducing an intrinsic cost might be to adapt the vector in vitro to host cells expressing the effects of manipulating the inoculum on immunity to the vaccine. small inocula that contain vaccine plus revertant are more prone to reduced immunity levels than are large inocula with little revertant. composition of the vaccine has the larger effect for the inoculm sizes and initial revertant fractions shown, as indicated by the contours being more horizontal than vertical. an intrinsic fitness cost of c = . was set for these trials. smaller c values would lead to higher vaccine and immunity levels across the graphs. equations, initial conditions and parameter values not shown here are given in s appendix; r code is included in s file. https://doi.org/ . /journal.pcbi. .g antigen in trans, allowing compensatory mutations to evolve in response to the antigen before the transgene is cloned into the genome. this adapted vector would then be used as the vaccine backbone. another simple approach would be to compete several different vaccine designs in vitro and pick the design with highest retention of the transgene. any approach using in vitro adaptation needs to avoid adapting the vector to the extent that it compromises ability to grow in vivo. most of these possibilities are ways to reduce pre-host evolution and reduce revertant concentration in the inoculum. one may hope that vaccine designs which reduce pre-host evolution also reduce within-host evolution. measuring the intrinsic fitness effect of the transgene is likely to be an important step in vaccine design. for assessing vaccine evolution, the relevant biological realms are within the host and in vitro. in vitro growth environments are the more easily studied and may reveal much about a vaccine's intrinsic propensity to evolve loss of antigen expression. there are various ways intrinsic fitness effects and their evolutionary consequences might be studied. vaccine growth in tissue culture may reveal some aspects of intrinsic fitness effects and should be relatively easy to study. deletion of the transgene per se would be detectable by pcr, and the fitness advantage of revertant over vaccine could be measured from changes in revertant frequency. the quantitative relevance of an in vitro estimate to in vivo growth would be unknown, but the measure should allow qualitatively comparing engineering designs that improve intrinsic vaccine fitness. if vaccine reversion were due to down regulation of the transgene instead of deletion, fitness estimation would require knowing the mutations responsible and monitoring their frequencies. use of culture-wide antigen levels to measure fitness might provide a sense of whether vaccine evolution would lead to reduced antigen levels in vivo, but it would be less sensitive in measuring evolution than is measuring mutation frequencies. evolution is not the only consideration in designing a recombinant vector vaccine, and the model helps us identify vaccine properties that promote efficacy. first the vaccine should elicit an immune response that rapidly clears the pathogen (i.e. the rate constant for clearance of the pathogen, call it k p , is high). second, the vaccine should elicit a large response to this antigen. this requires that the antigen rapidly elicits immunity (i.e. has low ϕ x , and in terms of immunology it should be an immunogenic antigen), and also requires a high vaccine viral load to generate a large response. engineering this requires tackling a trade-off between avoiding vaccine clearance (i.e. having a low k x ) but allowing for rapid clearance of the pathogen (having a high k p ). vaccines designed to express the antigen in a form that is different from that in the pathogen might help solve this problem. thus, to elicit immunity to influenza, one might design secreted forms of the hemagglutinin or neuraminidase proteins. a recombinant hemagglutinin protein that is secreted rather than on the virion surface would prevent the antibody response to this protein from clearing the recombinant vector vaccine (have low k x ) without compromising the clearance of the influenza virus pathogen which has hemagglutinin on its surface (i.e. has high k p ). in this manner our model allows the identification and tuning of parameters that affect vaccine efficacy, and a comprehensive search of parameter space would identify ideal combinations of vaccine properties. in vitro assays may be useful in measuring intrinsic fitness effects, but in vivo-in the patient-is the ultimate environment for studying within-host evolution and its effects. not only are the dynamics of viral spread different between in vitro and in vivo environments, but most immune components will be in play only in vivo. furthermore, those components may vary across tissues within the host. sampling across this heterogeneity in vivo will be challenging but may be necessary to know whether, when, and where vaccine evolution is a problem. if revertant remains a minority of the population, we expect that vaccine evolution can be ignored. perhaps in vitro studies of vaccine evolution will provide most of the information relevant to in vivo evolution, but it is too early to know. we have focused on recombinant vector vaccines that cause acute infections. necessarily, our recommendations are based on simple models that are caricatures of the complex withinhost dynamics of acute infections. simple models are appropriate at this stage because of uncertainties at many biological levels, and under these circumstances simple models frequently generate more robust results than do complex models [ , ] . the generation of innate and adaptive responses can be modeled with different assumptions than used here, and those alternative processes may affect the conclusions. for example, time-lags in the activation of cells may dominate the time for the generation of an innate immune response, with virus density having a consequently smaller role than assumed here (as can be seen in [ ] and modeled in [ ] ). we have modeled that responses to different antigens are generated independently of each other and do not compete. we have done so because vaccines are likely to cause relatively mild infections during which the densities of pathogen and immune cells do not reach sufficiently high levels required for competitive interactions to be important. the adaptive immune response may be more influenced by recruitment which is followed by a period of proliferation even in the absence of antigen [ ] [ ] [ ] . both these scenarios would minimize the impact of evolutionary changes in the vaccine on the amount of immunity generated to the transgene. finally, it is easily appreciated that there are realms we do not consider, such as within-host spatial structure [ ] and recombinant vector vaccines based on viruses such as cytomegalovirus that cause persistent infections [ ] or that are transmissible. spatial structure may limit the impact of vaccine evolution on immunity (e.g., prevent mutants from taking over the entire population). in contrast, vaccines that cause persistent infections or are transmissible are likely to be more severely affected by evolution than are vaccines causing acute infections, as there is a longer timeframe for evolution to operate. with so little experience from recombinant vector vaccines, we can merely guess how commonly the neglect of within-host evolution will compromise vaccine efficacy. given that simple steps can be taken to reduce vaccine evolution, vaccine development programs should at least entertain the possibility that evolution can underlie failure. avoiding vaccine evolution may be easier than developing an entirely new vaccine. supporting information s appendix. equations and parameters. 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models with data. monographs in population biology uses and abuses of mathematics in biology analysis of in vivo dynamics of influenza virus infection in mice using a gfp reporter virus memory cd + t cell differentiation: initial antigen encounter triggers a developmental program in naive cells naive ctls require a single brief period of antigenic stimulation for clonal expansion and differentiation models of cd + responses: . what is the antigen-independent proliferation program causes and consequences of spatial within-host viral spread. viruses self-boosting vaccines and their implications for herd immunity key: cord- - gwbc z authors: zecha, judith a. e. m.; raber-durlacher, judith e.; laheij, alexa m. g. a.; westermann, anneke m.; epstein, joel b.; de lange, jan; smeele, ludi e. title: the impact of the oral cavity in febrile neutropenia and infectious complications in patients treated with myelosuppressive chemotherapy date: - - journal: support care cancer doi: . /s - - - sha: doc_id: cord_uid: gwbc z febrile neutropenia (fn) is an inflammatory response causing fever that may develop during cancer therapy-induced neutropenia. fn may herald life-threatening infectious complications and should therefore be considered a medical emergency. patients presenting with fn are routinely subjected to careful history taking and physical examination including x-rays and microbiological evaluations. nevertheless, an infection is documented clinically in only – % of cases, whereas a causative microbial pathogen is not identified in over % of fn cases. the oral cavity is generally only visually inspected. although it is recognized that ulcerative oral mucositis may be involved in the development of fn, the contribution of infections of the periodontium, the dentition, and salivary glands may be underestimated. these infections can be easily overlooked, as symptoms and signs of inflammation may be limited or absent during neutropenia. this narrative review is aimed to inform the clinician on the potential role of the oral cavity as a potential source in the development of fn. areas for future research directed to advancing optimal management strategies are discussed. fever is a manifestation of multiple pathways including the release of pro-inflammatory cytokines (including interleukin (il)- , il- , and tumor necrosis factor (tnf)-alpha) as a consequence of infection or inflammation [ ] . fever that develops during cytotoxic cancer therapy-induced neutropenia, or "febrile neutropenia" (fn), is a medical emergency. fn may be an early and only indication of a serious infection, since profoundly neutropenic patients are unable to mount a robust inflammatory response, and signs and symptoms of inflammation are typically attenuated or absent. in these patients, infections may rapidly progress into life-threatening complications (e.g., systemic inflammatory response syndrome (sirs), (severe) sepsis, and septic shock). it is critical to early recognize fn (oral temperature > . °c or two consecutive readings of > . °c for h and an absolute neutrophil count < μ/l or expected to fall below this threshold), and depending on the estimated risk to develop life-threatening complications, initiate empiric systemic broad-spectrum antibacterial therapy and other supportive care measures promptly [ ] . moreover, fn is associated with higher morbidity, unplanned or prolonged hospitalization, potential intensive care admission, and can necessitate chemotherapy (ct) dose reductions and/or treatment delays, which may lead to poorer clinical outcome and survival [ , ] . if a patient presents with fn while on myelosuppressive ct, one should assume that this is due to infection, particularly if there is no evidence for an alternative explanation (e.g., transfusion reactions, medication allergies and toxicities, vasculitis or other inflammatory conditions, and tumor(lysis)-related fever) [ ] . nevertheless, patients must be carefully evaluated for potential infection. these patients are routinely subjected to careful history taking, physical examination, chest xray, and microbiological evaluations. however, infections can be documented clinically in only - % of febrile episodes, whereas a causative microbial pathogen cannot be identified in the majority (> %) of cases [ ] [ ] [ ] [ ] [ ] [ ] , and the cause of the fever may remain unidentified (fever of unknown origin; fuo). neutropenia risk depends on patient-, tumor-, and treatment-related factors. although ct dose intensity and myelotoxicity represent significant risk factors, neutropenia and its complications may occur after treatment with almost any type of ct [ ] . however, not all neutropenic patients develop fever, and not all episodes of fn progress into lifethreatening sepsis. ct regimens can be classified as high, intermediate, and low risk for fn [ ] . those with > % fn risk are considered high risk. this includes, but is not limited to, patients treated with high-dose ct with or without hematopoietic stem cell transplantation (hsct). most ct regimens used for the treatment of adult solid tumors and lymphoma are rated as intermediate (between and %) risk for fn, whereas low-risk cytotoxic regimens are typically considered to be associated with fn risk < % [ , ] . tools aimed to identify patients with fn at low risk of serious complications have been developed [ ] . however, identification of high-risk patients remains a challenge [ ] . it is well recognized that oral mucositis (om) as well as gastrointestinal mucositis may cause fever [ , ] and predisposes to systemic translocation of microorganisms [ , ] . if a patient presents with fn, the physician routinely performs an oral inspection to assess whether ulcerative om or mucosal infections are present. however, fever may be also caused by inflammation and infection at other oral sites, including the periodontium, the dentition, and salivary glands. most frequently, this involves asymptomatic chronic infections that were present before the initiation of ct. these infections may exacerbate and present with redness, swelling, and pain [ ] , but they may also be asymptomatic or manifest with only minimal signs of inflammation. nevertheless, these predominantly asymptomatic infections (particularly those of the periodontium) may contribute to systemic inflammation [ ] and risk of bacteremia and are associated with fn [ ] [ ] [ ] . unfortunately, these infections likely remain undiagnosed by simple oral inspection [ ] . in order to ascertain diagnosis and management of these "hidden" oral infections, the national institute of health [ ] stated that all patients should receive a comprehensive oral examination by a dental professional and potential foci of infection should be eliminated prior to the start of ct. in most centers, however, this is may be only routinely performed in patients considered at high risk for complications: i.e., patients scheduled for hsct and those undergoing (chemo)radiation therapy to the head and neck, whereas patients treated with other myelosuppressive ct regimens are not typically referred for pre-treatment oral evaluation. it is thus conceivable that particularly in these patients, fn may have originated from an oral source that remained undiagnosed. this narrative review is aimed to inform the oncologist of current evidence of the contribution of oral infections and non-infectious conditions (e.g., om) to the development of fn and infectious complications associated with myelosuppressive ct. areas for future research directed to developing optimal management strategies are presented. oral homeostasis is maintained due to complex and finely tuned interactions between the host and resident microorganisms. having cancer, myelosuppressive cancer treatment regimens and concurrent medications, including antibiotics, disturb the equilibrium of the oral ecosystem via direct and indirect mechanisms, and contribute to the development of oral complications and infectious sequelae. the mouth houses a diverse microbial community, harboring over species of bacteria that colonize the hard surfaces of teeth and soft tissues as biofilms [ ] . residing microbes prevent colonization and overgrowth of opportunistic and pathogenic microorganisms. studies suggest that treatment with myelosuppressive ct induces alterations of the oral microbiome ( table ) . as a result of cytotoxic ct and other concurrent medications, patients may develop hyposalivation (reduced salivary flow) [ ] . saliva has a key role in maintaining a stable oral ecosystem as it contains peptides and proteins that have many different qualities. these include antibacterial, antifungal, and antiviral functions as well as lubricant, buffering, remineralization, and digestion properties [ ] . a lack of these salivary defense mechanisms may contribute to oral dysbiosis contributing to an increase of the incidence and severity of complications, including om, oral mucosal infections, and dental caries [ , ] . the oral cavity is the primary gateway to the human body as it is contiguous with the tonsils, pharynx, esophagus, eustachian tube, middle ear, trachea, lungs, nasal passages, and sinuses. microorganisms colonizing the oral cavity may migrate via contiguous epithelial surfaces to these anatomical sites [ ] . in addition, oral microorganisms and inflammatory products may translocate into the circulation through ulcerated periodontal pockets and breached epithelial lining of mucous membranes and disseminate systemically [ , ] . this may contribute to fever and infectious complications in neutropenic or otherwise immunocompromised patients [ , , ] and has been reported to contribute to increased risk to early non-tumorrelated mortality in hsct recipients [ ] [ ] [ ] . om is an inflammatory condition of the oral mucosa induced by ct and/or radiation therapy. most often, it manifests at the buccal and labial surfaces, ventral surface of the tongue, floor of the mouth, and the soft palate [ ] . mucosal changes can range from erythema to extensive ulcerations and hemorrhage. patients experience om as a painful condition associated with significant suffering and a negative impact on the quality of life [ , ] . ulcerative om may not only act as a portal of entry for microorganisms and inflammatory products, but as it often compromises food intake, oral medication use, and oral hygiene, it may also contribute indirectly to infection risk [ , ] . the majority of patients treated with high-dose ct regimens (with or without hsct) develop om (all grades), whereas its incidence in less intensive forms of ct is estimated to vary between and % [ , ] (summarized in table ). there is a temporal relationship between having ulcerative mucositis, fever, and neutropenia [ , [ ] [ ] [ ] . however, the role of neutrophils in the pathogenesis and resolvent of om has not yet been fully elucidated, and studies on granulocyte stimulating growth factors for the prevention and treatment of om show conflicting results [ ] . sonis suggested that cells rendered apoptotic or necrotic by ct, release ct-associated molecular patterns ("cramps"), which play an important role in initiating inflammation [ ] . bacteremia with cons may originate from oral mucosal membranes hsct [ ] immunocompromised patients including cancer patients [ ] hsct [ ] soga et al. [ ] mrsa was detected in the oral cavity following hsct, especially during om peak severity hsct ct chemotherapy, cons coagulase-negative staphylococci, hsct hematopoietic stem cell transplantation, mrsa methicillin-resistant staphylococcus spp., om oral mucositis when pattern recognition receptors (prrs) expressed by cells of the innate immune system within the mucosa (e.g., macrophages, neutrophils, and dendritic cells) are exposed to cramps, this results in the release of cytokines that act locally, and also may have distant effects by activating intracellular and intercellular signaling loops [ ] . these include the hypothalamic-pituitary-adrenal axis resulting in fever, and effects on the liver resulting in the generation of acute phase proteins. all of which can ultimately result in sirs and sepsis. indeed, blijlevens and colleagues reported that mucositis induces a systemic inflammatory response characterized by fever in neutropenic hsct recipients, even in the absence of bacteremia [ , ] . although om is not an infectious process, studies suggest that poor oral hygiene and pre-existing oral infections aggravate the severity and duration of toxicity induced mucosal reaction and thereby contribute to the risk to develop fn and infectious complications [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (table ) . in addition to fn associated with om, oral mucosal ulcerations with a non-mucositis etiology may induce fn. however, from a clinical perspective, it can be difficult to discriminate between such lesions as they may develop simultaneously. herpes simplex virus (hsv) is a major pathogen causing mucosal ulceration that may be confused with om or may aggravate this condition [ ] . most hsv infections occur as a result of reactivation of latent virus in hsv seropositive patients. upregulation of nf-κb resulting in loss of mucosal barrier in om may also induce signaling pathways that stimulate virus reactivation [ ] . hsv reactivation can be controlled with antiviral prophylaxis or treatment in most patients, although reactivation is still possible despite prophylaxis [ ] . herpetic lesions in patients receiving ct can be painful and may be associated with hemorrhage and necrosis, and bacterial and fungal superinfections. in myelosuppressed patients, hsv infection has atypical clinical manifestations, mainly manifesting as intraoral ulcerations with or without labial involvement. hsv infection may develop at sites, which are usually not affected by om (e.g., the dorsum of the tongue, gingiva, and the hard palate). herpetic lesions in compromised hosts may persist for extended periods until recovery from myelosuppression or appropriately treated. in addition, other herpesviruses, epstein-barr virus (ebv) in particular, may predispose for developing oral mucosal ulcerations during neutropenia and thus may directly or indirectly play a role in fn originating from the oral cavity [ ] . periodontal pockets [ ] and salivary glands seem important reservoirs of cytomegalovirus (cmv) [ ] . the virus can be shed (via gingival crevicular fluid and salivary glands) in whole saliva forming a main mechanism for transmitting cmv. cmv induces local production of pro-inflammatory cytokines and remains a major cause of morbidity in hsct recipients [ ] . oral candidiasis is typically caused by opportunistic overgrowth of candida albicans, c. krusei, c. tropicalis, c. dubliniensis, and other commensal oral yeasts. neutropenia, mucosal damage, depressed cell-mediated immunity, hyposalivation, immunosuppressive medications, and use of antibiotics are risk factors. the most common forms are pseudomembranous and erythematous candidiasis [ ] . during oral infection with candida spp., a large amount of inflammatory cytokines is generated in the oral mucosa, potentially inducing fever [ , ] . current prophylactic strategies have reduced systemic candidiasis, although oral and oropharyngeal candidiasis still may have serious consequences. good oral hygiene is important in addition to antifungal therapy. rarely, molds, such as zygomycetes, coccidiomycosis, histoplasmosis, and aspergillus spp., cause oral infections. typical lesions include ulcers in the oral cavity, tongue, or gingival regions that may progress into invasion of deep tissues, including bone. early recognition (and confirmation by biopsy), aggressive debridement, and antifungal therapy with extended spectrum azoles are paramount [ ] . pre-existing dental pathologies that may cause fn and infectious complications during myelosuppressive ct include periodontal disease, profound dental caries, and periapical pathology due to infection of the root canal, (partially) impacted teeth, and retained roots [ ] . gingivitis is a localized or generalized inflammation of the gingiva without loss of periodontal attachment, whereas periodontitis is a chronic inflammation of the gingiva, affecting the deeper parts of the periodontium. as dental plaque migrates in apical direction, periodontal attachment degrades, leading to ulcerative periodontal pockets, bone resorption, and tooth loss. about - % of adults has chronic gingivitis, around % suffer from moderate periodontitis, whereas the prevalence of severe periodontitis is estimated to range from epstein and schubert [ ] treatment of dental pathologies and reducing the oral microbial load resulted in reduction of incidence and severity of om hsct [ ] hematologic malignancies [ ] kashiwazaki et al. [ ] maximal crp levels in peripheral blood and incidence of fn is significantly lower in the group who performed intensive oral hygiene hsct santos et al. [ ] treatment of dental pathologies and reducing oral microbial load resulted in shorter duration of om hsct khan and wingard [ ] presence of oropharyngeal mucositis is an independent risk factor for bacteremia in neutropenic patients hsct, non-hodgkin lymphoma, leukemia laine et al. [ , ] association between the presence of oral mucosal ulcers and episodes of fever and sepsis lymphoma sonis et al. [ ] schuurhuis et al. [ ] om is associated with higher fn risk as compared with patients without om ruescher et al. [ ] elting et al. [ ] bochud et al. [ ] marron et al. [ ] om was most likely origin of bacteremia with oral viridans streptococci, frequently resulting in fever and potentially leading to ards and septic shock hsct [ ] patients treated with ct [ , ] hematologic malignancies [ ] ebinuma et al. [ ] maintenance of good oral hygiene may reduce the gene mediating methicillin resistance. the oral cavity may act as a reservoir for harboring this gene hsct fn febrile neutropenia, om oral mucositis, hsct hematopoietic stem cell transplantation, crp c-reactive protein, ards acute respiratory distress syndrome, ct chemotherapy to % in western populations [ ] . periodontitis risk increases with age (peaks between and years) and is more prevalent in men than in women. the severity of the periodontal disease depends on environmental and host factors (e.g., genetic susceptibility, comorbidities, oral hygiene, and smoking) [ ] . inflamed and infected periodontal tissues can expose up to - cm of connective inflamed tissue and may serve as a reservoir of endotoxin (lipopolysaccharide), pro-inflammatory cytokines, and proteolytic enzymes that may spread systemically via ulcerated pocket epithelium and contribute to endothelial dysfunction, a pro-coagulant state, and low-level inflammation [ , ] . periodontal infection has been associated with bacteremic seeding of heart valves and prosthetic devices [ ] , fuo, and a wide range of systemic conditions, including ischemic cardiovascular disease, metabolic syndrome and type diabetes, rheumatoid arthritis, and respiratory diseases, including aspiration pneumonia [ ] [ ] [ ] . table summarizes the literature on gingivitis and periodontitis and their association with bacteremia, fn and infectious complications. ulcerated periodontal pocket epithelium can allow translocation of microorganisms into the bloodstream [ ] . bacteremia may be caused by facultative or strictly anaerobic bacteria likely originating from the periodontium, including capnocytophaga spp. and fusobacterium nucleatum [ ] [ ] [ ] [ ] [ ] . however, bacteremia with these microorganisms is relatively uncommon, whereas bacteremias with viridans streptococci and cons occur frequently in profoundly myelosuppressed patients. increases of gingival viridans streptococci and cons have been documented during highdose ct [ ] , and one study reported that periodontitis may contribute to the risk of bacteremia with viridans streptococci and cons during the neutropenic phase of hsct [ ] . periodontal disease may be occult without symptoms and clinical signs that may only be detected upon comprehensive periodontal evaluation and not identified based on general oral examination. a number of studies on the potential role of odontogenic infections in the development of fn do not distinguish between periodontal disease and other infections related to the dentition (table ). there is evidence suggesting that infectious and inflammatory processes of the oral cavity may contribute to fn, particularly in patients treated with high-dose ct with or without hsct. however, most studies are retrospective and include only a small number of patients. well-powered, prospective observational studies in homogenous groups of patients, including those treated with myelosuppressive ct for solid tumors, are needed to obtain a better understanding of the contribution of om and oral infections to fn and its potential sequelae. particularly, om [ , ] and periodontal disease [ , , , , , , , [ ] [ ] [ ] seem important in the development of fn, whereas the literature on other potential oral sources of fn (such as mucosal infections, periapical pathologies, and pericoronitis) is scarce. some studies report raber-durlacher et al. [ ] presence of periodontitis is associated with the development of bacteremia. hsct laine et al. [ ] in % of the febrile episodes, mild to moderate gingivitis was present in % of the febrile episodes, severe gingivitis was present lymphoma hong et al. [ ] . % of patients had severe gingivitis pediatric malignancies peterson and overholser [ ] greenberg et al. [ ] overholser et al. [ ] bergmann et al. [ ] laine et al. [ ] meurman et al. [ ] soga et al. [ ] gingivitis and periodontitis were found to be associated with bacteremia, fever, and sepsis in neutropenic patients russi et al. [ ] fn may be induced by periodontal inflammation and infection head and neck cancer fn febrile neutropenia, hsct hematopoietic stem cell transplantation infrequent conversion of chronic dental disease to an acute state during ct and conclude that there is no need for the treatment of "asymptomatic" chronic dental infections prior to ct or conditioning therapy for hsct [ , ] . however, studies should not focus solely on these acute exacerbations, associated with swelling and pain, since the inflammatory response may be muted or absent due to myelosuppression. it is essential that endpoints of future studies should also include the presence of "asymptomatic" disease, particularly chronic periodontitis which requires comprehensive examination including periodontal probing and appropriate dental imaging. there is convincing evidence from the periodontal literature that gingivitis as well as periodontitis induces frequent episodes of bacteremia during daily activities such as chewing and tooth brushing [ , ] . in non-myelosuppressed individuals, these episodes of bacteremia are typically transient. in myelosuppressed and otherwise immunocompromised cancer patients, however, bacteremia risk is likely increased as in addition to breached epithalial barriers, these patients are less capable of clearance of microorganisms from the blood stream, and infectious complications may develop. in addition to hematogenous spread of oral microorganisms and inflammatory mediators, there is evidence that poor oral and dental conditions may predispose for aspiration pneumonia [ , ] . systemic dissemination of oral microorganisms should be investigated in more detail in cancer patients treated with myelosuppressive ct. future investigations, using new open-end next-generation sequencing techniques, should aim to further characterize the role of the oral/periodontal microbiome in the pathobiology of mucositis as well as in systemic infectious complications. among other factors, the use of prophylactic antibiotics has changed the spectrum of isolated pathogens from the bloodstream as the most common causes of bacteremia in myelosuppressed cancer patients are coagulasenegative staphylococci, viridans group streptococci, and enterococci [ ] , which may all originate from the periodontium and oral mucosa [ , , , [ ] [ ] [ ] [ ] [ ] . particularly, the contribution of staphylococcus epidermidis originating from the oral cavity should be studied. an intriguing hypothesis that needs further exploration is the concept that inflammatory complications induced by cytotoxic cancer therapies may have a common pathobiological background and share genetically determined risk factors [ ] . inflammation, particularly an imbalance between upregulated greenberg et al. [ ] patients who did not receive dental treatment prior to ct: -in % of patients, sepsis occurred. - % of patients developed acute exacerbations of oral bacterial infections. patients in which dental infections were treated before ct: -in % of the patients, sepsis occurred. -no acute exacerbations of oral bacterial infections occurred. laine et al. [ ] patient with febrile episodes had more severe dental pathologies than those without fever ( . % vs . %) toljanic et al. [ ] incidence of acute dental problems during febrile episodes of % solid and hematologic malignancies akintoye et al. [ ] out of positive blood cultures with microorganisms were likely to originate from the oral cavity; of these bacteremias the periodontium was the most likely origin. hsct cullen et al. [ ] in % of febrile episodes an oral infection was thought to be the focus of infection hong et al. [ ] weighted prevalence of dental infections of . % patients treated with cytotoxic ct hong et al. [ ] weighted prevalence of dental infections was . %; pericoronitis was present in . % of patients akashi et al. [ ] development of septic shock in patients of which it was assumed that this was associated with a chronic odontogenic infection. no bacterial evidence was found from the blood cultures. fn febrile neutropenia, ct chemotherapy, hsct hematopoietic stem cell transplantation pro-inflammatory cytokine pathways and suppressed antiinflammatory cytokines, may be a major common driver of these complications [ ] . in addition, macrophages have been identified to play a critical role in the potential bidirectional links between periodontal and systemic inflammatory diseases [ ] . it has been proposed that the presence of inflammation (anywhere in the body) primes for a dysregulated and exaggerated inflammatory response following a subsequent inflammatory stimulus [ ] . this "two-hit model" has been hypothesized to underpin a potential association between periodontitis and om [ ] . moreover, it has been suggested that patients with periodontitis are more prone to developing fever and septic shock syndrome, since endotoxins derived from periodontal pockets may lead to precipitating conditions [ ] . these intriguing findings and hypotheses deserve further exploration. an important clinical question concerns the management of potential oral foci of fn. there is a consensus that patients planned to be treated with high-dose ct with or without hsct, or those who will be treated with curative (chemo)radiation for head and neck cancers should be referred to a dental professional for a comprehensive oral and periodontal pre-treatment evaluation with appropriate treatment. in patients with head and neck cancers, rigorous elimination of potential foci of infection (i.e., in most cases by extraction) is mandatory in patients with at-risk conditions in the radiation volume [ ] , whereas in other patient categories, protocols may be less rigid. there is substantial evidence suggesting that intensive oral care performed before and during cancer treatment results in a reduction of infectious complications [ , , , , [ ] [ ] [ ] . elad and coworkers [ ] developed a predictive model suggesting that no dental treatment increased the probability of dying due to a dental/periodontal infection in hsct recipients. in selected cases, treatment of periodontal infections by debridement and reducing the oral bacterial load, and endodontic treatments may be preferred over extractions. in addition, approaches aimed at resolving periodontal inflammation hold significant promise [ ] . these interventions may also positively affect om. when fn (likely) originating from the oral cavity develops during ct treatment, antimicrobial therapy should be preferably based on microbiological evaluation of swabs and/or oral rinsing samples. in conclusion, current recommendations for the prevention of complications include dental evaluation and work up of high-risk patients as an integrated part of the patient workup and management. nevertheless, large longitudinal studies are needed to 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retrospective evaluation. radiotherapy and oncology : journal of the champlin r ( ) oral mucositis in patients undergoing bone marrow transplantation a prospective study to evaluate a new dental management protocol before hematopoietic stem cell transplantation progress of oral care and reduction of oral mucositis-a pilot study in a hematopoietic stem cell transplantation ward a decision analysis: the dental management of patients prior to hematology cytotoxic therapy or hematopoietic stem cell transplantation periodontitis: a host-mediated disruption of microbial homeostasis publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgements we kindly acknowledge support from the eklund foundation. conflict of interest the authors declare that they have no conflict of interest. key: cord- -z ekgtv authors: babu, tara m; perera, ranawaka a p m; wu, joseph t; fitzgerald, theresa; nolan, carolyn; cowling, benjamin j; krauss, scott; treanor, john j; peiris, malik title: population serologic immunity to human and avian h n viruses in the united states and hong kong for pandemic risk assessment date: - - journal: j infect dis doi: . /infdis/jiy sha: doc_id: cord_uid: z ekgtv background: influenza a pandemics cause significant mortality and morbidity. h n viruses have caused a prior pandemic, and are circulating in avian reservoirs. the age-related frequency of current population immunity to h viruses was evaluated. methods: hemagglutinin inhibition (hai) assays against historical human and recent avian influenza a(h n ) viruses were performed across age groups in rochester, new york, and hong kong, china. the impact of existing cross-reactive hai immunity on the effective reproduction number was modeled. results: one hundred fifty individual sera from rochester and from hong kong were included. eighty-five percent of patients born in rochester and hong kong before had hai titers ≥ : against a/singapore/ / , and > % had titers ≥ : against a/berkeley/ / . the frequency of titers ≥ : to avian h n a/mallard/england/ / and a/mallard/netherlands/ / in subjects born before was % and %, respectively. there were no h hai titers > : in individuals born after . these levels of seroprevalence reduce the initial reproduction number of a/singapore/ / or a/berkeley/ / by %– %. a basic reproduction number (r( )) of the emerging transmissible virus < . predicts a preventable pandemic. conclusions: population immunity to h viruses is insufficient to block epidemic spread of h virus. an h n pandemic would have lower impact in those born before . influenza pandemics may occur when influenza a viruses of animal origin with a novel hemagglutinin (ha, or h) with or without neuraminidase (na, or n) subtypes to which the human population has little or no immunity infect humans and transmit efficiently from person to person. there were influenza pandemics in the th century. in , an influenza a virus of the h n subtype emerged and caused widespread disease; subsequently, h n viruses caused seasonal epidemics until . in a new influenza a virus of the h n subtype, sometimes referred to as the "asian" influenza, emerged to cause a second pandemic, and subsequently h n viruses replaced h n viruses as the cause of seasonal influenza from to . in a third pandemic was caused by an h n virus, which replaced h n viruses and continues to circulate in humans to the present day. influenza a pandemics are associated with significant mortality, morbidity, and financial burden. for example, the pandemic of - resulted in at least million influenza-related deaths [ ] , while the pandemics of (h n ) and (h n ) combined had estimated economic losses around us $ billion (estimated in dollars) [ ] . although not designated a pandemic, the reemergence of h n in also shared some characteristics with the other pandemics. the h n pandemic was caused by a virus subtype that was then circulating in humans and was unexpected because it was generally assumed that that population immunity would prevent emergence of a pandemic virus of a subtype currently endemic in humans. there continues to be concern regarding the potential for new influenza a viruses to be transmitted to humans, with documented severe zoonotic disease caused by influenza a h and h infections. however, because h n virus has already caused a pandemic, and h subtype viruses are currently circulating in wild and domestic birds [ ] , reemergence of an h n virus is one of the most likely scenarios for a new pandemic. anti-ha antibody, anti-na antibody, and cell-mediated immunity have all been correlated with protective immunity in both experimental animals and in humans [ ] [ ] [ ] [ ] . anti-ha antibody in peripheral blood sera, as assessed by the hemagglutination inhibition (hai) assay, has a strong correlation with protection against influenza infection and disease. although complicated by significant interlaboratory variation, an hai titer of : is generally accepted as a marker of reduced susceptibility [ ] . therefore, analysis of the population level of hai antibody can be used to estimate the population susceptibility to infection [ ] . for example, the seroprevalence of hai antibody to ph n in individuals older than years correlated with a significantly decreased influenza-associated mortality for these individuals during [ ] . it has been suggested that exposure to antigenically related h n influenza virus - years earlier provided older adults with some degree of immunity against the h n pdm virus [ ] . similarly, individuals who were exposed to h n viruses during the period from to may have persistent antibody to these viruses and be relatively protected from an emerging h n virus. however, more than two-thirds of the world population in was born after [ ] , suggesting that there may be substantial susceptibility to these viruses. because pandemics spread across the world within weeks after emergence [ ] , much faster than the process of developing and rolling out a vaccine to the newly emerged pandemic virus, which takes > months [ ] , attention has recently focused on developing systematic risk assessment algorithms for animal viruses of possible pandemic threat so that preemptive preparations including the development of vaccine seed strains can be initiated in advance. examples of these include the influenza risk assessment tool and the tool for influenza pandemic risk assessment [ ] . an integral aspect of this risk assessment process is assessment of population immunity to the relevant virus. in this study, we evaluated population immunity using hai assays against human and avian h n influenza strains in different age groups in the united states and hong kong. we then estimated the impact of existing cross-reactive hai immunity on reducing the effective reproduction number (r) of a potentially pandemic h subtype virus and characterized the minimum basic reproduction number (r ) that such a virus must possess to cause a pandemic. samples in rochester were collected between january and march from nonimmunosuppressed individuals who were healthy or had stable medical conditions and were from months to years of age. in the united states, influenza activity typically peaks in january or february. according to the centers for disease control and prevention, in - influenza activity peaked in early february and in - it remained low through february and did not peak until mid-march. in - , - , and - , influenza activity peaked in late december with some variability [ ] . sera from children and adults in hong kong were collected as part of a previous serological study between august and december , prior to the winter influenza season, which typically commences around february-march in hong kong [ ] . the preceding influenza season peaked in february-march and the dominant virus subtype was pandemic h n . two hundred ninety-five serum samples from this study were selected for testing in age strata. selection of viruses for hai testing was based on phylogenetic lineages of the h influenza virus subtypes: human and avian eurasian lineages. viruses were selected from each lineage to represent temporal and geographic diversity. a/singapore/ / (h n ) and a/berkeley/ / (h n ) represented the human lineage, whereas a/mallard/ england/ / (h n ) and recent h n virus isolate a/ mallard/netherland/ / (h n ) were of the eurasian avian lineage [ ] . the a/mallard/england/ / virus was generated by plasmid-based reverse genetics with ha and na of a/ mallard/england/ / and other internal virus genes of a/ puerto rico/ / origin. antigenic relatedness of the selected test viruses was determined by reciprocal hai assays shown in supplementary table . viruses with pandemic potential were handled in a us department of agriculture-approved animal biosafety level (absl )-enhanced facility. β-propiolactone (bpl)-inactivated virus using standard procedures for use as antigen in the hai test was prepared. the bpl-treated virus preparation was inoculated into -day-old hen's eggs following standard virus culture procedures to confirm complete inactivation of the virus. the antigen was then removed from the absl -enhanced laboratory for hai analysis. in rochester and hong kong, hai studies were performed in biosafety level conditions. serology hai tests were performed using turkey red blood cells in rochester, and chicken red blood cells in hong kong, otherwise the laboratories used the same procedure for the test. sera were pretreated with receptor-destroying enzyme (denka seiken co ltd) and tested at a starting dilution of : . hai was performed using "v" bottom microtiter plates as previously described [ ] . the positive control sera used were ferret hyperimmune sera against a/kruitt/ , a/mallard/netherlands/ / , a/swine/ missouri/ / , a/mallard/netherlands/ / , and a/bakker/ viruses, and goat hyperimmune sera against a/ japan/ / and a/singapore/ / . negative controls consisted of antigen alone wells and the reagent control contained phosphate-buffered saline with red blood cells. statistical significance was analyzed using graphpad prism software (graphpad, san diego, california) using -way analysis of variance, followed by bonferroni post hoc analysis. p values <. were considered statistically significant. the reproduction number of each virus in each population was calculated as follows. we partitioned the population into n = age groups ( - , - , - , - , - , - , - , ≥ ) and m = hai titer levels (< : , : , : , ≥ : ). let p i be the proportion of population in age group and s ij be the proportion of age group i with the jth hai titer. the age distribution p i was based on the most recent census data from the united states ( ) and hong kong ( ). to estimate s s for each age group, we used bayesian inference with dirichlet y i was the number of individuals in age group i in our serologic study and x ij was the number of subjects in age group i with the jth hai titer level [ ] . we assumed noninformative priors, that is, all priors were dirichlet distributions with parameters a j = for j m = ¼ , , , and hence the joint posterior distributions of s s we assumed that an hai titer of < : , : , : , and ≥ : reduced susceptibility by %, %, %, and %. as such, the proportion of the population that were immune was ij j , where z j was the reduction in susceptibility conferred by the jth hai titer level (ie, , and z = . ). the basic reproduction number r was the largest eigenvalue of the matrix q ij { } , where q ij was the average number of secondary cases in age group i generated by an infector in age group j when everyone in the population was susceptible [ ] . we constructed the matrix q ij { } using the united kingdom social contact matrix [ ] because analogous data are not available from the united states and hong kong. because the immune proportion of age group i was table . the demographics of the study population matched the demographics of the source population (data not shown). the results of hai testing of the sera from rochester and hong kong gave very similar results, despite the populations and different laboratories. the geometric mean titers (gmts) of antibody in the populations against the test viruses by decade of birth are shown in table . as expected, there were substantial levels of anti h hai antibody in the sera of persons old enough to have been infected with h n viruses between and , and essentially no detectable antibody in persons born after . among persons born before , the gmt of antibody against the early human a/singapore/ / was significantly higher compared with titers against the later human a/berkeley/ / virus, whereas in persons born from to there was a trend toward relatively higher titers against the a/berkeley/ / virus. titers against the avian h n viruses were lower. there were no significant differences in the gmt against a/singapore/ / , a/berkeley/ / , or a/ mallard/england/ / in sera tested in hong kong and rochester, but sera tested in rochester had significantly higher titers against a/mallard/netherlands/ / than the sera tested in hong kong. the prevalence of titers ≥ : against the test viruses is shown for sera from rochester and hong kong in persons born before the h n pandemic, during the years that h n circulated ( - ), or after is shown in figure . ninetyeight percent of individuals from rochester and hong kong born before had titers ≥ : against the a/singapore/ / virus, whereas > % of subjects born between and had titers ≥ : . in contrast, none of those born after had titers > : to a/singapore/ / . generally, persons born prior to had a lower prevalence of titers ≥ : to a/ berkley/ / , while the prevalence of titers ≥ in persons born during circulation of these viruses was similar against a/ ( ) ( ) - ( ) ( ) - ( ) ( ) - ( ) ( ) - ( ) ( ) - ( ) ( ) or later ( ) ( ) data are presented as no. (%). singapore/ and a/berkley/ . the prevalence of titers ≥ : against the avian h n viruses tested were significantly lower in these groups. no person born after had titer > : against any of the h viruses. after combining the results from both populations, the cumulative distribution of antibody titers against the test viruses in these age groups is shown in figure . eightyfive percent of individuals born in both the united states and hong kong before had hai titers ≥ : against a/ singapore/ / ( figure ). more than % of subjects had hai titers ≥ : to a/berkeley/ / if born before . the frequency of titers to avian h n viruses was %, and % of subjects born before had titers of ≥ : to a/mallard/ england/ / and a/mallard/netherlands/ / , respectively, with such titers seen in % and % of individuals born from to . successful pandemic emergence and spread of a virus depends on the proportion of the population that is immune and the initial r of the potentially pandemic strain. the median r for the a/h n pandemic was . (interquartile range [iqr], . - . ) [ ] . to assess the impact of these age-dependent population immunity profiles on the pandemic potential of each of these viruses if they were to emerge in the human population, we computed the impact of population immunity in reducing r (figure ). the current population immunity in the united states and hong kong would reduce the initial r of a/singapore/ / by around % ( %- %) and that of a/ berkeley/ / by around % ( %- %). as such, a pandemic of a/singapore/ / and a/berkeley/ / would be prevented if initial r of the emerging virus was below . ( . - . ) and . ( . - . ), respectively. the threshold r below which a pandemic with the avian subtype h viruses a/mallard/england/ / and a/mallard/netherlands/ / would be prevented was slightly lower. the comparability of data from geographically separated areas of the world, rochester and hong kong, argues for the representativeness and generalizability of such studies that aim to assess population immunity to viruses of pandemic concern. our study suggests that those individuals born prior to or during the period of h n virus circulation were more likely to have higher hai titers against the human h n viruses than those born after when h infection in humans had been displaced by the h n virus. in our study, we also confirmed evidence of cross-reactive hai antibodies to unrelated avian h viruses, albeit at lower prevalence and titer. the prevalence of such cross-reactive antibodies was higher in those born prior and the gmt of these cross-reactive antibodies was higher in those born prior to than in the birth cohort of - . this is possibly because those who were infected in the early pandemic waves of the h n virus in - were reinfected some years later by antigenically drifted h n viruses, possibly broadening cross-reactive immunity. as reported by others, we also observed low hai titers < : spanning across the age groups and this could be due to nonspecific data are presented as geometric mean titer ( % confidence interval). a samples in hong kong and rochester were tested at a starting dilution of < : and negative tests are given an imputed value of . inhibition by the sera. however, we checked the sera for nonspecific agglutinins where about % of the samples contained nonspecific inhibitors. since the assays were run on sera which were confirmed not to contain nonspecific inhibitors, these low titers may be due to a certain type of antigen exposure that needs further investigation. sera from those born after had higher hai titers to a/berkeley/ / than to a/singapore/ / whereas the converse was true in those born before . the older group of individuals who were first infected by a/singapore/ / like viruses likely had a boost of these titers when they were subsequently infected by later drift variants (ie, a/ berkley/ / -like viruses), the phenomenon known as "original antigenic sin" [ ] . the broader cross-reactivity may also occur due to targeting different antigenic sites, as a recent study points out that antibody response against h is mainly to the receptor binding domain resulting in a greater degree of cross-reactivity whereas for h or h viruses it is the hypervariable regions of ha resulting in a lesser cross-reactivity [ ] . soon after the h n pandemic of , studies done on sera collected prior to this pandemic were carried out and it was reported that people born prior to had detectable hai antibody to h viruses. it was therefore suggested that the historical pandemic that was believed to have occurred in - was likely caused by an h subtype virus [ ] . this prior exposure has also shown to elicit cross-reactive antibody for h strains that are currently circulating in animals, potential candidate pandemic stains [ ] . sera tested in rochester yielded higher titers against a/mallard/ netherlands/ / than the sera tested in hong kong. the differences between laboratories are not unexpected given the known laboratory-to-laboratory variation in the hai test, and what remains remarkable is the closeness of results. one technical difference between the laboratories was the source of erythrocytes for the hai test; rochester used turkey erythrocytes whereas hong kong used chicken erythrocytes. it is not known if this may contribute to this difference in hai test results. an alternative reason for the discrepancy in titers between countries may be attributed to variation in vaccination rates. vaccine uptake in hong kong is lower than in the united states. potentially, repeated vaccination may increase cross-reactive antibody to this avian h strain. assuming that the h seroprevalence in rochester and hong kong reflects global population seroprevalence, we modeled the impact of such immunity on possible emergence of an h n pandemic. this model was based on the candidate pandemic h strain originating from escape of the human adapted h n virus or from the avian gene pool that acquires transmissibility in humans. we estimate that the current levels of population immunity would reduce r of a/singapore / / - a recent risk assessment for viruses currently known to be circulating in wild birds has been carried out and the risk of these viruses acquiring transmissibility in humans was assessed to be low; most isolates replicated in human bronchial epithelial cells and ferrets. several did transmit between ferrets by direct contact, but all has retained a preference for avian-like α , -linked sialic acid receptors [ ] . these viruses still remain primarily in wild birds and have not been established in mammalian species including swine. our study provides the systematic assessment of the impact of human population immunity that goes toward such an overall assessment of pandemic risk. a limitation of the study is that other potential contributors to population immunity, such as cross-reactive na-inhibiting antibody, stalk-specific antibody, cell-mediated immunity, and heterosubtype reactive hai or neutralizing antibody [ ] [ ] [ ] [ ] ] , were not assessed. furthermore, in many parts of the world (especially in developing countries), the proportion of individuals born in or later might be larger than that in rochester and hong kong. the level of immunity against a(h n ) in these populations would be lower than that estimated here and the pandemic potential of a(h n ) in these populations would thus be higher than estimated here. in summary, we find that levels of population immunity to h subtype viruses are not substantial enough to block epidemic spread of an h virus that had acquired efficient transmissibility between humans. furthermore, the existing levels of population immunity to h viruses will continue to decline with new birth cohorts being added to the human population. given that human-adapted h n viruses that arose subsequent to the pandemic are present in many laboratories worldwide, these findings support the need to have preparedness for h viruses as a credible pandemic threat. the approach used in the case study of h viruses is more broadly applicable in defining the impact of population immunity to viruses to which there is some level of cross-reactive hai antibodiesfor example, swine h and h viruses and avian h n viruses [ ] . reliable methods for assessing population immunity are a key to reliable risk assessment of viruses for pandemic threat. it was the lack of such risk assessment of population immunity to the h n triple reassortant swine viruses that led to these viruses not being recognized as potentially pandemic viruses prior to ; indeed, it was the triple reassortment swine ha that was the key protective antigen for the h n pandemic virus. supplementary materials are available at the journal of infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. figure . estimations of overall population-level immunity against h viruses and the potential impact of population immunity on reproduction number, using potential titer cutoffs for % immunity. bars updating the accounts: global mortality of the - "spanish" influenza pandemic origin of the pandemic h influenza a virus and the persistence of its possible progenitors in the avian reservoir correlates of protection to influenza virus: where do we go from here? antibody to influenza virus neuraminidase: an independent correlate of protection preexisting influenza-specific cd + t cells correlate with disease protection against influenza challenge in humans cellular immune correlates of protection against symptomatic pandemic influenza seroprevalence to influenza a(h n ) virus-where are we? complex patterns of human antisera reactivity to novel h n and historical h n influenza strains cross-reactive antibody responses to the pandemic h n influenza virus vaccinate for the next h n pandemic now the infection attack rate and severity of pandemic h n influenza in hong kong pandemic preparedness and the influenza risk assessment tool (irat) relative incidence and individual-level severity of seasonal influenza a h n compared with pandemic h n adaptation of pandemic h n influenza a viruses in humans world health organization. manual for the laboratory diagnosis and virological surveillance of influenza the bugs book: a practical introduction to bayesian analysis inferring influenza infection attack rate from seroprevalence data social contacts and mixing patterns relevant to the spread of infectious diseases estimates of the reproduction number for seasonal, pandemic, and zoonotic influenza: a systematic review of the literature original antigenic sin responses to influenza viruses host versus flu: antibodies win a round? pre-epidemic antibody against , strain of asiatic influenza in serum of older people living in the netherlands studies on the content of antibodies for equine influenza viruses in human sera risk assessment of h n influenza viruses from the avian reservoir safety and antigenicity of whole virus and subunit influenza a/hong kong/ / (h n ) vaccine in healthy adults: phase i randomised trial key: cord- - qct authors: kummer, susann; avinoam, ori; kräusslich, hans-georg title: ifitm clusters on virus containing endosomes and lysosomes early in the influenza a infection of human airway epithelial cells date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: qct interferon-induced transmembrane proteins (ifitms) have been shown to strongly affect influenza a virus (iav) infectivity in tissue culture. moreover, polymorphisms in ifitm have been associated with the severity of the disease in humans. ifitm appears to act early in the infection, but its mechanism of action and potential interactions with incoming iav structures are not yet defined. here, we visualized endogenous ifitm interactions with iav in the human lung epithelial cell line a and in primary human airway epithelial cells employing stimulated emission depletion super-resolution microscopy. by applying an iterative approach for the cluster definition and computational cluster analysis, we found that ifitm reorganizes into clusters as iav infection progresses. ifitm cluster formation started at - h post infection and increased over time to finally coat iav-containing endosomal vesicles. this iav-induced phenotype was due to the endosomal recruitment of ifitm rather than to an overall increase in the ifitm abundance. while the iav-induced ifitm clustering and localization to endosomal vesicles was comparable in primary human airway epithelial cells and the human lung epithelial cell line a , the endogenous ifitm signal was higher in primary cells. moreover, we observed ifitm signals adjacent to iav-containing recycling endosomes. influenza a virus (iav) is the major cause for a contagious illness of the upper and lower respiratory tract during the seasonal influenza epidemics with peaks in fall and winter for each hemisphere [ ] [ ] [ ] . besides the acute risk through circulating human specific strains, the zoonotic reservoir (e.g., birds, swine) poses a constant threat of new influenza pandemics [ ] [ ] [ ] [ ] . iav hijacks cellular import mechanisms to enter the host cell, thereby making use of multiple entry routes. the binding of iav by the sialylated receptor [ , ] is followed by clathrin-mediated endocytosis [ , ] or micropinocytosis [ ] . it is reported that iav entry is cell-type dependent [ ] and that subtypes with a filamentous particle shape preferentially enter host cells via macropinocytosis [ ] . the trimeric surface glycoprotein hemagglutinin (ha) is a key factor for several steps during viral entry via endocytosis [ , ] . irrespective of the entry mechanism, iav exploits the non-linear endosomal pathway with its multitude of branches and needs to pass different stages of the endocytic machinery, which is assembled and constantly renewed around the internalized virus particles [ ] [ ] [ ] . endosomal trafficking to the perinuclear region is crucial for ph-dependent membrane fusion [ ] [ ] [ ] , followed by the release of the viral genome into the cytosol and nuclear import [ ] . the iav genome is composed of eight single rna strands in a negative sense orientation [ ] , and each segment is complexed with the nucleoprotein np [ , ] , forming ribonucleoparticles (rnps; [ , ] ). viral genome replication, splicing and transcription then occur in the nucleus. iav replication is strongly inhibited by type interferons already at the early stage, with mx being identified as a crucial interferon-induced host protein blocking iav replication [ ] . genome-wide sirna screens have identified many host factors modulating influenza virus infection [ ] [ ] [ ] [ ] [ ] . brass et al., [ ] and shapira et al., [ ] reported the strong inhibition of iav infection by interferon-induced transmembrane proteins (ifitms). the ifitm variants - can be induced by interferon i and ii. their localization is cell type-or tissue-dependent and changes with the expression level with a preference for cytoplasmic vesicles in most monolayer cell lines [ ] [ ] [ ] [ ] [ ] . besides iav, ifitm proteins have also been reported to confer a basal resistance to members of the flaviviridae (dengue and west nile virus) [ ] , bunyaviridae (rift valley fever virus) [ ] , filoviridae (ebola virus) [ ] , coronaviridae (sars) and retroviridae (hiv) [ ] , showing an unusually broad activity against a wide variety of enveloped and some non-enveloped viruses [ ] . however, murine leukemia virus (mlv) is not restricted by ifitm despite being an enveloped virus [ ] . the relevance of the ifitm -mediated inhibition of iav infection received strong support when it was shown that ifitm polymorphisms correlated with the severity of iav disease in human infection [ ] . the relevance of the ifitm rs -c polymorphism for severe iav infection appears to be population-dependent [ ] [ ] [ ] [ ] [ ] [ ] , and a second ifitm single nucleotide polymorphism, rs -a, was also reported to influence the severity of iav infection in humans [ ] . however, the mechanism of the ifitm-mediated inhibition of iav infection remains under discussion. ifitms have been suggested to disrupt viral membrane fusion [ , ] by altering cellular membrane properties such as fluidity and curvature [ , , ] . it has also been discussed that ifitms alter the lipid/protein composition of acidic intracellular compartments such as endosomes and lysosome [ , ] . the lipid composition-based models of the ifitm function cannot easily explain the lack of antiviral effects on viruses like amphotropic mlv or arenaviruses [ , ] , which also enter by endocytosis and endosomal fusion. in the case of iav, it was recently reported that ifitm elevates the level of cholesterol on late endosomes and lysosomes, thereby restricting early iav infection [ ] . other hypotheses suggest that ifitm directly interferes with the iav fusion pore formation or redirects iav containing endosomes to a non-productive pathway [ ] . it was further demonstrated that the inhibition of the hemagglutinin-mediated membrane fusion required the amphipathic helix of ifitm [ ] . most findings about the antiviral action and localization of ifitm are based on studies using expression plasmids creating an over-expression and/or focusing at the late stages of infection (> h post-infection) [ , , ] . due to the enormous protein load in the overexpression situation, it is far more difficult to determine the subtle differences in the localization of individual molecules, and thus it is possible that these changes may be overlooked. in addition, proteins whose expression is enhanced artificially are more prone to be degraded via the lysosomal pathway. in this regard, it is difficult to judge whether this is a natural re-localization as part of the antiviral defense or just a degradation via lysosomes, particularly in the late stages of infection. we therefore investigated the role of endogenous ifitm in the viral uptake at an early stage. we analyzed the localization of endogenous ifitm through the course of iav infection using confocal and super-resolution (sted) microscopy. we observed a strong clustering of ifitm early after iav infection, which was distinct from the interferon alpha induced ifitm up-regulation. moreover, a two-color sted nanoscopy revealed a close proximity of ifitm with the iav np protein in rab positive compartments within a time range of - h after the iav addition. a and mdck cells were maintained in dulbecco's modified eagle medium (dmem) (invitrogen, karlsruhe, germany), supplemented with % foetal calf serum (fcs) (invitrogen) and penicillin ( u/ml)/streptomycin sulphate ( µg/ml) (capricorn scientific gmbh, ebsdorfergrund, germany). human small airway epithelial cells (hsaepcs) were obtained from promocell (c- ) and grown in a primary cell basal medium (promocell) without antibiotics. four days after thawing, the hsaepcs were seeded for immunofluorescence. the ifitm knock-down in a cells was induced by crispr-cas gene editing using a single guide rna targeting the polyadenylation side of ifitm [ , ] . a ifitm knock-down cells were selected by puromycin resistance ( µg/ml). the ifitm expression was checked by real-time quantitative pcr, as described by zhao et al., [ ] using the fast real time pcr system (applied biosystems, lifetechnologies, sigma-aldrich, steinheim, germany) and by immunofluorescence, as noted below ( figure s ). in general, the cells subjected to immunofluorescence were seeded in well plates on mm glass cover slips. for immunoblotting, cells were seeded in well plates. human interferon alpha (hifnα) (sigma-aldrich, steinheim, germany) was used at u/ml for h prior to infection. amantadine (sigma-aldrich) and bafilomycin a (merck, darmstadt, germany) were applied to the cells at µm and nm concentrations min prior to infection. the cells were treated with mouse anti-hifnα (catalogue no. ab , abcam, berlin, germany). influenza a virus a/hong kong/ / (hk/ / ), a/puerto rico/ / (pr/ / ) and a/regensburg/ d / (r/d / ) were recovered in a hek t-mdck-coculture using the eight plasmid reverse genetics systems, as previously described [ ] . in live-cell experiments, cell mask™ orange plasma membrane stain (catalogue no. c , life technologies, sigma-aldrich, steinheim, germany) was directly added to the cell supernatant prior to the virus purification, following the manufacturer's guidelines. the virus stocks were titrated, making use of a standard plaque assay with minor changes, as previously described [ ] . briefly, serial -fold dilutions of each virus stock were prepared in dmem supplemented with . % bovine serum albumin (bsa, sigma-aldrich, steinheim, germany), mm hepes (sigma-aldrich, steinheim, germany), penicillin ( u/ml)/streptomycin sulphate ( µg/ml) (capricorn scientific gmbh, ebsdorfergrund, germany) and tpck-trypsin ( µg/ml) (sigma-aldrich, steinheim, germany). mdck cell monolayers were incubated with µl of each dilution for h at • c in a well plate format. then, the cells were overlaid with ml of . % avicel (fmc) in aqua dest. and × dmem (merck, darmstadt, germany) in a : ratio. after days, the overlay was removed, the cells were fixed with ml/well of % ethanol for min at room temperature and stained with ml/well of . % crystal violet (sigma-aldrich, steinheim, germany) in % methanol for min at room temperature. the cells were fixed using % paraformaldehyde (pfa) in × phosphate buffered saline (pbs) for min at • c and subsequently treated with . % triton-x in pbs for min. blocking was performed using % bsa in pbs for min. the following primary antibodies were used for immunofluorescence: mouse anti-np (catalogue no. mab , merck, darmstadt, germany), rabbit anti-ifitm (catalogue no. - -ap, proteintech, manchester, united kingdom), mouse anti-eea (catalogue no. , bd transduction laboratoriestm, san jose, ca, usa), mouse anti-rab (catalogue no. ab , abcam, berlin, germany), mouse anti-rab (catalogue no. sc- , santa cruz, dallas, texas, usa), and mouse anti-lamp (catalogue no. ab , abcam, berlin, germany). the cells were incubated with the indicated primary antibodies in a blocking solution for h at room temperature, and washed three times in pbs. the fixation, cellular membrane permeabilisation, and blocking were repeated between the first and secondary antibody incubation, as described above. for sted microscopy, goat anti-mouse star red (catalogue no. - - - , abberior, göttingen, germany) and goat anti-rabbit atto (catalogue no. abin , antibodies-online gmbh) were used as the secondary antibodies. for the triple staining, including the non-diffracted nm channel, donkey anti-goat alexa (catalogue no. ab , abcam, berlin, germany) was used. the secondary antibody staining was performed for h at room temperature under exclusion of light. after repeated washing in pbs, cover slips were mounted on the objective slides using mowiol. whole-cell lysates were prepared using a × lysis buffer ( % sucrose, . % bromophenol blue, mm edta ph . , mm tris ph . , % sds, and % beta-mercaptoethanol). protein samples were resolved by . % sds-page at volt for . h. then, the proteins were transferred to immobilon-fl membranes (merck, darmstadt, germany). the membranes were blocked in li-cor blocking buffer for min at room temperature and probed with first antibodies (rabbit anti-ifitm , catalogue no. - -ap, proteintech, manchester, united kingdom, and rabbit anti-actin, catalogue no. a , sigma-aldrich, steinheim, germany, mouse anti-tubulin no. t , sigma-aldrich, steinheim, germany, or mouse anti-gapdh, catalogue no. sc , santa cruz, dallas, texas, usa) in a blocking solution diluted : at • c overnight. after three repeated washings in × tbst for min, the membranes were incubated with a secondary donkey anti-mouse antibody (catalogue no. - , li-cor) or secondary donkey anti-mouse antibody (catalogue no. - , li-cor, lincoln, nebraska usa), for h at room temperature in the dark. the protein bands were visualized using the li-cor odyssey scanner (li-cor, lincoln, nebraska usa). fluorescence imaging was performed using a two-colour-sted microscopy instrumentation (abberior instruments gmbh, göttingen, germany). for confocal and sted microscopy, a × olympus uplansapo (na . ) oil immersion objective was used. for excitation (λ= nm and λ = nm), a nominal laser power of % was applied, and for sted (λ = nm, max. power = . w), a nominal laser power of % was applied. the pixel size was set to nm (confocal) and nm (non-diffracted), respectively. minor adjustments of contrast and brightness of the acquired images as well as the richardson-lucy deconvolution with a regularisation parameter of . (stopped after iterations) were carried out with the imspector software ( . . -win -aifpgav , abberior instruments gmbh, göttingen, germany). the d object counter and coloc plugin in fiji software (https://fiji.sc/, -bit version)were used for the cluster and mander's coefficient correlation (mcc) analyses, respectively. mcc was performed using the costes regression threshold. the defined objects were assigned as a cluster being larger than nm based on sted images with approximately nm resolution. all graphs show the mean values of three independent experiments plotted with the standard error calculated as a two tailed unpaired t-test. a statistical analysis was performed using graphpad prism software (version ). to visualize the subcellular localization of endogenous ifitm , we performed immunofluorescence microscopy of the uninfected and iav infected a cells. the basal ifitm levels of the uninfected cells showed weak cytosolic signals upon immunofluorescence staining. following infection with iav a/hong kong/ / (hk/ / ) for h, most cells exhibited strong and clustered ifitm signals in the extranuclear space ( figure a ). the ifitm signal increase and clustering correlated with the overall dim np signals in the extranuclear space and no nuclear np signals in the respective cells. in contrast, the productively iav infected a cells (exhibiting a strong nuclear np signal) in the same field of view did not exhibit an increased ifitm signal intensity or clustering ( figure a , right panels). given that we used a relatively high moi of one and that dim np signals were generally detected in these cells, these observations strongly argue that iav infection was abortive in the cells exhibiting an early accumulation of ifitm . ifitm is induced by type interferon [ ] . therefore, we analyzed its distribution upon the treatment of a cells with human interferon alpha (hifnα) for h and compared this to a cells which had been pretreated with hifnα and subsequently infected with iav ( figure b ). the cells treated for h with hifnα exhibited an increased ifitm signal intensity in the extranuclear ifitm is induced by type interferon [ ] . therefore, we analyzed its distribution upon the treatment of a cells with human interferon alpha (hifnα) for h and compared this to a cells which had been pretreated with hifnα and subsequently infected with iav ( figure b ). the cells treated for h with hifnα exhibited an increased ifitm signal intensity in the extranuclear space, both in the uninfected a cells and in the iav-infected cells with a dim or absent np signal. importantly, the productively infected cells exhibiting strong nuclear np signals again exhibited a weak ifitm signal, even after the pretreatment with hifnα. comparing the non-treated and hifnα-pretreated iav-infected a cells lacking a strong nuclear np signal, we observed an increased ifitm signal in both cases, partially colocalizing with vesicular structures. vesicular localization appeared more obvious in the absence of a hifnα pretreatment with a less diffuse cytosolic signal. blocking ifnα with a neutralizing antibody during the iav infection of a cells resulted in increased infection rates, and a concomitant minimal increase of the ifitm signal ( figure c ). to determine whether an iav-induced viral membrane fusion and genome uncoating are required for the observed ifitm signal increase upon iav infection, we performed experiments in the presence of bafilomycin a , specifically inhibiting endosomal acidification, or in the presence of amantadine, specifically blocking the tetrameric m channel of iav, thereby preventing genome uncoating. in both cases, no increase in the ifitm signal intensity and no ifitm clustering were observed ( figure d ,e). these results indicate that iav-induced membrane fusion and genome uncoating are required for an ifitm signal increase in a cells. the mechanism by which ifitm impedes iav infection was divergent in previous studies, and inhibition was mostly described upon ifitm over-expression [ , , ] . it was demonstrated that an ifitm knockdown in a cells increases infection rates [ ] . to determine whether endogenous ifitm is relevant for iav infection in a cells under the experimental conditions of our study, we performed iav infection in a ifitm knock-down cells ( figure s a ,b). the ifitm expression was reduced by a factor of (on the mrna level) in these cells ( figure s c ). the iav infection rate was significantly increased in the knock-down situation compared to the a wildtype cells (figure a ). viruses , , x for peer review of space, both in the uninfected a cells and in the iav-infected cells with a dim or absent np signal. importantly, the productively infected cells exhibiting strong nuclear np signals again exhibited a weak ifitm signal, even after the pretreatment with hifnα. comparing the non-treated and hifnαpretreated iav-infected a cells lacking a strong nuclear np signal, we observed an increased ifitm signal in both cases, partially colocalizing with vesicular structures. vesicular localization appeared more obvious in the absence of a hifnα pretreatment with a less diffuse cytosolic signal. blocking ifnα with a neutralizing antibody during the iav infection of a cells resulted in increased infection rates, and a concomitant minimal increase of the ifitm signal ( figure c ). to determine whether an iav-induced viral membrane fusion and genome uncoating are required for the observed ifitm signal increase upon iav infection, we performed experiments in the presence of bafilomycin a , specifically inhibiting endosomal acidification, or in the presence of amantadine, specifically blocking the tetrameric m channel of iav, thereby preventing genome uncoating. in both cases, no increase in the ifitm signal intensity and no ifitm clustering were observed ( figure d ,e). these results indicate that iav-induced membrane fusion and genome uncoating are required for an ifitm signal increase in a cells. the mechanism by which ifitm impedes iav infection was divergent in previous studies, and inhibition was mostly described upon ifitm over-expression [ , , ] . it was demonstrated that an ifitm knockdown in a cells increases infection rates [ ] . to determine whether endogenous ifitm is relevant for iav infection in a cells under the experimental conditions of our study, we performed iav infection in a ifitm knock-down cells ( figure s a ,b). the ifitm expression was reduced by a factor of (on the mrna level) in these cells ( figure s c ). the iav infection rate was significantly increased in the knock-down situation compared to the a wildtype cells ( figure a ). the increase in the ifitm signal intensity in a cells upon iav infection ( figure a ) and interferon treatment ( figure b ) could be caused either by higher expression levels or by ifitm clustering, yielding viruses , , of a higher density of the signal. to distinguish between these possibilities, we determined the ifitm abundance by an immunoblot analysis of the a cells at different time points after iav infection or interferon treatment, respectively. no significant increase in the ifitm expression was observed during the first h of iav infection ( figure b) , indicating that the signal changes observed by fluorescence microscopy (examples are shown in figure ) during this period were caused by the re-localization of constitutively expressed ifitm rather than by an induced ifitm expression. a strong increase in the ifitm expression levels was observed at late time points ( h p.i.; figure s b ), in accordance with previous studies [ ] . an increased ifitm expression was also observed following the interferon treatment, but starting already at h after the interferon addition ( figure s ), as previously reported [ ] . accordingly, the immunofluorescence phenotype after the interferon treatment ( figure b) is likely to be caused by an increased expression rather than by clustering. the increase in the ifitm signal intensity in a cells upon iav infection ( figure a ) and interferon treatment ( figure b ) could be caused either by higher expression levels or by ifitm clustering, yielding a higher density of the signal. to distinguish between these possibilities, we determined the ifitm abundance by an immunoblot analysis of the a cells at different time points after iav infection or interferon treatment, respectively. no significant increase in the ifitm expression was observed during the first h of iav infection ( figure b) , indicating that the signal changes observed by fluorescence microscopy (examples are shown in figure ) during this period were caused by the re-localization of constitutively expressed ifitm rather than by an induced ifitm expression. a strong increase in the ifitm expression levels was observed at late time points ( h p.i.; figure s b ), in accordance with previous studies [ ] . an increased ifitm expression was also observed following the interferon treatment, but starting already at h after the interferon addition ( figure s ), as previously reported [ ] . accordingly, the immunofluorescence phenotype after the interferon treatment ( figure b) is likely to be caused by an increased expression rather than by clustering. to unravel the distribution pattern of ifitm in a cells at early stages of infection, we made use of super-resolution microscopy. the blurring effect of diffraction limited imaging can obfuscate the potential clustering of ifitm . stimulated emission depletion (sted) super-resolution microscopy was therefore applied to resolve ifitm clusters in early iav-infected a cells. cells infected with iav hk/ / were fixed at - h post infection (h p.i.), stained with anti-ifitm and subjected to confocal and sted microscopy (resolution < nm) of the same region ( figure a ). an increased ifitm signal intensity was again observed in the early phase of the iav infection, and individual ifitm clusters could be resolved by sted microscopy. we computationally analysed the ifitm subcellular localization based on raw sted images using a d cluster analysis (objects counter). the clusters were defined as extranuclear ifitm signal accumulations with a size of > nm . the threshold was chosen by an iterative approach searching for the proportion of clusters, with the critical size being altered upon the infection progression. the analysis of uninfected and hk/ / -infected a cells at different time points revealed a very low proportion of large clusters in uninfected cells or up to h p.i. at h p.i. and later, we observed a significant increase in cluster abundance ( figure b although a cells are an established model cell line for iav research, adaptation to cell-culture conditions may have occurred; hence, we verified our results in primary human respiratory epithelial cells. to determine whether iav infection also causes ifitm clustering in primary cells, we infected human small airway epithelial cells (hsaepcs) with iav pr/ / for different periods of time and performed an indirect immunofluorescence analysis for ifitm and np (from incoming iav particles) using confocal and sted microscopy ( figure , uninfected example see figure s ). the iav infected hsaepcs revealed a co-localization of ifitm and iav np signals at apparently vesicular structures as early as h p.i. (figure a , white arrowheads). at later time points ( h p.i.), this was more obvious, with a clear ifitm clustering on the np-containing vesicular structures ( figure b ). ifitm often exhibited a ring-like appearance, suggesting the coating of endosomal vesicles, and this phenotype became more obvious at later time points ( h p.i; figure c ). some ifitm -positive vesicles exhibited strong np signals (e.g., figure b ), suggesting ifitm -coated vesicles carrying multiple iav particles. a strong ifitm clustering with a ring-like appearance indicating vesicle coating was observed in both iav-infected a cells ( figure a ) and hsaepcs at h p.i. (figure c ; white arrowheads indicating ring-like structures). in the case of the a cells, the ifitm signals in uninfected cells were weak and mostly diffuse. upon iav infection, > % of cells lacking a strong nuclear np signal displayed ifitm clustering (e.g., the right cell in figure a ), while this was only observed in < % of cells with a strong nuclear np ( figure b ). given the high infection rate in this cell line and the fact that cytoplasmic np was also observed in cells lacking the bright nuclear np of replicating iav ( figure a) , we suggest that early ifitm clustering at vesicular structures correlates with an abortive infection in this cell type, and that only cells that do not induce the vesicular clustering of ifitm become infected. in general, the ifitm signals were stronger in hsaepcs compared to a cells ( figure a,c) , and ifitm clusters apparently coating cytoplasmic vesicles were also present in productively iav-infected cells exhibiting a strong nuclear np signal in this case ( figure d ). no ifitm clustering at vesicular structures was observed in hsaepcs in the absence of iav infection, while the overall ifitm signal intensity was much higher in these primary cells compared to a cells without infection ( figure s ). a strong ifitm clustering with a ring-like appearance indicating vesicle coating was observed in both iav-infected a cells ( figure a ) and hsaepcs at h p.i. (figure c ; white arrowheads indicating ring-like structures). in the case of the a cells, the ifitm signals in uninfected cells were weak and mostly diffuse. upon iav infection, > % of cells lacking a strong nuclear np signal displayed ifitm clustering (e.g., the right cell in figure a ), while this was only observed in < % of cells with a strong nuclear np ( figure b ). given the high infection rate in this cell line and the fact that cytoplasmic np was also observed in cells lacking the bright nuclear np of replicating iav (figure a) , we suggest that early ifitm clustering at vesicular structures correlates with an abortive infection in this cell type, and that only cells that do not induce the vesicular clustering of ifitm become infected. in general, the ifitm signals were stronger in hsaepcs compared to a cells ( figure a,c) , and ifitm clusters apparently coating cytoplasmic vesicles were also present in productively iav-infected cells exhibiting a strong nuclear np signal in this case ( figure d ). no ifitm clustering at vesicular structures was observed in hsaepcs in the absence of iav infection, while the overall ifitm signal intensity was much higher in these primary cells compared to a cells without infection ( figure s ). viruses , , x for peer review of the ring-like appearance of ifitm clusters suggested the vesicular recruitment of this protein to iav-carrying endosomal structures. we therefore analysed whether ifitm clusters co-localize with early endosomal (eea ), late endosomal (rab ), or lysosomal (lamp ) marker proteins at early stages of iav infection in a cells ( figure , figure s ). we observed typical staining for early and late endosomes and for lysosomes with a weak ifitm signal in uninfected cells. the ifitm signal intensity increased at h and h p.i., as seen in the previous experiments, with some apparent colocalization with the endosomal marker proteins. to determine the degree of co-localization, we calculated the mander's correlation coefficient (mcc) [ ] . only ifitm clusters defined as objects with a size of > nm were included in this analysis. we observed a highly significant increase of ifitm cluster co-localization with early endosomes at h p.i., which was subsequently lost in the course of infection ( figure a ). in contrast, a significant increase of co-localization with the late endosomal marker ( figure b ) and some increased co-localization with the lysosomal marker ( figure c ) was seen at h, but not at h p.i. these results suggest that ifitm clustering on iav carrying endosomes already occurs early after endocytosis, prior to the ph-induced fusion and release from late endosomes, and probably persists as the endosomes mature. the ring-like appearance of ifitm clusters suggested the vesicular recruitment of this protein to iav-carrying endosomal structures. we therefore analysed whether ifitm clusters co-localize with early endosomal (eea ), late endosomal (rab ), or lysosomal (lamp ) marker proteins at early stages of iav infection in a cells ( figure , figure s ). we observed typical staining for early and late endosomes and for lysosomes with a weak ifitm signal in uninfected cells. the ifitm signal intensity increased at h and h p.i., as seen in the previous experiments, with some apparent co-localization with the endosomal marker proteins. to determine the degree of co-localization, we calculated the mander's correlation coefficient (mcc) [ ] . only ifitm clusters defined as objects with a size of > nm were included in this analysis. we observed a highly significant increase of ifitm cluster co-localization with early endosomes at h p.i., which was subsequently lost in the course of infection ( figure a ). in contrast, a significant increase of co-localization with the late endosomal marker ( figure b ) and some increased co-localization with the lysosomal marker ( figure c ) was seen at h, but not at h p.i. these results suggest that ifitm clustering on iav carrying endosomes already occurs early after endocytosis, prior to the ph-induced fusion and release from late endosomes, and probably persists as the endosomes mature. endocytosed material is generally sorted to different destinations: reusable ligands and receptors are returned to the cell surface via recycling endosomes, while the material destined for degradation traffic to lysosomes [ ] . previous studies reported a substantial co-localization between virus-containing compartments and rme- that is thought to be associated with sorting and recycling endosomes [ ] . furthermore, recent reports have suggested that rab -associated recycling endosomes are required for vrnp trafficking, assembly, and virion budding at the cellular surface in the late phase of iav infection [ ] [ ] [ ] [ ] . to determine whether iav and ifitm clustering can be detected on recycling endosomes, we compared uninfected and iav-infected a cells at different time points. some co-localization of np with ifitm and rab signals was observed on apparently vesicular structures in the extranuclear space of iav infected a cells at - h p.i. (figure a-c) . however, the co-localization of ifitm and rab was already present in the uninfected a cells, with no significant change of co-localization during the course of infection, as shown by the mcc analysis ( figure s ). the uptake of iav particles into recycling endosomes is expected to lead to an abortive infection, as recycling endosomes do not undergo acidification during trafficking. it is likely that rare events of an enhanced endocytosed material is generally sorted to different destinations: reusable ligands and receptors are returned to the cell surface via recycling endosomes, while the material destined for degradation traffic to lysosomes [ ] . previous studies reported a substantial co-localization between virus-containing compartments and rme- that is thought to be associated with sorting and recycling endosomes [ ] . furthermore, recent reports have suggested that rab -associated recycling endosomes are required for vrnp trafficking, assembly, and virion budding at the cellular surface in the late phase of iav infection [ ] [ ] [ ] [ ] . to determine whether iav and ifitm clustering can be detected on recycling endosomes, we compared uninfected and iav-infected a cells at different time points. some co-localization of np with ifitm and rab signals was observed on apparently vesicular structures in the extranuclear space of iav infected a cells at - h p.i. (figure a-c) . however, the co-localization of ifitm and rab was already present in the uninfected a cells, with no significant change of co-localization during the course of infection, as shown by the mcc analysis ( figure s ). the uptake of iav particles into recycling endosomes is expected to lead to an abortive infection, as recycling endosomes do not undergo acidification during trafficking. it is likely that rare events of an enhanced ifitm and rab the ifitm -mediated inhibition of iav infection has been suggested to be caused by changes in the membrane tension resulting in a block of fusion pore formation [ ] , and may be due to constitutively expressed protein or interferon induction [ , , , ] . applying confocal and superresolution imaging of endogenous ifitm , we show that pre-existing ifitm clusters at early and late endosomal structures carry iav at early time points of infection, prior to interferon induction. as shown previously [ ] and in our study ( figure s ), interferon alpha led to increased protein levels of ifitm at later time points. on the other hand, no increase in the ifitm protein levels was observed up to h p.i. in iav infected a cells. a semi-automated image analysis of iav a/hk/ / (pdmh n ) and iav a/r/d / (pdmh n ) infected a cells revealed ifitm clustering early after infection in both cases, with few differences in kinetics, which may reflect strain specific replication kinetics [ ] . the cd domain of ifitm , which is composed of the intermembrane domain and the intracellular loop, contains two phenylalanine residues mediating the physical association between ifitms; these residues are strongly connected with the antiviral function [ ] . combining this finding with our observation of the increasing levels of larger ifitm clusters, we suggest a direct relation between cluster formation and ifitm `s antiviral activity on endosomal vesicles. the ifitm clustering on apparently vesicular structures started early after infection ( - h p.i.), with an initial co-localization of ifitm with an early endosomal marker and a later co-localization with a late endosomal marker, reflecting the early to late endosomal pathway of iav. it appears likely, therefore, that ifitm the ifitm -mediated inhibition of iav infection has been suggested to be caused by changes in the membrane tension resulting in a block of fusion pore formation [ ] , and may be due to constitutively expressed protein or interferon induction [ , , , ] . applying confocal and super-resolution imaging of endogenous ifitm , we show that pre-existing ifitm clusters at early and late endosomal structures carry iav at early time points of infection, prior to interferon induction. as shown previously [ ] and in our study ( figure s ), interferon alpha led to increased protein levels of ifitm at later time points. on the other hand, no increase in the ifitm protein levels was observed up to h p.i. in iav infected a cells. a semi-automated image analysis of iav a/hk/ / (pdmh n ) and iav a/r/d / (pdmh n ) infected a cells revealed ifitm clustering early after infection in both cases, with few differences in kinetics, which may reflect strain specific replication kinetics [ ] . the cd domain of ifitm , which is composed of the intermembrane domain and the intracellular loop, contains two phenylalanine residues mediating the physical association between ifitms; these residues are strongly connected with the antiviral function [ ] . combining this finding with our observation of the increasing levels of larger ifitm clusters, we suggest a direct relation between cluster formation and ifitm 's antiviral activity on endosomal vesicles. the ifitm clustering on apparently vesicular structures started early after infection ( - h p.i.), with an initial co-localization of ifitm with an early endosomal marker and a later co-localization with a late endosomal marker, reflecting the early to late endosomal pathway of iav. it appears likely, therefore, that ifitm clusters, initially recruited to early endosomes and then coating endosomal vesicles through their trafficking pathway, may mediate the antiviral activity of ifitm and block the release of vrnps. this hypothesis is further supported by the identification of a critical sorting signal that is essential for the ifitm localization to endosomes and its anti-viral activity [ , ] . similar to iav, arena-(including lasv, lymphocytic choriomeningitis virus, and macv) and alphaviruses (including the chikungunya virus, sindbis virus, and venezuelan encephalitis virus) also fuse from late endosomes and lysosomes at a similar acidic ph, but are not restricted by ifitm [ ] . it will be of interest, therefore, whether a similar ifitm clustering can be observed on endosomal vesicles carrying these viruses or whether they can escape from ifitm clustering. importantly, a similar phenotype was observed in human primary respiratory cells from healthy donor tissue (hsaepcs). the basal levels of ifitm were much higher in these primary cells, compared to a cells in the absence of iav infection. however, ifitm clustering was not observed in the naïve primary cell population, but rapidly increased again upon iav infection and was even stronger than in a cells. ifitm clustering on cytoplasmic vesicles was strongly induced in both a cells and hsaepcs exhibiting weak extranuclear signals for iav np, which most likely reflects an incoming input virus. in contrast, no such ifitm clusters were seen in productively infected a cells with a strong nuclear np signal, and we speculate that infection of this cell type mainly occurs in cells that have not managed to block iav entry by inducing ifitm clustering. alternatively, ifitm clusters may be abrogated once productive infection has occurred and may thus no longer be visible in these cells. live-cell microscopy using labelled ifitm will be required to distinguish between these possibilities. a different phenotype was observed in primary hsaepcs, where ifitm clustering on vesicular structures was also observed in productively infected cells with a strong nuclear signal. despite having a much higher basal level, ifitm clustering in hsaepcs does not appear, therefore, to efficiently block iav infection in these primary cells. from our findings we assume two putative and presumably additive mechanisms for ifitm mediated antiviral action: (i) the first contact with iav particles after an endocytic uptake in (early) endosomes (role of recycling endosomes is discussed below) triggers the continuous recruitment of pre-existing ifitm proteins to iav containing compartments as a fast antiviral defense in the initial phase of infection. (ii) ifitm protein levels are increased by interferon signaling, further propagating its antiviral effect and possibly synergizing with additional ifn-induced antiviral factors [ ] . continuing studies aim to identify the modalities of the direct or indirect interaction between iav and ifitm . we have to admit that deficient particles might have the potential to fuse with the plasma membrane but fail to replicate. an abortive and ineffective infection might trigger the ifitm activation and recruitment to endosomes in the same way as can be seen for an efficient iav infection. various members of the endosomal network fulfil crucial functions in the viral entry, trafficking, and genome release. rab is mainly assigned to recycling endosomes [ ] . we made the observation that rab -positive vesicles loaded with ifitm often carried iav np early during infection. previous reports using live-cell imaging suggested that ifitm mediates the directed re-localization of viral particles to lysosomes [ ] . our results argue that the internalization of iav via recycling endosomes may trap iav in a non-productive pathway, as the membrane fusion is precluded in recycling endosomes having only a mildly acidic ph of . [ ] . it is conceivable that both mechanisms exist synergistically. in general, viruses being restricted by ifitm fuse at a lower ph in late endosomes or lysosomes [ ] . ifitm on recycling endosomes is thus unlikely to restrict the endosomal entry of iav (or of other viruses described to be restricted by ifitms), but may be an important defense against other pathogens entering via recycling endosomes [ ] . supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , figure s : ifitm knock-down validation, figure s : ifitm abundance in the absence and presence of hifnα and upon iav infection ( h p.i.) determined by western blotting, figure s : ifitm proteins cover early, late and recycling endosomes, figure s : ifitm abundance stays constant in recycling endosomes up to h p.i. in iav infected a cells, figure s : indirect immunofluorescence analysis of naïve human small airway epithelial cells (hsaepcs) using anti-ifitm and anti-np. the annual impact of seasonal influenza in the us: measuring disease burden and costs influenza a pandemics of the th century with special reference to : virology, pathology and epidemiology a history of influenza characterization of a novel influenza a virus hemagglutinin subtype (h ) obtained from black-headed gulls human infection with highly pathogenic h n influenza virus detection of novel swine origin influenza a virus (h n ) by real-time nucleic acid sequence-based amplification the influenza virus enigma differential infection of receptor-modified host cells by receptor-specific influenza viruses influenza a virus entry into cells lacking sialylated n-glycans infectious entry pathway of influenza virus in a canine kidney cell line entry of influenza a virus: host factors and antiviral targets dissection of the influenza a virus endocytic routes reveals macropinocytosis as an alternative entry pathway differential infectious entry of human influenza a/nws/ virus (h n ) in mammalian kidney cells influenza virus can enter and infect cells in the absence of clathrin-mediated endocytosis the biology of influenza viruses changes in the conformation of influenza virus hemagglutinin at the ph optimum of virus-mediated membrane fusion endocytosis and the recycling of plasma membrane assembly of endocytic machinery around individual influenza viruses during viral entry the first five seconds in the life of a clathrin-coated pit activation of influenza virus by acidic media causes hemolysis and fusion of erythrocytes fusion mutants of the influenza virus hemagglutinin glycoprotein anti-peptide antibodies detect steps in a protein conformational change: low-ph activation of the influenza virus hemagglutinin transport of incoming influenza virus nucleocapsids into the nucleus the viruses and their replication. fields virol structure of influenza virus rnp. i. influenza virus nucleoprotein melts secondary structure in panhandle rna and exposes the bases to the solvent does the higher order structure of the influenza virus ribonucleoprotein guide sequence rearrangements in influenza viral rna? cell interferon-inducible protein mx inhibits influenza virus by interfering with functional viral ribonucleoprotein complex assembly genome-wide rnai screen identifies human host factors crucial for influenza virus replication alteration of protein levels during influenza virus h n infection in host cells: a proteomic survey of host and virus reveals differential dynamics quantitative proteomic analyses of influenza virus-infected cultured human lung cells a quantitative proteomic analysis of lung epithelial (a ) cells infected with pandemic influenza a virus using stable isotope labelling with amino acids in cell culture the ifitm proteins mediate cellular resistance to influenza a h n virus, west nile virus, and dengue virus a physical and regulatory map of host-influenza interactions reveals pathways in h n infection inhibition of proliferation by - u in interferon-alpha-responsive and non-responsive cell lines association of rat with fyn protein kinase via lipid rafts is required for rat mammary cell differentiation in vitro distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus ifitm inhibits influenza a virus infection by preventing cytosolic entry the antiviral effector ifitm disrupts intracellular cholesterol homeostasis to block viral entry the cd domain of ifitm is required for both ifitm protein association and inhibition of influenza a virus and dengue virus replication ifitm- and ifitm- but not ifitm- restrict rift valley fever virus ifitm restricts influenza a virus entry by blocking the formation of fusion pores following virus-endosome hemifusion ifitm restricts the morbidity and mortality associated with influenza evaluation of ifitm rs association with severe pediatric influenza infection ifitm and susceptibility to respiratory viral infections in the community pilot screening study of targeted genetic polymorphisms for association with seasonal influenza hospital admission ifitm and severe influenza virus infection. no evidence of genetic association population genetics of ifitm in portugal and central africa reveals a potential modifier of influenza severity interferon-induced transmembrane protein inhibits hantaan virus infection, and its single nucleotide polymorphism rs influences the severity of hemorrhagic fever with renal syndrome snp-mediated disruption of ctcf binding at the ifitm promoter is associated with risk of severe influenza in humans palmitoylome profiling reveals s-palmitoylation-dependent antiviral activity of ifitm ifitm-family proteins: the cell's first line of antiviral defense ifitms restrict the replication of multiple pathogenic viruses ifitm proteins restrict viral membrane hemifusion interaction of polyene antibiotics with membrane lipids: physicochemical studies of the molecular basis of selectivity different mechanisms of cell entry by human-pathogenic old world and new world arenaviruses caveola-dependent endocytic entry of amphotropic murine leukemia virus late endosomal/lysosomal cholesterol accumulation is a host cell-protective mechanism inhibiting endosomal escape of influenza a virus ifitm requires an amphipathic helix for antiviral activity genome-scale crispr-cas knockout screening in human cells improved vectors and genome-wide libraries for crispr screening ellagic acid inhibits human pancreatic cancer growth in balb c nude mice generation of recombinant influenza virus from plasmid dna rapid accumulation of virulent rift valley fever virus in mice from an attenuated virus carrying a single nucleotide substitution in the mrna a practical guide to evaluating colocalization in biological microscopy the endocytic pathway: a mosaic of domains the ubiquitin-vacuolar protein sorting system is selectively required during entry of influenza virus into host cells rab regulates exocytosis of recycling vesicles at the plasma membrane a rab -and microtubule-dependent mechanism for cytoplasmic transport of influenza a virus viral rna apical transport of influenza a virus ribonucleoprotein requires rab -positive recycling endosome clustering of rab vesicles in influenza a virus infected cells creates hotspots containing the viral ribonucleoproteins interferon-induced transmembrane protein blocks fusion of sensitive but not resistant viruses by partitioning into virus-carrying endosomes the interferon-induced transmembrane proteins, ifitm , ifitm , and ifitm inhibit hepatitis c virus entry variation and infectivity neutralization in influenza identification of an endocytic signal essential for the antiviral action of ifitm phosphorylation of the antiviral protein interferon-inducible transmembrane protein (ifitm ) dually regulates its endocytosis and ubiquitination klf is involved in regulation of ifitm , , and genes during h n virus infection in a cells hang, h.c. ifitm directly engages and shuttles incoming virus particles to lysosomes emerging roles of recycling endosomes we thank kathleen börner (university hospital, heidelberg) for advice using crispr-cas gene editing, marino zerial (max planck institute of molecular cell biology and genetics, dresden) for the prab _egfp plasmid and jürgen stech (friedrich-loeffler-institute, insel riems) for the recovery plasmid system of influenza a/hong kong/ / , a/puerto rico/ / and a/regensburg/d / . we are grateful to vibor laketa (idip, heidelberg), bärbel glass and vera sonntag-buck (technical assistance) and carola sparn (student assistance) for excellent assistance. the authors declare no conflict of interest. key: cord- - l kyypm authors: chapman, colin a. title: a road for a promising future for china’s primates: the potential for restoration date: - - journal: zool res doi: . /j.issn. - . . sha: doc_id: cord_uid: l kyypm china is one of the most dynamic countries of the world and it shelters some amazing levels of biodiversity, including some very special primate species. however, primarily as a result of forest loss, most of which occurred in historical times, approximately % of china’s primate species have less than individuals. here i evaluate one road for future conservation/development that could produce very positive gains for china’s primates; namely forest restoration. i argue that for a large scale restoration project to be possible two conditions must be met; the right societal conditions must exist and the right knowledge must be in hand. this evaluation suggests that the restoration of native forest to support many of china’s primates holds great potential to advance conservation goals and to promote primate population recovery. the world is changing rapidly and china represents one of the most dynamic countries on earth and it shelters some amazing levels of biodiversity (i.e., > species of vascular plants (behind only brazil and colombia),~ species of terrestrial vertebrates (liu et al., ) ). globally, biodiversity is being lost at an accelerating rate, with current extinction rates approximately times higher than background rates (pimm et al., ) . recent estimates suggest that - species are lost each year and that surviving vertebrate species have declined in abundance by % since (dirzo et al., ) . humans are clearly responsible for this accelerating loss of biodiversity, including the endangerment of primates. between and , . million km of forest was lost globally and in the tropics forest loss increased each year (hansen et al., ) . to put this in perspective, this area is approximately the size of mexico. global estimates of the extent of wildlife over-exploitation are very poor. however, bennett et al. ( ) estimated that six million mammals were hunted annually in malaysian borneo. with respect to climate change, temperatures are predicted to increase by . • c by the end of the st century (ipcc, ) and using moderate greenhouse gas emission estimates, it is projected that by % of all tropical forests present in will experience temperatures that are higher than the temperatures presently supporting closed canopy forests (peres et al., ; wright et al., ) . china follows some of these patterns, but many aspects how china has changed are unique; they represent different challenges and most importantly different opportunities. for example, since , china's gross domestic product has increased by approximately % and china is now the world's largest economy (ahrends et al., ) . the economy has to support a population approaching . billion people (deng et al., ; wei & ye, ) . both the economic and population growth have come at an environmental cost. it is estimated that china has lost between . and . million km of its original forest in the last years (based on models of habitat suitability; ahrends et al., ) ; this is an area approximately the size of the democratic republic of congo. this has cost china in terms of primate diversity and population size. at least three species have been extirpated from china (pygathrix nemaeus, hylobates lar yunnanensis, nomascus leucogenys) and the hainan (nomascus hainanus) and cao-vit gibbons (nomascus nasutus) will almost certainly not see the turn of the century without very effective conservation action (fan, ; turvey et al., ) . gibbons were once found from xi'an in central china east to shanghai and all the way south to the border (fan, ; turvey et al., ; zhou & zhang, ) ; now they are isolated in a few forest fragments to the south. even orangutans were found in southern china only years ago (husson et al., ; steiper, ) , but they are no longer found on mainland asia. a recent analysis considering of the primate species in china (fan & ma, ) suggests that of the species have less than individuals and that % of the populations of all chinese primates are declining (estrada et al., ) . these sorts of statistics clearly indicate that china has lost a great deal of ground in the battle to conserve its biodiversity, but they also illustrate that china has great potential in terms of primate conservation. the objective of this opinion article for this special issue on primates and primatology in china is to evaluate one road for future conservation/development that could produce very positive gains for china's primates; namely forest restoration. there is clearly the need for restoration, as many of china's primates are only found in small isolated forests; thus expanding their habitat and connecting fragments is clearly vitally needed. for a large scale restoration project to be possible two conditions must be met. first, there must be the right societal conditions to make restoration possible and second the knowledge must be in hand to carry out such projects. in terms of the societal conditions, it appears that the timing is right for restoration projects. in november , government of china enacted the "law of the people's republic of china on the protection of wildlife" to facilitate the protection and management of wildlife. this law is the first truly comprehensive law to protect wildlife in china. however, since , the chinese government has enacted several national biodiversity conservation regulations, such as the natural forest protection project and ecological forest compensation, which have been effective in improving environmental conditions in many areas xu et al., ) . government financing for protected areas has also increased following the launch of the wildlife conservation and nature reserve construction project and the special fund for capacity building of national-level nature reserves. china is investing substantially in reforestation and tree planting efforts and this has totalled more than us$ billion in the past decade alone (ahrends et al., ; viña et al., ; zhang et al., ; zhang, ) . china now has the world's largest plantation area (approx. km (approximately the size of mozambique, ahrends et al., ) . at the same time, china is trying to reduce pressures on natural forests through strict bans on logging in primary forests and a massive expansion of its forest reserves to a current total more than reserves covering . million km (this area includes . % of the country, which is approximately the size of iran or twice the state of texas). lastly, over the last two decades there has been a large movement of people from rural areas (i.e., next to the reserves where china's primates are found) to the cities. in fact, the urbanization rate rose from . to . % between and and currently more than half of china's population live in cities (deng et al., ) . in terms of the right societal conditions to make restoration possible, an area still requiring a great deal of effort is that of hunting. in many areas of southern china, where forest cover is still substantial, primate populations can be dramatically reduced because of hunting (harrison et al., ) . even though some primates have class i protected animals in the chinese wildlife conservation law and hunting guns have been outlawed and confiscated, illegal hunting still frequently occurs as it has been a traditional practise that is promoted by poverty in local communities, the use of wildlife for medicinal uses, and poor knowledge and enforcement of the laws (fan et al., ) . for example, in south-west guangxi province, francois' langur (trachypithecus francoisi) populations declined by % between the early s and early s, at which time the total population size was estimated to be only approximately individuals found in isolated populations. the researchers conducting the later survey concluded that the primary threat to the langur was hunting, primarily for traditional medicine (li et al., ) . the second requirement is that the knowledge must be available to effectively carry out a large restoration projects. globally, there are only a handful of studies about the response of primate communities to forest regeneration; however, these studies suggest that forests, and the primate community they support, can rebound very rapidly when left to recover or encouraged to recover. for example, baya & storch ( ) surveyed a site in korup national park, cameroon that was abandoned - years previously and found populations of all eight species of diurnal primates that occur in the region; in addition, sighting frequency in this recovering area was not significantly different from other sectors of the park (linder, ) . in kibale national park, uganda, seven years after an area of grassland was replanted with trees as part of a carbon offset program (omeja et al., ) , all species of diurnal primates were present in high numbers, including endangered red colobus and chimpanzee. such studies give hope for the future. within china a great deal of research has accumulated over the last two or so decades that provides exactly the type of information needed for restoration/conservation efforts. to start such efforts accurate information on the state of the species to be targeted must be known. there is extensive survey information on the current size and threats to the country's primates (e.g., chen et al., ; cui et al., ) . the synthesis of this information will be vital in determining the locations to be prioritized in restoration efforts; however, other local aspects, such as the willingness of the local population to participate in restoration and to not hunt primates must also be considered. next, information must be available on the habitat requirements of the species targeted for help from a forest restoration project and again, there are many detailed projects focusing on primate habitat use (fan et al., (fan et al., , guo et al., ; li & rogers, ; liu et al., ) . of particular importance if the restoration project is to involve active replanting of trees is detailed information on the diet of the primates. with this information, either the food trees of the animals can be planted, or species with similar nutritional traits can be used in the restoration effort. there have been a number of high quality studies done on the nutritional ecology of china's primates (e.g., liu et al., ; ma et al., ; hou et al., ) . to make restorations efforts more effective, it is critical to understand the severity and geographical nature of threats to primates in china other than habitat loss (e.g., live capture for trade, bushmeat, and tourism; li et al., ; xia et al., ; yang et al., ; zhu et al., ) . in addition, information on conservation genetics is needed to determine the nature of corridors that can allow population mixing (liu et al., ) . with respect to conservation genetics, information concerning past interpopulational gene flow and landscape barriers on both short (e.g., satellite and aerial photography) and long time frames (e.g., river barriers), while rare (but see wang et al., ) , will be particularly valuable in determining the dispersal capabilities of primates relative to different types of barriers and can be used to provide guidance as to the nature of corridors that can be constructed to facilitate population mixing (guo et al., ; wang et al., wang et al., , . while this existing information is extremely valuable, what is lacking is data specific to restoration efforts and studies documenting behavioral patterns and responses of primates to the regeneration of forests. for restoration projects to be most effective, information on the survival strategies used in regenerating forests of all the major primate groups, including prosimians, macaques, colobines, and gibbons will be vital. while this is a formidable task, it also represents an exciting challenge to academics and universities; one where the value of research can be illustrated to the government and public. what now remains to be done is to pull this societal potential and information together to facilitate large-scale forest restoration efforts that is critically needed for primate conservation. since so many primate species in china are only hanging on as small remnant populations that are often only composed of a few groups (e.g., the cao vit gibbon population is estimated to only involve groups occupying forest patches of only hm with only - groups in china (fan et al., ) ) the only way to effectively promote conservation of these primates in the wild is through restoration. this will likely require some sort of well financed coordinating agency that is able to rally national and international scholars, conservation organizations, government agencies, and the public to first provided the needed scientific information and then to use this information to promote both natural regeneration and reforestation efforts on a very large scale. primates are very charismatic and are generally liked by both the chinese people and international groups, thus they can act as "flagship" or "guardian angel" species to promote the value of restoring these lands to a native forest (bicca-marques & de freitas, ; simberloff, ) . it is my opinion that this is an exciting time to integrate restoration into conservation strategies to make informed and effective conservation and management decisions for the primates of china. the authors declare that they have no competing interests. c.c. investigated and wrote the manuscript, but he benefited from talking to many friends and colleagues at the china primatological society in xi'an. china's fight to halt tree cover loss status of diurnal primate populations at the former settlement of a displaced village in cameroon saving borneo's bacon: the sustainability of hunting in sarawak and sabah hunting for sustainability in tropical forests the role of monkeys, mosquitoes, and humans in the occurrence of a yellow fever outbreak in a fragmented landscape in south brazil: protecting howler monkeys is a matter of public health preliminary study of the newly discovered primate species rhinopithecus strykeri at pianma, yunnan, china using infrared camera traps distribution and conservation status of shortridge's capped langurs trachypithecus shortridgei in china impact of urbanization on cultivated land changes in china defaunation in the anthropocene impending extinction crisis of the world's primates: why primates matter extant primates and development of primatology in china: publications, student training, and funding gibbons under seasonal stress: the diet of the black crested gibbon habitat and food choice of the critically endangered cao vit gibbon (nomascus nasutus) in china: implications for conservation behavioral responses of cao vit gibbon (nomascus nasutus) to variations in food abundance and temperature in ecological extinction of the critically endangered northern white-cheeked gibbon nomascus leucogenys in china the past, present, and future of gibbons in china response of a group of sichuan snub-nosed monkeys to commercial logging in the qinling mountains the mating system of the sichuan snub-nosed monkey (rhinopithecus roxellana) high-resolution global maps of st-century forest cover change impacts of hunting on tropical forests in southeast asia dietary and nutritional adaptions to extremes of northern-living by rhinopithecus roxellana orangutan distribution, density, abundance and impacts of disturbance climate change : synthesis report. contribution of working groups i, ii and iii to the fifth assessment report of the intergovernmental panel on climate change changes in distribution of the snub-nosed monkey in china dramatic decline of françois' langur trachypithecus francoisi in guangxi province current status and recent trends in financing china's nature reserves food items consumed by white-headed langurs in fusui the impact of hunting on primates in korup national park, cameroon: implications for primate conservation protecting china's biodiversity foods eaten by the sichuan snub-nosed monkey (rhinopithecus roxellana) in shennongjia national nature reserve, china, in relation to nutritional chemistry implications of genetics and current protected areas for conservation of endangered primates in china food selection in relation to nutritional chemistry of cao vit gibbons in jingxi biomass accumulation in tropical lands with different disturbance histories: contrasts within one landscape and across regions dispersal limitation induces long-term biomass collapse in overhunted amazonian forests the biodiversity of species and their rates of extinction, distribution, and protection effectiveness of china's national forest protection program and nature reserves flagships, umbrellas, and keystones: is single-species management passé in the landscape era? population history, biogeography, and taxonomy of orangutans (genus: pongo) based on a population genetic meta-analysis of multiple loci how many remnant gibbon populations are left on hainan? testing the use of local ecological knowledge to detect cryptic threatened primates effects of conservation policy on china's forest recovery low genetic diversity and strong population structure shaped by anthropogenic habitat fragmentation in a critically endangered primate, trachypithecus leucocephalus low genetic diversity and strong geographical structure of the critically endangered white-headed langur (trachypithecus leucocephalus) inferred from mitochondrial dna control region sequences urbanization, urban land expansion and environmental change in china. stochastic environmental research and risk assessment the future of tropical species on a warmer planet behavioural responses of yunnan snub-nosed monkeys (rhinopithecus bieti) to tourists in a provisioned monkey group in baimaxueshan nature reserve china's progress toward the significant reduction of the rate of biodiversity loss changes in attitudes toward wildlife and wildlife meats in hunan province, central china, before and after the severe acute respiratory syndrome outbreak special section: balancing conservation and development to preserve china's biodiversity china's forest policy for the st century distribution and vicissitude of gibbons (hylobatidae) in china during the last years potential pathogen transmission risk in non-human primate ecotourism: a case study at mt. huangshan, china i would like to thank rong hou and peng-fei fan for helpful comments on the manuscript. key: cord- - nj f authors: ambrose, rebecca k.; gravel, jennifer l.; commins, margaret a.; fowler, elizabeth v.; mahony, timothy j. title: in vivo characterisation of five strains of bovine viral diarrhoea virus (subgenotype c) date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: nj f bovine viral diarrhoea virus (bvdv- ) is strongly associated with several important diseases of cattle, such as bovine respiratory disease, diarrhoea and haemoragic lesions. to date many subgenotypes have been reported for bvdv- , currently ranging from subgenotype a to subgenotype u. while bvdv- has a world-wide distribution, the subgenotypes have a more restricted geographical distribution. as an example, bvdv- subgenotypes a and b are frequently detected in north america and europe, while the subgenotype c is rarely detected. in contrast, bvdv- subgenotype c is by far the most commonly reported in australia. despite this, uneven distribution of the biological importance of the subgenotypes remains unclear. the aim of this study was to characterise the in vivo properties of five strains of bvdv- subgenotype c in cattle infection studies. no overt respiratory signs were reported in any of the infected cattle regardless of strain. consistent with other subgenotypes, transient pyrexia and leukopenia were commonly identified, while thrombocytopenia was not. the quantity of virus detected in the nasal secretions of transiently infected animals suggested the likelihood of horizontal transmission was very low. further studies are required to fully understand the variability and importance of the bvdv- subgenotype c. bovine respiratory disease (brd) is the most important disease of intensively finished cattle. while multiple factors contribute to the likelihood of cattle developing brd, a generally accepted model for brd development is a primary viral infection predisposing cattle to more severe secondary bacterial infections. several viruses have been associated with an increased risk of brd, including bovine herpesvirus (bohv- ), bovine viral diarrhoea virus (bvdv- ), bovine respiratory syncytial virus and bovine parainfluenza virus. of the key brd associated viruses, bvdv- is the most genetically diverse with subgenotypes, bvdv- a to bvdv- u, having been reported [ ] . yesilbag et al. [ ] recently reviewed the geographical distribution of the bvdv- subgenotypes. this analysis highlighted several unusual trends in the distribution of the bvdv- subgenotypes. as an example, the vast majority of genotyped strains identified in australia have been classified within the subgenotype c [ , ] . this is in contrast to the usa where the subgenotype b is dominant, but the subgenotype a is also frequently reported [ ] . while in europe the most common subgenotypes are a and b, also common are subgenotypes d, e, f and h [ ] . however, the distribution of subgenotypes can vary between countries within europe, for example subgenotype b and d were recently reported to be the most frequently identified subgenotypes in germany [ ] . strains of the subgenotype c have been rarely reported in the usa or europe. the drivers of these distributions are unclear, but do not appear to be influenced by vaccine use [ ] . several studies have suggested that the subgenotypes, at least in part, also reflect the antigen diversity between strains using cross-neutralisation assays [ , , ] . clearly the degree of cross-protection afforded by the subgenotype(s) included in a bvdv- in a vaccine against the subgenotypes circulating in a cattle population is an important issue. the overall biological importance of the bvdv- subgenotypes remains to be fully elucidated since the majority of published studies have focused on the bvdv- a and bvdv- b genotypes. it is reasonable to accept that the subgenotypes of bvdv- share similar properties with respect to their capacity to infect susceptible ruminants, and their contribution to the development of more severe clinical disease outcomes such as brd and the birth of persistently infected calves following transplacental infection. all of these outcomes are reported in cattle populations regardless of what subgenotype is present. the evaluation of any variation of the in vivo properties of bvdv- subgenotypes has received little attention. to assist in developing a better understanding of the importance of the bvdv- subgenotypes, the aim of this study was to assess the in vivo properties of five bvdv- strains from the poorly studied subgenotype c. no overt clinical scores were recorded for any of the trial animals, challenged or unchallenged with the bvdv- c strains used in this study (data not shown). for all groups, except the cattle infected with bvdv- c strain ns , there was a general trend for an increase in the average rectal temperature on day of the experiment, prior to any viral exposure, compared to the temperatures from day to day (figure a -f). the reason(s) for this increase are unclear. the cattle had been inducted into the containment facility seven days prior to the commencement of the trial and were therefore considered to have been well adapted/acclimatised to the environment. however, on day there were extra staff and equipment present while the cattle were being inoculated which included extra handling of the animals/cattle potentially contributing to this apparent increase. due to this anomaly, the day temperatures were used as the reference point in the subsequent statistical comparisons within each treatment group. the daily rectal temperatures were monitored for the trial cattle for days following experimental infection. overall the mean rectal temperature results were variable ( figure ). animals (n = ) infected with bvdv- c strain pi exhibited a significant temperature elevation on day post infection (p < . , figure a ). for animals infected with bvdv- c strain trangie (n = ), the temperature increase was gradual from day to day post infection, although the increase was only statistically significant on day (p < . , figure b ). animals infected with bvdv- c strain ao (n = ) exhibited elevated mean temperatures on day and day post infection, with only the day mean temperature being statistically significant (p < . , figure d) . a similar temperature profile was observed for bvdv- c strain ns (n = ), with elevated temperatures on day and day , with only day being statistically significant (p < . , figure d ). although there was a suggestion of an elevated mean temperature on day post-infection for the cattle (n = ) infected with vr this was not statistically significant ( figure e ). the rectal temperatures of the contact animals (n = ) did not exceed . • c during the experiment (figure f ). no statistical comparisons were undertaken for this group due the low animal numbers. animals infected with bvdv- c strain trangie (n = ); (c) animals infected with bvdv- c strain ao (n = ); (d) animals infected with bvdv- c strain ns (n = ); (e) animals infected with bvdv- c strain vr (n = ); (f) uninfected animals (n = ). the asterisks above selected days indicate significant differences compared to day post-infection for that group of cattle. level of significance; * p < . ; ** p < . ; **** p < . . the course of the bvdv- c nasal shedding by the trial cattle was assessed by testing extracts from nasal swabs collected from day to day and serum samples collected on day and day post-infection with a bvdv- specific quantitative real-time (qpcr). a summary of these results expressed as the threshold cycle (ct) is shown in table . overall, the distribution of positive nasal swabs was variable between and within the groups of cattle infected with the different strains of bvdv- c. the earliest that virus was detected in nasal swabs was day post-infection for animals infected with pi ( of animals), trangie ( of animals) and ao ( of animals). one nasal swab from an animal infected with strain pi tested positive at day post infection. the bvdv- c strain ao was the virus most consistently detected in the nasal swabs with all infected animals reacting with the qpcr at day and day post infection, and three of the four animals also reacted in the qpcr on day ( table ). the serum samples collected from animals infected with ao at day post-infection also reacted and were deemed to be positive for bvdv- c (table ). (b) animals infected with bvdv- c strain trangie (n = ); (c) animals infected with bvdv- c strain ao (n = ); (d) animals infected with bvdv- c strain ns (n = ); (e) animals infected with bvdv- c strain vr (n = ); (f) uninfected animals (n = ). the asterisks above selected days indicate significant differences compared to day post-infection for that group of cattle. level of significance; * p < . ; **** p < . . the course of the bvdv- c nasal shedding by the trial cattle was assessed by testing extracts from nasal swabs collected from day to day and serum samples collected on day and day post-infection with a bvdv- specific quantitative real-time (qpcr). a summary of these results expressed as the threshold cycle (ct) is shown in table . overall, the distribution of positive nasal swabs was variable between and within the groups of cattle infected with the different strains of bvdv- c. the earliest that virus was detected in nasal swabs was day post-infection for animals infected with pi ( of animals), trangie ( of animals) and ao ( of animals). one nasal swab from an animal infected with strain pi tested positive at day post infection. the bvdv- c strain ao was the virus most consistently detected in the nasal swabs with all infected animals reacting with the qpcr at day and day post infection, and three of the four animals also reacted in the qpcr on day ( table ). the serum samples collected from animals infected with ao at day post-infection also reacted and were deemed to be positive for bvdv- c (table ) . table . detection of bovine viral diarrhoea virus subgenotype c in extracts from cattle samples using quantitative real time pcr (qpcr). nasal swabs (n) and serum (s) samples were analysed using qpcr. reactive samples were deemed positive for bvdv- c with the cycle threshold value shown (shaded cells). samples which did not react (> ) with the qpcr were deemed to be negative (−) for the virus. with respect to the other groups, bvdv- c was consistently detected in nasal swabs collected on day , day and day post-infection from two of the six animals infected with bvdv- c strain pi ( table ) . one of the animals in this group tested positive (nasal swabs) from day to day post-infection and the swabs from day and day were also positive. for cattle infected with bvdv- c strain trangie, one of the four animals tested positive on day post-infection, while all the other samples were negative throughout the sampling period (table ) . of the animals infected with bvdv- c strain ns , two animals had positive nasal swabs. one of these animals was positive on day . the strain ns was detected sporadically between day and in samples from animal . the serum samples from day and day from this animal were both reactive with the qpcr assay (table ) . two animals infected with bvdv- c strain vr tested positive for the virus in a sporadic manner. the remaining animals did not return any positive results ( table ) . none of the sample extracts from the two contact animals reacted with the qpcr during the sampling period (table ) . bvdv- c was not detected via qpcr in the nasal swab or serum samples collected from all animals on day , day , day and day post-infection and were deemed to be negative (data not shown). attempts were made to isolate the bvdv- c strains from the nasal swabs collected at day post-infection from selected animals. no bvdv- c was detected in any of the culture supernatants by qpcr after three passages of animal (pi infected and tested positive from day to day ), animal (trangie infected), animal (ao infected) or animal (ao infected). with respect to the other groups, bvdv- c was consistently detected in nasal swabs collected on day , day and day post-infection from two of the six animals infected with bvdv- c strain pi ( table ) . one of the animals in this group tested positive (nasal swabs) from day to day post-infection and the swabs from day and day were also positive. for cattle infected with bvdv- c strain trangie, one of the four animals tested positive on day post-infection, while all the other samples were negative throughout the sampling period (table ) . of the animals infected with bvdv- c strain ns , two animals had positive nasal swabs. one of these animals was positive on day . the strain ns was detected sporadically between day and in samples from animal . the serum samples from day and day from this animal were both reactive with the qpcr assay (table ) . two animals infected with bvdv- c strain vr tested positive for the virus in a sporadic manner. the remaining animals did not return any positive results ( table ) . none of the sample extracts from the two contact animals reacted with the qpcr during the sampling period (table ) . bvdv- c was not detected via qpcr in the nasal swab or serum samples collected from all animals on day , day , day and day post-infection and were deemed to be negative (data not shown). attempts were made to isolate the bvdv- c strains from the nasal swabs collected at day postinfection from selected animals. no bvdv- c was detected in any of the culture supernatants by qpcr after three passages of animal (pi infected and tested positive from day to day ), animal (trangie infected), animal (ao infected) or animal (ao infected). monocytes: seven days post-infection, animals infected with bvdv- c strain pi had significantly reduced concentration of monocytes compared to the pre-infection sample (p < . , figure a ). no significant changes in the numbers of monocytes were detected for any of the animals infected with the remaining bvdv- c strains or the contact animals ( figure ). (e) (f) (e) (f) (e) (f) platelets: overall, the concentration of platelets in infected animals were reduced for most bvdv- c strains on day and/or day post infection compared to day . the only significant reduction was for the animals infected with bvdv- c strain ns on day (p < . , figure d) . a reduction in platelet numbers was also apparent on day for animals infected with strain pi , trangie and vr , but these differences were not statistically significant (figure a,b,e) . in contrast to other infected groups, the concentration of platelets in cattle infected with bvdv- c strain ao appeared stable for the duration of the experiment, apart from a significant increase on day postinfection (p < . , figure c ). platelets: overall, the concentration of platelets in infected animals were reduced for most bvdv- c strains on day and/or day post infection compared to day . the only significant reduction was for the animals infected with bvdv- c strain ns on day (p < . , figure d) . a reduction in platelet numbers was also apparent on day for animals infected with strain pi , trangie and vr , but these differences were not statistically significant (figure a,b,e) . in contrast to other infected groups, the concentration of platelets in cattle infected with bvdv- c strain ao appeared stable for the duration of the experiment, apart from a significant increase on day post-infection (p < . , figure c ). platelets: overall, the concentration of platelets in infected animals were reduced for most bvdv- c strains on day and/or day post infection compared to day . the only significant reduction was for the animals infected with bvdv- c strain ns on day (p < . , figure d) . a reduction in platelet numbers was also apparent on day for animals infected with strain pi , trangie and vr , but these differences were not statistically significant (figure a,b,e) . in contrast to other infected groups, the concentration of platelets in cattle infected with bvdv- c strain ao appeared stable for the duration of the experiment, apart from a significant increase on day postinfection (p < . , figure c ). (a) (b) (c) (d) all trial animals were monitored for the development of bvdv- specific antibodies throughout the course of the experiment. virus specific antibody was first detected on day post-infection with of the infected animals testing positive. however, none of the animals infected with strains trangie or ao had detectable antibodies by this time point. by day post-infection, of the animals had detectable bvdv- specific antibody ( table ). all the bvdv- c challenged animals had detectable bvdv- antibodies by day post-infection ( table ). one of the animals infected with strain ao was positive on day but subsequently tested negative on day (table ) . no bvdv- antibody was detected in the serum samples from either of the two contact animals at any of the sampling time points (table ) . table . serological responses (igg) of cattle challenged with one of five strains of bovine viral diarrhoea virus genotype c. the level of virus specific igg were determined using a commercial elisa and assigned to one of six arbitrary categories, negative (−) or positive (+, ++, +++, ++++, or +++++). all trial animals were monitored for the development of bvdv- specific antibodies throughout the course of the experiment. virus specific antibody was first detected on day post-infection with of the infected animals testing positive. however, none of the animals infected with strains trangie or ao had detectable antibodies by this time point. by day post-infection, of the animals had detectable bvdv- specific antibody ( table ). all the bvdv- c challenged animals had detectable bvdv- antibodies by day post-infection ( table ). one of the animals infected with strain ao was positive on day but subsequently tested negative on day (table ) . no bvdv- antibody was detected in the serum samples from either of the two contact animals at any of the sampling time points (table ) . table . serological responses (igg) of cattle challenged with one of five strains of bovine viral diarrhoea virus genotype c. the level of virus specific igg were determined using a commercial elisa and assigned to one of six arbitrary categories, negative (−) or positive (+, ++, +++, ++++, or +++++). animal id infection pi − − − + ++ +++ ++ − − + +++ ++++ +++++ ++++ − − + +++ ++++ ++++ +++ − − − ++ +++ ++++ ++++ − − − ++ ++ ++++ ++++ − − − +++ +++ ++++ ++++ trangie − − − +++ ++++ ++++ +++ − − − ++ +++ +++ +++ − − − +++ +++ ++++ +++++ − − ++ +++ +++++ +++++ +++++ ao − − ++ +++ ++++ +++++ ++++ − − +++ − +++++ +++++ +++++ − − +++ ++++ +++++ +++++ +++++ − − − ++ ++++ +++++ ++++− − + + +++++ +++++ ++++ − − − − ++++ ++++ ++++ − − + ++++ +++++ +++++ +++++ vr − − − − ++ ++ ++ − − +++ +++ +++++ +++++ +++++ − − − + + ++ ++ contact − − − − − − − − − − − − − − several studies have explored the potential links between the subgenotypes and antigenic variation through cross neutralisation studies [ , , ] . clearly, knowledge of any such relationship is important as it would facilitate the selection of vaccine components to match the circulating bvdv- subgenotypes, while also enabling the ongoing monitoring of field strains to detect any change in the dominant subgenotype. the importance of the bvdv- subgenotypes with respect to the in vivo biology has received minimal attention and more research is required to define commonalities and divergences between each group such as virulence. several of the parameters measured in this study showed similar effects of the bvdv- c strains on their bovine hosts. these commonalities were not unexpected as the strains used in this study were all bvdv- subgenotype c. currently, there are no specific criteria proposed to evaluate bvdv- virulence, however clinical signs (respiratory and/or digestive), biphasic pyrexia, biphasic leukopenia and thrombocytopenia have been reported for bvdv- subgenotypes a, b, d, e and k from various countries [ ] [ ] [ ] [ ] [ ] . in the current study, no respiratory or digestive clinical signs were observed in the bvdv- c inoculated cattle. while a significant pyrexia was identified in cattle infected with four of the five bvdv- strains, however, there was no evidence of a biphasic pyrexia (figure ). four of the five bvdv- infected groups exhibited significant leukopenia at day post-infection, three of which were biphasic (figure ). only the group infected with vr did not have detectable leukopenia. as vr was the only cytopathic bvdv- strain included in the current study, further research is required to determine why this was the case. with respect to thrombocytopenia, only the cattle infected with strain ns had a significant loss of platelets that was detected days after infection (figure d ). collectively these data suggest the bvdv- subgenotypes c evaluated in this study have low virulence in transiently infected animals under the experimental conditions utilised. one additional parameter which is commonly considered in the assessment of viral virulence is transmission capacity [ ] . there is general agreement that there is either no or limited horizontal transmission of bvdv- between transiently infected cattle [ ] [ ] [ ] . sarrazin et al. [ ] concluded that the field strains of bvdv- subgenotype a and b evaluated in their study were unlikely to play an important role in transmission. the detection of bvdv- c in the nasal swabs of infected cattle the current study was sporadic and where detected the results suggested low quantities of virus ( table ). the range of ct values from nasal swabs and serum samples in the current study were . to . and . to . respectively (table ) . previous studies have evaluated the use of qpcr to differentiate persistently and transiently infected animals. hanon et al. [ ] estimated that a ct value below . from a blood sample would identify all persistently infected animals, although this value was likely to misclassify some transiently infected animals as being persistently infected. while hay et al. [ ] estimated that a ct value below from serum was indicative of an animal being persistently infected with bvdv- . the results of the current study, suggest a ct value of is too high for the differentiation of transiently and persistently infected animals based on a single sample. noting that both prior studies utilised field samples for these estimates, thus direct comparison to the current study requires caution. the use of these estimates, would also require sample preparation and analyses to be comparable, particularly volume of sample extracted and subsequently used in the qpcr assay. associated with these results, the two uninfected control animals included in the study did not test positive for bvdv- or seroconvert to bvdv- over the course of the experiment. unchallenged animals could only be included in one of the containment rooms of the study for logistical reasons. consequently, there were no uninfected animals penned with the animals infected with bvdv- c strain ao or strain pi , the viruses most consistently detected in nasal swabs and in the highest quantities (table ) . collectively, these results suggest that minimal amounts of the challenge viruses were present in the nasal secretions of the infected animals and as a result the risk of virus transmission to other animals was very low for these bvdv- c strains. evans et al. [ ] recently reported the absence of horizontal transmission from sheep experimentally infected with an australian strain of bvdv- subgenotype c to sentinel sheep. the possibility of animals transiently infected with the bvdv- c strains used in these studies producing and shedding sufficient quantities of virus to facilitate transmission if subjected to stressful conditions cannot be excluded. it has recently been demonstrated that the bvdv- strain h (subgenotype a) was only transmitted to sentinel animals when the infected animals were immunosuppressed with dexamethasone [ ] . if the low risk if transmission from transiently infected animals extends to all bvdv- subgenotype c it could have important implications in the implementation of effective bvdv- control plans with persistently infected animals as the sole source of virus [ , [ ] [ ] [ ] . further research is required to determine if any bvdv- subgenotype c strains replicate sufficiently in the nasal epithelia at levels to facilitate transmission to susceptible sentinel animals. the bvdv- c strains pi and ao would be excellent candidate viruses for these studies. while the detection of virus in nasal swab and serum samples was sporadic, the cattle were clearly infected as demonstrated by the serological analyses. infected animals started to seroconvert by day post-infection with of the infected animals testing positive for bvdv- specific antibody. the number of positive animals increased by day , with all infected animals being antibody positive by day . these data are consistent with other bvdv- infection studies [ , ] . there did not appear to be anything specific to the bvdv- c strains used in this study in relation to the serological data with animals from all groups becoming seropositive in a fourteen day period and all animals being seropositive by day . the impacts of some bvdv- c strains on cattle in the current study may have been under-estimated due to the smaller number of cattle in each group, particularly for strain vr where one animal was withdrawn from the experiment immediately prior to commencement of the trial for ethical reasons (lameness). the number of cattle in this study was constrained by the capacity of the facility and need to evaluate the properties of several bvdv- subgenotype c strains. another potential limitation of the current study was the viral inoculums used were quantified using rt-qpcr of the final cell culture supernatants. while previous studies have demonstrated correlations between rt-qpcr results and measures of in vitro infectivity such as plaque forming units and/or % cell culture infectious dose (tcid ) for other viruses, such relationships have not been reported for bvdv- as yet [ ] [ ] [ ] [ ] . future studies which aim to directly compare the in vivo properties of the bvdv- c strains used in the study would need to establish the relationship between rt-qpcr results and measures of in vitro infectivity to enable the standardisation of the challenge doses. future studies will be required to better understand the relationships between the bvdv- subgenotypes and virulence. strong et al. [ ] also identified that the challenge dose can influence the clinical outcomes of cattle challenged with bvdv- a. data was also reported which suggested an influence of calf age on clinical outcome. as a consequence, future studies aiming to characterise the interactions of bvdv- and its bovine host should aim to do so under a standardised challenge system, including route of infection, challenge dose (where possible multiple doses) and age of animals. it is imperative that the subgenotype of the bvdv- isolate(s) used also be included. while the current study has focused on the respiratory component of the bvdv- c infection, it is well accepted the virus can have profound impacts on the reproductive capacity of individual animals and cattle herds overall. the bvdv- c isolate trangie was used in several earlier studies that reported the capacity of this virus to significantly impair bovine reproductive function [ ] [ ] [ ] [ ] . the capacity of specific bvdv- strains and/or subgenotype groups to cause both respiratory and reproductive disease are yet to be investigated, and may be required to fully understand this important cattle pathogen. this study is the first to characterise the in vivo properties of bvdv- strains confirmed as belonging to the subgenotype c. interestingly, the overall impacts of the infection of the different strains on the infected cattle were in general similar to those reported for other bvdv- subgenotypes with transient pyrexia, leukopenia, and quantities of virus in nasal swabs which are unlikely to facilitate horizontal transmission [ ] [ ] [ ] [ ] [ ] ] . of the bvdv- c strains examined in this study pi had the most consistent impact on the experimentally infected cattle and is a strong candidate for use in cattle studies to further define the in vivo properties of the subgenotype c, including direct comparisons to strains of other subgenotypes. all experimental procedures involving animals were reviewed and approved by the university of queensland animal ethics committee, approval number qaafi/ / /mla. the bvdv- c isolates used in the cattle trial are described in table . viral inoculums were prepared by adding µl of primary stock of each bvdv- c strain to culture medium of subconfluent monolayers of mdbk cells in tissue culture flasks ( cm ) and incubated at • c in a % co atmosphere for seven days. the supernatants were clarified at g, aliquoted and stored at − • c until required. as the aims of this study did not include intergroup statistical comparisons, the viral supernatants were used as harvested to inoculate cattle at the maximum possible titre. cattle were sourced by veterinary health research ltd. pty (armidale, nsw, australia). the animals were black angus and to months of age. prior to enrolment into the study, cattle were tested multiple times and confirmed negative for serological evidence of prior bvdv- exposure/infection. elisas were performed using the bio k elisa, as described by the manufacturer (bio-x diagnostics, jemelle, belgium). seven days prior to commencement of the trial, cattle (n = ) were moved into the large animal pc facility at the queensland animal science precinct (gatton, qld, australia). the cattle were randomly assigned to one of four pens ( × ) with two pens per room ( table ). the rooms are operated independently, including separate air handling systems. to minimise the risk of virus transmission between rooms, separate teams of staff were used to maintain/care for and collect samples from the animals in each room daily. on day , the rectal temperature for each animal was recorded and two blood samples collected via the jugular vein. the cattle groups were inoculated with one of the bvdv- viral strains as shown in table . briefly, the animal was restrained with the nose elevated and the viral inoculum ( ml) was slowly dripped into each nostril. the nose was held in this position for to s and the animal then released. two animals in room were not inoculated with virus. clinical assessments: cattle were monitored from day to day post-infection for clinical signs in respect to nasal discharge, coughing, behavior/demeanour and loss of appetite (feed residue). temperature: the rectal temperatures for each animal was recorded from day to day . the expected rectal temperature of healthy cattle was . • c [ ] . nasal swabs were collected from day to day , day , day , day , day and day . nasal swabs were immediately placed on ice for transport back to the laboratory for storage at • c until required. nasal swabs were immersed in µl of pbs containing × antibiotic-antimycotic (thermofisher scientific, waltham, ma, usa) and gently agitated. the swab was subsequently removed and discarded. a µl aliquot of this resuspension was used for total nucleic acid extraction using the dneasy blood & tissue kit (qiagen, hilden, germany) as described by the manufacturer, except for the exclusion of rnase a. total nucleic acid extracts were also prepared from aliquots ( µl) of cattle serum samples, collected as described below, using the same methodology. sample extracts prepared from nasal swabs and sera were analysed by qpcr for the presence of bvdv- rna as previously described [ ] . samples yielding a ct value ≥ were deemed to be negative for bvdv- . an aliquot of the resuspended nasal swab from day post-infection from selected animals were utilised for virus isolation. aliquots, µl and µl of the nasal swab resuspension diluted : with pbs were added directly to culture medium of subconfluent monolayers of mdbk cells in well plates and incubated at • c in a % co atmosphere for seven days. the monolayers were freeze/thawed once and a µl aliquot of the culture supernatant added to new subconfluent monolayers of mdbk cells in well plates and incubated at • c in a % co for seven days. this process was repeated three times. total nucleic acids were extracted from a µl aliquot of each culture supernatant and tested using qpcr for the presence of bvdv- as described previously. blood sampling: blood for serum harvesting ( ml bd vacutainers™, bd biosciences, franklin lakes, nj, usa) and blood cell count analyses ( ml edta bd vacutainers™, bd scientific, franklin lakes, nj, usa) were collected on day , day , day , day , day , day , day and day post infection. serum samples were tested for the presence of bvdv- specific antibodies using the bio k elisa, as described by the manufacturer (bio-x diagnostics, jemelle, belgium). the level of virus specific igg in each serum sample was assigned to one of six arbitrary categories, negative (−) or positive (+, ++, +++, ++++, or +++++) according to the manufacturer's instructions. whole blood samples were submitted to the veterinary science diagnostic services (school of veterinary science, university of queensland, gatton, qld, australia) for analyses of the cell populations using standard blood smearing and cell counting methodologies. data generated from the animal trial were analysed 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bovine viral diarrhea virus infection around the time of insemination on the reproductive performance of cattle a single amino acid is critical for the expression of b-cell epitopes on the helicase domain of the pestivirus ns protein a manual for the primary animal health care worker; food and agriculture organization (fao multiplex real-time rt-pcr detection of three viruses associated with the bovine respiratory disease complex the authors declare no conflict of interest. the funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results". key: cord- -mhlqnj authors: wang, qi; hagedorn, curt; liu, shuanghu title: adapted hcv jfh variant is capable of accommodating a large foreign gene insert and allows lower level hcv replication and viral production date: - - journal: int j biol sci doi: . /ijbs. sha: doc_id: cord_uid: mhlqnj infectious hcv carrying reporter genes have further applications in understanding the hcv life cycle including replication, viral assembly and release. in this study, a full-length bp lacz gene was inserted into the derivative of jfh -am to develop an additional reporter virus. the results showed that the recombinant reporter virus jfh -am -lacz can replicate and produce lower titers of infectious virus. however, insertion of the lacz gene in the c-terminal region of the ns a in hcv jfh -am -lacz decreased viral replication and dramatically impaired the production of infectious viral particles. moreover, the jfh -am -lacz reporter virus lost the lacz gene after serial passage. nevertheless, the jfh -am -lacz reporter virus displayed the entire life cycle of hcv, from replication to production of infectious virus, in huh . cells. this study demonstrates that the ns a region of hcv jfh -am has the capacity to accommodate large foreign genes up to , bp and suggests that other relatively large gene inserts can be accommodated at this site. hepatitis c virus (hcv) is a member of the flaviviridae family and is a single-stranded positive-sense rna. it infects approximately % of the worldwide population ( , ) . hcv can cause acute and chronic hepatitis, and lead to fibrosis, cirrhosis and hepatocellular carcinoma ( ) . although much progress has been made in developing highly effective hcv antivirals, an effective vaccine has yet to be developed ( , ) . an internal ribosome entry site (ires) of hcv drives rna translation to produce a polyprotein of approximately , amino acids (aa) that encodes both structural and nonstructural proteins ( ) ( ) ( ) . the development of hcv replicon systems advanced the understanding of hcv replication, viral protein processing and viral-host cell interactions ( , ). an establishment of an infectious hcv cell culture system using a genotype a isolate (jfh strain) of hcv and huh- cells was a major achievement ( ) ( ) ( ) . in this system, infectious hcv particles are secreted in an envelope glycoproteindependent manner and enable a variety of questions to be answered regarding hcv biology and cell infection. however, virus titers of jfh released from infected cells are relatively low and limit some applications of this system. studies have identified adaptive or compensatory mutations that enhance infectious virus particle production from either wild-type jfh or intergenotypic chimeras ( ) ( ) ( ) ( ) ( ) ( ) ( ) . non-structural protein a (ns a) of hcv is a phosphoprotein, which has nt and a calculated molecular mass of kda. it migrates as -and -kda species in sds-polyacrylamide gels and is ivyspring international publisher organized into three domains: i (aa - ), ii (aa - ), and iii (aa - ). domain i coordinate a single zinc atom, and domains ii and iii are less well characterized but are important for rna replication and/or virion assembly ( ) ( ) ( ) ( ) . previous studies have demonstrated that the hcv jfh ns a c-terminal is a flexible region which is capable of accommodating foreign gene inserts (such as egfp, bp and rennilla luciferase [rluc] , bp) and still permit hcv replication and viral production ( , , ( ) ( ) ( ) ( ) ( ) ( ) . vesicular stomatitis virus (vsv) is negative-stranded rna virus that has been used as a gene expression vector. it can accommodate a gene insert as large as bases ( ) . one of the more common reporter genes, the e. coli lacz gene, is bp in length and encodes the protein β-galactosidase (β-gal). the expression of lacz can be identified by staining with x-gal substrate to produce a blue color as a measure of enzyme activity ( ) ( ) ( ) . human hepatoma cells (huh ) can express β-galactosidase using adenoviral vectors carrying the lacz gene ( ) . it is unclear whether the hcv jfh ns a c-terminal is capable of accommodating a large foreign gene such as lacz, and still permit hcv replication and production of infectious virus. in a previous study we demonstrated that there was no viral production after insertion of egfp ( bp) and rennilla luciferase (rluc; bp) into the c-terminus of ns a of wild type jfh ( ) . however, a higher titer of hcv reporter virus was produced after inserting egfp and rluc into ns a c-terminus of an adaptively mutated jfh strain designated jfh -am ( ) . in this study, we used the jfh -am as a vector to explore if infectious reporter virus would be produced following insertion of lacz gene that was three time larger than rluc, into the ns a c-terminus. the result showed that the ns a region of hcv jfh -am has the capacity to accommodate large foreign genes up to , bp and suggests that other relatively large gene inserts can be accommodated at this site. human hepatoma cells, huh . , were generously provided by charles rice ( ) (rockefeller university) and maintained in dulbecco's modified eagle's medium (dmem) (invitrogen) supplemented with u/ml of penicillin, µg/ml of streptomycin, nonessential amino acids, and % fetal bovine serum (fbs) (invitrogen) at ˚c in % co . all experiments described in this study were performed using these cells. the monoclonal antibody to the ns a protein ( e ) was a gift from charles rice ( ) . goat anti-mouse conjugated with horseradish peroxidase (hrp) (sigma), goat anti-mouse igg conjugated with alexa fluor (invitrogen), x-gal (genlantis, cat#a k), mammalian β-gal activity assay kit (thermofisher, # ), renilla luciferase assay system (promega) were obtained commercially. plasmids constructs were based on jfh-am that has adaptive mutations previous reported ( ) and jfh-am based on the consensus sequence of hcv pjfh , which was kindly provided by dr. wakita ( ) . jfh-am -egfp and jfh-am -rluc had been described previously ( ) . the egfp and renilla luciferace (rluc) genes were inserted in frame into a unique rsrii site located in the amino acid codon of jfh -am . jfh-am -lacz was also constructed by inserting the lacz gene ( bp) into the rsrii site of the ns a c-terminal region of jfh-am . the lacz fragment was amplified from vector psv-β-galactosidase control (promega, e ) using the primers lacz-foward ( '-tctcggtc cgatcgatcccgtcgttttaca- '), lacz-reverse ( '-cttacggaccgcctttttgacaccagaccaa ct- '). the new construct was sequenced and the correct full-length clone, jfh-am -lacz, was used for the subsequent experiments. to generate full-length genomic rna, pjfh -am -lacz and controls of pjfh -am , pjfh -am -egfp, pjfh -am -rluc and were linearized at the ' end of the hcv cdna with xbai. the linearized plasmid dna was purified and used as a template for t in vitro transcription (megascript; ambion, austin, tx). in vitro transcribed rnas were transfected into cells by electroporation as described previously ( , ) . briefly, trypsinized cells were washed twice and resuspended with serum-free opti-mem (invitrogen) at the concentration of x cells per ml. ten micrograms of rna were mixed with . ml of the cells in a -mm cuvette. a bio-rad gene pulser system was used to deliver a single pulse at v and µf and the cells were plated in t costar flasks (corning). transfected cells were cultured in complete dmem for the indicated times. cells were passaged every three days and the presence of hcv in the corresponding supernatants was determined by immunofluorescence assays (ifa) for the ns a protein or x-gal staining. cells infected by hcv or hcv reporter virus were washed with pbs, fixed with % paraformaldehyde, and permeabilized with . % triton x- . fixed cells were blocked with % bovine serum albumin and % normal goat serum in pbs. ns a protein was detected in cells with an ns a-specific monoclonal antibody and visualized with a secondary goat anti-mouse antibody conjugated with alexa fluor (invitrogen, : , dilutions). cover slips were mounted onto slides with dapi (vector labs) and immunostaining visualized by fluorescence microscopy (nikon e ). the titer of infectious hcv was determined by the number of cell foci staining for ns a as previously described in detail ( ) . briefly, cell supernatants were serially diluted -fold in complete dmem. the supernatant was used to infect × naive huh . cells per well in -well plates. the inocula were incubated with cells for three hours at o c and then supplemented with fresh complete dmem. the level of hcv infection was determined three days postinfection by immunofluorescence staining for hcv ns a and ns a-rluc, or x-gal staining for ns a-lacz and directly visualized the egfp for ns a-egfp. the viral titer is expressed as focus-forming units per milliliter of supernatant (ffu/ml). x-gal positive cells were identified using an x-gal staining assay kit (genlantis, cat#a k) and visualized by bright field microscopy (nikon) following the manufacturer's instructions. briefly, × naive huh . cells per well were seeded in -well plates and were transfected with rna of jfh-am -lacz or infected with the supernatant collected from transfected cells of rna of jfh-am -lacz or controls. hours post-transfection or infection, cells were fixed with formaldehydeglutaraldehyde buffer for minutes and stained the cells with x-gal staining solution for three hours. blue stained cells were identified by bright field microscopy (nikon e ). the viral titer of jfh-am -lacz was expressed as focus-forming units per milliliter of supernatant (ffu/ml). colocalization of x-gal pictures and immunofluorescence images were processed by photoshop cc software. the β-galactosidase activity was measured using a mammalian β-galactosidase assay kit (thermofisher, # ) and microplate reader (bio-tek) following the manufacturer's instructions. briefly, huh . cells were transfected with rna of jfh-am -lacz and controls of jfh-am , jfh-am -egfp and jfh-am -rluc and the cells were incubated for hours. then cells were washed once, μl of β-galactosidase assay reagent was added to each well, and cells were incubated for minutes at °c. reactions were stopped by adding μl of β-galactosidase assay stop solution to each well and absorbance at nm was measured. the hcv-transfected huh . cells were lysed in a radioimmuno-precipitation assay buffer ( mm tris-hcl, ph . , mm sodium chloride, % nonidet p , . % sodium deoxycholate) containing a cocktail of proteinase inhibitors (roche). the total protein for each sample was measured with a standard protein assay (bio-rad). twenty-five micrograms of total protein for each sample was analyzed by % sds-page and transferred to nitrocellulose membranes. the membranes were blocked by incubating them with % skim milk. hcv proteins were detected with monoclonal antibodies specific to ns a, horseradish peroxidase-conjugated goat anti-mouse immunoglobulin g (bio-rad) and a chemiluminescence substrate (pierce). β-actin was used as a control and was detected with an anti-β-actin monoclonal antibody (sigma). total rna was extracted with trizol (invitrogen) and hcv rna was measured by qpcr analysis as described previously ( ) . the relative quantity of hcv rna in control and hcv samples was calculated with the comparative ct (cycling threshold) method. a reference gene (β-actin) was used as the control. huh . cells were transfected with the rna of jfh -am -lacz and controls of jfh -am , jfh -am -egfp and jfh -am -rluc and the cells were passaged at every three days for a total of days. the total rna in the huh . cells were extracted with trizol reagent (invitrogen). the rna was reverse transcribed using superscript iii reverse transcriptase (invitrogen) and random primers. the resulting cdna was used as a template for subsequent pcr with platinum® pfx dna polymerase (invitrogen) and the following primers: jfh- -for, '-ttaattcctatgctgtcgggtcc cagct- '; jfh- -rev, '-gtgcgttgtacagta caccttgttatgg- '. the pcr amplicons ( bp for ns a wild type) were analyzed by % agarose gel electrophoresis and sequenced using standard methods. the mean and standard deviation of the mean for data were determined and the results for different experimental conditions were compared using the students t -test. the p value< . was considered to be significant. a previous study ( ) demonstrated hcv jfh wild type is capable of producing infectious virus but the titer is very low. it also cannot bear the foreign genes of egfp and rluc to produce viable reporter viruses ( ) . therefore, we used the adaptively mutated jfh strain, jfh -am , that produces higher titers of infectious virus as a vector for these studies ( ) . on this strain, egfp and renilla luciferace (rluc) genes were inserted in frame into a unique rsrii site located in the amino acid codon of ns a and reporter viruses of jfh -am -egfp and jfh-am -rluc were produced in huh . ( ) . the jfh -am -lacz was constructed by in frame inserting full lacz gene fragment ( bp) to the same rsrii site of jfh-am ns a c-terminal region and the lacz fragment was amplified from vector psv-β-galactosidase control (promega, e ) using primers lacz-f( '-tctcggtccgatagatcc cgtcgttttaca- ') and lacz-r( '-cttacggacc gcctttttgacaccagaccaact- '). the new clone was sequenced and the correct full-length clone was chosen for the subsequent experiments. all the constructs used in this study are shown in fig. . in this study the lacz gene ( bp) was inserted into the ns a c-terminal region of jfh -am , designated jfh -am -lacz. jfh -am , jfh -am -egfp and jfh -am -rluc were used as controls. rnas were transcribed from plasmids of jfh -am , jfh -am -egfp, jfh -am -rluc and jfh -am -lacz and were transfected to huh . cells to determine whether cells transfected with the rnas from each plasmid expressed hcv proteins and hcv rna. three days after rna transfection of cells, wild-type ns a ( kd) and the ns a-egfp ( kd), ns a-rluc ( kd) and ns a-lacz ( kd) fusion protein with the predicted molecular mass were detected in cell lysate by western blotting (fig. a) . however, the band of ns a-lacz protein was obviously weaker than ns a and other fusion proteins. the hcv rnas were also quantified by qpcr. the results showed that hcv rna amounts (fig. b ) correlated with changes in protein levels by western blotting. these results indicated that the replication level of jfh -am -lacz was significantly decreased after insertion of lacz gene into the ns a c-terminal of jfh -am . however, jfh -am -lacz can still replicate in huh . cells even though the lacz gene is bp in length. fig. . schematic representation of hcv constructs used in this study. jfh -am , jfh -am -rluc and jfh -am -egfp were generated in our previous study ( ) . egfp and renilla luciferace (rluc) genes were inserted in frame into a unique rsrii site located in the amino acid codon of ns a of jfh -am . jfh-am -lacz was generated by in frame insertion of full lacz gene, which was amplified from vector psv-β-galactosidase control (promega, e ) to the same rsrii site. the detailed protocol was described in material and method. jfh-am -lacz and jfh-am , jfh-am -egfp and jfh-am -rluc, the total rna was extracted from cells and qpcr analysis of hcv rna was performed. experiments were performed three times and the data presented as the mean ± sd (* p< . , * * p< . ). the lacz gene encodes β-galactosidase (β-gal) and the protein levels of this gene can be determined via staining with x-gal substrate and measuring the activity of the β-galactosidase enzyme. the protein levels and enzyme activity of β-galactosidase should be detected in jfh -am -lacz infected huh . cells. to verify this statement, the rna of jfh -am -lacz was transfected into huh . cells. rnas of jfh -am , jfh -am -egfp and jfh -am -rluc were used as controls. three days post transfection, β-galactosidase activity was measured using a mammalian β-galactosidase assay kit (thermofisher). the results showed only the sample of jfh -am -lacz had detectable β-galactosidase activity. all other transfected rnas were negative (fig. a ). using the same method, additional cells were transfected with jfh-am -lacz rna or control rnas and fixed with paraformaldehyde, stained using an x-gal staining assay kit (genlantis) and visualized the cell with blue color under bright field microscope. blue cells were only seen in cells transfected with jfh -am -lacz rna (fig. b) and there was not any on the other three transfected rnas (data not shown). these results confirmed the jfh -am -lacz could replicate in huh . cells. to examine the concurrent expression of hcv ns a and the β-galactosidase fusion protein in huh . cells after transfection of jfh -am -lacz rna, the colocalization of the ns a protein and β-galactosidase protein were determined by staining with ns a immunofluorescence assays and x-gal three days after transfection. cells were fixed with % paraformaldehyde and immunostained with anti-ns a antibody (red) and nuclei counterstained with dapi (blue). images were taken by immunofluorescence microscopy ( x). the coverslip was then washed and x-gal staining was performed, followed by visualizing and imaging by bright field microscopy ( x). colocalization assay was performed with photoshop cc software (fig. c) . images showed that ns a positive cells (red) were also with the blue color of x-gal staining. this result provided evidence that fusion protein of ns a and β-galactosidase can be co-expressed in huh . cells after transfection of jfh -am -lacz rna. to determine if transfection of huh . cells with jfh -am -lacz rna resulted in production of infectious viral particles, we inoculated naïve huh . cells with supernatants collected at th days post-transfection with jfh -am -lacz rna. supernatants collected from the cells transfected with rnas from jfh -am , jfh -am -egfp and jfh -am -rluc were used as controls. at three days post-infection, the cells infected with the supernatant of jfh-am -lacz were stained by x-gal and blue color cells were detected, while all of the others were negative. egfp positive cells were only found in jfh-am -egfp infected cells. jfh -am and jfh -am -rluc infected cells expressed ns a and ns a-rluc, as determined by the immunofluorescence assay (ifa). the viral titers are expressed as focus-forming units per milliliter of supernatant (ffu/ml). the titer of jfh -am , jfh -am -egfp, jfh -am -rluc and jfh -am -lacz were . x ffu/ml, . x ffu/ml, . x ffu/ml and . x ffu/ml, respectively. the viral titers of jfh -am -egfp and jfh -am -rluc were about times lower than jfh-am , however, the titer of jfh -am -lacz was dramatically lower (more than times) compared to jfh-am (fig. ) . these results demonstrated that the insertion of lacz in ns a c-terminal region of jfh -am significantly impaired the production of infectious viral particles. this result provided direct evidence that long exogenous genes can influence the capability of infectious hcv virus production at difference levels with a gene length dependent manner. fig. . analysis of ns a-lacz activity followed rna transfection of jfh -am -lacz. (a) huh . cells were transfected with the rna of jfh -am -lacz and controls of jfh -am , jfh -am -egfp and jfh -am -rluc as described in methods. three days post-transfection, the absorption peaks of β-galactosidase activity were measured using a bio-tec plate reader at nm. the values were relative to jfh -am -lacz. experiments were performed three times and the data presented as the mean ± sd. (b) three days post-transfection of huh . cells in -well plates with rna of jfh -am -lacz, cells were fixed with % paraformaldehyde followed x-gal staining. cover slips were visualized and images were taken ( x) by bright field microscopy. experiments were performed two times and representative results are shown. (c) co-localization of ns a and β-galactosidase. three days post-transfection of huh . cells in -well plates with rna of jfh -am -lacz, cells were fixed with % paraformaldehyde and were immunostained with anti-ns a antibody (red) and nuclei were counterstained using dapi (blue). images were taken by immunofluorescence microscopy ( x). then the coverslip was washed and were processed with x-gal staining followed visualizing and imaging by bright field microscopy ( x). colocalization assay was performed by photoshop cc software. experiments were performed three times and representative results are shown. fig. . infectivity assay of virus particles followed rnas transfection of jfh -am -lacz and controls. the rna of jfh -am -lacz and controls of jfh -am , jfh -am -egfp and jfh -am -rluc were electroporated into huh- . and the infectivity titers in the cultured supernatants at the th day were measured (described in material and method). the viral titer is expressed as focus-forming units per ml of supernatant (ffu/ml) as determined by the average number of ns a-positive foci detected by immunofluorescence for ns a (mock, jfh-am and jfh-am -rluc), or directly visualized egfp positive cells(jfh -am -egfp) and detected blue color cells after x-gal staining (jfh -am -lacz). assays were performed three times and the data are presented as mean ± standard deviation. (** p< . , *** p< . ) fig. . kinetics assay of the jfh -am -lacz hcv reporter after multiple passages. the cells transfected with the rna of jfh -am -lacz were passaged at every three days for a total of days. supernatants collected were designated p to p . the double titrations were carried out by x-gal staining for β-galactosidase and immunofluoresence staining for ns a protein (see materials and methods). experiments were performed three times and the data presented as the mean ± sd. our data show that jfh -am -lacz can replicate and produce a low titer of jfh -am -lacz hcv reporter virus in huh . cells. however, the genetic stability of a reporter virus is essential for its further application. to explore the durability of the jfh -am -lacz reporter in huh . cells subculture, a kinetics assay was performed by passaging the cells every three days for a total of days. supernatants were collected and designated p to p . double titrations were carried out by x-gal staining for β-galactosidase and immunofluorescence staining for ns a protein (see materials and methods). the titer measured by these two methods was different. the titer after ns a antibody staining was increased from p to p . the titer after x-gal staining was increase from p to p then decreased from p to p and reached zero at p and p (fig. ) . the discrepancy of these result indicated the lacz gene may be deleted and one strain lost lacz gene became a dominant and produced a higher titer virus. to confirm these results, western blotting was carried out for cells transfected with jfh -am -lacz rna and control rnas at p and p . the results show no band of ns a-lacz ( kd) in p cells and the ns a fragment reverting to the size of wild-type ns a ( and kd) (fig. a ) compared to the much larger ns a-lacz fusion protein in p cells ( fig. a) . ns a-egfp and ns a-rluc did not change after the same number of passages ( fig. a & a) . to verify these results, rt-pcr was performed on p and p cells and agarose gel analysis showed the predicted size pcr products ( fig. b and c ). the size of the ns a-wt, ns a-egfp and ns a-rluc pcr products did not change at p and p . however, the size of ns a-lacz pcr product was reverted to the same size of the ns a-wt product at p . this pcr product was sequenced and only six nucletitides of the lacz gene remained, while the other nucleotides of the lacz gene were absent, providing direct evidence for the loss of the lacz gene with multiple passaging of cells. on the fig. a and b , a weak band of similar size to wild-type ns a were seen in the lacz lane. this suggests that the deletion of the lacz gene may occur early in the first passage. these results indicated the jfh -am -lacz reporter virus was unstable after progressive cell culture. some rna viruses can tolerate the insertion of relatively large exogenous genes without disrupting replication and the production of functional recombinant viruses ( , ) . vesicular stomatitis virus (vsv), has been used as a gene expression vector and can accommodate the insertion of a foreign gene as large as bp ( ) . previous reports have shown that the hcv adapted jfh- strain can tolerate the insertion of exogenous genes less than bp, such as egfp ( bp) and rluc( bp), into the c terminus of ns a without disrupting viral replication and the production of infectious virions ( , , , , ) . a reporter gene as large as the lacz gene ( bp), has not been successfully inserted into the hcv genome with production of infectious virions. our results demonstrate that the lacz reporter gene can be inserted into the ns a c-terminus of hcv jfh -am and will express the predicted ns a-lacz fusion protein, which can be detected by western blotting three days after rna transfection of cells. however, compared to the control vectors of jfh -am , jfh -am -egfp and jfh -am -rluc, there was an obvious decrease in the expression of the ns a fusion protein ( fig. a) . β-galactosidase was also detected in these cells by several measures (fig. ) and the supernatants of these cells produced infectious virus of jfh -am -lacz (fig. ) . however, the infectious virus titer was much lower, . x ffu/ml compared to . x ffu/ml of parent jfh -am virus. in addition, the titer of infectious hcv jfh -am -lacz progressively decreased with serial passaging of cells, with the eventual loss of x-gal positive cells (fig. ) . this observation is consistent with western blot and ns a pcr analysis indicating the loss of the lacz reporter gene (fig. ) . the reason for the loss of the inserted lacz reporter gene is unclear. there may be three possible mechanisms on it. first, the hcv genome rna might have the capability of repairing itself and removing exogenous genes. second, the hcv ns a gene has a limit to it's carrying capacity for exogenous genes, and lacz is too large for stable expression. third, the combined effects of viral rna repair, expression stability and replication might result in lacz depletion in some strains, which could become the dominant strains during subsequent replication process, and this is consistent with the basic principle of natural adaptive mutation selection. . stability assay of jfh -am -lacz and controls followed the rna transfection. (a) huh . cells were transfected with the rna of jfh -am -lacz and controls of jfh -am , jfh -am -egfp and jfh -am -rluc and the cells were passaged at every three days for a total of days. the cells of passage ( fig. a) and passage were lysed for western blotting using anti-ns a and anti-β-actin bodies as described in methods.the experiment was performed twice and representative example are shown. (b & c) detection of hcv rna. huh . cells were transfected with the rna of jfh -am -lacz and controls of jfh -am , jfh -am -egfp and jfh -am -rluc and the cells were passaged at every three days for a total of days. the total rna was extracted and rt-pcr was performed described as in methods. the pcr products were analyzed by % agarose gel electrophoresis. experiments were performed two times and a representative experiment is shown (b showed passage st result and c showed passage th result). although hcv-egfp and hcv-rluc reporter viruses we have described previously were quite stabile with repeated passages, resulted in relatively high titers of infectious virions and have been used in a number of studies ( , ( ) ( ) ( ) , it is still unclear the capacity of hcv to accommodate large foreign genes. the production of infectious jfh -am -lacz virions reported here demonstrate that much larger gene inserts can be engineered into the c terminus of ns a of jfh -am for other applications and approaches. however, the instability of this reporter virus and the loss of lacz gene are likely to limit its use. nevertheless, the fact that a reporter gene up to bp in size can be inserted into the c terminus of ns a indicates that additional studies can be performed to guide further engineering of jfh -am in the research of hcv. global burden of hepatitis c: considerations for healthcare providers in the united states clinical practice. chronic hepatitis c infection hepatitis c virus to hepatocellular carcinoma sofosbuvir and velpatasvir for hcv genotype , , , , and infection oral direct-acting agent therapy for hepatitis c virus infection: a systematic review turning hepatitis c into a real virus structural biology of hepatitis c virus overview of hepatitis c virus genome structure, polyprotein processing, and protein properties efficient initiation of hcv rna replication in cell culture replication of subgenomic hepatitis c virus rnas in a hepatoma cell line production of infectious hepatitis c virus in tissue culture from a cloned viral genome robust hepatitis c virus infection in vitro complete replication of hepatitis c virus in cell culture cell culture adaptation of hepatitis c virus and in vivo viability of an adapted variant persistent hepatitis c virus infection in vitro: coevolution of virus and host robust production of infectious viral particles in huh- cells by introducing mutations in hepatitis c virus structural proteins advantages of a single-cycle production assay to study cell culture-adaptive mutations of hepatitis c virus a cell culture adapted hcv jfh variant that increases viral titers and permits the production of high titer infectious chimeric reporter viruses direct visualization of hepatitis c virus-infected huh . cells with a high titre of infectious chimeric jfh -egfp reporter virus in three-dimensional matrigel cell cultures the effects of ns a inhibitors on ns a phosphorylation, polyprotein processing and localization identification of residues required for rna replication in domains ii and iii of the hepatitis c virus ns a protein the ns a protein of hepatitis c virus is a zinc metalloprotein structure of the zinc-binding domain of an essential component of the hepatitis c virus replicase insertion of green fluorescent protein into nonstructural protein a allows direct visualization of functional hepatitis c virus replication complexes compensatory mutations in ns and ns a proteins enhance the virus production capability of hepatitis c reporter virus monitoring the antiviral effect of alpha interferon on individual cells insertion and deletion analyses identify regions of non-structural protein a of hepatitis c virus that are dispensable for viral genome replication analysis of hepatitis c virus superinfection exclusion by using novel fluorochrome gene-tagged viral genomes measuring antiviral activity of benzimidazole molecules that alter ires rna structure with an infectious hepatitis c virus chimera expressing renilla luciferase genetically modified vsv(nj) vector is capable of accommodating a large foreign gene insert and allows high level gene expression expression of a preproinsulin-beta-galactosidase gene fusion in mammalian cells characterization of the nuclear localization signal and subcellular distribution of hepatitis c virus nonstructural protein ns a augmentation of antitumor effects of p gene therapy by combination with hdac inhibitor constitutive activation of nf-kappab in human hepatocellular carcinoma: evidence of a cytoprotective role highly permissive cell lines for subgenomic and genomic hepatitis c virus rna replication enhancement of hepatitis c virus rna replication by cell culture-adaptive mutations rna-sequencing analysis of ' capped rnas identifies many new differentially expressed genes in acute hepatitis c virus infection coronaviruses as vectors: stability of foreign gene expression interferon-alpha inducible protein impairs egfr activation by cd and inhibits hepatitis c virus infection interferon lambda expression is suppressed by the host during viral infection activation of perk-nrf oncogenic signaling promotes mdm -mediated rb degradation in persistently infected hcv culture we thank dr. t. wakita for providing the plasmid containing the hcv jfh plasmid. we also thank dr. c.m. rice for providing huh . cells and the anti-ns a monoclonal antibody. this work was supported in part by nih grant ca to c.h.h. the authors have declared that no competing interest exists. key: cord- -vmmlxr o authors: zhu, yang; ma, yuanmei; lu, mijia; zhang, yu; li, anzhong; liang, xueya; li, jianrong title: efficient production of human norovirus-specific igy in egg yolks by vaccination of hens with a recombinant vesicular stomatitis virus expressing vp protein date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: vmmlxr o human norovirus (hunov) is responsible for more than % of outbreaks of acute nonbacterial gastroenteritis worldwide. despite major efforts, there are no vaccines or effective therapeutic interventions against this virus. chicken immunoglobulin y (igy)-based passive immunization has been shown to be an effective strategy to prevent and treat many enteric viral diseases. here, we developed a highly efficient bioreactor to generate high titers of hunov-specific igy in chicken yolks using a recombinant vesicular stomatitis virus expressing hunov capsid protein (rvsv-vp ) as an antigen. we first demonstrated that hunov vp protein was highly expressed in chicken cells infected by rvsv-vp . subsequently, we found that white leghorn hens immunized intramuscularly with rvsv-vp triggered a high level of hunov-specific yolk igy antibodies. the purified yolk igy was efficiently recognized by hunov virus-like particles (vlps). importantly, hunov-specific igy efficiently blocked the binding of hunov vlps to all three types (a, b, and o) of histo-blood group antigens (hbgas), the attachment factors for hunov. in addition, the receptor blocking activity of igy remained stable at temperature below °c and at ph ranging from to . thus, immunization of hens with vsv-vp could be a cost-effective and practical strategy for large-scale production of anti-hunov igy antibodies for potential use as prophylactic and therapeutic treatment against hunov infection. the virus family caliciviridae contains five established genera (norovirus, sapovirus, lagovirus, vesivirus, and nebovirus) and at least six proposed genera (recovirus, valovirus, bavovirus, nacovirus, minovirus, and salovirus) that infect many different animal species including humans. most of these agents are enteric pathogens whose replication and chief clinical manifestations are gastroenteritis and potentially life-threatening diarrhea. examples of these viruses include human norovirus (hunov), porcine norovirus, bovine norovirus, human sapovirus, porcine sapovirus, and the recently discovered tulane virus. hunov is the major food-and water-borne virus that accounts for more than % of nonbacterial acute gastroenteritis worldwide, but this percentage may be underestimated due to in this study, we developed a highly efficient bioreactor for large-scale production of chicken egg yolk igy antibodies using rvsv-vp as an antigen. we found that hunov vp was highly expressed in chicken cells infected by rvsv-vp and that hens vaccinated rvsv-vp induced a high level of hunov-specific igy in egg yolks. interestingly, intramuscular vaccination of rvsv-vp produced approximately three times more hunov-specific igy than combination of intramuscular and nasal drop vaccination route. importantly, hunov-specific igy produced by rvsv-vp vaccination was capable of blocking the binding of hunov vlps to type a, b, and o hbga receptors. in addition, egg yolk igy antibodies remained stable at temperature below • c and at ph ranging from to . these data demonstrated that recombinant rvsv-vp is a highly effective antigen for large-scale production of hunov-specific igy for passive immunization and therapeutic agent. recombinant vesicular stomatitis virus (rvsv) expressing human nov capsid protein (rvsv-vp ) was previously constructed in our laboratory [ ] . working stocks of rvsv-vp were propagated in confluent bsrt cells. briefly, bsrt cells in t flask were infected by rvsv-vp at a multiplicity of infection (moi) of . after h of absorption, the inoculum was removed, and the cells were washed twice with dulbecco's modified eagle's medium (dmem). after addition of ml fresh dmem (supplemented with % fetal bovine serum), the infected cells were incubated at • c in co incubator. when extensive cytopathic effects (cpe) were observed, cell culture fluid was harvested, and virus titer was determined by plaque assay in vero cells. thirty-five-millimeter dishes were seeded with bsrt- or df- cells (kindly provided by dr. qingzhong yu at usda ars, athens, ga) and were infected with rvsv-vp at a multiplicity of infection (moi) of . after h of absorption, the inoculum was removed, the cells were washed twice with dmem, ml of fresh dmem (supplemented with % fetal bovine serum) was added, and the infected cells were incubated at • c. at the indicated intervals, -µl aliquots of the cell culture fluid were removed and the same amount of fresh dmem was added back to the virus-infected cells. virus titers were determined by plaque assay in vero cells. six-well plates were seeded by bsrt or df- cells ( × cells per well). after h incubation at • c in co incubator the cells were infected with rvsv-vp at an moi of . at the indicated time points, cells were lysed in lysis buffer containing % β-mercaptoethanol, . % np- , and % sodium dodecyl sulfate (sds). proteins were separated by % sds-page and transferred to a hybond ecl nitrocellulose membrane (amersham, piscataway, nj, usa) in a mini trans-blot electrophoretic transfer cell (bio-rad, hercules, ca, usa). the blot was probed with guinea pig anti-human nov vp antiserum (a generous gift from dr. xi jiang) at a dilution of : , followed by horseradish peroxidase-conjugated goat anti-guinea pig igg secondary antibody (santa cruz biotechnology, inc., dallas, tx, usa) at a dilution of : , . the blot was developed with supersignal west pico chemiluminescent substrate (thermo scientific, waltham, ma, usa) and exposed to kodak biomax mr film (kodak, rochester, ny, usa). the harvested cell lysates were also subjected to western blot using anti β-actin antibody (proteintech, rosemont, il, usa), which recognizes β-actin from multiple species, including human, mouse, hamster, and chicken. the protein bands were scanned and quantified using a typhoon phosphorimager and imagequant tl software (ge healthcare, piscataway, nj, usa). the vp protein band of each time point was normalized by the respective β-actin band. the time point ( h postinfection in bsrt cells) with highest vp expression was set as %. the percentage of vp expression at other time points was normalized by the vp expression at h postinfection in bsrt cells. the animal study was conducted in strict accordance with usda regulations and the recommendations in the guide for the care and use of laboratory animals of the national institutes of health, and was approved by the ohio state university institutional animal care and use committee (animal protocol no. a ). chickens were housed in cages inside high-security isolation rooms provided with hepa-filtered intake and exhaust air at the ohio agriculture research and development center, the ohio state university. the animal care facilities at the ohio state university are aaalac accredited. before animal study, blood samples were collected from each chicken to confirm that they were negative for hunov antibody. six, -week-old, healthy white leghorn chickens were provided by dr. lilburn, department of animal sciences, the ohio state university and were randomly divided into two groups (three chickens per group). prior to the study, these chickens were negative for vsv and hunov antibody. chickens in group i were immunized intramuscularly by injecting µl of dmem containing × pfu of rvsv-vp into three different locations of the pectoral muscle. chickens in group ii were immunized by combination of intramuscular and intranasal routes. specifically, µl of rvsv-vp ( × pfu) was injected into three different locations of the pectoral muscle, and the remaining µl of rvsv-vp ( × pfu) was used for nasal drop vaccination. at week postimmunization, chickens in groups i and ii were boosted with × pfu of rvsv-vp via intramuscular and combination of intramuscular and intranasal routes, respectively. after immunization, eggs were collected daily until week post-booster vaccination. in addition, hens were observed daily for any abnormal reactions. eggs that were collected one week before immunization were used as negative control. eggs were stored at • c before igy extraction. purification of vlps from insect cells was described previously with some minor modifications [ ] . spodoptera frugiperda (sf ) cells were infected with baculovirus expressing hunov vp at an moi of , and the infected sf cells and cell culture supernatants were harvested at days postinoculation. the vlps were purified from cell culture supernatants and cell lysates by ultracentrifugation through a % (w/v) sucrose cushion, followed by cscl isopycnic gradient ( . g/cm ) ultracentrifugation. purified vlps were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) followed by coomassie blue staining. the protein concentrations of the vlps were measured by using the bradford reagent (sigma chemical co., st. louis, mo, usa). igy was extracted and purified from egg yolks using polyethylene glycol (peg , sigma, st. louis, mo, usa) precipitation method [ ] with some modifications. briefly, egg yolks were diluted in three volumes of pbs (ph . ) and mixed, and peg was added to a final concentration of . %. after vortexing, the mixture continued to roll on a rolling mixer for min. the mixtures were centrifuged at , × g for min at • c, and the precipitated debris were removed. subsequently, peg was added to the supernatant to a final concentration of . %, and the samples were mixed on a rolling mixer for min. the mixtures were centrifuged again at , × g for min at • c. the precipitated pellets containing igy were dissolved in ml of pbs and then precipitated again with % of peg using the same procedures described above. the final pellets was dissolved in . ml of pbs, filtered through a . µm filter, and stored at − • c. the purity of the igy was determined by sds-page followed by coomassie blue staining. standard elisa measured hunov-specific igy antibody titers. briefly, -well microtiter plates were coated with µl of purified hunov vlp antigen ( ng/well) and incubated overnight at • c. after blocking with % nonfat milk, times serially diluted chicken igys were added to the antigen-coated wells and incubated at • c for h. after washing with pbst (pbs containing . % tween), goat anti-chicken igy-hrp ( : ) (santa cruz biotechnology) was added for h. plates were washed and developed with µl of , ', , '-tetramethylbenzidine (tmb), and the optical density (od) at nm was determined using an enzyme-linked immunosorbent assay (elisa) plate reader. the igys from pre-immunized chicken yolks were used as controls. to calculate the amount of total igy and hunov-specific igy, a standard curve was set-up: wells were coated with µl of serially diluted pure chicken igy (promega, madison, wi, usa) at a concentration from . µg/ml to µg/ml. after washing with pbst, µl of goat anti-chicken igy-hrp (santa cruz biotechnology) at a dilution of : ) were added and incubated at • c for h. the bound hrp was colorized by substrate reagent (kirkegaard and perry laboratories, inc., gaithersburg, maryland, usa), followed by a reading of the signal intensity at nm (epoch micro-volume spectrophotometer system, biotek, winooski, vt, usa). the resulting standard curve of absorbance was used to quantify the relative concentration of total igy and hunov-specific igy from the egg yolks by coating plates with either hunov vlp or rabbit anti-chicken igy antibodies ( µg/ml, sigma) to capture the total igy or hunov-specific igy. the purified igys from egg yolks were analyzed by sds-page. samples were boiled for min in loading buffer containing % sds, . % β-mercaptoethanol, . mm tris-hcl (ph . ), and % glycerol and loaded onto a % polyacrylamide gel. proteins were visualized by coomassie blue staining. specific reactions of the chicken igy with hunov capsid protein were examined by western blot analysis. the purified hunov virus-like particles (vlps) were separated by conventional % sds-page and transferred to a hybond ecl nitrocellulose membrane (amersham) in a mini trans-blot electrophoretic transfer cell (bio-rad). after blocking with % nonfat milk, the membrane was incubated with anti-hunov-specific igy or nonspecific igy ( : ) in % nonfat milk-pbs at • c overnight, followed by horseradish peroxidase (hrp)-conjugated goat anti-chicken igy secondary antibody (santa cruz biotechnology) at a dilution of : . the blot was developed with supersignal west pico chemiluminescent substrate (thermo scientific) and exposed to kodak biomax mr film (kodak). the saliva-based hbga binding and blocking assays were performed as described previously [ , , ] . to avoid potential hunov-specific antibodies in the saliva that may interfere with the receptor-binding assay, saliva samples were boiled before being used in the assays. the boiled human saliva samples with known hbga phenotypes (a, b, or o) were diluted -fold and coated on -well microtiter plates at • c overnight. after blocking with % nonfat milk in pbs, hunov vlps were added to a final concentration of µg/ml. the bound vlps were detected by using serially diluted igys (from : to : , ), followed by the addition of hrp-conjugated goat anti-chicken igy at a dilution of : . the color was then developed by adding tetramethylbenzidine peroxidase liquid substrate (kirkegaard and perry laboratory) and stopped after min of incubation at • c by adding mol/l sulfuric acid. optical density (od) was measured at nm with the use of an epoch micro-volume spectrophotometer system (biotek, winooski, vt, usa). hbga blocking assay was performed to determine the inhibitory activity of igy against the binding of hunov vlps to the hbga antigens [ ] . the boiled human saliva samples with known hbga phenotypes (a, b, or o) were diluted -fold and coated on -well microtiter plates at • c overnight. the hunov vlps were preincubated with serially diluted igys for h at • c, and igy-vlp solutions were added to the saliva-coated wells. plates were washed times with . mol/l sodium phosphate buffer (ph, . ). then, a guinea pig anti-hunov vlp antiserum at a dilution of : was added and incubated for h at • c. the plates were washed again, and hrp-conjugated goat anti-guinea pig igg (at dilution of : ) was added and incubated for h at • c. the color was then developed by adding tetramethylbenzidine peroxidase liquid substrate (kirkegaard and perry laboratory) and stopped after min of incubation at • c by adding mol/l sulfuric acid. the blocking rates were calculated by comparing the optical densities (ods) measured with and without blocking by the chicken igys. the igys from chickens before immunization were used as controls [ ] . blank wells were incubated with buffer instead of igy-vlp and served as negative control whereas vlp binding to carbohydrates in the absence of igy sample was used as a positive control. for the ph stability, the purified total igy solution ( ml, : , ph . ) was diluted in . mol/l sodium phosphate buffer (ph . ). the ph of the solution was adjusted using either hcl or naoh to a final ph ranging from to . the solution was incubated at • c for h, followed by neutralization by adding × pbs (ph . ) to a final ph of . the hbga blocking assays were performed to measure the activity of igy, as described above. to determine heat stability of hunov-specific igy, the purified total igy solution ( ml, : , ph . ) was treated at temperature ranging from to • c for up to min. after heat treatment, the samples were cooled quickly on ice. the hbga blocking assays were performed to measure the activity of igy as described above. quantitative analysis was performed by either densitometric scanning of autoradiographs or by using a typhoon phosphorimager and imagequant tl software (ge healthcare, piscataway, nj, usa). each experiment was performed three to six times. statistical analysis was performed by one-way multiple comparisons using spss software . (spss, chicago, il, usa). a p-value of < . was considered statistically significant. vsv replicates efficiently in a wide range of mammalian cell lines. we previously showed that rvsv-vp replicated to a high titer in bsrt cells, baby hamster kidney cells [ ] . before chicken vaccination, we first determined whether rvsv-vp replicated efficiently in df- cells, which are derived from chicken embryo. briefly, bsrt and df- cells were infected with rvsv-vp at an moi of and the kinetics of viral replication was determined at time points from to h postinfection. as shown in figure a , rvsv-vp replicated efficiently in both df- and bsrt cells. viral titer gradually increased at h postinfection and reached a peak titer of . × pfu/ml at h postinfection. however, in bsrt cells, the virus titer started to increase at h postinfection, and reached a peak titer of . × at approximately h postinfection. in addition, virus titer in df- cell decreased figure c ). we next examined the kinetics of hunov vp expression in bsrt and df- cells. briefly, bsrt and df- cells were infected with rvsv-vp at an moi of and cell lysates were harvested at the indicated times. the expression of vp was determined by western blot. β-actin was loaded as an internal control. the vp expression was detectable at h postinfection in both cell lines, and reached a peak between and h postinfection ( figure b) . hunov vp was highly expressed in both bsrt and df- cells during - h postinfection. vp expression in df- decreased after h postinfection due to the cell death. quantitation of three independent experiments showed there were no significant difference in vp expression between bsrt and df- cells at time points from to h postinfection (p > . ) ( figure c ). we previously showed that rvsv-vp was not only attenuated in vitro and in vivo, but also triggered a high level of mucosal, humoral, and cellular immunities in a mouse model [ ] . since rvsv-vp can grow in chicken cells, we hypothesis that chickens will be susceptible to rvsv-vp infection and thus produce hunov-specific igy in eggs. to do this, rvsv-vp was inoculated into chickens by two routes: intramuscular or combination of intramuscular and intranasal route. after vaccination, the safety of rvsv-vp in chickens was monitored daily. no abnormal reaction was observed in chickens vaccinated by both routes. recombinant rvsv-vp vaccination did not affect feed intake and egg production. this result suggests that rvsv-vp was safe to chickens. after rvsv-vp vaccination, eggs from each hen were collected daily. to determine whether rvsv-vp triggers hunov-specific igy, total igy was purified from each egg collected at weeks , , , and postvaccination. to examine the purity of total igy, µl of total igy from one egg collected at week postvaccination was analyzed by sds-page. as shown in figure a , two protein bands with molecular weight of and kda were observed, which were consistent with the size of heavy and light chain of igy, respectively. to next determined whether purified total igys contain hunov-specific igy. purified hunov vlps (approximately kda) were separated by sds-page ( figure b ), followed by western blotting using total igy or serum igg. as a positive control, a band of kda protein was detected when anti-hunov vp serum antibody raised in guinea pig was used ( figure c ). as a negative control, no protein bands were identified in western blot using total igy from eggs prior to rvsv-vp vaccination ( figure d ). as shown in figure e , hunov vp was detected by western blot when total igy from eggs collected at week after rvsv-vp vaccination. thus, these results confirmed that rvsv-vp vaccination triggered hunov-specific igy in egg yolks. blotting using total igy or serum igg. as a positive control, a band of kda protein was detected when anti-hunov vp serum antibody raised in guinea pig was used ( figure c ). as a negative control, no protein bands were identified in western blot using total igy from eggs prior to rvsv-vp vaccination ( figure d ). as shown in figure e , hunov vp was detected by western blot when total igy from eggs collected at week after rvsv-vp vaccination. thus, these results confirmed that rvsv-vp vaccination triggered hunov-specific igy in egg yolks. we next determined the kinetics of hunov-specific igy responses following rvsv-vp vaccination. briefly, -well plates were coated with ng of purified hunov vlp antigen in each well and were reacted with serially diluted chicken igy at • c for h. after reacting with hrp labeled goat anti-chicken igy followed by addition of substrate reagent, an elisa reader read the od value at nm. subsequently, the amount of total igy and hunov-specific igy was quantified using standard curve generated by commercially available igy. as shown in figure a , the amount of hunov-specific igy gradually increased after rvsv-vp immunization. interestingly, at weeks , , and postvaccination, the levels of hunov-specific igy in intramuscular vaccination group were significantly higher than those in the combined intramuscular and nasal drop vaccination group (p < . ). at week postvaccination, hunov-specific igy in intramuscular group reached . mg/yolk whereas only . mg/yolk hunov-specific igy was detected in intramuscular and nasal drop group. using a similar method, the amount of total igy in each egg yolk was determined. as shown in figure b , there was no significant difference in total igy between the two vaccination groups (p > . ). in addition, there was no significant difference in total igy from eggs collected before and after rvsv-vp vaccination (p > . ). under our experimental condition, the amount of total igy in each yolk ranged from . ± . to . ± . mg. hunov utilizes hbgas as attachment factors. hunov vlps possess authentic receptor binding activity that can be detected by a hbga binding assay. in this assay, human saliva containing hbga receptors (types a, b, or o) or synthetic hbgas can be recognized by hunov vlps, which can be further detected by anti-hunov serum and a hrp-conjugated second antibody. therefore, we determined whether total igy derived from rvsv-vp vaccinated hens could be used as the primary antibody for the hbga binding assay. briefly, -well plates were coated with type a, b, or o human next, we calculated the percentage of hunov-specific igy in total igy in each egg yolk. as shown in figure c , the percentage of hunov-specific igy gradually increased from . % to % in the intramuscular immunization group. in contrast, the percentage of hunov-specific igy ranged from . % to % in the combined immunization group. collectively, these results showed that both immunization routes were capable of producing hunov-specific igy, and intramuscular injection alone was more effective in triggering hunov-specific igy than combination of intramuscular and nasal drop route. hunov utilizes hbgas as attachment factors. hunov vlps possess authentic receptor binding activity that can be detected by a hbga binding assay. in this assay, human saliva containing hbga receptors (types a, b, or o) or synthetic hbgas can be recognized by hunov vlps, which can be further detected by anti-hunov serum and a hrp-conjugated second antibody. therefore, we determined whether total igy derived from rvsv-vp vaccinated hens could be used as the primary antibody for the hbga binding assay. briefly, -well plates were coated with type a, b, or o human saliva, and ng of hunov vlps were added to bind the hbga receptor. after -h incubation, a serial dilution of total igy was added followed by addition of hrp-conjugated goat anti-chicken igy. after the addition of substrate reagent, an elisa reader measured od . purified total igy from rvsv-vp vaccinated groups strongly reacted with hunov vlps in the saliva-based hbga binding assays ( figure a,b) . in contrast, control igy from pre-immunized hens was negative in this assay. overall, total igy from intramuscular vaccination group had significantly higher od values than those in combined vaccination group (p < . , compare the od value in figure a ,b). in addition, it appears that vlps had a stronger binding activity to type a saliva compared to types b and o saliva ( figure a,b) . these results demonstrated that purified total igy induced by rvsv-vp vaccination could be used as a primary antibody to measure binding of hunov vlp to all three types of saliva in hbga binding assays. saliva, and ng of hunov vlps were added to bind the hbga receptor. after -h incubation, a serial dilution of total igy was added followed by addition of hrp-conjugated goat anti-chicken igy. after the addition of substrate reagent, an elisa reader measured od . purified total igy from rvsv-vp vaccinated groups strongly reacted with hunov vlps in the saliva-based hbga binding assays ( figure a,b) . in contrast, control igy from pre-immunized hens was negative in this assay. overall, total igy from intramuscular vaccination group had significantly higher od values than those in combined vaccination group (p < . , compare the od value in figure a ,b). in addition, it appears that vlps had a stronger binding activity to type a saliva compared to types b and o saliva ( figure a,b) . these results demonstrated that purified total igy induced by rvsv-vp vaccination could be used as a primary antibody to measure binding of hunov vlp to all three types of saliva in hbga binding assays. next, we determined whether the purified igy has potential antiviral activity that can be developed as a therapeutic agent against hunov infection. recently, a hbga blocking assay, which measures the ability of antibody blocks the binding of hunov vlps to the attachment factors (hbgas), has been developed [ , ] . presumably, blockage of viral receptor binding activity will inhibit viral attachment, entry, and subsequent viral infection. thus, this assay can serve as a useful surrogate assay for serum-virus neutralization assay. briefly, -well plates were coated with a known saliva type (a, b, or o) at °c overnight. the vlps were preincubated with the serially diluted igy at °c for h before adding to the saliva-coated wells. then, a guinea pig anti-hunov vlp antiserum was added, followed by the addition of hrp-conjugated goat anti-guinea pig igg. after the addition of substrate reagent, od was measured by an elisa reader. percent of blocking activity was calculated by comparing the od values measured with or without blocking by the chicken igys. as shown in figure , total igy antibodies isolated from egg yolks of rvsv-vp vaccinated groups were capable of blocking the binding of hunov vlp to hbgas (a, b, or o next, we determined whether the purified igy has potential antiviral activity that can be developed as a therapeutic agent against hunov infection. recently, a hbga blocking assay, which measures the ability of antibody blocks the binding of hunov vlps to the attachment factors (hbgas), has been developed [ , ] . presumably, blockage of viral receptor binding activity will inhibit viral attachment, entry, and subsequent viral infection. thus, this assay can serve as a useful surrogate assay for serum-virus neutralization assay. briefly, -well plates were coated with a known saliva type (a, b, or o) at • c overnight. the vlps were preincubated with the serially diluted igy at • c for h before adding to the saliva-coated wells. then, a guinea pig anti-hunov vlp antiserum was added, followed by the addition of hrp-conjugated goat anti-guinea pig igg. after the addition of substrate reagent, od was measured by an elisa reader. percent of blocking activity was calculated by comparing the od values measured with or without blocking by the chicken igys. as shown in figure , total igy antibodies isolated from egg yolks of rvsv-vp vaccinated groups were capable of blocking the binding of hunov vlp to hbgas (a, b, or o antigen) in a dose-dependent manner with a bt (a serum dilution with % blocking activity) of approximately : , : , and : , respectively. as controls, igy purified from egg yolks of chickens before immunization did not have detectable blocking activity. again, total igy from intramuscular group had a significantly higher blocking activity compared to the intramuscular and nasal drop group (p < . ). therefore, igy from rvsv-vp vaccinated hens specifically blocked the binding of hunov vlps to the hbgas. nasal drop group (p < . ). therefore, igy from rvsv-vp vaccinated hens specifically blocked the binding of hunov vlps to the hbgas. to investigate thermal stability of igy, igy solution was incubated at temperature ranging from to °c for up to min, and the receptor blocking activity was measured by the saliva-based hbga blocking assay. as shown in figure a -c, hunov-specific igys retained wildtype level of to investigate thermal stability of igy, igy solution was incubated at temperature ranging from to • c for up to min, and the receptor blocking activity was measured by the saliva-based hbga blocking assay. as shown in figure a -c, hunov-specific igys retained wildtype level of blocking ability to all three types of saliva at the temperatures below • c. however, the blocking activity significantly impaired at the temperatures above • c (p < . ). blocking ability to all three types of saliva at the temperatures below °c. however, the blocking activity significantly impaired at the temperatures above °c (p < . ). next, we determined whether hunov-specific igy is stable in acid and alkaline environments. to do this, purified igy was diluted times in pbs solution and the ph of the solutions was adjusted with either hcl or naoh to a final ph of - . after incubation at °c for h, the solution next, we determined whether hunov-specific igy is stable in acid and alkaline environments. to do this, purified igy was diluted times in pbs solution and the ph of the solutions was adjusted with either hcl or naoh to a final ph of - . after incubation at • c for h, the solution was adjusted to neutral ph, and the receptor blocking activity was measured by hbga blocking assay. as shown in figure a -c, the blocking activity of the chicken igy remained stable at ph - for h. however, a significant decrease of blocking activity was observed when the ph is lower than or higher than . viruses , , x for peer review of assay. as shown in figure a -c, the blocking activity of the chicken igy remained stable at ph - for h. however, a significant decrease of blocking activity was observed when the ph is lower than or higher than . in this study, we developed a highly efficient bioreactor to produce hunov-specific igy in egg yolks by immunization of white leghorn chickens with recombinant rvsv-vp . we found that intramuscular immunization alone was more efficient in triggering hunov-specific igy compared in this study, we developed a highly efficient bioreactor to produce hunov-specific igy in egg yolks by immunization of white leghorn chickens with recombinant rvsv-vp . we found that intramuscular immunization alone was more efficient in triggering hunov-specific igy compared to the combination of intramuscular and nasal drop immunization in hens. we demonstrated that igy antibodies strongly reacted with hunov vlps in elisa, western blot, and hbga binding assays. the igy antibodies were capable of blocking hunov-hbga receptor interactions, and remained stable in temperature below • c and at ph ranging from to . our results suggest that chicken egg yolk igy antibodies (igy) is a promising prophylactic and therapeutic agent for hunov. replication competent rvsv-vp is an excellent antigen for production of hunov-specific igy in chickens. the natural hosts of vsv are cattle, horse, deer, and pig. however, vsv has a broad tissue tropism and can replicate to a high titer in many mammalian cell lines, insect cell, yeast, worm, and c. elegans [ , ] . in this study, we found that rvsv-vp replicated to a high titer in df- cells, a continuous cell line derived from chicken embryo fibroblasts. the virus yields in df- cells were comparable to those produced in bsrt cell. in addition, rvsv-vp produced similar amounts of vp protein in both df- and bsrt cells. prior to our study, replication capability of vsv in avian species in vivo was poorly understood. the only report of vsv infection in chickens came from the study of rvsv-based influenza virus vaccine. it was found that chickens vaccinated with rvsv expressing the ha antigen of highly pathogenic avian influenza virus (h n ) triggered a high level of serum neutralizing antibody and provided complete protection against lethal challenge of avian influenza h n [ ] . in our study, we found that hens vaccinated with rvsv-vp triggered a high level of hunov-specific igy in yolks, further supporting that chickens were susceptible to vsv infection. we also showed that immunization route affected igy production in hens. we found that intramuscular vaccination triggered a higher hunov-specific igy than combination of intramuscular and nasal drop vaccination although the total igy levels were similar between two vaccination routes. our rationale to include nasal drop vaccination is that it may trigger a higher mucosal immunity since egg yolk is developed in reproductive tract of chickens. in fact, our previous mice study showed that oral and intranasal vaccination of rvsv-vp trigged a high level of both serum and mucosal antibody [ , ] . one of the major concerns of rvsv-based vaccine is the safety, particularly since vsv is neurotropic. vsv infection in ruminant animal causes vesicular lesions in the mouth, teats, and feet. in mice, wild type vsv can cause acute brain infection and fatal encephalitis [ ] . however, insertion of hunov vp into vsv vector significantly attenuated the virus in a mouse model [ , ] . in this study, rvsv-vp did not cause any abnormal reactions, feed intake, or egg production of chickens, which further proved that rvsv-vp was attenuated in vivo. although two recent reports showed that hunov-specific igy can be induced in hen vaccinated with purified vlps or p particles from insect cells using a baculovirus expression system [ , ] , our study represents the first report of using a live attenuated recombinant virus to generate hunov-specific igy. there are many potential advantages of using live attenuated rvsv-vp for igy production. first, rvsv-vp grows to a high titer in a wide range of cell lines including chicken cells. second, replication of rvsv-vp in chickens resulted in synthesis of large amount of vlps that in turn triggered a high level of hunov antibody. third, it is an economical and time-saving approach. it does not require purification of vlps or p particles. thus, rvsv-vp is a promising vaccine antigen for large-scale production of igy in chickens. hunov-specific igy is a potential passive immunization and therapeutic agent for hunov. hunov is a leading cause of viral gastroenteritis worldwide. despite significant social, health, and economical burden it causes, no fda-approved vaccine or therapeutic strategy is available. epidemiology studies showed that hunov could cause lethal infection in humans, particularly in high-risk populations, such as infants, young children, the elderly, and immunocompromised individuals. thus, there is a need to develop a safe and effective therapeutic strategy. we found that hunov-specific igys isolated from egg yolks were biologically functional in vitro. first, hunov-specific igys can react with vlps in elisa and western blot. second, similar to serum igg, hunov-specific igys can be used as a primary antibody in hbga binding assay. third, hunov-specific igys, but not the control igy, were capable of blocking the binding of hunov vlps to type a, b, and o hbgas in human saliva. although it is unknown whether hunov-specific igy can directly neutralize the infectious hunov, blockage of virus-receptor interaction will likely block the infectivity of hunov, which will prevent hunov infection and illness. in , reeck et al. found a direct correlation between the ability of an antibody to block vlp-hbga binding and protection against hunov infection and illness in a hunov human challenge study [ ] . in addition, nurminen et al., ( ) showed that children could be protected from a gii. hunov infection due to the pre-existing hbga blocking antibodies [ ] . thus, the igys is a promising passive immunization approach to prevent and treat hunov infection and illness. future study will determine whether igy can directly neutralize infectious hunov using the recently developed b cell culture system [ ] or human enteroids culture system [ ] . the concept of igy passive immunization has been developed in rotavirus in vivo animal models. it was found that passive immunization of igy could protect neonatal calves from bovine rotavirus -induced diarrhea [ ] . in a mouse model, it was found that igy could prevent murine rotavirus infection [ ] , bovine rotavirus-induced diarrhea [ ] , and human rotavirus-induced gastroenteritis [ ] . recently, human rotavirus-specific igy administered orally as a milk supplement passively protects neonatal pigs against an enteric human rotavirus infection [ ] . porcine epidemic diarrhea virus (pedv) is an enteric coronavirus which causes severe diarrhea, vomiting, and mortality in young piglets. kweon et al. ( ) found that igy passive immunization can protect piglets against pedv infection [ ] . future experiments will investigate whether hunov-specific igy can protect gnotobiotic piglets from hunov-induced gastroenteritis and viral shedding. chicken as a "factory" for large-scale production of antigen-specific igy. antibody-based passive immunization and therapy has been shown to be an effective strategy to prevent infectious diseases in many animals [ ] . however, preparation of serum antibody from mammals is expensive and time-consuming. thus, large-scale application of serum antibody has been limited. igy egg yolk immunoglobulins derived from hyperimmunized hens represent an economically feasible and practical strategy which has been explored for the passive treatment of enteric diseases. chicken igy production is a much easier, faster, and cheaper method for polyclonal antibody production than from any other sources. it is easy to raise chickens and collect eggs without involvement of any stressful procedures (such as bleeding). white leghorn chickens are highly productive in laying eggs, and they can continuously produce eggs containing antigen-specific antibodies in their yolks for a long time period after immunization [ ] . nguyen et al. ( ) demonstrated that chicken usually lays eggs/year and each egg yolk normally contains - mg of igy, which has to % antigen-specific antibodies [ ] . in addition, extraction of antibody from egg yolk is simple and noninvasive without affecting the immunized chickens. therefore, a chicken is an excellent "factory" for igy antibody production. in order to be used as immunological supplements in infant formulas and other foods, it is important to investigate the stability of igy during storage or following processing methods, involving thermal treatments, such as pasteurization, sterilization, or spray-drying. based on the hbga blocking assay, hunov-specific igy remains stable at temperature below • c. thus, it is safe for pasteurization (below • c) of igy for human consumption. however, the receptor blocking activity of igy significantly decreased when temperature reached above • c, suggesting that igy may be denatured at this temperature. for oral administration, igy should ideally be stable in acid or alkaline environment. our results showed that the receptor blocking activity of igy decreased at ph below or above . since the stomach ph is~ - , it may be necessary to encapsulate the igy in acid resistant capsules so that it can be released in intestines for neutralizing the infectious virus particles. for example, chang et al. ( ) demonstrated that addition of sugars, glycerol, or glycine to immunoglobulin solutions was effective to protect igys. in addition, film coating with gum arabic was proven to be effective in reducing the degree of hydrolysis of igy [ ] . in summary, we have developed a highly efficient bioreactor to generate a high titer of hunov-specific igy by vaccination of hens with rvsv-vp . hunov-specific igy was biologically active in capturing hunov antigen and blocking the interaction between vlps and hbgas. this study will facilitate the large-scale production and purification of hunov-specific igy for virus detection, diagnosis, passive immunization, and therapy. noroviruses everywhere: has something changed? foodborne viruses: an emerging problem food-related illness and death in the united states human noroviruses: recent advances in a -year history foodborne illness acquired in the united states-major pathogens winter vomiting disease inactivation of caliciviruses enteric bacteria 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gastroenteritis using immunoglobulin igy antibodies protect against human rotavirus induced diarrhea in the neonatal gnotobiotic piglet disease model immunoprophylactic effect of chicken egg yolk immunoglobulin (ig y) against porcine epidemic diarrhea virus (pedv) in piglets prophylactic and therapeutic efficacy of avian antibodies against influenza virus h n and h n in mice productivity and some properties of immunoglobulin specific against streptococcus mutans serotype c in chicken egg yolk (igy) this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the ohio state university owns the patent on recombinant vesicular stomatitis virus expressing vp of human norovirus (rvsv-vp ) (patent no. ). we are grateful to members of the j. li laboratory for critically reading the manuscript. we thank xi jiang's laboratory at cincinnati children's hospital for providing human norovirus antibody and saliva samples with known hbga phenotypes for this study. we thank michael lilburn for providing hens for this study. the authors declare no conflicts of interest. key: cord- - krf yxz authors: li, xi; huang, yongbo; xu, zhiheng; zhang, rong; liu, xiaoqing; li, yimin; mao, pu title: cytomegalovirus infection and outcome in immunocompetent patients in the intensive care unit: a systematic review and meta-analysis date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: krf yxz background: cytomegalovirus (cmv) infection is common in immunocompetent patients in intensive care units (icus). however, whether cmv infection or cmv reactivation contributes to mortality of immunocompetent patients remains unclear. methods: a literature search was conducted for relevant studies published before may , . studies reporting on cmv infection in immunocompetent patients in icus and containing × tables on cmv results and all-cause mortality were included. results: eighteen studies involving immunocompetent patients admitted to icus were included in the meta-analysis. the overall rate of cmv infection was % ( %ci – %, i( ) = %, n = ) and the cmv reactivation was % ( %ci – %, i( ) = %, n = ). the odds ratio (or) for all-cause mortality among patients with cmv infection, compared with those without infection, was . ( %ci . – . , i( ) = %, n = ). moreover, upon exclusion of studies in which antiviral treatment was possibly or definitely provided to some patients, the association of mortality rate with cmv infection was also statistically significant (or: . , %ci . – . , i( ) = %, n = ,). for cmv seropositive patients, the or for mortality in patients with cmv reactivation as compared with patients without cmv reactivation was . ( %ci . – . , i( ) = %, n = ). patients with cmv infection required significantly longer mechanical ventilation (mean difference (md): days ( % ci – , i( ) = %, n = )) and longer duration of icu stay (md: days ( % ci – , i( ) = %, n = )) than patients without cmv infection. when analysis was limited to detection in blood, cmv infection without antiviral drug treatment or reactivation was not significantly associated with higher mortality (or: . , %ci . – . , i( ) = %, n = ; or: . , i( ) = %, n = ). conclusion: critically ill patients without immunosuppression admitted to icus show a high rate of cmv infection. cmv infection during the natural unaltered course or reactivation in critically ill patients is associated with increased mortality, but have no effect on mortality when cmv in blood. more studies are needed to clarify the impact of cmv infection on clinical outcomes in those patients. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. human cytomegalovirus (cmv) is a prototypic member of the β herpes virus subfamily [ ] . the prevalence of cmv seropositivity in human populations is roughly - % [ ] [ ] [ ] and highest amongst older people [ ] . cytomegalovirus infection induces innate immune responses (eg. natural killer cells) and adaptive immunity (eg. cd +/cd + t cells). however, the virus can evade host detection by expressing genes that interfere with both the innate and adaptive immune systems. eventually, cmv is able to establish latency in which either the host fails to eliminate the virus or the virus cannot replicate. however, cmv can become reactivated during periods of host immune suppression [ ] . it is well known that cmv infection is common in canonical immunodeficiency patients, such as those with human immunodeficiency virus infection, solid organ or stem cell transplantation and patients undergoing chemoor radiotherapy [ ] [ ] [ ] . with the development of more sensitive detection method, the rate of cmv detection is high in intensive care units (icus) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, so far, there is no convincing research to support the use of antiviral treatment when critically ill but immunocompetent patients present with cmv infection. furthermore, whether cmv is a contributor or simply a bystander to the severity of illness remains under debate [ ] [ ] [ ] . whether cmv infection is associated with increased mortality in immunocompetent icu patients remains controversial [ ] [ ] [ ] [ ] . a previous meta-analysis published in demonstrated that cmv infection was associated with a higher mortality rate, nearly twice that observed in patients without cmv infection [ ] . however, this study did not consider the influence of antiviral drugs on clinical outcomes. moreover, many clinical studies about cmv have been reported in recent years. thus, to acquire a better understanding of the potential role of cmv infection in contributing to mortality in critically ill patients, especially those not receiving antiviral agents and cmv detected in blood, we performed a meta-analysis of data available in the literature, focusing on the outcome in immunocompetent icu patients with cmv infection. a literature search for relevant publications included within the electronic databases pubmed, embase and the cochrane library was performed using combinations of the keywords "cytomegaloviruses", "salivary gland viruses", "herpes virus", "cytomegaloviral infection", "hhv ", "intensive care", "critical care", "critical illness", "mechanical ventilation", and "pulmonary ventilator". all searches were updated on may , . no language restriction was enforced. we also consulted relevant reference articles and searched using google scholar. two researchers (lx and hyb) performed data extraction independently, and any discrepancies were addressed by discussion and reevaluation until consensus was achieved. observational studies were eligible if they reported on cmv infection in immunocompetent patients in the icu, and if a × table could be constructed based on cmv results and all-cause mortality. all patients were over years of age. the systematic review included only studies in which all patients were tested for cmv. an episode of cmv infection was defined by one of the examination cmv viral culture, polymerase chain reaction (pcr), cmv antigen (pp ) in blood, tracheal aspirates, urine, or a combination of these. a case was defined by the presence of reactivation, where the patient had cmv infection and was seropositive. immunocompetent patients were defined as those patients who did not receive a solid organ or hematopoietic stem cell transplant, did not receive immunosuppressive treatment, did not have human immunodeficiency virus infection, did not have primary immunodeficiency, and did not receive chemotherapy or radiotherapy before icu admission. we obtained information on basic study characteristics (author, year of publication, country of origin, study period, setting, and study design), characteristic population, the site and detection method of sample, cmv seropositivity, cmv infection incidence, all-cause mortality, length of icu/hospital stay, length of mechanical ventilation, and administration of antiviral drugs. the newcastle-ottawa scale, developed for evaluating the quality of observational studies (additional file : table s ) [ ] , was used to assess the validity of included studies. continuous variables are reported as mean or median values and categorical variables are reported as n (%). meta-analytic pooling was performed for outcome variables with a logit transformation approach, reporting results as summary point estimates ( % confidence interval, ci). we used the mantel-haenszel method to obtain odds ratios (ors) and % ci. when only the median, range, or interquartile range of length of mechanical ventilation and the length of icu stay were reported, we used simple formulas to estimate the mean and standard deviation [ ] . between-study heterogeneity was examined using the i measure of inconsistency and the chi-square test of heterogeneity. to evaluate publication bias, we constructed a funnel plot and used the egger test. sensitivity analyses of the begg's test were additionally conducted to ascertain the robustness of our findings. all meta-analyses were performed with r software (version . . for windows) and spss (ibm, armonk, ny, usa). the initial database search identified potentially relevant studies. following this, assessment of the full text yielded studies suitable for analysis. another publication was incorporated after examining references from the extracted articles [ ] . consequently, our meta-analysis consisted of articles (fig. ) , including one case-control [ ] and cohort studies [ - , - , ] . most studies were conducted in the united states and europe, except one cohort study in egypt [ ] , and were published between and (table ) . overall, the studies were well done, with a median score of (range - ) on the newcastle-ottawa scale for appraising the quality of observational studies. a total of patients were included, having been admitted to the icu for a variety of reasons, with a median age of years. the median period of prospective studies was months, ranging broadly from to months. all studies used cmv blood assays, and studies also assayed sputum samples. most studies indicated that the frequency of sample collection was once a week. in our analysis, the methods used to assess cmv infection were virus culture, pp antigen detection and pcr detection of cmv dna in ten, three and two studies, respectively, and combinations of two diagnostic methods in the remaining three studies. as shown in fig. , the overall detection rate of cmv was % ( % ci - %, i = %, n = ). as compared with patients without cmv infection, the all-cause mortality of patients with cmv infection was significantly higher (or: . ; % ci . - . , i = %, n = ) (fig. a) . when analysis was limited to cmv detection in blood, there was still statistical significance in mortality rate between patients with cmv infection (or: . , % ci . - . , i = %, n = ) compared with patients without infection (additional file : figure s ). to rule out the impact of antiviral drugs on patients with cmv infection, four studies in which patients received antiviral drugs during their icu stay and eight studies that did not specify the use of antiviral drugs were excluded. the remaining six studies of patients without antiviral treatment during the course of icu stay were analyzed [ , , , , , ] . the difference in mortality rates between patients with cmv infection remained significant (or: . , % ci . - . , i = %, n = ) compared with patients without infection (fig. b) . when analysis was limited to cmv detection in blood, there was no statistical significance in mortality rate between patients with cmv infection (or: . , % ci . - . , i = %, n = ) as compared with patients without infection (additional file : figure s ). the mean difference in mechanical ventilation days and duration of icu stay was an increase of days ( % ci - , i = %, n = ) and days ( % ci - , i = %, n = ), respectively, between patients with and without cmv infection ( fig. a and b) . when analysis was limited to cmv detection in blood, there was still a statistically significant difference in length of mechanical ventilation and icu stay between patients with cmv infection as compared with patients without infection (md: days ( % ci - , i = %, n = ) and md: days ( % ci - , i = %, n = )), respectively (additional file : figure s and additional file : figure s ). the cmv seropositivity rate, which represents previous infection, was % ( % ci - %, i = %, n = ) in immunocompetent icu patients (fig. a) . patients with cmv reactivation, which represents cmv detected among seropositive patients, was % ( % ci - %, i = %, n = ) (fig. b) . the or for mortality in patients with cmv reactivation as compared with patients without cmv reactivation was . ( % ci . - . , i = %, n = ) (fig. c) . but for patients of cmv infection in blood, the reactivation was not associated with higher mortality (or: . , % ci . - . , i = %, n = ) (additional file : figure s ). we also analyzed the rate of cmv and mortality thought categorized by the detection methods (additional file : figure s , additional file : figure s : additional file : figure s and additional file : figure s ). we used the egger test to detect publication bias. there was no publication bias either in the overall cmv prevalence analysis (t = . , p = . ) or in the all-cause cmv mortality analysis (t = − . , p = . ). we also used begg's test to detect sensitivity analysis, and the results showed that the analyses were robust. in this meta-analysis, we have demonstrated that cmv infection frequently present in critically ill immunocompetent patients at icu admission. the overall rate of cmv infection was %, which was higher than the % presented in a previous meta-analysis [ ] , because eight recent studies detecting cmv infection by pcr assay were included in our meta-analysis [ - , , ] . polymerase chain reaction has been demonstrated to be the most sensitive method of cmv detection [ ] , but even so, the cmv infection rate may still be underestimated because we chose only the studies containing × tables on cmv results and all-cause mortality. we excluded studies where either the rate of cmv infection or mortality was zero and we also excluded some studies with a % infection rate that used early monitoring of cmv, often fewer than days after admission to the icu [ , [ ] [ ] [ ] . we believe this could have led to underestimation of the cmv infection rate because the transition to cmv infection requires time for the complete lytic virus cycle to develop from the latent phase [ ] . we found that the detection rate of cmv by culture, pp and pcr was , and %, respectively. desachy for cmv infection were obtained in a median of days by pcr compared with days by pp antigen detection after onset of sepsis [ ] . therefore, pcr facilitates earlier diagnosis of an episode of cmv infection than any other method. we then analyzed the association between cmv positivity and mortality, stratified by detection method. we also found that patients with cmv infection detected by pcr had higher mortality than patients without cmv infection (or: . , % ci . - . , i = %, n = ). however, when compared with other methods, the association with mortality was marginally less strong using pcr. we may think that viral burden of cmv is determinant of pathogenesis, and higher cmv loads is correlated with progression of some cmv infection disease [ , ] . the presence of cmv seropositivity, representing previous infection, was found in % of immunocompetent icu patients and the incidence of cmv reactivation was high, observed in % of seropositive patients in our meta-analysis. there are several factors that might explain the high prevalence. first of all, the rate of cmv seropositivity increases with advancing age [ ] and in our analysis, the median age was years. second, to inhibit the reactivation of cmv, as many as % of all peripheral cd + and cd + t cells are constantly required for immune surveillance to maintain functional latency [ ] . sepsis is associated with immunoparalysis, as apoptosis of cd + and cd + t cells is increased [ , ] . furthermore, some patients in the icu may be immunosuppressed after trauma and major surgery [ ] . in addition, treatments commonly received in the icu, such as massive transfusion, corticosteroids, or catecholamines may transiently compromise host immunity [ ] . it has also been reported that the use of heart-lung machines can lead to temporary systemic immunosuppression [ ] . therefore, patients in the icu may show transient immunoparalysis [ ] , potentially resulting in the observed cmv reactivation. third, some inflammatory cytokines including tumor necrosis factor alpha and interleukin- β, can stimulate reactivation of latent cmv [ ] . thus, significant numbers of immunocompetent patients harboring latent virus are susceptible to cmv reactivation during critical illness. when the mortality analysis was limited to cmv detection in blood, cmv infection without antiviral drug treatment or reactivation was not significantly associated with higher mortality. this maybe explained that the presence of high peripheral levels of functional cmv-specific cd + and cd + t cells in immunocompetent patients, which can suppress cmv during episodes of reactivation [ ] . it was observed that cmv infection was not associated with mortality in cmv colitis. in steroid-refractory patients with ulcerative colitis, cmv was found in the colon by histopathology, which is also not associated with adverse clinical outcomes [ ] . indeed, there has been no research to demonstrate that immunocompetent critically patients with cmv infection could benefit from antivirus therapy. and there are a number of side effects of antiviral drugs, such as hematologic complications (neutropenia, anemia and thrombocytopenia), renal dysfunction, mental disorders [ ] . therefore, giving antiviral drugs to critically ill patients should be considered cautiously in terms of advantage-disadvantage ratio. to address this issue, there are two ongoing, blinded, randomized placebo-controlled clinical trials of an antiviral drug with activity against cmv in critically ill patients in the icu (nct , nct ). patients with sepsis have the highest incidence of cmv infection [ ] . early in 's, bacterial sepsis was considered to trigger cmv reactivation [ ] . the reactivation associated with sepsis was consequence of inflammatory stimulation, transient immune compromise, and maybe involving some component of epigenetic regulation of viral dna [ ] . there are five limitations in this study. first, we observed large heterogeneity in many of our analyses. however, little or no heterogeneity was observed in the meta-analysis of mortality outcome. second, most studies were not blind, thus reducing the reliability of the results. third, lack of sufficient data on clinical parameters (eg: severity of illness, cause of icu admission, comorbidity) meant that stratified analyses based on such clinical characteristics were not possible. fourth, the definition of the state of cmv infection was inconsistent and maybe restrictive to capture the dynamics of cmv infection. as such, we could not conduct meta-analysis with outcome data and this is a major limitation of our meta-analysis. finally, one [ ] cannot discount the effect of unmeasured confounders given the observational nature of the body of evidence comprising this meta-analysis. cytomegalovirus: pathogen, paradigm, and puzzle seroprevalence of cytomegalovirus (cmv) and risk factors for infection in adolescent males cytomegalovirus seroconversion rates and risk factors: implications for congenital cmv cytomegalovirus seroprevalence in the united states: the 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pp antigenaemia selective reactivation of human herpesvirus variant a occurs in critically ill immunocompetent hosts reactivation of human herpesvirus type in multiple organ failure syndrome evaluation by polymerase chain reaction of cytomegalovirus reactivation in intensive care patients under mechanical ventilation cytomegalovirus infection in patients with bacterial sepsis diagnostic approaches to cytomegalovirus infection in bone marrow and organ transplantation a real-time taqman pcr for routine quantitation of cytomegalovirus dna in crude leukocyte lysates from stem cell transplant patients broadly targeted human cytomegalovirus-specific cd + and cd + t cells dominate the memory compartments of exposed subjects sepsis-induced immunosuppression: from cellular dysfunctions to immunotherapy sepsis: a roadmap for future research acquired immunologic deficiencies after trauma and surgical procedures immunologic abnormalities in patients receiving multiple blood transfusions pediatric cardiac surgery with cardiopulmonary bypass: pathways contributing to transient systemic immune suppression sir isaac newton, sepsis, sirs, and cars lipopolysaccharide, tumor necrosis factor alpha, or interleukin- beta triggers reactivation of latent cytomegalovirus in immunocompetent mice cytomegalovirus in inflammatory bowel disease: pathogen or innocent bystander? treating hsv and cmv reactivations in critically ill patients who are not immunocompromised: con our findings suggests that there is a high incidence of cmv seropositivity and cmv infection in critically ill patients without immunosuppression. this study suggest that cmv infection without antiviral drug treatment or reactivation in critically ill patients is associated with increased mortality, and is not associated with mortality when cmv infection is detected in blood. further research is necessary to determine the full role of cmv in this vulnerable patient demographic. additional file : table s . the newcastle-ottawa scale (pdf kb) additional file : figure s availability of data and materials all data generated or analyzed during this study are included in this published article.authors' contributions lx conducted the literature search, extracted data, performed statistical analysis, and drafted the manuscript. hyb conducted the search, extracted the data, and revised the manuscript. xzh performed the statistical analysis and edited the manuscript. zr conducted the literature search and extracted data. lxq interpreted the data. mp designed the study, interpreted data, and revised the manuscript. lym conceived and designed the study and revised the manuscript. all authors have read and approved the final manuscript. key: cord- -hl ysaxj authors: samy, ahmed; naguib, mahmoud m. title: avian respiratory coinfection and impact on avian influenza pathogenicity in domestic poultry: field and experimental findings date: - - journal: vet sci doi: . /vetsci sha: doc_id: cord_uid: hl ysaxj the avian respiratory system hosts a wide range of commensal and potential pathogenic bacteria and/or viruses that interact with each other. such interactions could be either synergistic or antagonistic, which subsequently determines the severity of the disease complex. the intensive rearing methods of poultry are responsible for the marked increase in avian respiratory diseases worldwide. the interaction between avian influenza with other pathogens can guarantee the continuous existence of other avian pathogens, which represents a global concern. a better understanding of the impact of the interaction between avian influenza virus and other avian respiratory pathogens provides a better insight into the respiratory disease complex in poultry and can lead to improved intervention strategies aimed at controlling virus spread. avian influenza viruses (aivs), caused by influenza a viruses, are members of the orthomyxoviridae family [ ] . aivs infect both domestic poultry and wild birds; in addition, many reports have described their natural transmission to humans and occasionally to other mammals [ , ] . aivs are further classified, based on the presence of multiple basic amino acids at the cleavage site of their hemagglutinin (ha) protein and/or their virulence in chickens, as low pathogenic avian influenza (lpai) or high pathogenic avian influenza (hpai) viruses [ ] . lpaivs continue to spread worldwide and have been isolated frequently from apparently normal migrating waterfowl, shorebirds, and domestic poultry [ ] . lpaiv has become endemic in domestic poultry in different countries in asia and the middle-east, causing subclinical infections, mild respiratory symptoms, and/or drops in egg production [ ] . since , hpaiv h n of the eurasian lineage has spread worldwide in a very short time, causing continuous emerging threats mainly to the middle east and asian countries [ , ] . recently, frequent reassortments of hpaiv h have been noted, with other co-circulating aivs in different countries in europe, north america, east asia, and the middle east [ ] [ ] [ ] . in poultry, hpaiv induces a very high mortality rate and a sharp decline in productivity both under field conditions and experimental trials using specific pathogen-free (spf) chickens. in contrast, lpaiv induces minimal or no clinical signs in controlled spf challenge experiments; however, field observations associated with lpaiv h n infections showed slight mortality rates and a loss in egg productivity. one of the most important reasons for this difference is the mixed infection with other the vast majority of aivs are lp, causing mild respiratory signs or drops in egg production, while some birds display no clinical signs at all. hpaiv is a contagious infectious disease of poultry, producing necrosis, hemorrhage, and inflammation in multiple visceral organs, as well as in the brain and the skin [ ] . co-infections with both hpaiv and lpaiv were reported in many natural cases in different countries, such as co-infection of lpaiv h n with hpaiv h n in china, israel, bangladesh, and egypt [ ] . reassortment can emerge when two different aiv subtypes co-infect the same cell at the same time. several reassortment strains of aiv subtypes have been reported in different countries in europe, north america, east asia, and the middle east [ ] [ ] [ ] . in addition, experimental studies revealed that lpaiv h n a/chicken/hk/g / plays a role in protecting chickens from a lethal challenge of a hpaiv h n infection [ ] . however, infected chickens shed hpaiv h n in their feces (higher levels) and trachea (lower level), while the signs of the co-infected chickens were usually mild and included sneezing, nasal discharge, and ruffled feathers. it was suggested that chickens were protected against hpaiv h n through the cross-reactive cellular immunity produced by lpaiv h n influenza viruses. another study, described by khalenkov, et al. [ ] , reported that the chickens, previously inoculated with one of the israeli lpaiv h n genotype g , could show up to % survival after inoculation with the lethal h n virus. both findings suggest that co-circulation of h n with h n can contribute to the perpetuation of lethal hpaiv h n . in addition, a recent study revealed that previous infection, not vaccination with lpaiv, modulates the course of subsequent infection with the egyptian hpaiv h n [ ] . infectious bronchitis (ib) is an acute rapidly spreading disease of chickens characterized by respiratory signs, drop in egg production, and poor egg quality or nephritis/nephrosis [ , ] . numerous distinct genotypes have been reported in many countries worldwide [ ] . ibv is considered one of the most economically important respiratory viral diseases, and it threatens the poultry industry worldwide, along with the hpaiv and velogenic newcastle disease virus (vvndv) [ ] . ibv was described as being associated with both hpaiv and/or lpaiv as a natural infection in different countries in asia and the middle-east [ , ] . ibv initially infects the epithelial surface of the respiratory tract of chickens, then spreads via viremia in a range of other tissues for further replication (such as kidney, gastrointestinal tract, and oviduct) [ , ] . a trypsin-like serine protease domain, encoded by the open reading frame orf a of the ibv, has been reported; ibv infection may provide this enzyme and enhanced lpaiv h n pathogenicity in chickens [ ] . the literature on coinfections of ai virus with ib virus is sparse on co-infection with ib vaccine or ib field stain / . the ibv vaccine is used extensively in chicken farms in many countries worldwide where both ibv and lpaiv h n are endemic [ , ] . an experimental study with lpaiv h n (a/chicken/iran/sh- / (h n )) and an ibv live vaccine (h strain) was conducted concerning clinical signs, gross lesions, viral shedding, and mortality. it was reported that the interference between lpaiv h n and the ibv live vaccine increased the severity of lpaiv h n clinical signs (depression, ruffled feathers, respiratory distress (coughing, sneezing, and dyspnea), swelling of the periorbital tissues and sinuses, conjunctivitis, and nasal and ocular discharge), as well as the severity of gross lesions (such as tracheal congestion, lung hyperemia and exudation of the trachea with tubular cast formation in the tracheal bifurcation). in addition, this led to a prolonged shedding period of lpaiv h n that extended from day to day compared to day to day in chickens infected only with lpaiv h n ; it even caused death in the infected birds [ ] . to rule out the effect of the co-infection of lpaiv h n and ibv field strain, an experimental study was performed in chickens by seifi, et al. [ ] . chickens, co-infected simultaneously with lpaiv h n (a/chicken/iran/sh / ) and ibv (ibv/ / ), reported severe clinical signs (respiratory distress, facial edema, conjunctivitis, depression, lacrimation, ruffled feathers, whitish watery diarrhea, and nasal discharge which continued until eight days post-infection), gross lesions (tracheal congestion, air saculitis, lung hyperemia, tubular cast formation in the tracheal bifurcation which extended to the lower bronchi, swollen kidney, and hemorrhagic pancreas and intestine), and mortality rate ( %), which were significantly different when compared with chickens infected separately with the same virus. in addition, a significantly higher hi titer against aiv infection was noticed in the co-infected group, which may influence the diagnosis process of this virus in the field [ ] . these findings showed that ibv co-infection enhanced lpaiv h n pathogenicity through protease enzymes. the newcastle disease virus (ndv) is a member of the genus avulavirus in the paramyxoviridae family [ ] . ndv possesses different pathotypes based on the sequences of the protease cleavage site of the fusion (f) protein and virulence in chicken [ ] . the original classification of ndv was based on its virulence, either as low virulence (lentogenic), moderately virulent (mesogenic), or virulent (velogenic) [ ] . these viruses transmit from their natural reservoirs (wild birds) to domestic birds, initially producing subclinical infections, and occasionally causing upper respiratory disease and a decrease in egg production. ndv and aiv are two of the most economically important viruses that affect poultry sectors worldwide [ ] . frequent co-infections/interferences were reported in many areas of the world, especially where both viruses are endemic in many forms, like (a) lentogenic ndv with either lpaiv or hpaiv and (b) velogenic ndv with either lpaiv or hpaiv [ ] . ndv and aiv can replicate in the upper respiratory and intestinal epithelial cells by binding to the sialic acid-containing receptors on the cell surface (through the hn or ha protein of ndv or ai respectively) [ ] . virus replication may also be affected by the previous replication of another virus in the same site through the active antiviral immune responses including immunomodulators, induced interferon, or recruitment of immune cells [ ] . the impact of such co-infections on several host responses including clinical signs, viral shedding, and gross lesion was addressed in various studies. a co-infection study on lpaiv subtype h n (a/chicken/pakistan/udl/ ) and lndv vaccine strains (lasota) in chicken showed a significant reduction in virus replication. lower virus sheddings were observed in the first days post infection as compared to singly inoculated chickens. furthermore, significantly higher titer for lpaiv and lower for lndv was detected in oropharyngeal and cloacal swabs, and no clinical signs were observed [ ] . another co-infection study on evolved lpaiv h n (a/turkey/va/sep/ / ) and lndv (lasota) was performed in chicken. no clinical signs were observed in co-infected chickens, and the same shedding pattern was observed as in the previous study [ ] . natural co-infections with both hpai and vndv strains were reported in many countries, although both diseases are clinically indistinguishable [ ] . an experimental co-infection model reported that the virulent ndv (velogenic ca/ ) interferes with the replication of hpaiv (a/chicken/queretaro/ - / (h n )). reduction in the number of birds shedding hpaiv was observed. however, the death of all birds was recorded within . to . days with no difference in the clinical signs between the single infected or co-infected group in high dose of vndv. however, it has been noted that a low dose of vndv increased the survival of the co-infected chickens [ ] . conversely, chickens infected with the less virulent mesogenic ndv prior to hpaiv (with a lower dose) revealed reduced hpaiv replication and increased survival rates. in conclusion, viral interference depends on viruses' titer, virulence, and time of infection [ ] . co-infection and interference between aiv and vndv was also demonstrated in ducks [ ] . no clinical signs were observed in infected or co-infected ducks with vndv (apmv- /duck/vietnam (long bien)/ / ) and lpaiv (a/mallard/oh/ / h n ). however, co-infection decreased the number of birds shedding vndv, while it did not affect the number of ducks shedding lpaiv (except at dpi low virus shedding was observed in the co-infected group than the group infected only with lpai). ducks simultaneously infected with vndv and hpai (a/duck/vn/ncvd- / (h n )) survived fewer days compared to those that received the vndv two days prior to the hpaiv infection. moreover, reduced transmission of vndv to naïve contact ducks was reported. this confirms that infection with one virus can interfere with the replication of another virus, affecting its pathogenesis and transmission [ ] . an experimental study was carried out to investigate the effect of coinfection with lndv (mallard/us(mn)/ai - / ) and lpaiv (a/mallard/mn/ / (h n )), in which one-monthold mallards were inoculated with lndv and lpaiv simultaneously on the same day or sequentially two or days apart. co-infection revealed less productive lndv shedding and higher lpaiv shedding in cloaca swabs. minimal effects were observed with lndv and lpaiv co-infection, which might reflect the adaptation of both viruses in common waterfowl, and this could stabilize virus replication and transmission in wild ducks [ ] . staphylococcus is a gram-positive bacterium that affects a wide range of avian species and widely spreads in poultry rearing environments, and presents itself as a part of normal flora of mucous membranes. staphylococcus has several pathogenicity markers, including a clumping factor that correlates with clinical cases of staphylococcus in avian species. protein a, present on the bacterial cell surface, can bind to the fragment crystallizable region (fc) fragment of immunoglobulin and subsequently inhibit the phagocytosis of the bacteria. regarding the coinfection with aivs, staphylococcus sp. produces soluble proteases that are able to activate the ha of aivs [ ] . in the same context, with in vitro treatment of aivs with staphylococcus aureus proteases, the infectivity of most strains is enhanced by at least fold [ ] . furthermore, indirect activation of the ha could occur by staphylokinase that could activate chicken plasminogen into plasmin [ ] . experimentally, pre-infection of chickens with s. aureus, three days before infection with lpaiv h n (a/chicken/aq-y- / and a/chicken/beijing/ / ), leads to severe clinical signs. viruses were recovered from the blood of all co-infected chickens with extensive replication in respiratory tissue, which may explain the dissemination of the virus through the chicken body and the observed severe clinical signs. in contrast, chickens infected with same lpaiv h n strains alone showed no clinical signs, no virus was recovered from their blood, and there was lower replication efficiency in respiratory tissues [ ] . under field conditions, staphylococcus sp. are commonly recovered from chicken's respiratory tract tissues with respiratory clinical symptoms, with a higher prevalence among other causative agents ( . % as reported by türkyilmaz [ ] and . % as reported by popy, et al. [ ] ). ornithobacterium rhinotracheale (ort) is gram negative, non-motile, rod shaped, non-sporulating bacteria. ort is reported in many countries worldwide as being associated with respiratory signs and is isolated from a wide variety of hosts such as pheasant, pigeon, rook, duck, ostrich, goose, guinea fowl, turkey, chicken, red-legged partridge, and falcon; in particular, in chickens and turkeys it causes airsacculitis and pneumonia [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the pathogenicity of the ort depends on the route of inoculation, virulence of the strain, environmental factors, the immune status of the host, and the presence of the concurrent infection [ ] . ort spreads horizontally through aerosols and drinking water with longer survival rates at lower temperature, which also explains its dissemination during winter months and concurrent infection with other respiratory diseases common during winter season [ ] . in china, % of serum collected from birds with respiratory manifestation were seropositive for ort, and % of serum collected from apparent healthy birds were seropositive for ort; five of six ort strains recovered were associated with lpaiv h n infection [ ] . furthermore, experimental preinfection with ort and secondary infection with lpaiv h n (a/chicken/shandong/ (h n )) three days later induced the highest mortality rate with development of severe pneumonia and airsacculitis. on the other hand, a lower mortality was induced by coinfection and pre-infection with lpaiv h n than in association with a secondary infection of ort [ ] . lower mortality rate was also recorded by azizpour, et al. [ ] with coinfection of ort and lpaiv h n but with a different route of inoculation. altogether, ort as a preinfection, concurrent infection, or secondary infection is able to exacerbate the virulence of lpaiv h n as compared to infection with lpaiv h n alone. however, pre-infection with ort and secondary infection with h n induces a higher mortality rate with unique histopathological lesions represented by severe pulmonary fibrosis [ ] . mycoplasmas are prokaryote that are characterized by its very small size, absence of cell wall, small genome, and being surrounded by plasma membrane. almost mycoplasma species have been isolated from various species of birds [ ] . some mycoplasma species are able to penetrate the host cells as mycoplasma gallisepticum (mg) and mycoplasma synoviae (ms), and these two species are also able to hemagglutinate chickens and turkeys' rbcs [ ] . mg and ms can display little to no clinical signs in avian species unless they are complicated by other respiratory pathogens [ ] . stipkovits et al. [ ] demonstrated that chickens pre-infected with mg aerosol one week before challenge with lpaiv h n (a/mallard/hungary/ / ) exhibit clinical signs and pathological lesions along the entire respiratory system (tracheitis, bronchitis, airsacculitis, and pneumonia), as well as a significant reduction in body weight gain comparing to mg alone, while lpaiv h n infection did not induce clinical signs or result in a reduction in body weight gain. furthermore, pre-infection with mg accompanied with post challenge of a lpaiv h n revealed a decrease in the anti-mg antibodies level as compared to the chicken group infected with mg only [ ] . additionally, the host pathogen-pathogen interaction of mg and another lpaiv candidate like h n (a/chicken/saudi arabia/cp / ) was also investigated using tracheal organ cultures (toc) model as described by sid et al., [ ] . results revealed that mg can modify the pathogenesis of lpaiv h n depending on the interval between the two infections. the longer time of incubation with mg before the lpaiv h n challenge enhanced ciliostasis and significant down regulated the antiviral innate immune response that subsequently enhanced the effect of h n . in contrast, incubation with mg for h followed by a lpaiv h n infection promotes both viral and bacterial replications, while longer incubation promotes only bacterial growth and viral replication, which are significantly decreased as compared to lpaiv h n infection alone. this could be attributed to mg-inducing destruction of cilia, hyperplesia of epithelial cells, and desialytion of the tracheal epithelial cells. these findings could explain the ability of subclinical mg infection in birds, under field conditions, to develop severe respiratory signs with other concurrent live respiratory vaccines. these infections could be attributed to the immune modulation mediated by mg [ ] . a high prevalence of lpaiv and mg and/or ms coinfection has been frequently reported in intensified poultry production areas with respiratory manifestation [ , ] that increases the severity of the clinical signs with fibro necrotic cast in the tracheal bifurcation [ ] . escherichia coli (e. coli) is a gram-negative, non-spore-forming, rod-shaped bacteria that grows both aerobically and anaerobically, and many strains are motile and have peritrichous flagella. e. coli is a ubiquitous micro-organism that is wildly spread in poultry environments and is a normal inhabitant in poultry microflora. some e. coli strains such as avian pathogenic e. coli (apec) cause collibacillosis that could be either systemic or localized. it is widely accepted that collibacillosis is the most common infectious bacterial diseases of poultry of all ages [ ] . pre-infection of chickens with lpaiv h n and secondary inoculation with e. coli four days later was reported to induce significantly higher aiv antibodies weeks post-infection (wpi) compared to secondary infection with ib or ort. furthermore, in the pre-infected group, a prolonged virus shedding up to dpi was observed as compared to only dpi in the group inoculated with h n alone [ ] . chickens pre-infected with lpaiv h n before being inoculated intrathoracically with . × cfu/ bird of e. coli (bekaa valley of the lebanon (bvl-strain) three days later showed significant early mortality with more predominant clinical signs (conjunctivitis, diarrhea, ocular exudates, and rales) and gross lesions (abdominal airsacculitis, left thoracic airsacculitis, pericarditis, right thoracic airsacculitis and tracheitis) compared to groups that received lower e. coli count in the challenge [ ] . the articles discussed in this review recapitulate the adverse impacts of co/secondary viral and/or bacterial infections on aivs infection in poultry, as well as the synergy between different pathogens (table ) . moreover, this review provides important insights into the variation in the rates of severe morbidity and/or mortality that subsequently occur in the case of co-infection or pre-infection with another bacterial or viral pathogen. coinfection with aivs and a bacterial pathogen can exacerbate the course of the viral or bacterial disease. it has also been documented that ai-related bacterial and viral infections overall may account for up to a remarkable percent of reported cases under field conditions in different countries. in developing countries, where less biosafety measures are applied, this percentage is much higher, leading to severe economic losses in the poultry industry. this could also blur syndromic surveillance. we recommend the diagnosis of more pathogens during the inspection of an infected poultry flock that could be varied due to co-infection or pre-infection history. moreover, the application of good biosafety and biosecurity measures is likely to reduce the severity of co-infection, and can restrict the widespread transmission of those bacterial and viral pathogens. in conclusion, this review may hopefully contribute to future knowledge regarding the diagnosis and control of avian disease among different poultry sectors. table . summary of the impact of co-infection/interference of different bacterial and viral pathogens on avian influenza viruses. strain name impact reference a/chicken/hk/g / had a role in protecting chickens from lethal challenge of hpaiv h n infection with mild clinical sign include sneezing, nasal discharge, and ruffled feather with higher shedding in fecal material comparied to tracheal. [ ] israeli lpaiv h n genotype g some israeli strains showed up to % survival after inoculation with lethal h n virus while others not. [ ] egyptian lpaiv h n the egyptian lpai h n virus infection can modulate the course of subsequent infection with hpaiv h n . [ ] ib h increase the severity of lpaiv h n (a/chicken/iran/sh- / ) clinical signs that include depression, ruffled feathers, respiratory distress, swelling of the periorbital tissues and sinuses, and conjunctivitis, and nasal and ocular discharge), as well as gross lesions (as tracheal congestion, lung hyperemia and exudation of the trachea with tubular cast formation in the tracheal bifurcation). in addition, this led to a prolonged shedding period of lpaiv h n . it even can cause death of the infected birds. [ ] ibv/ / chickens, co-infected simultaneously with lpaiv h n (a/chicken/iran/sh / ) showed severe clinical signs (respiratory distress, facial edema, conjunctivitis, depression, lacrimation, ruffled feathers, whitish watery diarrhea, and nasal discharge), gross lesions (tracheal congestion, air saculitis, lung hyperemia, tubular cast formation in the tracheal bifurcation which extended to the lower bronchi, swollen kidney, and hemorrhagic pancreas and intestine), and mortality rate ( %) with significant higher hi titer. [ ] ndv lndv (lasota) co-infection with lpaiv-h n (a/chicken/pakistan/udl/ ) in chickens showed a significant reduction in virus replication and virus shedding compared to singly inoculated chickens. further significantly higher titer for lpaiv and lower for lndv was detected in oropharangeal and cloacal swabs, and no clinical signs were observed. co-infection with lpaiv (a/mallard/mn/ / (h n )) revealed less productive lndv shedding and higher lpaiv shedding and in the cloaca swabs minimal effects were observed with lndv and lpaiv co-infections. [ ] staphylococcus sp. experimentally, pre-infection of chickens with s. aureus, days before infection with lpaiv h n (a/chicken/aq-y- / and a/chicken/beijing/ / ), lead to severe clinical signs. viruses recovered from blood of all co-infected chickens with extensive replication in the respiratory tissue. [ ] ornithobacteriumrhinotracheale (ort) ort/chicken/ shandong/ experimental preinfection with ort and days later secondary infection with lpaiv h n (h n /chicken/shandong/ ) induced the highest mortality rate with development of severe pneumonia and airsacculitis with unique histopathological lesions represented by severe pulmonary fibrosis. on the other hand, a lower mortality rate induced by coinfection and pre-infection with lpaiv h n then secondary infection with ort. [ ] avian mycoplasmosis chickens pre-infected with mg aerosol one week before challenge with lpaiv h n (a/mallard/hungary/ / ) exhibit clinical signs, pathological lesions along the entire respiratory system (tracheitis, bronchitis, airsacculitis and pneumonia) as well as reduction in body weight gain significantly comparing to single infection with decrease in the anti-mg antibodies level comparing to group infected with mg only. [ , ] mg s lab. strain mg modifies the pathogenesis of lpaiv h n (a/chicken/saudi arabia/cp / ) using tracheal organ cultures (toc) model depending on the interval between the two infections.the longer time incubation with mg before lpaiv h n challenge the more enhancement of ciliostasis and significant down regulation of antiviral innate immune response that subsequently enhances the effect of h n . while longer incubation promote only bacterial growth while viral replication significantly decreased. [ ] pre-infection of chickens with lpaiv h n (a/chicken/pakistan/ / ) and days later secondary inoculated with e. coli induce higher aiv antibodies at wpi comparing to secondary infection with ib or ort. furthermore, in the pre-infected group, a prolonged virus shedding up to dpi compared to only dpi in the group inoculated with h n alone was recorded. [ ] bekaa valley of the lebanon (bvl-strain) chickens pre-infected with lpaiv h n then days later inoculated with . × cfu/ birds of e. coli intrathoracic showed significant early mortality with more predominant clinical signs (conjunctivitis, diarrhea, ocular exudates and rales) and gross lesion (abdominal airsacculitis, left thoracic airsacculitis, pericarditis, right thoracic airsacculitis and tracheitis) comparing to groups that received lower e. coli count used in the challenge [ ] avian influenza: a review highly pathogenic) avian influenza as a zoonotic agent transmission of influenza a/h n viruses in mammals diseases of poultry is low pathogenic avian influenza virus virulent for wild waterbirds? control of low pathogenicity avian influenza avian influenza virus (h n ): a threat to human health molecular epidemiology of h n avian influenza highly pathogenic avian influenza viruses and generation of novel reassortants molecular characterization of h n influenza viruses: were they 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standardisation of a new model of h n /escherichia coli challenge in broilers in the lebanon we acknowledge tomas edgren for his fruitful discussion and helpful comments on this written work.author contributions: ahmed samy and mahmoud naguib collected the data and wrote the paper. the authors declare no conflict of interest. key: cord- - rtk ppu authors: pickett, julie e.; thompson, john m.; sadowska, agnieszka; tkaczyk, christine; sellman, bret r.; minola, andrea; corti, davide; lanzavecchia, antonio; miller, lloyd s.; thorek, daniel lj title: molecularly specific detection of bacterial lipoteichoic acid for diagnosis of prosthetic joint infection of the bone date: - - journal: bone res doi: . /s - - -y sha: doc_id: cord_uid: rtk ppu discriminating sterile inflammation from infection, especially in cases of aseptic loosening versus an actual prosthetic joint infection, is challenging and has significant treatment implications. our goal was to evaluate a novel human monoclonal antibody (mab) probe directed against the gram-positive bacterial surface molecule lipoteichoic acid (lta). specificity and affinity were assessed in vitro. we then radiolabeled the anti-lta mab and evaluated its effectiveness as a diagnostic imaging tool for detecting infection via immunopet imaging in an in vivo mouse model of prosthetic joint infection (pji). in vitro and ex vivo binding of the anti-lta mab to pathogenic bacteria was measured with octet, elisa, and flow cytometry. the in vivo pji mouse model was assessed using traditional imaging modalities, including positron emission tomography (pet) with [( )f]fdg and [( )f]naf as well as x-ray computed tomography (ct), before being evaluated with the zirconium- -labeled antibody specific for lta ([( )zr]sac ). the anti-lta mab exhibited specific binding in vitro to lta-expressing bacteria. results from imaging showed that our model could reliably simulate infection at the surgical site by bioluminescent imaging, conventional pet tracer imaging, and bone morphological changes by ct. one day following injection of both the radiolabeled anti-lta and isotype control antibodies, the anti-lta antibody demonstrated significantly greater (p < . ) uptake at s. aureus-infected prosthesis sites over either the same antibody at sterile prosthesis sites or of control non-specific antibody at infected prosthesis sites. taken together, the radiolabeled anti-lta mab, [( )zr]sac , may serve as a valuable diagnostic molecular imaging probe to help distinguish between sterile inflammation and infection in the setting of pji. future studies are needed to determine whether these findings will translate to human pji. total joint arthroplasty is a common orthopedic surgical procedure with not only significant benefits but also challenging complications, especially related to infection. prosthetic joint infection (pji), which is an infection involving the implant and adjacent bone and joint tissues, affects up to % of all primary arthroplasties, resulting in an estimated cases and costs exceeding $ . billion. the current standard of care therapy for chronic pji of the hip and knee involves complete removal of all infected tissue and implants with eventual hardware replacement after prolonged antibiotic therapy. successful treatment occurs in ( - )% of cases, but failure often requires further treatments, surgeries, or even amputation. , as the total number of total hip and total knee arthroplasties in the united states is projected to exceed million by the year , these complications will only become more common. differentiating pji from sterile inflammation in the postoperative period remains a challenge for contemporary diagnostic techniques, such as commonly used imaging modalities, laboratory tests, and bacterial cultures. noninvasive imaging modalities are attractive options, as they offer the potential for diagnosing, monitoring, and assessing treatment longitudinally if sufficient sensitivity and resolution can be achieved. x-ray and magnetic resonance imaging (mri) reveal gross morphological changes but unfortunately produce implant-related artifacts (especially involving metallic implants) that obscure relevant changes in the surrounding tissue. positron emission tomography (pet) generates threedimensional ( d) tracer concentration images with the potential for excellent sensitivity and without implant artifacts suffered by computed tomography (ct) and mri. the sensitivity of this modality has the ability to detect infectious burden and molecular changes that occur prior to macroscopic anatomical alterations. widely used conventional tracers, including [ f]fdg ( -fluoro- deoxy-d-glucose), [ f]naf, and [ ga]citrate, have been investigated for pet localization and evaluation of pji with variable success. - however, current imaging approaches assess indirect markers of infection-methods that are often hindered by low specificity and/or sensitivity-rather than directly targeting the infectious agent. an alternative approach to improve diagnosis of pji may be to use targeted probes, such as antibodies or peptides. radiolabeled antibodies for immunopet have long been used for investigational and clinical studies for oncological applications. prior infection-related investigations have focused on antibodies to human targets such as white blood cells or other indirect markers of infection. , in this study, we assessed an antibody directly targeting gram-positive bacteria such as staphylococcus aureus and staphylococcus epidermidis (the bacteria commonly responsible for pji). the antibody is directed against lipoteichoic acid (lta), a surface-associated component of the cell wall of gram-positive bacteria. expressed by the bacteria at high levels, this antigen is a promising target for directed imaging and therapeutic agents. by directly targeting the infectious agent, we demonstrate improved diagnostic capabilities of noninvasive imaging, which may guide improved patient management. in vivo model of a s. aureus pji previous work with other bioluminescent strains has demonstrated that bioluminescent signals closely approximate and correlate with the bacterial burden, as measured by ex vivo colony-forming unit (cfu) counting. [ ] [ ] [ ] [ ] [ ] in vivo bioluminescence imaging (bli) of mice demonstrated that the average signal from the s. aureus-infected postoperative legs ( . ± . ) × p/s/cm / sr (n = ) was greater than for mice that had surgery performed without bacterial inoculation ( . ± . ) × p/s/cm /sr (n = , p < . ) (fig. a, b) . the infection remained locally at the original site of inoculation as bioluminescent signals were not detected above background at other regions of the body. in addition, this model of pji resulted in expansion and damage of the distal femur as the infection progressed, which was confirmed by ct and high-resolution x-ray imaging (fig. c) . the anatomical changes in the distal femur of the s. aureus-infected postoperative legs can be seen clearly compared with the contralateral, non-operative leg. [ f]fdg imaging [ f]fdg is a glucose analog that is taken up by cells with high glucose avidity, including tumors, inflammation, and bacteria. volumes of interest were defined manually around areas of focal [ f]fdg uptake in the distal femurs of both the postoperative and contralateral, non-operative legs of the mice after saline injection or s. aureus infection. to control for variability in mouse size, data are presented as averages of the fold increase of the postoperative leg measurement over the contralateral, non-operative leg. in the s. aureus-infected postoperative legs, there was a large mean increase ( . ± . , n = ) compared with a doubling ( . ± . , n = ) for the sterile postoperative legs (fig. ) . we see a significant uptake of in s. aureus-infected surgical postoperative legs compared to contralateral non-operative legs (p < . ), suggesting that the probe specifically localized to infected surgical sites. however, the glucose avidity of metabolically active cells, such as immune cells at sites of inflammation following sterile implantation surgery, complicate diagnosis of infection. fig. in vivo bioluminescent imaging in a mouse model of pji. a titanium kirschner-wire was surgically placed in the mouse femur with or without an inoculum of a bioluminescent s. aureus strain in the knee joint prior to closure. a representative in vivo bioluminescent signals at the s. aureus-infected postoperative site. b maximum radiance values for individuals (mean ± s.e.m.) with sterile control implants or s. aureusinfected postoperative distal femur sites at a representative time point ( ± vs ± , respectively). c ct (top) and x-ray (bottom) imaging demonstrating the location of the kirschner-wire (false-colored pink in the ct image) in the distal femur. *p < . bone scans with [ f]naf [ f]naf is incorporated within the bone matrix at sites of increased bone turnover such as that with natural growth at the physis as well as at sites of reactive bone changes that occur in response to infection. [ ] [ ] [ ] [ ] [ f]naf uptake was evaluated in this pji model, and greater [ f]naf uptake was observed in the s. aureus-infected postoperative legs ( . ± . fold, n = ) than in the sterile postoperative legs ( . ± . fold, n = , p < . ) (fig. a, b) . of note, the fold increase observed in the sterile postoperative leg compared with the contralateral non-operative leg was > . , which was likely due to modest bone turnover from the naturally occurring inflammatory response from the implant alone. ct imaging femoral volumes were defined from ct scan data. to control for variability in the size of the femurs between the individual mice, data are presented as averages of the fold increase of the measurement of the postoperative femur over the contralateral, non-operative femur. the femur volumes of s. aureus-infected postoperative legs had a doubling of bone volume for the entire femur ( . ± . , n = ) compared with sterile, postoperative legs, which had a slight increase over their non-operative counterpart ( . ± . , n = , p < . ), the large size and structural differences of which can be seen in representative scans ( fig. a-e) . taken together, ct data analysis showed that the s. aureus-infected operative legs had a marked expansion of the femur volume compared with sterile postoperative legs, which is consistent with our previous observations. , specificity of the anti-lta monoclonal antibody (mab) in order to specifically measure the infectious entity itself, we developed an immunopet agent against lta, a cell-surface component of gram-positive bacteria. an anti-lta mab (sac ) was identified from memory b cells isolated from a patient recovering from an s. aureus skin infection. this human immunoglobulin g (igg ) was evaluated for binding specificity and selectivity for lta-positive strains of pathogenic bacteria with clinical significance. affinity measurements by octet of sac binding to immobilized lta showed strong binding for the target (fig. a) . sac was also found by whole-cell enzyme-linked immunosorbent assay (elisa) to bind specifically to lta-positive bacteria, including s. aureus and s. epidermidis, but not to gramnegative bacteria (specifically escherichia coli and pseudomonas aeruginosa), whereas the control igg did not bind to any of the lta-negative bacterial species tested ( fig. b) sac also bound to in vitro grown s. aureus and s. epidermidis as measured by flow cytometry in the presence of human sera (fig. c ), thus indicating that the binding epitope for this anti-lta mab is not obscured by the presence of human sera. similarly, this anti-lta mab retained binding to the bacteria ex vivo, indicating that lta is expressed, and its epitope is accessible for antibody binding on bacteria harvested from infected mice (fig. d) . these results indicate that sac is a valid candidate to specifically detect lta-expressing bacteria in an infected animal. radiolabeling and immunopet imaging of sac anti-lta and isotype control igg were covalently conjugated to deferoxamine (dfo) via an isothiocyanate-amine reaction covalent linkage and subsequently radiolabeled with zirconium- . post-purification radiotlc confirmed purity by showing the > % radiopurity of the labeled constructs (fig. ). immunopet imaging of infected implant mice with [ zr]sac and control [ zr]igg, as well as control sterile implant mice was conducted over h. on day post injection, animals receiving the anti-lta antibody had a greater signal uptake ratio than infected implant mice receiving control igg or sterile implant mice receiving the anti-lta antibody (fig. a ). this was a surprising finding as the pharmacokinetics of full igg, which may circulate for days or weeks, are such that the antibody typically accumulates over days as background signal reduces. as shown in the representative animals in fig. b , early time points enabled clear distinction of the infected site using the specific antibody over the non-specific control or in the sterile infection model (p < . up to day ). the specific contrast decreased over the days of the experiment as nonspecific zirconium- uptake in the skeleton increased. a correlation analysis between of the uptake ratio of [ zr]labeled antibody and [ f]fdg and [ f]naf was performed, in addition to the suvmean values of [ zr]-labeled antibody and bli in the leg that had undergone surgical implantation of the pin. there was no significant correlation between the different pet imaging tracers [with r values (and two-tailed p-values) of . ( . ) and . ( . ), respectively]. a weak but statistically significant correlation between the standardized uptake values (suvmean) and the bioluminescent signal yielded an r value of . (p = . ). these correlation results indicate specificity of the antibody-targeted approach with bacterial luminescence, while there is a lack of direct association between radiopurity. radiopurity of the immunopet conjugates were determined by radio-instant thin layer chromatography.after purification, the activity for both sac and r conjugates is retained at the origin, with no evidence of free zirconium migrating up the plate, indicating that injected material does not contain free zirconium- . radiolabeled probes. while further work is required, this data indicate that a bacterial-specific tracer such as [ zr]sac may have considerable value in evaluating preclinical models and could assist in detection of pji in the clinical setting. diagnosis of pji in the setting of background inflammation is complex. commonly used x-ray, ct, and mri may not reliably distinguish sterile joint loosening from infection. development of a bacteria-specific imaging probe would allow for differentiation of infectious processes from sterile inflammation. the sensitivity of pet imaging would be particularly valuable in settings of low bacterial burden. here we evaluated a human mab directed against lta, a component of the cell wall of gram-positive bacteria (implicated in > % of pji). the high-affinity mab, sac , exhibited specific binding to gram-positive s. aureus and s. epidermidis not only after in vitro growth (fig. b, c) but also after in vivo passage in mice (fig. d) . with the intent to specifically and noninvasively image the bacterial pathogen directly and to facilitate improved sensitivity of detection, we have developed [ zr]sac . this immunopet agent provided statistically significant distinction of both the presence of infection over sterile implant sites as well as targeted over non-specific antibody. typically, immunopet imaging has been conducted several days after administration as background blood pool clearance of the antibody is required to distinguish the site of interest. consequently, we utilized the relatively long-lived isotope zirconium- . however, we observed that the signal ratio of [ zr] anti-lta mab at the s. aureus-infected postoperative legs vs contralateral, non-operative legs was greatest at day after administration. we speculate that this may be in part due to the improved immune system recognition of the bacteria after antibody binding, leading to the clearance of both the bacteria and the tracer from the site. shortening the time between injection and imaging would improve the logistics of utilizing this technology in patients and may allow for the use of shorter-lived isotopes, resulting in reduced radiation exposure. sac was compared to conventional pet tracers and ct imaging. [ f]fdg has previously been explored as a non-specific probe for evaluating pji with varying success. pet provides insight into the metabolism of host tissue, as cells that utilize radioglucose trap the tracer locally. pet provides millimeter-scale tomographic images of tracer distribution throughout the whole body, while ct provides higher-resolution attenuation maps of use for anatomical localization, abnormal tissue findings, and to identify sites of bone degeneration. we have shown that fdg uptake in the surgical distal femur of infected mice was much greater than that of the contralateral, nonsurgical femur and also correlated with the bli findings in the surgical vs nonsurgical sites. however, we also found increased fdg uptake in the sterile surgical leg (fig. b) , suggesting that some uptake is due to inflammation caused by the surgery. while [ f]fdg may easily identify robust pji, indolent infections with low bacterial burdens, or those being actively treated, may be indistinguishable from sterile background inflammation. [ f]naf has previously been used to assess the impacts of tuberculosis and total hip arthroplasty, as it is taken up by the surrounding bone matrix at these degenerative sites. [ ] [ ] [ ] while gross morphological changes were seen in this study after months of infection in the mouse model, such changes may not be distinguishable in the absence of long-term infection. remodeling of the bone due to sterile inflammation may again preclude the distinction of indolent infection vs sterile inflammation, as confirmed when comparing the sterile implant to the contralateral knee (fig. b) . while ct and [ f]naf pet may not aid in early diagnosis of pji, they may be useful once substantial remodeling has occurred. a correlation analysis across the pet imaging probes and that of bacterial-specific bioluminescence indicates the specificity of the antibody-targeted pet imaging approach. this study was limited in part by the fact that the physes in mice are continually active and do not fuse, as occurs in humans. this results in sustained bone turnover and signal at the ends of the long bones by both [ f]fdg and [ f]naf. however, this confound was controlled for by comparing postoperative joints to their contralateral, non-operative sites, which also have an active physis. there are also practical considerations that are relevant when comparing different imaging tracers or modalities of translational import. while it is finding greater acceptance in the cancer-imaging realm, [ zr]-labeled antibody pet imaging is still performed at only a small number of sites worldwide. ct imaging, [ f]fdg, and [ f]naf pet are routinely performed and can provide same day (outpatient) logistics. often with mabs, the long clearance time after injection requires several days between administration and imaging. intriguingly, here we have observed that the optimal [ zr]sac imaging time point was only h after injection. bli of bacteria in small animals is generally feasible but requires genetic manipulation of the infectious organism that may alter properties of the model and is not suitable for larger animal models or human use (as the light-producing proteins are highly immunogenic and provide signal sufficient for superficial imaging). in conclusion, we have demonstrated the ability of an anti-lta mab immunopet imaging probe to discriminate infection from non-specific inflammation in a preclinical mouse model of pji. we have further highlighted the potential and shortcomings of conventional pet tracers in imaging of these models. we plan to examine the role of molecularly specific imaging to monitor efficacy of the sac mab as a targeted therapeutic to clear lta-positive infections. investigation in larger animal models and in patients is needed to determine whether the [ zr]sac probe can be translated to diagnose pji and other infections in clinical practice. the monoclononal antibody (mab) specific for lta (anti-lta, sac ) was identified from igg + memory b cells selected from a patient convalescing from a s. aureus skin infection (erysipelas), as previously described. a human igg mab (r , directed against an unrelated hiv-gp antigen) was used as an isotype control human igg . antibody labeling the heterobifunctional chelate, p-scn-bn-dfo, was from macrocyclics (dallas, tx). reagents and chemicals were from sigma-aldrich (st. louis, mo) unless otherwise stated. zirconium- was from the mallinckrodt institute of radiology (washington university medical school, st. louis, mo). conjugating the chelator (dfo) to the antibody to antibody in . mol · l − hepes at ph . , µl of dfo in dimtethyl sulfoxide ( . µmol · l − ) was added three times followed by mixing to a final dfo:antibody ratio of : . the reaction was mixed at room tempterature for - min. excess unreacted dfo was removed by centrifugation using amicon ultra . ml centrifugal filters ultracel k regenerated cellulose nmwl (emd millipore, billerica, ma). labeling with zr to zirconium- , an excess of m oxalic acid was added. slowly na co was added to bring the ph to - . . zr was added to the dfo-conjugate antibody, and the reaction was mixed at room temperature for min. to chelate free zr, mmol · l − edta, ph , was used and removed by centrifugation using sterile saline in amicon ultra . ml centrifugal filters ultracel k regenerated cellulose nmwl (emd millipore, billerica, ma). instant thin layer chromatography (itlc) was performed using silica impregnated filter paper (pall corporation, port washington, ny). the itlc was run in mmol · l − edta, ph , and subsequently imaged and quantified using the phosphorimager and the autoquant software package, respectively (packard, perkinelmer, hopkinton, ma). for both specific sac and control antibodies, specific activities were in the range of . - . mci · g − . bacteria and inoculum preparation community-acquired methicillin-resistant staphylococcus aureus (ca-mrsa) strain sf usa ca-mrsa was kindly provided by binh diep of ucsf (san francisco, ca). staphylococcus epidermidis strain , p. aeruginosa strain pao , and escherichia coli strains were obtained from american type culture collection (atcc, manassas, va). s. aureus and s. epidermidis were streaked for isolation on trypticase soy agar (tsa) plates (vwr international, radnor, pa) and a single colony was inoculated into tryptic soy broth (tsb) and grown overnight. p. aeruginosa was grown overnight on a tsa plate and resuspended in tsb to the appropriate optical density (od) nm . a bioluminescent ca-mrsa strain (sap ) was derived from the parent nrs strain as previously described. sap possesses the bioluminescent lux construct in the bacterial chromosome so that only live and actively metabolizing bacteria will produce light, and light production is maintained in all progeny without selection. mid-logarithmic phase bacteria were obtained after overnight culture in tsb at °c with shaking ( r · min − ) followed by a h subculture at : dilution. the desired inoculum of bacteria was washed and reconstituted in sterile phosphatebuffered saline (pbs). whole-cell elisa binding bacteria from an overnight culture were washed in ice-cold pbs (vwr international) and adjusted to an od nm~ . in ice-cold pbs. elisa plates (nunc maxisorp, nalge nunc international, rochester, ny) were coated with µl of bacteria and incubated overnight at °c. plates with s. aureus were then blocked with µg · ml − rabbit igg (sigma-aldrich, st. louis, mo), and plates with other bacterial species were blocked with % bovine serum albumin (bsa; sigma-aldrich, st. louis, mo) in pbs for h at °c and then washed × with . % tween- (vol/vol) in pbs (pbst). serial dilutions of µl of sac or isotype control r were then applied to the plates and incubated for h at °c. the plates were washed times with pbst, and bound antibody was detected with µl of µg · ml − horseradish peroxidase-conjugated goatanti-human igg (jackson immunoresearch laboratory, west grove, pa) and , ′, , ′-tetramethylbenzidine substrate (kpl, seracare, milford, ma). the reaction was stopped after min with μl . mol · l − h so , and the od was measured in a spectrophotometer (molecular devices, sunnyvale, ca). flow cytometry-in vitro grown bacteria flow cytometry on in vitro grown bacteria was conducted on overnight bacterial cultures washed in ice-cold pbs, adjusted to od nm~ . , and blocked with % or % human sera for h at °c. after one wash in pbs with . % bsa, pellet was incubated with µl of alexa fluor (thermofisher scientific, waltham, ma)-labeled sac or r at µg · ml − in pbs in the presence of % or % human sera. following two washes, mab binding was quantified by flow cytometry with an lsrii flow cytometer (becton-dickinson, franklin lakes, nj). flow cytometry-in vivo grown bacteria six-to-eight-week-old female cd mice (harlan, indianapolis, in) were infected by intraperitoneal injection of × cfu of s. aureus or s. epidermidis. four hours postinfection, the animals were euthanized, and blood obtained after cardiac puncture was pooled from mice into ice-cold sodium citrate at . % (weight:vol) final concentration. eukaryotic cells were lysed with % np (thermo fisher scientific, waltham, ma), and bacteria were recovered by centrifugation ( min) at r · min − at °c . the bacterial pellet was sonicated in ml ice-cold pbs, washed once in pbs, and transferred to a -well u-bottom plate (thermo fisher scientific). first, non-specific binding to protein a was blocked with rabbit anti-protein a immune sera ( : ) for min at °c. bacteria were then incubated with anti-lta mab or igg ( µg · ml − ) for h at °c, washed in pbs, and incubated with alexa fluor (thermo fisher scientific)-conjugated goat antihuman igg for min at °c (jackson immunoresearch laboratory, west grove, pa). following one wash, live bacteria were stained for min at room temperature with bodipy fl vancomycin and mab binding was quantified as above. octet affinity measurement anti-lta mab-binding kinetics to purified lta were analyzed using an octet instrument with slanted well plates (fortebio, menlo park, ca). an aminopropylsilane biosensor was first loaded with µg · ml − of purified s. aureus lta (sigma-aldrich) for s. anti-lta mab ( . - nmol · l − ) association was measured for s, followed by a s dissociation into kinetic buffer (fortebio). all steps were performed using a -mm sensor offset with . hz sensitivity. data were exported to prism (graphpad, la jolla, ca) for global association/dissociation affinity curve fitting. mouse model of pji all animal procedures were approved by the johns hopkins university animal care and use committee. six-week-old male c bl/ mice (jackson laboratories, bar harbor, me) were used in all experiments. a mouse model of pji was performed as previously described. , briefly, a medial parapatellar approach was used to access the right distal femur. after dislocating the patella, a -gauge needle was used to ream the distal femoral medullary canal. thereafter, a medical-grade kirschner-wire ( . mm diameter × mm in length; modern grinding, port washington, wi) was inserted in retrograde fashion with approximately . mm protruding into the knee joint to which was added µl sterile saline for the sterile control or directly inoculated with × cfu. the patella was relocated and the knee was surgically closed using two interrupted absorbable sutures. in vivo bli postsurgical infection was confirmed by direct bli of bacteria using the ivis lumina iii imaging system (perkinelmer, hopkinton, ma). imaging (large binning and a min exposure) was performed immediately on anesthetized mice ( % isoflurane) before surgery and then on postoperative days , , , and . signal was quantified using the living image software within a region of interest (roi) of . × . cm centered over the surgical knee and measured as maximum radiance (photons/second/cm /steradian). the limit of detection was . × photons/s/cm /sr. after an infection was established, the right hind limb was cleared of hair, and a bioluminescent image was acquired (ivis spectrum; perkin elmer). an elliptical roi of . × . cm was used to measure persistent infection. injectate was assayed using a sodium iodide crc- dose calibrator and a calibration factor of (capintec, ramsey, nj). mice were anesthetized by isoflurane ( %) inhalation and injected intravenously via the retro-orbital sinus with μci average activity of tracer diluted to a final volume of μl in isotonic saline. for [ f]fdg, pet/ct was performed following a h uptake period under continuous isoflurane anesthesia. for [ f] naf, the mice were scanned following a min uptake period without anesthesia. radiolabeled antibodies were prepared as detailed above, and injectate was assayed using a well counter with a calibration factor of , as determined previously. in a volume of µl, µci of radiolabeled antibody (approximately µg) was administered intravenously via the retro-orbital sinus. mice were serially scanned ( - min acquisitions, - kev energy window) on days , , , and on a micropet r system (concorde microsystems inc., knoxville, tn) followed by faxitron mx- -dc digital x-ray imaging system (faxitron bioptics, llc, tucson, az). pet/ct imaging sequential pet ( min acquisition, - kev energy window) and ct scans were performed using the superargus small-animal integrated pet/ct scanner (sedecal systems, buffalo grove, il). ct parameters for acquisition were kvp and . ma with mm aluminum filtration. the d imaging analysis platform amira (version . ; fei, hillsboro, or) was used to merge the pet and ct data sets to visualize [ f]-fdg uptake in relation to the surgical site. pet and high-resolution x-ray imaging the dedicated micropet r system was used to acquire pet scans for radiolabeled antibodies. list-mode data were acquired using a gamma-ray energy window of - kev and a coincidence timing window of ns. pet image data were corrected for detector nonuniformity, dead time, random coincidences, and physical decay. for all static images, scan time was between and min. data were sorted into d histograms by fourier rebinning, and transverse images were reconstructed using a maximum a priori algorithm to a × × ( . mm × . mm × . mm) matrix. data sets were analyzed using the asipro vm micropet analysis software. volumes of interest were manually defined around the distal femur, and the injected activity per gram was calculated. an empirically determined system calibration factor for mice was used to convert voxel count rates to activity concentrations (in µci per ml of tissue). figures were generated using amira (version . ; fei, hillsboro, or). directly after pet scanning, while in the same position, planar x-ray images of the mice were acquired using the faxitron mx- -dc digital x-ray imaging system (faxitron bioptics, llc, tucson, az). statistical analysis all data with statistics are expressed as mean ± s.e.m. data were analyzed using an unpaired one-tailed (bli and [ f]fdg and [ f] naf uptake) or two-tailed (ct volume) student's t-test with welch's correction or the holm-sidak method, with alpha = . , and each row was analyzed individually, without assuming a consistent standard deviation. analysis of variance followed by bonferroni's multiple comparison test was used to assess [ zr]antibody uptake; outliers were removed via analysis of absolute variance relative to the mean. pearson's correlation analysis was performed in prism (graphpad version . d) between pet imaging of [ zr]-antibody (using the ratio of uptake in the surgical vs contralateral leg and fold increase uptake of [ f]fdg and [ f] naf) and the standardized uptake value (suvmean) of [ zr]antibody in the surgical pin leg vs bioluminescent flux values from the same limb. p-values < . were considered significant. clinical practice. infection associated with prosthetic joints projections of primary and revision hip and knee arthroplasty in the united states from to economic burden of periprosthetic joint infection in the united states two-stage exchange arthroplasty for infected total knee arthroplasty: predictors of failure accuracy of diagnostic tests for prosthetic joint infection: a systematic review a molecular imaging primer: modalities, imaging agents, and applications bacterial infection probes and imaging strategies in clinical nuclear medicine and preclinical molecular imaging nuclear medicine methods for evaluation of skeletal infection among other diagnostic modalities molecular imaging of bacterial infections in vivo: the discrimination of infection from inflammation radionuclide imaging of musculoskeletal infection: a review comparison of f-fdg and ga pet imaging in the assessment of experimental osteomyelitis due to staphylococcus aureus antibody positron emission tomography imaging in anticancer drug development stably luminescent staphylococcus aureus clinical strains for use in bioluminescent imaging mouse model of chronic post-arthroplasty infection: noninvasive in vivo bioluminescence imaging to monitor bacterial burden for long-term study a mouse model of post-arthroplasty staphylococcus aureus joint infection to evaluate in vivo the efficacy of antimicrobial implant coatings monitoring bacterial burden, inflammation and bone damage longitudinally using optical and μct imaging in an orthopaedic implant infection in mice vancomycin-rifampin combination therapy has enhanced efficacy against an experimental staphylococcus aureus prosthetic joint infection fdg pet of infection and inflammation imaging chronic tuberculous lesions using sodium [( )f]fluoride positron emission tomography in mice use of f- fluoride pet to differentiate septic from aseptic loosening in total hip arthroplasty patients review of the role of dynamic f-naf pet in diagnosing and distinguishing between septic and aseptic loosening in hip prosthesis use of f-fluoride pet to determine the appropriate tissue sampling region for improved sensitivity of tissue examinations in cases of suspected periprosthetic infection after total hip arthroplasty combined in vivo optical and µct imaging to monitor infection, inflammation, and bone anatomy in an orthopaedic implant infection in mice prosthetic joint infection )i]fiau: human dosimetry and infection imaging in patients with suspected prosthetic joint infection in vivo radiometric analysis of glucose uptake and distribution in mouse bone an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus identification of anti-alpha toxin monoclonal antibodies that reduce the severity of staphylococcus aureus dermonecrosis and exhibit a correlation between affinity and potency a recommendation for revised dose calibrator measurement procedures for zr and i this study was supported in part by national institutes of health t ar (to j. e.p. and j.m.t.), ro ca (d.l.j.t.) as well as the mrb molecular imaging service center (p ca ). the online version of this article (https://doi.org/ . /s - - -y) contains supplementary material, which is available to authorized users. the authors declare that they have no conflict of interest.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/ . /. key: cord- -lk ny wd authors: cantoni, diego; rossman, jeremy s. title: ebolaviruses: new roles for old proteins date: - - journal: plos negl trop dis doi: . /journal.pntd. sha: doc_id: cord_uid: lk ny wd in , the world witnessed the largest ebolavirus outbreak in recorded history. the subsequent humanitarian effort spurred extensive research, significantly enhancing our understanding of ebolavirus replication and pathogenicity. the main functions of each ebolavirus protein have been studied extensively since the discovery of the virus in ; however, the recent expansion of ebolavirus research has led to the discovery of new protein functions. these newly discovered roles are revealing new mechanisms of virus replication and pathogenicity, whilst enhancing our understanding of the broad functions of each ebolavirus viral protein (vp). many of these new functions appear to be unrelated to the protein’s primary function during virus replication. such new functions range from bystander t-lymphocyte death caused by vp -secreted exosomes to new roles for vp in viral particle formation. this review highlights the newly discovered roles of ebolavirus proteins in order to provide a more encompassing view of ebolavirus replication and pathogenicity. ebolaviruses are negative-sense single-stranded rna (ssrna) viruses capable of causing acute haemorrhagic fever. the prototypical ebola virus (ebov; zaire ebolavirus) was responsible for the recent west africa outbreak that resulted in , cases and , deaths between and [ ] . the reservoir of this lethal pathogen has not been conclusively proven, though there is good evidence suggesting fruit bats as a primary source of exposure [ ] . furthermore, survivors of ebolavirus disease (evd) can carry the virus in immunologically privileged sites for over one year, post-recovery from the acute infection [ , ] . resultantly, there is an a a a a a increased awareness of the risk of alternate forms of transmission in evd as survivors may carry ebov in semen for over months, causing sexual transmission, or in breast milk, which can lead to infection of newborns [ , ] . in , funding from project bioshield (united states department of health and human services) spurred research that has led to the development of novel therapeutics to prevent and treat evd, resulting in a very promising vaccine that was evaluated during the outbreak and showed % efficacy in disease prevention [ , ] . following project bioshield and the west africa outbreak, there has been extensive research into the ebolaviruses that has greatly expanded our understanding of viral replication and pathogenesis. ebolavirus is a genus within the family filoviridae, which also includes the genus marburgvirus (e.g., marburg virus: marv) and cuevavirus (e.g., lloviu virus) [ ] . the ebolavirus genus contains five species: zaire ebolavirus, sudan ebolavirus, taï forest ebolavirus, bundibugyo ebolavirus, and lastly, reston ebolavirus, the only member that is nonpathogenic in humans for reasons that are still unclear [ ] . the majority of research has focused on zaire ebolavirus, as this species has been associated with the greatest number of outbreaks and has the highest case-fatality rates of all the ebolaviruses ( %- %, depending on the specific outbreak), though as a consequence, many novel or species-specific functions of ebolavirus proteins may be undiscovered. the kb viral genome encodes for seven main proteins: nucleoprotein (np), glycoprotein (gp), l-polymerase (l) protein, viral protein (vp) , vp , vp , and vp (fig ) [ ] . l is an rna-dependent rna polymerase (rdrp) and forms the rdrp complex with vp that is responsible for viral genome transcription and replication. vp and vp inhibit interferon (ifn) signalling and facilitate evasion of the host immune response. np encapsidates the viral genome into the nucleocapsid, whilst vp drives viral assembly and budding. gp is the only protein on the surface of the virion and is essential for binding to target cells and subsequently mediating membrane fusion and the release of the viral genome. however, recent research has uncovered a multitude of new, often overlapping roles for ebolavirus proteins, and it is not possible to view viral replication as a one-protein-one-function process (table ). in this review, we examine the collection of recently identified secondary functions of ebolavirus proteins in order to provide a more comprehensive understanding of roles of each protein in viral replication and pathogenicity. vp is one of the most studied filovirus proteins, with the majority of studies focusing on its primary role in inhibiting the host ifn response. vp is known to inhibit ifn-α/β and ifn-γ activation by binding to key host proteins from the karyopherin α family (karyopherin α , α , and α ) preventing their binding to and the subsequent nuclear import of signal transducer and activator of transcription (stat ) [ ] . in addition, vp blocks ifn signalling by directly binding to stat , preventing its phosphorylation, nuclear import, and transcription of ifn-stimulated genes (isg) [ ] [ ] [ ] . recent data suggest vp is also able to block ifn induction by supressing nuclear factor-kappa b (nf-kb) activation following tumour necrosis factor alpha (tnf-α) stimulation [ ] and by supressing retinoic acid-inducible gene i (rig-i)-dependent activation of ifn-γ gene expression [ ] . furthermore, the innate response antagonist domains (irad) in ebov vp and vp have been implicated in preventing the maturation of ebov-infected dendritic cells by modulating global gene expression [ , ] . this effect required both vp and vp irad domains, though the vp irad domain alone was sufficient to down-regulate cytokine signalling pathways [ ] . these results highlight the importance of cooperative action of multiple viral proteins for evasion of the host immune response. vp has also recently been seen to function in capsid assembly [ ] . np and vp are known to be the major components of the viral nucleocapsid, though vp is weakly associated and may act as a catalyst for particle formation [ ] . further evidence for a structural role of vp comes from the observation that n-or c-terminal deletions in vp inhibited the formation of nucleocapsid-like structures mediated by vp , vp , and np coexpression [ ] . it was suggested that the n-terminal domain of vp facilitates capsid formation by mediating protein-protein interactions. this is supported by the observation that mutation of the vp n-terminal domain results in protein aggregation [ ] . a recent study confirmed the interaction between vp and np, showing that vp residues v and n are located on a highly conserved exposed loop that interacts with np during nucleocapsid assembly [ ] . coexpression of vp and vp results in a greater production of virus-like particles (vlps) than when vp is expressed alone [ ] . similarly, in live ebov infection, vp small interfering rna (sirna) knockdown decreases viral budding and increases the retention of viral proteins within the cell [ ] . further evidence suggests that vp binds to vp on the outer surface of the nucleocapsid where it organises the adjacent np layer, promoting nucleocapsid stability and explaining the observed interactions between np, vp , and vp during nucleocapsid formation [ ]. rna editing by addition of us occurs on the gp transcript in order to produce gp and the ssgp. post-translational modification of the gp protein results in two gp subunits after cleavage by host furin. slight differences in gene overlaps and the presence of intergenic regions are evident between the different ebolavirus species, though the functional consequence of these differences is not clear at present. ebov in addition to its role in nucleocapsid stability, vp may be necessary for incorporation of the viral rna (vrna) genome into the nucleocapsid. several studies have shown that vp , together with vp , induce conformational changes in np that are necessary for rna encapsidation [ , , ] . it was shown that vp may be directly involved in length-dependent vrna interactions and packaging [ ] . in the study, transcription-and replication-competent virus-like particles (trvlps) were analysed for rna content, and a significant reduction of packaged rna was observed when vp was knocked down with an interfering micro-rna (mirna). the trvlps that were produced showed a -fold reduction in rna content and a -fold reduction of infectivity, suggesting that vp may play an essential role in rna packaging. furthermore, disruption of the vp -np interaction reduced rna packaging and resulted in a significant reduction in reporter activity, highlighting the importance of vp in rna packaging [ ] . in addition, trvlp reporter gene activity was significantly affected by the presence of vp in a genome-length-dependent manner. using the trvlp tetracistronic genome system, the presence of vp during vlp production resulted in a -fold increase in reporter gene activity upon subsequent infection, whereas the presence of vp had no effect when monocistronic minigenome systems were used in the trvlp, suggesting a genomelength-dependent role for vp in rna packaging and vlp infectivity. surprisingly, vp may also have a length-dependent role in transcriptional regulation. it was observed that vp moderately inhibited the expression of reported genes from monocistronic minigenome plasmids [ ] . however, vp expression had no effect on protein expression from the trvlp tetracistronic genomes [ ] . whilst the impact of vp on protein expression is not clear, vp itself may be subject to length-dependent transcriptional regulation. recent work has implicated the length of the intergenic region (ir) between vp and vp as having a significant as with vp , vp is primarily known for its multifaceted ability to suppress the host cell immune response. vp is a type-i ifn antagonist, inhibiting the activation of interferon regulatory factor (irf)- via double-stranded rna (dsrna) binding and reducing ifn-α/β production by inhibiting rig-i signalling [ ] [ ] [ ] . vp also blocks ifn production by increasing protein inhibitor of activated stat (pias )-mediated sumoylation of irf- , thus inhibiting ifn production following toll-like receptor (tlr) and rig-i activation [ ] . lastly, vp is a suppressor of rna silencing, functionally equivalent to the human immunodeficiency virus (hiv- ) trans-activator of transcription (tat) protein and important for viral evasion of the innate immune response [ ] . together, there is significant evidence demonstrating vp 's intricate ability to inhibit innate immune signalling and the host antiviral response (fig ) . recent work suggests that vp may have more diverse functions during virus replication, as vp was shown to interact with l and facilitate genome transcription through the formation of the rdrp complex and genome packaging through association with np [ - ]. the first residues of vp appear to be essential for binding to l and thus rdrp function, whereas the c-terminus associates with np, thus linking np and l during nucleocapsid assembly [ , ] . n-terminal deletions in vp block these interactions and were sufficient to inhibit the replication and transcription of an ebov minigenome system [ ] . the role of the vp c-terminus in capsid assembly is perhaps surprising, as this region contains the ifn inhibitory domain responsible for its main role in immune evasion. however, this domain contains several conserved stretches of basic residues involved in dsrna binding and ifn inhibition, whereas a preceding stretch mediates interaction with np [ ]. further research found the vp -np interaction controls the switch between rna-bound np and free np, thus switching between genome replication and genome packaging in the nucleocapsid [ ] . an n-terminal peptide derived from the vp np-binding protein region (npbp) binds np with high affinity, causing the release of rna from np and resulting in the activation of genome transcription and the inhibition of np oligomerisation (fig ) [ ]. additional investigation of vp -np binding showed two further interaction sites. hydrophobic vp -np binding at these sites inhibited np oligomerisation and prevented np-rna binding by blocking access to the rna binding domain [ ] . work by leung and colleagues suggests that during nucleocapsid formation, the npbp peptide first disassociates from np, then rna binds to np, followed by np oligomerisation. in contrast, kirchdoerfer and colleagues show that monomeric np has no significant affinity for rna, suggesting that the npbp peptide would be displaced by an additional np molecule, causing np oligomerisation that would then allow for rna binding. however, in either process, vp -np interactions are crucial for virus replication and are being explored as targets for future therapeutics [ , ] . vp also undergoes further protein-protein interactions that may affect viral genome transcription through the interaction with the cytoplasmic dynein light chain (lc ) [ ] . lc is a highly conserved kda subunit of the cytoplasmic dynein motor complex but can also exist as a dimer in soluble form, which can affect viral transcription and assembly [ , ] . lc was seen to stabilise vp n-terminal oligomerisation in a dose-dependent manner and enhance viral genome synthesis [ ] . it was noted that lc functions mostly in the early stages of infection, enhancing early viral gene expression before the host cells are able to establish the antiviral state. thus, vp modulation of vrna transcription can facilitate virus replication while simultaneously enhancing immune evasion. the minor nucleoprotein vp has the primary role of initiating ebov transcription [ ] . it is dynamically phosphorylated, whereby upon phosphorylation, transcription is negatively regulated, enabling binding to np [ , ] . in turn, this permits interactions that regulate vrna synthesis [ ] . vp binds zinc ions due to the presence of an unconventional zinc-binding motif, facilitating rna binding and increasing viral genome transcription [ - ]. in addition, vp blockade of irf and irf phosphorylation inhibits the production of ifn-β. recent studies have also highlighted the importance of vp in regulating np-rna association. during viral genome replication, the vp n-terminal peptide binds to np, enabling the vrna to associate with the rdrp complex for replication. during virus assembly, vp disassociates, enabling np to oligomerise, bind rna, and form the nucleocapsid. 'ppp, ' triphosphate; dsrna, double-stranded rna; ifn, interferon; ikk, inhibitor of nuclear factor kappa b kinase subunit epsilon; irf, interferon regulatory factor; mavs, mitochondrial antiviral-signalling protein; mda , melanoma differentiation-associated protein ; np, nucleoprotein; pact, protein activator of the interferon-induced protein kinase; rdrp, rna-dependent rna polymerase; rig-i, retinoic acid-inducible gene i; tank, tumour necrosis factor-receptor-associated factor family member-associated nuclear factor kappa b activator; tbk , tumour necrosis factor-receptor-associated factor family member-associated nuclear factor kappa b activator binding kinase ; traf , tumour necrosis factor-receptor-associated factor ; vp, viral protein; vrna, viral rna. https://doi.org/ . /journal.pntd. .g in addition to its rna-binding role in transcription, vp also interferes with cellular rna silencing [ ] . in the presence of sirna, vp was seen to interact with the essential rna interference (rnai) protein dicer, though the vp n-terminus rna binding domain was not required for the interaction or for the suppression of rnai (fig ) . as with the rna silencing suppressor activity of vp , the exact role of rnai in antiviral immunity is not clear, nor is the consequence on ebov replication of blocking mirna/sirna processing as mediated by vp [ ]. however, despite the fact that rna binding is not required for rna silencing suppression, vp was seen to bind to a variety of nonviral rnas. vp -rna binding required specific base composition and structure of the target rna molecule [ ], though it is not clear if there is a function of vp binding to nonviral rna or if this is a consequence of its necessary binding to vrna during transcription. the matrix protein vp has roles predominantly in virus assembly and budding [ , ] . vp can assemble either as a hexamer, which appears to be involved in budding, or as an octamer that functions in genome replication and rna binding [ , ] . most research has focused on the role of vp in assembly and budding; however, recent studies have begun to elucidate novel roles. vp is known to be sufficient to mediate the formation and budding of vlps; however, recent results have demonstrated that vp induces the formation for exosomes that are capable of inducing bystander cell death [ , ] . exosome release has been seen in virally infected cells, and vp expression was seen to increase the expression of several endosomal sorting complex required for trafficking (escrt) proteins involved in exosome biogenesis, including tumour susceptibility gene (tsg ), vacuolar protein-sorting-associated protein (vps ), and vps [ , ] . this is consistent with previous reports showing vp utilising escrt proteins to aid viral budding, though how vp switches between budding and exosome release is not clear [ ] [ ] [ ] . transfer of vp -induced exosomes to naïve t lymphocytes and monocytes induced apoptosis and significantly reduced cell viability similarly to that seen when exosomes from virally infected cells were used [ , ] . however, pleet and colleagues noted that the presence of vp in the exosomes caused a down-regulation of mirna machinery in both the donor and recipient cells, including a reduction in the expression of dicer, argonaute- , and drosha ( fig ) . it was previously noted that vp , vp , and vp are capable of interacting with the mirna/rnai pathway [ ]; however, this was the first demonstration that the suppression of rna silencing can be transferred to naïve cells in the absence of virus. currently, the precise mechanism in which vp interacts with mirna machinery has yet to be characterised, and it is not yet known if the action of vp on the mirna machinery directly causes apoptosis or if the exosomes contain other proteins or rnas that may be responsible for causing the induction of apoptosis. however, the repression of key proteins in the mirna pathway has been previously linked to the induction of apoptosis; thus, the suppression of rna silencing may serve both to directly counteract the innate cellular immune response and to induce apoptosis of bystander immune cells, blocking the activation of adaptive immunity [ , , ] . due to the essential role of vp in viral assembly and budding, several studies have looked at inhibiting vp for the creation of new antiviral therapeutics [ , ] . as vp also plays a role in immune evasion (via rnai suppression and exosome-bystander cell death), there is increased motivation for developing therapeutics that target one or multiple functions of vp . it is known that viral replication requires vp phosphorylation at tyrosine by the cellular tyrosine kinase abelson murine leukaemia viral oncogene homologue (c-abl ) [ ] . in addition, recent results showed that cyclin-dependent kinase (cdk ) in complex with cyclin a or cyclin e phosphorylated exosomal vp at serine- [ ] . thus, the inhibition or modulation of vp phosphorylation may be a target for new therapeutics. lastly, pleet and colleagues showed that treatment with the fda-approved drug oxytetracycline reduced vp exosome release and significantly increased donor cell viability upon treatment with vp exosomes, further suggesting that targeting the secondary functions of vp may be a new approach for developing antivirals for evd. the gp gene has been shown to encode for three different products due to transcriptional editing by l: full length gp, which consist of gp (receptor binding) and gp (viral fusion) subunits; soluble gp (sgp), which lacks the transmembrane domain; and small soluble gp (ssgp) [ , ] . due to furin cleavage of sgp, a smaller cleaved fragment is also produced, called Δpeptide [ ] . gp is the only viral protein located on the surface of the virion and has a critical role in attachment and fusion [ ] [ ] [ ] . ebolaviruses are thought to predominantly enter cells via gp-dependent macropinocytosis, though other mechanisms have been reported, depending on factors such as host cell type [ , ] . after entry, gp directs fusion between the viral membrane and endolysosomes that contain the viral receptor niemann-pick c (npc ) and two-pore segment channel (tpc ), enabling release of the viral genome [ , ] . during the - outbreak, a mutation in gp at a v was detected with high frequency [ ] [ ] [ ] . this mutation increased gp membrane fusion activity and increased infectivity in a variety of cell types, including chimpanzee fibroblasts (s ), rhesus epithelial (frhk ), african green monkey epithelial (vero), and human dendritic cells. the authors propose that this mutation is likely a result of ebov adaptation to the human host, as several viral variants have been seen to increase human cell infectivity while decreasing virus entry in nonhuman primates [ ] [ ] [ ] . thus, the specific nature of protein function needs to be considered in the context of the given host. gp has been shown to have a multitude of secondary roles beyond attachment and fusion that affect both virus replication and pathogenicity. several studies have shown that gp contributes to ebov virulence; however, it is not sufficient on its own to be defined as a virulence marker, despite having marked effects beyond entry and fusion [ ] . ebov gp expression has been well established as having a cytotoxic effect on host cells [ ] [ ] [ ] . gp cytotoxicity is mediated through the mucin-like domain and its effect on the extracellular signal-regulated kinase (erk) mitogen-activated protein kinase (mapk) pathway [ ] . ebov gp reduces the phosphorylation and catalytic activity of erk , resulting in the loss of cell adherence, cell rounding, and the induction of non-apoptotic cell death. the decrease in erk activity was also necessary for gp-induced down-regulation of αv integrin expression, further impairing cell adherence and tight junction formation. in addition, the sgp cleavage product, Δ-peptide, may play a role in ebov pathogenicity by acting as a viroporin [ ] . Δ-peptide is able to form pores in the plasma membrane of mammalian cells, increasing ion permeability and causing cytotoxicity [ ] . however, it is not known if the Δ-peptide can be released from cells or if its induction of cytotoxicity is limited to within the infected cell. in order to regulate the toxicity caused by gp and its cleavage products at early stages of infection, gp expression is dynamically regulated [ ] . the balance and timing of ebov gp/sgp/ssgp/shed-gp/Δ-peptide expression has been found to be pivotal in virus replication, affecting not only cell death but viral assembly and budding [ ] . whilst vp expression is sufficient to produce vlps, gp expression enhances vlp generation, suggesting a possible secondary role for gp in viral egress [ ] . recent work suggests that gp does not directly affect viral assembly or budding, but rather counteracts the cellular budding restriction factor tetherin [ ] . in the absence of gp, vlps assemble and bud but are retained on the cell surface through the antiviral tethering actions of the tetherin protein. gp expression enabled vlp release but did not affect tetherin cell-surface localisation, nor was a specific gp-tetherin interaction found [ ] . it was shown that the gp glycosylation and its receptor-binding domain (rbd) were critical for anti-tetherin activity, though mutation of the rbd did not affect interactions with tetherin, and inhibition of gp-npc binding did not affect the anti-tetherin activity [ ] . instead, it is thought that gp is specifically able to block the association between vp and tetherin, though the nature of tetherin-vp interaction and the mechanism of gp inhibition is not known [ ] . ebov infection causes significant impairment of the endothelial barrier function. gp repression of erk activity reduces integrin expression and cell adherence; however, gp also induces endothelial cell activation, further decreasing endothelial barrier functions [ ] . during ebov infection of endothelial cells, cell adhesion molecules (cam) icam- and vcam- were found to be transcriptionally upregulated, with increased cell surface expression of cams. the activity occurs with cellular gp expression as well as following the transfer of gpcontaining vlps; viral replication does not appear to be required. endothelial activation was not observed in the absence of gp, nor with the gp transcriptional variant sgp or the gp cleavage product Δ-peptide [ ] . gp-induced endothelial cell activation may facilitate decreased barrier function, whilst the up-regulation of cams may facilitate adhesion and subsequent infection of immune cells, such as macrophages. a model was proposed whereby activated endothelial cells result in increased leukocyte recruitment, resulting in thrombomodulin release, resulting in an activated, leukocyte-rich endothelium in a procoagulant state [ ] . whether this is an unintended consequence of gp or has a role in increased viral spread and infectivity is yet to be investigated. ebov gp has also been implicated in modulation of the host immune system. gp on the cell plasma membrane has been shown to be cleaved by tnf-α-converting enzyme (tace), resulting in the release of a soluble cleaved product called shed gp that is missing the transmembrane domain [ ] . shed gp was seen to activate uninfected macrophages and dendritic cells, resulting in the production of multiple pro-and anti-inflammatory cytokines, including tnf-α, with subsequent effects on vascular permeability [ ] . it is thought that shed gp activates macrophages by binding to and activating toll-like receptor (tlr ) in a manner requiring gp glycosylation. recently it was also shown that full-length gp on vlps can also trigger the activation of tlr in macrophages, resulting in a similar activation phenotype [ ] . in contrast, gp binding to tlr on t lymphocytes directly triggers cell death through an up-regulation of caspase , even in the absence of infection [ ] . in dendritic cells, gp was found to interact with the liver and lymph node sinusoidal endothelial cell c-type lectin (lsectin), a ctype lectin that contains two amino acids, residues n and n , that bind gp in a ca + -dependant manner, triggering the activation of spleen tyrosine kinase (syk) signalling and the production of inflammatory cytokines tnf-α and interleukin- (il- ) [ ] . as shed gp retains most of the structure of full-length gp, this soluble molecule is capable of binding to and neutralising circulating anti-gp antibodies, facilitating viral immune evasion [ ] . similarly, sgp has been implicated in evading the immune system via antigenic subversion [ ] . it was found that boosting gp-immunised mice with sgp biased b-cell response towards epitopes that were shared between sgp and gp, reducing gp-specific antibody production and possibly impeding the immune-mediated clearance of ebov infection. the structure of sgp in complex with antibodies was recently solved and highlights differences in antibody reactivity between gp and sgp [ ] . whilst gp is trimeric, sgp oligomerises into a parallel homodimer. cross-reactive c c antibody epitopes are presented similarly on gp and sgp, though the authors report that one c c antibody binds to one gp trimer, whereas multiple different immune-complexes were formed with sgp, ranging from rectangular complexes with a : ratio of c c antibody to sgp dimer up to pentagonal : antibody:sgp complexes [ ] . this further supports the hypothesis that sgp enhances viral immune evasion by biasing the antibody response towards sgp binding. whilst the multitude of gp secondary effects have a significant impact on ebov infection, pathogenesis, and immune evasion, gp remains the immunodominant protein on the ebov virion, and vaccination with the gp protein on pseudotyped viruses, recombinant vesicular stomatitis virus with zaire ebolavirus gp (rvsv-zebov), has been highly effective in preventing evd [ ] . np has a distinct function in the replication cycle as it is a key component of the viral ribonucleoprotein complex and has critical roles in protecting vrna from degradation and in mediating genome encapsidation during virus assembly [ ] . at present, all research has focused on these primary activities of np, and any secondary roles remain to be determined [ , , ] . similarly, the rna-dependent l-polymerase is an essential component of the rdrp complex and required for viral genome transcription and replication [ ] . it has been observed that l can also edit mrna, as seen with the gp gene, where l-editing results in the production of the gp transcript instead of sgp [ ] . l-editing may also regulate the different expression levels of gp, sgp and ssgp. during serial passage in tissue culture cells, l was found to add a single uridine (u) residue to a site consisting of us in the gp gene, changing the expression ratio of gp:sgp to : . a single passage in guinea pigs caused reversion of the genome back to us and changed the gp:sgp expression ratio back to : , which may facilitate immune evasion during in vivo replication [ ] . in contrast, during viral replication in the human hepatocarcinoma cell line (huh ), a u variant was seen that retained the high level expression of sgp but had enhanced expression of ssgp [ ] . it is speculated that these rapid alterations in the gp gene may act as a regulatory mechanism, enabling efficient virus replication in different host environments. at present, no other roles for the l protein have been postulated. for many years now, the fundamental principles of ebolavirus replication have been known, with each viral protein playing a specific role: l and vp form the rdrp and mediate viral genome transcription and replication, np packages the vrna genome and forms the nucleocapsid, vp mediates virion assembly and budding, gp mediates virion attachment to and fusion with the host cell, and vp and vp enable evasion of the host immune system. however, in recent years, our understanding of ebolavirus replication and pathogenesis has significantly increased, and we now know that each viral protein plays a multitude of overlapping roles during virus replication. vp and vp were thought to primarily mediate immune evasion but have now been seen to function in viral replication and in the formation of the nucleocapsid. similarly, many of the viral proteins that were thought to play functional roles in replication (i.e., vp , vp , gp) have now been discovered to have significant roles in immune evasion. this broadens our understanding of the ways in which ebolaviruses can evade and subvert the host immune system. of particular interest is the observation that vp , vp , and vp all appear to supress the host rna silencing system, though the relevance of this to viral replication is not yet clear (fig ) . in addition, many ebolavirus proteins are seen to interact with immune cells, causing cell activation and/or cell death and facilitating both viral replication and spread (e.g., by recruiting monocytes to infected cells or by increasing vascular leakage) as well as enabling immune evasion (e.g., antibody neutralisation by sgp and by cleaved gp), roles that were previously solely attributed to vp and vp . since project bioshield was initiated in , there has been a substantial increase in our understanding of ebolavirus replication and pathogenesis. several vaccines and antiviral therapeutics have been developed and were used during the - west africa ebola virus outbreak. however, the outbreak was difficult to contain and highlighted the fact that there are many significant aspects of ebov that we do not yet understand (e.g., ebov persistence in immunologically privileged tissue) and that there is a pressing need for continued research to better understand and combat this deadly pathogen. • viral structural proteins, such as vp and gp, have been found to cause cell death in a wide range of cell types, including bystander t lymphocytes. • viral immune modulating proteins, such as vp and vp , now have significantly expanded functions during virus replication, including the regulation of viral particle formation. . guito after ebola in west africa-unpredictable risks, preventable epidemics fruit bats as reservoirs of ebola virus persistence and genetic stability of ebola virus during the outbreak in kikwit, democratic republic of the congo ebola rna persistence in semen of ebola virus disease survivors-final report ebola virus in breast milk efficacy and effectiveness of an rvsv-vectored vaccine expressing ebola surface glycoprotein: interim results from the guinea ring vaccination cluster-randomised trial efficacy and effectiveness of an rvsv-vectored vaccine in preventing ebola virus disease: final results from the guinea ring vaccination, open-label, cluster-randomised trial (ebola {Ç }a suffit!) proposal for a revised taxonomy of the family filoviridae: classification, names of taxa and viruses, and virus abbreviations risks posed by reston, the forgotten ebolavirus the pathogenesis of ebola virus disease structural characterization and membrane binding properties of the matrix protein vp of ebola virus the role of exosomal vp in ebola virus disease oligomerization and polymerization of the filovirus matrix protein vp structural rearrangement of ebola virus vp begets multiple functions in the virus life cycle a ppxy motif within the vp protein of ebola virus interacts physically and functionally with a ubiquitin ligase: implications for filovirus budding ebola vp in exosomes can cause immune cell dysfunction human fatal zaire ebola virus infection is associated with an aberrant innate immunity and with massive lymphocyte apoptosis overlapping motifs (ptap and ppey) within the ebola virus vp protein function independently as late budding domains: involvement of host proteins tsg and vps- ebola virus matrix protein vp interaction with human cellular factors tsg and nedd alix rescues budding of a double ptap/ppey l-domain deletion mutant of ebola vp : a role for alix in ebola virus egress inducing cell proliferation inhibition and apoptosis via silencing dicer, drosha, and exportin in urothelial carcinoma of the bladder could the ebola virus matrix protein vp be a drug target the natural compound silvestrol is a potent inhibitor of ebola virus replication productive replication of ebola virus is regulated by the c-abl tyrosine kinase the nonstructural small glycoprotein sgp of ebola virus is secreted as an antiparallel-orientated homodimer a new ebola virus nonstructural glycoprotein expressed through rna editing delta-peptide is the carboxy-terminal cleavage fragment of the nonstructural small glycoprotein sgp of ebola virus tyro family-mediated cell entry of ebola and marburg viruses ebolavirus glycoprotein structure and mechanism of entry ebola virus entry: a curious and complex series of events ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependent manner ebola virus enters host cells by macropinocytosis and clathrin-mediated endocytosis ebola virus and severe acute respiratory syndrome coronavirus display late cell entry kinetics: evidence that transport to npc + endolysosomes is a rate-defining step ebolavirus glycoprotein directs fusion through npc + endolysosomes ebola virus glycoprotein with increased infectivity dominated the - epidemic human adaptation of ebola virus during the west african outbreak functional characterization of adaptive mutations during the west african ebola virus outbreak spontaneous mutation at amino acid of the ebola glycoprotein potentiates virus entry and selection in tissue culture biochemical basis for increased activity of ebola glycoprotein in the - epidemic a polymorphism within the internal fusion loop of the ebola virus glycoprotein modulates host cell entry the ebola virus glycoprotein contributes to but is not sufficient for virulence in vivo identification of the ebola virus glycoprotein as the main viral determinant of vascular cell cytotoxicity and injury ebola virus glycoprotein-mediated anoikis of primary human cardiac microvascular endothelial cells ebola virus glycoprotein toxicity is mediated by a dynamin-dependent protein-trafficking pathway the erk mitogen-activated protein kinase pathway contributes to ebola virus glycoprotein-induced cytotoxicity modeling of the ebola virus delta peptide reveals a potential lytic sequence motif ebola virus delta peptide is a viroporin ebola virus glycoprotein gp is not cytotoxic when expressed constitutively at a moderate level less is more: ebola virus surface glycoprotein expression levels regulate virus production and infectivity tetherin-mediated restriction of filovirus budding is antagonized by the ebola glycoprotein ebola virus glycoprotein counteracts bst- /tetherin restriction in a sequence-independent manner that does not require tetherin surface removal the tetherin antagonism of the ebola virus glycoprotein requires an intact receptor-binding domain and can be blocked by gp -specific antibodies ebola virus glycoprotein promotes enhanced viral egress by preventing ebola vp from associating with the host restriction factor bst /tetherin effects of ebola virus glycoproteins on endothelial cell activation and barrier function ebola hemorrhagic fever: novel biomarker correlates of clinical outcome ectodomain shedding of the glycoprotein gp of ebola virus shed gp of ebola virus triggers immune activation and increased vascular permeability ebolaviruses associated with differential pathogenicity induce distinct host responses in human macrophages ebola virus glycoprotein directly triggers t lymphocyte death despite of the lack of infection the myeloid lsectin is a dap -coupled receptor that is crucial for inflammatory response induced by ebola virus glycoprotein antigenic subversion: a novel mechanism of host immune evasion by ebola virus structures of ebola virus gp and sgp in complex with therapeutic antibodies gp mrna of ebola virus is edited by the ebola virus polymerase and by t and vaccinia virus polymerases genomic rna editing and its impact on ebola virus adaptation during serial passages in cell culture and infection of guinea pigs key: cord- - vkhptas authors: wu, tong; perrings, charles title: the live poultry trade and the spread of highly pathogenic avian influenza: regional differences between europe, west africa, and southeast asia date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: vkhptas in the past two decades, avian influenzas have posed an increasing international threat to human and livestock health. in particular, highly pathogenic avian influenza h n has spread across asia, africa, and europe, leading to the deaths of millions of poultry and hundreds of people. the two main means of international spread are through migratory birds and the live poultry trade. we focus on the role played by the live poultry trade in the spread of h n across three regions widely infected by the disease, which also correspond to three major trade blocs: the european union (eu), the economic community of west african states (ecowas), and the association of southeast asian nations (asean). across all three regions, we found per-capita gdp (a proxy for modernization, general biosecurity, and value-at-risk) to be risk reducing. a more specific biosecurity measure–general surveillance–was also found to be mitigating at the all-regions level. however, there were important inter-regional differences. for the eu and asean, intra-bloc live poultry imports were risk reducing while extra-bloc imports were risk increasing; for ecowas the reverse was true. this is likely due to the fact that while the eu and asean have long-standing biosecurity standards and stringent enforcement (pursuant to the world trade organization’s agreement on the application of sanitary and phytosanitary measures), ecowas suffered from a lack of uniform standards and lax enforcement. highly pathogenic avian influenzas have become a major threat to human and livestock health in the last two decades. the h n panzootic ( ongoing) has been one the most geographically widespread and costly, resulting in the loss of hundreds of millions of poultry in countries [ ] and over human deaths worldwide-a mortality rate of percent [ , ] . for h n , and other h subtypes, most countries reporting poultry outbreaks also report evidence of the disease in wild bird populations, and the mechanisms for the spread of h n have been identified as a combination of wild bird transmission and the live poultry trade [ , ] . plos risk factors. our primary interest is in the role of live poultry imports as a source of traderelated avian influenza risk at the regional level. we note that other poultry products, such as packaged meat and eggs, do pose a risk, but it is significantly lower. although avian influenza can persist in frozen meat, contact with that meat is unlikely to cause infection [ ] . furthermore, since hpais are lethal to egg embryos, eggs are not a potential source of transmission [ ] . the data comprise an unbalanced panel covering countries over years; the lack of balance is due to the fact that membership of the eu changed over the timeframe. the response variable in all models estimated was a log transformation of the number of h n poultry outbreaks in a given country in a given year, obtained from the emergency prevention system for animal health (empres), a joint project of the fao and oie [ ] . the log transformation was applied to account for the wide disparities in the numbers of the outbreaks across countries. in , for example, indonesia recorded outbreaks while romania, the only eu country to be infected that year, had only . in addition to reflecting the differing directions and intensities of risk factors, this also reflects differences in reporting conventions for h n at the international level [ ] . a series of outbreaks may be reported separately in one country, but be treated as a single event in another. data on trade in live poultry were obtained from the united nations' comtrade database (comtrade.un.org) and resourcetrade.earth, a project of the royal institute of international affairs (www.chathamhouse.org). these report the total imports of live poultry into a given country in a given year by weight (kg). the data on trade in live poultry did not distinguish between different types of domestic birds, such as chickens, duck, and geese, but grouped them under a single commodity category of "live poultry." with respect to wild bird migration as a pathway for h n spread, we used the density of wild bird habitat as a proxy for the presence and scale of migratory bird populations, and the likelihood that wild and domestic birds will mix. lakes, wetlands, and (irrigated) agricultural areas have been consistently identified as wintering and breeding grounds for migratory birds, and as places where wild birds may come into contact with free-ranging poultry [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the indicator for wild bird habitat used in this study was the set of "important bird and biodiversity areas" (ibas) for "migratory and congregatory waterbirds" identified by birdlife the live poultry trade poses different avian influenza risks in different regions of the world table . the distribution of h n poultry outbreaks between - across the member states of asean, ecowas, and eu. "-" signifies that the country was not a member of its associated trade bloc in that given year . brunei cambodia indonesia laos international (datazone.birdlife.org). in their analysis of h n spread, kilpatrick, chmura ( ) also identified ibas as a proxy for migratory birds and the infection risks they pose. country-level statistics on socioeconomic and agro-ecological conditions were taken from the united nations' food and agriculture organization (www.fao.org/faostat/en/) and the world bank (data.worldbank.org). agricultural land cover was reported as a percentage of total land area of the country. per-capita gdp was reported in purchasing power parity terms as current international dollars. data for for these two variables were missing for certain countries. in these cases, the gaps were filled by extrapolating the missing data as a linear trend of the preceding years. we assume that agricultural land-where free-ranging chickens, ducks, and geese are commonly raised in all three regions-also acts as a relevant proxy for susceptible poultry. data on the biosecurity measures targeting avian influenza undertaken by each country were obtained from the world organisation for animal health (oie) (www.oie.int). these report a standardized series of biosecurity controls targeting wildlife and livestock diseases, including those related to surveillance, vaccination, border checks, and management of wild disease reservoirs, and whether or not a given country undertook them in a given year. we chose a subset of these biosecurity measures we considered most relevant to h n avian influenza risks for inclusion in our model. additionally, in any given year, there were to countries that did not provide a report of biosecurity measures to the oie; we assumed that this indicates an absence of action, and the dataset records these cases as zeroes. our modeling approach relied on generalized linear models (glm) to analyze a panel of data on disease outbreaks and associated risk factors. in this we follow others who have sought to predict the spread of h n at both national and international levels [ ] [ ] [ ] or h n [ ] [ ] [ ] . glms are well suited to epidemiological studies because of their flexibility regarding data type and the distribution of response variables, their simplicity of application, and their frequency of use [ ] . our identification strategy involved the selection of three specifications for each of two estimators. we adopted both random and fixed effects estimators. hausman tests conducted at the all-regions level favored a random effects estimator, as the p-value exceeded the % threshold below which fixed-effects regression is conventionally considered necessary. some factors that influence the likelihood and number of outbreaks in a given country or region are not likely to change significantly over the course of several years, or even a decade. in our dataset, netherlands for example, the amount of land covered by wild bird habitat is time-variant, while agricultural land and even per-capita gdp for many countries experienced relatively modest variations over the timeframe of the study. in this case, and as the hausman diagnostics indicate, a random effects estimator is more appropriate. nevertheless, since we wished to control for timeinvariant characteristics of regions and countries we also implemented fixed effects estimators at both the aggregate and trading bloc levels, implicitly assuming no changes in the trade or biosecurity environment at the bloc level that we are unable to control for. our first specification (model ) included a number of factors related to disease risk but excluded both live poultry imports and biosecurity measures. included predictors were land area, human population, per-capita gdp in purchasing power terms, agricultural area, wild bird habitat area, and the live chicken population. our second specification (model ) added intra-regional trade bloc and extra-bloc imports of live poultry. our third specification (model ) added four main biosecurity measures: border precautions, general surveillance, vaccination prohibition, and wild disease reservoir management. all are categories of oie-reported biosecurity measures taken against avian influenza. the general forms of the estimated random and fixed effects models were: where y it denotes the number of poultry outbreaks in country i in year t, x includes the predictors for model , z includes the additional predictors for model , u includes the additional predictors for model , ecowas and asean are dummy variables for the two titular regional trade blocs (the eu is the reference group), and u it and ε it are the "between" and "within" errors respectively. to account for heteroskedasticity, we used robust standard errors. finally, since the data used in this analysis are reported annually, and h n has been a conspicuous and fast-moving epidemic (meaning the effects of an outbreak are unlikely to persist over a long period of time) among poultry, we did not use a lag structure in our statistical analysis. therefore, we assumed that the factors driving an outbreak in a given year are contemporaneous with it (e.g., an outbreak that occurred in were modelled using trade volumes from ). we were also constrained by data availability in our use of annual increments: although monthly data exist for outbreaks, they do not for important predictor variables such as percapita gdp, human and poultry populations, the volume of live poultry traded, and biosecurity. regressions results from all models, including both random and fixed effects, are reported in tables - . at the all-regions level, the results for the random-and fixed-effects models were very similar, with the same set of predictor variables being statistically significant (i.e., p-values below the % or %) and the same direction of impact on the response variable. this set of predictors was human population (positive direction), per-capita gdp (negative direction), intra-trade bloc live poultry imports (negative direction), extra-trade bloc live poultry imports (positive direction), and the biosecurity measure of surveillance (negative direction). additionally, although the coefficient values for the same predictor differed between the two estimators, all pairs were within the same order of magnitude. the only exception to this was migratory waterbird habitat variable-the percent of land area covered by ibas for migratory and congregatory waterbirds. this was statistically significant and negative (i.e., had a mitigating impact on h n poultry outbreaks) for the fixed-effects model but was not significant for the random-effects model. the overall r-squared for the random-effects model was significantly higher than that for the fixed-effects model ( . vs. . ). the "between rsquared" value was particularly high ( . ) in the random effects model, signaling the importance of variation among countries (as opposed to "within r-squared," which measures the variation within countries over time). as we had expected, we found significant differences across trade regions. in the randomeffects model, ecowas diverged from all-regions conditions and from asean with respect to per-capita gdp and extra-bloc imports: while the two predictors were, respectively, riskdecreasing and risk-increasing at the all-regions level and in asean, they had the opposite impacts in ecowas. furthermore, ecowas differed from the all-regions level and from the eu in terms of intra-bloc imports: while this was risk-decreasing for the former two, it was risk-increasing for ecowas. finally, there were predictors that were statistically insignificant at the all-regions level but had a significant effect within different regions. for asean, agricultural land cover was a mitigating factor for outbreaks while wild disease reservoir management showed a strong positive relation with outbreaks. for ecowas, wild waterbird habitats and border precautions had a mitigating effect on outbreaks while vaccination prohibition and wild reservoir management had a positive effect. in the eu, the population of live chickens had a strong negative relation table . results from the regression models of h n outbreak risk factors for member states in all three regions; regressor coefficients are reported and statistically-significant factors are marked by asterisks. a blank space signifies that the variable was not included in the given model. units model model model with outbreaks, while vaccination prohibition, similar to the case with ecowas, was positively related. following liang, xu ( ), there is a perception that the long distance transmission of highly pathogenic avian influenza h n was largely due to wild bird migration, with the live poultry trade playing a minor and more localized role in some cases. our concern here has been to identify the nature of the risk posed by the live poultry trade in different regions of the world, and the conditions affecting that risk. our measure of development status, per-capita gdp, is simultaneously a proxy for modernization, biosecurity, consumption, and value-at-risk. as a proxy for modernization, it reflects risk-reducing differences in production methods. industrial livestock production methods typically include on-farm biosecurity measures that protect poultry from contact with disease-carrying wild birds. unlike traditional methods of free-range or "backyard" husbandry, factory production minimizes the likelihood of poultry intermingling with wild birds or being exposed to environmental pathogen pollution. for all its epidemiological, ecological, and ethical problems, industrial livestock production allows for more timely and widespread disease surveillance and vaccination, and for greater compliance with animal health regulations [ ] . at the same time, per-capita gdp growth is also associated with risk-increasing changes in meat consumption, and hence poultry production. indeed, the highest income elasticity of demand for meat and fish has been found in the poorest households and the poorest countries [ ] . in developing countries, % of the additions to meat consumption are from pork and poultry, with poultry dominating pork [ ] . absent changes in on-farm biosecurity, increased table . results from the regression models of h n poultry outbreak risk factors for the association of southeast asian nations (asean); regressor coefficients are reported and statistically-significant factors are marked by. a blank space signifies that the variable was not included in the given model. units model model model production implies increased risk. across all regions, the net effect of income growth is to reduce risk, dominating risk-increasing changes. in the ecowas region-the lowest income region-the effect is the opposite. the risk-increasing effects of income growth dominate the risk reducing effects (table ) . amongst the landscape variables-land area, the proportion in agriculture, and the proportion in ibas-our results reveal no uniform relation to h n outbreaks. at the all-regions level we found a weakly negative relation between outbreaks and the proportion of the land area in ibas (table ). this was driven by the european union, which includes the highest proportion of land area in ibas, but also the most industrialized forms of poultry production. the degree to which poultry production is industrialized also shows up in the coefficients on poultry numbers, which are negative and significant only for the eu (table ). while spatial heterogeneity at the landscape scale is important in terms of avian ecology, we were unable to take explicit account of these more detailed considerations in a country-scale analysis. the impacts of regional differences in biophysical conditions that are not directly controlled for are, however, included in bloc-level fixed effects. our primary concern is with the role of the live poultry trade, and how that differs between regions. across all regions we find that live poultry imports into a trade bloc are risk increasing. this is consistent with past studies that have shown that extra-bloc live poultry imports may be a significant source of additional avian influenza risk where they do not meet bloc sanitary and phytosanitary standards. the eu's common market and the asean free trade regime in particular have long-standing and standardized protocols, in accordance with the world trade organization's agreement on the application of sanitary and phytosanitary measures. but the two blocs have quite different exposures to external risk. a study of highly pathogenic avian influenza introductions to vietnam, for example, found that extra-asean imports of table . results from the regression models of h n poultry outbreak risk factors for the economic community of west african states (ecowas); regressor coefficients are reported and statistically-significant factors are marked by asterisks. a blank space signifies that the variable was not included in the given model. units model model model population # people . x - . x - �� . x - . x - �� . live poultry increased the risk of introduction [ ] . this is also what our study finds for the asean region (table ) . we do not see an equivalent effect for the eu (table ), reflecting differences in both import volumes and the biosecurity measures applied to imports. the eu imports less and applies stricter biosecurity measures to those imports. the ecowas story is different. extra-bloc live poultry imports are risk reducing, not risk increasing (table ). it is likely that imports from outside the bloc reduce avian influenza risk in the region in part because they meet biosecurity standards that are more stringent than the standards applied in the region. the effects of intra-bloc trade in live poultry mirror the effects of extra-bloc trade. in the eu and asean, intra-bloc trade is risk reducing (tables and ). this may reflect a "substitution effect" in which imports of safer intra-bloc poultry crowds out riskier extra-bloc imports. other studies have come to similar conclusions. eu-derived live poultry imports to spain, for example, were found to pose no threat of avian influenza introduction [ ] . once again, eco-was is the exception. extra-ecowas imports of live poultry are risk reducing while intrabloc imports are risk increasing (table ). this is likely due to poor internal biosecurity, such as lax standards and inconsistent execution of inspections. regulatory standards within the ecowas trade bloc have been weak for the whole of the study period [ ] . while harmonized sanitary and phytosanitary standards for the member states of ecowas were in principle adopted in , most ecowas states had yet to submit legislation for international certification by [ ] . failure to adopt and enforce unified standards may be partly due to income constraints in ecowas countries. in ppp terms, the bloc's per-capita gdp in was less than half that of asean and approximately / th that of the eu, meaning it had less resources available for biosecurity policies and institutions. political instability may be another important obstacle: a table . results from the regression models of h n poultry outbreak risk factors for the european union (eu); regressor coefficients are reported and statistically-significant factors are marked by asterisks. a blank space signifies that the variable was not included in the given model. units model model model the live poultry trade poses different avian influenza risks in different regions of the world number of ecowas member states, including nigeria, niger, sierra leone, mali, liberia, and cote d'ivoire have suffered from civil wars and armed insurgencies over the past two decades. such fraught geopolitical conditions are not conducive to the establishment and enforcement of cross-border regulations. it goes without saying, though, that certification of sanitary and phytosanitary legislation in ecowas states, and the establishment of enforcement agencies to bring states into compliance with the sps agreement and codex alimentarius is a necessary condition of improving regional trade-related biosecurity. in terms of biosecurity measures more specifically, we did not have direct measures of onfarm biosecurity (but conjecture that biosecurity is increasing in per-capita gdp), but we did have measures of four biosecurity policies at the national level. these include: ( ) border precautions (measures applied at airports, ports, railway stations or road check-points open to international movement of animal, animal products and other related commodities, where import inspections are performed to prevent the introduction of the disease, infection or infestation); ( ) general surveillance (surveillance not targeted at a specific disease, infection or infestation); ( ) prohibition of vaccination (prohibition of the use of a vaccine to control or prevent the infection or infestation); and ( ) management of wildlife reservoirs (measures to reduce the potential for wildlife to transmit the disease to domestic animals and human beings). the management of wild disease reservoirs differs widely across countries, but techniques include vaccination, treatment of infections with drugs, isolation of infected populations, population translocation, reproduction reduction, culling, and control (draining, flooding, or burning) of wild disease reservoir habitat [ ] . of these measures, only general surveillance was significant at the all-regions level, while at the bloc level the effects of the different measures were frequently ambiguous. in the eu, for example, only the prohibition of vaccination was significant, and then in positive relation to outbreaks. for poultry, vaccination may be prohibited because the practice makes it difficult to distinguish infected from vaccinated flocks. this makes it a concomitant of policies centered on livestock culling as the primary response to outbreak risk [ ] . no other biosecurity policy was found to have a statistically significant relation to outbreaks in the region. the same set of policies had opposite effects in asean and ecowas. the prohibition of vaccination and the management of wild reservoirs were positively related to outbreaks in ecowas but negatively related to outbreaks in asean, while border protection measures were negatively related to outbreaks in ecowas but positively related to outbreaks in asean. this may reflect regional disparities in the quality of implementation not captured in the data. but it may also reflect the greater importance of trade in the transmission of the disease in ecowas. in their survey of the international spread of h n in the early years of the global epidemic, kilpatrick, chmura ( ) found that transmission into europe was by wild birds, that transmission into southeast asia was by the poultry trade, and transmission into africa by a balance of both. our results suggest that after introduction, inter-country spread had differing dynamics in each region. while intra-bloc trade facilitated h n spread among west african countries, it did not in either europe or southeast asia. in these areas, greater risk was posed by out-ofregion live poultry imports. in recent decades, avian influenzas have emerged as a major threat to human and animal health across the world. in particular, hpai h n , which was first isolated in , has been the most widespread and among the most devastating in terms of livestock and human mortality. it has inflicted severe losses to poultry stocks and caused hundreds of human deaths. even today, as other avian influenzas have become epidemic, h n remains in circulation among wildlife and livestock. identifying and quantifying the mechanisms of its international spread can help lay the groundwork for prediction and mitigation. it may also provide an instructive framework for the management of other avian influenzas. in this study, we considered the risk posed by the international trade in live poultry and the effects of associated biosecurity measures. differing agro-ecological and socioeconomic conditions across the trade regions were shown to influence epidemic dynamics in different ways, with certain factors being risk-enhancing or risk-decreasing in one region but having the opposite effect, or no significant effect, in another. in policy terms, there is no one-size-fits-all solution to mitigating avian influenza spread. the particular conditions, including those related to the trade agreements and associated regulatory standards, of a given region need to be carefully considered. but overall, biosecurity measures are potentially effective at controlling h n risks, and should be undertaken as a means to forestall spread-in general, mitigation of epidemics is significantly more cost-efficient than suppression [ ] . on-farm and other forms of domestic biosecurity may be more important than trade-related measures, but where the protection of trade pathways is weak, the risk of avian influenza spread is clearly higher. supporting information s file. detailed information on data sources. the public sources of the data used in this study, and how they were acquired, are described. 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global strategy to address the pandemic threat we would like to thank ann kinzig, jim collins, ben minteer, and peter daszak for their insightful comments and discussions on the research presented here. conceptualization: tong wu, charles perrings. key: cord- -vclnb eh authors: de almeida, carlos podalirio borges; ziegelmann, patrícia klarmann; couban, rachel; wang, li; busse, jason walter; silva, denise rossato title: predictors of in-hospital mortality among patients with pulmonary tuberculosis: a systematic review and meta-analysis date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: vclnb eh background: there is uncertainty regarding which factors are associated with in-hospital mortality among patients with pulmonary tb (ptb). the aim of this systematic review and meta-analysis is to identify predictors of in-hospital mortality among patients with ptb. methods: we searched medline, embase, and global health, for cohort and case-control studies that reported risk factors for in-hospital mortality in ptb. we pooled all factors that were assessed for an association, and presented relative associations as pooled odds ratios (ors). results: we identified , records, of which we retrieved in full text; cohort studies that evaluated , patients proved eligible. moderate quality evidence suggested an association with co-morbid malignancy and in-hospital mortality (or . ; % ci . – . ). low quality evidence showed no association with positive sputum smear (or . ; % ci . – . ), or male sex (or . , % ci . – . ), and very low quality evidence showed no association with diabetes mellitus (or . , % ic . – . ), and previous tb infection (or . , % ci . – . ). conclusion: co-morbid malignancy was associated with increased risk of in-hospital death among pulmonary tb patients. there is insufficient evidence to confirm positive sputum smear, male sex, diabetes mellitus, and previous tb infection as predictors of in-hospital mortality in tb patients. mortality and worse outcomes compared with women , . previous tb with multiple treatments has also been associated with in-hospital mortality [ ] [ ] [ ] . furthermore, patients with malignant tumors are immunocompromised and can have unusual clinical presentations, both related to delayed diagnosis and high mortality [ ] [ ] [ ] . in tb program monitoring, tb deaths are crucial indicators of the impact of tb control measures [ ] [ ] [ ] [ ] [ ] , especially in areas with high hiv and tb prevalence. data on tb deaths should provide us with a better understanding of the factors associated with these deaths and help guide interventions to reduce mortality; however, there is uncertainty regarding which factors are associated with in-hospital mortality among patients with pulmonary tb . we therefore conducted a systematic review and meta-analysis to establish predictors of in-hospital mortality among patients with pulmonary tb. search strategy. we used a multimodal search strategy focused on bibliographical databases (medline, embase and global health). an experienced librarian (rc) used medical subject headings, adding terms and keywords from a preliminary search to develop the database search strategies. in each database, the librarian used an iterative process to refine the search strategy through testing several search terms and incorporating new search terms as new relevant citations were identified. there were no language restrictions. the search included the following databases from inception to november : medline, embase and global health. the search consisted of three concepts combined using the and operator : tuberculosis , hospitalization and mortality (appendix ). the protocol of this study was published elsewhere . study selection. eligibility criteria. eligible trials met the following criteria : cohort or case-control design ; explored risk factors for in-hospital mortality among patients with pulmonary tb in an adjusted analysis. assessment of study eligibility. two reviewers (cpba and drs) trained in health research methodology screened, independently and in duplicate, the titles and abstracts of all citations identified in our search. the same reviewers screened all full text articles for eligibility; disagreements were resolved by consensus, with consultation of a third investigator (jwb) when resolution could not be achieved. we measured agreement between reviewers with the kappa statistic to assess the reliability of full-text review using the guidelines proposed by landis and koch : < . as slight agreement, . - . as fair agreement, . - . as moderate agreement, . - . as substantial agreement and > . as almost perfect agreement. assessment of study quality. two reviewers (cpba and drs) assessed risk of bias for each eligible study, independently and in duplicate, using the newcastle-ottawa quality assessment scale (nos) for cohort studies . the scale consists of nine items that cover three dimensions : patient selection ( items) ; comparability of cohorts on the basis of the design or analysis ( items); and assessment of outcome ( items). a point is awarded for each item that is satisfied by the study. the total score therefore ranges from zero to nine, with higher scores indicating higher quality. a total score ≥ represents high quality. data extraction and analysis. two reviewers (cpba and drs) extracted data from each eligible study, including demographic information (e.g. sex, age, race), methodology, and all reported predictors. we performed meta-analysis for all predictors that were reported by more than one study. we used odds ratios (ors) with associated % ci to measure the association of binary predictors and in-hospital mortality. we used random effects models for all meta-analyses. if a study reported more than regression model, we used data from the most fully adjusted model presented. we also presented the results from the predictors explored by the studies but that were not eligible for meta-analysis. we evaluated heterogeneity for all pooled estimates through visual inspection of forest plots, because statistical tests of heterogeneity can be misleading when sample sizes are large and cis are therefore narrow . we used the software r. publication bias. for meta-analyses with at least studies, we assessed publication bias by visual assessment of asymmetry of the funnel plot and performed the begg rank correlation test . quality of evidence. we used the grading of recommendations assessment, development and evaluation (grade) approach to summarize the quality of evidence for all meta-analyses . we categorized the confidence in estimates (quality of evidence) as high, moderate, low or very low, on the basis of risk of bias , imprecision , indirectness, inconsistency and publication bias . we used grade evidence profiles to provide a succinct, easily digestible presentation of the quality of evidence and magnitude of associations . in case of doubt or missing details about the studies, authors were contacted for clarification. ethics and dissemination. this study is based on published data, and therefore ethical approval was not a requirement. this systematic review and meta-analysis is expected to serve as a basis for evidence to reduce in-hospital mortality in tb patients, and as a guide for future research based on identified knowledge gaps. it is anticipated that findings from this review will be useful for informing policy, practice and research priorities, improving the management of in-hospital tb patients. we also plan to update the review in the future to monitor changes and guide health services and policy solutions. search results and study characteristics. we identified , unique records, of which we retrieved english and non-english language articles in full text; cohort studies, published between and , that evaluated , patients proved eligible. figure shows the study selection flow diagram. there was substantial agreement (κ = . ) at the titles and abstract screening stage and perfect agreement (κ = . ) between reviewers at the full-text review stage. all eligible studies , , , - were single-center and there was one non-english (chinese) study included in our analysis. two studies , were conducted in japan, two , in taiwan, three , , in korea, one in germany, one in israel, one in iran and one in china. one study used tb-related mortality as defined by the world health organization (the number of tb patients who died during treatment, irrespective of cause) , two , used all-cause mortality, and eight , , , , , , , used tb-related mortality as judged by the investigators. the majority ( of ) , , , , - , , acquired data from medical records, with eight retrospective cohorts , , - and one prospective cohort study (table ) . overall, the quality, evaluated by the nos checklist for the outcome "mortality", was high (table ) . we did not have a sufficient number of studies in our meta-analyses to assess publication bias. a total of studies, involving a total of patients, reported the association of factors with in-hospital mortality , , , [ ] [ ] [ ] [ ] [ ] [ ] . on the basis of our criteria, we conducted meta-analyses for predictors of in-hospital mortality : acid-fast bacilli (afb) smear positive , diabetes mellitus , malignancy , history of previous tb, and . ; table ). table presents the associations with in-hospital mortality for the factors that were not amenable to meta-analysis. we found moderate quality evidence that co-morbid malignancy was associated with increased in-hospital mortality among tb patients. low quality evidence showed that sex and afb smear positive were not associated with in-hospital mortality, and very low quality evidence showed no association with previous tb infection and diabetes mellitus. our review has a number of strengths. our search, which had no language restrictions, was designed and implemented by a research librarian, and literature screening and data extraction were performed independently and in duplicate by two reviewers using pretested, standardized extraction forms. the main limitation of our review was the small numbers of events that contributed to our meta-analyses, resulting in wide estimates of precision for our pooled measures of association. other studies - also found that malignancy increases the risk of death in tb patients. patients with malignant tumors are immunocompromised due to the local or systemic effects of the disease itself, as well as to the treatment regimens, which can impair the immune system and make these patients particularly susceptible to developing tb . in addition, tb can have an unusual clinical presentation, making diagnosis more difficult in these patients, contributing to delay in diagnosis and high mortality rates , . while not significantly associated with mortality in our review, previous tb has been reported to be associated with in-hospital mortality in many studies , - . patients who undergo multiple treatment regimens for tb can develop resistance to drugs with the subsequent emergence of mdr-tb and xdr-tb, conditions highly associated with greater risk of death . further, in settings other than hospitals, studies , have demonstrated that smear positive patients have a better prognosis regarding mortality than smear negative patients. indeed, indicators of atypical manifestations, such as smear-negative sputum, were associated with delayed diagnosis and recently, a retrospective cohort study from brazil reported a high mortality rate during hospitalization ( . %), and negative sputum smear microscopy was an in-hospital mortality predictor in the population studied. however, patients with pulmonary and extrapulmonary tb were included in this study. we did not find a significant association between male sex and in-hospital mortality among pulmonary tb patients. worldwide tb notification data show that far more men than women have tb . some studies showed that mortality rates are higher in females during their reproductive years, but after that they are higher in men , . diabetes was also not associated with mortality in pulmonary tb patients in this study. only one study included in this meta-analysis showed that diabetes was a predictor of mortality in tb patients, possibly because they included a larger number of diabetes patients ( % of the enrolled individuals). some studies , - have found that diabetes increases risk of early mortality during tb treatment. this effect may be explained by impaired tb treatment response . in conclusion, the presence of malignancy was significantly associated with in-hospital death in pulmonary tb patients. other predictors were not associated with in-hospital mortality in tb patients, probably due to the small number of events. further research should explore promising predictors of in-hospital mortality in large prospective studies. table . unpooled predictors for in-hospital mortality among tb patients. factors associated with mortality in tuberculosis patients prognostic factors in tuberculosis related mortalities in hospitalized patients mortality of patients hospitalized for active tuberculosis in israel high mortality in adults hospitalized for active tuberculosis in a low hiv prevalence setting factors associated with mortality in hospitalized patients with newly diagnosed tuberculosis geneva: world health organization. available at: www.who tuberculosis in hospitalized patients: clinical characteristics of patients receiving treatment within the first h after admission time delays in diagnosis of pulmonary tuberculosis: a systematic review of literature hospitalizations for tuberculosis in the united states in : predictors of in-hospital mortality quality of life in tuberculosis: patient and provider perspectives the impact of comorbidity on mortality following in-hospital diagnosis of tuberculosis experience with a medical-psychosocial inpatient unit delay in diagnosis among hospitalized patients with active tuberculosis-predictors and outcomes the impact of nutritional deficit on mortality of in-patients with pulmonary tuberculosis diabetes is a strong predictor of mortality during tuberculosis treatment: a prospective cohort study among tuberculosis patients from mwanza, tanzania impact of diabetes and smoking on mortality in tuberculosis diabetes mellitus is associated with increased mortality during tuberculosis treatment: a prospective cohort study among tuberculosis patients in south-eastern amahra region a review of sex differences in the epidemiology of tuberculosis social and cultural dimensions of gender and tuberculosis mortality among mdr-tb cases: comparison with drug-susceptible tuberculosis and associated factors differences between risk factors associated with tuberculosis treatment abandonment and mortality independent predictors of tuberculosis mortality in a high hiv prevalence setting: a retrospective cohort study initial presentations predict mortality in pulmonary tuberculosis patients-a prospective observational study tuberculosis mortality: patient characteristics and causes excess mortality due to tuberculosis and factors associated to death in and annual cohort of patients diagnosed of tuberculosis predictors of in-hospital mortality among patients with pulmonary tuberculosis: a protocol of systematic review and meta-analysis of observational studies the measurement of observer agreement for categorical data the newcastle-ottawa scale (nos) for assessing the quality of non-randomised studies in meta-analyses. ottawa hospital research institute undue reliance on i( ) in assessing heterogeneity may mislead operating characteristics of a rank correlation test for publication bias grading quality of evidence and strength of recommendations grade guidelines: . rating the quality of evidence-study limitations (risk of bias) grade guidelines . rating the quality of evidence-imprecision grade guidelines: . rating the quality of evidence-inconsistency grade guidelines: . rating the quality of evidence-publication bias characteristics and outcome of patients with active pulmonary tuberculosis requiring intensive care development and validation of a tuberculosis prognostic score for smear-positive in-patients in japan risk factors related with mortality in patient with pulmonary tuberculosis patient mortality of active pulmonary tuberculosis requiring mechanical ventilation predictive factors for mortality among non-hiv-infected patients with pulmonary tuberculosis and respiratory failure hypoalbuminemia and lymphocytopenia are predictive risk factors for in-hospital mortality in patients with tuberculosis prognostic factors in pulmonary tuberculosis requiring mechanical ventilation for acute respiratory failure global tuberculosis programme. a framework for effective tuberculosis control the risk of tuberculosis in patients with cancer solid-organ malignancy as a risk factor for tuberculosis risk factors for and attributable mortality from tuberculosis in patients with hematologic malignances the directly observed therapy short-course (dots) strategy in samara oblast, russian federation risk factors for death during tuberculosis treatment in orel, russia deaths from pulmonary tuberculosis in a low-incidence country all authors made substantial contributions to conception and design. c.p.b.a. designed the study, collected data, and wrote the manuscript. r.c. designed the search strategy. l.w. designed the study and collected data. p.z. analyzed data and wrote the paper. j.b. designed the study and wrote the paper. d.r.s. designed the study, collected data, and wrote the paper. all authors provided final approval of the version to be published. supplementary information accompanies this paper at https://doi.org/ . /s - - - . the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -d hetoq authors: meng, xiangzhi; zhang, fushun; yan, bo; si, chuanping; honda, hiroaki; nagamachi, akiko; sun, lu-zhe; xiang, yan title: a paralogous pair of mammalian host restriction factors form a critical host barrier against poxvirus infection date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: d hetoq host restriction factors constitute a formidable barrier for viral replication to which many viruses have evolved counter-measures. human samd , a tumor suppressor and a restriction factor for poxviruses in cell lines, is antagonized by two classes of poxvirus proteins, represented by vaccinia virus (vacv) k and c . a paralog of samd , samd l, is also encoded by some mammals, while only one of two paralogs is retained by others. here, we show that samd l functions similarly to samd as a restriction factor and that the two paralogs form a critical host barrier that poxviruses must overcome to establish infection. in mice, which naturally lack samd , overcoming samd l restriction with viral inhibitors is essential for poxvirus replication and pathogenesis. while a vacv deleted of both k and c (vk l(-)c l(-)) was restricted by mouse cells and highly attenuated in mice, its replication and virulence were completely restored in samd l(-/-) mice. in humans, both samd and samd l are poxvirus restriction factors, although the latter requires interferon induction in many cell types. while knockout of samd with crispr-cas was sufficient for abolishing the restriction for vk l(-)c l(-) in many human cells, knockout of both paralogs was required for abolishing the restriction in interferon-treated cells. both paralogs are antagonized by vacv k , c and c homologs from diverse mammalian poxviruses, but mouse samd l is resistant to the c homolog encoded by a group of poxviruses with a narrow host range in ruminants, indicating that host species-specific difference in samd /samd l genes serves as a barrier for cross-species poxvirus transmission. emerging and reemerging infectious diseases have continued to pose a major threat to public health. in particular, zoonotic viral infections have caused such lethal human diseases as sars, avian influenza, human monkeypox, and ebola [ ] . for many viruses, including coronaviruses and influenza viruses, host species-specific difference in viral entry receptors presents a major hurdle for cross-species transmission [ ] . poxviruses, however, can enter nearly any animal cell [ ] . why many poxviruses show strict host species specificity and what it would take for them to jump to new hosts are less clear [ ] . poxviruses include many lethal animal and human pathogens [ ] , the most infamous of which is the smallpox-causing variola virus. smallpox was successfully eradicated mainly through a global immunization program with vaccinia virus (vacv), and routine vacv vaccination had since discontinued. the human population is now vulnerable to zoonotic orthopoxvirus infection, as some extant poxviruses related to variola virus are capable of infecting a wide variety of wild and domestic animals. there are also many poxviruses with a more restricted host range [ ] . for example, capripoxviruses, consisting of sheeppox virus, goatpox virus, and lumpy skin disease virus, have a very narrow host-range in ruminants, causing economically significant diseases in sheep, goats, and cattle, respectively. host-restricted poxviruses have been exploited as safe vectors for vaccines, gene therapy or oncolytic viral therapies, although the basis for their host restriction is largely unknown [ ] . poxvirus host range at the cellular level is governed by a group of poxvirus genes referred to as the host range genes [ , ] . the first discovered and perhaps the most important host range genes are k l and c l of vacv [ , ] . vacv replication in most mammalian cell lines requires either k l or c l [ ] , and the deletion of both genes from vacv aborts the replication prior to viral late gene expression [ ] . k l is only present in vacv and a few related orthopoxviruses, but a c l homolog that functions nearly identically to vacv c l is present in most mammalian poxviruses [ ] . samd (sterile alpha motif domain-containing ) was found to be the restriction factor in human cell lines that blocked the replication of poxvirus mutants that lack k l and c l-like genes [ ] . k and c are structurally distinct [ , ] , but k and many c homologs independently bind and inhibit human samd [ , , ] . samd was initially identified as a tumor suppressor whose loss-of-function mutations in humans cause normophosphatemic familial tumoral calcinosis (nftc) [ , ] . humans and many other mammals also encode a chromosomally adjacent paralog of samd named samd -like (samd l). human samd and samd l are ubiquitously expressed in many tissues [ ] , and their expression can be further induced by interferons [ , ] . evolutionary analysis suggested that the two paralogs derived from the duplication of an ancestral gene early in mammalian evolution and that some mammalian species suffered a loss of either samd or samd l [ ] . notably, mice lack samd , while many ruminants lack samd l. mouse samd l is also a tumor suppressor, and haploinsufficiency of mouse samd l resulted in myeloid malignancies [ ] . the molecular functions of samd family of proteins remain largely elusive, but recent sequence analysis predicted a complex domain architecture suggestive of a regulative function of a putative signal transduction network [ ] . although samd has been established as a critical poxvirus restriction factor in human cell lines, whether this restriction is important at the organismal level and in other mammalian species is unknown. moreover, whether samd l plays a role in host defense is unknown. we studied the function of samd l from a host that lacks samd (mice) as well as a host that maintains both paralogs (humans). we found that samd l functions nearly identically to samd as a host restriction factor and that overcoming samd /samd l (samd /l) restriction is essential for poxvirus replication and pathogenesis. we also discovered some host species-specific difference in samd /l and some poxvirus species-specific difference in antagonizing samd /l, suggesting that these differences contribute to the barrier for crossspecies poxvirus infection. the importance of human samd as a poxvirus restriction factor became evident only when viral inhibitors of samd were removed from poxviruses, so we used a vacv mutant deleted of both k l and c l (vk l -c l -) as the model poxvirus to study poxvirus restriction factors in different host species. mouse is one of the mammalian species that have lost samd gene, and a previous study suggested that mouse samd l was not a functional paralog of human samd in terms of the tumor suppressor function [ ] . however, mouse cells, such as nih t cells and mouse embryonic fibroblasts (mefs), restricted the replication of vk l -c l - [ ] . to test whether mouse samd l (msamd l) might be the restriction factor for vk l -c l -, we used the crispr-cas technology to knock out msamd l from t cells. to control for potential off-target effect, we performed two independent knockouts with different guide sequences. to confirm the gene knockout, cell clones were isolated, and the msamd l genotype of representative clones from the two knockouts (named Δmsamd l# and Δmsamd l# ) was determined by sequencing. all msamd l alleles of the cell clones were found to contain indels, resulting predominantly in frameshift (s a fig) . while vk l -c lwas unable to replicate in the parental t cells, it replicated well in Δmsamd l# ( fig a) and Δmsamd l# (s b fig) cells, resulting in nearly -fold increase of viral titer after hours of infection. moreover, while vk l -c lwas sensitive to interferons (ifns) in permissive human cells [ , ] , its growth in Δmsamd l cells was not inhibited by pretreating the cells with ifn-β ( fig a) . as an alternative to the crispr-cas knockout, we prepared mefs from samd l +/+ and samd l -/mice. while vk l -c lfailed to grow in samd l +/+ mefs, its replication was completely restored in samd l -/-mefs (fig b) , confirming that msamd l is the restriction factor for vk l -c lin mouse cells. vk l -c lis highly attenuated in mice [ ] . to determine whether msamd l functions as a restriction factor at the organismal level, we tested whether vk l -c lwould regain virulence in samd l -/mice. intranasal infection of samd l +/+ and samd l +/mice with plaque-forming unit (pfu) of vk l -c ldid not cause any disease symptom or sustained body weight loss, similar to the mock infection (fig a) . the infected mice developed an antibody response to vacv (s a fig), indicating that they were properly infected. in contrast to their samd l +/+ and samd l +/littermates, all samd l -/mice lost close to % of body weight by day post infection and had to be euthanized. a similar lethal effect on samd l -/mice was observed when the infectious dosage was reduced to or pfu (fig b) , indicating that vk l -c lwas highly virulent in samd l -/mice. in mice, samd l haploinsufficiency caused myeloid malignancies [ ] . to test whether samd l haploinsufficiency would increase susceptibility to vk l -c linfection, we infected samd l +/+ and samd l +/mice with the highest dosage of vk l -c lwe could reasonably obtain ( pfu in μl). again, neither group of mice developed any disease symptoms or had sustained body weight loss (fig c) , and no virus was detected in lungs when the mice were euthanized at five days post infection (s b fig), indicating that one copy of msamd l gene is sufficient for restricting vk l -c l -. altogether, the results from samd l knockout cells and mice demonstrate that msamd l constitutes an essential host barrier for vk l -c lreplication and pathogenesis. we previously used vk l -c las the parental virus in constructing a panel of recombinant viruses that expressed different c homologs from representative mammalian poxviruses [ , ] . this panel of viruses were used for comparing the function of different c homologs in the same viral background, and they served as a surrogate model for different mammalian poxviruses in terms of their ability to overcome samd restriction. studies of these viruses showed that the c homologs from many mammalian poxviruses, including myxoma virus (myxv; infect rabbits) myxv-m , yaba-like diseases virus (yldv; infect monkeys) yldv- , swinepox virus (swpv) swpv- , and sheeppox virus (sppv) sppv- , could bind human samd and overcome its restriction [ ] . all these c homologs, except for sppv- , could also overcome the restriction of vk l -c lby mouse cell lines [ ] . to determine vk l -c lvirulence was restored in samd l -/mice. littermates from samd l +/mice were grouped according to their samd l genotypes and infected intranasally with vk l -c lor mock infected (pbs). the body weight change following the infection (average and standard deviation) is shown for each group. " à " indicates statistically significant difference (p < . by two way anova analysis). n denotes group size. wt vacv wr strain was used as a control in c. (d,e,f). the c homolog from sheeppox virus (sppv- ) has a host-species specific defect in mice. same as a except the viruses used were vsppv- (d), vswpv- (e), or vvacv-c (f), which are vk l -c l --derived viruses that expressed sppv- , swpv- or vacv-c , respectively. whether the defect of sppv- in mouse cell lines reflects a host species-specific defect, we studied the mouse virulence of vsppv- , the vk l -c l --derived recombinant virus that expressed sppv- . swinepox virus swpv- is the closest homolog of sppv- , so vswpv- , the vk l -c l --derived recombinant virus that expressed swpv- , was used as the control. similar to vk l -c l -, vsppv- did not result in significant body weight loss in samd l +/or samd l +/+ mice but caused lethal intranasal infection in samd l -/mice ( fig d) . in contrast, the same dose of vswpv- was lethal to samd l +/+ and samd l +/mice as well as to samd l -/mice (fig e) . in a separate experiment, vvacv-c , the vk l -c l -derived recombinant virus that expressed vacv-c , was also lethal to samd l +/+ and samd l +/mice ( fig f) . as sppv- can overcome samd restriction in human cells, these data show that sppv- has a host species-specific defect in mice. vacv k , c and c homologs from diverse poxviruses could bind msamd l, but the sheeppox virus c homolog has a specific defect due to the variation of two amino acids a biochemical basis for samd antagonism by the poxvirus proteins is their binding of samd [ ] , so we next studied the binding of msamd l by the viral proteins. due to the lack of a specific antibody to msamd l, a plasmid encoding a flag-epitope tagged msamd l was used in transfection of cells, which were subsequently infected with recombinant viruses expressing different v -tagged viral proteins. pulldown of vacv-k , vacv-c , myxv-m , swpv- or yldv- also precipitated msamd l ( fig a) . however, sppv- failed to co-precipitate msamd l. as expected, myxv-m and myxv-m , which are two additional c homologs from myxv previously known not to bind human samd [ ] , also failed to co-precipitate msamd l. residue and of sppv- were previously shown to be largely responsible for the defect of sppv- in mouse cells, and substituting them with the corresponding ones found in swpv- and vacv-c was sufficient to restore the host-range function in mouse cells without compromising the function in human cells [ ] . the same substitution was found to result in the binding of sppv- with msamd l ( fig b) . as controls, individual substitutions of two neighboring residues (residue or ) did not result in msamd l binding, correlating with their lack of effect on the host-range function in mouse cells [ ] . interestingly, residues and are only adjacent to the three conserved loops of the c -like proteins ( fig b) that are critical for the binding with samd [ ] . the finding that mouse samd l functions similarly to human samd as a poxvirus restriction factor raised the question about the function of human samd l (hsamd l). since knockdown of human samd (hsamd ) was sufficient for abolishing the host restriction for vk l -c lin hela and a cells [ , ] , hsamd l was not previously suspected to play a role in host restriction. to find out whether hsamd l expression might be impaired in cell lines, we performed western blot on hela cells and normal human fibroblasts derived from foreskin, and found that both hsamd and hsamd l were constitutively expressed and their expression was further induced by ifn-β ( fig a) . to study hsamd l function independent of hsamd , we knocked out hsamd from hela cells with crispr-cas . in a hela cell clone with hsamd knockout (Δhsamd ), no hsamd was detected by western blot even after the cells were treated with ifn-β ( fig a) . Δhsamd cells were fully permissive for the replication of vk l -c l -, as reported previously https://doi.org/ . /journal.ppat. .g [ ] . however, pretreating the Δhsamd cells with ifn-β restored the host restriction for vk l -c l -, reducing the viral yield by more than -fold compared to that in untreated cells (fig b) . to test whether hsamd l was responsible for the restriction, we knocked out hsamd l from Δhsamd and the parental hela cells. both the hsamd l single-knockout cells (Δhsamd l) or hsamd and hsamd l double knockout cells (Δhsamd &l) had no detectable level of hsamd l protein in untreated cells and only a trace amount of the protein after ifn-β stimulation (fig a) . in contrast to Δhsamd cells, Δhsamd &l cells remained permissive for vk l -c lafter ifn-β treatment (fig b) , and the growth of vk l -c lwas similar to that of the wild type (wt) vacv wr strain (s fig), indicating that ifn-β-induced hsamd l restricted the replication of vk l -c lin the absence of hsamd . Δhsamd l cells were similar to the parental hela cells in restriction of vk l -c l - (fig b) . to ensure that the hela cell results are representative of the phenotype in human cells, we performed similar knockout studies in a variety of human cells that we identified to be restrictive of the replication of vk l -c l -. these include normal human foreskin fibroblasts (hffs) and cancer cells derived from skin (a ), breast (hs t and mda-mb- ), cervix (ht- ), prostate (pc- ) and ovary (skov ). we transduced these cells with a lentivirus expressing a grna targeting either hsamd or hsamd l and pooled the stably transduced cells. the samd and samd l protein level in the pooled cells was reduced but not eliminated as in the clonally selected hela knockout cells (s a & s a figs) , presumably because the targeted gene was repaired with in-frame indels in a fraction of the cells. thus, the cells were conservatively speaking gene knockdown instead of knockout cells. nevertheless, the results were similar to that in knockout hela cells. the hffs were less stringent than the hela cells in restricting vk l -c l -, allowing the viral titer increase by~ -fold after thus, in all human cells that we have tested, the basal level of hsamd as well as ifninduced hsamd l are both capable of restricting vk l -c lreplication. vacv k , c and c homologs from diverse poxviruses could overcome hsamd l restriction, but the sheeppox virus c homolog has reduced potency due to the variation of two residues to find out whether mammalian poxviruses could overcome the restriction of hsamd l, we induced hsamd l expression in Δhsamd hela cells with u/ml of ifn-β and then infected the cells with our panel of vk l -c l --derived vacvs. viruses that expressed vacv-k , vacv-c , myxv-m , sppv- , swpv- and yldv- (but not myxv-m and myxv-m ) grew in ifn-treated Δhsamd cells (fig a) , indicating that all known samd antagonists could also antagonize hsamd l. we then studied these viral proteins for binding of hsamd l. while myxv-m (s fig) and myxv-m did not co-precipitate hsamd l, vacv-k , vacv-c , myxv-m , yldv- , sppv- and swpv- co-precipitated hsamd l (fig b) . among them, sppv- precipitated a lower amount of hsamd l, indicating a reduced affinity. this defect was again due to residue and of sppv- , as substitution of these two residues increased the precipitation of hsamd l without affecting the precipitation of hsamd (s fig). to determine whether the weaker hsamd l binding affinity by sppv- resulted in reduced inhibitory potency, we induced an increasingly higher level of hsamd l from Δhsamd hela cells with ifn-β and then infected the cells with either vsppv- or vswpv- . the two viruses grew equally well in untreated Δhsamd cells, reaching similar titers after hours of infection (fig c) . the viral yields were gradually reduced by the increasing concentrations of ifn-β, and the magnitudes of the reduction were significantly larger for vsppv- than for vswpv- when the interferon concentration was greater than u/ml, indicating that sppv- is less effective than swpv- at antagonizing hsamd l. discussion samd was recently identified as a restriction factor for poxviruses in human cell lines [ , ] . however, whether samd is important for host defense against poxvirus pathogenesis and whether similar antiviral defense exists in other mammals, especially those that lack a samd ortholog, were not established. in this study, we showed that the samd paralog, samd l, from mammalian species as diverse as mice and humans, functions similarly to human samd as a poxvirus restriction factor. since at least one of the two paralogs is present in all mammals with a completely sequenced genome [ ] , our finding indicates that samd and/or samd l-mediated antiviral defense is conserved in mammals. this conservation allowed us to use the mouse model to reveal the critical role of samd /l in host defense against poxvirus pathogenesis. studies of samd /l from two different mammalian species and the samd /l inhibitors from diverse mammalian poxviruses revealed some host speciesspecific difference in samd /l, which could serve as a barrier for cross-species poxvirus infection. this knowledge could be useful in assessing the potential of a given poxvirus in switching or expanding its host range. mouse is one of the mammalian species that have lost samd but maintained samd l. mouse samd l is % and % identical to human samd and samd l at amino acid level, respectively. through gene knockout with both the conventional technique as well as the crispr-cas technique, we found that mouse samd l was essential for restricting the replication of a model poxvirus, a vaccinia virus mutant deleted of both k l and c l (vk l -c l -). t cells with crispr knockout of samd l as well as samd l -/-mefs were permissive for the replication of vk l -c l -. furthermore, vk l -c lcaused lethal intranasal infection only in samd l -/mice but was completely avirulent in samd l +/+ and samd l +/mice, demonstrating the potency of samd l as a restriction factor at the organismal level. human is one of the mammalian species that have both samd and samd l, located head-to-tail in adjacent positions of the same chromosome. previous phylogenetic studies suggested that samd and samd l originated from a common ancestor through an ancient gene duplication event [ ] . gene duplications have a major role in evolution of new biological functions [ ] . with about % amino acid sequence identity, human samd and samd l could have diverged to take on different functions. this idea would be consistent with the previous findings that loss-of-function mutations in only samd cause nftc in humans [ ] and that knockdown of samd was sufficient for abolishing the restriction for vk l -c lin several human cell lines [ , ] . we were thus initially surprised to find that human samd l functioned similarly to samd as a restriction factor for poxviruses. the advent of crispr-cas genome editing technique made it possible to readily knock out one or both of the human paralogs from various human cells. the knockouts in tumor cell lines from various tissues and the normal human foreskin fibroblasts showed that both samd and samd l, when sufficiently expressed, could inhibit vk l -c lreplication. in many human cells, the basal level of samd is sufficient for restricting vk l -c l -, but samd l has to be induced by ifn to have the same effect. this probably only reflects a difference in gene regulation of the two paralogs in human cells but not any real difference in their potency or mechanism of action. mouse samd l is also an interferon-stimulated gene [ ] , but the basal level of samd l in mouse cells is sufficient for restricting vk l -c l -, again suggesting that the role of ifn in samd l function is merely to induce samd l to a sufficient level in some cell types. similarly, the only contribution made by k and c in vaccinia virus antagonism of ifn or irf [ , ] is likely the inhibition of ifn-or irf -induced samd /l, as vk l -c lwas not sensitive to ifn in cells that had samd /l knockout. both human samd and samd l have strong anti-proliferative function, and gain-offunction (gof) mutations of samd or samd l cause multisystem developmental disorder. gof samd mutations cause mirage (myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes, and enteropathy) disorder [ ] , while gof samd l mutations cause a similar disorder characterized with cytopenia, immunodeficiency, and neurological symptoms [ , ] . in both cases, the gof mutations predispose to myelodysplastic syndrome by facilitating the selection for a loss of the chromosome that contain the mutations [ , ] . so why are both samd and samd l kept in humans and many other mammals? we can only postulate that the duplication of samd gene and the divergence of sequence and expression pattern represent a strategy for retaining two drastically different "alleles" of samd /l in the same host without the deleterious effect of both genes being constitutively active. having two different alleles of samd /l would have given the hosts more flexibility to rapidly evolve samd /l to evade the viral inhibitors, perhaps during a time of mammalian evolution when the challenge from poxviruses was particularly rampant. phylogenetic analysis has showed that both samd and samd l genes have been subjected to sustained positive selection [ ] . corresponding to the conservation of samd /l in mammals, the distribution of samd / samd l inhibitors in mammalian poxviruses are also very broad. nearly all mammalian poxviruses encode a c homolog that provides a host-range function similar to vacv c [ , , ] , and a few poxviruses also encode k (fig ) . all the functional c homologs and k were previously shown to bind and inhibit human samd [ , , ] . the breadth of their antagonistic activities is now expanded to include human and mouse samd l. among the c homologs from a wide variety of different mammalian poxvirus, the one from sheeppox virus, sppv- , stands out as the only one that displays a specificity for samd . sppv- could bind and inhibit human samd , but it has reduced potency against human samd l and failed to inhibit mouse samd l. remarkably, its binding to mouse samd l could be restored simply by substituting two residues, suggesting that subtle difference between samd and samd l might be responsible for their different ability in resisting sppv- . interesting, these two residues are only adjacent to the "three-fingered molecular claw" that is critical for samd binding [ ] , indicating that they may modulate the conformation of "the claw" to influence its binding specificity. sheeppox virus, goatpox virus, and lumpy skin disease virus (lsdv) are members of the capripoxvirus genus with a narrow host-range in ruminants. all capripoxviruses encode a nearly identical c homolog (> % identical) as sppv- , with the same residues at position and , suggesting that they all preferentially antagonize samd . this correlates with the loss of samd l in ruminants. thus, one evolutionary scenario is that the ancestor of sppv- could tolerate genetic drift that only disrupted samd l binding, after the ancestor of extant capripoxviruses established a niche host in ruminants and was no longer restricted by samd l. while the resulted failure of capripoxviruses in antagonizing mouse samd l could account for the host restriction of capripoxviruses in mice, the resulted reduction in potency against interferon-induced human samd l could only partly explain the host restriction of capripoxviruses in humans. we suspect that similar genetic drift might have occurred to other capripoxvirus host-range genes, resulting in their specialization to the ruminant hosts and altogether contributing to the host restriction of capripoxviruses in non-ruminant hosts. this idea is supported by a previous report that the sheeppox virus homolog of vacv host range gene e failed to provide the host range function in hela cells [ ] . that mouse samd l is completely resistant to sppv- while human samd l is only partially resistant could have been the result of a stronger positive selection in rodents, perhaps by some poxviruses that have similar inhibitory profile as the capripoxviruses. overall, our studies suggest that host species-specific difference in samd /l gene repertoire contributes to the barrier for cross-species poxvirus transmission. the animal studies reported in this paper were approved by the institutional animal care and use committee at the university of texas health science center at san antonio (protocol no. - ) , and pc- (atcc crl- ) were originally from atcc. hek ft was from thermo fisher scientific (cat. no. r ). mouse embryonic fibroblasts (mefs) were generated from embryos of samd l −/− and samd l +/+ mice according to standard protocols. human foreskin fibroblasts (hffs), described in [ ] , were kindly provided by dr. zhilong yang. wt vacv wr strain, k l and c l deletion vacv (vk l -c l -) and a panel of vk l -c l -derived recombinant viruses expressing vacv-k (vvacv-k l) or a c homolog from different poxviruses (vvacv-c l, vyldv- r, vmyxv-m r, vmyxv-m r, vmyxv-m r, vsppv- and vswpv- ) were described before [ , , ] . vsppv- with mutations at residue , , & of sppv- were also described before [ ] . all viruses were propagated and titrated on vero cells. the plasmids used for the gene knockout were constructed from lenticrisprv (a gift from feng zhang, addgene plasmid # ) according to the published protocol [ ] . in brief, len-ticrisprv was digested with bsmbi and ligated with a pair of oligonucleotides with the specific guide sequence. for each target gene, two guide sequences were chosen from the human or mouse genome-wide sgrna library [ ] . they were as follows: '-tatccgggaaccac ggttcg- ' (msamd l # ), '-acaccaacaatttccccgtg- ' (msamd l # ), '-ttgactgaacaagacgtgaa- ' (hsamd # ), '-gagaatttgttcttcgatac- ' (hsamd # ), '-cctgaccagttagacgacgc- ' (hsamd l # ), and '-ggctagctc tagggatatcc- ' (hsamd l # ). the lenticrisprv -derived plasmid was either transfected directly to the target cells or used in making lentiviruses for transduction of the target cells. in the latter case, the lentiviral plasmid and the packaging plasmids pmd .g and pspax (gifts from didier trono, addgene plasmid # & ) were transfected into hek ft cells with lipofectamin (thermo fisher scientific). hr post transfection, culture supernatants were collected, clarified by centrifugation, passed through a . μm filter, and used for transduction. for lentiviral transduction, the lentiviruses and the target cells in medium containing μg/ml polybrene were centrifuged at , rpm for hr. hr after either transfection or transduction, the cells were subjected to puromycin selection for (transfection method) or (transduction method) days. the puromycin concentrations are μg/ml for hela, μg/ml for t , and μg/ml for all other cells. for each gene knockout in t and hela cells, two separate knockouts with different guides were performed. clones of the knockout cells were isolated and validated by western blotting for hsamd or hsamd l or genotyping for msamd l. for genotyping, the genomic dna of the clones was extracted using the quickextract dna extraction kit (epicentre).~ bp of dna flanking the target site was pcr-amplified with the primer pair ( '-ggccactcaatctcattgacccaat- ' and '-tgcccaggatattcttagagctag c- ') and cloned to pgem vector (promega). the sequence of - pgem clones was determined by sanger sequencing. for knockout of hsamd or hsamd l in additional human cell lines and hffs, only guide # described above was used for the knockout. the stably transduced target cells were pooled without clonal selection, and the knockout was validated by western blot. mice with different samd l genotypes used in the infection experiment were generated from breeding pairs of samd l +/mice [ ] . at around to weeks old, the mice were infected intranasally with viruses in μl pbs as described previously [ ] . the viruses used were purified through a sucrose cushion sedimentation according to the standard protocol [ ] . individual mice were weighed every day and euthanized when % of the body weight was lost. statistical comparisons of body weight changes among groups were analyzed by two way anova using graphpad prism . . values of p < . were considered statistically significant. the anti-vacv serum antibody titers of some infected mice were determined by elisa with purified vacv virions as described previously [ ] . all mouse protocols were approved by uthscsa iacuc. msamd l expression plasmid msamd l orf was pcr-amplified with the primer pair ( '-agtggacaagtaactcaac caaaattg- ' and '-gatcacttttatgccatatgcc- ') from cdna synthesized from t cellular mrnas. a xflag tag sequence and a ha tag sequence were then appended to the ' and ' end of the msamd l orf by recombinant pcr, and the final pcr product was inserted between kpni and sacii sites of the pcdna . /v -his-topo vector (thermo fisher scientific). the msamd l orf was completely sequenced and found to have one amino acid difference (val instead of ile at ) compared to the msamd l reference sequence in gen-bank (np_ . ). the difference was not corrected, as samd l from most rodents also has val at this position. cells in -well plates were incubated with pfu per cell of different viruses for h at room temperature. following adsorption, the cells were washed twice with phosphate-buffered saline (pbs). one set of the cells was harvested immediately as the hr post infection sample, while the other set were moved to ˚c incubator to initiate viral entry and harvested at hr post infection. the viral titers in the cell lysates were determined by plaque assays on vero cells. for testing the effect of ifn, human or murine cells were treated with u/ml of human or murine ifn-β (pbl biomedical laboratories) for hr, before the cells were infected with vacv as described above. for assessing viral late protein expression, western blot with a mab against the vacv late protein wr (clone he ) [ ] was performed. for immunoprecipitation of hsamd or hsamd l, hela cells or ifn-treated Δhsamd hela cells were infected with different vacv viruses. for immunoprecipitation of msamd l, ft cells were transfected with the msamd l expression plasmid and then infected with different vacvs at hr post transfection. after hr of infection, the cells were lysed on ice with a lysis buffer ( . % (w/v) np- , mm tris, ph . , mm nacl) supplemented with protease inhibitor cocktail tablets (roche molecular biochemicals). the cleared cell lysates were mixed with μl of % (vol/vol) v -agarose beads (sigma-aldrich) for min at ˚c. after washing with lysis buffer, the beads were resuspended in sds sample buffer, the eluted proteins were resolved by sds-page and detected with western blot as described previously [ ] . the detection antibodies were mouse monoclonal antibodies (mab) against v (sigma-aldrich; clone v - ), flag tag (sigma-aldrich) or hsp (santa cruz), and rabbit polyclonal antibodies against hsamd (sigma-aldrich, hpa- ) and hsamd l (proteintech, - -ap). supporting information s fig. related to fig a. (a) . genotyping Δmsamd l t cells. the msamd l knockout cell lines (Δmsamd l) were constructed by transient transfection of t cells with a plasmid encoding cas and a grna targeting msamd l (guide# or guide# ). the targeted samd l sequence is underlined with the pam sequence in bold italics and the encoded amino acid sequence shown above the dna sequence. shown below the target are the genomic sequences from representative cell clones (Δmsamd l# and Δmsamd l# ). gray and crossed-out sequence indicates deletion.^indicates insertion. the number after the + and − denotes the number of indels, and the number before the "x" denotes the number of times the sequence was detected from a total of - cloned pcr products. (b). the restriction of k l and c l deletion vaccinia virus (vk l -c l -) in t cells was abolished by knocking out msamd l with crispr-cas . a validated msamd l knockout cell clone (Δmsamd l # ) and the parental t cells were infected with vk l -c lat an moi of pfu/cell. viral growth was determined by measuring viral titers at and hour-post-infection (hpi). (pdf) s fig. (a) . vk l -c linfection induced anti-vacv antibody response in mice. mice described in fig a were emerging viral diseases: confronting threats with new technologies cross-species virus transmission and the emergence of new epidemic diseases poxvirus cell entry: how many proteins does it take? viruses poxviridae: the viruses and their replication poxviruses and the evolution of host range and virulence vaccinia virus host range genes localization and sequence of a vaccinia virus gene required for multiplication in human cells the cowpox virus host range gene, cp , affects phosphorylation of eif alpha and vaccinia viral translation in apoptotic hela cells identification from diverse mammalian poxviruses of host-range regulatory genes functioning equivalently to vaccinia virus c l m is a host range factor essential for myxoma virus pathogenesis and functions as an antagonist of host samd in human cells structural basis for antagonizing a host restriction factor by c family of poxvirus host-range proteins structure function studies of vaccinia virus host range protein k reveal a novel functional surface for ankyrin repeat proteins identification of restriction factors by human genome-wide rna interference screening of viral host range mutants exemplified by discovery of samd and wdr as inhibitors of the vaccinia virus k l-c l-mutant a deleterious mutation in samd causes normophosphatemic familial tumoral calcinosis normophosphatemic familial tumoral calcinosis is caused by deleterious mutations in samd , encoding a tnf-alpha responsive protein human sterile alpha motif domain , a novel gene identified as down-regulated in aggressive fibromatosis, is absent in the mouse sterile alpha motif containing domain is involved in death signaling of malignant glioma treated with inactivated sendai virus particle (hvj-e) or type i interferon gain-of-function samd l mutations cause a syndrome of cytopenia, immunodeficiency, mds, and neurological symptoms evolution and divergence of the mammalian samd /samd l gene family haploinsufficiency of samd l, an endosome fusion facilitator, causes myeloid malignancies in mice mimicking human diseases with monosomy the complex domain architecture of samd family proteins, predicted stand-like ntpases, suggests new links to inflammation and apoptosis mouse samd l is not a functional paralogue of the human samd , the gene mutated in normophosphataemic familial tumoral calcinosis c l family of poxvirus host range genes inhibits antiviral activities induced by type i interferons and interferon regulatory factor vaccinia virus k l and c l inhibit antiviral activities induced by type i interferons samd is an innate antiviral host factor with stress response properties that can be antagonized by poxviruses gene duplication as a mechanism of genomic adaptation to a changing environment interferome v . : an updated database of annotated interferon-regulated genes samd mutations cause a novel multisystem disorder, mirage syndrome, and are associated with loss of chromosome ataxia-pancytopenia syndrome is caused by missense mutations in samd l the poxvirus c l host range factor superfamily comparative analysis of poxvirus orthologues of the vaccinia virus e protein: modulation of protein kinase r activity, cytokine responses, and virus pathogenicity the '-poly(a) leader of poxvirus mrna confers a translational advantage that can be achieved in cells with impaired cap-dependent translation vaccinia virus k l protein supports viral replication in human and rabbit cells through a cell-type-specific set of its ankyrin repeat residues that are distinct from its binding site for acap improved vectors and genome-wide libraries for crispr screening genome-scale crispr-cas knockout screening in human cells preparation of cell cultures and vaccinia virus stocks. current protocols in protein science / editorial board enhancement of immune response to an antigen delivered by vaccinia virus by displaying the antigen on the surface of intracellular mature virion. vaccine generation and characterization of a large panel of murine monoclonal antibodies against vaccinia virus we thank jouni uitto for shipping the samd +/mice. we thank zhilong yang and nicholas wallace for providing human foreskin fibroblasts. conceptualization: yan xiang. key: cord- -yy bennu authors: penna, fabio; ballarò, riccardo; beltrá, marc; de lucia, serena; costelli, paola title: modulating metabolism to improve cancer-induced muscle wasting date: - - journal: oxid med cell longev doi: . / / sha: doc_id: cord_uid: yy bennu muscle wasting is one of the main features of cancer cachexia, a multifactorial syndrome frequently occurring in oncologic patients. the onset of cachexia is associated with reduced tolerance and response to antineoplastic treatments, eventually leading to clinical conditions that are not compatible with survival. among the mechanisms underlying cachexia, protein and energy dysmetabolism play a major role. in this regard, several potential treatments have been proposed, mainly on the basis of promising results obtained in preclinical models. however, at present, no treatment yet reached validation to be used in the clinical practice, although several drugs are currently tested in clinical trials for their ability to improve muscle metabolism in cancer patients. along this line, the results obtained in both experimental and clinical studies clearly show that cachexia can be effectively approached by a multidirectional strategy targeting nutrition, inflammation, catabolism, and inactivity at the same time. in the present study, approaches aimed to modulate muscle metabolism in cachexia will be reviewed. cancer-induced muscle wasting is one of the hallmarks of cachexia, a multifactorial syndrome that represents one of the most important comorbidities in oncologic patients. the occurrence of cachexia markedly complicates the management of cancer patients, negatively impinging on the tolerance and response to antineoplastic treatments, worsening the quality of life, and reducing survival. in particular, about % of deaths by cancer are due to cachexia, rather than to the tumor itself [ ] . few years ago, a classification of cachexia was proposed, defining three different stages: precachexia, cachexia, and refractory cachexia [ ] . prognosis progressively worsens going from patients with precachexia to those with refractory cachexia. in this regard, the earlier anticachexia treatments are set up, the better. for this reason, the research on cachexia is focused on two main goals: (i) to find out biomarkers useful to the early identification of a condition of still latent cachexia and (ii) to define treatment protocols useful to delay the progression from precachexia to refractory cachexia. skeletal muscle wasting in cancer patients has a good prognostic value, being predictor of reduced tolerance to chemotherapy and/or surgery, decreased ability to perform daily activities, and shortened survival. in addition, recent data report that loss of muscle mass negatively affects quality of life in cancer patients [ , ] ; such correlation might occur irrespectively of survival rates [ ] . being poor quality of life one of the most prominent and invalidating consequences of cancer cachexia, to investigate the mechanisms underlying cancer-induced muscle wasting is even more relevant to design therapeutic strategies that also take into account patient well-being. the possibility to underestimate the occurrence of muscle mass depletion exists, since the first approach to clinically evaluate a patient is to obtain information about body weight and body mass index (bmi). however, in face of no body weight loss and/or normal bmi, reduced muscle mass might well occur, being masked by fat or water content. another relevant point that frequently is poorly taken into consideration is that the loss of muscle mass is likely exacerbated by anticancer treatments. at present, different mechanisms have been proposed to lead to muscle wasting in cancer hosts, among which there are altered protein and energy metabolism and impaired myogenesis [ ] . several factors may contribute to these alterations, such as reduced calorie intake, hormonal unbalance, and systemic inflammation. cancer-driven production of proinflammatory cytokines plays a relevant role in tumor progression and markedly contributes to cachexia. indeed, in cancer patients, systemic inflammation correlates with increased resting energy expenditure and with reduced survival rate [ ] . along the same line, increased circulating levels of tumor necrosis factor α (tnfα), interleukin- (il- ), γ-interferon (inf), and, more recently, growth and differentiation factor (gdf ) have been reported in cachectic cancer patients [ , ] . the link existing between cytokines and cachexia has led to the inclusion of anti-inflammatory drugs in treatment protocols [ ] . the present review will focus on strategies able to modulate metabolism that may reveal useful to prevent/delay cancer-induced muscle wasting. altered protein turnover is a general feature of muscle wasting in cancer cachexia. in particular, protein breakdown rates are persistently increased, while protein synthesis rates can be reduced, unchanged, or even increased, depending on the model system [ ] . the different reaction kinetics that characterizes protein synthesis and degradation rates, zero and first order, respectively, implies that if degradation is higher than normal, the loss of total protein cannot be antagonized by simply increasing the rate of synthesis. taking this assumption for true, any anabolic approach should be associated with anticatabolic strategies in order to achieve a beneficial effect on muscle protein mass. . . protein degradation. several pieces of evidence suggest that the intracellular proteolytic systems in the skeletal muscle of cancer hosts are poised towards activation above the physiological levels ( figure ). particularly relevant in this regard are the pathways dependent on ubiquitinproteasome and autophagy. while the former mainly degrades short-lived and regulatory proteins, the latter gets rid of structural proteins and organelles [ ] . both experimental and human cancer cachexia were associated with increased activity of the ubiquitinproteasome pathway [ ] . of interest, alterations in molecular and biochemical markers of proteasome activation were observed in gastric cancer patients before any evidence of body weight loss, supporting the need to detect cachexia as early as possible [ ] . modulations of the ubiquitinproteasome proteolytic system, however, are not a general finding in cancer cachexia, as shown by studies reporting that it is not differently activated with respect to controls in the muscle of patients affected by non-small-cell lung cancer (nsclc; [ ] ) or esophageal cancer [ ] . the involvement of the autophagic-lysosomal proteolysis in muscle wasting was recognized just in the last fifteen years. two main reasons account for such delay: (i) autophagy was not considered as typically operated by the skeletal muscle as a response to stress conditions. such belief was definitely abandoned when autophagy was clearly demonstrated in fasted mice overexpressing green fluorescent protein- labeled lc [ ] . (ii) the study and detection of autophagy was not easy since the atg genes were not discovered [ ] . a number of studies reported that the autophagic system was overactivated, without reaching complete cargo degradation, in the muscle of both tumor-bearing animals and cancer patients [ , [ ] [ ] [ ] . in particular, despite autophagic flux was enhanced in mice bearing the c tumor, autophagosomes accumulated, likely due to exhaustion of the lysosomal compartment [ ] . a similar pattern could also be observed in cancer patients, as suggested by lc b-ii and p accumulation [ , ] . both proteasome and lysosomes, however, cannot directly degrade intact myofilaments. in this regard, a preliminary cleavage was proposed to be operated by other proteolytic systems, such as those dependent on caspases or calpains. these latter are ca + -dependent cysteine proteases, normally inactive and localized in the cytosolic compartment. when intracellular ca + concentrations increase, inactive calpains translocate to the cell membrane and become activated by autoproteolysis [ ] . the system also includes calpastatin, a physiological inhibitor, which is a substrate of active calpain itself. increased calpain expression was reported in the muscle of tumor-bearing animals [ ] , while rats transplanted with the yoshida ah- hepatoma showed a progressive reduction of calpastatin levels and increased in vitro cleavage of specific fluorogenic substrates [ ] . more recently, calpain activation was also demonstrated in mice bearing the c colon carcinoma [ ] . both increased or unchanged muscle calpain expression were reported in cancer patients [ , ] . several lines of evidence show that proinflammatory cytokines act as triggers, or at least as contributors, of cancer-induced protein hypercatabolism [ ] . briefly, data obtained in both experimental models and human pathology have demonstrated that cytokines such as tnfα and il- lead to reduced rates of protein synthesis paralleled by enhanced protein breakdown, both accounting for the loss of muscle mass [ ] . such effects depend, at least in part, on activation of the transcription factor nf-κb, as shown in both experimental and human cancer cachexia patients [ , ] . cancer-induced muscle wasting has also been associated with another proinflammatory cytokine, namely, tnf-like weak inducer of apoptosis (tweak) [ ] . the therapeutic approaches mainly pursued by researchers to counteract enhanced muscle protein breakdown have long been those specifically targeting the different proteolytic systems. the results, however, did not really clarify the issue. since the discovery of muscle-specific ubiquitin ligases, these were considered a good target to interfere with protein breakdown, being involved in determining both substratespecificity and proteasome degradation rate. among the enzymes belonging to this family, the first described were mafbx/atrogin- and murf /trim , respectively, involved in the degradation of structural proteins and of proteins contributing to cell proliferation, differentiation, and survival [ ] . subsequently, other members came out, such as trim and fbxo . more recently, fbxo / musa was shown to contribute to denervation-and fasting-mediated muscle loss [ ] , as well as to cancerinduced muscle wasting (unpublished data). genetic approaches specifically targeting these ubiquitin ligases proved effective in protecting the muscle against protein depletion [ ] ; however, at present, the use of these enzymes as therapeutic targets for muscle wasting is not validated yet. on the other side, the inhibition of proteasome activity by means of pharmacological inhibitors was effective just in few models of muscle atrophy but totally unable to protect tumor-bearing animals against muscle wasting [ ] . in contrast with these findings, few years ago, a study reported that inhibition of the ubiquitin-proteasome pathway by mg was able to improve experimental cancer cachexia [ ] . however, mg is a rather unspecific inhibitor, being able to block also calpains and autophagy [ , ] . finally, carfilzomib, an irreversible selective inhibitor of proteasome chymotrypsin-like activity, was shown to improve cachexia in tumor-bearing mice by inhibiting muscle protein breakdown [ ] . such improvement, however, was associated with reduced tumor burden, which could be the real mechanism underlying the beneficial effect of the treatment. several lines of evidence proposed that modulations of autophagy could be useful to improve cancer-associated muscle wasting. in this regard, muscle-specific gene strategies aimed at silencing beclin- , one of the proteins involved in autophagosome formation, showed that suppression of autophagy in the c hosts was unable to rescue myofiber diameter (unpublished data). in addition, pharmacological inhibition of autophagy in mice hosting the c tumor lead to death of the animals, suggesting that lysosomal degradation is mandatory to sustain the requirement of both energy and substrates in tumor hosts, at least when they are facing the terminal phase of cancer growth [ ] . the other way round, excessive stimulation of muscle autophagy, experimentally obtained by the overexpression of tp inp / dor, exacerbated muscle atrophy in tumor-bearing mice (unpublished data), while activation of autophagy by means of the mtor inhibitor rapamycin was shown to positively affect the skeletal muscle in the c hosts [ ] . such discrepancy might depend on the fact that while mtor inhibition affects stress-induced autophagy, tp inp hyperexpression targets basal autophagy. despite the literature report data supporting the involvement of ca + -dependent proteolysis in the pathogenesis of cancer-induced muscle wasting, protein hypercatabolism was not downregulated in preparations of isolated muscles obtained from tumor-bearing animals and incubated in the presence of calpain inhibitors [ , , ] . more recently, both pharmacological and genetic approaches aimed at inhibiting the ca + -dependent proteolytic system were not able to prevent or delay cancer-induced muscle wasting [ ] , although contrasting results were reported in this regard [ ] . while interfering with specific proteolytic systems does not seem to be an appropriate approach to prevent/delay cancer-induced muscle wasting, the modulation of bulk protein turnover appears more promising. in this regard, anti-inflammatory approaches revealed able to improve muscle protein turnover in tumor-bearing mice [ ] . more recently, administration of formoterol, a β -adrenergic agonist, to tumor-bearing animals revealed able to reverse muscle wasting [ ] . such an effect is mainly exerted by stimulating protein synthesis and inhibiting protein degradation rates. in particular, both the ubiquitin-proteasome and the autophagic-lysosomal proteolytic systems were downregulated in formoterol-receiving animals ( [ ] ; unpublished data). at present, only one study evaluated the effectiveness of formoterol, combined with megestrol acetate, in cachectic cancer patients [ ] . the results suggest that both muscle size and strength can be improved by the treatment, although more trials are needed to draw clear-cut evidence. . . protein synthesis. as reported above, depending on the situation, reduced, normal, or even increased muscle protein synthesis rates were shown in cancer cachexia. due to the rapid development of cachexia, tumor-bearing animals frequently show reduced protein synthesis rates, although this is not a general finding. indeed, rats bearing the ah- hepatoma, that usually die about days after tumor transplantation, showed muscle protein synthesis rates comparable to those of healthy animals [ ] . the situation is more complex when studying human pathology. reduced muscle protein synthesis was reported many years ago in patients affected by different types of tumors [ ] and more recently in prostate cancer patients [ ] . on the contrary, van dijk et al. [ ] reported that baseline protein synthesis rates were higher than control values in cachectic patients affected by pancreatic cancer. also, intermediate results are available in the literature: myofibrillar protein synthesis rates were analyzed in healthy people and weight-stable and weight-losing gastrointestinal cancer patients and found comparable among the different groups. similarly, no changes in whole body protein synthesis were reported in nsclc patients [ ] . the possibility to modulate protein synthesis in order to correct muscle atrophy or simply to provide an environment permissive for the maintenance of muscle mass was long studied. many approaches were tested, most of them consisting in nutritional strategies or in molecular modulations aimed at pushing muscle metabolism towards anabolism. most of these approaches revealed unsuccessful, giving rise to the idea that anabolic resistance occurs in cancer cachexia. just to provide few examples, conventional nutritional supplementation or the infusion with an amino acid cocktail did not stimulate muscle protein synthesis in advanced cancer patients [ ] . along this line, studies aimed at stimulating the anabolic pathway depending on igf- , by both pharmacological and genetic means, did not succeed in improving muscle wasting in tumor-bearing animals [ , ] . recently, however, patients not yet considered as refractory cachectic were proposed to display an anabolic window that could be exploited with nutritional interventions [ , , ] or with other anabolism-inducing strategies. as an example, patients with stage iii and iv nsclc showed a normal anabolic response to hyperaminoacidemia but not to isoaminoacidemia, suggesting that high substrate availability is relevant to induce anabolism in cancer hosts [ ] . consistently, muscle protein synthesis could be stimulated in advanced cancer patients by a high protein formula versus a conventional nutritional supplement [ , ] . these observations point out the possibility to overcome the anabolic resistance that occurs in cancer patients by providing specifically enriched nutritional supplements. stimulation of anabolism can be exerted by several means. particularly interesting in this regard is ghrelin, a mediator released by the stomach during fasting or caloric restriction. modulations of ghrelin levels exert remarkable effects on both energy and protein metabolism, such as the inhibition of autophagy in conditions characterized by systemic inflammation [ ] . the administration of ghrelin to tumor-bearing animals improved food intake, body weight, lean body mass, and chemotherapy-induced toxicity [ ] . both ghrelin and ghrelin analogues are currently being tested in clinical trials. among these, anamorelin was recently shown to improve lean body mass, total body mass, and hand grip strength in patients affected by nsclc [ ] . other studies, however, showed that anamorelin administration to cancer patients increased body weight and improved faact scores while did not enhance hand grip strength [ ] . acids. in addition to be necessary to synthesize proteins, free amino acids (faa) also act as regulators of protein metabolism. in particular, plasma faa, that even represent a small fraction of the total amino acid pool, are the main source of metabolically active nitrogen compounds. among faa, the essential amino acids were reported to stimulate protein synthesis and to inhibit protein degradation. such a role is mainly played by the three branched chain amino acids (bcaa), leucine in particular. alterations of amino acid metabolism are frequent features in cancer-induced muscle wasting. reduced amino acid uptake was generally observed in cancer patients, mainly due to the occurrence of anorexia, which also leads to decreased insulin secretion. both decreased amino acid availability and insulin levels inhibit the anabolic pathway dependent on mtor, resulting in downregulation of protein synthesis rates and stimulating protein degradation. inhibition of mtor signaling in cancer cachexia is enforced by proinflammatory cytokines [ ] . reduced amino acid uptake in the muscle was also reported to derive from altered amino acid transport. indeed, in the soleus muscle of tumor-bearing rats, the activity of system a was decreased, while no changes were observed for systems l and asc [ ] . of interest, tnfα was shown to impair amino acid transport in tumor-bearing rats [ ] . plasma glutamine levels were shown to be significantly reduced in tumor-bearing rats with respect to healthy animals [ ] . of interest, reduced glutamine availability could activate the metabolic sensor adenosine monophosphateactivated protein kinase (ampk, see below; [ ] ). finally, leucine oxidation was markedly increased in the muscle of rats bearing the yoshida ah- hepatoma [ ] . consistently, enhanced activity of the bcaa dehydrogenase was reported in rats bearing the walker carcinoma [ ] . several studies have proposed amino acid supplementation as a mean to improve cancer-induced muscle wasting. in experimental models of cancer cachexia, bcaa were shown to attenuate the loss of muscle mass. the underlying mechanisms of such effect are not clear, although downregulation of protein degradation and stimulation of protein synthesis were hypothesized [ ] . more recently, metabolomic alterations were proposed to be the basis of the positive effects exerted by a leucine-rich diet on cachexia in rats bearing the walker carcinoma, in the absence of effects on tumor mass [ ] . as for clinical studies, bcaa were proposed to improve anorexia [ ] , thus removing, partially at least, one of the mechanisms accounting for reduced amino acid uptake. other studies supported a beneficial role of bcaas on muscle protein metabolism, although this should be confirmed by larger randomized, blind, placebocontrolled trials [ ] . beta-hydroxy-beta-methylbutyrate (hmb), a metabolite of leucine, was shown to improve muscle wasting in experimental cancer cachexia, mainly by inhibiting protein degradation rather than stimulating protein synthesis [ ] . recently, hmb was proposed to be more effective than leucine in preventing body weight loss in tumor-bearing animals [ ] . such effect, however, could depend on the model system chosen, since hmb does not appear able to modulate muscle mass in mice bearing the c tumor (costelli et al., unpublished observations). the situation is even more confused in human cachexia. increase of both hemoglobin levels and fat-free mass were reported in advanced cancer patients administered a nutritional supplement containing hmb, arginine, and glutamine [ , ] . another study, however, was not able to demonstrate a beneficial effect of the same supplement in cancer patients [ ] , suggesting that the effectiveness of hmb in the clinical practice is still unclear and deserves further investigation. glutamine supplementation was reported to attenuate muscle protein wasting in cancer patients [ ] , as well as to improve the energy balance in rats bearing the walker tumor [ ] . finally, promising data are available about the possibility to treat cancer hosts with l-carnitine, an amino acid derivative that plays a role in fatty acid metabolism and energy production [ ] . a negative energy balance, generally resulting from both reduced production and increased expenditure, is a frequent occurrence in cancer patients. while resting energy expenditure (ree) frequently increases, likely due to enhanced thermogenesis, the occurrence of reduced physical activity, particularly in advanced cancer patients, paradoxically leads to a net decrease of total energy expenditure. the increased thermogenesis is consistent with the observation that in cachectic tumor-bearing animals, the expression of the brown adipose tissue-(bat-) specific uncoupling protein (ucp ) is higher than in controls, while ucp (ubiquitous) and ucp (expressed in bat and muscle) levels increase in the skeletal muscle only [ ] . similarly, muscle ucp mrna levels were higher in weight-losing than in non-weight-losing cancer patients or controls [ ] . the increase of ree in cancer cachexia is not a new observation; however, just recently, the underlying mechanisms start to be clarified. a central point in this regard is played by muscle mitochondria compartment, which is markedly affected in tumor hosts. indeed, both morphological [ , ] and functional alterations [ , ] were reported in experimental tumor-bearing animals. in particular, mitochondrial uncoupling and reduced oxidative capacity were associated with myofiber shift from oxidative to glycolytic fibers [ ] . impairment of the mitochondrial compartment results in decreased atp production, leading to an energy deficit that becomes even worse since it is coupled with steadily increased ree. consistently, reduced atp levels and increased activity of the energy sensor ampk were shown in the muscle of tumor-bearing animals [ , ] . the lack of an appropriate energy supply results in compromised cell function and reduced contractile force generation, leading to loss of muscle mass and strength. several factors can lead to mitochondrial alterations in the skeletal muscle. among these, proinflammatory cytokines play a major role. indeed, the activation of nf-κb induced by tnfα was reported to reduce both muscle oxidative capacity and the expression of factors regulating mitochondrial biogenesis. similar observations were reported when other inflammation-driven pathways such as the il- /stat or tgfβ/smad are activated above physiological levels [ ] . in addition to proinflammatory mediators, also oxidative stress, due to reactive oxygen and nitrogen species levels exceeding the compensative capacity of the intracellular antioxidant systems, contributes to mitochondrial function impairment. in this regard, there are several studies sustaining the involvement of oxidative stress in cancerinduced muscle wasting, although a clear-cut causative evidence is still lacking [ ] (ballarò et al., unpublished data; figure ). being mitochondria crucial to the maintenance of muscle oxidative metabolism, emergency routes can be activated in order to avoid mitochondrial dysfunction. in particular, mitochondrial biogenesis and dynamics as well as the disposal of damaged organelles, mainly by mitophagy, are promoted ( figure ). the other way round, impaired function of the emergency routes themselves could trigger the accumulation of altered mitochondria resulting in reduced muscle oxidative metabolism. in this regard, the expression of the peroxisome proliferator-activated receptor-γ (ppar-γ) coactivator- α (pgc- α), the master regulator of mitochondrial biogenesis and oxidative metabolism, was shown to be reduced in the skeletal muscle of tumorbearing mice [ ] , although this is not a constant finding [ , ] . mitochondria dynamics, representing the balance between fission and fusion processes, was shown to be altered in both experimental cancer cachexia and in cancer patients [ , ] . in this regard, impaired mitochondrial dynamics could drive the hyperactivation of muscle protein breakdown, likely through pathways depending on ampk and foxo, eventually leading to muscle wasting [ , ] . autophagy (mitophagy) is the main mechanism responsible for disposal of altered mitochondria. also, mitophagy was reported to be impaired in cancer cachexia, as shown by the observation that bnip l and parkin mrna increased in the muscle of cancer patients [ ] . similarly, bnip l protein levels were increased in the muscle of mice bearing the lewis lung carcinoma [ ] . on the whole, these observations suggest that in addition to mitochondria biogenesis and dynamics, also their disposal is perturbed in the skeletal muscle of tumor hosts, thus contributing to mitochondrial dysfunction and reduced muscle oxidative metabolism. several strategies were proposed to improve energy metabolism by acting on mitochondria. the first and perhaps simplest one, in theory at least, is exercise training, in particular a combination of both resistance and endurance exercise. these two types of training affect different but complementary targets, being able to improve force production and metabolic adaptations, respectively. of particular relevance is the observation that endurance training was reported to increase the number of mitochondria and to drive myofiber-type shift from glycolytic to oxidative, thus specifically targeting alterations that characteristically occur in the skeletal muscle of tumor hosts. however, these potentially favorable effects can be exploited just systematically practicing exercise, even if at a moderate level. this may not be an easy task in cancer patients that frequently present with chronic fatigue and comorbidities eventually leading to exercise intolerance. this point is supported by the observation that c -bearing mice did not benefit from exercise training, suggesting that the effort to exercise in already compromised animals was damaging rather than protective [ ] . consistently, excessive endurance exercise was associated with increased mitochondrial fission in the absence of mitophagy induction [ ] . in the last years, the possibility to mimic the effects of exercise by drugs has been gaining a growing consensus. the positive side is that this strategy will allow to overcome both poor patient compliance to exercise training and possible occurrence of exercise intolerance. the negative part is that generally exercise mimicking drugs do not totally recapitulate the effects of exercise itself. in this sense, these drugs do not properly hit the target; however, they could be a good compromise when exercise training cannot be proposed to the patient. at present, several options were investigated as exercisemimicking strategies. while most of them are pharmacological, also a genetic approach was proposed. this latter consists in manipulations able to increase the levels of pgc- α in the skeletal muscle. in this regard, improved exercise capacity and oxidative metabolism were reported in mice specifically overexpressing this factor in the muscle, resembling the phenotype induced by endurance training [ ] . pgc- α overexpression was shown to interfere with muscle atrophy induced by activation of the tweak-fn pathway [ ] and to improve cancer-induced muscle wasting in tumor-bearing mice [ , ] , although contrasting data were previously reported [ ] . several classes of drugs were proposed to modulate energy metabolism, among which are activators of ampk, sirtuin (sirt ), and trimetazidine (tmz). different compounds such as resveratrol, metformin, quercetin, and aicar can activate ampk [ ] . in this regard, aicar administration was shown to impinge on exercise capacity, oxygen consumption, and fatty acid oxidation [ ] . muscle atrophy induced by angiotensin ii was prevented by treatment with aicar. this drug also revealed able to activate autophagy and to improve muscle phenotype in both dystrophic mdx mice and animals bearing the c tumor [ , ] . metformin administration was shown to improve sarcopenia of aging and muscle wasting in severely burned patients [ , ] and was proposed to be useful to treat muscle wasting in cancer cachexia [ ] . ampk activation can also be induced by resveratrol, as demonstrated by the observation that ampkα -or ampkα -deficient mice were refractory to resveratrol-induced increase of both mitochondrial biogenesis and endurance exercise capacity [ ] . consistently, obese men receiving resveratrol showed improved inflammation, ampk activation, and increased expression of pgc- α and sirt protein levels [ ] . finally, an ampk-stabilizing peptide was reported to improve white adipose tissue wasting in tumor hosts [ ] . sirt belongs to a class of deacetylases deregulated in aging and in different chronic diseases, including cancer. sirt is also involved in the regulation of energy homeostasis; its expression is induced in response to caloric restriction [ ] and can be activated in the skeletal muscle by ampk [ ] . specific overexpression of sirt in the muscle resulted in a fast-to-slow myofiber type transition, producing an oxidative phenotype. consistently, muscle-specific sirt transgenic mice exposed to fasting or denervation showed a reduced expression of atrogenes in comparison with wildtype littermates [ ] . finally, improved muscle phenotype was reported in mdx/sirt double transgenic mice [ ] . in addition to ampk, resveratrol also activates sirt . in this regard, part of the above-described effects exerted by resveratrol derive from sirt -dependent modulations of pgc- α acetylation state [ ] . synthetic selective sirt activators such as srt are also available [ , ] . plasma lipid profile and insulin sensitivity were improved in healthy volunteers by administration of srt [ , ] . very few studies are actually available about srt action on both muscle mass and function. in this regard, srt appeared to reduce the depletion of muscle mass due to fasting or inactivity [ ] , at least in part by increasing pgc- α expression [ ] . tmz is a metabolic modulator that blocks fatty acid oxidation, shifting atp production to glucose oxidation and improving cell energy metabolism. indeed, atp synthesis through fatty acid β-oxidation requires more oxygen than glucose oxidation [ ] . along this line, the shift to glucose oxidation improves the use of the available oxygen, possibly increasing metabolic efficiency and skeletal muscle function. tmz was shown to increase the size of cultured myotubes [ ] and to improve both heart metabolism and exercise capacity in patients suffering from chronic stable angina [ ] . when administered to aged animals, tmz resulted in increased muscle strength [ ] . finally, treatment of c -bearing mice with tmz resembled some of the benefits triggered by exercise, among which fast-to-slow myofiber phenotype shift, pgc- α upregulation, oxidative metabolism enhancement, and grip strength increase (molinari et al., unpublished). the relevance of fatty acid oxidation to cachexia is also supported by a recent study showing that several tumor cell lines are able to release proinflammatory mediators, resulting in enhanced fatty acid oxidation and activation of p -dependent signaling in the skeletal muscle, well before tissue wasting occurs. in addition, the same study also showed that treatment of tumor-bearing animals with etomoxir, an inhibitor of fatty acid oxidation, rescued both muscle mass and body weight [ ] . a complex network of metabolic alterations sustained by hypercatabolism, energy deficit, and systemic inflammation is the milieu underlying cancer cachexia. while becoming overtly detectable in advanced cancer patients, such perturbations likely take place very early in the course of the disease, at least at the molecular level. protein and energy dysmetabolism in cachexia are quite well recognized; however, the available therapeutic strategies, although frequently promising from the preclinical point of view, have not yet reached validation to be used in the clinical practice. several drugs identified by experimental studies are currently tested in clinical trials for their ability to improve muscle metabolism in cancer patients. other emerging strategies are those aimed at interfering with the intestinal microbiota, previously reported to improve cachexia in a preclinical model [ ] . the available results of both experimental and clinical studies, however, have clearly indicated that single-targeted therapies will hardly be successful in the treatment of cachexia. in this regard, the view of a multidirectional approach, selectively tailored and, whenever possible, personalized, is gaining a growing consensus. such an approach should not just rely on nutritional counseling and pharmacologic treatment with anti-inflammatory and anticatabolic drugs but also include exercise training and/or exercisemimicking agents. in this regard, exercise mimetics could not only merely replace exercise training in depleted patients but also improve exercise tolerance and effectiveness in precachectic individuals, thus amplifying the beneficial action of exercise itself. last but not least, treatments aimed at preventing/correcting the metabolic alterations underlying cancer-induced muscle wasting might also impinge on tumor-targeted therapies improving their effectiveness and/ or enhancing patient tolerance to chemotherapy. in addition, metabolic modulators could also directly affect tumor growth. this is the case, for example, of exercise, that was shown to prevent or at least delay tumor growth [ ] . cancer cachexia: understanding the molecular basis definition and classification of cancer cachexia: an international consensus muscle mass and association to quality of life in non-small cell lung cancer patients sarcopenia is associated with quality of life and 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survival of male mice on a standard diet and preserves bone and muscle mass the antiischemic effect of trimetazidine in patients with postprandial myocardial ischemia is unrelated to meal composition the metabolic modulator trimetazidine triggers autophagy and counteracts stress-induced atrophy in skeletal muscle myotubes trimetazidine improves exercise performance in patients with peripheral arterial disease improvement of skeletal muscle performance in ageing by the metabolic modulator trimetazidine excessive fatty acid oxidation induces muscle atrophy in cancer cachexia synbiotic approach restores intestinal homeostasis and prolongs survival in leukaemic mice with cachexia molecular mechanisms linking exercise to cancer prevention and treatment the authors declare that there is no conflict of interest regarding the publication of this paper. key: cord- -p h p bm authors: lindqvist, richard; upadhyay, arunkumar; Överby, anna k. title: tick-borne flaviviruses and the type i interferon response date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: p h p bm flaviviruses are globally distributed pathogens causing millions of human infections every year. flaviviruses are arthropod-borne viruses and are mainly transmitted by either ticks or mosquitoes. mosquito-borne flaviviruses and their interactions with the innate immune response have been well-studied and reviewed extensively, thus this review will discuss tick-borne flaviviruses and their interactions with the host innate immune response. tick-borne flaviviruses (tbfv), flaviviridae family, includes many pathogens causing severe human disease, ranging from mild fever to encephalitis and hemorrhagic fever. there are more than viruses in the genus flavivirus, and they are transmitted by arthropods such as mosquitoes (dengue virus (denv), japanese encephalitis virus (jev) and west nile virus (wnv), yellow fever virus (yfv), and zika virus (zikv) and ticks (tick-borne encephalitis virus (tbev), langat virus (lgtv), kyasanur forest disease virus (kfdv), omsk hemorrhagic fever virus (ohfv), powassan virus (powv), and louping-ill virus (liv)) [ ] [ ] [ ] [ ] [ ] . among the tbfvs, tbev, powv, and liv are encephalitic, whereas ohfv and kfdv are hemorrhagic viruses. there are general features that distinguish tbfv from mosquito-borne. one such feature is that tick-borne viruses tend to persist in certain stable foci whereas mosquito-borne viruses may suddenly emerge and they can rapidly spread to new areas and continents causing large epidemics [ ] [ ] [ ] [ ] [ ] [ ] . mosquito-borne flaviviruses are transmitted horizontally from mosquito to vertebrate to mosquito, and for some viruses, humans act as the amplifying host [ ] [ ] [ ] . mosquitoes have a short life compared to ticks, as it can take several years for a tick to develop from egg to adult [ ] . ticks also only take one blood meal at each of their life stages, larvae, nymph, and adult, this means that tick-borne viruses need to be maintained in infected ticks during a very long time [ ] . thus, replication in ticks needs to be lower compared to mosquitoes, which have higher viral turnover and a faster generation cycle, this also contributes to the high mutation rate in mosquito-borne flaviviruses, and relative stable genomes of tbfvs [ , ] . infection of tick-borne flaviviruses usually cause a short viremia in humans and larger vertebrates. this route of transmission has been described as less important in tick-borne flavivirus infection [ ] , instead tbev has been shown to be transmitted by viremic rodents and among co-feeding ticks without viremia [ ] . humans act as dead-end hosts since they do not produce high enough viremia to transmit the virus to new ticks. according to phylogenetic differences, tbev has been divided into three different subtypes, european, siberian, and far eastern. the european subtype is mainly transmitted by ixodes ricinus, whereas the siberian and far eastern subtypes are primarily transmitted by ixodes persulcatus [ , ] . tbev is found in central, eastern, and northern europe and asia ( figure ) and correlates with the presence of infected ticks. ixodes ricinus is found throughout europe, whereas ixodes persulcatus is found in eastern europe in the west, and china and japan in the east [ ] . tbev is considered one of the most important arboviruses in central and eastern european countries and in russia, with about , estimated human cases annually [ ] . in fact, over the last decade there has been an approximately % increase in the number of tbe cases in europe [ ] , and tbev is currently spreading into new regions in france, sweden, norway, and italy [ ] [ ] [ ] [ ] . this increase is thought to be due to growth in population and spread of ticks, which is promoted by factors including climate change, social and political change, and changes in the land use [ , ] . the increased expansion in europe also poses an increased risk for the population engaged in outdoor activities. tbev is a zoonotic disease and the natural cycle of tbev is dependent and maintained in a complex cycle involving ticks as the vector and reservoir of the virus and small rodents as hosts for ticks [ ] . humans are not part of the natural transmission cycle of tbev and are the incidental host when infected by a bite from an infected tick [ ] . transmission through consumption of unpasteurized milk has also been reported for tbev [ , ] , as well as transmission via solid organ transplant [ ] . during the tick bite, the virus is inoculated into the skin of the vertebrate host. the initial replication is believed to occur locally in the dendritic cells. this is followed by infection of the draining lymph nodes, resulting in the primary viremia and subsequent infection of the peripheral tissues, where further replication maintains the viremia for several days [ ] [ ] [ ] [ ] . the disease course of tbev is biphasic; the initial phase is characterized by flu-like symptoms and is followed by a second phase involving cns infection, with meningitis, encephalitis, or meningoencephalitis [ ] [ ] [ ] . the mortality rate of tbev varies from to % depending on the subtype, in which the european tbev subtype has shown lower mortality rates compared to the siberian and far eastern [ , , ] . among the patients that experience neuroinvasive tbev infection, approximately - % of the survivors suffer from long lasting neurological sequelae [ , ] . no antivirals are available for treatment of tbev infection but there is an effective vaccine [ , ] . lgtv, which is closely related to tbev, is found in south east asia and russia [ ] . lgtv has not been associated with human disease under natural infections although it shares % sequence identity with tbev [ ] . because of its avirulence in humans and close similarity to tbev, lgtv is often used as a model virus for tbev under biosafety level- conditions. powv is found in russia and north america, and is the only tbfv present in america ( figure ) [ ] . it is transmitted by ixodes scapularis, ixodes cookei, and several other ixodes tick species, to small and medium size mammals, whereas humans are accidental dead-end hosts. milk-borne powv transmission might also be possible since powv virus has been found to be secreted in milk under experimental settings [ ] . although not much is known about powv pathogenesis, recent studies in mice have found that tick saliva was important to enhance powv transmission and the outcome of disease [ ] . furthermore, it has been demonstrated that powv infects macrophages and fibroblasts in the skin, shortly after the tick bite, also, other unidentified cells were shown to be infected [ ] . interestingly, macrophages were found to be the primary target for powv in the spleen [ ] , and in the cns, which is the main target site for powv infection, neurons have been shown to be the primary target for powv in mice and humans [ , ] . during the last years there has been an increase of powv in the usa with approximately reported cases [ , ] . the recent rise in incidence could be due to increased surveillance and diagnosis of powv, or it may represent a true emergence of the disease in endemic areas, or both [ ] . the incubation period ranges from week to month. the symptoms of powv infection may include fever, headache, vomiting, weakness, confusion, seizures, and memory loss with a case fatality rate of % [ ] . approximately half of the survivors experience permanent neurological symptoms, such as recurrent headaches, muscle wasting, and memory problems (https://www.cdc.gov). there are no antiviral treatments or vaccines available against powv. liv is mainly distributed in the uk and ireland, but it has also been detected in sheep in norway and on the bornholm island in denmark ( figure ). liv is most commonly known as pathogen of sheep and red grouse although humans can also get infected [ , ] . animals develop a febrile disease, which can progress to fatal encephalitis [ ] . like tbev, the vector of liv is the tick species ixodes ricinus [ ] . ticks transmit the virus to animals, however, natural exposure to humans is rare [ ] . instead humans that are exposed to infected animals such as veterinarians, farmers, butchers, and abattoir workers, as well as laboratory scientists have acquired liv infection [ ] [ ] [ ] [ ] . in between the years of and , human cases of liv was described [ ] . in humans, liv causes a disease that closely resembles tbev, with initial flu-like symptoms which can progress to severe neurological disease. distinct but closely related viruses are also found in spain (spanish sheep encephalomyelitis virus), turkey (turkish sheep encephalitis virus), and greece (greek goat encephalitis virus) [ ] . ohfv distribution is restricted to western siberia ( figure ) [ ] . the main vector of ohfv is the meadow tick, dermacentor reticulatus, which can also transmit the virus to humans. however, humans are mainly infected after contact with infected muskrats (ondatra zibethicus) which are very sensitive to the infection and often succumb to the infection [ ] . muskrats develop high viremia which can last for several weeks. human infection occurs through contact with urine, feces, and blood [ ] . secretion of ohfv in unpasteurized goat milk has been reported but no milk-borne outbreaks have been observed [ ] . the exact number of annual cases are uncertain because of misdiagnoses and unreported cases, but cases were reported between and [ ] . ohfv may cause a biphasic disease; the initial phase is characterized by high fever, bleeding from the nose, mouth, and uterus. thirty to fifty percent of the cases experience a second phase characterized by high fever and reappearance of the symptoms from the initial phase. case fatality rates range from . to . % [ ] . no antiviral treatments are available against ohfv, instead treatment is focused on supportive care to minimize hemorrhage and other complications [ ] . kfdv is only found in india (figure ), although a similar genetic variant, alkhurma hemorrhagic fever virus (ahfv) has been detected in saudi arabia [ , ] . kfdv is mainly transmitted by ticks belonging to the genus hemaphysalis but other tick genera have also been shown to be able to transmit kfdv [ , ] . during hemorrhagic kfdv infection, the initial phase is similar to tbev infection with fever and flu-like symptoms, but it may also include bleeding from the nose, mouth or gastrointestinal tract [ , ] . the second phase, which is experienced by - % of the cases, includes neurological symptoms such as headache, mental disturbance, and tremor, however, no evidence of meninges or encephalitis have been found [ ] . there are about - reported annual cases of kfdv [ ] , with case fatality rates ranging from to % [ , ] . in a kfdv vaccine was developed, although it provided some protection, due to low efficacy, the number of cases still increased from to [ ] [ ] [ ] . treatment of kfdv is limited to supportive care [ ] . tbfvs are enveloped viruses around nm in diameter. the envelope carries two surface proteins, the envelope (e) protein and the membrane (m) protein. the latter is derived from a precursor protein, prm. the nucleocapsid (nc) lies inside the viral envelope and consists of multiple copies of capsid (c) protein and the viral genome. the tbfv genomes are single stranded, positive-sense rna of approximately , nucleotides. it has a -cap with a single open reading frame (orf). the orf is flanked by and untranslated regions (utrs). the viral protein is encoded by the orf as a single polyprotein, which is co-and post-translationally cleaved by cellular and viral proteases into individual viral proteins (three structural and seven non-structural). the polyprotein is arranged in the order -c-prm-e-ns -ns a-ns b-ns -ns a-ns b-ns - [ , ] . the first step of virus replication starts when the virus binds to its receptor and is taken up into the cell by receptor-mediated endocytosis. the attachment is mediated by viral e protein and entry receptor on the host cell. the entry receptor for tbfvs has not been identified, but attachment to heparan sulphate and glycosaminoglycan, which are present in abundance on many cell types of both vertebrates and ticks, is predicted to play a role during binding and entry [ ] . once the virus has entered the cell, it is transported to endosomes, where the acidic environment of these vesicles leads to reorganization and conformational change of the e protein, resulting in the fusion of the viral and endosomal membrane and the release of the viral capsid into the cytoplasm [ ] [ ] [ ] . the viral rna (vrna) also functions as mrna, associating with ribosomes to produce the polyprotein. the transmembrane domain of the viral protein is recognized as a signal peptide and recruits the vrna/ribosomes/nascent polypeptide complex to the er membrane, where it is co-translationally translocated into the er membrane [ , ] . the nascent polypeptide is then processed by the cellular and viral proteases into structural and non-structural (ns) viral proteins. some of the viral proteins such as ns b, ns a, and ns b integrate and alter the membrane of the er to form a membrane vesicular structure, with a small pore connecting the interior of the vesicle to the cytoplasm [ ] [ ] [ ] [ ] [ ] . these vesicles are the site for the formation of replication complexes (rc) and rna replication [ , , ] . following rna replication, genomic rna is proposed to exit the vesicle through the vesicular pore and is packaged by c proteins into the nc on the cytoplasmic side of the er membrane [ , , ] . during the process of budding of nc from the cytoplasm into the er, it acquires the lipid envelope along with e and prm proteins, which are associated with the er membrane. the mechanism that ensures efficient incorporation of nc into the er membrane with the e and prm protein is poorly understood. following budding into the er lumen, the immature virions are transported through the cellular secretory pathways in a copi and copii dependent manner [ ] to the extracellular medium. the immature virion has heterodimers of e and prm that completely cover the lipid bilayer to form a spiky proteinaceous coat. during the transport through the golgi, the e protein is glycosylated [ ] . the acidic environment of the golgi induces conformational changes in the e and prm proteins, which exposes a cleavage site on prm. cleavage of prm by the cellular protease furin results in the formation of a mature virion [ , ] . mature virions are then released by exocytosis, which completes the viral life cycle [ ] [ ] [ ] (figure ). in arthropods, such as mosquitos and ticks, rna interferences (rnai) are the most important antiviral defense and tick cells have been shown to mount rnai responses against lgtv and tbev [ ] . proteins involved in the rnai mediated response such as argonaute (ago ), ago and dicer (dcr) were subsequently identified as inhibitors of lgtv [ ] . furthermore rna-seq and mass spectrometric analysis revealed that when challenged by tbev infection, tick cells upregulated genes involved in immunity and metabolism, whereas genes involved in cellular stress were downregulated [ ] . using gene silencing approaches, this study confirmed the antiviral effect of ago and dcr in tick cells. furthermore, novel antiviral genes such as complement factor h, heat shock protein (hsp) and and trypsin was found to inhibit lgtv in tick cells [ ] . taken together, studies so far have identified activation and antiviral actions of the rnai mediated response in tick cells as well as other inhibitory proteins such as hsp , hsp , and trypsin. the innate immune response provides the first line of defense against viral infections. this defense includes physical and chemical barriers such as the skin and mucous membranes and the acidity of the stomach. they serve as an initial barrier that protects the host from infection. once these barriers have been breached, the innate immune response relies on a set of germline-encoded receptors, known as pathogen recognition receptors (prrs), which recognize pathogen specific molecular patterns that are sensed as a danger signal by the host cell [ , ] . engagement of prrs results in the activation of several defense mechanisms including, production of interferon (ifn), induction of phagocytosis, cytokines, chemokines, antimicrobial peptides, antiviral proteins, as well as activation of leukocytes and t-cells. furthermore, the innate immune response harbors cellular components, such as dendritic cells, macrophages, and natural killer (nk) cells [ ] . in isaacs and lindenmann discovered ifn as a secreted factor that interfered with viral replication [ ] . in the early pioneer work on ifn in the fifties and sixties, tbev served as a model system, and tbev was shown to induce ifn after infection and was also sensitive to pretreatment of ifns [ , [ ] [ ] [ ] [ ] . pretreatment of ifn was also found to inhibit kfdv, ohfv, and powv titers in a cells (adenocarcinomic human alveolar basal epithelial cells) [ ] , thus ifn pretreatment induced broad spectrum inhibition of tbfvs. ifn has also been found to be strongly induced in the brains of powv infected peromyscus lecuopus, which are known as a natural host of the virus [ ] . similarly, ifn was found to be induced in sheep infected with liv [ ] , and ifn-stimulated genes were induced in brains of kfdv infected mice, indicating an activated ifn response [ ] . three distinct classes of ifn, type i (ifn α and β), type ii (ifnγ), and type iii ifn (ifnλ) have been described. the expression of the ifnλ-receptors and ifnγ are restricted, but the type i ifns can be expressed by most cell types and their receptor, the interferon-α/β receptor (ifnar) is expressed on all nucleated cells [ ] . there are different type i ifns in humans; these cytokines are produced by cells upon recognition of pathogen-associated molecular patterns, which are foreign to the host cell [ , ] , and mediates antiviral activity by autocrine and paracrine signaling through ifnar [ , ] . signaling through ifnar induces an antiviral state by the expression of hundreds of interferon stimulated genes (isgs) [ , ] . the host cell detects invading pathogens through prrs, which recognize foreign molecular patterns that are generated during infection. the particular prr involved in the recognition of the pathogen depends on the infecting virus [ ] . in flavivirus infection, the most important prrs are toll-like receptor (tlr) , tlr , tlr , retinoic acid-inducible gene i (rig-i), and melanoma differentiation-associated protein- (mda- ). tlr recognizes double stranded (ds) rna, which is formed as an intermediate during flavivirus replication [ , ] . tlr and , which are located in the endosomes, recognize single stranded (ss) rnas, such as the genomic rna of flaviviruses [ , [ ] [ ] [ ] . rig-i and mda- recognize viral rna in the cytoplasm of infected cells; rig-i recognizes short dsrna and triphosphorylated and diphosphorylated ssrna, whereas mda- recognizes long dsrna [ ] [ ] [ ] . upon recognition, the different prr will recruit distinct adaptor molecules. tlr recruits tir-domain-containing adapter-inducing interferon-β (trif) [ ] , tlr and recruit myeloid differentiation primary response (myd ) [ ] , whereas the rig-i-like helicases, rig-i and mda- , recruit interferon-beta promoter stimulator- (ips- ) (also known as mitochondrial antiviral-signaling protein (mavs), virus-induced signaling adapter (visa) and card adapter-inducing ifn-beta (cardif)) [ ] [ ] [ ] [ ] . ligation of prrs and downstream recruitment of the adapter molecules result in the activation of transcription factors nf-κb and interferon regulatory factor (irf) and irf , which translocate to the nucleus and induces the expression and subsequent secretion of type i ifn [ ] . although the innate antiviral response has been well-studied in mosquito-borne flavivirus infection, the responses to tbfvs are less investigated, and most studies have used tbev which therefore will be the main focus of the remainder of this review. in tbev infection, ifnβ induction has been shown to correlate with amount of intracellular viral rna, and the ips- pathway has been shown to protect mice from lethal lgtv infection and prolong survival after tbev infection [ , ] . absence of ips- resulted in a lower systemic ifnα response, which correlated with higher viral replication in the peripheral tissues [ ] . furthermore, ips- was shown to be of particular importance for the ifn production locally within the brain, where ips- −/− mice had lower induction of ifnβ within the olfactory bulb despite higher viral burdens [ ] . furthermore, ifnβ induction was shown to be completely dependent on ips- and irf in mouse embryonic fibroblasts and viral recognition through the ips- -pathway was shown to be dependent on rig-i and not mda- in human osteosarcoma cells (u os) [ , ] . interestingly, rig-i has been shown to co-localize with stress granules (sg) during tbev infection [ ] . sg contains ribonucleoprotein aggregates with translationally stalled mrnas, s ribosomes, and several rna-binding proteins. sg function to prevent the generation of defective proteins [ ] . sg are induced during tbev infection and sg components tia- /tiar was found to bind viral rna and inhibit viral translation [ ] . little is known about the role of the tlr / -myd pathway in tick-borne flavivirus infection; however, tlr was shown to suppress lgtv replication within neurons of the brain although it did not affect pathogenesis [ ] . what we are aware of; no animal experiments have shown any significance of the tlr -trif pathway in tick-borne flavivirus infection. however, several studies have looked at the prevalence of polymorphisms in the tlr gene in tbe patients [ ] [ ] [ ] [ ] . in particular, one polymorphism has been investigated the t allele in rs . this polymorphism has been shown to reduce tlr signaling by about % [ ] . although the data regarding tlr is somewhat conflicting, grygorczuk et al. hypothesized that a functional tlr facilitates the onset of neurological disease [ ] by supporting the penetration through the blood brain barrier, but has a protective effect during the established cns infection [ ] . the differences between studies might also be connected to the different subtypes of the tbev strain and the genetic background of the studied populations [ ] . taken together, studies so far have demonstrated the importance of the rig-i-like-ips- pathway in tick-borne flavivirus infection, whereas the role of the tlr pathways remains unclear. after secretion, type i ifn signals in an autocrine and paracrine manner by binding to the heterodimeric ifnar, which consists of two subunits, ifnar and ifnar [ ] . these subunits are associated with janus activated kinases (jak) localized in the cytoplasm; ifnar interacts with tyrosine kinase- (tyk ), whereas ifnar interacts with jak . ligation of ifnar results in activation of tyk and jak , which subsequently phosphorylate signal transducers and activators of transcription (stat)- and stat- . phosphorylated stat- and stat- then form a heterodimer, which associates with irf to form ifn-stimulated gene factor- (isgf ) that binds to the ifn-stimulated response element (isre) in the promoter of many isgs to enhance the transcription of several hundreds of ifn-stimulated genes [ , , ] . together, these isgs act to coordinate an antiviral response that is able to inhibit almost any step in the viral life cycle [ ] . the ifn response in vivo in mice is very important to protect mice from lethal infection with lgtv. mice lacking ifnar all succumbed within days of infection, whereas % of wt mice ( - -week-old) survived the infection. interestingly, all ifnβ −/− mice survived the infection, demonstrating that the ifnαs can compensate for loss of ifnβ in lgtv infection. furthermore, ifnar was shown to be a critical determinant of lgtv tropism as lgtv rna was found in all organs in the absence of ifnar, whereas in wt, only low viral burdens can be detected in the olfactory bulb [ , , ] . using transgenic mice, it was further shown that the ifn response was needed both in the peripheral tissue, as well as locally in the cns, in order to clear lgtv infection [ , ] . within the cns, lgtv mainly infected neurons [ , ] , which is also the case in lethal tbev infection of humans [ ] . although neurons remain the main target cell of tbev, astrogliosis have been shown in post mortem human brains [ , ] . in vitro studies on primary cortical astrocytes show a fast up regulation and secretion of ifn after tbev infection, which is able to protect neighboring astrocytes and neurons from infection already and h post infection, respectively [ ] . astrocytes have been shown to be resistant to tbev-induced cytopathic effects [ , ] , and it was later shown that ifnar expression protected the astrocytes from the virus-induced cytopathic effects [ ] . pretreatment of cells with ifns strongly inhibits growth of most tbfvs in cell culture [ , , [ ] [ ] [ ] [ ] and this is due to the upregulation and concerted actions of several hundreds of isgs. only a few isgs have been identified to play a role in tbev and lgtv infection [ , , , , ] . one of them, the - -oligoadenylate synthetase ( - -oas) (oas) is activated by double-stranded rna, leading to the oas protein polymerization into - -linked oligoadenylates ( - as) [ , ] . these - as activate rnase l, resulting in the degradation of viral rna [ ] . several polymorphisms in the oas genes have been shown to correlate with severe forms of tbe in patients [ ] . also, the murine isoform oas b, which is often lacking in inbred mice strains, confers strain dependent resistance against neurovirulence from far eastern tbev [ ] . two other isgs which have been shown to be antivirally active against tbev are virus inhibitory protein endoplasmic reticulum associated interferon inducible (viperin) and tripartite motif- α (trim α). the trim proteins are a family of proteins able to mediate antiviral activity against many different viruses [ ] . the rodent specific trim α was identified to interact with lgtv ns in a yeast two-hybrid screen. trim α expression inhibited viral infection of lgtv and tbev by mediating lysosomal dependent degradation of ns . interestingly this mechanism was quite specific to tick-borne flaviviruses as the mosquito borne wnv was not inhibited by trim α nor did trim α interact with ns of wnv [ ] . viperin is highly conserved in evolution and was first identified as an ifn-inducible protein with antiviral activity against human cytomegalovirus in [ ] . over the last years, viperin has gained lot of attention and was shown to exhibit broad antiviral activity [ ] . viperin is also known as one of the most highly upregulated genes after viral infection [ , ] . within the family of flaviviridae, viperin has been shown to inhibit several members, such as wnv [ ] , denv [ ] , zikv [ ] , hepatitis c virus [ ] , and tbev [ , , , ] . viperin is an iron sulphur protein with three domains; a n-terminus amphipathic alpha-helix which mediates the intracellular localization to the er, a s-adenosylmethionine (sam) radical domain [ , , ] homologous to a family of proteins that use sam as a cofactor [ ] , and a highly conserved c-terminal domain, important for iron sulphur (fe/s) maturation [ , ] . although viperin is able to inhibit several different viruses, the mechanism of action, the important motif in viperin and the step of viral life cycle inhibited differs for different viruses [ ] . viperin interacts with many viral and host factors for its antiviral function. tbev replication is strongly inhibited by viperin, our research shows that viperin has no effect on the binding or entry of tbev. however, viperin targets genome replication, packaging, and release of tbev (figure ) [ , , ] . viperin specifically targets the plus-sense rna synthesis with no significant effect on the negative-sense rna of tbev during genomic replication [ ] . viperin's fe/s maturation is dependent on ciao [ , ] . the fe/s cluster and a functional sam domain of viperin is essential to inhibit the synthesis of the plus-sense rna of tbev [ ] . however, the target for the radical sam activity important for the antiviral activity of tbev is currently unknown. the structure of viperin was recently characterized [ ] , and based on similarities to other radical sam enzymes, several different hypothesis have been put forward regarding the substrate of viperin and its antiviral activity [ ] [ ] [ ] . however, none of these have shown importance in the context of mammalian viral infection. although the exact mode of action against tbev is not well understood, recent data indicates that viperin interacts with several viral proteins; both structural prm and e and non-structural ns a, ns b, and ns . these interactions lead to a viperin-ns dependent degradation of viral proteins. the degradation of tbev ns was shown to be proteasome dependent [ ] (figure ) . interestingly, ifn treatment induces an increase of tbev capsid particles and this effect was found to be dependent on viperin. viperin mediated this effect by interacting, via its n-terminus, with the cellular protein golgi brefeldin a resistant guanine nucleotide exchange factor (gbf ) [ ] . gbf is a key protein in the cellular secretory pathway and essential in the life cycle of many rna viruses, which utilize vesicular trafficking in their replication cycle and assembly process [ ] [ ] [ ] [ ] (figure ). viperin targets the ns protein for proteasomal degradation which inhibits the synthesis of + strand rna. furthermore, ns interacts with e, ns a, and ns b and these proteins are degraded by viperin in a ns -dependent manner. viperin also interferes with particle assembly by inducing secretion of c particles in a copii-dependent manner, independent of copi. viperin mediates this effect by interacting and sequestering gbf . red arrow = secretory pathway, blue arrow = "?" vesicular transport via unknown pathway. although viperin has been demonstrated to be antiviral active against many different viruses in vitro, few studies have investigated viperin's role in vivo. in tbfv infection, viperin was shown to control lgtv dissemination and replication in the brain after intraperitoneal administration, and viperin promoted survival after intracranial infection [ ] . interestingly, viperin has been shown to be expressed in the brain at the basal state [ ] , with high basal expression in primary astrocytes [ , ] . viperin's role within the brain during neurotropic lgtv infection was further mapped to certain brain regions, as viperin inhibited viral replication in the olfactory bulb and cerebrum, but not in the cerebellum or brainstem. this correlates very well with tbev infection since viperin strongly inhibited tbev replication in primary neurons and astrocytes from the cerebrum, but not in granular cell neurons isolated from the cerebellum. interestingly, cortical neurons were completely dependent on viperin for ifn-mediated inhibition of tbev, whereas in astrocytes, in which the ifn-mediated antiviral activities are strongly dependent on viperin, other isgs could partly compensate for loss of viperin [ ] . taken together, viperin has been shown to be an isg that strongly inhibits tick-borne flaviviruses in vivo and in vitro. this strong antiviral effect on tbev is mediated by targeting the virus at multiple steps of the life cycle. interestingly, viperin targeting the synthesis of plus-sense rna is dependent on the sam domain or the c-terminal domain, while the n-terminal domain of viperin is responsible for interfering with virus assembly and release. in order to establish an infection, a pathogen needs to overcome or evade the innate immune response. to breach the very first line of defense, the skin-tbfvs use the tick to deliver the virus through the skin via tick saliva. furthermore, tick saliva contains immunomodulatory compounds that enhance viral transmission and dissemination [ , ] . although there are several mechanisms in which mosquito-borne flaviviruses actively suppress the induction of type i ifn, no such mechanism has been identified in tick-borne flaviviruses so far [ ] . instead, tbev, like other flaviviruses, utilizes a passive evasion mechanism in which the virus hides its dsrna intermediates in vesicular structures inside the er membranes, and thus delaying the recognition by the cytosolic rig-i like receptors and subsequent irf phosphorylation and ifn induction ( figure a ) [ , , ] . the most conserved inhibition of the type i ifn system within the flaviviridae family is the antagonism of ifnar signaling carried out by ns ; this mechanism is conserved between several mosquito and tick-borne flaviviruses [ , [ ] [ ] [ ] [ ] [ ] . in lgtv infection, ns was shown to inhibit the jak-stat pathway and ns was shown to interact with the ifnar receptor [ ] . similarly, it was shown that ns of tbev interacts with scribble (hscrib) which mediates ns localization to the plasma membrane and this interaction enables ns to inhibit type i and type ii ifn mediated jak-stat signaling [ ] . knockdown of hscrib altered ns cellular localization and reversed the inhibition of the jak-stat signaling [ ] . further studies revealed that ns of tbev inhibited the cell surface expression of ifnar by binding to prolidase (pepd) [ ] . pepd is a peptidase that is needed for ifnar maturation and subsequent cell surface expression. ns binding of pepd prevented maturation of complex n-linked oligosaccharides on ifnar , which in turn disrupted its surface expression ( figure b ) [ , ] . in kfdv infection, ifn treatment failed to reduce viral titers when added after infection, this effect was also found to be mediated by the ns proteins antagonism [ , ] . during tbfv infection, a subgenomic noncoding rna is formed, called subgenomic flavivirus rna (sfrna) [ , , ] . it is produced as a product of incomplete degradation of genomic viral rna by cellular - exoribonuclease xrn [ ] . the ability to produce sfrna in wnv was shown to be needed for efficient viral growth in vitro and for pathogenicity in mice [ ] . interestingly, denv sfrna was found to bind to trim to inhibit rig-i-induced type i interferon expression in huh- cells [ ] . furthermore, denv and wnv sfrna was found to suppress the rnai response in both mammalian and insect cells [ ] . similarly, in tbev infection, sfrna has been demonstrated to inhibit the antiviral rnai response in tick cells [ ] . pepd is needed for maturation and subsequent transport of ifnar to the plasma membrane. ifnar and ifnar heterodimer on plasma membrane can be activated by ifnα/β which leads to the signaling cascade and phosphorylation and translocation of stat / -irf into the nucleus and upregulation of isgs. right panel: ns interferes with ifn signaling. ns protein interacts with pepd thus preventing ifnar plasma membrane localization (red t). ns also prevents stat phosphorylation (red t). arrows indicate protein transport. even though recent studies have shed light on the role of innate immunity during tick-borne flavivirus infection, much remains unknown. for example, the role of the tlr-trif and tlr-myd pathway in pathogenesis and viral recognition. furthermore, most studies were performed using lgtv and tbev, and although they are closely related to powv, liv, kfdv, and ohfv, their interactions with the innate immune response might differ. although ifn has been shown to strongly control viral tropism and pathogenesis of tick-borne flaviviruses, few antiviral isgs have been identified. viperin has been shown to be the most important isg in cortical neurons, however, other isgs that target tbev 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by flaviviruses is required for pathogenicity noncoding subgenomic flavivirus rna: multiple functions in west nile virus pathogenesis and modulation of host responses dengue subgenomic rna binds trim to inhibit interferon expression for epidemiological fitness noncoding flavivirus rna displays rna interference suppressor activity in insect and mammalian cells we would like to acknowledge yong-dae gwon for constructing figure , and harry tracy for critically reading the final version of the manuscript. the authors declare no conflict of interest. key: cord- -zwvesrct authors: thiessen, lindsey d.; neill, tara m.; mahaffee, walter f. title: development of a quantitative loop-mediated isothermal amplification assay for the field detection of erysiphe necator date: - - journal: peerj doi: . /peerj. sha: doc_id: cord_uid: zwvesrct plant pathogen detection systems have been useful tools to monitor inoculum presence and initiate management schedules. more recently, a loop-mediated isothermal amplification (lamp) assay was successfully designed for field use in the grape powdery mildew pathosystem; however, false negatives or false positives were prevalent in grower-conducted assays due to the difficulty in perceiving the magnesium pyrophosphate precipitate at low dna concentrations. a quantitative lamp (qlamp) assay using a fluorescence resonance energy transfer-based probe was assessed by grape growers in the willamette valley of oregon. custom impaction spore samplers were placed at a research vineyard and six commercial vineyard locations, and were tested bi-weekly by the lab and by growers. grower-conducted qlamp assays used a beta-version of the smart-dart handheld lamp reaction devices (diagenetix, inc., honolulu, hi, usa), connected to android . enabled, bluetooth-capable nexus tablets for output. quantification by a quantitative pcr assay was assumed correct to compare the lab and grower qlamp assay quantification. growers were able to conduct and interpret qlamp results; however, the erysiphe necator inoculum quantification was unreliable using the beta-smart-dart devices. the qlamp assay developed was sensitive to one spore in early testing of the assay, but decreased to > spores by the end of the trial. the qlamp assay is not likely a suitable management tool for grape powdery mildew due to losses in sensitivity and decreasing costs and portability for other, more reliable molecular tools. molecular techniques, such as pcr, are capable of being used to detect specific pathogens in air samples with high sensitivity and specificity (carisse, bacon & lefebvre, ; carisse et al., b; falacy et al., ; thiessen et al., ; west et al., ) . the detection of airborne pathogen inoculum has been improved through the development of quantitative pcr (qpcr) assays that allow for near real-time monitoring of inoculum concentration (carisse et al., b; rogers, atkins & west, ; temple & johnson, ; thiessen et al., ) . despite the utility of qpcr to monitor pathogens, it is often impractical due to requirements for experienced laboratory staff and expensive equipment to accurately assess pathogen concentration (notomi et al., ; west et al., ) . loop-mediated isothermal amplification (lamp) assays could be an inexpensive alternative for detection in the field or at remote facilities. lamp can use relatively inexpensive and mobile equipment and utilizes the bst polymerase that has a high tolerance to reaction inhibitors , which allows for quick, minimal dna extraction protocols. these traits make lamp useful in field detection assays (harper, ward & clover, ; kubota et al., ; temple & johnson, ; tomlinson, barker & boonham, ; tomlinson, dickinson & boonham, ) . loop-mediated isothermal amplification has been developed for monitoring inoculum in numerous plant pathosystems, including grape powdery mildew (erysiphe necator), fire blight of pear (erwinia amylovora), and gray mold (botrytis cinerea) (temple & johnson, ; thiessen et al., ; tomlinson, dickinson & boonham, ) . traditional lamp assays produce a magnesium pyrophosphate precipitate when dna is amplified that can be detected with the human eye; however, in low concentrations of target dna, precipitate may be difficult to observe kubota et al., ; thiessen et al., ) or require expensive equipment (temple & johnson, ) . several dyes have been explored to improve detection including sybr green (notomi et al., ) , hydroxynaphthol blue (cardoso et al., ) , and other synthetic dyes (fischbach et al., ) , but the dyes have the potential to inhibit lamp reactions or require the use of spectrophotometers, which increase labor and equipment costs. the use of a fluorescence resonance energy transfer (fret)-based probe, allows for specific detection of lamp products and target quantification from field samples without inhibiting amplification , and several portable fluorescence-reading lamp devices have been made commercially available, such as the genie (optigene ltd., west sussex, uk) and bioranger (diagenetix, inc., honolulu, hi, usa). using a fluorescent probe also reduces potential classification error from visual detection of lamp products, which may improve the accuracy of pathogen detection and allow for quantification. grape powdery mildew, caused by e. necator, causes damages to grape (vitis vinifera l.) wherever it is produced. this disease requires numerous applications of fungicides, which are either applied on a calendar schedule from bud break (bbch ) until véraison (bbch ) or based on disease risk models (carisse et al., a; gadoury & pearson, ; thomas, gubler & leavitt, ) . more recently, fungicide applications have been reduced using inoculum detection systems (thiessen, neill & mahaffee, ; thiessen et al., ) ; however, these systems do not provide in-field inoculum concentration for producers. additionally, the lamp assay that was successfully designed for field use in the grape powdery mildew pathosystem had numerous false negatives or false positives, which may have been caused by difficulty in perceiving the magnesium pyrophosphate precipitate, reducing the predictive values of the lamp assay (thiessen et al., ) . a timely and cost-effective system that improves detection of e. necator inoculum throughout the growing season is needed to allow growers to accurately time fungicide applications early in the growing season and adjust application intervals based on inoculum concentration. the purpose of this research was to develop a quantitative molecular assay for commercial implementation that could be used by growers or vineyard consultants for the detection and quantification of airborne e. necator inoculum. the specific objectives of this project were to ( ) develop a real-time, quantitative lamp (qlamp) assay that was sensitive and specific to e. necator, and ( ) test field use of a mobile, qlamp device by growers. sample rods were created by cutting stainless-steel lsi welding rods ( . mm in diameter) (weldcote metals, kings mountain, nc, usa) to mm lengths, then sterilized and prepared according to thiessen et al. ( ) . to produce a standard curve, conidial suspensions were generated by suspending e. necator conidia from v. vinifera cv. "chardonnay" vines in a . % tween (sigma-aldrich, st. louis, mo, usa) and nuclease-free water solution then pipetting the conidial suspension onto rod sets resulting in rods with , , , or , conidia per sample. rods with one or spores were created by transferring individual spores with eyelash brush. a total of six independent spore dilution series were used to generate the standard curve for the quantitative assay. additionally, a set of sample rods containing conidia was also generated using the conidial suspension to act as a positive control for all dna extractions and molecular reactions. the rods were air dried prior to processing. quantitative lamp assay dna for qlamp analysis was extracted using a quick extraction method modified from thiessen et al. ( ) . spore rods were transferred to -ml screw-cap tubes containing ml of % chelex (sigma-aldrich, st. louis, mo, usa) in molecular grade, depc-treated water. tubes containing rods were vortexed for s then placed in boiling water for min. tubes were removed from boiling water and vortexed another s. the tubes were boiled for another min, and then removed and allowed to cool at room temperature for min. samples were centrifuged at , g for min to collect the contents in the tube. rods were aseptically removed in a laminar flow hood prior to the pellet being processed using the chelex dna extraction process (described below). after dna were extracted and amplified, samples were stored at - c for further analyses. the qlamp reaction is a modified assay from thiessen et al. ( ) and kubota et al. ( ) , which was optimized to generate a quantification standard curve (described above). a fret-based probe was designed using the forward loop primer region with a fam reporter ( -carboxyfluorescein) and a quencher strand . each reaction contained . ml of isothermal master mix with no dye (optigene ltd, west sussex, uk), internal primers fip en and bip en ( . mm), external primers f en and b en ( . mm), forward loop primer fam strand (fl-f, . mm), and quencher strand (q-strand, . mm) to create a ml reaction (table ) . lab-conducted qlamp (l-qlamp) reactions were carried out on an abi stepone plus qpcr machine (applied biosystems, grand island, ny, usa). reaction conditions were c for min followed by c for min. all reactions were run in triplicate. the reaction time threshold (r t ) values, measured in minutes, of the spore standards were averaged and used to create a log-linear standard curve against which unknown samples were compared (fig. ) . a log-linear curve is required to describe the assay because lamp amplification rate is faster than exponential due to concatenation of amplicon (mori et al., ) . a -conidia extraction control, and , -conidia positive controls, as well as non-template controls were included in all reaction setups. unknowns were compared to the standard curve to determine relative spore quantity. positive control samples were also compared to the standard curve to determine extraction efficiency and amplification efficiency. unknown sample r t values were adjusted based on positive control r t values if the positive controls showed poor alignment to the standard curve. to test the l-qlamp sensitivity to target dna, separate spore concentration series were created and tested for positive amplification. growers were provided with all equipment and supplies to conduct the dna extraction and the qlamp reaction protocol described above. dna extraction and qlamp assays were conducted in any location growers deemed appropriate (i.e., office space, winery hallway, tractor barn, kitchen table). for the grower-conducted qlamp assay (g-qlamp), frozen aliquots of qlamp master mix were stored in insulated cryoboxes (vwr north america, radnor, pa, usa) at - c until reactions were conducted. all reactions were conducted in beta-version smart-dart handheld lamp reaction devices (diagenetix, inc., honolulu, hi, usa), which connected to android . enabled, bluetooth-capable nexus tablets for output (google, mountain view, ca, usa). all g-qlamp reactions were conducted in duplicate including -conidia positive controls and non-template controls. reaction conditions followed the protocol described above. smart-dart lamp devices provided amplification curves and the r t values associated with amplification curves. growers were asked to determine if samples were positive, as indicated by the presence of a sigmoidal amplification curve, or negative, no amplification observed, based on the output from the handheld lamp device. the dna from collected spore sampler rod pairs was extracted using the powersoil Ò dna extraction kit (mo bio laboratories, inc., carlsbad, ca, usa) following the manufacturer's protocols. in each set of dna extractions, a set of positive control rods containing e. necator conidia was included as an extraction efficiency control. e. necator primers developed by falacy et al. ( ) were paired with a taqman Ò probe with a minor groove binder (thiessen et al., ) . all qpcr reactions contained . ml perfec t a Ò qpcr toughmix Ò (quanta biosciences, gaithersburg, md, usa), nm final concentrations of each e. necator forward and reverse primers and probe (table ) , and . ml extracted sample dna for a ml total volume. reactions were carried out using an abi stepone plus qpcr machine (applied biosystems, foster city, ca, usa). all qpcr reactions were performed in triplicate, and each reaction plate contained the conidia extraction control, and , conidia positive reaction controls, and template-free negative control. cycle threshold (c t ) analysis was conducted using abi stepone tm software according to protocols by thiessen et al. ( ) . spore concentrations were determined for field samples by identifying the average c t value for each triplicate reaction, and comparing this value to the standard curve described below. average c t values of positive controls figure sensitivity of qlamp assay to erysiphe necator as a function of percent amplification (y-axis) and spore + log concentrations (x-axis). each point represents the amplification of separate extractions created from different e. necator conidia dilution series ( , , and conidia concentrations), one and conidia eyelash transferred spore rods, and conidia-free spore rods (n = ). full-size  doi: . /peerj. / fig- ( , , and , conidia) from each set of qpcr reactions were used to confirm the efficiency and to the suitability of the standard curve for determining conidia concentration of unknowns. the standard curve was generated by creating five independent, -fold conidial dilution series on the stainless-steel sampling rods to  conidia (described above), dna was extracted using the powersoil kit (described above), and the average c t values for each conidia quantity from the five independent dna extractions was used to fit a linear curve. custom impaction spore samplers (thiessen et al., ) , were placed at a research vineyard and six commercial vineyard locations within the willamette valley of oregon. each spore sampler contained a pair of sample rods described above. spore samplers were run continuously, sampling l/min, and sample rods were replaced daily or every monday and thursday (bi-weekly). three spore samplers were placed at each of the six commercial vineyards that were collected by growers bi-weekly. the growers completely maintained one trap, processing all sample rods derived from that trap. sample rods from the other two traps were collected by the growers and transported to the lab for processing with the l-qlamp assay and the qpcr assay. at the oregon state university botany and plant pathology research farm vineyard, paired spore samplers, one for the qpcr assay and one for the qlamp assay, were collected and processed by laboratory personnel on a daily and bi-weekly schedule. spore samplers for the l-qlamp and the qpcr assays were deployed on april , and april , and sample rods were collected from bud break until véraison (bbch ). spore samplers for the g-qlamp assay were deployed april , and were collected until july , . estimates of airborne inoculum concentration derived using qpcr and qlamp were compared to assess the accuracy of the qlamp procedure. the g-qlamp assay detection results were compared to the l-qlamp assay and qpcr detection data as described below. data was analyzed using r . . . detections from samples collected and quantified with l-qlamp assay were compared to qpcr assay detections using a student's t-test. the g-qlamp detection results were compared to l-qlamp detection results using a  contingency table whereby the l-qlamp results were assumed correct. both the l-qlamp and g-qlamp spore detections were evaluated using a  contingency table whereby the qpcr assay results were assumed correct. the qlamp assay detection accuracy, true positive proportion, true negative proportion, fisher's exact test, and chi-squared test were assessed comparing the qlamp detection results to the qpcr detection results. the qlamp assay showed high sensitivity to e. necator conidia dna when separate spore dilution series were tested ( fig. ) with % of one conidia samples amplifying using the qlamp assay. all other spore quantities tested showed % amplification sensitivity within the qlamp assay. the qlamp assay standard curve development resulted in a standard curve (r = . ) when fit with a log-linear curve (fig. ) . a log-linear curve was fit to the log spore quantity to account for the number of primers used in the assay, and the amplicon produced concatenates resulting in greater than an exponential rate of amplification. this curve was used to quantify the l-qlamp samples collected from the botany and plant pathology research farm vineyard. the l-qlamp spore quantification was significantly lower than the qpcr quantification when daily samples were collected in (p < . ) (fig. a) , but the biweekly l-qlamp and qpcr sample quantification was not significantly different in (p = . ) (fig. b) . the l-qlamp assay significantly underrepresented spore levels for both the daily collections (p < . ) (fig. a ) and the biweekly collections (p = . ) (fig. b ) compared to the qpcr assay in . utilizing l-qlamp for detection of e. necator showed similar results to qpcr assay detections in both and (p < . ) ( table ). the l-qlamp assay detection results were % and % accurate in and , respectively compared to the qpcr assay detection results. the l-qlamp assay detection results showed true negative proportions of % and % and true positive proportions of % and % in in and , respectively. there was an unexplained loss of sensitivity in sample testing that was extensively examined (see below). grower-conducted qlamp assay the software provided with the mobile lamp device used auto-adjusting threshold values to account for noise of fluorescence readings which significantly reduced accurate quantification by growers. the g-qlamp assay for the detection of e. necator was not correlated to the qpcr detection results (p = . ) ( table ). the g-qlamp detection results showed % accuracy compared to the qpcr assay results, respectively. the g-qlamp detection results show true negative proportions of %, and true positive proportions of % compared to the qpcr detection results. due to loss of sensitivity of the qlamp assays to e. necator observed during assay testing in , extensive troubleshooting was conducted. primer purification, polymerase used (bst; new england biolabs, ipswich, ma or iso- ; optigene ltd, west sussex, uk), master mix distributer, assimilating probe removal, primer and assimilating probe manufacturer, inhibitor removal compounds in the master mix, dna extraction and clean up, adjustment of reaction temperature, and replacement of reagents and primers were all tested. primer purification was tested prior to the implementation of the experiment, and during the observed degradation of qlamp sensitivity with no observable difference between reaction efficiency of hplc or desalted primers. regardless of polymerase used, bst or iso- , reaction efficiency and sensitivity to e. necator dna was reduced compared to assays conducted prior to implementation of field testing. different distributers of the optigene isothermal mastermix were also tested to determine if the decreased sensitivity was caused by storage or shipping errors; however, there was no difference among master mix vendors. it was not possible to test previous lots of the master mix prior to the observed decrease in sensitivity. the assimilating probe was removed and gel electrophoresis was used to compare with and without probe presence, and no difference was observed in amplification. there was also no difference between different primer and probe manufacturers, which also suggests there were no differences in manufacturing process. the concentrations of inhibitor removal compounds within the master mix were assessed, including % polyvinylpyrrolidone (pvp) , edta, and bsa concentrations, to determine if inhibitor presence was causing decreased reaction efficiency, and no differences were observed for inhibitor removal compounds. in addition to testing master mix removal of inhibitors, three dna extraction methods (extractions with ph . , mm tris- . mm edta buffer (affymetrix, santa clara, ca, usa), pvp (sigma-aldrich, st. louis, mo, usa) in depc-treated water, and powersoil Ò dna extraction kit (mo bio laboratories, inc., carlsbad, ca, usa)) were assessed with separate field collected spore samples. no differences were observed in amplification time or efficiency when testing each side-by-side extraction method. to test the optimal reaction temperature of the polymerase, temperatures between and c were examined to find the optimal reaction temperature. lower spore quantities ( spores or less) amplified at c. a last effort to determine if the effect notes: a "positive" and "negative" indicate the number of samples for which e. necator dna was detected and not detected, respectively, as tested by l-qlamp (n = in and n = in ) assays as described in the text. b g-qlamp (n = in ) assessed by growers using mobile qlamp devices (diagenetix, inc., honolulu, hi, usa) as described in the text. c qpcr results based on taqman Ò probe with minor groove binder for detecting e. necator dna. "positive" and "negative" indicate the number of samples for which e. necator dna was detected and not detected, respectively. qpcr detection data based on quantitative data from (thiessen, neill & mahaffee, ) . d fisher's exact test was used to assess the null hypothesis that each lamp assay was significantly different from the qpcr assay. * significant chi-squared test at p < . of qlamp and qpcr assays. was due to degradation of reagents or primers during the growing season, all reagents, primers, and probe were replaced; however, the decreased sensitivity to e. necator dna was still observed. despite targeting various portions of the reaction and extraction, the cause for loss of qlamp assay sensitivity remains undetermined. a highly sensitive qlamp assay was successfully developed using a simple dna extraction method for use by growers or crop consultants to use as a decision aid for timing fungicide applications similar to thiessen et al. ( ) and thiessen, neill & mahaffee ( ) . the qlamp assay developed was sensitive to e. necator dna with one spore amplifying % (n = ) using the simplified dna extraction. this sensitivity indicated that the assay should be suitable to detect inoculum (i.e., ascospores) at low concentrations (< spores) and aid management decisions. however, the qlamp assay consistently underrepresented spore quantities later in the growing season compared to the qpcr assay, which may be due to an increase in the presence of pcr inhibitors (such as pollen, humic acids from soil, spider webs, etc.) found in air samples (wilson, ) that may not have been removed by the rapid chelex dna extraction. in early dna extraction testing prior to qlamp sensitivity loss, the powersoil Ò extracted dna showed more consistent amplification of field samples than the other extraction methods (thiessen et al., ) ; however, the powersoil Ò dna extraction kit requires a larger time commitment and several steps that may not be feasible for in-field dna extractions. the lamp assay has been widely described as more tolerant to inhibitors than qpcr (francois et al., ; kaneko et al., ) , but it appears that the lamp assay tolerates different inhibitors than the qpcr assay (nixon et al., ) . additionally, the qlamp r t variance from one to spore samples (fig. ) was so great that they cannot be distinguished. this variance is likely due to using dna extractions of each spore concentration as opposed a dilution from higher spore concentration as is typically done (mahaffee & stoll, ) . because the lamp assay is not limited by temperature cycles, annealing is reliant on proximity of dna to the polymerase and primer set (notomi et al., ) , and the improved sensitivity with lower annealing temperatures is likely the result of lower specificity of primers rather than optimal reaction temperature. in reactions with lower quantities of dna (e.g., one and spores), more time may be required for the polymerase, primers, and target dna to meet, which may explain the variability of r t values of low spore quantities (fig. ) . the inhibition of the field qlamp assay and the difficulty of differentiating low spore quantities indicates that the assay currently has more utility as a qualitative inoculum detection tool as opposed to quantitative assessment of inoculum availability. the g-qlamp results were significantly different from the qpcr detection results (p = . ) ( table ). this may be due to difficulty in assessing positive detections from the output of the mobile device. the curve smoothing algorithm used by the device application (g-qlamp) often produced curves that drifted linearly with r t values reported even though there was no detectable amplification using gel electrophoresis. growers conducting the q-lamp assay were directed to ignore curves that ascended linearly due to curve smoothing; however, this may have caused growers to be overlyconservative in determining positive detections. additionally, the grower-conducted q-lamp occurred in when the loss of q-lamp sensitivity was observed and there was very low disease. the l-qlamp assay detection results were similar to qpcr assay detection results in both and , but true positive and true negative proportions were variable between years. this variability may be due to the presence of inhibitors. in , the source of stainless-steel rod material was changed from previous testing, and significant inhibition of dna amplification was observed. after troubleshooting various rod cleaning processes and dna extraction techniques, a hexane soak was added to the steel rod cleaning protocol to remove oils prior to sterilization and % chelex was used as the extraction buffer. after the hexane wash step addition, the accuracy of samples was improved to %, and the misclassification rate was reduced from % to %. in addition to inhibitors from the rods, the variability of inhibitors from field collections may have caused inconsistencies in qlamp assay detection results compared to the qpcr assay detection results. early in the growing season, the weather in the region is characterized by frequent precipitation events that limit pollen and insect flight. later in the growing season, pollen, insects, birds, and soil particulates are abundant in the air, and subsequently on the sampling rods. rnases, dnases, humic acids, and other heavy metals may not be removed when using the chelex dna extraction (qlamp template), but are removed during the powersoil dna extraction (qpcr template). the results from the qlamp had lower true positive proportions and true negative proportions than that of turbidimetric lamp previously developed (thiessen et al., ) . these reductions may be due to other factors besides amplification inhibitors, such as manufacturer differences, degradation of polymerase, inclusion of probes, or buffering of the qlamp reaction (corless et al., ; roux, ). using the qlamp assay for field detection and quantification of fungal pathogens may not be as feasible as previously thought due to the random loss of assay sensitivity and potential inhibition of polymerase activity by environmental contaminants. redesigning primers was another potential approach to examining the cause of the reduced sensitivity; however, the primer set used here was the result of two previous redesigns during development and testing and there was not sufficient heterogeneity in other regions of the its. additionally, the lamp assay quantification was also affected by numerous inhibitors, such as soil, pollen, or insect debris, found in field collected samples. lamp is capable of tolerating some inhibitors that affect pcr assays (francois et al., ) ; however, to determine the extent that lamp assays are capable of tolerating inhibitors, each potential inhibitor should be tested (nixon et al., ) . other lamp assays developed have utilized more complex dna extractions to reduce the effect of inhibitors on amplification for quantitation of dna (harper, ward & clover, ; kubota et al., ; mori et al., ) ; however, complex dna extraction techniques are likely to preclude field implementation of lamp assays and increase assay costs. the observed inconsistency indicates that the developed qlamp assays might not be robust enough for commercial implementation. the lamp assay was developed due to reports of high sensitivity and specificity to target dna, tolerance of the reaction to the presence of reaction inhibitors, and the potential for use by growers or crop consultants using handheld lamp devices such as the bioranger (diagenetix, inc., honolulu, hi, usa) or the genie ii and iii (optigene ltd, west sussex, uk) (kubota et al., , mori et al., mori et al., , notomi et al., ; temple & johnson, ; tomlinson, dickinson & boonham, ) ; however, field testing of the qlamp assay for e. necator revealed an unidentifiable degradation of the sensitivity of the assay to the target dna. the qlamp assay may still be a useful tool for field inoculum detection, but further analysis of the system is required to determine the specific cause of the degradation of the assay. at the time this research was initiated the lamp technology was the most advanced for inexpensive field application and thus selected for investigation over other potentially suitable technologies. however, other dna amplification techniques have since become more accessible for field use (marx, ) , including qpcr (biomeme, inc., philadelphia, pa, usa), recombinase polymerase amplification (piepenburg et al., ) , and helicase-dependent isothermal dna amplification (vincent, xu & kong, ) . these assays require minimal dna preparation, are capable of real-time data, and may be easily adapted to the air samples used here but require evaluation. there are several reviews that discuss the advantages and disadvantages of these technologies (craw & balachandran, ; gill & ghaemi, ; mahaffee, ; niemz, ferguson & boyle, ; yan et al., ) . a highly sensitive qlamp assay was developed using a simple dna extraction method for use by growers or crop consultants utilizing inoculum detection; however, the qlamp assay consistently underrepresented spore quantities later in the growing season compared to the qpcr assay. additionally, the qlamp assay lost sensitivity to low spore quantities (< spores) in the sampling period, and the cause was not determined during the course of this study. grower-conducted inoculum monitoring technologies, like the qlamp assay developed in this study, may provide an inexpensive tool for producers to apply targeted fungicide applications based on inoculum presence and concentration. given the limitations described herein, more assessment of the qlamp assay degradation is necessary before utilizing it as a monitoring tool for e. necator inoculum concentrations. visual detection of turkey coronavirus rna in tissues and feces by reverse-transcription loopmediated isothermal 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cinerea by loop-mediated isothermal amplification helicase-dependent isothermal dna amplification pcr to predict risk of airborne disease inhibition and facilitation of nucleic acid amplification isothermal amplified detection of dna and rna we thank the technical support of andy albrecht, cole provence, chris gorman, and jim eynard. we also thank anonymous reviewers for their helpful suggestions to improve the manuscript. we especially thank the numerous vineyard managers that collaborated on the project. the use of trade, firm, or corporation names in this publication is for the information and convenience of the reader. such use does not constitute an official endorsement or approval by the united states department of agriculture or the agricultural research service of any product or service to the exclusion of others that may be suitable. this work was supported by the american vineyard foundation, the oregon wine board, and usda-ars cris - - - d. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. the following grant information was disclosed by the authors: american vineyard foundation. oregon wine board. usda-ars cris: - - - d. the authors declare that they have no competing interests. lindsey d. thiessen conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft. tara m. neill conceived and designed the experiments, performed the experiments, contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper, approved the final draft. walter f. mahaffee conceived and designed the experiments, authored or reviewed drafts of the paper, approved the final draft. the following information was supplied regarding data availability:the raw ct values and positive negative values of detection events, as well as standard curve development and sensitivity of assay, are provided in supplemental dataset files. supplemental information for this article can be found online at http://dx.doi.org/ . /peerj. #supplemental-information. key: cord- - e u authors: paploski, igor adolfo dexheimer; corzo, cesar; rovira, albert; murtaugh, michael p.; sanhueza, juan manuel; vilalta, carles; schroeder, declan c.; vanderwaal, kimberly title: temporal dynamics of co-circulating lineages of porcine reproductive and respiratory syndrome virus date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: e u porcine reproductive and respiratory syndrome virus (prrsv) is the most important endemic pathogen in the u.s. swine industry. despite control efforts involving improved biosecurity and different vaccination protocols, the virus continues to circulate and evolve. one of the foremost challenges in its control is high levels of genetic and antigenic diversity. here, we quantify the co-circulation, emergence and sequential turnover of multiple prrsv lineages in a single swine-producing region in the united states over a span of years ( – ). by classifying over , prrsv sequences (open-reading frame ) into phylogenetic lineages and sub-lineages, we document the ongoing diversification and temporal dynamics of the prrsv population, including the rapid emergence of a novel sub-lineage that appeared to be absent globally pre- . in addition, lineage was the most prevalent lineage from to , but its occurrence fell to . % of all sequences identified per year after , coinciding with the emergence or re-emergence of lineage as the dominant lineage. the sequential dominance of different lineages, as well as three different sub-lineages within lineage , is consistent with the immune-mediated selection hypothesis for the sequential turnover in the dominant lineage. as host populations build immunity through natural infection or vaccination toward the most common variant, this dominant (sub-) lineage may be replaced by an emerging variant to which the population is more susceptible. an analysis of patterns of non- synonymous and synonymous mutations revealed evidence of positive selection on immunologically important regions of the genome, further supporting the potential that immune-mediated selection shapes the evolutionary and epidemiological dynamics for this virus. this has important implications for patterns of emergence and re-emergence of genetic variants of prrsv that have negative impacts on the swine industry. constant surveillance on prrsv occurrence is crucial to a better understanding of the epidemiological and evolutionary dynamics of co-circulating viral lineages. further studies utilizing whole genome sequencing and exploring the extent of cross-immunity between heterologous prrs viruses could shed further light on prrsv immunological response and aid in developing strategies that might be able to diminish disease impact. porcine reproductive and respiratory syndrome virus (prrsv), the etiological agent of prrs, is one of the most important endemic viruses affecting the swine industry in the united states (holtkamp et al., ) and globally (stadejek et al., ; vanderwaal and deen, ) . the economic impact of the disease in the united states has been estimated at $ million annually (holtkamp et al., ) . clinical signs in affected farms vary by viral variant and according to the farm's production stage (e.g., breeding or growing herd), herd management, immune status, and other factors (goldberg et al., ) . premature farrowing can occur in - % of sows in an affected farm, and up to % of piglets are stillborn during an outbreak (christianson and joo, ) . piglets may be born with low weight and can present with lethargy and anorexia, which can lead to a mortality of more than % among piglets (pejsak et al., ) . prrsvinfected pigs are also susceptible to secondary infections leading to poor average daily gain and feed conversion, further increasing production loss (solano et al., ; xu et al., ) . up to % of united states breeding herds experience outbreaks annually (tousignant et al., a) and control of the disease in the united states, europe, and globally is challenging due to high levels of antigenic variability and its rapidly expanding genetic diversity (frossard et al., ; brar et al., ; guo et al., ; smith et al., ) . porcine reproductive and respiratory syndrome virus was first recognized almost simultaneously in europe (wensvoort et al., ) and north america (collins et al., ) in the late s and early s, but genetic differences suggested a much earlier evolutionary divergence between the north american and european viral types. thus, prrsv is divided into two major phylogenetic clades, prrsv type (more prevalent in europe) and type (more prevalent in north america) (shi et al., a,b; stadejek et al., ) . within each clade, high levels of genetic and antigenic diversity exist and cross-protection is only partial (roberts, ; kim et al., ; correas et al., ) . genetic similarities between prrsv isolates have been used as a tool to understand disease transmission and epidemiology (kapur et al., ; wesley et al., ) , and several different strategies have been used for classifying isolates of prrsv into epidemiologically meaningful groups. for prrsv type , the most commonly used classification system is based on restriction fragment length polymorphisms (rflp) and sequencing, both of which are typically based on the open reading frame (orf ) portion of its genome (kapur et al., ; wesley et al., ) . the orf gene encodes for the major envelope protein (gp ), which plays a role in inducing virus neutralizing antibodies and cross-protection among prrsv variants (dea et al., ; kim et al., ) . rflps have been broadly adopted by the u.s. swine industry despite shortcomings, such as the fact that the genetic relationship between different rflp types is unclear, the potential for two distantly related viruses to share the same rflp type, and the instability of rflp-typing when assessing isolates related to each other by as few as animal passages (cha et al., ) . in , a classification system based on the phylogenetic relatedness of the orf portion of the virus's genome was proposed (shi et al., a,b ). this classification system aggregates isolates into phylogenetic lineages based on the ancestral relationships and genetic distance among isolates. using this system, nine different lineages were described within prrsv type , each of which was estimated to have diverged between and (shi et al., b) . phylogeny-based classification of organisms is seen as the most powerful and robust instrument for distinguishing between variants of a viral population (hungnes et al., ) and has been used in the study of other viral diseases (liu et al., ) . phylogeny-based classification of prrsv, rather than rflp profiling, is expected to provide fewer ambiguities and more insight into the evolutionary relatedness amongst different variants. while the existence of prrsv lineages is well established, the dynamics of their cocirculation within a given region has not been well documented. vaccination is often used as a tool to mitigate clinical impact and viral shedding (holtkamp et al., ) . although specific practices vary across farms, gilts are typically vaccinated before entering the herd, and sometimes the sow herd is mass vaccinated during the year. most commercial prrsv vaccines currently sold in the united states are considered "modified live vaccines" (mlv), which means that the vaccine is an attenuated live virus. vaccines against prrsv show different degrees of protection against homologous and heterologous challenges (cano et al., ; díaz et al., ; geldhof et al., ) ; the exact definition of what constitutes a homologous or heterologous challenge is often not clear, especially taking into consideration the genetic diversity existing within prrsv type (shi et al., b) . five major prrsv vaccines are commercialized in the united states, each developed using a different wild prrsv isolate (lineages , , , and , with the lineage vaccine being the most widely used historically). porcine reproductive and respiratory syndrome virus is known to possess a high mutation rate (hanada et al., ; brar et al., ) . genetic mutations for prrsv are thought to result from rna polymerase errors (murtaugh et al., ) and from the lack of proofreading (kappes and faaberg, ) . coupled to that, genetic recombination events can contribute to prrsv diversity (forsberg et al., ) . thus, the emergence of new variants of prrsv is expected to occur potentially through both mutation and recombination. viral variants can quickly emerge in animals (goldberg et al., ) even after inoculation with a single variant (chang et al., ) . thus, the viral population within an animal can be referred to as a viral cloud or swarm (lauring and andino, ) , which suggests that mutation has a considerable impact in virus diversification even on short time scales. in addition, it is assumed that the immune response removes genetic variants of the virus that it recognizes with high specificity, potentially creating selection pressure favoring antigenically divergent prrsv variants (murtaugh et al., ) . hypervariable portions of the viral genome may be subject to immune selective pressure (chen et al., ) ; variation in proteins coded by those sites may play a role in evasion of host immune defenses (ansari et al., ; darwich et al., ) . prrsv vaccines are known to diminish the severity of clinical signs once an infection occurs, but not to prevent an infection from occurring (lyoo, ) . at the population scale, it can be expected that most animals have some level of immunity because of the high prevalence of natural infection and widespread use of vaccine. this creates the potential for immune-mediated selection to be a driver of prrsv diversification and evolution (murtaugh et al., ) . the identification of point mutations that are undergoing positive selective pressure is often interpreted as evidence of increased evolutionary fitness (kryazhimskiy and plotkin, ) . one way to identify such sites is to evaluate dn/ds ratios, which measure the rate at which substitutions at non-synonymous sites (dn) occur relative to substitutions in synonymous sites (ds). substitutions in synonymous sites are thought to be mostly neutral, but a higher occurrence of substitutions in nonsynonymous sites can be interpreted as evidence of selective processes that favor changes in the protein sequence (kosakovsky pond and frost, ) . positive selective pressure in sites that code for epitopes recognized by the host immune system are of special interest, because they suggest that the origin of such selective pressure, if present, could be driven by the host immune response. the rapid evolution of prrsv coupled with the periodic emergence of new and sometimes more virulent viral variants creates a need to continually update our knowledge on circulating prrsv variants. reports that show the waxing and waning of different viral types in the whole north america (shi et al., b) are helpful when understanding continent-wide status of prrsv lineages. however, understanding viral dynamics on a regional scale could provide important insights into local evolutionary and ecological dynamics of prrsv, including an improved understanding of how often new variants emerge or re-emerge within the region. here, we describe the temporal dynamics of prrsv occurrence in a swine-dense region of the united states, characterizing these patterns according to orf genetic lineages and sub-lineages. we quantify the contemporary occurrence of each lineage, investigate the temporal dynamics and turnover of lineages, identify emerging sub-lineages, and examine evolutionary patterns for evidence of positive selective pressures. monitoring project (mshmp) were used for this analysis. briefly, mshmp is an ongoing voluntary producer-driven nation-wide monitoring program for endemic swine diseases that affect the u.s. swine industry. based at the university of minnesota (umn), this program collects weekly reports on the infection status of sow farms from participating swine-producing companies, veterinary practices, and regional control programs, which serves to capture the occurrence of infectious diseases in the country (tousignant et al., a,b; perez et al., ) . infection status data classifies farms into the following categories (holtkamp et al., ) : status : positive-unstable, status : positive-stable, either through use of live virus inoculation ( lvi) or use of vaccines ( vx); status : provisional negative; and status : negative. the main difference between positive-unstable (status ) and positive-stable (status vx or lvi) is that unstable herds have an active clinical outbreak and are weaning prrsv rt-pcr positive piglets. in contrast, prrsv may be still present in positive-stable herds (through use of field virus inoculation or modified live vaccine) but clinical disease is controlled and piglets weaned from such farms are prrsv-negative as a result of herd immunity, decreased shedding, and maternal antibodies (holtkamp et al., ) . mshmp collects farm-level data from approximately . million sows, which represents approximately . % of the united states breeding herd population (national agricultural statistics service [nass] , agricultural statistics board, and united states deparment of agriculture [usda], ). specific production systems (companies involved in pig production) participating in the project also share the orf prrsv sequences identified on their farms as part of routine veterinary management. for example, samples may be submitted by veterinary practitioners to determine if circulating prrsv on the farm is the same or different from the vaccine virus or a previous variant present on the farm. for this analysis, we analyzed , sequences reported between and from mshmp participants located in a relatively isolated swine-dense region in the united states with an approximate area of thousand square kilometers. production systems operating in this region account for ∼ % of the united states sow population. approximately % of farms within this region participate in mshmp and in this project in particular. sequences used in this study came mostly from sow ( . % of sequences), nursery ( . %) and finisher farms ( . %), followed by boar stud farms ( . %) and sequences without a description of their origin ( . %). sequences shared with us by project participants were sequenced according to standardized protocols adopted by laboratories at sdsu (animal disease research and diagnostic laboratory et al., ), isu (zhang et al., ) and eurofins genomics. of the orf gene sequences used in this analysis, seven had fewer than nucleotides. these were deemed incomplete and were excluded from further analysis. we also included orf gene sequences previously classified into nine different genetic lineages (shi et al., a,b) and added these to the collection of mshmp sequences. these sequences, assembled from a database of sequences that spanned from to , were used as guides to classify the mshmp sequences into the previously described genetic lineages, and will be referred to here as "anchor" sequences. we also obtained the orf gene sequences for five vaccines (ingelvac prrsv atp -genbank id dq . , ingelvac prrsv mlv -genbank id af . (both from boehringer ingelheim), fostera prrsv from zoetis -genbank id kp . , prime pac prrsv rr from merck -genbank id dq . , and prevacent, from elanco -genbank id ku . ). the ingelvac prrsv atp and fostera vaccines use isolates belonging to lineage , while ingelvac prrsv mlv uses a lineage isolate, prime pac a lineage isolate and prevacent a lineage isolate. we also obtained two prrsv prototypes (lelystad -genbank id nc_ . , and vr -genbank id ef . , which represent the prototypical european type and north american type viruses, respectively). the sequence dataset used here is sequences were aligned using the muscle algorithm implemented in aliview (larsson, ) using default settings. the alignment was then examined for the presence of recombinants using the recombinant detection program version (martin et al., ) , followed by removal of potential recombinants. in addition, duplicated sequences (with % nucleotide similarity) were identified and set aside for the allocation of sequences into lineages. the aligned and cleaned dataset was imported into mega (kumar et al., ) , where the genetic pairwise distance was measured as a percentage nucleotide difference. using stata (statacorp, ), each of the mshmp sequences were assigned to the lineage that had the smallest genetic distance to an anchor. after sequences were classified into lineages, the duplicated sequences were allocated to their respective lineage group according to the sequence with % similarity that was kept in the lineage classification process. a flow-chart of these steps can be seen in figure . a maximum likelihood phylogenetic tree illustrating genetic relatedness of sequences was constructed based on , bootstraps, adopting the tamura-nei model for substitution of amino acids (tamura and nei, ; kumar et al., ) . clusterpicker software was used to further stratify the most abundant lineage into sub-lineages (ragonnet-cronin et al., ) , in a matter that seemed consistent with the tree main branches while still returning epidemiological meaningful sublineages. the phylogenetic tree was then colored according to the lineage classification and source of sequences (anchor versus mshmp) using microreact (argimón et al., ) . traditional bootstrap support is estimated based on resampling and replication, which tends to yield low support particularly on deep branches and in large trees with hundreds or thousands of sequences (lemoine et al., ) . branch support on the phylogenetic tree thus was evaluated using the bootstrap support by the transfer method (lemoine et al., ) . this method circumvents issues of traditional bootstrapping by assigning a gradual "transfer" index to each clade within the tree rather than a binary presence/absence index for the presence of a clade in each bootstrap (i.e., a clade is considered absent in the bootstrap replicate if the sequences found within the clade is different by even a single member). temporal changes in the frequency of different lineages was tabulated by quarter of the year. graphs representing the relative frequency of prrsv lineages over time were constructed using stata . the frequency with which each lineage occurred over different years was compared using trend analysis for proportions (using the ptrend command) in stata (statacorp, ) . for this test only, lineages with fewer than sequences overall were grouped. the ratio of synonymous to non-synonymous mutations (dn/ds) for all sites in the orf gene region was calculated using the single-likelihood ancestor counting protocol (kosakovsky pond and frost, ) , implemented on the datamonkey webserver . because the analysis can only be performed on sequences at a time, the analysis was repeated on ten random subsets of sequences (after removal of % identical sequences). sites were considered under positive selective pressure if the p-value associated with a higher rate of non-synonymous versus synonymous mutations was smaller than . . the dn/ds (re-scaled for branch length) of all sites from different runs were averaged and the percentage of runs in which each codon was identified as under significant positive selection was calculated. after removal of the seven inadequately sized and two recombinant sequences from the mshmp data, the remaining , mshmp sequences were classified in five different lineages. . % ( , sequences) were classified as lineage , . % ( ) as lineage , . % ( ) as lineage , . % ( ) as lineage , and . % ( ) as lineage . a group of . % ( ) of the mshmp sequences were genetically closer to the european prototype (lelystad) reference, and were thus classified as type prrsv sequences. lineage was further separated into five sub-lineages (a to e). out of the total , sequences in lineage , . % ( ) were classified in lineage a, . % ( ) in lineage b, . % ( ) in lineage c, . % ( ) in lineage d and . % ( ) in lineage e. the phylogenetic tree with all sequences used in the analysis can be seen on figure . using the booster method (lemoine et al., ) , branch support on main branches (lineages and sub-lineages) was above %. the within-and between-lineage nucleotide pairwise genetic distance is shown in table . in general, between lineage/sub-lineage distances are higher than within lineage variation. the distances between sublineages of lineage seem to be smaller between them than between other lineages. broad tree topology was similar when the tree was constructed using nucleotides or amino acids alignment (supplementary figure s ) . on average, the total number of sequences reported to mshmp increased by each year (supplementary table s ), and there was a clear seasonal pattern (figure b ). the first quarter of each year (january -march) was the one with highest number of sequences reported in all but year. the relative frequency of each lineage changed through time ( figure a and supplementary table s ), and specific patterns are noteworthy. first, the absolute and relative occurrence of lineage decreased over time from . % ( sequences) in to < % ( sequences) in the years - . as lineage occurrence to determine whether changes in sampling effort across time impacted general patterns observed here, we repeated the analysis five times, each time randomly sampling orf sequences per quarter. general patterns of lineage occurrence did not change, suggesting that patterns of lineage occurrence were not affected by sampling effort in each quarter (supplementary figure s ) . the visual patterns and turnover of lineages apparent in figure a were shown to be statistically significant. the increase in the frequency of lineages a, , , and type (p < . ) was significant, and changes in the grouped frequency of other lineages (a sum of lineages d, e, and , p = . ) was also significant, but with a difficult interpretation since this is an aggregate of several uncommon lineages. lineages b and c increased in frequency and then decreased (p < . ). lineage frequency decreased over time (p-value < . ), while lineage occurrence remained unchanged (p-value = . ). a total of sites were identified as under positive selection in at least one single-likelihood ancestor counting run (figure ) . some sites were identified as under positive selection in all runs, while others were only identified in some runs. those identified in all runs (with the largest p-value across all runs), were sites (p-value = . ), (p-value = . ), (pvalue < . ), (p-value < . ), (p-value < . ), (p-value < . ), (p-value = . ), and (p-value = . ). a list of all sites identified as under positive selection in at least one run can be found in the caption of figure . most of the sites positively selected were located in the first third of the prrsv orf . the infection status of farms part of mshmp in the studied area over the study time span is shown in figure . this data show two periods in which vaccine usage increased, the first one in mid- , and a second in approximately mid- . not all farms that reported its status to mshmp contributed to sequences to this analysis. we documented the circulation, emergence and sequential turnover of multiple prrsv lineages in a single united states swine-producing region over a span of years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . by classifying over , prrsv orf contemporary sequences into phylogenetic lineages based on pre- data (shi et al., a,b) , we illustrated the continual diversification and temporal dynamics of the prrsv population. through further stratifying lineage into three main sub-lineages, we also describe the rapid emergence of a sub-lineage ( a), which was absent in the pre- analysis even though that dataset was based on > sequences from across the world (including the region in which we collected our samples) (shi et al., b) . we also identified sites within prrsv orf gene and resultant orf protein that showed evidence of positive selective pressure, indicating that non-synonymous mutations that lead to amino acid changes in the protein at these sites are favored. from to , lineage was the most prevalent genetic group observed in our dataset. shi et al., a,b showed that lineage was rapidly increasing in genetic diversity, which is a proxy for the effective population size of the virus, from to , and reached a peak from to . our data suggests that, at least for our study region, the occurrence of lineage peaked pre- , after which it rapidly declined and was replaced mostly by lineage variants. from to , three different major sub-lineages within lineage emerged, two of those being the most prevalent lineage in certain years ( c from to , a from to ). the emergence of sublineage a, beginning in and peaking in was perceived by veterinarians in the studied area as being a noteworthy event coinciding with the spread of the - - rflp-type. in our dataset, . % of the sequences belonging to the a sub-lineage were rflp-typed as - - (followed by . % of sequences with rflp - - and less than % of - - , - - , - - , - - and several others with less than % -see supplementary table s ) . while the failure to achieve consistent and reliable prrsv control and prevention through vaccination demonstrates gaps in our understanding of prrsv immunology (murtaugh, ) , based on current understanding, prrsv vaccines are expected to better protect against wild viral variants that have a higher degree of similarity to the original parental isolate used for vaccine development (cano et al., ; díaz et al., ; geldhof et al., ) . despite our limited understanding of heterologous cross-protection for prrsv, the emergence and sequential dominance of different variants leading to lineages and sub-lineages is consistent with the theory of multi-strain dynamics (gupta et al., ; kucharski et al., ) . immune responses, whether originating from human interventions or accumulation of immunity toward wild variants, can exert selective pressure that can ultimately lead to the emergence of new pathogen sub-populations (gupta et al., ) . as a virus evolves, immune responses generated against a past variant are expected to become less effective, resulting in a highly complex system, with different lineages interacting through the partial cross-immunity that they generate in the host population (gupta et al., ; kucharski et al., ) . theory predicts that due to frequency-dependent selection amongst co-circulating viral variants, rare antigenic variants are expected to spread more widely in the host population but then subsequently decline as herd immunity rises. such dynamics have been more thoroughly understood for influenza a (webster et al., ; mccullers et al., ; ferguson et al., ; nelson et al., ) and hiv (mcmichael et al., ) . for prrsv, recent research demonstrates that antibodies can exert a strong selective pressure to viral pathogens by targeting specific viral sub-populations, while allowing for the establishment of other sub-populations (wang, ) . when comparing prrsv genetic diversity before and after vaccine adoption in south korea, prrsv vaccination was suggested to increase viral genetic heterogeneity and the emergence of new glycosylation sites in viral populations (kwon et al., ) . however, the extent in which prrsv immunity, whether from natural infection or vaccination, can potentially drive the evolution of the virus in the field remains largely unanswered. our data does show a dominance of non-vaccine related lineages over time, which leads to speculation that these lineages have partially escaped the immunity induced by commercial vaccines or natural infection by variants in other lineages. prrsv vaccines do not protect against infection (scortti et al., ) , but diminish clinical signs and improve animal performance (cano et al., ) . since our project did not evaluate clinical signs of animals, it is difficult to assess the effects of vaccination in that regard. however, despite high region-wide vaccine usage from onward (figure ) , lineage a spread widely in the studied region, suggesting that vaccination and other biosecurity measures were insufficient to limit the transmission of lineage a. lineages shown (figure ) and discussed here and elsewhere are based on phylogenetic relationships in the orf region, and might not be predictive of cross-protection and immunological responses developed by hosts when faced with viruses belonging to different lineages. despite that, the lineage classification protocol used in this study did reveal temporal patterns consistent with what is expected based on epidemiological theory related to the spread of disease in immunologically naive populations. for example, epidemic-shaped curves of occurrence of different prrsv populations were seen, a pattern consistent with the spread of new pathogens (or subtypes) within a naive population. new (sub-) lineages may potentially be able to become the dominant prrsv in the population if they are sufficiently immunologically distinct to overcome herd immunity, and herds with different levels of immunity induced by pre-exposure protocols or natural infections might create selective pressure that changes how fast a new viral variant is selected in that population. for prrsv, it is apparent that protection against homologous prrsv is more robust than against heterologous variants, though the definition of what constitutes a heterologous virus is highly variable (cano et al., ; díaz et al., ; geldhof et al., ) . at the same time, genetic distance has not been shown to correlate with cross-protection, perhaps because pairwise nucleotide identity fails to capture key mutations that impact cross-protection. studies that further explore the immunological cross-reactivity among prrsv lineages are needed. with the data available in this study, it was not possible to investigate the occurrence of specific lineages with vaccination use and more precisely to which vaccine each farm/system used or to which virus was circulating previously on a specific farm. mshmp data of farms from systems that contributed sequences to this paper ( figure ) show two periods in which vaccine usage increased. the first increase in mid- , and a second in approximately mid- . the second spike in vaccine usage coincided with when lineage a began spreading in the study area. it is possible that this second spike in vaccine usage was a reaction to the shift in circulating lineages (more specifically, to the emergence of lineage a prrsv). it is also possible that the increased use of vaccines onward (shown on figure ) and the occurrence of lineages b and c (shown on figure ) immunologically selected sequences in a manner that allowed for the emergence of lineage a in . by mid- , a proportion of farms began using live virus inoculation (lvi). this strategy refers to the use of controlled exposure in gilts through inoculation with live virus isolated from recent clinical outbreak(s) at the farm (desrosiers and boutin, ) . the rationale is that by exposing gilts to virus found in a farm, gilts will mount "homologous" immunity to that specific wildtype virus and contribute to herd immunity and thus stability. according to veterinarians in the area, the increased use of lvi was due to the circulating virus being "different enough" from the viruses used in commercial vaccines. the practice of lvi in the systems here reported began primarily in (figure ). it is difficult to assess the impact that lvi might have on immunologically selecting for specific viral populations within specific lineages, especially with the aggregated data used in this analysis. while the inability of vaccination to control the spread of prrsv lends credence to immunological selection as a driver of prrsv diversification (murtaugh et al., ) , the impacts that immune-driven selection could have on long term prrsv evolution remain unknown. recording exposure procedures (lvi or vaccine use) within farms is crucial when trying to interpret longitudinal patterns of occurrence of prrsv. in future research aimed at more robustly testing hypotheses about immunity as a driver of evolutionary change, this crucial information would allow for investigation of frequencies in which specific lineages occur in farms pre-and post-vaccine/lvi adoption. within orf , we found sites under positive selective pressure within or near two hypervariable regions (figure ; hanada et al., ; delisle et al., ) located near the principal neutralizing epitope (pne). the pne is located between amino acids - and forms an ectodomain which triggers antibodies development during prrsv infection (plagemann et al., ; hanada et al., ) . the flanking hypervariable regions can be linked to the development of an immune response that block accessibility of antibodies to the pne (popescu et al., ) , including n-linked glycosylation sites such as n , n , and n (ansari et al., ) . in general terms, glycosylation may modulate protein-protein interactions, whether these proteins involve the humoral or cellular immune response of the host (lisowska, ) . in prrsv, there is evidence that these glycosylation sites play a role in glycan shielding, which is an important mechanism by which the virus evades neutralizing immune responses (vu et al., ) . while our findings do not explicitly explain the change in lineage, it does raise one hypothesis of the mechanism behind such change. further studies on how specific portions for the genome, both within orf and the whole genome, modulate immune recognition and possibly selective pressure are needed. we also consistently identified positive selective pressure within the pne region, specifically for amino acid . the identification of positive selective pressure in this region suggests that viral variants with different amino acid composition in that region may experience higher fitness and thus are favored. since this region seems to be the primary binding site of neutralizing antibodies developed during prrsv infection (plagemann et al., ; kim et al., ) , this suggests that the reason for such selective pressure could be immune in nature. such a scenario has been considered as a possible explanation for long-term evolution of rna viruses (domingo et al., ; pérez-sautu et al., ) . additional in vitro research is necessary to further clarify the immunological importance of sites identified in our analysis. however, our results suggest the plausibility of a scenario where prrsv variants with mutations in key immunological regions are able to evade immune responses and thus persist and spread within host populations with partial immunity (figure ) . further studies to investigate the role of an incomplete immunity on the evolution of prrsv are required. other mechanisms that might change the ability of the virus to infect hosts have also been proposed. non-muscle myosin heavy chain (myh ) is a molecule that has been shown to be an essential host factor for prrsv infection (gao et al., ) . myh interacts with prrsv glycoprotein (coded for by orf ), changing cell susceptibility to infection. further studies that investigate the contribution that molecules such as myh have on the infection of different orf prrsv variants are needed. additionally, non-neutralizing antibodies can delay the induction of neutralizing antibodies (ostrowski et al., ) in prrsv infection. indeed, the mean level and duration of viremia in pigs was greater among animal injected with sub-neutralizing prrsv-specific igg antibodies (yoon et al., ) , suggesting the existence of an antibody-dependent enhancement (ade) effect in prrsv. the extent in which prior exposures to the virus can elicit such effect, and how this may relate to emergence of new viral variants, also remains uncertain. as an epidemiologic study relying on secondary data generated at the population level, this study has several limitations. our sequence data were generated by different production systems that differ in number of farms, number of samples submitted, management practices, and health monitoring protocols. because of that, information may be incomplete and interpretation of data might not always be straightforward. for example, the reason for sample collection (clinical outbreak or routine monitoring), sample composition (single versus pool of animals) and type of sample (serum or tissues) is not always clear. the lack of a denominator (total amount of animals sampled in a farm, total number of farms tested) does not allow for the calculation of risk indicators for disease occurrence. data contribution by each system also varies with time. however, restricting the data to only the periods in which all systems contributed to the dataset would limit our ability to visualize long-term trends. additionally, the production system that was responsible for % of all sequences was present in the study for the entire study period. therefore, we believe that biases introduced by this issue were likely small and would not have changed the conclusions of our work. in this united states region, systems that participate in the mshmp represent approximately % of the swine farms. the remaining % of farms belong to smaller systems in the area or independent farmers. by having data from systems that represent the vast majority of farms in this region, we expect our data to be reasonably representative of prrsv occurrence in the region as a whole. additionally, despite the shortcomings mentioned above, the usage of mshmp data allows us to work with data directly from the systems, which might suffer less bias toward diseased animals than usual veterinary diagnostics laboratories data do. another limitation of this analysis involves the data generation process for the sequences analyzed here. production systems usually collect samples and send them to different diagnostic laboratories. laboratory details on quality of sequence reads were not available. these sequences most likely represent a consensus of viral sub-populations present within the host (goldberg et al., ; lauring and andino, ) , but further information that could help in assessing the quality of the read and the variability of sub-populations is not available. the sequences used here are from the orf gene alone and may not fully represent evolutionary dynamics elsewhere in the genome, since the orf gene represents approximately % of the whole genome of prrsv. studies that further explore whole genome sequencing as a tool to understand prrsv epidemiological and evolutionary patterns are required. factors affecting prrsv dynamics in specific farms are not clearly understood. we show overall temporal dynamics of prrsv in a swine-producing region of the united states, however, we have limited farm-level information. thus, we have limited ability to track turnover of viral variants within farms, though we expect this to be influenced by management practices, such as the vaccination protocol adopted by farms, the movement figure | we hypothesize that prrsv evolution is partially driven by immune-mediated selective pressure. immune-mediated pressure (either within an animal or during transmission between animals/farms) selects for escapee viral variants (inset). over time, the selection of escapees may allow for emergence of a heterologous viral populations (i.e., strains, genetic groups, or lineages) which are able to spread within the host population. in scenarios in which some method of pre-exposure is adopted, prevalence of immunity against specific types of prrsv is high (often artificially through vaccination or live virus inoculation) despite high population turnover, possibly favoring the occurrence of immune-mediated selection. of animals and personnel to and between farms, the proximity to other swine producing farms, how neighboring farms manage their animals, etc. pig production in the u.s. swine industry is characterized by multi-site pig production, which refers to segregating the breeding herd from the growing herd such that animals in each stage of production are housed at separate locations. multi-site production results in the movement of animals between different production sites, which can be located in different states within the united states (valdes-donoso et al., ; kinsley et al., ) . the role of animal movement in shaping the temporal dynamics of prrsv lineages is outside the scope of this study, but is an area of active research. in addition, the commingling of animals from different sources, which might have been previously exposed to different viral populations, may allow for the introduction of viral types prevalent in other parts of the country and also exacerbate the potential for recombination of viral populations. still, in our dataset we found evidence for recombination in only two mshmp sequences. immune interaction between infections of differing prrsv isolates remains poorly understood in swine. the vast adoption of control protocols that rely on imperfect immune response aimed mostly at reducing severity of upcoming infections (such as pre-exposure protocols with commercial vaccines or with lvi) suggests that a better understanding of the cross-immunity generated by infection with different isolates of the virus would be valuable to the industry as a whole. prospective studies that obtain sera from sow farms under different pre-exposure regimens and follow the farms through time recording prrsv occurrence would provide valuable information of potential cross-immunity in field conditions. of interest also is the better understanding of how the spread different lineages/sublineages are related to epidemiological data, for example, animal movement data and farm proximity. this might allow for a better comprehension of drivers for prrsv transmission while allowing for the evaluation of the effectiveness of practices aimed at reducing prrsv risk (dead animals disposal, manure composting, filtering the air of farms, to name a few). this study reflects data from a single united states region, which possibly does not reflect prrsv diversity and temporal dynamics of the whole swine industry in the country (shi et al., b) . that being said, the data presented here reflects a substantial portion of the u.s. swine industry in a region that is relatively spatially discontinuous from other swine producing regions in the united states. in addition, the general pattern of emergence and turnover of different lineages over time observed here describe an evolutionary phenomenon that is expected to also occur in other united states regions. a better understanding of the natural history of prrsv can provide insights that can potentially aid in mitigating the impact of the emergence of new viral variants as well as serving as a basis for further work exploring the evolution of prrsv and the effect this has on disease control, management and impact on the industry. here, we describe the occurrence of prrsv over years in a single united states region. we identified the emergence and turnover of different lineages and sub-lineages in the commercial pig population. such rapid turnover in the dominant lineage through time suggests that temporal patterns of prrsv occurrence are characterized by multi-strain dynamics, where different prrsv variants potentially interact through immune-mediated competition or selection. however, cross-immunity between different prrsv lineages elicited by natural or intentional infection is not fully understood, which hinders the effectiveness of disease control. more research is needed on drivers of evolution and emergence of new sub-lineages in order for the industry to be able to predict, prevent, and mitigate the impacts of prrsv. ongoing surveillance for prrsv using molecular epidemiological methods is invaluable to characterize the evolution of the virus but also to identify recent and historical trends that help understanding the natural history of prrsv in the united states. the sequence dataset used here is 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the united states temporal and spatial dynamics of porcine reproductive and respiratory syndrome virus infection in the united states using machine learning to predict swine movements within a regional program to improve control of infectious diseases in the us global trends in infectious diseases of swine role of animal movement and indirect contact among farms in transmission of porcine epidemic diarrhea virus immune evasion of porcine reproductive and respiratory syndrome virus through glycan shielding involves both glycoprotein as well as glycoprotein immunological selection as a driver of porcine reproductive and respiratory syndrome virus evolution and ecology of influenza a viruses mystery swine disease in the netherlands: the isolation of lelystad virus differentiation of a porcine reproductive and respiratory syndrome virus vaccine strain from north american field strains by restriction fragment length polymorphism analysis of orf secondary infection with streptococcus suis serotype increases the virulence of highly pathogenic porcine reproductive and respiratory syndrome virus in pigs antibody-dependent enhancement (ade) of porcine reproductive and respiratory syndrome virus (prrsv) infection in pigs high-throughput whole genome sequencing of porcine reproductive and respiratory syndrome virus from cell culture materials and clinical specimens using next-generation sequencing technology we gratefully thank the contributions that emily smith and andres perez made on early stages of the project. we would like to acknowledge the industry partners who contributed to data for this analysis and to shic and mshmp in general, especially to emily geary, involved in the mshmp data curation. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmicb. . /full#supplementary-material key: cord- -f uvohf authors: malmlov, ashley; bantle, collin; aboellail, tawfik; wagner, kaitlyn; campbell, corey l.; eckley, miles; chotiwan, nunya; gullberg, rebekah c.; perera, rushika; tjalkens, ronald; schountz, tony title: experimental zika virus infection of jamaican fruit bats (artibeus jamaicensis) and possible entry of virus into brain via activated microglial cells date: - - journal: plos negl trop dis doi: . /journal.pntd. sha: doc_id: cord_uid: f uvohf the emergence of zika virus (zikv) in the new world has led to more than , human infections. perinatal infection can cause severe neurological complications, including fetal and neonatal microcephaly, and in adults there is an association with guillain-barré syndrome (gbs). zikv is transmitted to humans by aedes sp. mosquitoes, yet little is known about its enzootic cycle in which transmission is thought to occur between arboreal aedes sp. mosquitos and non-human primates. in the s and ‘ s, several bat species were shown to be naturally and experimentally susceptible to zikv with acute viremia and seroconversion, and some developed neurological disease with viral antigen detected in the brain. because of zikv emergence in the americas, we sought to determine susceptibility of jamaican fruit bats (artibeus jamaicensis), one of the most common bats in the new world. bats were inoculated with zikv prvabc but did not show signs of disease. bats held to days post-inoculation (pi) had detectable antibody by elisa and viral rna was detected by qrt-pcr in the brain, saliva and urine in some of the bats. immunoreactivity using polyclonal anti-zikv antibody was detected in testes, brain, lung and salivary glands plus scrotal skin. tropism for mononuclear cells, including macrophages/microglia and fibroblasts, was seen in the aforementioned organs in addition to testicular leydig cells. the virus likely localized to the brain via infection of iba (+) macrophage/microglial cells. jamaican fruit bats, therefore, may be a useful animal model for the study of zikv infection. this work also raises the possibility that bats may have a role in zika virus ecology in endemic regions, and that zikv may pose a wildlife disease threat to bat populations. introduction zika virus (zikv) was first isolated from a sentinel rhesus macaque in uganda in and subsequently from aedes africanus mosquitoes in the same location [ ] . the first human cases were identified in in nigeria and serosurveys found evidence of a broad geographic distribution for zikv throughout africa and asia with sporadic cases in humans [ , ] . the first recognized zikv epidemic occurred in yap state, federated state of micronesia in . an estimated % of residents were infected, and of those % presented with clinical disease [ ] . in , a second epidemic occurred in french polynesia with , cases reported. during the latter outbreak, the incidence rate of guillain-barré syndrome (gbs) increased -fold and first indication of a connection between zikv infection and gbs was established [ ] . the virus spread to brazil in [ , ] and has since disseminated throughout much of tropical south america, central america, the caribbean, and the southern united states, with more than , confirmed cases [ ] . zikv can also cause congenital zika syndrome (czs) in naïve populations and is therefore a virus of high concern [ ] . zika virus is maintained in an urban cycle, transmitted between an aedes mosquito vector and humans thereby maintaining endemicity [ ] . it is generally accepted that the virus transmits between non-human primates and vectors in a sylvatic cycle; however, the sylvatic cycle has not been well characterized in the old world and little is known about a new world sylvatic cycle [ , ] . molecular analysis of zikv to better understand viral phylogenetics suggests that animal hosts affected viral evolution and therefore may play an important role in viral ecology [ ] . in the s and ' s, the susceptibility of bats to zikv was investigated. shepherd and williams [ ] screened wild bats from different species in uganda for antibodies against zikv and found / little free-tail bats (tadarida pumila) and / angolan freetail bats (t. condylura) were seropositive by hemagglutination inhibition assay. additionally, two angolan free-tail bats were experimentally inoculated with zikv and serially bled to test for viremia. both animals were viremic on days , and as determined by paralysis in mice inoculated with the sera from those two bats [ ] . simpson and o'sullivan [ ] experimentally inoculated three straw-colored fruit bats (eidolon helvum), three egyptian fruit bats (rousettus aegyptiacusi), and five angolan free-tail bats. two of the straw-colored fruit bats were viremic and had seroconverted. one of the egyptian fruit bats was viremic and two had seroconverted. the angolan free-tail bats were euthanized on days , , , and days post inoculation and screened for viral tropism. at one day post infection, a kidney was trace positive [ ] . finally, reagan et al. [ ] inoculated new world little brown bats (myotis lucifigus) by different routes: intracranial, intraperitoneal, intradermal, intrarectal and intranasal. bats in all groups, with the exception of the intranasal group, developed fatal neurological disease - days post inoculation. brain tissue was virus-positive in all animals with clinical disease, determined by inoculation of mice with brain homogenate suspension [ ] . considering the evidence that african bats are naturally susceptible to zikv and that little brown bats develop disease, the question emerged: could bats serve as a natural reservoir host for zikv in the new world? to test this hypothesis, we inoculated jamaican fruit bats (artibeus jamaicensis), among the most abundant bats in the caribbean, central america and mexico, with zikv to examine virology, immunology and pathology of the infection. although virus was detected in several organs, including the testes and brains, no overt clinical signs were detected, and substantial viremia or viruria was not evident. these results suggest that jamaican fruit bats are unlikely to serve as amplification hosts but that zikv infection may constitute a wildlife disease threat to bats. bats for this project were obtained from the colorado state university breeding colony approved by the institutional animal care and use committee (protocol - a). two experimental infections were conducted; a pilot study and a time course study. in the pilotstudy, three male bats (aj-z , aj-z , aj-z ) were intradermally inoculated with . x plaque forming units (pfu) zikv, strain prvabc ; a high dose to assess susceptibility. no signs of disease were apparent during this day experiment; however, all three bats had antibody titers of on day (table ) . after demonstration of susceptibility in the pilot study, a time course study was conducted. six male bats (aj-z through aj-z ) were identically inoculated and two were euthanized at , and days post inoculation (dpi). no conspicuous signs of disease were observed in any of the inoculated bats. necropsies immediately followed euthanasia and no significant gross pathology was evident. quantitative probe-based reverse transcription pcr (qrt-pcr) was performed on seruminoculated vero cell supernatants, serum, brain, lung, liver, spleen, kidney, urinary bladder, prostate and testes from bats from both studies. in addition, urine collected during the time course study was similarly assayed. urine from bats aj-z at dpi and aj-z at dpi had low levels of vrna whereas bat aj-z , euthanized at dpi, had low levels of vrna in its brain ( fig ) . all other samples were negative. sera from aj-z at dpi, and aj-z and aj-z at dpi were negative by elisa. sera were blind passaged on vero e cells in an attempt to isolate zikv and all were negative for cpe and pcr. hematoxylin and eosin stain (h&e). heart, lung, liver, kidney, testes, prostate, urinary bladder, and brain were collected from all animals as well as salivary glands from / bats. all samples were blindly read by one pathologist. a summary of the consistent histopathology findings is listed in table . for the time course study, aj-z at dpi showed mild pulmonary congestion with multifocal areas of interstitial pneumonia, mild intra-alveolar hemorrhage and mild atelectasis. terminal airways had slightly increased amounts of mucus. kidneys had multifocal interstitial infiltrates of small numbers of lymphocytes. all other tissues were within normal limits. in aj- z at dpi, lungs showed milder pathology than aj-z with minimal interstitial to perivascular infiltrates predominately lymphocytes and macrophages with a band of collapsed air spaces subjacent to the pleural surface. there were focal lesions in the left ventricle of the heart where there was individual cell loss or else fragmentation of the sarcoplasm of scattered cardiomyocytes. degenerate/necrotic cardiomyocytes were accompanied by infiltrations of small numbers of macrophages, lymphocytes and satellite cells. all other tissues were within normal limits. lungs from aj-z at dpi had minimal focal interstitial histiocytic pneumonia with atelectasis. kidneys showed multifocal chronic lymphohistiocytic pyelitis with a few degenerate and detached epithelial cells accumulating in the renal pelvis and infiltration of pelvic stroma by small numbers of mixed inflammatory cells. mandibular salivary gland showed focal moderate cellular infiltrates of periductular lymphocytes and macrophages. affected salivary ducts contained detached and degenerate epithelial cells and leukocytes. occasional ducts were encircled by granulation tissue and a few heterophils. rare apoptosis was evident in the lining epithelium of such ducts. all other tissues were within normal limits. aj-z at dpi had lungs with minimal alveolar septal infiltrates scattered within collapsed lung parenchyma along with multifocal microscopic hemorrhages. kidneys had multifocal areas of mineralization. in the outer medulla and at the cortico-medullary junction were rare perivascular infiltrates of lymphoplasmacytes. esophagus and lymphoid tissue associated with palatine salivary gland showed focal mild lymphoplasmacytic inflammation. moderate numbers of lymphocytes and plasma cells were arranged in columns parallel to the respiratory mucosal epithelium of the nasophayrnx. the lumen contained increased amounts of mucus and a few inflammatory cells, mainly heterophils and lymphocytes. in the testicles, there was focal testicular degeneration manifested by presence of giant spermatids in the lumina of affected seminiferous tubules and accumulation of a small numbers of interstitial lymphocytes and macrophages. all other tissues were within normal limits. lungs from aj-z at dpi had minimal interstitial to perivascular infiltrates with multifocal atelectasis and microscopic hemorrhages. the left papillary muscle of the heart showed rare multifocal cardiomyocyte necrosis characterized by rounding up of individual cardiomyocytes. necrotic cardiomyocytes appeared with hypereosinophilic cytoplasm, devoid of cross striations or fragmented and rarely vacuolated. minimal interstitial hypercellularity due to increased activity of satellite cells and infiltration of small numbers of lymphocytes was observed in the vicinity of degenerate/necrotic cardiac muscle fibers. kidneys had an area of focal lymphoplasmactyic pyelitis. additionally, there was a focal area of mineralization and inflammation in the inner medulla. all other tissues were within normal limits. aj-z at dpi had occasional focal inflammation and cardiomyocyte degeneration in the left ventricle and interventricular septum. area ca of the hippocampus in the brain showed focal pyrimidal neuronal necrosis with a focal area of mineralization around a vessel in the cerebral cortex along with focal gliosis and individual neuronal necrosis (fig ) . all other tissues were within normal limits. testicular, neural and salivary glands' lesions are believed to be associated with zikv infection as they were not seen with other viral infections. in the pilot study bats, aj-z at dpi had more prominent interstitial pneumonia with congestion of the lungs compared to earlier time points. the heart had minimal cardiomyocyte degeneration and necrosis with hypercellular interstitium and increased amounts of mature fibrous connective tissue. the kidney had focal interstitial infiltrates of the cortical and outer medullary interstitium. the brain showed degenerate neurons in area a of the hippocampus. all other tissues were within normal limits. aj-z had minimal focal testicular degeneration (fig ) . all other tissues were normal. aj-z had perivascular lymphocyte pulmonary infiltrates and atelectasis. heart demonstrated locally extensive lymphocytic and histiocytic pericarditis. kidneys showed multifocal interstitial lymphocytic infiltrates. brain had focal, perivascular infiltrates of small numbers of lymphocytes at the subfornical commissure. the reticular formation showed multifocal neuronal degeneration/necrosis. immunohistochemistry and immunofluorescence. tissues were stained with a polyclonal antibody for zikv (cdc, fort collins). ajz- at dpi with inflammation of the mandibular salivary gland had moderate immunoreactivity in the lumen of affected ducts (fig ) . aj-z at dpi had immunoreactive cells in the brain and mononuclear cell immunoreactivity in the testes ( fig ) . additionally, aj-z demonstrated immunoreactivity in purkinje cells of the cerebellum (fig a) . aj-z at dpi had immunoreactive cells around the pulmonary arteries in the lungs ( fig a) . aj-z also had immunoreactivity perivascullarly in the tunica albuginea of the testes (fig a) . scrotal skin had focal lymphocytic dermatitis with immunoreactive mononuclear cells ( fig d) . cell morphology consistently identified mononuclear cells compatible with macrophages and fibroblasts as the primary cell types showing immunoreactivity against zikv antigen. brain and testicular tissues stained with both goat polyclonal goat anti-iba (green) and monoclonal g- flavivirus e specific antibodies (red) showed co-localization (yellow) of zikv antigen in cytoplasm of activated microglial cells with their characteristic morphology in the cerebral cortex of infected bats dpi in the time course study and day dpi in the pilot study (fig ) . increased microgliosis was noted in the vicinity of co-localization sites. the gliosis was also prominent in the cerebellum and hippocampus especially around dead neurons. in the testicles, occasional macrophages showed similar co-localization similar to that noted in the brain in the testicular interstitium, inner layer of tunica albuginea and scrotum. cells consistent in morphology with leydig cells were similarly highlighted by zika viral antigen only showing strong immunoreactivity using polyclonal anti-zikv antibody. two bat infection experiments were conducted in this investigation; ) a pilot study to determine susceptibility of jamaican fruit bats to zikv infection, and ) a time course study to better understand pathophysiology and chronology of events pertaining to the dynamics of viremia, viral tropism, replication and shedding of the virus in a new world bat species. the goal was to determine whether bats can be used as an animal model for zikv pathogenesis and to assess the possible role of bats in zikv ecology in the new world. in the pilot experiment, no signs of disease were apparent during the -day study. sera collected at euthanasia indicated modest antibody titers of for each bat by elisa (table ) , whereas the human -convalescent control serum titer was � , . bats typically have low to modest antibody titers, perhaps due to limited somatic hypermutation and affinity maturation [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . concerning viremia, cell-serum supernatants, blind passage supernatants, and neat serum results were all negative. although serum is routinely used for zikv diagnostics in humans, it may not be the most suitable sample [ ] [ ] [ ] [ ] [ ] . in one investigation zikv patient had negative serum sample for the duration of the study, whereas whole blood yielded positive qrt-pcr results from days to [ ] . one possible explanation for the phenomenon of negative serum in human patients is that the virus during acute infection disseminates via a cell-associated viremia or as novel findings suggest that the virus gets phagocytized in neutrophils and therefore whole blood is a more sensitive diagnostic sample than serum. viruria is commonly detected in zikv-infected humans [ ] ; therefore, urine may be an equally important diagnostic sample with higher viral load in early infection when compared to blood in humans and other primates [ ] [ ] [ ] [ ] . although urine collection from bats was challenging, we collected urine from some of the inoculated bats in the time course study. aj-z exhibited viruria only at dpi, and aj-z was equivocal only at dpi, corroborating the findings in other mammals that urine may be a route of viral shedding early in infection. urine from one human patient was positive from the first time point ( dpi) through dpi and again on day . similarly, saliva from that same patient was positive from day nine through day and again on day [ ] . another investigation compared diagnostic samples of infected patients and showed that urine was positive in of them, whereas serum was only positive in patients by qrt-pcr. the study concluded that viral loads in urine were tenfold higher compared to serum and that uremia lasted longer [ ] . these data corroborated the first study that identified zikv shed in urine in which there was a higher viral load in urine for longer duration compared to serum [ ] . zikv rna in plasma was detected in the bats by qrt-pcr between and dpi, but between to dpi in urine [ ] . the lack of detectable viremia in the serum of bats is congruent with some of the human and nhp investigations in that viremia is low and short-lived. detached renal pelvic urothelial cells and degenerate salivary gland ductular epithelium as seen in the current study will make urine and saliva equally important fluids to collect in order to maximize detection of zikv in the acute and established stages of infections. for this experiment, all male bats were used because female bats are prioritized for colony expansion. zikv exhibited tropism for the testes with strong immunoreactivity in reproductive organs (figs & ) . histologically, minimal focal testicular degeneration in two bats ( fig ) suggests viral related pathology may be minimal. in humans it has yet to be completely elucidated what reproductive organs harbor zikv, it has been determined that semen contains zikv both in both vasectomized and unvasectomized men [ , ] . this suggests that zikv is sequestered in the testes and/or accessory sex glands. mouse models have demonstrated zikv infection and associated pathology in the testes [ ] [ ] [ ] of humanized blt mouse model with infection primarily targeting macrophages and leydig cells [ ] . limited investigation has been done relating to infection of accessory sex glands in mouse models, but one study that assessed the prostate found no virus, possibly due to differential expression of the receptor candidate in the testes but not in the prostate [ ] . for this experiment the finding of viral antigen and viral rna in the testes but not in the prostate is consistent with published animal models and may suggest the potential for bats to serve as another animal model. three bats had histopathological alterations in the hippocampus at later time points and one bat had viral nucleic acid present in the brain as determined by qrt-pcr demonstrating tropism for the cns, a tissue predilection also documented in humans and animal models. zikv has a predilection for nervous tissue in animal studies and disease manifestation in humans. as a neurological teratogen, zikv has been detected in the brain mononuclear cells in human newborns with fatal microcephaly and fetal miscarriages. histological lesions are varied but may include parenchymal calcification, microglial nodules, gliosis, cell degeneration, mononuclear infiltration and necrosis [ ] [ ] [ ] . in non-human animal models, evidence for viral tropism has been found in brain and/or peripheral nervous tissue [ ] [ ] [ ] [ ] . in immunocompromised mouse models, the virus has a predilection for the brain but with the mice engineered for specific immune traits it is difficult to know to what extent this recapitulates natural zikv pathophysiology [ ] . in the bats used in this experiment, evidence of zikvinduced pathology in the brain is consistent with what has been seen in human newborns and fetuses. the novel finding of co-localizing zikv antigen in bat iba + microglial/macrophage cells lends support to the earlier evidence of microglial cell infection via axl ligand bridging zikv particles to glial cells [ ] . iba (aka, allograft inflammatory factor , aif ) is a microglia/macrophage-specific calcium-binding protein, which has actin-bundling activity that participates in membrane ruffling and phagocytic activity of activated microglia. activated microglial cells appeared with increased ability of cell migration and phagocytosis, which is controlled by remodeling of membrane cytoskeleton [ ] . the morphology of cells with co-localization in the brain of infected bats is consistent with activated microglia depicting prominent branched processes. recent primate models in rhesus and cynomolgus macaques demonstrated similar viral distribution of zikv antigen to that in bats, described herein. high-level of zikv was evident in cerebellar neurons and the same studies documented involvement of iba positive microglial cells in cns infections. in primate models there is increasing evidence that zikv antigen was detected in individuals with the highest peak plasma viremia, which in part implies that zikv may initially seed the cns by a passive spillover from circulating monocytes to resident microglial cells. this is further substantiated in all of human and animal studies, which did not show any evidence of disruption to bbb or viral distribution reminiscent of circumventricular distribution seen in alphavirus animal models [ ] . in addition to brain and testes immunoreactivity, scrotal skin and mandibular salivary gland also harbored viral antigen. distribution of viral antigen in bat tissues suggests that infection in this species recapitulates human infection, which is thought to start with infection of epidermal and dermal cells with subsequent dissemination to multiple organs including salivary glands as viral rna can be detected in human saliva [ , ] . the histopathology for aj- zika virus infects new world bats z , dpi showed sialoadenitis and the presence zikv antigen by ihc (fig ) . this suggests zikv may be shed in the saliva, although additional animal experiments need to be performed to confirm such a route of shedding. the results presented here suggest that jamaican fruit bats may be a suitable animal model for examining zikv infection to elucidate its pathogenesis. jamaican fruit bats may also serve as a model to ascertain sexual transmission, in utero transmission, teratogenesis and neurological pathophysiology. it may be that zikv is a wildlife disease threat for bats that could lead to infertility in some males, which could impact bat populations. zikv is thought to be maintained in two different distinct cycles: sylvatic-cycling between non-human primates (nhp) and mosquito species, and urban-cycling between humans and mosquito species [ ] . while there are limited data on what mosquito species feed on jamaican fruit bats, evidence for natural flavivirus infection has been identified in wild new world bats. dengue virus (denv) rna and antibodies to denv were detected in multiple species of bats, including jamaican fruit bats, in mexico [ ] . additionally, antibodies to denv were detected in multiple bat species including those of the artibeus genus in costa rica and ecuador [ , ] . these data indirectly provide evidence for mosquito-bat interactions in the wild; either through consumption of bat-blood meals taken by mosquitoes or bat consumption of infected mosquitoes. as it pertains to a wildlife reservoir, wild nhps have antibody to zikv including several monkey species trapped near ziika forest [ ] , and wild and semi-captive orangutans in borneo [ ] . not only have nhp been found to be seropositive, but also many other mammals, including rodents, horses, cows, and goats [ , ] . furthermore, experimental inoculation of various north american species resulted in seroconversion (cottontail rabbits, boar goats, pigs, and leopard frogs) and demonstrated viremia (nine-banded armadillo and leopard frogs) [ ] . molecular epidemiology suggests animals play an important role in an enzootic cycle [ ] . much about the enzootic cycle of zikv has yet to be understood but it stands to reason that bats may be capable of maintaining the virus in nature. jamaican fruit bats are found in northern south america, central america, and the caribbean-areas that now have zikv potentially exposing bat populations to the virus [ , ] . however, the data presented here suggest it is unlikely that jamaican fruit bats can serve as amplification hosts of zikv, unless virus sequesters in some as-yet unidentified way that could lead to periodic shedding of virus. it may also be that some bats become persistently infected and can transmit sexually to maintain virus within populations of bats. further experimental and field studies will be necessary to fully understand the ecological role of bats in zikv maintenance. all animal procedures were approved by the colorado state university (csu) institutional animal care and use committee (protocol - a) and were in compliance with u.s. animal welfare act. bats csu has a captive colony of jamaican fruit bats (artibeus jamaicensis), a neotropical fruit bat indigenous to much of south america, central america and the caribbean [ ] . colony bats are kept in a free flight room measuring 'w x 'l x 'h. roosting baskets are hung from the ceiling throughout the room and drapes of different cloth material are positioned for hanging and roosting. ambient temperature is maintained between ˚c and ˚c, with humidity between % and %, and a hour light/ hour dark light cycle via a computer-controlled system. diets consist of a combination of fruits (shamrock foods, fort collins, co), tekald primate diet (envigo, huntington, uk), molasses, nonfat dry milk and cherry gelatin that are placed in multiple feeding trays around the room once a day. fresh water is provided. in addition, fruit is hung around the room to stimulate foraging behavior and serve as enrichment. for infection experiments, bats were trapped using a butterfly net and placed in an "d x "w x "h cage for hours prior to inoculations to allow for acclimation. hanging clothes were provided for roosting and coverage. food and water are placed in open trays in the bottom of the cage and changed daily. tray liners were changed every two days, and cages and hanging clothes are changed every two weeks. due to the social nature of these bats, minimums of two bats were kept in cages at all times to mitigate potential stress. two sets of experiments were performed; a pilot study and a time course study. zika virus strain prvabc . prvabc was isolated in by centers for disease control and prevention (fort collins, co) from an infected individual who traveled to puerto rico (genbank accession no. hq ). the virus stock titer is x plaque forming units (pfu) per ml of media, and the fourth passage was used for both studies. for the pilot study, three male bats were anesthetized with % to % isoflurane to effect with an oxygen flow rate of . l/min, administered with a gas mask. animals were placed on a heating pad to maintain body temperature and respirations continuously monitored. the dorsum of each animal was disinfected with % ethanol and ul containing . x p.f.u of virus was administered subcutaneously (sc) at the level of the scapula with a sterile hypodermic gauge needle in a biosafety cabinet. when procedures were finished, bats were removed from isoflurane and placed back in the cage in ventral recumbency. respirations were monitored until animal was fully awake and ambulated normally. bats were identified as aj-z , aj-z and aj-z . animals were euthanized at days post-inoculation (dpi). for the time course study, six male bats were anesthetized under the same protocol as the pilot study. animals were placed in ventral recumbency. after disinfecting the dorsum of each animal with % ethanol, . mls of % lidocaine was administered sc at the level of the last rib with a gauge sterile hypodermic needle as a local anesthetic. iptt transponders (biomedic data systems, inc., seaford, de) were inserted sc at the level of the caudal edge of the scapula. twenty-five microliters containing . x p.f.u of virus was administered sc at the level of the cranial edge of the scapula. recovery followed the same protocol as for the pilot study bats. animals were identified as aj-z through aj-z . aj-z and aj-z were euthanized at two dpi. aj-z and aj-z were euthanized at dpi. aj-z and aj-z were euthanized at dpi. female bats were excluded from the study because they are prioritized for breeding to sustain and expand upon the colony. for the pilot study, bats were visually monitored twice daily for fourteen days, and then monitored once a day for an additional fourteen days. for the time course study, bats were monitored twice a day throughout the experiment. for both studies, energy levels, behavior, ability to ambulate, respirations, presence of oral or nasal discharge, and fecal consistency were all assessed. during the time course study urine was collected at , , and dpi from as many bats as possible. urine was collected by allowing bats to grasp screen cloth with their feet and then the bat was placed in a clear solo cup (dart container, lake forest, il) with the screen covering the top of the cup as a lid, and kept in place with a rubber band. this allowed the bats to hang in a clear container. bats were monitored for minutes. if they urinated, bats were removed from the collection contraption and placed back in the cage without disrupting the urine. urine collection was attempted on all remaining bats at each time point, but not all bats would urinate at each collection attempt. urine was successfully collected as follows: two dpi from aj-z and aj-z ; three dpi from aj-z , aj-z and aj-z ; five dpi from aj-z , aj-z , aj-z and aj-z ; and ten dpi from aj-z and aj-z . urine was pipetted off the surface of the cup with a sterile pipette tip and put in a . ml microcentrifuge tube and stored at - ˚c for future use. urine volume ranged between ul and ul. bats were deeply anesthetized and maintained with % isoflurane and an oxygen flow rate of . l/min. deep pain was assessed by firmly pinching skin and toes with forceps and assessed for any response. a thoracotomy was then performed with sterile standard scissors to puncture through the skin, muscle and diaphragm just caudal to the sternum and cut through the wall of the chest cavity caudally to cranially-removing and preventing negative pressure from building in the thorax. cardiac blood was collected with a gauge sterile needle inserted into the apex of the heart. a maximum blood volume of between and . mls is collected in a syringe and transferred to a red top tube (rtt). rtts sat at room temperature for one hour to allow a clot to form and then centrifuged at x g for min at room temperature. serum was removed from the clot, placed in a new microcentrifuge tube and stored at - ˚c. serum from bats at and dpi were used to assess for viremia. serum from dpi and the dpi pilot study bats were used to determine antibody titers. because blood draws yield a small volume of blood ( μl whole blood for a non-terminal blood draw, μl whole blood for terminal blood draw) it was necessary to prioritize samples to optimize data retrieved. in order to assay the serum for viral rna and perform serology, earlier time points were used to assess for viremia and later time points for seroconversion. along with sample partitioning for data maximization, the small blood volume led to concerns that there would be an undetectably small viral load. to circumvent this issue, neat serum and : diluted serum were inoculated onto vero cells to amplify any virus that may have been present at low levels. one blind passage on vero cells was done and cell supernatants assayed by qrt-pcr. the remaining serum from three of the four bats was assayed directly for zikv rna. necropsies were performed immediately after euthanasia. bats were assessed for gross pathology. the following tissues were collected for both experiments: heart, lung, liver, spleen, kidney, urinary bladder, prostate, testes, and brain. a portion of tissues were collected and kept at - ˚c for rna extraction, and a portion placed in % buffered formalin for histology at a : weight to volume ratio for histology. for a negative control animal a male bat was trapped from the colony and euthanized under the same protocol as the experimental infection bats. vero e cells (atcc) were propagated to % confluency in a -well tissue culture plate and infected with zikv strain prvabc at an m.o.i. of . . after a one hour incubation period, unbound virus was removed and replaced with % fbs-dmem and incubated for a maximum of three days. media was then replaced with % acetone for minutes at - ˚c to fix virusinfected cells to plate and serve as an antigen for enzyme-linked-immunosorbent assay (elisa). plates were stored at ˚c until use and used within two weeks. plates were washed x with . % tween -pbs and blocked with superblock t (tbs) blocking buffer (thermo fisher scientific, waltham, ma) for one hour at room temperature. serum from an uninfected bat was used for a negative control. a convalescent human serum sample (kindly provided by b. foy, csu) was used as a positive control. a two-fold serial dilution was used starting at : to : . diluted serum was placed in wells and incubated for two hours at room temperature. serum was removed and plates washed. hrp-conjugated protein a/g (thermo fisher scientific, waltham, ma) was added at a concentration of μg/ml to each well, and incubated for minutes at room temperature. hrp-conjugated protein a/g was used in place of a secondary antibody as it targets the fc portion of an antibody, which is highly conserved and therefore can be used for multiple animal species [ ] . plates were washed and μl of abts peroxidase substrate ( component) (kpl, gaithersburg, md) added according to manufacturers' instructions, incubated at room temperature for minutes, and then μl of abts peroxidase stop solution (kpl, gaithersburg, md) added. plates were read on an emax plus microplate reader (cambridge scientific, watertown, ma). absorbance was measured at nm and the limit of detectable response was set at three standard deviation values above mean negative control serum. trizol reagent was used for rna extraction from serum-cell supernatants, serum, urine and tissues according to ambion, life technologies protocol. for tissues, approximately mg of tissue was homogenized with one ml of trizol reagent. a mm stainless steel bead (qiagen, valencia, ca) was used with a tissuelyser lt (qiagen, valencia, ca) at hz for minutes. one ml of trizol was added to urine to to μl of urine. one ml of trizol was added to μl of serum from aj-z , aj-z , and aj-z . two-hundred microliters of serum-cell supernatants were added to one ml of trizol. samples were then incubated at room temperature for minutes. chloroform (thermo fisher scientific, waltham, ma) was added, samples were mixed, incubated for minutes at room temperature and centrifuged at , x g for minutes at ˚c. the aqueous phase was removed, μg of glycogen (thermo fisher scientific, waltham, ma) and % molecular grade isopropanol added (thermo fisher scientific, waltham, ma). samples were incubated at room temperature for minutes and then centrifuged at , x g for minutes at ˚c. supernatant was removed and % molecular grade ethanol (thermo fisher scientific, waltham, ma) was added to rna pellet. samples were vortexed and centrifuged at x g for minutes at ˚c. wash was removed and air-dried. rna was resuspended in rnase-free water and stored at - ˚c for future use. vero cells were grown to to % confluency in a -well tissue culture plate with % fbs-dmem. media was removed and ul of bat serum from dpi bats and dpi bats was inoculated onto cells. additionally, serum from each bat was diluted -fold in % fbs (millipore sigma) pbs supplemented with % calcium and magnesium, and inoculated onto cells. samples were incubated for one hour at ˚c. inoculum was removed and cells washed twice in sterile pbs. two-percent fbs-dmem was added to wells and plates were incubated at ˚c, % co . cells were assessed daily for cytopathology (cpe) through day but none was observed. two-hundred microliters of the supernatant was removed on day and used for rna extractions. an additional μl of supernatant was blind passaged onto vero cells at to % confluency. cells were incubated for one hour at ˚c, washed twice with sterile pbs and % fbs-dmem added. on day seven, supernatant was removed and trizol extractions performed for rna recovery. serum was treated as such in an attempt to amplify viral load and increase assay sensitivity serum may not be the most sensitive diagnostic sample [ ] [ ] [ ] [ ] . if any serum was remaining it was directly used for trizol rna extractions. serum samples remained from aj-z at dpi, and aj-z and aj-z at dpi. no serum remained from aj-z . roche real time ready rna virus master kit (roche, indianapolis, in) was used on rna extracted from serum-cell supernatants, serum, urine and tissue to assay for zikv rna according to manufacturers' instructions. primers used were zikv (ccgctgcccaa cacaag) and zikv c (ccactaacgttcttttgcagacat). probe was zikv -fam (agcctaccttgacaagcagtcagacactcaa) [ ] . two-hundred nanograms of sample rna was added to each reaction. reactions were performed in duplicate. standards were a non-infectious clone of full length zikv strain prvabc by which concentration was determined through optical density. molecular weight of the genome sequence was used to calculate copy number [ ] . a log dilution series of the standard was made and linear regression used to determine copy number equivalents of positive samples. amplification was performed according to manufacturers' protocol for roche real time ready rna virus master kit (roche diagnostics corporation, indianapolis, in) with pcr conditions as follows: min at ˚c, s at ˚c, and cycles of s at ˚c, s at ˚c and s at ˚c. tissues fixed in %-buffered formalin were cut in and submitted to colorado state university veterinary diagnostic laboratory (csu vdl, fort collins, co) for paraffin embedding, sectioning and staining with hematoxylin and eosin, as well as immunohistochemistry (ihc). tissues cut in on bats to assess for histology included: heart, lung, liver, kidney, testes, prostate, urinary bladder and brain. additionally, for aj-z and aj-z mandibular salivary gland was cut in. aj-z had esophagus and lymphoid tissue that included palatine salivary gland cut in. antibody for ihc was a polyclonal rabbit antibody that targets prem and e proteins of zikv and was provided by csu vdl's pathology department. the bond-iii automated instrument (leica biosystems, wetzlar, germany) was used for ihc staining. all slides were blindly read by a diplomat of the american college of veterinary pathologists. brain tissues was prepared for immunohistochemical and immunofluorescence staining as previously reported [ ] . tissue was dehydrated by using a graded ethanol series of % ethanol for h, % overnight, % for h and % for h. brain tissues were then post-fixed in dimethylbenzene for min and embedded in dimethylbenzene-paraffin at ˚c for h, after which samples were embedded in a metal frame. sagittal sections were collected at um thick. all dewaxing, antigen retrieval and immunofluorescence staining was automated using a leica bond rxm. in short, sections were dewaxed using ethanol and then boiled in antigen retrieval solution for minutes. the cooled sections were incubated in % h o for min at room temperature and then blocked with % donkey and goat serum (millipore sigma) for hour. rabbit anti-iba (wako chemicals usa, irvine, ca) and g- flavivirus e specific monoclonal antibodies (cdc, fort collins) were diluted in tbs to final concentrations of : and : , respectively. sections were incubated in primary antibodies concurrently at room temperature for one hour. following removal of unbound primary antibodies by washing, goat anti-rabbit secondary (alexafluor- ) and donkey anti-mouse secondary (alexafluor- ) was added and incubated for hour at room temperature. finally, dapi counterstain (vector laboratories, burlingame, ca) was applied and sections were washed with tbs prior to cover slipping for imaging. stained sections were imaged on a ziess lsm with airyscan laser-scanning confocal microscope (ziess, oberkochen, germany) using a × oil immersion objective. each field of view was imaged as a z-stack ( - planes, . -μm step size) transformed into a single maximum projection image using the ziess zen (blue) imaging software. zika virus. i. isolations and serological specificity zika virus: a report on three cases of human infection during an epidemic of jaundice in nigeria zika virus: history, emergence, biology, and prospects for control zika virus outbreak on yap island, federated states of micronesia rapid spread 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overview of the current status of zika virus pathogenesis and animal related research axl mediates zika virus entry in human glial cells and modulates innate immune responses microglia/macrophage-specific protein iba binds to fimbrin and enhances its actin-bundling activity entry sites of venezuelan and western equine encephalitis viruses in the mouse central nervous system following peripheral infection detection of zika virus in saliva biology of zika virus infection in human skin cells denge virus in mexican bats neotropical bats that co-habit with humans function as dead-end hosts for dengue virus detection of dengue virus neutralizing antibodies in bats from costa rica and ecuador sylvatic transmission of arboviruses among bornean orangutans zika virus, vectors, reservoirs, amplifying hosts, and their potential to spread worldwide: what we know and what we should investigate urgently a sero-epidemiological survey for certain arboviruses (togaviridae) in pakistan investigating the potential role of north american animals as hosts for zika virus. vector borne zoonotic dis mammalian species: artibeus jamaicensis chimeric igg-binding receptors engineered from staphylococcal protein a and streptococcal protein g genetic and serologic properties of zika virus associated with an epidemic, yap state, micronesia rapid and specific detection of asian-and african-lineage zika viruses fluoxetine prevents lps-induced degeneration of nigral dopaminergic neurons by inhibiting microglia-mediated oxidative stress the authors would like to than brent davis, cdc, fort collins, co for supplying anti-zikv polyclonal and specific monoclonal antibodies. key: cord- -a acr o authors: koch, r. m.; diavatopoulos, d. a.; ferwerda, g.; pickkers, p.; de jonge, m. i.; kox, m. title: the endotoxin-induced pulmonary inflammatory response is enhanced during the acute phase of influenza infection date: - - journal: intensive care med exp doi: . /s - - - sha: doc_id: cord_uid: a acr o background: influenza infections are often complicated by secondary infections, which are associated with high morbidity and mortality, suggesting that influenza profoundly influences the immune response towards a subsequent pathogenic challenge. however, data on the immunological interplay between influenza and secondary infections are equivocal, with some studies reporting influenza-induced augmentation of the immune response, whereas others demonstrate that influenza suppresses the immune response towards a subsequent challenge. these contrasting results may be due to the use of various types of live bacteria as secondary challenges, which impedes clear interpretation of causal relations, and to differences in timing of the secondary challenge relative to influenza infection. herein, we investigated whether influenza infection results in an enhanced or suppressed innate immune response upon a secondary challenge with bacterial lipopolysaccharide (lps) in either the acute or the recovery phase of infection. methods: male c bl/ j mice were intranasally inoculated with × ( ) pfu influenza virus (ph n , strain a/netherlands/ / ) or mock treated. after (acute phase) or (recovery phase) days, mg/kg lps or saline was administered intravenously, and mice were sacrificed min later. cytokine levels in plasma and lung tissue, and lung myeloperoxidase (mpo) content were determined. results: lps administration days after influenza infection resulted in a synergistic increase in tnf-α, il- β, and il- concentrations in lung tissue, but not in plasma. this effect was also observed days after influenza infection, albeit to a lesser extent. lps-induced plasma levels of the anti-inflammatory cytokine il- were enhanced days after influenza infection, whereas a trend towards increased pulmonary il- concentrations was found. lps-induced increases in pulmonary mpo content tended to be enhanced as well, but only at days post-infection. conclusions: an lps challenge in the acute phase of influenza infection results in an enhanced pulmonary pro-inflammatory innate immune response. these data increase our insight on influenza-bacterial interplay. combing data of the present study with previous findings, it appears that this enhanced response is not beneficial in terms of protection against secondary infections, but rather damaging by increasing immunopathology. patients with influenza infection often suffer from severe secondary bacterial infections, which are associated with high morbidity and mortality rates [ , ] . a striking example of this relationship was provided by bacteriological and histopathological analysis of infected lung tissue obtained from people who died of influenza during the - "spanish flu" pandemic, in whom bacterial pneumonia was found to be the predominant cause of death [ ] . these data suggest that an influenza infection profoundly influences the immune response upon a secondary bacterial infection. several studies have evaluated immunological interactions between influenza and bacterial infections, including infections with gram-negative bacteria [ ] . in vitro studies in which influenza-infected alveolar macrophages were subsequently stimulated with bacterial lipopolysaccharide (lps), a bacterial compound that induces a profound innate immune response, revealed increased levels of pro-inflammatory cytokines tumor necrosis factor (tnf) α, interleukin (il)- β, and il- [ ] [ ] [ ] [ ] [ ] , indicative of a priming effect on these cells by influenza. data from in vivo animal studies are ambiguous. similar to the in vitro data, some report enhanced responses. for instance, influenza infection in mice was shown to enhance the inflammatory response and neuropathogenicity resulting from lps administration on days and after influenza inoculation [ ] . likewise, murine influenza infection resulted in increased levels of pro-inflammatory cytokines in both plasma and lungs, and enhanced pulmonary neutrophil influx upon pneumococcal infection days later [ ] . similar results were observed in mice days after influenza infection [ ] . however, two otherwise largely comparable studies demonstrated reduced pulmonary pro-inflammatory cytokine concentrations upon streptococcus pneumoniae and staphylococcus aureus infections in mice infected with influenza days before, indicative of influenza-induced immunosuppression [ , ] . these equivocal results may be due to differences in the severity or kinetics of the influenza infection or the use of different bacteria as secondary challenges, thereby targeting various complex multi-receptor signaling pathways. also, the use of live bacteria could have contributed to these ambiguous results. for instance, if influenza would induce immunosuppression and thereby facilitate outgrowth of bacteria upon a secondary live infectious challenge, the increased bacterial burden can eventually result in fulminant inflammation, which would wrongfully suggest influenza-induced augmentation of the immune response. in the present study, we investigated whether influenza infection results in an enhanced or suppressed innate immune response upon a secondary challenge with lps. furthermore, we assessed the kinetics of these influenza-induced effects by performing the lps challenges in either the acute or the recovery phase of influenza infection. all procedures described were in accordance with the requirements of the dutch experiments on animals act, the ec directive / , and approved by the animal ethics committee of the radboud university nijmegen medical center (ru-dec - ). forty-eight male c bl/ j mice (charles river, sutzfield, germany) aged - weeks and weighing - g were used. mice were housed in individually ventilated cages, with five mice per cage at the central animal facility of the radboud university. at day , six groups of eight mice (total n = ) were anesthetized by isoflurane and intranasally inoculated with a sublethal dose of influenza virus (ph n , strain a/ netherlands/ / , × pfu) or mock treated (nacl . %) in a volume of μl. following infection, all mice were monitored and weighed daily. the temperature was recorded with an infrared thermometer on the skin, and physical condition was scored using a scoring and weight sheet (weight, body temperature, ruffled coat, hunched back, reduced mobility, and moribund). at either day (acute phase) or day (recovery phase), mice were placed in a temperature-controlled chamber to receive lps (e coli, serotype :b , mg/kg) or nacl . % by intravenous injection in the tail vein. ninety minutes after lps or nacl administration, mice were deeply anesthetized with isoflurane and exsanguinated through orbital extraction, followed by cervical dislocation after which organs were collected. ethylenediaminetetraacetic acid (edta)-anticoagulated blood was centrifuged at ×g for min at room temperature after which plasma was stored at − °c until analysis. subsequently, perfusion of the lungs was performed by intracardiac injection with phosphate-buffered saline (pbs), after which lung lobes were harvested and snap frozen in liquid nitrogen and stored at − °c until homogenization. lung tissue was placed in ml lysis buffer containing pbs, . % triton x- , and a protease inhibitor cocktail (complete edta-free tablets, roche, woerden, the netherlands, tablet per ml lysis buffer). subsequently, lung lobes were homogenized at hz, using a polytron homogenizer, and subjected to two rapid freeze-thaw cycles using liquid nitrogen. finally, homogenates were centrifuged ( min, , ×g, °c), and the supernatant was stored at − °c until cytokine analysis. concentrations of tnf-α, il- β, il- , and il- in plasma and lung homogenates were measured using a luminex assay (milliplex, millipore, billerica, ma) according to the manufacturer's instructions. the lower detection limit of the assay was pg/ml for all cytokines. plasma il- β levels were below the detection limit in the majority of animals. lung homogenate cytokine concentrations were normalized to total protein content determined by bicinchoninic acid assay (bca protein assay; thermo fisher scientific). myeloperoxidase (mpo) content was measured in lung homogenates using an enzyme-linked immunosorbent assay (hycult biotech, uden, the netherlands) according to the manufacturer's instructions. concentrations were normalized to total protein content as described above. all data were normally distributed according to the shapiro-wilk test. the grubbs test (extreme studentized deviate method) was used to exclude significant outliers from analysis (maximum of one exclusion per dataset). to determine the number of animals required per group, we performed a power calculation based on a minimal detectable difference of % in lps-induced plasma tnf-α levels between influenza-infected and non-influenza-infected mice. mean ± sd ( ± pg/ml) tnf-α plasma levels were obtained from previous work from our group, in which male c bl/ j mice were also injected intravenously with mg/kg lps and sacrificed min later [ ] . using a two-sided α of . and a power of % (β of . ) in an unpaired t test design, six animals per group were required. to account for potential loss of animals due to influenza infection, eight animals per group were used. the effect size was based on previous work [ ] , in which influenza infection modulated the plasma cytokine response to lps administration by at least %. comparisons were analyzed using unpaired student's t tests and repeated measures one-way analysis of variance (anova) as indicated in the figure legends. statistical analyses were performed in graphpad prism . for windows (graphpad software, san diego, ca). two-tailed p values < . were considered statistically significant. all influenza-inoculated mice showed clinical signs of infection, including weight loss, lethargy, and pyrexia. four influenza-infected mice were prematurely taken out of the experiment because of signs of severe infection. body weight decreased in all influenza-infected mice in the acute phase of infection, whereas it remained stable in mock-inoculated mice (fig. ) . from day onwards, body weight started to increase, marking the recovery phase of influenza infection (fig. ) . influenza infection by itself did not result in increased plasma levels of any of the cytokines measured at both and days post-infection (fig. ) . expectedly, lps fig. body weight of influenza-or mock-inoculated mice. data are presented as mean with sem. dagger indicates the two time points at which mice in the respective groups were sacrificed administration led to profoundly increased plasma concentrations of tnf-α, il- , and il- . although tnf-α and il- plasma levels appeared to be somewhat higher in mice challenged with lps days after influenza infection compared with mock-inoculated mice, this did not reach statistical significance (p = . and p = . , respectively). plasma concentrations of the anti-inflammatory cytokine il- were however significantly enhanced in mice challenged with lps days after influenza infection. no differences in any of the plasma cytokine levels were measured between influenza-infected and mock-inoculated mice at days. in lung homogenates, influenza by itself caused mildly elevated levels of tnf-α, il- β, il- , and il- at days post-infection and to a lesser extent at days after infection (fig. ) . similar to what was found in plasma, lps challenge also led to increased concentrations of all measured cytokines in lung tissue. a synergistic increase of all pro-inflammatory cytokines in the lungs was found in influenza-infected mice challenged with lps days later and, to a lesser extent, in mice challenged with lps days post-influenza infection. for il- , the potentiating effect was additive rather fig. plasma levels of tnf-α, il- , and il- in mice that received influenza/mock followed by lps/nacl or days later. data are presented as scatter-dot plots with horizontal lines indicating the mean value. *p < . , **p < . , ***p < . (calculated by unpaired student's t tests) fig. levels of tnf-α, il- β, il- , and il- in lung homogenates of mice that received influenza/mock followed by lps/nacl or days later. data are presented as scatter-dot plots with horizontal lines indicating the mean value. *p < . , **p < . , ***p < . , # p = . - . (calculated by unpaired student's t tests) than synergistic, only observed at days post-influenza infection, and reached a trend towards statistical significance. in accordance with pulmonary cytokine levels, influenza infection by itself led to increased mpo content in the lungs days after infection and tended to result in increased mpo content days post-infection (fig. ) . again, lps administration also resulted in increased mpo levels in lung tissue, and there was a trend towards enhanced mpo content in influenza-infected mice challenged with lps days after infection. in the present study, we demonstrate that a systemic lps challenge in the acute phase of influenza infection ( days post-infection) results in an enhanced pulmonary, but not systemic pro-inflammatory cytokine response. this effect was synergistic rather than additive, indicating that influenza infection actually modulates the immune response to a subsequent challenge with lps. furthermore, this effect remained present, although less pronounced, in the recovery phase of influenza infection ( days post-infection). the lps-induced increase in mpo content in lung homogenates, reflecting pulmonary neutrophil influx or sequestration, tended to be enhanced in the acute phase of influenza infection as well. these results suggest that influenza infection, especially in the acute phase, may cause a more pronounced pulmonary pro-inflammatory immune response upon a secondary bacterial infection. our results are in accordance with in vitro data reporting a cellular priming effect of influenza observed upon secondary stimulation with lps [ ] [ ] [ ] [ ] [ ] , as well as with other murine in vivo studies that report increased inflammation and pulmonary neutrophil influx or sequestration upon a secondary bacterial infection or lps challenge in the acute phase of influenza infection [ , ] . for example, a preceding influenza infection in mice gravely enhanced lung injury induced by a secondary infection with streptococcus pneumoniae days later, resulting in a severe necrotic pneumonia accompanied by increased mortality [ ] . also, fig. mpo content in lung homogenates of mice that received mock/influenza followed by nacl/lps or days later. data are presented as scatter-dot plots with horizontal lines indicating the mean value. *p < . , **p < . , ***p < . , # p = . - . (calculated by unpaired student's t tests) the increased mpo content observed in our study is an important hallmark of acute respiratory distress syndrome (ards) [ , ] , a severe complication of influenza infection caused by excessive pulmonary inflammation. these and our study reveal that the enhancing effect on the pro-inflammatory innate immune response is most evident in the lungs, probably because the influenza-induced damage and consequent inflammatory effects are most pronounced at this site. in this context, our data are in line with the recommendation to use corticosteroids in patients with severe influenza infections in the intensive care unit to counteract the pulmonary hyperinflammatory response causing ards. several underlying mechanisms may contribute to the observed effects. at the cellular level, studies have shown that influenza and certain bacterial pathogens, such as haemophilus influenzae and streptococcus pneumoniae, utilize similar immunological pathways and that the overlap in the inflammatory mediators produced thereby creates augmentation of the immune response during sequential infection, in turn causing immunopathology [ , ] . furthermore, it has been hypothesized that influenza stimulates tnf-α gene transcription activators or may interfere with labile transcription repressor proteins and stabilizes tnf-α mrna by delaying its degradation [ ] . alternatively, the increased lung mpo levels observed do not necessarily reflect pmn infiltration into the lungs, but may (also) result from pmns trapped in the vasculature, as circulating activated neutrophils become rigid and can be trapped within the small capillaries of the lung [ ] . as such, increased entrapment of leukocytes in the pulmonary vasculature during influenza infection could also contribute to the enhanced inflammatory cytokine levels upon lps challenge. we can only speculate on this, because no histological data are available, which represents a limitation of this work. it may be argued that the enhanced pro-inflammatory immune response induced by influenza serves as a means to efficiently eliminate the primary pathogen and to enhance host defense towards a secondary infection. for instance, pro-inflammatory cytokines are induced in influenza-infected cells to limit viral replication and to initiate downstream immune responses [ ] . however, this is not supported by previous work, where an increased bacterial burden was observed irrespective of an enhanced or suppressed response [ ] [ ] [ ] . several explanations for this observation may be put forward. first, next to potentiating pro-inflammatory cytokine responses, the present study and work by others [ ] have shown that influenza infection also potentiates production of the key anti-inflammatory cytokine il- , which was demonstrated to be crucial in facilitating bacterial outgrowth upon secondary challenge with streptococcus pneumoniae [ ] . second, influenza may on the one hand prime for production of innate cytokines produced by myeloid cells, but impair t cell-derived cytokines that are instrumental for the adaptive immune response. this was elegantly demonstrated by kudva et al., who showed that, in line with our results, infection with staphylococcus aureus days after influenza resulted in increased pulmonary levels of innate cytokines such as il- and mcp- , and increased neutrophil influx to the lungs, but decreased concentrations of t-cell-derived il- and il- , which were demonstrated to play a pivotal role in fending off the staphylococcal infection [ ] . whereas the enhancing effects of influenza on pro-inflammatory innate immune parameters were less pronounced at days post-infection, a suppressed response was neither evident. this could be partly biased by the exclusion of two mice in both recovery groups due to severe influenza infection. however, it might also be argued that days post-infection is too soon for these effects to manifest. for example, profound desensitization towards lps and flagellin, another toll-like receptor (tlr) ligand, was observed in alveolar macrophages obtained from mice up to weeks after influenza infection [ ] . furthermore, the direction of the response upon a secondary challenge is probably highly dependent on the pathogen or stimulus used, each using distinct intracellular signaling pathways. with regard to this, it is well-known that influenza virus particularly predisposes to aspergillus fumigatus, which is present in % of all influenza patients [ , ] , causing infections such as invasive pulmonary aspergillosis that are associated with very high mortality rates. as different mechanisms may be important in host defense towards various pathogens, the specific response towards aspergillus fumigatus could be suppressed by a preceding influenza infection. the use of corticosteroids may be another important factor in the observed vulnerability towards particular secondary infections, as steroid use was shown to be independently associated with the presence of aspergillus fumigatus in sputum of cystic fibrosis patients [ ] and with a substantially increased risk of community-acquired staphylococcus aureus bacteremia [ ] . furthermore, a meta-analysis revealed that the use of corticosteroids was significantly associated with nosocomial infections [ ] . to the best of our knowledge, these putative detrimental effects of corticosteroid treatment during influenza on secondary infections have yet to be studied systematically in animal models. in any case, it remains to be determined whether the overall effects of corticosteroid treatment are beneficial or not, as they may lead to increased susceptibility in a subset of influenza virus-infected patients but may also provide health benefits in another subset of influenza virus-infected patients. an lps challenge in the acute phase of influenza infection results in an enhanced pulmonary pro-inflammatory innate immune response. these data increases our insight concerning viral-bacterial interplay. combined with previous findings, it appears that this enhanced pro-inflammatory response does not lead to protection against secondary infections but rather causes immunopathology leading to damage, and thereby to organ failure. predominant role of bacterial pneumonia as a cause of death in pandemic influenza: implications for pandemic influenza preparedness secondary bacterial infections associated with influenza pandemics influenza-induced type i interferon enhances susceptibility to gram-negative and gram-positive bacterial pneumonia in mice infection of macrophages by influenza a virus: characteristics of tumour necrosis factor-alpha (tnf alpha) gene expression the potentiating effect of lps on tumor necrosis factor-alpha production by influenza a virus-infected macrophages cytokine release from human peripheral blood leucocytes incubated with endotoxin with and without prior infection with influenza virus: relevance to the sudden infant death syndrome tumor necrosis factor-alpha production of influenza a virus-infected macrophages and potentiating effect of lipopolysaccharides influenza a virus infection of macrophages. enhanced tumor necrosis factor-alpha (tnf-alpha) gene expression and lipopolysaccharidetriggered tnf-alpha release lipopolysaccharide treatment and inoculation of influenza a virus results in influenza virus-associated encephalopathy-like changes in neonatal mice induction of pro-and anti-inflammatory molecules in a mouse model of pneumococcal pneumonia after influenza il- is an important mediator of the enhanced susceptibility to pneumococcal pneumonia after influenza infection inhibition of pulmonary antibacterial defense by interferon-gamma during recovery from influenza infection influenza infection leads to increased susceptibility to subsequent bacterial superinfection by impairing nk cell responses in the lung spleen-derived ifn-gamma induces generation of pd-l (+)-suppressive neutrophils during endotoxemia cxcl -cxcr enhances the development of neutrophil-mediated fulminant lung injury of viral and nonviral origin contribution of neutrophil-derived myeloperoxidase in the early phase of fulminant acute respiratory distress syndrome induced by influenza virus infection insights into the interaction between influenza virus and pneumococcus patterns in bacterial-and viralinduced immunosuppression and secondary infections in the icu the neutrophil in vascular inflammation new fronts emerge in the influenza cytokine storm influenza a inhibits th -mediated host defense against bacterial pneumonia in mice sustained desensitization to bacterial toll-like receptor ligands after resolution of respiratory influenza infection invasive pulmonary aspergillosis is a frequent complication of critically ill h n patients: a retrospective study influenzaassociated aspergillosis in critically ill patients inhaled corticosteroids and aspergillus fumigatus isolation in cystic fibrosis use of glucocorticoids and risk of community-acquired staphylococcus aureus bacteremia. a population-based case-control study mayo corticosteroids for the treatment of human infection with influenza virus: a systematic review and meta-analysis. clinical microbiology and infection: the official publication of the european society of clinical microbiology and infectious diseases the authors thank fred van opzeeland, elles simonetti, francine van der poll, ilona van de brink, and jelle gerretsen for their help with the mouse experiments and laboratory analyses. this work was supported by an efro (dutch: "europees fonds voor regionale ontwikkeling," english: "european regional development fund") grant ( - ). efro had no role in the design of the study; in the collection, analysis, and interpretation of data; and in writing the manuscript. data sharing is not applicable to this article as no reusable datasets were generated or analyzed during the current study.authors' contributions rk designed and conducted the study, analyzed and interpreted the data, and drafted the manuscript. dd and gf aided in the study design and conduct, interpreted the data, and critically revised the manuscript. pp and mdj supervised the study, interpreted the data, and critically revised the manuscript. mk designed and supervised the study, interpreted the data, and critically revised the manuscript. all authors read and approved the final manuscript. all procedures described were in accordance with the requirements of the dutch experiments on animals act, the ec directive / , and approved by the animal ethics committee of the radboud university nijmegen medical center (ru-dec - ). not applicable. the authors declare that they have no competing interests. key: cord- - eeykk authors: mao, changyi; yu, zhihui; li, chengliang; jin, yongguo; ma, meihu title: the functional properties of preserved eggs: from anti-cancer and anti-inflammatory aspects date: - - journal: korean j food sci anim resour doi: . /kosfa. . . . sha: doc_id: cord_uid: eeykk preserved egg, a kind of alkaline-fermented food, is a traditional egg product in china. here, we investigated the nutritional functions of preserved eggs by in vivo and in vitro experiments. the results of in vivo studies showed that the levels of triglycerides (tg), total cholesterol (tcho) and low-density lipoprotein cholesterol/high density lipoprotein cholesterol (ldl-c/hdl-c) were significantly decreased (p< . ) in the liver of rats treated with preserved eggs. meanwhile, the levels of two important cancer markers, interleukin- (il- ) and tumor necrosis factor-α (tnf-α), were also significantly decreased (p< . ) in treated rats. in vitro studies were performed on caco- cells, a human epithelial colorectal adenocarcinoma cell line. it demonstrated that the gastrointestinal (gi) digests of preserved eggs significantly accelerated (p< . ) the apoptosis by upregulating caspase- in the caco- cells. besides, after treated with preserved eggs, the half maximal inhibitory concentration (ic ) of preserved eggs digests to caco- cells was . mg/ml, indicating the significant inhibition of cell proliferation provided by preserved eggs (p< . ). the results shown in this study demonstrated that preserved eggs may be a novel functional food involved with antilipemic, anti-inflammatory activity as well as the effect on accelarating the apoptosis of caco- cells. inhibit the growth and invasion of tumor cell in vitro (corder et al., ) . studies have shown that preserved egg white hydrolysates show anti-inflammatory effects in vitro and in vivo experiments and can significantly decrease the expression levels of il- and tnf-α (zhao et al., ) . in addition, ace-inhibitory peptides isolated from preserved eggs proved their antioxidant activity in vitro (yang, ) . numerous studies have indicated that alkaline foods, such as kelp (yuan and walsh, ) , grape (renaud and de, ) , and bitter gourd (rajasekhar et al., ) , can inhibit the growth of various human cancer cell lines. the results showed that the algae polyphenols extracted from kelp can inhibit the rancidity of fish oil and the antioxidant efficiency was . -fold higher than that of butylated hydroxytoluene (bht) (yan et al., ) . researchers found that seaweed polyphenols can significantly inhibit the proliferation of tumor cells, and the inhibition effect was linearly correlated to the total level of phenol in seaweed (yuan and walsh, ) . resveratrol in grapes was shown to have inhibitory effects on cancer cell initiation, proliferation and development (corder et al., ) . at present, the research on the preserved eggs is limited on the optimization of processing technology. in this paper, the functional characteristics of preserved eggs were monitored using complementary in vitro and in vivo models. the levels of liver lipid and inflammatory factors in rats before and after treatment, as well as the ability of preserved eggs to inhibit tumor cells in vitro were investigated. the objective is to explore the nutritional and anticancer values of preserved eggs. our findings will contribute to provide the theoretical basis for understanding the relationship between preserved eggs and human health. all chemicals, unless otherwise stated, were purchased from sinopharm chemical reagent co., ltd (china). the tg assay kit, tcho assay kit, hdl cholesterol assay kit and ldl cholesterol assay kit were provided by nanjing jiancheng biology engineering institute (china). the rat il- elisa kit and rat tnf-α elisa kit were provided by neobioscience biology engineering institute (china). the caspase- activity assay kit was purchased from beyotime institute of biotechnology (china). annexin v-fitc/pi kit was supplied by zoman biotechnology co., ltd (china). caco- cells were obtained from the cell bank of chinese academy of sciences (china). fresh duck eggs ( - g) were obtained from hubei shendan healthy food co., ltd (china). preserved eggs were obtained using clear material soaking method (zhao et al., b) with some modifications. duck eggs soaked with a kind of special solution containing . % of naoh, % of nacl, . % of cuso , and . % of chinese black tea at ℃. after d, preserved eggs were obtained. after peeling, the preserved eggs were stirred in a homogenizer and lyophilized to obtain preserved egg powders. twenty-five female sprague dawley (sd) rats aged - wk were obtained from tongji medical college, huazhong science and technology university. all rats were kept and treated according to the requirements of the laboratory animal ethics review committee (animal welfare no. ). sufficient food and water were provided and the room temperature and relative humidity were controlled in the range of ± ℃ and ± %, respectively. after one week of adaptive feeding, five rats were sacrificed as -day control group. the rest were labeled with % picric acid solution (yellow) and . % neutral magenta solution (red). after labeling, the rats were randomly divided into experiment groups and rats contained in each group. the rats in control group were intragastric administrated with distilled water while the other groups were administrated with the preserved egg powder at concentrations of . mg/ml (low-dose group), . mg/ml (middle-dose group) and . mg/ml (high-dose group) in distilled water of ml respectively. the specific measurement was based on human and rat dose conversion formula, low dose, medium dose and high dose equivalent to every adult eats , and preserved eggs a day. the experiment period was d. the behavior of the rats was observed every day, including their body weight, food & water intake, mood and stool status. the weight of each rat was measured every five days and the food intake was determined every three days. the rats were sacrificed by cervical dislocation and the liver was extracted with tweezers after fasted overnight at the end of the experiment ( d after treatment). we collected rats blood of the retro-orbital plexus and put the livers in a centrifuge tube followed by homogenization with ml of methanol and acetone mixture ( : ). then the mixture was centrifuged at , ×g for min, and the supernatant was collected and stored at - ℃ for analysis of lipids, il- and tnf-α. tg, tcho, ldl cholesterol and hdl cholesterol were mesured by the tg assay kit, tcho assay kit, hdl cholesterol assay kit and ldl cholesterol assay kit following the manufacture's instructions. il- and tnf-α were mesured by the rat il- elisa kit and rat tnf-α elisa kit following the manufacture's instructions. simulated gi digestion using in vitro pepsin-pancreatin hydrolysis was conducted as described by you et al ( ) with some modifications. the powder was dissolved ( % w/v, in distilled water) and the ph was adjusted to . with m hcl. then pepsin ( . % of the powder, w/w) was added. the mixture was incubated at ℃ for h. the ph was then adjusted to . with the m nahco solution. pancreatin was added ( % of the powder, w/w). and the mixture was further incubated at ℃ for h. to terminate the digestion, the samples were kept in boiling water for min. then the gi digests were cooled to room temperature and centrifuged at , ×g for min. the supernatant was lyophilized, sealed in plastic bags and stored at - ℃ before use. to investigated the changes in cell proliferation inhibition rate of preserved eggs during simulated gi digestion, aliquots of gi digests were removed at (pepsin digestion after h), , , , h (switch from h to h during pancreatin digestion) during digestion procession. the human colon cell line caco- was purchased from the type culture collection of the chinese academy of sciences, shanghai, china. cells were placed into cm tissue culture flasks and grown at ℃ under a humidified % co atmosphere in dmem medium with high glucose, % fbs, units/ml penicillin and units/ml streptomycin. the cells were seeded on the six-well plate and adhered for h, followed by treatment with gi digests for further h. the morphology, size and rupture of the cell were observed by fluorescence uptake microscope (ix , olympus co., ltd, japan). approximately × cells/well of caco- cells were placed in -well plates and incubated for h. then the cells were exposed to . , . , . , , mg/ml of the gi digests for h. the medium was removed at the end of incubation and each well was incubated with μl of cck- solution (no bubbles) for h, the absorbance at nm was measured using a microplate reader (imark, lenovo biological technology co., ltd, china). a: absorbance of sample group a : absorbance of control group approximately . × cells/well of caco- cells were placed in -well plates and incubated for h. the cells were exposed to . , , mg/ml of the gi digests for h after carefully washed with pbs, set the cell without the digests as a blank control. after incubation, the cells were immediately collected and washed times by pbs, then centrifuged at a speed of , ×g for min to remove the supernatant, μl binding buffer, μl annexin v-fitc and pi were added to suspend the cells, respectively. mixtures were finally incubated in dark at room temperature for min prior to investigation by flow cytometry (facscalibur, bd co., ltd, usa) for h. approximately × cells/well of caco- cells were placed in -well plates and exposed to . , , mg/ml of the gi digests for h. we set the cell without the digests as a blank control. after incubation, the cells were immediately collected and washed times by pbs, then centrifuged at a speed of , ×g for min to remove the supernatant. the cells were collected after the lysate was added to the cells at a rate of μl/ × cells, and the remaining steps were performed based on the instructions from caspase- activity assay kit. the results were analyzed using software of ibm spss statistics . all data were expressed as the mean±standard error. statistical significance was assessed using the duncan's multiple range test, where p< . was considered significant, and the as shown in fig. , the body weight of rats treated with preserved eggs for days was not significantly different at the end of treatment from that of control rats (p> . ). the gain of body weight was normal in preserved egg-treated rats and there was no significant difference between control and preserved egg-treated rats. as shown in fig. , the tg and tcho contents of rats treated with the preserved eggs were significantly decreased (p< . ) compared with the control group after days (fig. ) . in the high-dose group ( . mg/ml), the tg and tcho contents in the liver were found to decrease by . % ( fig. a ) and . % (fig. b ) respectively compared with the control group. however, as the dose raised up to high level ( . mg/ml), the tg levels in rats increased compared to treatment with low doses of preserved eggs. on the one hand, the increase of tg levels may be due to the upregulation of the expression of fatty acid synthase (fas) in livers after treated with high doses of preserved egg (kakuma et al., ) . fas is the key enzyme in liver fatty acid synthesis, tg increases with the increasement of the fas (postic and girard, ) . on the other hand, the level of cholesterol was mg per g preserved egg (ge et al., ) . as egg yolk is rich in tg, the intake of high dose of preserved eggs elevated tg levels in rats. so, the tg levels were observed to be elevated by high dose preserved eggs ( fig. a) . actually, our body health has been proved to be favored by an appropriate of eggs intake daily because it exerts protective effect on our heart. the ingredients in the eggs can also regulate the absorption and metabolism of cholesterol and counteract the damage caused by excessive intake on the body (yang, ) . this could partly explain the fact that preserved egg could reduce the content of tg in a proper range ( fig. a) . as shown in fig. , the preserved eggs significantly decreased the level of ldl-c and ldl-c/hdl-c in rat's liver after -days treatment (p< . ), by . % (fig. a ) and . % (fig. b) in the low-dose group, . % (fig. a ) and . % (fig. b ) in high-dose group compared with control group. after treatment with preserved egg, the level of ldl was significantly inhibited compared with the control, and an increase of ldl level was observed as the concentration of preserved egg increased. it may be due to tg invoved in preserved eggs upregulating ldl-c levels (cullen, ; ji et al., ) , as the dosage of preserved eggs increased, the level of ldl-c increased. this result corresponds to the previous explanation for tg. these results showed that long-term intake of preserved eggs may help to reduce the risk of atherosclerosis (as), cerebrovascular disease (cvd) and heart disease. as shown in the fig. , the level of il- and tnf-α in rat's liver were significantly decreased compared to control group (p< . ) after treaments. in low-dose group ( . mg/ml), the il- and tnf-α values in liver were decreased . % (fig. a) and . % (fig. b ) respectively, indicating that preserved eggs contribute to reduce the risk of inflammation in rats. however, at . mg/ml of preserved eggs, the production of il- was rather increased than control. this may be limited by the amout of inflammatory cytokine receptor. due to the inflammatory factor -receptor reaction, the most appropriate amount of preserved eggs may be around . mg/ml. therefore, the optimal value is shown at . mg/ml. when it exceeds . mg/ml, the reaction rate decreases. the in vitro anticancer efficiency study as shown in intestinal digestion. consequently, we chose the sample that digested h by pepsin and h by trypsin to advance further experiments with the concentration of . mg/ml, mg/ml, and mg/ml. as shown in fig. , the cells in the control group ( mg/ml of gi digests) were closely arranged, complement in morphology, and good in growth status. in the pictures, it was observed that the cell morphology was destroyed under the treatment with the gi digests. notably, when the gi digests concentration increased up to mg/ml, cells were cleaved into fragments, and the cell morphology was collapsed. as shown in table , the apoptosis behavior of cells greatly dependended on the dose of gi digests. the significantly enhanced cell death rate was observed (fig. ) , accompanied by the delay in most of the apoptotic cells in high dose treatment. this result suggested that the gi digests could cause apoptosis in caco- cells. as shown in fig. , the activity of caspase- in caco- cells was significantly increased with a dose dependent. in addition, the activity of caspase- in the high concentration group ( mg/ml) was . times of the control group (p< . ). caspase is an important mediator of cell apoptosis. they are induced by various stimuli and contribute to cell apoptosis by cutting various cell substrates (fan et al., ; nuñez et al., ; riedl and shi, ) . the results showed that preserved egg digests can significantly induced the cell apoptosis by up-regulating caspase- levels in caco- cells. in present study, we found that preserved eggs decreased the levels of tg, tcho, and ldl/hdl in rat's liver, which are related to lipid metabolism in rats (van der made et al., ). we found that preserved eggs could significantly reduce the tg mg/ml . mg/ml mg/ml mg/ml fig. . cell morphology of caco- exposed to gastrointestinal digests in different concentration. concentration (mg/ml) . apoptosis rate (%) d . c . b . a a-d means (n= ) with different concentration, cell apoptosis in caco- exposed to gi digests were significantly different (p< . ). mg/ml . mg/ml mg/ml mg/ml fig. . cell apoptosis in caco- exposed to gastrointestinal digests in different concentration. korean journal for food science of animal resources vol. , no. , and tcho contents in liver. in the low-dose group, tg and tcho contents were decreased by . % and . % respectively, indicating that the preserved eggs are conductive in antilipemic capacity. this could be involved with several major influencing factors: ) some active substance such as apolipoprotein e (apoe) genotype (miettinen et al., ; kesäniemi et al., ) and angiotensin converting enzyme (ace) genotype (morise et al., ) may promote the lipoprotein metabolism and transformation in the liver; ) the substances above may inhibit the intestinal absorption of cholesterol, further reducing the synthesis of cholesterol and improving the decomposition rate of tg in the liver . furthermore, results also showed that the preserved eggs can significantly reduce the ldl-c and ldl-c/hdl-c level in rat's liver. after treatment with preserved eggs of low dose, the ldl-c levels in the liver were reduced by . % compared with the control group. in previous studies, ldl and ldl / hdl could be used to predict cvd, and the risk of cvd can be reduced lowering by % if ldl-c wad reduced by mmol/l (baigent et al., ) . based on this, we suspected that active ingredients such as lecithin in preserved eggs may have the potential to reduce the risk of cvd, as, and other diseases by inhibiting the synthesis of ldl-c and promoting the synthesis of hdl-c (poutanen et al., ) . in future, we will do further experiments to find out the exactly active ingredients. furthermore, the intake of preserved eggs depressed the production of il- and tnf-α, and these indicators are significantly related to the occurrence of inflammation and cancer in rats (kim et al., ; suganuma et al., ) . cell experiments results showed that the gi digests of the preserved eggs could inhibit the growth of tumor cells caco- and accelerated cell apoptosis after gastrointestinal digestion. in vivo animal experiments, we found that fed with low-dose of preserved eggs could reduce the contents of il- and tnfα in rat's cytokines contents effectively, which are served as the key substances to induce cell transformation and malignancy in the course of chronic inflammation (tang et al., ; zhu et al., ) . our results are consistent with the evidences from tumor cell experiments. it could be ascribed to that the active components in preserved eggs can repressed the release of il- and tnf-α. this result is consistent with the results of other studies (zhao et al., ) . combinning with the results of cell apoptosis, we suspected that this is more likely to be related to mitochondrial pathways (dassé et al., ; lanvin et al., ) . therefore, we will focus on the mechanism of mitochondrial pathways in the following studies. furthermore, the gi digests of the preserved eggs could inhibit the proliferation of caco- cells and induce cell apoptosis. apoptosis is a procedural and physiological form of death. the characteristics of apoptosis include cell morphological changes, the formation of dna fragments, etc. (arends et al., ; bortner et al., ) . tumor development is not only the result of cell proliferation out of control, but more likely to be blocked due to apoptosis, cell proliferation and apoptosis caused by imbalance (bhattacharjee et al., ; sun and liu, ) . cell accumulation and aggressive development of viable cells into tumors will occur if cell apoptosis fails (hoeppner et al., ) . caco- cells are derived from human colonic adenocarcinoma cells, which can be spontaneously epithelized and closely linked with the culture environments. morphological and functional expression of the marker enzymes are similar to those of human intestinal mucosa (bonnesen et al., ) . in present study, the triggering of caco- apoptosis by the gi digests has been successfully evaluated. to determine the dose of gi digests, we used the method of cck- to calculate the ic value ( . mg/ml) and most of the apoptosis in late apoptotic was highlighted. therefore, the digests of the preserved eggs may play an important role in the late stage of apoptosis in terms of the triggering. caspase- is an important regulatory proteases involved in multiple signaling pathways such as p and jak-stat (dassé et al., ; lanvin et al., ) . as a pro-apoptotic protein, the activity of caspase- was significantly upregulated in caco- cells after treated with preserved egg digests compared to the control group. anti-cancer drugs, hormonal preparations, radiotherapy, etc., are generally induced by the induction of apoptosis of the cells to achieve the purpose of treatment. therefore, triggering of cell apoptosis has become the main treatment for anti-tumor purpose (hong and sporn, ; kelloff et al., ) . this suggests that preserved egg digests could be used in the medical field cause it could inhibit cancer by inducing the apoptosis of caco- cells. the current research on the preserved eggs has been limited on the effect of diverse processing methods and quality modification but is not involved in the functional properties changes. the main results in present study showed that the preserved eggs could inhibit the release of inflammatory factors and induce apoptosis of tumor cells. moreover, from nutritional aspect, it was also interesting that the preserved eggs possessed effective antilipemic capacity as evidence by the lipid level in rat's liver. in conclusion, the preserved eggs may play a potential role in human health and can be developed as a kind of functional food in the future. our next investigation would be focus on the relationship between preserved eggs and antilipemic capacity and its mechanism of action. furthermore, characterization of active substances derived from the preserved eggs such as functional peptides are also deserved to be investigated to give more insights into the structureactivity relationship in a nutritional and functional level. apoptosis. the role of the endonuclease efficacy and safety of cholesterol-lowering treatment: prospective meta-analysis of data from , participants in randomised trials of statins classification of human lung carcinomas by mrna expression profiling reveals distinct adenocarcinoma subclasses dietary indoles and isothiocyanates that are generated from cruciferous vegetables can both stimulate apoptosis and confer protection against dna damage in human colon cell lines the role of dna fragmentation in apoptosis health: endothelin- synthesis reduced by red wine evidence that triglycerides are an independent coronary heart disease risk factor tissue inhibitor of metalloproteinase- promotes hematopoietic differentiation via caspase- upstream the mekk /mek /p α pathway caspase family proteases and apoptosis effects of different processing methods on cholesterol level of eggs programmed cell death: from development to disease. meeting report recent advances in chemoprevention of cancer egg preservation in china chemical and structural changes in preserved white egg during pickled by vacuum technology leptin, troglitazone, and the expression of sterol regulatory element binding proteins in liver and pancreatic islets progress in cancer chemoprevention: development of diet-derived chemopreventive agents intestinal cholesterol absorption efficiency in man is related to apoprotein e phenotype the function of preserved eggs in vivo and in vitro role of the il- -jak -stat -oct- pathway in the conversion of non-stem cancer cells into cancer stem-like cells interleukin- induces apoptosis of pre-b cells expressing dominant-negative forms of stat : evidence for caspase-dependent and -independent mechanisms serum cholesterol response to dietary cholesterol and apoprotein e phenotype angiotensin-converting enzyme polymorphism and essential hypertension caspases: the proteases of the apoptotic pathway african fermented foods: from art to science contribution of de novo fatty acid synthesis to hepatic steatosis and insulin resistance: lessons from genetically engineered mice identification of severe acute respiratory syndrome in canada corrigendum to "isolation and characterization of a novel antihyperglycemic protein from the fruits of momordica cymbalaria wine, alcohol, platelets, and the french paradox for coronary heart disease molecular mechanisms of caspase regulation during apoptosis human gastric cancer development with tnf-αinducing protein secreted from helicobacter pylori apple phytochemical extracts inhibit proliferation of estrogen-dependent and estrogen-independent human breast cancer cells through cell cycle modulation hmgb -il- -il- -il- -stat axis promotes tumor growth in murine models of melanoma consuming a buttermilk drink containing lutein-enriched egg yolk daily for year increased plasma lutein but did not affect serum lipid or lipoprotein concentrations in adults with early signs of age-related macular degeneration effects of allicin on lipid metabolism and antioxidant activity in chickens prevention of fish oil rancidity by phlorotannins from sargassum kjellmanianum study on nutritional effects of egg cholesterol and screening of lipid regulatory elements and the regulation mechanism of cholesterol separation ace inhibition peptides from preserved eggs by decompression method and study its in vivo activity changes in the antioxidant activity of loach (misgurnus anguillicaudatus) protein hydrolysates during a simulated gastrointestinal digestion antioxidant and antiproliferative activities of extracts from a variety of edible seaweeds study on protein changes during processing of preserved eggs changes of fatty acids during the processing of preserved eggs physicochemical and nutritional characteristics of preserved duck egg white simulated gastrointestinal digest from preserved egg white exerts antiinflammatory effects on caco- cells and a mouse model of dss-induced colitis tnf-α promotes gallbladder cancer cell growth and invasion through autocrine mechanisms the authors would like to thank meihu ma for his contribution towards the experiments in this study. this research was supported by national natural science foundation (project code no. ) and the special fund for modern agro-korean journal for food science of animal resources vol. , no. , industry technology research system (project code no. cars- -k ). key: cord- - ozhye q authors: yu, haijing; liu, yang; wang, hongwu; wan, xiaoyang; huang, jiaquan; yan, weiming; xi, dong; luo, xiaoping; shen, guanxin; ning, qin title: clara cell kda protein alleviates murine hepatitis virus strain -induced fulminant hepatitis by inhibiting fibrinogen-like protein expression date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: ozhye q background: fulminant hepatitis (fh) is a serious threat to human life, accompanied by massive and rapid necroinflammation. kupffer cells, the major immune cell population involved in innate immune responses, are considered to be central for fh. fibrinogen-like protein (fgl ) is a pro-coagulant protein that is substantially induced in macrophages upon viral infection, and fgl depletion represses murine hepatitis virus strain (mhv- ) infection. clara cell kda (cc ) protein is a secretory protein with anti-inflammatory properties in allergic rhinitis and asthma. however, its mechanisms of action and pathogenic roles in other disease are still unclear. in this study, we aimed to determine the role of cc in fh and the regulation of fgl by cc . methods: a mouse fh model was established by peritoneal injection of mhv- . the mice received cc protein through tail vein injection before viral infection. survival rate, liver function, liver histology, fibrin deposition, and necrosis were examined. the regulatory effect of cc on fgl expression was investigated using thp- cells and mouse peritoneal macrophages in vitro. results: in the mouse fh model induced by mhv- , the survival rate increased from to . % in the cc group compared to that in the saline-only control group. meanwhile, the levels of alt and ast in serum were significantly decreased and liver damage was reduced. furthermore, hepatic fgl , tnf-α, and il- β expression was obviously downregulated together with fibrin deposition, and hepatocyte apoptosis was reduced after administration of cc protein. in vitro, cc was found to significantly inhibit the expression of fgl in ifn-γ-treated thp- cells and mhv- -infected mouse peritoneal macrophages by western blot and real-time pcr. however, there was no direct interaction between cc and fgl as shown by co-immunoprecipitation. microarray investigations suggested that hmg-box transcription factor (hbp ) was significantly low in cc -treated and ifn-γ-primed thp- cells. hbp -sirna treatment abrogated the inhibitory effect of cc on fgl expression in human umbilical vein endothelial cells (huvecs). conclusion:cc protects against mhv- -induced fh via suppression of fgl expression in macrophages. such effects may be mediated by the transcription factor hbp . fulminant hepatitis (fh) is a serious life-threatening disease characterized by massive hepatocyte necrosis, severe liver damage, and high mortality. the underlying mechanisms and the pathogenesis of fh are not clear. however, accumulating evidence suggests that, regardless of the pathogenesis of fh, the host's inflammatory responses contribute to liver microcirculatory disorders and injuries. accordingly, it has been shown that immune cell activation and inflammatory cytokines play an important role in fh ( ) . in recent years, our laboratory has conducted extensive research on the pathogenesis of fh and found that immune cells play a key role in it. kupffer cells, natural killer (nk) cells ( , ) , cytotoxic t-lymphocytes (ctls), and double negative t-cells (dnt) ( ) ( ) ( ) in liver and the cytokines that are produced by these cells cause liver damage. prothrombinase fgl belongs to the fibrinogen superfamily and is produced by activated macrophages or endothelial cells, transforming prothrombin directly into thrombin, so as to quickly initiate the process of coagulation. this promotes the conversion of fibrinogen into fibrin, resulting in thrombosis ( ) ( ) ( ) ( ) ( ) ( ) . our study found that fgl was highly expressed in peripheral blood mononuclear cells (pbmcs) and in liver tissue of humans or mice with severe viral hepatitis, and was positively related to the severity of the disease ( , ) . gene therapy targeting fgl silencing showed that the survival rate of fulminant hepatitis mice increased from to . % ( ) . thus far, the discovery and related research involving fgl have provided new insights into the molecular mechanism of hepatocyte necrosis in fh. in view of the important role of fgl in severe viral hepatitis, investigations concerning the regulation of fgl will be beneficial in the search for new strategies for treatment of severe hepatitis. clara cell kda protein (cc ), also considered to be uteroglobin, clara cell secretory protein, is one of members of secretoglobin superfamily. expressed in mucosal epithelial cells of organs (including lungs and nose) that communicated with the outside world ( ) . cc has immunomodulatory and anti-inflammatory effects. compared to wild-type mice, cc -knockout mice exhibited excessive airway inflammation abbreviations: fh, fulminant hepatitis; mhv- , murine hepatitis virus strain ; fgl , fibrinogen-like protein ; cc , clara cell kda protein; alf, acute liver failure; pfu, plaque-forming units; pbs, phosphate-buffered saline; alt, alanine aminotransferase; ast, aspartate aminotransferase; pca, pro-coagulant activity; hrp, horseradish peroxidase; tunel, terminal deoxynucleotidyl transferase dutp nick end labeling. caused by allergic reaction and bacterial and viral infections ( ) . reduced levels of cc are associated with inflammatory and allergic airway diseases, including sinusitis, asthma and allergic rhinitis ( ) ( ) ( ) ( ) . previous studies and published articles show that cc protein can not only inhibit th cell responses by inhibiting expression of related molecules of dendritic cells and cytokines in mice with allergic rhinitis, but also can inhibit chitosan- like protein ( , ) . moreover, cc inhibits the expression of an important immune regulator, osteopontin (opn), in models of allergic rhinitis ( ) . in this study, we investigated the role of cc in hepatitis virus strain (mhv- )-induced fh in mice and explored whether cc protein could regulate fgl in the disease process. female balb/cj mice (shanghai shilaike animal seed center, shanghai, china), - weeks of age, with a body weight of . - . g, were kept in tongji hospital with food and water. mice were divided into two groups: cc group (experimental group) and phosphate-buffered saline (pbs) group (control group). this study was carried out in accordance with the recommendations of the guidelines of the national institutes of health and the animal experiment committee of tongji hospital. this study was reviewed and approved by the animal experiment committee of tongji hospital. the human monocyte cell line thp- was purchased from the cell institute of the chinese academy of sciences (shanghai, china). human umbilical vein endothelial cells (huvecs) were obtained from the biology treasure center of wuhan university, china. the chinese hamster ovary (cho) cell line was acquired from the typical culture preservation commission cell bank, the chinese academy of sciences (shanghai, china). human umbilical vein endothelial cells (huvecs) and cho cells were cultured in dulbecco's modified eagle's medium (dmem), and thp- cells were maintained in rpmi , containing % heat inactivated fetal bovine serum (fbs, gibco life technologies, usa), u/ml penicillin, and mg/ml streptomycin and cultured at • c, ml/l co and % humidity. peritoneal exudative macrophages (pems) were obtained from balb/cj mice. cells were resuspended in rpmi , supplemented with % fbs at - × cells/ml in a -well plate and incubated for h. they were then washed with rpmi medium and non-adherent cells discarded. the adherent cells were macrophages and were incubated for a further h. peritoneal exudative macrophages (pems) were divided into two groups. one group was supplemented with cc protein ( ng/ml) and in the other group, pbs was added. after h of stimulation, , plaque forming units (pfus) of mhv- was added to the cells, which were then cultured for h. peritoneal exudative macrophages (pems) were harvested and lysed for real-time pcr and western blotting analysis. cell apoptosis was detected by the terminal deoxynucleotidyl transferase dutp nick end labeling (tunel) method with a tunel apoptosis detection kit (roche, switzerland). briefly, µm sections were deparaffinized, dehydrated through an alcohol series and incubated with proteinase k for min at • c. after stopping the proteinase k digestion reaction with pbs, the samples were incubated with terminal deoxynucleotidyl transferase end-labeling cocktail (a mixture of terminal deoxynucleotidyl transferase and dutp at a ratio of : , respectively), for h at • c in an immunohistochemistry wet box. following washing and blocking, each section was supplemented with reagent (converter-pod) to cover the tissues and incubated for min at • c in a wet box. then, the liver tissue sections were washed with pbs, and colored with diaminobenzidine (dab) subsequently. hepatocytes with nucleus stained brownish yellow were considered to be apoptotic cells. the expression of fgl on thp- cells was measured by flow cytometry (bd facs canto ii, usa). briefly, cells ( × per tube) were incubated with human trustrain fcx (fc receptor blocking solution, biolegend, usa) for min at room temperature and then incubated in the dark with mouse anti-fgl antibody ( : , abnova,) or normal goat serum (an isotype control) at • c for min. cells were washed with pbs and incubated in the dark with pe-conjugated goat anti-mouse igg antibody ( : , biolegend, usa) at • c for min. cells were then washed with pbs and resuspended in µl pbs for study. liver slices were fixed in % paraformaldehyde and then embedded in paraffin. immunohistochemistry of liver tissues was performed using sp- splink detection kits (biotin-streptavidin hrp detection systems) (zsgb-bio, beijing, china) according to the manufacturer's instructions. for immunohistochemistry staining, the expression of fgl , fibrinogen, fas and tnf-receptor in mouse liver tissues was detected with polyclonal rabbit anti-mouse fgl antibody ( : , proteintech, usa), polyclonal rabbit anti-mouse fibrinogen antibody ( : , , abcam, england), polyclonal rabbit antimouse fas antibody ( : , abcam, england), and polyclonal rabbit anti-mouse tnf-receptor antibody ( : , abcam, england), respectively. after incubation with an horseradish peroxidase (hrp)-labeled goat igg fraction to rabbit igg fc, the target protein was detected using a dab kit (zsgb-bio, beijing, china). the slides were then counterstained with hematoxylin and visualized under a microscope (olympus, tokyo, japan). liver tissue and cells were homogenized in ripa lysis buffer with phenyl methane sulfonyl fluoride (pmsf) protease inhibitor. protein lysates were separated by sds-page, and western blotting was performed using a monoclonal mouse antihuman/mouse fgl ( : , abnova), a monoclonal mouse antihuman hbp ( : , santa cruz, usa), and a monoclonal rabbit anti-human/mouse β-actin ( : , , cell signaling technology, usa). liver tissues were collected from mhv- -infected balb/cj mice at h, and total rna was extracted using trizol reagent (invitrogen, usa) and then reverse transcribed into cdna by using revertra ace qpcr rt kit (toyobo, japan). the cdna was then amplified by rt-pcr by using dream taq green pcr master mix ( ×) (thermo scientific, usa). realtime quantitative pcr (qpcr) with sybr green real-time pcr master mix (toyobo, japan) was performed using a cfx real-time pcr detection system (bio-rad, usa) and mrna levels were normalized with reference to those of the house keeping gene gapdh. primer sequences for qpcr amplification were as follows: mtnf-α forward, ′ -ttt gag atc cat gcc gtt gg- ′ ; mtnf-α reverse, ′ -gcca cca cgc tct tct gt- ′ ; mil- β forward, ′ -tgt aat gaa aga cgg cac acc- ′ ; mil- β reverse, ′ -tct tct ttg ggt att gct tgg- ′ . mfgl forward, ′ -gcc aaa tgt gag tcc ctg gaa- ′ ; mfgl reverse, ′ -ttc cac cca aga gca cgt tta ag- ′ ; hfgl forward ′ -aca gtt cag gct ggt ggt- ′ ; hfgl reverse, ′ -ggc tta aag tgc ttg ggt- ′ ; hbp forward, ′ -tga agc aga agc tgg gagt- ′ ; hbp reverse, thp- cells were treated with ng/ml phorbol -myristate -acetate (pma) (sigma, usa) for h to induce differentiation toward adherent macrophage-like cells as reported previously ( ) . the cc group was supplemented with cc protein ( ng/ml). after h of stimulation, ifn-γ ( ng/ml) was added to these cells, which were then cultured for h before they were collected for western blotting and real-time pcr studies. the chinese hamster ovary (cho) cells were cultured in cm cell culture dishes with dmem supplemented with % fbs until - % confluence. next, µg pcdna . -hfgl (constructed in our lab) was mixed with µg pcdna . -hcc in serumfree dmem. the mixture was then combined with lipofectamine , (invitrogen, usa) and mixed gently. after incubation at • c for min, the solution was added to cho cells and incubated at • c in % co . four to six hour after transfection, the medium was removed and fresh medium containing % fbs was added. at h after transfection, the cells were collected for co-immunoprecipitation analysis to evaluate the interaction of cc with fgl . both huvec and thp- cells express fgl . however, in the transfection experiments, it is difficult to transfect the thp- cells with sirna, so we use huvec instead of thp- . human umbilical vein endothelial cells (huvecs) were cultured in figure | cc protein increased survival rate and reduced liver damage in mice. (a) the survival rate of cc group is higher than the control group comprised of mhv- -infected balb/cj mice treated with saline. cc protein ( µg) or saline were injected into mice by tail vein. balb/cj mice then received pfu of mhv- intraperitoneally h later to develop fulminant viral hepatitis. then, cc protein ( µg) or saline were injected into mice by tail vein following mhv- infection h later. the survival rate was observed for days (n = /group). representative data from three independent experiments are shown. the survival curve was analyzed by using the log-rank test. ***p < . compared with saline group. (b) histopathology of liver tissues (h&e staining; original magnification, × , n = /group) at h post-mhv- infection was evaluated in the two groups of mhv- -infected balb/cj mice. livers were collected from saline-treated (a) and cc -treated (b) balb/cj mice at h after mhv- infection. arrows point to inflammatory cell infiltration areas or necrotic regions with inflammation. (c) effect of cc on serum alt and ast levels (n = - /group). values represent means and standard error of three independent experiments performed in triplicate. **p < . compared with the saline group. six-well plates with dmem supplemented with % fbs until - % confluence. pmol hbp -sirna was mixed with µl serum-free dmem. two microliter lipofectamine , was gently mixed with serum-free dmem. after incubation at • c for min, the solution was added to huvecs and incubated at • c. four hour after transfection, the medium was removed and fresh medium containing % fbs was added. at h after transfection, cells were collected for real-time pcr and western blot analysis to evaluate the effects of hbp on fgl . at h after transfection, the cc group was supplemented with the cc protein ( ng/ml). after h of stimulation, ifn-γ ( ng/ml) was added to these cells. these cells were then cultured for h before they were harvested for real-time pcr studies to evaluate the effects of cc on fgl by hbp . negative control was used as a control. to detect whether there was a potential interaction between cc protein and fgl , cho cells were transfected with pcdna . -hcc and pcdna . -hfgl for h. cells transfected with empty plasmid pcdna . (mock) were used as negative controls for cc gene transfection. immunoprecipitation and immunoblotting were performed by using pierce co-immunoprecipitation kit (pierce, usa). total cell proteins were extracted as previously described ( ) . the proteins were immunoprecipitated by mouse anti-human fgl antibody ( : , abnova). for co-immunoprecipitation experiments, western blotting was performed using both rat anti-human uteroglobin/scgb a antibody ( : , r&d, usa) frontiers in immunology | www.frontiersin.org and mouse anti-human fgl antibody ( : , abnova). control isotype rat igg was used as a negative control for primary antibodies. the human cc coding region gene, including a bp sequence, was amplified from homogenized human turbinate tissue by rt-pcr. in this study, the sequences of pcr primers for cc were as follows: hcc -forward, ′ -ccc tcc acc atg aaa ctcg- ′ ; hcc -reverse, ′ -tga gat gct tgt ggt tta ttg aag- ′ . the pcr products were cloned into peasy-t cloning vector (transgen, beijing, china) and then subcloned into hindiii/xbai site of pcdna . vector (invitrogen, usa) to form eukaryotic expression plasmids pcdna . -hcc . microarray analysis was used to screen changes in genome-wide gene expression patterns in thp- cells with or without cc protein. the changes in over , human gene expression patterns were assessed using affymetrix gene microarrays (human genome u plus . ) (capitalbio co.,ltd., beijing, china). three replicates were used for microarrays analysis. data obtained from the experiments are expressed as means ± sem. survival curve comparisons were performed with the log rank test. multiple group analyses for data were evaluated by one-way analyses of variance. analyses of two group results were performed using student's t-test to evaluate the statistical significance of differences. values of p < . indicated significance. to establish an animal model of mouse fh, mhv- was injected intraperitoneally to balb/cj mice ( mice/group). to further study the role of cc in fh, recombinant mouse cc protein ( µg/mouse) or saline was administrated into the tail vein h prior to mhv- infection. the same dose of cc protein or saline was then administered h later. the survival rate of the cc and saline groups was observed for days. the results showed that mice in the two groups began to die at h after injection of mhv- and exhibited symptoms of horripilation, slow activity, and reduced food consumption. in the cc group mice were alive on day after infection, mice alive on day , and of ( . %) mice recovered from fulminant viral hepatitis. at the same time, in saline treated group, there were mice alive on day , mice alive on day after infection, and no mice survived to day . that is to say, the mice in the saline group died within or days. three of ( . %) mice of the cc group recovered from fulminant viral hepatitis ( figure a) . to better understand the mechanisms underlying the biological effects of the cc protein, liver function (alt and ast levels in serum) and liver histology in mice of mhv- -infected was performed. liver tissues were harvested h following mhv- infection, and liver histology was detected by h&e staining. these results showed that there was substantial inflammatory cell infiltration and widespread necrosis of hepatocytes in the liver tissue of the saline group mice (figure ba ). there were rare or no infiltrating inflammatory cells, and few or no hepatocyte necrosis in the livers of mice in the cc group h after mhv- infection (figure bb) . serum alt and ast levels in mice were observed h after mhv- infection. the results showed that serum alt and ast levels in the saline group reached a peak h after mhv- infection, but there was no significant increase in the cc group compared to the levels in the control group (p < . , figure c) . these results suggested that cc protein has a role in protection against mhv- -induced liver injury in mice. to further elucidate the mechanisms of reduced liver injury following cc protein injection, we investigated the cytokines tnf-α and il- β expression. because these two cytokines play a crucial role in the liver damage of fh. they are characterized by an increase in apoptosis. levels of tnf-α and il- β in liver tissues were markedly reduced in the cc group (as shown in figure a) . hepatic apoptosis (figure b ) was significantly reduced in the cc group. we and collaborators have a long standing interest in studying the role of fgl in viral hepatitis. fgl has been verified to play an essential role in the progression of fulminant viral hepatitis as we appreciate from previous reports. we have provided liver pathology figures and liver function for mhv- infected mice with a fgl gene knockout as shown in supplementary figure . the data was comparable with previous reports from our center and collaborators. from this current study we shown that cc plays a protective role in liver damage.to study the related molecules of cc in mhv- -induced fh mice, we evaluated whether there was crosstalk between fgl and cc . we found that the expression of fgl in the liver of mice was reduced h after mhv- infection and treatment with cc protein (figures a,b) . furthermore, fibrin deposition, an indicator of liver injury associated with fgl expression in fh, was also decreased in the livers of cc -treated mice compared to that in controls (figure c ). this indicates that cc treatment reduced liver injury after viral infection by inhibiting fgl expression. we examined the effect of increasing doses of cc protein ( , , , and ng/ml) on ifn-γ-induced fgl expression in thp- cells. cc treatment showed a . % decrease in thp- cells compared to that in control after stimulation with ng/ml ifn-γ for h. cc protein inhibited fgl expression between doses of ng/ml and ng/ml (figure a ). in particular, ng/ml cc protein had the strongest inhibitory effect on fgl expression among the doses, and we chose this dose for the following experiments. we explored the effect of different time points of stimulation with a concentration of ng/ml cc protein. after stimulation with cc protein for , , and h compared to the pbs control, the strongest inhibitory effect on fgl expression was noted at h; hence, we chose this time point for the following studies ( figure b ). an increasing number of studies suggest that macrophages are the primary source of fgl . in order to ascertain that cc has a direct effect on macrophages, we treated thp- cells with recombinant cc and assessed the expression of fgl . unlike in controls, ifn-γ induced a significant increase in fgl expression. this effect was attenuated when cells were treated with cc protein (figures c,d) , revealing that cc directly reduces the levels of fgl in macrophages. to further explore the possibility that cc protein directly acts on macrophages, we infected murine pems with mhv- in the presence of recombinant cc and determined fgl expression. compared to levels in the controls, mhv- infected macrophages exhibited a significant increase in fgl production, and this effect was abolished by using cc protein (figures a,b) , indicating that cc directly modulates fgl production in macrophages. in order to determine genes that were downregulated after stimulation by cc protein, we used dna microarray analysis to screen for differentially expressed genes. thp- cells were cultured and pma was added to induce differentiation into macrophages. the production of fgl was stimulated by ifnγ. the experimental group was treated with cc protein for microarray detection of differentially expressed genes. the results showed that the most obviously downregulated genes were ube w, hectd , mir , atrx, sox , hbp , and fgl (supplementary table ) . and then these genes were tested by qpcr. however, ube w, hectd , mir , atrx, and sox was not differentially expressed by qpcr, while hbp and fgl were still down-regulated genes. dna microarray analysis identified hbp as a down-regulated gene involved in the pathological processes of the regulation of cc . recently, very limited studies have explored the role of hbp in fh. nevertheless, the mechanistic functions of hbp in fh remain largely unexplored. therefore, we selected this gene for further study. qpcr analysis confirmed that mrna levels of hbp were significantly decreased in thp- cells after cc protein stimulation compared to that in the pbs control group (figure a ). we knocked down hbp using hbp -sirna. then, transfection of hbp -sirna into huvecs was detected by qpcr and western-blotting methods. as expected, hbp knockdown led to significantly decreased expression of hbp (figures b,c) . furthermore, hbp knockdown impaired expression of fgl (figure d ), suggesting that hbp was able to activate fgl . hbp -sirna was used to transfect huvecs. then, ifn-γ was added to induce the expression of fgl followed by stimulation with cc protein ( ng/ml) after h. finally, we explored the expression of fgl by qpcr. the results showed that hbp -sirna treatment abrogated the inhibitory effect of cc on fgl expression in huvecs (figure ) . that is to say, cc could suppress fgl expression in macrophages. such an effect may be mediated by the transcription factor hbp . it is well-known that cc protein can suppress the immune response. in animal models of allergic diseases of the respiratory tract, most of evidences confirm this inhibition ( ) . its function in fh has not been investigated yet. here, we used a murine fh model established by mhv- infection to explore the effects of cc in this disease process. to determine the role of cc in the pathogenesis of fh, cc protein was injected into a mouse fh model established by mhv- infection. mhv- -induced liver injury in cc -treated mice occurred rarely and the areas of lesions were much fewer than those in saline-treated control mice. in summary, these results suggested that cc could reduce pathological liver damage in this fh model together with lower mortality rates followed by mhv- infection. mhv- induced fulminant viral hepatitis progresses rapidly and infected mice die within - days. previous studies suggested fgl played a vital role in this process with a - % increase of survival when fgl was deleted ( , , , ) . multiple inflammatory factors or mediators including tnf-α and ifn-γ, il- β and c ar have been demonstrated to promote fh progression with significant discrepancies between liver damage and survival rate ( ) ( ) ( ) ( ) , which is accordant with our observation that cc substantially alleviated liver injury though survival rate improved mildly. the survival rate based on hours may be more accurate to examine the effect of cc on fh. it is speculated that fgl can mediate lethality in mhv- -induced fh. this is due to the fact that fgl induces the deposition of fibrinogen, which leads to activation of the coagulation cascade and induction of procoagulant activity ( ) . to determine whether the tissue necrosis was mediated by fgl in cc -treated mice following infection, fgl expression was observed. results suggested that the expression of fgl was significantly increased in mhv- -induced fh mice and cc treatment significantly reduced the production of fgl in the infected liver and serum. in addition, decreased fibrinogen deposition was also observed in the livers of cc -treated mice. therefore, our research results strongly clarify that the lower mortality of cc -treated mice after mhv- infection is due to the lower levels of fgl and decreased fibrinogen deposition. indeed, it has been reported that fgl is expressed on macrophages, and the expression of fgl is believed to be induced by ifn-γ and tnf-α ( ) . cultured thp- cells activated by ifn-γ or il- have been demonstrated, with induction of fgl expression and enhanced activation of human prothrombin ( ) . therefore, in this study, we explored this cell line to investigate the modulation of cc on fgl . surprisingly, we found that cc directly inhibited ifn-γ-induced fgl expression in thp- cells. as we know, ifn-γ has proved to be the main cytokine that leads to the development and progression of fh. also, it was shown that ifn-γ might exert its own proinflammatory biological function through enhancing fgl expression. therefore, in our study, cc might counter the effect of ifn-γ in the setting of fh, which substantiates its role in fh. these results demonstrated that cc regulates the expression of fgl in macrophages. in the current study, we used co-immunoprecipitation to analyze binding between cc and fgl . in this study, we investigated possible protein-protein interactions between cc and fgl in vitro. the chinese hamster ovary (cho) cells transfected with pcdna . -hcc and pcdna . -hfgl . cellular proteins were immunoprecipitated with anti-cc antibody or anti-fgl antibody. immunoblotting was performed with anti-fgl and anti-cc antibodies. immunoprecipitation of protein extracts from pcdna . -cc and pcdna . -fgl co-transfected cho cells with anti-fgl or anti-cc antibody followed by western blotting with fgl and cc antibodies indicated that cc did not co-immunoprecipitate with fgl , showing that there is no direct relationship between cc and fgl (data not shown). the results showed that cc has no direct interaction with fgl . from our previous study the gene of fgl contributed profoundly in mhv- induced fulminant hepatitis and is extensively expressed in macrophages and endothelium ( , ) . our microarray indicated a cc down-regulated fgl expression and this is further confirmed by qpcr and western blotting in vivo (peritoneal macrophages) and in vitro (thp- , macrophage cell line). therefore, it is reasonable to focus on macrophages to display the effect of cc on fgl expression and eventually mice survival. we entirely agree there may be other possibilities for a protective effect of cc to contribute to the disease process. this is worth further studies. the potential receptor of cc has not been revealed yet. our previous study have demonstrated that cc have effect of dendritic cells in allergic rhinitis ( ) . in this research, we evaluated the effect of cc on macrophages functions and found fgl was substantially down-regulated upon cc treatment, therefore, we speculate that potential cc receptor may be also expressed on macrophages. the potential target of cc on other immune cells cannot be excluded. dna microarray analysis is one of the most powerful approaches for the potential identification of unexpected genes involved in pathogenic processes. by using this approach, hmgbox transcription factor (hbp ) was found to be one of the most downregulated genes after cc treatment of thp- cells. hbp is a well-described transcriptional repressor that modulates expression of genes involved in cell cycle progression. in a recent study, it was found that hbp is a direct target of mir- and confirmed that hbp modulates the inhibitory function of mir- -aso in hepatosteatosis and carcinogenesis simultaneously ( ) . hbp is an endogenous inhibitor of the wnt signaling pathway in both normal and cancer cells. the tumor suppressor role of hbp has been reported in some malignancies, such as oral cancer and glioma ( ) . however, an association between hbp and fgl has not been investigated yet. the current study clearly demonstrated that cc protects against mhv- induced fh via suppression of fgl expression. such effects might be mediated by hbp . however, the functional status of hbp in the cc pathway requires further research, and such studies are conducting in our laboratory. in conclusion, we demonstrated that cc could limit the immunopathological damage in mhv- -induced fh mice. our results suggest that enhancing cc expression by an immunotherapeutic approach might be an effective treatment for fh. hy performed all the described experiments and wrote the manuscript. yl assisted with some experiments, analyzed experimental results, and edited the manuscript. hw analyzed experimental results. xw reviewed and 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can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material table | list of down-regulated genes by dna microarray. in order to determine genes that were downregulated after stimulation by cc protein, we used dna microarray analysis to screen for differentially expressed genes. briefly microarray analysis was used to screen changes in genome-wide gene expression patterns in thp- cells with or without cc protein. the changes in over , human gene expression patterns were assessed using affymetrix gene microarrays (human genome u plus . ) (capitalbio co. ltd., beijing, china). three replicates were used for microarrays analysis. thp- cells were cultured and pma was added to induce differentiation into macrophages. the production of fgl was stimulated by ifn-γ. the experimental group was treated with cc protein for microarray detection of differentially expressed genes. the results showed that the most obviously downregulated genes were ube w, hectd , mir , atrx, sox , hbp , and fgl . key: cord- -mowj wyl authors: zhou, xuezhong; lei, lei; liu, jun; halu, arda; zhang, yingying; li, bing; guo, zhili; liu, guangming; sun, changkai; loscalzo, joseph; sharma, amitabh; wang, zhong title: a systems approach to refine disease taxonomy by integrating phenotypic and molecular networks date: - - journal: ebiomedicine doi: . /j.ebiom. . . sha: doc_id: cord_uid: mowj wyl the international classification of diseases (icd) relies on clinical features and lags behind the current understanding of the molecular specificity of disease pathobiology, necessitating approaches that incorporate growing biomedical data for classifying diseases to meet the needs of precision medicine. our analysis revealed that the heterogeneous molecular diversity of disease chapters and the blurred boundary between disease categories in icd should be further investigated. here, we propose a new classification of diseases (ncd) by developing an algorithm that predicts the additional categories of a disease by integrating multiple networks consisting of disease phenotypes and their molecular profiles. with statistical validations from phenotype-genotype associations and interactome networks, we demonstrate that ncd improves disease specificity owing to its overlapping categories and polyhierarchical structure. furthermore, ncd captures the molecular diversity of diseases and defines clearer boundaries in terms of both phenotypic similarity and molecular associations, establishing a rational strategy to reform disease taxonomy. disease taxonomy plays an important role in defining the diagnosis, treatment, and mechanisms of human diseases. the principle of the current clinical disease taxonomies, in particular the international classification of diseases (icd), goes back to the work of william farr in the nineteenth century and is primarily derived from the differentiation of clinical features (e.g. symptoms and micro-examination of diseased tissues and cells) (council et al., ) . despite its extensive clinical use, this classification system lacks the depth required for precision medicine with the limitations of its rigid hierarchical structure and, moreover, it does not exploit the rapidly expanding molecular insights of disease phenotypes. for example, many diseases (e.g. cancer, chronic inflammatory diseases) in the current disease taxonomies have either high genetic heterogeneity (bianchini et al., ; mcclellan and king, ) or manifestation diversity (arostegui et al., ; jeste and geschwind, ; mannino, ) , which give little basis for tailoring treatment to a patient's pathophysiology. furthermore, disease comorbidities (hu et al., ; lee et al., ; hidalgo et al., ) , temporal disease trajectories (jensen et al., ) in clinical populations, various molecular relationships between disease-associated cellular components and their connections in the interactome (blair et al., ; goh et al., ; barabasi et al., ; rzhetsky et al., ; zhou et al., ) , and many successful drug repurposing cases (li and jones, ; chong and sullivan jr., ; ashburn and thor, ; wu et al., ; evans et al., ) altogether demonstrate the vague boundary between different diseases in current disease taxonomies. moreover, the deep understanding of diseases based on the advances in disease biology, bioinformatics, and multi-omics data necessitates the reclassification of disease taxonomy (mirnezami et al., ) . in the past decade, efforts to reclassify diseases based on molecular insights have increased with studies related to molecular-based disease subtyping in different disease conditions, such as acute leukemias (golub et al., ; alizadeh et al., ) , colorectal cancer (dienstmann et al., ) , oesophageal carcinoma (cancer genome atlas research et al., ) , pancreatic cancer (bailey et al., ) , cancer metastasis (chuang et al., ) , neurodegenerative disorders (mann et al., ) , autoimmunity disorders (ahmad et al., ) , multiple cancer types across tissues of origin (hoadley et al., ) , and a network-based stratification method for cancer subtyping (hofree et al., ) . further insights will arise from integrating all types of biomedical data with a single framework to exploit diseasedisease relationships. data integration methods that utilize multiple types of data, including ontological and omics data, have been used to classify and refine disease relationships (gligorijevic and przulj, ; menche et al., ; gligorijevic et al., ) . despite these efforts, the development of a molecular-based disease taxonomy that links molecular networks and pathophenotypes still remains challenging hofmann-apitius et al., ; jameson and longo, ) . here, we aim to refine a widely used clinical disease classification scheme, the icd. to achieve this, we first quantify the category similarity among the icd chapters using ontology-based similarity measures and investigate the molecular connections of disease pairs in the same icd chapters. furthermore, we seek the correlation between category and molecular similarity, and check for the heterogeneity of molecular specificity and correlated boundary between categories in icd taxonomy. finally, we construct a new classification of diseases (ncd) with overlapping structures. the aim is to provide clear boundaries between distinct diseases belonging to different categories using a new disease classification scheme ( fig. & fig. s ). fig. . overview of the new disease taxonomy construction and validation. a. similarity calculation between the disease pairs in icd taxonomy, including the calculation of ) category similarity; ) phenotype similarity (based on icd-mesh term mapping) and ) molecular profile similarities (based on icd-umls term mapping) of disease pairs in icd; b. module or community annotations of disease association network by chapters in icd or ncd. we generate disease association network, in which nodes represent diseases and the link weights represent their corresponding phenotype or molecule profile similarities. the module annotations of the disease network correspond to icd chapters or ncd categories; c. construction of integrated disease network (idn) and generation of ncd. the links of idn are fused from the multiple similarities (e.g. phenotype similarity and shared gene similarity). based on idn, ncd is generated by community detection algorithms with overlapping disease members; d. quality evaluation and validation of icd and ncd. the molecular specificity (or inverse molecular diversity) and network modularity are used for evaluation and comparison of the quality of two disease taxonomies. furthermore, we validate the robustness of ncd with two independent phenotype-genotype association datasets, namely gwas and phewas. in this work, large curation efforts are performed to generate the related data sources (details see supplementary materials (sm) section ). we obtained the updated text version of icd- -cm ( ) and extracted the list of icd codes with their hierarchical structures. while we recognize the improvements of the currently used icd- over icd- , nevertheless, we chose to use icd- -cm as the adoption of icd- has been slow in the united states (butler, ) and since it was still being widely used at the time of the data collection for this paper (blair et al., ; wang et al., ) . furthermore, although icd- does have more codes than icd- -cm, the structure is kept almost the same. we obtained the high-quality phenotype-genotype (disease-gene) associations from disease connect database ( version) (liu et al., ) , leaving out the less reliable text mining entries and focusing only on genome-wide association study (gwas), online mendelian inheritance in man (omim) and differential expression evidence types, and manually mapped those diseases in unified medical language system (umls) codes to icd and mesh codes (sm section . ). to calculate the molecular network and phenotype characteristics related to disease phenotypes, a high-quality subset of human proteinprotein interactions was filtered from string v . (franceschini et al., ) using the score threshold at ≥ , as well as a wellestablished disease-phenotype (disease-symptom) association dataset (i.e. disease network with symptom similarity, hsdn) derived from pubmed bibliographic records and the gene ontology annotations from ncbi gene database are adopted. to ensure the results are not biased by computational predictions in the string database, we replicated the classification pipeline with manually curated ppi networks , which rely only on physical protein interactions with experimental support, and found that the results are robust (sm section . ). in addition, to validate the robustness of our results from independent data sources, we filtered the gwas and phenome wide association studies (phewas) data from university of california santa cruz (ucsc) genome browser (tyner et al., ) and phewas catalog (denny et al., ) respectively, and performed additional icd mapping task to prepare the data for validation analysis. the gwas evidence of the diseaseconnect database, which we used to build the disease associations, comes from the national human genome research institute (nhgri) gwas catalog (welter et al., ) , whereas for validation, we used the ucsc-gwas genome browser. we have ensured that the gwas data used to build the networks and to validate them have a very small overlap (sm section ). here, we systematically evaluated the consistency of disease categories in icd taxonomy from both clinical phenotype and molecular profiles (details are in sm section ). we investigated the quality of icd disease taxonomy by evaluating the correlation between the closeness of disease pairs in the disease taxonomy and the underlying molecular connections (and symptom phenotype similarities) between disease pairs. for example, if two disease pairs have close positions (e.g. have a low level common parent disease) in the disease taxonomy, then we would expect that those disease pairs might have common genes or shared protein-protein interactions or similar phenotypes. we calculated the category similarity between disease pairs using a widely used semantic similarity measure (i.e. lin measure using information content) (lin, ; pesquita et al., ) to represent the closeness of disease pairs located in the icd taxonomy. information theoretic measures such as information content have been used in the context of icd- -cm previously (dahlem et al., ) . the category similarity measure takes as input two concepts c and c and outputs a numeric measure of similarity. if two icd codes have a very specific common parent code in the taxonomic tree structure, then the category similarity would be~ . the molecular and phenotype similarity between disease pairs are calculated by evaluating the shared genes and their go annotations, molecular network similarities, and shared phenotypes by established similarity measures (e.g. cosine measure and jaccard measure). in particular, to propose a more robust representation of molecular network profiles of diseases, we partitioned the string network into topological modules (data s ) and used them to construct the relevant module vectors of diseases using odds ratio (or) as weighting measure. for example, an icd disease code would be represented with a -dimensional vector, which has a value of w ij if its related gene is in a module or otherwise. suppose we have n genes in total and m i genes of a module i. now for a disease d j with n j genes, which has k ij overlapping genes with the module i, we calculated the value of w ij as the following equation, we used the cosine measure to calculate the molecular module similarity between disease pairs after the molecular module vector (i.e. or weighting) of each disease was constructed. furthermore, as icd taxonomy proposes a framework for organizing the diseases, it is expected that there should overlapping molecular interactions or phenotype relationships between the diseases of the same chapters than those of the different chapters. thus, we assumed that when we collapse the icd chapters as the module annotations, such that all the diseases in one chapter would be considered as members of a same module, the modularity of the disease association networks, i.e. the disease networks with molecular or phenotype associations as links, would reflect the quality of icd disease taxonomy. this means that the higher the modularity, the higher the quality of the icd chapters as a disease category framework. to evaluate the quality of community structures in complex network, the modularity measure (newman, ) was proposed to quantify the extent to which the connection in communities is above the random expectation in the whole network. let a network have m edges and a vw be an element of the adjacency matrix of the network. suppose the vertices in the network are divided into communities such that vertex v belongs to community c v . then the modularity q is defined as: where the function δ(i,j) is if i = j and otherwise, and k v is the degree of vertex v. the value of the modularity lies in the range [− / , ]. it is positive if the number of edges within groups exceeds the number expected on the basis of chance. otherwise, it would be negative. we use it to measure the consistency of disease categories (icd chapter or ncd) as an annotation of topological module (or community) structures within disease networks. we hypothesize that if a disease category framework captures the molecular or phenotypic profiles of diseases, then there would be more links existing between the disease members in a category than random expectation. as a quantification of the molecular diversity (or the inverse specificity) of a disease, we calculated the maximum betweenness of diseaserelated genes in the ppi network (data s ). betweenness (freeman, ) is a widely used centrality measure to quantify how many shortest paths run through a given node. in particular, bridging nodes that connect disparate components of the network often have a high betweenness. the betweenness centrality of a node v is given by: where n st (v) denotes the number of shortest paths from s to t that pass through v and g st is the total number of shortest paths from s to t. we will adopt the convention that nstðvÞ g st ¼ if both n st (v) and g st are zero. we assume the molecular diversity of diseases would largely lie on the related genes with maximum betweenness. for example, to quantify the molecular diversity (in terms of maximum betweenness) of alzheimer's disease (ad), we calculated all the betweenness values for the ad-related genes, such as app, apoe, tnf and nos . finally, we considered the molecular diversity of ad as . e- since we found that app has the maximum betweenness of . e- among those genes (see fig. s a ). in fact, this kind of measurement has been successfully used in a previous study to evaluate the diversity of diseases, which indicated that the diversity of disease manifestations has a strong positive correlation with the molecular diversity of diseases. for disease taxonomy with good quality, we would expect it to have its lowest level diseases (the leaf nodes in the tree-structure disease taxonomy) with similar molecular diversities. we calculated the edge density to quantify the molecular interactions between icd chapters. to further detect the significant interactions between diseases in different chapters, we find an approach to obtain the diseases that have significant interactions with diseases in chapters other than their own. given a disease d i for investigation, we evaluate whether the proportion of interactions (i.e. edge density) of d i to the disease set d c k of a chapter c k is significantly larger than the average proportion of interactions between the diseases in c k (fig. s ) . we use binomial test to filter the significant interacting disease-chapter pairs, in which the edge density of the disease to the chapter is significantly higher than the average edge density of the diseases in the corresponding chapter (details are in sm section ). the results showing positive correlations between category similarity and molecular similarity, and the high molecular diversity of many diseases imply that it would be possible to predict the multi-category map for each disease using its underlying molecular connections. to demonstrate a pilot method for multiple disease category prediction by integrating molecular module and shared gene similarities, we provided a novel algorithm to generate the possible associated additional disease categories for a given disease with the corresponding molecular association scores. (details are in sm section , fig. s ). in this algorithm, we integrated the correlation between category similarity and module similarity with significant disease-chapter associations (which are based on the shared gene similarity) to predict the additional chapters for a given disease. we divide the disease pairs in the same chapter to three subsets, which correspond to those pairs with shared root parents, shared second-level intermediate parents and shared third-level intermediate parents, respectively, to help predict to what degree a pair of diseases would be located closely in the disease taxonomy. the principle of the algorithm adheres to the positive correlation between category similarity (or the closeness of position of the disease pairs in icd disease taxonomy) and molecular profile similarity of disease pairs, which means that strong molecular profile similarity between disease pairs would indicate close locations of them in the disease taxonomy. to ensure detecting the significant disease-chapter associations, we next filtered the predicted disease-chapter associations with positive association scores by the significant disease-chapter interactions based on shared genes. to integrate disease associations derived from both molecular and phenotype features, we performed several sequential analytical steps to generate a highly reliable disease network with strict filtering criterions of the disease links (details are in sm section ). firstly, we generated three disease association networks: disease network with module similarity (msdn) with , links and nodes, disease network with shared genes (sgdn) with , links and nodes, and disease network with symptom similarity (hsdn) with , , links and nodes (fig. s & table s ) according to shared genes, shared phenotypes and molecular module similarity, respectively. to reduce the possible noise and bias of disease related data sources, we applied a multi-scale backbone algorithm (serrano et al., ) to obtain high reliable disease links (with significantly high weights than the random expectations) from the three disease networks. we finally obtained , , and , high reliable links for msdn, sgdn and hsdn, respectively and retained most nodes ( for msdn, for sgdn and for hsdn) of these networks. to further reduce the possible weak associations (the disease pairs with high module similarity but no direct protein interactions) derived from module similarity, we calculated the minimum length of the shortest paths (mspls) between each disease pairs and used it as a filtering criterion (with mspl≤ ) for msdn, which resulted in a more biological meaningful subset of msdn with , links and diseases. sgdn would capture strong associations between disease pairs if they have high degree of shared genes even their related genes are not forming functional modules. however, msdn would give high weights for disease links if the disease pairs have similar co-locations on the topological modules of molecular network even they have no shared genes. therefore, msdn and sgdn are actually two complementary molecular association evidences for disease pairs and we finally obtained the union of the subset of msdn and sgdn as the molecular association disease network (madn), which contains , links and nodes with the weights derived from the two original networks. next, we adopted a highly strict criterion to obtain an integrated disease network (idn) from the fusion of madn and hsdn links, which contains , disease links and nodes. finding the overlapping disease categories could be transformed to the task of detecting the overlapping communities (i.e. modules) from the idn. bigclam (yang and leskovec, ) is a state-of-the-art overlapping community detection algorithm based on a variant of nonnegative matrix factorization, which achieves near linear running time and comparable high quality community results. we used the bigclam algorithm, which is packaged in snap complex network software (http://snap.stanford.edu/snap/) to automatically detect overlapping communities from idn network. finally, we obtained overlapping disease communities with distinct icd disease codes. these disease subcategories contain different numbers of icd codes, ranging from to (fig. s & data s ). to obtain a top-level category framework of diseases corresponding to the chapters in icd taxonomy, we calculated the overlapping degree of the disease sub-categories by using jaccard similarity to measure the common number of diseases held by two given disease categories. this generated a disease category network with links representing shared icd codes (a link is established if two disease categories share at least an icd code and the weights of links correspond to the jaccard similarity) and nodes representing disease categories. after that, we clustered the disease sub-categories additionally by a widely used non-overlapping community detection algorithm (considering the link weight and setting the resolution parameter as . ) into top-level categories (which corresponds to the number of original chapter-level categories in icd, which we named as new chapters, ncs) using the shared icd codes (fig. s c & data s ). the modularity of these top-level categories (this makes a good comparable partition with icd chapters) in the network of sub-categories is . , which means a rather good partition of the network. these ncs contain different numbers of sub-categories ranging from to or of diseases ranging from to (fig. s c & data s ), covering diseases from all of the chapters of icd taxonomy (table s ) . these ncs would still contain overlapping disease codes since the disease subcategories have overlapping disease codes. therefore, ncs with disease sub-categories form a disease taxonomy consisting of two hierarchical levels with polyhierarchical categories although with a limited number ( ) of disease members. to validate the robustness of ncd, we obtained two external phenotype-genotype data sources (i.e. ucsc-gwas and phewas catalog), which have not been integrated yet for generating ncd for further investigation. by measuring whether the disease members in the subcategories in ncd tend to incorporate the associations of shared genes from these two data sources, we would be able to validate the quality of ncd. if the diseases in such ncd sub-categories would tend to involve shared genes, then the diseases would be more likely associated with one another than other diseases. to test this hypothesis, we obtained the overlapping disease codes (odc) in both ncd and the two external phenotype-genotype association databases and evaluate the degree of these odc disease links in each ncd sub-category when considering two diseases linked if they share common genes. in detail, we firstly obtained the common disease codes involved in both ncd and ucsc-gwas or phewas database. then we generated a disease network with shared genes derived from the two datasets, in which two diseases linked if they shared at least one common gene. after that for each ncd sub-category, we generated a complete disease network with the odc diseases in it and overlaid the network on the disease network with shared genes. finally, the overlapping percentage of disease links would be calculated for evaluating the degree of molecular associations involved in diseases in each ncd sub-categories (details are in sm section , figs. s -s ). we use r . . as the main statistical tool in our work. the comparison of two percentages was calculated by binomial test or chi-squared test. wilcoxon rank sum test was used for compare two independent list of values (e.g. two types of molecular diversities and two groups of mspls). all the correlations between two variables were calculated by pearson's product moment correlation coefficient. due to the incompleteness and bias of disease-related data (i.e. disease-gene associations and disease-symptom associations), we need to distinguish the information from the background noise. therefore, for comparison with random expectation, we reshuffle ( random permutations) the symptom features and the related genes of each disease using the fisher-yates method (fisher and yates, ) . the calculations from random permutations were used for the correlation between category similarity and molecular similarity, as well as phenotype similarity. in addition, this was used for detection of the disease categories with high molecule diversity. we curated distinct icd disease codes (table s ) from the level tree structure of , icd- -cm codes, as well as high confidence protein-protein interactions consisting of , nodes and , edges (franceschini et al., ) . we compiled , distinct diseasegene associations between distinct diseases in umls codes and , genes reported in the diseaseconnect database (liu et al., ) (fig. s ) . next, by manually mapping the diseaseconnect identifiers to icd codes, we obtained , disease-gene records involving distinct icd codes and , genes (figs. s - and data s ). to evaluate the closeness of two diseases in the icd tree structure, we applied an established semantic similarity algorithm named category similarity (see methods, sm section . ). this similarity measure is based on the information content, which quantifies the specificity of a term and can be applied to any categorization scheme that has a rooted tree structure, including the icd- -cm disease classification. we then created a disease network comprising nodes (representing icd codes) and , edges. the edge weight reflects the category similarity values and higher values reflect higher similarity between diseases whose code positions are adjacent in the icd tree. the category similarity distribution showed that most disease pairs ( , , . %) had similarities between . and . (fig. s a) . disease pairs within this range mostly belong to different disease subcategories in the same chapter, such as diseases of other endocrine glands and disorders of thyroid gland. for example, type diabetes (icd: . ) and simple goiter (icd: . ), which are in icd chapter , have a category similarity of . . however, there do exist disease pairs with high category similarities, such as type diabetes (icd: . ) and type diabetes (icd: . ) with a category similarity . . overall, this measure indicates the capability of icd in bringing together similar diseases in its tree structure, and the overrepresentation of lower similarity scores is indicative of its limitations in doing so. while the icd classification was derived from clinical manifestations (including symptoms and signs) and does not necessarily reflect the connections among the molecular components of diseases, it is informative to quantify to what extent it carries molecular information. we investigated the correlations of category similarity of disease pairs with ) the degree of shared genes and shared clinical phenotypes, ) go term (cell component, molecular function, biology process) similarity (mistry and pavlidis, ) , and ) topological similarity (i.e., minimum shortest path length and molecular module similarity) among them (methods, sm section . ). we found that close disease codes (disease pairs with a high category similarity) actually have higher clinical phenotype similarity (methods, sm section . ), which adheres to the construction principle of icd taxonomy based on symptom phenotypes (fig. s b , pcc = . , % ci = [ . , . ], p = . e- ). furthermore, we observed strong correlations between higher category similarity bins for molecular profiles, compared to lower category similarity bins (fig. s c-i and table s . see methods, sm section for detailed information). in particular, we observed that in addition to the strongly positive correlations, the percentage overlap of disease pairs with shared genes was generally larger than the random controls ( fig. s c and d, see methods, sm section . ). the top disease pairs with the largest number of shared genes are all from chapter , which consists of cancer types. this might reflect the fact that cancers are the most studied and complex disease phenotypes involving various gene mutations (table s , see methods, sm section for detailed information). overall, these findings indicate that diseases in the same icd chapter tend to have a higher degree of shared genes, and the closer their positions in the icd tree, the higher is the degree of shared genes. we measured the maximum betweenness of disease-related genes in the protein-protein interaction (ppi) network to quantify the molecular diversity (the inverse of specificity) of each disease, as described previously ) (see methods, sm section ). a high maximum betweenness indicates a high molecular diversity. for example, c c c c c c c c c c c c c c the molecular diversity of alzheimer's disease could be represented by the maximum betweenness of its related genes (i.e., the betweenness of the app gene) in the ppi network (fig. s a) . we observed that the molecular diversity of diseases in the icd taxonomy is heterogeneous, with molecular diversity values varying from − to − with a median value of . e- ( fig. a and data s ). the top two disease chapters with the highest median molecular diversity were chapter ( . e- ) and chapter ( . e- ) (fig. b) . furthermore, we found that neoplasm (chapter ) and infectious disease (chapter ) categories tended to have higher molecular diversity compared to their complementary categories (neoplasms vs. non-neoplasms p b . e- , infectious diseases vs. non-infectious diseases p = . e- , fig. s b-c) and random controls. we also found that disease categories annotated as "other/unspecified" categories (sm section . ) had higher molecular diversity compared to disease categories with specific conditions (p = . e- , fig. s d , data s ; see sm section . ) and its random control. these results indicate that the diseases in neoplasms, infectious diseases, and "other/unspecified diseases" categories should be further investigated for molecular subtypes. a detailed discussion of disease cases is offered in sm section , data s & tables s -s . in the current icd taxonomy, we observed many instances where there exists a significant number of links between diseases in different chapters, comparable to the number of links between diseases within the same chapter (table s & fig. d , see methods & sm section ). for example, strong shared-gene relationships were detected between respiratory diseases (chapter ) and mental, behavioral, and neurodevelopmental disorders (chapter ) ( fig. c-d , more examples shown in sm section , tables s - ). in addition, by calculating the shared molecular connections between diseases in the context of chapters, we could detect diseases with a significant number of shared genes with diseases other than those in their own chapters (data s & sm section ). to further quantify the molecular boundaries between the disease categories in icd disease taxonomy, we evaluated the modularity, a structural measure of the tendency of the network to form close-knit communities (see methods, sm section . ), generated by either shared molecular profiles or shared phenotypes. when we mapped icd chapters as grouping annotations on the disease networks filtered by with appropriate weight thresholds, and calculated their modularity, we observed very low modularity values (fig. e) . since modularity is a widely used measure to validate the quality of partitions/module structures in complex networks, this means that the grouping of icd chapters does not agree with the natural topological groupings of their corresponding molecular networks (disease modules). this finding gives strong evidence for the blurred disease boundaries of the icd taxonomy, possibly arising from the complexity of the underlying molecular mechanisms, in particular the possible overlap of their respective subnetworks, or disease modules, in the interactome. furthermore, although the modularity of disease networks with shared phenotypes (similarity ≥ . ) is slightly positive, the weak correlation (pcc = . , p-value = . ) between phenotypic similarity and category similarity of disease pairs in each chapter (fig. f) indicates that icd taxonomy does not adequately incorporate phenotype similarity knowledge into disease category structures. these observations indicate that the strict tree structures in the icd taxonomy wherein terms can only have one lineage (cimino, ) may be inefficient for disease classification given the contemporary knowledge of disease pathobiology, and should therefore be refined to be polyhierarchical in structure. it has been proposed that if two disease modules overlap in the molecular interaction network, local perturbations in one disease might disrupt the biological pathways in the other disease, resulting in shared pathobiological characteristics . we observed a strong positive correlation between category similarity and module similarity (see methods, sm section . ) of diseases, indicating that two diseases with higher module similarity would be more closely localized in the disease category (fig. a, here, we utilize the module similarity between disease pairs to predict the categories of similar diseases. in particular, we determine the taxonomic closeness (sm section . ) of each given disease pair to predict the additional categories of diseases, by applying heuristic rules incorporating the positive correlation between category similarity and module similarity (see methods, sm section . ). in particular, using the , disease pairs with positive module similarity (data s ), we generated predicted additional category results for out of disease codes ( . %) in which each disease code had~ categories on average (data s & ). we found that the number of predicted categories positively correlated with the molecular diversity of the original disease codes (fig. c , pcc = . , % ci = [ . , . ], p b . e- ; for external validations see sm . ). this indicates that diseases with multiple pathogenic pathways could be captured by polyhierarchical mapping. for example, the diseases in chapter (i.e. diseases of the respiratory system) have been predicted to belong to over five additional chapters, such as neoplasms, infectious diseases, and diseases of the skin and subcutaneous tissue (fig. d) , which is consistent with the heterogeneous pathogenesis of copd and asthma (grainge et al., ; sharma et al., ) . a detailed discussion on the polyhierarchial map of the mental disorders is offered in sm section . (fig. s ) . furthermore, we found that the predicted category framework, which is based only on molecular module similarity, also had higher phenotype similarity than diseases with shared root codes in the original icd chapters (see sm section . , median: . vs. . ; mean: . vs . ; p b . e- , fig. e ). this observation helps to establish that the predicted category results are of higher quality than icd with respect to their phenotype homogeneity. to extend and redefine disease concepts by discovering additional categories of a disease, we generated a novel disease taxonomy by constructing an integrated disease network (idn) with: (a) shared clinical phenotypes including shared symptoms; (b) shared molecular profiles including (i) shared genes and molecular module similarity and (ii) shortest path lengths in the ppi network, based on a systematic integration process to filter out possible false positive associations (see methods, sm section , fig. s and fig. s a) , which includes diseases and , links (data s ). next, we applied high performance community detection algorithms to identify overlapping community structures in the idn fig. . lack of molecular specificity in icd taxonomy and the blurred boundary between disease categories in icd taxonomy. a. the distribution of molecular diversity of icd diseases; b. the boxplot of molecular diversity of icd chapters (ordered by median values); c. the disease network with shared genes in which the diseases belong to chapter and chapter . the icd codes , , in chapter have dense relationships to the icd codes in chapter ; d. the disease category network with shared genes. the nodes indicate the disease chapters and the weights of edges represent the edge densities between disease chapter pairs; the nodes with same color are considered as a chapter cluster, which is detected by community detection algorithm; e. modularity of disease networks with chapter as module annotations; f. the correlation between category similarity and phenotype similarity of icd chapters. (see methods, sm section and fig. s a ). in particular, we first used bigclam (see methods) since this method is able to detect overlapping communities whereby a disease can belong to multiple communities, in line with our main premise of creating a molecular based flexible disease classification. this resulted in disease subcategories with overlapping diseases as members ( fig. s a and data s ), which included distinct diseases from the icd taxonomy. these disease sub-categories contain different numbers of icd codes, ranging from to (fig. s ) , therefore, they represent different levels of disease categories similar to icd chapters and their sub-categories. to further develop a more unified view of the disease category quality, we used the established bgll method (blondel et al., ) , which detects non-overlapping communities, to cluster these subcategories further into non-overlapping, distinct parts, such that these represent the new chapter-level categories (called new chapters, or ncs) using the shared icd codes (see methods, sm section . , fig. s b ). overall, this clustering order effectively ensures distinct top-level categories that have overlapping subcategories. the resulting ncs contain different numbers of sub-categories ranging from to , or of diseases ranging from to (fig. s c) . we denote the ncs together with their disease sub-categories as our new overlapping disease classification (ncd). each of the resulting ncs reflects the shared features of integrative molecular and phenotypic profiles (sm section . ; fig. s c & table s , data s - ). for example, nc could be denoted as the "limbic system development-vision disorders-related diseases" since the most enriched ppi module (p = . e- , relevance ratio = . ) of its constituent diseases was mainly related to biological process; "limbic system development" (p = . e- ), and . % ( / ) of diseases in nc shared the phenotype, "vision disorders" (p = . e- ) (tables s -s ). to confirm the phenotypic and molecular cohesiveness of our overlapping disease categories, we compared the modularity of ncd with that of the icd taxonomy. we found that the ncs consistently have much higher modularity than the original icd chapters for all types of disease association networks (sm section . ; fig. a-h, fig. s ). this finding indicates that the phenotypic and molecular links between the diseases of a category in ncd are much denser compared to icd taxonomy. furthermore, to ensure that the performance of ncd is indeed due to the combined effect of the molecular and phenotypic profiles, we performed a controlled experiment where we determined the new disease categories based on molecular-based networks and phenotype-based networks only by running the entire analytical pipeline and applying the same category prediction algorithm (sm section . ). we found that ncd outperforms both molecular-based categories and phenotype-based categories in capturing the gene similarity, go term similarity and phenotypic similarity (figs. s -s ). this suggests the importance of integrating both clinical phenotypes and molecular profiles to obtain a high-quality disease taxonomy. furthermore, we found that the minimum shortest path lengths in the ppi network between disease pairs that belong to the same ncd categories had a larger percentage of low values (i.e., [ , ]) compared to icd (fig. i, . % vs . %, p b . e- ; sm section . ). this result indicates that diseases within an ncd category have a significantly higher degree of shared genes (or shorter path lengths) in comparison to diseases within a category in icd. on the other hand, the mspls between disease pairs in different ncd categories had a significantly lower percentage of low values than those in the icd ( . % vs . %, p b . e- , fig. i & fig. s polyhierarchical map of the disease codes in chapter , indicating that the disease codes in chapter have significant associations with two disease category clusters: ) chapter (infectious disease) and chapter (neoplasms); ) chapter (endocrine, nutritional and metabolic diseases and immunity disorders), chapter (mental disorders), chapter (nervous diseases), chapter (digestive diseases), chapter (skin and subcutaneous disease) and chapter (musculoskeletal system and connective tissue diseases); e. the boxplots of phenotype similarity of predicted disease pairs and original icd disease pairs in the same top-level chapters (p b . e- , wilcoxon test). which indicates a lower degree of shared genes (or shorter path lengths) between diseases from different categories in ncd than in icd. these findings demonstrate that our ncd framework has clearer boundaries between distinct diseases belonging to different categories than those in the original icd disease taxonomy. moreover, to validate the robustness of ncd predictions, we calculated the degree of associations in terms of network density among the diseases in each sub-category of ncd. to this end, we investigated the overlaps with the disease pairs connected by shared genes using two independent phenotype-genotype association databases, gwas and phewas (see methods, sm section . , . & ). we found that for the sub-categories in ncd, network density was significantly higher compared to random controls (gwas: p-value = . e- , fig. j ; phewas:p-value = . e- , fig. k ). this means that the diseases in the sub-categories in ncd would tend to have shared genes. for example, the new chapter: nc in ncd, including sub-categories and icd diseases (belonging to eight icd chapters), is enriched with respiratory and airway diseases (e.g. copd and asthma). we obtained overlapped diseases from the gwas database, which have a high degree of shared genes with the diseases in each sub-category of the nc (fig. a) . in particular, the sub-categories, such as nc .m (p-value = . e- ), nc .m (p-value = . e- ) and nc .m (p-value = . e- ) have significantly higher density than those of the whole gwas disease network (fig. s ) . furthermore, we found that the overlapping subcategories of the ncd are able to differentiate between different components (i.e. asthma/allergy vs. copd) of the same broad group of diseases (i.e. respiratory diseases) (see fig. s for a detailed example). indeed, in the nc disease chapter chiefly containing respiratory diseases, the two sub-categories, namely nc .m and nc .m , overlap in the underlying molecular interaction network while still containing the respective disease (asthma and copd, respectively) genes separately (fig. a) . a detailed discussion is offered in sm section (with results in data s - , tables s - & figs. s -s ). in addition, in ncd, a disease can be classified into multiple categories, and the number of categories of a disease positively correlates with its molecular diversity (fig. l , pcc = . , % ci = [ . , . ], pvalue b . e- ; external validations in sm . ). for example, we reclassified neoplastic diseases into multiple categories due to their high molecular diversity. two hundred and fifty-eight neoplastic diseases in our ncd were divided into sub-categories and ncs (figs. s & s ) . thirty-nine out of sub-categories ( . %) were enriched with "neoplasm" diseases (data s , p-value = . e- ). there were mainly ncs (i.e., nc , nc , nc , and nc ) containing these sub-categories and "neoplasm" disease codes (fig. b) , where . % ( / ) of the neoplastic diseases were classified into n sub-category, ranging from to (data s & table s ). the neoplasm with the highest molecular diversity, "malignant neoplasm of connective and other soft tissue" (icd: ; molecular diversity: . ), was reclassified into sub-categories, and "malignant neoplasms of thyroid gland" (icd: ; molecular diversity: . ) was assigned to sub-categories. furthermore, related diseases had been reclassified together in ncd, such as the well-known diseasecorrelations among h. pylori infection (icd: . ), stomach cancer (icd: ), and duodenal ulcer (icd: ) or peptic ulcer (icd: ) (fig. b) (sitas, ; graham, ) . more interestingly, some diseases, like viral hepatitis c (icds: arthritis (icds: / . ), each from different chapters in icd taxonomy, were classified together into a unique ncd sub-category (nc .m ) since % ( / ) of these diseases share a ppi module related to immune response (sm section . , fig. c , data s - , tables s - ). in addition, diseases originally in the same icd chapter, such as viral pneumonia (icd: ) and influenza (icd: ) from respiratory system-related diseases (chapter ), were reclassified into different categories in the ncd (nc , nc ). influenza shared more phenotype profiles with "episodic mood disorders" (icd: ) in nc .m , rather than viral pneumonia in nc (fig. s & data s ) , which is in accordance with recent epidemiological studies between episodic mood disorders and influenza (okusaga et al., ; canetta et al., ) , and, furthermore, we also found that influenza shared some molecular profiles with "episodic mood disorders" (icd: ) in nc .m (fig. s , data s ). these findings suggest that ncd offers a promising integrative framework incorporating both clinical phenotypes and molecular profiles for disease taxonomy that has very practical implications for the precise investigation of disease subtyping and etiologies. given the molecular network mechanisms (barabasi et al., ; zanzoni et al., ) , genetic pleiotropy (solovieff et al., ) , as well as complicated genotype-phenotype associations underlying diseases, the establishment of a molecular-based disease taxonomy with clear boundaries is essential but challenging. from the molecular network perspective, we first investigated the utility, shortcomings, and inconsistencies of icd- -cm, the established disease taxonomy for clinical settings. we found that there exist a considerable number (~ % of our investigated diseases) of diseases, for example, cancer and infectious diseases, that have diverse molecular network mechanisms and tend to interact more with diseases from other chapters. it is also these molecularly diverse diseases that mainly contribute to the blurred boundary of icd disease taxonomy (see methods, sm section & ). upon exploring the molecular diversity and cross-chapter interactions between diseases, we propose a novel disease classification system based on the integration of the clinical and molecular profiles of diseases. in particular, we integrate disease networks taking into account molecular and phenotypic connectivity among diseases, predict the multiple disease categories that diseases belong to, and finally validate the biological cohesiveness of our ncd by network topological measures such as modularity and shortest path length. our findings indicate that although general correlations exist between disease closeness in icd taxonomy and underlying molecular profiles, icd still displays significant limitations with regard to the heterogeneity of molecular diversity and clear category boundaries. in our ncd, a disease with a high molecular diversity tends to be classified into multiple disease categories, which indicates that there exist more disease subtypes for that disease. for example, "malignant neoplasm of the pancreas" was reclassified into sub-categories and ncs, which is consistent with a recent study wherein phenotypic subtypes of pancreatic cancer were enriched for distinct molecular mechanisms (bailey et al., ) . therefore, we believe that the new disease classification system may help facilitate precise clinical diagnosis and correct prognosis (jameson and longo, ) , and does so in alignment with refined molecular network diagnostics. furthermore, the molecular network underpinnings and overlapping disease categories of ncd provide a credible relationship map between diseases and disease categories that may radically transform our current understanding of diseases and relevant treatment paradigms. on the one hand, our approach accurately links diseases with all possible underlying mechanisms in the molecular interaction network. on the other hand, it presents a promising approach to the identification of targeted drugs for the treatment of related diseases. for example, breast cancer and influenza (both in nc .m ) may share potential drug targets (park, ) . as another example, metformin, widely prescribed to treat metabolic syndrome (in nc .m ), could alter the gut microbiome composition and function, improve gut microbial dysbiosis (forslund et al., ; cabreiro et al., ) , and also prevent colorectal cancer (also in nc .m ) through microbiomeinfluenced immune response modification (nakatsu et al., ) . here, it is important to note that while a considerable number of diseases have a strong environmental component, our main focus has been the many diverse molecular determinants. in the future, additional environmental factors such as epigenetic changes can be added into the data integration scheme to further refine the classification. there exist several potential limitations of this work. although we have aimed to address the possible confounders by constructing random controls and using external evaluations, the incompleteness and bias incorporated in the integrated data sources are likely to influence the generalization of our results. for example, diseaseconnect yields an incomplete disease-gene database: icd diseases could be mapped (table s ) , leading to only diseases included in the ncd. furthermore, as with other studies that rely on literature-based and ontological knowledge, investigation bias remains an issue, where the molecular mechanisms (e.g. related genes and their interactions) of some diseases (e.g. cancer) being more intensively studied than others may influence the results . we expect the results of similar works to be more refined in the future as biomedical datasets become more complete. the incorporation of more comprehensive disease-gene data sources, such as diseases (pletscher-frankild et al., ) and malacards (rappaport et al., ) , could result in an improved study. while we have chosen to keep individual external gene expression datasets outside the scope of this study since gene expression is highly tissue-and cell-type dependent, it presents an interesting future direction and could potentially improve the quality of the resulting disease categories if exhaustive lists of tissue-specific expression datasets are used in dedicated studies. in addition, our ncd merely delivers a two-level taxonomy framework without elaborated hierarchical structures in the same disease categories, which could be further refined or optimized through methods like hierarchical clustering algorithms (murtagh and contreras, ) and systematic posteriori ontology engineering method (gessler et al., ) . finally, high-quality ontologies, such as the human phenotype ontology (kohler et al., ) and disease ontology (kibbe et al., ) , can be used for external validation, or for further integration to obtain more robust and extensive ncds. in this big-data era, the dramatically increasing multi-omics databases, as well as clinical data from electronic health records (ehr) involving phenotypic, therapeutic and environmental factors information (jensen et al., ) , should also be incorporated into the new disease taxonomy refinement for patient stratification and disease treatment. at this point, a realistic assumption is that the translation of this classification to the clinic will need some time. that said, while fig. . biological insights of new disease taxonomy. a. the new chapter containing airway diseases (nc ) consists of sub-categories and icd diseases belonging to icd chapters. the subcategories overlap in the underlying molecular interaction network, while still separately including the disease genes (asthma and copd, respectively) that characterize each subcategory; b. the disease network of neoplasms in ncd. the sub-categories significantly representing neoplasms are divided into ncs (g ). helicobacter pylori [h. pylori] ( . ), malignant neoplasm of stomach ( ), duodenal ulcer ( ), peptic ulcer, site unspecified ( ), which have significant relationships, are clearly clustered into a subcategory (nc . m ) (g ); c. a sub-category (nc .m ) in ncd, which includes diseases from different icd chapters with shared molecular mechanism and phenotypes. fifty percent ( / = %) of the diseases in nc .m share a ppi module, the biological function of which is enriched with immune system response, while over % ( / = . %) of the shared common phenotype of this module is "pain". the icd is originally made "by clinicians for clinicians", it is now widely used by biomedical researchers as well to gain a deeper understanding of human diseases. we therefore believe that researchers will be the first and direct beneficiaries of our approach. in conclusion, our study provides valuable insights into the polyhierarchical network-based disease classification beyond the traditional tree structure. our integrated disease network approach is sufficiently powerful to elucidate the tangled underpinnings of human diseases and uncover distinct disease boundaries. our work may provide a new framework for the disease taxonomy reform based on bigdata fusion, so as to generate further the robust infrastructure needed for precision medicine. supplementary data to this article can be found online at https://doi. org/ . /j.ebiom. . . . the work was supported by national natural science foundation of china ( , and ), national science and technology major project for new drugs research and development of china ( zx - , zx - - ), national key r&d project ( yfc ), and the fundamental research funds for the central public welfare research institutes (zz , jbm , dut zd , dut zd , dut zd ). we also acknowledge the support by national institutes of health (nih) grants p - hg -cegs, mapgen grant (u hl ) and p hl , u hl , p hl , r hl , p hl - and rc hl . the funders had no role in study design, data 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( ) on helicobacter pylori and stomach cancer risk pleiotropy in complex traits: challenges and strategies the ucsc genome browser database: update classification of common human diseases derived from shared genetic and environmental determinants the nhgri gwas catalog, a curated resource of snp-trait associations an ancient, unified mechanism for metformin growth inhibition in c. elegans and cancer overlapping community detection at scale: a nonnegative matrix factorization approach a network medicine approach to human disease human symptoms-disease network the authors declare that they do not have any competing financial interests. key: cord- -v w wi c authors: rahmatpanah, farah; agrawal, sudhanshu; jaiswal, natasha; nguyen, hannah m.; mcclelland, michael; agrawal, anshu title: airway epithelial cells prime plasmacytoid dendritic cells to respond to pathogens via secretion of growth factors date: - - journal: mucosal immunol doi: . /s - - - sha: doc_id: cord_uid: v w wi c plasmacytoid dendritic cells (pdcs) are critical for defense against respiratory viruses because of their propensity to secrete high levels of type i interferons (ifn). the functions of pdcs in the lung can be influenced by airway epithelial cells. we examined the effect of human primary bronchial epithelial cells (pbecs) on pdc functions by performing rna-sequencing of pdcs after co-culture with air liquid interface differentiated pbecs. functional analysis revealed that pdcs co-cultured with pbecs displayed upregulation of type i ifn production and response genes. upregulated transcripts included those encoding cytosolic sensors of dna, zbp- ,irf- , and nfkb as well as genes involved in amplification of the ifn response, such as ifnar , jak/stat, isg . in keeping with the rna-seq data, we observe increased secretion of type i ifn and other cytokines in response to influenza in pdcs co-cultured with pbecs. the pdcs also primed th responses in t cells. the enhanced response of pdcs co-cultured with pbecs was due to the action of growth factors, gmcsf, gcsf, and vegf, which were secreted by pbecs on differentiation. these data highlight possible mechanisms to enhance the production of type-i ifn in the airways, which is critical for host defense against respiratory infections. plasmacytoid dendritic cells (pdcs) are a subset of dendritic cells, which are found in circulation and other organs. they are distinguished by their capacity to produce copious amounts of type i interferons (ifn) , via stimulation of toll like receptor and with nucleic acids or viruses. because of this property, they play a crucial role in fighting viral infections. , however, the cytokine secretion by pdcs is not restricted to ifns as they can also secrete other cytokines and chemokines including il- , il- , cxcl , cxcl , ccl , and ccl . similar to conventional myeloid dcs, they also express mhc class ii and costimulatory molecules and migrate to prime t cell responses. , production of type i ifns along with il- by pdcs primes cd + t cells and ifn-γ secreting th cd + t cells. pdcs can also induce t regulatory cells via induction expression of ido. , in addition, type i ifn production by pdcs enhances b cell activation, plasma cell generation and antibody secretion. tnf-related apoptosis inducing ligand (trail) and granzyme b serve as immunoregulatory factors that endow pdcs with the capacity to kill tumor cells, induce apoptosis of infected cd + t cells and suppress t cell proliferation. , pdcs also play an important role in pulmonary immune responses. pdcs in the lung have been reported to be immature, expressing low levels of mhc ii and costimulatory molecules but high levels of pdl- . these characteristics make them poor inducers of t cells and that is why they are thought to play an immune-regulatory role in the lung. depletion of pdcs in a mouse model of asthma enhanced the inflammation against harmless antigens. similarly, in acute lung injury (ali) and acute respiratory distress syndrome (ards), activated pdcs are protective and have been shown to prevent recruitment of pro-inflammatory monocytes into the lung. in contrast, pdcs were reported to be key drivers of immune-inflammatory cascade during asthma exacerbations via boosting of th responses. thus, pdcs can be inflammatory or tolerogenic depending on the context. activated pdcs are major drivers of protection against respiratory infections such as influenza and rsv by virtue of production of type i ifns and other cytokines. the functions of lung dc are influenced by many soluble factors such as growth factors, chemokines, and cytokines that are released from different cells within the lung. these soluble factors regulate the intensity and duration of immune response by stimulating or inhibiting activation and function of dc. for example, previous studies from our group and others have demonstrated that pbecs enhance the inflammatory and immune-surveillance capacity of mdcs. [ ] [ ] [ ] however, their effect on pdcs is not elucidated. this knowledge is essential for manipulation of pdc response to respiratory pathogens. here we examined the changes introduced in human pdcs by primary bronchial epithelial cells at gene and functional level. peripheral blood samples were obtained from healthy volunteers ( - years) , approved by the institutional review board of the university of california (irvine, ca, usa). written, informed consent was obtained. obtained from lonza inc. (basel, switzerland) and were differentiated at the ali on transwell plates as described. briefly, × pbecs per insert were seeded into -well transwell plate with apical chamber coated with the rat tail collagen (bd biosciences, san jose, ca, usa). cells were seeded in µl b-ali™ growth medium onto the collagen coated trans well inserts; µl of b-ali™ growth medium was added to the basal chamber of wells containing the inserts. on day after seeding, once the monolayer of pbec was confluent, media were removed from the apical chamber and allowed for air-liquid differentiation. the media in the bottom chamber were changed every two days. approximately days post-differentiation, pbecs were tested for the presence of cilia by staining for beta-tubulin and mucus secretion (data not shown). isolation and culture of human plasmacytoid (pdcs) with pbecs for rna sequencing pdcs were purified from the peripheral blood mononuclear cells (pbmcs) of young subjects by negative selection using a dc enrichment kit (stemcell sep, vancouver). enriched dc population was subsequently sorted using facsaria (bd biosciences) to obtain a pure pdc population. purity was above %. (cd +/cd c−) pdcs from different donors were added to the bottom chamber of the transwell with ali-differentiated pbecs for h. subsequently, the pdcs were collected for rna-seq and other studies. pdcs cultured in pbec medium without pbecs were used as controls. rna isolation from pdcs total rna was isolated from cultured pdc cells from four individuals using ffpe rna/dna purification plus kit (cat # , norgen biotek corp) with minor modifications. this kit is suitable for challenging samples such as formalin fixed paraffin embedded tissue in, which the recovery of quality rna is difficult. we applied this kit to isolate rna from pdcs cells since the numbers of cells were small. the rna quality and quantity was assessed using agilent bioanalyzer and qubit fluorimeter respectively. high yield and quality rna was obtained. the illumina truseq rna access library prep kit was used to study gene expression profiling in stimulated and unstimulated pdcs. the access method of illumina is based on sequence specific capture of coding rna. this method is suitable for wide range of rna quality and low rna input. briefly, ng of rna was used for library preparation. the total rna is fragmented into small pieces using divalent cation at o c. then cdna is synthesized using the cleaved rna fragments using random priming during the first and the second synthesis. sequencing adaptors are ligated to the dscdna fragments. the quality of libraries was assessed after the first pcr amplification. the coding regions of the transcriptome are then captured from the library using sequence specific probes to create the final libraries (as described by the manufacturer). samples were assayed after the second pcr amplification on the agilent bioanalyzer. qualification assay (qpcr) was performed using kapa sybr fast library quantification kit (illumina) universal qpcr mix ref # . samples were sequenced at × cycle at the uci high throughput genomic core facility (https://biochemghtfuci.wordpress.com/). sequencing reads were aligned to genome reference files from ensembl using strand ngs . (http://www.strand-ngs.com/) sequencing analysis package. approximately - % of mapped reads were aligned to protein coding regions as shown by the pie chart plot (supplementary fig. ). reads were filtered on duplicates to remove pcr artifacts. reads with better mapping quality and base average were retained (we set the number of duplicated reads to be permitted at " ") (excel supplementary data). deduplicated sequencing reads for each sample was quantified for gene expression, which is the measurement of the association of reads to genes and exons "raw reads". to normalize the raw reads we used reads per kilo million (rpkm) method in strand ngs program. we measured the variations in ′ to ′ end coverage along each transcript using gene body coverage analysis in strand ngs. uniform transcript coverage was observed among all samples. pooled analysis was performed using the audic claverie test (ac), which pools together raw counts across four pdcs and pdcs co-cultured with epithelial cells, separately, and tests the differences between them based on a poisson assumption on distribution of counts. multiple testing correction: benjamini hochberg fdr of . and the maximum p value cut off . was used (excel supplementary data). the genes of ac test were ranked based on p value adjusted (fdr corrected) and cut off was set at p adj < . to select for statistically significant differentially expressed genes between the pdcs co-cultured with epithelial cells and pdcs. the methods used for rna isolation and rna seq library construction worked with samples with small number of cells. for co-culture experiments purified pdcs were cultured without or with pbec differentiated at ali. after overnight co-culture, pdcs from both groups were collected and put together with purified cd t cells at a ratio of : (pdc:cd t) in serum free aim v medium (invitrogen) for days. subsequently, the cells were collected and stained with specific antibodies for cd , cd and foxp (rnd systems). gated cd cells were analyzed for the expression of cd and foxp using flow jo. supernatant collected was assayed for ifn-γ and il- by elisa. for co-culture experiments purified pdcs were cultured without or with pbec differentiated at ali. after overnight co-culture, pdcs from both groups were collected and stimulated with influenza (heat killed influenza a strain a/pr/ / -(flu, charles river, north franklin, ct) at µg/ml) for h. culture supernatants were collected h post stimulation and run on multiplex kit. the specific kit used was milliplex human -plex cytokine panel (cat# hcytmag- k-px ), which contains the following analytes: gm-csf, g-csf, ifnγ, il- α, il- rα, il- β, il- , il- , il- , il- , il- , il- , il- , il- , il- p , il- p , il , il , il- , mcp- , tnfα, tnfβ, eotaxin, type i ifn, ip- , mip- α, mip- β, egf, vegf, and rantes. the procedure followed was according to manufacturer's protocol. briefly, supernatant was mixed with premixed beads ( cytokines) overnight and after incubation with detection antibodies and streptavidin-pe for h each, the plate was run on magpix to identify specific cytokines. culture of pdcs with pbecs/growth factors purified pdcs were cultured with and without gmcsf ( ng/ml), g-csf ( ng/ml) or vegf ( ng/ml), il- ( ng/ml), il- ( ng/ml), mcp- ( ng/ml) (rnd systems) or a cocktail of these in various combinations and stimulated with influenza for h. supernatant collected was assayed for inflammatory mediators with multiplex as described above. culture of pdcs with blocking antibodies pdcs cultured without or with pbec were stimulated with influenza in the presence or absence of specific blocking antibodies against gmcsf (invitrogen), gcsf, vegf (rnd systems) or a mixture of the three antibodies at µg/ml. after h supernatant was collected and assayed for type i ifn, tnf-α, il- β, il- using multiplex. percent inhibition in the antibody treated groups was calculated compared to control group without antibody treatment. pbecs from three different subjects were used for this experiment. pbecs were allowed to differentiate at ali. the conditioned basal airway epithelial cells prime plasmacytoid dendritic cells to respond to. . . f rahmatpanah et al. medium in the bottom chamber was collected once the aec differentiation was complete. pbec differentiation media alone was used as control. multiplex detection of factors (same as above) was performed to determine the factors secreted by pbecs using milliplex. statistical analysis for cell culture experiments was performed using graphpad prism (graphpad inc., san diego, ca, usa). one way anova followed by tukeys multiple comparison test was used for the analysis. a p-value of < . was considered statistically significant. the effect of pbecs on pdcs gene expression changes was investigated using the transwell co-culture system. to investigate the effect of aecs in the form of pbecs on pdcs, the pbecs were cultured at ali till differentiation was achieved. pdcs purified from the blood were then added to the basal side of the monolayer to model the airways. , an aliquot of the pdcs was also cultured without pbecs but with the media. twenty-four later pdcs were collected and rna was extracted. pdcs from four different individuals were used. the description is provided in supplementary table . the viability was comparable between pdcs cultured with and without pbecs (data not shown). because of the low quantity of rna, we used the access method of library construction from illumina, which is based on sequence specific capture of coding rna. this method is suitable for wide range of rna quality and low rna input. paired end rna sequencing (rna-seq) was performed to determine the gene expression differences between pdc and pdcs co-cultured with epithelial cells. we observed a uniform reads coverage in the gene body for all rna seq data plot. we did not observe very large increases or decreases in transcript abundance, which was to be expected because pdcs were exposed to pbecs and there was no stimulation with a pathogen. pdcs in the airways are reported to induce tolerance via the expression of ido. , we found increased expression of ido- (fig. a) in the pdcs cultured with pbecs compared to pdc alone. in addition, the expression of costimulatory molecules, cd and cd was decreased. however, expression of other costimulatory molecules including, cd , ox , icos, icam was upregulated. the expression of the inhibitory costimulatory molecule, pdl- was also decreased. the pdcs therefore displayed a mixed phenotype in terms of costimulatory molecules. we performed functional analysis to confirm whether the pdcs were tolerized by pbecs. pdcs exposed to pbecs induced significantly increased levels of cd + cd + foxp + t regulatory cells to controls (p = . ), fig. b, c) . the secretion of ifn-γ was significantly decreased (p = . ) while il- was increased (p = . ) in the pdcs exposed epithelial cell group compared to control. these data suggest that pbecs enhance the tolerogenic capacity of pdcs at homeostasis. rna-seq data indicates pbecs enhance the production and response of type i ifn in pdcs pdcs in the airways are also important for host defense against infections because of their capacity to secrete high levels of type i ifns. our results indicate that the major pathways altered in pdcs in response to pbecs are the type i ifn related pathways such as the type i ifn signaling, interferon stimulated gene (isg ), cytosolic sensors of dna as well as jak-stat (fig. a-d) . in the type i ifn pathway, the expression of ifnar as well as jak-stat genes such as jak , stat , stat , which signify activation, are upregulated while socs -and , which inhibit the pathway, are downregulated ( fig. a-d) . furthermore pathways involved in the interferon response such as isg also displayed alterations after co-culture (fig. b) . among the innate sensors, the expression of cytosolic dna sensor, zbp- was upregulated on pdcs after co-culture with apcs (fig. c) . the expression of irf- and nfkb , which are required for signaling via this receptor, was also upregulated. in contrast, the negative regulators of the pathway, nfkb a, ikbkb are downregulated. interestingly, the majority of the genes in the prostaglandin pathway which is a major negative regulator of ifn production were downregulated (fig. e) . in addition to ifn related pathways, g-protein coupled receptor (gpcr) signaling pathway also displayed major changes in pdcs co-cultured with pbecs compared to pdcs (fig. f) . several of the chemokine receptor genes, such as ccr , ccr , ccr , ccr , cx cr , cxcr were upregulated indicating an activated state of pdcs. genes in the il- pathway also displayed changes (fig. g) . il- is known to promote pdc activation and survival. in summary, rna seq data suggests that the capacity of pdcs to produce and amplify type i ifn production is upregulated in pdcs after co-culture with pbecs. pbecs enhance the cytokine and chemokine response of pdcs to influenza to confirm the rna-seq data we performed functional assays to determine the alterations in the capacity of pdcs to produce type i ifn after exposure to pbecs. briefly, pdcs co-cultured with/without pbecs differentiated at ali were stimulated with influenza and cytokines and chemokines were assayed in the supernatants. conditioned pbec medium was used as control. pdcs exposed to pbecs secreted significantly higher levels of type i ifns (p = . ) on stimulation with influenza as compared to pdcs cultured alone (fig. a) , which is in keeping with the rna-seq data. tnf-α secretion was also significantly increased (p = . ) in influenza stimulated aec-exposed pdcs as compared to unexposed pdc (fig. a) . secretion of il- α (p = . ) and il- β (p = . ) was also increased significantly in pdcs co-cultured with pbecs with and without stimulation with influenza (fig. a) . no type i ifn, tnf-α, il- α, and il- β was detected in the conditioned medium. secretion of other mediators including anti-inflammatory cytokine, il- was below the limit of detection (data not shown). these data indicate that pbecs enhance the inflammatory response of pdcs to pathogens as suggested by the rna-seq data. to confirm that the aec-exposed pdcs were indeed primed to induce enhanced responses to influenza, the pdcs co-cultured with/without pbecs and influenza were cultured with purified t cells for five days and secretion of t cell specific cytokines was determined by multiplex. culture of t cells with pdc exposed to pbecs and stimulated with influenza induced significantly higher level of il- (p = . ) as compared to pdcs stimulated with influenza without aec exposure (fig. b) . enhanced il- secretion indicated increased proliferation. in keeping with the increased proliferation, the levels of tnf-α (p = . ) and ifn-γ (p = . ) secretion by these t cells was also significantly enhanced (fig. b) . other cytokines such as il- , il- , il- , il- were not induced by influenza primed pdcs and were comparable between the t cells cultured with pdc with and without pbecs (data not shown). these results suggest that pbecs enhance the inflammatory response of pdcs to influenza, which leads to increased induction of th responses. mechanisms used by pbecs to prime pdcs, a multiplex analysis was performed to determine the soluble factors in the conditioned medium from pbecs differentiated at the ali. the medium for differentiation was used as control. remarkably, out of mediators tested (see methods) we observed only six factors, which were significantly upregulated in the medium of ali differentiated pbecs (fig. ) . differentiated pbecs secreted significant levels of growth factors g-csf (p = . ), gmcsf (p = . ) and vegf (p = . ). secretion of cytokines, il- (p = . ) as well as chemokines, cxcl- (il- , p < . ), ccl- (p = . ) was also upregulated. ali differentiated pbecs might be priming the pdcs via secretion of the growth factors and the cytokines, chemokines. to identify the specific mechanism that leads to pdc priming in presence of pbecs, the effect of the factors found to be significantly enhanced in differentiated pbecs were explored. initial experiments were performed with three cocktails, growth factor and cytokine, chemokine cocktail (gmcsf, vegf, gcsf, il- , il- , ccl- ), growth factor cocktail (gmcsf, vegf and gcsf) and cytokine, chemokine cocktail (il- , il- , ccl- ). purified pdcs were cultured in the presence or absence of these cocktails and activated with influenza. culture of pdcs with combination cocktail as well as growth factor cocktail enhanced the secretion of type i ifn while the cytokine, chemokine cocktail had no significant effect (supplementary fig. ). further experiments were then done with growth factors. we tested the individual effects of the growth factors to determine whether this was due to a single factor or a synergistic effect of all factors. both gcsf (p = . ), gmcsf (p = . ) led to increase in type i ifn secretion by pdcs in the response to influenza (fig. a) . the increase by vegf was not significant (p = . ). the secretion of type i ifn was maximal when a cocktail of all three growth factors was used indicating a synergistic effect. both gmcsf and gcsf were also able to induce significantly increased (p = . -gmcsf; p = . -gcsf; fig. a ) levels of tnf-α. tnf-α was also significantly increased in the cocktail (p = . ). il- β displayed no significant increase with the growth factors as well as the cocktail suggesting that the growth factors do not induce il- β in pdcs. to further confirm the action of the growth factors, experiments using blocking antibodies against gmcsf, vegf and gcsf as well as a combination of all three antibodies were performed. pdcs cultured in the presence of pbec conditioned medium were stimulated with influenza in the presence of the antibodies and percent inhibition of type i ifn, tnf-α and il- β secretion by pdcs was calculated. as is evident from fig. b conditioned media and stimulated with influenza (data not shown). these data suggest that though gmcsf and gcsf can enhance the induction of type i ifn and tnf-α in pdcs, the synergistic action of all three factors is really what is responsible for the increase of these cytokines observed in pdc cocultured with pbecs. type i ifn secretion by pdcs plays a critical role in fighting viral infection at the respiratory mucosa. here we demonstrate for the first time that the capacity of pdcs to secrete type i ifn is enhanced by growth factors secreted by pbecs. previous studies suggest that pdcs may play a tolerogenic role in the respiratory mucosa via induction of t regulatory cells. , a very recent study in mice by lynch et al. has demonstrated that depletion of pdcs early in life enhanced features of asthma such as airway hyper-responsiveness, allergic inflammation and airway remodeling due to an impaired ability to expand tregs and maintain tolerance in the airways. our data indicates that exposure to pbecs induces tolerance in pdcs at homeostasis but concomitantly also primes pdcs to enhance their response to infections (figs. , ) . the induction of tregs by pdcs is enhanced which could be due to the increased expression of ido. the factors which lead to the increase in tolerogenic capacity of pdcs remain to be explored. in keeping with the activation genes in rna-seq, we also observed increased secretion of type i ifn from influenza stimulated pdcs co-cultured with pbecs. the expression of tlr and zbp was upregulated. zbp has recently been shown to be the innate sensor of influenza virus. it signals via irf and nfkb both of, which were upregulated in pdcs exposed to pbecs. the rna-seq data did not display significant changes in pathways known to induce ifn secretion in pdcs such as the irf . however, increased nfkb related genes may play a role in tlrdependent ifn production. , interestingly, pge has been reported to be a negative regulator of type i ifn secretion by human dcs upon stimulation with tlrs and prostaglandin pathway was downregulated in pdcs exposed to pbecs (fig. e) . the expression of another gene, fcɛriγ/fce a is significantly downregulated in pdcs co-cultured with pbecs and it has been demonstrated to inhibit tlr mediated type i ifn production in human pdcs via itam-mediated signals. similarly socs , a key negative regulator of myd -dependent type i ifn signaling , is also downregulated in pbecs exposed pdcs. in addition to negative regulators, pdcs co-cultured with also pbecs displayed increased expression of genes involved in type-i-ifn signaling. type i ifn can act both in an autocrine and a paracrine manner. it can amplify its own secretion via autocrine actions. increased expression of ifnar would enhance the autocrine action of ifns. therefore, pbecs seem to prime pdcs to enhance type i ifn secretion. this is supported by the increase in the genes of the isg pathway. isg is not only an interferon response pathway but is also reported to bind to irf- and enhance ifn responses. , furthermore, isg pathway can also affect important signaling pathways such as ifn, nfκb, and jnk pathways, all of, which are upregulated in pdcs after exposure to pbecs. together the gene expression changes suggest that pbecs enhance the capacity of pdcs to produce type i ifn via inhibiting the negative regulators as well as by enhancing the expression of molecules, which can amplify the production of type i ifns. we had observed similar priming of mdcs by pbecs earlier. , even though rna-seq data suggests that pdcs cocultured with pbecs express type i ifn at homeostasis, we were unable to detect type i ifn in our supernatants without stimulation with influenza. this could be due to lack of sensitivity of the elisa which may not be able to detect the various type i ifn subtypes. it is also possible that the type i ifn is bound to the receptor and therefore not detectable. high level of type i ifn secretion in the absence of infection is not desirable since its upregulation under such conditions is usually associated with autoimmunity. the tolerogenic nature of pdcs at homeostasis may therefore prevent secretion of the type i ifn to prevent inflammation. it is also possible that the changes visible at gene level are not transcribed to proteins as it is well established that only about % of genes are transcribed to proteins. this is especially true for cytokines. we also find that growth factors, gmcsf, gcsf, and vegf secreted by pbecs help prime pdc responses to influenza (fig. ) . interestingly, these factors signal via the jak-stat pathway, which displays significant upregulation in the rna-seq data (fig. d) . some of the growth factors have already been reported to modulate dc function. for example, knockdown of gcsf receptor in dcs has been reported to reduce the expression of at least proinflammatory cytokines and antigen presenting molecules, while the expression of pdl and pdl was increased. gmcsf is also considered as a dc activation agent and has been demonstrated to enhance the ifn-α production from pdcs in lupus patients. moreover, gmcsf secretion by kidney epithelial cells has also been reported to enhance maturation and type i ifn production in pdcs. additionally, gmcsf has been shown to synergize with tlr-dependent microbial stimuli to upregulate of proinflammatory cytokines. interestingly, bdca- , which functions as a marker for pdcs is a receptor on endothelial and tumor cells for vascular endothelial growth factor (vegf-a). studies examining the effect of vegf on lung dc subtypes in vegf-transgenic mice have reported an increase in lung mdc and pdc populations. an increase in inflammatory responses of mdc was also reported in the same study. the effect of vegf on pdc responses has not been examined. our results indicate a possible synergistic effect of vegf with other growth factors on type i ifn production by pdcs. the present study only examines effect of soluble factors from pbecs on pdc functions. information regarding cell to cell contact between pbecs and pdc is missing which can also play an important role in modulation the functions of pdcs. this is because existing technology only allows the differentiation of pbecs at ali on a transwell insert which prevents contact with pdcs. development of systems which can allow pbec differentiation on surfaces which are capable of providing cell to cell contact would be very valuable in studying such interactions. in summary, rna-seq data indicates that exposure of pdcs to pbecs both tolerizes and activates pdcs. pbecs exposed pdcs induce increased levels of tregs at homeostasis. however, the pdcs are also primed by pbecs to respond to infections. in keeping with the rna-seq data, we observe increased secretion of type i ifn and other cytokines in response to influenza in pdcs cocultured with pbecs. the pdcs also primed th responses in t cells. the enhanced response of pdcs co-cultured with pbecs was due to the action of growth factors, gmcsf, gcsf and vegf, which were secreted by pbecs on differentiation. to our knowledge this is the first report, which investigates the crosstalk between human pdcs and pbecs. these data highlight possible mechanisms to enhance the production of type-i ifn in the airways, which is critical for host defense against respiratory infections. plasmacytoid dendritic cells: sensing nucleic acids in viral infection and autoimmune diseases unraveling the functions of plasmacytoid dendritic cells during viral infections, autoimmunity, and tolerance the dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting the multifaceted biology of plasmacytoid dendritic cells essential role of lung plasmacytoid dendritic cells in preventing asthmatic reactions to harmless inhaled antigen plasmacytoid dendritic cells control lung inflammation and monocyte recruitment in indirect acute lung injury in mice plasmacytoid 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eprostanoid receptor engagement plasmacytoid dendritic cell-specific receptor ilt -fc epsilonri gamma inhibits toll-like receptor-induced interferon production socs regulates the ifn but not nfkappab pathway in tlrstimulated human monocytes and macrophages cross-regulation of two type i interferon signaling pathways in plasmacytoid dendritic cells controls anti-malaria immunity and host mortality self-priming determines high type i ifn production by plasmacytoid dendritic cells proteomic identification of proteins conjugated to isg in mouse and human cells positive regulation of interferon regulatory factor activation by herc via isg modification beyond isglylation: functions of free intracellular and extracellular isg isg enhances the innate antiviral response by inhibition of irf- degradation dendritic cell-airway epithelial cell cross-talk changes with age and contributes to chronic lung inflammatory diseases in the elderly gcsf receptor regulates antigen uptake and expression of cytokines and costimulatory molecules in dendritic cells systematic cytokine receptor profiling reveals gm-csf as a novel tlr-independent activator of human plasmacytoid predendritic cells regulation of the interferon-alpha production induced by rna-containing immune complexes in plasmacytoid dendritic cells human plasmacytoid dendritic cells acquire phagocytic capacity by tlr ligation in the presence of soluble factors produced by renal epithelial cells cutting edge: granulocyte-macrophage colony-stimulating factor is the major cd +t cell-derived licensing factor for dendritic cell activation bdca- , bdca- , and bdca- : three markers for distinct subsets of dendritic cells in human peripheral blood lung vascular endothelial growth factor expression induces local myeloid dendritic cell activation this study was supported by grant from the nih ag (to aa), and from the national center for research resources and the national center for advancing translational sciences # ul tr . the content is solely the responsibility of the authors and does not necessarily represent the official views of the nih. we are grateful to icts uc irvine for providing the blood samples. the online version of this article (https://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users.competing interests: the authors declare no competing interests. key: cord- -yu mw authors: chen, pei; chen, ely; chen, luonan; zhou, xianghong jasmine; liu, rui title: detecting early‐warning signals of influenza outbreak based on dynamic network marker date: - - journal: j cell mol med doi: . /jcmm. sha: doc_id: cord_uid: yu mw the seasonal outbreaks of influenza infection cause globally respiratory illness, or even death in all age groups. given early‐warning signals preceding the influenza outbreak, timely intervention such as vaccination and isolation management effectively decrease the morbidity. however, it is usually a difficult task to achieve the real‐time prediction of influenza outbreak due to its complexity intertwining both biological systems and social systems. by exploring rich dynamical and high‐dimensional information, our dynamic network marker/biomarker (dnm/dnb) method opens a new way to identify the tipping point prior to the catastrophic transition into an influenza pandemics. in order to detect the early‐warning signals before the influenza outbreak by applying dnm method, the historical information of clinic hospitalization caused by influenza infection between years and were extracted and assembled from public records of tokyo and hokkaido, japan. the early‐warning signal, with an average of ‐week window lead prior to each seasonal outbreak of influenza, was provided by dnm‐based on the hospitalization records, providing an opportunity to apply proactive strategies to prevent or delay the onset of influenza outbreak. moreover, the study on the dynamical changes of hospitalization in local district networks unveils the influenza transmission dynamics or landscape in network level. despite current approaches to prevention and control, seasonal influenza remains a significant cause of morbidity and mortality worldwide. being infected by influenza virus, people especially elderly and children are at a high-risk for further deterioration including circulatory diseases, severe respiratory illness, and other life threatening complications. , influenza pandemic also causes considerable economic burden including direct medical costs and indirect loss such as substantial workplace absenteeism. the estimated average direct medical costs of influenza in the united states reaches $ . billion each year, and the actual annual cost would be more. early detection and recognition of upcoming influenza outbreak, and timely public health prevention including vaccination schedule and control strategy, are critical in reducing the pandemic magnitude and distribution. , however, it is usually a challenging task to achieve the real-time prediction of influenza outbreak due to its complex dynamics involving both biological systems and social systems. in addition, surveillance capacity for such detection can be costly, and many countries lack the public health infrastructure to identify outbreaks at their earliest stages. furthermore, there may be economic incentives for countries to not fully disclose the nature and extent of an outbreak. , therefore, a new computational method is required to predict the outbreak of epidemic diseases only based on available data, thus simplifying information gathering and monitoring processes. the dynamic network marker/biomarker (dnm/dnb) is our recently proposed method. it is a generalized methodology to identify the tipping point or pre-transition state which is a critical state before the catastrophic event, , by mining the dynamical information from both horizontal high-dimensional data and longitudinal historical records. regarding the influenza outbreak as a tipping point at which the system undergoes a critical transition, then there is a common understanding that the dynamical process of the system can generally be expressed by three states ( figure b) , that is, a normal state with high resilience, a pre-outbreak state (the critical state) with low resilience, and an after-outbreak state with possible high resilience. the normal state is a steady stage, during which there are no many clinic visiting patients. the pre-outbreak state is defined as the limit of the normal state immediately before the tipping point. in this pre-outbreak stage, the process is usually reversible to the normal state if appropriately treated, implying the criticality of the preoutbreak state. unlike the traditional detection of the after-outbreak state, the dnm enables the identification of the pre-outbreak state or critical state that generally has no clear abnormalities but with future trending of deterioration or critical transition. this method has recently been successfully applied to a variety of biological progresses to detect the early-warning signals to an irreversible catastrophic stage, such as the cell differentiation process, the process of cell fate decision, the critical transition in the immune checkpoint blockade-responsive tumour, the multi-stage deteriorations of t d, acute lung injury, hcv induced liver cancer, cancer metastasis, and many others. [ ] [ ] [ ] [ ] in this study, dnm method was employed to explore the dynamical information based on a combination of city network and the high-dimensional clinic hospitalization records, which are from over clinics distributed in wards in tokyo, japan, and clinics distributed in districts in hokkaido, japan. the results show that the dnm method successfully identified the critical state just before the outbreak of influenza as a realtime surveillance system. such a system may enable a rapid response for the preventive care or the implementation of interventions to a health epidemic. in addition, this work unveils the influenza transmission dynamics or landscape in a local district network level, based on the measured data. the advantage and effectiveness of the dnmbased system is also demonstrated by the comparison between dnm and other surveillance systems of flu pandemic. influenza viruses circulate around the world every year, causing financial losses, suffering, and death. the dynamical process of flu outbreak can be modeled by three states or stages ( figure ) similar to disease progression : the before-outbreak state, which is a stable state with high resilience or high robustness to perturbations; the pre-outbreak/critical state, which is the tipping point just before the catastrophic shift into the outbreak state and is thus characterized by low resilience or low robustness due to its critical dynamics, but is still reversible to the before-outbreak state with appropriate control management; and the outbreak state, which is another stable state with high resilience or high robustness. clearly, it is of great importance to identify the pre-outbreak state, which holds the key to apply effective control management to prevent the catastrophic flu outbreak. however, different from the outbreak state in which there are obvious signs including huge amount of outpatient visits, it is a difficult task to identify the pre-outbreak state because there are generally no significant signs or differences between the before-outbreak state and the pre-outbreak state. on the other hand, the dynamic network marker/biomarker (dnm/dnb) method was developed to quantitatively identify the tipping point or the critical state during the dynamic evolution of a complex system based on the observed data. theoretically, when a complex system is near the critical point, there exists a dominant group (a dominant group of variables or members) defined as the dnm features, which satisfy the following three necessary conditions based on the observed data : • the correlation (pcc in ) between any pair of members in the dnm group rapidly increases; • the correlation (pcc out ) between one member of the dnm group and any other non-dnm member rapidly decreases; • the standard deviation (sd in ) or coefficient of variation for any member in the dnm group drastically increases. in other words, the above conditions can be approximately stated as: the appearance of a strongly fluctuating and highly correlated group of features implies the imminent transition into the flu outbreak. then, these three conditions are adopted to quantify the tipping point as the early-warning signals of diseases, and further, the identified dominant group of features consists of dnm members. the -fold change threshold is usually applied to recognize the significant changes in dnm score and obtain the warning signal. the dnm theory has been applied to a number of analyses of disease progression and biological processes to predict the critical states as well as their driven factors. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in this work, by considering the flu outbreak process as a non-linear dynamics process, we further applied the dnm method to detect the tipping point or the earlywarning signal of flu outbreak. to quantify the critical state, the following criterion i dnm was used as the signal of the critical point by combining the above three statistical conditions: thus, from the observed data of a sample, whenever there is a group of features appearing with a high i dnm score, this group of features is the dnm group and the state of this sample is considered to be near the tipping point. therefore, from the hospitalization records of each sample, we can identify the dnm members and further quantify whether or not this sample is near the critical state using the i dnm score. to further reliably identify the critical state of flu outbreak, we developed a new method called the landscape dnm, which explores f i g u r e schematic illustration to detect early-warning signals of influenza outbreak based on the dnm method. a, the historical information of clinic hospitalization caused by influenza infection between january and december was extracted and assembled from public records of tokyo and hokkaido, japan. b, according to the dnm theory, the process of a time-dependent non-linear system is divided into three states, including a normal state, a pre-outbreak state and an after-outbreak state. the abrupt increase in the dnm score indicates the pre-outbreak state, ie, the tipping point just before the upcoming catastrophic influenza outbreak that results in a boost of clinic-visiting patients. c, based on the historical and current clinic records, and regional geographic characteristics of a city, the dnm score is able to provide the early-warning signals of the upcoming influenza outbreak as a real-time indicator monitoring both the local and global records as well as the network structure, and the detailed algorithm is provided below. given a network structure for the observed variables, an efficient method to detect dnm, called the landscape dynamic network marker (or landscape dynamic network biomarker), is proposed by employing the local-landscape method on the basis of the three dnm statistic properties. specifically, first we mapped the historic records of flu patients to the city network ( figure a) . second, the network was partitioned into many local networks. each local network contained a centre node/ward and all of its first-order neighbours based on the network structure. the local-network index iscore of a centre node at time point t for a local network with n members (ie, one centre node with n- first-order neighbouring nodes) was then calculated through the following definition: where jΔsd t ðinÞj ¼ ∑ n i¼ jsd t ðiÞ À sd tÀ ðiÞj n is the average differential standard deviation (in absolute value) of the nodes inside the local network; ;j¼ jpcc t ði; jÞ À pcc tÀ ði; jÞj n  n is the average differential pearson's correlation coefficient (in absolute value) inside the local network, i.e., both nodes i and j are in the same local network; jΔpcc t ðoutÞj ¼ ∑ n i¼ ;j¼ jpcc t ði; jÞ À pcc tÀ ði; jÞj n  n is the average differential pearson's correlation coefficient (in absolute value) between a member (node i) in the local network and that (node j) outside. theoretically, when the system approaches the tipping point, ie, t ∈critical state, and t- ∉ critical state, there are three cases for the local network of a centre node: • in the local network, all the nodes (or nodes) are dnm members; • in the local network, there are dnm and non-dnm members; • in the local network, all the nodes are non-dnm members. according to the three cases respectively, there are critical behaviours for a centre node shown as in table . thus, the network based index, i t , can quantitatively characterize the criticality of the state for each dnm member or node. clearly, each node has an i t value, and hence those i t scores for all of nodes with the time evolution construct a landscape as shown in figure . when the system approaches the critical state, i t of each dnm node increases drastically based on the three statistic conditions of dnm, while i t of other non-dnm node may have no significant change. obviously, during the critical transition, the dnm group has an abil- for each ward or district, the raw data were averaged in terms of the total number of clinics within the ward/district. this normalization process is directly related to the population of each ward/district, since the population is roughly proportional to the number of clinics. the raw data were processed through window shift where window breadth is set as , that is, both the standard deviation and correlation coefficient are calculated based on the data within every weeks. the flu transmission dynamics before sudden outbreak is usually too complicated to be fully expressed mathematically in high-dimensional spaces involving both biological systems and social systems. the drastic or a qualitative transition in a local system or network, from a normal state to an after-outbreak state, corresponds to a so-called bifurcation point in dynamical systems theory. , if the system is approaching a bifurcation point, it will eventually be constrained to a one-or two-dimensional space (ie, the centre manifold in a generic sense), in which a dynamical system can be expressed in a very simple form. this is the theoretical basis for developing a general indicator that can detect the critical state of flu outbreak only based on the observed data. as shown in figure n→ notation: the system is near a tipping point, ie, it moves from time point t- to t, with t ∈ critical state, and t- ∉ critical state. . "↗" represents the increase of the index; "↘" represents the decrease of the index; "→" represents that there is no significant change in the index. . "d" stands for the dnm members, or the pcc with dnm members; "n" stands for the non-dnm nodes, or the pcc with non-dnm members. . sd t is the average standard deviation at time t; pcc t (in) is the average pearson's correlation coefficient between two nodes inside the local network; pcc t (out) is the average pearson's correlation coefficient between a node inside the local network and a node outside. (table s ). the dynamical evolution of network shows that the dnm-based system uncovers the epidemic situation and transmission trends, which better present the transmission dynamics at a system network level. as another dnm application to the influenza outbreak, we also applied the dnm to detect the early-warning signals against flu outbreak in hokkaido region, which is shown in figures s -s . it is seen from figure s figures s and s ). figure s shows the dynamics of the region network of year in terms of local dnm scores. the performances of dnm score is compared to other systems using machine learning algorithm ( figure ). specifically, a popular surveillance system of flu pandemic is based on logistic regression. [ ] [ ] [ ] it is clear from figure that given only hospitalization records, the dnm-based system performs better than a system based on logistic regression. actually, the dnm method has natural advantage comparing with traditional machine learning algorithm in the following aspects. f i g u r e the performance of dnmbased and machine-learning-based methods. it is seen that using only the hospitalization records, the dnm-based surveillance system performs better than the logistic regression. the auc of dnm is . while that of logistic regression is . . the performance comparison is carried out based on the data of tokyo. note that the dnm-based method generally has no overfitting problem due to the three statistic conditions of dnm without the training data, in contrast to the machine-learning-based methods that depend on the training data influenza-related mortality trends in japanese and american seniors: evidence for the indirect mortality benefits of vaccinating schoolchildren influenza and its relationship to circulatory disorders estimates of deaths associated with seasonal influenza-united states the annual impact of seasonal influenza in the us: measuring disease burden and costs simulation suggests that rapid activation of social distancing can arrest epidemic development due to a novel strain of influenza our strategies for fighting severe acute respiratory syndrome (sars) early detection of disease outbreaks using the internet official versus unofficial outbreak reporting through the internet detecting early-warning signals for sudden deterioration of complex diseases by dynamical network biomarkers early diagnosis of complex diseases by molecular biomarkers, network biomarkers, and dynamical network biomarkers single-cell-based analysis highlights a surge in cell-to-cell molecular variability preceding irreversible commitment in a differentiation process cell fate decision as highdimensional critical state transition dynamic versus static biomarkers in cancer immune checkpoint blockade: unravelling complexity detecting tissue-specific early warning signals for complex diseases based on dynamical network biomarkers: study of type diabetes by cross-tissue analysis identifying critical transitions of complex diseases based on a single sample identifying critical transitions and their leading biomolecular networks in complex diseases dynamic network biomarker indicates pulmonary metastasis at the tipping point of hepatocellular carcinoma detecting early-warning signals of type diabetes and its leading biomolecular networks by dynamical network biomarkers detecting critical state before phase transition of complex biological systems by hidden markov model detecting the tipping points in a three-state model of complex diseases by temporal differential networks identifying critical differentiation state of mcf- cells for breast cancer by dynamical network biomarkers mathematical biology catastrophe theory for scientists and engineers an analysis of the spatial and temporal patterns of highly pathogenic avian influenza occurrence in vietnam using national surveillance data predicting influenza infections during epidemics with use of a clinical case definition environmental factors contributing to the spread of h n avian influenza in mainland china additional supporting information may be found online in the supporting information section at the end of the article. how to cite this article the work was supported by the national key r&d program of the authors declare that they have no competing interests. rl and ec conceived the project; rl, lc, and xz supervised the project. ec, pc, and rl performed computational and analysis. all authors wrote the manuscript. all authors read and approved the final manuscript. key: cord- -tkqxb ql authors: toman, miroslav; celer, vladimir; kavanová, lenka; levá, lenka; frolichova, jitka; ondráčková, petra; kudláčková, hana; nechvátalová, kateřina; salat, jiri; faldyna, martin title: dynamics and differences in systemic and local immune responses after vaccination with inactivated and live commercial vaccines and subsequent subclinical infection with prrs virus date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: tkqxb ql the goals of our study were to compare the immune response to different killed and modified live vaccines against prrs virus and to monitor the antibody production and the cell mediated immunity both at the systemic and local level. in the experiment, we immunized four groups of piglets with two commercial inactivated (a —progressis, a —suivac) and two modified live vaccines (b —amervac, b —porcilis). twenty-one days after the final vaccination, all piglets, including the control non-immunized group (c ), were i.n., infected with the lelystad strain of prrs virus. the serum antibody response (igm and igg) was the strongest in group a followed by two mlv (b and b ) groups. locally, we demonstrated the highest level of igg antibodies in bronchoalveolar lavages (balf), and saliva in group a , whereas low iga antibody responses in balf and feces were detected in all groups. we have found virus neutralization antibody at dpv (days post vaccination) and higher levels in all groups including the control at dpi (days post infection). positive antigen specific cell-mediated response in lymphocyte transformation test (ltt) was observed in groups b and b at dpv and in group b at dpv and in all intervals after infection. the ifn-γ producing lymphocytes after antigen stimulation were found in cd (−)cd (+) and cd (+)cd (+) subsets of all immunized groups days after infection. after infection, there were obvious differences in virus excretion. the virus was detected in all groups of piglets in serum, saliva, and occasionally in feces at dpi . significantly lower virus load was found in groups a and b at dpi . negative samples appeared at dpi in b group in saliva. it can be concluded that antibodies after immunization and infection, and the virus after infection can be detected in all the compartments monitored. immunization with inactivated vaccine a —progressis induces high levels of antibodies produced both systemically and locally. immunization with mlv-vaccines (amervac and porcilis) produces sufficient antibody levels and also cell-mediated immunity. after infection virus secretion gradually decreases in group b , indicating tendency to induce sterile immunity. the goals of our study were to compare the immune response to different killed and modified live vaccines against prrs virus and to monitor the antibody production and the cell mediated immunity both at the systemic and local level. in the experiment, we immunized four groups of piglets with two commercial inactivated (a -progressis, a -suivac) and two modified live vaccines (b -amervac, b -porcilis). twenty-one days after the final vaccination, all piglets, including the control non-immunized group (c ), were i.n., infected with the lelystad strain of prrs virus. the serum antibody response (igm and igg) was the strongest in group a followed by two mlv (b and b ) groups. locally, we demonstrated the highest level of igg antibodies in bronchoalveolar lavages (balf), and saliva in group a , whereas low iga antibody responses in balf and feces were detected in all groups. we have found virus neutralization antibody at dpv (days post vaccination) and higher levels in all groups including the control at dpi (days post infection). positive antigen specific cell-mediated response in lymphocyte transformation test (ltt) was observed in groups b and b at dpv and in group b at dpv and in all intervals after infection. the ifn-γ producing lymphocytes after antigen stimulation were found in cd − cd + and cd + cd + subsets of all immunized groups days after infection. after infection, there were obvious differences in virus excretion. the virus was detected in all groups of piglets in serum, saliva, and occasionally in feces at dpi . significantly lower virus load was found in groups a and b at dpi . negative samples appeared at dpi in b group in saliva. it can be concluded that antibodies after immunization and infection, and the virus after infection can be detected in all the compartments monitored. immunization with inactivated vaccine a -progressis porcine reproductive and respiratory syndrome (prrs) is the most economically significant infectious disease currently affecting swine worldwide. typical clinical symptoms of prrs are mild to severe respiratory disease in infected newborn and growing pigs, and reproductive failure in pregnant sows. two genotypes of the prrs virus (prrsv) have been identified: european (type ) and north american (type ). there are considerable genetic and virulence differences between and within prrsv genotypes ( - ) correlated with a lack of cross-protection by vaccines ( ) ( ) ( ) ( ) ( ) . highly pathogenic strains of prrsv (hp-prrsv) have been identified within both genotypes ( ) ( ) ( ) . depending on viral strain and immune status of the host, some swine farms may have pigs subclinically infected, whereas others experience severe reproductive, and/or respiratory disease. infection with both "classical" and highly pathogenic strains is associated with aberrant host immune response ( , ) . swine are the only known natural host of prrsv and the primary target cells for replication of prrsv are porcine alveolar macrophages (pams) ( ) . the first stage is represented by acute infection, resulting in viremia - h post-infection (pi), and lasting for several weeks despite the presence of circulating antibodies. in the second, persistent stage of infection, the virus is no longer detected in blood and lungs, and pigs no longer exhibit signs of clinical disease. in this stage, viral replication is primarily localized in lymphoid organs, including tonsils, and lymph nodes ( ) . infection with prrsv elicit poor innate and adaptive immune responses associated with immune modulation and incomplete viral clearance in most of the pigs, depending on their age, and immune status ( , ( ) ( ) ( ) . infection with certain prrsv strains induced significant suppression of nk cell cytotoxic activity ( ) . the quantity of pro-inflammatory cytokines is significantly lower than in other viral infections and is strain dependent ( ) . prrsv is also a poor inducer of ifn-α. infection with prrsv induces an antibody response (production) by - dpi but with no evidence of protection against prrsv infection; serum neutralizing antibodies appear only later, typically ≥ days pi ( ) . the virus also evades host cell-mediated immunity most likely by the promotion of immunosuppressive cytokines il- and tgf-β resulting in delayed onset of th immune response ( ) . similarly, an immunosuppressive function of prrsv was shown to probably be mediated by the cytokines il- and tgf-β and action of treg ( ) ( ) ( ) . immunosuppression induced by prrsv facilitates other viral and bacterial infections ( , , ) . vaccination is the principal means used to control and treat prrsv infection. several comprehensive review articles have been published recently. they critically evaluate different vaccination approaches against the prrs virus and indicate the main weaknesses of current vaccines and vaccination strategies ( ) ( ) ( ) ( ) . among others the problem are caused by high heterogeneity and occurrence of highly pathogenic strains and therefore efforts have been made to develop vaccines with a broad spectrum of effects ( , , , ( ) ( ) ( ) ( ) . however, the opinion still prevails that vaccination is more cost-beneficial over other health interventions ( ) ( ) ( ) . our study had the following three aims: ) to establish complex immune response characteristics using several methodological approaches; ) to monitor the dynamics in different compartments and in a time-dependent manner after vaccination and the challenging infection; ) to compare the types of immune responses after vaccination with inactivated or live attenuated vaccines and subsequent challenge using a homologous strain. twenty-five weaned piglets aged weeks and weighing - kg of the large white breed from a prrsv negative herd were used. the negative status of the animals was confirmed by serology using commercial elisa kit (idexx labs). the use of animals was approved by the branch commission for animal welfare of ministry of agriculture of the czech republic (approval protocol no. mze- ) as a part of project as a part of project respig (qj ). four commercial vaccines were used. their characteristics are in table . the virus was clarified by centrifugation, and its concentration was determined by plaque assay. the concentration of stock virus used in experiments was × plaque forming units per ml. twenty-five piglets were used in the experiment. the piglets were assigned to five groups of five animals each according to weight and gender. the animals were housed in bsl isolation rooms, keeping animals from only one experimental group in each room. the animals were left to acclimate for days after stocking. all piglets were clinically healthy at the time the experiment started. on day (d ) two groups of piglets (a and a ) were immunized. each animal was administered ml of inactivated vaccine by an intramuscular (i.m.) injection. after days (d ), piglets in these groups were revaccinated with the same dose, and piglets from the other two groups (b and b ) were immunized with ml of a mlv vaccine. the health status of piglets was monitored on a regular basis, including temperature measurements, and samples of blood and other body fluids were taken for respective examinations at preset time intervals. after an additional days (d ), all pigs, including control group (c ), were infected with ml of the live prrs virus. the piglets were monitored for another days and then slaughtered (d ). euthanasia was performed by exsanguination after combined anesthesia with a tkx (telazol-ketamin-xylazin) mixture containing . mg/ml tiletamine and . mg/ml zolazepam (telazol, virbac, carros, france), . mg/ml ketamine (vetoquinol, lure, france), and . mg/ml xylazine (bioveta, ivanovice na hane, czech republic), administered intramuscularly in a final volume of . ml/kg body weight. as well as collection of blood and other body fluids (intestinal contents, bronchoalveolar lavage), an autopsy was performed and organs (lung parenchyma, spleen, lymph nodes,...) were collected for virological examination. blood samples for serum and heparin-treated blood samples were taken from the jugular vein. group saline samples were collected using ropes which were left in the hutch for h. individual fecal samples were collected when handling the animals. bronchoalveolar lavage fluid (balf) sampling was performed for the first time on live animals and for the second time after slaughter. the intravital lavage was performed with the animals under general anesthesia (a mixture of xylazine and ketamin) without the use of an endoscope by a method described earlier ( ) . pigs were positioned in the sternal recumbency. an endotracheal tube was inserted into the trachea and ml of sterile pbs (ph . ) was injected into the distal parts of the airways, toward the bronchus. about % of the infused saline was recovered as balf aspirate and was filtered and centrifuged for min at g. supernatant was stored at − • c prior to serological analyses. total rna from experimental samples of sera, oral fluids, and bal ( µl) was extracted using a nucleospin r rna ii kit (macherey-nagel), in accordance with the manufacturer's instructions (protocol for total rna preparation from biological fluids). the rna obtained was eluted in µl rnase-free water and immediately used for qrt-pcr amplification. remaining rna was frozen at − • c for subsequent use. isolated rna was used for qrt-pcr amplification by ez-prrsv tm mpx . real time rt-pcr kit (tetracore), in accordance with the manufacturer's instructions. quantification of the virus genome copies was based on quantification standards included in the kit. for the evaluation of systemic and local antibody production two elisa methods were used. all swine sera tested were examined by commercially available elisa test ingezim prrs universal (ingenasa), in accordance with the manufacturer's instructions, to examine for the presence of n protein specific igg antibodies. all swine sera, oral fluids, and bal tested were examined by home-made indirect elisa test based on recombinant nucleocapsid protein n of prrs virus (developed previously in our department) for detection of specific igm, igg, and iga antibodies. optimal antigen, serum, and antibodies concentrations were determined by checkerboard titration of positive and negative porcine sera. the cut-off value was determined by defining the upper prediction limit based on the upper tail of the t-distribution of negative control od readings, at a confidence level of . %. positive serum with an absorbance corresponding to the calculated cut-off was included in all test plates. the recombinant n protein diluted in mm bicarbonate-carbonate buffer ph . to a final concentration of . µg/ml was coated on -well-microtiter plates (maxisorp ii r immunoplates, nunc, denmark) overnight at • c. the wells were then blocked with % skimmed milk in pbs for min at • c and then washed with pbs. positive and negative controls were included in each test plate. each sample diluted in % skimmed milk in t-pbs (pbs with . % tween and . m nacl) was added in duplicates on antigen-coated wells with some differences among different types of samples. one hundred microliters of serum samples diluted : (for detection of igm antibodies) or µl of non-diluted samples of bal (for detection of igg and iga antibodies) were incubated for min at • c. two hundred and fifty microliters of oral fluids diluted : were incubated h at • c (for detection of igg antibodies). subsequently the plates were washed three times with t-pbs and antibody binding was detected by incubation for min at • c with µl of anti-pig igm peroxidase conjugate ( : , , bethyl), anti-pig igg peroxidase conjugate ( : , , sigma), or with anti-pig iga peroxidase conjugate ( : , , bethyl) separately (diluted in t-pbs with % skimmed milk). after washing the plates as described above, µl per well of the tmb-complete (test-line) substrate was added. the optical density (od) was measured at nm after an incubation time of - min at room temperature. the virus neutralization test for detection of prrsv neutralization antibodies was performed as follows. samples of sera were diluted : in dmem medium (sigma-aldrich) supplemented with % fbs. then, heat inactivated sera ( • c for min) were diluted -fold serially in flat-bottom -well-microplate (nunc). next, equal volume ( µl) of media containing prrsv pfu (lelystad-capm v- ) was added to each well. following incubation ( min at • c) marc- cells were added to each well ( × per well) in µl media per well. after days of cultivation ( • c, % co ), the cytopathic effect (cpe) of prrsv on marc- was evaluated by optical microscopy. the reciprocal value of the last sera dilution causing % reduction of cpe was defined as virus neutralization antibody titer. the lymphocyte transformation test was performed according to the method published earlier ( ) . peripheral blood mononuclear cells (pbmc) were obtained by gradient centrifugation post infection d ++ ++ + + + + + + -+ + + + + + ++ + + + + + + ++ the end of experiment d + ++ ++ + ++ + + + ± -+ + ++ + frontiers in immunology | www.frontiersin.org (histopaque- , sigma-aldrich). concentration of the cells was adjusted to , cells in µl of rpmi- medium supplemented with % of autologous serum, iu/ml penicillin, µg/ml streptomycin, and µg/ml gentamicin). they were incubated with the µg of optimal concentration of the specific antigen (moi . ) for days at • c in % co . negative controls were incubated with rpmi- medium only. all samples were evaluated in triplicate. h-thymidine was added on the last day of cultivation. subsequently, the cells were harvested (filtermate harvestor, packard bioscience company, usa), and h-thymidine incorporation was measured by a microplate scintillation and luminescence counter (topcount nxt tm , packard bioscience company) in counts per minute (cpm). the results were expressed in terms of stimulation indexes (si), which were calculated as the ratio of cpm in stimulated samples vs. cpm in non-stimulated controls. the number of ifn-γ producing cells was calculated by elispot techniques. commercially available porcine ifn-γ elispot kit ( - hpw- , mabtech) was used in accordance with the manufacturer's instructions. the number of cells used in the test was × /well. prrsv was used for stimulation of the antigen-specific response in multiplicity of infection (moi) . . mitogen cona at a concentration µg/ml was used as a positive control. cells without stimulation were used as a negative control. incubation lasted for h. spots were detected using elispot reader system elro tl (aid, germany). the results were recalculated to the number of cd + lymphocytes. the × of pbmc per well was stimulated with prrs virus in moi . for h. the × of cultured pbmc were pelleted and µl of primary monoclonal antibody cocktail containing anti-cd (igg b, clone - - , wsu, monoclonal antibody center, usa), anti-cd (igg a, clone - - , wsu, usa), and anti-γδ tcr (igg , clone pgbl a, wsu, usa) and µl of heat-inactivated goat serum was added. the cells were incubated for min at • c and then rinsed twice with cell washing solution. then, µl of goat anti-mouse secondary antibody cocktail (anti-igg b: dylight , anti-igg a: alexa fluor , and anti-igg : pe-cy ) was added and the cells were incubated for another min at • c. the cells were rinsed and then µl of anti-cd antibody (igg , clone ppt , southern biotech, pre-stained with alexa fluor dye using zenon antibody labeling kit, invitrogen) was added and the cells were incubated, rinsed twice, and fixation and permeabilization for subsequent intracellular staining was performed by solutions a and b of intra stain kit (dako cytomation, usa) ( ) . finally, µl of rpe-conjugated anti-ifn-γ antibody (clone cc , abd serotec uk) was added and the cells were incubated for min. the cells were measured as soon as possible using bd lsr fortessa flow cytometer (becton-dickinson, usa). at least , events were acquired. the post-acquisition analysis of data was performed using the facs diva software (becton-dickinson, usa). the following lymphocyte subpopulations were identified: (cd + ) γδ + cd + , (cd + ) γδ + cd − , (cd + γδ − ) cd + cd + , (cd + γδ − ) cd − cd + , (cd + γδ − ) cd + cd − , (cd + γδ − ) cd − cd − , and cd − cd + . the percentage of ifn-γ-positive cells was established for each subpopulation. the normality of data distribution were confirmed. experimental groups were compared using non-parametric man-whitney test. data from different dates were compared using non-parametric wilcoxon test for paired samples. after vaccination with inactivated vaccines (a and a ) the first igm in the serum started to appear days after the first dose in some piglets, and days after the second dose in all animals of the a group ( figure a and table ). igg antibodies appeared in all animals of both groups days after the second dose ( figure b) . the level of antibodies in the a group was significantly higher than in the group given the a vaccine. in groups of piglets vaccinated with mlv vaccines (b and b ), both igm and igg antibodies appeared days after vaccination. on day after immunization, their antibody responses were comparable to that of the a group. after infection, we identified a further increase in antibodies in the vaccinated groups. for the a group, a further increase in serum igg antibodies was observed after week and especially at and days after infection, when this antibody level significantly exceeded the values in the mlv immunized groups (b and b ) and the control one (c ). the a group showed a sharp increase on post infection days and , and the level of serum igg antibodies at these intervals was comparable to a . in groups immunized with mlv (b and b ), serum igg levels increased after , , and days post infection, but did not reach the a group values. in the control, non-immunized group, the first igm antibodies appeared days after infection, with a significant increase on day and . igg antibody levels appeared and days after infection but were lower than in the immunized groups. the virus neutralization antibody was detected in sera of animals days after vaccination (figure c) . these antibodies were detected in some animals in the groups a and a only. at the end of experiment (d , days post-infection) the high level of virus neutralization antibodies were detected in all groups except b group, in which significantly lower level was found ( figure c) . local antibodies in the balf performed days post vaccination we detected low levels of iga in all immunized groups (figure a) and igg in a , a , and b (none in b ) with the level in the a group being significantly higher (figure b) . days after infection we detected low levels of iga antibodies in a , b , b , and control group (c ) and low levels of igg antibodies in all immunized groups (none in c ). local antibodies were detected in the saliva of the a group in low concentrations and days after the second vaccination and in all groups after infection with variability in individual intervals and with no statistically significant between-group difference ( figure c) . in the feces, local antibodies (iga) were detected from week after the second immunization dose in groups a and a , and from week after mlv immunization in groups b and b ( figure d ) increased levels of antibody were detected in all groups including control after infection with no statistically significant betweengroup difference. a positive cell-mediated response after lymphocyte stimulation with specific antigen in vitro (stimulation index in ltt above ) was observed in the b group after and days post vaccination and and days post-infection (figure ) . a positive stimulation index was detected in the b group at days post vaccination only as a non-specific basal stimulation occurred from days post vaccination in this group (figure a) . groups immunized with inactivated vaccines a and a showed a marked non-specific stimulation of cells even without using antigen ( figure a ) and, therefore, it was impossible to demonstrate the effect of antigen addition and thus cell-mediated immune response. cell-mediated immune response after challenge infection was positive in all vaccinated groups and in the control group after days post infection, using elispot in pbmc from bronchoalveolar lavages. the results were recalculated to the number of cd + lymphocytes. the differences between individual animals, but no significant differences between groups were detected ( figure d) . we detected ifn-γ producing lymphocytes after prrs antigen stimulation. the most marked differences from control were found in cd − cd + and cd + cd + (and partly also in cd − + and γδ + + ) subsets of all immunized groups days after infection. in the groups vaccinated with live vaccines (b and b ) , the virus load was demonstrated in serum and saliva from day after immunization, in balf days after immunization (the only time point when the lavage was taken), and in feces occasionally days after vaccination, then in all piglets days after immunization (data not shown). no clinical signs were observed in piglets after infection. elevated body temperature was occasionally found in the first days, independent of the experimental group. however, viral shedding was noted, with between-group differences. the virus appeared in serum, saliva, and feces in all groups including the control group days after infection (figures a,b,d) . the virus was detected in balf days after infection in a , a , and c groups (figure c) . virus shedding was decreased in immunized groups and days after infection with the level in the a and b group being significantly lower compared to control group days after infection. negative samples appeared days after infection in saliva (in b group) and in feces (b and b groups). the goals of our study were ( ) to establish comprehensive immune response characteristics using several methodological approaches and monitor the dynamics in different compartments and in a time-dependent manner after vaccination and the challenging infection and ( ) to compare the immune response to different killed and modified live vaccines against prrs using these methodological tools. in order to compare the immune response after vaccination with different vaccines, we used a model of vaccinations of young piglets (beginning at weeks of age) and given vaccination intervals and subsequent infections, regardless of the fact that manufacturers' recommendations were different (especially in progressis). there are only a few papers published providing a comprehensive picture of immune response after vaccination against prrsv ( ) ( ) ( ) ( ) because the majority of the existing studies are based mainly on the evaluation of the vaccination effectiveness by monitoring the immune responses found in the blood ( , , , ( ) ( ) ( ) . our results show that antibodies after immunization and infection, and the virus after infection, can be detected in all the monitored compartments (blood, respiratory tract, intestine). by repeated sampling and simultaneous monitoring of the antibody and cell-mediated immunity and virus shedding systematically and locally, we have managed to get comprehensive information about the dynamics of the immune response after vaccination or prrs virus infection. in practical diagnostics of field samples is an effort to seek simple approaches to obtain tentative information on the epidemiological situation of the herd. one current trend is the monitoring of antibody levels and shedding of the virus in the oral fluid ( ) ( ) ( ) . in our experiment, the antibody detection rate in the oral fluid collected with ropes in pens was sufficient. the levels of antibodies detected after vaccination were low, but they increased after challenge infection. these findings confirms the possibility of using this approach for preliminary characteristics in the herd. it was interesting to observe the dynamics of antibody levels and viral shedding in feces too. this is an approach which is not often used for prrsv infection monitoring but is used in other situations where feces samples are more readily available than samples from other sources ( , ). we were wondering, among other things, to what extent infections occurring systemically, or locally in the respiratory tract occur in this remote compartment. our findings show that fecal samples can also be used for prrsv infection monitoring. detection of both the viruses and antibodies is not entirely consistent, because they appear in individual animals, and cease at later intervals, therefore, it is necessary to consider these findings as approximate. they can be used for herd-or pen-level testing, but not for establishing a diagnosis in individual animals. it appeared to be technically difficult to demonstrate specific cell-mediated immunity. the partial results were provided by each of the three methods used and a comprehensive picture could be obtained by compiling this information. therefore, it was not possible to use only data of ifn-γ production in elispot, although it is currently the most commonly used method for cmi ( , , , , , ) . a positive cell-mediated response after lymphocyte stimulation with specific antigen in vitro (in lymphocyte transformation test) was observed in mlv groups and especially in the b group as a non-specific basal stimulation occurred from days post-vaccination in b group. the strong non-specific stimulation of pbmc without specific antigen were detected in groups a and a immunized with inactive types of vaccine. this non-specific stimulation of cells in vivo masks the overall picture, and thus specific cell-mediated immunity cannot be demonstrated. this effect is attributed to the use of strong adjuvants in inactivated types of vaccines. in the test of ifn-γ production and detection with elispot, which is very often used to identify cmi both in experimental studies ( , ) , and in the field ( , ) , we have shown an increase in both blood and cells acquired by lavage, but the individual variability among the animals was too high and, consequently, there were no differences found between the groups under study. we detected also ifn-γ producing lymphocytes after prrs antigen stimulation in all immunized groups days after infection. the most marked differences from control were found in cd − cd + and cd + cd + (and partly also in cd − cd + and γδ + cd + ) subsets of lymphocytes. the cd − cd + subpopulation belongs to cytotoxic groups of cells, cd + cd + is considered a group of th memory cells ( ) . in another study the expression of cytotoxic cd + cd + and cd + cd − was described which help to recover from prrs infection ( ) . there were qualitative and quantitative differences in the immune responses to the inactivated vaccines and to mlv ones. after immunization with the inactivated vaccine (especially a -progressis), high levels of antibodies were produced generally (serum), which were mostly of the igm, and igg isotypes, and also locally (saliva, balf), both igg and iga. nevertheless it should be noted that we have applied progressis to piglets in our study, while the manufacturers declare the use of this vaccine for gilts and sows. cell-mediated immunity was detected only after infection, high non-specific cell stimulation was detected after vaccination and therefore any specific response could not be demonstrated in these intervals. the antigen specific cell mediated immunity after inactivated vaccine is rarely described ( ) . most work describes low or no cmi after vaccination with inactivated vaccine. after immunization with mlv vaccines, sufficient levels of antibodies in serum and balf (igg) were also produced, but lower than after the inactivated vaccine administration. the levels of iga antibodies in balf were comparable but low. low levels of virus neutralization antibodies after vaccination can be explained by a short interval between vaccination and infection, since neutralizing antibodies after vaccination or prrs infection occur within days ( ) . the dynamics of virus shedding after vaccination and infection is often used for monitoring vaccine efficacy ( , , ) . the decrease in virus secretion was observed days after mlv immunization and disappearance in days ( ) . in another study the excretion of virus was described still for days after vaccination with for porcilis or amervac vaccine ( ) . demonstration of cell-mediated immunity and reduction in viral load correlate with studies by other authors and support the preferred use of mlv vaccines in the control of prrs infection ( , ) . the question is to what extent these results are influenced by the composition of vaccines from different manufacturers and to what extent different types of vaccines (inactivated vs. live attenuated). there was an obvious difference in the quality between the inactivated vaccines, whereas the character of the immune response to both mlv vaccines was similar with only partial differences in the time-related response dynamics. the vaccine b (amervac) showed a more pronounced decrease in virus secretion and a tendency to induce sterile immunity, while b (porcilis) vaccine had a more pronounced of cmi in lymphocyte transformation test. it should be noted that the strain used in porcilis had a higher genetic link with the lelystad strain compared to the strain of amervac ( , ) which, however, probably did not significantly affect the above characteristics. despite the fact that many studies focused on prrs immunoprophylaxis have already been published and many procedures are implemented in the agricultural industry, a universal model does not yet exist ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the use of live attenuated vaccines is generally preferred as was also confirmed in our field study ( ) . in this study, we controlled the infection by a repeated blanket immunization with mlv vaccine (porcilis), followed by targeted immunization of gilts, and sows. the success of the strategy selected and evidence of virus eradication from the given herd were demonstrated by introducing sentinel animals into a fattening herd. based on this result, we believe that control programs can be adopted even in herds with continual throughput housing without interrupting production. however, in this case, vaccination is only one of the necessary preconditions and the introduction of very strict principles of good biosecurity is of no less importance. the use of animals was approved by the branch commission for animal welfare of ministry of agriculture of the czech republic (approval protocol no. mze ) as a part of project as a part of project respig (qj ). mt, vc, js, and mf designed the study. mt, ll, js, and kn performed the experiments. ll, hk, lk, po, and jf performed the lab work and analyzed the data. lk produced the figures and statistical analysis. mt wrote the manuscript. js and mf participated in manuscript preparation. all authors read and approved the final manuscript. the study was supported by projects no. qj , qj , and ro of the ministry of agriculture of the czech republic and by project lo with financial support from the ministry of education, youth, and sports of the czech republic under the npu i program. porcine reproductive and respiratory syndrome virus strains of exceptional 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molecular epidemiology of prrsv: a phylogenetic perspective genomic characterization and pathogenicity of a strain of type porcine reproductive and respiratory syndrome virus elimination of prrs virus (eu-type) from farrow-to finish breeding farm by vaccination with ingel-vac r prrs mlv under unfavourable conditions microbial ecology of swine farms and prrs vaccine vaccination strategies development and control of an acute prrs field virus infection in an endemic prrs breeding herd after vaccination with modified live vaccine different porcine reproductive and respiratory syndrome (prrs) vaccine regimes and its effect on pig immunity status at south asia pig farms evaluation of the effect of a porcine reproductive and respiratory syndrome (prrs) modifiedlive virus vaccine on sow reproductive performance in endemic prrs farm successful elimination of prrs virus from an infected farrow-to-finish herd by vaccination key: cord- -ml mgyf authors: huang, linna; zhang, wei; yang, yi; wu, wenjuan; lu, weihua; xue, han; zhao, hongsheng; wu, yunfu; shang, jia; cai, lihua; liu, long; liu, donglin; wang, yeming; cao, bin; zhan, qingyuan; wang, chen title: application of extracorporeal membrane oxygenation in patients with severe acute respiratory distress syndrome induced by avian influenza a (h n ) viral pneumonia: national data from the chinese multicentre collaboration date: - - journal: bmc infect dis doi: . /s - - -x sha: doc_id: cord_uid: ml mgyf background: evidence concerning the efficacy and safety of extracorporeal membrane oxygenation (ecmo) in patients with influenza a (h n ) has been was limited to case reports. our study is aimed to investigate the current application, efficacy and safety of ecmo in for severe h n pneumonia-associated acute respiratory distress syndrome (ards) in the chinese population. methods: a multicentre retrospective cohort study was conducted at hospitals that admitted patients with avian influenza a (h n ) viral pneumonia patients’ admission from provinces in china between october , , and march , . data from the national health and family planning commission of china, including general conditions, outcomes and ecmo management, were analysed. then, successfully weaned and unsuccessfully weaned groups were compared. results: a total of patients, aged ± years, were analysed; . % of patients were male with % mortality. all patients underwent invasive positive pressure ventilation (ippv), and rescue ventilation strategies were implemented for cases ( . %) with an average ippv duration of ± d, pao( )/fio( ) of ± mmhg, tidal volume (vt) of ± ml and plateau pressure (p(plat)) of ± cmh( )o pre-ecmo. after h on ecmo, pao( ) improved from ± mmhg to ± mmhg and paco( ) declined from ± mmhg to ± mmhg. haemorrhage, ventilator-associated pneumonia (vap) and barotrauma occurred in . %, % and . % of patients, respectively. compared with successfully weaned patients (n = ), the unsuccessfully weaned patients had a longer duration of ippv pre-ecmo ( ± d vs. ± d, p < . ) as well as a higher p(plat) ( ± cmh( )o vs. ± cmh( )o, p < . ) and vt ( ± ml vs. ± ml, p < . ) after h on ecmo support. furthermore, the unsuccessfully weaned group had a higher mortality ( % vs. . %, p < . ) with more haemorrhage ( . % vs. . %, p < . ). conclusions: ecmo is effective at improving oxygenation and ventilation of patients with avian influenza a (h n ) induced severe ards. early initiation of ecmo with appropriate ippv settings and anticoagulation strategies are necessary to reduce complications. conclusions: ecmo is effective at improving oxygenation and ventilation of patients with avian influenza a (h n ) induced severe ards. early initiation of ecmo with appropriate ippv settings and anticoagulation strategies are necessary to reduce complications. keywords: extracorporeal membrane oxygenation (ecmo), avian influenza a (h n ), acute respiratory distress syndrome (ards), complications, mortality background avian influenza a (h n ) viral pneumonia can manifest with varying degrees of dyspnea and is associated with a mortality of~ % [ ] . in particular, % of patients develop rapidly progressive pneumonia and % progress to acute respiratory distress syndrome (ards). the mortality of severe ards is as high as % [ ] . timely and effective respiratory support is particularly important to treat severe ards caused by avian influenza a (h n ) pneumonia. however, severe ards induced by avian influenza a (h n ) pneumonia might manifest as refractory hypoxaemia even with appropriate invasive positive pressure ventilation (ippv) support. extracorporeal membrane oxygenation (ecmo) is the ultimate respiratory support method and directly improves the oxygenation and ventilation of patients as well as enables implementation of the "lung protective ventilation strategy" [ ] . ecmo was the breakthrough treatment for the severe avian influenza a (h n ) outbreak of and reduced mortality from this outbreak [ ] [ ] [ ] . therefore, we believe that ecmo could also be effective for other types of severe viral pneumonia. existing studies of ecmo treatment for avian influenza a (h n ) pneumonia are primarily limited to case reports [ ] [ ] [ ] , and no study has systematically reviewed the efficacy or safety of ecmo to treat such diseases. therefore, it is particularly important to understand the current application of ecmo for avian influenza a (h n ) pneumonia-induced severe ards, investigate the application timing and management strategies of ecmo, and explore the possible reasons for treatment failure. based on the current study, we expect to standardize the management of ecmo and provide a description of our experiences using ecmo to treat patients with avian influenza a (h n ) pneumoniainduced severe ards. patients who had laboratory-confirmed avian influenza a (h n ) virus-induced pneumonia were included in this study. patients were admitted to hospitals in provinces of china between october , , and march , , and were reported to the national health and family planning commission of china. we included patients aged > ys who were supported by ecmo. patients who were lacking key detailed records of parameters during ecmo, such as ventilator or laboratory findings, were excluded. the included patients were divided into groups, namely, the "successfully weaned group" and "unsuccessfully weaned group". the former refers to a group of patients whose condition improved and were weaned from ecmo for at least h; the "unsuccessfully weaned group" refers to those who died or voluntarily discontinued treatment due to lack of improvement during ecmo support. the general conditions included age, gender, pregnancy status, underlying disease, time from onset to antiviral drug administration, vasoactive drug administration pre-ecmo, duration of ippv pre-ecmo, whether rescue ventilation strategies (including lung recruitment maneuvre, prone-position ventilation, and high-frequency oscillation ventilation) were implemented pre-ecmo, disease severity score, total duration of ecmo and ippv. we collected the ecmo blood flow at , , , and h on ecmo. improvement in circulatory and respiratory physiological indicators were considered, as well as ippv parameters at h pre-ecmo and , , and h on ecmo. furthermore, anticoagulation indicators during ecmo, including the types of anticoagulant drugs and methods of use; the maximum and minimum values of the activated coagulation time (act) and activated partial thromboplastin time (aptt); and the differences between the maximum and minimum act and aptt at , , and h on ecmo were recorded. finally, data regarding complications during ecmo therapy, including ecmo and ippv-related complications and nosocomial infections, were collected. the primary outcome was in-hospital mortality. the secondary outcomes were the length of stay in the intensive care unit (icu) and total length of hospitalization. three methods were used for a laboratory diagnosis, namely, the real-time reverse transcription-polymerase chain reaction (rt-pcr), viral isolation, and serological testing for the avian influenza a (h n ) virus using a modified haemagglutinin inhibition assay [ ] [ ] [ ] . we defined ards according to the berlin definition in [ , ] . pneumonia was diagnosed as an acute illness with fever, cough, or dyspnea/tachypnea, and at least one new focal chest sign that was supported by a finding of lung shadowing on a chest radiograph and without other noninfectious causes. the primary criteria for severe pneumonia were as follows: < > need for tracheal intubation and mechanical ventilation (mv) and < > need for vasoactive drugs after the active fluid resuscitation due to septic shock. the secondary criteria were as follows: < > respiratory rate ≥ times/ min; < > pao /fio ≤ mmhg; < > multiple lobe infiltration; < > disturbances of consciousness, disorientation, or both; < > blood urea nitrogen ≥ . mmol/l; and < > systolic blood pressure ≤ mmhg that required active fluid resuscitation. patients who met one primary criterion or at least three secondary criteria were diagnosed as having severe pneumonia [ ] . the criteria for the diagnosis of vap are in accordance with the european centre for disease prevention and control [ ] and included the following: < > two or more sequential chest x-rays or ct scans with a suggestive image of pneumonia for patients with underlying cardiac or pulmonary disease, or one definitive chest x-ray or ct scan in patients without underlying cardiac or pulmonary disease; < > a fever greater than °c and/ or leukocytosis greater than or equal to , wbc/ mm or leukopenia less than or equal to wbc/ mm ; and < > at least one of the following: <a > new onset of purulent sputum or change in the characteristics of the sputum; <b > cough, dyspnea, or tachycardia; <c > auscultatory findings, such as rales, bronchial breath sounds, ronchi, or wheezing; or <d > worsening gas exchange (e.g., oxygen desaturation or increased oxygen requirements or increased ventilation demand). for all included patients, we first described the general conditions, ecmo model and parameters, ippv parameters, the changes in circulation and respiratory physiological indicators from pre-ecmo to on ecmo status, anticoagulation on ecmo, and complications during ecmo therapy in all included patients. then, we compared patients who were successfully or unsuccessfully weaned from ecmo with regard to above items. all of the analyses were performed using spss . software. normally distributed continuous variables are expressed as the means ± sd and were compared using the t-test or chi-square test. non-normally distributed continuous variables are expressed as medians and quartiles and were compared using the wilcoxon rank-sum test. categorical variables were compared using the x test. p-values < . were considered significant. a total of patients were diagnosed with avian influenza a (h n ) virus-related pneumonia. patients were admitted to hospitals in provinces of china between october , , and march , , and were reported to the national health and family planning commission of china. the medical records of patients were available, and patients were reported to be supported by ecmo. one of the patients lacked ippv and ecmo parameters pre-ecmo and on ecmo and was eliminated as a participant; therefore, patients were ultimately selected ( fig. ) . data from patients ( . % males), with an average age of ± years, were analysed. there was no patient under the age of . a total of patients had underlying diseases, patients were treated with steroids and immunosuppressive agents within month of admission to the hospital, and patient was pregnant. the sequential organ failure assessment (sofa) score was ± points, and the murray score was . ± . points. the time from onset to antiviral drug administration was approximately ± d, and the time from onset to ecmo support was approximately ± d. high-dose vasoactive drugs [ ] were needed to maintain blood pressure in patients ( . %). the duration of ippv pre-ecmo was approximately ± d. rescue ventilation strategies, including the recruitment manoeuvre (rm), prone-position ventilation (pp) and high frequency oscillatory ventilation (hfov), were needed for patients ( . %) pre-ecmo. the total durations of ippv and ecmo were approximately ± d and d ( - d), respectively. of the patients, ( %) were successfully weaned from ecmo, and the other patients died due to an uncontrolled haemorrhage ( patients), septic shock ( patients due to progressive lung infection, patients due to bloodstream infection), heart failure ( patients) and discontinuation of treatment because of no improvement ( patients). one of patients showed an aggregated lung infection after weaning and eventually died due to septic shock. the inhospital mortality was %. the length of icu stay was ± d, and the total length of hospitalization was ± d (table ) . of the patients, were treated using the veno-venous ecmo (v-v ecmo) model. a total of patients with severe cardiac insufficiency and cardiogenic shock were treated using the venous-arterial ecmo (v-a ecmo) model. the ecmo equipment was mainly provided by maquet (shanghai) medical equipment co., ltd. and sorin (shanghai) medical equipment co., ltd. the pump from sorin was the stockert centrifugal pump system (scp/scpc), and the oxygenator was the d eos ecmo. the pump from maquet was the rota-flow, and the oxygenator was the quadrox pls. changes in ippv parameters and physiological indicators in patients on ecmo the ventilator parameters, including fio , positive end-expiratory pressure (peep), p plat , and vt, were significantly decreased in patients on ecmo. the vital signs, which included the heart rate, respiratory rate, and spo , and the arterial blood gas analysis (abg), which included the ph, paco , and pao levels, were improved in patients after ecmo support ( table ) . monitoring of anticoagulation all patients received a continuous infusion of unfractionated heparin for anticoagulation. however, heparin was discontinued for patients with cerebral haemorrhage and with active gastrointestinal haemorrhage. the act was ± to ± s at h, ± to ± s at h, and ± to ± s at h on ecmo. the aptt was ± to ± s and ± to ± s at and h on ecmo, respectively. complications during ecmo therapy in this study, the rates of gastrointestinal haemorrhage, cerebral haemorrhage, brain death, renal insufficiency, disseminated intravascular coagulation (dic), hyperglycaemia, and ecmo oxygenator thrombosis were higher compared to the relevant data from the ecls registry report [ , ] . new cases of vap developed in patients during ecmo, with an incidence rate of %. new cases of barotrauma occurred in patients, accounting for . % of cases. in addition, patients had a urinary infection, with an incidence rate of . %, and patients had a catheter-related blood stream infection (crbsi), with an incidence rate of . % (table ) . comparison between the patients successfully and unsuccessfully weaned from ecmo group contained patients who were successfully weaned from ecmo, and group included patients who were unsuccessfully weaned from ecmo. compared with patients successfully weaned from ecmo, the unsuccessfully weaned group had a higher mortality ( % vs. . %, respectively, p < . ), and was older ( ± years vs. ± years, respectively, p = . ), and more likely to have diabetes mellitus ( . % vs. . %, respectively, p < . ), had more frequent severe conditions (sofa: ± points vs. ± points, respectively, p < . ) pre-ecmo. meanwhile, they had a longer duration of ippv ( ± d vs. ± d, respectively, p < . ), had lower pao /fio levels ( . ± . mmhg vs. . ± . mmhg, respectively, p < . ), and higher rate of rescue ventilation strategies ( % vs. . %, respectively, p < . ) before ecmo support. no significant differences were found in the total duration of ippv, total duration of ecmo, length of icu stay and length of hospitalization between the two groups (table ) . ecmo blood flow did not significantly differ between the two groups during the initiation of ecmo support. however, in the successfully weaned group vs. the unsuccessfully weaned group, a significant decrease in blood flow correlated with an increase in the duration of support, which was . ± . l/min vs. . ± . l/min, respectively, (p < . ) at h on ecmo and . ± . l/ min vs. . ± . l/min, respectively, (p < . ) at h on ecmo (additional files and ). in the successfully weaned group compared to the unsuccessfully weaned group, fio was ± % vs. ± (fig. , additional file ). the vital signs were improved but did not significantly differ between the two groups pre-ecmo and during ecmo support (additional file ). patients who were unsuccessfully weaned from ecmo compared to patients who were successfully weaned from ecmo had severe acidosis (ph: . ± . vs. . ± . , respectively, (p < . ), a higher paco ( . ± . mmhg vs. . ± . mmhg, respectively, (p < . ), and a higher lactate concentration ( . ± . mmol/l vs. . ± . mmol/l, p < . ) pre-ecmo. ph and paco did not differ significantly between the two groups during ecmo support, while patients who were eventually successfully weaned from ecmo had a gradual ascending tendency of pao at and h on ecmo and a sustained low level of lactate (fig. , additional file ) . during the early stage of ecmo ( and h), the successful weaning group showed smaller differences between the act max and act min than the unsuccessful weaning group, which was ± s vs. ± s at h (p < . ) and ± s vs. ± s at h (p < . ). however, this trend was not found with regard to the difference between the maximum and minimum aptt. there were no differences between the two groups in mechanical complications associated with ecmo, vap and barotrauma. the successfully weaned group compared to the unsuccessfully weaned group had a lower haemorrhage rate ( . % vs. . %, respectively, p < . ), lower rate of renal insufficiency ( . % vs. . %, respectively, p < . ), lower rate of liver failure ( % vs. . %, respectively, p < . ) and lower heart failure rate ( . % vs. . %, respectively, p < . ). this study was the first to systematically and comprehensively discuss as well as elaborate on the current application of the efficacy and safety of ecmo in patients with h n pneumonia-related ards. a few studies [ , , , ] have shown that the mortality of ph n -induced ards was reduced to - % following ecmo treatment. presently, no studies with large samples have investigated the mortality of h n -induced ards, while the in-hospital mortality was as high as % in our study. late initiation of ecmo, inappropriate ippv settings during ecmo, and more ecmo complications might explain the relatively high mortality. moreover, as a multicentre collaboration study, the experiences of ecmo varied among the centres (additional file ), which might be another reason for the high mortality. according to the extracorporeal life support organization (elso) data [ , ] , ecmo is indicated when death risk exceeds %, i.e., when pao /fio < mmhg on fio > % and the murray score is - . our patients met the indications for ecmo support. the duration of mv for more than days pre-ecmo is an important prognostic factor for death [ ] . for patients in the successfully weaned group, the duration of ippv pre-ecmo was ± d; however, the duration was even longer among patients in the unsuccessfully weaned group ( ± d). moreover, rescue ventilation strategies were implemented for most patients before ecmo, which partially delayed the timing of ecmo. in comparison, ecmo was initiated at h ( - h) after ippv among patients with ph n in australia and new zealand in [ ] , which was significantly shorter than that in our cases. therefore, we emphasized early implementation of ecmo in our patients. the principle of ippv during ecmo is the "lung rest strategy" [ ] . the reva registry study examined patients with ph n -induced ards [ ] and showed that the high p plat ( cmh o) on day of ecmo was related to high mortality. in our study, the pre-ecmo p plat level was high ( ± cmh o). high p plat can lead to overdistension of the alveoli and cause lung volutrauma. the shear force between the overdistended and collapsed alveoli further aggregates vili [ ] , which ultimately increases mortality. although the p plat values [ ] showed that a high peep level within d of being on ecmo was related to decreased mortality. although no difference was observed in the peep levels between the two groups, we speculated that the down-regulation of peep during ecmo might have further aggravated the occurrence of collapse-induced injury, which led to atelectasis and sputum discharge obstacles. therefore, the ippv parameters, including high p plat and vt levels and low peep settings, might have been unreasonable in our study; lung rest or the maintenance of open alveoli was not achieved. the incidence of an ecmo oxygenator thrombus, haemorrhage, and organ failure in our study was high, which suggests that some problems existed in the anticoagulation management and organ supportive treatment of ecmo. we found that the unsuccessfully weaned group had larger fluctuations in act (the difference between act max and act min were larger) during the early stage of anticoagulation. this effect might suggest relatively unstable anticoagulation and a higher risk of haemorrhage. moreover, the incidence rate of vap during ecmo was as high as % and was partially attributed to the long course of h n pneumonia and the prolonged duration of ippv. therefore, intensification of airway management was extremely necessary. our study had limitations. the nature of the study required the collection of data at multiple consecutive time points to evaluate the efficacy of ecmo. as a retrospective study with some missing data, we were unable to successfully collect data at h pre-ecmo and , , and h post-ecmo. additionally, the number of subjects was too small to perform a multiple regression analysis to explore the risk factors for unsuccessful weaning from ecmo. ecmo is effective at improving oxygenation and ventilation of patients with avian influenza a (h n )-induced severe ards. early initiation of ecmo with appropriate ippv settings and anticoagulation strategies are necessary to reduce complications. fig. comparison of ippv parameters and abgs between two groups of patients on ecmo. for the successfully weaned group compared to the unsuccessfully weaned group, fio was ± % vs. ± %, respectively, at h (p < . ) and ± % vs. ± %, respectively, at h (p < . ). the monitored p plat was ± cmh o vs. ± cmh o, respectively, at h (p < . ) and ± cmh o vs. ± cmh o, respectively, at h (p < . ). the monitored vt was ± ml vs. ± ml, respectively, at h (p < . ) and ± ml vs. ± ml, respectively, at h (p < . ) after ecmo support. however, there were no differences in peep during ecmo between the two groups. patients who were in the unsuccessfully weaned group compared to patients in the successfully weaned group had severe acidosis (ph: . ± . vs. . ± . , respectively, (p < . ), a higher paco ( . ± . mmhg vs. . ± . mmhg, respectively, (p < . ), and a higher lactate concentration ( . ± . mmol/l vs. . ± . mmol/l, respectively (p < . ), pre-ecmo. the ph and paco did not significantly differ between the two groups during ecmo therapy, while patients who eventually weaned successfully from ecmo had a gradual ascending tendency in pao at and h on ecmo and a sustained low level of lactate after ecmo therapy people's republic of china. department of intensive care unit, the first affiliated hospital of wannan medical college people's republic of china. department of intensive care unit clinical findings in cases of influenza a (h n ) virus infection the new definition for acute lung injury and acute respiratory distress syndrome: is there room for improvement? extracorporeal support for patients with acute respiratory 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scholars (grant number /h ) and grants from the national natural science foundation of china ( / h and /h ), and the national program for the prevention and control of human infections by avian-origin h n influenza a virus (kjyj- - - ). the funding sources had no role in the design, conduct, or reporting of the study or the decision to submit the manuscript for publication. the remaining authors have disclosed that they do not have any potential conflicts of interest. the datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request. department of infectious diseases, henan provincial people's hospital, zhengzhou, henan province, people's republic of china. additional file : blood flow during ecmo, changes in ippv parameters and physiological indicators pre-ecmo and during ecmo. (docx kb) additional file : blood flow during ecmo between the two groups. in the successfully weaned group vs. the unsuccessfully weaned group, a significant decrease in ecmo blood flow correlated with an increase in the duration of support, which was . ± . l/min vs. . ± . l/ min, respectively, at h (p < . ) and . ± . l/min vs. . ± . l/ min, respectively, at h (p < . ). (tiff kb) additional file : changes in vital signs pre-ecmo and during ecmo between the two groups. vital signs were improved and did not significantly differ between the two groups during ecmo. authors' contributions all authors made substantial contributions to the conception and design of the study, data acquisition, analysis or interpretation of data, and review and approval of the final manuscript. drs. lh, wz, yy, ww and wl contributed equally to the article. drs. bc and qz assumed full responsibility for the integrity of the submission and publication and were involved in the study design. drs. wz, yy, ww, wl, hx, hz, yunfu wu (yw), js, lc, and ll were responsible for caring for the influenza a (h n ) cases and have been involved in gathering data. drs. lh, bc, qz, cw, dl, and yw had full access to all of the data in the study, assume responsibility for the integrity of the data and the accuracy of the data analysis and were responsible for data verification and analysis, as well as the drafting of the manuscript.ethics approval and consent to participate all patients gave written informed consent before ecmo treatment. as a highly pathogenic disease, the chinese national health and family planning commission approved the collection of the data from the patients with h n virus-induced pneumonia. the informed consent was waived to allow for exploration of the characteristics of the emerging infectious disease after rigorous contemplation and discussion by the chinese national health and family planning commission. not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. submit your next manuscript to biomed central and we will help you at every step: key: cord- -swlkjtyo authors: arzt, jonathan; branan, matthew a.; delgado, amy h.; yadav, shankar; moreno-torres, karla i.; tildesley, michael j.; stenfeldt, carolina title: quantitative impacts of incubation phase transmission of foot-and-mouth disease virus date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: swlkjtyo the current investigation applied a bayesian modeling approach to a unique experimental transmission study to estimate the occurrence of transmission of foot-and-mouth disease (fmd) during the incubation phase amongst group-housed pigs. the primary outcome was that transmission occurred approximately one day prior to development of visible signs of disease (posterior median hours, % ci: . – . ). updated disease state durations were incorporated into a simulation model to examine the importance of addressing preclinical transmission in the face of robust response measures. simulation of fmd outbreaks in the us pig production sector demonstrated that including a preclinical infectious period of one day would result in a % increase in the median number of farms affected ( additional farms and , pigs euthanized) compared to the scenario of no preclinical transmission, assuming suboptimal outbreak response. these findings emphasize the importance of considering transmission of fmd during the incubation phase in modeling and response planning. interventions . the proportion of transmission that occurs during the incubation phase is commonly denoted by theta (θ) , . a low value of θ indicates correspondingly low extent of transmission prior to observation of clinical disease. although a low θ is relatively uncommon, this has been associated with infectious diseases for which control or eradication programs have been highly successful such as smallpox and rinderpest , . at the other end of the spectrum are diseases for which θ is remarkably high, such as hiv/aids, which are notoriously difficult to control . closely related to the concept of θ is the temporal disparity between incubation and latency which represents the period of infectiousness included within the incubation period, defined herein as ω (omega; fig. ). while θ is intrinsically linked to the total length of the infectious period (often difficult to measure), ω can be estimated without having knowledge of the termination of infectiousness. additionally, the basic reproduction number (r ) of a pathogen is frequently cited to project and characterize an infectious disease epidemic. this quantity is defined as the average number of secondary cases generated by a typical primary case during its entire period of infectiousness, in a completely susceptible population and in the absence of control measures , . thus, in addition to aspects intrinsic to the specific host-pathogen combination, r will also be affected by numerous extrinsic factors at the population level. combining estimates for r , θ, and ω provides a robust background for evaluating the importance of preclinical transmission in successful modeling of disease spread and control interventions. this current study focused on investigating the concept of θ and ω for direct contact transmission of a virulent strain of foot-and-mouth disease virus (fmdv) amongst juvenile domestic pigs, and examining the impact of pre-clinical transmission on simulated outbreak size and severity within a us swine production system assuming either optimal or suboptimal response conditions. fmdv is a highly contagious viral pathogen of cloven-hoofed domestic and wild animals (genus: aphthovirus, family: picornaviridae) , . the virus, which is the causative agent of foot-and-mouth disease (fmd), has been associated with multiple major disease epidemics in previously free regions in recent years [ ] [ ] [ ] . given the highly contagious nature of the disease, the ability to predict potential fmd dissemination in a naïve population and assess the effectiveness of control interventions can result in critical improvements in preparedness and emergency response for food animal production systems . however, data-driven modeling is intrinsically and precariously dependent upon the appropriateness of the data used for parameterization. recent work examining incubation phase transmission of fmdv amongst cattle has been inconsistent between two studies suggesting either a high value of θ for dairy cattle or a low value for calves , . in contrast to this, a similar investigation demonstrated the occurrence of substantial transmission of fmdv during the incubation phase in group-housed pigs . this current report provides novel modeling approaches based upon the data obtained from this recent transmission study . the findings presented herein suggest a difference between the duration of the latent-and incubation periods (pre-clinical infectiousness; ω) in fmdv-infected pigs that is statistically significantly greater than , and a resultant θ that is larger than previously suggested in studies of fmdv transmission in cattle. additionally, simulating fmd outbreak scenarios using fixed and incrementally increasing values of ω demonstrated significant influence of incubation phase transmission on the size and duration of fmd outbreaks in us pig production systems, even in the presence of rigorous control strategies. fmdv transmission study. the experimental basis for the analyses presented herein consisted of a transmission study in which groups of naïve pigs were exposed to a group of fmdv-infected "donor pigs" through successive eight hour time periods (fig. ) . successful transmission from donors was determined by confirmed occurrence of fmd in contact-exposed pigs subsequent to exposure. the progression of infection was monitored and quantitated in both donors and contact-exposed pigs through measurements of viral genomic rna in serum and oropharyngeal fluids (opf). in brief, all pigs in the donor group were confirmed to have been infected by needle-free oropharyngeal inoculation with fmdv and were shedding fmdv rna in opf throughout the figure . definitions used to characterize distinct periods of infectious diseases. the latent period (e; green arrow) begins at the time of infection and ends at the onset of the infectious period. (i; red arrow). the incubation period (c; blue arrow) starts at the time of infection and ends at the appearance of clinical signs of disease (purple arrow). the difference between incubation and latency is denoted by ω (omega), and can have either a positive or negative value depending on whether transition to the infectious period occurs before or after appearance of clinical signs. the ratio of transmission occurring during incubation, and transmission occurring through the total infectious period is denoted by θ (theta; not shown). contact transmission trial. viremia in donor pigs was first detected at hours post inoculation (hpi), whereas fever (rectal temperatures > °c) and vesicular lesions were observed simultaneously at hpi ( supplementary fig. s ). there were no transmission events between the donors and contact groups or , which had been exposed to the donor pigs from to hpi, and to hpi, respectively. contrastingly, all pigs in contact groups through , corresponding to exposure from through hpi of the donors, developed clinical fmd (fig. ) . on this basis it was experimentally demonstrated that transmission from donor pigs had occurred at least hours prior to detection of clinical disease . a bayesian model was used to estimate the length of the latent, incubation, and infectious periods of the donor pigs based on data from the transmission trial . the model estimated the latent period to last slightly longer than one day (median hours, % ci - hours) ( table , fig. ). the incubation period was estimated to be approximately days (median hours, % ci: - ), and the total infectious period was estimated to be approximately days (median hours, % ci: - hours). the posterior median latent period was shorter than the prior median latent period (fig. a ). using the prior information, the latent period ended approximately hours after inoculation. by the posterior information, the length of the latent period was updated to last about hours (table ) . thus, the latent period is likely longer than a day and the effect of the observed data was large enough to influence the diffuse prior information (table ; figure . experimental design, fmdv transmission study. seven groups of five pigs each were exposed to five fmdv-infected donor pigs through successive eight hour exposure periods. contact groups were housed in separate isolation rooms before and after exposure to the donor pigs. the time points in the figure represent beginning (green) and end (red) of exposure for each contact group in relation to inoculation of the donor pigs. there was no transmission of infection to contact groups and (exposed from - , and - hours post infection of donors, respectively). all pigs in contact groups through developed clinical fmd after exposure to the donors. , incubation (c), and infectious (i) periods and their means (μ e, μ c, and μ i ) are expressed in terms of hours for a typical member of the donor animal group. parameter estimates for θ are in terms of a proportion, and β is expressed in terms of transmission rate. www.nature.com/scientificreports www.nature.com/scientificreports/ fig. a) . in contrast to this, the durations of the incubation and total infectious periods by posterior distributions were not significantly different from the prior distributions (fig. b,c) . a specific interest of this part of the analysis was to determine the duration of infectiousness during the incubation period (ω) as well as the proportion of transmission that occurred during this subclinical infectious period relative to total transmission (θ). the estimation of θ relies on the length of the total infectious period, which is comprised by both subclinical and clinical phases. assuming a median total infectious period of hours as estimated by the bayesian model, θ was estimated to be . . the duration of the subclinical phase of infectiousness corresponds to the difference between incubation and latent periods, both of which were modeled based upon directly measured experimental data. given the posterior medians of the latent ( hours) and incubation ( hours) periods, the difference between these values indicates a median duration of subclinical infectiousness (ω) of approximately hours ( % ci: . - . hours; table ). estimating the sensitivity of θ to variations in duration of infectious period. theta (θ) represents the fraction of transmission that occurs during the incubation phase in relation to transmission occurring through the total infectious period and is thereby intimately linked to the duration of the total infectious period. yet, no study has effectively measured the duration of infectiousness in fmdv-infected animals experimentally; rather, modeling studies have universally used proxies to generate estimates for this variable. in order to explore the sensitivity of θ to variations of the infectious period, θ was modeled using step-wise increasing durations of infectiousness ranging from to days, while keeping ω at the modeled estimate of . hours. the maximum resultant value of θ, which was based on modeling an infectious period of day was . ( % ci . - . ; supplementary table s ). contrastingly, if modeling a duration of infectiousness of days, the resulting estimate of θ was . ( % ci . - . ; supplementary table s ). as expected, θ varied inversely with duration of infectiousness throughout the sensitivity analysis. because transmission is rarely directly quantified in experimental or field studies, we tested the ability of our bayesian model to characterize transmission using various forms of more commonly available proxy measures for table ) . in order to test the reliability of these proxies to predict transmission, the bayesian model of donor pig infection dynamics was repeated to compare the model outcomes when the onset of infectiousness in donor pigs was defined based on the proxy measures rather than confirmed transmission events (cte-standard). the most noteworthy findings from this approach were the substantial changes in the modeled estimates of the duration of latency and subclinical infectiousness (ω) when defining the onset of infectiousness by either the occurrence of clinical signs of fmd in donor pigs or by any detection of fmdv rna in opf. defining the onset of infectiousness by the occurrence of clinical signs in donors led to a hour discrepancy of latency compared to the cte-standard, with a prolonged latent period of hours ( % ci - hours), and an ω of − . hours (fig. , supplementary table s ) . similarly, detection of (any) fmdv rna in opf as indicator of infectiousness resulted in a shortened latent period lasting only hours ( % ci - hours) and a resultant duration of subclinical infectiousness (ω) of hours ( % ci - hours; fig. , supplementary table s ). defining the onset of infectiousness by detection of fmdv rna in opf above a threshold of . log gcn/ml led to marginally decreased durations of latency and ω, whereas use of the proxy, detection of fmdv in serum led to the closest estimate to the cte-standard (fig. , supplementary table s ). our bayesian modeling of infection dynamics in fmdv-infected pigs estimated the occurrence of a subclinical infectious period (ω) of hours (table ). the practical ramification of this finding is that pigs that are infected with fmdv are capable of transmitting disease for approximately day prior to the development of any visible signs of infection, which could lead to further disease spread through animal movements and indirect contacts before a producer realizes through clinical observation that there is a health problem in the herd. in order to assess the impact of the duration of the subclinical infectious period in fmdv-infected pigs on outbreak size and duration, we performed a series of fmd outbreak simulations with the us national fmd model (interspread plus (isp) version . model software ), which can account for the complex movements and interactions seen in intensive pig production systems. simulations were run on a reduced farm population file composed of , swine operations with different production types and a total of , , pigs, in the eastern united states ( supplementary fig. s ). surveillance and control measures were modeled including passive and active surveillance in zones around infected premises, movement restrictions in the k zone around infected premises, and depopulation of infected premises. under optimal conditions, detection of the index case was based on the onset of clinical signs within the herd and depopulation occurred within to days (depending on herd size) at a rate of farms/day. for the suboptimal response, initial detection was delayed days, and depopulation was delayed days. for both the optimal and suboptimal responses, the duration of subclinical infectiousness was varied from to days, and the effect on outbreak size and duration examined. overall, incremental increases of subclinical infectious period (ω) led to substantial increases in the size and severity of simulated outbreaks under both categories of outbreak responses. when outbreak response conditions were assumed to be optimal, a -day subclinical infectious period (ω = day) as compared to absence of subclinical infectiousness (ω = day), resulted in a % increase of the median number of infected farms, necessitating euthanasia of , more pigs, and an increase of the median outbreak duration from to days ( fig. and supplementary table s ). when the outbreak response was assumed to be suboptimal, the effects of increasing ω were more profound, with a one-day subclinical infectious period resulting in median outbreak size increasing by farms ( %), outbreak duration increasing from days to days, and euthanasia of , additional pigs required ( fig. and supplementary table s ). further increases of ω resulted in corresponding increases in outbreak size and severity, under both optimal and suboptimal outbreak response conditions (fig. ) . specifically, each incremental increase in omega led to a longer duration of the outbreak, with some increments having significant effect. the greatest magnitude of effect that was estimated occurred with a -day subclinical infectious period (ω = day) combined with suboptimal outbreak response which resulted in . % of farms, and . % of all pigs in the population ( , , pigs) becoming infected. numeric values between and , with . increments corresponding to observations of lesions in contact-exposed pigs table . proxy measures used to determine the onset of infectiousness. www.nature.com/scientificreports www.nature.com/scientificreports/ effective control of infectious disease outbreaks is dependent upon rapid identification of infected individuals and mitigation of risks that could otherwise cause widespread dissemination of contagion. in the absence of an outbreak, data-driven mathematical models that can estimate disease spread and the impacts of control interventions can offer valuable insights to enable planning and preparedness . the ultimate goal of such modeling is to guide and assess the effectiveness of control measures to minimize disease impacts, quantitated as morbidity, mortality, economic loss, or other relevant metrics , . the overarching objective of the current investigation was to use detailed data from a carefully executed experimental study of fmdv transmission to model the durations of distinct stages of disease in fmdv-infected pigs, and to highlight the importance of addressing incubation phase (subclinical) transmission in fmd models. in order to explore the concept of disease spread before clinical detection, it was of particular interest to estimate the latent period (time from infection until onset of infectiousness) in relation to the incubation period (time from infection until appearance of clinical signs of disease). the estimated parameters were subsequently used to explore the impact of alterations in the duration of subclinical infectiousness on simulated fmd outbreaks in commercial swine production systems under both optimal and suboptimal response conditions. disease modeling is critically dependent upon input parameters that closely reflect the intrinsic properties of the infectious agent as well as externally variable features of the modeled scenario (e.g. population composition and density, contact networks and patterns, topographical characteristics, meteorological variations and availability of resources) , . the simulations in this current investigation utilized the national fmd model developed by the us department of agriculture to simulate fmd outbreaks within the us pig production system. this the proxy measures consisted of detection of fmdv rna in blood ("viremia"), detection of fmdv shedding in oropharyngeal fluid (opf) either above the assay lower limit detection ("any" shedding), or above a defined threshold of . log fmdv rna copies per ml ("threshold" shedding), or detection of clinical signs of fmd. the duration of latency (a) was underestimated compared to the cte-standard, when using fmdv shedding in opf ("any" or "threshold" shedding) to define the onset of infectiousness. detection of viremia as a proxy of infectiousness led to an estimated latent duration that was close to the cte-standard, whereas defining infectiousness by detection of clinical signs overestimated the duration of latency. the duration of subclinical infectiousness (b) and the proportion of transmission during the incubation phase (c) were underestimated when the onset of infectiousness was based on detection of clinical signs. the estimates based on the remaining four proxy-measures were less dispersed. for both parameters, detection of viremia provided the estimates closest to the cte-standard, whereas detection of fmdv shedding in opf provided slightly higher estimates. www.nature.com/scientificreports www.nature.com/scientificreports/ model is adapted to specific conditions within the us agricultural system, and parameterized to reflect the complex and overlapping outbreak response measures identified in the us national fmd outbreak response plan . additionally, the model is flexible and allows for adjustment of select parameters, as was performed in the current study to explore the effect of preclinical transmission on simulated outbreaks. similar to other disease spread models, this model is built upon assumptions concerning specific characteristics of the us production system for which it was designed. extrapolation of the modeled output should therefore be done with caution. however, under the given circumstances, the outcome of the outbreak simulations included herein serve to emphasize the critical impact that the occurrence of disease transmission during the incubation phase may have on the magnitude of an fmd outbreak. parameters for modeling of disease outbreaks are usually derived from meta-analyses figure . output of fmd outbreak simulations based on increasing durations ( - days) of subclinical infectiousness under optimal and suboptimal outbreak response conditions. ribbon plots for the cumulative number of infected pigs (a) and ridge plots for the epidemic curve (b) when modeled using incrementally increasing durations of subclinical infectiousness (ω) and assuming optimal (left panels) and suboptimal (right panels) outbreak responses. the lower edge, central line, and upper edge of the plots represent th percentile, median, and th percentile, respectively for the specific duration of the subclinical infectiousness (ω). www.nature.com/scientificreports www.nature.com/scientificreports/ of published experimental investigations that were not originally designed to assess disease transmission , . bayesian methods offer an alternative approach to inferring epidemiologic parameters from transmission experiments, which can improve our understanding of the latent and infectious periods , . regardless of the method used, in the absence of data explicitly describing actual disease transmission, different proxy measures must be used to define the transition between distinct stages of disease. the use of proxies often results in the assumption that the onset of infectiousness is defined either by the first evidence of infection in a given individual (pathogen detection), or by the appearance of visible clinical signs of disease. in the current investigation, the proxy measure of high-threshold shedding was interpreted to be the most relevant predictor of transmissibility of fmdv. onset of viremia was also correlated with infectiousness, which is likely due to the role of viremia in causing systemic dissemination and shedding of virus. as demonstrated in this investigation, inappropriate use of proxy measures to define the onset of infectiousness in replacement of actual transmission data can lead to drastic misinterpretations of the transmission potential of infected animals. these misinterpretations may in turn affect fmd outbreak simulations, resulting in underestimation of outbreak size and severity, leading to unrealistic estimates of resource needs and misguided guidance on the appropriate application of control interventions. specifically, underestimating the potential for disease transmission during the incubation phase may lead to wider dissemination of the outbreak than anticipated, and subsequent failure of reactive control interventions such as vaccination. contrastingly, overestimating disease transmission can promote excessively aggressive countermeasures and lead to destruction of large numbers of healthy animals and associated economic losses. this may result in substantial animal welfare issues and added economic losses as products and animals from unaffected farms cannot enter the production chain. appropriately balanced interventions are thus critical to effectively control disease spread while striving to maintain business continuity. the current investigation demonstrated that the modeled onset of infectiousness in fmdv-infected pigs occurred approximately one day prior to appearance of clinical signs of disease, as was consistent with the primary descriptive data from these experiments . this is consistent with earlier work which demonstrated fmd transmission during the incubation phase in pigs, lambs, and cattle . however, one published work contradicts these findings by suggesting that fmd is unlikely to be transmitted before the onset of clinical disease . although there is limited published information on this subject, the consensus of available data suggests that subclinical transmission of fmd does occur, indicating that incubation is longer than latency and the resultant ω is greater than zero. this result was concluded based on the significant disparity between the durations of incubation and latency which indicated a distinct period of subclinical infectiousness that was demonstrated by experimentation and verified by modeling. due to the limited group sizes and ubiquitous infection in groups to which transmission occurred, it was not possible to estimate r in the current investigation. however, previous investigations have estimated the basic reproduction ratio, r , for fmdv within groups of non-vaccinated pigs in experimental settings to be as high as . ( % ci: . - . ) or ( % ci: - ) . the combination of a generally high r for within-group transmission of fmdv in pigs, and a positive value for ω as determined in the current study suggests that the potential for fmdv transmission during the incubation phase should be recognized when modeling fmd outbreak scenarios. our estimate for the proportion of preclinical transmission of fmdv that occurs in pigs was %, falling between values reported for sars (θ < %) and smallpox ( < θ < %) . fraser et al. identified a relationship between r , θ, and the effectiveness of control interventions to bring an outbreak under control. as r and θ increase, control interventions must be highly effective, and multiple interventions are required in order bring the outbreak under control. estimated values of r and θ for fmdv suggest that control of epidemics is dependent on using multiple, highly effective control interventions. interestingly, these results may also suggest that isolation and contact tracing, if conducted nearly perfectly, could eventually be sufficient to prevent epidemic propagation. however, in the absence of significant technological advances, such interventions are unlikely to be implemented perfectly in livestock populations, and the timelines required for disease control could be much longer than when more interventions are enacted simultaneously. the potentially profound consequences of fmdv incursions into regions previously free of fmd can, to a great extent, be attributed to the highly contagious nature of the virus. this was demonstrated in the current study by the maximum impact simulated example of infection of over , , pigs when high ω was combined with a suboptimal outbreak response. although fmdv can be transmitted via a multitude of both direct and indirect routes , movement of infected animals has been identified as the most significant risk for dissemination of infection during the early phase of an outbreak . this current investigation demonstrated the relevance of fmdv transmission during the incubation period in group-housed pigs. furthermore, it was demonstrated that the duration of subclinical infectiousness had significant effects on spread and duration of simulated fmd outbreaks. the data used for modeling of disease stage durations in the current study were derived from experiments in which pigs were infected with one specific fmdv strain, under experimental conditions. the virus strain and host criteria were chosen based upon extensive experience in our laboratory with these conditions and the assumption that the virus and conditions were representative of most virulent fmdvs. it is possible that disease dynamics in the field may differ due to strain-specific variations in virulence, as well as differences in animal age, health status, and housing conditions. thus, it should be emphasized that the modeled output presented herein is based on experimental conditions, and represents our best estimate of what could be expected to occur under natural conditions in the field. the findings presented herein demonstrate the importance of considering and elucidating the intricacies of key epidemiologic parameters, including preclinical infectiousness, the importance of understanding the relationships between proxy measures of disease status and infectiousness, and the subsequent value of incorporating these detailed parameters into disease spread models. additionally, improved understanding of the influence of animal-level disease dynamics upon dissemination of fmd outbreaks may lead to improved approaches to www.nature.com/scientificreports www.nature.com/scientificreports/ surveillance and diagnostic testing to further refine control measures and maximize effectiveness while limiting undesired consequences for the agricultural industries. animal experiment. the data used in this current investigation were derived from an experimental trial designed to evaluate the onset of infectiousness in relation to the appearance of clinical disease in fmdv-infected pigs (fig. ) . a detailed description of the experimental study and clinical findings has been published previously . animal experiments were carried out within bsl -ag facilities at plum island animal disease center, new york. all procedures were carried out in accordance with guidelines specified within the associated experimental protocol (protocol - -r), and were approved by the plum island animal disease center institutional animal care and use committee. in brief, the study included groups of pigs of approximately - weeks of age (~ kg), of which one group was infected with fmdv a cruzeiro through simulated-natural inoculation . the remaining groups of pigs (contact groups - ) were sequentially exposed to the infected donor pigs through hours of successive co-habitation within a designated isolation room (fig. ) . after exposure to the donor pigs, contact-exposed pigs were moved into separate isolation rooms and were monitored for development of fmd. samples collected were oropharyngeal swabs to assess virus shedding and blood samples to measure viremia. clinical examinations and sample collection were done at - hour intervals after exposure. the choice to use fmdv a cruzeiro for these experiments was based upon numerous previous experiments in our laboratory which had demonstrated that this strain was consistently virulent and transmissible in pigs , - . data analysis. definitions. the end of latency for the donor pigs was defined as the beginning of the hour contact exposure period during which the first successful transmission event occurred. it was not possible to attribute transmission events to specific individuals as donors and contact pigs were allowed to move freely within the exposure room. thus, the earliest observed transmission event was used to define the transition from latent to infectious periods for all donor pigs. the end of the incubation period for the donor pigs was determined for each pig individually by the first detection of vesicular lesions, which for all donor pigs coincided with detection of fever (rectal temperature ≥ °c). fmdv shedding was defined by continuous detection of fmdv rna in opf. viremia was defined by detection of fmdv rna in serum. a bayesian model was fitted to the data for the purpose of estimating the length of three distinct periods the donor pigs were expected to traverse: the latent, incubation, and infectious periods. a modeling approach was adapted from that published by charleston et al. . this model describes the relationship among the observed transmission successes and the unobserved latent, incubation, and infectious periods as well as the hyperparameters describing the distribution of those periods in the following way: : start/end time of challenge i of donor j. p ij : probability of successful transmission in challenge i of donor j. β: transmission rate. t ij : time during challenge i for which donor j is infectious. e j : latent period for donor j. c j : incubation period for donor j. i j : infectious period for donor j. μ e , μ c , μ i , σ e , σ c , σ i , ρ ec : hyperparameters for latent, incubation, and infectious period prior distributions (in order; the means for the three periods, the standard deviations for the three periods, and the correlation between the latent and incubation periods). α e , η e , α c , η c , α i , η i : hyperparameters for the means and standard deviations for the latent, incubation, and infectious period prior distributions (in order, the mean and standard deviation for the mean of the prior latent distribution, the mean and standard deviation for the mean of the prior incubation distribution, the mean and standard deviation for the mean of the prior infectious distribution). the likelihood above describes the relationship between the observed transmission event data with the unobserved latent, incubation, and infectious periods, while the prior information (table ) was based on accumulated data from previous investigations carried out under similar conditions [ ] [ ] [ ] [ ] , or were left diffuse in the absence of such information. these sources of information were combined and the posterior distribution over the parameters (latent, incubation, and infectious periods along with the means of the three periods) given the observed data ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports www.nature.com/scientificreports/ (the outcomes of the transmission events) was estimated. this distribution is proportional to the product of the likelihood and the joint prior distribution for all of the parameters. as the posterior conditional distributions for at least the latent period, infectious period, and the transmission rate were intractable, a numerical rather than an analytic approach was pursued. the model was coded using just another gibbs sampler (jags) software designed to perform markov chain monte carlo (mcmc) simulations . this version of the model required two sacrifices: the latent and incubation periods could not be jointly lognormal, and the prior hyperparameter variance terms (σ e , σ c, and σ i ) were held fixed due to mcmc chain convergence issues as a result of unidentifiability issues. the result of the former being that the latent and incubation periods are assumed independent a priori. the proportion of infection that occurs prior to onset of clinical disease, denoted as θ, is a function of the quantities estimated by the model described above. using the jags version of the model adapted from charleston et al. and assuming that latent and incubation periods are independent a priori, θ is described in as follows: in addition to θ, we propose herein a distinct parameter (ω; omega) that allows more precise and direct inference about pre-clinical infectiousness to be made regardless of the total duration of the infectious period by representing the disparity between incubation and latency (fig. ) . estimating fluctuations of θ due to variations in duration of infectious period. as total duration of infectiousness was not experimentally evaluated in the current study, information about the length of the infectious period is drawn almost solely from the prior information. in order to evaluate the sensitivity of θ to variations in the total infectious period, θ was estimated as described above, but using incrementally increased integer values of infectious duration ranging from to days. modeling infection dynamics using different proxy measures to determine the onset of infectiousness. four different proxy measures (table ) were evaluated for their ability to predict disease transmission. the outcome of modeling transmission using the defined proxy measures was compared to the standard model, which was based on confirmed transmission of fmdv to contact-exposed pigs (confirmed transmission event (cte) -standard). the four evaluated proxies were defined as follows: (a) detection of fmdv rna in serum, (b) detection of any fmdv rna in opf, (c) detection of fmdv rna in opf above a threshold of . log gcn/ml, which had previously been associated with successful transmission of fmdv (d) clinical signs of fmd. the data were input into a bayesian model via five distinct indicator matrices, one for each measure of transmission, in which the number of rows equaled the number of contact animals-hours-post-inoculation combinations and the number of columns represented the five donor pigs. each entry in the matrices was either a , if the donor animal met the criteria for successful transmission for the given transmission metric (table ) during exposure to the contact animal represented by that row, or a if the donor animal did not meet the criteria for successful transmission given transmission metric at that time. for the cte-standard transmission metric, which was defined by confirmed transmission to contact-exposed pigs, the vector for all donors was identical as the effect of individual donors on contact animal could not be determined as both sets of animals were exposed to one another in groups. simulation modeling of an fmd outbreak using estimated transmission parameters. the objective of the fmd outbreak simulations described herein was to evaluate the effect of altering the duration of the subclinical infectious period (omega; ω) on spread and duration of an outbreak of fmd in a us pig production sector. estimates of disease stage durations were derived from modeling of animal-level infection dynamics ( table ). the originally modeled output consisted of estimates for the durations of latent (e), incubation (c) and infectious (i) periods. the durations of the subclinical infectious period (ω = c − e) and clinical infectious period (i c = i − c) were derived from these outputs. in addition to the baseline scenario (ω = day), five omega values ( day, days, days, days, and days) were subjectively selected to evaluate the effect on altering the duration of the subclinical infectious period on the spread and duration of simulated outbreaks. while choosing different omega values, the durations of latent and clinical infectious periods were kept constant (similar to baseline scenario) except for the scenario in which ω = day of omega where the latent duration was set to days (supplementary table s ). the within-herd (wh) software version . . available through the north american animal disease spread model was used to estimate herd-level parameters based on the modeled animal-level fmd disease stage durations. a spatial microsimulation model called the farm location and agricultural production simulator (flaps) was used to generate a synthetic population file of , farms with , , total pigs representative of pig production systems in the eastern united states (supplementary fig. s ). the us pig farms (with essential attributes for isp such as identification number, herd size, type of farms, and cartesian coordinates) located in the great lake, north east and south east regions were included in the model scenarios. about % (n = , ) of the total pig farms in the united states are located in these regions. amongst these farms, % were commercial farms with a median herd size of pigs (range: to , ) and % were small-scale enterprises with a median herd size of pigs (range: to ). farm-type specific movement parameters and contact rates were assigned to reflect differences in movements between commercial and small-scale enterprises. fmd outbreak simulations were performed using interspread plus (isp) version . model software . the isp www.nature.com/scientificreports www.nature.com/scientificreports/ is a state-transition, stochastic and spatial modeling tool for the simulation of fmd and other similar diseases . twelve fmd outbreak scenarios were developed in isp representing optimal and suboptimal outbreak response conditions for each of distinct values for the duration of subclinical infectiousness: day, day, days, days, days, and days of omega, respectively (supplementary table s ). the unit of interest in the model was the individual farms. fmd epidemics were initiated from single farms. after exposure to fmdv, the susceptible farms were modeled to transit into latent, subclinically infectious, clinically infectious, and depopulated states. the spread of fmdv from an infected to susceptible farms was modeled to occur through direct contact, indirect contact, and local spread (supplementary note). the daily probability of transmission of fmd virus from infected farms to susceptible farm was calculated as the hypergeometric probability of shipping at least one infected animal off of an infected farm given the average herd size, shipment size, and the number of infected animals in a herd on a given day ( supplementary fig. s , supplementary note). once an infected farm was detected, several control strategies were imposed simultaneously as is typically performed in response to outbreaks in fmd-free areas. control strategies included zoning of control areas, tracing of animal movements, animal movement restrictions, depopulation of the infected farms, and surveillances as delineated in the national response plan for fmd (supplementary note). for each of the omega scenarios, two overall control strategies (optimal and suboptimal) were separately simulated. in the optimal control strategy, the detection of infected farms through passive surveillance was modeled to occur just after onset of clinical signs, which was further delayed by days in suboptimal control category. additionally, the delay in depopulation of detected farms was (small farms) to days (big farms) in the optimal control category, which was delayed by more days to represent the suboptimal control. the major outputs parameters were outbreak size (number of infected farms and pigs), epidemic duration (days from onset of infection to end of epidemic), time between onset of infection to detection, and daily new infected farms due to each of the incorporated omega values. the median and interquartile range of outbreak size and epidemic duration were reported. the data analyses were performed using sas (sas institute inc., nc, usa, ) and microsoft excel (microsoft excel, redmond, washington, ). the kruskal-wallis test with bonferroni corrections was performed for multiple group comparisons for the outcomes from various omega values. a p-value of ≤ . % was considered for statistical significance. all data generated or analyzed during this study are included in this published article and its supplementary information files. table . prior distributions and estimates used to model animal level infection dynamics. posterior estimates for the latent (e), incubation (c), and infectious (i) periods, their means (μ e, μ c, and μ i ), and their standard deviations (σ e , σ c , and σ i ) are expressed in terms of hours for a typical member of the donor animal group. parameter estimates for θ are in terms of a proportion, and β is expressed in terms of transmission rate. mathematical prediction in infection models of foot-and-mouth disease transmission dynamics and control of ebola virus disease (evd): a review data-driven models of foot-and-mouth disease dynamics: a review decision-making for foot-and-mouth disease control: objectives matter bayesian inference of epidemiological parameters from transmission experiments simulation modelling of a hypothetical introduction of foot-andmouth disease into alberta managing complexity: simplifying assumptions of footand-mouth disease models for swine transmission of foot-and-mouth disease virus during the incubation period in pigs planning for smallpox outbreaks factors that make an infectious disease outbreak controllable relationship between clinical signs and transmission of an infectious disease and the implications for control global eradication of rinderpest. yea or nay? infectious diseases of humans: dynamics and control on the definition and the computation of the basic reproduction ratio r in models for infectious diseases in heterogeneous populations the pathogenesis of foot-and-mouth disease ii: viral pathways in swine, small ruminants, and wildlife; myotropism, chronic syndromes, and molecular virus-host interactions foot-and-mouth disease description of recent foot and mouth disease outbreaks in nonendemic areas: exploring the relationship between early detection and epidemic size reemergence of foot-and-mouth disease the foot-and-mouth disease epidemic in japan. the journal of veterinary medical science/the foot and mouth disease virus transmission during the incubation period of the disease in piglets, lambs, calves, and dairy cows quantifying the value of perfect information in emergency vaccination campaigns parameter values for epidemiological models of footand-mouth disease in swine parameterization of the duration of infection stages of serotype o foot-and-mouth disease virus: an analytical review and meta-analysis with application to simulation models a meta-analysis quantifying transmission parameters of fmdv strain o taiwan among non-vaccinated and vaccinated pigs the pathogenesis and diagnosis of foot-and-mouth disease relative risks of the uncontrollable (airborne) spread of fmd by different species infection dynamics of foot-and-mouth disease virus in pigs using two novel simulated-natural inoculation methods early events in the pathogenesis of foot-and-mouth disease in pigs; identification of oropharyngeal tonsils as sites of primary and sustained viral replication detection of foot-and-mouth disease virus rna and capsid protein in lymphoid tissues of convalescent pigs does not indicate existence of a carrier state direct contact transmission of three different foot-and-mouth disease virus strains in swine demonstrates important strain-specific differences american animal disease spread model (naadsm) development team simulating the distribution of individual livestock farms and their populations in the united states: an example using domestic swine (sus scrofa domesticus) farms interspread plus: a spatial and stochastic simulation model of disease in animal populations pauszek are thanked for supporting sample processing and laboratory analyses. c.s. and j.a. conceived and coordinated the study, executed the animal experiments and drafted the manuscript. m.b. performed the bayesian analysis and contributed to data interpretation. k.m. and s.y. performed simulation modeling and contributed to data interpretation. a.d. coordinated and oversaw data analyses and contributed scientific content. m.t. critically reviewed data analyses and contributed scientific content. all authors have reviewed and revised the manuscript and approved the final product. supplementary information accompanies this paper at https://doi.org/ . /s - - - .competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -zy m xc authors: garcía-belmonte, raquel; pérez-núñez, daniel; pittau, marco; richt, juergen a.; revilla, yolanda title: african swine fever virus armenia/ virulent strain controls interferon beta production through the cgas-sting pathway date: - - journal: j virol doi: . /jvi. - sha: doc_id: cord_uid: zy m xc african swine fever virus (asfv) is a complex, cytoplasmic double-stranded dna (dsdna) virus that is currently expanding throughout the world. currently, circulating virulent genotype ii armenia/ -like viruses cause fatal disease in pigs and wild boar, whereas attenuated strains induce infections with various levels of chronic illness. sensing cytosolic dsdna, mainly by the key dna sensor cyclic gmp-amp synthase (cgas), leads to the synthesis of type i interferon and involves signaling through sting, tbk , and irf . after phosphorylation, sting translocates from the endoplasmic reticulum to the golgi compartment and to the perinuclear region, acting as an indispensable adaptor connecting the cytosolic detection of dna to the tbk -irf signaling pathway. we demonstrate here that attenuated nh/p , but not virulent armenia/ , activates the cgas-sting-irf cascade very early during infection, inducing sting phosphorylation and trafficking through a mechanism involving cgamp. both tbk and irf are subsequently activated and, in response to this, a high level of beta interferon (ifn-β) was produced during nh/p infection; in contrast, armenia/ infection generated ifn-β levels below those of uninfected cells. our results show that virulent armenia/ asfv controls the cgas-sting pathway, but these mechanisms are not at play when porcine macrophages are infected with attenuated nh/p asfv. these findings show for the first time the involvement of the cgas-sting-irf route in asfv infection, where ifn-β production or inhibition was found after infection by attenuated or virulent asfv strains, respectively, thus reinforcing the idea that asfv virulence versus attenuation may be a phenomenon grounded in asfv-mediated innate immune modulation where the cgas-sting pathway might play an important role. importance african swine fever, a devastating disease for domestic pigs and wild boar, is currently spreading in europe, russia, and china, becoming a global threat with huge economic and ecological consequences. one interesting aspect of asfv biology is the molecular mechanism leading to high virulence of some strains compared to more attenuated strains, which produce subclinical infections. in this work, we show that the presently circulating virulent armenia/ virus blocks the synthesis of ifn-β, a key mediator between the innate and adaptive immune response. armenia/ inhibits the cgas-sting pathway by impairing sting activation during infection. in contrast, the cgas-sting pathway is efficiently activated during nh/p attenuated strain infection, leading to the production of large amounts of ifn-β. our results show for the first time the relationship between the cgas-sting pathway and asfv virulence, contributing to uncover the molecular mechanisms of asfv virulence and to the rational development of asfv vaccines. p athogen associated molecular patterns (pamps) are recognized by pattern recognition receptors, which activate signal transduction pathways in order to induce innate immune responses such as type i interferon (ifn) production. cytoplasmic double-stranded dna (dsdna) acts like a potent pamp, sensed by cyclic gmp-amp synthase (cgas). cgas detects cytoplasmic dsdna and catalyzes the synthesis of gmp-amp cyclic dinucleotide (cgamp) ( , ) . cgamp acts as a second messenger and binds to the stimulator of interferon genes protein (sting), which traffics from the endoplasmic reticulum (er) to the trans-golgi network (tgn), where tank-binding kinase (tbk ) is recruited and phosphorylated. this event allows the recruitment of irf that is subsequently phosphorylated by tbk and then translocates to the nucleus, where it acts as a transcription factor for type i ifn-␤ gene expression ( ) ( ) ( ) . african swine fever virus (asfv) causes a lethal disease, which poses important economic consequences to the pig industry and to the ecosystems in affected countries ( ) . because there is no suitable vaccine, at present, the spread of asfv is uncontrolled. asfv expansion started in in the republic of georgia, then reached russia, eastern and central europe, and recently china ( ) , where more than outbreaks have been declared so far. asfv, the only member of the asfaviridae family ( ), is an enveloped, cytoplasmic dsdna virus that encodes more than proteins in infected macrophages, the natural target cell population ( ) , including proteins that have various roles in virus-host interactions and in the modulation of the immune response ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . however, the function of many viral gene products remains unknown ( ) . in africa, wild suidae, such as warthogs and bush pigs, are also infected with asfv; however, they show only subclinical infections and can act as virus carriers. in contrast, acute asf in domestic pigs or the european wild boar is characterized by hemorrhages in lymph nodes and internal organs and high temperatures, resulting in the death of the animal in about to days. different strains of the virus exhibit different virulence, ranging from peracute to acute to subclinical and chronic forms of the disease (reviewed in reference ) . the fact that asfv strains display different virulence patterns, suggests a distinctive activation of the immune system (reviewed in reference ) , resulting in a complex scenario of virus-host interactions ( ) ( ) ( ) and type i ifn cascade ( ) . our studies show, for the first time, that virulent asfv armenia/ strain has acquired specific mechanisms to control ifn-␤ production during infection of porcine alveolar macrophages. these mechanisms involve the inhibition of (i) cgas-dependent viral dna sensing, (ii) cgamp-mediated phosphorylation of sting, (iii) sting trafficking, and (iv) tbk /irf activation. the inhibition and control of ifn-␤ synthesis, one of the most important antiviral immune factors, is most likely an essential feature for the virulent asfv armenia/ strain. on the other hand, the induction of ifn-␤ by nh/p could further explain its in vivo attenuation. virulent asfv armenia/ infection inhibits mrna production and secretion of ifn-␤. asfv strains can either cause chronic, subclinical, or fatal, acute asf disease. in order to study whether differences in asfv virulence are related to differences in the activation of the innate immune response, we analyzed the level of ifn-␤ produced by porcine alveolar macrophages infected either with nh/p (attenuated) or with armenia/ (virulent) asfv strains. for this purpose, a time course experiment in macrophages at , , , and h postinfection (hpi) was performed. figure a shows a higher production of ifn-␤ mrna in cells infected with nh/p compared to those infected with armenia/ , starting at hpi with a maximum at hpi, a time point where ifn-␤ mrna was very low in cells infected with armenia/ . interestingly, we observed a significant increase of ifn-␤ mrna in cells infected with nh/p from to hpi, indicating that cellular signaling leading to ifn-␤ transcription is activated during the course of the infection with the attenuated virus. next, the amount of ifn-␤ secreted during attenuated versus virulent infections was determined. supernatants from either nh/p -or armenia/ -infected macrophages were collected, and ifn-␤ levels were measured by enzyme-linked immunosorbent assay (elisa) (fig. b) . the results show a strong decrease of secreted ifn-␤ at hpi, after an initial increase at hpi, during the infection with the virulent strain, whereas in nh/p -infected macrophages the amount of ifn-␤ increases over time. these data correlate well with the results obtained regarding ifn-␤ mrna production (fig. a ). this indicates that virulent asfv shuts down the ifn-␤ synthesis pathway several hours after infection (fig. a) , although ifn-␤ mrna production is significantly above the control level at hpi. this effect also translates to ifn-␤ cytokine production, which initially increases and is totally abolished at hpi, when macrophages are infected with virulent asfv (fig. b) . in addition, we analyzed the expression of ifit- and cxcl , two ifn-␤-dependent genes ( , ) at , , , and hpi. fig. c and d, show that armenia/ does not activate either ifit- or cxcl mrna synthesis during the course of the infection, whereas, in contrast, ifit- and cxcl mrna level increases during nh/p infection. finally, and as shown in fig. e and f, the p mrna and protein levels were equivalent during the infection with both attenuated and virulent strains, thus demonstrating that same amount of viruses have been used. taking together, these results reveal two important aspects regarding ifn-␤ production by attenuated versus virulent asfv strains. first, the attenuated nh/p virus induces the production of ifn-␤ in infected macrophages, which in turn stimulates the expression of ifn-␤-dependent genes such as ifit- and cxcl . second, the virulent armenia/ strain is able to efficiently block ifn-␤ mrna synthesis and production in infected macrophages. the sting pathway is blocked by virulent armenia/ , while it is activated by attenuated nh/p . given the results described above, we set out to dissect the molecular mechanism of ifn-␤ inhibition/induction by different asfv strains in infected porcine alveolar macrophages. it has been shown that induction of ifn-␤ synthesis depends mainly on the cgas-sting pathway, which is triggered by the sensing of dsdna in the cytoplasm ( ). we therefore studied the correlation between ifn-␤ production and cgas-sting pathway activation in macrophages after infection with attenuated or virulent asfv. hpi, and lysates were separated by to % sds-page, followed by immunoblotting with anti-asfv p (viral early p ) and anti-actin antibodies. the data are means Ϯ the standard errors of the mean (sem; n ϭ ). data were statistically analyzed by using a student t test (*, p Ͻ . ; **, p Ͻ . ; ***, p Ͻ . ). as seen in fig. a , infection with nh/p , but not with armenia/ , induces phosphorylation of sting and irf at hpi, indicating that the sting pathway is activated in response to the infection with the attenuated, but not with the virulent, asfv strain. sting phosphorylation occurs as early as hpi, reaches a peak at to hpi and decreases by hpi during nh/p infection (fig. b) . meanwhile, irf phosphorylation, which controls ifn-␤ transcription ( ) , starts at hpi and increases until hpi (fig. b) . these data indicate that sting activation occurs early after nh/p infection and before irf phosphorylation and is maintained throughout h of infection; this supports the ifn-␤ production data presented in fig. . in contrast, infection of macrophages with armenia/ produces only a weak phosphorylation signal of sting after and hpi, which decreases at later time points and is almost negative at hpi (fig. c) . a similar activation profile was seen for irf (fig. c ). this demonstrates that-after initial moderate activation-virulent armenia/ is able to inhibit sting and irf phosphorylation at later time points after infection; similar results were observed in regard to ifn-␤ synthesis (fig. b) . the specific inhibitor of cgas, ru , inhibits the activation of sting mediated by nh/p and armenia/ infection. cgas is a cellular sensor for cytoplasmic dsdna detection, which further induces sting pathway activation ( ) ( ) ( ) . to test the role of cgas in sensing of asfv dsdna, porcine alveolar macrophages were mock treated or treated with increasing concentrations of the specific cgas inhibitor ru ( ) for min. cells were then mock infected or infected with nh/p and armenia/ at a multiplicity of infection (moi) of for h or h, a time by which psting was shown to be maximum for attenuated and virulent strains, respectively ( fig. b and c) . the cells were then collected, and the sting phosphorylation level was analyzed by western blotting. as fig. shows, sting phosphorylation was not detected in mock-infected cells, independent of ru treatment. as expected by this time of infection, during both nh/p and armenia/ infections sting was phosphorylated in the nontreated cells; however, interestingly, the level of sting phosphorylation decreased when ru at concentrations of and g/ml was added at the beginning of the infection. these results indicate the involvement of cgas in the activation of the sting pathway triggered, at early times after infection, either by nh/p or armenia/ asfv strains. infection of nh/p phosphorylates irf and induces its binding to chromatin. as mentioned above, we have observed that infection with nh/p is able to substantially activate the cgas-sting pathway. it has been described previously that the activation of sting and tbk induces the phosphorylation and then translocation of phosphorylated irf (p-irf ) to the nucleus ( , ) , where it acts as a transcription factor for ifn-␤. in order to analyze the phosphorylation status and cellular localization of irf during attenuated versus virulent asfv infections, we used a specific antibody to detect p-irf in different subcellular locations of the cell. to achieve this, mockinfected, nh/p -infected, or armenia/ -infected macrophages were fractionated after hpi, a time point when both sting and irf were found to be phosphorylated by nh/p but not by armenia/ ( fig. b and c). cytoplasmic (s ) and nuclear (p ) fractions were obtained from the respective whole-cell extracts (wce). next, the p fraction was further separated into nuclear soluble fraction (s ) and chromatin fraction (p ) (fig. a ). we used specific markers for each cell fraction to confirm the accuracy of the activation of sting by nh/p and armenia/ is dependent on the cgas sensor. porcine alveolar macrophages were untreated or treated with , , , or g/ml of ru and mock infected or infected with nh/p or armenia/ ( pfu/cell). at or h after armenia/ or nh/p infection, respectively, the cells were lysed in ripa buffer, and lysates were separated by to % sds-page, followed by immunoblotting with anti-p-sting, anti-sting, or anti-actin antibodies as a control. numbers below the bands indicate the relative level of phosphorylated sting. densitometry was performed using imagej. the fractionating procedure: actin for the soluble fraction, histone (h ) for the soluble nuclear fraction, and ␤ -laminin for the chromatin fraction. as shown in fig. b and p-irf is found in wce from nh/p infected cells, whereas it was not found in mock-infected cells or only in small amounts in armenia/ -infected cells. after cell fractionation, p-irf is mainly detected in the cytoplasmic fraction of alveolar macrophages infected with nh/p , as well as in the soluble nuclear fraction (s ) (fig. c ). p-irf was also detected in the chromatin fraction (p ) of nh/p -infected cells, suggesting that at h after nh/p infection, functional p-irf binds to chromatin, possibly acting as transcription factor for ifn-␤. only weak or no bands corresponding to p-irf were detected either in the chromatin fraction (p ) or in the nuclear soluble fraction (s ) in uninfected cells or in cells infected with armenia/ . these data suggest that the cgas-sting pathway is strongly activated during nh/p infection, resulting in the activation and translocation of p-irf to the nuclear chromatin fraction, where it activates synthesis of ifn-␤. in contrast, asfv armenia/ displays mechanisms which almost completely inhibit p-irf activation and translocation to the nucleus; this resulted in weak or no induction of ifn-␤ synthesis. armenia/ -induced blockage of the cgas-sting pathway happens early during viral infection. asfv displays a very accurate temporal kinetics of replication in the infected cell, producing early-early, early-late, and late viral mrnas. these differently timed messenger rnas are the products of viral genes whose transcription is regulated before or after viral dna replication ( ) . in order to determine at what step of the armenia/ replication the cgas-sting route is blocked, we used cytosine arabinoside (arac), an inhibitor of viral dna replication, widely used to prevent the expression of late asfv genes ( ) . in order to determine whether the cgas-sting inhibition produced by armenia/ occurs at early or late times of asfv replication, we infected alveolar macrophages in the presence or absence of arac and analyzed the phosphorylation of both sting and irf . the results showed that the cgas-sting pathway inhibition by armenia/ occurs at a time point prior to the replication of viral dna. no phosphorylation of sting and irf was observed in the presence of arac (fig. ), indicating that early viral genes and/or gene products are involved in this inhibition exerted by the virulent asfv strain. as a control for both the infection and the effects of arac on viral dna replication, we employed a specific antibody to assess the expression of the viral protein p , a late viral product, which should be only expressed in the absence of arac. as shown in the fig. , p is only found in the lanes corresponding to infected cells in the absence of arac. the blocking of the cgas-sting pathway mediated by armenia/ depends on the products of viral early gene(s). porcine alveolar macrophages were mock infected or infected with nh/p or armenia/ strain ( pfu/cell) and untreated or treated with g/ml of cytosine arabinoside (arac). at h postinfection, the cells were lysed in ripa buffer, and lysates were separated by to % sds-page, followed by immunoblotting with anti-p-sting, anti-sting, anti-p-irf , anti-irf , anti- , and antiactin antibodies, respectively. in contrast, nh/p infection induces activation of the cgas-sting route in the presence and absence of arac, since phosphorylation of both sting and irf are observed. this indicates that the replication of nh/p viral dna is not necessary to activate the cgas-sting pathway. armenia/ blocks sting and tbk phosphorylation by a cgamp-dependent mechanism. immediately after sensing cytoplasmic dsdna, cgas is activated to synthesize cgamp from atp/gtp; cgamp binds sting, inducing its phosphorylation, dimerization, and activation ( ) . in order to analyze the mechanism that armenia/ uses to block sting phosphorylation and to determine whether cgamp is involved, we infected macrophages with increasing mois of armenia/ (fig. a ). at hpi, a time point by which armenia/ fully inhibits sting phosphorylation (see fig. c ), cgamp ( g/ml) was added to the cultures. after an two additional hours of incubation ( hpi), the phosphorylation of both sting and tbk was analyzed by western blotting. as shown in fig. a , treatment of the noninfected cells with cgamp induces phosphorylation of sting and tbk . in the presence of cgamp, a decrease in the amounts of both p-sting and p-tbk was observed when cells were infected with armenia/ at an moi of ; this indicates that the virulent virus is able to reverse the sting and tbk activation induced by cgamp in a dose-dependent manner, probably also involving a c-gas-dependent mechanism. this result also shows that the viral process to inhibit sting/tbk activation can be overcome by cgamp, suggesting that cgamp is a critical component of the activation pathway. a specific monoclonal antibody against the asfv protein p was used as a control for asfv infection of the cells. in an additional experiment, cgamp ( g/ml) was added after h of infection with the attenuated or the virulent asfv strains and then incubated for two additional hours with or without arac (fig. b) . exogenous cgamp caused the phosphorylation of sting in noninfected cells, which is also observed during nh/p infection. interestingly, the inhibition of sting phosphorylation induced by armenia/ ( fig. c and ) was reversed by exogenous cgamp, confirming that armenia/ inhibits sting phosphorylation by a mechanism that involves cgamp. to assess whether these events are dependent on viral dna replication, cells were treated with g/ml of arac. there was no difference whether the cells were treated or not with arac, suggesting that both the sting blockage by armenia/ and the rescue by cgamp are mechanisms independent of viral replication, suggesting in turn that very early or early viral factors are involved. sting localizes at perinuclear microsomes in cells infected with nh/p but not in cells infected with armenia/ . as mentioned above, armenia/ blocks the cgas-sting pathway by using early viral effectors downstream of cgas activation. sting activation requires posttranslational modifications, dimerization, and transport from the er through the golgi to perinuclear structures, which are critical for its function ( ) . to investigate the localization of sting during asfv infection, porcine alveolar macrophages were mock infected or infected at an moi of for h with armenia/ or nh/p . cgamp-treated macrophages were used as a positive control of sting activation ( g/ml), and the samples were then processed for immunofluorescence by using a specific anti-sting antibody. as observed in fig. a , cgamp-treated cells and cells infected with nh/p show sting localization in punctuate perinuclear structures (or microsomes; highlighted by arrows). sting localization in microsomes indicates its activation and has been described as necessary for further activation of downstream components of the sting pathway ( ) . in contrast, cells infected with armenia/ present a more diffuse sting distribution pattern throughout the cytoplasm, similar to that found in mock-infected cells. the latter results indicate a nonactivation status of sting and confirms the small amount of p-sting we found during armenia/ infection (fig. c ). both armenia/ and nh/p infections were confirmed by the presence of viral factories, which are stained by dapi ( =, =-diamidino- -phenylindole) as small dna accumulations next to the cell nucleus (arrowheads). we quantified sting distribution pattern in infected cells, which were identified by the presence of viral factories. this analysis revealed that in more than % of nh/p -infected cells sting is localized in punctuated perinuclear microsome structures (fig. b ). in contrast, sting was localized in microsomes in less than % of armenia/ -infected cells (fig. b) . taken together, these results indicate that armenia/ induces a blockage in the activation and intracellular transport of sting, whereas nh/p infection does not, resulting in a typical punctuate pattern surrounding the nucleus characteristic of sting activation. differences in sting trafficking during nh/p versus armenia/ infection. sting undergoes posttranslational modifications necessary for its activation during its traffic through the golgi network. in particular, during the passage through the trans-golgi network (tgn), activated sting induces tbk activation, which is a key component in the pathway to activate ifn-␤ production ( ) . taking into account the inhibition found on sting trafficking mediated by virulent armenia/ (fig. ) , we further assessed the impact that virulent strain exerts on the cellular transit of sting. we hypothesized that the activation of sting, and therefore its traffic through the tgn, should be an event occurring at early times after infection, since we have found sting phosphorylation in the presence of arac. thus, we compared sting distribution either at "very early" ( hpi) or at "early" ( hpi) times after infection with attenuated or virulent asfv strains. to do this, we used either nh/p or armenia/ at an moi of to infect porcine alveolar macrophages, together with cgamp-treated macrophages as a positive control of sting activation; the cells were then processed for immunofluorescence, and specific antibodies against sting and against the ap complex (marker for tgn [ , ] ) were applied. as shown in fig. a similar colocalization pattern between sting and ap was found in cgamp-treated macrophages (fig. ) . this indicates that both attenuated and virulent asfv strains "activate" sting trafficking very early after infection. by hpi, sting accumulates in punctuated perinuclear structures in cells infected with nh/p but not with armenia/ (see fig. , lower panels). furthermore, sting partially colocalizes with ap in cytoplasmic structures outside from perinuclear structures in nh/p -infected cells. importantly, this colocalization pattern was not detected in cells infected with armenia/ (fig. , lower panels) . ap is located mainly around the asfv viral factories (arrowhead) in both armenia/ -and nh/p -infected cells, a localization that was previously described ( ) . the degree of colocalization pattern observed under these conditions (fig. a ) was quantified to determine the fraction of sting overlapping ap (mander's coefficient). as shown in fig. b , the highest colocalization between sting and ap was found in both nh/p -and armenia/ -infected cells after h of infection, similarly to cgamptreated cells. this colocalization degree strongly increases if quantification is restricted to cells where sting is activated, forming punctuate structures (fig. a , see arrows, and fig. c ). these data emphasize that sting activates and traffics through the tgn during both armenia/ and nh/p infection at the very early time of hpi. however, the sting-ap colocalization significantly decreased at hpi and more specifically in armenia/ -versus nh/p -infected cells, as shown in fig. b and c. altogether, these results suggest that whereas both virulent and attenuated asfv strains trigger the cgas-sting pathway at very early times postinfection, armenia/ is able to inhibit sting activation during later times of infection, thus impairing its activation and traffic to perinuclear microsomes. the manner how two different asfv strains ranging in virulence affect ifn-␤ production of infected cells through the control of the cgas-sting pathway has been addressed in detail in the present study. one asfv strain is nh/p , a nonfatal, naturally occurring, nonhaemadsorbing virus, isolated from a chronically infected pig ( ) , and the other asfv strain is armenia/ , a highly virulent virus currently circulating in europe, russia, and china ( ) . our results demonstrate that whereas armenia/ inhibits the synthesis of ifn-␤ (and its dependent genes ifit- and cxcl ) by inhibiting irf activation and sting phosphorylation, nh/p does not. infection of porcine alveolar macrophages with nh/p results in (i) the induction of significant levels of ifn-␤, (ii) phosphorylation of sting, (iii) traffic of sting through the golgi to perinuclear punctuated structures, and (iv) localization of irf to the nuclear soluble fraction and binding to chromatin. furthermore, inhibition of cgas by the specific inhibitor ru ( ) impairs nh/p -induced sting phosphorylation, suggesting that cgas is the main dna sensor activating the sting cascade by the attenuated asfv nh/p isolate. in contrast, cgamp-induced phosphorylation of sting was prevented by infection of cells with armenia/ . also, sting trafficking to perinuclear punctuated structures was severely impaired in armenia/ -infected cells, indicating that the virulent asfv strain prevents the activation and trafficking of sting. differences in the ifn-␤ mrna expression levels in infected cells have been reported for other virulent and attenuated asfv strains ( ) . similar observations were made in previous reports describing the inhibition of ifn-␣ production during virulent asfv strain infection ( , ) . in contrast, portugal et al. reported recently that ifn-␣ inhibition was exerted by both virulent and attenuated asfv strains ( ) . similarly, when ifn-␣ and ifn-␤ were analyzed in sera from animals infected with virulent asfv strains ( , ) , induction of ifn-␣ and ifn-␤ was observed, which was attributed to cytokine production by non-asfv-susceptible cells. since there are most likely several pathways and virulence factors involved in asfv virulence, this result is not surprising. it is noteworthy that whereas our data show a strong inhibition of ifit- mrna expression during armenia/ infection, in comparison with the expression during nh/p infection, the amount of ifn-␤ secreted after hpi with armenia/ does not affect ifn-␤-dependent gene ifit- expression. one possible explanation would be that virulent strain may encode genes functionally homologous to type i ifn receptors as has been described for vaccinia and other poxvirus ( ) , in such a way that the produced ifn is "neutralized" at this time point of the virulent infection ( hpi), impairing the activation of ifn-dependent genes. the cgas-sting pathway, which leads to the production of ifn-␤, is critical for the innate immune response and is therefore targeted by several viruses ( ) . here, we show for the first time that ifn-␤ production during asfv infection is controlled via the cgas-sting pathway. importantly, early after infection the cgas-sting pathway was induced by both attenuated and virulent asfv, but later on, was effectively inhibited only by virulent armenia/ . our data reveal a clear correlation between asfv virulence and virus ability to inhibit the cgas-sting pathway. similar results have been obtained with vaccinia virus, an asfv-related, dsdna cytoplasmic virus. the attenuated modified vaccinia virus ankara (mva) induces ifn-␤ in infected cells via the activation of the cgas-sting pathway ( ) , while virulent vaccinia strains prevent cgas-sting activation and hence ifn-␤ production in infected cells ( ) . our results, therefore, reinforce the idea that asfv virulence versus attenuation, two concepts controversially discussed in the asfv field, may be phenomena associated with the control of the cgas-sting pathway. activation of the cgas-sting pathway involves the phosphorylation and translocation of sting from the er through the golgi compartment, the phosphorylation of both tbk and irf , and the subsequent translocation of p-irf to the nucleus. using the viral dna replication inhibitor arac, we showed that sting phosphorylation is impaired by armenia/ in the presence of arac, thus involving events happening before viral dna replication. in contrast, sting phosphorylation is triggered as early as h after nh/p infection and leads to the phosphorylation of irf , a transcription factor regulating ifn-␤ synthesis ( ) . the different time points at which irf is phosphorylated during attenuated versus virulent strains suggest that a faster transition between activated sting and phosphorylated irf occurs during virulent strain infection. phosphorylated irf then translocates to the nucleus and binds to the chromatin fraction to induce ifn-␤ production. the latter happens during nh/p infection, whereas during armenia/ infection, irf was not phosphorylated and consequently absent from the nuclear and chromatin fractions of the infected cells. interestingly, it has been recently reported ( ) that sting phosphorylation is required to further activate irf . similarly, sting activation and p-irf have been found in the nuclear fraction in cells infected with influenza virus ( ) and the gammaherpesvirus rhesus rhadinovirus ( ) , which are both able to induce ifn-␤. furthermore, during mva versus virulent poxvirus wr infection, an opposing regulation of the ifn-␤ has been reported. the authors showed that attenuated mva virus induced activation of the cgas-sting pathway, whereas inhibition of this pathway was observed during virulent wr infection ( , ) ; this behavior is similar to what we report here in relation to the regulation of the cgas-sting pathway by nh/p and armenia/ in infected cells. sting is a prototype downstream effector, which can be modulated by several viral products in order to counteract the cgas-sting pathway and type i ifn production ( ) ( ) ( ) ( ) ( ) ( ) ( ) . activation of sting during viral infection can occur through the triggering of different cytoplasmic dsdna sensors ( ) ( ) ( ) . cgas was identified as the main sensor of this pathway in both humans and mice ( ) ( ) ( ) , playing an important role in type i ifn response against dna viruses, including hsv- , kshv, and vaccinia virus ( , , ) . importantly, we have been able to identify cgas as the main dna sensor involved in sting activation upon asfv infection in porcine alveolar macrophages. in fact, we found that a specific inhibitor of cgas, ru ( ) , blocked the activation of sting produced by nh/p infection. however, we cannot exclude the involvement of other sensors such as ifi or sensors triggered in the absence of active cgas ( , ) . in addition, we showed that cgamp, the cgas-activated product mediating the activation of sting, was counteracted during armenia/ but not nh/p infection, suggesting that the viral mechanism displayed by armenia/ to inhibit sting activation somehow competes with cgamp. it has been described that various viruses can act at different levels to counteract the ifn-␤ activation pathway ( ) . here, we show that virulent armenia/ is able to counteract cgamp-dependent sting activation, even in the presence of exogenous cgamp. however, alternative inhibition modes exerted by armenia/ or by other virulent asfv strains cannot be excluded, and future experiments are planned to assess this interesting possibility. it is remarkable that the activation of sting, after phosphorylation and dimerization, involves trafficking from the er through the golgi compartment to perinuclear punctuated structures, called microsomes ( , , ) . significantly, sting appears in these microsomes in most of the nh/p -infected cells at hpi, whereas at that time postinfection with armenia/ , sting is mainly found throughout the cytoplasm. transit of sting through the golgi, prior to its translocation into microsomes, has been shown to be a key event for sting activation, since sting undergoes posttranslational modifications and interaction with molecules such as tbk ( , ) . therefore, to further assess the virulence mechanism of asfv, we investigated whether the transit of sting through the golgi is blocked during armenia/ infection, as reported for other viruses such as hsv- or human cytomegalovirus ( , ) . at very early times after both nh/p and armenia/ infection, sting traffics through the tgn in a similar manner than in cgamp-treated cells, while at hpi this traffic is prevented in armenia/ -infected cells but not in nh/p -infected cells, thus impairing the ability of sting to reach microsomes. these results suggest that at very early times of infection, sting is activated in both nh/p and in armenia/ infected cells, but several hours later, the virulent asfv strain is able to block activation of sting. the involvement of the cgas-sting pathway in the control of ifn-␤ production during asfv infection has not been described so far, and therefore the potential viral candidate genes and/or the respective molecular mechanisms involved in blocking of the cgas-sting pathway are not yet known. from the results presented here, possible viral gene candidates could be very early genes, such as products of the so-called multigene families ( ) . in this regard, very recently, studies in human embryonic kidney (hek) cells, have postulated that the transfected dp r gene of the chinese virulent strain / could have a role in the control of the cgas-sting pathway ( ) . however, these results should be discussed cautiously, since (i) hek cells are not susceptible to asfv infection and (ii) these studies have not been performed in the context of an active asfv infection. in addition, the dp r gene is present and identical in both attenuated and virulent strains of the same genotype, such as nh/p and ba (both genotype i) and has % identity between attenuated and virulent strains of different genotypes, such as nh/p (genotype i) and armenia/ (genotype ii). therefore, the experiments performed above with dp r most likely do not explain the differences in the cgas-sting pathway modulation exerted by virulent and attenuated asfv strains. future in vitro and in vivo studies are necessary to elucidate the molecular mechanisms of control of the cgas-sting pathway by virulent strains of asfv. cell culture and virus infections. porcine alveolar macrophages (pam) were prepared by bronchoalveolar lavage as described previously ( ) and grown in dulbecco modified eagle medium (dmem) supplemented with mm l-glutamine, u/ml gentamicin, nonessential amino acids, and % porcine serum. cells were grown at °c in a % co atmosphere saturated with water vapor. cos- cells (from african green monkey kidney) were obtained from the american type culture collection (atcc) and grown in dmem supplemented with mm l-glutamine, u/ml gentamicin, nonessential amino acids, and to % fetal bovine serum (invitrogen life technologies). the asfv isolates armenia/ and nh/p were propagated on pam. briefly, subconfluent pam cells were cultivated in p plates and infected with asfv at an moi of . pfu/cell in dmem- % porcine serum. at hpi, the cells were recovered and centrifuged at , rpm for min. the cell pellet was discarded. the supernatant containing the viruses was clarified at , rpm for h at °c, resuspended in medium, and stored at Ϫ °c. the viruses were then titrated by plaque assay on cos- cells as previously described ( ) . infection was performed after asfv adsorption at °c for min, when the inoculum was removed, and fresh medium was added. the cells were then incubated at °c for the times indicated (in hpi). antibodies and reagents. polyclonal rabbit anti phospho-sting antibody (ser , catalog no. ), monoclonal rabbit anti phospho-irf antibody (ser , catalog no. ), and monoclonal rabbit anti phospho-tbk /nak antibody (ser , catalog no. ) were purchased from cell signaling. monoclonal mouse anti-irf antibody (sl- , sc- ), anti-m-igg secondary antibody (bp-hrp, sc- ), polyclonal goat anti actin antibody (i- , sc- ), and polyclonal goat anti ␤-laminin (s- ) were purchased from santa cruz biotechnology. polyclonal rabbit anti tmem /sting antibody ( - -ap) was purchased from proteintech. polyclonal rabbit anti-histone h antibody (ab ) was acquired from abcam. antiserum was kindly provided by e. tabarés; monoclonal mouse anti-p (s- d ) was kindly provided by s.-y. sunwoo. anti-rabbit/alexa fluor and anti-mouse/alexa fluor antibodies were purchased from invitrogen. anti-rabbit and anti-mouse immunoglobulin g coupled to peroxidase and ecl prime western blotting detection reagent were acquired from amersham biosciences. monoclonal mouse anti-␥-adaptin antibody (ap- , a ), anti-goat antibody g coupled to peroxidase, and digitonin were acquired from sigma-aldrich. cellular fractionation. pam were seeded in p plates ( ϫ cells/plate) and mock or asfv infected ( pfu/cell) for h. the wce, cytoplasmic fraction (s ), soluble nuclear fraction (s ), and chromatin fraction (p ) were isolated as described previously ( ) . briefly, cells were resuspended in buffer a ( mm hepes [ph . ], mm kcl, . mm mgcl , . m sucrose, % glycerol, mm dithiothreitol [dtt], . mm phenylmethylsulfonyl fluoride [pmsf] , and protease and phosphatase inhibitors [roche]) and triton x- were added (separating a part for wce), followed by incubation for min on ice. the nuclei were then centrifuged at , rpm min at °c. the first supernatant (s ) was centrifuged at , rpm min at °c, and the second supernatant was stored (s ). the first pellet (p ) was washed with buffer a and later was resuspended in buffer b ( mm edta, . mm egta, mm dtt, . mm pmsf, and protease and phosphatase inhibitors [roche]), incubated min on ice, and centrifuged at , rpm for min at °c. the third supernatant (s ) was stored, and the third pellet (p ) was washed with buffer b and finally resuspended in loading buffer. cgamp and ru treatment. cells were treated with a specific concentration of = =-cgamp (invivogen) or cgas inhibitor ru (invivogen) in digitonin permeabilization buffer ( mm hepes [ph . ], mm kcl, mm sucrose, mm mgcl , . % bovine serum albumin [bsa], mm atp, . mm dtt, and g/ml digitonin) ( ) . the cells were incubated for min at °c, and then the cells were washed with phosphate-buffered saline (pbs), and fresh medium or virus inoculation was added as indicated. for the ru -treated samples, cells were incubated for h with fresh dmem before being infected. elisa. pam were seeded in -well plates ( ϫ cells/well) and mock or asfv infected ( pfu/cell) in dmem- % porcine serum. asfv viral adsorption to cells was performed at °c for min. the inoculum was then removed, and l of fresh dmem medium without porcine serum was added. at the indicated times postinfection, cell culture supernatants were collected and assayed for porcine ifn-␤ using a porcine ifn-␤ elisa kit (mybiosource), as described by the manufacturer. immunofluorescence. pam were grown on coverslips and treated or untreated with cgamp ( g/ml) or mock or asfv infected at the indicated moi. at the indicated times postinfection ( or h after cgamp treatment, the cells were fixed with % paraformaldehyde for min at room temperature, permeabilized with . % triton x- for min, and blocked with pbs- % bsa for min. the cells were stained with the primary antibodies anti-sting antibody ( / ) and anti-␥-adaptin (ap ) antibody ( / ) diluted in pbs- % bsa for h. the cells were then washed with pbs and incubated with the fluorescence-conjugated secondary antibodies anti-rabbit images were acquired by using a clsm lsm coupled to an inverted axioobserver microscope (zeiss) with a ϫ oil immersion objective lens and analyzed using imagej. colocalization between sting and ap was quantified by calculating mander's coefficient (m ) using the plugin jacop pam were seeded in p plates ( ϫ cells/plate) and mock or asfv infected with gene expression levels were normalized to the housekeeping gene ( s rrna), and these values were then normalized to the mock-treated sample. the primers used were =-ggccc gaggttatctagagtc- = and =-tcaaaaccaacccggtca- = for porcine s rrna detection, =-tcagg gcaaagagagcctta- = and =-ggccattttgtctgaatgct- = for porcine ifit- detection, =-caatgaa aaagaatggggaga- = and =-cctttctttgctaattgctttca- = for porcine cxcl detection, =-aaaa atgataatgaaaccaatgaatg- = and =-atgagggctcttgctcaaac- = for viral p detection, and =-gtggaacttgatgggcagat- = and =-ttcctcctccatgatttcctc- = for porcine ifn-␤ detection. western blot analysis. pam were cultivated as indicated and mock or asfv infected. at the specified times postinfection, the cells were collected, washed with pbs, and lysed with radioimmunoprecipitation assay (ripa) buffer supplemented with protease and phosphatase inhibitors (roche) for min at °c, sonicated, and centrifuged at , rpm for min at °c. the supernatants were collected, and equal amounts of protein per sample were used. samples were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) and transferred to immobilon-p membranes (millipore). the membranes were incubated with the following specific primary antibodies: anti-sting antibody / , ) diluted in tris-buffered saline (tbs) supplemented with % milk. membranes were washed three times with tbs and exposed h to specific peroxidase-conjugated secondary antibodies: anti-rabbit and anti-mouse immunoglobulin g coupled to peroxidase ( / , and / , , respectively) from amersham biosciences and anti-m-igg secondary antibody ( / , ) from santa cruz biotechnology structural mechanism of cytosolic dna sensing by cgas cyclic gmp-amp synthase is a cytosolic dna sensor that activates the type i interferon pathway cgas-cgamp-sting: the three musketeers of cytosolic dna sensing and signaling cyclic gmp-amp is an endogenous second messenger in innate immune signaling by cytosolic dna african swine fever virus biology and vaccine approaches molecular characterization of african swine fever virus, china ictv virus taxonomy profile: asfarviridae african swine fever virus transcription the viral protein a l inhibits tnf-␣ expression through a cbp/p transcriptional coactivators pathway a l inhibits nf-atc , nf-b, and c-jun activation through a novel mechanism involving protein kinase c-theta-mediated upregulation of the amino-terminal transactivation domain of p regulation of inducible nitric oxide synthase expression by viral a l-mediated inhibition of p /rela acetylation and p transactivation the african swine fever virus dynein-binding protein p induces infected cell apoptosis the african swine fever virus lectin ep r modulates the surface membrane expression of mhc class i antigens the cd v protein of african swine fever virus interacts with the actin-binding adaptor protein sh p african swine fever virus iap homologue inhibits caspase activation and promotes cell survival in mammalian cells cd v interacts with adaptor protein ap- during african swine fever infection characterization of the african swine fever virus decapping enzyme during infection novel virulence and host range genes of african swine fever virus pathology of african swine fever: the role of monocytemacrophage porcine immune responses to african swine fever virus (asfv) infection virulence in african swine fever: its measurement and implications isolation of a non-haemadsorbing strain of african swine fever (asf) virus from a natural outbreak of the disease antigenic relationships among strains of african swine fecre virus inhibition of cgas-sting-tbk signaling pathway by dp r of asfv china / interferon-beta is a potent inducer of interferon regulatory factor- / -dependent ip- /cxcl expression in primary human endothelial cells interferon induced ifit family genes in host antiviral defense cgas produces a =- =-linked cyclic dinucleotide second messenger that activates sting pivotal roles of cgas-cgamp signaling in antiviral defense and immune adjuvant effects pan-viral specificity of ifn-induced genes reveals new roles for cgas in innate immunity small molecule inhibition of cgas reduces interferon expression in primary macrophages from autoimmune mice virus-dependent phosphorylation of the irf- transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation sting specifies irf phosphorylation by tbk in the cytosolic dna signaling pathway sting regulates intracellular dnamediated, type i interferon-dependent innate immunity activation of sting requires palmitoylation at the golgi visualization of tgn to endosome trafficking through fluorescently labeled mpr and ap- in living cells the non-haemadsorbing african swine fever virus isolate asfv/nh/p provides a model for defining the protective anti-virus immune response african swine fever virus isolate, georgia deletion of african swine fever virus interferon inhibitors from the genome of a virulent isolate reduces virulence in domestic pigs and induces a protective response african swine fever virus multigene family and genes affect host interferon response the low-virulent african swine fever virus (asfv/nh/p ) induces enhanced expression and production of relevant regulatory cytokines (ifn␣, tnf␣, and il p ) on porcine macrophages in comparison to the highly virulent asfv/l modulation of type i interferon signaling by african swine fever virus (asfv) of different virulence l and nhv in macrophage host cells sensitivity of african swine fever virus to type i interferon is linked to genes within multigene families and interferon status and white blood cells during infection with african swine fever virus in vivo a virus-encoded type i interferon decoy receptor enables evasion of host immunity through cell-surface binding the cgas-sting defense pathway and its counteraction by viruses modified vaccinia virus ankara triggers type i ifn production in murine conventional dendritic cells via a cgas/sting-mediated cytosolic dna-sensing pathway virulent poxviruses inhibit dna sensing by preventing sting activation cgas in action: expanding roles in immunity and inflammation mapk phosphatase expression induced by influenza and other rna virus infection negatively regulates irf activation and type i interferon response a rhesus rhadinovirus viral interferon (ifn) regulatory factor is virion associated and inhibits the early ifn antiviral response denv inhibits type i ifn production in infected cells by cleaving human sting sars coronavirus papain-like protease inhibits the type i interferon signaling pathway through interaction with the sting-traf -tbk complex hsv- degrades, stabilizes, requires, or is stung by sting depending on icp , the us protein kinase, and cell derivation dna tumor virus oncogenes antagonize the cgas-sting dna-sensing pathway hepatitis b virus polymerase disrupts k -linked ubiquitination of sting to block innate cytosolic dna-sensing pathways modulation of the cgas-sting dna sensing pathway by gammaherpesviruses herpes simplex virus ␥ . protein inhibits sting activation that restricts viral replication dai (dlm- /zbp ) is a cytosolic dna sensor and an activator of innate immune response ifi is an innate immune sensor for intracellular dna the helicase ddx senses intracellular dna mediated by the adaptor sting in dendritic cells ifi is required for dna sensing in human macrophages by promoting production and function of cgamp zcchc is a co-sensor of cgas for dsdna recognition in innate immune response atg a controls dsdna-driven dynamic translocation of sting and the innate immune response irhom is essential for innate immunity to dna viruses by mediating trafficking and stability of the adaptor sting human cytomegalovirus protein ul inhibits dna sensing of cgas to mediate immune evasion identification and utility of innate immune system evasion mechanisms of asfv production and titration of african swine fever virus in porcine alveolar macrophages chromatin association of human origin recognition complex, cdc , and minichromosome maintenance proteins during the cell cycle: assembly of prereplication complexes in late mitosis we thank sun-young sunwoo for providing the anti-p antibody. we also thank the imaging facilities of the centro de biología molecular severo ochoa for their excellent assistance.this study was funded by the u.s. department of homeland security under grant award number dhs- -st- -ag for the center of excellence for emerging and zoonotic animal disease (ceezad) and the state of kansas national bio and agro-defense facility (nbaf) transition fund and by grant cup j c from sardinia region (italy). r.g.-b. was funded by the programa operativo de empleo juvenil from autonomous community of madrid (spain). the funders had no role in the study, design, data collection and interpretation, or the decision to submit the work for publication. key: cord- -hjx d rq authors: márquez-jurado, silvia; nogales, aitor; Ávila-pérez, ginés; iborra, francisco j.; martínez-sobrido, luis; almazán, fernando title: an alanine-to-valine substitution in the residue of zika virus ns a protein affects viral rna synthesis and attenuates the virus in vivo date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: hjx d rq the recent outbreaks of zika virus (zikv), its association with guillain–barré syndrome and fetal abnormalities, and the lack of approved vaccines and antivirals, highlight the importance of developing countermeasures to combat zikv disease. in this respect, infectious clones constitute excellent tools to accomplish these goals. however, flavivirus infectious clones are often difficult to work with due to the toxicity of some flavivirus sequences in bacteria. to bypass this problem, several alternative approaches have been applied for the generation of zikv clones including, among others, in vitro ligation, insertions of introns and using infectious subgenomic amplicons. here, we report a simple and novel dna-launched approach based on the use of a bacterial artificial chromosome (bac) to generate a cdna clone of rio grande do norte natal zikv strain. the sequence was identified from the brain tissue of an aborted fetus with microcephaly. the bac clone was fully stable in bacteria and the infectious virus was efficiently recovered in vero cells through direct delivery of the cdna clone. the rescued virus yielded high titers in vero cells and was pathogenic in a validated mouse model (a mice) of zikv infection. furthermore, using this infectious clone we have generated a mutant zikv containing a single amino acid substitution (a v) in the ns a protein that presented reduced viral rna synthesis in cell cultures, was highly attenuated in vivo and induced fully protection against a lethal challenge with zikv wild-type. this bac approach provides a stable and reliable reverse genetic system for zikv that will help to identify viral determinants of virulence and facilitate the development of vaccine and therapeutic strategies. zika virus (zikv) is a recently emerged mosquito-borne member of the family flaviviridae, which was declared by the word health organization (who) as a global public health emergency on february [ , ] . like other flaviviruses, the viral particle is constituted by an inner nucleocapsid composed of the capsid (c) protein associated with the viral genomic rna (grna), surrounded by a lipid bilayer that contains the structural membrane (m) and envelope (e) proteins, which are arranged nucleus from the cmv promoter with a second amplification step in the cytoplasm driven by the viral polymerase. the recombinant virus rescued from the bac clone was fully infectious in vitro and in vivo. the zikv-rgn infectious clone was further used to evaluate the effect of a single amino acid change (alanine to valine) at residue of the ns a protein on viral rna synthesis and pathogenesis in vivo. we found that this unique single amino acid substitution impairs viral rna synthesis in cell culture and results in viral attenuation in a mice. remarkably, a single dose of the mutant virus was sufficient to induce protection against challenge with the parental wild-type (wt) zikv. these results demonstrate the reliability and potential of our bac approach to study zikv biology and to facilitate the development of vaccine and antiviral strategies. vero (a kidney epithelial cell line from an african green monkey) and a (an human adenocarcinomic alveolar epithelial cell line) cells were purchased from the american type culture collection (atcc, ccl- ) and were grown and maintained at • c and % co in growth medium, consisting in dulbecco's modified eagle's medium (dmem) supplemented with % fetal bovine serum (fbs) (hyclone, thermofisher scientific, madrid, spain), mm l-glutamine (sigma-aldrich, madrid, spain), % nonessential amino acids (sigma-aldrich), u/ml penicillin (sigma-aldrich) and µg/ml streptomycin (sigma-aldrich). the recombinant zikv-rgn wt (rzikv-rgn) or ns a mutant (rzikv-rgn-mns a) viruses were propagated in vero cells with virus growth medium (dmem supplemented with % fbs, mm l-glutamine, % nonessential amino acids, u/ml penicillin and µg/ml streptomycin) at • c and % co . for virus stocks preparation, to % confluent monolayers of vero cells were infected with a multiplicity of infection (moi) of . plaque forming units (pfu) per cell in virus growth medium and incubated at • c under % co . after - days of infection, the tissue culture supernatants were collected, clarified by centrifugation at × g for min, and stored in small aliquots at − • c. the bac plasmid pbelobac [ ] , kindly provided by h. shizuya (california institute of technology, pasadena, ca, usa), was used to assemble the zikv-rgn infectious cdna clone. this plasmid is a synthetic low-copy-number plasmid (one copy per cell) based on the escherichia coli (e. coli) f-factor [ ] that minimize the toxicity problems in the bacteria of exogenous sequences. e. coli dh b cells (invitrogen, thermofisher scientific) were used to amplify the bac plasmids. electrocompetent dh b cells (invitrogen, thermofisher scientific) were transformed by electroporation using a micropulser unit (bio-rad, madrid, spain), according to the manufacturer's instructions. bac-based plasmids were isolated and purified using the large-construct kit (qiagen, hilden, germany), following the manufacturer's specifications. we have assembled a zikv infectious cdna clone in the bac plasmid pbelobac , based on the data of the full-length sequence of the zikv clinical strain rgn [ ] deposited in the genbank (accession number ku ). this strain was selected because the full-length sequence was obtained directly from the virus-infected brain tissue of an aborted fetus with microcephaly in brazil in and therefore represents a good candidate to study zikv pathogenesis. the first step for the assembly of the full-length cdna clone was the selection of the restriction sites pmli, afei, and bstbi (genomic positions , and , respectively), which are unique in the viral genome ( figure a ). after that, four overlapping dna fragments covering the entire viral genome (zikv to zikv ) and flanked by the appropriate restriction sites, were generated by chemical synthesis (bio basic, inc., toronto, canada) ( figure b ). zikv fragment contained the cmv promoter precisely fused to the first nucleotides of the viral genome flanked at the '-end by apali and asci (absent in the viral genome) sites and at the '-end by a multiple-cloning site containing the selected restriction sites (pmli, afei and bstbi) followed by mlui (absent in the viral genome) and bamhi. fragments zikv (flanked by pmli and afei) and zikv (flanked by afei and bstbi) covered the genomic regions - and - , respectively. zikv fragment contained the restriction site bstbi, the last nucleotides of the viral genome, the hepatitis delta virus (hdv) ribozyme, the bovine growth hormone (bgh) termination and polyadenylation sequences, and the mlui restriction site. the infectious clone was assembled into pbelobac by sequential cloning of these four overlapping dna fragments. briefly, fragment zikv was digested with apali and bamhi and cloned into pbelobac −afei (a pbelobac without the afei restriction site) digested with the same enzymes, to generate the intermediate plasmid pbac-zikv . then, this plasmid was used as the backbone for the sequential cloning of the remaining overlapping dna fragments (zikv to zikv ) into the multicloning site of the intermediate plasmid (contains the restriction sites selected, pmli, afei, bstbi and mlui) to generate the full-length cdna clone pbac-zikv-rgn ( figure b) . the genetic integrity of the cdna clone was verified throughout the assembly process by extensive restriction analysis and sequencing. in all cases, the bacterial strain dh b (invitrogen, thermofisher scientific) was used as the e. coli host for all the cloning steps and the propagation of the bac cdna clone. to recover the infectious virus, vero cells on -well plates were grown to % confluence in growth medium without antibiotics, and transfected with µg of the bac cdna clone using µl of lipofectamine (invitrogen, thermofisher scientific), following the manufacturer's specifications. after h of incubation at • c, the transfection medium was replaced with fresh growth medium and the cells incubated at • c. aliquots of the culture supernatants were collected at h intervals for virus titer determination by plaque assay on vero cells. after five to seven days of transfection, when the cytopathic effect (cpe) was clear, cell culture supernatants were harvested and the recovered virus was cloned by three rounds of plaque purification. to determine the complete genome sequence of the rescued viruses, virions from supernatant of infected vero cells (moi of . pfu/cell) were purified through a % (w/v) sucrose cushion. viral rna was isolate from the purified virus with the qiaamp viral rna minikit (qiagen) following the manufacturer's instructions and deep-sequenced at the university of rochester genomics research center using illumina miseq (illumina, san diego, ca, usa). briefly, . µg of total viral rna was fragmented by controlled sonication and a dna library was generated using the nebnext mrna library prep master mix set for illumina (new england biolabs, ipswich, ma, usa), according to the manufacturer's instructions. after analyzing the library for size and quality (bio-analyzer; agilent technologies, inc., santa clara, ca, usa), deep-sequencing was performed using miseq (illumina) and the raw sequencing reads analyzed using swarm custom software. the genomic 'and '-terminal sequences were determined by the rapid amplification of cdna ends (race) using the '/ ' race second generation kit (roche, basilea, switzerland) with a polya-tail added to the cdna prior to the ' race reaction using polya polymerase (new england biolabs), following the manufacturer's instructions. to analyze the genetic stability of the recombinant zikv harboring the point mutation a v in the coding region of the ns a protein (rzikv-rgn-mns a), total rna was purified from vero cells infected with viruses from passage (p ) to passage (p ) using the rneasy minikit (qiagen), according to the manufacturer's specifications. purified rna ( ng) was reverse transcribed (rt) with random hexamer primers using the high-capacity cdna transcription kit (life technologies, thermofisher scientific), and the cdna was amplified by pcr with the forward primer zikv- vs viruses , , of ( '-gaggaatggtgctgcagg- '), spanning nucleotides to of the viral genome, and the reverse primer zikv- rs ( '-gcttgacatctccccag- ') , complementary to nucleotides to of the viral genome. finally, the amplicons generated covering the region encoding ns a and ns b proteins (genomic region - ) were sequenced by sanger sequencing (macrogen europe, amsterdam, netherlands) using specific oligonucleotides. vero cells seeded into -well plates at - % of confluence were infected with µl of serial -fold dilutions of the virus in virus growth medium without fbs for h at • c. after viral absorption, the viral inoculum was removed and the cells overlaid with ml of virus growth medium containing % deae-dextran (sigma-aldrich) and . % agar noble (difco, thermofisher scientific). after - days of incubation at • c under % co , the cells were fixed with % formaldehyde for h at room temperature, the overlaid removed, and the viral plaques visualized by staining with . % crystal violet in % methanol or by immunostaining with µg/ml of the pan-flavivirus e protein monoclonal antibody (mab) g (bei resources; nr- ) using the vectastain abc kit (vector laboratories inc., burlingame, ca, usa). visible plaques were counted and virus titers were calculated as pfu/ml. vero and a cells seeded into -well plates at % of confluence were infected with the indicated viruses diluted in virus growth medium without fbs at the specified mois. after h of absorption at • c in % co , the virus inoculum was removed, the cell monolayers washed twice with pbs, and . ml of fresh virus growth medium was added to each well. cells were incubated at • c under % co and at selected time points, aliquots of tissue culture supernatants were collected and virus titers determined by plaque assay in vero cells as described above. viral rna synthesis was evaluated by quantitative rt-pcr (rt-qpcr). total intracellular rna from uninfected or infected vero cells was purified using the rneasy minikit (qiagen) and total cdna was synthetized from ng of purified rna using random hexamer primers and the high-capacity cdna transcription kit (life technologies, thermofisher scientific), following the manufacturer's specifications. using this cdna, the level of viral rna was further quantified by qpcr using a custom taqman assay specific for zikv-rgn rna. this taqman assay is constituted by the forward primer '-gaagagcatccagccagagaa- ' (spanning nucleotides to of the viral genome), the reverse primer '-ctgggagccatgaactgaca- ' (complementary to nucleotides to of the viral genome), and the probe '-fam-tggagtaccggataatg- iabkfq- ' (covering nucleotides to of the viral genome). to normalize for differences in rna sampling, the expression of the histone h b (reference housekeeping gene) was analyzed using a specific taqman gene expression assay (rh _s ; life technologies, thermofisher scientific). data were acquired with a real-time pcr system (life technologies, thermofisher scientific) and analyzed with abi prism software v . . . the relative quantifications were performed using the cycle threshold ( −∆∆ct ) method [ ] . all experiments and data analysis were miqe (minimum information for publication of quantitative real-time pcr experiments) compliant [ ] . the expression of zikv e protein was analyzed by indirect immunofluorescence assay (ifa). vero cells grown on coverslips in -well plates at - % of confluence were infected with the rescued rzikvs at the indicated mois. at selected time points post-infection, cells were fixed with % paraformaldehyde in mm hepes ph . during min at room temperature and then permeabilized with . % triton x- in pbs for min. after that, cells were treated for h at room temperature with blocking solution ( % fbs in pbs) and incubated with µg/ml of the pan-flavivirus e protein mab g (bei resources; nr- ) in blocking solution for h at room temperature. after three washed with pbs, cells were incubated at room temperature for h with donkey anti-mouse antibody conjugated to alexa fluor (invitrogen, thermofisher scientific) diluted : in blocking solution, extensively washed with pbs, and incubated for min with dapi ( ', '-diamidino- -phenylindole) (sigma-aldrich) diluted : in pbs for nuclear staining. finally, coverslips were mounted in prolong gold antifade reagent (invitrogen, thermofisher scientific) and analyzed on a leica sp confocal microscope. immunofluorescence acquired images were processed and analyzed with imagej . b software [ ] . for the evaluation of the virus-specific antibodies levels present in the sera of vaccinated mice, enzyme-linked immunosorbent assays (elisas) were performed as previously described [ ] . briefly, -well plates were coated with cell lysates from mock-or zikv-infected vero cells and incubated overnight at • c. the coated wells were washed with pbs, blocked with % bsa in pbs, and then incubated with two-fold dilutions (starting dilution of : ) of mice sera for h at • c. after that, plates were washed with water and incubated with hrp-conjugated goat anti-mouse igg ( : ; southern biotech, birmingham, al, usa) for h at • c. reactions were developed with tetramethylbenzidine (tmb) substrate (biolegend, san diego, ca, usa) for min at room temperature, quenched with n h so , and read at nm in a vmax kinetic microplate reader (molecular devices, san jose, ca, usa). the in vivo studies were performed in type-i interferon (ifn) receptor deficient (ifnr-/-) a mice (the jackson laboratory, bar harbor, me, usa) maintained in the animal care facility at the university of rochester under specific pathogen-free conditions. in this animal model, subcutaneous (s.c.) or intraperitoneal (i.p.) infection with zikv induces neurological disease and the animals succumb to viral infection, with high viral load in blood, brain, spin cord, and testes, consistent with manifestations of zikv infection in humans. although deficient in innate ifn responses, a mice retain their adaptive immunity and have been successfully used as a suitable model for testing antivirals and vaccines [ ] [ ] [ ] . to evaluate virus pathogenicity, female -to- -week-old a mice (n = ) were first anesthetized i.p. with a mixture of ketamine ( µg per gram of body weight) and xylazine ( µg per gram of body weight), and then mock-infected (pbs) or infected s.c. in the footpad with the indicated doses of rzikv-rgn or rzikv-rgn-mns a diluted in pbs in a final volume of µl. after viral infection, animals were monitored daily for morbidity (body weight loss and disease signs, including hunching, ruffling and hind limb paralysis) and mortality (survival) over days. mice showing more than % of body weight loss or severe paralysis were considered to have reached the experimental endpoint and were humanely euthanized. to correlate development of clinical symptoms and death with virus replication, -to- -week-old mice (n = ) were infected as described above and the viral titers in serum were determined at days (n = ) and (n = ) by plaque assay and immunostaining using the pan-flavivirus e protein mab g as indicated before. to evaluate the protection efficacy of the rzikv-rgn-mns a, female -to- -week-old a mice (n = ) were first anesthetized i.p. as indicated above, and then mock-immunized (pbs) or immunized s.c. in the footpad with pfu of rzikv-rgn-mns a diluted in pbs in a final volume of µl. at days post-immunization, mouse sera were collected by submandibular bleeding and the presence of total antibodies against zikv-rgn was evaluated by elisa. twenty-four hours after bleeding, mice were challenged s.c. in the footpad with pfu of rzikv-rgn and their morbidity and mortality monitored over days as previously described. to determine viral replication, challenged -to- -week-old a mice (n = ) were bleeding at days (n = ) and (n = ) post-challenge and viruses , , of zikv viremia was determined by plaque assay and immunostaining using the pan-flavivirus e protein mab g as previously described. for quantitative analyses, a two-tailed, unpaired student t test was used to analyze differences in mean values between groups. all results were expressed as mean ± standard deviations of the means. p values of < . were considered significant. for mice experiments, the meier log-rank test was used to compare survival data and the reed and muench method to determine the mouse lethal dose (mld ). graphpad prism v . software was used for all statistical analysis. all animal protocols were approved by the university of rochester committee of animal resources (protocol number: ucar- - / ; approval date: / / ) and complied with the recommendations in the guide for the care and use of laboratory animals of the national research council [ ] . to overcome the toxicity problems associated to several flavivirus sequences during its propagation in bacteria, we used the bac plasmid pbelobac (a single-copy plasmid derived from the e. coli f-factor) [ ] to assemble a zikv infectious cdna clone, based on the genome sequence of the rgn strain of zikv (genbank accession number ku ) [ ] (figure ). this zikv-rgn strain was selected because it has no laboratory passage history and the full-length genome sequence was obtained from a zikv-infected fetus with microcephaly in [ ] , constituting a good candidate to further study zikv pathogenesis. after appropriate selection of unique restriction sites in the zikv-rgn genome ( figure a ), four overlapping dna fragments (zikv to zikv ), spanning the full-length viral genome and flanked for the selected restriction sites, were chemically synthesized, and sequentially cloned into pbelobac to generate the infectious cdna clone pbac-zikv-rgn ( figure b ). fragment zikv contained the cmv immediate-early promoter to allow the expression of the viral rna in the nucleus by the cellular rna polymerase ii [ ] and fragment zikv was flanked at the '-end by the hdv ribozyme followed by the bgh termination and polyadenylation sequences to produce synthetic rnas bearing authentic '-ends of the viral genome. this dna-lunched system ensures capping of the viral rna and allows the recovery of infectious virus from the transfected cdna clone without the need of an in vitro transcription step. once assembled, the full-length sequence of the zikv-rgn bac clone was determined and no changes were detected to that reported for the zikv-rgn strain (genbank accession number ku ). finally, to confirm the stability of this synthetic infectious cdna clone in bacteria, the bac clone was passaged in e. coli dh b cells for more than two hundred generations and the genetic integrity of the passaged infectious clone analyzed by restriction endonuclease analysis and sequencing. no differences were detected, demonstrating that the zikv-rgn bac clone was fully stable in bacteria and that the bac approach is a reliable and simple method to generate zikv infectious cdna clones. to recover the infectious virus ( figure ), vero cells were transiently transfected with the bac cdna clone using lipofectamine and virus production analyzed during seven days. in contrast to mock-transfected cells, increasing amounts of infectious virus were detected in the tissue culture supernatant of cells transfected with the infectious clone, with peak titers around pfu/ml on day five ( figure a ). to further confirm the identity of the rescued virus, vero cells were infected with an moi of . pfu/cell of the rescue virus and monitored for cpe induction and viral e protein expression by ifa using the pan-flavivirus e protein mab g ( figure b ). the rescued virus induced a clear cpe, characterized by the presence of rounded and birefringent cells, and high levels of e protein expression were detected in the perinuclear region of infected cells. these results demonstrated that the zikv-rgn infectious bac cdna clone produces high titers of rzikv-rgn directly after transfection of susceptible vero cells. to recover the infectious virus ( figure ), vero cells were transiently transfected with the bac cdna clone using lipofectamine and virus production analyzed during seven days. in contrast to mock-transfected cells, increasing amounts of infectious virus were detected in the tissue culture supernatant of cells transfected with the infectious clone, with peak titers around pfu/ml on day five (figure a ). to further confirm the identity of the rescued virus, vero cells were infected with an moi of . pfu/cell of the rescue virus and monitored for cpe induction and viral e protein expression by ifa using the pan-flavivirus e protein mab g ( figure b ). the rescued virus induced a clear cpe, characterized by the presence of rounded and birefringent cells, and high levels of e protein expression were detected in the perinuclear region of infected cells. these results demonstrated that the zikv-rgn infectious bac cdna clone produces high titers of rzikv-rgn directly after transfection of susceptible vero cells. once the identity of the rescued virus was confirmed, it was cloned by three round of plaque purification, and its phenotypic and genotypic properties were determined. analysis of the growth kinetics revealed that the rzikv-rgn replicated efficiently in both vero and a cells, reaching peak titers of approximately and pfu/ml at hpi, respectively ( figure a ). in addition, the rescued virus generated homogeneous plaques of about mm in size after four days of infection in vero cells ( figure b ). finally, the genetic identity of the virus was analyzed by deep-sequencing of two independent clones. full-genome sequencing of both viral clones revealed that both clones presented the same sequence that the cdna clone. overall, these results demonstrate the feasibility of generating infectious rzikvs using a bac-based approach. once the identity of the rescued virus was confirmed, it was cloned by three round of plaque purification, and its phenotypic and genotypic properties were determined. analysis of the growth kinetics revealed that the rzikv-rgn replicated efficiently in both vero and a cells, reaching peak titers of approximately and pfu/ml at hpi, respectively ( figure a ). in addition, the rescued virus generated homogeneous plaques of about mm in size after four days of infection in vero cells ( figure b ). finally, the genetic identity of the virus was analyzed by deep-sequencing of two independent clones. full-genome sequencing of both viral clones revealed that both clones presented the same sequence that the cdna clone. overall, these results demonstrate the feasibility of generating infectious rzikvs using a bac-based approach. once the identity of the rescued virus was confirmed, it was cloned by three round of plaque purification, and its phenotypic and genotypic properties were determined. analysis of the growth kinetics revealed that the rzikv-rgn replicated efficiently in both vero and a cells, reaching peak titers of approximately and pfu/ml at hpi, respectively ( figure a ). in addition, the rescued virus generated homogeneous plaques of about mm in size after four days of infection in vero cells ( figure b ). finally, the genetic identity of the virus was analyzed by deep-sequencing of two independent clones. full-genome sequencing of both viral clones revealed that both clones presented the same sequence that the cdna clone. overall, these results demonstrate the feasibility of generating infectious rzikvs using a bac-based approach. to determine whether the rescued rzikv-rgn was pathogenic in vivo (figure ) , groups of five female -to- -week-old a mice (ifnr-/-) were inoculated s.c. in the footpad with pbs (as negative control) or with different doses of rzikv-rgn ( , and pfu per animal) and the morbidity (body weight loss and disease signs) and survival were monitored daily over days ( figure a ). as expected, weight loss and survival correlated with the inoculated dose. mice infected with pfu did not show disease symptoms, only a slight reduction in body weight was detected on days to , and all of them survived. in the case of mice infected with pfu, they presented some symptoms of disease (hunching and reduced mobility) and weight loss from days seven to nine (with a maximum of % on day nine), but all of them recovered the initial body weight and survived. in contrast, animals infected with pfu showed hind limb paralysis, rapidly lost weight, and all of them succumbed to viral infection between days seven and eight post-infection ( figure a ). using the reed & muench method we determined that the mld of rzikv-rgn was approximately × pfu. to further analyze whether the virulence observed correlated with viral replication, viral titers in mouse sera were analyzed at days two and four post-infection ( figure b ). as expected, the viremia in the infected animals was dose dependent, reaching the highest titers at day two after inoculation. at day four post-inoculation a significant reduction of the viral titers was observed. in the case of animals infected with or pfu, viremia was not detected or only detected in one of the three infected mice, respectively ( figure b ). overall, these results indicated that the rzikv-rgn recovered from the infectious clone is virulent in mice but only at high ( pfu) dose. to determine whether the rescued rzikv-rgn was pathogenic in vivo (figure ) , groups of five female -to- -week-old a mice (ifnr-/-) were inoculated s.c. in the footpad with pbs (as negative control) or with different doses of rzikv-rgn ( , and pfu per animal) and the morbidity (body weight loss and disease signs) and survival were monitored daily over days ( figure a ). as expected, weight loss and survival correlated with the inoculated dose. mice infected with pfu did not show disease symptoms, only a slight reduction in body weight was detected on days to , and all of them survived. in the case of mice infected with pfu, they presented some symptoms of disease (hunching and reduced mobility) and weight loss from days seven to nine (with a maximum of % on day nine), but all of them recovered the initial body weight and survived. in contrast, animals infected with pfu showed hind limb paralysis, rapidly lost weight, and all of them succumbed to viral infection between days seven and eight post-infection ( figure a ). using the reed & muench method we determined that the mld of rzikv-rgn was approximately × pfu. to further analyze whether the virulence observed correlated with viral replication, viral titers in mouse sera were analyzed at days two and four post-infection ( figure b ). as expected, the viremia in the infected animals was dose dependent, reaching the highest titers at day two after inoculation. at day four post-inoculation a significant reduction of the viral titers was observed. in the case of animals infected with or pfu, viremia was not detected or only detected in one of the three infected mice, respectively ( figure b ). overall, these results indicated that the rzikv-rgn recovered from the infectious clone is virulent in mice but only at high ( pfu) dose. week-old a mice (five mice per group) were mock-infected (pbs) or infected s.c. in the footpad with the indicated pfu of rzikv-rgn, and body weight loss (expressed as the percentage of starting weight, left panel) and survival (right panel) were monitored daily during days. mice that lost more than % of their initial body weight or presented hind limb paralysis were humanely euthanized. error bars represent standard deviations of the mean for each group of mice. asterisks indicate that the differences between viral doses of and are statistically significant when data are compared using the unpaired t test (*, p < . ; **, p < . ). (b) viral titers in mice sera. female -to- -week-old a mice (six mice per group) were infected with the indicated pfu of rzikv-rgn as described above, and viral titers in sera were determined at days two and four after infection (three animals per time point) by plaque assay and immunostaining using the pan-flavivirus e protein mab g . symbols represent data from individual mice and bars the geometric means of viral titers. asterisks indicate that the differences in viral titers between experimental samples are statistically significant when data are compared using the unpaired t test (**, p < . ; ***, p < . ). &: virus not detected in two mice; nd: virus not detected. the detection limit of the assay ( pfu/ml) is indicate as a dashed line. during the assembly of the pbac-zikv-rgn infectious clone, we detected the presence of a point mutation in the ns a protein, which was introduced during the chemical synthesis of fragment zikv . this mutation consists in a cytosine-to-thymidine substitution at genomic position , resulting in an alanine-to-valine change in the residue of the ns a protein (a v). because this mutation consists of a conservative amino acid change, we decided to explore the possibility of using this mutation as a genetic marker. to this end, the infectious clone pbac-zikv-rgn-mns a was generated by replacing the zikv wt fragment for that containing the ns a a v mutation. this infectious clone was fully stable in bacteria and no additional mutations were observed after sequencing the full-length clone. after that, vero cells were transfected with the mutant infectious clone and the recovery efficiency of the rzikv-rgn-mns a mutant virus was compared to that of the parental rzikv-rgn virus ( figure ). although the infectious virus was recovered in both cases, virus production was one logarithm lower in the case of the mutant rzikv-rgn-mns a, reaching maximum titers of pfu/ml at seven days post-transfection ( figure a ). when the plaque phenotype was analyzed, we found that the plaque size of the mutant rzikv-rgn-mns a was smaller (more than a -fold reduction) than that of the parental rzikv-rgn ( figure b ), indicating that the a v mutation, despite of being a conservative substitution, caused reduction in plaque size and virus production. in addition, an in silico analysis was performed to evaluate the frequency of amino acid residues of the ns a protein in more than zikv strains sequences deposited in the database [ ] (https://www.viprbrc.org/brc/home.spg?decorator=flavi). this analysis indicated that amino acid a is highly conserved, since the % of the analyzed zikv sequences contained an alanine residue at this position. to further confirm the effect of the ns a a v mutation on virus production, the growth kinetics at high ( pfu/cell) and low ( . pfu/cell) moi of the mutant virus were compared to those of the parental virus ( figure c ). again, a reduction of about one logarithmic unit in virus production was detected in vero cells infected with the mutant virus both at high and low moi ( figure c ). taken into consideration that flavivirus ns a protein is involved in regulation of rna replication and virus assembly [ ] , we further analyzed whether the reduction in plaque size and virus production of the mutant virus was associated with reduced viral rna synthesis. to this end, the production of viral rna in vero cells infected with either the parental or mutant viruses at an moi of . pfu/cell was analyzed at and hpi by rt-qpcr using a custom taqman assay specific for zikv-rgn genome ( figure d ). at both times, a -fold reduction in the levels of viral rna was observed in cells infected with the mutant virus ( figure d ), confirming that ns a a v mutation at least impairs viral rna synthesis. in agreement with these data, a reduction in the expression levels of zikv e protein was observed by ifa in vero cells infected with the mutant virus in comparison to cells infected with the parental virus ( figure e ). finally, to discard the presence of other undesired mutations, the full-length sequence of the mutant virus was determined by deep-sequencing, and no mutations other than ns a a v were detected. collectively, these results indicated that ns a a v mutation alone affected zikv growth in vero cells at least by impairing viral rna synthesis. to investigate whether the reduced rna synthesis of rzikv-rgn-mns a in vero cells could result in viral attenuation in vivo, the ability of the mutant virus to induce pathogenesis was analyzed in a mice and compared with that of the parental rzikv-rgn ( figure ). to that end, groups of five female -to- -week-old a mice were inoculated s.c. in the footpad with pfu of either rzikv-rgn or rzikv-rgn-mns a, or with pbs as a negative control, and the body weight loss and survival were monitored daily over days. in contrast to mice infected with rzikv-rgn that quickly lost weight and all of them died at day eight after infection, mice infected with the mutant rzikv-rgn-mns a did not presented any clinical signs of infection or weight loss and all of them survived to viral infection ( figure a ). to further analyze the correlation of the attenuation of the mutant virus with viral replication, presence of the virus in mice sera was analyzed at days two and four post-infection. in agreement with the pathogenicity data, mice infected with the mutant virus presented lower viremia than mice infected with the parental virus ( figure b ). the mutant virus was only detected at day two after infection and at lower titers (approximately . logarithms lower) than the parental virus. as an internal control of the experiment, the plaque phenotype of the viruses recovered from the blood of infected mice were analyzed. as expected, rzikv-rgn formed big plaques while the mutant rzikv-rgn-mns a formed small plaques ( figure c ). these results indicated that rzikv-rgn-mns a was highly attenuated in mice, as compared to rzikv-rgn, and that this attenuation may be due to a lower replication of the rzikv-rgn-mns a mutant virus. after elucidating that rzikv-rgn-mns a was attenuated in vivo, its ability to induce protection against a challenge with the parental rzikv-rgn was analyzed (figure ) . to that end, groups of five female -to- -week-old a mice were vaccinated s.c. in the footpad with pfu of rzikv-rgn-mns a or mock-vaccinated with pbs. twenty days after vaccination, blood samples were collected to evaluate the humoral response. one day later, mice were challenged with a lethal dose ( pfu) of rzikv-rgn and the body weight loss and survival were analyzed daily over figure . pathogenesis of rzikv-rgn-mns a in a mice. (a) weight loss and mortality. female -to- -week-old a mice (five mice per group) were mock-infected (pbs) or infected s.c. in the footpad with pfu of rzikv-rgn or rzikv-rgn-mns a, and body weight loss (expressed as the percentage of starting weight, left panel) and survival (right panel) were monitored daily during days. mice that lost more than % of their initial body weight or presented hind limb paralysis were humanely euthanized. error bars represent standard deviations of the mean for each group of mice. (b) viral titers in mice sera. female -to- -week-old a mice (six mice per group) were infected with pfu of rzikv-rgn (wt) or rzikv-rgn-mns a (mut) as described above, and viral titers in sera were determined at days two and four after infection (three animals per time point) by plaque assay and immunostaining using the pan-flavivirus e protein mab g . symbols represent data from individual mice and bars the geometric means of viral titers. asterisks indicate that the differences between rzikv-rgn and rzikv-rgn-mns a are statistically significant when data are compared using the unpaired t test (***, p < . ). nd: virus not detected. the detection limit of the assay ( pfu/ml) is indicate as a dashed line. (c) plaque phenotype. vero cells at % confluence ( -well plate format) were infected with pfu of rzikv-rgn (left) or rzikv-rgn-mns a (right) recovered from infected mice at day two post-infection and the plaque size evaluated by plaque assay and immunostaining using the pan-flavivirus e protein mab g . after elucidating that rzikv-rgn-mns a was attenuated in vivo, its ability to induce protection against a challenge with the parental rzikv-rgn was analyzed (figure ) . to that end, groups of five female -to- -week-old a mice were vaccinated s.c. in the footpad with pfu of rzikv-rgn-mns a or mock-vaccinated with pbs. twenty days after vaccination, blood samples were collected to evaluate the humoral response. one day later, mice were challenged with a lethal dose ( pfu) of rzikv-rgn and the body weight loss and survival were analyzed daily over days. as expected, mice vaccinated with pbs lost weight rapidly, showed clear symptoms of disease, and all of them succumbed to challenge with rzikv-rgn. in contrast, mice vaccinated with rzikv-rgn-mns a did not lose weight and all of them survived the challenge with rzikv-rgn ( figure a ), indicating that a single immunization dose with rzikv-rgn-mns a is enough to induce full protection against zikv-rgn. in agreement with these data, a strong humoral response against zikv-rgn was observed in mice vaccinated with rzikv-rgn-mns a ( figure b ) and no viremia was detected in sera samples of vaccinated mice at days two and four after challenge ( figure c ). figure a ), indicating that a single immunization dose with rzikv-rgn-mns a is enough to induce full protection against zikv-rgn. in agreement with these data, a strong humoral response against zikv-rgn was observed in mice vaccinated with rzikv-rgn-mns a ( figure b ) and no viremia was detected in sera samples of vaccinated mice at days two and four after challenge ( figure c ). once confirmed that rzikv-rgn-mns a was attenuated in vivo and induces protection against zikv-rgn in mice, the genetic stability of the mutant virus was analyzed in vero cells, in order to test the possible use of this mutant virus as a base for the development of a live-attenuated zikv vaccine (figure ). to this end, both mutant (rzikv-rgn-mns a) and parental (rzikv-rgn) viruses were passaged five times in vero cells (p to p ) and the virus plaque phenotype, growth kinetics and the sequence of ns a were analyzed for each passage. analysis of the plaque phenotype showed that the parental virus presented the expected plaque size throughout all passaging. in contrast, for the mutant virus a reversion to parental plaque phenotype was observed. this reversion started at p ( % of big plaques), clearly increased at p ( % of big plaques) and was complete at p ( figure a ). after that, we analyzed whether this plaque size reversion of the mutant rzikv-rgn-mns a correlated with an increase in virus replication to levels of that of the parental virus. to that end, the growth kinetics (moi of pfu/cell) of the mutant virus from p and p were compared to those of the parental virus. growth curve analysis showed that in contrast to the mutant virus from p , the virus from p replicated to the same levels as the parental virus ( figure b ), suggesting that the mutant virus reverted to the wt sequence during its propagation in vero cells. to confirm these observations, the ns a coding region of mutant viruses from p to p was amplified by rt-pcr and sequenced. sequence analysis confirmed the reversion of the a v mutation to the wt sequence. although the instability and reversion of the mutant virus to the wt sequence during its propagation in vero cells limits the use of this mutant for vaccine development, these data further support the importance of this ns a residue for virus replication. at hpi, cell culture supernatants were collected and used to infect fresh vero cells. this process was repeated four more times and virus stocks of passages to (p to p ) were generated. (a) plaque size. vero cells were infected with the different passages (p to p ) of rzikv-rgn or rzikv-rgn-mns a, and at four days post-infection the viral plaques were visualized by immunostaining using the pan-flavivirus e protein mab g . (b) growth kinetics. vero cells at % confluence ( -well plate format; triplicates) were infected (moi of pfu/cell) with p and p of rzikv-rgn or rzikv-rgn-mns a and at the indicated hpi, virus titers were determined by plaque assay. error bars represent standard deviations of the mean from three experiments. asterisks indicate that the differences between rzikv-rgn-mns a p and the experimental samples, rzikv-rgn p , rzikv-rgn p and rzikv-rgn-mns a p , are statistically significant when data are compared using the unpaired t test (***, p < . ). analysis of the plaque phenotype showed that the parental virus presented the expected plaque size throughout all passaging. in contrast, for the mutant virus a reversion to parental plaque phenotype was observed. this reversion started at p ( % of big plaques), clearly increased at p ( % of big plaques) and was complete at p ( figure a ). after that, we analyzed whether this plaque size reversion of the mutant rzikv-rgn-mns a correlated with an increase in virus replication to levels of that of the parental virus. to that end, the growth kinetics (moi of pfu/cell) of the mutant virus from p and p were compared to those of the parental virus. growth curve analysis showed that in contrast to the mutant virus from p , the virus from p replicated to the same levels as the parental virus ( figure b ), suggesting that the mutant virus reverted to the wt sequence during its propagation in vero cells. to confirm these observations, the ns a coding region of mutant viruses from p to p was amplified by rt-pcr and sequenced. sequence analysis confirmed the reversion of the a v mutation to the wt sequence. although the instability and reversion of the mutant virus to the wt sequence during its propagation in vero cells limits the use of this mutant for vaccine development, these data further support the importance of this ns a residue for virus replication. moreover, these data also suggest that zikv ns a protein represents a good target for the development of antivirals against zikv infection. the significance of zikv to public health due its association with guillain-barré syndrome and fetal abnormalities [ ] [ ] [ ] [ ] [ ] [ ] , together with the lack of approved antiviral agents or vaccines, have triggered a global effort to study this flavivirus in order to develop effective strategies to prevent and control zikv infection in humans. in this respect, the development and implementation of reverse genetic approaches for zikv provide investigators with a novel and powerful experimental tool to study both the biology and pathogenesis of zikv as well as the development of attenuated forms of zikv for their implementation as live-attenuated vaccines. however, as described for other flaviviruses, the generation of zikv infectious clones using traditional approaches are very difficult due to the toxicity and instability of some viral sequences when they were propagated as cloned cdna in bacteria [ ] [ ] [ ] . in the past two years, several approaches that overcomes this toxicity problem have been applied for the successfully generation of zikv infectious clones. these include the use of low-copy plasmids [ , ] , insertion of introns to disrupt toxic sequences [ ] [ ] [ ] , mutational silencing of cbps present in the viral genome [ ] , in vitro ligation of cdna fragments [ , , ] , the isa method [ , ] , and the cper approach [ ] . although very useful, some of these approaches are laborious, time consuming and present several disadvantages. for instance, most of them need in vitro ligation and transcription steps that complicate the assembly and reduce the recovery efficiency. moreover, these low recovery efficiencies increase the presence of undesired mutations that could result in in vitro and/or in vivo attenuation, limiting the use of these infectious clones for certain studies. others, such as the mutational silencing of cbps, in which a high number of silent mutations have to be introduced, could affect viral fitness. finally, the use of low-copy plasmids has been shown to be effective for several zikv strains but not for others, probably due the different degrees of toxicity of rna sequences of different strains [ , , , ] . here, we describe a powerful approach for the generation of an infectious cdna clone of the zikv-rgn strain in a single plasmid, based on the use of a combination of synthetic biology and bacs. the full-length cdna copy of the zikv-rgn strain was generated from four synthetic dna fragments and cloned in the bac plasmid pbelobac [ ] under the control of the cmv promoter, which allows the expression of the viral rna in the nucleus [ ] , and flanked at the '-end by the hdv ribozyme and the bgh polyadenylation and termination sequences to produce synthetic rnas bearing authentic '-ends of the viral genome. the bac cdna clone was fully stable during its propagation in bacteria and the functional infectious virus was rescued after direct transfection of susceptible vero cells that was pathogenic in a mice. a zikv-rgn infectious clone generated using the cper approach has been recently reported [ ] . however, in contrast to our results, the rescued virus was asymptomatic and nonlethal in female -to- -week-old a mice infected with doses of to ccid ( % cell culture infective doses) via the s.c. route. whether the differences in pathogenicity among this rzikv-rgn and ours are related to the experimental approach (cper versus bac), the age of the mice ( -to- -week-old versus -to- -week-old) or the moi used to infect the mice ( - ccid versus pfu) remain to be evaluated. although other zikv reverse genetic systems have been reported (discussed above), the bac approach constitutes an useful alternative that presents important advantages: (i) the bac plasmids present a strictly controlled replication leading to only one plasmid per cell and therefore minimize the toxicity associated with several flavivirus sequences when amplified in bacteria [ ] . this allows the easy and direct manipulation of the viral genome for molecular studies; (ii) similarly to other approaches using polii-driven promoters, the bac approach results in intracellular expression of the viral rna [ , , , , ] , allowing the capping of the viral rna and the recovery of infectious virus without the need of an in vitro transcription step. although some splicing events could occur during the nuclear expression of the viral genome, mainly due to the presence of donor and acceptor putative sequences in the viral genome, the efficiency of this phenomenon is very low and does not affect the recovery of infectious viruses [ ] ; (iii) like other systems based on transfection of dna constructs [ , , , , ] , bac cdna clones present a higher efficiency of transfection than rna transcripts in mammalian cells. this allows higher efficiencies of virus recovery, reducing the passages in cell culture to get a viral stock and therefore, the possibility of introducing undesired mutations by cell culture adaptation; (iv) the manipulation of bac cdna clones is relatively easy and similar to that of conventional plasmids with slight modifications due to the presence of only one plasmid copy per cell. in addition to standard protocols, the bac cdna clones could also be efficiently modified into e. coli by homologous recombination using a two-step approach that combine the red recombination system and counterselection with the homing endonuclease i-scei [ ] [ ] [ ] [ ] ; and (v) the bac approach has been successfully used to engineer infectious clones of other flaviviruses, including denv [ ] , and several coronaviruses that contain the largest viral rna genome known and similar toxicity problems to those described for flaviviruses [ , [ ] [ ] [ ] [ ] . these data highlight the potential of the bac approach for the rapid and reliable construction of stable infectious clones of emerging flavivirus and other similar rna viruses with unstable viral genomes when amplified as cdnas in bacteria. the zikv reverse genetic system described in this article was further used to study the effect of a single amino acid substitution (a v) in the viral ns a protein on virus growth in cultured cells and pathogenesis in vivo. our results suggested that this single amino acid change impaired viral rna synthesis and virus production in cell culture and highly attenuated the virus in mice. however, we cannot discard that this mutation in the ns a protein could affect other steps in the replication cycle of the virus. flavivirus ns a protein is a -kda hydrophobic protein associated with the endoplasmic reticulum that contains eight transmembrane domains. it is a multifunctional protein that has been involved in viral rna synthesis [ , ] , virus assembly [ , ] , membrane rearrangement [ ] , and immunomodulation of innate immune response [ ] [ ] [ ] . by homology with the denv ns a topology, the zikv ns a a v mutation maps in the last transmembrane domain, for which no specific function has been reported. therefore, our data constitute the first evidence of a role of this ns a domain in viral rna synthesis. on the other hand, it is important to note that the ns a a v mutation promoted a -fold reduction in viral rna synthesis and more than -fold reduction in virus production. this reduced virus production could be a consequence of the rna synthesis impairment. however, since the reduction in virus production is higher than that observed in rna synthesis and that flavivirus ns a protein is also involved in virus assembly, we cannot discard an additional effect of a v mutation in zikv assembly. in addition, we have found that the mutant virus was attenuated in a mice. this attenuation could be explained as a consequence of the lower rna synthesis of the mutant virus. however, an additional effect of the a v mutation on the putative immunomodulatory role of ns a protein [ ] [ ] [ ] , leading to virulence attenuation, cannot be discarded. future studies will be required to determine whether this mutation affects only viral rna synthesis or also virus assembly and immunomodulation of the host defenses. importantly, we have shown that immunization with a single dose of pfu of the mutant rzikv-rgn-mns a induced protection against a lethal challenge with the parental rzikv-rgn, suggesting the potential implementation of this ns a mutant as the base of a live-attenuated vaccine. unfortunately, the mutant rzikv-rgn-mns a was instable and reverted to the wt sequence during its propagation in vero cells, limiting the use of this mutation alone for vaccine development. however, this instability and the high conservation of the amino acid a of the ns a protein among zikv strains highlights the importance of this ns a residue for virus replication, and therefore the potential use of ns a protein as a good target for antiviral development against zikv infection. in summary, we have developed a powerful zikv reverse genetic system based on the use of bacs that has allowed us to identify a single point mutation in the ns a protein that attenuates the virus in vitro and in vivo. this infectious clone system provides a valuable tool to the research community to explore zikv molecular biology, viral determinants of zikv pathogenesis, virus-host interactions, and vaccine and antivirals developments. an update on zika virus infection who calls off global zika emergency the . å resolution cryo-em structure of zika virus probing molecular insights into zika virus(-)host interactions zika virus: a report on three cases of human infection during an epidemic of jaundice in nigeria simultaneous outbreaks of dengue, chikungunya and zika virus infections: diagnosis challenge in a returning traveller with nonspecific febrile illness the global threat of zika virus to pregnancy: epidemiology, clinical perspectives, mechanisms, and impact zika virus outbreak on yap island, federated states of micronesia zika virus, french 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virus-host interactions and vaccine development development and characterization of recombinant virus generated from a new world zika virus infectious clone simple reverse genetics systems for asian and african zika viruses a robust method for the rapid generation of recombinant zika virus expressing the gfp reporter gene a reverse genetics system for zika virus based on a simple molecular cloning strategy de novo generation and characterization of new zika virus isolate using sequence data from a microcephaly case development of a novel dna-launched dengue virus type infectious clone assembled in a bacterial artificial chromosome complete nucleotide sequence of two generations of a bacterial artificial chromosome cloning vector cloning and stable maintenance of -kilobase-pair fragments of human dna in escherichia coli using an f-factor-based vector analysis of relative gene expression data using real-time quantitative pcr and the (−∆∆ c(t)) method. methods the miqe guidelines: minimum information for publication of quantitative real-time pcr experiments an open-source platform for biological-image analysis canine influenza viruses with modified ns proteins for the development of live-attenuated vaccines a susceptible mouse model for zika virus infection a mouse model of zika virus pathogenesis characterization of a novel murine model to study zika virus committee for the update of the guide for the care and use of laboratory animals sindbis virus dna-based expression vectors: utility for in vitro and in vivo gene transfer virus pathogen resource (vipr), faviviridae. available online engineering the largest rna virus genome as an infectious bacterial artificial chromosome targeted modification of a human β-globin locus bac clone using get recombination and an i-scei counterselection cassette a highly efficient escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of bac dna two-step red-mediated recombination for versatile high-efficiency markerless dna manipulation in escherichia coli a new logic for dna engineering using recombination in escherichia coli construction of a severe acute respiratory syndrome coronavirus infectious cdna clone and a replicon to study coronavirus rna synthesis engineering a replication-competent, propagation-defective middle east respiratory syndrome coronavirus as a vaccine candidate molecular characterization of feline infectious peritonitis virus strain df- and studies of the role of orf abc in viral cell tropism recovery of a neurovirulent human coronavirus oc from an infectious cdna clone subcellular localization and some biochemical properties of the flavivirus kunjin nonstructural proteins ns a and ns a mutations in west nile virus nonstructural proteins that facilitate replicon persistence in vitro attenuate virus replication in vitro and in vivo mutations in the yellow fever virus nonstructural protein ns a selectively block production of infectious particles role of nonstructural protein ns a in flavivirus assembly analysis of adaptive mutations in kunjin virus replicon rna reveals a novel role for the flavivirus nonstructural protein ns a in inhibition of β interferon promoter-driven transcription a single amino acid substitution in the west nile virus nonstructural protein ns a disables its ability to inhibit α/β interferon induction and attenuates virus virulence in mice subversion of interferon by dengue virus we are grateful to carla gómez and snezhana dimitrova for technical assistance in the bac clone generation and mice experiments, respectively. we also thank sylvia gutiérrez and ana oña at the cnb advanced microscopy facility for their valuable support in immunofluorescence microscopy analysis. the authors declare no conflict of interest. the funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, and in the decision to publish the results. key: cord- -i ztypg authors: chow, eric j.; doyle, joshua d.; uyeki, timothy m. title: influenza virus-related critical illness: prevention, diagnosis, treatment date: - - journal: crit care doi: . /s - - - sha: doc_id: cord_uid: i ztypg annual seasonal influenza epidemics of variable severity result in significant morbidity and mortality in the united states (u.s.) and worldwide. in temperate climate countries, including the u.s., influenza activity peaks during the winter months. annual influenza vaccination is recommended for all persons in the u.s. aged months and older, and among those at increased risk for influenza-related complications in other parts of the world (e.g. young children, elderly). observational studies have reported effectiveness of influenza vaccination to reduce the risks of severe disease requiring hospitalization, intensive care unit admission, and death. a diagnosis of influenza should be considered in critically ill patients admitted with complications such as exacerbation of underlying chronic comorbidities, community-acquired pneumonia, and respiratory failure during influenza season. molecular tests are recommended for influenza testing of respiratory specimens in hospitalized patients. antigen detection assays are not recommended in critically ill patients because of lower sensitivity; negative results of these tests should not be used to make clinical decisions, and respiratory specimens should be tested for influenza by molecular assays. because critically ill patients with lower respiratory tract disease may have cleared influenza virus in the upper respiratory tract, but have prolonged influenza viral replication in the lower respiratory tract, an endotracheal aspirate (preferentially) or bronchoalveolar lavage fluid specimen (if collected for other diagnostic purposes) should be tested by molecular assay for detection of influenza viruses. observational studies have reported that antiviral treatment of critically ill adult influenza patients with a neuraminidase inhibitor is associated with survival benefit. since earlier initiation of antiviral treatment is associated with the greatest clinical benefit, standard-dose oseltamivir ( mg twice daily in adults) for enteric administration is recommended as soon as possible as it is well absorbed in critically ill patients. based upon observational data that suggest harms, adjunctive corticosteroid treatment is currently not recommended for children or adults hospitalized with influenza, including critically ill patients, unless clinically indicated for another reason, such as treatment of asthma or copd exacerbation, or septic shock. a number of pharmaceutical agents are in development for treatment of severe influenza. annual seasonal influenza epidemics of variable severity result in significant morbidity and mortality in the united states (u.s.) and worldwide [ ] [ ] [ ] . in temperate climate countries, including the u.s., influenza activity peaks during the winter months whereas in tropical regions influenza activity may be more variable [ ] [ ] [ ] . most persons with symptomatic influenza virus infection have self-limited uncomplicated upper respiratory tract illness. one study estimated that during - , approximately . % of the u.s. population experienced symptomatic influenza each year [ ] . however, complications may result in severe illness, including fatal outcomes. during - , an estimated . - million medical visits, , - , hospitalizations, and , - , deaths were associated with influenza each year in the u.s. [ ] . another study estimated that , - , influenza-related intensive care unit (icu) admissions occur annually in the u.s. [ ] . there are an estimated , - , respiratory deaths attributed to seasonal influenza each year worldwide [ ] . here, we review strategies for prevention, diagnosis, and treatment of influenza virus infections in the icu (table ) . influenza vaccination is the primary method for preventing influenza and reducing the risk of severe outcomes. in the u.s., the advisory committee on immunization practices (acip) recommends annual influenza vaccination for all persons aged months and older and prioritizes those at higher risk for influenza complications [ ] . high-risk groups include adults aged > years [ , ] , children aged < years (particularly those aged < years) [ , ] , pregnant women (up to weeks postpartum) [ ] [ ] [ ] [ ] , persons with certain chronic medical conditions, native americans/alaska natives, and residents of nursing homes and other long-term care facilities (table ) . studies have specifically highlighted that those with chronic pulmonary, cardiovascular, renal, hepatic, neurologic, hematologic or metabolic disorders, immunocompromised persons, children and adolescents receiving aspirin-or salicylate-containing medications and who might be at risk for experiencing reye syndrome with influenza virus infection, and those who are extremely obese (bmi > ) are at increased risk for influenza-related complications [ , [ ] [ ] [ ] [ ] [ ] . many studies evaluated risk factors for severe influenza during the h n influenza pandemic. adult icu patients with influenza a(h n )pdm virus infection were primarily non-elderly, were obese [ ] [ ] [ ] [ ] [ ] , and had higher odds of death, invasive mechanical ventilation, acute respiratory distress syndrome (ards), septic shock, and multi-lobar pneumonia when compared with seasonal influenza patients [ , ] . in children, independent risk factors for influenza a(h n )pdm -related mortality included chronic neurologic condition or immune compromise, acute myocarditis or encephalitis, and early presumed mrsa co-infection of the lung [ ] . female gender was also identified as a risk factor; however, there was no gender difference in overall mortality. bacterial coinfection was identified in approximately one third of fatal influenza a(h n )pdm cases in the largest autopsy case series [ ] . bacterial co-infections in the interpandemic period are also common in critically ill influenza patients [ ] . one study identified past or current tobacco use as a risk factor associated with icu admission [ ] . a recent multicenter cohort study reported that mortality was higher in immunosuppressed patients with influenza a(h n )pdm than in immunocompetent patients [ ] . severity of influenza seasons varies from yearto-year based on the predominant influenza viruses, and between seasonal and pandemic influenza [ , ] . one study reported that patients with influenza a(h n ) pdm had higher odds of severe disease than patients with either influenza a(h n ) or influenza b virus infections [ ] . however, influenza b virus infection has been shown to increase the odds of in-hospital mortality in children compared with influenza a virus infection [ ] . influenza vaccination is recommended each fall for all persons aged > months in the u.s. and should continue • there are an estimated , - , seasonal influenza-associated respiratory deaths every year worldwide. • annual influenza vaccination is the primary method of preventing influenza and influenza-related complications, especially in high-risk persons. • influenza molecular diagnostic testing is recommended for all patients requiring hospitalization with suspected influenza. • influenza antiviral treatment should be started as soon as possible in hospitalized patients with suspected influenza, including critically ill patients, and should not be delayed while awaiting results of influenza diagnostic tests. • enterically administered oseltamivir is recommended for influenza patients except for those with contraindications (e.g., gastric stasis, ileus, malabsorption). • repeat virologic testing in lower respiratory tract specimens may be required to determine therapeutic endpoints in ventilated patients with influenza • corticosteroids are not recommended for the routine treatment of influenza except when indicated for treatment of underlying medical conditions (e.g., copd or asthma exacerbation) or septic shock. [ ] , and reducing in-hospital mortality and icu admissions for those aged - years and > years compared to unvaccinated individuals [ ] . one study reported that duration of icu hospitalization was reduced a half-day in patients aged - years who had received influenza vaccination compared with unvaccinated patients [ ] . a study across all age groups in spain reported influenza ve of % in reducing the risk of severe influenza requiring hospitalization [ ] . a southern hemisphere study reported influenza ve of % in reducing influenza-associated icu admissions among adults [ ] while a study in spain showed an adjusted influenza ve of % in preventing icu admission and death [ ] . despite the benefits of influenza vaccination, there continues to be low vaccine coverage among adults admitted to the icu who often have a high prevalence of high-risk comorbidities [ , ] . in children, low influenza vaccination coverage has also been reported among those admitted to pediatric icus, even among those with underlying high-risk conditions [ ] . full influenza vaccination was shown to result in a % reduction in pediatric icu admissions compared to unvaccinated or partially vaccinated influenza patients [ ] . furthermore, one study showed that influenza ve was % in reducing the risk of mortality in children aged months to years in the u.s. [ ] . these data further emphasize the benefits of influenza vaccination in reducing severe influenza complications, especially in high-risk persons. persons with uncomplicated influenza typically experience acute onset of respiratory symptoms (cough, rhinorrhea, congestion), myalgias, and headache with or without fever. during influenza season, clinicians should also consider influenza when there is only fever present or in patients who are afebrile and have respiratory symptoms [ ] . complications of influenza vary by age, underlying comorbidities or high-risk conditions such as pregnancy, and immune function; elderly and immunocompromised persons may not always manifest fever. critically ill patients may be admitted with respiratory or multi-organ failure, exacerbation of an underlying condition such as chronic lung disease [ , ] , heart failure [ ] , or other extrapulmonary complications including stroke, encephalopathy, or encephalitis [ , , ] . influenza testing is recommended for all patients requiring hospitalization with suspected influenza, including those admitted to the icu during influenza season with acute respiratory illness and community-acquired pneumonia, without a clear alternative diagnosis. furthermore, all individuals requiring critical care outside of influenza season should be tested for influenza if there is a possible epidemiological link to an individual with recent influenza, such as travel to areas with influenza activity or exposure to an institutional influenza outbreak. special consideration should be given to elderly and immunocompromised patients, as influenza virus infection may not present with typical acute respiratory illness signs and symptoms (e.g., absence of fever). the infectious diseases society of america (idsa) influenza clinical practice guidelines also recommend influenza testing for patients at high risk of complications such as exacerbation of chronic cardiopulmonary disease [ ] . diagnosis of influenza should be made as soon as possible in critically ill patients, and initiation of antiviral treatment should not be delayed while awaiting results of diagnostic tests. studies have reported an increase in mortality of icu patients with influenza a(h n )pdm virus infection when diagnosis was delayed [ ] , and shorter hospital length of stay when antiviral treatment was initiated within h of admission [ ] . several kinds of influenza diagnostic tests are available in clinical settings with variable sensitivities and specificities, including antigen detection assays, and molecular assays (nucleic acid detection) using respiratory tract specimens (table ) . within each of these testing categories, there is a wide range of available tests with varying diagnostic accuracy, and understanding the limitations of each diagnostic tool will allow the clinician to properly interpret their results. most studies of influenza diagnostic accuracy have been conducted on specimens from patients with uncomplicated influenza, and few have assessed the performance of influenza tests in critically ill patients. the idsa guidelines recommend molecular influenza assays for testing respiratory specimens from all hospitalized patients with suspected influenza because of their high sensitivity, specificity, and time to results ( min to several hours) [ ] . the use of rapid influenza molecular diagnostic testing can result in better outcomes for patients and reduce the amount of resources required to care for patients in the emergency room [ ] . serology and viral culture are not recommended for clinical decision making, because timely results will not be available to inform clinical management. serology requires collection of appropriately paired acute and convalescent sera performed at specialized public health reference laboratories, and results based upon a single serum specimen are not interpretable [ ] . although viral culture can confirm the presence of infectious virus with very high sensitivity and specificity, it must be performed at public health laboratories and requires - days to yield results. a recent meta-analysis reported that influenza antigen detection tests that produce rapid results had very high specificities (> %), but sensitivities were highly variable compared with rt-pcr [ ] . rapid influenza diagnostic tests (ridts) without an analyzer device had only moderate sensitivity ( - %), ridts that utilize an analyzer device (digital immunoassays) had moderately high sensitivity ( - %), and rapid influenza molecular assays (nucleic acid detection) had high sensitivity ( - %) [ ] . low sensitivity of ridts for detecting influenza virus in icu patients has been reported [ ] . recently, a systematic analysis of rapid influenza molecular tests from studies reported pooled sensitivity and specificity of . % and . %, respectively [ ] . therefore, antigen detection assays, such as rapid influenza diagnostic tests and immunofluorescence assays, are not recommended for hospitalized patients with suspected influenza because of their lower sensitivities, unless molecular assays are not available [ ] . negative results for influenza based on tests with low sensitivity (e.g., ridts, immunofluorescence assays) should not be used to make clinical decisions. instead, negative test results should be followed up with reverse transcription polymerase chain reaction (rt-pcr) or other influenza molecular assays to confirm results, and antiviral treatment should continue until results are available. preferred respiratory specimens for influenza testing in hospitalized patients without lower respiratory tract disease include nasopharyngeal, mid-turbinate nasal, or combined nasal-throat swabs. collection of lower respiratory tract specimens should be considered in hospitalized patients with suspected influenza if upper respiratory tract specimens are negative and a positive test would result in a change of clinical management [ ] , because viral replication in the lower respiratory tract may be ongoing and prolonged after virus is no longer detectable in the upper respiratory tract [ , ] . influenza a(h n )pdm virus in particular has been shown to have affinity for infecting the lower respiratory tract [ , ] . in hospitalized patients receiving invasive mechanical ventilation in whom influenza is suspected, but not yet diagnosed, influenza testing should be performed on endotracheal aspirate specimens instead of those collected from the upper respiratory tract [ ] . molecular testing, including rt-pcr for influenza viruses can also be performed on bronchoalveolar lavage (bal) fluid if collected for the testing of other pathogens. blood, plasma, serum, cerebrospinal fluid, urine, and stool samples have very low diagnostic yield and are not recommended for influenza testing [ ] . diagnostic test results on specimens collected from non-respiratory sites should not be used for clinical decision making even for patients with extra-pulmonary complications of influenza. novel influenza a viruses are typically of animal origin, differ antigenically and genetically from currently circulating seasonal influenza a viruses (including h n pdm and h n subtypes) and have infected at least one person. novel influenza a viruses can cause a wide clinical spectrum of illness, ranging from asymptomatic infection, uncomplicated illness, to fulminant pneumonia, ards, and multi-organ failure [ ] and human infection with a novel influenza a virus is of public health concern. in the u.s., human infection with a novel influenza a virus is nationally reportable to the centers for disease control and prevention; globally, treatment of severe influenza presents multiple challenges. the mainstay of therapy for patients with influenza is initiation of antiviral medication as soon as possible after illness onset [ ] . currently available fdaapproved antiviral medications include neuraminidase inhibitors (nais) (e.g., oral oseltamivir, inhaled zanamivir, and intravenous peramivir); cap-dependent endonuclease inhibitor (baloxavir marboxil); and adamantanes (e.g., amantadine and rimantadine) ( table ). nais and baloxavir have activity against both influenza a and b viruses. adamantanes only have activity against influenza a viruses and are not recommended for treatment of influenza due to widespread resistance among currently circulating strains of seasonal influenza a viruses. notably, fda-approved antiviral medications for treatment of influenza are approved for early treatment of uncomplicated influenza in outpatients based upon randomized placebo-controlled clinical trials conducted among previously healthy outpatients. meta-analyses of randomized placebo-controlled clinical trials of early oseltamivir treatment of influenza in pediatric and adult outpatients have reported clinical benefit in reducing duration of illness and risk for some complications associated with influenza [ , ] . no completed randomized, placebo-controlled trials of antiviral treatment have been conducted in hospitalized influenza patients to establish the efficacy of oseltamivir or other nais. a number of observational studies have reported clinical benefit of neuraminidase inhibitors in hospitalized patients, including reduction in duration of hospitalization and risk of death, including in icu patients [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . additionally, a systematic review of published reviews/meta-analyses reported survival benefit of nai treatment in hospitalized patients [ ] , although another meta-analysis of observational studies did not [ ] . in particular, a large pooled individual patient-level meta-analysis of observational studies from countries identified a % reduction in risk of mortality in critically ill adults and those aged ≥ years old when comparing early nai treatment (< h) with later treatment (> h), and a % reduction in mortality risk between influenza patients receiving early nai treatment and those who did not receive nais [ ] . the mortality risk reduction of nai treatment at any time versus no treatment was % for critically ill patients aged ≥ years old; while a similar reduction in mortality was identified in critically ill children aged < years, the result was not statistically significant [ ] and was likely underpowered because death is less common in hospitalized children with influenza than in adults. although studies have shown the greatest clinical benefit when antivirals are started within days of illness onset, some observational studies have shown clinical benefit of neuraminidase inhibitors when started up to days following symptom onset [ , , , ] . the large metaanalysis mentioned above also identified a significantly reduced mortality risk reduction ( %) in critically ill patients aged ≥ years old who received nai treatment > h after symptom onset compared with those who did not [ ] . a cohort study of early versus late oseltamivir treatment reported a significant reduction in mortality and median duration of icu hospitalization in severely ill patients with influenza a(h n ), but not a(h n pdm ) or b virus infection in greece [ ] . one french study reported delays in initiation of oseltamivir treatment prescribed to hospitalized influenza patients and suggested empiric administration of oseltamivir treatment in the emergency department for patients being admitted with lower respiratory tract disease during influenza season [ ] . overall, based upon available observational data to date in hospitalized patients with influenza, including icu patients, initiation of neuraminidase inhibitor antiviral treatment is recommended as soon as possible for hospitalized patients with suspected or confirmed influenza. data on optimal dosing and duration of therapy with neuraminidase inhibitors are limited in critically ill influenza patients. enterically administered oseltamivir is the preferred treatment for most hospitalized patients, given the lack of data for intravenous peramivir in this population. the use of inhaled zanamivir is not recommended in in critically ill patients due to the lack of data in hospitalized patients and the risk of bronchospasm in patients with underlying lung disease. studies indicate that oseltamivir administered orally or via oro/naso-gastric tube is well absorbed in critically ill patients and reaches plasma levels comparable to those in ambulatory patients [ ] . similarly, several observational studies indicate that enteric oseltamivir reaches comparable plasma concentrations to non-critically ill patients in those receiving extracorporeal membrane oxygenation (ecmo) and renal replacement therapy [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , although dosing should be reduced in patients with significant renal impairment. there is scant evidence that increased nai dosing (e.g., twice daily dosing) in critically ill patients provides additional clinical benefit than standard dosing [ , [ ] [ ] [ ] [ ] [ ] . of note, studies also suggest that increased oseltamivir dosing does not provide additional clinical benefit in obese adults, including extreme obesity (bmi > ) [ , ] . duration of therapy can be difficult to define, as prolonged influenza viral replication and shedding from the both upper and lower respiratory tract can occur in critically ill patients [ , ] . for this reason, it may be beneficial to continue antiviral therapy beyond days, and repeat virologic testing may be beneficial in determining appropriate therapeutic endpoints [ ] . continuing antiviral treatment in critically ill patients until virus is not detectable in the lower respiratory tract may also help reduce the pro-inflammatory dysregulated cytokine response triggered by influenza virus infection and reduce nosocomial influenza virus transmission to healthcare personnel in the icu. consultation with a specialist with training in infectious diseases for the potential emergence of antiviral resistant virus infection should be considered for icu patients with evidence of persistent influenza viral replication after nai treatment, particularly in severely immunocompromised patients [ , ] . for patients who cannot tolerate or absorb enteric oseltamivir due to gastric stasis, malabsorption, or other gastrointestinal processes, intravenous peramivir may be an alternative [ , ] ; however, studies have not identified an advantage for intravenous peramivir in comparison with enteric oseltamivir [ ] . notably, a randomized trial conducted in three influenza seasons found similar clinical outcomes between iv peramivir and enteric oseltamivir in hospitalized adult influenza patients [ ] ; a separate trial did not identify significant additional clinical benefit of peramivir in combination with standard-of-care therapy (which often included an nai) [ ] . a more recent, multicenter randomized controlled trial also found similar clinical benefit between enteric oseltamivir and intravenous peramivir in hospitalized influenza patients [ ] . in , a novel antiviral agent, baloxavir marboxil, was fda-approved for early treatment of uncomplicated influenza in outpatients aged ≥ years old. baloxavir acts via inhibition of the influenza virus cap-dependent endonuclease, a different mechanism than the neuraminidase inhibitors, and can treat nai-resistant influenza virus infections. randomized controlled trials of single-dose oral baloxavir showed similar clinical benefit to days of twice-daily oral oseltamivir [ ] . however, because these studies were limited to patients with uncomplicated influenza, the role of baloxavir monotherapy or in combination with an nai for treatment of hospitalized influenza patients is unclear. specifically, optimal dosing, duration of therapy, and appropriate endpoints have yet to be determined for baloxavir treatment of hospitalized influenza patients. in the outpatient rct, patients treated with single-dose baloxavir showed significant reduction in influenza viral levels in the upper respiratory tract at h compared with those receiving placebo or oral oseltamivir [ ] . however, it is unknown whether this reduction in influenza viral shedding correlates with reduced transmissibility. a potential concern for the use of baloxavir in critically ill patients is the rapid development of resistance observed during the outpatient clinical trials [ ] . a trial to assess the efficacy and safety of baloxavir in combination with oseltamivir versus oseltamivir monotherapy in hospitalized influenza patients is currently enrolling participants [ ] . there are no completed randomized clinical trials of adjunctive corticosteroid treatment in influenza patients. a trial of corticosteroid therapy was planned during the h n pandemic, but was halted due to limited number of enrolees [ ] . one observational study in china during the h n pandemic reported that administration of parenteral glucocorticoids within h of illness onset tripled the risk of developing critical illness or death from influenza a(h n )pdm virus infection [ ] . a re-analysis of prospectively collected data on influenza patients admitted with primary influenza pneumonia to icus in spain during - using propensity scoring matching reported that corticosteroid use was significantly associated with icu mortality [ ] . meta-analyses of observational studies have concluded that that corticosteroid treatment of hospitalized influenza patients does not result in better outcomes and may be associated with adverse outcomes including increased mortality [ ] [ ] [ ] . similarly, a retrospective observational study conducted on critically ill children during the h n pandemic found that high-dose (equivalent to mg/kg per day of methylprednisolone) corticosteroid treatment was associated with mortality in the icu, although a causative relationship was not determined [ ] . a selection of individual observational studies in critically ill children and adults have also reported potential association between corticosteroid treatment and adverse influenza outcomes [ , , ] . a recent cochrane review of available observational studies suggested increased mortality when adjunctive corticosteroid therapy is used for influenza patients; however, the available evidence was of low quality and the authors suggest interpreting these results with caution [ ] . multiple studies have reported that corticosteroid treatment is associated with prolonged influenza viral shedding in hospitalized patients [ ] [ ] [ ] , including in sporadic human infections with avian influenza a(h n ) virus in china [ ] , and increased rates of secondary bacterial and fungal co-infections [ , ] , which may lead to adverse clinical outcomes. however, there is some evidence to suggest that the increased risk attributed to corticosteroid treatment is a result of bias in observational studies. a large, retrospective study of critically ill adults in canada found an increased risk of mortality in patients receiving corticosteroids; however, after adjusting for time-dependent differences between groups, no significant differences in mortality were observed with corticosteroid treatment [ ] . moreover, potential differences between low-dose and medium-/ high-dose corticosteroid treatment are not well understood. one observational study of hospitalized patients with viral pneumonia due to avian influenza a(h n ) virus infection in china reported that high-dose, but not low or moderate-dose corticosteroids, was associated with increased -day and -day mortality [ ] . currently, on the basis of available observational data to date, adjunctive corticosteroid treatment is not recommended for children or adults hospitalized with influenza, including critically ill patients, unless clinically indicated for another reason, such as treatment of asthma or copd exacerbation or septic shock [ ] . further studies are required to understand the clinical benefit or harms associated with corticosteroid treatment of critically ill influenza patients. although neuraminidase inhibitors (oseltamivir) are currently recommended for antiviral treatment of influenza in hospitalized patients based on observational studies, including in critically ill patients, there are a number of novel strategies and products for treating influenza in various stages of development. one approach under investigation is triple-combination antiviral drug (tcad) therapy, which combines amantadine, ribavirin, and oseltamivir for treatment of influenza in critically ill and high-risk patients. unfortunately, studies to date have not shown a benefit of tcad over oseltamivir monotherapy [ ] [ ] [ ] . several novel antiviral compounds are in various stages of investigation, including small-molecule polymerase inhibitors such as pimodivir [ ] and favipiravir [ ] . a number of monoclonal and polyclonal antibodies, targeted against a variety of influenza viral proteins, are also in development [ ] [ ] [ ] [ ] . similarly, convalescent plasma has shown potential benefit in the treatment of severe influenza, and further trials are underway [ ] [ ] [ ] . another area of intense interest is the modification of the host antiviral response to influenza virus infection. there are ongoing preclinical and clinical studies of a variety of other immunomodulatory agents for treatment of influenza, including celecoxib [ ] , statins, etanercept, pioglitazone, azithromycin [ ] , and interferons [ ] . influenza vaccination can reduce the risk of complications from influenza, including reducing illness severity and the risks of hospitalization, icu admission, and death. the elderly, young children, pregnant women, and those with underlying medical conditions are most at risk for severe complications of influenza. a diagnosis of influenza should be considered in critically ill patients admitted with complications such as exacerbation of underlying chronic comorbidities, community-acquired pneumonia, and respiratory failure during influenza season. influenza molecular assays are recommended for testing upper respiratory tract specimens in patients without signs of lower respiratory tract disease. however, because critically ill patients with lower respiratory tract disease may have cleared influenza virus in the upper respiratory tract, but have prolonged influenza viral replication in the lower respiratory tract, an endotracheal aspirate (preferentially) or bronchoalveolar lavage fluid specimen (if collected for other diagnostic purposes) should be tested by molecular assay. antiviral treatment with standard-dose oseltamivir delivered orally or enterally by oro or naso-gastric tube is recommended as soon as possible for patients with suspected influenza without waiting for testing results. corticosteroids should not be routinely administered for treatment of influenza and should only be given for other indications (e.g., exacerbation of asthma or chronic obstructive pulmonary disease, or septic shock), because of the risk for prolongation of influenza viral shedding and ventilator-associated pneumonia in critically ill influenza patients with respiratory failure. future directions for treatment of influenza in critically ill patients include novel antiviral compounds, combination antiviral treatment with drugs with different mechanisms of action, immunomodulatory agents, and strategies for multimodality, combination antiviral, and host-directed immunomodulatory therapies. endnotes these risk factors are included in the u.s. cdc's advisory committee on immunization practices recommendations for influenza vaccination. this may also apply to indigenous people from other countries, including indigenous australians and first nations people. burden of medically attended influenza infection and cases averted by vaccination -united states, / through / influenza seasons estimates of global seasonal influenza-associated respiratory mortality: a modelling study estimated 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adjunctive macrolide treatment in adults hospitalized with influenza: a randomized controlled trial the role of adjuvant immunomodulatory agents for treatment of severe influenza not applicable. no external funding was received. the authors were supported by their work at the centers for disease control and prevention (cdc). not applicable. the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention. key: cord- -hfnht wa authors: berto, a.; pham, h. a.; thao, t. t. n.; vy, n. h. t.; caddy, s. l.; hiraide, r.; tue, n. t.; goodfellow, i.; carrique-mas, j. j.; thwaites, g. e.; baker, s.; boni, m. f. title: hepatitis e in southern vietnam: seroepidemiology in humans and molecular epidemiology in pigs date: - - journal: zoonoses public health doi: . /zph. sha: doc_id: cord_uid: hfnht wa viral pathogens account for a significant proportion of the burden of emerging infectious diseases in humans. the wellcome trust-vietnamese initiative on zoonotic infections (wt-vizions) is aiming to understand the circulation of viral zoonotic pathogens in animals that pose a potential risk to human health. evidence suggests that human exposure and infections with hepatitis e virus (hev) genotypes (gt) and results from zoonotic transmission. hypothesising that hev gt and gt are circulating in the vietnamese pig population and can be transmitted to humans, we aimed to estimate the seroprevalence of hev exposure in a population of farmers and the general population. we additionally performed sequence analysis of hev in pig populations in the same region to address knowledge gaps regarding hev circulation and to evaluate if pigs were a potential source of hev exposure. we found a high prevalence of hev gt viral rna in pigs ( . % in faecal samples and . % in rectal swabs) and a high hev seroprevalence in pig farmers ( . %) and a hospital-attending population ( . %) in southern vietnam. the hospital population was recruited as a general-population proxy even though this particular population subgroup may introduce bias. the detection of hev rna in pigs indicates that hev may be a zoonotic disease risk in this location, although a larger sample size is required to infer an association between hev positivity in pigs and seroprevalence in humans. emerging infectious diseases have an important impact on human health. it is well established that viruses account for a significant proportion of emerging infections in humans and the majority are have a zoonotic origin, as highlighted by the recent ebola epidemic in africa and the repeated mers cov outbreaks in the middle east. hepatitis e virus (hev) is a public health concern, as it causes an estimated million human infections annually, with over three million symptomatic hev cases and , deaths worldwide (kamar et al., ) . mortality in immunocompromised patients and pregnant women can approach % for those infected with genotype (kamar et al., ) . hev infections are responsible for > % of cases of acute viral hepatitis in endemic countries (kamar et al., ) . hev is generally associated with a self-limiting hepatitis, as the infection clears within months from onset of symptoms. recently, it was observed that infection with hev gt can become chronic in immunocompromised patients, such as organ transplant recipients or those infected with hiv (galante et al., ) . hev is the sole member of the hepeviredae family of the orthohepevirus a genus. it has only one serotype and four genotypes (gts), although a reclassification into seven gts has been proposed (smith et al., ) . of the four genotypes, gt and gt are able to sustain human-to-human transmission and sporadically cause large human outbreaks in endemic regions due to faecal contamination of the water supply. genotypes and are typically zoonotic; infection can occur from eating undercooked pork or venison, via direct exposure to animal faeces, and sporadically through blood transfusion or liver transplantation (berto et al., ; christou & kosmidou, ; lu, li, & hagedorn, ; renou et al., ; shen et al., ; sridhar, lau, & woo, ; tedder et al., ) . there is now evidence that hev gts and infections mainly originate in swine, and local zoonotic transmission of hev has been recorded worldwide (christou & kosmidou, ) . recent initiatives to increase and improve the surveillance for hev disease in humans have shown that it may be more common than hepatitis a; however, the true incidence of hev infections in humans is still not known (kamar et al., ; da silva et al., ) . it has been shown that seroprevalence of hev in human populations varies between % and % in blood donors, depending on the geographical location (dalton et al., ; scotto et al., ; slot et al., ; vollmer et al., ) . furthermore, it is possible that the majority of patients with unexplained and undiagnosed hepatitis may be hev positive as hev is generally not considered as a potential cause especially in developing countries, due to the lack of diagnostic tests (kamar et al., ; da silva et al., ) . the seroprevalence of hev in pigs in various european countries varies between . % and % (berto et al., ; mccreary et al., ) , and antibodies against hev have also been detected in several other species worldwide, such as rabbit, rats, cattle, sheep, goats, chickens and dogs (drexler et al., ; johne et al., ; sun et al., ) . to date, there are only two published studies describing the circulation of hev in vietnam. one reported a hev seroprevalence rate of % in patients with elevated alanine aminotransferase (alt) (buchy et al., ) , while the second reported that nine of human serum samples obtained from patients with acute sporadic hepatitis in hanoi were infected with hev genotype (dieu ngan et al., ) . vietnam, along with other southeast asian countries, is considered a global hotspot for zoonotic infections (jones et al., ) . the wt-vizions (wellcome trust-vietnamese initiative on zoonotic infections) programme is aiming to gather data on the circulation of viral zoonotic pathogens in animals that pose a risk to human health. hypothesising that hev gt and gt are common zoonotic pathogens in vietnam, we aimed to estimate (i) the hev seroprevalence in a hospital-attending population, as a proxy for the general population, (ii) the hev seroprevalence in individuals working in close contact with pigs (farmers, family members of farmers, animal workers, veterinarians and abattoir workers) and (iii) the prevalence of hev infection in pigs. all collected farm samples (faeces/rectal swabs and human plasma) were collected from dong thap province (southern vietnam). furthermore, we retrospectively tested for presence of anti-hev igg in a hospital population sample of , human serum samples collected between and from dong thap. the wt-vizions is a descriptive observational farm-based and community-based study of zoonotic infection, diseases in domestic livestock and pathogens circulating among individuals with significant occupational or residential exposures to domestic livestock, wildlife and/or animal products. the study enrolled cohort members from approximately different sites in dong thap province. the wt-vizions study design aimed at having an average of - cohort members per farm and - cohort members per abattoir/market rabaa et al., ) . selection of dong thap province for this substudy was coordinated with a complementary long-term seroepidemiology study from dong thap provincial hospital (dtph), with residual samples collected from the hospital haematology laboratory; this was part of an ongoing seroepidemiology study being conducted in southern vietnam (boni et al., ; nhat et al., ) . additionally, dong thap province was selected because the high density of domestic livestock and/or farmed wildlife populations, the diversity of local agroecosystems, and pre-existing frameworks for collaborative research between oxford university clinical research unit and sub-departments of preventive medicine and animal health (pmc and sdah, respectively). the vizions study team, working with the sdah, identified the individual farms and abattoirs within dong thap province. the provincial coordinator (pc) of dong thap organised a first site visits to confirm whether inclusion criteria were met. at each study site, the person with primary management responsibility was identified (farmer, farm manager or abattoir manager), informed of the study objectives, and asked to obtain written informed consent to sample animals and human blood. the study sites were included in the study if they fulfilled the following criteria: permission from local authorities (peoples' committee and institutional management bodies), farmers provided written permission, minimum two consenting participants per site and access to a cell phone with texting capacity. sites were excluded if they were difficult to reach, if the participant was less than year of age, or if the participant had any known immunosuppressive condition or ongoing receipt of any immunosuppressive therapy. the study was approved by the university of oxford tropical research ethics committee (oxtrec no. ) and the scientific and ethical committee of the hospital for tropical diseases in ho chi minh city. written informed consent was required from patients and parents or legal guardians if children under age of prior to participation in the study. pig faeces samples from pigs were collected on farms during , while pig rectal swabs were collected in - from farms. from each farm up to individual animals were sampled. as this study was a part of the wt-vizions project, which aimed to assess the circulation of many different pathogens in addition to hev, the number of animals to sample from each species was a function of the relative number of animals of each species present on the farm. for the above reason, a scoring system was developed to assign different weight to each species and calculate the number of individuals to sample. therefore, for each farm between and , pigs were randomly sampled depending on the total number of different animals species present on the farm rabaa et al., ) . the total number of pigs for each farm was not recorded. the age of the pigs varied between weeks and years, but the precise age of the individual sampled pig was not recorded, although the majority of the animals were adult pigs. in all farms, samples of a minimum of g of faeces were collected aseptically in a sterile plastic container and resuspended in ml of viral transport medium (vtm); rectal swabs were placed in tubes containing ml of vtm. the collected samples were maintained at °c (max. hr), subsequently transported to oucru and frozen at − °c until processing. human plasma samples were collected from sampling sites: the farms sampled in - from which pig rectal swabs were tested, additional farms from which no pig rectal swabs were available, three rodent trading sites, two poultry markets, two pheasant trading sites and one abattoir. an average of five human plasma samples were collected from each site ( in total), and the plasma sampling on the farms with pig rectal swabs was concurrent with the pig sampling. at enrolment, participants were asked to consent to a ml whole blood specimen collection ( ml for children aged < years). blood was allowed to clot at room temperature, transported at ambient temperature and centrifuged within hr. berto et al. page zoonoses public health. author manuscript; available in pmc july . europe pmc funders author manuscripts the primary coordinator coordinated the storage and shipment of human specimen materials to oucru laboratory in hcmc. all specimens were separated into three equal aliquots and preserved at − °c until processing. serum samples from the hospital population were collected from dtph from the hospital haematology laboratory, as part of an on-going seroepidemiology study currently being conducted in southern vietnam. in this study, samples are collected at each collection time point (between three and six per year), in order to allow for the lower range of seroprevalences to be inferred accurately; with this design, a % seroprevalence can be inferred with a % confidence interval from . % to . %. in each collection, a minimum of samples were required from each of the age bands - , - and +. for the subsample used in this study, , samples were selected. the sampling design was approximately per year from to , with a : gender ratio. thirty samples were chosen from each of the age bands - , - , - and - . fifteen samples were chosen from the age bands . - and - . the entire sample set is smaller than the intended , as some age groups did not have enough samples. each collected specimen contained approximately . ml of serum. samples for this seroepidemiology study are stored at − °c and transported on dry ice twice yearly to ho chi minh city where they are stored at − °c as part of a central serum bank for southern vietnam (boni et al., ; nhat et al., ; thuy et al., ) . plasma from the farmers and farmers' family members, veterinarians, animal workers and abattoir workers (n = , farms plus eight other sites) and serum from the hospital population (n = , ) from dong thap (figure ) were screened for anti-hev antibody (igg) using the wantai elisa kit (sanbio, china), following the manufacturer's instructions (izopet et al., ) . all faecal samples and rectal swabs from pigs were screened for hev rna using quantified pcr (qpcr) to detect hev rna as described by jothikumar, cromeans, robertson, meng, and hill ( ) . the qpcr-positive samples were amplified using a nested pcr targeting a bp fragment of orf as previously described by li et al. ( ) . all pcr amplicons ( bp) were visualised on % agarose gels under ultraviolet (uv) light and sequenced by sanger sequencing using an abi sequencer in both forward and reverse directions using the primers from the second round nested pcr. the hev sequences were aligned with a subset of publicly available reference sequences from all seven recently new proposed classification of hev genotypes (smith et al., ) , yielding a -reference sequences data set (both orf and whole genome) and vietnamese sequence data set from pigs ( bp). all sequences were aligned with mega . maximum-likelihood phylogeny was inferred using raxml v . . (stamatakis, ) with bootstrap replicates. the tree was visualised and annotated using figtree (version . . ). a standard loess curve (local polynomial regression fitting; smoothing parameter = . ) was used to construct an age-seroprevalence curve with % confidence intervals. a spearman rank correlation coefficient was used to determine the correlation between the hev rna-positive pigs and the hev seropositivity of farmers. all confidence intervals in the text are presented as exact binomial confidence intervals. the figure ). all sequences obtained from the hev pig samples (genbank accession numbers from kx to kx ) were found to belong to gt and clustered into three main clades ( figure ). all vietnamese pig sequences clustered separately from the previously identified gt sequences circulating worldwide and the subgenotypes did not cluster with any previously known subgenotype (figure ). the anti-hev igg unadjusted seroprevalence was lower in the farmer cohort ( . %; % ci: . %- . %) than the general population ( . %, % ci: . %- . %) in dong thap (table , figure ). there was no difference in seroprevalence by gender in either sample set. although the seroprevalence in both populations increased with age, the prevalence of anti-hev igg was higher in children in the hospital population than in children enrolled in the farm cohort study (figure ). breaking the comparisons into year-age bands, the hospital population had higher hev seroprevalence for individuals aged - (both p < . ; one-way test on elisa optical density); hev seroprevalence was indistinguishable between the two groups when considering individuals aged > years (all p > . ) (figure ). hev seroprevalence did not vary through time in the hospital population cohort. due to the small sample size (n = ) of farms with both molecular diagnostics on pigs and farmer plasma samples, there was insufficient statistical power to determine if there was a positive correlation between hev rna-positive pigs and seropositive farmers (spearman pvalue = . ). this study aimed to measure the prevalence of faecal hev shedding on pig farms and the seroprevalence of hev in the human population in southern vietnam (dong thap province). we report an observed prevalence of hev rna in pigs in southern vietnam that is similar to countries in europe and asia (liu et al., ) . the hev prevalence observed in pig rectal swab samples was . %, which is lower in comparison with previous studies, but the sensitivity of pcr screening is considerably lower using rectal swabs than faecal samples. we additionally found that all sequences obtained from pig samples were hev gt . despite all samples being collected in the same province, these data show that hev sequences clustered into three distinct clades within gt , and the clusters were not associated with the farm or year of collection. further, although the sequences clustered with hev gt , they did not cluster with previously known subgenotypes. these data confirm the substantial genetic variability that exists within the various hev genotypes (cao, dianjun, & meng, ) . the hev seroprevalence identified in the farmer cohort ( . %) was comparable to other asian and european countries (drexler et al., ; johne et al., ; sun et al., ) . the retrospective serology from the general population identified an unadjusted seroprevalence of . %. despite using the same elisa as other studies, this figure is lower when compared to previous data from vietnam ( %) and other endemic regions such as nepal ( . %) and bangladesh ( . %) (izopet et al., ; tran et al., ) ; nevertheless, the results show that a large number of individuals in southern vietnam are exposed to hev. while the participants studied in the farmer cohort were healthy individuals, the samples from the "general-population proxy" are best described as a sample from the potentially hospital-attending population. the same type of comparison is performed in other epidemiological studies where blood donors, for logistic reasons (e.g., easier recruitment), are recruited as a general-population proxy even though this particular population subgroup may introduce bias. as our study assessed the hev seroprevalence in the hospital-attending population, it is important to highlight this distinction because some socioeconomic or occupational groups may have more reason to attend a hospital than others. thus, these samples may represent individuals that have higher disease risk or vulnerability, to hev or to infections in general, and hence may not be representative of the population at large. in particular, children in this sample are likely to be more vulnerable than children in a true general-population sample. the observed low seroprevalence in young children from the farmer cohort is in agreement with previous published data from endemic and non-endemic countries, such as nepal, bangladesh and france (izopet et al., ) , contrary to what was observed in the same age group in the hospital population sample in dong thap. despite not knowing the exact representativeness of the general-population samples, we believe that the results of this study provide unique and valuable insights into the epidemiology of hev in the south of vietnam that could be used to investigate associations of hev seroprevalence and putative risk factors by a nationwide cross-sectional study. in particular, despite hev gt being a zoonotic infection, a higher seroprevalence was not found in adult farmers than in other adults. this suggests that additional hev risk factors need to be identified and evaluated. confounders associated with the high hev seroprevalence detected in both farmers and the hospital-attending population may be due to other risk factors rather than just direct contact with pigs. one potential risk factor might be consumption of internal pig organs, which is common in vietnam. other risk factors associated with high seroprevalence in human may be due to household flooding, drinking contaminated water or poor household hygiene. future population-based studies, using random controls chosen by geographic area, should be performed to confirm or contradict these results. farmers do not have a higher hev seroprevalence than adults in the dtph sample; however if they did, the relative risk of zoonotic hev genotypes (i.e., gt or gt ) would be impossible to estimate using this seroprevalence assay. our initial hypothesis was that individuals living on pig farms would have higher hev seroprevalence than the general population. we did not find evidence to support this hypothesis. however, our study had certain limitations; first no viral characterisation was performed in the two human cohorts, as no individuals presented with symptoms of liver diseases or jaundice during the study. therefore, we could not link human hev genotypes to pig hev genotypes; only presence of anti-hev igg was measured, and direct evidence of hev zoonotic infection was not identified. furthermore, it was not possible to determine the genotype of hev exposure in humans because hev has only one serotype, and the elisa cannot distinguish the different gts. our farm-based individuals work and live in close contact with pigs, and dong thap is a semi-rural region with a high density of pig farms; therefore, we speculate that the majority of the individual anti-hev igg-positive individuals have been exposed to hev gt rather than other hev genotypes (only hev gt was identified in the pigs in our sample). in conclusion, this study demonstrates for the first time that hev is circulating in the pig population in southern vietnam, and also that the human population in the same location has high seroprevalence to hev. therefore, this study provides data that may be used for hev risk factor assessment to prevent and reduce hev infection and transmission. more studies, incorporating testing blood donors or an alternative healthy general-population sample, are required to better understand hev dynamics in human and pig populations in vietnam in order to assess the implication for human health. seroprevalence in both general population and framers cohort groups analysed in this study. the graphs represent the total seroprevalence observed in the general population (left panel) and in the farmer cohort (right panel) stratified by age. y-axis shows the seroprevalence percentage of people anti-hev igg positive, while the x-axis indicates the age groups; individuals were grouped into -year-age bands for maximum informativeness and clarity. the red line shows the inferred seroprevalence curve, and the pink shaded area shows the % confidence band. the grey dots show the seroprevalence for each -year-age group, and the size of the dot is proportional to the sample size. prevalence and transmission of hepatitis e virus in domestic swine populations in different european countries hepatitis e virus in pork liver sausage, france. emerging infectious diseases 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pigs of different ages and its presence in slurry stores in the united kingdom structure of generalpopulation antibody titer distributions to influenza a virus the vietnam initiative on zoonotic infections (vizions): a strategic approach to studying emerging zoonotic infectious diseases possible zoonotic transmission of hepatitis e from pet pig to its owner global policy report on the prevention and control of viral hepatitis in who member states seroprevalence of hepatitis e virus among blood donors in a district of southern italy cloning of full genome sequence of hepatitis e virus of shanghai swine isolate using race method prevalence of hepatitis e virus antibodies in individuals exposed to swine in mato grosso, brazil. memórias do instituto oswaldo cruz silent hepatitis e virus infection in dutch blood donors proposed reference sequences for hepatitis e virus subtypes hepatitis e: a disease of reemerging importance raxml version : a tool for phylogenetic analysis and post-analysis of large phylogenies genetic identification of avian hepatitis e virus (hev) from healthy chicken flocks and characterization of the capsid gene of avian hev isolates from chickens with hepatitis-splenomegaly syndrome in different geographical regions of the united states hepatitis e risks: pigs or blood-that is the question tetanus in southern vietnam: current situation prevalence of hepatitis virus types b through e and genotypic distribution of hbv and hcv novel approach for detection of hepatitis e virus infection in german blood donors key: cord- -byxuruk authors: fritsch, annemarie; schweiger, brunhilde; biere, barbara title: influenza c virus in pre-school children with respiratory infections: retrospective analysis of data from the national influenza surveillance system in germany, to date: - - journal: euro surveill doi: . / - .es. . . . sha: doc_id: cord_uid: byxuruk introduction: recent data on influenza c virus indicate a possible higher clinical impact in specified patient populations than previously thought. aim: we aimed to investigate influenza c virus circulation in germany. methods: a total of , samples from to year-old children presenting as outpatients with influenza-like illness (ili) or acute respiratory infection were analysed retrospectively. the samples represented a subset of all samples from the german national surveillance system for influenza in this age group in – . the presence of influenza c virus was investigated by real-time pcr. for positive samples, information on symptoms as well as other respiratory virus co-infections was considered. retrieved influenza c viral sequences were phylogenetically characterised. results: influenza c viral rna was detected in ( . % of) samples, including during the / season. the majority ( / ) of influenza c-positive patients had ili according to the european union definition, one patient had pneumonia. viruses belonged to the c/sao paulo and c/kanagawa lineages. most ( / ) samples were co-infected with other respiratory viruses. conclusion: our data are the first on influenza c virus circulation in germany and notably from a european national surveillance system. the low detection frequency and the identified virus variants confirm earlier observations outside a surveillance system. more virus detections during the / season indicate a variable circulation intensity in the different years studied. influenza c virus can be considered for ili patients. future studies addressing its clinical impact, especially in patients with severe disease are needed. influenza viruses are a major threat to human health and are therefore in the focus of national and international health authorities. among these, influenza virus types a and b are the main considered, as they cause annual epidemics with high morbidity and considerable mortality [ ] . in contrast, influenza c virus has been regarded as a pathogen of minor relevance, causing mild or clinically unapparent disease [ , ] . nevertheless, in recent years, detections of influenza c in hospitalised young children with (severe) lower respiratory tract disease were reported [ ] [ ] [ ] [ ] [ ] [ ] . thus, the clinical and epidemiological significance of this virus species might have been underestimated and needs to be reassessed. in europe, the burden of influenza c virus infection in children and adults is largely unknown, as no systematic surveillance data are available. the few studies published mainly focus on clinical data, mostly from hospitalised children [ , , ] . in germany, no surveillance data and no sequence information on circulating influenza c viruses have ever been reported. therefore, we decided to search for influenza c in our outpatient sample collection assembled for the purpose of influenza virus surveillance in germany. as young children are described to have the highest infection rates [ , , ] , we confined our study to the - year-old age group. we furthermore sequenced the haemagglutinin esterase (he) gene from influenza c-positive samples to phylogenetically characterise the detected viruses. all samples were collected from practitioners participating in the national influenza surveillance, who are distributed over the complete german territory and represent a statistically valid proportion of the german population [ ] . these practitioners continuously collect nasal or throat swabs from non-hospitalised patients presenting with symptoms of influenza-like illness (ili) according to the european union (eu) definition or an acute respiratory infection (ari). an ili case is defined by a sudden disease onset with at least one of four systemic symptoms (fever or feverishness, malaise, headache, myalgia) and at least one of three respiratory symptoms (cough, sore throat, shortness of breath) [ ] , while ari is an acute respiratory disease with at least one of the four following symptoms: fever, cough, rhinorrhoea or sore throat. the samples are sent to the german national influenza centre, accompanied by a completed questionnaire on patient characteristics, sampling date, disease symptoms, influenza vaccination status and therapeutic intervention i.e. antiviral treatment. they are routinely analysed for influenza virus types a and b, human respiratory syncytial virus (rsv) as well as -since april -human adenovirus (adv), metapneumovirus (hmpv) and rhinovirus (hrv). all samples are stored at - °c afterwards. for this study, a subset of , samples ( . %) was selected from a total number of , samples taken from children ≤ years of age in the years - as described in the supplementary file (supplement s ). briefly, at least every second sample in a chronological order was retrospectively analysed for influenza c virus. to extend the basis for the co-infection data, all influenza c-positive samples were additionally tested for human parainfluenza viruses types - and coronaviruses oc , nl , hku and e. positive samples taken before april were furthermore retrospectively examined for adv, hmpv and hrv. the conduct of a sentinel surveillance is covered by german legislation ( § , § , protection against infection act). the german national surveillance of influenza and other respiratory viruses was furthermore approved by an ethical committee of the charitè berlin (application number ea / / ). additionally, for all samples a written consent was given for their inclusion in research studies. all analyses were done with pseudonymised data. after their arrival in the laboratory, ml of cell culture medium (minimum essential medium (mem) with for sequence analyses, cdna was synthesised with the accuscript hi-fi reverse transcriptase (agilent, santa clara, us) and a primer that binds to the conserved ' end of the rna gene segments (uni , see table ). assay validation was performed with synthetic double stranded dna strings (gblocks; idt, skokie, us) in singleplex as well as duplex format (including the internal control fcv) on -well as well as -well plates. for the determination of the linear detection range and the correlation (r ) of quantification cycle (c q ) values, a -fold dilution series ( - copies per reaction) was examined in duplicates. pcr efficiency was calculated by inserting the slope value of the standard curve into the formula e = (- /slope)- . the limit of detection (lod) was established as % detection probability, calculated by probit analyses of the results of a -fold examination of low copy numbers ( - . genome equivalents per reaction) applying the ibm spss statistics software. for intraassay precision, gblocks were examined sixfold in a single run, while for interassay precision the intraassay data were extended by two additional runs with double reactions. all reproducibility runs were performed on consecutive days in independent experiments, and precision was described as standard deviation of the observed c q values. conventional pcr for sequence determination of the he gene was carried out in a total reaction volume of µl. the reaction contained x extaq buffer, . mmol/l dntp (thermo fisher scientific, waltham, us) with dutp (ge healthcare, chicago, us), . u extaq polymerase (takara, kusatsu, japan), nm primers (metabion, planegg, germany) as listed in table , and µl of the prediluted cdna. alternatively, the superscript monthly distribution in - year-old children of (a) the number of samples tested for influenza c, as well as testing coverage among samples received by table ) were used for amplicon sequencing in cases where the nested pcr primers did not yield a sequence spanning the complete amplicon. all he sequences were processed and assembled in the geneious software before their deposition at the global initiative on sharing all influenza data (gisaid; www. gisaid.org) database (epi -epi ). the applied amino-acid numbering includes the signal peptide. all he sequence analyses were performed with geneious version . . . multiple sequence alignments were compiled on the basis of the mafft algorithm. the n-terminal sequences including the signal peptide sequence (mffslllmlgltea [ ] ) as well as the c-terminal region with incomplete sequence information (last nt including the stop codon) were excluded. the alignment for phylogenetic analyses thus covered the nt to , of the complete coding sequence and was calculated including reference sequences downloaded from the gisaid database (see supplement s ). maximum likelihood trees were constructed applying the hky model and the spr tree topology search. branching reliability was estimated by performing , bootstrap replicates. trees were manually edited in corel draw x . a qpcr assay for the detection of influenza c viruses was established as singleplex as well as duplex qpcr including our routine internal control fcv. the assay proved to be a robust and sensitive tool and furthermore did not show any cross-reactivity to a variety of viral respiratory pathogens and to human genomic dna (validation results summarised in table ). the duplex qpcr approach was applied to retrospectively examine , throat or nasal swabs, of which , samples gave valid qpcr results, i.e. yielded either an influenza c or a fcv signal (or both) in duplex qpcr runs. twenty samples ( . %) were found positive for influenza c virus rna, with c q values ranging from to . the positive samples predominantly were taken between october and april , reaching an average positivity rate of . % ( / ) in these months ( . - . % per month, figure ). outside of this particular winter season, viruses were identified only sporadically with detection rates of . % ( / , january-april ) or . % ( / , october -april . no virus detection was achieved from may to september of any year studied. also, no particular age distribution could be observed ( figure ). all influenza c-positive samples were additionally examined by qpcr to identify other respiratory viruses. more than half of the influenza c-positive patients ( / ; %) proved to be co-infected with diverse other respiratory pathogens (table ) , with influenza c c q values covering the complete range of to . all patients with influenza c virus infection reported fever and cough. fifteen patients reported a maximum temperature between . °c and . °c, while for the remaining five patients the maximum temperature was not provided. additionally, a sudden disease onset ( / ) , rhinitis ( / ) , sore throat ( / ) and muscular pain and/or headache ( / ) were predominant symptoms. clinical signs of pneumonia were reported for one patient with an influenza c c q value of , but also low amounts of influenza a(h n ) were detected in this sample. in patients with a sole influenza c virus infection, the sudden disease onset ( / ), the maximum fever ( . °c - . °c in patients), rhinitis ( / ), sore throat ( / ) and muscular pain and/or headache ( / ) were reported in similar proportions. the sequencing of the he gene was achieved for samples, of which three yielded only partial sequences. two of the incomplete sequences covered a consecutive stretch of , and , nt, respectively, while the three incomplete sequences were only characterised based on the nt homologies to other sequences. the two fragments of sample - ( nt, nt) are % identical to our sample sequence - , which belongs to the c/victoria/ / subgroup of the c/sao paulo clade. similarly, sample sequence - ( , nt) is % identical to the sequence of c/sao paulo samples - and - , which group into the c/miyagi/ / subgroup of the c/aichi/ / subclade. sample sequence - has a similarity of > . % to the same sao paulo lineage cluster, while the similarity to c/victoria/ / ( . %) and the prototype strain sequences for the other five he lineages is lower (≤ . %). although influenza c virus was discovered years ago, there is only little knowledge on the biology and epidemiology of this virus type. some studies indicated a low clinical impact with only mild symptoms [ , , ] , and in spite of a high seroprevalence in the population, virus detections were rare [ , , ] . these findings led to the conclusion that influenza c infection is common, but clinically inapparent or too mild to require a visit to a doctor [ ] . additionally, the low detection rate may be in part due to the fact that in earlier times virus diagnostics were mainly based on virus culture, which is difficult for influenza c [ , ] and necessitates conditions that differ from influenza a and b virus cultivation [ ] . as a consequence, influenza c virus diagnostics were restricted to specialised laboratories and correspondingly rare [ ] . with the introduction of molecular methods, influenza c has been increasingly included into studies on respiratory pathogens and clinical diagnostics. thereby, the low detection rates in the general population were confirmed, but a higher clinical impact for paediatric patients was indicated, as influenza c was described to also cause lower respiratory tract disease [ ] [ ] [ ] [ ] [ ] . in a -month prospective study (december -may including japanese children with communityacquired pneumonia, bronchiolitis or bronchitis, influenza c infection was identified even with a prevalence approximating those of influenza a or hmpv [ ] . further studies, mostly in children, described the symptoms of influenza c infection to be indistinguishable from influenza a and b infections [ , ] , although the maximum body temperature may be lower and the fever shorter compared with influenza a [ , , ] . in finnish military recruits, influenza c virus caused common cold-like symptoms, but occasionally resulted in pneumonia and bronchitis [ ] . for europe, only little information on influenza c circulation has been published. in adults, a seroprevalence of ca % and more was found in france [ ] , finland [ ] , and united kingdom [ ] . applying pcr on samples from all age groups, a virus detection rate of ≤ % was reported for normandy/france [ ] , scotland [ ] and spain [ ] , but higher detection rates of . - . % were found in two adult studies from finland [ , ] . outside europe, a similar seroprevalence as well as comparable detection rates have been described for australia, canada, japan, nigeria, the philippines, peru and the us [ ] [ ] [ ] , , , , , [ ] [ ] [ ] . in view of the lack of knowledge on influenza c virus circulation in germany, we decided to generate the first systematic data on the basis of our national influenza virus surveillance. we chose to examine the age group of - years, as young children have been shown to have the highest infection rates [ , , , ] . we analysed a representative subset of the , samples received in this age group between and ( . - % of all samples in the corresponding month). first, we validated a previously published qpcr [ ] and duplexed it with our routine internal control, fcv. in an extensive validation effort according to international standards [ ] , we found the singleplex as well as the duplex format to perform with high sensitivity, specificity and precision. we therefore applied it to our sample compilation and identified influenza c rna in of , samples with valid qpcr results ( . %). the vast majority of virus detections ( of ) was found in samples that were collected between october and april , signalling a more pronounced virus circulation during these months with positivity rates of up to . % ( / ) in november . as these samples were collected in of german federal states, virus circulation was not confined to a region, but widespread, maybe even nationwide. the virus prevalence however was markedly lower during the other winter seasons observed in this study, and no virus could be detected during the summer months. this absence of summer circulation is in congruence with reports from japan, france, finland and spain [ , , , , ] , but is in contradiction to a report from catalonia in spain, in which the majority of positive samples were taken in august and september of the observed time span [ ] . an upsurge of influenza c virus circulation in the spring of was also observed in the philippines [ ] , but did not occur in japan, from where virus circulation in even numbered years was reported, including the years and [ , ] . however, a biennial pattern of virus circulation with increased or time-shifted profile has also been described for other respiratory viruses in germany [ , ] and therefore is conceivable, but remains open in our study due to the short study period, which presents a limitation. in total, the proportion of influenza c-positive patients was small, but within the expected range. it needs to be emphasised though, that the obtained overall positivity rate is largely based on only few months during the winter season / with substantial virus circulation in our study population. because of our limited access to clinical data, only few conclusions can be drawn with regard to the clinical relevance of influenza c virus. the vast majority of patients ( / ) carrying the virus fulfilled the eu ili definition. although, due to our study design, there may be a bias to ili cases during periods of influenza a and b virus circulation, our findings are in concordance with other studies, in which ili was described for the majority or all of influenza c infected patients [ , ] . bronchitis or bronchiolitis was not reported for any patient, but one child (infected also with an influenza a virus) presented with symptoms of pneumonia. however, the proportion of pneumonia in our influenza c-positive samples does not differ considerably from that of our complete sample collection of this age group spanning the years to (data not shown). in our ambulant setting, we thus do not see an indication for an accumulation of lower respiratory tract disease in influenza c infected patients, but an influenza-like clinical presentation is common. interestingly, a substantial share of influenza c-positive samples showed co-infection with other pathogens, as reported also in other studies [ , , , ] . in these cases, the cause for ili symptoms cannot clearly be attributed to influenza c. yet, we used qpcr assays with comparable performance characteristics (lod and efficiency), so that a comparison of the obtained c q values can be semiquantitatively interpreted for the different pathogens within one sample. in our study, the majority of co-infected samples ( / ; . %) exhibited the highest viral load for influenza c, including the pneumonia case for whom it was ca , -fold higher than that of influenza a at the time point and the sample site examined. in four samples, the influenza c c q was close to the detection limit (≥ ) and thus influenza c was presumably of minor relevance. repetitive sampling from the same patients and continuous parallel assessment of the patients clinical presentation could clarify the role of the single pathogens in the disease course, but is not included in our routine influenza surveillance system. therefore, we cannot judge on the temporal dynamics of virus replication and the clinical impact of each virus detected. due to the slow evolution and thus high antigenic homology of influenza c virus [ ] , we decided to characterise the german sequences only on the basis of the he gene sequences. the he glycoprotein has a variety of functions in the viral replication cycle and greatly determines the antigenicity of the virus [ ] . based on antigenic and phylogenetic characteristics of this protein, distinct virus lineages have been described that were named after their prototype strains c/taylor/ / , c/kanagawa/ / , c/mississippi/ , c/aichi/ / , c/ yamagata/ / and c/sao paulo/ / [ , ] . all influenza c lineage clusters are comprised of isolates from a multitude of continents, indicating a global circulation of virus lineages [ ] . however, four lineages seemingly disappeared (c/taylor, c/aichi, c/ mississippi, c/yamagata), and only the c/kanagawa and c/sao paulo lineage have been detected within the last decade [ , , , , , , ] . from our positive samples, a total of partial and near full-length he sequences could be generated. we almost exclusively detected c/sao paulo lineage viruses, and only two c/kanagawa viruses were identified. our c/ sao paulo sequences add to both lineage subclades described by matsuzaki et al. and represented by c/ aichi/ / and c/victoria/ / [ ] . a total of sequences ( complete, incomplete) group into the c/aichi/ / subclade and were sampled between march and april , while three additional sequences ( complete, incomplete) group into the c/ victoria/ / subclade and were sampled between november and march . thus, viruses of both subclades co-circulated during the / winter season. in contrast, the two c/kanagawa lineage viruses were sampled in march and april , thus outside of the period with increased infection rates. they form a distinct cluster within the c/kanagawa clade, most closely related to c/miyagi/ / . both c/ kanagawa viruses are almost identical to each other showing a nt homology of . %, although they were sampled with a -year distance. their closest neighbour, c/miyagi/ / even has a homology of . % and . % on the nt level. this further supports the described genetic stability of this virus type compared with influenza a and b viruses [ ] , possibly also reflecting their antigenic properties. to summarise, our study is the first report on influenza c circulation in the context of a nationwide outpatient influenza surveillance system in europe. we found influenza c in a proportion of samples that was in accordance with previous reports. an increased and widespread virus circulation was observed during the winter and spring months of / , with viruses predominantly belonging to c/aichi/ / subclade of c/ sao paulo lineage viruses. infected patients showed symptoms of ili including upper as well as lower respiratory tract infection, although its association to the observed clinical symptoms remain uncertain in the majority of cases due to the identified co-infections. further knowledge is needed about the virus epidemiology, its transmission patterns, its role in sole and mixed infections as well as the associated disease burden, especially in young children and patients with lower respiratory tract disease. bootstrap analyses were performed applying the maximum likelihood algorithm with , replicates as described in the methods section. the tree is rooted at c/taylor/ / . only bootstrap values greater than are displayed at the branch nodes. prototypes of influenza c virus lineages are in bold font and blue colour, subclade representatives are bold and underlined. the german sequences are marked in bold italic letters influenza (seasonal) fact sheet. geneva: who production of common colds in human volunteers by influenza c virus influenza c virus infection in military recruits--symptoms and clinical manifestation prospective study of influenza c in hospitalized children influenza c virus and human metapneumovirus infections in hospitalized children with lower respiratory tract illness clinical features of influenza c virus infection in children community-acquired influenza c virus infection in children isolation and characterization of influenza c viruses in the philippines and japan influenza c virusassociated community-acquired pneumonia in children. influenza other respir viruses study of influenza c virus infection in france epidemiological information regarding the periodic epidemics of influenza c virus in japan ( - ) and the seroprevalence of antibodies to different antigenic groups bericht zur epidemiologie der influenza in deutschland saison on the communicable diseases and related special health issues to be covered by epidemiological surveillance as well as relevant case definitions diagnostic approach for the differentiation of the pandemic influenza a(h n )v virus from recent human influenza viruses by realtime pcr detection and quantification of influenza c virus in pediatric respiratory specimens by real-time pcr and comparison with infectious viral counts influenza c virus hemagglutinin: comparison with influenza a and b virus hemagglutinins age distribution of the antibody to type c influenza virus detection of influenza c virus by a real-time rt-pcr assay. influenza other respir viruses isolation of influenza c virus recombinants influenza c virus surveillance during the first influenza a (h n ) pandemic wave in catalonia, spain a novel real-time rt-pcr assay for influenza c tested in peruvian children influenza c virus infection in france detection of influenza c virus but not influenza d virus in scottish respiratory samples detection by reverse transcriptionpolymerase chain reaction of influenza c in nasopharyngeal secretions of adults with a common cold specific viruses detected in nigerian children in association with acute respiratory disease influenza c infections in western australia and victoria from to . influenza other respir viruses sensitive diagnostics confirm that influenza c is an uncommon cause of medically attended respiratory illness in adults minimum information necessary for quantitative real-time pcr experiments genetic lineage and reassortment of influenza c viruses circulating between and human metapneumovirus: insights from a ten-year molecular and epidemiological analysis in germany genetic variability of group a human respiratory syncytial virus strains circulating in germany from to hemagglutinin-esterase-fusion (hef) protein of influenza c virus analyses of evolutionary characteristics of the hemagglutinin-esterase gene of influenza c virus during a period of years reveals evolutionary patterns different from influenza a and b viruses we thank susi hafemann, nathalie tollard and uwe kozian for excellent technical assistance. we also acknowledge all laboratories that contributed influenza c virus sequences to the gisaid database and thereby enabled our analyses. none declared. af: screening and sequencing of patient samples, manuscript preparation; bs: design of study, manuscript preparation; bb: design of study, data analyses, manuscript preparation. this is an open-access article distributed under the terms of the creative commons attribution (cc by . ) licence. you may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence and indicate if changes were made.any supplementary material referenced in the article can be found in the online version. key: cord- - yhoiv authors: singleton, courtney d.; humby, monica s.; yi, hyun ah; rizzo, robert c.; jacobs, amy title: identification of ebola virus inhibitors targeting gp using principles of molecular mimicry date: - - journal: j virol doi: . /jvi. - sha: doc_id: cord_uid: yhoiv a key step in the ebola virus (ebov) replication cycle involves conformational changes in viral glycoprotein (gp ) which facilitate host-viral membrane fusion and subsequent release of the viral genome. ebola gp plays a critical role in virus entry and has similarities in mechanism and structure to the hiv gp protein for which inhibitors have been successfully developed. in this work, a putative binding pocket for the c-terminal heptad repeat in the n-terminal heptad repeat trimer was targeted for identification of small molecules that arrest ebov-host membrane fusion. two computational structure-based virtual screens of ∼ . m compounds were performed (dock program) against a gp five-helix bundle, resulting in commercially available compounds purchased for experimental testing. based on assessment of inhibitory activity, cytotoxicity, and target specificity, four promising candidates emerged with % inhibitory concentration values in the to μm range. molecular dynamics simulations of the two most potent candidates in their dock-predicted binding poses indicate that the majority of favorable interactions involve seven highly conserved residues that can be used to guide further inhibitor development and refinement targeting ebov. importance the most recent ebola virus disease outbreak, from to , resulted in approximately , individuals becoming infected, which led to over , causalities worldwide. the particularly high pathogenicity of the virus makes paramount the identification and development of promising lead compounds to serve as inhibitors of ebola infection. to limit viral load, the virus-host membrane fusion event can be targeted through the inhibition of the class i fusion glycoprotein of ebolavirus. in the current work, several promising small-molecule inhibitors that target the glycoprotein gp were identified through systematic application of structure-based computational and experimental drug design procedures. ular modeling tools and experimental characterization. specifically, large-scale virtual screening of ϳ . million small molecules was performed with gp using the program dock ( ) . we hypothesize that small molecules that interact with the gp nhr pocket will interfere with assembly of the hb required for ebov-host membrane fusion (fig. ) . interfering with hb formation is a strategy previously employed successfully against hiv ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) through targeting an analogous pocket on the viral protein gp ( , ) . the computational screening resulted in the prioritization and purchase of compounds for experimental characterization, which led to hits that inhibit viral entry in both ebov-gp-pseudotyped virus and ebov transcription-and replicationcompetent virus-like particle (trvlp) systems. compounds were further evaluated to assess (i) potential activity artifacts using detergent-containing experiments, (ii) specificity for ebov using a vesicular stomatitis virus glycoprotein (vsv-g)-pseudotyped virus particle counterscreen, and (iii) step(s) within the ebov replication cycle where they exerted the majority of inhibitory activity using time-of-addition (toa) analysis. results suggest that of the compounds act to specifically inhibit ebov entry after attachment but prior to virus-host membrane fusion. molecular dynamics (md) simulations in conjunction with genome analysis identified highly conserved residues across different ebola virus strains (e .a, a .a, l .a, f .a, t .c, l .c, and l .c) that contribute a majority of the favorable interactions between the compounds and gp . virtual screening outcomes. the goal of this study was to identify molecules that inhibit ebov infection by interfering with the interactions required for formation of the gp six-helix bundle ( hb). since the conformational change required to produce the postfusion structure is dependent on chr binding the nhr region of gp (fig. ), a virtual screen of approximately . million compounds was conducted to a five-helix bundle model of gp constructed by the removal of one chr from a high-resolution postfusion structure (pdb entry ebo [ ] ) (see materials and methods, below). compound prioritization led to candidates purchased for experimental testing (fig. ) and employed five distinct scoring functions: dce sum (dock cartesian van der waals and electrostatic energy), fps vdw (footprint comparison of the van der waals energy of the reference peptide and selected ligands), fps es (footprint for electrostatic energy), fps sum (footprint for both van der waals and electrostatic energy), and ts (total score; the combination of dce sum and fps sum ). a large number of molecules was prioritized based on their structural and spatial similarity to the reference ligand composed of a segment of the chr that made the most favorable interactions with our model of a gp five-helix bundle. as a rule, all compounds chosen for experimental testing showed good overlap with the reference (fig. a) . however, those selected based on favorable footprint similarity (fps) have somewhat better overlap than those selected based on dce or ts (fig. b) . consistent with visual inspection (fig. ) , molecules in each of the five groups share similar size and flexibility, with a mean molecular weight (mw) distribution of . g/mol and number of rotatable bonds of . (table ) . compounds purchased based on similarity in electrostatic (es) interaction profiles (fps es ) had the overall smallest mw ( . g/mol) and fewer numbers of rotatable bonds ( . ) , while those selected from the ts list were largest ( . g/mol) ( table ) . as expected ( , ) , compounds selected using a specific scoring function (table , scoring function column) generally showed the best average score with regard to that specific chemical or physical property (table , property columns). for example, compounds prioritized using the dce sum function yielded a more favorable (lower) average dce sum energy (Ϫ kcal/ mol) than those obtained using other functions (Ϫ to Ϫ kcal/mol). likewise, molecules selected using fps sum resulted in a more favorable average fps sum score ( . ) than the other groups ( . to . ). for compounds prioritized using fps es and fps vdw footprint components, the scores were the lowest ( . ) and second lowest ( . ), respectively, among their respective fps es and fps vdw groups. for the dce sum -selected group, the favorable scores can be attributed to strong es interactions resulting in an average dce es score of Ϫ . kcal/mol, over -fold greater than the ensemble average (Ϫ . kcal/mol). the overall strength of the dce sum scores, in conjunction with being the second smallest group in terms of mw and number of rotatable bonds ( . ), suggests that the dce sum list compounds are highly polar. in contrast, the ts list interactions are dominated by strong vdw interactions due to their larger size (mw ϭ g/mol) ( table ) . consistent with the fact that fps sum is a part of the ts scoring function, the fps score components are better than those observed using dce sum . however, the overlap is relatively moderate (fps sum ϭ . , fps vdw ϭ . , fps es ϭ . ); therefore, future work could explore increasing the contribution of the fps component of ts. in summary, molecular property analysis confirms that the purchased candidates are similar in size and flexibility but diverse in terms of interaction energy and overlap the reference peptide. nine molecules from the initial in silico screen inhibit ebov-pseudotyped virus entry in vitro. the compounds identified from the aforementioned in silico screen were tested for their ability to inhibit ebov entry and for cytotoxicity at m ( , , ) . ebov (hiv- /ebov)-pseudotyped virus entry into t cells was quantified by luciferase signal normalized by cytotoxicity and dimethyl sulfoxide (dmso) control to yield the infectivity signal per cell as a fraction of the maximum (see materials and methods). encouragingly, nine compounds resulted in a normalized luciferase signal of Յ . (fig. , blue) . additionally, the observed luciferase signal for the nine compounds was approximately . standard deviations below the average infectivity signal for all purchased molecules, . Ϯ . . although the two compounds with the most activity (i and i ) were also the most cytotoxic (fig. , lower, blue), all nine hits with activity were retained and used as starting points for identification of structurally related analogs in a secondary computational screen (see discussion). secondary similarity screen. to identify additional compounds with enhanced activity, a second similarity-based computational screen was conducted to explore the chemical search space around the nine initial hits. each of the hits in turn was used to rescore and rerank the top , docked molecules from the initial screen to identify compounds with similar functionality and three-dimensional ( d) shape using the dock hungarian similarity (hms) scoring function ( ) . the top-scoring molecules from the nine unique lists were further interrogated using five additional functional methods to assess energy score (dce) and similarity to the initial hit (footprint [fps], pharmacophore [fms], volume overlap [vos], and tanimoto). figure compares docked geometries for four of the initial hits (gray) overlaid with two representative compounds each (orange) from the secondary screen. in these examples, with the exception of i , the compounds generally showed strong overlap and made residue-based interaction patterns similar to those of their respective references (fig. ), corresponding to a high average vos score of ϳ . and a low average fps score of ϳ . . despite the overall similarity of ligand scaffolds within each group, the use of different dock functions generally resulted in the selection of chemically diverse molecules at the atomic level. in some cases, however, the same ligand was the top-ranked candidate across the different groups. for example, rank ordering by pharmacophore or volume overlap yielded the same top-scored results for i (fms ϭ . , vos ϭ . ), which suggests high structure and functional similarity with the initial hit (fig. , fms and vos). overall, the secondary virtual screen resulted in the selection of additional candidates, which were subsequently evaluated for inhibition and cytotoxicity at m against ebov-pseudotyped virus. a luciferase signal of Յ . , which was more than standard deviation below the population mean luciferase signal of . Ϯ . , was used to identify additional hits with moderate to low cytotoxicity ( fig. , green, s prefix). dose-response characterization of candidates against hiv/ebov-gp-pseudotyped virus. to further explore the most promising candidates identified from the two in silico screens ( initial plus secondary), in terms of reducing infectivity and their effects on cell viability, the dose-dependent activity for each was measured. of the tested from fig. , compounds exhibited generally well-behaved entry inhibition compared to that of the known control inhibitor, e , seemingly independent of cytotoxicity, especially at the observed % inhibitory concentration (ic ) values, as shown in fig. . the structures of the compounds, with code names, are shown in fig. . encouragingly, of the molecules, exhibited ic values under m, comparable to the results observed for the control inhibitor e (ic ϭ . Ϯ . m) under the same conditions ( fig. and table ). specifically, the ic values for i , i , and s were less than m, and the ic values for s , s , s , and s were less than m (fig. and table ). an accurate cytotoxic concentration that results in % cell death (cc ) could be obtained for of the compounds. for s , s , and e , the computed cc values had large standard deviations, although examination of the cytotoxicity curves suggests minimal impact on cell viability. the two most potent molecules in this assay, i and i , displayed cc values of approximately to m ( table ). all other hits had observed cc values of m or greater. selectivity index (si ϭ cc /ic ) values were also calculated. the higher the si ratio, the more potent and the safer the compound is projected to be in vivo. examination of the data showed a range of si values from to for pseudotyped virus ( table ) . of the compounds with computable si, the two hits with the greatest si were s and s , which have si values around (table ) . to test the effects of the inhibitors in an ebov system that utilizes virus particles of a size and shape similar to that of native ebov ( ), the compounds were assessed for inhibitory effect against the ebov trvlp system at various concentrations ( fig. ) . notably, of the yielded ic s under m (table ) . of particular interest, comparative linear regression analysis between the ic s observed for each candidate against ebov-pseudotyped virus and trvlps yielded an r value of . (n ϭ ), which increased to . (n ϭ ) with the removal of the outliers i and s ( table ) . as expected, based on the good correspondence between the two dose-response assays, i remained the most potent compound, with an ic of . Ϯ . m (fig. and table ). furthermore, s , which exhibited an ic of approximately m in the pseudotyped experiment, was the only compound found to have an ic greater than m. the range of si values from the trvlp experiments was between . and . , with five candidates yielding selectivity indices greater than that of e (table ). of the aforementioned five inhibitors, the two compounds with the largest si values are s ( . ) and s ( . ) ( table ). in summary, the results indicate good reproducibility between pseudotyped virus and trvlp assays, affirming the observed activity of the tested hits. specificity of candidates for ebov-gp. the hits were also examined using computational and experimental methods to ascertain if the observed activity involved nonspecific effects as a result of colloidal aggregation, pan-assay interference compound (pains) liabilities ( - ), or promiscuity. as an initial step to assess whether activity was a result of colloids, the compounds were screened for structural similarity to known aggregators using aggregation advisor (http://advisor.bkslab.org) ( ) . eight candidates exhibited no known similarity to compounds in the current database. the three remaining compounds (i , s , and s ) were found to have %, %, and % structural similarity to a known aggregator. as described by irwin and shoichet ( ), the addition of detergent should lead to a decrease in activity if a compound inhibits exclusively due to colloidal aggregation. thus, activity was also tested in the presence of . % tween (table ) . compounds here were defined as not sensitive to detergent if their ic values with and without detergent were similar, if their ic ranges with and without detergent overlapped, or if their activity increased. based on these criteria, none of the hits appeared to be sensitive, although s was classified as ambiguous due to the absence of a computable error associated with the ic value. the compounds were also subjected to an evaluation for pains alerts using distinct computational filters (cbligand [ ] , fafdrugs [ ] , and swissadme [ ] ). i was the only compound with a pains warning, which occurred for all three programs due to the possibility of mannich reaction ( ) . despite this warning, we opted to retain compound i at this early stage given the fact that multiple fda-approved drugs elicit pains alerts ( ) . finally, pubchem ( ) was searched to assess if any of the compounds fig dose-response infectivity of ebov trvlps with the treatment of hits. the most promising candidates identified from assays using pseudotyped virus were retested against ebov trvlps. molecules from the initial and secondary screens are labeled with the prefixes i and s, respectively. dose-response curves (black) and cytotoxicity results (red) were generated from replicate experiments (n Ն ). ic s are displayed above each graph with the number of biological replicates performed to calculate the viral entry results. were previously reported as being active against multiple targets (i.e., whether or not they were promiscuous inhibitors). results were only available for i , which had been tested in independent studies. in these prior works, i was reported as active in studies to different targets, as an inconclusive inhibitor in experiments, and as a nonspecific inhibitor of steroidogenic acute regulatory protein (bioassay aid ; https://pubchem.ncbi.nlm.nih.gov/bioassay/ ) ( ) . due to its apparent promiscuity, i was not considered further. a counterscreen using vsv (hiv- /vsv-g) was performed to experimentally determine the specificity of the prioritized set of compounds. in a procedure similar to that of the ebov-pseudotyped virus screen (fig. ) , cells were treated with dmso, the ebov inhibitor e , the nonspecific endosome acidification inhibitor bafilomycin a ( ), or the candidates (fig. ). compound i was tested at m due to its low cc (table ) , while the other candidates were tested at m. notably, all compounds showed less inhibitory activity against the vsv-g screen (fig. ) than the initial ebov-gp screen (luciferase signal, Յ . ) (fig. ) . the four compounds with the least average inhibitory activity against vsv-g and, therefore, likely higher specificity for ebov were i , s , s , and s (fig. ) . these hits showed minimal effects on cell viability. based on the aforementioned analysis, although other compounds shown in fig. would also be promising to explore, at this stage only i , s , s , and s were selected for further characterization. candidate compounds exhibited maximal inhibition postattachment and before membrane fusion. to explore the stage in the ebov entry cascade at which the candidates act, time-of-addition (toa) experiments ( , , , ) were performed ( fig. ) for the four compounds showing the most specificity, as suggested by the averaged activity results depicted in fig. . in this toa assay, t cells were treated with the four candidates and the cathepsin inhibitor e d at various time points postinfection. compounds were tested at the concentration required to reach maximum inhibition without a significant effect on cell viability as described by the dose-response curves against pseudotyped virus (fig. and table ). importantly, the four candidate molecules exhibited an activity trend similar to that of the known control e d, where maximum inhibition occurred up until the -min time point and then began to decrease (fig. ). the fact that the compounds track with e d suggests they act after pinocytosis, after cleavage to the npc binding form, but prior to the fusion step, as expected for molecules targeted to disrupt the interaction between the chr and nhr necessary for hb formation. i and s exhibit reproducible pose stability in md simulations. experimental characterization through concentration-dependent analysis, counterscreening, and toa experiments suggested that i , s , s , and s were the most potent, specific inhibitors of the premembrane fusion stage of ebov entry identified from virtual screening. to more fully explore the energetic and geometric compatibility of these inhibitors with gp at the proposed pocket, all atom md simulations of the dockpredicted poses were executed. as previously described ( , , ) , six replica -ns simulations for each candidate-gp complex were performed in explicit solvent, where each replica employed a different random seed. ligand movement was quantified using rmsds (root mean squared deviations) that accounted for translation, rotation, and differences in internal geometry relative to the initial predicted pose. analysis of the trajectories showed that of the four compounds simulated, i and s maintained their dock-predicted poses more closely across all six simulations, as observed by the reproducible average rmsds of . Ϯ . Å and . Ϯ . Å, respectively (fig. ) . since the average rmsds of i and s were less than or equal to . Å, which is close to the typical benchmark ( . Å) commonly used in redocking validation tests ( ) , additional characterization for these two compounds was performed as described further below. in contrast, s and s adopted a wider variety of ligand poses during md simulations, resulting in a larger range of rmsds (fig. ) . visual inspection showed s adopted two overall geometries during its md simulations, one closer to the original dock pose, which contributed to its bimodal rmsd histogram (fig. ) . in general, compound s showed a much larger overall spread in rmsds (mean of Ͼ . Å) as a result of larger changes in internal geometry and/or movement in the pocket. footprint interaction analysis. as a step toward understanding the hypothesized mechanism of action inhibiting six-helix bundle formation, the interactions of i and s with gp were characterized. to determine which residues had the greatest contribution to the ligand-receptor interactions across both hits, footprint interaction profiles were generated for each compound from the energies obtained over the md trajectories (fig. ) . overall, the footprints showed striking similarity to the reference, especially in terms of the vdw profile (fig. ) , suggesting good molecular mimicry of the chr region. moreover, i and s maintained strong contacts to a similar degree with the same residues, consistent with their overlap in the binding site and structural similarity. the residues with the most favorable interactions across the two candidates, which resulted in combined average energies greater than Ϫ . (fig. ) . regarding the es energies, the reference profile contains two es peaks corresponding to e .a and q .a; however, e .a was the only consensus residue with a combined average energy (Ϫ . Ϯ . kcal/ mol) of less than Ϫ . kcal/mol (fig. ) . notably, s also had a considerable interaction with q .a (Ϫ . Ϯ . kcal/mol) (fig. ). further inspection of the individual footprint profiles of i and s showed that s interacted slightly more favorably with the ebov five-helix bundle than i across multiple residues in addition to q .a. for instance, s had stronger predicted interactions with e .a in both the vdw (Ϫ . Ϯ . kcal/mol) and es (Ϫ . Ϯ . kcal/mol) plots than i (vdw, Ϫ . Ϯ . kcal/mol; es, Ϫ . Ϯ . kcal/mol). although simulation of s resulted in slightly greater energies over of the key residues (fig. ) , the energies of the candidates are within one standard deviation from the means and therefore are insignificantly different, highlighting e .a, a .a, l .a, f .a, t .c, l .c, and l .c as the key gp residues that interact with the reference ligand, i , and s . of the corresponding residues, notable favorable vdw interactions were visualized at f .a and t .c. specifically, f .a was involved in strong nonspecific vdw interactions with the -methyloxy, -carboxylphenyl substituent of i and the phenyl substituent of s (fig. ) . additionally, although both hits interact with t .c, i was the only compound to exhibit a vdw interaction with t .c throughout approximately . % of the simulations. regarding es interactions, the two inhibitors established and maintained strong es contacts with e .a across one main substituent throughout the majority of their md simulations. for instance, the protonated nitrogen of the methylpiperidine substituent of i maintained water-mediated hydrogenbonding interactions (ϳ %) with the backbone and sidechain of e .a and direct hydrogen-bonding interactions with the sidechain of e .a about % of the time (fig. ) . on the other hand, s retained water-mediated interaction with e .a through approximately % of the simulations and direct hydrogen-bonding interactions for a total of approximately % of the simulations (fig. ). in summary, results suggest that i and s have the potential to establish and retain strong vdw and es interactions with the predicted gp binding site. sequence conservation across the key residues. to assess whether the inhibitors have the potential to interact favorably with other ebolavirus species and related filoviridae viruses, a comprehensive sequence alignment study was conducted. specifically, human sample sequences containing the complete gp genome for the five known ebolavirus species, zaire, bundibugyo, reston, sudan, and tai forest, were selected via the virus pathogen resource (vipr) database (www.viprbrc.org; nih). an additional virus sequences were selected using blast ( ) based on similarity to the core gp sequence (pdb entry ebo_a) used to conduct the virtual screens. multiple-sequence alignment was then performed using cobalt ( ) to align the above-mentioned , gp -containing sequences to the full-genome sequence of gp (zaire ebolavirus strain mayinga- ; genbank accession number ahc ). ultimately, sequences seen in humans and nonhuman primates were retained with fragmented or complete gp sequences, which were used for sequence comparison analysis (fig. ) . consistent with previous studies ( , , ) , there is high sequence identity for the core region of gp (g -f ), with the exception of an intentional cys ala mutation introduced into ebo_a to facilitate crystallization ( ) . comparison of genomes to a complete gp sequence (residues to ) exhibited approximately % conservation (fig. , bottom) . notably, the zaire sequences, which are the most common and pathogenic ( ) ( ) ( ) , showed % sequence similarity. the seven key residues identified from the md-based footprint analysis of i and s with gp (e .a, t .c, a .a, l .c, l .a, f .a, and l .c) showed greater than % conservation across all sequences, and for zaire in particular there was ϳ % conservation. overall, the high sequence conservation among the subset of surveyed genomes for the five ebolavirus species, for which a representative example is shown in fig. , suggests that i and s have the potential to interact with the seven key residues in analogous gp binding sites (fig. , shaded bars) and thereby inhibit sequence variants of zaire ebolavirus and different ebolavirus species. however, experimental testing would be required to characterize the activity of the small molecules against the different viruses. ebov particles enter the cell through macropinocytosis ( ) , where they are later trafficked to the endosome and a conformation change is induced in the viral envelope protein gp that leads to membrane fusion ( ) ( ) ( ) ( ) . during this conformational change, the three chr regions bind to the nhr trimer, forming a six-helix bundle ( hb) and mediating host-virus membrane fusion ( ) . due to the current lack of fdaapproved therapeutics available to treat evd and the key involvement of gp in virus entry, this study focused on the identification of small-molecule leads to inhibit the formation of the hb necessary for virus entry by targeting gp at the interface where the chr interacts with the nhr. it is important, however, to note that our gp docking model is only an approximation of the ebov prehairpin and, thus, is not likely to reflect all of the subtleties inherent in the actual biological system. nevertheless, as the approach was successfully used by our group in prior work ( , , ) and led to the identification of entry inhibitors targeting hiv gp , we believe that adapting the methods to target ebola is a reasonable strategy. in this work, an initial virtual screen followed by a second similarity screen were performed to prioritize molecules with energetically favorable interactions with the gp nhr pocket. this led to a total of compounds for experimental testing, of which appeared promising in an ebov-pseudotyped virus entry assay. subsequent doseresponse analyses narrowed down the group to inhibitors with low to moderate cytotoxicity. to further validate activity, the hits were tested against ebov trvlps, which are more similar in shape and size to the native virus. the trvlp results correspond well with those obtained using pseudotyped virus, affirming the hits are promising ebov inhibitors. to probe specificity, the hits were also tested using vsv-g-pseudotyped virus-like particles (fig. ) . at this stage, four compounds (i , s , s , and s ) were prioritized for additional analysis given their strong inhibition, low cytotoxicity, and apparent specificity for ebov. in the time-of-addition assay, the control curve for e d showed the maximal level of inhibitory activity occurring between time and up to the -min time point at which inhibition starts to decrease (fig. ) . this is consistent with other studies ( ) that have a lag in the ebov entry pathway compared to that of influenza virus due to trafficking to the late endosome/lysosome. the timing of loss of inhibition of e d and the experimental compounds, as reported in mingo et al. ( ) , occurred with full restoration of infectivity by the -h time point. in contrast, full infectivity was not restored in our system until approximately h. this could be due to differences in vlps, cell types, or the readout assay. importantly, all four hits (i , s , s , and s ) exhibited a time-of-addition trend similar to that of e d, suggesting that they are acting late in entry at a step that is after npc binding. although the hits are hypothesized to prevent the collapse of the metastable intermediate into the stable hb, it is possible they interact with an earlier gp /gp prefusion conformation. they could also disrupt interactions with other partner proteins, the lipid bilayer, or bilayer components or disrupt the putative e d-sensitive cleavage step ( ) . additional mechanistic investigation, such as site-directed mutagen-esis and structural studies, will be required to confirm our hypothesis that the hits are inhibiting hb formation. further study of the candidate compounds and future work on analogs and additional target sites could uncover important details about the fusion trigger. as an initial step, to help validate that the inhibitors prevent hb formation, the steric and energetic compatibility of the hits were explored via md simulations. for the two hits with the most reproducible ligand poses (lower rmsds), the md analysis identified seven key gp residues (e .a, a .a, l .a, f .a, t .c, l .c, and l .c) engaged in significant favorable protein-ligand interactions (fig. ) . notably, these residues are highly conserved (fig. ) across different ebolavirus species, suggesting the hits have the ability to inhibit different types of evd-causing viruses. the compounds identified in this work have efficacy similar to that of other reported inhibitors of virus entry. specifically, we identified compounds with ic values of less than m, and three of the hits had ic values of less than m. previously reported inhibitors include zmapp, which is a combination of three antibodies, two of which appear to prevent conformational changes in the npc- -primed gp that are necessary for progression to late-stage entry ( ) . the estimated ic value for zmapp is to m (estimated from literature values reported by holtsberg et al. [ ] of . to . g/ml). other examples include c-peptide inhibitors ( ) designed on the concept of the successful hiv peptides t (enfuvirtide) and c , which prevent hb collapse ( ) . in contrast to hiv c-peptides, ebov c-peptides showed weak or insignificant antiviral activity due to their inability to access the endosomal compartment ( ) . however, inhibition was significantly improved when researchers added the hiv tat protein transduction domain (ptd), for which the resulting ebo-tat hybrid showed % inhibition at m ( ). other peptide-based inhibitors include prehairpin intermediate mimics reported by clinton et al. ( ) , which showed mid-nanomolar inhibition in a pseudotype assay and a series of cyclopeptides ( ) with ic values ranging from . to . m. in terms of small molecules, basu et al. ( ) reported a benzodiazepine derivative hypothesized to bind in a pocket observed in a prefusion conformation of gp /gp that inhibited entry with an ic of . m. another study identified that the g protein-coupled receptor (gpcr) antagonist benztropine inhibited ebov with an ic of . m. subsequent crystallographic studies by stuart and coworkers ( , ) showed that benztropine and other compounds, including bepridil, paroxetine, sertraline, toremifene, and, interestingly, ibuprofen, bound to the gp /gp site and are thought to destabilize the protein complex ( ) . in contrast, the present compounds are hypothesized to stabilize a gp fusion intermediate, which prevents conformational changes required for formation of the hb. notably, an investigation of drug synergy reported by dyall et al. ( ) using fda-approved drugs showed that the majority of pairs identified as synergistic inhibitors of ebola virus included an entry inhibitor. this suggests it is worthwhile to determine if there is synergy between the entry inhibitors identified in this work and other compounds. in summary, this study has demonstrated the utility of computer-aided modeling, in conjunction with experimental testing, to identify four compounds (i , s , s , and s ) that appear to be specific inhibitors of ebov entry. we targeted a previously unexploited site on ebov gp in a conformation representative of a prehairpin intermediate and utilized protein mimicry to select for small-molecule gp mimics. the identified inhibitors, hypothesized to prevent formation of the critical hb, serve as proof of principle for this technique and as a starting point for further gp -targeted studies. computational methods. in this work, several computational methods were employed to target gp , which can be arranged into five distinct protocols: (i) gp binding site and reference ligand designation through hot-spot identification, (ii) receptor and reference preparation, (iii) dock receptor setup, (iv) dock virtual screening protocols and compound prioritization, and (v) md simulations. the work employed several software packages, including antechamber, tleap, cpptraj ( ) , sander, and pmemd from the amber suite of programs (university of california san francisco) and dms, grid ( ) , and sphgen ( ) , which are part of the dock suite of programs (university of california san francisco). divided into chunks of at most , molecules. compounds were flexibly (flx) ( ) docked to the gp five-helix bundle in parallel, using the mpi version of dock . (university of california san francisco). for each docked compound, the best scoring pose was retained, which was then energy minimized using the standard dock cartesian energy (dce) function to further fine-tune the interactions between the receptor and candidate ligands and permit footprint similarity scoring ( , ) , where the similarity in vdw and es interaction profiles between the reference and screened molecules was quantified using euclidean distance. key descriptors were computed with the program moe for the , top-scoring molecules based on dce score, including the number of lipinski violations, number of chiral centers, and logp, to aid in compound prioritization. the moe maccs clustering method was concurrently employed, using a best-first approach, to group compounds into structurally related families with the best dce scored compound per family to serve as a clusterhead. to promote diversity in compound selection, the top-scored clusterheads were rank ordered using five distinct scoring criteria: (i) the sum of the van der waals and electrostatic dock cartesian energy score (dce sum ), (ii) the van der waals fps score (fps vdw ), (iii) the electrostatic fps score (fps es ), (iv) the sum of the fps vdw and fps es scores (fps sum ), and (v) the combined dce sum and fps sum scores (total score, or ts) ( ) . following d visual inspection of the top-scoring members from each of the five lists, compounds, referred to with the prefix i (initial screen), were purchased for experimental testing. a second set of ligands, referred to with the prefix s (secondary screen), was purchased based on similarity comparisons to hits identified in the initial screen. similarity was computed using the following dock scoring functions: hungarian similarity ( ), footprint similarity ( ), pharmacophore similarity ( ) , and volume overlap. for both screens, additional ligand properties considered included central location in the pocket, number of chiral centers (less than ), formal charge between Ϫ and ϩ , favorable overall score with respect to the particular rank-order method, and favorable electrostatic score. md simulations and analysis. for the most promising candidates, md simulations were performed to assess geometric and energetic stability. the amber accessory programs antechamber and tleap were used to protonate, solvate, assemble, and assign force-field parameters for the protein receptor (ff sb) ( ) , solvent (tip p) ( ) , and ligand (gaff) ( ) . ligand partial charges were obtained from those preassigned by the zinc database ( ) . the five-helix bundle was capped where the n terminus was capped with ace and the c terminus was capped with nme. as previously described ( ), a nine-step protocol was used to equilibrate each solvated ligandprotein complex. briefly, all simulations were performed using the cuda-accelerated version of pmemd ( ) ( ) ( ) in amber . in short, first the solvent and protein-ligand hydrogens were minimized with a restraint weight of . kcal mol Ϫ Å Ϫ on all complex heavy atoms for , cycles. second, the restraint was lifted and the entire complex was minimized for , cycles. third, over ps, the system was heated from to k. fourth, a short md simulation of ps, with an all-atoms restraint weight of . kcal mol Ϫ Å Ϫ , was performed to optimize the water box density to . lastly, each complex underwent five equilibration steps, each ps in length, with lessening restraint weights of all protein and ligand heavy atoms. for the protein, the restraint weights were (i) visualization of md trajectories was conducted using vmd ( ) and chimera ( ) . the amber accessory program cpptraj ( ) and in-house protocols were utilized to extract vdw and es energies (with distance-dependent dielectric) and compute molecular footprints, rmsds (root mean squared deviations), and hydrogen-bonding interactions of each compound throughout its md trajectories ( , frames for each simulation). as previously described ( , ) , predicted interaction energies from all six replica md trajectories were used to calculate the mean vdw and es energies between the small molecule and each residue of the five-helix bundle. residues with energies of less than Ϫ . kcal/mol for the reference ligand and experimentally verified gp entry inhibitors were used to select key gp residues involved in an interaction energy. to compute ligand rmsds, a two-step protocol was executed ( ) . first, the protein-ligand complex in each frame of the trajectory was aligned using cpptraj so that the protein's alpha carbons overlapped. second, atomic-level small-molecule translation and rotation compared to that of the docked pose was quantified. for interpretation, rmsds were binned based on frequency using cpptraj and plotted using python (python software foundation). the amber accessory program cpptraj was used to extract the direct and water-mediated hydrogen-bonding interactions from each trajectory and provide a frequency, location, and frame. experimental methods. the experimental methods to characterize the inhibitory activity of the small molecules identified from in silico screening are described below. three different assays were employed: (i) pseudotyped hiv- /ebov-gp was utilized to assess viral entry, (ii) pseudotyped hiv- /vsv-g was utilized to assess inhibitor specificity, and (iii) ebov trvlp was utilized as a second confirmatory assay of viral entry. cell lines and plasmids. the following reagents were obtained through the aids reagent program, division of aids, niaid, nih: tzm-bl cells (number ; from j. c. kappes and x. wu) ( ) and replication-defective hiv vector pnl - .luc.r-e-(number ; from n. landau) ( ) . the following reagent was obtained through bei resources, niaid, nih: vector pcdna . containing zaire ebolavirus glycoprotein nr- ( ) . plasmid pcmv-vsv-g was a gift from e. freed (nci-frederick). the ebov trvlp transfection plasmids pcaggs-vp , pcaggs-np, pcaggs-vp , pcaggs-l, pcaggs-t , p cis-vrna-rluc, and pcaggs-tim were a gift from h. feldmann (nih) ( ) . t cells (atcc crl- ) and tzm-bl cells were cultured in dulbecco's modified eagle's medium (dmem; corning) supplemented with % heat-inactivated fetal bovine serum (fbs; gemini bio-products) containing g/ml of streptomycin and u/ml of penicillin (dmem-ps- % fbs) in a °c incubator with % co atmosphere. replicationincompetent pseudotyped virus containing the replication machinery of hiv- and the outer glycoproteins of either ebola (hiv- /ebov-gp) or vesicular stomatitis (hiv- /vsv-g) virus were prepared by a standard transfection method using polyethylenimine (pei) max (polysciences) ( , ) . specifically, h prior to transfection, ϫ cells of t cells were seeded per -mm dish. the cells were cotransfected with equal amounts ( . g) of hiv- core plasmid (pnl - .luc.r-e-) and envelope protein plasmid using g pei transfection reagent per plate. twenty-four h posttransfection the medium was replaced, and pseudotyped virus was harvested from the supernatant at and h posttransfection. the supernatant was clarified by low-speed centrifugation followed by filtration with a . -m-pore-size filter (millipore). the filtered supernatant was centrifuged ( , rpm) at °c for h, and the pellet was resuspended in dulbecco's phosphate-buffered saline (dpbs) and stored at Ϫ °c until needed ( ) . infectious titers of virus stocks were quantified by -bromo- -chloro- -indolyl-␤-d-galactopyranoside staining in tzm-bl cells ( , ) . ebov trvlp preparation. a transient-transfection-based transcription-and replication-competent system that models the entire replication cycle at biosafety level was utilized to confirm inhibition. this system is more physiologically relevant than pseudotyped virus due to the native size and shape of the ebov particles. preparations of ebov trvlps were prepared as previously described ( , ) . briefly, t cells were seeded in ml in a -well plate at ϳ % confluence. twenty-four h postseeding, the cells were transfected with the following plasmids per well: ng pcaggs-vp , ng pcaggs-np, ng pcaggs-t , ng pcaggas-vp , g pcaggs-l, and ng p cis-vrna-rluc, using . g pei transfection reagent. twenty-four h posttransfection, medium was replaced with ml dmem-ps- % fbs. seventy-two h posttransfection, the supernatant containing the trvlps was pooled, clarified by lowspeed centrifugation, and stored at °c. screening of in silico-selected compounds in viral entry assays. viral entry was measured using a luciferase reporter. testing of selected compounds and controls against all three types of virus particles, ebov (hiv- /ebov-gp) pseudotyped, vsv (hiv- /vsv-g) pseudotyped, and ebov trvlp, was performed in a similar procedure. t cells were seeded at ϫ cells/well in -well tissue culture-treated white-bottom plates (greiner) that were precoated with g/ml linear pei (sigma). for ebov trvlp infection, helper ribonucleoprotein (rnp) components must be provided in trans through expression plasmid transfection h postseeding (amounts of helper rnp plasmids per well were . ng pcaggs-vp , . ng pcaggs-np, . ng pcaggs-vp , . ng pcaggs-l, and . ng pcaggs-tim , with . ng pei transfection reagent). twenty-four h postseeding (pseudotyped virus particles) or posttransfection (trvlps), t cells were pretreated with selected compounds or controls for h at °c. the medium then was removed and the cells were infected with virus particles that had also been pretreated for h at °c. after h the inoculum was removed, the cells were washed briefly with pbs, and fresh medium was added. plates were incubated for h, and viral entry was measured using the luciferase reporter. the experiment was also performed in the absence of virus to determine the toxicity of the selected compounds and controls. viral entry and cell viability were measured using one-glo ϩ tox luciferase reporter and cell viability assay (promega) according to the manufacturer's protocol using a spectra max m plate reader (molecular devices). luciferase signal was normalized to the cell viability and then further normalized to the luciferase signal in the dmso-treated samples ( ) . compounds with infectivity signal per cell as a fraction of the maximum below . were considered active hits in the initial screening. additionally, for the dose-response assays, % inhibitory concentration (ic ), % cytotoxicity concentration (cc ), and % confidence intervals (ci ) were computed, and ic was plotted using prism . c (graphpad software, la jolla california usa). cc s were reported if a standard deviation within -fold of the cc could be calculated. as previously described ( ), selected controls were dissolved in dmso. cathepsin inhibitor e (millipore) is a cysteine protease inhibitor that prevents cleavage events that are necessary specifically for ebov fusion with the endosomal membrane. it is used as a positive control for inhibition in hiv/ebov-gp and ebov trvlp assays and as a negative control in vsv-g assays, as it does not inhibit vsv-g fusion. e d has the same action as e but is cell permeable. bafilomycin a (calbiochem) is a vacuolar atpase inhibitor that prevents both ebov and vsv entry by alkalinizing the endosome and is used as a positive control for inhibition in both assays. cells were infected with either ebov-or vsv-g-pseudotyped virus at a multiplicity of infection (moi) of . or with l of ebov trvlps. where indicated, . % tween (sigma) was also added to the assay to test for colloidal aggregation. time-of-addition assay. t cells were seeded at ϫ cells/well in pei-precoated -well tissue culture-treated white-bottom plates. the next day, ebov-pseudotyped virus was added to the cells at an moi of . . the plates were centrifuged for h at °c at , ϫ g to allow the virus to attach to the cells and to synchronize the infection. the plates were washed with pbs to remove unbound virus. the plates were then moved to °c to allow for viral entry ( h). small molecules i ( m), s ( m), s ( m), and s ( m) and the e d control ( m; millipore) were added to the plates at various time points as indicated. cell viability and viral entry were measured and analyzed h postinfection as described above. report of an international commission fifteen countries are at risk of ebola outbreak, says who clinical management of ebola virus disease in the united states and europe characteristics and survival of patients with ebola virus infection, malaria, or both in sierra leone: a retrospective cohort study chemical and structural aspects of ebola virus entry inhibitors identification of a small-molecule entry inhibitor for filoviruses a new player in the puzzle of filovirus entry basic clinical and laboratory features of filoviral hemorrhagic fever ebola virus disease in west africa-clinical manifestations and management emergence of zaire ebola virus disease in guinea bioterrorism: management of major biological agents ebola (ebola virus disease) treatments filoviridae: marburg and ebola viruses 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envelope glycoprotein gp from ebola virus at . -Å resolution designed protein mimics of the ebola virus glycoprotein gp ␣-helical bundle: stability and ph effects c-peptide inhibitors of ebola virus glycoprotein-mediated cell entry: effects of conjugation to cholesterol and side chain-side chain crosslinking design and characterization of ebolavirus gp prehairpin intermediate mimics as drug targets mechanism of binding to ebola virus glycoprotein by the zmapp, zmab, and mb- cocktail antibodies novel small molecule entry inhibitors of ebola virus inhibition of ebola and marburg viral entry by g protein-coupled receptor antagonists toremifene interacts with and destabilizes the ebola virus glycoprotein potent neutralizing monoclonal antibodies against ebola virus infection antibody treatment of ebola and sudan virus infection via a uniquely exposed epitope within the glycoprotein receptor-binding site target identification and mode of action of four chemically divergent drugs against ebola virus infection identification of diaryl-quinoline compounds as entry inhibitors of ebola virus identification of ellagic acid from plant rhodiola rosea l. as an anti-ebola virus entry inhibitor molecular docking based screening of predicted potential inhibitors for vp from ebola virus cysteine cathepsin inhibitors as anti-ebola agents inhibition of heat-shock protein reduces ebola virus replication enfuvirtide dock : impact of new features and current docking performance development of peptide and small-molecule hiv- fusion inhibitors that target gp computer-aided approaches for targeting hivgp small molecule inhibitors of hivgp n-heptad repeat trimer formation footprint-based identification of viral entry inhibitors targeting hivgp development of indole compounds as small molecule fusion inhibitors targeting hiv- glycoprotein- structure-based identification of small molecule antiviral compounds targeted to the gp core structure of the human immunodeficiency virus type development of hiv entry inhibitors targeted to the coiled-coil regions of gp n-substituted pyrrole derivatives as novel human immunodeficiency virus type entry inhibitors that interfere with the gp six-helix bundle formation and block virus fusion design, synthesis, and structureactivity relationship of a novel series of -aryl -( -oxo- -phenethyl- -thioxothiazolidinylidenemethyl)furans as hiv- entry inhibitors development of smallmolecule hiv entry inhibitors specifically targeting gp or gp evidence that a prominent cavity in the coiled coil of hiv type gp is an attractive drug target inhibition of human immunodeficiency virus type infectivity by the gp core: role of a conserved hydrophobic cavity in membrane fusion identification of small molecule inhibitors of botulinum neurotoxin serotype e via footprint similarity implementation of the hungarian algorithm to account for ligand symmetry and similarity in structure-based design a novel life cycle modeling system for ebola virus shows a genome length-dependent role of vp in virus infectivity the ecstasy and agony of assay interference compounds phantom pains: problems with the utility of alerts for pan-assay interference compounds docking screens for novel ligands conferring new biology an aggregation advisor for ligand discovery new substructure filters for removal of pan assay interference compounds (pains) from screening libraries and for their exclusion in bioassays faf-drugs : a web server for compound property calculation and small-molecule inhibitors of ebola virus entry journal of virology chemical library design swissadme: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules pubchem substance and compound databases pubchem bioassay: update vesicular stomatitis virus g protein acquires ph-independent fusion activity during transport in a polarized endometrial cell line ebola virus and severe acute respiratory syndrome coronavirus display 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identification of combinations of approved drugs with synergistic activity against ebola virus in cell cultures ptraj and cpptraj: software for processing and analysis of molecular dynamics trajectory data automated docking with gridbased energy evaluation using shape complementarity as an initial screen in designing ligands for a receptor binding site of known threedimensional structure grid-based molecular footprint comparison method for docking and de novo design: application to hivgp implementation and evaluation of a docking-rescoring method using molecular footprint comparisons comparison of multiple amber force fields and development of improved protein backbone parameters strategies for lead discovery: application of footprint similarity targeting hivgp docking validation resources: protein family and ligand flexibility experiments ucsf chimera-a visualization system for exploratory research and analysis fast, efficient generation of high-quality atomic charges. am -bcc model. i. method fast, efficient generation of high-quality atomic charges. am -bcc model. ii. parameterization and validation development and testing of a general amber force field zinc-a free database of commercially available compounds for virtual screening pharmacophore-based similarity scoring for dock ff sb: improving the accuracy of protein side chain and backbone parameters from ff sb comparison of simple potential functions for simulating liquid water routine microsecond molecular dynamics simulations with amber on gpus. . generalized born routine microsecond molecular dynamics simulations with amber on gpus. . explicit solvent particle mesh ewald spfp: speed without compromise-a mixed precision model for gpu accelerated molecular dynamics simulations vmd: visual molecular dynamics effects of ccr and cd cell surface concentrations on infections by macrophagetropic isolates of human immunodeficiency virus type human immunodeficiency virus type viral protein r (vpr) arrests cells in the g phase of the cell cycle by inhibiting p cdc activity the intracellular cargo receptor ergic- is required for the production of infectious arenavirus, coronavirus, and filovirus particles optimization of lentiviral vector production using polyethylenimine-mediated transfection transient mammalian cell transfection with polyethylenimine (pei) production, concentration and titration of pseudotyped hiv- -based lentiviral vectors detection of replication-competent and pseudotyped human immunodeficiency virus with a sensitive cell line on the basis of activation of an integrated beta-galactosidase gene permanent inhibition of viral entry by covalent entrapment of hiv gp on the virus surface clomiphene and its isomers block ebola virus particle entry and infection with similar potency: potential therapeutic implications. viruses :e development of therapeutics for treatment of ebola virus infection ebola virus entry: a curious and complex series of events key: cord- -gw cow d authors: gray, darren w.; welsh, michael d.; mansoor, fawad; doherty, simon; chevallier, olivier p.; elliott, christopher t.; mooney, mark h. title: diva metabolomics: differentiating vaccination status following viral challenge using metabolomic profiles date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: gw cow d bovine respiratory disease (brd) is a major source of economic loss within the agricultural industry. vaccination against brd-associated viruses does not offer complete immune protection and vaccine failure animals present potential routes for disease spread. serological differentiation of infected from vaccinated animals (diva) is possible using antigen-deleted vaccines, but during virus outbreaks diva responses are masked by wild-type virus preventing accurate serodiagnosis. previous work by the authors has established the potential for metabolomic profiling to reveal metabolites associated with systemic immune responses to vaccination. the current study builds on this work by demonstrating for the first time the potential to use plasma metabolite profiling to differentiate between vaccinated and non-vaccinated animals following infection-challenge. male holstein friesian calves were intranasally vaccinated (pfizer rispoval(®)pi +rsv) and subsequently challenged with bovine parainfluenza virus type- (bpi v) via nasal inoculation. metabolomic plasma profiling revealed that viral challenge led to a shift in acquired plasma metabolite profiles from day to p.i., with metabolites identified whose peak intensities were significantly different following viral challenge depending on vaccination status. elevated levels of biliverdin and bilirubin and decreased -indolepropionic acid in non-vaccinated animals at day p.i. may be associated with increased oxidative stress and reactive oxygen scavenging at periods of peak virus titre. during latter stages of infection, increased levels of n-[( α, β, α)- , -dihydroxy- , -dioxocholan- -yl]glycine and lysophosphatidycholine and decreased enterolactone in non-vaccinated animals may reflect suppression of innate immune response mechanisms and progression to adaptive immune responses. levels of hexahydrohippurate were also shown to be significantly elevated in non-vaccinated animals from days to p.i. these findings demonstrate the potential of metabolomic profiling to identify plasma markers that can be employed in disease diagnostic applications to both differentially identify infected non-vaccinated animals during disease outbreaks and provide greater information on the health status of infected animals. introduction bovine respiratory disease (brd) is a multifactorial disease characteristic of a viral-bacterial synergistic infection with predisposition from environmental stressors [ ] . the disease constitutes a major source of economic loss through mortality, clinical disease and the associated treatments and long lasting reduced growth performance of infected young stock [ , ] . the annual cost of brd is estimated at $ billion in the usa, with preventative measures contributing a further $ billion [ , ] . vaccines are commonly used for controlling brd viral pathogens [ ] , but despite seasonal vaccination, animals can become infected with each new outbreak [ ] , maintaining the infection within the population. the viral pathogens associated with brd [bovine parainfluenza virus type- (bpi v), bovine respiratory syncytial virus, bovine viral diarrhoea virus and bovine herpes virus- ] impair immune responses in infected animals and damage the respiratory tract allowing the establishment of secondary infections, that may develop further into bacterial pneumonia [ ] . however, vaccinated animals can successfully clear viral infections faster than non-vaccinated animals through immune memory response, reducing the associated viral tissue damage or impairment of immune functions preventing the establishment of secondary bacterial and mycoplasma infections [ ] . during disease outbreaks, identification of unvaccinated animals at the early stages of infection could provide a window for effective treatment and facilitate the removal of animals that pose a greater risk of becoming infected and transmitting the infection to more susceptible juvenile stock. furthermore, halting viral disease progression to more severe and costly secondary bacterial infections through the identification of vaccine failure animals during infection outbreaks would reduce the level of antibiotic use in the agricultural industry. the only definitive method for successfully identifying vaccinated animals in the presence of an active viral infection is to determine the rate of viral shedding by virus isolation, cytokine/interleukin profiling or virus neutralization assay [ ] . these types of analysis require repeated sampling, a period for seroconversion and are expensive compared to serology based elisa, and are therefore not routinely employed during endemic viral infection outbreaks. differentiating infected from vaccinated animals (diva) marker vaccines (e.g. a modified wild type virus with a gene deletion resulting in the absence of a particular diagnostic antigen) can be employed to differentiate vaccine antibody responses from that of wild type virus. companion serology based tests rely on seroconversion, and upon exposure to wild type virus the antibody response to diva vaccines will be masked by that of the wild type virus. vaccine diva functionality is often limited to large viruses with increased potential for gene deletion and removal of redundant expressed antigens. therefore, for viruses with small genomes such as paramyxoviruses (e.g. bpi v and bovine respiratory syncytial virus of the brd complex) where gene deletion of neutralizing antigens may reduce vaccine efficacy, alternative approaches are required to provide diva functionality. one approach is to design molecular diva vaccines that contain a marker nucleotide sequence differing from the wild type virus that can be employed in combination with pcr-based molecular diagnostics to differentiate between vaccine and wild virus strains [ , ] . successful differentiation of vaccinated from non-vaccinated animals using this technique requires concurrent vaccination and infection [ , ] , with a narrow diagnostic window post-infection for detection of diva vaccine and viral genetic material. furthermore, detection of vaccine genetic material only demonstrates exposure to the vaccine and not the successful generation of immune protection, limiting functionality in assessment of herd level immunity. consequently, there is a clear need for alternative diagnostic methods that can assess efficacy of vaccines and vaccination status of animals exposed to brd viral pathogens at the early stages of infection prior to seroconversion and which do not require repeated sampling. additionally, the lower initial exposure rates to viral infections in field settings combined with variation in strain nucleotide sequences and short periods of virus secretion highlights the requirement for a diva approach with a long diagnostic window which is not strain specific. a potential approach that can meet these needs is based on the application of metabolomics to identify metabolites or 'small molecules' in biological samples that are signatures that correlate or provide some evidence of immune protection. these metabolites are often the end stage products of biological processes and therefore provide an accurate representation of an organism's homeostatic status at time of sampling [ , ] . metabolomic analysis of bio-fluids has provided new insights to the understanding of the patho-physiological processes involved in disease establishment, development and diagnosis [ ] [ ] [ ] [ ] . whilst metabolomics has had limited application in the field of veterinary research, several studies have demonstrated the potential of this technique in the prediction of brd disease outcome [ ] , differentiation of stress from viral infection responses [ ] , and characteristic of immune responses following vaccination [ ] . this study focuses specifically on bpi v due to its endemnicity within cattle populations and absence of clinical symptoms which still predispose animals to more severe bacterial infections [ ] . due to its small genome and absence of non-redundant proteins suitable for removal in diva vaccines, bpi v is an excellent model for assessing the potential of metabolomics to establish vaccination status in infected animals. the aims of the current study were therefore to assess the performance of reverse phase (rp) and hydrophobic interaction liquid chromatography (hilic) separation methods for ultra performance liquid chromatography-mass spectrometry (uplc-ms) metabolomic profiling of bovine plasma and identify plasma metabolomic markers capable of differentiating between vaccinated and nonvaccinated calves following intranasal challenge with bpi v. this work for the first time reports the metabolomic responses following challenge with bpi v and demonstrates how the application of metabolomic profiling may help overcome current limitations in diva diagnostics by identifying markers capable of differentiating between vaccinated and non-vaccinated animals, and importantly allow the development of better tools to assess the performance of vaccines. assessment of clinical findings of animals post bpi v vaccination and challenge have been reported in detail previously [ ] . briefly, animals were healthy throughout the duration of the study with no clinical signs of disease in vaccinated or non-vaccinated study groups. calves were sourced from respiratory disease free farms with no history of vaccination against brd. prior to the commencement of vaccination all calves tested seropositive for anti-bpi v igg. these residual levels of maternally derived anti-bpi v immunoglobulin are in keeping with other studies employing calves of the same age [ ] . at the commencement of vaccination there was no significant difference in anti-bpi v igg observed between treatment groups. vaccination with rispoval pi +rsv resulted in a significant increase in anti-bpi v igg with no significant increase in non-vaccinated controls. following bpi v challenge vaccinated calves had significantly higher plasma anti-bpi v igg relative to non-vaccinated animals. post-bpi v challenge, anti-bpi v igg remained elevated in vaccinated animals. although no significant differences in anti-bpi v igg levels were observed in non-vaccinated animals at days - post-infection (p.i.), igg was elevated in / animals relative to day levels, with the remaining animal demonstrating elevated levels from days to day p.i. there was a significant increase in lymphocyte counts in non-vaccinated animals from day to p.i., and a significant decrease from days to , with no significant variations observed in vaccinated animals. there were no significant differences in neutrophil counts between study groups at sampling points post-infection, however significant (p < . ) temporal variations were observed with a decrease from day to in non-vaccinated animals, and an increase from day to in both study groups. at day p.i., animals per group were prepared for gross postmortem analysis. two of animals in the non-vaccinated group showed gross inflammatory lesions, with some peri-vascular cuffing consistent with pneumonia. no significant gross / histological abnormalities were detected in / of the vaccinated calves at day p.i. at postmortem. preliminary metabolomic analysis was performed to select a suitable method for the comprehensive profiling of metabolites within bovine plasma. rp and hilic chromatographic methods were compared using day p.i. samples (n = ), which corresponds to peak viral titre [ ] . despite improved resolution of poorly retained hydrophobic compounds eluting between - min by hilic separation, increased chromatographic peaks corresponding to plasma derived compounds (relative to blank injections) were observed in rp acquired profiles (fig a and b) relative to hilic profiles (fig c and d ). observable differences between plasma samples from vaccinated and non-vaccinated animals within chromatogram profiles were only present using rp (s fig) . furthermore, upon data extraction and processing (metabolites with cv > % in qc pools), increased numbers of accurate mass retention time pairs (amrtps) were observed following rp (n = ) relative to hilic (n = ) separation of plasma. unsupervised pca analysis of plasma profiles acquired by rp-uplc-ms resulted in clear separation between study groups when assessing principle components (pc) and ( . % of systemic (r x) variation within the dataset) as illustrated in fig a. in contrast, hilic-uplc-ms metabolomic profiles facilitated only partial separation between study groups based on the pca scores plot (pc and pc , . % of systemic (r x) variation) ( fig b) . due to the small number of chromatographic peaks, fewer amrtps and poor unsupervised pca separation of plasma profiles acquired following hilic-uplc-ms chromatography, rp-uplc-ms was selected as the method of choice for metabolomic profiling of remaining study plasma samples. plasma samples from days , , and p.i. were extracted, analysed and combined with day p.i. data for multivariate analysis. mass accuracy and retention time deviation of reference compounds between analysis runs was excellent (s table) and within marker lynx extraction parameters ( . da and . min respectively) ensuring accurate peak matching. amrtps (n = ) with %cv less than % in inter-run quality control plasma pools were used to construct of pca models (pre-filtered from amrtps extracted from raw data). the effect of bpi v challenge on the metabolite profile of all animals irrespective of vaccination status, was illustrated in un-supervised pca scores plot ( fig a) by separation in pre-(day p.i.) and post-bpi v challenge samples (days - p.i.) when observing pc and pc ( . % r x). the greatest separation was observed between day pre-and day p.i. post-challenge stages in vaccinated and non-vaccinated animals. differentiation of vaccinated from non-vaccinated animals based on metabolite profiles following challenge with bpi v was investigated using un-supervised (pca) and supervised (opls-da) multivariate analysis. prior to bpi v challenge metabolite profile variation between vaccinated and non-vaccinated animals was low, evidenced by no separation at day ( fig b) when observing all pcs. with study progression to post-challenge stages, the variation between vaccinated and non-vaccinated animal metabolite profiles increased from partial separation at day p.i. (fig c, . % r x with pcs and ) to clear separation at days , and , with % (fig a) , . % ( fig d) and . % ( fig e) variation (r x) respectively in pcs and . opls-da analysis was employed for the selection of marker amrtps that could discriminate between vaccinated and non-vaccinated animals post-infection. fig a- d illustrates opls-da score (inset) and s-plots for supervised discriminate analysis of plasma metabolite profiles from bpi v infected vaccinated and non-vaccinated animals at days , , and p. i. respectively. the amount of variation responsible for differentiation of animals of different vaccination status (r x) was . %, . %, . % and . % in models generated for days , , and p.i. respectively, with excellent fit (r y > . %) and good cross-validated prediction (q > %). as opls-da is known to over-fit, particularly in megavariate datasets where the number of variables are higher than the sample size we employed permutation and false discovery rate testing to reduce the chances of selecting false positives. leave-one-out cross validation was performed to assess the performance of the models generated. briefly, all technical replicates from a single biological sample (test sample) were removed and the remaining dataset employed to generate a predictive opls-da model (vaccinated vs non-vaccinated). this predictive opls-da model was employed to predict the vaccination treatment group of the test sample. this cross-validation was permeated until all biological samples had been assessed for treatment classification. the results (s table) indicated that all biological replicates were accurately classified to their respective treatment groups upon cross validation prediction model testing. amrtps which contributed to class discrimination were selected on a criterion of a variable importance score (v.i.p.) score > from opls-da discriminate analysis. amrtps were further filtered to select only those with a fold change (fc) > . and significant difference (anova with bonferroni post-hoc test) p < . between study groups. final selection of amrtps was performed by assessment of raw mass spectrometric data to determine those with good peak shape and consistent peak intensity (height) in replicate injections. unique potential markers combined from all opls-da analysis ( at day p.i., at day p.i., at day p.i., and at day p.i.) were selected for further refinement to remove fragments and adducts for parent ion identification. the selected panel of unique amrtps (s table) differentiating animals of different vaccination status at various time-points post-bpi v challenge were deconvoluted to identify parent ion mass, adducts and low energy fragments using low and high energy data (function and respectively), yielding parent ions for elemental composition determination. nonparametric mann whitney was also employed on the selected panel of metabolites from day , and p.i. time points, with all markers attaining significance levels within thresholds comparative with anova. the false discovery rate was calculated from the cv ( %) filtered dataset via the benjamini-hochberg procedure with a threshold of %. all selected markers passed this threshold and met further selection criteria (v.i.p. score > and fc > . ) were selected as potential markers. retention times and accurate masses of potential markers post-bpi v challenge (days , , and p.i.) are shown in table this is the first study to report the potential to differentiate between infected animals with differing vaccination status through the use of metabolomic profiling techniques (diva metabolomics). previous work [ ] by the authors has demonstrated that metabolomics can identify metabolites associated with immune responses to vaccination, and the current study has advanced this concept by applying metabolomics analysis of plasma to identify unique metabolite marker profiles that are capable of distinguishing between vaccinated and non-vaccinated animals following infection. calves vaccinated (rispoval pi +rsv) and infection-challenged (bpi v) demonstrated a significantly stimulated anti-bpi v igg post-vaccination response which remained elevated post-challenge. lung histology and haematology confirmed that the bpi v inoculum employed for viral challenge was sufficient to induce some evident respiratory pathology in non-vaccinated animals, with an absence in vaccinated animals. the subsequent challenge in primed vaccinated animals also resulted in maintenance of pre-challenge elevated anti-bpi v igg (indicating response to inoculum), with absence of respiratory pathology. following preliminary analysis of a subset of samples (day p.i.), rp-uplc-ms analysis demonstrated increased capacity to profile bovine plasma metabolites relative to hilic-based chromatographic separation and was subsequently used for extensive metabolomic plasma profiling. unsupervised pca analysis of acquired metabolomic profiles of preand post-challenge plasma revealed clear and distinguishable variation in plasma metabolites between vaccinated and non-vaccinated animals. considering that elevated anti-bpi v igg typically occurs at weeks post-challenge [ , ] , variation in plasma metabolite profiles as early as day p.i. illustrates the capacity of metabolomic profiling methods to detect changes stimulated by immune responses at early post-infection phases. supervised multivariate discriminant analysis of plasma metabolomic profiles yielded potential metabolites (amrtps) that were significantly different in the plasma of vaccinated compared to non-vaccinated animals following bpi v challenge. following de-convolution (with removal of adducts and fragment ions), parent metabolite ions were identified and shown to be present at different levels within plasma from vaccinated and non-vaccinated animals from days - p.i. despite no significant differences in the parent ion intensity between treatment groups at day p.i. plasma metabolite profiles differed between vaccinated and nonvaccinated animals through supervised opsl-da. identities of of these parent metabolites were revealed via database searching, in silco fragmentation and spectral matching. divergent plasma metabolite profiles have previously [ ] been reported following vaccination, with altered metabolites shown to be associated with primary or secondary immune responses to vaccination. the metabolomic profiling performed here in this study on post-bpi v challenge acquired samples, has identified a unique panel of plasma metabolites which differ between vaccinated and non-vaccinated animals, and significantly are involved in recognised immune response mechanisms. at day p.i., increased biliverdin (fc = . ), bilirubin (fc = . ) and decreased -indolepropionic acid (fc = - . ) levels were observed in plasma of non-vaccinated animals. biliverdin is degraded from heme by heme-oxygenases, and is further reduced to bilirubin by bilirubin reductase [ ] . heme-oxygenase- exerts anti-inflammatory effects as demonstrated by reduced tumour necrosis factor alpha release in lipopolysaccharide-stimulated macrophages [ ] . -indolepropionic acid is a reactive oxygen species scavenger [ ] , produced in the microbiome [ ] . phagocyte activation by rna viruses results in both increased reactive oxygen species release and the production of pro-oxidant cytokines (tumour necrosis factor alpha and interleukin- ) which promote iron uptake by the mononuclear phagocyte system [ , ] . decreasing plasma -indolepropionic acid levels in concert with elevated biliverdin and bilirubin levels (via heme-oxygenase- action) in non-vaccinated animals at day p.i. maybe indicative of -indolepropionic acid scavenging of reactive oxygen species produced by phagocytes, or increased downstream ros production from cytokine signalling. fluctuations in the levels of a number of bile acids ( -oxocholic acid, cholic acid and ndgca) were observed in plasma of animals at various stages in the study. -oxocholic acid and ndgca were found to be significantly increased (fc = . and . respectively) in the plasma of non-vaccinated animals at day and p.i respectively, whereas cholic acid was significantly higher in vaccinated animals (fc = - . ) at day p.i. altered plasma bile acid profiles at distinct study phases suggest that bile acid metabolism and conjugation is associated with different immune response mechanisms. bile acid driven farnesoid x receptor activation (expressed in pulmonary endothelial cells [ , ] ) enables a regulatory immune response as demonstrated by dendritic cell modulation [ ] , natural killer t-cell inhibition, reduced proinflammatory osteopontin production [ ] and reduced lung permeability, suppressing leukocyte movement to sites of tissue inflammation [ ] . when transported into cells ndgca is a potent farnesoid x receptor activator [ ] and elevated ndgca at day in non-vaccinated animals suggests an association with the switching and/or suppression of innate inflammatory responses towards adaptive immune responses. cholic acid, the primary metabolite of cholesterol, was found to significantly increase from day to p.i. in vaccinated animals. cholesterol, a component of lymphocyte lipid rafts, supports b-and t-cell receptor signalling [ ] . reverse cholesterol transport is associated with immunosuppression via reduction in b-and t-cell receptor signalling, lymphocyte activation and proliferation [ , ] , and elevated cholic acid may indicate increased cholesterol metabolism via reverse cholesterol transport in proliferating lymphocytes, down-regulating the secondary immune response to viral challenge during later stages of infection (day p.i.). lysophosphatidylcholine produced from phosphatidylcholine [ ] [ ] [ ] stimulates dendritic cell maturation through the action of a g-protein-coupled receptor with further ability to stimulate interleukin- and interferon-γ production in t cells [ ] indicating a role in innate-toadaptive immune response progression. plasma levels of six lysophosphatidylcholine derivatives were significantly up-regulated (fc > . ) in non-vaccinated animals at day p.i. significantly higher plasma levels suggest involvement in systemic immune responses to primary bpi v antigen stimulation with dendritic cell recruitment to lymph nodes and subsequent dendritic cell driven t-cell activation. decreased plasma peak intensity of enterolactone (fc = - . ), a lignan metabolite formed in ruminants through the action of colonic bacteria on secoisolariciresinol diglucoside [ ] , was observed in non-vaccinated animals at days and p.i. enterolactone crosses the intestinal barrier and exerts anti-inflammatory functions by suppressing nuclear factor-κb signalling, tumour necrosis factor alpha production [ ] and interleukin- β release [ ] . as enterolactone is dietary derived it cannot be readily replenished or up-regulated during periods of high demand, therefore decreased enterolactone in non-vaccinated animals during the latter stages of bpi v infection (days and post-challenge) may reflect its metabolism to induce anti-inflammatory effects associated with the transition of acute to adaptive immune responses. from a diagnostic perspective, those plasma metabolite markers found to be altered at early stages of infection prior to sero-conversion and which persist to the later stages of infection are of particular interest. hexahydrohippurate, despite not showing significantly altered plasma levels until day p.i., remained elevated in vaccinated animals (fc > - . ) for the duration of the study. significantly lower levels of n-methylhippuric acid (fc = . ) and higher n-(cyclohex- -en- -ylcarbonyl)glycine (fc = - . ) were also observed in vaccinated calves at day p.i. n-(cyclohex- -en- -ylcarbonyl)glycine, n-methylhippuric acid and hexahydrohippurate are formed through the action of glycine n-acyltransferase on cyclohexanecarboxy-coa, cyclohexene- -carboxyl-coa or benzoyl-coa, produced during shikimate and phenylalanine metabolism [ , [ ] [ ] [ ] [ ] . with no significant variation in the plasma levels of phenylalanine (which would attribute post-bpi v challenge acyl-glycine conjugate to dietary variations), elevated hexahydrohippurate and n-(cyclohex- -en- -ylcarbonyl)glycine levels may be a consequence of an increased demand for coa in the liver due to the need to free coa otherwise sequestered in cyclohexanecarboxy-coa or cyclohexene- -carboxyl-coa. increased coa demand may therefore be associated with increased rate of b-and t-cell proliferation and antibody production following an adaptive memory response to bpi v challenge (as illustrated previously by post-parental immunization [ ] ). assessment of hexahydrohippurate concentrations in plasma could allow for differentiation of vaccination status in infected calves with a wider diagnostic window to that observable currently through monitoring differences in anti-bpi v igg levels. hexahydrohippurate occurs at high abundance in plasma offering potential for use as a realistic marker that could be measured using on-site based testing methods. in conclusion, this study highlights the potential of untargeted uplc-ms metabolomics to differentiate the vaccination statuses of virus challenged animals (i.e. diva metabolomics). the differential pre-challenge immune status of vaccinated and non-vaccinated animals resulted in divergent plasma metabolite profiles following bpi v challenge, evident as early as days p.i., with increasing variation from time post challenge. the metabolites identified were associated with immune cell regulation mechanisms, including t/b-cell proliferation and phagocyte activation and maturation. the wide diagnostic window for hexahydrohippurate combined with metabolite markers altered at distinct time periods associated with specific immune response mechanism (e.g. lysopc, biliverdin, bilirubin or -indolepropionic acid), could find application in staging of infection. the effectiveness of these efforts is reflected in the large magnitude fold-change and low intra-group variation of statistically significantly metabolites identified at days and p.i. similar to cytokine profiling, metabolomic based diagnostics are unlikely to match the pathogen specificity of molecular and serological testing but instead provide greater information on physiological health status, with future disease diagnostics potentially employing multiple methods to improve disease management decisions. a limitation of this study, as with many other biomarker discovery investigations involving large bovine animals, is the small cohort size (i.e. n = per group; n = at days and p.i.) which can be incorporated effectively into experimental groups. this potentially may impact on how findings from resulting analysis can be accurately translated to that observed within the wider more variable herd population. to mitigate against such issues, animals were extensively screened with regards to health and maternal antibody status prior to study group inclusion, and post-metabolomics profiling, stringent metabolite selection criteria (fc > . ; p < . ; v.i.p. score > ; % false discovery rate) were applied to select only the most robust metabolites as marker candidates. as such, further investigation using a larger, more comprehensive sample set (differing sexes, breeds and ages of animals) with alternative routes of vaccination, vaccination failure (degradation or immunosuppression) and challenge (with multiple pathogens) may determine a panel or 'fingerprint' of metabolites with greater diagnostic specificity. a number of unidentified markers showed promising expression profiles and as metabolomics is still a relatively new field, lacking the level of database curation compared to genomics and proteomics for marker identification, these markers may be identified in the future. further studies are required to expand upon this initial proof-of-concept to determine if the observed diva metabolite markers are robust. importantly these studies have identified potential markers that would also be of benefit in screening and assessing new vaccine formulations and allow identification of trails indicative of protective immune responses. hplc grade acetone and analytical standards were purchased from sigma aldrich (dorset, uk). lc-ms grade acetonitrile, water, methanol and chloroform were purchased from fisher scientific (loughborough, uk). all animal studies were carried out in accordance with the uk animals (scientific procedures) act and with the approval of the agri-food and biosciences institute northern ireland ethical review committee. calves were vaccinated with rispoval pi +rsv as previously reported [ ] . briefly, male holstein friesian calves aged between and weeks were sourced commercially from farms with no history of prior respiratory disease outbreaks. the calves had no prior vaccination for brd and were clinically examined and declared fit for the study on day by the named veterinary surgeon. claves were divided into two study groups (n = ) and assigned as non-vaccinated and vaccinated calves. vaccinated calves were treated with pfizer rispo-val pi +rsv intranasal vaccine (designated vaccinated animals) as per manufacturer's instructions, and non-vaccinated calves treated with empty poly-(lactic-co-glycolic) acid nanoparticles (designated non-vaccinated) prepared using standard double emulsification solvent evaporation technique (w/o/w) [ ] . calves received two dosages of vaccine formulation at and days prior to intranasal bpi v challenge (post-infection = p.i.) (inoculation with ml of virus suspension (tcid of . /ml) per nostril). calves were screened weekly following vaccination and at days , , , and post-bpi v infection (p.i.) for the presence of bpi v igg in blood serum using svanovir-pi v-ab kit (boehringer ingelheim svanovir, uppsala, sweden) as per manufacturer's instructions. at day p.i. animals per group were sacrificed for viral isolation and histology after sampling. blood samples drawn via jugular venepuncture at days , , , and p.i. into ml plastic k edta vacuette tubes (greiner bio-one, stroudwater, uk) were processed at random to platelet poor plasma via a double centrifugation method [ ] optimized for metabolomic analysis within hours of initial blood drawing and plasma stored at - ˚c prior to use. samples processing order was randomized to negate processing bias on sample metabolite profiles. μl of plasma was added to . ml of ice cold acetone, vortexed for sec and placed on ice for min. the sample was then deproteinated by centrifugation at , g at ˚c for min. . ml of supernatant was removed and dried under nitrogen for min at ˚c using turbovap lv (caliper life sciences, hopkinton, usa). resulting residue was reconstituted in μl of ultra-pure h o and liquid/liquid extraction of lipids performed by addition of μl of ice-cold methanol:chloroform ( : v/v) and vortexing for sec followed by centrifugation at , g at ˚c for min. liquid/liquid extraction was repeated and after centrifugation μl of the aqueous layer was removed and dried under nitrogen. the residue was reconstituted in μl ultra-pure h o and filtered by centrifugation at , g, ˚c using . μm costar spin-x centrifuge tube filter for mins. μl of plasma was added to a well in an ostro protein precipitation and phospholipid removal plate (waters corporation, milford, ma, usa). μl of % formic acid/acetonitrile (v/v) was added to the sample well, the plate shaken for sec and extracted metabolites drawn through under vacuum into a well ml collection plate. rp-uplc-ms analysis was performed using an acquity uplc system coupled to a xevo g q-tof (waters corporation, milford, ma, usa). a test mix of acetominophen, sulfaguanidine, sulfadimethoxine, val-tyr-val, verapmil, terfenadine, leucine-enkephalin, reserpine and erythromycin was injected to ensure calibration of mass spectrometer mass accuracy and uplc performance prior to analysis. pooled samples (comprising a μl aliquot from all study samples) were injected times before the start of each run for column conditioning [ ] and intermittently throughout the run to validate instrument performance. the run-order of samples entering the mass spectrometer was constructed using a randomized sample list comprising technical replicates per biological sample. no other samples were injected during the analysis run. μl of prepared sample extracts were injected onto an acquity uplc hss-t column ( mm x . mm i.d., . μm; waters corporation, milford, ma, usa). column and autosampler temperature were maintained at ˚c and ˚c respectively. chromatographic separation was carried out at a flow rate of μl/min with mobile phase consisting of . % h o/ . % formic acid (a) and . % acetonitrile/ . % formic acid (b). the elution gradient was as follows: - min isocratic at % of b, - . min linear gradient form - % of b, . - . min isocratic at % of b, and finally . - min linear gradient at - % of b. mass spectrometry was performed in positive-ion mode (esi+) with the capillary voltage set to v and the sampling cone voltage v. the desolvation and cone gas flows were set at l/h and l/h respectively. source and desolvation temperatures were ˚c and ˚c respectively. leucine enkephalin ([m+h] + = . da, and [m+h] + = . da) was used for accurate lockmass calibration during data acquisition. lockmass acquisition setting were: . sec scan time, sec interval, scan average, mass window +/- . da. centroid data were acquired in positive mode using resolution mode. collision energy was only applied on function , with ramping between ev and ev. for hilic-uplc-ms/ms analysis the test mix was cytosine, o-acetyl-l-carnitine and lvaline. pooled samples (comprising a μl aliquot from all study samples) were injected times before the start of each run for column conditioning [ ] and intermittently throughout the run to validate instrument performance. the run-order of samples entering the mass spectrometer was constructed using a randomized sample list comprising technical replicates per biological sample. no other samples were injected during the analysis run. μl of prepared sample extracts were injected onto an acquity uplc beh hilic column ( . mm x mm i.d., . μm; waters corporation, milford, ma, usa). column and autosampler temperature were maintained at ˚c and ˚c respectively. chromatographic separation was carried out at a flow rate of μl/min with mobile phase consisting of mm ammonium formate ph . (a) and acetonitrile with . % formic acid (b). the elution gradient was as follows: - min isocratic at % of a, - min linear gradient form - % of a, - min linear gradient from - % of a, - min isocratic at % of a, - . min linear gradient from - % of a, . - min isocratic at % of a. mass spectrometry was performed using a waters xevo g q-tof (milford, ma) operating in positive-ion mode (esi+) with the capillary voltage set to v and the sampling cone voltage v. the desolvation and cone gas flows were set at l/h and l/h respectively. source and desolvation temperatures were ˚c and ˚c respectively. leucine enkephalin was used for accurate lockmass calibration during data acquisition, with acquisition settings the same as with rp-uplc-ms analysis. centroid data were acquired in positive mode using resolution mode. collision energy was only applied on function , with ramping between v and v. total ion count (tic) chromatograms and spectra were acquired with masslynx version . (waters corporation, milford, ma, usa) in centroid format and metabolite data was processed using the markerlynx software. the markerlynx method for data extraction and deconvolution was as follows. ions were extracted from function data using peak detection analysis of retention time window . - . min, with a mass range of da to da. the xic window for data collection was . da and apex peak tracking parameters were set to automatic with no smoothing. data collection parameters consisted of an intensity threshold (counts) of , a mass window of . da with a retention time window of . min. a noise elimination level of was applied and isotopes were removed. peak heights for extracted ions were normalized against the total peak height of all extracted ions and standardized to a total ion count of , . the results were exported in .csv format as a two dimensional data table in which rows and columns respectively represented analysed samples and the relative normalized peak heights of each detected mass spectrometric signal, i.e. as an accurate mass (m/z) and retention time (min) pair (amrtp). extracted and processed data submitted for downstream multivariate and statistical analysis for metabolite marker selection can be found in supplementary data (s table) . temporal changes in bpi v antibody titre were analysed using two-tailed paired t-test, and significant differences between treatment groups at sampling stages was assessed using two-tailed heteroscedastic t-test. simca-p+ version . (umetrics, sweden) was used for multivariate metabolite marker selection. the dataset was pre-filtered to exclude amrtps with coefficient of variation greater than % in inter-run quality control pools (generated from equal aliquots of all extracted samples and injected intermittently throughout the analysis run). all centroid data were pareto scaled and analysed by unsupervised principle component analysis (pca) and supervised discriminatory analysis by orthogonal projections of latent structures-discriminant analysis (opls-da). unsupervised pca models were generated at each sampling day to reveal potential relationships between treatment groups. supervised analysis by opls-da was performed to reveal potential markers of response to treatment in vaccinated calves compared to non-vaccinated calves at each sampling day. robustness of final opls-da discriminative models was assessed by setting a predictive model of each case in which / of the data (known treatment) was used to predict the remaining / (unknown treatment). significance of the identified markers at all time points was determined using anova with bonferroni post-hoc test and non-parametric mann whitney for day and p.i. time points. the elemental composition of selected parent compounds was determined in masslynx using both positive and negative mode data. mass uncertainty was set to mda, odd and even electron state, carbon isotope filter of +/- % and elements included were c, h, o, n, p and s. where applicable na and k adduct elemental composition were determined with the respective element included in the analysis parameters. elemental compositions were searched against pubchem and chemspider online databases, and where possible function fragments were matched against metlin, hmdb or massbank databases. where fragmentation spectra for the analyte in question was not available, in silico fragmentation was performed using metfrag and function fragmentation data was validated against potential in silico fragments. identified compounds were validated using mass spectrum and retention time relative to authentic analytical standards analysed under identical experimental conditions (s fig). pooled plasma samples and individual analytical standards ( μm) were analysed under identical uplc and mass spectrometric run conditions as utilized previously [ ] , and metabolite identities confirmed by matching retention time and function and function spectra (including low and high energy fragments and adducts). putative annotations were acquired for compounds with spectral similarity to public spectral libraries (metlin, hmcb or massbank). table. cross-validation of opls-da models. leave-one-out (loo) cross validation was performed to assess the performance of the models generated. all technical replicated from a single biological sample were removed and an opls-da model containing the remaining biological and technical replicates employed to predict class representation. (xlsx) s table. selected amrtps of plasma metabolomic markers of response bpi v challenge in vaccinated and non-vaccinated animals. combined list of amrtps selected using opls-da at days , , and post-bpi v infection filtered to exclude those with p < . , fc < . and v.i.p. score < . (xlsx) s table. processed raw data employed for downstream metabolomic analysis. markerlynx was employed for data extraction and processing. amrtp levels are reported as peak height. the medicine and epidemiology of bovine respiratory disease in feedlots factors of respiratory disease: review of management factors. bovine respiratory disease: total health management use of treatment records and lung lesion scoring to estimate the effect of respiratory disease on growth during early and late finishing periods in south african feedlot cattle economic impact associated with respiratory disease in beef cattle. veterinary clinics of north america-food animal practice a dynamic range compression and three-dimensional peptide fractionation analysis platform expands proteome coverage and the diagnostic potential of whole saliva duration of immunity of a quadrivalent vaccine against respiratory diseases caused by bhv- , pi v, bvdv, and brsv in experimentally infected calves iowa: iowa state 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effects of gut microflora on mammalian blood metabolites human leukocyte pyrogen induces release of specific granule contents from human neurtophils oxidative stress during viral infection: a review. free radical biology and medicine downregulation of endothelin- by farnesoid x receptor in vascular endothelial cells fxr-mediated regulation of enos expression in vascular endothelial cells ursodeoxycholic acid suppresses eosinophilic airway inflammation by inhibiting the function of dendritic cells through the nuclear farnesoid x receptor the bile acid sensor farnesoid x receptor is a modulator of liver immunity in a rodent model of acute hepatitis fxr protects lung from lipopolysaccharide-induced acute injury endogenous bile acids are ligands for the nuclear receptor fxr bar lipid rafts and signal transduction high-density lipoproteins and the immune system hdl in innate and adaptive immunity hydrolysis of phosphatidylcholine during ldl oxidation is mediated by platelet activating factor acetylhydrolase serum lysophosphatidic acid is produced through diverse phospholipase pathways differential hydrolysis of molecular species of lipoprotein phosphatidylcholine by groups iia, v and x secretory phospholipases a mature dendritic cell generation promoted by lysophosphatidylcholine weekly excretion of the mammalian lignan enterolactone in milk of dairy cows fed flaxseed meal enterodiol and enterolactone modulate the immune response by acting on nuclear factor-kappa b (nf-kappa b) signaling flaxseed, and the lignan enterolactone increase stroma-and cancer cell-derived il- ra and decrease tumor angiogenesis in estrogen-dependent breast cancer metabolism of cyclohexanecarboxylate in rat glycine conjugation activity of benzoic-acid and its acinar locaalization in the perfused-rat-liver benzoic acid glycine conjugation in the isolated perfused rat-kidney. drug metabolism and disposition the origin of urinary aromatic compounds excreted by ruminants. . the metabolism of quinic, cyclohexanecarboxylic and non-phenolic aromatic acids to benzoic acid the preparation and characterization of poly(lactide-co-glycolide) microparticles. . the entrapment of a model protein using a (water-in-oil)-in-water emulsion solvent evaporation technique peptidomic analysis of human blood specimens: comparison between plasma specimens and serum by differential peptide display global metabolic profiling procedures for urine using uplc-ms we acknowledge the staff at afbi animal services unit vsd (stormont) for their help in the animal study and the staff at the advanced asset centre igfs (qub) for their excellent technical assistance. key: cord- -o j xq s authors: tan, shanfeng; wang, kai; sun, fuguang; li, yang; gao, yisheng title: cxcl promotes prostate cancer progression through inhibition of cytokines from t cells date: - - journal: mol med rep doi: . /mmr. . sha: doc_id: cord_uid: o j xq s chemokines have been demonstrated to serve an important role in a variety of diseases, particularly in tumor progression. there have been numerous studies that have reported that t cells serve major roles in tumor progression. however, the function of cxc motif chemokine ligand (cxcl ) in prostate cancer remains unknown. the present study aimed to investigate the role of cxcl in prostate cancer. a prostate cancer mouse model was generated by treating c /bl- and b .cg-selplgtm fur/j mice with , ′-dimethyl -aminobiphenyl (dmab). hematoxylin and eosin staining detected the histopathological alterations of mouse prostate tissues. immunohistochemistry (ihc) staining determined cell proliferation of the mice. flow cytometry was used to detect the alterations of t cells in c +dmab or cxcl +dmab mice. immunofluorescence revealed that there was positive expression of interleukin- (il- ) and transforming growth factor (tgf)-β in the mouse tissues. the survival rates of c +dmab and cxcl +dmab mice was analyzed. the association of cxcl expression and clinical stages was also evaluated. results revealed that prostate cancer pathology and cell proliferation in cxcl +dmab mice were significantly greater compared with the c +dmab mice. compared with c +dmab mice, the number of t cells in peripheral blood and spleen of cxcl +dmab mice was significantly reduced. ihc demonstrated that the expression of il- and tgf-β was significantly downregulated in the cxcl +dmab mice. the survival rate of cxcl +dmab mice was significantly decreased compared with the c +dmab mice. in addition, reverse transcription-quantitative polymerase chain reaction analysis demonstrated that cxcl mrna expression in clinical samples was positively associated with clinical pathological stages of prostate cancer. in conclusion, cxcl may promote prostate cancer progression via inhibition of cytokines from t cells. prostate cancer is a malignant tumor that occurs in prostate tissue, which is the result of abnormal dysplasia of prostate acinar cells. in regions such as europe and the united states, prostate cancer is the most common male malignancy; the mortality rate has exceeded lung cancer. the incidence of prostate cancer has gradually increased with the arrival of china's aging population and the extension of life and diet changes ( ) . it is also known that prostate cancer-associated fatalities frequently occur in patients with metastatic castration-resistant prostate cancer. although several novel drugs for castration-resistant prostate cancer have been approved, each of these has prolonged survival by just a few months. therefore, novel treatments of prostate cancer are required to extend life expectancy even further. currently, immunotherapy has been reported to be an effective treatment for cancer patients ( ) . several strate- ( ) . several strate- ( ) . several strategies, including cancer vaccines and immune checkpoint inhibitors, have been investigated in clinical studies for cancer patients ( ) ( ) ( ) . however, t cell immunotherapy of prostate cancer is still at an early stage of clinical development. it has been reported that several molecules are potent t cell checkpoint inhibitors which reverse immunologic tolerance in many types of cancer, including prostate cancer ( ) . therefore, the molecules which serve crucial roles in t cell activity during prostate cancer may be useful for the treatment of this disease. cxc motif chemokine ligand (cxcl ) is a chemo- (cxcl ) is a chemo- (cxcl ) is a chemokine, which regulates the host's response to inflammation by recruiting leukocytes to the inflammatory environment. chemokines serve important roles in immune responses of the body. chemokines are expressed by almost all monocytes, neutrophils, eosinophils and natural killer (nk) cells but are expressed to a lesser degree by macrophages, b cells and t cells ( ) ( ) ( ) . it has been reported that the absence of cxcl affects leukocyte migration and tissue infiltration ( ) . as cxcl has the ability to interact with a variety of ligands, it serves a certain biological role in the inflammatory response. in addition, inflammation is closely associated with the occurrence and development of tumors ( , ) . the present study aimed to investigate the effect of cxcl on t cells in prostate cancer. clinical data. prostate cancer tissue specimens (n= ) were obtained between july and december from linyi people's hospital (linyi, china), from patients aged between and years who were diagnosed with prostate cancer without any therapy. these tissue samples were collected with the consent of the patients. the study was approved by the medical ethics committee of the linyi people's hospital. the clinical stages of the patients were clarified according to the tumor nodes metastasis staging system. adenoma (n= ), stage /i (n= ), ii (n= ), iii (n= ) and iv (n= ). mice, cell and prostate cancer models. c bl/ black mice ( weeks old; male n= ; female n= ; weight ~ - g), were purchased from sun yat-sen university experimental animal center (guangzhou, china) and were used as control mice. a total of four b .cg-selplgtm fur/j mice, ( weeks old; male n= ; female n= ; weight ~ - g), which highly express cxcl , were purchased from jackson laboratory (ben harbor, me, usa). in total, mice were used in this study. they were maintained under specific pathogen-free conditions with -h light/ -h dark cycles at - ˚c and - % humidity and a normal diet (including % fat and . % cholesterol) and water ad libitum. dmab ( , '-dimethyl -aminobiphenyl), was used as a chemical carcinogenic agent. the mice were enrolled and initiation with subcutaneous injection of dmab ( mg/kg b.w.) once a week for weeks to construct the prostate cancer model. the mice were anesthetized by % chloral hydrate solution ( ml/kg, sigma-aldrich; merck kgaa, darmstadt, germany), and then the blood samples were collected. then the mice were sacrificed by cervical dislocation, and the prostate tissues were collected and stored. animal experiments were approved by the medical ethics committee of linyi people's hospital. hematoxylin-eosin (h&e) staining. the prostate tissues of each mouse were fixed using % formalin at room temperature for h. subsequently, the tissues were cut into paraffin sections ( µm) and stained with hematoxylin for mins and eosin for sec at room temperature, mounted in a % glycerol solution in distilled water to prevent autofluorescence (mikrochem, bratislava, slovak republic) and evaluated with an axiophot microscope (zeiss, oberkochen, germany; magnification, x ) equipped with a cellscan system (scanalytics; bd biosciences, franklin lakes, nj, us). immunohistochemistry. immunohistochemistry was performed according to a conventional protocol ( ) . the paraffin-embedded prostate tissue sections were incubated in a dry oven at ˚c for h. de-paraffinization and rehydration were then performed. the sections were incubated with - µl anti-il- (sab ), anti-tgf-β (sab ) and anti-pcna (wh m ) antibodies (dilution : ; sigma-aldrich; merck kgaa) overnight at ˚c. the sections were washed with pbs buffer three times, each time for min. a total of - µl hrp conjugated il- (a ), tgf-β (a ) and pcna (af ) secondary antibodies ( : , ; beyotime, shanghai china) was added to the tissue section and incubated at ˚c for h. the sections were washed with pbs buffer three times, each time for min. excess liquid was dried around the tissue and then placed flat into the moist chamber. a working solution of , '-diaminobenzidene ( - µl; sigma-aldrich; merck kgaa) was added to the sections and incubated at room temperature for - min. after the color was developed, the sections were rinsed with distilled water to stop the reaction. a positive results was defined as the presence of staining in % or more of cells. the stained tissue sections were reviewed and scored separately by two pathologists blinded to the clinical parameters. flow cytometry. blood samples were collected from different mouse organs, such as, orbital cavity ( ml), caudal vein ( - µl), spleen ( - µl) and bone marrow ( - µl). then red blood cell lysis buffer (beyotime institute of biotechnology, shanghai, china) was added to the blood samples, and centrifuged at x g for min at ˚c. the supernatant was discarded and the bottom part of the precipitation was washed with . % bovine serum albumin (bsa; beyotime institute of biotechnology) times at room temperature, each time for min. anti-cd -fitc antibody (f ; : ; sigma-aldrich; merck kgaa) was added in the dark and incubated for min and centrifuged at x g for min at ˚c. the supernatant was discarded and the precipitation was washed times with . % bsa, the supernatant was removed and the precipitation was re-suspended in µl . % bsa. the protein was detected using facscanto™ ii (becton dickinson, franklin lakes, nj, usa). immunofluorescent staining. the prostate tissue was fixed with % formaldehyde for min at room temperature, then washed times with pbs before permeabilization with . % triton x- (pbs) for min, also at room temperature. the paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated in a graded series of ethanol solutions and then incubated for min in % h o to quench the endogenous peroxidase activity. next, the sections were heated in target retrieval solution (dako; agilent technologies, inc., santa clara, ca, usa) for min in a microwave oven (oriental rotor, tokyo, japan) to retrieve the antigen. subsequently, the sections were blocked with % bsa at ˚c for min (d ; beyotime institute of biotechnology) and incubated with cd + (af ) and cd + (af ) primary antibodies ( : ; beyotime institute of biotechnology) overnight at ˚c, and stained with a fluorescein-conjugated secondary antibody anti-cd +-fitc antibody (f ) and anti-cd +-fitc antibody (f ; : ; sigma-aldrich; merck kgaa) for h at room temperature. finally, images were captured with leica sp aobs confocal microscope (leica microsystems gmbh, wetzlar, germany), and the number of positive cells and field area ratio were calculated with image j software version . (national institutes of health, bethesda, md, usa) ( ) . western blotting. tissues were lysed in laemmli sample buffer (bio-rad laboratories, inc., hercules, ca, usa) with a protease inhibitor, complete edta-free (roche diagnostics gmbh, mannheim, germany). the protein lysate sample was extracted from tumor tissue of mice, and then centrifuged at , x g for - min at ˚c. the supernatant was collected, and the protein concentration was measured using bca protein assay kits (pierce; thermo fisher scientific, inc., waltham, ma, usa). the protein sample were electrophoresed on % polyacrylamide gels (bio-rad laboratories) and transferred to immobilon-psq polyvinylidene difluoride membranes (emd millipore, billerica, ma, usa). then the membrane was blocked in % bsa, at ˚c for min with tbs containing % skimmed milk and . % tween- , and incubated with the anti-il- (sab ) and anti-tgf-β (sab ) antibodies (sigma-aldrich; merck kgaa) at ˚c overnight. finally, the membrane was incubated with hrp conjugated il- (a ) and tgf-β (a ) secondary antibodies ( : , ; beyotime institute of biotechnology) at room temperature for - min. the protein was visualized using an enhanced chemiluminescence reagent (bio-rad laboratories, inc., hercules, ca, usa). the proteins were semi-quantified using image j software version . (national institutes of health). reverse transcription-quantitative polymerase chain reaction (rt-qpcr). total rna was extracted from prostate tissues of the patients by using trizol reagent (invitrogen; thermo fisher scientific, inc.). reverse transcription was performed with random primers using the first strand cdna synthesis kit (takara biotechnology co., ltd., dalian, china). rt-qpcr was performed with sybr-green pcr master mix (thermo fisher scientific, inc.). pcr cycles were run on the icycler iq multi-color detection system (bio-rad laboratories, inc.) and the cycling conditions were ˚c for sec, cycle; ˚c for sec and ˚c for sec, cycles. the primers used for amplification were: cxcl f, '-agg gtc ggc tgt tcc tgc atc- ' and r, '-ttc aca tct gct gaa tct ggg ttt a- '; gapdh f, '-gca ccg tca agg ctg aga ac- ' and r, '-tgg tga aga cgc cag tgg a- '. relative expression level was calculated using the -∆∆cq method ( ) . gapdh was employed as an endogenous control. the data analysis was performed based on the sample threshold cycle (ct) value from three independent experiments. statistical analysis. all data were analyzed using spss version . statistical software (ibm corp., armonk, ny, usa). the results of immunohistochemistry were analyzed by image pro plus software version . (media cybernetics, inc., rockville, md, usa). p< . was considered to indicate a statistically significant difference. one-way analysis of variance was conducted followed by bonferroni method as a post hoc test for multiple comparisons. kaplan-meier survival plots were generated and comparisons were constructed with log-rank statistics. p< . was considered to indicate a statistically significant difference. high expression of the cxcl gene promotes the pathogenesis of prostate cancer mice. in order to determine whether cxcl overexpression affects the pathogenesis of prostate cancer in mice, prostate cancer was induced in c bl/ (c ) and b .cg-selplgtm fur/j (cxcl -overexpressed) mice. h&e staining (fig. a) revealed that the prostate cancer pathology of cxcl -overexpressed mice was significantly greater than c mice and cxcl -overexpressed mice had developed differentiated adenocarcinoma at weeks. the nuclear staining was significantly greater, the ducts were irregular and glandular cavity became smaller (black arrows). at weeks, cxcl -overexpressed mice exhibited a clear pathology of prostate cancer with a neuroendocrine differentiation phenotype (fig. a) . therefore, the mice at weeks were selected for subsequent experiments. immunohistochemical staining (magnification, x ) of proliferating cell nuclear antigen (pcna) revealed that proliferation levels in cxcl +dmab mice were increased compared with c +dmab mice (fig. b) . the aforementioned results suggested that cxcl overexpression serves an important role in promoting progression of prostate cancer and accelerates the pathogenesis of prostate cancer. high expression of cxcl inhibits t cell activation. as chemokines serve an important role in the tumor microenvironment, it was hypothesized that cxcl regulates t cell-mediated immunity. the present study examined the alterations of t cells in peripheral blood and spleen in c +dmab mice and cxcl +dmab mice. the results showed a reduction in the number of t cells in peripheral blood and spleen of cxcl +dmab mice compared with c +dmab mice ( fig. a) (q quadrant) . the infiltration number of t cells in tumor tissues was also examined in cxcl +dmab mice and c +dmab mice. the results suggested that high expression of cxcl significantly inhibited t cell infiltration into the tumor microenvironment (fig. b) . based on the above findings, it was hypothesized that cxcl may regulate the in order to verify whether cxcl overexpression affects the cytokines secreted in the tumor microenvironment, the expression levels of il- and tgf-β in the prostate tissues were examined. the results demonstrated that high expression of cxcl downregulated the levels of il- and tgf-β in tumor tissues compared with c +dmab mice (fig. a and b) . therefore, it was concluded that cxcl reduced the secretion of il- and tgf-β via the reduction of the number of t cells in immune organs and the tumor microenvironment, and promoted the development of prostate cancer. high expression of cxcl accelerates the pathogenesis of prostate cancer. to address the association between cxcl overexpression and progression of prostate cancer, the survival rates of c +dmab mice and cxcl +dmab mice were measured. the results suggested that the survival rate of cxcl +dmab mice was lower than c +dmab mice (fig. a ). in addition, the mrna expression levels of cxcl in clinical samples were measured. the mrna levels of cxcl in patients at stage iii was greater than at stage ii (p= . ), and the mrna levels of cxcl in patients at stage iv was greater than at stage iii (p= . ). the results suggested that the mrna expression levels of cxcl were positively associated with clinical staging (fig. b ). prostate cancer is a highly malignant tumor with complex pathogenesis. abnormal expression levels of chemokines have been identified in prostate cancer ( ). many chemokines may have inhibitory effects on the tumor, and certain chemokines may promote tumor progression ( ) . several chemokines, including cxcl and cxcl , have been used in translational medicine ( ) . in a previous study, an association between cxcl and cancer was demonstrated; however, the effect of cxcl in prostate cancer has not been reported. a previous studies revealed that cxcl is highly expressed in human digestive organs, such as gastrointestinal organs, however the expression of cxcl in developmental organs has not been studied ( ) . in the present study, cxcl -overexpressed mice and c mice were selected for investigation. chemical induction was used to induce prostate cancer in the mice, and it was demonstrated that cxcl may serve an important role in promoting tumorigenesis. in addition, the effect of inhibition or knockdown of the cxcl gene in tumorigenesis is important to fully illustrate the effect of cxcl . however, a limitation of the present study was that these experiments were not performed due to the difficulty of gene knock-out mouse construction; however, this will be investigated in further studies. the underlying mechanism of cxcl in promoting the pathogenesis of prostate cancer was investigated in the present study. chemokines serve an important role in the tumor microenvironment, and it was hypothesized that cxcl serves an indirect role in affecting white blood cells. the results suggested that the t cells in the spleen were signifi -cells in the spleen were signifi -cells in the spleen were significantly reduced in cxcl +dmab mice. a previous study reported that cxcl regulates the homing of lymphocyte thymus and affects t cell maturation ( ) . in the tumor mouse model, the function of cxcl in regulating t cell maturation and reducing the number of t cells requires further investiga-cells requires further investiga-cells requires further investigation. il- is produced by activated t cells and has anti-tumor effects. the anti-tumor biological activity of il- primarily includes: (i) promoting the proliferation and killing activity of nk, and promoting nk cells to secrete interferon and express the il- receptor agonist chain; (ii) inducing lympho-inducing lympho-inducing lymphocyte-activated killer cells, which may dissolve a variety of tumor cells ( , ) . the present study demonstrated a reduc- ( , ) . the present study demonstrated a reduc- ( , ) . the present study demonstrated a reduction in the number of t cells in peripheral blood and spleen in cxcl +dmab mice compared with c +dmab mice. tgf-β is a cytokine secreted by immune cells and has the ability to inhibit tumor cell growth and induce tumor degeneration ( , ) . studies have revealed that the anti-tumor effect of tgf-β depends on its secretion by t helper cd-positive cells to inhibit tumor growth and angiogenesis in receptor-interacting serine/threonine protein kinase (rip)-tag model mice ( ) . a mouse model where the rip promoter may be controlled has been obtained from the hybridization of rip-tag model mice and lmcv mice. it has been demonstrated that cd -positive t cells serve an important role in inhibiting tumor growth ( ) . the present study concluded that t cells and expression levels of il- and tgf-β were reduced by increased levels of cxcl , resulting in enhanced growth of tumors in hybrid mice. cxcl overexpression reduced the number of t cells in immune organs and the tumor microenvironment, and reduced the secretion of il- and tgf-β , and thereby promoted the development of prostate cancer. it wa s ob s e r ve d t h at t h e su r v iva l r at e of cxcl -overexpressed mice was significantly reduced compared with c mice. therefore, the targeted inhibition of cxcl expression may be applied in a clinical setting. rt-qpcr detected the mrna expression of cxcl in prostate cancer patients at different pathological stages, and the results revealed that the mrna expression levels of cxcl were positively associated with clinical staging in clinical samples, suggesting that the patients with high expression of cxcl exhibited more advanced pathological features. in conclusion, the present study suggested that expression of cxcl in prostate cancer is associated with clinical stage. cxcl may inhibit the expression of il- and tgf-β in prostate cancer model mice, however the underlying regulatory mechanism of the development of prostate cancer requires further investigation. customized viral immunotherapy for hpv-associated cancer molecular insights into the development of t cell-based immunotherapy for prostate cancer cancer immunotherapy: moving forward with peptide t cell vaccines vaccines targeting helper t cells for cancer immunotherapy enhanced cancer immunotherapy by chimeric antigen receptor-modified t cells engineered to secrete checkpoint inhibitors different host cell proteases activate the sars-coronavirus spike-protein for cell-cell and virus-cell fusion hmgb acts on microglia mac to mediate chronic neuroinflammation that drives progressive neurodegeneration mesenchymal stem cell-derived ccl- and ccl- promote mammary tumor cell invasion 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regulated by p-selectin and its ligand psgl- interleukin- : a bone marrow stromal cell paracrine signal that induces neuroendocrine differentiation and modulates autophagy in bone metastatic pca cells tgf-β induces maturation of immature human intestinal epithelial cells and inhibits inflammatory cytokine responses induced via the nf-κb pathway tgf-β induces epithelial-mesenchymal transition in cultured human lens epithelial cells through activation of the pi k/akt/mtor signaling pathway endotoxin-induced endothelial fibrosis is dependent on expression of transforming growth factors β and β pathogenesis of prostatic small cell caricinma involves the inactivation of the p pathway tnf-alpha is critical for antitumor but not antiviral t cell immunity in mice the authors declare that they have no competing interests. key: cord- - rxv fy authors: welch, david; buonanno, manuela; grilj, veljko; shuryak, igor; crickmore, connor; bigelow, alan w.; randers-pehrson, gerhard; johnson, gary w.; brenner, david j. title: far-uvc light: a new tool to control the spread of airborne-mediated microbial diseases date: - - journal: sci rep doi: . /s - - -w sha: doc_id: cord_uid: rxv fy airborne-mediated microbial diseases such as influenza and tuberculosis represent major public health challenges. a direct approach to prevent airborne transmission is inactivation of airborne pathogens, and the airborne antimicrobial potential of uvc ultraviolet light has long been established; however, its widespread use in public settings is limited because conventional uvc light sources are both carcinogenic and cataractogenic. by contrast, we have previously shown that far-uvc light ( – nm) efficiently inactivates bacteria without harm to exposed mammalian skin. this is because, due to its strong absorbance in biological materials, far-uvc light cannot penetrate even the outer (non living) layers of human skin or eye; however, because bacteria and viruses are of micrometer or smaller dimensions, far-uvc can penetrate and inactivate them. we show for the first time that far-uvc efficiently inactivates airborne aerosolized viruses, with a very low dose of mj/cm( ) of -nm light inactivating > % of aerosolized h n influenza virus. continuous very low dose-rate far-uvc light in indoor public locations is a promising, safe and inexpensive tool to reduce the spread of airborne-mediated microbial diseases. cells in a particular field of view, while green fluorescence indicated the integration of live influenza a (h n ) viruses into the cells. results from the zero-dose control studies (fig. , top left) confirmed that the aerosol irradiation chamber efficiently transmitted the aerosolized viruses through the system, after which the live virus efficiently infected the test mammalian epithelial cells. figure shows the surviving fraction, as a function of the incident -nm far-uvc dose, of exposed h n aerosolized viruses, as measured by the number of focus forming units in incubated epithelial cells relative to unexposed controls. linear regressions (see below) showed that the survival results were consistent with a classical exponential uv disinfection model with rate constant k = . cm /mj ( % confidence intervals . - . cm / mj). the overall model fit was good, with a coefficient of determination, r = . , which suggests that most of the variability in virus survival was explained by the exponential model. the rate constant of . cm /mj corresponds to an inactivation cross-section (dose required to inactivate % of the exposed viruses) of d = . mj/cm ( % confidence intervals . - . mj/cm ). we have developed an approach to uv-based sterilization using single-wavelength far-uvc light generated by filtered excilamps, which selectively inactivate microorganisms, but does not produce biological damage to exposed mammalian cells and tissues [ ] [ ] [ ] . the approach is based on biophysical principles in that far-uvc light can traverse and therefore inactivate bacteria and viruses which are typically micrometer dimensions or smaller, whereas due to its strong absorbance in biological materials, far-uvc light cannot penetrate even the outer dead-cell layers of human skin, nor the outer tear layer on the surface of the eye. here we applied this approach to test the efficacy of the -nm far-uvc light to inactivate influenza a virus (h n ) carried by aerosols in a benchtop aerosol uv irradiation chamber, which generated aerosol droplets of sizes similar to those generated by human coughing and breathing. aerosolized viruses flowing through the irradiation chamber were exposed to uvc emitting lamps placed in front of the chamber window. as shown in fig. , inactivation of influenza a virus (h n ) by -nm far-uvc light follows a typical exponential disinfection model, with an inactivation cross-section of d = . mj/cm ( % ci: . - . ). for comparison, using a similar experimental arrangement, but using a conventional nm germicidal uvc lamp, mcdevitt et al. found a d value of . mj/cm ( % ci: . - . ) for h n virus. thus as we , and others [ ] [ ] [ ] reported in earlier studies for bacterial inactivation, -nm far-uvc light and -nm broad-spectrum germicidal light are also comparable in their efficiencies for aerosolized viral inactivation. other recent work comparing viral inactivation across the uvc spectrum has shown variations in efficiency are expected, but in general both regions of the spectrum are effective in inactivation, though the precise cause of inactivation may differ , . however as discussed above, based on biophysical considerations and in contrast to the known human health safety issues associated with conventional germicidal -nm broad-spectrum uvc light, far-uvc light does not appear to be cytotoxic to exposed human cells and tissues in vitro or in vivo [ ] [ ] [ ] . if these results are confirmed in other scenarios, it follows that the use of overhead low-level far-uvc light in public locations may represent a safe and efficient methodology for limiting the transmission and spread of airborne-mediated microbial diseases such as influenza and tuberculosis. in fact the potential use of ultraviolet light for airborne disinfection is by no means new, and was first demonstrated more than years ago , . as applied more recently, airborne ultraviolet germicidal irradiation (uvgi) utilizes conventional germicidal uvc light in the upper part of the room, with louvers to prevent direct exposure of potentially occupied room areas . this results in blocking more than % of the uv radiation exiting the uvgi fixture, with substantial decrease in effectiveness . by contrast, use of low-level far-uvc fixtures, which are potentially safe for human exposure, could provide the desired antimicrobial benefits without the accompanying human health concerns of conventional germicidal lamp uvgi. a key advantage of the uvc based approach, which is in clear contrast to vaccination approaches, is that uvc light is likely to be effective against all airborne microbes. for example, while there will almost certainly be variations in uvc inactivation efficiency as different influenza strains appear, they are unlikely to be large , . likewise, as multi-drug-resistant variants of bacteria emerge, their uvc inactivation efficiencies are also unlikely to change greatly . in conclusion, we have shown for the first time that very low doses of far-uvc light efficiently inactivate airborne viruses carried by aerosols. for example, a very low dose of mj/cm of -nm light inactivates > % of airborne h n virus. our results indicate that far-uvc light is a powerful and inexpensive approach for prevention and reduction of airborne viral infections without the human health hazards inherent with conventional germicidal uvc lamps. if these results are confirmed in other scenarios, it follows that the use of overhead very low level far-uvc light in public locations may represent a safe and efficient methodology for limiting the transmission and spread of airborne-mediated microbial diseases. public locations such as hospitals, doctors' offices, schools, airports and airplanes might be considered here. this approach may help limit seasonal influenza epidemics, transmission of tuberculosis, as well as major pandemics. far-uvc lamps. we used a bank of three excimer lamps containing a kr-cl gas mixture that predominantly emits at nm , . the exit window of each lamp was covered with a custom bandpass filter designed to remove all but the dominant emission wavelength as previously described . each bandpass filter (omega optical, brattleboro, vt) had a center wavelength of nm and a full width at half maximum (fwhm) of nm and enables > % transmission at nm. a uv spectrometer (spm- -bt , photon control, bc, canada) with a sensitivity range between nm and nm was utilized to verify the nm emission spectrum. a deuterium lamp standard with a nist-traceable spectral irradiance (newport model , irvine, ca) was used to radiometrically calibrate the uv spectrometer. an sm- ozone monitor (aeroqual, avondale, auckland, new zealand) measured the ozone generation from the lamps to be < . ppm, which is not a significant level to provide an antimicrobial effect to aerosolized viruses . far-uvc dosimetry. optical power measurements were performed using an -uv/db low-power uv enhanced silicon photodetector with an -r optical power meter (newport, irvine, ca). additional dosimetry to determine the uniformity of the uv exposure was performed using far-uvc sensitive film as described in our previous work , . this film has a high spatial resolution with the ability to resolve features to at least µm, and exhibits a nearly ideal cosine response , . measurements were taken between experiments therefore allowing placement of sensors inside the chamber. scientific reports | ( ) : | doi: . /s - - -w a range of far-uvc exposures, from . µj/cm up to . mj/cm , were used to define a response calibration curve. films were scanned as bit rgb tiff images at dpi using an epson perfection v photo flatbed scanner (epson, japan) and analyzed with radiochromic film analysis software to calculate the total exposure based on measured changes in optical density. measurements using both a silicon detector and uv sensitive films were combined to compute the total dose received by a particle traversing the exposure window. the three vertically stacked lamps produced a nearly uniform dose distribution along the vertical axis thus every particle passing horizontally through the irradiation chamber received an identical dose. the lamp width ( mm) was smaller than the width of the irradiation chamber window ( mm) so the lamp power was higher near the center of the irradiation chamber window compared to the edge. the uv sensitive film indicated a power of approximately µw/cm in the center third of the window and µw/cm for the outer thirds. the silicon detector was used to quantify the reflectivity of the aluminum sheet at approximately % of the incident power. combining this data allowed the calculation of the average total dose of . mj/cm to a particle traversing the window in seconds. additionally, the silicon detector was used to confirm the attenuation of -nm light through a single sheet of plastic film was %. the addition of one or two sheets of plastic film between the lamps and the irradiation chamber window yielded average doses of . mj/cm and . mj/cm , respectively. benchtop aerosol irradiation chamber. a one-pass, dynamic aerosol / virus irradiation chamber was constructed in a similar configuration to that used by ko et al. , lai et al. , and mcdevitt et al. , . a schematic overview of the system is shown in fig. and is pictured in fig. . aerosolized viruses were generated by adding a virus solution into a high-output extended aerosol respiratory therapy (heart) nebulizer (westmed, tucson, az) and operated using a dual-head pump (thermo fisher - - fk, waltham, ma) with an input flow rate of l/min. the aerosolized virus flowed into the irradiation chamber where it was mixed with independently controlled inputs of humidified and dried air. humidified air was produced by bubbling air through water, while dry air was provided by passing air through a desiccant air dryer (x - - , wilkerson corp, richland, mi). adjusting the ratio of humid and dry air enabled control of the relative humidity (rh) within the irradiation chamber which, along with the nebulizer settings, determined the aerosol particle size distribution. an optimal rh value of % resulted in a distribution of aerosol particle sizes similar to the natural distribution from human coughing and breathing, which has been shown to be distributed around approximately µm, with a significant tail of particles less than µm - . after combining the humidity control inputs with the aerosolized virus, input flow was directed through a series of baffles that promoted droplet drying and mixing to produce an even particle distribution and stable humidity . the rh and temperature inside the irradiation chamber were monitored using an omega rh meter (omega engineering inc., stamford, ct) immediately following the baffles. a hal technologies hal-hpc particle sizer (fontana, ca) was adjoined to the irradiation chamber to allow for sampling of particle sizes throughout operation. during uv exposure, the -nm lamps were placed cm from the irradiation chamber window. the lamps were directed at the cm × . cm chamber window which was constructed of -µm thick uv transparent plastic film (topas x , topas advanced polymers, florence, ky), and which had a transmission of ~ % at nm. the wall of the irradiation chamber opposite the transparent window was constructed with polished aluminum in order to reflect a portion of the uvc light back through the exposure region, therefore increasing the overall exposure dose by having photons pass in both directions. the depth of the irradiation chamber between the window and the aluminum panel was . cm, creating a total exposure volume of . l. flow of the aerosols continues out of the irradiation chamber to a set of three way valves that could be configured to either pass through a bypass channel (used when no sampling was required), or a biosampler (skc inc, eighty four, pa) used to collect the virus. the biosampler uses sonic flow impingement upon a liquid surface to collect aerosols when operated at an air flow of . l/min. finally, flow continued out of the system through a final hepa filter and to a vacuum pump (wp , emd millipore, billerica, ma). the vacuum pump at the end of the system powered flow through the irradiation chamber. the flow rate through the system was governed by the biosampler. given the flow rate and the total exposure volume of the irradiation chamber, . l, a single aerosol droplet passed through the exposure volume in approximately seconds. the entire irradiation chamber was set up inside a certified class ii type a biosafety cabinet (labconco, kansas city, mo). all air inputs and outputs were equipped with hepa filters (ge healthcare bio-sciences, pittsburgh, pa) to prevent unwanted contamination from entering the chamber as well as to block any of the virus from releasing into the environment. irradiation chamber performance. the custom irradiation chamber simulated the transmission of aerosolized viruses produced via human coughing and breathing. the chamber operated at a relative humidity of % which resulted in a particle size distribution of % between . µm and . µm, % between . µm and . µm, and % > . µm. a comparison to published ranges of particle size distributions is shown in table . aerosolized viruses were efficiently transmitted through the system as evidenced from the control (zero exposure) showing clear virus integration (fig. , top left) . , ( ) where k is the uv inactivation rate constant or susceptibility factor (cm /mj). the regression was performed with the intercept term set to zero, which represents the definition of % relative survival at zero uv dose. bootstrap % confidence intervals for the parameter k were calculated using r . . software . the virus inactivation cross section, d , which is the uv dose that inactivates % of the exposed virus, was calculated as d = −ln[ − . ]/k. global, regional, and national life expectancy, all-cause mortality, and cause-specific mortality for causes of death, - : a systematic analysis for the global burden of disease study aerosol transmission is an important mode of influenza a virus spread evidence of airborne transmission of the severe acute respiratory syndrome virus control of air-borne microorganisms by ultraviolet floor irradiation ultraviolet germicidal irradiation handbook: uvgi for air and surface disinfection viability of b. coli exposed to ultra-violet radiation in air the effects of ultraviolet radiation on antibioticresistant bacteria in vitro principles of selective inactivation of viral genome. i. uv-induced inactivation of influenza virus wavelengths effective in induction of malignant melanoma ultraviolet radiation and cataract -nm uv light -a promising tool for safe low-cost reduction of surgical site infections. i: in vitro studies -nm uv light-a promising tool for safe low-cost reduction of surgical site infections. ii: in-vivo safety studies germicidal efficacy and mammalian skin safety of -nm uv light efficiency of krcl excilamp ( nm) for inactivation of bacteria in suspension comparison of the disinfection effects of vacuum-uv (vuv) and uv light on bacillus subtilis spores in aqueous suspensions at , and nm aerosol susceptibility of influenza virus to uv-c light wavelength-dependent damage to adenoviral proteins across the germicidal uv spectrum comparison of uv-induced inactivation and rna damage in ms phage across the germicidal uv spectrum the history of ultraviolet germicidal irradiation for air disinfection upper-room ultraviolet germicidal irradiation (uvgi) for air disinfection: a symposium in print spatial distribution of fluence rate from upper-room ultraviolet germicidal irradiation: experimental validation of a computer-aided design tool dielectric-barrier-discharge excilamp in mixtures of krypton and molecular chlorine krcl barrier-discharge excilamps: energy characteristics and applications a new ozone-based method for virus inactivation: preliminary study unlaminated gafchromic ebt film for ultraviolet radiation monitoring measurement of uv emission from a diffusing optical fiber using radiochromic film radiation technology for polymers angular dependence of the efficiency of the uv sensor polysulphone film on multichannel film dosimetry with channel-independent perturbations influence of relative humidity on particle size and uv sensitivity of serratia marcescens and mycobacterium bovis bcg aerosols size and uv germicidal irradiation susceptibility of serratia marcescens when aerosolized from different suspending media characterization of uvc light sensitivity of vaccinia virus characterization of expiration air jets and droplet size distributions immediately at the mouth opening the size distribution of droplets in the exhaled breath of healthy human subjects size distribution and sites of origin of droplets expelled from the human respiratory tract during expiratory activities the log transformation is special r: a language for data analysis and graphics this work was supported by the shostack foundation and also nih grant r ai - . we thank dr. rea dabelic from the department of environmental health sciences, mailman school of public health at columbia university for her expertise and training with viral cell culture. d.w., m.b. and v.g. designed and performed experiments, analyzed the data, and wrote the manuscript; i.s. analyzed the data; c.c. and a.w.b. designed the irradiation chamber; g.w.j. constructed the irradiation chamber; d.j.b and g.r.-p. supervised, contributed conceptual advice, and wrote the manuscript. all authors discussed the results and commented on the manuscript. competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - t xyayf authors: he, hangyong; wang, hao; li, xuyan; tang, xiao; sun, bing; tong, zhaohui title: successful management of refractory respiratory failure caused by avian influenza h n and secondary organizing pneumonia: a case report and literature review date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: t xyayf backgroud: organizing pneumonia (op) is a rare complication of influenza infection that has substantial morbidity. we report the first case of op associated with avian influenza h n infection that had significant improvement with corticosteroid treatment. case presentation: a -year-old male admitted to intensive care unit because of respiratory failure. he was diagnosed as severe pneumonia caused by avian influenza h n viral infection. after initial clinical improvement supported by extracorporeal membrane oxygenation (ecmo), the patient’s condition worsened with persistent fever, refractory hypoxemia. chest x-rays and computed tomographies showed areas of consolidation and ground glass opacification. although op was suspected and mg/kg methylprednisolone was used, the patient’s condition didn’t improved considerably. an open lung biopsy was performed, and histopathological examination of the specimen was compatible with op. the patient was treated with methylprednisolone . mg/kg for days. ecmo was weaned on day , and he was discharged on day with good lung recovery. conclusions: to the best of our knowledge, this was the first case of successful management of refractory severe respiratory failure caused by avian influenza h n infection complicated with op. refractory hypoxia with clinical manifestation and radiological findings compatible with op, a differential diagnosis should be considered among patients at the second or third week of influenza h n infection, especially in patients with clinical condition deteriorated after the primary influenza pneumonia was controlled. and a steroid dose of methylprednisolone . mg/kg may be suggested for treatment of op associated with avian influenza h n infection. human infected with avian influenza a h n virus were first confirmed on march th, in china [ , ] , with high incidence of severe respiratory failure, high intensive care unit (icu) admission and mortality. the development of organizing pneumonia (op) has been reported in patients with influenza a, h n and influenza b, and a high incidence of more than % of op in influenza a h n was reported in one case series [ ] [ ] [ ] . in this report, we describe the first case of op associated with avian influenza h n infection. a year-old male, non-smoker, with a history of poultry contact days before, was admitted to emergency room with fever and cough for days (considered as day for the case timeline). physical examination showed bilateral moist crackles. laboratory tests showed white blood cell count (wbc) was . × /l (and it became to . × /l two days later), c reactive protein (crp) was . mg/l, and procalcitonin (pct) was< . ng/ml. chest x-ray and chest computed tomography (ct) showed bilateral ground-glass opacities (ggo) and consolidation (fig. ) . moxifloxacin mg daily was administered for two days. and his condition deteriorated with dyspnea and severe respiratory failure, and the blood gas analysis showed pao was mmhg under oxygen mask with a fio of . . he was transferred to our intensive care unit (icu) supported with noninvasive ventilation (niv) and intubated h later. mechanical ventilation with peak inspiratory pressure (pip) of cm h o, positive end expiratory pressure (peep) of cm h o and fio of . could not maintain the oxygenation. as his pao /fio ratio less than mmhg lasted for h, the venovenous-extracorporeal membrane oxygenation (vv-ecmo) was established. the microscopic examination, culture and galactomannan detection from serum and bronchial-alveolar lavage fluid (balf) for virus pcr, fungal, and the culture for bacteria and the microscopic examination for bacteria and tuberculosis were done at icu admission. the nucleic acid polymerase chain reaction (pcr) for influenza h n virus of sputum specimen turned out to be positive and oseltamivir phosphate was initiated ( mg twice daily for weeks after the pcr of the virus were negative for two consecutive tests.). as the pct rose to . ng/ml, and the galactomannan detection was positive ( . from serum sample and . from balf sample), vancomycin, imipenem cilastatin and caspofungin were applied. hydrocortisone mg daily for days was also used for septic shock. ecmo was weaning off on day (the time of ecmo supporting was days) when his blood flow of ecmo was decreased to less than l/min with a significant improvement on his chest x-ray. the patient turned to high fever in the following days. repeated pcr for h n virus were tested and show continuous negative in the lower respiratory samples after a week of icu admission. advanced antibiotics and antifungal agents were administered, no positive pathogenic result was emerged, and pct level remained downtrend. the chest ct on day shows bilateral ggo with aggravating consolidation on new areas (fig. ) , compatible with organizing pneumonia (op). considering no underlying cause of op existed other than virus infection, therefore, op associated with h n influenza virus infection was suspected. methylprednisolone mg ( mg/kg) daily was applied on day for days with tapering. with clinical improvement, the patient was extubated on day , and supported with niv with a fio of . . the chest ct on day showed obvious remission of consolidation with patchy ggo and fibrotic changes. however, the clinical condition of the patient deteriorated again on day with high fever to °c, refractory hypoxemia (pao :fio = ) and a mild leukopenia (wbc was . × /l). the patient was reintubated and supported with invasive mechanical ventilation. methylprednisolone mg daily was applied at the beginning as a suspicion of the relapse of op. and chest ct on day revealed progression of consolidation especially in the lower lobe. as the patient's respiratory failure and condition did not improve after days of daily use of methylprednisolone mg, histological examination was done via open lung biopsy (olb) on day , and op was confirmed with the presence of intraluminal plugs of granulation tissue within alveolar ducts and surrounding alveoli associated with chronic inflammation of the surrounding lung parenchyma. the therapy of steroid was changed to methylprednisolone mg ( . mg/kg) for days, mg for days, mg for days. the oxygenation improved, and the patient was extubated on day and discharged on day . a time line of the steroids use, white cell count and ratio of pao /fio is illustrated in fig. . the following-up for months from onset of primary virus infection showed gradually improvement, with mild interlobular septal thickening, traction bronchiectasis and consolidation in chest ct on the ninth month ( fig. ) . as shown in tables , previously published cases and the current case of op associated with influenza virus infection were reviewed [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . with available information, the age range was -year-old to year-old with % ( / ) female. influenza a constituted the majority ( %, / ), with cases were identified to h n , and the current case, h n . the other one case was op complicated with co-infection of influenza b and streptococcus. respiratory failure associated with op was reported in % ( / ) patient. fever ( %, / ), dyspnea ( %, / ), cough ( %, / ) were the most common symptoms reported. especially, clinical condition deteriorated after controlling of primary disease can be found in % ( / ) patients. ggo and consolidation were the main findings on high resolution computed tomography (hrct), shown in %( / ) and %( / ) of cases, with release of primary opacity associated with influenza infection in some cases. autopsy were applied at about two weeks and most transbronchial lung biopsy (tblb) or open lung biopsy (olb) were applied over three weeks. steroid was the main treatment, varying from prednisolone mg/day to methylprednisolone mg/day pulse therapy, with or without tapering. most patients react well to the treatment with clinical and radiological improvement, excepting the relapsing of op in our case. since the cases infected with avian influenza h n virus were first confirmed in in china [ , ] , five seasonal epidemics were observed, with an earlier start and a steep increase in infected number in the latest epidemic [ ] . in a clinical study including cases confirmed h n virus infection, . % were admitted to an icu and % died. the median time from the exposure to disease onset was five days, then, . days to acute respiratory distress syndrome (ards). in dead cases, the median time from disease onset to death was days [ ] . half of the patients required mechanical ventilation and % need ecmo for severe respiratory failure [ , ] . however, refractory severe respiratory failure caused by op secondary to avian influenza h n virus infection was first reported in this case. op was originally described by davison et al. in [ ] ( ), and in , epler et al. described the same entity as the term "bronchiolitis obliterans organizing pneumonia" (boop) [ ] . now, for avoiding confusion with bronchiolitis obliterans (bo), the term organizing pneumonia (op) is preferred [ ] . in fact, op is an nonspecific inflammatory response from human body towards acute lung injuries. it can be with idiopathic considering symptomatology, physical signs, laboratory and pulmonary function tests, radiologic or histomorphological findings, there is no obvious difference between cop and secondary op, excepting the latter may have higher mortality [ ] . op has been linked to multiple influenza viral infections including influenza a and b, but this is the first report of avian influenza h n -associated op with relapse occurrence and severe respiratory failure. the main clinical features of our avian influenza h n -associated op is similar to the cases of influenza a cases. in these cases reported, op onset mostly at the second to third week in the course of influenza, and occurred after the releasing of primary virus infection; and the op is complicated with respiratory failure, and no evidence of other pathogen was found; and the main findings on hrct for this kind of op were ggo and consolidation. biopsy were done via tblb or olb at the third week. in cases with influenza associated op, other than common findings of op, diffuse alveolar damage (dad), alveolar hemorrhage and edema and bronchiolitis can also be found, showing the lung injury of primary virus infection. however, in our case, the patient present with high fever as a main manifestation of op, and had a relapse of respiratory failure associated with op. a differential diagnosis of op should be considered at the second to third week after the primary infection among patients with influenza h n infection. furthermore, there was no clear evidence of bacterial and fungus infection during the beginning of hospitalization. however, the levels of pct and galactomannan showed significant increase after the patient was established with ecmo. the op may also be caused by the nosocomial infection which was frequently complicated with severe influenza pneumonia. the majority of patients with op show rapid responding to steroids. the introduced initial dose is prednisone . - . mg/kg, with tapering over - month. however, up to one-third patients may relapse in tapering period. for the current patient with avian influenza h n associated op, methylprednisolone mg ( mg/kg) daily was applied for days with tapering in the beginning of suspicion of op. the patient showed rapid clinical and radiological improvement and was extubated on the fifth day of steroids applying. however, the patient deteriorated with high fever and refractory hypoxemia days later. with the confirmation of histological examination, steroid dose was increased to methylprednisolone mg ( . mg/kg) daily for days. and op was finally controlled without relapsing in follow-ups. insufficient initial dose of steroid may contributed to the relapsing of op before final diagnosis. therefore, post avian influenza h n infection op may responsive to a brief course (weeks) of moderate-to-high dose prednisone therapy. to the best of our knowledge, this was the first case of op associated with avian influenza h n infection. with clinical manifestation and radiological findings compatible with op, a differential diagnosis should be considered among patients with influenza h n infection at the second or third week after the initial viral infection, especially in patients with clinical condition deteriorated after controlling of primary influenza pneumonia. and a steroid dose of methylprednisolone . mg/kg maybe suggested for treatment of op associated with avian influenza h n infection. our case provide clinical insight into refractory respiratory failure with lung involvement due to avian influenza h n . op should be considered on the differential diagnosis of patients with fever and respiratory failure after severe influenza a h n infection, where steroids might be useful. human infection with a novel avian-origin influenza a (h n ) virus human infections with the emerging avian influenza a h n virus from wet market poultry: clinical analysis and characterisation of viral genome organizing pneumonia in patients with severe respiratory failure due to novel a (h n ) influenza influenza a-associated bronchiolitis obliterans organizing pneumonia mimicking wegener's granulomatosis influenza b/streptococcal coinfection complicated by organizing pneumonia chest ct findings of influenza virus-associated pneumonia in adult patients. influenza other respir viruses clinicopathological findings of four cases of pure influenza virus a pneumonia influenza a (h n ) virus-associated pneumonia: high-resolution computed tomography-pathologic correlation influenza a (h n ) organising pneumonia organizing pneumonia associated with swine-origin influenza a h n viral infection sudden increase in human infection with avian influenza a(h n ) virus in china clinical findings in cases of influenza a (h n ) virus infection cryptogenic organizing pneumonitis bronchiolitis obliterans organizing pneumonia european respiratory society international multidisciplinary consensus classification of the idiopathic interstitial pneumonias organizing pneumonia: a kaleidoscope of concepts and morphologies publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations no acknowledgement. all authors made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; reviewed and approved the final manuscript; and contributed significantly to this study. hh takes full responsibility for the integrity of the submission and publication, and was involved in study design. hw and hh had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis, and was responsible for data verification, analysis and drafting of the manuscript. bs, zt had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. xt and xl were responsible for the data collection. all authors read and approve the final manuscript. not applicable.availability of data and materials not applicable. we did not commence any experimental use of a novel procedure or tool in this case, and all therapy was approved by an ethics committee of our hospital. written informed consent was obtained from the patient for publication of this case report and any accompanying images. a copy of the written consent is available for review by the editor of this journal. the authors declare that they have no competing interests. key: cord- -uvfppirt authors: gornati, laura; zanoni, ivan; granucci, francesca title: dendritic cells in the cross hair for the generation of tailored vaccines date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: uvfppirt vaccines represent the discovery of utmost importance for global health, due to both prophylactic action to prevent infections and therapeutic intervention in neoplastic diseases. despite this, current vaccination strategies need to be refined to successfully generate robust protective antigen-specific memory immune responses. to address this issue, one possibility is to exploit the high efficiency of dendritic cells (dcs) as antigen-presenting cells for t cell priming. dcs functional plasticity allows shaping the outcome of immune responses to achieve the required type of immunity. therefore, the choice of adjuvants to guide and sustain dcs maturation, the design of multifaceted vehicles, and the choice of surface molecules to specifically target dcs represent the key issues currently explored in both preclinical and clinical settings. here, we review advances in dcs-based vaccination approaches, which exploit direct in vivo dcs targeting and activation options. we also discuss the recent findings for efficient antitumor dcs-based vaccinations and combination strategies to reduce the immune tolerance promoted by the tumor microenvironment. introduction vaccines represent one of the most effective copernican revolutions for humankind and world health. this innovative discovery by edward jenner in the late years of the xviii century allowed for control or complete eradication of infectious diseases as smallpox ( ) and rinderpest virus ( ) ( ) . this immunization strategy posed the bases for current remarkable therapeutic approaches against not only infections but also cancer. in evolutionary terms, pathogens have acquired the capability to circumvent the immune system with several evasion mechanisms, revised elsewhere ( ) , that prevent pathogen clearance and the establishment of immune memory. vaccines represent the unique tool we have to impede pathogen spread; therefore, the urgent need for efficient vaccines is as relevant as before. mycobacterium tuberculosis, which causes tuberculosis, is currently one of the most feared infectious agent due to its capability to evade the immune system, leading to death of more than one million of people per year. unbelievably, the only licensed vaccine against mycobacterium tuberculosis is bacillus calmette-guérin (bcg) conceived about years ago. nonetheless, bcg has displayed some degree of inefficacy in humans, thus raising the need for new tailored vaccination strategies that are currently under investigation ( ) . moreover, every year, new cases of human immunodeficiency virus (hiv) infections lead to the necessity of a vaccine to control and prevent the spread of the virus. up to now, vaccines against hiv have not passed phase ii clinical trials due to poor protection conferred, requiring revision of delivered antigens (ags) and strategy to improve t cell response ( ) . moreover, the recent outbreaks of ebola virus and zika virus infections clearly demonstrate that still nowadays more than few infectious diseases need to be overwhelmed, as reported by the world health organization. on the other hand, vaccines represent also a therapeutic tool against cancer. one of the hallmarks of cancer is the capability of tumor cells to evade immune-mediated destruction ( ) by promoting a tolerant milieu. therefore, the immune system has to be pushed to respond specifically and robustly against tumors cells. to address this purpose, it is becoming more and more evident that dendritic cells (dcs) stand out as a potent tool in our hands, being the mediators of cellular and humoral responses ( ) . dcs have been discovered in by r. steinman and z. cohn that divided phagocytic cells (discovered by e. metchnikoff in ) in macrophages and dcs on the basis of different effector functions: microbial scavenging activities for macrophages and antigen-presenting function for dcs ( , ) . since then, dcs have emerged as the most potent antigen-presenting cells capable of shaping adaptive responses both during infections and cancer. moreover, the broad spectrum of dcs activation makes them suitable for fine shifting of the type of response the context needs. taking advantage of new adjuvants, innovative ags-delivery carriers and targeting strategies, it is now feasible to optimize the activation and ag presentation processes by the specific dcs subset that is the most effective in the initiation of the adaptive response needed in a given context. here, we discuss the diverse phenotypical and functional properties of dcs subtypes that are exploited by recently developed vaccine approaches, dealing with advances in the use of ags, adjuvants, carriers and dcs-expressed molecules, object of targeting. dendritic cells are the primary professional antigen-presenting cells (apcs) that reside in both lymphoid and non-lymphoid organs ( ) ( ) ( ) . dcs encompass several heterogeneous subsets whose subdivision relies on ontogeny, expression of surface-receptors, and transcription factors ( ) ( ) ( ) . much effort has been done in the identification and characterization of tissue-specific dc subsets to unravel the correlation between phenotype, localization, and functional properties, both in health and disease. initially, dcs have been classified into conventional dcs (cdcs) and plasmacytoid dcs (pdcs). briefly, cdcs prime naïve t cells and orchestrate ag-specific adaptive responses, while pdcs intervene during viral infections producing type i interferons (ifns). advanced approaches have extremely pushed our understanding of dc biology, resulting in a recent readapted taxonomy ( , , ) . indeed, villani and colleagues identify six subsets of dcs and monocytes in human (figure ): dc (clec a + cd + dcs), dc and dc (cd c + dcs), dc (fcgr a/cd + dcs), dc (axl + siglec + dcs) and dc (pdcs). dc represent the cross-presenting cd + /bdca + dcs while d and d correspond to cdcs displaying antigen uptake and processing capabilities. dc seem to be more prone to respond to viruses and are phenotypically close to monocytes. dc represent a newly defined subset that share features with both pdcs and cdcs, even though they appear to be functionally different from pdcs and more similar to cdcs. indeed, dc localize in t cell zone of tonsils, probably promoting fast adaptive immunity. due to this fine clustering, dc correspond to a more pure pdcs population ( ) . this precise classification opens the way for a more accurate view of dcs role in pathologies and provides cues for more specific targeting in immunotherapies. indeed, it is reasonable to assume that this extreme phenotypical diversity correlates with different intrinsic functional properties of dcs, as emerged in villani's work ( , , ) . in addition, environmental cues dictate dc activation and drive specific t cell responses ( , ) . indeed, dcs display a plethora of pattern recognition receptors (prr) that are specifically bound by microbe-or damage-associated molecular pattern (pamp and damp, respectively) ( ) . upon receptors engagement in peripheral tissues, the transduction signals lead to dc maturation with the upregulation of co-stimulatory molecules (referred to as "signal ") and the pivotal chemokine receptor ccr that allows dcs migration through afferent lymphatic vessels to the draining lymph node (ln) ( ) ( ) ( ) . in parallel, dcs mediate ag proteolysis to present intracellular peptides on major histocompatibility complex (mhc) class i to cd + t cells and exogenous peptides on mhc ii to cd + t cells (referred as "signal "). dcs can present exogenous ags on mhc class i through the so-called cross-presentation, allowing them to induce cd + cytotoxic t lymphocytes (ctls) against viruses and tumor cells. indeed, once in the ln, mature dcs encounter cognate naïve t cells and initiate adaptive responses ( ) . in the absence of maturation, as in steady-state conditions, the ag presentation and consequent migration to ln promote peripheral tolerance via t cell anergy or regulatory t cell formation ( ) ( ) ( ) . depending on the receptors engaged, dcs display different maturation states and produce different inflammatory mediators (often referred to as "signal ") that impact on the following cellular and humoral responses. the three signals released by dcs drive t helper (th) cell differentiation. briefly, dcs educate cd + t cells against intracellular bacteria by promoting their polarization into ifn-γ-producing th type (th ) cells. upon infection by multicellular parasites, dcs, with the help of basophils, polarize cd + t cells into th type (th ) cells that produce mainly il- . for specialized mucosal and skin immunity, dcs drive the activation of th type (th ) ( ) . thus, polarization of t cells is a crucial event that provides mechanisms specifically orchestrated to restore physiological homeostasis. dcs undergo apoptosis once they have fulfilled their functions. the rapid dc turnover after activation is necessary to avoid excessive t cell activation ( ) and to maintain self-tolerance ( , ) . t lymphocyte activation culminates with the establishment of the immunological memory, providing the host with t cells more prone and efficient in responding to a reinfection by the same pathogen or upon tumor relapses ( ) . besides, dcs are key players in humoral responses too. indeed, they directly interact with b cells and indirectly support them by activating cd + t cells, leading to humoral memory. all these notions strengthen the idea that dcs represent an optimal target for immunotherapies and vaccines, acting at the interface of innate and adaptive immunity. to harness robust responses through dc-targeting vaccinations, dc maturation is essential. adjuvants become compulsory complement of inactivated or subunit vaccines that may promote suboptimal responses. furthermore, they improve dc migration, ag availability, and specific targeting. although it seems clear that immunization could benefit from adjuvant uses, the solely adjuvant licensed in clinics, until recently, was alum ( ) . despite alum has been used in vaccination practice since the beginning of the last century, the mechanism through which it activates innate immunity for the subsequent activation of adaptive immune responses remains elusive. the adjuvant properties of alum were initially attributed to the activation of nlrp inflammasome ( , ) ; nevertheless, further studies have clearly shown the dispensability of nlrp and caspase- for the generation of responses in the presence of this adjuvant ( , ) . tlr signaling is also dispensable for alum adjuvanticity ( ) as well as mast cells, eosinophils, or macrophages ( ) . recently, it has been proposed that upon contact with alum, dcs produce il- through the activation of src and syk kinases, ca + mobilization, and nfat nuclear translocation. il- , in turn, is required for optimal t cell priming, activation, and antibody production ( ) . in addition to alum, other chemical adjuvants have been tested in preclinical models, showing a clear heterogeneity in the responses driven by different adjuvants, independently of the ag ( ) . this underlies the need of deepening our knowledge on these powerful tools to drive immune responses. indeed, mf , an oil-in-water emulsion adjuvant, that allows long-lasting ag retention in draining ln and enhanced ag uptake by ln-resident dcs, promotes robust humoral responses via follicular dc activation ( ) and cd + t cell immunity induction ( ) . conversely, ic adjuvant, which consists of an antibacterial peptide and a synthetic oligodeoxynucleotide (odn), elicits ifn-β release by human dcs via engagement of endosomal tlrs supporting immunity against intracellular pathogens and cancer ( ) . in the last decades, attention has been focused on tlr ligands as adjuvants. currently, several compounds are under investigation: pam csk , pam csk , or analogs as tlr / or tlr / ligands ( , ) , poly(i:c) and similar compounds acting on tlr ( , ) , tlr agonists ( ), flagellin acting on tlr ( ), imiquimod and other tlr ligands ( , ) , tlr agonists ( ), cpg odn binding tlr ( , ) . due to the possible reactogenicity that may be induced by administering tlr agonists, some compounds are chemically modified to reduce toxicity or are delivered specifically to the dc subsets of interest, avoiding tlr ligand dissemination. monophosphoryl lipid a, a low-toxicity molecule derived from lipopolysaccharide (lps), displays promising effects for vaccine design ( ) even though it promotes terminal differentiation of cd + cells, leading to reduced memory protection ( ) . another lps-analog is -acyl lipid a that has emerged as potent inducer of ifn-γ-mediated ag-specific responses when co-delivered with poorly immunogenic tumor ags ( ) . to improve the effectiveness and strength of immunity, in addition to the efficiency of apc, activation and ag processing and presentation of other aspects should be taken into account. the importance of dc-derived il- in the activation of adaptive responses has been shown not only in alum-driven immune responses and in mouse models of infections ( , ) but also in tests of human t cell priming in the presence of activatory dcs. during the first few hours after interaction with t cells, activatory monocyte-derived dcs (modcs stimulated with the cytokine cocktail, tnf-α, il- , il- β, and pge ) produce il- and cd ( ) . dc-derived il- is, in turn, trans-presented to t cells at the immunological synapse via cd . since naïve t cells start to express cd only many hours after ag encounter, the dc-mediated presentation of the il- /cd complex is indispensable for an efficient t cell priming ( ) . it has been proposed that this is the reason why approved therapies based on the use of anti-cd antibodies to avoid the acute phases of autoimmune diseases, or acute rejection of kidney, heart, and hand transplants, are so efficient in interfering with t cell priming or t cell reactivation ( ) . since il- is produced in nfatdependent manner to improve the adjuvanticity of prr agonists for vaccination purposes, the capacity of selected prr agonists to induce nfat signaling pathway activation and il- production should be considered. many prr ligands have been shown to activate the nfat transcription factor family members in innate immune cells ( ) . the nfat pathway is activated in neutrophils, macrophages, and dcs in response to curdlan ( , ) , it is also activated in dcs in response to lps ( ) downstream of cd , it is activated in response to mannose-capped lipoarabinomannan (man-lam), a major lipoglican of mycobacterium tuberculosis ( ) , and downstream of tlr in response to β-glucan bearing fungi ( ) . the production of il- by innate immune cells during inflammatory responses is relevant not only for an efficient t cell priming but also for the skewing of t cell activation toward type i responses. in mice, dc-derived il- is one of the cytokines required to elicit ifn-γ production from nk cells both in lpsmediated inflammatory conditions and during fungal infections ( ) ( ) ( ) . ifn-γ potently activates macrophages and favors th commitment of cd + t cells. therefore, early ifn-γ release by nk cells is not only crucial for controlling a variety of primary bacterial and fungal infections but also for the induction of type i immunity and memory, fundamental for the protection against bacterial, fungal, and viral infections and in antitumor immune therapies. another important reason for considering the capacity to activate the nfat pathway in adjuvant selection tests is represented by the fact that nfats regulate also the production of the prostanoid pge by activated dcs ( ) . pge promotes activated dc migration ( ) and sustains vasodilation and local edema formation during the inflammatory process. this is particularly relevant for vaccination purposes since the increase of the interstitial pressure generated by the edema forces the fluids into the afferent lymphatics and favors a first wave of antigen arrival to the draining ln ( ) . intriguingly, ln drainage of proteins or antigens occurs very rapidly after subcutaneous, intradermal, and intramuscular immunization ( ) ( ) ( ) , thus permitting an extremely fast uptake by phagocytes strategically localized in close proximity to the subcapsular sinus or lymphatic sinus of draining ln ( ) ( ) ( ) ( ) . antigen-presenting cells in ln then maintain the homeostasis of ln themselves and activate adaptive immune responses. in the last decades, the long-held paradigm of migratory dcs, resident in peripheral tissues as the skin, as unique apcs involved in t cell immunity has dramatically changed. indeed, cd + subcapsular sinus macrophages, medullary macrophages, and ln-resident dcs are ln sentinels that avoid excessive pathogen dissemination ( , ) and mediators of immune responses ( ) . concerning migratory dcs and considering the skin, which represents the site of utmost importance for vaccination strategies due to the ease accessibility and the extremely high presence of dcs, skin-resident dcs have been subdivided into epidermalresident langerhans cells (lcs), which are langerin + and two diverse subsets of dermal (d)dcs: langerin + cd + and langerin − cd − ( , ) . upon infection, ddcs migrate to the ln within - h while lcs within - h, supporting long-lasting ag-presentation. several works reveal the intrinsic differences between the two subsets in inducing th or ctl responses, due to the particular cross-presenting capabilities of cd + ddcs, for instance ( , , ) . once in the ln, whose strategical architecture enhances the probability of encounter between migratory dcs and cognate naïve t cell, adaptive immunity is initiated. of note, ln-resident dcs are sufficient to promote early adaptive responses independently of migratory dcs when pathogens or antigens directly access the lymphatic conduits ( , , ) . in antiviral responses, cd α + ln-resident dcs play a crucial role, thanks to their intrinsic capability of cross-presentation to cd + ctl ( , ) that may be supported by pdcs ( ) . in herpes simplex virus (hsv) skin infection, cd α + ln-resident dcs uptake cargo-antigens, ferried by skinresident migratory dcs in order to elicit ctl ( ) . indeed, lcs and ddcs synergize with cd α + ln-resident dcs, which stand out as the most potent ctl inducers, preferentially sustaining cd + th responses both in influenza ( ) and hsv cutaneous infections ( ) . in addition to cd α + ln-resident dcs, cd + ddcs display intrinsic capability of cross-presentation, as their human counterpart, clec a + cd + dcs ( ) ( ) ( ) ( ) ( ) . besides, some authors demonstrated that blocking dc migration from the skin hinders cd + t cell activation in response to subcutaneous bacterial ( ) or soluble antigen challenge ( ) . ablating langerin + ddcs reduced t cell immunity strength, corroborating the notion that migratory dc complement ln-resident dc effects on adaptive responses ( ) ( ) ( ) . nonetheless, the roles of lcs in activating t cells are still uncertain, probably due to the controversial functional properties of this innate subset ( ) ( ) ( ) . despite this, the synergic effects of ln resident and migratory dcs seem to be undoubted ( , ) . indeed, allenspach and colleagues reported that ag presentation by ln-resident dcs few hours after the infection is required to entrap ag-specific t cells in the draining ln and to favor an optimal activation of t cells by migratory dcs that arrive at the ln many hours later ( ) . it emerges, therefore, that another aspect to be considered for the identification of efficacious adjuvants concerns the type of dc subset to be targeted and the consequential effects that adjuvants imprint on that subset. adjuvants play a pivotal role in determining tissue-resident dc mobility to draining ln and efficiency of t cell polarization. indeed, ddcs acquire mobility after subcutaneous injection of th -specific adjuvants as cpg and lps, but not with th -specific ones, as papain, or following contact sensitization with dibutyl phthalate and acetone. moreover, ddcs are sufficient to promote th and th responses, while lcs are only supportive of th ( ) . this evidence underscore that, in addition to the polarizing capabilities of adjuvants, also the targeted dc subset must be considered to elicit specific adaptive immunity. indeed, antonialli and colleagues reported differential immune responses when cd α + and cd α − dcs were targeted with the same ag and adjuvant, either cpg odn or flagellin ( ) . in addition, to enhance the efficacy of vaccination, the coincident delivery of ag and prr adjuvants to apcs plays a crucial role. encouraging evidence highlights the importance of conjugation of ag with prr adjuvants, since it improves ag uptake, humoral and cellular responses when compared to vaccination with ag codelivered with free tlr ligands ( ) . these findings strengthen the notion that adjuvants are formidable chiefs in shaping immune responses and must be selected for the outcomes they promote, in chemical association to the ag of interest. the traditional vaccination approaches consisted in the adminis tration of live or attenuated micro-organisms. up to now, several innovative strategies have emerged to address the need for efficient vaccines, especially against diseases that are critical to treat, as cancer and the infectious diseases already mentioned. the main purpose is to convey ag, adjuvant, and targeting-molecule in a unique compound to increase the efficacy of the ag-specific immune response. to address this issue, different approaches have been explored or are currently under investigation, as shown in figure . recombinant antibody (rab) represents a feasible option. this approach exploits the possibility to chemically fuse peptide ag, adjuvant, and targeting-molecule to ab to tailor dcs targeting ( ) ( ) ( ) . in addition to rab, single-chain fragments variable (scfv) revealed to be an appealing strategy due to their reduced size and enhanced infiltration into tissues, as in solid tumors ( ) . other approaches involve the use of nano-carries as vehicles. the most promising solution to target phagocytes is indeed the use of particulate materials ( , ) . nanoparticles (nps) are the best candidates as delivery system, since they can be manipulated to efficiently and predominantly target phagocytes. this is possible, thanks to the versatility of nps due to: (i) the large amounts of existing different nanomaterials; (ii) the possibility to adjust their size, morphology, and deformability with great precision; (iii) the possibility to load virtually any different type of drug molecules ( ) . viral vectors-based vaccines or virus-like particles rely on the intrinsic capability of viruses to infect cells and exploit their protein-encoding machinery, allowing expression in the cytosol of the engineered plasmid-genes, as ag, costimulatory molecules, cytokines, and adjuvants, providing the bases for strong ctl induction ( ) . on the other hand, naked dna can be directly injected or conjugated to nano-carriers to favor specific targeting. the easy designing of nano-carriers-based vaccines along with their multi-component loading feature improve targeting of specific subsets ( ) and shape immune responses ( , ) , favoring their application in several fields. in a cancer setting, nano-carriers allow to avoid killing of healthy cells, by delivering tumor ags or dna encoding these peptides to apcs, inducing specific antitumor responses. indeed, nps allow endocytosis and mhc presentation on both class i and ii ( ) eliciting broad adaptive immunity, even against cancer cells. rosalia and colleagues designed a polymer-based biodegradable poly(lactic-co-glycolic acid) plga nps loaded with ag, pam csk , and poly(i:c) and coated with an agonistic αcd -monoclonal ab (np-cd ). this multi-functional strategy resulted in efficient and selective delivery of nps to dcs in vivo upon s.c. injection and induced priming of cd + t cells against tumor associated ags, increasing tumor-bearing mice survival ( ) . plga nps carrying the poorly immunogenic melanoma-derived antigen tyrosinase-related protein along with -acyl lipid a, manage in breaking the immunotolerance acting against tumor-antigens. indeed, administration of the abovementioned nps resulted in antigen-specific cd + ctl responses, characterized by ifn-γ production and increase of pro-inflammatory cytokines in the tumor microenvironment (tme) ( ) . another nano-carrier-based approach relies on liposome, self-assembled vesicles composed by lipid bilayers with high functionalizing properties. besides, maji and colleagues reported that after uptake by dcs, cationic liposomes localize in endosomal compartments that allow ag presentation preferentially on mhc i but do not exclude mhc ii ag presentation ( ), suggesting a crucial role in antitumor or antiviral immunity supported by th responses. in addition to the use of nps, targeting dc-specific receptors has become an attractive strategy for vaccine development due to the enforced efficiency of immune responses when compared to generic-delivering approaches. here, we report the more characterized dcs receptors, currently under investigation in the scenario of tailored-vaccination, as shown in table . clec a or dngr is a c-type lectin receptor that mediates endocytosis, but not phagocytosis, with low ph endosomes promoting the drift toward cross-presentation. importantly, clec a binding of antigens induces antigen presentation on both mhc i (cross-presentation) and mhc ii. it is highly and specifically expressed on cd c + cd + xcr + cdcs and cd + cd − monocytes in human and in murine pdcs and xcr + cd a + ln resident but not cd + xcr + migrating dcs ( , ) . indeed, cd + xcr + dcs constitute the human counterpart of cd α + xcr + murine dcs ( ) . they share xcr , the receptor of xcl . xcl is released by activated t cells and the axis xcr -xcl is necessary for robust ctl responses ( ) . cd + xcr + dcs are the main cross-presenting dcs in human, thus they appear promising for ctl-mediated responses, in tumors and viral infections ( ) . this specific subset is characterized by the expression of tlr that may be exploited to fully activate clec a + xcr + dcs since antibody binding of clec a leads to its rapid internalization but not tlr-pathway activation, preventing pro-inflammatory cytokine production and full maturation of dcs ( ) . conversely, caminschi and li independently demonstrated the potentiality of targeting clec a that resulted in enhanced humoral immunity independently of trif-myd or tlr pathway, even in the absence of adjuvants ( , ) . targeting clec a induces enhanced cd + t cell proliferation in vivo, which supports b cell immunity, when compared to the targeting of another endocytic receptor, discussed later, dec- , independently of the use of adjuvants as cpg ( ) . some years later, different authors demonstrated that this strong humoral response is endorsed by the establishment of follicular t helper cells memory, even upon vaccination with glycoprotein d of hsv, both in mice and non-human primates ( , , ) . these promising results were confirmed also in a human in vitro setting, on cd + dcs ( ) . finally, the efficacy of targeting clec a has been evaluated in the delivery of poorly immunogenic virus-derived antigens. park and colleagues managed in conferring specific humoral response, protective upon reinfection ( ) . thus, exploiting the specific expression of this receptor on the most specialized dcs in cross-presentation in combination with tlr ligands, will enhance antiviral and anticancer responses ( ), combined with robust humoral immunity. dec- or cd is a kda endocytic receptor that has a cysteine-rich domain, a fibronectin type ii domain, and c-type lectin-like domains, as well as an internalization sequence in its cytoplasmic tail ( ) . thus, it mediates cross-presentation through clathrin-and dynamin-dependent receptor-mediated endocytosis. indeed, it is expressed by the most professional cross-presenting dcs, the cd α + dcs subtype, while cd α − dcs display very low level of this receptor. in addition, dec- is found on dermal/interstitial dcs and lcs ( ), thus guaranteeing ag delivery to both skin-resident and ln-resident professional apcs. in humans, dec- is shared among cdcs, monocytes, and b cells, while pdcs, granulocytes, nk cells, and t lymphocytes express low levels of this receptor ( ) . in addition, dec- regulates molecule recycling through late endosomes, promoting also mhc ii presentation to cd + t cells in lcs ( ) . steinman and nussenzweig have addressed this molecule to improve vaccine efficacy since ( ) . by taking advantage of anti-dec- rab conjugated to ova peptide, they demonstrated that s.c. injections of this compound lead to a strong ifn-γ and il- mediated immunity only when dcs activation was supported by αcd mab, otherwise, tolerance against the ova peptide occurs ( ) . indeed, diversely from prr agonists, antibody crosslinking the dec- does not induce dcs maturation ( ) . furthermore, few years later, the combined strategy of anti-dec- and αcd was reported to confer protection against melanoma and intranasal influenza infection ( ) . in a viral setting, anti-dec- rab chemically coupled with hiv p gag protein tested in vitro on blood cells derived by hiv-infected donors has revealed efficient expansion of ifn-γ-producing cd + t lymphocytes ( ) from all the different donors. this indicated that dcs and cd can lead to the generation of different peptides from a single protein. moreover, vaccines based on the filamentous bacteriophage fd presenting an αdec- scfv, efficiently induce dcs maturation via the activation of the tlr -myd pathway ( ) , without adjuvants and further elicit potent antitumor responses when compared to non-tailored ag delivery ( ) . intriguingly, dec- , orphan of a specific ligand, has been proven to be necessary for cpg uptake and eventual dc activation ( ) . cd is a molecule belonging to the tnf receptor family, expressed by several cell types and among these, dcs. it has emerged as a receptor for the human chaperone heat shock protein (hsp) that mediates the internalization of peptides bound to hsp itself ( ) . moreover, upon activation, t cell transiently expresses cd l allowing cross-linking of cd on dcs and completing their maturation. from these notions, cd appeared an interesting molecule to target for dc-based vaccination strategies. indeed, by engineering antibody chemical structure, schjetne and colleagues demonstrated the efficacy of cd engagement conferring protection against myeloma-and lymphoma-derived ags ( ) . moreover, through the co-administration of two dna-based vaccines encoding either cd and the foot-and-mouth disease-derived ags, the transient increase of endogenous αcd antibodies allows an efficacious dcs activation and an efficient development of ag-specific t cell immunity, if compared to the administration of dna encoding ags alone ( ) . further promising results have been obtained in a vaccine against cyclin-d that is overexpressed by mantle cell lymphoma (mcl). thanks to algorithm analysis, chen and colleagues identified three cyclin-d -derived peptides that efficiently bind to mhc class i of dcs, potentially overexpressed in all mcl patients. by generating a rab targeting cd , they efficiently delivered these tumor associated ags to dcs and mounted ifn-γ-specific t cell responses in patients-derived peripheral blood mononuclear cells ( ) . thus, cd represents a specific dc-targeting molecule that has been used in combination with other targeting approaches to support specific dcs activation, avoid tolerance, and induce robust t cell immunity ( , ) . when evaluating vaccination strategies for cancer patients, it is compulsory to take into account one of the hallmarks of cancer: avoiding immune destruction by promoting tolerance and disarming the immune system ( ) . the orchestration of antitumor responses involves multiple protagonists and mediators, among these, cytotoxic t cells and nk cells, whose activation is supported by dcs ( ) . furthermore, dcs-based vaccines has emerged as more efficient in promoting t cell immunity if compared to peptide-based vaccination approaches ( ) . thus, much effort has been made to improve strategies of dcs-based vaccination in neoplastic diseases, to ameliorate the prognosis or eradicate both primary tumor and metastases. up to now, two different approaches have been addressed: ex vivo generation of autologous pulsed dcs and direct in vivo targeting of dcs, as previously discussed. the former strategy provides a better control of the maturation and activation state of dcs and a specific load of the ag to the selected dcs subset. despite this, intense work is needed to generate this vaccine, since it is personalized for each patient and only few subsets of dcs are feasibly generated in vitro or collected ex vivo, limiting the access of ags to other more functionally driven subsets. diversely, the in vivo targeting methods allow the generation of large amount of vaccine in a one-step procedure, and the targeting of diverse dcs subsets in their natural environment. once the dcs-based vaccine is generated, the efficacy of antitumoral responses has to be evaluated. it is mainly related to (i) the capability to establish specific antitumor-associated ag (taa) immunity and (ii) the overcome of the tolerogenic status promoted by the tme. to select highly immunogenic ags, multiple solutions have been tested: whole tumor lysate or killed tumor cells, synthetic long peptides (slps), full length proteins, transfection or electroporation with dna or mrna coding for taa, transduction with lentiviral vectors and neoantigens. the availability of an elevated number of antigens through the incubation of dcs with whole tumor lysates or autologous tumor cells allows the presentation of multiple epitopes, loaded on both mhc class i or ii, which leads to th and cytotoxic responses. indeed, several clinical trials are currently evaluating the benefits obtained by using this approach (nct ; nct ; nct ). slps are - aa long peptides cross-presented by dcs ( ), currently under investigation in both preclinical and clinical setting. compared to short synthetic peptides, the use of slps lacks the necessity to know the patients' hla haplotype, thus permitting their full exploitation in a larger cohort of people. moreover, slps administration to dcs leads to an enhanced cd + t cells activation since, once engulfed, they rapidly escape from the endolysosome to follow the path of mhc class i presentation, fundamental in antitumor responses. indeed, slps and dcs-based vaccines are showing promising results in terms of safety and immunogenicity, in both preclinical and clinical settings ( ) . they have gained attention in the context of human papilloma virus cervical ( ) , ovarian ( ) , and colorectal cancer ( , ) , displaying immunogenic capacities, in terms of antibody production and cd + and cd + t cell activation, when delivered with adjuvants, as poly iclc, montanide-isa- (nct ), and ifnα. when comparing slps and full length proteins, it has emerged that dcs process slps better that full length protein, due to the slower processing route the latter display ( ) . concerning transfection or electroporation of dcs with mrna or dna encoding, not only for taa but also for costimulatory molecules and cytokines, to enforce adaptive immunity, has proven to be efficacious in inducing antitumor cd + and cd + t cells expansion, mediated by dcs targeting ( ) . a similar approach regards in vivo lentiviral transduction of dcs, which displays versatility for gene delivery and efficient transduction for non-dividing cells, as dcs. indeed, bryson et al. conceived a multifunctional vaccine composed by a modified lentivirus, whose glycoproteins can directly target dc-sign on dcs, loaded with breast cancer ags, alpha lactalbumin, and erb-b receptor tyrosine kinase . single injections of the compound provided tumor self-ags-specific cd + t cell immunity, reducing tumor growth ( ) . despite the improvements derived by these advanced strategies, in the last years, neoantigens are becoming more and more appealing ( ) . during tumor progression, cancer cells give rise to neoantigens, novel ags different from the self-tumor ags, derived by the tumor-specific mutations. therefore, prediction tools, rna mutanome, and deep-sequencing have allowed the identification of specific non-self-ags that are fundamental in strong t cell immunity ( ) ( ) ( ) . indeed, several clinical trials are currently investigating the potential of neoantigens (nct ; nct ; nct ; nct ; nct ). as emerged, different strategies of ags selection have been explored and, even though one strategy may result in a more enforced antitumor immunity if compared to another, still the issue of the tme negative influence on the immune system has to be faced. indeed, the tme actively suppresses the activation of the immune system. tumor cells secrete immunosuppressive cytokines, as vascular endothelial growth factor ( , ) , macrophage colony-stimulating factor ( ), transforming growth factor β (tgf-β) ( ) , and il- ( , ) . even though some of these cytokine display controversial roles, depending on the pathological context, they generally promote dcs tolerogenicity, by limiting their activation and increasing their expression of pro-tumor molecules, such as programmed cell death (pd- ) and indoleamine , -dioxygenase (ido). therefore, tolerogenic dcs lead to t cells anergy, tregs expansion, and th responses inhibition. phenotypical characterization of immune cells isolated from breast cancer patients, highlighted the functional alteration in dcs, t, and nk cells in promoting antitumor responses ( ) . furthermore, tumor cells retain dcs into the tme, preventing their migration to draining lns and promoting metastatization ( ) . to address this issue, some ex vivo generated dcs-based vaccines are directly administered intranodally, as for the cd c + dcs pulsed with hla-a . -restricted tumor peptides administered to patients with stage iv melanoma (nct ), which generated tumor-specific cd + t cells responses and further improvement of survival ( ) . to reduce the tolerogenic influence of the tme on dcs, the positive role of gm-csf in improving dcs survival and responsiveness is currently exploited in some clinical trials like a phase i/ii trial with a dc/tumor cell fusion vaccine administered in association with gm-csf to treat renal cancer (nct ). similarly, others are focusing their attention on fms-like tyrosine kinase -ligand (flt l), another crucial dcs growth factor, in combination with other compounds (nct ; nct ; nct ; nct ). flt l has, indeed, been shown to increase the efficacy of proteins-and rna-based vaccines, due to a maturation effect on dcs ( ) ( ) ( ) . additional efforts made to counteract the tolerogenic influence of the tme include the use of pd- and ido inhibitors. co-administration of anti-pd- molecules increases the efficacy of dcs-based vaccines, in terms of enforced intratumoral cd + t cell responses and trafficking of cd + memory t cells, as observed in a preclinical model of glioblastoma ( ) . in parallel, several clinical trials are aiming at evaluating the efficacy of dcs-based vaccines combined with anti-pd- agents (nct ; nct ; nct ). the other tolerogenic marker addressed in cancer immunotherapy and dcs-based vaccine is ido. indeed, silencing approaches to reduce the expression of ido in dcs for vaccination in preclinical models, have resulted in decreased t cell apoptosis, reduced numbers of tregs, decreased tumor size when compared to mice that had received ags-loaded dcs without ido silencing ( ) . ido inhibitors in dcs vaccination are currently being tested in phase ii clinical trials (nct ; nct ). all these approaches have explored different scenarios to evaluate the more efficient therapeutic combination that seems to move toward personalized vaccinations for cancer patients. in this review, we have underscored the crucial role of dcs in orchestrating immune responses and; therefore, the great interest in targeting these cells in novel vaccination strategies. we have reported examples of different approaches aimed at amplifying the efficiency of immunizations against cancer or infectious diseases. indeed, the urgent need of vaccines is as relevant as before because of newly emerging diseases with ineffective current therapies. deepen the mechanisms underlying these pathologies may provide cues on the more appropriate design of vaccines and by merging diverse tailoring strategies we could enforce the immune system. as a matter of fact, it is suggested to act on different fronts when designing new vaccines, since several factors must be considered: (i) targeting dc subsets specialized in initiating the desired cellular or humoral immunity/memory; (ii) adjuvants that strengthen and drive t and b cell responses; (iii) fine and optimized selection of the immunogenic ags to drive enforced responses; (iv) novel strategies to convey ags and adjuvants to dcs; (v) route of administration. starting from these notions, in the last decades, enormous efforts have been made to tailor vaccination strategies. new technologies as well as recent advances have allowed extreme flexibility in designing vaccines and shaping the following outcomes. nowadays, researchers do have smart tools to manipulate immune responses with prophylactic or therapeutic vaccinations. the abovementioned findings pave the way for possible therapeutic approaches, theoretically applicable to all pathological contexts. despite this encouraging evidence, several limitations or issues still have to be overcome. indeed, more than a few vaccines do not pass phases i of clinical trials either for toxicity issues and lack of immunogenicity in some individuals. what is missing? part of the answer to this question could sit on human genetics and population variability. syngeneic animal models are ideal settings in which the systems are pushed although they constitute a necessary and useful step preceding clinical trials. moreover, when translating vaccine testing from in vivo experiments on animals to ex vivo on human cells, often the opted choice are blood human cells, while in most of the cases vaccines will be administered in the skin, having a complete different dcs-based milieu ( ) . crucially, idoyaga and colleagues dissected the interindividual variability in skin-resident dcs, stressing the need of shedding light on the effects that genetics and environment imprint on dcs. it is compulsory to decode the complex scenario of human diversity to provide personalized therapies with increased efficacy. in the omics era, systems biology and computational modeling integrate huge data-sets to address the urgent need of information on the global behavior. indeed, genome-wide association studies have provided insights into human genetics variants associated to the immunogenicity of vaccines ( , ) . therefore, integration of "wet" evidence and "dry" notions may fasten the designing process and provide both efficient vaccine strategies and their predictive efficacy. all authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication. 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dcs use different chemokine receptor ccr -dependent routes for entry into the lymph node and intranodal migration distinct dendritic cell populations sequentially present antigen to cd t cells and stimulate different aspects of cell-mediated immunity migratory, and not lymphoid-resident, dendritic cells maintain peripheral self-tolerance and prevent autoimmunity via induction of itreg cells dendritic cells induce peripheral t cell unresponsiveness under steady state conditions in vivo tolerogenic dendritic cells th cells in mucosal immunity and tissue inflammation cd regulates the dendritic cell life cycle after lps exposure through nfat activation dendritic cell apoptosis in the maintenance of immune tolerance elimination of antigen-presenting cells and autoreactive t cells by fas contributes to prevention of autoimmunity most microbe-specific naïve cd + t cells produce memory cells during infection alum: an old dog with new tricks cutting edge: alum adjuvant stimulates inflammatory dendritic cells through activation of the nalp inflammasome cutting edge: inflammasome activation by alum and alum's adjuvant effect are mediated by nlrp the nlrp inflammasome is critical for aluminum hydroxide-mediated il- β secretion but dispensable for adjuvant activity dna released from dying host cells mediates aluminum adjuvant activity adjuvantenhanced antibody receptor signaling alum induces innate immune responses through macrophage and mast cell sensors, but these sensors are not required for alum to act as an adjuvant for specific immunity the syk-nfat-il- pathway in dendritic cells is required for optimal sterile immunity elicited by alum adjuvants different human vaccine adjuvants promote distinct antigen-independent immunological signatures tailored to different pathogens vaccine adjuvant mf promotes retention of unprocessed antigen in lymph node macrophage compartments and follicular dendritic cells vaccine adjuvant mf promotes the intranodal differentiation of antigen-loaded and activated monocyte-derived dendritic cells the two-component adjuvant ic ® boosts type i interferon production of human monocyte-derived dendritic cells via ligation of endosomal tlrs pegylation of a tlr -agonist-based vaccine delivery system improves anti gen trafficking and the magnitude of ensuing antibody and cd +t cell responses the tlr / ligand pam csk is a th polarizing adjuvant in leishmania major and brugia malayi murine vaccine models defined tlr -specific adjuvant that induces nk and ctl activation without significant cytokine production in vivo using tlr ligands as a combination adjuvant induces qualitative changes in t cell responses needed for antiviral protection in mice the science of vaccine adjuvants: advances in tlr ligand adjuvants functional properties of flagellin as a stimulator of innate immunity novel adjuvant alum-tlr significantly potentiates immune response to glycoconjugate vaccines vaccine composition formulated with a novel tlr -dependent adjuvant induces high and broad protection against staphylococcus aureus a formulated tlr / agonist is a flexible, highly potent and effective adjuvant for pandemic influenza vaccines a tlr agonist enhances the anti-tumor immunity of peptide and lipopeptide vaccines via different mechanisms a dual tlr agonist adjuvant enhances the immunogenicity and protective efficacy of the tuberculosis vaccine antigen id the toll-like receptor agonist monophosphoryl lipid a augments innate host resistance to systemic bacterial infection tlr ligands lipopolysaccharide and monophosphoryl lipid a differentially regulate effector and memory cd + t cell differentiation co-delivery of cancer-associated antigen and toll-like receptor ligand in plga nanoparticles induces potent cd +t cell-mediated anti-tumor immunity inducible il- production by dendritic cells revealed by global gene expression analysis regulation and dysregulation of innate immunity by nfat signaling downstream of pattern recognition receptors (prrs) a role for interleukin- trans-presentation in dendritic cell-mediated t cell activation in humans, as revealed by daclizumab therapy immunity and cancer: a transcription factor comes of age dectin- stimulation by candida albicans yeast or zymosan triggers nfat activation in macrophages and dendritic cells calcineurin regulates innate antifungal immunity in neutrophils dectin- is a direct receptor for mannose-capped lipoarabinomannan of mycobacteria phagocytosis-dependent activation of a tlr -btk-calcineurin-nfat pathway co-ordinates innate immunity to aspergillus fumigatus skin infections are eliminated by cooperation of the fibrinolytic and innate immune systems il- cis presentation is required for optimal nk cell activation in lipopolysaccharide-mediated inflammatory conditions a contribution of mouse dendritic cell-derived il- for nk cell activation cd and nfat mediate lipopolysaccharide-induced skin edema formation in mice prostaglandin e is generally required for human dendritic cell migration and exerts its effect via ep and ep receptors a spatially-organized multicellular innate immune response in lymph nodes limits systemic pathogen spread capture of influenza by medullary dendritic cells via sign-r is essential for humoral immunity in draining lymph nodes conduits mediate transport of low-molecular-weight antigen to lymph node follicles strategically localized dendritic cells promote rapid t cell responses to lymph-borne particulate antigens histo-cytometry: a method for highly multiplex quantitative tissue imaging analysis applied to dendritic cell subset microanatomy in lymph nodes subcapsular sinus macrophages prevent cns invasion on peripheral infection with a neurotropic virus the conduit system transports soluble antigens from the afferent lymph to resident dendritic cells in the t cell area of the lymph node lymph node macrophages restrict murine cytomegalovirus dissemination subcapsular sinus macrophages limit acute gammaherpesvirus dissemination tracking and quantification of dendritic cell migration and antigen trafficking between the skin and lymph nodes dynamics and function of langerhans cells in vivo: dermal dendritic cells colonize lymph node areasdistinct from slower migrating langerhans cells lymph-node resident cd a+ dendritic cells capture antigens from migratory malaria sporozoites and induce cd + t cell responses lymph node resident rather than skin-derived dendritic cells initiate specific t cell responses after leishmania major infection cross-presentation, dendritic cell subsets, and the generation of immunity to cellular antigens cross-presentation of cell-associated antigens by mouse splenic dendritic cell populations cd +t cells orchestrate pdc-xcr +dendritic cell spatial and functional cooperativity to optimize priming migratory dendritic cells transfer antigen to a lymph node-resident dendritic cell population for efficient ctl priming multiple dendritic cell populations activate cd + t cells after viral stimu lation cross-presentation of viral and self antigens by skin-derived cd + dendritic cells imaging of the cross-presenting dendritic cell subsets in the skin-draining lymph node resident cd + and migratory cd + dendritic cells control cd t cell immunity during acute influenza infection human tissues contain cd hicross-presenting dendritic cells with functional homology to mouse cd +nonlymphoid dendritic cells peripheral cd + dendritic cells form a unified subset developmentally related to cd α + conventional dendritic cells selective expression of the chemokine receptor xcr on cross-presenting dendritic cells determines cooperation with cd + t cells ccr -dependent recruitment of blood phagocytes is necessary for rapid cd t cell responses to local bacterial infection expression of the self-marker cd on dendritic cells governs their trafficking to secondary lymphoid organs tumor immunotherapy by epicutaneous immunization requires langerhans cells langerin expressing cells promote skin immune responses under defined conditions langerin + dermal dc, but not langerhans cells, are required for effective cd -mediated immune responses after skin scarification with vaccinia virus langerhans cells: not your average dendritic cell langerhans cells suppress contact hypersensitivity responses via cognate cd interaction and langerhans cell-derived il- the balance between immunity and tolerance: the role of langerhans cells robust anti-viral immunity requires multiple distinct t cell-dendritic cell interactions migratory and lymphoid-resident dendritic cells cooperate to efficiently prime naive cd t cells selective and site-specific mobilization of dermal dendritic cells and langerhans cells by th -and th -polarizing adjuvants cpg oligodeoxinucleotides and flagellin modulate the immune response to antigens targeted to cd α+and cd α-conventional dendritic cell subsets cd -targeted dendritic cell delivery of plga-nanoparticle vaccines induce potent anti-tumor responses intensified and protective cd + t cell immunity in mice with anti-dendritic cell hiv gag fusion antibody vaccine antigen targeting to dendritic cells elicits long-lived t cell help for antibody responses in vivo targeting of antigens to maturing dendritic cells via the dec- receptor improves t cell vaccination antibody constructs in cancer therapy: protein engineering strategies to improve exposure in solid tumors magic bullets" for targeting the immune system drug nanocarriers to treat autoimmunity and chronic inflammatory diseases nanoparticles and innate immunity: new perspectives on host defence overall survival analysis of a phase ii randomized controlled trial of a poxviral-based psa-targeted immunotherapy in metastatic castrationresistant prostate cancer a novel antigen delivery system induces strong humoral and ctl immune responses vaccine nanocarriers: coupling intracellular pathways and cellular biodistribution to control cd vs cd t cell responses synthetic vaccine nanoparticles target to lymph node triggering enhanced innate and adaptive antitumor immunity plga microspheres for improved antigen delivery to dendritic cells as cellular vaccines a lipid based antigen delivery system efficiently facilitates mhc class-i antigen presentation in dendritic cells to stimulate cd +t cells superior antigen cross-presentation and xcr expression define human cd c + cd + cells as homologues of mouse cd + dendritic cells clec a is a novel activation c-type lectin-like receptor expressed on bdca + dendritic cells and a subset of monocytes characterization of human dngr- + bdca + leukocytes as putative equivalents of mouse cd α + dendritic cells the role of xcr and its ligand xcl in antigen cross-presentation by murine and human dendritic cells the c-type lectin receptor clec a mediates antigen uptake and (cross-) presentation by human blood bdca + myeloid dendritic cells antibodies targeting clec a promote strong humoral immunity without adjuvant in mice and non-human primates the dendritic cell subtype restricted c-type lectin clec a is a target for vaccine enhancement targeting antigen to mouse dendritic cells via clec a induces potent cd t cell responses biased toward a follicular helper phenotype targeting antigen to clec a primes follicular th cell memory responses capable of robust recall evolution of b cell responses to clec a-targeted antigen targeting clec a delivers antigen to human cd + dc for cd + and cd +t cell recognition enhancing vaccine antibody responses by targeting clec a on dendritic cells tumor therapy in mice via antigen targeting to a novel, dc-restricted c-type lectin the receptor dec- expressed by dendritic cells and thymic epithelial cells is involved in antigen processing cd (dec- ): a recognition receptor for apoptotic and necrotic self expression of human dec- (cd ) multilectin receptor on leukocytes the dendritic cell receptor for endocytosis, dec- , can recycle and enhance antigen presentation via major histocompatibility complex class ii -positive lysosomal compartments a monoclonal antibody to the dec- endocytosis receptor on human dendritic cells efficient targeting of protein antigen to the dendritic cell receptor frontiers in immunology | www.frontiersin.org in the steady state leads to antigen presentation on major histocompatibility complex class i products and peripheral cd + t cell tolerance dec- mediates antigen uptake and presentation by both resting and activated human plasmacytoid dendritic cells dec- receptor on dendritic cells mediates presentation of hiv gag protein to cd + t cells in a spectrum of human mhc i haplotypes antigen delivery by filamentous bacteriophage fd displaying an anti-dec- single-chain variable fragment confers adjuvanticity by triggering a tlr -mediated immune response vaccination with filamentous bacteriophages targeting dec- induces dc maturation and potent anti-tumor t-cell responses in the absence of adjuvants dec- is a cell surface receptor for cpg oligonucleotides cd , an extracellular receptor for binding and uptake of hsp -peptide complexes delivery of antigen to cd induces protective immune responses against tumors cd -expressing plasmid induces anti-cd antibody and enhances immune responses to dna vaccination a novel vaccine for mantle cell lymphoma based on targeting cyclin d to dendritic cells via cd hematology & oncology prolonged contact with dendritic cells turns lymph node-resident nk cells into anti-tumor effectors peptide-pulsed dendritic cells have superior ability to induce immune-mediated tissue destruction compared to peptide with adjuvant superior induction of anti-tumor ctl immunity by extended peptide vaccines involves prolonged, dc-focused antigen presentation dendritic cells process synthetic long peptides better than whole protein, improving antigen presentation and t-cell activation vaccination against hpv- oncoproteins for vulvar intraepithelial neoplasia phase i trial of overlapping long peptides from a tumor self-antigen and poly-iclc shows rapid induction of integrated immune response in ovarian cancer patients addition of interferon-α to the p -slp v vaccine results in increased production of interferon-c in vaccinated colorectal cancer patients: a phase i/ii clinical trial induction of p -specific immunity by a p synthetic long peptide vaccine in patients t reated for metastatic colorectal cancer vaccination with mrna-electroporated dendritic cells induces robust tumor antigen-specific cd + and cd + t cells responses in stage iii and iv melanoma patients breast cancer vaccines delivered by dendritic cell-targeted lentivectors induce potent antitumor immune responses and protect mice from mammary tumor growth neoantigens in cancer immunotherapy personalized cancer vaccine effectively mobilizes antitumor t cell immunity in ovarian cancer personalized rna mutanome vaccines mobilize poly-specific therapeutic immunity against cancer an immunogenic personal neoantigen vaccine for patients with melanoma vascular endothelial growth factor impairs the functional ability of dendritic cells through id pathways vascular endothelial growth factor inhibits the function of human mature dendritic cells mediated by vegf receptor- high co-expression of il- and m-csf correlates with tumor progression and poor survival in lung cancers tumor-derived tgf-β reduces the efficacy of dendritic cell/tumor fusion vaccine serum il- predicts worse outcome in cancer patients: a meta-analysis il- : master switch from tumor-promoting in flammation to antitumor immunity immune cell dysfunctions in breast cancer patients detected through whole blood multi-parametric flow cytometry assay inhibition of dendritic cell migration by transforming growth factor-b increases tumor-draining lymph node meta stasis effective clinical responses in metastatic melanoma patients after vaccination with primary myeloid dendritic cells classical flt l-dependent dendritic cells control immunity to protein vaccine flt ligand enhances the cancer therapeutic potency of naked rna vaccines dendritic cell-specific delivery of flt l by coronavirus vectors secures induction of therapeutic antitumor immunity pd- blockade enhances the vaccination-induced immune response in glioma silencing ido in dendritic cells: a novel approach to enhance cancer immunotherapy in a murine breast cancer model searching for the human genetic factors standing in the way of universally effective vaccines global analyses of human immune variation reveal baseline predictors of postvaccination responses key: cord- -kje lvgl authors: pigeyre, laetitia; schatz, malvina; ravallec, marc; gasmi, leila; nègre, nicolas; clouet, cécile; seveno, martial; el koulali, khadija; decourcelle, mathilde; guerardel, yann; cot, didier; dupressoir, thierry; gosselin-grenet, anne-sophie; ogliastro, mylène title: interaction of a densovirus with glycans of the peritrophic matrix mediates oral infection of the lepidopteran pest spodoptera frugiperda date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: kje lvgl the success of oral infection by viruses depends on their capacity to overcome the gut epithelial barrier of their host to crossing over apical, mucous extracellular matrices. as orally transmitted viruses, densoviruses, are also challenged by the complexity of the insect gut barriers, more specifically by the chitinous peritrophic matrix, that lines and protects the midgut epithelium; how capsids stick to and cross these barriers to reach their final cell destination where replication goes has been poorly studied in insects. here, we analyzed the early interaction of the junonia coenia densovirus (jcdv) with the midgut barriers of caterpillars from the pest spodoptera frugiperda. using combination of imaging, biochemical, proteomic and transcriptomic analyses, we examined in vitro, ex vivo and in vivo the early interaction of the capsids with the peritrophic matrix and the consequence of early oral infection on the overall gut function. we show that the jcdv particle rapidly adheres to the peritrophic matrix through interaction with different glycans including chitin and glycoproteins, and that these interactions are necessary for oral infection. proteomic analyses of jcdv binding proteins of the peritrophic matrix revealed mucins and non-mucins proteins including enzymes already known to act as receptors for several insect pathogens. in addition, we show that jcdv early infection results in an arrest of n-acetylglucosamine secretion and a disruption in the integrity of the peritrophic matrix, which may help viral particles to pass through. finally, jcdv early infection induces changes in midgut genes expression favoring an increased metabolism including an increased translational activity. these dysregulations probably participate to the overall dysfunction of the gut barrier in the early steps of viral pathogenesis. a better understanding of early steps of densovirus infection process is crucial to build biocontrol strategies against major insect pests. the transmission of parvoviruses predominantly occurs by horizontal routes through inhalation or oral exposure, making interaction with mucosal epithelia a crucial part of their pathogenesis (for review [ ] ). the oral route represents a major challenge for viruses as they need to overcome a diversity of barriers to invade their host. indeed, most animal epithelia are covered in their apical surface by a carbohydrate-rich meshwork of various complexity and thickness, the glycocalyx, which can be coated by an additional layer of secreted mucus [ ] . these structures constitute successive protective surfaces where viruses aggregate and either access to attachment factors and receptors at the surface of the epithelial cells or are eliminated by luminal or cilia movements [ ] . this dual fate depends on the virus affinity for glycans, which must allow to escape the trap of the mucus. the diversity of glycans present on the epithelial surfaces vary between and within species and therefore constitute an important component of the innate immunity and of the species barrier [ ] . members of the parvoviridae family are non-enveloped viruses that have a simple capsid with t = icosaedral symmetry protecting a - kb linear, single stranded (ss) dna genome [ ] . they can cause diseases of various severity in a wide range of animals. parvovirus interest in human and animal health or in biomedicine as vectors for gene transfer, has long focused research to understand their cell tropism and entry mechanisms. a number of cellular attachment factors and receptors have been characterized, mostly for vertebrates' parvoviruses. they highlight the importance of glycans in capsid recognition and binding specificity [ ] [ ] [ ] . however, how capsids interact with epithelia remains poorly known, mainly due to difficulties to reconstitute in cellular models the complexity of animal epithelial systems [ , ] . insect parvoviruses, named densoviruses, can be highly pathogenic, a feature that can both represent threats for insect mass rearing or opportunities for biocontrol against harmful insects as alternative to chemicals. developing methods against infections or tools for biocontrol requires a deep understanding of the mechanisms driving host range and pathogenesis. like their vertebrate counterparts, densoviruses are mainly transmitted orally, gut recognition and binding constitute the primary step of their pathogenesis. the mechanisms determining densovirus specificity are poorly known. depending on species, densovirus replication can be either restricted to or exclude the gut, viral particles being then transported across the epithelium by transcytosis to reach internal organs where replication goes [ ] [ ] [ ] [ ] . only one cellular receptor has been so far characterized for a "gut restricted" densovirus. it is a mucin of the gut of the silkworm bombyx mori, whose inactivation makes silkworms resistant to infection with the bombyx mori densovirus type (bmdv) [ ] . we have previously reported that gut transcytosis of the junonia coenia densovirus (jcdv) involves a gut specific receptor-dependent mechanism in caterpillars [ ] . however, the mechanisms used by viral particles to overcome the successive intestinal barriers of different structures and composition remain elusive. in insects, the intestinal tract is covered by a chitinous acellular layer, which has specific features due to the dual embryonic origin of the gut. indeed, anterior and posterior extremities of the gut are ectodermal and the acellular layer is covered by an impermeable cuticle. the midgut section is endodermal and has no cuticle but is covered in most insects by a semi-permeable membrane, named the peritrophic matrix (pm) (for review [ ] ). the pm forms a highly organized lattice of chitin fibrils associated with glycoproteins, mainly peritrophins that have a chitin-binding domain [ , ] . the midgut is thus the portal of entry for most pathogens and their interaction with the pm a critical step of their pathogenesis. the pm forms pores whose size varies between insect species (e.g., - nm in lepidoptera and up to nm in coleoptera) and developmental stages [ , ] . large entomopathogenic viruses have developed specific mechanisms to pass through extracellular matrices including virus-encoded enzymes and specific proteins that are associated with the viral particles of baculo-and entomopoxviruses [ ] [ ] [ ] [ ] . how densoviruses cope with the physical barriers that constitute the gut and in particular the pm is so far unknow. due to their small size, it was initially thought they could diffuse passively across the pores of the matrix, but measures of the size of pores, the complexity of the pm and the nature of the interactions between components make this hypothesis unlikely [ , ] . we previously reported that following oral infection, viral particles of jcdv, a type species ofthe ambidensovirus genus, aggregate on the pm of spodoptera frugiperda caterpillars as a first step of the infection process [ ] . such rapid virus concentration on a carbohydrate-rich surface suggested a lectin-like activities of the capsids. although there is no sequence similarity of the unique protein making the surface of the jcdv with lectin domains, its structure displays similarities with cellular carbohydrate binding proteins including lectins, which suggests that capsids could indeed recognize and bind carbohydrates [ ] . herein, we used a combination of approaches, including microscopy, biochemistry, proteomics and transcriptomics, to decipher the interaction of jcdv with pm major components (i.e., chitin, glycans and proteins). we found that capsids affinity for the pm might result from multiple interactions with different glycans including chitin and glycosylated proteins. in addition, we showed that jcdv early infection results in (i) an arrest of n-acetylglucosamine (glcnac) secretion by epithelial cells associated with a disorganization of the pm structure mimicking the effect of chitin-binding plant lectin; (ii) substantial changes in the expression of gut genes, which may also contribute to an early gut dysfunction and participate to viral pathogenesis. caterpillars were reared under controlled conditions ( ± • c, to % relative humidity [rh], -h light, -h dark photoperiod) on a wheat germ-based artificial diet. jcdv was amplified by oral infection. at death, larvae were crushed and virus extraction was processed by clarification and filtration on . µm to constitute a semi-purified virus stock (jcdv). to obtain a purified viral stock (p_jcdv), the semi-purified virus stock was loaded on optiprep tm (sigma-aldrich, lyon, france) density gradient and dialyzed against phosphate-buffered saline (pbs) x as previously described in [ ] . viral concentrations were estimated by quantitative pcr (qpcr) as described in [ ] and expressed as viral equivalent genomes ([veg]). virus titers were determined by the tissue culture assay method ( % tissue culture infective dose (tcid ) in the permissive ld cells as previously described [ ] . calcofluor white m r (calcofluor; f ), n-acetyl-d-glucosamine (glcnac; a ), n-acetyl-d-galactosamine (galnac; a ), d-(+)-fucose (fucose; f ), d-(+)-mannose (mannose; m ), mucin from porcine stomach (porcine mucin; m ) and wga-fitc conjugate (l ) were all purchased from sigma-aldrich (lyon, france). for in vivo bioassays, third-instar (l ) s. frugiperda caterpillars were individually infected by feeding with jcdv ( veg/caterpillar) or by intrahemocelic injection ( veg/caterpillar). each treatment was applied to a cohort of caterpillars and three independent experiments were performed. calcofluor ( . % to %) was concomitantly administrated with the virus to l or l caterpillars (as described in the figures). for competition assays, jcdv was incubated with glycans (glcnac, galnac, fucose or mannose at µm and/or mm; for one hour before feeding or injection. in all experiments, control larvae were fed or injected with pbs. caterpillars mortality was recorded each day during days and results were presented as survival rates per day. the time to death was assessed by comparing the survival curves using the kaplan meier method (graphpad prism software, version ). the significance between groups were analyzed using log-rank (mantel-cox) tests and gehan-breslow-wilcoxon tests. rabbit erythrocytes cells were diluted at % (v/v) with mm nacl and treated with . mg/ml of trypsin (sigma) for min at • c. after washes with mm nacl, µl serially two-fold dilutions of viral inocula (jcdv or p_jcdv; started from veg/µl) were mixed with µl of erythrocytes in well microtiter plates for min at • c. positive controls of hemagglutination was performed using µl of wga ( mg/ml). fifty µl of erythrocytes were subsequently added to each well for min at • c. hemagglutination was read under a light microscope. for calcofluor experiments, pms were isolated from sixth-instar (l ) s. frugiperda caterpillars, previously anesthetized on ice, then opened and washed with phosphate buffered saline (pbs) to remove the food bolus. pms isolated from caterpillars treated with calcofluor were fixed h with % paraformaldehyde (pfa), then dehydrated with increasing concentrations of ethanol ( % to %) and in : ( % ethanol and hexamethyldisilazane [hmds]) for min and finally in % hmds for min. after overnight evaporation, samples were ultimately coated with platinium and observed with a scanning electron microscope (sem) hitachi s- , with an acceleration voltage of kv and a working distance of mm. for ex vivo infection experiments, pms were isolated as described above and incubated for min at room temperature (rt) with jcdv ( veg/µl; µl), or with jcdv pre-incubated for h with glycans (glcnac, galnac, fucose or mannose; µm to mm) before infection. the pms were then washed with pbs and fixed with % pfa as above. immunolabelling of jcdv particles was performed using a specific rabbit anti-capsid antibody ( : , ; eurogentec) incubated for h at rt and an anti-rabbit secondary antibody (alexa fluor ® ; : ; invitrogen, carlsbad, ca, usa) incubated for min at rt. labeled pms were then mounted in dako (sigma) on coverslips for observation. for epifluorescence measurements, we worked on a zeiss axioimager z , equipped with suitable filters for the dyes, using a ×/ . plan-apochromat oil ph and a ×/ . plan-apochromat oil ph objectives. for structured illumination images, the zeiss apotome-slider was introduced into the field-stop plane of the microscope to improve resolution of images. we used the zen software to operate the microscope and took at least images ( fields per pm) for each treatment. all images were taken with a cmos orca flash . b&w camera. images were processed with fiji software. the intensity of fluorescence (arbitrary unit) were measured on epifluorescence images. statistical analyses were performed using the non-parametric kruskal-wallis test (graphpad prism software, version , graphpad software, san diego, ca, usa). each experiment was repeated at least three times, and each independent experiment gave similar results. l s. frugiperda caterpillars were individually infected by feeding as in section . . twenty four hours later, caterpillars were anesthetized on ice and sacrificed. in parallel, caterpillars were kept until death to insure their infection status. sacrificed caterpillars were fixed in % pfa + . % glutaraldehyde for h at • c and dehydrated by incubations in increasing concentrations of ethanol ( % to %) before progressive embedding in % unicryl resin (bb international) as described in [ ] . semi-thin sections ( µm) were cut in the central part of the caterpillars corresponding to the midgut. after min of incubation with wga-fitc ( : ), to label glcnac and the pm, or with phalloidin-fitc ( : ; sigma), to label actin cytoskeleton, and min with dapi ( µg/ml; invitrogen) to label nuclei, semi-thin sections were mounted and observed with a zeiss axioimager z microscope using the structured illumination module zeiss apotome-slider as in . . images were taken with a cmos orca flash . b&w camera using fixed parameters for all treatments and processed with fiji software. ten µl of chitin beads (new england biolab) were washed three times with pbs and added in µl of virus suspension ( veg/µl in pbs). after h of incubation with gentle rotation, beads were pulled-down by centrifugation ( × g for min à • c), washed five times with pbs, then resuspended in laemmli buffer × ( % sds, % glycerol, % -mercaptoethanol, . % bromophenol blue and . m tris hcl, ph ) and heated at • c for min before western blot analysis. two µl ( % of the pull down and % of the initial input) was loaded on - % polyacrylamide tris-hcl gel (mini-protean ® tgx™ precast gels, biorad, hercules, ca, usa) and separated by sds-page for h at v. next, samples were transferred to pvdf membrane (immobilon-p, merck) for h at ma. subsequently, the membrane was saturated with % of milk in pbs/tween . % (pbst), then incubated h at rt with the primary anti-capsid antibody ( : , ; see above). after washes in pbst, the membrane was incubated h at rt with an anti-rabbit secondary antibody hrp-conjugated ( : ; biorad, hercules, ca, usa). proteins were revealed by enhanced chemiluminescence (millipore, burlington, ma, usa) using a chemidoc imager (biorad). peritrophins were extracted from pools of five pms from sixth instar s. frugiperda in µl of laemmli buffer x, then boiled and heated at • c for min [ ] . after centrifugation at × g for min • c, the supernatant was collected and / was loaded on - % polyacrylamide tris-hcl gel and separated by sds-page as above. thirty µg of porcine mucins were also loaded on the same gel. gel was stained with page blue (thermo fisher scientific, waltham, ma, usa) to analyze total proteins or with periodic acid-schiff (pas) as described in [ ] to analyze glycosylated proteins. proteins were also transferred on nitrocellulose membranes (biorad) for h at v for viral protein overlay binding assay [ ] . briefly, the membrane was saturated with % bsa in pbst for h at rt, then incubated ovn at • c with jcdv ( veg diluted in pbst containing % bsa). after washes in pbst, the membrane was incubated with the rabbit anti-capsid antibody ( : ; see above), then the anti-rabbit secondary antibody hrp-conjugated, and proteins were revealed by enhanced chemiluminescence as above. protein bands revealed by vopba were cut in sds-page gel stained with page blue ( replicates, bands) and destained with three washes in % acetonitrile and mm triethylammonium bicarbonate (teabc). after protein reduction (with mm dithiothreitol in mm teabc at • c for min in the dark) and alkylation ( mm iodoacetamide teabc at room temperature for min) proteins were digested in-gel using trypsin ( . µg/band, gold, promega, madison, wi, usa) as previously described [ ] . digested products were dehydrated in a vacuum centrifuge and resuspended with . % trifluoroacetic acid (tfa) and % acn. the generated peptides were analyzed online by nano-flow hplc-nanoelectrospray ionization using a q-exactive plus mass spectrometer (thermo fisher scientific, waltham, ma, usa) coupled to an ultimate rslc (thermo fisher scientific). desalting and pre-concentration of samples were performed on-line on a pepmap ® pre-column ( . mm × mm, dionex, sunnyvale, ca, usa). the capillary reverse-phase column ( . mm × mm, acclaim pepmap ® c , thermo fisher scientific) fitted with an uncoated silica picotip emitter (new objective, woburn, ma, usa) was first equilibrated in solvent a ( . % formic acid) and a multistep linear gradient of acetonitrile consisting of - % of solvent b ( . % formic acid in % acetonitrile) for min, - % for min and % for min, at nl/min was used to elute peptides from. spectra were acquired with the instrument operating in the information-dependent acquisition mode throughout the hplc gradient. survey scans were acquired in the orbitrap system with resolution set at a value of , . up to twelve of the most intense ions per cycle were fragmented and analyzed using a resolution of , . peptide fragmentation was performed using nitrogen gas on the most abundant and at least doubly charged ions detected in the initial ms scan and an active exclusion time of s. for all full scan measurements with the orbitrap detector a lock-mass ion from ambient air (m/z . ) was used as an internal calibrant as described [ ] . analysis was performed using the maxquant software (version . . . ) [ ] . all ms/ms spectra were searched using andromeda against a decoy database consisting of a combination of s. frugiperda databases [ ] and classical contaminants, containing forward and reverse entities. the following settings were applied: spectra were searched with a mass tolerance of ppm (ms) and . th (ms/ms). enzyme specificity was set to trypsin. up to two missed cleavages were allowed and only peptides with at least seven amino acids in length were considered. carbamidomethylation was set as fixed cystein modification and oxidation was set as variable methionine modification for searches. fdr was set at . for peptides and proteins. sequences which found homology were annotated according to the gene ontology (go) terms and classified using blast go software (https://www.blast go.com/; [ ] ). the enrichment in go terms compared to the s. frugiperda reference (predicted proteins from the ogs . genome; gouin et al., ) was analyzed with the same software (fdr set at . ). fourth instar s. frugiperda caterpillars were orally infected or not with jcdv ( veg per caterpillar; twenty caterpillars per condition). at -day p.i. or days p.i., caterpillars were anesthetized on ice and dissected. the midguts were washed in pbs to eliminate the food bolus and the pms. trachea, malpighi tubes and visceral muscles were removed and the epithelia were incubated with . % trypsin for min to dissociate the tissues. after washes, gut cells were lysed in µl of trizol ® reagent (invitrogen) for total rna extraction according to the manufacturer's instructions. total rna amount and purity were checked by using spectrophotometrer nanodrop nd- (thermo scientific) and the integrity of total rna was analyzed by capillary electrophoresis ( bioanalyzer instrument, agilent, santa clara, ca, usa). we used digital gene expression (dge) method that generates short sequences (tags) specific for mrna [ ] [ ] [ ] . four dge libraries were constructed from midgut total rna extracted from s. frugiperda caterpillars infected (or not) for and days. sequence tag preparation was done with illumina's digital gene expression tag profiling kit according to the manufacturer's protocol (version . b) as described in [ ] . for fourth libraries, µg of total rna were incubated with oligo-dt beads. first-and second-strand cdna syntheses were performed using superscript ii reverse transcription kit according to the manufacturer's instructions (invitrogen). the cdnas were cleaved using the nlaiii anchoring enzyme. subsequently, digested cdnas were ligated with the gex adapter containing a restriction site for mmei. the second digestion with mmei was performed, which cuts bp downstream of the catg site. at this point, the fragments detach from the beads. the gex adapter was ligated to the end of the tag. a pcr amplification with cycles using phusion polymerase (finnzymes) was performed with primers complementary to the adapter sequences to enrich the samples for the desired fragments. the resulting fragments of bp were purified by excision from a % polyacrylamide tbe gel. the dna was eluted from the gel using spin-x cellulose acetate filter ( . µm), precipitated, resuspended in mm tris-hcl (ph . ) and quantified using nanodrop spectrophotometer. cluster generation was performed after applying pm of each sample to the individual lanes of the illumina g flowcell. after hybridization of the sequencing primer to the single-stranded products, cycles of base incorporation were carried out on the g analyzer according to the manufacturer's instructions. image analysis and base calling were performed using the illumina pipeline, where sequence tags were obtained after purity filtering. we could assign % of the tags (supplementary materials tables s -s ) out of which % correspond to multiple matches and were discarded from functional analysis with go. functional annotation was performed using biotag software (skuld-tech, grabels, france). the statistical value of dge data comparisons, as a function of tag counts, was calculated by assuming that each tag has an equal chance of being detected. differential expression of the tag counts of the infected vs. mock conditions was performed to obtain a list of up-and down-regulated tags for each condition. tags for which differential expression was ≥ fold change were assigned using the reference databases for s. frugiperda [ , ] . sequences with homology were annotated according to the go terms and classified using blast go software (https://www.blast go.com/; [ ] ) and represented at level of biological process and molecular function. the enrichment in go terms compared to the s. frugiperda reference (predicted transcripts from the ogs . genome) was analyzed with the same software (fdr set at . ). the junonia coenia densovirus (former jcdnv) corresponds to the complete genome of the oxford isolate (genbank accession number kc . ). early studies have estimated that the average pore size of the pm is around nm in s. frugiperda caterpillars, which might exclude nm densovirus particles [ ] . to assess the pm barrier function against densovirus infection, we disrupted its structure by feeding third instar (l ) larvae with sub lethal doses ( . %) of the chitin-binding agent calcofluor white prior jcdv infection [ ] [ ] [ ] [ ] [ ] . we then measured larval mortality rates daily. results showed that jcdv infected larvae pre-treated with calcofluor displayed a significant shorter median time to death (lt ) compared to untreated infected larvae ( vs. days p.i. for control larvae; p < . ) ( figure a and supplementary materials figure s ), supporting that the pm can limit jcdv infection. noteworthy, an increased larval mortality was also observed at late time post-treatment with calcofluor in mock-infected caterpillars compared to untreated controls ( % at days p.i.), confirming a detrimental inhibition of chitin assembly on larval development [ ] . larvae (n = ) were infected individually by feeding with jcdv ( veg/caterpillar) concomitantly with . % ( µg) of calcofluor or pbs as a control. caterpillar mortality was recorded once a day during days and results were presented as survival rates per day. three independent experiments were performed, each independent experiment gave similar results, one is represented here. the log-rank (mantel-cox) and the gehan-breslow-wilcoxon tests were used to determine statistical significance. p values of less than . were considered significant (**, p < . ). pbs refers to control (pbs-treated and non-infected) caterpillars; calcofluor refers to calcofluor-treated and non-infected caterpillars; jcdv refers to jcdv-infected caterpillars and jcdv+calcofluor to calcofluor-treated and jcdv-infected caterpillars. (b) calcofluor disrupts the pm integrity. sem images of pm ultrastructure isolated from l caterpillars fed with pbs ( %), . % ( µg) or % ( µg) of calcofluor. endoperitrophic face is shown (bars, nm). we analyzed the effect of calcofluor on the pm integrity by scanning electron microscopy (sem). because the pm of l larvae has a gel-like structure that cannot be manipulated, we took l larvae for this experiment as the pm is thick and solid at this stage and can be easily dissected. l caterpillars were fed with up to % calcofluor (not lethal at h post treatment) and pms were isolated h post-treatment and prepared for sem analysis. as shown by figure b , pms from control larvae displayed a highly organized structure, similar to pms from caterpillars treated with . % calcofluor. by contrast, pms from caterpillar fed with % calcofluor had a clear disrupted structure with enlarged pores, confirming that calcofluor binding to chitin fibrils compromised the integrity of the matrix. the rapid recognition of the pm by jcdv capsids suggests that their affinity for glycans is important for the oral infection process. to test this hypothesis, we first assayed the capsid ability to agglutinate erythrocytes, a feature displayed by vertebrate parvoviruses [ , ] . we performed a typical hemagglutination assay, adding serial dilutions of the virus inoculum to rabbit erythrocytes ( figure ). the first dilutions ( : and : ) of jcdv triggered a strong hemolysis of erythrocytes suggesting a toxic effect of the viral inoculum, ie capsids or some host-derived component associated with the inoculum. it is worthy to note that we use semi-purified inoculum as it mimics naturally occurring infections. jcdv was therefore further purified on a density gradient (p_jcdv) and similarly assayed for hemagglutination. a clear hemagglutination was obtained with p_jcdv, supporting that toxicity is likely due to a host-derived component that can be eliminated during the purification process. hemagglutination with p_jcdv was obtained up to the third dilution (hemagglutination titer of : ), which indicates a rather weak interaction of the capsids with glycans at the surface of (mammalian) erythrocytes. to better understand capsid affinity for glycans, we performed a competition bioassay using monomeric glycans as jcdv-binding competitors. jcdv binding was revealed with an immunofluorescence staining on the pms using a specific anti-capsid antibody. we quantified this fluorescence as a proxy of binding and competition. we first performed competition ex vivo on isolated pms incubated with jcdv in the presence of four monosaccharides commonly found in insects [ ] , ie n-acetyl-d-glucosamine (glcnac), which is the monomeric unit of chitin, n-acetyl-d-galactosamine (galnac), d-fucose and d-mannose (figure ). we first verified with a dot blot assay that capsids interaction with monosaccharides were not interfering with antibody recognition, which validated the competition bioassay (supplementary materials figure s ). as shown in figure , jcdv binding resulted in an intense fluorescence signal on the pms (left panel), which was similarly competed away by the four monosacharides and within a similar concentration range ( . mm to mm). we noted that fluorescence quantification did not result in a strictly linear dose-dependent effect. to further test the role of glycans in jcdv pathogenesis, we carried out these competition bioassays in vivo (figure and supplementary materials figure s ). we mixed jcdv with each monosaccharide prior infection, fed caterpillars with these inocula and calculated mortality rates daily as in figure . results showed that oral infection with mm (but not µm) of each monosaccharide significantly delayed the median time to death (lt ) of caterpillars ( vs. days; p < . for mm) ( figure a and supplementary materials figure s a ,b), further supporting that pm recognition is the first step of jcdv oral infection. to confirm these results and reveal jcdv binding and competition for binding the pm in vivo, we carried out midgut semi-thin sections and immunofluorescence as above. caterpillars were infected with jcdv mixed or not with mm glcnac and sacrificed at h p.i. for midgut isolation and preparation. as shown in figure b , we observed a red fluorescence signal in untreated infected caterpillars that typically lines the pm. in addition, labelling was also observed in the lumen, likely revealing jcdv interaction with food bolus and/or microbial components. both signals were strongly and specifically decreased following competition with glcnac ( figure b) , showing that different glcnac-containing glycans in the gut lumen can recognize the capsids. control caterpillars were fed with pbs. three independent experiments were performed, each independent experiment gave similar results, one is represented here. the log-rank (mantel-cox) and the gehan-breslow-wilcoxon tests were used to determine statistical significance. p values of less than . were considered significant (ns, non-significant; * p < . ; ** p < . ; *** p < . ). (b) immunolabeling of midgut semithin transversal sections h after ingestion of jcdv alone or jcdv ( veg/caterpillar) incubated for h with mm of glcnac before oral infection. control caterpillars were fed with pbs. the pm is shown by an arrowhead. phalloidin-fitc is in green, jcdv is in red, and nuclei are labeled with dapi (blue). bars, µm. lum, midgut lumen; hemol, hemolymphatic compartment. (c) survival curves of caterpillars (n = ) infected by injection of jcdv alone ( veg/caterpillar) or of jcdv incubated for h with mm of each glycan (glcnac, galnac, fucose or mannose) before infection. control caterpillars were injected with pbs. three independent experiments were performed, each independent experiment gave similar results, one is represented here. the log-rank (mantel-cox) and the gehan-breslow-wilcoxon tests were used to determine statistical significance, p > . were considered non-significant (ns). pbs refers to control (pbs-treated and non-infected) caterpillars; 'jcdv' to jcdv-infected caterpillars; 'jcdv + glcnac', 'jcdv + galnac', 'jc + fucose' and 'jc + mannose' refer to caterpillars infected with jcdv incubated with glcnac, galnac, fucose or mannose, respectively, before infection. last, we studied whether such "stickiness" was specifically required by the densovirus to cross the gut, i.e., for oral infection. jcdv infection of target cells (eg epidermis, trachea, hemocytes) proceeds by a receptor-dependent mechanism different from intestinal cells [ ] . these cells express glycan structures of various complexity that are attached to the cell surface or secreted and forming extracellular matrices, whose glycans might be similarly targeted by jcdv for attachment. we performed competition bioassays in vivo, bypassing the midgut by injecting caterpillars with jcdv mixed or not with mm of each monosaccharide. interestingly, the median time to death was similar for all conditions ( figure c and supplementary materials figure s c ; p > . ), showing that none of the monosaccharides competed with jcdv infection proceeding by the systemic route. altogether these results show that jcdv capsid is a carbohydrate-binding protein and this feature is required for oral infection to target the pm of caterpillars. we next wanted to determine which component of the pm, i.e., chitin and/or glycosylated proteins were involved in capsid interactions, using biochemical assays. we first tested capsid physical interaction with chitin using a pull-down assay with chitin beads. jcdv from purified or semi-purified inocula were incubated with chitin beads, pulled-down by centrifugation and subsequently revealed by western blot using a specific jcdv anti-capsid antibody. figure a shows jcdv pull-down by chitin beads and we did not observed difference between inocula (purified vs. semi-purified), which confirmed that capsids can interact directly with chitin. second, we tested virus interaction with pm proteins using a viral overlay binding assay (vobpa). total proteins were extracted from isolated pms, separated with sds-page and either stained with page blue and periodic acid schiff (pas) in order to visualize total and glycosylated proteins respectively, or blotted onto nitrocellulose membranes for vopba. we included porcine mucins as a control of highly (o-)glycosylated proteins. at the first glance, vopba revealed that jcdv binds to most if not all the pm proteins labelled by page blue and pas combined, although with different intensities ( figure b ). interestingly, no binding was observed with the porcine mucins which might support some specificity for insect glycans. more specifically, a set of jcdv-interacting proteins was identified at high molecular weights (> kda) including proteins with a pattern similar to porcine mucins. these proteins were labelled with page blue and pas or only with pas suggesting that they are mostly and probably highly glycosylated ( figure b ). interestingly, proteins at kda displayed a higher intensity as they are in a relative lower amount (according to page blue), which suggests higher affinity for jcdv. proteins interacting with jcdv were detected at - kda and - kda, and corresponding to proteins with low or no glycosylation (according to pas staining). thirty µg of porcine mucins were also loaded in the gel as a control of highly o-glycosylated proteins. proteins were then stained with page blue or periodic acid schiff (pas, pink) to visualize total or glycosylated proteins, respectively, and transferred to nitrocellulose membranes for probing with jcdv and anti-jcdv capsid antibody. proteins interacting with jcdv capsids were finally revealed by enhanced chemiluminescence (black arrowheads on the vopba jcdv membrane); the corresponding positions of these bands were reported on the page blue and pas gels and indicated as well by black arrowheads on the right of these gels. in total, bands representing jcdv interacting proteins are reproducibly obtained with vopba. noteworthy, each band probably include several proteins and/or isoform/glycoform of the same proteins. proteins corresponding to these bands were next analyzed by lc-ms/ms mass spectrometry. we only considered proteins that were shared between replicates ( figure a) , out of which were annotated in the reference genome of s. frugiperda [ ] . these proteins are pm structural proteins (i.e., peritrophins including intestinal mucins) and pm-associated proteins (enzymes, i.e., serine proteases and aminopeptidases n (apn) (supplementary materials table s ). gene ontology (go) annotation confirmed the enrichment in proteolytic activities (particularly serine-type endopeptidases) and chitin synthesis, which are consistent with the pm composition and the gut function ( figure b ). interestingly among the set of proteins > kda, we identified intestinal mucins, an atp binding cassette a type (abca ) transporter and aminopeptidases n (supplementary materials table s ); the latter being known receptors for a number of viruses and for the cry toxins from bacillus thuringiensis [ ] [ ] [ ] [ ] . [ ] . (b) go terms enrichment for the common annotated pm proteins interacting with jcdv (in green), compared to the reference in grey (predicted proteins from ogs . s. frugiperda genome) (fdr set at . ). specific enrichment in jcdv interacting proteins is considered when the green bars exceed the greys (controls). the common pm proteins were assignated to the go terms using blast go software. these results show that jcdv capsids can recognize and bind to the different components of the pm including chitin and several highly glycosylated proteins, both structural components of the pm (mucin, peritrophins) or associated proteins (enzymes). jcdv recognition and binding to glycans of the pm concentrates viral particles close to the epithelial surface, which raised questions about the mechanism involved to cross over and reach the midgut receptor(s). we hypothesized that capsids aggregation on the matrix can result in its disorganization, in a way similar to chitin-binding wheat germ agglutinin (wga) lectin or calcofluor [ ] . to test this hypothesis, we used fluorescent wga-labelling (wga-fitc) to label chitin and thus examine chitin fibrils formation and pm organization. third-instar caterpillars were fed with jcdv and then sacrificed at day p.i. to dissect and prepare midguts for semi-thin sections and wga labelling. as shown in figure , the labelling of the pm (green) lined the apical surface of the epithelium in non-infected larvae (pbs condition, figure , upper panel). in addition, we observed a specific labelling at the apex of columnar cells, probably corresponding to microvillar secretion of glcnac from these cells. by contrast, the labelling lining the epithelium appeared discontinuous following infection (figure , lower panel) , displaying a disorganized pattern reminiscent of the pm structure observed for caterpillars fed with the wga lectin [ ] . moreover, intracellular labelling was no longer observed in the sections from infected caterpillars suggesting an arrest of glcnac secretion from the cells following early infection, i.e., before we can detect virus replication in subepithelial tissues [ ] . these results thus support the hypothesis that jcdv binding on the pm and transcytosis is associated with a loss in its integrity, which might reveal gut dysfunction. to determine the midgut response following jcdv break in, we analyzed the transcriptomic response. we used digital gene expression (dge) based on the serial analysis of gene expression approach [ ] . this method involves the sequencing and quantification of end tagged short cdna fragments (i.e., tags), which enables quantitative differential gene-expression analysis. we built four cdna libraries from midguts of mock-and infected larvae (supplementary materials tables s and s ) . tag sequences were mapped to the genome and transcriptome of s. frugiperda ( [ , ] ) and to the jcdv genome. none of the tags were assigned to viral transcripts, which is consistent with jcdv pathogenesis excluding replication in midgut cells [ ] . pie charts represented go assignment corresponding to unique transcripts at and days p.i. that displayed a differential expression at least -fold up-or down-regulated (supplementary materials figure s a,b) . interestingly, the distribution of go terms was roughly similar at and days p.i., suggesting that the overall intestinal response to jcdv oral infection was poorly affected by virus replication going on in subepithelial tissues (supplementary materials figure s a ,b). we observed only go terms enrichment for the over-represented transcripts at -day p.i., more specifically in functions involved in metabolic processes including translation (i.e., regulation of biological processes, response to stimuli and signaling) at day p.i., that might indicate that jcdv intrusion induces a rapid metabolic response in the gut (figure ). interestingly, these changes did not change significantly at days p.i. suggesting that the gut response is rapidly initiated by jcdv transcytosis and was not affected by the viral replication that takes place in underlying tissues. we did not observe any significant activation of genes involved in "inflammation" nor in the canonical gut immune response. however, we observed an increased expression in cytochrome p and catalase genes that might indicate a response of the cells to the ongoing infection. table s ), we only observed few changes including a trehalose transporter and an intestinal mucin, both down-regulated from day p.i., and a chitinase up-regulated at day p.i.. except for an aminopeptidase n, no gene corresponding to the jcdv interacting proteins identified by vopba displayed a transcriptional change. we did not observe any significant activation of genes involved in "inflammation" nor in the canonical gut immune response. however, we observed an increased expression in cytochrome p and catalase genes that might indicate a response of the cells to the ongoing infection. altogether these results show that jcdv infection induces rapid changes in the gut, particularly in translation and metabolism, within h p.i.. both increased molecular activities might favor viral invasion by supporting the increased energetic demand associated with virus replication in target tissues. interestingly, the canonical midgut immune system did not detect jcdv break in and transport across the epithelium. how densoviruses cope with the forest of glycans that constitute extracellular matrices and decorates insect cell surfaces has been so far a neglected step of their early pathogenesis. results presented here show that jcdv capsids display carbohydrate-binding properties that insure recognition of the peritrophic matrix and determines caterpillars oral infection. we found that capsids can bind to the different components of the pm and their agglutination on the pm surface is associated with the disruption of its organization. furthermore, we showed that this primary step of infection of caterpillars results in a series of physiological changes in the midgut including an arrest of chitin synthesis by epithelial cells. the pm is an obligatory binding platform for capsids to avoid elimination and get closer to the epithelial cell surface where receptor recognition can occur. however, strong attachment to glycans composing the pm would trap capsids there and thus impair their physical connection with the receptor(s). therefore, a first hypothesis is that the "stickiness" of the capsids is balanced to bind and unbind glycans. we used competitions assays with monosaccharides to test this "bind and release" hypothesis and our results showed that indeed, capsids have an affinity for glycans, although the concentration range of the monosaccharides (mm) we tested likely indicates their poor affinity for the capsids. as these monosaccharides could compete capsids away from the pm further suggests that glycan-capsids interaction are probably of low affinity. the issue for bound viral particles is then to move across the pm. our experiments show that capsid binding results in a structural disorganization of the pm similar to effects induced by chitin-binding lectin wga and calcofluor. such capsid-induced disruption of the pm thus favors a second hypothesis, involving a "saturate and pass through" mechanism, where bound capsids are not released but open a way for viral particles to cross over. such "cooperative" mechanism of the capsids to overcome the pm is supported by the fact that pm disruption is enhanced by virus concentration and decreased as caterpillars age. such developmental resistance of s. frugiperda has been also reported following baculovirus infections and a high synergism with calcofluor was obtained at late instars (e.g., > -fold at th instar vs. to -fold at nd and rd instars) [ ] .the structure and the composition of the pm can vary as caterpillars grow and feed, or between populations, which might impact virus-pm interactions and consequently insect susceptibility [ , , ] . whether pm disruption results from mechanical stresses on chitin fibers similar to calcofluor and probably wga lectins or from an enzymatic activity of the capsids (i.e., of vp ) remains to be analyzed more thoroughly. understanding the early interaction of jcdv with the glycans of the pm within species, i.e., along the larval development is of importance to develop biocontrol strategies against insect pests. whether or not the pm could contribute to the species barrier against densovirus infection is unknown. a better understanding of the structure and the glycan composition of the pm in s. frugiperda together with comparative studies in different lepidopteran species are essential to go further on the role of the pm in densovirus infection. structure-fonction studies of the capsid of parvoviruses infecting vertebrates, in particular for species in the genera protoparvovirus and dependovirus, have highlighted the importance of glycans recognition on tissue tropism, pathogenicity, and host range adaption [ , , [ ] [ ] [ ] [ ] . regarding densoviruses, information and capsid structure-function studies is poor [ ] . we performed preliminary assays with a glycan array from the consortium for functional glycomics (http: //www.functionalglycomics.org/fg/, as gosselin-grenet, unpublished data). although this array represents mammalian glycans, the specific recognition of jcdv capsids by paucimannose, which are particularly abundant in insects, suggests some specificity in the interaction of the capsid with glycans. an "insect array" would be of interest to explore glycan ligands affinity and specificity for densovirus capsids. morevover, insects are particularly "handly" animal models for structure-function assays in vivo. we showed that jcdv early infection triggers an arrest of glcnac secretion, which might considerably weaken the pm and explain the disorganized pattern observed with chitin-labelling at day p.i. interestingly, these changes are observed before we could detect jcdv transcription/replication in primary targeted cells [ ] , suggesting that these effects are induced as a consequence of the transport of the viral particles across the epithelium. it has been reported that specific drugs that disorganize microtubules induce an arrest of chitin synthesis [ , ] . we speculate that jcdv transcytosis might induce some stress on the cytoskeletal network, similar to microtubules disorganizing drugs. it is worthy to note that chitin synthesis arrest was not associated with a drop in the expression of chitin synthase genes. however, we cannot exclude to have missed enzymes due to some lack in the annotation of the genome of s. frugiperda [ ] . last, our results showed that jcdv capsids can also interact with components of the luminal compartment including food and bacteria. the competition of the capsid with monosaccharides for binding the pm, suggests that food components could interfere with infection. indeed, plants contain compounds that can interfere with pm synthesis, which has been shown to consequently affect baculovirus infection [ ] (chen et al., ) . regarding bacteria, it has been shown recently that the pm controls commensal bacteria, and conversely that its synthesis and integrity can be microbiota-dependent, i.e., the gut microbiota inducing the expression of components of the peritrophic matrix [ , ] . so, it is plausible that food and microbiota can modify the outcome of the densovirus infection, either by directly competing for binding the pm, or indirectly by modulating the composition of the pm. the binding of densovirus capsids to a wide array of glycans questioned about the role of this "stickiness" in the whole infection cycle including transmission. groundbreaking articles have shown the role of microbiota polysaccharides including glcnac, on the infectivity and thermostability of picornaviruses [ ] [ ] [ ] , whose capsid share structural similarity with parvoviruses [ ] . it is tempting to speculate that densovirus "stickiness" can similarly impact their transmission, which might participate to their success if only among arthropods that occupy extremely diversified ecosystems [ ] . such consideration could also apply to parvoviruses going through faecal-oral route and environmental contamination [ , ] . more generally, stickiness is a major issue for most viruses to and mathematical models have been applied to influenza. they predict that a maximum stickiness favors a maximum fitness [ ] . however, trade-off probably exists in biological systems with an optimal "stickiness" that must be found to infect and leave a host for transmission. densoviruses can be highly pathogenic for insect pests and vectors, which have long stimulated their interest as biocontrol agents or genes vectors [ ] . they are considered today with a renewed interest as solutions to control harmful insects are lacking, which encourages efforts to understand their pathogenesis and their specificity. altogether our results suggest that pm glycans are crucial interacting components of the early jcdv pathogenesis. exploring their diversity and their complexity in insects can also provide important cues on the extend of the mechanisms that determine densovirus specificity. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , figure s . calcofluor increases s. frugiperda caterpillars susceptibility to densovirus oral infection. figure s . virus interaction with monosaccharides did not interfere with anti-capsid antibody recognition. figure s . replicates of survival curves of caterpillars infected by jcdv. figure s . jcdv oral infection induces midgut gene expression modulation. table s . annotation of pm proteins interacting with jcdv in vopba. table s . characteristics of dge libraries generated from caterpillars orally infected with jcdv. table s . characteristics of annotated tags and transcripts. table s . annotation of regulated intestinal transcripts following oral jcdv infection. the authors declare no conflict of interest. the mucosal barrier at a glance extracellular matrix in plants and animals: hooks and locks for viruses ruvoën-clouet, n. host-pathogen co-evolution and glycan interactions twenty-five years of structural parvovirology an essential receptor for adeno-associated virus infection binding site on the transferrin receptor for the parvovirus capsid and effects of altered affinity on cell uptake and infection aav transcytosis through barrier epithelia and endothelium secreted and transmembrane mucins inhibit gene transfer with aav more efficiently than aav detailed investigation of the sequential pathological changes in silkworm larvae infected with bombyx densovirus type densovirus crosses the insect midgut by transcytosis and disturbs the epithelial barrier function pathogenesis of junonia coenia densovirus in spodoptera frugiperda: a route of infection that leads to hypoxia diversity of small, single-stranded dna viruses of invertebrates and their chaotic evolutionary past a single amino acid substitution in the bombyx-specific mucin-like membrane protein causes resistance to bombyx mori densovirus new insights into peritrophic matrix synthesis, architecture, and function the roles of mucus-forming mucins, peritrophins and peritrophins with mucin domains in the insect midgut the origin and functions of the insect peritrophic membrane and peritrophic gel analysis of the ultrastructure and formation pattern of the peritrophic membrane in the cupreous chafer, anomala cuprea (coleoptera: scarabaeidae) insect digestive enzymes: properties, compartmentalization and function structural basis for the enhancement of virulence by viral spindles and their in vivo crystallization barriers to success: how baculoviruses establish efficient systemic infections disintegration of the peritrophic membrane of silkworm larvae due to spindles of an entomopoxvirus spatial distribution of orally administered viral fusolin protein in the insect midgut and possible synergism between fusolin and digestive proteases to disrupt the midgut peritrophic matrix multiple origins of viral capsid proteins from cellular ancestors a titration procedure of the junonia coenia densovirus and quantitation of transfection by its cloned genomic dna in four lepidopteran cell lines modeling the structure of the type i peritrophic matrix: characterization of a mamestra configurata intestinal mucin and a novel peritrophin containing chitin binding domains glycoprotein staining following electrophoresis on acrylamide gels identification of protein interactions by far western analysis enhanced detection of cns cell secretome in plasma protein-depleted cerebrospinal fluid parts per million mass accuracy on an orbitrap mass spectrometer via lock mass injection into a c-trap maxquant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification two genomes of highly polyphagous lepidopteran pests (spodoptera frugiperda, noctuidae) with different host-plant ranges blast go: a universal tool for annotation, visualization and analysis in functional genomics research deepsage-digital transcriptomics with high sensitivity, simple experimental protocol and multiplexing of samples whole transcriptome profiling of successful immune response to vibrio infections in the oyster crassostrea gigas by digital gene expression analysis serial analysis of gene expression establishment and analysis of a reference transcriptome for spodoptera frugiperda properties of the digestive enzymes and the permeability of the peritrophic membrane of spodoptera frugiperda (lepidoptera) larvae calcofluor white and congo red inhibit chitin microfibril assembly of poterioochromonas: evidence for a gap between polymerization and microfibril formation chitin synthesis inhibitors: old molecules and new developments calcofluor disrupts the midgut defense system in insects turnover, and functions of chitin in insects optical brightener m r destroys the peritrophic membrane of spodoptera exigua (lepidoptera: noctuidae) larvae functional implications of the structure of the murine parvovirus, minute virus of mice analysis of the cell and erythrocyte binding activities of the dimple and canyon regions of the canine parvovirus capsid diversity and functions of protein glycosylation in insects role of the phosphatidylinositol- -kinase/akt/target of rapamycin pathway during ambidensovirus infection of insect cells further characterization of aminopeptidase-n as a receptor for coronaviruses porcine aminopeptidase n is a functional receptor for the pedv coronavirus in vitro evidence supports membrane alanyl aminopeptidase n as a receptor for a plant virus in the pea aphid vector feline aminopeptidase n serves as a receptor for feline, canine, porcine, and human coronaviruses in serogroup i effect of wheat germ agglutinin on formation and structure of the peritrophic membrane in european corn borer (ostrinia nubilalis) larvae effect of optical brighteners on the insecticidal activity of a nucleopolyhedrovirus in three instars of spodoptera frugiperda the effect of diet on midgut and resulting changes in infectiousness of acmnpv baculovirus in the cabbage looper protoparvovirus cell entry host-selected amino acid changes at the sialic acid binding pocket of the parvovirus capsid modulate cell binding affinity and determine virulence host-specific parvovirus evolution in nature is recapitulated by in vitro adaptation to different carnivore species four amino acids of an insect densovirus capsid determine midgut tropism and virulence chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases microbiota-induced peritrophic matrix regulates midgut homeostasis and prevents systemic infection of malaria vector mosquitoes pgrp-ld mediates a. stephensi vector competency by regulating homeostasis of microbiota-induced peritrophic matrix synthesis intestinal microbiota promote enteric virus replication and systemic pathogenesis interactions between enteric bacteria and eukaryotic viruses impact the outcome of infection bacteria and bacterial envelope components enhance mammalian reovirus thermostability discovery of parvovirus-related sequences in an unexpected broad range of animals transmission ecology of canine parvovirus in a multi-host, multi-pathogen system how sticky should a virus be? the impact of virus binding and release on transmission fitness using influenza as an example viral delivery of dsrna for control of insect agricultural pests and vectors of human disease: prospects and challenges we are grateful to c. gibard, r. bousquet and g. clabot for their great help with insect rearing and the piq quarantine plateform from vectopole sud. we especially acknowledge e. jublanc and c. cazevieille for their skillful technical assistance and the imaging facility mri, member of the national france-bioimaging infrastructure supported by the french national research agency (anr- -inbs- , pia «investments for the future»). we warmly thank m. agbandje-mckenna from the cfg consortium (university of florida) for the glycan array and p. clair from the qphd platform (montpellier genomix). m. younes, and d. piquemal are also acknowledged for their support in the sage analysis. special thanks to p.a. lafon for his help for statistical analyses. l. pigeyre is a doctoral fellow of the french ministry for higher education and research/ephe. key: cord- -cpbbjm authors: georgakouli, kalliopi; fatouros, ioannis g.; draganidis, dimitrios; papanikolaou, konstantinos; tsimeas, panagiotis; deli, chariklia k.; jamurtas, athanasios z. title: exercise in glucose- -phosphate dehydrogenase deficiency: harmful or harmless? a narrative review date: - - journal: oxid med cell longev doi: . / / sha: doc_id: cord_uid: cpbbjm objectives: glucose- -phosphate dehydrogenase (g pd) deficiency, theoretically, renders red blood cells (rbc) susceptible to oxidative stress. g pd deficiency has also been found in other types of cells than rbc, such as leukocytes and myocytes, where an inefficient protection against oxidative stress may occur too. glutathione (gsh), a significant antioxidant molecule, levels are lower in g pd individuals, and theoretically, the probability of oxidative stress and haemolysis due to exercise in individuals with g pd deficiency is increased, whereas dietary supplementation with antioxidants may have beneficial effects on various aspects of this enzymopathy. methods: a search of the available literature was conducted using the keywords glucose- -phosphate dehydrogenase (g pd), deficiency, disease, exercise, muscle, antioxidant, vitamin, supplement, and supplementation. the search was limited to publications in english, conducted on humans, and published until august . after screening, only relevant articles were included. results: there is little evidence indicating that g pd deficiency can cause perturbations in redox status, haemolysis, and clinical symptoms such as fatigability and myoglobinuria, especially after intense exercise, compared to individuals with normal enzyme levels. conclusions: exercise could be used by g pd-deficient individuals as a tool to improve their quality of life. however, there is a lack of training studies, and assessment of the effects of regular and systematic exercise in g pd-deficient individuals is warranted. finally, since gsh levels are lower in g pd deficiency, it would be interesting to examine the effects of antioxidant or cysteine donor supplements on redox status after exercise in these individuals. glucose- -phosphate dehydrogenase (g pd) is an enzyme that helps cells to counterbalance oxidative stress, which is an imbalance between antioxidant defence (enzymatic and nonenzymatic) and the production of reactive oxygen and nitrogen species (rons) in favour of rons. among other functions, g pd is involved in the regeneration of reduced glutathione (gsh) from its oxidized form (gssg), a reaction catalysed by glutathione reductase [ ] . gsh is a major endogenous antioxidant that protects cells against oxidative damage in several ways. therefore, g pd-deficient activity, and consequently lower gsh levels, theoretically, renders cells susceptible to oxidative stress. g pd deficiency is a genetic disorder and the most common enzymopathy, affecting more than million people worldwide [ ] . more than g pd variants have been identified and are responsible for different levels of the enzyme activity. the world health organization (who) classifies these variants into five classes, according to the level of residual enzyme activity and the associated clinical manifestations [ ] . although in most cases g pd deficiency causes no symptoms, it may lead to diseases such as neonatal jaundice (hyperbilirubinemia) and acute or chronic haemolysis [ ] as red blood cells (rbc) are susceptible to oxidative stress and consequently destruction [ ] . frequency and severity of haemolysis vary depending on the g pd variant and exposure to various oxidative agents [ , , [ ] [ ] [ ] . such agents include oxidative drugs, chemicals, foods (fava beans), and even some vitamins (e.g., vitamin k). moreover, infection is thought to be another common cause of haemolytic anaemia in g pd-deficient individuals [ , ] . redox imbalance has been suggested to contribute to viral replication and virulence [ ] [ ] [ ] , and haemolytic episodes have been reported after various viral and bacterial infections in g pd-deficient individuals. there is a growing concern that g pd deficiency may be involved in the pathogenesis of various diseases mediated by oxidative stress. although g pd deficiency is well studied in rbc, there is a paucity of information about the activity of g pd in other cells such as white blood cells and myocytes. prevalence of diseases and associated complications such as neonatal jaundice, diabetic ketoacidosis, acute renal failure, cataract, and cancer have been claimed to be increased among g pd-deficient individuals, suggesting that g pd deficiency may be a risk factor for a wide range of pathological conditions. reports indicate that g pd deficiency is present in both rbc and the muscle and that a positive relationship exists between the two tissues in g pd activity [ ] . since the musculature is heavily utilized during exercise and the generation of reactive oxygen species can over exceed the theoretically reduced antioxidant capacity of the g pd-deficient individual, exercise could have harmful effects on various health aspects in these individuals. furthermore, since rbc are extremely active during intense exercise [ ] , g pddeficient rbc might not be able to withstand the exerciseinduced oxidative stress, and acute haemolytic anaemia could develop. on the other hand, it is well known that exercise confers multiple protective effects on the human body. thus, advising g pd-deficient individuals to refrain from participating in exercise without substantial evidence of the harmful effects of exercise to them does not seem prudent. the purpose of this review was to examine whether exercise has any positive or negative effects on individuals with g pd deficiency. we believe that the topic is very interesting since it relates with the most common enzymopathy and could generate interest for more research that could potentially lead to exercise guidelines being formulated for these individuals. a search of the available literature was conducted in the following databases: pubmed, scopus, and google scholar. the keywords used were glucose- -phosphate dehydrogenase (g pd), deficiency, disease, exercise, muscle, antioxidant, vitamin, supplement, and supplementation. the search was limited to publications in english, conducted on humans, and published until august . then, the authors screened the abstracts of the identified articles to remove those that were obviously irrelevant. studies that included exercise as a stimulus to generate perturbations on health, redox status, and performance indices were included. the literature search revealed a few case report studies on g pd deficiency and its association with muscle g pd activity and exercise ( table ) . two of these articles report cases of g pd individuals with clinical symptomatology following exercise [ , ] whereas the other one, which describes the case of a world class runner, did not report any adverse results. table summarizes results from five experimental studies that examined the effects of exercise on various haematological and/or blood redox status indices [ ] [ ] [ ] [ ] [ ] . none of these methodologically sound experimental studies report unwanted results following different modes and types of exercise. although g pd deficiency in rbc has gained a lot of attention and a number of studies have enlightened many aspects of this enzyme defect, research on the deficient activity of g pd in muscle cells is scarce. bresolin and colleagues [ ] investigated g pd activity in muscle biopsy specimens of four g pd-deficient patients with the mediterranean variant. all patients were found to have a very low g pd activity in the muscle and other tissues. also, g pd activity in myoblasts, myotubes, and skin fibroblasts of three patients was tested and found to be residual. these findings reinforced the view that g pd deficiency is present not only in rbc but also in several cell types, while muscle g pd deficiency was reported for the first time. concerning muscle g pd deficiency, a relation with muscular symptomatology after exercise was indicated; however, only one patient developed myoglobinuria after intense exercise, while another patient reported moderate exercise intolerance and recurrent episodes of myalgia and muscle fatigability. despite the heterogeneity and small number of subjects, a first concern on the involvement of muscle g pd deficiency in clinical symptoms after exercise was raised. adding to this concern is the well-documented significant perturbation on redox status following exercise and sports involvement. in theory, since g pd-deficient individuals are susceptible to oxidative stress and exercise results in excess of rons generation, g pd-deficient individuals would be more predisposed to rons cell destruction and therefore should avoid performing heavy physical exercise [ ] . during strenuous physical exercise, there is accelerated production of rons in exercising muscles, heart, and other tissues [ ] , which can cause damage to biomolecules (dna, protein, and lipids) and fatigue. a major antioxidant molecule of great importance for rbc and other animal cells is gsh. as mentioned before, g pd-deficient individuals have lower levels of gsh in rbc and other cells, and they may be predisposed to increased oxidative stress. as mentioned earlier, there are reports in the literature that come from case studies with no rigid research designs, which report that heavy exercise in g pd-deficient individuals caused clinical signs of haemolysis, muscle degeneration, myalgia, and myoglobinuria, which may be attributed to increased oxidative stress [ , , [ ] [ ] [ ] . however, more recent and better methodologically designed experimental studies indicate that different intensities of exercise do not cause oxidative stress or haemolysis in g pd-deficient individuals to a greater extent than expected for their nondeficient counterparts [ ] [ ] [ ] [ ] [ ] . individuals. the responsible mechanisms for the oxidative stress observed after exercise, especially that of high intensity, have been extensively discussed elsewhere [ ] . regarding g pd-deficient individuals, there is some concern about the possibility of negative effects of intense exercise on health due to their increased susceptibility to oxidative stress; however, research on this topic is scarce and does not support that concern. nikolaidis and colleagues [ ] examined oxidative stress after exercise until exhaustion in g pd-deficient individuals. nine g pd-deficient males and nine controls matched for age and maximal oxygen consumption (vo max) participated in two exhaustive treadmill exercise protocols of different duration. the first trial lasted for about min where vo max was determined, and the second trial lasted for about min ( min at - % vo max and then at % till exhaustion) with a -day washout period between the two trials. gsh, gssg, haematocrit (hct), and haemoglobin (hb) were significantly lower in the g pd-deficient group than the control group at the baseline. after the two exercise protocols, hct and hb levels did not change, gsh decreased, and thiobarbituric acid reactive substances (tbars), protein carbonyls (pc), catalase, and total antioxidant capacity (tac) increased in both groups. gssg increased and the gsh/gssg ratio decreased after the long-duration trial in both groups. the results of this study showed that exercise until exhaustion did not lead to higher oxidative stress and increased haemoglobin oxidation in g pd-deficient males in comparison to their normal counterparts, despite the lower baseline gsh levels in g pd-deficient males. the effect of moderate intensity exercise on markers of oxidative stress and haemoglobin oxidation in g pddeficient males was studied by jamurtas and colleagues [ ] . nine males with g pd deficiency and nine agematched control males run at approximately % of their maximum heart rate (mhr) for min. gsh and hct were significantly lower in the g pd-deficient group compared to the control group at the baseline. no changes in gsh, gssg, gsh/gssg, or lipid peroxides were found for any group after exercise. also, heinz body formation (an index of haemolysis) was not detected before or after exercise in either group. the results from this study indicate that moderate intensity exercise at approximately % mhr may not cause oxidative stress or haemolytic anaemia in g pddeficient individuals. in another study from the same laboratory, theodorou and colleagues [ ] investigated the effect of high-intensity muscle-damaging exercise on markers of muscle function, blood redox status, and haemolysis in g pd-deficient males. nine g pd-deficient males and nine control males matched for age and maximal isometric torque performed an isokinetic eccentric contraction session of the knee extensors of both legs. gsh and gssg were lower in the g pddeficient group compared to the control group at the baseline. immediately and till days after exercise, changes in indices of muscle function, redox status, and haemolysis in the g pd-deficient group were similar to those in the control group. gsh and gssg were always lower in the g pddeficient group. these results suggest that high-intensity muscle-damaging exercise may not cause different fluctuation pattern on muscle function, blood redox status, and haemolysis in g pd-deficient males in comparison to their normal counterparts. finally, an experimental study reported that min of moderate intensity ( - % of vo max) did not perturbate redox status indices (tbars, gsh, and gssg) in a g pd male individual [ ] . however, it has to be mentioned that this individual was well-trained since his vo max was more than ml/kg/min. therefore, the limited literature of experimental studies indicates that exercise does not seem to perturbate the redox status of g pd-deficient individuals to a greater extent than individuals with normal enzyme levels. furthermore, even though earlier case studies indicated an increased probability for increased haemolysis, more recent experimental studies do not support such findings. even though it seems that there is less chance for haemolysis with intense exercise, it is unknown whether the assumed compromised ability of the immune system of the exercising g pd-deficient individual will lead to increased predisposition for upper respiratory tract infection under stressful conditions. also, it has to be mentioned here that there is a complete lack of studies that have examined the exercise training effects on redox and health status in general on g pd-deficient individuals. finally, other factors that should be taken into consideration are the coexistence of another red blood cell defect (e.g., sickle cell trait) [ ] , and the g pd variant of the studied subjects as different variants cause different levels of enzyme activity in various types of cells. all these factors could potentially render rbc more susceptible to oxidative stress, resulting in severe haemolysis after intense exercise. individuals. exposure of g pd-deficient individuals to oxidative stress, such as consumption of fava beans, use of certain drugs, and maybe infections, can lead to haemolytic anaemia [ ] . drug-induced haemolysis is thought to be the most common adverse clinical consequence of g pd deficiency. for that reason, g pd-deficient individuals are advised to avoid certain drugs. theoretically, the lower levels of gsh in g pd-deficient individuals could make them more susceptible to increased oxidative damage due to lower inherent antioxidant activity. regarding the use of dietary supplements, only a few studies have examined their effects on various health aspects in this population. a systematic review by lee and colleagues [ ] concluded that the use of herbal or dietary supplements at therapeutic doses is not likely to induce haemolysis in g pd-deficient individuals. from the dietary or herbal substances that were assessed, only henna (lawsonia inermis) was found to increase the possibility for haemolysis [ ] . in addition to that, dietary supplements may help in the prevention or improvement of haemolytic anaemia, as well as other adverse effects of oxidative stress. cysteine and other sulphur-containing compounds may result in increased levels of gsh; therefore, it could be hypothesized that supplementation with such compounds would restore gsh redox status in g pd-deficient individuals, making their cells less susceptible to oxidative stress. four weeks of supplementation with α-lipoic acid, a sulphurcontaining compound and potent antioxidant, has shown to improve some indices of redox status in g pd-deficient individuals with no signs of haemolytic anaemia [ ] . moreover, vitamin e may protect the membranes of rbc from oxidation and consequent haemolysis. dietary supplementation of iu of vitamin e per day for - months resulted in improvement of indices of chronic haemolysis in g pd-deficient individuals [ , ] . since redox status in g pd deficiency may be disturbed, there is a potential application of antioxidant dietary supplementation in physically active individuals with this deficiency. to the best of our knowledge, no study has looked into the effects of supplementation on health aspects in g pd-deficient individuals after exercise. one study by tsakiris and colleagues [ ] investigated the effect of exercise training and alpha-tocopherol supplementation on g pd activity in erythrocytes of non-g pd-deficient individuals. ten basketball players participated in a game (forced training) before and days after alpha-tocopherol supplementation, with blood samples obtained before and immediately after each game. alpha-tocopherol supplementation resulted in increased pre-and postgame total antioxidant status (tas). postgame g pd activity was also increased after alpha-tocopherol supplementation compared to postgame g pd activity at the baseline. these results indicate that alpha-tocopherol supplementation could potentially lead to increased antioxidant capacity in g pd-deficient individuals after a period of strenuous training. further research is needed to assess whether supplementation with antioxidants or cysteine donors would increase gsh levels and therefore help cells counterbalance exercise-induced oxidative stress, an effect that could benefit g pd-deficient individuals that participate in exercise. in conclusion, the limited available experimental data indicate that g pd-deficient individuals may safely participate in exercise of various intensities. this conclusion stems from results from full-scale controlled research trials that employed the most rigorous and demanding types of exercise, using a large number of parameters that showed no harmful effects after several modes and intensities of exercise on g pddeficient individuals. this is of great importance if someone takes into account that exercise has pleiotropic positive effects on health, and g pd deficiency is the most common enzymopathy. excluding individuals with this enzymopathy from participating in exercise a priori or based on some case studies does not seem a prudent thing to do. there are some reports of clinical signs of haemolysis, muscle degeneration, myalgia, and myoglobinuria after heavy exercise in g pd-deficient individuals that come from case studies, and they should be viewed with caution when interpreting them. deficient g pd activity in skeletal muscles and other tissues than rbc observed in some individuals may play a role in this phenomenon. therefore, future studies should examine the responses to exercise of individuals with different levels of g pd activity in various tissues. finally, since gsh levels are lower in g pd deficiency, it would be interesting to examine the effects of antioxidant or cysteine donor supplements on redox status after acute exercise or chronic training in these individuals. glucose- -phosphate dehydrogenase deficiency glucose- -phosphate dehydrogenase deficiency diagnosis and management of g pd deficiency effect of age on the enzyme activity in erythrocytes molecular cloning and nucleotide sequence of cdna for human glucose- -phosphate dehydrogenase variant a(-) variants of glucose- -phosphate dehydrogenase are due to missense mutations spread throughout the coding 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and glucose- -phosphate dehydrogenase deficiency glucose -phosphate dehydrogenase deficiency in an elite long-distance runner exerciseinduced oxidative stress in g pd-deficient individuals effect of exercise on oxidative stress in individuals with glucose- -phosphate dehydrogenase deficiency comparison between glucose- -phosphate dehydrogenasedeficient and normal individuals after eccentric exercise cell-derived microparticles after exercise in individuals with g pd viangchan exercise-induced oxidative damage in a person with glucose- -phosphate dehydrogenase deficiency muscle glucose -phosphate dehydrogenase (g pd) deficiency and oxidant stress during physical exercise muscle g pd deficiency glucose- -phosphate dehydrogenase lodi^ c: a study on its expression in blood cells and muscle muscular g pd deficiency: a definite clinical syndrome? exerciseinduced oxidative stress in humans: cause and consequences adverse effects of herbal or dietary supplements in g pd deficiency: a systematic review Α-lipoic acid supplementation up-regulates antioxidant capacity in adults with g pd deficiency the vitamin e status among glucose- phosphate dehydrogenase deficient patients and effectiveness of oral vitamin e reduced chronic hemolysis during high-dose vitamin e administration in mediterranean-type glucose- -phosphate dehydrogenase deficiency α-tocopherol supplementation restores the reduction of erythrocyte glucose- -phosphate dehydrogenase activity induced by forced training the authors declare that there is no conflict of interest regarding the publication of this paper. key: cord- - ta fg authors: van norman, gail a. title: expanding patient access to investigational new drugs: overview of intermediate and widespread treatment investigational new drugs, and emergency authorization in public health emergencies date: - - journal: jacc basic transl sci doi: . /j.jacbts. . . sha: doc_id: cord_uid: ta fg individual patients with life-threatening or severely debilitating diseases can petition the u.s. food and drug administration (fda) through their physicians to have expanded access (ea) to drugs that are in clinical trials but have not reached full fda approval (the “single-patient” investigational new drug [ind] application). additionally, recent state and federal laws—so-called “right to try legislation”—allow patients to approach drug companies directly for access prior to fda approval. while these pathways provide potential access for individual patients to investigational drugs, different ea pathways permit entire groups of certain patients to access investigational drugs prior to fda approval. this review focuses on special categories of ea inds intended for multiple patients—the intermediate-group ind and the widespread-treatment ind—as well as emergency authorization for use of investigational drugs and biological products (e.g., vaccines) in public health emergencies. for patients with life-threatening or severely debilitating disease, the wait for approval is simply too long, and can both abolish hope for those who diseases will be quickly fatal, and lead to sustained or even permanent disability for those whose diseases linger but are without effective proven therapies. spurred by patient advocacy during the early days of the acquired immunodeficiency syndrome (aids) epidemic in the late s, and facilitated by subsequent legislative efforts over the next years, regulatory initiatives permit the fda to release drugs for use in individual patients through expanded access (ea) inds ( , ) , in many cases allowing emergency treatment with nonapproved drugs within hours of application, and nonemergency treatment within an average of days ( ) . further, most states have enacted so-called "right to try" legislation, permitting "compassionate use" of investigational drugs by individual patients through applications directly to the manufacturer ( ) . it should be noted that although the terms "compassionate use" or "preapproval access" are often used informally to refer to the use of an investigational drug to treat a patient outside of a clinical trial, these terms are not defined or described in fda regulations, which simply refer to expanded access to investigational drugs. the call for ea is not limited to individual patients. advocacy organizations have pressed for groups of patients with rare and/or "orphan" diseases, for example, to be able to access promising new therapies prior to their approval. indeed, social media is increasingly becoming a consumer/patient advocacy tool for implementing fda regulatory changes and promoting access to investigational therapeutics ( ) . in addition, once a drug has completed phase testing and is awaiting approval, patients who have benefited from in-trial treatments may want continued therapy, and such use requires some form of "bridging approval" from the fda to allow potentially large groups of patients to continue treatment while final fda approval is pending. a previous review discussed individual patient emergency and nonemergency access to investigational drugs ( ) . this review will focus on fda ea for intermediate-sized groups of patients (the "intermediate-sized ind") and ea for entire classes of patients (the "widespread treatment use" ind), as well as emergency release of investigational drugs and biologics for use in public health emergencies. releasing investigational new drugs to individual patients who are facing certain death or disability seems to be a relatively uncomplicated decision, but allowing ea to entire groups of patients for treatment with an investigational new drug presents more complex regulatory, logistical, and ethical challenges for scientists, commercial entities, and the fda. the current regulatory process from ind filing to drug approval has evolved and includes not only the fda's historical primary mission of ensuring patient safety, but also, since the latter half of the th century, the newer mission of ensuring that marketed drugs are actually efficacious for their advertised/approved use. ea for a single patient may not present much of a challenge to the assertion that a drug's benefits outweigh the risks, because as presumably the patient requesting compassionate use faces an otherwise dismal clinical future, taking even significant risks with a new drug still presents potential benefits to a patient without other options. early in a drug's regulatory pathway, however, it is not usually possible to ensure that a drug has a reasonable risk/benefit ratio for all patients, including those in the early stages of disease. drug companies face bigger issues when the seeker of ea is a group of patients or an entire class of patients. before marketing, manufacture of the drug for clinical studies is nearly an "all cost" proposition for the commercial entity; the drug cannot be marketed to cover its costs. thus, companies generally only manufacture sufficient quantities (plus a small margin) to cover the requirements of clinical studies, rather than devote resources to manufacturing large quantities of a drug which has a < % chance of ever making it to market ( , ) . the fda approval process begins with the filing of an investigational new drug (ind). making the drug available to groups or classes of patients who might then deplete the supply of drug for clinical studies could compromise the very research that would more completely disclose a drug's risks and benefits; thus, it could possibly impede full market approval that would make the drug more widely available. companies have also expressed concern about how data from such "compassionate use" may be applied in the approval process. for cmv infection ( ) . later studies also showed that ganciclovir patients were living longer ( ). the fda refused approval of ganciclovir for treatment of cmv retinitis, because they had no animal studies for that use, nor significant human placebocontrolled trials on which to base a marketing application. many questioned whether the use of ganciclovir was wise, or safe ( , ) . but, because of ganciclovir's known efficacy, it became paradoxically impossible to carry out human controlled trials, because such trials are only ethically justifiable if investigators are honestly uncertain about whether net positive benefits over placebo exists ( ) . furthermore, neither patients nor doctors were willing to risk assignment to placebo and further loss of eyesight after the results were published. syntex then sought approval to study ganciclovir for treatment of cmv colitis, knowing that once marketing approval of the drug was obtained, fda rules would allow "off-label" use for retinitis ( ) . at this time, ea of nonapproved drugs for treatment of more than patient at a time is achievable only through the fda, in contrast with access for individual patients, which technically can be legally obtained without the fda by applying to the manufacturer directly ( ) . as with individual ea inds, specific conditions for group patient access apply: ) the patients must have a serious or immediately life-threatening disease or condition with no comparable therapy or satisfactory alternative therapy; ) the potential benefit must justify the potential risks of the treatment; and ) providing the treatment must not compromise or interfere with the ongoing fda drug development program, such as by critically depleting a limited supply of investigational drug that is also needed for an ongoing study or a future study that is in the planning stages ( ). an "immediately life-threatening condition or disease" is defined by the fda as "a stage of disease in which there is reasonable likelihood that death will occur within a matter of months or in which premature death is likely without early treatment." a serious disease or condition is defined as being "associated with morbidity that has substantial impact on day-to-day functioning." furthermore, while short-lived or self-limited morbidity will usually not be a sufficient qualifying condition, the morbidity "need not be irreversible, provided it is persistent or recurrent." the fda states that whether a condition is serious or not "is a matter of clinical judgment, based on its impact on such factors as survival, day-to-day functioning, or the likelihood that the disease, if left untreated, will progress from a less severe condition to a more serious one" ( ) . and other animal species such as cats, armadillos, guinea pigs, swine and ferrets in which thalidomide had been tested, teratogenic effects had been induced only occasionally" ( ) . in fact, when human birth defects began to appear in the offspring of women who had ingested thalidomide during pregnancy as a sedative and to treat nausea, researchers pointed out that thalidomide had failed to demonstrate teratogenicity in rats, and at first insisted that thalidomide could not be the culprit. in germany, where the drug was first developed, thalidomide was held to be so safe that no prescription was required for its use, it was advertised for use in pregnant women ( ) , and the drug company distributed free samples to its factory employees ( , ) . given the most severe rating for drugs that contribute to fetal deformities, and for drugs whose risks prioritizing those who should receive treatment, such as women and children ( ) . the fact that the treatment was made available to u.s. citizens and not to africans, who comprised most of its victims, engendered anger over the social justice of such decisions, providing, as enserink ( ) points out, a tragic validation to the satirical yet somewhat prophetic paper that had appeared in the onion only weeks before titled "experts: ebola vaccine at least white people away" ( ) . as of now, products have been approved under the animal rule, of which were issued quickly after the guidance was published ( table there is a reasonably well-understood pathophysiological mechanism of the toxicity of the toxic substance and its prevention or substantial reduction by the product. the effect is demonstrated in more than animal species expected to react with a response predictive for humans, unless the effect is demonstrated in a single animal species that represents a sufficiently well-characterized animal model for predicting the response in humans. the animal study endpoint is clearly related to the desired benefit in humans, generally the enhancement of survival or prevention of major morbidity. the data or information on the kinetics and pharmacodynamics of the product or other relevant data or information, in animals and humans, allows selection of an effective dose in humans. drugs, devices, and the fda: part understanding fda regulatory requirements for investigational new drug applications for sponsor-investigators policy and procedures: office of new drugs: ind clinical holds expanded access (compassionate use) early access programs: benefits, challenges and key considerations for successful implementation expanding patient access to investigational drugs: single patient inds and the "right to try going "social" to access experimental and potentially life-saving treatment: an assessment of the policy and on-line patient advocacy environment for expanded access compassionate use: a story of ethics and science in the development of a new drug -propoxymethyl)guanine in patients with aids and other immunodeficiencies oral ganciclovir for the prevention of cytomegalovirus disease in persons with aids blinded by science: dhpg and the conflict between "clean data" and humane health care (abstract th.g.p. ). paper presented at: int conf aids medicine: drugs from the underground the question of clinical equipoise and patients' best interests off-label" and investigational use of marketed drugs, biologicals and medical devices-information sheet expanded access to investigational drugs for treatment usequestions and answers; guidance for industry food and drug administration. food and drug administration amendments act (faaa) of administrationamendmentsactof /default.htm fda basics webinar: a brief overview of risk evaluation and mitigation strategies (rems) drugs as teratogens the death and afterlife of thalidomide. retro report. the new york times changing the face of medicine use of thalidomide in leprosy years after thalidomide: why regulation matters kefauver-harris amendments revolutionized drug development thalidomide approved to treat leprosy, with other uses seen. the new york times a comprehensive program for controlling and monitoring access to thalidomide the embryo project encyclopedia. us regulatory response to thalidomide project bioshield act of project bioshield: what is it, why is it needed, and its accomplishments so far regulatory information: emergency use authorization of medical products summary of process for eua issuance considerations for use of investigational drugs in public health emergencies food and drug administration how two u.s. patients changed the debate about using untested ebola drugs world health organization. ethical considerations for use of unregistered interventions for ebola virus diseases: report of an advisory panel to who. who, geneva switzerland _eng.pdf?ua¼ . accessed experts: ebola vaccine at least white people away fda product development under the animal rule: guidance for industry working with the u.s. food and drug administration to obtain approval of products under the animal rule food and drug administration ebola virus disease survivors: clinical and immunologic follow-up prevail iv: double-blind, randomized two-phase, placebo-controlled phase ii trial of gs- to assess the antiviral activity, longer-term clearance of ebola virus and safety in male ebola survivors with evidence of ebola virus persistence expanded access (compassionate use): ind submissions medicine, university of washington, th avenue west, seattle, washington . e-mail: lbsparrow@ yahoo.com. key: cord- -iq e qp authors: otxoa-de-amezaga, amaia; miró-mur, francesc; pedragosa, jordi; gallizioli, mattia; justicia, carles; gaja-capdevila, núria; ruíz-jaen, francisca; salas-perdomo, angélica; bosch, anna; calvo, maria; márquez-kisinousky, leonardo; denes, adam; gunzer, matthias; planas, anna m. title: microglial cell loss after ischemic stroke favors brain neutrophil accumulation date: - - journal: acta neuropathol doi: . /s - - - sha: doc_id: cord_uid: iq e qp stroke attracts neutrophils to the injured brain tissue where they can damage the integrity of the blood–brain barrier and exacerbate the lesion. however, the mechanisms involved in neutrophil transmigration, location and accumulation in the ischemic brain are not fully elucidated. neutrophils can reach the perivascular spaces of brain vessels after crossing the endothelial cell layer and endothelial basal lamina of post-capillary venules, or migrating from the leptomeninges following pial vessel extravasation and/or a suggested translocation from the skull bone marrow. based on previous observations of microglia phagocytosing neutrophils recruited to the ischemic brain lesion, we hypothesized that microglial cells might control neutrophil accumulation in the injured brain. we studied a model of permanent occlusion of the middle cerebral artery in mice, including microglia- and neutrophil-reporter mice. using various in vitro and in vivo strategies to impair microglial function or to eliminate microglia by targeting colony stimulating factor receptor (csf r), this study demonstrates that microglial phagocytosis of neutrophils has fundamental consequences for the ischemic tissue. we found that reactive microglia engulf neutrophils at the periphery of the ischemic lesion, whereas local microglial cell loss and dystrophy occurring in the ischemic core are associated with the accumulation of neutrophils first in perivascular spaces and later in the parenchyma. accordingly, microglia depletion by long-term treatment with a csf r inhibitor increased the numbers of neutrophils and enlarged the ischemic lesion. hence, microglial phagocytic function sets a critical line of defense against the vascular and tissue damaging capacity of neutrophils in brain ischemia. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. neutrophil infiltration under conditions of sterile inflammation can contribute to tissue injury. neutrophils are transiently detected in the brain after stroke since they are rapidly attracted to the injured brain peaking between and days post-ischemia [ , , , ] . compelling evidence suggests that neutrophils are contributors to tissue damage after ischemic stroke [ , , , , ] , in spite of the fact that diverse experimental strategies inhibiting neutrophil activation or depleting neutrophils provided conflicting results [ , ] . likely, the differences between experimental studies depend on the efficacy and potential side effects of the diverse neutrophil depleting or inhibiting strategies, status of capillary reperfusion, lesion severity, and integrity of the blood-brain barrier (bbb). moreover, several aspects of neutrophil infiltration after acute ischemic brain damage remain controversial. neutrophils accumulate in perivascular spaces in murine and human strokes [ , ] . the presence of neutrophils in the brain parenchyma has been reported in rodent models of permanent ischemia [ , , ] , but it is more controversial in experimental models of transient ischemia [ , ] . several studies reported the presence of neutrophils in the brain parenchyma in postmortem samples of patients deceased between day and [ ] , or days after stroke onset but not at other time points [ , ] . in other studies, neutrophils were not detected in the brain parenchyma of stroke patients [ ] . therefore, the molecular determinants underlying perivascular neutrophil accumulation and the conditions facilitating the potential access of neutrophils to the brain parenchyma need further clarification. the observation that microglia phagocytose neutrophils in the ischemic brain [ ] [ ] [ ] led us to hypothesize that microglia function may be critical to explain neutrophil accumulation in the injured brain tissue. microglial cells react to brain ischemia in different ways depending on the regional location and temporal course of the lesion. microglial cells are vulnerable to ischemia and previous reports showed death of microglia after oxygen and glucose deprivation in tissue slices [ ] and cell cultures [ , ] . in addition, microglial reduction has been reported after transient mcao [ ] , and microglial dysfunction and loss was detected in classical neuropathological studies of brain ischemia in rodents and primates [ , ] . classical histopathological studies have shown long-lasting microgliosis surrounding the infarction several days after ischemic stroke onset. however, the progression of this reaction from the very acute phase of stroke is less precisely determined mainly due to the fact that microglia and infiltrating macrophages show many common features and markers leading to the frequent terminology of microglia/macrophages to describe the mononuclear myeloid cell reaction that follows stroke. microglia have a unique transcriptomic signature distinguishable from that of macrophages or monocytes [ , ] . therefore, reactive microglia and infiltrating macrophages likely play different functions in the injured brain tissue. current developments allow the distinction between these cells with antibodies against more specific microglia markers [ , ] , availability of fluorescent reporter mice [ ] , or transfer of fluorescent reporter leukocytes [ ] . by exploiting some of these novel experimental possibilities, we investigated the neutrophil-microglia crosstalk after brain ischemia. the results show that microglial cells effectively remove brain-infiltrating neutrophils, hence microglia dysfunction or death is associated with neutrophil accumulation into the injured brain tissue. we used adult male mice on the c bl/ background. mice expressing tamoxifen-inducible cre recombinase under the direction of the cx cr promoter in the mononuclear phagocyte system (cx cr cre/ert ) [ ] (# jax ® mice) were crossed with either ai mice harboring a loxp-flanked stop cassette that prevents transcription of the red fluorescent protein tdtomato (tdt) (b .cg-gt(rosa) sortm (cag-tdtomato)hze/j (# jax ® mice) [ ] , or colony stimulating factor receptor (csf r) +/flox mice (b .cg-csf r tm jwp /j, # jax ® mice). we used heterozygous catchup ivm mice expressing tdt in ly g +/− neutrophils [ ] . homozygous catchup ivm (ly g −/− ) mice were crossed with cx cr gfp/gfp mice to obtain double heterozygous mice with red fluorescent neutrophils and green fluorescent microglia [ , ] . we also obtained cells from dsred mice constitutively expressing the red fluorescent protein dsred under the control of the actin promoter [ ] . wildtype mice were obtained from a commercial source (janvier, france). mice were maintained in the animal house of the school of medicine of the university of barcelona under controlled spf conditions. animal work was conducted with the approval of the ethical committee of the university of barcelona (ceea) and the direcció general de polítiques ambientals i medi natural, departament de territori i sostenibilitat de la generalitat de catalunya. studies complied with the "principles of laboratory animal care" (nih publication no. - , revised ), and the spanish national law (real decreto / ). the brains of six patients suffering from acute ischemic stroke who died between and days after stroke onset at the stroke unit of the hospital clinic of barcelona were used after obtaining written consent from their relatives or legal representatives for tissue removal after death at the neurological tissue bank of the biobank-hospital clinic-institut d'investigacions biomèdiques august pi i sunyer (idibaps). the ethics committee of this hospital approved the study. online resource shows a summary of patient characteristics. the elapsed time from death to autopsy was - h. an expert neuropathologist dissected the ischemic core, periphery, and a portion of non-ischemic tissue (control) obtained from a region distant to infarction, as described [ ] . samples were embedded in oct and immediately frozen in liquid nitrogen for sectioning at µm in a cryostat. the bone marrow of transgenic dsred mice [ ] was used to generate chimeric mice, as reported [ ] . in brief, recipient adult ( -month old) wild-type mice received three intraperitoneal injections of the chemotherapeutic agent busulfan ( mg/g body weight) , and days prior to transfer via the tail vein of five million bone marrow cells from dsred donor mice. mice were used weeks after grafting and reconstitution was assessed by flow cytometry analysis. to impair microglial function, mice received a daily oral administration by gavage of the csf r inhibitor gw [ ] ( mg/kg body weight in a volume of . ml) (#s , selleckchem) for days, which is a dosing regimen that does not challenge microglial survival [ ] . treatment controls received the same volume of the vehicle ( . % hydroxypropylcellulose, . % tween- ). treatment started h prior to induction of ischemia, it was randomly allocated, and was administered in a blinded fashion. for microglia depletion, mice received the csf r inhibitor plx (plexxikon) following previously reported protocols [ , , ] . the inhibitor was mixed into ain- a standard chow at ppm (brogaarden, denmark). mice ( -week-old) received the diet ad libitum for weeks prior to induction of ischemia and the diet was maintained until the mice were killed. treatment controls received ain- a diet for the same period of time. both diets were given in parallel in groups of five animals per cage. surgery was carried out under isoflurane anaesthesia and mice received analgesia (buprenorphine, µl of a . mg/ml solution, via s.c.). permanent occlusion of the middle cerebral artery (mcao) was induced by coagulation of the distal portion of the right mca together with ligation of the ipsilateral common carotid artery. this experimental model induces a focal cortical lesion in the ipsilateral hemisphere. a subset of mice receiving the above diets (control or plx ) was used to study the volume of the lesion day after induction of ischemia by t w mri in a . t biospec / horizontal animal scanner (bruker biospin, ettlingen, germany), as reported [ ] . sample size was calculated using g*power . software (university of dusseldorf) with an alpha level of . , statistical power of . , and estimating a size effect of . based on sd of previous results from our laboratory and published data on the effect of microglia depletion on infarct volume in other stroke models [ ] . one mouse died (control diet), and one mouse was excluded (plx diet) due to surgical problems. bromodeoxyuridine (brdu) ( mg/ml) (# , bs pharmingen) was daily injected ( μl) via i.p. into mice starting day after mcao until day . one-hour after the last brdu administration mice were killed and processed for immunofluorescence. brdu was detected in brain tissue sections using a rat monoclonal fitc-anti-brdu antibody ( : , #ab , abcam, cambridge, uk) [ ] . mouse blood and brain tissue were processed for flow cytometry as described [ ] . fc receptors were blocked by previous incubation for min with cd /cd (clone . g , bd pharmingen) in facs buffer (pbs, mm edta, % fbs) at °c. live/dead aqua cell stain (molecular probe, invitrogen) was used to determine the viability of cells. cells were incubated with the following mix of microglia cells from adult mice ( - weeks old) were isolated and cultured using immunomagnetic separation (miltenyi biotec, germany). mice were perfused via the left ventricle with ml of cold saline and collected in hanks' balanced salt solution (hbss) buffer without calcium/magnesium (# - ; life technologies). the brain tissue was enzymatically dissociated using the neural tissue dissociation kit-p (# - - ; miltenyi biotec). the gentlemacs™ dissociator with heaters (# - - ; miltenyi biotec) was used for mechanical dissociation steps during min at °c. the digested tissue was filtered ( µm) with hbss buffer with calcium and magnesium (# - ; life technologies) and prepared for myelin removal process (myelin removal beads ii, # - - ; miltenyi biotec). then, cells were magnetically labeled with cd b microbeads (# - - ; miltenyi biotec) diluted in pbs supplemented with . % bsa for min in the dark in the refrigerator ( - °c) . cd b + cells were collected using magnetic field columns (miltenyi biotec). cell suspensions ( μl) were then plated in complete medium consisting of dmem medium (# ; gibco-brl) supplemented with % fetal bovine serum (fbs; gibco-brl) containing u/ml penicillin and μg/ml streptomycin (# ; gibco-brl) added as a drop in the middle of each well of a poly-l-lysine (#p ; sigma) pre-coated -well plate (µ-slide well, ibidi # ). cells were incubated for min at °c and then µl of complete medium were carefully added to each well. twenty-four hours later, we replaced % of complete medium, and we did a full medium change at day . the cells were maintained at °c in a humidified atmosphere of % co for div. we obtained human microglial cells from the ischemic tissue of one patient deceased days after fatal stroke. fresh brain tissue (about mg) was harvested at autopsy ( h after death) and was placed in a falcon tube with sterile cold rpmi medium (# - , gibco). visible meninges were removed, the tissue was cut in small pieces using a scalpel and incubated in a . % trypsin-edta solution in pbs at rt for min. then, dmem/f (# ; gibco-brl) with % fbs and dnase i ( units/ml) was added ( : ), the tissue was disaggregated, centrifuged for min at ×g and the pellet was re-suspended in ml dmem/f supplemented with % fbs, % l-cell conditioned medium obtained from the l cell line, and u/ml penicillin/ μg/ml streptomycin (# ; gibco-brl). cells were seeded in poly-l-lysine coated t flasks, incubated in % co at °c and allowed to adhere. culture medium was changed twice a week and at div the cells were scrapped and seeded in a -well plate (µ-slide well, ibidi # ) previously coated o/n with poly-l-lysine. a time-lapse microscopy study was initiated h later after addition of fresh bone marrow neutrophils. afterwards, we fixed the cells for an immunofluorescence study with antibodies against the purinergic receptor p y, g-protein coupled, (p ry ) ( : , #as a, anaspec). neutrophils were obtained from the bone marrow of adult ( - weeks old) mice. the bone marrow was flushed using a -gauge needle with rpmi (# - , gibco) supplemented with % fbs onto a ml falcon tube through a -μm cell strainer. cells were centrifuged at ×g for min. the supernatant was discarded and cells were then incubated for min with an erythrocyte lysis solution ( mm nh cl, mm khco , . mm edta). after washing with cold pbs supplemented with % fbs, cells were incubated at °c for min with a mix of fcblock ( / ; clone . g ; bd pharmingen; bd bioscience), and the antibody ly g (clone a , fitc; bd pharmingen) with µl/ cells. cells were washed with pbs- . % bsa, and were then incubated with anti-fitc microbeads (# - - , miltenyi biotec) for min at °c with µl microbeads/ cells. after washing, the fraction of positive ly g cells was magnetically collected and prepared for immediate use or cells were frozen in fbs serum with % of dmso until the day of the experiment. human neutrophils were isolated from the blood by density gradient centrifugation. human and mouse neutrophils were stained with celltracker™ green cmfda (#c ; ther-mofisher scientific). fig. localization of microglia and infiltrating leukocytes. we generated chimeric mice by administering dsred fluorescent bone marrow cells to -month old wild-type receptor mice (n = ). after months, we induced ischemia and days later we studied the brain by immunofluorescence (n = ) (a, b) and flow cytometry (n = ) (c, e). a cd hi cd b hi cells infiltrating the ipsilateral hemisphere are mostly dsred + whereas cd dim cd b dim microglial cells are dsred − . b flow cytometry shows an increase in infiltrating dsred leukocytes (cd b hi cd hi ) (mann-whitney test, **p = . ). c immunostaining of astrocytes (gfap, green) showed the presence of dsred cells at the border and core of the lesion separated from the peripheral area that shows a prominent astroglial reaction. d microglial cells were stained with an antibody against p ry (green), which did not co-localize with dsred + leukocytes. the morphology of the microglia in the different regions is illustrated with representative images. cell nuclei were stained with to-pro (blue). e p ry + microglial cell at the border of the lesion nearby dsred + infiltrating leukocytes. f schematic representation of the distribution of microglia (green) and infiltrating leukocytes (red) in the different regions of the ischemic hemisphere. scale bar c µm; d, e µm . c-f analysis of microglial morphology in the core and periphery of the ipsilateral cortex and the contralateral cortex (contral.) (n = - cells per region of different mice represented by the different colors) using imagej tools. g, h imaris analysis of d-reconstructions of the above cells (b). i counting the number of microglial cells per area showed an increased microglial density in the periphery and a decrease in the core (n = - fields in different mice). statistical analyses in c-i were carried out with the kruskal-wallis test followed by the dunn's test. **p < . and ***p < . vs. contralateral microglia; && p < . and &&& p < . vs. peripheral microglia. j flow cytometry after dissecting out the core and periphery regions of the ipsilateral hemisphere and mirror regions of the contralateral hemisphere and days after mcao in an independent group of mice (n = mice per time point). for comparative purposes the number of microglial cells (cd low cd b + ) is expressed by mg of brain tissue. data analysis was conducted with two-way anova by region (p < . ) and time point (p = . ) followed by the bonferroni test. the number of microglia was higher in the periphery than the core of infarction day ( && p < . ) and days ( &&& p < . ) after mcao. furthermore, the number of microglia decreased in the core of infarction versus the corresponding contralateral region day (**p < . ) and days (*p < . ) postischemia. in the periphery, the number of microglia cells increased in the ipsilateral versus the corresponding contralateral hemisphere at day ( # p < . ). scale bar μm isolated and stained neutrophils ( , cells/ml) were added to the adult microglia cultures at div. automated multiposition live cell imaging was carried out using a leica tcs sp confocal microscope (leica microsystems, heidelberg, germany) equipped with adaptive focus control to keep the specimen in focus and an incubation system with temperature and co control. cells were subjected to a timelapse study while maintained at °c in a humidified atmosphere of % co . all images ( - z sections) were acquired using a apo × (numerical aperture . ) glycerol immersion objective lens, pinhole set at . airy units. images of cmfda and dsred were acquired sequentially line by line using and laser lines and detection ranges at - and - , respectively. simultaneously, bright field images were acquired. multiposition confocal images were acquired every min during - h, with an image matrix of × pixel; hz; × line average and autofocus control. manual analysis was performed using fiji software (version . . -rc- / . d). we recorded - timelapse videos per well and analysed - frames in each video. in every frame, manual tracking of neutrophils was performed using the mtrackj plugin [ ] to identify phagocytosis of neutrophils by microglial cells. we studied in parallel four wells per genotype (csf r +/+ or csf r +/− microglial cells) in each independent experiment and conducted five independent experiments. the analysis was performed in a blinded fashion by assigning a code to each video that did not reveal the identity of the genotype. we used green fluorescent zymosan a bioparticles (#z- ; thermo fisher scientific) in the phagocytosis assay. at div, microglial cells were exposed to zymosan fluorescent beads ( , particles/ml) for h. following - washes to remove all the non-phagocyted particles, cells were fixed with cold % paraformaldehyde for min, permeabilized with . % triton x- (sigma) in pbs . m for min, blocked with % goat serum in pbs for h, and incubated overnight at °c with the primary rabbit antibody against the p ry receptor ( : , #as a, anaspec). the next day, cells were washed and incubated with red fluorescence alexa fluor ® dye-labelled goat anti-rabbit igg antibody (#a , life technologies) for h at room temperature. dapi (#d , life technologies) stained was performed to visualize the cell nuclei. cells were then covered using fluoromount-g ® (southern biotech, birmingham, al, usa). images were obtained with a fluorescence inverted microscope (leica ctr ). mice were perfused via the left heart ventricle with ml of cold saline ( . %) followed by ml of cold % paraformaldehyde (pfa) diluted in phosphate buffer (pb) ph . . the brain was removed, fixed overnight with the same fixative, and immersed in % sucrose in pb for cryoprotection for at least h until the brains were completely sunk to the bottom of the tube. after that, brains were frozen in isopentane at − °c. cryostat brain sections ( -μm thick) were fixed in ethanol %, blocked with % normal serum, and incubated overnight at °c with primary antibodies: rat monoclonal antibodies against ly g cell nuclei were stained with dapi or to-pro (invitrogen). cryostat sections from human brain tissue were processed for immunofluorescence as described above with a rabbit polyclonal antibody against p ry ( : , # a, anaspec) and a mouse monoclonal antibody against ki ( : , # , cell signaling tech). consecutive sections were stained with thionine for examination of the lesion at the light microscope. confocal images were obtained (tcs-spe-ii or sp microscopes from leica microsystems; or a zeiss lsm microscope) and were not further processed except for enhancing global signal intensity in the entire images for image presentation purposes using las software (leica), imagej, or adobe photoshop. for estimation of the density of p ry + cells and ki + cells in human brain sections, images were obtained ( × objective), the number of immunostained cells and cell nuclei per image were counted in ten different fields per brain region of each subject, and average values per region and time group were calculated. for cell counting in mouse brain sections, we obtained - confocal images of the immunostaining ( × objective) in three different brain sections per mouse. microglia morphology was assessed using fiji software (version . . -rc- / . d) and imaris software (ima-ris bitplane v. . ). basic shape descriptors such as the circularity index (ci) or the area were performed with the plugin shape descriptors [ ] ; other parameters, such us the ramification index (ri), were obtained using the sholl analysis plugin [ ] . parameters such as volume or sphericity index were measured using imaris software after creating a d surface in the maximum intensity projection image. then, microglial cells were thresholded by the huang method [ ] to generate a binary mask (with a . mean filter). the ci parameter was calculated by the shape descriptors plugin ( p[area]/[perimeter] ). the highest count of intersections (max inters) reflects the highest number of processes in the cell. two-group comparisons were carried out with the mann-whitney u test. for multiple group comparisons we used the kruskal-wallis test followed by the dunn's test. comparisons were two-sided. comparisons of groups by brain region and time were carried out with two-way anova followed by the bonferroni post-hoc analysis. twoway anova by genotype and experiment, with an experiment-matched design, was used to analyze quantification of in vitro studies. statistical analyses were performed with graphpad software. the specific test used in each experiment and n values are reported in the figure legends. to unequivocally distinguish microglia from infiltrating leukocytes we generated chimeric mice with the hematopoietic system derived from fluorescent (dsred) donor mice where microglia remained non-fluorescent, whereas a high proportion of peripheral myeloid cells were dsred + (fig. a; online resource ). most myeloid cells infiltrating the ipsilateral brain hemisphere were dsred + days after mcao, as assessed by flow cytometry (fig. a, b) , and they were preferentially located in the lesion core where the expression of gfap is lost (fig. c) . we stained microglia with anti-p ry antibodies [ , , ] and verified that the infiltrating dsred + cells were not p ry + . microglia and infiltrating dsred + leukocytes co-existed at the border of infarction, whereas microglia were abundant at the infarct periphery but scarce in the core of the lesion (fig. d-f ). microglia acquired a reactive phenotype at the periphery and border of infarction with thicker ramifications compared to microglia of the contralateral hemisphere. in contrast, microglial cells in the infarcted core showed a dystrophic morphology since the cell body became smaller and there were only a few long ramifications showing an appearance of discontinuity as if they were broken or beaded, and the density appeared to be reduced (fig. d) . to ensure that in wild-type mice we did not miss microglial cells in the infarcted core due to downregulation of the markers used to label microglia, i.e. iba and p ry , we studied the cx cr cre/ert mice [ ] crossed with floxed rosa :tdt reporter mice [ ] , which express the red fluorescent protein in microglia (fig. a, b; online resource ) obtaining the same findings as in wild-type mice. next, we analysed microglia morphology (online resource ) in the contralateral hemisphere, and in different zones of the ipsilateral hemisphere, i.e. periphery and infarcted core, days after mcao (fig. c-h) . shape descriptors showed increased circularity and reduced area of microglia in the ipsilateral hemisphere that was more marked in cells located in the core region (fig. c, d) . a sholl analysis showed that ischemia reduced the number of ramifications, and maximal intersections per microglial cell, and again the changes were greater in the core (fig. e, f) . analysis of d-reconstructions of the cells showed a reduced volume and higher sphericity index after ischemia, particularly in the core (fig. g, h) . furthermore, the quantification of cells per area showed that while microglial cell density increased in the periphery, it was reduced in the core of the lesion (fig. i) . this result was confirmed by flow cytometry after excising the core and periphery of the ipsilateral hemisphere and mirror regions of the contralateral hemisphere and days after mcao. microglia cell number was severely reduced in the core of the lesion at and days post-ischemia (fig. j) . altogether, these findings show that microglial cells are sensitive to persistent ischemic conditions and are lost in the infarcted core. fig. microglial proliferation after ischemic stroke in mouse and human brain. immunofluorescence in mouse (a-c) and human (d-g) brain tissue. a-c brdu incorporation (green) in dsred − ramified microglial cells expressing p ry (blue). d human tissue of stroke patients deceased at different time points after stroke onset. the brains were grouped according to the day of death after stroke in ' - days' (n = ) and ' - days' (n = ) groups. the number of p ry + cells per area tended to decrease in the core of the lesion in both groups and to increase at the periphery of infarction in the ' - days' group. **p < . vs. periphery. the percentage of ki + cells among the p ry + cell population increased at the periphery of infarction in the ' - days' group (***p < . vs. control and vs. core). e-g representative images of double-immunopositive p ry (green) and ki (red) microglial cells (arrowheads) at the periphery of infarction from different stroke patients deceased (e-f) or (g) days after stroke. nuclei are stained with to-pro (blue). scale bar µm in contrast to the lesion core, the microglial cell number increased at the periphery of infarction days post-ischemia (fig. i, j) . this effect was attributable to microglial proliferation as shown in dsred chimeric mice that received injections of the cell proliferation marker brdu after mcao (fig. a-c) . by cell counting, we calculated that . ± . % (mean ± sd, n = mice) of the microglial cells at the periphery of infarction incorporated brdu suggesting microglial proliferation, in agreement with previous reports [ , ] . in contrast, microglial cells were brdu − in the contralateral non-ischemic hemisphere. notably, brdu was found in ramified microglial cells suggesting that microglia could undergo cell division without regression of their differentiation status. we extended the results to the human brain by showing an increased number of microglial cells and proliferating ki + microglia at the periphery of infarction in post-mortem human brain tissue of stroke patients (fig. d-g) . we also found reduced microglial cell numbers in the lesion core of stroked human brains (fig. d) thus supporting that the findings in mice might be relevant to human stroke. our data identified the edge of the lesion as a site for possible interactions between microglia and leukocytes since both cell types were abundant in this zone. indeed, we observed numerous microglial cell processes surrounding the blood vessels and getting into contact with infiltrating dsred + leukocytes by sampling the dsred + cells adjacent to the vascular endothelium (online resource ). furthermore, we detected microglial processes surrounding dsred + cells suggestive of engulfment of leukocytes (fig. a-c; online resource ). d-reconstructions of confocal images showed phagosomes completely wrapping dsred + leukocytes (fig. b, c) . the csf r signaling pathway is critical for survival of microglia and maintenance of their functions [ ] , including endocytic processes [ ] . consequently, by interfering with microglial function after oral administration of the csf r inhibitor gw [ ] for days, we found more dsred + cells at the periphery of infarction not surrounded by microglia or in apparent contact with microglia, suggesting that microglia dysfunction impaired the process of phagocytosis of leukocytes (fig. d) . neutrophils showed bright dsred fluorescence in the dsred chimeric mice, as assessed by flow cytometry and immunofluorescence days post-ischemia (fig. a) . we detected ly g + dsred + neutrophils shrouded by microglia at the periphery of infarction (fig. b, c) . furthermore, processes of microglial cells located nearby the surface of the cortex seemed to traverse the external cortical basement membrane and surround neutrophils located in the subpial space (fig. d) . catchup ivm mice crossed with transgenic cx cr gfp/gfp mice allowed obtaining heterozygous double reporter mice with red fluorescent neutrophils and green fluorescent microglia [ , ] . in these mice, we observed microglia (green) adjacent to the basal lamina of blood vessels and surrounding extravasated neutrophils (red) after mcao (fig. e- to further investigate the phagocytosis of neutrophils by microglia, we carried out an in vitro study using time-lapse confocal microscopy. we isolated microglia from adult mouse brains, cultured the cells in vitro for days, and exposed them to freshly isolated bone marrow neutrophils (stained with cmfda) to study phagocytosis. microglial cells phagocytosed neutrophils in vitro (fig. a, b , and timelapse microscopy movie in online resource ). by following neutrophil trajectories along time (time-lapse microscopy movie in online resource ), we counted the number of neutrophils phagocytosed by microglia. we validated that allogenicity in the phagocytosis experimental design was not interfering with the assay by comparing phagocytosis of neutrophils obtained from different mice with the phagocytosis of neutrophils from the same mouse. we found no differences, ruling out prime effects of allogenicity in the fig. microglia cells phagocytose infiltrating dsred leukocytes. a-c chimeric mice were generated by bone marrow transfer from donor dsred mice to recipient wild type mice and were subjected to ischemia (n = ). immunofluorescence showing iba + dsred − microglial cells engulfing dsred leukocytes at the periphery of infarction at day after mcao. a confocal images of an iba + (green) dsred − microglial cell engulfing a dsred leukocyte. the images correspond to different z planes. b d-reconstructions of iba + dsred − microglial cells (blue) engulfing dsred + leukocytes (red). c microglial cell (iba + , blue) with engulfed red cells in several prolongations. nuclei are stained with dapi (white). magnified details of engulfed dsred cells are shown on the right hand side. f dsred chimeric mice received oral administration of either the csf r inhibitor gw or the vehicle (n = per group) starting h prior to mcao and then daily for days. the brain was studied days post-ischemia by immunofluorescence using anti-p ry antibody to label microglia (green), dsred for infiltrating leukocytes, and to-pro- for staining the cell nuclei (blue). the image shows a microglial cell at the periphery of infarction engulfing dsred leukocytes. schematic representation of this cell illustrating: (a) a dsred cell completely surrounded by a microglial process; (b) a microglial process making apparent contact with a dsred leukocyte (touching); and (c) a dsred leukocyte separated from microglia (free). counting the number of dsred cells in the above status (a-c) in relation to microglia showed that the csf r inhibitor increased (***p < . ) the number of free dsred cells. two-way anova by treatment and condition followed by the bonferroni test. scale bar corresponds to a, b µm; c µm for left image and µm for the magnifications on the right; d µm ◂ phagocytosis of neutrophils by microglia (online resource ). given that we observed signs of impaired phagocytosis of neutrophils in vivo after ischemia in mice after shortterm treatment with a csf r inhibitor (fig. d) , we investigated in vitro the role of csf r in this process. to this end, we obtained microglia from adult mice with heterozygous csf r +/− microglia (obtained by crossing cx cr cre/ ert with csf r flox/+ mice). less neutrophils were phagocytosed by csf r +/− microglia compared to csf r +/+ microglia, as assessed by time-lapse microscopy where we counted the neutrophils phagocytosed by microglia during the -h duration of the experiment (fig. c ). in addition, csf r +/− microglia showed reduced phagocytic activity in a phagocytosis assay with fluorescent beads (fig. d-f ). we then studied the potential relevance of these findings for human cells by obtaining human microglia from the post-mortem brain of an ischemic stroke patient deceased days after stroke onset. after maintaining the human cells in culture for days, we exposed the cells to neutrophils obtained from blood of a donor control subject. we observed very active phagocytosis of neutrophils (fig. g, h and timelapse microscopy movie in online resource ). by immunofluorescence after the ex vivo assay, we detected p ry expression in the cells and observed that they contained material from neutrophils (fig. i) . neutrophil numbers were higher in the core of infarction than the periphery and days after mcao, as assessed by flow cytometry after dissection of these brain regions (fig. a) . neutrophils were seen in perivascular spaces of venules (fig. b) but also in the parenchyma outside the basement membrane in the core of the lesion (fig. c, d) . strikingly, neutrophils were found in the brain parenchyma in zones of the core where microglia was absent or showed signs of dystrophy, as assessed in immunostained brain sections of wild-type mice and reporter mice, including the cx cr cre/ert -rosa :tdt mice (fig. e ) and the catchup ivm -cx cr +/− double reporter mice (fig. f-h) . at the periphery of infarction neutrophils were surrounded by reactive microglia, suggesting that microglia phagocytosed neutrophils thereby preventing their accumulation in this region. in contrast, in the core of infarction, dysfunction or loss of microglia might facilitate the accumulation of neutrophils. to obtain further evidence that microglia remove neutrophils after ischemia, we depleted microglia by feeding the mice for weeks with a diet containing a csf r antagonist (plx ) [ , , ] . plx diet caused a strong reduction ( %) of the microglia population (fig. a ) but did not affect blood leukocyte counts (online resource shows the gating strategy and cell quantifications are shown in online resource ), in agreement with previous reports [ , ] . however, we detected a reduction of a minor subset of blood ly c − monocytes (online resource ), which is dependent on csf r [ ] . in brain tissue, plx diet reduced the numbers of infiltrating monocytes ( %) and f / + macrophages ( %) versus the control diet days post-ischemia (online resource ), in agreement with previous findings suggesting a role for microglia in the recruitment of monocytes into the brain [ ] . in contrast, the plx diet increased the numbers of neutrophils in the brain tissue after ischemia, as assessed by flow cytometry at day (fig. b) . immunofluorescence in cx cr cre/ert :r -tdt mice showed extravasated neutrophils that seemed more abundant in the absence of microglia (fig. c, d) . by counting the number of neutrophils located either in the parenchyma or associated with blood vessels (hence not crossing the parenchymal basal lamina) we found an increase in the percentage of parenchymal neutrophils in the absence of microglia and days post-ischemia (fig. e) . of note, neutrophils were often observed on the fig. microglia engulf neutrophils. a-c chimeric mice generated by transfer of dsred bone marrow cells to wild type recipients. a flow cytometry shows cd b + ly g + dsred neutrophils in the ipsilateral but not the contralateral hemisphere (mann-whitney test, **p = . , n = ) days post-ischemia. infiltrating neutrophils (nimp-r + , ly g + ) are dsred + . b, c p ry + microglial cells (blue) engulf neutrophils at the periphery of infarction. images obtained days after mcao representative of n = chimeric mice. d image of a cx cr cre/ert :rosa -tdt mouse days after mcao showing a microglial cell (red) sending a process across the external basal lamina of the cortex (α -laminin, blue) to trap a neutrophil (ly g + , green) in the subpial space. e-i images obtained from cx cr gfp/+ /catchup mice, which have gfp + microglia (green) and tdt + neutrophils (red) day after mcao. sequence of confocal z-images showing the basal lamina (pan-laminin, blue) of a capillary with a neutrophil in the lumen and an extravasated neutrophil, surrounded by a microglial cell (e). z projections of one plane and confocal projection illustrating interaction of microglia with basal lamina and neutrophils (f-g). d-reconstructions showing the microglial cell (green) attached to the capillary (blue) and intraluminal (h) and extravasated (i) neutrophils (red). nuclei are stained with dapi (white). images were obtained from superficial cortical layers (a-c, e-i) and the brain surface (d) in zones corresponding to the core of the lesion (a), border of the lesion (d-i), and periphery (b, c). scale bar μm ◂ basement membrane of capillaries (fig. d) and venules (fig. f) suggesting a possible interaction of these cells with basal lamina components. in line with this, microglia depletion increased the size of the ischemic lesion (fig. g) , further supporting a beneficial effect of microglia at the periphery of the infarction. this study supports the concept that microglia phagocytose and remove neutrophils after brain ischemia [ , [ ] [ ] [ ] and demonstrates that neutrophil accumulation in the brain parenchyma is associated with reduced microglial phagocytic activity, attributable to ischemia-induced microglial cell dysfunction due to loss or dystrophy. morphometric analysis of microglia showed changes in the periphery of the lesion compatible with microglial reactivity and similar to those reported [ ] . overall, morphological changes of microglia within the lesion core were larger than in the periphery, for instance regarding the notable loss of ramifications and reduced cell size. such profound morphological changes of microglia in the lesion core might indicate a further process of transformation from reactive microglia to dystrophic microglia, potentially associated with cell dysfunction. furthermore, we found reduced microglial cell numbers in the core of infarction. while our results support microglial degeneration in the infarcted core, microglial cells proliferated and accumulated at the periphery of infarction in mouse and human brain, in agreement with previous findings in the mouse brain [ , ] . these reactive microglial cells at the periphery of infarction phagocytosed neutrophils, suggesting that the phagocytic activity of microglia prevented neutrophil accumulation in this region. accordingly, the numbers of neutrophils were higher in the core than the periphery of the lesion. microglial activity and survival are critically dependent on csf r [ ] . consequently, drug-induced inhibition of csf r or genetic reduction of csf r expression in microglia impaired their phagocytic capacity in vivo and in vitro. previous studies reported that csf- promotes phagocytosis of ab - peptide by primary human microglia in vitro [ ] , and it regulates cell motility in macrophages [ ] . csf r is a tyrosine kinase that upon activation shows phosphorylation of several intracellular tyrosine residues [ ] . upon activation, csf r associates with several signaling molecules, notably phosphoinositide -kinase (pi k) [ ] . csf r also activates akt [ ] , and it induces erk / -mediated signaling in microglia [ ] . akt [ , ] and erk / [ ] are involved in the phagocytic process. however, the specific signaling molecules downstream of csf r participating in phagocytosis in microglia after brain ischemia, and the precise step(s) of the phagocytic process affected by csfr remain to be identified. microglia depletion induced by long-term inhibition of csf r in vivo [ , , , ] increased the numbers of neutrophils in the ischemic brain tissue, further supporting the view that microglial cells contribute to neutrophil removal. neutrophils are attracted to the injured brain after acute stroke [ , , , , , ] . thereby, neutrophils adhere to venules and migrate through the vessel wall to reach perivascular spaces [ ] . in addition, neutrophils access perivascular spaces of penetrating cortical vessels from the leptomeninges [ ] . accumulation of neutrophils in the leptomeninges might be due to extravasation from pial vessels. in addition, neutrophil migration from the skull bone marrow through direct anatomic connections [ ] might explain the presence of neutrophils in the subarachnoid space, although migration of neutrophils from there to perivascular spaces of cortical vessels still needs further investigation. subpial neutrophils are separated from the brain parenchyma by the basement membrane and glia limitans. likewise, the parenchymal basal lamina and surrounding astrocyte end-feet separate perivascular cells from the brain parenchyma. interestingly, we observed ramifications of microglial cells apparently crossing the basal lamina suggesting the possibility that reactive microglia might sample the perivascular space and also the subpial space after brain ischemia. using intravital fig. microglia phagocytose neutrophils in vitro under the control of microglial csf r expression. microglial cells were obtained from adult mice (a-f) or a human stroke patient deceased days after ischemic stroke (g-i). after days in culture, the cells were exposed to corresponding control mouse or human neutrophils and studied by time-lapse confocal microscopy for h. a, b microglia were obtained from dsred mice (red) and neutrophils were stained with cmfda (green). d-reconstructions of image sequences ( ) ( ) ( ) ( ) ( ) of the time-lapse video (see supplementary video and ) illustrating the phagocytosis of neutrophils by mouse microglia (a). the time point of each image is indicated (hours:minutes). an original confocal image is shown b for illustrative purposes. c microglia cultures were obtained from csf r +/+ (wt) and csf r +/− littermate mice (n = mice per genotype) and the cell cultures were exposed to neutrophils and studied by time-lapse microscopy, where microglial cells were seen by phase contrast and neutrophils were stained with cmfda and detected by green fluorescence. we studied four wells per mouse in each independent experiment (n = ), recorded - videos of different fields per well, and analysed - frames per time-lapse video. quantification of the number of neutrophils phagocytosed by microglia (normalized by the number of microglia in each well) shows that heterozygous csf r +/− microglia phagocytose less neutrophils than wt microglia. two-way anova by genotype and experiment, with an experiment-matched design, show a genotype effect ***p < . . d-f cultures of wt and csf r +/− microglia were exposed to green fluorescent zymosan beads (n = independent experiments). at the end of the experiment cells were fixed and immunostained with anti-p ry antibodies (red) and the number of cells containing fluorescent beads was counted. compared to wt microglia (d), csf r +/− microglia (e) also shows a reduced capacity to phagocytose zymosan beads (green) (f). two-way anova by genotype and experiment, as above, shows a significant genotype effect **p < . . g sequential snapshot images ( - ) of human microglia obtained during the h time-lapse microscopy study in which frames were studied. the images illustrate neutrophil phagocytosis by human microglia at the indicated times (hours:minutes). h magnification of the indicated part of sequence in g is shown to illustrate the engulfment of a neutrophil by a human microglial cell. i after the time lapse-experiment, human cells were fixed and stained with anti-p ry antibodies (red). nuclei are shown in blue (dapi). the microglial cells contain material (green fluorescence) derived from digested neutrophils. scale bar μm ◂ microscopy, we previously found evidence that microglia phagocytosed neutrophils before they extravasated to the brain parenchyma [ , ] . however, further studies are required to demonstrate whether microglia can really cross the external cortical basement membrane after brain ischemia. although we detected engulfment of complete cells by microglia, some of the images suggest that microglia may take portions of the neutrophils while they are located in the perivascular or subpial spaces, potentially through a process of trogocytosis [ ] . the blood vessel glycocalyx and basement membrane composition varies between organs and inflammatory conditions suggesting that leukocytes may have to use diverse strategies to access different inflamed tissues [ ] . in the brain, neutrophils cross the endothelial cell layer and the endothelial basal lamina of venules to reach the perivascular spaces after ischemia [ ] . then, they accumulate in the perivascular spaces because they do not seem to readily cross the parenchymal basal lamina [ ] , at least not at the same pace as they transmigrate through the former layers. however, the precise molecular determinants of this process remain to be identified. the different molecular composition of the two layers of basal lamina surrounding the perivascular spaces, local molecular diversity, and the finding that certain basal lamina components inhibit leukocyte transmigration [ ] , might explain why neutrophils have more difficulty to cross the parenchymal than the endothelial basal lamina after brain ischemia. in a model of transient ischemia, there is evidence suggesting that neutrophils are kept in the perivascular spaces without infiltrating the brain parenchyma [ ] , whereas other studies suggested that neutrophils reach the brain parenchyma [ ] . it is plausible that stroke severity, status of microglia function, and time point of the study are critical determinants of the presence of neutrophils in the brain parenchyma. neutrophils located in the perivascular spaces might damage the basement membrane by releasing proteolytic enzymes and/or undergoing netosis [ ] . however, at this stage we cannot exclude the possibility that neutrophils gained access to the brain parenchyma in a passive fashion after loss of vessel integrity in the ischemic core. several lines of evidence support that after brain ischemia neutrophils release proteolytic enzymes, promote matrix metalloproteinase (mmp) activation, and cause bbb breakdown [ , , , , , ] . accordingly, blocking neutrophils or neutrophil-derived mmp- is markedly protective in models of systemic inflammation and stroke, e.g. [ , ] . furthermore, pharmacological inhibition of neutrophil elastase or genetic deficiency of this enzyme reduced bbb disruption and vasogenic edema after transient mcao [ ] suggesting that neutrophils contributed to vascular damage following stroke. in this study we showed that, after permanent ischemia, neutrophils gained access to the brain parenchyma of the lesion core when it was already severely damaged and microglia was lost due to persistent ischemia. under these conditions, parenchymal neutrophils might be bystanders of severe tissue damage. therefore, it is likely that preventing the access of neutrophils to the brain parenchyma in this model would not have a major impact on the size of the brain lesion since the damage is already established by the time the cells reach the parenchyma and the core of infarction will not recover. this possibility agrees with the finding that inhibition or deficiency of neutrophil elastase was not protective in models of permanent mcao [ ] . in contrast, inhibiting microglial phagocytic activity in this model might bear negative effects by favoring neutrophil accumulation in the ischemic periphery. accordingly, detrimental effects of neutrophils became apparent in our study after microglia depletion causing an abnormal increase in neutrophils and larger ischemic lesions. a limitation of our study is that we did not assess stroke outcome in the long term. future work should investigate how microglia depletion affects the progression of the ischemic brain lesion and the neurological deficits. the results highlight an aspect of microglia phagocytic function that may be beneficial for the ischemic tissue. nonetheless, several mechanisms can contribute to the detrimental effect of microglia depletion and csf r deficiency. for instance, pioneer studies demonstrated increased ischemic lesions related to reduced production of neurotrophic factors after depleting proliferating microglia [ ] , and neuroprotective functions mediated by csf r [ ] . moreover, we previously identified that absence of microglia significantly augmented infarct size in a model of transient ischemia, in part mediated by dysregulation of fig. the presence of neutrophils in the brain parenchyma is associated with dystrophy and loss of microglia. a flow cytometry analysis of cd b + ly g + neutrophils in the core and periphery of infarction and in mirror regions of the contralateral hemisphere and days after mcao (n = mice per time point) shows that ischemia increases the number of neutrophils in the core of infarction more than the periphery (two-way anova, bonferroni test, ***p < . ). b-d brain confocal images day after mcao (n = ) show ly g + neutrophils (green) in the perivascular space of a venule (b) and the parenchymal side of capillaries (c, d). the basal lamina is stained with pan-laminin (red) and nuclei are stained with to-pro (blue). e neutrophil (ly g + , green) accumulation is higher at the border (dotted line) of the infarcted core than the periphery, whereas microglia (red cells, cx cr cre/ert :rosa -tdt mice) accumulate in the infarct periphery and are scarce in the core. the vascular basal lamina is shown in blue (pan-laminin). f-h images obtained from catchup mice crossed with cx cr gfp/gfp mice at day (f, g) and day (h) postischemia. extravasated neutrophils (red) away from the vascular basal lamina (pan-laminin, blue) are seen in the core of the lesion where microglia (green) is absent or dystrophic, whereas neutrophils are surrounded by reactive microglial cells in the periphery (f). g examples ( - ) of microglial cells sending prolongations towards neutrophils day after pmcao. neutrophils located in perivascular spaces extend protuberances crossing the basal lamina ( ). extravasated neutrophils are seen near vessels with discontinuous basal lamina ( ). they are surrounded by reactive microglia at the border of the lesion ( - ), but dystrophic microglia seem to be unable to fully reach the extravasated neutrophils ( ) . h at day , microglial cells sending prolongations towards neutrophils are seen at the border of the lesion ( ). extravasated neutrophils are seen in zones where microglia is dystrophic ( - ) or absent ( ) ( ) ( ) . the vascular basal lamina is hardly detected at places where the neutrophils are located ( ) . arrowheads indicate neutrophils, whereas arrows indicate microglia. the cell nuclei (dapi, white) are shown in g ( ) ( ) and h ( - ). scale bar μm ◂ neuronal activity [ ] . the latter model caused moderate leukocyte infiltration and we failed to observe a significant impact of microglia depletion on bbb injury and leukocyte recruitment, at least at the times examined [ ] . in contrast to the findings suggesting beneficial effects of microglia in brain ischemia, several lines of evidence support that the phagocytic activity of microglia could exert negative effects by removing viable neurons through phagoptosis [ , , , ] . it is possible that any negative consequences of phagoptosis of neurons might predominate under mild ischemic conditions where the inflammatory response is low, vascular integrity is preserved, and neutrophil attraction to the brain is negligible. collectively, our results support a model where microglia removes neutrophils from the parenchyma and perivascular and subpial spaces after brain ischemia. severe ischemic conditions induce local microglia loss/dystrophy facilitating the presence of neutrophils in perivascular spaces first and in the brain parenchyma later. overall, this study shows that reactive microglial cells phagocytose and remove neutrophils, whereas microglial loss or dysfunction enhances neutrophil accumulation in the ischemic lesion. our results, hence, suggest that microglia function is critical to prevent neutrophil infiltration to the brain parenchyma and to minimize the negative impact of neutrophils in the vascular bed after ischemic stroke. fig. microglia depletion increases the number of neutrophils in the brain parenchyma. a schematic representation of the experimental design. eight-week old mice received a control or plx -containing diet for days. then mcao was induced and the animals continued with the corresponding diet one or four more days until the end of the study. b flow cytometry (n = mice per group) showed the microglia (cd low cd b + cells) depleting effect of the plx diet, and an increased number of neutrophils (cd b + ly g + ) in the ipsilateral hemisphere of mice with depleted microglia. two-way anova by treatment and brain hemisphere, and bonferroni posthoc test, **p < . . c control or plx -containing diet was given to cx cr cre/ert :rosa -tdt mice with the same dosing regimen. microglia is shown in red, neutrophils in green (ly g + ), the vascular basal lamina is stained with pan-laminin (blue), and nuclei (dapi) are shown in white. the absence of microglia is associated with an increased presence of extravasated neutrophils (arrowheads). d is a magnified image showing extravasated neutrophils after a plx containing diet. e, f distribution of neutrophils in parenchymal versus perivascular or intravascular locations, as assessed by cell counting in brain sections immunostained with ly g, pan-laminin and to-pro . mice treated with the plx diet show a higher (mann-whitney test) percentage of parenchymal neutrophils day (**p = . ) and days (**p = . ) post-ischemia (n = - mice per group). f illustrates the previous staining after plx diet. g brain lesion volume was measured with t w mri h post-ischemia in both diet groups (n = per group). the brain lesion was larger in mice depleted of microglia (mann-whitney test, *p = . ). scale bar μm (c); μm (e, f) ◂ new tools for studying microglia in 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microglia in the adult brain: review of novel microglia depletion systems microglia are required for protection against lethal coronavirus encephalitis in mice microglia remodel synapses by presynaptic trogocytosis and spine head filopodia induction blood-brain barrier breakdown in acute and chronic cerebrovascular disease ischemic vulnerability of primary murine microglial cultures fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis neutrophil migration into the infected uroepithelium is regulated by the crosstalk between resident and helper macrophages dominant role of microglial and macrophage innate immune responses in human ischemic infarcts csf- receptor structure/function in maccsf r −/− macrophages: regulation of proliferation, differentiation, and morphology acknowledgements aoda had a predoctoral fellowship from the mineco-fpi program and fmm had a peris award by the health department of generalitat de catalunya. part of this work was performed at the centre de recerca biomèdica cellex, barcelona. the cerca programme of generalitat de catalunya supports the institut d'investigacions biomèdiques august pi i sunyer (idibaps). plx was provided by plexxikon under materials transfer agreement. we acknowledge the cytomics and image platforms of idibaps for access to equipment. we would like to thank elisenda coll (advanced optical microscopy-ccitub) for excellent technical assistance. we thank the neurological tissue bank of the biobank-hospital clinic-idibaps for sample and data procurement, and to patient's relatives for giving consent to sample use for research purposes. key: cord- - s ojc authors: park, jeong-in; song, kyung-hee; jung, seung-youn; ahn, jiyeon; hwang, sang-gu; kim, joon; kim, eun ho; song, jie-young title: tumor-treating fields induce raw . macrophage activation via nk-κb/mapk signaling pathways date: - - journal: technol cancer res treat doi: . / sha: doc_id: cord_uid: s ojc objective: tumor-treating fields are currently used to successfully treat various cancers; however, the specific pathways associated with its efficacy remain unknown in the immune responses. here, we evaluated tumor-treating fields–mediated initiation of the macrophage-specific immune response. materials and methods: we subjected raw . mouse macrophages to clinically relevant levels of tumor-treating fields ( . v/cm, khz) and evaluated alterations in cytokine expression and release, as well as cell viability. additionally, we investigated the status of immunomodulatory pathways to determine their roles in tumor-treating fields–mediated immune activation. results and discussion: our results indicated that tumor-treating fields treatment at . v/cm decreased cell viability and increased cytokine messenger rna/protein levels, as well as levels of nitric oxide and reactive oxygen species, relative to controls. the levels of tumor necrosis factor α, interleukin β, and interleukin were markedly increased in tumor-treating fields–treated raw . cells cocultured with t murine mammary carcinoma cells compared with those in t or raw . cells with or without tumor-treating fields treatment. moreover, the viability of t cells treated with the conditioned medium of tumor-treating fields–stimulated raw . cells decreased, indicating that macrophage activation by tumor-treating fields effectively killed the tumor cells. moreover, tumor-treating fields treatment activated the nuclear factor κb and mitogen-activated protein kinase pathways involved in immunomodulatory signaling. conclusion: these results provide critical insights into the mechanisms through which tumor-treating fields affect macrophage-specific immune responses and the efficacy of this method for cancer treatment. tumor-treating fields (ttfs) therapy, in which small electric fields are pulsed through the skin to disturb mitosis, represents a noninvasive, regional treatment modality that was recently approved for the therapy of recurrent glioblastoma (gbm) by the us food and drug administration and gained a ce mark in europe. [ ] [ ] [ ] the clinical concept is that ttfs therapy delivers low-intensity ( . - v/cm), intermediate-frequency ( - khz), and alternating electric fields to the tumor using transducer arrays placed on the skin around the tumor site. the proposed mechanisms of action involve disruption of the metaphase of tumor cells by interfering with mitotic spindle formation and disruption of anaphase by the dielectrophoretic dislocation of intracellular constituents, resulting in an overall effect of apoptosis. the antimitotic effect of ttfs treatment has been showed in various cell lines as well as in gbm by tuning the appropriate frequency to specific cancer cell types. [ ] [ ] [ ] a phase iii trial of ttfs combined with temozolomide established that this treatment protocol led to minimal toxicity, increased the overall survival rate, and improved patient quality of life with equivalent therapeutic efficacy to chemotherapy. moreover, in addition to its combination with chemotherapy, synergistic enhancement of the cellular response was reported when ttfs were administered in combination with radiation therapy. thus, with these merits, ongoing and future trials will evaluate ttfs to treat various cancer types, including newly diagnosed gbm, solid tumor brain metastases, nonsmall cell lung cancer, ovarian cancer, and pancreatic cancer. emerging evidence has demonstrated that the tumor microenvironment may greatly contribute to the final outcome of cancer treatment modalities. several types of cells including leukocytes, fibroblasts, and vascular endothelial cells form the tumor microenvironment, which has immune cells as its major component. these immune cells interact with tumor cells to influence the initiation, proliferation, elimination, invasion, and metastasis of tumors. in particular, tumor-associated macrophages are immune cells that orchestrate various factors in the tumor microenvironment through the polarization of m or m macrophages. , , tumor-associated macrophages play a crucial role in the link between inflammation and cancer to produce inflammatory cytokines, such as interleukin (il) b, interferon g, il- , and il- , along with anti-inflammatory mediators (il- , il- , and il- ). [ ] [ ] [ ] [ ] despite increasing studies of the effect of ttfs on cancers, the influence of ttfs on immune cells has not yet been determined. therefore, to further understand the mechanism underlying the beneficial effects of ttfs therapy and further improve this treatment modality, we investigated the influence of ttfs exposure on the function of murine macrophage raw . cells, a frequently used in vitro model to investigate the immune response and inflammation. tumor-treating fields were generated using a pair of insulated wires connected to a functional generator and a high-voltage amplifier, which generated sine-wave signals ranging from to v and resulted in an applied electric field intensity and frequency of . v/cm and khz, respectively. we used . v/cm as the field intensity to match the application in clinical settings. for irradiation treatment, the cells were plated in -mm dishes and incubated at c under humidified conditions in a % co atmosphere until reaching % to % confluence. cell culture and preparation of conditioned medium raw . mouse macrophages and t murine mammary carcinoma cells were purchased from american type culture collection (manassas, virginia) and cultured in dulbecco modified eagle medium (dmem; welgene, seoul, korea) supplemented with % heat-inactivated fetal bovine serum (fbs) and antibiotics ( u/ml penicillin and mg/ml streptomycin; welgene) at c in a humidified % co incubator. for the preparation of conditioned medium (cm), raw . cells were treated with ng/ml of lipopolysaccharide (lps; sigma, st louis, missouri) as an inflammatory stimulator control or ttfs; nontreated cells were used as the control group. after and hours, the supernatants were harvested and filtered through . mm membrane (millipore, massachusetts). coculture of raw . and t cells t cells (  cells/ml) were plated in -well plates, and the -hour ttfs-treated raw . cells were added to the wells for coculture at different densities:  cells/ml,  cells/ml, or  cells/ml. the cells were cocultured in dmem supplemented with % fbs and % antibiotics for hours at c with % co . cell-free supernatants derived from the coculture of raw . and t cells were stored at À c prior to measurement of cytokine production. the amounts of il- (invitrogen, carlsbad, california), il- b (bd science, san diego, california), and tumor necrosis factor a (tnf-a; r&d system, minneapolis, minnesota) produced in the cm were determined with commercial enzyme-linked immunoassay kits according to the manufacturer's protocol. the absorbance at nm was obtained using a -well microplate reader (thermo scientific, rockford, illinois). cell viability was determined by a trypan blue exclusion assay and -( , -dimethylthiazol- -yl)- , -diphenyltetrazolium bromide (mtt; amresco life science, philadeplhia, pennsylvania) assay. an equal volume of trypan blue reagent was added to the cell suspension, and the percentage of viable cells was evaluated by microscopy. t cells were plated in -well plates (  cells/well) for various times after treatment of the raw . cell cm. then, mtt ( mg/ml) reagent was added to each well for hours, and absorbance was measured at nm using a microplate reader (multiskan ex, thermo labsystems, waltham, massachusetts). assays were performed in triplicate. reactive oxygen species (ros) levels in macrophages were monitored using the fluorescent ros indicator c , -dichlorodihydrofluorescein diacetate ( mmol/l; molecular probes, eugene, oregon). cell-associated fluorescence was detected by fluorescence-activated cell signaling (facs) using a facs-calibur flow cytometer (bd biosciences, san diego, california) with flowjo software v . . (tree star inc, ashland, orogen). images of fluorescent cells were acquired under a confocal microscope (carl-zeiss, germany). raw . cells at a density of  cells/well were treated with ttfs or ng/ml of lps from escherichia coli :b (l ; merck kgaa, darmstadt, germany) as a positive control for the activation of macrophages. after incubation, the culture supernatants of raw . cells were collected, and each culture supernatant was mixed with the same amount of griess reagent and incubated at room temperature for minutes. the absorbance of the mixture was determined at nm using a microplate reader (multiskan ex, thermo fisher scientific, waltham, massachusetts). all measurements were performed in triplicate. the amounts of nitrites were determined using a standard curve established with nano . total rna was extracted from raw . cells using trizol reagent (invitrogen) and reverse transcribed using ecodry premix (clontech laboratories, mountain view, california) according to the manufacturer's recommendations. quantitative reverse transcription polymerase chain reaction experiments were performed using maxima sybr green qpcr master mix (thermo scientific) in lightcycler real-time pcr system (roche diagnostics, mannheim, germany). using gapdh as an internal reference, the relative gene expression was calculated based on the ddct method. the following primer pairs were used: inducible nitric oxide synthase (inos), -cga aac gct tca ctt cca a- (forward) and -tga gcc tat att gct gtg gct- (reverse); il- b, -tga agg gct gct tcc aaa cct ttg acc- (forward) and -tct cca ttg agg tgg aga gct ttc agc- (reverse); tnf-a, -atg agc aca gaa agc atg atc cgc- (forward) and -cca aag tag acc tgc ccg gac tc- (reverse); and gapdh, -acc aca gtc cat gcc atc ac- (forward) and -tcc acc acc ctg ttg ctg ta- (reverse). experiments were repeated at least times. total proteins from raw . cells were extracted in tnn buffer ( mm tris-cl, ph . ; % np- ; mm nacl, and mm edta) supplemented with protease inhibitors ( mmol/ l phenylmethylsulfonyl fluoride [pmsf], mg/ml aprotinin, mg/ml leupeptin, and mmol/l na vo ) and quantified using the bradford method. protein samples ( mg) were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. after blocking nonspecific antibody binding sites, the membrane was incubated overnight at c with mouse monoclonal antibodies against inos, extracellular signal-regulated kinase (erk), phosphorylated erk (perk), c-jun n-terminal kinase (jnk), pjnk, p , p-p , ikb-a, pikb-a, and gapdh. after incubation with peroxidase-conjugated secondary antibodies at c for hour, the protein bands were visualized using enhanced chemiluminescence reagent (ge healthcare biosciences, pittsburgh, pennsylvania) and detected using the amersham imager (ge healthcare biosciences). the relative levels of protein expression were calculated with reference to the levels of gapdh. all results are expressed as the mean (standard deviation). significant differences among groups were determined by analysis of variance and tukey post hoc comparisons using graph-pad software version (graphpad, la jolla, california). statistical significance was defined as p values <. , and the individual p values in the figures denoted by asterisks (*p < . ; **p < . ; ***p < . ). to investigate the effect of ttfs on macrophages, raw . cells were treated with ttfs ( . v/cm) or lps, which is a representative activator of macrophages. the morphological changes were observed under a phase contrast microscope ( figure a ). the normal morphology of raw . cells was round in the control group. in contrast, lps-treated cells showed polygonal and dendritic-like morphology, indicating that lps effectively activated raw . cells. in ttfstreated cells, the dendritic-like shape was observed in a portion of the population, but the number of cells seemed to be decreased at hours compared to that in other groups. therefore, the cell viability was determined by a trypan blue dye exclusion assay. as shown in figure b , lps-stimulated and unstimulated control cells continuously proliferated, whereas raw . cells treated with ttfs exhibited cell growth inhibition but without a statistically significant influence on cell viability. nitric oxide (no) is an important inflammatory mediator that can be produced by activated macrophages to kill tumors and pathogens as well as convey intracellular signals. as shown in figure a , lps-or ttfs-treated cells increased the messenger rna (mrna) and protein expression of inos compared with those of the control. the production of no in ttfs-treated raw . cells was slightly increased, whereas lps stimulation significantly enhanced no production ( figure b ). it has been demonstrated that activated macrophages exhibit increased ros accumulation under inflammatory conditions; therefore, we evaluated whether ttfs affect the generation of ros. as shown in figure c , ros levels were increased in raw . cells when exposed to ttfs for hours. concomitantly, lps-induced ros generation was also evident. these results indicate that ttfs increased the production of no and ros in activated raw . macrophages. among the various cytokines, tnf-a can activate macrophages in an autocrine manner to induce the expression of other inflammatory and immunomodulatory mediators. raw . cells were treated with ttfs or lps for hours, and the mrna expression levels of proinflammatory cytokines such as il- b and tnf-a were determined ( figure a) . the mrna levels of il- b and tnf-a were significantly upregulated by the administration of ttfs, and further increase was observed in lps-activated raw . cells. we further investigated the release of some key proinflammatory cytokines in the coculture medium. compared with that in other groups, il- b, tnf-a, and il- levels in the coculture of ttfs-treated raw . cells with t cells were significantly increased at hours in a manner dependent on the ratio of the cells ( figure b ). to evaluate the cytotoxic activity of the cm of ttfs-treated raw . cells against t cells, an mtt assay was performed. as shown in figure c , t cells exposed to the cm of unstimulated raw . cells did not show statistically significant decrease in viability, whereas t cells exposed to cm from ttfs-treated raw . cells exhibited decreased viability after or hours contact with the cm. as expected, the cm of lps-treated raw . cells also significantly decreased the viability of t cells. these results suggested that ttfs activate macrophages and promote the expression of proinflammatory cytokines. p mitogen-activated protein kinase and nuclear factor-kb signaling pathways in ttfs-administered raw . cells to investigate the signaling pathways involved in the activation of raw . cells by ttfs, mitogen-activated protein kinase (mapk) and nuclear factor-kb (nf-kb) signaling pathways, which are vital targets for regulating inflammatory responses, were examined using western blot analysis. the expression of map kinase family members, namely, erk, jnk, and p mapk, was determined. the phosphorylation levels of p mapk were significantly increased in ttfsadministered raw . cells at hours after treatment. however, activation of mapk in lps-treated raw . cells was not observed at that time point as lps treatment activated raw . cells more quickly and much more strongly than ttfs treatment. under normal cell conditions, nf-kb exists as an inactive cytoplasmic complex with its inhibitor ikb-a. tumor-treating fields-administered raw . cells showed increased phosphorylation of ikb-a and p , indicating that the p subunit of nf-kb was released from ikb-a, allowing for its translocation to the nucleus to regulate the transcription of many genes that activate macrophages (figure ). tumor-treating fields have recently been reported as a promising and noninvasive therapeutic approach for cancer therapy with good clinical results. the underlying mechanical mechanisms include the disruption of mitosis and selective killing of rapidly proliferating cells by delivering continuous (> h/d) low-intensity, intermediate-frequency, alternating electric fields to the tumor site. tumor-treating fields ultimately lead to caspase-dependent or caspase-independent-induced apoptosis. [ ] [ ] [ ] tumor-treating fields have been shown to apply its antimitotic effects in preclinical systems of various cancers, including gbm through the similar molecular mechanisms of activity. , , , furthermore, ttfs have been tried in preclinical systems combined with cytotoxic, chemotherapeutic agents in order to enhance the general antitumor effects. combined treatment of ttfs with these chemotherapeutic agents (microtubule inhibitors, nucleoside analogues, folate antimetabolites, alkylating agents, and immune checkpoint inhibitors) demonstrated an additive cytotoxic effect. taxanes and ttfs showed a synergistic effect against cancer cells. in addition, in in vitro preclinical models, ttfs were shown to expose calreticulin to the cell surface, which ultimately significantly decreased the tumor volume in vitro and to considerably decrease the tumor volume in combination with antiprogrammed t-cell death to significantly increase antigenpresenting cell infiltration of the tumor. these events show the potentiation of immunogenic cell death by the treatment of ttfs, which needs further investigation. although a large number of studies using ttfs as an anticancer therapy are ongoing, the influence of ttfs on normal tissues in the tumor microenvironment remains largely unknown. thus, in the current study, we demonstrated that ttfs exert potential immunostimulatory activity via regulation of nf-kb/mapk signaling pathways in raw . macrophages. macrophages are crucial for host defense against infections and in inflammatory processes through the release of molecules, such as no, tnf-a, and il- . on the basis of our results, the study showed that ttfs increase macrophage activation in raw . cells. furthermore, ttfs significantly enhanced the production of no in raw . cells in vitro. after its release from macrophages, no acts as an intracellular messenger to mediate the nonspecific immune response and has also been suggested to be a critical factor in the immune response. nitric oxide is a gaseous, free radical and acts as a signaling molecule in biological systems, recruiting leukocytes to affected tissues. , inducible nos is the inducible enzyme for no production and is responsible for increased levels of no. accumulating evidence indicates that intracellular ros also serves as a second messenger in inflammatory signal transduction by modulating the release of other inflammatory mediators and stimulating mapk activity. [ ] [ ] [ ] in our result, we observed that the stimulation of raw . cells by ttfs increases inos expression as well as the production of no and ros, although the increase was much lower than that observed in lps-treated cells. therefore, ttfs may have immune mediating/modulating effects, including activation of macrophages and mediating their biological functions such as tumoricidal activity through no-dependent pathways. once activated, macrophages release abundant cytokines that act as signals to control homeostasis via regulating cell differentiation, proliferation, apoptosis, and immune responses. figure . upregulation of inflammatory cytokines in ttfs-treated raw . cells. a, cells were treated with ttfs ( . v/cm) or lps ( ng/ml) for hours, and mrna levels of il- b and tnf-a were evaluated by qrt-pcr. b, raw . cells were treated with ttfs ( . v/cm) for hours, and the cells were cocultured with t cells at the indicated ratios ( t :ttfs-raw . ). t cells, untreated-or ttfs-treated raw . cells were designated as t , ct, and ttf, respectively. co-cultivations were performed for hours, and the levels of proinflammatory cytokines (il- b, tnf-a, and il- ) were determined by elisa. **p < . , ***p < . compared with the ttf group. c, raw . cells were treated with ttfs ( . v/cm) or lps ( ng/ml) for hours, and the culture medium (cm) of raw . cells was used to treat t cells for and hours. the untreated raw . cells were designated as ct. the percentage of cell viability was determined by the mtt assay and calculated relative to that of untreated t cells (none). data represent the mean (standard deviation) of triplicate samples. **p < . , ***p < . compared with the hours none group. yy p < . compared with the hours none group. elisa indicates enzyme-linked immunoassay; il, interleukin; lps, lipopolysaccharide; mrna, messenger rna; mtt, -[ , -dimethylthiazol- -yl]- , -diphenyltetrazolium bromide; tnf, tumor necrosis factor; ttfs, tumor-treating fields; qrt-pcr, quantitative reverse transcription polymerase chain reaction. some of these key cytokines that play critical roles in immune and inflammatory processes include tnf-a, which is also produced by t-lymphocytes and natural killer cells, and il- b, which facilitates the release of activated macrophages. , in the present study, ttfs markedly treated raw . cells to release tnf-a and il- b, indicating that ttfs-stimulated macrophages contribute to provoking an inflammatory response. the levels of tnf-a, il- b, and il- were markedly higher in ttfs-treated raw . cells cocultured with t murine mammary carcinoma cells than in t or raw . cells with or without ttfs. moreover, the viability of t cells treated with the cm of ttfs-stimulated raw . cells decreased, indicating that macrophage activation by ttfs effectively killed the tumor cells. as essential pathways of inflammation, we therefore evaluated the effect of ttfs on nf-kb and mapks activation in raw . cells. nuclear factor-kb is a ubiquitous transcription factor that plays a crucial role in inflammatory processes via the induction of a wide variety of genes, including inflammatory cytokines, mediators, and chemokines. the phosphorylation-induced degradation of ikbs activates and dissociates from nf-kb. as demonstrated in the present result, ttfs significantly increased the phosphorylation of ikb-a in raw . cells, thereby inducing nuclear translocation and transcriptional activation of nf-kb in macrophages. moreover, mapks regulate various inflammatory mediators, including tnf-a, il- b, il- , il- , cyclooxygenase , and inos, [ ] [ ] [ ] suggesting that the inflammatory activity of ttfs is associated with its effect on the mapk signaling pathway, a key upstream signaling pathway in the regulation of inflammatory mediators. mitogen-activated protein kinases, including erk, p , and jnk, participate in the activation of nf-kb to regulate the gene expression and protein synthesis of inflammatory mediators. interleukin b, a major proinflammatory cytokine, can easily activate mapk pathways. , thus, we investigated whether the inflammatory effects of ttfs involved the mapk regulation. indeed, p mapk signaling pathways were obviously activated in ttfs-treated macrophages. therefore, our results indicate that ttfs may increase inflammatory responses by inducing p mapk pathways in macrophages. administration of ttfs did not cause cell death in raw . cells; however, cell growth inhibition was clearly observed, suggesting that highly proliferating cancer cells are selectively targeted by ttfs therapy. lipopolysaccharidetreated cells were morphologically changed from a roundshape to dendritic-like shape, and the number of cells was figure . activation of raw . cells via mapk and nf-kb signaling pathways. a, raw . cells were treated with ttfs ( . v/cm) or lps ( ng/ml) for hours. levels of the indicated proteins were determined with western blotting, and gapdh was used as the internal control. a representative result of independent experiments is shown. b, band intensities corresponding to the indicated proteins were quantified by densitometry using image j software, normalized to the gapdh, and expressed as the fold-change from each control. data represent the mean (standard deviation) of triplicate samples. *p < . compared with the control group. lps indicates lipopolysaccharide; mapk, mitogen-activated protein kinase; nf-kb, nuclear factor-kb; ttfs, tumor-treating fields. decreased at the early stages but was subsequently recovered. therefore, the effect of ttfs on various types of normal cells should be further investigated, and in vivo assays are also needed. although this study cannot be extrapolated to in vivo conditions, it may provide a theoretical and experimental basis for immune responses specific to ttf treatments in the future. in conclusion, this is our first result to demonstrate the role of the p mapk/nf-kb pathway in the ttfs-induced inflammatory action in raw . cells. a clearer biological understanding of the functional significance of this treatment may constitute a potential new therapeutic option. thus, these results can provide a preclinical basis for ttfs therapy and expand research geared toward the development of novel anticancer modalities. the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. the author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: this study was supported by a grant from the korea institute of radiological and medical sciences (kirams), which was funded by the ministry of science, ict, republic of korea ( - ). eun ho kim, phd https://orcid.org/ - - - x novottf- a versus physician's choice chemotherapy in recurrent glioblastoma: a randomised phase iii trial of a novel treatment modality maintenance therapy with tumor-treating fields plus temozolomide vs temozolomide alone for 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folium inhibits interleukin- beta-induced expression of matrix metalloproteinases and inflammatory mediators by suppressing the activation of nf-kappab and p mapk in sw human chondrocytes icariin inhibits mmp , mmp and mmp expression through mapk pathways in il beta stimulated sw chondrosarcoma cells key: cord- - t mklwi authors: wendelboe, aaron m.; mccumber, micah; erb-alvarez, julie; mould, nicholas; childs, richard w.; regens, james l. title: managing emerging transnational public health security threats: lessons learned from the west african ebola outbreak date: - - journal: global health doi: . /s - - -z sha: doc_id: cord_uid: t mklwi background: pandemics pose significant security/stability risks to nations with fragile infrastructures. we evaluated characteristics of the west african ebola outbreak to elucidate lessons learned for managing transnational public health security threats. methods: we used publically available data to compare demographic and outbreak-specific data for guinea, sierra leone, and liberia, including key indicator data by the world health organization. pearson correlation statistics were calculated to compare country-level infrastructure characteristics with outbreak size and duration. results: hospital bed density was inversely correlated with longer evd outbreak duration (r = − . ). country-specific funding amount allocations were more likely associated with number of incident cases than the population at-risk or infrastructure needs. key indicators demonstrating challenges for guinea included: number of unsafe burials, percent of evd-positive samples, and days between symptom onset and case hospitalization. sierra leone’s primary key indicator was the number of districts with ≥ security incident. liberia controlled their outbreak before much of the key-indicator data were collected. conclusion: many of the country-level factors, particularly the who key indicators were associated with controlling the epidemic. the infrastructure of countries affected by communicable diseases should be assessed by international political and public health leaders. the nexus between risks posed by infectious disease pandemics to the stability of fragile states and international security was demonstrated dramatically by the largest reported ebola virus disease (evd) outbreak which occurred in west africa between and [ ] [ ] [ ] [ ] [ ] . underscoring its severity, the west african ebola outbreak was larger than all previous evd outbreaks combined. the epidemic began in december and the world health organization (who) was notified on march , . the who declared it a public health emergency of international concern on august , [ ] . further demonstrating the security threat posed by this public health emergency, the united nations security council resolution ( ) adopted on september , declared the outbreak was "a threat to international peace and security in the region" [ ] . the international community's response acting in cooperation with the immediately affected countries included diagnosing the disease as early as possible, contact tracing, patient isolation and care, infection control, ring vaccination, safe burial practices, and public education (e.g., messages on billboards and radio describing symptoms, hand hygiene, school closures, and avoiding close contact to prevent transmission). indeed, many of these factors comprise the majority of the who ebola situation reports' key performance indicators [ ] . the outbreak officially ended on march , . ten countries were directly impacted, three of which experienced significant outbreaks (guinea, liberia, and sierra leone), while seven countries reported one or more evd cases without widespread human-to-human transmission (italy, mali, nigeria, senegal, spain, the uk, and the us) [ , ] . we aim to systematically examine country-specific factors within the context of the larger global pandemicwith the greatest emphasis on guinea, liberia, and sierra leone. specifically, we ) conduct a quantitative analysis of country-specific factors in guinea, liberia, and sierra leone and ) conduct a qualitative analysis of patterns of disease incidence and transmission among all countries with ≥ case of evd to draw lessons learned from the west african ebola outbreak for managing emerging transnational health security threats. by march , , who reported , cases and , deaths from the west african ebola outbreak, [ ] with an overall case fatality rate of · %. cases were observed in countries across three continents (africa, europe, and north america). table summarizes the number of cases, deaths, contacts followed, and date the country was first declared ebola-free. epidemic curves for the three widely affected countries are shown in fig. . at the epidemic's peak, the doubling time was - days for cases in liberia and - days for sierra leone and guinea [ ] . although four new confirmed cases were diagnosed in guinea during march - , the who director-general declared on march , the end of the public health emergency of international concern regarding the evd outbreak in west africa [ ] . the first case likely occurred december , , but not identified as ebola until march , [ ] . the index case was traced to a two-year-old boy in meliandou, a village near gueckedou, guinea [ ] . evidence suggests his exposure was likely an infected angolan free-tailed bat [ , ] . although guinea had the highest case fatality rate ( · %, fig. ) and was the last to be declared ebola-free, it had the lowest number of cases among the widely-affected countries. the peak number of weekly cases reported was in november [ ] . eight ( · %) of the prefectures in guinea did not report any ebola cases [ ] . by march , , there were evd cases, deaths [ ] (table ) and cases among health care workers (hcws), of which died [ ] . the first sierra leone case was retrospectively identified as a female guest in the index case's home in meliandou, guinea, but returned to sierra leone and died in early january [ ] . it was not until april , that sierra leone "stepped up vigilance for imported cases when two members of the same family who had died from ebola virus disease in guinea were repatriated to sierra leone for burial" [ ] . by november , evd had been reported in each district in sierra leone [ ] and the peak number of weekly cases reported was [ ] . sierra leone had the highest number of cases ( , ) but the lowest case fatality rate ( · %). the cumulative number of deaths was [ ] (table ) and evd cases among hcw, of which died [ ] . to "implementing the full package of control interventions, including community engagement, acceptance, and ownership of the response" [ ] . ultimately there were , cases and deaths (case fatality rate = · %) [ ] . all districts in the country reported cases of evd [ ] . liberia had the highest number of hcw evd cases (n = ), but fewer deaths (n = ) [ ] than sierra leone. comparison of widely-affected countries table illustrates important demographic differences between these three countries. guinea's population ( , , ) is approximately twice sierra leone's ( , , ) and liberia's ( , , ), yet has the lowest health and education expenditures and literacy rate. guinea also has the lowest ethnic diversity. generally, we failed to detect correlations between the health indicator data (infant mortality rate, health expenditures as % of gdp, physician density, and hospital bed density) and evd-related outcomes (i.e., duration of the evd outbreak and the evd-specific mortality rate). the only statistically significant correlation was between outbreak duration and hospital bed density (r = − · , p < · ); fewer hospital beds per population was correlated with longer evd outbreak duration. country-specific information regarding the distribution of evd-related financial resources is opaque. initially, guinea received approximately half the funding sierra leone and liberia received. according to a who budget [ ] for july-december , guinea received $ , , , sierra leone received $ , , , and liberia received $ , , . nigeria, which ultimately was not widely affected, initially received nearly as much funding ($ , , ) as guinea. a who financial report covering october , reported the following geographic allocation of donor funds: guinea %, liberia %, sierra leone %, regional %, and other % [ ]. by the outbreak's end, approximately $ · billion had been dedicated to the ebola response during - [ ] . money allocation more closely correlated with country-specific incident evd cases than population size. the evd case and population-at-risk distribution was: guinea = · % cases, · % population-at-risk, sierra leone = · % cases, · % population-at-risk, and liberia = · % cases, · % population-at-risk. comparing the key indicator data provides limited insights into differences between guinea and sierra leone's country-specific epidemics; the epidemic was largely controlled in liberia (fig. ) by the time they were systematically collected. three indicators showed worse problems in guinea (number of unsafe burials, percent of tested samples that were evd positive, and days between symptom onset and case hospitalization) and one showed worse problems in sierra leone (number of districts with at least one security incident); the remaining indicators were nearly equivalent (fig. ) . evd cases were exported to seven additional countries and details regarding the country-specific outbreaks are organized by date of first confirmed evd case. of these, nigeria, senegal, and mali are west african countries with relatively high potential for ongoing transmission, particularly nigeria. however, a robust public health response prevented widespread transmission in these countries [ ] . the us, spain, the uk, and italy also had ≥ imported evd case. only the us reported in-country evd transmission. all of these countries reported their evd cases in , except italy, which was in may . table summarizes the number of cases, deaths, contacts followed, and date first declared ebola-free. the first case was reported july , in the lagos district and imported from liberia [ ] . this marks the first recorded time ebola spread to another country via airline travel. [ ] although the index case in nigeria transmitted the disease to at least five people who died, no transmission occurred on the flight, despite the infected adult male vomiting enroute [ ] . port harcourt was nigeria's second district to be affected with an evd case reported on august . nigeria was declared ebola-free on october , with a final case count of , eight deaths [ ] and ≥ contacts requiring follow-up [ ] . an adult male who travelled by road from his home in guinea to dakar was senegal's only evd case and reported on august , [ ] . he survived and senegal was declared ebola-free on october , . on september , , the first evd case in the us was reported in dallas, tx. an adult male travelling from liberia developed symptoms four days after arriving and expired on october . he transmitted evd to two female nurses. the first was diagnosed on october and discharged from the hospital on october . the second was diagnosed on october and discharged on october . she took two flights but no transmission was identified [ ] . the fourth case was in a male physician living in new york city who worked with doctors without borders. he was diagnosed october and discharged november . this physician potentially exposed multiple individuals during the first day of his illness at public places like restaurants and a bowling alley, but no transmission was identified [ ] . of these four cases, one died, two were imported, and two were from local transmission [ ] . there were an additional six evd cases acquired in west africa but treated in the us [ ] . of these, there were three hcws, an aid worker, a missionary, and a cameraman. all but one recovered. the u.s. was declared ebola-free on december , . health officials in mali investigated six suspect cases in april which helped prepare for the introduction of their first confirmed case in october . this evd case was a two-year-old female from guinea (reported on october ) [ ] . being symptomatic during her journey to mali, the girl died the following day. on october , a grand imam from siguiri prefecture in guinea was admitted to a clinic in bamako for acute kidney failure. ebola was not initially recognized and he died on october . because his symptoms began in guinea, he is reported as a guinean case. that case led to seven additional cases, with five deaths [ ] . by january , , when mali was declared ebola-free, the final case count was eight with six deaths [ ] . kayes and bamako were the only districts affected [ ] . on december , a female hcw returning from sierra leone was reported as the only evd case in the uk [ ] . she developed symptoms as she arrived in london. the people on a shared flight were classified by at least one source as exposed contacts, but were fig. indicator data from who ebola situation report treated as low risk. all passengers on board the shared flight (at from casablanca to london) and crew members were successfully contacted. public health england advised passengers sitting in the two rows adjacent (comprising passengers) to take their temperature twice daily for days [ ]. the patient survived and there were no additional cases [ ] . on may , , a case of ebola in a male italian volunteer hcw was reported. he returned from sierra leone on may , became symptomatic on may , and hospitalized on may . no inflight contact tracing was done. there were contacts to this case [ ] . on july , , italy was declared ebola-free having no additional evd cases and no deaths [ ] . who declared the ebola epidemic over on march , with , cases and , deaths across countries and three continents. of the three countries most widely affected, liberia was first to successfully control the epidemic (february ), which was before the key indicator data were collected (march ). the epidemic in sierra leone and guinea was largely controlled by april , but experienced sustained transmission into november . using the data from the present study, the key indicators that seemed to be substantially different between sierra leone and guinea were ) the number of unsafe burials, ) the percent of tested samples that were evd positive, ) the number of days between symptom onset and case hospitalization, and ) the number of districts with at least one security incident. there are a number of additional factors highlighted in the who ebola situation reports (and substantiated in the published literature). burial practices were cited as the single strongest risk factor for transmission; - % of all cases reported an exposure to a traditional burial [ ] . while previous outbreaks were largely controlled by using contract tracing, [ , ] this is the first evd pandemic with a prolonged duration where treatment and supportive care played an unquantified role in the response [ , , ] . another key difference was the strong stigma associated with evd infection in this outbreak as demonstrated by community attempts to hide cases from authorities. these areas came to be known as "shadow zones" [ ] and were accompanied by angry mobs attacking health care facilities and workers. the first such occurrence was on april , in macenta, guinea, [ ] and differences in events between sierra leone and guinea are shown in fig. . the pearson correlation estimates were limited by small sample size (three countries). the strongest country-level health indicator was between hospital bed density and the duration of the outbreak (r = − · ). increasing bed capacity was shown to contribute to controlling the ebola pandemic by two modeling studies, [ , ] however, the belated timing of the escalation of bed capacity likely limited its impact [ ] . we suggest that helping countries increase health infrastructure, particularly by having more hospital beds, as well as ongoing infection control training and capacity building, may help combat severe diseases such as evd. diagnostic equipment, largely unavailable during the early part of the outbreak, was also key for effective patient management. this pandemic highlights the need for the international community to respond to transnational outbreaks in a timely manner. despite the heroic efforts of early responders (e.g., local hcws, personnel with médecins sans frontières, cdc, and others), on a global scale, the response time was inadequately slow. this is possibly due to international politics and the sluggish turning wheels of governments who strive to do the right thing while meeting the needs of their citizens. the response by the us arguably began in march when cdc deployed personnel to investigate ebola cases in guinea, and further on july , with the activation of cdc's emergency operations center. however, because evd was largely out of the public's eye until the fall of , its response was similarly delayed. that is, the peak number of evd cases in liberia was september , ; one week after president obama's announcement to commit troops and provide additional aid to the ebola response effort [ ] and one month after who declared it a public health emergency of international concern. these events unlikely contributed substantially to controlling the evd pandemic [ ] . the us's response was impressive in terms of funding and personnel. by april , the us spent $ . billion on the ebola response effort. during - , the hhs global and domestic response mobilized more than personnel [ ] . approximately active duty commissioned corps officers in the us public health service established, operated and staffed the monrovia medical unit, an ebola treatment unit having more sophisticated medical capabilities than those in conventional ebola treatment units. the monrovia medical unit provided advanced supportive care to responders who became ill during this west african crisis and were the only us (non-private) personnel to provide direct patient care to persons infected with evd. this study is subject to certain limitations. perhaps the most substantial of which is the difficulty to quantify many factors affecting control of the outbreak. for example, the west african people educated their communities, facilitating the change of social practices, such as greetings with physical contact and modifying long-standing traditional burial practices [ , ] . education messages on fighting the epidemic were communicated through messaging in radio, billboards, etc. in conclusion, the west african ebola outbreak devastated three west african countries and propagated to seven additional countries on three continents. the public health response and subsequent control of the outbreak was different across guinea, liberia and sierra leone. the results from both the analysis of country-level data and the qualitative analysis generate hypotheses that inadequate infrastructure at the country level contributed to the delayed control of evd within the affected countries and contributed to lower survival rates. this ebola epidemic, as many in the past (such as hiv, west nile virus, sars, and cholera in haiti), shows us that epidemics can have a devastating global impact, and therefore should be considered everyone's problem. the recent zika virus epidemic followed many similar patterns highlighting that we all share the responsibility in responding to infectious outbreaks. many of the country-level factors, particularly the who key indicators were associated with controlling the epidemic. international political and public health leaders should assess the infrastructure of countries affected by communicable diseases. we used publicly available data for guinea, liberia, and sierra leone for the following demographic attributes: ) population and population density, ) median age (years), ) infant mortality rate (deaths/ live births), ) health expenditures (% of gdp), ) physician density (physicians/ population), ) hospital bed density (beds/ population), ) literacy (age + can read and write), ) education expenditures (% of gdp), ) urbanization (% urban), ) religions (% distribution), and ) ethnic groups (% distribution). pearson correlations were calculated and associated scatterplots were generated to assess associations between healthcare indicators for each country (health expenditures, physician density, hospital bed density, and infant mortality rate) and the duration of the evd outbreak (in days) and the evd-specific mortality rate. the outbreak-specific data were obtained from the weekly who ebola situation reports during which the key indicator data were uniformly published ( march , -october , . key indicators included ) number of confirmed cases, ) number of confirmed deaths, ) number and proportion of evd-positive reported community deaths, ) number of samples tested and the percent of positive evd results, ) percent of new confirmed cases from negative contacts, ) time between symptom onset and hospitalization (in days), ) case fatality rate among hospitalized cases, ) number of newly infected health workers, ) number of unsafe burials and the reported number of community deaths, ) number of districts with at least one security incident or other form of refusal to cooperate. line graphs for each key indicator were generated to assess differences between guinea, liberia, and sierra leone. we also summarize the impact of evd in the seven additional countries with limited evd transmission. the number of cases, deaths, case fatality rate, case-contacts requiring monitoring, date of first evd case, and date first declared ebola-free were summarized for each country. data were collected using index mundi, the who situation reports and internet searches, focusing on government sources, to identify relevant ebola-related information within the scope of outcome measures listed above. health and national security: a contemporary collision of cultures the routledge handbook of new security studies. london: routledge health security as a public health concept: a critical analysis the coming plague pandemics on the radar screen: health security, infectious disease and the medicalisation of insecurity ebola response team. ebola virus disease in west africa--the first months of the epidemic and forward projections security council approves threemonth extention for united nations mission in liberia accessed ebola situation report ebola situation report accessed ebola response team. after ebola in west africa--unpredictable risks, preventable epidemics estimating the future number of cases in the ebola epidemic--liberia and sierra leone one year into the ebola epidemic: a deadly accessed investigating the zoonotic origin of the west african ebola epidemic bat-filled tree may have been ground zero for the ebola epidemic ebola virus disease--sierra leone and guinea cdc classification of countries with reported ebola cases for evaluation of persons in the united states accessed ebola situation report who strategic action plan for ebola outbreak response: annex accessed accessed who: ebola response roadmap update cases of ebola diagnosed in the united states accessed how many ebola patients have been treated outside of africa accessed ebola virus disease -united kingdom accessed ebola situation report - accessed world health organization. factors that contributed to undetected spread of the ebola virus and impeded rapid containment. in: one year into the ebola epidemic. geneva: world health organization ebola --new challenges, new global response and responsibility the ebola outbreak, - : old lessons for new epidemics measuring the impact of ebola control measures in sierra leone impact of bed capacity on spatiotemporal shifts in ebola transmission impact of interventions and the incidence of ebola virus disease in liberiaimplications for future epidemics empty ebola clinics in liberia are seen as misstep in u.s. relief effort engaging 'communities': anthropological insights from the west african ebola epidemic ebola situation report accessed who: ebola response roadmap situation report ebola situation report - accessed accessed this research was supported in part by the defense intelligence agency, grant # hhm - - - (pi: regens). the views and conclusions contained herein are those of the authors and should not be interpreted as necessarily representing the official policies or endorsements, either expressed or implied, of dia, the indian health service, or the us government. funding was provided through the defense intelligence agency, grant # hhm - - - (pi: regens). the data are publically available and the sources are referenced within the manuscript. the data collected from the weekly who situation reports were summarized in an ms excel file which will be made publically available pending acceptance of the manuscript for publication.authors' contributions amw, mm, and jlr contributed to study conception and design. amw and mm were primarily responsible for data analysis. all authors contributed to interpreting the data. all authors contributed to the drafting/revising of the manuscript for important intellectual content. all authors have provided final approval of the submitted manuscript. all authors agree to be accountable for the accuracy and integrity of the work.ethics approval and consent to participate not applicable. we did not seek approval from an ethics board/institutional review board because the data were from publically available sources. the authors declare that they have no competing interests. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - uqa j authors: cervera, héctor; ambrós, silvia; bernet, guillermo p; rodrigo, guillermo; elena, santiago f title: viral fitness correlates with the magnitude and direction of the perturbation induced in the host’s transcriptome: the tobacco etch potyvirus—tobacco case study date: - - journal: mol biol evol doi: . /molbev/msy sha: doc_id: cord_uid: uqa j determining the fitness of viral genotypes has become a standard practice in virology as it is essential to evaluate their evolutionary potential. darwinian fitness, defined as the advantage of a given genotype with respect to a reference one, is a complex property that captures, in a single figure, differences in performance at every stage of viral infection. to what extent does viral fitness result from specific molecular interactions with host factors and regulatory networks during infection? can we identify host genes in functional classes whose expression depends on viral fitness? here, we compared the transcriptomes of tobacco plants infected with seven genotypes of tobacco etch potyvirus that differ in fitness. we found that the larger the fitness differences among genotypes, the more dissimilar the transcriptomic profiles are. consistently, two different mutations, one in the viral rna polymerase and another in the viral suppressor of rna silencing, resulted in significantly similar gene expression profiles. moreover, we identified host genes whose expression showed a significant correlation, positive or negative, with the virus' fitness. differentially expressed genes which were positively correlated with viral fitness activate hormone- and rna silencing-mediated pathways of plant defense. in contrast, those that were negatively correlated with fitness affect metabolism, reducing growth, and development. overall, these results reveal the high information content of viral fitness and suggest its potential use to predict differences in genomic profiles of infected hosts. fitness is a complex parameter often used by evolutionary biologists and ecologists to quantitatively describe the reproductive ability and evolutionary potential of an organism in a particular environment (linnen and hoekstra ; orr ) . despite this apparently simple definition, measuring fitness is difficult and most studies only measure one or more fitness components (e.g., survival to maturity, fecundity, number of mates, or number of offspring produced) as proxies to total fitness (linnen and hoekstra ; orr ). in the field of virology, it has become standard to measure fitness by growth-competition experiments in mixed infections with a reference strain (holland et al. ; wargo and kurath ) . with this experimental set up, fitness is just the relative ability of a viral strain to produce stable infectious progeny in a given host (cell type, organ, individual, or species) when resources have to be shared with a competitor (domingo and holland ) . regardless its limitations, this approach provides a metric for ranking viral strains according to their performance in a particular environment/host. such a fitness measure has been pivotal for quantitatively understanding many virus evolution processes: the effect of genetic bottlenecks and accumulation of deleterious mutations (chao ; duarte et al. ; de la iglesia and elena ) , the rates and dynamics of adaptive evolution into novel hosts , the pleiotropic cost of host range expansion (novella, clarke, et al. ; turner and elena ; lali c et al. ) , the cost of genome complexity (pesko et al. ; willemsen et al. ) , the cost of antiviral escape mutations (novella et al. ; westerhout et al. ; mart ınez-picado and mart ınez ) , the topography of adaptive fitness landscapes (da silva and wyatt ; lali c and elena ; cervera et al. ) , and the role of robustness in virus evolution (codoñer et al. ; sanju an et al. ; novella et al. ) . but differences in viral fitness should also matter in genome-wide studies seeking to understand the mode of action of the viruses (i.e., the precise way they interact with their hosts). it has been argued that an integrative systems biology approach to viral pathogenesis would result in a better understanding of pathogenesis and in the identification of common targets for different viruses, therefore serving as a guide to a more rational design of therapeutic drugs (tan et al. ; viswanathan and früh ; bailer and haas ; barab asi et al. ; friedel and haas ; elena and rodrigo ; finzer ) . pioneering studies have ignored the high genetic variability of viruses in fitness and in mode of action. experimental evidence supports that even single nucleotide substitutions have significant effects on viral fitness regardless of whether they are synonymous or nonsynonymous, or they affect coding or noncoding genomic regions (sanju an et al. ; carrasco, de la iglesia, et al. ; domingo-calap et al. ; peris et al. ; acevedo et al. ; bernet and elena ; visher et al. ) . a common trend among all these studies is that, whenever fitness is evaluated in the standard host, the distribution of mutational effects is highly skewed toward deleterious effects, with a large fraction of mutations being lethal. furthermore, increasing evidence suggests that the distribution of fitness effects increases as the genetic divergence among two hosts (e.g., a tested host and the natural one) increases (lali c et al. ; vale et al. ; lali c and elena ; cervera et al. ) . together, all these observations suggest that the fitness of a given viral genotype depends not only on its own genetic background but also on the host where fitness is evaluated. arguably, differences in viral fitness reflect differences in the virus-host interaction. as cellular parasites, viruses need to utilize all sort of cellular factors and resources, reprogram gene expression patterns into their own benefit, and block and interfere with cellular defenses. all these processes take place in the host complex network of intertwined interactions and regulations. interacting in suboptimal ways with any of the elements of the host network may have profound effects in the progression of a successful infection and therefore in viral fitness; inefficient interactions may result in attenuated or even abortive infections. little is known about how viral fitness informs about the underlying changes occurring in host gene expression and protein function at a genome-wide scale. in this work, we have investigated the potential association between viral fitness and host transcriptional regulation upon infection as a first step into this direction. we have characterized the transcriptomic profiles of nicotiana tabacum l. var xanthi nn plants inoculated with a collection of genotypes of tobacco etch virus (tev; genus potyvirus, family potyviridae) that differ in their fitness in this natural host. analyses of gene expression data allowed us to characterize differential gene expression upon infection with different tev genotypes, as well as to identify sets of candidate genes whose expressions positively or negatively correlated with the magnitude of tev fitness. differences in expression for representative genes from these two categories were experimentally validated by an alternative method. differences in viral fitness and host symptomatology figure shows relevant information about the seven tev genotypes used for this study. the mutant genotypes differ from the wild-type (wt) genotype in a rather limited number of nonsynonymous mutations ( or ). however, their fitness values and the severity of symptoms induced differ widely. in this study, the fitness of each mutant genotype was estimated as the ratio of malthusian growth rates of the mutant and the wt (see materials and methods for details). significant differences existed among the fitness values of the seven selected b ). figure c illustrates the differences in symptoms induced by each one of the seven genotypes. symptoms ranged from the asymptomatic infection or local chlorotic spots characteristic of mutant as , the mild etching of mutant cla and the severe etching induced by the wt and the other mutants. no correlation exists between virus fitness and symptoms, a finding previously reported for this experimental system (carrasco, de la iglesia, et al. ). differences in viral fitness are associated with differences in the magnitude of the change in the host transcriptome first, we sought to test whether differences in tev fitness might be associated with differences in the gene expression profiles of infected plants. we hypothesized that viral fitness results from a particular interaction between virus and host factors and assumed that the outcome of infection of a wt virus in its natural host results from an optimal (from the virus perspective) modulation of the host's gene expression profile. as viral fitness is reduced, interactions are less optimal and, consequently, the gene expression profile of the plant will be increasingly different from that resulting from the infection with the wt virus. to test this hypothesis, we infected n. tabacum plants with each one of the seven tev genotypes described earlier. eight-day postinoculation (dpi) symptomatic tissues were collected for all mutants except for the very low fitness mutant as , for which tissues were collected dpi because the delay in symptoms appearance and severity ( fig. c ). total rnas were extracted, normalized, and used to hybridize n. tabacum gene expression  k microarrays (agilent). slides were handled as described in the materials and methods section; intensity signals were normalized using tools in babelomics (alonso et al. ) . normalized expression data are contained in supplementary file s , supplementary material online. figure a shows the clustering (unweighted average distance method; upgma) of average expression data for those genes that significantly changed expression ( -fold) among plants infected with the seven viral genotypes ( -way anovas with false discovery rate (fdr) correction; overall p < . ) relative to the mock-inoculated plants. regarding individual genes, two major clusters can be distinguished, one corresponding to the overexpression of genes related to stress response and a second one corresponding to the underexpression of genes involved with metabolism and plant development. to further explore the similarity in the perturbation induced by each viral genotype into the plants' transcriptome, we computed all pairwise pearson productmoment correlation coefficients (r) between the mean expression values for all genes in the microarray. these correlations were used as a measure of similarity to build a upgma dendrogram. the rationale for this analysis is as follows: the more correlated two expression profiles are, the more similar the effects induced in infected plants. when comparing expression profiles from a pair of infected plants, a high correlation may indicate that genes that changed expression relative to the mock-inoculated plants, are exactly the same in both samples, showing a similar expression pattern. conversely, if genes with differential expression do not match in the two profiles being compared, then the correlation will be low. figure b shows both the heat-map of the correlation coefficients and the resulting dendrogram. three clusters result from this analysis ( fig. b ). the first cluster is constituted by the three viral genotypes with the higher fitness values, that is, wt, pc , and pc . genotypes of intermediate fitness cla , pc , and cla constitute a second cluster. finally, plants infected with as show the most dissimilar gene expression profile. the heat-map is shown with viral genotypes ordered according to the upgma clustering. correlations decreased as the distance in the cladogram increases. within clusters, r > . , whereas between clusters the correlations ranged between . < r < . , except for plants infected with as , whose similarity with other infected plants was r < . . next, to further investigate the similarity between expression profiles of plants infected with different tev genotypes, we performed a principal components (pc) analysis of all the gene expression data. the percentage of total observed variance explained by the first three components was $ % (the first pc itself explained %). figure c shows the distribution of values in the space defined by the three first principal components. results are equivalent to those obtained with the two previous clustering methods, where genotypes are classified into three groups. wt, pc , and pc are closer in the space and characterized by positive values of first pc but negative values of the second and third pcs. cla , pc , and cla form a second group, with positive values of first and second pcs but negative values of the third. as before, as effect on host transcriptome is clearly different, and has negative values of the first and second pcs but positive of the only genes that show significant differences among all infections (oneway anova with fdr, adjusted p < . ) are included in the heat-map. hierarchical clustering of genes done with upgma by using the correlations between all pairs of mean profiles as distance metric. genes down-regulated (in blue) mainly correspond to metabolic and developmental processes, whereas genes up-regulated (in red) mainly correspond to stress responses. (b) tev genotypes clustered (upgma) according to the similarity of the mean expression profiles of plants infected with each one of them. the heat-map represents the value of the pearson's correlation coefficient between pairs of mean profiles. (c) representation of the three major principal components from the data shown in panel (b) . the three first pcs explain up to % of the total observed variance. lines link each genotype with the centroid of the d space. the arrow represents a putative trajectory of increasing viral fitness. (d) association between viral fitness and the magnitude of the perturbation (vs. mockinoculated control plants) both relative to wt (p ¼ . ). (e) association between viral fitness and the distance of each genotype to the wt (p ¼ . ), from the dendrogram shown in panel (b). cervera et al. . doi: . /molbev/msy mbe third. interestingly, figure c shows that genotypes are located in this principal component space following a trajectory of increasing fitness values (indicated by the arrow in fig. c ). along this trajectory, pcs switch sign in different directions. this transition suggests that the over-or under-expression of a set of genes is associated with particular levels of viral fitness: low fitness as is characterized by a positive third pc and a negative first pc while high fitness viruses are characterized by the opposite sign. that is, over-or underexpressed genes are not progressively accumulated as long as viral fitness changes. these genes will be evaluated in the following sections. following from our working hypothesis, if the wt virus has evolved to optimize its interaction with the host, it is logical that small departures in viral fitness will be associated with small deviations between the transcriptomes of plants infected with the wt virus and with viruses whose fitness is close to the wt. conversely, the less similar fitness between the wt and mutant viruses, the more dissimilar would be the transcriptional profiles of infected plants. to test this prediction, we have explored the following: ) the correlation between the similarity of transcriptional profiles of plants infected with the wt tev and with each mutant (again using pearson's r) and fitness and ) the correlation between the distance from wt in the cladogram shown in figure b and fitness. the results of these analyses are shown in figure d and e. as expected, both correlations were significant (r ¼ . , p ¼ . and r ¼ À . , p ¼ . , respectively; in both cases df) and of the expected sign. viral fitness and perturbation of host's transcriptomes . doi: . /molbev/msy mbe plants. the number of down-expressed degs ranges between (for as ) and , (for cla ), while in the case of upexpressed degs the range is slightly narrower: from (as ) to , (cla ). figure b illustrates the number of degs in common between all pairs of transcriptomes from infected plants. the heat-map shows a pattern of modularity, with three well-defined modules. the first module contains the three viruses with highest fitness (wt, pc , and pc ), the second module contains the three viruses with intermediate fitness (pc , cla , and cla ), and the very low fitness genotype as is the only member of the third module. the number of shared degs within each of these modules is > % of total. the number of shared degs between modules drops < %. next, following the same rationale as the previous section, we sought to determine whether the number of degs also depends on the difference in fitness from wt. in this case, we hypothesized that the overlap in the lists of degs must be similar for wt and viruses of equivalent fitness (e.g., pc or pc ), whereas the magnitude of the overlap between deg lists would decrease as differences in fitness became larger. figure c shows the counts of degs that are differentially expressed (for up-and down-expressed genes) between wt and the other six viral genotypes. as expected, pc , pc , and pc alter the same genes, though in a different magnitude (as shown in the previous section). the number of genes that are not in common with wt increases from cla ( ), cla ( ), and as ( ) of particular interest is the similarity between pc , a mutant of the replicase nib gene, and cla , a mutant of the vsr hc-pro gene. these two mutations led to close fitness values ( fig. a ), but also resulted in significantly similar gene expression profiles ( fig. b ). at first sight, one may argue that their impact in transcriptomic profiles should be different since these mutations affect virus proteins that are functionally unrelated. however, our results suggest that the effects on the overall virus-host interaction of each mutant are canalized in the same way. this clearly shows that viral fitness contains high information about the virus-host interaction. lists of genes are difficult to interpret and functional analyses provide a good tool to cluster genes into groups with related functions. to this end, we performed an analysis of enriched functional categories (go terms) for each viral genotype. figure illustrates the way that plants infected with each one of the seven tev genotypes differ in the functional categories significantly overrepresented relative to the mockinoculated plants. figure shows the go terms ordered from the highest to the lowest viral fitness. the upper plane shows the functional categories altered in plants infected with wt tev, with metabolic process (go: ) containing the largest number and photosynthesis (go: ) the smallest. regulation of response to biotic stimulus (go: ), defense response (go: ), immune system process (go: ), protein modification process (go: ), hormone-mediated signaling (go: ), and cell death (go: ) are all enriched in up-expressed degs, while photosynthesis, lipid metabolic process (go: ), and regulation of nitrogen compound metabolic process (go: ) are categories significantly enriched in down-expressed degs. categories such as metabolic process, unspecific response to stress (go: ), response to stimulus (go: ), response to abiotic stimulus (go: fig. . functional analysis associated to differential gene expression. artwork of meaningful biological processes (in a plane). categories that are overrepresented in the two lists of degs for each tev genotype (up-and down-regulated), either in one of them or in both, are indicated with different colors. in red, we represent categories that are significantly enriched by up-expressed degs, in blue categories that are significantly enriched by down-expressed degs, and in pink categories enriched in both types of degs; the surface of each circle is proportional to the number of degs included in each category. enrichments were evaluated by fisher's exact tests with fdr (adjusted p < . ). the different planes are organized according to viral fitness. we considered infected versus mock-inoculated control plants (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.). cervera et al. . doi: . /molbev/msy mbe ), localization (go: ), or transport (go: ) are enriched in both types of degs. therefore, overall speaking, genes involved in different aspects of plant defense pathways and response to infection are up-expressed, whereas genes involved in metabolism and photosynthesis are down-expressed. the second fitness plane corresponds to genotypes pc and pc , both of mild effect and carrying mutation in the ci gene. the most remarkable difference between these two genotypes and the rest of genotypes is the significant enrichment in up-expressed degs related to signal transduction (go: ) and regulation of gene expression (go: ). drifting down in the virus fitness scale, the next plane in figure corresponds to genotype pc , which shows a similar distribution of gos as the wt except for a lack of enrichment in up-expressed degs in the regulation of response to biotic stress category. next plane corresponds to genotypes cla and cla hyposuppressors of moderate fitness and carrying point mutations in the hc-pro gene. plants infected with these two viral genotypes differ from plants infected with the wt virus in three main functional categories: the loss of significant enrichment in the hormonemediated signaling category, and a significant enrichment in up-expressed degs into the localization and transport categories. finally, the bottom plane in the fitness scale corresponds to genotype as ( fig. ), which has a very week vsr activity, very low fitness and induces no symptoms or very mild symptoms. these differences in fitness and severity of symptoms have a direct translate into the enrichment of the different functional categories. compared with wt, metabolic process is enriched with down-expressed degs while nonspecific response to stress is very much enriched now in up-expressed degs, and response to stimulus, protein modification process, localization, and transport are not enriched in any particular type of degs. moreover, no significant enrichment in the hormone-mediated signaling module was found in plants infected with as , or for the other hc-pro mutant genotypes cla and cla . taken together, the results shown in the previous sections suggest that the transcriptomic response of plants to infection varies with the fitness of the virus being inoculated. this observation motivated us to identify genes whose expression significantly correlates with viral fitness; that is, systematic changes in virus fitness are associated with an increase or decrease in the expression level of a particular gene. this is a correlation analysis and as such does not assume a functional dependence between viral fitness and the expression of individual host genes. yet, it may provide a list of candidate genes to be considered as determinants of viral fitness. we computed a nonparametric spearman's correlation coefficient between viral fitness and the normalized degree of expression (z-score) for each one of the previously characterized degs correlation plots between host gene expression and viral fitness for those genes that significantly vary across all viral infections (one-way anova with fdr, adjusted p < . ), and that exhibit a significant positive (upper panel; red dots) or negative (lower panel; blue dots) trend (spearman's correlation test, p < . ). expression data represented as z-scores. (b) pie charts of biological and molecular functions. on the top, for genes whose expression increases with tev fitness (red dots in panel a); on the bottom, for genes whose expression decreases with fitness (blue dots in panel a). (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.). viral fitness and perturbation of host's transcriptomes . doi: . /molbev/msy mbe next, we sought to explore which functional categories and molecular functions, if any, were enriched among these two subsets of degs. results are shown in figure b and functional annotations are all reported in the supplementary file s , supplementary material online. there are significant differences in the distribution of positively and negatively correlated degs into different functional categories ( fig. b , left column; homogeneity test: v ¼ . , df, p < . ), although the difference in magnitude is moderate (cram er's v ¼ . ). among degs whose expression is positively correlated with viral fitness, biological regulation (go: ) and developmental processes (go: ) are strongly enriched compared with the negatively correlated degs. by contrast, negatively correlated degs disproportionally contribute more than positively correlated ones to the categories response to stimulus, localization, metabolic processes, and cell death. focusing on molecular functions ( fig. b , right column), a significant difference also exists among degs whose expression is positively-and negatively correlated with viral fitness (homogeneity test: v ¼ . , df, p < . ), with a magnitude of the difference in the moderate to large magnitude range (cram er's v ¼ . ). on the one hand, among positively correlated degs, nucleic acid binding (go: ) shows the largest departure from negatively correlated ones. on the other hand, catalytic activity (go: ) and transporter activity (go: ) are the two molecular functions that appear to be enriched among negatively correlated degs. together, these results suggest that positively correlated degs play a role in the transcriptional regulation of host defenses. by contrast, degs with negative correlation between expression and tev fitness participate more in catalytic and transport activities than genes with positive correlation, suggesting a redirection of resources by the host that is not independent of viral fitness. normalized expression data used in figure were estimated from changes in spot intensity in the n. tabacum gene expression  k microarrays (agilent). to validate these results with rt-qpcr, we selected four positively correlated and five negatively correlated degs that cover the entire range of observed significant spearman's correlation coefficients (supplementary file s , supplementary material online). they represent different biological functions and are expressed at different developmental stages and under different environmental situations (see below). the four positively correlated degs selected were (ordered according to the observed r s values): dicer-like gene (dcl ; r s ¼ . ), the gene encoding for the vq motif-containing protein (vq ; r s ¼ . ), the gene encoding for the gast protein homolog (gasa ; r s ¼ . ), and a gene encoding for a member of the lipase/lipoxygenase plat/lh family (plat ; r s ¼ . ). dcl is involved in defense response to viruses, maintenance of dna methylation and production of ta-sirnas involved in rna interference (parent et al. ) . vq is a negative transcriptional regulator of lightmediated inhibition of hypocotyl elongation that likely promotes the transcriptional activation of phytochrome interacting factor (pif ) during early seedling development, and participates in the jasmonic acid-mediated (ja) plant basal defense, as the vq proteins interact with wrky transcription factors . gasa encodes for a gibberellin-and brassinosteroid-regulated protein possibly involved in cell elongation (bouquin et al. ) , also reported to be involved in resistance to abiotic stress through ros signaling (o'brien et al. ) . plat encodes for a lipase/lipoxygenase that promotes abiotic stress tolerance (hyun et al. ) , is a positive regulator of plant growth, and regulates the abiotic-biotic stress cross-talk. the negatively correlated degs selected for validation are the adenosine kinase gene (adk ; r s ¼ À . ), the gene encoding for the small b chain of the rubisco (rbcs b; r s ¼ À . ), the agamous-like gene (agl ; r s ¼ À . ), the factor of dna methylation gene, (fdm ; r s ¼ À . ), and the granule-bound starch synthase gene (gbss ; r s ¼ À . ). adk encodes for an adenosine kinase involved in adenosine metabolism, including the homeostasis of cytokinines (schoor et al. ) , controls methyl cycle flux in a s-adenosyl methioninedependent manner and plays a role in rna silencing by methylation. rbcs b is involved in carbon fixation during photosynthesis and in yielding sufficient rubisco content (zhan et al. ) . agl is a dna-binding mads-box transcription activator modulating the expression of homeotic genes involved in flower development and maintenance of inflorescence meristem identity, transitions between vegetative stages of plant development and in tolerance to cold (lee et al. ) . fdm is an sgs -like protein that acts in rna-directed dna methylation participating in the rna silencing defense pathway (xie et al. ) . gbss is involved in glucan biosynthesis and responsible of amylase synthesis that is essential for plant growth and other developmental processes (denyer et al. ) . rt-qpcr-based relative expression data were calculated using the ddc t method normalized by each one of the two reference genes and then averaged (see materials and methods). to make expression data by microarray readings and rt-qpcr readily comparable, they were both transformed into z-scores. figure a shows the comparison of the two expression measures for the four degs with positive correlation with tev fitness and figure b for the five degs with negative correlation with tev fitness. two different plots are presented for each gene. in all cases, the left plot illustrates the relationship between the expression z-scores obtained with the microarray method (x-axis) and with rt-qpcr method (y-axis) for each one of the seven tev genotypes; the solid lines indicate the best linear fit between these two data sets. in this representation, a regression line of slope is expected if both quantification methods provide identical z-scores. in all nine cases, both expression z-scores are highly and significantly correlated; pearson's r values ranged from . (vq ) to . (gasa ) (in all cases df, -tailed p . ). if a more stringent holm-bonferroni correction of the overall significance level is taken, then vq would not remain significant. for each gene, the right plot shows both expression z-scores as a function of tev fitness; solid lines represent mbe the best linear fit between normalized expressions and tev fitness. in this representation, the more overlap between the two regression lines, the better the agreement between both quantitative methods. in this representation, vq and adk show the largest departure between both regression lines, though even in these extreme cases, the difference was not large enough as to be significant in a nonparametric wilcoxon's signed ranks test (p ! . in all nine cases) or in a student's t-test for the comparison of regression coefficients (p ! . ). thus, we conclude that, at least for the sample of genes analyzed, the observed correlations between host's gene expression and viral fitness are consistent for both experimental methods used to evaluate levels of gene expression. we further delineated a picture of virus-plant interaction reflected in precise alterations of transcriptomic profiles and regulatory networks. plant-virus interactions result from the confrontation of two players with opposed strategies and interests. from the plant perspective, activation of basal defenses, immunity, hormone-regulated pathways, and rna-silencing (some of which are not virus-specific) will result in an immediate benefit to control virus replication and spread. we found that some plant defense responses are expressed upon infection regardless the fitness of the virus, whereas other defenses are induced progressively as viral fitness increases. consistent with the first mode, we observed the activation of the genes eds and pad , components of r gene-mediated disease resistance with homology to lipases, in every infection (fig. a ). these are master regulators of plant defenses that connect pathogen signals with salicylic acid signaling (cui et al. ). salicylic acid is involved in resistance to a broad spectrum of pathogens, and in particular viruses (alamillo et al. ; conti et al. ) . consistent with the second mode, we observed the activation of many genes involved in defenses in proportion to tev fitness ( fig. a ). for example, the dcl and ago genes-key for the rna silencing response-, genes modulating resistance to pathogens such as the subtilisin-like protease (sbt . ), or genes expressing proteins involved in hormone-regulated defenses such as gasa and vq , brassinosteroids (e.g., brassinosteroid enhanced expression bee , brassionosteroid-signaling kinases and , brassinosteroid bak bri -associated receptor kinase), ethylene response factors (e.g., erf b, erf , or cytokine response factor crf ), and members of abscisic acid perception pathway (e.g., pyl -rcar , a regulatory component of the abscisic acid receptor family). likewise, genes involved in methylation-mediated stress responses, such adk , fdm or the methionine adenosyltransferase mat reduce their expression as virus replication is more efficient, thus resulting in less methylation and increased expression of genes that participate in apoptosis and posttranscriptional gene silencing (schoor et al. ) . in this way, the overexpression of genes that modulate histone acetylation or chromatin organization, such as the histone acetyltransferase hac and the chromatin remodeling factor r (chr ) would regulate differentiation, apoptosis, transcriptional activation, or ethylene response just as viral fitness increases. however, these activations have a cost, mainly in terms of resources that can be invested into secondary metabolism and development. consistent with this idea is the fact that many genes participating in metabolic processes (e.g., cysc , a cysteine synthase) are highly repressed upon infection ( fig. a ). there are also central genes for the plant metabolism whose repression correlates with viral fitness, such as gbss , photosystem components, or assembly factors (e.g., lhcb . and hcf ), rubisco subunits and atpases, catalases, transketolases, nucleotide and phosphate transporters, synthases involved in flavonoid, isoprenoid, ascorbate, or tryptophan biosynthesis, and gapdh ( fig. ). diverting host cell resources and reprograming the metabolic machinery to support rna metabolism and atp production is a general strategy both of plant ) and animal viruses (tang et al. ; tiwari et al. ) . tev achieves this reprogramming by altering the expression of a series of genes to its own benefit. for example, we found the expression of genes involved in actin cytoskeleton organization such as adf and pfn to be negatively correlated with tev fitness. the profilin pnf is an actinbinding protein and adf participates in the depolymerization of actin filaments that results from microbial-associated molecular patterns being recognized by the corresponding pattern-recognition receptors (henty-ridilla et al. ) . therefore, by downregulating this function, longer and more stable actin filaments are produced that virions can use to move around the cell from the er-associated replication factories to plasmodesmata. another example is the repression observed for the ubp b gene, a negative regulator of potyvirus translation, that would allow for a more optimal virus accumulation (hafr en et al. ) . genes involved in nonsense-mediated decay (nmd) defenses (wachter and hartmann ) , such as the atp-dependent rna helicase upf , also show reduction in levels of expression. other group of proteins that show alteration during viral infection are those involved in protein degradation, via ubiquitination and downstream into the proteasome pathway (e.g., ubiquitin-protein ligase , upl ; ubiquitin-conjugating enzyme , ubc ; ubiquitin e variant b, mmz ; ein -binding f box protein , ebf) or via autophagy (e.g., the atp-driven chaperone cdc c and the plant autophagy adaptor nbr ). moreover, tev activates in a fitness-dependent manner the expression of genes rh , an rna helicase, and pcap , a fig. . continued measured by microarray and in blue by rt-qpcr) and viral fitness. the solid lines represent linear models; the closer the slopes of both lines, the more similarity between microarray and rt-qpcr expression data. (a) genes whose expression increases with viral fitness (cases from fig. a , red dots). (b) genes whose expression decreases with viral fitness (cases from fig. b, blue dots) . bidimensional error bars represent sd. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) cervera et al. . doi: . /molbev/msy mbe membrane-associated cation-binding protein, also required by potyviruses for cell-to-cell movement (vijayapalani et al. ) (fig. a ), and of a diversity of transcription factors including global (e.g., gra , gte ), sequence-specific (e.g., sacl and spl ), gata/nac family members (e.g., gata , nac , nac ), bzip g-box finding factors (e.g., gbf and bzip ), and involved in homeotic gene expression (e.g., agl and homeobox- ). we also found genes related to genome integrity, (e.g., the cohesins syn and smc , and the chromatin protein spt ), dna replication and nucleosome assembly, alternative splicing (e.g., sf , an homolog nuclear splicing factor), chromatin transition (e.g., spt , an histone chaperone involved in transcription elongation from rnapolii promoters and regulation of chromatin transitions; or the histone acetyltransferase hac , a coactivator of gene transcription with a major role in controlling flowering time and also essential for resistance to bacterial infections), dna replication, and cell division (e.g., the mitotic cohesin rad and the cyclin-dependent protein kinase cych ). however, not all host factors recruited by the virus present alterations in their expression. according to our data, the translation initiation factor eif e, known to be exploited by tev for its own translation (robaglia and caranta ) , was found to be unperturbed ( fig. a ) while eif a and eif g expression is positively correlated with tev fitness. in essence, there are genes that are significantly altered (up or down) upon infection irrespective of the ability of the virus to replicate, genes whose expression correlates with this ability (positively or negatively), and genes that remain unaltered. nevertheless, this picture of virus-plant interaction may be biased by the limited number of viral genotypes analyzed in this work. three out of six genotypes correspond to hc-pro mutants. as a multifunctional protein, it is not surprising that different fitness levels can be reached by introducing mutations in different functional domains. but, certainly, more mutants should be analyzed in future work to provide a comprehensive picture and avoid bias toward certain virus proteins. in addition, we here focused on the transcription regulation, but other interlinked networks exist in the cell (e.g., metabolism, protein-protein interactions, . . .). to provide an insight on these other networks, we constructed the interactome ( fig. b ) of hc-pro with the host proteins known to interact with this virus protein (revers and garc ıa ) . we then contextualized our gene expression data over multiple tev infections. many of the cellular functions in which hc-pro participate (protein degradation, translation, redox processes, and cation signaling) are not regulated transcriptionally upon infection (or regulated marginally). presumably, the virus exploits these processes for its own benefit (mainly to enhance replication and movement within a cell), and the normal expression of the corresponding genes is sufficient for such subversion. by contrast, rna silencing and methylation are functions involved in defense against pathogens that are quantitatively regulated, as a sort of control strategy exerted by the plant, as long as they are needed, that is, according to viral fitness. biological systems and processes can be analyzed and modeled at every scale of complexity. it is expected that components of each level of complexity may contribute to determine the behavior of processes at other levels. the complexity at the molecular level (i.e., the lowest level of biological organization) is astonishing both in terms of possible elements (genes, functional rnas, proteins, and metabolites) and of interactions among them (barab asi et al. ) . thus, if the components at lower scales of complexity, presumed to be more accessible experimentally, are informative enough about the underlying processes, they result in excellent proxies to understand biological systems. in the case of a disease (in plants or animals), the symptoms exhibited by the organism have been traditionally used as macroscopic indicators of what occurred within the organism. this allows diagnosing diseases without the need to perform further analyses. however, symptoms are generally uncoupled from the magnitude of the perturbation at the molecular level in the host (with respect to a healthy state) (barab asi et al. ; finzer ) . this is particularly true in the case of a virusinduced disease, a paradigmatic example of a system-wide perturbation (tan et al. ; viswanathan and früh ; bailer and haas ; friedel and haas ; elena and rodrigo ) . here, we have studied for the first time the use of viral fitness as an indicator of the molecular changes occurring in the host upon infection. after all, the progress of a viral infection depends on the fitness of the virus mutant swarm. classically, viral fitness has been evaluated by means of parameters describing the absolute growth and accumulation, by competition experiments (holland et al. ; wargo and kurath ) , or even by correlating it with the development of host's symptoms (carrasco, de la iglesia, et al. ; wargo and kurath ) . we focused on the infections exerted by different genotypes of a given virus in the same host. fitness differences among genotypes are due to several causes. first, they may be a direct consequence of the effect of mutations on viral proteins, perhaps even resulting in altered folding, and thus jeopardizing their functions. second, in the case of mutations affecting regulatory regions (e.g., rna stems and loops), the effect may be due to altered structural configurations that impede the binding of virus own proteins or of cellular factors. plenty of examples illustrate the effect of mutations via these two mechanisms (bernet and elena ) . a third, more intriguing, yet poorly explored possibility is that mutated viral components (i.e., rnas and proteins) may interact in nonoptimal ways with the complex network of genetic and biochemical interactions of the cell as a whole. interacting in nonoptimal ways with any of the elements of the host regulatory and biochemical networks may have profound effects in the progression of a successful infection and therefore of viral fitness. in this work, we considered mutations affecting the ci protein (with rna helicase, atpase, and membrane activities), the viral replicase nib, and the hc-pro protein (vsr, protease, and helper-component during transmission by aphids). our results point out that fitness, irrespective of what type of mutation is introduced, is a good indicator of how a given mutant reprograms gene expression patterns, to its own benefit or as a consequence of cellular defenses (e.g., fig. c ). despite the interest of this hypothesis, none of the early studies tackled the relationship between genotype and fitness of the virus and transcriptomic profiles of the host in a systematic manner, but rather focused on comparing two viral genotypes. evolution experiments simulating the spillover of tev from its natural host n. tabacum into a novel, poorly susceptible one, arabidopsis thaliana, have shown that adaptation of tev to the novel host (i.e., concomitant to large increases in fitness) was associated with a profound change in the way the ancestral and evolved viruses interacted with the plant's transcriptome, with genes involved in the response to biotic stresses, including signal transduction and innate immunity pathways, being significantly underexpressed in plants infected with the evolved virus than in plants infected with the ancestral one (agudelo-romero et al. ) . further evolution experiments into different ecotypes of a. thaliana that differed in their susceptibility to infection illustrated a pattern of adaptive radiation in which viruses were better adapted to their local host ecotype than to any alternative one, but with viruses evolved into more restrictive ecotypes being more generalists than viruses evolved in the more permissive ones (hillung et al. ) . interestingly, these differences in fitness had a parallelism with differences in the transcriptomic profiles of plants from different ecotypes; the more generalist viruses altering similar genes in every ecotype, whereas the more specialist viruses altered different genes in different ecotypes (hillung et al. ) . similarly, a. thaliana plants infected either with a mild or a virulent isolate of turnip mosaic potyvirus also showed profound differences in the genes and functional categories altered (s anchez et al. ) . in this case, the more virulent strain mainly altered stress responses and transport functions compared with the mild one (s anchez et al. ) . in a recent study, the transcriptomic alterations induced in nicotiana benthamiana plants infected either with a wt tobacco vein banding mosaic potyvirus or a genotype deficient in the vsr were compared (geng et al. ) . both transcriptomes differed in many aspects, including repression of photosynthesis-related genes, genes involved in the rna silencing pathway, the jasmonic acid signaling pathway, and the auxin signaling transduction (geng et al. ) . altogether, the results reported in this study illustrate the complex interaction between viruses and their native host plants, and how the outcome of this interaction, in terms of viral replication and accumulation, correlates with the expression of host genes ( fig. a ). our observation that viral fitness correlates positively or negatively with the expression of certain genes is of particular interest. by simply measuring the fitness of the virus infecting a given host, we may predict the whole genomic profile of the host cell to characterize its state (molecular impact of infection). moreover, by specifically targeting host genes that are essential for high fitness virus variants but not for milder ones, we may prevent the cervera et al. . doi: . /molbev/msy mbe spreading of the former variants, whereas still allowing mild variants to replicate and, perhaps, act as attenuated vaccines that enhance the antiviral response of the plant. the infectious clone pmtev contains a full copy of the genome of a wt tev strain isolated from tobacco ( fig. a ; genbank accession dq ) (bedoya and dar os ) . six tev mutant genotypes were constructed by sitedirected mutagenesis starting from template plasmid pmtev as described in torres-barcel o et al. ( ) (mutants as , cla , and cla ) and in carrasco, de la iglesia, et al. ( ) (mutants pc , pc , and pc ) . figure a shows the characteristics of the seven genotypes used in the study. the pmtev-derived plasmids contain a unique bglii restriction site. after linearization with bglii, each plasmid was transcribed with mmessage mmachine sp kit (ambion), following the manufacturer's instructions, to obtain infectious -capped rnas. transcripts were precipitated ( . volumes of diethyl pyrocarbonate [depc]-treated water, . volumes of . m licl, mm edta), collected, and resuspended in depc-treated water (carrasco, dar os, et al. ) . rna integrity and quantity were assessed by gel electrophoresis. in addition, each transcript was confirmed by sequencing of a ca. -bp fragment circumventing the mutation site as described elsewhere (lali c et al. ) . in short, reverse transcription (rt) was performed using m-mulv reverse transcriptase (thermo scientific) and a reverse primer outside the region of interest to be pcr-amplified for sequencing. pcr was then performed with phusion dna polymerase (thermo scientific) and appropriate sets of primers for each transcript. sequencing was performed at ibmcp sequencing service. templates were labelled with big dyes v . and resolved in an abi xl machine (life technologies). nicotiana tabacum l. cv. xanthi nn plants were used for production of virus particles of each of the seven genotypes ( fig. a ). the -capped rna transcripts were mixed with a : volume of inoculation buffer ( . m k hpo , mg/ml carborundum). batches of -week-old n. tabacum plants were inoculated with $ mg of rna of each viral genotype by abrasion of the third true leaf. inoculations were done in two experimental blocks, the first one including as , cla , cla , pc , and their controls, and the second one including pc , pc , and their corresponding controls. all plants were at similar growth stages. afterward, plants were maintained in a biosafety level- greenhouse chamber at c under a -h natural sunlight (supplemented with w high-pressure sodium lamps as needed to ensure a minimum light intensity of par lmol/m /s) and h dark photoperiod. all infected plants showed symptoms - dpi, except the as infected plants, which remained asymptomatic and only showed erratic chlorotic spots. at dpi virus-infected leafs and apexes from each plant were collected individually in plastic bags (after removing the inoculated leaf), with the exception of the as infected plants that were collected at dpi. next, plant tissue was frozen with liquid n , homogenized using a mixer mill mm (retsch), and aliquoted in . ml tubes ( mg each). these aliquots of tevinfected tissue were stored at À c. rna extraction from mg of fresh tissue per plant was performed using agilent plant rna isolation mini kit (agilent technologies) following the manufacturer's instructions. the concentration of total plant rna extract was adjusted to ng/ml for each sample. each rna sample was resequenced again at this stage to ensure the constancy of the genotypes as described earlier. viral loads were measured by absolute real-time rt-quantitative pcr (rt-qpcr), using standard curves (lali c et al. ) . standard curves were constructed using ten serial dilutions of the wt tev genome, synthetized in vitro as described earlier, in total plant rna obtained from healthy tobacco plants treated like all other plants in the experiment. quantification amplifications were done in a -ml volume, using a gotaq -step rt-qpcr system (promega) following the manufacturer's instructions. the forward (q-tev-f -ttggtcttgatggcaacgtg- ) and reverse (q-tev-r -tgtgccgttcagtgtcttcct- ) primers were chosen to amplify a -nt fragment in the end of tev genome and would only amplify complete genomes (lali c et al. ). amplifications were done using an abi stepone plus realtime pcr system (applied biosystems) and the following cycling conditions: the rt phase consisted of min at c and min at c; the pcr phase consisted of cycles of s at c, s at c, and s at c; and the final phase consisted of s at c, min at c, and s at c. amplifications were performed in a -well plate containing the corresponding standard curve. three technical replicates per infected plant were done. quantification results were examined using stepone software version . . (applied biosystems). total rna was extracted and virus accumulation quantified by rt-qpcr as described earlier and detailed previously (lali c et al. ) . virus accumulation (expressed as genomes/ng of total rna) was quantified dpi for all genotypes with the exception of as , that was quantified dpi. these sampling times assure that viral populations were at a quasi-stationary plateau in n. tabacum (carrasco, dar os, et al. ) . these values were then used to compute the fitness of the mutant genotypes relative to that of the wt genotype using the expression w ¼ (r t /r ) /t , where r and r t are the ratios of accumulations estimated for the mutant and wt viruses at inoculation and after t days of growth, respectively (carrasco, de la iglesia, et al. ) . fitness (w) data were fitted to a generalized linear model with a normal distribution and an identity link function. the model incorporates two random factors, the tev genotype (g) and the replicate plants (p), with the second nested within the first: where l is the grand mean value and e ijk is the error associated with individual measure k (estimated from the three technical viral fitness and perturbation of host's transcriptomes . doi: . /molbev/msy mbe replicates of the rt-qpcr reaction). the statistical significance of each factor was evaluated using a likelihood ratio test that asymptotically follows a v probability distribution. this statistical analysis was performed with ibm spss version . total rna was isolated as described earlier and its integrity assessed using a bioanalyzer (agilent) before and after hybridization. the rna samples were hybridized onto a genotypic designed n. tabacum gene expression  k microarray (amadid: ), which contained , probes ( -mer oligonucleotides) and was used in a onecolor experimental design according to minimum information about a microarray experiment guidelines (brazma et al. ) . three biological replicates for each of the six tev mutant genotypes, four replicates for the wt tev, plus four mock-inoculated negative control plants were analyzed. sample rnas ( ng) were amplified and labeled with the low input quick amp labeling kit (agilent). the one-color spike-in kit (agilent) was used to assess the labeling and hybridization efficiencies. hybridization and slide washing were performed with the gene expression hybridization kit (agilent) and gene expression wash buffers (agilent) as detailed in the manufacturer's instructions kits. after washing and drying, slides were scanned with a genepix b (axon) microarray scanner, at mm resolution. image files were extracted with the feature extraction software version . . (agilent). microarray hybridizations were performed at ibmcp genomics service. interarray analyses were performed with tools implemented in the babelomics webserver (alonso et al. ) . firstly, all agilent files were uploaded together to standardize the expression-related signals using quantile normalization (bolstad et al. ). this process resulted in a matrix of normalized expression with genes in rows and samples (tev genotypes, controls, and their replicates) in columns, provided as supplementary file s , supplementary material online. to compare the expression profiles of two tev genotypes, the expression level corresponding to mockinoculated plants (control) was first subtracted. secondly, differential expression was carried out by comparing two different samples, including replicates (against mock-inoculated or wt tev-infected plants), by using the limma test (smyth ) with fdr according to benjamini and hochberg ( ) (adjusted p < . ). an additional criterion of at least -fold change in mean expression, that is jlog (fold change)j > , was imposed to discard genes presenting minimal increases or decreases. lists of degs, up-or down-regulated, provided in supplementary file s , supplementary material online. thirdly, one-way anova tests were performed to identify genes that vary across all conditions (with fdr as above, adjusted p < . ). to identify the genes shown in figure a , the test was done over all samples, including the control. by contrast, to identify the genes shown in figure a , the test was done over all samples corresponding to infections with distinct tev genotypes. an additional criterion of significant spearman's correlation between mean fitness and mean expression (p < . ) was imposed in this latter case. lists of genes whose expressions correlate with viral fitness, either positive or negative, provided in supplementary file s , supplementary material online. the similarity between expression profiles of plants infected with different tev genotypes was evaluated with a principal components (pc) analysis with matlab version r b (mathworks) with default parameters (singular value decomposition). the annotation of the individual probes in the agilent's tobacco microarray (files _d_aa_ .txt and _d_genelist_ .txt provided by agilent) was updated by blasting the oligo sequence file ( _d_fasta_ .txt) against the most recent version of the n. tabacum mrna database (ntab-bx_awok-ss_basma.mrna.annot.fna) available at the sol genomics network (fern andez-pozo et al. ) . sequences that did not return a significant blast hit were removed from the output. a total of , annotated probes were generated. in , cases, more than one probe pointed to the same n. tabacum gene (e.g., probes a_ _p and a_ _p were both complementary to gene eb ), and in those cases the target appeared twice in the output. each one of the hits could be associated with an alternatively spliced mature mrna in the sol genomics network database. we then proceeded to generate the list of n. tabacum orthologous genes in the a. thaliana genome. to do so, we used blast against the tair version ten database of a. thaliana cdnas (lamesch et al. ) , with a cutoff e-value < À . the resulting mapping between n. tabacum and a. thaliana orthologues is listed in supplementary file s , supplementary material online. the determination of the gene ontology (go) categories overrepresented within the lists of degs was carried out in the agrigo webserver ) by using the fisher's exact test (with fdr adjusted p < . according to benjamini and yekutieli [ ] criterion). for the graphical representation, we constructed a plane involving the most relevant categories, depicted as circles with size proportional to the total number of host genes belonging to that category (in log scale). in addition, with the lists of genes whose expression correlates with viral fitness, we calculated the pie charts associated to the following: ) biological function and ) molecular function in the panther webserver (mi et al. ). total rna was extracted from mg of fresh tissue of plants infected with each one of the seven tev genotypes as described earlier. the concentration of total plant rna was adjusted to ng/ml. nine candidate genes were selected to validate the effect of each tev genotype on expression. specific primers were cervera et al. . doi: . /molbev/msy mbe designed for each gene that amplified the matured version of their corresponding mrnas. primers were designed using oligo primer analysis software version (www.oligo.net). gene expression was quantified by rt-qpcr relative to the expression of two housekeeping genes (schmidt and delaney ) . the first housekeeping gen encodes for the l ribosomal protein (genbank accession l ). forward ntl -f ( -cccctcaccacagagtctgc- ) and reverse ntl -r ( -aagggtgttgttgtcctcaatctt- ) primers were chosen to amplify a -nt long fragment. the second housekeeping gen encodes for the elongation factor a (genbank accession af ). for this second gene, forward ntef a-f ( -tgagatgcaccacgaagctc- ) and reverse ntef a-r ( -ccaacattgtcaccaggaagtg- ) primers were chosen to produce a -nt long amplicon. amplifications were done in ml volume, using gotaq -step rt-qpcr system (promega) following the manufacturer's instructions. the forward and reverse primers for each target gene were chosen to amplify a - nt fragments in the corresponding tobacco mature mrna. amplifications were done using an abi stepone plus realtime pcr system (applied biosystems) and the following cycling conditions: the rt phase consisted of min at c and min at c; the pcr phase consisted of cycles of s at c, s at c, and s at c; and the final phase consisted of s at c, min at c, and s at c. amplifications were performed individually for each target gene (with the corresponding set of primers) in a -well plate containing three biological replicates and two technical replicates per infected plant. in addition, each plate incorporates the two housekeeping genes. since each plate served for the quantification of a single mature mrna together with the two housekeeping reference genes, a baseline value of . , resulting from averaging the threshold baselines of all plates analyzed, was used as default threshold. quantification results were examined using the stepone version . . software (applied biosystems). details on the primers used for amplifications, the size of the amplicons, the genbank identification ids, and rt-qpcr threshold crossing (c t ) values for the nine degs and the corresponding internal reference genes from the same samples are all reported in supplementary file s , supplementary material online. the microarray data that support the findings of this study have been deposited at ncbi geo with accession number gse . processed data are presented in the supplementary material online. all other relevant data are available from the corresponding author on request. supplementary data are available at labarchives under doi: . /h 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domain-mediated complex formation for its function in rna-directed methylation cosupression of rbcs b in arabidopsis leads to severe photoinhibition caused by ros accumulation viral fitness and perturbation of host's transcriptomes we thank francisca de la iglesia and paula agudo for excellent technical assistance, the evolsysvir lab members for help, comments and discussions, rachel whitaker for english proofreading, and lorena latorre (ibmcp genomics service) and javier forment (ibmcp bioinformatics service) for their assistance. this research was supported by grants from spain's agencia estatal de investigaci on-feder (bfu - and bfu - -p to s.f.e. and bfu - -p to g.r.) and generalitat valenciana (prometeoii/ / ). key: cord- -bswndfvk authors: lalle, eleonora; biava, mirella; nicastri, emanuele; colavita, francesca; di caro, antonino; vairo, francesco; lanini, simone; castilletti, concetta; langer, martin; zumla, alimuddin; kobinger, gary; capobianchi, maria r.; ippolito, giuseppe title: pulmonary involvement during the ebola virus disease date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: bswndfvk filoviruses have become a worldwide public health concern, especially during the – western africa ebola virus disease (evd) outbreak—the largest outbreak, both by number of cases and geographical extension, recorded so far in medical history. evd is associated with pathologies in several organs, including the liver, kidney, and lung. during the – western africa outbreak, ebola virus (ebov) was detected in the lung of infected patients suggesting a role in lung pathogenesis. however, little is known about lung pathogenesis and the controversial issue of aerosol transmission in evd. this review highlights the pulmonary involvement in evd, with a special focus on the new data emerging from the – ebola outbreak. ebolavirus is part of the filoviridae family, which consists of three genera: marbugvirus, cuevavirus, and ebolavirus. there are currently six known, genetically distinct, species of ebolavirus-ebola virus (ebov), sudan ebolaviurs (sudv), tai forest ebolavirus (tafv), bundibugyo ebolavirus (bdbv), reston ebolavirus (restv), and bombali ebolavirus (bomv) [ , ] . no virus has triggered fear in the general population more than the filovirus ebolavirus [ ] . ebov is categorized among the deadliest viruses, with mortality rates up to %. the zoonotic origin of outbreaks are often the result of transmission from primates, although the suspected natural reservoir for ebov, bats, is still being questioned. since it was first identified in in zaire (the actual democratic republic of congo), confirmed outbreaks, mainly in the central part of africa, have occurred, and each outbreak was accompanied by high case fatality rates up to %, including the new declared outbreak ongoing in the north kivu province of the democratic republic of the congo [ ] [ ] [ ] . the - ebola outbreak is the largest (both by number of cases and geographical extension) ebolavirus outbreak ever reported, resulting . tlr stimulates irf- and nf-κb via myd activation, leading to the release of proinflammatory cytokines and the production of ifn-α, -β, and -λ, respectively. secretion of proinflammatory cytokines and chemokines activate the immune system, through recruitment of eosinophils, neutrophils, macrophages, dendritic cells, t cells, and nk cells. most respiratory viruses have developed strategies to escape antiviral defense, mainly by interfering with the ifn system or by affecting the epithelium barrier, with the consequence of a loss of integrity and protection. furthermore, respiratory viruses can perturb (skewed or exaggerated) inflammatory responses and production of soluble mediators. ebov infection is acquired through direct contact with bodily fluids. the virus enters blood circulation through breaks in the skin and mucosa and spread to different organs, causing systemic manifestation of cardiovascular, coagulation, or inflammatory disturbances [ ] . the terminal stages of evd usually involve massive tissue injury and hemorrhage, resulting in multiorgan failure and shock, the main cause of exitus in evd patients [ ] . during evd, respiratory symptoms such as chest pain, shortness of breath, cough, and nasal discharge are signals of the multisystem involvement, but, so far, lung damage has not been directly linked to ebov replication in the respiratory tract. however, new evidences collected during the recent - ebola outbreak hypothesized shedding of the virus in the lung and identified viral replication markers in sputum samples collected from ebov infected patients [ ] . on the other hand, the high virulence of ebov is attributed in large part to the ability of this virus to interfere with the host immune response, and the high degree of variation in lung pathogenesis is usually linked to indirect damage due to endothelial and epithelial inflammation and the hyper-activation of the immune system subsequent to ebov infection. in fact, viral direct damages are always associated with indirect damages, caused by inflammatory and immune reactions elicited by the viruses through the activation of soluble mediators (cytokines and chemokines) as part of the immune response ( figure ). the acute inflammation process is characterized by increasing blood flow, which enables plasma and leukocytes to reach extra-vascular sites of injury. even though inflammation may be often restored, in evd, severe inflammation is associated with a cytokine storm and more serious pathological changes are observed. for instance, ebov in vitro infection of monocytes and macrophages triggers the robust expression of inflammatory mediators, including il- β, il- , il- , mip- a, mip- β, mcp- , and tnf-α [ , ] , whereas the dysregulation of immune mediators in humans has been associated with the secretion of other inflammatory mediators, such as il- β, il- , ccl , ccl , ccl , cxcl , cxcl , cxcl , cxcl , il , mif, spp [ ] [ ] [ ] . in addition, severe inflammatory upon entrance into the cell, viruses are recognized by the toll-like receptor (tlr) on either cell membrane or in endosomes. tlrs activate interferon regulatory factors (irfs) leading to ifn-α and ifn-β release via the toll/il- receptor domain-containing adaptor (trif). tlr stimulates irf- and nf-κb via myd activation, leading to the release of proinflammatory cytokines and the production of ifn-α, -β, and -λ, respectively. secretion of proinflammatory cytokines and chemokines activate the immune system, through recruitment of eosinophils, neutrophils, macrophages, dendritic cells, t cells, and nk cells. most respiratory viruses have developed strategies to escape antiviral defense, mainly by interfering with the ifn system or by affecting the epithelium barrier, with the consequence of a loss of integrity and protection. furthermore, respiratory viruses can perturb (skewed or exaggerated) inflammatory responses and production of soluble mediators. ebov infection is acquired through direct contact with bodily fluids. the virus enters blood circulation through breaks in the skin and mucosa and spread to different organs, causing systemic manifestation of cardiovascular, coagulation, or inflammatory disturbances [ ] . the terminal stages of evd usually involve massive tissue injury and hemorrhage, resulting in multiorgan failure and shock, the main cause of exitus in evd patients [ ] . during evd, respiratory symptoms such as chest pain, shortness of breath, cough, and nasal discharge are signals of the multisystem involvement, but, so far, lung damage has not been directly linked to ebov replication in the respiratory tract. however, new evidences collected during the recent - ebola outbreak hypothesized shedding of the virus in the lung and identified viral replication markers in sputum samples collected from ebov infected patients [ ] . on the other hand, the high virulence of ebov is attributed in large part to the ability of this virus to interfere with the host immune response, and the high degree of variation in lung pathogenesis is usually linked to indirect damage due to endothelial and epithelial inflammation and the hyper-activation of the immune system subsequent to ebov infection. in fact, viral direct damages are always associated with indirect damages, caused by inflammatory and immune reactions elicited by the viruses through the activation of soluble mediators (cytokines and chemokines) as part of the immune response ( figure ). the acute inflammation process is characterized by increasing blood flow, which enables plasma and leukocytes to reach extra-vascular sites of injury. even though inflammation may be often restored, in evd, severe inflammation is associated with a cytokine storm and more serious pathological changes are observed. for instance, ebov in vitro infection of monocytes and macrophages triggers the robust expression of inflammatory mediators, including il- β, il- , il- , mip- a, mip- β, mcp- , and tnf-α [ , ] , whereas the dysregulation of immune mediators in humans has been associated with the secretion of other inflammatory mediators, such as il- β, il- , ccl , ccl , ccl , cxcl , cxcl , cxcl , cxcl , il , mif, spp [ ] [ ] [ ] . in addition, severe inflammatory cytokines/chemokines may spill over into the circulation and result in systemic cytokine storms, which are responsible for multi-organ dysfunction and for the impairment of the vascular system and disseminated intravascular coagulation [ , ] . dendritic cells (dcs) play an essential role in the link between the innate and adaptive immune response, and their maturation is essential for the correct functionality of dcs, such as the migration, processing, and presentation of viral antigens to t-and b-cells for their activation and correct viral clearance [ , ] . ebov infection has been shown to influence these mechanisms through impairment of dcs in upregulating co-stimulatory molecules (cd , cd , and cd ) and major histocompatibility complex (mhc) class ii, as well as soluble chemokines and cytokines [ ] . ebov infection is also able to influence the adaptive immune response: severe lymphopenia and the destruction of lymphoid tissue is one of the hallmarks of ebov infection. fatal cases showed a more marked reduction of nk cells and γδ t-cell frequency, as well as a loss of peripheral blood cd + and cd + t cells [ , ] . moreover, a recent study showed that patients with fatal outcome presented lower, or often absent, levels of both ebov-specific igm and igg, which, when detected, appeared later than in survivors [ ] . overall, the alteration of the innate and adaptive response explains the paralysis of the immune system and its inability to initiate and maintain a protective immune response. at the pulmonary level, many of the pathological changes are, in fact, secondary to systemic alterations, correlating with general pathogenic mechanisms, which are the major causes of severe disease in humans, even at the respiratory level [ , ] . evd is a viral hemorrhagic fever (vhf) characterized by acute systemic manifestations with vascular damage, plasma leakage, severe inflammation, and disruption of the immune system [ ] . evd transmissibility seems to vary depending on the stage of disease [ ] . a high-level of ebov replication, associated with systemic dissemination to multiple cell types, results in a complex pathogenesis, which is linked to an increased risk of infection transmission [ ] . as stated above, these pathogenic mechanisms include detrimental immune suppression and over-activation of the immune response, disordered coagulation, and tissue damage due to direct viral and indirect host-mediated effectors. in the absence of adequate supportive care, these processes commonly result in multiple organ failure and death within about days of symptom onset in humans. it is well recognized that ebov infection is acquired by direct contact with bodily fluids. notably, studies conducted in animal models have instilled doubts about possible airborne/droplet transmission (see section . ). however, this route of infection in humans is still debated. piercy and colleagues evaluated the actual stability of the virus particles in aerosol droplets [ ] . they created ebola-containing aerosol droplets and, according to the decay rates, estimated that ebov and restv can survive in aerosols for roughly and min, respectively, at % to % relative humidity and ± • c [ ] . therefore, a key additional question to ask is whether primary pulmonary infection of ebov could be a potential scenario for the future. a fair amount of studies, based on animal experiments (table ) and clinical evidence collected during the outbreaks ( table ), suggest that pulmonary infection may be a possibility. this possibility will be fully investigated below. after its first discovery in in cynomolgus macaques imported to reston, virginia, restv was detected in domestic swine in the philippines in a co-infection with the porcine reproductive and respiratory syndrome virus (prrsv, family arteriviridae, genus arterivirus) and porcine circovirus type (pcv- ; family circoviridae) [ , ] . later on, restv was identified to cause asymptomatic infections with mild respiratory symptoms, which may result in severe mortality in cases of co-infections with other viral pathogens like viruses in the families arteriviridae and circoviridae. the virus was first isolated in lung and lymphoid tissues in the original disease investigation [ ] . however, the massive presence of the virus in the lungs may be due to the fact that restv infection in pigs has been mostly associated with other infections of the respiratory tract, which may contribute to the specific localization of the virus and the respiratory symptoms of the disease. marsh et al. [ ] conducted an experimental study to rule out the effect of other pathogens affecting pigs, using a philippines swine isolate of restv. specifically, five-week-old pigs were exposed (via the oro-nasal or subcutaneous route) to the virus, and the subsequent viral replication in internal organs and shedding of the virus from the nasopharynx was observed. the researchers detected the highest levels of virus replication in lung and lymphoid tissues, confirming previous results [ ] . the detection of restv in domestic swine raised important biosecurity concerns about the potential for the disease's emergence in humans and other livestock, mainly in animals for food consumption [ , ] . the evidence of restv seropositive individuals further increased the concern for human infections and the worries of researchers, farm owners, and the public at large (world health organization. who, , available online: https://www.who.int/csr/resources/publications/hse_ epr_ _ .pdf). interestingly, so far restv has not been seen to result in any human disease, even if there is concern that its passage through swine may allow restv to diverge and shift its potential for pathogenicity [ ] . on the other hand, several studies investigated if other ebola viruses may be transmitted through the aerosol route and may result in primary pulmonary infection [ , , ] . researchers reviewed the different animal models and offered an overview regarding the possibilities of ebola viruses causing aerosol infections in non-human primates (nhps) and other animals. experimental studies analyzed the respiratory tract involvement in filovirus infections when the animals were exposed to the virus through different aerosol routes (artificially aerosolized virus or natural aerosol transmission) [ , , , ] . in these experimental studies conducted on nhps and pigs, ebov was inoculated via the aerosol route, and, following mucosal exposure, ebov replicated, reaching high concentrations, mainly in the respiratory tract, with the development of severe lung pathology. interestingly, weingartl et al. demonstrated that piglets inoculated oro-nasally with ebov and then transferred to a different room housing macaques in an open inaccessible cage system resulted in ebov infection of all macaques, suggesting a need to revise prevention and control measures during outbreaks [ ] . viral replication was observed within alveolar spaces [ , ] , in type i pneumocytes and macrophages [ ] , and in type ii pneumocytes, bronchiolar epithelial cells, and endothelial cells [ ] , supporting the respiratory involvement. the upper and lower respiratory tract, the lymphoid tissues, and the mediastinal lymph nodes showed infection signs, as well [ ] . similarly, in experiments on cynomolgus macaques placed separately in cages with experimentally infected piglets [ ] , and on guinea pigs exposed via aerosols to a guinea pig-adapted ebov strain [ ] , viral antigens were detected within alveolar and septal macrophages, pneumocytes, epithelial cells, endothelial cells, fibroblasts, and other interstitial cells of the respiratory tree [ ] . considering the pathology of the respiratory system, the expression of disease in the lungs and the patterns of lesions seem to be influenced by the exposure routes (aerogenous or hematogenous). broncho-interstitial pneumonia, characterized by injury to both the bronchiolar and the alveolar epithelium, is commonly caused by aerogenous viral infections [ ] . moreover, such pathological features were generally not evidenced following the inoculation of ebov by other routes in nhps and laboratory animals [ , ] . as shown in animal studies, primary pulmonary infections could occur and cause active viral shedding from the respiratory tract, thus potentially setting up a cycle of ongoing respiratory transmission in humans [ , ] . overall, experimental works conducted so far have shown that ebov infection induces respiratory complications, that the virus can be shed via the respiratory secretions, and that it can cause similar pulmonary lesions both in animals exposed to aerosols and in those kept nearby in separate cages with no close contact. the pathophysiological mechanism of pulmonary disease in patients with evd is unknown. notably, autopsies were performed on a limited number of humans (about cases), primarily during the sudv and ebov evd outbreaks and revealed interesting characteristics at microscopic level. during the first known sudv outbreak, chest pain was almost universal ( % of patients), often accompanied by a dry cough. autopsies were further performed on two patients and thickening of the alveolar walls due to proliferative accumulations of alveolar cells was found [ ] . furthermore, a possible pathogenetic role of the virus in the respiratory tract was suggested by the fact that viral inclusions within alveolar macrophages and free viral particles within alveolar space were found in the lungs from fatal evd cases who showed congestion, focal intra-alveolar edema, diffuse alveolar damage, and hemorrhaging. [ , ] . one of the most common symptoms in evd patients is a cough (up to %), especially during the progression of the disease, when viral loads in serum significantly increase, and the virus is copiously emitted in most body fluids, as well as in aerosol particles of various sizes [ , ] . among the reported evd cases in the literature, respiratory symptoms were commonly reported with a wide range of symptoms, such as a cough (from % [ , ] to % [ ] ), dyspnoea or breathless (detected from % [ ] to % [ ] ), and chest pain (from . % [ ] to . % [ ] ). moreover, a who study on the first months of the epidemic in western africa found that nearly % ( out of ) of the patients experienced coughing and . % ( of ) had a bloody cough [ , ] . a study of ebov-positive patients of the - outbreak in western africa, treated in europe and usa, reported that cough and dyspnoea were present at admission in seven ( %) and five ( %) evd patients, respectively. at symptom onset, only a cough was reported in one patient. furthermore, during hospitalization, patients ( %) experienced hypoxemia while they were breathing ambient air, patients ( %) had pulmonary oedema, seven patients ( %) had pneumonia, patients ( %) had respiratory failure, and six patients ( %) had a diagnosis of acute respiratory distress syndrome (ards). of these patients, four patients ( %) received non-invasive mechanical ventilation, and seven patients ( %) received invasive mechanical ventilation [ ] . notably, the first ebov-positive patient treated in italy, mechanically ventilated for respiratory insufficiency for days, had high levels of ebov rna in the lower respiratory tract secretions. the authors concluded that the absence of other identified respiratory pathogens in broncho-alveolar lavage fluids and aspirates supported the hypothesis of a direct contribution to the lung tissues damage by ebov. notably, ebov rna was detected in bronchial aspirate fluids when the ebov rna concentration in the concomitant blood samples was barely detectable. furthermore, the blood ebov rna concentrations in the previous days were significantly lower than the concentrations detected in the bronchial aspirate samples. these findings suggest that this ebov infection is unlikely a spillover from the blood compartment, eventually accompanied by delayed clearance. instead, the most plausible explanation is that the virus actually replicated into the lower respiratory tract [ ] . in the second ebov-patient treated in italy, our group investigated the presence of ebov genetic material in the lungs and blood during the patient's treatment and recovery. the patient showed a persistence of ebov replication markers within the respiratory tract, with a prolonged detection of ebov viral rnas (negative and positive sense rnas: neg-rna and pos-rna, respectively), known to be associated with ebov replication, in the lower respiratory tract for up to five days after the ebov viral load in blood was already undetectable. these results suggest that ebov may replicate in the lungs, although it is possible that the lungs simply provided a protective environment that allowed rna to linger longer than it did in the plasma. nevertheless, the detection of pos-rna together with neg-rna in the sputum (until day and of the hospital stay, respectively) supports the concept of active viral replication within the respiratory tract, rather than plasma spill-over or prolonged rna stability [ ] . overall, the pathophysiological mechanisms of pulmonary disease in patients with evd are still uncertain, but there could be multiple contributing factors, including vascular leak from endothelial infection, cytokine dysregulation, or direct damage to ebov-infected cells (figure ). viruses , , x for peer review of of pos-rna together with neg-rna in the sputum (until day and of the hospital stay, respectively) supports the concept of active viral replication within the respiratory tract, rather than plasma spill-over or prolonged rna stability [ ] . overall, the pathophysiological mechanisms of pulmonary disease in patients with evd are still uncertain, but there could be multiple contributing factors, including vascular leak from endothelial infection, cytokine dysregulation, or direct damage to ebov-infected cells (figure ). our understanding of ebov transmission in humans mainly relies on epidemiological observations. contact with bodily fluids from evd patients remains the most likely route of transmission. notably, the number of past outbreaks and associated epidemiological studies hat carefully examine transmission patterns is small. therefore, conclusions about transmission are based on relatively limited data sets [ ] . interestingly, ( . %) of the cases in the sudv outbreak in nzara, sudan, and ( . %) of the cases during the ebov outbreak in kikwit, drc, had no direct or physical contact with an infected person or known infected dead body [ , ] , thus pointing to other possible routes of transmission, e.g., human to human respiratory tract infection through droplet and aerosols. during the - western africa epidemic, more than health care workers (hcw) were infected, with a case fatality rate of % [ ] , whereas during our understanding of ebov transmission in humans mainly relies on epidemiological observations. contact with bodily fluids from evd patients remains the most likely route of transmission. notably, the number of past outbreaks and associated epidemiological studies hat carefully examine transmission patterns is small. therefore, conclusions about transmission are based on relatively limited data sets [ ] . interestingly, ( . %) of the cases in the sudv outbreak in nzara, sudan, and ( . %) of the cases during the ebov outbreak in kikwit, drc, had no direct or physical contact with an infected person or known infected dead body [ , ] , thus pointing to other possible routes of transmission, e.g., human to human respiratory tract infection through droplet and aerosols. during the - western africa epidemic, more than health care workers (hcw) were infected, with a case fatality rate of % [ ] , whereas during the current - outbreak in drc, as of july , hcw have been already affected ( . % of total cases) [ ] . currently, full body protection is recommend by who and cdc [ , ] . all hcw involved in the care of evd patients must receive training and demonstrate competency in performing all ebola related infection control practices and procedures, specifically in proper donning and doffing ppe even if using an n mask or a powered air-purifying respirator (papr). the risk of infection via inhalation of contaminated aerosols from exposed individuals has not been documented. however, droplets containing ebov that have become aerosolized (e.g., from coughing sneezing, vomiting, invasive medical or surgical procedures, or surfaces) may have the potential to come into contact with a person's mucous membrane in their nose or mouth or non-intact skin. therefore, respiratory protection may be helpful in providing a barrier to help prevent infectious materials from contacting a wearer's mucous membranes. finally, the epidemiologic and viral evidence of ebov detection and replication in the respiratory tract raise concerns on the need of strict application of cough etiquette for patients and of droplet and/or respiratory precautions for all hcw involved in the clinical management of evd suspected and confirmed cases. acute respiratory tract infections (artis) remain a leading cause of mortality, morbidity, and economic loss, and viruses are one of the main causes of such disease. who estimates that artis cause nearly four million deaths per year, a rate of more than deaths/ , people [ ] . the microbial etiology of aris is varied, with viruses being the most common cause in humans [ ] , leading to a high level of awareness and the necessity to develop countermeasures to control them (table ) . filoviruses are not commonly considered to be viruses responsible for aris, even if respiratory symptoms may be present as a consequence of diffuse systemic alterations. interestingly, evidence collected in animal studies, in the epidemiological analysis of transmission chains, and in the most recent ebola outbreaks suggests that ebov may be able to cause primary pulmonary infection. this evidence highlights the ability of the virus to be shed in the lung, suggesting a role in lung pathogenesis. specifically, the relevant proportion of evd patients without any epidemiologic link to the exposure to contaminated biological samples or fomites, or to any contact with evd patients; the evidence of respiratory signs and symptoms commonly reported all over the clinical course; the abundance of viral antigens in the lungs in animal necropsies; the prolonged persistence of ebov detection and replication within the respiratory tract days after undetectable ebov viral load in plasma; and similar clinical patterns in several other viral respiratory tract infections are all different parameters with consistent evidence of a major role in the pathogenesis of evd in respiratory tissues [ ] . on the other hand, there is no evidence of aerosol transmission in evd. however, different studies addressing this issue have been performed [ , ] , and aerosol transmission was considered a possibility as a consequence of epidemiological observations in past outbreaks, where people showed signs of evd even in the absence of a direct or physical contact with an infected person or known infected dead body [ , ] . this hypothesis was corroborated by other studies, in which the presence of free viral particles in alveoli and within intra-alveolar macrophages demonstrated a pulmonary involvement [ ] . from a clinical point of view, the - ebov outbreak underlined the lung involvement in evd pathogenesis. in fact, only a few patients treated in europe and usa had a cough and difficulty breathing at admission. nevertheless, during the clinical progression, half of the patients experienced hypoxemia while breathing room air, one third had respiratory failure, and one fourth received invasive or non-invasive mechanical ventilation [ ] . in the italian experience at the national institute for infectious diseases "l. spallanzani" (inmi), respiratory symptoms were present in both patients, in the absence of other common respiratory pathogens [ , ] . one case required mechanical ventilation and the other presented ebov replication markers in the lungs even after clearance of the virus from the blood. the inmi experience suggests a direct role of the virus in lung pathogenesis. although lung pathogenesis in evd may be secondary to systemic alterations (correlating with general pathogenic mechanisms) the direct presence of the virus is undisputable in the lung, and its interaction with the immune system, whose hyper-activation may be the most likely explanation of the lung damage, is also indisputable. further research will be 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of the viral hemorrhagic fevers dengue viruses can infect human primary lung epithelia as well as lung carcinoma cells, and can also induce the secretion of il- and rantes pathogenesis of lassa fever. viruses pathophysiology of hantavirus pulmonary syndrome in rhesus macaques this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -uo hdrgc authors: de vries, rory d.; rennick, linda j.; duprex, w. paul; de swart, rik l. title: paramyxovirus infections in ex vivo lung slice cultures of different host species date: - - journal: methods protoc doi: . /mps sha: doc_id: cord_uid: uo hdrgc in vivo experiments in animal models of disease are of crucial importance for viral tropism and pathogenesis studies. however, these experiments must be complemented with in vitro and ex vivo experiments. here, we describe a protocol for the preparation and ex vivo infection of lung slices from different mammalian host species with various respiratory paramyxoviruses expressing fluorescent reporter proteins, and suggest follow-up experiments including immunohistochemistry, flow cytometry and confocal microscopy. ex vivo models provide an important bridge between in vitro and in vivo experiments. the use of agarose-inflated lung slices for respiratory virus pathogenesis studies has been described previously [ ] [ ] [ ] [ ] [ ] [ ] . here, we describe a protocol in which agarose-inflated lung slices can be kept viable in culture for at least seven days post-necropsy of an experimental animal. the combination of these viable lung slices with recombinant viruses expressing fluorescent reporter proteins [ ] [ ] [ ] allows for accurate, sensitive and reproducible assessment of respiratory virus infection and dissemination over time. furthermore, use of these recombinant viruses allows for real time monitoring of infection processes, using multiple methods for measurement of fluorescence (e.g., flow cytometry and confocal laser scanning microscopy). lung slices are also suitable for analysis by immunohistochemistry, thereby visualizing virus cell tropism and spatial localization of infected cells within the tissue. we have validated this technique by infecting lung slices of multiple host species (cotton rats, ferrets, dogs and macaques) with various paramyxoviruses expressing fluorescent reporter proteins (measles virus (mv), canine distemper virus (cdv), human respiratory syncytial virus (hrsv) and human metapneumovirus (hmpv)) [ ] . this technique, however, is directly transferable to different host species and different viruses [ ] . this protocol describes the ex vivo agarose inflation of lungs and generation of slices for virus infection. lungs should be taken from the experimental animal during necropsy, keeping the material as intact as possible and attached to the primary bronchus and trachea. using a (blunt-end) needle or flexible catheter, the fresh lungs are inflated through the trachea (or primary bronchus, if inflation of a half lung or single lobe is desired) with low-melting point agarose mixed with culture medium. after solidification on ice, slices can be prepared by hand using microtome blades and cultured in -or -well plates with culture medium, dependent on the size of the slices. these lung slices are subsequently inoculated with infectious virus (or could be used for any other experimental purpose) and can be followed in time by phase-contrast inverted light microscopy or fluorescence, when reporter viruses expressing fluorescent proteins are used (see figure ). in addition, emigrant cells in the culture medium can be harvested in time and analyzed by flow cytometry. although these emigrant cells could be present due to tissue degradation, they can be detected at early time-points after refreshing the culture medium, suggesting they are mobile emigrant cells. we have detected cells from lymphoid, myeloid and epithelial origin using this method. at the end of the experiment, slices can be used as desired; potential applications include fixation followed by paraffin embedding and immunohistochemistry, or generation of single cell suspensions to be analyzed via flow cytometry. . after solidification on ice, thin slices of approximately -mm thick can be cut by hand (c) and are transferred to -or -well plates pre-filled with culture medium (d). slices can subsequently be inoculated with the desired virus (e) and infection of the slices is followed in time (f). in this example in panel f, a macaque lung slice infected with recombinant measles virus is shown. all animals used in these studies were housed and experiments were conducted in strict compliance with european guidelines (eu directive on animal testing / /eec) and dutch legislation (experiments on animals act, ). protocols were approved by the independent animal experimentation ethical review committee dcc in driebergen, the netherlands. animal welfare was observed on a daily basis. critical step prepare % (w/v) low-melting point agarose by dissolving agarose in phosphate-buffered saline (pbs) and boil for several minutes (e.g., using a microwave oven, be careful for boiling over). as an alternative, agarose can be prepared by autoclaving. be sure to allow agarose to cool down to • c (e.g., in a water bath) before inflating the lungs. the volume of low-melting point agarose should be adjusted according to the size of the lungs. resect the lungs, including the distal part of the trachea, from the experimental animal. critical step resection should be performed within h after euthanasia, preferably sooner. critical step be sure not to damage the lungs while resecting, as this will interfere with the inflation process. locate the trachea (for small species) or primary bronchus (for larger species) through which you wish to inflate. for smaller mammals, inflation through the trachea works best, but for larger animals (body weight > - kg) inflation through the primary bronchus is more efficient. fill a syringe with a • c pre-warmed : mixture of % (w/v) low-melting point agarose and lung slice medium (dmem/ham's f medium supplemented with % (v/v) fbs, penicillin, streptomycin, l-glutamine and amphotericin b). final agarose percentage is % (w/v). critical step avoid the formation of air bubbles in the agarose. decide on the size of the syringe dependent on the size of the trachea or bronchus through which you will inflate. either the pipette tip, blunt-end needles, catheters, cannulas the end of the syringe should be a tight fit into the trachea or bronchus. insert the end of the syringe into the trachea or primary bronchus, enforce placement inside the trachea or bronchus by clamping (or use sutures as an alternative) the trachea or bronchus around the needle. critical step be careful not to damage the trachea or bronchus while inserting the needle into the trachea or bronchus. we advise the use of pipette tips, blunt-end needles, catheters or cannulas instead of sharp needles at the end of the syringe. . inject the proper amount of agarose into the lungs. check inflation while injecting; keep adding agarose until the lungs are completely inflated (see video s ). critical step it is not always easy to determine the best level of inflation. in our experience, the volume used for inflation should be slightly higher than the tidal volume of the animal, which is usually around ml per kg body weight. if you are inflating just one lobe of the lung, the volume should be adapted accordingly. remove needle but keep the clamp positioned on the trachea or bronchus and allow the inflated lungs to solidify on ice ( min for smaller species and - min for larger species). transfer lungs to biosafety cabinet, remove the clamp, prepare a surface for cutting (cutting board or petri dish) and microtome blade. prepare the lungs for slicing by finding anatomically interesting locations. critical step dependent on the focus of the experiment, you have to decide which part is anatomically interesting. decide on whether you want to use for example the left or right side, and either the upper or lower parts of available lobes. to check viability, we advise to include a cut of the primary bronchus in the slice, to determine the beating of cilia indicating viable epithelial cells. start with an initial cut to ensure that a straight edge is available for cutting. now manually prepare slices of approximately mm thickness. if desired, the lungs can be attached to the cutting board by needles (see video s ). as a rule of thumb, slices are thin enough for culture if you can see through the slice while cutting it and actually see the microtome blade. gently transfer cut slices into -, -, -, or -well plates (dependent on size of the slices) that were pre-filled with lung slice culture medium. infection of slices can be performed immediately. pause step slices can also be infected after h of culture at • c in % (v/v) co . we have never observed discrepancies between obtained results after either direct infection or after overnight culture. transfer the lung slices into empty -, -, -, or -well plates (dependent on size of the slices). gently add virus (preferably a recombinant virus expressing a fluorescent reporter protein) in a drop wise fashion onto the lung slice. typically, we used an inoculation volume of µl; always include a mock infection as negative control. we typically use - × tissue culture infectious dose- (tcid ) per slice; variations are possible. . incubate at • c in % (v/v) co for h. after h, add an appropriate volume of lung slice medium (inoculum can be washed away) and return plates to • c in % (v/v) co . . in our experience, slices can be held viable (based on beating cilia in epithelium) and followed in time for a maximum of days. culture medium has to be replaced to ensure viability every other day (i.e., day , and ). critical step when replacing culture medium, medium should be taken off and directly replaced, not allowing the lung slices to dehydrate. beating of cilia is the best measure for viability, however when beating of cilia is no longer observed slices start disintegrating. depending on the goal of the experiment, non-viable slices should be removed or kept in culture. slices should be checked for viability on daily basis: find a bronchus with cilia under a normal light microscope. movement (see video s ) of cilia is indicative of viable epithelial cells, suggesting there are live cells in culture. if fluorescent reporter viruses were used, slices should be checked for fluorescence on a daily basis (see video s and figure ). use a macroscopic imaging lamp as described previously [ ] in combination with an emission filter to visualize fluorescence macroscopically. shine lamp directly on lung slices and find fluorescent spots. photographs can be acquired if desired. use an inverted uv microscope or confocal laser scanning microscope to identify fluorescent-positive cells. during infection and culture, effluent cells can be harvested and checked for expression of fluorescent proteins by flow cytometry if desired. harvest supernatants from slices (e.g., on day , and while replacing culture medium), transfer supernatant to tubes and centrifuge ( - min, × g). transfer pellets to -wells v-bottom plate, wash once in facs buffer (pbs + . % (v/v) bsa + mm edta). optional step to determine which subsets of cells are infected, a fluorescence activated cell sorter (facs) staining for specific cell types can be performed at this point. cells that migrate out of the lung slices are mainly lymphoid, myeloid or epithelial in origin. to study the tropism of infection you can selectively stain for example t-cells, b-cells, natural killer (nk) cells, neutrophils, granulocytes, dendritic cells, macrophages or epithelial cells, or combinations thereof. acquire cells on flow cytometer to determine number of virus-positive cells. multiple experiments can be performed at the end of culture or at desired time points. slices can be directly stained for confocal laser scanning microscopy (section . . ), fixed for immunohistochemistry (section . . ) or used to generate a single cell suspension for flow cytometry (section . . ). critical step not all fluorophores are well-preserved by pfa fixation. sensitivity of fluorophores to fixation should be tested beforehand, and the fixative of mounting medium can be adjusted accordingly. wash slices after min and permeabilize in . % (v/v) triton x- for min. wash slices and subsequently stain with fluorescent antibodies of choice. critical step to understand tissue morphology, it is important to counterstain nuclei (for example with to-pro- or nucblue) and add a marker of choice. we found it useful to stain cilia using an antibody against class iv β-tubulin. transfer slices to thin-bottom/glass-bottom dishes. view fluorescence by confocal laser scanning microscopy (using an inverted microscope, see figure ). since slices are approximately -mm thick, z-stacks can be generated and from those d images can be rendered. in addition to using lung slices as an ex vivo culture and infection system, the method can also be used to screen the lungs of in vivo virus-infected animals for infection and determine the viral tropism (e.g., see reference [ ] ). two approaches are possible: instead of inflating lungs with the : mixture of % (w/v) low-melting point agarose and lung slice medium, prepare a : mixture of % (w/v) low-melting point agarose and % (w/v) pfa in pbs. the rest of the protocol is identical, after preparing lung slices these can be screened by fluorescence microscopy directly, virus-positive slices can be processed further as desired (immunohistochemistry, staining for confocal laser scanning microscopy, preparation of single cell suspensions for flow cytometry). as an alternative, slices can be inflated with : mixture of % (w/v) low-melting point agarose and lung slice medium and kept in culture for a period of time after necropsy, to evaluate viral tropism and dissemination [ ] . with the protocol described here, dependent on the size of the species used in the experiment, viable lung slices from potentially any species can be obtained for ex vivo experiments. we have observed viability of these slices for up to seven days post resection; however, this could be dependent on the species and culture conditions. infections with respiratory viruses of lung slices are normally relatively successful: figures and show examples of ex vivo paramyxovirus-infected lung slices. in these experiments, macaque lungs were inflated and infected with mv, which is clearly able to infect the macaque lung slices. of course, viruses corresponding to the target species should be chosen to obtain positive results. in the family of paramyxoviridae for example, viruses exclusively infecting primates or carnivores exist. measles virus, a virus of primates, successfully infected non-human primate but not dog lungs, whereas cdv, a virus of carnivores, behaved vice versa. using viruses expressing fluorescent reporter proteins allows for sensitive detection of virus-infected cells in these experiments; however, these experiments can also be performed with non-fluorescent viruses. staining should still make infected cells visible in immunohistochemistry, confocal laser scanning microscopy or flow cytometry. the model described here of course has some limitations, as a single region of the lung is directly infected, often with a relatively high inoculum. therefore, in vivo experiments in animal models of disease are of crucial importance for viral tropism and pathogenesis studies. however, these experiments must be complemented with proper in vitro and ex vivo experiments, indicating the potential of ex vivo experiments in these cultured lung slices. % agarose: prepare correct weight of hydroxyethylagarose in dpbs; • lung slice medium: dmem/ham's nutrient mixture f powder, glutamax, fbs, penicillin/streptomycin, amphotericin; • fluorescence activated cell sorter buffer: pbs + . % (v/v) bsa + mm edta (use stock of . m in distilled water); • paraformaldehyde: dissolve appropriate weight in pbs with high ph by initially supplementing with naoh. after pfa has completely dissolved, ph should be re-adjusted to . . always prepare fresh or store aliquoted at − • c. do not use buffered formalin when directly screening fluorescence, as this will disrupt the fluorescence of most reporter proteins. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s . video s : inflation of cotton rat lungs. syringe is filled with agarose mixed with lung slice medium and inserted into the trachea, kept in place by a suture. lungs are slowly inflated by injecting agarose and medium into the trachea; video s : slicing of cotton rat lungs. solidified cotton rat lungs are places on petri-dish and subsequently sliced. an initial cut is made for a straight edge followed by the preparation of a single lung slice; video s : viability screening of lung slice. a bronchus consisting of viable epithelial cells with beating cilia; video s : fluorescence screening of live lung slice infected with a paramyxovirus expressing a fluorescent reporter protein (green). the movie initially shows a combination of normal light and fluorescent light, followed by a shutdown of the normal light, indicating the identical area but now with fluorescence light only. time for completion: h . wash slices twice in hank's balanced salt solution (hbss) transfer slices into petri dishes and manually cut in small pieces transfer pieces into -well plate pre-filled with hbss + collagenase ( units/ml) + dnase ( . mg/ml) incubate for h on rocking platform at • c in % (v/v) co prepare single cell suspension by straining small pieces over µm cell strainer centrifuge and lyse red blood cells by adding ml red blood cell lysis buffer to pellet, incubate • c in % (v/v) co for three minutes centrifuge, resuspend pellet in facs buffer and perform facs staining of choice acetylcholine-induced calcium signaling and contraction of airway smooth muscle cells in lung slices responsiveness of individual airways to methacholine in adult rat lung explants early target cells of measles virus after aerosol infection of non-human primates a prominent role for dc-sign + dendritic cells in initiation and dissemination of measles virus infection in non-human primates pb -f modulates early host responses but does not affect the pathogenesis of h n seasonal influenza virus long-term maintenance of mature pulmonary parenchyma cultured in serum-free conditions predominant infection of cd + lymphocytes and dendritic cells during measles virus infection of macaques an improved plaque reduction virus neutralization assay for human metapneumovirus observation of measles virus cell-to-cell spread in astrocytoma cells by using a green fluorescent protein-expressing recombinant virus paramyxovirus infections in ex vivo lung slice cultures of different host species asymptomatic middle east respiratory syndrome coronavirus infection in rabbits the authors wish to thank geert van amerongen for providing excellent technical assistance during the establishment of this technique. we acknowledge nih for their ongoing funding of respiratory virus pathogenesis studies, specifically grant ai to w.p.d. the authors declare no conflict of interest. key: cord- - m vyxq authors: jayathilaka, p. g. n. s.; mendis, a. s. v.; perera, m. h. m. t. s.; damsiri, h. m. t.; gunaratne, a. v. c.; agampodi, suneth buddhika title: an outbreak of leptospirosis with predominant cardiac involvement: a case series date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: m vyxq background: severe leptospirosis is known to cause multi organ dysfunction including cardiac involvement. in the clinical setting with limited resources, high degree of suspicion is needed to diagnose cardiac involvement including myocarditis. although myocarditis is not reported as a common complication due to lack of diagnostic facilities, there are evidence to support myocarditis is more prevalent in post mortem studies of patients died due to leptospirosis. we present a case series of severe leptospirosis with cardiac involvement observed during a period of one month at colombo-north teaching hospital, sri lanka. case presentation: we report here five patients with severe leptospirosis complicated with cardiac involvement, admitted to a single medical ward, colombo-north teaching hospital, sri lanka during a one-month period. out of six suspected leptospirosis patients admitted during that period, five in a raw developed severe leptospirosis with cardiac involvement. in this case series, four patients were confirmed serologically or quantitative pcr and one patient had possible leptospirosis. all patients developed shock during their course of illness. two patients developed rapid atrial fibrillation. one patient had dynamic t wave changes in ecg and the other two had sinus tachycardia. two patients had evidence of myocarditis in d echocardiogram, whereas other two patients had nonspecific findings and one patient had normal d echocardiogram. all five patients had elevated cardiac troponin i titre and it was normalized with the recovery. all five patients developed acute kidney injury. four patients needed inotropic/vasopressor support to maintain mean arterial pressure and one patient recovered from shock with fluid resuscitation. all patients were recovered from their illness and repeat d echocardiograms after recovery did not show residual complications. one patient had serologically proven dengue co-infection with leptospirosis. conclusions: myocarditis and cardiac involvement in leptospirosis may be overlooked due to non-specific clinical findings and co-existing multi-organ dysfunction. atypical presentation of this case series may be due to micro-geographic variation and unusual outbreak of leptospirosis. co-infection of dengue with leptospirosis should be considered in managing patients especially in endemic areas. leptospirosis is a well-known zoonosis which causes outbreaks particularly in tropical countries. the causative organism is a spirochete of the genus leptospira. history of leptospirosis is likely to extend to ancient times which is evident by chinese texts describing "rice field jaundice" [ ] . in , adolph weil describes a syndrome consists of jaundice, splenomegaly, renal dysfunction, conjunctivitis, and skin rash [ ] and few years later, inada described the causative organism of spirochetosis icterohaemorrhegica [ ] now known as leptospirosis. the classical untreated disease is described as a biphasic illness with initial acute leptospireamic phase followed by immune phase. most cases are self-limited, but some patients develop fatal complications with severe disease. jaundice and renal failure ("weil's disease"), pulmonary hemorrhage, acute respiratory distress syndrome (ards), uveitis, optic neuritis, peripheral neuropathy, myocarditis, and rhabdomyolysis are well known complications [ ] . after the resolution of febrile phase with the clearance of leptospiremia, the immune phase can occur in less than % of patients. however, atypical presentations are reported more frequently in the recent history [ ] . in sri lanka, these differences of clinical presentations has been observed and attributed to micro-geographic changes [ ] . there are more than serovars of leptospira which have been classified in to more than serogroups and the different clinical manifestations are partially attributed to specific serovars. understanding and identifying the varying clinical presentations of leptospirosis mimicking other diseases is important in clinical practice for early treatment and management. in this case series, we describe a series of male patients with severe leptospirosis with cardiac involvement, presented to a single medical ward during a period of one month. here we define cardiac involvement as positivity of at least one of following criteria. they are, ) transient echocardiogram abnormalities during the illness ) elevated troponin i titer which came down with the recovery of illness, ) transient electrocardiogram changes during the illness. we present five patients who were treated for leptospirosis with complications. all are male patients admitted to a single medical ward at north colombo teaching hospital, sri lanka during a one month period starting from to - . data were collected by direct interview of patients, during admission and follow up visits, and from hospital records. fifty-eight years old previously healthy mason admitted to the hospital on / / with fever for three days. fever was associated with chills, rigors, headache, body aches, faintishness, mild cough producing whitish sputum for two days, dysuria, two episodes of loose stool on day of illness, and loss of appetite with poor intake. urine output was normal up to the day of admission. he had a history of cleaning a drainage system one week prior to onset of symptoms. on examination, he was febrile ( °f) and dehydrated. he had low volume pulse with a rate of bpm, blood pressure of / mmhg. examination of other systems were unremarkable except, few basal crepitations in the right lung. inward uss abdomen was performed and there was no free fluid indicative of dengue hemorrhagic fever. initial investigations revealed neutrophil leukocytosis with thrombocytopenia (table ) , high c-reactive protein level (table ) , high serum creatinine with marginally elevated liver transaminases (ast > alt). urine analysis showed microscopic hematuria and ecg showed sinus tachycardia. he was resuscitated with intravenous crystalloids. despite adequate resuscitation he remained in shock and oliguric acute renal failure. after five hours of admission he was started on intravenous noradrenalin infusion and later dobutamin also added to the therapy (table ) . clinical diagnosis was made as leptospirosis and intravenous cefotaxime was started in the meanwhile. urine output was improved with the rise of mean arterial pressure. but patient was dependent on ionotrope and vasopressor. d echocardiogram showed mild global hypokinesia with ejection fraction - % and concluded as possible myocarditis. troponin i titre became positive. on day of illness, patient developed rapid atrial fibrillation with shock requiring electrical cardioversion to achieve sinus rhythm. by day five of illness, he became heamodyanemically stable without inotropic/ vasopressor support. during the recovery, he developed asymptomatic hypokalemia and potassium was replaced. by day eleven of illness he was completely recovered clinically and full blood count, liver function tests, renal function tests and ecg were normal. c-reactive protein and troponin i titer were coming down and patient was discharged. after three weeks of illness, d echocardiogram was performed and it was completely normal. leptospira was detected in qpcr (quantitative polymerase chain reaction) performed on day five of illness and leptospirosis antibody test on day seven of illness (mat) was positive. (titre- : ) his urine and blood cultures, dengue antigen were negative. a years old previously healthy male, a retired clerk presented to the medical casualty with a history of fever for three days. it was associated with arthralgia, myalgia, headache and loss of appetite. he did not have respiratory, urinary symptoms and bowel habits were normal. he denied any history of exposure to leptospirosis or contact history of fever. on admission, his general examination was normal with a heart rate of bpm and blood pressure of / mmhg. other system examination was unremarkable. after admission it was noted that his urine output is low while he was on maintenance fluid. initial investigations revealed neutrophilia with normal white blood cell count, thrombocytopenia, elevated blood urea, serum creatinine, c-reactive protein and ast. urine analysis showed - pus cells, - red cells with granular casts. clinical diagnosis of leptospirosis was made on high index of suspicion although there was no significant history of exposure to leptospirosis. patient was started on intravenous cefotaxime. by the day five of illness, he developed confusion (gcs- / ), low blood pressure ( / mmhg) with tachycardia ( bpm), high fever spike ( f), and mild dyspnea with spo % on air. ecg showed sinus tachycardia, non-contrast ct brain was normal, d echocardiogram revealed ejection fraction of > %, chest x ray-pa was normal, and troponin i titer was marginally positive. ultrasound abdomen showed renal parenchymal changes with normal sized kidneys. serum creatinine was rising. patient was started on inotropic and vasopressor support to maintain blood pressure. even after achieving mean arterial pressure > mmhg patient went in to anuric acute renal failure. meanwhile he developed rapid atrial fibrillation which was settled with electrical cardioversion. he was given hemodialysis on day of illness. on day of illness again patient developed rapid atrial fibrillation and it did not respond to electrical cardioversion and started on iv amiodarone infusion and patient regained sinus rhythm and could tail off inotrope and vasopressor. since day , he gradually improved clinically with good urine output, hemodynamic stability and confusion settled. but he did not recover from acute kidney injury and renal functions remained rising again. he was given another hemodialysis on day of illness. then his renal functions slowly improved and discharged on day of illness with a follow up plan at nephrology clinic. on discharge patient had normal platelet count, c-reactive protein, liver transaminases, ecg. serum creatinine was static around micromol/l. repeat d echocardiogram which was done three weeks after recovery was normal. leptospirosis antibody titre (mat) on day of illness was positive. ( : ). a year old male patient presented with fever for two days. fever was associated with chills, rigors, arthralgia, myalgia, frontal headache, faintishness, lower back pain, loss of appetite, vomiting, loose stool - times/day for two days. patient denied a significant exposure to leptospirosis. there was no contact history of fever. he was a manual worker. on admission he was ill looking, febrile ultrasound scan of abdomen showed acute renal parenchymal changes and there was no evidence of free fluid in the abdomen. initial investigations revealed neutrophil leukocytosis with thrombocytopenia, high c-reactive protein ( mg/l), high blood urea ( mg/dl) and serum creatinine ( micromol/l), marginally elevated liver transaminases (ast > alt), microscopic hematuria, ecg showed sinus tachycardia with mild t inversions in v -v . chest x ray was normal. possible diagnosis of leptospirosis was made on clinical grounds and he was started on intravenous cefotaxime. his blood pressure was improved after fluid resuscitation and he had good urine output. his d echocardiogram was normal, but his troponin titer increased and then came down. patient was discharged from the ward on day of illness with complete recovery and normal full blood count, renal and liver function tests. crp and trop i titer was coming down. d echocardiogram which was performed after three weeks of recovery was normal. his dengue antigen test, blood and urine cultures were negative. the leptospirosis qpcr test performed on day three of the illness was reported as not detected though one out of triplicate samples was positive. patient was clinically diagnosed as a "possible" case of leptospirosis. a year old male laborer presented with fever for four days duration. he was previously diagnosed to have diabetes mellitus, but he was not taking treatments. fever was associated with arthralgia, myalgia, headache, lower back pain, dysuria and reduced urine output for two days, cough for one week producing scanty amount of whitish sputum. he had a history of muddy contact within one week prior to symptom onset. on admission, patient was febrile (temp- f), ill looking, mildly dehydrated and had conjunctival suffusion. his pulse rate was bpm with blood pressure of / mmhg. other system examination was unremarkable. initial laboratory work up showed neutrophilia with normal white cell count, thrombocytopenia, high c-reactive protein ( mg/l), high serum creatinine ( micromol/l) and normal liver transaminases. ecg showed sinus tachycardia and chest x ray-pa was normal. depending on clinical grounds, diagnosis was made as leptospirosis and started on intravenous cefotaxime while fluid resuscitation is being carried out. despite adequate fluid resuscitation patient developed shock with low urine output on the same day of admission. (day of illness-pulse rate- bpm, bp- / ) then vasopressor support was given and small dose of frusemide infusion was started after achieving normal blood pressure with noradrenalin. d echocardiogram was performed on d of illness and it showed mild global hypokinesia with ejection fraction - %, dilated left ventricle with concentric left ventricular hypertrophy and concluded as hypertensive heart disease with or without myocarditis. cardiac troponin i titre became positive and had rising titre when repeated and then came down by the time of recovery. us scan of abdomen revealed bilateral renal parenchymal changes with normal sized kidneys. noradrenalin was tailed off within h and urine output was improved with maintenance fluid therapy. patient had rising serum creatinine till day of illness and then started to come down. serum electrolytes were normal throughout and there was no acidosis. patient was improved dramatically and was discharged from the hospital by day of illness. on discharge he had rising platelet count, normal serum creatinine and dropping troponin i titre and crp. d echocardiogram was repeated after weeks of discharge and his ejection fraction was improved to % and there was mild left ventricular hypertrophy with grade i diastolic dysfunction. his diabetes was controlled with soluble insulin during acute illness and changed to oral hypoglycemic treatment with the recovery. leptospirosis antibody titre (mat) done on day of illness was positive ( : ). a years old male patient presented with fever for days. it was high fever associated with arthralgia, myalgia and mild difficulty in breathing. he also complained of reduced urine output and loose stool (two episodes) for one day. there were no other respiratory or urinary symptoms. he denied a significant exposure to leptospirosis. he had a past history of hypertension for which he was not taking treatment and past history of renal calculi for which he has undergone surgery several years back. on admission he was ill looking, febrile (temp- f), and anicteric. pulse rate was bpm and blood pressure / mmhg. other system examination was unremarkable. initial investigations revealed marked thrombocytopenia, neutrophilia with low normal white blood cell count, high c-reactive protein ( mg/l), high serum creatinine ( micromol/l), elevated liver transaminases (ast > alt), urine analysis showed pus cells - , red cells - and albumin + (urine culture became negative). chest x ray-pa was normal. possibility of dengue fever could not be excluded with his full blood count and clinical presentation, but all other initial investigations were supportive towards leptospirosis although there was no history of significant exposure to leptospirosis. on admission ultrasound scan of the abdomen was performed inward and there was no evidence of fluid leakage. therefore, patient was started on intravenous cefotaxime in addition to hydration with maintenance fluid. patient had low urine output and went in to shock (pr- , bp- / mmhg) despite of adequate fluid resuscitation (on day of illness). he was started on iv noradrenalin to maintain blood pressure. ultrasound scan of the abdomen revealed right side scarred kidney with left side renal parenchymal changes with normal size kidney. there was no evidence of leaking by the time of developing shock. d echocardiogram showed severe mitral regurgitation with and there was no evidence of myocarditis. troponin i titer became marginally positive and later came down. ecg showed sinus tachycardia. histological diagnosis or cardiac mri to diagnose cardiac involvement was not accessible due to lack of resources in the hospital. noradrenalin could be tailed off within h. (on day of illness). by day five of illness urine output was gradually improved but serum creatinine remained rising with normal serum electrolytes. dengue ns antigen was negative, but igm and igg antibodies were positive with dropping platelet count and white cell count (neutrophilia persisted). dengue pre-critical monitoring was continued while giving maintenance fluid therapy. daily ultrasound scans were performed to exclude fluid leakage. patient remained hemodynamically stable and platelet and white cell count started to increase by day of illness and serum creatinine started to come down by day of illness. he was discharged from the hospital on day of illness with a plan to be followed up in nephrology clinic for possible chronic kidney disease. d echocardiogram was repeated after three weeks of recovery and it was normal other than trivial mitral regurgitation. leptospirosis antibody titer done on day of illness was positive. ( : ). severe leptospirosis is characterized by multiple organ dysfunction including liver, kidney, lungs and brain. it is also known to cause cardiac involvement as well. cardiac manifestations range from non-specific electrocardiographic changes and arrhythmias to myocarditis, pericarditis, endocarditis and cardiogenic shock [ ] [ ] [ ] . but the pathophysiology behind it is less well understood and the magnitude of the problem is under-reported [ ] . all five patients included in this case series had evidence of acute kidney injury. the most striking feature of these five patients admitted to a single unit within a month was cardiac involvement. all five patients developed shock with low blood pressure during their course of illness. except case number , all other patients needed vasopressor/inotropic support to maintain blood pressure. case number showed evidence of myocarditis in d echocardiogram at the time of shock. case number had possible evidence of myocarditis whereas case number , had normal echo findings. case number had severe mitral regurgitation in his d echocardiogram. all these echocardiogram were performed while the patients were in shock. repeat d echocardiograms performed after three weeks of recovery were completely normal except in case number and . number had mild left ventricular hypertrophy with grade diastolic dysfunction and number had trivial mitral regurgitation. in addition to these various echo findings all of these five patients had more or less positive cardiac troponin i titre which came down with the recovery of illness. case number one and two developed atrial fibrillation which needed intervention for normalization. case number three had mild t wave inversions in anterior leads which was dynamic in serial electrocardiograms. case number and had only sinus tachycardia. all five patients had shock by definition and the most probable explanation is cardiogenic shock due to cardiac involvement of leptospirosis. though not commonly reported, myocarditis in severe leptospirosis may not be a rare complication. the european society of cardiology working group on myocardial and pericardial diseases has developed clinical and diagnostic criteria, when present myocarditis should be suspected. presence of unexplained cardiogenic shock, positive cardiac troponins, variable ecg changes are included for these criteria in addition to several other criteria [ ] . definitive diagnosis of myocarditis ideally should be established by histopathological, immunological and immunohistochemical criteria for which myocardial biopsy is required. this is not practical in most settings as these investigations are not routinely done and not required for patient management. in this case series none of the patients underwent histopathological or cardiac mri diagnosis of cardiac involvement due to lack of resources in the hospital. due to wide variability in presentation and non-specific clinical findings, many cases of myocarditis likely to go undetected. as an example, study conducted examining hearts from patients who had died due to leptospirosis has revealed myocarditis in % of cases histologically. endocardial inflammation had been observed in % of cases [ ] . in sri lanka, myocarditis has been reported previously as a complication of leptospirosis [ , ] and around - % of confirmed cases are being reported as having this complication [ , , ] . however, in most of the previous studies, the details of diagnosis of myocarditis was not clearly given. in our case series, histological diagnosis or cardiac mri to diagnose cardiac involvement was not possible due to lack of resources in the hospital. there is another phenomena coming up in the recent literature to explain the shock in leptospirosis. according to julie cagliero et al. dys-regulation of inflammatory mechanisms in severe leptospirosis can lead to cytokine storm causing sepsis like picture [ ] . systemic inflammatory response syndrome (sirs) is supposed to occur in severe leptospirosis [ ] . sirs itself can cause elevated cardiac troponins [ , ] . therefore, pure cardiac involvement in leptospirosis becomes more difficult to diagnose. all these five patients presented during one-month period in a raw and we had only six total suspected (notified) cases of leptospirosis during that month. observing cardiac involvement in five out of six probable cases of leptospirosis may be due to an outbreak caused by a different strain of a leptospira. as previously observed, outbreaks of leptospirosis with uncommon complications such as pancreatitis [ ] needs more investigations and explanations. however these patients did not have evidence of pulmonary involvement which is a known complication to occur in severe leptospirosis. case number patient had serological evidence of leptospirosis and co-infection with dengue virus. co-infection of leptospirosis and dengue is a known phenomenon in endemic countries with subtropical and tropical climates. a study conducted in malaysia has concluded that there is a considerable prevalence of leptospirosis and dengue co-infection with overlapping demographic, clinical and laboratory presentations [ ] . in sri lanka [ ] as well as in many other places [ ] [ ] [ ] , a co-infection of these two had been reported earlier and possible due to high endemicity of both diseases. it is crucial to consider co-infection with dengue where clinical suspicion arise even in the presence of enough supportive evidence for leptospirosis. because close monitoring and fluid management are the lifesaving principles of management of dengue hemorrhagic fever which must be done timely. developing severe leptospirosis in five out of six cases during same period may be due to outbreak of uncommon strain of leptospirosis. cardiac manifestations of leptospirosis are possibly under-diagnosed due to co-existence with other multi-organ involvement. diagnosis of myocarditis is difficult due to lack of imaging facilities, lack of specificity of available tests as well as unavailability of non-invasive gold standard diagnostic test. to assess the significance of cardiac troponins in diagnosing cardiac involvement in leptospirosis further studies are required. co-infection of dengue in a patient with leptospirosis should be considered especially in endemic areas. Über eine eigenthümliche mit milztumor, icterus und nephritis einhergehende acute infectionskrankheit areport on the discovery of the causative organism (a new spesies of spirochete) of weil's disease. tokyo ijishinshi leptospirosis in humans severe leptospirosis and pancreatitis; a case series from a leptospirosis outbreak in anuradhapura district, sri lanka regional differences of leptospirosis in sri lanka: observations from a flood-associated outbreak in cardiac manifestations in leptospirosis. apropos of cases observed in new caledonia cardiac involvement in severe leptospirosis cardiac and pulmonary involvement in leptospirosis current state of knowledge on aetiology, diagnosis, management, and therapy of myocarditis: a position statement of the european society of cardiology working group on myocardial and pericardial diseases cardiac findings in leptospirosis myocarditis causing severe heart failure -an unusual early manifestation of leptospirosis: a case report co-existent facial palsy and myocarditis in a -year old farmer diagnosed with probable leptospirosis: a case report predictors of the development of myocarditis or acute renal failure in patients with leptospirosis: an observational study leptospirosis outbreak in sri lanka in : lessons for assessing the global burden of disease demographic, clinical and laboratory features of leptospirosis and dengue co-infection in malaysia fatal co-infection with leptospirosis and dengue in a sri lankan male fatal leptospira spp./zika virus coinfection-puerto rico sero-epidemiology study of leptospirosis in febrile patients from terai region of nepal clinical predictors of dengue fever co-infected with leptospirosis among patients admitted for dengue fever -a pilot study we acknowledge the staff of ward of colombo-north teaching hospital, ragama in making this study a success.funding sba is supported through u.s. public health service grants u ai . the funders have played no role in the research.availability of data and materials all data contained within the article.authors' contributions nj perceived the study and prepared the first draft of the manuscript. nj, asvm, mhmtsp, hmtd, avcg provided patient care, followed up the patients, collected and interpreted clinical data. sba involved in design, analysis, interpretation of data and preparing the manuscript. all authors contributed, read and approved the final manuscript.ethics approval and consent to participate not applicable. written informed consent was obtained from all patients for publication of their individual details. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -ycdaw vh authors: maslow, joel n. title: zika vaccine development—current progress and challenges for the future date: - - journal: trop med infect dis doi: . /tropicalmed sha: doc_id: cord_uid: ycdaw vh zika virus is an emergent pathogen that gained significant importance during the epidemic in south and central america as unusual and alarming complications of infection were recognized. although initially considered a self-limited benign infection, a panoply of neurologic complications were recognized including a guillain–barré-like syndrome and in-utero fetal infection causing microcephaly, blindness, and other congenital neurologic complications. numerous zika virus vaccines were developed, with nine different vaccines representing five different platforms entered into clinical trials, one progressing to phase ii. here we review the current landscape and challenges confronting zika virus vaccine development. the zika virus, discovered in uganda in [ ] , was shown to be endemic through sub-saharan africa and tropical areas of southeastern asia in studies through the second half of the th century [ ] . isolated outbreaks occurred in yap island in [ ] and on french polynesia in [ ] . starting in mid- , zika virus infection achieved epidemic status, spreading rapidly through south america, central america, and the caribbean islands [ ] . it was soon recognized that zika virus infection occurring during pregnancy caused microcephaly and other congenital disorders in the developing fetus, the latter being the primary reason for the world health organization (who) labelling zika as an international threat in early . beginning in late , numerous academic labs and pharmaceutical companies initiated work to develop a vaccine against zika, however, by the time the first vaccines entered clinical trials, the zika epidemic had started to wane creating significant challenges to vaccine assessment [ ] that has engendered discussion of other regulatory pathways to licensure [ ] . in this paper, we provide a brief update of current progress in zika virus vaccine development and explore the challenges to vaccine assessment and eventual licensure. zika virus infection presents with a symptom complex consisting of a diffuse maculo-papular rash, fever, asthenia, myalgias, arthralgias, headache, and retroorbital pain. the frequency and degree of symptomatology has, however, varied between studies. a retrospective study immediately following the outbreak on yap island found that only % of survey participants reported symptoms consistent with zika virus infection [ ] , a figure that has been cited to suggest that zika virus infection is asymptomatic in as many as % of individuals. however, of individuals who provided blood samples, representing % of households, % were symptomatic [ ] . a second retrospective study of the larger outbreak in french polynesia [ , ] , similarly appeared to have a low rate of symptomatology based on a sample of blood donors [ ] . however, a subsequent seroprevalence study found that % of those with evidence of prior infection had symptoms consistent with zika virus infection [ ] . a more recent meta-analysis of studies noted that between and % of cases were reported as asymptomatic, although as the authors note, assessment of the prevalence of symptoms was not the goal of many studies [ ] . in adults, the most common reported complication of zika virus infection is a guillain-barré-like illness that had a prevalence of . cases per cases of infection in the french polynesian epidemic [ ] . of interest, a recent meta-analysis has questioned this causal association [ ] . other less common reported neurologic complications in adults include meningoencephalitis [ ] and an adem illness. in contrast, infection during pregnancy has been associated with fetal microcephaly and a number of other congenital illnesses including visual deficits, hearing disorders, neural calcifications, learning disabilities, and arthrogryposis that may affect as many as % children born to mothers infected during pregnancy [ ] [ ] [ ] [ ] [ ] [ ] . additionally, zika can directly invade the placenta and has been associated with prolonged maternal viremia [ ] [ ] [ ] . zika virus can also be transmitted through sexual contact, first reported for a researcher returning from senegal in [ ] . numerous subsequent case reports were published among travelers as part of the epidemic affecting the americas [ ] . the frequency of sexual transmission in endemic regions is unknown and could not be differentiated from mosquito-borne transmission. zika virus carriage in the male urogenital tract is common through days post infection and may persist in some to months [ ] . in mice, zika virus infection causes testicular atrophy and significantly decreases spermatic function and fertility rates [ ] [ ] [ ] . in one study of a zika virus dna vaccine, the adverse effects in the male reproductive system were prevented by vaccination [ ] . the question of whether zika virus can adversely affect human reproductive potential and, if so, whether such effects would be age-related is unknown. females may also excrete zika virus rna for extended times. a prospective study of five women showed that zika rna was detected in vaginal fluid for as long as months and a month or more in three of the five [ ] . murine studies showed that zika virus caused infection of the ovaries of non-immunosuppressed c bl/ mice and induced a t-cell inflammatory reaction, but without affecting reproductive potential [ ] . in contrast to the above human study, female macaques rapidly cleared virus from the genital tract [ ] . thus, while zika virus infection is mildly symptomatic and self-limited for the vast majority of individuals, with infrequent neurologic complications in adults, vaccination of the general population may not be warranted. however, vaccination of females at or entering reproductive age and their male partners is prudent. as discussed previously, vaccination during pregnancy is non-ideal due to the time to generate protective immunity and unknown vaccine safety [ ] . the one unknown aspect is whether vaccination of males of any age may be beneficial to protect against testicular complications. calls for generalized vaccination programs have, however, been put on hold due to the decreasing incidence of zika infection rates, as discussed in the next section. in late and through mid- , global concern for this spreading epidemic was increasing, however, the experience in the two prior epidemics was predictive of the subsequent epidemiology in the americas. the zika virus outbreaks in yap island and french polynesia were characterized by rapid onset and rapid resolution over a span of - months [ , ] . the zika epidemic in the americas was characterized by a longer time to reach peak incidence, taking approximately one year for brazil [ ] presumably related to the larger and more varied geography, with attack rates decreasing greater than -fold the next year [ ] . data published by the pan american health organization (paho) found similar patterns through south and central america (https://www.paho.org/). the almost complete disappearance of zika has created significant barriers to ongoing vaccine studies as attack rates have fallen below those need to meet reasonable sample sizes [ ] . the who removed the designation of zika a virus of global concern in as case rates have fallen. in march , the who and the national institutes of allergy and infectious diseases (niaid) convened a meeting of academics, representatives from industry, and regulators to discuss the approach to zika virus vaccine development in an era of waning incidence [ ] . although zika virus remains endemic in asia, africa, and central and south america, current transmission rates, coupled with high background herd immunity, would require an extremely large and logistically difficult study. as population immunity wanes, future outbreaks are likely. the ability to conduct a meaningful future clinical trial may be dependent upon having pre-existing site and regulatory approvals with an established vaccine network to enable rapid response to new reports suggesting increased disease activity. vaccine development against the zika virus began in earnest in late following the reports of microcephaly in fetuses and infants in brazil. of note, the first demonstration of immunoprotection was as part of a study to define the ultrastructural characteristics of zika virus, that found intramuscular vaccination of mice with infectious viral filtrates protected against cerebral infection [ ] . poland et al. provide a comprehensive listing of almost zika virus vaccines in development as of [ ] . diamond et al. discuss potential immunoreactive epitopes on the zika envelope and provide further information on those vaccines that progressed into clinical trials [ ] . the large number of delivery platforms include live attenuated and inactivated whole-viruses; viral-vectored vaccines utilizing adeno-associated virus, vesicular stomatitis virus, measles virus, and dengue or yellow fever chimeric vaccines; dna and mrna vaccines; and peptide and protein subunit vaccines. most have been assessed in animal models utilizing non-human primates and/or lethal challenge experiments involving immunosuppressed mice [ ] . as of , six vaccines had advanced to phase i studies [ , , ] . since that time, two additional vaccines have entered into clinical trials (table ) . a total of three dna vaccines have entered into human testing [ , ] , including one that has advanced to phase ii (table ) . gls- , a dna vaccine encoding for the zika virus prme genes designed as a consensus based on available zika virus sequences through december [ ] , was the first to enter into clinical trials. in pre-clinical studies, vaccinated mice and non-human primates were shown to develop b and t-cell immune responses against the zika virus envelope and protected against development of neurologic disease and death in immunosuppressed, interferon α, β receptor deficient (ifnar) mice [ ] . moreover, histologic sections of brain tissue showed that vaccinated mice were without inflammatory infiltrates evident in non-immunized mice [ ] . subsequent studies showed that the vaccine protected against testicular damage, testicular atrophy, spermatozoal damage, and infertility in mice [ , ] . a phase i study evaluated gls- administered via intradermal injection (id), followed by electroporation (ep) at doses of either or mg per vaccinations followed immediately by electroporation at baseline, weeks and weeks [ ] . there were no serious adverse events (sae) reported as part of the study, with the most frequent adverse events related to discomfort, pain, or swelling at the injection site. seroconversion was observed in % of individuals after two vaccinations and % after three vaccinations with gls- [ ] . neutralizing antibodies were detected for % of participants in a vero-cell (monkey kidney cell) assay, however, % of participants demonstrated the ability to neutralize infection of u mg neuroblastoma cells [ ] . vaccine responses were maintained through a year of follow-up. there was no difference in responses based on dose level. notably, passive transfer of immune serum from vaccinated participants was able to protect % of ifnar mice against lethal infection independent of the presence of vero cell neutralizing antibodies [ ] . gls- is being evaluated as part of a second double-blind, placebo-controlled clinical trial (nct ) performed in puerto rico. analysis of the latter study is in progress. two additional dna vaccines, based on the french polynesian h/pf/ strain were developed as chimeras that included the jev prm signal sequence followed by the zika envelope (e) gene (vrc ) or a similar construct with the terminal amino acids of e, representing the stem and transmembrane regions, exchanged for the analogous jev sequence (vrc ) [ ] . both vaccines were immunogenic for mice and nhps and protected > % of nhps against viremia at a dose of either or mg given twice [ ] . both vaccine candidates were advanced into clinical trials with mg of administered intramuscularly at weeks , and with vaccine vrc administered by needle and syringe while vrc was administered either as a single dose or split dose by needle and syringe or as a split dose given by the pharmajet needle-free device [ ] . seroconversion was % in the group administered vaccine with the needle-free device, less for vaccine administered as split-dose by needle and syringe, and lowest for vaccine given as a single injection with needle and syringe. the vrc vaccine was advanced into phase ii studies in the americas that utilized clinical sites with clinical site selection guided by epidemiologic modeling [ ] . long-term follow-up has not been reported. three inactivated vaccines have entered into clinical studies, of which clinical data for only zpiv vaccine has been published [ ] . zpiv is a whole inactivated virus vaccine of puerto rican strain prvabc [ ] . studies in mice showed that a single vaccination given intramuscularly with alum generated antibody titers of approximately . log and fully protected balb/c immunocompetent mice from viremia, whereas unvaccinated mice were unprotected and subcutaneously vaccinated mice were only partially protected against the zika brazil strain [ ] . a subsequent study in non-human primates vaccinated twice at four-week intervals with alum generated binding and microneutralization antibody titers of . and . log , respectively, and complete protection against viremia and viruria following challenge with either brazilian or puerto rican strains of zika virus [ ] . zpiv safety and immunogenicity was tested in three clinical trials to assess zika vaccine responses relative to dose level, vaccination schedule, or following vaccination with either the yellow fever yf-vax or japanese encephalitis virus (jev) ixaro vaccines to assess responses in a flavivirus-exposed population [ ] . there were no vaccine-associated saes reported. the most common adverse events were pain and tenderness at the injection site; no neurologic events were reported. seroconversion was % using a cutoff for peak geometric mean titer of : and % using a titer of : . response rates after a single immunization were % and . % using cutoffs of : or : , respectively. vaccine responses were observed through day . passive transfer of purified igg derived from zika immunized participants to groups of balb/c mice per participant provided sterilizing immunity against viremia for % ( of ) of mice overall, with viremia observed for one or more mice per group inoculated with sera from five ( %) individuals [ ] . clinical trial data for the other vaccines has not yet been reported as of the date of this monograph, however, pre-clinical data has been reported for three candidate vaccines. an mrna vaccine that incorporates the prm-e genes of a micronesian strain of zika virus was created incorporating with or without four-point mutations in the fusion-loop segment of the dii region of the envelope gene that abolished binding of antibodies directed against the fusion-loop region to reduce the potential risk for antibody-dependent enhancement of infection [ ] . immunization of ag mice with un-modified lipid nanoparticle-encapsulated vaccines mrna was immunogenic and protective against lethal infection; immunization of c bl/ immunocompetent mice followed by treatment with anti-ifnar blocking antibody showed protection against viremia in approximately % of animals [ ] . pizv, an inactivated vaccine derived from puerto rican strain prvabc selected as without passage-related mutations, protected against lethal zika virus challenge in ag mice when administered with alum adjuvant [ ] . a measles-vectored vaccine encoding the zika virus prme was shown to lessen viremia in pregnant ifnar mice and prevented clinical disease in mouse pups [ ] . preclinical data for vla- inactivated viral vaccines and the rzikv/d ∆ - live virus vaccine has not yet been reported. a number of animal models have been assessed for vaccine development and have been reviewed elsewhere [ ] . immunocompetent mice can develop transient viremia following infection and may represent models to test sterilizing immunity. interferon α/β receptor knockout mice (ag or ifnar -/-) develop lethal infection following zika virus infection and have been used as a more stringent measure of protection. non-human primates develop a self-limited mild illness associated with viremia and can transmit virus in utero to primate fetus [ ] . the logistical difficulties in pursuing standard vaccine evaluation have created significant interest in the possibility of controlled human infection models (chim). the conduct and planning for human challenge trials raises unique medical and ethical considerations. for zika virus trials, one must balance the relative benign and self-limited infection experienced by the vast majority against the risk for less benign complications and transmission risks. as reviewed above, published studies provide estimates that zika virus infection is relatively asymptomatic and self-limited for the majority of individuals, however, methodology in these studies varies widely. in adults, two sets of complications warrant consideration for a proposed chim study. as reviewed above, a guillain-barre-like syndrome occurs in approximately of zika virus infections [ ] , whereas other neurologic complications such as meningoencephalitis, myelitis [ , , ] , and acute disseminated encephalomyelitis [ ] are rare. second, and perhaps more important, is the risk of transmission to a sexual partner and the potential for infection during pregnancy. zika virus commonly persists in the male urogenital tract for months, and may persist in some individuals for up to months [ ] . some have considered limiting studies to non-pregnant females as zika virus colonization of the female genital tract may be temporally limited. the fact that zika virus is known to cause testicular atrophy in mice [ ] [ ] [ ] , raises yet another as yet theoretical concern for humans. these questions as well as the theoretical potential for vector-borne transmission were debated in detail in late with the conclusion that the benefits of a human challenge infection did not outweigh the risks [ ] , however, this analysis was performed just as the initial epidemic wave in the americas was ending. the group published a follow-up in [ ] . despite the recognition that conducting a placebo-controlled vaccine trial had become significantly more difficult due to declining case rates, the group's conclusion was essentially unchanged. to address safety concerns, there has been work to develop attenuated viral strains deleted for potential neurotropic regions [ ] with a goal to prevent viremia. whether the attenuated viral strain (rzikv/d ∆ - ) being tested in a phase i study will serve as putative challenge strain is as yet undetermined. in summary, zika vaccine development continues with multiple candidate vaccines in clinical trials. because of the significant decline in incidence, evaluation of vaccine efficacy is increasingly difficult. there has been renewed interest in animal model and human infection models of infection. the author is an employee of geneone life science, inc., a developer of dna-based vaccines including a vaccine against the zika virus. walter reed army institute of research niaid national institutes of allergy and infectious disease vrc vaccine research center zika virus: (i). isolations and serological specificity zika virus: following the path of dengue and chikungunya? lancet zika virus outbreak on yap island, federated states of micronesia european centre for disease prevention and control. rapid risk assessment: zika virus infection outbreak, french polynesia zika virus in the americas-yet another arbovirus threat steep drop in zika cases undermines vaccine trial demonstrating vaccine effectiveness during a waning epidemic: a who/nih meeting report on approaches to development and 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demise increased hospitalizations for neuropathies as indicators of zika virus infection, according to health information system data guillain-barré syndrome, acute disseminated encephalomyelitis and encephalitis associated with zika virus infection in brazil: detection of viral rna and isolation of virus during late infection ethical considerations for zika virus human challenge trials; national institutes of health bystander risk, social value, and ethics of human research zika vaccines: role for controlled human infection key: cord- -mjkixqhs authors: szilasi, anna; dénes, lilla; krikó, eszter; heenemann, kristin; ertl, reinhard; mándoki, míra; vahlenkamp, thomas w; balka, gyula title: prevalence of feline immunodeficiency virus and feline leukaemia virus in domestic cats in hungary date: - - journal: jfms open rep doi: . / sha: doc_id: cord_uid: mjkixqhs objectives: feline immunodeficiency virus (fiv) and feline leukaemia virus (felv) are retroviruses affecting cats worldwide. the objectives of the study were to estimate the prevalence of these retroviruses in domestic cats in hungary and to characterise the phylogenetic relationships of fiv strains. methods: a total of anticoagulated whole-blood samples obtained from both a healthy and ill cat population were examined for the presence of fiv and felv with two methods: elisa and pcr. statistical analysis was carried out to analyse the data obtained. sequencing and phylogenetic analysis of partial polymerase (pol) gene sequences was performed to describe circulating fiv subtypes. results: statistical analysis showed . % and . % true prevalence of felv and fiv, respectively, with elisa. the apparent prevalence calculated from the pcr results were . % for felv and . % for fiv. phylogenetic analysis of partial pol gene sequences obtained from fiv strains showed that all observed hungarian strains belonged to fiv subtype b. the strains were grouped into several monophyletic subgroups reflecting the geographic locations of the origin of the samples. the overall mean genetic similarity between the analysed strains was . %. conclusions and relevance: we report the first thorough overview of the prevalence of felv and fiv in hungary, which is relatively high, and give insight into the genetic diversity of hungarian strains of fiv. feline leukaemia virus (felv) is an enveloped singlestranded rna virus belonging to the retroviridae family, genus gammaretrovirus, and it infects feline species. the predominant subgroup, designated felv-a, represents the transmissible form of the virus spread cat to cat in nature and from which the other subgroups, felv-b, , felv-c and felv-t, arise de novo during the course of infection. [ ] [ ] [ ] the infection is common worldwide and is transmitted by secretions via the oronasal route, predominantly by prolonged contact, but also by direct inoculation. feline immunodeficiency virus (fiv) is a member of the lentivirus genus within the retroviridae family and it infects species of the families felidae and hyaenidae. it causes an acquired immune deficiency syndrome (aids) in cats, resembling aids caused by hiv in humans. transmission is usually by direct inoculation (eg, bite and scratch wounds). the strains are grouped into seven phylogenetic subtypes a-f and u-nzenv. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] distribution of discovered subtypes is illustrated in figure . , [ ] [ ] [ ] [ ] [ ] [ ] [ ] to test the infection status of cats, point-of-care elisa tests are widely used, which detect antibodies against the p protein of fiv and the p antigen of felv. the most frequent test used to confirm elisa results, or in case of false/non-interpretable results, is pcr. studies report a relative low prevalence of these viruses worldwide. in north america, felv prevalence ranges between . % and . %, and is % in australia, whereas in europe it is slightly higher ( . - . %). fiv prevalence levels are quite similar: . - . % in north america, % in australia and . - . % in europe. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the aim of this study was to estimate the prevalence of these retrovirus infections in domestic cats in hungary, to evaluate the main factors affecting the infection rate and to examine the phylogenetic relations of the fiv strains detected. a total of randomly selected, client-owned domestic cats, presented in clinics, over a period of years ( - ) were tested from all over hungary ( figure ). no free-roaming or sheltered cats were included in the survey. age, sex, patient history, vaccination status and the household status of cats were registered. general physical examination was performed in each case and edta-anticoagulated blood samples were drawn as a part of the routine diagnostic or screening process. a witness felv-fiv elisa test (zoetis) was performed immediately, according to the manufacturer's instructions. the rest of the blood samples were frozen and sent to the department of pathology, at the university of veterinary medicine, budapest, and they were stored at - °c until further examination. the point-of-care tests used in this study detect the presence of felv p antigen ( . % sensitivity and . % specificity) and fiv antibodies against p antigen ( . % sensitivity and . % specificity). , sensitivity and specificity values were given according to data provided by zoetis (used in the statistical analysis), although slightly different values can be found in some field studies (eg, . %/ . % for felv and . %/ % for fiv). one drop ( . ml) of edta-anticoagulated whole blood was used as per the manufacturer's instructions (zoetis). from the stored whole-blood specimens, nucleic acid extraction was carried out in a qiacube instrument (qiagen) with the use of qiamp cador pathogen mini kit (qiagen), according to the manufacturer's instructions. nucleic acid was eluted in μl rnase-free distilled water (qiagen). the preparation of endpoint pcr for fiv, toptaq master mix kit (qiagen) was used in accordance with manufacturer's instructions: μl master mix, . μl forward primer ( μm), . μl reverse primer ( μm), μl coralload concentrate, μl rnase-free water and μl template dna, and were mixed together for each sample. the hot-start pcr amplification with the given protocol was carried out as follows: °c for mins; °c for s, °c for s and °c for min ( cycles); and °c for mins. the fiv primers used in the study amplify early reverse transcription products (process of reverse transcription figure worldwide distribution of feline immunodeficiency virus subtypes (map scheme: www.outline-world-map.com). subtypes a and b are spread widely, subtypes c, d, e, f and u-nzenv are distributed only regionally rna to dna); both bind in the long terminal repeat (ltr) region, which enables the detection of low-dose and/or recent infections (primer sensitivity was - cell-associated virus). , for the detection of felv, a one step rt-pcr kit (qiagen) was used. a master mix containing . μl rnase-free water, μl × buffer, . μl dntp, . μl enzyme mix, . μl rnase inhibitor, . μl forward primer ( μm) and . μl reverse primer ( μm) were added into tubes for each sample ( . μl each). the amplification was °c for mins and °c for mins (reverse transcription and initial denaturation); °c for s, °c for min and °c for min ( cycles); and °c for mins. felv primers were obtained from a publication. the base pair (bp) (fiv) and bp (felv) long amplicons were visualised by electrophoresis in . % agarose gel. statistical calculations were performed in an r environment (r core team, vienna, austria) and a logistical regression model was used to detect the relationship between the examined variants and retroviral infection status. the epi.prev function was used to calculate prevalences. cohen's kappa (κ) was counted to show possible cross-compliance between the elisa and pcr methods. phylogenetic analysis in fiv-positive cases, partial proviral pol genes were amplified with the previously described protocol of adams et al. pcr products were subjected to gel electrophoresis, amplicons of bp length were cut out manually and purified by a qiagen gel extraction kit (qiagen). bidirectional sanger sequencing reaction was performed with the corresponding primers and the capillary electrophoresis was made by a commercial provider (microsynth). in two cases (i/ / and ii/ / ) repeated sequencing did not result in clean sequences; thus, these pcr products were cloned into a pjet . blunt vector and subsequently transformed into escherichia coli, with the help of a clonejet pcr cloning kit (thermofisher scientific). after proofreading of the obtained sequences, they were assembled using the e-ins-i method of the online software mafft version , and aligned against available fiv genomes downloaded from genbank that represent the overall diversity of the virus. maximum likelihood analyses were carried out and the trees were visualised and edited with mega using the tamura-nei model with bootstrap replicates and gamma distribution. pairwise genetic analyses were conducted using the kimura -parameter model in mega . altogether, samples from domestic cats were included in the study. all cats had owners -no stray or shelter animals were included. the mean age of cats in this study was . years (range months to years). a total of females ( . %) and males ( . %) were included in the research. of the females, ( . %) were intact and ( . %) were spayed; of the males, ( . %) figure origin of the samples collected in this study (map scheme: www.terkepek.net). on the map of hungary, the locations from where the blood samples were taken are indicated (red and green pinpoints). cities highlighted with green pinpoints are the locations of partially sequenced fiv strains. beside the name of city, number in brackets indicate how many sequences were obtained from that location were intact and ( . %) were castrated. seventy-seven cats were kept strictly indoors ( . %) and had outdoor access ( . %). a total of cats were found to be clinically healthy during the physical examination. we observed an overall low prevalence of vaccination: only ( . %) were immunised at some point of their lifespan, usually with a combined vaccine, and among these cats, only ( . %) were vaccinated against felv (however, two of them proved to be felv-infected). of was observed in the case of fiv infection for every year older the cats were. we could not find significant correlation of infection status with sex (although males were . times more likely to have fiv and/or be felvinfected than females [ % ci . - . , p = . ]). the same was observed for neutering status: there was a . times higher risk for intact males to be infected ( % ci . - . ; p = . ) and a . higher risk for intact females ( % ci . - . ; p = . ), but these data were not statistically significant. altogether, we were able to sequence partial pol genes out of the fiv pcr-positive cases. we suspect that the rest of the samples contained insufficient amounts or poor quality nucleic acids. multiple alignments and maximum likelihood tree reconstructions revealed that all of the strains were clustered into subtype b (figure ) . within clade b, the hungarian sequences were located into a monophyletic group that also included a strain originating from joinville, brazil, (acc. number: ky . ). within this 'hungarian' cluster, the strains formed several subgroups of closely related sequences that mostly reflected the geographic location of their origin. the subgroups were supported by relatively high bootstrap values. pairwise genetic analyses revealed that the overall mean genetic similarity between the analysed strains was . % (the lowest similarity was . % and the highest was . %). only two strains originated from indoor cats and the majority of these cats were males ( males, six females). the samples represent most of geographic regions of hungary (figure ). genbank accession numbers are mn -mn . our results show a relatively high rate of infection of felv in owned domestic cats in hungary vs the most recent data from surrounding (or more distant) countries (with the exclusion of prevalence data including free-roaming or shelter cats). the prevalence rate of fiv does not show such a difference. data regarding vaccination status showed that there is a strong need for improving in the vaccination prevalence in hungarian cat populations. two of the immunised cats had felv-positive results, both determined by elisa and pcr, but their vaccinations were not regular, which could be an answer to possible unprotected periods of time and the ability to become infected. moreover, . % of cats had outdoor access and most of them were not neutered. previous publications and our statistics show a higher occurence of retroviral infection in intact (usually male) outdoor cats. another possible explanation for the higher observed prevalence data could be the weak positive predictive value of every diagnostic test that does not have % specificity in the case of low prevalence rates in a population ('false positive paradox'). in relation to the discordant results between elisa and pcr in some cases, where only one test method was positive, there can be various explanations. in the case of fiv, the witness test detects antibodies, whereas the pcr amplifies and detects part of the ltr region of the virus. in the early stages of infection there are no detectable antibodies (most cats produce antibodies within weeks of exposure); moreover, there might be extremely low levels of antibodies in the terminal phase of the disease, causing a false-negative elisa result, whereas the pcr result will be positive as a result of increased viral load in the circulation. however, a positive elisa and negative pcr result can suggest extremely low levels of circulating proviral dna/viral rna (which can be the case in the asymptomatic phase of fiv infection) or viral genetic diversity (where nucleotide sequence dissimilarity can result in failure of primer binding). the presence and interference of maternal antibodies with the elisa can be excluded in our cases, as the youngest animals included in the study ( - months of age) were fiv negative. maternal antibodies can be present in kittens up to months of age. in the case of felv, the witness test detects the p antigen, whereas the pcr amplifies and detects the u part of the ltr region. a negative elisa and positive pcr result can also suggest an early phase of infection or illness in a regressive stage, when small amounts of proviral dna/viral rna are already present in the circulation, but the amount of the antigen is still below detectable levels. regarding the phylogenetic analysis of the fiv sequences, all the sequenced samples belonged to subtype b, and they form a unique cluster of strains that includes hungarian sequences and also a brazilian one. the monophyletic pattern suggests a common ancestor for all the hungarian (and the single brazilian) sequences analysed in the study. based on our data, it can be speculated that there was a single event of virus introduction into the country and the genetic diversity observed is the result of divergent local evolution of the strains. it must be highlighted that our sequences are insufficient to give a complete picture of the circulating fiv strains in hungary, but it can be suggested that mainly subtype b strains are present in hungary. this result is largely in harmony with data published from surrounding countries, where mostly subtypes a and b were discovered. this is the first report of a thorough investigation on retroviral prevalence and phylogenetic analyses of fiv strains in hungary, and suggests that fiv subtype b is the most prevalent in this country. this data set and prospective phylogenetic analyses of further fiv figure phylogenetic tree reconstructed from the partial pol gene sequences of hungarian feline immunodeficiency virus strains and selected reference sequences from genbank. maximum likelihood bootstrap support values (⩾ ) are shown as percentages above the branches. the two indoor cats are marked with red dots; all the others had outdoor access. strains displayed on the phylogenetic tree are coded differently in the case of hungarian strains, the code of sample and city of the cat's origin are shown. in case of reference sequences obtained from genbank, accession number, year of publication and country of collection are displayed strains can give useful information for vaccine developers as the only existing fiv vaccine, which is not available in hungary (fel-o-vax fiv; boehringer ingelheim), contains inactivated whole virus subtypes a and d, which will not confer a sufficient protective effect against subtype b. , identification of envelope determinants of feline leukemia virus subgroup b that permit infection and gene transfer to cells expressing human pit or pit host range and receptor binding properties of vectors bearing feline leukemia 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infection in lions recent developments in the mafft multiple sequence alignment program mega : molecular evolutionary genetics analysis version . for bigger datasets mega : molecular evolutionary genetics analysis version . a simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences efficacy of an inactivated felv vaccine compared to a recombinant felv vaccine in minimum age cats following virulent felv challenge seroprevalences of feline leukemia virus and feline immunodeficiency virus in cats with abscesses or bite wounds and rate of veterinarian compliance with current guidelines for retrovirus testing infectious diseases of the dog and cat effects of passive transfer of immunity on results of diagnostic tests for antibodies against feline immunodeficiency virus in kittens born to vaccinated queens development of the dual-subtype feline immunodeficiency virus vaccine acknowledgements we wish to thank all the veterinarians who provided blood samples and the background data of the cats, and also zoetis hungary, with special thanks to zsolt varga for providing the witness elisa tests. kitti schönhardt, molecular biologist of the department of pathology, university of veterinary medicine, budapest, was generous with her time helping with the pcr procedures. ethical approval this work involved the use of nonexperimental animals only (owned or unowned), and followed internationally recognised high standards ('best practice') of individual veterinary clinical patient care. ethical approval from a committee was not necessarily required.informed consent informed consent (either verbal or written) was obtained from the owner or legal custodian of all animal(s) described in this work for the procedure(s) undertaken. no animals or humans are identifiable within this publication, and therefore additional informed consent for publication was not required. https://orcid.org/ - - - reinhard ertl https://orcid.org/ - - - key: cord- -sw qbbj authors: aylward, ryan e.; van der merwe, elizabeth; pazi, sisa; van niekerk, minette; ensor, jason; baker, debbie; freercks, robert j. title: risk factors and outcomes of acute kidney injury in south african critically ill adults: a prospective cohort study date: - - journal: bmc nephrol doi: . /s - - - sha: doc_id: cord_uid: sw qbbj background: there is a marked paucity of data concerning aki in sub-saharan africa, where there is a substantial burden of trauma and hiv. methods: prospective data was collected on all patients admitted to a multi-disciplinary icu in south africa during . development of aki (before or during icu admission) was recorded and renal recovery days after icu discharge was determined. results: of admissions, the mean age was . years and mean saps score was . . comorbidities included hypertension ( . %), hiv ( . %), diabetes ( . %), ckd ( . %) and active tuberculosis ( . %). the most common reason for admission was trauma ( %). aki developed in ( . %). male gender, illness severity, length of stay, vasopressor drugs and sepsis were independently associated with aki. aki was associated with a higher in-hospital mortality rate of . % vs . % in those without aki. age, active tuberculosis, higher saps score, mechanical ventilation, vasopressor support and sepsis were associated with an increased adjusted odds ratio for death. hiv was not independently associated with aki or hospital mortality. ckd developed in of ( . %) patients with stage aki; none were dialysis-dependent. conclusions: in this large prospective multidisciplinary icu cohort of younger patients, aki was common, often associated with trauma in addition to traditional risk factors and was associated with good functional renal recovery at days in most survivors. although the hiv prevalence was high and associated with higher mortality, this was related to the severity of illness and not to hiv status per se. acute kidney injury (aki) is commonly encountered in the intensive care unit (icu) [ ] , but with a widely variable reported incidence due to non-standardization of its definition [ ] . regardless of the definition used, aki is a well-recognized independent risk factor for mortality, is associated with substantial morbidity and is a current major cause for global concern [ ] [ ] [ ] [ ] . furthermore, aki requiring dialysis is now recognized as a risk factor for end stage kidney disease in the long term [ , ] and is associated with poor long-term quality of life after icu discharge [ , ] . aki has been well characterized in high income (hi) countries and appears to be increasing in incidence [ , [ ] [ ] [ ] . however, there is a marked paucity of data from african icu's concerning the incidence, aetiology and effect of aki on mortality and functional renal recovery, where the prevalence of hiv and trauma is high and where resources are often limited [ , , ] . renal replacement therapy is an expensive [ ] and scarce resource in south africa [ ] and in particular, in the rest of sub-saharan africa [ , ] . the international society of nephrology has boldly called for by : the elimination of preventable deaths from aki by [ ] . timely diagnosis and prevention remain the most important strategies. accordingly, understanding the epidemiology of aki in lower and middle income (lmi) countries must be a key step in tackling this problem. our aim therefore, was to describe the epidemiology of aki in patients admitted to our south african multidisciplinary icu, where there is currently a high prevalence of hiv and traumaassociated admissions. we sought to characterize the factors associated with the development of aki, the effect of hiv on aki as well as survival and to report on the -day renal function outcome of all who developed aki. an observational prospective design was used. cohorts were divided into those who did and did not develop aki (prior to and/or after admission to the icu) as well as by hiv status where known. all patients older than years admitted to the livingstone hospital icu between january and january were included. patients who died within h of being admitted to icu, those who were brain-dead awaiting organ harvesting, and patients with known or presumed end stage kidney disease were excluded from the study. the livingstone hospital adult icu is a tertiary service, closed, multi-disciplinary -bed unit serving a catchment area of . million people. the hospital is government-funded and is located in the nelson mandela bay metropole in south africa where . million people live in an urban setting, . % of whom live in informal shack dwellings and . % of whom are unemployed. the balance of , people live in surrounding rural areas within a radius of km [ ] . full time consultant supervision is provided by two intensivists and two nephrologists. the provision of dialysis is also government-funded and not restricted within the icu or by hiv status. however, general prognosis and current resource limitations are taken into consideration prior to the admission of any patient to the icu [ ] . the modality of dialysis is chosen by the treating consultant based on clinical status with a preference for intermittent haemodialysis or sustained low efficiency daily dialysis (sledd) due to cost constraints. continuous renal replacement therapy (crrt) is reserved for severely haemodynamically unstable patients and for those with raised intracranial pressure. referring disciplines include medicine, trauma, general surgery, urology, neurosurgery, orthopaedics, obstetrics and gynaecology. elective cardiology and cardio-thoracic surgery have their own dedicated icu. obstetrics also has their own high care although patients with advanced organ dysfunction are referred to our unit. aki was diagnosed and staged according to the kidney diseases improving global outcomes (kdigo) definition [ ] . a normal serum creatinine not older than days was assumed to be the baseline where available, as recommended [ ] . the cause of aki was determined by the treating intensivist/nephrologist and more than one cause could be assigned. aki was recorded as resolved once creatinine improved to the known or presumed baseline. if renal function had not recovered by hospital discharge, patients were followed up for at least days following icu discharge or until renal recovery. patients who had not recovered their renal function by this time were deemed to have chronic kidney disease (ckd). approval for the study (protocol number: / ) was granted by the walter sisulu university human research ethics unit. since we were conducting a nonexperimental study that would not influence clinical decision-making or patient management, the need for study participant consent was waived by the ethics unit. demographic data including age, sex, race and details related to co-morbidities were recorded. for patients known with hiv, a premorbid cd count and viral load was recorded, where available. the simplified acute physiology score (saps ) [ ] was calculated within the first hour of icu admission. the sequential organ failure assessment (sofa) [ ] was calculated h after admission and every third day thereafter, or sooner if the patient's condition deteriorated. vasopressor and mechanical ventilation requirements were also recorded. cause of aki, renal replacement modality and creatinine on admission, peak and discharge were recorded for patients in the aki cohort. sepsis was defined using sepsis- criteria [ ] . data were exported from the research electronic data capture (redcap) hosted at the university of cape town [ ] and analyzed with rstudio, (version . . ). hypothesis tests were considered significant if the two-sided p-value < . . continuous data were tested for normality using the kolmogorov-smirnov, shapiro-wilk, anderson-darling and pearson's chi-squared tests. normally distributed data are reported as means (standard deviation) and skewed data as medians (interquartile range). discrete data are presented as numbers (percentages). the student's t-test and the mann-whiteney u test were used to compare continuous data and the chisquare and fischer's exact test were used for discrete data, as appropriate. missing outcome data (n = ) were analysed using multiple imputation. hazard ratios for mortality by aki and hiv status were calculated using the cox proportional hazards model. multivariate logistic-regression models were used to determine associations of developing aki and dying. variables by bivariate analysis with an alpha level < . between aki and non-aki cohorts as well as known predictors for aki (age, sex, hypertension, active malignancy and admission saps score) were included in the model. kdigo stages , and were compared to patients who did not develop aki as reference. a total of patients were admitted to the icu during the study period and were excluded from the analysis; fig. details the reasons for exclusion. vital status after icu discharge could not be established for patients due to in-patient transfers to other hospitals and the unavailability of further records; six were in the aki cohort, and outcomes were imputed. in-hospital mortality was . % while mortality in icu was . %. the most common diagnoses admitted were: assault ( %), motor and pedestrian vehicle accidents ( %), acute abdomen ( %), pneumonia ( %; including cases later identified as tuberculosis) and self-inflicted drug/toxin overdose ( . %). admissions included surgical emergencies (n = , . %), medical emergencies (n = , . %), surgical elective cases (n = , %) and obstetric emergencies (n = , . %). table shows the baseline characteristics of all patients admitted to the icu and by aki status. aki developed in patients ( . %), . % of whom were diagnosed with aki on admission to the icu. the maximum kdigo stage was stage in ( . %), stage in ( . %) and stage in ( . %) patients respectively. figure shows the main causes identified for developing aki. herbal ingestion was only documented in patients and patients were exposed to antituberculous drugs (rifampicin and isoniazid). of the hiv positive patients who developed aki, had received tenofovir prior to aki diagnosis. histology was obtained in situations where the cause of aki was not clear or the clinical course of the patient was unclear. kidney biopsies were performed in patients ( % of the aki cohort), only of whom had hiv which showed interstitial hiv associated nephropathy. the others were in hiv negative subjects; showed features of mesangiocapillary glomerulonephritis, of which was crescentic and had features of ascending pyelonephritis. risk factors associated with aki are presented in table . on multivariate analysis, male gender, increased sofa and/or saps score, increased length of stay, the need for vasopressor drugs and eighty-eight patients were dialyzed ( . % of patients with stage aki, . % of the cohort with aki and . % of the entire cohort), . % of whom were fig. ). the most common indications for dialysis initiation were life-threatening hyperkalaemia ( . %), uraemic symptoms such as encephalopathy or seizures ( . %) and refractory metabolic acidosis ( . %). the development of aki was associated with a higher in-hospital mortality rate of . % compared to . % in those without aki (hazards ratio . , % ci . ; . ; logrank p < . (fig. ) ). further, the odds of dying increased stepwise with increasing kdigo aki stage (fig. ) in the aki cohort, an increased adjusted odds ratio for death was observed with increasing age, active tuberculosis, higher saps score, receipt of mechanical ventilation, receipt of vasopressor support and in those with sepsis (table ) . hiv infection was associated with worse survival in those with aki (fig. ) . however, on multivariate analysis, hiv was not found to be an independent risk factor for mortality (table ) . patients with hiv and aki were more severely ill on admission with mean saps score (sd) of . ( . ) versus . ( . ), p = . and sofa (iqr) of ( ; ) versus ( ; ), p = . . they also had a higher likelihood of having active tuberculosis [or ( %ci) = . ( . ; . ), p = . ]. the median premorbid cd count was significantly lower in those with aki than without ( . cells/microliter versus cells/microliter, p = . ) but viral suppression was similar in both groups (viral load log . vs . respectively, p = . ). although icu length of stay was less in hiv patients with aki (median icu days vs , p = . ), the proportion of patients with hiv who died within the first h was higher at . % compared to . % in those who were hiv negative, p = . . this is the largest prospective african study of aki in critically ill adults with an hiv seropositive rate of . %. similar studies include reports from morocco (n = ) [ ] , the democratic republic of congo (drc, n = ) [ ] and egypt (n = ) [ ] . however, only the drc study reported hiv prevalence, which was low at . %. consistent with other lmi countries, patients were younger than in hi country cohorts (mean age . years) with lower comorbidity rates [ , , , [ ] [ ] [ ] [ ] , mostly of black african ethnicity, and were representative of the local community that we serve [ ] . as in other african [ ] [ ] [ ] and hi country [ ] icubased studies, aki was very common in our cohort and affected nearly two thirds ( . %) of all patients admitted to the icu. aki was associated with male gender, higher severity of illness, more sepsis, longer icu stay and the need for vasopressors. pre-existing ckd was negatively associated with aki but reflects an admission bias against very ill patients with ckd to the unit due to a lack of resources to continue with chronic renal replacement therapy in most. increased age, severity of illness, sepsis, mechanical ventilation, the use of vasopressors and the presence of tuberculosis were independently associated with mortality in those with aki. furthermore, increasing stage of aki showed a stepwise increase in the risk of mortality. although our unit admits emergency medical as well as elective surgical cases, just over a quarter of all admissions were trauma related, reflecting the alarmingly high levels of interpersonal violence and road traffic accidents prevalent in south africa. in , interpersonal violence and road injury combined were the second leading cause of death and disability adjusted life years lost in south africa, after hiv which was the leading cause [ ] . consequently, a large proportion of aki was attributable to hypovolaemic shock ( . %) and rhabdomyolysis ( . %) in keeping with the degree of trauma-related admissions. two recent retrospective studies in south africa also highlighted aki in trauma victims as a major contributor to morbidity and mortality [ , ] . this reflects a major change in the epidemiology of aki in south africa where older reports have not highlighted trauma as a major cause of aki [ , , ] and has important public health implications for health administrators. sepsis was also a common precipitant of aki at . % which is similar to that reported in other lmi country studies in the critically ill [ , , ] . herbal and traditional medicine use is not a prominent cause of aki in our region compared with prior reports from other regions [ , ] . tropical diseases such as malaria are not endemic in our region and are therefore also not common precipitants of aki. severe aki (kdigo stage ) was common, affecting . % of all those with aki and . % of all admissions. the number of patients dialyzed ( . % of all admissions) was slightly lower than in the large acute kidney injury-epidemiologic prospective investigation (aki-epi) study where . % of all admissions were dialyzed [ ] and may be explained by local practice to usually delay the initiation of dialysis until more classic indications exist. notwithstanding this fact, most patients requiring dialysis were initiated early (within h of icu admission) reflecting the advanced state of organ dysfunction at admission and late presentation that is common in lmi countries [ ] . although the aki-epi study was multinational, only patients from africa were included. continuous renal replacement therapies are available in our center but cost in excess of times more than intermittent dialysis. as such, crrt is reserved for specific indications and the rate of crrt use was much lower in our study at . % compared to . % in the aki-epi study. of those who developed aki and survived, a significant proportion of patients with stage aki did not recover renal function fully ( . %). south africa has a very high hiv burden of . million people living with hiv as well as the largest number of people on antiretroviral therapy in the world [ ] . the hiv seropositive rate of . % in our cohort is consistent with the known background prevalence of hiv in our province of . % in adults aged - when measured in [ ] . this is vastly different to a retrospective study in a south african medical icu in [ ] where only three patients ( . %) in the aki cohort were hiv positive. in our study, active tuberculosis was very common, affecting in of all admissions. this is likely in part due to the high prevalence of hiv, but also due to the high reported background incidence of tuberculosis in the community of cases/ per year [ ] . whilst tuberculosis was not associated with aki per se, it was independently associated with mortality. many cases of active tuberculosis were diagnosed during icu admission through microbiological means and many were not the primary cause of admission. hiv infection was associated with higher mortality, as well as the presence of sepsis and active tuberculosis. it was also associated with increased severity of illness and the need for emergency admission. length of stay was shorter for those with hiv, but reflects earlier mortality in those with increased severity of illness. as in other studies in the post-haart era [ , , [ ] [ ] [ ] , it would appear from our data that traditional predictors of mortality such as higher severity of illness are implicated in predicting mortality and not hiv status, cd count, viral suppression nor the use of haart. proportionally few ( . %) hiv positive patients that developed aki were receiving tenofovir-based haart at the time of icu admission. although there was little difference in viral suppression between groups, the cd count was significantly lower in the group that developed aki thereby placing patients at risk for the immune reconstitution inflammatory syndrome (iris) [ ] . while tenofovir has been shown to be nephrotoxic [ ] , we hypothesize that the pathogenesis of aki in at least some of our hiv patients was related to the often recent initiation of haart with the development of an unmasking tuberculosis-associated iris [ ] and consequent tuberculosis sepsis syndrome with associated aki. this has previously been reported [ ] and is likely to be under-recognized. this study needs to be viewed in the context of its limitations. we may have underestimated the incidence of aki since we were unable to reliably utilize the kdigo urine output criterion for diagnosis as patients admitted to the icu were not always weighed or catheterised. secondly, . % of all patients admitted to the icu were not tested for hiv. all patients who were able to consent were encouraged to have an hiv test; however, patients that were moribund or confused were not tested for hiv without indication or consent. on the other hand, to our knowledge, this is the largest published prospective cohort of critically ill adults with hiv and aki [ ] . the study was inclusive of all major disciplines with the exception of cardiothoracics and the loss to follow up was low at . %. standardised criteria for the diagnosis of all stages of aki were used and -day renal recovery data was obtained. in this large prospective multidisciplinary icu cohort of younger patients in a lmi country with a high hiv prevalence and many trauma related admissions, aki was frequently encountered, and was associated with a high mortality, but good functional renal recovery in most survivors. while hiv infection was associated with higher mortality, this was due to increased severity of illness, not hiv status per se. epidemiology of acute kidney injury in critically ill patients: the multinational aki-epi study timing and outcome of aki in critically ill patients varies with the definition used and the addition of urine output criteria incidence, outcomes, and comparisons across definitions of aki in hospitalized individuals small acute increases in serum creatinine are associated with decreased long-term survival in the critically ill acute kidney injury: an increasing global concern raising awareness of acute kidney injury: a global perspective of a silent killer five-year risk of end-stage renal disease among intensive care patients surviving dialysis-requiring acute kidney injury: a nationwide cohort study acute kidney injury and chronic kidney disease as interconnected syndromes six-month outcome in acute kidney injury requiring renal replacement therapy in 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the use of traditional medicines the fifth south african national hiv prevalence, incidence, behaviour and communication survey acute renal failure in the medical icu still predictive of high mortality. south african medical journal =. suid-afrikaanse tydskrif vir geneeskunde nationwide and regional incidence of microbiologically confirmed pulmonary tuberculosis in south africa, - : a time series analysis acute kidney injury among hivinfected patients admitted to the intensive care unit hiv-positive patients in the intensive care unit: a retrospective audit. south african medical journal =. suid-afrikaanse tydskrif vir geneeskunde clinical characteristics, outcomes and risk factors for death among critically ill patients with hiv-related acute kidney injury a practical approach to the diagnosis and management of paradoxical tuberculosis immune reconstitution inflammatory syndrome tenofovir nephrotoxicity: acute tubular necrosis with distinctive clinical, pathological, and mitochondrial abnormalities tuberculosis-associated immune reconstitution inflammatory syndrome and unmasking of tuberculosis by antiretroviral therapy acute kidney disease due to immune reconstitution inflammatory syndrome in an hiv-infected patient with tuberculosis the outcome of hiv-positive patients admitted to intensive care units with acute kidney injury springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank all the rotating doctors who helped collect data. noline van vuuren was invaluable in data checking and cleaning. authors' contributions ra wrote the protocol, collected and cleaned data and wrote the manuscript. evdm assisted with the protocol, consultancy and manuscript writing. sp cleaned data and performed the statistical analysis. mvn collected and cleaned data. je assisted with consultancy, following up patients in the renal unit and appraisal of the final manuscript. db assisted with data collection, consultancy and appraisal of the final manuscript. rf assisted with the protocol, consultancy, manuscript writing and follow up of patients in the renal unit. all authors approved the final version of the manuscript. the authors gratefully acknowledge roche south africa for an unrestricted research grant that was used to fund data capturing, analysis, presentation and publication of the study (ref: sa/nonp/ / ). the funder did not contribute to the design, conduct of the study, analysis or interpretation of results. not applicable. the authors declare that they have no competing interests.author details adult critical care unit, livingstone hospital, port elizabeth, south africa. key: cord- -tazd dvm authors: yang, kun; jin, ming-ji; quan, zhe-shan; piao, hu-ri title: design and synthesis of novel anti-proliferative emodin derivatives and studies on their cell cycle arrest, apoptosis pathway and migration date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: tazd dvm emodin is a cell arrest and apoptosis-inducing compound that is widely distributed in different plants (rhubarb, aloe), lichens and terrestrial fungi, and also isolated from marine-derived fungi and marine sponge-associated fungi. in this study, we designed and synthesized a novel series of emodin derivatives by binding emodin to an amino acid using linkers of varying lengths and composition, and evaluated their anti-proliferative activities using hepg cells (human hepatic carcinoma), mcf- cells (human breast cancer) and human normal liver l cells. most of these derivatives showed moderate to potent anti-proliferative activities. notably, compound a exhibited potent anti-proliferative activity against hepg cells with the half maximal inhibitory concentration (ic( )) value of . µm, which was enhanced . -fold compared to the parent compound emodin (ic( ) = . µm), and it also exhibited better selective anti-proliferative activity and specificity than emodin. moreover, further experiments demonstrated that compound a displayed a significant efficacy of inducing apoptosis through mitochondrial pathway via release of cytochrome c from mitochondria and subsequent activation of caspase- and caspase- , inducing cell arrest at g /g phase, as well as suppression of cell migration of tumor cells. the preliminary results suggested that compound a could be a promising lead compound for the discovery of novel anti-tumor drugs and has the potential for further investigations as an anti-cancer drug. emodin ( -methyl- , , -trihydroxyanthraquinone, figure ) is a naturally occurring anthraquinone derivative that is widely distributed in different plants (rhubarb, aloe), lichens and terristrial fungi, that has also been isolated from marine-derived fungi and marine sponge-associated fungi [ ] [ ] [ ] [ ] . it is also an active ingredient in chinese medicinal herbs used in the treatment of constipation, jaundice, gastro-intestinal hemorrhage, and ulcers. emodin has the same tricyclic planar chromophore skeleton as certain anti-tumor antibiotics such as daunorubicin, doxorubicin, epirubicin, and several synthetic analogs distributed such as mitoxantrone and pixantrone ( figure ) ; all of which are in used for clinical treatment of various cancers. many reports have demonstrated that emodin possesses a wide spectrum of pharmacological effects such as anti-tumour [ ] [ ] [ ] , anti-inflammatory [ , ] , antiviral [ ] , antibacterial [ ] , anti-allergic [ ] , anti-osteoporotic [ ] , anti-diabetic [ , ] , immunosuppressive [ ] , neuroprotective [ ] , and hepatoprotective [ , ] activities, etc. among these effects, anti-tumor anti-osteoporotic [ ] , anti-diabetic [ , ] , immunosuppressive [ ] , neuroprotective [ ] , and hepatoprotective [ , ] activities, etc. among these effects, anti-tumor activity is the most widely reported. in recent decades, pharmacological studies have shown that emodin is capable of inhibiting cellular proliferation [ ] , inducing of cell differentiation [ ] and apoptosis [ ] , and activating of caspase cascade pathway [ , ] and mitochondrial death pathway [ , ] in different cancer cells. chemical transformation of bioactive compounds of natural products is one of the most common approaches in drug discovery to improve therapeutic properties. to date, the anti-tumor activity of emodin has been improved by structural modification, mainly to the side chain including methyl, hydroxyl, and/or aryl ring groups. for example, wang et al. reported that emodin quaternary ammonium salt derivatives showed significant anti-cancer activities against hepatoma cells [ ] [ ] [ ] . xing et al. reported the effects of emodin rhamnoside derivatives against human cancer cells [ ] , and tan et al. reported dna-binding pyrazole emodin derivatives [ ] . however, emodin is not an ideal chemotherapeutic agent for cancer due to its poor bioavailability, low solubility, and high toxicity in vivo. the bioavailability of a drug is positively correlated with its solubility; thus amino acids, which possess the carboxylic and amino functionality, are ideal as a moiety for the structural modification of bioactive natural products. amino acids can interact with other biomolecules via secondary interactions such as hydrogen bonding to improve their pharmacological profiles in both potency and bioavailability [ ] . for example, conjugation of an amino acid to betulinic acid is able to improve its water-solubility as well as its anti-melanoma activity [ ] . these findings prompted us to design and synthesize a series of novel emodin derivatives linked with various amino acids as tfa salt ( figure ). among these derivatives, compound b derived from (r)- -aminopropanoic acid exhibited the most anti-proliferative activity against both hepg cells and mcf- cells. this prompted us to screen the different length of diol linker between -aminopropanoic acid and emodin. our results also suggest that compared to compound a, compound a exhibited a slightly more potent chemical transformation of bioactive compounds of natural products is one of the most common approaches in drug discovery to improve therapeutic properties. to date, the anti-tumor activity of emodin has been improved by structural modification, mainly to the side chain including methyl, hydroxyl, and/or aryl ring groups. for example, wang et al. reported that emodin quaternary ammonium salt derivatives showed significant anti-cancer activities against hepatoma cells [ ] [ ] [ ] . xing et al. reported the effects of emodin rhamnoside derivatives against human cancer cells [ ] , and tan et al. reported dna-binding pyrazole emodin derivatives [ ] . however, emodin is not an ideal chemotherapeutic agent for cancer due to its poor bioavailability, low solubility, and high toxicity in vivo. the bioavailability of a drug is positively correlated with its solubility; thus amino acids, which possess the carboxylic and amino functionality, are ideal as a moiety for the structural modification of bioactive natural products. amino acids can interact with other biomolecules via secondary interactions such as hydrogen bonding to improve their pharmacological profiles in both potency and bioavailability [ ] . for example, conjugation of an amino acid to betulinic acid is able to improve its water-solubility as well as its anti-melanoma activity [ ] . these findings prompted us to design and synthesize a series of novel emodin derivatives linked with various amino acids as tfa salt ( figure ). anti-osteoporotic [ ] , anti-diabetic [ , ] , immunosuppressive [ ] , neuroprotective [ ] , and hepatoprotective [ , ] activities, etc. among these effects, anti-tumor activity is the most widely reported. in recent decades, pharmacological studies have shown that emodin is capable of inhibiting cellular proliferation [ ] , inducing of cell differentiation [ ] and apoptosis [ ] , and activating of caspase cascade pathway [ , ] and mitochondrial death pathway [ , ] in different cancer cells. chemical transformation of bioactive compounds of natural products is one of the most common approaches in drug discovery to improve therapeutic properties. to date, the anti-tumor activity of emodin has been improved by structural modification, mainly to the side chain including methyl, hydroxyl, and/or aryl ring groups. for example, wang et al. reported that emodin quaternary ammonium salt derivatives showed significant anti-cancer activities against hepatoma cells [ ] [ ] [ ] . xing et al. reported the effects of emodin rhamnoside derivatives against human cancer cells [ ] , and tan et al. reported dna-binding pyrazole emodin derivatives [ ] . however, emodin is not an ideal chemotherapeutic agent for cancer due to its poor bioavailability, low solubility, and high toxicity in vivo. the bioavailability of a drug is positively correlated with its solubility; thus amino acids, which possess the carboxylic and amino functionality, are ideal as a moiety for the structural modification of bioactive natural products. amino acids can interact with other biomolecules via secondary interactions such as hydrogen bonding to improve their pharmacological profiles in both potency and bioavailability [ ] . for example, conjugation of an amino acid to betulinic acid is able to improve its water-solubility as well as its anti-melanoma activity [ ] . these findings prompted us to design and synthesize a series of novel emodin derivatives linked with various amino acids as tfa salt ( figure ). among these derivatives, compound b derived from (r)- -aminopropanoic acid exhibited the most anti-proliferative activity against both hepg cells and mcf- cells. this prompted us to screen the different length of diol linker between -aminopropanoic acid and emodin. our results also suggest that compared to compound a, compound a exhibited a slightly more potent among these derivatives, compound b derived from (r)- -aminopropanoic acid exhibited the most anti-proliferative activity against both hepg cells and mcf- cells. this prompted us to screen the different length of diol linker between -aminopropanoic acid and emodin. our results also suggest that compared to compound a, compound a exhibited a slightly more potent anti-proliferative activity against hepg cells and mcf- cells. following this, the anti-cancer mechanism of compound a was evaluated against hepg cells. a novel series of emodin derivatives linked with amino acids were designed and synthesized. the synthesis strategies for these emodin derivatives are outlined in schemes - . for the synthesis of compounds a- z, compound was subjected to an alkylation reaction with -iodoethanol and cs co in dmf that resulted in compound . then coupling reactions under the conventional coupling condition (dcc/dmap) between compound and various commercially available n-boc protected amino acids resulted in the formation of condensation products. after removal of the protecting group usinng % tfa in dcm, the target compounds a- z were obtained as tfa salts. molecules , , x for peer review of anti-proliferative activity against hepg cells and mcf- cells. following this, the anti-cancer mechanism of compound a was evaluated against hepg cells. a novel series of emodin derivatives linked with amino acids were designed and synthesized. the synthesis strategies for these emodin derivatives are outlined in scheme - . for the synthesis of compounds a- z, compound was subjected to an alkylation reaction with -iodoethanol and cs co in dmf that resulted in compound . then coupling reactions under the conventional coupling condition (dcc/dmap) between compound and various commercially available n-boc protected amino acids resulted in the formation of condensation products. after removal of the protecting group usinng % tfa in dcm, the target compounds a- z were obtained as tfa salts. following the dcc/dmap coupling condition and deprotection under % tfa in dcm for the synthesis of compounds a- z, the target compounds a and b were obtained as tfa salts (scheme ). scheme . synthesis of compounds a- z. reagents and conditions: (a) -iodoethanol, cs co , dmf, • c, %; (b) (i) various n-boc amino acids, dcc, dcm, • c; (ii) % tfa in dcm, r.t., %- % over two steps. following the dcc/dmap coupling condition and deprotection under % tfa in dcm for the synthesis of compounds a- z, the target compounds a and b were obtained as tfa salts (scheme ). these products were then coupled with formaldehyde ( % aqueous solution) and reduced utilizing nabh cn to provide a and b as tfa salt in % and % yields, respectively. as shown in scheme , by employing the route for the synthesis of compounds a- z, compounds a- l were obtained as tfa salts. scheme . synthesis of compounds a- l. reagents and conditions: (a) various hydroxyalkyl bromides or iodides, cs co , dmf, °c, %- %; (b) (i) r/s-n-boc-ala-oh, dcc, dcm, °c; (ii) % tfa in dcm, r.t., %- % over two steps. the in vitro anti-proliferative activities of all the novel synthesized compounds were evaluated against hepg cells and mcf- cells by celltiter-glo ® luminescent cell viability assay, using paclitaxel (a clinically used drug) as a positive control. emodin ( ) and compound were also included for comparison. as shown in table , emodin ( ) showed weak inhibitory activity against hepg cells and mcf- cells with ic values of . ± . µm and . ± . µm, respectively. compared to emodin ( ), compound showed reduced activity due to the introduction of the hydroxyethyl group at the -position of emodin. among compounds a- z containing amino acid groups, it is worth noting that most of the compounds displayed more potent activity than emodin ( ) . compound a with the gly group displayed near double the activity of emodin ( ) against hepg cells, but had slightly decreased activity against mcf- cells. compound b with the d-ala group displayed the most potent anti-proliferative activity against hepg cells and mcf- cells with ic values of . ± . µm and . ± . µm, respectively. compound c with the l-ala group displayed slightly weaker activity than compound b, but still displayed stronger activity than emodin ( ). compounds d- j containing alkyl chain amino acid groups also displayed stronger inhibitory activities than emodin ( ) . overall, with the exception of compound n, compounds k- m with cyclic alkyl amino acid groups displayed worse inhibitory activities than emodin ( ); especially compound m containing a heterocycle. interestingly, compound n which contains a cyclopentane amino acid group displayed higher anti-proliferative activity against hepg cells and mcf- cells with ic values of . ± . µm scheme . synthesis of compounds a, b, a and b. reagents and conditions: (a) (i) boc-n-me-r/ s-ala-oh, dcc, dcm, • c; (ii) % tfa in dcm, r.t., %- % over two steps; (b) formaldehyde ( % aqueous solution), nabh cn, meoh, r.t., %- %. these products were then coupled with formaldehyde ( % aqueous solution) and reduced utilizing nabh cn to provide a and b as tfa salt in % and % yields, respectively. as shown in scheme , by employing the route for the synthesis of compounds a- z, compounds a- l were obtained as tfa salts. these products were then coupled with formaldehyde ( % aqueous solution) and reduced utilizing nabh cn to provide a and b as tfa salt in % and % yields, respectively. as shown in scheme , by employing the route for the synthesis of compounds a- z, compounds a- l were obtained as tfa salts. the in vitro anti-proliferative activities of all the novel synthesized compounds were evaluated against hepg cells and mcf- cells by celltiter-glo ® luminescent cell viability assay, using paclitaxel (a clinically used drug) as a positive control. emodin ( ) and compound were also included for comparison. as shown in table , emodin ( ) showed weak inhibitory activity against hepg cells and mcf- cells with ic values of . ± . µm and . ± . µm, respectively. compared to emodin ( ), compound showed reduced activity due to the introduction of the hydroxyethyl group at the -position of emodin. among compounds a- z containing amino acid groups, it is worth noting that most of the compounds displayed more potent activity than emodin ( ) . compound a with the gly group displayed near double the activity of emodin ( ) against hepg cells, but had slightly decreased activity against mcf- cells. compound b with the d-ala group displayed the most potent anti-proliferative activity against hepg cells and mcf- cells with ic values of . ± . µm and . ± . µm, respectively. compound c with the l-ala group displayed slightly weaker activity than compound b, but still displayed stronger activity than emodin ( ). compounds d- j containing alkyl chain amino acid groups also displayed stronger inhibitory activities than emodin ( ) . overall, with the exception of compound n, compounds k- m with cyclic alkyl amino acid groups displayed worse inhibitory activities than emodin ( ); especially compound m containing a heterocycle. interestingly, compound n which contains a cyclopentane amino acid group displayed higher anti-proliferative activity against hepg cells and mcf- cells with ic values of . ± . µm the in vitro anti-proliferative activities of all the novel synthesized compounds were evaluated against hepg cells and mcf- cells by celltiter-glo ® luminescent cell viability assay, using paclitaxel (a clinically used drug) as a positive control. emodin ( ) and compound were also included for comparison. as shown in table , emodin ( ) showed weak inhibitory activity against hepg cells and mcf- cells with ic values of . ± . µm and . ± . µm, respectively. compared to emodin ( ), compound showed reduced activity due to the introduction of the hydroxyethyl group at the -position of emodin. among compounds a- z containing amino acid groups, it is worth noting that most of the compounds displayed more potent activity than emodin ( ) . compound a with the gly group displayed near double the activity of emodin ( ) against hepg cells, but had slightly decreased activity against mcf- cells. compound b with the d-ala group displayed the most potent anti-proliferative activity against hepg cells and mcf- cells with ic values of . ± . µm and . ± . µm, respectively. compound c with the l-ala group displayed slightly weaker activity than compound b, but still displayed stronger activity than emodin ( ). compounds d- j containing alkyl chain amino acid groups also displayed stronger inhibitory activities than emodin ( ) . overall, with the exception of compound n, compounds k- m with cyclic alkyl amino acid groups displayed worse inhibitory activities than emodin ( ); especially compound m containing a heterocycle. interestingly, compound n which contains a cyclopentane amino acid group displayed higher anti-proliferative activity against hepg cells and mcf- cells with ic values of . ± . µm and . ± . µm, respectively. compounds o and p with d-and l-ala groups exhibited similar inhibitory activity to compound n. compared to emodin ( ), compounds q- z with aryl (phenyl, substituted phenyl, benzyl, substituted benzyl, etc.) amino acid groups displayed enhanced activity in varying degrees when tested against two cancer cell lines, with ic values ranging from . ± . to . ± . µm. following our initial results, we performed further modification of compounds b and c to improve the anti-proliferative activity, thus mono-and dimethylation of the amino moiety in the amino acid afforded compounds a, b, a and b. unfortunately, this modification led to decreased activity to varying degrees against both the hepg and mcf- cell lines, with ic values ranging from . ± . to . ± . µm, as shown in table . notably, compound b displayed a -fold activity reduction against mcf- cell lines compared to emodin ( ). the results revealed that "nh " was the moiety that favors improving the anti-proliferative activity. the preliminary explanation was that amino group can interact with other biomolecules to improve the anti-proliferative activity via formation of hydrogen bond. mono-or dimethylation of amino group might weaken the trend of hydrogen bonding and decrease the anti-proliferative activity. next, as shown in table , the linking group of compounds b and c was further optimized by increasing the linker length in compounds b and c by one additional methylene group. the resulting compounds, a and b, displayed further enhanced potency at inhibiting cell growth against both hepg cells and mcf- cells. however, compounds c- f initially failed to increase the potency of cell growth inhibition against both tested cancer cell lines; this was successfully addressed by increasing the linker length using additional methylene bridge. on the other hand, elongation of the linker by two to four ethanediol groups resulted in compounds g- l, which showed decreased activity compared to compounds b and c. three pairs of compounds possessing stronger anti-proliferative activities were screened out and their cytotoxic activities were preformed against human normal liver cells (l ) in vitro. as shown in table , the parent compound emodin exhibited stronger cytotoxic activity against l cells with an ic value of . ± . µm than against cancer cell lines (ic = . ± . µm against hepg cells and ic = . ± . µm against mcf- cells). however, all the six compounds exhibited weaker cytotoxic activities against l cells compared with their corresponding cancer cell lines. above all, we finally chose to investigate the mechanism of action for compound a because it had the strongest anti-proliferative activity against hepg cells. lack of selective cytotoxicity is the main factor that hinders conventional chemotherapeutic agents. thus, to evaluate the selective anti-proliferative activity of the compound a, the selectivity index (si) between cancer and normal cells was calculated and the results are summarized in table . the si was calculated by dividing the ic values in normal cells by the ic values in cancer cells. emodin displayed stronger cytotoxic activity, with si values of . (hepg ) and . (mcf- ), respectively, indicating that both normal cells and cancer cells would be killed. however, compound a exhibited weaker cytotoxic activity with si value of . (hepg ) and . (mcf- ), respectively. it means that a exhibited a better selective anti-proliferative activity and specificity than emodin. in order to verify whether compound a is able to induce apoptosis in hepg cells, we utilized fitc-annexin v/pi staining and estimated the percentage of apoptotic cells by flow cytometry. we noted a concentration-dependent increase in the percentage of apoptotic cells when the cells were treated with compound a for h at concentrations . , , and µm. as shown in figure a , few ( . %) apoptotic cells were present in the control panel. in contrast, the percentage of apoptotic cells increased to . % after treatment with compound a at µm for h and further increased to . % after treatment with a at the concentration of µm. as illustrated in figure b , the quantitative analysis of apoptosis strongly suggests that treatment with compound a effectively induced apoptosis in hepg cells in a concentration-dependent manner in comparison to the control. in order to verify whether compound a is able to induce apoptosis in hepg cells, we utilized fitc-annexin v/pi staining and estimated the percentage of apoptotic cells by flow cytometry. we noted a concentration-dependent increase in the percentage of apoptotic cells when the cells were treated with compound a for h at concentrations . , , and µm. as shown in figure a , few ( . %) apoptotic cells were present in the control panel. in contrast, the percentage of apoptotic cells increased to . % after treatment with compound a at µm for h and further increased to . % after treatment with a at the concentration of µm. as illustrated in figure b , the quantitative analysis of apoptosis strongly suggests that treatment with compound a effectively induced apoptosis in hepg cells in a concentration-dependent manner in comparison to the control. statistical significance is determined by two-tailed student t-test: "***" denote p < . , "**" denote p < . , respectively (supplementary table s ). (c, e) western blot analysis effect of compound a on the levels of bax, bcl- , cytochrome c, procaspase- , caspase- and procaspase- expression in hepg cells. (d, f) an equal amount of protein was loaded on sds-page gel for western blot analysis. data are expressed as means ± sd of the percentages of apoptotic cells from three independent experiments. statistical significance is determined by two-tailed student t-test: "***" denote p < . , "**" denote p < . , "*" denote p < . , respectively (supplementary table s ). to verify the molecular mechanisms of apoptosis induction of compound a, we performed a western blot assay. it is well known that the bcl- family of pro-apoptotic and anti-apoptotic the quantitative analysis of apoptosis. data are expressed as means ± sd of the percentages of apoptotic cells from three independent experiments. statistical significance is determined by two-tailed student t-test: "***" denote p < . , "**" denote p < . , respectively (supplementary table s ). (c, e) western blot analysis effect of compound a on the levels of bax, bcl- , cytochrome c, procaspase- , caspase- and procaspase- expression in hepg cells. (d, f) an equal amount of protein was loaded on sds-page gel for western blot analysis. data are expressed as means ± sd of the percentages of apoptotic cells from three independent experiments. statistical significance is determined by two-tailed student t-test: "***" denote p < . , "**" denote p < . , "*" denote p < . , respectively (supplementary table s ). to verify the molecular mechanisms of apoptosis induction of compound a, we performed a western blot assay. it is well known that the bcl- family of pro-apoptotic and anti-apoptotic proteins regulates the mitochondrial pathway of apoptosis. these bcl- family proteins stimulate the permeabilization of the mitochondrial outer membrane, which results in the release of cytochrome c into the cytosol and in turn promotes the activation of the caspase cascade. the activation of the caspase cascade ultimately leads to the induction of apoptotic cell death. as shown in figure c and d, in comparison with the control cells, compound a induced an increase in the levels of bax and a decrease in the expression of bcl- in a concentration-dependent manner. meanwhile, the release of cytochrome c from mitochondria increased after the treatment of compound a, while procaspase and procaspase decreased after treatment with a, indicating that the caspase and caspase were activated. as shown in figure e ,f, the increased expression of cleaved caspase- after treatment with a provided a further evidence that compound a induced cell apoptosis through mitochondrial pathway in a concentration-dependent manner. the apoptosis process can be summarized as follows: the mitochondrial apoptosis-induced channel (mac) of hepg cells was formed by pro-apoptotic protein bax after the treatment of compound a. the formation of mac led to the releasing of cytochrome c from mitochondria. once cytochrome c was released, it binded with apoptotic protease activating factor- (apaf- ) and atp, which then binded to procaspase- to create a protein complex known as apoptosome. the apoptosome cleaved the pro-caspase- to its active form of initiator caspase- , which in turn activated procaspase- and then the effector caspase- and finally resulted in cell apoptosis. to further examine how compound a suppressed the growth of hepg cells, the effect of compound a on cell cycle distribution with different concentrations was investigated by flow cytometric analysis following staining the dna with propidium iodide (pi). the results of a typical experiment are shown in figure a . as determined by flow cytometry, the exposure of hepg cells to compound a for h resulted in an obvious increase in the percentage of cells in g /g phase in comparison with the control. treatment with compound a resulted in an increase of g phase cells ( . %) compared to the control ( . %). inversely, s phase cell population decreased to . % compared to the control ( . %). therefore, compound a resulted in a significant g /g phase arrest in a concentration-dependent manner with a concomitant decrease in the number of cells in the s phase of the cycle. proteins regulates the mitochondrial pathway of apoptosis. these bcl- family proteins stimulate the permeabilization of the mitochondrial outer membrane, which results in the release of cytochrome c into the cytosol and in turn promotes the activation of the caspase cascade. the activation of the caspase cascade ultimately leads to the induction of apoptotic cell death. as shown in figure c and d, in comparison with the control cells, compound a induced an increase in the levels of bax and a decrease in the expression of bcl- in a concentration-dependent manner. meanwhile, the release of cytochrome c from mitochondria increased after the treatment of compound a, while procaspase and procaspase decreased after treatment with a, indicating that the caspase and caspase were activated. as shown in figure e ,f, the increased expression of cleaved caspase- after treatment with a provided a further evidence that compound a induced cell apoptosis through mitochondrial pathway in a concentration-dependent manner. the apoptosis process can be summarized as follows: the mitochondrial apoptosis-induced channel (mac) of hepg cells was formed by pro-apoptotic protein bax after the treatment of compound a. the formation of mac led to the releasing of cytochrome c from mitochondria. once cytochrome c was released, it binded with apoptotic protease activating factor- (apaf- ) and atp, which then binded to procaspase- to create a protein complex known as apoptosome. the apoptosome cleaved the pro-caspase- to its active form of initiator caspase- , which in turn activated procaspase- and then the effector caspase- and finally resulted in cell apoptosis. to further examine how compound a suppressed the growth of hepg cells, the effect of compound a on cell cycle distribution with different concentrations was investigated by flow cytometric analysis following staining the dna with propidium iodide (pi). the results of a typical experiment are shown in figure a . as determined by flow cytometry, the exposure of hepg cells to compound a for h resulted in an obvious increase in the percentage of cells in g /g phase in comparison with the control. treatment with compound a resulted in an increase of g phase cells ( . %) compared to the control ( . %). inversely, s phase cell population decreased to . % compared to the control ( . %). therefore, compound a resulted in a significant g /g phase arrest in a concentration-dependent manner with a concomitant decrease in the number of cells in the s phase of the cycle. table s ). to evaluate the effect of compound a on cancer migration, a wound healing assay was conducted to determine whether compound a could prevent hepg cell migration. after culturing hepg cells for h in the presence and absence of compound a at , . , . , and µm, a pipette tip was streaked through the cell culture, resulting in a cell deficient space. the territory recovered by the hepg cells was used to observe the migration inhibition capability of compound a. the empty space reduced significantly in size in the absence of compound a because of cell proliferation and migration and results indicate compound a suppressed the mobility of tumor cells in a concentration-dependent manner ( figure ). cell cycle distribution after treatment of compound a. data are expressed as means ± sd of the percentages of apoptotic cells from three independent experiments. statistical significance is determined by two-tailed student t-test: "***" denote p < . , "*" denote p < . and ns means no significance, respectively (supplementary table s ). to evaluate the effect of compound a on cancer migration, a wound healing assay was conducted to determine whether compound a could prevent hepg cell migration. after culturing hepg cells for h in the presence and absence of compound a at , . , . , and µm, a pipette tip was streaked through the cell culture, resulting in a cell deficient space. the territory recovered by the hepg cells was used to observe the migration inhibition capability of compound a. the empty space reduced significantly in size in the absence of compound a because of cell proliferation and migration and results indicate compound a suppressed the mobility of tumor cells in a concentration-dependent manner ( figure ). table s ). the starting materials and reagents, purchased from commercial suppliers, were used without further purification. emodin extracted from polygonum cuspidatum was purchased from china xi'an sino-herb bio-technology co., ltd. (xi'an, china). all reactions were monitored by thin-layer chromatography (tlc) on aluminum sheets (silica gel -f , e. merck, darmstadt, germany). compounds were visualized by uv light. column chromatography was carried out using silica gel ( - mesh). all reaction solvents were dried prior to use according to standard procedures. all primary reagents were commercially available. silica gel chromatography solvents were of analytical grade. nmr spectra were recorded in dmso-d on a bruker- spectrometer (bruker biospin, fällanden, switzerlahd), at mhz for h-nmr, mhz for c-nmr and mhz for f-nmr with tms as the internal standard. chemical shifts were expressed in δ (ppm) and coupling constants (j) in hz. multiplicity was indicated as follows: s (singlet), d (doublet), t (triplet), p (quintet), dd (doublet of doublets), brs (broad singlet), etc. mass spectra were obtained on an agilent series lc/msd trap mass spectrometer (esi-ms, agilent, santa clara, ca, usa). table s ). the starting materials and reagents, purchased from commercial suppliers, were used without further purification. emodin extracted from polygonum cuspidatum was purchased from china xi'an sino-herb bio-technology co., ltd. (xi'an, china). all reactions were monitored by thin-layer chromatography (tlc) on aluminum sheets (silica gel -f , e. merck, darmstadt, germany). compounds were visualized by uv light. column chromatography was carried out using silica gel ( - mesh). all reaction solvents were dried prior to use according to standard procedures. all primary reagents were commercially available. silica gel chromatography solvents were of analytical grade. nmr spectra were recorded in dmso-d on a bruker- spectrometer (bruker biospin, fällanden, switzerlahd), at mhz for h-nmr, mhz for c-nmr and mhz for f-nmr with tms as the internal standard. chemical shifts were expressed in δ (ppm) and coupling constants (j) in hz. multiplicity was indicated as follows: s (singlet), d (doublet), t (triplet), p (quintet), dd (doublet of doublets), brs (broad singlet), etc. mass spectra were obtained on an agilent series lc/msd trap mass spectrometer (esi-ms, agilent, santa clara, ca, usa). , -dihydroxy- -( -hydroxyethoxy)- -methylanthracene- , -dione ( ) to a mixture of emodin ( . g, . mmol) in dry dmf ( ml) were added cs co ( . g, . mmol) and -iodoethanol ( . g, mmol) at room temperature. after stirring for h at • c, the resulting mixture was evaporated under reduced pressure and then mixed with water ( ml). the ph value of aqueous phase was adjusted to around with % hydrochloric acid solution. the yellow precipitate was collected and washed with water to give the crude product, which was in further purification by triturating twice with ethyl acetate ( ml) and filtered to afford compound ( . g, %) as a brown solid; general procedure a for preparation of compounds a- z, a- b, and a- l to a mixture of compound ( . mmol) in dry dichloromethane ( ml) were added various n-boc amino acids ( . mmol), dicyclohexyl carbodiimide (dcc) ( . mmol) and -(n,n-dimethlyamino) pyridine (dmap) ( . mmol) at • c. after stirring about min to h at • c, tlc analysis showed the complete consumption of compound , and then the resulting mixture was added dropwise tfa ( ml) at the same temperature and kept stirring for anther about h. the insoluble side product was filtered out and the filtrate was evaporated to give the residue, which was purified by reverse phase flash chromatography with the following conditions: column: spherical c , - µm, g; mobile phase a: water (plus mm tfa); mobile phase b: acn; flow rate: ml/min; gradient: % b gradient in min, % b- % b gradient in min; detector: nm. the fractions containing the desired product were collected at around % b and concentrated under reduced pressure to afford compounds a- z in % to % yields. -( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)- -oxoethanaminium , , -trifluoroacetate ( a). according to the general procedure a, compound was treated with n-boc-glycine and then purified by reverse phase flash chromatography to give compound a: yellow solid; yield, %; (s)- -( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)- -oxopropan- -aminium , , -trifluoroacetate ( c). according to the general procedure a, compound was treated with n-boc-l-alanine and then purified by reverse phase flash chromatography to give compound c: yellow solid; yield, %; h-nmr (s)- -( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)- -hydroxy- -oxopropan- -aminium , , -trifluoroacetate ( d). according to the general procedure a, compound was treated with n-boc-o-tert-butyl-l-serine and then purified by reverse phase flash chromatography to give compound d: yellow solid; yield, %; (r)- -( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)- -methyl- -oxobutan- -aminium , , -trifluoroacetate ( e). according to the general procedure a, compound was treated with n-boc-d-valine and then purified by reverse phase flash chromatography to give compound e: yellow solid; yield, %; . , . , . , . , . , . , . , . , . , . , . , . , . , . , . , . , . , . . , . , . , . , . , . , . , . , . , . , . , . -( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)- -methyl- -oxopropan- aminium , , -trifluoroacetate ( j). according to the general procedure a, compound was treated with n-boc-aib-oh and then purified by reverse phase flash chromatography to give compound j: yellow solid; yield, %; h-nmr δ . (br, h), . (br, h), . (s, h), . - . (m, h), -(( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)carbonyl)cyclopropan-aminium , , -trifluoroacetate ( k). according to the general procedure a, compound was treated with -(boc-amino)cyclopropanecarboxylic acid and then purified by reverse phase flash chromatography to give compound k: yellow solid; yield, %; h-nmr δ . (brs, h), . , . , . , . , . , . , . , . , . , . , . , . , . , . , . , . , . -(( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)carbonyl)cyclobutan-aminium , , -trifluoroacetate ( l). according to the general procedure a, compound was treated with -(boc-amino)cyclobutanecarboxylic acid and then purified by reverse phase flash chromatography to give compound l: yellow solid; yield, %; h-nmr δ . (brs, h), -(( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)carbonyl)oxetan- -aminium , , -trifluoroacetate ( m). according to the general procedure a, compound was treated with -boc-amino- -oxetanecarboxylic acid and then purified by reverse phase flash chromatography to give compound m: yellow solid; yield, %; h-nmr δ . (brs, h), -(( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)carbonyl)cyclopentan-aminium , , -trifluoroacetate ( n). according to the general procedure a, compound was treated with n-boc-aminocyclopentanecarboxylic acid and then purified by reverse phase flash chromatography to give compound n: yellow solid; yield, %; h-nmr δ . (brs, h), (r)- -(( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)carbonyl)-pyrrolidinium , , -trifluoroacetate ( o). according to the general procedure a, compound was treated with n-boc-d-proline and then purified by reverse phase flash chromatography to give compound o: yellow solid; yield, %; h-nmr δ . (d, j = . (s)- -(( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)carbonyl)-pyrrolidinium , , -trifluoroacetate ( p). according to the general procedure a, compound was treated with n-boc-l-proline and then purified by reverse phase flash chromatography to give compound p: (r)- -( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)- -oxo- -phenylethanaminium , , -trifluoroacetate ( q). according to the general procedure a, compound was treated with n-boc-d-phenylglycine and then purified by reverse phase flash chromatography to give compound q: yellow solid; yield, %; h-nmr δ . (brs, h), (r)- -( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)- -( -hydroxyphenyl)- -oxoethanaminium , , -trifluoroacetate ( s). according to the general procedure a, compound was treated with n-boc-d- -hydroxyphenylglycine and then purified by reverse phase flash chromatography to give compound s: yellow solid; yield, %; h-nmr δ . (brs, h), . (s)- -( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)- -( -hydroxyphenyl)- -oxoethanaminium , , -trifluoroacetate ( t). according to the general procedure a, compound was treated with n-boc-l- -hydroxyphenylglycine and then purified by reverse phase flash chromatography to give compound t: yellow solid; yield, %; h-nmr δ . (brs, h), . (r)- -( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)- -( -fluorophenyl)- -oxoethanaminium , , -trifluoroacetate ( u). according to the general procedure a, compound was treated with (r)-n-boc- -fluorophenylglycine and then purified by reverse phase flash chromatography to give compound u: yellow solid; yield, %; h-nmr δ . (brs, h), (s)- -( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)- -( -fluorophenyl)- oxoethanaminium , , -trifluoroacetate ( v). according to the general procedure a, compound was treated with (s)-n-boc- -fluorophenylglycine and then purified by reverse phase flash chromatography to give compound v: yellow solid; yield, %; h-nmr ( mhz, dmso-d ) δ . (brs, h), (r)- -( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)- -oxo- -phenyl-propan- -aminium , , -trifluoroacetate ( w). according to the general procedure a, compound was treated with n-boc-d-phenylalanine and then purified by reverse phase flash chromatography to give compound w: yellow solid; yield, %; h-nmr δ . brs, h), (s)- -( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)- -oxo- -phenyl-propan- -aminium , , -trifluoroacetate ( x). according to the general procedure a, compound was treated with n-boc-l-phenylalanine and then purified by reverse phase flash chromatography to give compound x: yellow solid; yield, %; h-nmr δ . (brs, h), (r)- -( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)- -( -hydroxyphenyl)- -oxopropan- -aminium , , -trifluoroacetate ( y). according to the general procedure a, compound was treated with n-boc-o-tert-butyl-l-tyrosine and then purified by reverse phase flash chromatography to give compound y: yellow solid; yield, %; h-nmr δ . (s, h), . (d, j = . (s)- -( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)- -( -hydroxyphenyl)- -oxopropan- -aminium , , -trifluoroacetate ( z). according to the general procedure a, compound was treated with n-boc-l-tyrosine and then purified by reverse phase flash chromatography to give compound z: yellow solid; yield, %; h-nmr δ . (s, h), . (d, j = . (r)- -( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)-n-methyl- -oxo-propan- -aminium , , -trifluoroacetate ( a). according to the general procedure a, compound was treated with boc-n-methyl-d-alanine and then purified by reverse phase flash chromatography to give compound a: yellow solid; yield, %; h-nmr δ . (brs, h), . (s)- -( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)-n-methyl- -oxo-propan- -aminium , , -trifluoroacetate ( b). according to the general procedure a, compound was treated with boc-n-methyl-l-alanine and then purified by reverse phase flash chromatography to give compound b: yellow solid; yield, %; h-nmr δ . (brs, h), . (brs, h), . general procedure b for preparation of compounds a and b to a solution of compound a or b ( mg, . mmol) and paraformaldehyde ( % aqueous solution, mg, . mmol) in meoh ( ml) was added nabh cn ( mg, . mmol) at • c. after stirring h at room temperature, the reaction was quenched by tfa ( . ml). the resulting reaction solution was used directly in purification by reverse phase flash chromatography with the following conditions: column: spherical c , - µm, g; mobile phase a: water (plus mm tfa); mobile phase b: acn; flow rate: ml/min; gradient: % b gradient in min, % b- % b gradient in min; detector: nm. the fractions containing the desired product were collected at around % b and concentrated under reduced pressure to afford compounds a or b in % or % yield, respectively. (r)- -( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)-n,n-dimethyl- -oxopropan- -aminium , , -trifluoroacetate ( a). yellow solid; yield, %; general procedure c for preparation of compounds a- f to a mixture of emodin ( mmol) in dry dmf ( ml) were added cs co ( mmol) and hydroxybromides or iodides ( mmol) at room temperature. after stirring for h at • c, the resulting mixture was evaporated under reduced pressure and then mixed with water ( ml). the ph value of aqueous phase was adjusted to around with % hydrochloric acid solution, extracted with dichloromethane ( × ml). the combined organic layer was washed with brine ( ml), dried over anhydrous sodium sulfate and evaporated to dryness. the crude product was purified by silica gel column chromatography with %- % ethyl acetate in petroleum to afford compounds a- f. total cell lysates from cultured hepg cells treated with different concentrations of compound a for h were obtained by lysing the cells in ice-cold ripa buffer ( pbs, % np- , . % sodium deoxycholate and . % sds) containing mg/ml pmsf, mg/ml aprotinin, mg/ml leupeptin, mg/ml pepstatin and mg/ml naf. after centrifugation at , rpm for min, the protein in the supernatant was quantified by the bradford method (bio-rad, hercules, ca, usa) using a multimode varioscan instrument (thermo fischer scientific, waltham, ma, usa). twenty micrograms of protein per lane was applied in % sds polyacrylamide gel. after electrophoresis, the protein was transferred to a polyvinylidine difluoride membrane (amersham biosciences, marlborough, ma, usa). the membrane was blocked at room temperature for h in tbst containing % blocking powder (santa cruz, dallas, tx, usa). the membrane was washed with tbst for min, and the primary antibody was added and incubated at • c overnight (o/n). bax ( , cst, danvers, ma, usa), bcl- ( , cst), cytochrome c ( , cst), procaspase- (ab , abcam, cambridge, ma, usa), procaspase- (ab , abcam), caspase- ( - -lg, proteintech group, rosemont, il, usa) and gapdh (ab , abcam) antibodies were employed. after three tbst washes, the membrane was incubated with the corresponding horseradish peroxidase-labeled secondary antibody ( : ) (santa cruz) at room temperature for h. membranes were washed with tbst for min five times and the protein blots were visualized with chemiluminescence reagent (thermo fischer scientific ltd.). the x-ray films were developed with a developer and fixed with fixer solution. the grey levels were analyzed using imagequant las system (ge, marlborough, ma, usa). the hepg cells were treated with indicated concentrations of compound a. after incubation for h, cells were washed twice with ice-cold pbs, fixed and permeabilized with ice cold % ethanol at − • c overnight. the cells were treated with µg/ml rnase a at • c for min, then washed with ice-cold pbs and finally stained with mg/ml pi in the dark at • c for min. the cellular dna content for the cell cycle distribution analysis was performed with the system software (cellquest; bd biosciences), plotting at least , events per sample. the percentage of cells in the g , s and g phases of the cell cycle were determined using the modfit lt version . software package (verity software, topsham, me, usa). hepg cells were grown in dmem medium containing growth factors at a cell density of × cells/ml for h. a disposable ml plastic pipette tip was used to scratch the monolayer of cells in a streaking motion. compounds were added to the streaked cell culture at the indicated concentrations. the streaked cells were then cultured in serum-free medium for an additional h and photographed. to quantify the experimental results, the % cell inhibitory rate was calculated by the equation: cell inhibitory rate (%) = ( − d drug /d control ) × %, where d drug is the mean distance of cell migration in drug group and d control is the mean distance of cell migration in control group. pictures of the initial wounded monolayers were compared with the corresponding pictures of cells at the end of the incubation, and data were presented as mean ± sd from three independent experiments. in conclusion, a novel series of emodin derivatives via the introduction of an amino acid were designed and synthesized. their in vitro anti-proliferation tests revealed that these derivatives exhibited moderate to potent anti-proliferative activity against hepg cells and mcf- cells. among these compounds, the most potent compound, a, exhibited better selective anti-proliferative activity and specificity than emodin, displayed a significant effect in inducing cell cycle arrest at g /g phase and inducing cell apoptosis in hepg cells via release of cytochrome c and subsequent activation of caspase- and caspase- , which revealed that the possible molecular mechanism of a apoptosis induction may mainly through the mitochondrial death pathway. moreover, compound a also resulted in inhibition of hepg cells migration in the wound healing assay. these preliminary molecular mechanism results suggest that compound a could be a promising lead compound for the development of novel antitumor drugs and has the potential for further investigations as an anti-cancer drug. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , tables s -s : biochemistry analytical data from three independent experiments; figures s -s : h-nmr, c-nmr and f-nmr spectra of these compounds. according to the general procedure c, emodin was treated with -iodopropan- -ol and then purified by silica gel column chromatography to give compound a: brown solid; yield, %; h-nmr δ . (s, h), . (s, h), . (s, h according to the general procedure c, emodin was treated with -bromobutan- -ol and then purified by silica gel column chromatography to give compound b: brown solid; yield, %; h-nmr δ . (s, h), . (s, h), . (s, h) according to the general procedure c, emodin was treated with -bromopentan- -ol and then purified by silica gel column chromatography to give compound c: brown solid; yield, %; h-nmr δ . (s, h), . (s, h) according to the general procedure c, emodin was treated with -( -bromoethoxy)ethanol and then purified by silica gel column chromatography to give compound d: brown solid; yield, %; h-nmr δ . (s, h), . (s, h), . (d, j = -bromoethoxy)ethoxy)ethanol and then purified by silica gel column chromatography to give compound e: brown solid; yield, %; h-nmr δ . (s, h) -bromoethoxy)ethoxy)ethoxy) ethanol and then purified by silica gel column chromatography to give compound f: brown solid h-nmr δ . (s, h), . (s, h), . (d, j = . hz, h), . (t, j = . hz, h), . (d, j = . hz, h) according to the general procedure a, compound a was treated with n-boc-d-alanine and then purified by reverse phase flash chromatography to give compound a: yellow solid; yield, %; h-nmr δ . (d, j = according to the general procedure a, compound a was treated with n-boc-l-alanine and then purified by reverse phase flash chromatography to give compound b: yellow solid according to the general procedure a, compound b was treated with n-boc-d-alanine and then purified by reverse phase flash chromatography to give compound c: yellow solid according to the general procedure a, compound b was treated with n-boc-l-alanine and then purified by reverse phase flash chromatography to give compound d: yellow solid hz, h), . (s, h), . - . (m, h), . (d, j = . hz, h) according to the general procedure a, compound c was treated with n-boc-d-alanine and then purified by reverse phase flash chromatography to give compound e: yellow solid; yield, %; h-nmr δ . (brs, h), . (brs, h), . (d, j = according to the general procedure a, compound c was treated with n-boc-l-alanine and then purified by reverse phase flash chromatography to give compound f: yellow solid (s, h), . - . (m, h), . - . (m, h), . - . (m, h), . (d, j = . hz, h) according to the general procedure a, compound d was treated with n-boc-d-alanine and then purified by reverse phase flash chromatography to give compound g: yellow solid; yield, %; h-nmr δ according to the general procedure a, compound d was treated with n-boc-l-alanine and then purified by reverse phase flash chromatography to give compound h: yellow solid according to the general procedure a, compound e was treated with n-boc-d-alanine and then purified by reverse phase flash chromatography to give compound i: yellow solid; yield, %; h-nmr δ according to the general procedure a, compound e was treated with n-boc-l-alanine and then purified by reverse phase flash chromatography to give compound j: yellow solid; yield, %; h-nmr δ according to the general procedure a, compound f was treated with n-boc-d-alanine and then purified by reverse phase flash chromatography to give compound k: yellow solid; yield, %; h-nmr δ . (brs, h), . (d, j = . hz, h) according to the general procedure a, compound f was treated with n-boc-l-alanine and then purified by reverse phase flash chromatography to give compound l: yellow solid; yield, %; h-nmr δ ) containing % fetal bovine serum (fbs) and % penicillin ( units/ml)-streptomycin ( µg/ml) in a humidified incubator at • c under % co and % relative humidity (rh) atmosphere. the cells were harvested for metabolomics experiments using cell scrapers cell viability assay hepg cells and mcf- cells in µl dmem supplemented with % fbs were seeded at a density of cells/well in -well cell culture plate ( , corning), respectively. after h, the medium was substituted with fresh medium containing various sample solutions in dmso usa) was added to each well containing cells and the contents were mixed for min on an orbital shaker to induce cell lysis. the plates were then incubated at room temperature for min to stabilize the luminescent signal. the luminescence was recorded on an envision cells were seeded at × /well in % fbs-dmem into -well plates and treated with compounds a for h. the cells were washed twice with cold phosphate buffered saline (pbs) and then analysis is as follows: lower left quadrant, viable cells (annexin v−/pi−); lower right quadrant, early apoptotic cells (annexin v+/pi−); upper right quadrant, late apoptotic cells (annexin v+/pi+); upper left quadrant, necrotic cells a new diphenyl ether from marine-derived fungus aspergillus sp b-f- a: a new antimicrobial anthraquinone from a sea urchin-derived fungus monodictys sp a new ergosterol analog, a new bis-anthraquinone and anti-obesity activity of anthraquinones from the marine sponge-associated fungus talaromyces stipitatus kufa . mar. drugs bis-indolyl benzenoids, hydroxypyrrolidine derivatives and other constituents from cultures of the marine sponge-associated fungus aspergillus candidus kufa emodin and aloe-emodin suppress breast cancer cell proliferation through er alpha inhibition. evid.-based complement emodin suppresses wnt signaling in human colorectal cancer cells sw and sw emodin suppresses hyperglycemia-induced proliferation and fibronectin expression in mesangial cells via inhibiting cflip emodin suppresses inflammatory responses and joint destruction in collagen-induced arthritic mice emodin blocks the sars coronavirus spike protein and angiotensin-converting enzyme interaction effect of emodin on the cariogenic properties of streptococcus mutans and the development of caries in rats emodin, a naturally occurring anthraquinone derivative, suppresses ige-mediated anaphylactic reaction and mast cell activation ct imaging biomarker for evaluation of emodin as a potential drug on lps-mediated osteoporosis mice formula optimization of the jiashitang scar removal ointment and antiinflammatory compounds screening by nf-kappa b bioactivity-guided dual-luciferase reporter assay system emodin protects against diabetic cardiomyopathy by regulating the akt/gsk- beta signaling pathway in the rat model emodin prolongs recipient survival time after orthotopic liver transplantation in rats by polarizing the th /th paradigm to th emodin induces neurite outgrowth through pi k/akt/gsk- beta-mediated signaling pathways in neuro a cells emodin ameliorates ethanol-induced fatty liver injury in mice emodin protects against concanavalin a-induced hepatitis in mice through inhibiting activation of the p mapk-nf-kappa b signaling pathway emodin suppresses cell proliferation and fibronectin expression via p mapk pathway in rat mesangial cells cultured under high glucose emodin accelerates osteoblast differentiation through phosphatidylinositol -kinase activation and bone morphogenetic protein- gene expression emodin-induced apoptosis through p -dependent pathway in human hepatoma cells emodin azide methyl anthraquinone derivative triggers mitochondrial-dependent cell apoptosis involving in caspase- -mediated bid cleavage aloe-emodin induces apoptosis of human nasopharyngeal carcinoma cells via caspase- -mediated activation of the mitochondrial death pathway emodin inhibits tnf-alpha-induced human aortic smooth-muscle cell proliferation via caspase-and mitochondrial-dependent apoptosis emodin induces apoptosis in human lung adenocarcinoma cells through a reactive oxygen species-dependent mitochondrial signaling pathway synthesis and antitumor activity of emodin quaternary ammonium salt derivatives synthesis and biological activity, evaluation of emodin quaternary ammonium salt derivatives as potential anticancer agents synthesis, sar and pharmacological characterization of novel anthraquinone cation compounds as potential anticancer agents antitumor effects and mechanism of novel emodin rhamnoside derivatives against human cancer cells in vitro synthesis, dna binding and cytotoxicity of new pyrazole emodin derivatives synthesis and cytotoxic activities of beta-carboline amino acid ester conjugates preparation of amino acid conjugates of betulinic acid with activity against human melanoma this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest. key: cord- -bdw ke i authors: guo, hongbo; rabouw, huib; slomp, anne; dai, meiling; van der vegt, floor; van lent, jan w. m.; mcbride, ryan; paulson, james c.; de groot, raoul j.; van kuppeveld, frank j. m.; de vries, erik; de haan, cornelis a. m. title: kinetic analysis of the influenza a virus ha/na balance reveals contribution of na to virus-receptor binding and na-dependent rolling on receptor-containing surfaces date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: bdw ke i interactions of influenza a virus (iav) with sialic acid (sia) receptors determine viral fitness and host tropism. binding to mucus decoy receptors and receptors on epithelial host cells is determined by a receptor-binding hemagglutinin (ha), a receptor-destroying neuraminidase (na) and a complex in vivo receptor-repertoire. the crucial but poorly understood dynamics of these multivalent virus-receptor interactions cannot be properly analyzed using equilibrium binding models and endpoint binding assays. in this study, the use of biolayer interferometric analysis revealed the virtually irreversible nature of iav binding to surfaces coated with synthetic sialosides or engineered sialoglycoproteins in the absence of na activity. in addition to ha, na was shown to be able to contribute to the initial binding rate while catalytically active. virus-receptor binding in turn contributed to receptor cleavage by na. multiple low-affinity ha-sia interactions resulted in overall extremely high avidity but also permitted a dynamic binding mode, in which na activity was driving rolling of virus particles over the receptor-surface. virus dissociation only took place after receptor density of the complete receptor-surface was sufficiently decreased due to na activity of rolling iav particles. the results indicate that in vivo iav particles, after landing on the mucus layer, reside continuously in a receptor-bound state while rolling through the mucus layer and over epithelial cell surfaces driven by the ha-na-receptor balance. quantitative bli analysis enabled functional examination of this balance which governs this dynamic and motile interaction that is expected to be crucial for penetration of the mucus layer and subsequent infection of cells by iav but likely also by other enveloped viruses carrying a receptor-destroying enzyme in addition to a receptor-binding protein. introduction specificity, avidity and dynamics of influenza a virus (iav)-receptor interactions are determining factors in host tropism and pathogenesis. virus attachment to sialic acid (sia) receptors on host cell surfaces and decoy mucins is mediated by hemagglutinin (ha) [ ] [ ] [ ] , while neuraminidase (na) removes receptors by cleaving sias [ ] [ ] [ ] . a precisely tuned functional ha/na balance [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] is required for efficient infection of, and replication in, a specific host. ha and na properties affect host-and cell-specificity but have been studied in much more detail for ha because of the relative lack of accurate na assays [ ] . iavs that infect humans bind preferentially to α , sialosides, having an α , -linkage between sia moieties and the penultimate residue, whereas avian iavs prefer binding to α , -linked sias [ ] [ ] [ ] . cell surface glycan composition, as well as branching, modification and linkage-type of the internal carbohydrate chain residues, is variable between host and cell type and also strongly affects binding affinity (reviewed in [ ] ). nas of avian iavs are highly active and prefer cleavage of α , -linked sialoglycans, while human virus nas cleave α , -and α , -linked sias with lower activity [ ] [ ] [ ] . the ha/na balance is important for initiating iav infection. abundantly sialylated mucins in the mucus layer covering the airway epithelial cells bind ha and may trap iavs before they reach the epithelial cells [ , ] . this needs to be counteracted by na activity sufficiently matching ha binding specificity. this is also required to prevent the virus-trapping at sites on the cell surface that do not support efficient endocytosis [ , ] . na activity-dependent de-sialylation of the surface of the infected cell and the virus envelope glycoproteins facilitates budding and release of new virus particles and prevents virus aggregation [ ] . as a consequence, viral fitness depends on continuous fine-tuning of the ha/na balance during virus evolution. replication of viruses, harboring mismatched pairs of ha and na or propagated in the presence of inhibitory decoy receptors or na inhibitors, could be rescued by adaptive mutations in the ha, na or both proteins [ , , , ] . cross-species transmission events, leading to human pandemics in the past, required adaptions in both ha and na [ , ] . when infecting a novel host species, a non-optimal ha/na balance is for instance adapted by evolving increased na activity as well as decreased binding by ha to the decoy glycan-receptor repertoire on mucins present in the mucus, and which differs between different hosts. [ , [ ] [ ] [ ] . there is no straightforward method to determine the ha/na balance of iavs. hemagglutination of erythrocytes, which harbor poorly defined receptor repertoires, does not well reflect binding avidity and dynamics. current setups of solid phase binding assays (e.g. elisas, glycan arrays) as endpoint binding assays do not handle well the polyvalent aspects of virus binding by masking the dynamics of virus binding, even more so as precise variation of receptor density is difficult. moreover, besides a wide range of synthetic glycans, only very few glycoproteins (mostly fetuin) are being employed in current binding studies, thus limiting studies on the effects of diversity and clustering of glycans as naturally presented on glycoproteins. na activity assays often use soluble substrates poorly reflecting natural sialosides [ , ] . the soluble substrate -( -methylumbelliferyl)-α-d-n-acetylneuraminic acid (munana) can be used to determine the na activity on a soluble substrate but ignores the effects of the virion context on na activity on a receptor-coated surface (e.g. binding of the virion to the polyvalent substrate via its ha). thus, methods that determine the dynamic effects of ha and na activity urgently need improvement to allow integration into a model that accurately provides a quantitative description of the dynamic interaction between iav particles and (decoy) receptors. biolayer interferometry (bli) is increasingly being used for analyzing virus-receptor interactions [ ] [ ] [ ] [ ] , but standard methods for quantifying kinetic parameters describing iavreceptor interactions are lacking. here we have used this label-free real-time binding analysis method to study the dynamics of iav-receptor interactions. our results indicate that the initial virus binding rate is the prime, and physiologically most important kinetic parameter of ha-dependent iav particle binding to synthetic as well as natural glycoprotein receptors. na was shown to contribute to virus binding and to be absolutely required for virus dissociation. in turn, na activity is critically dependent on the ability of virions to bind to a receptor-coated surface. the ha/na balance of different virus-receptor combinations was determined by measuring empirical virus binding and elution parameters. prior to virus elution, na-dependent morphological changes in receptor-associated virus particles were observed as well as rolling of virus particles over a receptor-coated surface in a na activity-dependent manner. bli is a potentially versatile tool to obtain mechanistic and quantitative insight into the dynamics of virus-receptor interaction by real-time recording of virion binding to, and release from, receptor-coated surfaces. these interactions are highly polyvalent by nature and expected to be poorly described by equilibrium binding models. in addition, receptor density and identity are key determinants of the interaction. we therefore established a flexible experimental set-up, using synthetic sialoside receptors as well as n-linked or o-linked sialoglycoproteins which carry sialoglycan receptors attached in the natural linkage-conformation that is encountered in vivo. we first evaluated the applicability of the set-up to a quantitative description of iav-receptor binding kinetics. to assist interpretation of the results, the approximate geometric properties of a streptavidin (sa) bli sensor surface, to which biotinylated glycans or glycoproteins can be tightly attached (k d = − ), and of iav virions are depicted in s fig (fig ) . virus association was performed in the presence of the na inhibitor oseltamivir carboxylate (oc) and the lack of virus dissociation after transfer of sensors to buffer containing oc ( fig a and fig b) showed that virus binding is virtually irreversible (off-rate constant k off % ) due to highly polyvalent interactions between virus particles and the receptor-coated bli sensor surface. pr mtsin is a faster binder than pr cam , (fig a and fig b) , but reached a maximum binding signal of~ nm at the highest concentration only after hours (s a fig and s b fig) . a fully occupied sensor surface theoretically accommodates . e+ spherical virus particles ( nm diameter; s fig and [ ] [ ] [ ] [ ] ). this fitted well with the experimentally determined number of virus particles bound to a maximally loaded sensor as determined by quantification of na activity (s c fig). importantly, sensor-regeneration followed by re-binding of virus from the same well could be consecutively repeated at least times yielding identical curves (s fig) . thus, substantial virus decay during the assay or binding of a limited subset of virus particles with particular properties did not occur. analysis of iav-receptor interactions has predominantly been focused on quantification of an apparent dissociation constant (k d = k off /k on ) by measuring polyvalent binding of virus particles or artificial ha polymers (e.g. by antibody complexation) after a fixed binding time to geometrically poorly characterized receptor-coated surfaces with often unknown receptor density and orientation. however, as virus binding is virtually irreversible, equilibrium binding models do not apply and the binding rate equation ( ) and virus concentration was observed for the fast (pr mtsin ; r = . , p = . ) and the slow binding (pr cam , ; r = . , p = . ) virus ( fig c) . the effect of receptor density on iav association and dissociation rates can be precisely determined as bli provides quantitative recording of receptor loading levels. lowering the receptor density, resulting in less potential ha-receptor contacts between virus and sensor surface, gradually reduced maximum binding levels ( fig d and s d and s e fig) as well as the v obs ( fig e) . remarkably, irreversible binding was observed at any receptor density that supported binding (fig d) . a~ -fold higher fractional receptor loading was required for the weaker binder pr cam , ( . ± . ) than for pr mtsin ( . ± . ) to reach % of the maximum fractional initial binding rate (p< . ) ( fig e) . pr mtsin reached a maximum binding rate at~ % receptor loading whereas the weaker binder pr cam - reached its maximum binding rate close to maximum receptor density. a~ -fold reduction in receptor loading was already sufficient to reduce the v obs from maximal to zero for both viruses. this suggests a narrow margin between the number of interacting ha-receptor pairs, below which binding is undetectable due to high reversibility (high k d ) and above which binding is irreversible (see fig d, no dissociation at any receptor density). we conclude that v obs , which is directly related to virus concentration and receptor density, is the most suitable parameter for quantification of virus binding strength over a range of receptor densities and is likely to reflect in vivo virus binding, which probably occurs to levels very far from saturation. as the initial binding rate is linear with virus concentration, the relative specificity for two receptors (v rel = v obs / v obs ) is independent of virus concentration and the relative-binding specificity of viruses can therefore be quantitatively compared without the need for elaborate virus quantitation procedures. as an example, we quantified the effect on receptor-specificity of amino acid substitution e d in ha of pr which is predicted to cause a shift in binding from α , -to α , -linked sia receptors in h [ ] . indeed glycan array analysis of the ha proteins of lab strains pr cam , and pr cam , , which only differ by substitution d e, showed an absolute specificity shift (fig a) . in contrast, virus binding quantified by bli showed a relative specificity shift (v rel = v 'slnln /v 'slnln ) of . ± . for pr cam , to . ± . for pr cam , ( fig b and fig c) . remarkably, strain wsn ha, which carries e plus some additional mutations in comparison to pr cam , , displayed efficient binding to α , -as well as α , -linked synthetic glycans on the microarray, but wsn wt virus did not bind to synthetic glycans in the bli assay. possibly, soluble ha proteins can have access to short glycans on glycan arrays whereas virus envelope-embedded has cannot, depending on virus strain, bind to the same glycans on bli sensors. to test this, we examined binding to glycoproteins carrying α , -or α , -linked sias on n-linked sialoglycans ( 'n fetuin or 'n transferrin bt; fig d and fig e) . pr cam , virus displayed absolute specificity for 'n fetuin. however, an absolute α , -to α , -linked sia specificity shift by mutation e d, as observed above by glycan array analysis, did not occur , pr cam , or wsn wt was determined by glycan array analysis. binding to mono-, bi-and tri-antennary glycans carrying one, two or three lacnac repeats terminated with a α , -or α , -linked sia is indicated. means of independent replicates are graphed, standard errors of the means are indicated. biotinylated 'slnln or 'slnln for virus binding to glycoproteins (v rel = v 'n fetuin /v 'n transferrin = . ± . for pr cam , ), similar to what was observed for the synthetic glycans ( fig b and fig c) . glycoproteins, in contrast to synthetic glycans, supported wsn wt virus binding to bli sensors which displayed dual binding specificity (v rel = . ± . ) in agreement with the glycan array results for wsn ha proteins. in conclusion, determination of v obs allows quantification of relative receptor specificity differences for virus particles, which is independent of virus concentration (s g fig, s h fig and s i fig) . the application of (tailor-made) glycoproteins enables to elucidate quantitative differences in iav receptor-binding fine specificity, which is a step forward to mimicking virus-receptor interactions that occur in vivo. the versatility of the latter is further illustrated by showing the relative specificity for n-linked versus o-linked glycans, using engineered fetuin constructs (s fig). o-linked glycans are abundantly present on soluble mucins present in respiratory mucus. as an initial experiment to explore potential differences in binding dynamics to o-or n-linked glycans we expressed fetuin variants containing three n-linked and/or three olinked glycans and compared the binding of pr cam , and pr mtsin to these receptors (s a- na activity contributes to the dynamic interaction of virions with receptor-coated surfaces. we first determined whether non-bound virions are able to cleave receptors loaded on bli sensors. we used viruses carrying the same na but a different ha (pr mtsin (fig b) . in the presence of na activity, the binding rate of tx namtsin became increasingly lower in time, presumably reflecting virion release due to ongoing receptor depletion. regeneration of sensors, which removes all virus particles but leaves the biotinylated glycans on the sensors, was followed by a second round of tx namtsin binding in order to determine the extent of de-sialylation that occurred in the first round of binding ( fig c) . only tx namtsin binding in the first round in absence of oc led to receptor desialylation as detected by inefficient binding of tx namtsin in the second round. in contrast, α , specific pr mtsin did not bind to the sensor and thereby could not remove sias, as shown by efficient re-binding of tx namtsin upon regeneration. we conclude that non-bound virions do not contribute to sialidase activity and do not have to be taken into account when studying the effect of virion-associated na activity on iav receptor binding dynamics by bli. we determined the quantitative effect of na activity on the dynamics of iav-receptor interactions of two viruses carrying the same ha, but different na proteins (wsn hamtsin and pr mtsin ; s e fig) . whereas the viruses displayed similar binding to both 'n fetuin and 'sln when na was inhibited (fig a and d ), only pr mtsin binding was strongly reduced by its associated na activity (fig b and e ). this observation corresponded well to thẽ -fold lower na activity of wsn hamtsin per virus particle (s i fig and s j fig) . we conclude that, in addition to ha binding properties, na activity drastically affects virus binding dynamics in the absence of na inhibitors. the binding curves reflect the ha/na balance which is quantifiable by empirical parameters like initial binding rate, the area under the curve (unit is min nm) and x,y coordinates of the peak (time and binding level). na activity-dependent self-elution of viruses bound in presence of oc provides quantification of other descriptive parameters that characterize the balance between ha, na and receptor. of note, the concentration of released particles is too low for their re-association to take place. as expected, pr mtsin eluted much faster from 'n fetuin or 'sln than wsn hamt-sin (which does not elute from 'sln). for both viruses the self-elution rate from 'n fetuin was higher than from 'sln ( fig c and fig f) . this likely results from differences in specific na-dependent influenza a virus rolling on receptor surfaces activity of the nas for these receptors but, as ha and na are in competition for binding/cleavage of the same receptor, might also be affected by the k d of monovalent ha-receptor interactions and thus be a reflection of ha/na balance on a specific receptor. this possibility is further corroborated by an experiment where we compared the elution rates from 'n fetuin and 'n transferrin bt for two viruses (pr cam , and wsn wt ) carrying the same na (from wsn wt ) but a different ha (s fig). both viruses bind at similar, but relatively low, rate to 'n transferrin bt ( fig e) whereas wsn wt displays a~ -fold faster binding rate to 'n fetuin than pr cam , ( fig d) . ha clearly affects the self-elution rate as, in combination with the same na, self-elution from 'n fetuin is much more efficient in companion of the weaker binding ha of pr cam , (s b fig thus, whereas α , sia versus α , sia specificity of na is seemingly opposite for pr cam , and wsn wt , this is not caused by the na itself (which is identical for both viruses) but by differences in their has. also a change of receptor (in this case to fucosylated 'sln, giving sialyl-lewis x ; sle x ) was shown to have differential effect on the observed binding rate ( fig g) and na-driven self-elution ( fig h and i ). while v obs is similar for sle x and 'sln in the absence of na activity ( fig g) , an active na resulted in a lower v obs and maximum binding level and a smaller area under the curve for 'sln ( fig h) and in a faster virion self-elution from 'sln ( fig i) . thus, receptor binding dynamics on 'sln and sle x differ due to a lower specific na activity towards sle x which shifts the relative ha/na balance on these receptors. in conclusion, bli can be used to quantify changes in the dynamics of receptor binding due to an altered ha/ na/receptor balance. in a small increase in reflection resulting in negative virus dissociation values, which cannot be explained by additional virus particle binding, was consistently observed during the initial phase of self-elution (e.g. fig c, f and i). the effect was most prominent when na activity was low. the possible role of na activity herein was further examined by performing self-elution of pr mtsin from 'sln and wsn hamtsin from 'n fetuin in the presence of an oseltamivir concentration range (fig a and b ). duration and magnitude (maximally~ . nm) of the increase in the apparent binding level, which was not observed when na was fully inhibited, depended on the degree of na activity. the effect must reflect a neuraminidase activitydependent change in the receptor-bound virus particles as free virus particles that can bind are absent. cleavage of sias by na is expected to gradually decrease the number of ha-sia contacts between receptor-coated surface and virus until the particle dissociates. relaxation of virus particle binding (by less contacts) could result in an altered binding conformation of a particle that, at the maximal number of contacts, might be tightly squeezed against the receptor surface. such a morphological change likely explains the observed, na-activity dependent, changes in reflection. remarkably, the initial binding rate of wsn hamtsin to 'n fetuin was higher in absence than in presence of a high concentration of oc (compare fig a and b ). several avian na genotypes have been shown to possess a nd sia-binding site, alternatively referred to as hemadsorption site [ , ] , but such a site is not conserved in wsn na and oc binding to this site has never been demonstrated. however, active site mutations that abolish catalytic na activity have resulted in na-dependent hemagglutination, which could be inhibited by oc [ , ] . we therefore examined the effect of oc on binding of wsn hamtsin (low na activity) and pr mtsin (high na activity) virions to 'sln and 'n fetuin. complete na inhibition gave binding curves displaying a continuous increase of virus binding (fig c- f , blue lines). however, the v obs of wsn hamtsin increased at lower oc concentrations in a concentrationdependent way (fig d and fig f) . in time, the curves bent down by depletion of sia receptors due to na activity. in contrast, the v obs of pr mtsin , which has a highly active na in comparison to wsn hamtsin , directly decreased strongly at lower concentrations of oc ( fig c and fig e) . the results imply that nas with a relatively low cleavage rate (low k cat ) contribute to the v obs by binding with their active site to the sia receptor. to strengthen this conclusion, we quantified the enhancement of the initial binding rate by wsn wt viruses carrying the same na but four different has ( fig g and s fig) . whereas all four viruses displayed an effect of na on v obs , the virus with the lowest ha-dependent initial binding rate (pr cam , ), was relatively most enhanced in initial binding rate by na-dependent binding (s e fig and s f fig) . this demonstrates that the degree of the contribution of na to v obs is influenced by its ha partner. in conclusion, the na protein contributes to the initial binding rate balance by binding with its active site to sia receptors and the magnitude of the effect is probably influenced by the ha/na balance. all four viruses showed faster dissociation when more virus was loaded, indicating that there is positive cooperativity between viruses in respect to self-elution rate. the only plausible mechanism by which viruses can assist each other in self-elution is by exerting na activity on a larger surface area than their own contact area, implicating that virus particles move over the receptor-coated surface. to test this hypothesis a concentration series ( . pm to pm) of pr mtsin, harboring a highly active na, was bound to 'n fetuin in presence of oc resulting in virus saturation levels on the sensor surface ranging from~ % to~ % (fig e) , based on a maximal binding level of nm after prolonged binding (s a fig). subsequent na-dependent self-elution ( fig f) confirmed that higher virus loading levels result in faster self-elution as shown by plotting fractional virus dissociation against time (fig g) . we next determined whether virus particles are ) for minutes in presence of μm oc after which the sensors were washed in pbs and dissociation was examined at a range of oc concentrations as indicated in the panels. (c-f) pr mtsin (c, e) and wsn hamtsin (d, f), carrying the same ha mtsin but either na mtsin (high na activity) or na wsn (low na activity), respectively, were bound at pm concentration to biotinylated 'sln (c, d) or fc-tagged 'n fetuin (e, f) loaded to maximum level. binding was performed in absence (red lines) or in the presence of a range of oc concentrations as indicated in the panels. significant differences between initial binding curves shown in panels c-f were analyzed by ibm spss statistic . in panel c, there was no significant difference between curves with μm oc and nm oc (p> . ), whereas the curves of μm oc and nm oc were significantly different from nm, . nm and nm (p< . ). in panel d, the curves of μm oc and nm were significantly different from those of nm, . nm and nm. there also was a significant difference between nm and . nm. in panel e, the nm curves significantly differed from the other three oc concentrations. in panel f, there were significant differences between the nm (and nm) and . nm and nm curves. (g) the ratio (initial virus binding rate v obs in absence of oc)/(initial virus binding rate v obs in presence of μm oc) of four viruses carrying the wsn wt na in the background four different has was plotted (the individual binding curves are shown in s fig) . standard deviations and significant differences between the mean initial binding rate ratios are indicated ( à indicates p< . ). indeed able to clear sias from a larger surface area than their own contact area during self-elution. after self-elution, sensors were regenerated and re-bound with a high concentration ( pm) of virus ( fig h) . clearly, even at a level where only~ % of the surface was bound with virus, self-elution created a surface to which only very limited re-binding of virus particles could take place indicating extensive removal of sias from the complete surface. as the concentration of virus released from the surface is too low for re-association and non-bound virus does not contribute to receptor cleavage, we conclude that virus particles exert na activity while moving over the receptor-coated surface. migration of attached iav particles over a receptor surface necessarily depends on the very high k d of monovalent ha-sia interactions resulting in their rapid formation and dissociation. we hypothesize that na, in combination with the highly dynamic formation and release of individual ha-sia interactions, drives virus rolling by the generation of a receptor gradient due to its receptor destroying activity. rolling of virus particles is predicted to be faster for virus particles with higher na activity. this was tested by comparing the binding and dissociation dynamics of pr mtsin and wsn hamtsin , which have the same ha but have high and low na activity, respectively. the experimental set-up shown in fig a links sensors to bli graphs by color coding. first (fig b) , biotinylated 'n+o fetuin -coated sensors were loaded with wsn hamtsin to a~ % saturation level in the presence of oc (blue and red) or incubated in buffer (green). in the next step (fig c) , these sensors were incubated with pr mtsin in the presence or in the absence of oc. in presence of oc (blue) efficient binding of pr mtsin to the large virus-free area takes place. in the absence of oc, high na activity prevents long na-dependent influenza a virus rolling on receptor surfaces lasting binding of pr mtsin to an empty sensor (green). the slightly negative slope of the wsn hamtsin loaded sensor (red) represents minor dissociation of wsn hamtsin in absence of oc, as expected on basis of its low na activity. this also implicates that wsn hamtsin elution is not appreciably assisted by pr mtsin . in the last step of the experiment (fig d) , the sensors were regenerated to remove all bound viruses followed by probing the residual sia content by its association capacity with wsn hamtsin in the presence of oc. as expected, the blue sensor, to which both viruses were bound in presence of oc, was as efficiently bound by wsn hamtsin as in the first round. also as expected, the green sensor to which no virus was bound in the first step and pr mtsin in absence of oc in the second step was efficiently cleared from sias, as demonstrated by its poor capacity to re-bind to wsn hamtsin . in contrast, binding of wsn hamtsin to~ % of sensor surface in the first step (red) prevented complete removal of sias by pr mtsin in the second step as demonstrated by considerable re-binding of wsn hamt-sin in the third step (red). this implies that wsn hamtsin , by marginal rolling over the surface because of its low na activity, protects its contact surface with the sensor against the na activity of pr mtsin , which cleaves sias of all the non-protected surface area. we conclude that na activity is the driver of virus rolling on a receptor-coated surface. the ménage a trois between iav ha, na and (decoy) receptors largely determines viral fitness and host cell tropism. here, by studying their highly dynamic but poorly understood interplay using bli, we obtained novel mechanistic and quantitative insights into iav-host interactions. we combined the key findings into a schematic model (fig ) . the initial monovalent ha-sia interaction is virus concentration-dependent and governed by a binding rate constant k on (m − s − ) and a dissociation constant k off (s − ). its high k d (k on /k off % . to~ mm [ ] [ ] [ ] cannot be easily determined and makes monovalent virus-receptor interactions undetectable by bli too. in combination with picomolar virus concentrations, this high k d inevitably results in the observed low virus binding rate. subsequent ha binding steps are intramolecular (determined by a different k on and k off with the unit s − ), resulting in multivalent binding. remarkably, and counterintuitive to its receptor destroying activity, na can contribute to the initial binding rate. receptor residence time in the na substrate binding site is determined by the binding constant (k d = k on /k off , range is μm~ μm [ ] and catalytic rate constant (k cat ). obviously, a low k cat promotes the chance on secondary binding events (mostly of ha), which will also be affected by the na/ha virus incorporation ratio (s k fig) . bli-detectable virus binding is, due to multivalency, virtually irreversible but highly dynamic as individual ha-and na-sia interactions are rapidly formed and broken in a virus concentration independent mode. the number of simultaneous interactions required for virtually irreversible (k off % ) binding is low and logically depends on receptor density and the k off and k on values of a virus (fig e) . the rapid interconversion of binding states via the association/dissociation events shown in fig enables a virus to roll over the surface. na activity (curled arrows) is required to drive rolling, most likely by creating a receptor gradient that forces a virus to roll away from the empty receptor positions. receptor cleavage eventually results in virus dissociation when receptor density becomes too low to support tight multivalent binding. this model clearly sketches important biological as well experimental consequences. quantification of iav-receptor binding usually focuses on determination of the dissociation constant k d . however, equilibrium binding models did not apply, even at low receptor densities, and binding curves were dominated by concentration-independent avidity effects resulting in virtually irreversible binding. the binding curves are in agreement with a random sequential adsorption model [ , ] . in such models irreversible particle binding proceeds to a plateau at~ % occupation of the binding surface (the jamming limit). however, iav particle binding could slowly proceed to complete saturation of the surface (fig and s a fig). we attribute this to virus rolling over the surface, a mechanism by which at an eventually very low rate sufficient space for new binding events is created. current, inherently complex, models for polyvalent binding lack general applicability [ , ] and cannot be used to determine the kinetic constants of the different binding events shown in part of the viral envelope, containing ha (red symbol) and na (blue symbol), and the sensor surface, coated with sia (purple diamond)-containing receptors (r), is shown. kinetically different steps lead to multivalent interaction between iav and a receptor-coated surface. the initial ha-dependent binding event (step in red) is a virus concentration-dependent intermolecular process governed by a binding rate constant k on (with the unit m − s − ) and a dissociation constant k off (with the unit s − ). subsequent ha binding steps (e.g. steps and ) are intramolecular with a k on (not necessarily the same as in the first step) and k off (both with the unit s − ). for iav, having a k d (k off /k on ) of~ . to~ mm for a monovalent ha-receptor interaction, binding at pm concentrations inevitably results in a low binding rate that is mostly determined by the first binding event. na can also contribute to the initial binding rate. this contributory effect can be inhibited by oc and is therefore attributed to binding of receptor to a na catalytic site. the contribution of na to receptor binding is determined by a dissociation constant (k d = k off /k off ) for the substrate (step i in blue) and a catalytic rate constant (k cat ; bold blue arrow) determining the receptor cleavage rate. a lower k cat will result in prolonged receptor binding before cleavage or dissociation takes place. this will enhance the chance for additional binding events (mostly by ha, which is present at higher density than na), thereby promoting the cascade of multivalent interactions responsible for tight virus binding. given the lower k d ( μm~ μm) [ ] of na, in comparison to ha, for interaction with sialosides, a considerable contribution to the initial binding rate by na is expected even whereas the na/ha ratio of a virus particle is generally quite low. longer lasting, bli-detectable, binding requires the formation of additional ha-and/or na-sia bonds, which is indicated by the grey shaded area. initial binding events will be hardly detected due to the low levels of equilibrium binding in step and i. during the bli-detectable phase of binding, ha-and na-sia interactions are formed and broken in a virus concentration independent mode with the result that all binding states can rapidly interconvert via binding/dissociation events to and ii to iv. the number of simultaneous interactions that can occur is logically dependent on receptor density and k off /k on ratios but how many simultaneous interactions suffice to keep a virus particle bound to the surface remains unknown. the experiments shown in fig suggest the number of interactions required is very low. theoretically, the dynamics of ha-sia interactions allow a virus to roll over the surface but experiments shown in fig show that na activity strongly stimulates rolling (and eventually leads to virus dissociation). this is schematically indicated by the curled arrows where na cleavage activity creates receptor-free positions on the surface. the receptor gradient caused in this way is probably the driving force for virus rolling but the direction in which the virus rolls (away from the empty position or "reaching over" the empty position) still needs further research. an endpoint binding assay (shown for our data in s f fig) falsely assumes an equilibrium binding model and further errors into apparent k d determinations are introduced by low binding rates (as observed for weak binders or at low virus concentration, s a fig and s b fig) that prevent reaching a binding plateau within min. iav binding to cells is poorly reversible [ ] and adherence of only a few particles is already sufficient for productive cell infection. thus, where equilibrium binding levels or binding to saturation are not an issue, the initial binding rate v obs is a physiologically relevant parameter for iav binding. it can be determined by bli, but not easily by endpoint assays like conventional glycan arrays, more recently developed shotgun glycan arrays [ ] or receptor-coated -well plate based assays. direct proportionality between virus concentration and v obs during the initial binding phase allows quantitative comparison of viruses of unknown concentration by determination of the relative binding rate constants for different receptor pairs. coating of sensors with recombinant glycoproteins, in a homogeneous orientation by virtue of their nterminal tag (e.g. fc-tag, biotinylated bap-tag), provides analysis of receptor surfaces on which the sialoglycans receptors are attached in protein-linked conformations as encountered in vivo. genetic engineering enables binding studies to glycoproteins carrying specific glycantypes (n-linked, o-linked) or glycan-density whereas further tuning of glycan structure can be accomplished by glycoprotein expression in cell lines with specific differences in their glycosyltransferase expression patterns. this will enable a much more refined analysis than the often used biotinylated polyacrylamide molecules carrying randomly distributed glycans and supposed to adopt a spherical configuration with a~ nm diameter [ ] . the recent emergence of h n strains displaying na-dependent hemagglutination hint at a role for na in determining the changes in receptor binding that accompany and/or drive virus evolution. this phenomenon is thought to reflect the gained capacity of na to bind receptors that are refractory to cleavage by na [ , , ] . by using highly sensitive bli assays we showed that the na of strain wsn wt contributes to the initial binding rate even though it is capable of receptor cleavage leading to virus elution. this effect is exerted by binding of the receptor to the na catalytic site, as demonstrated by the inhibitory effect of oc, and is negatively correlated with the specific activity of the na. changes in ha-receptor binding specificity and avidity are thought to be prime factors in causing, or responding to, antigenic change [ , ] or infection of an altered host cell range. secondary changes in na have been proposed to restore a critical ha/na functional balance. now, considering the evidence for a role of na in receptor binding, more complex scenarios should be considered. for instance, changes in na might facilitate (transient) functional changes in ha by (temporarily) contributing to the initial virus binding rate. bli allows quantification of such effects by determination of the relative increase by na of ha-dependent virion binding (fig and s fig). a functional ha/na balance has primarily been described by weighing separately determined ha binding and na activity properties, using isolated proteins or virions. bli enables quantification of the separate contributions of ha and na to virus binding rates. the strength of bli lays in studying the simultaneous effect of na and ha, and thereby their balance, on the dynamics of virus-receptor interactions. virtually irreversible binding is the result of multiple ha-sia interactions that rapidly associate and dissociate, thereby providing access for na to temporarily unbound sias. sia cleavage by na creates a receptor density gradient that drives virus rolling, most likely away from the receptor-free spot in the direction of higher receptor density, or alternatively, by taking "a large step" to a site beyond the de-sialylated receptor. ultimately, when receptor density becomes too low, virus dissociation will occur. the resulting binding/dissociation profiles can be recorded by bli and are a quantitative reflection of the ha/na balance. yet lacking a mathematical model, these profiles can be described by empirical parameters (initial binding rate, area under the curve and x,y coordinates of the peak). kinetic binding rate constants for ha and na (k on and k off ) and the na catalytic rate constant (k cat ) will determine virion rolling characteristics (fig ) . in addition, the ha/na ratio and distribution pattern in the virus envelope will determine the frequency at which na will be present in the contact area between virus and receptor surface where receptor cleavage can take place. as such, ha/na ratio and distribution pattern are additional variables that can be involved in functional evolution of the ha/na balance and its effect on virus rolling. whereas mostly a random distribution of ha over the viral envelope has been observed, the less abundant na has been shown to occur as singular tetramers as well as small clusters thereof. the latter has mostly been observed for iavs with a filamentous morphology [ ] [ ] [ ] . whereas iavs harvested from cell culture usually display a spherical (~ to nm diameter) or slightly pleomorphic shape, filamentous morphology is frequently, but not always, observed in clinical samples (reviewed in [ , ] ) although the loss of a filamentous phenotype is not absolutely required for adaptation to growth in cell culture. filamentous iavs usually have a diameter of to nm and a length up to μm. patches of na clusters at the tip and the base of long filaments have been described [ , ] but other reports suggest the presence of na along the length of the filament [ ] . several functions for a filamentous morphology have been proposed (reviewed in [ , ] ) including a role in clearance of sias from mucus by long budding filaments that have not pinched of from the cell surface [ ] . results obtained in this study are confined to the behavior of spherical iavs and additional studies are required to see if filamentous iavs can move over a surface by lateral rolling, crawling or a caterpillar-like motion. interestingly, unidirectional motility of a filamentous influenza c virus over a receptor-coated glass-slide was recently demonstrated by microscopy [ ] where as a more random movement of spherical iav particles was observed on fetuin-coated glass slides by total internal reflection microscopy. directional movement of virus particles at two different velocities, both dependent on na activity, was reported [ ] . earlier, the combination of a receptor-binding and a receptor-destroying enzyme has been proposed to enable iav movement over a cell surface by a mechanism of repeated cycles of receptor binding, receptor release and receptor cleavage [ ] . spherical and filamentous iav particles probably both occur in vivo and may very well have different functions. whereas na-dependent motility of filamentous particles through a mucus layer might be difficult, they might be better suited for spreading the infection to neighboring cells or clearing the mucus layer from their sia content. spherical iav particles on the other hand, are likely obtained during in vivo infection of humans by inhalation of aerosols containing a relatively low number of virions that somewhere get stranded on the mucus layer covering epithelial cells of the respiratory tract. thus, whereas quantitative measurements of spherical iav binding kinetics by bli should yield valuable data for modeling and testing the movement of such particles through a mucus layer and over epithelial cell surfaces, additional experiments are required to determine whether this can be extended to filamentous iav particles. iav was shown to require na activity for penetrating through a mucus layer in vitro [ ] . the irreversible but highly dynamic binding mode that leads to virus rolling results in virus self-elution from a receptor-coated surface only upon efficient clearance of receptors from the complete surface. considering the low amount of virions confronted by a large amount of mucus and epithelial cells, we propose that virions, once attached to the mucus, remain receptor bound for their entire extracellular life. soluble, densely sialylated mucins in the mucus layer ( - μm thickness) form, by polymerization, a mesh-like structure with an average pore-size of~ nm [ ] through which the virions need to move to reach the pericilliary layer. in this layer, the cilia of columnar epithelial cells ( . to . μm in diameter; to μm in length) are covered by membrane-spanning mucins and tethered muco-polysaccharides that protrude into the narrow (~ nm) space between cilia from which soluble mucins or nm beads were shown to be excluded [ ] . we consider it likely that rolling is necessary for gaining the directionality and even traction required for efficiently penetrating this heavily sialylated maze to reach sites on the epithelial cell membrane that permit virus entry. whether, and to which extent, rolling also takes place over the epithelial cell surface in order to arrive at a site that permits entry by endocytosis remains an open question. the overall density of sias on the cell surface is estimated to be high [ , ] , but also very heterogeneous. iav entry by clathrin-mediated endocytosis was shown to take place by de novo formation of clathrincoated pits at sites where a virus particle was bound [ ] . thus, it seems likely that in case of rolling the virus becomes arrested at a specific place in time. tight binding to specific receptors might be required for this but these have so far not been identified. note that the virus may also surf together with a tightly-bound glycoprotein over the cell surface. rolling might also be important for cell-to-cell virus spread. virions budding from de-sialylated cells, resulting from na activity, may even utilize sialylated mucins covering the epithelial cell layer as a rolling track to neighboring cells. in this perspective, an optimal ha/na balance should be considered as the combination of ha and na kinetic parameters, including factors like ha/na ratio and distribution, that optimally supports virion rolling over distinct surfaces coated with a diversity of receptors. also protective antibodies targeting iav receptor binding sites will function within this context with an important role for the ha/na/receptor balance as they will compete with receptors for interaction with iav [ , ] . the bli-based methodology that we have established here is well-suited to quantify such processes using a highly versatile, modifiable and controllable receptor repertoire. [ ] as well as all other viruses were grown in mdck-ii cells as described previously [ ] and stored at − ˚c. virus titers were determined by measuring the tcid on mdck-ii cells. sequences of the ha and na genes were confirmed by sequence analysis (macrogen). for quantification, virus samples were concentrated by tca precipitation [ ] and applied to standard % sds-page gels for separation of viral proteins followed by silver staining. silver-stained polymerase bands were quantified by densitometry on silver staining gels as outlined and shown in supplementary s a fig, s b fig and s c fig. ha and na amounts were quantified by western blotting of standard % sds/page gels. monoclonal antibodies used for detection and quantification by densitometry were fi (for quantification of ha) [ ] , n - d (for quantification of pr mtsin n ) [ ] and gt (for quantification of wsn n ). recombinant purified ha and na proteins (see below) were used for standard curves (s d- s i fig) . before the application of samples, -mesh copper grids with a pure carbon film were exposed to a glow discharge in air for s to make them hydrophilic. ten microliters of the virus preparations was applied to the grids and incubated for min. excess sample was blotted with a filter paper. for negative staining, μl of % phosphotungstic acid at ph . was applied. after min, excess stain was blotted and grids were left to dry. the specimens were examined in a jeol jem transmission electron microscope at kv and images were taken at a magnification of . x with a matataki k x k camera. human codon-optimized h -encoding and n -encoding cdnas of pr mtsin and wsn wt (accession no. p . for wsn ha, acf . for wsn na, adx . for pr ha, and p . for pr na) were cloned in pcd (ha) or pfrt (na) expression vectors flanked by cd signal peptide-, gcn -isoleucine-zipper trimerization (for ha) or tetramerization (for na) domains, and strep-tag ii-encoding sequences similarly as described previously [ , ] . codon-optimized cdna fragments encoding the variable domains of the heavy and light chains of antibody fi [ ] were synthesized by genscript usa inc and cloned inframe into pcaggs vectors containing human igg heavy and light constant domains, respectively, similarly as described previously [ ] . codon-optimized bos taurus fetuin-encoding cdna (accession no. np_ . ) was cloned in the pcaggs expression vector fused in frame to sequences encoding a human fc-tag with or without a tandem repeat of the -amino-acid biotin acceptor peptide (bap)-tag [ ] . biotinylated fetuin is referred to as fetuin bt. mutations for knocking out o-linked glycosylation sites were introduced into the fetuin gene by using the q site-directed mutagenesis kit (neb) and confirmed by sequencing. human codon-optimized cdna encoding biotin protein ligase (bira) [ ] was cloned in pcd in frame with sequences encoding a cd signal peptide and a his tag. expression vectors were transfected into hek t (atcc crl- ),cho k (atcc ccl- ) and gnti-deficient cho b cells [ ] (from ineke braakman, utrecht university, the netherlands) using polyethyleneimine i (pei) similarly as described previously [ , ] . cho cells are deficient in α , sialyltransferases and therefore exclusively synthesize , sia linkages [ , ] . tissue culture supernatants were harvested - days post transfection. recombinant ha and na proteins were purified using strep-tactin sepharose beads according to the manufacturer's instructions (iba, germany). fc tag-containing proteins were purified using protein a sepharose beads (ge healthcare), similarly as described previously [ ] . for an overview of the different fetuin constructs see s c fig. the concentration of purified protein was determined by using a nanodrop spectrophotometer (isogen life sciences) according to the manufacturer's instructions and analyzed by % sds-page followed by visualization of protein bands using a colloidal blue staining kit (invitrogen). biotinylated transferrin (sigma) contains two glycan chains exclusively carrying α , sias [ , ] (referred to as 'n transferrin bt). the activity of iav virus particles as well as recombinant soluble nas was determined by using a fluorimetric assay similarly as described previously [ ] . in short, viruses and na preparations were subjected to -fold serial dilutions in reaction buffer ( mm tris-hcl, mm cacl , ph . ) in a flat-bottom -well black plate (greiner bio-one). subsequently, a similar volume of reaction buffer containing μm -( -methylumbelliferyl)-α-d-n-acetylneuraminic acid (munana; sigma) was added to each well, mixed well, and incubated at ˚c for min. the reaction was terminated with the stop solution ( . m glycine, % ethanol, ph . ). the fluorescence of the -mu reaction product was immediately determined in relative fluorescence units (rfus) using a fluostar optima plate reader (bmg labtech, mornington, australia) with excitation and emission wavelengths at and nm, respectively. microarrays were printed as described previously [ ] . the glycan array analysis of the ha proteins was performed as previously described [ ] . briefly, μg/ml recombinant ha was precomplexed with a horseradish peroxidase-linked anti-streptavidin tag antibody and an alexa fluor anti-mouse antibody ( : : molar ratio) for min at ˚c, prior to incubation for min on the microarray slide under a microscope cover glass in a humidified chamber at room temperature. after repeated washes with phosphate-buffered saline (pbs) with . % tween, pbs, and deionized water, the slides were immediately subjected to imaging. bli analysis was performed on an octet qk machine using standard streptavidin (sa) or protein a bio-sensors. pbs with calcium and magnesium (pbs+/+) was used as standard assay buffer. receptor loading was performed by loading biotinylated receptors (synthetic glycans or proteins) to sa sensors or fc-tagged glycoprotein receptors to protein a sensors. unless otherwise specified, sensor were loaded with receptor to maximum levels (no further increase in reflection) using nm synthetic glycan or μg/ml glycoprotein as loading sample concentration. after loading sensors were washed in pbs+/+ until a stable baseline was obtained. virus binding was performed by moving receptor-loaded sensors to wells containing μl virus sample at the indicated concentrations (the use and concentration of oc is indicated where applicable). then virus-loaded sensors are usually moved to pbs+/+ in presence of μm oc to examine virus dissociation (consistently producing a flat line), or washed times for seconds in pbs+/+ to remove oc and next transferred to pbs+/+ without oc to measure na activity-driven self-elution. alternatively, in order to determine simultaneous action of ha and na virus binding was analyzed from the start in pbs+/+ in the absence of oc. regeneration of sensors, preserving the binding of biotinylated receptors but removing all bound virus, was performed by dipping sensors briefly in mm tris/glycine buffer ph . pr mtsinspecific antibody ( / from nibsc) was used for detection of virus binding in presence of oc with or without prior sensor generation. fetuin-coated sensors were also analyzed for their lectin-binding properties. to this end, fetuin-coated sensors were incubated with different lectins ( ng/ul; sna, mal-i, mal-ii, eca, all from vector labs) and lectin-binding curves were obtained. each bli experiment was repeated at least twice. representative experiments were graphed. initial binding rates, corresponding to the sloops of the binding curves during the first few minutes of the virus-binding experiments, were determined by second order polynominal equation (graphpad prism . ). the correlation between virus particle numbers and the initial binding rate was determined by linear regression and pearson r analysis using graphpad prism . software. significant differences between curves were analyzed by univariate analysis of variance model using ibm spss statistic . fractional receptor densities correlating with half maximum initial binding rates were determined by non-linear regression analysis using graphpad prism . software. significance analysis was based on two tailed unpaired t test or one way anova followed by tukey's multiple comparisons test (graphpad prism . ). streptavidin-coated (sa) biosensors contain biotin binding sites (pall-fortebio). sa tetramers carry four biotin binding sites, ordered two by two at opposing planes of the cubic structure [ ] . only two binding sites (spaced at . nm distance as determined by x-ray crystallography [ ] ) on one side of a surface-coated sa tetramer ( nm center to center distance assuming regular hexagonal packaging) are assumed to have exposed biotin binding sites [ ] . ha trimers are closely packaged on the virus surface (s fig, in agreement with [ ] [ ] [ ] [ ] ) and the center to center distance has been determined at nm [ ] [ ] [ ] [ ] . a fully loaded streptavidin can, in principle, form a bivalent interaction with an ha-trimer in which the sia-binding sites are spaced at nm distance [ , ] . lowering the receptor-density results in a non-homogenous sensor surface with streptavidins carrying , or receptor molecules. as a result, increasing amounts of surfacearea will have a receptor density too low to bind virus at decreasing receptor concentrations thus contributing to the observed decrease in maximum binding levels and initial binding rate when lowering receptor density (fig d and fig e) . (b) labstrains pr and wsn wt are spherical viruses with a diameter of~ nm (s fig) [ ] [ ] [ ] [ ] . when virus particles can be flattened for . times the radius % of the virus surface will be in contact with the sensor. (c) when % of the virus surface is in contact with the sensor,~ ha trimers can interact with receptor-loaded sa molecules at the virus-sensor contact interface (inner red circle). in principle two receptor molecules on a sa molecule can interact with an ha trimer but whether this occurs simultaneously will depend on the exact geometry of the specific glycan that was loaded. (d) at saturating levels of virus binding (hexagonal packaging) the majority of sa molecules are not present at the contact interface and therefore cannot be cleaved by na without virus movement. [ , ] . biotinylated fetuin was made by expressing a construct encoding a bap-tag fused to fetuin that, by co-transfection with a plasmid carrying a biotinylation enzyme, yields c-terminally biotinylated 'n+o fetuin ( 'n+o fetuin bt) upon expression in cho k cells. (d) confirmation of sia linkage-type specificity of glycoproteins using lectin binding. the glycoproteins were analyzed for linkage type specificity of their sialic acids using lectins mal i (specific for siaα , galα , glcnac linkages abundantly present on n-linked glycans), mal ii (specific for siaα , galα , galnac linkages abundantly present on o-linked glycans), sna (specific for siaα , galα , glcnac linkages abundantly present on n-linked glycans), and eca (specific for terminal galα , glcnac epitopes present on non-sialylated n-linked glycan antennae). (e) viruses used for binding to receptor-loaded sensors are wild type pr mtsin , wild type wsn wt and recombinant viruses carrying the ha encoding segments of pr mtsin (wsn hamtsin ) or pr cam (pr cam , ) in the background of seven wsn segments. pr cam , is identical to pr cam , except for a substitution (d e) that was introduced in ha to obtain a shift from α , to α , linkage-type binding specificity. tx namtsin carries the ha encoding segment of a/bilthoven/ / (h n ) in the background of seven pr mtsin segments [ ] . whereas both pr mtsin and pr cam , bound to fetuin containing n-glycans (a and b), only pr cam , was able to bind fetuin containing o-glycans, albeit at a -fold lower initial binding rate than to n-glycosylated fetuin (c). when n-and o-linked glycans are both present (a), the initial binding rate seems to be determined by the stronger binding to n-linked glycans as binding of pr cam , is not accelerated despite its ability to bind α , sialylated o-linked glycans. (e-g) confirmation of presence of n-and/or o-glycan on recombinant fetuin. the glycoproteins were treated with pngase f (neb) for hours at ˚c under non-denaturing conditions, which effectively removes almost all n-linked oligosaccharides from glycoproteins. the glycoproteins were analyzed for linkage type specificity using lectins mal i (specific for siaα , galα , glcnac linkages abundantly present on n-linked glycans), mal ii (specific for siaα , galα , galnac linkages abundantly present on o-linked glycans), and eca (specific for terminal galα , glcnac epitopes present on non-sialylated n-linked glycan antennae). (e) after pngase f treatment, the binding level of mal i to ' n+o (blue) and ' n fetuins (red) significantly reduced, whereas the binding level to ' o fetuin (green) remains the same, indicating the presence of sialylated n-linked oligosaccharides specifically on ' n+o and ' n fetuin. (f) after pngase f treatment, the binding level of eca to ' n+o (blue) and ' n fetuins (red) dramatically decreased, whereas the binding level to ' o fetuin (green) remains the same, indicating the presence of non-sialylated n-linked oligosaccharides specifically on ' n+o and ' n fetuin. (g) similar binding of mal ii to glycoproteins was observed after treatment with pngase f, indicating that o-linked oligosaccharides were not removed by pngase f treatment. reliable determination of v obs depends on precise determination of virus concentration. hemagglutination titers or infectious titers do not reveal absolute or relative particle of different viruses. quantitative pcr or western blotting (usually targeting np) are sensitive to variation caused by rna or protein contamination of virus preparations and, when comparing different viruses, to differences in specificity of probes or antibodies. therefore, densitometric quantification of the polymerase content of a virus preparation was used, assuming that every particle carries eight c) and (e). the obtained numbers/particle fit well to numbers obtained by different methods by others [ , ] . (g, h, i) quantification of na by similar procedures as applied for ha in panel d-f. antibodies gt -gtx (wsn) and n - d (pr ) were used and quantification was calibrated using a western blot of a standard concentration series of recombinant soluble na of pr mtsin and wsn expressed in hek t cells run in parallel. the na proteins were expressed similarly are described previously [ ] . (j) na activity of pr mtsin and wsn wt virus particles and expressed recombinant na soluble tetramers was determined using a two-fold dilution series in a soluble substrate na activity assay (munana assay). the activity of recombinant wsn na was . -fold lower than of pr mtsin na, whereas the na activity of the cognate virus particles showed a . -fold lower activity for wsn wt particles. this difference is explained by the . -fold higher incorporation level of na in pr mtsin particles as determined by western blotting (g-i). the ha content of both viruses was similar (d-f). (k) ha/na ratio was determined from panels f and i. both viruses bind at similar, but relatively low, rate to 'n transferrin bt (fig e) whereas wsn wt displays a~ -fold faster binding rate to 'n fetuin than pr cam , ( fig d) . ha clearly affects the self-elution rate as, in combination with the same na, self-elution from 'n fetuin is much more efficient in companion of the weaker binding ha of pr cam , (b) than in companion of the stronger binding ha of wsn wt (a). self-elution rates from 'n transferrin bt are more similar for both viruses. thus, whereas α , versus α , sia specificity of na is seemingly opposite for pr cam , and wsn wt , this is not caused by the na itself (which is identical for both viruses) but by differences in their has. in absence of oc, ongoing receptor cleavage reduces receptor density in time and thus the binding rate of additional virus particles, resulting in bending of the curves. as receptor cleavage by na is in competition with receptor binding by ha, the 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pleiomorphy characterized by cryoelectron tomography polypeptides specified by the influenza virus genome i. evidence for eight distinct gene products specified by fowl plague virus mutation of the second sialic acid-binding site, resulting in reduced neuraminidase activity, preceded the emergence of h n influenza a virus oseltamivir carboxylate was kindly provided roche. recombinant pr virus tx namtsin was kindly provided by dr. r. fouchier (erasmus medical center, rotterdam, the netherlands). n - d was generously provided by dr. x. saelens (molecular virology unit, university of ghent). cho b cell was kindly provided by ineke braakman (utrecht university, the netherlands). key: cord- -m czkya authors: pinto, marlene cavaleiro; craveiro, hélder; johansson wensman, jonas; carvalheira, júlio; berg, mikael; thompson, gertrude title: bornaviruses in naturally infected psittacus erithacus in portugal: insights of molecular epidemiology and ecology date: - - journal: infect ecol epidemiol doi: . / . . sha: doc_id: cord_uid: m czkya background: the genus orthobornavirus comprises non-segmented, negative-stranded rna viruses able to infect humans, mammals, reptiles and various birds. parrot bornavirus to (pabv- to ) causes neurological and/or gastrointestinal syndromes and death on psittacines. we aimed to identify and to produce epidemiologic knowledge about the etiologic agent associated with a death of two female psittacus erithacus (grey parrot). methods and results: both parrots were submitted for a complete standardised necropsy. tissue samples were analysed by pcr. the findings in necropsy were compatible with bornavirus infection. analysis revealed pabv- related with genotypes detected in captive and in wild birds. the n and x proteins of pabv- were more related to avian bornaviruses, while phosphoprotein was more related to variegated squirrel bornavirus (vsbv- ). within the p gene/phosphoprotein a highly conserved region between and within bornavirus species was found. conclusions: portugal is on the routes of the intensive world trade of psittacines. broad screening studies are required to help understanding the role of wild birds in the emergence and spread of pathogenic bornaviruses. pabv- phosphoprotein is closer to vsbv- associated with lethal encephalitis in humans than with some of the avian bornaviruses. the highly conserved p gene/phosphoprotein region is a good target for molecular diagnostics screenings. bornaviruses are enveloped, to nm in diameter with a non-segmented genome of single-stranded negative sense rna of around nucleotides in length, belonging to the order mononegavirales [ ] . bornaviruses replicate in the nucleus of the nerve cells of various organs and establish persistent, non-cytolytic infections by exploiting the cellular splicing mechanisms to efficiently use its genome, organized into six open reading frames (orfs) [ ] (figure ). alternative transcription start and stop sites and splicing produce mrnas that are translated to produce the viral-encoded proteins [ ] (figure ). the first transcription unit contains an orf for the nucleoprotein (n), the second transcription unit contains two overlapping orfs for the phosphoprotein (p), and the x protein (x) [ ] (figure ). the third transcription unit is spliced differently, and also has different transcription initiation and termination signals, enabling polymerase read-through during transcription, which results in expression of the matrix protein (m), the glycoprotein (g), and the rna-dependent rna-polymerase (l) [ ] ( figure ). the p and x orfs overlap; as well as, the m and g orfs [ ] (figure ). the immunogenicity of phosphoprotein [ ] , as well as, the degree of conservation of the phosphoprotein and its gene, within and between bornavirus species, make them good candidates as universal targets in laboratory diagnosis [ ] . once the family bornaviridae is expanding speedily [ ] , producing knowledge about highly conserved regions within phosphoprotein will be useful for the development of sensitive laboratory diagnostic tools. so far, genus carbovirus, cultervirus and orthobornavirus have been identified, which comprise species [ ] . from those, species are associated with the development of severe neurological and/or gastrointestinal disease and death of its hosts [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (figure ). the disease has been reported in humans, several species of pets, production and wild animals [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . namely, two species infect mammals (mammalian to orthobornavirus), five infect birds (passeriform to orthobornavirus, psittaciform to orthobornavirus and waterbird orthobornavirus) and three infect reptiles (queensland carbovirus, southwest carbovirus and elapid orthobornavirus) [ ] (figure ). however, some mammalian orthobornavirus showed the ability to also infect farmed ostriches and wild birds (mallards and jackdaws) [ , ] . wild birds are hosts of avian bornaviruses (e.g. strains of waterbird orthobornavirus and psittaciform orthobornavirus) [ ] [ ] [ ] ] and mammalian bornaviruses (e.g. genotypes of mammalian orthobornavirus) [ ] . therefore, co-infection may play a role in the emergence of new pathogenic and zoonotic bornavirus species; once x and p proteins of pabv- look like to have different ancestors [ ] . in psittaciformes, parrot bornavirus to (pabv- to ) can cause proventricular dilatation disease (pdd), characterized by a flaccid and distended proventriculus impacted with feed, as a result of the inability of seeds' digestion (however throughout the gastrointestinal tract can occur variable distention) [ ] . psittaciformes can also show uncoordinated movements, postural disorders, apathy, blindness, and behavioural disorders, such as loss of appetite [ ] . the genomic map is similar for all bornaviruses, as far as known, except for queensland carbovirus and the southwest carbovirus, which have the gene order ʹ-n-x-p-g-m-l- ʹ, representing a transposition of the g and m genes [ ] . figure . phylogeny shows the species and strains belonging to the bornaviridae family, which were associated with the development of neurological and/or gastrointestinal disease and/or death of its hosts. the phylogenetic relationship between species and strains was based on a segment of the m gene. sequences identified by genbank® accession numbers and abbreviated name of virus. bornaviruses marked with asterisks are not classified following the currently accepted taxonomy [ ] . abbv- to = aquatic bird bornavirus to , bodv- to = borna disease virus to , cnbv- to = canary bornavirus to , esbv- = estrildid finch bornavirus , jcpv = jungle carpet python virus. lgsv- = loveridge's garter snake virus , swcpv = southwest carpet python virus, pabv- to = parrot bornavirus to , vsbv- = variegated squirrel bornavirus . and self-mutilation (resulting from lesions in the central nervous system) [ ] . within captive birds, the virus has become relevant for psittaciformes housed in reserves, in breeding projects of rare species, in private collections and zoos, because of the severe effects it may cause for bird welfare, economy, and biodiversity levels. analyses of molecular epidemiology suggested that a world trade of psittacines without biosafety measures has been carried out [ ] . there is, to the best of our knowledge, no publications identifying and characterizing the avian bornaviruses infecting pet parrots in portugal. moreover, there are still unresolved questions on the epidemiology of bornaviruses, such as the role of waterbirds in the emergence and dissemination of new pathogenic and zoonotic species, and the localization of highly conserved regions within p gene inter-and intraspecies of bornaviruses. the aim of this study was to identify and phylogenetically characterize the etiologic agent associated with clinical signs and necropsy findings consistent with avian bornavirus infection in two pet parrots in portugal, as well as to produce molecular epidemiologic knowledge on bornaviruses. two female psittacus erithacus (grey parrot) from different owners died with an interval of months between them. one of the parrots (case number b ) did not show clinical signs prior to sudden death. the other parrot (case number b ) had a history of clinical signs compatible with bornavirus infection and had been examined (physical examination and x-ray) at the veterinary hospital prior to its death. the owners gave their informed consent for the necropsy, as well as for the collection of tissue samples and information on the clinical and life history of the parrots (by applying a structured questionnaire designed for the study). both parrots were submitted for a complete standardised necropsy by a veterinarian specialized in exotic birds. tissue samples from adrenal gland, brain, cecum, colon, crop, heart, ileum, jejunum, kidneys, liver, lung, ovary and proventriculus were collected and frozen at − °c. the laboratory diagnosis was directed to search for avian bornavirus rna based on ) the clinical history of the captive flock (weight loss, sudden death, undigested seeds in faeces, diarrhoea, poor condition of plumage) from which the parrot b originated; ) the clinical signs, physical examination and x-ray of the parrot b ; and ) the macroscopic findings reported during the necropsy, for both parrots. bornavirus nucleic acid detection by polymerase chain reaction (pcr) we used tissue samples from brain, liver, kidney, spleen, ventricle, lung, heart, duodenum and pancreas, to search for avian bornavirus rna. extraction of total rna from the tissue samples was done as previously described [ ] . briefly, the trizol/chloroform method (trizol reagent, life technologies) was used followed by purification with the rneasy® kit (qiagen, hilden, germany), as recommended by the manufacturer's instructions. estimation of the total rna concentration was achieved using nanodrop® nd- uv-vis spectrophotometer (thermo scientific, usa), according to the manufacturer's instructions. five hundred nanograms of total rna were reverse transcribed into cdna as previously described [ ] . the cdna was amplified by two conventional pcr protocols, one using a set of primers specific for a fragment of the n gene (the forward primer: ʹ-catgaggctatwgattggatta- ʹ and the reverse primer ʹ-tagccngccmktgtwggrttyt- ʹ) and the other was carried out with a set of primers specific for a fragment of the m gene (forward primer: ʹ-ca aggtaatygtycctggatgg- ʹ and the reverse primer: ʹ-accaatgttccgaagmcgaway- ʹ). in both protocols, the thermal cycling parameters and the reaction composition of pcr were carried out as previously described [ ] . the expected length of the conventional products was base pairs (bp) for the fragment of the n gene and bp for the fragment of the m gene. from the positive samples, for both genes, selected tissues were used to conduct an additional pcr, combining the forward primer targeting the n gene and the reverse primer targeting the m gene, under previously reported experimental conditions [ ] . the expected length of the pcr product was bp (covering a partial region of the n gene, a total region of the x and p genes and a partial region of the m gene). the visualization of the pcr products was in electrophoresis using a % agarose gel, staining with gel red tm (biotium glowing products for science tm , hayward, ca) at . %. the pcr products from all tissue samples tested were purified using the grs pcr & gel band purification kit (grisp, porto, portugal) and both strands of dna were analysed by sanger sequencing. the nucleotide sequences obtained in this study were submitted to genbank® database under accessions numbers mk and mk . the identification of bornaviruses was conducted by finding regions of similarity between the nucleotide sequences obtained (from all tissues tested) and sequences from the genbank® database. throughout the x and p protein, conserved regions between bornavirus species were searched, comparing the amino acid sequences produced with the selected sequences (from genbank® database we selected two amino acid sequences from each species of bornaviruses, when available). the identification of bornaviruses, as well as the search for conserved regions throughout the x and p proteins were conducted using the basic local alignment search tool (blast®) (https://blast.ncbi.nlm.nih.gov/blast.cgi), the per cent identity (id) and its statistical significance (e-value) was collected. the relationship between the genotypes detected in the present study and the parrot bornavirus (pabv- ) genotypes reported was evaluated by phylogenetic analysis, based on n gene segment. therefore, some of the nucleotide sequences of pabv- from each of the five previously identified genotype clusters [ ] were included, as well as the new sequences deposited in genbank® database. the output of the analysis was divided into clusters, the strains were classified to belong to the same cluster when the per cent identity between them were ≥ %. since not all sequences identified by blast® search covered completely the pcr products produced, all sequences were trimmed to the length of the shortest sequence included in the respective phylogenetic analysis. to evaluate the rule of waterbirds in the emergence and dissemination of new pathogenic bornaviruses, the evolutionary relationship between the pabv- and bornaviruses detected in wild birds (aquatic bird bornavirus to and borna disease virus ), as well as bornaviruses linked to infection in humans (borna disease virus and variegated squirrel bornavirus ), was determined. therefore, sequences representative of virus genotypes were selected from genbank® database and included in the analysis. phylogenetic analyses were conducted in mega software [ ] using nucleotide sequences deposited in genbank®. one of the parrots (case number b ) had suddenly died without previous clinical history compatible with bornavirus infection. the parrot belonged to a private collection in which infection by avian bornaviruses was the cause of death of several parrots years ago. since the outbreak, the parrot b was the only case of illness, reported by the owner. the parrot was born in captivity in spain and lived in the northern region of portugal until its death. the necropsy of the bird showed a severe cachexia, with splenomegaly and hepatomegaly. the other organs were grossly normal, including a normal proventriculus without dilatation. the other parrot (case number b ) had a clinical history compatible with bornavirus infection, displaying regurgitation, seeds not digested in the faeces, cachexia and prostration. the x-ray revealed a severe dilatation of the proventriculus ( figure ). the bird, at necropsy, showed a reduced muscle mass and the proventriculus showed a severe dilatation with a ruptured wall ( figure ). the other organs were grossly normal. the parrot had always lived apparently healthy, as a pet in the central region of portugal during years. the owner reported that the parrot had been donated to him and was unaware of its origin. the two parrots never had contact with each other during their lifetime. all tested tissue samples from the two psittacus erithacus were positive for bornavirus rna, with amplification of fragments of the n gene (with bp) and of the m gene (with bp). additionally, from tissue samples of brain and lung of each psittacus erithacus, nucleic acid fragments (with base pairs) comprising partially the n and m genes and totally the x and p genes were amplified. the bioinformatics analysis revealed that both parrots were infected with parrot bornavirus (pabv- ), based on the genetic segment that contained the partial n and m genes and the complete x and p genes ( base pairs for case number b and base pairs for case number b ). the highest per cent identity (id) found between pabv- reported in the genbank® database and the virus linked to b was % (e-value = . ) and % (e-value = . ) to b . additionally, the nucleotide composition of the two virus sequences in the present study, differed by % from each other (id = %, e-value = . ). when the analysis was restricted to the x and p genes, they differed from each other by % (id = %, e-value = e − ) and % (id = %, e-value = . ), respectively. the genetic differences found between the two pabv- resulted into % and % of variation of amino acid composition of x protein (id = %, e-value = e − ) and p protein (id = %, e-value = e − ), respectively (table ) . similar results were observed when comparing parrot bornavirus (pabv- ) genotypes detected in the present study with worldwide distributed bornaviruses (mammalian, reptile and avian bornaviruses) (table ) . specifically, the per cent identity found for x protein was lower (id = ( - )%, e-value = ( e − - e − )) than the identity found for p protein (id = ( - )%; e-value = ( e − - e − )), within viruses of the same species (within psittaciform orthobornavirus) and between viruses of different species (psittaciform orthobornavirus, passeriform and orthobornavirus, mammalian and orthobornavirus and elapid orthobornavirus) ( table ) . within x protein, the higher variation of identity occurred in the region shared with the p protein (id = ( - )%, e-value = ( e − - e − )) ( table ) . moreover, in the non-shared region of x protein the identity between the two pabv- genotypes and mammalian bornaviruses (mammalian and orthobornavirus (id = %, e-value = e − )) was the same for some avian bornaviruses (passeriform orthobornavirus (canary bornavirus and (e-value = e − )) and water orthobornavirus (aquatic bird bornavirus (e-value = e − )) ( table ) . concerning p protein, the amino acid sequence region non-shared with x protein revealed higher variation (id = ( - )%, e-value = ( e − - e − . )) than the shared region (id = ( - )%, e-value = ( e − - e − )), within and between species of bornaviruses (table ). in the region shared between the two proteins, the identity found between the two pabv- genotypes and members of mammalian orthobornavirus species was higher than the per cent identity found with some members of avian bornaviruses (table ) . namely, the case number b showed a higher identity with mammalian orthobornavirus members (id = %, e-value = e − ) than with some members of waterbird orthobornavirus (aquatic bird bornavirus , (id = %, e-value = e − )) and passeriform orthobornavirus (canary bornavirus , (id = %, e-value = e − )) ( table ) . also, the case number b showed a higher per cent identity with mammalian orthobornavirus members (id = %, evalue = e- ) than with some members of waterbird orthobornavirus (aquatic bird bornavirus , (id = %, e-value = e − )) ( table ) . additionally, only in the region shared by both proteins, a statistically significant similarity was found between the two pabv- genotypes reported and members of reptilian bornaviruses (id = ( - )%, e-value = ( e − - e − )), ( table ) . the results obtained for x and p proteins, described above, were complemented by the phylogenetic analysis based on the segment of n gene, since the reported genotypes, in the present study, showed to be phylogenetically adjacent to pabv- genotypes worldwide distributed ( figure ). namely, they were mostly related with pabv- genotypes detected in several species of pet psittacines, in countries of asia (israel and japan), europe (austria, germany, hungary, sweden and switzerland) and north america (canada and usa) and south america (brazil) ( figure ) . moreover, the analysis of the n segment revealed that the detected pabv- genotypes, in pet parrots sampled in portugal, were also phylogenetically adjacent to those found in wild birds in japan (such as anas spp., emberiza spp. and grus spp.) ( figure ) . besides, the analysis showed that pabv- genotypes circulating in portugal were closely related to genotypes which have been circulating in the last years, all over the world (according to the earliest sampling date reported in genbank® for pabv- ) ( figure ). the second phylogenetic approach, based on the n gene segment, confirmed the results obtained for the relationship between the pabv- genotypes and bornaviruses (of avian and mammalian) in bioinformatics analysis of x protein (table ) ; moreover, it added information about their evolutionary relationship (figure a) . namely, phylogenetic analysis revealed that pabv- genotypes, detected in the present study, showed to be more closely related to avian bornaviruses than to mammalian bornaviruses ( figure ). within waterbird orthobornavirus species, the aquatic bird bornavirus (abbv- ) genotypes are phylogenetically closer to pabv- than to aquatic bird bornavirus (abbv- ) genotypes ( figure (a) ). regarding mammalian bornaviruses, the mammalian orthobornavirus members were more related to pabv- than to mammalian orthobornavirus members (figure (a) ). specifically, the variegated squirrel bornavirus (vsbv- ) genotypes detected in squirrels (from zoos and breeding collections) and in humans (with a fatal limbic encephalitis) are phylogenetically more close related to pabv- than to borna disease virus (bodv- ) detected in humans (associated with a lethal encephalitis) and in wild birds (figure (a) ). when the phylogenetic analysis was conducted based on the amino acids encoded by the segment of n gene, the main evolutionary relationship persisted between parrot bornavirus (pabv- ), aquatic bornavirus (abbv- , abbv- ), variegated squirrel bornavirus (vsbv- ) and borna disease virus (bodv- ) (figure (b) ). the third phylogenetic approach, based on the p gene segment, revealed that members of mammalian orthobornavirus species are the most related to pabv- genotypes than of all bornaviruses detected and reported in wild birds, other than parrots (figure (a) ). that is, the pabv- genotypes are evolutionarily closer to the vsbv- genotypes, linked to the infection of squirrels (from zoos and breeding collections) and humans than members of waterbird orthobornavirus (abbv- and abbv- ) and of mammalian orthobornavirus (bodv- ), detected in samples of wild birds (figure ) . namely, within genotypes detected in wild-bird samples, borna disease virus (bodv- ) is the most evolutionarily distant from parrot bornavirus (pabv- ) (figure (a) ). however, within borna disease virus (bodv- ) the genotype associated with lethal encephalitis in humans (accession nº mh . ) is evolutionarily more distant from pabv- than the variants detected in samples of wild anas spp. (figure (a) ). when the phylogenetic analysis was conducted based on the amino acids encoded by the segment of the p gene, the main evolutionary relationship remained between bornaviruses (figure (b) ). the findings obtained through the several analyses conducted suggest that within parrot bornavirus (pabv- ) genotypes the n and p gene had different ancestors. since the n gene of pabv- is evolutionarily more close to the n gene of avian bornaviruses (figure (a,b) ), while p gene is more related to mammalian bornaviruses, specifically with the zoonotic variegated squirrel bornavirus (vsbv- ) genotypes (figure (a,b) ). additionally, the findings showed that the borna disease virus (bodv- ) detected in samples of wild birds (accession nº af . , af . and af . ) are intermediate variants between the two bodv- genotypes figure . phylogenetic relationships between pabv- genotypes identified in the present study and pabv- detected in wild birds as in pet parrots, regarding n gene. the evolutionary history was inferred using the neighbor-joining method [ ] . the confidence probability was estimated using the bootstrap test ( replicates) and is shown above the branches [ , ] . the tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. the evolutionary distances were computed using the kimura two-parameter method [ ] and are in the units of the number of base substitutions per site. the analysis involved nucleotide sequences. all positions containing gaps and missing data were eliminated. there was a total of positions in the final dataset. evolutionary analyses were conducted in mega [ ] . sequences identified by genbank® accession numbers, abbreviation name of virus and its hosts, year and geographic origin of sampling. pabv- = parrot bornavirus . the sequences marked with a circle were produced during this study and triangles marked nucleotide sequences identified from wild birds. (accession nº lt . and mh . ) linked with lethal encephalitis of humans, on the evolutionary line (figures (b) and (a,b) ). although parrot bornavirus to (pabv- to ) are recognized as the etiologic agent of proventricular dilatation disease (pdd) in psittacines, there are still unanswered questions regarding its epidemiology. this is the first study that characterizes, at genetic and protein level, genotypes of psittaciform orthobornavirus, circulating in portugal. the sequencing of more than base pairs of the viral genome allowed the identification of parrot bornavirus (pabv- ) with statistically significant probability (percent identity (id) = ( . - . )%, figure . phylogenetic relationships between pabv- genotypes identified in the present study and bornaviruses detected in mammalian and in wild birds, based on n gene (a) and on n protein (b). the evolutionary history was inferred using the neighbor-joining method [ ] . the confidence probability (multiplied by ) was estimated using the bootstrap test ( replicates) and is shown next to the branches [ , ] . both trees were drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. both trees were rooted with a vaccine strain (accession nº dq . ). evolutionary analyses were conducted in mega [ ] . (a) the evolutionary distances were computed using the kimura two-parameter method [ ] e-value = . ), as the etiological agent associated with the death of two pet parrots (psittacus erithacus). none of the pabv- identified in portugal share % identity with genotypes reported so far, when the recovered genome segment with more than nucleotides was analysed. the complete p protein of both pabv- genotypes differs from proteins deposited in the genbank® database (id = ( . - . )%, e-value = e − - e − ). regarding x protein, one of the identified pabv- (accession nº b ) share % of identity (e-value = e − ) with genotypes detected in germany and sweden, according to genbank® database. the x protein of the other pabv- genotype differ from proteins deposited in genbank® database (id = . %, e-value = e − ). regarding the parrot b , the source of infection is unknown; however, we suspect that parrot bornavirus (pabv- ) entered via the olfactory pathway and migrated to brain [ ] , causing a lethal encephalitis; without neurological signs perceptible by the owner. the absence of clinical signs in bornavirus-infected parrots with brain lesions has previously been reported [ ] . therefore, in this case, probably the virus followed one of the postulated pathways, which consist of the centrifugal spread of virus from brain, passing down the spinal cord, affecting the parasympathetic and sympathetic nerves and the organs innervated by them (e.g. lung, heart, proventriculus and intestine) [ ] . concerning the figure . phylogenetic relationships between pabv- genotypes identified in the present study and bornaviruses detected in mammalian and wild birds, based on p gene (a) and on p protein (b). the evolutionary history was inferred using the neighbor-joining method [ ] . the confidence probability (multiplied by ) was estimated using the bootstrap test ( replicates) and is shown next to the branches [ , ] . both trees were drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. both trees were rooted with a vaccine strain (accession nº dq . ). evolutionary analyses were conducted in mega [ ] . (a) the evolutionary distances were computed using the kimura two-parameter method [ ] and are in the units of the number of base substitutions per site. the analysis involved nucleotide sequences. all positions containing gaps and missing data were eliminated. there were a total of positions in the final dataset (b). the evolutionary distances were computed using the poisson correction method [ ] and are in the units of the number of amino acid substitutions per site. the analysis involved amino acid sequences. all positions containing gaps and missing data were eliminated. there were a total of positions in the final dataset. sequences identified by genbank® accession numbers, abbreviation name of virus and its hosts, year and geographic origin of sampling. abbv- = aquatic bird bornavirus , abbv- = aquatic bird bornavirus , bodv- = borna disease virus , pabv- = parrot bornavirus , vsbv- = variegated squirrel bornavirus . the sequences marked with a circle were produced during this study and triangles marked nucleotide sequences identified from samples of wild birds. parrot b , the source of infection is unknown; however, it is probable that virus entered through oral route and spread from proventriculus to the central nervous system, as postulated in the literature [ ] , based on the clinical picture characterized by the development of gastrointestinal signs followed by neurological signs at the final stage of disease. in the present study, we produced additional and detailed information to the previous reported insights about the degree of conservation of x and p protein within and between species of bornaviruses [ ] . as previously reported, the p protein (phosphoprotein) showed to be more conserved between and within species of bornaviruses than the x protein [ ] . however, the present study showed that, within and between bornaviruses, the degree of conservation throughout the p protein was not constant (table ) . namely, intra-and inter-species of bornaviruses, the identity percent of the first amino acid was higher than the following amino acid of p protein ( table ). the region showing a higher identity percentage is a shared amino acid sequence with x protein. in contrast, throughout the x protein, the lower identity percent (intra-and inter-species of bornaviruses) comes from the region shared with p protein (table ) . therefore, the first amino acid of the sequence revealed to be more conserved within x protein (table ) . nevertheless, when comparing both conserved regions of the two proteins, the x protein showed a smaller variability range of the amino acid sequence (table ) . however, its most conserved region represents about . % of the x protein, which corresponds approximately to nucleotides of the gene, making it less advantageous as a target region (in the design of tools) for molecular diagnostics, compared to the more conserved region of the p protein. in the case of p protein, the most conserved region corresponds approximately to nucleotides, among species of family bornaviridae, allowing more options in the design of molecular tools, aiming to decrease the occurrence of false negatives. in some cases, despite all findings of clinical diagnosis, the inability to amplify the viral nucleic acid might be a consequence of the presence of unknown species of bornaviruses in samples [ ] . thus, a decrease in the number of false negatives is expected, since the highly conserved regions in evolutionarily more distant viruses will be more identical to the bornaviruses described so far. therefore, the highly conserved region is a good candidate as a target in laboratory diagnostics for screening purposes of potential reservoirs or asymptomatic carriers, as well as to confirm the clinical diagnosis. the information produced about the highly conserved region is thus useful, since the diversity of members of the family bornaviridae is expected to be higher than reported, considering the evolution of the scientific knowledge produced. in fact, between the end of twentieth century and the beginning of twenty-first century, the family bornaviridae comprised only the mammalian orthobornavirus species [ ] . from then to the present, the family bornaviridae includes species and strains [ ] . moreover, within each of the strains, there are several genotypes described, as, for example, the taxonomic group pabv- that integrates more than genotypes [ ] . the evidence produced by the phylogenetic analysis restricted to the parrot bornavirus (pabv- ) genotypes suggests that portugal is on the trade route of psittacines; because both genotypes cluster together with genotypes worldwide distributed and detected from captive parrots [ ] . over the years, the international trade, without biosafety measures [ ] , is expected to have had negative implications on biodiversity. whereas, in the last years (based on the oldest data collection ( figure )), bornavirus infection has contributed to the reduction of endangered specimens [ , ] . the psittacus erithacus is one of the endangered species (according to the international union for conservation of nature (iucn)) [ ] included in the list of the convention on international trade in endangered species of wild fauna and flora (cites) [ ] . in portugal, the lawful trade of endangered species of parrots is supervised by several national entities working with cites [ ] . the present study further considered parrot bornavirus (pabv- ) genotypes detected in wild birds (anas spp., emberiza spp. and grus spp.) and in pet psittacines of japan and thailand ( figure ), more inclusive and updated than in the previous study [ ] , resulting in a broader view of the geographic dimension of the psittacines trade. also, it suggests there are at least two factors (the wild birds and the world trade of psittacines) that, simultaneously or singularly, might be a factor for the dissemination of the pabv- infection. some of the pabv- genotypes detected in pet parrots are phylogenetically adjacent to some genotypes detected in wild bird samples ( figure ). in our analytical approach, it was not possible to include the genotypes of pabv- detected in wild parrots from brazil [ ] , because the nucleotide sequences were not available in the genbank® database. over the last years, the evidence that wild birds could be reservoirs of bornaviruses has been accumulated [ ] [ ] [ ] [ ] [ ] . in addition to the parrot bornavirus , cases of infection in wild birds with several genotypes of parrot bornavirus (such as anas spp., grus spp., haliaeetus spp., larus spp., galidris spp., calidris spp. and emberiza spp.), borna disease virus (such as anas spp., and coloeus spp.) and aquatic bird bornavirus to (such as cygnus spp., greylag spp., canda spp. and anas spp.) have been reported [ ] [ ] [ ] [ ] [ ] . the co-infection was reported to occur in wild birds by different strains and genotypes belonging to psittaciform orthobornavirus [ ] . it could then be expected that all members of the family bornaviridae could be involved in co-infection events, since there are species of wild birds that may be hosting a wide range of genotypes from several species of bornaviruses (such as waterbird orthobornavirus [ ] [ ] [ ] , psittaciform orthobornavirus [ ] and mammalian orthobornavirus [ ] ). therefore, co-infected reservoirs can play a role in the emergence of new virus variants, providing conditions for the occurrence of changes in virulence and in host range by two major ways: through recombination exchange between virus strains and through accumulation of amino acid changes that are under pressure by the immune system (antigenic drift event). both events have been reported to occur within viruses belonging to the order mononegavirales [ ] [ ] [ ] [ ] [ ] . a previous study reported findings of borna disease virus (bodv- ) genome segments in samples from wild birds (such as anas spp. and coloeus spp.) in uppsala, sweden, which showed an intra-species divergence [ ] . namely, they revealed to be divergent from each other, as they were divergent from the borna disease virus (bodv- ) genotypes detected from naturally infected cat, lynx and horse in the same area [ ] . in the present study, the bioinformatics approach suggests that the n and p genes of parrot bornavirus (pabv- ) have had different ancestors (figure (a,b); and (a,b) ). namely, within pabv- genotypes, the n gene showed to be evolutionarily close to avian bornaviruses (members of waterbird orthobornavirus) and p gene revealed to be closer to mammalian bornaviruses (members of mammalian orthobornavirus) (figures and ) . moreover, the phylogeny conducted based on the p gene segment revealed that the mammalian orthobornavirus members (vsbv- (accession nº ln . and mf . )) associated with lethal encephalitis of humans in germany are evolutionarily closest related to pabv- (figure (a) ). on the other hand, the mammalian orthobornavirus genotypes associated with death of humans in germany, (accession nº mh . and lt . ), are evolutionarily closer to bornaviruses detected in wild birds (such as abbv- , abbv- and bodv- in denmark, germany, sweden and usa) (figure (a) ). furthermore, the borna disease virus (bodv- ) variants detected in samples of wild anas spp. (accession nº af . and af . ) are phylogenetically adjacent to bodv- genotypes associated with lethal outcomes in humans (accession nº lt . and mh . ), as well as to the vaccine strain (accession nº dq . /rabbit, germany, ) (figure (a) ). in fact, the borna disease virus (bodv- ) detected in samples of wild anas spp. are intermediate genotypes, positioned in the evolutionary line, between the two genotypes that were the cause of death of humans (accession nº mh . and lt . ) (figure (a) ). when the analysis was conducted based on the amino acid sequence encoded by the p gene segment, the main phylogenetic relationship remained between aquatic bird bornavirus (abbv- to ), parrot bornavirus (pabv- ), variegated squirrel bornavirus (vsbv- ) and borna disease virus (bodv- ) genotypes (figure (b) ). however, their evolutionary relationship changed when the phylogenetic analysis was based on the segment of the n gene (figure (a) ). the findings revealed that the n gene of pabv- genotypes shared their ancestor with avian bornavirus (abbv- and abbv- ) detected in wild birds ( figure (a) ). furthermore, the parrot bornavirus (pabv- ) and variegated squirrel bornavirus (vsbv- ) genotypes diverged earlier in the evolutionary line, regarding n gene (figure (a) ). however, parrot bornavirus (pabv- ) remains evolutionarily closer to variegated squirrel bornavirus (vsbv- ) than to the borna disease virus (bodv- ) detected in samples of wild birds (accession nº af . , af . and af . ) and in human tissues (accession nº mh . and lt . ) (figure (a) ). interestingly, from an evolutionary point of view, the borna disease virus (bodv- ) detected in wild birds remains as intermediate variants between the two genotypes linked to the death of humans, based on their nucleoprotein profile ( figure (b) ). in addition, wild birds can also disseminate viruses along their migration routes [ , ] . according to published studies, the flow of migratory birds occurs within and between the four main migratory routes (pacific north of america/atlantic north of america, east asian/australian, central asia and black sea/ mediterranean) [ , ] , covering the geographical regions where the species waterbird orthobornavirus (e.g. abbv- ), mammalian orthobornavirus (e.g. bodv- strains) and psittaciform orthobornavirus (e. g. pabv- and pabv- strains) were detected in samples of anas spp [ , , ] . particularly the anas spp. are considered long-distance transporters of virus, within and between continents [ , ] . therefore, more screening studies of some species of wild birds may be promising to understand the contribution of these species in the epidemiology of bornaviruses. in conclusion, this is the first study characterizing at genetic and protein level that parrot bornavirus (pabv- ) is circulating in portugal. the characterized viruses were phylogenetically related with pabv- worldwide distributed in captive psittacines, suggesting that portugal is on the route of the international trading, which occurs without biosafety measures. moreover, the pabv- genotypes detected in portugal are evolutionarily closer to some genotypes found in wild birds in japan, than with some of those found in pet psittacines in europe. more studies are needed to clarify the role of wild birds in the introduction of bornaviruses to captive parrots. the n and x proteins of pabv- are more related with avian bornaviruses detected in samples of wild migratory waterbirds, while phosphoprotein is phylogenetically closer to mammalian bornaviruses, as the variegated squirrel bornavirus (vsbv- ) genotypes linked to lethal encephalitis of humans. wild migratory birds may be a key element in understanding the intraand inter-continental emergence and dissemination of pathogenic variants of mammalian and bird bornaviruses. the role of co-infected wild reservoirs in the emergence of new virulent variants and in the host range modification should be addressed in future studies. for screening purposes, the highly conserved p gene/ protein region is a good candidate as universal target in laboratory diagnostics of bornaviruses. no potential conflict of interest was reported by the authors. borna disease virusfact and fantasy distribution of viral antigen and inflammatory lesions in the central nervous system of cockatiels (nymphicus hollandicus) experimentally infected with parrot bornavirus detection and phylogenetic analysis of parrot bornavirus identified from a swedish blue-winged macaw (primolius maracana) with unusual nonsuppurative myositis taxonomy of the order mononegavirales: update analysis of exotic squirrel trade and detection of human infections with variegated squirrel bornavirus borna disease virus infection in racing horses with behavioral and movement disorders markers of borna disease virus infection in cats with staggering disease serological markers of bornavirus infection found in horses in iceland borna disease virus infection in cats borna disease in ostriches sero-epidemiological analysis of vertical transmission relative risk of borna disease virus infection in dairy herds a variant form of feline borna disease pathology and diagnosis of avian bornavirus infection in wild canada geese (branta canadensis), trumpeter swans (cygnus buccinator) and mute swans (cygnus olor) in canada: a retrospective study characterization of a new genotype of avian bornavirus from wild ducks aquatic bird bornavirus in wild geese wild birds as a possible natural reservoir of borna disease vírus parrot bornavirus- and − rna detected in wild bird samples in japan are phylogenetically adjacent to those found in pet birds in japan the pathogenesis of proventricular dilatation disease divergent bornaviruses from australian carpet pythons with neurological disease date the origin of extant bornaviridae prior to the end-cretaceous extinction mega : molecular evolutionary genetics analysis version . for bigger datasets the neighbor-joining method: a new method for reconstructing phylogenetic trees estimating errors and confidence intervals for branch lengths in phylogenetic trees by a bootstrap approach a simple method for estimating and testing minimum evolution trees a simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences evolutionary divergence and convergence in proteins taxonomy of the order mononegavirales: update the iucn red list of threatened species the cites species. cites appendices i, ii and iii. convention on international trade in endangered species of wild fauna and flora (cites) convention on international trade in endangered species of wild fauna and flora (cites) avian bornavirus in free-ranging psittacine birds generation and characterization of a recombinant newcastle disease virus expressing the red fluorescent protein for use in co-infection studies antigenic drift defines a new d subgenotype of measles virus evolution of subgroup a respiratory syncytial virus: evidence for progressive accumulation of amino acid changes in the attachment protein a possible occurrence of genome reassortment among bipartite rhabdoviruses homologous recombination is a force in the evolution of canine distemper virus avian influenza virus, streptococcus suis serotype , severe acute respiratory syndrome-coronavirus and beyond: molecular epidemiology, ecology and the situation in china anas platyrhynchos) in the spread of avian influenza: genomics, population genetics, and flyways key: cord- - ahxau k authors: lu, yujie; liu, wenjuan; zhang, man; deng, yanfang; jiang, min; bai, gang title: the screening research of nf-κb inhibitors from moutan cortex based on bioactivity-integrated uplc-q/tof-ms date: - - journal: evid based complement alternat med doi: . / / sha: doc_id: cord_uid: ahxau k inflammation is a common and important pathological process, and nuclear factor-κb (nf-κb) is a key mediator of it. moutan cortex (mc), the dried root cortex of paeonia suffruticosa andr., is widely used as a remedy for the treatment of inflammatory diseases in asian region. however, there are few studies on the systematic identification of nf-κb inhibitors of mc. in this study, the effect of inhibiting nf-κb activation of mc was assessed at the cellular level using a tumor necrosis factor-α (tnf-α) induced inflammatory model. subsequently, ultra-performance liquid chromatography-quadrupole/time of flight-mass spectrometry (uplc-q/tof-ms) combined with biological activity assay was established to screen and identify potential anti-inflammatory ingredients in mc. the results revealed that mc significantly inhibited the activation of nf-κb. seven potential nf-κb inhibitors were screened from mc, including oxypaeoniflorin, paeoniflorin, galloylpaeoniflorin, benzoyloxypaeoniflorin, mudanpioside c, gallic acid, and paeonol. among them, the nf-κb inhibitor activity of galloylpaeoniflorin, benzoyloxypaeoniflorin, and mudanpioside c is first reported here. in conclusion, the anti-inflammatory activity of mc was associated with the seven components mentioned above. and the bioactivity-integrated uplc-q/tof which contains both chemical and bioactive details is suitable for screening active ingredients from natural medicines. inflammation is a multicomponent response to injury, tissue stress, and infection, associated with most diseases, and it can occur in various tissues and organs of the organism [ ] . clinically, there are two main categories of anti-inflammatory drugs: steroidal drugs and non-steroidal anti-inflammatory drugs (nsaids). steroidal drugs generally refer to adrenocortical hormones, which have strong anti-inflammatory effect, but have obvious side effects such as water-sodium retention, puffiness, and osteoporosis [ ] . and nsaid is one of the most widely used drugs in the world and are mainly classified into salicylates, propionic acids, indoles, fenamic acids, acetic acids and pyrazolones [ ] . however, it has potential cardiovascular and gastrointestinal bleeding risks [ ] . due to the strong toxic and side effects of many synthetic drugs, and because traditional chinese medicine (tcm) has the advantages of abundant resources, definite therapeutic effectiveness, and fewer side effects, the discovery of novel anti-inflammatory drugs from natural compounds has gradually become a research hotspot [ ] . moutan cortex (mc) is the dried root cortex of paeonia suffruticosa andrews and commonly used for removing blood stasis, dredging meridian, expelling pus, and eliminating inflammation in tcm prescriptions [ , ] . some pharmacological studies have showed that mc could inhibit the production of no and tumor necrosis factor-(tnf-) induced by lipopolysaccharide/recombinant interferon-(lps/rifn-) [ ] . in animal experiments, mc could significantly decrease the level of cytokines including interleukin- (il- ), macrophage inflammatory peptide- (mip- ), il- , and il- , which apparently inhibited lps-induced acute lung injury in rat models [ ] . available evidence revealed that mc had the potential nf-b inhibitory activity and clinical anti-inflammatory efficacy; however, previous studies primarily focused on the single component, and the integral evidence-based complementary and alternative medicine active components and their mechanisms of action have not yet been fully clear. therefore, we mainly conducted a systematic study of the potential nf-b inhibitors in mc. in the current study, dual-luciferase reporter assay integrated uplc-q/tof-ms was utilized to screen out potential nf-b inhibitors of mc extract. and the anti-inflammatory activity of some components was confirmed through in vitro experiments. additionally, the mechanism of active compounds was predicted through network pharmacology methods. ) , ultrasonic extraction of min at ∘ c, centrifuge and collect the supernatant. the extract was dried into powder by vacuum freeze-drying. culture. the hek cells were purchased from american type culture collection (rockville, md, usa) and were cultured in dmem high glucose with % fbs, iu/ml penicillin, and mg/ml streptomycin at ∘ c and % co in a thermostatic incubator. beas- b, derived from human bronchial epithelial cells, was also purchased from the american type culture collection (rockville, md, usa). their culture conditions were the same as those of hek cells, except for dmem/f- (ham) : instead of dmem high glucose. group, and mc-l ( . mg/ml) group. all the cells except for cells in the control group were stimulated with tumor necrosis factor-(tnf-, ng/ml) and simultaneously given with drugs for h. among them, dex was used as a positive control and could decrease the expression of nf-b in hek cells. relative content of nf-b in each group was represented as relative light unit (rlu) ratio following the manufacturer's instructions. a waters acquity uplc system (waters co., milford, ma, usa) equipped with a photo diode array detector (pdad) was used to analyze samples. the chromatographic column was an acquity beh c -column ( . mm × mm, . m; waters co.). the flow rate was . ml/min and the column temperature was maintained at ∘ c. the test sample injection volume was l. the optimal mobile phase was composed of a ( . % formic acid in water) and b (acetonitrile) and the ratio was as follows: - . min, % to % b; . - . min, % to % b; . - . min, % to % b; . - . min, % to % b; . - . min, % to % b; . - . min, % to % b; . - . min, % to % b; . - . min, % to % b; . - . min, % to % b. accurate mass and ms/ms measurements were performed by a waters q/tof micro synapt high definition mass spectrometer (waters ms technologies, manchester, uk) with a dual electrospray ionization (esi) system. the optimal analytical condition was set as follows: the source temperature was ∘ c; the capillary voltage was . kv in positive ion mode and . kv in negative ion mode; the sample and extraction cone voltage were v and . v, respectively; the flow rate of the desolvation gas was l/h at a desolvation temperature of ∘ c; the cone gas flow was l/h. the ms spectra scanning range in the wide-pass mode was da to da. leucine enkephalin amide acetate (lea, ng/ml) was used as the lock mass ([m+h] + = . , [m-h] + = . ). and a flow rate was set at l/min. based on the ms/ms information, peaks of interest were confirmed by the molecular weight and structure of the contained constituent. some peaks with the similar ms/ms information could be identified by their different retention behaviors. the freeze-dried powder of mc extract was accurately weighed and ultrasonic dissolved in methanol ( mg/ml). after column separation, the % fractions were transported to the q-tof/ms system for components identification and the % fractions were collected every . min into a deep -well plate and vacuum dried at ∘ c. the residues were dissolved in dmem ( l) for dual-luciferase assay. the operation process was the same as above. screening out fractions that could reduce the level of nf-b was performed for further structural analysis. activity. dualluciferase assay system was used to verify the antiinflammatory activity of the monomeric compounds. the operation steps were the same as above. additionally, evidence-based complementary and alternative medicine human il- and il- elisa kits were used, respectively, to measure the concentrations of inflammatory factors (il- and il- ) in the culture supernatants of beas- b cells after the stimulation of drugs and tnf-. the absorbance of each sample was measured at nm using a bio-rad model microplate reader. data processing followed the instructions of elisa kits. analysis. the test results were represented with mean ± sem. and t-test was used for comparison of significant differences among different groups. spss v. . statistical analysis software (spss inc., chicago usa) was used for statistical analysis. results with values of p < . were considered statistically significant. to verify the effects of mc on anti-inflammatory, the level of nf-b in tnfinduced hek cells was investigated using a dualluciferase reporter assay system. as shown in figure , the different doses of mc (mc-l, . mg/ml; mc-m, . mg/ml; mc-h, mg/ml) not only significantly inhibited nf-b production, but also showed a dose-dependent inhibition (p< . ). the result confirmed that mc contained some components with potential anti-inflammatory activity. to identify the anti-inflammatory components in mc, we performed a dual-luciferase reporter assay integrated uplc-q/tof-ms. representative chemical components of mc and the total ion current chromatograms in positive and negative esi modes, respectively, are shown in figures (a) and (b) . in total, seven fractions showed potential nf-b inhibitor activity (figure (c) ). we analyzed the seven fractions, and compounds they contain were identified by exact molecular weights and diagnostic fragment ions (figure (d) ). the detailed results and ms/ms information were shown in table . the seven potential nf-b inhibitors could be classified into two types according to their chemical structures: monoterpenes (oxypaeoniflorin, paeoniflorin, ( ) gallic acid ( ) galloylpaeoniflorin, benzoyloxypaeoniflorin, mudanpioside c) and phenolic acids (gallic acid, paeonol). among them, the nf-b inhibitory activities of galloylpaeoniflorin, benzoyloxypaeoniflorin, and mudanpioside c are first reported here. we then tested and verified the anti-inflammatory activity of these monomers. compounds. oxypaeoniflorin, galloylpaeoniflorin, benzoyloxypaeoniflorin, and mudanpioside c have the same structural characteristics, all of which have paeoniflorin as the mother nucleus structure. since the generation of drug efficacy mainly depends on their chemical structure, we selected paeoniflorin, the mother nucleus of the four compounds, as a representative component to study their anti-inflammatory activity. for verification of nf-b inhibitory activity, varying concentrations ( − mol/l, − mol/l, and − mol/l) of three ingredients (gallic acid, paeoniflorin, and paeonol) were chosen for dual-luciferase reporter assay. as shown in figure , gallic acid, paeoniflorin, and paeonol all showed significant nf-b inhibitory effects. for further verification of anti-inflammatory activity, gallic acid, paeoniflorin, and paeonol with the same concentration the transcription factor nf-b acts a key role in the process of immune response and it can usually be activated when exposed to inflammatory cytokines such as tnf-, viral infection, ultraviolet irradiation, and other physiological and nonphysiological stimuli [ ] [ ] [ ] . however, the activated nf-b signaling pathway participates not only in immune regulation and inflammation, but also in infection, cell cycle regulation, cell differentiation, and apoptosis [ ] [ ] [ ] . if the activation cannot be eliminated in time, it may lead to serious pathological reactions, such as rheumatoid arthritis, systemic lupus erythematosus, septic shock, atherosclerosis, and cancer [ ] [ ] [ ] . consequently, inhibitors of nf-b activation are of primary significance in protecting cells from the potential damage of inflammation. based on the efficient separation and analysis functions of uplc-q-tof-ms and cell biological method, seven compounds with nf-b inhibitory activity were screened from mc. among them, gallic acid could inhibit nf-b activation by prevention of rela acetylation [ ] . and paeoniflorin could significantly inhibit nf-b by reducing the expression of the phosphorylation of i b and p [ ] . additionally, paeonol could suppress nf-b signaling through blocking mapk/p signaling pathway [ , ] . furthermore, oxypaeoniflorin could inhibit the elevation of the expression levels of nf-b although the mechanism remained unclear [ ] . additionally, nf-b inhibitory activity of galloylpaeoniflorin, benzoyloxypaeoniflorin, and mudanoside c has not been reported in previous studies. in the present study, those three compounds could inhibit the activation of nf-b induced by tnf-a and could be considered as novel nf-b inhibitors. in general, the efficacy of drugs depends mainly on their chemical structures [ ] . according to our results, other monoterpenoids with paeoniflorin as the core structure may also have nf-b inhibitory activity, which can be used as a lead compound for the study of innovative drugs. furthermore, the results demonstrated that the antiinflammatory activity of mc was related to the process of various components acting on multiple targets ( figure ) , which was consistent with the characteristics of tcms with multiple components, multiple pathways, and multiple targets [ , ] . in addition to its anti-inflammatory effects, mc also has cytotoxicity to cancer cells. recent research has showed that mc extract could reduce cell viability with ic within ∼ mg/ml in bladder cancer cells [ ] . and after treatment for h with paeonol ( g/ml), one of the active ingredients of mc, the ratio of apoptotic cells reached . % [ ] . this study demonstrated that, at a concentration of . mg/ml, the extract of mc already had anti-inflammatory activity. taking paeonol as an example, it showed significant nf-b inhibitory effects at a concentration of − mol/l, which was much lower than its toxic content. this finding was in agreement with the characterization of most drugs as playing a therapeutic role in a certain dose range. the quality marker (q-marker) representing the quality of tcm should not only take the content of certain components as an index, but also be able to reflect its efficacy [ , ] . consequently, determining the content of paeonol as the only approach to evaluate the quality of mc in chinese pharmacopeia is unilateral. according to our results, oxypaeoniflorin, paeoniflorin, galloylpaeoniflorin, benzoyloxypaeoniflorin, mudanpioside c, gallic acid, and paeonol were related to the anti-inflammatory effect of mc and could be considered as a reference standard for evaluating the quality of mc. in conclusion, mc showed significant efficacy in inhibiting nf-b activation, and seven bioactive components were screened by a dual-luciferase reporter assay integrated uplc-q/tof-ms. according to their structural characteristics, the potential nf-b inhibitors could be categorized into two types: monoterpenes (oxypaeoniflorin, paeoniflorin, galloylpaeoniflorin, benzoyloxypaeoniflorin, mudanpioside c) and phenolic acids (gallic acid, paeonol). thereinto, galloylpaeoniflorin, benzoyloxypaeoniflorin, and mudanpioside c were first reported to the effects of inhibiting the activation of nf-b. the present study demonstrates that mc contains a variety of structurally diverse anti-inflammatory active ingredients, acting on different targets, which provides a basis for the discovery of novel anti-inflammatory drugs with fewer side effects. and this article may provide a useful reference for improving the quality standards of mc in the future. additionally, these experimental results also showed that the bioactivity-integrated uplc-q/tof which contain both chemical and bioactive details is suitable for screening active ingredients from natural medicines. the data used to support the findings of this study are included within the article. the authors declare that they have no conflicts of interest. transcriptional control of the inflammatory response consensus document on treatment of type diabetes in patients with chronic kidney disease nonsteroidal antiinflammatory drugs nsaids nsaids and risk of lower gastrointestinal bleeding osthole: a promising lead compound for drug discovery from a traditional chinese medicine (tcm) 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function t cell receptor/carma / nf-b signaling controls t-helper (th) differentiation nf-b directly regulates fas transcription to modulate fas-mediated apoptosis and tumor suppression genetic interaction between genes involved in nf-b signaling pathway in systemic lupus erythematosus negative regulation of nf-b and its involvement in rheumatoid arthritis suppression of cyclooxygenase- and inducible nitric oxide synthase expression by epimuqubilin a via ikk/i b/nf-b pathways in lipopolysaccharide-stimulated raw . cells gallic acid suppresses lipopolysaccharide-induced nuclear factor-b signaling by preventing rela acetylation in a lung cancer cells paeoniflorin attenuates pressure overload-induced cardiac remodeling via inhibition of tgf /smads and nf-b pathways paeonol attenuates isoflurane anaesthesia induced hippocampal neurotoxicity via modulation of jnk/erk/p mapk pathway and regulates histone acetylation in neonatal rat anti-inflammatory and antioxidative activities of paeonol and its metabolites through blocking mapk/erk/p signaling pathway antioxidative and antiinflammatory activities of paeoniflorin and oxypaeoniflora on ages-induced mesangial cell damage chemical structure and concentration of intratumor catabolites determine efficacy of antibody drug conjugates the pathological effects of sleep deprivation on coronary heart disease and treatment using chinese medicine tranquilization comparison of efficacy and toxicity of traditional chinese medicine (tcm) herbal mixture lq and conventional chemotherapy on lung cancer metastasis and survival in mouse models antitumor effect of extracts from moutan cortex on dld- human colon cancer cells in vitro promotion of quality standard of chinese herbal medicine by the integrated and efficacy-oriented quality marker of effect-constituent index discovery of quality control markers from traditional chinese medicines by fingerprintefficacy modeling: current status and future perspectives this work was supported by a grant from the national natural science foundation of china (no. ). key: cord- -yyzbv ys authors: arslan, mehboob; yang, xin; santhakumar, diwakar; liu, xingjian; hu, xiaoyuan; munir, muhammad; li, yinü; zhang, zhifang title: dynamic expression of interferon lambda regulated genes in primary fibroblasts and immune organs of the chicken date: - - journal: genes (basel) doi: . /genes sha: doc_id: cord_uid: yyzbv ys interferons (ifns) are pleiotropic cytokines that establish a first line of defense against viral infections in vertebrates. several types of ifn have been identified; however, limited information is available in poultry, especially using live animal experimental models. ifn-lambda (ifn-λ) has recently been shown to exert a significant antiviral impact against viral pathogens in mammals. in order to investigate the in vivo potential of chicken ifn-λ (chifn-λ) as a regulator of innate immunity, and potential antiviral therapeutics, we profiled the transcriptome of chifn-λ-stimulated chicken immune organs (in vivo) and compared it with primary chicken embryo fibroblasts (in vitro). employing the baculovirus expression vector system (bevs), recombinant chifn-λ (rchifn-λ ) was produced and its biological activities were demonstrated. the rchifnλ induced a great array of ifn-regulated genes in primary chicken fibroblast cells. the transcriptional profiling using rna-seq and subsequent bioinformatics analysis (gene ontology, differential expressed genes, and keggs analysis) of the bursa of fabricious and the thymus demonstrated an upregulation of crucial immune genes (viperin, ikkb, ccl , il β, and ap ) as well as the antiviral signaling pathways. interestingly, this experimental approach revealed contrasting evidence of the antiviral potential of chifn-λ in both in vivo and in vitro models. taken together, our data signifies the potential of chifn-λ as a potent antiviral cytokine and highlights its future possible use as an antiviral therapeutic in poultry. viral pathogens pose significant threats to the poultry industry around the globe. this necessitates the development of novel and alternative antiviral therapies to contain the impacts of pathogens. avian influenza viruses (aivs) are a particular threat, which cause severe damage to the poultry industry, especially in developing countries where huge monetary losses are incurred [ , ] . public health is also threatened by aivs, owing to their zoonotic importance. active preventive strategies would minimize the risk of viral transmission to humans and also benefit the poultry industry. interferons (ifns) are pleiotropic functional cytokines with antiviral, antitumor, and natural immune-boosting effects. ifns play a significant role in eliciting an antiviral state in vertebrates [ ] . ifns are broadly categorized into three distinct types based on their molecular structure, receptor specificity, and induction pathway [ ] . type i ifns include ifn-α, ifn-β, ifn-ε, ifn-κ, and ifnω, and all signal via common cell surface receptors (ifnαr- ) and (ifnαr- ), which are situated on a broad range of cells [ ] . type ii ifns consist of ifn-γ, which is activated through highly specific ligand interactions with distinct ifn-γ receptors (ifn-γr ) and (ifn-γr ). the third family of ifns consists of ifn lambda, which interacts with a heterodimeric receptor complex (il- rα and il- β). ifn-λ was first discovered in mammals and subdivided into ifn-λ (also known as il- ), ifn-λ (il- a), ifn-λ (il- b), and ifn-λ [ ] . ifns are crucial in an innate immune response, as their expression and antiviral potential is dependent on their cognate receptor interaction in a particular system [ ] . in chickens, type i ifns primarily interact in fibroblasts, whereas epithelial cells (gastrointestinal and respiratory tract) are the primary site for the actions of type iii ifns [ ] . despite morphological diversity, ifns share integrated, interconnected, and a precisely coordinated cascade in immunity pathways [ ] . ligand recognition and interaction by ifn receptors results in rapid activation of janus kinase/signal transducers and activators of transcription (jak-stat pathway). this leads to phosphorylation of stat and stat , activation of interferon stimulated gene factor (isgf ), binding of ifn-stimulated response elements (isres), and expression of ifn stimulated genes (isgs) [ ] . once expressed, these isgs demonstrate an essential role in the antiviral response. it is evident from published data that ifns upregulate identical sets of isgs, which in turn express antiviral proteins. ifn-induced transmembrane protein (ifitms), viperin and myxovirus resistance protein (mx) are some of the potent antiviral proteins expressed in response to viral infections [ ] . once expressed, these isgs control viral replication, which provides an antiviral atmosphere to limit viral propagation in infected cells. compared to the mammalian ifn-λ repertoire (ifn-λ , ifn-λ , ifn-λ , and ifn-λ ), chicken ifn-λ is the sole member in birds and demonstrates structural identity with human ifn-λ . ifn-λ is chiefly involved in protection against viral infection of the respiratory and gastrointestinal tract epithelia (aiv, ndv, ibv), and due to the distribution of il- rα in epithelium-rich organs, ifn-λ demonstrates significant potential to limit viral propagation [ ] . while most of the current studies in chickens are mainly focused on type i and type ii ifns, we investigated the potential of type iii ifns in innate and adaptive immunity. previously, it was established that chifn-α presented a significant delay in the propagation of rous sarcoma virus and confirmed in vivo [ ] . it was also revealed that chifn-α treatment ameliorates infection progression in experimental chickens with highly pathogenic influenza a virus (hpaiv) subtype h n [ ] . compared to type i ifns, chifn-λ has also been shown to elicit moderate antiviral response in both the chicken macrophage cell line hd and primary chicken embryo fibroblasts (cef) [ ] . another published study demonstrated that cefs treated with recombinant chifn-λ induced isgs in a temporal fashion [ ] . however, the antiviral potential of chifn-λ in live animals (e.g., chickens) has not yet been investigated, which could provide evidence for the potential of chifn-λ in animals per se. to investigate the impact of exogenous chifn-λ on the innate immune system in chickens, we first expressed chifn-λ in a silkworm bioreactor platform utilizing a baculovirus expression vector system (bevs) [ ] . compared to the autographa californica nucleopolyhedrovirus (acmnpv)-sf cell expression system, the bombyx mori nucleopolyhedrovirus (bmnpv)-silkworm system possesses greater post-translational modifications and enhanced expression efficiency [ , ] . comparative transcriptomic profiling revealed the key mechanisms, signaling pathways, and expression patterns of genes involved in interferon-induced immunity. our results highlight the dynamics of chifn-λ roles in chicken innate immunity. bm cells (bombyx mori-derived cell line) were cultured and maintained at • c with % fetal bovine serum (fbs, gibco, usa) in tc (insect cell culture medium) (applichem, darmstadt, germany) as per the published literature [ ] . for co-transfection, bm cells were cultured at a constant density of × cells per well in six well plates for hours with tc media containing fbs. tc media without fbs was used to wash the cells twice and a mixture of transfection and co-transfection was introduced to cells. between - h post-transfection, fbs was introduced to the cell culture media. for viral amplification and expression, cells were infected with a multiplicity of infection (moi) of . for - h. the ensembl chicken genome database (ftp://ftp.ensembl.org/pub/release- /fasta/gallus_ gallus/dna/) was extensively screened for homologues of chifn-λ by employing the blast algorithm (http://www.ncbi.nlm.nih.gov/blast/). a stretch of sequences demonstrating high sequence identity was identified and characterized. ] were acquired from the national center for biotechnology information (ncbi) and aligned using the clustalw program, and phylogenetic analysis was performed using the neighbor-joining method with bootstrap n = in mega software (version ). amino acid sequences of ifn-λ from multiple species were aligned using the clustalw algorithm. the espript . (http://espript.ibcp.fr/espript/cgi-bin/espript.cgi) was utilized to analyze the sequences. in our previous study, we developed a novel defective-rescue recombinant bombyx mori bacmid (rebmbac) expression system [ ] . we used this in-house built and developed system to express chifn-λ. the rebmbac-silkworm expression system was employed to construct chifn-λ (interferon lambda- [gallus gallus]; sequence id: xp_ . ; length: ). briefly, in order to enhance expression efficiency by codon optimization, chifn-λ genes were optimized for expression in the silkworm (bombyx mori) and synthesized by genscript company (china). plasmid-containing orf + with gene of interest (chifn-λ) and pph as a promoter was co-transfected with rebmbac in the bm cell line. recombinant virus containing the chifn-λ gene was harvested - days post co-transfection. expression product was acquired after - days of silkworm/pupae infection. the plaque assay was performed to evaluate the recombination efficiency [ ] . luciferase assay kit (promega, usa) was employed to analyze expression quantity of luciferase in µg of protein lysate. the bradford method was used to measure the amount of protein [ ] . antiviral activity of chifn-λ was assayed in the gfp-reduction assay using recombinant vesicular stomatitis virus (vsv-gfp) [ ] . cefs were prepared from - days old specific pathogen free (spf) chicken eggs and maintained in cell culture flasks [ ] . after hours, cefs were stimulated with chifn-λ and cells were harvested after hours post treatment, snap frozen, and stored at − • c for further processing. all experiments were performed in triplicate. the present study was conducted in accordance with animal ethics guidelines and approval was given by the beijing administration office of laboratory animals, china. a total of newly hatched spf chicks were obtained from beijing arbor acre company ltd., p.r. china. chicks were reared in cages (n = birds/cage) and placed in six cages in a temperature-controlled environment at the biotechnology research institute, chinese academy of agricultural sciences (caas), p.r. china. birds were offered standard commercial feed obtained from cp group ltd., p.r. china. unrestricted access to water was provided via nipple drinker lines and ad libitum feed was offered. a treatment group of -day old chicks were injected daily with chifn-λ ( , iu/kg body weight) ( iu/ml). phosphate buffer saline (pbs) was injected intramuscularly to the control group. the bursa of fabricious and thymus were obtained by euthanizing the chickens at five days post-treatment. tissue samples were rapidly collected, snap-frozen in liquid nitrogen, and stored at − • c for further processing. total rna was extracted from virus-infected or mock-treated cefs (in triplicates), as per manufacturer's guidelines [ ] . similarly, a total of five immune organs (bursa of fabricious and thymus) were pooled (in duplicates) from randomly selected chicken from each virus-or mock-infected group. total rna extraction was performed as per manufacturer's instructions [ ] . extracted rna quality was analyzed by employing % agarose gel and rna integrity was assured using rna nano assay kit from bioanalyzer system (agilent technologies, ca, usa). extracted samples were sent to novogene beijing for sequencing. samples were sequenced using hiseq x ten (ilumina) and pe platforms. rna-seq generated from cef, bursa of fabricious and thymus samples of chicken (both chifnλ-treated and control groups) are presented in supplementary table s . reads were mapped to the reference genome database (ftp://ftp.ensembl.org/pub/release- /fasta/gallus_gallus/dna/). individually mapped reads for each sample were assembled by stringtie (v . . b) using a reference-based approach. featurecountsv . . -p was utilized to estimate read numbers mapped to each gene. fragments per kilo base of transcript sequence per million base pairs sequenced (fpkm) of each gene was analyzed on the basis of length of gene and read count mapped to this gene. differential expression analysis was accomplished by employing deseq r package ( . . ). using benjamini and hochberg's approach, p-values were adjusted for controlling false discovery rate (fdr). genes with (p < . , |log fold change|> ) observed by deseq were designated as differentially expressed. for differentially expressed genes, both gene ontology (go) enrichment analysis and kyoto encyclopedia of genes and genomes (kegg) pathway enrichment was conducted using the clusterprofiler r package. go terms with adjusted p-values < . were considered as significantly enriched (http://www.genome.jp/kegg/). using the chicken ifn gene as a query, we constructed the phylogenetic tree by employing the neighbor joining method (bootstrap n = ). this demonstrates the relationship of chifnλ with its mammalian orthologues by illustrating that chifn-λ is distinct in its evolution. furthermore, this revealed the contrasting consensus sequence from databases including ensembl and genbank. chifn-λ encodes a putative protein of amino acids and further demonstrates typical characteristics of type iii ifns. a pairwise blast analysis demonstrated that chifn-λ shares %, %, %, % and % sequence similarity with recently characterized pig, mouse, human, cattle, and frog ifn-λ, respectively. based on amino acid homology, conserved amino acids among distinct avian and mammalian ifn-λ are identified. taken together, this comparative characterization further shows that chifn-λ shares characteristic features of type iii ifns (supplementary figure s a ,b). in order to construct chifn-λ, we employed a bevs study. in order to determine the expression efficiency, we used a luciferase reporter gene for quality control as we described previously [ ] . the luciferase gene was acquired from pgl -basic vector by employing bglii/xbai digestion and insertion into the bamhi/xbai-digested pvl vector to construct pvl -luc vector. a combination of pvl -luc and rebmbac dna was co-transfected in bm cells (figure ) . a viral plaque assay was used to determine a suitable virus strain with which to express luciferase. supernatant from bm cells containing recombinant bmnpv (rebm-luc) was harvested five days post-transfection before inoculation into silkworms. after four to five days, protein was harvested from silkworms and µg protein from lysed larval haemolymph was subjected to luciferase assays. luminescence detected from silkworm larval haemolymph was approximately . ± . × relative light units (rlu), compared to - rlu from luc-negative virus-infected samples. pcr amplification (qpcr) further verified and validated the chifn-λ gene expression in bevs (supplementary figure s c ). in order to construct chifn-λ, we employed a bevs study. in order to determine the expression efficiency, we used a luciferase reporter gene for quality control as we described previously [ ] . the luciferase gene was acquired from pgl -basic vector by employing bglii/xbai digestion and insertion into the bamhi/xbai-digested pvl vector to construct pvl -luc vector. a combination of pvl -luc and rebmbac dna was co-transfected in bm cells (figure ) . a viral plaque assay was used to determine a suitable virus strain with which to express luciferase. supernatant from bm cells containing recombinant bmnpv (rebm-luc) was harvested five days post-transfection before inoculation into silkworms. after four to five days, protein was harvested from silkworms and μg protein from lysed larval haemolymph was subjected to luciferase assays. luminescence detected from silkworm larval haemolymph was approximately . ± . × relative light units (rlu), compared to - rlu from luc-negative virus-infected samples. pcr amplification (qpcr) further verified and validated the chifn-λ gene expression in bevs (supplementary figure s c) . in order to investigate the possible biological, cellular, and molecular mechanisms involved in the cascade of interferon-induced immunity, we performed transcriptomic analysis on chicken embryo fibroblasts and organs of live chickens. transcriptomes from the bursa of fabricious and thymus (most important immune organs in chicken) were compared with the control group to identify differentially expressed genes (degs) among all groups. experimentation started at day post-hatch as this is a phase of rapid growth and development, and we hoped to achieve biologically active transcriptional changes. the differences in degs observed in the present study control cellular architecture, immune function, metabolic pathway, and muscular function. it has previously been established that huifn-λ signals via il- and il- r exhibit typei-like antiviral potential [ ] . protection from simian foamy virus (sfv) and avian influenza (ai) augments the antiviral functioning and further postulates its diverse antiviral potential against avian pathogens. in this context, we stimulated chickens with silkworm-expressed chifn-λ and profiled the gene expression in immune organs (thymus and bursa) and compared it with that in primary chicken fibroblasts using rna-seq. an overall low isg expression was noticed in chifn-λstimulated cef; out of , genes, were degs ( upregulated and downregulated) (p< . , │ log fold change │ > ) (figure a) . although cef do not possess receptors for ifn-λ, slight temporal expression of degs in response to chifn-λ treatment signifies its antiviral potential in primary cells. next, we monitored the gene expression in the thymus and bursa. between the chifn-λ-treated and non-treated thymus, a total of , genes were expressed. among them, genes were degs, in which genes were upregulated and genes were downregulated (figure b) . in the bursa of fabricious, out of , genes were differentially expressed ( upregulated and in order to investigate the possible biological, cellular, and molecular mechanisms involved in the cascade of interferon-induced immunity, we performed transcriptomic analysis on chicken embryo fibroblasts and organs of live chickens. transcriptomes from the bursa of fabricious and thymus (most important immune organs in chicken) were compared with the control group to identify differentially expressed genes (degs) among all groups. experimentation started at day post-hatch as this is a phase of rapid growth and development, and we hoped to achieve biologically active transcriptional changes. the differences in degs observed in the present study control cellular architecture, immune function, metabolic pathway, and muscular function. it has previously been established that huifn-λ signals via il- and il- r exhibit typei-like antiviral potential [ ] . protection from simian foamy virus (sfv) and avian influenza (ai) augments the antiviral functioning and further postulates its diverse antiviral potential against avian pathogens. in this context, we stimulated chickens with silkworm-expressed chifn-λ and profiled the gene expression in immune organs (thymus and bursa) and compared it with that in primary chicken fibroblasts using rna-seq. an overall low isg expression was noticed in chifn-λ-stimulated cef; out of , genes, were degs ( upregulated and downregulated) (p < . , |log fold change|> ) (figure a ). although cef do not possess receptors for ifn-λ, slight temporal expression of degs in response to chifn-λ treatment signifies its antiviral potential in primary cells. pathways [ ] (figure ) . due to the induction of a distinct subset of genes, a lower level of antiviral activity is observed as compared to type-i ifns. it is speculated that the activation of chifn-λ is similar to type-i ifns but they are diverse in functional capability. the chifn-λ have particular significance in viral infections of epithelial origin, where they are optimally active by eliciting a broad antiviral state. using conventional approaches, we have confirmed the expression of selected genes as shown in supplementary figure s a and s b. next, we monitored the gene expression in the thymus and bursa. between the chifn-λ-treated and non-treated thymus, a total of , genes were expressed. among them, genes were degs, in which genes were upregulated and genes were downregulated ( figure b ). in the bursa of fabricious, out of , genes were differentially expressed ( upregulated and downregulated) ( figure c ). interestingly, a relatively low number of genes overlapped among these three groups ( figure d ). in order to confirm the expression of degs, we used a conventional approach (qpcr) and show (supplementary figure s a,b) a scenario corresponding to the rna-seq data. on the basis of abundance and fold change, degs were further characterized (supplementary table s ). cumulatively, a significant upregulation of crucial cytokine and chemokine genes (il -β, ccl , ccl , and cx cl ) was observed. these are broadly involved in antiviral response, apoptosis, cellular proliferation and differentiation, cytokine-cytokine receptor interaction and inflammation pathways [ ] (figure ). due to the induction of a distinct subset of genes, a lower level of antiviral activity is observed as compared to type-i ifns. it is speculated that the activation of chifn-λ is similar to type-i ifns but they are diverse in functional capability. the chifn-λ have particular significance in viral infections of epithelial origin, where they are optimally active by eliciting a broad antiviral state. using conventional approaches, we have confirmed the expression of selected genes as shown in supplementary figure s a degs were further analyzed for go terms and the kegg pathway by utilizing deseq [ ] . of go terms associated with chifn-λ-treated cef, go terms were significant (p < . ) ( figure a ). in the bursa, among biological processes, we observed the wnt signaling pathway (wif /camk a), cytokine-cytokine receptor interactions (tnfsf ), the apelin signaling pathway (ryr /myl ), and the significant antiviral pathway (novel gene) in cellular components (figure b) . in the thymus, out of go terms, we observed significant, and in the bursa, out of go terms, were significant (p < . ). in order to understand the biological functions associated with degs, we further analyzed the data in three distinct categories, including biological processes (bp), cellular components (cc), and molecular functions (mf) (figure c) . degs were further analyzed for go terms and the kegg pathway by utilizing deseq [ ] . of go terms associated with chifn-λ-treated cef, go terms were significant (p < . ) ( figure a ). in the bursa, among biological processes, we observed the wnt signaling pathway (wif /camk a), cytokine-cytokine receptor interactions (tnfsf ), the apelin signaling pathway (ryr /myl ), and the significant antiviral pathway (novel gene) in cellular components ( figure b ). in the thymus, out of go terms, we observed significant, and in the bursa, out of go terms, were significant (p < . ). in order to understand the biological functions associated with degs, we further analyzed the data in three distinct categories, including biological processes (bp), cellular components (cc), and molecular functions (mf) ( figure c ). further to gene ontology and differential expression, we investigated kegg pathway enrichment. in cefs, significant enrichment was seen in pathways including the mapk signaling pathway ( further to gene ontology and differential expression, we investigated kegg pathway enrichment. in cefs, significant enrichment was seen in pathways including the mapk signaling pathway (fos/il b/fosb), the toll-like receptor signaling pathway (fosb, il l , il b, fos, ccl ), influenza a (il l /il b/ccl ), cytokine-cytokine receptor interactions (ccl /il l /il b/cx cr /ccl ), salmonella infection (fosb/il l /il b/fos), the nod-like receptor signaling pathway (il l /il b/ccl ), and herpes simplex infection (fosb/il b/fos/ccl ) ( figure a ). in bursa, wnt signaling (wif ), the apelin signaling pathway (ryr ), and the calcium signaling pathway (ryr ) were significantly observed ( figure b ). for the thymus, the nod-like receptor signaling pathway (plcb /mapk ), the mapk signaling pathway (srf/mapk ), influenza a (rsad /mapk ), and mapk (salmonella, toll-like, herpes simplex infection) were observed ( figure c ). collectively, apoptosis (jun/birc /ctsc/actg ), rna degradation (eno /btg /c d), the tca cycle (mdh /idh a), the p signaling pathway (perp /ccnb ), biosynthesis of amino acid (eno /idh a), influenza a (rsad /jun/actg ), and the toll-like receptor signaling pathway (jun) were among the most significant. here, we present the first comprehensive report on cloning and expression of chifn-λ by employing bevs and demonstrate that it is biologically active in both cef (in vitro) and live chickens here, we present the first comprehensive report on cloning and expression of chifn-λ by employing bevs and demonstrate that it is biologically active in both cef (in vitro) and live chickens (in vivo). the identification of this potentially significant ifn among the ifn family advances fundamental aspects and functionality of chifn-λ in avian type-iii ifns. it is evident from the data that this ifn, like human interferon lambda (huifn-λ) , demonstrates similar type-i ifn-like properties. however, a distinct pattern of expression of isgs in chifn-λ contrasts it from other type-i ifns. knowledge regarding ifns is fundamental as rapid outbreaks of viral pathogens cause huge economic losses to the poultry industry every year. the present study investigates the isgs and signaling pathways associated with avian immunity and will bring new horizons to target problematic viral pathogens, e.g., aivs, circulating within the poultry industry. interferon lambda is a biologically active type-iii interferon which primarily acts on epithelial tissues [ ] . studies have demonstrated the antiviral potential of ifn-λ against highly pathogenic avian influenza by eliciting a broad antiviral state [ ] . ifn-λ is structurally peculiar as it possesses five exonic regions located on chromosome , contrary to type-i ifns, which are intronless and situated on the z sex chromosome in chicken [ , ] . this is in agreement with human ifn-λ subfamily which are anatomically identical by possessing five exonic regions on chromosome of the human genome [ ] . furthermore, % of amino acids are identical between huifn-λii and chifn-λ, which signifies the similarity of these two ifns. however, unlike mammals, only one member exists in chicken (chifn-λ). this is in agreement with the other types of chicken ifns, which have fewer members compared to mammalian ifns [ ] . reduced expression of isgs in response to chifn-λ in our experiment demonstrates the fact that cefs are optimally less receptive to ifn-λ, which is in agreement with published reports [ ] . one study revealed that chifn-λ can actively inhibit the viral replication of ai in primary embryonic tracheal organ cultures and clec- (chicken lung cell line). it is further postulated that with treatment of chifn-λ, isgs are expressed significantly, especially mx gene, which is primarily expressed in epithelial rich organs (i.e., trachea, lungs, and intestine) was also observed in the present study [ ] . furthermore, studies have also revealed that a high degree of cell type specificity in receptor-ligand interactions make avian ifns distinct from mammalian ifns. recently, it has been established that chicken ifn-λ inhibits low pathogenic influenza virus replication in cefs; however, as compared to chifn-γ and chifn-β, higher doses are required to induce isgs and maintain the strong antiviral state in the cells [ ] . go and kegg analysis of each experimental group demonstrated overlapping biological functions. an important gene involved in the host response of infected samples is rsad , also termed viperin, which is one of the potent interferon stimulated genes (isgs) responsible for eliciting a broad antiviral state against a variety of viral and bacterial pathogens [ ] . in mammals, it is highly expressed in response to invading viral infections [ ] . elevated expression of viperin in chifn-λ-treated organs further augments the expression of isgs in response to injected ifn in vivo. viperin was upregulated in response to chifn-λ treatment, which is symbolic for all isgs. ifn-inducible transmembrane protein- (ifitm- ) is one of the potent isgs expressed in response to either type of ifn and plays an antiviral role by blocking cytoplasmic entry [ ] . it is further demonstrated that ifitm alters membrane fluidity, hence producing curvature in the outer leaflets of the membrane or by interfering with intracellular cholesterol homeostasis [ , ] . significant upregulation of ifitm in the chifn-λ-treated thymus augments the temporal expression of isgs in response to ifn treatment. further studies are needed to investigate the possible future role of chifn-λ as a potent and novel therapeutic in the poultry industry. although the immune response elicited by type iii ifns is still not very clear, in the present study we also found some novel genes involved in the cascade of the avian immune response. furthermore, in vitro exposure of cef to chifn-λ demonstrated a rapid surge of pro inflammatory cytokines. considering their vital role in immune pathways, cytokine gene expression is widely employed as an indicator for the immune response. we did observe some genes that were previously illustrated in publications; one such example is chemokine (c-c motif) ligand (ccl , ensgalt ) [ ] . chemokines are secreted chemotactic cytokines that play a fundamental role in the recruitment and migration of lymphoid and myeloid cells in target tissues, and hence govern the avian immune response [ ] . ccl is a chemokine secreted by monocytes that is capable of activating macrophages and t lymphocytes [ ] . ccl , like its mammalian orthologue, is responsible for recruiting lymphoid cells and is involved in the early immune response in chickens [ ] . likewise, ccl , ccl , and ccl were also upregulated in cef and are chiefly involved in the innate avian immune response. the present study describes the transcriptomic analysis of differential gene expression following exposure to chifn-λ and the resultant pro-inflammatory response in both cef and chicken tissues. this response ostensibly is due to rapid and sustained signaling via cell surface receptors and a surge of chemokines and cytokines, which in turn create an antiviral environment. a contrasting feature of the present study is the upregulation of the toll-like receptor (tlr) signaling pathway in all three treatment groups, where it is evident that numerous genes are upregulated in tlr mediated cytotoxicity. tlr , a unique chicken receptor expressed on the surface of fibroblasts, heterophils, and macrophages, shares % sequence identity with tlr [ ] . it is evident from experimentation that tlr is a broad spectrum tlr that has the capability to recognize heat stable components of both gram-positive and gram-negative bacteria, cpg oligonucleotides, lipopolysaccharide (lps), and tripalmitoylated lipopeptide [ ] . tlr , an avian-specific tlr, plays a significant role in avian immune responses against bacterial and viral pathogens. recently, it has been demonstrated that diacylated lipopeptide from mycoplasma synoviae activated tlr and regulated innate immune responses [ ] . similarly, significant upregulation of tlr , observed in the present study, highlights a possible role of chifn-λ against mycoplasma infections in chicken. however, it warrants future studies to delineate the molecular processes. it has also been established by repeated experimentation that chifn-λ has been seen to cause delay in viral excretion and the spread of highly pathogenic avian influenza (hpai) h n [ ] . it is evident that in mammals, ifn-λ elicits a protective antiviral response toward ai, whereas ifn-λ plays a minor role in lung epithelia [ ] . similarly, in the respiratory tract of chickens, not all mucosal cells are responsive to chifn-λ. therefore, treatments can only delay, but do not significantly support the complete removal of viral loads of h n or halt the virus crossing the epithelial barrier [ ] . however, for low-pathogenic avian influenza (lpai), it is evident that chifn-λ has demonstrated significant antiviral activities [ ] . recent reports revealed another contrasting feature of ifn-λ, where it significantly elicited strong antiviral potential on intestinal epithelial cells to control murine rotaviruses [ , ] . it will be fascinating to investigate in the future whether the same antiviral phenomena occurs, and chifn-λ might also demonstrate epitheliotropism like rotaviruses and halt viral pathogens of the gastrointestinal tract in chickens. nuclear factor kappa-b (nf-kb) is the most significant, evolutionarily conserved, pleiotropic, inducible transcription factor responsible for regulating genetic expression in a variety of fundamental processes, including apoptosis, growth, immune response, inflammation, stress response, etc. [ ] . notably, the upregulation of nf-kb in response to chifn-λ treatment on cef signifies their potent role in the immune response. activator protein (ap- ) is a transcription factor complex highly responsive for cytokine signaling and growth promotion [ ] . formed through noncovalent dimerization between the fos and jun family of nuclear oncogenes, this complex activates ap- -dependent genes, hence controlling cell proliferation, differentiation, and apoptosis [ ] . consistent with these observations, our study demonstrated that many genes associated with this pathway were upregulated. this finding suggests a link between ap and the transcriptional cascade associated with recombinant interferon treatment. overall, transcriptomic analysis revealed significant upregulation of fos and jun in cef and bursa, and thymus of chicken. the innate immune response is a highly complex, precise, interconnected, and integrated response that relies on many factors. the genes investigated in our study control direct protein interactions and are significantly involved in the avian innate immunity cascade. however, further validation of a broad set of immunity-related genes will also be required to elucidate the mechanism of interferon-induced immunity. a more comprehensive study including a larger set of immune genes and multiple recombinant ifns, which will correlate their integrated role, will enable researchers to provide comprehensive insight into the avian innate response. other future studies involving backyard poultry to assess whether similar patterns of innate immunity prevail in indigenous breeds in response to chifnλ are also important and will further develop our understanding of avian immunity. in the current study, we employed rna-seq to illustrate vital transcriptomes involved in the cascade of avian biology and observed divergent results in recombinant interferon-treated chickens compared to a control group chickens. our data suggest that significant antiviral, cell cycle regulators, and biologically active genes are expressed in response to administered chicken ifn. functional characterization of these vital genes warrants further investigation to determine the future possible role for recombinant chicken ifn in the poultry industry. the authors declare no conflict of interest. phylogeography and evolutionary history of reassortant h n viruses with potential human health implications h n avian influenza virus in korea: evolution and vaccination avian interferons and their antiviral effectors characterization and transcriptional analysis of the mouse chromosome cytokine receptor gene cluster ifn-λs mediate antiviral protection through a distinct class ii cytokine receptor complex lambda interferon (ifn-λ), a type iii ifn, is induced by viruses and ifns and displays potent antiviral activity against 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agonists diacylated lipopeptide from mycoplasma synoviae mediates tlr induced innate immune responses ifn-λ: a new spotlight in innate immunity against influenza virus infection differential responses of innate immunity triggered by different subtypes of influenza a viruses in human and avian hosts type iii interferon gene expression in response to influenza virus infection in chicken and duck embryonic fibroblasts distinct roles of type i and type iii interferons in intestinal immunity to homologous and heterologous rotavirus infections interferon-λ and interleukin act synergistically for the induction of interferon-stimulated genes and control of rotavirus infection interferons and viral infections innate immune sensing of dna viruses neuronal activity-dependent local activation of dendritic unfolded protein response promotes expression of brain-derived neurotrophic factor in cell soma key: cord- -kjkuet authors: lópez-camacho, césar; chan kim, young; blight, joshua; lazaro moreli, marcos; montoya-diaz, eduardo; t huiskonen, juha; mareike kümmerer, beate; reyes-sandoval, arturo title: assessment of immunogenicity and neutralisation efficacy of viral-vectored vaccines against chikungunya virus date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: kjkuet chikungunya virus (chikv) has caused extensive outbreaks in several countries within the americas, asia, oceanic/pacific islands, and europe. in humans, chikv infections cause a debilitating disease with acute febrile illness and long-term polyarthralgia. acute and chronic symptoms impose a major economic burden to health systems and contribute to poverty in affected countries. an efficacious vaccine would be an important step towards decreasing the disease burden caused by chikv infection. despite no licensed vaccine is yet available for chikv, there is strong evidence of effective asymptomatic viral clearance due to neutralising antibodies against the viral structural proteins. we have designed viral-vectored vaccines to express the structural proteins of chikv, using the replication-deficient chimpanzee adenoviral platform, chadox . expression of the chikv antigens results in the formation of chikungunya virus-like particles. our vaccines induce high frequencies of anti-chikungunya specific t-cell responses as well as high titres of anti-chikv e antibodies with high capacity for in vitro neutralisation. our results indicate the potential for further clinical development of the chadox vaccine platform in chikv vaccinology. chikungunya virus (chikv) is transmitted by aedes mosquitoes and it is an important growing human health concern in many countries, causing significant outbreaks of acute febrile illness and long-term arthropathy [ ] . its origins are traced to africa as an enzootic spillover, belonging to the semliki forest complex of the alphavirus (togaviridae) [ ] and was first isolated from a human patient in tanzania in [ ] . it currently exists as four lineages. the two african enzootic lineages are the west african (wa) and the east central south africa (ecsa) lineages. the additional two lineages originated from the ecsa lineage and are called the asian and the recently emerged indian ocean lineage [ , [ ] [ ] [ ] . chikv is composed of a positive, single-stranded genomic rna of kilobases, encoding four non-structural (ns) and five structural (s) proteins [ , ] . the non-structural proteins, nsp , nsp , nsp and nsp , are required for virus replication [ ] . a sub-genomic rna encodes the structural proteins: capsid (c), e , e , k and e , and thus a polyprotein is produced which is then processed by the capsid auto-proteinase and signalases [ ] . the chikv surface is mainly composed by e -e heterodimers [ ] where e glycoproteins mediate fusion [ ] and e glycoproteins interact with the host receptor [ ] . since its discovery the virus has spread to asia, oceania/pacific, europe, and mostly recently to the americas, where there have been more than a million reported cases since its detection in late [ ] [ ] [ ] [ ] , whilst major outbreaks continue to be recorded in the asian and african continents. although chikungunya vaccines have been under development for several decades, no licensed option is yet available and chikv still causes substantial morbidity, overwhelming public health systems and contributing to poverty [ ] . in the meantime, current control strategies rely on reducing human exposure to potentially infected mosquito vectors. several institutions are now engaged in the development of a safe and cost-effective chikv vaccine; such vaccines [ ] [ ] [ ] are based on live-attenuated or inactivated chikv, chimeric chikv, dna, subunit, virus-like particle (vlp) and viral-vectored platforms. they are mainly designed to induce humoral responses against the structural viral protein e , as well as e , due to strong correlates of protection with neutralising antibodies against structural chikv proteins in asymptomatic cases [ ] . in addition to that, passive transfer of sera from convalescent humans to mice prevented infection [ ] whilst neutralising antibodies against e and e were able to protect immunocompromised mice [ ] . in humans, an early igg neutralising response is associated with reduced clinical symptoms [ ] and asymptomatic cases have been associated with high neutralising antibody titres [ ] . here, we describe the design and development of chikv vaccines based on the clinically relevant adenoviral vector, chadox and the modified vaccinia ankara (mva) platforms [ ] [ ] [ ] [ ] [ ] . viral vector expression of the schikv proteins is able to form chikungunya viral particles, thus mimicking a real exposure of chikv, whilst being an in silico designed mosaic protein, aiming to represent all four chikv lineages. in mice, chadox vaccines candidates expressing schikv antigens are able to induce strong humoral and cellular responses upon a single and non-adjuvanted immunisation approach. importantly, we present evidence of in vitro neutralising activity in sera from vaccinated mice. finally, whilst durability of humoral responses was achieved upon a single immunisation, mva vaccines expressing schikv were produced to be used as an alternative heterologous booster regime (chadox /mva), in order to increase neutralising antibody titres. taken together, a viral vectored vaccine for chikv, based on the chadox platform is a good candidate for further pre-clinical and clinical studies. full length structural polyprotein sequences for chikv from all lineages were collected from the ncbi protein database (txid ), aligned using clustal omega [ ] , and a neighbour-joining tree created (juke-cantor, bootstraps). conservation within clades (intra-clade) and subsequently between clades was calculated using our in-house developed software based on a sliding window approach with a sequence weighting method to enable equal representation of all lineages and variants (manuscript in preparation). the structural cassette chikv sequence derived from various chikv lineages was codon optimised. to improve initiation of translation a kozak consensus sequence was included before the end of the transgene. finally, the transgene design included the required enzymatic restriction sites to allow the in-frame cloning of the transgene between the cmv promoter and the polya sequence region contained in our shuttle and expression vector (pmono). a synthetic gene cassette was produced by geneart ® (fisher scientific, regensburg, germany) and was named schikv. the schikv plasmid was used as dna template to further generate the e ,e , k,e cassette with no capsid by pcr-cloning; this variant was named schikv ∆c. forward primer: atggaggaatggtccctggctatc. reverse primer: tcatcagtgccggctgaag. the plasmid containing the schikv cassette (c,e ,e , k,e ) and the schikv ∆c (e ,e , k,e ) were digested with kpni and noti restriction enzymes (neb, ipswich, ma, usa) to allow in-frame ligation between the cmv promoter and the poly(a) regions contained in the shuttle plasmid (pmono). the recombinant dna plasmids were expanded and purified from e. coli using the midiprep kit (qiagen, hilden, germany). resulting plasmids were verified by restriction analysis and and flanking sequencing. to generate chadox vaccines, the shuttle plasmids containing attl regions sequences were each recombined with those attr regions contained in the destination vector chadox using an in vitro gateway reaction (lr clonase ii system, invitrogen tm , carlsbad, ca, usa). successfully recombined chadox schikv and chadox schikv ∆c plasmids were verified by dna sequencing using flanking primers (forward promoter primer and poly-(a) reverse primer). standard cell biology and virology techniques were performed to generate the non-replicative adenoviral vectors [ ] . to generate mva based vaccines both the schikv and the schikv cassette ∆c were digested with kpni and xhoi to allow in-frame ligation between the p . promoter and the tkr locus, contained in the entry plasmid mva. ligated dna plasmids were expanded in e. coli and a midiprep (qiagen) was used for plasmid purifications. resulting plasmids were verified by restriction analysis and and flanking sequencing, and co-transfected to produce mva schikv and mva schikv ∆c, using the methodology as previously described [ ] . control chadox and mva vectors comprised non-structural (ns) regions from chikv and they were named chadox ns and mva ns, respectively. female inbred balb/c (h- d), ( - weeks) were used for the assessment of immunogenicity (n = mice per group). mice were purchased from envigo rms inc. (bicester, g.b.). the experimental design took into account the r reduction (replacement, reduction, refinement). no randomisation was used in this work. for chimpanzee adenoviral-vectored vaccines, mice were vaccinated intramuscularly with a single dose of × infectious units (iu). for boosting, mva vaccines were administered at a dose of × plaque-forming units (pfu) per mouse. all vaccines were diluted in endotoxin-free pbs. vero cell were grown following the atcc conditions. hek- cells (atcc ® , crl- tm) were grown in dmem media supplemented with % fbs, % l-glutamine and % non-essential amino acids. baby hamster kidney- (bhk- ) cells were maintained in glasgow´s minimum essential medium (gmem) supplemented with % fetal bovine serum (fbs), % l-glutamine, % tryptose phosphate broth, mm hepes ph . , u/ml penicillin and . mg/ml streptomycin at • c and % co . the cell number and viability were calculated by trypan blue staining using the countess automated cell counter (life technologies, carlsbad, ca, usa). cell lines are described in the ncbi biosample database. the expression screening was carried out in vero cells by transduction of chadox schikv, using a moi = . three days after transduction, the cells and supernatants were harvested, the supernatant was concentrated with a kda amicon ® spin column (millipore uk, ltd). the x concentrated fraction, the non-concentrated fraction (input) and flow through fraction was also recovered. cells and supernatants samples were boiled at • c for five minutes in laemli buffer. equal amounts of total cell extract and cell supernatants were resolved by sds/page and transferred to pvdf membranes. blots were blocked with x pbs-tween- % milk and incubated with an anti-chikv e antibody (az , aalto bioreagents, dublin, ireland) at : dilution, and anti-chikv envelope seropositive mice sera ( : ), followed by incubation with hrp-conjugated secondary antibody ( : ). chemiluminescence (perkin-elmer life sciences, boston, ma, usa) was visualised using the chemidoc srs device (biorad, watford h., uk). formvar/carbon mesh cu grids were glow-discharged in air and loaded with . µl of the sample from chadox schikv vaccine stock or purified vlp of chikv by sucrose gradient ( - %). excess liquid was removed, and the grids were washed times with milliq water. finally, grids were stained with % uranyl acetate for s, excess uranyl acetate was carefully removed using filter paper. the grids were air dried and analysed with a t transmission electron microscope (fei, eindhoven, the netherlands). elispots were carried out using peripheral blood mononuclear cells (pbmcs). briefly, maip elispot plates (millipore uk, ltd) were coated with anti-mouse ifnγ antibody (mab an , mabtech), after h blocking with complete dmem media ( % fcs). isolated pbmcs (using ack buffer solution) were plated alongside with -mer specific chikv structural peptides overlapped by a.a. ( µg/ml) and . × splenocytes from naïve mice per well. after h incubation, cells were discarded, and plates washed with pbs. following this, µl of biotinylated anti-mouse ifnγ mab (mab r - a , mabtech) ( : in pbs) was added to each well and incubated for h. after washing, plates were incubated with µl of streptavidin-alp (mabtech) at : dilution in pbs for h. after another washing step, developing solution (biorad, watford h., u.k.) was used. once spots were visible, the reaction was stopped by rinsing the plates with water. spots were acquired using an elispot reader. spot forming cells (sfc)/ pbmcs producing ifnγ were calculated. for expression and purification of the chikv e protein, the codon-optimised gene of e (a.a. - ) was cloned into the phlsec vector [ ] . which is flanked by the chicken β-actin/rabbit β-globin hybrid promoter with a signal secretion sequence and a lys-his tag. in order to improve secretion of the e protein, the c-terminal region of e (a.a. - ) was deleted. the phlsec chikv e plasmid ( µg) was transfected in hek- t cells using polyethyleneimine (pei) in roller bottles (surface area of , cm ) under standard cell culture conditions. five days after transfection cells were discarded and media was filtered through . µm disposable filters. the secreted protein was purified from the supernatant by ni sepharose affinity chromatography (histrap tm , ge healthcare), using the Äkta start chromatography system and eluted with imidazole mm. finally, the eluted protein was dialysed using slide-a-lyzertm cassette (thermo fisher scientific, rockford, il, usa) against × pbs. antibody binding to chikv e was measured by an igg enzyme linked immunosorbent assay (elisa). briefly, mice sera were diluted in nunc maxisorp immuno elisa plates coated with e diluted in pbs to a final concentration of µg/ml and incubated at room temperature overnight. plates were washed times with pbs/ . % tween (pbs/t) and blocked with µl with pierce tm protein-free (pbs) blocking buffer (thermo fisher scientific, waltham, ma, usa) for h at rt. mice serum was added and serially diluted -fold down in pbs/t with µl per well as final volume and incubated for h at rt. following washing times with pbs/t, bound antibodies were detected following a h incubation with µl of alkaline phosphatase-conjugated antibodies specific for whole mouse igg (a - ml, sigma aldrich, st. louis, mo, usa). following additional washes with pbs/t, development was achieved using µl of -nitrophenylphosphate diluted in diethanolamine buffer and the absorbance values at od were measured and analysed using a clariostar instrument (bmg labtech, aylesbury, gb). serum antibody endpoint titres were defined by an absorbance value three standard deviations greater than the average od of the control. titres of neutralising antibodies were determined as described previously using chikv replicon particles (vrps) expressing gaussia luciferase (gluc) [ ] . briefly, bhk- cells were seeded in -well plates at × cells per well. the next day, vrps (moi of . ) were preincubated with -fold serial dilutions of serum samples for h at • c before the mixture was added to the -well plates. after incubation for h at • c, the inoculum was removed, cells were washed with pbs, and the medium was added. readout of secreted gaussia was performed at h post infection using a renilla luciferase assay system (promega, southhampton, uk). neutralisation potency was determined as a percentage of measured gluc activity compared to the gluc readout after vrp application without serum. results are presented as % neutralisation (nt ) titres. all animals and procedures were used in accordance with the terms of the uk home office animals act project license. procedures were approved by the university of oxford animal care and ethical review committee (ppl / ). the authors declare that the data supporting the findings of this study are shown in the article. in order to generate a structural gene cassette (c,e ,e , k, e ) from chikv, a protein sequence mosaic was constructed using full-length structural polyprotein sequences collected from the ncbi protein database. a neighbour-joining tree was created which identified three distinct clades (a, b, c; figure a ). subsequently conservation was assessed within each clade (intra-clade; figure b ) using a sliding window approach, in which a conservation value between and was assigned, being fully conserved. anything lower than the first quartile (q ) was classed as conserved. lastly, the number of windows conserved across each clade and the degree of conservation were identified and used to create a normalised mosaic consensus. figure c illustrates the level of shared conservation between clades, from to - , with - being fully conserved. the resulting sequence mosaic schikv was synthesised (figure a) . we constructed chimpanzee adenoviral vector vaccines expressing the full structural cassette (chadox schikv), the capsid-deleted structural cassette (chadox schikv ∆c) and a non-structural sequence from chikv (chadox ns), the latter being used as a control vaccine (figure b ). in addition, modified vaccinia ankara (mva) viral-vector was used to construct the mva schikv, the mva schikv ∆c and the control mva ns (figure c ). after cloning and production of chadox vaccines encoding the genomic schikv cassette, we verified the formation of adenoviral particles by transmission electron microscopy ( figure a) . because it has been described that cellular expression of the full structural chikv cassette is capable to self-assemble vlps for chikv, as well as for other alphaviruses [ ] [ ] [ ] [ ] , we collected supernatant from hek cells expressing the schikv antigen. the supernatant was then subjected to sucrose gradient showing a characteristic sedimentation band, which is representative of vlp accumulation (figure b, left) . further electron microscopy preparations revealed particles of approximately nm in size (figure b, right) , which resembled wild-type chikv particles. alternatively, we transduced vero cells with chadox schikv and chadox schikv ∆c, and verified their expression in cells and supernatants. specific detection was achieved by western blot, using an anti-chikv mice serum and an anti-e monoclonal antibody (figure c,d, respectively) . in cells transduced with chadox schikv, we detected bands at approximately kda in the supernatants (figure c, lane , , ) , with a band of approximately kda only in the concentrated supernatant (figure c, lane ) . in cellular extracts, we detected specific bands of and kda (figure c, lane ) . in cells transduced with chadox schikv ∆c we also detected bands at approximately kda in supernatants (figure c, lane , , ) , but no bands of kda or kda were found in the concentrated supernatant nor in the cell extracts. therefore, it is suggested that the kda or kda band may represent the capsid protein, as previously demonstrated [ ] , which are not present in chadox schikv ∆c-transduced cells. as a positive control, we used a commercially available chikv e protein (figure c, lane ) ; therefore, it is suggested that the kda band detected in our blots is e protein. in addition, a western blot was performed using a monoclonal antibody against chikv e (figure d ); we detected a specific band in the positive control e protein (lane ), as well as a kda band in all the supernatant fractions from both chadox schikv-and chadox schikv ∆c-transduced cells. no signal in cell extracts was detected (figure d, lane , ) , probably due to conformational epitope masking. taken together, our results suggest that our adenoviral-vectors are able to express the schikv antigens which are able to induce the formation of chikungunya viral-like particles. thus, we aim to mimic a real antigen exposure of chikv particles. to assess the specific immunogenicity of our viral-vectored vaccines, we immunised groups of balb/c animals (n = ) with a single and non-adjuvanted vaccine dose of chadox schikv, chadox schikv ∆c, and control chadox ns, respectively. two weeks after immunisation, t cell responses were quantified by ex vivo ifnγ elispot, using a specific pool of overlapping peptides spanning the full chikv structural cassette, as well as a pool of overlapping peptides related to the control ns vaccines (figure ) . balb/c mice receiving vaccines encoding schikv antigens showed high t cell frequencies after stimulation with the schikv peptide pool (chadox schikv = mean of spot-forming cells (sfc)/ peripheral blood mononuclear cells (pbmcs) and chadox schikv ∆c = mean of , sfc/ pbmcs); with no statistical difference between both groups, using one-way anova analysis (figure a ). the control group chadox ns showed low specific t cell responses against the ns peptide pool and no responses to specific schikv peptide pool. in addition, we analysed the responses towards specific peptide sub-pools for c, e , e , k, e and ns (figure b ). schikv peptide deconvolution elispot allowed the identification of an immunodominant region located in the k region which is induced upon chadox schikv and chadox schikv ∆c vaccination ( , and , sfc/ pbmcs, respectively). as expected, we detected specific cellular responses towards the capsid in animals vaccinated with chadox schikv (mean= sfc/ pbmcs) in comparison with those animals vaccinated with chadox schikv ∆c in which low background responses were recorded (figure b ). modest responses were detected in pbmcs against the e and e peptide pools. taken together, we conclude that a single injection of chadox schikv or chadox schikv ∆c is immunogenic and able to induce specific t cell responses in balb/c mice. to assess humoral responses, -well plates were coated with chikv-e protein. we performed elisa assays to identify the immunogenic properties of chadox schikv and chadox schikv ∆c, upon a single and unadjuvanted vaccination strategy ( figure a) . two weeks after a chadox schikv or chadox schikv ∆c vaccination, specific igg antibody binding to chikv e protein was detected in sera from vaccinated animals ( figure a, left) . six weeks after prime ( figure a , right) all the animals increased the levels of od binding to the e protein. of particular interest, months after a single immunisation, sera from vaccinated animals showed specific igg antibody binding to chikv e , similar to that of six weeks prime group ( figure a, bottom) . chadox ns was used as control as it does not generate antibody responses towards the schikv antigens. log reciprocal antibody titre analysis (figure b) showed that levels of anti-e antibodies increased over time for both chadox schikv or chadox schikv ∆c prime-vaccinated animals and remarkably they were maintained after months upon a single vaccination strategy. interestingly, our results indicated that the deletion of capsid in the chadox schikv ∆cvaccinated group did not impair the ability to elicit anti-e chikv antibody titres. finally, modified vaccinia ankara (mva) viral-vector technology has been shown to be a potent booster of immune responses [ , ] . therefore, we have constructed mva schikv and mva schikv ∆c to test their boosting immunogenic potential in a prime-boost heterologous strategy. mice immunised with either chadox schikv or chadox schikv ∆c were boosted with mva schikv and mva schikv ∆c at six weeks after prime, respectively; and sera was collected two weeks after boost. prime-boost vaccinated mice showed high endpoint titters of > of anti igg-e antibodies (figure b , prime-boost section). mva ns was used as control to boost animals vaccinated with chadox ns vaccines. taken together, we conclude that a single vaccination with no adjuvant strategy is able to induce early, specific and long-lasting b responses in balb/c mice towards chikv-e protein. we tested balb/c mice sera obtained after an early prime time-point and in a prime-boost vaccination to assess chikv antibody neutralisation activity in vitro. the method we used is based on the virus replicon particle-based (vrp) chikungunya virus neutralisation assay to determine nt values [ ] . naïve and control chadox ns sera were included alongside the assay. at two weeks post-vaccination, sera from the chadox schikv-and chadox schikv ∆c-vaccinated groups showed high neutralisation activity against vrp chikv with a nt titre of . x and . x respectively (figure a) . in a prime-boost regime (figure b ), chadox schikv/mva schikv showed a further . -fold increase of the nt titter ( . x ), in comparison to the prime vaccination regime of chadox schikv. a similar increase of nt titre was found in the sera from mice vaccinated with a prime-boost chadox schikv ∆c/mva schikv ∆c, with a -fold increase ( . x ), when compared to that of the nt titre from single chadox schikv ∆c vaccinated animals (figure b ). these results indicate that when the capsid is deleted from the chikv structural genes, a decrease in the antibody nt titre is observed, upon a single chadox vaccination. conversely, when a prime-boost regime is used, the deletion of the capsid is beneficial to increase the chikv nt antibody titres. taken together, our results confirm that our viral-vectored vaccines elicit functional neutralising antibodies against chikv-infective particles, tested by an in vitro model. in this work we have constructed chikv vaccines based on viral-vector platforms, such as chadox and mva vectors. the immunogen was designed based on a mosaic consensus sequence from the asian, east, central and south african and west african regions. although individual strains are antigenically related and chikv vaccines previously reported have been developed using heterologous strains [ ] , a mosaic-based approach ensures a high coverage aimed at raising neutralising antibodies against all variants circulating worldwide. here, we have developed two different vaccine immunogen candidates: a vaccine encoding the full structural chikv polyprotein (schikv) and a vaccine in which the capsid antigen was deleted (schikv ∆c). one of the functions of the capsid is to auto-cleave from the e -e - k-e polyprotein. this proteolytic process exposes a signal leader which is contained in the amino-terminal region of e and promotes the entry of the polyprotein into the endoplasmic reticulum [ ] . there are several advantages in comparing these two different antigenic cassettes. genetic vaccines, such as dna vaccines, rna vaccines, and viral-vectored vaccines, are often limited by the number of nucleotides that can be inserted into the antigenic cassette. exploring the minimum sequence length able to induce immunogenicity and efficacy (either by in vitro neutralisation or by sterile protection after a viral challenge), will inform the vaccine development of new generation of vaccine candidates against chikv. in addition, optimising the minimum number of genetic antigens could allow the inclusion of further antigens to create combined vaccines (either homologous or heterologous) to induce immune response against different pathogens, all in a single genetic vaccine approach. based on this assumption, efforts are underway to create chadox vaccines expressing shorter versions of the chikv polyprotein, such as e and e proteins from chikv and other alphaviruses. e protein is involved in receptor recognition, while e protein is involved in membrane fusion [ , ] . therefore, exploring single-protein chikv vaccines in chadox or in other viral-vectored platforms seems feasible and promising, since anti-e and anti-e antibodies are high in convalescent chikv-immune populations [ ] . however, ensuring proper antigenic folding in such developments, identical or similar to those antigenic structures in virions from chikv is paramount. here, we have demonstrated by transmission electron microscopy, that our adenoviral vaccine chadox schikv is capable of expressing chikungunya-vlps, which promotes the correct conformational antigens to be presented upon vaccination, therefore mimicking a real exposure to wild-type chikv. it will be interesting to explore and compare the neutralisation efficacy between encoded chikungunya-vlps, e or e proteins in viral-vectored vaccines. in this work we have tested the immunogenicity of our viral-vectored vaccines by assessing the cellular and humoral responses in balb/c mice. our data shows that the t cell immunogenicity elicited by chadox schikv and chadox schikv ∆c is directed primarily to a peptide region located in the k protein, by ifn-γ production. it will be interesting to explore the development of t-cell based vaccines against chikv. the role of t cell immunity in alphavirus infection, especially in chikv is not very well understood. importantly, antigen design and the modulation of its intracellular expression will play a major role at inducing t cell responses directed to the antigen. a clear example is the comparison between chikv and chikv ∆c elispots, in which t cell responses directed to capsid were detected in the chikv groups but it was ablated in the chikv ∆c groups. it will be interesting to explore the protective and immunological relevance of the t cell response directed by the capsid in pre-clinical models. although we identified k as the immunodominant region in balb/c, this observation may differ in other inbred or outbred mouse strains; or in the case that vaccines comprise only e , e or e antigens. for example, chikv-specific cd +t cells were directed mainly against e and e proteins in c bl mice [ ] [ ] [ ] . t cells induced after chikv infection in c bl/ mice were not essential to control viremia but cd t cells were involved in chikv pathology [ ] . furthermore, t cell adoptive transfer did not confer protection against chikv challenge [ ] . finally, t cell responses in human chikv infection is also poorly understood. t cell responses (cd + > cd + ) have been found in chronic and convalescent chikv patients [ ] , in which specific responses were detected for e , capsid and ns protein (nsp ). further studies using chikv challenge models would be informative to further dissect the role of cellular responses in protective efficacy using ours or other vaccine platforms. in terms of humoral responses, we have assessed the binding of antibodies from vaccinated mice by elisa, coating with e protein. e protein has been shown to be a primary target for neutralising antibodies, since e functions for recognition and attachment to cellular receptors. here, we have demonstrated that our vaccines are able to mount anti-e antibody responses. the level of antibody responses after a single immunisation of chadox schikv or chadox schikv ∆c were maintained for up to months, highlighting the suitability of our vaccine approach to promote long-lasting immune responses against chikv. nonetheless, we have tested a boosting strategy using mva chikungunya vaccines to further increase the anti-e antibody titres. although we have used an elisa assay to detect anti-e antibodies, we are possibly detecting only antibodies against monomeric e . however, our vaccine platform assembles vlp for chikv, in which conformational epitopes might be physiologically relevant to confer protective efficacy in pre-clinical models. finally, we do not rule out the possibility of inducing responses other structural components. the chadox schikv and the chadox schikv ∆c prompt responses in both, b-and t-cell compartments. in vitro neutralisation assays are an excellent standard to test the capacity of chikv sero-positive serum to neutralise chikv. here we utilised a widely used neutralisation assay, using a non-infectious chikv particle which is fully capable of packing chikv replicon rna, which carries the gaussia luciferase (gluc) gene located in the subgenomic rna region [ ] . therefore, infected cells secreting gluc can be detected as a surrogate detection of chikv infection. in this study, we tested a rather early time-point after a chadox schikv or a chadox schikv ∆c prime vaccination, with the hope to demonstrate efficacy of neutralisation after two weeks post-vaccination, as vaccines should be aimed to guarantee protection in a shortest window of time between vaccination and pathogen exposure. conversely, we have tested the neutralisation capacity upon a booster vaccination using mva schikv and mva schikv ∆c, respectively. our results demonstrate that our chadox chikv and chadox chikv ∆c were able to induce > nt titres. since the nt assay presented here has been used in several vaccine development studies for chikv, we are able to afford a comparative assessment between our nt titters and other vaccine developments. garcia-arriaza et al. reported a mva-chikv vaccine expressing the full structural cassette, in which the mean of nt titters were < in a -week prime post-immunisation strategy [ ] . hallengärd et al., developed an attenuated chikv vaccines in which they achieved > when using the vaccine ∆ nsp [ , ] . finally, chikv neutralisation titres have been assessed in human sera yielding nt titres of . x , . x and x in three different chikv patients returning from mauritius, la reunion and seychelles, [ , ] respectively. therefore, the mean of the nt titres we have achieved in both prime (> ) and prime-boost regimes (> ) reflect the capacity of our viral-vectored vaccines to induce physiologically relevant nt titres. no licensed vaccine is yet available to prevent chikv infection. from > experimental vaccines, only have reached clinical trials and three are still active and leading the field [ ] . a leading vaccine consists of a virus-like particle that presents antigens similar to chikv but without replicating, stimulating immune responses but requiring multiple injections to induce antibody titres [ ] , thus increasing costs and logistics for its use in in low-income countries. another development consists of a recombinant live attenuated measles virus (mv) expressing surface chikv antigens [ ] . the vaccine was immunogenic and safe in humans, but it also requires > doses. regarding adenoviral vectors, only one human adenovirus expressing chikv structural antigens has been published [ ] . unfortunately, human adenoviral-vectored vaccines are not suitable to vaccinate humans, due to the widespread presence of wild-type adenovirus eliciting anti-adenovirus immunity and thus decreasing vaccine efficiency [ ] ; hence the use of chimpanzee adenovirus circumvent this problem. in the ideal chikv vaccine development, safety and immunogenicity should be achieved by a single vaccine dose and no adjuvant requirement. such a simple and robust development could benefit low-income settings, in which affordability and simplification of logistics are essential to deliver the vaccine to a target population. however, in the event that a booster vaccine is needed, an mva-based vaccine for chikv could be used. given the suitably of chadox in the clinical setting, the capability to produce millions of 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island attenuated and vectored vaccines protect nonhuman primates against chikungunya virus prime-boost immunization strategies against chikungunya virus chikungunya fever in travelers returning to europe from the indian ocean region safety and tolerability of chikungunya virus-like particle vaccine in healthy adults: a phase dose-escalation trial immunogenicity, safety, and tolerability of a recombinant measles-virus-based chikungunya vaccine: a randomised, double-blind, placebo-controlled, active-comparator, first-in-man trial chimpanzee adenovirus antibodies in humans, sub-saharan africa we would like to thank the jenner institute's vector core facility for producing and supplying the recombinant viral vectors, janett wieseler for excellent technical assistance and phillip kemlo for software development. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord- -d i sur authors: kim, hyung-jun; jeong, euiseok; choe, pyoeng gyun; lee, sang-min; lee, jinwoo title: intensive care unit relocation and its effect on multidrug-resistant respiratory microorganisms date: - - journal: acute crit care doi: . /acc. . sha: doc_id: cord_uid: d i sur background: infection by multidrug-resistant (mdr) pathogens leads to poor patient outcomes in intensive care units (icus). contact precautions are necessary to reduce the transmission of mdr pathogens. however, the importance of the surrounding environment is not well known. we studied the effects of icu relocation on mdr respiratory pathogen detection rates and patient outcomes. methods: patients admitted to the icu before and after the relocation were retrospectively analyzed. baseline patient characteristics, types of respiratory pathogens detected, antibiotics used, and patient outcomes were measured. results: a total of adult patients admitted to the icu, months before and after the relocation, were included. of them, were admitted to the icu before the relocation and afterward. baseline characteristics, including age, sex, and underlying comorbidities, did not differ between the two groups. after the relocation, the incidence rate of mdr respiratory pathogen detection decreased from . to . cases per , patient-days, but that difference was statistically insignificant. the use of colistin was significantly reduced from . days ( % confidence interval [ci], . to . days) to . days ( % ci, . to . days). furthermore, the duration of hospital stay was significantly reduced from a median of days (interquartile range [iqr], to days) to days (iqr, to days). conclusions: incidence rates of mdr respiratory pathogen detection were not significantly different before and after icu relocation. however, icu relocation could be helpful in reducing the use of antibiotics against mdr pathogens and improving patient outcomes. given the extensive use of antibiotics in intensive care units (icus) [ ] , nosocomial infections with multidrug-resistant (mdr) pathogens in an icu have become a major problem. in a previous prospective surveillance study of asian countries, resistance to imipenem was observed in . % of acinetobacter isolates and . % of pseudomonas aeruginosa isolates, with mdr rates of % and . % among patients with hospital-acquired pneumonia and ventilator-associated pneumonia, respectively [ ] . the mortality rates of hospital-acquired pneumonia and ventilator-associated pneumonia are higher than those of community-acquired pneumonia and healthcare-associated pneumonia [ ] . furthermore, mdr pathogens can increase the mortality rates of icu-acquired pneumonia [ ] . therefore, controlling the pre valence of mdr pathogens in the icu is mandatory for patient safety. efforts have been made to decrease the risk of mdr pathogen transmission. methods such as active surveillance cultures, room placement, cohorting, hand hygiene, use of clean gloves and gowns, and enforcing strict adherence to precautions have been used to decrease the incidence rates (irs) of mdr pathogen infections [ ] [ ] [ ] . however, all those precautions focus on person-to-person transmission. the importance of environmental contamination is not well understood. in the present study, we elucidate the effects of an environmental change, the relocation of a medical icu (micu), on respiratory mdr microorganisms in the icu. between november and july , patients admitted to the micu in a tertiary referral center in seoul national university hospital were screened. all adult patients ( ≥ years) were included. because of a remodel of the center's micu, patients had to be moved to a newly-built temporary micu on the th of march, . prior to its relocation, the micu had open beds and two isolation beds, and the average space was . m per patient. among the open beds, four belonged to the department of neurology and were excluded from the analysis. after its relocation, the micu had open beds with two isolation beds, all of which belonged to the department of internal medicine, and the average space was . m per patient. patients were retrospectively analyzed in terms of baseline demographics (sex, age, underlying comorbidities, and acute physiology and chronic health evaluation [apache] ii scores) before and after the relocation. the use of medications with potential effects on respiratory microbiota, such as antacids and steroids, and antibiotic use were also reviewed. as clinical outcomes, we compared the use of antibiotics, length of micu stay, length of hospital stay, and in-hospital mortality. days of medication use and ventilator use were calculated in calendar days. this study was conducted in accordance with the amended declaration of helsinki and was approved by the institutional review board of seoul national university hospital (protocol no. h- - - ). the requirement for informed consent was waived because of the retrospective study design and because all patients records and information were anonymized prior to analysis. ■ multi-drug resistant respiratory pathogens tended to decrease after relocation of the intensive care unit, although without statistical significance. ■ use of colistin was significantly reduced after the relocation. ■ duration of hospital stay was shorter after the relocation. to maximize the sensitivity of respiratory microbiome detection, several respiratory specimen culture results were reviewed. noninvasive respiratory specimens from sputum and endotracheal aspirate, along with invasive respiratory specimens such as bronchial washings and bronchoalveolar lavage fluids were included. sputum was collected in a sterile conical tube after coughing effort from the patient, and endotracheal aspiration was performed by the attending nurse with a sterile catheter and saline. bronchoscopic specimens were collected by a board-certified pulmonary specialist with a sterile bronchoscope and saline. active surveillance cultures of carbapenem-resistant acinetobacter baumannii (crab) were routinely performed from sputum or endotracheal aspirate specimens upon micu admission and were also included in the analysis. crab, methicillin-resistant staphylococcus aureus (mrsa), carbapenem-resistant pseudomonas aeruginosa (crpa), extended-spectrum β-lactamase (esbl)-producing gram-negative enterobacteriaceae, and stenotrophomonas maltophilia were considered as the mdr pathogens [ , ] . the ir of mdr respiratory pathogens was compared before and after the relocation and also described monthly. the microorganisms were considered to be micu-acquired if they were first detected after micu admission, or if they first appeared after days of micu stay. isolation beds were isolated with walls and a door, whereas open beds were divided by curtains. the patient to nurse ratio was : during the day and evening and : at night. routine cleaning of the floors was performed every day with % sodium hypochlorite and was done more frequently when visual contamination was observed. when a patient left the icu bed, every surface of the patient's medical equipment was cleaned with % benzalkonium chloride. medical professionals were required to wash their hands with soap and water and with . % chlorhexidine gluconate in % or % ethanol before and after attending a patient. if any mdr microorganism was detected in a patient, wearing acute and critical care november ( ): - clean nonsterile gloves and gowns was obligatory while attending to that patient. also, separate items such as thermometers, stethoscopes, tourniquets, and blood pressure cuffs were used. adherence to those precautions was monitored on a weekly basis, and the precaution protocols did not change before and after the micu relocation. categorical variables are described as numbers and percentages and compared using either the chi-square test or fisher exact test. numerical variables are described using the median and interquartile range (iqr) and compared using the mann-whitney u-test. irs are described as numbers per , patient-days with % confidence intervals (cis) and compared using poisson regression analyses or negative binomial regression analyses. all statistical analyses were performed using stata version . (stata corp., college station, tx, usa). the p-values of less than . were considered statistically significant. a total of patients were admitted to the micu during the study period. among them, patients were admitted to the micu before its relocation, and the other were admitted afterward. patients were predominantly male ( %), with a median age of years (iqr, to years) and a median apache ii score of (iqr, to ). among the patients, ( . %) were diagnosed with sepsis, and ( . %) were diagnosed with pneumonia. the prevalence of sepsis increased significantly after the micu relocation ( . % to . %, p= . ). the most common underlying comorbidity was hematologic malignancy ( . %), followed by solid organ malignancy ( . %), diabetes mellitus ( . %), heart failure ( . %), end-stage renal disease ( . %), liver cirrhosis ( . %), solid organ transplant ( . %), and bronchiectasis ( . %). the proportion of patients who were transferred from other icus was . %, which did not differ before and after the relocation (p = . ). antacids the monthly irs of mdr respiratory microorganisms were specified to further assess the effect of the micu relocation. in contrast to a high ir of mdr respiratory microorganisms in february ( . per , patient-days), the ir in march , the month in which the relocation of the micu occurred, decreased to . per , patient-days. the ir further decreased to . per , patient-days in may , and the difference between february and may was statistically significant (p = . ) ( figure ). however, the ir rose again to . per , patient-days in june , when of patients ( . %) were diagnosed with at least one detectable mdr pathogen. this detection rate was significantly higher than that observed for the other months, including april ( / patients, . %) and may ( / patients, . %). high rates of crab ( / patients, . %) and mrsa ( / patients, . %) detection were the main factors contributing to the increase in mdr microorganisms. other than crab and mrsa, crpa (two patients), and esbl-producing gram-negative enterobacteriaceae (two patients) were also detected in june . we reviewed the most commonly used antibiotics and found that carbapenem was most often used, for . days per , patient-days before the relocation and . days after the relocation (p = . ). the use of other antibiotics, piperacillin/ tazobactam ( . vs. . days per , patient-days, p= . ), quinolone ( . vs. . days per , patient-days, p= . ), glycopeptide ( vs. . days per , patient-days, p= . ), and linezolid ( . vs. . days per , patient-days, p= . ), did not differ significantly according to the micu relocation. however, the use of colistin decreased significantly from . days ( % ci, . to . days) to . days ( % ci, . to . days) per , patient-days (p= . ) (figure ). we compared the clinical outcomes before and after the micu relocation. among the patients included in the study, the overall in-hospital mortality rate was . %. the median length of micu stay was days (iqr, to days), and the median length of hospital stay was days (iqr, to days). the length of micu stay (median, vs. days, p = . ) and inhospital mortality ( . % vs. . %, p = . ) did not differ significantly between the two groups. however, the length of hospital stay was shortened after the relocation from a median of days (iqr, to days) to a median of days (iqr, to days), which was statistically significant (p = . ) ( table ). in this retrospective, single-center study, relocation of the micu led to nonsignificant changes in the ir of mdr respiratory microorganisms, including crab and mrsa. however, the use of colistin was significantly reduced, and the length of hospital stay was shortened. this is the first study to describe the importance of environmental change to infections by mdr pathogens, including crab, one of the respiratory microorganisms most commonly found in icus. the detection rate of mdr respiratory microorganisms and the use of antibiotics in our study are comparable with those reported in previous studies. in a prospective study from spain [ ] , the detection rate of mdr respiratory pathogens in icu patients with pneumonia was . %. in our study, of patients ( . %) had at least one detectable mdr respiratory microorganism, and of patients with pneumonia ( . %) had detectable mdr respiratory microorganisms. a prospective study from south korea reported that the detection rate of crab in active surveillance cultures was % [ ] . in our study, crab was detected in of patients ( . %). a prospective study from china reported mrsa incidence to be . cases per icu admissions [ ] , and a retrospective study from canada reported mrsa incidence to be . - . cases per , patient-days [ ] . our study revealed . mrsa cases per admissions (or . cases per , patient-days). the use of piperacillin/tazobactam, glycopeptide, and quinolone was similar to that reported in previous studies, but in our center, the use of carbapenem was higher and that of third-generation cephalosporin was lower than previously reported [ , ] . our results showed nonsignificant changes in the ir of mdr respiratory microorganisms after the relocation. the ir seemed to drop right after the relocation but increased again as time passed, especially in june , when the ir of mdr respiratory microorganisms was the highest in our study period, mostly crab and mrsa. although the exact reason for the sudden outbreak of crab and mrsa during june could not be clarified due to the study's retrospective design, some factors could be considered. among the patients admitted to the micu during june, a higher proportion were placed on mechanical ventilators ( / patients, . %) than in other months ( / patients, . %). the duration of ventilator application also tended to be slightly longer in june (mean, . days) than in the other months (mean, . days) although that difference was not statistically significant. the application of mechanical ventilators and more ventilator-days has previously been associated with a higher risk of mdr acinetobacter baumannii colonization [ ] . once colonization has been established, the next patient admitted to the same environment might have been exposed to an elevated risk of developing that infection, especially with acinetobacter species [ ] . furthermore, contamination of healthcare workers' protective gowns and gloves during routine management of patients can be frequent with acinetobacter species and mrsa [ ] . those factors could explain why the colonization rate of mdr microorganisms increased with time, even after relocation. this study demonstrated a significant decrease in colistin use, despite a nonsignificant change in the ir of crab from respiratory specimens. this can be explained by the fact that crab is a common environmental colonizer [ , ] and that acinetobacter species are known to cause only . % to . % of the nosocomial infections caused by gram-negative bacilli [ ] . it is therefore possible that a significant proportion of patients might have had crab "colonization" in their respiratory system rather an infection with "pathogenic" crab, resulting in a decrease in the use of colistin. we also found a shortened hospital stay among patients after the micu relocation. because the baseline characteristics (age, sex, apache ii score, and underlying disease) did not differ significantly before and after the relocation, this difference in clinical outcome could result from changes associated with the micu relocation, including the reduced use of colistin. several retrospective studies [ , ] have reported that acinetobacter baumannii infection is associated with a longer hospital stay. inappropriate use of antibiotics during the initial days of a hospital-stay is known to be associated with longer hospital stays [ ] , and that inappropriate use is most commonly associated with acinetobacter species [ ] . although mortality rates did not differ after the relocation in the present study, there are contradicting reports on differences in mortality rates according to acinetobacter infections [ , , , ] . the inconsistency between the duration of hospitalization and mortality rates could be caused by the low virulence of acinetobacter species [ ] . this study has several limitations. first, this was a retrospective study from a single center. information about recent antibiotics use, adequate antibiotics use, or a history of mdr infections could not be obtained. second, it was difficult to discriminate between colonizers and pathogens due to the study's retrospective nature. our results must be replicated in future prospective studies to be generalizable to other clinical settings. third, due to the center's training curriculum, the annual turnover of resident doctors occurs in march. therefore, resident doctors in february had nearly years of practice as physicians, whereas those in march had only year of practice and no experience as icu doctors. that turnover might have affected the clinical outcomes after the micu relocation [ ] . however, the number of mdr pathogens detected in the previous year did not reveal significant monthly variability (supplementary table ), which implies that the annual turnover of residents might not have affected the rates of mdr pathogen detection. fourth, environmental sampling of the micu before and after the relocation was not conducted. collecting that information in future studies could support our results. in conclusion, the ir of mdr respiratory microorganisms seemed to decrease after the micu relocation but then increased in subsequent months, thereby leading to a nonsignificant difference in the -month ir before and after the relocation. nonetheless, the use of colistin use was significantly reduced. although a difference in overall mortality was not observed, length of hospital stay was shortened. our findings emphasize the importance of environmental contamination control, especially for crab. prevalence of antimicrobial use in us acute care 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the association between physician turnover (the "july effect") and survival after in-hospital cardiac arrest no potential conflict of interest relevant to this article was reported. we would like to thank the staff of the medical intensive care unit and infection control center for their devotion in their daily practice to decrease the incidence rate of multidrug-resistant microorganisms. key: cord- -xogzl lv authors: alsuheel, ali mohammed; ali, abdelwahid saeed; al-hakami, ahmed musa; shati, ayed abdullah; chandramoorthy, harish c.; al-qahtani, saleh mohammed title: human metapneumovirus in pediatric patients with acute respiratory tract infections in the aseer region of saudi arabia date: - - journal: saudi j med med sci doi: . /sjmms.sjmms_ _ sha: doc_id: cord_uid: xogzl lv background: human metapneumovirus (hmpv) is a paramyxovirus known to cause acute respiratory tract infections in children and young adults. to date, there is no study from the aseer region of saudi arabia determining the proportion and severity of hmpv infection among pediatric hospitalized patients with respiratory infections. objectives: the objective of this study is to determine the presence of hmpv antigens in the nasopharyngeal secretions of pediatric patients hospitalized with respiratory tract infections in the aseer region of saudi arabia. materials and methods: this prospective, serological hospital-based study included all pediatric patients who were admitted to aseer central hospital, abha, saudi arabia, from july to november with upper and/or lower respiratory tract infections. basic demographics of patients and their clinical data on and after admission were recorded. direct fluorescent antibody assay was used to detect the presence of hmpv antigens in the obtained nasopharyngeal secretion specimens. results: during the study, pediatric patients were hospitalized due to upper and/or lower respiratory tract infections, of which . % were positive for hmpv. these patients were aged months to years, were from abha city or its surrounding localities and were mostly ( . %) hospitalized during autumn or winter. the most common diagnosis on admission was bronchopneumonia ( . %) and aspiration pneumonia ( . %), and some patients also had underlying chronic conditions such as chronic heart disease ( . %) and bronchial asthma ( . %). conclusions: the results obtained indicated that hmpv is a potential etiologic factor for the commonly occurring acute respiratory infections in hospitalized children from the aseer region of saudi arabia. hmpv infection was also found to be associated with complicated respiratory conditions such as bronchopneumonia, chronic heart disease and bronchial asthma. human metapneumovirus (hmpv) is a single-stranded rna-enveloped virus, recently classified in the order mononegavirales, family pneumoviridae, genus metapneumovirus and species hmpv. it was first isolated in the netherlands by van den hoogen et al., [ , ] and is now a known causative agent of upper and lower respiratory tract infections in children and adults. hmpv infections have been reported in australia, [ ] canada, [ ] the united states, [ ] the united kingdom, [ ] hong kong, [ ] south africa, [ ] mexico, [ ] spain [ ] and peru. [ ] however, in the middle east, there have only been few reports of hmpv infections, mainly as sporadic infections in egypt, [ ] jordan, [ ] kuwait [ ] and saudi arabia. [ ] along with respiratory syncytial virus (rsv) infections, hmpv is now recognized as a primary etiologic agent for acute upper and lower respiratory tract infections in pediatrics. [ , ] in a study from mexico, it was found that the number of hmpv infections increased in children aged - months as compared with those in younger age groups, whereas rsv infections were inversely proportional to increase in age. [ ] co-infection with both viruses can also occur, resulting in a more complicated and serious clinical disease. [ , ] in addition to the pediatric population, studies have also found hmpv to infect adults [ ] and elderly people. [ ] in terms of transmission, hmpv spreads through contact with contaminated secretions, i.e., droplet, aerosol or fomites. hospital-acquired hmpv infections have also been reported. [ ] in young children, infections are usually not asymptomatic, and bronchiolitis, with or without pneumonia, is the most common associated presentation. [ , , ] other reported conditions include bronchial asthma exacerbation, otitis media, pneumonitis, flu-like illness and community-acquired pneumonia. [ ] in adults, hmpv has been associated with bronchitis, pneumonia and exacerbations of both bronchial asthma and chronic obstructive pulmonary disease. [ , ] in terms of detection, reverse transcriptase-polymerase chain reaction (rt-pcr) is a sensitive and commonly used method to detect hmpv. [ ] real-time rt-pcr is also commonly used for detecting hmpv in clinical specimens with many genomic target sequences. [ , ] a touch-down genomic amplification protocol for the diagnosis of acute viral respiratory tract infections has also been used previously. [ ] the enzyme-linked immunosorbent assay for hmpv diagnosis is a simple and specific serological test for anti-hmpv antibodies detection. [ ] immunofluorescence using specific antibodies is routinely used for detecting hmpv antigens, particularly in epidemiological studies. [ ] however, cell culturing techniques have a low sensitivity in detecting hmpv from respiratory tract secretions, as the virus exhibits extremely limited types of cell tropism. [ , , , ] in saudi arabia, there is a paucity of data regarding the occurrence of hmpv and its role in complicated clinical cases of commonly reported respiratory infections. therefore, the current study aimed to determine the role of hmpv in the respiratory tract infections' severity and complications among hospitalized children in the aseer region, where no such study has previously been conducted. the study was conducted after obtaining approval from the ethical committee of college of medicine, king khalid university, saudi arabia (kku research ethics committee meeting no. rec # - - ; dated january , ) this prospective, serological study included pediatric patients who were admitted to aseer central hospital, abha, kingdom of saudi arabia, from july to november with upper and/or lower respiratory tract infections. aseer central hospital is the largest tertiary care referral hospital in the aseer region, and thus its sample is representative of the area. data such as age, gender, clinical presentation and current medications were collected using an objectively prepared questionnaire. informed consent was obtained from the parents/guardians of all patients before sample collection. nasopharyngeal secretions were collected from all hospitalized patients included in this study using the standard collection method. briefly, physicians collected the nasopharyngeal secretions with the help of a sterile feeding tube connected to a vacuum pump. following the vacuum application, the tip of the tube was cut and placed into a sterile container labelled with the patient's name and identification number. the container was then transported to the virology laboratory at the department of microbiology and clinical parasitology, college of medicine, king khalid university, abha, saudi arabia, and either processed on the same day or stored at − °c. specimens were processed according to the manufacturer's instructions for the direct fluorescent antibody (dfa) kit (oxoid ltd., cambridge, uk) with minor modifications. samples were transferred to an eppendorf tube containing ml of phosphate-buffered saline (pbs; ph . ). the specimens were gently vortexed for s to reduce the viscosity and dilute the mucus. samples were then centrifuged at rpm for min to separate the cells from the mucus. the supernatant was removed, and the cells in the pellets were used for dfa staining. the authors chose to use dfa because it has been found to be a useful technique for wider hmpv epidemiological studies. [ ] preparation of cells the cell suspension was washed several times with pbs and the final cell deposit was resuspended in ml pbs (ph . ). the cells were then gently agitated by pipetting up and down until the cellular material was released from the mucus. additional pbs was added until a smooth suspension was obtained, and any visible flecks of mucus were removed. after the cell separation process was completed, the obtained cell suspension was centrifuged at room temperature ( °c- °c) for min at rpm and the supernatant was discarded. the final cell deposit was resuspended in pbs to dilute any remaining mucus and maintain high cell density. a volume of µl of the resuspended cell deposit was placed in slides with -mm-diameter wells. the specimens were then allowed to air dry thoroughly and fixed with fresh acetone at room temperature ( °c- °c) for min. the slide was air-dried after fixation. a volume of µl of imagen™ hmpv reagent (oxoid ltd., cambridge, uk), which contains monoclonal antibodies against hmpv conjugated to fluorescein isothiocyanate, was added to the fixed cell preparation on the slide to cover the wells. the same amount was also added to the positive control slide. the slides were then incubated with the reagent in a moist chamber for min at °c. following incubation, excess reagent was washed off with pbs, and the slide was gently washed in an agitating bath containing pbs for min. the pbs was drained off, and slide was allowed to air dry at room temperature ( °c- °c). one drop of imagen™ hmpv mounting fluid was added to the center of each well, and cover-slip was placed over the mounting fluid and specimen to ensure that there are no trapped air bubbles. the stained slides were immediately examined under epifluorescence microscope at × and then × . apple-green fluorescence was observed in the cells infected with hmpv, whereas non-infected cells appeared as red color because they were stained with the evans blue counterstain. images of these cells were captured using a microscopic camera (nikon-ds-fi , nikon corp., tokyo, japan) and archived. during the study, a total of pediatric patients were hospitalized based on upper and/or lower respiratory tract infections. of these ( . %) patients tested positive for hmpv antigens, as demonstrated by dfa from the nasopharyngeal secretions [ figure ]. table provides the demographic and clinical presentation data of all hmpv-positive patients. the age of these patients ranged from months to years and all were saudi nationals except one infant, who was a jordanian by nationality but was born and raised in saudi arabia. from the patient's demographics, it was observed that hmpv antigens were detected not only in patients from abha but also among those from its bordering areas, namely, algahama, bilahmar, ahud rufida, sarat abeedah, khamis mushait and bilasmer. three of the nine positive cases were found from abha ( . %), and one positive case from each of the previously mentioned six cities was reported ( . %). of the nine hmpv-positive patients, seven ( . %) were hospitalized during the autumn and winter of - . in the hmpv-positive patients, the symptoms included fever ( . %), cough ( . ), shortness of breath ( . %), nasal congestion ( . %), cyanosis ( . %) and stridor ( . %). these patients also had underlying chronic illnesses such as chronic heart disease ( . %) and bronchial asthma ( . %), and most had tachypnea ( . %). on physical examinations, bilateral crepitation and wheezing were found to be the major findings along with bronchopneumonia ( . %) and aspiration pneumonia ( . %). in saudi arabia, although few studies have reported the incidence, epidemiological elements and genetic diversity of hmpv in some regions, [ , , ] there are no reports on the association between hmpv infections and the clinical presentations of respiratory tract infections among pediatrics. the current study found that in the aseer region of saudi arabia, about % of hospitalization among pediatrics with respiratory tract infections between july and november was due to hmpv infections. in addition, this study also found that hmpv infections were associated with presentation of acute respiratory symptoms, thereby highlighting the role of the infection in complicating the course of the disease. the results of the present study concur with several other studies demonstrating an association between hmpv infection and acute respiratory conditions such as bronchopneumonia and pneumonia. [ , ] a previous study implicated hmpv as a causative agent for severe and acute respiratory infections among pediatric patients, [ ] which is similar to the findings of the current study. another study found that children with hmpv infection are likely to have immunodeficiency; however, the current study was not able to substantiate these findings as none of the patients were found to be immunocompromised. [ ] the prevalence of hmpv infection of the current study (about %) was similar to that reported in studies from saudi arabia and kuwait. [ , ] however, other similar studies that used direct immunofluorescence assays for the detection of hmpv antibodies in sera of patients revealed much higher prevalence rates. [ ] this suggests that different results can be obtained with different diagnostic techniques for hmpv infection. in the current study, we used dfa with monoclonal antibody, which has previously been shown to have % specificity, thereby indicating the reliability of our results. [ ] the current study did not find any gender predilection in terms of the infections; these findings are in accordance with that of bastien et al. [ ] and kahn. [ ] in this study, all nine hmpv-positive children were from abha or its surrounding areas. these areas are known for their high altitude, low temperature and low oxygen tension. it was well known that the prevalence of viral respiratory infections is high in cold and dry areas. [ ] in addition, almost % of the hmpv-positive cases were in children who had been admitted during the autumn and winter seasons. it is also known that the majority of the viruses responsible for bronchiolitis and bronchopneumonia have their peak infectivity during winter and late autumn, with only sporadic cases through other seasons. [ , ] the hmpv-positive patients in the current study had fever, cough, nasal congestion, cyanosis, stridor and shortness of breath, while some also had underlying chronic illnesses such as chronic heart disease and bronchial asthma; similar hmpv-associated illnesses were observed in another study. 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metapneumovirus infections in croatia genetic diversity, seasonality and transmission network of human metapneumovirus: identification of a unique sub-lineage of the fusion and attachment genes human metapneumovirus infection plays an etiologic role in acute asthma exacerbations requiring hospitalization in adults human metapneumovirus infection in children hospitalized for wheezing there are no conflicts of interest. key: cord- - r q authors: dzimianski, john v.; beldon, brianna s.; daczkowski, courtney m.; goodwin, octavia y.; scholte, florine e. m.; bergeron, Éric; pegan, scott d. title: probing the impact of nairovirus genomic diversity on viral ovarian tumor domain protease (votu) structure and deubiquitinase activity date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: r q post-translational modification of host and viral proteins by ubiquitin (ub) and ub-like proteins, such as interferon stimulated gene product (isg ), plays a key role in response to infection. viruses have been increasingly identified that contain proteases possessing deubiquitinase (dub) and/or deisgylase functions. this includes viruses in the nairoviridae family that encode a viral homologue of the ovarian tumor protease (votu). votu activity was recently demonstrated to be critical for replication of the often-fatal crimean-congo hemorrhagic fever virus, with dub activity suppressing the type i interferon responses and deisgylase activity broadly removing isg conjugated proteins. there are currently about known nairoviruses classified into fourteen species. recent genomic characterization has revealed a high degree of diversity, with votus showing less than % amino acids identities within the family. previous investigations have been limited to only a few closely related nairoviruses, leaving it unclear what impact this diversity has on votu function. to probe the effects of votu diversity on enzyme activity and specificity, we assessed representative votus spanning the nairoviridae family towards ub and isg fluorogenic substrates. this revealed great variation in enzymatic activity and specific substrate preferences. a subset of the votus were further assayed against eight biologically relevant di-ub substrates, uncovering both common trends and distinct preferences of poly-ub linkages by votus. four novel x-ray crystal structures were obtained that provide a biochemical rationale for votu substrate preferences and elucidate structural features that distinguish the votus, including a motif in the hughes orthonairovirus species that has not been previously observed in otu domains. additionally, structure-informed mutagenesis provided the first direct evidence of a second site involved in di-ub binding for votus. these results provide new insight into nairovirus evolution and pathogenesis, and further enhances the development of tools for therapeutic purposes. introduction nairoviruses are negative sense single stranded rna [(-) ssrna] viruses within the order bunyavirales. initial classification of nairovirus species relied on antigenic cross-reactivity, leading to the clustering of viruses into seven serogroups; however, with the recent increase in the number of available viral sequences the classifications have shifted to a comparative genomics approach. this not only confirmed the diversity observed based on the serogroup classification, but also further accentuated how these viruses vary across the nairoviridae family. the family nairoviridae now consists of approximately viruses that are currently classified into species (fig ; [ - ] ). most nairoviruses are tick-borne viruses infecting multiple vertebrate host species they parasitize in nature. several have been implicated in human disease, the most notable being crimean-congo hemorrhagic fever virus (cchfv), which has reported case fatality rates in humans that can exceed % [ ] . other nairoviruses associated with human disease include dugbe virus (dugv), nairobi sheep disease virus (nsdv) and the asian variant ganjam virus (ganv), erve virus (ervev), issyk-kul virus (iskv) and kasokero virus (kasv). these viruses have been reported to cause a myriad of symptoms, some of which include fever, headache, and diarrhea [ ] [ ] [ ] [ ] [ ] [ ] . nairoviruses have also been observed to cause fatal animal disease. for example, nsdv has been reported to have a > % mortality rate in sheep and goats making it a significant economic as well as human health concern [ ] . a recently characterized nairovirus, leopards hill virus (lphv), was isolated from bats and causes severe gastroenteric hemorrhaging and hepatic disease in mice [ ] . hazara virus (hazv) was isolated from ticks collected from the royle's mountain vole and has been proposed as a model system to study cchfv based on its ability to cause similar fatal disease in interferon (ifn)-receptor knockout mice [ , ] . beyond these viruses causing disease in mammalian hosts, other nairovirus have been associated with a broad taxonomic diversity of vertebrate hosts such as birds, fish, and reptiles. for example, viruses in the hughes orthonairovirus species, such as farallon virus (farv), have been implicated in infecting birds [ ] . nairoviruses possess a tripartite genome consisting of small (s), medium (m), and large (l) segments that encode the viral nucleoprotein, glycoproteins, and rna-dependent rna the tree was constructed utilizing the jones-thornton-taylor model in the mega program [ ] . current species groupings are indicated by colored ovals, and the assigned species denoted. previous serogroup classification, if applicable, is shown in parentheses. virus votus included in this study are denoted by red lettering. inset is a structure-based phylogenetic tree of votus, with the mammalian cezanne, a , and otulin otus included for comparison. the tree was constructed using pdb ids prp, hxd, jze, dx , dx , dx , dx , lrv, lrx, and znz in the multiseq module of vmd [ ] . sequence accession numbers are included in s table. polymerase, respectively. interestingly, the nairoviral l segment also encodes a viral homologue of the ovarian tumor protease (otu) at the n-terminus. this feature uniquely distinguishes the nairoviridae family and genus tenuivirus from other members of the order bunyavirales. the viral otu (votu) does not appear to play a direct role in genome replication and is dispensable in minigenome replication systems [ ] . instead, the votu's primary function appears to be the reversal of post-translational modifications by ubiquitin (ub) and the ub-like protein interferon stimulated gene product (isg ) . this votu-encoded deubiquitinase (dub) and deisgylase activity has been implicated in evading the innate immune response [ ] [ ] [ ] . ub is an . kda protein that is involved in a wide range of cellular processes, including key regulatory functions in innate immunity. ub is conjugated to target proteins by means of a three step process involving activating (e ), conjugating (e ), and ligating (e ) enzymes, and can either occur as a single ub moiety (mono-ub) or in polymeric and branched forms (poly-ub). these chains can be formed by linkage through either the n-terminus (linear) or one of seven lysine residues in ub (k , k , k , k , k , k , and k ), with different forms often mediating different downstream effects. the most thoroughly studied forms, k and k , play important roles in regulation of the innate immune responses. specifically, k -mediated proteasomal degradation has been associated with feedback control, while k polyubiquitination is required for pathway activation, including retinoic acidinducible protein i (rig-i), mitochondrial antiviral signaling protein (mavs), tumor necrosis factor (tnf) receptor associated factor (traf ), tank binding kinase (tbk ), and ifn regulatory factor (irf ). this signaling cascade leads to the production of ifn-α/β, which ultimately results in the upregulation of numerous ifn-stimulated genes, including isg [ , ] . the role of isg is complex and not well understood but is generally associated with mediating and regulating antiviral responses both as a co-translational modification and as free isg in the cytosol and secreted form inducing the secretion of ifn-γ and il- by binding cell surface receptor lfa- [ ] [ ] [ ] [ ] [ ] [ ] . initial studies on nairoviruses, including cchfv, dugv, and nsdv, established the potential immune modulatory effects of votu activity based on overexpression of the respective isolated otu domain in cell culture [ ] [ ] [ ] . the ability to probe the specific role of the votu during the viral replication cycle remained elusive, however, until the recent development of a reverse genetics system for cchfv. these studies revealed distinct roles for dub versus deisgylating activity during the course of a cchfv infection [ ] . specifically, that cchfv votu dub activity is not as promiscuous towards ubiquitinated host proteins as it first seemed based on the overexpression studies, but appears to be restricted to a targeted subset of cellular substrates associated with suppression of rig-i-mediated early cellular responses to infection. in particular, wildtype cchfv was able to reduce the induction of several immune components, including rig-i, while cchfv with a votu specifically lacking dub activity resulted in enhanced cellular responses to infection and establishment of a cellular antiviral state that reduced viral titers. in contrast, deisgylating activity appears to play a role in later stages of cchfv infection. a recent study demonstrated a similar impact of dub activity in viral immune suppression during the replication cycle of severe acute respiratory study aligned using the t-coffee sequence alignment program [ ] . percentages show the sequence identity relative to cchfv votu. generic votu secondary structure based on define secondary structure of proteins (dssp) algorithm calculations for the votus is shown in reddish orange, with the α and α helices of farv votu shown in teal. the catalytic triad is boxed in black and the selectivity pocket in orange. mutation sites related to the selectivity pocket are shown by yellow stars, sites related to differences in how farv votu engages mono-ub by blue stars, and the dgkv votu catalytic triad mutant by a green star. mutation sites for the second ub binding site in farv votu are denoted by red stars. the region deleted in the farv votu Δ - construct is indicated by a bracket. syndrome coronavirus (sars-cov) [ ] . specifically, when the dub activity of the sars-cov papain-like protease (plpro) was selectively disrupted, the virus showed increased sensitivity to ifn and slower growth kinetics. furthermore, domain exchanges of plpro's between different sars-cov variants supported this observation, establishing dub activity to be a distinguishing virulence trait. these emerging insights into the impact of dub activity in the cchfv votu and sars-cov plpro during viral replication emphasizes the importance of robust dub activity among pathogenic viruses. the demonstrated votu-associated dub/ deisgylase activity of other nairoviruses such as dugv, ervev, and nsdv/ganv, further highlights a potentially substantial role of the votu in viral replication and immune suppression for viruses in the nairoviridae family [ ] [ ] [ ] ] . remarkably, the nairoviral votu domain shows a great degree of sequence diversity, with sequence identities that can drop below % between species (fig ) . a particularly striking case of this diversity is found in members of the hughes orthonairovirus species, such as farv, which possess - additional residues in the middle of the otu domain ( fig b) . these sequence differences between votus suggest a plasticity in the otu domain that could play a role in evolutionary adaptation. currently, exploration into the phenotypical effects of this diversity has been restricted to only a few taxa that include cchfv, dugv, nsdv/ ganv, and ervev [ , ] . these studies revealed that votus possess different enzymatic and structural characteristics. in particular, votus display a wide degree of variation in the efficiency with which they engage ub and isg that is driven by specific sequence and structural features. these substantial differences in viruses within closely related taxa raises questions on the impact of votu diversity across the nairoviridae family. specifically, how votus from viruses in each species vary in structure and activity, and the implications of this for the potential to suppress the innate immune response and affect viral pathogenesis and host tropism. to better understand the impact of votu diversity, we sought to obtain a more complete perspective of the functional and structural features of votus within the nairoviridae family. in vitro assays revealed that votus across diverse taxa possess ub activity, but that activity towards isg appears more restricted. further characterization of votu activity uncovered distinct trends and preferences for specific poly-ub linkages. to better understand the molecular mechanisms driving ub activity and specificity, novel x-ray crystal structures were solved revealing features that distinguish the votus from each other, including a pocket that correlates with ub specificity. additionally, a structure of the farv votu provides details into the structural nature of the additional residues in hughes orthonairovirus votus. structureinformed mutagenesis of farv votu identified residues involved specifically in di-ub binding, representing the first report of the role of a second site involved in di-ub binding in nairovirus votus. this novel enzymatic and structural data not only provides insight into the nature of votu diversity, but also lays a foundation for understanding the impact of the votu interaction with the innate immune response and its connection to viral pathogenesis. to gauge votu diversity across the nairoviridae family, viruses representing the divergent species were selected and the otu domain recombinantly expressed. initially selected based on the traditional serogroups as well as emerging genetic characterization, these viruses include members of the most distantly related taxa and represent of the currently recognized species in the dynamic classification landscape of nairoviruses (fig ) . included were the votus from cchfv, nsdv and ganv, dugv and kupe virus (kupev), hazv, taggert virus (tagv), ervev, farv, dera ghazi khan virus (dgkv), huángpí tick virus (hptv- ), lphv, qalyub virus (qybv), and iskv (fig ) . to better understand the global diversity of nairoviral engagement with ub and isg substrates, these votus were assessed for activity towards ub and human isg fluorogenic substrates. these specific activities were measured by the accumulation of the fluorescent molecule -amino- -methylcoumarin (amc) as a result of cleavage from the c-terminus of ub or isg (fig ) . intriguingly, the votus showed a diverse range of activity towards ub. in general, votus can be divided into groups possessing high (cchfv, hazv, nsdv/ganv, tagv), moderate (dugv, kupev, farv, qybv, iskv), or low activity (ervev, dgkv, lphv, hptv- ) (fig a) . for some of these votus, their deubiquitination activity mirrors that observed in dub-deficient cchfv mutants that impact cellular ubiquitination levels leading to an impaired ability to suppress the ifn response [ , ] . to a large degree, viruses more closely related phylogenetically with cchfv possess the most robust activity (figs a and a ). beyond this, there is not an obvious phylogenetic trend to how well the votus cleave ub-amc, with disparate taxa showing similar low to mid-range activity. overall, engagement with ub is observed to be a feature that can be present in diverse species in the nairoviridae family, with some taxa demonstrating enhanced activity. the patterns of activity for ub are in stark contrast to those of isg -amc, which shows a more dichotomous pattern as a substrate for the votus (fig b) . specifically, there appears to be an abrupt break phylogenetically between groups that contain votus with deisgylating activity, compared to others for which activity is almost negligible. this break appears to exist at the node separating the thiafora, artashat, sakhalin, crimean-congo hemorrhagic fever, nairobi sheep disease, dugbe, and hazara orthonairovirus species from the remaining seven ( fig a) . interestingly, the presence of isg activity does not encompass every votu in these species, suggesting individual factors may have driven the development or retention of isg activity for viruses within this clade. naturally, this also implies that dub activity could be a more broadly utilized mechanism to evade cellular responses. this led us to further explore the dynamics of different nairovirus votu's interactions with ub. while the ub-amc assay provides general information on the ability of votus to engage monomeric ub, cellular substrates are typically modified by poly-ub chains through various linkage types [ ] . additionally, dubs in general and votus in particular have been observed to prefer some linkage types over others [ , [ ] [ ] [ ] [ ] [ ] . to assess the patterns of ub linkage preferences of diverse votus, a subset of the votus were analyzed against di-ub fret-tamra substrates ( fig a) . hazv votu and ganv votu were selected because they have been considered to be a potential model system for cchfv and have significant health and economic impact, respectively. tagv votu represents a more distantly related votu that also has substantial dub activity, while the votus encoded by qybv, farv, and dgkv display diminished activity towards mono-ub. to reduce the influence of interactions with the fret pairs that may disrupt interaction, multiple fret pair configurations were assessed when available and the one displaying the highest activity selected (two positions each for k and k ; s fig) . comparison of votu activities towards different di-ub fret substrates reveals that each species' votu has distinct preferences for specific di-ub linkages. while hazv votu and ganv votu both possess notable activity for k and k di-ub, there appears to be more substantial activity towards k . tagv votu, on the other hand, prefers k and k to a greater extent, while not possessing as much activity towards k . for farv votu, the opposite is observed, with k being preferred. dgkv votu, consistent with its low ub-amc activity, possesses very low activity for the di-ub fret substrates, regardless of the linkage. similar to the pattern observed for hazv and ganv, qybv votu shows the most activity towards k , though at lower overall levels. additionally, qybv votu shows a more pronounced difference in the relative preference for k versus k linkages, with substantially more activity towards k . regrettably, the commercial availability of fret-tamra di-ub substrates is restricted to these tested linkages. additionally, limitations are known to exist due to how the positions of diversity of nairovirus votu structural fold and substrate preference the donor-quencher pairs affects binding of these substrates with the proteases. to gain a more complete and natural perspective of di-ub linkage preferences, these votus were also assessed by sds-page for the ability to cleave unlabeled di-ub substrates of all eight linkage types ( fig b) . as expected, the votus did not show equal preferences for the different linkages. intriguingly, some of the results appeared to differ from the fret analysis. specifically, for both hazv votu and farv votu the gel cleavage assay would suggest the k activity to be the highest with k and k roughly equal, suggesting that the positions of the donorquencher pairs may have hindered binding to some of the substrates. as expected, the hazv, ganv, and tagv votu showed substantial cleavage of several di-ub substrates that is consistent with their high ub-amc activity, while the dgkv and qybv votus showed low level of substrate cleavage over the same time course. intriguingly, farv votu showed substantial cleavage of some of the substrates, despite not possessing high ub-amc activity. this may be reflective of differences in the assays in measuring dub activity. alternatively, it may also suggest the existence of an additional site of interaction with the proximal ub that enhances the efficiency of di-ub cleavage. assimilation with previously reported data reveals interesting trends and points of divergence between the votus ( [ ] ; fig c) . linear and k -linked di-ub does not show any sign of cleavage with any votu. votus demonstrate varying levels of low/detectable activity on k -linked and k -linked di-ub. in contrast, votus show a consistent pattern of higher activity towards k , k , k , and k di-ub. while most of the votus show some level of enhanced activity towards these linkages, the specific linkage most preferred can differ. the cchfv, hazv, tagv, and farv votus all show a distinct preference for k over k linkages, while dugv and qybv votu show more activity towards k . ganv votu shows approximately equal preference for these linkages. interestingly, ganv votu also shows more activity towards both k and k di-ub at approximately equal levels. the high degree of preference towards these substrates extends to the majority of the votus, as even for dgkv votu, which shows minimal or no cleavage of most of the substrates, cleavage of k -linked di-ub can be identified within an hour ( fig b) . the other votus, with the exception of ervev, all show detectable levels of k cleavage, with most also cleaving k . the cchfv, hazv, dugv, and farv votus all showed a greater relative preference for k over k , while tagv votu was the opposite. qybv votu, similar to ganv, showed approximately equivalent activity towards k and k , though overall activity was lower. overall, these patterns of activity suggest that votus do not merely cut any ub moiety, but that they are specific to a subset of linkages that may influence specific aspects of cellular biology. to gain a better understanding of how sequence diversity translates into structural differences, x-ray crystal structures were sought of votus from divergent species. the votus from dgkv, qybv, and tagv readily crystallized and were solved to . Å, . Å, and . Å, respectively (table ) . these votus represent diverse nairovirus species, and possess extensive variation in ub activity with the dgkv, qybv, and tagv votus possessing low, medium, and high activity towards ub-amc, respectively (fig ) . tagv provides a glimpse into the sakhalin orthonairovirus species, a taxon that is more closely related to the ervev and cchf/nsd/dugbe/hazara cluster, while dgkv and qybv are from the dera ghazi khan and qalyub orthonairovirus species, respectively, and are much more distantly related (fig ) . these structures reveal global similarities among the votus (fig and s fig) . each votu possesses a seven-stranded beta sheet as the core feature, with five major alpha helices framing the rest of the structure. the catalytic triads perfectly superimpose over each with the exception of dgkv votu ( fig a) . in dgkv votu, aspartate is replaced by a glutamate that alters the spatial dynamics of the catalytic triad, possibly contributing to a less rigid structure that allows the histidine to adopt the alternate conformation. while atypical for the votus, it does not appear to be the cause of dgkv's low dub activity, as mutating the glutamate to an aspartate only further diminished activity. looking beyond the catalytic triad, a structural overlay of the votus highlights a point of difference in the overall structure that distinguishes the proteases from each other. specifically, there appears to be substantial variability in the region encompassing the α ("selectivity") helix that has been associated with substrate preference, and the loop between the β and β a strands ( fig a; [ , ] ). comparing the root mean square deviation (r.m.s.d.) for the positions of the main chain atoms of these different structures further emphasizes how they deviate from structure to structure (s fig). closer examination of the structures reveals distinct molecular interactions that account for these observed structural differences within the votus. specifically, particular amino acid differences can be identified that form interactions that would promote the observed conformation of each protease, suggesting these differences to not merely be a consequence of dynamics diversity of nairovirus votu structural fold and substrate preference or crystal packing. intriguingly, these residues are not limited to just the selectivity helix and β -β a loop but extend to other secondary structural elements in local proximity, forming an ensemble of interactions that drive the noticeable variability of the α region (fig b and c ). the first area of prominent influence centers around position of cchfv votu (fig c, panel i). this position is strongly conserved in possessing an aromatic residue, consisting of a phenylalanine or a tyrosine in cchfv and more closely related viruses, while consisting of a tryptophan in the rest of the votus studied ( fig b) . while subtle, this change results in distinctly different local interactions that influence the positioning of the selectivity helix. in the cchfv votu, tyr forms hydrogen bonds with the backbone of leu within the α -α loop. in contrast, the tryptophan residues in the other votus fill into a hydrophobic cleft that involves residues within the selectivity helix. in dgkv votu, trp packs with the methylene group of ser on one side and the aliphatic portion of lys 's side chain on the other. lys itself is stabilized in this permissive conformation by hydrogen bond pairing with the carbonyl of gly . in qybv votu, trp packs with pro . additionally, it is in proximity to his , suggesting potential stacking of the rings. such an interaction could have an indirect effect on the positioning of pro at the surface of the votu-substrate interface. in tagv votu, trp fits between leu and thr . the second area centers around helix α and shows a great degree of variation between the votus (fig c, panel ii) . it can encompass interactions that can extend to the α and α helices as well as the β a sheet with the potential to influence the local structural architecture. cchfv votu possesses a number of interactions within this region, including unique lysine pairings consisting of lys , glu , lys , and glu that accommodate hydrophobic packing with tyr and phe . along with hydrogen bond pairing of glu with thr , these work in conjunction in orienting the position of the selectivity helix, a region that has been implicated in substrate preference [ , ] . the other votus, in contrast, possess fewer interactions but still could influence the structure. in dgkv votu, gln within the selectivity helix is central to the interaction, pairing with both ser and tyr by hydrogen bonding. similarly, for tagv votu thr and glu form electrostatic interactions with gln and tyr , respectively. in contrast, for qybv votu there do not appear to be any direct interactions with the selectivity helix. instead, lys and ser form electrostatic interactions with asp and asn , respectively, suggesting a role in manipulating the positioning of the β -β a loop. the third region consists of the selectivity helix and β -β a loop themselves ( fig c, panel iii). specifically, direct interactions, or the lack thereof, work in conjunction with the other interactions to complete the structural features. this is most notable in qybv votu, in which phe appears to pack with pro . in cchfv votu, the corresponding residue is ile , which does not appear to be able to bridge the distance and form an interaction. in tagv votu, asn and thr are in general proximity, but appear to be too distant to form a strong interaction with each other. similarly, dgkv votu appears to lack any direct interactions. overall, these interactions contribute to structural features that influence spatial and chemical presentation of the votu interface, potentially affecting how these votus engage substrates. binding with ub often centralizes around the specific hydrophobic residues leu , ile , and val [ ] . looking at x-ray crystal structures of the cchfv and dugv votus bound to ub reveals that leu in particular has to be spatially accommodated in a pocket deep within the interaction interface (fig a) . to confirm that this interaction with leu is likewise involved in ub binding with these other votus, isothermal titration calorimetry (itc) was performed using the tagv votu to determine the relative binding efficiency of alanine and asparagine ub mutants (ub-l a and ub-l n) compared to wt ub (table , s a fig) . this revealed a stark difference in the affinity. while wt ub bound strongly with a dissociation constant (k d ) of . ± . μm, ub-l a showed no detectable binding under similar conditions. the ub-l n mutant faired only slightly better than the ub-l a mutant with a times weaker dissociation constant, k d of . ± . μm, compared to wt. these results further underscore the importance of votus being able to accommodate ub leu in order to have robust deubiquitinating activity. examining the analogous residues in the other votus reveals a diverse composition for this pocket that could influence how well leu can be accommodated. in tagv votu, this pocket is largely hydrophobic possessing two tyrosines as well as a valine. in contrast, the dgkv and qybv votus possess more polar residues, including asparagine, glutamate, and threonine for dgkv and glutamine and lysine in qybv. when considering the activity towards ub based on the amc assay, a general trend emerges that correlates the degree of an enzyme's ability to engage mono-ub with the hydrophobicity of this pocket. looking more closely at this interface suggests an additional nuance to the ability to accommodate particular substrates. specifically, spatio-chemical characteristics could largely influence what defines a good or acceptable pocket composition for binding a given substrate. in the votus that most effectively engage with ub, such as cchfv, hazv, nsdv/ganv, and tagv, the residue that most directly interfaces with ub's leu is an isoleucine, valine, or threonine that corresponds to position in cchfv votu (figs b, and a ). this correlation is consistent across the nairoviridae family. despite being phylogenetically distant from cchfv and the other robust votu dubs, qybv demonstrates substantial ub activity and possesses ile that could pack with ub's leu . in other votus that have poor ub activity this residue is typically polar, such as ervev's asn (fig b, panel i) . this creates an environment that would discourage binding with ub. mutation of this residue in ervev to the corresponding hydrophobic residues in cchfv has been observed to generate robust ub activity [ ] . the farv and iskv votus appear to have similar characteristics, with both encoding a glutamine at this position. intriguingly, other votus may go even further in discouraging ub binding. these include lphv and htv- , which possess an arginine and lysine, respectively, at their equivalent positions to asn . modeling in an arginine at this position, such as what lphv votu possesses, reveals that this type of change would be prohibitive for ub binding (fig b, panel ii) . to test the central role of this pocket for ub activity, a series of mutants were made in the dgkv, qybv, and tagv votus and tested against ub-amc ( fig c) . as expected, the disruptive mutants i r in qybv and y r and v r in tagv completely knocked out the ability to process ub-amc, in keeping with a previous report demonstrating that the presence of arginine hindered ervev votu ub activity [ ] . further, increasing the hydrophobicity of the pocket in qybv votu was able to enhance ub-amc cleavage, boosting activity by % and % for the q v and q a mutants, respectively. interestingly, changing the pocket in dgkv votu failed to improve activity. this suggests that some votus lacking any outright dub activity may have evolved to a degree that prevents the generation of this activity through simple changes to better accommodate leu in ub. this leaves the presence of a ub leu accommodating pocket in votus as a major marker for deubiquitinating activity and, if present, the ability to dictate variable levels of activity based on the hydrophobicity. table ). inspection of the structure immediately revealed the impact of this additional sequence on the protease's structure ( fig a) . while possessing the familiar core domain and secondary structure features, the protease possesses extended α /α helices that are connected by several intervening residues. thirteen residues could not to be built due to a lack of well-defined electron density in the crystal structure, and those that could be modeled possess high b factors, suggesting this region to have a high degree of flexibility. this contrasts with the votus from cchfv and other nairoviruses that possess relatively small α /α helices connected by a short loop (fig b) . additionally, the α /α helices of farv votu appear to interact with each other in a manner resembling a coiled-coil motif. this is facilitated by hydrophobic packing between ile , val , ala , and the aliphatic portion of the arg sidechain. this relationship between the helices is further promoted by electrostatic interactions, including a salt bridge between arg and asp , as well as a hydrogen bond between tyr and asp . beyond this interaction, tyr is also positioned to hydrophobically interact with tyr , which together create an environment in which trp can insert. trp further promotes the interaction between these helices through a hydrogen bond with asn , as well as through additional hydrophobic packing with lys and leu . the presence of the extra sequence/structural motif in the hughes orthonairovirus species raises the question of whether it could be involved in substrate interaction. a model of how farv votu could interact with ub further accentuates this possibility, suggesting the α /α helices to be in close enough proximity to participate in binding (fig ) . such an interaction could potentially offset other factors in farv votu are not ideal for binding. looking at the coot. (b) the extra space (black arrow) existing in the ervev-mouse isg structure (pdb id jze), with the cchfv votu bound to ub included for comparison (panel i). qybv votu with ub and mouse isg (green) modeled in based on votu secondary structure alignments, with an arginine also modeled into the space opened up by the mouse isg conformation (panel ii). (c) activities for ub-amc for mutant dgkv, qybv, and tagv votus relative to wt. error bars represent the standard deviation of two independent experiments. https://doi.org/ . /journal.ppat. .g selectivity pocket of farv votu reveals it to possess more of a hydrophilic character and contains a relatively bulky gln residue in the equivalent site to position in cchfv (fig a, panel i) . additionally, farv votu possesses a potential steric hindrance to efficient binding with the presence of arg (fig a, panel ii) . this residue may be less accommodating for the leu in ub than other votus, such as cchfv and tagv which contain a histidine at this site. further, farv votu may lack a significant interaction that cchfv votu possesses with arg of ub (fig a, panel iii) . in contrast to gln in cchfv votu that is able to form a hydrogen bond, farv votu possesses a leucine that is unable form this interaction. to test the influence of these sites on dub activity, mutations were made to arg and leu in farv votu to histidine and glutamine, respectively. as anticipated, r h was able to improve ub-amc activity, boosting it by~ %. making the reverse mutation in tagv votu, h r, essentially knocked out this activity suggesting this residue to have a key impact in diminishing farv votu's activity compared to other votus. interestingly, the l q mutation in farv votu led to a large reduction in ub-amc cleavage. looking more closely at this region shows that leu is in the middle of a large hydrophobic region in farv votu (s b fig) . the swap to a large polar residue may impact the structural integrity of the β -β a region, further underscoring the nuances created by the variability of this region. to probe the potential significance of the α /α motif in offsetting these other effects in farv votu, a construct was synthesized lacking residues - ("farv votu Δ - ") and assessed for activity against ub substrates (fig b) . removing this region reduced activity towards ub-amc by almost %, suggesting that this motif could play a significant role in ub binding (fig b, panel i) . interestingly, when tested against k and k fret di-ub substrates a more modest reduction in activity is observed, with only about a % and % reduction in activity, respectively. this is further borne out with unlabeled di-ub, with there being no substantial difference between the wt and Δ - votus over the longer reaction time course (figs b and b, panel ii). although the di-ub cleavage assays are able to differentiate linkage preference, the structural architecture is still relatively simple. to gauge whether this motif can engage with more complex poly-ub structures, the wt and Δ - farv votus were tested with k and k linked tri-ub (fig b, panel iii) . interestingly, both constructs showed a clear preference for k over k tri-ub. this is in contrast to the gel cleavage assay for di-ub, which showed a slight preference for k di-ub (figs b and b, panel ii) . beyond this, both constructs showed similar patterns of activity for these substrates. despite possessing low to moderate activity towards ub-amc, farv votu possesses substantial activity towards some di-ub linkages (figs and ) . this suggests an additional site of interaction with the proximal ub molecule that substantially increases the overall efficiency. to ascertain where this site may be located, a model of how farv votu may bind di-ub was generated (fig ) . examining the potential interface with the proximal ub, two residues in farv votu, arg and lys , immediately stand out as potential contributors. these residues are just beyond the active site, and are part of a region that likely forms the closest contact with the proximal ub. beyond these two residues, thr of farv votu also stands out as being in an area with a higher r.m.s.d. between the votu structures, which in farv votu positions it closer to the general area of the proximal ub (fig b and s fig) . to assess whether these sites could play a role in the farv votu's interaction with di-ub, mutations at these positions were designed in an attempt to alter activity towards k and k fret di-ub substrates. as a control, each mutant was also run with mono-ub substrates. due to the proximity of arg and lys to the space that would be occupied by the fluorogenic molecule, assays were performed with both ub-amc and ub-rhodamine (rh ) to mitigate artifacts. excitingly, these mutations substantially altered the rate of di-ub cleavage, often towards both substrates (fig ) . individually mutating arg and lys to leucine reduces activity towards k di-ub to - % of wildtype and activity towards k to - %. although the mono-ub activity appears to suffer as well in the case of r l, the~ % difference in the ub-amc versus ub-rh mono-ub substrates suggests this to be an artifact of interactions with the amc fluorophore. otherwise, these mutants had little or no effects on mono-ub activity. when a charge flip was introduced at position , activity was reduced to % for k and % for k while not substantially altering the activity for mono-ub. a charge flip at position had the most pronounced effect, driving it down to % for k and % for k . however, there is also a substantive corresponding reduction in both the amc and rh mono-ub substrates, indicating a potentially large disruptive interaction with the hydrophobic fluorophores or possible influence on the local fold. interestingly, while mutating thr to valine did not appreciably change the activity, introducing an arginine at this position increased the activity by % for k and doubled it for k , suggesting the longer sidechain may be able to form a new interaction. ub is among the most conserved and important cellular regulatory components, influencing almost every key aspect of cell biology. ub itself is tightly regulated by an array of endogenous dub enzymes that specifically curb and tailor its effects. the realization that viruses also possessed enzymes with dub activity introduced a paradigm in which these normal regulatory mechanisms could be manipulated to suppress immune responses and enhance viral propagation [ , [ ] [ ] [ ] [ ] . further investigation into these mechanisms continues to uncover how these viral dubs disrupt cellular responses to infection. in particular, the role of robust dub activity in promoting viral replication and conferring virulence in cchfv and sars-cov emphasizes the impact of the respective proteases and highlights the emerging importance of understanding their effects when considering potential pathogenicity and therapeutic strategies. with the almost perfectly conserved sequence of ub, it is not surprising that tick-borne nairoviruses from disparate taxa possess notable dub activity. such a mechanism could provide broad utility in infecting hosts beyond the primary arthropod reservoir by enabling a route of horizontal as well as vertical transmission that amplifies viral replication. the diversity in the observed activity, however, raises questions as to the specific effects relating to arthropod versus vertebrate hosts. in general, the votus from viruses most closely related to cchfv appear to have the most substantial dub activity based on the ub-amc assay (figs and ) . these viruses are known to cause viremia in vertebrate hosts, including mammals. this raises the prospect that increased ub activity may be an adaptive mechanism allowing these nairoviruses to infect a wider host range. while ticks are known to possess rnai and toll sensingmediated antiviral responses, there is little information pertaining to whether ub plays a significant role in arthropod responses to viral infection [ ] [ ] [ ] . further characterization of ub systems in arthropods will be needed to shed light into these questions and would clarify the significance of votu enzymatic diversity in nairovirus adaptation for arthropod versus vertebrate hosts. in contrast to some mammalian dubs, otu proteases generally show poly-ub linkage specificity that ranges from moderate to highly specific [ ] . nairovirus votus reflect this tendency, possessing activity towards poly-ub linkages that is neither highly promiscuous nor completely selective for a single linkage type with each votu possessesing its own respective preferences for the different linkages. while showing individual variation, the votus consistently show the ability to process k , k , k , and k linked di-ub. the fact that nairovirus votus generally show activity towards k and k di-ub is significant. these well-studied forms of di-ub have clearly established roles for cellular processes in general, as well as in antiviral responses specifically. it can be easily envisioned how disruption of k and k -mediated functions could dampen antiviral responses. it is intriguing, however, that votus as a whole also possess substantial activity towards k and k linkages. these forms of ub have been studied much less extensively, with roles that have typically been associated with dna damage responses and cell cycle regulation [ , ] . as part of the l protein, votu activity would be restricted to the cytosol, raising questions as to whether these observed activities of votus are incidental, or if there are important cytosolic functions of k and k linkages that could be manipulated. recent studies have begun to expand knowledge of these linkages. specifically, k poly-ub has been associated with tnf signaling, providing a direct link to the innate immune response [ ] . even more recently, k has emerged as a key component in regulating mitophagy [ , ] . given the key role of mitochondria in innate immunity, this raises an interesting question of how votu activity could impact this process, and whether such manipulation could provide benefits for the virus [ ] . what other functions remain to be identified for these linkages is still an open question, as well as how votus may engage with them to modify cellular responses. the differences in linkage preferences between votus implies potential differences in the degree to which specific viruses may influence these pathways. alternatively, it's possible that the relative importance of the linkages may differ in different hosts, and that different votu preferences reflects virus adaptation to their specific preferred hosts. the new votu structures reveal an array of conserved and divergent features. the conserved elements of nairovirus votu structure distinguishes these from other otu proteases, as highlighted by how they cluster together in a structure-based phylogenetic tree (fig a, inset; [ , , ] ). most notably, this includes the presence of two additional beta sheets and a helix at the n-terminus of votus that are absent from eukaryotic otus. while possessing these characteristic features of the votu fold, the nairoviruses show distinguishable differences from each other that can be traced to specific residue differences. this is particularly noteworthy when looking at the relationship between the selectivity pocket and the observed ub activity by a given protease. it is significant that votus possessing the highest activity for ub all possess highly hydrophobic residues in this region. while many of the votus possessing robust dub activity are closely related phylogenetically, the presence of substantial activity in the more distantly related qybv votu demonstrates that it is not exclusive to this subset of viruses. this suggests the votu fold to be a flexible platform that has allowed dub activity to evolve independently to the benefit of each virus. beyond the central role of this pocket that is deep in the binding interface, the votus also display structural diversity in more peripheral regions. this includes areas that have been observed to influence substrate binding in votus, such as the α selectivity helix, suggesting a potential impact on how votus engage with other proteins. viruses in the hughes orthonairovirus species possess a motif previously unobserved for otu domains. this raises the question of whether this structural feature could have a functional impact. in particular, whether this motif could impact engagement with substrate. the effect of removing this motif from the farv votu on ub-amc activity suggests that it can at least contribute to mono-ub binding. this is consistent with what is observed when comparing farv votu to a ub-bound structure of cchfv votu, where elements of this motif are in proximity for potential interactions with ub ( fig a) . this additional interface provided by the motif likely compensates for the presence of other less optimal factors for ub binding, including an arginine that hinders interaction with the tail of ub. overall, these structural features suggest a mixture of elements that either promote or hinder interaction, with some that may carry a more dominant effect. the involvement of the structural motif in farv votu formed by the two helices and intervening loop, which we also refer to as a substrate interacting bundle (sib), suggests it may form a region that introduces potential to engage with otherwise inaccessible surfaces. interestingly, removing the sib motif from farv votu appears to have a lesser impact on di-ub activity compared to mono-ub (fig b) . this could be accounted for by the presence of an additional site of interaction in farv votu that interacts with the proximal ub. the existence of one or more subsites has been postulated as a mechanism for discriminating different di-ub linkages based on the proximal ub, and has been demonstrated in several mammalian otus [ , ] . while not definitively observed in votus, the ability to distinguish between different linkages implies a similar mechanism. the farv votu mutants provide the first reported direct evidence identifying such a site in a votu, confirming that votus can utilize this mechanism to distinguish various linkages. in addition to supplying potential leads for elucidating such sites in other votus, it also demonstrates a case where this site can have a major impact on activity towards substrate, even when factors hindering binding with the distal ub are present. although di-ub wouldn't directly interact with the sib motif in a manner that would directly influence cleavage, it is possible that a more complex poly-ub structure could engage with it. modeling how tri-ub might bind suggests that a k linkage could place one of the ub molecules in close proximity to the structural motif (fig c) . in contrast, for the other linkages farv votu most readily cleaves-k , k , and k -this ub would likely be too distant to form any interaction. surprisingly, removal of the sib motif has no noticeable impact on k tri-ub cleavage, despite the apparent proximity the tri-ub could have. it's possible that tri-ub may not possess a large enough architecture to be influenced by the sib motif, and that a longer poly-ub may interact with it. additionally, ub is able to form complex chains consisting of multiple linkage types [ ] . it may be that the sib motif can engage more effectively with these "heterotypic" ub chains. alternatively, the primary role of the sib motif may go beyond ub and facilitate interactions with other binding partners. the votu domain exists in the context of the multifunctional l protein. apart from the votu domain, the structural features and dynamics of the nairovirus l protein are currently unknown. this leaves open the possibility that the sib motif could be involved in binding another feature of the l protein to stabilize the overall architecture, or in facilitating interactions with other proteins. in addition, the sib motif could potentially bind to other host factors in the innate immune system. viruses in the hughes orthonairovirus species have been isolated from birds or from ticks that infest them. the immune system of birds, including antiviral responses, possesses considerable differences from mammals in terms of what elements are present and how they are regulated (reviewed in [ ] and [ ] ). this includes the apparent absence of an isg homologue in birds. these differences from mammals raises the possibility that the sib motif could play a role in adaptation to the avian innate immune system, perhaps by facilitating interactions with proteins other than ub. in addition, the lack of isg in birds leaves open the possibility that the motif could engage with other ub-like entities that are involved in regulating the innate immune response. while divergent votus possess the ability to engage with ub, it is possible that this may not be the only, or even predominant function of all votus. in the case of ervev, it has been observed that it possesses poor activity towards ub, while showing potent ability to engage with isg (fig ; [ , ] ). this raises the possibility that other votus that possess poor ub activity may be able to engage with other ub-like entities. while none of the new votus assessed possess notable deisgylase activity, the availability of amc-derived substrates is limited to human isg (hisg ). in contrast to ub, isg shows considerable species-species variances that have been shown to impact binding with viral proteins, including votus from nairoviruses [ , , ] . this leaves open the possibility that votus, while not engaging with hisg , may still possess the ability to interact with isg from species they productively infect. the presence of arginine, lysine, or glutamine in the selectivity pocket of several of the votus, while not ideal for ub, may still allow them to engage with other substrates. the structure of the ervev votu bound to mouse isg (misg ) has a gap in the area that ub's leu would typically occupy (fig b, panel i) . modeling suggests that this feature would also be more permissive of binding with votus possessing a bulky residue such as arginine at position (fig b, panel ii) . this gap is caused by a pairing of glu with lys in misg that pulls the sidechain of glu away from the interface, and suggests a possible mechanism that could allow votus with hydrophilic or bulky residues to effectively engage with non-ub moieties. as highlighted by the lack of isg in birds, however, it's also possible that votus, particularly in the hughes orthonairovirus species, may engage with other ub-like entities that can modulate the immune response. the lack of either ub or isg activity in a number of votus further accentuates this possibility, implying possible biochemical functions that have yet to be characterized among votus. further developments shedding light on these questions could yield key insights into these influential virus-host interactions. the recent increase in genomic characterization of nairoviruses has uncovered a wealth of diversity among them. while our knowledge of nairoviral sequence diversity has expanded, much is still unknown on how this variability affects virus-host relationships. the exact range of vertebrate hosts and their disease state upon infection is not presently known for all members of the nairoviridae family. this novel characterization of nairovirus votus reveals a diversity in the ability to engage mono-and poly-ub that mirrors the genomic diversity. additionally, this study uncovers motifs that appear to play a predominant role in determining these preferences, making it feasible to begin predicting dub activity in uncharacterized or newly discovered nairoviruses. given the presence of robust dub activity in nairoviruses known to infect humans, including cchfv and nsdv, this could serve as an early flag for assessing the risk posed by emerging viruses, and may shed light on the evolutionary trends leading to some viruses to having this capability over others. further, these new structure and activity insights provide a platform to continue the development of robust tools, such as poly-ub specific votus, that can be paired with reverse genetics systems to better understand the role of the votu in the course of a viral infection and how differences in certain activities impact nairoviruses. such knowledge could help propel the field in fully elucidating the detailed functional mechanism of the votu in the viral life cycle, potentially aiding in the development of better disease model systems. in addition, it provides insight that will further gauge the prospects of the votu as a therapeutic target for nairovirus-caused diseases such as cchf, either through the development of specific inhibitors or live attenuated virus vaccines. further, the diversity of the votu suggests a potential relationship with viral host adaptation, and that the role of the votu may extend beyond its well-known function in engaging with ub and/or isg . the votus were constructed and expressed as previously described in published methods [ , ] . purification of qybv, tagv, and dgkv were carried out as previously described. for farv, a slightly different approach altered from the previously described method was used to optimize the expression. e. coli strains with votus from farv were grown at ˚c in l of luria-bertani broth with μg/ml ampicillin. once the optical density reached . - . , . mm isopropyl-β-d-thiogalactopyranoside (iptg) was added to induce gene expression. the temperature was then dropped to ˚c and expression continued overnight. the culture was subsequently centrifuged at xg for minutes and the pelleted cells stored at - ˚c. assays were carried out as described previously [ , ] . briefly, assays were run in mm nacl, mm hepes [ph . ], . mg/ml bsa, mm dithiothreitol (dtt) at ˚c. reactions were run in -well plates with a μl reaction volume using a clariostar plate reader (bmg labtech, inc.). for ub-amc, all votus were assessed at a final enzyme concentration of nm. for isg -amc, votus were assessed at a final enzyme concentration of nm with the exception of nsdv, ganv, and ervev, which were run at a final enzyme concentration of nm due to the high activity towards the substrate. both ub-amc and isg -amc assays were run at a final substrate concentration of μm. assays with ub-rh were run under the same reaction conditions as ub-amc with instrument settings adjusted to optimize detection of the fluorophore. for dgkv votu additional assays were run with the wt and e d mutant using the peptide z-rlrgg-amc (bachem) substrate with protease concentrations of μm and a substrate concentration of μm. assays with fret tamra/qxl pair tagged k , k , and k di-ub substrates were performed as previously described with nm votu and μm substrate [ ] . untagged poly-ub cleavage assays were adapted from the previously published method. briefly, nm of each votu was tested against μm linear (m ), k , k , k , k , k , k , and k linked di-ub (boston biochem, ma). reactions were initiated by the addition of votu and incubated at ˚c in reaction buffer ( mm nacl, mm hepes [ph . ], mm dtt). the reactions were stopped at the time points indicated by mixing μl of each reaction with x laemmli sample buffer and heat killed by boiling at ˚c for minutes. the cleavage over time was visualized using - % mini-protean tgx precast gels (bio-rad) by coomassie staining. assays with k and k linked tri-ub were run in the same manner except that tri-ub was present at μm. all four votus were screened against a series of qiagen nextal suites in a -well hanging drop format with a ttp labtech mosquito (ttp labtech, herfordshire, united kingdom). qybv votu was screened at . mg/ml, tagv votu at . mg/ml, dgkv votu at . mg/ml, and farv votu at . mg/ml. initial hits were optimized along salt, precipitant, and ph gradients as applicable. the tagv and farv votu hits were also optimized with an additive ht screen from hampton research. peg as the cryoprotectant. for selenomethionyl (se-met) derivative qybv votu crystals, bacterial cells were grown in minimal media to od . and induced with . mm iptg at ˚c for hrs. prior to induction, the cultures were supplemented with eight amino acids (leu, ile, val, and trp at . g/l; thr, lys, phe, and cys at . g/l) as well as selenomethionine ( . g/l). cells were harvested and protein purified as previously described. final crystals were grown in . m magnesium acetate, % peg , in drops formed from μl of solution and ul of . mg/ml protein. native datasets of the qybv, dgkv, tagv, and farv votus were collected at a wavelength of Å. a se-met single anomalous dispersion (sad) dataset for qybv votu was collected at the absorption edge of se at . Å. the data sets were indexed, integrated and scaled with hkl- [ ] . experimental phasing of the se-met-sad dataset was performed using the phenix suite of programs [ ] . hyss was utilized to locate the se-met sites, with phaser solving the experimental phases [ ] [ ] [ ] . initial model building was performed using autobuild, with subsequent cycles of refinement and model building carried out in phenix and coot ( [ , , ] . this structure was then used as a search model to solve the qybv votu native dataset by molecular replacement in phaser [ ] . the other votus were solved by molecular replacement. a qybv votu-based homology model was used to solve dgkv votu, while homology models based on dugv votu (pdb entry hxd) were used to solve tagv votu and farv votu. all the structures were built with autobuild, followed by alternating manual building and refinement in coot and phenix. structures were validated using the molprobity server [ ] . mutations were made using the quikchange lightning kit according to the manufacturer's protocol (agilent technologies, inc.). the pcr-generated plasmids were transformed into escherichia coli neb- α cells by heat shock. the mutant plasmids were confirmed by sequencing and transformed into t express cells (new england biolabs). t express cells expressing ub, ub-l a, and ub-l n in pet- b were grown to od . - . at ˚c. expression was induced with . mm iptg and continued at ˚c overnight. the cells were pelleted and stored as described above. the pellet was resuspended in mm nacl, mm tris [ph . ] supplemented with lysozyme at ˚c for minutes. the cells were sonicated on ice at % power with a % duty cycle for a total of minutes, followed centrifugation at , xg for minutes. the supernatant was filtered through a . μm and applied to a gravity flow ni-nta column (goldbio) pre-equilibrated with mm nacl, mm tris [ph . ]. the column was washed with the same buffer containing mm imidazole, followed by elution with mm imidazole. thrombin was added to cleave the x his-tag and the elution dialyzed overnight in mm nacl, mm hepes [ph . ], mm dtt at ˚c. after dialysis the protein was filtered through a . μm membrane and run over a superdex column (ge healthcare) equilibrated with mm nacl, mm hepes [ph . ], mm dtt. the fractions were pooled based on the chromatogram and concentrated to~ - . mm, supplemented with % glycerol, and flash frozen in liquid nitrogen followed by storage at - ˚c until further use. tagv votu was purified as previously described and dialyzed alongside ub and ub-l n in mm nacl, mm hepes [ph . ], mm tcep overnight at ˚c. itc was performed with a microcal peaq-itc (malvern, worcestershire, uk). ub or ub-l n were titrated into the cell in series of injections, μl each with a spacing of seconds. the temperature was kept constant at ˚c with a reference power ranging from - μcal/s. final protein structures were deposited in the protein data bank with ids dwx, dx , dx , dx , and dx for se-met qybv votu, native 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dirasantha, obaiah; feldman, emily r.; wilkerson, gregory k.; sawyer, sara l. title: selective use of primate cd receptors by hiv- date: - - journal: plos biol doi: . /journal.pbio. sha: doc_id: cord_uid: deqiets individuals chronically infected with hiv- harbor complex viral populations within their bloodstreams. recently, it has come to light that when these people infect others, the new infection is typically established by only one or a small number of virions from within this complex viral swarm. an important goal is to characterize the biological properties of hiv- virions that seed and exist early in new human infections because these are potentially the only viruses against which a prophylactic hiv- vaccine would need to elicit protection. this includes understanding how the envelope (env) protein of these virions interacts with the t-cell receptor cd , which supports attachment and entry of hiv- into target cells. we examined early hiv- isolates for their ability to infect cells via the cd receptor of different primate species. primates were the original source of hiv- and now serve as valuable animal models for studying hiv- . we find that most primary isolates of hiv- from the blood, including early isolates, are highly selective and enter cells through some primate cd receptor orthologs but not others. this phenotype is remarkably consistent, regardless of route of transmission, viral subtype, or time of isolation post infection. we show that the weak cd binding affinity of blood-derived hiv- isolates is what makes them sensitive to the small sequence differences in cd from one primate species to the next. to substantiate this, we engineered an early hiv- env to have high, medium, or low binding affinity to cd , and we show that it loses the ability to enter cells via the cd receptor of many primate species as the binding affinity gets weaker. based on the phenotype of selective use of primate cd , we find that weak cd binding appears to be a nearly universal property of hiv- circulating in the bloodstream. therefore, weak binding to cd must be a selected and important property in the biology of hiv- in the body. we identify six primate species that encode cd receptors that fully support the entry of early hiv- isolates despite their low binding affinity for cd . these findings will help inform long-standing efforts to model hiv- transmission and early disease in primates. introduction compatibility with primate cd receptors might be dynamic, given that cd is highly diverged in its protein sequence at the interaction interface with hiv- env (fig a) . we began by cloning the cd genes of primate species, plus human cd . using retroviral transduction, we stably introduced these genes into cf th cells (dog thymocytes) that had also been engineered to express the human c-c motif chemokine receptor (ccr ) coreceptor for hiv- entry (s fig). expression levels of these cd receptors on cf th cells were similar to endogenous expression levels of cd seen on human immortalized t cells and on primate primary t cells that we isolated directly from blood (s fig). our goal was then to infect these cells with an early hiv- isolate. for the sake of this study, we refer to all viruses or env clones that were isolated < days post infection as "early" and those isolated > days post infection as "chronic" isolates. this nomenclature is consistent with the fiebig scale classification, for which fiebig stages i-v (typically lasting until to days post infection) capture early infection stages and fiebig stage vi indicates progression into chronic infection [ , ] . this is also consistent with other studies on transmitted or early hiv- , which have used viruses isolated from human blood weeks to months after infection [ , , , , ] . the properties of all of the hiv- env isolates used in this study are summarized in s table. the hiv- envelope clone "bg " has risen to prominence in the study of early hiv- . the overbaugh group amplified bg env from an infant approximately weeks after delivery and showed that this env sequence was nearly identical to the two other env sequences amplified from this baby at the same time point [ ] . this indicated that the infection probably started from a single virion and that the captured env sequence closely resembles the env of this transmitted virion. a cocrystal of bg env has been solved in complex with cd ( fig a) [ , [ ] [ ] [ ] , and the alternate folding conformations of bg env have been characterized in depth [ ] . we prepared hiv- pseudotyped with the bg env by cotransfecting t cells with two plasmids: one expressing the bg env, and another expressing an hiv- green fluorescent protein (gfp) reporter virus genome (q Δenv-gfp, representing a blood-derived clade a isolate from year after seroconversion [ , ] ). cell lines expressing human ccr and different primate cd receptors were then infected with this bg virus (similar infection results were obtained in cell lines expressing matched cd and ccr from each species; s fig). forty-eight hours post infection, cells were harvested and analyzed by flow cytometry. samples were first gated for live cells and further gated such that all samples were narrowed to the same log decade of cd receptor expression (s fig). gfp-positive (i.e., infected) cells were then enumerated within this population. we identified three categories of cd receptors that support hiv- entry to varying degrees ( fig b) . the cd receptors of some species (shown with purple bars: white-cheeked gibbon, olive baboon, gelada, black snub-nosed monkey, colobus, and owl monkey) behave similarly to human cd when challenged with bg hiv- . these species are not our closest relatives but rather are distributed throughout the primate phylogeny ( fig b) . a second class of species (shown with gray bars: chimpanzee, gorilla, orangutan, and sooty mangabey) encodes cd receptors that support entry of this virus but at a level that is approximately -fold lower than human cd . a third class of species (macaques, mandrills, sabaeus monkeys, and squirrel monkeys) encodes cd receptors that support entry at a level -fold or more reduced compared to human cd . with some of the cd receptors in the latter class, infection was reduced by more than -fold compared to human cd . while the chimpanzee cd allele tested here has a semipermissive phenotype, we have recently shown that some chimpanzee cd alleles are highly resistant to hiv- entry [ ] . as a control, we determined that infection did not change the surface expression level of human or any primate cd receptor (s fig) . this allows us to conclude that the observed differences in infection are due to defects in entry and that our gating strategy did not exclude infected cells because they no longer express cd . from this data, we can conclude that cd receptors are functionally variable from one primate species to the next with regards to supporting entry of an early hiv- isolate. prior to this, there was only one primate species known to encode a cd receptor compatible with early hiv- isolates, owl monkeys [ ] , but now we can add five additional species to that list: white-cheeked gibbon, olive baboon, gelada, black snub-nosed monkey, and colobus monkey. hiv- entry requires engagement of the amino-terminal "d " domain of the cd receptor (residues . this is evidenced by numerous env-cd cocrystals [ ] [ ] [ ] and mapping of d -domain residues that affect interaction with hiv- env [ , [ ] [ ] [ ] . for instance, the macaque cd receptor can be rendered functional for hiv- entry by introducing a single amino acid substitution in the d domain (isoleucine " i" mutated to the human residue, asparagine " n") [ ] . likewise, owl monkey populations circulate cd alleles that are both non-functional ( i) and functional ( n) for hiv- entry [ ] . from the panel of primate cd s that we have tested, we can now build on this observation that residue in cd is particularly important for hiv- engagement. in fig b, the residues encoded at cd position in each species are listed at the tips of the phylogeny, along with the total number of amino acid differences in the d domain compared to human cd . from this, it appears that cd must encode an asparagine ("n") at position in order to have any appreciable function as a receptor for bg hiv- . indeed, position in cd has experienced positive selection during primate speciation [ , ] , and this may be because mutations that replace the asparagine protect individuals against infection. however, there are clearly sequence determinants outside of position that also matter because not all receptors with an asparagine at position function equally well. we next sought to determine whether the selective use of only some primate cd receptors is a property specific to early hiv- isolates. from this point forward, we focused on cd receptors from a subset of primate species, in particular the african primate species involved in the zoonotic transmissions of hiv- (chimpanzees and gorillas) and hiv- (sooty mangabeys) [ ] , and the macaque species that serve as the current laboratory model for hiv- (expression histograms for these cell lines are shown in s fig) . all of these receptors were partially or highly defective for entry mediated by bg env (fig b) . we next tested envs isolated from the blood of four individuals chronically infected with hiv- at time points more than months after the original infection [ ] . these envs were pseudotyped onto q Δenv-gfp, as described above. these viruses were then used to infect stable cell lines expressing human ccr and cd from different primate species. two of the four envs demonstrated the specific use of human cd that we have described (fig a, left two panels) . the other two demonstrated a weakening of this selective cd phenotype but still a preference for human cd (fig a, right two panels) . this suggests that the phenotype of selective cd usage may be retained during later stages of disease and may not be unique to early hiv- isolates. to address this further, we looked at how cd receptor usage may change over time as infection progresses from early-to late-stages of disease. to test this, we made use of sets of molecular clones that had been amplified by single-genome amplification from patient blood early after infection and then again from the same patient months later [ , , , ] . from four such sets of molecular clones, we cloned the rev-env cassettes into a mammalian expression vector, which was then used to pseudotype each env onto q Δenv-gfp, as described above. these viruses were tested for their ability to enter cell lines stably expressing human or primate cd receptors, along with human ccr . we observed no significant difference in receptor usage between the early env (grey bars) and its matched -month counterpart (black bars) (fig b) . in fact, for each patient, the envs derived from the two different time points ( months apart) behaved almost identically. infection mediated by all of these envs was reduced in cells expressing chimpanzee or macaque cd receptors when compared to human cd . one exception was seen with virus from patient ch , whose env did mediate entry somewhat better via chimpanzee and macaque cd compared to other envs, although still less well than with human cd . therefore, selective use of primate cd receptors appears to describe many hiv- isolates from the blood, including those isolated just after infection (weeks), later at months (fig b) , or even during chronic stages of infection (fig a) . we next examined the transmission bottleneck directly by testing matched donor-recipient hiv- env pairs. hiv- was pseudotyped with envs from mother-infant pairs, of which the mother was chronically infected and her baby was newly infected (< weeks) [ ] . for seven such pairs, we found that mother hiv- envs (black bars) were not different from infant hiv- envs (grey bars), in that all demonstrated the selective use of human cd (fig c) . in fact, corresponding mother and baby envs behaved very similarly. when the seven pairs were grouped, no significant differences were noted between maternal (chronic) and infant (early) envs in terms of their ability to infect cells bearing chimpanzee or rhesus macaque cd receptors (mann-whitney test, p > . ). the tested pairs represented most major hiv- group m subtypes, suggesting that selective use of cd may describe all or most globally circulating forms of hiv- . we next verified this by challenging cell lines expressing different cd cells stably expressing human ccr and human or primate cd (x-axis) were infected with q Δenv-gfp bearing (a) chronic envelopes (envs) [ ], (b) early (newly infected; grey bars) and chronic ( months post infection; black bars) env pairs derived from the same patient [ , , , ] , shown for one patient in each panel, or (c) envs derived from maternal (chronic; black bars)/infant (newly infected; grey bars) transmission pairs [ ] , with one mother-baby pair shown in each panel. the percent cells infected (gfp-positive) was measured by flow cytometry and normalized to the percent infected with human cd . error bars represent the mean + sem from two independent experiments, each with two to three technical replicates. data associated with this figure can be found in the supplemental data file (s data). ccr , c-c motif chemokine receptor ; gfp, green fluorescent protein. receptors with early hiv- isolates representing each of the four major group m (pandemic) hiv- subtypes. these four envs, isolated from patient blood shortly after initial infection [ , , ] , were pseudotyped onto the q Δenv-gfp virus, as described before. while human cd supported entry of all of these viruses, primate cd orthologs supported levels of hiv- entry that ranged from -to -fold lower ( fig a) . collectively, our study has explored a breadth of hiv- isolates taken from patient blood, representing all of the major subtypes found globally, and has found that they can all be phenotypically described by their selective use of only certain primate cd receptors. next, we wanted to understand more about why blood-derived hiv- env isolates demonstrate this selective use of only some primate cd receptors. thus far, all of the envs that we have tested are from ccr -tropic viruses isolated from the blood. there are three types of hiv- in the body of an infected person: ccr t cell-tropic (the most abundant form in the blood, utilizing the ccr coreceptor), cxcr t cell-tropic (in the blood but using an alternate coreceptor, c-x-c chemokine receptor type [cxcr ]), and ccr macrophage-tropic (rare in the blood, usually found in the central nervous system) [ , - ]. only the first of these transmits to new individuals, whereas the latter two types arise in special evolutionary niches within the human body during the course of chronic infection and rarely transmit [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in this study, we did not consider or test cxcr -utilizing viruses. we next tested hiv- pseudotyped with envs from four macrophage-tropic viruses [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] for their ability to infect cells bearing primate cd receptors ( fig b) . in stark contrast to the blood-derived isolates tested above (figs , and a), macrophage-tropic viruses were promiscuous in their use of all cd receptors tested. for each primate cd tested, the median relative infection for all early hiv- envs (fig a) was significantly lower than macrophage-tropic envs (fig b; p < . ; mann-whitney u test). macrophage-tropic hiv- env is known to bind cd with higher affinity compared to other forms of hiv- env [ ] [ ] [ ] . this is a selected property that these virions possess because macrophages have lower densities of cd molecules on their surface [ ] . based on this, we hypothesized that weak cd binding affinity is what makes most blood-derived hiv- envs sensitive to small sequence differences in cd receptors from different primates. to test this, we first purified a soluble version of the human cd receptor for use in neutralization assays (scd , consisting of the d and d domains of cd ) (s fig) . when scd is preincubated with hiv- , it is known to competitively inhibit virus interaction with the cd expressed on the cell surface and block virus entry [ ] . our purified human scd behaved identically to a commercially available version (s fig). hiv- pseudotyped with early or macrophage-tropic envs was preincubated with increasing concentrations of scd and then used to infect tzm-bl indicator cells (hela cd /ccr cells that express luciferase in response to hiv- tat expression). hiv- pseudotyped with either of two different early envs (bg or ac . . ) was neutralized in a dose-dependent manner by human scd (fig c) . as expected, hiv- pseudotyped with either of two macrophage-tropic envs (bal or sf ) was neutralized by human scd to an even greater extent, consistent with these viruses having a higher affinity for cd ( fig d) . the ic values are shown within each panel and are approximately -fold higher for early envs than for macrophage-tropic envs. in conclusion, the broadened ability to enter cells through the cd receptors encoded by all primates tested ( fig a and b ) correlates with tighter cd binding (fig c and d) . macrophage-tropic hiv- isolates, which are known to bind human cd more tightly, are promiscuous in their use of primate cd receptors. (a, b) cells stably expressing various primate cd receptors (x-axis), along with human ccr , were infected with q Δenv-gfp pseudotyped with (a) early hiv- envelopes (envs) or (b) envs from common macrophagetropic hiv- isolates. the percent cells infected (gfp-positive) was measured by flow cytometry and normalized to the percent infected with human cd . error bars represent the mean + sem from two independent experiments, each with two to three technical replicates. values above error bars represent fold decrease in viral entry relative to human cd , only indicated for those samples that passed significance thresholds (one-way anova for repeated measures effect, dunnett's test; p < . ). (c and d) infection of tzm-bl cells with q Δenv-gfp pseudotyped with early (c) or macrophage-tropic (d) envs. each virus was preincubated with increasing concentrations of human soluble cd (scd ) as a competitive inhibitor. tzm-bl cells are hela cd /ccr cells that express luciferase in response to hiv- tat expression, read in relative light units (rlus). error bars represent the mean + sd from one biological replicate (n = to technical replicates), representative of two independent experiments. error bars are not plotted in instances in which the size of the error bar is less than the symbol size. ic values from the soluble cd neutralization assays are shown within each panel, for which the mean and sd values reflect the variation of the ic calculations from two independent experiments. (e and f) same as c and d, only neutralization assays were performed with chimpanzee and rhesus macaque scd instead of human scd . data associated with this figure can be found in the supplemental data file (s data). ccr , c-c motif chemokine receptor ; gfp, green fluorescent protein. we next wanted to confirm that weak binding to human cd receptors results in a loss of binding to primate cd receptors. we purified chimpanzee and rhesus macaque scd (s fig) and tested the ability of these proteins to neutralize different classes of hiv- . viruses bearing macrophage-tropic envs (bal or sf ) were neutralized by chimpanzee and macaque scd (fig f) , while viruses bearing envs from early hiv- isolates (bg or ac . . ) were not neutralized to an appreciable degree ( fig e) . collectively, these data suggest that most early hiv- isolates from the blood, which have lower affinity for human cd compared to macrophage-tropic viruses, do not bind well to chimpanzee and macaque cd . this shows that our entry assay for selective versus promiscuous use of primate cd s is essentially a readout of cd binding affinity. to further test this premise, we next created a panel of envs engineered to have high, medium, or low binding affinity for cd . bg env, the prototypic early env discussed above, was engineered to have two different point mutations, one at position and the other at position . these mutations in env have been previously shown to increase binding affinity to cd , although only one of them has been characterized within the bg env background [ , ] . consistent with these previous reports, the a v mutation subtly increased binding to human scd relative to wild type (demonstrated by increased neutralization by scd ), while the s y mutation resulted in a more substantial increase ( fig a) . while both of these mutations were originally characterized because they increase entry via macaque cd [ , ] , we found that both mutations also improve entry via all of the primate cd receptors tested, with increasing improvement as the binding affinity for cd increases (left to right in fig b) . this pattern of increased binding affinity for human cd and increased ability to enter cells via primate cd s also correlated to increased binding affinity for primate cd receptors, as read in a neutralization assay with purified chimpanzee and rhesus macaque scd proteins (left to right in fig c) . finally, we engineered two additional mutations into hiv- bg env that have been shown to increase cd binding and to increase entry via macaque cd [ ] . these mutations also make env able to more efficiently mediate entry via all primate cd s tested, not just that of macaques ( fig d) . these data support the general premise that promiscuous use of primate cd s can be created by mutations in env that increase cd binding affinity. further, the promiscuous use of primate cd receptors is not just a property of macrophage-tropic viruses but instead may be a common property of viruses engineered or adapted to bind cd more tightly. the fact that virtually no blood-derived hiv- isolate that we have tested has this phenotype (promiscuous use of primate cd receptors) suggests that this phenotype is strongly selected against in the human body. the reasons for this are unknown. we then employed a virus-free approach to confirm that the mechanism of selective use of certain primate cd receptors stems from weak cd binding, as opposed to other interactions between hiv- and cd that may occur during the course of an infection. cells expressing hiv- env on their surface were mixed with cells expressing human ccr and various primate cd receptors ( fig a) . cellular fusion was assessed using a split luciferase reporter system in which, upon cell-cell fusion, the two halves of luciferase merge and produce a functional enzyme [ ] . we used this assay to test the interactions between four different envs and six different cd receptors, as shown in fig b. in this system, the phenotypes seen in our previous infection assays were largely recapitulated despite the fact that this assay is virus-free and only requires three proteins (env and cd /ccr ). the early envs mediated fusion most efficiently with cells expressing human cd , while the macrophage-tropic envs broadly engaged and fused with diverse primate cd receptors. this assay, when taken together with the experiments employing purified cd described above, reveal that weak binding to cd is an early env engineered to have weak, medium, or tight binding to cd becomes progressively more promiscuous in its use of primate cd receptors. (a-c) amino acid substitutions were introduced into the bg envelope (env) at position and . the headers at the top describing these mutations pertain to panels a, b, and c. in each column, the indicted env was pseudotyped onto q Δenv-gfp. (a) each of the three viruses was preincubated with increasing concentrations of human soluble cd (scd ) and then used to infect tzm-bl cells, which are hela cd /ccr cells that express luciferase in response to hiv- infection and tat expression, read in relative light units (rlus). error bars represent the mean + sd from one biological replicate, representative of two independent experiments. error bars are not plotted in instances in which the size of the error bar is less than the symbol size. ic values are listed on each panel, for which the mean and sd values reflect the variation of the ic calculations from two independent experiments. (b) cells stably expressing the indicated cd s (x-axis) and human ccr were infected with each virus. the percent cells infected (gfp-positive) was measured by flow cytometry and normalized to the percent infected with human cd . error bars represent the mean + sem from two independent experiments, each with three technical replicates. values above error bars represent fold decrease relative to human cd for those samples that passed significance thresholds (one-way anova for repeated measures effect, dunnett's test; p < . ). (c) same as panel a but with chimpanzee and rhesus macaque scd instead of human scd . (d) two additional single point mutations were introduced into the bg env, which have also been shown to increase binding affinity for cd [ ] . these envs were pseudotyped onto q Δenv-gfp and used to infect cells stably expressing the indicated cd (top of each graph) and human ccr . the percent cells infected (gfp-positive) was measured by flow cytometry and normalized to the percent infected with human cd . error bars represent the mean + sem from two independent experiments, each with two to three technical replicates. values above error bars represent fold increase relative what makes most blood-derived hiv- isolates (i.e., those fueling the global pandemic) selective in their use of only certain primate cd receptors. interestingly, macaques (both rhesus and pig-tailed) encode a cd receptor that is not permissive for entry of most blood-derived hiv- isolates (figs , , and ) , consistent with previous reports [ , , ] . since macaques (predominantly rhesus macaques) serve as the current animal model for hiv- , we sought to determine whether some allelic variants of rhesus macaque cd might encode receptors that better support hiv- infection. we sequenced the cd gene from captive indian-origin rhesus macaques and identified alleles encoding five unique cd protein variants ( fig a) . all of these allelic versions of rhesus macaque cd behaved identically and were nonfunctional for entry of virus pseudotyped with bg env to wildtype bg env for those samples that passed significance thresholds (one-way anova for repeated measures effect, dunnett's test; p < . ). data associated with this figure can be found in the supplemental data file (s data). ccr , c-c motif chemokine receptor ; gfp, green fluorescent protein. https://doi.org/ . /journal.pbio. .g [ ] . these two cell types were then cocultured to induce membrane fusion, which was detected by a luminescent product following the addition of the renilla luciferase substrate. (b) the percentage of fusion was calculated relative to what was observed with human cd . error bars represent the mean + sem from three to four independent experiments, each with four technical replicates. values above error bars represent fold decrease relative to human cd for those samples that passed significance thresholds (one-way anova for repeated measures effect, dunnett's test; p < . ). data associated with this figure can be found in the supplemental data file (s data). ccr , c-c motif chemokine receptor . and also with env from another early hiv- isolate, cap . . .e ( fig b) . therefore, while owl monkey and chimpanzee populations circulate cd alleles with dramatically different functions as receptors for hiv/siv entry [ , , ] , it appears that the function of macaque cd is identical from one allele to the next. in this study, we have shown that envs from over early and chronic hiv- isolates from human blood (mother-infant pairs, etc.) all demonstrate selective entry via only some primate cd receptors. we next tested additional envs (n = total, four of which were previously tested in infection assays herein; s table) using the cell-cell fusion assay described above. these envs were universally poor at mediating fusion with cells expressing rhesus macaque cd and ccr proteins (fig c) . similar data have been generated in other large screens of early hiv- envs [ , ]. collectively, it seems that virtually no hiv- isolated from the bloodstream is compatible with the macaque version of cd . if we now understand the inability of hiv- to use macaque cd to be a reflection of weak cd binding, this leads us to understand that virtually all early-and late-stage hiv- isolates in the human bloodstream experience continual selection to retain weak cd binding. total rna was isolated from the blood of rhesus macaque individuals. cd was amplified from cdna, and pcr products were sequenced to yield genotype information for each individual ( chromosomes). genotype information was used to phase snps using phase . [ , ] in dnasp v [ ] . the table summarizes the unique cd alleles identified. for each allele, the residue encoded at each variable amino acid positions is noted. unique alleles were cloned and further verified by sanger sequencing. (b) dog thymocytes (cf th cells) stably expressing human ccr and the indicated rhesus macaque cd alleles (x-axis) were infected with hiv- (q Δenv-gfp) bearing a subtype a (bg ) or subtype c (cap . . .e ) envelope (env). the percent cells infected (gfp-positive) was measured by flow cytometry hours post infection. error bars represent the mean + sem from two independent experiments, each with three technical replicates. values above error bars represent fold decrease relative to human cd for those samples that passed significance thresholds (one-way anova for repeated measures effect, dunnett's test; p < . ). (c) t cells transiently expressing diverse hiv- envs (see s table) and cf th cells stably expressing the indicated cd /ccr receptors were each transfected with plasmids encoding half of a split renilla luciferase [ ] . these two cell types were then cocultured to induce membrane fusion, which was detected by a luminescent product following the addition of the renilla luciferase substrate. a total of envs were tested from the following major hiv- subtypes: subtype a (n = ), subtype b (n = ), subtype c (n = ), subtype d (n = ), and subtype a/d (n = ). the percentage of fusion was calculated relative to what was observed with human cd /ccr . error bars represent the mean + sd, for which n = (two technical replicates from one experiment). data associated with this figure can be found in the supplemental data file (s data). nt, nucleotide; aa, amino acid; ccr , c-c motif chemokine receptor ; gfp, green fluorescent protein. https://doi.org/ . /journal.pbio. .g selective use of primate cd receptors by hiv- we find that many hiv- isolates from the blood, including early hiv- isolates, enter cells poorly through a subset of primate cd receptors. this finding is highly analogous to what has been observed with primate restriction factors that block hiv- infection, in that they too are highly species-specific in their interaction with hiv- [ , ] . like cd , most restriction factors are also evolving under intense positive selection in primates [ ] [ ] [ ] [ ] , explaining why these proteins vary in protein sequence on virus-binding surfaces from one host species to the next. it appears that sivs, and probably other viruses as well, have put intense selective pressure on these genes to evolve in a way that changes the protein sequence on virus-binding surfaces. the selection and retention of new cd alleles that block virus entry could protect a species from infection, at least until the virus population counter adapts. when this type of arms race is playing out in multiple host species independently, it drives divergence in protein sequence at host-virus binding interfaces and has the long-term effect of making it difficult for viruses to move between species [ ] . indeed, a prevailing theme that has emerged in recent years is that receptor sequence divergence serves as a potent barrier to the movement of viruses between species [ , [ ] [ ] [ ] [ ] [ ] . likewise, this study suggests that hiv- entry is blocked by the cd receptor of some primate species that it might encounter, whether that be in the wild or in the lab. on the other hand, we identify several primate species that encode cd receptors compatible with unmodified isolates of hiv- from the human bloodstream, including early isolates. these species could possibly be useful as animal models of hiv- transmission and infection, assuming that restriction factor blocks can be overcome in these species. we show that selective use of only certain primate cd receptors occurs when hiv- env has weak binding affinity for cd . this weak affinity makes hiv- env sensitive to sequence differences in the cd molecule. the strongest evidence in favor of this model is that viruses that have been selected or engineered to have tight cd binding affinity become agnostic to sequence differences in primate cd and can more efficiently enter cells using all cd orthologs that we have tested. while neutralization assays to assess the cd binding affinity of a given virus are laborious, our assay for selective or promiscuous primate receptor usage allowed us to screen a large number of hiv- isolates from human blood. based on this, we show that weak cd binding is a nearly constant property of hiv- circulating in the human bloodstream, including but not specific to early-stage virions. this underscores the need to further understand the significance of cd binding affinity to hiv biology. while it has long been known that different hiv types have different affinities for cd , the functional consequences of this are unclear. this is important to understand because weak cd binding seems to be selected for in the human bloodstream, yet some of our current primate models use envs that are engineered for tight cd binding affinity so that they more efficiently engage the nonpermissive macaque cd receptor. for example, mutations at hiv- env , when engineered into shivs, render virons compatible with macaque cd [ ] . however, mutations at this position do not emerge in vivo when hiv- envs are adapting to macaque cd , whereas mutations at env do [ ] . based on our data, we can now speculate that envs with high binding affinity to cd have a fitness penalty in vivo. given that nearly all hiv- in the human bloodstream seems to be selected to bind cd weakly, why would this be preferable? although the reasons for this are currently unclear, there have been numerous speculations put forward [ ] . it is possible that more efficient use of cd requires changes in env conformation that would increase susceptibility to antibody neutralization. indeed, arrildt and colleagues have shown that when compared to patient matched t-cell tropic isolates from the blood, macrophage-tropic isolates appear to be more sensitive to certain cd binding site antibodies [ ] . thus, any conformational change that increases antibody neutralization sensitivity would likely be purged from the viral population. alternatively, tighter cd binding is known to enhance hiv- tropism for cells of the monocyte-macrophage lineage [ , , ] . however, replication of hiv- in these cells appears to be slowed due to cell type-specific blocks, including the restriction factors samhd and apobec a [ , ] , delayed reverse transcription and integration [ , ] , and transcriptional repression [ ] . these blocks to replication in macrophages may put high-affinity cd binders at an evolutionary disadvantage during early stages of infection. finally, highly efficient macrophage infection may directly promote disease via inflammatory processes. hiv- infection of central nervous system macrophages coincides with severe inflammation and neuronal impairment [ ] [ ] [ ] [ ] . additionally, respiratory and cardiac dysfunction during latestage hiv- infection has been associated with chronic inflammation, of which macrophages may be playing a central role [ , ] . viral infection is a delicate balance between replicating efficiently enough to transmit, yet limiting disease to maintain the health of the host. altering cellular tropism via high-affinity cd binding may be disadvantageous to the virus, disproportionately skewing infection toward a more diseased state. herein, as in many other studies, comparative genetics between humans and primates has revealed to us new lessons about hiv's interaction with its host. this has been a tremendously powerful tool in the hiv field, having led to the original identification of, and mechanistic insights into, many of the host factors that control hiv- biology (for only a few examples, see [ , [ ] [ ] [ ] [ ] [ ] ). as primate research becomes more tenuous [ ] , we need to keep in mind how powerful these comparative approaches have been in the study of hiv- and in the study of other viruses as well (for only a few examples, see [ ] [ ] [ ] [ ] ). hek t (atcc crl- ), tzm-bl (nih arp # ), and canine thymocytes (cf th) (atcc crl- ) were cultured in dulbecco's modified eagle medium (invitrogen) with % fbs, mm l-glutamine, and % pen strep (complete medium). all cells were maintained at ˚c and % co . . then, diluted blood was separated in density gradient media (ficoll-pacque plus ge - - ) by x g centrifugation for minutes. the peripheral blood mononuclear cell layer (i.e., pbmcs or buffy coat) was removed and washed twice in three volumes of balanced salt solution per sample. each donors' pbmcs were then immediately subjected to a cd -positive selection kit (easysep human cd positive selection kit ii cat # ) to isolate cd + t cells. rhesus macaque and owl monkey cd + t cells were then activated for three days in rpmi supplemented with u il /ml (recombinant human-il ; sigma # ) and μg/ml or . μg/ml pha-p (sigma l ), respectively. cd + tcell cultures were subsequently maintained in rpmi + u il /ml media. cd + t cells isolated from rhesus macaque or owl monkey whole blood (as described above) or human t cells (hut cells) were analyzed for cd expression by flow cytometry (beckman coulter cyan adp high-performance flow cytometer). cells were first incubated in fc receptor (cd monoclonal antibody ( g ), ebioscience cat # - - ) blocking buffer (pbs + % fcs + mm edta + . % bsa) on ice for hour. these cells were then fixed in % pfa and stained for cell surface expression of cd (bd cd mouse anti-human, apc, clone: l ). on the cytometer, only live cells were gated and histograms of cd expression determined by unstained and isotype (bd mouse igg , , cat # ) controls. the following env clones were obtained from the nih aids reagent program, division of aids, niaid, nih: env clones from early infection (qf . m [ , , , ]) were gifts from dr. beatrice hahn. we amplified the rev-env cassettes from these by pcr, ta-cloned them into pcr (clontech), and then used gateway cloning to move them into a gateway converted pcdna . (invitrogen) mammalian expression vector. further description of these env clones, and those used in the fusion assay screens, are described in s table. nucleotide changes producing s y/w/h and a v were introduced into bg .w m.b by site directed mutagenesis using overlapping pcr primers containing the mutation of interest, following standard methods. cd and/or ccr was amplified from rna isolated from the following sources: jurkat t cells (human cd , genbank mk ), immortalized b-lymphocytes (rhesus macaque ccr , genbank nm_ . ), whole blood (rhesus macaque cd alleles - , genbank mf -mf ; owl monkey cd , genbank mk ), and fibroblasts (gorilla cd , genbank mk ). pig-tailed macaque cd and ccr were subcloned into the pcr /gw/ topo ta plasmid from pbabe retroviral vectors provided by dr. julie overbaugh [ ] . human ccr was obtained through the nih aids reagent program, division of aids, niaid, nih (human ccr expression vector pcccr # from dr. nathaniel landau) and subcloned into the pcr /gw/topo ta plasmid. the remaining constructs were purchased as gene block fragments (idt), pcr amplified (phusion hifi master mix; thermo fisher), and ta cloned into the pcr /gw/topo ta plasmid (thermo fisher). the genbank sequences used for gene block synthesis are as follows: chimpanzee cd (nm_ . ) and ccr (u . ), sooty mangabey cd (nm_ . ) and ccr (xm_ . ), gorilla ccr (af . ), sabaeus cd (xm_ . ), gelada cd (xm_ . ), mandrill cd (xm_ . ), colobus cd (xm_ . ), baboon cd (xm_ . ), black snub-nosed monkey cd (xm_ . ), white-cheeked gibbon cd (xm_ . ), squirrel monkey cd (af . ), marmoset cd (af . ), and orangutan cd (xm_ . ; incomplete cds, missing sequence ( nucleotides) was substituted with human cd residues). following ta cloning, an lr clonase ii reaction (invitrogen) was used to shuttle inserts into gateway-converted plpcx (cd ) or plhcx (ccr ) retroviral packaging vectors (clontech). to produce retroviruses for transduction, hek t cells plated in antibiotic free media ( x cells/well in a -well plate) were transfected with a μg of transfer vector (plpcx [cd ] or plhcx [ccr ] retroviral vector), μg pcs -mgp (mlv gag/pol), and . μg pc-vsvg (vsv-g envelope) using a : ratio of transit (mirus) transfection reagent to micrograms of dna, according to manufacturer's directions. forty-eight hours post transfection, retrovirus vlps were collected, filtered through . -μm cellulose acetate filters, and stored at − ˚c in single-use aliquots. cf th cells were plated at x cells/well of a -well dish (approximately % confluent) and hours later, transduced with μl of retroviral supernatant by spinoculation at , x g for minutes in the presence of μg/ml polybrene. the following day, the cells were placed in complete medium containing antibiotic ( μg/ml puromycin for plpcx or μg/ml hygromycin-b for plhcx) and cultured until stable outgrowth was noted (> week). stable cell lines were maintained indefinitely in selection media. to produce hiv- reporter viruses, x hek t cells were seeded into a -cm dish in antibiotic free media and hours later transfected with . μg of q Δenv-gfp and . μg of env plasmid. q Δenv-gfp, encoding an approximately -bp deletion in env, and with gfp inserted into the nef gene (therefore destroying the function of nef), was obtained from dr. julie overbaugh and described previously [ ] . fourty-eight hours post transfection, the cell supernatant was harvested, concentrated (approximately -fold) using amicon ultracell k filters (millipore), and stored at − ˚c in single use aliquots. virus titers were determined by facs analysis for gfp-positive cells, using a range of virus dilutions on cf th cells stably expressing human cd and ccr . for infection assays, cf th cells stably expressing cd and ccr were plated at x cells/well of a -well plate (approximately % confluent) hours prior to infection. the cells were then infected with hiv- pseudoviruses at a multiplicity of infection (moi) of approximately . in the presence of μg/ml of polybrene by spinoculation at , x g for minutes. fourty-eight hours post infection, the cells were harvested from the plate and fixed in % paraformaldehyde for minutes. the fixed cells were washed three times with pbs and resuspended in μl facs buffer ( x pbs buffer containing % fbs and mm edta) antibody cocktail. all antibody pairs were used at : dilutions, and incubated at ˚c with cells for minutes as follows: for primate cd /primate ccr (s fig), pecy mouse α-human cd clone l (bd # ) and apc mouse α-human cd (ccr ) clone a (bd # ); for all other studies, the combination of apc mouse α-human cd clone l (bd # ) and pecy mouse α-human cd (ccr ) d (bd # ) was used. stained cells were analyzed on a cyan adp (beckman coulter) flow cytometer. following live-cell gating, cd and ccr expressing gates were drawn and then the percent gfp positive cells was enumerated within the double positive population. the data from approximately x live cells was analyzed using flowjo version . paxgene (bd # ) preserved whole blood was obtained from animals housed at the kccmr per blood collection protocols approved by the university of texas, md anderson cancer center iacuc. total dna/rna was extracted using the allprep dna/rna mini kit (qiagen). extracted rna was used as a template for oligo (dt) primed reverse transcription (superscript iii rt; thermo fisher). pcr amplification of cd was performed using pcr supermix hifi (thermo fisher) containing - ng of cdna and . μm final concentration of each pcr primer cd -forward aagcagcgggcaagaaagacg and cd -reverse caagttcctgccctctgtgg in a final volume of μl. pcr cleanup was performed using exonuclease-i and shrimp alkaline phosphatase treatment (affymetrix) for minutes at ˚c, and then the cleaned-up products were sanger sequenced using the following cd -forward ggagttcaaaatagacatcg and cd -reverse cagacacttcct tgttcttc sequencing primers. the resulting genotype information was used to phase snps using phase . [ , ] in dnasp v [ ] . five unique rhesus macaque cd alleles were identified, ta cloned into the pcr gateway topo ta cloning vector (thermo fisher), and then subcloned into the gateway converted plpcx retroviral vector (clontech). all constructs were further verified by sanger sequencing. allele represented the major allele circulating in this population and was used in all experiments except where designated otherwise. human, chimpanzee, and rhesus macaque cd cloned into the pcr plasmid (described above) served as a template for soluble cd (scd ) pcr. the d /d domain (nucleotide positions - , minus signal peptide) was amplified by pcr and cloned into phl-sec [ ] (addgene # ), which has been optimized for protein production in mammalian cells. x t cells (three -cm dishes) were transfected with μg of scd expression plasmids using transit- (mirus), and cell supernatant containing secreted scd was harvested at days three and six post transfection. cell supernatant was spun down at , x g for minutes to remove cell debris, filtered using a . -μm cellulose acetate membrane, and mixed : with wash buffer ( mm na po ph . , mm nacl, mm imidazole). cleared cell supernatant was then incubated with -ml bed volume of ni-nta agarose beads (qiagen, # ) equilibrated in wash buffer for hours at ˚c. the mixture was then added to a gravity flow chromatography column and the flow-through was passed through the column a second time. the ni-nta beads were washed with ml of wash buffer. bound protein was eluted with wash buffer containing mm imidazole in -ml fractions. fractions containing scd were pooled, washed three times with pbs, and concentrated to μl using an amicon ultra- ml centrifugal filter with a kda molecular weight cutoff (emd millipore, #ufc ). the sample was purified to homogeneity on a superdex increase / gl (ge healthcare, # - - ) in pbs. purified samples were concentrated to mg/ml using an amicon ultra . -ml centrifugal filter with a -kda molecular weight cutoff, aliquoted for single use, and flash-frozen in liquid nitrogen. for the neutralization assays, each virus (normalized by equivalent tdu/ml) was incubated with serial -fold dilutions of scd (range μm to approximately μm final concentration) at ˚c for hour. human, chimpanzee, and rhesus scd were produced in this study (methods above), and commercially available human scd was obtained through the national institutes of health aids reagent program: scd - # (from pharmacia, inc.). following incubation, the scd treated and untreated viruses were spinoculated ( , x g for minutes) onto x tzm-bl cells plated in -well plates, in the presence of μg/ml of polybrene. following spinoculation, the cells were washed three times with pbs and given fresh media. at hours post infection, infectivity was measured by firefly luciferase assays following the manufacturers protocol (promega). luminescence was determined using the bmg clariostar plate reader. cf th cells stably expressing human or primate cd and human ccr , and t cells, were plated at x and x cells/well of a -well dish, respectively. the next day, ) cf th cells were transfected with . μg of dna ( . μg of ½ renilla luciferase [ ] and . μg of pcdna . filler dna) using lipofectamine (invitrogen) and ) t cells were transfected with . μg of dna ( . μg of ½ renilla luciferase and . μg of the env expression plasmid) using transit (mirus). twenty-four hours post transfection, the cells were removed from the plate using an enzyme free x citric saline solution ( x solution, . m kcl, . m sodium citrate) diluted in pbs, counted, and resuspended to a final volume of x cells/ml. μl of transfected cells in quadrupilicate ( x t and cf th cells) were mixed : and incubated for hours in -well flat bottom plates. following incubation, fusion was assessed using a renilla luciferase assay (promega). luminescence was determined using the bmg clariostar plate reader. all data were plotted, and one-way anova and mann-whitney u tests were performed where indicated, using prism version . a for mac (graphpad). ic values were determined by first normalizing the relative luminescence units as follows: % and % inhibition was defined by wells receiving the highest concentration of scd and absence of scd , respectively. a nonlinear regression curve was fit to the normalized data, and ic values were calculated using prism (graphpad) software. the blood collections performed on the animals in this study were approved as protocol number -rn by the university of texas, md anderson cancer center, institutional animal care and use committee (mdacc-iacuc). the protocol approved by the mdac-c-iacuc adhered to the recommendations provided in the guide for the care and use of laboratory animals [ ] and also adhered to the blood collection volumes recommended by the guidance document of the association of primate veterinarians (https://www.primatevets. org/guidance-documents) entitled guidelines for blood sampling in nonhuman primates (also known as blood sampling guidelines for nonhuman primates in biomedical research). hiv- transmission biology: selection and characteristics of infecting viruses human immunodeficiency virus type population genetics and adaptation in newly infected individuals identification and characterization of transmitted and early founder virus envelopes in primary hiv- infection quantitating the multiplicity of infection with human immunodeficiency virus type subtype c reveals a non-poisson distribution of transmitted variants envelope-constrained neutralization-sensitive hiv- after heterosexual transmission differences in the selection bottleneck between modes of sexual transmission influence the genetic composition of the hiv- founder virus wide variation in the multiplicity of hiv- infection among injection drug users a substantial transmission bottleneck among newly and recently hiv- -infected injection drug users in st petersburg, russia neutralization escape variants of human immunodeficiency virus type are transmitted from mother to infant transmitted/founder and chronic hiv- envelope proteins are distinguished by differential utilization of ccr characterization of glycosylation profiles of hiv- transmitted/founder envelopes by mass spectrometry env length and n-linked glycosylation following transmission of human immunodeficiency virus type subtype b viruses antigenicity and immunogenicity of transmitted/founder, consensus, and chronic envelope glycoproteins of human immunodeficiency virus type resistance of transmitted founder hiv- to ifitm-mediated restriction relative resistance of hiv- founder viruses to control by interferon-alpha how host genetics dictates successful viral zoonosis origins of hiv and the aids pandemic animal models for hiv/aids research modern-day siv viral diversity generated by extensive recombination and crossspecies transmission rapid evolution by positive darwinian selection in t-cell antigen cd in primates positive selection of primate genes that promote hiv- replication dual host-virus arms races shape an essential housekeeping protein evidence for ace -utilizing coronaviruses (covs) related to severe acute respiratory syndrome cov in bats evolutionary reconstructions of the transferrin receptor of caniforms supports canine parvovirus being a reemerged and not a novel pathogen in dogs computational and functional analysis of the virus-receptor interface reveals host range trade-offs in new world arenaviruses nonmacrophage-tropic human immunodeficiency virus type r envelopes predominate in blood, lymph nodes, and semen: implications for transmission and pathogenesis macrophage tropism of human immunodeficiency virus type and utilization of the cc-ckr coreceptor generation and structural analysis of soluble oligomeric gp envelope proteins derived from neutralization-resistant and neutralization-susceptible primary hiv type isolates effect of major deletions in the v and v loops of a macrophage-tropic hiv type isolate on viral envelope structure, cell entry, and replication distinct biological and serological properties of human immunodeficiency viruses from the brain complete nucleotide sequence, genome organization, and biological properties of human immunodeficiency virus type in vivo: evidence for limited defectiveness and complementation characterization of antibody responses elicited by human immunodeficiency virus type primary isolate trimeric and monomeric envelope glycoproteins in selected adjuvants the role of mononuclear phagocytes in htlv-iii/lav infection hiv- infects multipotent progenitor cells causing cell death and establishing latent cellular reservoirs an infectious molecular clone of an unusual macrophage-tropic and highly cytopathic strain of human immunodeficiency virus type macrophage-tropic hiv- variants from brain demonstrate alterations in the way gp engages both cd and ccr quantification of entry phenotypes of macrophage-tropic hiv- across a wide range of cd densities macrophage entry mediated by hiv envs from brain and lymphoid tissues is determined by the capacity to use low cd levels and overall efficiency of fusion blocking of hiv- infectivity by a soluble, secreted form of the cd antigen envelope residue substitutions in simianhuman immunodeficiency viruses enhance cd binding and replication in rhesus macaques a single gp residue can affect hiv- tropism in macaques conformational changes of the hiv- envelope protein during membrane fusion are inhibited by the replacement of its membrane-spanning domain cd receptor diversity in chimpanzees protects against siv infection a new statistical method for haplotype reconstruction from population data a comparison of bayesian methods for haplotype reconstruction from population genotype data dnasp v : a software for comprehensive analysis of dna polymorphism data hiv restriction factors and mechanisms of evasion. cold spring harb perspect med macaque-tropic human immunodeficiency virus type : breaking out of the host restriction factors positive selection of primate trim alpha identifies a critical species-specific retroviral restriction domain ancient adaptive evolution of the primate antiviral dna-editing enzyme apobec g species-specific activity of hiv- vpu and positive selection of tetherin transmembrane domain variants ancient adaptive evolution of tetherin shaped the functions of vpu and nef in human immunodeficiency virus and primate lentiviruses the evolution and genetics of virus host shifts human viruses: discovery and emergence animal origins of the severe acute respiratory syndrome coronavirus: insight from ace -s-protein interactions novel insights into cell entry of emerging human pathogenic arenaviruses parvovirus family conundrum: what makes a killer? phenotypic correlates of hiv- macrophage tropism the hiv env variant n enhances macrophage tropism and is associated with brain infection and dementia samhd is the dendritic-and myeloid-cell-specific hiv- restriction factor counteracted by vpx apobec a is a specific inhibitor of the early phases of hiv- infection in myeloid cells kinetics of human immunodeficiency virus type reverse transcription in blood mononuclear phagocytes are slowed by limitations of nucleotide precursors characterization of the early steps of infection of primary blood monocytes by human immunodeficiency virus type regulation of hiv- transcription in cells of the monocyte-macrophage lineage hiv- target cells in the cns hiv- replication in the central nervous system occurs in two distinct cell types neurocognitive dysfunction predicts postmortem findings of hiv encephalitis updated research nosology for hiv-associated neurocognitive disorders preferential destruction of interstitial macrophages over alveolar macrophages as a cause of pulmonary disease in simian immunodeficiency virus-infected rhesus macaques the macrophage: the intersection between hiv infection and atherosclerosis the cytoplasmic body component trim alpha restricts hiv- infection in old world monkeys species-specific vulnerability of ranbp shaped the evolution of siv as it transmitted in african apes non-human primate schlafen inhibits production of both host and viral proteins a trim -cyclophilin a fusion protein found in owl monkey kidney cells can restrict hiv- cyclophilin a retrotransposition into trim explains owl monkey resistance to hiv- monkey kingdom nuclear trim specifically targets influenza virus ribonucleoproteins to block the onset of rna chain elongation an intrinsically disordered region of the dna repair protein nbs is a species-specific barrier to herpes simplex virus in primates dengue viruses cleave sting in humans but not in nonhuman primates, their presumed natural reservoir. elife species-specific deamidation of cgas by herpes simplex virus ul protein facilitates viral replication hiv type variants transmitted to women in kenya require the ccr coreceptor for entry, regardless of the genetic complexity of the infecting virus a time-and cost-efficient system for high-level protein production in mammalian cells committee for the update of the guide for the care and use of laboratory animals we thank julie overbaugh, soraya shehata and joe timpona for comments on the manuscript. key: cord- -fz ucwwm authors: freundt, eric c.; drappier, melissa; michiels, thomas title: innate immune detection of cardioviruses and viral disruption of interferon signaling date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: fz ucwwm cardioviruses are members of the picornaviridae family and infect a variety of mammals, from mice to humans. replication of cardioviruses produces double stranded rna that is detected by helicases in the rig-i-like receptor family and leads to a signaling cascade to produce type i interferon. like other viruses within picornaviridae, however, cardioviruses have evolved several mechanisms to inhibit interferon production. in this review, we summarize recent findings that have uncovered several proteins enabling efficient detection of cardiovirus dsrna and discuss which cell types may be most important for interferon production in vivo. additionally, we describe how cardiovirus proteins l, c and l(∗) disrupt interferon production and antagonize the antiviral activity of interferon effector molecules. picornaviridae is an important family of single-stranded, positive-polarity rna viruses that includes > genera with over species (zell et al., ) . within picornaviridae, the genus cardiovirus includes encephalomyocarditis virus (emcv), theiler's murine encephalomyelitis virus (tmev) and saffold viruses (safv). although emcv has been described as a potential zoonotic agent, safvs are the only cardioviruses known to regularly infect humans, with the vast majority of people showing evidence of infection (zoll et al., ; carocci and bakkali-kassimi, ) . emcv has been found to infect over host species and contains one serotype, mengo virus, which was isolated in in the mengo district of uganda (dick et al., ) . tmev was discovered in by max theiler and is found in wild mice and rats worldwide. tmev can cause different diseases, depending on the virus strain and host genetics, ranging from fatal encephalitis to a chronic demyelinating disease that has served as a model for multiple sclerosis (brahic et al., ) . the genome of cardioviruses is approximately . - . kb and contains and untranslated regions (figure ) . translation of the genome gives rise to a polyprotein that is cleaved by the c protease, leading to the production of proteins. two additional proteins, l * and b * , are expressed from alternate open reading frames. l * is only expressed by tmev and is important for infection of macrophages, persistence of the virus in mice and inhibiting rnase l (van eyll and michiels, ; sorgeloos et al., ) , b * results from a frameshifting mechanism conserved in cardioviruses that regulates the ratio of structural and non-structural proteins translated over time. protein b * itself is only thought to be important for replication of emcv, as mutants that abolish its expression had a small plaque phenotype. b * in tmev and safv is unlikely to act as a protein as it is predicted to encode a peptide of amino acids (loughran et al., ) . in this review, we focus on the ways in which cardioviruses trigger the innate immune response and the efficient mechanisms that they have evolved to suppress these signaling pathways. we consider which cell types may be most important for production of interferon (ifn) in vivo, and also describe how cardioviruses disrupt the functions of interferon effectors. double-stranded rna (dsrna) is a necessary product of picornavirus replication, as positive-stranded genome is copied to produce a negative-stranded, full-length template, which is in turn used to produce additional genomes. dsrna is recognized by several sensor proteins within the cell and triggers a signal transduction pathway that results in transcription of the type i ifn (ifn-α/β) genes, as well as ifn-λ. in the endosome, dsrna is detected by toll-like receptor (tlr ), which signals through the adaptor protein trif to activate ifn regulatory factor (irf- ) and nuclear factor kappa b (nf-κb) (yamamoto et al., ) . cytoplasmic dsrna is detected by the rig-i-like receptor (rlr) family of proteins, which includes rig-i (retinoic acid-induced gene i) and mda (melanoma differentiation-associated gene ). upon recognition of dsrna, these proteins undergo a conformational change that exposes n-terminal caspase activation and recruitment domains (cards). the rlrs are then capable of stimulating the mitochondrial antiviral signaling (mavs) protein, also known as ips- , cardif, and visa, which in turn activates tank-binding kinase- (tbk ), inducible i-κb kinase (ikk-ε) and irf- , which then translocates to the nucleus to facilitate transcription of ifn genes (reviewed in gebhardt et al., ) . although rig-i and mda both detect dsrna within the cytosol, their functions are non-redundant. rig-i recognizes relatively short dsrna species (< kb) with ppp or pp, which are produced in certain virus infections (hornung et al., ; pichlmair et al., ) . in contrast, mda recognizes long dsrna, which is present during picornavirus replication (kato et al., ; pichlmair et al., ) . thus, while rig-i is activated during infection with flaviviruses, paramyxoviruses, influenza, and others, mda is responsible for detection of picornaviruses (gitlin et al., ; kato et al., ; pichlmair et al., ; wang et al., ; feng et al., ) . the importance of mda for control of cardioviruses was demonstrated in mda -deficient mice, which failed to control emcv infection and did not efficiently produce ifn (gitlin et al., ; kato et al., ) . in addition to rig-like helicases described above, which activate the mavs pathway, another ifn-inducible rna helicase, moloney leukemia virus homolog (mov ) was reported to enhance ifn induction (cuevas et al., ) . mov expression in hek cells restricted emcv replication. interestingly, mov acts through irf- activation, in a rlr and mavs-independent way and signals require ikk-ε but not tbk . such mavs-independent pathways are likely not critical for global ifn production in emcv infected mice, given the major impact of mda or mavs deficiency in mice, but they may be important in specific cell types or in conditions where the other pathways may be less potent. laboratory of genetics and physiology (lgp ), also known as dhx , is also a member of the rlr family but lacks a card domain (yoneyama et al., ) . given its structural similarity and lack of a card domain, lgp was initially thought to negatively regulate dsrna recognition by rig-i, as its overexpression limited ifn induction by sendai virus and newcastle disease virus (rothenfusser et al., ) . however, although the negative effect of lgp on rig-i remained controversial, later studies have established that lgp acts as a co-activator of mda . mice deficient for lgp were impaired in responding to rna ligands for mda or to emcv infection (venkataraman et al., ; satoh et al., ) . lgp was also shown to increase the rate of mda interaction with rna and downstream signaling by facilitating the formation of numerous, shorter mda filaments (bruns et al., ) . thus, it appears that lgp can act as both a positive and negative regulator of rlr signaling, with the outcome likely dependent on the concentration of lgp (bruns and horvath, ) . however, recombinant mda was directly activated as measured by an atp hydrolysis assay by the replicative form of dsrna coxsackievirus b , showing that lgp is not essential for activation of mda in vitro by dsrna (feng et al., ) . both lgp and mda are important for detecting cardiovirus replication. mefs deficient for either protein produce lower amounts of ifn-β when infected with emcv (deddouche et al., ) . intriguingly, lgp may enhance activation of mda during emcv infection by binding to rna complementary to the leader (l) gene and forming a complex with mda . this rna sequence from l was also shown to be a potent activator of mda in the absence of virus infection (deddouche et al., ) . although dsrna can bind and activate recombinant mda in the absence of other proteins, it is likely that additional partners are required for efficient activation of mda in vivo. for example, mda is phosphorylated in resting cells, and requires dephosporylation by pp α/γ (wies et al., ; takashima et al., ) . additional proteins that participate in recognition of cardiovirus dsrna have recently been described, including a study showing that tar rna binding protein (trbp) interacts with lgp in a yeast two-hybrid screen (komuro et al., ) . lgp was found to interact with trbp in co-immunoprecipitation experiments and depletion of trbp by sirna reduced interferon production induced by tmev and emcv. moreover, trbp enhanced ifn induction by tmev and emcv when overexpressed. depletion of trbp did not affect induction of ifn by sendai virus, which is recognized by rig-i. this study establishes that trbp participates in detection of cardiovirus dsrna by lgp /mda but the mechanism and its importance in vivo remain to be elucidated. as trbp is a component of the rnai machinery (chendrimada et al., ; haase et al., ) , other molecules involved in rnai were assessed for their role in dsrna detection and protein activator of pkr (pact) was found to also participate in activation of ifn signaling by cardioviruses. when pact was depleted by sirna, interferon production was decreased during infection of both tmev and emcv. overexpression of pact also increased ifn activation when lgp was co-expressed with mda . intriguingly, single-stranded tmev genome enhanced the association of lgp and pact, which suggests that a secondary structure in the tmev genome might facilitate this interaction (miyamoto and komuro, ) . in a separate report, pact was shown to be required for induction of ifn by emcv but not sendai virus. this study also demonstrated that pact and mda were recruited to dsrna (poly(i:c)) but not single stranded rna, and that pact expression increased the amount of mda oligomerization (lui et al., ) . both trbp and pact have also been reported to bind to double stranded rna-dependent protein kinase (pkr), and pact can bind rig-i (park et al., ; patel and sen, ; kok et al., ) . at the present time, it is not clear if these interactions are important for mediating recognition of cardioviruses. yet another partner in detecting dsrna in cardiovirus infection was recently uncovered. a cdna screen to identify genes involved in regulating ifn signaling revealed that dhx expression increased transcription of an ifn-β reporter plasmid in response to high molecular weight (hmw) poly(i:c) (zhu et al., ) . depletion of dhx resulted in decreased phosphorylation of tbk and irf- in response to hmw poly(i:c) and emcv as well as decreased production of ifn-β. the authors also show that dhx binds to mda but not mavs or rig-i, and binding could only be detected when mda was activated by emcv or hmw poly(i:c). mechanistically, this study demonstrated that dhx mediated rna binding of mda by interacting with mda through its n-terminus and rna through its dexd and helicase domains, and that dhx promotes formation of mda filaments, which are required for activation (zhu et al., ) . dhx was also independently described to interact with rig-i (sugimoto et al., ) , although it may be of greater importance for activation of mda (zhu et al., ) . together, these studies show that multiple proteins facilitate recognition of dsrna by mda during cardiovirus infection (figure ). thus far, these proteins include lgp , dhx , pact and trbp. additional helicases are also likely to participate in rlr-dsrna complex formation but their activities remain to be clarified (oshiumi et al., ) . moreover, recent discoveries have identified a novel role for pkr in recognition of dsrna and activation of mda , which will be discussed below. as the number of proteins mediating mda activation grows, so does the number of questions about how this pathway functions. for example, what role does each of the proteins play and how do they work together mechanistically to activate mda ? do they play non-redundant roles, or do they function in the same way but in different cell types? resolving these questions will allow for deeper understanding of the first line of immune defense against rna viruses. upon recognition of dsrna, pkr controls virus infection by phosphorylating eukaryotic initiation factor (eif α), which inhibits translation (farrell et al., ) . in this way, an infected cell can suppress production of viral proteins. phosphorylation of eif α also leads to stress granule formation and induces autophagy, and both pathways are commonly observed during virus infection (paul and munz, ; poblete-duran et al., ) . not surprisingly, many viruses have evolved strategies to inhibit the activation or function of pkr (reviewed in garcia et al., ) , as evidenced by the fact that pkr-deficiency did not modify the survival time of emcv infected mice (yang et al., ) . in addition to its role in inhibiting translation, recent evidence has emerged to show that pkr participates in production of ifn. for example, pkr appears to be important for nuclear translocation of irf- following mda activation (pham et al., ) . pkr was found to bind to mda and this interaction was not disrupted by nuclease treatment, indicating that binding does not depend on the presence of rna. in pkr-deficient cells, emcv infection failed to induce irf- translocation to the nucleus. moreover, a constitutively active mutant of pkr induced ifn through mavs. the effect of pkr on induction of ifn required its catalytic activity but did not depend on phosphorylation of eif α (pham et al., ) . in a separate report, pkr was shown to be important for normal processing of ifn-β mrna, suggesting that pkr may function at multiple points in the ifn pathway (schulz et al., ) . additionally, activation of pkr by hmw poly(i:c) was shown to be inhibited in cells depleted of mda and mavs, suggesting that mavs influences activation of pkr. moreover, mavs and pkr were found to interact in co-ip experiments, and the interaction depended on the card domain in mavs and the dsrna binding domain of pkr (zhang et al., ) . together, these studies clearly indicate a role for pkr in mda -dependent ifn induction, although several mechanisms may be involved, which require clarification. the role of pkr in ifn production after cardiovirus infection remains to be resolved. in one study that examined mengo virus infection, knockdown of pkr led to decreased induction of ifn-β in hela cells, suggesting that pkr may play a role in the mda pathway during cardiovirus infection (langereis et al., ) . this observation fits with the model proposed by onomoto et al. ( ) , which was based on influenza virus infection and suggests that pkr triggers the formation of "antiviral stress granules" that serve as a recruitment platform for dsrna and rig-like helicases, thereby enhancing ifn production. pkr is, however, not essential for ifn production in cardiovirus-infected cells because mengo virus possessing a deletion in the zinc-finger of l, which abrogates its functions as an interferon antagonist, was found to induce interferon in the cells lacking pkr and rnase l (feng et al., ) . cardioviruses may also inhibit pkr through activity of the l protein, as stress granule formation was prevented by the l protein of mengo, tmev, and safv- during virus infection (borghese and michiels, ) . however, direct inhibition of pkr by l remains to be demonstrated. during infection, viral dsrna can also be released into the extracellular milieu, either non-specifically during lysis of infected cells or possibly intentionally by exocytosis to trigger innate immunity by uninfected cells. dsrna can then be endocytosed by neighboring cells and infiltrating immune cells and lead to ifn production. tlr recognizes dsrna in endosomes and signals through trif to activate irf- and nf-κb. the importance of tlr in context of cardiovirus infection may depend on the model of infection. for example, mice deficient for myd or tlr were not significantly more susceptible than wild-type mice to emcv infection . in a separate report, however, tlr -deficient mice had higher viral loads in the liver and heart and were more susceptible to infection (hardarson et al., ) . also, a study that evaluated the role of tlr in controlling a strain of emcv with tropism for β cells of the pancreas found that tlr protected mice from a fatal infection and that tlr -deficent mice produced less ifnβ early in infection ( and h post-infection). however, this deficiency was transient and mice lacking tlr produced levels of ifn-β equivalent to wild-type animals at h post-infection. in the same experiments, the authors demonstrated that mice lacking mda succumbed to the infection more rapidly than tlr -deficient mice and produced less ifn-β at h postinfection (mccartney et al., ) . finally, a recent report using intracerebral inoculation of the gdvii strain of tmev evaluated the importance of these molecules for control of virus replication and induction of ifn. trif-/-, myd -/-, and mice lacking both trif and myd showed wild-type levels of ifn induction, while mavs-deficient animals were slightly but significantly impaired. when trif, myd and mavs were all depleted, however, mice were unable to induce ifn. only mice lacking myd and trif, or mice lacking myd , trif and mavs showed increased titers of gdvii in the brain (pfefferkorn et al., ) . thus, both tlr and rlrs contribute to controlling virus replication in vivo, although the relative importance of these pathways may depend on the virus and route of inoculation. surprisingly, mda is also responsible for the vast majority of ifn produced from extracellular dsrna in vivo. mice deficient for mda produced substantially less ifn when administered polyi:c, whereas tlr -deficient mice responded like wild-type (gitlin et al., ) . these results raise the question of how dsrna taken up through endocytosis could gain access to the cytoplasm. the mechanism by which dsrna could be internalized and then access the cytoplasmic rlrs has been unresolved until a recent discovery identified sidt , the mammalian ortholog of the sid- dsrna transporter in caenorhabditis elegans, as a transporter of dsrna from the endosome to the cytoplasm. sidt was shown to be important for mediating detection of dsrna in the context of emcv infection in vivo. mice that were deficient for sidt failed to control replication, produced less ifn-β, and succumbed to infection (nguyen et al., ) . these data suggest that a crucial pathway for innate signaling in emcv infection is release of viral rna into the extracellular milieu, endocytosis, and subsequent transfer of viral rna to the cytoplasm to access mda . two proteins encoded by cardioviruses were shown to counteract ifn production in infected cells: protease c, which is responsible for the processing of the virus-encoded polyprotein, and the leader protein (l), which corresponds to the n-terminal peptide of the polyprotein. c is a cysteine proteinase with a trypsin-like serine protease fold, responsible for most cleavages occuring during the maturation of the viral polyprotein (pelham, ) . like c proteases of other picornaviruses that were shown to target critical factors involved in ifn induction such as rig-i (barral et al., ) , emcv c was reported to cleave traf family member-associated nf-kb activator (tank) in infected cells, thus disrupting the complex involving tbk , ikke and irf and limiting type i ifn production (huang et al., ) . likewise, emcv c was shown to target mov , an rna helicase that acts in a mavs-independent way, as a possible innate immune evasion mechanism (cuevas et al., ; figure ). l is a small, multifunctional protein of - amino acids expressed by all cardioviruses (figure ). l contains an n-terminal zinc finger motif (cys-his-cys-cys), an acidic domain, a serine/threonine rich domain, and a c-terminal theilo domain, which is present in safv and tmev but absent in emcv. l was shown to be dispensable for replication of tmev in cell culture but its loss inhibits spread in cells that have a functional interferon response, like l cells, and also impairs the viral persistence in vivo (van pesch et al., ) . l deletions in figure | interferon antagonism by the cardiovirus c protease. the c protease is responsible for cleavage of the cardiovirus polyprotein produced from translation of the genome. in addition, the cardiovirus c protease cleaves host proteins, such as tank and mov , to prevent the cell from producing ifn. frontiers in microbiology | www.frontiersin.org emcv prevent the virus from shutting off host protein synthesis and enable interferon production (zoll et al., (zoll et al., , . mengo virus containing a mutation in the zinc finger of l failed to inhibit ifn synthesis and its replication was inhibited during low moi infections in vitro. in mice lacking the ifn α/β receptor, the mutant virus behaved as wild-type, but in wild-type mice, replication of the l mutant virus was impaired and it failed to cause disease, demonstrating that activity of l is important for pathogenesis in vivo (hato et al., ) . mutations in the zinc finger domain or the theilo domain of tmev or safv l inhibit its ability to antagonize interferon signaling (ricour et al., ) . a critical step in production of ifn following detection of viral replication by mda and other molecules is nuclear translocation of irf- and nf-κb. these proteins enable transcription of the ifn genes to produce mrna, which must then be exported from the nucleus for translation. since picornaviruses do not replicate within the nucleus, many viruses within this family disrupt nucleocytoplasmic trafficking, which results in inhibition of ifn production and translocation of nuclear proteins to the cytosol to benefit viral replication (reviewed in yarbrough et al., ; flather and semler, ) . the mechanism of how l interferes with ifn production may be due to its abilities to disrupt nucleocytoplasmic trafficking, activation of irf- , and assembly of stress granules in infected cells (figure ) . each of these activities will be explored below. the l protein of cardioviruses perturbs the function of the nuclear pore complex (npc) (porter et al., ) . in mammals, the npc consists of approximately different proteins, called nucleoporins (nups) and enables transit across the nuclear membrane (gorlich and kutay, ; wente and rout, ) . while small molecules and ions are able to diffuse through the npc, molecules larger than approximately - kda require active transport, which is regulated by transport receptors called karyopherins (yarbrough et al., ) . transport into the nucleus requires a short amino acid motif, called a nuclear localization sequence (nls) that can interact with either the α or β subtypes of karyopherins, depending on the sequence of the protein's nls. binding and dissociation of nls-containing proteins by karyopherins is also regulated by small gtpase ran. in the cytosol, ran is bound to gdp and can bind cargo proteins. once in the nucleus, however, ran is converted to the gtp bound form by the ran guanine nucleotide exchange factor (rangef) and dissociates from cargo. export then requires a nuclear export sequence (nes) that binds to karyopherins bound to rangtp, and dissociation of this complex occurs in the cytoplasm when a ran gtpase-activating protein (rangap) hydrolyzes gtp to gdp. in this way, the rangdp/gtp gradient regulates directional transport into and out of the nucleus. localization of l to the nucleus depends on expression of a, which contains a nls in its c-terminus (groppo et al., ) . upon nuclear localization, l interacts with ran with high affinity and a is displaced as the binding sites for a and ran partially overlap (petty et al., ) . l from emcv, tmev, and safv induce hyper-phosphorylation of nups including nup and nup (ricour et al., ; ciomperlik et al., ) , likely by recruiting and activating a kinase, which may be facilitated by l binding of exportins crm and cas (ciomperlik et al., ) . chemical inhibition of erk and p was able to block l-mediated hyper-phosphorylation of nups (porter et al., ) . additionally, l of emcv is phosphorylated by casein kinase (ck ) and this phosphorylation is required for nup phosphorylation, although ck did not phosphorylate l of safv or tmev . it is possible, although it remains to be shown, that these kinases also play a role in inhibition of nucleocytoplasmic trafficking by l of tmev and safv. in addition to its role in disrupting nucleocytoplasmic trafficking, tmev and mengo l prevent production of type i ifn in infected cells by interfering with irf- dimerization and tmev l also prevents export of mrna from the nucleus (delhaye et al., ; ricour et al., ) . for both tmev and mengo virus, dimerization of irf- was impaired despite the protein having been phosphorylated. inactivation of irf- occurs despite reports that it accumulates in the nucleus of infected cells (delhaye et al., ) . these data suggest that dsrna is detected in cardiovirus infected cells leading to activation of mavs and downstream kinases, but that irf- is unable to induce ifn transcription. stress granules can form in cells during virus infection and often result from inhibition of translation following phosphorylation of eif α by pkr (white and lloyd, ) . the l protein of mengo, tmev, and safv- inhibits stress granule formation during infection and ectopic expression of l was able to prevent thapsigargin-and arsenite-induced stress granules (borghese and michiels, ) . stress granules formed during infection with viruses containing deletions in the zinc-finger domain or a mutation in the theilo domain of l, indicating that these motifs are also important for inhibition of stress granules (borghese and michiels, ) . while l inhibits nucleocytoplasmic trafficking, stress granule formation, and possibly pkr activation, it has not been possible to uncouple these events using l mutants. when one function of l is disrupted, all functions are simultaneously impaired. therefore, it remains possible that l inhibits ifn production by blocking pkr activation, by interfering with irf- dimerization or nucleocytoplasmic trafficking, or through a combination of these mechanisms (figure ) . the importance of these antiviral pathways in controlling infection is underscored by the variety of mechanisms that viruses have evolved to prevent their activity. for example, the l protein of foot-and-mouth disease virus (fmdv), a picornavirus in the genus aphthovirus, has proteolytic activity whereas the l protein of cardioviruses does not. despite the major differences in these proteins, they all still function to inhibit induction of ifn. fmdv l pro can cleave eif g (devaney et al., ; kirchweger et al., ; guarne et al., ) and therefore reduce translation of cellular mrnas, and can also perturb ifn transcription by cleaving nf-κb (de los santos et al., . however, in the once in the nucleus, l interacts with high affinity with ran gtpase, thus displacing a. the l-ran complex would activate a kinase and trigger nucleoporin hyper-phosphorylation, thereby leading to nuclear pore complex dismantling and to nucleocytoplasmic trafficking perturbation. on the other hand, l may inhibit pkr, thus preventing translation arrest through eif α phosphorylation and therefore block assembly of stress granules. inhibition of ifn gene transcription by l may result from irf- trafficking perturbation and/or from the absence of pkr-enhanced dsrna detection by mda . context of a chimeric mengo virus infection, fmdv l pro was less effective at inhibiting ifn induction in vitro and in vivo (hato et al., ) . similar convergent evolution is apparent when considering the a protein of picornaviruses. whereas a functions as a protease for most picornaviruses and cleaves mediators of type i interferon signaling, this activity is not present in cardioviruses. nevertheless, l still targets some of these same molecules for inactivation (agol and gmyl, ) . additionally, both a of enteroviruses and l of cardioviruses inhibit stress granule assembly (yang et al., ) . the functions of l appear to be sufficiently important to the virus so that it maintains high levels of l expression throughout infection. emcv and tmev undergo a frameshift during translation later in infection by a binding to a stem-loop structure in the genome (napthine et al., ) . this frameshift decreases expression of non-structural proteins bc- abcd by - % (finch et al., ) . a follow up study using metabolic labeling estimated the frameshifting to be - % efficient (ling and firth, ) . this mechanism may allow for cardioviruses, and perhaps other picornaviruses, to increase the translation of structural proteins later in infection. due to its position in the genome, however, l expression would remain high throughout infection despite it not having a structural role for virus assembly. thus, it may be important for cardioviruses to express sufficient levels of l to counteract the immune response throughout the replication cycle. with effective ways to inhibit the production of interferon during infection, control of cardioviruses likely depends on nearby uninfected cells to produce interferon. intriguingly, these pathways seem to also depend on mda , although tlr may also be important in certain cell types such as plasmacytoid dendritic cells (hornung et al., ) . as discussed, recent data suggest a model where viral dsrna is released, endocytosed, and then the rna is translocated to the cytosol where it is detected by mda . in the cns, astrocytes appear to be the primary producers of ifn-β for several neurotropic viruses that preferentially infect neurons, such as tmev and la crosse virus (kallfass et al., ; pfefferkorn et al., ) . using transgenic mice that expressed firefly luciferase under the control of the ifn-β promoter restricted to different cell types, the authors were able to determine that % of ifn-β production during a neurotropic tmev infection was from astrocytes, whereas only % was from neurons, which are the primary target of infection. in mice lacking mavs, ifn-β production was slightly but significantly reduced, suggesting that the rlr pathway is active during infection but may not be the only pathway activated by tmev in astrocytes. intriguingly, mice deficient for myd and trif did not show a significant decrease in ifn-β induction, although induction of ifn-β was completely abrogated in mice deficient for mavs, myd and trif. therefore, it appears that both rlr and tlr signaling are important for ifn-β production after tmev infection of the cns. astrocytes were also shown to be primary producers of ifn during infection with rabies virus and vesicular stomatitis virus. in the case of rabies virus, astrocytes are stimulated to produce ifn by an abortive infection. that the virus is unable to replicate fully may prevent expression of viral interferon antagonists and allow for robust production of ifn. how viral replication is prevented in these cells will be important to uncover and may lead to novel insights about viral control in vivo. it is likely that abortive infection of astrocytes occurs during infection by tmev. however, this remains to be demonstrated and viral rna may well be encountered by other means. mda is critical for induction of ifn against cardioviruses in the periphery as well. ex vivo, cells such as macrophages, conventional dendritic cells and fibroblasts depend on mavs for production of ifn in response to dsrna (sun et al., ) . similarly, mda was shown to be essential in these cells for type i ifn production after emcv infection, in contrast to pdcs which induce ifn production in a tlr-dependent fashion (gitlin et al., ; kato et al., ) . after emcv infection of mice, some ifn is produced through tlrs, likely by pdcs, but most ifn was produced by mda activation (gitlin et al., ; kato et al., ) . levels of ifn-i were strongly decreased in the serum of mda -deficient mice infected by emcv. whereas mda expression strongly influenced survival in response to infection, the effect of myd depletion had a modest effect and loss of trif or rig-i did not affect survival . thus, mda is essential for controlling emcv infection in the periphery. interferon secreted by infected cells binds to its receptor on surrounding cells, activating a signaling cascade that leads to expression of hundreds of interferon-stimulated genes (isgs). two of these isgs, pkr and oligoadenylate synthetases (oas) are part of the best-characterized interferon effector pathways. as described earlier, pkr is likely antagonized by the l protein, as l inhibits pkr-induced stress granule assembly. moreover, a recent study reported increased sumo conjugation figure | inhibition of rnase l activation by l * tmev l * binds rnase l ankyrin repeats and (numbered) through a direct protein-protein interaction, thereby preventing association of - a with rnase l monomers and the consequent dimerization and activation of the enzyme. of pkr in emcv-infected cells, which dampens pkr activation and promotes caspase-dependent pkr degradation (maarifi et al., ) . oligoadenylate synthetases are enzymes responsible for rnase l activation. cardioviruses have evolved two strategies to interfere with the oas-rnase l pathway. in an infected cell, oas are activated by dsrna and produce - oligoadenylates ( - a). binding of two - a molecules to the ankyrin domain of the latent endoribonuclease rnase l triggers its dimerization and activation (figure ) . active rnase l then cleaves viral and cellular ssrna leading to decreased viral replication and ultimately to apoptosis of the cell. interestingly, rna fragments generated by rnase l can amplify ifn production in a rig-i, mda and mavs-dependent way (malathi et al., ) . rnase l targets both viral and cellular mrna but is also predicted to cleave the genome of ssrna viruses, as reported for emcv (li et al., ) . in addition to -phosphodiesterases and phosphatases that tightly regulate the system by degrading - a within minutes of their synthesis, rnase l activity can be negatively regulated by the rnase l inhibitor (rli/abce) (bisbal et al., ) . rli/abce expression is induced by emcv and correlates with rnase l inhibition (martinand et al., ) . accordingly, overexpression of rli/abce inhibited the action of ifn against emcv (bisbal et al., ) . rnase l inhibition by emcv-induced rli is, however, partial as rnase l antiviral activity against emcv was demonstrated in vitro using dominant negative rnase l and oas overexpression (chebath et al., ; zhou et al., ) and in vivo, in rnase l-deficient mice, which presented increased emcv infection and mortality compared to wild-type mice (zhou et al., ) . the l * protein of tmev was found to potently inhibit rnase l through a direct protein-protein interaction (sorgeloos et al., ) . mechanistically, l * binds to rnase l ankyrin repeats and , thereby preventing - a binding to the enzyme and further activation steps (drappier et al., ; figure ). in wild-type macrophages, replication of l * -mutant was significantly impaired as compared to that of the wild-type virus (sorgeloos et al., ) . in contrast, l * -mutant and wildtype viruses replicated to the same level in rnase l-deficient primary peritoneal macrophages. moreover, l * was shown to be active in vivo in the context of mhv chimeric viruses; l * could substitute for another viral rnase l inhibitor, namely the ns phosphodiesterase of mhv, in the liver of infected mice (drappier et al., ) . the fact that the virus devotes one of its proteins to rnase l antagonism highlights the importance of this antiviral pathway against tmev. interestingly rnase l inhibition by l * is highly species-specific; l * of a mouse tmev strain inhibits mouse rnase l but not its orthologs from other species including rat (sorgeloos et al., ; drappier et al., ) . accordingly, l * of a rat tmev strain inhibits rat but not mouse rnase l. theiler's murine encephalomyelitis virus is the only cardiovirus expressing l * , and thus the only cardiovirus known to directly inhibit rnase l. this could stem from its tropism for macrophages, which are the main tmev target during the chronic phase of infection and in which the oas-rnase l system is particularly active (zhao et al., ) . however, macrophages were reported to play important roles in emcv pathogenesis, including for viral replication and dissemination in piglets (papaioannou et al., ) and as reservoir cells for emcv persistence in rats (psalla et al., ) . since emcv is sensitive to rnase l activity, it is possible that another emcv protein will have developed some rnase l antagonistic activity, which might be identified using the appropriate host-pathogen context. interactions between safvs and rnase l have yet to be described, but it is also likely that these viruses have evolved ways of inhibiting this pathway. given the many ways that picornaviruses inhibit interferon production and signaling in infected cells, it is not surprising that the most important producers of ifn would be uninfected or abortively infected cells. indeed, cardioviruses efficiently block ifn in infected cells but loss of mda in mice causes them to be more susceptible to virus infection. these data indicate that detection of cytoplasmic dsrna by mda occurs in cells that are not productively infected (gitlin et al., ) and the recent finding that sidt mediates this process opens many new exciting areas of research (nguyen et al., ) . which molecules might be important for release of viral rna? is there a role for exosomes in this process? how might these pathways be stimulated pharmacologically? preventing release of viral rna and subsequent detection by uninfected cells may represent selective pressure favoring non-lytic release. given the exquisite genetic malleability in response to natural selection displayed by viruses, it is likely that viruses will have evolved mechanisms of inhibiting detection of viral rna by uninfected cells, perhaps by restricting dsrna release or by secreting proteins that inhibit rna transport into uninfected cells. it will be exciting to see how discoveries unfold in this area of research. several recent studies involving cardioviruses have revealed a more complicated picture regarding initial detection of replicating rna and induction of ifn. while it is clear that the helicases lgp , dhx , pact and trbp work in concert with mda for detection of dsrna, it remains to be determined how these molecules coordinate and interact and whether they function in a cell-type specific manner. it will also be important to resolve the way in which pkr functions to activate ifn signaling. future studies in this area will likely have broad relevance for innate detection of viruses. finally, the mechanisms by which l disrupts nucleocytoplasmic trafficking, stress granule formation, and interferon production clearly require further clarification. mutational analysis of l has revealed that these activities are tightly coupled, suggesting that the l interacts with protein(s) that can serve as a common node in each of these pathways. as ifn signaling and stress granules are important for a variety of viral pathogens, answers to these questions may provide broadly relevant insight into host-pathogen interactions. ef, md, and tm wrote the manuscript and approved its final version. ef was supported by a david delo research grant. md was supported by action de recherches concertée (arc). research in the tm lab was supported by the belgian fund for medical research (frsm, pdr # t. . ) and by eos joint program of fonds de la recherche scientifique -fnrs and fonds wetenschapelijk onderzoek -vlaanderen -fwo (eos id: ). viral security proteins: counteracting host defences rig-i is cleaved during picornavirus infection encephalomyocarditis virus leader is phosphorylated by ck and syk as a requirement for subsequent phosphorylation of cellular nucleoporins cloning and characterization of a rnase l inhibitor. a new component of the interferon-regulated - a pathway the leader protein of cardioviruses inhibits stress granule assembly the genetics of the persistent infection and demyelinating disease caused by theiler's virus lgp synergy with mda in rlrmediated rna recognition and antiviral signaling the innate immune sensor lgp activates antiviral signaling by regulating mda -rna interaction and filament assembly the encephalomyocarditis virus constitutive expression of ( '- ') oligo a synthetase confers resistance to picornavirus infection trbp recruits the dicer complex to ago for microrna processing and gene silencing three cardiovirus leader proteins equivalently inhibit four different nucleocytoplasmic trafficking pathways cardiovirus leader proteins bind exportins: implications for virus replication and nucleocytoplasmic trafficking inhibition mov provides antiviral activity against rna viruses by enhancing rig-i-mavs-independent ifn induction the leader proteinase of foot-and-mouth disease virus inhibits the induction of beta interferon mrna and blocks the host innate immune response degradation of nuclear factor kappa b during foot-and-mouth disease virus infection a conserved domain in the leader proteinase of foot-and-mouth disease virus is required for proper subcellular localization and function identification of an lgp -associated mda agonist in picornavirus-infected cells the leader protein of theiler's virus interferes with nucleocytoplasmic trafficking of cellular proteins leader protein of foot-and-mouth disease virus is required for cleavage of the p component of the cap-binding protein complex mengo encephalomyelitis; a hitherto unknown virus affecting man a novel mechanism of rnase l inhibition: theiler's virus l * protein prevents - a from binding to rnase l phosphorylation of initiation factor elf- and the control of reticulocyte protein synthesis mda detects the double-stranded rna replicative form in picornavirus-infected cells characterization of ribosomal frameshifting in theiler's murine encephalomyelitis virus picornaviruses and nuclear functions: targeting a cellular compartment distinct from the replication site of a positivestrand rna virus the dsrna protein kinase pkr: virus and cell control discrimination of self and non-self ribonucleic acids essential role of mda- in type i ifn responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus transport between the cell nucleus and the cytoplasm mutational analysis of the emcv a protein identifies a nuclear localization signal and an eif e binding site structure of the foot-and-mouth disease virus leader protease: a papainlike fold adapted for self-processing and eif g recognition trbp, a regulator of cellular pkr and hiv- virus expression, interacts with dicer and functions in rna silencing toll-like receptor is an essential component of the innate stress response in virus-induced cardiac injury the mengovirus leader protein blocks interferon-alpha/beta gene transcription and inhibits activation of interferon regulatory factor differential ifn-alpha/beta production suppressing capacities of the leader proteins of mengovirus and foot-and-mouth disease virus '-triphosphate rna is the ligand for rig-i encephalomyocarditis virus c protease attenuates type i interferon production through disrupting the tank-tbk -ikkepsilon-irf complex visualizing production of beta interferon by astrocytes and microglia in brain of la crosse virus-infected mice length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-i and melanoma differentiation-associated gene differential roles of mda and rig-i helicases in the recognition of rna viruses foot-and-mouth disease virus leader proteinase: purification of the lb form and determination of its cleavage site on eif- gamma the double-stranded rna-binding protein pact functions as a cellular activator of rig-i to facilitate innate antiviral response the tar-rna binding protein is required for immunoresponses triggered by cardiovirus infection mda localizes to stress granules, but this localization is not required for the induction of type i interferon rnase l mediates the antiviral effect of interferon through a selective reduction in viral rna during encephalomyocarditis virus infection an analysis by metabolic labelling of the encephalomyocarditis virus ribosomal frameshifting efficiency and stimulators ribosomal frameshifting into an overlapping gene in the b-encoding region of the cardiovirus genome pact facilitates rna-induced activation of mda by promoting mda oligomerization differential effects of sumo and sumo on pkr activation and stability small self-rna generated by rnase l amplifies antiviral innate immunity rnase l inhibitor is induced during human immunodeficiency virus type infection and down regulates the - a/rnase l pathway in human t cells rna sensor-induced type i ifn prevents diabetes caused by a beta cell-tropic virus in mice pact is required for mda -mediated immunoresponses triggered by cardiovirus infection via interaction with lgp protein-directed ribosomal frameshifting temporally regulates gene expression sidt transports extracellular dsrna into the cytoplasm for innate immune recognition critical role of an antiviral stress granule containing rig-i and pkr in viral detection and innate immunity accessory factors of cytoplasmic viral rna sensors required for antiviral innate immune response pathogenesis of encephalomyocarditis virus (emcv) infection in piglets during the viraemia phase: a histopathological, immunohistochemical and virological study tar rna-binding protein is an inhibitor of the interferon-induced protein kinase pkr pact, a protein activator of the interferoninduced protein kinase, pkr autophagy and mammalian viruses: roles in immune response, viral replication, and beyond translation of encephalomyocarditis virus rna in vitro yields an active proteolytic processing enzyme binding interactions between the encephalomyocarditis virus leader and protein a abortively infected astrocytes appear to represent the main source of interferon beta in the virus-infected brain pkr transduces mda -dependent signals for type i ifn induction rig-i-mediated antiviral responses to single-stranded rna bearing '-phosphates activation of mda requires higher-order rna structures generated during virus infection who regulates whom? an overview of rna granules and viral infections a picornavirus protein interacts with ran-gtpase and disrupts nucleocytoplasmic transport nucleoporin phosphorylation triggered by the encephalomyocarditis virus leader protein is mediated by mitogen-activated protein kinases pathogenesis of experimental encephalomyocarditis: a histopathological, immunohistochemical and virological study in rats inhibition of mrna export and dimerization of interferon regulatory factor by theiler's virus leader protein the rna helicase lgp inhibits tlrindependent sensing of viral replication by retinoic acid-inducible gene-i lgp is a positive regulator of rig-i-and mda -mediated antiviral responses protein kinase r contributes to immunity against specific viruses by regulating interferon mrna integrity evasion of antiviral innate immunity by theiler's virus l * protein through direct inhibition of rnase l helicase proteins dhx and rig-i cosense cytosolic nucleic acids in the human airway system the specific and essential role of mavs in antiviral innate immune responses riok -mediated phosphorylation of mda interferes with its assembly and attenuates the innate immune response influence of the theiler's virus l * protein on macrophage infection, viral persistence, and neurovirulence the leader protein of theiler's virus inhibits immediate-early alpha/beta interferon production loss of dexd/h box rna helicase lgp manifests disparate antiviral responses mda and mavs mediate type i interferon responses to coxsackie b virus the nuclear pore complex and nuclear transport regulation of stress granules in virus systems dephosphorylation of the rna sensors rig-i and mda by the phosphatase pp is essential for innate immune signaling role of adaptor trif in the myd -independent toll-like receptor signaling pathway picornavirus a protease regulates stress granule formation to facilitate viral translation deficient signaling in mice devoid of double-stranded rna-dependent protein kinase viral subversion of nucleocytoplasmic trafficking shared and unique functions of the dexd/h-box helicases rig-i, mda , and lgp in antiviral innate immunity ictv virus taxonomy profile: picornaviridae ips- plays an essential role in dsrna-induced stress granule formation by interacting with pkr and promoting its activation antagonism of the interferon-induced oas-rnase l pathway by murine coronavirus ns protein is required for virus replication and liver pathology interferon action and apoptosis are defective in mice devoid of ' , '-oligoadenylate-dependent rnase l impact of rnase l overexpression on viral and cellular growth and death dhx functions as an rna co-sensor for mda -mediated emcv-specific antiviral immunity saffold virus, a human theiler'slike cardiovirus, is ubiquitous and causes infection early in life mengovirus leader is involved in the inhibition of host cell protein synthesis the mengovirus leader protein suppresses alpha/beta interferon production by inhibition of the iron/ferritin-mediated activation of nf-kappa b key: cord- - qrg lqu authors: wang, yingchen; dong, tuo; qi, guiyun; qu, lixin; liang, wei; qi, binbin; zhang, zhe; shang, lei; gao, hong; du, xiqiao; lu, bing; guo, yan; liu, zhenwei; yu, huisong; cui, qi; wang, xiaocen; li, ye; guo, weiyuan; qu, zhangyi title: prevalence of common respiratory viral infections and identification of adenovirus in hospitalized adults in harbin, china to date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: qrg lqu background: respiratory infections pose a great challenge in global health, and the prevalence of viral infection in adult patients has been poorly understood in northeast china. harbin is one of the major cities in northeast china, and more than half of any given year in harbin is occupied by winter. to reveal the viral etiology and seasonality in adult patients from harbin, a -year consecutive survey was conducted in harbin, china. methods: from january to december , specimens were obtained from adult patients admitted to the second affiliated hospital of harbin medical university with lower respiratory tract infections. sputum samples were examined by direct immunofluorescence assays to detect seven common respiratory viruses, including influenza virus (type a and b), parainfluenza virus (type to ), respiratory syncytial virus and adenovirus. adenovirus positive samples were seeded onto a cells to isolate viral strains. phylogenetic analysis was conducted on the highly variable region of adenoviral hexon gene. results: a total of , hospitalized adult patients with lower respiratory tract infections were enrolled, in which patients ( . %) were detected as having at least one viral infection. the co-infection rate in this study was . % ( / ). the dominant viral pathogen from to was parainfluenza virus, with a detection rate of . %, followed by influenza virus, respiratory syncytial virus and adenovirus. based on the climate seasons determined by daily average temperature, the highest overall viral detection rate was detected in spring ( . %, / ), followed by winter ( . %, / ), autumn ( . %, / ) and summer ( . %, / ). adenovirus type strains with slight variations were isolated from positive cases, which were closely related to the gb strain from the united states, as well as the harbin b strain isolated locally. conclusion: this study demonstrated that common respiratory viruses were partially responsible for hospitalized lower respiratory tract infections in adult patients from harbin, china, with parainfluenza virus as the dominant viral pathogen. climate seasons could be rational indicators for the seasonality analysis of airborne viral infections. future surveillance on viral mutations would be necessary to reveal the evolutionary history of respiratory viruses. background: respiratory infections pose a great challenge in global health, and the prevalence of viral infection in adult patients has been poorly understood in northeast china. harbin is one of the major cities in northeast china, and more than half of any given year in harbin is occupied by winter. to reveal the viral etiology and seasonality in adult patients from harbin, a -year consecutive survey was conducted in harbin, china. methods: from january to december , specimens were obtained from adult patients admitted to the second affiliated hospital of harbin medical university with lower respiratory tract infections. sputum samples were examined by direct immunofluorescence assays to detect seven common respiratory viruses, including influenza virus (type a and b), parainfluenza virus (type to ), respiratory syncytial virus and adenovirus. adenovirus positive samples were seeded onto a cells to isolate viral strains. phylogenetic analysis was conducted on the highly variable region of adenoviral hexon gene. results: a total of , hospitalized adult patients with lower respiratory tract infections were enrolled, in which patients ( . %) were detected as having at least one viral infection. the co-infection rate in this study was . % ( / ). the dominant viral pathogen from to was parainfluenza virus, with a detection rate of . %, followed by influenza virus, respiratory syncytial virus and adenovirus. based on the climate seasons determined by daily average temperature, the highest overall viral detection rate was detected in spring ( . %, / ), followed by winter ( . %, / ), autumn ( . %, / ) and summer ( . %, / ). adenovirus type strains with slight variations were isolated from positive cases, which were closely related to the gb strain from the united states, as well as the harbin b strain isolated locally. lower respiratory tract infections are a persistent public health problem, causing more than two million deaths per year worldwide, with a rate of deaths per , population (gbd causes of death collaborators, . the morbidity and mortality of respiratory infections could be even worse in developing countries, including china. viral infections played an important role in pediatric lower respiratory tract infections, and the corresponding common viral pathogens were influenza a and b virus (iav and ibv), parainfluenza virus (piv, type to ), respiratory syncytial virus (rsv) and human adenovirus (adv) (pavia, ) . these seven viruses were also common in respiratory infections of the adult population in shandong province, china (liu et al., ) . to the best of our knowledge, there were no commercial vaccines available for most of these viruses, except for influenza a and b viruses (lambkin-williams et al., ) . the etiology of respiratory infection in the adult population has been overlooked, at least in china, although there are plenty of reports on the epidemiology of respiratory viral infection in the pediatric population (wang et al., ) . china is a vast country with notable variations in climate characteristics among different regions. due to the differences in socioeconomic, geographic or climate factors, the epidemiological features of viral infection in the respiratory tract show dramatic variation among different study populations (sloan et al., ; lu et al., ; he et al., ; rehder et al., ) . harbin is one of the major metropolises in northeast china, hosting more than eight million people. the winter in harbin lasts for more than half a year, while the summer is short, resulting in the length of four seasons being unequal. viral respiratory infections in the pediatric population from harbin were reported by the authors' team in (zhang et al., ) , in which the adult population was not included. a clear picture of the viral etiology in hospitalized adults with respiratory infections ought to be critical for both public health policy makers and clinical practitioners. phylogenetic history of several respiratory viruses isolated from china in recent years have been well established, including the influenza virus yang et al., ) , the piv (pan et al., ) and the rsv zou et al., ) . the information about adenoviral evolutionary history in china was limited to outbreak reports and novel strains identification (lu et al., ; li et al., ) , leaving a gap for circulating stains among adult patients. human adenovirus is one of the common pathogens in respiratory infections and could be divided into seven species (ismail et al., ) . the most recent reported human adenovirus type is type in species d, which was isolated from japan (hashimoto et al., ) . human adenovirus species b type (hadv b ) was reported to be responsible for adult pneumonia outbreaks in beijing china during with the genome identity as . %, comparing to the prototype strain of hadv b in (cheng et al., ) . adenoviral infections in children from harbin were found to be adv species b, as reported by the authors, while the viral type among adult patients is still poorly understood. the adenoviral virion was composed of capsomeres, containing hexons and pentons, while each penton was anchored by one fiber (or two fibers in several types) (nemerow et al., ) . the knob region in adenoviral fiber was responsible for cellular attachment through host receptors including car, cd , dsg- and sialic acids (baker et al., ; lenman et al., ) . the rgd loop of penton could be recognized by the host cell's integrin (veesler et al., ) . the hexon protein is the major neutralizing antigen of adenoviruses (madisch et al., ) , while the hyper variable region (hvr) in hexon protein hosts its type specific epitopes reported by the authors' team (yuan et al., ) . experiments on chimeric adenoviral capsids showed that the replacement of hvrs in hexon could alter the viral serotypes (qiu et al., ; tian et al., ) . the hexon specific cd + and cd + t cells have been proved to be an effective treatment for human adenoviral infections in clinical settings (zandvliet et al., ; feucht et al., ) . recent reports indicated that antibodies against fiber knob or penton base may also be involved in the neutralization of adenoviruses tomita et al., ) . to the best of our knowledge, the hypervariable region of adenoviral hexon protein plays an essential role in viral type-specific immunity. in this report, the prevalence of common viruses in the lower respiratory tract infection of hospitalized adult patients from harbin, china was explored in hopes of revealing the clinical and pathogenic features of respiratory viruses. adenoviral strains were isolated from positive cases to identify potential molecular variations within its hypervariable regions. from january to december , sputum samples were collected from , adult patients hospitalized for lower respiratory tract infections in the second affiliated hospital of harbin medical university. all patients enrolled in this study lived in urban and suburban areas of harbin without travel histories within months before admission and sampling. all patients were older than years and suffering from at least one complaint, including fever with body temperature ≥ • c, cough, expectoration, hemoptysis, chest tightness (chest pain), or dyspnea. the sputum samples were expectorated spontaneously into sterile containers and delivered to the laboratory within h, tested immediately or stored at − • c prior to use. patients' clinical data, including symptoms, history of illness, clinical diagnoses and laboratory test results were surveyed through the hospital's health information system. this study had been approved by the ethical review committees of harbin medical university in accordance with the declaration of helsinki. both informed and written consents were obtained from each participant who provided specimens. the presence of common respiratory viruses, including piv type to , influenza virus a and b virus (iav and ibv), rsv, and human adv was determined by the d ultra tm dfa respiratory virus screening kit (diagnostic hybrids, inc., athens, oh, united states). virus isolation for the adv-positive specimens was performed using the a cell line (provided by the cancer hospital of harbin medical university) following the protocol described previously (smith et al., ; huang et al., ) . in brief, cells seeded with clinical samples were incubated at • c for days. if there was no observed cytopathic effect, two additional blind passages of the culture were performed to avoid false negative results. the cultures with adv-like cytopathic effects were passaged again to confirm viral presence. the viral dna of human adv was extracted from infected cells using the axyprep multisource genomic dna miniprep kit (axygen biosciences) according to the manufacturer's instructions and eluted in µl elution buffer. the highly variable region in the adenoviral hexon gene was amplified using the following primer pairs: forward, -caattcactcgctccta- and reverse, -gtggaaaggcacataacg- . all primers were synthesized by comate bioscience co., ltd. pcr was performed with the platinum pcr supermix (invitrogen) following the manufacturer's instructions. a total reaction volume of µl contained µl of × pcr buffer (takara bio premix ex taq tm ), nm of each primer, nm of each dntps, µl of each dna sample, . µl taq enzyme (takara bio premix ex taq tm ), and sterile water. the cycling conditions were min at • c, followed by cycles of s at • c, s at • c, and s at • c, with a final -min extension at • c. dna extracted from the a cell culture and sterile water were used as negative controls. the amplicons were bi-directionally sequenced using the sanger sequencing method with an abi prism genetic analyzer (comate bioscience co., ltd., jilin, china). the daily average air temperature from january st, to december st, in harbin city was kindly provided by the harbin meteorological observatory. historical temperature datasets covering - in harbin were accessible from the china meteorological data service center. climate seasons were determined based on the smoothed daily average temperature curve according to the chinese national standard no: qx/t - (administration, ) . in brief, spring starts when the daily average temperature permanently rose above • c, while summer comes when it rose above • c permanently. the autumn starts when the daily temperature drops to • c permanently and the winter begins at • c. "permanently" means the daily temperature has remained above or below the threshold for at least five consecutive days. the details can be found in the supplementary table s . statistical analysis was conducted using the r language (version . . ). the prevalence (detection percentage) of viruses was calculated by dividing the sum of positive cases by the number of cases in total. chi-square tests or fisher's exact tests were selected for comparing the cross tables of categorical variables. the wilcoxon rank-sum or kruskal-wallis tests were chosen for continuous variable comparisons as appropriate. datasets were visualized by an excel spreadsheet. the quality of sequencing data was checked manually via chromas software version . (technelysium pty ltd., south brisbane, qld, australia), before contigs for each isolate were assembled using ugene (protsyuk et al., ) software suite (version . ) guided by the published hexon gene sequence of human adenoviruses. similar strains to the isolates in this report were found from the genbank database via the blast search method (boratyn et al., ) . fifty-eight nucleotide sequences of the hexon gene covering all seven species of human adv were selected as the reference in this study. before the tree building process, multiple sequences were aligned by muscle (edgar, ) software (version . ). the unrooted neighbor-joining tree was reconstructed using the kimura parameters distance, whose robustness was tested by the bootstrap method with , replications implemented in mega (kumar et al., ) software (version . . ). the parsimony tree was also built on the same dataset. in total, , hospitalized adult patients with respiratory infection were enrolled from january to december in harbin city. among these patients, were male and were female, as shown in bronchiectasis, with asthma and with other infections. some clinical signs were more frequent among cases with certain diagnoses. in all, ( . %) pneumonia cases complained of fever, while only ( . %) cases with bronchitis had fever (p < . ). cough was the chief complaint of the bronchial infections (p < . ), including bronchitis ( . %), copd ( . %) and asthma ( . %). out of , enrolled patients and were diagnosed with chronic diseases, including chronic respiratory disease, cancer, diabetes mellitus, chronic cardiac diseases, cerebrovascular disease, chronic kidney disease, and other chronic diseases lasting more than year. among the , total patients, ( . %) cases were determined to be positive with at least one of seven viruses, including influenza virus (a and b), piv (type , , and ), rsv and adv. one hundred and forty patients were infected with only one virus as the single infection, and patients were co-infected with more than one virus. double infections were observed in cases, while triple infection was found in six cases. as shown in table , the most frequent single infection was piv ( cases) and the major double infection was combined influenza and parainfluenza viruses ( cases). the details were accessible from the supplementary table s . the median age of the viral positive group was (iqr - ) and the negative group was (iqr - ). the age difference between the viral infection positive and negative group was not statistically significant (p = . ). the viral detection rate varied from . % ( / ) in the middle age group to . % ( / ) in the senior citizen group, but the difference was not statistically significant (p = . ). there was no significant difference in the viral infections between genders (p = . ). the virus detection rate among the study years varied significantly (p = . ) from . % ( / ) in to . % ( / ) in , as shown in table . the detection rate was not equal among calendar seasons (p = . ) or climate seasons (p = . ). based on the climate seasons, the highest overall detection rate was in spring ( . %, / ), followed by winter ( . %, / ), autumn ( . %, / ), and summer ( . %, / ). for individual virus positive samples, certain viruses were more frequently detected in spring than in other seasons, including influenza virus ( . %, / ), piv ( . %, / ), and rsv ( . %, / ), as shown in table . adv was more frequently detected in winter, with a detection rate of . % ( / ). piv was the dominant viral pathogen during the study period, accounting for . % ( / ) of the viral infection cases in , . % ( / ) in , . % ( / ) in , and . % ( / ) in . as shown in table , viral co-infections were more not equally distributed among seasons, with the highest ratio in spring at . % ( / , p = . ). flu piv rsv adv cases total overall detected respiratory viral positive cases were plotted against daily average air temperature in figure . the low temperature over days was a strong indicator for respiratory viral infections. positive cases were rare in summer, which began late in june and ended in mid of august. one exception was found in the summer in which was related to a sudden drop of air temperature from . • c on th july to . • c on th july . the overall percentage of viral infection was significantly connected with clinical diagnoses (p = . ), as shown in table . the highest detection rate was observed in the pneumonia group ( . %, / ), followed by bronchitis ( . %, / ), while the lowest detection rate was found in the asthma group ( %, / ). chest tightness was the only symptom that showed statistically significant difference (p = . ) between the positive ( . %, / ) and negative ( . %, / ) group of viral infections. the respiratory viral infection did not prolong hospital stay statistically (p = . ), although the median days in the hospital for the positive viral infection group was (iqr - ), which was shorter than in the negative group (iqr - ). overall, the viral detection rate was relatively lower in the chronic diseases group ( . % vs. . %), with statistical significance (p = . ). detection rates varied among different chronic diseases, but the difference was not significant (p = . ). the viral co-infection ratio among different diagnosed groups was not significant (p = . ). two strains of human adv were isolated from two patients diagnosed with pneumonia, respectively. the harbin b strain ( came from a male patient aged whose samples were collected in december . the other strain, named harbin a, was isolated from a male patient aged whose samples were collected in january . based on the evolutionary tree reconstruction of the hexon gene sequencing data, both strains isolated in this study were identified as human adv species b type . these two strains were nearly identical, with the closest evolutionary distance to the strain gb (genbank: ay ; atcc: vr- ) from the united states as well as the harbin b strain from a previous local epidemic, as shown in figure . the parsimony tree was similar in topology with neighborjoining tree (figure not shown in the article). multiple sequence alignments indicated that, compared with the gb strain, the harbin a and harbin b strain shared a variable site in the nd codon (gb numbering) of the hexon gene hosting an a to c mutation, resulting in threonine (t) to proline (p) mutation of the amino acid sequences. the harbin b strain had a threonine on the same site in the hexon protein encoded by the codon acu, which was identical to the harbin a and harbin b strain. adult populations with respiratory infections bear heavy burdens of hospitalization due to severe respiratory illness, especially for senior citizens (muller-pebody et al., ) . the constantly changing nature of viral pathogens has made the surveillance of these infections critical to public health authorities (zou et al., ; li et al., ) . the key indicators for viral infections from this report and recent published surveys in china has been summarized in table . the overall detection rate of viral infection among hospitalized adult patients in this report is . %, which was consistent with the result of . % in the age group above years old by a national survey from to in china (feng et al., ) . the dominant viral pathogen throughout this study period was piv, with a detection rate of . %, followed by influenza virus. the national survey on hospitalized patients with respiratory infection in china from to showed that influenza virus had the highest detection rate of . % in adult patients. the detection rate of respiratory viruses in the over years age group was . % in shandong province of china (liu et al., ) and was dominated by influenza virus ( . %) from to . from to , the respiratory viral detection rate in hospitalized patients above years old within beijing and shandong china was reported to be . %, in which influenza virus had the highest frequency of . % (yu et al., ) . compared with recent surveys in beijing, shandong and other regions in china, this report showed that the piv dominated epidemic seasons from to in adult patients in harbin, which should be important for preparing future epidemic control strategies in public health. the viral infection rate within asthma subgroup of this report was %, and it was one patient with rsv among asthma patients in total as shown in table . asthma is related to viral infection, while the detection rates varies among different pathogens. a recently published meta-analysis showed that the mean prevalence of rsv, influenza virus, piv and adv was . %, %, . %, and . %, respectively (zheng et al., ) . the apparently low detection rate within asthma group in this report could be possibly caused by the sampling bias due to the limited subgroup size. air temperature was a major meteorological variable associated with respiratory viral infection (chan et al., ) . harbin city is located at degrees north latitude with an annual average temperature of . degrees celsius. climate seasons are not in equal length in harbin, according to historical statistics from to . the longest season in harbin is winter, which starts on the th of october and lasts days, followed by spring on the rd of april with days. starting on the rd of june, summers in harbin are relatively short at days, while autumn is days and is the shortest season, starting on the th of august. in routine epidemiological analysis, months in a year were usually divided into four seasons equally, which is not the case in harbin and possibly compromises contagious disease seasonality prediction. based on climate seasons defined by the daily average air temperature, influenza, parainfluenza and rsv detected in this report all presented significant seasonal peaks in spring, although similar phenomena were absent within the calendar seasons. sputum samples were acceptable both in viral culture and antigen detection in respiratory infections compared with nasopharyngeal swabs and aspirates (covalciuc et al., ) , although they were not frequently collected in recent surveys on lower respiratory tract infected adult patients from china. detection of respiratory virus in sputum samples has been proven effective on a small scale, either by the immunofluorescence method (honkinen et al., ) or the pcr method (branche et al., ) . this report provided a medium scale test on the validity or effectiveness of respiratory viral detection from sputum samples with , cases. in this report, two nearly identical strains were isolated from pneumonia patients in harbin, possessing high similarity with the local strain harbin b and the strain gb. the harbin b strain was isolated from a pediatric patient in harbin (zhang et al., ) . adv strain gb was named vr- in the atcc database, which was isolated from an adult patient with common cold in maryland, united states in (huebner et al., ) . the similarity of these strains across asian and american continents through several decades may result from the globalization movement in modern china. it could also be explained by another hypothesis based on the evolution history of adenoviruses. on the geological scale, homo sapiens inherit human adenoviruses from our ancestors within the hominidae, resulting in an evolutionary rate of human adv as low as . e- substitution per site per year (hoppe et al., ) . considering its kbp genome, we would have to wait a millennium (or years exactly) before a single site mutation within an adenoviral genome occurred naturally. there were a few limitations to this study. first, the years of investigation on a single city in this present report was relatively short and much more localized than other long-term or national surveillance, which may lead to a bias of the viral etiology. second, the enrolled population in this study consisted of adult patients only, while the samples from pediatric patients were not collected simultaneously. the comparison of the viral prevalence status between adult and pediatric populations in this study was difficult. third, the bacterial culture results of the clinical samples were not included in this report, which prevents us from drawing a comprehensive picture on the pathogens associated with lower respiratory tract infections. in this report, . % of lower respiratory tract infections in hospitalized adults from harbin were found to be related with common respiratory viruses. the piv dominated viral infection from to with detection rates of . %. climate seasons based on daily temperature records were found to be reliable in seasonality analysis. adv type strains with slight variations were isolated from positive cases. further surveillance would be important to continuously monitoring the viral etiology in adult patients both local and abroad. zq, yw, and td designed this study and prepared the manuscript. gq, yg, and wg collected the specimens. yw, td, and yg conducted the epidemiological investigation. td, lq, wl, bq, zz, ls, hg, xd, bl, zl, hy, qc, xw, and yl performed the laboratory experiments. yw and td analyze the data and made the figures and tables. all authors reviewed the manuscript. division of climatic season designer oncolytic adenovirus: coming of age blast: a more efficient report with usability improvements detection of respiratory viruses in sputum from adults by use of automated multiplex pcr hospitalization incidence, mortality, and seasonality of common respiratory viruses over a period of years in a developed subtropical city comparative genomic analysis of re-emergent human adenovirus type pathogens associated with adult severe community-acquired pneumonia reveals conserved genomes and capsid proteins comparison of four clinical specimen types for detection of influenza a and b viruses by optical immunoassay (flu oia test) and cell culture methods muscle: multiple sequence alignment with high accuracy and high throughput viral etiologies of 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and hemagglutination determinants of all human adenovirus prototypes as a basis for molecular classification and taxonomy modelling hospital admissions for lower respiratory tract infections in the elderly in england structure of human adenovirus human parainfluenza virus infection in severe acute respiratory infection cases in beijing, - : a molecular epidemiological study. influenza other respir viral infections of the lower respiratory tract: old viruses, new viruses, and the role of diagnosis shared bioinformatics databases within the unipro ugene platform serotype-specific neutralizing antibody epitopes of human adenovirus type (hadv- ) and hadv- reside in multiple hexon hypervariable regions detection of multiple respiratory viruses associated with mortality and severity of illness in children impact of pollution, climate, and sociodemographic factors on spatiotemporal dynamics of seasonal respiratory viruses identification of a critical and conformational neutralizing epitope in human adenovirus type hexon antibodies against adenovirus fiber and penton base proteins inhibit adenovirus vector-mediated transduction in the liver following systemic administration single-particle em reveals plasticity of interactions between the adenovirus penton base and integrin alphavbeta prevalence of respiratory viruses among children hospitalized from respiratory infections in shenzhen assessment of human-to-human transmissibility of avian influenza a(h n ) virus across five waves by analyzing clusters of case-patients in mainland china variation in influenza b virus epidemiology by lineage characteristics of neutralizing antibodies to adenovirus capsid proteins in human and animal sera comparison of the prevalence of respiratory viruses in patients with acute respiratory infections at different hospital settings in north china molecular modeling and epitopes mapping of human adenovirus type hexon protein combined cd + and cd + adenovirus hexon-specific t cells associated with viral clearance after stem cell transplantation as treatment for adenovirus infection respiratory viruses in hospitalized children with acute lower respiratory tract infections in harbin regional, age and respiratory-secretion-specific prevalence of respiratory viruses associated with asthma exacerbation: a literature review evolution and transmission of respiratory syncytial group a (rsv-a) viruses in guangdong we gratefully acknowledge the harbin meteorological observatory and the china meteorological data service center for providing air temperature datasets in this report. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmicb. . /full#supplementary-material key: cord- - bozjfv authors: cagliani, rachele; forni, diego; sironi, manuela title: mode and tempo of human hepatitis virus evolution date: - - journal: comput struct biotechnol j doi: . /j.csbj. . . sha: doc_id: cord_uid: bozjfv human viral hepatitis, a major cause of morbidity and mortality worldwide, is caused by highly diverse viruses with different genetic, ecological, and pathogenetic features. technological advances that allow throughput sequencing of viral genomes, as well as the development of computational tools to analyze such genome data, have largely expanded our knowledge on the host range and evolutionary history of human hepatitis viruses. thus, with the exclusion of hepatitis d virus, close or distant relatives of these human pathogens were identified in a number of domestic and wild mammals. also, sequences of human viral strains isolated from different geographic locations and over different time-spans have allowed the application of phylogeographic and molecular dating approaches to large viral phylogenies. in this review, we summarize the most recent insights into our understanding of the evolutionary events and ecological contexts that determined the origin and spread of human hepatitis viruses. human hepatitis viruses are extremely diverse and consequently belong to different viral families (table ). in recent years, the availability of high-throughput technologies has revealed that relatives of human hepatitis viruses can be found in a wide variety of animals. this finding, as well as the increasing availability of the genome sequences of human-infecting viruses sampled across different geographic areas, has largely expanded our knowledge about the genetic diversity and evolutionary origin of these human pathogens. in this review, we thus focus on the latest insights into the possible events and ecological contexts that determined the origin and spread of human hepatitis viruses. a short presentation of the most widely used approaches to estimate the ages of viral lineages is also provided to contextualize recent research efforts on these viruses. molecular dating analyses using virus genetic data can be particularly informative due to the rapid rate of evolution of many viral species. by converting genetic differences among sequences into time units, molecular dating provides information on the timing of viral spread or emergence. most molecular dating approaches are based on maximum-likelihood or bayesian phylogenetic frameworks [ , ] , and they usually exploit two strategies: molecular clock calibration using the sampling dates of the viral sequences (tip dating) and/or calibration using information on some internal nodes of the phylogeny. tip dating is well-suited to study relatively recent events (e.g., epidemics or intra-host evolution), but requires that a temporal signal is present in the dataset (i.e., that the sequences have accumulated a measurable amount of change between sampling times) [ ] . calibration using internal nodes can in principle allow to dig deeper into the past, but requires some a priori knowledge about the virus evolutionary history (e.g., host-virus co-evolution, paleontological information). the widespread use of molecular dating has however revealed that the relationship between genetic divergence and time is complex, as evolutionary rates tend to vary with the time frame of measurement. in particular, high evolutionary rates are observed in the short term, whereas low rates are inferred in long time span studies [ ] [ ] [ ] . this pattern was observed for many viral lineages and is sometimes referred to as the time-dependent rate phenomenon (tdrp) [ ] . failure to account for the tdrp can potentially lead to erroneous molecular dating results [ , ] . the tdrp reflects mutation rate in very short timescales and substitution rate in very long timescales, during which transient deleterious mutations are removed by the action of natural selection, leading to lower rate estimates [ ] . another factor most likely contributing to the tdrp is the saturation of nucleotide substitutions, which is extremely rapid in viral genomes, especially when the polymerase is error-prone [ ] . thus, recent analyses indicated that, for all baltimore classification groups, viral evolutionary rates tend to decrease continuously with the timescale of measurement [ , ] . because the rate of decay is consistent with a power law relationship between substitution rate and sampling timescale, a model using a simple regression was at first proposed to estimate the tdrp effect on viral phylogenies [ ] . very recently, this approach was implemented in a bayesian statistical framework, in which evolutionary rates can vary among different time epochs [ ] . before the introduction of such an approach, effective attempts to correct for the tdrp were performed by the use of nucleotide substitution models that allow site-and branch-specific variation in selective pressure (selectioninformed models). these models, which were applied to analyze the ancient evolution of some viral lineages, at least partially correct for the effects of both purifying selection and substitution saturation in branch length estimation [ ] [ ] [ ] . hav is mainly transmitted via the faecal-oral route through exposure to contaminated food or water, or through direct contact with infected people. hav is a single-strand, positive rna virus with a genome of approximately . kb in length ( table ). the hav genome contains a single orf flanked by a relatively long utr and a utr. the utr harbors an internal ribosome entry site that directs the cap-independent translation of hav proteins. the orf encodes a polyprotein processed in mature proteins: structural proteins involved in capsid formation (vp , vp , vp , vp , and px, deriving from p segment) and nonstructural proteins with a role in rna genome amplification ( b, c, a, b, cpro, and dpol, deriving from p and p segments) [ , ] . based on genomic structure, hav belongs to the family picornaviridae within the genus hepatovirus. nevertheless, many characteristics distinguish hav (and hepatoviruses in general) from other picornaviridae family members. some peculiar features include the primary tropism for hepatocytes, the ability to shed as nonenveloped virus in feces and as enveloped particles in blood, as well as some genomic features such as low g/c ratio, low cpg levels, and strong codon bias [ , ] . hav was identified as the etiologic agent of hepatitis a by feinstone and colleagues [ ] in . unlike hbv and hcv, which establish chronic infections in humans, hav infection is usually acute and generates lifelong immunity. this condition is able to determine the disappearance of the virus in small and isolated populations [ , ] and did not probably favor its maintenance in early human communities. it is thus legitimate to wonder how hav survived and evolved during early human history, a question that remains presently unanswered. for a long time, it was thought that hepatoviruses were restricted to humans and non-human primates (nhps), with genetically distinct variants classified as six main different genotypes [ ] : three isolated from humans (hav, genotype i-iii) and subclassified in subgenotypes (ia, ib, iia, iib, iiia, iiib) and three of simian origin (shav, genotype iv-vi). however, despite genetic heterogeneity, hav viruses belong to a single common serotype. in recent years, the advent of new sequencing approaches has led to an exponential increase in the identification of new viral spe- acute cies, including highly diverse non-primate hepatoviruses. several hav-related viruses were identified in different mammalian orders. in particular, a number of hav-like viruses were recovered in placental mammals, mainly in bats and rodents, but also in tree shrews, hedgehogs, seals and chinese woodchucks [ ] [ ] [ ] . recently, de oliveira carneiro and colleagues [ ] identified a novel hav-related virus in didelphis aurita, a brazilian common opossum, further extending the host range of mammal-infecting hepatoviruses. moreover, viruses related to mammalian hepatoviruses were detected in reptiles, amphibians, and fish [ ] . these advances allowed new insights into the evolutionary history of hav. the phylogenetic relationships among hepatovirus that infect small mammals only partially reflects those among their hosts, suggesting multiple, non-recent cross-species and cross-order host switches during hepatovirus evolution [ ] . this observation is supported by recombination events observed in hepatoviruses that have been identified in genetically and geographically distant hosts [ ] . these cross-species transmission events also involved the opossum hepatovirus, which most likely originated from an ancestral host switch from rodents into marsupials [ ] . conversely, hepatovirus phylogenies suggest no host switch involving a primate donor. this evidence, the absence of recombination events between havs and non-primate hepatoviruses, as well as the observation that primate hepatoviruses form, regardless of the genomic region considered, a monophyletic group in the topology of hepatovirus phylogenies, support the hypothesis that humans and nhp have acquired hepatoviruses from other animal reservoirs relatively recently [ , ] . however, if and when this hypothetical host-jump occurred into primates remains to be clarified. phylogenetic and ancestral state reconstruction suggested a likely cricetid rodent origin for primate havs and marsupial hepatoviruses, whereas a laurasiatherian host origin was proposed for all mammalian hepatoviruses [ , ] (fig. ) . in this scenario, the evolutionary history of hepatoviruses is evocative of that of hantaviruses, as the origin of mammalian hantaviruses is traced back to bats and insectivores [ ] . thus, the supposed origin of hepatoviruses in insectivorous laurasiatherian mammals, as well as the preservation of some structural and functional characteristics similar to present-day insect picorna-like viruses (dicistroviridae) [ ] led drexler and colleagues to hypothesize a more ancient evolutionary origin of havs, with an ancestry in a primordial insectborne virus [ ] . in conclusion, hav emergence in humans likely represents a relatively recent evolutionary event, probably of zoonotic origin. nonetheless, the ancestor of human hepatoviruses has yet to be identified. the characterization of other hepatoviruses in primates, and mammals in general, will be instrumental to the identification of the hav ancestors and will clarify the evolutionary history of hepatoviruses. hbv was the first human hepatitis virus to be isolated and identified in [ ] . hbv transmission varies depending on the prevalence of infection. in areas with a low prevalence (< %), the most common mode of transmission is through infected blood or high-risk behaviors (e.g. unprotected sex or injecting drug use). in high-and intermediate-prevalence areas, hbv is commonly spread through perinatal and horizontal (especially among children) routes [ ] . hbv belongs to the hepadnaviridae family, which comprises two genera: orthohepadnavirus (mammal-infecting viruses) and avihepadnavirus (bird-infecting viruses) ( fig. a) . its genome, a partially double-stranded circular dna of about . kb (table ) , is composed of four overlapping frameshifted open reading frames (orfs) [ ] . viral replication is carried out by a reverse transcriptase with no proofreading ability and considerable variability exists among strains. thus, at least nine genotypes (a-i) plus a tentative one (j), with a heterogeneous global distribution, have been described to date [ ] [ ] [ ] [ ] [ ] (fig. b) . despite its worldwide diffusion and the accumulation of detailed knowledge on the associated pathologies, the origin and evolutionary history of hbv are still debated [ , ] . hepadnaviruses were detected in several mammals, including nhps, rodents and bats, birds, fish, and reptiles [ , ] (fig. a) . recently, lauber et al. discovered a family of fish viruses with genomic features similar to those of hbv, dating the origin of the hepdnaviridae family to at least million years ago [ ] ; this finding, together with the discovery of endogenous hepadnavirus elements integrated in the genome of birds and reptiles [ ] [ ] [ ] [ ] , suggests a long and complex relationship between this viral family and its hosts. concerning hbv, different theories were proposed to explain its origin, but all of them have some sort of limitation. hbv was initially thought to have emerged quite recently in the new world from genotypes f/h infecting amerindians [ , ] ( fig. a and b). however, the discovery of an ancient strain in a th century asian mummy, as well as the worldwide diffusion of hepadnaviruses in nhps, questioned this hypothesis [ ] (fig. a ). an alternative theory suggests that hbv followed the out-of-africa migration of modern humans, which occurred approximately , years ago [ ] [ ] [ ] . in particular, paraskevis et al. found a good correspondence between the demographic histories of hbv and those of human populations [ ] . these authors also showed that the substantial divergence of the f and h genotypes ( fig. a) , a major evidence in favor of the new world origin hypothesis [ ] , was probably due to positive selection acting on those branches [ ] . nonetheless, the extensive application of ancient dna sequencing revealed a more complex scenario. in fact, two european neolithic hbv genomes did not cluster with any extant human strain in the phylogenetic tree, but did cluster with nhp viruses [ ] (fig. a ). other authors [ ] , who sequenced ancient hbv genomes of different ages ( - years old), obtained a similar result, with the ancient strains clustering with known modern genotypes or forming new clades ( fig. a) . this implies that some hbv lineages of the past went extinct ( fig. a) . moreover, muhlemann and coworkers showed that the geographic distribution of ancient samples does not match the modern genotype distribution [ ] . they thus suggested that early evolutionary scenarios can be concealed and overwritten by more recent migratory events [ ] . a third hypothesis for the origin of hbv posits that hepadnaviruses co-speciated with their primate hosts in the new world and in the old world. thus, multiple zoonotic transmissions would have originated hbv genotypes found in humans [ ] . this scenario is supported by the diffusion of hepadnaviruses in diverse primate species and by the inferred divergence time of the orthohepadnavirus and avihepadnavirus genera, that is very similar to that of their host classes [ ] . however, the recent identification of a novel hepadnavirus in capuchin monkeys confirmed that new world monkeys are infected by viruses that are very distantly related to hbv ( fig. a) , indicating that they do not represent the direct ancestors of genotypes h and f [ ] . instead, evolutionary analyses with human and nhp viral strains placed the origin of hbv ancestors in hominoid old world primates, preceding the formation of the human lineage [ ] . in summary, although considerable progress was achieved in recent years, a high level of uncertainty concerning the ultimate origin of hbv still exists. the particular organization of the viral genome (i.e. overlapping reading frames in a short genome) limits the variability of most of nucleotide positions (i.e. to avoid the introduction of nonsynonymous mutations) and results in a relatively slow mutation rate. this characteristic, along with the action of natural selection on particular genotypes [ ] , as well as the adaptation to different human populations [ ] , contributes to hbv variability and complicates inferences about its origin. finally, different studies [ , , ] have shown that, as for other viruses (see section ), hbv substitution rates are affected by viral sampling time frames. indeed, the evolutionary rates generated using information from ancient hbv genomes were shown to fit well with the tdrp regression line calculated for baltimore group vi and vii viruses (i.e., reverse-transcribing viruses) [ ] . crucially, these results indicate that, whereas tip calibration approaches have demonstrated to be useful in the reconstruction of recent epidemiological events [ ] [ ] [ ] , limiting analysis to extant strains for the reconstruction of ancient hbv evolution can be misleading [ ] and that approaches that correct for the tdrp should be envisaged. hcv is an enveloped virus belonging to the flaviviridae family (genus hepacivirus). in analogy to other members of the family, hcv has a . kb positive-stranded linear rna genome. the virus encodes a single polyprotein that is processed by cellular and viral proteases to yield at least mature products. hcv was identified in by houghton and colleagues as a cause of non-a and non-b hepatitis [ ] . if left untreated, hcv can persist lifelong in humans, often resulting in cirrhosis and hepatocellular carcinoma. presently, the hcv worldwide seroprevalence is estimated to be $ % [ ] , with about million persons living with chronic infection [ ] . the hcv epidemic apparently started recently, in the s- s, as a consequence of practices that determined parenteral or percutaneous exposure (e.g., blood transfusion, vaccination campaigns, and intravenous drug injection) [ ] [ ] [ ] . for instance, one of the most affected countries is egypt, where the virus was most likely disseminated through nationwide vaccination programs or contaminated blood-derived products [ ] . in fact, sexual or vertical transmission of hcv are relatively rare, and the overwhelming majority of infections occur via the parenteral/percutaneous route. thus, due to historical reasons and to the transmission pattern, a small number of so-called ''epidemic" hcv subtypes ( a, b, a, and a) account for most infections worldwide [ , ] . hcv is, however, genetically heterogeneous and the epidemic subtypes represent a minor fraction of viral diversity. eight major hcv genotypes (hcv- to - ) have been described, and these are further divided into at least subtypes (https://talk.ictvonline. org/ictv_wikis/flaviviridae/w/sg_flavi/ /hcv-classification) (fig. a) . several of these genotypes and subtypes were identified and classified only recently [ , ] , suggesting that a considerable proportion of hcv diversity remains undescribed. moreover, a number of natural inter-genotype recombinants were reported [ , ] was generated using phyml [ ] . hepatovirus host silhouettes are colored according to taxonomic order. the human hav subgenotypes are also reported. (https://talk.ictvonline.org/ictv_wikis/flaviviridae/w/sg_flavi/ / table- -recombinant-rf-hcv-genomes). hcv genotypes display antigenic variability and viral genetic diversity is geographically structured: in sub-saharan africa and south-east asia highly divergent subtypes of the same genotype dominate transmissions across contiguous areas (fig. a) . these subtypes are referred to as ''endemic" [ , ] and their presence is consistent with a long-standing association of hcv with populations living in these regions. because parenteral exposure became common only in the relatively recent past, several hypotheses were formulated to account for the maintenance of endemic hcv transmission. some authors proposed that traditional practices such as circumcision, tattooing, piercing or acupuncture facilitated and maintained hcv infection among human populations [ , ] . others indicated that sexually transmitted infections (stis) that disrupt mucosal integrity are responsible for increased sexual hcv transmission [ ] . this was indeed shown to be the case in modern high-risk populations [ ] and stis have probably been common throughout human history [ ] . an alternative scenario is that the bite of arthropods, especially those taking large blood [ ] ). graphical representation of genotype (gt) diversity. the number of distinct recognized subtypes is represented on the y axis. circle size is proportional to the average pairwise distance between subtypes. genotype is marked with an asterisk as only two subtypes are known. (b) phylogenetic tree of the rdrp domain of known hepaciviruses. the tree was obtained using raxml with bootstrap replicates [ ] . nodes with support equal to or higher than . are marked with a black dot. (c) hypothetical viral phylogenies that illustrate the effect of viral lineage extinction on the evolutionary inference about the origin of hcv and ehv/chv. in the left panel, a horse-to human transmission event is hypothesized, with the following extinction of the transmitted ehv lineage. in the right panel, a reverse zoonosis introduces a hcv-like virus in horse populations; the following extinction of several ehv lineages accounts for the low genetic diversity of extant strains. meals, can mechanically transmit hcv, possibly from other animal reservoirs such as horses [ , ] . hcv infection is in fact restricted to our species but, thanks to extensive field work, a number of hepaciviruses have been described in domestic and wild mammals, as well as in reptiles and fish [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (fig. b ). at present, the largest diversity of hepacivirus species seems to be hosted by rodents and bats (fig. b) . instead, as previously noted [ ] , the lowest genetic diversity is observed for hepaciviruses that infect cattle and horses (fig. b) , suggesting that husbandry practices may have resulted in the artificial selection of specific viral strains or facilitated recent viral transmission from some other animal source (e.g., commensal rodents). non-human primates also host hepaciviruses, but these are highly divergent from hcv. overall, the phylogenetic relationships among hepaciviruses poorly mirror those among their hosts (fig. b ), suggesting several crossspecies and cross-order host switches during viral evolution. up to now, the closest relatives of hcv were identified in horses/donkeys (equine hepacivirus, ehv) and dogs (canine hepacivirus, chv) (fig. b) . because chv is less genetically diverse than ehv, the canine virus possibility originated as a recent cross-species transmission from horses [ ] , hinting at the ability of hepacivirus to shift among genetically distant hosts. despite these advances, the events that led to the origin of hcv are still unknown. taking as a fact the relatedness of the human virus to ehv/chv, possible scenarios include that: i) hcv originated from a cross-species transmission of ehv, ii) that ehv was transmitted to horses by humans infected with hcv, which leaves the question on hcv origin open; iii) that hcv and ehv originated from the cross-species transmissions of the same (or similar) virus, with subsequent host adaptation and divergence. if this were the case, multiple crossspecies transmission events may have originated distinct hcv genotypes [ ] . teasing apart these possibilities clearly requires understanding of the timing and circumstances of hcv (and ehv) evolution. up to date, no archaeological sample carrying traces of hcv or ehv has been described and the oldest hcv sequence dates to [ ] ( for ehv [ ] ). thus, molecular dating efforts have relied on extant sequences, with the difficulties associated with the tdrp. studies that did not account for the tdrp provided estimates of the time to the most recent common ancestor (tmrca) of hcv genotypes in a range between $ and years ago [ , , , , ] ; the origin of the horse virus was dated around ce [ ] . a study that separately accounted for the rate of synonymous and nonsynonymous substitutions estimated hcv to have originated at least years ago [ ] . recently, a method based on an a selection-informed model was used to estimate the divergence time of hcv genotypes and the origin of extant ehv/chv strains [ ] . this approach, provided estimates of $ years ago for the tmrca of extant hcv genotypes (with a low-bound estimate of $ years before present) and of $ years ago for ehv/chv [ ] . if these dates are taken to provide at least an indication of the real evolutionary scenario, the possibility that hcv was transmitted to humans by horses infected with ehv can be excluded, an observation in line with the low diversity of ehv/chv [ ] . the origin of ehv/chv as reverse zoonosis (i.e., the transmission of a human virus to animals) seems also unlikely, as in this case horse viruses should cluster within hcv diversity, unless the hcv lineage that originated ehv went extinct in the last years. indeed, as the hbv story exemplifies, viral lineage extinction can occur and was previously documented for other human pathogens such as parvovirus b [ ] , and variola virus [ ] . as anticipated above, breeding practices may facilitate this process in the case of animal viruses. we know, for instance, that a minimum of two horse lineages went extinct during the domestication process and that horse genetic diversity has largely been shaped by events that occurred in the last few centuries [ ] . it is thus possible that human-mediated selection on the host also resulted in the artificial selection of viral lineages. this would explain the relatively recent origin of extant ehv strains and their low diversity. if viral lineage extinction did occur, the time frames of ehv and hcv evolution would be underestimated and the scenario of hcv originating as a zoonosis from horses (or ehv as a reverse zoonosis) may still hold (fig. c) . of course, the alternative possibility that ehv and hcv were transmitted independently to their present-day hosts by a third unknown reservoir is also in line with data on extant diversity and, if the transmission to horses occurred recently, does not require to postulate viral lineage extinction. thus, a number of open questions remain on the origin of hcv. hopefully, technological advances that allow sequencing of trace genetic material from ancient samples will provide information on viral strains hosted by humans and horses back in the past. at the same time, the extensive application of metagenomic approaches to different animal hosts across diverse geographic regions will expand our knowledge on hepacivirus diversity and eventually uncover the direct ancestor of hcv (if it still exists). indeed, the possibility that the different hcv genotypes derived from independent cross-species transmission events [ ] would imply that viruses related to hcv are relatively common in the wild, thus giving good chances to be recovered in large field surveys. hdv is a defective virus incapable of autonomous propagation [ ] . its genome, a self complementary circular rna of $ nucleotides, encodes a single protein (the hdv-encoded delta antigen) ( table ) . hdv requires hbv surface proteins, that are complexed with the delta antigen, to form transmissible virions [ ] . thus, hdv is usually considered a satellite of hbv, although recent data have shown that other enveloped viruses can promote hdv propagation, at least in vitro (e.g. hcv, dengue virus, vesicular stomatitis virus) [ ] . genetic heterogeneity among strains is quite high for hdv, which is thus classified in eight different genotypes (from to ), although a three major genogroup classification was recently proposed (group for previous genotype , group for genotypes and genotypes from to , and group for previous genotype ) [ ] hdv genetic diversity is highest in africa, suggesting that the defective virus emerged in and spread from this continent [ , ] . the evolutionary origin of hdv is nonetheless unknown. hdv-like circular rnas were only recently described in birds, reptiles, amphibians, fish and insects [ ] [ ] [ ] . however, these hdvlike elements were not found to be associated with hepadnavirus infection, reinforcing the idea the hdv is not necessarily only transmitted in conjunction with hbv-related viruses, at least in these animals [ ] . current evidence suggests that hdv evolved from the human cellular transcriptome [ ] . the first indications that a virus was responsible for waterborne, epidemic hepatitis came from studies of asian outbreaks in the - s and, in analogy to hcv, the agent was referred to as ''epidemic non-a, non-b hepatitis" [ ] [ ] [ ] . hepatitis e virus was eventually isolated and sequenced in the early s [ , ] . since then, a number of hev strains responsible for human infection were identified. hev is a positive-strand rna virus belonging to the hepeviridae family (table ) . in common with all other members of this viral family, the hev genome comprises three partially overlapping open reading frames (orfs): orf and orf encode a non-structural [ ] . the piscihepevirus branch is in red, orthohepevirus branches are in blue. the enlargement shows phylogenetic relationships for viruses belonging to the orthohepevirus a species, with representative hosts. (b) geographic distribution of anthropotropic (hev- and hev- ) and enzootic (hev- -hev- ) hev strains. genotypes were assigned to countries irrespective of their prevalence. thus, even if a single case was reported in a given country, the genotype was recorded as present. cases that could be clearly ascribed to migration/travels were excluded. data derive from forni et al. [ ] , with updates from [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . (c) time-scaled phylogenetic tree of a non-recombing orf region [ ] . branch lengths represent evolutionary time. the time-frames of historical events mentioned in the text are reported. the rabbit and camel silhouettes mark the split of the rabbit-infecting and camel/dromedary-infecting genotypes. the pig silhouette marks the human-restricted/enzootic genotype split. polyprotein and the viral capsid, respectively, whereas orf codes for a small phosphoprotein. a fourth orf (orf ), overlapping the helicase domain in orf , was recently described but seems to be specific for some hev genotypes (hev genotype , hev- ) [ ] . members of the hepeviridae family are currently classified into two genera, orthohepevirus and piscihepevirus [ ] (https://talk. ictvonline.org/ictv-reports/ictv_online_report/positive-sense-rnaviruses/w/hepeviridae). the piscihepevirus genus includes only one species (piscihepevirus a) with one member (cutthroat trout virus), whereas the orthohepevirus genus is divided into four species of viruses infecting mammals and birds (orthohepevirus a-d) [ ] (fig. a) . this classification is, however, likely to change in the near future following the identification of novel hepeviruses in fish other than trout and in amphibians [ ] (fig. a) . human-infecting hev strains are genetically heterogeneous and display distinct epidemiologic patterns, but all belong the orthohepevirus a species. orthohepeviruses a account for a minority of the overall diversity of hepeviruses that infect vertebrates and their closest relative is a presently unclassified virus detected in a swedish moose (fig. a ) [ ] . field surveys revealed a high prevalence of hev in moose populations from sweden and other baltic regions [ , ] . in general, ungulates represent major orthohepeviruses a reservoirs. at present, eight orthohepevirus a genotypes are recognized (hev- to - ) (fig. a ). hev- and hev- infect only humans and cause waterborne outbreaks mainly in tropical and subtropical regions (fig. b) . conversely, genotypes and account for the majority of hepatitis e human cases in industrialized countries. hev- and hev- also infect several other domestic (mainly pigs) and wild (e.g., ungulates and small carnivores) animals, their transmission to humans being usually zoonotic [ ] (fig. a) . phylogenetic analyses showed that hev- and hev- sequences derived from human cases are interspersed within those isolated from swine, indicating that pig-infecting hev- and hev- can easily cross the species barrier and infect humans [ ] . notably, though, evolutionary rates are higher for genotypes and than for the human-specific genotypes, suggesting cyclical adaptation to different mammalian hosts [ ] . a distinct hev- clade, mainly detected in rabbits (hev- ra), can also cause human hepatitis e [ ] [ ] [ ] [ ] (fig. a) . the remaining genotypes hev- /hev- and hev- /hev- have been detected in boars and camels, respectively [ ] (fig. a) . however, they are also thought to have zoonotic potential, as hev- and hev- can infect cynomolgus monkeys [ , ] and hev- was detected in a patient who consumed camel meat and milk [ ] . thus, viruses belonging to all hev genotypes seem to be transmissible to humans. conversely, experimental infection with hev- and hev- indicated that these viruses have a host range restricted to primates [ ] . hev genotypes are therefore usually referred to as enzootic (hev- and À ) or human-restricted/anthropotropic (hev- and - ). although several human hepatitis e cases have a zoonotic origin and orthohepeviruses a are found in diverse mammals, recent data indicated that one or more reverse zoonoses led to the emergence and radiation of hev genotypes [ ] . in fact, character state reconstruction on a large phylogeny revealed that humans were the most likely hosts of the ancestor of extant orthohepeviruses a [ ] . this notion is in line with the observation that most, if not all, hev genotypes can infect our species, whereas other animals are differentially susceptible to distinct hev genotypes. moreover, increasing evidence suggests that reverse zoonotic events (also known as zooanthroponoses) are all but rare, and examples include other rna viruses such as rotaviruses, enteroviruses, and human influenza viruses [ ] [ ] [ ] [ ] . for both swine influenza a viruses and swine vesicular disease virus onward transmission in pigs is well documented [ , ] and is facilitated by intensive husbandry practices. molecular dating using a selection-informed method inferred that the ancestor of extant orthohepeviruses a existed $ to $ years ago, most likely in east asia [ ] . these inferences well correlate with historical circumstances that may have favored hev emergence and host range expansion. in this period, sedentary agriculture promoted the appearance of large human settlements in several asian regions and pig husbandry practices started to intensify in east asia [ ] [ ] [ ] [ ] [ ] (fig. c) . crowded living conditions and poor sanitation possibly favored the emergence and spread of the waterborne, human-specific hev strains. the close contact between humans and pigs most likely promoted hev zooanthroponotic transmission and emergence of the enzootic strains (fig. c) . additional reverse zoonotic transmissions may have also originated the camel-infecting and rabbit-infecting strains. in fact, the estimated timing of hev- / emergence ( bce to bce) [ ] encompasses the time of domestication of bactrian and dromedary camels [ ] [ ] [ ] (fig. c) . as for hev- ra, it was estimated to have diverged from hev- around ce, in europe [ ] . this time frame corresponds to the middle ages, when historical evidence indicates that rabbits were kept in warrens and bred for meat [ ] (fig. c ). of course, these estimates do not necessarily imply that camels and rabbits acquired hev from humans, as the domestication process may have exposed these animals to various viral sources, including other domestic (e.g., pigs) and peridomestic mammals. these scenarios provide a credible framework for orthohepevirus a origin, as well as for the diversification of hev genotypes, and selection-informed methods should at least partially correct for the tdrp, as they explicitly model purifying selection [ ] [ ] [ ] . nonetheless, the above-mentioned data on hbv [ ] suggest caution in the inference of time and location of ancestral events based on extant viral diversity. also, pig infection with hev- and hev- is generally asymptomatic [ ] , possibly indicating a long-standing host-virus association that might even predate swine husbandry development. it should also be noted that the ultimate origin of orthohepevirus a remains unknown. humans may have acquired hev through cross-species transmission from other animals. however, known orthohepeviruses that infect mammals and birds are distantly related to orthohepevirus a, indicating that none of them represents the source of human-infecting hev. likewise, the origin and evolutionary relationship between the moose virus and human-infecting orthohepeviruses remain unclear. by allowing the large-scale identification of novel viral species, as well as the sequencing of viral genomes from archaeological samples, technological advances have largely expanded our knowledge on the evolution and origin of human hepatitis viruses. these insights have been paralleled by the development of computational tools and theoretical frameworks to analyze and mine viral sequence data (e.g., the above-mentioned recognition of the tdrp and the development of approaches to correct for it). the overall picture emerging from these studies clearly indicates that, with the possible exception of hdv, viruses related to human hepatitis viruses infect several other mammalian and non mammalian vertebrates. the specific events that originated these human pathogens remain, however, largely unknown. for hcv and hev, the evolutionary history of the human viruses is likely intertwined with that of related viruses that infect domestic animals. conversely, in the case of hav and hbv, the closest relatives of the human viruses are found in nhps. in general, these observations indicate that a deeper understanding of the evolutionary dynamics of human viruses has a relevance not only to improve our ability to treat and prevent present infections, but also to gain insight into the ecological contexts that may fuel the emergence of novel human pathogens, with particular reference to zoonotic ones. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. world health organization (who) zoonotic hepatitis e virus: classification, animal reservoirs and transmission routes hepatitis a: old and new type a viral hepatitis: a summary and update on the molecular virology, epidemiology, pathogenesis and prevention bayesian molecular dating: opening up the black box 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infection in different groups of estonian patients and people who inject drugs the worldwide burden of viral hepatitis in terms of death and disability is enormous. in , viral hepatitis caused . million deaths, the majority of which imputable to the effects of chronic hbv (hepatitis b virus) and hcv (hepatitis c virus) infection [ ] . an estimated % of hbv-infected persons are also co-infected with hdv (hepatitis delta virus), which worsens the clinical outcome compared to hbv monoinfection [ ] . less than % of overall hepatitis mortality is caused by hav (hepatitis a virus) and hev (hepatitis e virus), that are usually associated with acute, self-limiting disease [ ] . however, the case fatality rate of hev is particularly high in specific groups, notably pregnant women and elderly individuals [ ] . although rare, infection with hav can also cause acute liver failure and death, and the risk increases with age [ ] . despite the existence of a safe and effective vaccine, hav remains a common cause of acute viral hepatitis in many regions of the world [ ] . key: cord- -lg gvgwt authors: zhou, shaochuan; ge, xinna; kong, can; liu, teng; liu, aijing; gao, peng; song, jiangwei; zhou, lei; guo, xin; han, jun; yang, hanchun title: characterizing the prrsv nsp deubiquitinase reveals dispensability of cis-activity for replication and a link of nsp to inflammation induction date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: lg gvgwt the papain-like cysteine protease (plp ) within the n-terminus of the porcine reproductive and respiratory syndrome virus (prrsv) nsp replicase protein specifies a deubiquitinating enzyme (dub), but its biochemical properties and the role in infection have remained poorly defined. by using in vitro assays, we found that the purified plp could efficiently cleave k and k linked polyubiquitin chains ub - in vitro although displaying a differential activity in converting the respective ubiquitin dimers to monomer. the subsequent mutagenesis analyses revealed that the requirement for plp dub activity surprisingly resembled that for cis-cleavage activity, as several mutations (e.g., d r, d r, etc.) that largely ablated the dub function also blocked the cis- but not trans-proteolytic cleavage of nsp / polyprotein. moreover, the analyses identified key mutations that could differentiate dub from plp cis- and trans-cleavage activities. further reverse genetics analyses revealed the following findings: (i) mutations that largely blocked the dub activity were all lethal to the virus, (ii) a point mutation t g that selectively blocked the cis-cleavage activity of plp did not affect viral viability in cell culture, and (iii) an e q mutation that did not affect either of the plp activities led to rescue of wt-like virus but displayed significantly reduced ability to induce tnf-α production. our findings support the possibility that the plp dub activity, but not cis-cleavage activity, is essential for prrsv replication. the data also establish a strong link of nsp to pro-inflammatory cytokine induction during infection that operates in a manner independent of plp dub activity. protein ubiquitination is an important post-translational modification that regulates a variety of cellular biological processes [ ] [ ] [ ] , i.e., signaling transduction [ ] , protein turnover [ ] , cell cycle progression [ ] , and the immune and inflammatory responses [ , , ] , etc. this modification, however, can often be reversed by deubiquitinases (dubs) through removing various ubiquitin molecules from substrates [ ] . not surprisingly, many dna and rna viruses have evolved to encode dubs to manipulate diverse cellular pathways [ ] . the currently discovered viral dubs largely resemble their cellular counterparts in structure and generally fall into two major classes: ubiquitin-specific preventing it from efficient affinity purification in large scale ( figure b , lane ). in contrast, plp ( - )-strepii was well expressed, and a substantial amount was presented in the supernatants ( figure b, lane ) . moreover, this fragment in the sonicated supernatants could be subsequently purified to homogeneity by one-step affinity purification ( figure b , lane ) with a yield of - mg per ml culture. thus, we have successfully developed a strategy to realize high-level soluble expression of prrsv plp . the dub activity of purified plp was subsequently investigated in a series of in vitro assays. we first tested its ability to hydrolyze ub-amc, an ubiquitin conjugated aminomethylcoumarin fluorophore (amc). by monitoring the release of amc, the dub activity was measured. as shown in figure a , the purified plp exhibited dub property in vitro with better activity achieved at a higher concentration ( μg). next, we examined the cleavage of a specific type of polyubiquitin chains ( figure b and c) . overall, when the total amount of processed ub monomer and dimers (ub+ub ) more recently, prrsv plp was shown to possess deubiquitinating activity in transfected ft cells and can antagonize interferon signaling through inhibiting activation of nf-κb [ , , ] , a feature that is similar to the counterparts from eav and nairoviruses of the family of bunyaviridae [ , , ] . further, deaton et al. have reported the dub activity of prrsv plp by in vitro assay and found that the e. coli purified recombinant plp (aa. - ) is able to cleave both k and k poly-ubiquitin [ ] . on the other hand, although prrsv plp was shown to have deisgylation activity in transfected human cells [ ] , the recombinant plp showed little in vitro deisgylating activity toward isg of porcine origin [ , ] , leaving in question whether it can actually efficiently cleave swine isg conjugates in primary macrophages. in any case, it is clear now that the prrsv plp possesses at least cis-, trans-cleavage, and dub activities [ , , ] . however, their biochemical properties and the contribution to viral infection have remained poorly defined. in this study, we started with characterizing the plp dub activity of hp-prrsv strain jxwn by using in vitro and cell-based assays. by using site-directed mutagenesis, we were able to identify mutations that can differentiate the dub activity from cisand trans-cleavage activities and assess their roles in the context of prrsv infection by reverse genetics analysis. our results revealed novel biochemical aspects of prrsv plp and showed that the plp dub activity, but not the cis-cleavage activity, is likely important for prrsv replication. unexpectedly, we also revealed a strong link of prrsv nsp to virus-induced inflammation that occurs in a dub-independent manner. the findings further advance our understanding of prrsv nsp function and it is the regulation of host immunity and has implications in antiviral drug and vaccine development. marc- and hek ft cells were maintained in dulbecco's modified eagle's medium (dmem) (invitrogen, carlsbad, ca, usa) with % fbs (invitrogen, ca, usa). primary porcine pulmonary alveolar macrophages (pams) were obtained from specific-pathogen-free (spf) pigs as previously described [ ] and cultured in rpmi medium (invitrogen, ca, usa) containing % fetal bovine serum (fbs) (invitrogen, ca, usa). the chinese highly pathogenic prrsv strain jxwn infectious cdna clone was described previously [ ] . mouse anti-flag monoclonal antibody (f ), mouse anti-ha monoclonal antibody (h ), mouse anti-β-actin monoclonal antibody (a ), and rabbit anti-myc polyclonal antibody (c ) were all purchased from sigma-aldrich (st. louis, mo, usa). horseradish peroxidase (hrp)-conjugated goat anti-mouse polyclonal antibodies and hrp-conjugated goat anti-rabbit polyclonal antibodies were purchased from zsgb-bio (beijing, china). all the restriction enzymes used in this study were from new england biolabs inc. (beverly, ma, usa). the prokaryotic expression plasmids expressing plp ( - )-strepii and plp ( - )-strep ii were constructed by cloning the respective region coding for nsp aa. - and aa. - from hp-prrsv strain jxwn (accession number: ef ) into the vector pet- a (novagen, madison, wi, usa) at the sites of nco i and xho i with a strep ii tag attached to c-terminus for purification purpose. the mammalian plasmid expressing plp ( - )-myc was made by cloning the same plp sequence into the vector pegfp-n (clonetech, ca, usa) at the sites of bamh i and xho i in frame with c-myc epitope tag coding sequence. the plasmids pnsp -myc, pflag-ha-nsp - , and pflag-ha-nsp - ∆ - were generated by fusion expression of the indicated epitope tags with the corresponding coding region for nsp , nsp - or nsp - lacking the first amino acids in the vector pegfp-n (clonetech, mountain view, ca, usa) at the sites of bgl ii and bamh i. all genes in the created eukaryotic plasmids are under the control of cmv promoter, and a kozak core sequence is also included to allow optimal expression. for expression of the derivatives of plp , nsp , or nsp - , the point mutation mutants were created by quikchange site-directed mutagenesis. all the constructs were generated by standard recombinant dna procedure and verified by dna sequencing. bacterial strain bl (de ) containing the plasmid for plp ( - )-strepii or its derivative was cultured overnight at • c and then inoculated at : into ml of yeast extract-tryptone medium culture ( xyt). when the bacterial density at nm reached . to . , protein expression was induced • c for h by addition of iptg (isopropyl-d- -thiogalactopyranoside) (sigma) at the final concentration . mm. the cells were harvested by centrifugation at rpm for - min and then resuspended in pbs with the protease inhibitor cocktail (sigma, p ). the cells were sonicated and lysed for min at • c on ice with % triton x- (sigma, t ). the cell lysates were cleared by centrifugation at , rpm for - min. the supernatants were incubated with ul streptactin sepharose (ge healthcare, - - ) at • c overnight on the rocker. the streptactin sepharose beads were collected by centrifugation and washed six times with tris buffer ( mm tris, ph . ). the target protein was eluted with ul elution buffer ( mm tris, . mm desthiobiotin, % glycerol, ph . ). the dub activity of purified prrsv plp was assayed in µl reaction containing indicated amount of purified plp protein, µm ub-amc (boston biochem, u- ), and × reaction buffer ( mm nacl, . mm kcl, . mm na hpo , . mm kh po , and mm dtt) in a -well black microplate. the fluorescent intensity (excitation, nm, emission, nm) of each well was observed by infinite m pro plate reader (tecan, inc). the dub activity of purified prrsv plp was assayed in µl reaction containing indicated amount of purified plp protein, . µg k -(boston biochem, uc- ) or k -linked polyubiquitin chains ub - (boston biochem, uc- ), and × reaction buffer ( mm nacl, . mm kcl, . mm na hpo , . mm kh po , and mm dtt) in a µl volume ep tube. the mixture was incubated at • c for h and then subject to sds-page analysis on % tris-hcl gel. hek t cells grown to %- % confluence in six-well plates were transfected plasmids encoding ha-ubiquitin or plp -myc or nsp , nsp - or derivatives either singly or in combination via lipofectamine plus (invitrogen, ca, usa). at h post-transfection, the cells were treated with mg- (selleck, s ) at µg/ml for h before collection. the cells were washed by pbs for twice and lysed by ripa buffer on the shaker at • c for min. the cell lysates were subject to sds-page and western blot analyses with antibodies to ha or myc epitope. the amount of actin served as a loading control. for the trans-cleavage assay, hek t cells were transfected to express plp -myc, or nsp d n, or its derivatives together with the substrate nsp - ∆ - or nsp - ∆ - g g via lipofectamine plus (invitrogen, ca, usa). for cis-cleavage assay, plasmids coding for nsp - or its derivatives were transfected singly into t cells. at h post-transfection, the expression of enzyme plp or nsp was subject to western blot analysis by using the cell lysates, whereas the cleavage of substrate was analyzed by immunoprecipitation coupled with western blot. briefly, the cells were rinsed with cold pbs and lysed with ripa buffer. the cell debris was removed by centrifugation at , rpm for min and the supernatants were precleared with the protein a/g agarose (santa cruz, ca, usa) on the shaker at • c for min. after that, the cleared supernatants were incubated with ug antibodies to flag epitope and ul protein a/g agarose (santa cruz, ca, usa) at • c overnight. the immunocomplexes were washed for thrice by cold pbs, and the proteins were separated on sds-page and transferred to pvdf membrane. for western blot analysis, the membrane was blocked by pbst with % skimmed milk for h at room temperature and incubated with indicated primary antibody (antibody to ha epitope) at • c overnight. after being washed for thrice by pbst, the membrane was incubated with indicated secondary antibody for h at room temperature. after being washed three times, the membrane was developed with ecl western blot analysis system. for mutagenesis of the infectious clone of hp-prrsv strain jxwn (ef ), the mutations were first introduced into shuttle plasmid peasy-blunt (transgen, beijing, china) contain the fragment a [ ] by site-directed quikchange mutagenesis. after verification by sequencing, digested fragment a was transferred to the infectious clone backbone as described previously [ ] . marc- cells grown on six-well plates were transfected with plasmids of either wt or prrsv mutants. the virus-induced cpe was monitored daily and the cell culture was harvested at - days post-transfection. the cell lysates were passaged blindly onto fresh monolayers for three passages. the viability of mutants was determined by rt-pcr targeting orf and immunofluorescence to n protein. the rescued viruses of passage were titrated on marc- cells. for growth kinetics analysis, marc- cells or primary pams in six-well plates were infected with plp mutants and parental virus at an moi of . . for growth on marc- cells, after h of incubation at • c, the cells were first washed with acid buffer ( mm nacl, mm kcl, mm citric acid, ph . ), followed by once rinse with dmem. at indicated time points of infection, the whole culture was harvested and freeze-thawed three times to release cell-associated viruses. samples were then titrated on marc- cells by using the standard endpoint dilution assay according to the reed-muench method [ ] . primary pams in -well plates were infected with nsp mutants or parental virus at an moi of . . at h post-infection, total rnas were extracted from lysates of infected pams with trizol reagent (invitrogen, ca, usa) according to manufacturer's protocols and dissolved into rnase free water. the concentration of rna in samples was measured with a nanodrop lite spectrophotometer (thermol scientific, usa). µg of total rnas per sample was used for cdna synthesis by fastquant rt kit (tiangen, beijing, china). we conducted qpcr assays using real-time sybr master mix kit (applied biosystem, usa) on an ab real-time pcr system (applied biosystem, usa). primary pams in -well plates were infected with nsp mutants or parental virus at indicated moi. at indicated time points post-infection, tnf-α in cell culture supernatants was quantified using a commercial sandwich enzyme-linked immunosorbent assay (elisa) according to the manufacturer's protocol (cusabio, wuhan, china). the i-tasser online service tool was used to model the structures of prrsv strain jxwn plp [ ] . the plp structures were referred to eav plp ( ium) [ ] , and then its interaction with ubiquitin was analyzed by pymol and coot software. the in vitro cleavage efficiency of k and k -linked polyubiquitin chains was expressed as the ratio of the amount of ub plus ub over total ub (ub+ub +ub +ub +ub +ub +ub ) by measuring the band density in each lance of sds-page images. the ratio of ub over ub was calculated by band density of ub over ub ). the relative dub activity of plp mutants was measured by the corresponding band density of polyubiquitin conjugates and then normalized against actin and expression level of plp . all the data are shown as mean± sem with independent experiments. statistical significance was evaluated by using a two-way analysis of variance (anova). statistical analyses were performed using graphpad prism software (version . ). the quantitation of each protein band is measured by image j software (version . . ). flanking sequence is critical for the yield and solubility of prrsv plp protease domain in e. coli the n-terminal residues of prrsv jxwn nsp was recently reported to exhibit dub activity when expressed and purified from e. coli bl cells [ ] . this fragment, however, was expressed at a very low level in our hands, preventing further efficient purification. since the downstream flanking sequence (nsp aa. - ) is critical for prrsv nsp function during infection [ ] , we hypothesized that this region might be critical for the folding of plp domain, and if so, a c-terminal extension might improve the solubility and yield of plp . accordingly, we made two additional constructs to include prrsv strain jxwn nsp region aa. - and aa. - ( figure b ). these proteins were tagged with a strep ii epitope tag at the c-terminus to facilitate purification. when expressed in e. coli bl cells, plp ( - )-strep ii mostly existed in the pellet, preventing it from efficient affinity purification in large scale ( figure b , lane ). in contrast, plp ( - )-strepii was well expressed, and a substantial amount was presented in the supernatants ( figure b the dub activity of purified plp was subsequently investigated in a series of in vitro assays. we first tested its ability to hydrolyze ub-amc, an ubiquitin conjugated aminomethylcoumarin fluorophore (amc). by monitoring the release of amc, the dub activity was measured. as shown in figure a , the purified plp exhibited dub property in vitro with better activity achieved at a higher concentration ( µg). next, we examined the cleavage of a specific type of polyubiquitin chains ( figure b ,c). overall, when the total amount of processed ub monomer and dimers (ub+ub ) was calculated, prrsv plp exhibited similar efficiency in cleaving both k -and k -linked polyubiquitin chains ub - (% cleavage efficiency = ub +ub /ub - ) ( figure b ,c). however, prrsv plp display a differential activity converting k and k linked ubiquitin dimers into monomers. the results showed that the cleavage into monomer of k -linked ub - was relatively inefficient ( figure c ) with the reaction taking place in a dose and time-dependent manner ( figure c ). for example, at a lower dose of plp ( µg), k -linked ub - was mainly cleaved into ub dimers ( figure c , lane ), even if the incubation time was extended to two hours ( figure c , lane ). however, when the dose increased ( µg), plp could hydrolyze k ub - efficiently into monomers within h ( figure c , lane ). the difference in kinetics toward cleaving k versus k ub - was further investigated in a more detailed time-dependent manner from to min at a concentration of µg for plp . the result showed that plp was able to quickly cleave both k and k ub - within minutes ( figure d ,e), but the conversion from ubiquitin dimer to monomer was several fold faster for plp k dub activity ( figure d -f). to identify critical residues that are potentially important for plp dub activity, we modeled the structure of plp core domain with the online program i-tasser [ ] by using eav plp (pdb id: ium) as a model. the homology remodeling revealed that prrsv strain jxwn plp (aa. - ) has an overall similar structure to that of the counterpart eav plp , except for two minor structural differences as indicated by the red arrows ( figure a ). to identify critical residues that are potentially important for plp dub activity, we modeled the structure of plp core domain with the online program i-tasser [ ] by using eav plp (pdb id: ium) as a model. the homology remodeling revealed that prrsv strain jxwn plp (aa. - ) has an overall similar structure to that of the counterpart eav plp , except for two minor structural differences as indicated by the red arrows ( figure a) . the bioinformatics analysis also identified an acidic cluster (d dwatded ) that has potential contact with the positively charged c-terminus of ubiquitin ( figure b ). in particular, the ub residues r and r form a positively charged surface, whereas prrsv plp residue d , d , d , e and d form a negatively charged region. the modeling showed that the charged cterminus is inserted into a grove formed by negatively charged residues d , e , and d of plp ( figure b ). further, a closer look into the contact sites revealed that both plp d and d can potentially interact with r and r respectively through potential hydrogen bond and electrostatic interactions ( figure c ). in contrast, d and d are in a position that is far away from ub cterminus ( figure c ). the bioinformatics analysis also identified an acidic cluster (d dwatded ) that has potential contact with the positively charged c-terminus of ubiquitin ( figure b ). in particular, the ub residues r and r form a positively charged surface, whereas prrsv plp residue d , d , d , e and d form a negatively charged region. the modeling showed that the charged c-terminus is inserted into a grove formed by negatively charged residues d , e , and d of plp ( figure b ). further, a closer look into the contact sites revealed that both plp d and d can potentially interact with r and r respectively through potential hydrogen bond and electrostatic interactions ( figure c ). in contrast, d and d are in a position that is far away from ub c-terminus ( figure c ). for this identified acidic cluster, the residues d , d , w , a , d , and d are highly conserved among various prrsv strains, whereas the residues t and e are highly conserved among type ii prrsv strains but are replaced with the respective residues s and y in type i prrsv strains ( figure s ) [ ] . among these residues, both w and d have been shown to be critical for the trans-cleavage of nsp / [ ] . thus, we, therefore, selected other residues (d , d , t , e , d ) for further mutational analysis with a purpose to distinguish dub from its trans-cleavage activity. the acidic residues were changed to either the residue asparagine (n) or to the opposite charge residue arginine (r), whereas t was replaced with either serine (s), glycine (g), or arginine (r). the corresponding mutants were expressed and purified from e. coli bl cells, and their enzymatic activities were tested by the in vitro dub assay ( figure a ) and the quantified relative activity was shown at the right of corresponding figures. for this identified acidic cluster, the residues d , d , w , a , d , and d are highly conserved among various prrsv strains, whereas the residues t and e are highly conserved among type ii prrsv strains but are replaced with the respective residues s and y in type i prrsv strains ( figure s ) [ ] . among these residues, both w and d have been shown to be critical for the trans-cleavage of nsp / [ ] . thus, we, therefore, selected other residues (d , d , t , e , d ) for further mutational analysis with a purpose to distinguish dub from its trans-cleavage activity. the acidic residues were changed to either the residue asparagine (n) or to the opposite charge residue arginine (r), whereas t was replaced with either serine (s), glycine (g), or arginine (r). the corresponding mutants were expressed and purified from e. coli bl cells, and their enzymatic activities were tested by the in vitro dub assay ( figure a ) and the quantified relative activity was shown at the right of corresponding figures. the results showed that the mutations of the residues d and e (e.g., d n, d r, e r, e q, etc.) did not have much effect on plp dub activity, as the corresponding mutants could efficiently cleave k or k polyubiquitin chains into monomers ( figure a , lanes , , , and ). in contrast, mutation of the residue d to either n or r largely blocked the cleavage of ub - with only a very small portion cleaved into ub dimer or monomer ( figure a , lane and ). the mutational effect of d was amino acid-dependent. the d r mutation largely blocked the plp dub activity ( figure a , lane ), whereas the mutation d n did not affect the cleavage much ( figure a, lane ) . for the residue t , substitution with r (t r) largely ablated the dub activity ( figure a , lane ), whereas the mutation to serine (t s) did not have an effect ( figure a, lane ) . the t g mutation did not have a large effect on the ability of plp to process the k polyubiquitin chains ( figure a , lane , bottom panel), but it partially crippled the ability of plp to cleave the ubiquitin dimer ( figure a , lane , top panel), suggesting a differential sensitivity. we next used a cell-based assay to further check on the mutational effect on plp dub activity within mammalian cells ( figure b ). at the same time, the mutants plp c a and d n ( figure b , lanes and ), which have been shown to be defective of trans-cleavage activity [ ] , were used as negative control. the ha-tagged ubiquitin was transiently expressed in hek t cells together with plp or plp -derived mutants. at h post-transfection, the cell lysates were collected to assess the level of ubiquitination by western blot analysis with antibodies to ha epitope ( figure b ). the results showed that the mutants d r, d n, t s, e q, and d n were dub active ( figure b ). in contrast, the mutants d r, d n, t r, and d r lost the dub activity, together with the control mutants plp c a and d n ( figure b ). on the other hand, the mutations e r and t g partially crippled the plp dub activity ( figure b, lanes and ) . overall, the cell-based results are largely consistent with the in vitro dub assay. in addition, the results of mutagenesis studies are in accordance with the bioinformatics prediction, except for several mutations (e.g., t r, e r, and d r), which will be discussed in the discussion section. we next investigated whether the same mutation affects the plp cisor trans-cleavage activity, which can be important for the processing of prrsv polyprotein nsp - during infection. to address this question, we performed the cell-based assay by co-expressing plp or its derivatives with the substrate nsp - ∆ - lacking the plp domain in transfected ft cells ( figure a ). for a negative substrate control, the plp cleavage site at the nsp - junction was mutated to make the construct nsp - ∆ - g p ( figure a ). as shown in figure a , except for the control d n, all other plp mutants were capable of efficiently processing the nsp - polyprotein, suggesting that the mutations did not affect the trans-cleavage activity of plp . thus, the mutations (e.g., d n, d r, e r, d r, t r, etc.) that affected the dub activity can be used to differentiate the dub activity from trans-cleavage activity. viruses , , x for peer review of we next investigated whether the same mutations affect the plp cis-cleavage activity. since cis-cleavage is essentially an intramolecular interaction, the mutational effect was evaluated by using the full-length nsp - polyprotein precursor as reported previously [ ] . in addition, since both cisand trans-cleavage activities can direct the nsp - cleavage [ ] , the trans-cleavage has to be silenced in the first place before proceeding to test the effect on cis-activity. here, we took advantage of the d n mutation, which is able to decouple the nsp cisfrom trans-cleavage activity by using prrsv strain vr- [ ] . consistently, the d n mutation strongly reduced the trans-cleavage activity of plp to process nsp - ∆ - in trans in the cotransfection assay ( figure a, lane ) , and the cleavage efficiency was only about - %. to make sure the full-length nsp carrying d n mutation also behaves the same as seen for the plp fragment ( figure a, lane ) , we introduced this mutation into the full-length nsp of prrsv strain jxwn . as expected, the full-length nsp carrying d n mutation also failed to process in trans the polyprotein nsp - lacking the protease domain (nsp - ∆ - ) in the co-transfected cells ( figure b, lane ) , suggesting the trans-cleavage activity is blocked. when the same mutation was introduced into the nsp - precursor (nsp - d n) , it, however, did not affect the processing in cis of nsp from nsp - d n ( figure c, lane ) . thus, this result is in agreement with the previous report based on prrsv strain vr- [ ] . next, we introduced the corresponding acidic cluster point mutations into the background nsp - d n ( figure c ), which allows only cis-processing of nsp . to our surprise, the mutations (e.g., d n, d r, t r, and d r) that largely ablated the dub activity also blocked the cis-activity of plp ( figure c , lanes , , , and , respectively). in contrast, neither d n nor d r affected the cis-cleavage activity ( figure c , lane and ). notably, the mutation t g could selectively block the plp cis-cleavage activity ( figure c, lane ) , as the mutant plp t g was still equipped with trans-cleavage activity ( figure a , lane ) and largely dub activity ( figure a, lane ) . together, these results suggest that the requirement for plp dub activity somehow is more closely related to that for cis-cleavage activity, rather than the trans-cleavage activity. in addition, t g is a critical mutation that can differentiate the cis-cleavage activity from dub and trans-cleavage activity. the mutational effect on viral replication was tested by introducing the corresponding point mutations into the infectious cdna clone of hp-prrsv strain jxwn in a dna-launched system. the recombinant plasmids were transfected into marc- cells for virus recovery. our analyses by reverse genetics of plp mutants led to the findings as follows (table ). (i) mutations (d n, d r, t r, and d r) that largely blocked the dub activity in cell-based assay were all lethal to the virus, (ii) mutations (d r, d n, t s, and e q) that did not have an effect on dub activity did not affect viral viability, (iii) the point mutation t g that selectively blocked the plp cis-activity did not affect viral viability in cell culture, and (iv) the mutation d n and e r, which did not affect either of the plp activities, somehow did not allow the rescue of viable virus. together, our results strongly suggest a dispensable role of the plp cis-activity for prrsv viability. on the other hand, the fact that the cis-cleavage activity is not required for prrsv replication suggests that the lethal phenotype of several point mutations (d n, d r, t r, and d r), which blocked both dub and cis-activity, is not due to a block in cis-cleavage activity, but may be linked to the inactivation of plp dub activity. plp activities, somehow did not allow the rescue of viable virus. together, our results strongly suggest a dispensable role of the plp cis-activity for prrsv viability. on the other hand, the fact that the cis-cleavage activity is not required for prrsv replication suggests that the lethal phenotype of several point mutations (d n, d r, t r, and d r), which blocked both dub and cis-activity, is not due to a block in cis-cleavage activity, but may be linked to the inactivation of plp dub activity. four viable mutants of passage (p ) were chosen for growth kinetics analysis in both marc- cells ( figure a ) and primary pams ( figure b ). all the mutants tested showed relatively similar growth properties to the parental virus，except for the mutant t g, which exhibited reduced growth in marc- cells by about half a log and a slight decrease in pams although being statistically insignificant ( figure b ). to test the stability, we sequenced the plp mutation region at the end of experiments or p viruses. the results showed that the mutations were stable ( figure c ). in addition, we did not see any other compensating mutations arising within nsp . since prrsv nsp is involved in antagonizing host innate immunity and the plp mutants did not have an apparent growth defect in primary pams, we tested whether mutations have an effect on inflammatory cytokine production rather than on interferon signaling. in particular, we focused on the expression and secretion of tnf-α, il- β, and il- ( figure ) , the most potent inflammatory cytokines. to this end, primary pams were infected with wt or mutant viruses with an moi of . . at h postinfection, the mrna level of tnf-α, il- β, and il- were measured by relative quantitative rt-pcr normalized against peptidylprolyl isomerase a (ppia), a molecule that has been previously used for internal control [ ] [ ] [ ] . the results showed that prrsv strain jxwn significantly upregulated the mrna expression of both tnf-α and il- β ( figure a ). on the other hand, the effect on il- was not impressive ( figure a ). interestingly, the mutant e q exhibited greatly reduced ability to induce expression of both tnf-α and il- β, and to a lesser extent by the mutant t g ( figure a ). slightly blocked viral viability, iii) the point mutation t g that selectively blocked the plp cis-activity did not affect viral viability in cell culture, and iv) the mutation d n and e r, which did not affect either of the plp activities, somehow did not allow the rescue of viable virus. together, our results strongly suggest a dispensable role of the plp cis-activity for prrsv viability. on the other hand, the fact that the cis-cleavage activity is not required for prrsv replication suggests that the lethal phenotype of several point mutations (d n, d r, t r, and d r), which blocked both dub and cis-activity, is not due to a block in cis-cleavage activity, but may be linked to the inactivation of plp dub activity. four viable mutants of passage (p ) were chosen for growth kinetics analysis in both marc- cells ( figure a ) and primary pams ( figure b ). all the mutants tested showed relatively similar growth properties to the parental virus，except for the mutant t g, which exhibited reduced growth in marc- cells by about half a log and a slight decrease in pams although being statistically insignificant ( figure b ). to test the stability, we sequenced the plp mutation region at the end of experiments or p viruses. the results showed that the mutations were stable ( figure c ). in addition, we did not see any other compensating mutations arising within nsp . since prrsv nsp is involved in antagonizing host innate immunity and the plp mutants did not have an apparent growth defect in primary pams, we tested whether mutations have an effect on inflammatory cytokine production rather than on interferon signaling. in particular, we focused on the expression and secretion of tnf-α, il- β, and il- ( figure ) , the most potent inflammatory cytokines. to this end, primary pams were infected with wt or mutant viruses with an moi of . . at h postinfection, the mrna level of tnf-α, il- β, and il- were measured by relative quantitative rt-pcr normalized against peptidylprolyl isomerase a (ppia), a molecule that has been previously used for internal control [ ] [ ] [ ] . the results showed that prrsv strain jxwn significantly upregulated the mrna expression of both tnf-α and il- β ( figure a ). on the other hand, the effect on il- was not impressive ( figure a) . interestingly, the mutant e q exhibited greatly reduced ability to induce expression of both tnf-α and il- β, and to a lesser extent by the mutant t g ( figure a ). largely blocked - : active, -: blocked, nd: not determined, asmids were transfected into marc- cells for virus recovery. our analyses by plp mutants led to the findings as follows (table ) . i) mutations (d n, d r, at largely blocked the dub activity in cell-based assay were all lethal to the virus, , d n, t s, and e q) that did not have an effect on dub activity did not affect e point mutation t g that selectively blocked the plp cis-activity did not affect l culture, and iv) the mutation d n and e r, which did not affect either of the ehow did not allow the rescue of viable virus. together, our results strongly ble role of the plp cis-activity for prrsv viability. on the other hand, the fact activity is not required for prrsv replication suggests that the lethal phenotype tations (d n, d r, t r, and d r), which blocked both dub and cis-activity, ck in cis-cleavage activity, but may be linked to the inactivation of plp dub le . the association between various plp activity and virus viability. ced production of tnf-α and il- β is strongly associated with nsp that is dub activity tants of passage (p ) were chosen for growth kinetics analysis in both marc-) and primary pams ( figure b ). all the mutants tested showed relatively similar to the parental virus，except for the mutant t g, which exhibited reduced cells by about half a log and a slight decrease in pams although being icant ( figure b ). to test the stability, we sequenced the plp mutation region at nts or p viruses. the results showed that the mutations were stable ( figure c) . not see any other compensating mutations arising within nsp . since prrsv nsp onizing host innate immunity and the plp mutants did not have an apparent rimary pams, we tested whether mutations have an effect on inflammatory n rather than on interferon signaling. in particular, we focused on the expression f-α, il- β, and il- ( figure ) , the most potent inflammatory cytokines. to this s were infected with wt or mutant viruses with an moi of . . at h postlevel of tnf-α, il- β, and il- were measured by relative quantitative rt-pcr peptidylprolyl isomerase a (ppia), a molecule that has been previously used for - ] . the results showed that prrsv strain jxwn significantly upregulated the of both tnf-α and il- β ( figure a ). on the other hand, the effect on il- was ure a). interestingly, the mutant e q exhibited greatly reduced ability to f both tnf-α and il- β, and to a lesser extent by the mutant t g ( figure a ). four viable mutants of passage (p ) were chosen for growth kinetics analysis in both marc- cells ( figure a ) and primary pams ( figure b ). all the mutants tested showed relatively similar growth properties to the parental virus, except for the mutant t g, which exhibited reduced growth in marc- cells by about half a log and a slight decrease in pams although being statistically insignificant ( figure b ). to test the stability, we sequenced the plp mutation region at the end of experiments or p viruses. the results showed that the mutations were stable ( figure c ). in addition, we did not see any other compensating mutations arising within nsp . since prrsv nsp is involved in antagonizing host innate immunity and the plp mutants did not have an apparent growth defect in primary pams, we tested whether mutations have an effect on inflammatory cytokine production rather than on interferon signaling. in particular, we focused on the expression and secretion of tnf-α, il- β, and il- ( figure ) , the most potent inflammatory cytokines. to this end, primary pams were infected with wt or mutant viruses with an moi of . . at h post-infection, the mrna level of tnf-α, il- β, and il- were measured by relative quantitative rt-pcr normalized against peptidylprolyl isomerase a (ppia), a molecule that has been previously used for internal control [ ] [ ] [ ] . the results showed that prrsv strain jxwn significantly upregulated the mrna expression of both tnf-α and il- β ( figure a ). on the other hand, the effect on il- was not impressive ( figure a) . interestingly, the mutant e q exhibited greatly reduced ability to induce expression of both tnf-α and il- β, and to a lesser extent by the mutant t g ( figure a) . similarly, none of the mutants affected the level of il- ( figure a ). we also measured the secretion of these cytokines in the infected supernatants by elisa. consistent with a reduction at the mrna level, we observed a significant drop of tnf-α in the cell culture supernatant from primary pams infected with the mutants t g and e q ( figure b ). for il- β and il- , we tried several commercial kits, and unfortunately, they did not work well in our hands. to test whether the effect was contingent on the infection dose, we chose e q and t g for further analysis (figure ) . the results showed that either moi of . , . , or could significantly reduce the secretion of tnf-α in the infected supernatants ( figure a ). when compared with wt, this reduction is by more than - % for the mutants e q and t g at h post-infection or later, moreover, the e q mutant had a greater ability to reduce the production of tnf-α ( figure a ). similarly, none of the mutants affected the level of il- ( figure a ). we also measured the secretion of these cytokines in the infected supernatants by elisa. consistent with a reduction at the mrna level, we observed a significant drop of tnf-α in the cell culture supernatant from primary pams infected with the mutants t g and e q ( figure b ). for il- β and il- , we tried several commercial kits, and unfortunately, they did not work well in our hands (data not shown). to test whether the effect was contingent on the infection dose, we chose e q and t g for further analysis (figure ) . the results showed that either moi of . , . , or could significantly reduce the secretion of tnf-α in the infected supernatants ( figure a ). when compared with wt, this reduction is by more than - % for the mutants e q and t g at h post-infection or later, moreover, the e q mutant had a greater ability to reduce the production of tnf-α ( figure a) . finally, to further evaluate whether the observed effect on pro-inflammatory cytokine production is associated with altered plp dub activity, we introduced the corresponding mutations (e q, t g, d r, and t r) into the full-length nsp rather than merely into the plp fragment ( figure b ). the deubiquitinating ability of nsp or its derivatives was then tested in transfected t cells by co-expression with ha-ubiquitin. consistent with the phenotype exhibited by the truncated nsp , the mutant nsp e q ( figure b , lane ) behaved just like wt nsp ( figure b, lane ) , and nsp t g was largely dub-active ( figure b, lane ) . in contrast, the catalytic site mutation c a blocked the dub activity of the full-length nsp ( figure b, lane ) . together, the above data provided strong evidence on a critical role of prrsv nsp in inducing tnf-α and il- β during infection, and also this property of nsp is independent of the plp dub activity. in this report, we developed a strategy to obtain high-level soluble expression of prrsv strain it is now known that prrsv plp possesses at least cis-, trans-cleavage, and dub activities and that the trans-activity requires the nsp region aa. - [ ] . in this report, we went much further to reveal several important biological aspects of this particular protease. beginning with the in vitro purification, we found that the downstream flanking sequence (nsp aa. - ) was critical for the yield and solubility of plp domain in the prokaryotic expression system ( figure b) . although a previous study reported that the prrsv strain jxwn plp (nsp aa. - ) is sufficient for dub activity in vitro, but it did not provide description about the expression level and purification of this fragment [ ] . in our hand, this fragment was unfortunately poorly expressed in e. coli bl cells despite attempts for optimization of expression conditions. although truncation is usually the way for protein expression, we used an unconventional approach to solve the problem, making a cterminal extension dramatically improved the plp yield and solubility ( figure b) . our results suggest that the flanking sequence likely plays an important role in the folding of plp core domain, and this is consistent with our previous finding that deletion of nsp aa. - is lethal to prrsv strain vr . the realization of high-level soluble expression also paves the way for future structural studies and in vitro anti-plp drug screening. previous studies have shown that prrsv plp possesses differential dub activity toward cleaving k and k ubiquitin dimers into monomer. our results are in general in agreement with this finding [ ] . different from the previous study, we found that prrsv plp is able to cleave ubiquitin dimer but just in a dose and time-dependent manner. this difference is likely attributed to the plp flanking sequence (aa. - ) (figure ), which can promote the folding of plp core domain, leading to increased cleavage efficiency toward k polyubiquitin chains. in addition, we finally, to further evaluate whether the observed effect on pro-inflammatory cytokine production is associated with altered plp dub activity, we introduced the corresponding mutations (e q, t g, d r, and t r) into the full-length nsp rather than merely into the plp fragment ( figure b ). the deubiquitinating ability of nsp or its derivatives was then tested in transfected t cells by co-expression with ha-ubiquitin. consistent with the phenotype exhibited by the truncated nsp , the mutant nsp e q ( figure b , lane ) behaved just like wt nsp ( figure b , lane ), and nsp t g was largely dub-active ( figure b , lane ). in contrast, the catalytic site mutation c a blocked the dub activity of the full-length nsp ( figure b, lane ) . together, the above data provided strong evidence on a critical role of prrsv nsp in inducing tnf-α and il- β during infection, and also this property of nsp is independent of the plp dub activity. in this report, we developed a strategy to obtain high-level soluble expression of prrsv strain jxwn plp in e. coli bl cells for assaying its dub activity in vitro. the in vitro and cell-based assays uncovered important biochemical properties of plp , including efficient cleavage ability towards both k -linked and k -linked polyubiquitin chains and a surprisingly shared requirement for both cis-cleavage and dub activity. moreover, we identified mutations that could distinguish the three activities of plp . further reverse genetics analysis revealed dispensability of cis-activity for prrsv replication. in contrast, viruses carrying point mutations that largely blocked the dub activity were not viable, pointing to a potentially critical role of plp dub in prrsv replication. during characterizing the viral mutants, we also unexpectedly discovered a dub-independent regulation of pro-inflammatory cytokine production by prrsv nsp . together, these findings further deepen our understanding of the biological properties and function of prrsv plp and nsp in virus life cycle and have implications in understanding viral pathogenesis and in vaccine development. the relevant significance or insights are discussed below. it is now known that prrsv plp possesses at least cis-, trans-cleavage, and dub activities and that the trans-activity requires the nsp region aa. - [ ] . in this report, we went much further to reveal several important biological aspects of this particular protease. beginning with the in vitro purification, we found that the downstream flanking sequence (nsp aa. - ) was critical for the yield and solubility of plp domain in the prokaryotic expression system ( figure b) . although a previous study reported that the prrsv strain jxwn plp (nsp aa. - ) is sufficient for dub activity in vitro, but it did not provide description about the expression level and purification of this fragment [ ] . in our hand, this fragment was unfortunately poorly expressed in e. coli bl cells despite attempts for optimization of expression conditions. although truncation is usually the way for protein expression, we used an unconventional approach to solve the problem, making a c-terminal extension dramatically improved the plp yield and solubility ( figure b ). our results suggest that the flanking sequence likely plays an important role in the folding of plp core domain, and this is consistent with our previous finding that deletion of nsp aa. - is lethal to prrsv strain vr . the realization of high-level soluble expression also paves the way for future structural studies and in vitro anti-plp drug screening. previous studies have shown that prrsv plp possesses differential dub activity toward cleaving k and k ubiquitin dimers into monomer. our results are in general in agreement with this finding [ ] . different from the previous study, we found that prrsv plp is able to cleave ubiquitin dimer but just in a dose and time-dependent manner. this difference is likely attributed to the plp flanking sequence (aa. - ) (figure ), which can promote the folding of plp core domain, leading to increased cleavage efficiency toward k polyubiquitin chains. in addition, we found that prrsv strain jxwn plp can equally efficiently cleave k and k -linkd ub - into dimers and monomer. we also identified critical residues for prrsv plp dub activity. d , an amino acid that is highly conserved among prrsv strains [ ] , was revealed to be a key residue for prrsv plp dub activity (figure ). either a conserved (d n) or non-conserved (d r) substitution led to a significant destruction of plp dub activity ( figure ) . the mutational effect also appears specific, as similar mutations of the nearby residue d (d n and d r) that is also highly conserved did not have an effect (figure ). this result is also in accordance with the bioinformatics prediction ( figure ) . the mutational effect of other residues is contingent on the amino acids used for substitution. for example, the mutations d r and t r largely blocked the plp activity and e r partially blocked the dub activity ( figure ). we suspect that the mutational effect of d r is most likely indirect and could result from electrostatic interaction with e to change the local structure. in a similar manner, the mutation t r can potentially interact locally with e , d , or d , leading to local conformational change of plp , which may have an adverse effect on plp function. this also partially explains why t s, e q, and d e did not have an effect. most interestingly, we found that the t g mutation can selectively block the cis-cleavage activity ( figure c ), without affecting the trans-cleavage activity ( figure a ) and only partially blocking the dub activity in either truncated ( figure b ) or full-length form ( figure b ). this is the first time to report a point mutation that distinguishes cis-cleavage from other activities and paves the way to test the importance of cis-cleavage activity in prrsv infection. it has been viewed that hydrolysis of ubiquitin substrates takes advantage of the trans-activity of viral dubs. surprisingly, we observed an intriguing relationship between prrsv plp dub and cis-activity. that is, several mutations that largely blocked the plp dub activity were found to also ablate its cis-activity, and these include the mutants d n, d r, t r, and d r (figures and c ). in contrast, none of these mutations had an effect on the plp trans-activity cleaving nsp - polyprotein ( figure a ). thus, it seems that the requirement for prrsv plp dub activity is somehow more closely related to cis-activity, suggesting that they may share some intrinsic common mechanistic details in terms of recognition and cleavage of the substrates. to our knowledge, this is the first report revealing such intimate relationship between dub and cis-activity concerning otu cysteine proteases. the molecular basis for these observations is currently not clear, but hopefully, elucidation of the three-dimensional structure of prrsv plp may help resolve the puzzle in the future. a long-asked question is whether the plp dub and cis-cleavage activities are essential for prrsv infection. the inability to differentiate the enzymatic activities by point mutations has limited the evaluation of the contribution of individual enzymatic activities. despite the reports of several studies on the plp dub activities [ , , , ] , none of them established a strong correlation between dub and viral replication, as they did not rule out the mutational effect on both transand cis-cleavage activities. by taking advantage of site-directed mutagenesis, we in the first place provided strong evidence to suggest that the plp cis-activity is not necessary for viral replication ( figure ). this is evidenced by the specific point mutation t g, which selectively blocked the plp cis-cleavage activity but allowed the successful rescue of viable virus. moreover, this mutant exhibited similar growth rate to wt in primary pams ( figure ). in contrast, the mutations (e.g., d n, d r, t r, d r, etc.) that largely disarmed the plp dub activity were lethal to prrsv in marc- cells. although these mutations also crippled the cis-cleavage activity of plp at the same time, the successful rescue of t g mutant strongly argues against an essential contribution of inactive cis-activity to the non-viability phenotype. in addition, the lethal phenotype is less likely due to a potential defect of deisgylation activity of plp , as it has been reported that the hp-prrsv strain jxwn plp used in this study has very limited deisgylation activity [ ] and that isgylation was not observed in prrsv-infected marc- cells [ ] , a cell line that was used for virus rescue. thus, there appears to exist a strong correlation between the lethal phenotype of these mutations and the essential role of plp dub activity in prrsv infection. sun et al. recently characterized the nsp otu domain of a european prrsv strain sd - (type i strain) and reported that plp inhibits nf-κb activation through its dub activity [ ] . they also mutated the residues in the acidic cluster, including the residues d , d , s , d , d , etc., corresponding to the residues d , d , t g and d in this study, respectively [ ] . consistent with our studies, the mutations d a and d a were lethal to prrsv, whereas d a, s a, and d a allowed the successful rescue of the viable virus [ ] . in these studies, the mutant d a did not affect the viral growth, whereas s a and d a crippled the virus in marc- cells [ ] . sun et al. further correlated the growth property with the ability to inhibit nf-κb activation and found that the mutations that were lethal to the virus were these capable of completely inhibiting nf-κb activation, whereas the mutations s a and d a unable to inhibit the activation as wt lead to viable virus [ ] . however, they did not further test the mutational effect of these mutants on cisand trans-cleavage activity and the dub activity. our studies here suggest that the lethal phenotype is not linked to an impairment of cisor trans-cleavage activity, but rather related to an effect on plp dub activity. however, because these are negative results, we could not rule out the possibility that the dub-inactivating mutations may have other unexpected consequences on nsp , or both, which renders the virus non-viable. on the other hand, the mutations e r and d n, which did not affect much the plp dub activity, did not allow the rescue of viable viruses. this result suggests that the mutations may have an adverse effect on other functions of nsp , highlighting the complexity of mutational effect on the function of plp or nsp . another interesting observation is that the partial ablation (t g) of plp dub activity did not affect viral viability whereas a near-complete block (e.g., d n, d r, t r, d r, etc.) was lethal to the virus. these results may suggest that there exists a threshold of dub activity required for prrsv replication, in which the dub activity may be required to remove polyubiquitins from substrates. future study will be directed to investigate detailed mechanisms by which the prrsv plp plays during infection. infections by prrsv often cause de-regulation of inflammatory cytokine production [ ] . for the chinese hp-prrsv, it inhibits inflammation in early infection, but induces inflammatory storm during the late stage in pigs, contributing to lung injury and interstitial pneumonia [ , ] . the regulatory mechanisms of this process have remained poorly understood, but it is known that several nonstructural proteins, including nsp α, nsp β, nsp , and nsp , are capable of inhibiting inflammation response [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , whereas the structural proteins n and e promote inflammation [ , ] . prrsv nsp is a type i interferon signaling antagonist [ ] and recently implicated in the modulation of pro-inflammatory cytokine production during infection [ , , ] . liu et al. [ ] reported that mutants of hp-prrsv strain bb carrying deletion of nsp region aa. - or aa. - had the reduced ability to induce the expression of inflammatory cytokines il- β, tnf-α, and il- in pams. however, the corresponding mutants also exhibited reduced growth titer by . to log when compared with the parental virus. moreover, down-regulation is quite subtle (< %). on the other hand, the single-site substitution represents a nice approach by which it can minimize the risk of gross conformational alteration as a result of mutagenesis. at the beginning of this study, we did not mean to study the role of nsp in inflammatory cytokine induction, but with two independent point-mutation mutants (t g and e q) at hand, we unexpectedly established a strong link of nsp to prrsv-induced induction of pro-inflammatory cytokines (figures and ) . the mutants t g and e q retained the similar growth rate to the parental virus jxwn in primary pams, but exhibited significantly reduced ability to induce tnf-α and il- β at both mrna and protein levels (figures and ) . moreover, this reduction rate was by more than % at hpi or later ( figure a ), suggesting a decisive role of nsp in modulating the production of tnf-α and il- β during infection. different from the previous study [ ] , we did not observe a change in expression level of il- . the crippled ability to induce tnf-α and il- β cannot be attributed to an impairment of plp dub activity, as the mutant nsp e q was fully dub active ( figure b ). in addition, because viral dubs often negatively regulate host innate immunity, it is expected that inactivation of dub activity should promote the inflammatory cytokine production as reported by several cases [ , , , , ] . our results, however, showed the opposite, the mutant t g with partially crippled dub activity showed reduced production of tnf-α and il- β (figures and ). thus, with two independent nsp mutants (t g and e q) showing the similar phenotype, our results provide strong evidence to suggest a dub-independent regulatory mechanism by prrsv nsp in inducing tnf-α and il- β production. together, our findings further deepen our understanding of the biological properties and function of prrsv nsp and also provide clues into the pathogenic mechanisms of hp-prrsv. additionally, the results have implications in vaccine development by modifying nsp to reduce cytokine-induced lung injury. in the near future, it will be interesting to address molecular mechanisms of how the point mutations in nsp lead to crippled ability of prrsv to induce pro-inflammatory cytokines. breaking the chains: structure and function of the deubiquitinases atypical ubiquitylation-the unexplored world of polyubiquitin beyond lys and lys linkages the ubiquitin code a ubiquitin replacement strategy in human cells reveals distinct mechanisms of ikk activation by tnfalpha and il- beta a multiubiquitin chain is confined to specific lysine in a targeted short-lived protein k -linked 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differential tnf-alpha production induced by porcine reproductive and respiratory syndrome virus strains with different pathogenicity in vitro porcine reproductive and respiratory syndrome virus nonstructural protein antagonizes beta interferon expression by targeting the nf-kappab essential modulator we thank our colleague nianzhi zhang for his help on bioinformatics analysis of plp . the authors declare that they have no conflicts of interest with the contents of this article. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.viruses , , key: cord- - u ax authors: shrestha, ashish c.; wijesundara, danushka k.; masavuli, makutiro g.; mekonnen, zelalem a.; gowans, eric j.; grubor-bauk, branka title: cytolytic perforin as an adjuvant to enhance the immunogenicity of dna vaccines date: - - journal: vaccines (basel) doi: . /vaccines sha: doc_id: cord_uid: u ax dna vaccines present one of the most cost-effective platforms to develop global vaccines, which have been tested for nearly three decades in preclinical and clinical settings with some success in the clinic. however, one of the major challenges for the development of dna vaccines is their poor immunogenicity in humans, which has led to refinements in dna delivery, dosage in prime/boost regimens and the inclusion of adjuvants to enhance their immunogenicity. in this review, we focus on adjuvants that can enhance the immunogenicity of dna encoded antigens and highlight the development of a novel cytolytic dna platform encoding a truncated mouse perforin. the application of this innovative dna technology has considerable potential in the development of effective vaccines. vaccines represent an effective strategy in the fight against infectious diseases and recent estimates suggest that vaccination prevents - million deaths every year [ ] . the need for rapid and large scale vaccine production during epidemics against emerging pathogens is a major challenge in vaccine development [ ] , including effective vaccines for antigenically diverse and versatile pathogens that successfully subvert host immunity such as human immunodeficiency virus (hiv), hepatitis c virus (hcv), and malaria [ ] [ ] [ ] . dna vaccines can overcome some of these challenges, as it is relatively easy to produce large number of doses within a short period of time, and they are stable at ambient temperature and do not require cold chain transportation. they are also consistent between lots and have an excellent safety profile allowing for safety evaluations by regulatory authorities and distribution in a large scale [ , ] . importantly, dna vaccines can induce both humoral and cell-mediated responses in the vaccinated host [ ] [ ] [ ] . although they are safe and well tolerated, they are often poorly immunogenic and inefficacious in humans [ ] . therefore, recent studies on the advancements of dna vaccines are focused on effective delivery and increasing the immunogenicity of the encoded antigen/s of interest [ , ] . effective immunization with dna vaccines requires efficient transfection of host cells which is highly dependent on the delivery route and use of devices. conventional delivery routes to introduce the dna vaccine include intramuscular, intradermal, subcutaneous and oral routes [ ] . the preferred delivery route depends on the requirement to activate specific immune cells. the skin is rich in immune cells including local dendritic cells (dcs) and natural killer (nk) cells, and is therefore likely to be a more favorable site for vaccine delivery [ , ] . attempts to improve dna delivery have been made through other physical methods with the use of 'gene guns' or electroporation, which transiently permeabilizes the cell membrane to efficiently transfer the dna resulting in increased vaccine uptake by skin and muscle cells [ ] . although these methods have shown to increase dna uptake [ , ] , they require optimization to achieve increased efficiency and acceptance for clinical use. an alternative approach to improve transfection efficiency includes formulation of dna with liposomes or nanoparticles [ ] . liposomal delivery can be affected by pre-systemic (epithelial) and systemic barriers (enzymatic degradation, binding, and opsonization) [ ] . encapsulation of dna with nanoparticles has been reported to increase dna uptake or transfection efficiency [ , ] . some of the challenges in the use of nanoparticles with dna include encapsulation inefficiency, endocytosis by target cells and toxicity [ ] . the use of genetic adjuvants is one approach to enhance the immunogenicity of the antigen and can be used to complement other strategies (e.g., dna delivery) also designed to improve the immunogenicity of dna vaccines. upon immunization with a dna vaccine, the target cells uptake dna by endocytosis [ ] and the transfected cells express the dna-encoded protein antigen(s). when antigen-presenting cells (apcs) are directly transfected, the intracellular proteins are processed and immunogenic epitopes are then presented by mhc class i molecules, which can directly stimulate naïve cd + t cells [ , ] . the protein immunogen released from transfected cells can be endocytosed and/or phagocytosed by other apcs and are presented by mhc class ii molecules to activate naive cd + t cells [ , ] . if the proteins are expressed by stromal cells like keratinocytes, apcs can also indirectly capture secreted antigens and cross-present by mhc class i molecules to further stimulate cd + t cells [ ] . after dna vaccination, cd + t cells specific to the vaccine antigen undergo expansion, acquire effector functions and differentiates into memory cd + t cells [ , ] . the memory cells differentiate into effector memory t cells upon re-exposure to the antigen [ , ] . the ability of a dna vaccine to elicit t cell immunity is thus dependent on activating apcs to present antigen: mhc complexes to t cells [ ] and adjuvants can serve as an important costimulatory factor to enhance this process. in this brief review, based on our experience, we discuss the progress in the development of dna vaccines, approaches to improve delivery and genetic adjuvants used to enhance immunogenicity. we focus on an innovative cytolytic dna technology developed and patented in our laboratory. the immunogenicity of dna vaccines is enhanced by cpg motifs present in the plasmid backbone, which can activate apc via toll like receptors (tlr ) [ ] [ ] [ ] . unmethylated cpg motifs have been reported to induce b cell proliferation and secretion of immunoglobulin in vitro and in vivo [ ] . activation of macrophages and dcs results in upregulation of antigen presentation and costimulatory molecules, and secretion of cytokines (il- and il- ) involved in helper t cells (th ) response [ ] . thus, cpg motifs in dna plasmids serve as a 'natural adjuvant' for dna vaccines. plasmid dna can be designed to encode additional adjuvants with the antigen(s) of interest. molecular adjuvants such as fusion proteins including heat shock protein (hsp ), and vesicular stomatitis virus (vsvg) have been developed and used to enhance vaccine immunogenicity [ ] [ ] [ ] . the gene encoding such proteins as adjuvants is either fused with the gene encoding the vaccine antigen to produce a fusion protein driven by a same promoter or as separate proteins driven by different promoters in the same or different plasmid. co-encoding of genes creates a suitable cellular micro environment such as sustained antigen release and/or upregulation of cytokines, enhancing the immunogenicity of dna vaccines [ ] . a majority of the studies on experimental dna vaccines with genetic adjuvants have been studied in animal models such as mice (table ) , and very few of these have been clinically tested ( table ) . limited published data on clinical trials pose difficulty to compare efficacy between adjuvants in animals and humans. different cytokines, such as interleukins (il- , il- , il- ), chemokines, granulocyte/macrophage colony-stimulating factor (gm-csf), costimulatory molecules (cd , cd , and cd ), and signaling molecules (interferon regulatory factor - ) have been used as genetic adjuvants with dna vaccines [ , , [ ] [ ] [ ] , ] . genes expressing ifn-γ il- , il- , il- , and il- have been used to stimulate th responses [ , , ] , and il- , il- , il- , il- , for th stimulation [ , , [ ] [ ] [ ] . the inclusion of genes encoding cytokines, like il- or il- , as adjuvants for hiv- dna vaccines is known to increase cell mediated immunity (cmi) [ , ] . however, a bicistronic hiv dna encoding gp and il- elicited weaker specific immune response than monocistronic hiv- gp dna [ ] . combinations of genetic adjuvants like il- and il- with hiv- dna vaccine have also been used but no synergistic effect on the level of total antibody to hiv- antigen was reported [ ] . a phase i/iia trial showed that coadministration of dna vaccine encoding prostatic acid phosphatase (pap) with gm-csf elicited pap-specific cd + and/or cd + t cell responses [ ] . however, gm-csf was administered as a recombinant protein. hsp , a class of molecular chaperone, is known to induce maturation of dcs and activation of the th pathway [ ] [ ] [ ] . a fusion vaccine for multiple myeloma termed hdkk -hhsp was shown to be effective in inhibiting the targeted tumor and increased survival of vaccinated mice by eliciting tumor-specific humoral and cellular immune responses [ ] . however, a dna vaccine encoding hpv e fused with hsp , targeting hpv and cervical intraepithelial neoplasia / failed to enhance significant t cell responses in a phase i clinical trial [ ] . a bicistronic dna encoding hsp as a membrane bound or secreted protein has been used to improve the immunogenicity of a hiv gag [ ] . in this case, hsp expression was driven by a weaker sv promoter and hiv gag by a stronger cmv promoter. such a vaccine design enhanced gag-specific t cell responses, providing greater protection in mice challenged with ecohiv [ ] . ecohiv is a chimeric virus containing the envelope protein gp of mouse leukemia virus rather than hiv gp that can replicate in mouse leukocytes in vivo, thus representing a viable mouse challenge model for early assessment of hiv vaccines [ ] . the proposed mechanism of hsp as an adjuvant is that tlr / on dcs interacts with secreted or bound hsp , further attracting dcs to the site of antigen expression. this is followed by dc maturation, presentation of antigens by mhc molecules and secretion of cytokines and costimulatory molecules [ ] , thus enhancing t cell immune responses against the vaccine antigen. a chimeric version of the oligomerization domain from the chicken complement inhibitor (c bp) was used to produce an oligomeric form of vaccine antigens [ , ] . this protein, termed imx , forms a heptameric structure of the vaccine protein. this has been used to develop dna vaccines for tuberculosis, malaria and hiv to enhance humoral and/or cellular responses [ , , ] . vaccination with a dna vaccine encoding secreted hiv tat (tpa-tat imx ) induced higher humoral and cellular responses and improved protection against ecohiv challenge in mice [ ] . a phase i clinical trial of tuberculosis vaccine mva a-imx evaluated the vaccine to be safe and immunogenic, but cellular (ag a-specific ifn- [ ] . the proposed mechanism of hsp as an adjuvant is that tlr / on dcs interacts eted or bound hsp , further attracting dcs to the site of antigen expression. this is by dc maturation, presentation of antigens by mhc molecules and secretion of cytokines mulatory molecules [ ] , thus enhancing t cell immune responses against the vaccine en complement inhibitor imeric version of the oligomerization domain from the chicken complement inhibitor as used to produce an oligomeric form of vaccine antigens [ , ] . this protein, termed forms a heptameric structure of the vaccine protein. this has been used to develop dna for tuberculosis, malaria and hiv to enhance humoral and/or cellular responses [ , , ] . on with a dna vaccine encoding secreted hiv tat (tpa-tat imx ) induced higher and cellular responses and improved protection against ecohiv challenge in mice [ ] . a inical trial of tuberculosis vaccine mva a-imx evaluated the vaccine to be safe and enic, but cellular (ag a-specific ifn-Ƴ ) and humoral (mva-specific igg) responses were ficant [ ] . thus, the ability of such an adjuvant to enhance immunogenicity of dna n humans is still debated. g is a type iii viral fusion envelope protein, which mediates fusion of the virus envelope ost cell membrane [ ] . the use of fusogenic membrane glycoprotein (fmg) gene from vsv vaccine encoding the h protein of human papillomavirus type was shown to enhance ell responses and effectively control growth of tumors [ ] . the vsv g protein was also induce a t-response at low doses or a t-independent response at higher doses [ ] . fmg s into large multinucleated syncytia, which are then killed by a nonapoptotic mechanism ytiosomes are released from the membrane and present antigens efficiently to dcs [ ] . thus act as an adjuvant for any antigen expressed by a dna vaccine. vaccination of mice hepatitis c virus (hcv) nonstructural protein (ns ) together with vsvg resulted in frequency of ifn-γ, but not tnf-α-or il- -producing cd + cd + cd + effector memory lls [ ] . this dna vaccine was constructed in a bicistronic vector with vsvg co-encoded e plasmid with hcv ns . vsvg expression was driven by a weaker sv promoter and stronger cmv promoter [ ] . however, others have also reported an increase in the specific n the immunogen and vsvg were expressed from different plasmids [ , ] . tic protein rin (prf) is a pore forming protein released by immune cells including nk cells and t lymphocytes (ctl) [ ] . the -kilodalton prf protein oligomerizes to form pores that channel to release granzyme into the cytosol of target cells [ ] . suicide genes inducing of target cells have been used for cancer therapies, and the role of apoptotic cell death in n, whether immune-stimulatory or immune-suppressive, is debated [ ] . recently, a novel hnology has been developed--termed cytolytic dna technology-in which a truncated f is incorporated in a bicistronic dna vector to act as a vaccine adjuvant [ ] [ ] [ ] ] . lytic dna vaccines are based on a bicistronic dna plasmid constructed on a pvax (invitrogen) with a cytomegalovirus (cmv) promoter and a simian virus (sv ) promoter gene encoding the protein of interest as an immunogen is inserted downstream of the cmv and the gene encoding a truncated version of mouse prf downstream of the sv promoter . the sv is a weaker promoter compared to cmv and has been shown to result in -fold tein expression in transfected hek t cells in vitro [ , ] . ) and humoral (mva-specific igg) responses were not significant [ ] . thus, the ability of such an adjuvant to enhance immunogenicity of dna vaccines in humans is still debated. vsv g is a type iii viral fusion envelope protein, which mediates fusion of the virus envelope and the host cell membrane [ ] . the use of fusogenic membrane glycoprotein (fmg) gene from vsv in a dna vaccine encoding the h protein of human papillomavirus type was shown to enhance cd + t cell responses and effectively control growth of tumors [ ] . the vsv g protein was also shown to induce a t-response at low doses or a t-independent response at higher doses [ ] . fmg fuses cells into large multinucleated syncytia, which are then killed by a nonapoptotic mechanism [ ] . syncytiosomes are released from the membrane and present antigens efficiently to dcs [ ] . fmgs can thus act as an adjuvant for any antigen expressed by a dna vaccine. vaccination of mice with the hepatitis c virus (hcv) nonstructural protein (ns ) together with vsvg resulted in increased frequency of ifn-γ, but not tnf-α-or il- -producing cd + cd + cd + effector memory t (t em ) cells [ ] . this dna vaccine was constructed in a bicistronic vector with vsvg co-encoded in the same plasmid with hcv ns . vsvg expression was driven by a weaker sv promoter and ns by a stronger cmv promoter [ ] . however, others have also reported an increase in the specific cmi when the immunogen and vsvg were expressed from different plasmids [ , ] . perforin (prf) is a pore forming protein released by immune cells including nk cells and cytotoxic t lymphocytes (ctl) [ ] . the -kilodalton prf protein oligomerizes to form pores that serve as a channel to release granzyme into the cytosol of target cells [ ] . suicide genes inducing apoptosis of target cells have been used for cancer therapies, and the role of apoptotic cell death in vaccination, whether immune-stimulatory or immune-suppressive, is debated [ ] . recently, a novel dna technology has been developed--termed cytolytic dna technology-in which a truncated mouse prf is incorporated in a bicistronic dna vector to act as a vaccine adjuvant [ ] [ ] [ ] ] . cytolytic dna vaccines are based on a bicistronic dna plasmid constructed on a pvax backbone (invitrogen) with a cytomegalovirus (cmv) promoter and a simian virus (sv ) promoter [ ] . the gene encoding the protein of interest as an immunogen is inserted downstream of the cmv promoter and the gene encoding a truncated version of mouse prf downstream of the sv promoter ( figure ) . the sv is a weaker promoter compared to cmv and has been shown to result in -fold lower protein expression in transfected hek t cells in vitro [ , ] . the prf gene was modified to express a truncated version of prf (~ kda) lacking the final amino acid residues of the c terminus (unstructured region of prf) [ , , ] in order for prf to become cytolytic [ ] . the final c-terminal amino acids are required to export prf protein from the endoplasmic reticulum (er) to the golgi, from where glycosylated prf is then transported to the prf gene was modified to express a truncated version of prf (~ kda) lacking the final amino acid residues of the c terminus (unstructured region of prf) [ , , ] in order for prf to become cytolytic [ ] . the final c-terminal amino acids are required to export prf protein from the endoplasmic reticulum (er) to the golgi, from where glycosylated prf is then transported to secretory granules [ ] . removal of the c-terminal abrogates the export of prf from the er and its subsequent accumulation in the er is cytotoxic to the host cell [ , ] . different recombinant cytolytic dna-prf vaccines (rdna-prf) have been shown to elicit immune responses higher than those elicited by canonical dna vaccines (without prf) and the mechanism underlying this has been established [ ] . a previous study showed that coexpression of hcv ns and prf elicited nonapoptotic cell death in transfected cells, whilst immunization with ns -prf dna vaccine increased ns -specific t cell mediated responses as evidenced by increased ns -specific ifn-γ responses in an elispot assay and increased numbers of polyfunctional cd + t em cells that simultaneously secreted ifn-γ, il- , and tnf-α [ ] . cytolytic dna platform where the expression of immunogen is driven by a stronger promoter allows for sufficient antigen expression and accumulation within the target cells followed by nonapoptotic cell death due to lesser expression of prf driven by a weaker sv promoter; thus, balancing the level of antigen expression with the timing of cell death [ ] . necrosis is considered as the mechanism of cell death by prf as evidenced by release of lactate dehydrogenase (ldh) and low caspase activity [ , , ] . ldh release occurs after the rupture of cell membrane during secondary necrosis [ , ] . in contrast, ldh was not released by cells treated with doxorubicin (a proapoptotic drug) or cells transfected with ns wild type prf or ns del a prf (mutant and nontoxic prf) [ ] . expression of prf from a cytolytic dna, e.g., ns prf vaccine, thus results in necrotic cell death mediated by receptor-interacting protein- kinase activity, as evidenced by detection of uncleaved cytokeratin in huh- cells [ ] . necrosis releases damage associated molecular patterns (damps) which in turn activate dcs to migrate to the site of vaccination [ , ] . when purified dcs including the cd α + subset from naïve c bl/ mice were exposed to hek t cells (transfected with ovalbumin-prf), it resulted in upregulation of costimulatory molecules (cd /cd ), indicating maturation of the immune cells with the cytolytic dna [ ] . a significant increase in cd c + dcs and cross-presenting cd a + dcs, and upregulation of cd has been reported in mice vaccinated with a cytolytic dna hiv gag prf compared to a canonical dna vaccine [ ] . local and migrated dcs at the site of inflammation can take up antigens by endocytosis and are also exposed to damps. activated and matured dcs can then prime naïve cd + t cells (figure ) . antigen cross-presentation by dcs to cd + t cells has been shown to increase the number of proliferating cd + t cells by~ -fold with cytolytic dna compared to the noncytolytic prf dna [ ] . thus, a cytolytic dna vaccine has an inbuilt adjuvant to enhance the immunogenicity of the vaccine immunogen. whereas, the immunogenicity of canonical dna vaccines mostly depends on direct transfection of dcs and to a lesser extent cross-presentation of antigens shed from transfected cells and/or derived from transfected cells that have undergone spontaneous cell death [ ] . several studies have established that dcs exposed to necrotic or lytic cells expressing antigens mature and cross-present more efficiently than dcs exposed to antigens derived from a cellular milieu that comprise of apoptotic cells [ ] [ ] [ ] [ ] [ ] . comparative studies evaluating the ability of proapoptotic (e.g., rotavirus nonstructural protein (nsp ) and diphtheria toxin subunit a (dta)) and necrotic proteins (e.g., truncated prf) to enhance the immunogenicity of dna when encoded in plasmid dna vaccines showed that truncated prf is the most effective for this purpose [ ] [ ] [ ] . however, a caveat is that vaccine-encoded antigens need to accumulate significantly inside the cell before necrosis occurs following expression of truncated prf in order to activate dcs to cross-present vaccine-encoded antigens [ , ] . several studies have established that dcs exposed to necrotic or lytic cells expressing antigens mature and cross-present more efficiently than dcs exposed to antigens derived from a cellular milieu that comprise of apoptotic cells [ ] [ ] [ ] [ ] [ ] . comparative studies evaluating the ability of proapoptotic (e.g., rotavirus nonstructural protein (nsp ) and diphtheria toxin subunit a (dta)) and necrotic proteins (e.g., truncated prf) to enhance the immunogenicity of dna when encoded in plasmid dna vaccines showed that truncated prf is the most effective for this purpose [ ] [ ] [ ] . however, a caveat is that vaccine-encoded antigens need to accumulate significantly inside the cell before necrosis occurs following expression of truncated prf in order to activate dcs to cross-present vaccine-encoded antigens [ , ] . rdna-prf technology has been used in the development of hiv and hcv dna vaccines [ , , ] . direct comparison of the effects of the cytolytic prf and the apoptotic protein dta on the immunogenicity of the hiv- gag protein showed that prf activated dcs more efficiently, as evidenced by the increase in frequency of cross-presenting dcs and upregulation of activation marker (cd ) [ ] . in both dna vaccines, prf and dta were driven by sv promoter. immunization of mice with a dna vaccine encoding proapoptotic dta as an adjuvant in a hiv gag dta vaccine resulted in decreased dc activation, suggesting that dta-induced apoptosis attenuated immune response [ ] . furthermore, improved protection in the mouse ecohiv challenge model was achieved with rdna-prf encoding hiv gag compared to protection levels in mice vaccinated with a canonical gag dna vaccine [ ] . a rdna-prf vaccine encoding the hcv ns protein coexpressed with prf was shown to increase ns -specific cmi in mice and pigs, compared to ns coexpression with a proapoptotic protein, the rotavirus nsp protein [ ] . nsp is an enterotoxin that elicits a proapoptotic effect by disrupting the mitochondrial membrane and activating caspase- , - , and - [ , ] . this study showed that prf coexpression induced cell death by necrosis, and thus enhanced ns -specific immune responses, whereas, proapoptotic nsp reduced ns -specific response [ ] . importantly, hcv ns prf was more immunogenic than the canonical ns vaccine in pigs, demonstrating the translational potential of the cytolytic dna vaccines in human clinical trials [ ] . likewise, we have shown that a multi-antigenic hcv dna vaccine encoding genotype a proteins ns , ns a, ns b, and ns b coexpressed with prf induced robust cmi against the range of hcv ns proteins, compared to coexpression with vsvg [ ] . we also showed that multi-antigenic and multigenotypic (hcv genotype and a) dna cocktail vaccines encoding prf can significantly increase the magnitude and breadth of cmi responses to ns and ns b against both genotypes compared to those elicited by a single-genotype vaccine [ ] . rdna-prf technology has been used in the development of hiv and hcv dna vaccines [ , , ] . direct comparison of the effects of the cytolytic prf and the apoptotic protein dta on the immunogenicity of the hiv- gag protein showed that prf activated dcs more efficiently, as evidenced by the increase in frequency of cross-presenting dcs and upregulation of activation marker (cd ) [ ] . in both dna vaccines, prf and dta were driven by sv promoter. immunization of mice with a dna vaccine encoding proapoptotic dta as an adjuvant in a hiv gag dta vaccine resulted in decreased dc activation, suggesting that dta-induced apoptosis attenuated immune response [ ] . furthermore, improved protection in the mouse ecohiv challenge model was achieved with rdna-prf encoding hiv gag compared to protection levels in mice vaccinated with a canonical gag dna vaccine [ ] . a rdna-prf vaccine encoding the hcv ns protein coexpressed with prf was shown to increase ns -specific cmi in mice and pigs, compared to ns coexpression with a proapoptotic protein, the rotavirus nsp protein [ ] . nsp is an enterotoxin that elicits a proapoptotic effect by disrupting the mitochondrial membrane and activating caspase- , - , and - [ , ] . this study showed that prf coexpression induced cell death by necrosis, and thus enhanced ns -specific immune responses, whereas, proapoptotic nsp reduced ns -specific response [ ] . importantly, hcv ns prf was more immunogenic than the canonical ns vaccine in pigs, demonstrating the translational potential of the cytolytic dna vaccines in human clinical trials [ ] . likewise, we have shown that a multi-antigenic hcv dna vaccine encoding genotype a proteins ns , ns a, ns b, and ns b coexpressed with prf induced robust cmi against the range of hcv ns proteins, compared to coexpression with vsvg [ ] . we also showed that multi-antigenic and multigenotypic (hcv genotype and a) dna cocktail vaccines encoding prf can significantly increase the magnitude and breadth of cmi responses to ns and ns b against both genotypes compared to those elicited by a single-genotype vaccine [ ] . dna vaccines are still a promising option in the development of novel vaccination strategies. although they have many advantages, the ability to induce effective immune responses in humans required for protection has been challenging. these challenges include ineffective delivery and poor uptake of dna. consequently, a recent focus has been in the development of delivery methods and/or inclusion of genetic adjuvants. such genetic adjuvants are generally coexpressed with the antigen of interest or delivered through different plasmids. in the quest to develop and identify effective genetic adjuvants, a range of adjuvants was tested (hsp , vsvg, imx , dta, and prf) and a novel and promising cytolytic dna vaccine strategy has been developed. this cytolytic dna vaccine is unique as it is based on a bicistronic plasmid with the ability to coexpress antigen and prf in a balanced mechanism causing necrosis of vaccine-transduced cells, followed by increased activation of immune cells and cross presentation of vaccine immunogen. cytolytic dna vaccines encoding nonstructural proteins of hcv have been tested to enhance immunogenicity of vaccine antigen in mice [ , ] and in a large preclinical animal model, the pig [ ] . likewise, increased immunogenicity and improved protection against ecohiv challenge in mice with hiv gag prf [ ] demonstrate the effectiveness of cytolytic dna vaccines. adjuvants that provide effective costimulation for immune responses with specific immunogens may not have a similar effect with other immunogens, and therefore these need to be tested for their efficacy. use of a genetic adjuvant such as prf produces a suitable microenvironment for multiple/different immunogens and thus improves the delivery, immunogenicity and effectiveness of dna vaccines. this strategy has considerable potential in the development of dna-based vaccines against a range of infectious agents. world health organisation. facts on immunization the complexity and cost of vaccine manufacturing--an overview editorial: why vaccines to hiv, hcv, and malaria have so far failed-challenges to developing vaccines against immunoregulating pathogens a vision for vaccines against hiv, tuberculosis and malaria dna vaccines for emerging infectious diseases: what if? emerg advancements in dna vaccine vectors, non-mechanical delivery methods, and molecular adjuvants to increase immunogenicity effective humoral immune response from a h n dna vaccine delivered to the skin by microneedles coated with plga-based cationic nanoparticles differential humoral and cellular immunity induced by vaccination using plasmid dna and protein recombinant expressing the ns protein of dengue virus type vaccines for emerging infectious diseases: lessons from mers coronavirus and zika virus clinical applications of dna vaccines: current progress dna vaccines-how far from clinical use? dna vaccines: recent developments and the future. vaccines dose sparing with intradermal injection of influenza vaccine skin-resident antigen-presenting cells: instruction manual for vaccine development what you always needed to know about electroporation based dna vaccines gene transfer into muscle by electroporation in vivo electroporation-mediated gene delivery liposomes as vaccine delivery systems: a review of the recent advances barriers to liposomal gene delivery: from application site to the target plga nanoparticles for dna vaccination-waiving complexity and increasing efficiency solid lipid nanoparticles mediate non-viral delivery of plasmid dna to dendritic cells production and clinical development of nanoparticles for gene delivery specific endocytosis and degradation of naked dna in the endocardial cells of cod (gadus morhua l.) directly transfected langerin + dermal dendritic cells potentiate cd + ; t cell responses following intradermal plasmid dna immunization chemical adjuvants for plasmid dna vaccines programming, demarcating, and manipulating cd + t-cell memory duration of antigen expression in vivo following dna immunization modifies the magnitude, contraction, and secondary responses of cd + t lymphocytes secondary memory cd + t cells are more protective but slower to acquire a central-memory phenotype immunogenicity of infectious pathogens and vaccine antigens bacterial cpg-dna and lipopolysaccharides activate toll-like receptors at distinct cellular compartments cpg motifs in bacterial dna trigger direct b-cell activation cpg-oligonucleotides in vaccination: signaling and mechanisms of action enhancing immunogenicity and transmission-blocking activity of malaria vaccines by fusing pfs to imx multimerization technology a hiv-tat/c -binding protein chimera encoded by a dna vaccine is highly immunogenic and contains acute ecohiv infection in mice enhanced presentation of major histocompatibility complex class i-restricted human immunodeficiency virus type (hiv- ) gag-specific epitopes after dna immunization with vectors coding for vesicular stomatitis virus glycoprotein-pseudotyped hiv- gag particles mechanisms of action of adjuvants engineering dna vaccines via co-delivery of co-stimulatory molecule genes dna vaccine for cancer immunotherapy intracellular adhesion molecule- modulates beta-chemokines and directly costimulates t cells in vivo coimmunization with ifn-gamma or il- , but not il- or il- cdna can enhance th -type dna vaccine-induced immune responses in vivo coadministration of dna encoding interleukin- and hemagglutinin confers protection from influenza virus challenge in mice development of th and th populations and the nature of immune responses to hepatitis b virus dna vaccines can be modulated by codelivery of various cytokine genes modulation of amplitude and direction of in vivo immune responses by co-administration of cytokine gene expression cassettes with dna immunogens toll-like receptor adaptor molecules enhance dna-raised adaptive immune responses against influenza and tumors through activation of innate immunity activation of innate immunity, inflammation, and potentiation of dna vaccination through mammalian expression of the tlr agonist flagellin regulation of dna-raised immune responses by cotransfected interferon regulatory factors novel strategies to improve dna vaccine immunogenicity co-immunization with an optimized plasmid-encoded immune stimulatory interleukin, high-mobility group box protein, results in enhanced interferon-gamma secretion by antigen-specific cd t cells dlm- /zbp ) as a genetic adjuvant for dna vaccines that promotes effective antitumor ctl immunity mda can be exploited as efficacious genetic adjuvant for dna vaccination against lethal h n influenza virus infection in chickens development of a molecular adjuvant to enhance antigen-specific cd (+) t cell responses dna vaccines with single-chain fv fused to fragment c of tetanus toxin induce protective immunity against lymphoma and myeloma induction of antigen-positive cell death by the expression of perforin, but not dta, from a dna vaccine enhances the immune respons intradermal delivery of dna encoding hcv ns and perforin elicits robust cell-mediated immunity in mice and pigs a multiantigenic dna vaccine that induces broad hepatitis c virus-specific t-cell responses in mice calreticulin is an effective immunologic adjuvant to tumor-associated antigens development of a dna vaccine targeting human papillomavirus type oncoprotein e dna vaccines encoding membrane-bound or secreted forms of heat shock protein exhibit improved potency safety and immunogenicity of an hiv- gag dna vaccine with or without il- and/or il- plasmid cytokine adjuvant in healthy, hiv- uninfected adults vaccination with a plasmid dna encoding her- /neu together with low doses of gm-csf and il- in patients with metastatic breast carcinoma: a pilot clinical trial a phase i safety study of plasmid dna immunization targeting carcinoembryonic antigen in colorectal cancer patients timing of plasmid cytokine (il- /ig) administration affects hiv- vaccine immunogenicity in hiv-seronegative subjects safety and tolerability of hiv- multiantigen pdna vaccine given with il- plasmid dna via electroporation, boosted with a recombinant vesicular stomatitis virus hiv gag vaccine in healthy volunteers in a randomized, controlled clinical trial dna priming increases frequency of t-cell responses to a vesicular stomatitis virus hiv vaccine with specific enhancement of cd + t-cell responses by interleukin- plasmid dna safety and comparative immunogenicity of an hiv- dna vaccine in combination with plasmid interleukin and impact of intramuscular electroporation for delivery safety and immunological efficacy of a dna vaccine encoding prostatic acid phosphatase in patients with stage d prostate cancer a phase i trial of a human papillomavirus dna vaccine for hpv + cervical intraepithelial neoplasia / . clin antigen-specific humoral and cellular immune responses can be modulated in rhesus macaques through the use of ifn-gamma, il- , or il- gene adjuvants genetic adjuvants for dna vaccines intranasal immunization of a dna vaccine with il- -and granulocyte-macrophage colony-stimulating factor (gm-csf)-expressing plasmids in liposomes induces strong mucosal and cell-mediated immune responses against hiv- antigens cytokine molecular adjuvants modulate immune responses induced by dna vaccine constructs for hiv- and siv intranasal administration of human immunodeficiency virus type- (hiv- ) dna vaccine with interleukin- expression plasmid enhances cell-mediated immunity against hiv- enhancement of cell-mediated immunity against hiv- induced by coinnoculation of plasmid-encoded hiv- antigen with plasmid expressing il- augmentation and suppression of immune responses to an hiv- dna vaccine by plasmid cytokine/ig administration il- expression plasmid enhances cell-mediated immunity induced by an hiv- dna vaccine a mage /heat shock protein dna vaccine induces both innate and adaptive immune responses for the antitumor activity heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology human dkk and human hsp fusion dna vaccine induces an effective anti-tumor efficacy in murine multiple myeloma a mouse model for study of systemic hiv- infection, antiviral immune responses, and neuroinvasiveness encoded novel forms of hsp or a cytolytic protein increase dna vaccine potency fusion of the mycobacterium tuberculosis antigen a to an oligomerization domain enhances its immunogenicity in both mice and non-human primates the oligomerization domain of c -binding protein (c bp) acts as an adjuvant, and the fusion protein comprised of the -kilodalton merozoite surface protein fused with the murine c bp domain protects mice against malaria a first-in-human phase trial to evaluate the safety and immunogenicity of the candidate tuberculosis vaccine mva a-imx , administered to bcg-vaccinated adults vesicular stomatitis virus g protein transmembrane region is crucial for the hemi-fusion to full fusion transition combined administration with dna encoding vesicular stomatitis virus g protein enhances dna vaccine potency vesicular stomatitis virus indiana glycoprotein as a t-cell-dependent and -independent antigen fusogenic membrane glycoproteins as a novel class of genes for the local and immune-mediated control of tumor growth viral fusogenic membrane glycoproteins kill solid tumor cells by nonapoptotic mechanisms that promote cross presentation of tumor antigens by dendritic cells comparison of primary human cytotoxic t-cell and natural killer cell responses reveal similar molecular requirements for lytic granule exocytosis but differences in cytokine production the structural basis for membrane binding and pore formation by lymphocyte perforin dna vaccines and apoptosis: to kill or not to kill? cytolytic dna vaccine encoding lytic perforin augments the maturation of-and antigen presentation by-dendritic cells in a time-dependent manner systematic comparison of constitutive promoters and the doxycycline-inducible promoter protection from endogenous perforin: glycans and the c terminus regulate exocytic trafficking in cytotoxic lymphocytes perforin forms transient pores on the target cell plasma membrane to facilitate rapid access of granzymes during killer cell attack the roles of caspase- and bcl- in chemically-induced apoptosis but not necrosis of renal epithelial cells apoptosis, pyroptosis, and necrosis: mechanistic description of dead and dying eukaryotic cells tolerance, danger, and the extended family how dying cells alert the immune system to danger tumor immunogenicity is determined by the mechanism of cell death via induction of heat shock protein expression natural adjuvants: endogenous activators of dendritic cells consequences of cell death: exposure to necrotic tumor cells, but not primary tissue cells or apoptotic cells, induces the maturation of immunostimulatory dendritic cells necrotic but not apoptotic cell death releases heat shock proteins, which deliver a partial maturation signal to dendritic cells and activate the nf-kappa b pathway cross-presentation by dendritic cells induction of genotype cross-reactive, hepatitis c virus-specific, cell-mediated immunity in dna-vaccinated mice rotaviral enterotoxin nonstructural protein targets mitochondria for activation of apoptosis during infection porcine reproductive and respiratory syndrome virus nonstructural protein induces apoptosis dependent on its c-like serine protease activity we thank thrf and the donor community for supporting the development of our novel dna vaccines. ashish c. shrestha and danushka k. wijesundara are recipients of early career fellowships from thrf. the authors declare no conflict of interest. key: cord- -heo mtxk authors: stoian, ana m.m.; zimmerman, jeff; ji, ju; hefley, trevor j.; dee, scott; diel, diego g.; rowland, raymond r.r.; niederwerder, megan c. title: half-life of african swine fever virus in shipped feed date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: heo mtxk african swine fever virus is transmissible through animal consumption of contaminated feed. to determine virus survival during transoceanic shipping, we calculated the half-life of the virus in feed ingredients exposed to -day shipment conditions. half-lives ranged from . to . days, indicating that the feed matrix environment promotes virus stability. however, half-life calculations were based on the limited data available at the time, including time points representing inoculation dose and titers at the conclusion of the study and insufficient replicates from which to calculate ses or % cis around the half-life estimates. for this study, our objective was to improve the accuracy of asfv half-life estimates by increasing the number of time points and replicates in the same trans-atlantic model. we used feeds or feed ingredients for this study. we programmed an environmental chamber with the environmental conditions of humidity and temperature, which fluctuated every hours, over a -day simulated trans-atlantic shipment ( ) . we added g of each gamma-irradiated feed ingredient to -ml conical tubes before inoculating them with µl of % tissue culture infective dose (tcid ) of asfv. we used asfv georgia / ( ) because of its similarity to currently circulating isolates ( ) . negative controls consisted of complete feed samples in meal form with µl of sterile phosphate-buffered saline (pbs) added. positive controls consisted of ml of rpmi medium (gibco, https://www.thermofisher. com) lacking feed with µl of tcid asfv. after addition of virus or pbs, we vortexed samples for s and covered each tube with a vented cap for incubation. after removing the samples from the environmental chamber, we added ml of sterile pbs and replaced the vented caps with solid caps. we organized samples in duplicate into replicate batches representing time points and simulated the trans-atlantic shipping model over separate -day periods. we used titrations for the half-life calculations in feed ( time points × replicates = titers/feed ingredient) and duplicate titers over time points to calculate half-life in rpmi medium. we tested samples for asfv on days , , , and after contamination. the first sample was collected at day after contamination to allow the virus to stabilize within each matrix. asfv was quantified by virus titration as described previously ( ) . we vortexed samples for s and then centrifuged at , × g for min at °c. supernatant from each sample was stored at − °c. we collected porcine alveolar macrophages for virus isolation by lung lavage of - -week-old pigs and cultured for day in rpmi medium supplemented with % fetal bovine serum and antibiotics emerging in a °c % co incubator. we prepared -fold serial dilutions in rpmi medium in triplicate, added dilutions to monolayers of porcine alveolar macrophages in -well plates, and incubated for h at °c. cells were washed again and rpmi medium replaced. after days at °c, the cells were fixed with % acetone for min and stained with mouse anti-p primary monoclonal antibody ( : , dilution). we incubated plates at °c for h and washed times with pbs before addition of goat anti-mouse alexa fluor secondary antibody (invitrogen, https://www. thermofisher.com; : dilution), followed by -h incubation at °c. we viewed cells under a fluorescence microscope and calculated the log tcid /ml according to the spearman-karber method ( ) . for all sample types, we calculated the half-life and corresponding % ci. the half-life analysis was performed by fitting a linear regression model to the data by using r version . . (https://www.r-project.org), with the natural log of the virus endpoint titers as the response variables and time as the explanatory variables. we estimated the slope and se of the respective lines by using these regression models and half-lives calculated as −log e ( )/slope as previously described ( ) . we calculated the se for each half-life by multiplying the se of the slope by log( ) divided by the square of the slope. we calculated the upper and lower bounds of the % ci as the estimated half-life plus/ minus the product of the se times the critical value of a t distribution with quantile as . and degrees of freedom as n - , where n is the sample size for that ingredient ( ) . environmental conditions during the course of the trans-atlantic model (figure, panel a) were a mean + sd temperature of . ± . °c (range - °c) and a mean + sd humidity of . % ± . % (range %- %). negative control samples remained negative. all asfvinoculated samples showed detectable quantities of infectious asfv (figure, panel b) . the half-life estimate in the rpmi-positive control was shorter than that for all feed ingredients tested: . + . days ( % ci . - . days) ( feed ingredients were inoculated with tcid asfv based on previous half-life calculations ( , ) and the infectious dose in feed ( ) . tcid , % tissue culture infective dose. soybean meal) and . + . days (organic soybean meal). the relative stability in feed may be the result of variable protein, fat, or moisture content among ingredients. overall, the mean half-life for asfv in all animal feed ingredients was . days. although the high stability of asfv in contaminated pork products and blood has been appreciated for decades ( ) , the stability of asfv in plant-based feed has been recognized only recently ( ) . our previous estimation of the half-life of asfv in feed ingredients was based on the limited data we had available, including inoculation dose and titers quantified at time point during the -day model ( , ) . in this study, we quantified viral decay at several time points over the -day model and increased sample size, which enabled us to calculate ses and % cis around the half-life estimates. in general, this updated modeling approach resulted in longer half-life estimates across all matrices. this study provides quantitative data on the half-life of asfv georgia in animal feed ingredients exposed to moderate temperature and humidity conditions simulating transoceanic shipment. the longer virus half-lives in feed compared with half-lives in media support the concept that the feed matrix provides an environment that increases asfv stability. furthermore, these data provide additional evidence supporting the ability of plant-based feed ingredients to promote survival of asfv should these products become contaminated. comparative analysis of whole-genome sequence of african swine fever virus belgium / outbreak of african swine fever emergence of african swine fever in china swine enteric coronavirus disease: a review of years with porcine epidemic diarrhoea virus and porcine deltacoronavirus in the united states and canada survival of viral pathogens in animal feed ingredients under transboundary shipping models infectious dose of african swine fever virus when consumed naturally in liquid or feed african swine fever spread in china african swine fever virus introduction into the eu in : experience of latvia genome sequences derived from pig and dried blood pig feed samples provide important insights into the transmission of african swine fever virus in china correction: survival of viral pathogens in animal feed ingredients under transboundary shipping models correction: survival of viral pathogens in animal feed ingredients under transboundary shipping models african swine fever virus isolate statistical method in biological assay the use of half-lives and associated confidence intervals in biological research is there a risk for introducing porcine reproductive and respiratory syndrome virus (prrsv) through the legal importation of pork? we thank the staff of the biosecurity research institute and maureen sheahan for their assistance in the biosafety level laboratory. we acknowledge the kansas state university applied swine nutrition team for their past contributions to the area of feed risk. the asfv georgia / isolate was kindly provided by linda dixon at the pirbright institute and obtained through the generosity of david williams at the commonwealth scientific and industrial research organization's australian animal health laboratory.funding for this study was provided by the swine health information center (grant no. - ) and the state of kansas national bio and agro-defense facility fund. ms. stoian is a phd student in the college of veterinary medicine at kansas state university, manhattan, kansas. her research is focused on understanding virus replication in gene-edited animals and on feed biosecurity for prevention and control of foreign and endemic infectious diseases. key: cord- -npijn co authors: esfandiyari, reza; halabian, raheleh; behzadi, elham; sedighian, hamid; jafari, ramezan; imani fooladi, abbas ali title: performance evaluation of antimicrobial peptide ll- and hepcidin and β-defensin- secreted by mesenchymal stem cells date: - - journal: heliyon doi: . /j.heliyon. .e sha: doc_id: cord_uid: npijn co peptides are secreted by different cell types and are trendy therapeutic agents that have attracted attention for the treatment of several diseases such as infections. antimicrobial peptides exert various mechanisms such as changing cell membrane permeability which leads to inhibition or death of bacterial cells. mesenchymal stem cells (mscs) are key to produce antimicrobial peptides and to inhibit the growth of pathogens. these cells have been shown to be capable of producing antimicrobial peptides upon exposure to different bacteria. as a result, antimicrobial peptides can be considered as novel agents for the treatment of infectious diseases. the purpose of this review was to investigate the targets and mechanisms of antimicrobial peptides secreted by mscs. antibiotics are used to treat bacterial infections through either inhibiting or killing the target bacteria. the first antibiotic was discovered by alexander fleming in , called penicillin and saved the lives of millions of people. however, the discovery of penicillin as a modern medication was preceded by the management of microbial infections in egypt, greece and ancient china [ ] . shortly after the discovery of penicillin, the bacterial resistance to this antibiotic became one of the challenges in the treatment of bacterial infections. therefore, new generation of beta-lactam antibiotics was developed to overcome antibiotic resistance and to treat bacterial infections. however, the first case of methicillin-resistant staphylococcus aureus (mrsa) was reported in england in [ , ] . the production and discovery of new antibiotics continue to this day and new antibiotics are marketed to counteract the drug resistance problem. yet, the point should be raised that one day antibiotics can no longer affect bacteria and will no longer be able to control bacterial infections. consequently, in recent years, researchers have devised other means to treat bacterial infections. one of such approaches is the use of antimicrobial peptides (amps) or "peptide antibiotics" to kill pathogenic bacteria and to treat bacterial infections [ ] . over the past decades, antimicrobial peptides (peptide antibiotics) have been shown to be effective in innate immunity of various species, such as plants, invertebrates and vertebrates. the intrinsic immune system is the first line of defense against the attack of microorganisms, among which the antimicrobial peptide molecules are the most important ones. the cathelicidin family is important antimicrobial agents in mammals [ , ] . these peptides are mainly stored in lysosomes of macrophages (mq) and polymorphonuclear neutrophils (pmns) [ ] . cathelicidins have been isolated from many cell types including neutrophils to coordinate the immune system, but have been found in other immune cells such as epithelial cells and macrophages and have been shown to combat against bacteria, viruses and fungi. cathelicidins have a variety of sizes ( - amino acids) and also have a wide range of structures [ ] . the molecular mechanism of antimicrobial peptides has been investigated [ ] . stem cells have been the focus of research because they have shown good potential in the field of therapy [ ] . one of the features of the stem cells referred to in this review is antimicrobial activity of mesenchymal stem cells that perform this action through antimicrobial peptides such as ll- , hepcidin and β-defensin- [ ] . the purpose of this study is to briefly review mesenchymal stem cells and antimicrobial peptides and how these peptides function. in recent years, stem cells have been widely used in the treatment of many diseases. one of the most important stem cells is mesenchymal stem cells (mscs) that have been shown to play a role in regulating the immune system and suppressing deleterious properties. mscs have the ability to differentiate into mesenchymal tissues such as cartilage, bone, muscle and fat. mscs have been obtained from bone marrow, umbilical cord, blood, placenta, skeletal muscle and adipose tissue. recent studies have found that mscs play an important role in the treatment of diseases, including infections, by producing antimicrobial peptides [ , , ] . cathelicidin is a carrier that has a wide range of functional molecules (i.e. cysteine or non-cysteine). the presence of this peptide has been proven in cattle, rabbits, pigs and humans [ ] . due to the unique characteristics of antimicrobial peptides, these peptides are one of the main candidates in the treatment of bacterial diseases and are effective on antibiotic resistant strains and even cancer cells. these properties include rapid killing and a wide range of activity that perform antimicrobial action by pore-forming the cell membrane [ ] . but these peptides can also be toxic to the cells of the body, so using peptides with a wide range of lethality and low side effects can help cure bacterial infections [ ] . in , agerberth et al [ ] based on the protected section of cathepsin, derived human bone marrow cdna clones from an unspecified antibacterial peptide named fa-ll- . the peptide constitutes of monocyte [ ] amino acids whose n-terminal is fall and the name of the peptide was coined for fall. the helical structure of this peptide was investigated in a saline environment containing vitamin e upon synthesis and antibacterial activity was investigated [ ] . the peptide is specifically secreted in the secondary granules of neutrophils. it is also produced by many types of cells, including macrophages, natural killer (nk) cells, epithelial cells of the skin, airways, eyes and intestinal tract. also, the expression of the peptide ll- is controlled by inflammatory pathways, similar to the pathway of vitamin d [ ] . in addition to antimicrobial activity, this peptide has immunomodulatory roles. for example, exposure to μg/ml of ll- peptide during a monocyte-macrophage differentiation leads to a positive inflammatory response, resulting in a decrease in the level of interleukin and an adjustment of p . in addition, the peptide ll- leads to the phenotype m , which suggests that this peptide has an important role in the development of macrophages and cytokine production [ ] . other chemical properties of ll- is as follows: migration of neutrophils and eosinophils through the formyl-peptide receptor; transactivation of the epidermis growth factors by the peptide causes the migration of keratinocytes, which results in wound healing; mcp- ccl- is a monocyte extraction factor that is secreted by ll- after stimulation. in addition, transforming growth factor beta (tgfβ) released from the epithelial cells of the intestine after exposure to ll- has an effect on the migration of epithelial cells and improved response. it is concluded that the peptide ll- is spread at the site of the infection, causing inflammatory response and wound healing [ , , ] . ll- peptide has different activities, all of which lead to antimicrobial activity. these include regeneration and angiogenesis, which is done by the formyl-peptide receptor-like expressed on the endothelial cells [ ] . chemotaxis is also one of the activities of this peptide, which causes the migration of mast cells to the environment [ ] . in addition, degranulation of mast cells and the release of inflammatory mediators takes place by the ll- peptide [ ] . ll- peptide, on the other hand, induces many cytokines and induces the production of antibodies [ ] . ll- peptide from primary human keratinocytes and human keratinocytes (hacat) cells protect from apoptosis [ ] . the results have shown that fluid membrane-associated peptides increases the plasma membrane of the subcutaneous glandular cells [ ] . this peptide also considered as an endotoxin, which inhibits the responses of inflammatory proteins to the bacterial lipopolysaccharides (lps) in human cells (fig. ) [ ]. this peptide performs its actions by connecting to cell receptors ( table ) . as previously mentioned, ll- is actively involved in physiological responses to eukaryotic cells, which is essentially an antimicrobial peptide. this peptide is effective in people whose immune system is low, so that through transactivation of egfr and angiogenesis by fprl receptor and chemotaxis causes cell migration [ ] . in addition, the expression of transgenic ll- leads to a significant increase in proliferation of corpuscular cells in hacat and hek [ ] . with the presence of lymph node metastases in estrogen-receptor positive or er positive cancer, clinical trials have shown an increase in erb-b receptor tyrosine kinase (erbb ) signaling, indicating an increase in peptide ll- activity, which plays an important role in the treatment of cancer [ ] . this peptide also interacts with p x purinoceptor, which is expressed mainly in monocytes, macrophages and dendritic cells (dcs) and stimulates and releases cytokine il- β in human gingival fibroblasts (fig. ) [ ]). another antimicrobial peptide produced by mscs is called hepcidin, which plays an important role in the clearance of pathogens. this antimicrobial peptide is rich in cysteine, the precursor of which is secreted by the liver (fig. ) . hepcidin is present in three forms of prehormone ( amino acids), prohormone ( amino acids) and hormones ( amino acids) [ ] . this peptide was discovered in and was called leap , but after a while due to presence in the liver, the peptide was named "hepcidin". this peptide is effective on a wide range of fungi, bacteria and viruses. one of the most important effects of hepcidin in the body is the regulation of iron hemostasis. this peptide prevents iron absorption from the small intestine and releases iron from reticuloendothelial cells. in infectious diseases, macrophages and bacteria compete to absorb iron [ ] . macrophages interfere with the absorption of iron by bacteria. eventually, the pathogen does not grow and replenish. factors that cause hepcidin production are increased in bone marrow and anemia. other factors that increase the production of hepcidin are iron accumulation and inflammation [ ] . hepcidin is effective on iron transfer from macrophages. in the presence of hepcidin, ferritin is transmitted into the macrophage and is destroyed by lysosomes, resulting in storage of iron inside the cell. in low concentrations of hepcidin, ferritin is present in the cell membrane, allowing the release of iron. after leaving the cell, iron oxide is rapidly oxidized by ceruloplasmin, a copper-rich ferroxidase and converted into ferric iron and then bound to transferrin [ ] . hepcidin is bound to plasma alpha- macroglobulin (alpha m). evidence suggests that other cells may express the hepcidin mrna at a much lower level than the hepatocytes; the biological significance of the extra hepatic production of hepcidin remains uncertain. plasma hepcidin is freely treated through glomeruli and in animals with normal kidney activity it quickly passes through the urine. in addition, a part of hepcidin is cleansed through degradation along with ferritin [ ] (fig. ). cysteine-rich cationic proteins are found in vertebrates, invertebrates and plants, which range from to amino acids [ ] . the role of these peptides is to defend the host against bacteria, fungi and viruses. these peptides play a role in destroying the bacterial cell membrane. defensins were discovered for the first time in . the basic genes produced by these peptides are highly polymorphic. generally, defensins are classified in three main groups of alpha, beta and theta, each of which has different genetic symbols and is produced from different cells [ ] , as shown in table . in immature individuals, defensins (present in breast milk) play a very important role in protecting the disease due to the weak immune system. the defensin is produced by epithelial cells and leukocytes, as indicated in table . alpha defensin exists only in mammals, while beta defensin exists in many vertebrates, invertebrates and plants, but theta table defensin types [ , , ] . defensin- alpha defensin- β -defensin β β-defensin secreted by leukocytes and epithelial cells of many kinds. have been found only in the leukocytes of the rhesus macaque and the olive baboon, papio anubis, being vestigial in humans and other primates. fig. . mscs produce microbial peptides including hepcidin, ll- and β-defensin to fight against bacteria. as shown in the figure above, the antimicrobial peptide ll- is effective against staphylococcus aureus and escherichia coli bacteria, while the effect of β-defensin peptide on the bacteria of escherichia coli has been proven [ ] . defensin has been found in several species of ancient monkeys, but not in humans and other primates. the important point is that the mrna manages to denote the theta defensin, but the mutations cause the end codon to stop peptide production. one of the key characteristics of defensins is their antimicrobial activity, which is active against grampositive and gram-negative bacteria, fungi and viruses [ ] . in addition to antimicrobial activity, defensin exerts other functions that includes chemical absorption for monocytes, cytokines production by monocytes and epithelial cells, angiogenesis activity and response to hormones. antimicrobial effect on gram-positives is different from gram-negative bacteria. the reason is that, first, the cell wall structure of gram-positive bacteria is different from gram-negatives (the cell wall of gram-negative bacteria is rich in lipopolysaccharide (lps), which has a negative charge, while in gram-positive bacteria the cell wall have teichoic acids (ta) with less negative charge. secondly, the peptidoglycan layer is thick in gram-positive bacteria and the formation of pores by defensin on the surface of the gram-positive cells requires more time. therefore, defensin has weaker anti-microbial activity in gram-positive bacteria [ ] . defensin functions on the bacterial cells via the l-arginine cationic chain that are attached by electrostatic absorption to the membrane's anionic structure, then defensin is introduced into the membrane by using the electromotive force of the membrane, creating channels inside the membrane. ultimately, materials that are surrounded by membranes are leaked outside or inside the cell and causes bacterial cell lysis [ ] . attempts to design novel treatment strategies as alternatives for the current treatment of diseases such as sepsis, bacteremia, bloodstream infections and other bacterial infections have become a major challenge. one of the most important treatments used to kill pathogenic bacteria is the use of antibiotics, which has some disadvantages in addition to its benefits. antibiotic resistance is one of the biggest treatment problems with antibiotics. antibiotic resistance is on the rise and the number of resistant are constantly circulating in the communities and it can be said that it will not last long as antibiotics will no longer be effective against bacteria [ , ] . an emerging approach to treat infections is the use of antimicrobial peptides produced from various cells, including mscs. research has shown that mscs have antimicrobial properties and this property is related to the production of antimicrobial peptides (fig. ) [ ] . there are several barriers to using stem cells in treatment, such as the immune response to stem cells that can reduce the function of these cells, as well as their use may cause infection, poisoning, cancer and even death [ ] . as a result, it is better to use mesenchymal stem cells in treatment if the treatments that have been used so far are no longer effective. on the other hand, the use of mesenchymal stem cells as a treatment has many benefits, one of which is the lack of damage that is usually caused by chemical treatments on the body. the use of mesenchymal stem cells in the treatment of bacterial infections can reduce the spread of antibiotic resistance [ ] . in conclusion, mscs are very capable of eliminating bacterial infections. the use of these cells and their antimicrobial products can be a potential alternative to current treatments, while bacterial resistance can be avoided. author contribution statement reza esfandiyari, raheleh halabian, elham behzadi, hamid sedighian, ramezan jafari, abbas ali imani fooladi: conceived and designed the experiments; analyzed and interpreted the data; wrote the paper. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. a brief history of the antibiotic era: lessons learned and challenges for the future the antibiotic resistance crisis: part : causes and threats molecular mechanisms of antibiotic resistance antimicrobial peptides: an introduction antibacterial effect of human mesenchymal stem cells is mediated in part from secretion of the antimicrobial peptide ll- the role of cathelicidins in the innate host defenses of mammals cathelicidins, multifunctional peptides of the innate immunity human defensins and ll- in mucosal immunity the human gene fall and processing of the cathelin precursor to the antibacterial peptide ll- in granulocytes human embryonic stem cell-derived retinal pigment epithelium in patients with age-related macular degeneration and stargardt's macular dystrophy: follow-up of two open-label phase / studies antibacterial effect of mesenchymal stem cells against escherichia coli is mediated by secretion of beta-defensin- via toll-like receptor signalling concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing mesenchymal stem cells in tissue repair antimicrobial activity of mesenchymal stem cells: current status and new perspectives of antimicrobial peptide-based therapies purification and structural characterization of bovine cathelicidins, precursors of antimicrobial peptides a novel tool against multiresistant bacterial pathogens: lipopeptide modification of the natural antimicrobial peptide ranalexin for enhanced antimicrobial activity and improved pharmacokinetics membrane interactions of mesoporous silica nanoparticles as carriers of antimicrobial peptides fall- , a putative human peptide antibiotic, is cysteine-free and expressed in bone marrow and testis solution structure and peptide binding of the sh domain from human fyn ll- directs macrophage differentiation toward macrophages with a proinflammatory signature induction of keratinocyte migration via transactivation of the epidermal growth factor receptor by the antimicrobial peptide ll- little peptide, big effects: the role of ll- in inflammation and autoimmune disease an angiogenic role for the human peptide antibiotic ll- /hcap- a cathelicidin family of human antibacterial peptide ll- induces mast cell chemotaxis the cathelicidin ll- activates human mast cells and is degraded by mast cell tryptase: counter-regulation by cxcl serum levels of ll- and inflammatory cytokines in plaque and guttate psoriasis the human antimicrobial peptide ll- suppresses apoptosis in keratinocytes modulation by ll- of the responses of salivary glands to purinergic agonists aldose reductase mediates the lipopolysaccharide-induced release of inflammatory mediators in raw . murine macrophages new development in studies of formyl-peptide receptors: critical roles in host defense curcumin inhibits proliferation of interleukin- -treated hacat cells the human cathelicidin antimicrobial peptide ll- and mimics are potential anticancer drugs ll , a human antimicrobial peptide with immunomodulatory properties α- antitrypsin binds preprohepcidin intracellularly and prohepcidin in the serum iron homeostasis and the inflammatory response iron homeostasis in host defence and inflammation intracellular iron transport and storage: from molecular mechanisms to health implications hepcidin bound to α -macroglobulin reduces ferroportin- expression and enhances its activity at reducing serum iron levels antimicrobial proteins in intestine and inflammatory bowel diseases comparative genomics and evolution of the alpha-defensin multigene family in primates increased levels of α-defensin (- , - and - ) in type diabetic patients with nephropathy defensins. handbook of biologically active peptides rhesus theta-defensin prevents death in a mouse model of severe acute respiratory syndrome coronavirus pulmonary disease mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by α-helical antimicrobial and cell non-selective membranelytic peptides the tlr -par axis regulates bone marrow mesenchymal stromal cell survival and therapeutic capacity in experimental bacterial pneumonia review of antibiotic resistance in china and its environment mesenchymal stem cells and immunomodulation: current status and future prospects antimicrobial design of titanium surface that kill sessile bacteria but support stem cells adhesion hepcidin regulation: ironing out the details the authors declare no conflict of interest. no additional information is available for this paper. key: cord- -ml pbewf authors: manczinger, máté; boross, gábor; kemény, lajos; müller, viktor; lenz, tobias l.; papp, balázs; pál, csaba title: pathogen diversity drives the evolution of generalist mhc-ii alleles in human populations date: - - journal: plos biol doi: . /journal.pbio. sha: doc_id: cord_uid: ml pbewf central players of the adaptive immune system are the groups of proteins encoded in the major histocompatibility complex (mhc), which shape the immune response against pathogens and tolerance to self-peptides. the corresponding genomic region is of particular interest, as it harbors more disease associations than any other region in the human genome, including associations with infectious diseases, autoimmune disorders, cancers, and neuropsychiatric diseases. certain mhc molecules can bind to a much wider range of epitopes than others, but the functional implication of such an elevated epitope-binding repertoire has remained largely unclear. it has been suggested that by recognizing more peptide segments, such promiscuous mhc molecules promote immune response against a broader range of pathogens. if so, the geographical distribution of mhc promiscuity level should be shaped by pathogen diversity. three lines of evidence support the hypothesis. first, we found that in pathogen-rich geographical regions, humans are more likely to carry highly promiscuous mhc class ii drb alleles. second, the switch between specialist and generalist antigen presentation has occurred repeatedly and in a rapid manner during human evolution. third, molecular positions that define promiscuity level of mhc class ii molecules are especially diverse and are under positive selection in human populations. taken together, our work indicates that pathogen load maintains generalist adaptive immune recognition, with implications for medical genetics and epidemiology. a a a a a the major histocompatibility complex (mhc) genes in vertebrates encode cell surface proteins and are essential components of adaptive immune recognition [ ] . mhc proteins are endowed with highly variable peptide-binding domains that bind short protein fragments. the mhc region is one of the most polymorphic gene clusters in vertebrate genomes [ ] . co-evolutionary arms race with pathogens is considered largely responsible for the observed exceptionally high levels of genetic diversity [ ] [ ] [ ] [ ] , yet it cannot fully account for the observed geographic differences in human mhc genetic diversity [ , ] . this indicates that, beyond mhc allelic diversity, other mhc-related factors contribute to the capacity of human populations to withstand pathogens. in this paper, we argue that peptide-binding repertoire size (or, shortly, promiscuity) of mhc alleles is one important factor. recent empirical studies demonstrated that there is a substantial variation in the size of the bound and presented antigen repertoire across mhc class i alleles. certain mhc class i alleles appear to be promiscuous and are capable of binding an exceptionally large set of epitope peptide segments [ , ] . for example, paul and colleagues carried out bioinformatics analysis to predict the binding capacity of common hla-a and hla-b alleles to a set of , dengue virus-derived peptides [ ] . the analysis revealed over -fold variation in the number of peptides bound by the different alleles, indicating significant variation in epitope repertoire size across hla molecules. the authors selected three alleles for further study in an in vivo transgenic mouse model. immunization of the corresponding hla transgenic mouse strains with a set of dengue virus-derived peptides revealed a positive relationship between epitope repertoire size and immunogenicity. similarly, kosmrlj and colleagues computed the fraction of self-peptides that bind to various hla-b molecules, and found that this fraction varies extensively across four hla-b alleles [ ] . the authors then demonstrated that the self-peptidebinding repertoire of hla-b shapes the native repertoire of t-cell clones developed in the thymus, with implications for recognizing human immunodeficiency virus (hiv) epitopes. their results could explain why individuals carrying hla-b � alleles can maintain low hiv rna without therapy. remarkably, analogous mhc class i alleles with the hla-b � superfamily is widespread in chinese rhesus macaques, animals which show especially slow progression of simian immunodeficiency virus (siv)/hiv [ ] . finally, by focusing on seven chicken mhc class i haplotypes and four human hla-b alleles, chappel and colleagues demonstrated that mhc class i molecules that can bind a wide range of viral epitopes show lowered expression on the cellular surfaces of immune cells, such as monocytes and lymphocytes [ ] . the authors suggested that the breadth of epitope-binding repertoire shapes genetic susceptibility to marek's disease virus in chickens and hiv disease progression in humans. more generally, by recognizing more peptide segments, promiscuous mhc molecules may promote immune response against a broader range of pathogens and are hence generalists [ , ] . prior case studies in chicken indicate that this may be so [ ] [ ] [ ] [ ] . however, it remains to be established whether this relationship generally holds across mhc class i and ii alleles and a wide range of infectious diseases. specifically, we propose that in regions of high pathogen diversity, human populations should carry promiscuous mhc alleles. moreover, as migrating human populations have been exposed to changing sets of pathogens [ ] , shifts in mhc promiscuity level should have occurred repeatedly and in a rapid manner during the course of human evolution. to test these predictions, we first focused on the human hla class ii drb gene, for several reasons. first, drb is the most variable hla class ii locus, with over , registered alleles [ ] . together with hla-dra, hla-drb encodes the heterodimeric hla-dr protein complex, but hla-dra is basically invariant. second, drb shows the strongest general signature of selection among hla class ii loci [ ] , while at the same time showing the weakest evidence for divergent allele advantage, an alternative mechanism at the genotype level for presenting a broader set epitopes [ ] . third, drb has diversified very rapidly in the human lineage [ ] . many of the drb alleles appear to be human specific and most likely evolved after the migration of ancestral human populations out of africa [ ] . these periods have been associated with human populations encountering numerous new pathogens [ , ] . for other hla class ii loci, the level of genetic diversity is lower [ , ] , probably driven by selection for functions partly unrelated to pathogens. notably, hla-dq has a fundamental role in the development of immune tolerance [ , ] , while hla-dp contributes to the presentation of epitopes of intracellular origins [ ] [ ] [ ] . fourth, epitope-binding prediction algorithms show higher accuracy for drb than for other hla class ii loci [ , ] . finally, the abundance of drb on the cell surface is especially high compared with other hla class ii molecules [ ] [ ] [ ] . subsequently, we also evaluated promiscuity patterns of hla class i molecules. estimates on epitope-binding promiscuity were derived from two sources: experimental assays that measured individual peptide-mhc interactions in vitro and systematic computational predictions. in a series of analyses, we show that predictions of our hypothesis are upheld, regardless of how hla-drb promiscuity level is estimated. given that large-scale experimental assays to measure individual peptide-mhc interactions are extremely tedious, we first employed established bioinformatics tools to predict the binding affinities of experimentally verified epitope peptides for a panel of nonsynonymous hla-drb alleles, all of which are present at detectable frequencies in at least one human population [ ] [ ] [ ] . the set of investigated epitopes was derived from the immune epitope database (iedb) and contains , peptide epitopes of pathogens known to be bound by certain hla class ii alleles [ ] (s data). epitopes showing high levels of amino acid similarity to each other were excluded from the analysis (see methods). most included epitopes are to amino acids long and are found in only one of the pathogens (s fig, s data) . the netmhciipan algorithm was used to predict individual epitope-mhc interactions [ ] , not least because it outperforms other prediction algorithms [ ] . the breadth of epitope-binding repertoire or, shortly, the level of promiscuity of individual hla-drb alleles was estimated as the fraction of epitopes with a binding affinity stronger than nm to the given mhc molecule. this threshold corresponds to high-affinity binding, which is frequently observed in mhc molecules displaying immunodominance [ ] . we found large variation in promiscuity levels across hla-drb alleles (s fig, s data) . using a smaller dataset with information from both approaches, we show that the computationally predicted and the empirically estimated promiscuity values are strongly correlated with each other (spearman's rho: . , taking advantage of the confirmed reliability of computational predictions, we next investigated the geographic distribution of hla-drb alleles. we first collected high-quality hla-drb allele prevalence data of human populations residing in countries from two databases and an article [ ] [ ] [ ] . the weighted average of promiscuity level in each population was calculated based on the promiscuity values and allele frequencies of individual alleles in the population (see methods). the analysis revealed a large variation in mean promiscuity across geographical regions and the corresponding human populations (s table) . importantly, several distantly related but highly promiscuous alleles contribute to this pattern (s table) . notably, an especially high allelic promiscuity level was found in southeast asia, an important hot spot of emerging infectious diseases [ ] . to minimize any potential confounding effect of high genetic relatedness between neighboring populations, we merged populations with similar hla allele compositions for all further analyses (see methods). using the global infectious diseases and epidemiology network (gideon), we compiled a dataset on pathogen richness in the corresponding geographic regions [ ] . it consists of diseases caused by extracellular pathogens, including diverse bacterial species, fungi, protozoa, and helminthes. using the same protocol, we additionally compiled a dataset on the prevalence of diseases in the same regions caused by viral and other obligate intracellular pathogens. the dataset and methodology employed for the analysis are standardized and have been used previously in similar contexts [ , , ] . we report a strong positive correlation between extracellular pathogen diversity and mean promiscuity: hla-drb alleles that can bind epitopes from a broader range of pathogens are more likely to be found in regions of elevated pathogen diversity ( fig a) . this pattern is unlikely to be explained by confounding factors, such as country size or hla-drb genetic diversity across countries (s table) . by contrast, we found no significant association between hla-drb promiscuity level and diversity of intracellular pathogens (fig b) . we conclude that the geographical distribution of promiscuous hla-drb alleles has been mainly shaped by the diversity of extracellular pathogens. the above results hold-and are even stronger-when estimates on promiscuity were derived from empirical in vitro mhc binding data (shortly, in vitro promiscuity), downloaded from the iedb database [ ] (fig c and d , s table and s data). however, these results do not exclude the possibility that the geographical link between pathogen diversity and promiscuity is indirect. more direct support on the causal relationship between the two variables comes from analysis of prior human genetic studies. to investigate this issue, we focused on two geographically widespread allelic groups with exceptionally high (hla-drb � ) and exceptionally low (hla-drb � ) promiscuity values, respectively, and conducted literature mining on their reported associations with infectious diseases (s table) . as expected, hla-drb � was associated with protection against at least five infectious diseases, while hla-drb � was associated with susceptibilities to eight infectious diseases, which is highly unlikely by chance (fisher test, p = . ) (s table, s data). the data also indicate local adaptation towards elevated promiscuity under diverse pathogen pressure. the hla-drb � : allele is prevalent in specific regions of southeast asia. compared with other alleles detected in this region, hla-drb � : has a relatively high promiscuity value (top . indeed, this allele is associated with protection from recurrent pulmonary tuberculosis, recurrent typhoid fever, and hepatosplenic schistomiasis (s data), all of which are endemic diseases in southeast asia [ ] [ ] [ ] . remarkably, the frequency of this allele increases with extracellular pathogen diversity in this region ( fig b) . together, these observations support the hypothesis that promiscuous epitope binding of hla-drb alleles is favored by selection when extracellular pathogen diversity is high. an important unresolved issue is how promiscuity has changed during the course of human evolution. under the assumption that local pathogen diversity drives the evolution of epitope recognition of hla class ii alleles, promiscuity as a molecular trait should have evolved rapidly as human populations expanded into new territories. to investigate this issue, we combined an established phylogeny of hla-drb alleles [ ] with predicted epitope-binding promiscuity values. we found that alleles with a high promiscuity level have a patchy distribution across the tree (s fig), indicating that high promiscuity has multiple independent origins. to investigate this observation further, we selected a set of hla-drb alleles with a detectable frequency in at least one human population and appropriate sequence data (see methods). a comparison of all pairs of these alleles revealed that even very closely related alleles show major differences in promiscuity levels ( fig a) . for example, alleles belonging to the hla-drb � group show over % amino acid sequence identity to each other, but display as much as -fold variation in the predicted promiscuity levels. we conclude that the switch between high and low promiscuity levels has occurred repeatedly and in a rapid manner during the allelic diversification of the hla-drb locus. we next asked how selection on promiscuity has shaped the genetic diversity along the epitope-binding region of hla molecules. to quantify protein sequence variability at each amino acid position, we calculated the shannon entropy index based on the alignment of the selected hla-drb alleles from above. for each position, we also calculated promiscuity fragility, that is, the median impact of single amino acid substitutions on promiscuity (see methods). a strong positive correlation was found between shannon entropy and promiscuity fragility ( the above data suggest a link between allele promiscuity and hla-drb diversification, probably as an outcome of selection for locally optimized promiscuity levels. finally, we note that several variable molecular sites in the binding region of hla-drb affect epitope-binding characteristics without any major impact on promiscuity per se. for example, our computational analysis indicates that mutations at amino acid site numbers and do not seriously affect promiscuity level ( fig b) . however, several mutations at these sites are associated with binding self-peptides and thereby shape vulnerability to specific autoimmune diseases [ , ] . for all pairs of selected alleles, the predicted promiscuity difference between two hla-drb alleles is shown as a function of amino acid distance measured after excluding the epitope-binding region. large differences in promiscuity can be observed even between closely related pairs of alleles (e.g., at zero amino acid distance). as a result, there is no correlation between amino acid distance and promiscuity fold difference (spearman's rho = . , p = . ). amino acid distances were binned as shown on the figure (n = , , , , , , ). violin plots show the density function of promiscuity fold difference values for allele pairs in the given bin. white circles show median values; bold black lines show the interquartile range. (b) sequence variability of an amino acid site in the epitope-binding region of hla-drb (measured as shannon entropy) correlates positively with the site's promiscuity fragility, measured as the median predicted promiscuity fold difference caused by a random amino acid change at the given site (see inset, spearman's rho: . , p = . ). sites that have a larger impact on promiscuity are more diverse in human populations. line in inset represents linear regression between the two variables. the same result was obtained when promiscuity fragility was calculated based on nucleotide substitutions instead of amino acid substitutions (spearman's rho: . , p = . , see methods) or when sequence variability was measured as nonsynonymous nucleotide diversity (π a ) instead of sequence entropy (s fig). sites under positive selection as identified by furlong and colleagues [ ] show significantly higher promiscuity fragility (wilcoxon rank sum test, p = . ) and are marked with asterisks (see also s fig) . the underlying data for this figure can be found in s data. the relationship between pathogen diversity and epitope-binding promiscuity may be more general, as similar results hold for the hla-a locus. hla-a is one of the three types of classical human mhc class i molecules and is mainly involved in the presentation of epitopes from intracellular pathogens [ ] . in agreement with expectation, we report a positive correlation between local intracellular pathogen diversity and the hla-a promiscuity level of the corresponding human populations (s a and s b fig, s data) . no marked positive correlation was found for two other mhc class i genes (hla-b and hla-c, see s c to s f fig, and s data) . therefore, other unrelated evolutionary forces may shape the geographical distribution of promiscuous hla-b and hla-c alleles (s text). central players of the adaptive immune system are the groups of proteins encoded in the mhc. by binding short peptide segments (epitopes), mhc molecules guide both immune response against pathogens and tolerance to self-peptides. the genomic region encoding these mhc molecules is of special interest, for two reasons. it harbors more disease associations than any other regions in the human genome, including associations with infectious diseases, autoimmune disorders, tumors, and neuropsychiatric diseases [ , ] . a growing body of literature is now revealing that certain mhc class i alleles can bind a wider range of epitopes than others, but the functional implications of this variation remain largely unknown [ ] . by recognizing a larger variety of epitopes, such promiscuous mhc alleles promote immune response against a broader range of pathogens at the individual level. therefore, promiscuous epitope binding of mhc molecules should be favored by selection in geographic regions where extracellular pathogen diversity is high. importantly, this mechanism is conceptually distinct from the well-established concept of heterozygote advantage at the mhc [ ] , as it concerns individual alleles and not allele combinations or genotypes. to test this hypothesis, we combined data on the geographic distribution of human mhc class ii alleles and prevalence of extracellular pathogens, empirical/computational estimates of epitope-binding promiscuity, and phylogenetic analyses. our main findings, strongly supporting our hypothesis, are as follows. first, in geographical regions of high extracellular pathogen diversity, human hla-drb alleles have exceptionally high epitope-binding repertoires. this suggests that the geographical distribution of promiscuous hla-drb alleles has been shaped by the diversity of extracellular pathogens. the hla-drb � : allele highlights this point. hla-drb � : is a promiscuous allele that has been associated with protection from certain infectious diseases (s data). as expected, this allele is especially prevalent in regions of southeast asia with elevated pathogen load (fig b) . it is well established that antigens presented by hla class ii molecules derive mainly from extracellular proteins [ ] . however, hla class ii molecules have well-established roles in controlling immune response against viruses [ , ] . additionally, viral peptides are reported to be processed and presented also by the hla class ii pathway [ ] . therefore, it remains to be established why intracellular pathogen diversity has no major impact on the global distribution of hla-drb alleles. notably, the relationship between pathogen load and epitope-binding promiscuity may be more general, as similar results hold for the hla-a locus: we found a positive correlation between local intracellular pathogen diversity and the hla-a promiscuity level of the corresponding human populations (s a and s b fig, s data) . second, a phylogenetic analysis revealed major differences in promiscuity levels of very closely related hla-drb alleles. this suggests that high promiscuity level in hla-drb has evolved rapidly and repeatedly during human evolution. finally, amino acid positions with a prominent role in shaping hla-drb promiscuity level are especially variable in human populations and tend to be under positive selection. in sum, we conclude that hla promiscuity level is a human trait with paramount importance during adaptation to local pathogens. our work has important ramifications for future studies. mhc is the most variable region of the human genome, and the variation is associated with numerous infectious and immunemediated diseases [ , , [ ] [ ] [ ] [ ] [ ] . the impact of mhc promiscuity level on population allelic diversity is an interesting area for future research. in a similar vein, mhc allelic diversity is associated with olfaction-based mating preferences in human and other animals [ ] . the roles of mhc promiscuity in mating success and mating preferences are a terra incognita. we note that the most promiscuous hla-drb alleles are rare in certain human populations (s table; s data). this suggest that these alleles are not particularly favored by natural selection in these areas. why should it be so? first, high promiscuity may not be able to cope with the rise of novel and highly virulent pathogens. in such cases, displaying a particular epitope might be the most efficient way to achieve resistance, and high promiscuity might be suboptimal due to a reduced specificity [ , ] . second, high promiscuity level may elevate the risk of immune reactions against host tissues and non-harmful proteins [ , ] . clearly, future work should elucidate the evolutionary trade-offs between protection from pathogens and genetic susceptibility to autoimmune diseases. this will require high-throughput experimental methods to determine epitope-binding repertoire [ ] , and hla transgenic mice studies on the role of promiscuity in immune response [ ] . finally, genetic variation within particular mhc genes influences vaccine efficacy [ ] , rejection rates of transplanted organs [ ] , susceptibility to autoimmune diseases [ ] , and antitumor immunity [ , , ] . our work raises the possibility that, by altering the maturation and functionality of the immune system, the size of the epitope-binding repertoire of mhc alleles itself could have an impact on these processes. the exact role of mhc promiscuity in these crucial public health issues is an exciting future research area. the iedb has collected the results of individual and systematic studies on epitope binding by mhc alleles [ ] . the experimental studies include hla-binding assays, t-cell activation assays, and immunopeptidomic studies as well. epitopes of all available viral, bacterial, and eukaryotic pathogens known to be bound by at least one hla-i or hla-ii allele were extracted from iedb. reference proteomes of pathogenic species that carry at least one of the collected epitope sequences were retrieved from the uniprot database ( for hla-i and for hla-ii epitopes) [ ] . only epitopes of these species were analyzed further. all proteomes were scanned for each epitope sequence, and epitope sequences found in only one proteome (i.e., species-specific epitopes) were kept for further analysis. highly similar epitope sequences were identified using clustal omega [ ] and excluded as follows. a protein distance matrix was created and epitopes were discarded iteratively. in each iteration, the epitope pairs with the lowest k-tuple distance were identified. then, the epitope with the highest average similarity to all other sequences was excluded. iterations were repeated until distance values less than . (corresponding to greater than approximately % sequence identity) were eliminated from the matrix [ ] . note that this filtering procedure was carried out separately for epitope sequences bound by hla-i and hla-ii. binding affinities of the remaining , hla-i epitope sequences to hla-a, hla-b and hla-c alleles were predicted with the netmhcpan- . algorithm [ ] . the binding of , hla-ii epitope sequences to hla-drb alleles was predicted using the netmhciipan- . algorithm [ ] . all alleles are present in at least one of the human populations studied here (see below). the "pep" sequence input format was used for both hla-i and hla-ii epitope-binding prediction. a binding affinity threshold of nm was applied, yielding peptides that are likely to be immunodominant [ ] . for alternative binding threshold definitions, see s fig. for each binding threshold, epitope-binding promiscuity was defined as the fraction of the epitope set bound by a given allele. to determine the epitope-binding promiscuity of hla-drb alleles based on previously published experimental data, we used the iedb database [ ] . specifically, we downloaded all mhc ligand and t-cell assay data, which was available for hla-drb alleles. binding data of alleles screened for at least ligands each were further analyzed. the epitope set of each allele was filtered for highly similar sequences, as described above. as the majority of in vitro assay data were available in a binary format (i.e., presence or absence of binding), promiscuity was calculated as the fraction of positive binding assays for a given allele. to calculate population-level promiscuity values, we obtained hla allele frequency data from the allele frequency net database (afnd) and the international histocompatibility working group (ihwg) populations [ , ] . haplotype-level data of the th international hla and immunogenetics workshop (ihiw) populations were downloaded from dbmhc (national center for biotechnology information [ncbi]; ftp://ftp.ncbi.nlm.nih.gov/pub/mhc/mhc/final % archive). additionally, allele frequency data of the th and th ihiw populations, as published by riccio and colleagues [ ] , and populations in the afnd were used in the analyses. to avoid potential confounding effects of recent genetic admixture and migration, we focused on native populations, similarly to previous studies (s table) [ , ] . we excluded ihwg populations reported to deviate from hardy-weinberg equilibrium [ ]. among the afnd populations and ihwg populations without haplotype-resolution data ( th and th ihiw), those comprising less than genotyped individuals or those in which the sum of allele frequencies deviated from by more than % were excluded. populations reported in multiple databases were included only once in the analysis. for each hla loci, we calculated mean population promiscuity by averaging promiscuity values of alleles weighted by their relative frequencies in the populations. in all of these calculations, we used standardized (i.e., z-score) promiscuity values to make the in silico and in vitro values more easily comparable. finally, when calculating population-level promiscuity based on in vitro promiscuity data, we excluded populations for which in vitro promiscuity values could be assigned to less than % cumulative allele frequency. to tackle the issue of nonindependence of data points, we focused on populations instead of countries and grouped those populations that have highly similar hla allele compositions, based on standard measures of genetic distance (see below). we merged populations with highly similar hla allele compositions, allowing us to avoid pseudoreplication of data points while retaining informative allele frequency differences between populations residing in the same broad geographical areas. to this end, we first generated a genetic distance matrix between populations with the adegenet r library using allele frequency data of the examined locus. we used the rogers' genetic distance measure [ ] because it does not assume that allele frequency changes are driven by genetic drift only, an unlikely scenario for hla genes. next, populations were merged using a network-based approach. populations were treated as nodes and two nodes were connected if their genetic distance was under a cutoff value. populations were grouped in an iterative manner. in each iteration, all maximal cliques (i.e., subsets of nodes that are fully connected to each other) in the network were identified. maximal cliques represent groups of populations in which all populations have similar allele compositions to each other. then, mean genetic distance within each clique was calculated. the clique with the lowest average distance was selected and its populations were grouped together. then, this clique was deleted from the network. iterations were repeated until no maximal cliques remained in the network. grouping of populations was carried out using different distance value cutoffs ( st, th, th, and th rank percentile of all distance values). the resulting population groups and the individual populations that remained in the network were treated as independent data points in subsequent statistical analyses. mean promiscuity level in population groups was calculated by averaging population promiscuity values. unless otherwise indicated, all figures are based on population groups using the th percentile genetic distance cutoff value. importantly, using different cutoffs has no impact on our results (s data). finally, we note that genetic differences among human populations mostly come from gradations in allele frequencies rather than from the presence of distinctive alleles [ ] . therefore, traditional clustering of populations based on hla composition would have been ill-suited for our purposes. data on infectious diseases were collected from gideon [ ] . for each disease, the number of causative species or genera (when species were not listed for the genus) was determined using disease information in the gideon database, as described previously [ ] . causative agents were classified into obligate intracellular and extracellular pathogen groups based on a previous study [ ] and literature information. putative facultative intracellular pathogens were excluded from the analysis. diseases caused by agents that could not be clearly classified were also excluded from the analysis. extracellular and intracellular pathogen diversity (richness) of each country was approximated by the number of identified endemic extracellular and intracellular species, respectively. finally, we assigned country-level measures of pathogen and hla diversity to population groups as follows. for each population group, extracellular and intracellular pathogen counts were calculated by averaging the corresponding country-level values across the populations within the group. for example, if a population group contained two populations residing in neighboring countries, then we assigned the average pathogen diversity of the two countries to it. to examine associations between selected hla allele groups and infectious diseases, we carried out a systematic literature search on pubmed database using the following terms: "assoc � drb ", "assoc � drb ", "assoc � drb ", "assoc � drb ", "assoc � dr ", "assoc � drb � ", "assoc � drb ", "assoc � drb ", "assoc � dr ", "assoc � drb � ", "assoc � dr ", "infect � drb ", "infect � drb ", "infect � drb ", "infect � drb ", "infect � dr ", "infect � drb � ", "infect � drb ", "infect � drb ", "infect � dr ", "infect � drb � ", and "infect � dr ". "assoc � " and "infect � " refer to any word beginning with these letters. each resulting paper containing hla association data was examined, and statistically significant associations between allele groups (drb � or drb � ) or common alleles in allele groups (drb � : , drb � : , drb � : ) and infectious diseases were collected. associations with diseases caused by intracellular pathogens were excluded from the analysis. hla-disease associations were classified as beneficial or detrimental, if all related studies supported the beneficial or detrimental role of hla allele/allele group in the development or course of the given disease. otherwise, the association was classified as controversial. the results were summarized (s table) , and statistical association between beneficial/detrimental effects and high/low promiscuity across allele groups was determined by a fisher's exact test. we used amino acid distance as a proxy for phylogenetic distance between pairs of drb alleles. to this end, nucleotide sequences of drb alleles that contained full exon and regions were downloaded from the ipd-imgt/hla database [ ] . to limit our analyses to alleles that have an impact on the inferred promiscuity level of a population, we considered only those sequences that had a nonzero frequency in at least one human population (see above). from allele groups that code for the same protein sequence (synonymous differences, differentiated by the third set of digits in the hla nomenclature), one of the alleles was randomly chosen. this selection procedure resulted in alleles. multiple alignment of nucleotide sequences was performed using the muscle algorithm as implemented in the mega software [ ] and converted to protein sequence alignments. amino acid distance was calculated using the jones-taylor-thornton substitution model in mega [ ] (fig a) . epitope-binding region sites-as defined previously [ ]-were excluded when calculating amino acid distance. the rationale behind this exclusion is that these sites are known to be under positive selection [ , ] and are therefore less informative on evolutionary distance. additionally, by removing these sites, the amino acid distance remains independent of promiscuity predictions. finally, as intragenic recombination may distort the inference of evolutionary distance, we identified such events across all alleles following the protocol of satta and colleagues [ ] using gene-conv [ ] and rdp algorithms [ ] as implemented in the rdp software [ ] . recombinant alleles were removed when calculating amino acid distance. we first defined the epitope-binding region of hla-drb alleles, as previously [ ] . to estimate sequence diversity along the epitope-binding region, we employed two measures: standard shannon entropy [ ] and nucleotide diversity (π), a widely employed measure of genetic variation [ ] . using the protein sequence alignment of the alleles defined above, we calculated amino acid sequence variability as the shannon entropy of the given amino acid site as follows: where p i is the fraction of residues of amino acid type i at a given site, and m is the number of amino acid types observed at that site. nonsynonymous nucleotide diversity (π a ) measures the average number of nonsynonymous nucleotide differences per nonsynonymous site between two randomly chosen protein coding dna sequences from the same population [ , ] . π a was calculated for each amino acid site in the epitope-binding region for each population using dnasp software [ ] and custom-written r scripts. nucleotide sequences of drb alleles were downloaded from the ipd-imgt/hla database [ ] . the calculation is based on the work of nei and colleagues [ ] using the equation where x i and x j are the frequencies of the ith and jth alleles in the population, respectively, and p a ij is the number of nonsynonymous nucleotide differences per nonsynonymous nucleotide site between the two codon sequences of the given amino acid site in the ith and jth alleles. to calculate π a for each population, allele frequency data of human populations were obtained, as described earlier (see above). an overall nucleotide diversity index was calculated by averaging π a across populations. to calculate each amino acid site's impact on epitope-binding promiscuity (promiscuity fragility), promiscuity was predicted for each possible amino acid change along the epitopebinding region of each of alleles. the fold difference in promiscuity resulting from each amino acid substitution was calculated. the median promiscuity fold difference of each possible allele and amino acid change combination ( × ) was used to estimate promiscuity fragility at each amino acid position. as some of the possible amino acid changes are not accessible via a single nucleotide mutation, and the accessible amino acid changes can have different likelihoods based on the codon sequence of the site and the genetic code, we also calculated promiscuity fragility based on each nonsynonymous nucleotide substitution of the codon instead of each amino acid substitution of the site. all statistical analyses were carried out in r version . . [ ] . smooth curves were fitted using the cubic smoothing spline method [ ] . . we selected (i) epitope sequences from this dataset, for which binding affinity data to all the alleles were available, and (ii) , epitopes used for calculating in silico promiscuity throughout the paper, which were not used for the training of the netmhciipan algorithm. in vitro and predicted promiscuity of the alleles was determined at a nm binding threshold using the selected and , epitopes, respectively. we used standardized (i.e., z-score) allele promiscuity values for the comparisons. we report a strong correlation between the in silico and in vitro promiscuity . one might speculate that there might be no selection for elevated hla-b promiscuity level due to a dominant balancing selection on this locus (see s text) [ , ] . similarly, (e) the promiscuity level of hla-c molecules showed no significant correlation with intracellular pathogen diversity (spearman's rho: − . , p = . ). finally, (f) hla-c promiscuity level showed a marginally significant positive correlation with extracellular pathogen diversity (spearman's rho: . , p = . ). this is surprising, but this preliminary result needs to be considered with caution, and studied further in future works. for detailed explanation of these results, see s text. population groups were created using the th percentile genetic distance cutoff (see methods). for a list of populations assigned to each group, see s data. for results of multivariate models and obtained upon using alternative distance cutoff values, see s data. red curves indicate smooth curves fitted using cubic smoothing spline method in r (see methods). the underlying data for this figure can be found in s data. (tif) s table. list of populations and their mean hla-drb allele promiscuity. table. the relationship between promiscuity and pathogen diversity is independent of hla diversity and country size. table. results of hla association studies suggest a protective role of high allele promiscuity in infectious diseases. (docx) s (docx) s towards a systems understanding of mhc class i and mhc class ii antigen presentation the mhc, disease and selection how pathogens drive genetic diversity: mhc, mechanisms and misunderstandings the importance of immune gene variability (mhc) in evolutionary ecology and conservation from evolutionary genetics to human immunology: how selection shapes host defence genes adaptive value of novel mhc immune gene variants pathogen-driven selection and worldwide hla class i diversity distinct evolutionary strategies of human leucocyte antigen loci in pathogen-rich environments expression levels of mhc class i molecules are inversely correlated with promiscuity of peptide binding generalists and specialists: a new view of how mhc class i molecules fight infectious pathogens hla class i alleles are associated with peptide-binding repertoires of different size, affinity, and immunogenicity effects of thymic selection of the t-cell repertoire on hla class i-associated control of hiv infection peptide-binding motifs associated with mhc molecules common in chinese rhesus macaques are analogous to those of human hla supertypes and include hla-b -like alleles influences of major histocompatibility complex class i haplotypes on avian influenza virus disease traits in thai indigenous chickens the mhc of the chicken, genomic structure, gene products, and resistance to oncogenic dna and rna viruses. veterinary immunology and immunopathology association of the chicken mhc b haplotypes with resistance to avian coronavirus mhc gene control of growth of avian sarcoma virus-induced tumours in chickens: a study on the role of virus strain natural selection and infectious disease in human populations the ipd and imgt/hla database: allele variant databases current perspectives on the intensity of natural selection of mhc loci divergent allele advantage at human mhc genes: signatures of past and ongoing selection a human-specific allelic group of the mhc drb gene in primates understanding rare and common diseases in the context of human evolution intensity of natural selection at the major histocompatibility complex loci cell-surface mhc density profiling reveals instability of autoimmunity-associated hla nonsynonymous substitution rate heterogeneity in the peptide-binding region among different hla-drb lineages in humans. g (bethesda) diversifying and purifying selection in the peptide binding region of drb in mammals phenome-wide scanning identifies multiple diseases and disease severity phenotypes associated with hla variants hla-drb * : a significant risk factor for sarcoidosis in blacks and whites the mhc class i antigen presentation pathway: strategies for viral immune evasion major histocompatibility complex genomics and human disease excess of deleterious mutations around hla genes reveals evolutionary cost of balancing selection red queen processes drive positive selection on major histocompatibility complex (mhc) genes primary t cell expansion and differentiation in vivo requires antigen presentation by b cells association between an mhc class ii allele and clearance of hepatitis b virus in the gambia endogenous mhc class ii processing of a viral nuclear antigen after autophagy hla class ii sequence variants influence tuberculosis risk in populations of european ancestry class ii hla interactions modulate genetic risk for multiple sclerosis construction of a population-specific hla imputation reference panel and its application to graves' disease risk in japanese widespread non-additive and interaction effects within hla loci modulate the risk of autoimmune diseases polymorphisms of large effect explain the majority of the host genetic contribution to variation of hiv- virus load mhc class i peptides as chemosensory signals in the vomeronasal organ does intra-individual major histocompatibility complex diversity keep a golden mean high-throughput engineering and analysis of peptide binding to class ii mhc hla class ii transgenic mice as models of human diseases genetic variants within the mhc region are associated with immune responsiveness to childhood vaccinations indirect recognition of donor hla-dr peptides in organ allograft rejection mhc-i genotype restricts the oncogenic mutational landscape evolutionary pressure against mhc class ii binding cancer mutations uniprot: the universal protein knowledgebase fast, scalable generation of high-quality protein multiple sequence alignments using clustal omega performance comparison between k-tuple distance and four model-based distances in phylogenetic tree reconstruction netmhcpan- . : improved peptide-mhc class i interaction predictions integrating eluted ligand and peptide binding affinity data deriving phylogenetic trees from allele frequencies: a comparison of nine genetic distances genetic structure of human populations mega : molecular evolutionary genetics analysis version . for bigger datasets nucleotide substitution at major histocompatibility complex class ii loci: evidence for overdominant selection natural selection at major histocompatibility complex loci of vertebrates. annual review of genetics mhc class ii dqb diversity in the japanese black bear, ursus thibetanus japonicus statistical tests for detecting gene conversion rdp: detection of recombination amongst aligned sequences rdp : detection and analysis of recombination patterns in virus genomes bio d: an r package for the comparative analysis of protein structures mathematical model for studying genetic variation in terms of restriction endonucleases synonymous and nonsynonymous polymorphisms versus divergences in bacterial genomes dnasp v : a software for comprehensive analysis of dna polymorphism data r: a language for data analysis and graphics pathogen diversity drives the evolution of generalist mhc-ii alleles in human populations we wish to thank jim kaufman for the insightful comments we have received on an earlier version of the manuscript. we are also grateful to károly kovács for his suggestions on data analysis. key: cord- -bw tziup authors: perez-zsolt, daniel; martinez-picado, javier; izquierdo-useros, nuria title: when dendritic cells go viral: the role of siglec- in host defense and dissemination of enveloped viruses date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: bw tziup dendritic cells (dcs) are among the first cells that recognize incoming viruses at the mucosal portals of entry. initial interaction between dcs and viruses facilitates cell activation and migration to secondary lymphoid tissues, where these antigen presenting cells (apcs) prime specific adaptive immune responses. some viruses, however, have evolved strategies to subvert the migratory capacity of dcs as a way to disseminate infection systemically. here we focus on the role of siglec- , a sialic acid-binding type i lectin receptor potently upregulated by type i interferons on dcs, that acts as a double edge sword, containing viral replication through the induction of antiviral immunity, but also favoring viral spread within tissues. such is the case for distant enveloped viruses like human immunodeficiency virus (hiv)- or ebola virus (ebov), which incorporate sialic acid-containing gangliosides on their viral membrane and are effectively recognized by siglec- . here we review how siglec- is highly induced on the surface of human dcs upon viral infection, the way this impacts different antigen presentation pathways, and how enveloped viruses have evolved to exploit these apc functions as a potent dissemination strategy in different anatomical compartments. dendritic cells (dcs) are the most potent antigen presenting cells (apcs) found in humans [ , ] and their immune function is key to initiate immunity against invading viruses [ ] [ ] [ ] . these cellular sentinels patrol distinct mucosae and upon infection, viral sensing triggers rapid innate immune responses that might initially contain viral spread. dc activation also elicits cellular migration towards secondary lymphoid tissues, where dcs acquire a fully mature phenotype and become competent for antigen presentation, activation of naïve t cells, and expansion of antigen-specific adaptive t cell responses [ , ] . despite the immune activity exerted by dcs after viral infection, it has been known for decades that viruses evolved different strategies to escape dc antiviral activity [ ] [ ] [ ] . furthermore, certain viruses exploit the immune function of dcs as a way to colonize distant tissues and effectively disseminate systemically [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the discovery of the role of the receptor siglec- /cd , a sialic acid-binding ig-like lectin- expressed by dcs, has greatly contributed to our understanding of how viruses subvert dc activity. the siglec- receptor acts as an immuno-surveillance molecule [ ] but can also be effectively hijacked by distinct enveloped viruses, which either infect dcs directly or are effectively transferred to bystander target cells that become productively infected [ ] [ ] [ ] . hence, siglec- function on dcs clearly illustrates how these apcs can trigger antiviral immunity but also enhance viral spread via this receptor. siglec- is a type i transmembrane lectin with an amino-terminal v-set domain that interacts with sialylated ligands, preferentially n-acetylneuraminic acid (neu ac) in an α - linkage [ , ] . several enveloped viruses including human immunodeficiency virus (hiv)- [ ] [ ] [ ] and ebola virus (ebov) [ , [ ] [ ] [ ] incorporate such sialylated ligands within their membranes as an integral part of the gangliosides that are dragged from the plasma membrane when viruses bud from infected cells. although siglec- affinity for sialylated ligands is in the micromolar range, high-avidity binding can be achieved upon clustering of thousands of gangliosides in the viral membrane with their receptors on the cellular membrane [ , ] . moreover, as siglec- contains ig-like c -type extracellular domains that separate the ligand-binding site from the cell surface, it is available for interaction with external ligands and not bound in cis to cell-surface molecules, which is what usually happens with shorter siglecs that are also expressed by dcs [ , , ] . in vivo, the role of siglec- during viral infection has been mostly studied in murine models, focusing on resident tissue macrophages that express this lectin and play key immunomodulatory functions. siglec- -expressing macrophages are located in the subcapsular sinus of the lymph nodes, and they protect mice against vesicular stomatitis virus (vsv) infection by containing incoming viruses. viral sensing triggers cytokine release and promotes antigen presentation to b cells [ , ] . however, studies using different retroviruses to infect mice have shown that the protective function of these macrophages can be hijacked for efficient viral infection and dissemination within tissues. indeed, robust infection of a particular retrovirus in lymphoid tissues and spleen requires siglec- -expressing macrophages [ ] . the pathogenicity of the infecting retrovirus is key to tip the balance of these siglec- -expressing macrophages in favor of the protective immune function. the antiviral response dominates when the replicating virus has an expanded tropism [ ] , as it also happens in the case of the amphotropic vsv infection [ , ] . under these pathogenic conditions, viral capture via siglec- macrophages is necessary to elicit an effective antiviral cd + t cell response via antigen cross-presentation by dcs [ ] . overall, these murine studies explain how siglec- can contain viral replication and induce antiviral immunity against highly pathogenic viruses, but also favor viral spread within tissues when retroviruses have a limited tropism. yet, how these findings correlate with the pathogenesis of different siglec- -interacting human viruses, such as hiv- or ebov, remains largely unexplored. hiv- is the causative agent of acquired immunodeficiency syndrome (aids), a pandemic that has affected more than million people worldwide [ ] , while ebov is responsible for the intermittent outbreaks that produce a filovirus-associated disease (fvd) with high fatality rates [ ] . in this review, we discuss how siglec- is induced on human dcs upon viral infection, to what degree that impacts different viral antigen presentation routes, and in which ways distant enveloped viruses have evolved to exploit siglec- function as a dissemination strategy in distinct anatomical compartments. siglec- is a receptor codified by an interferon-stimulated gene and is therefore potently upregulated on distinct human dcs, monocytes, and macrophages when these cells sense type i interferons (ifns) such as ifnα [ ] [ ] [ ] . thus, infection with viruses such as hiv- or ebov tightly upregulates siglec- expression on apcs, as they directly trigger or indirectly promote the release of type i ifns via immune activating factors (figure ). hiv- induces secretion of type i interferons (ifns) by plasmacytoid dcs (pdcs) through toll-like receptor (tlr) - and - sensing, which upregulates siglec- on dcs in a paracrine manner. in addition, lipopolysaccharide (lps) from bacterial translocation upregulates siglec- on dcs via tlr sensing and autocrine type i ifn release. (b) during ebov infection, type i ifns might also play a central role in enhancing siglec- expression on dcs, although this needs further investigation. pdcs may produce type i ifns in response to ebov infection in vivo, while bacterial translocation was suspected during a case of gram-negative septicemia in an ebov-infected patient. in parallel, viral components such as secreted ebov glycoprotein may induce activation of myeloid cells through tlr signaling, providing an alternative stimulus of autocrine type i ifns during ebov infection. while solid arrows indicate established mechanisms, dotted arrows suggest processes that require further investigation. ifnar: ifnα/β receptor; sgp: secreted glycoprotein. in the case of hiv- infection, ifnα levels are potently boosted during acute infection, and sustained-although to a lower extent-throughout the chronic stage, which is characterized by a persistent immune activation [ ] [ ] [ ] . several dc types have been identified as the sources of ifnα production during the course of hiv- infection, and therefore contribute to siglec- induction. plasmacytoid dcs (pdcs) are considered the most potent type i ifn producers in blood [ ] , and their capacity to secrete ifnα in response to hiv- sensing has been demonstrated in vitro [ ] [ ] [ ] [ ] [ ] and in vivo [ ] [ ] [ ] , both during the acute and chronic phases of the disease [ , , ] . of note, pdc activation in response to hiv- sensing induces ifnα secretion through toll-like receptor (tlr) - and - signaling [ , ] and maturation of bystander myeloid dcs [ ] , and this ifnα response is more potent on pdcs derived from females [ ] . in turn, secretion of this cytokine can directly upregulate siglec- expression on dcs [ ] ( figure a ). in addition to pdcs, myeloid dcs also secrete type i ifns, although this release is mostly and indirectly triggered by immune activating signals present during the course of hiv- infection, which can induce the expression of ifn-stimulated genes on dcs in an autocrine manner [ ] [ ] [ ] [ ] . one of those factors is bacterial lipopolysaccharide (lps), which is increased in the plasma of hiv- -infected individuals due to the bacterial translocation that takes place in the gut-associated lymphoid tissue as a consequence of the gut epithelial barrier disruption occurring early after hiv- infection [ , , ] ( figure a ). lps induces siglec- expression on dcs [ ] . moreover, plasma from hiv- -infected individuals also stimulates siglec- expression on dcs signaling via type i ifn receptor [ ] . this explains why on circulating monocytes of hiv- infected individuals, siglec- expression correlates in vivo with the levels of plasma viremia, and why these levels only diminish after introduction of combined antiretroviral treatment [ ] . moreover, both types of siglec- -inducing factors are also present throughout the course of ebov infections ( figure b ). secretion of ifnα has been detected in humans and nonhuman primate models [ , ] , especially in lethal cases [ ] , while asymptomatic ebov infections are characterized by the absence of this cytokine [ ] . although in vitro pdcs exposed to ebov do not secrete ifnα [ ] , activated pdcs have been found in ebov-infected nonhuman primates, suggesting that these cells might produce ifnα in vivo [ ] ( figure b ). aside from pdcs, myeloid cells could contribute to ifnα secretion during ebov infection, as ebov-like particles induce ifnα production by murine bone marrow-derived dcs through tlr signaling [ ] . ebov glycoprotein interaction with human monocyte-derived macrophages induced tlr -dependent ifnα secretion by these cells [ ] . moreover, a cleaved and secreted form of ebov glycoprotein signals through tlr [ ] , although ifnα secretion in response to these glycoproteins remains unexplored ( figure b ). noteworthy, lps was also found in a case of ebov infection complicated with septicemia, possibly due to bacterial translocation [ ] , which might account for indirect ifnα secretion during ebov infection as described for hiv- ( figure b) . overall, the presence of type i ifns throughout the course of these viral infections is well-established, although both hiv- and ebov have evolved particular molecular mechanisms via viral antagonistic proteins that aid to evade cellular immune sensing [ ] [ ] [ ] [ ] . intriguingly, the protective role of type i ifn responses is controversial, since the apparent antiviral function during the earliest stages of infection may, in turn, fuel pathogenesis during the later stages of viral disease. that seems to be the case not only for hiv- [ ] , but also for ebov [ ] , where clinical data collected during human outbreaks have indicated that elevated levels of circulating ifnα, as well as upregulation of type i ifn-inducible genes, correlates with fatal disease outcome [ , [ ] [ ] [ ] . thus, hiv- and ebov infections trigger an immune activation state that upregulates siglec- expression on dcs, a situation that might favor early viral dissemination events in an otherwise antiviral environment [ ] . viral sensing enhances siglec- expression on apcs, and this facilitates hiv- infection of dcs and other myeloid cells (figure a , top), such as macrophages [ ] . although dcs are generally resistant to hiv- infection, a recent study showed that siglec- mediated hiv- productive infection of a population of human dc precursors known as pre-dcs [ ] . even though all dcs express the hiv- -interacting cellular receptor cd and the viral coreceptors [ , ] , being therefore susceptible to viral infection in vitro [ ] [ ] [ ] , infectivity was less prominent than in activated cd + t cells [ ] [ ] [ ] [ ] . importantly, the activation process that enhances siglec- expression on dcs further restricts viral infection, as mature dcs are -fold to -fold less susceptible to hiv- than immature dcs [ , , [ ] [ ] [ ] . infection of dcs with hiv- also appears to be uncommon in vivo, although it has been reported for both cutaneous and mucosal dcs, including vaginal epithelial dcs [ , , ]. yet, the role of siglec- in promoting hiv- infection of these dcs remains largely unexplored. in most dc subtypes, however, the restriction factor samhd inhibits reverse transcription, thus precluding immune sensing and the synthesis of viral antigens. bottom: conversely, hiv- encodes vpx, which counteracts samhd activity allowing reverse transcription of the viral genome, that can be sensed via cgas and trigger cytokine release. newly synthesized proteins lead to the production of viral particles, but also to proteosomal cleavage the de-phosphorylated form of the host factor sterile alpha motif histidine-aspartate domain-containing protein (samhd ) is the most potent restriction factor that limits hiv- infection in dcs [ ] (figure a, top) . however, if this lack of infectivity is actually beneficial for an immune control remains controversial, as the absence of viral replication impairs viral sensing and limits presentation of hiv- -specific antigens to prime adaptive immune responses [ ] [ ] [ ] . in contrast to hiv- , hiv- naturally replicates on dcs [ ] , which depends on the counteraction of samhd by the viral antagonist vpx [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (figure a, bottom) . hiv- genome replication in infected dcs is detected by the innate cytosolic dna sensor cyclic guanosine-adenosine monophosphate synthase (cgas), which triggers immune responses upon dna sensing [ , ] (figure a, bottom) . in contrast, hiv- restriction by samhd prevents viral dna (vdna) retrotranscription in the cytoplasm, impairing the induction of antiviral type i ifn responses [ ] (figure a, top) . thus, viral replication on dcs can provide an additional source of viral components to be detected via immune sensors or presented to t cells [ , ] (figure a, bottom) , but also compromise cell viability and release inflammatory factors that fuel viral pathogenesis, as previously described for sustained or chronic type i ifn responses. while hiv- replication on dcs remains hard to identify in vivo, it has been known for more than a decade that dcs are among the first target cells encountering ebov [ ] . dcs are highly susceptible to ebov infection [ , ] , and this is a complex process that involves several host factors whose function is still being identified [ ] . indeed, siglec- expressed on activated dcs has recently emerged as a new host factor implicated in ebov attachment, a mechanism that facilitates subsequent cytoplasmic viral entry [ ] ( figure b ). initial ebov attachment to the dc surface is mediated by several receptors that recognize different elements on the viral membrane and often have a redundant activity [ ] . c-type lectin receptors (clrs) such as the dendritic cell-specific intercellular adhesion molecule- -grabbing non-integrin (dc-sign) and the liver/lymph node sinusoidal endothelial c-type lectin (lsectin) mediate viral attachment through binding to viral glycoproteins [ , ] , while receptors of the tim/tam families (comprising the t cell immunoglobulin and mucin domain receptor along with tyro-axl-mer receptors) recognize phosphatidylserine lipids present on the viral envelope [ ] (figure b ). ebov incorporates sialylated gangliosides on their membrane [ ] , and we have recently shown that these viruses are effectively recognized by the siglec- receptor [ ] (figure b ). siglec- recognition of sialylated gangliosides on ebov modulates the binding, uptake, and trafficking of filoviral particles into a sac-like virus-containing compartment (vcc) continuous with the plasma membrane ( figure b ). viruses stored in this compartment can be redirected into the classical endosomal pathway and facilitate viral entry into the cytoplasm [ ] . indeed, ebov macropinocytosis allows trafficking into late endosomes, where cleavage of viral glycoproteins by cathepsin b (ctsb) facilitates the interaction with the endosomal receptor niemann-pick c [ ] [ ] [ ] (ncp ) that triggers cytoplasmic viral entry [ ] [ ] [ ] (figure b ). thus, siglec- -mediated attachment facilitates viral access to the cell cytoplasm [ ] . while siglec- contributes to ebov entry into dcs, filoviral replication within these cells compromises immune function and prevents adaptive immune responses by limiting cytokine secretion, downregulating the expression of major histocompatibility complex (mhc) and costimulatory molecules and also by reducing the ability of dcs to stimulate t cell proliferation [ , [ ] [ ] [ ] . these results suggest that ebov suppression of dc function prevents initiation of adaptive immune responses and facilitates uncontrolled systemic virus replication [ , ] through a mechanism that is enhanced by siglec- activity [ ] . however, clinical data gathered during the west african - outbreak showed strong and sustained t cell activation [ ] , challenging the in vivo relevance of this viral dc-escape mechanism [ ] . overall, both hiv- and ebov can exploit siglec- activity to boost dc infectivity, although ebov replication is more prominent in these cells. yet, viral infection poses a difficult balance for apcs. on the one hand, infectivity triggers antiviral immunity via viral sensing and antigen presentation, but on the other hand, it also promotes cell death and suppression of immune responses through the activity of particular viral antagonist proteins. recently, it has been suggested that this apparent paradox is overcome by a division of labor between distinct dc subsets [ ] . there is therefore a dissociation between viral infection and antigen presentation, which occurs in distinct dc subpopulations. by these means, susceptible infected dcs transfer viral antigens to resistant dcs, which remain competent to launch adaptive immune responses against viral infections [ ] . while several pathways allow for antigen transfer between dcs, secretion of extracellular vesicles bearing particular antigens is among the most effective ones. although the functional paradigm of dc biology states that the particular apc that interacts with incoming viruses in the mucosa would be the one processing these viruses and then traveling to the lymphoid tissue, these cells may not always be the only ones presenting the captured antigens. rather, these pathogen-interacting dcs may transfer captured antigens to other apcs by several mechanisms, including secretion of extracellular vesicles bearing antigen-loaded fragments, which can even be already processed and presented in mhc molecules (figure , top) . by these means, the number of dcs bearing viral-specific antigens can be increased very quickly upon infection, thus amplifying the initiation of primary adaptive immune responses [ ] [ ] [ ] . importantly, to induce naïve t cell stimulation in vitro, these extracellular vesicles require a competent activated dc to deliver the co-stimulatory signals to t cells [ ] (figure , top) . thus, antigen-containing extracellular vesicles do not overcome the need for a competent apc to activate naïve t cells. among the distinct cellular receptors expressed by dcs, siglec- is key to capture secreted extracellular vesicles through the same mechanism hijacked by enveloped viruses [ ] (figure ). siglec- interacts with extracellular vesicles via recognition of sialylated gangliosides packaged on the vesicle membrane [ ] , which assemble and bud from cellular membranes as viruses do [ , ] . this result has been confirmed not only in vitro [ , ] with extracellular vesicles derived from cell lines or primary cells but also in murine models where siglec- expressed on lymphoid tissues was required to trap extracellular vesicles in vivo [ ] . upon capture of extracellular vesicles on activated dcs via siglec- , these vesicles are trafficked along with the receptor towards a sac-like compartment invagination that is continuous with the plasma membrane and allows for extracellular vesicle retention [ , ] (figure , top) . the siglec- positive compartment formed within activated dcs may serve as an antigen depot, controlling and sustaining adaptive immunity even if the source of antigen is not directly in contact with the apc, that still can trigger antigen-specific immune responses. these antigens could maintain immunity for prolonged periods, as it happens when dcs control endosomal acidification to preserve antigen cross-presentation over time [ ] . although mature or activated dcs markedly downregulate their macropinocytic capacity, these cells are still able to capture, process, and present antigens internalized via endocytic receptors [ ] , and that may also be the case for siglec- via extracellular vesicle trapping. moreover, as dcs continue to capture and present antigens after maturation in vivo [ ] , they could also initiate responses to newly encountered antigens during the course of viral infections, a process that would be boosted by siglec- expression. overall, siglec- retention of distinct viruses on extracellular vesicle-containing compartments highlight how these cells might act as "trojan horses", capturing filoviruses or retroviruses in the peripheral mucosae and carrying them to secondary lymphoid tissues, where viruses can be effectively transmitted to target cells and contribute to the systemic spread of infection [ ] [ ] [ ] , ] . recently, we have identified the presence of siglec- -expressing cells in cervical mucosa. these dcs are capable of capturing hiv- and mediate viral transmission to target cd + cells via transinfection, even in a basal state where no apparent activation is detected [ ] (figure ) . indeed, dcs directly isolated from human ectocervix displayed a basal siglec- expression that was sufficient to mediate viral transfer. however, at the endocervix, where the expression of siglec- is lower than at the ectocervix, this capacity was enhanced upon ifnα stimulation (figure ) . hiv- is mostly acquired by sexual transmission, and in the cervical mucosa, there are two major sources of antiviral type-i ifn responses after retroviral infection: resident myeloid cells [ ] and pdcs, which are the most potent producers of ifnα [ ] and are soon recruited to the cervix [ ] ( figure ) . although increased antiviral ifnα secretion could limit initial viral infection, it could promote viral capture on cervical myeloid cells via siglec- induction as well. of note, in the cervical the fate of trapped extracellular vesicles on dcs is diverse, as they provide a source not only for antigen cross-presentation to cd + t cells, but also to stimulate antigen-specific naïve cd + t cell responses in vivo [ , ] . cd + t cell stimulation can take place either by reprocessing the antigens contained in the captured extracellular vesicles or by the direct presentation of previously processed functional epitope-mhc complexes exposed in the vesicle surface [ , ] . direct extracellular vesicle antigen presentation in the absence of lytic degradation within dcs was initially described using dc populations devoid of particular mhc-ii molecules, that were still able to activate cd + t cells because the necessary mhc-ii molecules were already presenting the antigen on the extracellular vesicles trapped by those dcs [ ] . thus, extracellular vesicles displaying previously processed functional epitope-mhc complexes on their surface can be recognized, retained, and directly transferred from dcs to antigen-specific cd + t cells [ ] (figure , top) . in turn, siglec- upregulation on activated dcs, which are competent apcs, could boost extracellular vesicle uptake and magnify antiviral immune responses. intriguingly, hiv- and other retroviruses exploit this antigen dissemination pathway usually engaged by extracellular vesicles to reach cd + t cells [ ] , which are the main cellular targets of this particular retrovirus (figure , bottom left) . siglec- directs captured hiv- particles to the same vcc where ebola viral particles are retained [ ] , that is in addition the same compartment where extracellular vesicles are trapped in activated dcs [ , ] (figure , bottom left) . however, in the case of hiv- recognition, viral entry via siglec- does not lead to the productive infection of dcs as it happens with ebov, but favors the transfer of trapped viruses to bystander cd + t cells. thus, trapped viruses are efficiently transmitted across infectious synapses to susceptible lymphocytes [ , , ] ( figure ). this mechanism of viral transmission is known as trans-infection [ ] and is mediated by siglec- on activated monocyte-derived dcs, monocytes, blood conventional dcs, pre-dcs, and primary myeloid cells isolated from lymphoid and cervical tissues [ ] [ ] [ ] , , ] . filoviral trans-infection from dcs to cd + t cells is improbable as lymphocytes are largely resistant to ebov infection [ ] . nonetheless, filoviruses display a broad cell tropism, infecting hepatocytes, adrenal cortical cells, and endothelial cells, among other cellular targets [ ] [ ] [ ] [ ] . thus, aside from lymphocytes, other cellular targets could be transinfected (figure , bottom right), as it was previously shown for a human cell line binding ebov that transinfected hela cells [ ] . however, further research will be required to determine in which anatomical context dcs trapping ebov via siglec- could transfer that infectivity to susceptible cellular targets in vivo. overall, siglec- retention of distinct viruses on extracellular vesicle-containing compartments highlight how these cells might act as "trojan horses", capturing filoviruses or retroviruses in the peripheral mucosae and carrying them to secondary lymphoid tissues, where viruses can be effectively transmitted to target cells and contribute to the systemic spread of infection [ ] [ ] [ ] , ] . recently, we have identified the presence of siglec- -expressing cells in cervical mucosa. these dcs are capable of capturing hiv- and mediate viral transmission to target cd + cells via trans-infection, even in a basal state where no apparent activation is detected [ ] (figure ) . indeed, dcs directly isolated from human ectocervix displayed a basal siglec- expression that was sufficient to mediate viral transfer. however, at the endocervix, where the expression of siglec- is lower than at the ectocervix, this capacity was enhanced upon ifnα stimulation ( figure ) . hiv- is mostly acquired by sexual transmission, and in the cervical mucosa, there are two major sources of antiviral type-i ifn responses after retroviral infection: resident myeloid cells [ ] and pdcs, which are the most potent producers of ifnα [ ] and are soon recruited to the cervix [ ] (figure ). although increased antiviral ifnα secretion could limit initial viral infection, it could promote viral capture on cervical myeloid cells via siglec- induction as well. of note, in the cervical biopsy of a viremic hiv- + patient, siglec- + cells harbored hiv- -containing compartments, demonstrating that in vivo, these cells can trap viruses [ ] . interestingly, similar vcc-like structures have been detected in urethral macrophages of hiv- -infected individuals under suppressive combination antiretroviral therapy [ ] , but if siglec- is implicated in the formation of these particular structures remains to be determined. siglec- allows transferring viruses to bystander cd + t cells in the mucosa, but also the systemic viral dissemination upon dc migration to lymphoid tissues ( figure ) . indeed, dcs bearing retroviruses are found in the draining lymph nodes of distinct animal models as soon as h after vaginal challenge [ , [ ] [ ] [ ] and these findings originally led to formulation of the trojan horse hypothesis, which states that dcs can serve as vehicles transporting the virus from the entry sites to distant tissues [ , ] . . proposed mechanism of hiv- dissemination from the female reproductive tract. in the vaginal/ectocervical mucosa, basal siglec- + dcs may mediate local hiv- trans-infection to target cd + t cells. at the endocervical mucosa, lower siglec- expression is boosted in response to ifnα released by recruited pdcs sensing hiv- . siglec- + dcs may also contribute to systemic hiv- spread due to their ability to migrate to secondary lymphoid tissues, where cd + t cells accumulate. while a similar mechanism could be exploited by ebov, further work needs to address this possibility. we hypothesize that the outcome of early interactions between dcs and enveloped viruses may be key to mount effective antiviral responses, but these early encounters also foster viral dissemination to distant tissues. the emerging roles of siglec- receptor on dcs clearly exemplify how the immune-mediated activity of this lectin can be effectively hijacked by unrelated viruses such as hiv- or ebov. future work should address if other enveloped viruses causing relevant human infectious diseases may contain sialylated gangliosides on their membranes and be recognized by siglec- , as it has been already shown for the henipavirus [ ] . moreover, the precise contribution of siglec- to viral immune containment or to pathogenic viral dissemination should be carefully evaluated, as understanding both mechanisms may provide novel avenues to combat infectious agents. the identification of individuals which naturally lack siglec- expression due to the presence of an early stop codon in the siglec gene in homozygosis [ ] indicates that the role of this protein is not essential and that it may be therefore a safe therapeutic target. developing such . proposed mechanism of hiv- dissemination from the female reproductive tract. in the vaginal/ectocervical mucosa, basal siglec- + dcs may mediate local hiv- trans-infection to target cd + t cells. at the endocervical mucosa, lower siglec- expression is boosted in response to ifnα released by recruited pdcs sensing hiv- . siglec- + dcs may also contribute to systemic hiv- spread due to their ability to migrate to secondary lymphoid tissues, where cd + t cells accumulate. while a similar mechanism could be exploited by ebov, further work needs to address this possibility. sexual transmission is not considered a major route of ebov infection. however, a case of sexually transmitted ebov has been well documented [ ] . moreover, a recent mathematical model is consistent with a significant contribution of sexual ebov transmission during the - outbreak in west africa [ ] . importantly, infectious viral particles are found in semen of ebov convalescent individuals several months following symptoms onset [ ] [ ] [ ] [ ] , and seminal fluid amyloids may enhance ebov infection [ ] . as the cytokine tgf-β is abundant in semen, and it also upregulates siglec- expression on dcs [ ] , the role of this receptor should be further assessed in the context of ebov sexual transmission. moreover, dcs are early and sustained targets of ebov that can disseminate infection from the portals of viral entry to the regional lymph nodes, spleen, and liver [ ] . since siglec- is expressed in all these ebov replicating-tissues [ , ] , this receptor could also boost systemic viral spread as previously suggested for hiv- [ , , , , ] . collectively, these data indicate that siglec- on dcs may not only participate in viral transmission at the mucosa, but also promote systemic viral dissemination to secondary lymphoid tissues. we hypothesize that the outcome of early interactions between dcs and enveloped viruses may be key to mount effective antiviral responses, but these early encounters also foster viral dissemination to distant tissues. the emerging roles of siglec- receptor on dcs clearly exemplify how the immune-mediated activity of this lectin can be effectively hijacked by unrelated viruses such as hiv- or ebov. future work should address if other enveloped viruses causing relevant human infectious diseases may contain sialylated gangliosides on their membranes and be recognized by siglec- , as it has been already shown for the henipavirus [ ] . moreover, the precise contribution of siglec- to viral immune containment or to pathogenic viral dissemination should be carefully evaluated, as understanding both mechanisms may provide novel avenues to combat infectious agents. the identification of individuals which naturally lack siglec- expression due to the presence of an early stop codon in the siglec gene in homozygosis [ ] indicates that the role of this protein is not essential and that it may be therefore a safe therapeutic target. developing such pharmacological agents to block siglec- interaction with viruses could pave the way to ameliorate viral systemic dissemination. moreover, exploiting the immune-surveillance function of siglec- receptor to induce antiviral immune responses may also prove valuable. in turn, dissecting how distinct viruses exploit common molecular pathways will advance future antiviral strategies to generate broad spectrum 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dendritic cell-mediated capture and transfer of hiv- and henipavirus identification of siglec- null individuals infected with hiv- this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -bp sray authors: hu, guangyu; han, xueyan; zhou, huixuan; liu, yuanli title: public perception on healthcare services: evidence from social media platforms in china date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: bp sray social media has been used as data resource in a growing number of health-related research. the objectives of this study were to identify content volume and sentiment polarity of social media records relevant to healthcare services in china. a list of the key words of healthcare services were used to extract data from wechat and qzone, between june and september . the data were put into a corpus, where content analyses were performed using tencent natural language processing (nlp). the final corpus contained approximately million records. records on patient safety were the most frequently mentioned topic (approximately . million, . % of the corpus), with the contents on humanistic care having received the least social media references ( . million, . %). sentiment analyses showed . %, . %, and . % of positive, neutral, and negative emotions, respectively. the doctor-patient relationship category had the highest proportion of negative contents ( . %), followed by service efficiency ( . %), and nursing service ( . %). neutral disposition was found to be the highest ( . %) in the contents on appointment-booking services. this study added evidence to the magnitude and direction of public perceptions on healthcare services in china’s hospital and pointed to the possibility of monitoring healthcare service improvement, using readily available data in social media. investigating public perception of healthcare services from different perspectives may generate inconsistent results. for example, patient-initiated violence against health workers [ ] [ ] [ ] [ ] [ ] [ ] , and the tension between doctors and patients for their dissatisfaction with the quality of healthcare [ , ] , were wildly covered in the chinese media. while patient experience surveys on the national level showed that patients were generally satisfied with both in-patient and out-patient services [ ] . such differences may result from biases rooted in the survey and media coverage; however, the inconsistency also pointed to the need for additional data sources to monitor public opinions on chinese healthcare services. it has been suggested that social media might be such a data source. rozenblum et al. pointed out that when patient-centered healthcare, the internet, and social media were combined, the current relationship between healthcare providers and consumers might face major changes-thus creating a "perfect storm" [ ] . users' posts on the social media platforms would generate a large volume of real-time data regarding public or private issues, among which healthcare related information scatters. therefore, the utilization of social media data for healthcare research becomes a dramatically growing field and already covered various medical and healthcare research fields [ , ] . sinnenberg and colleagues proposed four ways in which social media data were used in healthcare studies: ( ) content analysis, ( ) volume surveillance of contents on specific topics, ( ) engagement of users with others, and ( ) network analysis of users [ ] . for the content analysis, most studies focused on measuring public discussion on specific diseases [ ] [ ] [ ] , sentiment analysis for medical interventions (e.g., cancer screening) [ , ] , identifying safety concerns among health consumers [ ] , detecting adverse events of health products [ , ] . several researchers studied patient experience, based on the comments posted by patients from online health communities in china [ , ] , but few studies have been conducted to gather information on healthcare services related topics using social media data. meanwhile, although sentiment analysis has been wildly applied to process user sentiments associated with health-related text [ ] , the lexical resource and tools designed for doing health-related sentiment analysis in chinese language are few and far between. fast-advancing in technology and economy, social media users and their activities spiked in china, which made social media a promising source for healthcare service monitoring. in china, the internet penetration rate reached . % at the end of [ ] , with local providers dominating the market, rather than facebook and twitter, which are not accessible in china. chinese social media sites have a unique landscape, and it may not only be used as a communication software but also as an entry point for information. as subsidiaries of tencent holdings limited, shenzhen, china, wechat and qzone are two leading social media and networking services platforms. each of them reached more than million and million monthly active user accounts in the first quarter of [ ] . according to the wechat data report, typical users of wechat were born in the s or s [ ], representing a wide breadth of demographic group in china. besides providing multimedia communication and supporting social networking, wechat also has "official accounts", which serve as channels for publishing articles to the public. any individual or organization can apply for having their own official account to broadcast their ideas and believes. as for qzone, it is a platform bundled with qq, a popular online messaging application in china. qzone allows users to create their own personal page to write blogs and post updates. and users could be able to express their individual opinions and attitudes freely and instantly on the social media platforms. subject to the platforms' terms of service and privacy policy agreed upon by users [ , ] , three kinds of information were collected, stored, and used by the platforms: ( ) personal information; ( ) non-personal information; and ( ) shared information. the shared information refers to information that is voluntarily shared on the platforms by users freely and instantly, thus providing a valuable perspective and opportunity to gather public opinions on healthcare services. as such, we selected wechat and qzone as the social media platforms to conduct this exploratory study. the objectives of this study are to conduct volume and sentiment analyses base on the extracted social media contents on hospital healthcare services. the study could demonstrate the social media users' perceptions of hospitals healthcare and may shed light on the further utilization of social media as a data source for healthcare research in china. this study consisted of three phases. firstly, we utilized a predefined list of healthcare services categories to devise key words and search strategies accordingly. the data searching strategy would then be used to extract contents from a raw database, which contained publicized posts of wechat and qzone. the extracted materials were then put into a corpus. secondly, we applied natural language processing (nlp) techniques from tencent nlp platform to the corpus and calculated the volume of content concerning different healthcare services topics. thirdly, we conducted sentiment analysis to explore the sentiment polarity of chinese social media users on different healthcare service topics. the detailed process of data collection and analysis is presented in figure the raw databases used for this study come from wechat and qzone of the version only operated in mainland china. the user volumes and data inclusion criteria of the platforms were showed in table . publicly available posted information such as: posted blogs, reviews and articles that are voluntarily shared by individual users from june to september were collected from the two platforms. the data collection followed the privacy policy for users of tencent and was subject to the confidentiality and security measures that implemented by the platforms. and the data analyses were supported by technicians in the tencent. the nine healthcare service categories, used in this study, were derived from the objectives of the national healthcare service improvement initiative ( - ), which was dedicated to improving patient-centered healthcare and patient experience nationwide by the former national health and family planning commission of p.r. china (nhfpc) [ ] . the initiative operated under the leadership of the bureau of medical administration of nhfpc [ ] , which suggested that we used nine predefined categories to reflect the healthcare services in hospital (see table ). table . healthcare services categories and corresponding descriptions in the national healthcare service improvement initiative - (nhsii). objectives of nhsii optimize the layout of the facility and build a friendly service environment appointment-booking service promote utilization of clinical appointment services and guide patient flow service efficiency improve service efficiency and effectiveness by rational allocation of resources information technology take advantage of information technology to improve patient experience inpatient service promote inpatient service process reengineering and provide integrated healthcare service nursing service continuously improve quality of nursing care and enhance nursing workforce patient safety ensure patient safety by promoting adoption of standard operating procedures humanistic care strengthen humanistic care and provide medical social worker service doctor-patient relationship harmonize the doctor-patient relationship and reduce medical disputes in this study, we constructed a healthcare services corpus, in the chinese language from the social media data source, to enable further analyses. first, we constructed lexica of keywords and terms in accordance with the predefined service topics. for example, the lexicon for "information technology", used in this study indicate new information dissemination channels, based on information technology provided by hospital to improve patient experience of service information acquisition. and this lexicon contains six information technology service-related terms, namely, "weibo", "wechat", "website", as well as "self-service machine". second, we developed a set of searching strategy to extract the relevant data from the two sources based on the corresponding lexicon of topics. the entire list of search terms for each category and its corresponding searching strategy were provided in supplementary table s . finally, we applied the search strategies to the database of publicly posted materials to screen for posts related to the healthcare service categories to construct the corpus. the search and screening process were performed by qcloud. based on the healthcare services corpus, we classified the content to different healthcare services topics that predefined and measured the content volume of the topic. specifically, we used the open application programming interface (openapi) services provided by tencent nlp to analyze the retrieved contents. it is an open platform for chinese natural language processing (based on parallel computing and distributed crawling system) [ ] . such services enable us to split reviews and blogs into sentences, and each sentence was filtered to classify whether it contained target service topic keywords and terms. if the sentences, containing certain keywords and terms, belonged to the corresponding topic of healthcare services categories as listed in table s , then they would be divided into a certain category. by counting the appearances of each service topic keywords in terms of the number of sentences in the corpus, we can aggregate the counts at the topic level and calculate the proportion of different topics from the social media corpus. for the sentiment analysis tool in chinese, we also select tencent nlp, as its algorithm was trained by hundreds of billions of entries of internet corpus data in chinese and with successful application in other tencent products (https://nlp.qq.com). openapi with function of chinese batch texts automatic summarization and sentiment analysis of tencent nlp enable us to categorize the sentences on certain topic in the social media corpus into a sentiment polarity classification (i.e., neutral, positive, and negative). finally, each sentence was tagged and classified into different sentiment polarity. the social media corpus contained approximately million records from wechat and qzone, spanning the pre-defined categories, related to hospital healthcare services. table presents the content volume of each healthcare services topic by social media channel. among the social media content on healthcare services topics, patient safety was the most commonly encountered topic, both in wechat and qzone. the majority of the content related to patient safety issue, its approximately . million records and covered . % of the entire corpus. the proportion of contents related to other topics varied in the corpus: information technology ( . %), service efficiency ( . %), service environment ( . %), inpatient service ( . %), appointment-booking service ( . %), nursing service ( . %), doctor-patient relationship ( . %), and humanistic care ( . %). the results of the sentiment analysis of contents from the corpus found that, in all nine healthcare services topics, . % of the contents in the corpus have been recognized to reveal a positive disposition, . % neutral and . % negative. we found that topic comprising most positive contents was service environment ( . %), followed by patient safety ( . %). with regard to the topics that contained more negative contents than positive, the most one was doctor-patient relationship ( . %), followed by service efficiency ( . %), and nursing service ( . %). notably, over one third of contents in the appointment-booking service ( . %) revealed a neutral disposition. additionally, in contrast to the content volume distribution for the nine topics, the sentiment disposition of contents in corresponding healthcare services topics shows differences. for instance, table shows that the nursing service and doctor-patient relationship share an equal proportion ( . %) of contents in the corpus, however, we observed the disposition of contents from social media users to the two topics varied in figure . to our knowledge, this is the first study that has attempted to explore the public perceptions of healthcare services, using publicly posted materials, of two chinese social media platforms. our results showed that patient safety was the most significant topic for users of chinese social media platforms, followed by information technology and service efficiency. service environment was found to have the highest proportion of positive comments. the research assessed the application of content volume calculation and sentiment analyses on chinese social media data. the study is a crucial step to discovering the methodology on harnessing the social media data in china and an early attempt to track the perceptions of healthcare services in the public by analyzing a unique data source. this study found a large number of information technology and service efficiency, which might reflect the series of efforts made by both the government and the hospital in integrating information technology in healthcare services in china. several researchers have identified that health information technology services were used to enhance patient experience [ ] [ ] [ ] , and as a potential solution to shorten the lengthy waiting time in china's public hospital [ , [ ] [ ] [ ] . humanistic care was the least mentioned topic in the corpus complied by this study. it may suggest that chinese social media users are not very familiar with the idea of humanistic care. those who posted about it basically expressed a positive attitude. an alternative explanation might be this type of care has yet to reach the public only experienced by a few people. further empirical studies or controlled studies may be conducted to provide further insights. our research also explored the sentiment disposition of social media content on healthcare services: . % provided negative feedback. although this was only the initial results, it could be quite alarming to healthcare administrations and policymakers. despite the fact that patient surveys generally had favorable results in china [ ] , there was still a significant amount of negative comments to our knowledge, this is the first study that has attempted to explore the public perceptions of healthcare services, using publicly posted materials, of two chinese social media platforms. our results showed that patient safety was the most significant topic for users of chinese social media platforms, followed by information technology and service efficiency. service environment was found to have the highest proportion of positive comments. the research assessed the application of content volume calculation and sentiment analyses on chinese social media data. the study is a crucial step to discovering the methodology on harnessing the social media data in china and an early attempt to track the perceptions of healthcare services in the public by analyzing a unique data source. this study found a large number of information technology and service efficiency, which might reflect the series of efforts made by both the government and the hospital in integrating information technology in healthcare services in china. several researchers have identified that health information technology services were used to enhance patient experience [ ] [ ] [ ] , and as a potential solution to shorten the lengthy waiting time in china's public hospital [ , [ ] [ ] [ ] . humanistic care was the least mentioned topic in the corpus complied by this study. it may suggest that chinese social media users are not very familiar with the idea of humanistic care. those who posted about it basically expressed a positive attitude. an alternative explanation might be this type of care has yet to reach the public only experienced by a few people. further empirical studies or controlled studies may be conducted to provide further insights. our research also explored the sentiment disposition of social media content on healthcare services: . % provided negative feedback. although this was only the initial results, it could be quite alarming to healthcare administrations and policymakers. despite the fact that patient surveys generally had favorable results in china [ ] , there was still a significant amount of negative comments on the social media platforms. further and more detailed methodology is necessary to further understand the negative comments. in the topics investigated in this study, we found huge variations in the negative feedback as well as content volumes across topics. for instance, the contents related to doctor-patient relationship only take percentage of . % in the corpus, however . % of the content revealed negative feedback. the varied sentiment polarity distribution of the topics may have important policy implications for healthcare reform in china. for example, . % of the social media references to appointment-booking service reflected neutral feedback, which may suggest that the unsureness of the public on this novel service. patients have yet to be familiar with the services-even though it certainly aims to improve the convenience for patients as well as hospital efficiency. such feedback could be essential for hospitals to improve their service quality by enhancing patient education. further research might focus on what exactly were discussed in those negative posts so that targeted measures can be employed by the hospitals and responsible administrators to improve the services. in line with previous evidence [ , ] , our results show that social media could be a useful tool for health research in china, as well as english, and could be used to capture the public's perspective of healthcare [ , ] . however, it appeared that the most concerned issue of healthcare in social media is different from what has been found in patient surveys. findings from a recent qualitative study found that patients cared about the environment and facilities in hospital the most [ ] , whereas in our study patient safety issues had the greatest volume. another research examined the online doctor reviews in china revealed that most posts expressed positive attitudes towards the physicians [ ] . although the evidence on these issues are still not conclusive, it might suggest the perception difference between general public and patients. our research extends application of the natural language processing techniques to analysis of healthcare services related contents in china's social medial platforms and offers a new perspective of healthcare services in china's hospital. the results would be of benefit to healthcare providers and regulators benchmarking their performance on patient-centered healthcare delivery. this is important because the social media has been considered as a portal of health information acquisition for chinese netizens [ ] , the perspective of social media would be supplementary in understanding how consumer views the healthcare services in hospital besides the results from traditional paper-based surveys. this study has the following limitations. first, because the raw material was user-generated data, selection bias may have affected the data. for example, it was observed that most social media users were born in the s or s [ ], however we were unable to characterize the users social-demographic information in detail, since the user privacy policy of tencent currently prohibit such practice. moreover, considering the exploratory nature of the study, our study focused only on wechat and qzone as data sources, whereas other social media platforms in china may have the potential to conduct such analyses as well. second, since we derived the healthcare services categories and lexica based on the government document on nhsii and expert consultation, thus the corpus in this study may have failed to include certain amount of healthcare services related data. as a result, we may have underestimated the content volume of healthcare services from the two social media platforms. furthermore, although all the material in the databases are in chinese, and therefore most likely be generated by users from china, we are currently not able to determine whether the posts, containing the key terms on healthcare, were describing the chinese healthcare system or discussing foreign healthcare systems in chinese language. further research may strive to develop searching strategies that enable such distinction and increase the specificity of the results. third, although the consumer health vocabulary (chv) is the gold standard reference for retrieving the target data, it has been used in previous researches [ , ] , such open source of vocabulary list and its corresponding lexica are not available in chinese language. the accuracy and credibility of the sentiment analysis of this study also await further validation; however, it would require an alternative method to conduct sentiment analyses for chinese language and the possibility to apply such methods on the tencent data, which were publicly posted material but still under strict terms of utilization. another limitation concerned that we have no ability to confirm that the data supplied by tencent completely represent all users' data as there could be undocumented keyword filter on the platforms. these would inflict potential bias and limit the generalizability of our findings. weibo is another popular chinese social media platform considered to be the counterpart of twitter in china. future research could consider extend the analysis process to contents from weibo, to further explore users, and their views, that have not been covered in this study. both the quantitative approach, as shown in our research, and the qualitative approach, such as the face-to-face individual interview method, would be useful to better understand consumer care in healthcare services. there is a scarcity of empirical research exploring the latter issue at present. it has been proposed to complement public perspectives on healthcare services [ ] . furthermore, the popularity of consumers' unsolicited comments on healthcare providers in social media, prompts an important avenue for understanding patient experience, and has been demonstrated by previous researches [ , [ ] [ ] [ ] [ ] . future research for measuring patient experience based on social media data at hospital level would be help to better understand the landscape of healthcare quality in china. by analyzing shared information from wechat and qzone, this study showed that patient safety was the most concerned topic for users of chinese social media platform, followed by information technology and service efficiency, while the doctor-patient relationship was found to have the highest proportion of negative comments. this study explored the possibility of utilizing social media to monitor public perceptions on healthcare services. the findings provide an overview of public opinion on healthcare services, which could help regulators to set up the benchmark, on a national or regional level, to monitor the progress of healthcare improvements between comparator districts and services domains. it is also a necessary complement to the traditional paper-based consumer survey. the potential differences between social media perception and traditional consumer survey results would help regulators better understand the gap in quality of care services from various perspectives. further studies could also focus on extending the nlp method to a more content-based resource and to expand our understanding of mass opinion on healthcare services. the following are available online at http://www.mdpi.com/ - / / / /s , table s : list of keywords, terms, and text strings used for data searching. facing up to the threat in china violence against chinese health-care workers the danger of being a doctor in china the frequency of patient-initiated violence and its psychological impact on physicians in china: a cross-sectional study how to decrease violence against doctors in china? workplace violence against medical staff of chinese children's hospitals: a cross-sectional study media contribution to violence against health workers in china: a content analysis study of online media reports changing of china's health policy and doctor-patient relationship consumer satisfaction with tertiary healthcare in china: findings from the china national patient survey patient-centred healthcare, social media and the internet: the perfect storm? instagram and whatsapp in health and healthcare: an overview twitter as a tool for health research: a systematic review using twitter to measure public discussion of diseases: a case study social big data analysis of information spread and perceived infection risk during the middle east respiratory syndrome outbreak in south korea characterizing depression issues on sina weibo sentiment analysis of breast cancer screening in the united states using twitter using social media to characterize public sentiment toward medical interventions commonly used for cancer screening: an observational study social media in health-what are the safety concerns for health consumers? utility of social media and crowd-sourced data for pharmacovigilance: a scoping review protocol systematic review of surveillance by social media platforms for illicit drug use the development of online doctor reviews in china: an analysis of the largest online doctor review website in china unhappy patients are not alike: content analysis of the negative comments from china's good doctor website capturing the patient's perspective: a review of advances in natural language processing of health-related text china internet network information center the st china statistical report on internet development national health and family planning commission announcement of implementing the healthcare services improvement initiative national health and family planning commission the implementation strategy of the healthcare services improvement initiative social media landscape of the tertiary referral hospitals in china: observational descriptive study a way to understand inpatients based on the electronic medical records in the big data environment what predicts patients' adoption intention toward mhealth services in china: empirical study patient experience with outpatient encounters at public hospitals in shanghai: examining different aspects of physician services and implications of overcrowding discrete event simulation models for ct examination queuing in west china hospital questionnaire survey about use of an online appointment booking system in one large tertiary public hospital outpatient service center in china collecting and analyzing patient experiences of health care from social media what do patients care most about in china's public hospitals? interviews with patients in jiangsu province how the public uses social media wechat to obtain health information in china: a survey study predicting hcahps scores from hospitals' social media pages: a sentiment analysis web-based textual analysis of free-text patient experience comments from a survey in primary care use of sentiment analysis for capturing patient experience from free-text comments posted online harnessing the cloud of patient experience: using social media to detect poor quality healthcare we would like to acknowledge dalu wang and hongda wu from tencent for their technical support in data analysis. the authors declare no conflict of interest. int. j. environ. res. public health , , key: cord- -nuep nim authors: dewald, lisa evans; johnson, joshua c.; gerhardt, dawn m.; torzewski, lisa m.; postnikova, elena; honko, anna n.; janosko, krisztina; huzella, louis; dowling, william e.; eakin, ann e.; osborn, blaire l.; gahagen, janet; tang, liang; green, carol e.; mirsalis, jon c.; holbrook, michael r.; jahrling, peter b.; dyall, julie; hensley, lisa e. title: in vivo activity of amodiaquine against ebola virus infection date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: nuep nim during the ebola virus disease (evd) epidemic in western africa ( ‒ ), antimalarial treatment was administered to evd patients due to the high coexisting malaria burden in accordance with world health organization guidelines. in an ebola treatment center in liberia, evd patients receiving the combination antimalarial artesunate-amodiaquine had a lower risk of death compared to those treated with artemether-lumefantrine. as artemether and artesunate are derivatives of artemisinin, the beneficial anti-ebola virus (ebov) effect observed could possibly be attributed to the change from lumefantrine to amodiaquine. amodiaquine is a widely used antimalarial in the countries that experience outbreaks of evd and, therefore, holds promise as an approved drug that could be repurposed for treating ebov infections. we investigated the potential anti-ebov effect of amodiaquine in a well-characterized nonhuman primate model of evd. using a similar -day antimalarial dosing strategy as for human patients, plasma concentrations of amodiaquine in healthy animals were similar to those found in humans. however, the treatment regimen did not result in a survival benefit or decrease of disease signs in ebov-infected animals. while amodiaquine on its own failed to demonstrate efficacy, we cannot exclude potential therapeutic value of amodiaquine when used in combination with artesunate or another antiviral. antiviral activity of amodiaquine and artesunate and their metabolites in cell culture. the antimalarial treatment asaq that was administered to evd patients in , is a coformulation of artesunate (as) and amodiaquine (aq). as and aq are rapidly metabolized in the liver to dihydroartemisinin (dha) and desethylamodiaquine (deaq), respectively. prior to in vivo evaluation, the drug asaq, its components, and the metabolites were characterized for their inhibitory effects on ebov (makona variant, ebov/mak) replication in cell culture ( table ). the data confirm previous reports that both aq and the metabolite deaq block ebov replication with similar activity (ic = . to . µm) in huh and vero e cell lines (ic = . to µm). in primary human macrophages, both aq and deaq exhibited elevated cytotoxicity, which precluded any interpretation of antiviral activity. in contrast, activity of as and its metabolite dha was weak or undetectable. combinatorial testing of aq and as or the metabolites, deaq and dha, did not reveal in vitro synergistic effects against ebov replication (data not shown). based on the in vitro data, the decision was made to evaluate aq in the nhp model of ebov infection. nhps with an aq dosing regimen similar to that used for evd patients in ebola treatment centers in . treatment consisted of a -day course of as-aq with the dose determined according to age of the patient. the dose range for aq in humans is . to mg/kg corresponding to a rhesus macaque equivalent dose range of . to . mg/kg based on body surface area . a pharmacokinetic (pk) study in rhesus macaques ( groups of males and females) was performed to monitor plasma concentrations of aq (fig. a ) and the active metabolite deaq (fig. b) . the study evaluated three daily oral doses of aq, using mg/kg or mg/kg. pharmacokinetics were determined by collecting blood samples at , . , , , , , , , , and hours (h) following dosing on day and day ( fig. ) . after days of dosing with mg/kg in healthy male and female macaques, aq was well-tolerated. the higher dose of mg/kg resulted in a variety of clinical observations including hypoactivity, shivering (muscle tremors) and/or diarrhea. extreme hypoactivity was observed in a single male of the mg/kg group that resolved after dosing ceased. animals in both the mg/kg and mg/kg groups had lower blood pressure readings than at predose on both dosing days, typically at and/or h post-dosing. clinical chemistry and hematology parameters for both doses were analyzed and fell within normal ranges with the exception of liver enzymes. alanine aminotransferase (alt) and aspartate aminotransferase (ast) were elevated . -to . -fold on day at h after the rd dose. the increases in alt and ast were seen in both male and female animals at both the and mg/kg dose regimens, though changes were not dose-related. these increases were considered toxicologically significant. aq and the metabolite deaq reached peak concentrations at . to h (t max ) after administration for both day and day dosing (supplementary tables and ). the elimination phase half-life (t / ) varied among the individual animals, ranging from to h for aq and to h for deaq. plasma concentrations of the metabolite deaq were higher than concentrations of the parent drug resulting in auc last , and auc inf values that were . -to -fold higher for deaq than for aq. the mg/kg aq dose resulted in c max for aq ( . - . ng/ml) and deaq ( - ng/ml) that were in a similar range as reported for the mg/kg dose in humans (aq: c max = . ± . ng/ml and deaq: c max = . ± . ng/ml) . based on the results of the pharmacokinetics study, the mg/kg aq dose was chosen for a study to evaluate the in vivo effect of aq in the nhp model of evd (study outline, supplementary fig. ). rhesus macaques were challenged intramuscularly (im) with a target dose of plaque-forming units (pfu) of ebov/mak (measured dose of pfu) on study day . the control group (n = , female, males) received vehicle treatment on days , , , , and postexposure. treatment group (n = , females, males) received a -day course of mg/kg of aq by oral administration on days , and after exposure to ebov. treatment group (n = , females, males) received a -day course of mg/kg of aq by oral administration on days , , and after exposure to ebov. all animals were euthanized or succumbed to disease by day postexposure (fig. a) . median times to disposition were days for placebo and treatment group , and days for treatment group with no significant difference between the placebo or treatment groups versus the treatment group (p = . and . , respectively). the animals in all groups became febrile by day or postexposure, followed by a marked decrease in body temperature preceding death (fig. b) . no significant difference in febrile illness or weight loss was observed between control and treatment groups (fig. b,c) . nhps had a mild reduction in activity and responsiveness by www.nature.com/scientificreports www.nature.com/scientificreports/ day postexposure with a severe decrease observed for most animals on day postexposure ( table ). loss of appetite and lymphadenopathy began on day to followed by dehydration and rash (petechial, maculopapular) on day postexposure. clinical signs did not differ significantly between the groups. viral loads in plasma were not reduced in treated animals. plasma viremia was quantified by rt-qpcr, and viral titer was determined by plaque assay. viral rna was first detected in the serum of some animals days after exposure, with a rapid and substantial increase by day for both placebo-and aq-treated animals. rna levels at necropsy were in the range of - viral rna copies/ml for all nhps (fig. a) . infectious viral titers followed a similar trend for all groups with a sharp increase in viral titer starting on day postexposure and reaching a peak plateau by day before animal death (fig. b ). viremia correlated with the onset of clinical signs of disease such as loss of appetite, lymphadenopathy, and fever. aq treatment did not reduce plasma viral rna copies or infectious virus titer regardless of when treatment was initiated. no significant difference in viral titers was observed between groups. amodiaquine-treated animals present with abnormal hematology and biochemical profiles and pathological disease that corresponds with ebola virus disease. the animals showed a similar hematological profile regardless of treatment. complete blood counts and serum chemistry analysis were performed at scheduled sampling points, before (days - , - , and ) and after (days , , , and at necropsy) exposure (figs. , ) . the chemistry values for aq-treated nhps followed the same trends as the control animal values. similar hematological and biochemical profiles were observed for all animals regardless of treatment both in aq-and placebo-treated animals. concurrent neutrophilia and thrombocytopenia were observed for aqand placebo-treated animals on day postexposure with ebov (fig. ) . as animals reached endpoint criteria, neutrophil levels decreased (fig. a) . the low-level neutrophil values on day postexposure were from necropsy samples only. nhps from all treatment groups exhibited a concomitant increase in serum creatinine and blood urea nitrogen levels, indicative of renal dysfunction often noted in nhps with concurrent evd (fig. a,d) . other biochemical abnormalities consistent with evd were also observed beginning on day postexposure, including substantial elevation of serum alt, ast, and gamma-glutamyl transpeptidase (ggt) levels that indicate hepatocellular damage (fig. b ,e,f). necropsies were performed on all animals to evaluate pathological disease associated with ebov infection following aq or placebo treatment. the pathologist was blinded to the group identification during necropsy and tissue evaluation. the pathological findings were similar in type and severity between treated and untreated animals, and the gross and histopathologic findings in all animals were consistent with typical evd in rhesus macaques. common gross findings included a cutaneous rash on the face, often extending to other regions of the body, dehydration, discoloration of the liver with increased friability, turgid spleen, and enlarged, often congested kidneys. at the microscopic level, all animals exhibited lymphoid depletion of the axillary lymph nodes, lymphoid depletion and necrosis of the spleen, and hepatocellular degeneration and necrosis. panniculitis and necrosis were observed at the virus challenge site of animals. samples from infected animals collected on days , , , and postexposure and on day of necropsy (days , or ) were analyzed for determination of plasma levels of aq and its metabolite deaq. data indicate negligible levels of aq in all but five plasma samples, each from a different animal and day (fig. a) . as the samples were diluted by a factor of . for decontamination protocol, the concentrations were likely below the limit of detection. plasma levels of the metabolite deaq were higher than aq and detectable in all treated animals from both nhp nhp nhp nhp nhp table . clinical scores for responsiveness of animals. responsiveness of unanesthetized animals was scored using the following criteria: (white) = alert, responsive, normal activity, free of disease signs or exhibits only resolved/resolving disease signs; = slightly diminished general activity, subdued but responds normally to external stimuli; = withdrawn, may have head down, fetal or hunched posture, or reduced response to external stimuli; = recumbent but able to rise if stimulated, or moderate to dramatically reduced response to external stimuli; = persistently recumbent, severely or completely unresponsive, or may have signs of respiratory distress. www.nature.com/scientificreports www.nature.com/scientificreports/ groups and (fig. b) . deaq was evident through day postexposure and/or necropsy in of animals in group and in all animals in group . plasma levels of deaq in ebov-infected nhps were compared at different time points (fig. ) . animals that were treated on days , and (group , fig. a ), had plasma deaq levels ranging from to ng/ml on days , , and postexposure. the highest deaq levels were detected in necropsy samples on day ( and ng/ ml). for animals treated on days , and postexposure (group , fig. b ), plasma concentrations ranged from - ng/ml on day . higher concentrations were detected on day ( - ng/ml) and day postexposure ( - ng/ml). plasma levels of deaq in infected nhps and healthy nhps were compared at select time points that were identical in the pk and the efficacy study. plasma samples from infected group animals on day correspond to pk-plasma samples taken at h after the rd dose. healthy animals had higher plasma levels of deaq ( - ng/ml) than infected animals ( - ng/ml) (fig. a) . similarly, plasma samples on day from infected group animals were in a lower range (n = , - ng/ml) than those of healthy animals at the corresponding time point ( h after rd dose, n = , - ng/ml) (fig. b) . on day , h after the rd dose, deaq levels in the plasma of two animals from group were in a similar range (n = , - ng/ml) as found in healthy animals (n = , - ng/ml) (fig. c,d) . repurposing drugs continues to be of interest to the healthcare professional community for the treatment of emerging and re-emerging hemorrhagic fever viruses such as ebola, marburg, and lassa viruses. extensive efforts to screen approved and established drugs for antiviral activity led to a panel of drugs with broad-spectrum antiviral activity profiles that are available, affordable, have well-characterized pk/safety profiles and could be used under the emergency use authorization (eua) mechanism , , , . while a number of fda-approved compounds have proven to be efficacious against ebov in vitro or in murine models of disease, clinical evaluation or evaluation in more relevant disease models, such as nhps, has been limited. aq is a well-known antimalarial drug with wide usage in the countries that experience outbreaks of evd. the drug has activity against several human pathogens of other virus families including corona-, flavi-, and bunyaviruses [ ] [ ] [ ] [ ] . a recent report showed that entry of lassa-gp pseudotyped virus was blocked by aq, indicating that the drug may also have value for the treatment of other viral hemorrhagic fevers . aq is known to have in vitro antiviral activity against ebov - . multiple mechanisms for antiviral activity have been proposed. aq may block ebov entry, which depends on the acidification of endosomes. as a cationic amphiphilic drug, aq can accumulate in the late endo/lysosome and, as a weak base, neutralize the acidic environment, thereby inhibiting cathepsin b activation required for fusion of the viral and endosomal membrane . aq has also been reported to bind and inhibit cathepsin b directly . in silico binding studies revealed that aq may also bind to the viral protein vp . we found that aq inhibits ebov replication in multiple cell types, including huh and vero e cells and primary human macrophages, with ic 's ranging from . - . um depending on experiment and the cell type (table ) . aq is rapidly metabolized to the active metabolite, deaq, following oral administration. in vitro studies confirmed that the active metabolite also has anti-ebov properties in huh and vero e cells, and primary human macrophages, with ic 's ranging from . - . µm µm depending on experiment and the cell type. in contrast, as and its metabolite dha demonstrated minimal to no in vitro antiviral activity against ebov when tested side-by-side to aq using our assay parameters. when used in combination, as and aq did not have a synergistic effect on ebov replication in vitro. whereas as could possibly act with aq to impact the whole body-system in response to ebov in a way that cannot be replicated in the in vitro assays, this study assumed that aq was the main contributing factor of the drug combination resulting in anti-ebov activity. www.nature.com/scientificreports www.nature.com/scientificreports/ the goal of the study was to treat animals with aq using a similar dosing strategy as for human patients, with a target blood concentration range of the parent compound aq of . ± . ng/ml . indeed, we found that a mg/kg aq dose using the -day treatment regimen resulted in correlating plasma levels of . - . ng/ ml in healthy nhps. however, this treatment regimen did not result in a survival benefit or decrease of disease symptoms in ebov-infected animals. these results are disappointing and highlight the importance of utilizing relevant animal models of evd to evaluate potential antivirals, including detailed characterization of pharmacokinetics and tolerability in the context of evd. whereas aq treatment did not lead to detectable beneficial effects on evd progression in nhps, there may be value in investigating novel aq derivatives that have been improved for anti-ebov potency and/or improved tolerability . a possible impact from aq side effects needs to be considered. rare instances of acute liver injury usually after prolonged treatment have been reported , . for this reason, aq and the combination drug asaq are recommended for use as a malaria treatment in endemic areas, but not for prophylaxis against malaria. the onset of hepatic injury is often associated with agranulocytosis. elevated liver enzymes, alt and ast, were observed in the healthy nhps during the pharmacokinetics study. given that the evd patients in were given a co-formulation of aq and as, it would have been of interest to test the combined regimen rather than just aq to evaluate potential implications of the two drugs on disease progression. whereas as has no measurable anti-ebov effect when tested in combination with aq in vitro, the drug could have an in vivo effect that is not captured in cell culture assays. as and artemether are derivatives of artemisinin and are both metabolized to dha, the active metabolite for the treatment of malaria. however, as www.nature.com/scientificreports www.nature.com/scientificreports/ is water soluble, and reaches peak concentration more rapidly with higher plasma c max than artemether, which could impact in vivo activity against ebov. another consideration is that as and aq may affect each other in terms of pk, drug metabolism, or disposition. for example, as co-administration with aq has been reported to reduce exposure to aq . questions remain on the possible contribution of as to the beneficial effect of the asaq treatment observed in evd patients . therefore, testing the co-formulation would have value, but without testing each drug singly as well, attribution of any observed effect to a specific component will be challenging. other confounding factors not measured in the gignoux study could have accounted for the beneficial effect of asaq . the potential impact of concurrent malaria and the type of antimalarial used in evd patients was not addressed in our study. in conclusion, treatment with aq did not have a beneficial effect on survival or the symptoms of evd in rhesus macaques under the conditions tested. whereas aq on its own failed to demonstrate efficacy, we cannot exclude potential therapeutic value of aq when used in combination with another antiviral. www.nature.com/scientificreports www.nature.com/scientificreports/ cells and virus. vero e (atcc crl- , manassas, va), hela (atcc ccl- ), and huh (human hepatocellular carcinoma) cells were maintained following recommended protocols. human monocyte-derived macrophages (mdms) were generated as previously described . ebola virus/h.sapiens-tc/gin/ /makona-c (ebov/mak, genbank accession no. kx . ) was propagated in vero e cells (bei resources, niaid, nih: vero c (e ), african green monkey kidney, working bank #nr- ) as previously described . virus stock and challenge inoculum titers were determined by plaque assay on vero e cells as previously described . drugs and treatment preparation. in vitro studies. the antimalarial artesunate-amodiaquine (asaq winthrop) (sanofi-aventis, gentilly cedex, france) was solubilized by crushing the tablets and resuspending in dimethyl sulfoxide to prepare a solution with the aq component at a concentration of mm. the compounds, amodiaquine dihydrochloride dihydrate (#a ), artesunate (#a ), and dihydroartemisinin (#d ) were purchased from sigma-aldrich (saint louis, mo). n-desethylamodiaquine hydrochloride (#sc- ) was obtained from santa cruz biotechnology (dallas, tx). drugs were prepared as mm stocks in dimethyl sulfoxide. animal studies. source, formulation, and preparation of amodiaquine hydrochloride was the same for the pk and the efficacy studies. amodiaquine hydrochloride was obtained from us pharmacopeia (rockville, md; cat. ; lot j ) and a mg/ml aq (free base) solution was prepared in sterile water for injection (usp). cell-based testing of ebov antiviral agents. the cell-based ebov drug screen and cytotoxicity assays were performed as previously described . briefly, vero e and huh cells were seeded at × cells/well, and mdms at × cells/well in -well plates. after h, cells were treated with compounds at -fold dilutions starting from µm. the starting concentration of the asaq tablet suspension corresponded to µm of the aq base component. cells were infected with ebov/mak h after the addition of the drugs in biosafety level (bsl ) containment at multiplicity of infection (moi) of . - . . after h, plates were fixed with % neutral buffered formalin (richard-allan scientific), and ebov/mak was detected with a mouse antibody specific for ebov vp protein (#b-md -bd -ae , usamriid) followed by staining with alexa fluor ® goat anti-mouse igg (heavy + light chain) antibody (life technologies, grand island, ny) or with anti-mouse igg-peroxidase labeled antibody (kpl# - ). fluorescence or luminescence was quantified on a plate reader (infinite ® m pro, tecan us, morrisville, nc). the signal of treated infected wells was normalized to uninfected control wells and measured (in percent) relative to untreated infected wells. non-linear regression analysis was performed, and the % inhibitory concentrations (ic s) were calculated from fitted curves (log [agonist] versus response [variable slope] with constraint to remain above %) (graphpad software, la jolla, ca). the ebov drug screen assay was performed with three replicates for each drug concentration, and the assay was repeated at least twice for confirmation. to evaluate cytotoxicity, cells were treated with compounds as described above in absence of virus. at h after drug addition, cell viability was quantified using the celltiter glo luminescent cell viability assay kit (promega, madison, wi). www.nature.com/scientificreports www.nature.com/scientificreports/ pharmacokinetic analysis of amodiaquine in nonhuman primates. rhesus macaques (macaca mulatta) were obtained from covance research products (princeton, nj). two groups (n = , females and males per group) were dosed with or mg/kg amodiaquine hydrochloride (us pharmacopeia, rockville, md; cat. ; lot j ) orally via nasogastric intubation once daily for days. nhps were housed in stainless steel primary enclosures singly or in pairs if compatible. nhps were provided teklad certified global % protein primate diet (# c) and purified water ad libitum. clinical observations were conducted for days including the day of dosing. blood (maximal μl) was collected from cephalic vessels on day and day . on day , blood was collected pre-dose, . , , , , , , , and h post-dose (immediately prior to dosing on day ). on day , blood was collected h after dose (or h after dose ), immediately prior to dosing on day , and . , , , , , , , and h after dose . drug concentrations of aq and its metabolite, deaq, were determined in collected plasma samples using liquid chromatography with tandem mass spectrometry (lc-ms/ms). plasma samples (volume μl) were prepared by adding ml of methyl t-butyl ether. each tube was vortexed for approximately minutes (min) at maximal speed and centrifuged for min at , g to facilitate separation of the liquid phases. upper layers ( μl) were transferred to new tubes, and the solvent was removed under vacuum in a centrifugal evaporator. the dried residues were reconstituted with μl of internal standard solution ( ng/ml risperidone and ng/ml chloroquine in : [v:v] acetonitrile:water). the tubes were vortexed min and clarified by centrifugation ( , g) for min. the clarified extracts were transferred to high performance liquid chromatography vials containing glass inserts for subsequent lc-ms/ms analysis. lc-ms/ms was performed using a lc- ad pump system (shimadzu, columbia, md) and a qtrap mass spectrometer (sciex, concord, ontario, canada) in multiple reaction monitoring mode and a polaris c -a column ( × . mm, μm; agilent, ca), using gradient elution with . % formic acid in water and . % formic acid in acetonitrile as the mobile phase. the lower limit of quantitation for aq and deaq of the method was ng/ml. the following parameters and constants were determined: maximal plasma concentration (cmax), time to maximum plasma concentration (tmax), area under the plasma concentration-time curve to the last time point and extrapolated to infinity (auc last and auc inf ), terminal elimination half-life (t / ), apparent volume of distribution (v/f), and total clearance (cl/f) after oral administration. pk parameters were analyzed using phoenix ® winnonlin ® software (v . ; certara, princeton, nj) to perform noncompartmental data analysis for extravascular administration. for clinical pathology (serum chemistry, hematology), blood ( ml) was collected prior to dosing on day and approximately h after the final day dose. treatment and challenge of nonhuman primates. rhesus macaques of chinese origin (n = , adult, < years of age) were obtained from charles river laboratories (frederick, md). animals were singly housed in stainless steel primary enclosures and provided chow and purified (reverse osmosis) water ad libitum. the vehicle control group (group ; n = ) was treated with sterile water. two treatment groups were treated with aq orally once daily as a consecutive -day course of mg/kg free base ( mg/kg amodiaquine hydrochloride (us pharmacopeia, rockville, md; cat. ; lot j ). treatment began on the day of ebov exposure (group ; n = , females and males), or on day postexposure (group ; n = , females and males). all groups were challenged im with a target dose of pfu) of ebov/mak (actual dose = pfu). the plaque assay back-titration of the challenge material was initiated on the day of preparation (study day ) on vero e cells, and plaques were fixed with % formalin and stained with crystal violet days later. animals were observed and weighed daily. the vehicle control group (group , n = ) received an equivalent volume of sterile water (gibco, cat. a , lot ). weight was recorded on day - and day - , then daily starting one day before challenge until primates succumbed to disease. responsiveness of the animals was monitored following -point range, in which a score of met primary endpoint criteria and initiated secondary criteria evaluation (table ) . when nhps scored a for primary euthanasia criteria and their temperature was above °c, secondary euthanasia criteria were reviewed. an nhp had to meet at least two secondary criteria, blood urea nitrogen ≥ mg/dl, calcium ≤ . mg/dl, ggt ≥ u/dl, and/or creatinine ≥ . mg/dl, for required euthanasia. two rhesus macaques were euthanized based on their secondary criteria. six of the fifteen rhesus macaques were euthanized based on veterinary discretion, not based on their clinical score or secondary criteria. hematology and serum chemistry. complete blood count with leukocyte differential was performed from peripherally collected blood samples using vacuette k ethylenediaminetetraacetic acid (edta) tubes and a sysmex xt- iv hematology instrument using a preprogrammed monkey species profile (sysmex america, ny). plasma and serum were prepared by separation for min at ambient temperature with centrifuge set to rcf. serum chemistries were performed using the piccolo xpress analyzer and general chemistry discs (abaxis, abbott point of care, nj) from vacuette z serum clot activator tubes (greiner bio-one, monroe, nc). pathology and histology. all animals were humanely euthanatized in accordance with defined experimental endpoints, and gross necropsy was performed. tissue samples were inactivated by fixing for h in % neutral buffered formalin. following fixation and removal from the bsl- laboratory in accordance with standard operating procedures, tissue samples were routinely processed in a tissue-tek vip- automated vacuum infiltration processor (sakura finetek usa, torrance, california, usa) followed by paraffin embedding with a tissue-tek model tec unit (sakura finetek usa, torrance, ca, usa). using a standard semiautomated rotary microtome and lighted water flotation bath (leica biosystems, wetzlar, germany), tissue sections were cut at a thickness of µm and mounted on positively charged uncoated glass slides (thermofisher, waltham, ma, usa), air-dried at room temperature, stained with hematoxylin and eosin (h&e), and coverslipped for microscopic evaluation by the pathologist. plasma samples from rhesus macaques that had received a dose of aq at integrated research facility/ niaid (fort detrick, md) were analyzed for concentrations of aq and the metabolite deaq. prior to shipment to sri, the samples were diluted : . and irradiated at mrad using a cobalt- source to destroy any pathogens that may have been present in the plasma samples. a pilot study with spiked quality control standards confirmed that irradiation does not appreciably alter the level of aq in the plasma (data not shown). a liquid:liquid extraction method with methyl t-butyl ether was used to separate aq and deaq from rhesus macaque plasma. samples were analyzed by lc-ms/ms with a lower limit of quantitation (lloq) of ng/ml ( . ng/ml × . dilution factor) in plasma for both aq and deaq. aq and deaq concentrations were adjusted for the dilution factor of . for analysis in figs. and . the virus working stock ebov/makona used for exposure (genbank accession no. kx . , biosample: samn ). clinical management of patients with viral haemorrhagic fever effect of artesunate-amodiaquine on mortality related to ebola virus disease effect of mass artesunate-amodiaquine distribution on mortality of patients with ebola virus disease during west african outbreak the clinically approved drugs amiodarone, dronedarone and verapamil inhibit filovirus cell entry identification of compounds that block ebola virus-like particle entry via a repurposing screen of approved drugs a systematic screen of fda-approved drugs for inhibitors of biological threat agents evaluation of ebola virus inhibitors for drug repurposing identification of agents effective against multiple toxins and viruses by host-oriented cell targeting the antiviral activities of artemisinin and artesunate nonhuman primate models of ebola virus disease a simple practice guide for dose conversion between animals and human pharmacokinetics and tolerability of artesunate and amodiaquine alone and in combination in healthy volunteers identification of combinations of approved drugs with synergistic activity against ebola virus in cell cultures a screen of approved drugs and molecular probes identifies therapeutics with anti-ebola virus activity establishment of an antiviral assay system and identification of severe fever with thrombocytopenia syndrome virus inhibitors antiviral activities of selected antimalarials against dengue virus type and zika virus amodiaquine, an antimalarial drug, inhibits dengue virus type replication and infectivity repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection arbidol and other low molecular weight drugs that inhibit lassa and ebola viruses antiviral activity of cationic amphiphilic drugs a common feature pharmacophore for fda-approved drugs inhibiting the ebola virus novel amodiaquine derivatives potently inhibit ebola virus infection amodiaquine induced agranulocytosis and liver damage tolerability and safety of artesunate-amodiaquine and artemether-lumefantrine fixed dose combinations for the treatment of uncomplicated plasmodium falciparum malaria: two open-label, randomized trials in nimba county evaluation of the activity of lamivudine and zidovudine against ebola virus high dose sertraline monotherapy fails to protect rhesus macaques from lethal challenge with ebola virus makona pathology of experimental ebola-zaire (mayinga) virus infection transmitted to guinea pigs by oral, conjunctival and tonsillar routes the findings and conclusions in this report do not necessarily reflect the views or policies of the us department of health and human services or of the institutions and companies affiliated with the authors. the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to j.d.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - apf w j authors: wang, li; xia, tian; guo, tiantian; ru, yi; jiang, yanping; cui, wen; zhou, han; qiao, xinyuan; tang, lijie; xu, yigang; li, yijing title: recombinant lactobacillus casei expressing capsid protein vp can serve as vaccine against rabbit hemorrhagic disease virus in rabbits date: - - journal: vaccines (basel) doi: . /vaccines sha: doc_id: cord_uid: apf w j rabbit hemorrhagic disease virus (rhdv) is the causative agent of rabbit hemorrhagic disease (rhd). rhd, characterized by hemorrhaging, liver necrosis, and high morbidity and mortality in rabbits and hares, causes severe economic losses in the rabbit industry worldwide. due to the lack of an efficient in-vitro propagation system for rhdv, the current vaccine is produced via chemical inactivation of crude rhdv preparation derived from the livers of infected rabbits. inactivated vaccines are effective for controlling rhd, but the potential problems of biosafety and animal welfare have negative effects on the application of inactivated vaccines. in this study, an oral lactobacillus casei (l. casei) vaccine was used as an antigen delivery system to express rhdv capsid protein vp (vp )-egfp fusion protein. the expression of the recombinant protein was confirmed via western blotting and immunofluorescence (ifa). our results indicate that oral administration of this probiotic vaccine can stimulate secretory immunoglobulin a (siga)-based mucosal and igg-based humoral immune responses in rabbits. the immunized rabbits were completely protected against challenge with rhdv. our findings indicate that the l. casei expression system is a new strategy for the development of a safe and efficient vaccine against rhdv. rabbit hemorrhagic disease (rhd), characterized by severe necrotizing hepatitis and disseminated intravascular coagulation in the liver, spleen, kidney and other solid organs, is a highly contagious and lethal infection in rabbits [ , ] . rhd is peracute and often lethal hepatitis caused by the rabbit caliciviruses lagovirus europaeus gi. (previously called rabbit hemorrhagic disease virus-rhdv) and lagovirus europaeus gi. (previously called rhdv or rhdvb) [ , , ] . this disease was first reported in china in , and has, since, spread rapidly around the world in less than years, causing considerable economic losses in the rabbit industry and impacting the ecology of wild rabbit populations [ , , , ] . in , oie designated this illness as a viral hemorrhagic disease and added it to list b of the international animal health code [ ] . subsequently, a novel lagovirus, gi. , emerged in france in [ ] . gi. is now endemic in europe and australia, and appears to be replacing gi. strains in these regions [ ] [ ] [ ] . rhdv (rabbit hemorrhagic disease virus) is the etiologic factor of rhd. rhdv is a calicivirus in the genus lagovirus, family caliciviridae [ ] . rhdv is a non-enveloped icosahedral virus possessing a single-stranded positive sense rna genome that is approximately . kb in length [ , ] . the genome comprises a untranslated region (utr), a utr, and two overlapping open reading frames (orfs): orf and orf . orf encodes a polyprotein that is cleaved by the viral protease into seven nonstructural proteins (nsp - ) and a major structural capsid protein vp (presently vp ) at its c-terminus. vp (presently vp ) is a minor structural protein that is encoded by orf [ ] [ ] [ ] . vaccination is the main approach for controlling rhdv because no effective treatment is available for this disease. inactivated vaccines against rhdv were introduced in the early s, improving the survival of rabbits on rabbit farms [ , ] . however, rhdv inactivated vaccines are manufactured using the livers of rabbits infected with rhdv. this is because rhdv cannot grow in any continuous cell lines. therefore, biological risks, animal-welfare concerns, and high costs are the major bottleneck problems in the production and usage of tissue-inactivated vaccines [ ] . rhdv spreads mainly through the upper respiratory and digestive tracts. the initial steps leading to rhdv infection take place on mucosal surfaces [ , ] . it is generally believed that mucosal immunization is an effective approach for preventing systemic infection by pathogens present on mucosal surfaces [ ] . the gastrointestinal (gi) tract is the largest mucosal surface accessible via oral administration [ ] . oral vaccination can trigger a response involving neutralizing mucosal antibodies (iga) and cell-mediated immunity, and does not interfere with igg-based responses [ ] [ ] [ ] [ ] . additionally, oral vaccines show better safety and compliance profiles, and are simpler to manufacture and administer, than traditional injectable preparations [ , ] . however, the delivery of antigens for oral vaccination of the gi tract is hindered by multiple physicochemical and biological barriers; antigens can be subjected to early disintegration and advanced degradation by low ph and proteases present in the gi tract [ , ] . l. casei is a probiotic that is well known for its health-promoting properties, such as maintaining homeostasis and suppressing pathogens in humans and animals [ ] . l. casei has shown a good safety profile, can colonize the intestine, and exerts a nonspecific immunoadjuvant effect. for those reasons, oral vaccines using l. casei as a delivery system for pathogenic antigens have garnered much interest in vaccine development [ ] [ ] [ ] . currently, there is increasing interest in the development of l. casei oral vaccines, and this approach is significant for the effective induction of a mucosal immune response [ ] . the results to date have been confirmed that the safety and the effectiveness of l. casei were used as the oral vaccine vehicle, which were extensively used in protecting individuals against a variety of pathogens [ ] [ ] [ ] [ ] [ ] [ ] [ ] . developing an efficient and safe oral vaccine that can induce strong mucosal and systemic immune responses is desirable for effective prevention of rhdv. therefore, in our current study, we developed a recombinant l. casei expressing the major structural capsid protein vp (vp )-egfp fusion protein of rhdv. then we evaluated the humoral and mucosal immune responses to this recombinant l. casei, and assessed its immunogenic properties upon administration as an oral vaccine. lactobacillus casei atcc (lc ), kindly provided by jos seegers (nizo, ede, the netherlands), the vector ppg-t g -ppt transformed into competent lc cells by electroporation to generate l.casei ppg-t g (ppg/lc ). lactobacillus casei was cultured in de man, rogosa and sharp (mrs) broth at • c without shaking under anaerobic conditions. rhdv- was extracted from homogenates of liver samples obtained from rhdv-infected rabbits in our laboratory. the sequences of the primers used for pcr amplification of vp were as follows: -atggagggcaaaacccgcacagcgc- ; -tcagacataagaaaagccattggct- . ppg-t g -ppt, constructed in our previous study [ ] , was used as a constitutive expression plasmid in this study; it encoded resistance to chloramphenicol (cm) and contained an hce promoter, t g enhancer, pgsa anchor, and the rrnbt t terminator. plasmids pmd -t simple-egfp and pmd -t simple-vp were constructed in our laboratory previously. . . construction of recombinant l. casei (ppg-t g -egfp-vp /lc ) a schematic diagram of the recombinant dna plasmid is shown in figure . in brief, recombinant plasmid pmd -t simple-egfp was cleaved with saci and kpni restriction endonuclease and inserted into the corresponding sites of a pmd -t simple-vp expression vector digested using saci and kpni. this generated recombinant plasmid pmd -t simple-egfp-vp . the egfp-vp fragment, cleaved with saci and apai, was inserted into a constitutive expression vector ppg-t g -ppt, resulting in the recombinant plasmid ppg-t g -egfp-vp (cm + ). afterwards, the cm resistance gene in ppg-t g -egfp-vp (cm + ) was disrupted using muni and ncoi. finally, the recombinant plasmid ppg-t g -egfp-vp was obtained via blunt-end ligation and transformed into competent lc cells by electroporation to generate recombinant l. casei ppg-egfp-vp /lc . atggagggcaaaacccgcacagcgc- ′; ′-tcagacataagaaaagccattggct- ′. ppg-t g -ppt, constructed in our previous study [ ] , was used as a constitutive expression plasmid in this study; it encoded resistance to chloramphenicol (cm) and contained an hce promoter, t g enhancer, pgsa anchor, and the rrnbt t terminator. plasmids pmd -t simple-egfp and pmd -t simple-vp were constructed in our laboratory previously. a schematic diagram of the recombinant dna plasmid is shown in figure . in brief, recombinant plasmid pmd -t simple-egfp was cleaved with saci and kpni restriction endonuclease and inserted into the corresponding sites of a pmd -t simple-vp expression vector digested using saci and kpni. this generated recombinant plasmid pmd -t simple-egfp-vp . the egfp-vp fragment, cleaved with saci and apai, was inserted into a constitutive expression vector ppg-t g -ppt, resulting in the recombinant plasmid ppg-t g -egfp-vp (cm + ). afterwards, the cm resistance gene in ppg-t g -egfp-vp (cm + ) was disrupted using muni and ncoi. finally, the recombinant plasmid ppg-t g -egfp-vp was obtained via blunt-end ligation and transformed into competent lc cells by electroporation to generate recombinant l. casei ppg-egfp-vp /lc . with saci and kpni, was inserted into the corresponding sites of pmd -t simple-vp digested by saci and kpni; ②: egfp-vp fragment, cleaved with saci and apai, was inserted into constitutive expression vector ppg-t g -ppt; ③ : cm resistance gene in ppg-t g -egfp-vp (cm + ) was disrupted by muni and ncoi, and recombinant plasmid ppg-t g -egfp-vp was obtained by blunt end ligation. the recombinant l. casei ppg-egfp-vp /lc , ppg/lc , and lc were cultured overnight in mrs broth and harvested by centrifugation at × g for min at °c after cell lysis and centrifugation, the supernatant was subjected to % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and western blot assay. the proteins in the supernatant were separated by sds-page and electrotransferred onto pvdf membranes (millipore, milford, ma, usa). the immunoblot was then incubated with the following primary antibodies: rabbit polyclonal anti-vp (vp ) prepared in our laboratory (at the dilution of : ), and mouse monoclonal anti-egfp (at the dilution of : ) (abcam, cambridge, ma, usa) eat °c overnight. then, the immunoblot was incubated with horseradish peroxidase (hrp)-conjugated goat anti-rabbit or goat anti-mouse igg : egfp, cleaved with saci and kpni, was inserted into the corresponding sites of pmd -t simple-vp digested by saci and kpni; : egfp-vp fragment, cleaved with saci and apai, was inserted into constitutive expression vector ppg-t g -ppt; : cm resistance gene in ppg-t g -egfp-vp (cm + ) was disrupted by muni and ncoi, and recombinant plasmid ppg-t g -egfp-vp was obtained by blunt end ligation. the recombinant l. casei ppg-egfp-vp /lc , ppg/lc , and lc were cultured overnight in mrs broth and harvested by centrifugation at × g for min at • c after cell lysis and centrifugation, the supernatant was subjected to % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and western blot assay. the proteins in the supernatant were separated by sds-page and electrotransferred onto pvdf membranes (millipore, milford, ma, usa). the immunoblot was then incubated with the following primary antibodies: rabbit polyclonal anti-vp (vp ) prepared in our laboratory (at the dilution of : ), and mouse monoclonal anti-egfp (at the dilution of : ) (abcam, cambridge, ma, usa) eat • c overnight. then, the immunoblot was incubated with horseradish peroxidase (hrp)-conjugated goat anti-rabbit or goat anti-mouse igg secondary antibodies (at the dilution of : ) (sigma, ronkonkoma, ny, usa) at • c for min. protein expression was visualized using a chemiluminescent substrate reagent (thermo scientific, durham, nc, usa) according to the manufacturer's instructions. we used fluorescence microscopy and indirect immunofluorescence assay (ifa) to detect the expression of vp (vp ) and egfp. in brief, recombinant l. casei was cultured overnight in mrs at • c. cell pellets were washed thrice with pbs, incubated with rabbit anti-vp (vp ) hyper-immune serum(prepared in our laboratory), and then with tritc red fluorescently labeled anti-rabbit igg secondary antibody (diluted at : ) at • c for min. cell pellets were then washed thrice, resuspended in µl pbs, and smeared onto microscope slides. images were obtained via laser confocal microscopy (model lsm meta; zeiss, oberkochen, germany). two-month-old specific pathogen-free (spf) rabbits, obtained from liaoning changsheng biotechnology co., ltd. (liaoning, china) were housed under spf conditions with free access to standard chow diet and water. prior to oral administration, recombinant l. casei was cultured overnight in mrs medium, washed with pbs, and then resuspended in pbs at a final concentration of × cfu/ml. the rabbits were randomly separated into three groups, with six rabbits per group. the first group was injected with a single does of a commercial inactivated rhdv vaccine. the second group was orally administered ml l. casei ppg-egfp-vp /lc ( × cfu/ml). the third group, serving as unimmunized control, was inoculated with ml of orally administered pbs. the rabbits were immunized orally once a day for three consecutive days and boosted twice at -week intervals ( figure ). serum, nasal washes, vaginal lotions, tears, and fecal samples were obtained on days , , , , , and after immunization, and were stored at − • c until use. fecal samples were pretreated according to a previously published method [ ] . three groups of rabbits (n = ), immunized with ppg-egfp-vp /lc , inactivated vaccine, or pbs, were challenged with ml rhdv crude liver extract (at a viral rna copy number of . × . ) administered via intramuscular injection into the leg at d after the first vaccination ( figure ). the viral rna copy number was determined by real-time pcr. all rabbits were humanely euthanized and dissected at days post-infection (dpi). vaccines , , x of secondary antibodies (at the dilution of : ) (sigma, ronkonkoma, ny, usa) at °c for min. protein expression was visualized using a chemiluminescent substrate reagent (thermo scientific, durham, nc, usa) according to the manufacturer's instructions. we used fluorescence microscopy and indirect immunofluorescence assay (ifa) to detect the expression of vp (vp ) and egfp. in brief, recombinant l. casei was cultured overnight in mrs at °c. cell pellets were washed thrice with pbs, incubated with rabbit anti-vp (vp ) hyper-immune serum(prepared in our laboratory), and then with tritc red fluorescently labeled anti-rabbit igg secondary antibody (diluted at : ) at °c for min. cell pellets were then washed thrice, resuspended in μl pbs, and smeared onto microscope slides. images were obtained via laser confocal microscopy (model lsm meta; zeiss, oberkochen, germany). two-month-old specific pathogen-free (spf) rabbits, obtained from liaoning changsheng biotechnology co., ltd. (liaoning, china) were housed under spf conditions with free access to standard chow diet and water. prior to oral administration, recombinant l. casei was cultured overnight in mrs medium, washed with pbs, and then resuspended in pbs at a final concentration of × cfu/ml. the rabbits were randomly separated into three groups, with six rabbits per group. the first group was injected with a single does of a commercial inactivated rhdv vaccine. the second group was orally administered ml l. casei ppg-egfp-vp /lc ( × cfu/ml). the third group, serving as unimmunized control, was inoculated with ml of orally administered pbs. the rabbits were immunized orally once a day for three consecutive days and boosted twice at week intervals ( figure ). serum, nasal washes, vaginal lotions, tears, and fecal samples were obtained on days , , , , , and after immunization, and were stored at − °c until use. fecal samples were pretreated according to a previously published method [ ] . three groups of rabbits (n = ), immunized with ppg-egfp-vp /lc , inactivated vaccine, or pbs, were challenged with ml rhdv crude liver extract (at a viral rna copy number of . × . ) administered via intramuscular injection into the leg at d after the first vaccination ( figure ) . the viral rna copy number was determined by real-time pcr. all rabbits were humanely euthanized and dissected at days post-infection (dpi). the levels of anti-vp (vp )-specific igg in the sera, and those of siga in the feces, genital tract secretions, and intestinal mucusa, of the rabbits used in this study, were determined via elisa. briefly, polystyrene microtiter plates were coated with rhdv and incubated overnight at • c. after washing thrice with pbst (pbs containing % tween- ), the plates were blocked with % skimmed milk at • c for h. after washing thrice with pbst, the collected samples, prepared in triplicate, were diluted with pbs (sera diluted at : ; supernatants of feces, tears, vagina, and nasal cavity lavage diluted at : ), incubated at • c for h. then, an hrp-conjugated goat anti-rabbit igg or iga antibody (sigma, ronkonkoma, ny, usa) was added to each well (at : ), and incubated for h at • c. color was developed using tetramethylbenzidine (tmb) (sigma, ronkonkoma, ny, usa) as a substrate, and absorbance was measured at od . the liver, spleen, and small intestine obtained from the rabbits were subjected to histopathological examination. tissue samples were fixed in % neutral-buffered formalin, processed routinely, embedded into paraffin blocks, and sectioned at µm. the sections were stained with hematoxylin and eosin (h&e), and morphology was evaluated using light microscopy. all data are presented as mean ± standard deviation (sd). the samples from each group ( rabbits) were pooled and then analyzed as one sample in triplicate. data were analyzed using two-way anova with multiple comparison (lsd) test in spss. different letters (a vs. b, a vs. c, b vs. c) indicate significant differences (p < . ) at the same time point. the recombinant plasmid ppg-t g -egfp-vp was generated ( figure ) and transformed by electroporation into competent lc cells. egfp-positive recombinant l. casei cells were collected by flow cytometry and designated as ppg-egfp-vp /lc . to assess whether recombinant l. casei retained chloramphenicol resistance, recombinant l. casei were cultivated on mrs solid plates containing and not containing chloramphenicol. our results indicate that recombinant l. casei ppg-egfp-vp /lc grew normally on mrs medium without chloramphenicol, but did not grow on mrs medium containing chloramphenicol. this indicates that the chloramphenicol gene had been removed successfully (figure a) . we then examined the inherited stability of the recombinant l. casei. recombinant l. casei ppg-egfp-vp /lc was serially inoculated times. the th, th, th, th, th, th, th, and th generations of recombinant l. casei were cultured for h, and the plasmid was extracted. inherited stability was then assessed using pcr and sequencing. our results indicate that recombinant l. casei showed good inherited stability and no gene deletion (figure b ). vaccines , , x of recombinant l. casei ppg-egfp-vp /lc , ppg/lc , and lc cells were cultured in mrs broth without chloramphenicol antibiotic. cell lysates were analyzed by western blotting using mouse anti-egfp monoclonal antibody and rabbit anti-vp (vp ) polyclonal antibody. for both anti-egfp and anti-vp (vp ) antibodies, specific immunoreactive bands of the expected size were observed in recombinant l. casei ppg-egfp-vp /lc , and there was no band at the corresponding position in ppg/lc or lc (figure a ). confocal microscopy and ifa were used to determine the expression of the egfp-vp (vp ) fusion protein in recombinant l. casei. confocal microscopy was also used to observe the expression of egfp in l. casei. our results show the emission of green fluorescence in l. casei ppg-egfp-vp /lc , but not in ppg/lc (figure b, top) . for ifa, ppg-egfp-vp /lc and ppg/lc were incubated with rabbit anti-vp (vp ) polyclonal antibody and tritc-conjugated goat anti-rabbit igg secondary antibody, and then observed using laser confocal microscopy. emission of red fluorescence was observed in ppg-egfp-vp /lc , but not in ppg/lc (figure b, bottom) . these results indicate that the protein of interest was successfully expressed in the recombinant l. casei. recombinant l. casei ppg-egfp-vp /lc , ppg/lc , and lc cells were cultured in mrs broth without chloramphenicol antibiotic. cell lysates were analyzed by western blotting using mouse anti-egfp monoclonal antibody and rabbit anti-vp (vp ) polyclonal antibody. for both anti-egfp and anti-vp (vp ) antibodies, specific immunoreactive bands of the expected size were observed in recombinant l. casei ppg-egfp-vp /lc , and there was no band at the corresponding position in ppg/lc or lc (figure a ). confocal microscopy and ifa were used to determine the expression of the egfp-vp (vp ) fusion protein in recombinant l. casei. confocal microscopy was also used to observe the expression of egfp in l. casei. our results show the emission of green fluorescence in l. casei ppg-egfp-vp /lc , but not in ppg/lc (figure b, top) . for ifa, ppg-egfp-vp /lc and ppg/lc were incubated with rabbit anti-vp (vp ) polyclonal antibody and tritc-conjugated goat anti-rabbit igg secondary antibody, and then observed using laser confocal microscopy. emission of red fluorescence was observed in ppg-egfp-vp /lc , but not in ppg/lc (figure b, bottom) . these results indicate that the protein of interest was successfully expressed in the recombinant l. casei. next, we analyzed the immunogenicity of recombinant l. casei ppg-egfp-vp /lc and effectiveness of the inactivated virus vaccine in rabbits after oral and intramuscular immunization. systemic and mucosal immune responses were assessed by detecting the presence of anti-rhdv igg and iga antibodies; the levels of these antibodies were quantified using elisa. starting at day after primary vaccination, a significantly increased level of anti-vp (vp )-specific igg was detected in rabbits orally administered with ppg-egfp-vp /lc , and in those injected with the inactivated vaccine, compared with the responses of the pbs-treated control group (p < . ). additionally, there was less igg present for the l. casei vaccine in comparison to the inactivated vaccine ( figure ) . similarly, localized levels of anti-vp (vp )-specific siga in the feces, tears, vagina, and nasal cavity lavage of the immunized rabbits were measured via elisa. in brief, mucosal siga levels increased significantly in the feces (figure a ), vagina (figure b ), nasal cavity (figure c) , and tears (figure d ) of the group orally immunized with ppg-egfp-vp /lc compared with those of the pbs-treated control group (p < . ). the level of siga induced by oral immunization with ppg-egfp-vp /lc was higher than that induced by the inactivated vaccine. next, we analyzed the immunogenicity of recombinant l. casei ppg-egfp-vp /lc and effectiveness of the inactivated virus vaccine in rabbits after oral and intramuscular immunization. systemic and mucosal immune responses were assessed by detecting the presence of anti-rhdv igg and iga antibodies; the levels of these antibodies were quantified using elisa. starting at day after primary vaccination, a significantly increased level of anti-vp (vp )-specific igg was detected in rabbits orally administered with ppg-egfp-vp /lc , and in those injected with the inactivated vaccine, compared with the responses of the pbs-treated control group (p < . ). additionally, there was less igg present for the l. casei vaccine in comparison to the inactivated vaccine ( figure ) . similarly, localized levels of anti-vp (vp )-specific siga in the feces, tears, vagina, and nasal cavity lavage of the immunized rabbits were measured via elisa. in brief, mucosal siga levels increased significantly in the feces (figure a), vagina (figure b ), nasal cavity (figure c) , and tears (figure d ) of the group orally immunized with ppg-egfp-vp /lc compared with those of the pbs-treated control group (p < . ). the level of siga induced by oral immunization with ppg-egfp-vp /lc was higher than that induced by the inactivated vaccine. to assess immunoprotective efficacy, the three groups of rabbits administered ppg-egfp-vp /lc , inactivated vaccine, or pbs were infected with ml rhdv (rna copy number of . × . ) at days after the last booster. our results show that all rabbits in the pbs group died within three days post-challenge, while the rabbits immunized with recombinant l. casei or inactivated vaccine were completely protected against challenge with rhdv ( figure ) . to assess immunoprotective efficacy, the three groups of rabbits administered ppg-egfp-vp /lc , inactivated vaccine, or pbs were infected with ml rhdv (rna copy number of . × . ) at days after the last booster. our results show that all rabbits in the pbs group died within three days post-challenge, while the rabbits immunized with recombinant l. casei or inactivated vaccine were completely protected against challenge with rhdv ( figure ). , nasal cavity (c), and tears (d) was performed by elisa using rhdv as the coating antigen. the samples from each group ( rabbits) were pooled and then analyzed as one sample in triplicate. bars represent the mean ± standard deviation (sd) in each group. different letters (a vs. b, a vs. c, b vs. c) indicate significant differences (p < . ) at the same time point. to assess immunoprotective efficacy, the three groups of rabbits administered ppg-egfp-vp /lc , inactivated vaccine, or pbs were infected with ml rhdv (rna copy number of . × . ) at days after the last booster. our results show that all rabbits in the pbs group died within three days post-challenge, while the rabbits immunized with recombinant l. casei or inactivated vaccine were completely protected against challenge with rhdv ( figure ) . the liver, spleen, and small intestine, collected from the rabbits in different treatment groups, were subjected to histopathological evaluation. lesions on liver, spleen, and small intestine were more severe in the pbs-treated group than in the vaccinated groups. in the pbs-treated group, the hepatocytes in the liver presented a disordered appearance and clearly manifested degeneration and necrosis. splenic corpuscles appeared destroyed, and some had disappeared entirely, splenic sinus was congested, and intestine villi were necrotic and severely desquamated. there are almost no differences between the ppg-egfp-vp /lc -immunized group and inactivated vaccineimmunized group (figure ). the liver, spleen, and small intestine, collected from the rabbits in different treatment groups, were subjected to histopathological evaluation. lesions on liver, spleen, and small intestine were more severe in the pbs-treated group than in the vaccinated groups. in the pbs-treated group, the hepatocytes in the liver presented a disordered appearance and clearly manifested degeneration and necrosis. splenic corpuscles appeared destroyed, and some had disappeared entirely, splenic sinus was congested, and intestine villi were necrotic and severely desquamated. there are almost no differences between the ppg-egfp-vp /lc -immunized group and inactivated vaccine-immunized group (figure ). the liver, spleen, and small intestine, collected from the rabbits in different treatment groups, were subjected to histopathological evaluation. lesions on liver, spleen, and small intestine were more severe in the pbs-treated group than in the vaccinated groups. in the pbs-treated group, the hepatocytes in the liver presented a disordered appearance and clearly manifested degeneration and necrosis. splenic corpuscles appeared destroyed, and some had disappeared entirely, splenic sinus was congested, and intestine villi were necrotic and severely desquamated. there are almost no differences between the ppg-egfp-vp /lc -immunized group and inactivated vaccineimmunized group (figure ). rabbit hemorrhagic disease (rhd) is a comparatively new and economically important viral disease that affects adult rabbits [ ] . it is important to develop effective strategies for preventing and controlling the spread of rhdv. however, because there is no efficient in-vitro system for propagating rhdv, it is difficult to produce this virus as a source of vaccine antigens [ ] . at present, most commercial vaccines are produced using viral preparations obtained from the livers of experimentally infected rabbits [ ] . this strategy raises numerous concerns with respect to animal welfare and biological safety [ ] . to address these issues, and to develop an effective, safe, and convenient oral vaccine against rhdv, we constructed a recombinant l. casei expressing the rhdv vp (vp ) protein. antigens are key factors in vaccine development. previous studies have shown that capsid protein vp (vp ), a major immunogenic protein of rhdv, induces expression of a neutralizing antibody [ ] . rabbits inoculated with a sufficient amount of recombinant vp (vp ) are fully protected from wild-type rhdv, and vp is commonly used to develop recombinant engineered vaccines against rhdv [ , ] . therefore, vp (vp ) was selected as an immunogen for the development of a recombinant engineered vaccine in this study. recombinant engineered vaccines containing vp (vp ) have been developed using bacteria, yeasts, plants, poxvirus-based vectors, and insect cells as delivery vectors [ , [ ] [ ] [ ] . however, intramuscular injection, which is the main route of administering these recombinant engineered vaccines, requires trained personnel, generates a vast amount of biohazardous waste, and can elicit a stress response in the animals [ , ] . these issues can be readily solved by live vector-based oral vaccines [ ] . as an oral vaccine vector, l. casei offers many advantages, such as increased safety, resistance to the harsh gi environment, robust capacity for intestinal colonization, and nonspecific immunoadjuvant effect [ ] [ ] [ ] . previously, live vector-based oral vaccines were constructed using lc , a promising strain of l. casei that can deliver antigenic material to the mucosa and express numerous targeted proteins, such as vp protein of porcine parvovirus (ppv), glycoprotein e of bovine viral diarrhea virus (bvdv), spike protein (s) of transmissible gastroenteritis virus (tgev), and core neutralizing epitope (coe) antigen of porcine epidemic diarrhea virus (pedv) [ , [ ] [ ] [ ] . oral vaccination with these vaccines can efficiently induce mucosal, humoral, and cellular immune responses, indicating that lc is an excellent vector for use in oral vaccines. therefore, lc was selected as a vector in the genetically engineered l. casei oral vaccine developed in our present study. the main disadvantage of traditional plasmid expression systems is using antibiotic resistance genes as selective markers for genetically engineered bacteria [ , ] . although antibiotics are helpful for vector screening, using antibiotic resistance genes in practice can impact biosafety, contribute to antibiotic resistance, and present potential hazards to health and the environment [ ] . to avoid potential issues caused by antibiotic resistance markers, it is important to establish expression vectors without the use of antibiotic resistance genes. egfp has many advantages as a genetic marker and reporter gene. egfp expression is mostly non-toxic to cells, and egfp fluorescence can last for an extended time [ ] . in the present study, we constructed a recombinant l. casei ppg-t g -egfp-vp /lc using egfp as a selection marker and removed chloramphenicol resistance from the vector. analysis of inherited stability of ppg-t g -egfp-vp /lc demonstrated that recombinant l. casei was stably inherited for more than generations with no gene deletion. we designed the ppg-t g -ppt expression vector, a constitutive cell-surface expressed plasmid, to construct recombinant l. casei for the delivery of vaccine antigen. the ppg-t g -ppt vector contained a t g enhancer, derived from gene of bacteriophage t ; the t g enhancer can augment protein translation and expression [ ] . expression of the egfp-vp (vp ) fusion protein was confirmed via western blotting and ifa. our results indicate that a genetically engineered oral vaccine against rdhv, using l. casei without antibiotic resistance gene, was developed successfully. an effective oral vaccine should induce both systemic and mucosal immune responses. thus, we evaluated the immunogenicity of orally administered ppg-egfp-vp /lc in rabbits. the multiple immunization procedure of recombinant lactobacillus was employed in this study was due to the amounts of recombinant lactobacillus adhered to the intestinal mucosa were decreased with the time [ ] . the immune effect of the multiple immunization procedure of recombinant lactobacillus has been confirmed in the previous studies [ , ] . the rabbits were immunized with inactivated vaccine according to the instruction. serum-derived igg can appreciably contribute to immune defense by reducing the ability of pathogens to cross intestinal mucosa, thereby limiting the systemic spread of invasive organisms [ ] . we detected substantially increased levels of anti-rhdv igg in rabbits orally immunized with ppg-egfp-vp /lc ; these levels increased rapidly following booster immunization. siga, a major immunoglobulin in mucosal immune responses, protects against pathogen invasion at mucosal sites [ ] . therefore, siga levels can be used to evaluate the protective efficacy of vaccines [ ] . in the present study, we detected high levels of antigen-specific mucosal siga in the nasal washes, vaginal lotions, tears, and feces of rabbits orally immunized with ppg-egfp-vp /lc . recombinant l. casei ppg-egfp-vp /lc induced a higher level of siga compared with that induced by the inactivated virus vaccine, indicating that l. casei ppg-egfp-vp /lc was more efficient at eliciting mucosal immune responses, and there were almost no significant differences of siga and igg antibody levels in the experimental animals administered with ppg/l. casei and pbs, which have been confirmed in our previous studies [ , , ] . previous studies also have shown that oral vaccines elicit stronger mucosal immune responses than do injected vaccines [ ] . in our present study, we have shown that oral vaccination with recombinant l. casei ppg-egfp-vp /lc can strongly induce anti-rhdv systemic and mucosal immune responses. in this study, the protective efficacy of l. casei was analyzed using rabbits. our results show that survival rates of rabbits vaccinated with recombinant l. casei and inactivated virus vaccine were at % after the viral challenge; however, none of the rabbits in the pbs-treated group survived challenge with rhdv. this finding indicates that ppg-egfp-vp /lc , administered via oral immunization, effectively induced a sufficient immune response to protect rabbits against infection with rhdv. rabbits infected with rdhv often develop acute necrotizing hepatitis and disseminated intravascular coagulation in the liver, spleen, kidney, and other solid organs [ ] . histopathological analysis showed topical histopathological changes in rdh of the liver and spleen of the pbs-treated group after challenge with rhdv. however, these changes were not observed in the organs of rabbits immunized with recombinant l. casei ppg-egfp-vp /lc or inactivated virus vaccine. the villi of the small intestine were necrotic and severely desquamated in the pbs-treated group and the group treated with inactivated virus vaccine. the group administered l. casei ppg-egfp-vp /lc showed the intact structure of the villi in the small intestine. this finding indicates that l. casei protected the intestinal environment against the effects of rhdv. in conclusion, we developed an 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administration of lactobacillus casei producing porcine parvovirus vp protein mucosal immunity and vaccines secretory iga's complex roles in immunity and mucosal homeostasis in the gut up-regulation of mdp and tuftsin gene expression in th and th cells as an adjuvant for an oral lactobacillus casei vaccine against anti-transmissible gastroenteritis virus the authors declare no conflict of interest.vaccines , , key: cord- - ncj il authors: valencia-ramos, juan; arnaez, juan; calvo, sara; gomez, fernando; del blanco, isabel title: observational study of newborn infant parasympathetic evaluation as a comfort system in awake patients admitted to a pediatric intensive care unit date: - - journal: j clin monit comput doi: . /s - - - sha: doc_id: cord_uid: ncj il to compare the newborn infant parasympathetic evaluation system (nipe) scores with a validated clinical scale using two different nebulizers in children with bronchiolitis admitted to a picu. comfort was evaluated using the comfort-behavior scale (cbs) before (t ), during (t ) and after (t ) each nebulization. in order to compare nipe and cbs values during the whole t to t period, the variable dif-cbs was defined as the difference between maximal and minimal cbs scores, and dif-nipe as the difference between th and th percentile nipe values. analyses were carried out, firstly for the total of nebulizations and secondly comparing two different nebulization systems: a jet nebulizer (jn) and a nebulizer integrated in high flow nasal cannulas (nhf). nebulizations were recorded on patients with a median [ th– th percentile] age of months ( . – . ). a dif-cbs of points ( – ), as well as changes in cbs scores between t and t , defined the nebulization as a discomfort stimulus. the nipe system, represented as the dif-nipe, showed a median variation of points ( – ), and was poorly correlated to dif-cbs [r(s) . (p = . )]. discomfort during nebulization, assessed by cbs was greater with the jn system compared to nhf: ( – ) vs ( – ) (p = . ). nipe monitoring detected no significant differences between both nebulization systems (p = . ). nipe monitoring showed a variation in comfort during nebulization in the patient with bronchiolitis, though correlation with cbs was poor. further research is required before nipe can be suggested as a comfort monitoring system for the awake infant. bronchiolitis is an acute inflammatory injury of the bronchioles caused by a viral infection in infants [ ] . it is the most frequent cause of lower respiratory tract infection in infants and the predominant reason for admission of children under year of age [ , ] . of these, - % will require admission to a pediatric intensive care unit (picu) [ ] . unfortunately, specific therapies are not available and only a few symptomatic strategies are extensively used, including oxygen, hydration, and nasal suction [ , ] . nebulization with salbutamol, epinephrine, or % hypertonic saline is the most commonly used therapy in clinical practice, despite its doubtful efficacy, with the aim of reducing clinical severity [ , ] . however, nebulizations are not harmless because they are usually administered through a jet nebulizer that generates discomfort [ ] , due to its noise, with frequent awakening and struggling to remove it. this discomfort might be especially counterproductive in critically ill patients when trying to achieve desired stability [ ] . the gold standard to assess patient comfort is self-reporting, based on pain scales. however, self-reporting is unlikely in pre-verbal children, as well as in infants with altered consciousness [ ] . regardless of which tool is used to assess pain, perception of pain in pediatrics is complex, and entails physiological, behavioral, and developmental factors [ , ] , including the limitation of the experience of the examiner. in these instances, a cooperation-independent monitor for pain would be highly helpful [ ] . the newborn infant parasympathetic evaluation (nipe, mdms, loos, france) is a valid non-invasive system in children up to years of age based on the analysis of the respiratory fluctuations of heart rate that mainly reflect variability in the parasympathetic tone [ ] . the nipe monitor records the ecg signal continuously, enabling a quantitative assessment of the respiratory variability of heart rate, which decreases during nociceptive stimulation [ ] . to date, only a few studies have focused on this technology in children, the majority of them in the area of anesthesia [ ] [ ] [ ] . the aim of this study was ( ) to evaluate whether the nipe system could be a useful tool for the continuous monitoring of discomfort and pain, compared to the clinical comfort behavior scale when nebulizing children with bronchiolitis admitted to a picu; and ( ) to determine whether nipe values vary when two different nebulization systems are used. infants with bronchiolitis and treatment with high flow nasal cannulas (hfnc) and aerosol therapy were enrolled in this prospective observational study in the picu of a tertiary university hospital between january and may . the use policy for nebulizations in children with bronchiolitis in our picu is dependent on the physician responsible for the patient, an initial test generally being performed and prescribing nebulizations if the clinical response is suitable. children more than months old with arrhythmia or cardiac malformation were excluded from the study. this study respected the helsinki declaration and was approved by the local institutional review board (comité Ético de investigación clínica del Área de salud burgos y soria, ref: ceic- ). informed consent was requested from parents. after parental consent was obtained, subjects were randomized by a computer-generated random number list to begin the nebulization with a jet nebulizer (jn) or a nebulizer integrated in the hfnc system (nhf), alternating the nebulization device. the nipe monitor (nipe, mdms, loos, france) was connected to the electrocardiogram and data recording enabled until nebulizations ended during admission. the algorithm used for nipe computation has been previously described [ , ] . it expresses a numerical value between and ; high values correspond to high parasympathetic activity reflecting the wellbeing of the patient. the nipe calculates two indexes: averaged value (nipem) for chronic or prolonged pain and instantaneous reading (nipei) for acute pain. we used the nipei to evaluate this procedure. clinical comfort was assessed using the comfort behavior scale (cbs) by nurses who had received specific training in this area, at different times: t = min before, t = during, and t = min after the nebulization. each cbs item is scored from to , and the final score range is the sum of the six behavioral items. this made the lowest possible score (no pain) and the highest (the greatest pain) [ ] . our working hypothesis was that nipe would detect changes in the level of discomfort or pain experienced during nebulization, and that its values would vary as clinical assessment scale scores had varied, depending on the type of nebulization system used [ ] . the nipe system stores the values obtained in each minute in its internal memory, as well as the quality of the signal at that time (quality), such that ' ' reflects an adequate recording signal and ' ' reflects an inadequate signal. once nipe "zero quality" values had been removed from a total of nebulizations on a total of patients, the final sample size that was available for analysis amounted to nipe values from nebulizations performed on patients. these measurements were evenly distributed amongst both nebulization systems: nipe values from nebulizations using nhf and nipe values from nebulizations using jn. in order to analyze the changes in the comfort-discomfort, the 'difference' (dif) variables were used. these variables (dif-nipe and dif-cbs) were created so that nipe and cbs comfort measurements could be compared during the whole nebulization period (t to t ). "dif-cbs" was defined as the median of the differences between the maximum and minimum scores obtained when cbs was applied at each moment of the nebulization period (t , t and t ). dif-nipe represented the difference between the th and th percentiles of the nipe values during the same period as cbs (from t to t ). comfort levels were assessed using cbs and nipe for all nebulizations as a whole and also for each of the two nebulization systems used (jn and nhf). a correlation study between the two comfort monitoring systems (clinical scale and nipe monitoring) was made, and the two nebulization systems were compared (jn vs. nhf). other variables potentially associated with the comfortdiscomfort state were collected: date and time; food intake; administration of painkillers, whether the patient was being handheld by a parent or not; physiological variables (heart rate (hr), respiration rate (rr); and the administration of supplemental oxygen as fraction of inspired oxygen (fio )). absolute and relative frequencies were calculated for each qualitative variable, and differences based on nebulization system were identified using chi square or fisher's exact test. for quantitative variables, median [ th- th percentile] were obtained according to nebulization system and time period. spearman's correlation coefficient (r s ) was used to measure correlation among the cbs (dif-cbs) and nipe values (difnipe), and nonparametric testing (mann-whitney's u test) was used to compare the two nebulization systems in each time period. anova for repeated measures was used when cbs scores were compared during different moments within the same nebulization (t , t and t ) for each of the two nebulization systems (nhc and jn). when dif-cbs and dif-nipe were being compared, a "patient" variable was introduced in order to reflect the number of nebulizations administered to each individual patient. values of p < . were considered to be statistically significant. statistical analyses were performed using statistics software (pasw statistics . , spss, chicago, illinois). a total of nebulizations were recorded on patients with bronchiolitis admitted to the picu, (table ). cbs values increased during nebulization (t ) as compared to the period before (t ): median [ th- th percentile] of ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) vs ( - ) (p < . ), with a difference of points ( - ) when the whole period (from t to t ) was considered. as regards to nipe monitoring, dif-nipe was points ( - ) during the whole t to t period. the correlation coefficient between dif-nipe and dif-cbs was: r s . (p = . ) (fig. ) . considering the nebulizations, heart rate increased significantly from bpm ( ; ) before the nebulization (t ) to ( . ; . ) during aerosol therapy (t ) (p = . ). this variation between t and t for the cbs showed a correlation of r s . (p < . ). of the nebulizations, were performed with the nebulization system integrated in the hfnc (nhf), and with the conventional nebulizer (jn). there were no significant differences at baseline between the groups (nhf and jn) when analyzing the time since last food intake, being held in the arms of relatives, time of day (day time-night time), use of analgesia, oxygen saturation, fraction of breathed oxygen, the breathing frequency, or heart rate before nebulization. both nebulizers showed an increase in heart rate and breathing rate between t and t , which was statistically significant in the case of jn (p = . ) ( table ) . during nebulization (t ) the cbs showed higher scores using jn compared with nhf: ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) vs. ( - ), respectively (p = . ). jn cbs scores were higher (p = . ) than those obtained using nhf in all moments of the nebulization period (t , t , t ). the number of nebulizations with a cbs score < ( / ) was higher in the nhf group ( / ) than in the jn group ( / ) (p < . ). during the whole period (from t to t ), dif-cbs was greater for nhf compared with jn: ( - ) vs ( - ) (p = . ). no differences were found between the dif-nipe from t to t with nhf system and jn: ( - ) vs ( - ) (p = . ), and correlation comparing the dif-nipe variables with dif-cbs for each of the nebulizating systems was r s . (p = . ) and r s . (p = . ) for the nhf and jn systems, respectively. providing comfort in critically ill infants and children, and minimizing their anxiety, fear, and distress, are important parts of the daily activities in a picu. however, in spite of its frequency, pain in this group of population is often underestimated and undertreated [ ] . the aim of this study was to examine a tool that could shed some light in the challenging task of interpreting pain and discomfort in this patient group. the population chosen was infants with bronchiolitis admitted to picu, with demographics similar to those described in previous studies [ ] . the reason for choosing this disease, apart from its elevated incidence, was the frequent use in this group of treatments with potential discomfort, such as nebulizations [ ] . in this paper, the results from a smaller previous study are corroborated, since it has been consistently found that nebulizations constitute a scource of discomfort for patients. the degree of this discomfort, as measured using clinical scales, is higher when using a jn system compared to a nhf system [ ] . the comfort behavior scale (cbs) was designed for use in pediatric populations with a similar age to the present study [ ] , and it is a valid and reliable tool for assessing comfort in critical care pediatric patients [ ] . like other authors, we believe that pain scores are central to our ability to evaluate and titrate pain relief [ ] . however, the use of clinical scales has a series of disadvantages, such as the inability to perform them continuously, the degree of subjectivity, and the care burden involved for the health staff. thus, the clinical potential of a clinical monitoring system like the nipe. the nipe is a - non-invasive index, calculated from heart rate variability analysis, which provides a continuous measurement of the parasympathetic tone as a surrogate for the analgesia-nociception balance, with high values corresponding to maximal parasympathetic activity (analgesia) and low values corresponding to sympathetic activation (nociception) [ ] . its use has been validated in children up to years of age. in adult patients, this monitoring system, called the analgesia nociception index (ani), has demonstrated that the respiratory variability of heart rate decreases in response to painful stimuli during general anesthesia or in post-anesthesia care unit [ ] [ ] [ ] , and it has also been used to evaluate pain in awake surgical patients [ ] , and in pregnant women during labor [ ] . there are much less studies carried out in pediatric patients compared to adults with this monitoring system, the majority being focused on the field of anesthesia [ , , ] and very few in neonates and preterm neonates [ , ] . as far as we know, the data presented here are the first regarding the usefulness of the nipe in children with spontaneous breathing admitted to a picu with no surgery performed. in the present study, the nipe appears to be a sensitive tool to detect changes during nebulization, as a variation was detected in the nipe values of points during nebulization. however, these changes were almost identical between one nebulization system and the other. these results contrast with the variations in comfort that the clinical cbs detected during nebulization, as well as the greater clinical discomfort of the jn nebulization system compared to the nhf [ ] . if a score of less than on the cbs scale, like that determined by van dijk et al., is taken as a cut-off fig. the relationship between the difference in comfort levels measured using nipe (dif-nipe) and the difference in comfort levels measured using cbs (dif-cbs) from t to t . correlation between dif-nipe and dif-cbs during t -t period was: r s . (p = . ) point for the administration of analgesics [ ] , the number of patients with values below during nebulization was significantly higher with jn than with nhf. this lack of correlation between the comfort difference during the nebulization with the nipe and the measurement with the clinical scales has also been found in adult patients [ , ] . furthermore, there could be other explanations for these findings in our study. in the first place, an increase of points was found in the hr compared to the baseline, there being a slight correlation with the change in the clinical cbs. this increase of barely % difference from the increase found in other studies supports changes in the hr of - % as a good indicator of nociception [ , ] . in pediatrics, and in pediatric anesthetics in particular, it is normal practice to monitor changes in hr from baseline as an indicator of pain and discomfort [ , ] . as such, migeon et al. showed that ani measurements decreased after skin incision in children over years of age anesthetized with sevoflurane. this could be used in combination, or as an alternative to hr monitoring [ ] . ledowski et al. observed similar results, concluding that ani might provide a more sensitive assessment of nociception in anesthetized children than hemodynamic parameters or skin conductance [ ] . however, the percentage rise in hr required to identify a painful stimulus in children is debatable, some studies not finding changes in the hr in painful procedures [ , ] , or even emphasizing the need for caution in interpreting heart rate as an index of comfort [ ] . secondly, we had to exclude a number of nipe values with quality ' ' which meant not analyzing % of the nebulizations. however, the number of nebulizations rejected in each nebulization system was similar; thus this should not be the reason why we did not find any differences between the two nebulizers used. in any case, it is worth noting, given that the nipe system depends on the quality of the ecg signal, that it may have limitations in awake infants whose continuous movement interferes in the ecg trace. this fact could explain the contrast with the potential of this system in older pediatric patients. nonetheless, these are the children in whom pain assessment can be the most challenging. third, a limitation of our study is that, owing to its design, it does not permit determination of the nipe values at the exact same time as the clinical scales. the non-integration of the monitor as a routine tool in our unit meant that the exact time of the evaluation of the scale will not be noted correctly in the monitor, obliging us to rule out this line of study, and focus instead on analysis of the difference of the values obtained during the entire period. in future research we aim to measure not only the magnitude of nipe variation during nebulization, as was done in this present paper, but to investigate whether the direction of this change (increase or decrease) indeed correlates to the variations in comfort levels measured using clinical scales. finally, we cannot rule out the possibility that factors such as noise, and other environmental factors common to a picu, might have influenced the values recorded by the nipe [ , ] . although randomization of the nebulization of each system would have decreased this bias, these factors were not analyzed. in summary, this study demonstrates the variation in discomfort during nebulization in the patient with bronchiolitis with nipe monitoring, although it does not seem to correlate well with the clinical evaluation. clinical scales like the comfort behavior scale are good tools to evaluate the discomfort and pain generated by nebulization in nonsedated patients breathing spontaneously, presenting with bronchiolitis. more studies are needed before being able to recommend the nipe monitor as an alternative to the evaluation of comfort by more traditional methods, such as hr monitoring and the use of clinical 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deliveries: evaluation based on behavioral pain scoring and heart rate variability the com-fort behavior scale: a tool for assessing pain and sedation in infants postoperative pain after laparoscopic cholecystectomy is not reduced by intraoperative analgesia guided by analgesia nociception index (ani®) monitoring: a randomized clinical trial ultrasonographic guidance for sciatic and femoral nerve blocks in children epidemiology and morbidity of regional anesthesia in children: a follow-up one-year prospective survey of the french-language society of paediatric anaesthesiologists (adarpef) a comparison of pudendal block vs dorsal penile nerve block for circumcision in children: a randomised controlled trial. anaesthesia analgesia nociception index (ani) to predict intraoperative haemodynamic changes: results of a pilot investigation responsiveness to stimuli of bispectral index, middle latency auditory evoked potentials and clinical scales in critically ill children pain assessment in neonates and infants in the post-operative period following cardiac surgery physiologic correlates of comfort in healthy children the experience of critically ill children: a phenomenological study of discomfort and comfort we are grateful to mmdoloris for providing a nipe monitor for this study. key: cord- -esnyjoq authors: hu, zheng; wang, li; shi, zhaoying; jiang, jing; li, xiangning; chen, yonglong; li, kai; luo, dixian title: customized one-step preparation of sgrna transcription templates via overlapping pcr using short primers and its application in vitro and in vivo gene editing date: - - journal: cell biosci doi: . /s - - - sha: doc_id: cord_uid: esnyjoq overlap extension polymerase chain reaction (pcr) is a powerful technology for dna assembly. based on this technology, we synthesized dna templates, which were transcribed into sgrna in vitro, and further detected their efficiency of purified sgrnas with cas nuclease. the sgrnas synthesized by this approach can effectively cleave the dna fragments of interest in vitro and in vivo. compared with the conventional method for generating sgrna, it does not require construction of recombinant plasmids and design of primers to amplify sgrna core fragment. only several short primers with overlapped sequences are needed to assemble a dna fragment as the template of sgrna. this modified and simplified method is highly applicable and less time-consuming. overlap extension pcr efficiently joins different dna fragments through designing of overlapping primers, which adds an overlap sequence at the end of products and then connect the adjacent fragments with overlap regions. it was first reported that this technology could successfully generate site-directed mutations in a gene [ ] , and then was widely used in a variety of applications such as to synthesize sars genomic fragments [ ] . at present, this technology has more matured and played an important role in modifying dna, including site-specific mutations, synthesis of the long fragment of dna and gene knock out [ , ] . clustered regularly interspaced short palindromic repeats (crispr)/cas system is first found in bacterial and archaea, as an immune defense system to protect against the invasion of viruses and plasmids [ ] . it is composed of cas nuclease, crisprrna (crrna) and transactivating crrna (tracrrna), or a single guide rna (sgrna) fused with crrna and tracrrna. distinct from zfn (zinc finger nucleases) and talens (transcription activator-like effectors), crispr/cas system identifies the target site using sgrna and cleaves target sequence to generate dna double strand breaks (dsbs) with cas nuclease and then initiates different repair mechanisms of non-homologous end joining (nhej) or homologydirected repair (hdr) to produce different types of mutation [ ] . however, conventional methods of generating sgrna either direct synthesis sgrna or requires plasmid construction for pcr amplification to make transcription templates [ ] , which is either expensive or cumbersome and requires several steps to produces grnas. cell & bioscience in the present study, we described a simplified and customized in vitro synthesis approach utilizing multiple overlapping primers to synthesize dna fragment for six selected gene loci as the transcription templates of sgr-nas by a single-step sequential primer extension under appropriate conditions, and tested the cleave efficiency to targeted loci by using these sgrnas combined with cas nuclease in vitro and in vivo. the results demonstrate that the sgrnas produced by overlap extension pcr method could efficiently cleave targeted-dna by combined with cas nuclease, providing a highly applicable method to prepare sgran for targeted locus rapidly. several conventional methods of generating sgrna have been described and applied in gene functional researches ( fig. ). direct sgrna synthesis at commercial companies is a simple method to customize sgrnas exception to being expensive (fig. a) . the conventional method of generating sgrna is in vitro transcription using templates. the direct method to make the templates is to synthesize t -sgrna dna fragments at commercial companies as transcription templates (fig. b) . to generate sgrna transcription templates in lab usual either need a plasmid to construct a recombinant plasmid, or need to take plasmids as templates to amplify transcription templates through using a forward long primer and a reverse primer, which either is cumbersome and requires several steps to produces grnas or synthesis a long primer above bp each time (fig. c, d) . the synthesis of sgrnas transcription templates with overlapping primers consists of four steps and the dna template includes three parts (fig. ) . for each gene, we designed four primers that were connected in turn by overlapping parts as shown in fig. . after denaturation, annealing and extension, a final dna fragment with a fragment size of about bp was synthesized in a pcr instrument, as shown in fig. . the size of the fragment displayed by the electropherogram is consistent with the size of the theoretical primer synthesis, as shown in additional file : table s , demonstrating the feasibility of this method. the conventional methods to generate sgrnaare in vitro transcription using templates. b the direct method to prepare the templates is to dna synthesize t -sgrna fragments as transcription templates. c one of methods is to construct a recombinant plasmid with t -sgrna backbone sequence to be taken as the sgrna transcription template. d another method is to take plasmids with t -sgrna backbone as pcr templates to amplify transcription templates through using a forward long primer and a reverse primer the sgrna dna template synthesized by overlapping primers was shown on electrophoresis photograph (fig. ) . the size of dna template fragments of the three genes were around bp. in additional file : table s , the final combined fragment labeled by af -af -af -tracr-r, the longest products, was the template of sgrna and the size of expected dna fragments similarly assembled by this means were bp. in order to further verified the sequence of the templates, the pcr products were subcloned into pmd -t vector and we chose three e. coli colonies to confirmed by sanger's sequencing. the fig. a -c show the representative sequences of the transcription template of egfr-exon , pu - , pu - . we demonstrated that the dna templates of sgrna synthesized were practicable by using this method. however, it is noteworthy that the design and concentrations of primers in the whole experiment are the key factors affecting the quality of products. in the process of pcr reaction, the concentrations of primers should be adjusted and tested according to the difference in the length of primers. after optimization, the overlapping primers of af , af , af , tracr-r were mixed at the ratios of : : : in the present study, which generated a single dna fragment nearly with no intermediate products. these optimized parameters ease the synthesis of sgrna templates with the same quality as commercial kit, but with time saving and less costly. we detected the efficiency and specificity of sgrna combined with cas nuclease in vitro. the agarose gel indicated that egfr-exon fragment was cut at the target position, leading to the full length of bp cut to two segments with the size about bp and bp. the pu - and pu - were digested into products at the size of bp and bp and the products at the size of bp and bp respectively. these digested fragments were all located at the expected sites after the electrophoresis as shown in fig. a . furthermore, the digested products were subsequently confirmed by dna sequencing analysis. as shown in fig. b-d , the results demonstrated that the three genes were successfully cleaved at the target sites and produced products at the sizes as expected in vitro based on the crispr/cas system. the method established in the present study is feasible to explore gene function with site-specific mutation. in general, crispr/cas technology has been used extensively to elucidate gene function through the regulation of gene expression [ , ] . however, the conventional method of obtaining sgrna is that design primers to amplify dna fragment containing the target site from plasmid as template and then it is transcribed into sgrna, which is more cumbersome and need long primers to amplify [ ] . here, the reported method in the present study only requires several short primers, containing approximately bp overlapped sequence for assembling a complete fragment as template of sgrna. this reported method is faster and more straightforward than conventional one. we demonstrated this approach was an effective tool for functional sgrna synthesis in vitro. these in vitro synthesized sgrnas work well for in vitro digestion of target genes, and have applications in mutation analysis, particularly for the cancer hotspot mutations lacking particular restrictive endonucleases. furthermore, the sgrnas activity and specificity in vivo were tested using x. tropicalis, a frequently used model for study of development as model to assay [ ] . three gene loci, xt.rtbdn, xt.znf . and xt.znf , were selected for these experiments (additional file : tables s , s ). following cas mrna and sgrnas co-injected into embryos of x. tropicalis, the pcr products of xt.rtbdn, xt.znf . and xt.znf targeting sequences ( ) the two primers : tracr-r and af ; ( ) the three primers: tracr-r, af and af ; ( , , ) the templates of egfr, pu - and pu - amplified by four primers respectively. b-d represent the sequencing results of the transcription template of egfr-exon , pu - , pu - respectively. the underlined part represents the designed sequence of sgrna amplified from embryos genomic dna were digested with t endonuclease i, and the fragments of digestion were analyzed by agarose gel as shown in fig. a . the presence of mutated dna fragments were clearly observed and identified as there were two segments by t enzyme digestion on the agarose gel. to further confirmed the sgrnas efficiency, another method was applied to assay the targeted disrupted rate after coinjecting cas mrna/sgrnas into embryos. the pcr products of xt.rtbdn, xt.znf . and xt.znf targeting sequences amplified from embryos genomic dna were used for ta cloning and sequencing. twenty single colonies were sent to dna sequencing analysis. the frequency of indels mutations accumulated in the target site of xt.rtbdn, xt.znf . and xt.znf loci were calculated as described before [ , ] . as showing in fig. b -d, the frequency of indels mutations in xt.rtbdn, xt.znf . and xt.znf gene loci are . %, . % and . %, respectively. together, the results show injection of cas mrna combined with sgrnas generated by the four overlapped primers into embryos of x. tropicalis efficiently induced targeted mutations in vivo. summary, we demonstrated an approach by combining crispr/cas gene editing technology with multiple overlapping primers to synthesize dna fragment as the template of sgrna to substitute the conventional method. the simplified and optimized method is its simplicity as the sgrna dna template can be generated only by the single-step sequential primer extension for rapid production of sgrna, saving time and money fig. a the electropherogram about the result of detection of fragment cleavage site in vitro. egfr, pu - and pu - represent the pcr fragments with bp, bp and bp length respectively. as the negative control "−" indicates that the gene is not digested by cas /sgrna. "+" indicates that the result of digestion by cas /sgrna. b-d the representative sequencing results of the cleaved fragments of egfr, pu - and pu - respectively in comparing with purchasing commercial kit. despite the convenience using commercial kits from companies like as neb or thermo fisher, to generate sgrnas are expensive and inefficient. furthermore, we verified that the sgrna synthesized by this approach was highly efficient and specific in vitro and in vivo. finally, the present study also demonstrated the application of using synthesized sgrna in mutation analysis for cancer hotspot mutations. supplementary information accompanies this paper at https ://doi. org/ . /s - - - . additional file : table s . the targeting sequences of cas /sgrna. table s . overlapping primer sequences for sgrna transcription templates amplification. table s . the corresponding products from each step of the sequential primer extension. crispr: clustered regularly interspaced short palindromic repeats; zfn: zinc finger nucleases; talens: transcription activator-like effectors; crrna: crisprrna; tracrrna: trans-activating crrna; sgrna: a single guide rna; x. tropicalis: xenopus tropicalis; dsbs: dna double strand breaks; nhej: nonhomologous end joining; hdr: homology-directed repair. research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over m website views per year • at bmc, research is always in progress. learn more biomedcentral.com/submissions ready to submit your research ? people's republic of china. translational medicine institute site-directed mutagenesis by overlap extension using the polymerase chain reaction de novo synthesis of pcr templates for the development of sars diagnostic assay gene splicing and mutagenesis by pcr-driven overlap extension creating insertions or deletions using overlap extension polymerase chain reaction (pcr) mutagenesis multiplex genome engineering using crispr/cas systems efficient precise knockin with a double cut hdr donor after crispr/cas -mediated double-stranded dna cleavage efficient rna/ cas -mediated genome editing in xenopus tropicalis ligase iv inhibitor scr enhances gene editing directed by crispr-cas and ssodn in human cancer cells a single administration of crispr/cas lipid nanoparticles achieves robust and persistent in vivo genome editing publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. all data generated or analysed during this study are included in this published article [and its additional information files]. all experiments were carried out according to the guidelines set by the institutional animal ethics committee of the first people's hospital of chenzhou, hunan, p.r. china. not applicable. the authors declare that they have no competing interests. fig. a the disruption rate induced by injection of cas /sgrnas into embryos of x. tropicalis by t e method assay. "−" represents without t enzyme digestion; "+" represents plus t enzymedigestion; and "m" represents dna marker. b-d the disruption rate induced by injection of cas / sgrnas into embryos of x. tropicalis using ta cloning and sequencing analysis. after ta clonging, ta clones were randomly picked up for dna sequencing, and the disruption rate (%) was calculated based on the results of dna sequencing key: cord- - r z otz authors: nakamura, kojiro; kageyama, shoichi; kupiec-weglinski, jerzy w. title: the evolving role of neutrophils in liver transplant ischemia-reperfusion injury date: - - journal: curr transplant rep doi: . /s - - - sha: doc_id: cord_uid: r z otz purpose of review: hepatic ischemia-reperfusion injury (iri), an inevitable event during liver transplantation, represents a major risk factor for the primary graft dysfunction as well as the development of acute and chronic rejection. neutrophils, along macrophages, are pivotal in the innate immune-driven liver iri, whereas the effective neutrophil-targeting therapies remain to be established. in this review, we summarize progress in our appreciation of the neutrophil biology and discuss neutrophil-based therapeutic perspectives. recent findings: new technological advances enable to accurately track neutrophil movements and help to understand molecular mechanisms in neutrophil function, such as selective recruitment to ir-stressed tissue, formation of neutrophil extracellular traps, or reverse migration into circulation. in addition to pro-inflammatory and tissue-destructive functions, immune regulatory and tissue-repairing phenotype associated with distinct neutrophil subsets have been identified. summary: newly recognized and therapeutically attractive neutrophil characteristics warrant comprehensive preclinical and clinical attention to target iri in transplant recipients. liver transplantation (lt) has become the standard of care for patients with end-stage liver disease and those with liver malignancies [ ] . hepatic ischemia-reperfusion injury (iri), an inevitable event during lt, represents a major risk factor for the primary graft dysfunction as well as the development of acute and chronic rejection [ , ] . hence, minimizing iri is this article is part of the topical collection on immunology * jerzy w. kupiec-weglinski jkupiec@mednet.ucla.edu important not only for improving clinical outcomes but also for successful use of marginal liver grafts and expansion of donor organ pool available for transplantation. despite its obvious clinical importance, however, the mechanisms accounting for liver iri are only partially understood and effective preventive or therapeutic strategies remain to be established. in the initial stage of liver iri, ischemic insult renders hepatic cells sensitive to glycogen consumption, oxygen deprivation, ph changes, and adenosine triphosphate (atp) depletion [ ] . these are followed by enhanced production of reactive oxygen species (ros), higher intracellular calcium concentration, and organelle damage, leading to the initial parenchymal cell death [ ] . the reperfusion itself disturbs liver metabolism and evokes inflammatory cascades leading to aggravated hepatocellular damage. innate immune activation plays a central role in this inflammatory response via cytotoxic mechanisms and dynamic cross-talk with adaptive immune cell repertoires, ultimately converting immunologically quiescent hepatic milieu into an inflammatory organ [ , ] . in unstressed human liver, kupffer cells (liver-resident macrophages) account for about % of total hepatocellular population and - % of whole body macrophages [ ] . in iri-lt pathophysiology, both kupffer cells (donor-origin) and liver-infiltrating bone marrow-derived macrophages (recipient-origin) play dominant roles in priming innate immune responses [ ] [ ] [ ] , with the majority of studies focusing on macrophage regulation [ , ] . on the other hand, neutrophils are dominant immune cells in the steady-state blood circulation ( - % in human, - % in mouse [ , ] ), constantly patrolling and serving as the first line of defense against invading pathogens [ ] . likewise, in ir-stressed blood-perfused liver, neutrophils are recruited to the injury site, contributing to sterile inflammation and enhancing the hepatocellular damage. indeed, despite being long considered as "non-specialized" innate effector cells, neutrophil infiltration into hepatic sinusoidal lumen is now considered as one of reliable biomarkers of liver iri [ , ] . in this review, we first summarize progress in our appreciation of the neutrophil biology, and then discuss therapeutic prospects of their targeting for the treatment of inflammatory states, such as iri in lt recipients. neutrophils, the largest circulating fraction of leukocytes, are continuously generated from myeloid precursors in the bone marrow in a process of "granulopoiesis" (daily production reaches up to × cells) [ , ] . the crucial signals for neutrophil activation are provided by danger-associated molecular patterns (damps), i.e., endogenous molecules constitutively expressed in nuclear, cytoplasm, and extracellular matrix under basal conditions. although indispensable for homeostasis maintenance, once released in response to tissue injury, damps are detected by and become critical triggers of the inflammatory cell activation. a growing number of damps have been identified to date, such as atp, histone, high mobility group box (hmgb ), with complementary pattern recognition receptors (prrs) crucial in cell recognition and signaling pathways (representative are listed in table ). the initial parenchymal cell damage results in the release of damps from damaged/dead/moribund cells that stimulate local sentinel kupffer cells and liver sinusoidal endothelial cells (lsecs). kupffer cells sense neighboring cell death by receiving damps signals and produce il β, which up-regulates intercellular adhesion molecule- (icam- ) on lsecs. neutrophils are then recruited via integrin αmβ (mac )-dependent adhesion to endothelial icam- . this neutrophil adhesion mechanism requires atp-induced activation of p x receptor and nod-like receptor pyrin domaincontaining- (nlrp ) inflammasome [ ] . indeed, mice depleted of kupffer cells by clodronate showed reduced caspase, il , and neutrophil recruitment, whereas impaired p x r signaling was accompanied by equivalently impaired neutrophil accumulation in a liver thermal injury model [ ] . activated kupffer cells, along with parenchymal cells, also release chemokines detected by g protein coupled receptors (e.g., cxcr ) on neutrophils and thereby recruit them to the site of inflammatory damage. in addition, lsecs themselves can sense damps via tlr to release il β and il [ ] (fig. a) . because of the unusual hepatic microvasculature, circulating neutrophils can move directly to integrinmediated adhesion devoid of selectin-mediated rolling. in line with this scenario, mac- or icam- neutralizing antibodies effectively alleviated liver iri [ , ] . histone and hmgb are among the most tested damps in the model of liver iri. the levels of circulating histones are significantly increased in liver iri, and anti-histone neutralizing antibodies suppress tlr /myd signaling while decreasing inflammatory hepatic damage [ ] . treatment with hmgb neutralizing antibody alleviated iri in wt but not tlr deficient mouse recipients [ ] . likewise, recombinant thrombomodulin (hmgb inactivator) mitigated liver iri in wt but not tlr -knockout mouse [ •] . since broad spectrum of immune cells express tlrs, the hmgb -tlr inflammatory axis is unlikely to be exclusive for neutrophil regulation. indeed, hepatocyte-specific hmgb deficient mice showed decreased hepatic necrosis and neutrophil accumulation, whereas the number of their macrophages remained unchanged in acetaminophen-induced liver injury model [ ] . in contrast, hepatocyte-specific hmgb -deficient mice exhibited exaggerated liver ir-damage, implying hmgb is essential for hepatocyte resistance against hypoxia [ ] . clearly, extracellular damps are promising therapeutic targets to manage liver iri by alleviating innate, including neutrophil, cell responses. following adhesion to the endothelium, neutrophils are guided by kupffer cell-derived chemokines (cxcl , cxcl ), bind to cxcr on the luminal surface of lsecs, and form the chemotactic gradient at the injury site [ ] (fig. b) . consistent with such a mechanism, reparixin (an allosteric cxcr antagonist) decreased neutrophil infiltration and mitigated liver iri [ ] . in addition, cxcl blocking antibody alleviated hepatic infiltration in necrotic cellinduced neutrophil mobilization model [ ] , whereas in a carbon tetrachloride (ccl )-induced acute liver injury, defective cxcl expression in tlr -knockout or s a -knockout mice was accompanied by suppressed hepatic neutrophil recruitment [ ] . in the following dynamic stage, neutrophils moving on the lsecs luminal side migrate underneath the sinusoidal wall to the damaged site. mitochondrial n-formyl peptides (fmit), cleavage products of mitochondrial proteins, are critically involved in this process serving as chemoattractant damps. damaged cells release fmit and create a gradient that attracts neutrophils through neutrophilspecific formyl peptide receptor (fpr ) [ ] . honda neutrophils move on the lsecs luminal side toward the damaged site, guided by kupffer cell-derived chemokine (cxcl /cxcl ) gradient. c neutrophils detect mitochondrial n-formyl peptides (fmit) via formyl peptide receptor (fpr ) and migrate underneath lsecs toward the injury site. d neutrophils express and secret matrix metalloproteinase (mmp ) to degrade extracellular matrix (ecm) during migration reported that cyclosporine h (fpr antagonist) decreased the number and crawling velocity of neutrophils while alleviating liver iri and inhibiting the accumulation of neutrophils into laser irradiation-induced necrotic areas [ •] . interestingly, at this time point, neutrophils are attracted by two opposing chemoattractant gradients, cxcl mostly anchored on the luminal lsecs side and fmit emanated from parenchymal damaged cells. the final signaling via fmit-fpr overrides the cxcl /cxcl -cxcr axis, making neutrophils to preferentially migrate toward the fmit while ignoring the cxcl attraction (fig. c) . such a chemotactic hierarchy has been consistently observed in pathogen-triggered infection where bacterial formylated peptides are dominant to endogenous chemoattractant [ ] , as well as in sterile inflammation where neutrophils preferentially migrated toward injured cells rather than soluble chemokines [ ] . indeed, marques et al. demonstrated that cxcr and fpr antagonists alleviated neutrophil migration in acetaminophen-induced liver damage, while simultaneous cxcr and fpr antagonism achieved maximum protection [ ] . hence, both cxcl and fmit are critically involved in the hepatic neutrophil migration in a dynamic and sequential manner. the extracellular matrix (ecm), a network of proteins and sugars in all solid organs, is the essential scaffold and structural cell support as well as tissue regulator of cell adhesion, migration, differentiation, proliferation, and survival. once neutrophils migrate across lsec, they move toward damaged site while simultaneously degrading ecm. this process is dominated by matrix metalloproteinases (mmps), zincdependent endopeptidases involved in the breakdown of ecm, and tissue inhibitor of metalloproteinases (timps), which proteolytically regulate mmps activity. mmp and mmp are the most prominent in the pathophysiology of liver iri [ ] . in liver iri, mmp is predominantly expressed by ly g-positive neutrophils, while neutrophils are the only cells in the body which can release mmp free of its endogenous inhibitor timps, and therefore are uniquely capable of delivering highly active mmp to digest its fibronectin and collagen type iv substrates [ ] . fibronectin, a major ecm component of the subendothelial space of disse in normal liver, interacts with integrin receptor α β on activated neutrophils to induce mmp [ ] (fig. d) . hamada et al. demonstrated that mmp deficient neutrophils were impaired in their ability to migrate across fibronectin in a transwell culture system, whereas genetic disruption or exogenous blocking with mmp antibody decreased neutrophil infiltration and cytoprotection in a mouse hepatic iri model [ ] . meantime, ir-insult markedly increases timp expression, an endogenous mmp inhibitor. indeed, timp deficient mice had markedly increased mmp activity and massive neutrophil infiltration in their ir-stressed livers, which has led to % death. in contrast, virus-mediated gene transfer of timp increased timp expression primarily on parenchymal cells, suppressed gelatinolytic activity of neutrophil mmp , attenuated neutrophil infiltration, and alleviated the hepatocellular damage [ , ] . furthermore, lt patients experiencing acute rejection had elevated serum mmp activity at week, implying its role as a potential biomarker in clinical lt [ ] . neutrophils recruited or activated in liver sinusoids do not cause hepatic damage per se, as they can exert cytotoxicity only after migrating across the endothelium and in close proximity to the parenchymal cells [ ] . tissue damage is mediated primarily by ros and proteases, with kupffer cells being principal ros producers early after reperfusion, and neutrophil oxidative burst becoming the main source of ros in the later iri phase. indeed, by h of reperfusion (the peak of hepatocellular damage in a murine iri-lt model), infiltrating ly g-positive neutrophils elaborated large amounts of ros metabolite ( -hydroxynonenal) [ ] . neutrophils generate ros through nicotinamide adenine dinucleotide phosphate (nadph) oxidase, which consists of a rho gtpase, two membrane-bound phox (phagocytic oxidase) proteins (p phox and gp phox ), and three cytosolic phox proteins (p phox , p phox , and p phox ). patients encoded with hypofunctional mutations in gp phox or p phox were protected from transiently induced upper limb iri [ ] , while apocynin (gp phox inhibitor) suppressed ros metabolite ( hydroxy- -deoxyguanosine) and alleviated liver iri in methionine/choline-deficient diet conditioned rats [ ] . meanwhile, neutrophils contain large quantities of serine proteases in the azurophilic granules, such as neutrophil elastase, proteinase , and cathepsin g. after arriving at the damaged site, neutrophils degranulate and release them to exert, in combination with ros, their effector functions. neutrophil elastase, a kda chymotrypsin-like serine protease which degrades ecms causes organ damage, whereas sivelestat (neutrophil elastase inhibitor) is widely used in treating acute lung injury in humans. we and others have reported that sivelestat inhibited adhesion and migration of neutrophils to vascular endothelium and protected mouse livers against iri [ , ] . furthermore, a randomized clinical study in patients that underwent hepatic resection revealed sivelestat treatment reduced post-operative serum hmgb and il levels [ ] . myeloperoxidase (mpo), a heme protein synthesized during myeloid differentiation, is often used as a neutrophil activity marker. in the presence of physiological chloride concentrations, mpo reacts with hydrogen peroxide to catalyze formation of hypochlorous acid/hypochlorite and other oxidizing species [ ] , and thus, it is also considered as a cytotoxic molecule in liver pathophysiology. however, its cytotoxicity in liver iri remains to be rigorously re-evaluated as a recent study demonstrated mpo was required for hepatoprotection in endotoxin-induced inflammation [ •] . with recent data showing efficacy and tolerability of azd (an orally absorbed irreversible human mpo inhibitor) in parkinson patients [ ] , the thorough evaluation of mpo in liver lt settings seems warranted. activated neutrophils can enhance inflammatory tissue damage by releasing neutrophil extracellular traps (nets), i.e., extracellular scaffolds of dna fibers decorated with histone, granule-derived antimicrobial peptides, and enzymes, such as neutrophil erastase, cathepsin g, and mpo [ ] . generation of ros by nadph oxidase and activation of protein-arginine deiminase (pad ), an enzyme that converts arginine to citrulline on histones, are essential steps for chromatin decondensation in the net formation. ne and mpo translocated into the nucleus further unfold chromatin, leading to the nuclear membrane break, chromatin release into cytosol where it is further decorated with granular and cytosolic proteins, and the emission of these traps into extracellular space [ ] . nets function was originally described as the efficient means to immobilize, catch, and eliminate pathogens, whereas recent studies have recognized its critical involvement in noninfectious inflammatory states, including ir-stress. indeed, biopsies obtained from human kidney transplant recipients with post-transplant acute tubular necrosis exhibited increased nets formation [ •], while bronchoalveolar lavage fluid collected from human lung transplant recipients with primary graft dysfunction contained extensive nets [ ] . furthermore, several net-targeting agents successfully attenuated iri in murine models, including pad inhibitors (yw - , yw - ), which reduced nets formation and decreased severity of liver iri [ •]; pad inhibitor (clamidine), which suppressed nets and alleviated renal iri [ •]; or dnase i combined with recombinant tissue-type plasminogen activator, which inhibited nets and alleviated myocardial iri [ ] . additional functional aspects of nets are detailed below. in addition to pro-inflammatory and tissue-destructive functions, recent studies have identified previously unappreciated novel role of neutrophils to control inflammation and resolve tissue damage. first, neutrophils retain the potential to inhibit t cell cytotoxicity. arginase- (arg , a tertiary granule content) is a manganese-containing enzyme expressed in mouse myeloid cells, such as macrophages. however, in humans, it has been detected selectively in neutrophils [ , ] , whereas activated neutrophils release arg during degranulation process, which inhibit t cell proliferation and expansion [ , ] . neutrophil elastase (an azurophilic granule content) cleaves cd , cd , and cd on peripheral blood t lymphocytes, leading to reduced il production and suppressed t cell cytotoxicity [ ] . neutrophil-derived ros may also suppress t cells via inactivation of coffilin, an actin-remodeling protein, which impairs formation of the immune synapse and cell activation [ ] . moreover, as regulatory t cells (treg) are less sensitive to inhibition by ros [ ] , production of ros by neutrophils effectively creates a treg dominant environment [ ] . several lines of evidence indicate that neutrophil can limit inflammation response in other innate immune cells. selective neutrophil ablation or genetic mpo deletion in endotoxinchallenged mice unexpectedly enhanced inflammation responses and increased mortality, indicating that neutrophils and neutrophil-derived mpo can contribute to host resistance by limiting endotoxin-driven innate immune pathology [ •] . cathepsin g (an azurophilic granule content) cleaves nkp and impairs nkp -mediated responses of nk cells, including ifnγ production and cell degranulation [ ] . neutrophilderived microvesicles cause macrophages to generate lipid pro-resolving mediators and tgfβ, leading to counter inflammatory response [ ] . dead neutrophils secret cytosolic protein annexin a (anxa ), which limits neutrophil migration by interacting with formyl peptide receptor (fpr ) [ , ] and prompts neutrophil apoptosis [ ] . clearance of apoptotic neutrophils by macrophage efferocytosis may then switch macrophages to anti-inflammatory il /tgfβ dominant phenotype, leading to tissue-repairing environment as well as treg dominated immune tolerance. analogous to the concept of pro-inflammatory (m ) and anti-inflammatory (m ) macrophage phenotypes [ ] , tissuespecific microenvironment may promote neutrophil polarization into two distinct phenotypes [ ] . thus, n neutrophils are anti-tumoral/pro-inflammatory, having increased tnfα expression and reduced arginase/cxcr /mmp- /vegf levels, whereas n neutrophils are pro-tumoral/anti-inflammatory and characterized by decreased tnfα and increased arginase/cxcr /mmp- /vegf expression [ , ] . several cytokines may contribute to n /n polarization, with tgfβ promoting neutrophils to acquire n phenotype by simultaneously inhibiting n neutrophil generation [ ] . endogenous ifnβ imprints neutrophils to n phenotype, which restrict tumor angiogenesis and enhance inflammatory cytotoxicity [ ] . a recent study demonstrated that analogous to m /m macrophage polarization, lps and ifnγ drove peripheral blood neutrophils toward n phenotype while il polarized toward n phenotype [ ] . in addition, murine infarctedmyocardium was infiltrated by day with n dominant neutrophils expressing pro-inflammatory markers, whereas n neutrophils increased by day concomitantly with enhanced levels of anti-inflammatory markers, supporting putative role of n neutrophils in the resolution of inflammation and promoting tissue repair [ ] . noteworthy, rosiglitazone (peroxisome proliferator-activated receptor-γ agonist) treatment in a mouse brain stroke model shifted neutrophil population toward n phenotype ( % vs. %), suppressed inflammation, enhanced neutrophil clearance, and alleviated brain damage [ ] . although macrophage m /m plasticity is broadly accepted [ ] , neutrophils generally have short lifespan ( - h in blood stream), their plasticity remains unknown. future studies need to elucidate whether n /n polarization is dominated by re-programming of existing neutrophils or environmental influence on de novo neutrophil populations. by virtue of technological advances and efforts to identify neutrophil heterogeneity, additional antiinflammatory neutrophil subsets have been recognized. studies by tirouvanziam et al. identified a cd +/mhc-ii+/cd +/cd + human neutrophil subset in mature cystic fibrosis airway, which retained anabolic/pro-survival phenotype and suppressed t cell function [ , ] . pillay et al. found a subset of mature human cd bright /cd l dim neutrophils in endotoxin-challenged blood, which suppressed t cell proliferation via neutrophil mac- dependent manner [ ] . neutrophils may also contribute to tissue-repairing processes following infectious or sterile inflammatory organ damage [ ] . clinical observations support this concept, as patients with leukocyte adhesion deficiency type (an autosomal recessive disorder characterized by defects in neutrophil adhesion ability) experience delayed wound healing [ ] . in the beginning of tissue repair process, neutrophils acting as professional phagocytes remove tissue debris. indeed, in a thermal hepatic injury model, neutrophil depletion resulted in far more debris remaining at the injury site [ • ]. next, neutrophil apoptosis shifts the phagocytosing macrophages to the resolution phase (m ), which governs tissue-repairing environment. m macrophages secrete il ra, il , tgfβ, and vegf, leading to fibroblast differentiation into myofibroblasts, synthesis of interstitial fibrillar collagens by myofibroblasts, and expression of mmps/timps that control ecm turnover [ ] . since neutrophils can release mmp free of its endogenous inhibitors (timps), they are capable of delivering highly active mmp , a key proangiogenic factor degrading ecm, to release ecm-bounded vegf and other growth factors [ ] . a series of recent studies have deepened our understanding of that process. for instance, christoffersson et al. identified a cd b+/gr +/cxcr high neutrophil subset in a syngeneic mouse pancreatic islet transplant model, which was recruited via vegf-a depending pathway. it contained higher amount of mmp than those recruited to an inflammatory stimulus, and contributed to revascularization via mmp dependent mechanism [ ] . they also found an analogous cd d+/vegfr high /cxcr high neutrophil population in mouse and human, which was recruited by hypoxia-induced vegf-a [ ] . in a liver thermal injury model, limited period of neutrophil depletion (~ h) resulted in delayed revascularization at days and the presence of non-healing injury area at weeks, suggesting the requirement for neutrophil in liver homeostasis [ •] . likewise, in a transplantation model of bioengineered u-graft, which contained an unassembled suspension of vascular cells embedded in a hydrogel, host-derived alternatively polarized neutrophils (n ) contributed to graft revascularization [ • ]. in a successful response to an acute injury, it is crucial to prevent tissue damage by promoting the local resolution of inflammation through the removal of neutrophils from the stressed site [ ] . apoptosis, a highly organized programmed cell death program, is considered a favorable neutrophil death mechanism because the orderly cell elimination limits uncontrolled release of toxic neutrophil contents and damps, which potentially may cause further tissue damage [ ] . morphologically, apoptotic neutrophils show the condensation of chromatin and its migration to the nuclear periphery, fragmentation of nuclear dna, and the blebbing of cell membranes leading to apoptotic body formation. as discussed, engulfment of apoptotic neutrophils by macrophages occurs in the resolution phase of inflammation and repair from tissue injury by turning off production of pro-inflammatory cytokines and launching an anti-inflammatory transcriptional program characterized by the release of il and tgfβ [ ] . in contrast to the organized apoptotic cell death, neutrophil necrosis is a traumatic and highly detrimental event resulting in disintegration of the nuclear envelope and cytoplasmic membranes. neutrophils unavoidably undergo necrosis if the insult is too severe or the step-by-step apoptosis procedure fails to be achieved in a timely fashion, a process called a "secondary necrosis." necrotic neutrophil passively releases toxic effector molecules as well as damps leading to further damage and inflammation in neighboring tissues [ ] . netosis is the process by which neutrophils produce and release nets. during netosis, intracellular proteins become exposed to the extracellular space, which results in the potential presentation of autoantigens to the host immune system and the release of damps that amplify ongoing immune reactions [ ] . in this inflammatory aspect, the netosis seems closer to necrosis than apoptosis. on the other hand, the function of netosis to promote coagulation and thrombosis have been now recognized [ , ] , whereas a recent study from mcdonald et al. has revealed net-platelet-thrombin axis that promotes intravascular coagulation and microvascular dysfunction in endotoxin-induced liver injury model [ •] . considering neutrophils preferentially undergo necrosis/ netosis rather than apoptosis if the stress/infection is severe [ ] , the function of netosis to block blood flow to/from the severely damaged site seems to be favorable by averting the spread of damps/pathogens to remote organs and preventing distant organ injury. in this context, nakazawa et al. revealed nets inhibitor alleviated acute kidney injury while concomitantly reducing serum nets levels and mitigating remote organ injury in the lung, liver, heart, and brain [ • ]. in contrast, homozygous pad knockout in mrsa challenged mice was accompanied by decreased nets and reduced lung injury despite higher bacterial counts, increased levels of inflammatory cytokines, and equivalent overall survival as compared to wt counterparts. the heterozygous pad knockout mice unexpectedly showed improved survival as compared with wt counterparts or homozygous knockout mice [ •] ; in a mouse model of peritonitis, degradation of nets by dnase attenuated organ damage only when combined with antibiotics [ ] ; in an experimental model of a gout (acute sterile inflammatory reaction to monosodium urate crystals), netosis-deficient mice developed exacerbated and chronic disease that could be reduced by adoptive transfer of aggregated nets [ ] . therefore, the influence of nets inhibition depends on disease etiology and severity of the inflammation response. because of increased incidence of acute respiratory distress syndrome (ards) and acute kidney injury in lt patients [ , ] , it is important to evaluate potential clinical value of nets reducing therapeutic strategies. it has long been accepted that apoptosis is the most common and preferred cell death program to terminate neutrophil activity. however, recent evidence suggests that neutrophils do not necessarily die in an inflamed site, and instead can leave the site of tissue damage in a process termed as a "reverse migration." this phenomenon was first visualized in zebrafish larvae in which most of the recruited neutrophils leave a damaged site and traffic to a distal site [ , ] . a recent study combining intravital imaging and photoactivation techniques in a mouse thermal hepatic injury model demonstrated that neutrophils recruited to the stressed tissue neither underwent apoptosis, necrosis, nor phagocytosis by monocytes, but instead reversely migrated to the circulation, became arrested within the lung without causing local tissue injury, and then homed to the bone marrow where they ultimately underwent apoptosis [ •] . in contrast, in hepatic iri or acute pancreatitis model, activated neutrophils were redistributed to produce remote organ injury [ ] [ ] [ ] . it is noteworthy that neutrophils undergoing transendothelial migration in vitro expressed specific marker, icam high / cxcr low , which was resistant to apoptosis and produced more ros [ ] , whereas patients with acute pancreatitis who developed acute lung injury had more icam high / cxcr low expressing neutrophils in their circulation [ ] . this implies the potential for neutrophil reprograming or existence of active/inactive neutrophil subsets after reverse migration. however, there is no rigorous evidence as to why and how reversely migrating neutrophils can be toxic in some cases but inactive in other pathology states. neutrophils have long been recognized as important players in liver iri, while great majority of studies focused on their proinflammatory and tissue-destructive functions. indeed, neutrophil-targeting strategies mostly narrowed at reducing their target organ infiltration. table lists representative preclinical studies on neutrophil targeting in liver iri [ , , , •, , , , •, - ] . it is important to keep in mind that lt recipients are immunocompromised while neutrophils constitute the forefront of pathogen host defense. hence, therapies limiting neutrophil sequestration and/or effector functions may in turn increase the risk of infections in transplant recipients. despite preclinical studies showing therapeutic efficacy, global neutrophil depletion is unlikely to be clinically feasible. more specific maneuvers to inhibit neutrophil adhesion, migration, and effector functions are required, though it remains to be determined whether and how such therapies can achieve optimal balance between effective host immune surveillance while controlling cytodestruction. albeit non-specifically targeting neutrophil functions, the reduction of extracellular damps such as hmgb or histone seems favorable, because damps are the immune-activating signals released from host cells but not from pathogens. of note, one of the hmgb inactivators (art- , recombinant thrombomodulin), originally shown to mitigate liver iri in tlr depending manner [ •] , is now used clinically for disseminated intravascular coagulation [ , ] . in agreement with recent data [ •, •] , inhibition of nets formation by specifically targeting highly destructive neutrophil function seems biologically beneficial. however, futures studies need to evaluate potential risks, as this maneuver may spread abundant damps or pathogens, which otherwise should be isolated inside the nets areas. since dnase has been successfully applied for the treatment of cystic fibrosis patients and the nets digestion is considered to be a part of its beneficial effects [ , ] , these investigations are clearly warranted. although a number of recent studies revealed neutrophil immunoregulatory and tissue-repairing capabilities, little is known about these functions in liver iri. this might be due to the overwhelming focus on the "toxic" side of the neutrophil biology. although t cells, particularly of cd + phenotype, are indispensable for the activation/regulation of sterile inflammation in liver iri [ , ] , it seems that the presence of cd + t cells per se rather than ag-specific de novo t cell activation is needed for immune regulation [ ] . hence, the ability of neutrophils to reduce t cell activation may be insufficient to significantly impact iri outcomes. in addition, the severe hepatocellular damage produced in currently used murine iri models may lead to a failure in detecting discrete immunomodulatory and/or tissue-repairing neutrophil functions. future studies need to focus on neutrophil involvement in the resolution of inflammation and recreation of tissue homeostasis. experimental evidence indicates the antiinflammatory (n ) neutrophil polarization by tgfβ, il or rosiglitazone [ ] [ ] [ ] . although the neutrophil plasticity has not been rigorously proven, the n to n phenotype shifting should be of a potential therapeutic interest. neutrophil apoptosis is a favorable way of its functional termination, leading to an immunomodulatory shift of phagocytosing kupffer cells and prevention of the damps burst, while excessive induction of neutrophil apoptosis may cause unnecessary immune suppression. indeed, ectoine, a natural cell protectant, improved inflammatory resolution in a sterile inflammation lung model by preferentially prompting apoptosis in activated but not inactivated neutrophils, reducing neutrophil numbers and improving resolution of the lung pathology [ ] . furthermore, a recent randomized clinical trial in elderly patients highlight the efficacy of inhaled ectoine in neutrophilic lung inflammation [ ] . studies by christoffersson et al. have identified vegf-a-induced cxcr high unique neutrophil subset, which retained the ability of revascularization in a pancreatic islet transplant model [ , ] , while a study by wang et al. [ •] implied cxcr expressing noninflammatory neutrophil subset capable of homing back to bone marrow in the "reverse migration" mechanism [ ] . thus, one may envision a great potential for future regimens shifting neutrophils into tissue-resolving cxcr -positive dominant population. due to technological advances, a remarkable progress has been recently made in our appreciation of the neutrophil biology, especially its immune regulatory and tissue-repairing functions. live-cell and deep-tissue imaging combined with single cell flow cytometry analyses [ ] allow to accurately track neutrophil functions and interactions in real-time. unlike lysm reporter mice, unable to distinguish between neutrophil and monocyte/macrophage lineages, a newly developed murine strain with a more specific ly g promoter [ ] has enabled to investigate in vivo neutrophil functions more selectively and unequivocally. since efficient strategies against hepatic iri have not been yet developed, newly discovered and therapeutically attractive neutrophil functions, as discussed in this review, warrant future comprehensive preclinical and clinical attention in transplant recipients. conflict of interest the authors declare that they have no conflict of interest. human and animal rights and informed consent this article does not contain any studies with human or animal subjects performed by any of the authors. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. particular interest, published recently, have been highlighted as: • of importance challenges to liver transplantation and strategies to improve outcomes the utility of marginal donors in liver transplantation ischaemia-reperfusion injury in liver transplantation-from bench to bedside increased ischemic injury in old mouse liver: an atp-dependent mechanism mechanisms of hepatic injury and protective effects of nitric oxide liver ischemia and reperfusion injury: 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therapy molecular mechanisms of net formation and degradation revealed by intravital imaging in the liver vasculature the emerging role of t cell immunoglobulin mucin- in the mechanism of liver ischemia and reperfusion injury in the mouse t-cell immunoglobulin mucin- determines severity of liver ischemia/reperfusion injury in mice in a tlr -dependent manner cd t cells promote tissue inflammation via cd signaling without de novo activation in a murine model of liver ischemia/reperfusion injury recovery of neutrophil apoptosis by ectoine: a new strategy against lung inflammation reduction of neutrophilic lung inflammation by inhalation of the compatible solute ectoine: a randomized trial with elderly individuals neutrophil mobilization and clearance in the bone marrow turning on the lights inside neutrophils catchup: a mouse model for imaging-based tracking and modulation of neutrophil granulocytes key: cord- -y zpvw authors: tan, kai sen; andiappan, anand kumar; lee, bernett; yan, yan; liu, jing; tang, see aik; lum, josephine; he, ting ting; ong, yew kwang; thong, mark; lim, hui fang; choi, hyung won; rotzschke, olaf; chow, vincent t; wang, de yun title: rna sequencing of h n influenza virus-infected human nasal epithelial cells from multiple subjects reveals molecular pathways associated with tissue injury and complications date: - - journal: cells doi: . /cells sha: doc_id: cord_uid: y zpvw the human nasal epithelium is the primary site of exposure to influenza virus, the initiator of host responses to influenza and the resultant pathologies. influenza virus may cause serious respiratory infection resulting in major complications, as well as severe impairment of the airways. here, we elucidated the global transcriptomic changes during h n infection of human nasal epithelial cells from multiple individuals. using rna sequencing, we characterized the differentially-expressed genes and pathways associated with changes occurring at the nasal epithelium following infection. we used in vitro differentiated human nasal epithelial cell culture model derived from seven different donors who had no concurrent history of viral infections. statistical analysis highlighted strong transcriptomic signatures significantly associated with and h after infection, but not at the earlier -h time point. in particular, we found that the influenza infection induced in the nasal epithelium early and altered responses in interferon gamma signaling, b-cell signaling, apoptosis, necrosis, smooth muscle proliferation, and metabolic alterations. these molecular events initiated at the infected nasal epithelium may potentially adversely impact the airway, and thus the genes we identified could serve as potential diagnostic biomarkers or therapeutic targets for influenza infection and associated disease management. the global burden of inter-pandemic influenza is high. it is estimated to affect billion people annually, with - million severe cases requiring hospitalization or intensive care treatment, resulting in approximately . million deaths [ ] . worryingly, drug-resistant influenza strains are emerging at a rapid rate that will severely hamper the ability of our healthcare systems to contain influenza outbreaks [ ] . therefore, alternative strategies are needed against severe influenza infections during both seasonal and pandemic influenza outbreaks. the normal human airway epithelium is a pseudo-stratified layer of ciliated and non-ciliated columnar cells, goblet cells, club cells, and basal cells [ ] . the airway epithelium protects against airway infection via efficient mucociliary clearance (mcc), the production of inflammatory mediators and chemokines against viruses, and the recruitment of immune cells [ ] . when the influenza virus breaches the defense of the human airway epithelium, it causes a myriad of innate responses by the infected host in response to viral invasion [ , ] . among these changes are critical factors that can determine disease severity, and which may lead to the development of diagnostic, prognostic prediction markers, or anti-influenza therapies [ ] [ ] [ ] [ ] . however, few studies have hitherto been performed in relevant models [ ] and human models of influenza are not feasible due to potential severity of the infection. therefore, the mechanistic study of viral-induced airway changes using relevant models can lead to better understanding of the development of severe complications. additionally, we need greater clarity on the different immune responses in view of the rising prevalence of chronic diseases such as diabetes mellitus and asthma. patients with these disorders are especially susceptible to severe influenza complications compared to healthy subjects [ ] . thus, the establishment of a baseline response against influenza infection of "healthy" tissue is beneficial to facilitate future comparative studies to better manage influenza in patients with co-morbidities. although the study of host responses in influenza infection is not new, current in vitro cell lines cannot accurately represent human airway infection due to the lack of key mucociliary features [ ] . hence, we have previously developed an air-liquid interface (ali) human airway epithelial cell culture that is able to sustain influenza infection [ , ] . we have also further compared the transcriptomic responses of our infected human nasal epithelial cells (hnecs) with other in vitro and in vivo influenza infection transcriptomic studies [ ] . the comparison revealed that at their peak responses against influenza, the differential transcriptome signature in hnecs was highly similar to the signatures from other influenza infection models [ ] . interestingly, compared to the homogenous cell lines tested, our heterogenous hnec model exhibited a more comparable response to the clinical influenza studies, indicating that most responses were initiated at the nasal epithelium [ ] . therefore, in this study, we aim to further utilize the hnec model as a physiologically relevant in vitro model to clarify the nasal epithelial responses against influenza h n infection, which would then facilitate the identification of the key host factors that are significant for future studies. to establish host factors that are significantly altered in the nasal epithelium as a reference of early innate responses against influenza, the dynamic expression of the genes needs to be clearly elucidated. while there are many studies that utilize microarray analysis to identify the host responses against influenza, the limitation of the microarray is its inability to determine the full extent of gene changes due to its hybridization-based protocol [ ] . the aim of this study was to utilize rna sequencing (rnaseq) technology to not only reveal the hnec responses (from multiple individuals) against influenza infection, but also to identify those genes with high magnitude changes to serve as potential reference markers of the innate responses of influenza infection. given that rnaseq functions by reading virtually all the rnas present in the samples tested, we can also discern the magnitude of each rna change and mark them as the canonical responses. in addition, as rnaseq is not constrained by probe usage as in microarrays, they are therefore more reliable in detecting novel interactions during influenza infections of hnecs. hence, rnaseq analysis will further augment the transcriptomic data established previously by microarray analysis. the augmented baseline can then be applied to future clinical studies and practice against influenza infection, especially for comparison against patients with other underlying co-morbidities that may be affected by more severe disease. approval to conduct this study was obtained from the national healthcare group domain-specific board of singapore (dsrb ref: d/ / ) and the institutional review board of the national university of singapore (irb ref: . written consent was obtained from donors prior to the collection of the tissue biopsies. at the time of collection, all subjects were free of symptoms of urti. the medical backgrounds of the subjects are summarized intable s . the hnespcs were isolated and enriched from the tissue biopsies according to a previously standardized protocol [ , ] , which normalized the hnespcs to a baseline state that differentiates into hnecs resembling healthy tissues if they pass the quality control checks for their differentiation [ ] . following enrichment, the hnespcs were expanded further and subjected to ali culture in transwells for in vitro differentiation according to previous protocol as well [ , ] . briefly, primary cells were subjected to isolation for selection of hnespcs, which were enriched and expanded with dulbecco's modified eagle medium: nutrient mixture f- (dmem/f ) (gibco-invitrogen, carlsbad, ca, usa) containing ng/ml of human epithelial growth factor (egf, gibco-invitrogen, carlsbad, ca, usa), µg/ml of insulin (sigma, st. louis, mo, usa), . nm of cholera toxin (sigma, st. louis, mo, usa), . µg/ml of hydrocortisone (sigma, st. louis, mo, usa), nm/ml of , , -triiodo-l-thyronine (t ) (sigma, st. louis, mo, usa), µl/ml of an n- supplement (gibco-invitrogen) and iu/ml of antibiotic-antimycotic (gibco-invitrogen, carlsbad, ca, usa). the expanded hnespcs were then transferred onto -well . µm transwell inserts (corning, corning, ny, usa). once confluent, growth medium was discarded and µl of pneumacult™-ali medium with inducer supplements (stemcell technologies inc., vancouver, canada) was added to the basal chamber to establish ali conditions. the cells were cultured in ali culture for weeks, with media change every - days. after - weeks of differentiation, hnecs from a total of seven donors were then subjected to influenza h n virus infection. the influenza a strain used in this study is of the h n subtype (a/aichi/ / ) (atcc, manassas, va, usa). the virus was propagated using embryonated egg culture and used for all the infection in the hnecs. prior to infection, fully differentiated hnecs were washed with × dpbs and infected with the h n influenza virus at a multiplicity of infection (moi) of . and incubated for h at • c. after the h incubation, the viral inoculum was removed and the hnecs were incubated back in • c. the control hnecs were harvested for apical wash and rna prior to the infection at h post-infection (hpi). the infected hnecs were then harvested for the apical wash and rna following , , and hpi incubation at • c. at each infection time point, µl of x dpbs was added and incubated in the apical chamber for min at • c to recover progeny viruses as the apical wash. the plaque assay for viral quantification was performed using overnight mdck cultures (atcc, manassas, va, usa) at - % confluence in -well plates. the mdck cells were incubated with µl of serial dilutions (from − to − ) of virus from apical washes at • c for h, where plates were rocked every min to ensure equal viral distribution. after incubation, the inocula were removed and replaced with ml of avicel (fmc biopolymer, philadelphia, pa, usa) overlay, and incubated at • c for - h. after incubation, avicel overlay were removed, and cells were fixed with % formaldehyde in × pbs for h. formaldehyde was then removed, and cells were washed with × pbs prior to staining with % crystal violet for min before washing the stain away. the plaque-forming units (pfu) were calculated as follows: number of plaques × dilution factor = number of pfu per µl. at each time point after the collection of apical wash, the hnecs were lysed using rna lysis buffer. total rna was then extracted from the lysate using mirvana mirna isolation kit (life technologies, grand island, ny, usa). the extracted total rna was first subjected to nanodrop analysis to first ensure the rna quality, before being submitted for rnaseq analysis. then, ng from the remaining rna was subjected to cdna synthesis using maxima first-strand cdna synthesis kit (thermoscientific, pittsburgh pa, usa). after this, qpcr analysis was performed to evaluate the transcriptional levels of host response genes selected based on previous microarray analysis using pre-designed primers (sigma aldrich). each qpcr reaction was performed in duplicate using gotaq-qpcr master mix kit (promega, san luis obispo, ca, usa), and relative gene expression was calculated using the comparative method of -∆∆ct normalized to the housekeeping gene pgk . relative gene expression levels were presented as median values and interquartile ranges, while statistical significance was determined using the wilcoxon signed-rank test. all human rnas were analyzed on the agilent bioanalyzer (agilent, santa clara, ca, usa) or the perkin elmer labchip gx system (perkin elmer, waltham, ma, usa) for quality assessment with rna integrity number (rin) or rna quality score range from . - . and median of . . cdna libraries were prepared using ng of total rna and µl of a : , dilution of ercc rna spike in controls (ambion ® thermo fisher scientific, waltham, ma, usa) using smartseq v protocol [ ] , except for the following modifications: fastq files were mapped to the human genome build hg using star. gene counts were computed using featurecounts (part of the subread package) using annotations from gencode version . differential gene expression analysis was performed using edger in a paired fashion under r version . . . multiple testing correction was done using the method of benjamini and hochberg and p-values (false discovery rate; fdr) less than . was deemed to be significant. geneset enrichment analysis using data from gene ontology (go) was performed using the bioconductor package topgo, while the analysis using reactome pathway was performed using the vioconductor package reactomepa. both analyses were run in r version . . using multiple testing-corrected significant differentially-expressed genes. tnf-α, tnf-β, vegf, eotaxin/ccl , and pdgf-aa. samples and standards were incubated with fluorescent-coded magnetic beads which had been pre-coated with the respective capture antibodies. after an overnight incubation at • c, plates were washed twice. biotinylated detection antibodies were incubated with the complex for h, and streptavidin-pe was then added and incubated for another min. plates were washed twice again, then beads were re-suspended with sheath fluid before acquiring on the flexmap ® d (luminex) using xponent ® . (luminex) acquisition software. data analysis was done on bio-plex manager™ . . (bio-rad). standard curves were generated with a -pl ( -parameter logistic) algorithm, reporting values for both mfi and concentration data. results were then expressed as mean fold change compared with uninfected control, and p-values (fdr) of less than . were considered significant. prior to analysis, the responses of all seven hnecs donors following influenza infection were plotted on a principal component analysis (pca) plot. the pca plot indicated a degree of variability in the responses between donors and time points (figure ). nonetheless, the responses were clustered tightly enough following infection to signify their consistency of infection for further transcriptomic analysis-similar to those observed in our previous study [ ] . the respective capture antibodies. after an overnight incubation at °c, plates were washed twice. biotinylated detection antibodies were incubated with the complex for h, and streptavidin-pe was then added and incubated for another min. plates were washed twice again, then beads were resuspended with sheath fluid before acquiring on the flexmap ® d (luminex) using xponent ® . (luminex) acquisition software. data analysis was done on bio-plex manager™ . . (bio-rad). standard curves were generated with a -pl ( -parameter logistic) algorithm, reporting values for both mfi and concentration data. results were then expressed as mean fold change compared with uninfected control, and p-values (fdr) of less than . were considered significant. prior to analysis, the responses of all seven hnecs donors following influenza infection were plotted on a principal component analysis (pca) plot. the pca plot indicated a degree of variability in the responses between donors and time points ( figure ). nonetheless, the responses were clustered tightly enough following infection to signify their consistency of infection for further transcriptomic analysis-similar to those observed in our previous study [ ] . significant gene expression changes (fdr < . ) of infected hnecs were detected as early as hpi, and further increased at and hpi (table ; figure a ). also, the number of genes decreased in a linear fashion as the fold change in expression increased, as seen in the x fold change genes significant gene expression changes (fdr < . ) of infected hnecs were detected as early as hpi, and further increased at and hpi (table ; figure a ). also, the number of genes decreased in a linear fashion as the fold change in expression increased, as seen in the x fold change genes indicated in figure b , where about % of the significantly altered genes remained. at hpi, there were upregulated genes and downregulated genes. the major upregulated genes were the antiviral sensors and early response genes such as ifns, ifits, and ifis. interestingly enough, interferon lambda (ifnλ) gene ifnls was the earliest response interferon of infected hnecs, as opposed to interferons alpha or beta, at hpi. at later time points, the number of gene expression changes increased substantially, with upregulation of and genes, and downregulation of and genes at and hpi, respectively. there was augmented expression of antiviral effectors and inflammatory genes at both time points. ifnl remained the interferon gene with highest expression at both time points, while a marked elevation of cytokines such as cxcl and cxcl was also observed. considering downregulated genes, proliferative and transcriptomic functions appeared to be suppressed, with diminished expression of genes such as fmo , klk , and fosb. genes associated with metabolism, cell cycle, and dna repair were further suppressed following infection at and hpi. tables s -s list the complete set of significant gene expression changes, arranged according to their fold change (log fc). in addition, we have also verified that the genes showing major expression changes by rnaseq generally concurred with rt-qpcr analyses. of the genes tested by qpcr at hpi, all of them showed the same directional changes in expression as observed by rnaseq. hence, seven of these genes showed a p-value of < . (il i , ifnl (il ), cxcl , tnfsf , ifi , ccl , and cyp a ), one gene had a p-value of < . (ctgf), while only two genes were not statistically significant (tgfa and ano ) ( figure s ). indicated in figure b , where about % of the significantly altered genes remained. at hpi, there were upregulated genes and downregulated genes. the major upregulated genes were the antiviral sensors and early response genes such as ifns, ifits, and ifis. interestingly enough, interferon lambda (ifnλ) gene ifnls was the earliest response interferon of infected hnecs, as opposed to interferons alpha or beta, at hpi. at later time points, the number of gene expression changes increased substantially, with upregulation of and genes, and downregulation of and genes at and hpi, respectively. there was augmented expression of antiviral effectors and inflammatory genes at both time points. ifnl remained the interferon gene with highest expression at both time points, while a marked elevation of cytokines such as cxcl and cxcl was also observed. considering downregulated genes, proliferative and transcriptomic functions appeared to be suppressed, with diminished expression of genes such as fmo , klk , and fosb. genes associated with metabolism, cell cycle, and dna repair were further suppressed following infection at and hpi. tables s - list the complete set of significant gene expression changes, arranged according to their fold change (log fc). in addition, we have also verified that the genes showing major expression changes by rnaseq generally concurred with rt-qpcr analyses. of the genes tested by qpcr at hpi, all of them showed the same directional changes in expression as observed by rnaseq. hence, seven of these genes showed a p-value of < . (il i , ifnl (il ), cxcl , tnfsf , ifi , ccl , and cyp a ), one gene had a p-value of < . (ctgf), while only two genes were not statistically significant (tgfa and ano ) ( figure s ). we then further compared the transcriptomic alterations in the hnecs over time, following influenza h n infection. the number of gene expression changes mirrored the viral titer changes, which peaked at hpi, and were consistent between donors ( figure a ). approximately two thirds of genes at and hpi overlapped with other time points, while about one third of genes at hpi overlapped ( figure b ). the overlapping genes displayed similar directional consistency at the significant time points. in addition, congruent with the consistent viral titer with most gene expression changes at hpi, we also noted the most consistent alterations in expression of genes across donors. this is highlighted in figure c , which portrays the heatmaps of the top genes with the smallest p-value, together with their direction and magnitude of change. based on these analyses, we proposed that hpi represents the optimal time point for the subsequent pathway analysis to ascertain influenza-specific pathway changes. we then further compared the transcriptomic alterations in the hnecs over time, following influenza h n infection. the number of gene expression changes mirrored the viral titer changes, which peaked at hpi, and were consistent between donors ( figure a ). approximately two thirds of genes at and hpi overlapped with other time points, while about one third of genes at hpi overlapped ( figure b ). the overlapping genes displayed similar directional consistency at the significant time points. in addition, congruent with the consistent viral titer with most gene expression changes at hpi, we also noted the most consistent alterations in expression of genes across donors. this is highlighted in figure c , which portrays the heatmaps of the top genes with the smallest p-value, together with their direction and magnitude of change. based on these analyses, we proposed that hpi represents the optimal time point for the subsequent pathway analysis to ascertain influenza-specific pathway changes. we then further subjected the significant gene changes to gene set enrichment using both go and reactome databases. at time points , , and hpi, there were , , and significant (adjusted p-value < . ) go biological processes (table s ) and , , and significant (adjusted pvalue < . ) reactome pathways (table s ) , respectively. at the early time of hpi, interferon- we then further subjected the significant gene changes to gene set enrichment using both go and reactome databases. at time points , , and hpi, there were , , and significant (adjusted p-value < . ) go biological processes (table s ) and , , and significant (adjusted p-value < . ) reactome pathways (table s ) , respectively. at the early time of hpi, interferon-mediated antiviral responses were elevated as expected. at hpi, the pathways appeared to be more stabilized and consistent for both go and reactome analyses, despite displaying more gene expression changes at this time point. responses to influenza virus skewing towards type i immunity were predominant in the go analysis. the expected interferon-mediated functions by the epithelium validated the authenticity of our model, where we found enriched type i interferon (go) and rig-i (reactome) pathways with upregulation of nearly all significant gene members (data not shown). besides the interferon and antiviral pathways, we identified several functions of interest initiated by the nasal epithelium that may contribute to the pathology and pathogenesis of influenza. at hpi, go pathway enrichment analysis revealed that the nasal epithelium was actively involved in initial ifnγ signaling (go: ), despite not directly producing ifnγ. we also observed enriched function in apoptosis and necroptosis (go: and go: ), immune evasion (go: ), and other pathways that may lead to complication events such as smooth muscle proliferation (go: ) and response to fatty acid (go: ) ( table ). for the reactome pathway analysis, we selected pathways that were enriched with more than significant genes present in the enriched pathway, and these were generally in agreement with the go analysis (table ). in addition to ifnγ signaling ( ) and apoptosis ( ), it also revealed changes in epithelial-initiated b cell receptor signaling ( and ) and amino acid metabolism ( ) following influenza infection. it is noteworthy that these pathways were initiated at the epithelial level without the participation of immune cells, thus highlighting the relevant genes of interest for future studies. given that rnaseq analysis facilitates more accurate expression changes following infection compared to hybridization technology such as microarray, we conducted further analysis on the levels of gene expression changes to enable more stringent and accurate transcriptomic analyses for future studies. by comparing these results to a previous study that identified influenza-specific signatures, we verified that these genes were all expressed in infected nasal epithelium later at hpi, but not at hpi. furthermore, at both and hpi, all but one of the gene signatures exhibited elevated expression of > . -fold change (> . log fc) compared to uninfected control hnecs (table ). when we applied the higher fold change cutoff, the number of significant genes decreased by approximately % (figure a) , which was also congruent with the linear association observed earlier. therefore, future studies on early transcriptional alterations could consider adopting the . -fold change in expression as a more stringent threshold, which may be more feasible, especially for large transcriptomic studies that yield large numbers of data points. in addition, when compared to the previous microarray study on a similar hnec model [ ] , both rnaseq and microarray shared a high degree of overlap, with about one third and half of total genes from rnaseq and microarray overlapping, respectively ( figure b ). the overlap was generally observed in genes with highly altered expression, such as cxcl , cxcl , and rsad , which were changed to a similar magnitude in both rnaseq and microarray (table s ). when we compared the influenza signature genes, rnaseq revealed a more consistent increase in magnitude, i.e., at hpi, the magnitude of the gene change was generally higher than that of the microarray (table ). in addition, rnaseq was also able to detect novel genes with expression changes of high magnitude that were generally higher than those found by microarray only ( genes versus genes with elevated expression greater than . -fold). genes such as heatr , pdcd , il i , art , and kcnh were altered to a higher magnitude than the . -fold threshold. hence, rnaseq-based transcriptomic analysis may augment transcriptomic findings to identify novel gene responses against influenza in the future. from rnaseq and microarray overlapping, respectively ( figure b ). the overlap was generally observed in genes with highly altered expression, such as cxcl , cxcl , and rsad , which were changed to a similar magnitude in both rnaseq and microarray (table s ). when we compared the influenza signature genes, rnaseq revealed a more consistent increase in magnitude, i.e., at hpi, the magnitude of the gene change was generally higher than that of the microarray (table ). in addition, rnaseq was also able to detect novel genes with expression changes of high magnitude that were generally higher than those found by microarray only ( genes versus genes with elevated expression greater than . -fold). genes such as heatr , pdcd , il i , art , and kcnh were altered to a higher magnitude than the . -fold threshold. hence, rnaseq-based transcriptomic analysis may augment transcriptomic findings to identify novel gene responses against influenza in the future. after deriving the transcriptomes by rnaseq, we then further investigated whether the changes in expression of genes resulted in alterations in secretory cytokines and chemokines early in the infection of hnecs. initially, we detected significant reductions in multiple cytokines at hpi, with the exception of il- which was increased ( figure s ). this may reflect the initial immune suppression during influenza infection. however, at and hpi, less significant changes were observed, i.e., only increase in tnf-a and decrease in mdc and pdgf-aa were noted at hpi. this was followed by increase in ip- (cxcl ) and tgf-a and decrease in pdgf-aa seen at hpi. this analysis highlights changes in ip- , tgf-a, and pdgf-aa to be significant early responses in secretory cytokines/chemokines following influenza infection. our study has identified epithelium-initiated host responses which are found to be involved in both innate and adaptive responses. the finding is significant as we can now focus on the primary point of contact of influenza-the nasal epithelium in the study of early host responses for identifying host factors that can be utilized for diagnostic and therapeutic purposes [ ] . in addition, our study also showed that it is important for reference databases to use relevant human models like the hnecs model, which contains the mucociliary component of the airways, in order to provide closely representative host responses. while there exists a high number of microarray studies that showed the host responses using similar hnec models, there are only a small number of equivalent rnaseq studies. compared to microarrays, rnaseq analysis can provide a more comprehensive picture of the transcriptomic landscape, and is not limited by the hybrid library variant and concentrations [ ] . hence, in order to derive accurate magnitude of gene expression changes, we performed an rnaseq analysis of h n infection using the hnecs model. h n influenza virus was selected, given that it is a major circulating subtype over long periods of time. in addition, relatively lower efficacy of vaccines against this subtype prompted us to study its interactions with the primary host target to elucidate the immune responses and association with adaptive immunity [ , ] . this model has been previously evaluated to be a highly clinically-relevant model that can facilitate controlled infection of nasal cells from multiple individuals. in addition, we have also previously shown-by microarray analysis-that the nasal epithelium is responsible for the initiation of host responses following influenza infection [ ] . this renders the hnecs to serve as a valuable tool to analyze transcriptomics from different individuals infected under the same conditions to ensure consistent and relevant responses in humans. once the magnitude of gene expression changes was considered, several interesting findings emerged. firstly, the infected hnecs were observed with strong activation of antiviral genes and early inflammatory genes leading to type i immune responses. a large number of gene expression changes were of magnitude of over -fold difference (log to log fold change). most of the genes with high-magnitude expression changes were verified by qpcr, with statistical significance congruent with the rnaseq analysis. secondly, despite the absence of immune cells, the infected hnecs were able to generate strong type i responses that may likely aid the recruitment of cytotoxic cells to clear the infected cells. thirdly, in early responses of the hnecs, ifnλ genes, which represent type iii interferons, were more strongly induced than the more frequently observed type i interferons (ifnα and ifnβ), while type ii interferons were not produced by hnecs, in agreement with previous studies [ , ] . the induction of type iii interferons may reflect an important event within the hnecs where ifnλ, the initial responders against the infection, may be more beneficial in the antiviral response [ , ] . moreover, we also observed notable suppression of expression of certain genes following influenza infection, including suppression of proliferation and dna repair genes, which may contribute to the pathology and pathogenesis of influenza [ ] . finally, rnaseq also unraveled expression changes of certain newly-discovered genes in response to influenza infection of the upper airway cells. genes such as heatr [ ] , il i [ ] , tnfsf b (baff) [ ] , and pdcd (pd- ) [ ] are recently implicated in influenza pathogenesis and mucosal defense, thereby signifying the role of the nasal epithelium against influenza infection. furthermore, rnaseq identified altered expression of art and kcnh genes that were not previously detected in influenza transcriptomes. these findings hence further reiterate the value of rnaseq in enhancing data on influenza transcriptomes for reference in future studies. via pathway enrichment analysis, we have identified known antiviral pathways to validate the hnecs responses against influenza. in addition, we have also documented the potential pathways initiated by the nasal epithelium that may contribute to influenza pathogenesis as represented by the gene expression changes listed in tables and . by analyses using literature-inferred go and reactome databases, we have demonstrated that the nasal epithelium can play a role in the main antiviral signaling, i.e., ifnγ responses despite not being a direct producer of ifnγ. the pathway enrichment indicated that hnecs may serve as important regulators of type ii interferons. even though the effects of ifnγ are vital to the robust clearance of influenza viruses [ ] , there are reports of unregulated ifnγ being a contributor to inflammatory damage [ , ] . therefore, the over-production of ifnγ response factors such as icam and cd may contribute to inflammatory damage of the epithelium. hence, production of factors such as stat [ ] by the hnecs is also crucial in ensuring appropriate regulation of ifnγ-mediated expression of influenza response genes to modulate inflammation and to minimize damage. the primary contact of influenza virus with the nasal epithelium may subsequently lead to damage to the airway epithelium as well. this is apparent with the clear enrichment of the pathways of apoptosis, mitochondrial apoptotic processes, and necroptosis that contribute to cell death and mechanical barrier loss during infection [ , ] . genes such as ifi , bak , caps , tnfsf , and fas suggest active apoptotic cell death that not only destroys cells in the epithelial barrier, but may also serve to propagate the virus and to perpetuate the damage [ ] [ ] [ ] . furthermore, during virus infection, aberrant regulation of apoptosis may also lead to further injury to the epithelium and surrounding tissues [ ] . on the other hand, necroptosis pathways have also been observed to be enriched in influenza-infected hnecs. compared to apoptosis, the study of necroptosis in influenza infection is relatively new with contradicting findings [ ] . ripk /necroptosis studies appear to generate contradictory results as to whether necroptosis protects against or is detrimental during influenza infection [ , ] . hence, its increased expression during infection of hnecs warrants further investigation on its role in influenza-induced damage. in addition, we also noted enrichment of b-cell signaling pathways by the infected hnecs which may be vital for b-cell responses during the adaptive immune response [ ] . we noted that most genes enriched in the b-cell pathways were related to antigen recognition such as proteasome subunits (psme , psmb , psma , etc.) and b-cell receptor-associated genes such as dapp and card [ ] . however, changes in expression of certain growth factors (including ereg and fgfs) following influenza infection may lead to complications involving airway remodeling and recruitment [ , ] . further, the effects of the growth factors were further confirmed by the enrichment of pathways related to the proliferation of smooth muscle cells also induced by the infection. changes to airway smooth muscle cells are usually implicated in airway remodeling [ ] [ ] [ ] , and may also contribute to post-influenza complications. hence, the genes found in this study may be crucial for elucidating the nasal-initiated responses that may contribute to the pathology and pathogenesis of influenza infection of the airways. finally, another interesting pathway that may contribute to epithelial damage is the negative regulation of innate immune responses. these genes may serve as proviral factors and aid in immune evasion. for example, adar is a proviral factor that works in synergy with influenza ns to enhance viral replication [ ] . trafd is a negative regulator of toll-like receptor signaling which is upregulated in influenza-infected hnecs [ ] . dhx is a negative regulator of rig-i/mda signaling pathway [ ] . ceacam is involved in regulation of liver inflammation [ ] and its expression appears to exert antiviral effects on influenza virus [ ] . nmi binds to influenza virus ns and inhibits irf -mediated interferon signaling [ , ] . therefore, aberrant expression of genes in this signaling pathway may directly contribute to immune evasion of influenza, culminating in viral propagation and increased epithelial damage. we summarized the identified pathways (listed in tables and ) that alluded to immune evasion (negative regulation of innate immune responses), antigen processing (metabolism of amino acids and derivatives), and immunomodulation (interferon gamma signaling, b cell receptor signaling, and response to fatty acid) that may contribute to severity of influenza. there was evidence of direct pathway enrichment of potential influenza evasion strategies and/or immunomodulation with accompanying transcriptomic changes. the genes in the pathway may be analyzed for their immunomodulatory activity and whether their expression is beneficial to the virus (immune evasion) or the host (preventing cytokine storm). in addition, the infected hnecs also revealed modified responses associated with fatty acid, with many lipid signaling molecules such as leukotrienes that mediate antiviral responses and subsequent inflammation of the airway [ , ] . such modified responses may also determine the afforded in the airway and the severity of airway inflammation and damage. in addition, the modification may also affect the lower airway responses to inflammatory mediators; hence, the changes in these pathways may also suggest a potential mechanistic link to the pathogenesis of viral-induced exacerbation of chronic inflammatory diseases. lastly, we also noted enrichment of pathways related to amino acid metabolism, which is important in antigen processing and proteasomal degradation of foreign protein. the changes in these genes at the hnecs, the target site of influenza infection, may determine the effectiveness of antiviral responses mounted and may therefore influenza disease severity. in addition, we also compared our rnaseq analysis against previously reported influenza-specific signatures in order to improve future transcriptomic analysis [ ] . in vitro transcriptomic analysis yields a large number of differentially-expressed genes that would require additional criteria to identify functionally significant genes. by means of this comparison, we discovered that almost all influenza-specific signatures exhibited differences in expression of above . -fold. hence, we propose applying fold change of > . as a threshold for future in vitro transcriptomic systems analyses, in order to increase the stringency in detecting functionally significant gene changes. finally, we observed that, unlike the transcriptome, there were notably fewer cytokines that were readily secreted during the acute phase of infection. expression of cytokines was reduced at hpi, except for il- , which interestingly is implicated in influenza-induced acute lung injury [ ] . even fewer cytokines showed altered expression at later time points. among them, only tgf-a, ip- (cxcl ), and pdgf-aa were significantly altered at and hpi. these may be significant markers that can be detected in the secretion of influenza-infected mucosal surface that may influence the severity of influenza. ip- is a well-established ifnγ response gene, and serves as a useful marker for response against influenza [ , ] . tgf-a represents an important factor involved in the secretion of il- in response to influenza, and may determine the early appropriate innate responses to prevent severe disease [ ] . on the other hand, it is also involved in pulmonary fibrosis as a ligand of epidermal growth factor receptor (egfr) and may contribute to complications in the lower airway [ ] . pdgf-aa was found to be elevated in the cerebrospinal fluid of influenza-associated encephalopathy [ ] , but was consistently reduced in hnec secretory fluid, thus warranting further investigation into its role in the infected nasal mucosa. the establishment of a reference transcriptome based on early responses of the human nasal epithelium model serves a key role in research on critical host factors involved in influenza. as the primary host contact with the virus, not only are immune responses against influenza important, but also the alterations in non-immune functions such as metabolism, cell content, and cell cycle, which may contribute to disease severity. in terms of translational potential, the model system identified gene expression changes of significant magnitude and pathways that impact responses against influenza and its severity. these genes may represent novel targets for future diagnostic and therapeutic development. under controlled conditions, the hnecs clinically establish the baseline for "normal" innate immune responses of the nasal epithelium against influenza viral infection. such a baseline can be particularly crucial when studying the changes in innate immune responses against influenza, especially in patients with underlying chronic diseases who may have aberrant airway responses against influenza. their antiviral responses may differ from "normal" subjects, and this study thus provides the basis for comparing the differential responses that culminate in more severe infections in patients with co-morbidities such as diabetes and chronic airway inflammatory diseases. such comparative clinical studies can potentially enhance the management of influenza viral infection in patients with chronic diseases. in conclusion, rnaseq technology allowed us to accurately quantify the magnitude of gene expression changes, as well as the relevant enriched pathways during h n influenza virus infection of hnecs, which can serve as a baseline for future clinical studies. the establishment of this baseline under controlled condition elucidated the antiviral innate response by the infected nasal epithelium, and highlighted the molecular factors and abnormalities in the upper airway that may contribute to influenza severity. furthermore, this study also culminated in the identification of novel gene signatures and host factors that may be harnessed for future research to develop influenza diagnostic markers and therapeutic targets. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , figure s . real-time quantitative pcr validation of genes selected from rnaseq analyses. pcr data are expressed as log fold change using median and interquartile range. statistical significance was determined using wilcoxon signed-rank test. * p < . , # p < . ; figure s . luminex assay of secreted cytokines/chemokines in the apical supernatant. luminex data are expressed as mean fold change from uninfected control. statistical significance was determined using fdr. * p < . ; table s . information of seven donors of hnecs; table s . significant enriched pathways based on reactome pathway database analysis; table s . list of significant genes with altered expression at hpi of influenza h n infection of hnecs analyzed by 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expression in human nasal epithelium nf-kappab-dependent induction of tumor necrosis factor fas/fasl is crucial for efficient influenza virus propagation nucleoprotein of influenza a virus negatively impacts antiapoptotic protein api to enhance e f -dependent apoptosis and virus replication influenza a virus enhances its propagation through the modulation of annexin-a dependent endosomal trafficking and apoptosis programmed cell death in the pathogenesis of influenza dai senses influenza a virus genomic rna and activates ripk -dependent cell death zbp /dai is an innate sensor of influenza virus triggering the nlrp inflammasome and programmed cell death pathways the multifaceted b cell response to influenza virus influenza virus-induced type i interferon leads to polyclonal b-cell activation but does not break down b-cell tolerance neutrophils induce smooth muscle hyperplasia via neutrophil elastase-induced fgf- in a mouse model of asthma with mixed inflammation respiratory syncytial virus infection provokes airway remodelling in allergen-exposed mice in absence of prior allergen sensitization cd , a laminin receptor showing increased expression in asthma, contributes to airway hyperresponsiveness through calcium signaling regulation of human airway smooth muscle cell migration and relevance to asthma airway smooth muscle in asthma: phenotype plasticity and function. pulm pharm the interactomes of influenza virus ns and ns proteins identify new host factors and provide insights for adar playing a supportive role in virus replication yoshimura, a. fln , a novel interferon-and lps-inducible gene acting as a negative regulator of toll-like receptor signaling rna-and virus-independent inhibition of antiviral signaling by rna helicase lgp ceacam in liver injury, metabolic and immune regulation ceacam -mediated inhibition of virus production subcellular proteomic analysis of human host cells infected with h n swine influenza virus negative regulation of nmi on virus-triggered type i ifn production by targeting irf mast cells and influenza a virus: association with allergic responses and beyond leukotriene b enhances nod -dependent innate response against influenza virus infection multi-cohort analysis identifies conserved transcriptional signatures across multiple respiratory viruses interleukin- is critical in the pathogenesis of influenza a virus-induced acute lung injury influenza induces il- and gm-csf secretion by human alveolar epithelial cells through hgf/c-met and tgf-alpha/egfr signaling overactive epidermal growth factor receptor signaling leads to increased fibrosis after severe acute respiratory syndrome coronavirus infection vascular endothelial growth factor (vegf) and platelet-derived growth factor (pdgf) levels in the cerebrospinal fluid of children with influenza-associated encephalopathy this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we thank the surgeons and staff in the department of otolaryngology, national university hospital, singapore. we thank h.h. ong and t.t. he for the subject selection and recording. we thank m.c. phoon and s.h. lau for technical assistance in viral experiments. the authors would like to acknowledge the staff of the immunomonitoring platform at sign. the authors declare no conflicts of interest. key: cord- -qeao ghg authors: aris-brosou, stéphane; parent, louis; ibeh, neke title: viral long-term evolutionary strategies favor stability over proliferation date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: qeao ghg viruses are known to have some of the highest and most diverse mutation rates found in any biological replicator, with single-stranded (ss) rna viruses evolving the fastest, and double-stranded (ds) dna viruses having rates approaching those of bacteria. as mutation rates are tightly and negatively correlated with genome size, selection is a clear driver of viral evolution. however, the role of intragenomic interactions as drivers of viral evolution is still unclear. to understand how these two processes affect the long-term evolution of viruses infecting humans, we comprehensively analyzed ssrna, ssdna, dsrna, and dsdna viruses, to find which virus types and which functions show evidence for episodic diversifying selection and correlated evolution. we show that selection mostly affects single stranded viruses, that correlated evolution is more prevalent in dna viruses, and that both processes, taken independently, mostly affect viral replication. however, the genes that are jointly affected by both processes are involved in key aspects of their life cycle, favoring viral stability over proliferation. we further show that both evolutionary processes are intimately linked at the amino acid level, which suggests that it is the joint action of selection and correlated evolution, and not just selection, that shapes the evolutionary trajectories of viruses—and possibly of their epidemiological potential. humanity is regularly reminded of the epidemiological toll of viruses, in part due to recent and ongoing viral outbreaks of influenza [ ] , ebola [ ] , and zika [ ] . thanks to recent technological and analytical developments, it is now possible to elucidate, almost in real time [ ] , their epidemiological dynamics, and unravel their evolutionary dynamics [ ] . however, while the evolutionary dynamics of rna viruses are well documented [ ] , those of other viruses are not so well known, in particular in a unifying context including major viral types such as double-stranded (ds) and single-stranded (ss) dna and rna viruses. to date, one of the most salient evolutionary features shared by all viruses is the existence of a negative correlation between mutation rate and genome size [ ] . this is a critical result as it suggests that selection is driving the evolution of mutation rates, establishing a trade-off between mutational load and availability of adaptive mutations [ ] . however, the processes driving the evolution of different types of viruses are multiple. as already argued, both ssdna and ssrna viruses share small genome sizes, high mutation rates, but also large effective population sizes, little to no gene duplication or recombination, and overlapping reading frames [ ] . this last point suggests that a less frequently explored evolutionary process in viral studies, correlated evolution, could be as critical as positive selection. correlated evolution happens when mutations at two locations in a genome occur one after the other, in a quick succession [ ] , repeatedly [ ] . typical examples include drug resistance mutations that have a fitness cost, and that require a second mutation to compensate for the first one [ ] , or trnas that require a specific base-pairing to maintain their secondary and tertiary structures, so that a mutation in the stem region necessitates a second mutation to restore the correct, functional, structure [ ] . this process is of particular interest as correlated evolution can be underlain by epistasis, which occurs when the fitness effects of these two mutations are non-additive [ ] , as in the two examples above. as such, correlated evolution can help us understand the relationship between genotype and fitness, which is a key determinant of evolutionary trajectories [ ] . to date, however, correlated evolution has only sporadically been investigated in viral evolution, and these rare instances only focused on ssrna viruses. indeed, recent work uncovered pervasive evidence for correlated evolution in influenza viruses [ , ] , and both the zika [ ] and the ebola viruses [ ] . intriguingly, in this latter case (ebola), evidence was found that sites evolving in a correlated manner could also be under positive selection-bearing the question as to how frequently these two processes, correlated evolution and positive selection, occur, possibly jointly, and if this co-occurrence is limited to ssrna viruses, or can be generalized to all viruses. to better understand the role of correlated evolution and positive selection in the evolutionary dynamics of viruses infecting humans, we constructed a nearly exhaustive viral data set spanning all dsdna, dsrna, ssrna, and ssdna viruses deposited in genbank (as of august ), and conducted an extensive survey of correlated evolution and diversifying selection in these viruses, asking more specifically about the prevalence of these two processes in each viral type, independently or jointly, with the specific hypothesis that the genes affected by both processes encode functions that are most critical to each viral life cycle. lists of dsdna, dsrna, ssrna, and ssdna viruses infecting humans were retrieved from the virusite database [ ] in august (tables s -s ); subtypes/genotypes/clades were treated as independent data sets. although some of these viruses could be segmented or not, with circular or linear genome, with positive or negative strands, and with or without overlapping reading frames, accounting for these structural features would have led to smaller and smaller data sets, precluding any statistical analysis, so that the data were not split beyond viral type. each list contained the virus names, the length of their genome, their number of protein-coding genes (cds's), and was associated with a reference coding sequence (see query_sequences.zip at [ ] ). in order to obtain corresponding sequence alignments of orthologous genes, blastn searches were performed on a custom database limited to viral genes present in the national center for biotechnology information nucleotide database with blast- . . +. for this, all gbvrl*.seq.gz files were downloaded from reference [ ], while querying virusite, and were concatenated into a single genbank file, then converted into a fasta file with readseq to specifically extract cds's [ ] . this was done to avoid retrieving ' and ' untranslated regions that would cause problems to the downstream codon analyses. blastn searches were performed for each virusite viral sequence with a stringent e-value threshold of − , keeping a maximum of sequences with at least % coverage with each query; this ensured that subtype/genotype/clade boundaries were not crossed. as the viruses retrieved from the virusite also included viruses that require a vector (e.g., arboviruses such as the dengue and yellow fever viruses), or viruses that circulate in non-human hosts but that can lead to zoonoses (e.g., the camel alphacoronavirus leading to mers [ , ] ), these stringent thresholds also ensured that sequences contained in each alignment mostly came from a single host. only data sets with at least hits were kept for downstream analyses. sequences corresponding to each accession number were retrieved from the fasta file obtained with readseq [ ] . because this file contained partial sequences, each set of retrieved sequences was first aligned with muscle . . [ ] . each alignment was then quality checked ensuring that (i) its length is a multiple of three, (ii) it starts with an atg and stops with a stop codon. alignments failing at least one condition were discarded. within each alignment, mean numbers of indelsn indels were computed for each sequence, and those containing number of indels ≥n indels + sem (standard error of the mean) were eliminated. the remaining nucleotide sequences were then re-aligned with translatorx [ ] , at the amino acid level, using the muscle aligner and their heuristics to determine the correct reading frame. alignment files were then cleaned-up with gblocks . b at the codon level using the stringent default settings [ ] . both trees and alignments are available at [ ] . to obtain gene annotations, gene ontology (go) terms were retrieved from gene sequences with hmmer go ( [ ] ), relying on the hidden markov models in [ ] (ver. febuary ). this is equivalent to performing a blast go search [ ] , but without the limitations of proprietary software. individual mapping files coming from our four viral types (ssrna, dsrna, ssdna, and dsdna viruses) were then merged to create a custom annotation file, used for go term enrichment testing with topgo [ ] , based on fisher's exact test. the reference gene list was always the entire set of genes within each viral type. phylogenetic trees were reconstructed with fasttree . . [ ] under the gtr+Γ model of evolution [ ] ; note that fasttree was recompiled locally to use double-precision arithmetics, as recommended by its authors to estimate very short branch lengths accurately. those with a nonzero tree length were midpoint rerooted using the phytools package ver. . - [ ] in r ver. . . [ ] . patterns of correlated evolution among sites were identified with the bayesian graphical model (bgm) implemented in spidermonkey [ ] (sm), which is part of hyphy ver. . . [ ] . default hyphy scripts were slightly modified to read in codon data, which were used to reconstruct mutational paths under the mg × hky substitution model [ ] at nonsynonymous sites along each branch of the estimated trees. ambiguous reconstructions were resolved by considering all possible resolutions and averaging them. these reconstructed mutational paths were then recoded as a binary matrix, with rows corresponding to branches and columns to each site of the alignment. the bgm was then used to identify the pairs of sites that exhibit correlated patterns of nonsynonymous substitutions according to their posterior probability, estimated with a markov chain monte carlo sampler that was run for steps, with a burn-in period of , steps sampling every steps for inference [ ] . patterns of episodic selection were identified based on the mixed effects model of evolution [ ] (meme), also as implemented in hyphy. the default script from ver. . . was used, still with the . . hyphy engine, to infer nonsynonymous to synonymous rate ratios ω assuming that these rates can vary across lineages and among sites. in this implementation, two categories of sites were assumed, those for which ω neg ≤ , in proportion p, and those for which ω pos > that are under positive selection, in proportion − p. evidence for selection was derived by means of a likelihood ratio test between this model, and a null model where ω pos was constrained to take its value between and . linear models were fitted through robust regressions [ ] . all r scripts and hyphy source files are available from reference [ ] (file "api_scripts.zip," in "data"). first, in order to better understand the genomic characteristics of the four types of viruses known to infect humans, dsdna (n = ), dsrna (n = ), ssrna (n = ), and ssdna (n = ), and understand how these characteristics can impact the evolutionary dynamics of these viruses (methods; figure s ), we examined the distribution of four of their genomic features. while dsdna viruses had the largest and the most variable genome lengths, ssdna were the smallest genomes by at least an order of magnitude, with both dsrna and ssrna exhibiting intermediate sizes (figure a) . because of the compact structure of these genomes, these differences were also reflected in the number of genes, protein-coding genes, and rnas encoded by the genomes of these viruses ( figure s ). in turn, these characteristics suggest that, with their large genomes, dsdna viruses potentially have a higher degree of functional redundancy than ssdna or even ssrna viruses, possibly due to duplication events [ ] , and thereby be under less stringent selective pressures than viruses with smaller genomes. on the other hand, the extent of correlated evolution and its interaction with selection is more difficult to predict. to address these questions, we first tested for the presence of episodic diversifying selection in each viral type. altogether, we found extensive differences among all four viral types in terms of the number of genes under selection (figure b ; x = . , d f = , p < . × − ), and that single stranded viruses were more subject to selection than double stranded viruses (x = . , d f = , p < . × − ). indeed, and contra our original hypothesis, there were no differences between dsdna and dsrna viruses (x = . , d f = , p = . ), or between ssdna and ssrna viruses in terms of prevalence of diversifying selection (x = . , d f = , p = . ). note that these differences cannot be attributed to genetic diversity, as dsdna and dsrna, which have similar levels of selection, have however different levels of diversity ( figure s ). however, it is unlikely that "strandedness" (single vs. double stranded genetic material) alone drives selection, even if greater instability can be postulated in single-stranded nucleic acids [ ] . indeed, strandedness is negatively correlated with genome size (t = − . , d f = . , p = . × − ), which is itself correlated with mutation rates [ , ] . as a result, episodic diversifying selection is mostly driven by structural aspects of viral genomes, which condition mutation rates across all virus types [ ] . to understand if similar aspects drive intragenic correlated evolution, we counted in the same way the number of genes for which we could find evidence for interactions. here, again, we found extensive differences among all four viral types (figure c ; x = . , d f = , p < . × − ), with no difference between dsdna and ssdna viruses (x = . , d f = , p = . ), or between dsrna and ssrna viruses (x = . , d f = , p = . ). again, diversity is not driving these differences, as both dsdna/ssdna and dsrna/ssrna have significantly different levels of diversity ( figure s ). as a result, the prevalence of correlated evolution seems to be mostly driven by the nature of viral genetic material. at least two processes can underpin correlated evolution: linkage and epistasis [ ] . as recombination is pervasive in some dsdna viruses [ ] , linkage alone may not explain the high prevalence of correlated evolution in dna viruses (close to %: figure c ). rather, this pattern suggests that intragenic constraints are higher in dna viruses for unknown structural reasons, maybe due to protein structure [ ] , or in the same way that recombination in rna viruses may be driven by mechanistic constraints associated with genome structures and viral life cycles [ ] . while previous work showed that both diversifying selection and correlated evolution can affect the same gene and even the same site in a viral genome such as ebola's [ ] , an ssrna virus, the generality of this association is still unknown. at the gene level, we found strong heterogeneity among all four viral types for gene numbers showing evidence for selection, correlated evolution or both (x = . , d f = , p < . × − ). surprisingly, ssrna viruses are those that show the least overlap between selection and correlated evolution, with only % of the genes evolving under both mechanisms (figure d ). patterns are, however, much more difficult to extract here, mostly because the number of genes involved becomes quite small, in particular for the virus type with most overlap, the dsrna viruses (figure d: . %, i.e., five genes). to assess the extent to which some of these differences at the gene level are functionally driven, we extracted the gene ontology (go) annotations, or go terms, associated with the genes analyzed, as well as those under selection, correlated evolution, or both-focusing exclusively on the virus types for which we had the largest samples sizes, dsdna and ssrna viruses (figure e) . each of the three parts of the ontology (molecular function [mf], biological process [bp], and cellular component [cc]) was first limited to the second level of go in order to derive a high-level interpretation (low-level descriptions are shown in tables s -s ). figure e shows that go terms related to catalytic activity (mf), involved in the establishment or maintenance of a certain location (bp) at the membrane level (cc) are predominantly present in both dsdna and ssrna genomes. however, only a subset of these go terms is mostly under episodic diversifying selection: helicase activity/binding (mf) and replication (bp) in the host cell nucleus (cc) in dsdna, while it is mostly peptidase activity and methylation on the viral envelope in ssrna viruses (table s ; p < . ). in spite of these differences, we note that episodic diversifying selection mostly affects genes involved in viral replication (table s ). genes that are affected by correlated evolution include: helicase activity and binding at the interface of multiple compartments in dsdna viruses, or transferase activity and protein modifications on the envelope in ssrna viruses (table s ) . again, most of these functions and processes are involved in viral replication (table s ) . at the intersection of these evolutionary processes however, the genes that are jointly affected by selection and correlated evolution are involved in structural integrity and assembly within or outside a cell at the capsid level for dsdna viruses, or in interacting with host cell surface via antigen activity on the viral envelope for ssrna viruses-functions and processes that are mostly involved in "viral stability" (cell entry, integrity, assembly, immune escape; table s ). this suggests that, despite key differences in life history strategies adopted by dsdna and ssrna, there is a certain unity across viral types, where genes involved in replication are mostly under either selection or correlated evolution, while those involved in viral stability are mostly affected by both evolutionary processes-as has been shown for the influenza [ ] and the ebola [ ] viruses (both cases involved a glycoprotein required for cell entry). because these two evolutionary processes are required by epistasis, it is possible that the genes involved in viral stability are the most likely to show evidence for non-additive fitness effects-and hence shape viral fitness landscapes. this tension between replication and stability is evocative of the existence of trade-offs between capsid stability and proliferation within a host [ ] , or fecundity and lifespan [ ] in dsdna viruses, which supports the idea that it is the joint action of selection and of correlated evolution that shapes viral life histories. the previous analyses were at the gene level. one outstanding question is whether this relationship between selection and correlated evolution also holds at the amino acid level that is, if the amino acids under selection are also involved in correlated evolution. this question was addressed in two different ways, all virus types confounded (in order to have larger sample sizes), by focusing on one process at a time, and finding at what point evidence supporting the second process becomes significant. first, we searched the list of pairs of sites evolving in a correlated manner (the sm sites) to see if at least one pair member was potentially evolving under episodic diversifying selection (the meme sites), irrespective of its probability of being a meme site. for this, we identified pairs of sm sites, that is those with a posterior probability ≥ . , and plotted their posterior probability as a function of the − log probability of each pair member to be under selection (figure a ). both the least-square (ρ slope = . , t = . , p = . ) and the robust regressions (ρ slope = . , t = . , p = . ) have positive and significant slopes, hereby demonstrating the existence of a relationship between correlated evolution and episodic diversifying selection at the amino acid level-even if most of the sm sites show weak evidence of selection (at p ≤ . , gray broken line in figure a) . thus, to validate the existence of this relationship, we then took the list of meme sites, and searched the list of sm sites to see if at least one pair member was potentially a meme site, irrespective of its posterior probability of being in an sm site pair. for this, we identified the meme sites, that is those with a p-value ≤ . (≥ on a − log scale), and plotted this (on the x-scale) vs. their posterior probability of being in an sm pair (on the y-scale; figure b ). both the least-square (ρ slope = . , t = . , p = . ) and the robust regressions (ρ slope = . , t = . , p = . ) have positive and significant slopes, further confirming the existence of a relationship between correlated evolution and episodic diversifying selection at the amino acid site level. note, however, that this latter analysis is more informative than the former, as the density also shows that most of the sites under selection are involved in weak interactions. this is intriguingly reminiscent of the involvement of weakly interacting pairs of sites in severe outbreaks or pandemics [ ] . altogether, we showed that episodic diversifying selection is mostly found in single stranded viruses, while correlated evolution is more prevalent in dna viruses. more critically, we also showed that the genes affected by each process, when acting independently, are involved in viral replication. however, the genes that are jointly affected by both processes are mostly involved in viral stability (cell entry, integrity, assembly, immune escape), and that the same amino acid sites tend to be affected by both processes. in retrospect, this tight relationship between selection and correlated evolution may not be surprising, as correlated evolution can be underlain by epistasis [ , ] , which directly involves selection (in a non-additive way). epistasis being a key determinant of fitness landscapes, and hence of evolutionary trajectories [ ] , our results suggest that, in the long-term, both processes jointly shape the life history of viruses, favoring stability over proliferation. if so, analyzing viral evolution in the joint light of selection and correlated evolution might help us better predict how viruses that affect humans might evolve [ , ] -as predicting their evolution [ ] and epidemiology [ ] has a long history fraught with mixed success. we note however that we neglected some aspects of viral structure: indeed, viruses can be segmented or not, with a circular or linear genome, with positive or negative strands, overlapping reading frames, complications that we could not consider here due to the resulting small sample sizes, even if these factors can impact the mode of evolution of viruses [ ] . future work should however strive to address these limitations. we also neglected the population genetics context in which different viruses evolve, a context that can often be correlated to structural constraints [ , ] . furthermore, as we solely focused on intragenic interactions, and not intergenic or higher order correlations, it is not impossible that we missed higher-level constraints affecting viral evolution. in particular, it is possible that intergenic correlations reveal the nature of trade-offs shaping the life history strategies in viruses [ ] . future modeling [ ] and empirical [ ] work should probably focus on elucidating not only these constraints, but also the theoretical basis connecting correlated evolution, if not epistasis, to selection in shaping the evolutionary strategies of biological replicators, as current evidence linking these processes is currently limited to the influenza [ ] and the ebola [ ] viruses. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , figure s : overview of the experimental procedures, figure s : physical characteristics of the four types of viruses, figure s : viral diversity across viral types, table s : go enrichment tests for all the meme genes of interests, table s : go enrichment tests for all the sm genes of interests, table s : go enrichment tests for all the sm + meme genes of interests. we thank three anonymous reviewers for providing us with constructive comments, as well as compute canada and ontario's centre for advanced computing for giving us access to their servers. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. the origins and evolutionary genomics of the swine-origin h n influenza a epidemic genomic surveillance elucidates ebola virus origin and transmission during the outbreak zika virus in the americas: early epidemiological and genetic findings mobile real-time surveillance of zika virus in brazil unifying the epidemiological and evolutionary dynamics of pathogens the evolution and emergence of rna viruses what does virus evolution tell us about virus origins? prevalence of epistasis in the evolution of influenza a surface proteins the unsolved challenge to phylogenetic correlation tests for categorical characters stability-mediated epistasis constrains the evolution of an influenza protein compensatory evolution in mitochondrial trnas navigates valleys of low fitness the 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high? from molecular genetics to phylodynamics: evolutionary relevance of mutation rates across viruses molecular evolution of human species d adenoviruses constraints from protein structure and intra-molecular coevolution influence the fitness of hiv- recombinants why do rna viruses recombine? viruses' life history: towards a mechanistic basis of a trade-off between survival and reproduction among phages experimental selection reveals a trade-off between fecundity and lifespan in the coliphage qß darwinian evolution can follow only very few mutational paths to fitter proteins evolutionary footprint of epistasis predicting the emergence of h n influenza viruses reveals contrasted modes of evolution of ha and na antigens forecasting national and regional influenza-like illness for the usa coordinated evolution of influenza a surface proteins deep mutational scanning identifies sites in influenza nucleoprotein that affect viral inhibition by mxa key: cord- - dcnext authors: corpus, carla; williams, victoria; salt, natasha; agnihotri, tanya; morgan, wendy; robinson, lawrence; maze dit mieusement, lorraine; cobbam, sonja; leis, jerome a title: prevention of respiratory outbreaks in the rehabilitation setting date: - - journal: bmj open qual doi: . /bmjoq- - sha: doc_id: cord_uid: dcnext background: respiratory viral (rv) outbreaks in rehabilitation facilities can jeopardise patient safety, interfere with patient rehabilitation goals and cause unit closures that impede patient flow in referring facilities. problem: despite education about infection prevention practices, frequent rv outbreaks were declared each year at our rehabilitation facility. methods: before and after study design. the primary outcome was the number of bed closure days due to outbreak per overall bed days. process measures included delays in initiation of transmission-based precautions, rv testing and reporting of staff to occupational health and safety (ohs). balancing measures included the number of isolation days and staff missed work hours. interventions: based on comprehensive analysis of prior outbreaks, the following changes were implemented: ( ) clear criteria for initiation of transmission-based precautions, ( ) communication to visitors to avoid visitation if infectious symptoms were present, ( ) exemption of staff absences if documented due to infectious illness, ( ) development of an electronic programme providing guidance to staff about whether they should be excluded from work due to infectious illness. results: the number of bed closure days due to outbreak per overall bed days dropped from . % to . % during the intervention season and sustained at . % during the postintervention season (p< . ). there were fewer delays in initiation of droplet and contact precautions ( . % to . %, p= . ) and collection of rv testing ( . % to . %, p< . ), better reporting to ohs ( vs . reports per employees; p< . ) and fewer isolation days ( . % vs . %; p= . ) without a significant increase in missed work hours per hours worked ( . vs . ; p= . ). conclusion: this quality improvement study highlights the process changes that can prevent respiratory outbreaks in the rehabilitation setting. nosocomial transmission of respiratory viruses (rvs) can lead to unanticipated complications for patients during their contact with the healthcare system. in rehabilitation facilities, these outbreaks jeopardise patient safety, interfere with patient rehabilitation goals and cause unit closures that impede patient flow in referring facilities. multicomponent infection prevention and control (ipac) strategies including hand hygiene, early symptom identification, transmission-based precautions, use of personal protective equipment by healthcare personnel and environmental cleaning, can be successful in preventing nosocomial transmission of rvs. [ ] [ ] [ ] [ ] [ ] [ ] despite this, adherence to these best practices is frequently suboptimal and rv outbreaks in healthcare remain a common yet preventable occurrence. at our rehabilitation institution in toronto, canada, we experienced frequent rv outbreaks every season despite continued efforts to educate healthcare providers about best ipac practices. we hypothesised that rv outbreaks could be prevented using quality improvement (qi) methodology, beginning by a systematic understanding of the problem, engagement of key stakeholders and design of new processes that support improved ipac practices. our aim was to reduce bed closure days due to outbreak by over % during subsequent rv seasons. st. john's rehabilitation centre (sjr) is a -bed rehabilitation facility located in toronto, ontario, canada and is a tertiary care teaching hospital affiliated with university of toronto. the patient population includes cardiac, amputee, stroke, trauma, medical debility, burn and musculoskeletal patients. the average length of stay is approximately days and there are about admissions per year. at baseline, six rv outbreaks were declared in the / season resulting in bed closure days ( . % of all rehabilitation bed days). ipac strategies in place prior to the qi study included: mandatory core competency training for all clinical staff on hire and renewal every years; a healthy workplace policy that required healthcare workers to open access stay home if they were ill; droplet and contact precautions for patients with respiratory symptoms including patient placement (single room and cohorting); routine daily and terminal environmental cleaning of horizontal and high touch surfaces; monthly hand hygiene directly observed audits (compliance rate ~ %- %); multiplex rv testing via polymerase chain reaction (turnaround time ~ hours); antiviral treatment and prophylaxis for confirmed cases of influenza and exposed roommates and annual influenza vaccination campaign with uptake of % for staff and ~ % for patients. the rv season was defined as october to april during baseline ( / ), intervention ( / ) and postintervention ( / ) seasons. patients with respiratory symptoms were prospectively identified through active surveillance based on unit reporting and tracking of laboratory specimens. mid-turbinate (mt) swabs were collected from all patients with new or worsening onset of one or more respiratory symptom (rhinorrhoea, cough, sore throat, wheeze or dyspnoea). a case was considered nosocomial if symptoms developed > hours after admission. a rv outbreak was defined as two nosocomial cases (non-roommates) in a designated unit with symptom onset within hours and a lab-confirmed respiratory virus detected in a least one case. in the absence of laboratory confirmation of a respiratory virus, three nosocomial cases (non-roommates) within hours in one unit was considered a rv outbreak. rv outbreaks were declared over on day following the onset of the last nosocomial rv case, in accordance with local public health guidelines. any unit where a rv outbreak was declared was immediately closed to new admissions. accordingly, the census on the unit decreased throughout each outbreak as patients were discharged without new patient admissions. reducing the number of bed-closure days due to outbreak was thus chosen as the primary aim of this study because it reflected the impact of these outbreaks on our facility's ability to fulfil its mission of providing inpatient rehabilitation. the improvement team was convened by senior leaders of the institution in may and began by trying to identify the most important drivers of rv outbreaks during the two preceding seasons. first, a comprehensive retrospective review of the line listed cases and epidemiological curves were conducted to adjudicate primary precipitant of the prior rv outbreaks. this review identified that outbreaks resulted from a delay in initiation of droplet and contact precautions ( / , %), staff working while ill ( / , %), shared accommodations ( / , %) and no definite cause identified but sick visitor suspected ( / , %). second, a half-day interdisciplinary stakeholder meeting was held ( healthcare providers present) where they completed an ishikawa diagram aimed at identifying the contributing factors to rv outbreaks. this activity revealed additional contributors including: the facility's infrastructure (only % of all inpatient accommodations are private rooms) leading to higher threshold for initiation of transmission-based precautions, confusion about how many symptoms should trigger initiation of droplet and contact precautions, visitors coming into the facility with infectious symptoms due to lack of awareness about the consequences, healthcare worker perceptions about the implications of taking days off when sick on human resources attendance management and limited access to occupational health and safety (ohs) after regular work hours. between september and december , new processes were fully implemented to address the most important contributors of rv outbreaks. table summarises the four new processes developed and their relationship to the problems identified. first, clarity was achieved regarding the criteria for initiation of droplet and contact precautions to include any patient with any of the following symptoms: new or worsening cough, runny nose, congestion or sore throat (september ). second, communication to visitors to please not visit if they have any of those symptoms was included in two forms: an automated telephone message heard on calling the rehabilitation centre and signs placed in the lobby entrance (november ). third, communication to staff regarding exemption for illness due to infectious causes was communicated to all healthcare providers in an attempt to address the perceived barrier of not coming to work ill (october ). to mitigate the risk of abuse of this exemption policy, improved reporting to ohs was required, which in turn necessitated a more efficient process for reporting to ohs. the fourth intervention was the development of an electronic programme allowing staff to report illness to ohs and simultaneously receive guidance about whether they are excluded from work due to infectious illness (december ). this programme incorporated questions about specific symptoms and symptom-onset and provided a personalised recommendation about whether the healthcare provider should work or remain home, based on whether or not they were considered infectious (see online supplementary material). in the event that the system identified a staff person as being infectious, the recommendation could be forwarded to the manager of the unit in order to exempt the employee from the attendance management system. the primary outcome measure was the number of bed closure days due to outbreaks during a rv season adjusted for the facility's overall bed availability. this was defined as any bed-closure occurring during a rv outbreak. process measures included percentage of staff that received training about criteria for initiation of table description of factors contributing to viral respiratory outbreaks and the corresponding interventions implemented staff lack clarity regarding criteria that warrant initiation of transmission-based precautions ► min face to face education sessions using real patient story ► visual reminders (posters) on units listing the criteria for initiation of transmission-based precautions visitors coming into the facility with infectious symptoms ► bold and bright posters incorporating photos to alleviate language barriers were strategically posted at facility and unit entrances asking visitors not to enter if they have any of the depicted symptoms ► automated telephone message on calling the facility reminding visitors not to visit when ill with infectious symptoms ► unit managers empowered frontline staff to send visitors home if noted to be ill staff working while ill due to perceived implications on human resource attendance management ► clear communication to staff regarding exemption of any absences from human resources attendance management on condition that illness is documented to be infectious (through online or in-person reporting to occupational health & safety) lack of after-hours access to occupational health and safety to report infectious illness ► creation of electronic reporting system (occupational health & safety e-nurse (parklane-canada)) which allows healthcare workers to enter their symptoms and receive immediate recommendation about whether they are allowed to work. this system also provides printable documentation to managers transmission-based precautions, the proportion of patients with delays in initiation of droplet and contact precautions or collection of mt swabs (each defined as more than hours from onset of symptoms) and the number of healthcare providers reporting sick to ohs. all laboratory and clinical data were obtained from laboratory reports and prospective ipac surveillance documentation. all healthcare provider illness reporting was obtained from ohs documentation. to account for any unintended consequences associated with these new processes, balancing measures were assessed including number of isolation days adjusted to total patient days, number of mt swabs processed by the microbiology laboratory and non-physician inpatient staff missed work hours due to illness adjusted to staff worked hours. the number of isolation days included any patient managed on precautions in the facility including patients with confirmed rv infection and those with suspected rv infection for whom mt swab results were pending. descriptive statistics were calculated for all variables of interest. continuous measures were summarised using means and sd, or median and iqr if they did not pass the test for normality. categorical measures were summarised using counts and percentages. the χ² or fisher's exact test were used to detect difference in proportions. p< . was considered statistically significant. data were analysed using spss statistics v. software (ibm, markham, ontario, canada). this study was deemed to be quality improvement within the mandate of the ipac programme and therefore formal research ethics board review was waived. the interdisciplinary stakeholder meeting did not include a patient or public representative, but patients and visitors were engaged in the project during the implementation of the process changes. patient input was received regarding sign location and automated telephone notification. the impact of the quality improvement study was presented to the organisation's quality committee that includes representation from the public. during implementation of the four process changes, % ( / ) of nursing staff were trained regarding criteria for initiation of transmission-based precautions, including % ( / ) of full-time staff. figure depicts the monthly proportion of patients placed on droplet and contact precautions and figure shows the monthly proportion tested with mt swabs for respiratory viruses greater than hours after onset of symptoms. a shift in practice was noted following training with significant reductions in delays in both initiation of droplet and contact precautions ( . % vs . %; p= . ) and viral testing ( . % vs . %; p< . ) in the intervention respiratory season and onward. table summarises the family of measures during the intervention and postintervention seasons compared with the baseline season. the number of bed closure days due to outreak adjusted for overall bed days dropped from . % to . % during the intervention season and was sustained at . % during the postintervention season (p< . ). staff reporting to ohs increased from to . per employees (p< . ). in terms of unintended consequences, earlier initiation of droplet and contact precautions did not lead to increased isolation days. in fact, the proportion of isolation days per patient days decreased during the intervention season as compared with baseline ( . % vs . %; p= . ). with regard to potential for increased absenteeism, there was no significant change in the number of missed work hours for employees ( . missed hours/ worked hours at baseline vs . missed hours / worked hours during intervention; p= . ). our rehabilitation centre reduced the number of bed closure days due to rv outbreaks to less than a third for two consecutive seasons through process changes that supported earlier initiation of transmission-based precautions for symptomatic patients, discouraged visitation by people with potentially infectious symptoms and made it easier for healthcare workers to stay home when potentially infectious. despite evidence supporting ipac practices to prevent nosocomial transmission of rv infection each rv season, the adoption of these practices following education alone is often suboptimal leading to preventable nosocomial outbreaks. there is a paucity of literature around using qi strategies to address these gaps and to our knowledge, none have been undertaken to prevent rv outbreaks in rehabilitation settings. in the paediatric population, nosocomial transmission has been shown to be prevented through screening of patients and cohorting nurses with positive children along with the use of transmission-based precautions. in a population of stem cell transplant patients, universal use of surgical masks by healthcare providers was associated with a greater than % reduction in nosocomial rv infection. rehabilitation settings are environments conducive to nosocomial rv transmission due to the nature of the patient population, which is being mobilised daily in common spaces. our approach was to better understand the barriers to adherence to ipac practices within the rehabilitation context. we used epidemiological data to identify important triggers of outbreaks and then drilled down to understand the main drivers. for example, delays in testing and initiation of droplet and contact precautions were occurring due to lack of clarity around syndromic criteria which was addressed through systematic training. lack of private rooms was an additional barrier noted by our staff, but our experience showed that earlier initiation of transmission-based precautions actually led to the same number of isolation-days presumably due to reduced nosocomial transmission. finally, we uncovered a number of system problems that made it easier for staff to work while sick rather than staying home. these included the perceived pressure from human resources attendance management and the challenge in accessing ohs. once these were addressed through open access redevelopment of an ohs electronic software reporting programme, better reporting of illness was seen. these examples underscore the impact of process changes that are linked to the specific barriers to best practice rather than relying on education alone. a striking finding in our study is that despite making it easier for staff to report to ohs, we observed no significant increase in the average number of missed work hours. one explanation for this finding is that the additional absences for staff staying home when potentially infectious was offset by a reduction in staff becoming infected while at work due to fewer encounters with infectious colleagues and fewer unprotected encounters with nosocomial rv cases. these data argue against the perception that staff staying home when symptomatic could increase staff shortages and should empower organisations to create similar models of care that make staying home when sick the easier thing to do. despite improvement in ipac practices, we continued to observe sporadic nosocomial transmission of rvs at our facility. one potential explanation is that increased viral testing resulted in ascertainment of more nosocomial cases even though there was reduced transmission through earlier use of droplet and contact precautions. another possibility is that our intervention could not fully prevent nosocomial transmission as visitors continued to enter with infectious symptoms since we could not monitor this practice accurately. finally, even though we reduced symptomatic visitation, either healthcare providers or visitors could have continued to transmit rv through asymptomatic viral carriage. some studies found limiting visits by children under the age of during the winter months, may reduce nosocomial transmission of viral respiratory infection. however, this approach may be a challenging and costly to operationalise as active screeners have to be strategically posted throughout the season. our study has several important limitations. we compared only three rv seasons where interyear differences could be explainable by differences in seasonality. however, the documented practice changes that coincided with the lower outbreak-days suggest that these improvements were related to the intervention. implementation of multiple strategies simultaneously made it difficult to assess the effectiveness of individual components, which would have been useful to direct resources more efficiently. on the other hand, the impact on prevention of rv outbreaks was close to predicted based on the individual drivers identified. our study highlights the impact of new processes of care that address barriers to following ipac practices on the prevention of nosocomial outbreaks in the rehabilitation setting. these strategies have the potential to improve both patient and staff safety across these institutions. nosocomial respiratory syncytial virus infections: the cost-effectiveness and costbenefit of infection control nosocomial respiratory syncytial virus infection: impact of prospective surveillance and targeted infection control nosocomial respiratory syncytial virus infections: prevention and control in bone marrow transplant patients risk of nosocomial respiratory syncytial virus infection and effectiveness of control measures to prevent transmission events: a systematic review prospective controlled study of four infection-control procedures to prevent nosocomial infection with respiratory syncytial virus ontario agency for health protection and promotion, provincial infectious diseases advisory committee. routine practices and additional precautions in all health care settings. rd edition. toronto, on: queen's printer for ontario infectious diseases protocol: appendix b: provincial case definitions for diseases of public health significance. disease: respiratory infection outbreaks in institutions and public hospitals universal mask usage for reduction of respiratory viral infections after stem cell transplant: a prospective trial advancing infection prevention and antimicrobial stewardship through improvement science working with symptoms of a respiratory infection: staff who care for high-risk individuals transmission of influenza: implications for control in health care settings respiratory virus infections after marrow transplant: the fred hutchinson cancer research center experience acknowledgements we would like to thank all staff at st. john's rehabilitation centre for their engagement in the development of these practice changes as well as the support from infection prevention & control, occupational health & safety, human resources, unit managers and the senior leadership team.contributors cc, ns, ta, wm, lr, lmdm and jal contributed to study concept and design. acquisition, analysis or interpretation of data performed by cc, vw, wm and jal. manuscript was drafted by cc, vw and jal. critical revision of the manuscript received from all authors.funding the authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.competing interests none declared.patient consent for publication not required.provenance and peer review not commissioned; externally peer reviewed.data availability statement all data relevant to the study are included in the article or uploaded as supplementary information.open access this is an open access article distributed in accordance with the creative commons attribution non commercial (cc by-nc . ) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. see: http:// creativecommons. org/ licenses/ by-nc/ . /. key: cord- -htgjwcgs authors: sachse, sven; hunger, iris title: lage – krise – katastrophe. eine konzeptualisierung biologischer gefahrenlagen date: - - journal: bundesgesundheitsblatt gesundheitsforschung gesundheitsschutz doi: . /s - - - sha: doc_id: cord_uid: htgjwcgs unusual biological threats demand adequate preparedness efforts, as demonstrated, for example, by the ebola virus disease outbreak in west africa in / and pandemic influenza in / . in germany, responsibilities for such efforts are located in different governmental authorities and differ from state to state. as a result, there are many different preparedness approaches using divergent core terminology. in this article a common definition for the term “unusual biological incident” is proposed. to do so, a literature review as well as semi-structured expert interviews with representatives of central actors in germany were conducted. the understanding of “unusual biological incident” was not consistent among experts; four approaches to qualify a biological incident as “unusual” were identified. these were merged in a comprehensive system-oriented approach that focuses on the health system’s resilience and on shortages of knowledge and material resources during incidents. based on this approach, we suggest a stage model for the categorization of biological threats as “incident,” “crisis,” “severe crisis,” or “disaster.” the need for central coordination is a defining characteristic to qualify a biological incident as “unusual.” based on the identified shortages, the necessary response strategies can be derived. nicht nur der ausbruch des ebolafiebers in westafrika / und die pandemische influenza / zeigen, dass außergewöhnliche biologische gefahrenlagen einer vorsorgeplanung bedürfen, um die geografische ausbreitung des krankheitsausbruchs so gering wie möglich zu halten und die negativen auswirkungen zu begrenzen. die erwartungshaltung der bevölkerung gegenüber den staatlichen stellen hat hinsichtlich einer vorsorgeplanung im bereich krisen und katastrophen allgemein und damit auch hinsichtlich einer gesundheitsbedrohung in den letzten jahren immer weiter zugenommen [ , ] . auch im öffentlichen sektor wird davon ausgegangen, dass "irgendjemand die verantwortung übernehmen wird und dann auch weiß, was zu tun ist" [ ] . bedrohungen der gesundheit der bevölkerung ändern sich in deutschland seit einigen jahren: die wahrscheinlichkeit des auftretens bisher in europa unbekannter krankheiten steigt, die geschwindigkeit der (weltweiten) ausbreitung von krankheiten im ausbruchsfall nimmt zu und die bedrohung durch den absichtlichen einsatz von biologischen agenzien (bioterrorismus) besteht fort [ ] [ ] [ ] . als reaktion darauf sind in deutschland auf bundesebene eine reihe von zwischen bund und ländern abgestimmten krankheitsspezifischen und generischen vorsorgeplänen entwickelt worden. die zuständigkeit hinsichtlich der medizinischen versorgung und des ausbruchsmanagements im rahmen der vorsorgeplanung liegt hierbei beim gesundheitsressort, wohingegen die abwehr bioterroristischer gefahren dem innenressort zuzuordnen ist (zu den zuständigkeiten ausführlicher: [ ] ). eine strikte trennung der vorsorgeplanung nach diesen zuständigkeiten ist nicht zweckmäßig unter berücksichtigung der tatsache, dass sowohl die zum management notwendigen ressourcen als auch das betroffene schutzgut der bevölkerungsgesundheit in beiden fällen (bioterrorismus und natürliches seuchengeschehen) identisch sind. "gesundheitlicher bevölkerungsschutz auf bundes-und landesebene in deutschland [erfordert] gemeinsame konzepte sowie nationale und internationale kooperation und koordination" [ ] . ressourcengerechte vorsorgeplanung muss darüber hinaus auch generisch ausgerichtet, das heißt flexibel auf unvorhersehbare und unbekannte biologische gefahrenlagen anwendbar sein. die erstellung "gemeinsamer konzepte" in der schnittmenge von gesundheits-und sicherheitssektor ist unter dem aspekt zukunftsorientierter, ressourcenadaptierter und somit effektiver vorsorgeplanung gewissermaßen unabdingbar. grundvoraussetzung für "gemeinsame konzepte" ist die klärung der grundbegriffe des zu bearbeitenden sachverhalts und damit des eigentlich durch vorsorgeplanungpräventiv zubearbeitenden problems. ein begrifflicher konsens zwischen den beteiligten akteuren verringert kommunikationsbarrieren und ist für den effektiven ablauf von bewältigungsmaßnahmen unerlässlich. es ist daher problematisch, dass begrifflichkeiten gleichlautend sind, jedoch nicht zwangsläufig gleichbedeutend verwendet werden. gleichzeitig erscheint es unmöglich, für zukünftige, zum teil rein hypothetische lagen vorzusorgen, wenn schutzgegenstand und schutzziel terminologisch unklar sind und konzeptionell nicht sauber festgelegt werden. die vielfalt an unterschiedlichen ansätzen der konzeptualisierung biologischer gefahrenlagen sowohl auf nationaler als auch internationaler ebene zeigt einen klärungsbedarf auf. (zu der gleichermaßen im katastrophenschutz bestehenden problematik terminologischer abweichungen siehe [ ] .) im folgenden soll auf grundlage der gemeinsamkeiten und unterschiede bestehender vorsorgeplanungen und der untersuchung des expertenverständnisses der begriff der "außergewöhnlichen biologischen gefahrenlage" für den deutschsprachigen raum näher bestimmt werden. ziel ist eine harmonisierung des verständnisses und der nutzung des begriffs, um vorsorgeplanungen eindeutiger und damit effektiver zu machen. zusätzlich wurden neun semistrukturierte experteninterviews durchgeführt. ein interviewleitfaden diente als primäres erhebungsinstrument. die zentrale frage bezog sich auf das begriffsverständnis der "außerordentlichen biologischen gefahrenlage". unter "experten" wurden personen verstanden, die auf nationaler ebene für den entwurf, die implementierung oder kontrolle der umsetzung von vorsorgeplänen verantwortlich sind oder über einen privilegierten zugang zu derartigen informationen verfügen. bei der auswahl der interviewpartner wurden die besonderheiten des durch den föderalismus geprägten deutschen öffentlichen gesundheitssystems berücksichtigt. neben den akteuren des bundes (bundesministerium für gesundheit und robert koch-institut) wurden auch akteure auf landes-und kommunaler ebene befragt. zusätzlich wurden experten aus der gesundheitsversorgenden praxis (sonderisolierstation eines maximalversorgenden krankenhauses) und der medizinischen forschung (helmholtz-zentrum für infektionsforschung) interviewt. die identifizierung der zuständigen abteilungen bzw. organisationseinheiten erfolgte anhand der öffentlich zugänglichen organigramme und, falls vorliegend, der geschäftsverteilungspläne. der interviewleitfaden wurde den experten im vorfeld zugeschickt. eine mögliche verzerrung durch gezielte vorberei-tung der befragten im vorfeld wurde in kauf genommen, um eine möglichst hohe informationsdichte bei der begrenzten anzahl an geführten interviews zu erhalten. die interviews wurden aufgezeichnet und in anlehnung an die regeln von gläser und laudel zur auswertung transkribiert [ ] . einen ersten anhaltspunkt zur definition der "außergewöhnlichen biologischen gefahrenlage" bietet fock [ ] . nach ihm zeichnen sich außergewöhnliche biologische gefahrenlagen in abgrenzung von anderen gefahrenlagen durch erschwerte wahrnehmbarkeit, enorme variabilität des gefahrenpotenzials (agens, ausbringungsform), weitere verbreitung des agens durch dritte sowie das potenzial einer massenhysterie in der bevölkerung aus. hierbei handelt es sich um eine "maximaldefinition", die in jüngerer vergangenheit aufgetretene außergewöhnliche biologische gefahrenlagen (z. b. ebolafieberausbruch / und ehec (enterohämorrhagische escherichia coli)/o -ausbruch ) nicht erfüllen. die literaturrecherche identifizierte auf bundesebene vier bestehende vorsorgepläne. in reaktion auf die wachsende besorgnis um bioterroristische anschläge entstand im jahr mit dem "bund-länder-rahmenkonzept zu notwendigen fachlichen vorbereitungen und maßnahmen zur seuchenbekämpfung nach bioterroristischen anschlägen (teil pocken)" ein erstes umfassendes konzept zum management außergewöhnlicher biologischer gefahrenlagen [ ] . zugrunde liegt dem konzept die annahme, dass jedes auftreten von pocken, selbst wenn es sich nur um wenige fälle handelt, eine außergewöhnliche biologische gefahrenlage darstellt. im pockenrahmenplan heißt es: "wenn eine absichtliche ausbringung wahrscheinlich ist, [muss] von einer realen bedrohung für die sicherheit der bundesrepublik deutschland ausgegangen werden" [ ] . nachdem europa im zuge der globalen polioeradikationsinitiative durch die weltgesundheitsorganisation für poliofrei erklärt wurde, ist der "polioleitfaden" entwickelt worden [ ] . dieser legt vor allem maßnahmen für die aufrechterhaltung eines erreichten zustandes fest und ist damit nicht primär ein notfallplan. aber auch hier gilt das auftreten bereits eines einzigen falls als außergewöhnliche biologische gefahrenlage, da dies den erfolg der eradikationsinitiative gefährdet. der globale aufruf der weltgesundheitsorganisation zur pandemischen influenzavorsorgeplanung mündete in dem nationalen influenzapandemieplan für deutschland [ ] , der zuletzt im jahr aktualisiert wurde [ ] . ursprünglich orientierte sich die vorsorgeplanung für eine pandemische situation strikt am influenzastufenmodell der weltgesundheitsorganisation, welches je nach schweregrad der pandemie eine mehr oder weniger hohe alarmstufe definierte, welche konkrete maßnahmen nach sich zog. erfahrungen mit der "schweinegrippe" / haben zu einer flexibilisierung dieses stufenmodells geführt [ ] [ ] [ ] . als reaktion auf den ausbruch des ebolafiebers in westafrika / wurde das "rahmenkonzept ebolafieber" erarbeitet [ ] . in diesem fall wurde originäre vorsorgeplanung betrieben, um bei möglichen nationalen fällen schnellstmöglich eine einheitliche handlungsgrundlage zu etablieren und denkbare probleme vorab zu adressieren. wie bei den pocken würde auch jegliches auftreten von ebolafieber in deutschland als "außergewöhnliche biologische gefahrenlage" eingeschätzt werden. entsprechende vorsorgeplanung wurde daher schon bei sehr geringem einschleppungsrisiko und der reinen möglichkeit des auftretens "einer infizierten person im einzelfall" betrieben [ ] . die in deutschland bestehende föderale zuständigkeitsteilung im gesundheitswesen weist die vorsorgeplanung für biologische gefahrenlagen als grundsätzliche aufgabe des öffentlichen gesundheitsdienstes (Ögd) den ländern zu. somit existieren innerhalb der bundesrepublik deutschland neben den planungen auf ebene des bundes weitere, landesspezifische vorsorgepläne, die teilweise unterschiedliche konzeptansätze aufweisen. ein herausragendes beispiel für vorsorgeplanung auf landesebene ist der "generische plan für biologische gefahrenlagen" des landes berlin. dieser umfasst fünf verschiedene szenarien unterschiedlichen schweregrades, die idealtypisch für verschiedene gefahrensituationen stehen. zu den szenarien gehören der "einzelfall einer hochkontagiösen lebensbedrohlichen erkrankung" (z. b. ebolafieber oder sonstige virale hämorrhagische fieber), "lokale epidemie(n)" (z. b. salmonellosen oder masern), "überregionale epidemie(n)" (z. b. ehec), pandemien (z. b. influenza, schweres akutes respiratorisches syndrom [sars], pocken) und das "auffinden von verdächtigen materialien (z. b. pulverfund mit verdacht auf anthrax)" [ ] . bei der einordnung der schwere einer situation wurden im rahmen der dort erfolgten konzeptualisierung die parameter "anzahl betroffener", "morbidität/letalität", "örtliche ausbreitung", "ausbreitungswahrscheinlichkeit" und "ausbreitungsdynamik" berücksichtigt [ ] . die gesetzlichen vorgaben kommen weitgehend ohne eindeutige definitionen aus. zwar wird nach der jüngsten Änderung des infektionsschutzgesetzes (ifsg) der begriff der "bedrohlichen übertragbaren krankheit" legaldefiniert, doch bezieht sich diese definition nur auf die unbestimmten tatbestandsmerkmale der "klinisch schweren verlaufsform" oder einer erforderlichen "schwerwiegenden gefahr für die allgemeinheit" (vgl. § nr. a ifsg). diese definition gibt keine klaren maßstäbe für die objektive bewertung oder qualifikation einer gefahrenlage als besonders schwerwiegend vor, sondern bezieht sich durch unbestimmte rechtsbegriffe ausschließlich auf abstrakte und interpretationsbedürftige eigenschaften der übertragbaren krankheit. im rahmen der regelungen des bund-länder-informationsverfahrens (siehe § ifsg) wird von "epidemisch bedeutsamen fällen" gesprochen, diese werden jedoch an keiner stelle -auch nicht in der da- basierend auf diesem ansatz schlagen wir ein stufenmodell zur kategorisierung biologischer gefahrenlagen in "lage", "krise", "schwere krise" und "katastrophe" vor. die notwendigkeit zentraler koordination kann darin als bestimmendes merkmal der "außergewöhnlichkeit" einer biologischen gefahrenlage identifiziert werden. aus der identifizierung der konkret bestehenden mängel lassen sich die erforderlichen bewältigungsstrategien ableiten. biologische gefahrenabwehr · cbrn-lage · gesundheitskrise · ressourcenmanagement · vorsorgeplanung unusual biological threats demand adequate preparedness efforts, as demonstrated, for example, by the ebola virus disease outbreak in west africa in / and pandemic influenza in / . in germany, responsibilities for such efforts are located in different governmental authorities and differ from state to state. as a result, there are many different preparedness approaches using divergent core terminology. in this article a common definition for the term "unusual biological incident" is proposed. to do so, a literature review as well as semi-structured expert interviews with representatives of central actors in germany were conducted. the understanding of "unusual biological incident" was not consistent among experts; four approaches to qualify a biological incident as "unusual" were identified. these were merged in a comprehensive system-oriented approach that focuses on the health system's resilience and on shortages of knowledge and material resources during incidents. based on this approach, we suggest a stage model for the categorization of biological threats as "incident, " "crisis, " "severe crisis, " or "disaster. " the need for central coordination is a defining characteristic to qualify a biological incident as "unusual. " based on the identified shortages, the necessary response strategies can be derived. biological incident response · cbrn threat · health crisis · resource management · preparedness planning zugehörigen verwaltungsvorschrift zur ifsg-koordinierung -definiert. das begriffsverständnis der "außergewöhnlichen biologischen gefahrenlage" war unter den experten nicht einheitlich. viele experten empfanden ihn als "sehr polizeilichen begriff, der sehr stark durch . . . biologische sicherheit . . . geprägt" sei und eher assoziationen mit dem themenfeld "bioterrorismus" nahelegen würde. grundsätzlich ließen sich je nach aufgaben-und zuständigkeitsbereich des experten unterschiedliche schwerpunktsetzungen in der auslegung identifizieren. je "niedriger" ein experte in der zuständigkeitenkette angesiedelt war, desto pragmatischer war die begriffsinterpretation. ein experte stellte fest, dass nur vier verschiedene biologische gefahrenlagen unterschieden werden könnten: der einzelfall, die epidemie bzw. pandemie, der laborunfall und die intentionale ausbringung. der begriff "außergewöhnliche biologische gefahrenlage" sei schon eine einschränkung, denn "es gibt biologische gefahrenlagen, und dann gibt es außergewöhnliche". die expertenmeinungen, hinsichtlich der entscheidungskriterien zur einstufung einer biologischen gefahrenlage als "außergewöhnlich", können grob in vier gruppen unterteilt werden. es kann auf spezifische eigenschaften des auslösenden biologischen agens mittels vorab festgelegter einstufungskriterien abgestellt werden ("erregerspezifischer ansatz"). als solche kriterien könnten u. a. herangezogen werden: die hohe infektiosität und virulenz des erregers, die hohe letalität oder kontagiösität der resultierenden krankheit, eine schlechte therapierbarkeit und gravierende seuchenhygienische maßnahmen sowie komplizierte diagnostik -insgesamt also eine ausgelöste hohe populationsbasierte krankheitslast. als beispiel für eine derartige einstufung wur- im englischenwirdindiesem zusammenhang häufig von "high consequence infectious diseases" gesprochen. den die "dirty dozen, die ja bekannt" seien, genannt. einige experten führten zur differenzierung den ursprung einer biologischen gefahrenlage an, bei welchem zwischen beabsichtigter und unbeabsichtigter ausbringung biologischer agenzien unterschieden wird ("verursacheransatz"). so sei eine beabsichtigte ausbringung immer außergewöhnlich, denn auch bekannte erreger könnten sich dann "nicht so verbreite[n], wie wir es kennen, und dadurch die gefahrenlage außergewöhnlich" machen. weiterhin gilt das "ausmaß" einer lage, also die anzahl und geografische verteilung der betroffenen ("quantitativgeografischer ansatz"), unter den experten als maßstab. durch die auswertung bestehender vorsorgeplanungen und die darin zum ausdruck kommenden definitionen kann die für die konzeptualisierung biologischer gefahrenlagen grundlegende unterscheidung zwischen "normal" und "außergewöhnlich" nicht aussagekräftig vorgenommen werden. es muss davon ausgegangen werden, dass die entwickler der bestehenden vorsorgepläne selbst ein durch ihre individuelle vorbildung geprägtes grundlegendes, inhärentes verständnis des themenkomplexes voraussetzen. bedingt durch eine derartig individuell geprägte auslegung von begrifflichkeiten unterliegt ein unstrukturierter, lediglich auf dem wortlaut beruhender vergleich unweigerlich einem terminologischen interpretationsbias. bei den experteninterviews wurden daher spezifisch fragen zum begriffsverständnis der "außergewöhnlichen biologischen gefahrenlage" gestellt. jedoch konnte der begriff auch durch die experteninterviews nicht eindeutig geklärt werden. da es sich um den kernbegriff vieler konzepte (wenn auch in anderer terminologie ausgedrückt) handelt, soll unter berücksichtigung der im rahmen der interviews genannten merkmale eine grundsätzliche definition gefunden werden. die dynamik biologischer gefahrenlagen verlangt es auch, als definition kein starres gerüst zu schaffen. vielmehr ist ein dynamischer und systemorientierter ansatz zu wählen, der auf die bestehende bewältigungskapazität des betroffenen systems abstellt (resilienz des gesundheitssystems, in anlehnung an [ , ] ; zu der aufkommenden bedeutung in der risikoforschung siehe [ ] , auch im englischsprachigen raum [ ] [ ] [ ] die "außergewöhnliche biologische gefahrenlage" wurde in den interviews als situation, die das system "überfordert", beschrieben. eine Überforderung bezeichnet in dem fall das notwendige abweichen von routineprozessen, das erforderlich wird, wenn bestehende prozesse nicht auf eine konkrete (ausnahme-)situation anwendbar sind oder für eine solche noch nicht existieren. im verständnis des hier vorgestellten systemorientierten ansatzes sind bei objektiven oder absoluten mängeln durch die zentrale koordination neues wissen und neue ressourcen zu schaffen. bei subjektiven beziehungsweise quantitativen mängeln kann auf woanders bestehende ressourcen zurückgegriffen werden, wobei diese im rahmen einer zentralen koordination "lediglich" umdisponiert werden müssen. allgemein wird sich originäre ressourcengenerierung schwieriger gestalten als eine ressourcenreorganisation, weshalb objektive bzw. absolute mangelsituationen tendenziell als schwerwiegender anzusehen sind. neben der beurteilung außergewöhnlicher biologischer gefahrenlagen anhand der genannten punkte wird zusätzlich vorgeschlagen, verschiedene stufen biologischer gefahrenlagen zwecks gefahrenbewertung zu unterscheiden. den ausgangspunkt bildet die "lage" (stufe ), welche sich durch das vorhandensein einer biologischen gefahr auszeichnet. bei stufe entsteht ein zentraler koordinationsbedarf entweder durch wissensbasierte oder durch ressourcenbasierte mängel. im ersten fall werden managementprozesse eher auf informations-strukturen, den aufbau von fachwissen oder kommunikationsmaßnahmen abzielen. im zweiten fall wird der schwerpunkt auf der ressourcengenerierung liegen. solche außergewöhnlichen biologischen gefahrenlagen können auch als "krise" bezeichnet werden. bei stufe sind wissen und materielle ressourcen gleichzeitig nicht ausreichend vorhanden. eine solche lage wird, sollte sie auftreten, noch intensivere zentrale koordination erfordern, da maßnahmen zur beseitigung der bestehenden mängel auf wissens-und ressourcenebene parallel ablaufen müssen. außergewöhnliche biologische gefahrenlagen der stufe können auch als "schwere krise" bezeichnet werden. eine mangels bewältigungskapazität (temporär) nicht zu überwindende gefahrenlage, die mit einem gewissen systemversagen einhergeht, kann als "katastrophe" (stufe ) bezeichnet werden. im rahmen des managements der biologischen gefahrenlagen muss eine kontinuierliche lagebewertung (revision) stattfinden, die über den fortbestand einer einmal getroffenen einstufung entscheidet. ist eine beseitigung von mängeln im laufe des bewältigungsprozesses möglich und eine zentrale koordination nicht mehr nötig, kann eine ursprünglich außergewöhnliche biologische gefahrenlage zu einer gewöhnlichen (stufe ) werden. umgekehrt kann während einer gewöhnlichen gefahrenlage das auftreten von mängeln eine zentrale koordination nötig machen und damit die gefahrenlage zu einer außergewöhnlichen machen. eine zusammenfassende Übersicht bietet . abb. . zusammenfassend kann die frage nach einer definition unter zuhilfenahme des systemorientierten ansatzes be- der begriff "katastrophe" wird hier im sinne einer ressourcenbasierten Überforderungssituation verwendet. definitionen des begriffs "katastrophe" und rechtliche folgen des ausrufens eines katastrophenfalls nachdem katastrophenschutzrecht der länder bleiben unberührt. antwortet werden: eine biologische gefahrenlage ist immer dann "außergewöhnlich", wenn das fehlen von wissen oder materiellen bzw. personellen ressourcen einen zentralen koordinationsbedarf zur aufrechterhaltung der bewältigungskapazität des gesundheitssystems erforderlich macht. außergewöhnliche biologische gefahrenlagen können dabei insbesondere entstehen, wenn sie besonders gefährliche krankheiten betreffen, intentional verursacht werden oder nach geografischer ausdehnung oder zahl der betroffenen besonders "groß" sind. es wird die einteilung von biologischen gefahrenlagen in stufen vergleichend zum status quo (der "routinebewältigungskapazität") vorgeschlagen. so bildet die routine das "alltägliche" geschehen ab. besteht eine biologische gefahr, handelt es sich zunächst um eine lage (stufe ), die je nach schweregrad zu einer krise (stufe ), schweren krise (stufe ) oder letztlich zu einer katastrophe (stufe ) eskalieren kann. die föderalistisch bedingte fragmentierung der zuständigkeiten ist ressourcenaufwendig. der bund ist originär für den zivilschutz und subsidiär für landesübergreifende gefahrenlagen zuständig, die länder mit ihren öffentlichen gesundheitsdiensten gleichermaßen für den gesundheitlichen bevölkerungs-und katastrophenschutz im lokalgebiet. gerade durch diese trennung von zuständigkeiten und die vielzahl von vorsorgeplänen werden konzeptabweichungen immer vorhanden sein. es bleibt daher eine herausforderung, bei geteilter planungszuständigkeit kongruenz und optimale bewältigungskapazität zu erreichen. es muss auch überlegt werden, ob in zukunft die bisher noch vorherrschende trennung zwischen gewöhnlichen und außergewöhnlichen biologischen gefahrenlagen in der vorsorgeplanung sinnvoll ist. wenn stringent einer systemorientierten betrachtung gefolgt wird, sind immer dieselben ressourcen betroffen. ein unterschied zwischen gewöhnlichen und außergewöhnlichen lagen ergibt sich nur noch bei der aktivierung von plänen im ereignisfall und der damit einhergehenden ressourcengenerierung oder -allokation. katastrophenschutzkonzept der landesregierung die einbindung der bevölkerung in das krisen-und katastrophenmanagement in deutschland (der brd) nach dem zweiten weltkrieg analyse der bedeutung des subjektiven sicherheitsgefühls von verwaltungsmitarbeitern für die funktionsfähigkeit der öffentlichen verwaltung im pandemiefall jahrhundert: epidemien, pandemien und bioterrorismus konferenzbericht zum heidelberger dialog zur internationalen sicherheit : epidemien und pandemien als unkalkulierbares risiko? regulierung von gesundheit und sicherheit in einer vernetzten welt globalisierung und gesundheit rahmenkonzeption für den cbrn-schutz (abc-schutz) im bevölkerungsschutz krisenmanagement im bereich des gesundheitswesens terminologische normierungen und diskussion gesundheitssicherstellung in außergewöhnlichen biologischen gefahrenlagen. masterarbeit (unveröffentlicht) experteninterviews und qualitative inhaltsanalyse als instrumente rekonstruierender untersuchungen außergewöhnliche biologische gefahren vorbereitung auf eine biologische großschadenlage: der pockenrahmenplan das bund-länder-rahmenkonzept zur vorbereitung auf biologische gefahrenlagen (beispiel: pocken) leitfaden für gesundheitsämter zum vorgehen bei fällen von poliomyelitis in der bundesrepublik deutschland: erarbeitet von der nationalen kommission für die polioeradikation in der bundesrepublik deutschland in zusammenarbeit mit dem nationalen referenzzentrum für poliomyelitis und enteroviren am robert koch-institut expertengruppe influenza-pandemieplanung ( ) influenzapandemieplanung: nationaler influenzapandemieplan. bericht der expertengruppe nationaler pandemieplan teil i: strukturen und maßnahmen und teil ii: wissenschaftliche grundlagen recommendations for good practice in pandemic preparedness identified through evaluation of the response to pandemic (h n ) world health organization ( ) global influenza programme guidance revision frequently asked questions rahmenkonzept ebolafieber: vorbereitungen auf maßnahmen in deutschland generischer plan für biologische gefahrenlagen: anhand von szenarien rising from the ashes: development strategies in times of disaster environmental health in emergencies and disasters: a practical guide. world health organization resilienz in der risiko-und katastrophenforschung: perspektiven für disziplinübergreifende arbeitsfelder global catastrophic biological risks: toward a working definition building community disaster resilience: perspectives from a large urban county department of public health risk communication and generic preparedness: from agent-based to action-based planning-a conceptual framework key: cord- -ocnce z authors: torres, antoni; chalmers, james d.; dela cruz, charles s.; dominedò, cristina; kollef, marin; martin-loeches, ignacio; niederman, michael; wunderink, richard g. title: challenges in severe community-acquired pneumonia: a point-of-view review date: - - journal: intensive care med doi: . /s - - -y sha: doc_id: cord_uid: ocnce z purpose: severe community-acquired pneumonia (scap) is still associated with substantial morbidity and mortality. in this point-of-view review paper, a group of experts discuss the main controversies in scap: the role of severity scores to guide patient settings of care and empiric antibiotic therapy; the emergence of pathogens outside the core microorganisms of cap; viral scap; the best empirical treatment; septic shock as the most lethal complication; and the need for new antibiotics. methods: for all topics, the authors describe current controversies and evidence and provide recommendations and suggestions for future research. evidence was based on meta-analyses, most recent rcts and recent interventional or observational studies. recommendations were reached by consensus of all the authors. results and conclusions: the idsa/ats criteria remain the most pragmatic tool to predict icu admission. the authors recommend a combination of a beta-lactam/beta-lactamase inhibitor or a third g cephalosporin plus a macrolide in most scap patients, and to empirically cover pes (p. aeruginosa, extended spectrum beta-lactamase producing enterobacteriaceae, methicillin-resistant s. aureus) pathogens when at least two specific risk factors are present. in patients with influenza cap, the authors recommend the use of oseltamivir and avoidance of the use of steroids. corticosteroids can be used in case of refractory shock and high systemic inflammatory response. during recent decades, the number of patients requiring intensive care management due to severe community-acquired pneumonia (scap) has increased globally, especially among the elderly, patients with comorbidities and the immunocompromised [ ] . a large populationbased surveillance study on hospitalized cap patients found that % of patients required intensive care unit (icu) admission, with % of them needing mechanical ventilation [ ] . scap hospital mortality is still high, ranging from % to more than % [ , ] . since delays from hospitalization to icu admission have been related to increased mortality [ ] , several scoring systems have been evaluated in order to promptly identify patients requiring intensive care management and to guide empiric antibiotic therapy [ ] . streptococcus pneumoniae remains the main pathogen responsible of cap, regardless of age and comorbidities [ ] . however, approximately % of cap are caused by antibiotic-resistant pathogens [ ] . furthermore, the implementation of multiplex polymerase chain reaction (pcr) techniques has identified respiratory viruses, mainly influenza virus and rhinovirus, as important cap causative pathogens [ ] . early adequate antibiotic administration is crucial in scap management [ ] ; however, the optimal strategy is still far from being established. initial antimicrobial therapy lacking activity against the offending pathogens has been associated with greater mortality [ ] . the cluster rct from postma et al. [ ] showed the same efficacy when comparing beta-lactam monotherapy with betalactam plus macrolide or quinolone. the constant debate regarding the superiority of β-lactam plus macrolide compared to β-lactam plus fluoroquinolones in scap is still open [ ] . septic shock is the most lethal complication of scap. corticosteroids are recommended in refractory septic shock, although some controversies still remain. due to the emergence of pathogens outside the core microorganisms of cap [ ] , new antibiotics are urgently needed. in this point-of-view review paper, a group of experts discuss the current main controversies regarding scap: severity scores, pathogens outside the core microorganisms of cap (pes pathogens), viral scap, empirical treatment, septic shock and the potential role of new antibiotics. all the topics include four sections: the current controversy, the evidence, suggested recommendations and suggestions for future research. the evidence was based on meta-analyses, most recent rcts and recent interventional or observational studies that the panel considered important for the question. recommendations were reached by consensus of all the authors and are summarized in table . severity assessment is an essential component of the initial evaluation of cap patients [ ] . to date, there is no consensus on the optimal assessment tool or how it should be applied in clinical practice [ , ] . some "real-world" problems may complicate the interpretation of studies that investigate scores for icu admission prediction [ ] . in one study, / of patients presenting to hospital had advanced directives or do not attempt resuscitation (dnar) orders in place that made icu admission inappropriate [ ] . second, many studies include patients who require mechanical ventilation or vasopressor treatment at admission in "prediction" studies, making a prediction score moot [ ] . third, the number of adult icu beds [ ] , the threshold for icu admission and the characteristics of patients admitted to icu are highly variable across different healthcare systems. finally, there is still relatively little evidence that implementation of severity tools into clinical practice results in improved outcomes [ ] . the two most widely used severity assessment tools in cap, the pneumonia severity index (psi) and the curb score, perform well to predict -day mortality, but are less useful in identifying scap requiring icu admission [ ] . this reflects the strong influence of age on both scoring systems, and the low value provided to respiratory failure and other organ dysfunctions which are often a major driver of icu admission. alternative scoring systems have been proposed that are more focused on organ dysfunction. the idsa/ats criteria (table ) predict both mortality and future requirements for mechanical ventilation and vasopressor support as a surrogate of icu admission [ ] . simplification of these criteria with the removal of less common organ dysfunctions is possible without losing prognostic accuracy. lim et al. [ ] conducted a before and after implementation study in which the idsa/ats criteria were used to triage patients. this resulted in a reduced mortality (from . to . %), an increased use of icu resources ( . % of patients admitted to the icu vs.. a group of experts discuss current controversies regarding severe community-acquired pneumonia and provide a summary of recommendations. the idsa/ats criteria remain the most pragmatic and robust tools to predict patients requiring icu admission we recommend empirically covering pes pathogens in scap when at least two specific risk factors are present we recommend the use of prompt therapy with oseltamivir in patients with influenza cap and avoidance of the use of steroids. zanamivir can be used in cases of treatment failure and/or confirmed oseltamivir resistance we recommend a combination of a beta-lactam/beta-lactamase inhibitor or a third g cephalosporin plus a macrolide in most scap patients patients with scap and septic shock should be managed with current practice guidelines. corticosteroids can be used in cases of refractory shock and high systemic inflammatory response based on available data, new antibiotics providing existing limitations in empiric therapy (including macrolide resistant species and mrsa) are needed . % previously) and reduced delayed icu admissions. similar criteria are included in the smart-cop tool [ ] . recently, it has been shown that sepsis- criteria can also help to identify patients at risk of icu admission, although disease-specific tools still have the best discrimination for mortality [ ] . the idsa/ats criteria remain the most pragmatic and robust tools for predicting patients requiring icu admission. major criteria identify patients requiring immediate icu care, while minor criteria (either the simplified or standard version) identify patients with a higher likelihood of requiring icu care and benefiting from more aggressive therapy or closer observation [ , , ] . psi and curb should not be used to guide icu care as they can be misleading. biomarkers such as c reactive protein, proadrenomedullin, procalcitonin and others have been suggested to provide additional information about cap prognosis [ , ] . none are currently fully validated and ready for implementation in clinical practice. we need data demonstrating the utility of severity scores to predict a complicated course of cap, to help improving patient allocation (need for icu admission) and to identify patients likely to respond to specific therapies, including corticosteroids or macrolides [ ] [ ] [ ] . finally, we need data demonstrating a lower mortality rate when these scoring systems are used. guidelines for cap recommend empiric therapy for pathogens outside the core microorganisms of cap, including methicillin-resistant s. aureus (mrsa), p. aeruginosa and other drug-resistant gram-negatives, in selected patients with severe illness [ ] . however, the incidence of these pathogens in cap is low and often varies with geography and patient characteristics. the healthcare-associated pneumonia (hcap) definition is not a good predictor of these pathogens [ ] . identifying patients at higher risk could avoid the overuse of broadspectrum empiric therapy. in one study of icu-admitted cap patients, one in six pseudomonas were resistant to third-generation cephalosporins with antipseudomonal activity. common pathogens included e. coli ( . %), p. aeruginosa stenotrophomas maltophilia ( . %) and acinetobacter baumannii ( . %) [ ] . in one review, the incidence of gram-negative cap was estimated to be between and %, but not all these organisms were resistant and not all patients were in the icu [ ] . shindo et al. reported . % of cap and . % of hcap caused by drug-resistant pathogens [ ] ; however, the number of patients treated in icu was not stated. similarly, in another study, . % of cap and . % of hcap were caused by pathogens outside the core microorganisms of cap, but only of were treated in icu [ ] . one recent development is the concept of pes (p. aeruginosa, extended-spectrum beta-lactamase producing enterobacteriaceae and mrsa) pathogens. pes pathogens have been identified in % of cap patients with an etiologic diagnosis, with p. aeruginosa being the most common; - % of patients with pes pathogens required icu admission, more often than those without these pathogens [ ] . in another study of cap patients, pes pathogens were found in . % patients but icu admission was needed in only . % cases, a rate similar to those without pes pathogens [ ] . risk factors for pes pathogens have been identified, although most studies are not specific to icu patients (table ). webb et al. divided risk factors into therapyrelated (extrinsic factors), patient-related (intrinsic factors) and those related to selective antibiotic pressure [ ] . in one study, risk factors were prior antibiotic therapy, gastric acid-suppressive therapy, tube feeding and non-ambulatory status [ ] , while in another study were cap severity, prior antibiotic therapy, recent hospitalization, poor functional status, dialysis and immune suppression [ ] . in a study of bacteremic cap due to pes pathogens, risk factors for these pathogens were prior antibiotic therapy, low c-reactive protein (crp) and the absence of pleuritic chest pain [ ] . some studies have focused on risks for specific pathogens. a multinational study of patients found p. aeruginosa in . % and antibiotic-resistant p. aeruginosa in % [ ] . risk factors for p. aeruginosa were in another study, risk factors for p. aeruginosa cap included male sex, chronic respiratory diseases, lower crp and higher psi. however, the only risk factor for antibiotic-resistant p. aeruginosa was prior antibiotic therapy [ ] . risk factors for mrsa included many of the above plus chronic dialysis, prior mrsa infection/colonization, recurrent skin infections and severe comorbidities [ , ] . the studies that investigate risk factors for pes pathogens often use the term "multidrug resistent" (mdr), although they include indistinctly mdr and non-mdr microorganisms, mainly p. aeruginosa. in this manuscript we decided to use the acronym pes because we believe it reflects better the need for a different antibiotic treatment covering these pathogens (carbapenems +/linezolid) compared to the standard one required for the "core" cap pathogens. we recommend covering pes pathogens when specific risk factors are present, including prior antibiotic therapy, recent hospitalization, recent p. aeruginosa or mrsa infection or colonization, poor functional status and immune suppression. when patients have at least risk factors, the frequency of pes pathogens can exceed %, thus requiring empiric therapy against these pathogens. [ , ] . we need prospective studies using invasive sampling methods and new molecular diagnostic tests in a population of cap patients treated exclusively in icu. we need to identify patients at higher risk of pes pathogens through accurate scoring systems and to determine a threshold above which empiric therapy for these pathogens is justified. finally, we need to be aware of scap microbiology future changes induced by influenza and pneumococcal vaccination, in both adults and children. before the appearance of influenza pandemics, respiratory viruses were uncommonly diagnosed and affected essentially patients with comorbidities [ ] . in fact, influenza virus a is the most frequent respiratory virus identified, followed by human rhinovirus, human respiratory syncytial virus (rsv) and influenza b virus. rsv is now recognized as a significant problem in the elderly, persons with cardiopulmonary diseases and immunocompromised hosts [ ] . a major controversy in patients with suspected severe viral cap (svcap) is twofold: the use of unnecessary antibiotics when the primary cause of pneumonia is viral without co-infection, and possible treatments with antiviral agents. currently, recommendations for patients with svcap are focused on rapid recognition of the pathogen and antiviral treatment with neuraminidase inhibitors (nais). recommendations regarding nais administration are controversial. the cochrane review of the topic in concluded that oseltamivir did not reduce hospitalizations and complications due to influenza [ ] . two systematic reviews and meta-analyses found that benefits in patients who were otherwise healthy did not outweigh its risks [ , ] . however, another meta-analysis found that oseltamivir was effective in the prevention of influenza at individual and household levels. in critically ill patients, observational studies have found a benefit to a prompt use of oseltamivir [ ] . on the other hand, zanamivir has been proposed by different guidelines, especially in immunosuppressed patients, based on a potential antiviral resistance to oseltamivir among circulating influenza viruses that is currently low [ ] . inhaled zanamivir is not recommended because of the lack of data regarding its use in patients with severe influenza disease. the use of corticosteroids has re-emerged in patients with scap based on recent randomized control trials (rct) and systematic review and meta-analysis [ , ] . in patients with svcap, the use of corticosteroids has not been associated with survival benefit but with an increased risk of nosocomial infections [ ] . a recent observational study found that corticosteroid administration as adjuvant therapy to standard antiviral treatment in critically ill patients with severe influenza pneumonia was associated with increased icu mortality [ ] . regarding rsv, not many treatment options are available, while a phase b rct of presatovir for the treatment of rsv in lung transplant recipients has been recently published with no positive results. we suggest maintaining an active communication with sentinel national and continental centers, and a local routine surveillance program in hospital settings [ ] . we advocate diagnosing svcap in accordance with a seasonal activity pattern. we encourage prompt treatment with oseltamivir in patients with svcap within the first h from influenza diagnosis. we recommend not using zanamivir regularly and only on the basis of treatment failure and confirmed oseltamivir mutations. a rct has not demonstrated a superior effect of zanamivir compared to oseltamivir; all treatments had a similar safety profile in hospitalised patients with severe influenza [ ] . we recommend avoiding the use of steroids in patients with svcap due to futile effect and an increased risk of super-infections in all subgroups of patients including the immunosuppressed. in cases of rsv, there is no available treatment at the present time. the best preferable evidence to determine the effect of nais should come from rcts. currently, only very few patients with high severity rates and a psi above have been enrolled in rcts for scap [ ] . in addition, in patients with infections, performing a rct with or without antibiotics will foremost be inappropriate and unethical. although there is sufficient evidence that antivirals decrease viral loads, their use in scap is still a matter of controversy [ ] . studies analyzing the timing of nais administration could provide further positive results. regarding the use of corticosteroids, a rct could be conducted in svcap patients with high inflammation and severity. no rct has specifically targeted scap. only one allowed enrollment of mechanically ventilated patients, while the rest specifically excluded scap patients [ ] . conversely, epidemiologic data suggest that scap patients may have a different etiologic spectrum than patients hospitalized outside the icu [ , ] , including a high incidence of viral infection. therefore, whether antibiotics appropriate for non-icu patients are safe and efficacious in scap is unclear. moreover, rapid diagnostic tests offer the possibility for specific treatment. if they demonstrate high sensitivity for atypical pathogens, fluoroquinolone monotherapy may even be superior to macrolides. whether other effects of macrolides are beneficial in cases other than s. pneumoniae is debatable, and beta-lactams are clearly not needed for atypicals. the controversy is threefold: ( ) is beta-lactam/macrolide combination therapy superior to other beta-lactam treatments? ( ) are additional antibiotics required for pes pathogens? and ( ) is prolonged antibiotic therapy needed for all patients with only positive viral testing? non-interventional trials suggest that beta-lactam/macrolide combination therapy is associated with lower mortality, especially in patients with pneumococcal bacteremia [ ] . however, the study by postma et al. [ ] found no difference in -day mortality when comparing beta-lactam alone with beta-lactam/macrolide or quinolones. the study exhibits two important limitations: first, % of patients had no chest x-ray confirmation; second, most patients had a low-grade severity pneumonia, as measured by the psi scale. another rct study [ ] found a lower rate of readmissions and a higher rate of clinical cure only in patients with psi categories iv and v pneumonia receiving beta-lactam plus macrolide. observational studies of beta-lactam/quinolone combination therapy for scap suggest better outcome than beta-lactam monotherapy. one prospective study found combination therapy with an early quinolone was slightly superior to a cephalosporin alone [ ] . three theories support the benefit of empirical macrolide combination: ( ) better coverage of atypical pathogens, including legionella, ( ) suppression of exotoxin production from s. pneumoniae [ ] , and ( ) host immunomodulatory effects. the latter two clearly differentiate between macrolides and quinolones, although both are effective against atypical pathogens. the underlying assumption that most of these culture-negative cases are s. pneumoniae is questionable with greater use of the highlyeffective conjugate pneumococcal vaccines [ ] . some data support the use of quinolones for proven severe legionella [ ] . methicillin-sensitive strains are likely covered adequately with standard empirical therapy. however, empirical coverage of mrsa for all scap patients does not improve outcomes [ ] . gross hemoptysis, leukopenia, skin rashes, and rapidly progressive or necrotizing infiltrates are relatively distinctive for the toxigenic community-acquired strain [ ] . observational studies suggest a better outcome with the use of antibiotics that interfere with ribosomal synthesis, such as linezolid or clindamycin [ ] . whether more rapid killing associated with the cephalosporin ceftaroline obviates the need for toxin suppression is unknown [ ] . patients with scap who are at risk for pathogens usually considered nosocomial represent a therapeutic dilemma. unfortunately, piperacillin/tazobactam, the most commonly prescribed antibiotic for suspected drug-resistant pathogens, has recently been shown to have adverse outcomes in patients with e. coli and k. pneumoniae bloodstream infection and ceftriaxone resistance [ ] . in cases of svcap, the overwhelming majority of patients receive empirical antibiotics despite infrequently documented bacterial superinfection. short-course prophylactic antibiotics may prevent bacterial superinfection while prolonged courses predispose to nosocomial infections, disrupting gut and lung microbiomes. it is worth pointing out that some scap cases require longer antibiotic administration. these include scap caused by s. aureus, patients with pleural effusions, pulmonary abscess and, patients with initial inadequate antibiotic treatment. we recommend a combination of a beta-lactam/betalactamase inhibitor or a third g cephalosporin plus a macrolide for most scap patients. legionella, if documented, should be treated with a quinolone. empirical linezolid should be reserved to patients with risk factors for community-acquired mrsa. empirical broader spectrum therapy for gram-negative pathogens should be limited to patients with several risk factors for pes pathogens. we need a rct of usual treatment (cephalosporin/ macrolide) with additional empirical coverage for pes pathogens versus pathogen-specific therapy based on rapid diagnostic testing. we need interventional studies investigating the duration of scap antibiotic treatment according to procalcitonin and rapid molecular diagnostic techniques. finally, we need a rct of short-course antibiotic therapy for scap patients with only viral detection on molecular testing. pneumonia is the most common cause of septic shock [ ] . despite improvements in the overall survival from severe sepsis, mortality from scap remains high-up to % in some studies [ ] . reasons for this discrepancy remain unclear, but it suggests the possibility that scap represents a unique subset of septic shock that deserves a unique set of guidelines for management. the high mortality in scap, despite early and adequate antibiotic treatment, may be a result of inadequate infection control and/or dysregulated inflammatory responses. the latter possibility raises the perennial question in the management of scap of whether or not to employ systemic corticosteroid therapy. current strategies to manage patients with scap and shock include the identification of pathogens using available diagnostics [ ] , early and appropriate (including combination) antimicrobial administration [ ] , hemodynamic resuscitation [ ] , and, for some patients, appropriate management of acute respiratory failure or ards [ ] . two recent rcts, the adrenal and the approc-chss, supported the use of adjunct corticosteroid therapy in septic shock (table ) , both studies demonstrating a reduction in the number of vasopressor-and ventilatordependent days [ , ] . in these studies, % and % of patients had pulmonary infections, respectively. the approcchss demonstrated a small mortality benefit, a feature some authors attributed to the inclusion of mineralocorticoids in the treatment protocol. however, this finding may be better explained by the higher baseline mortality in the latter trial, which would fit with the general trend in steroid trials dating back to (including the french trial, hypress, and corticus), which showed the greatest benefit of therapy in the sickest populations [ ] [ ] [ ] . a recent network meta-analysis of septic shock studies supported with strong evidence the role of corticosteroids in shock reversal [ ] . the use of steroids in scap (with or without shock) remains controversial [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , although many studies have shown significant reductions in length of stay and time to clinical stability. increasing evidence suggests that patients with strong inflammatory responses, such as those with highly elevated crp, may represent a subset of scap patients who would benefit from such corticosteroid treatment [ ] . conversely, corticosteroid use in patients with versus cap has been related to increased mortality [ ] . there is currently insufficient evidence to support other adjuvant therapies in scap, such as immunoglobulins, g-csf or statins [ ] . patients with scap and shock should be managed according to current practice guidelines. adjunctive therapy, including systemic corticosteroids, should be reserved for scap patients with refractory septic shock or with high systemic inflammatory response (as measured by crp). studies are still needed to clarify why scap mortality remains high despite improvements in overall sepsis outcomes. host inflammatory responses (both of the lung and systemic) require better characterization to determine the potential role of immune modulators in scap. finally, additional studies are needed to better assess patients' immune phenotype and to determine who should receive steroids and other immunosuppressive therapies. treatment success in scap rests on prompt delivery of antibiotics targeting the likely causative organisms. an important controversy is whether existing antibiotics are adequate therapies or whether new antimicrobials are needed. initial inappropriate empiric therapy in scap is primarily driven by the failure to cover a specific pathogen (e.g., mrsa) or the presence of a resistant bacterial pathogen (e.g., macrolide-resistant s. pneumoniae) [ ] . the need to empirically cover both "typical" bacterial pathogens (s. pneumonia, haemophilus influenza, mmsa) and "atypical" pathogens (mycoplasma pneumoniae, legionella [ , ] . based on the available data, it appears that new antibiotics providing coverage for the currently existing limitations in empiric therapy are needed (table ) . lefamulin is a novel semisynthetic pleuromutilin that inhibits bacterial growth by binding to the peptidyl transferase center of the s ribosomal subunit [ ] . pleuromutilins are not typically affected by resistance to other antibiotic classes (including macrolides, fluoroquinolones, and tetracyclines). two phase trials (leap intravenous to oral lefamulin; leap -oral only) have demonstrated comparable (non-inferior) outcomes to moxifloxacin (https ://inves tors.nabri va.com/stati c-files / c b - cc- -b d -d ea c d b (accessed july ). omadacycline is from the aminomethylcycline class created by chemical modification of minocycline. it inhibits protein synthesis by binding to the s ribosomal subunit. chemical modifications enable it to be active against the two main forms of bacterial resistance to the tetracyclines: efflux and ribosomal protection. results from the phase optic trial comparing once-daily oral and intravenous omadacycline to oral and intravenous moxifloxacin demonstrated noninferiority (https ://globe newsw ire.com/news-relea se/ / / / / /en/parat ek-annou nces-posit ive-phase - -study -of-omada cycli ne-in-commu nity-acqui red-bacte rial-pneum onia.html (accessed july ). delafloxacin (baxdela ™ ) is a potent fluoroquinolone with structural differences allowing it to move better than other fluoroquinolones through an acidic medium facilitating transmembrane passage into bacteria. delafloxacin has a high affinity for both topoisomerase iv and dna gyrase targets, giving it activity against gram-positive and gram-negative bacteria, as well as anaerobes and intracellular microorganisms [ ] . the results of a phase trial comparing delafloxacin to moxifloxacin for hospitalized patients with cap are awaited. solithromycin (solithera ™ ) is a fourth-generation macrolide and the first fluoroketolide in clinical development. solithromycin has potent in vitro activity against the most common cap pathogens, including fluoroquinolone-resistant isolates of s. pneumoniae. two phase trials of oral and intravenous to oral therapy for cap demonstrated comparable results to moxifloxacin [ ] . however, due to concerns over potential liver toxicity, the fda recommended that the company initiate a new clinical study to better evaluate the drug's safety profile in patients. nemonoxacin is a novel nonfluorinated quinolone with a wide antimicrobial spectrum covering gram-positive cocci and gram-negative bacilli, including the common cap pathogens. one published phase trial and two unpublished phase trials suggest that nemonoxacin is non-inferior to levofloxacin for the treatment of cap [ , ] . ceftaroline fosamil (teflaro ™ ) is an n-phosphonoamino water-soluble prodrug cephalosporin with the active form, ceftaroline, possessing broad-spectrum in vitro antimicrobial activity. the spectrum of activity includes typical cap bacterial pathogens and its high affinity for pbp a allows coverage of mrsa [ ] . the high superiority of ceftaroline compared to ceftriaxone in bacterial pneumonia was demonstrated in the focus and trials [ ] . the current role of the new antibiotics in scap is almost unknown, since the majority of them have not been studied in this specific subgroup of patients. ceftaroline could be added to the list of beta-lactams for the empirical or targeted treatment of scap. we need observational and/or rct studies of new antibiotics in the specific scap population. non-traditional agents, such as monoclonal antibodies, that may minimize or avoid the emergence of resistance should also be explored. scap is a major challenge in icu due to its high mortality, complications, short and long-term consequences. however, the optimal care is still not well standardized. scap remains a small section of general cap recommendations, and performing interventional and rcts in this subgroup of patients may be difficult. in this point-of-view review paper, we provide literature evidence, suggested recommendations and suggestions for future research regarding six seminal questions of scap management: ( ) who needs to be admitted to icu? ( ) when should pes pathogens be suspected? ( ) how should severe viral cap be managed? ( ) what is the optimal empirical antibiotic treatment for scap? 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campaign: international guidelines for management of sepsis and septic shock management of ards in adults adjunctive glucocorticoid therapy in patients with septic shock hydrocortisone plus fludrocortisone for adults with septic shock hydrocortisone therapy for patients with septic shock effect of treatment with low doses of hydrocortisone and fludrocortisone on mortality in patients with septic shock effect of hydrocortisone on development of shock among patients with severe sepsis: the hypress randomized clinical trial corticosteroids in septic shock: a systematic review and network meta-analysis corticosteroid therapy for severe community-acquired pneumonia: a meta-analysis corticosteroids in patients hospitalized with community-acquired pneumonia: systematic review and individual patient data metaanalysis efficacy of corticosteroid treatment for severe community-acquired pneumonia: a meta-analysis adjunctive therapies for community-acquired pneumonia: a systematic review corticosteroids in the treatment of community-acquired pneumonia in adults: a meta-analysis corticosteroid therapy for patients hospitalized with community-acquired pneumonia: a systematic review and meta-analysis adjunctive systemic corticosteroids for hospitalized community-acquired pneumonia: systematic review and meta-analysis adjunctive corticotherapy for community acquired pneumonia: a systematic review and meta-analysis efficacy and safety of corticosteroids for community-acquired pneumonia: a systematic review and meta-analysis corticosteroids as adjunctive therapy in the treatment of influenza nonantibiotic adjunctive therapies for community-acquired pneumonia (corticosteroids and beyond): where are we with them? a case series of macrolide treatment failures in community acquired pneumonia the effect of macrolide resistance on the presentation and outcome of patients hospitalized for streptococcus pneumoniae pneumonia macrolide-resistant mycoplasma pneumoniae prevalence and clinical aspects in adult patients with communityacquired pneumonia in china: a prospective multicenter surveillance study in vitro activity of lefamulin tested against streptococcus pneumoniae with defined serotypes, including multidrug-resistant isolates causing lower respiratory tract infections in the united states in vitro activity of delafloxacin against contemporary bacterial pathogens from the united states and europe spotlight on solithromycin in the treatment of community-acquired bacterial pneumonia: design, development, and potential place in therapy a randomized, double-blind, multicenter phase ii study comparing the efficacy and safety of oral nemonoxacin with oral levofloxacin in the treatment of communityacquired pneumonia managing community acquired pneumonia in the elderly-the next generation of pharmacotherapy on the horizon ceftaroline fosamil for the treatment of community-acquired pneumonia: from focus to capture key: cord- -iaktm a authors: soto-quintero, albanelly; guarrotxena, nekane; garcía, olga; quijada-garrido, isabel title: curcumin to promote the synthesis of silver nps and their self-assembly with a thermoresponsive polymer in core-shell nanohybrids date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: iaktm a this work presents a simple one-pot protocol to achieve core-doped shell nanohybrids comprising silver nanoparticles, curcumin and thermoresponsive polymeric shell taking advantage of the reducing properties of phenolic curcumin substance and its ability to decorate metallic surfaces. silver nanoparticles were synthesized, via sodium citrate and silver nitrate addition into a boiling aqueous solution of curcumin, monomers and surfactant. curcumin and sodium citrate promoted silver nucleation, acting as reducing and stabilizing agents. these curcumin-capped agnps enabled, after adding the radical polymerization initiator, the assembling of the growing polymer chains around the hydrophobic agnp surface. the resultant core-doped shell nanohybrids exhibit plasmonic, luminescent and volume thermoresponsive properties, with improved possibilities to be used as successful therapeutic platforms. in fact, the possibility to nanoconfine the synergistic antioxidant, antiviral, antibacterial features of silver and curcumin in one bioavailable hybrid paves the way to promising applications in the biomedical field. synthesis of ag@cur-p(meo ma) core-doped shell hybrid nps. ag@cur-p(meo ma) hybrid nanoparticles, from now on also called as (ag@cur-g), were synthesized through one-pot two-steps method (fig. ) . letter g denotes to thermoresponsive p(meo ma) polymer participation. a typical procedure for the synthesis is described below for sample ag@cur-g b (table ). in the first step (fig. b) , ag@cur nps (silver nps decorated with curcumin (table ) (fig. b) . the mixture was vigorously stirred for an additional min at reflux and then allowed to cool down slowly to room temperature. in the second step (fig. c) , p(meo ma) shell formation around ag@cur core was endowed by free radical precipitation polymerization (frpp) of meo ma and tegdma crosslinker. the solution was degassed by n gas for min at room temperature and polymerization was initiated by adding μl of aps solution ( . m) after temperature was raised to °c. the reaction was allowed to proceed for h at °c under stirring and n gas inlet (fig. c ). after that, the mixture was cooled down in an ice-cold water bath while it was exposed to the air. the resultant core-doped shell nps (ag@cur-g b) were purified by three centrifugation cycles by using successive decreasing centrifugal rates ( , and rpm) at °c for min. subsequent centrifugation rendered three supernatant fractions (f , f and f ) and a fourth fraction (f ) corresponding to the final precipitate from the third cycle. this procedure ensured the complete removal of empty polymer particles and excess of reactants. finally, nps pellet was redispersed in milli-q water and kept at °c in a refrigerator immediately prior to use. during the synthesis, different reaction parameters such as reaction temperature (first step, fig. b ) and curcumin and sodium citrate concentration were considered in order to study their influence on size, shape and concentration of agnps. ag@citrate nps without curcumin, as control samples, were also synthetized. synthesis of cur-p(meo ma) nps or (cur-g). the p(meo ma) crosslinked nanogels in the presence (cur-g) and absence (g , control sample) of curcumin were synthesized through precipitation polymerization strategy in water. a typical procedure for the synthesis is described below for sample cur-g (table ). in a pyrex tube equipped with a magnetic stirrer, μl of a solution of curcumin in ethanol ( . mmol), meo ma monomer ( . mmol), μl of tegdma crosslinker solution in ethanol ( . mmol), μl of sds aqueous solution ( . - mmol) and ml of milli-q water were added. after min n gas purge, polymerization was initiated by heated up to °c, followed by addition of μl of aps solution ( . m). after around min, the solution turned cloudy, indicating that polymerization started, and the solution was left to react for h. to stop the reaction, the solution was cooling down in an ice-cold water bath while the tube was opened to air. afterward, the sample was purified by centrifugation ( rpm, min, °c) and then the pellet redispersed in water. the final product was stored at °c until further use. methods for characterization. ftir spectra were recorded by perkin elmer spectrum two spectrometer using attenuated total reflectance (atr) accessory. uv-vis absorption spectra, at controlled temperature, table . summary of curcumin concentration, ζ-potential, z average diameter and polydispersity (pdi) by dls, volume temperature induced phase transition (vptt), swelling ratio (q), and maximum of curcumin absorbance (λ abs ) of cur-p(meo ma) nanogels. (a) curcumin wt% is the feed curcumin/polymer ratio, (b) ζ values were determined at °c were carried out in a cary bio-varian uv-vis spectrophotometer equipped with a peltier temperature control device. the particle size and zeta potential (ζ) of core-shell nps and agnps were determined by a zetasizer nano zs instrument (malvern instruments ltd, uk) equipped with a mw he-ne laser operating at a light source wavelength of nm and a fixed scattering angle of ° for detection. malvern dispersion software was used for data acquisition and analysis, applying the general purpose algorithm for calculating the size distribution. transmission electron microscopy (tem) images were recorded with a field emission scanning electron microscope (fesem) hitachi su- operated at kv in transmitted electron imaging mode (s-tem). energy-dispersive x-ray spectroscopy (edx) analysis to determine the elemental composition of the hybrid samples was performed using a bruker nano with x-flash detector coupled to the sem. data were recorded at an accelerating voltage of kv. the progress of sample purification by centrifugation was verified by uv-vis spectroscopy (nanodrop one thermo-scientific spectrometer). the centrifugation was performed by refrigerating micro centrifuge r (eppendorf ™ ) to obtain ag@cur-g and cur-g nanoparticle pellets. to determine the luminescence features, uv-vis absorption and fluorescence spectra were recorded on a perkin elmer lambda- and perkin elmer ls ob spectrophotometer, respectively. emission spectra of dilute solutions of ag@cur-g core-doped shell nanohybrids and cur-g nanogels in water and dilute solutions of fluorescein in naoh . m were recorded keeping absorbance at the excitation wavelength (λ exc = nm) less than . . quantum yields were obtained by comparing the studied samples with fluorescein standard by the following equation applicable for dilute solutions. where Φ f and Φ s are the photoluminescence qy of the sample and that of the standard fluorescein (Φ s = . ) , respectively; i e and i e(s) are the integrated intensity of the emission curves corresponding to the sample and standard; η and η (s) are the refractive indices of the sample and reference solution; and i a and i a(s) are the fraction of light absorbed by the sample and standard respectively estimated as: where a is the absorbance at λ exc . synthesis and characterization of ag@cur-p(meo ma) core-doped shell nanohybrids. the ag@cur-p(meo ma) core-doped shell hybrid nps were prepared by free radical precipitation polymerization (frpp) of the stimuli-responsive meo ma monomer in the presence of tegdma (crosslinking agent), using curcumin-decorated ag@cur nps as seeds (fig. ) . the presence of sds guaranteed the control of np size. among all the reagents involved in the process, silver nitrate and sodium citrate followed standard concentration procedures , . however, the dual key-role of curcumin, as reducing agent and growth-polymerization promoter in this specific synthesis, required additional investigation to understand and optimize the chemical variables (solubility, concentration and reaction temperature) in order to achieve homogeneous, monodisperse and mononuclear ag@cur-p(meo ma) core-shell nanohybrids. indeed, frpp is a useful and common strategy to synthesize particles of thermoresponsive polymers, as long as monomers are soluble at the reaction temperature and polymer precipitates when their chains reach a critical size. then, since water solubility of curcumin is rather poor; pure curcumin compounds needed to be first dissolved into an organic phase of ethanol, meo ma monomer and crosslinker (fig. a) . a subsequent injection of aqueous solution of sds surfactant followed by dropwise addition of water resulted in a good dispersion of monomers and curcumin. a color change from cloudy (at room temperature) to transparent yellowish dispersion (at the reaction temperature), indicated that both curcumin and monomers had been dissolved. note that curcumin solubility increases with temperature . when the desired temperature was reached, sodium citrate solution was added to the reaction mixture already containing curcumin; and finally, in the presence of the two reducing agents, silver nitrate was added to the reaction (fig. b) . agnps synthesis (outlined in fig. b ) was completed at two different temperatures, °c and °c (series -a and -b in table , respectively). after min of reaction, the flask was removed from the silicon bath and allowed to cool to room temperature. the as-synthesized silver nanocores had an average size around nm ( °c, series -a) and nm ( °c, series b) in diameter, respectively. to elucidate the role of curcumin as stabilizing agent, the sample obtained in this first step was purified by centrifugation to eliminate the excess of reagents that are not protecting the silver core. the presence of curcumin close to the agnp surface, attained in this first step, was confirmed by atr-ftir spectroscopy (fig. s ). as it can be observed in this figure, the spectrum corresponding to ag@cur (green line) is very similar to that of curcumin (blue line). the most prominent vibration band at ∼ cm − assigned to highly mixed vibration including c=o stretching appears in both spectra as well as other characteristic vibration bands of curcumin at cm − (mixed c=c and c=o stretching), cm − (deformation of ch ), cm − (deformation of phenyl rings), cm − (c-h out-of-plane aromatic motions). in this fig. s , spectra corresponding to sodium citrate and ag@citrate nps are also displayed. as it can be seen, two strong bands corresponding to the asymmetric and symmetric stretching of co − groups at cm − and at cm − , respectively appear with nearly similar intensity for the free sodium citrate spectrum (violet line); whereas for ag@citrate spectrum (orange line), the most prominent band corresponds to the symmetric stretch at cm − . in the spectrum corresponding to ag@cur nps (green line), this vibration band at cm − is www.nature.com/scientificreports www.nature.com/scientificreports/ absent and only the asymmetric stretching (∼ cm − ) can be hardly intuited overlapped with vibration bands from curcumin (blue line); these facts indicate that the affinity of sodium citrate for the silver surface is lower than that of curcumin. in addition, the bands attributed to c-h ( - cm − ) and c=o stretching vibrations of meo ma appear very weak, also suggesting a low interaction of the monomer with the silver surface. figure c outlines the polymer shell preparation. this second step was done without previous ag@cur sample purification. for the purpose of thermoresponsive-shell polymerization, ag@cur was first flushed with n for min at the lowest possible temperature (room temperature), with the aim of reducing water evaporation during nitrogen flow. subsequently, aps initiator was added and incubated for h in a closed flask under stirring at °c. the presence of hydrophobic curcumin nearby the metallic surface led to precipitation-polymerization of p(meo ma) around the agnps previously formed (fig. b,c) ; and the resulting ag@cur-p(meo ma) nanoparticles were born negatively charged due to the persulfate groups from the aps initiator, which promotes their colloidal stability. the successful encapsulation of ag@cur nps within p(meo ma) nanogels can be anticipated by the atr-ftir spectrum shown in fig. s corresponding to f of ag@cur-g b sample (table ) , which displays typical vibration bands attributed to both curcumin and p(meo ma). it should be noted that an attempt to encapsulate agnps synthesized solely using sodium citrate caused irreversible aggregation in the second polymerization step (fig. s ). this fact evidences the role of hydrophobic curcumin to provide both a protection to the agnps and a good interface between the polymer and the metal surface. well-defined ag@cur-p(meo ma) nanohybrids (table ) were obtained by selective removal of the silver-free nanogels by centrifugation (see experimental section). it should be taken into account that the number of empty polymer particles created during the polymerization process exceeded the number of agnps in the solution by about two orders of magnitude; and they should be removed from. as illustration, uv-vis spectra at different purification steps by centrifugation of the ag@cur-g b crude sample are displayed in fig. . initially, the spectrum of the crude shows an intense band at nm resulting from a combined contribution of curcumin absorbance and surface plasmon resonance (spr) due to the collective oscillations of conductions electrons of silver nps. note that spr maximum for spherical agnps can be typically tuned from to nm depending on their np size and shape as well as their surrounding dielectric medium. then, after the first and second centrifugation cycles, the supernatant fractions (f and f respectively) revealed absorption maxima around nm, indicating that the most predominant absorbance is due to curcumin embedded in p(meo ma) nps. the third centrifugation cycle, however, on the basis of the supernatant fraction (f ) and its corresponding precipitate (f ) evidenced only core-doped shell ag@cur-p(meo ma) nanoparticles content (absorption www.nature.com/scientificreports www.nature.com/scientificreports/ maxima at nm). therefore, the core-doped shell nanohybrids used for further characterization and analysis correspond to f fraction from each crude sample (table ) . moreover, curcumin trapped-polymeric nanogels (cur-p(meo ma)) were synthesized through a similar precipitation-polymerization strategy reported for polymer encapsulating of ag@cur (fig. c , ag@ cur-p(meo ma)) and using similar curcumin/polymer ratios (see experimental section and table ). the chemical structure of both, polymeric nps (cur-g) and hybrid nps (ag@cur-g) can be elucidated from the atr-ftir spectra shown in fig. . typical vibration bands (c=o stretching at cm − and c-o stretching at cm − ) corresponding to p(meo ma) are present in both types of nps, in addition to vibration bands at , , and cm − attributed to curcumin molecule. surprisingly, when comparing both cur-g and ag@cur-g nps, for similar curcumin/polymer ratios (tables and ), qualitative differences were observed in their respective spectra evolution (fig. ) . consistently with curcumin data (tables and ) , curcumin related vibration bands seemed to be differently affected by its content. cur-g series exhibited an increase tendency of intensity (fig. a,b) ; whereas ag@cur-g series (fig. c,d) barely varied. this observation could be explained through the key-implication of curcumin in our nuclei-growth approach (fig. ) which determines, at the end, the morphology of ag@cur-g nanohybrids. in fact, the specific silver-nucleation strategy (fig. b) makes feasible that certain amount of curcumin be preferably confined at the metal surface, being subsequently enclosed at higher concentration during p(meo ma) polymer growing shell onto silver core nps (ag@cur-g) than during polymer assembling along silver-free p(meo ma) nanogels formation (cur-g). s-tem micrographs in fig. evidence a core-shell morphology of the ag@cur-g hybrid nps with silver core formed at °c (series ag@cur-g#a, fig. a ) and at °c (series ag@cur-g#b, fig. b) . a mere glance to s-tem pictures revealed almost concentric nanostructuration of polymer shell coverage around metal core for both series of nanohybrids. both series were synthesized with similar curcumin/polymer ratios ( . , . , . and . wt%, table ). the resultant mean values of ag-core diameter ( nm at °c and nm at °c) for both series are collected in table . these differences can be attributed to the faster nucleation rate with increasing temperature that leads to smaller nps. edx spectra in fig. s confirm the presence of ag in two representative hybrid samples with nm (ag@cur-g a) and nm (ag@cur-g b) core diameter, the determined elemental composition is in agreement with ther core size of both nanohybrids. tables and . moreover, it seems that samples synthesized with the two highest curcumin ratios ( . wt%, fig. aiv -biv and . wt%, fig. av -bv) exhibit a more homogeneous spherical shape and a quasi monodistribution, especially, when synthesized at °c (fig. biv-v) . this is in contrast to what happens with the lowest curcumin concentration ( fig. aii and bii) where two silver np populations appeared during the synthesis. these morphological changes were found to affect the spr of the resulting core-doped shell au@cur-g hybrid nps. spr bands of ag@cur-g#a samples synthesized at °c (uv-vis, fig. ai ) are broader and red-shifted compared to those of ag@cur-g#b hybrid nps obtained at °c (uv-vis, fig. bi ). in fact, these wavelengths variations resulted from changes of refractive index of the medium surrounding the metal surface, due to polymer/curcumin reorganization around the two different ag-core sizes. correlative changes in color were also observed when different sizes of metal core were used by keeping all the other reaction parameters constant (table and uv-vis insets of fig. ). larger silver nps exhibited a visual reddish color and a wavelength red-shift. in fact, the λ max for ag@citrate of and nm are and nm respectively , so that red shifts of about - nm collected in table are in accordance to the polymer coating with shell thickness values shown in table . aware of the importance of chemical variables, as curcumin and citrate reducing agents, on the silver-nuclei and their impact on the polymer-growth, an additional experiment with a higher amount of sodium citrate was programmed. at this time, μl of a citrate solution . m was added to the reaction mixture instead of . m, in the presence of . wt% of curcumin, at °c. as expected, it resulted in two highly differentiated ag-core populations ( nm and nm) shown in fig. s . interestingly, agnps (∼ nm) were mostly formed at the limit around the polymeric vesicle boundary but also closely packed inside polymer networking-shell of nm of size (fig. s ). this behavior is attributed to the high nucleation of the nanoparticles due to the high concentration of citrate that causes a greater ion exchange and increases the total ionic strength in the solution, promoting the formation of particle agglomerates in some cases. then, in the regime of low curcumin concentration, phase separation can take place, being the smallest spherical cores produced by sodium citrate and the almost rod-like agnps promoted by curcumin. so, we hypothesize that an appropriate balance among both reducing agents (sodium citrate and curcumin) is needed to obtain more uniform agnp population. thermoresponsive properties. micro/nanogels of crosslinked p(meo ma) in aqueous solution exhibit a reversible volume decrease when raising temperature above the lower critical solution temperature (lcst) of linear p(meo ma). this temperature is known as temperature-induced volume phase transition (vptt) which can be followed by measuring the particle hydrodynamic size by dls. in fig. representative z-average vs temperature curves are shown for the series of ag@cur-g#b nps synthesized at °c ( fig. ai and table ) and of the cur-g# nanogels (fig. aii and table ). as can be observed the vptt values, calculated from the inflection www.nature.com/scientificreports www.nature.com/scientificreports/ point of the curve (fig. ai) , are around °c for the two hybrid nps series (ag@cur-g, table ), showing no dependence on the curcumin content. these results are in good agreement with the atr-ftir spectra (fig. ) which indicated rather similar curcumin content for ag@cur-g hybrids nps. on the contrary, cur-g nanogels (fig. aii ) exhibit slightly decreasing vptt values with increasing the curcumin content (table ). this implication is clearly evidenced at low temperature, where the decrease of the nanogel size is affected by the hydrophobicity feature of curcumin (fig. aii) . interestingly, hybrid nanoparticles exhibit a higher nps size than polymeric nanogels. a plausible explanation of this behavior can lie on the impact of metal core which acts as seed during the precipitation-polymerization; since hybrid nps may need to grow more in order to decrease the polymer-metal interfacial energy and to reach then the stabilization. swelling ratio (q) is an important parameter affecting nanogel properties that can be determined by dls. it can be directly calculated from the ratio between the nanogel volume (v) at swollen state and at the collapsed state : while for polymeric nanogels the volume is directly calculated from the particle radius (r) as v = / π r , for spherical hybrid nps, shell volumes at both temperatures are calculated as v(shell) = / π [r p -r c ]. r p is the hydrodynamic radius of the hybrid particle and r c the hydrodynamic radius of the ag core. table shows the swelling ratios for the silver-polymer (ag@cur-g) hybrid nps and table for the polymeric (cur-g) nanogels investigated. the observed temperature-responsive swelling ratios (q) were close to for the most of the hybrid nps and slightly higher for polymeric nanogels. indeed, for polymeric nanogels ( table ) a decrease of q results from the increase of curcumin in the polymeric nanogels, which could be attributed to a lower swelling ability since the hydrophobic balance increases. on the other hand, the fact that for ag@cur-g hybrid nps, q values show no dependence on the feed composition, could indicate that the final composition of the polymer p(meo ma) shell is rather similar, corroborating again the atr-ftir results (fig. d) . some cyclic heating-cooling experiments were done (data not shown), after observing the reversible collapse-swelling behavior of the samples (fig. ) . at low temperature, the steric protection by the hydrophilic (table ) and (aii) cur-p(meo ma) nanogels (table ) with different curcumin content. (b) ζ-potential determined by dls at °c and °c for colloidal samples of (bi) p(meo ma) nanogels (g ), (bii) agnps functionalized with . wt% curcumin content (ag@cur) before and (biii) after (meo ma) polymerization, ag@cur-p(meo ma) hybrid nps (ag@cur-g b). (c) uv-vis spectra (below and above vptt) of ag@cur-p(meo ma) hybrid nps with ag core synthesized at °c (ci) and °c (cii), and cur-p(meo ma) nanogel (ciii). all samples shown (c) were synthesized with . wt% curcumin/polymer ratio (table and table www.nature.com/scientificreports www.nature.com/scientificreports/ p(meo ma) shell provides good stability in the aqueous medium; whereas at higher temperature, the driving force of colloidal stability is the electrostatic repulsion among the particles revealed by ζ-potential measurements. tables and show ζ-potential values corresponding to ag@cur, ag@cur-g hybrid nps and cur-g nanogels at °c. all types of nps synthesized with curcumin exhibit a fairly high negative ζ-potential that would confer stability against aggregation, especially at temperature above the vptt, where the increase of hydrophobic balance in the polymer shell suddenly produces water expelling from the nanogel. the negative ζ-potential of the synthesized nps can be attributed to several origins. it is well well-known that during polymerization process, incorporation of initiator groups (anionic persulfate) into polymer chains introduces negative charges into micro/nanogel particles synthesized by frpp. additionally, in the specific synthesis of the nanohybrids with silver core explored in this work, the contribution of the remanent sodium citrate cannot be neglected. even more, the negative ζ-potential may arise from curcumin, as it has been reported by some authors , . certainly, in previous works, we found an increase of the ζ-potential at temperature above polymer collapse for both, thermoresponsive polymeric nanogels and hybrid nps with metallic au-core . this effect was attributed to the decrease of the particle size, which increases the charge density at the np surface. nevertheless, in the present investigation, we only observed this temperature-dependent effect on nanoparticles without curcumin (g in fig. bi) . indeed, hybrid nps exhibited a tunable ζ-potential temperature-dependent behavior before (ag@ cur) and after polymerization (ag@cur-g). so, initial ζ-potential diminution values with increasing temperature (fig. bii ) rapidly transited to high and almost invariant ζ-potential values with temperature (fig. biii) as polymerization progressed. these observations (table and fig. b ) so far can be explained through the antagonistic tendencies of the polymer shell and curcumin. the ability of the polymer shell to swell or collapse as function of temperature can tune the optical and luminescent properties of the synthesized nanoparticles. figure c displays representative uv-vis spectra (below and above vptt) for two nanohybrid samples with different core size (ag nm@cur-g a, fig. ci and ag nm@ cur-g b, fig. cii ) and nanogels (cur-g , fig. ciii ) synthesized with . wt% of curcumin content (table ). in uv-vis absorbance spectra, spr band of the nanohybrid with a higher silver diameter (fig. ci) red-shifted while broadening considerably with respect to spr band of the nanohybrid with a smaller silver diameter (fig. cii) . obviously, the spr band position and width was affected by the core-size and the temperature-induced variation of the electromagnetic field in its neighboring environment. the cur-g sample containing silver-free nanogel (fig. ciii) only showed the curcumin effect on nanogel swelling behavior which determines the refractive index of the polymer-gel with the surrounding aqueous medium. in addition, a reversible red-blue shift was observed, when the temperature increase-decrease the vptt of the polymer shell (fig. ) . when the external temperature rises above the vptt, the nanogel shell expels water dictated by the strengthening of hydrophobic interactions giving rise to (i) a decrease of the polymer shell thickness and (ii) an increase of the refractive index. at that point, the effect of refractive index augmentation exceeds to that of the shell thickness diminution; therefore a red-shift should be observed above the vptt for plasmonic-core@thermoresponsive-shell nanohybrids. fig. ci and cii illustrate this behavior for our ag-based thermoresponsive-polymer (core@shell) hybrid nps which correlates well with similar results reported for au-core based thermoresponsive core@shell nanogels . furthermore, the change of the spr maximum with temperature is more pronounced for larger silver core (fig. ci) as previously observed for core-shell nanohybrids with gold core . indeed for the nanohybrids with silver core of nm (series ag@cur-g#a), the Δλlspr max is about - nm whereas for the nm silver core ones (series ag@cur-g#b) the Δλlspr max is about - nm ( table ). the cur-g nanogel sample, without metallic core intervention (fig. ciii) , exhibits no so remarkable λ max absorption change with temperature, at similar curcumin/polymer ratio. www.nature.com/scientificreports www.nature.com/scientificreports/ luminescent properties. curcumin is a tautomeric compound that can occur in diketo and keto-enol forms which ratio strongly varies with the solvent. according to manolova et al. , in solvents as ethanol, only the enol-keto tautomer is present; whereas the addition of water leads to appearance of a new spectral band (about nm), which was attributed to the diketo tautomeric form. the keto-enol form, due to a strong intramolecular hydrogen bond, undergoes transformation into a totally delocalized π-system. due to the hydrophobic character of curcumin, it presents luminescence properties in organic solvents but it is almost non-fluorescent in aqueous solution. however, by complexation through hydrophobic interactions with proteins or with amphiphilic polymers as pluronic , or encapsulating curcumin in hydrophobic polymer nanoparticles , fluorescence enhancement in aqueous medium has been reported. previous to the luminescence investigation of the ag@cur-g hybrid nps, we investigated the absorption and emission of curcumin encapsulated in the thermoresponsive nanogels, cur-g (table and fig. s ). hydrophobic curcumin was engulfed by thermoresponsive nps during the polymerization process, based on the precipitation of the growing polymer at °c due to its unfavorable interaction with the water solvent above lcst (fig. ) . absorbance spectra for curcumin embedded in p(meo ma) nanogels (cur-g) at the polymer concentration of mg ml − show an increase of intensity proportional to the curcumin concentration (fig. s ). in agreement with previous reports of curcumin entrapped in hydrophobic or amphiphilic block copolymers , a main absorption band at - nm in addition to a shoulder about nm appear in fig. s . some authors attributed the signal at nm to the keto-enol tautomer , , whereas the band at nm, associated to the keto form and that typically appears in water, is absent in the present case. fig. s inset shows the linear dependence of the curcumin absorption maxima at nm vs molar concentration of curcumin according to the beer-lambert law for cur-g nanogels. table ). samples were excited at nm. ( fig. a emission spectra in water for the synthesized cur-g nanogels excited at nm evidence remarkable luminescence due to curcumin. in table , the emission maxima (λ em ) and qy, calculated with fluorescein standard, are collected. the emission maxima are about - nm in good agreement with the values reported by banerjee et al. for curcumin encapsulated in polymeric nanoparticles. the position of the emission maxima of curcumin is strongly dependent on the solvent; so by increasing the polarity from chloroform to water, the emission maximum is red-shifted and broadened from nm to nm . both the qy values and the emission maxima position for the present cur-g nanogels seem to indicate that curcumin is well protected by the hydrophobic environment provided by the polymer. with respect to the qy values, two facts have been observed: first, the qy value increases with the decrease of curcumin concentration in the nanogels (fig. a and table ) ; and, second, an amazing fluorescence enhancement is noticed by the temperature increase (fig. b) . the effect of curcumin concentration could be related to the protection that hydrophobic domains of the polymer offer to curcumin, which is enhanced at low curcumin/polymer ratio. the increase of luminescence with increasing temperature should be associated to the water expelled out of the nanogels when temperature rises above vptt. indeed, at lower curcumin ratio (fig. b) , the remarkable increase in absorbance at °c can be attributed to the higher polymer contribution. this reversible change in luminescence with temperature has table ). samples were excited at nm. ( www.nature.com/scientificreports www.nature.com/scientificreports/ been previously reported for fluorescent dyes as bodipy in thermoresponsive p(meo ma) linear polymers and hydrogels . in fig. and fig. s a different behavior can be found for hybrid nps. in first place, analyzing the qy values collected in table , it can be seen that nanohybrids´ fluorescence values are lower than those measured for cur-g nanogels. on the other hand, for both series of ag@cur-g hybrid nps, an increase of luminescence with increasing of curcumin concentration is detected, in opposite to what happened in cur-g nanogels (table and fig. a ). however we know that it can be an ¨apparent increase¨ since the absorption band consideration for estimating the qy values was not properly drawn up. note that this absorption band reflects the additive contribution from the overlapped silver and curcumin spr bands. then, on the basis of these considerations, at lower curcumin concentration, the real curcumin contribution to the final absorbance will be lower. the same miscalculation could arise when comparing qys of hybrid nps with those of polymeric nanogels, because for hybrids an important part of the absorbance arises from the spr of agnps. in last place, the temperature effect also departs from that effect observed for cur-g nanogels, where an increase of fluorescence occurred after polymer collapse on account of hydrophobicity augmentation. in the case of hybrid nps, a slight decrease of fluorescence is detected by moving away from room temperature. this experimental evidence suggests a change on the curcumin-polymer interaction due to the silver presence, or in this specific case, that the interaction of curcumin with silver played a major role, since the specific silver-nucleation strategy causes that certain amount of curcumin be confined at the metal surface. the fluorescence diminution that accompanies the temperature decrease may find its origin in the hydrophilicity-gain experienced by the thermoresponsive polymer shell and the subsequent quenching of curcumin emission induced by water. whereas, when increasing temperature, the polymer increases its hydrophobic balance, which should contribute to protect curcumin against water; nevertheless this positive effect could be overcome by the fact that after polymer collapse, curcumin can get too close to the metal surface and this effect should lead to a further emission quenching. similar observation was reported on the strong curcumin emission-quenching for other silver np-based hybrid systems by other authors , . conclusion in this work, we focused on the knowledge and application of one-pot protocol to obtain core-shell hybrid nanoparticles with the ability to encapsulate hydrophobic, bioactive, antioxidant, and low bioavailability molecules. the aim is to draw on the reductive capacity of some bioactive molecules as curcumin for the in situ formation of metallic nps, meanwhile their hydrophobic character leads the polymer nanostructuration in aqueous medium in the presence of the inorganic metal-core. therefore, new core-doped shell nanohybrids based on silver plasmonic core, thermoresponsive polymer shell and embedded luminescence and bioactive curcumin are easily obtained with potential bioapplications such as antimicrobial systems. furthermore, polymeric nanogels encapsulating curcumin were also achieved, which luminescent properties are strongly enhanced by increasing temperature. this last property could be tuned even with the minimum ratio of bioactive compound. functional microgels and microgel systems temperature-and ph-tunable plasmonic properties and sers efficiency of the silver nanoparticles within the dual stimuli-responsive microgels stimuli-responsive nanogel composites and their application in nanomedicine facile synthesis of gold/polymer nanocomposite particles using polymeric amine-based particles as dual reductants and templates a simple approach to obtain hybrid au-loaded polymeric nanoparticles with a tunable metal load versatile thiolated thermosensitive polymers synthesized by atrp of meo ma and acsema, a new methacrylic monomer with a protected thiol group plasmonic gold-poly(n-isopropylacrylamide) core-shell colloids with homogeneous density profiles: a small angle scattering study encapsulation and growth of gold nanoparticles in thermoresponsive microgels controlled nucleation for regulation of particle-size in monodisperse gold suspensions a study of the nucleation and growth processes in the synthesis of colloidal gold au@pnipam colloids as molecular traps for surface-enhanced, spectroscopic, ultra-sensitive analysis au@pnipam thermosensitive nanostructures: control over shell cross-linking, overall dimensions, and core growth microgels loaded with gold nanorods: photothermally triggered volume transitions under physiological conditions synthesis of au@polymer nanohybrids with transited core-shell morphology from concentric to eccentric emoji-n or optical and swelling stimuli-response of functional hybrid nanogels: feasible route to achieve tunable smart core@shell plasmonic@polymer nanomaterials smart core-shell hybrid nanogels with ag nanoparticle core for cancer cell imaging and gel shell for ph-regulated drug delivery catalytic reduction of -nitrophenol using silver nanoparticles with adjustable activity core-shell hybrid nanogels for integration of optical temperature-sensing, targeted tumor cell imaging, and combined chemo-photothermal treatment silver-decorated polymeric micelles combined with curcumin for enhanced antibacterial activity in situ formation of curcumin stabilized shape-selective ag nanostructures in aqueous solution and their pronounced sers activity green synthesis and catalytic application of curcumin stabilized silver nanoparticles rapid green synthesis and characterization of silver nanoparticles arbitrated by curcumin in an alkaline medium in situ synthesis and surface functionalization of gold nanoparticles with curcumin and their antioxidant properties: an experimental and density functional theory investigation synthesis of au nanorods through prereduction with curcumin: preferential enhancement of au nanorod formation prepared from ctab-capped over citrate-capped au seeds encapsulation of curcumin nanoparticles with mmp -responsive and thermos-sensitive hydrogel improves diabetic wound healing antibacterial adhesive injectable hydrogels with rapid self-healing, extensibility and compressibility as wound dressing for joints skin wound healing multisite inhibitors for enteric coronavirus: antiviral cationic carbon dots based on curcumin nanocurcumin is superior to native curcumin in preventing degenerative changes in experimental cerebral malaria curcumin modified silver nanoparticles for highly efficient inhibition of respiratory syncytial virus infection effect of encapsulation of curcumin in polymeric nanoparticles: how efficient to control esipt process? fluorescence quantum yields and their relation to lifetimes of rhodamine g and fluorescein in nine solvents: improved absolute standards for quantum yields ¶ adsorption and surface-enhanced raman of dyes on silver and gold sols raman response of dithiolated nanoparticle linkers temperature-dependent spectroscopic evidences of curcumin in aqueous medium: a mechanistic study of its solubility and stability dft and experimental studies of the structure and vibrational spectra of curcumin a rapid method to estimate the concentration of citrate capped silver nanoparticles from uv-visible light spectra interfacial tension of turmeric nanoparticles optical properties of responsive hybrid au@polymer nanoparticles the effect of the water on the curcumin tautomerism: a quantitative approach fluorescence study of the curcumin-casein micelle complexation and its application as a drug nanocarrier to cancer cells organic additive, -methylsalicylic acid induces spontaneous structural transformation of aqueous pluronic triblock copolymer solution: a spectroscopic investigation of interaction of curcumin with pluronic micellar and vesicular aggregates synthesis of a self organizable curcumin derivative and investigation of its interaction with metals in % aqueous media bodipy-conjugated thermo-sensitive fluorescent polymers based on -( -methoxyethoxy)ethyl methacrylate the authors gratefully acknowledge the financial support provided by the spanish ministerio de ciencia, innovación y universidades (grant mat - -r and pgc - -b- ). a.s.-q. acknowledges conacyt for the grant epe - . the authors would like to thank david gómez vargas assistance with the sem measurements. key: cord- -if ba pk authors: rodríguez, e. title: a propósito de un caso de neumonía redonda date: - - journal: semergen doi: . /j.semerg. . . sha: doc_id: cord_uid: if ba pk nan mujer de años, fumadora de cigarros/día, antecedentes de diabetes, depresión e hipotiroidismo, consulta por dolor costal izquierdo de características mecánicas de una semana de evolución que no cede con analgesia. a la exploración física presentaba dolor a la palpación a punta de dedo en espacio intercostal . • - . • auscultación pulmonar estertores crepitantes en el lóbulo inferior izquierdo, hipertermia, saturación de oxígeno % (fio ), dificultad respiratoria y astenia. se solicita radiografía de tórax de urgencias con la siguiente imagen ( fig. ). imagen hipodensa circular de unos cm de diámetro, con contorno bien definido, localizada en hemitórax izquierdo, lóbulo inferior con base pleural, aunque por imagen sugiere lesión pulmonar (menos probable origen pleural). la paciente ingresa para estudio con el diagnóstico de neumonía redonda. analítica al ingreso con aumento de reactantes de la fase aguda (pcr: , mg/dl y vsg: mm/h) leucocitos , mmc, neutrófilos: , % y linfocitos: , %. no obstante se solicita tac torácico con contraste para descartar proceso neoplásico ( fig. ). condensación pulmonar basal izquierda, de morfología triangular y base pleural con broncograma aéreo y engrosamiento pleural asociado. se pauta tratamiento antibiótico con amoxicilina/clavulánico, analgesia con metamizol y tramadol, con buena evolución clínica, analítica (antígeno de legionella y streptococcus en orina: negativos, serología de figura imagen hipodensa circular de unos cm de diámetro, con contorno bien definido, localizada en hemitórax izquierdo, lóbulo inferior con base pleural, aunque por imagen sugiere lesión pulmonar (menos probable origen pleural). gérmenes frente a coxiella burnetii, mycoplasma, legionella y chlamydophila pneumoniae negativos, vsg, pcr y serie blanca normal) y radiografía de tórax con resolución de la condensación por lo que se da el alta a los días de su ingreso con el diagnóstico de neumonía redonda basal izquierda. condensación pulmonar basal izquierda, de morfología triangular y base pleural con broncograma aéreo y engrosamiento pleural asociado. la neumonía redonda es un subtipo raro de neumonía lobular debida a un defecto del desarrollo del tejido conectivo (poros de köhn y canales de lambert). en la infancia es bien conocida (afecta principalmente a los niños menores de años), en adultos raramente está descrita. se estima que el % de las neumonías del adulto presenta este comportamiento radiológico. los síntomas y las alteraciones analíticas están relacionados con el tamaño y suelen ser: fiebre, tos, disnea, dolor torácico, leucocitosis, elevación de la proteína c reactiva y velocidad de sedimentación globular, pero con frecuencia estas neumonías pasan inadvertidas en los adultos y aparecen como hallazgo casual en pacientes asintomáticos. radiológicamente , la neumonía redonda puede presentarse como una imagen de hasta cm de diámetro máximo, localizado en los lóbulos inferiores y posteriores, adyacente a la pleura, a veces con broncograma aéreo y de bordes lisos o no bien definidos. los microorganismos más comunes son: s. pneumoniae, k. pneumoniae, h. influenzae y m. tuberculosis. otros trabajos han mostrado que c. psittaci , c. burnetti y el coronavirus son responsables del síndrome de distrés respiratorio agudo severo y pueden tener este comportamiento radiológico. el diagnóstico diferencial , incluye las neoplasias (carcinoma bronquioloalveolar, metástasis o linfomas), otras infecciones (quistes hidatídicos, parásitos o embolias sépticas), causas inmunológicas (sarcoidosis o wegener), metabólicas, vasculares (malformaciones arteriovenosas) y enfermedades profesionales. en la mayoría de los casos la evolución suele ser favorable, ya sea autolimitada o tras instaurar tratamiento antibiótico, y se resuelve por completo. los autores declaran que han seguido los protocolos de su centro de trabajo para la publicación de imágenes de pacientes y han solicitado los permisos correspondientes. clinical manifestations and chest radiographic and ct findings of round pneumonia in adults round pneumonia: imaging findings in a large series of children pulmonary mass in tachypneic, febrile adult a frequent error in etiology of round pneumonia round pneumonia in adults, an inusual presentation of lung parenchymal infection eight cases of severe acute respiratory syndrome presenting as round pneumonia key: cord- - h vvim authors: chen, xiang-fan; li, xiao-li; liu, jin-xin; xu, jing; zhao, yan-yan; yang, min; zhang, yan title: inhibition on angiotensin-converting enzyme exerts beneficial effects on trabecular bone in orchidectomized mice date: - - journal: pharmacol rep doi: . /j.pharep. . . sha: doc_id: cord_uid: h vvim background: this study aimed to study the osteo-preservative effects of captopril, an inhibitor on angiotensin-converting enzyme (ace), on bone mass, micro-architecture and histomorphology as well as the modulation of captopril on skeletal renin-angiotensin system (ras) and regulators for bone metabolism in mice with bilateral orchidectomy. methods: the orchidectomized (orx) mice were orally administered with vehicle or captopril at low dose ( mg/kg) and high dose ( mg/kg) for six weeks. the distal femoral end, the proximal tibial head and the lumbar vertebra (lv) were stained by hematoxylin and eosin, safranin o/fast green and masson-trichrome. micro-computed tomography was performed to measure bone mineral density (bmd). results: treatment with captopril increased trabecular bone area at distal metaphysis of femur, proximal metaphysis of tibia and lv- , moreover, high dose of captopril significantly elevated trabecular bmd of lv- and lv- . the mrna expressions of renin receptor, angiotensinogen, carbonic anhydrase ii, matrix metalloproteinase- , and tumor necrosis factor-alpha were significantly decreased in tibia of orx mice following treatment with captopril. the administration with captopril enhanced the ratio of opg/rankl mrna expression, the mrna expression of transforming growth factor-beta and the protein expression of bradykinin receptor- . conclusions: the inhibition on ace by captopril exerts beneficial effects on trabecular bone of orx mice. the therapeutic efficacy may be attributed to the regulation of captopril on local ras and cytokines in bone. osteoporosis is a disease that thins and weakens bones to the point that they become fragile and easily break. it is well-known that osteoporosis-related fractures due to low-trauma or fragility result in heavy health-related costs, substantial disability and mortality among postmenopausal women and older men [ ] . osteoporosis is now recognized as a major threat to health in aging people. men sustain bone loss of approximately . - % per year from the sixth decade despite not undergoing a menopausal transition as women do [ ] . overall, the trend of age-adjusted prevalence of osteoporosis was similar between women and men [ , ] . however, the mortality and morbidity caused by osteoporotic fractures are greater in men with testosterone deficiency-induced osteoporosis than those in women with postmenopausal osteoporosis [ , ] . bone metabolism is normally modulated by regulators and cytokines in bone micro-environment. the expression ratio of osteoprotegerin (opg) and receptor activator of nuclear factor-kb ligand (rankl), both of which are secreted by osteoblasts, determines the maturation of osteoclasts. matrix metalloproteinase (mmp)- and carbonic anhydrase (ca)ii produced from osteoclasts are responsible for resorbing organic proteins and inorganic minerals, respectively. bradykinin manages bone metabolism through modulating osteogenesis and osteoclastogenesis by binding to its receptor. recently, various cytokines like tumor necrosis factor (tnf)-α and transforming growth factor (tgf)-β are found to play major roles in bone health. the renin-angiotensin system (ras) components exist in tissues and organs, namely tissue ras, participating in kinds of pathophysiologic processes [ , ] . recent animal studies indicated the existence of ras components in trabecular bone [ , ] , and cell studies found the expression of angiotensin receptors in primary osteoblasts [ ] , suggesting there locally exist ras components in bone micro-environment. the previous functional studies indicated that the skeletal ras displays key biological actions on mediating bone metabolism [ ] [ ] [ ] . angiotensin ii, key effector in ras, is produced from angiotensin i by catalyzation of angiotensin-converting enzyme (ace), a vital molecule in ras. the clinical studies found that ace inhibitor (acei) was capable of elevating bone mineral density (bmd) and reducing fracture risk in patients [ , ] . in line with the clinical observations of the bone-preservative properties following ace inhibition, animal studies showed that treatment with acei repressed estrogen deficiency-induced decrease in bone density [ , ] and accelerated bone healing [ , ] . while, in a contrary to the beneficial effects of acei on bone health, the recent emerging evidences indicated that the use of acei might potentially accelerate bone loss [ ] [ ] [ ] . therefore, we are keen to know whether the inhibition on ace could preserve bone tissue of mice with testosterone deficiency-induced osteoporosis. this study investigated the osteo-preservative effects of captopril on bone density, histology and micro-structure of trabecular bone as well as the modulation of captopril on skeletal ras and regulators for bone metabolism in mice with bilateral orchidectomy. thirty-two three-month-old male icr mice (slac laboratory animal, shanghai, china) received commercial diet and distilled water ad libitum during experimental period. the acclimatized mice underwent either bilateral laparotomy (sham, n = ) or bilateral orchidectomy (n = ). after recovery for week, the orchidectomized (orx) mice were randomly divided into three groups: orx mice with vehicle treatment (n = ), orx mice with orally administration of captopril with low dose (orx-l, mg/kg, n = ) or high dose (orx-h, mg/kg, n = ) by intragastric gavage. the dosage for captopril used in this study was referred as described previously [ , ] . six weeks after drug administration, tibias, femurs and lumbar vertebras were immediately collected for a variety of analyses. the animal experiment complied with the arrive guidelines, and the study protocol was approved by the animal ethics committee. the femurs, tibias, and lumbar vertebras were fixed in % formaldehyde in pbs (ph . ), decalcified in edta ( . m, ph . ), and embedded in paraffin. the bone sections with mm were cut on a microtome. safranin o (sigma-aldrich)/fast green staining was performed on femurs and tibias. additionally, both the hematoxylin and eosin staining and the masson-trichrome staining were performed on lumbar vertebra (lv)- . the images for all stained slides were captured under microscope (leica dm- ). trabecular bone area (%) and thickness were measured using an osteomeasure system (osteometrics inc., decatur, ga, usa) as described previously [ ] and the whole process was performed in a blind manner. the lv- and lv- without decalcification were scanned with micro vivact system (scanco medical, bassersdorf, switzerland). the detecting parameters were set as previously described [ ] . trabecular bone was determined by a fixed threshold. after images were captured ( ma), slices were established as the volume of interest. trabecular bmd was obtained. tibias were crushed under liquid nitrogen condition and total rna was isolated by trizol (invitrogen, carlsbad, ca, usa). agarose gel electrophoresis was performed to verify rna integrity. moloney murine leukemia virus reverse transcriptase (invitrogen, usa) was used to synthesize cdnas by reverse transcription reactions with mg of total rna. dna engine (abi) was applied for regular pcr with cdnas as the template. glyceraldehyde- phosphate dehydrogenase (gapdh) was applied as a reference control to measure the amount of pcr products. the primer sequence utilized in this study was referred as previously described [ ] . the femur was homogenized and extracted in laemmli buffer (boston bioproducts, worcester, ma, usa), followed by min boiling and centrifugation to obtain the supernatant. protein concentration was determined by bio-rad protein assay kit. mg protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes, which was blocked with % (w/v) nonfat dry milk in tbst containing . % (w/v) tween , and incubated with one of the following primary antibodies (santa cruz biotechnology, usa) at c overnight: mouse anti-renin monoclonal antibody, goat anti-bradykinin receptor polyclonal antibody. after washes with tbst, the membranes were incubated with secondary antibody with dilution of : , . the protein bands were captured by odyssey infrared imaging system (li-cor, usa), quantified (odyssey application software version . ) and normalized to β-actin signal using mouse monoclonal anti-β-actin antibody (sigma, usa). the results were expressed as mean ae standard error of mean (sem). the analysis for statistical difference was carried out with prism version . (graphpad). inter-group differences were analyzed by one-way anova, and followed by tukey's multiple comparison tests which were applied to compare the values between groups and the difference with p value of less than . was considered statistically significant. the affection of captopril on trabecular bone was evaluated by safranin o/fast green staining at the distal end of femur (fig. a ) and the proximal metaphysis of tibia (fig. b) . the loss of bone mass and the deterioration of network connection were clearly shown at spongiosa zones at both femur (fig. c , p < . ) and tibia (p < . ) in orchidectomized (orx) group as compared to those in sham group. treatment with low dose of captopril (orx-l) increased the trabecular bone area at proximal tibial metaphysis (p < . ), and treatment with high dose of captopril (orx-h) significantly elevated (p < . ) the area of trabecular bone at both femur and tibia in orx mice. histological staining on trabecular bone at lumbar vertebra great amount of trabecular bone exists at spine bone, thus, hematoxylin and eosin (he) staining ( fig. a) and massontrichrome staining (fig. b) were performed on lumbar vertebra (lv)- . similarly as observed at femur and tibia, the orchidectomy induced the loss of bone mass and the impairment of cancellous bone at lv- as demonstrated by the decrease of trabecular bone thickness (fig. c, p < . ) and trabecular bone area (fig. d , p < . ). captopril with low dose (p < . ) and high dose (p < . ) dramatically increased the trabecular bone area of lv- in orx mice. captopril dose-dependently enhanced the thickness of trabecular bone even though there was no statistical difference between captopril-treated groups and orx group. the measurement by micro-computed tomography (fig. ) showed that the orchidectomy alone induced significant decrease in bmd at both lv- (p < . ) and lv- (p < . ). the treatment with high dose of captopril dramatically (p < . ) increased bmd at both lv- and lv- , which was well consistent with the histological result at lumbar vertebra. to clarify that tissue ras was involved in the action of captopril on bone, the mrna expressions of ras components in tibia, such as angiotensinogen (agt) and renin receptor (renin-r), were determined (fig. ) . the expressions of renin-r (fig. b , p < . ) and agt (fig. b , p < . ) were significantly up-regulated in tibia of vehicle-treated orx mice. gene expression of renin-r was down-regulated by captopril with both doses (p < . ) and mrna expression of agt was markedly reduced only in orx-h group (p < . ) when compared to those in orx group. the expression ratio of osteoprotegerin (opg) and receptor activator of nf-kb ligand (rankl) plays vital role in formation and maturation of osteoclasts, thus, the expression of opg and rankl and the ratio of opg/rankl were determined (fig. ) . the mrna expression of opg was lower in orx group than that in sham group (fig. c, p < . ) , and captopril significantly increased opg mrna expression as compared to that of orx group (p < . ). the statistical difference for rankl mrna expression was not found among groups. while, the expression ratio of opg/rankl was decreased in orx group (p < . ) and this ratio in orx mice after treatment with low and high dose of captopril was recovered to the level in sham group (p < . ). the mrna expressions of markers for bone resorption were measured in tibia for further evaluating the potential effect of captopril on bone resorptive activity (fig. ) . the orchidectomy significantly increased (fig. d , p < . ) the mrna expression of caii. the captopril treatments with low dose and high dose dramatically reversed this change (p < . ). the captopril treatment at high dose significantly down-regulated the mrna expression of mmp- as compared to that in vehicle-treated orx group (p < . ). thus, this study indicated the potential regulation of captopril on osteoclastic activity. in addition, the mrna expressions of two cytokines, tnf-α and transforming tgf-β, were determined in tibia (fig. ) . the mice after orchidectomy showed the increased expression of tnf-α (fig. e , p < . ) and the decreased expression of tgf-β (p < . ). treatment with captopril reduced the tnf-α expression by % (p < . ) and enhanced the tgf-β expression by % (p < . ) as comparison with those in orx group. orchidectomy significantly induced the down-regulation of bradykinin receptor- (b r) protein (fig. b, p < . ) , but did not alter the protein expression of renin in femur. treatment with low dose and high dose of captopril in orx mice markedly increased the expression of b r (p < . ). of noted, the protein expression of renin was higher (p < . ) in orx-h group than that in vehicletreated orx group. angiotensin-converting enzyme inhibitors (aceis) are classically applied for anti-hypertension treatment [ ] , and are currently in wide use for the treatment of diabetic complications [ , ] . given the key role of tissue renin-angiotensin system (ras) in maintaining bone health, it is vital to study the effect of aceis on bone injury induced by testosterone deficiency. in the present study, we investigated the therapeutic effect of captopril, one typical acei, on trabecular bone histology and bone mass at long bone and spine bone as well as studied the modulation of captopril on regulators for bone metabolism in orchidectomized (orx) mice. this study demonstrated that the treatment with captopril effectively attenuated the orchidectomy-induced pathological alterations of micro-structure of trabecular bone at lumbar vertebra (lv)- , distal metaphysis of femur and proximal metaphysis of tibia as observed by histological staining, moreover, bone mineral density (bmd) at both lv- and lv- was significantly enhanced in orx mice in response to captopril treatment for weeks. these results revealed the preventive effects of inhibiting angiotensin-converting enzyme (ace) on decrease of bone mineral density and impairment of trabecular bone structure at axial and appendicular bones. the beneficial effect of captopril on bone of orx mice shown in this study was well consistent with previous animal studies, which demonstrated that the intervention of tsukuba hypertensive mice with acei enalapril attenuated osteoporosis [ ] as well as the ovariectomized (ovx) rats treated with captopril showed the increased area of trabecular bone at lv- and the improved mechanical properties at lv- [ ] . additionally, the patients after treatment with aceis had an increase in bmd and a reduced risk in fracture [ ] . thus, the preclinical and clinical studies fully suggested the potential of acei in exerting osteoprotective efficacy. of noted, the recent studies demonstrated that captopril repaired cortical morphometric features [ ] and improved cortical bone thickness [ ] in ovx rats, while, the potential effect of captopril on cortical bone in orx animals, such as the middle-shaft of tibia and femur, require further investigation. it is evident that the activation of skeletal ras would result in bone injuries [ , ] . this study revealed that orchidectomy alone increased the mrna expressions of renin receptor (renin-r) and angiotensinogen (agt) in bone, and the similar experimental results were found in tibia of rats [ ] and mice [ ] with diabetes. upon to the treatment with captopril, the abnormal expression of agt and renin-r in orx mice was almost reduced to the level of sham group. thus, this study indicated the biological role of tissue ras in development of testosterone deficiency-induced osteoporosis and suggested that the skeletal ras may a candidate target in drug discovery for anti-osteoporosis of elderly males. our results clearly showed that the expressions of genes involved in bone resorption (caii, mmp- ) were significantly induced and the ratio of opg/rankl expression (a marker for osteoclastogenesis) was decreased in tibia of orx mice, a typical feature of bone turnover associated with testosterone deficiency. treatment of orx mice with captopril significantly suppressed these changes of caii, mmp- and opg/rankl. similar results were reported by others that captopril increased the ratio of opg/rankl in bone [ ] and serum [ ] of ovx rats. both mmp- and caii, produced from osteoclasts, are enzymes to dissolve organic component and inorganic substance of bone, respectively [ ] . thus, our results clearly indicated that captopril exerted the suppression on bone resorption by which captopril protected against bone deteriorations induced by testosterone deficiency in male mice. besides the local ras in bone tissue, bradykinin, the major active peptide in the kallikrein-kinin system (kks), is able to regulate bone metabolism through acting on bradykinin receptor- (b r) [ ] . the expression of bradykinin receptor was decreased in osteoblasts with exposure to high glucose level and in bone of ovx mice [ , ] . this study demonstrated that captopril effectively reversed orchidectomy-induced down-regulation of b r protein expression in mice, indicating that bradykinin receptor was involved in management of captopril on bone metabolism in orx mice. tnf-α, one of inflammatory markers, could activate osteoclasts, consequently stimulating bone resorption and resulting in osteoporosis. in vivo studies showed that the administration of animals with ras inhibitors could reduce tnf-α expression in tissue [ , ] , similarly, in this study captopril dramatically decreased mrna expression of tnf-α in bone of orx mice. in addition, tgf-β is an important modulator for bone formation and repair. the present study showed the down-regulation of tgf-β in bone of orx mice, which was in consistent with early study [ ] . the increase in tgf-β expression in response to captopril treatment was found in orx mice in this study, and the similar action of captopril on tgf-β was also reported in human lung fibroblasts [ ] . our study indicated that the regulation of captopril on tnf-α and tgf-β might be involved in its protective effects on bone tissue of orx mice. although treatment with captopril attenuated bone injury in orx mice, we found that captopril at high dose in this study induced the elevation of renin protein expression in bone. the compensatory increase in renin expression is a common problem for aceis due to the disruption of the feedback inhibitory loop in renin production [ ] . the increase in ras activity might ultimately limit the therapeutic effectiveness of ace inhibition. thus, the clinical practice currently suggests the application of ras blockers with aliskiren, one renin inhibitor which could suppress the first and rate-limiting step within ras [ ] . while, whether the combination of acei with aliskiren could synergistically ameliorate bone impairments induced by testosterone deficiency need to be further clarified. taken together, this study clearly showed the beneficial effect of captopril on trabecular bone in orchidectomized mice with testosterone deficiency, and the underlying mechanism may, at least partially, be attributed to the regulation of captopril on skeletal ras and local cytokines. pyrroloquinoline quinone prevents testosterone deficiency-induced osteoporosis by stimulating osteoblastic bone formation and inhibiting osteoclastic bone resorption effects of body size and skeletal site on the estimated prevalence of osteoporosis in women and men prevalence of osteoporosis and low bone mass in older chinese population based on bone mineral density at multiple skeletal sites prevalence of osteoporosis and related lifestyle and metabolic factors of postmenopausal women and elderly men: a cross-sectional study in gansu province male osteoporosis: new trends in diagnosis and therapy pathophysiology of age-related bone loss and osteoporosis the effect of renin angiotensin system on tamoxifen resistance role of angiotensin-converting enzyme in cardiac hypertrophy induced by nitric oxide synthase inhibition angiotensin ii type receptor blockade increases bone mass role of the local bone renin angiotensin system in steroid induced osteonecrosis in rabbits activation of renin-angiotensin system induces osteoporosis independently of hypertension involvement of the skeletal renin-angiotensin system in age-related osteoporosis of ageing mice early molecular responses of bone to obstructive nephropathy induced by unilateral ureteral obstruction in mice effects of angiotensinconverting enzyme inhibitor, captopril, on bone of mice with streptozotocininduced type diabetes evolution of the bone mass of hypertense menopausal women in treatment with fosinopril angiotensin converting enzyme inhibitor use is associated with higher bone mineral density in elderly chinese prevention of osteoporosis by angiotensin-converting enzyme inhibitor in spontaneous hypertensive rats ace- / ang - /mas cascade mediates ace inhibitor, captopril, protective effects in estrogen-deficient osteoporotic rats inhibition of angiotensin-converting enzyme stimulates fracture healing and periosteal callus formation -role of a local renin-angiotensin system the effects of the angiotensin converting enzyme inhibitor enalapril and the angiotensin ii type receptor blocker losartan on fracture healing in rats does the use of ace inhibitors or angiotensin receptor blockers affect bone loss in older men the effect of angiotensin-converting enzyme inhibitor use on bone loss in elderly chinese differential response of bone and kidney to acei in db/db mice: a potential effect of captopril on accelerating bone loss effect of ace inhibitors and at receptor antagonists on pentylenetetrazole-induced convulsions in mice the angiotensin converting enzyme inhibitor lisinopril improves muscle histopathology but not contractile function in a mouse model of duchenne muscular dystrophy involvement of skeletal renin-angiotensin system and kallikrein-kinin system in bone deteriorations of type diabetic mice with estrogen deficiency renin inhibitor aliskiren exerts beneficial effect on trabecular bone by regulating skeletal reninangiotensin system and kallikrein-kinin system in ovariectomized mice ace phenotyping as a guide toward personalized therapy with ace inhibitors effect of angiotensinconverting enzyme inhibitors and angiotensin ii receptor blockers on all-cause mortality, cardiovascular deaths, and cardiovascular events in patients with diabetes mellitus: a meta-analysis targeting inflammation: new therapeutic approaches in chronic kidney disease (ckd) captopril improves osteopenia in ovariectomized rats and promotes bone formation in osteoblasts antihypertensive medications, bone mineral density, and fractures: a review of old cardiac drugs that provides new insights into osteoporosis a comparative study between the effect of -β estradiol and angiotensin converting enzyme inhibitor on osteoporosis in ovariectomized rats local bone interaction between renin-angiotensin system and kallikrein-kinin system in diabetic rat bradykinin regulates osteoblast differentiation by akt/erk/nfkb signaling axis bradykinin receptors and ephb /ephrinb pathway in response to high glucose-induced osteoblast dysfunction and hyperglycemia-induced bone deterioration in mice inhibition of aneurysm progression by direct renin inhibition in a rabbit model effect of angiotensin-converting enzyme inhibitor on cardiac fibrosis and oxidative stress status in lipopolysaccharide-induced inflammation model in rats orchiectomy markedly reduces the concentration of the three isoforms of transforming growth factor beta in rat bone, and reduction is prevented by testosterone profibrosing effect of angiotensin converting enzyme inhibitors in human lung fibroblasts rationale for combining a direct renin inhibitor with other reninangiotensin system blockers. focus on aliskiren and combinations clinical role of direct renin inhibition in hypertension this work was supported in part by jiangsu specially-appointed professor program and university of shanghai for science and technology ( kjfz ). the authors declare no conflict of interest. key: cord- -n ncamqh authors: donaldson, braeden; lateef, zabeen; walker, greg f.; young, sarah l.; ward, vernon k. title: virus-like particle vaccines: immunology and formulation for clinical translation date: - - journal: expert rev vaccines doi: . / . . sha: doc_id: cord_uid: n ncamqh introduction: virus-like particle (vlp) vaccines face significant challenges in their translation from laboratory models, to routine clinical administration. while some vlp vaccines thrive and are readily adopted into the vaccination schedule, others are restrained by regulatory obstacles, proprietary limitations, or finding their niche amongst the crowded vaccine market. often the necessity to supplant an existing vaccination regimen possesses an immediate obstacle for the development of a vlp vaccine, despite any preclinical advantages identified over the competition. novelty, adaptability and formulation compatibility may prove invaluable in helping place vlp vaccines at the forefront of vaccination technology. areas covered: the purpose of this review is to outline the diversity of vlp vaccines, vlp-specific immune responses, and to explore how modern formulation and delivery techniques can enhance the clinical relevance and overall success of vlp vaccines. expert commentary: the role of formation science, with an emphasis on the diversity of immune responses induced by vlp, is underrepresented amongst clinical trials for vlp vaccines. harnessing such diversity, particularly through the use of combinations of select excipients and adjuvants, will be paramount in the development of vlp vaccines. virus-like particles (vlp) are a type of subunit vaccine based on virus-derived proteins, assembled to form a particle. vlp hold several advantages over other particulate subunit vaccines. these include a morphological resemblance to their parent virus, a highly repetitive immunogenic surface structure, and the retention of cell uptake and immune processing pathways associated with their parent virus [ ] . vlp themselves are nonpathogenic, devoid of an intact virus genome, and are incapable of infection or replication. the noninfectious nature of vlp significantly improves their safety profile over live-attenuated vaccines, while also possessing advantages when compared to other forms of subunit, killed, or particulate vaccines. commercially successful vlp vaccines include hepatitis b virus (hbv) vlp, such as recombivax hb (merck), engerix-b (glaxosmithkline), elovac b (human biologicals institute), genevac b (serum institute) and shanvac b (shantha biotechnics), hepatitis e virus (hev) vlp, such as hecolin (innovax), and human papillomavirus (hpv) vlp, including gardasil (merck) and cervarix (glaxosmithkline). vlp vaccines currently under investigation in clinical trials include influenza a virus (iav) vlp (nct , nct ) [ , ] , chikungunya virus vlp (nct ) [ ] , human cytomegalovirus (hcmv) vlp (nct ) [ ] , and human norovirus (hunv) vlp (nct , nct , nct ) [ ] [ ] [ ] . recent and current vlp clinical trials registered in the united states of america and the european union are summarized in table . vlp can be bulk expressed in bioreactor cultures, utilizing advanced in vitro protein expression systems for producing vaccine-grade vlp suitable for clinical administration. the manufacture of some vlp also includes additional processing steps, such as disassembly and reassembly of vlp. the manufacture of the hpv vlp utilizes disassembly and reassembly to improve vlp morphology and stability [ ] . similarly, the manufacture of qβ vlp includes this method [ ] . different vlp expression systems and the applications of vlp disassembly and reassembly was explored in a recent review [ ] . compatibility with commercial upscaling technology, gmp production, and with minimal postproduction manipulation or modification supports large-scale use of vlp vaccines; however, some vlp vaccines struggle in their translation from laboratory research and development, to clinical trials and routine public access [ ] [ ] [ ] . open accessibility to the molecular or genetic components of derivative constituents may place some limitations on the commercialization of specific vlp vaccines. this may be circumvented through tactical use of proprietary modification, or by utilizing the vaccine as a constituent within a composite formulation [ ] . the development of some vlp vaccines can be challenged by issues with stability and longevity, which may be alleviated with formulation excipients, or other vaccine additives that facilitate vaccine distribution and storage [ , ] . the purpose of this review is to explore some of the challenges in the translation [ ] , and negative-sense rna viruses such as human parainfluenza virus type [ ] . variety can be observed in vlp size, ranging from ms bacteriophage vlp at around . nm in diameter [ ] , to hpv vlp at around nm [ ] , and influenza vlp at around nm [ , ] . vlp also vary in structural complexity, as illustrated in figure , including mono-layer vlp such as hbv vlp formed from hbv surface antigen (hbsag) or hbv core antigen (hbcag) [ ] [ ] [ ] , and multi-layer vlp such as rotavirus vlp [ , ] . vlp can be encapsulated within a phospholipid bilayer envelope to resemble their parent virus, such as hiv [ ] or sendai virus vlp [ ] . the envelope itself can also form the primary particle structure of some vlp, with recombinantly expressed virus envelope-stabilizing proteins embedded within the membrane, such as with iav virosomes [ ] . some vlp are compatible with the formation of polyvalent or mosaic vlp, derived from multiple virus strains [ ] . while this increases the diversity of vlp vaccines beyond the variety of parent viruses, the increased complexity of polyvalent or mosaic vlp may require post-production manipulation to facilitate stable particle formation [ , ] . in addition to facilitating the development of complex vlp vaccine constructs, introduction of postproduction manipulation has also been demonstrated to improve the consistency and stability of some standard structure vlp vaccines [ , ] . the diversity of vlp can also be characterized based on the variety of diseases these vaccines have been developed to prevent or treat. these include vlp vaccines developed for both human and veterinary pathologies, with some examples including an hbv hbcag core particle-based vaccine for her + cancer [ ] , an adenovirus vlp-based vaccine for placental malaria [ ] , and a qβ vlp-based vaccine for leishmania infection [ ] . a selection of recently developed and investigated novel vlp vaccines are provided in table . vlp can be produced using a variety of expression systems, usually involving vlp assembly by spontaneous polymerization upon the expression of virus protein constituents. vlp can be expressed in cells derived from bacteria, yeast, insects and mammals, in cell-free expression systems, and in live organisms such as silkworm pupae and various plants [ ] [ ] [ ] . selection of an appropriate expression system is important, as each expression system can differ in their efficacy for expressing specific vlp, their post-translational modification (e.g. phosphorylation, glycosylation), and their potential for contamination with biologically compatible zoonotic viruses. protein expression for the production of vlp is highly amenable to modification, such as substituting strain-specific amino acid sequences [ ] , or inserting foreign peptide sequences or proteins [ , ] . manipulation of this process can facilitate the engineering of recombinant chimeric vlp containing xenogeneic antigens, inducing an immune response against targets other than the parent virus [ ] . vlp can also present self-antigens in an immunogenic context, which can be particularly useful in immunotherapeutic vaccination for cancer [ ] . a diverse range of applications for this type of modification have been investigated prophylactically and therapeutically for various conditions, including protection against infection from other organisms [ ] , autoimmune inflammatory disease [ ] , and as an antitumor immunotherapy [ , , ] . chimeric vlp have been investigated as a potential therapy for conditions not usually associated with vaccination, such as atherosclerosis [ ] , type ii diabetes mellitus [ ] , and nicotine addiction [ ] . some of these vlp vaccines utilize chemical conjugation for incorporation of antigenic sequences as opposed to recombinant insertion. vlp can serve as an effective immunogenic scaffold for chemical conjugation, compatible with a broad range of conjugation chemistries. the applications of chemical conjugation with vlp has been comprehensively reviewed in recent articles [ , [ ] [ ] [ ] ; however, it is worth noting that introducing unique forms of chemical conjugation may provide some proprietary claim over methods involving vlp vaccines already in the public domain. vlp are a form of subunit vaccine, inducing a characteristic immune response shared amongst exogenous antigens. while vlp are taken up by cells through non-specific pathways such as phagocytosis and macropinocytosis, some vlp can also utilize specialized uptake pathways inherited from their parent virus. in general, protein-based subunit vaccines like vlp are internalized into antigen-presenting cells (apcs), digested within the phagolysosome, and the resulting antigen peptides are presented loaded on major histocompatibility complex (mhc) ii molecules to cd + t helper cells (t h cells). t h cells recognize epitopes presented on mhc-ii through their specific t cell receptor (tcr), and activate dependent on the strength of this interaction, in addition to the degree of signaling through costimulatory receptors and cytokines. these additional signals are usually supplied by the apc, which is activated to increase the presentation of costimulatory receptors and the release of cytokines in response to a variety of stimulatory signals. these stimulatory signals correspond with the activation of pattern recognition receptors (prrs), such as tolllike receptors (tlrs), nod-like receptors (nlrs), and rig -like receptors (rlrs), which each recognize specific pathogenassociated molecular patterns (pamps) and damage-associated molecular patterns (damps). as vlp vaccines usually contain minimal or negligible amounts of these stimulatory molecules, these signals are instead induced by vaccine adjuvants. manipulation and selective targeting of specific apcs or other target cell populations may be an effective means of developing a novel proprietary vlp vaccine. chemical conjugation of receptor ligands onto the surface of compatible vlp should promote binding and uptake into cells expressing the corresponding receptor, while primary vlp uptake and processing pathways should continue unperturbed. this type of modification may be particular effective when targeting a specific apc population is desired for the induction of a specific type of immune response. for example, chemical conjugation of mannoside-based saccharides on the surface of rabbit hemorrhagic disease virus (rhdv) vlp selectively targets the mannose receptor expressed on the surface of apcs, inducing increased uptake and alteration of antigen cross-presentation in murine dendritic cells [ ] . chemical conjugation of unmethylated cpg oligonucleotides on the surface of rhdv vlp has also been investigated for targeting uptake through the dec receptor [ ] . similarly, coating of hiv vlp with phosphatidylserine-laced liposomes enhanced uptake into macrophages by mimicking apoptotic bodies [ ] . although the immune response to vlp may be generalized due to similarities shared with other subunit vaccines, there are some unique components of a vlp-induced immune response. the pathways for uptake of vlp into apcs vary depending on the size and morphology of each vlp, including the potential retention of receptor binding motifs. particles smaller than nm, such as norovirus p particles [ ] , can drain directly from the vaccination site into the lymph through pores in the fenestrated lymphatic vessels [ ] . trafficking of molecules within the lymphatics is likewise size dependent, with soluble proteins smaller than kda or nm transported by specialized small antigen conduits [ , ] . vlp are larger than nm, and many are instead transported passively to the lymph nodes bound on the surface of myeloid immune cells [ ] [ ] [ ] . vlp larger than nm cannot drain directly through the fenestration pores into the lymph, and instead require active transport following internalization by apcs at the vaccination site, such as in dendritic cells (dcs), macrophages and b cells [ ] . uptake of virus antigens into antigen presenting cells can occur through multiple pathways, including phagocytosis, macropinocytosis, caveolae-mediated uptake, clathrinmediated uptake and clathrin noncaveolae-mediated uptake [ ] . vlp have been identified to utilize a similar repertoire of uptake pathways, including phagocytosis [ ] , size-dependent macropinocytosis [ ] , and clathrin-dependent or independent forms of receptor-mediated endocytosis [ ] [ ] [ ] . the retention of receptor-mediated endocytosis indicates that some vlp can inherit functional receptor binding domains, and are compatible with the corresponding uptake pathways derived from their parent virus. these mechanisms of receptor-mediated uptake can also be introduced to vlp through post-expression modification, such as the chemical conjugation of superantigen on hbv vlp for internalization through mhc-ii molecules [ ] , or the chemical conjugation of transferrin on qβ vlp for internalization through the transferrin receptor, which is upregulated in some cancer cells [ ] . non-specific uptake into apcs subjects vlp to standard components of exogenous antigen processing pathways, including lysosomal fusion, enzymatic digestion, mhc-ii loading and antigen presentation (figure (a) ). parent viruses of vlp internalized through these pathways engage additional mechanisms to progress toward virus replication, including capsid disassembly or uncoating, exportation of the virus genomic material, and disruption of vesicular fusion. while the retention of intracellular processing mechanisms resembling those utilized by parent viruses has been investigated for some vlp [ , ] , it remains unclear whether vlp universally mimic the intracellular processing of their parent virus. vlp can retain access to cross-presentation pathways, facilitating presentation of antigens on mhc class i (mhc-i) for induction of a cd + t cell-mediated cytotoxic immune response. there are several known mechanisms of cross-presentation in apcs, including antigen escape from the early endosome, fusion of the endosome with the endoplastic vlp can be internalized and processed through a variety of intracellular pathways. (a) vlp internalized through non-specific pathways such as phagocytosis and macropinocytosis can be processed, with peptides presented on mhc-ii as exogenous antigen. (b) some vlp can also utilize cross-presentation pathways, facilitating the presentation of peptides on mhc-i [ ] . (c) influenza vlp, in this example an influenza virosome, is internalized through receptor-mediated endocytosis prior to fusion of its envelope with the endosomal membrane [ , , ] . (d) jcv vlp is thought to utilize the processing pathway of its parent virus to facilitate delivery of exogenous nucleic acids, including clathrin-dependent endocytosis, nuclear trafficking, and uncoating of the capsid within the nucleus [ , ] . reticulum, and recycling of mhc-i receptors from the cell surface. rhdv vlp are known to utilize the mhc-i receptor recycling pathway of cross-presentation [ ] (figure (b) ). additional examples of vlp known to induce cross-presentation of antigens include vlp derived from hbv [ , ] , hepatitis c virus (hcv) [ ] , hpv [ ] , papaya mosaic virus [ ] , and parvovirus [ ] . the ability to induce cross-presentation is important when a cellmediated cytotoxic immune response is the primary desired outcome of a vlp vaccine. the retention of receptor-mediated internalization in vlp illustrates that these mechanisms of uptake in their parent virus are autonomous, independent of the integrity of the complete virion. vlp derived from influenza a or b viruses are prime examples of this process. influenza vlp can be produced in several forms, including enveloped vlp formed by expressing the influenza matrix (m ) protein [ ] , retroviral gag proteins [ ] , or as independent virosome particles without a traditional protein core [ , ] . each of these constructs includes the expression of influenza hemagglutinin (ha) with or without neuraminidase (na). these influenza proteins are crucial for the induction of an influenza-specific immune response, and they also play essential roles the budding of influenza virus-like particles [ , , ] , and in receptor-mediated endocytosis. influenza vlp can bind to sialylated glycoproteins and glycolipids on the surface of cells from the inherent activity of ha, and are internalized through clathrin-mediated endocytosis [ , ] (figure (c)); however, alternative endocytic pathways have also been identified [ ] [ ] [ ] [ ] . as the early endosomal ph lowers, the envelope fuses with the endosome membrane. ha and na proteins distributed along the internal surface of the endosome membrane are enzymatically degraded, with peptides presented as exogenous antigens on mhc-ii (figure (c) ). specialized vlp vaccines designed to deliver a genetic payload add further complications to vlp uptake and processing. retention of the ability to deliver dna/rna requires conservation of intracellular processing pathways utilized by the parent virus for replication, or the induction of an alternative method of nucleic acid translocation. john cunningham virus (jcv) vlp is a polyomavirus vlp that has received significant attention over its ability to facilitate gene delivery and genetic manipulation [ , , ] . jcv is internalized by clathrin-dependent endocytosis upon binding to an undefined plethora of receptors and molecules known to include the serotonin receptor and sialic acid [ , ] . the virus then colocalizes to endosomes along with transferin, prior to cytosolic translocation, and subsequent trafficking through nuclear pores, mediated by the n-terminus of the vp capsid protein [ ] . the virus then uncoats, exposing the genomic material within the capsid. jcv vlp are thought to mimic this process due to their ability to deliver dna plasmids or other nucleic acids to the nucleus [ , ] (figure (d) ). vlp that demonstrate this ability to deliver nucleic acids likely utilize similar processing pathways to their parent virus [ , ] , but vlp can also be modified to utilize alternative processing pathways [ , ] . the primary desired outcome of most commercial vlp vaccines is the production of antibodies specific for the vlp parent virus. anti-vlp antibodies produced by these vaccines correspondingly neutralize the parent virus, protecting against infection. the production of anti-vlp antibodies involves the activation of a humoral immune response. b cells that specifically recognize vlp surface domains through their b cell receptor (bcr) bind and internalize vlp. binding of vlp to the bcr provides a stimulatory signal that primes the b cell for activation. most vlp consist of structurally identical capsid proteins arranged in a repetitive quasicrystalline pattern, which can crosslink multiple bcrs to provide a stimulatory advantage over other types of subunit vaccines. crosslinking of bcrs is important for inducing a robust humoral response, as b cells can ignore some monomeric soluble antigens [ ] . potent stimulation through bcr crosslinking can override inherent tolerogenic mechanisms, and can activate unresponsive or anergic b cells [ ] . highly repetitive structures also promote binding to low-affinity bcrs through multivalent, or highavidity interactions [ ] . antigen primed b cells receive additional activation signaling induced by pamps delivered with the vlp vaccine. these signals are usually provided by vaccine adjuvants, but may also be induced by other virus-associated or expressionderived endogenous molecules associated with the vlp. vlpderived antigens are presented to t h cells loaded on mhc-ii, inducing the cytokine and costimulatory receptor signaling from the t h cell that continues b cell activation. activated b cells initially become plasmablasts, a transient extrafollicular activation state that can provide some immediate antibody production [ ] . plasmablasts migrate to the follicular region of lymph nodes to form germinal centers, a specialized region where activated b cells proliferate. plasmablasts within germinal centers under affinity maturation and immunoglobulin class switching, guided by signaling from t follicular helper (t fh ) cells [ , ] . this specialized t fh cell guidance results in the development of high-affinity antibody-producing plasma cells, and long-lived memory b cells. rapid t cellindependent activation can also be induced in marginal zone b cells and b cells, providing an alternative pathway for activation and antibody production over the predominant follicular b cell population [ ] . some vlp have been identified utilizing this alternative form of b cell activation [ ] . the production of anti-vlp antibodies has been investigated and characterized amongst a broad range of vlp. this process can be influenced by various vaccine components, constituents, and even unexpected molecules, such as those derived from the vlp expression system. for example, the presence of bacterial rna packed inside qβ vlp plays a role in the induction of igg a/c antibodies in mice, and igg antibodies in humans [ , ] . qβ vlp can also induce the production of iga class antibodies in immunocompetent mice when immunized through the intranasal route [ ] , while t h cell-deficient mice required subcutaneous rather than intranasal vaccination to induce robust iga production through t-cell independent b cell activation [ ] . tlr- , simulated by singlestranded rna in endosomes, was also identified as crucial for the induction of both igg c and iga antibodies against qβ vlp in mice. this further indicates the importance of endogenous bacterial rna present in these vlp. the induction of anti-hpv vlp antibodies has also been extensively studied. although the production of secretory iga antibodies is desired for protection against viral infection at mucosal surfaces, the predominant antibody class found in secretions from the female genital tract following vaccination with hpv vlp is igg [ ] [ ] [ ] . while the presence of igg in these secretions may be due to transudation from the serum, some active transportation or local production in the mucosa may be possible [ , ] . vaccination with hpv vlp induces the production of both igg and iga antibodies in the serum and cervical secretions of humans [ ] . while serum titers of anti-hpv antibodies may initially decline post-vaccination, these levels plateau and remain stable to provide prolonged immunity [ ] . while the induction of a potent humoral immune response and the subsequent production of anti-vlp antibodies is the primary desired outcome of most commercial vlp vaccines, these is increasing appreciation for the role of vaccine-induced cell-mediated immunity [ ] [ ] [ ] . measurements of anti-vlp titers can provide an important indication of vaccine efficacy with respect to the neutralization of a virus challenge, a cellmediated immune response also plays an important role in antivirus immune defense [ , ] . activation of a cellmediated immune response can also be the primary desired outcome of vlp vaccines, particularly for chimeric or other modified vlp vaccines that target non-virus pathologies. a potent cell-mediated response to vlp vaccines is dependent upon cross-presentation of vlp-derived antigens on mhc-i. cd + t cytotoxic (t c ) cells can recognize through mhc-i antigen complexes through their tcr, and become primed for activation. these antigen-primed t c cells require additional signaling for activation, including interaction with costimulatory receptors, and cytokines from t h cells. while many vlp may be capable of cross-presentation, some may be more effective at inducing cross-presentation due to uptake and processing pathways inherited from their parent virus [ , ] . the induction of a potent cell-mediated immune response is particularly important for immunotherapeutic cancer vaccines. tumor cell-specific antibodies can enhance the inherent cytotoxic activity of natural killer cells through mechanisms such as antibody-dependent cell-mediated cytotoxicity, or directly through complement-dependent cytotoxicity; however these mechanisms tend to be restricted to passive vaccination with monoclonal antibodies, such as the her -specific monoclonal trastuzumab [ ] . a cell-mediated immune response combines target specificity with the in vivo expansion of effector cell populations, with the capacity to establish prolonged memory. rhdv vlp are particularly effective as a vector for cancer vaccines, capable of inducing the cross-presentation of vlp-derived antigens [ ] , and compatible with recombinant insertion and chemical conjugation [ ] . rhdv vlp vaccines have been investigated in models of hpvinfected cervical cancer [ ] , melanoma [ ] , lewis' lung carcinoma [ ] , and colorectal cancer [ ] . additional examples of vlp that can induce cross-presentation and a potent cellmediated immune response include qβ vlp [ ] , ms vlp [ ] , and alphavirus vlp [ ] . preexisting immunity in the form of preformed anti-vlp antibodies can be an important consideration for some vlp vaccines [ , ] . binding of anti-vlp antibodies may interfere with the normal uptake and presentation pathways of vlp vaccines. anti-vlp antibodies may be present in unvaccinated individuals due to environmental exposure to prevalent strains of the parent virus, or induced following vlp vaccination. while the presence of preexisting antibodies may decrease the efficacy or alter the immune recognition of some vlp vaccines [ , ] , they can also be associated with measures of enhanced vaccine efficacy [ ] . this may be due to accessing fc receptor-mediated uptake pathways, the formation of antibody-vlp complexes, or antigenic boosting of specific b cells. the presence of anti-vlp antibodies have also been found to have no observable effect on the intended vaccine outcome for several vlp vaccines, such as polyomavirus [ ] , and rhdv vlp [ ] . when anti-vlp antibodies present deleterious interference, alternating between different vlp vaccine vectors may be a suitable solution. alternating between rotavirus and adenovirus vlp has been found to enhance both the humoral and cell-mediated immune responses against target antigens in comparison to repeat delivery with the same vlp vector [ ] . similarly, alternating between closely related vlp such as rhdv and human norovirus (hunv) vlp can be sufficient to enhance vaccine immunogenicity [ ] . further complication can arise when vlp vaccines induce a phenomenon referred to as carrier-induced epitopic suppression (cies), in which the intended immune response is outcompeted by a strong anti-vlp response [ ] . cies may be prevented by masking vlp surface antigens, or by avoiding recognition through recombinant modification. for example, recombinant insertion of the p domain of hiv into hev vlp was identified to avoid recognition by anti-hev antibodies [ ] . the formulation of a vaccine refers to the constituents that make up the final administrable solution, including the vaccine vector, adjuvants, and excipients. for vlp vaccines, these excipients can include a variety of salts and compounds prepared as an aqueous solution or emulsion, which maintains the physical stability of vlp for enhanced shelf life of the vaccine. appropriate use of buffers can limit fluctuations in ph, while protection from fluctuations in temperature and desiccation may be provided by thermoprotectants and lyoprotectants, respectively. formulation science involves investigating the various components of a vaccine under different environmental conditions, with the intention of formulating a stable vaccine product suitable for the route of administration, and with maximized immunogenicity. the combination of formulation components, vlp vaccine preparation states and routes of administration is outlined in figure . the majority of vlp vaccines currently on the market and under clinical evaluation are liquid suspensions, ready for administration. this places strict limitations on vlp vaccine storage and distribution for safe and compliant administration. for example, the commercial hpv vaccine gardasil (merck) must be refrigerated at - °c, and protected from light. gardasil cannot be frozen, and guidelines for the use of gardasil advise that the vaccine must be used within h when removed from refrigeration at temperatures below °c, or when stored at - °c. these guidelines mirror those of many other commercial vlp vaccines, and outlines the primary stability, storage and distribution challenges for formulation science to investigate. maintaining the integrity and stability of vlp in solution is largely dependent on the combination of salts, and the buffering chemicals and compounds used. optimization of buffer ph, ionic strength, and other stabilizing components is imperative to developing a marketable, stable, liquid vlp vaccine [ ] . an investigation into the stability of ev vlp identified that sodium phosphate-based buffers were superior to citrate or tris buffered solutions, with vlp stored in sodium phosphate buffer remaining stable for month at both and °c [ ] . such prolonged stability at room and core body temperature suggests that this vlp may be suitable for importation into countries where maintaining cold-chain storage and distribution can be difficult. other important considerations for the selection of an appropriate buffer include compatibility with downstream applications, such as chemical conjugation, and the availability of scavengable nutrients that may promote the growth of potentially harmful organisms in an improperly stored vaccine. various additive molecules, particularly carbohydrates such as trehalose, sucrose and glycerol, have been investigated extensively in vlp vaccine formulations. the addition of these molecules to vaccine formulations demonstrated enhancement in vlp stability the role of formulation science in vlp vaccine manufacture includes the chemical composition of buffers, preservatives, additives and other stabilizing compounds for maintaining intact vlp. this includes protecting vlp from chemical or physical instability, and enzymatic degradation. formulations can also include targeted delivery compounds, such as muco-adhesives, and immunogenic components such as adjuvants. storage and distribution of vlp vaccines, and the subsequent route of administration are also important considerations in formulation science, critical in determining the efficacy and immunogenicity of the vaccine. within a liquid suspension of norwalk virus vlp [ ] and rotavirus vlp [ ] . other than sugar-based formulation additives, polyanionic solutions can also stabilize vlp that would otherwise be unstable at neutral ph, such as chikungunya virus vlp [ ] . the majority of commercial vlp vaccines are distributed as a liquid suspension, requiring a cold chain maintaining - °c throughout distribution and storage. even under stable cold chain conditions, the longevity of vlp can be limited. this can be prolonged by storage at temperatures at or below − °c, stabilized with the addition of a cryopreservative such as glycerol or trehalose [ ] ; however, this only further exacerbates the issue of delivering these vaccines intact where they are needed, such as in developing countries without reliable cold-chain delivery infrastructure. alternative storage methods such as freeze-drying, or lyophilization, can have variable effects on the stability of vlp. mechanical damage induced by ice crystallization, or exposure to varying salt concentrations and ph changes during the freezing process may adversely affect the integrity of vlp [ ] . exposure to these factors can be limited through the addition of specific cryoprotectants and lyoprotectants. for example, red-spotted grouper nervous necrosis virus (rgnnv) vlp [ ] reportedly retain stable particles and remain immunogenic when freezedried in the presence of sorbitol, but were adversely affected in the presence of mannitol. qβ vlp [ ] , murine polyomavirus (mupyv) vlp [ , ] , and hbv vlp [ ] have likewise demonstrated some capacity to survive varying freeze-drying methodologies. spray-drying has also gained traction as an alternative vaccine storage mechanism, avoiding the potential mechanical damage induced by freeze-drying by instead forming a dry powder formulation through a combination of nebulization and dehumidification [ ] ; however, spray-drying involves exposure to elevated temperatures, which can be similarly damaging to thermolabile vlp. hpv vlp suspended in a formulation containing mannitol, trehalose, dextran, l-leucine and inositol are capable of surviving this procedure [ ] [ ] [ ] . spray-dried ms - l vlp stored at room temperature for months were found to induce high titer anti-hpv l igg antibodies, and significantly protected vaccinated mice from challenge with hpv [ ] . air or vacuum drying methods have also been reported for coating microneedles with influenza vlp suspended in a formulation containing carboxymethylcellulose (cmc). microneedle-based delivery of dried h n (a/pr ) [ ] and h n (a/aichi) [ ] were found to induce immune responses comparable to intramuscular injection, but with the advantage of prolonged longevity in storage. investigation of similar formulation and preparation strategies with different types of vlp may uncover some universality in their application, with the potential for these methods to eradicate the cold-chain limitation imposed on many vlp vaccines. as has been previously described, many vlp possess structural or molecular features that can confer some auto-immunostimulatory properties. these properties facilitate the induction of immune responses by vlp without the need for adjuvants; however, the use of adjuvants with vlp vaccines may enhance vaccine immunogenicity, and promote the activation of a specific type of immune response. currently licensed adjuvants for use with vaccines include aluminum sulfate salts (alum), proprietary combination adjuvants such as as (glaxosmithkline), as (glaxosmithkline) and mf (novartis), thermo-reversible oil-in-water immersions, and montanide isa [ ] . while many standard vaccine adjuvants may be suitable for vlp vaccines where the generation of anti-vlp antibodies is the desired outcome, these adjuvants may fail to overcome immunotolerance and induce antitumor immunity in cancer vaccine formulations [ ] ; however, where traditional adjuvants may fail, novel tlr agonist adjuvants have had success. the tlr agonist adjuvant imiquimod was recently approved for use with vaccines for melanoma, and another clinical trial is assessing its candidacy for treatment of bladder cancer [ ] . tlr / stimulate anti-viral interferon pathways in apcs, promoting the activation of cd + t cells and nk cells [ , ] . the use of the adjuvant gardiquimod in combination with vaccination has been found to induce tumor regression in murine models of melanoma [ ] , human hepatocellular carcinoma [ ] and pancreatic cancer [ ] . tlr agonist adjuvants such as unmethylated cpg oligonucleotides have drawn considerable interest due to their inherent ability to associate with some vlp [ , ] . similarly, the presence of other nucleic acids can have downstream ramifications on the immune response induced by vlp. for example, the presence of rna inside qβ vlp can skew the humoral response induced in mice to produce igg a antibodies, while the removal of this rna results in the production of igg antibodies [ ] . qβ vlp can also contain additional adjuvant molecules derived from their expression system, encapsidated during particle formation. a recent study in mice investigated the use of various adjuvants in conjunction with a filovirus vlp vaccine, including the tlr agonist adjuvant poly-iclc (hiltonol), tlr agonist adjuvant monophospholipid a (mpla), cpg odn and alhydrogel [ ] . poly-iclc was found to induce a predominantly t h response, with an igg c subtype antibody production that correlated with protection from virus challenge. in comparison, administration of the vlp vaccine with alhydrogel elicited a strong t h response with high antibody titers, but conferred no protection from virus challenge. this study highlighted the importance of carefully considering what kind of adjuvant is suitable to accompany a vlp vaccine, as the most immunogenic adjuvant may not necessarily provide the desired vaccine outcome. vlp vaccines approved for clinical use have utilized various administration modalities, including vaccination subcutaneously, intradermally, intramuscularly or at mucosal surfaces [ ] . live attenuated vaccines are traditionally injected subcutaneously, while inactivated or subunit vaccines are often administered intramuscularly. in infants, intramuscular injection of vaccines tends to confer enhanced immunogenicity over subcutaneous administration [ ] . modern vaccine delivery technologies, such as intradermal microneedle patches, have also been investigated for their potential applications with vlp vaccines [ , , ] . while the majority of recent clinical trials involving the administration of vlp favor intramuscular injection, alternative routes of administration are being investigated based on differential immunogenicity at different delivery sites. for example, an investigation into the routes of administration of hunv vlp found that intranasal inoculation of mice induced the production of mucosal anti-hunv igg and iga antibodies, while intramuscular injection only induced igg production [ ] . the study identified that only mucosal iga was suitable for neutralization of infectious hunv virions, rendering intramuscular injection unsuitable for administration of hunv vlp. a similar finding was identified regarding the administration of respiratory syncytial virus (rsv) vlp, with the desired t h response, neutralizing mucosal antibodies and cd + t cell responses identified following intranasal inoculation, rather than intramuscular injection [ ] . vaccination with vlp at mucosal surfaces also requires higher doses of vlp in comparison to parental administration. oral delivery of virus-like particles is also being investigated as an alternative, convenient route of administration. vlp expressed in plant-based systems represents a particularly efficient means of both production and delivery, with expression in an edible plant species potentially forgoing the need for vlp purification and vaccine formulation. examples of this concept include the expression of hbv and norwalk virus vlp in solanum tuberosum potato and lycopersicon esculentum tomato plants [ , ] . nicotiana benthamiana tobacco are also used as an efficient plant expression system for production of vlp. hbv vlp purified from n. benthamiana are capable of withstanding a simulation of the acidic environment within the stomach [ ] ; however, the hbcag polypeptide of hbv vlp was digested when exposed to porcine pepsin. the protection of protein-based vlp from enzymatic digestion is a significant hurdle for oral delivery, despite the promise of this vaccination route. the translation of vlp vaccines from preclinical research to routine clinical administration is a multifaceted task combining studies into stability, formulation science, immunogenicity, and clinical vaccine efficacy. these issues are not necessarily unique to the development of vlp vaccines, encompassing the breadth of research and development necessary for bringing any vaccine candidate to market. the expression of stable vlp is only the first step toward the development of a novel vlp vaccine, which may eventually include various excipients, adjuvants and other compounds in the administrable form. advancements in virology and vaccinology that have facilitated the development of novel vlp vaccines seemingly parallel regulatory and proprietary restrictions placed upon new vaccines; however, vlp vaccines also possess unparalleled potential due to their balance between clinical vaccine efficacy and safety, and their versatility as a vaccine vector. the field of vlp vaccines has continued to experience significant growth over the past decade, both in the diversity of vaccines and in the translation of vaccines toward routine clinical administration. our research focuses upon the development of rhdv vlp as a versatile vaccine scaffold, particularly for immunotherapeutic vaccination as an alternative treatment option for cancer. the translation of vlp vaccine constructs from proof of concept models toward clinical administration is no menial feat. we have observed a corresponding shift toward focusing on clinical translation amongst our colleagues in the field. focal points of discussion highlighted by the process include the relevance and translatability of research animal models, adaptation to the established dogma within the field of human vaccination, and the establishment of a developmental and commercial niche conducive to the advancement of novel vaccines throughout the rigors of clinical trials and regulatory approval. the immune response to vlp vaccines tends to strike a desirable balance between outcomes indicative of optimal vaccine efficacy, such as the induction of high-titer mucosal iga antibodies, while also maintaining an almost unparalleled vaccine safety profile. the inherent inability of vlp to infect or replicate alleviates potential vaccine risks, such as spontaneous reversion to pathogenesis, or incomplete inactivation. retention of the ability to facilitate cross-presentation of associated antigens, and the induction of a potent cell-mediated immune response can also have significant implications for vlp vaccines, diversifying the field to cover a broader range of disorders and diseases. the intracellular processing pathways remain largely unexplored for many vlp, and elucidation of the underlying mechanisms involved in some of the more advanced applications of vlp vaccines, such as gene delivery and immunomodulation. the role of formulation science appears underrepresented amongst clinical trials and clinical reports for vlp vaccines, with at times minimal elucidation of the vaccine administration route, the excipients included, and even exclusion of adjuvant delivered with the vaccine. the importance of formulation science and the relevance of excipients and adjuvants may be expectedly underappreciated during earlier phases of research and development, where establishment of vaccine efficacy may be paramount; however, early adoption of these vital constituents may ease clinical translation, potentially expanding the repertoire of marketable vlp vaccines. novelty, proprietary design, and versatility are almost as important in the development of vlp vaccines as the actual vaccine efficacy itself. maintaining compatibility with the upscaling of production and gmp manufacture pipelines is another important component in the development of a vlp vaccine for routine clinical administration. the benefit of such foresight is clear, and the promotion of clinical translation will be advantageous across the field of vlp vaccines. upon review across the field including current trends in new and emerging research, the progression of established vaccine research, and clinical trial schedules, vlp vaccine research is experiencing a period of rapid growth. this includes both the repertoire of organisms and conditions being targeted with vlp vaccines, and the advancement of established vaccines through clinical trials, translation, and clinical applications. over the next five years, we predict that there will be a significant increase in the diversity of vlp vaccines in active circulation. we expect that vlp will be used in more complex applications than as a vaccine against the cognate virus from which they were derived. we also predict the development of novel vlp vaccine constructs for human and zoonotic pathogenic organisms and conditions with or without current vaccination options, and the incorporation of advanced vaccination methodologies and techniques into routine vlp vaccine production, distribution and administration. in particular, we predict increased incorporation of alternative vaccine-storage techniques such as freeze-dried or spray-dried methods. the development of vlp vaccines is often focused on facilitating distribution amongst populations and communities where these vaccines are most needed. we predict that over the next five years, the capability to support vlp vaccine distribution will increase by limiting the current reliance on cold-chain delivery. these predictions outline an exciting future over the next five years for vlp vaccine research and development. • virus-like particles consist of virus-derived proteins and associated molecules that spontaneously form a particulate structure. • vlp vaccines have the safety profile of a subunit vaccine, but with efficacy and vaccination outcomes that can be comparable to killed or live attenuated vaccines, depending on the particular vlp vaccine. • while the predominant desired immune response amongst the majority of vlp vaccines is a potent humoral response producing high titer antibodies, some vlp vaccines can induce a potent cytotoxic immune response for selective elimination of cells currently infected by the target virus, or target tumor cells. • formulation science plays a major role in the development of vlp vaccines, facilitating the selection of appropriate combinations of excipients and adjuvants. excipients are used to increase vlp particle stability in the vaccine formulation, while adjuvants enhance vaccine efficacy, and help to select for a specific desired immune response and outcome. this research was funded by the university of otago. interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. peer reviewers on this manuscript have no relevant financial or other relationships to disclose. braeden donaldson http://orcid.org/ - - - vernon k. ward http://orcid.org/ - - - papers of special note have been highlighted as either of interest (•) or of considerable interest (••) to readers antigen delivery by virus-like particles for immunotherapeutic vaccination this review covers the structural diversity of vlp, post-production modification, and the immune response to vlp vaccines with a specific focus on immunotherapeutic vaccine development immunogenicity of a quadrivalent virus-like particles (vlp) influenza vaccine in healthy 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particles: a review this review covers the breadth of expression systems available for vlp vaccine production, while also discussing vlp structural diversity and characterization mannosylation of virus-like particles enhances internalization by antigen presenting cells coupling the adjuvant cpg oligonucleotides to rhdv vlp. dunedin: university of otago αenv-decorated phosphatidylserine liposomes trigger phagocytosis of hiv-virus-like particles in macrophages norovirus p particle: a subviral nanoparticle for vaccine development against norovirus, rotavirus and influenza virus nanoparticles target distinct dendritic cell populations according to their size conduits mediate transport of low-molecular-weight antigen to lymph node follicles the humoral immune response is initiated in lymph nodes by b cells that acquire soluble antigen directly in the follicles virus-like particle (vlp) lymphatic trafficking and immune response generation after immunization by different routes follicular shuttling of marginal zone b cells facilitates antigen transport b cells acquire particulate antigen in a macrophage-rich area at the boundary between the follicle and the subcapsular sinus of the lymph node a molecular assembly system that renders antigens of choice highly repetitive for induction of protective b cell responses. vaccine dissecting virus entry via endocytosis despite differences between dendritic cells and langerhans cells in the mechanism of papillomavirus-like particle antigen uptake, both cells cross-prime t cells. virology cross-presentation of epitopes on virus-like particles via the mhc i receptor recycling pathway the length of vesicular stomatitis virus particles dictates a need for actin assembly during clathrin-dependent endocytosis single-particle tracking of murine polyoma virus-like particles on live cells and artificial membranes differential uptake and crosspresentation of human papillomavirus virus-like particles by dendritic cells and langerhans cells 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vaccine followed by one dose of varicella vaccine in children aged months- years or - years primed with measles-mumps-rubella (mmr) vaccine long-term protective immunity from an influenza virus-like particle vaccine administered with a microneedle patch mucosal antibodies induced by intranasal but not intramuscular immunization block norovirus gii. virus-like particle receptor binding a single intranasal administration of virus-like particle vaccine induces an efficient protection for mice against human respiratory syncytial virus virus-like particle expression and assembly in plants: hepatitis b and norwalk viruses expression of norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice plant-expressed hepatitis b core antigen virus-like particles: characterization and investigation of their stability in simulated and pig gastro-intestinal fluids key: cord- -c ytamge authors: da fonseca pestana ribeiro, jose mauro; park, marcelo title: less empiric broad-spectrum antibiotics is more in the icu date: - - journal: intensive care med doi: . /s - - -z sha: doc_id: cord_uid: c ytamge nan antibiotics are administered in approximately % of patients who are admitted to the intensive care unit (icu) and have helped to save millions of lives [ ] . however, up to half of all antibiotic prescriptions may be unnecessary [ ] . antibiotic overuse has contributed to alarmingly high levels of global antibiotic resistance, which is increasing at a rate faster than that at which novel antibiotics are produced. therefore, finding a fine balance between the appropriate use and avoidance of unnecessary administration is crucial to prevent the renaissance of a new world without antibiotics [ ] . antibiotics largely reduce mortality associated with moderate and severe infections, with a historical numberneeded-to-treat estimated in . for severe pneumonia patients [ ] . infection progressing to sepsis is the leading cause of death in icu patients and can be potentially treated using antibiotics, along with organ dysfunction support, and infection source control [ ] . from the cognitive dimension, the fear of patient deterioration due to sepsis favours the empirical use of antibiotics in icu patients. once a severe infection is diagnosed, the early administration of broad-spectrum antibiotics is recommended to decrease the risk of death [ ] . this intuitive recommendation is also based on observational studies that have been carried out in the emergency department [ , ] . in contrast, a randomized study, showed that the pre-hospital administration of antibiotics in septic patients did not reduce the mortality [ ] . furthermore, in a prospective cohort of icu patients with bacteremia, early initiation and appropriateness of antibiotic intervention were not found to impact mortality when adequately adjusted for confounders [ ] . additionally, a pooled analysis of the current literature failed to demonstrate a survival benefit related to antibiotic administration within the first hour or within the first h following a diagnosis of sepsis [ ] . in surgical and trauma patients, a quasi-experimental before and after study demonstrated that more aggressive antibiotic use had similar outcomes and higher antibiotic exposure compared to conservative use [ ] . interestingly, when antibiotics were administered following the diagnosis of shock (mean arterial pressure < mmhg), the mortality of the aggressively-treated group was higher than that of the conservatively-treated group ( % vs. %, p < . ). the authors presented several plausible factors to explain these findings. the adequacy of initial antibiotic treatment was lower in the aggressivelytreated group, which therefore extended the antibiotics exposure. moreover, the waiting time for blood cultures and observation of the clinical course may also disclose alternative diagnosis to infections. at last, up to % of patients initially diagnosed as septic shock did not have an identified infection h after their initial diagnosis [ ] . from the physiological point of view, there is no plausibility that minor time differences in antibiotic administration reduce the intensity of the inflammatory response, and may even be associated with a transient worsening after administration. lastly, it is difficult to differentiate the effect of early antibiotic use per se from the awareness of critical illness and the timely institution of high quality-of-care [ ] . several adverse effects related to antibiotic use are described in the literature; with acquired multidrug resistance (mdr) being the most concerning effect. since *correspondence: marcelo.park@hc.fm.usp.br intensive care unit, emergency department, hospital das clínicas, university of são paulo medical school, são paulo, brazil , antibiotic resistance has been a major fear of sir alexander fleming. currently, mdr bacteria are largely spread across the world [ ] . the real impact of mdrs on the outcomes of icu patients is debatable, but despite this controversy, the incidence of mdrs is related to poor quality-of-care, as an expression of reduced compliance to hand hygiene [ ] , and a high burden of antibiotic exposure [ ] . de-escalation approach, in which the antibiotic spectrum is narrowed or even withdrawn after re-evaluation, has been implemented to reduce exposure to antibiotics. de-escalation has proved to be safe in terms of survival; however, it is associated with an increased icu lengthof-stay, without reducing the incidence of mdrs [ ] . de-escalation decreases the time of antibiotic use, but a short exposure still exists; in this way, a single antibiotic dose may be enough to treat severe infections such as fig. two different mindsets in the decision making process to initiate antibiotics to critically ill patients who are getting worse. a aggressive mindset, in which the antibiotics are initiated as soon as possible to avoid further clinical deterioration; and b conservative mindset, in which antibiotics are only initiated with the infection diagnosis, or in shock patients without non-infectious alternative suspicion. mdr denotes multidrug resistant bacteria. atms denote antimicrobials. kpc denotes klebsiella pneumonia carbapenemase. cre denotes carbapenem resistant enterobacteriaceae. *in the intensive care unit, patients have h of close clinical observation. # the gram-positive cocci absence in the tracheal aspirate has a high negative predictive value to staphylococcus aureus growing in patients with high clinical probability of ventilator associated pneumonia and clinical worsening-new fever, hypothermia, unexplained tachycardia and hyperventilation. laboratorial worsening-leukocytosis, leukopenia, increased c-reactive protein and increased procalcitonin. red boxes⇒no evidence-no randomized study or cohort evaluation on favor the practice, or randomized study against the practice. yellow boxes⇒some evidence-at least one cohort evaluation on favor the practice. green boxes⇒clinical evidence-at least one randomized study on favor the practice meningococcal meningitis [ ] , and to promote profound and sustained microbiome unbalances, therefore facilitating opportunistic infections and damping the potential benefit of the de-escalation approach [ ] . the main step toward the reduction of antibiotic use is the adequacy of hand hygiene in healthcare professionals [ ] . an antibiotic stewardship focusing on feedback, monitoring, persuasion, and audit after each drug prescription is associated with a long term reduction in healthcare associated infections, antibiotic prescriptions, and health care costs, without the deleterious effects on length-of-stay, readmissions, and in-hospital mortality [ ] . furthermore, the decrease in the use of carbapenems, has been associated with an overall reduction in the incidence of mdrs [ ] to ensure patient safety, the early aggressive administration of broad-spectrum antibiotics in the icu setting is common practice [ ] . however, maintaining a conservative mindset with respect to antibiotic use and safety is fundamental to both the patient and environment. mindset modification accomplishes many dimensions; for instance, the reset model which has been applied to dairy cattle farms resulted in a reduction in antibiotic use in this area [ ] . reset dimensions are ( ) (r)ulesan external motivation to reduce antibiotic prescription; ( ) (e)ducation-showing that antibiotic prescriptions are unnecessarily excessive, expensive, and paradoxically unsafe; ( ) (s)ocial pressure-ensuring societal awareness that unnecessary use of antibiotics is dangerously growing; ( ) (e)conomics-the awareness of economic consequences of reduced use of antibiotics to save costs; and ( ) (t)ools-ways to spread knowledge regarding the conscious use of antibiotics. a schematic, aggressive, and conservative mindset to commence antibiotics is presented in fig. . there are several reasons why aggressive early use of broad-spectrum antibiotics should be avoided in icu patients. presence of shock without an alternative diagnosis other than infection, and a diagnosis of infection based on cultures, bacterioscopic examinations, and imaging results for the initiation of antibiotics is currently considered safe practice. furthermore, clinicians can consider investigating feasible alternative diagnosis for shock in unstable icu patients before antibiotics initiation. consideration of antibiotic use in our icus is essential, and if necessary; there is great plausibility in changing our mindset to restrict antibiotic use. the authors declare that they have no conflicts of interest. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. international study of the prevalence and outcomes of infection in intensive care units ready for a world without antibiotics? the pensières antibiotic resistance call to action treatment of pneumonia with -(p-aminobenzenesulphonamido) pyridine surviving sepsis campaign: international guidelines for management of sepsis and septic shock the timing of early antibiotics and hospital mortality in sepsis empiric antibiotic treatment reduces mortality in severe sepsis and septic shock from the first hour: results from a guideline-based performance improvement program prehospital antibiotics in the ambulance for sepsis: a multicentre, open label, randomised trial antibiotic use and impact on outcome from bacteraemic critical illness: the bacteraemia study in intensive care (basic) the impact of timing of antibiotics on outcomes in severe sepsis and septic shock: a systematic review and meta-analysis aggressive versus conservative initiation of antimicrobial treatment in critically ill surgical patients with suspected intensivecare-unit-acquired infection: a quasi-experimental, before and after observational cohort study septic shock with no diagnosis at hours: a pragmatic multicenter prospective cohort study association between state-mandated protocolized sepsis care and in-hospital mortality among adults with sepsis clinical epidemiology of the global expansion of klebsiella pneumoniae carbapenemases interventions to reduce colonisation and transmission of antimicrobial-resistant bacteria in intensive care units: an interrupted time series study and cluster randomised trial effect of short-term carbapenem restriction on the incidence of nonpseudomonal multi-drug resistant gram-negative bacilli in an intensive care unit de-escalation versus continuation of empirical antimicrobial treatment in severe sepsis: a multicenter non-blinded randomized noninferiority trial ceftriaxone as effective as long-acting chloramphenicol in short-course treatment of meningococcal meningitis during epidemics: a randomised non-inferiority study profound alterations of intestinal microbiota following a single dose of clindamycin results in sustained susceptibility to clostridium difficile-induced colitis long-term effects of phased implementation of antimicrobial stewardship in academic icus the reset mindset model applied on decreasing antibiotic usage in dairy cattle in the netherlands key: cord- -rloey j authors: harel, noam; meir, moran; gophna, uri; stern, adi title: direct sequencing of rna with minion nanopore: detecting mutations based on associations date: - - journal: nucleic acids res doi: . /nar/gkz sha: doc_id: cord_uid: rloey j one of the key challenges in the field of genetics is the inference of haplotypes from next generation sequencing data. the minion oxford nanopore sequencer allows sequencing long reads, with the potential of sequencing complete genes, and even complete genomes of viruses, in individual reads. however, minion suffers from high error rates, rendering the detection of true variants difficult. here, we propose a new statistical approach named associvar, which differentiates between true mutations and sequencing errors from direct rna/dna sequencing using minion. our strategy relies on the assumption that sequencing errors will be dispersed randomly along sequencing reads, and hence will not be associated with each other, whereas real mutations will display a non-random pattern of association with other mutations. we demonstrate our approach using direct rna sequencing data from evolved populations of the ms bacteriophage, whose small genome makes it ideal for minion sequencing. associvar inferred several mutations in the phage genome, which were corroborated using parallel illumina sequencing. this allowed us to reconstruct full genome viral haplotypes constituting different strains that were present in the sample. our approach is applicable to long read sequencing data from any organism for accurate detection of bona fide mutations and inter-strain polymorphisms. a major goal of genetics today is the characterization of genetic diversity in a population. in microbes, such diversity is generated in particular by high mutation rates, which may generate both nucleotide substitutions as well as point insertions or deletions (indels) ( ) . longer indels may also occur ( ) , as may events of genetic recombination. disease pathogenesis, progression, management and epidemiology are all affected by the genetic diversity created in microbial populations ( ) ( ) ( ) ( ) ( ) ( ) ( ) . thus, characterizing this diversity is of utmost importance in clinical as well as research settings, which makes the development and improvement of suitable sequencing technologies crucial ( ) ( ) ( ) . the availability of second-generation dna sequencing technologies ( ) , with the illumina platform currently at the forefront, has made the sequencing of genomes conventional. in particular, this technology has dramatically furthered the study of viruses, whose relatively small genomes allow in depth characterization of a population of viruses ( , ) . illumina-based sequencing allows the detection of minor variants that the standard sanger-based method often missed. however, illumina short-read sequencing technologies all share one major limitation, the short length of each read, which typically ranges between and bp (for a paired end read). this means that a complete viral genome sequence cannot be obtained in a single read, impairing the ability to link distant mutations in an individual viral genome. another illumina limitation is that rna cannot be sequenced directly. during library preparation, rna is reverse-transcribed into cdna and amplified by pcr. this creates multiple problems that have been extensively discussed but not resolved ( ) : first, reverse transcription and pcr may introduce errors during early stages of amplification that will be carried on to later stages ( ) ( ) ( ) . second, some molecules may be preferentially amplified over others, a term known as pcr bias. third, pcr and reverse transcription reactions can often result in chimeric dna sequences that originate from different molecules ( ) ( ) ( ) . together, these problems make the inference of haplotypes from pcr-based libraries that are sequenced with illumina extremely limited. currently, single-molecule third-generation sequencing systems, such as oxford nanopore technologies, provide a promising alternative for sequencing full-length single viral genomes ( ) . in fact, these technologies now allow directly sequencing either dna or rna. the long reads provided by these methods have the potential to allow for the inference of up to an entire genome of a typical rna virus, whose genome is generally shorter than , bp ( ) ( ) ( ) ( ) ( ) . however, one of the major shortcomings of the third gener-ation technologies are their relatively high error rates, with the proportion of errors on a read often exceeding % ( , ) . this high error rate makes the detection of true single-nucleotide variants very difficult ( , , ) . here, we devised a simple statistical procedure called as-socivar that allows weeding out real mutations from technical errors using only the minion sequencing results. we focused our analysis on the ms bacteriophage, an extremely small ( bases) and fast evolving +ssrna virus ( , ) that is highly amenable to direct sequencing with oxford nanopore minion. we sequenced virus populations in parallel using both minion and illumina, allowing us to corroborate the inferences of associvar. this then allowed us to directly infer relationships between mutations and to deduce the entire genome sequences of viral strains in the population. we were also able to use associvar to analyze a yeast mrna sample and data of mixed strains of zika virus. this illustrates the generality of our approach which can be applied to other organisms as well. during the course of an evolve-and-resequence experiment, we performed serial passaging of the phage ms (meir et al., in preparation). briefly, clonal ms stock was propagated from a single plaque that was the precursor of all the evolutionary lines established in this work. we performed serial passages at • c with two biological replicates (hereby denoted as a and b). the serial passages were performed as follows: ml cultures of naive escherichia coli c- were grown up to an optical density of od = . (corresponding to a density of about cells/ml). each passage was infected with ml of phages from the previous passage. the cultures were grown for h at each temperature with shaking, and the e. coli cells were then removed by centrifugation. the supernatant was subjected to filtration with . m filter (stericup ® filter, emd millipore) to remove any remaining residues. naive hosts were provided for each passage. the new phage stock was then stored at • c. aliquots of these phage stocks were used for measuring the concentration of phages by plaque assay and infecting the next serial passage. we then determined the population frequency of each mutation at passage and passage through whole genome deep sequencing as described below, using illumina and minion. library preparation was performed according to our lab's accungs sequencing protocol ( ) with some modifications: the reverse transcription reaction was performed using superscript ® iii reverse transcriptase (thermo scientific), the reaction was performed on ng of rna phage with l of dntp mix ( mm), l of r primer (tg ggtggtaactagccaagcag) ( m) and l of sterile distilled water. the mixture was incubated for min at • c followed by incubation on ice for min. after brief centrifugation, l of x first-strand buffer were added along with l dtt ( . m), l of rnase out (thermo scientific) and l of superscript™ iii rt ( units/l). the mixture was incubated for mins at • c followed by inactivation for mins at • c. the last step was adding l of rnase h to the reaction according to manufacturer instructions. cdna from the reverse transcription reaction was directly used as a template for the pcr amplification of the full ms genome in three overlapping fragments. pcr reactions were performed using 'phusion high-fidelity dna-polymerase' (thermo scientific) according to manufacturer instructions with the primers: f (gggtggga cccctttcgg), r (tttttctagagagccgttgc ct), f (ggcccaaatctcagccatgc), r (cgtg tctgatccacggc), f (ggcacaagttgcagga tgca), r (tgggtggtaactagccaagcag), see supplementary figure s . after pcr, the three amplicons were purified with a pcr clean up kit (promega). purified amplicons were quantified with qubit assays (q , life technologies), diluted and pooled in equimolar concentrations. the illumina nextera xt library preparation protocol and kit were used to produce dna libraries, according to manufacturer instructions with some modifications. briefly, the tagmentation reaction was performed with . ng/l of dna, l td buffer, l atm enzyme and up to l ddw. the mixture was incubated for min at • c and then directly used as a template for l pcr reaction using 'phusion high-fidelity dna-polymerase' (thermo scientific) according to manufacturer instructions with the index primers from the nextera xt kit. after pcr, a double sized selection was performed using ampure beads to remove short and long library fragments, since bp fragments were required. we collected the supernatant and read l from each sample in the tapestation (agilent high sensitives d ) to verify fragment size. the yeast enolase sample was prepared as described for the phage rna. rt and pcr amplification were performed using primers: e (atggctgtctctaaagtttacg cta) and e (ttacaacttgtcaccgtggtgg). libraries were sequenced on an illumina miseq using the × miseq reagent kit (illumina, ms- - ) for pairedend reads. bioinformatics processing of the data was performed using the accungs pipeline ( ) with the default parameters (minimal %id = , e-value threshold = e− and q score cutoff of ). briefly, this pipeline is based on (a) mapping the reads to the reference genome using blast, (b) searching for variants that appear on both overlapping reads, (c) calling variants with a given q-score threshold and inferring their frequency. all libraries attained a mean coverage of ∼ , reads/base. the reference genome was determined by comparing the consensus of passage to gen-bank id v . (differences noted in supplementary table s ). when examining the results of the control sequence (a plasmid bearing the ms genome), we noted a high error rate at several positions that resided near the primer sites, and accordingly positions from each end of the genome were excluded from downstream analysis. the oxford nanopore minion was used to sequence the ms rna directly. we sequenced the three samples (p a, nucleic acids research, , vol. , no. e p b and p a) in three separate runs. we prepared direct rna libraries according to manufacturer library prep instructions with some modifications. we altered the supplied reverse transcriptase adapter (rta) ( ) , which has a t overhang, to specifically target the ms genome with nucleotides complementary to the ms conserved end (supplementary figure s ). the ligation reaction was performed with ng rna in l, nm costume adapter in l, l of nebnext quick ligation buffer and . l of t dna ligase enzyme (neb). the mixture was incubated for min at • c followed by incubation on ice for min and then directly used as a template for the next step of the library prep which is cdna synthesis according to manufacturer instructions. the cdna synthesis step was performed in order to maintain the rna fragments integrity during minion library prep and sequencing. the library was cleaned up each time using l of ampure xp dna beads per l of sample and we added l of rnase out (thermo scientific) to protect the rna. the rna was directly sequenced on the minion nanopore sequencing device using a flo-min flow cell equipped with the r . chemistry. the minknow control software version . . was used and was allowed to proceed for h. the basecalling was performed locally by the miniknow software as well, and the data was written out in the fastq format. reads were filtered with the miniknow default cutoff of a minimum average q-score of . the accungs pipeline ( ) described above was next applied to the data in order to determine variant frequencies. blast parameters were modified to minimal % identity = , e-value threshold = e− and no q-score cutoff, to allow the highly variable minion reads to map. as with the illumina results, positions from each end of the genome were excluded from downstream analysis. this also solved the problem of very low coverage areas in the minion sequencing. variant frequency distributions for minion and miseq were calculated by using the accungs pipeline results. for substitutions and deletions, the distribution is straightforward and accounts for the results for all bases. for insertions, we focused on point insertions, defined as the first insertion after any position, in order to create the frequency distributions. positions close to the ends of the genome that show a high error rate in miseq due to primer proximity were removed from this analysis. to calculate the error rate, we sequenced a control sample from the beginning of the passaging experiment (p a), and the mean, th percentile and th percentile errors were calculated for every error type. associvar searches for strong associations between variants as an indication that these represent bona fide mutations. the method is based on five stages: (a) detecting non-random associations. for each pair of positions, a read is classified into four categories based on whether the read bears the wt nucleotide (i.e., identical to the reference genome) or non-wt nucleotide (i.e., different from the reference genome) at each of the two given positions. we use this to create a × contingency matrix of observed counts, which is then used as the input for a chi-square test and a resulting chisquare statistic (see supplementary text). we focus only on contiguous reads that spanned the entire genome. notably at this stage we focus only on wt versus non-wt assignments (rather than the exact identity of the non-wt allele) for computational tractability. this is relaxed later on. (b) removing proximal positions. since we observed that positions that are highly proximal (< bp apart) often tended to be highly associated, and we suspected this is an artifact of the sequencing machine, chi square results for all such proximal positions were removed from the analysis. to each other tend to present spurious high associations, due to transitivity we expect a position next to a real mutation to also be highly associated with other positions with real mutations. in other words, if positions p and q are highly associated because they are real mutations, and positions q and q + are highly associated because they are proximal, we will see a high association between positions p and q + as well. however, we expect the association between the real mutations to be the highest, i.e. to be a local maximum in the surroundings of a given pair. a normalized chi score's surroundings is defined as the four neighboring normalized chi scores when the data is regarded as a two-dimensional matrix. for example, the normalized chi score for ( p, q) is required to be higher than the normalized chi scores for ( p, q − ), ( p, q + ), ( p − , q) and ( p + , q). (e) use of a control sequence. in order to create a cutoff for the normalized chi-square statistic, we used the values obtained for a control sequence (supplementary figure s ). we know that our control sample was not completely homogenous, since it contained two mutations at a frequency slightly higher than %. nevertheless, it served as a valid control when setting a confidence rate of . %, i.e. calculating the normalized chi score that allows . % of the positions in our control sample to be identified as significant (allowing for three 'false positive' positions in our case). after stages (a) through (e), associvar infers n positions with real mutations in the population. the last stage is to identify the identity of the mutations (a, c, g, t, −), in a similar way to that described in (a). insertions are ignored here. every position has four possible alternative variants (the three nucleotides that differ from the reference, or a deletion), and we test these variations against each other using chi-square tests, leading to n × (n− ) tests, where n is the number of positions previously identified as having real mutations. again, we created a × contingency matrix of observed frequencies, which is then used as the input for the chi-square test. for every position, we choose the variant with the highest average chisquare statistic for all the tests for pairs containing that variant. we begin by focusing only on the variants inferred as bona fide mutations in the last stage. this means that in principle there are n possible haplotypes bearing these mutations. we filter out reads with variants that do not match our inference (for example, if one of the inferred mutants is a g, we filter out reads with the nucleotides c, t or a deletion at position ). we then use our inferred percentile error thresholds (table ) to deduce which combinations of mutations are likely to be true and which may have been created by the technical error rate. we use an iterative approach to classify which of the n haplotypes is reliable. first, for every single nucleotide variant, we group together all of the haplotypes that include this base variant (a haplotype can appear in more than one group). second, in each group, we compute the relative frequency of each haplotype as its proportion of all haplotypes in the group. we iterate through the haplotypes from highest frequency to lowest, classifying each haplotype as reliable or not. the first haplotype is automatically classified as reliable. for every haplotype, we compare its relative frequency with the probability that it is created by technical errors from the closest haplotype classified as reliable, called its parent haplotype, using the inferred error threshold. for example, if a haplotype has an additional deletion and substitution when compared to its parent haplotype, we require that its relative frequency be higher than the product of . × . = . to be classified as reliable (using the th percentile error frequencies in table ). we iterate through the haplotypes until classifying all the haplotypes in each group. if reads for a wt haplotype exist, the wt is also treated as a base for a group -which in this case will include all of the observed haplotypes. haplotypes may appear in more than one group, if a haplotype appears as reliable in at least one group it will be classified as reliable overall. see supplementary text for a visual summary of the algorithm we employ. the code we provide produces a file with all the variant combinations observed, the calculations described here and whether a haplotype is reliable or not. it also produces a file with the haplotypes classified as reliable, with their proportion in the population recalculated appropriately. the sequencing data created and used in this study is available in the sequencing read archive (sra, https://www. ncbi.nlm.nih.gov/sra), under bioproject prjna . the accompanying code can be found at https://github. com/sternlabtau/associvar. we set out to sequence two evolved populations of the ms coliphage. both populations were derived by fifteen serial passages performed at • c (denoted as a and b) as part of an evolve-and-resequence experiment (methods). we first performed deep sequencing of both populations at passage with the illumina miseq platform ( ) . this revealed several segregating mutations (figure ), some of which shared similar frequencies. however, due to the short-read nature of the sequencing it was impossible to infer whether these mutations co-occurred on the same genome. we next sequenced the same two populations of rna viruses from passage using oxford nanopore's minion. importantly, we employed direct rna sequencing, without using reverse transcription or pcr amplification, and without any shearing of the genomes. the only requirement for library preparation was the ligation of an adaptor to the of the rna genome, allowing the to enter the sequencing pore. each replica was sequenced independently, denoted as p a and p b. we also sequenced a sample from line a passage using both illumina miseq and minion. as this was a mostly unevolved and homogenous population, we used this as a control sample. a total of , , , and , reads were produced for the minion-p a, minion-p b and the control runs, respectively. in order to map the reads to the ms reference genome we ran our computational pipeline (materials and methods) ( ) , which infers the proportion of each point mutation (a, c, g, t or '-') at each position in the genome. over % of the reads were mapped to the reference, yet often sequencing terminated before it reached the end in both the evolved and control populations (supplementary figure s ). nevertheless, ∼ % of the reads (between ∼ , and ∼ , ) covered the entire ms genome. we next focused on the frequency of an observed variant, defined here as any base called differently from what is present in the reference sequence, at any position. we expect such variants to be the sum of two independent processes: real biological variations derived from evolutionary processes in the phage populations, and technical errors introduced by the sequencing process. comparing between the variant distributions of illumina and minion, it was evident that minion suffers from a very high technical error rate (figure ) . notably, in the control population the number of variants exceeding a frequency of % in the illumina sequencing was , whereas with minion we observed variants in positions exceeding %. this allowed us to infer that the vast majority of minion variant frequencies are technical errors, and further allowed us to roughly estimate the various types of minion error rates for our experiment (table ) . notably, we observed that the point deletion and point insertion rates together exceeded the substitution rate, reinforcing previous observations ( , ) . we attempted to use the inferred minion error rates as thresholds that can distinguish between real mutations from errors, by setting the th percentile obtained for the control sample as an error threshold for each type of error (table ). this naïve approach that is often used, quickly turned out to be invalid, as corroborated by our parallel illumina results. for example, we knew from the illumina results that only mutations in line a and mutations in line b exceeded a frequency of % at passage ( figure ). however, the minion results showed mutations in positions and mutations in positions exceeding % in both replicas, respectively. we sought a strategy to weed out the technical errors from the real mutations in the minion results independently of the illumina results. we calculated the conditional probabilities of observing one variant given another variant observed on the same read ( figure ). when observing the pattern of conditional probabilities, we noted two distinctly different patterns. some variants co-occurred more or less randomly with all other variants, manifested as more or less the same probability of observing one variant given any other variant (similar colors across a given column in figure a) . on the other hand, some variants displayed a nonrandom pattern, where the probability of observing variants together depended very much on which pair of variants was examined (different colors across any given column in figure b) . importantly, the variants that displayed a non-random pattern were variants that we knew were true mutations based on the illumina data. this led us to realize that random technical errors are expected to display a different pattern than real biological mutations: we expect technical errors to be associated randomly with any other technical error, whereas a pair or more of real biological mutations are expected to be non-randomly associated with each other. this is a reflection of evolutionary processes operating on genomes. while mutations created from replicative polymerases will be mostly randomly distributed along the genome, selection and genetic drift will lead to the fact that specific combinations of mutations reach higher frequency. thus, true mutations that are prevalent in a population will tend to be either present with some other mutations on the same genome/read, or not present with some other mutation on the same genome. both these properties (tendency to be present or not present with other mutations) reflect non-random association between mutations. one of the most commonly used methods to test for associations between two properties is the chi-square test: here we use this test to see whether the observed joint variant counts deviate from what is expected when variants are counted independently. to this end, each variant was classified as either wild-type (wt) or non-wt, based on whether it was identical or not to the reference genome. notably, this led to ( we began by inspecting all associations between all pairs of positions. this allowed us to make a few general observations. first, we observed that proximal pairs of positions (residing up to bases apart from each other) tended to be highly associated. we postulate that this is a reflection of minion errors, and also the high deletion rate, which could cause slight misalignment of reads covering positions proximal to the deletions. second, we observed a very similar pattern of associations among the three samples. this suggests that minion sequencing has a tendency towards specific a pattern of errors for a given genome that is sequenced (supplementary figure s ) . we devised a method called associvar that detects the real variants in the data, based on the following properties: (a) the method searches for the strongest non-random associations, (b) the method takes into account that pairs of proximal positions (i.e. up to bases apart) that have high associations between them are likely false positives induced by the minion machine itself, (c) in order to make the different positions comparable, the method normalizes the distribution of chi square scores per position, essentially searching for outliers from all the associations of a given po-sition, (d) the method also takes into account that because proximal positions are highly associated, a position next to a real mutation may be associated with other real mutations due to transitivity. however, we expect the two real mutations to display the highest association, i.e. we require an association to be a local maximum in a given window. finally, (e) the method uses a control sample to set a cutoff for the highest associations (materials and methods). associ-var hence calculates a normalized chi-square statistic and infers the positions where 'true' variation occurs, based on the above properties ( figure ) . after applying associvar to the data, we were able to identify five out of the six mutations appearing at a frequency above % in the illumina results in p a, and all eight positions within the p b sample (figure , supplementary table s ). notably, associvar also often correctly identified mutations segregating at lower frequencies quency lower than % according to illumina. all in all, the results indicate that our association approach has the power to resolve real variants from technical errors based on the minion data alone. we began the analysis by classifying mutations into wt and non-wt, for computational tractability. next, identifying the specific nucleotide variant in our samples after having identified the positions with real mutations is easy enough using a similar approach. every position has four possible variants (the three nucleotides different that the reference and a deletion for that base), and we test these variations against each other, again--under the assumption that the most highly associated variant for each position is the real one (materials and methods). for the positions identified by our association analysis and verified as correct by the illumina sequencing, all but one of the positions were matched with their correct variant with this method (position in p b was identified as a deletion instead of nucleotide a). the minion rna sequencing kit comes with a control sequence of the enolase ii yeast gene. we ran our association analysis on the enolase results, originally to verify that we pick up no variation in this gene. however, we were surprised to see two positions with a very high and outstanding association ( figure ) . we thus sequenced the same sample with the illumina miseq platform. reassuringly, the results verified the findings of associvar and showed that these two variants do appear in the sample and are the only two variants that ap- . notably, other high associations were ruled out as induced by proximity with the local maximum analysis (materials and methods). since we do not have a control sample for this gene, we cannot use it to infer a cutoff. however, the association between these two positions is so prominent when compared to the rest of the data that we were able to conclude they are between positions with 'true' variation (as later verified by illumina). pear there at a frequency higher than %. this suggests that our method can be used (a) as a general approach and not only for virus populations, and (b) in the absence of a control. we next tested our method on a sample of zika virus genomes ( ) . in this study, two different strains that differed at several positions had been artificially mixed and sequenced using minion. we ran associvar on one of the sequenced amplicons, in the absence of a control sequence. at least five of the six true mutations in this amplicon stood out as having highly prominent associations (supplementary figure s ). finally, we tested how our method fares in the absence of a control for our ms data, and compared the true positive rate versus false positive rate as a function of (a) thresholds set for the normalized chi-square statistic, and (b) a frequency threshold from the illumina results that determine when we define a mutation as true or false. we further compared this to the use of a 'naïve' approach where we use a varying frequency threshold for the minion results (as described above). our results show that associvar inference is consistently much more accurate that the naïve approach, and moreover, can be used even to detect mutations at a frequency of %, at the risk of some false positives ( figure ). one of the main goals of minion sequencing, in particular in the context of rna virus evolutionary experiments, is the detection of haplotypes and identification of distinct strains in the population. we thus set out to use the approach we devised to infer the composition of strains in our ms samples. notably, this is challenging on two fronts: first, our association approach can tell us which mutations in the min-ion data are real, and which pairs are associated, but it does not tell us what their frequency is (or rather, we do not trust the observed frequencies given the very high error rate). second, we are interested in inferring haplotypes, i.e. which mutations reside together regularly on the same genomes and which do not. once again, the high error rate makes this extremely tricky since we observe reads bearing almost all possible combinations of mutations. in fact, most reads bore so many variants, that ∼ - % of the bases called per read were different than the reference (supplementary figure s ). in this case, we used a two-pronged approach: we first focused only on variants inferred as true mutations using our method, associvar, as described above. second, we used the inferred error threshold to infer the probability of two or more variants residing erroneously on the same genome, utilizing an iterative approach in which we compare a given haplotype to haplotypes already classified as reliable (materials and methods). in our case, because the ms populations bore many mutations at low frequencies, we also limited the analysis to variants that appeared at a frequency of at least % in the minion sequencing results. the summary of inferred strains is shown in table and provides a few interesting insights into our populations. first, seventeen and ten different strains were identified in each of the a and b populations, respectively. second, a g and t -, both of which rose to high frequencies in both replicates, were found to be mutually exclusive in both replicates. on the other hand, g -(which we know from the illumina data to actually be g a) was found to be tightly linked to t -, in line with the very similar frequencies of these mutations in p b. all of these results were unobtainable with the illumina results alone and highlight the added value of using minion for inferring viral genotypes. we have developed here a simple and intuitive approach, associvar, to (a) detect bona fide mutations from minion population sequencing, and (b) infer the set of haplotypes (strains) present in a population. our approach is based e nucleic acids research, , vol. , no. page of figure . receiver operating characteristic (roc) of associvar versus a naïve method. performance of prediction of mutations is assessed using roc curves, where each curve is plotted as a function of the normalized chi-square statistic threshold for associvar (solid lines), or frequency threshold for the naïve method (dashed lines). the illumina results are used as the gold standard test to define a mutation as true or false, and the three different colors represent different thresholds for this definition. for example, for the blue line labeled as %, only mutations at a frequency higher than % according to illumina are defined as true. on the notion that sequencing errors will be randomly dispersed along the reads, whereas real mutations tend to associate with specific genetic backgrounds. in the case where technical error rates are high (such as occurs with min-ion), this allows one to focus on the real genetic diversity that is hidden in the vast array of technical errors generated by this method. notably, our approach is general enough so that it can be used for any type of long read sequencing. we applied associvar to sequencing data from an evolved population of phages where illumina sequencing was available, allowing us to corroborate whether mutations we found based on analysis of the minion data alone were indeed real. strikingly, all but one of the high frequency mutations observed in the p a and p b data (> %) were picked up using associvar, despite the fact that the th percentile for technical errors was as high as % (table ). in fact, despite the very high deletion rate, associvar accu-page of nucleic acids research, , vol. , no. e rately identified the one real deletion mutation present in our populations, suggesting a very high sensitivity of the method. our approach also shows high specificity, with a false positive rate lower than . %. finally, we have shown that using a naïve approach based on a frequency threshold as a cutoff to separate real mutations from errors results in extremely high false positive rates, demonstrating the value of our approach. originally, when observing the data in figure , as a first approximation it seemed likely to assume that mutations with a similar frequency would be mutations shared on the same genomes. accordingly, we had hypothesized that at least two clusters of mutations in line b (t -/g a/a g and a g/ a g/t c/g a/a g) would be present on the same genomes. this turned out to be only partially true: mutations with similar frequency were sometimes indeed on the same genomes (e.g. t -/g a), but sometimes completely not (the former two and a g) ( table ) . these results illustrate the utility of minion to resolve the relationships among mutations, and its advantage for differentiating variants with mutations displaying similar frequencies. we further used our approach to perform the reverse analysis: when analyzing the mrna of the yeast gene enolase, our analysis suggested that the mrna population sequenced was not homogenous. this was then precisely verified by illumina sequencing of the same population. remarkably, this analysis shows that (a) associvar can be used to analyze different types of data, ranging from virus genomes to mrna of any organism, and (b) associvar can be used without sequencing a control sequence. we note that this requires more caution, since our analysis of ms showed that spurious associations between mutations may be created artifactually by the sequencing process itself. use of associvar without a control sequence requires the user to specify the threshold of the normalized chi square statistic. as with all methods, the specificity of associvar comes at the cost of sensitivity, and vice versa ( figure ). nevertheless, it seems the best strategy we can suggest is to use a very high threshold, which is extremely effective for variants at a frequency higher than or %. it is important to delineate the limitations of our approach. we note that we cannot distinguish haplotypes/strains that differ from each other at one position only, because our method relies on the association between two positions containing real mutations. similarly, if two strains differ at very proximal loci, associvar will also fail, since we filter out associations between mutations that are < base pairs apart. we postulate that the presumably artifactual associations we observed between proximal loci are induced by the rna (or dna) passing through the pore of the sequencer. finally, we also noted specific patterns of mutations that were reproduced between our control sequence and the two evolved populations of ms . this suggests two possibilities: first, perhaps sequence context and/or rna secondary structure induce specific errors in minion, and second, it is possible that ms genomes undergo rna modifications and these are the cause of these specific errors. minion direct rna sequencing records the raw electric signal produced by the rna going through the pores, and this potentially offers the opportunity to identify rna modifications using a newly developed tool called tombo (version . ) by oxford nanopore (https://nanoporetech.github.io/tombo/). unfortunately, we could not conclusively determine the presence or effect of rna modifications and its relationship to associated mutations. our results suggest that tombo still suffers from a high false positive rate, while the true positive rate of the method has not yet been determined ( ) . the former was demonstrated herein by a high number of presumably modified sites found in the enolase yeast gene, despite the fact this gene was created synthetically in vitro, where modifications would not likely occur. we nevertheless analyzed our ms samples and found a similar pattern of presumable modifications among the three ms samples, yet there was no correlation between sites with a high rate of modification and sites with high normalized chi scores by associvar (see supplementary text, supplementary figures s -s ). while we cannot rule out that rna modifications are responsible for the pattern of errors in minion, we conclude that further research is required to determine which factors induce these errors. although our method is ideal for direct rna or direct dna sequencing, we also used the method for cdna that was amplified from rna in the case of the zika virus analysis ( ) (supplementary figure s ) . when we tried to reconstruct the known haplotypes present in this sample, our method did not fully succeed to recapitulate the haplotypes (data not shown). one possible explanation for this is that during the amplification step, either chimeric sequences of both strains were created, or pcr recombination occurred, breaking down some of the linkage between sites. in such cases, the use of associvar is limited to the detection of mutations only, and this further suggests that direct rna/dna sequencing may be preferable. to summarize, we anticipate that due to its ease of use and advantages listed above, direct long read sequencing using minion will be increasingly valuable in the field of virus genetics and in additional diverse fields such as transcriptome studies, cancer genetics, and microbiology. the associvar approach we suggest herein is simple and applicable to any organism, and as such we hope it will be a useful addition to the genomics toolbox in multiple fields. viral mutation rates the defective component of viral populations early minion™ nanopore single-molecule sequencing technology enables the characterization of hepatitis b virus genetic complexity in clinical samples viral phylodynamics minority hiv- drug resistance mutations are present in antiretroviral treatment-naïve populations and associate with reduced treatment efficacy evolutionary analysis of the dynamics of viral infectious disease highly accurate-single chromosomal complete genomes using iontorrent and minion sequencing of clinical pathogens antimicrobial resistance prediction and phylogenetic analysis of neisseria gonorrhoeae isolates using the oxford nanopore minion sequencer field investigation with real-time virus genetic characterisation support of a cluster of ebola virus disease cases in dubreka distinguishing low frequency mutations from rt-pcr and sequence errors in viral deep sequencing data evolution of foot-and-mouth disease virus intra-sample sequence diversity during serial transmission in bovine hosts minion nanopore sequencing identifies the position and structure of bacterial antibiotic resistance determinants in a multidrug-resistant strain of enteroaggregative escherichia coli coming of age: ten years of next-generation sequencing technologies evaluating the accuracy and sensitivity of detecting minority hiv- populations by illumina next-generation sequencing genomics and outbreaks: foot and mouth disease examining sources of error in pcr by single-molecule sequencing pcr amplification introduces errors into mononucleotide and dinucleotide repeat sequences insight into biases and sequencing errors for amplicon sequencing with the illumina miseq platform a general method to eliminate laboratory induced recombinants during massive, parallel sequencing of cdna library minimizing dna recombination during long rt-pcr dna recombination during pcr direct rna sequencing of the coding complete influenza a virus genome highly parallel direct rna sequencing on an array of nanopores the oxford nanopore minion: delivery of nanopore sequencing to the genomics community multiplex pcr method for minion and illumina sequencing of zika and other virus genomes directly from clinical samples minion nanopore sequencing of an influenza genome long-read sequencing-a powerful tool in viral transcriptome research oxford nanopore sequencing, hybrid error correction, and de novo assembly of a eukaryotic genome assessing the performance of the oxford nanopore technologies minion an amplicon-based sequencing framework for accurately measuring intrahost virus diversity using primalseq and ivar from squiggle to basepair: computational approaches for improving nanopore sequencing read accuracy genomewide patterns of substitution in adaptively evolving populations of the rna bacteriophage ms effect of deleterious mutation-accumulation on the fitness of rna bacteriophage ms accurate in vivo population sequencing uncovers drivers of within-host genetic diversity in viruses the asqc basic references in quality control: statistical techniques improved data analysis for the minion nanopore sequencer characterization of minion nanopore data for resequencing analyses direct rna nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants and enables modification analysis we thank david burstein and tzachi hagai for critical reading. supplementary data are available at nar online. key: cord- -ohdukhqt authors: patil, shital p.; goswami, ashutosh; kalia, kiran; kate, abhijeet s. title: plant-derived bioactive peptides: a treatment to cure diabetes date: - - journal: int j pept res ther doi: . /s - - -z sha: doc_id: cord_uid: ohdukhqt abstract: recent advances in analytical techniques have opened new opportunities for plant-based drug discovery in the field of peptide and proteins. enzymatic hydrolysis of plant parent proteins forms bioactive peptides which are explored in the treatment of various diseases. in this review, we will discuss the identified plant-based bioactive proteins and peptides and the in vitro, in vivo results for the treatment of diabetes. extraction, isolation, characterization and commercial utilization of plant proteins is a challenge for the pharmaceutical industry as plants contain several interfering secondary metabolites. the market of peptide drugs for the treatment of diabetes is growing at a fast rate. plant-based bioactive peptides might open up new opportunities to discover economic lead for the management of various diseases. graphic abstract: [image: see text] biomolecules play an important role in any ligand-target interaction and their recognition in any biological system starts the cascade of signalling steps, which is an important part of the normal body function. nucleic acids, carbohydrates, and lipids are the important ligands that bind and cause conformational changes in targets (receptors) but the messengers that release after binding and conformation changes are peptides. any defect in these signal transduction processes cause diseases (otvos and wade ; gautam ) . proteins, under the influence of proteolytic enzymes get fragmented at particular catalytic sites to form bioactive peptides. bioactive peptides are made up of - amino acids and lack protein-protein interaction because of their small size. they have properties like tissue affinity, specificity, and efficiency (moller et al. ) . the structure and the overall charge and hydrophobicity/hydrophilicity of the any bioactive peptide depends on the nature of amino acids, their sequences in the peptide backbone along with the n and c terminals. the relation between structure and biological activity of the bioactive peptides are yet not confirmed but the structure is an important arbitrator for their activity (li and yu ) . bioactive and nutraceutical peptides have a positive effect in the regulation of the health of an individual (kitts and weiler ) . they boost the quality of human health by preventing various ailments and by improving medical conditions related to lifestyle, metabolism, and immunity (vitetta et al. ) . peptide and protein drugs are available from the different sources for therapeutic uses, but as with many drugs, they have several advantages and disadvantages of their own. peptide drugs are effective and specific to their biological target but devoided of some of the essential qualities of a drug to withstand in the current market of therapeutics. proteins have a large chemical structure which makes their synthesis complex, tedious and expensive, the further low oral bioavailability of proteins limit their oral delivery hence they are administered through parenteral route (uhlig et al. ) . the worldwide peptide therapeutics market size valued at usd , . million in and assumes to grow at cagr of . % over the period to . ( ) . insulin is the first therapeutic peptide which has been used widely and shares good peptide market. it was first isolated by frederick banting and charles best from pancreatic islet extracts, further, j.b. collip developed the method for extraction and purification of insulin from other animal sources (quianzon and cheikh ) . eli lilly began producing insulin from the animal pancreas, later on, biosynthetic insulin was introduced in with the name humulin. novo nordisk, sanofi, and eli lilly together share . percent of the global insulin market. lantus (basal insulin) is a product of sanofi accounts for approximately . % of the total sales of top antidiabetic drug brands, in sale of lantus was usd . billion (dezzani ) . small molecules and peptide-based drug candidate belongs to the category of new chemical entities (nces) according to the food and drug administration, on the other hand, proteins used for the treatment of various diseases are classified under new biological entities (nbes). in recent years, therapeutic peptides are approved for the mitigation of various ailments mainly tumors, immunological disorders, blood disorders and metabolic disorders such as diabetes, obesity ( fig. ) (loganathan ) . the peptide drug, liraglutide (victoza) is a glucagon-like peptide- receptor (glp- r) agonist and used for the treatment of t dm has gained popularity as a top-selling drugs in recent past, which is marketed by novo nordisk (lund et al. ) . glatiramer acetate (copaxone), developed by teva is an immunomodulator and used to treat multiple sclerosis (duda et al. ) . leuprolide (lupron), developed by abbott is useful for the treatment of cancer and estrogendependent conditions that respond to hormone therapy (brito et al. ) . goserelin (zoladex) from astra zeneca is a gonadotropin-releasing hormone agonist (gnrh agonist) that suppresses the release of different sex hormones and is useful in the treatment of breast and prostate cancer (magon ) . other innovator companies include amgen, eli lilly, roche, and pfizer have peptide and protein-based drugs in different developmental stages will reach the market soon for the treatment of various complicated diseases (loganathan ) . till date, plant secondary metabolites, mainly small molecules are the established source of new drugs to treat various diseases (verpoorte ) , however, advancement in analytical technique, sophisticated purification methodology and in vitro assay system pointed out the researchers fig. uses of therapeutic peptides and proteins in various disease conditions (loganathan ) to look beyond the small molecules. identification of plant primary metabolites including proteins and peptides for the management of diseases have opened up a new horizon in the development of plant-based peptides as a drug candidate (otvos ) . this review summarizes various plant-based peptides reported for the treatment of diabetes, challenges with the development of peptide-based drugs and the future prospect of peptides as a drug. diabetes is a metabolic disorder and can hit anyone at different stages of life. though it is considered as one of the top health killers, its treatment is a huge challenge. diabetes can be characterized with symptoms of prolonged hyperglycemia, glucose intolerance, disturbance in the regulatory systems for storage and utilization of metabolic energy, including catabolism and anabolism of carbohydrate, lipids, and proteins in a diabetic individual due to lack of insulin production and impaired insulin receptor functioning or both (piero ; votey and peters ) . based on the causes and clinical survey, diabetes mellitus is categorized into four types as mentioned in table (deepthi et al. ) . ordinarily, the people below age suffer from the type diabetes, whereas type diabetes mainly occurs in adult and old age group. according to the world health organisation, approximately % of all diabetes cases are type , and the remaining % cases of diabetes worldwide are of type (baynes ) . diabetes is ruining the life of peoples worldwide. population suffering from diabetes is increasing and the imminence is expected to rise even more because of the current lifestyle issues. till the year , million people were affected by type diabetes and the number is expected to increase up to million people worldwide by (fig. ) . the data indicates that approximately % of diabetic patients will increase in the next two decades. the threat is every s a person dies with diabetes (roglic ) . from the statistics, it is clear that the population suffering from type diabetes is increasing and it requires more promising therapies. still, no promising drug is available which has the ability to completely cure type diabetes. generally, to treat type diabetes, oral hypoglycemic agents are used. the first line treatment is metformin, which is assisted with other hypoglycemic agents from different categories like thiazolidinediones, alpha-glucosidase inhibitors, sulphonylurea, dipeptidyl peptidase-iv (dpp-iv) inhibitors, sodium glucose cotransporter (sglt ) inhibitors. the risk associated with these therapies include hypoglycemia, weight gain, tiredness, diarrhea, and anemia risk, etc. over the period of time, these side effects lead to further complications for a visceral system like cardiovascular system and central nervous system (nathan et al. ). peptide drugs used for the treatment of diabetes are derived from different sources, like exendin- was initially isolated from the heloderma suspectum (gila monster) and its synthetic version had come to the market in the form of exenatide (aramadhaka et al. ) . liraglutide and semaglutide are structurally very close to glp- with improved half-life and resistant to dpp-iv mediated degradation (edmonds and price ) . nowadays, these peptides are being produced in large scale by using recombinant dna technology. the peptides which were obtained via synthesis or by using the recombinant technology are comparatively expensive than small molecules and are not affordable to most of the patients. the interest in health-promoting products from the natural origin and pharmaceutical formulations involving bioactive peptides are remarkably increasing (henninot et al. ) . numerous scientific reports have been published on bioactive peptides obtained from animal proteins. expedition by researchers in the field of bioactive peptides is of great interest as it opens the way to find economic lead from plants for the better care of diabetic patients (daliri et al. ). plentiful bioactive peptides have been reported from the animal and plant sources. most of the peptides that had bioactivity are being derived from animal products such as milk, eggs, meat, and fish. plant peptides are still under exploration and few bioactive peptides are reported from soy, wheat, etc (hartmann and meisel ) . in animals, proteins are associated with high-fat content and lead to diseases, including high blood pressure and heart diseases if consumed in the large amount. on the other hand, plant proteins neither have associated fat nor have any side effects (nehete et al. ) . previously, plant hormones were considered the only player through which cells communicate, but from the last decade as research was driven towards an understanding of plants signaling, secreted peptides, small rnas and transcription factors emerging as a new and well-defined player in the cell to the cell communication network (lindsey et al. ; van norman et al. ; murphy et al. ) . however, it is now clearly defined that signaling peptides of plant and animal origin are biosynthesized through the pathway that is evidently similar, although some aspects of the biosynthesis are still not identified. peptides secreted by plants have well recognized role in preliminary processes of development like the growth of meristem, organ shedding, cell elongation, cell multiplication, cell differentiation, geotropism and protection from the invader (ghorbani ) . the ubiquitous presences of proteins, peptides as mediator components convey to the proposals that these messenger peptides may have emerged evolutionary in microbes and were further integrated into the complicated mediator systems of complex organisms during evolution (roth et al. ) . existence of hormone-like peptides in plants including insulin-like peptide in spinacia oleracea l. (spinach) and lemna gibba g- , prolactin-like inhibitor in alfalfa, a substance with luteinizing hormone-releasing activity from leaves of avena sativa l. (oat) and somatostatin-like material in spinach support this assumption, it is speculated that the signaling peptides in plants existed from about billion years ago (collier et al. ; fukushima et al. ; leroith et al. ; morley et al. ) . plant proteins containing essential amino acids are important in the food chain to fulfill human physiological needs. the proteins from plants are derived from leaves, seeds, and fruits, out of which seeds are considered as an economical source of proteins. as compared to vegetables and fruits, seeds (legumes and cereals) contain a higher amount of protein (creighton ) . proteins in a mature seed represent - % in pisum sativum l. (pea), vicia faba l. (faba) bean and till - % in lupinus albus l. (lupin) and glycine max l. (soybean) (gueguen and cerletti ) . many peptides are reported from different plants for the treatment of diabetes as shown in tables and through various known targets such as (a) alpha-glucosidase inhibitors (b) alpha-amylase inhibitors (c) dipeptidyl peptidase-iv inhibitors (d) inhibitors of the glucose transporter system (e) insulin mimetics alpha-glucosidase inhibitors competitively inhibit small intestinal enzyme alpha-glucosidase, which converts nonabsorbable polysaccharides into absorbable monosaccharide, these effects together postpone and minimize the rise in postprandial plasma glucose level. inhibitors of this enzyme decrease the glucose toxicity (improves insulin sensitivity), decreases stress on beta cells (decreases post-meal hyperglycemia), increases glucagon like peptide- (glp- ) production hence increases insulin secretion (scheen ; mooradian and thurman ) . in general, normalization of postprandial hyperglycemia is difficult as compared to fasting hyperglycemia, inhibitors act specifically on the postprandial part of h blood glucose curve and causes a reduction in diabetes-associated hyperglycemia which is responsible for macrovascular complication in patients (mooradian and thurman ; ceriello et al. ). glycaemic control is achieved with these agents reduces glucotoxicity which increases insulin secretion from the beta cell (scheen ; salvatore and giugliano ) . all marketed alpha-glucosidase inhibitors are from natural origin, however, none of them belongs to peptide class. few research groups have reported peptides from plants having alpha-glucosidase inhibitory activity. peptides obtained from cannabis sativa l. (hemp) seeds contains hydrophobic amino acids (pro and leu), essential amino acids and branched chain amino acids in their structure and have reported to have enzyme inhibitory activity for this target (ren et al. ) . oat seed proteins hydrolysate obtained by protease (alcalase) digestion gives bioactive peptides which inhibits this enzyme. in vivo study also revealed that oat peptides, at a higher dose, showed the hypoglycaemic effect on stz-induced diabetic mice, reduces the food intake, stimulates insulin secretion, improves insulin sensitivity and elevate glycogenesis (zhang et al. ) . walnut hydrolyzed peptides (whps) from the fruit proteins of juglans mandshurica maxim. (walnut) showed anti-diabetic activity by inhibiting the enzyme. in vitro study of whps showed that peptides of molecular weight ( - kda) inhibit alpha-glucosidase with the inhibitory rate of ( . %) and they remarkably raise extracellular glucose consumption in insulin-resistant hepg cells. whereas, in vivo results showed that whps reduce . % of fasting blood glucose level by increasing insulin secretion, liver glucokinase, and glycogen level by . %, . %, and . % respectively (wang et al. ) . vigna angularis wild. (adzuki bean) proteins have reported enzyme inhibitory properties in mice model kk-ay of diabetes. the extract reduces the postprandial ( ) hook.f. in vitro and in vivo marella et al. ( ) cucurbita pepo l. in vitro (vaštag et al. ) lam. in vitro (gonzalez garza et al. ) l. in vitro arise ( ) theobroma cacao l. in vivo sarmadi et al. ( ) l. in vitro mojica et al. ( b) willd. in vitro nongonierma et al. ( ) l. in vivo and in vivo yibchok-anun et al. ( ) momordica charantia l. insulin mimetic in vivo (khanna et al. ) momordica charantia l. seed protein expressed in e.coli stimulated the phosphorylation of pdk and akt, enhanced expression of glut- , stimulated both the uptake of glucose in cells and the clearance of glucose in vitro and in vivo lo et al. ( ) hook.f. in vivo rajasekhar et al. ( ) hook.f. molina. duchesne. in vivo teugwa et al. ( ) zea mays l. enhance the secretion of glp- in vivo hira et al. ( ) glycine max l. increases the glucose uptake, enhance the expression of p-ir, p-irs , p-akt and membrane glut protein improve the insulin resistance hypoglycemic in vitro and in vivo lu et al. ( ) oryza sativa l. improve insulin resistance in vivo boonloh et al. ( ) roxb. in vivo poovitha et al. ( ) blood glucose in two sucrose challenge model i.e. normal and streptozocin-treated rats by . % and . % respectively. in vitro study of extruded adzuki bean proteins at mg/ml, concentration inhibits . % of rat intestinal alpha-glucosidase (yao et al. amylase (alpha- , -glucan- -glucanohydrolase) is an important enzyme that speeds up the hydrolysis of (alpha- , ) glycosidic linkage of carbohydrate and starch present in the diet and helps in absorption of non-absorbable polysaccharides after their conversion into absorbable monosaccharide (alagesan et al. ). inhibitors of amylase minimize the hydrolysis of (alpha- , ) glycosidic linkage and help in slow digestion of carbohydrate and thus delay the absorption of glucose and reduces the postprandial glucose level in blood (jayaraj et al. ). the pinto beans are a rich source of proteins and other nutrients but are not well explored for its therapeutic benefits. pinto bean protein fraction was digested enzymatically using protease (protamex) at ph . , and for the incubation period of -h with (e/s) ratio of : at °c, hydrolysed fraction was dialysed using molecular weight cutoffs and the fraction obtained with molecular weight < kda showed . ± . % of enzyme inhibition (ngoh and gan ) . similarly digestion with protease (bromelain) and dialysis with molecular weight cut-off < kda showed . ± . % of enzyme inhibition (oseguera-toledo et al. ) . the peptide obtained from cuminum cyminum l. (cumin) seeds, cumin seed peptide (csp ) had shown . % of alphaamylase inhibition (siow and gan, ) . in vitro study of triticum aestivum l. (wheat) albumin extract showed alphaamylase inhibition, wheat albumin restrained the peak of postprandial blood glucose levels in a dose-dependent manner. it showed the reduction in postprandial blood glucose by %, %, and % after administering . g, . g, and . g of wheat albumin respectively. in long-term administration study, . g of wheat albumin did not affect fasting blood glucose levels, while it reduces hemoglobin a c levels which reflects the metabolic control of individual suffering from diabetes (kodama et al. ) . a study revealed that different protein fractions isolated from the hordeum vulgare l. (barley) flour have been subjected to pancreatic hydrolysis and the obtained peptides have shown - % alpha-amylase inhibitory activity (alu'datt et al. ). cucurbitin protein was obtained from the cucurbita pepo l. (pumpkin) seeds, alcalase and pepsin hydrolysed fraction of it showed the enzyme inhibition (vaštag et al. ) . oligopeptides from the mulberry showed highest enzyme inhibition with ic . µg/ml. protein hydrolysates prepared from the seeds of moringa oleifera lam. by enzymes like trypsin, chymotrypsin and pepsin-trypsin for . and h. pepsin-trypsin digested fraction showed alpha-amylase inhibition and the ic was found to be . and . µg/ml for . h and h of hydrolysis respectively (gonzalez garza et al. ) . protein hydrolysate of citrullus lanatus l. (watermelon) seeds was prepared by using enzymes like pepsin, trypsin and alcalase where alcalase hydrolysate gives the highest enzyme inhibition and the ic was found to be . mg/ml (arise ) . autolysates of theobroma cacao l. fruits were prepared at ph . and . . autolysate at ph . showed highest inhibition of alpha-amylase as well as helps to release the insulin, in vivo study with autolysates showed decrease in the blood glucose level (sarmadi et al. ) . quinoa proteins were enzymatically hydrolyzed to obtain bioactive peptides, hydrolyzed fraction thus obtained showed . % inhibition of alpha-amylase at µm concentration (vilcacundo et al. ). two main gut-derived hormones glp- and glucosedependent insulinotropic polypeptide (gip), also known incretin hormones are responsible for the attainment of normoglycemia. they stimulate insulin secretion, decrease glucagon, inhibit gastric emptying, reduce food intake and appetite (drucker and nauck ) . in diabetic individuals, reduced secretion of incretin hormones have been observed. these gut hormones bind with their respective receptors and show their biological action (barnett ) . treatment of diabetes requires to restore either normal secretion or reduce the degradation of these hormones. dipeptidyl peptidase-iv (dpp-iv) is a protease enzyme that cleave the glp- hormone within minutes after its secretion and makes it inactive for biological function. thus, inhibition of dpp-iv has the potential to revert the hyperglycaemic condition. dpp-iv inhibitors block the rapid degradation of glp- and enhance the postprandial level of active glp- which further reduces the liver glucagon production and stimulate beta cells of the pancreas and increase insulin level (ahrén ) . dipeptides obtained from oryza sativa l. (rice) bran protein hydrolysates showed inhibition of dpp-iv enzyme and have ic . ± . mg/ml (hatanaka et al. ) . in another study, rice bran protein fractions have been defatted and hydrolyzed with umamizyme g and bioprase sp. the dipeptides obtained after digestion with umamizyme g showed inhibition of enzyme and the ic was found to be . ± . mg/ml (hatanaka et al. ) . the proteins extracted from amaranthus hypochondriacus l., have been subjected to simulated gastrointestinal digestion resulted into bioactive peptides. these peptides fraction have shown dose-dependent enzyme inhibition and have ic . mg/ml (velarde-salcedo et al. ) . glycine max l. (soybean) and lupinus albus l. (lupin) protein hydrolysate have been screened for the presence of bioactive peptides, and soy and lup were found to be efficient inhibitors of dpp-iv with ic values and μm respectively (lammi et al. ) . peptides obtained (cpgnk and ggglhk) from the protein digestion of common beans showed enzyme inhibitory activity and the ic (mg dw ml − ) for the pure peptides were found to be . ± . and . ± . respectively (mojica et al. b) . gastrointestinal digestion of proteins obtained from phalaris canariensis l. (canary) seed showed . % inhibition of dpp-iv (estrada-salas et al. ) . enzymatic digestion of quinoa proteins with papain showed enzyme inhibition and the ic for the hydrolysate-papain was found to be . ± . mg/ml (nongonierma et al. ) . study was performed on quinoa protein simulated duodenal digestion and the fraction (˃ kda) obtained after min of digestion showed highest dpp-iv inhibition and the ic (mg protein/ml) was found to be . ± . (vilcacundo et al. ). glucose, the metabolic fuel of any living cell, unable to cross lipid bilayer of the membrane due to its polar nature and membrane proteins (glucose transporter) associate with lipid bilayer helps glucose to travel across (abdul-ghani et al. ) . glut transporters facilitate the transport of glucose passively across the membrane along with its concentration gradient and do not require energy to operate, on the other hand sglt transporters actively transport glucose across the membrane against the concentration gradient thus these types of transporters require energy to operate. in order to receive energy for operation, sodium is transported along with its concentration or electrochemical gradient thus provide the needed amount of energy for the transportation of glucose across the membrane (musso et al. ; brown ) . in the human body, at least different glut transporters and sglt transporters exist which belongs to membrane class of proteins. excreted glucose is reabsorbed up to % in a convoluted segment of proximal tubule via high capacity, low-affinity sglt transporter while remaining % of glucose is reabsorbed in the distal segment of proximal tubule via high affinity, low capacity sglt transporter. in the case of diabetes, hyperglycemia causes the high load of glucose in order to compensate for this high load of glucose, an increase in expression of a glucose transporter gene in proximal tubule occurs (abdul-ghani et al. ) . plant peptides targeting glucose transporters are less explored. however, peptides having amino acid sequences aksplf, atnplf, feeln, and lsvsvl, isolated from the black beans have shown the ability to block the glucose transporters glut- and sglt- . the results of in vitro studies by using the caco- cell model have shown reduced glucose re-absorption by . % after h treatment of these bioactive peptides. the oral glucose tolerance test in rats has shown a . % decrease in postprandial glucose (p < . ) ( mg hydrolyzed protein isolate/kg bw) (mojica et al. a ). insulin hormone trigger several signaling events that control the metabolic fate of nutrients. insulin binds with alphasubunit of insulin receptor which causes the conformational changes in β-subunit of the insulin receptor, and these conformational changes in receptor activate the cytoplasmic tyrosine kinase domain. this activation further leads to the auto-phosphorylation which in turn trans-phosphorylate various intracellular substrate of the insulin receptor. these effects collectively control metabolic and mitogenic processes (nankar and doble, ) . tyrosine kinase domain activation by insulin mimetic, cause the auto-phosphorylation of receptor and trigger downstream signaling requires for metabolic activity of insulin. the other mechanism by which insulin mimetic (vanadate) works is by inhibiting the dephosphorylation of the insulin receptor via inhibition of tyrosine phosphatase (stapleton ) . the reports suggest that plant extracts of spinach and lemna gibba g- mimics like mammalian-insulin, which was confirmed by the radioimmunoassay. in an in vivo study on young rats indicated that the plant insulin-like material binds to insulin receptors on im- lymphocytes and stimulates glucose oxidation and lipogenesis in isolated adipocytes (collier et al. ) . vigna unguiculate l. (cowpea) containing bio-molecules have contributed in reducing the blood glucose level and enhancing the antioxidant status of patients. study on l rat skeletal muscle cells was performed where cells were treated with cowpea peptides at different doses ( . , , and ng) for h or insulin ( nm) for min. further, from the treated cells proteins were isolated for western blot analysis to check the phosphorylation of akt (a form of protein kinase b; pkb). the results of the study showed that the cowpea peptides have the ability to phosphorylated akt in the cell culture. this observation suggests that the treatment of cowpea peptides to the skeletal muscle cells can initiate the insulin signaling cascade. there is a possibility that cowpea peptides have the ability to mimic like insulin by inducing the same signaling cascade (uruakp, ) . linum usitatissimum l. (flax) seed peptides obtained from the protein hydrolysate ( kd) increases glucose uptake in l cells ). mc - - peptide fraction was obtained from momordica charantia l. showed hypoglycaemic effect in in vivo study. mc - - fraction decreases the blood glucose level by . % and . % in alloxan-induced diabetic mice with a time interval of and h respectively (yuan et al. ) . study on protein from the pulp of m. charantia l. showed increase glucose uptake in c c myocytes and t l adipocyte, the in vivo studies showed that the protein obtained from pulp helps in secretion of the insulin as well as act like insulin to lower the glucose level (yibchok-anun et al. ). polypeptide-p was isolated from the m. charantia l. acts like insulin mimetic (khanna et al. ) . another -residue insulin receptor (ir)-binding protein (mcirbp) was reported from the plant upon in vitro digestion yields mcirbp- , spanning residues - of mcirbp which increases the binding of insulin to its receptor, stimulate the phosphorylation of pdk and akt, induces the expression of glucose transporter , and stimulate both the uptake of glucose in cells and the clearance of glucose in diabetic mice (lo et al. ) . a novel protein called mcy having molecular weight kd was isolated from momordica cymbalaria l. fruit which showed hypoglycemic effect in in vivo study (rajasekhar et al. ) . the peptide fraction obtained from the soybean showed increase in glucose uptake in muscular cells, it is reported that obtained peptide fraction enhances the glucose uptake via amk activation (roblet et al. ) . protein extract of cucurbitaceae family containing seeds like telfairia occidentalis hook.f., citrullus lanatus l., lagenaria siceraria molina., and cucurbita moschata duchesne., showed a significant decrease in blood sugar level in in vivo study (teugwa et al. ) . peptide reported from the leaves of bauhinia variegata l, mimics like insulin which is evidently proved from the immunological reactivity and via in vivo study (azevedo et al. ) . zea mays l. (zein) seed protein hydrolysate increase the secretion of glp- directly in the ileum and indirectly in the duodenum in the rat and also confirmed with the help of glutag cell line study (hira et al. ). aglycine which is a bioactive peptide obtained from the soy bean increases the glucose uptake in c c cell, whereas in in vivo study on diabetic model it was observed that treatment with aglycine increases the expression of p-ir, p-irs , p-akt and membrane glut protein which results into hypoglycemia (lu et al. ) . soymorphin- also called µ-opioid obtained from soy bean decreases the blood glucose level via activation of adiponectin and pparα systems in diabetic kkay mice (yamada et al. ) . rice bran protein hydrolysate helps to improve insulin resistance and prevent metabolic diseases (boonloh et al. ) . protein extracted from the fruit pulp of momordica dioica roxb. decreases the blood glucose level in a diabetic model (poovitha et al. ) . plant scientist believe that there are more plants left unexplored which may contain insulin-like peptides or peptides which will mimic insulin (xavier-filho et al. ) . the success of any therapy and its acceptability depends on the ease of its delivery. the oral route of drug administration remains the most accepted route of drug delivery and most of the available drugs in the market are delivered through the oral route (morishita and peppas ) . an orally active drug should be stable enough to withstand the gastric microenvironment and also it should be permeable through the gastrointestinal membrane. small molecules are well tolerant to the gastric ph and permeated through the membrane whereas the large macromolecules are vulnerable to degradation in the gastrointestinal tract and are not permeable in their intact form to reach specific target successfully without being degraded in git (morishita and peppas ) . as the medical field advances, the peptide drugs are widely explored for their therapeutic potential in the treatment of various diseases. though these drugs are specific to their target, efficacious and potent, the oral bioavailability of peptide and peptide-based drugs always remains a challenge that limits their wide acceptance and market value. high cost of therapy is a major limitation associated with peptide drugs (ayoub and scheidegger ) . with the advancement of biotechnology, the recombinant synthesis of a peptide of interest opens a door for cheap and cost-effective peptides. also, the commercial interest for the production of bioactive peptides from the natural sources is elevated to reduce the cost but due to the lack of suitable technologies which helps to retain or increase the activity of bioactive peptides, the large-scale production gets hampered (craik et al. ) . another limitation associated with peptide-based drugs is their short half-life. when the peptides come in contact with intestinal mucosa, gastric acid in stomach and peptidases that are present in the blood convert peptides into amino acids and gets quickly eliminated from the body, thereby reducing its half-life. in order to achieve prolonged action and to reduce the frequent dosing, it is necessary to increase the half-life of the peptides. as peptide therapeutics advances, several non-degradable polymers and polymeric matrix are available to increase the half-life of peptides and proteins thus reducing the frequent dosing (brown ) . another strategy to increase the half-life of the peptide is to modify the amino acids which are susceptible to cleavage by enzymes (adessi and soto ) . oral delivery of the peptides is explored in past years but limits its use as peptides are degraded in the git tract (lau et al. ) . novo nordisk developed a semaglutide oral formulation which is currently in clinical trial phase . other non-invasive and suitable routes for the peptide and protein-based drugs are under investigation includes the nasal and pulmonary route which directly deliver the drugs to the blood component hence, reduces the overall drug degradation which is the main drawback associated with oral delivery of peptides (davies et al. ). different therapeutic peptides have made their way to the market in recent years, and it is assumed that the market demand will increase further as peptides are specific, selective and safe in comparison to small molecules (fosgerau and hoffmann ; loffet ) . traditional synthesis and drug design approaches are used by researchers and pharmaceutical companies for the development of the peptide-based drug candidate. the recombinant protein is another tool for the synthesis of the desired peptide in bacteria, yeast, and fungi. the market of peptides has now moved from the isolation of peptides from an animal source that can mimic the endogenous peptide to synthetic, semi-synthetic and recombinant peptides. further, development in this area is drug conjugated peptides and multifunctional peptides (fosgerau and hoffmann ) . peptides are susceptible to degradation when given orally, which is supposed to be the most convenient route of administration. the preferred route for the peptide delivery is intravenous, however, more research is directed for the delivery of peptides. recently, alternative routes are explored, which include oral, transdermal and intranasal route. as peptides are excellent biomarkers, they can also be utilized for diagnosing diseases. further, peptides are also found to be advantageous in vaccine development (brown ; sheridan ). plant peptides have been isolated and characterized in different ways as described in fig. . x-ray crystallography and nmr have contributed enormously in the field of structure elucidation of pure peptides (gomathi and subramanian fig. the general scheme of isolation, purification, and characterization of plant peptides (gomathi and subramanian ; krishnan and rupp ; sinz et al. ; wysocki et al. ) ; krishnan and rupp ) . other techniques like electron microscopy, fluorescence resonance energy transfer, chemical cross-linking emerged in recent year for the characterization of proteins and peptides (sinz et al. ) . mass spectrometry is frequently used technique in analyzing peptide mixtures and identifying their proposed structures. amino acid sequencing in mass spectrometry can be performed by two methods namely top-down sequencing and down-top sequencing. in top-down sequencing, the proteins analysis is performed without hydrolysis and the molecular weight can be detected by fragmentation pattern of the whole protein. a common approach is down-top sequencing in which proteolytic digestion is performed followed by ms analysis. software based on the algorithm of the amino acid sequence is available for protein identification (wysocki et al. ). plants remain a valuable source for developing a new drug candidate. they are still sharing their fair share of the new drug candidates and lead. as research interest is now shifted from small molecules to bio-molecules by virtue of several added benefits, plants are now under exploration for the presence of proteins and bioactive peptides for the disease management. though plant proteins and their functions have not been defined earlier, plant hormones were only considered through which cells communicate, as our understanding to these signalling pathways become clear, several secreted peptides and small rnas now known which regulates molecular recognition and thus, controls cell communication. from our diet we consume proteins which after gastrointestinal digestion get converted to bioactive peptides with more tissue affinity to specific receptor. several research publications in recent years claimed plant proteins and their bioactive peptides as a potential candidate in the treatment of various diseases specially in metabolic and life style borne diseases including cancer, diabetes and obesity. sophisticated instrument and advancement in technology leads easy isolation, purification and characterization of proteins and peptides in a short period of time, which further reduces the overall time of discovery. apparently, most of the bioactive peptides available for the treatment of diabetes have emerged from the synthetic route or derived from recombinant technology, which further adds in the overall cost of the treatment. to make peptide therapy economic there is a demand to explore plant-based biomolecules. this review discusses bioactive peptide isolated from various plant parts like leaves, fruits and seeds. the trend indicates that most of the bioactive peptides are obtained from seeds, although reports are available where bioactive peptides have been also isolated from leaves, whole fruits and pulps. it was observed in various in vitro and in vivo studies that protein hydrolysate obtained from aqueous extracts of plant are capable of inhibiting the enzymes and transporter systems responsible for the diabetes. expedition to find molecular targets and mechanism of action of plant based bioactive peptides is needed to use these bioactive peptides as a potential drug candidate. epidemiological data reveals that type diabetes requires promising therapy as the available synthetic drugs for the treatment have moderate to severe adverse effects. plant-derived bioactive peptides inhibit the enzymes like alpha-glucosidase, alpha-amylase, dipeptidyl peptidase-iv and glucose transporter systems involved in type diabetes. in vivo studies of a certain plant peptides fraction showed insulin-mimetic action in animal models. it is clear that plant-derived bioactive peptides are not explored to their full potential in comparison to other classes of natural products. the reason might be the lack of suitable instrumental techniques to identify, purify and characterize the peptides from plant source. however, the recent advancement in lcms, lc-nmr, and x-ray crystallography has bridged the gap and this would be the right time to embark on plant peptides. in future, systematic studies 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diabetic kk-ay mice slow acting protein extract from fruit pulp of momordica charantia with insulin secretagogue and insulinomimetic activities purification and characterisation of a hypoglycemic peptide from momordica charantia l. var. abbreviata ser peptides derived from oats improve insulin sensitivity the authors want to thank niper ahmedabad, under the aegis of the ministry of chemicals and fertilizers, department of pharmaceuticals for providing facilities and research fellowships. key: cord- -sbds sda authors: portran, philippe; jacquet-lagreze, matthias; schweizer, remi; fornier, william; chardonnal, laurent; pozzi, matteo; fischer, marc-olivier; fellahi, jean-luc title: improving the prognostic value of ∆pco( ) following cardiac surgery: a prospective pilot study date: - - journal: j clin monit comput doi: . /s - - - sha: doc_id: cord_uid: sbds sda conflicting results have been published on prognostic significance of central venous to arterial pco( ) difference (∆pco( )) after cardiac surgery. we compared the prognostic value of ∆pco( ) on intensive care unit (icu) admission to an original algorithm combining ∆pco( ), ero( ) and lactate to identify different risk profiles. additionally, we described the evolution of ∆pco( ) and its correlations with ero( ) and lactate during the first postoperative day (pod ). in this monocentre, prospective, and pilot study, patients undergoing conventional cardiac surgery were included. central venous and arterial blood gases were collected on icu admission and at , and h postoperatively. high ∆pco( ) (≥ mmhg) on icu admission was found to be very frequent ( % of patients). correlations between ∆pco( ) and ero( ) or lactate for pod values and variations were weak or non-existent. on icu admission, a high ∆pco( ) did not predict a prolonged icu length of stay (los). conversely, a significant increase in both icu and hospital los was observed in high-risk patients identified by the algorithm: . ( . – . ) days versus . ( . – . ) days (p = . ) and . ( . – . ) versus . ( . – . ) days (p < . ), respectively. an algorithm incorporating icu admission values of ∆pco( ), ero( ) and lactate defined a high-risk profile that predicted prolonged icu and hospital stays better than ∆pco( ) alone. tissue perfusion after cardiac surgery may become impaired due to multiple factors and in turn induce organ dysfunction, organ failure, prolonged stay in intensive care unit (icu) and in hospital and increased mortality [ ] . unfortunately, the adequacy of tissue perfusion remains difficult to assess. surrogate markers like central venous to arterial pco difference (Δpco ), oxygen extraction ratio (ero ) and lactate are used to evaluate this adequacy [ , ] . elevated arterial lactate is a commonly used marker of global anaerobic metabolism and even mild hyperlactatemia has recently found to be correlated both with microcirculatory flow abnormalities and a worse outcome [ ] [ ] [ ] . however, lactate is not a pure marker of anaerobic metabolism and non-hypoxic causes of hyperlactatemia are common in septic shock or after cardiopulmonary bypass [ ] [ ] [ ] . accordingly, additional markers of tissue perfusion have been explored. ero can be calculated on basis of oxygen arterial saturation (s a o ) and central venous saturation (s cv o ) using the following formula: ero = (s a o −s cv o )/s a o . hemodynamic monitoring guidelines recommend to monitor this ratio after cardiac surgery and a s cv o ≥ % is often considered a target for optimal hemodynamic resuscitation [ , ] . however, because of the potential extraction defect some patient might have microcirculatory impairment with a normal or supranormal s cv o . in this context Δpco has been proposed as a global marker of tissue perfusion adequacy [ ] [ ] [ ] . Δpco is a marker of the venous blood flow ability to remove the excess co produced in tissue. an impaired tissue perfusion, due to low cardiac output or microcirculatory alteration, is therefore the main determinant of an elevated Δpco [ ] . yet, the prognostic significance of Δpco after cardiac surgery remains unclear and conflicting results have been published [ , ] . moreover, limited prospectively reported data on Δpco after cardiopulmonary bypass (cpb) are available. finally, in clinical practice Δpco and lactate or ero values frequently appear contradictive, which makes the interpretation of an elevated Δpco difficult. several authors already suggested that interpreting Δpco with s cv o improve the prognostic significance of these markers [ , , ] . de backer in a recent review on hemodynamic in shock suggests an algorithm combining Δpco , s cv o and lactate [ ] . this multiparametric approach could better discriminate different cardiovascular profiles and improve our understanding of apparently contradictive patterns. still, no clinical study has yet evaluated algorithms combining these three markers following cardiac surgery. in this pilot study, we evaluate the prognostic value of Δpco at the time of icu admission and compare it to an original algorithm combining Δpco , ero and lactate to identify different risk profiles after elective conventional cardiac surgery. additionally, we describe the evolution of Δpco and its correlations with ero and lactate on the first post-operative day (pod ). all adult patients scheduled for elective cardiac surgery with cpb were eligible for this monocentric, prospective, observational study, which was approved by the local ethics committee (comité de protection des personnes, reference cpp: a -d -vol. ). according to french law and because data were collected during routine care, authorization was granted to waive written informed consent. however, verbal consent was obtained from all study participants before surgery. all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the helsinki declaration and its later amendments or comparable ethical standards. from april to june , patients who were scheduled for cardiac surgery at caen university hospital, caen, france, were eligible for participation in the study. inclusion criteria were as follows: patients > years old who were admitted to the surgical icu following elective conventional cardiac surgery (coronary artery bypass graft (cabg) and/or aortic or mitral valvular surgery) with cpb. exclusion criteria were age < years or patient under tutorship, off pump surgery, cardiac transplantation, ventricular assist device implantation and any emergency situations. the anesthesia was standardized (target-controlled infusion of propofol and remifentanil) and adjusted to obtain a bispectral index value between and . immediately after the induction of general anesthesia and tracheal intubation in the operating room, a radial artery catheter and a right jugular central venous catheter were inserted. more advanced hemodynamic monitoring was left at the anesthetist's discretion. patients were ventilated at - ml kg − of ideal body weight, positive end expiratory pressure was set to - cm h o. the ventilator was switched off during cpb. anticoagulation was obtained during cpb with an initial bolus of heparin ( ui kg − ) to maintain activated coagulation time more than s. reversion was systematically per-formed with protamine at the end of cpb. cpb was performed under normothermia and myocardial protection was achieved by intermittent cold blood cardioplegia. boluses of ephedrine and/or phenylephrine were given intraoperatively to maintain mean arterial pressure between and mm hg. the heart was defibrillated after aortic unclamping, if sinus rhythm did not resume spontaneously. after the termination of cpb, norepinephrine was used to maintain the mean arterial pressure greater than mm hg, and the trigger for transfusion of packed erythrocytes was set to a hematocrit of % in all patients and complied with routine practice at the study institution. on arrival in the icu, all pressure monitors were zeroed at the mid-axillary line upon arrival and the position of the tip of the central venous catheter in the upper part of the right atrium was verified by chest radiography. discontinuation of invasive ventilation, administration of blood products, management of hemodynamics and fluid balance, icu and hospital discharge followed institutional standards. data elements included demographic variables: age, gender, body mass index (kg/m ), euroscore (%), baseline serum creatinine value (μmol/l); intra operative data: type of surgery, cpb time, cross clamp time, use of epinephrine, norepinephrine or dobutamine, preoperative. the first blood sample was collected on admission to the icu (t ) and h (t ), h (t ) and h (t ) after. venous and arterial blood gas were drawn simultaneously from radial arterial catheter and central venous catheter. central venous and arterial lactate serum level, co partial pressure, oxygen saturation and content were obtained from the blood gases. Δpco was calculated as p cv co minus p a co . the patient's heart rate, mean arterial pressure (map), central temperature and oxygen saturation were measured simultaneously to the blood samples. the primary outcome was icu length of stay. at the icu discharge, we also collected duration of mechanical ventilation, the need for epinephrine, norepinephrine or dobutamine on pod and total postoperative chest tube drainage. secondary outcomes included sequential organ failure assessment (sofa) score on pod , acute kidney injury (aki) on first and second postoperative day (pod and pod ) and hospital length of stay [ ] . aki was defined according to the acute kidney injury network criteria as stage or higher (increase in peak postoperative serum creatinine level to > % or ≥ . μmol/l from baseline value) [ ] . hyperlactatemia was defined as an arterial lactate level above . mmol/l [ , ] . we choose a Δpco > mmhg to define an elevated Δpco and a normal ero as a level of % or lower. these cutoff values were chosen according to previous studies [ , ] . we constructed an algorithm combining Δpco , ero and lactate. this algorithm helped us identifying a low-risk profile and a high-risk profile (fig. ). in the algorithm, lactate elevation was considered a marker of global anaerobic metabolism in the presence of altered values of Δpco or ero . if Δpco and ero were both normal, lactate elevation was considered to be non-hypoxic hyperlactatemia. Δpco elevation was considered to reflect low cardiac overall, ( %) patients had high-risk profile whereas ( %) patients experienced a low-risk profile output (co) in case of high ero or microcirculatory dysfunction with impaired oxygen extraction if ero was normal. an elevated ero in presence of a normal Δpco was considered to reflect a decrease in do non-related to a low co (anemia, hypoxemia). patients with global anaerobic metabolism, non-hypoxic hyperlactatemia or microcirculatory dysfunction with impaired oxygen extraction as determined by the algorithm were considered to have a high-risk profile. the icu length of stay (los) of high-risk profile patients was then compared to the rest of the population. the number of patients included in that pilot study was fixed empirically to . all data were tested for normal distribution with the kolmogorov-smirnov test. normally distributed data were displayed as mean ± standard deviation and not normally distributed data were displayed as median with th percentile and th percentile. comparisons were performed using fisher's exact test or chi squared test for categorical data according to the distribution. independentsamples t test was used to test the differences in normally distributed variables and mann-whithney u test for not normally distributed variables. trends in the parameters over time in two groups were compared with repeated-measures anova. pairwise comparisons were corrected for multiple testing with the bonferroni procedure. pearson or spearman correlation coefficients for data normally distributed and not normally distributed, respectively, were used to evaluate the relation between two variables. we analyzed correlation between values of the whole dataset and markers pod variations. to calculate markers pod variations we identified, among the whole dataset, the successive samples of lactate, Δpco and ero measured within to h intervals in a given patient and calculated the markers variations as following: marker variation = % × (second marker value−initial marker value)/initial marker value. we used the log-rank test to compare the length of stay in icu according to the patient risk group on admission. for all tests, a two-tailed p value less than . was considered significant. statistical analyses were performed using r software (r foundation for statistical computing ). twenty-five consecutive adult patients were included in the study ( % cabg, % valvular surgery and % combined surgery). baseline characteristics and surgery-related parameters in the whole cohort of patients and in both lowand high-risk groups are shown in table . no significant difference was found between groups with the exception of the icu los. at the time of admission, % of the patients had elevated Δpco (fig. ) . on pod a Δpco ≥ mmhg was reported at least once in all patients. Δpco did not significantly decrease on pod and patients with high Δpco on admission did not have significantly different Δpco at t , t and t compared with patients with normal Δpco (fig. ) . correlations between Δpco and lactate or ero for pod values and variations are shown in table . a weak correlation was found between Δpco and ero both for pod absolute values (r = . , p < . ) and variations (r = . , p < . ). correlation between s cv o and ero was excellent (r = − . , p < . ). other correlations between markers were weak or non-significant ( table ). s cv o was not significantly different in patients with normal or elevated Δpco ( ( - ) % versus ( - ) %, respectively, p = . ). at the time of icu admission an elevated Δpco did not predict prolonged icu and hospital stays ( fig. a and b) . the algorithm combining Δpco with ero and lactate identified patients with a low-risk profile and patients with a high-risk profile at the time of admission. out of the patients with elevated Δpco upon admission, ( %) were classified as low-risk profile while ( %) were identified as high-risk profile. conversely, out of the patients with low Δpco upon admission, ( %) were identified as high-risk profile and ( %) as low-risk profile. the precise incidence of the different hemodynamic patterns is further described in fig. . temperature on icu admission was not different in patients with normal and elevated Δpco ( . ± . °c vs. . ± . °c respectively, p = . ) and between high and low risk groups ( . ± . °c vs. . ± . °c respectively, p = . ). the high-risk patient group at the time of admission had icu los twice as long as those in the low-risk patient group. (table and fig. c ) hospital length of stay was also significantly longer for patient in the high-risk group compared to the low risk group ( . ( . - . ) versus . ( . - . ) days respectively, p < . ). (figure d ) we found significantly more aki in patients of the high-risk group compared to the low risk group (( ( %) vs. ( %), respectively, p = . ) and serum creatinine on pod and pod were significantly higher ( table ). all aki were stage according to the acute kidney injury network. need for prolonged mechanical ventilation, inotropic support and sofa on pod were not significantly different between groups ( table ). the icu los was not significantly different across patients with low or elevated lactate ( . ( . - . ) days versus . ( . - . ) days, respectively, p = . ) and/or low or elevated ero ( . ( . - . ) days versus . ( . - . ) days, respectively, p = . ) at the time of admission. the main results of this prospective pilot study are as: -high Δpco (≥ mmhg) on admission and on pod of conventional cardiac surgery was found to be very frequent and did not predict an elevated Δpco at t , t or t . correlations between pod values and pod variations for Δpco and ero or lactate were weak or non-existent. -at the time of admission an elevated Δpco alone did not predict a prolonged icu stay. conversely, icu los increased by -fold in the high-risk patient group identified with the algorithm. high-risk patients also had significantly more postoperative aki and longer hospital los. limited prospectively reported data on Δpco after cpb are available. as previously reported in retrospective studies, our prospective study demonstrates that a widening in Δpco on pod after conventional elective cardiac surgery is quite frequent [ , ] . the reason why Δpco remains elevated on icu admission and on pod is unclear. an adequate venous blood flow is the main contributor of Δpco and depends on both cardiac output and tissue perfusion [ , ] . it has been demonstrated that Δpco after cardiac surgery is only poorly correlated to cardiac output or regional blood flow [ ] . it has been suggested that impaired microcirculation could be responsible for the widening in Δpco especially when it is associated with normal s cv o [ , ] . in these studies the pattern of a high Δpco with a normal s cv o was associated to further post-operative complications, impaired splanchnic function or elevated lactate. yet, it is still uncertain whether a high Δpco after cardiac surgery is related to microcirculatory hypoperfusion and further studies are needed. we found no strong correlations between Δpco and ero or lactate, which is concordant with previous studies in the settings of cardiac surgery and septic shock [ , , [ ] [ ] [ ] . this particular lack of strong correlation is not surprising, since Δpco , ero and lactate provide information on different hemodynamic mechanisms. for example, high lactate is a marker of global anaerobic metabolism whereas high Δpco indicates decreased blood flow that can occur without anaerobic metabolism [ ] . conversely, tissue hypoxia from non-ischemic cause will not be detected by Δpco . but will induce a rise in lactate value [ ] . concerning ero , its elevation is a normal adaptation mechanism that can also occur without tissue hypoxia [ ] . we did not find any significant prognostic value of an elevated Δpco at the time of admission. similarly, ero and lactate level taken alone at the time of admission did not predicted a prolonged icu stay. it is important to not misinterpret these results. we underline that the prognostic value of hyperlactatemia or abnormal ero after cardiac surgery has been demonstrated in several studies [ , , , ] . concerning Δpco , an elevation ≥ mmhg at admission has also been shown to be associated with poor prognosis in high surgical risk patients but the results are conflicting in cardiac surgery [ , , ] . in a recent retrospective study, high Δpco after cardiac surgery was not associated with a worst outcome but the authors analyzed Δpco alone [ ] . conversely, habicher et al. found that in presence of normal s cv o ≥ %, a high Δpco (Δpco ≥ mmhg) was associated to further post-operative complications [ ] . the combination of these two markers seem to better predict complications. similarly, our study suggests that an algorithm combining Δpco with ero and lactate improved prognostic signification of these markers at admission. indeed, in our study, % of patients with high Δpco at the time of admission were classified as low-risk group and % patients with low Δpco were eventually categorized in the high-risk group. consequently, we think that Δpco , ero and lactate should not be interpreted separately but together using an algorithm. our study population was at low risk according to the euroscore evaluation. however, when associating to this preoperative scoring system the Δpco , ero and lactate measures immediately at the icu admission almost half of our population was eventually classified as highrisk group. interestingly, although our study was lacking of power for prognosis evaluation, icu and hospital stays were significantly longer and patients had more acute renal failure in the high-risk group compared to the low-risk group while their euroscore did not differ significantly. we think that our algorithm-based evaluation at the time of icu admission may have led to the identification of clinically significant intraoperative complications. the design of our interpretation algorithm is quite similar to the algorithm which was recently published by de backer in a review on hemodynamic in shock [ ] . yet, some differences should be discussed. for example, de backer considers hyperlactatemia with normal ero and Δpco as a profile of high cardiac output with dysoxia (i.e. sepsis). in our algorithm we consider this pattern as non-hypoxic hyperlactatemia. indeed, in the setting of cardiac surgery, the occurrence of hyperlactatemia without evidence of inadequate oxygen delivery (do ) has been reported [ ] . although, the pathogenesis of this disorder remains unclear, according to the authors it should be considered as reflecting a type b lactic acidosis instead of an anaerobic metabolism. these patients with non-hypoxic hyperlactatemia were still considered as high risk. indeed, an increased lactate level with a normal tension difference/arteriovenous o content difference ratio (Δpco /Δconto ; another anaerobic metabolism marker) was shown to be correlated to poor prognosis in a medical icu [ ] . another difference of interpretation relates to the pattern of an increased ero with normal lactatemia and Δpco . both our algorithm and de backer's findings agree on a decrease in do but de backer associates it with dysoxia while we consider a rise in ero to be a normal adaptation mechanism [ ] . nevertheless, we regard both algorithms as useful tools for clinicians to improve comprehension of the patterns drawn by these routine markers of systemic perfusion. this study is the first to describe the incidence of the different hemodynamic profiles defined by these algorithms. there are several limitations to our study. first, the circulatory profiles defined by the algorithm were not externally validated by, for example, a measurement of cardiac output and an evaluation of microcirculation by video microscopy. further studies are needed to assess both macro and microcirculation in the suggested hemodynamic profiles. another important limitation was the small size of the study population and the moderate severity of disease within it. we used central instead of mixed-venous blood to assess ero and co derived variables; our results may have differed if a pulmonary artery catheter (pac) had been used [ ] . fig. length of stay in icu stay according to Δpco alone (a) or according to risk group (Δpco in combination with ero and lactate) (b) at the time of admission. Δpco central venous to arterial pco difference, icu intensive care unit. patients with global anaerobic metabolism, non-hypoxic hyperlactatemia or microcircula-tory dysfunction with impaired oxygen extraction as determined by the algorithm were considered to have a high-risk profile (fig. ) . patients with normal tissue perfusion or decreased oxygen delivery without anaerobic metabolism were considered to have low-risk profile however, pac is no longer used in conventional cardiac surgery. hypothermia is also a potential confounder for Δpco measurement. it may decrease cellular respiration and co generation, especially for very low temperature [ ] . nevertheless none of our patients had profound hypothermia on icu admission and normothermia was rapidly achieved for all of our patients. finally, lactate, Δpco or ero surely have different physiological kinetics, with the clearance of lactate probably slower than that of ero and co derived variables. this makes it difficult to interpret a snapshot of those markers. that being said, taking the kinetics variations of the markers into account would also be very difficult. we designed our algorithm to provide clinicians with an everyday tool for the interpretation of arterial and central venous blood gases, and it appears to correlate with a clinical reality as it predicts occurrence of post-operative aki and longer icu and hospital stays. in this original pilot study on patients who underwent standard cardiac surgery high Δpco (≥ mmhg) on admission and on pod of conventional cardiac surgery was found to be very frequent and high Δpco on admission did not predict an elevated Δpco at t , t or t . correlations between pod values and pod variations for Δpco and ero or lactate were weak or non-existent. an algorithm incorporating the icu admission values of Δpco , ero and lactate defined a high-risk profile that predicted prolonged icu and hospital stays better than Δpco alone. relation between oxygen consumption and oxygen delivery in patients after cardiac surgery blood lactate and mixed venous-arterial pco gradient as indices of poor peripheral perfusion following cardiopulmonary bypass surgery frequency, risk factors, and outcome of hyperlactatemia after cardiac surgery mildly elevated lactate levels are associated with microcirculatory flow abnormalities and increased mortality: a microsoap post hoc analysis even mild hyperlactatemia is associated with increased mortality in critically ill patients lactate/pyruvate ratio as a marker of tissue hypoxia in circulatory and septic shock type b lactic acidosis following cardiopulmonary bypass lactate is an unreliable indicator of tissue hypoxia in injury or sepsis mixed venous oxygen saturation predicts short-and long-term outcome after coronary artery bypass grafting surgery: a retrospective cohort analysis s guidelines for intensive care in cardiac surgery patients: hemodynamic monitoring and cardiocirculary system central venous-arterial pco difference identifies microcirculatory hypoperfusion in cardiac surgical patients with normal central venous oxygen saturation: a retrospective analysis central venous o saturation and venous-to-arterial co difference as complementary tools for goal-directed therapy during high-risk surgery central venous-to-arterial carbon dioxide difference as a prognostic tool in high-risk surgical patients ) difference during regional ischemic or hypoxic hypoxia high veno-arterial carbon dioxide gradient is not predictive of worst outcome after an elective cardiac surgery: a retrospective cohort study a prospective, randomized study of goal-oriented hemodynamic therapy in cardiac surgical patients detailing the cardiovascular profile in shock patients the sofa (sepsis-related organ failure assessment) score to describe organ dysfunction/failure. on behalf of the working group on sepsis-related problems of the european society of intensive care medicine acute kidney injury network: report of an initiative to improve outcomes in acute kidney injury central venous-arterial carbon dioxide difference as an indicator of cardiac index venoarterial co gradient after cardiac surgery: relation to systemic and regional perfusion and oxygen transport high central venous saturation after cardiac surgery is associated with increased organ failure and long-term mortality: an observational cross-sectional study low and "supranormal" central venous oxygen saturation and markers of tissue hypoxia in cardiac surgery patients: a prospective observational study central venous-to-arterial carbon dioxide difference: an additional target for goal-directed therapy in septic shock? combination of arterial lactate levels and venous-arterial co to arterial-venous o content difference ratio as markers of resuscitation in patients with septic shock no agreement of mixed venous and central venous saturation in sepsis, independent of sepsis origin acknowledgements the proofreading of this article was supported by the bibliothèque scientifique de l'internat de lyon and the hospices civils de lyon. conflict of interest the authors declare that they have no conflict of interest.ethical approval all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the helsinki declaration and its later amendments or comparable ethical standards.informed consent according to the french law and because data were collected during routine care, authorization was granted to waive written informed consent. however, verbal consent was obtained from all study participants before surgery. key: cord- - xgurlue authors: ozer, tugba; geiss, brian j.; henry, charles s. title: review—chemical and biological sensors for viral detection date: - - journal: j electrochem soc doi: . / . jes sha: doc_id: cord_uid: xgurlue infectious diseases commonly occur in contaminated water, food, and bodily fluids and spread rapidly, resulting in death of humans and animals worldwide. among infectious agents, viruses pose a serious threat to public health and global economy because they are often difficult to detect and their infections are hard to treat. since it is crucial to develop rapid, accurate, cost-effective, and in-situ methods for early detection viruses, a variety of sensors have been reported so far. this review provides an overview of the recent developments in electrochemical sensors and biosensors for detecting viruses and use of these sensors on environmental, clinical and food monitoring. electrochemical biosensors for determining viruses are divided into four main groups including nucleic acid-based, antibody-based, aptamer-based and antigen-based electrochemical biosensors. finally, the drawbacks and advantages of each type of sensors are identified and discussed. nucleic acids with a transducer that can detect the interaction of the analyte, and can be applied for medical diagnosis, environmental monitoring, food, water and agricultural product processing. the goal of this review is to explore recent developments in electrochemical biosensors for viruses and viral infections. the review is divided by analyte type and is intentionally not all inclusive given the large numbers of publications in this field. individual viral particles usually include either an rna or a dna genome. a dna biosensor is based on the immobilization of a single stranded oligonucleotide on a transducer surface to detect its complementary dna sequence due to surface hybridization. then, the hybrid formed on the electrode surface is transformed into an analytical signal via a transducer. electrochemical dna biosensors have drawn attention due to their advantages such as portability, simplicity, cost-effectiveness, fast response time, high sensitivity, high selectivity, and compatibility with miniaturized detection technologies. the working principle of an electrochemical dna biosensor depends on two steps including the interaction or hybridization between dna-dna, dna-rna, and protein-ligand molecules, or conversion of the changes in the dna structure or the assembly into an electrochemical signal. [ ] [ ] [ ] in addition, there are indirect techniques that use electrochemical active dna intercalators, enzymes, redox mediators and particles for the amplification of signal. , [ ] [ ] [ ] a variety of electrochemical dna biosensors have been developed for detecting different viruses. [ ] [ ] [ ] [ ] [ ] [ ] west nile virus (wnv) is a member of the flaviviridae family (genus flavivirus) that is endemic in many parts of the world and transmitted by culex mosquito vectors. wnv commonly presents with flu like symptoms but can cause severe neurological symptoms and even death in a small subset of patients. detection of active viral infection can help with disease management as well as helping detect wnv present in mosquito populations to reduce transmission. wang et al., developed a label-free capacitance-based dna capacitance sensor for the kunjin subtype of wnv using interdigitated au electrodes ( figure a ). a -nucleotide dna probe based on a west nile virus sequence was immobilized on a pre-cleaned gold coated interdigitated electrode (ide) followed by inactivation of vacant gold sites with the use of -mercapto- -undecanol (mcu). due to the hybridization of nucleic acid targets with ssdna probe oligomers on microelectrodes, the capacitance changes were measured and found as nf in response to as few as complementary dna targets ( . attogram) at a concentration of . am. capacitance changes had excellent linear behavior toward complementary target with concentration of to million target dna molecules, which enables sample analysis in a typical clinical application environment. the biosensor advantages include low detection limit and better reproducibility compared to other non-faradaic capacitive biosensors. moreover, specificity tests of the biosensor were performed. unlike non-complementary target dnas, complementary dna showed a large capacitance change in the biosensor. ebola (family filoviridae) infection can lead to fatal hemorrhagic fever, but death can be prevented with various therapies if the infection is detected in time. [ ] [ ] [ ] [ ] according to the world health organization, there are over , cases and , fatalities caused by ebola virus in guinea, liberia and sierra leone in . [ ] [ ] [ ] due to the resource-poor regions of africa where ebola outbreaks generally occur, developing inexpensive point-of-care detection devices is highly desirable. screen-printed electrodes (spes) are often used for fabrication of biosensors since they possess advantages such as disposability, mechanically robustness, inexpensiveness, stability and reproducibility for mass production. for instance, a novel electrochemical-based nucleic acid sensor was fabricated for ebola virus rna detection (figure b) . in the first step, the gold surface of screen-printed electrode was functionalized with a thiolated dna capture probe sequence via s-au bonding. then, biotinylated target strand dna was immobilized to the electrode surface for hybridization of thiolated dna single strand capture. subsequently, the streptavidin-alkaline phosphatase enzyme was exposed to the biotinylated hybrid via biotin-streptavidin conjugation bond and, covered with -aminophenyl phosphate solution. electrochemical impedance spectroscopy (eis) was used to monitor dna immobilization and differential pulse voltammetry (dpv) method was used to detect the enzymatically-produced -aminophenol. the electrical signal of the fabricated biosensor exhibits a linear relationship between nm- nm ebola virus complementary target strand with . nm limit of detection under optimum conditions. however, this method has not been applied for detecting ebola virus rna in real samples yet and would require amplification for diagnostic use. similarly, a novel label-free electrochemical method for rapid detection of hepatitis a virus (hav) was developed by manzano et al. hav is a picornavirus (family picornaviridae) that possesses a small positive strand rna genome. hav infection, which commonly occurs through a fecal oral route with patients having consumed contaminated food, causes a range of symptoms including nausea, diarrhea, and liver disfunction. the early symptoms of hav can often be mistaken for influenza infection, so positive diagnosis can help avoid inappropriate treatment with anti-influenza drugs. a screen-printed gold electrode was incubated with thiolated-ssdna probe. cyclic voltammetry (cv) was performed using the indicator, tripropylamine (tpa), to observe cv peak current that occurred because of hybridization of complementary synthetic target dna sequences on the electrode surface. the fabricated sensor demonstrated a lod of . pm and . fg/μl for the complementary ssdna and cdna sequences characteristic for hav target, respectively. the target hav cdna was also quantified by nested real-time pcr and limit of detection was found as . fg/μl. the dna electrochemical biosensor is advantageous relative to traditional and molecular methods such as pcr-based methods due to its cost and fast detection of hav. , hepatitis b virus (hbv), a circular double-stranded dna virus, infects millions of people and causes diseases such as liver cirrhosis and hepatocellular carcinoma. an estimated million people are infected with hbv globally, and almost one million people die every year due to hbv induced liver disease. , huang et al. developed an electrochemical biosensor for detection of target dna derived from the hbv genome. rolling circle amplification (rca) and molecular beacon (mb) mediated circular strand displacement (csd), which are isothermal dna amplification methods, were used for electrochemical detection of hbv dna sequences. the gold surface electrode was modified with mb. rca was employed to amplify the dpv current signal response. since methylene blue specifically binds to the guanine bases of dna molecules, it was used as the electrochemical redox probe. the biosensor demonstrated a linear dna concentration range from am to am with a . am detection limit under optimal experimental conditions. using the rca and mb mediated csd approaches, the biosensor sensitivity was improved relative to traditional methods. , various nanomaterials have been utilized in nucleic acid-based biosensors due to properties such as large surface area, high conductivity, and strong affinity toward bioreceptor probes with reactive groups such as thiols leading to high sensitivity and low limits of detection. these nanomaterials could be gold nanoparticles, carbon nanomaterials, magnetic nanoparticles or magnetic beads (mbs), silica nanoparticles, quantum dots (qds) and hybrid nanostructures. khater et al. developed a rapid, label-free impedimetric biosensor for citrus tristeza virus (ctv) detection using gold nanoparticles ( figure c ). ctv (family closteroviridae) is a filamentous positive strand rna virus that infects citrus trees (sour orange) and is transmitted through insect transmission by aphids. ctv causes stunting, stem pitting, low yields, and poor fruit quality from infected trees and is of major concern to the citrus industry. traditional methods for ctv detection use dot-blot hybridization, which is slow and labor intensive in terms of analysis time and quantitative analysis. kather et al. used gold nanoparticles (aunps) deposited on the surface of a screen-printed carbon electrode (spce). then, poly (at)-thiolated ssdna probes were covalently attached onto the aunps-modified electrode surface in the presence of mercaptohexanol (mch). here, poly (at) and mch were used to enhance sensor selectivity by preventing any non-specific binding of nucleobases. the hybridization process was investigated by eis in fe(cn ) − /fe(cn ) − red-ox system. the sensor demonstrated logarithmic behavior in the concentration range between . - μm of ctv-related dna with lod of nm and a min assay time. the biosensor was applied in leaf extracts from healthy citrus plants spiked with various target dna concentrations and detected the target dna concentration as low as nm. while real samples would likely never have this high of dna concentration, it shows method viability in a complex matrix. chikungunya virus (chikv) is a small positive strand rna virus that is transmitted by aedes species mosquitoes. chikv exploded out of the indian subcontinent in , and rapidly spread through africa, europe, and south and central america over the next two years. chikv infection can cause debilitating arthralgia disease that, while rarely fatal, can incapacitate infected individuals for weeks to months. singhal and coworkers proposed a novel paperbased dna biosensor utilizing fe o @au nanocubes for detection of chikv. some metal oxide nanomaterials such as zinc oxide (zno), iron oxide (fe o ), titanium dioxide (tio ), copper oxide (cuo) have been employed with aunps to synthesize au-metal oxide composites for attaining better electrochemical performance. [ ] [ ] [ ] [ ] to fabricate the paper-based device, screen-and wax-printing methods were used. aunps and magnetic nanoparticles (fe o ) were mixed to synthesize fe o @au nanocubes, which has ability to improve sensitivity of the sensor due to their higher surface area. the working electrode was modified with the prepared fe o @au nanocubes which is responsible for electron transfer between probe dna. after that, chikv probe dna was immobilized onto this modified electrode. cv and dpv were used to analyze the hybridization. the biosensor showed lod of . nm in a linear range between . nm- μm. the chik probe dna/ fe o @au/epad platform was also used for detection of chikv target rna in serum sample. the advantages of the biosensor include being simple to fabricate, reduced costs, and disposability of the used tests due to the use of paper. human papilloma viruses (family papillomaviridae) are small double-stranded dna viruses that are common in the human population due to sexual transmission. certain hpv types are strongly associated with cancer (hpv- , hpv- , hpv- , and hpv- ), and detection of specific hpv types can help physicians provide appropriate patient care. au nanotubes have drawn attention due to their outstanding properties like being physicochemically stable and capable of sensitively detecting complex analytes. the -d nanostructured-based templates are suitable for dna detection since they offer nanofluidic channel structures. shariati et al. reported a human papilloma virus dna biosensor with the lack of labelling. a gold nanotube decorated nanoporous polycarbonate (aunts-pc) template was created by electrodeposition and used as the working electrode. then, thiolated -mer ssdna probe was immobilized on the surface of the aunts-pc electrode. eis was used to observe the dna hybridization using external electric field bias, which helps probes and targets orientate flat on the surface. due to the presence of aunts and the electric field, this dna-based sensor displayed a wide linear detection range of . pm- μm with a very low lod of fm. the selectivity of the fabricated sensor was confirmed by mismatch, non-complementary and complementary dna oligonucleotides. influenza virus is a common human pathogen that causes a high level of morbidity and mortality. the world health organization (who) reported that influenza leads to , - , deaths annually all around the world through respiratory infections, and young children and the elderly are particularly at risk. approximately % of the population is affected by pandemics caused by influenza a, whereas influenza b and c lead to mild outbreaks. , it becomes challenging to control influenza a due to the propagation through aerosol particles, its short incubation time, and yearly mutation. , the influenza a/h n strain has resulted in more than , deaths and , hospitalizations while costing billions of dollars for health care in the united states alone. , moreover, there is concern that pathogenic h n has the potential to be used as a biological weapon, and developing sensitive and robust point-of-care diagnostics is critical for rapid response. , tran et al. fabricated carbon nanotube basedfield effect transistors (cntfets)-based dna sensor for detecting influenza virus rna. synthetic dna matching the rna sequence was used as a model. carbon nanotubes (cnts) have been employed for fabrication of electrodes because of their large surface to volume ratio and excellent electronic characteristics as well. the surface of carbon nanotubes was modified with strong nitric acid. after the pretreatment, probe dna strands were deposited to the cnt network followed by immersing in a solution of the target dna for the dna hybridization. the sensor exhibited high reproducibility and fast response time of less than a minute. they reported a selectivity and lod of pm to nm and pm, respectively. the biosensor has a promising potential for detection of influenza type a dna. recently, nanoparticle (np)carbon nanomaterials (cnms) hybrid structures have become popular owing to their physical and mechanical properties, and excellent electrical conductivity. cnts are the most widely used nanomaterials to generate au based-nanocomposites hybrid structure since they can be easily modified and have distinguished properties. au-cnt nanocomposites can be produced either by attaching au nanostructures directly to cnts or by forming some binding between au nanostructures and cnts via au-s bond or π-π stacking. , over kinds of foodborne pathogens caused million foodborne illnesses and , deaths globally with norovirus responsible for approximately % of these illnesses. , therefore, there is great interest in rapid and sensitive detection of noroviruses. lee et al. developed an electrical resistance biosensor based on multi functionalized-cnts for detection of influenza a and norovirus target dna. first, gold/magnetic iron-oxide nanoparticle (mnp)-decorated cnts (au/mnp-cnt) were synthesized and deposited on the surface of a commercial planar pt-interdigitated electrode using an external magnetic field. then, this electrode was conjugated with thiol (-sh)modified probe dnas for influenza virus and norovirus. linear sweep voltammetry was used for target dna detection. while single and fully mismatched dna sequences were used to examine the selectivity of the sensor toward influenza a virus, zika virus rna and influenza a rna were used to investigate the selectivity for norovirus against rna viruses. the au/mnp-cnt-based dna sensing platform demonstrated high selectivity against target rnas. electrical resistance of the sensing channel displayed a linear behavior with the concentration of target dna in the range from pm to nm with lod of . pm and . pm for influenza virus and norovirus, respectively. while these levels are low, they are still well above what would be required for detection without amplification. challenges with current nucleic acid biosensors.-the development of new nucleic acid biosensors that can accurately and sensitively detect specific sequences is quite encouraging, and many of the detection modalities provide potential ways to sense dna and rna. however, there several challenges remain for nucleic acid biosensors. first, the effective use of nucleic acid biosensors requires that the target nucleic acid be accessible to the biosensor surface. virus nucleic acids in patient samples (serum, saliva, urine, etc.) are generally protected from their environments inside viral particles. for the biosensor to detect the nucleic acid, the virus particles need to be disrupted in a way that releases the nucleic acid from the viral particle. disruption of virus particles can be performed via heating or chemical treatment, which adds additional sample preparation procedures into the testing paradigm needed to detect target nucleic acids. heat disruption is the gentlest way of disruption particles, although solvent conditions during heating need to be well controlled to avoid base-mediated hydrolysis of rna genomes. chemical disruption using detergents to dissolve membranes (sodium dodecyl sulfate, triton x , etc.) and chaotropic agents to unfold proteins (urea, guanidinium hydrochloride, etc.) can be effective, but can affect subsequent steps in analyte detection and must be removed. protein removal is particularly important in negative sense rna viruses, such as influenza and ebola viruses, as the genomes present in viral particles are tightly bound to nucleoproteins that must be removed before sequence-specific probes can recognize viral rnas. nucleic acid purification is essential for detecting viral genomic nucleic acids in real patient samples, but increases the time and effort required to sense nucleic acid analytes. another critical issue with nucleic acid biosensors is viral genome structure. many of the nucleic acid sensors listed above test their system using short oligonucleotide dnas that contain viral sequences. short rna or dna sequences purchased from commercial vendors are inexpensive, easy to work with, and available at high concentrations but are very different than purified viral genomes. long singlestranded viral genomes can adopt a wide range of thermodynamically stable secondary structures (including stems, loops, and pseudoknots) and these structures can strongly interfere with sequence-specific dna probes from recognizing the target nucleic acid. hybridization of dna probes with long single-stranded nucleic acids requires the target first being completely denatured to remove inhibitory secondary structure, followed by time for limited numbers of denatured target genomes to interact with surface-bound probe dnas. hybridization in this context is similar to hybridization performed in nuclease protection assays, which use long rna probes ( - bases) with a short °c denaturation step followed by - hr incubations at high temperatures ( - °c) in high ionic strength solutions to reduce secondary structure formation and achieve efficient and specific hybridization. these conditions are not required for the short oligonucleotide targets but are critical for detecting longer genomes. incorporation of rapid and efficient hybridization conditions are essential for nucleic acid biosensors to be useful at the point of care. the use of molecular crowding chemicals (such as polyethylene glycol) or applying electrophoretic potentials to biosensors to increase local concentrations of dna probes and target nucleic acids to increase hybridization rates may help decrease hybridization time and increase efficiency. double-stranded rna and dna genome structures (such as reoviruses and papilloma viruses, respectively) also pose similar issues. base-paring between both strands of dsrna and dsdna viral genomes generally precludes biosensor-bound probe molecules from binding in a sequence-specific manner, making recognition impossible. variants of traditional nucleic acids, such as peptide nucleic acids (pnas) and bridged nucleic acids (bnas), have the potential to undergo strand invasion and perform local denaturation of nucleic acids, but these tend to be relatively expensive. additionally, while it is possible to denature double-stranded nucleic acids with heat or chemical treatment, the affinity of the long single-stranded genome strands for each other is generally much higher than the affinity for short dna probes bound on biosensor surfaces. this leads to complementary strands of viral genomes preferentially re-hybridizing to each other rather than to the biosensor-bound dna, reducing the sensitivity of the biosensor. finally, the amount of viral genomes present in clinical samples is generally fairly small and in many cases much lower than the lod of reported biosensors. for example, faye et al. performed a retrospective study of viremia (viral genome copies/ml plasma) in ebola virus infected individuals and compared viral loads to disease outcomes. this study found that patients with less than genome copies/ml had a low probability of death, patients with viral titers between and had moderate probability of death, and patients with titers over had a high probability of death. viral titers over genome copies/ml were not observed in patients. genome copies/ml appears to be quite a large number, but actually only represents approximately attomoles of viral genomes per ml of plasma. biosensors that demonstrate nanomolar and picomolar lods are to orders of magnitudes less sensitive than necessary to detect ebola virus even in very highly infected patients as described above. for nucleic acid biosensors to be useful for detecting viral genomes without additional amplification, they need to be able to detect between genomes/ml ( . femtomoles) and molecules/ml ( . zeptomoles). in other words, detection limits for biosensors should be below ∼ am for the biosensor to be able to detect clinically relevant infections. each of the factors listed above can significantly degrade the ability of a nucleic acid biosensor from binding and detecting viral genomes in real-world patient samples, and often multiple of these factors make detection in real patient samples impossible. testing biosensors with short target oligonucleotides is only the first step in diagnostic development, and using more authentic genomes for later-stage testing is critical. additionally, appropriate sample preparation and nucleic acid handling is absolutely essential for any diagnostic assay to function correctly, so for dna-based biosensors to continue to evolve into useful diagnostic platforms and be used in detecting viral genomes in diagnostic settings, these issues need to be addressed early in the development process. the working principle of an electrochemical immunosensor is based on the generation of an electrical signal due to the specific interaction between antibody and antigen in the presence of an electrochemical transducer. antibodies are attached to the biosensor surface with either covalent or non-covalent bonding. biotin-streptavidin or conductive polymers (polypyrrole) are commonly used examples for immobilizing antibodies on electrodes for these applications. after the capture antibodies are immobilized on a sensor surface, a complex between the immobilized capture antibodies, the antigen, and detection antibodies are used for sandwich-type immunosensors. immobilizing antibodies on a solid surface can also improve their stability. , different immunoassay electrochemical sensing platforms have become available owing to their high affinity and sensitivity. [ ] [ ] [ ] [ ] [ ] assays targeting viral antigens.-haji-hashemi et al. presented the first label-free electrochemical immunosensor to detect ctv antigens. first, a gold electrode was modified using -mercaptoundecanoic acid (mua) and -mercaptopropionic acid (mpa). then, the terminal carboxylic groups on surface of the modified electrode were activated using n-( -dimethylaminopropyl)-nethyl carbodiimide hydrochloride (edc) and n-hydroxysuccinamide (nhs). the modified gold electrode was incubated with the antibody against the coat protein (cp) of ctv. after that, a solution of bsa was dropped on the electrode to block the non-specific sites. dpv method was used to evaluate the antibody-antigen interaction on the electrode surface in the presence of [fe(cn) ] −/ − . the detection limit was found to be . nm, which is lower than elisa and other immunoassay techniques for ctv detection. , in another example of ctv detection, a microfluidic electrochemical device (μfed) was proposed by freitas et al. the μfed included an array of eight screen-printed electrodes fabricated. poly(diallyldimethylammonium chloride solution) (pdda) was applied to electrode surface to create a positively charged film. next, the electrodes were modified with aunps resulting in formation of a pdda/aunp bilayer. monoclonal antibodies specific for the capsid protein of ctv (ab ) were covalently immobilized on aunps by eds/nhs coupling procedure. to block the non-activated carboxyl groups, bsa was used. after that, horseradish peroxidase enzyme (hrp) and polyclonal capture antibody (ab ) were conjugated to carboxyl-functionalized magnetic beads (mbs). the prepared ab /mb/hrp was used to capture cp-ctv biomarker from sample. the μfed array modified with ab was incubated with the cp/ab /mb/hrp, forming a sandwich-type immunoassay. the electrochemical detection of the magnetically captured biomarker was achieved by simultaneously applying a potential of − . v to the -immunosensors via amperometry. the detection mechanism is based on the enzymatic activity of hrp using h o and hydroquinone (hq), and shown as following: an lod of . fg ml − for the detection of ctv was obtained. the immunosensor was successfully used to detect citrus tristeza virus with the use of small volumes of healthy and infected plant samples. graphene (gr), which is a monolayer of carbon atoms formed in a honeycomb lattice, has received attention because of its high electrical conductivity, fast electrode transfer kinetics, and strong mechanical strength. graphene-based biosensors are more advantageous than conventional carbon electrodes since they have higher sensitivities, lower lods, faster response time and longer life time than traditional carbon surfaces. islam and coworkers demonstrated a smart graphene-based field-effect transistors (fets) to detect human immunodeficiency virus (hiv) biomarkers ( figure a) . hiv is a globally important virus that infects patients t-cells and can result in a severe immunodeficiency syndrome. according to world health organization recent data, . million people have been affected by hiv worldwide. according to recent studies, it is possible to decrease the transmission up to % by early antiretroviral treatment so rapid and accurate point-of-care (poc) detection of hiv infection status is critical for protecting patients. for fet fabrication, graphene was exfoliated on sio /si substrate and then electron beam lithography and thermal evaporation techniques were applied to pattern the electrodes. graphene was reacted with edc and nhs to activate its carboxylic groups on the surface. anti-p antibody for hiv was covalently conjugated by dropping on the activated graphene fet. while the specific antibody bound the p antigens, the graphene layer converted the chemical signal from antibody-antigen interactions into a measurable electrical signal. the sensor exhibited a linear response to p protein from fg/ml to μg/ml with an lod of fg/ml. the graphene-based fet immunosensor exhibited better response time and sensitivity compared to traditional methods but does require more expensive fabrication methods. graphene oxide (go) provides enhanced electron transport, mechanical, and chemical characteristics for biosensors. go contains large number of oxygen with functional groups so that it can be assembled with biomolecules on the sensor. , a screen-printed graphene oxide textile biosensor for point-of-exposure detection of influenza a virus was developed by kinnamon and coworkers. after the electrode array was screen printed using conductive silver ink on textile, the silver electrodes were patterned with a layer of go. -pyrenebutyric acid-n-hydrosuccinimide ester (panhs) as crosslinker was attached on surface of the sensor via π-π stacking. subsequently, influenza protein-specific antibodies were incubated on the textile-biosensor surface leading to an amide bond with the panhs. eis was used to detect influenza a in a biofluid analog buffer and the sensor displayed a linear range from ng/ml to μg/ml with a limit of detection of ng/ml. application to real samples was not demonstrated. lim et al. developed a sensitive, label-free electrochemical biosensor to detect the dengue fever biomarker, ns . m phage display technique was used to identify high affinity peptides for dengue virus type ns (denv ns ), which is a protein biomarker for specific detection of dengue fever. while phage peptides are not the same as antibodies, they work in many of the same ways from a binding perspective. gold electrodes were coated with the peptides and their affinity tested using cv and eis. due to its high content of basic residues (two his and arg), the r # phage peptide with amino acid sequence ehdrmhayyltr was chosen to modify the electrode surface to bind denv ns protein. the resulting lod was . μg/ml, which is below the normal level of ns in bloodstream from dengue patients ( μg/ml). however, the biosensor was not validated with real samples. particles can provide information to clinicians about the phase of the infection. traditionally, viral particle concentrations (or "titers") have been determined by culturing patient samples on permissive mammalian or insect cell lines and looking for cell death. this approach is slow and is not amenable to poc applications. the henry and geiss groups developed a microfluidic paper-based analytical device with integrated microwire au electrodes for trace detection of west nile virus particles ( figure b ). the au microwire electrodes were treated with lipoic acid for attachment of the carboxyl terminated alkane dithiol. next, the au microwires were modified with amine-peg biotin, edc, and nhs to obtain carbodiimide cross-linking for capturing antibodies. for characterization of the device, biotin-modified microwires were utilized to bind to streptavidin modified microparticles to optimize performance. west nile virus particles were then detected with the antibody-modified electrodes and the lod was found as . particles in μl of cell culture media. the assay sensitivity was verified in the presence of . × particles ml − sindbis virus particles. the detection platform has better performance characteristics when compared to current methods. avian leukosis virus (alv) is an avian retrovirus that can infect and cause cancers in poultry, and . % excess mortality in chicken flocks has been attributed to alv-j subtype in commercial broiler-breeder flocks. there is no therapeutic for alv, so detection of the virus in new animals introduced to flocks is the only way of controlling infection in naïve flocks. ning and coworkers developed a simple, sensitive electrochemical immunosensor to detect alv virus particles. a ferrocene-functionalized gold nanoparticle (fc-aunp) nanocomposite was prepared. then, β-cyclodextrin (β-cd) was covalently bound to secondary antibodies (ab ) and incubated with fc-aunp resulting in fc-aunp-β-cd-ab bioconjugates. next, perylene- , , , tetracarboxylic acid (ptca) was combined with graphene via π-π stacking interactions to overcome the limitations of graphene itself for primary antibody (ab ) attachment. while gr-ptca was utilized as the sensing platform to capture ab , fc-aunp-β-cd -ab was used the label. dpv was performed to obtain the amperometric responses of the immunosensor. the au nps improved the sensitivity of immunosensor due to their ability to bind multiple fc molecules. the immunosensor exhibited a linear current response against alv-j in the range from . to . tcid /ml with a detection limit of . tcid /ml. bhardwaj et al. developed a novel a vertical flow assay (vfa)based paper immunosensor to detect influenza h n viruses electrochemically and colorimetrically. a double pore size (dp) sample pad, a conjugate pad, and a nitrocellulose (nc) membrane strip were attached together for fabrication of the vfa sensor. wax printing was used to pattern the nc membrane strip. a gold paper electrode, carbon paste electrode and ag/agcl electrode were used as working, counter and reference electrodes, respectively. the dp sample pad included both large pores and small pores that allowed rapid detection (∼ min) via concentrated antigen-antibody complexes on the conjugate pad. in addition, small particles like viruses can pass through the sample pad, which gave the ability for virus detection in air samples. an lod of . plaque forming units (pfu)/ml and . pfu/ml (saliva) was obtained using eis. the integrated device is useful for sensitive, onsite detection of h n viruses in real samples while decreasing false results using simultaneous quantitative and qualitative analysis. han et al. proposed a nano-flow electrochemical immunosensor chip with three different sensors for simultaneous detection of h n , h n , and h n influenza viruses ( figure c ). first, su- was used to coat a bare si wafer for fabrication of master mold. then, pdms was poured onto the master mold and cured to create a microfluidic chamber. then, zno nanorods (nrs) were grown in the three sensor regions of the pdms channel via hydrothermal method and lift-off process. after the zno nrs growth, o plasma treatment was applied to adhere zno nrs to the pdms sensor. an immunosensor chip with three detection zones was achieved by photolithography. subsequently, capture antibodies of h n , h n , and h n were immobilized onto the zno nrs grown on three sensor regions of the pdms surface. finally, h n , h n , and h n antigens were incubated on sensors followed by injection of detection antibodies conjugated with horseradish peroxidase (hrp). , , , -tetramethylbenzidine (tmb) substrate was oxidized by hrp enzyme resulting in signal production. h n , h n , and h n influenza viruses were simultaneously detected in a mixture of three virus antigens using three-gold electrodes via amperometry. the limit of detection of each virus using this technique was pg/ml. real samples were not tested using the system. hushegyi et al. developed a glycan-based impedimetric biosensor for detection of influenza virus. a gold electrode was modified with self-assembled monolayer consisting of oligoethylene glycol (oeg)-cooh and oeg mixture of with a ratio of : . next, edc-nhs was used to activate the cooh to covalently link an amine-terminated glycan to the electrode surface. lectin maackia amurensis agglutinin (maa), which is a glycan binding protein, was analyzed in the presence of two nonspecific probes namely datura stramonium lectin (dsl) and human serum albumin (hsa). the glycan-based biosensor was able to sensitively detect maa using eis in the concentration range of am- . nm with an lod of am. influenza h n and h n viral particles were used to evaluate the selectivity of biosensor as well. the biosensor had an lod of viral particles in μl for h n viruses, it was capable of selectively detecting h n viruses with a sensitivity ratio of over influenza viruses h n . baek et al. fabricated a highly sensitive and selective peptide basedbiosensor for detection of human norovirus. gold screen-printed electrode (au-spe) was functionalized via au-s covalent bond by separately applying eight novel synthetic noroviral peptides. among these peptides, au electrode modified with the norobp peptide showed the best selectivity toward norovirus using eis technique with a detection limit of . copies/ml, which is -fold lower than the previously reported methods. finally, electrochemical behavior of gold electrode coated with . mg/ml norobp peptide was successfully evaluated due to the increased impedance signal with increasing concentration of norovirus extracted from oyster and showed lod of . copies/ml. challenges with antibody-based biosensors.-there are numerous challenges associated with antibody-based biosensor that can largely be divided between reagent and target. the first challenge is generation of consistent high affinity antibodies that selectively target the analyte of interest. antibodies are notoriously variable from lot to lot, meaning sensors can work with one batch but not the next. monoclonal antibodies generally have better reproducibility but bind only a single epitope giving rise to potential problems with number of binding sites. another antibody related problem is immobilization on the surface of the electrode. if care is not given to the method, a significant fraction (up to %) can be either denatured or immobilized in a manner that makes antigen binding impossible. careful consideration of immobilization techniques and blocking for non-specific adsorption is required. finally, antibodies can lack stability relative to dna/rna reagents and often require cold storage to maintain shelf life. increased temperatures, ionic concentrations, and reducing agents can inactivate antibodies on sensor surfaces and dramatically reduce the efficacy of the sensor. therefore, appropriate buffer composition and temperature control are critical considerations in the use of antibody-based sensors. with regards to the target, the most significant challenge comes from the biology itself. viruses are notorious for both mutating to change their coat proteins and for having coat proteins that are very similar. for the former, one need only consider influenza. new flu vaccines are needed each year because a new influenza variant appears almost every year. this makes creating antibodies against the mutating species very difficult. likewise, the similarity of coat proteins across virus serotypes is a significant challenge for selective antibody-based sensors. for example, there are four main dengue virus, and creating antibodies that can distinguish between these serotypes is non-trivial. careful selection and treatment of antibodies used on biosensors is critical for successful detection. aptamers are single-stranded oligonucleotides or peptides possessing high affinity toward antigens and they are derived from the sequential exponential enrichment (selex) method. compared to antibodies, aptamers have advantages including low cost, reproducibility, simple production, high specificity, stability, and low toxicity. aptamers are capable of binding to their target molecules via molecular shape complementarities, electrostatic or van der waals interactions, stacking of aromatic rings, and hydrogen bonding. aptamers have good stability and can repetitively denature and refold. aptamer-based biosensors (aptasensors) has been developed for sensitive and selective virus detection. [ ] [ ] [ ] [ ] aptasensors with high affinity and specificity aptamers have advantageous such as sensitive, selective, real-time, fast, label-free and low-cost analysis. ghanbari et al. developed an electrochemical aptasensor for sensitive detection of hepatitis c virus (hcv) core antigen ( figure a) . at first, graphene quantum dots (gqd) were synthesized according to their previous work. a glassy carbon electrode (gce) was modified with gqd and the aptamer was adsorbed to the surface of gqds due to the interactions between hydroxyl and carboxyl groups at gqds edges and amine groups of the aptamer. eis was used to detect hcv core antigen using ferricyanide/ferrocyanide as a re-dox probe. the aptasensor exhibited two linear concentration ranges - pg ml − and - pg ml − with a detection limit of . pg ml − . the aptasensor was used for detection of hcv core antigen concentration in human serum samples. the aptasensor is more advantageous than previously reported aptasensors for detection of hcv that it is easy to fabricate, rapid, cost effective and has low detection limit. wang et al. reported a label-free nanowell-based quartz crystal microbalance aptasensor for detecting h n avian influenza virus (aiv). a nanoporous gold film was prepared using metallic corrosion technique and immobilized onto a gold electrode surface. then, a mixture of mercaptohexadecanoic acid (mhda) and , hexanedithiol (hdt) were applied on the gold electrode surface to form a nanowell-based electrode. moreover, edc and nhs were used for further treatment to immobilize nh -aptamers via amide bond on the electrode surface. the immobilization of aptamers was enhanced fivefold using a nanowell-based electrode compared to the electrode without the nanowells. the aptasensor detected aiv h n as low as − hemagglutination units (haus)/ μl and had linear range − to haus/ μl. the aptasensor specificity was demonstrated with non-target aiv subtypes h n , h n , h n , and h n and was verified by detection of aiv h n in chicken tracheal swab samples. the detection time using the nanowell-based qcm aptasensor was reduced to min from hr using a label-free assay compared to other reported qcm immunosensors/aptasensors for detection of aivs. bai et al. developed an electrochemical impedance aptasensor to detect h n virus particles ( figure b ). the aptamer selective to h n was identified and used on the surface of modified gold electrode. the detection limit was . pg/μl with the higher probe density. since the affinity of the selected aptamer depended on probe density, the biosensor demonstrated response toward h n and influenza b virus. therefore, this work was not suitable for subtyping. however, bhardwaj and coworkers reported more specific label-free electrochemical aptasensor for subtyping of influenza a h n virus. a ssdna aptamer, which enables to detect a wide range of influenza a h n subtypes, was developed. indium tin oxide (ito)-coated glass strips were pretreated with nh oh, h o , and h o to generate hydroxyl groups on the ito surface. the electrode was modified with % polyethylenimine followed by aptamer adsorption. dpv was performed for detection of the h n virus using [fe(cn) ] −/ − as the redox probe between the potential − . v and + . v with a scan rate of mvs − . the lod of the aptasensor for h n viruses was . pfu ml − . the selectivity of the aptasensor was confirmed by distinguishing six strains of h n viruses from four different subtypes of influenza a viruses. the detection of h n virus using this aptasensor is faster than the standard elisa and nucleic acid-based techniques. challenges with aptamer-based biosensors.-aptasensors have the advantages of dna/rna based sensors in terms of chemical stability and the ability to be reproducibly synthesized making them attractive for targeted binding relative to antibodies. aptamers have the benefit of being relatively easy to mass produce through chemical synthesis techniques and provide reasonably high specificities and binding affinities, similar to antibodies. synthetic oligonucleotide aptamers can be easily produced with multiple different attachment chemistries (thiol, nhs, amines, carboxylates, etc.) at the ' or ' ends of the aptamer or on internal bases if desired, allowing very precise control of the orientation that the aptamer binds to the sensor surface. oligonucleotide-based aptamers have the added benefit of being able to re-fold into their active structures if the sensor surface encounters harsh conditions (increased ionic strength solutions, denaturing reagents, increased temperatures). this can allow sensors using aptamers as the affinity reagent to potentially be re-used, increasing the utility and decreasing the costs associated with the assays. therefore, aptamers possess significant advantages as sensor-coupled affinity reagents compared to protein-based affinity reagents. however, several challenges do exist with the use of aptamers that should be considered. the most significant problem with aptamers, however, is the lack of known aptamers for virus detection related sensing. the number of high affinity aptamers is very limited relative to the number of antibodies and development of new aptamers takes time and specialized knowledge limiting the rate at which new reagents are reported. another issue that should be considered with nucleic acidbased aptamers, especially rna based aptamers, is degradation by rnase enzymes. rnases are relatively ubiquitous and care should be taken to protect rna aptamers from degradation by rnases, which would reduce the sensitivity of the sensor. this can be mitigated by addition of rnase inhibitors (such ribolock) with the analyte during sensing. dna and 'o-modified aptamers tend to be less sensitive to nuclease digestion and can be used without additional nuclease inhibitors. another strategy for virus detection is to modify electrodes with antigens to detect circulating antibodies. wang et al. developed a sensitive method for detecting zika virus specific antibodies using non-faradaic capacitance sensing ( figure a ). zika virus (zikv) is a member of the flaviviridae family (genus flavivirus) that has been rapidly spreading across the globe through aedes mosquito vector and sexual transmission. zikv was declared a public health emergency of international concern in february by the world health organization (who) due to the expanded outbreaks throughout much of the americas. , zikv has been detected in blood, semen, urine, saliva and breast milk of infected patients. the biosensor consisted of a glass substrate with a polydimethylsiloxane (pdms) layer, and a two electrode system including gold and ag/agcl microwires. the gold microwires were modified with mua. the electrode surface was activated using nhs/edc followed by functionalizing with zika virus (zikv e) or chikungunya virus (chikv e) envelope antigen. the change in the total capacitance was measured due to the binding of antibody to antigen. the detection limit was approximately antibody molecules in a μl sample and the sensor was able to distinguish antibody isotypes. also, the platform was used for specific, sensitive detection of polyclonal anti-zikv antibodies available in mouse serum. the developed technique is more advantageous than other immunosensors or elisa assays because of its superior detection limit. palomar et al. developed an impedimetric biosensor to detect the dengue virus antibody ( figure b ). dengue is a major health issue due to its transmission by mosquitoes, and it is estimated that there are to million new cases every year according to who. in this work, multi-walled cnt were deposited on a glassy carbon electrode and then functionalized with pyrrole-nhs via electropolymerization. dengue virus ns glycoprotein was subsequently immobilized via amide coupling. the gce/cnt/poly (pyrrole-nhs)/dengue virus ns sensor was used to sensitively detect the antibody of dengue virus ns in pbs and bovine plasma using eis in the presence of [fe(cn) ] −/ − . the biosensor had a linear range between − and − g ml − with a detection limit of − g ml − in pbs and a linear range between − and − g ml − with a detection limit of − g ml − in plasma. mikula et al. reported an electrochemical biosensor for sensitive detection of anti-hemagglutinin antibodies. a gold electrode was modified with -mercapto- -butanol (mbt) and synthesized dipyrromethene derivatives (dpm). cu (ii) was attached to the electrode surface with a surface coverage of . ± . × − mol cm − due to affinity of dpm toward transition metal ions. the recombinant histidine-tagged hemagglutinin (ha) from the h n pdm influenza virus (his -h ha) was immobilized on the dpm-cu (ii) redox activated electrode. the change in cu (ii)/cu (i) redox current was measured via osteryoung square-wave voltammetry to detect antihemagglutinin h monoclonal antibodies against swine virus present in mice sera vaccinated with mixture of his -h ha in monomeric and oligomeric form. it was possible to detect the antibodies in sera sample diluted fold and this sensitivity was a few order of magnitude better than elisa test. challenges with antigen-based biosensors.-antigen-based sensors share many of the challenges associated with antibody-based sensors such as reagent stability and immobilization concerns. in addition, synthetic proteins or peptides are often used as antigens and may not have the correct structure for which the antibody was generated in the body, so careful validation of these synthetic peptides and antigens in non-biosensor assays is important. these factors can make creating a viable sensor difficult, but consideration of these factors at the start of development will significantly improve the probability of a successful assay being produced. electrochemical biosensors for early detection of viral infections have drawn attention due to their selectivity and sensitivity as well as wide dynamic range, simple fabrication, fast analysis and small sample volumes. this review describes the recent developments in fabrication and application of electrochemical biosensors for detection of viruses. these examples prove that electrochemical biosensors can offer advantages over conventional methods such as enzyme-linked immunosorbent assay, polymerase chain reaction or western blot due their characteristics such as simplicity, portability, miniaturization, disposability, fast response, real-time analysis. also, radioactive labels, toxic dyes, and expensive instrumentation are not necessary with electrochemical biosensors. nowadays various nanomaterials such as gold nanoparticles, carbon nanomaterials, magnetic nanoparticles or magnetic beads, quantum dots and hybrid nanostructures have been employed for fabrication of biosensors to enhance their sensitivity and stability. also, microarrays integrated with microfluidic systems usually provide low-cost fabrication and simultaneous viral detection. although electrochemical biosensors demonstrate promising features, some of the reported biosensors need to be validated in real samples. bioinspired materials for medical applications the lancet infectious diseases analytical abstracts clinical infectious diseases physica e: low-dimensional systems and nanostructures advances in natural sciences: nanoscience and nanotechnology world health organization world health organization, dengue and dengue haemorrhagic fever present work was supported by funding from the national science foundation (che- ) and the national institutes of health (nih/niaid r ai and r ai ). charles s. henry https://orcid.org/ - - - key: cord- -mky fufk authors: brauchle, m.; wildbahner, t. title: zielgruppengerechte krisenintervention – angehörige und team date: - - journal: med klin intensivmed notfmed doi: . /s - - - sha: doc_id: cord_uid: mky fufk families find themselves in an exceptional situation after the sudden death of someone close. anxiety, aggression, rage, incomprehension, and distraction are only a few feelings of the concerned people which intensive care staff must take care of. crisis intervention, developed in the middle of the last century, offers a framework with its concepts for the healthcare staff of how to work with the bereaved people during the first few hours. the basis model is a sort of counseling technique that guides nurses and physicians: bonding and urging the acceptance of the facts, providing structure and information, and securing backup support networks. professionals who offer help need a high level of empathy and compassion for their work. but it is essential to offer help only in situations where advice is possible. otherwise, physicians and nurses are at high risk to develop compassion fatigue. the right training, advanced education, and supervision are necessary, so that healthcare professionals can support people in crisis. ehemann in salzburg verstarb und fr. f. ininnsbruckintensivmedizinischbetreut wurde, konnte sie sich von ihm nicht verabschieden. es war aufgrund der verletzungen auch nicht möglich, fr. f. im bett zu ihrer -jährigen tochter zu fahren. ein hohes maß an flexibilität und teamgeist ermöglichten es, den leichnam der tochter auf die station zu bringen, damit sich fr. f. und ihr sohn von dem verunglückten mädchen verabschieden konnten. das berühren des bereits erkalteten körpers machten den tod für fr. f. greifbar, wohingegen das ableben ihres mannes bis heute mit unverständnis verbunden ist [ ] . am beispiel der familie f. wird deutlich, wie wichtig verabschiedungsrituale sind, um den tod eines nahen angehörigen verarbeiten zu können. und welches maß an schmerz über jahre bleibt, wenn diese möglichkeit nicht gegeben war. ziel dieses leitthemenbeitrags ist es, das ba-sis-modell der ki vorzustellen, das einen rahmen vorgibt, wie angehörige speziell auf intensivstationen begleitet werden können, wenn eine nahestehende person verstorben ist. ki professionelle angehörigenarbeit ist belastend und erfolgt überwiegend durch faktoren, nämlich gefühlsansteckung, empathie und mitgefühl [ ] : bei der gefühlsansteckung ergreift die stimmung des anderen vom beobachter selbst besitz und wird dabei zu dessen eigenstem gefühl. dies kann nicht willentlich kontrolliert werden, da gefühlsansteckung ein angeborener prozess ist, der bereits im kleinkindalter beobachtet werden kann [ ] . empathie erfolgt entwicklungsmäßig erst später und ist ein erkenntnisvermittelnder prozess. empathie beinhaltet die erfahrung, die gefühlslage des anderen nachzuempfinden und sie dadurch zu verstehen. dabei muss im gegensatz zur gefühlsansteckung nicht zwingend eine identifikation mit der anderen person erfolgen. empathie bedeutet somit auch, die belastenden negativen gefühle der gefühlsansteckung und des mitgefühls zu regulieren, damit intensivpersonal von diesen negativen gefühlen nicht überschwemmt wird. mitgefühl wiederum ist ein phänomen, das von der empathie insofern abzugrenzen ist, als mitgefühl zusätzlich die sorge um die andere person oder die anteilnahme an deren situation miteinschließt. gefühle sind ansteckend und können auch beim intensivpersonal als enorm belastend erlebt werden. die psychologie spricht in diesem zusammenhang von gefühlsansteckung. dabei wird davon ausgegangen, dass bei einer person, die eine andere person beobachtet, auch jene emotionalen reaktionen auftreten, die den tatsächlichen oder antizipierten reaktionen der beobachteten person entsprechen. die Übernahme der gefühle von anderen personen erfolgt unbemerkt, ist deshalb nicht steuerbar und kann als basisprozess empathischen verhaltens betrachtet werden. von besonderer bedeutung ist dies für das intensivpersonal, das sich mit der angst, dem entsetzen, der hilflosigkeit, dem erfahrenen leid und der trauer von patienten, angehörigen oder hinterbliebenen auseinandersetzen muss [ ] . krisenintervention · team · empathie · intensivstation · posttraumatische reifung · basis-modell families find themselves in an exceptional situation after the sudden death of someone close. anxiety, aggression, rage, incomprehension, and distraction are only a few feelings of the concerned people which intensive care staff must take care of. crisis intervention, developed in the middle of the last century, offers a framework with its concepts for the healthcare staff of how to work with the bereaved people during the first few hours. the basis model is a sort of counseling technique that guides nurses and physicians: bonding and urging the acceptance of the facts, providing structure and information, and securing backup support networks. professionals who offer help need a high level of empathy and compassion for their work. but it is essential to offer help only in situations where advice is possible. otherwise, physicians and nurses are at high risk to develop compassion fatigue. the right training, advanced education, and supervision are necessary, so that healthcare professionals can support people in crisis. crisis intervention · team · empathy · intensive care unit · posttraumatic growth · basis model in der vergangenheitsform gesprochen wird [ ] . beispiel. wollen sie mir von ihrem angehörigen erzählen? wie war er so? wann haben sie ihn das letzte mal gesehen? angehörige fragen immer wieder nach, ob es denn sein kann, dass der patient tatsächlich verstorben ist. solche fragen sind ehrlich, mit einem "ja, er ist tot" zu beantworten. angehörige fragen auch nach, wann die situation erträglicher oder leichter wird, oder wann der schmerz aufhört. dazu kann gesagt werden, dass das erste jahr ver-mutlich das schwerste sein wird. alles im jahreslauf, ob geburtstage, feiertage, weihnachten, ostern, jahrestage u. ä., wird ohne den geliebten angehörigen erlebt und niemand weiß im vorfeld, wie sich das anfühlt. sind jedoch die feierlichkeiten einmal ohne den geliebten menschen durchlebt, kennen die betroffenen das gefühl und lernen, damit umzugehen. nicht umsonst gab es früher ein offizielles trauerjahr. auch eine wichtige maßnahme um die anerkennung des tods zu fördern ist es, eine verabschiedung zu ermöglichen. wo (auf der station oder beim bestatter) angehörige fragen auch immer wieder, ob sie nicht etwas "zur beruhigung" haben können. es gilt zu vermeiden, angehörigen diverse tranquilizer zu geben. dies lässt angehörige meist handlungsunfähig werden und führt nur zu einer verzögerten trauerreaktion. im nachhinein wird dieses "nichts-tun-können" als sehr belastend empfunden. beispiel. "ich war so unruhig . . . da hat mir der hausarzt etwas zur beruhigung gespritzt . . . danach war ich wie in watte gepackt . . . ich konnte nichts tun, außer auf der couch zu sitzen und zuzuschauen, wie die anderen familienangehörigen das begräbnis planten. im nachhinein war ich sehr traurig, dass ich nicht fähig war, aktiv die beerdigung meines mannes mitzugestalten." Über das leben, den tod und das ewige band der liebe symptomatology and management of acute grief. die entdeckung der katastrophe als betätigungsfeld -kritische betrachtungen über helfer als helden am beispiel der lawinenkatastrophe von galtür. in: hermanutz m, buchmann k-e (hrsg) texte der fachhochschule krisenintervention auf intensivstationen persistierende dissoziation als prädiktorposttraumatischerbelastungsstörungen bei psychosozialen fachkräften spiegelbild und empathie. die anfänge der sozialen kognition hrsg) krisenkompass. orientierung für den umgang mit schweren krisen im kontext schule. schulverlag blmv achtung, ansteckungsgefahr! emotionale belastungen von pflegekräften das gletscherbahnunglück von kaprun in Österreich im jahr : maladaptive copingstrategien, intrusionen und posttraumatische a preliminary analysis of compassion satisfaction and compassion fatigue with considerations for nursing unit specialization and demographic factors posttraumatische belastungsstörung bei angehörigen von verstorbenen the relationship between resiliency and posttraumatic growth following the death of someone close posttraumatische reifung posttraumatic growth: conceptual foundations and empirical evidence ki oder die professionelle hilfestellung von Ärzten und pflegepersonal ist keine therapie, sondern versteht sich vielmehr als emotionale erste hilfe. ziel ist es, das soziale netz der angehörigen wieder zu reaktivieren, sodass sie sich gegenseitig unterstützen und auch miteinander über das geschehene sprechen können.in vielen fällen kann die unterstützung durch das personal sehr praktisch orientiert sein: key: cord- -h omskec authors: uber, amanda m.; sutherland, scott m. title: acute kidney injury in hospitalized children: consequences and outcomes date: - - journal: pediatr nephrol doi: . /s - - - sha: doc_id: cord_uid: h omskec over the past decade, the nephrology and critical care communities have adopted a consensus approach to diagnosing acute kidney injury (aki) and, as a result, we have seen transformative changes in our understanding of pediatric aki epidemiology. the data regarding outcomes among neonates and children who develop aki have become far more robust and aki has been clearly linked with an increased need for mechanical ventilation, longer inpatient stays, and higher mortality. though aki was historically thought to be self-limited, we now know that renal recovery is far from universal, particularly when aki is severe; the absence of recovery from aki also carries longitudinal prognostic implications. aki survivors, especially those without full recovery, are at risk for chronic renal sequelae including proteinuria, hypertension, and chronic kidney disease. this review comprehensively describes aki-related outcomes across the entire pediatric age spectrum, using the most rigorous studies to identify the independent effects of aki events. acute kidney injury (aki) describes a phenomenon marked by a rapid decline in renal function, reduced elimination of waste products, dysregulated electrolyte and acid-base balance, and impaired fluid homeostasis [ ] . it has become a common complication among hospitalized children and we are fortunate that recent advances in the diagnostic approach have improved our understanding of pediatric aki epidemiology [ ] [ ] [ ] [ ] . acute kidney injury is now clearly associated with poorer early outcomes and those who develop aki frequently experience adverse consequences while hospitalized ( fig. ) [ , [ ] [ ] [ ] . additionally, a connection between aki and long-term sequelae has been established (fig. ) ; persistent renal morbidity and chronic kidney dysfunction are highly prevalent among aki survivors [ ] [ ] [ ] [ ] [ ] [ ] [ ] . our epidemiologic progress has extended to neonatal aki as well which is significant given the unique diagnostic challenges in this population. we know that a neonate's serum creatinine at birth is a reflection of maternal renal function and, depending on gestational age, can take days or weeks to achieve true steady state. additionally, neonates have impaired urinary concentrating ability which can mask oliguria [ ] [ ] [ ] [ ] . however, the last few years have seen tremendous advances in our ability to accurately diagnose and study neonatal aki. in this context, the goal of our review is to comprehensively describe aki-related outcomes across the entire pediatric age spectrum while focusing on studies which have employed a standard, consensus aki definition. when reviewing aki outcome studies, it is essential to interpret the data within the context of several general concepts. understanding these concepts allows one to more effectively compare analyses and extrapolate findings. first, it is important to identify how aki is diagnosed; this has a significant impact on the type of aki events identified and the resultant outcomes. historically, the absence of a standard aki definition hampered our ability to understand aki epidemiology and fully explore aki-related outcomes. in , the acute dialysis quality initiative (adqi) group created the risk, injury, failure, loss, esrd (rifle) criteria, establishing the first consensus approach to aki identification and ushering in the modern era of aki outcomes research [ ] . since then, however, several iterations of rifle have been developed and employed; these include the acute kidney injury network (akin), the pediatric risk, injury, failure, loss, esrd (prifle), and the kidney disease: improving global outcomes (kdigo) classification systems [ ] [ ] [ ] [ ] . although all these definitions have the same foundation, the subtle differences which exist between them can lead to significantly different epidemiologic findings. for example, prifle has been described as more sensitive and akin as more specific, suggesting that the severity of aki identified by the two definitions will be less and more severe, respectively [ ] [ ] [ ] ] . indeed, when the two definitions were applied to the same population, the mortality risk of stage akin defined aki (or . , th ci . - . ) was significantly higher than stage prifle defined aki (or . , th ci = - . ) [ ] . given that kdigo is the most current evolutionary form of the consensus approach, we hope that studies will move towards employing it preferentially as this will allow outcomes to be compared between adults and children, across diseases, and within different patient populations. second, outcomes among children with aki are dependent on the population examined. as an example, one study examined mortality rates among children with stage aki who were and were not receiving critical care; patients who developed aki while in the intensive care unit (icu) had an increased risk of death (likelihood ratio . , th ci . - . ) whereas those on acute care wards did not (likelihood ratio . , th ci . - . ) [ ] . third, aki outcomes tend to be associated with severity of illness. for example, among neonates who experienced encephalopathy, aki was associated with increased length of stay [ ] . however, that effect was mitigated when severity of illness parameters was included in the model, and it is common for those who are most ill to have the poorest aki-related outcomes [ , ] . indeed, one of the most important aspects of aki outcomes research is determining the independent effect attributable to aki itself. the best available data demonstrate that in hospitalized children, aki is associated with increased need for mechanical ventilatory support, longer hospital and icu stays, and higher mortality (fig. ) . despite the aforementioned relationship between disease complexity, severity of illness, and aki, multivariate analysis continues to find that aki is associated with poorer in-hospital outcomes, even after adjusting for disease outcomes among children who develop acute kidney injury (aki). children who develop aki while hospitalized are at risk for poorer short-and mid/long-term outcomes. across both acute and critical care populations, aki is associated with longer lengths of stay, non-recovery of baseline renal function, and chronic renal disease including proteinuria, hypertension, and chronic kidney disease (ckd). children receiving critical care who develop aki are more likely to require prolonged mechanical ventilation support and experienced higher mortality. egfr estimated glomerular filtration rate, los length of stay severity and confounders. this is true not only in children and adolescents, but neonates as well; the independent effects of aki on outcomes have been found across the entire pediatric age spectrum. across nearly all critically ill populations, aki has been associated with an increased need for mechanical ventilation as well as longer a longer duration of support [ , , [ ] [ ] [ ] [ ] [ ] [ ] . for example, the development of aki was associated with . additional days of ventilator support (p < . ) across a general critical care population; notably, those with kdigo stage aki received mechanical ventilation for . additional days (p < . ), suggesting a dose dependent effect [ ] . within a similar population, a single center study found that the development of aki more than doubled the length of mechanical ventilation ( . vs. . days, p < . ) [ ] . the multicenter, prospective assessment of worldwide aki, renal angina, and epidemiology (aware) study, which is the largest such study performed to date, was able to demonstrate a stepwise increase in mechanical ventilation use which correlated with aki severity (table ) ; patients with stage , stage , and stage aki required mechanical ventilation . %, . %, and . % of the time, respectively (vs. no aki at . %) [ ] . the same effect has been seen in neonates; selewski and colleagues demonstrated that neonates who develop aki after experiencing perinatal asphyxia require mechanical ventilation for more days (p < . ) than those who do not develop aki [ ] . aki has also been linked with longer hospital and icu lengths of stay (los). in the original prifle (pediatric rifle or pediatric risk, injury, failure, loss, esrd) study, patients who experienced aki had longer hospital los ( . ± . days vs. . ± . days, p = . ) than those without aki [ ] . this finding is highly relevant since, as previously discussed, prifle is a more sensitive aki definition which identifies milder aki events, underscoring the strength of the association [ , ] . aware corroborated this finding as patients with aki (increase of . - . days, p < . ) and severe aki (increase of . - days, p < . ) had longer icu los even after adjusting for severity of illness (table ) [ ] . this association is also seen in neonates across all gestational ages. two single center reports found that aki increased hospital los among neonates by . days (p = . ) and . days ( th ci . - . ), respectively [ , ] . the first study was performed in term infants experiencing asphyxia (mean gestational age ± . weeks, mean birth weight ± g) [ ] . the second study, which found the larger impact on los, examined aki in very low birth weight (vlbw) neonates (mean gestational age . ± weeks, mean birth weight ± g) [ ] . these two studies not only demonstrate the significance of aki among newborns, they also highlight the influence that the patient population and severity of illness can have on aki related outcomes. importantly, the association between aki and los is evident even in studies focused on non-critically ill children. for example, zappitelli and colleagues examined children receiving acute care who were administered aminoglycosides [ ] . even after adjusting for confounders, children with aki had longer los. this effect was more pronounced for children who experienced more severe aki (stage and ), which again suggests a dose-dependent effect. rheault et al. investigated aki in children admitted with nephrotic syndrome [ ] . they too found that aki was independently associated with prolonged hospitalization. thus, the association between aki and longer los can be found across all pediatric age groups and in both intensive and non-intensive care settings. [ ] . chawla et al. found that aki events were associated with a mortality rate more than double that of myocardial infarctions [ ] . in children, aki has also been independently associated with reduced survival. in a two-center, retrospective study, alkandari et al. found that children admitted to the intensive care unit who developed aki were - times more likely to die than those who did not; those with severe aki (kdigo stage / ) experienced mortality rates that were - times higher, even after adjusting for severity of illness and intergroup differences. [ ] . these findings were corroborated by aware, the aforementioned multicenter, prospective study, which found that kdigo stage / aki was associated with higher mortality even after adjusting for covariates (table ) [ ] . these findings have been extended to neonates by a number of analyses [ , , , [ ] [ ] [ ] . for example, in vlbw infants, aki was associated with a nearly two-fold increased risk of death after adjusting for confounders [ ] ; similar results have been found in term and near term infants [ ] . perhaps, the best illustration of the mortality impact of aki among neonates is the assessment of worldwide acute kidney injury epidemiology in neonates (awaken) study which evaluated the incidence of and outcomes following aki across newborns from pediatric institutions (table ) [ ] . after adjusting for relevant confounders and intergroup differences, aki was associated with a . -fold increased risk for death ( th ci . - . , p < . ) [ ] . it is important to note that in children, the impact of aki on morality has been confined to critically ill patients. in one representative study, aki was significantly associated with higher mortality in the pediatric icu; however, this was not the case in patients receiving acute care (fig. ) [ ] . while the best available data are not yet able to demonstrate a causative effect of aki on survival, it certainly demonstrates that critically ill children who develop aki experience higher mortality independent of underlying disease severity. the data available to date demonstrate that aki is associated with significant adverse short-term consequences. more recently, however, data regarding mid-and long-term outcomes have begun to emerge. we now know that chronic renal dysfunction is common following aki events and proteinuria, hypertension, and chronic kidney disease (ckd) are more highly prevalent among aki survivors than in the general population (fig. ) . moreover, renal recovery, or the lack thereof, seems to play an important prognostic role in children who develop aki in the hospital. renal recovery is a newer concept which remains inconsistently defined, and over the past to years, studies have used different approaches to characterize recovery [ ] [ ] [ ] . indeed, renal recovery has been described as a post-aki nadir serum creatinine of less than mg/dl, a nadir serum creatinine that is less than . x, . x, or . x baseline, a creatinine that is within . mg/dl of baseline, no longer requiring renal replacement therapy (rrt), or no longer meeting any aki criteria [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this has created heterogeneity among the data and complicated our interpretation; however, the data which are available clearly demonstrate the prognostic importance of this outcome concept. given the paucity of data regarding renal recovery in children, it is helpful to examine the relevant adult findings. one french multicenter study found that among , adults with dialysis requiring aki, renal recovery occurred . % of the time [ ]. this is a relatively high rate of recovery, likely reflecting the fact that they defined recovery as no longer needing rrt. long et al. evaluated , adults who experienced aki while receiving either acute or critical care [ ] . overall renal recovery, when defined as a serum creatinine < . x baseline (no longer meeting stage aki criteria), was %. not surprisingly, the study found a dose-dependent effect and recovery was less common as aki severity increased; renal recovery occurred in %, %, and % of adults with stage , stage , and stage aki, respectively. finally, a study of adults undergoing transcatheter aortic valve replacement found that renal recovery, or a lack thereof, carries prognostic implications. of those with aki, % experienced full renal recovery, defined as no longer meeting any stage aki criteria at discharge [ ] . while those with full recovery experienced greater -year mortality (adjusted hr . , th ci . - . ) than those without aki at all, patients with only partial recovery (discharge creatinine > . x baseline but not requiring rrt) experienced even higher -year mortality (adjusted hr . , th ci . - . ). not surprisingly, those who continued to require rrt at discharge had the highest -year mortality risk of all (hr . , rth ci . - . ). pediatric data, though not as extensive, are consistent with the adult findings described above. for example, basu et al. described the post aki course of children who developed aki and required rrt [ ] . when defined as non-dialysis dependence month after aki, renal recovery occurred in % of patients. however, only % of patients had an estimated glomerular filtration rate (egfr) > ml/min/ . m by that time, suggesting that even though many patients may not need rrt indefinitely following aki, a far smaller proportion experience complete recovery. hessey et al. examined pediatric icu admissions and found that the likelihood of recovery was highly dependent on how it was defined [ ] . for example, . % of patients with aki recovered function when it was defined as a discharge creatinine < . x baseline, but recovery only occurred in . % when it was defined as discharge creatinine < . x baseline. hollander et al. examined children who underwent cardiac transplantation and found that % experienced renal recovery (creatinine < . x baseline months after the aki event) [ ] . notably, recovery was more common in the setting of mild aki (stage recovery % vs. stage / recovery %, p < . ), echoing the dose-dependent effect of the adult findings described above. perhaps most interestingly, this study found that non-recovery was a risk factor for the subsequent development of ckd. while none of the patients who survived aki and recovered developed ckd, % of those who did not recover from aki developed ckd (defined as egfr < ml/min/ . m , p = . ). the concept of renal recovery is relatively new and few adult studies and fewer pediatric studies have been performed. in fact, no studies examining recovery in neonates exist at all. however, the data we do have clearly underscore the prognostic relevance of renal recovery and the importance of standardizing the definition. the association between aki and long-term renal sequelae in children has been described for over a decade. one of the earliest studies performed in hospitalized children examined patients with aki (based upon diagnostic coding) and found that proteinuria ( %), hypertension ( %), and an estimated gfr of less than ml/min/ . m ( . %) were common following aki [ ] . buysse and colleagues studied children who developed aki (defined as creatinine x normal value) in the setting of septic shock and found nearly identical rates of hypertension and proteinuria years after the aki event [ ] . hingorani et al. found that aki (defined as a serum creatinine x baseline) increased the risk for ckd (egfr < ml/min/ . m ) by % in children undergoing stem cell transplantation [ ] . mammen et al. reviewed aki survivors (akin stage or greater) - years after icu discharge. they found only moderate rates of hypertension ( . %) and proteinuria ( . %); however, nearly % of these children had an egfr < ml/min/ . m [ ] . although these data are compelling, the studies are plagued by several issues which were well illustrated by a meta-analysis performed in [ ] . this analysis found that the studies performed up to that point had widely variable follow-up timeframes, diagnosed aki in different ways, defined outcomes dissimilarly, and almost universally failed to include a non-aki comparator group. since then, the majority of studies have used a consensus definition for aki, improving our ability to compare findings. additionally, many have compared outcomes between aki and non-aki cohorts. as an example, one such study examined hypertension among children undergoing stem cell transplantation. they found that high blood pressure was common across the entire population; however, aki (defined as doubling of serum creatinine, equivalent to kdigo stage or greater) was associated with a . -fold increased risk for the [ ] . in acute care environments, mortality among children with and without aki was similar regardless of aki severity (p > . ). in children receiving critical care, mortality was higher among children who experienced aki than those who did not. there was a dose-dependent effect as mortality was higher at each successive severity stage. the increases at stage and stage were statistically significant when compared with the prior stage (p < . ). all stages had significantly higher mortality than patients without aki (p < . ) development of hypertension [ ] . menon et al. examined children who developed nephrotoxic aki and found impressively high rates of proteinuria ( . %), hypertension ( . %), and an egfr < ml/min/ . m ( . %) [ ] . when compared with matched non-aki controls, those who experienced aki had significantly lower egfr, more proteinuria, and a higher incidence of hypertension [ ] . there have been two prospective studies performed specifically in the setting of congenital heart disease. the first is a -year follow-up of the translational research investigating biomarker endpoints in aki (tribe-aki) study; the authors found that hypertension ( %), proteinuria ( %), and a egfr < ml/ min/ . ( %) were common following cardiac surgery; however, these sequelae were not more common among the children who experienced perioperative aki [ ] . the second, entitled bfollow-up renal assessment of injury longterm after aki (frail-aki),^compared renal findings in children years after undergoing cardiopulmonary bypass. the aki and non-aki patients had similar rates of proteinuria and hypertension as well as comparable egfrs [ ] . they did note that those with aki had higher urinary levels of il- and liver-type fatty acid binding protein (l-fabp) than non-aki patients and healthy controls; this suggests that patients who experience aki may have subtle evidence of chronic renal injury even in the absence of overt ckd. interestingly, a subsequently published study did find that cardiac surgery-associated aki was associated with a greater risk for ckd stage or greater [ ] . the -year cumulative incidence of ckd for patients with cardiac surgeryassociated aki was %, higher than the % seen in those without aki (adjusted hr . , th ci . - . ). while this study was retrospective in nature, it was large, used a consensus definition for aki, and a rigorous definition of ckd. the data in neonates suffer from similar issues to those described above. namely, the studies describing the relationship between aki and the subsequent development of ckd have been observational, small, focused on specific patient cohorts, and often use disparate diagnostic criteria for both aki and chronic renal dysfunction [ ] . however, chaturvedi et al. examined eight observational studies and noted rates of ckd up to % in various populations; furthermore, proteinuria was seen in - % of patients and hypertension was seen in > % of patients in six of the eight studies analyzed [ ] . one of these studies is particularly relevant because they used a consensus definition for aki (rifle), applied an appropriate definition for ckd, and included a non-aki comparator group [ ] . this analysis, performed by zwiers and colleagues, examined children who underwent extracorporeal membrane oxygenation (ecmo) during the neonatal period; they found that aki was associated with increased risk for the subsequent development of ckd (or . , th ci . - . , p = . ) [ ] . in summary, although we are in need of more rigorous and larger analyses, the studies performed to date clearly suggest that, across the entire pediatric age spectrum, the development of aki puts children at risk for chronic renal disease. over the past decade, we have seen an increase in the quality and generalizability of aki research primarily due to the development of a consensus aki definition. the creation of a standard approach to aki identification has given us a more detailed and accurate understanding of the manner in which aki affects short-and long-term outcomes in children. the best available data now suggest that neonates, infants, children and adolescents with aki will require mechanical ventilation for longer durations, remain in the hospital for greater periods of time, and experience higher mortality. though some work remains to fully develop a consensus definition around renal recovery, we now know that recovery is far from universal, less likely when aki is more severe, and has prognostic implications for long-term renal function. the studies performed to date demonstrate that proteinuria, hypertension, and reduced excretory function are highly prevalent following aki and greatly exceed the rates seen in the general pediatric population. additionally, there seems to be a dose-dependent association between aki and ckd risk. thus, children who develop aki are at risk for long-term renal sequelae and these children warrant long-term observation and monitoring. review questions (answers are provided following the reference list) . true or false? studies which include a more severe phenotype of aki are likely to find a stronger association with poor outcomes? . aki is associated with higher mortality in children who are receiving a. intensive care b. acute care c. both intensive and acute care d. neither intensive or acute care . aki has been associated with a greater risk for chronic renal disease in children who are receiving a. intensive care b. acute care c. both intensive and acute care d. neither intensive or acute care . renal recovery is a. a concept in need of a unifying diagnosis b. less common with more severe aki c. potentially associated with chronic kidney disease d. all of the above conflict of interest the authors declare that they have no conflicts of interest. aki in hospitalized children: comparing the prifle, akin, and kdigo definitions improving global outcomes (kdigo) acute kidney injury work group. kdigo clinical practice guideline for acute kidney injury modified rifle criteria in critically ill children with acute kidney injury acute dialysis quality initiative w ( ) acute renal failure -definition, outcome measures, animal models, fluid therapy and information technology needs: the second international consensus conference of the acute dialysis quality initiative (adqi) group acute kidney injury network ( ) acute kidney injury network: report of an initiative to improve outcomes in acute kidney injury epidemiology of acute kidney injury in critically ill children and young adults aki in hospitalized children: epidemiology and clinical associations in a national cohort acute kidney injury is an independent risk factor for pediatric intensive care unit mortality, longer length of stay and prolonged mechanical ventilation in critically ill children: a two-center retrospective cohort study acute kidney injury and chronic kidney disease as interconnected syndromes the severity of acute kidney injury predicts progression to chronic kidney disease chronic kidney disease after acute kidney injury: a systematic review and meta-analysis elevated bp after aki long-term risk of ckd in children surviving episodes of acute kidney injury in the intensive care unit: a prospective cohort study - year longitudinal follow-up of pediatric patients after acute renal failure acute kidney injury associated with high nephrotoxic medication exposure leads to chronic kidney disease after months neonatal acute kidney injury update on acute kidney injury in the neonate acute kidney injury in the neonate incidence and outcomes of neonatal acute kidney injury (awaken): a multicentre, multinational, observational cohort study a comparison of the systems for the identification of postoperative acute kidney injury in pediatric cardiac patients acute kidney injury in neonatal encephalopathy: an evaluation of the awaken database acute kidney injury in children incidence, risk factors, and outcomes of acute kidney injury after pediatric cardiac surgery: a prospective multicenter study western canadian complex pediatric therapies follow-up g ( ) acute kidney injury after heart transplant in young children: risk factors and outcomes fluid overload and mortality in children receiving continuous renal replacement therapy: the prospective pediatric continuous renal replacement therapy registry congenital heart surgery in infants: effects of acute kidney injury on outcomes sepsis prevalence o, therapies study i, pediatric acute lung i, sepsis investigators n ( ) acute kidney injury in pediatric severe sepsis: an independent risk factor for death and new disability validation of the kdigo acute kidney injury criteria in a pediatric critical care population acute kidney injury in asphyxiated newborns treated with therapeutic hypothermia recognition and reporting of aki in very low birth weight infants acute kidney injury in non-critically ill children treated with aminoglycoside antibiotics in a tertiary healthcare centre: a retrospective cohort study midwest pediatric nephrology c ( ) aki in children hospitalized with nephrotic syndrome acute kidney injury after cardiac surgery according to risk/injury/failure/loss/end-stage, acute kidney injury network, and kidney disease: improving global outcomes classifications association between aki and long-term renal and cardiovascular outcomes in united states veterans epidemiology of acute kidney injury in critically ill patients: the multinational aki-epi study acute kidney injury reduces survival in very low birth weight infants fluid overload and mortality are associated with acute kidney injury in sick near-term/term neonate acute kidney injury is independently associated with mortality in very low birthweight infants: a matched case-control analysis acute kidney disease and renal recovery: consensus report of the acute disease quality initiative (adqi) workgroup renal recovery at different ages renal recovery renal recovery and long-term survival following acute kidney injury after coronary artery surgery: a nationwide study acute kidney injury recovery pattern and subsequent risk of ckd: an analysis of veterans health administration data predicting renal recovery after liver transplant with severe pretransplant subacute kidney injury: the impact of warm ischemia time efficacy and outcomes of continuous peritoneal dialysis versus daily intermittent hemodialysis in pediatric acute kidney injury recovery from acute kidney injury and ckd following heart transplantation in children, adolescents, and young adults: a retrospective cohort study improved long-term survival and renal recovery after acute kidney injury in hospitalized patients: a year experience the association between renal recovery after acute kidney injury and long-term mortality after transcatheter aortic valve replacement renal function follow-up and renal recovery after acute kidney injury in critically ill children long-term health status in childhood survivors of meningococcal septic shock chronic kidney disease in long-term survivors of hematopoietic cell transplant long-term risk of chronic kidney disease and mortality in children after acute kidney injury: a systematic review hypertension in long-term survivors of pediatric hematopoietic cell transplantation kidney outcomes years after pediatric cardiac surgery: the tribe-aki study follow-up renal assessment of injury long-term after acute kidney injury (frail-aki) cardiac surgery in patients with congenital heart disease is associated with acute kidney injury and the risk of chronic kidney disease the path to chronic kidney disease following acute kidney injury: a neonatal perspective ckd and hypertension during long-term followup in children and adolescents previously treated with extracorporeal membrane oxygenation answers: .true; studies which include a more severe phenotype of aki are likely to find a stronger association with poor outcomes? .a key: cord- - crtevc authors: moreno sancho, federico; tsakos, georgios; brealey, david; boniface, david; needleman, ian title: development of a tool to assess oral health-related quality of life in patients hospitalised in critical care date: - - journal: qual life res doi: . /s - - - sha: doc_id: cord_uid: crtevc aims and objectives: oral health deteriorates following hospitalisation in critical care units (ccu) but there are no validated measures to assess effects on oral health-related quality of life (ohqol). the objectives of this study were (i) to develop a tool (ccu-ohqol) to assess ohqol amongst patients admitted to ccu, (ii) to collect data to analyse the validity, reliability and acceptability of the ccu-ohqol tool and (iii) to investigate patient-reported outcome measures of ohqol in patients hospitalised in a ccu. methods: the project included three phases: ( ) the development of an initial questionnaire informed by a literature review and expert panel, ( ) testing of the tool in ccu (n = ) followed by semi-structured interviews to assess acceptability, face and content validity and ( ) final tool modification and testing of ccu-ohqol questionnaire to assess validity and reliability. results: the ccu-ohqol showed good face and content validity and was quick to administer. cronbach’s alpha was . suggesting good internal consistency. for construct validity, the ccu-ohqol was strongly and significantly correlated (correlation coefficients . , . and . , p < . ) with global ohqol items. in the validation study, . % of the participants reported a deterioration in self-reported oral health after ccu admission. finally, . % and % of the participants reported considerable negative impacts of oral health in their life overall and quality of life, respectively. conclusions: the new ccu-ohqol tool may be of use in the assessment of oral health-related quality of life in ccu patients. deterioration of ohqol seems to be common in ccu patients. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorised users. during the last years, much emphasis has been placed on the importance of patient-reported outcome measures (prom) in research investigating patients hospitalised in critical care [ ] . it is well established that oral health status is one of the determinants of quality of life [ ] . poor oral health has been shown to negatively impact quality of life across different populations [ ] [ ] [ ] [ ] [ ] [ ] . therefore, it is of concern that recent evidence suggests that hospitalisation is associated with deterioration in oral health as shown by increased plaque levels, gingival bleeding and xerostomia [ , ] . the decline in oral health during hospitalisation is of the utmost electronic supplementary material the online version of this article (https ://doi.org/ . /s - - - ) contains supplementary material, which is available to authorised users. importance: this population is more vulnerable to oral disease, this deterioration may potentially affect their quality of life and poor oral health may result in a greater risk of ventilator associated pneumonia (vap) as it is reported that vap-associated pathogens may translocate from the oral cavity into the lungs. when patients develop vap, this infection results in an increase of mortality, length of ccu stay and cost. a systematic review that included trials and patients concluded that "oral decontamination of mechanically ventilated adults using antiseptics is associated with a lower risk of ventilator associated pneumonia" indicating that fourteen patients would need to receive this intervention to prevent one case of vap [ ] . poor oral health might add an additional burden, affecting the patient's comfort and limiting the ability to eat and speak in these already compromised patients [ ] . therefore, deterioration of oral health in ccu patients might constitute a public health issue, as it could reduce quality of life, with evidence indicating that it also confers an increased risk for nosocomial infections [ ] . a major limiting factor to further research is the absence of a validated tool to measure oral health-related quality of life in ccu patients. commonly used generic ohqol questionnaires include a large number of questions which are irrelevant for the critically ill given their life circumstances, limiting their applicability in this setting. furthermore, these questionnaires take a long time to complete and are not feasible if the patients are frail and severely compromised, as it is the case in a ccu, because of the burden placed on patients and/or healthcare givers. therefore, the aim of this study was to develop and validate a suitable tool to assess the impact of critical care on oral health-related quality of life (ohqol) and to investigate patient-reported outcome measures of ohqol in patients hospitalised in a ccu. the study was conducted in accordance with the principles of the declaration of helsinki (ethical approval nres rec london -fulham, iras project id ). we followed a multi-stage approach similar to that described by guyatt and co-workers [ ] . the project included three phases: (a) initial tool development, (b) pilot study and (c) validation study; see fig. ( ) admitted to ccu for at least h ( ) received level two or level three care as described by the intensive care society standards ( ) able to communicate in english to the nurse/assessor (glasgow coma scale (gcs) of ; richmond agitation sedation scale (rass) score between and ) ( ) at least years of age conceptually, for the development of the questionnaire, we used the definition of ohqol from locker and allen: "the impact of oral disorders on aspects of everyday life that are important to patients and persons, with those impacts being of sufficient magnitude, whether in terms of severity, frequency or duration, to affect an individual's perception of their life overall" [ ] . a literature search was performed which ascertained that no research was available in relation to ohqol in ccu nor were there any validated tools to measure the ohqol of patients at a ccu. we reviewed the literature to identify the most common oral health problems experienced by those hospitalised in ccu. we mapped the items and domains obtained from the literature and sought advice from a panel of experts, including consultants and nurses in critical care and experts in oral care and public health, to compare them against existing ohqol tools validated in other settings. the domains and items were revised and re-presented to the expert panel until consensus was reached. the comprehension of each of the items was initially assessed using the question understanding aid tool (quaid) and items modified as required [ ] . as a result of this initial research, an initial ccu-ohqol tool was created (supplementary files-initial tool). the domain and item revision involved administering the tool in a pilot study involving ccu patients before discharge. the patients assessed the interpretability, relevance and acceptability of the ccu-ohqol. after the questionnaire was completed, the nurses conducted a short interview comprising a series of open-ended questions with regard to the validity of the domains and items of the questionnaire and to identify possibly missing items. the initial tool was modified as necessary. the final version of the ccu-ohqol tool (supplementary files-ccu-ohqol questionnaire) was administered to a larger sample, to assess construct validity, reliability and acceptability. as per the intended final use, the questionnaire was self-administered. there are no definitive criteria for the required sample size in a validation study of this kind. however, previous literature suggests that a sample size of to patients should be sufficient for the proposed analysis [ ] . content validity was assessed through the frequency of endorsement for each item amongst the patients included in the pilot study. for construct validity, the correlation between previously validated global ohqol items [ ] and the score for the items in our questionnaire was analysed using the non-parametric spearman's rank correlation coefficient. furthermore, associations between the ccu-ohqol overall score grouped in categories (i.e. good/fair/poor ohqol) and global ratings of ohrqol were tested using kruskal-wallis non-parametric test. the reliability of the tool was also assessed: internal consistency was measured using cronbach's alpha coefficient and by analysis of the item-total correlation and cronbach's alpha if item deleted. test-retest reliability was tested via intraclass correlation coefficients. differences in self-reported oral health before hospitalisation and at ccu discharge were assessed by wilcoxon signed rank tests. overall scores for the ccu-ohqol were calculated by summing up the response codes since ordinal values were coded for each question as presented in supplementary files-ccu-ohqol questionnaire. the distribution of the participants within categories ("good", "fair" or "poor" ohqol) was investigated as well as the distribution of responses for all individual items. we also assessed three summary variables: severity, prevalence and extent; in a similar fashion to that defined by slade and co-workers [ ] and modified to suit the characteristics of our questionnaire: • severity the sum of ordinal responses • prevalence the percentage of participants answering one or more item with ordinal values or . (i.e. "very bothered", "extremely bothered", "dissatisfied"strongest negative impacts) • extent the number of items per subject answered with ordinal values or the correlation between the global rating qol items, the self-reported oral health items and the score for the items in our questionnaire were analysed using the nonparametric spearman's rank correlation coefficient. the purpose of assessing these correlations was to explore the relationships between self-reported oral health, ohqol and qol overall. furthermore, the associations between self-reported changes in oral health during ccu stay and overall ccu-ohqol score were tested using one-way analysis of variance. analysis was carried out using spss statistics for windows (version . , ibm corp, ny). a total of and subjects were recruited for the pilot and validation study, respectively. the demographic characteristics of the study population for both parts b and c are presented in table . the expert panel agreed that the tool should include items for the following domains: "satisfaction with oral health", "functional limitations", "oral symptoms", "social impact", "self-care" and "psychological impact". subsequently, a pool of items was scrutinised until consensus was reached for a total of items to be included in the initial tool (supplementary files-initial tool, items to ). questions assessing self-reported oral health, three global ohqol items and a global qol item were also included. the analysis of the frequency of endorsement of each item revealed very few items left unanswered ( . %) and a good distribution of endorsement amongst the available responses for each item. in addition, "floor" and "ceiling" effects were not present when the questionnaire was analysed as a composite score. during the short interviews, . % (n = ) of the sample reported that the questions were relevant and participants ( . %) reported that the items related well or very well to their experience during their ccu stay. the addition of questions asking about tooth brushing frequency and bad breath was suggested by five subjects each. the recruitment rate was . % of all subjects asked to participate in the study. the mean time for questionnaire completion was min s (ci % min s- min s). the majority of the sample ( . %) thought that the time to complete the questionnaire was reasonable and they would do it again while reporting that the wording of the questions was good ( . %, n = ) and easy to understand ( . %, n = ). following data collection and analysis of the pilot study, the results were reviewed by the expert panel in critical care and oral health that contributed to the development of the initial tool. as a result, two items relating to bad breath and toothbrushing frequency were incorporated. the final tool was completed by ccu patients. the correlation coefficient between the ccu-ohqol score and the overall score of the global items of ohqol was . (p < . ). furthermore, the ccu-ohqol score was strongly or moderately correlated with all three global items individually (item : . , item : . and item : . ) being all also statistically significant (p < . ). there was an association between the categorised version of the ccu-ohqol tool and the mean rating of the global ohqol tool which was statistically significant (p < . : kruskal-wallis non-parametric test). similar associations and gradients were also observed with each of the global ohqol items when assessed individually (see fig. ). cronbach's alpha coefficient was . . all of the items of the ccu-ohqol tool showed an item-total correlation between . and . (supplementary files-item-total correlation) with the exception of questions ( . ) and question (− . ). these two questions were also the only items where their deletion resulted in an overall increase in cronbach's alpha (item : . ; item ( . ). test-retest reliability was assessed by asking a subset of the subjects (n = ) to complete the questionnaire twice, days apart. the mean intraclass correlation coefficients (iccs) for the scale overall was . ( % ci . - . ). a total of patients were approached to take part in part c of the study and participants were recruited, translating into a recruitment rate of . %. on average, the questionnaires took the participants min and s (ci % min s- min s) to complete. the dimensionality of the tool was assessed using principal component exploratory factor analysis (efa). we retained factors with eigenvalues > and rotated them with "varimax", which is an orthogonal rotation method. items were assigned to retain rotated factors when they had a loading of ≥ . in absolute value. the magnitude of factor loadings, distribution of variance amongst the factors, and the relative correlation of the items with the different factors were assessed. after varimax rotation, the first component explained % of the variance, and the second principal component added . % to the variance explained (see table ). the third, fourth, and fifth components had eigenvalues of . , . , and . , and explained variances were . %, . %, and . %, respectively. fourteen out of the items loaded highly on no more than one particular dimension (see table ). the four items in the first rotated principal component describe functional limitations and include four out of the five items which were included by our expert panel within this dimension and therefore maintained the name of the original dimension. items referring to psychosocial impacts (items and ) were found in the second factor only. in addition, this factor also included items and which were included in the oral symptoms dimension. item may well have been considered by patients as an indicator of a social interaction and this factor was therefore named "psychosocial impacts". the third dimension/factor included the two items measuring symptoms of dry mouth and was therefore named "xerostomia". the main item tapping into the th factor was question . the second highest factor loading (although just short of the cut-off . value) in this dimension was item , which was also the only other item with a factor loading above . for this component. since items and were the two items originally included in the dimension "self-care", this component retained this name. finally, the fourth factor referred to questions and . question was included as an item for "satisfaction with oral health" and seems unrelated to question ; therefore, it is difficult to discern to which specific dimension these items may be tapping and remained unnamed. table . in addition, nearly % of the participants (n = ) experienced negative impacts that bothered them at least "somewhat", . % reported that the negative impacts affected their life overall at least "somewhat" and . % had negative impacts that affected their quality of life at least "somewhat". those who reported a deterioration in self-reported oral health showed a higher mean ccu-ohqol score ( . , ci % . - . ) compared to those whose self-reported oral health did not change ( . , ci % . - . ) although this was not statistically significant (p = . ). the ccu-ohqol showed clear face and content validity. for construct validity, the tool achieved very strong correlations with global ohqol (p < . ) in the expected direction with higher ccu-ohqol scores indicating worse ohqol. furthermore, participants with a "poor ohqol" showed greater impacts in their overall life and quality of life. reliability was adequate with a cronbach's alpha of . , above the acceptable threshold of . [ ] , and adequate test-retest intraclass correlation coefficient of . ( % ci . - . ). therefore, these data suggest that the ccu-ohqol has appropriate characteristics to be used for the stated purpose. according to our results, hospitalisation in ccu has a negative effect on the self-perceived oral health of patients. more interestingly, those who reported a deterioration in self-perceived oral health, also showed a higher mean ccu-ohqol score (poorer ohqol). although the correlation marginally failed to show statistical significance (p = . ), there was a clear trend with nearly a twofold increase in the overall ccu-ohqol score which may have reached significance in a larger sample. this could be understood as initial evidence to suggest that changes in oral health after hospitalisation, as perceived by the patients, may indeed have an effect on their quality of life. we also obtained a cross-sectional view of the ohqol amongst the participants. we use estimates of prevalence, extent and severity as they provide important complimentary information when interpreting ohqol data [ ] . these estimates show significant impacts of oral health on quality of life in our sample. the impacts seem to be more prevalent and intense in the "functional limitations", "oral symptoms" and "self-care" domains and relatively more modest for "psychosocial impact". in addition, the overall qol items revealed that a significant proportion of the sample had impacts that affected their life overall. to date, there are no other studies exploring self-reported oral health and ohqol in the critically ill which precludes comparison of these results. the current results suggest a very strong impact of "xerostomia" in the ohqol of the critically ill. this is consistent with previous evidence reporting that intubated ccu patients have a significantly reduced salivary flow during hospitalisation [ ] . in addition, there are some studies suggesting a deterioration of ohqol in patients admitted to hospital outside ccus [ , ] . similar to our findings, hospitalisation resulted in worsening of oral health and low ohqol in other hospitalised populations [ ] [ ] [ ] . yu and co-workers showed that for chinese geriatric patients, the disruption of the normal daily living routine as a result of hospitalisation translated to a worsening of oral health and low ohqol [ ] . additionally, schimmel and co-workers compared the ohqol of hospitalised stroke patients who presented with hemi-facial and/or limb palsy at the university hospital of geneva (switzerland) to that of subjects with similar age, gender and dental status [ ] . the results also indicated a lower ohqol in the hospitalised population compared with the control group. the ohip-edent [ ] mean score was . ± . in the control group and . ± . in the hospitalised population (p = . ), with higher scores indicating lower ohqol. also in accordance with the previous studies, cornejo et al. [ ] in a cross-sectional study amongst institutionalised elderly in barcelona (spain) found that % of the participants had poor ohqol and this was associated with self-reported poor oral health. all in all, it seems that the decline of quality of life due to a worsening of the oral health might be more marked in unwell and hospitalised patients. extrapolating what we know about the impact of critical care on the hqol and the impact of poor oral health on the ohqol of medically compromised patients as discussed above in addition to the results of our study, it could be possible that some of the deterioration in hqol of the critically ill is due to changes in oral health. in order to improve patient care, we need to know what ohqol means to patients and what experiences they relate to their stay in ccu. experiences of critically ill patients are an important aspect of the quality of the care and are essential to guide bedside decisions in the ccu; they should be placed at the centre of public health debate. the strengths of the study include the systematic process followed to develop the tool tailored to our target population. the ccu-ohqol tool is "patient-centred and incorporates aspects of daily living that patients deem to be important" as the patients themselves were involved in the development of the tool [ ] . ideally, participants could have been involved earlier in the process via interviews at the stage of item generation to further limit the risk of critical items being overlooked. the aim was to develop a questionnaire that would not be burdensome on patients or staff to complete. the time for completion of the questionnaire was short and % of the patients reported they would be happy to repeat the questionnaire if needed. however, we recruited a relatively low number of subjects in the final phase of the project and the low recruitment rates may suggest some degree of selection bias; based on the data presented in table , the population in our study seems to be representative of the patients seen at the ccu at uch. forty-five patients are at the lower end of what would be considered acceptable [ ] . in addition, the sample size for test-retest analysis was also small, precluding meaningful interpretation. completion of the second questionnaire is logistically difficult since many patients may be discharged from the ccu to their homes or to other hospitals. in addition, in the initial development and validation of the tool we did not complete an oral examination to assess objectively the oral health of the participants but rather used a previously validated self-reported measure of oral health. further studies using the ccu-ohqol and including objective clinical assessments of the oral health of the participants would allow assessment of the responsiveness of the tool and also provide an opportunity to compare the questionnaire against other generic ohqol tools. finally, the cross-sectional analysis of the ohqol of the participants should be interpreted with caution since the study was designed with the primary aim of developing and validating the ccu-ohqol tool. without a control group, the changes might have been due to secular effects not related to ccu. it is not clear what the ideal control group should be. nonetheless, assessing the oral health and ohqol of patients admitted to other hospital wards or institutionalised in nursing homes might allow meaningful comparisons even if not representing a true control group [ , ] . it is also worth noting that it has been proposed that some of the changes in ohqol and qol may be due to psychological adjustments such as changes in expectations due to illness which may be particularly relevant in the ccu due to the "response shift" that patients hospitalised in critical care may suffer. further evaluation of the ccu-ohqol tool is needed to test its psychometric properties and its applicability in other ccu settings. future studies should provide information regarding test-retest stability and the ability of the tool to discriminate between groups with different levels of oral health assessed by traditional clinical measures. finally, the responsiveness of the questionnaire could be studied through administration as part of a randomised controlled trial [ ] . as perceived by the patients themselves, our results contribute to the body of evidence suggesting a deterioration in oral health following hospitalisation. more importantly, this deterioration was associated with poorer ohqol indicating that these changes may indeed have an impact in the life and well-being of those hospitalised in a ccu. since the care received at the ccu would ultimately influence the oral health of the critically ill, this is an extremely important finding encouraging the search of new interventions and care pathways to maintain oral health following admission to a ccu, being this an important public health matter. by including this ohqol tool in future research, influential data will be provided to decision makers for the development of health promotion and may help to identify a group of patients for whom new care pathways aimed at improving oral health are needed [ ] . simple ccu nurse-led interventions have already been shown to maintain oral health [ ] and therefore this is an achievable objective. ( ) this study is the first to develop a tool to measure ohqol in patients hospitalised in a ccu: the ccu-ohqol. the new tool has good acceptability, clear face, content and construct validity and satisfactory internal consistency reliability. ( ) for a large proportion of the patients, self-rated oral health deteriorated following hospitalisation in a ccu and this deterioration seems to be associated with poorer oral health-related quality of life. ( ) overall, there were substantial negative effects of oral health on the patient's quality of life during their stay at the ccu with a high prevalence, extent and severity of impacts in the patient's daily living and quality of life. quality of life after intensive care: a systematic review of the literature the concept of positive health: a review and commentary on its application in oral health research effect of arch length on the functional well-being of dentate adults impact of prosthodontic status on oral wellbeing: a cross-sectional cohort study key factors associated with oral health-related quality of life (ohrqol) in hong kong chinese adults with orofacial pain effects of severe dentoalveolar trauma on the qualityof-life of children and parents quality of life and psychological well-being among endodontic patients: a case-control study impact of oral health on the life quality of periodontal patients the impact of hospitalization on oral health: a systematic review the impact of hospitalization on dental plaque accumulation: an observational study oral decontamination for prevention of pneumonia in mechanically ventilated adults: systematic review and meta-analysis guidelines for oral health care for long-stay patients and residents measuring disease-specific quality of life in clinical trials what do measures of 'oral health-related quality of life' measure? quaid: a questionnaire evaluation aid for survey methodologists measuring functioning and well-being. the medical outcomes study approach to what extent do oral disorders compromise the quality of life? impacts of oral disorders in the united kingdom and australia health measurement scales: a practical guide to their development and use interpreting oral health-related quality of life data. community dentistry and oral epidemiology oral health status and development of ventilator-associated pneumonia: a descriptive study impact of oral health status on oral health-related quality of life in chinese hospitalised geriatric patients oral health-related quality of life in hospitalised stroke patients oral health-related quality of life in institutionalized elderly in barcelona (spain) developing short-form measures of oral health-related quality of life changes in dental plaque following hospitalisation in a critical care unit: an observational study measuring change over time: assessing the usefulness of evaluative instruments oral health-related quality of life: what, why, how, and future implications acknowledgements we thank dr jean e. suvan, deborah smyth, magda rocha, georgia bercades, jung hyun ryu, alicia san jose and alison macklin for their help for study coordination, recruitment and collection of data. this project was supported by researchers at the national institute for health research university college london hospitals biomedical research centre.open access this article is distributed under the terms of the creative commons attribution . international license (http://creat iveco mmons .org/licen ses/by/ . /), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. conflict of interest the authors declare no conflict of interest. ucl and/or uclh and uclh cbrc paid salaries as part of their research/ clinical roles to all authors and researchers involved in the project.ethical approval all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the helsinki declaration and its later amendments or comparable ethical standards (ethical approval nres rec london -fulham, iras project id ).informed consent informed consent was obtained from all individual participants included in the study. key: cord- -gcmpatlb authors: errecaborde, kaylee myhre; macy, katelyn wuebbolt; pekol, amy; perez, sol; o’brien, mary katherine; allen, ian; contadini, francesca; lee, julia yeri; mumford, elizabeth; bender, jeff b.; pelican, katharine title: factors that enable effective one health collaborations - a scoping review of the literature date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: gcmpatlb advocates for a one health approach recognize that global health challenges require multidisciplinary collaborative efforts. while past publications have looked at interdisciplinary competency training for collaboration, few have identified the factors and conditions that enable operational one health. through a scoping review of the literature, a multidisciplinary team of researchers analyzed peer-reviewed publications describing multisectoral collaborations around infectious disease-related health events. the review identified factors that support successful one health collaborations and a coordinated response to health events across three levels: two individual factors (education & training and prior experience & existing relationships), four organizational factors (organizational structures, culture, human resources and, communication), and six network factors (network structures, relationships, leadership, management, available & accessible resources, political environment). the researchers also identified the stage of collaboration during which these factors were most critical, further organizing into starting condition or process-based factors. the research found that publications on multisectoral collaboration for health events do not uniformly report on successes or challenges of collaboration and rarely identify outputs or outcomes of the collaborative process. this paper proposes a common language and framework to enable more uniform reporting, implementation, and evaluation of future one health collaborations. ongoing and emerging health challenges such as infectious disease epidemics, bioterrorism, antimicrobial resistance, and natural disasters require a coordinated response from a highly diverse, collaborative, and trained health workforce. "one health" is a concept and approach intended to meet such demands. though loosely defined, a broadly accepted definition of one health describes it as "the integrative effort of multiple disciplines working to attain optimal health for people, animals, and the environment [ ] [ ] [ ] [ ] world organisation of animal health (oie), n.d.; world health organization, n.d). a one health approach recognizes that complex health challenges are beyond the purview of any one sector or discipline working in isolation [ ] and that a resilient health workforce must be capable of effective and collaborative prevention and detection of, as well as response to emerging health challenges. a one health approach, therefore, calls for collaboration across disciplines, sectors, organizations, and national borders in support of increasingly complex health challenges [ ] [ ] [ ] [ ] [ ] [ ] [ ] . while one health advocates increasingly support collaborative and multi-sectoral approaches to health challenges, no common language or metrics exist to uniformly describe and evaluate such efforts. few studies explicitly analyze the factors and conditions that support effective one health practices and collaborations. this hinders the ability of health professionals to learn from past experiences and improve upon current and future one health policies, partnerships, and practices. this paper seeks to address this gap by analyzing and identifying factors that enable effective multisectoral collaboration and response to health events. in this study, a multidisciplinary team of researchers reviewed a broad scope of literature describing collaborative and multi-sectoral approaches to past health events to understand how such collaborations are commonly described and evaluated and to identify and synthesize enabling factors for one health collaborations. this paper identifies twelve factors related to effective one health implementation and collaboration and concludes with a proposed framework for evaluating future multisectoral one health collaborations. the ultimate aim of this work is to support and improve multisectoral preparedness and response efforts. although its conceptual foundations date back hundreds of years, the formal global health construct known today as one health wasn't officially recognized by international and scholarly bodies until [ ] . the hiv/aids pandemic in the s and the hanta virus outbreak in , made clear that emerging disease threats can cross national borders, cultures and species. with that came a broader recognition that animal and zoonotic diseases pose a serious threat not only to human health but to global health security. policy makers and health practitioners looked to collaborative health efforts as a response to these emerging challenges [ ] . the subsequent decades which were marked by unprecedented global interconnectedness and human mobility [ ] were associated with threats to global health security, including manmade threats, such as the use of anthrax as a bioweapon, and emerging diseases like sars and avian influenza. these challenges necessitated the need for a more formal coordinated action from countries, regions, and the global health community at large to address such health threats. in order to address the afore-mentioned challenges, there have been emergence of major health initiatives and frameworks such as the global health security agenda, the international health regulations-joint external evaluations (ihr-jee), the world health organization (who)-world organisation for animal health (oie) operational framework for good governance at the human-animal interface [ ] , and the world bank's one health operational framework [ ] . a common thread among these initiatives is the emphasis on multisectoral and transdisciplinary collaboration and a call for strengthening human, animal and environmental health systems through a one health approach. the global health community, including those already engaged in one health, continue to grapple with the fundamental questions of what characterizes a successful one health approach, including how to set goals, establish frameworks, facilitate collaborative work, and how to process and measure outcomes [ ] . efforts to measure one health programmatic outcomes and operations are necessary for the improvement of collaborative efforts. this article supports such efforts by ) identifying key factors that support effective collaboration around health events and ) proposing a framework for documenting and evaluating one health collaborations in a more uniform and systematic manner. collaboration is an inherent and explicit part of the one health approach which calls for the active engagement of institutions, managers, and health practitioners across disciplines and sectors [ ] [ ] [ ] [ ] . despite widespread recognition of the importance of a one health approach, there exists a gap in the literature regarding what constitutes a successful one health collaboration. this study draws upon the existing public affairs literature on collaborative, or 'crossboundary' collaborations to understand which factors enable successful collaboration around health events. review of the literature on collaboration. scholars of public policy, organizational partnerships, team science, and multisectoral collaboration have produced a series of theoretical frameworks to describe cross-boundary collaborations and identify which practices make them successful [ ] [ ] [ ] . the focus on collaboration and partnership is not unique to any one discipline, yet there is very little cross-fertilization of research across disciplines. this research builds upon the existing literature on cross-boundary collaborations and applies it to one health collaborations. the conceptual framework for this study focuses on three critical phases of a successful cross-boundary collaboration: adequate starting conditions, an effective process of collaboration, and attention to the outcomes of collaboration [ ] [ ] [ ] [ ] [ ] . starting conditions. there is a general consensus in the literature on cross-boundary collaborations that starting conditions-the conditions in place before any collaborative process begins-impact the process, structures and outcomes of collaborative engagement. these include prior history (e.g. successes, failures, existing partnerships), the environment (e.g. resource imbalances, stakeholder incentives), and relational dynamics (e.g. balances of power, who convenes or facilitates the collaboration, and how and by whom problems are defined) [ , ] . the presence or absence of such conditions influences successes and challenges encountered during the collaborative process. process. beyond starting conditions, many scholars point to the process of collaboration itself and the structures in place to support effective collaboration [ , , , ] . although the terms used for collaboration vary, scholars focusing on the process of collaboration point to the importance of leadership, shared goals, trust and mutual understanding, institutional structures and resources, communication, and data management. measuring outcomes. a review of the literature on collaboration suggests a lack of validated metrics for measuring collaborative effectiveness and performance. several scholars of cross-boundary collaborations, citing works published between and , highlight the importance of measuring the outcomes of collaboration and lament the challenges of describing and evaluating collaborations in a uniform way [ , , , , , [ ] [ ] [ ] . this underscores the importance of understanding which factors support collaborative efforts, and how teams can evaluate their performance and outcomes in association with these factors. the literature on cross-boundary collaborations and its attention to the starting conditions, processes, and outcomes of collaborative approaches have informed this study on the factors that enable effective one health collaborations. the following questions guided this study: what factors (systems, structures, processes, skills, competencies, decisions, and actions) enabled two or more disciplines or sectors to collaborate effectively in a health event? a scoping literature review was conducted to identify key factors that facilitate multisectoral collaborations around major health events such as disease outbreaks using published accounts of actual health events. a scoping review, in contrast to a systematic review, is well-suited for a field such as one health that is still relatively new and evolving, as the method allows for assessment of emerging evidence, as well as a first step in research development [ ] [p. ]. due to the lack of a common language and framework for describing one health collaborations, this scoping review builds that foundation by providing a broad overview of one health collaborations and supporting the synthesis of key concepts, evidence, and research gaps [ , ] . the scoping review was initiated by a multidisciplinary team in january . the team members were composed of individuals with expertise in veterinary medicine, public health, public policy, organization and management leadership studies, international development, monitoring and evaluation, and education. because the researcher is central to the methods and analysis of qualitative research, it was important to select a transdisciplinary research team that could work effectively to address the research questions and to illustrate the disciplines that were represented in the transdisciplinary approach employed for this scoped review. selection of relevant articles. the search included peer-reviewed articles available todate in the u.s. national library of medicine's pubmed database that were searched using specified mesh (medical subject headings) terms. although the multidisciplinary research team has extensive experience in one health, they were not trained in sensitive search strategies [ ] . the research team thus elected to work with a university of minnesota research librarian to develop mesh terms for this study. table provides a list of the key terms used to identify articles discussing multisectoral health events and collaborations. to avoid tautology, it was a deliberate decision to not use "one health" as a search term. instead, drawing upon the researchers' extensive experience in one health, various terms were used to describe one health and similar multidisciplinary and cross-sectoral health collaborations. the underlying assumption was that any articles explicitly addressing one health would be captured using these key terms. this initial mesh search identified , non-duplicated articles. this scoping review was an inductive study of the literature and was conducted in order to support more hypothesis driven research for one health. by design, the authors elected to limit this literature review to the pubmed database at the outset of the study. pubmed is peer-reviewed and peer-led database. articles are selected and included based on scholarly and quality criteria by literature review committees and are tagged by keyword and by article structure, contributing to more accurate retrieval than other databases (e.g. google scholar); accurate retrieval supports the search results are reproducible and reportable, which is critically important for a scoping review of the literature in which it is important for other researchers, no matter their location, to repeat the study. the decision to use one database reflects the exploratory nature of this study and the author's intent to propose further hypothesis-driven research that may include additional databases. this methodological choice is in line with arksey and o'malley ( ) who attest that decisions must be made at the outset of a study to clarify reasons for the coverage of the review in terms of time span and language [ ] [p. [ ] [ ] . initially, citations and abstracts of these articles were screened in two phases. the articles were reviewed for inclusion based on the criteria outlined in table . in the first screening, abstracts met initial inclusion criteria and full articles were procured and reviewed. in the second phase of screening, two further criteria were included to better achieve scoping review objectives. the research team divided into transdisciplinary pairs which included a reviewer from the health sciences and one from the social sciences. each of the articles that met the initial inclusion criteria were divided among the team members and then independently reviewed according to the modified screening criteria. articles were included if both reviewers agreed that they met all initial requirements. in instances where the transdisciplinary reviewers did not agree, the articles were brought to a full research team meeting and reviewed jointly until consensus among all researchers was achieved. this same method of collaborative review was used for the second round of screening and resulted in articles for the final analysis. the prisma diagram below (fig ) illustrates the article search, screening, and review process. . the health event discussed involved an infectious disease challenge; and . the case or event involved at least two sectors or disciplines. inclusion criteria: . articles met initial screening criteria and were included if they met the following targeted requirements: . the article provides a retrospective analysis of an actual health event; . the case or health event involved a noteworthy (describing successes or challenges encountered during health event) interaction among at least two sectors or disciplines. exclusion criteria: articles were excluded if they failed to discuss any specific aspects of collaboration, even if they generally acknowledged the importance of multisectoral collaboration. https://doi.org/ . /journal.pone. .t analysis. the interdisciplinary team conducted an analysis of the articles that explicitly addressed multisectoral collaboration in response to an actual health event. each reviewer coded approximately - selected articles using the qualitative data analysis software, maxqda [ ] . descriptive codes were identified in advance to ensure that baseline data reflected the one health aspects of the articles reviewed. all other codes emerged from the data using a grounded theory approach [ , ] . preliminary and axial coding procedures are outlined in the following section and ensured that inductive and deductive thinking could be related. preliminary coding. a set of predetermined, descriptive codes were used to denote the location and nature of the health event in the articles, including specific infectious agents, factors that enable one health collaboration: a scoping review relevant disease vectors or hosts, and the various entities involved in the collaboration. each paper was coded for the predetermined codes outlined in table . predetermined codes were also used to identify the entities involved in each health event response. the team used the code "roles" to identify individuals or groups who participated in the coordinated response in a formal role based on individual expertise and formal training. while many of these roles represent professions in the health sciences, this category also included representation from the social sciences, media and community relations, government, and engineering. other articles focused on types of training, identified by the research team as "disciplines," (e.g., clinical epidemiology [ ] or food safety [ ] ), or specific professions (e.g., toxicologist [ ] or information technology specialist [ ] versus specific professions). a third type of classification in the literature was more general categorization of sectors involved, such as the traditional designations of public, non-profit, and private/for-profit. axial coding. axial coding was used to construct linkages between "data sets" or, in this scoping review, articles regarding intersectoral collaboration. axial coding is a qualitative research technique to relate data together in order to reveal codes, categories, and subcategories, as well as patterns in the data [ ] . this grounded theory is an iterative process that combines inductive and deductive thinking. each article was first coded to identify any area of text where authors analyzed collaboration around a specified health event. in this process, it became quickly apparent that the review team would need to differentiate between actual and hypothetical forms of collaboration reported. all articles included in the analysis at this stage were retrospective analyses of actual health events, yet many were actually prospective in their analysis and discussion. as an example, several of these articles included suggestions based on what should happen in an ideal scenario, rather than what occurred in practice, thus leaving out key details of the actual event. therefore, a first round of organizing codes differentiated between collaborations that actually happened versus ideal scenarios and hypothetical lessons, allowing the research team to focus the analysis on what actually happened ( table ). the text was further coded to reflect whether the authors were reporting a success factor of collaboration, or a challenge of collaboration. both the successes and challenges reported in the literature were related during the grounded theory thematic analysis and informed the final thematic results reported. after the first round of axial coding was conducted to organize the data, the authors employed a deductive framework developed from the review of literature on multisectoral collaboration [ ] . aligned with this framework, the research team distinguished between starting conditions for collaboration, the process of collaboration itself, and the outcomes of collaboration (table ) . finally, the review team re-examined the passages coded as "actually happened" and "successes". these codes were then related to the deductive codes of starting conditions and process-based factors. an excel table was used to organize axial codes into a table of final results. limitations. the primary limitations of this scoping review are three-fold. first, the literature analysis relies on peer reviewed publications alone, which may have underrepresented collaborative efforts that are more commonly encountered in grey literature. future work may be expanded to include these types of sources. second, there was no consistent framework or language for reporting the successes or challenges of collaboration, and thus, important content may have been missed during the search and review [ ] . the scoping review team tried to overcome this with two strategies, which included building an expanded list of search terms and conducting an iterative review process using two independent transdisciplinary reviewers. both methods offset this limitation and might have minimized the likelihood of missing specific content. third, the researchers could not identify specific metrics for evaluating performance and collaboration in the literature. this meant that an evaluation baseline was not present. however, the research team believes that the final subset of articles represents a diverse crosssection of transdisciplinary efforts around emerging health events. the scoping review yielded peer-reviewed publications explicitly addressing multisectoral collaboration in response to an actual health event. this section describes the nature of the one health collaborations analyzed as well as the various factors that enable one health collaboration. types of one health events analyzed include natural disasters, infectious disease outbreaks, endemic disease, bioterrorism, and biosecurity preparedness. in each of these cases, the underlying multisectoral collaboration was either a preparedness (planned or ongoing collaboration) or response (emergency or ad hoc collaboration) effort. the sample included one health events from around the world. most articles addressed health events in europe/eurasia ( %), the americas ( %), and asia ( %). less represented in this sample were health events taking place in africa ( %), oceania ( %), and the middle east ( %). most health events involved a specific infectious agent ( %), while the remaining % focused on infectious disease challenges such as hospital infections, pest management, or tsunami response. a total of different infectious agents were coded. among the infectious agents identified, % were bacterial, % were viral, and % were protozoal (e.g. malaria). % of these agents primarily affect humans and % are predominantly animal-related. % of the agents were food and water-related, % were insect related, and an additional % were related to environment. overall, % of the infectious agents were considered zoonotic, meaning they spread between humans and animals. relevant disease vectors or hosts represented in more than one publication included bats, cattle, poultry, horses, swine, humans, mosquitoes and midges. involved parties or entities played varied roles and represented diverse disciplines and sectors, as illustrated in table . thematic findings are presented according to the one health collaboration framework, which distinguishes between individual, organizational, and network factors that enable multisectoral and transdisciplinary collaboration at the onset and in the process of addressing a one health event. the team ultimately created organizing categories that reflected the individual, organizational and network levels of collaboration (table ) . these categories were informed by a review of the literature; for the purposes of this discussion, the definition of network is provided by emerson and nabatchi [ ] , and is defined as "the processes and structure of public policy decision making and management that engage people constructively across the boundaries of public agencies [organizations], levels of government, and or the private and civic spheres in order to carry out a public purpose that could not otherwise be accomplished," [p. ]. within each level, the review team created groups of subcategories to further organize codes. the research team identified key factors that support successful multi-sectoral collaborations around major health events. at the individual level, these factors include ) education & training and ) prior experience & existing relationship. organizational factors include ) organizational structures, ) organizational culture, ) human resources, and ) communication. finally, network-level factors include ) network structures, ) relationships, ) leadership, ) management, ) available & accessible resources, and ) the political environment. these final individual, organizational and network factors were then further characterized factors that enable one health collaboration: a scoping review according to their relevance at the start of a collaboration "starting condition" or during the process "process-based" factors of collaboration. the researchers identified that the organizational thematic factors were relevant to both starting conditions and process-based factors so were not separated. the final results of this literature review are thus presented in table . the researchers also coded each paper for outcomes. of all the articles coded, only articles reported on outcomes of collaborations. the outcomes reported included: ( ) cost reduction; ( ) decreased mortality; ( ) decreased morbidity, ( ) multisectoral development opportunities resulted from the collaboration; ( ) improved safety; ( ) effective use of available resources. table . final axial coding process included both inductive and deductive codes and reflects emerging themes for successful collaboration. starting condition factors initial deductive code (table ) process factors initial deductive code (table ) individual (emergent axial code table ) preemptive technical training/ continuing education [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] disease specific technical training [ , , ] preemptive collaborative training [ , ] strong public-sector led training [ ] training and capacity building provided a platform for better collaboration for outbreak [ ] ngos support gov. through staff training [ ] participatory epidemiology training [ ] prior experience & existing relationships (informal/formal) pre-existing multisectoral relationships [ , [ ] [ ] [ ] [ ] previous experience collaborating for health events [ , , ] ad hoc "just-in-time" training shared training & organizational alignment; aggressive, rigorous, just-in-time, and critical trainings for key positions and critical events with monthly follow-up meetings to support compliance [ , ] training and capacity building provided a platform for better collaboration for outbreak response [ ] instituting multisectoral disease specific training; ongoing training-for new and existing systems [ , ] organizational (emergent axial code table ) shared response guidelines [ , ] structures frequently included policies/protocols [ , ] reporting -management protocol -task management -response plan -communications/ communication strategy [ , ] infection planning, control and traceback procedures [ ] systems reporting, laboratory systems [ ] surveillance systems [ , , ] planning; iterative improvement of systems [ , , ] information management system/ database [ , , , ] information sharing (data available and useful) [ , ] ) tool sharing during response [ ] lab systems in place [ ] online system for hr recruitment [ ] intentional multidisciplinary engagement, collaborative capacity [ , , , ] standard operating procedures [ ] interoperability [ ] needs assessment and prioritization [ , ] culture leadership, accountability, ownership, trust, transparency of processes, systems based thinking, cultural awareness and engagement leadership to support the iterative and developmental review of collaborative processes [ ] strong, engaged leadership [ , , , ] accountability; ownership [ , ] cultural engagement; engagement; diversity/ involvement of community [ , ] trust [ , , , ] transparency [ , , ] need to understand each other's' processes [ , ] systems based thinking/ approach [ , ] cultural awareness; engagement of diverse stakeholders to reflect community needs [ ] credibility [ ] existing relationships [ ] institutional knowledge (experience and relationships) [ , ] revise and revisit mandates based on lessons learned [ , ] clearly defined roles and responsibilities [ , , ] resources available and accessible (including human resource allocation) [ , ] informed staff/ staff are aware of systems in place, increased engagement of staff [ , ] reflexive workforce reflexive human resource protocol to ensure positions are adequately filled & workers are incentivized [ , ] reflexive approach [ , ] adaptability to rapidly changing context [ ] rapid start-up response; shared response guidelines [ ] (continued ) factors that enable one health collaboration: a scoping review multi sectoral coordinating mechanisms/ platforms for engagement [ , , , , ] memoranda of understanding, terms of reference or bilateral agreements to support the development of existing relationships that promote ongoing engagement [ , , , ] in this discussion, the research team suggests thematic factors that may be used by practitioners involved in one health activities to more systematically assess the successes and challenges of multisectoral collaboration, including those contributing to successful outcomes. further research is needed to refine and validate these factors and ultimately support more uniform and rigorous assessments of one health collaborations. the axial coding process allowed for factors reported to facilitate or discourage successful collaboration to be categorized as either a relevant starting condition of collaboration, or as relevant to the process of collaboration. during the data analysis, certain themes within each category of individual, organizational and network factors emerged as relevant to "setting the stage" for effective collaborative processes, while other factors were essential to maintaining the process of collaboration itself. the researchers found that this distinction was critical in our understanding of how successful collaborative processes are initiated. the starting conditions presented in this paper represent the collaborative preparedness and planning necessary to support effective one health processes. in addition, the process of collaboration allows for the emergence of new ways of collaborating. this symbiotic relationship between starting conditions and process, allows us to view the entire system of collaboration. in this system, starting conditions influence the process of collaboration, and the process itself can lead to improvement of structures and processes that will now inform improved starting conditions. this cyclical and emergent process is inherent in collaboration and must be accounted for when considering evaluation and systems-based improvements. individual factors. relevant success factors at the onset of a one health event include an individual's education and training, as well as prior experience and existing relationships. many authors identified existing or previous education and training as enabling factors for collaborative success [ , [ ] [ ] [ ] , , , ] . formal technical education and training of individual workers prior to a health event was critical to prepare the necessary human resources for response efforts. authors noted that foundational technical training during an event was often not possible [ , ] , but that preemptive and collaborative planning did support the development of key relationships, and in some cases, the development of shared protocols used in the response. the absence of formalized training opportunities before an event, both individual technical training and collaborative, were frequently reported as a gap and a challenge to effective one health response [ ] [ ] [ ] ] . shared competencies were suggested as a strategy for standardizing protocols and performance across multiple individuals and organizations [ ] . multiple sources also reported the importance of prior experience in collaborative response efforts and how this established existing relationships to support the work, both formal (i.e. required communication through standard operating procedures) and informal (i.e. loosely structured and based on personal relationships and ongoing professional engagement) [ , , , , , ] . when instituted before a health event occurs, these starting conditions were reported to support a more effective collaboration processes. individual factors that supported the process of collaboration were most frequently reported as workers having access to necessary education and training that was available ad hoc or as "just in time" training to support operations during the health event. examples reported include the use of shared training across organizations to additionally support institutional alignment and partnership with community-level organizations to provide training [ , , ] . many of these trainings were reported to be rigorous and responsive with continuous follow-up to support compliance [ , , ] . williams et al [ ] discussed how ongoing multisectoral disease specific training supported workers to operate within new and existing systems while simultaneously sharpening their technical competence. these training and capacity-building opportunities were reported to provide a platform for better collaboration for outbreak response [ ] . however, ad hoc trainings do not replace or diminish the need for foundational technical training, as formalized education and training were reported as a critical starting condition to facilitate quick mobilization in the case of a health event. our literature review uncovered the role for both strong university-based education and training, and the role that ad hoc or "just-in-time" training can play to meet immediate operational needs during process-based response. organizational factors. factors reported to enable organizational-level collaboration were broadly applicable to both the starting conditions and the processes of collaboration. organizational structures, culture and resources were cited as important elements for creating an enabling environment for effective one health collaboration. the organizations serve to connect the individual worker with a network of one health actors. the organizational structures that support collaboration were often discussed as a success factor. these structures include, but are not limited to, the policies and protocols or systems established within organizations to support technical implementation and collaborative efforts. policies and protocols reported to be supportive included technical guidelines and standard operating procedures, as well as management, response and communication strategies and protocols [ , , , , , ] . in addition, organizations reported the need for functional systems for information and resource management and sharing and reporting both surveillance and laboratory results [ , , , , , , , , , ] . these systems were reported to benefit from being adaptive, flexible/reflexive and improved through iterative feedback and monitoring and evaluation [ , , , ] . organizational culture was reported in multiple key areas [ , , , , , , , , [ ] [ ] [ ] ] . the role of organizational leadership was discussed at length in many of the reviewed publications. authors recognized and identified the importance of having strong and engaged leadership [ , , , ] and the need for leadership to support the iterative and developmental review of collaborative processes [ ] . in addition, organizations benefited from having a culture that supported accountability, ownership, cultural engagement and diversity [ , ] . trust and credibility were consistently reported as a key element of organizational success [ , , , ] , as was the need for both an understanding of each other's processes and systems based thinking [ , , , ] . authors reflected on the importance of cultural awareness, transparency of communication processes [ , , , ] and the engagement of diverse stakeholders who were able to reflect community needs [ ] . human resource-related factors appeared in all three levels of analysis. research suggests that workers need to be trained at an individual level, have defined roles and responsibilities at an organizational level, and need to be able to mobilize their efforts at a network level. at an organizational level, human resources are made up of individual contributors and also function as collective entities that reflect employees' prior experiences, existing relationships, and the collective institutional knowledge of its members [ , , , ] serve to benefit the organizations in which they work. clear roles and responsibilities were consistently reported [ , , ] , as well as awareness of systems in place to support ongoing engagement, operations and information sharing [ , ] . additionally, several authors highlighted the importance of a reflexive workforce, i.e. human resources that were readily available and could be mobilized quickly and efficiently to respond to health event in a rapidly changing context [ , , , ] . network factors. starting condition factors reported to enable collaboration at the network level included network structures, existing relationships, available resources in the face of a health event, and the political environment in place to support these efforts. pre-existing network structures were reported to provide a foundation for effective collaborative efforts to occur across participating organizations. established multisectoral coordinating mechanisms (mcms), also referred to as one health platforms or joint task forces, were often reported as key to assisting with collaboration across a network [ , , , , , , , ] . organizational and network structures provided operational standards that crossed relational and organizational boundaries at all levels of the system-individual, organizational and network-which supported the development of formal relationships at each level. analysis suggested that these systems and relationships need to be in place before the health event. mcms provide a formalized operating foundation in which organizations and individuals could contribute, and formalized roles and responsibilities supported effective human resource mobilization in both organizations and networks [ , , , ] . these structures were often supported by formal policies or agreements such as bilateral agreements or memoranda of understanding (mous) [ , , ] . in addition, operating procedures such as the incident command system (ics) also supported effective mobilization of multiple organizations within the mcm [ ] . finally, the importance of formal structures were repeatedly emphasized as a response to "lessons learned" during challenging responses. on the contrary, lack of existing structures was reported to prevent efficient multisectoral engagement in the preparedness and response to health events [ , , ] . several sources indicated that reporting structures and policies at local, regional, national and international levels support continuity of response and effective implementation in response to health events [ , , , , , , , , ] . these reporting structures and policies allowed for information flow between stakeholders, and the coordination of response efforts across a diversity of individuals and organizations participating in preparedness or response efforts [ , , , , ] . established structures created a foundation for network relationships that support effective outbreak response to a health event [ , , , , , , ] . development of formal and informal relationships prior to a health event allowed individuals, organizations and networks to more effectively respond once an emergency arose. the existence of structural agreements in any form such as mcms, mous, shared standard operating procedures (sops) or bilateral agreements were reported to support the further development of existing relationships to promote ongoing engagement prior to and throughout a health event [ , ] . preemptive planning for potential disease threats was reported to strengthen connections and relationships and support multisectoral disease training, sometimes leading to shared protocols [ ] . additionally, the creation of common goals [ , ] and clearly defined, previously established, roles and responsibilities for individual actors and network partners were reported as necessary in network operations [ , , , , , ] . cultural awareness and the inclusion of diverse stakeholders from government, nonprofit, and private sectors from the national to community level, was consistently reported as a success factor for collaborative efforts if included from the start [ , , , , , , , , , ] . availability of resources, including human resources, that can be easily and efficiently mobilized in a health event was considered an important factor for response [ , , , , , , , , , , ] . authors also noted the importance of a supportive political environment to aid in the development and institutionalization of effective collaborative structures [ , , , ] . a supportive political environment was reported to enable the flow of available financial, human and material resources and empowered decision making [ ] . readily available resources supported rapid mobilization of collaborative efforts when a health emergency occurred. this is particularly impactful given that the absence of these resources and actions was noted across the literature as challenges to effective health response. network leadership and management processes were critical to effective multisectoral response efforts. leadership engagement during a health event allowed for the mobilization and needed support for management processes. by utilizing existing structures and decision-making power, leaders and lead agencies can support managers and the process of management across organizations and networks. emergency response protocols, such as the ics, were frequently reported as mechanisms to this end, by concretely providing a leadership and management structure to support ongoing multi-organizational response. it was particularly useful for identifying a lead agency and establishing structures for regular meetings and communications. in the process of collaboration, relevant network factors included network leadership, management, and the effective and efficient mobilization of resources for response. for example, strong and engaged network leadership was noted as an important success factor for collaboration. when established prior to a health event, factors reported to support network collaborations included identifying a lead agency [ , ] , promoting information sharing and joint decision-making across a network [ , , ] , and convening regular multisectoral meetings [ , , ] . in addition, strong leadership was integral for strategic risk communication across the network [ ] . effective network management during an outbreak was reported in the areas of management practices, monitoring and evaluation (m&e), communication, awareness and ongoing stakeholder engagement. management practices included case and task management through the mcm [ ] , regularly scheduled meetings [ , , ] and development of shared response guidelines and management protocols across the network [ , ] . these management practices, when paired with existing structures, can support rapid start up response in the face of health events [ ] . monitoring and evaluation allowed for the iterative review of the collaborative processes during response efforts, as well as the outcomes of the collaborative process [ , , , , , , ] . monitoring and evaluation was reported as integral in being able to show the outcome of interventions as beneficial or not [ , , ] . the importance of communications cannot be overemphasized and was repeatedly reported as an integral factor for building relationships, trust and supportive organizational culture, and for contributing to effective response processes. both the characteristics and the methods of communications were highlighted as important. characteristics of successful communication included the need to be frequent and honest [ , ] ; timely and consistent [ ] ; reflexive and flexible [ ] and prioritized (risk-based) [ ] , and streamlined [ , ] . additionally, characteristics included the need for communications that build trust [ ] and have a clear purpose [ , , ] . these elements were widely reported to support effective communication within and across organizations [ , , , , , , , , , , , , , ] . communication was deemed most effective when it was regular, frequent, and designed to foster awareness and support the engagement of a range of stakeholders, from local through national, regional and international levels. the mcms, or the use of ics, were often cited as important organizing structures for ongoing communication during a health event, supporting meetings, data collection and information sharing [ , , , ] , underscoring the importance of starting conditions to support communications. multiple methods of communication were reported including electronic communications, list-servs, contact lists and regular meetings; in many cases these were supported through existing mcms [ , ] monthly meetings [ , , ] and establishing clear lines of communication [ , , , , ] were reported as critical. these methods were supported by the use of a variety of methods and platforms such as press briefings, websites, television, newspaper, teleconferencing, listserv, available contact lists, local/ regional/ cross-border meetings and periodic reporting [ , , , , , , ] . additionally, leadership and management processes played a key role in supporting or challenging communication; high-level support, resource allocation, and use of good practices across an organization are foundational for good internal and external communication. closely linked with communication was the reported importance of building shared awareness and diverse stakeholder engagement. awareness included information sharing, education campaigns, jointly coordinated communications and public release of reports with all members of the network and with the public [ , , , , , , , , ] . engagement of diverse stakeholders before, during, and after the response was reported as essential; these stakeholders included community and local actors, national governments, intergovernmental organizations, and operating partners [ , , [ ] [ ] [ ] ] . to facilitate communication across stakeholder groups, adams et al. [ ] and butler et al. [ ] underscored the importance of transparent joint communications specifically between responders and community leaders for efficient and effective response. butler et al. [ ] further reported the success of joint interviews held with stakeholders to support shared understanding of response needs. diverse partners, including foreign militaries, were reported to support foundational infrastructure that allowed other international partners to stay involved when supporting a response effort when they would not have been able to serve effectively on their own [ , , ] . common goals, common interests, and perspective sharing amongst stakeholders were reported to support an effective response to a health event [ , , ] . resource mobilization and allocation during an event, relies heavily on the starting conditions, as well as the communication, leadership and management during the process of collaboration. a number of authors pointed to the importance of being able to mobilize both the material and human resources. once again, the involvement of diverse stakeholders, the use of mcms and management systems such as ics were attributed with the ability to draw upon existing resources. processes characterized as successful included establishing a supply chain with standardized access, delivery, allocation and flow [ , , , ] . human resource mobilization benefited from online recruitment [ ] as well as reflexive human resource protocols to ensure that positions were filled and workers are incentivized and rewarded for participation [ , ] . outcomes reported. although the researchers created a code to capture reported outcomes of collaborative efforts, only a small number of authors reported outcomes of their collaborative processes. outcomes were consistently missing or under-reported in the literature reviewed, and this is likely a result of one health outcomes being difficult to characterize. the few reported included the outcomes of cost reduction and improved safety [ ] , decreased mortality [ ] , reduction in mrsa (methicillin-resistant staphylococcus aureus) cases [ ] , increased stakeholder buy-in [ ] , and a report that multisectoral professional development opportunities resulted from the response [ ] . however, implementation of m&e activities was one of the major gaps in the reports of one health collaboration. the majority of articles reviewed never discussed the evaluation of either the process of collaboration or the resulting outputs or outcomes. this creates a pivotal challenge in understanding how to improve one health operations. the authors noted that outcomes of collaborative efforts were consistently missing or underreported in the literature reviewed. language in collaboration. language used to describe one health work continues to be a challenge when working across disciplines. each discipline contributing, and specifically those authors reporting on these interactions, bring their own nomenclature and vernacular [ ] . as also discussed in the limitations of this work, we encountered challenges in how authors reported on which entities were involved in the response to a health event. organizations, sectors and disciplines were characterized in different ways, making is difficult to find a standard classifying system for the coding. considerations for the evaluation of one health. despite an emphasis on the importance of iterative improvements to collaboration, the implementation of monitoring and evaluation activities was one of the major gaps in the reports of one health collaboration. the majority of articles reviewed never discussed the evaluation of either the process of collaboration or the resulting outputs or outcomes. this creates a pivotal challenge in understanding how to improve one health operations. it became clear in the literature review that there was no standard framework for how to evaluate one health processes [ , ] . although networks and collaborators such as the network on the evaluation of one health are making important advances, practical evaluation tools are still needed [ ] . some authors from public affairs, such as emerson and nabatchi ( ) et al. [ ] have proposed a framework for evaluating outputs, outcomes, and what they refer to as "adaptations" of collaborative processes [ , ] . their work is one of the first to propose an integrated framework that captures collaborative results at all levels of the system, from the target population to the participating organizations, and the network as a whole. the results of this scoping review are intended to support the next steps in supporting one health evaluation. using the factors uncovered in this review, the authors have outlined a reporting framework ( table ) that may help practitioners consider their activities in light of important collaborative starting conditions and process-based factors. the researchers propose this to the one health factors that enable one health collaboration: a scoping review community as a tangible next step that may lead to more effective reporting and potentially evaluation of one health efforts in the future. the proposed framework in table recognizes that each factor will be operationalized within the context of the health event and that flexibility in reporting is imperative. this framework may be useful in providing a common language on how practitioners discuss and report on their one health efforts. in the process of conducting this research, the research team encountered many of that same collaborative challenges as described in the articles reviewed. the research team had to negotiate and re-negotiate ways of working, integrate differing points of view and assign roles in ways to leverage expertise but not reinforce bias. additionally, the researchers had to establish and meet internal standards while also achieving the outward facing objective of finishing the analysis and writing of this article. finally, as with any transdisciplinary work, language was consistently a problem. the inherent challenge of interdisciplinary work is in how we talk about collaboration and the terminology we use to describe both theory and practice. for the research team, creating clear definitions supported the development of a common language. differing approaches can be a significant barrier when active collaboration is not structurally supported, valued, and continuously monitored for health and effectiveness. our efforts reinforce the need for training for those skill sets that fall beyond technical sector-specific training. when grappling with the question of which skills were most important in our collaborative process, we determined that the shared objective of collaboration was the foundation for our ability to integrate the differing expertise that each team member brought to the process. simply, we took continual action to achieve our combined goal including reading new literature, considering new frameworks, learning new things, and asking many questions. the subsequent challenge is, of course, that there are very few formal opportunities to gain access to training around these competencies and mindsets in one health teams. most often, as in our case, it is an ad hoc process that rests on the motivations, shared values, and time available within a team to develop in this way. our review suggests that, while this approach worked for us, it would not be a time or resource-effective approach within the context of a health emergency. thus, one health approaches need to be evaluated to help practitioners decide when and how to most effectively collaborate for their intended purposes. of the , article abstracts screened, only met initial inclusion criteria and the full research articles were obtained. of that subset of articles, only discussed the successes, challenges and lessons learned from operational one health response to a health event. a majority of the articles focused broadly on the need for collaboration between multiple sectors or disciplines with little attention to what factors enable an effective one health response effort. the low number of included articles reflects a broader challenge for the one health community, suggesting the necessity that one health researchers move beyond discussing the inherent need for one health, to actually reporting on the processes, outputs and outcomes of their collaborative efforts. as such, no consistent framework or language was found to report on the process, outputs or the outcomes of one health work in the articles reviewed. in the analysis, the research uncovered factors that supported successful health event response. the researchers were able to make important advances by characterizing these factors as important at the start of collaboration or relevant to the process of collaboration. using these factors, the researchers propose a one health reporting framework which when used to report on one health collaborations, can support the further refinement and identification of success factors for one health. these factors may serve as the basis for developing evaluation metrics and the iterative improvement of 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sharing and public communication public health communication with frontline clinicians during the first wave of the influenza pandemic department of defense west nile virus surveillance in collaboration, cholera, and cyclones: a project to improve point-of-use water quality in madagascar a collaborative strategy to tackle tuberculosis in england north india: delivering dots via collaboration with private providers and non-governmental organizations a blueprint to evaluate one health. front public heal key: cord- -ulqajban authors: jiang, hai title: the expanding vulnerabilities of being utxless date: - - journal: signal transduct target ther doi: . /s - - -z sha: doc_id: cord_uid: ulqajban nan in a recent study. published in signal transduction and targeted therapy, dr. yu liu and collaborators report that the differentiation block in utx-null leukemia cells can be reverted by an lsd inhibitor, highlighting additional ways of targeting utx-deficient malignancies. utx is an important epigenetic regulator, and many human cancers harbor mutations or deletions in this gene. a series of recent studies have established the role of utx as a tumor suppressor in leukemia, lymphoma, pancreatic, and lung cancers. [ ] [ ] [ ] [ ] [ ] in particular, it was also demonstrated that utx escapes from x chromosome inactivation, therefore females have more functional copies of this tumor suppressor than do males, and different dosages of utx in male and females contribute to cancer sex bias. , biochemically, utx is a histone demethylase that removes methyl groups from tri-and di-methyl h k , which negatively regulates gene expression. the loss of utx, therefore, has been demonstrated to affect gene expression, cellular differentiation, and embryonic development. in many cancer models, the role of utx as a tumor suppressor has been linked to epigenetic changes associated with utx loss. , significant changes in enhancer and chromatin accessibility were observed in utx knockout cancer models, and an enzyme activity-independent role has also been proposed for utx as a tumor suppressor. , several studies have shown that utx-deficient cancers are more aggressive and can lead to poor patient survival. , , an important question is how to develop specific strategies to more effectively treat these utx-deficient cancers. several lines of evidence have been established (fig. ) . first, since utx is a h k demethylase, several research groups have shown that inhibitors of ezh , the h k methyltransferase, can strongly inhibit the growth of utxdeficient cancers. , second, in pancreatic cancer models it was found that utx-deficient cancer is sensitive to bet inhibitors, which restrain gene expression from super-enhancers that are altered by utx loss. third, two separate studies suggest that the cellular sensitivity to cytarabine, a cytosine analog that inhibits dna synthesis, is potentially affected by the h k methylation status. in aml, the loss of the h k methyltransferase ezh induced resistance to cytarabine, whereas in lymphoma models the loss of the h k demethylase utx sensitized the cells to this drug. it remains interesting to determine whether the above findings, if applied in clinics, could enhance the treatment outcome of utx-deficient tumors. in an interesting paper in this issue of signal transduction and targeted therapy, research conducted by dr. yu liu's group has further expanded the weaponry against utx-deficient malignancies. to further explore the potential pharmacological weakness of being utx-deficient, an elegant model of utx-null hematopoietic stem and progenitor cells (hspcs) was employed. in human patients, utx mutation in this cell type causes a differentiation block that contributes to the development of mds and aml. while screening for small molecules that are able to release such a differentiation block, the authors identified sp , a selective inhibitor of the h k demethylase lsd . ensuing studies demonstrated that sp promoted the differentiation of utxnull hspcs but not wild type hspcs. gene signatures in utx-null hspcs were also reverted by this drug. importantly, from a cancer treatment perspective, sp promoted the differentiation of utx-deficient aml cells in vivo and extended mice survival. these findings convincingly demonstrated that h k methylation is crucially involved in the differentiation block caused by utx deficiency. it was also the first time that the h k demethylase inhibitor was suggested for fighting utxless cancers. fig. loss of utx in cancer cells results in an altered epigenetic state that renders cancer cells vulnerable to several small molecules and anticancer drugs (in red) interestingly, utx often coexists with two h k methyltransferases mll /mll in the compass complex. this complex, by coordinately removing the repressive h k methylation marker and establishing the active h k methylation marker, mediates crucial decisions in gene expression and cellular differentiation. indeed, in the paper by wu et al., a utx knockout was shown to decrease h k methylation in hspcs, likely through the compass-like complex, and the lsd inhibitor sp decreased the self-renewal ability of utx-null hspcs. together with previous findings, it seems that utx-deficient cancer cells dance delicately on the balance between h k and h k methylation status, and pharmacological weakness can be specifically explored for such an altered balance within utxdeficient cancer cells. the novel findings by wu et al. not only established the relevance of the compass complex for the maintenance of the utx-deficient cancer state but also highlighted additional enzymatic targets in the fight against utxless malignancies (fig. ) . epigenetic drug library screening identified an lsd inhibitor to target utx-deficient cells for differentiation therapy utx mutations in human cancer contrasting roles of histone lysine demethylases in acute lymphoblastic leukaemia loss of kdm a activates super-enhancers to induce gender-specific squamous-like pancreatic cancer and confers sensitivity to bet inhibitors utx is an escape from x-inactivation tumor-suppressor in b cell lymphoma in vivo crispr screening unveils histone demethylase utx as an important epigenetic regulator in lung tumorigenesis utx-mediated enhancer and chromatin remodeling suppresses myeloid leukemogenesis through noncatalytic inverse regulation of ets and gata programs tumor-suppressor genes that escape from x-inactivation contribute to cancer sex bias utx/kdm a loss enhances the malignant phenotype of multiple myeloma and sensitizes cells to ezh inhibition loss of the histone methyltransferase ezh induces resistance to multiple drugs in acute myeloid leukemia open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/ . /. key: cord- -cu tl authors: dondorp, arjen m.; hoang, mai nguyen thi; mer, mervyn; dünser, martin w.; mohanty, sanjib; nakibuuka, jane; schultz, marcus j.; thwaites, c. louise; wills, bridget title: management of severe malaria and severe dengue in resource-limited settings date: - - journal: sepsis management in resource-limited settings doi: . / - - - - _ sha: doc_id: cord_uid: cu tl this chapter summarizes recommendations on important aspects of the management of patients with severe malaria and severe dengue. severe falciparum malaria requires rapid parasitological diagnosis by microscopy or rapid diagnostic test (rct) and prompt initiation of parenteral artesunate. fluid bolus therapy should be avoided in patients without hypotensive shock, and we suggest initial ( h) crystalloid fluid therapy of – ml/kg/h, which may subsequently be reduced to ml/kg/h in patients receiving additional fluids, e.g., through enteral tube feeding. in the minority of those patients presenting with hypotensive shock, we suggest fluid bolus therapy ( ml/kg) with an isotonic crystalloid and early initiation of vasopressor support. enteral feeding in non-intubated adult patients with cerebral malaria can start after h, to avoid aspiration pneumonia. there are insufficient data to suggest this in pediatric cerebral malaria. the diagnosis of severe dengue is commonly with a combined dengue antigen (ns ) and antibody rdt. no antiviral treatment is currently available. dengue shock results from capillary leakage, although hemorrhage or depression of myocardial contractility can contribute. the world health organization guidelines recommend restoration of the circulation guided by pulse pressure, capillary refill time, hematocrit, and urine output. large (> ml/kg) rapid (< min) fluid boluses should be avoided, but prompt fluid administration with crystalloids is essential and should be restricted as soon as the critical phase is over to avoid pulmonary edema. corticosteroids are not recommended, neither is platelet transfusion for thrombocytopenia in the absence of active bleeding or other risk factors. sepsis in resource-limited settings will often have different etiologies to those in western settings, including severe malaria, severe dengue, viral hemorrhagic fevers, melioidosis, typhus, and leptospirosis. the surviving sepsis campaign (ssc) guidelines [ ] are mainly based on evidence from studies on bacterial sepsis. these guidelines are widely applicable, but there are also exceptions. we here focus on disease-specific recommendations for the management of severe falciparum malaria and severe dengue. an international team with extensive practical experience in resource-limited intensive care units (icus) identified key questions concerning the ssc's management recommendations on these diseases. pertinent evidence from resource-limited settings was evaluated using the grading of recommendations assessment, development, and evaluation (grade) tools. severe falciparum malaria is a multiorgan disease caused by plasmodium falciparum transmitted by anopheles mosquitoes. the highest transmission and disease burden is in sub-saharan africa, where severe malaria is largely a pediatric disease, as older children and adults become partly immune. in asia and south america, all age groups may be affected. independent of age, the presenting symptoms with the strongest prognostic significance are coma (cerebral malaria), metabolic (lactic) acidosis, and renal dysfunction. acute respiratory distress syndrome is a common and often fatal complication in adult patients with severe malaria. hypotension occurs infrequently (~ % of cases), and should raise a suspicion of concomitant bacterial sepsis. one of the main pathophysiologic differences of severe falciparum malaria compared to bacterial sepsis is microcirculatory impairment caused by sequestration of parasite-infected erythrocytes, red cell rigidity, and red cell clumping. severe dengue is caused by dengue virus transmitted by aedes mosquitoes. approximately - % of patients will develop severe manifestations. the defining feature is a vasculopathy with increased capillary permeability, causing plasma leakage, reduced intravascular volume, and if severe life-threatening hypovolemic shock [ ] . this "critical phase" typically starts during the period of defervescence and lasts for approximately h. bleeding complications and organ involvement of the brain, liver, kidney, and heart may be additional features and occur more frequently in adult cases [ ] . recommendations and suggestions are summarized in table . . severe malaria is an old disease, and historically, the guidance for fluid management has been to "keep them dry." this approach was subsequently challenged when it was recognized that severe malaria is a severe sepsis syndrome with signs of tissue hypoperfusion and thus might benefit from fluid bolus therapy. the ssc guidelines recommend in patients with sepsis-induced tissue hypoperfusion and suspicion of hypovolemia an initial fluid challenge of minimal ml/kg of crystalloids, to be completed within h, of which a portion may be albumin equivalent; this applies to patients with hypotension or a plasma lactate ≥ mmol/l [ ] . it was shown by various techniques that both children and adults with severe falciparum malaria are intravascular dehydrated [ ] [ ] [ ] although this was debated by some [ ] . small trials in african children with severe malaria suggested a benefit from fluid bolus therapy, in particular with albumin [ ] [ ] [ ] [ ] , as recently reviewed [ ] . however, a subsequent large trial on fluid bolus therapy in african children with severe infections and compensated shock, of which % had falciparum malaria, showed overall a % increase in mortality with fluid bolus therapy ( ml/kg or ml/kg with either saline or albumin). in the children with severe p. falciparum malaria, mortality in the bolus groups was % higher (rr . [ . - . ]) than without fluid bolus therapy [ ] . in the same study, febrile patients with hypotensive ("decompensated") shock were randomized between and ml/kg fluid bolus therapy with either saline or albumin; % of the children ( of ) in the albumin bolus group and % ( of ) in the saline-bolus group died (p = . ). in asian studies in adult severe malaria, rapid fluid resuscitation did not improve metabolic we recommend not to use fluid bolus therapy in normotensive patients with severe falciparum malaria ( a). we suggest that patients receive maintenance isotonic crystalloid fluid therapy ( - ml/kg/h), which may subsequently be reduced to ml/kg/h in patients receiving additional fluids, e.g., through enteral tube feeding ( d). we suggest that in patients with hypotensive shock, fluid bolus therapy ( ml/kg) with isotonic crystalloids be commenced (ungraded) and, if available, early initiation of vasopressor medication (ungraded) timing of enteral feeding in cerebral malaria we suggest initiating enteral feeding in non-intubated adult patients with cerebral malaria after h, in order to limit the possibility of aspiration pneumonia ( b). there are insufficient data to make this recommendation for children with cerebral malaria permissive hypercapnia in ventilated cerebral malaria we suggest not to use a strategy of permissive hypercapnia to achieve ventilation with low tidal volumes in patients with cerebral malaria, because of the high incidence of brain swelling in these patients (ungraded) fluid management in severe dengue we recommend that fluid resuscitation in severe dengue is executed promptly and guided by pulse pressure, capillary refill time, hematocrit, and urine output according to who guidelines and that fluid therapy should be restricted as soon as the critical phase of the disease is over to avoid pulmonary edema ( c). we recommend that rapid administration of large fluid boluses should be avoided, unless the patient is hypotensive ( d). we recommend that in dengue patients with compensated shock, colloid fluids are not used ( a) use of corticosteroids in severe dengue we recommend not to use corticosteroids in the treatment of severe dengue ( b) we recommend not to use prophylactic platelet transfusion for thrombocytopenia in the absence of active bleeding complications or other risk factors (uncontrolled arterial hypertension, recent stroke, head trauma or surgery, continuation of an anticoagulant treatment, existing hemorrhagic diathesis) ( b) acidosis [ , ] , and transpulmonary thermodilution-guided rapid fluid resuscitation resulted in pulmonary edema in / ( %) patients [ ] . one observational study showed no deterioration in renal function or plasma lactate with maintenance fluid therapy between . and . ml/kg/h [ ] . a recent systematic review concluded that fluid bolus therapy with either crystalloid or albumen is not beneficial in severe falciparum malaria [ ] . we recommend not to use fluid bolus therapy in normotensive patients with severe falciparum malaria ( a). we suggest not to use colloid therapy, including albumin % ( c). in normotensive patients, we suggest initial crystalloid fluid therapy of - ml/kg/h ( d). in patients receiving enteral fluids, e.g., through enteral tube feeding, we suggest that this can be reduced to ml/kg/h ( d). this is slightly more conservative than the recommendation in the management guidelines for severe malaria issued by the world health organization, recommending - ml/ kg/h [ ] . there are no data on the benefit of balanced fluids over normal saline. we suggest fluid bolus therapy ( ml/kg) with an isotonic crystalline in patients with hypotensive shock and, if available, early start of vasopressive medication (ungraded). hypotensive shock in a patient with severe malaria could indicate concomitant bacterial sepsis, and be evaluated and treated accordingly. the ssc guidelines suggest administering oral or enteral (if necessary) feeds, as tolerated, rather than either complete fasting or provision of only intravenous glucose within the first h after a diagnosis of severe sepsis/septic shock (grade c) [ ] . early enteral feeding is thought to preserve gut integrity and function, maintain bile secretion and secretory iga, maintain gut-associated lymphoid tissue (galt) resulting in reduced translocation, improve splanchnic blood flow, and act prophylactically against stress ulceration. in patients with severe malaria, malnutrition is common, as is concomitant invasive bacterial infection [ ] . therefore, the recommendation for early start of enteral feeding seems valid for patients with severe malaria, including intubated patients with cerebral malaria. however, in resourcelimited settings, endotracheal intubation of comatose patient is often not practiced, and there might be an increased risk of aspiration pneumonia. we could identify one randomized clinical trial on the timing of enteral feeding in patients with cerebral malaria [ ] . this trial in (mainly) adult bangladeshi patients with cerebral malaria who were not on mechanical ventilation, and thus had an unprotected airway, showed that early (< h) enteral feeding was associated with aspiration pneumonia in / ( %) versus / with late start after h (p = . ). this despite proper positioning of patients, and pre-feed inspection of gastric retention. no difference in the incidence of hypoglycemia was observed. we suggest starting enteral feeding in non-intubated adult patients with cerebral malaria after h ( b). there are insufficient data on pediatric patients with cerebral malaria from african settings. acute respiratory distress syndrome (ards), or pulmonary malaria, is a feared complication of severe falciparum malaria and can also complicate the course of vivax malaria [ ] . the incidence of ards in adult patients with severe malaria is estimated to % and up to % in pregnant women; ards is thought to be rare in pediatric severe malaria [ ] . to protect the lung from the damaging effects of mechanical ventilation, the ssc recommends targeting a tidal volume of ml/kg predicted body weight in patients with sepsis-induced acute respiratory distress syndrome (ards), that plateau pressures be measured in patients with ards and that the initial upper limit goal for plateau pressures in a passively inflated lung be < cm h o [ ] . there are no randomized clinical trials to evaluate this recommendation specifically for ards in the context of severe malaria. however, given the large benefit of this ventilation strategy in patients with other causes of ards, this recommendation should also be valid in severe malaria. the ssc guidelines also suggest that to facilitate the use of a lung protective ventilatory strategy, permissive hypercapnia can be used. it should be noted that availability of blood gas or endtidal co monitoring is limited in resource-limited settings, compromising its safe implementation. there are no randomized clinical trials on the use of permissive hypercapnia in mechanically ventilated patients with severe falciparum malaria. however, in cerebral malaria, brain swelling is common, caused by an increase in intracerebral blood volume including the sequestered parasitized red blood cell mass, vasogenic edema, and cytotoxic edema, and is more prominent in pediatric cases [ ] [ ] [ ] [ ] . because hypercapnia will further increase intracranial pressure, we suggest against the use of permissive hypercapnia to achieve the goal of low tidal volume ventilation in patients with cerebral malaria, as cerebral malaria is associated with brain swelling and variably increased intracranial pressure (ungraded). severe dengue can be defined as a sepsis syndrome. yet, important aspects of the pathophysiology of the circulatory changes are distinct from bacterial sepsis. dengue shock syndrome is characterized by a vasculopathy during the critical phase of the disease, with a plasma leak and hemoconcentration, causing important intravascular volume depletion [ ] . this initially leads to a compensated shock with signs of tissue hypoperfusion and a decreased pulse pressure with preserved systolic blood pressure. this can be followed by life-threatening hypotensive shock. hemorrhage, in particular from the gastrointestinal tract, and more rarely myocarditis, can contribute to circulatory shock. the onset is usually more gradual than with bacterial sepsis. management of patients with severe dengue relies largely on careful monitoring, including early recognition of vascular leakage and proper fluid replacement, combined with prompt but carefully guided volume resuscitation for patients who develop dengue shock syndrome. the ssc guidelines advocate fluid bolus therapy for patients with sepsis-induced tissue hypoperfusion and suspicion of hypovolemia [ ] , which might not be appropriate for patients with severe dengue and compensated shock. in addition, because of the prominent plasma leak, the use of colloids might be beneficial in dengue with hypotensive shock, as opposed to its use in patients with bacterial sepsis. the who guidelines for the management of patients with severe dengue distinguish patients with compensated shock from those with decompensated (hypotensive) shock [ , ] . in compensated shock, recommended initial fluid therapy is with isotonic crystalloid solutions at - ml/kg over h, which can be tapered every few hours if the patient improves guided by the pulse pressure, capillary refill time, hematocrit, and urine output. prudential fluid therapy is important throughout the disease, but in particular fluid administration should be restricted as soon as the critical phase of the disease is over to avoid pulmonary edema. in the same guidelines, it is recommended in patients with hypotensive shock, to resuscitate with crystalloid or colloid solution at ml/kg as a bolus given over min. no randomized clinical trials to support the who fluid resuscitation recommendations could be identified. fluid bolus therapy, and liberal fluid management more in general, was a risk factor for respiratory distress in a large prospective observational study in latin american and asian patients with dengue [ ] . a large prospective observational study in vietnamese children with laboratory-confirmed dengue shock syndrome practiced an initial fluid regimen of ringer's lactate solution at ml/kg over h, with colloid solutions reserved for children presenting with decompensated shock [ ] . the observed case fatality rate with this approach was / children ( . %). we recommend to follow the current who guidelines on fluid management in severe dengue/dengue shock syndrome ( c). we recommend that rapid (< min) administration of large (> ml/kg) fluid boluses should be avoided, unless the patient is hypotensive ( d). there are several randomized clinical trials comparing crystalloid with colloid fluid management for the treatment of patients with severe dengue and compensated shock. in a vietnamese trial, children with moderately severe dengue shock syndrome were randomized to fluid therapy with either ringer's lactate, % dextrose, or % hydroxyethyl starch in a : : ratio [ ] . the need for rescue resuscitation with a colloid or the proportion of children with shock recurrence (which carries a worse prognosis) was similar between treatment arms. an additional two other randomized trials did not show better outcome parameters with (more expensive) colloids over crystalloid fluids [ , ] . a quasi-randomized study from the philippines with alternate allocation of starch versus crystalloid fluids also did not show an additional benefit of colloid therapy [ ] . we recommend that in dengue patients with compensated shock, colloids are not used for initial resuscitation ( a). there is insufficient evidence to recommend fluid choice in severe dengue with hypotensive shock, but there is discussion among experts whether there is a role for colloids in severe dengue patients with hypotension, given the prominent role of capillary leak it its pathogenesis. since current evidence strongly suggests that all hydroxyethyl starches (hes) increase the risk of acute kidney injury and renal replacement therapy [ ] , we suggest not to use hes for fluid resuscitation in patients with severe dengue (ungraded). both humoral and cellular immune responses are thought to be implicated in the pathogenesis of vasculopathy, which is central in the pathogenesis of dengue shock syndrome [ ] . the risk for developing severe disease is increased in secondary heterotypic infections, in which antibody-dependent enhancement (ade) of infection and cross-reactive memory t cells are thought to play a role. these insights have led to the use of immunomodulatory therapy with corticosteroids in severe dengue infection. a cochrane review on patients with dengue shock syndrome identified four randomized or quasi-randomized trials comparing corticosteroids with no corticosteroids or placebo involving participants with dengue shock syndrome [ ] . corticosteroids did not reduce the number of deaths (rr . , % ci . - . ; participants, trials), the need for blood transfusion (rr . , . - . ; participants, trials), or the number of serious complications (convulsions and pulmonary hemorrhage, trial). the evidence was rated low quality as most studies were underpowered or lacked stringent randomization or allocation concealment. corticosteroids were administered after the onset of shock. a more recent vietnamese randomized trial in children with dengue fever evaluated early oral prednisolone therapy ( mg/kg versus . mg/kg versus placebo for days) [ ] . the use of oral prednisolone was not associated with prolongation of viremia and was considered safe. however, no reduction in the development of dengue shock syndrome or other complications was observed with early prednisolone therapy, although the trial was not sufficiently powered to assess efficacy. an additional analysis of the same trial focusing on immunological endpoints did not show an important attenuation of the host immune response with prednisolone treatment [ ] . an additional cochrane review of trials on the early use of corticosteroids in patients with dengue fever identified four studies (including the study discussed above), enrolling a total of children and adults, showing no benefit of corticosteroids regarding mortality or dengue complications, although the evidence was considered low to very low quality [ ] . with the current level of evidence, the use of corticosteroids is not recommended in the treatment of severe dengue ( b). bleeding is a feared complication of severe dengue infection. thrombocytopenia with a thrombocytopathy is invariably present in patients with severe dengue infection. however, vasculopathy is a central and important additional contributor to the bleeding risk [ ] . prophylactic transfusion of platelets is a common practice in dengue-endemic countries [ ] . platelet transfusion is not without risks, since it can cause allergic reactions and transmission of blood-borne pathogens. an open-label randomized study in patients with dengue and a platelet count below , /μl did not show decreased incidence of severe bleeding with prophylactic platelet transfusion [ ] . a non-randomized singaporean study in dengue patients with thrombocytopenia < , /μl, of whom were given prophylactic platelet transfusion, also did not show decreased bleeding episodes in the treatment group [ ] . an observational study from martinique during a dengue outbreak evaluated a conservative strategy to prophylactic platelet transfusion (only if platelets count < /μl, or in case of additional risk factors). a poor correlation between thrombocytopenia and the occurrence of severe bleeding during admission was observed, and the followed conservative transfusion strategy was considered safe [ ] . the who guidelines do not recommend prophylactic platelet transfusion in severe dengue. the results of the adult dengue platelet study (adept, clinicaltrials.gov: nct ), a prospective randomized open-label trial to examine the safety and efficacy of prophylactic platelet transfusion in singaporean adults with severe dengue-related thrombocytopenia (platelet count below , / μl but no bleeding), are pending. in resource-limited settings, the availability of safe pathogens vs. screened blood products can be limited, and platelet transfusion can have important cost implications, supporting restrictive use of platelet transfusion. we do not recommend platelet transfusion for thrombocytopenia in the absence of active bleeding complications or other risk factors such as the use of anticoagulants, existing hemorrhagic diathesis, uncontrolled arterial hypertension, recent stroke, head trauma or surgery ( c). in case of bleeding complications, we suggest transfusion of fresh-frozen plasma (or cryoprecipitate) and platelet concentrate (ungraded). although most recommendations in the ssc guidelines are also applicable for the management of severe malaria and severe dengue, there are some important exceptions, in particular regarding fluid management. open access this chapter is licensed under the terms of the creative commons attribution . international license (http://creativecommons.org/licenses/by/ . /), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license and indicate if changes were made. the images or other third party material in this chapter are included in the chapter's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the chapter's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock in: book dengue: guidelines for treatment, prev control. geneva: world health organization cardiac function and hemodynamics in kenyan children with severe malaria severe p. falciparum malaria in kenyan children: evidence for hypovolaemia fluid resuscitation of adults with severe falciparum malaria: effects on acid-base status, renal function, and extravascular lung water assessment of volume depletion in children with malaria pre-transfusion management of children with severe malarial anaemia: a randomised controlled trial of intravascular volume expansion randomized trial of volume expansion with albumin or saline in children with severe malaria: preliminary evidence of albumin benefit volume expansion with albumin compared to gelofusine in children with severe malaria: results of a controlled trial response to volume resuscitation in children with severe malaria choice of fluids for 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swelling and mannitol therapy in adult cerebral malaria: a randomized trial brain swelling and death in children with cerebral malaria handbook for clinical management of dengue. geneva: who vascular leakage in dengue-clinical spectrum and influence of parenteral fluid therapy clinical characteristics of dengue shock syndrome in vietnamese children: a -year prospective study in a single hospital comparison of three fluid solutions for resuscitation in dengue shock syndrome fluid replacement in dengue shock syndrome: a randomized, double-blind comparison of four intravenous-fluid regimens acute management of dengue shock syndrome: a randomized double-blind comparison of intravenous fluid regimens in the first hour a comparative study of the efficacy of % haes-steril and ringer's lactate in the management of dengue shock syndrome hydroxyethyl starch (hes) versus other fluid therapies: effects on kidney function recent advances in dengue pathogenesis and clinical management corticosteroids for treating dengue shock syndrome effects of short-course oral corticosteroid therapy in early dengue infection in vietnamese patients: a randomized, placebocontrolled trial corticosteroids for dengue-why don't they work? corticosteroids for dengue infection prophylactic platelets in dengue: survey responses highlight lack of an evidence base effectiveness of platelet transfusion in dengue fever: a randomized controlled trial lack of efficacy of prophylactic platelet transfusion for severe thrombocytopenia in adults with acute uncomplicated dengue infection prospective observational study of low thresholds for platelet transfusion in adult dengue patients key: cord- -lw vluxm authors: avila-calderón, eric daniel; flores-romo, leopoldo; sharon, witonsky; donis-maturano, luis; becerril-garcía, miguel angel; arreola, ma. guadalupe aguilera; reynoso, beatriz arellano; güemes, francisco suarez; contreras-rodríguez, araceli title: dendritic cells and brucella spp. interaction: the sentinel host and the stealthy pathogen date: - - journal: folia microbiol (praha) doi: . /s - - - sha: doc_id: cord_uid: lw vluxm as dendritic cells (dcs) are among the first cells to encounter antigens, these cells trigger both innate and t cell responses, and are the most potent antigen-presenting cells. brucella spp., which is an intracellular facultative and stealthy pathogen, is able to evade the bactericidal activities of professional phagocytes. several studies have demonstrated that brucella can survive and replicate intracellularly, thereby provoking impaired maturation of dcs. therefore, the interaction between dcs and brucella becomes an interesting model to study the immune response. in this review, we first will describe the most common techniques for dcs differentiation in vitro as well as general features of brucellosis. then, the interaction of dcs and brucella, including pathogen recognition, molecular mechanisms of bacterial pathogenesis, and intracellular trafficking of brucella to subvert innate response, will be reviewed. finally, we will debate diversity in immunological dc response and the controversial role of dc activation against brucella infection. dendritic cells (dcs) are professional antigen (ag)-presenting cells (apcs) distributed throughout an animal's body that exhibit major histocompatibility complex (mhc) on their surface and are a good source of pro-inflammatory cytokines such as il- (janeway and medzhitov ) . dcs are involved in two main roles: ag uptake and processing and linking innate and adaptive immunity. infectious agents and inflammatory products can induce dc activation, upon which dcs migrate to regional lymphoid tissue, such as lymph nodes, spleen, and peyer's patches (banchereau and steinman ; pulendran et al. ) . in peripheral tissues, dcs are present as immature cells with a poor capacity to stimulate t cells but are highly equipped to capture ag (banchereau and steinman ) . when immature dcs capture microbial agents or their products by phagocytosis, they travel away from the infection site and translocate to the t cell areas of the proximal lymph nodes (pulendran et al. ) . dcs interact with a variety of t cells and drive the immune response (colonna et al. ) . for instance, dcs expressing mhc-i interact with cd + t cells and induce a cytotoxic immune response, while mhc-ii + dcs interact with cd + t cells and induce a mixed th /th immune response (itano et al. ; mantegazza et al. ) . cd + t cells, in the presence of mature dcs and il- , become interferon-γ (ifnγ)-producing t cells. ifnγ activates microbicidal macrophage properties and promotes an inflammatory (th ) response (itano et al. ; pulendran ). on the other hand, il- produced by dcs induces cd + t cells to differentiate into th cells. th cells secrete il- and il- and subsequently activate eosinophils as well as help b cells make agspecific antibodies (hochrein et al. ) . dcs originate in bone marrow from a common precursor for macrophages and dcs, the macrophage and dc precursor (mdp). mdp gives rise to the common dc precursor (cdp), which serves as a common progenitor for conventional dcs (cdcs) and plasmacytoid dcs (pdcs) (poltorak and schraml ) . in vivo, the development of all dcs is mostly dependent on fms-like tyrosine kinase ligand (flt l). in bone marrow, flt l acts on mdp and/or cdp and activates different transcription factor cascades to give rise to different dc subsets in a steady state (poltorak and schraml ) . granulocytemacrophage colony stimulating factor (gm-csf) is the other important growth factor for dcs development. although gm-csf does not have a major role such as flt l in dc development, it seems to balance dc subsets. for example, gm-csf decreases pdcs and cd α + dc differentiation by blocking interferon regulatory factor- (irf ) via signal transducer and activator of transcription- (stat ) (zhan et al. a ). gm-csf and flt l have been used to generate dc subsets in vitro. bone marrow cells stimulated with recombinant flt l give rise to three dc subsets (referred to as fl-dcs): pdcs and dc equivalents to the cdc subset. although fl-dcs do not express the same markers as their in vivo cdcs counterparts, they use the same transcription factors, produce similar cytokine and chemokine profiles, and have similar efficiencies for ag presentation as splenic cd α + and/or cd α − dcs (brasel et al. ; naik et al. ) . generally, gm-csf is used in combination with il- to generate immature dcs from peripheral cd + monocytes or bone marrow cells, and a further maturation step with a cytokine cocktail (tnfα, or bacterial-ag is required to maintain a dc phenotype (soruri and zwirner ) . gm-csf-derived dcs (referred as to gm-dcs) are equivalent to myeloid dcs (cd b high , cd c + , d + , and cd α − ) and differentiation is independent of stat (zhan et al. b) . gm-csf plus il- are broadly used for the generation of dcs; however, alternative methodologies such as the addition of inflammatory cytokines have been developed to obtain dcs. for example, il- allows the generation of dcs equivalents to gm-dcs from cd + monocytes without the use of gm-csf (saikh et al. ). differences in surface marker molecules on dcs subsets might be indicative of differences in the nature of the t cell response (pulendran et al. ). cd c and mhc-ii seem to be constitutively expressed in most dc subsets; however, these surface markers do not display all different phenotypes and functions of dc subsets. dc subsets are classified based on different phenotypic characteristics such as the expression of intracellular and surface markers, transcription factors used for their differentiation, and anatomic location (schlitzer et al. ) . in the mouse, two well-characterized cdc subsets have been described: the cdc subset includes dcs expressing cd α + (in lymphoid organs) or cd (in peripheral organs), while the cdc subset is comprised of cd + cd b + dcs in the spleen and cd + cd b + dcs in nonlymphoid tissues such as the lung, intestine, and dermis. the cdc subset requires irf and is the only group that expresses toll-like receptor (tlr ), whereas cdc subsets require transcription factors such as irf . phenotypic and functional studies have revealed similarities between mouse and human cdcs; in humans, cd + dcs and cd c + dcs resemble mouse cdc and cdc , respectively (guilliams et al. ; schlitzer et al. ) . pdcs are the other major subset of dcs that develop from the cdp in bone marrow. pdcs belong to a different lineage of dcs; pdcs express b , cd , cd , and cd and produce large amounts of type- interferon (ifn-α) (ifn-producing cells or ipcs) (seillet and belz ) . in lymphoid and nonlymphoid tissues, different subsets of dcs have been found. for example, in the spleen, two separate subsets of dcs have been described: lymphoid dcs (cd α + cd b − cd + cd + ; referred to as cd α + ) located at the t cell zone in the spleen and myeloid dcs (cd α − cd b + cd + d + ; referred to as cd α − ) in the red pulp at the marginal zone. these dcs subsets not only differ in their surface markers and location but also in their capacity to process ag and cytokine production; (i.e., il- production is restricted to lymphoid dcs) (hey and o'neill ) . dcs take up and process a great variety of ags, including those derived from bacterial pathogens. once dcs process bacterial ags, they trigger different signaling pathways to induce the appropriate immune response. however, bacterial pathogens, especially those able to live in an intracellular niche, have developed a plethora of strategies to subvert dc responses. here, we focused especially on the interaction between the bacterial pathogen brucella spp. and dcs. brucella species are intracellular facultative gram-negative pathogens that reside inside various host cell types, including dcs. brucella species avoid the host immune response by utilizing several clever strategies such as avoiding intracellular destruction mechanisms and inhibiting dc maturation, ag presentation, and t cell activation (de figueiredo et al. ) . thus, dcs could serve as a model to understand the brucella pathogenesis and to identify new targets for vaccine development against brucellosis. in the following sections, we describe some important characteristics of the interaction between brucella spp. and dcs. the stealthy pathogen: brucella spp. brucella species are facultative intracellular gram-negative bacteria, noncapsulated and nonspore-forming. brucella causes the zoonosis named brucellosis, also known as malta fever. brucellosis is endemic in many countries of the world, including latin america, the middle east, africa, central asia, and the mediterranean (pappas et al. ) . to date, brucella species have been described and classified according to the preferred animal host: b. melitensis (sheep and goats), b. abortus (cattle), b. suis (swine), b. canis (dogs), b. ovis (sheep) and b. neotomae (woodrats), b. ceti (dolphins, porpoises and whales), and b. pinnipedialis (seals) (pappas ) . in the last few years, new species have been found in different sources: b. microti was found in voles and foxes, b. inopinata was isolated from a breast implant infection, and recently, b. vulpis was isolated from red foxes (scholz et al. (scholz et al. , (scholz et al. , . in both humans and animals, brucella targets the mucosa mainly through the respiratory epithelium or conjunctiva, and then the bacterium is internalized by phagocytes leading to systemic dissemination. however, little is known about the specific cells that allow bacterial internalization. in humans, brucella infection is mainly acquired through the ingestion of unpasteurized dairy products containing the bacteria; unpasteurized cheese and raw milk are commonly involved in brucellosis outbreaks in underdeveloped countries. brucellosis is associated with some occupational groups such as farmers, veterinarians, ranchers, and meat-packing employees who may have contact with sick animals (seleem et al. ) . the clinical manifestations of human brucellosis comprise nonspecific symptoms; the most common include undulant fever, myalgia, arthralgia, chills, headache, and weakness. approximately - % of brucellosis patients will develop chronic infection and/or some complications such as spondylitis, arthritis, endocarditis, or neurobrucellosis (dean et al. ; guzmán-hernández et al. ) . in animals, brucella causes spontaneous abortion, stillbirths, and decreased fertility and milk production in females, whereas males experience orchitis, epididymitis, and fertility issues (seleem et al. ) . brucella evades the innate immune response, impairing dc maturation and subverting macrophage and neutrophil killing; indeed, brucella resists and survives the bactericidal actions of neutrophils but does not replicate inside these cells (barquero-calvo et al. ). at the initial stage of infection, brucella induces an adaptive immune response involving the microbicidal activity of macrophages (activated by ifn-γ of cd + and cd + t cells), and then infected macrophages are eliminated by cytotoxic t cells, and brucella opsonization/ phagocytosis is induced by igg antibodies (ahmed et al. ). in the case of chronic brucellosis, cd + and cd + t cells increase in the spleens of infected mice, which exert an immunosuppressive state regulating t cell activity. in patients with chronic brucellosis, high levels of tgf-β have been found in their sera, and furthermore, no t cell proliferation after stimulation with brucella antigens has been observed, indicating suppressed t cell function mediated by tgf-β (elfaki and al-hokail ) . the chronicity of brucellosis is due to brucella's ability to survive inside professional phagocytic cells and to evade the host immune response. brucella can infect different cell types, including epithelial cells, trophoblasts, b cells, dcs, macrophages, synoviocytes, and t cells (baldwin and goenka ; giambartolomei et al. ; goenka et al. ; velásquez et al. ) . brucella persistence occurs in mononuclear phagocytic system tissues, but the bacteria can also be found in reproductive organs, bones, and joints (ahmed et al. ). once infection is established, brucella uses infected cells as a replicative niche and reservoir, inhibiting apoptosis and promoting chronic infection (baldwin and goenka ) . omp from b. melitensis contributes to the apoptosis inhibition induced by tnfα in raw . macrophages by the classical and mitochondrial pathway (zhang et al. ) , while b. abortus induces the a protein in raw . macrophages and promotes apoptotic pathway inhibition (wei et al. ) . in mice infected with brucella, the spleen is the main organ in which the pathogen persists. susceptible il- p ko (knock out)-mice infected intranasally with b. melitensis showed spleen infection after days postinoculation. the bacteria were located in t cells on the white pulp of the spleen, and the main cells harboring brucellae were cells resembling m a macrophages. these reservoir cells infected with brucella expressed cd c + , cd + , and arg + ; these markers were observed also in lung cells infected with mycobacterium tuberculosis (hanot mambres et al. ) . the deficient th response in ko mice favoring the differentiation of the m a macrophages population is indicative of a th environment. previously, xavier et al. found that m a macrophages were induced during chronic infection provoked by b. abortus; interestingly, macrophages containing high numbers of bacteria were observed (xavier et al. ) . because m a macrophage polarization is induced by il- / il- via stat , bacterial colonization in the spleen and impairment of m a macrophage surface markers in double ko mice (il- p −/− stat −/− ) were expected. however, hanot-mambres et al. found that neither brucella infection in il- p −/− stat −/− mice nor the surface markers expressed in macrophages were affected. based on the observations of xavier et al. and hanot-mambres et al., it is probable that myeloid cells are the critical reservoir in the spleen that may promote chronicity during brucellosis. there is controversy over the ability of brucella to invade multiple sites of the gastrointestinal tract; for example, b. abortus has been isolated from the gastrointestinal tracts of calves previously infected by the oral route with spiked milk (carpenter ) . additionally, rossetti et al. inoculated b. melitensis ( × ufc) in the intestinal loops of calves, and while the bacteria were recovered after -min and -h postinoculation in the peyer's patches, no histopathological damage was observed in the tissue (rossetti et al. ). on the other hand, von bargen et al. showed that mice infected with b. melitensis m ( bacteria) by the oral route induced the formation of granulomas composed of epithelioid cells and a few neutrophils in the cervical lymph nodes (cln), although no dissemination was observed (von bargen et al. ) . at the onset of brucella infection in mice (from to days postinfection), there are no morphological or cell distribution changes in the spleen; additionally, the bacteria may be detected in the liver at the sinusoids inside the kuppfer macrophages. in the acute phase (from the third day to the secondthird week postinfection) the number of bacteria increases in the organs, and cell infiltration is apparent. in the liver, granulomas are evident in the first week after infection; granulomas are composed of macrophages and dcs, accompanied by plasma cells and lymphocytes (de figueiredo et al. ; grilló et al. ; yoneyama and ichida ) . the spleen in the acute phase, after the first week of infection, shows increased size with macrophage and neutrophil infiltration, while the bacterial burden remains constant (enright et al. ). at the end of the acute phase, the numbers of macrophages, neutrophils, and b and t cells increase slightly, as well as levels of il- , il- , and ifnγ murphy et al. ). in the chronic phase (ranging from to weeks postinfection), granuloma size in the liver increases, and multinucleated giant cells and polykarions are observed in the centers of the granulomas (cheville et al. ) . similarly, granuloma number and size in the spleen increase. the initial chronic brucellosis phase starts around the second-third week, and bacterial burden slowly decreases at approximately days postinfection. moreover, splenomegaly decreases, and macrophage infiltration is reduced (enright et al. ; grilló et al. ) ; however, the liver shows clearance at approximately - weeks postinfection, whereas the spleen is highly colonized during the chronic stage . from outside to inside: how do dcs allow brucella entry? once ingested, brucella spread during transport through the digestive tract. some research has focused on studying oral infection of brucella in vivo. one such report by rossetti et al. involved healthy calves infected with cfu of b. melitensis in the distal jejunum and ileum (ligated ileal loop model). the authors demonstrated that b. melitensis modulates inflammatory responses, limiting intestinal histopathological lesions, invading peyer's patches, and disseminating to the mesenteric lymph nodes to induce bacteremia (rossetti et al. ) . likely, brucella translocates through the mucosal epithelium and is taken up by the dc subset present in the subepithelial dome of the ileal peyer's patch. this dc subset secretes high levels of lysozyme (termed lysodcs) and is highly efficient at capturing salmonella enterica serovar typhimurium (hereafter referred as s. typhimurium) (lelouard et al. , ; however, further investigation is needed to clarify whether lysodcs are involved in brucella uptake. in a retrospective study analyzing patients diagnosed with brucellosis acquired via the ingestion of unpasteurized contaminated dairy products, most bacteria were found in the cervical lymph nodes (clns) ( - %) and occasionally disseminated to other organs. clns drain the oral cavity, and most of the bacteria acquired by the oral route reach this regional lymph node (von bargen et al. ; yamazaki et al. ) . upon arrival at the clns, bacteria are captured or disseminated. therefore, clns represent an efficient trap and reservoir for pathogens able to cause chronic infections, and the tissues drained by cnls are the most successful route of entry to the host for pathogens acquired orally. as mentioned above, ingestion of unpasteurized contaminated dairy products is the most common route by which people acquire brucella infection; however, it has been demonstrated experimentally that this is not the most efficient route since the bacterial burden required to infect mouse ranges from to cfu. in infections caused by pathogens transmitted via the oral route, regional lymphadenitis is more common than diarrhea, indicating that the upper mucosa is the normal site of entry for these pathogens instead of the gastrointestinal tract. taking into consideration these observations, it has been proposed that brucella is not a proper enteropathogen (gorvel et al. ; von bargen et al. ) . another report described the oral infection of mice with b. melitensis m ( bacteria/mouse) using spiked milk. oral infection led to early colonization of the cln, where brucella proliferated and persisted up to days postinfection (chronic steady state). moreover, local inflammation with upregulation of ifnγ and monocyte/macrophage was observed, but dc infiltration was not (von bargen et al. ) . under steady-state condition, the airways and trachea contain four specific dc subsets: intraepithelial dcs (cd c high cd b low cd + ), dcs localized at the submucosa (cd b high cd − cd c high ), monocytic dcs (cd b high cd − mhc-ii + ), and pdcs. intranasal infection of mice with s. typhimurium increases the percentage of dcs located at the submucosa level, whereas intraepithelial dcs decrease. however, both of these dc subsets upregulate the expression of costimulatory molecules such as cd and cd , indicating clear activation of the immune system in the lung. since brucella is considered highly infectious, the inhalation of brucella aerosols from aborted placenta or through laboratory exposures also leads to infection. in the case of the intranasal route, mice inoculated intranasally with × b. abortus were compared with a group of mice infected with s. typhimurium. the results showed that pulmonary b. abortus infection did not change the distribution of pulmonary dc subsets in the lung nor upregulate the expression of costimulatory molecules from h up to days postinfection (onset to acute infection) (archambaud et al. ; del rio et al. ; smither et al. ; traxler et al. ; vermaelen and pauwels ) . alveolar macrophages have a major role in protecting against b. abortus infection; macrophages inhibit dc action, and only when the macrophages are overwhelmed will dcs interact with the pathogen. upon partial macrophage depletion, the induction of inflammatory response by dcs was observed (archambaud et al. ) . brucella was able to survive and replicate inside macrophages and dcs; alveolar macrophages along with dcs transport brucella to the mediastinal lymph node, contributing to the dissemination of infection from the lung to other parts of the infected host (de figueiredo et al. ) . another common route for brucella infection in humans and animals linked to bacterial aerosols is the conjunctival route. although the eye is considered an immune-privileged organ, a subset of immature and mature dcs were found in the human and mouse cornea, at the epithelium and stromal zone. at the normal cornea, cdcs and langerhans dcs have been observed: cd + (cdc ) and cd c + (cdc ) in humans and cd + (cdc ) and cd b + (cdc ) in mice (knickelbein et al. (knickelbein et al. , . however, it is not clear which dc subset is involved in brucella infection. although the conjunctiva is a less common route of natural infection with brucella, this route has been used for vaccination to control caprine and ovine brucellosis, employing a b. melitensis rev. live vaccine (blasco ) . the conjunctiva is related to the conjunctiva-associated lymphoid tissue (calt); an interconnection in the mucosal system allowing a distal immune mucosal response when the local mucosa is stimulated has been demonstrated, and m cells might be involved in the translocation of antigens (pulendran and ahmed ) . for example, when sheep were infected by the conjunctival route with × cfu of virulent b. melitensis h , the animals developed acute systemic brucellosis located in the peripheral lymph nodes but eradicated the bacteria at the local site of inoculation (suraud et al. ). the first step in establishing an intracellular lifestyle is entry of the bacterium into host cell, followed by avoidance of any intracellular destruction mechanisms. some brucella surface molecules are involved in host cell entry. one of these molecules is the sp protein, found in b. melitensis, b. abortus, and b. suis; sp is encoded by the bmei locus from b. melitensis m, and its function is to mediate entry of the pathogen into epithelial cells (castañeda-roldán et al. ; hernández-castro et al. ) . moreover, the efp gene and the pathogenicity island bab _ - from b. abortus promote adhesion and internalization into macrophage and epithelial cells (czibener and ugalde ; iannino et al. ) . inside host cells, especially dcs, brucella modulates the expression of surface molecules related to maturation, costimulation, migration and/or ag presentation, as well as influences cytokine production (billard et al. a; elfaki et al. ; fabrik et al. ). there is contradictory information in the literature regarding the effects of brucella on dc maturation/activation. therefore, we divided the following section into two parts: the first will show evidence of dc activation/maturation by infection/stimulation with brucellae or their antigens (outer membrane proteins (omps), lipopolysaccharide (lps) or heat-killed bacteria), and the second part will describe reports demonstrating impaired activation/maturation. moreover, we include a table which summarizes all these findings ( table ) . zwerdling et al. ( ) observed the activation and maturation of gm-dcs infected with b. abortus. infected gm-dcs upregulated cd , cd , cd , cd , ccr , mch-ii, and mhc-i surface molecules and induced cytokines such as il- , il- , il- , and tnfα (required for the induction and modulation of adaptive immune response). gm-dc maturation was independent of bacterial viability since heat-killed b. abortus (hkba) induced upregulation of costimulatory molecules, chemokines receptors, and cytokines. gm-dcs maturation was attributed to brucella lipoproteins; gm-dcs stimulated with lipidated omp induced the upregulation of surface molecules and cytokines, whereas purified b. abortus lps or unlipidated omp did not (zwerdling et al. ). using murine bone marrow-derived dcs (differentiated with gm-csf plus il- ; referred as bmgm-dcs), macedo et al. observed the upregulation of costimulatory molecules (cd and cd ), mhc-ii, as well as significant cytokine production (tnfα and il- p ) at -h poststimulation with hkba relative to unstimulated cells (macedo et al. ) . b. melitensis, b. abortus, and b. suis are the main species causing infection in humans and are mainly transmitted zoonotically (guzmán-hernández et al. ) . a large number of studies have focused on these brucella species and their interactions with the host due to the importance, incidence/ frequency, and virulence of infection. compared with the three species mentioned above, b. canis and b. ovis are the only two natural rough brucella species that infect their animal host. b. canis, with a lower incidence of human cases, also represents a public health risk; however, until now, no studies examining its interaction with dcs have been reported (marzetti et al. ) . in humans, b. canis causes a mild or asymptomatic infection, whereas in dogs, like other animals, brucellosis induces abortion in females and epididymitis/ orchitis in males, as well as some complications such as diskospondylitis (wanke ) . it is possible that differences in human and canine brucellosis may be attributed to differences in the immune response. when infected with b. canis, human and canine gm-dcs upregulate the expression of cd , cd , and class ii molecules -h postinfection. on the other hand, cytokine gene profile expression differs: while canine gm-dcs overexpress il- a, il- , ifnγ, il- p , human gm-dcs overexpress il- p , tnfα, and il- β. at the protein level, the cytokine profiles are very similar; high production of infγ and il- a in canine gm-dcs and il- and tnfα in human gm-dcs. clearly, b. canis elicits a th response in human gm-dcs that is essential for the pathogen eradication, and this is likely the reason symptoms in humans are less severe. gm-dcs infected with b. canis, elicit a th /th cytokine profile (pujol et al. ) . the role of the th response in brucellosis is not clear yet; il- has been implicated in osteoarticular complications in brucellosis. additionally, th cells producing ifnγ/il- are related to inflammatory disorders (giambartolomei et al. ) . another intracellular pathogen, m. tuberculosis induces a combined th /th response and the formation of granulomas (bystrom et al. ; lyadova and panteleev ) . it is likely that the ifnγ/il- cytokine profile elicited by b. canis in canine dcs is associated with osteoarticular complications in dogs such as diskospondylitis. despite the stealthy ability of brucella to reside inside professional phagocytes and impair the immune response, the role of brucella in dcs activation/maturation seems to be contradictory. human monocyte-derived gm-dcs infected with smooth b. suis or b. abortus decreased the expression of maturation surface markers (chemokine receptor ccr , cd ), costimulatory molecules (cd , cd ), and mhc-ii relative to gm-dcs infected with rough mutants. moreover, brucella infection decreased ag presentation, as well as the production of the inflammatory cytokines il- and tnfα (billard et al. b) . tnfα is essential for dc maturation; in a previous report, b. suis omp impaired tnfα production on thp- monocytes (jubier-maurin et al. ) . recently, it was demonstrated that b. suis omp dysregulates microrna expression on porcine alveolar and murine macrophages, leading to the inhibition of nf-κb signaling and tnfα suppression (billard et al. b; luo et al. ) . the decrease in inflammatory cytokines and the poor t cell stimulation of brucellainfected gm-dcs avert the induction of a th immune response, which is essential for eradicating intracellular bacteria (billard et al. b) . salcedo et al. ( ) observed decreased expression of cd , cd , cd , mhc-ii, and low cytokine concentrations of il- p , tnfα, il- , and ifn-β in bmgm-dcs infected with live b. abortus relative to bmgm-dcs infected with s. typhimurium. these results suggest dc maturation impairment in cells infected with brucella (salcedo et al. ) . in a comparative study, human monocyte-derived gm-dcs were infected with different intracellular bacterial pathogens: orientia tsutsugamushi the etiologic agent of scrub typhus, coxiella burnetii responsible for q fever, and brucella. although these bacterial pathogens have an intracellular lifestyle, they have different preferential niches for their replication; while b. abortus and c. burnetii reside in an intracellular vacuole, o. tsutsugamuchi is located in the cytoplasm (benoit et al. ; gorvel et al. ; tantibhedhyangkul et al. ) . gorvel et al. ( ) infected gm-dcs with b. abortus , c. burnetii, and o. tsutsugamuchi for h. compared with gm-dcs infected with c. burnettii or o. tsutsugamushi, b. abortus-infected gm-dcs showed impaired immune responses. however, the expression of cd (a dc maturation marker), and costimulatory molecules (cd and cd ) was relatively higher in o. tsutsugamushi-infected gm-dcs, than b. abortus -and c. burnetii-infected cells (gorvel et al. ) . despite the intracellular lifestyle of these pathogens, notable differences in gm-dc immune responses were observed; further analysis involving transcriptomics was performed on infected gm-dcs. transcriptomic analysis revealed high levels of ifn-β in gm-dcs infected with o. tsutsugamushi, whereas ifn-β expression was impaired in brucellaand c. burnetti-infected gm-dcs. according to the results obtained, ifn type i was impaired due to defective activation of p in gm-dcs infected with b. abortus and c. burnetii (gorvel et al. ). compared with human or murine dcs, bovine monocytederived gm-dcs are not permissive for b. abortus proliferation. heller et al. ( ) found low expression of costimulatory molecules (cd and cd ) and cytokines (il p and ifnγ) in bovine monocyte-derived gm-dcs infected with b. abortus, but no bacterial proliferation was observed (heller et al. ) . while the proliferation of bacteria in macrophages is commonly observed, intracellular bacterial proliferation in dcs is poor, probably because phagolysosome acidification is decreased in these cells. it is well known that brucella requires phagolysosome acidification for the expression of crucial virulence factors for intracellular survival (starr et al. ). it is evident that there are discrepancies between the results reported in the literature regarding dc activation/maturation, probably due to the dc subsets used in experiments, the species of brucella or antigens, and the time points used for stimulation/infection, among others. billard et al. ( b) found low activation levels of cd , costimulatory molecules, and chemokine receptors at -h postinfection in human gm-dcs infected with b. suis, whereas zwerdling et al. ( ) observed high expression levels of surface molecules at early time points ( -h postinfection) in human gm-dcs infected with b. abortus. likely, the induction of surface molecules reaches its maximum intensity at different time points after infection, explaining why differences were observed between these reports. moreover, billard et al. ( b) performed gm-dc differentiation for days, while zwerdling et al. ( ) did so for days. additionally, zwerdling et al. ( ) induced a further maturation step using different antigens (escherichia coli lps, b. abortus lps, hkba, pam cys, lipidated-omp , and unlipidated-omp ), whereas billar et al. stimulated the cells after differentiation with cytokines. despite the impaired gm-dc maturation observed in the experiments of billard et al., brucella infection was slightly detected based on the immune response, since low cytokine levels were induced. based on the observation of brucella infection in dcs, it can be hypothesized that dc activation mediated by brucella is present at the onset of the immune response, while at later time points, the pathogen might avert a th response by establishing chronic infection through different host immune system evasion mechanisms (billard et al. a (billard et al. , b zwerdling et al. ). reports of murine dcs infected with brucella have presented contrasting information. macedo et al. ( ) used heat-killed b. abortus ( bacteria/cell) to infect murine dcs, observing upregulation of costimulatory molecules, as well as significant cytokine production. salcedo and colleagues used living b. abortus strain ( - bacteria/ cell) and observed decreased expression of surface molecules and low cytokine concentrations. the stealthy nature of brucella hampers the immune response of dc subsets human and mouse gm-dcs have been the most commonly used model to study brucella-dc interaction. since brucella can disseminate and reach different lymphoid tissues such as the spleen, the bacteria can interact with different dc subsets. papadopoulos et al. ( ) demonstrated that brucella can infect different bone marrow-derived dc subsets. bone marrow cells were differentiated in gm-dcs, fl-dcs (pdcs, and cd α + , and cd b + equivalent to cdcs), gm/fl-dcs (gm-csf plus flt l dcs), and gm/ -dcs (gm-cfs plus il- dcs) and then infected with virulent b. abortus strain. in the case of gm/fl-dcs, gm-csf decreased cd α + dcs and pdcs, while cd b + dcs increased these subsets. on the other hand, gm/ -dcs produced higher levels of ifnγ and il- (promoting th response) than gm-dcs (brasel et al. ; naik et al. ; papadopoulos et al. ) . when dc subsets were infected with brucella, different infection kinetics were observed; for example, fl-dcs and gm/ -dcs showed higher infection levels at early time points than gm/fl-dc or gm-dcs. brasel et al. ( ) observed the similar efficiency of fl-dcs and gm-dcs for ovalbumin (ova) uptake; however, brucella uptake may be mediated by different mechanisms (i.e., lipid rafts and/or phagocytosis). at late times postinfection, brucella uptake levels were similar in all different dc subsets. on the other hand, gm-dcs and gm/fl-dcs showed late maturation (high levels of cd , cd , and mhc-ii at h), while gm/ -dcs and fl-dcs (only cd α + and cd b + dcs) showed early maturation at -h postinfection. gm-dcs and gm/fl-dcs induced high levels of il- corresponding to late maturation of cells. fl-dcs and gm/ -dcs showed higher expression of infγ, il- , il- , ifnβ, and il- β as well as higher levels of chemokine and chemokine receptor (ccl and ccr ) corresponding to early maturation. moreover, in dc subsets with inflammatory cytokine profiles, brucella replication was lower than that in gm-dcs and gm/fl-dcs (papadopoulos et al. ) . during brucella infection, il- production is commonly observed; mouse gm-dcs produce il- at -h postinfection and splenocytes from infected mice produced high levels of this cytokine up to weeks postinfection. in this sense, dcs, cd + t cells, and macrophages from the spleen are the main il- producers . il- is considered an immunoregulatory cytokine that is able to suppress il- production and modulate an excessive th response (couper et al. ) . during brucella infection, il- production at early times is crucial for evolution to chronic brucellosis, which includes the inhibition of pro-inflammatory cytokines. xavier et al. ( ) found that at early times during brucella infection, il- produced by cd + cd + t cells has a regulatory effect during the acute phase, which decreases inflammation and tissue damage; this immune response protects bacteria from being eliminated. valuable data were obtained in experiments using il- ko mice infected with b. abortus. il- ko mice showed lower bacterial loads than wild-type mice. additionally, gm-dcs from il- ko mice produced higher levels of proinflammatory cytokines than gm-dcs from wild-type mice. altogether, these results indicate that il- has a detrimental effect on the protective immune response against brucella (couper et al. ); xavier et al. ) . the stealthy nature of brucella is attributed mainly to the smooth lps on its surface. typical lps is composed of lipid a, a core oligosaccharide, and an o-side chain polysaccharide. brucella lps is an unbranched homopolymer ranging from to glycosyl subunits; the o-side chain is linked to a core oligosaccharide, and the lipid a is linked to the core oligosaccharide (cardoso et al. ) . lps plays an important role in brucella pathogenesis, protecting from cellular cationic peptides, reactive oxygen species, and complement-mediated lysis and is involved in invasion. brucella lps is less cytotoxic than enterobacterial lps (cardoso et al. ; von bargen et al. ) . there is some controversy as to whether or not brucella lps hampers the dc immune response; as mentioned above, some authors have reported dc maturation/activation inhibition by smooth brucella strains, whereas dcs infected with rough strains showed maturation/ activation and proinflammatory cytokine profiles. outer membrane vesicles (omvs) are nanovesicles released from the outer membrane of gram-negative bacteria. omvs contain proteins from the outer membrane, periplasm, and cytoplasm, as well as lps. these vesicles have been reported in b. melitensis and b. abortus. purified vesicles from b. melitensis m and the rough mutant b. melitensis vtrm were used to measure the levels of cytokine expression in murine bmgm-dcs at different time points. the results revealed early expression of il- , ifnγ, and tnfα in bmgm-dcs stimulated with omvs from b. melitensis rough strain but not expression with omvs from a smooth strain (avila-calderón et al. ). these differences could be attributed to the o-side chain lacking the lps of omvs from the rough strain. conde-Álvarez et al. ( ) found a relationship between the core oligosaccharide and immune response impairment in dcs. in their experiments, the authors used the b. abortus wadc mutant; the wadc gene encodes a mannosyltransferase that is important for the assembly of complete lps. the b. abortus wadc mutant exhibited a partially defective core oligosaccharide but an intact lipid a and o-side chain. bmgm-dcs were stimulated with lps purified from the wadc mutant and wildtype strain. the mutant lps induced higher production of il- , tnfα, and cd and mhc-ii overexpression, but cells stimulated with the wild-type lps did not (conde-Álvarez et al. ) . similar experiments were performed by zhao et al. ( ) using human monocyte-derived gm-dcs stimulated with b. melitensis-wadc mutant lps and wild type lps, the production of il- p , il- , and tnfα was observed only in cells stimulated with mutant lps . clearly, immune response impairment in gm-dcs is due to the core oligosaccharide in brucella lps, but this is not the rule in all dcs subsets. for example, in the case of fl-dcs stimulated with b. melitensis wild-type lps, the cd b + and cd α + , but not pdc, subsets upregulated the expression of cd , cd , cd , and mhc-ii molecules and secrete significant amounts of tnfα, il- p , il- , and il- . additionally, upregulation of surface molecules and cytokine production was observed in fl-dcs dcs stimulated with b. melitensis-wadc mutant lps but was higher than that observed in fl-dcs stimulated with wild-type lps. however, despite the activation profiles elicited in cd b + and cd α + cells stimulated with wild-type lps, these dcs were not able to induce the proliferation of cd + or cd + t cells . it has been reported that human or mouse gm-dcs treated with b. abortus or pure wild-type lps hampered proinflammatory cytokine profiles and demonstrated an inability to upregulate costimulatory molecules ( molecular interaction: how does brucella avoid dcs immune response? brucella spp., are recognized by tlrs from dc subsets and activate intracellular pathways for cytokine production tlrs are involved in recognizing pathogen-associated molecular patterns (pamps), and in some cases, dc activation is initiated by these receptors. tlrs have an extracellular leucine-rich repeat (lrr) domain, and the intracytoplasmic domain toll/il- receptor domain (tir domain) homologous to the il- receptor family (il- r) is crucial for intracellular signaling. myeloid differentiation factor (myd ) is an adaptor protein which possesses a tir domain and associates with il- r family members including tlrs (kaisho and akira ) . based on their observations, zwerdling et al. ( ) proposed that brucella signals through tlr , since gm-dcs stimulated with pure brucella omp impaired cytokine production when this receptor was blocked. on the other hand, huang et al. ( ) observed a prominent role for tlr and myd in cd c + dcs isolated from the spleen for il- production after the cells were treated with hkba (huang et al. ; zwerdling et al. ) . tlr and tlr are superficial receptors at the cytoplasmic membrane and are recruited at the site of the pathogen interaction during phagocytosis, whereas tlr is an intracellular receptor that recognizes distinct patterns of nucleic acids in endosomes (kaisho and akira ) . zhang et al. ( ) observed that tlr , but not tlr is required for hkba phagocytosis in cd α − and cd α + dcs isolated from mouse spleen. hkba phagocytosis induced the production of tnfα and il- ; tlr was important for tnfα production, while tlr was related to il- production and was myd signaling-dependent. however, tlr was not required for the production of any cytokines in both dc spleen subsets. additionally, it was demonstrated that tlr -tlr cooperation was necessary for the production of pro-inflammatory cytokines. hkba interacts with tlr in splenic dcs and signals through p , leading to phagocytosis, and signals for tnfα production, resulting in phagolysosome fusion; then tlr interacts with bacterial dna in late endosomes, signaling for il- production (zhang et al. ). on the other hand, in bmgm-dcs infected with b. abortus, tlr expression was higher than tlr expression, and tlr was found to be more important for il- and tnfα production . to identify the differential roles of tlrs in brucella elimination in a pulmonary infection model, bmgm-dcs from tlr , tlr , and tlr ko mice were infected with rough vaccine b. abortus rb and smooth virulent b. abortus . bmgm-dcs infected with strain rb showed higher levels of mhc-ii, cd , and cd expression mediated by tlr , , and , as well as il- production mediated by tlr and tlr , than the smooth virulent strain. gm-dcs from wild-type mice showed impaired activation. dc activation was induced using moi = in the case of b. abortus , while moi = was necessary in the case of the rough strain to activate cells (surendran et al. ) . clearly, il- production is necessary for an appropriate th response against brucellosis. tlr is the only receptor that signals through both trif (tir-domain-containing adapter-inducing interferon-β) and myd upon stimulation and acts synergistically in dcs for il- production (krummen et al. ) . in this sense, zhang et al. ( ) demonstrated il- production-independent tlr signaling by splenic dcs infected with b. abortus; however, surendran et al. ( ) proposed tlr participation in il- production during b. abortus infection. recently, tlr and tlr have been implicated in sensing brucella rna. b. abortus rna-induced il- , il- , and tnfα production in murine bmgc-dcs; this cytokine production was tlr-dependent and occurred via mapk/nf-κb signaling. although tlrs were required to sense bacterial rna and cytokine production, they were not necessary for brucella eradication in vivo (campos et al. ) . almost all tlrs signal through myd , but alternative downstream signaling pathways could be involved in cytokine production. for example, zhang et al. ( ) observed tnfα production mediated by tlr via p activation. on the other hand, tlr also activates erk / and exerts negative feedback for il- production mediated by tlr . upon activation, tlrs signal through the common adaptor molecule myd (except tlr ) and recruit interleukin- (il- ) receptor-associated kinase (irak) and tumor necrosis factor (tnf) receptor-associated factor (traf ). once tlrs interact with myd , irak is activated by tak (transforming growth factor-activated kinase) in a phosphorylation-dependent manner. activated irak associates with traf , leading to the activation of jnk (janus kinase) and culminating in iκb degradation and nf-κb translocation to the nucleus for the transcription of proinflammatory cytokine genes such as il- and tnfα (kawai and akira ; takeda and akira ; underhill and ozinsky ) . additionally, irak- was found to be involved in the production of il- and tnfα in macrophages and dcs infected by brucella. bone marrow-derived macrophages and bmgm-dcs from irak- −/− mice were stimulated with live b. abortus , hkba, tlr , and tlr agonists, irak- deficient macrophages and bmgm-dcs showed impaired production of tnfα and il- , demonstrating an essential role for irak- in the production of pro-inflammatory cytokines upon brucella recognition by tlrs (oliveira et al. ). a more detailed study of the interaction of mouse gm-dcs and b. abortus was performed using microarrays. functional expression analysis showed that not only type i ifn response but also mapk p activation were impaired (gorvel et al. ). it has been reported that the o-side chains of lps from b. melitensis m, b. abortus , and b. suis are able to restrict the activation of p in murine j a. macrophages. additionally, the production of inflammatory cytokines and chemokines depends on p mapks (jiménez de bagüés et al. ) . taking into account these data, we can show that brucella modulates dc maturation by impairing the type i ifn and the immune response, allowing bacterial intracellular persistence and chronic infection. another strategy used by brucella to avoid the host immune response involves blocking or impairing tlrs signaling pathway through bacterial tir homologs (rana et al. ). cirl et al. identified bacterial tir-domain proteins in uropathogenic e. coli cft and brucella species, referred as tir-domain-containing proteins c (tcpc) and b (tcpb, also known as btpa), respectively (cirl et al. ). the btpa gene was found in b. melitensis and b. abortus, but not in the b. suis genome; the genetic evidence proposes that these genes were acquired recently via a phage-mediated integration event. the btpa protein interferes with nf-κb activation mediated by tlr and tlr signaling, as well as cytokine production and dc maturation (radhakrishnan et al. ). for tlrs, activation adaptor proteins are recruited, one of which is toll-interleukin- receptor domain-containing adaptor protein (tirap, also known as mal; myd adaptor protein). tirap is an adaptor molecule for the myd dependent pathway derived from tlr and tlr -signaling (kawai and akira ; underhill and ozinsky ) . radhakrishnan et al. ( ) observed no interaction between btpa, tlr , tlr , or tirap to inhibit nf-kb activation in hek cells transfected with the btpa gene. interestingly, btpa was found to mimic tirap function during tlrs signaling; btpa likely competes with myd and blocks downstream signaling (radhakrishnan et al. ). on the other hand, recombinant btpa was able to induce the degradation of tirap, while other tlr components were not affected. it has been suggested that the interaction of btpa and tirap facilitates the ubiquitination of tirap for its degradation ( fig. ) (sengupta et al. ) . myd possesses an amino-terminal death domain (dd) involved in cell death and signaling. through the interaction of their dds, myd recruits the irak- and irak- kinases (loiarro et al. ). according to a fragment complementation assay, it was possible to determine that btpa interacts directly with myd and tirap through the dds. the interaction between btpa and myd was stronger than that observed with btpa-tirap. although btpa interacts strongly with myd , the interaction does not impair downstream signaling (fig. ) (chaudhary et al. ) . a second tir-domain-containing protein (bab _ ) in the brucella genome was discovered and designated as btpb. btpb is a potent inhibitor of tlr , tlr , tlr and tlr signaling. infection of murine bmgm-dcs with b. abortus btpb mutant led to the reduction of tnfα and il- . some differences were observed between btpa and btpb; for example, btpa, but not btpb impaired tnfα production, while btpb decreased the expression of mhc-ii, cd , cd , and cd in bmgm-dc cells. however, both proteins contributed to the control of dc activation during brucella infection (salcedo et al. ) . based on this evidence, btpa may affect tirap functions but could also interact directly with myd in a dd-domain dependent manner, although not with the tir domain. btpb also showed stronger myd -binding and likely blocks tlrs that are dependent on myd signaling (fig. ) (salcedo et al. ). brucella invades and replicates in a variety of host cells. a high percentage of the studies to examine brucella pathogenesis, host-pathogen interaction, virulence, etc., are performed in macrophages, and in some cases, these cells are the preferential niche for intracellular replication. however, there is no doubt that dcs are an intracellular niche for brucella species, and their migratory properties allow bacterial dissemination. with the exception of bovine-derived dcs, this pathogen infects and replicates inside dcs. brucella recognition at the outside of the eukaryotic cell is mediated by tlrs and signaling triggers the immune response, as shown by cytokine production. despite discrepancies regarding dc activation/ maturation after brucella infection, it is clear that brucella infection is detected by murine or human gm-dcs since the bacteria drive low pro-inflammatory cytokine levels. unlike other intracellular pathogens such as salmonella, which induces high levels of pro-inflammatory cytokines and the expression of activation/maturation surface molecules, brucella is able to subvert or bdelay^the dc response, impairing the inflammatory response or the expression of surface molecules. at later times postinfection, brucella exerts immunomodulatory mechanisms to avoid a protective immune response, promoting intracellular trafficking and reaching its intracellular niche by blocking tlr signaling and cytokine production. it is evident that there is a controversy regarding whether infection with brucella prevents or promotes the activation/ maturation of dcs. in part, this contradiction is due to a large number of variables that are managed in experiments using dcs, for example, the bacterial strains used, the bacterial dose and the time for infection/stimulation. it is impossible to homogenize all methodologies, since one dc subset cannot be used because, as we have emphasized in this review, there are several dc phenotypes. on the other hand, the genetic variation of brucella strains, even though the same bacterial species are used, including references or clinical isolated strains, may affect the results. similarly, it is impossible to control the physiological and genetic variations of the animals and donors used to obtain dcs. undeniably, the nature of the stimulus used causes variations in the results, since stimulation performed with a purified protein, purified lps, heat-killed bacteria or a live mutant strain is not the same. for example, in the case of subunit stimuli, although they are fundamentally important to determine the role of a specific macromolecule, they do not contain all elements encompassed by the whole bacterium and act together at the time of infection. however, some variables can be controlled, such as the dose and time of infection/stimulation with brucella or brucella antigens, the time of dc subset differentiation, the strain and the age of the animals used. thanks to studies of the interaction between brucella and dcs, it has been possible to identify important virulence factors that may be key therapeutic targets for the control of brucellosis, for example, the proteins btpa and btpb, the core fig. schematic representation of brucella-dc interaction. lipid raftsmediated interaction between brucella and dcs has been reported. tlr , tlr , tlr , tlr , tlr , and tlr have been involved in brucella recognition. however, brucella phagocytosis involved recruitment of tlr but not tlr at the cytoplasmic membrane, and almost % of the ingested brucella are eliminated by professional phagocytes; the fusion of late endosomes with intracellular receptors such as tlr allows type i ifn induction. once inside, brucella survive inside phagocytic vacuole and evade late endosomal traffic to reach intracellular niche. brucella tir proteins are translocated into cytoplasmic compartment. btpa impairs dc activation/maturation through myd and tirap interaction (chaudhary et al. ; radhakrishnan et al. ; sengupta et al. ) , and btpb binds to various eukaryotic tir-proteins (tlr , tlr , tlr myd , tirap, etc.) (salcedo et al. ) . moreover, brucella impairs type i interferon family expression. blue solid arrows indicate intracellular pathways activated via brucella recognition by tlrs. red dashed arrows indicate impaired cytokine pathways by brucella tir proteins and subsequent dcs maturation region of lps or the wadc gene. these elements fundamentally affect the immune response induced by dendritic cells. establishment of chronic infection: brucella's stealth strategy contrasting roles of macrophages and dendritic cells in controlling initial pulmonary brucella infection characterization of outer membrane vesicles from brucella melitensis and protection induced in mice host immune responses to the intracellular bacteria brucella: does the bacteria instruct the host to facilitate chronic infection? dendritic cells and the control of immunity brucella abortus uses a stealthy strategy to avoid activation of the innate immune system during the onset of infection coxiella burnetii, the agent of q fever, stimulates an atypical m activation program in human macrophages interaction of brucella suis and brucella abortus rough strains with human dendritic cells brucella suis prevents human dendritic cell maturation and antigen presentation through regulation of tumor necrosis factor alpha secretion a review of the use of b. melitensis rev vaccine in adult sheep and goats generation of murine dendritic cells from flt -ligand-supplemented bone marrow cultures harnessing the therapeutic potential of th cells tlr and tlr sense brucella abortus rna to induce proinflammatory cytokine production but they are dispensable for host control of infection brucella spp. noncanonical lps: structure, biosynthesis, and interaction with host immune system bacterium abortem invasion of the tissues of calves from the ingestion of infected milk characterization of sp , a surface protein of brucella associated with adherence and invasion of host epithelial cells the brucella tir-like protein tcpb interacts with the death domain of myd persistence of brucella abortus in the livers of t cell-deficient nude mice subversion of toll-like receptor signaling by a unique family of bacterial toll/interleukin- receptor domain-containing proteins dendritic cells at the hostpathogen interface the lipopolysaccharide core of brucella abortus acts as a shield against innate immunity recognition lack of endogenous il- enhances production of proinflammatory cytokines and leads to brucella abortus clearance in mice il- : the master regulator of immunity to infection identification of a unique gene cluster of brucella spp. that mediates adhesion to host cells toll-like receptor plays an important role in host innate resistance to brucella abortus infection in mice pathogenesis and immunobiology of brucellosis: review of brucella-host interactions clinical manifestations of human brucellosis: a systematic review and meta-analysis development and functional specialization of cd + dendritic cells transforming growth factor beta production correlates with depressed lymphocytes function in humans with chronic brucellosis host response to brucella infection: review and future perspective comparative histopathology in balb/c mice infected with virulent and attenuated strains of brucella abortus serving the new mastersdendritic cells as hosts for stealth intracellular bacteria brucella and osteoarticular cell activation: partners in crime b lymphocytes provide an infection niche for intracellular bacterium brucella abortus is brucella an enteric pathogen? intracellular bacteria interfere with dendritic cell functions: role of the type i interferon pathway what have we learned from brucellosis in the mouse model? dendritic cells, monocytes and macrophages: a unified nomenclature based on ontogeny brucellosis: a zoonosis of importance in mexico in situ characterization of splenic brucella melitensis reservoir cells during the chronic phase of infection in susceptible mice characterization of brucella abortus infection of bovine monocyte-derived dendritic cells the bmei gene of brucella melitensis is required for internalization in hela cells murine spleen contains a diversity of myeloid and dendritic cells distinct in antigen presenting function interleukin (il)- is a major regulatory cytokine governing bioactive il- production by mouse and human dendritic cells th -like cytokine induction by heat-killed brucella abortus is dependent on triggering of tlr brucella abortus efp gene is required for an efficient internalization in hela cells distinct dendritic cell populations sequentially present antigen to cd + t cells and stimulate different aspects of cell-mediated immunity innate immune recognition regulation of the mitogen-activated protein kinases by brucella spp. expressing a smooth and rough phenotype: relationship to pathogen invasiveness major outer membrane protein omp of brucella suis is involved in inhibition of tumor necrosis factor alpha production during infection of human macrophages dendritic-cell function in toll-like receptor-and myd -knockout mice tlr signaling stratification of antigen-presenting cells within the normal cornea antigen-presenting cells are stratified within normal human corneas and are rapidly mobilized during ex vivo viral infection human corneal antigenpresenting cells release of il- by dendritic cells activated by tlr ligation is dependent on myd signaling, whereas trif signaling is indispensable for tlr synergy pathogenic bacteria and dead cells are internalized by a unique subset of peyer's patch dendritic cells that express lysozyme peyer's patch dendritic cells sample antigens by extending dendrites through m cell-specific transcellular pores identification of critical residues of the myd death domain involved in the recruitment of downstream kinases brucella downregulates tumor necrosis factor-α to promote intracellular survival via omp regulation of different micrornas in porcine and murine macrophages th and th cells in tuberculosis: protection, pathology, and biomarkers central role of myd -dependent dendritic cell maturation and proinflammatory cytokine production to control brucella abortus infection presentation of phagocytosed antigens by mhc class i and ii recent trends in human brucella canis infection immune control of brucella abortus infections in balb/c mice cutting edge: generation of splenic cd + and cd -dendritic cell equivalents in fms-like tyrosine kinase ligand bone marrow cultures interleukin- receptor-associated kinase is essential for initial host control of brucella abortus infection brucella discriminates between mouse dendritic cell subsets upon in vitro infection the changing brucella ecology: novel reservoirs, new threats the new global map of human brucellosis fate mapping of dendritic cells variability in the response of canine and human dendritic cells stimulated with brucella canis modulating th /th responses with microbes, dendritic cells, and pathogen recognition receptors immunological mechanisms of vaccination distinct dendritic cells subsets differentially regulate the class of immune response in vivo sensing pathogens and tuning immune responses brucella tir domain-containing protein mimics properties of the toll-like receptor adaptor protein tirap bacterial tir-containing proteins and host innate immune system evasion systems biology analysis of brucella infected peyer's patch reveals rapid invasion with modest transient perturbations of the host transcriptome il- -induced conversion of monocytes to mature dendritic cells brucella control of dendritic cell maturation is dependent on the tir-containing protein btp btpb, a novel brucella tir-containing effector protein with immune modulatory functions dendritic cells and monocyte-derived cells: two complementary and integrated functional systems brucella microti sp. nov., isolated from the common vole microtus arvalis brucella inopinata sp. nov., isolated from a breast implant infection brucella vulpis sp. nov., isolated from mandibular lymph nodes of red foxes terminal differentiation of dendritic cells brucellosis: a reemerging zoonosis subversion of innate immune responses by brucella through the targeted degradation of the tlr signaling adapter, mal development and characterization of mouse models of infection with aerosolized brucella melitensis and brucella suis dendritic cells: limited potential in immunotherapy brucella intracellular replication requires trafficking through the late endosomal/ lysosomal compartment acute infection by conjunctival route with brucella melitensis induces igg+ cells and ifn-gamma producing cells in peripheral and mucosal lymph nodes in sheep role of tlrs in brucella mediated murine dc activation in vitro and clearance of pulmonary infection in vivo toll-like receptors orientia tsutsugamushi, the causative agent of scrub typhus, induces an inflammatory program in human macrophages a literature review of laboratory-acquired brucellosis toll-like receptors: key mediators of microbe detection brucella abortus induces apoptosis of human t lymphocytes pulmonary dendritic cells cervical lymph nodes as a selective niche for brucella during oral infections canine brucellosis a promotes brucella intracellular growth via inhibition of macrophage cell death and activation cd + t cellderived il- promotes brucella abortus persistence via modulation of macrophage function dendritic cells from oral cavity induce foxp + regulatory t cells upon antigen stimulation recruitment of dendritic cells to pathological niches in inflamed liver the inflammatory cytokine, gm-csf, alters the developmental outcome of murine dendritic cells the regulation of the development and function of dendritic cell subsets by gm-csf: more than a hematopoietic growth factor tlr signaling subpathways regulate tlr signaling for the effective induction of il- upon stimulation by heat-killed brucella abortus omp of brucella melitensis m impairs the apoptosis of macrophages triggered by tnf-α immunomodulatory properties of brucella melitensis lipopolysaccharide determinants on mouse dendritic cells in vitro and in vivo brucella lipoproteins mimic dendritic cell maturation induced by brucella abortus acknowledgments the authors thank sm boyle for editing and provide key: cord- -qtwcbn m authors: gao, yaning; tai, wanbo; wang, ning; li, xiang; jiang, shibo; debnath, asim k.; du, lanying; chen, shizhong title: identification of novel natural products as effective and broad-spectrum anti-zika virus inhibitors date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: qtwcbn m zika virus (zikv) infection during pregnancy leads to severe congenital zika syndrome, which includes microcephaly and other neurological malformations. no therapeutic agents have, so far, been approved for the treatment of zikv infection in humans; as such, there is a need for a continuous effort to develop effective and safe antiviral drugs to treat zikv-caused diseases. after screening a natural product library, we have herein identified four natural products with anti-zikv activity in vero e cells, including gossypol, curcumin, digitonin, and conessine. except for curcumin, the other three natural products have not been reported before to have anti-zikv activity. among them, gossypol exhibited the strongest inhibitory activity against almost all zikv strains tested, including six recent epidemic human strains. the mechanistic study indicated that gossypol could neutralize zikv infection by targeting the envelope protein domain iii (ediii) of zikv. in contrast, the other natural products inhibited zikv infection by targeting the host cell or cell-associated entry and replication stages of zikv. a combination of gossypol with any of the three natural products identified in this study, as well as with bortezomib, a previously reported anti-zikv compound, exhibited significant combinatorial inhibitory effects against three zikv human strains tested. importantly, gossypol also demonstrated marked potency against all four serotypes of dengue virus (denv) human strains in vitro. taken together, this study indicates the potential for further development of these natural products, particularly gossypol, as the lead compound or broad-spectrum inhibitors against zikv and other flaviviruses, such as denv. zika virus (zikv) is a mosquito-borne flavivirus in the same genus as other important human pathogens, including dengue virus (denv), west nile virus (wnv), yellow fever virus (yfv), japanese encephalitis virus (jev), and tick-borne encephalitis virus (tbev) [ ] . zikv was originally isolated in a rhesus macaque in [ ] , but this virus has only recently claimed worldwide attention owing to its close association with congenital zika syndrome (czs), as represented by microcephaly, fetal demise, central nervous system abnormalities, and other neurological complications [ ] [ ] [ ] [ ] [ ] . no antiviral therapeutics for the treatment of zikv-associated human diseases, particularly congenital syndrome a plaque reduction inhibition assay was carried out to measure the inhibitory activity of natural products in a natural product collection library from microsource discovery systems (gaylordsville, ct, usa) against infections of zikv and denv, as previously described [ ] [ ] [ ] [ ] . briefly, zikv (strain pan , ~ plaque-forming unit (pfu); multiplicity of infection (moi) ~ . ) was incubated with -fold serial dilutions of natural products (including curcumin, digitonin and conessine, as well as anti-zikv compound control, bortezomib) or dmso ( . % vol/vol) control at °c for h. the compound-virus mixtures were then transferred to vero e cells ( /well) and incubated at °c for h. for gossypol, zikv (strain pan , ~ . × pfu; moi ~ . ) was incubated with serial dilutions of this natural product at °c for h, and the unbound gossypol was removed by centrifugation after addition of % peg- . gossypol-treated zikv was then incubated with vero e cells at °c for h. the cells were washed thoroughly with pbs and overlaid with dmem containing % carboxymethyl cellulose and % fbs, followed by in vitro culture at °c for - days, and then stained with . % crystal violet. the inhibitory activity of all natural products tested against denv- - , as described above, was performed following the above procedures, except that llc-mk cells were used for infection, and cells were cultured at °c for - days before staining with . % crystal violet. the % inhibitory concentration (ic ) and ic of natural products were calculated based on the concentrations at % and % plaque reduction, respectively, using the calcusyn computer program, as described before [ ] . the cytotoxicity of natural products in vero e for zikv or llc-mk cells for denv- - was evaluated using the cell counting kit- (cck , sigma, st. louis, mo, usa), according to the manufacturer's instructions. briefly, -fold serial dilutions of each of the natural products ( l/well) were added to equal volumes of cells ( . × /well) in -well plates and cultured at °c for days. the cells were then incubated with cck solution and absorbance was measured at nm (a value) using a microplate reader (infinite f pro, tecan, morrisville, nc, usa). the % cytotoxic concentration (cc ) of natural products was calculated based on the percent cytotoxicity a plaque reduction inhibition assay was carried out to measure the inhibitory activity of natural products in a natural product collection library from microsource discovery systems (gaylordsville, ct, usa) against infections of zikv and denv, as previously described [ ] [ ] [ ] [ ] . briefly, zikv (strain pan , ~ plaque-forming unit (pfu); multiplicity of infection (moi) . ) was incubated with -fold serial dilutions of natural products (including curcumin, digitonin and conessine, as well as anti-zikv compound control, bortezomib) or dmso ( . % vol/vol) control at • c for h. the compound-virus mixtures were then transferred to vero e cells ( /well) and incubated at • c for h. for gossypol, zikv (strain pan ,~ . × pfu; moi~ . ) was incubated with serial dilutions of this natural product at • c for h, and the unbound gossypol was removed by centrifugation after addition of % peg- . gossypol-treated zikv was then incubated with vero e cells at • c for h. the cells were washed thoroughly with pbs and overlaid with dmem containing % carboxymethyl cellulose and % fbs, followed by in vitro culture at • c for - days, and then stained with . % crystal violet. the inhibitory activity of all natural products tested against denv- - , as described above, was performed following the above procedures, except that llc-mk cells were used for infection, and cells were cultured at • c for - days before staining with . % crystal violet. the % inhibitory concentration (ic ) and ic of natural products were calculated based on the concentrations at % and % plaque reduction, respectively, using the calcusyn computer program, as described before [ ] . the cytotoxicity of natural products in vero e for zikv or llc-mk cells for denv- - was evaluated using the cell counting kit- (cck , sigma, st. louis, mo, usa), according to the manufacturer's instructions. briefly, -fold serial dilutions of each of the natural products ( µl/well) were added to equal volumes of cells ( . × /well) in -well plates and cultured at • c for days. the cells were then incubated with cck solution and absorbance was measured at nm (a value) using a microplate reader (infinite f pro, tecan, morrisville, nc, usa). the % cytotoxic concentration (cc ) of natural products was calculated based on the percent cytotoxicity using the calcusyn computer program [ , ] . the combinatorial cytotoxicity of gossypol with other natural products to vero e cells was detected using a similar approach as described above, except for mixing gossypol with one of the natural products (curcumin, digitonin, conessine, or bortezomib) throughly before adding them to the cells. this experiment was carried out as previously described, with some modifications [ ] [ ] [ ] [ ] [ ] [ ] [ ] . briefly, vero e cells ( /well) or zikv were incubated at different infection steps as described below, with or without the tested natural products at the specified concentrations of µm for gossypol, µm for curcumin, . µm for digitonin, or µm for conessine for h before zikv infection, h after infection, or the same time during infection. anti-zikv compounds, such as temoporfin [ ] , -hydroxycholesterol [ ] , bortezomib [ ] , and nitd [ ] , were included as controls for these steps. after the culture of the zikv-or compound-treated cells at • c for - days, plaques were visualized with crystal violet staining, as described above, and the percent inhibition of natural products was calculated. specifically, the following six stages of zikv infection were tested: (a) pretreatment of zikv (pan ,~ . × pfu; moi~ . ) with natural products at • c for h before incubation with cells; (b) pretreatment of cells with natural products at • c for h before incubation with zikv (pan ,~ pfu; moi~ . ); (c) cotreatment of cells with zikv (pan ,~ pfu; moĨ . ) and natural products at • c for h; (d) cotreatment of cells, zikv (pan ,~ pfu; moĨ . ), and natural products at • c for h; (e) preincubation of cells with zikv (pan ,~ pfu; moi~ . ) at • c for h and then incubation with natural products at • c for h; and (f) preincubation of zikv (pan ,~ pfu; moi~ . ) and cells at • c for h, followed by incubation with natural products at • c for h. the binding between natural products and zikv full-length e protein (aviva systems biology, san diego, ca, usa) or envelope protein domain iii (ediii) protein (e residues - fused with a c-terminal human fc) [ ] was carried out by elisa, as previously described [ , , , ] . briefly, elisa plates were precoated with the proteins described above at • c overnight and blocked with % fat-free milk at • c for h. serial dilutions of natural products or dmso (negative control) were then added to the plates and incubated at • c for h. the plates were washed with pbs containing tween- (pbst) and incubated at • c for h with zikv ediii-specific human monoclonal antibody (mab) zka -lala ( . µg/ml) for binding to zikv full-length e and ediii proteins. the plates were washed with pbst and incubated with horseradish peroxidase (hrp)-conjugated anti-human igg-fab ( : , abcam, cambridge, ms, usa) antibody at • c for h. the , , , -tetramethylbenzidine (tmb) substrate (sigma) was added to the plates, and the reaction was stopped by n h so . absorbance at nm (a value) was measured by elisa microplate reader (tecan). ec ( % effective concentration) was calculated based on the calcusyn computer program, as described above [ ] . to determine the ability of gossypol to inhibit binding between zikv ediii protein and ediii-specific human mabs (smzab , zka -lala, zv- , or z ) or edi/ii-specific human mab control (zka ) [ ] , elisa was carried out, as described above, except that serially diluted gossypol or dmso (negative control) was added in the presence of mabs ( . µg/ml), followed by sequential incubation with hrp-conjugated anti-human igg-fab antibody and tmb substrate and detection for a value. the percent inhibition of natural products was calculated, and ic (concentration causing % reduction in ediii-mab binding) was obtained using the calcusyn computer program [ ] , as described above. the interactions between natural products and zikv full-length e were analyzed at • c using the biacore t system (ge healthcare, port washington, ny, usa), as previously described [ , ] . briefly, zikv e was immobilized on a sensor chip (cm ) using the amine coupling kit (ge healthcare). natural products at various concentrations were injected as analytes, and pbs-p ( mm phosphate buffer containing . mm kcl, mm nacl, and . % surfactant p , ph . ) was used as the running buffer. the data were analyzed using biacore evaluation software (t version . ), and the curve was fitted with a : binding model. the potential combinatorial effect of gossypol with other natural products was carried out as previously described [ , ] . briefly, zikv (strain pan , flr, or prvabc , . × pfu; moĨ . ) was incubated with serially diluted gossypol at • c for h, and the unbound gossypol was removed by centrifugation after addition of % peg- . gossypol-treated zikv was incubated with vero e cells at • c for h in the presence of dmem containing serial dilutions of each of the other three natural products identified, such as curcumin, digitonin, and conessine, or anti-zikv compound control (bortezomib). the unbound viruses and natural products were removed, the cells were cultured at • c for - days, and stained with . % crystal violet. the natural products without combinations were used as controls. the ic values of natural products were calculated using the calcusyn computer program [ ] , as described above. the natural products were then analyzed for combinatorial effects based on the combination index (ci) [ ] and ic values using the calcusyn computer program, as previously described [ , , ] . specifically, ci values < and > indicate synergy and antagonism, respectively. synergy was further identified as five different categories: ci values < . , . - . , . - . , . - . , and . - . indicate very strong synergism, strong synergism, synergism, moderate synergism, and slight synergism, respectively. fold enhancement of anti-zikv potency is expressed as the ratio of molar concentrations of natural products tested alone and in the mixture using the formula ((ic alone)/(ic in the mixture)- ). using a plaque-based assay, we initially screened natural products (at µm of concentration) from a natural product library for their inhibitory activity against infection of a recent zikv human strain (pan ). based on table , four "hit" natural products, including gossypol, curcumin, digitonin, and conessine ( figure a -d), were selected, since they demonstrated inhibitory activity against zikv infection with no obvious cytotoxicity in vero e cells when observed under a microscope. these natural products were subsequently ordered from sigma with ≥ % purity, and tested for their cytotoxicity using a cell-based cytotoxicity assay (cck kit), with cc values ranging from . to . µm (table , figure s ). among these natural products, curcumin has been previously reported to inhibit zikv infection [ ] , whereas the other three natural products have not been previously reported to have anti-zikv activity. gossypol, curcumin, digitonin, and conessine were retested to confirm their anti-zikv (strain pan ) activity, with ic values of . , . , . , and . µm, respectively ( table ). the ic values of these natural products against zikv pan strain were also calculated, equal to about . , . , . , and . µm, respectively, for gossypol, curcumin, digitonin, and conessine ( figure s ), which were slightly higher than the respective ic values, but much lower than the corresponding cc values. table . initial screening of a natural product library for identification of potential anti-zikv inhibitors. natural products from microsource discovery systems for screening primary hits (with inhibition against zikv strain pan ) a ( . ) primary hits (with high cytotoxicity in vero e cells) b ( . ) specific hits (primary hits with low cytotoxicity) ( . ) specific hits (available to purchase) ( . ) specific hits (ordered from sigma and retested to confirm anti-zikv activity) ( . ) specific hits displaying ic < cc ( . ) a a total of primary hits inhibited zikv (strain pan ) infection by more than % at µm, whereas the negative control (dmso) had inhibitory activity less than %. b observed cytotoxicity of natural products (at µm) under a microscope. the cytotoxicity was recorded as grades (−, ±, +, ++, +++, ++++), and the natural products with cytotoxicity greater than or equal to grade ++ were referred to as "high cytotoxicity in vero e cells", and discarded for further testing. viruses , , of table . initial screening of a natural product library for identification of potential anti-zikv inhibitors. no. (%) natural products from microsource discovery systems for screening primary hits (with inhibition against zikv strain pan ) a ( . ) primary hits (with high cytotoxicity in vero e cells) b ( . ) specific hits (primary hits with low cytotoxicity) ( . ) specific hits (available to purchase) ( . ) specific hits (ordered from sigma and retested to confirm anti-zikv activity) ( . ) specific hits displaying ic < cc ( . ) a a total of primary hits inhibited zikv (strain pan ) infection by more than % at μm, whereas the negative control (dmso) had inhibitory activity less than %. b observed cytotoxicity of natural products (at μm) under a microscope. the cytotoxicity was recorded as grades (−, ±, +, ++, +++, ++++), and the natural products with cytotoxicity greater than or equal to grade ++ were referred to as "high cytotoxicity in vero e cells", and discarded for further testing. the identified natural products were further studied for their broad-spectrum activity against nine additional zikv strains, including those isolated from different hosts, namely humans, mosquitos, and rhesus macaques, at different time periods in different countries, including mexico, panama, columbia, honduras, puerto rico, thailand, nigeria, and uganda ( figure ). the results showed that these natural products could inhibit infections of all nine zikv strains tested with various ic values ( table ). gossypol exhibited the most potent inhibitory activity for ic values, ranging from . to . m, against all nine zikv strains tested (table ) . it was also more potent than bortezomib, the anti-zikv previously reported compound as an active compound control, against all zikv strains tested ( table ). the ic values of these natural products against the selected zikv strain, prvabc , were calculated, among which gossypol had the lowest ic value (about . m, slightly higher than its ic value but lower than its cc value) ( figure s ), confirming its strong and broad-spectrum anti-zikv activity. the identified natural products were further studied for their broad-spectrum activity against nine additional zikv strains, including those isolated from different hosts, namely humans, mosquitos, and rhesus macaques, at different time periods in different countries, including mexico, panama, columbia, honduras, puerto rico, thailand, nigeria, and uganda ( figure ). the results showed that these natural products could inhibit infections of all nine zikv strains tested with various ic values (table ). gossypol exhibited the most potent inhibitory activity for ic values, ranging from . to . µm, against all nine zikv strains tested (table ) . it was also more potent than bortezomib, the anti-zikv previously reported compound as an active compound control, against all zikv strains tested ( table ). the ic values of these natural products against the selected zikv strain, prvabc , were calculated, among which gossypol had the lowest ic value (about . µm, slightly higher than its ic value but lower than its cc value) ( figure s ), confirming its strong and broad-spectrum anti-zikv activity. comparison of zikv e protein sequences revealed that most of the amino acid sequences were highly conserved, but that some variations occurred among the zikv strains used for evaluation of the inhibitory activity of natural products, including the pan strain tested earlier ( figure s ). the above data demonstrate that the identified natural products, particularly gossypol, were able to block infections of divergent human, mosquito, and monkey zikv strains isolated from different time periods and countries, including six recent zikv human strains, confirming their broad-spectrum anti-zikv activity. to identify which steps of zikv infection in its life cycle were blocked by these natural products, we carried out a time-of-addition experiment by incubating natural products with zikv or cells at different time points during zikv and cell interaction, and then calculated the percent inhibition based on the number of plaques formed [ , , , ] . to test whether a natural product can neutralize zikv infection or inhibit viral entry by targeting the viral proteins, zikv was pretreated with the natural product at • c before incubation with the host cells ( figure a(a) ). to evaluate whether a natural product can bind to the cellular receptors or cofactors to block virus-receptor binding, cells were pretreated with the natural product at • c before incubation with zikv ( figure a(b) ). to determine whether a natural product can inhibit attachment of zikv to target cells, but cannot block the virus-cell membrane fusion, cells were cotreated with zikv at • c in the presence of the natural product ( figure a (c)). to assess whether a natural product can inhibit attachment of zikv to target cells and subsequent virus-cell membrane fusion, the cells were cotreated with zikv and the natural product at • c ( figure a(d) ). to investigate whether a natural product can inhibit zikv fusion with the cell membrane and then entry into the cell, cells were pretreated with zikv at • c first and then incubated with the natural product at • c ( figure a (e)). to study whether a natural product can inhibit zikv infection at postentry stages (i.e., viral replication, virion assembly, or release), cells were pretreated with zikv and then incubated with the natural product at • c ( figure a (f)). after completing the above approaches, we gained insight into the potential mechanisms of the natural products responsible for inhibiting zikv (pan ) infection. after pretreatment of zikv with gossypol at • c before incubation with the target cells, zikv completely lost its infectivity, whereas it maintained its infectivity after other treatments described above ( figure b ), suggesting that gossypol can effectively neutralize zikv infection by targeting the virus, rather than the cell or cell-associated entry or replication stages. the results from curcumin revealed that about - % of zikv infection was blocked when curcumin was incubated with zikv only at • c, or coincubated with zikv and cells at • c or • c, whereas there was low to no impact on zikv infection when curcumin was pretreated with cells or postincubated with zikv-treated cells at • c and • c, respectively ( figure c ). these results suggest that curcumin inhibits zikv infection at the early stages of viral entry, particularly the viral attachment stage. pretreatment of vero e cells with digitonin and then with zikv or cotreatment of cells with zikv and digitonin at • c significantly (≥ %) blocked zikv infection, whereas preincubation of cells with zikv and then with digitonin at • c, pretreatment of cells with zikv at • c and then with digitonin at • c, or cotreatment of cells with digitonin and zikv at • c inhibited about - % of zikv infection ( figure d ). in contrast, preincubation of digitonin and zikv had no effect on zikv infection ( figure d ). these results suggest that digitonin could not directly neutralize zikv infection, but inhibited zikv infection by binding to the viral receptors or inhibiting viral entry (i.e., attachment and membrane fusion or postentry steps). the data from conessine indicated that coincubation of cells with conessine and zikv at • c or postincubation of conessine with zikv-treated cells at • c or • c resulted in - % inhibition of zikv infection, whereas pretreatment of cells with conessine before zikv incubation blocked about % of zikv infection. nevertheless, preincubation of conessine and zikv at • c or cotreatment of cells with conessine and zikv at • c had very low to no effect on zikv infection ( figure e ). these data suggest that conessine does not block zikv attachment to the host cell, but inhibits zikv infection by targeting virus-cell fusion or postentry step. the above steps of inhibition of zikv infection were further proven inhibition of zikv infection were further proven by the respective anti-zikv compound controls ( figure f-i) . therefore, the above data confirm the potent inhibitory activity of the identified natural products, particularly gossypol, in blocking zikv infection at various stages of the viral life cycle. anti-zikv inhibitor, temoporfin [ ] , was used as a control for step a. (g) an anti-zikv entry (especially in inhibition of the internalization/fusion step) inhibitor, -hydroxycholesterol [ ] , was used as a control for steps b and e. anti-zikv compound bortezomib was used as a control for step d (h), and a replication inhibitor, nitd [ ] , for step f (i). the natural product curcumin (c), which has been previously reported to inhibit the attachment of zikv to host cells [ ] , was used as a control for stage c. the percent inhibition was calculated in the presence or absence of serially diluted natural products. the data are expressed as mean ± s.e.m. (n = ). the experiments were performed in vero e cells and repeated three times, with similar results. (f) a potent anti-zikv inhibitor, temoporfin [ ] , was used as a control for step a. (g) an anti-zikv entry (especially in inhibition of the internalization/fusion step) inhibitor, -hydroxycholesterol [ ] , was used as a control for steps b and e. anti-zikv compound bortezomib was used as a control for step d (h), and a replication inhibitor, nitd [ ] , for step f (i). the natural product curcumin (c), which has been previously reported to inhibit the attachment of zikv to host cells [ ] , was used as a control for stage c. the percent inhibition was calculated in the presence or absence of serially diluted natural products. the data are expressed as mean ± s.e.m. (n = ). the experiments were performed in vero e cells and repeated three times, with similar results. to identify the potential binding regions of the natural products in zikv proteins, we first carried out an elisa-based approach by coating the plates with zikv full-length e or ediii proteins. we then tested for binding affinity using a zikv ediii-specific mab, zka -lala, for e or ediii binding. results revealed that gossypol bound potently to the full-length e and ediii proteins, with ec values of . and . µm, respectively, whereas curcumin had much lower binding affinity to zikv full-length e and ediii proteins ( figure a,b) . otherwise, digitonin, conessine, bortezomib (anti-zikv compound control), and dmso (negative control) did not bind to any zikv proteins tested ( figure a,b) . we then evaluated the binding of gossypol using an spr assay, and the result showed that it had binding affinity values of . µm to zikv e protein ( figure c ). zikv full-length e and ediii proteins ( figure a,b) . otherwise, digitonin, conessine, bortezomib (anti-zikv compound control), and dmso (negative control) did not bind to any zikv proteins tested ( figure a,b) . we then evaluated the binding of gossypol using an spr assay, and the result showed that it had binding affinity values of . m to zikv e protein ( figure c ). since gossypol bound to zikv e protein, potentially in the ediii region, we further carried out an elisa completion assay to identify its possible binding site(s) in the ediii. accordingly, zikv ediii protein was coated on the plates, and binding between zikv ediii and ediii-specific mabs (smzab , zka -lala, zv- , or z ), or a zikv edi/dii-specific mab control (zka ) was evaluated in the presence of serially diluted gossypol. the results showed that gossypol potently blocked binding of ediii-specific mabs smzab , zka -lala, zv- , or z to ediii protein in a dose-dependent manner, with ic values of . , . , . , and . m, respectively, whereas the dmso control showed no blockage of this binding ( figure d ). in the meantime, there was no binding between the control mab zka and ediii protein, so no obvious inhibition of gossypol was detected ( figure d ). the above zikv ediii-specific mabs have been previously shown to potently neutralize zikv infection [ ] and recognize epitopes, including the lateral ridge, such as residues - , - , , , , and - of zikv ediii protein [ ] [ ] [ ] . therefore, the above data suggest that gossypol most likely binds to the lateral ridge of the zikv ediii protein to block the ediii-mab binding. the ability of gossypol to inhibit the binding between zikv ediii and ediii-specific neutralizing mabs. the concentrations of zikv ediii and mabs were . and . µg/ml, respectively. the percent inhibition in the ediii-mab binding was measured in the presence or absence of serially diluted gossypol using the formula (( -(ediii-mab-gossypol)/(ediii-mab) × ), which, in turn, formed the basis for calculating % inhibitory concentration (ic ) values. zikv edi/dii-specific mab (zka ) and dmso were used as controls. the data are expressed as mean ± s.e.m. (n = ) . the experiments were repeated twice, with similar results. since gossypol bound to zikv e protein, potentially in the ediii region, we further carried out an elisa completion assay to identify its possible binding site(s) in the ediii. accordingly, zikv ediii protein was coated on the plates, and binding between zikv ediii and ediii-specific mabs (smzab , zka -lala, zv- , or z ), or a zikv edi/dii-specific mab control (zka ) was evaluated in the presence of serially diluted gossypol. the results showed that gossypol potently blocked binding of ediii-specific mabs smzab , zka -lala, zv- , or z to ediii protein in a dose-dependent manner, with ic values of . , . , . , and . µm, respectively, whereas the dmso control showed no blockage of this binding ( figure d ). in the meantime, there was no binding between the control mab zka and ediii protein, so no obvious inhibition of gossypol was detected ( figure d ). the above zikv ediii-specific mabs have been previously shown to potently neutralize zikv infection [ ] and recognize epitopes, including the lateral ridge, such as residues - , - , , , , and - of zikv ediii protein [ ] [ ] [ ] . therefore, the above data suggest that gossypol most likely binds to the lateral ridge of the zikv ediii protein to block the ediii-mab binding. the data described here demonstrate that the identified natural product gossypol bound strongly to zikv e protein, potentially the conserved ediii, thus blocking ediii-mab binding at important neutralizing epitopes and inhibiting viral entry into the target cell. these data reasonably explain the potent broad-spectrum antiviral activity of gossypol against infections of multiple zikv strains. since gossypol demonstrated the highest antiviral activity individually against all zikv strains tested, we next investigated the potential combinatorial effects of the combination of gossypol with three other natural products identified, namely curcumin, digitonin, and conessine, as well as anti-zikv compound control (bortezomib). results demonstrated significant combinatorial inhibitory effects against three zikv strains (pan , flr, and prvabc ) tested when combining gossypol with any of these natural products, and the ci values ranged from . to . µm, . to . µm, and . to . µm for zikv pan , flr, and prvabc strains, respectively (tables - ). the combinations of gossypol with each of these natural products also resulted in the highest enhancement of anti-prvabc activity among the three zikv strains tested (table ). these data show that gossypol can be combined with other inhibitors described above to further increase overall inhibitory activity against current and future emergent zikv strains. the above results on the combinatorial effects against zikv infection could not exclude the possibility that the observed decrease in ic in the presence of another natural product might result from the enhanced cell death caused by the natural product in combination. therefore, we also assessed the change of cc of the natural products alone and in combination, and compared the enhancement of cc with that of ic against zikv strain prvabc . as shown in table , the cytotoxicity of gossypol in combination with curcumin, digitonin, or conessine was not enhanced. the cytotoxicity of gossypol in combination with bortezomib was slightly increased ( . -fold), but it was much lower than the enhancement in anti-prvabc activity of gossypol ( . -fold) in combination with bortezomib. in addition, the cytotoxicity of curcumin and bortezomib, respectively, in combination with gossypol was not enhanced. the cytotoxicity of digitonin or conessine in combination with gossypol was slightly increased by about . -and . -fold, respectively, while they were still lower than the enhancement in anti-prvabc activity of digitonin ( . -fold) or conessine ( . -fold) in combination with gossypol. moreover, the ci values of gossypol with any of the four natural products tested were greater than ( table ), indicating that there was no combinatorial effect on the cytotoxicity to the test cells. these results suggest that the observed decrease in the ic values of the natural products in combination was not due to the enhancement in cytotoxicity of these natural products. note: gossypol with one of the natural products, including curcumin, digitonin, conessine (three lead natural products) or bortezomib (anti-zikv compound control), were first mixed according to the molar ratio identified in the above combinational experiment against zikv strain prvabc , and then added to vero e cells to determine cytotoxicity. the combinatorial cytotoxicity of natural products to vero e cells is expressed as cc identification of broad-spectrum anti-flavivirus inhibitors is crucial to treat infections caused by zikv and other flaviviruses, such as denv. hence, using a plaque assay similar to zikv, we evaluated the antiviral activity of natural products against infections of four serotypes of denv human strains in llc-mk cells. even though all four natural products could inhibit denv- - infections, results showed that gossypol had the highest potency against denv- , denv- , and denv- infections, with ic values of . , . , and . µm, respectively (table ) . also, the anti-denv- activity of gossypol (ic value: . µm) was only slightly higher than that of curcumin (ic value: . µm) ( table ) . the cytotoxicity of these natural products on llc-mk cells was investigated by a cytotoxicity assay, with the cc values ranging from . to . µm ( table ) . the above data indicate the potent anti-denv activity of the natural products, particularly gossypol, against infections of four denv human strains with low to no cytotoxicity. the experiments were performed on llc-mk cells, and the cytotoxicity of natural products in this cell line is expressed as cc . the inhibitory activity of natural products against infections of denv- - is expressed as ic . the data are expressed as mean ± s.e.m. (n = ). the experiments were repeated twice, with similar results. as described earlier, gossypol targeted zikv e protein, potentially ediii. although a number of variations have been identified in the amino acid sequences of e proteins of zikv and denv strains tested in this study ( figure s ), gossypol could still inhibit all zikv and denv strains tested, suggesting that it potentially targeted the conserved sequences in zikv and denv ediii proteins. our data further explain the potent, broad-spectrum activity of gossypol against infections of at least two flaviviruses, including zikv and denv. development of safe and effective antiviral therapeutics is urgently needed to treat zikv infections and zikv-caused diseases, particularly zikv-associated microcephaly, fetal death, or neurological diseases [ , [ ] [ ] [ ] [ ] . here, we have identified four small-molecule-based natural products, namely gossypol, curcumin, digitonin, and conessine, with robust anti-zikv activities in vero e cells. among them, gossypol had the greatest potency to block infections of zikv for almost all strains isolated from to from nine countries and three hosts, including six recent human strains associated with congenital zika syndrome and other neurological malformations. time-of-addition-based mechanistic studies indicated that gossypol could neutralize zikv infection by targeting the virus rather than the cell or cell-associated zikv entry or replication steps. inhibition assays revealed that gossypol strongly bound to zikv e protein, particularly the conserved ediii, and blocked the binding between zikv ediii and ediii-specific neutralizing mabs, rationalizing its efficacious and broad-spectrum anti-zikv activity. it appears that the binding between gossypol and zikv e/ediii protein might be nonspecific, since gossypol is also active against other enveloped viruses, including herpes simplex virus type , parainfluenza virus type , influenza virus, and hiv- [ ] [ ] [ ] [ ] [ ] . because of the potential toxicity and side effects of gossypol to humans [ ] [ ] [ ] , it might not be a good idea to use this natural product as a drug to treat human diseases, including zikv-associated microcephaly and other flavivirus-caused diseases. nevertheless, the goal of this study is to identify the best active, natural product, then modify it to improve its antiviral activity and drug-like properties and reduce its toxicity. due to the symmetric nature of the gossypol molecule, there will be better opportunity to defragment its structure to smaller drug-like molecules with higher activity and lower toxicity. therefore, it is suggested not to use gossypol as the lead molecule, but rather the final drug. unlike gossypol, digitonin and conessine did not neutralize zikv directly, but instead inhibited zikv infection at various stages of the life cycle, including viral attachment, membrane fusion, or postentry steps. notably, digitonin is a glycoalkaloid saponin detergent. it is widely used as a cell membrane permeabilizing agent [ , ] and a detergent for selective solubilization of membrane proteins [ ] [ ] [ ] ; thus, the antiviral activity of this natural product is likely to be very nonspecific. by comparison, conessine appears to be a good candidate with a decent anti-zikv activity and very low cytotoxicity (higher selectivity). however, it is a steroidal alkaloid [ ] , which might not be ideal for further optimization. similar to gossypol, curcumin may directly neutralize zikv infection, but it seems to mainly inhibit the early stage (attachment) of virus infection, the mechanism of which is similar to that seen in a previous report using different approaches [ ] . our data have also demonstrated strong in vitro ability of these natural products-gossypol in particular-against four serotypes of denv human strains with low to no cytotoxicity, and the ic values against denv were similar to those against zikv. we have found that gossypol bound to zikv e protein, potentially ediii, suggesting that it may recognize highly conserved regions and sites in the e proteins of zikv and denv. previously, curcumin was shown to potentially inhibit denv- infection through direct effects on viral particle production or various cellular systems [ ] . we have not found studies in the literature reporting on the antiviral activity of curcumin on other serotypes of denv or the inhibitory effects of gossypol, digitonin, and conessine on zikv and other flaviviruses. thus, the present study provides a rationale for further understanding of anti-denv and anti-zikv mechanisms of these natural products and identifying their potential binding sites in the viral proteins. except for demonstrating the individual in vitro anti-zikv activity of natural products identified in this study, particularly gossypol, we have confirmed important combinatorial anti-zikv effects of gossypol in combination with other natural products. it is interesting to note that combining gossypol with curcumin, digitonin, or conessine resulted in increased combinatorial effect against prvabc strain compared to the flr strain of zikv, potentially because gossypol had more potent anti-flr activity than anti-prvabc activity when tested individually. also, such combinations enhanced the individual antiviral activity of curcumin, digitonin, and conessine against the test viruses. we have shown that the combination of gossypol and bortezomib, a licensed proteasome inhibitor previously reported inhibiting zikv infection [ , ] , led to significant combinatorial activity against the three epidemic zikv human strains tested. therefore, our study demonstrates the possibility of combining gossypol with other natural products or compounds to enhance antiviral activity. overall, this study has evaluated the antiviral activity of four natural products in vitro, among which three are newly identified natural products with strong anti-zikv ability. future observations will be needed to evaluate the protective efficacy of these natural products individually or in combination, or using their defragmented, small drug-like molecules (in the case of gossypol) against zikv-caused congenital infection and fetal demise in available animal models. modification of the structure of gossypol and identification of its derivatives with better antiviral activity, but without cytotoxicity, would be essential for developing a safe and effective anti-zikv agent for human use. also, future studies to evaluate the antiviral activity of modified gossypol or its derivatives against other flaviviruses, both in vitro and in vivo, will be helpful for identification of an effective and safe pan-flavivirus inhibitor. taken together, the broad-spectrum ability of the identified natural products, especially gossypol, against zikv and denv infections indicates the potential for further development of these small molecules or their derivatives as the lead compounds or effective anti-flavivirus inhibitors. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s . figure s : association between inhibitory activity of natural products against zikv strain pan and their cytotoxicity. figure s : association between inhibitory activity of natural products against zikv strain prvabc and their cytotoxicity. figure s : multiple sequence alignment of amino acid (aa) sequences of e protein of zikv strains and denv- - human strains used in this study. author contributions: s.j., a.k.d., and l.d. designed the study. y.g., w.t., n.w., and x.l. performed the experiments and analyzed the data. y.g. and n.w. carried out sequence alignment analyses. y.g., s.j., a.k.d., l.d., and s.c. wrote and revised the manuscript. zika virus: new clinical syndromes and its emergence in the western hemisphere zika virus (i). isolations and serological specificity zika virus infection in pregnancy: maternal, fetal, and neonatal considerations implications of zika virus 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detergents specific release of membrane-bound annexin ii and cortical cytoskeletal elements by sequestration of membrane cholesterol anti-malarial property of steroidal alkaloid conessine isolated from the bark of holarrhena antidysenterica clinical use of proteasome inhibitors in the treatment of multiple myeloma this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no competing interests. key: cord- -svidp ar authors: tomori, shouhei; morishima, satoko; nishi, yukiko; nakachi, sawako; tamaki, keita; morichika, kazuho; tedokon, iori; shimabukuro, natsuki; hanashiro, taeko; kitamura, sakiko; uchibori, sachie; miyagi, riko; miyagi, takashi; karimata, kaori; ohama, masayo; yamanoha, atsushi; tomoyose, takeaki; karube, kennosuke; fukushima, takuya; masuzaki, hiroaki title: transplant-related complications are impediments to the success of allogeneic hematopoietic stem cell transplantation for adult t cell leukemia patients in non-complete remission date: - - journal: bone marrow transplant doi: . /s - - -z sha: doc_id: cord_uid: svidp ar outcomes of allogeneic hematopoietic stem cell transplantation (allo-hsct) for patients with adult t cell leukemia/lymphoma (atl) are not satisfactory, particularly in patients in non-complete remission at transplantation (pt-non-cr). we conducted a regional retrospective study in the atl endemic area of okinawa, japan. of atl patients, received allo-hsct in cr and in non-cr. the -year overall survival ( yos) rate and median survival time for the whole cohort was . % and . months, respectively. the yos of pt-non-cr was significantly lower than that of patients in cr (pt-cr) ( . % vs. . %, p = . ). transplant-related mortality (trm) was significantly higher in pt-non-cr than in pt-cr ( . % vs. . %, p = . ), while there was no significant difference in disease-associated mortality (dam) between pt-non-cr and pt-cr. multivariable analysis for pt-non-cr revealed that poor performance status (poor-ps) and higher sil- r level (high sil- r) adversely affected os. poor-ps was associated with higher trm, but not with higher dam in pt-non-cr. high sil- r did not affect trm or dam in pt-non-cr. overall, high trm rates rather than dam contribute to the poor outcomes of pt-non-cr, suggesting that not only disease control but also management of transplant-related complications is required for allo-hsct in atl patients. adult t-cell leukemia/lymphoma (atl) is a malignancy of peripheral t lymphocytes caused by human t-cell leukemia virus type (htlv- ), which was the first retrovirus to be isolated from a human malignant disease [ ] [ ] [ ] . htlv- shows a puzzling geographical distribution around the world, and southwestern japan (kyushu and okinawa) is one of several areas with a high prevalence of infection [ ] . the incidence of atl is closely linked to the prevalence of htlv- infection, and thus okinawa is an endemic area of atl [ ] . atl is divided into four clinical subtypes: acute, lymphoma, chronic, and smoldering [ ] . these clinical subtypes are closely related to prognosis, which is extremely poor for the aggressive subtypes [ ] . although the best clinical results are achieved by systemic chemotherapy, the median survival time is only . months and complete response is achieved in only % of treated cases [ ] . most of these patients eventually relapse and have a median progression-free survival time of - months. moreover, the treatment options are extremely limited for those who do not respond to the initial chemotherapy. new immunotherapy or immunomodulatory agents, such as mogamulizumab (anti-ccr monoclonal antibody) [ , ] and lenalidomide (an oral immunomodulatory drug) [ ] have recently been used as treatments for atl in japan and are effective in some patients. however, the long-term clinical outcomes remain unclear. allogeneic hematopoietic stem cell transplantation (allo-hsct) has been available for patients with aggressive atl since [ ] and it is now considered a promising treatment option for these patients [ ] [ ] [ ] [ ] . a nationwide retrospective study of allo-hsct for the treatment of atl [ ] demonstrated several pretransplantation factors that are associated with poor survival rates, such as poor eastern cooperative oncology group performance status (ecog-ps) rating, higher age, male sex, non-complete remission (non-cr) at transplantation, and the use of unrelated cord blood as the stem cell source. in that study, non-cr at transplantation was also identified as a risk factor for diseaseassociated mortality (dam). although disease status at transplantation is known to be an important factor associated with outcome after allo-hsct for atl, it is often difficult to achieve cr in atl patients. as a consequence, some atl patients are compelled to receive allo-hsct despite their non-cr status. indeed, in daily practice, we often encounter patients whose atl tumor cells become chemoresistant during their planned chemotherapy. we therefore think that it is essential to improve the allo-hsct treatment strategy in atl patients, especially those in non-cr. here, we conducted a regional retrospective study in the endemic atl area of okinawa prefecture to clarify the factors affecting transplant outcomes of atl patients, focusing on patients in non-cr at transplantation (pt-non-cr). we retrospectively collected data from patients with aggressive atl who had received allogeneic transplantation at university of the ryukyus hospital and heartlife hospital in okinawa prefecture between september and january . since all allo-hsct procedures are performed in these two centers in okinawa, the patients analyzed in the current study included all patients with aggressive atl who underwent allo-hsct in this area. informed consent was obtained in accordance with the declaration of helsinki. this study was conducted with the approval of the institutional review board of the university of the ryukyus. the primary endpoint of this study was overall survival, defined as the time from the date of transplantation until the date of death from any cause. the secondary endpoints were cumulative incidences of dam and transplant-related mortality (trm). reported causes of death were reviewed and categorized into disease-associated or transplantassociated deaths. disease-associated deaths were defined as deaths from relapse or progression of atl. transplantrelated deaths were defined as any death without relapse or progression of atl. descriptive statistics were used to summarize variables related to patient demographic and transplant characteristics. comparisons between pt-non-cr and patients in cr at transplantation (pt-cr) were performed with the fisher's exact test for categorical variables and the mann-whitney u test for continuous variables. the probability of overall survival was estimated according to the kaplan-meier method, and univariable comparisons among the groups were made using the logrank test. data on patients who were alive at the time of last follow-up were censored. fine and gray's proportionalhazards model for subdistribution of a competing risk was used to analyze the cumulative incidences of trm and dam. for dam, transplant-related deaths were competing events; for trm, disease-associated deaths were competing events. gray's test was used for group comparisons of cumulative incidence [ ] . cox's proportional-hazards regression model [ ] was used to evaluate variables potentially affecting overall survival. variables considered were recipient age group (< years and ≥ years); recipient sex (female and male); lines of chemotherapy prior to transplantation ( and ≥ ); donor source (related and unrelated); human leukocyte antigen (hla) matching (matched and mismatched); disease status before transplantation (cr and non-cr); type of conditioning regimen (reduced-intensity conditioning [ric] and myeloablative conditioning [mac]); ecog-ps before transplantation (ecog-ps, - , and - ); and soluble interleukin- receptor (sil- r) level (sil- r < u/ml and ≥ u/ml). conditioning regimens were classified as myeloablative when total-body irradiation was > gy, oral busulfan was ≥ mg/kg, intravenous busulfan was ≥ . mg/kg, or melphalan was > mg/m , in accordance with the report by giralt et al. [ ] . hla matching between patient and donor was defined according to the results of serological or molecular typing for hla-a, b, and dr antigens. results were expressed as hazard ratios with % confidence interval (ci). all tests were two-sided, and a p value of less than . was considered to indicate statistical significance. all statistical analyses were performed with ezr (saitama medical center, jichi medical university), which is a graphical user interface for r (the r foundation for statistical computing, vienna, austria) [ ] or stata version (statacorp llc, college station, tx). table shows the characteristics of the patients. of the patients, ( %) received allo-hsct while in cr and ( %) while in non-cr. the patients who received transplants in non-cr had higher ecog-ps values, higher sil- r levels, and shorter follow-up periods. among the ptnon-cr, of were ecog-ps - (ps , n = ; ps , n = ), while there was only one patient with ecog-ps - among the pt-cr (ps , n = ). at transplantation, none of the pt-cr had sil- r levels of ≥ u/ml, while of the pt-non-cr had sil- r levels of ≥ u/ml. pt-cr had received a median line (range - ) of chemotherapy, while pt-non-cr had received a median lines (range - ) of chemotherapy prior to allo-hsct. chemotherapy regimens prior to transplant and detailed transplant procedures are shown in tables s and s , respectively. conditioning regimen, graft-versus-host disease (gvhd) prophylaxis, and infection prophylaxis were similar in both transplant centers. cyclophosphamide + total-body irradiation was used as the myeloablative conditioning regimen, while fludarabine + melphalan-based or fludarabine + busulfan-based regimen was used as the ric regimen. for gvhd prophylaxis, cyclosporine a + shortterm methotrexate was used in patients transplanted from an hla-matched related donor, while tacrolimus + short-term methotrexate was used in patients transplanted from an unrelated or hla-mismatched related donor. antimicrobial prophylaxis with levofloxacin, antifungal prophylaxis with fluconazole or voriconazole, varicella-zoster virus prophylaxis with acyclovir, and pneumocystis jirovecii pneumonia prophylaxis with trimethoprim-sulfamethoxazole were standard prophylaxis for infection. the ratio of pt-non-cr to pt-cr was higher in the university of the ryukyus hospital (pt-non-cr, n = ; pt-cr, n = ) than in heartlife hospital (pt-non-cr, n = ; pt-cr, n = ) (p = . ). the majority of pt-non-cr underwent transplantation at the university of the ryukyus hospital before (table s ) . of the patients included in the study, were alive after a median follow-up of . days (range, - days). (fig. b) . the cumulative incidence of neutrophil engraftment within days after transplantation was % in pt-cr and . % ( % ci, . - . %) in pt-non-cr (p = . ) (fig. s ) . median time to neutrophil recovery in pt-cr and pt-non-cr was days ( - days) and days ( - days), respectively. univariable analyses for the whole cohort revealed five factors that adversely affected overall survival ( table ) : age ≥ years (hazard ratio [hr], . ; % ci, . - . ; p = . ), lines of chemotherapy prior to transplantation ≥ (hr, . ; % ci, . - . ; p = . ), non-cr at transplantation (hr, . ; % ci, . - . ; p = . ), ecog-ps - (hr, . ; % ci, . - . ; p < . ), and sil- r ≥ u/ml at transplantation (hr, . ; % ci, . - . ; p < . ). because disease status at transplantation, sil- r level, and lines of chemotherapy prior to transplantation co-vary, sil- r and lines of chemotherapy were not examined in multivariable analysis. in multivariable analysis, ecog-ps - (hr, . ; % ci, . - . ; p < . ), age ≥ years (hr, . ; % ci, . - . ; p = . ), and non-cr status at transplantation (hr, . ; % ci, . - . ; p = . ) were significantly associated with worse os (table ) . we performed subgroup analysis of the non-cr patient group to analyze the effect of pretransplantation factors on overall survival in pt-non-cr. univariable analysis of survival in non-cr patients identified two factors, which adversely affected overall survival: ecog-ps - (hr, . ; % ci, . - . ; p < . ) and sil- r ≥ u/ml at transplantation (hr, . ; % ci, . - . ; p = . ) ( table ). multivariable analysis also revealed that higher ecog-ps values (hr, . ; % ci, . - . ; p < . ) and sil- r (hr, . ; % ci, . - . ; p = . ) were associated with poorer overall survival ( table ). the -year overall survival rates in pt-non-cr with ecog-ps - and those with ecog-ps - were . % ( % ci: . - . %) and . % ( % ci: . - . %), respectively (p < . ) (fig. a) . the -year overall survival rates in ptnon-cr with sil- r levels of < u/ml and those with sil- r ≥ u/ml were . % ( % ci: . - . %) and . % ( % ci: . - . %), respectively (p = . ) (fig. b ). overall, ( . %) patients died from transplant-related complications. the cumulative incidence of trm was . % ( % ci, - . %) for the whole cohort (fig. s ). the cumulative incidence of trm in pt-non-cr was significantly higher than that in pt-cr ( . % [ % ci, . - . %] vs. . % [ % ci, . - . %], p = . ) (fig. a) . death from progression of atl occurred in ( . %) patients. the cumulative incidence of dam was . % ( % ci, . - . %) for the whole cohort (fig. s ) . the cumulative incidence of dam in patients who received transplants in non-cr and in those who received transplants in cr were . % ( % ci, . - . %) and . % ( % ci, . - . %), respectively. there was no significant (fig. s b ). the sil- r level at transplantation did (fig. s ) . the causes of death after transplantation in atl patients are summarized in table this study on allo-hsct in atl patients demonstrated that the extremely poor outcomes in pt-non-cr were attributable to trm rather than dam. contrary to our initial expectations, the dam rate of pt-cr almost equaled that of pt-non-cr. disease status other than cr at transplantation in patients with aggressive atl is associated with a low survival rate [ , ] , and it is widely accepted that disease progression might contribute to the poor survival rate after allo-hsct in non-cr atl patients. to our knowledge, however, there have been no reports focusing on the prognostic impact of disease status at transplantation on trm following allo-hsct in atl patients. in this study, high ecog-ps values and high sil- r levels were significantly associated with poor survival in ptnon-cr. in atl patients, a high level of sil- r ( u/ ml or higher) at transplantation is known to be a significant risk factor for poor overall survival and disease progression after allo-hsct [ ] . in our cohort, none of the pt-cr had high levels of sil -r, indicating that the level of circulating sil- r closely reflects the disease status of atl. although sil- r levels correlate with tumor burden in atl [ , ] , in the current study, high sil- r levels at transplantation were not associated with dam in pt-non-cr. these findings indicate that sil- r levels would not provide sufficient information to enable a decision to be made on whether to proceed with additional chemotherapy and/or immunotherapy before allo-hsct in non-cr patients because intensive therapies before transplantation can give rise to various complications after allo-hsct. the major causes of death in pt-non-cr were not due to disease progression, and infection was the most common cause of death among a variety of complications. in contrast to our results, relapse and disease progression are the predominant causes of treatment failure and mortality after allo-hsct in patients with refractory acute myeloid leukemia (aml) [ , ] . a low number of naive tlymphocytes may underlie the mechanism of immunodeficiency in htlv- infected individuals [ ] . indeed, atl patients are susceptible to various opportunistic infections and it has been reported that infection-related mortality is significantly higher than in patients with aml and acute lymphoblastic leukemia (all) [ ] . therefore, complications caused by infections may have a stronger impact on survival after allo-hsct in patients with atl than in patients with other hematological malignancies. the high rate of complications among pt-non-cr raises the question as to why transplant-related complications cause more severe problems for pt-non-cr than for pt-cr. kozako et al. reported that the expression of programmed death- in cd + t-cells, including in cytomegalovirus-and epstein-barr virus specific cytotoxic tcells, was significantly higher in patients with atl than in htlv-i carriers and control individuals [ ] . it is tempting to speculate that the compromised cellular immunity in atl patients is attributable to t-cell [ ] . therefore, compared with those in cr, patients with atl in non-cr may show reduced immune responses to various pathogens. we also noticed that a certain number of pt-non-cr died of transplant-related complications other than infection, but we could not clarify whether residual atl gave rise to these complications (table ) . a nationwide retrospective study of allo-hsct in atl patients revealed transplantation outcomes similar to that of the whole cohort in our study, that is, -year overall survival, cumulative incidence of trm, and disease-associated death rates were %, %, and %, respectively [ ] . it also showed that transplant-related events were the principal causes of early death, while disease-associated deaths were more common in the later phases [ ] . we demonstrated that transplant-related deaths in the early phase of allo-hsct in atl patients were prominent among pt-non-cr ( fig. and table ). interestingly, the cumulative incidence of diseaseassociated death of pt-cr at transplantation was roughly equivalent to that of pt-non-cr in the current study. these results suggest that disease progression after transplantation in pt-non-cr cannot be evaluated properly due to the early deaths of the patients. shigematsu et al. reported that the -year overall survival rate of patients with aggressive atl who received allo-hsct in cr was more than % [ ] , while in the current study, the -year overall survival rate of atl pt-cr was only . %. patients with aggressive atl who do not receive allo-hsct in okinawa prefecture show poorer clinical outcomes than those patients in other areas of japan [ ] . the difference in clinical outcomes between patients with atl in okinawa and those in other areas of japan might be partly attributed to the different distribution of the htlv- tax genotype in okinawa from mainland of japan [ ] . further study is needed to clarify the impact of geographical factors and/or genetic backgrounds of atl patients on transplantation outcomes after allo-hsct in okinawa. since this study was an observational retrospective study and included a small patient population, we cannot draw definitive conclusions about factors affecting the outcomes of allo-hsct in all atl patients. however, our study included all patients with aggressive atl who received allo-hsct in okinawa prefecture during the study period. therefore, the results of our study reflected actual conditions of allo-hsct for atl in okinawa. because patients received transplantation without strict transplant eligibility criteria, patients with poor ecog-ps were included in this study. indeed, there was one patient with an ecog-ps of three in pt-cr and four patients with an ecog-ps of three in pt-non-cr. no patients had ecog-ps in either group. even after excluding patients with ecog-ps of three, similar results were seen in these patients ( fig. s and table s ). in this study, pt-non-cr included patients with partial response, patients with stable disease, and patients with progressive disease at transplantation (table ) . worse disease status was associated with lower overall survival and higher trm, but did not affect dam (fig. s ) . intensive chemotherapy for patients with atl is effective for the first several courses of treatments. however, it is difficult to complete planned treatments because of toxicity and/or loss of effectiveness of the chemotherapy [ ] . in cases of dismal outcomes after intensive chemotherapy in atl patients, hematologists often consider allo-hsct as a treatment option for patients with aggressive atl in non-cr. indeed, in the current study, and in other retrospective studies of allo-hsct for aggressive atl, the number of ptnon-cr tends to be more than double that of pt-cr [ , ] . since the early application of allo-hsct is considered to reduce trm and improve overall survival in patients with aggressive atl, we should consider both the disease status at transplantation and the optimal timing of allo-hsct. furthermore, we should also consider more effective treatment strategies to reduce disease progression and relapses after allo-hsct. in conclusion, we revealed that high trm rates in the early posttransplantation phase contribute considerably to the poor survival rate of patients with atl who received allo-hsct while in non-cr. even after overcoming complications in the early phase, disease progression and relapse remain important problems in patients with atl both in non-cr and in cr at transplantation. our findings suggest that not only treatment for disease control but also intensive management to prevent transplant-related complications is required in order to improve the success rate of transplantation in atl patients who cannot achieve cr before allo-hsct. furthermore, more effective therapeutic strategies for atl are required to attain cr in patients undergoing allo-hsct. adult t-cell leukemia: clinical and hematologic features of cases detection and isolation of type c retrovirus particles from fresh and cultured lymphocytes of a patient 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subdistribution of a competing risk regression model and life tables reduced-intensity conditioning regimen workshop: defining the dose spectrum investigation of the freely available easy-to-use software 'ezr' for medical statistics high level of serum soluble interleukin- receptor at transplantation predicts poor outcome of allogeneic stem cell transplantation for adult t cell leukemia soluble interleukin receptors in sera of japanese patients with adult t cell leukemia mark activity of disease significance of soluble interleukin- receptor levels for evaluation of the progression of adult t-cell leukemia factors that contribute to long-term survival in patients with leukemia not in remission at allogeneic hematopoietic cell transplantation allogeneic stem cell transplantation for refractory acute myeloid leukemia: a single center analysis of long-term outcome impaired production of naive t lymphocytes in human t-cell leukemia virus type i-infected individuals: its implications in the immunodeficient state distinct clinical features of infectious complications in adult t cell leukemia/lymphoma patients after allogeneic hematopoietic stem cell transplantation: a retrospective analysis in the nagasaki transplant group pd- /pd-l expression in human t-cell leukemia virus type carriers and adult t-cell leukemia/lymphoma patients aberrant pd-l expression through ′-utr disruption in multiple cancers characterization of patients with aggressive adult t-cell leukemia-lymphoma in okinawa, japan: a retrospective analysis of a large cohort human t-cell leukemia virus type i tax genotype analysis in okinawa, the southernmost and remotest islands of japan: different distributions compared with mainland japan and the potential value for the prognosis of aggressive adult t-cell leukemia/lymphoma conflict of interest the authors declare that they have no conflict of interest.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - no e authors: jeannoël, m.; lina, g.; rasigade, j. p.; lina, b.; morfin, f.; casalegno, jean sebastien title: microorganisms associated with respiratory syncytial virus pneumonia in the adult population date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: no e respiratory syncytial virus (rsv) has been recognized as responsible for severe respiratory illness in adults, especially in the elderly. while pneumonia is commonly observed during rsv infection, the burden and epidemiology of bacterial superinfection is poorly understood. the aim of this study was to identify microorganisms associated with rsv-positive pneumonia in adults. a retrospective study was conducted during three consecutive winters (october to april – ) in the university hospital of lyon, france. during rsv circulation periods, a systematic rsv screening was performed by reverse-transcription pcr on all respiratory samples collected from adults. records of rsv-positive patients were subsequently analyzed to identify radiologically confirmed pneumonia cases. bacteria were identified by standard bacteriology cultures or urinary antigen screening and classified as potentially causative of pneumonia if quantification was above the specific threshold as defined by the european manual of clinical microbiology. overall, , adult respiratory samples were screened for rsv detection by pcr. in total, had a positive rsv detection ( . %) among which presented with pneumonia including bacterial superinfections ( . %) with streptococcus pneumonia, haemophilus influenza, staphylococcus aureus, pseudomonas aeruginosa, and moraxella catarrhalis. most patients were elderly ( . %) and patients with comorbidities ( . %). a more severe outcome was observed for rsv-bacteria-associated pneumonia compared with rsv pneumonia: length of stay was significantly longer ( days vs days) and icu hospitalization more frequent ( . % vs . %) (p < . ). in conclusion, we did not observe major differences in the epidemiology of bacterial superinfections in rsv-positive pneumonia compared to reports on post-influenza pneumonia. respiratory syncytial virus (rsv) is now recognized as an important cause of respiratory illness in adults, especially in the elderly [ ] . secondary bacterial pneumonia after or during an rsv infection is among the more severe complications reported in this population [ ] . few studies have described superinfection or coinfection with bacteria among adults with rsv-positive pneumonia in inpatient setting [ , ] . diagnostic testing for rsv is usually not routinely performed in this population. however burden and epidemiology of rsv-positive pneumonia in adults is important to assess in regards of the current antibiotic therapy recommendations [ ] . the aim of our study was to gain insights in the epidemiology of bacterial superinfections associated with rsv pneumonia in adults. we conducted a retrospective, monocenter, and observational study on three successive winter periods (october to april, april, - . in the university hospital of lyon (hospices civils de lyon, france), upper and lower respiratory tract samples from patients with respiratory symptoms are systematically screened for rsv and metapneumovirus (hmpv) using a reverse transcriptase polymerase chain reaction (rt-pcr). we included all adult patients (≥ years old) positive for rsv with radiologically confirmed pneumonia as diagnosed in the medical chart. hospital-acquired pneumonia (hap) was defined as radiologically confirmed pneumonia that manifests ≥ h after the patient's admission to the hospital, and not present at the time of intubation. data collected included laboratory results, demographics (age, sex), underlying conditions (cardiovascular disease, pulmonary disease, diabetes mellitus, and immunodeficiency), as well as clinical outcomes (length of stay, acute respiratory distress syndrome (ards), intensive care unit (icu) admission, in-hospital death). for virus detection, clinical specimens comprised nasopharyngeal swabs, endotracheal aspirates, and broncho-alveolar lavages. respiratory specimens were extracted using easymag (biomerieux, marcy l'étoile, france). rsv detection was performed using a rt-pcr (abi ; thermofisher, illkirch, france), or duplex rt-pcr detecting rsv and hmpv (r-gene detection kit, biomérieux-argene, france) as previously described [ ] . for bacteria detection, pathogens were classified as potentially causative of pneumonia if identified in pleural fluid or blood, or if identified within a sputum specimen, endotracheal aspirate, and broncho-alveolar lavage with good-quality criteria and if quantification was above the appropriate emcm decision threshold. semi-quantitative bacterial culture process and interpretation was performed following the european manual of clinical microbiology (emcm) guidelines [ ] . maldi-tof (vitek-ms) was used for species identification. statistical analyses were performed using graphpad prism software (version . , graphpad software, la jolla, ca, usa). univariate analysis was used to compare rsvbacteria-associated pneumonia and rsv-positive pneumonia. quantitative variables (age and length of stay) were expressed as median and interquartile range (iqr). difference between the age groups was assessed by student's t test. difference between lengths of stay was assessed by non-parametric mann-whitney test. qualitative variables were expressed as number and percentage. a fisher's exact test was used for univariate comparisons. a p value < . was considered significant. during the study period, viral rt-pcr was performed on , respiratory samples collected from adult patients. influenza was detected in patients ( . % of all respiratory samples), rhinovirus in patients ( . % of all respiratory samples), rsv in patients ( . % of all respiratory samples), and hmpv in patients ( . %). the median age of patients tested positive for rsv was years (interquartile range to ), with a sex ratio of ( . % were men). among rsv-positive patients, . % of case records were retrieved (n = / ). primary discharge diagnoses in patients without respiratory symptoms were as follows: . % (n = / ) of systematic sampling without acute respiratory infection in immunosuppressed patients, . % (n = / ) of acute heart failure, . % (n = / ) of sepsis, and . % (n = / ) of neurological disorders. . % (n = / ) patients presented with respiratory disorders, including . % (n = / ) of respiratory failure, . % (n = / ) of acute respiratory infection, . % (n = / ) of acute respiratory distress syndrome, and . % (n = / ) radiologically confirmed pneumonia. among these pneumonia, cases were associated with bacteria classified as potentially causative (rsv-bacteria-associated pneumonia) and cases had no bacterial documentation (rsv-positive pneumonia). among these rsvpositive pneumonia, cases were associated with another virus ( rsv and influenza coinfections, rsv and picornavirus coinfection, rsv and bocavirus coinfection, and rsv and cmv coinfection). among the rsv-bacteria associated pneumonia, hospital-acquired pneumonia (hap) was observed mostly in adults ≥ years old (n = / ) whereas community-acquired pneumonia (cap) occurred mostly in to -year-old adults (n = / ). most of the patients presenting a pneumonia were elderly (median age years (interquartile range to ) and had underlying conditions ( . %) ( table ). only . % were resident of nursing homes. demographic characteristics did not differ between patients with an rsv-bacteria-associated pneumonia and patients with an rsv-positive pneumonia. among these pneumonia, patients received an empiric antibiotic treatment and immunosuppressed patients received an antiviral treatment: patient received ribavirin, patient received polyvalent immunoglobulins, and patient received ribavirin and polyvalent immunoglobulins. in total, patients presented an ards, patients were admitted to icus and in-hospital mortality occurred in adults of which all were ≥ years old (n = / ) or immunosuppressed (n = / ). rsv and bacteria coinfection was statistically associated with a more severe outcome than rsv-positive pneumonia as length of stay was significantly longer ( days vs days) and icu hospitalization more frequent ( . % vs . %) (p < . ). however no significant difference was observed regarding in-hospital mortality ( table ) . among the cases of rsv-positive pneumonia with superinfection, the most frequent bacteria were streptococcus pneumoniae, enterobacteriaceae ( kleb siella p neu monia , en teroba cte r cloa ca e , citrobacter koseri), pseudomonas aeruginosa, haemophilus influenza, staphylococcus aureus, and moraxella catarrhalis. in two patients, more than one bacterial pathogen was detected. in patients hospitalized f o r c a p, t he m o s t f r e qu en t ba ct er i a w e re s . pneumoniae, h. influenzae, and s. aureus whereas in hap the most frequent bacteria were p. aeruginosa and enterobacteriacae (table ). in this study, detection rates of the screened viruses were consistent with previous reports describing the epidemiology of respiratory viruses in hospitalized adults [ ] [ ] [ ] . rsv detection in our study was low ( . %) compare to rates of rsv detection reported in previous studies ranging from % of hospitalized adult patients to % of hospitalization for acute respiratory illness in an elderly population [ ] [ ] [ ] . it is probably due to the systematic testing strategy associated to a species distribution of pathogenic bacteria involved in rsv-positive pneumonia (cap) and hospital-acquired pneumonia (hap) sampling bias toward influenza-like illness. those symptoms are indeed poorly predictive of an rsv infection [ ] . among rsv infected patients, . % presented a radiologically confirmed pneumonia and . % had an rsv-bacteriadocumented pneumonia. despite the large number of rsv testing among the adult population, we identified only a few rsv radiologically confirmed pneumonia. although this is a low prevalence, it is in agreement with the current literature in which to % of rsv-bacteria co-infections were reported [ ] . in our study, a higher prevalence was noted in the elderly ( / ), immunosuppressed, and patients with comorbidities ( / ). as it has been previously described, we observed that rsv-bacteria-associated pneumonia have a more severe outcome than rsv-positive pneumonia [ ] . enterobacteriaceae and pseudomonas aeruginosa were the most frequently detected pathogens in hap and did not differ from the pathogens usually responsible for hap in icu [ ] . s. pneumonia and h. influenza were the most frequently detected pathogens in cap. in a previous study in adults with cap, s. pneumonia and h. influenza were also mainly reported in rsv-bacteria coinfections [ ] . we did not observed major differences to influenza-bacterial coinfections [ ] . those results suggest that there is no main difference between the microbiological epidemiology of rsv-bacterial coinfections and influenza-bacterial coinfections. this observation needs to be confirmed by other studies. further research should focus on estimating the rsv cap burden in adult as well as understanding the underlying mechanisms of copathogenesis. conflict of interest the authors declare that they have no conflict of interest. ethical approval this article does not contain any studies with human participants performed by any of the authors. informed consent for this type of study formal consent is not required. risk of mortality associated with respiratory syncytial virus and influenza infection in adults viral-bacterial coinfection affects the presentation and alters the prognosis of severe community-acquired pneumonia respiratory syncytial virus infection in elderly and high-risk adults incidence and characteristics of viral community-acquired pneumonia in adults community-acquired pneumonia genetic characterization of respiratory syncytial virus highlights a new ba genotype and emergence of the on genotype in lyon european manual of clinical microbiology. basel: european society for clinical microbiology and infections diseases : société française de microbiologie can analysis of routine viral testing provide accurate estimates of respiratory syncytial virus disease burden in adults? prevalence of respiratory viruses among adults, by season, age, respiratory tract region and type of medical impact of viral multiplex real-time pcr on management of respiratory tract infection: a retrospective cohort study respiratory syncytial virus hospitalization in middle-aged and older adults rates of hospitalizations for respiratory syncytial virus, human metapneumovirus, and influenza virus in older adults respiratory syncytial virus infection in adults clinical characteristics and outcome of respiratory syncytial virus infection among adults hospitalized with influenza-like illness in france elucidation of bacterial pneumonia-causing pathogens in patients with respiratory viral infection hospital-acquired pneumonia in icu the co-pathogenesis of influenza viruses with bacteria in the lung key: cord- -h d z z authors: wichmann, ole; schaade, lars title: impfen im kontext globaler herausforderungen date: - - journal: bundesgesundheitsblatt gesundheitsforschung gesundheitsschutz doi: . /s - - -y sha: doc_id: cord_uid: h d z z nan liebe leserin, lieber leser, unter der federführung des bundesministeriums für gesundheit wird derzeit eine neue "strategie der bundesregierung zu globaler gesundheit" entwickelt. damitsoll aufneue globale herausforderungen reagiert und eine aktualisierung der ziele des deutschen engagements vorgenommen werden [ ] . ein internationales beratergremium wurde eingerichtet und hat mitte seine empfehlungen vorgelegt [ ] . zusätzlich haben verschiedene deutsche akteure positionspapiere erstellt, in denen weitere Überlegungen und anregungen zur neuen strategie zusammengefasst sind. impfungen spielen bei den Überlegungen zur verbesserung der globalen gesundheit eine entscheidende rolle, gehören sie doch zu den wirksamsten und auch effizientesten maßnahmen der modernen medizin. durch sie können erkrankungen erfolgreich eliminiert und allgemeine gesundheitsziele erreicht werden, wie die senkung von kindersterblichkeit, gesundes altern oder die reduzierung von ungleichheiten in der gesundheit. die bundesregierung beteiligt sich beispielsweise an der weltweiten ausrottung der poliomyelitis -seit hat sie mehr als mio. € zu ihrer bekämpfung beigesteuert [ ] . auch die globale impfallianz (gavi) wird gefördert als teil des engagements der bundesregierung im bereich der gesundheitssystemstärkung und kindergesundheit. für die finanzierungsperiode bis gehört deutschland mit einer zusicherung von mio. € zu den vier größten gavi-geldgebern (nach dem vereinigten königreich, usa und norwegen) [ ] . deutschland ist zudem gründungsmitglied der impfstoffinitiative "coalition for epidemic preparedness innovations" (cepi) und wird diese bis mit bis zu mio. € unterstützen. cepi ist eine non-profit-organisation, die impfstoffe gegen erreger mit hohem pandemiepotenzial entwickeln soll. trotz der fortschritte in der prävention, diagnostikund therapie vonmalaria, tuberkulose und dengue stellen diese erkrankungen global weiterhin ein relevantes public-health-problem dar. die forschung zu neuen impfstoffen gegen diese erreger wurde in den letzten jahrzehnten vorangetrieben und hat dazu geführt, dass sich aktuell diverse impfstoffkandidaten in der entwicklung befinden, ein erster dengueimpfstoff zugelassen wurde und ein erster zugelassener malariaimpfstoff in großen pilotprojekten in afrika hinsichtlich seiner effekte auf die gesundheit der bevölkerung getestet wird. im globalen aktionsplan der weltgesundheitsorganisation (who) zum kampf gegen antimikrobielle resistenzen ist die investition in neue impfstoffe als strategisches ziel aufgeführt [ ] . verschiedene impfstoffkandidaten, die gegen bakterielle erreger mit einer resistenzproblematik gerichtet sind (z. b. clostridium difficile, staphylococcus aureus, mycobacterium tuberculosis), werden derzeit entwickelt [ ] . aber auch etablierte impfstoffe (z. b. gegen pneumokokken, typhus und influenza) tragen bereits jetzt zur reduzierung des antibiotikaverbrauchs und damit zur reduzierung antimikrobieller resistenzen bei und könnten es -bei höheren impfquoten -in noch größerem maße tun. impfungen spielen auch bei der eindämmung drohender pandemien und epidemien eine wichtige rolle und tragen so zu globalem gesundheitsschutz und globaler gesundheitssicherheit bei. im aktuellen ebolafieberausbruch, der seit april in der demokratischen republik kongo grassiert, konnte in der betroffenen region durch den frühen beginn einer impfkampagne mit einem zu diesem zeitpunkt noch experimentellen impfstoff das risiko, an ebola zu erkranken, um % gesenkt werden [ ] . der grund, warum die impfkampagne nicht noch erfolgreicher war, lag primär nicht am impfstoff, sondern an den herausforderungen bei der umsetzung der impfstrategie, vor allem an der kritischen örtlichen sicherheitslage. mittlerweile ist der impfstoff von den europäischen behörden zugelassen und hat auch von der who im november eine präqualifizierung erhalten. damit auch für andere bedrohliche erreger im epidemiefall zeitnah impfstoffe zum einsatz kommen können, müssen diese im vorfeld entwickelt und möglichst bis zu ihrer zulassung gebracht werden. da solche impfstoffe aber vergleichsweise selten benötigt werden und somit für multinationale hersteller eher unattraktiv sind, wurden initiativen wie die in diesem themenheft beschriebene cepi gegründet; sie investiert aktuell u. a. in die entwicklung von impfstoffkandidaten gegen lassa, nipha und das middle east respiratory syndrome (mers). nationale impfprogramme gibt es in allen ländern der welt. sie haben zur senkung der kindersterblichkeit maßgeblich beigetragen. in den meisten ländern unterstützen nationale impfkommissionen durch evidenzbasierte entscheidungsprozesse die einführung neuer impfungen und die entwicklung nationaler impfstrategien und werden dabei von den expertengremien der who begleitet. um möglichst hohe impfquoten zu erreichen und damit das potenzial der impfstoffe für die gesundheit der bevölkerung voll ausschöpfen zu können, müssen die verantwortlichen akteure die ursachen von impfakzeptanz und -ablehnung sowie die bedürfnisse der lokalen bevölkerung kennen. das who-regionalbüro europa hat daher, wie hier im themenheft beschrieben, ein konzept zur erfassung der impfakzeptanz und der entwicklung zielgerichteter interventionen erstellt, das sogenannte tailoring immunization programme (tip). ungleichheiten beim impfen können zwischen ländern, aber auch innerhalb eines landes bestehen. so weisen länder mit mittlerem einkommen die niedrigste zahl an neu eingeführten impfungen in ihren nationalen impfprogrammen auf und im durchschnitt auch die niedrigsten impfquoten bei etablierten impfungen im vergleich zu ländern mit hohen, aberauchniedrigeneinkommen [ ] . letztere profitieren von externen geldgebern bzw. der bereitstellung von impfstoffen zu günstigen preisen. innerhalb einzelner länder können ungleichheiten bei der inanspruchnahme von impfungen z. b. aufgrund des sozioökonomischen status oder des bildungstands auftreten. selbst in deutschland gibt es solche unterschiede, insbesondere in bezug auf region (z. b. liegen die hpv-impfquoten im osten mittlerweile bei über % und im süden nur bei %) oder migrationsstatus (z. b. mit niedrigeren impfquoten gegen tetanus oder masern bei menschen, die nicht in deutschland geboren sind oder die nicht gut deutsch sprechen). strategien, wie impfprogramme als integraler bestandteil funktionierender gesundheitssysteme zu einer ge-rechterenbereitstellung und inanspruchnahme von impfungen und damit zu einer verringerung gesundheitlicher ungleichheit beitragen können, sind daher von besonderer bedeutung. reisende können zur globalen verbreitung von infektionserregern beitragen. impfungen gegen gelbfieber, meningokokken oder poliomyelitis können bei ein-bzw. ausreise vorgeschrieben sein; reiseimpfungen sind aus diesem grund im kontext globaler herausforderungen mit zu betrachten. eine zunehmende herausforderung für reisemedizinisch tätige Ärzte ist dabei die impfung von besonderen risikogruppen, wie zum beispiel senioren oder menschen mit bestimmten grundkrankheiten (inkl. immunsuppressiver oder immunmodulatorischer therapie), die das risiko für schwere krankheitsverläufe erhöhen und die wirksamkeit der verfügbaren impfstoffe reduzieren können oder aber eine anwendung gar nicht erlauben. in diesem themenheft werden zwei grundlegende arten von herausforderungen behandelt: einerseits die vielfältigen herausforderungen im bereich der globalen gesundheit, die mit wirksamen impfstoffen adressiert werden können, und andererseits die barrieren bei der umsetzung der impfstrategien. das spektrum, wie impfungen zur globalen gesundheit beitragen, hat sich dabei über die letzten jahrzehnte zunehmend verbreitert. nicht nur, weil es mittlerweile impfstoffe gegen deutlich mehr erreger gibt (einschließlich solcher, die krebs verursachen, wie hepatitis b oder humane papillomviren), sondern auch, weil impfen inzwischen breiter gedacht wird und als integraler bestandteil eines funktionierenden gesundheitssystems mit relevanz für alle altersgruppen gilt. viele dieser aspekte sollen in der künftigen "immunization agenda " verankert werden, die aktuell von der who im rahmen eines größeren konsultationsprozesses erarbeitet wird und im mai verabschiedet werden soll. barrieren, die die umsetzung von impfstrategien und die entfaltung des tatsächlichen public-health-potenzials der verfügbaren impfungen behindern (wie impfskepsis, niedrige impfquoten und eine ungleichheit bei der inanspruchnahme von impfungen auf subnationaler ebene oder durch risikogruppen), bleiben probleme, mit denen fast alle länder konfrontiert sind. nur durch den internationalen austausch von expertise und erfahrungen sowie durch innovative impfstrategien und ein gemeinsames vorgehen von nord und süd bei der entwicklung und bereitstellung wirksamer, sicherer und preisgünstiger impfstoffe können wir den hier beschriebenen herausforderungen begegnen. wir hoffen, dass dieses themenheft, das wichtige entwicklungen auf den genannten gebieten zusammenfasst, zum verständnis der bedeutung und des potenzials von impfungen für die globale gesundheit beitragen kann. globale gesundheitspolitik gestaltengemeinsam handeln -verantwortung wahrnehmen internationales beratergremium zu globaler gesundheit (iab) ( ) erklärung des internationalen beratergremiums zu globaler gesundheit the vaccine alliance ( ) how we work together: quick start guide for new members of the vaccine alliance global action plan on antimicrobial resistance impact of vaccines on antimicrobial resistance ebola vaccination in the democratic republic of the congo world health organization regional office for key: cord- -i yhaagz authors: zhang, zhi-hao; he, jun-qiu; zhao, ying-yong; chen, hua-chao; tan, ning-hua title: asiatic acid prevents renal fibrosis in uuo rats via promoting the production of d-pgj , an endogenous ligand of ppar-γ date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: i yhaagz renal fibrosis is an inevitable outcome of all kinds of progressive chronic kidney disease (ckd). recently, asiatic acid (aa), a triterpenoid compound from chinese medicine centella asiatica, has been found to attenuate renal fibrosis. in the current study, we explored the mechanisms underlying antifibrotic effect of aa on uuo model. sd rats and icr mice were subjected to unilateral ureteral occlusion (uuo) surgery. prior the surgery, rats were administered aa ( mg·kg(− ) per day, ig) for days, whereas the mice received aa ( mg·kg(− ) per day, ig) for days. uuo group displayed significant degree of renal dysfunction, interstitial fibrosis, oxidative stress, and activation of the tgf-β/smad and wnt/β-catenin signaling pathway in the kidney, these pathological changes were greatly ameliorated by pretreatment with aa. in addition, we found that co-treatment with gw , a selective ppar-γ antagonist ( mg·kg(− ) per day, ip) for days, abolished the protective effects of aa. we further revealed that aa pretreatment did not significantly change the expression levels of ppar-γ in the kidney, but markedly increase the plasma levels of d-pgj , an endogenous ligand of ppar-γ. in uuo mice, pretreatment with d-pgj ( μg·kg(− ) per day, ip, for days) produced similar protective effect as aa. moreover, aa pretreatment upregulated the expression levels of active, nuclear-localized srebp- (nsrebp- ), whereas fatostatin, a specific inhibitor of srebp- , decreased the expression of nsrebp- , as well as the level of d-pgj . these results provide new insight into the antifibrotic mechanism of aa and endogenous metabolites might become a new clue for investigation of drug mechanism. chronic kidney disease (ckd) affects nearly % of adults in the united states, and the incidence and prevalence of this disease are increasing worldwide [ ] . ckd progresses to end-stage renal disease as renal function declines gradually when a critical number of functional nephrons are lost. treatment options for ckd are limited and only offer partial protection against ckd progression [ , ] . the development of more effective drugs to halt ckd progression is therefore a critical challenge for public health. natural small molecules remain promising drug sources. asiatic acid (aa), a triterpenoid compound, is isolated from centella asiatica, which is a traditional chinese medicine. previous studies have shown that aa has a variety of pharmacological effects, including anti-inflammation [ ] , antioxidation [ ] , and antitumor effects [ , ] . few studies have evaluated the mechanism of aa for the treatment of renal fibrosis. although previous studies have demonstrated that aa produces a significant effect on the inhibition of renal fibrosis by regulating tgf-β/smad signaling [ , ] , the details remain elusive. it is unclear whether aa regulates key proteins in the tgf-β/smad signaling pathway by direct binding or through other mediators. it has been reported that thiazolidinediones, peroxisome proliferator-activated receptor-γ (ppar-γ) agonists, attenuate renal interstitial fibrosis, and inflammation through a reduction in tgf-β [ ] . we hypothesize that aa attenuates renal injury and fibrosis by activating ppar-γ. to test this hypothesis, we investigated whether the selective and irreversible ppar-γ antagonist gw counteracts the protective effects against renal injury and fibrosis afforded by aa treatment in animals. our results showed that aa upregulated the expression of nuclear-localized sterol regulatory element-binding proteins- (nsrebp- ), enhanced d-pgj , activated ppar-γ, and consequentially attenuated renal damage in unilateral ureteral occlusion (uuo) models. chemicals and reagents lc-grade methanol and chloroform were purchased from merck (darmstadt, germany). internal standards (myristic acid), n, o-bis (trimethylsilyl) trifluoroacetamide (bstfa), methoxyamine hydrochloride, and pyridine were supplied by sigma-aldrich (st. louis, mo, usa). -deoxy-Δ , -prostaglandin j ( d-pgj ) was supplied by cayman chemical (ann arbor, mi, usa). aa was obtained from feiyu biotech ltd co (nantong, china), and gw was purchased from abmol bioscience (houston, tx, usa). fatostatin was supplied by targetmol (shanghai, china). the chemicals used in this study were of analytical grade, and their purity was above . %. all animal experiments were approved by the ethics committee for animal experiments of china pharmaceutical university. after acclimatization for week, rats and mice were used in the experiments. male sprague-dawley rats (weight - g) and male icr mice (weight - g) were given free access to water and regular chow. an animal model of uuo was established as described previously [ ] . in brief, under chloral hydrate anesthesia, the left ureter was double-ligated with a - silk suture following a midline abdominal incision. sham-operated animals underwent the same surgical intervention except for ureteral ligation. to investigate the effect of aa, the rats were randomly separated into the following four groups: the uuo group (n = ), the sham-operated group (control group, n = ), the uuo + aa group (n = ) (rats that were orally administered aa ( mg·kg − per day, once a day continuously for days) h prior to uuo surgery), and the uuo + aa + gw group (n = ) (rats that were administered gw ( mg·kg − per day, ip, once a day continuously for days) a half hour prior to the oral administration of aa). all rats were operated in the morning and sacrificed at : a.m. days later. for the verification test, male icr mice were used and randomly separated into the following four groups: the uuo group (n = ), the control group (n = ), the uuo + aa group (n = ) (uuo mice that were given aa ( mg·kg − per day, once a day continuously for days)), and the control + aa group (n = ) (sham-operated mice that were given aa ( mg·kg − per day, once a day continuously for days)). all mice were operated on in the morning and sacrificed at : a.m. days later. to verify the role of the alterative metabolite d-pgj in renal injury, we also used a mouse model, and mice were randomly separated into the uuo group (n = ), the control group (n = ), the uuo + d-pgj group (n = ) (mice that were administered d-pgj ( μg·kg − per day, ip, once a day continuously for days)), and the uuo + d-pgj + gw group (n = ) (mice were administered gw ( mg·kg − per day, ip, once a day continuously for days) a half hour prior to the administration of d-pgj ). all mice were operated on in the morning and sacrificed at : a.m. days later. to investigate the relationship between aa and the generation of d-pgj , a mouse model was used, and male icr mice were randomly separated into the uuo group (n = ), the control group (n = ), the uuo + aa group (n = ) (mice that were given aa ( mg·kg − per day, once a day continuously for days)), and the uuo + aa + fatostatin group (n = ) (mice that were given fatostatin ( mg·kg − per day, ip, once a day continuously for days) a half hour prior to the administration of aa). all mice were operated on in the morning and sacrificed at : a.m. days later. under chloral hydrate anesthesia, plasma was collected for biochemical detection or gc/ms analysis, and the left kidney was harvested and stored at − °c until analysis. blood parameters, histology, and western blotting blood analyses were performed on an olympus au automatic analyzer. the histological protocol was described in detail previously [ ] . the western blot protocol was described previously [ ] . the following primary antibodies were used: anti-collagen i (ab , abcam, cambridge, uk), anti-fibronectin gc-ms sample preparation internal standard solutions ( µl of myristic acid in methanol, mg/ml) were added to μl of plasma. the mixed solution was extracted with µl of methanol and chloroform ( : , v/v) and vortexed for s. the mixture was stored at room temperature for min and centrifuged at × g for min at °c. the resulting μl of supernatant was transferred to a sample vial for vacuum drying at room temperature. the residue was redissolved in μl of a methoxyamine solution ( mg/ml in pyridine) and vortexed for min. an oximation reaction was performed at °c for . h. then, μl of bstfa (containing % tmcs) was added to the solution, and the solution was vortexed for s. the sample was kept at °c for h and vortexed for s. the supernatant was transferred to a sample vial for gc-ms analysis. the samples were analyzed using an agilent chromatograph coupled with a b ms system (agilent technologies, santa clara, ca, usa). separation was achieved on a db- ms capillary column coated with % dimethyl % diphenyl polysiloxane ( m × . mm i.d., . -µm film). the initial gc oven temperature was set at °c for min, followed by a °c/ min oven temperature ramp to °c, which was maintained for min. the temperature of the inlet, transfer line, and ion source was set to , , and °c, respectively. the injection volume was μl with a split ratio of : . helium was used as the carrier gas with a constant flow rate of . ml/min. measurements were made with electron impact ionization ( ev) in full scan mode (m/z − ). the raw gc-ms data were converted and prepared for multivariate analysis according to the previously published methods [ ] . the peak area was normalized to the internal standard. pca analysis was used to visualize general clustering, trends and outliers among the observations. opls-da was used to validate the pca model and identify the differential metabolites. fold changes of the arithmetic mean values (each group/the control group) were calculated. student's t test was used to analyze the statistical significance of the results. the p values obtained from student's t test were further adjusted by a false discovery rate method based on the hochberg-benjamini method. differential metabolites were identified by a library search (nist and fiehn) and confirmed by available references. representative photographs of the left kidneys of the rats are shown in fig. a . as shown in fig. b , the uuo rats exhibited significant increases in serum creatinine (scr), blood urea nitrogen asiatic acid ameliorates renal fibrosis via ppar-γ zh zhang et al. ). *p < . , **p < . , ***p < . (compared with control group); # p < . , ## p < . (compared with uuo group) (bun), and triglyceride (tg) levels, suggesting a significant degree of renal dysfunction. treatment with aa group significantly attenuated the increase in bun and scr levels. however, the attenuation of bun and scr levels observed after aa treatment was reversed by the administration of gw , an antagonist of ppar-γ (fig. b) . histological analysis suggested that kidney tissues from the uuo rats exhibited severe tubular dilatation, tubular atrophy, and widened interstitial space with severe inflammatory cell infiltration (fig. c) . the oral administration of aa attenuated the interstitial injury, whereas the attenuation of interstitial damage was reversed by gw (fig. d, e) . uuo resulted in a significant increase in collagen i (col i), fibronectin (fn), and alpha-smooth muscle actin (α-sma) levels, as well as collagen iii levels, suggesting interstitial fibrosis (fig. a, b) . the treatment of rats with aa produced a significant attenuation of the uuo-mediated increase in col i, fn, and α-sma levels, indicating improvement in interstitial fibrosis (fig. b) . the administration of gw in aa-treated rats produced an increase in the expression of the abovementioned proteins similar to that in uuo rats and thus abolished the protective effect mediated by aa (fig. a, b) . wnt/β-catenin signaling is closely associated with renal fibrosis [ ] as well as the classic tgf-β/smad signaling axis. we next examined the effects of gw on the wnt/β-catenin and tgf-β/ smad signaling pathways in uuo rats treated with aa. uuo caused a significant increase in dvl , dvl , and β-catenin expression, which indicated the activation of wnt/β-catenin signaling. treatment with aa mediated a significant attenuation of the uuo-induced increase in dvl , dvl , and β-catenin expression, while the administration of gw abolished the protective effect of aa (fig. c) . meanwhile, we found that uuo caused a significant increase in tgf-β , smad , and smad expression and a decrease in smad expression, which indicated the activation of tgf-β/smad signaling (fig. d ). after treatment with aa, the upregulation of tgf-β , smad , and smad expression was significantly reduced; the downregulation of smad expression was significantly increased (fig. d) . the administration of gw in aa-treated rats produced a marked increase in tgf-β , smad , and smad expression as well as a marked decrease in smad expression, suggesting that it abolished the improvement in the expression of these proteins mediated by aa (fig. d) . in addition to fibrosis-related proteins and pathways, aa also affected oxidative stress in uuo rats. as shown in fig. f , the expression levels of reactive oxygen species (ros)-generating molecules including nox , rac , and -lox were significantly increased in uuo rats. treatment with aa produced a significant attenuation of the uuo-mediated increase in nox , rac , and -lox expression, and the administration of gw abolished the antioxidant effect mediated by aa (fig. f) . since an antagonist of ppar-γ, gw , can reverse the renal protective effects of aa, we hypothesized that aa may ameliorate renal fibrosis via activating ppar-γ. therefore, we investigated the expression of ppar-γ in kidneys from different group of rats. uuo rats induced a significant increase in the expression of ppar-γ compared with that in sham-operated rats (fig. e) . when compared with that in uuo rats, the expression of ppar-γ did not significantly change in rat kidneys after treatment with aa or gw (fig. e) , which indicates that aa activates ppar-γ through other processes instead of directly increasing its protein expression. untargeted gc-ms-based metabolomics identified important differential plasma metabolites typical total ion chromatograms of the representative samples obtained are presented in supplementary fig. s . a pca score plot of the plasma metabolites from the control and uuo groups is shown in fig. a . there was a clear separation observed in the plot of the plasma samples from the control and uuo groups, and a supervised method, opls-da, was also used to show the metabolic distinction between the two groups (fig. b) . the opls-da model parameters r y and q (cum) were . and . , suggesting good fitness and predictive ability of the opls-da model. pca and opls-da score plots of plasma metabolites in the control, uuo and uuo + aa groups are shown in fig. c , d. the uuo + aa group was more similar to the control groups than the uuo group was. twenty-two plasma metabolites were identified ( table ). out of the metabolites, were differentially expressed in uuo rats. then, a heatmap was created to visualize the relative levels of the differential metabolites in each group (supplementary fig. s ) . notably, of these identified metabolites, there were two ppar-γ ligands, namely, d-pgj and linoleic acid. both of them were significantly increased in aa-treated rats compared with uuo rats (fig. e) . targeted gc-ms-based metabolomics validated the production of d-pgj to validate whether aa can enhance the production of d-pgj , the plasma level of d-pgj in mice was measured by gc-ms. mice treated with aa showed a higher level of d-pgj than that exhibited by controls. in addition, uuo mice treated with aa showed an enhanced level of d-pgj compared with that shown by uuo mice (fig. f) . these results demonstrate that aa indeed increases the plasma level of d-pgj . d-pgj mediated similar therapeutic effects as those of aa in uuo mice to prove our hypothesis that aa ameliorates renal fibrosis via enhanced formation of d-pgj , we administered d-pgj to uuo mice to test whether d-pgj shows similar therapeutic effect as those of aa. uuo mice exhibited significant increases in bun and scr levels, suggesting a significant reduction in renal function. treatment with d-pgj significantly attenuated the increase in bun and scr levels. however, the attenuation of bun and scr levels observed after d-pgj treatment was reversed by the administration of gw (fig. a, b) . kidney tissues from uuo mice showed severe interstitial fibrosis and inflammatory cell infiltration. the administration of d-pgj significantly attenuated the interstitial damage, whereas the attenuation was reversed by gw ( figs. c and a ). in addition, uuo mediated a significant increase in the expression of type i collagen (col i), fn, and α-sma, which are marker proteins in fibrosis (fig. b) . the treatment of mice with d-pgj produced a significant attenuation of the uuo-induced increase in col i, fn, and α-sma expression, indicating an improvement in interstitial fibrosis, while the administration of gw abolished the protective effect mediated by d-pgj (fig. b) . similar to aa, d-pgj inhibited the activation of wnt/βcatenin and tgf-β/smad signaling pathways, and the effect was blocked by gw (fig. c, d) . taken together, these data suggest that d-pgj shows similar therapeutic effects as those of aa in the uuo animal model and that the renoprotective effects of d-pgj can be abolished by gw . aa regulated the formation of endogenous d-pgj through srebp- it was demonstrated that aa activates ppar-γ by promoting the production of endogenous d-pgj , the natural ligand of ppar-γ. to investigate how aa regulates d-pgj , we measured the expression of srebp- , which is of importance in the synthesis of fatty acids since d-pgj contains a long-chain fatty acid structure. we found that the expression of active nsrebp- was significantly increased in uuo rats treated with aa compared with model rats, while the expression of full-length srebp- was slightly decreased (fig. a) , and total srebp- mrna levels quantitative measurements of protein expression in kidneys from each group as indicated. *p < . , **p < . , or ***p < . remained unchanged between the two groups (fig. b) , suggesting that aa mediated srebp- activity at the posttranscriptional level. to validate whether srebp- regulates the formation of d-pgj , we gave uuo + aa mice fatostatin to test whether the level of d-pgj was decreased. the treatment of mice with aa significantly increased the expression of nsrebp- compared with that in uuo mice, while the administration of fatostatin, which is an inhibitor of srebp- , in aa-treated mice produced a decrease in the expression of nsrebp- (figs. c and s ) . the plasma level of d-pgj in the mice was measured by gc-ms. uuo mice treated with aa showed a higher level of d-pgj compared with that in the uuo group, while fatostatin significantly decreased the level of d-pgj compared with that in uuo mice treated with aa (fig. d) , which was consistent with the expression of nsrebp- . moreover, the expression of fibrotic proteins was decreased in aa-treated mice and upregulated after the administration of fatostatin (supplementary fig. s ). these results demonstrated that aa might regulate the level of endogenous d-pgj in mice through srebp- and play an antifibrotic role. in this study, we demonstrated that the upregulation of nsrebp- mediated by aa enhances the level of endogenous d-pgj , which activates ppar-γ and protects against renal damage and renal fibrosis (fig. e) . our major novel findings include the following: ( ) aa attenuates renal injury, oxidative stress, and fibrosis induced by the activation of ppar-γ through increasing its fig. a pca scores plot from control and uuo groups. b opls-da scores plot from control and uuo groups. c pca scores plot from control, uuo, and uuo + aa groups. d opls-da scores plot from control, uuo, and uuo + aa groups. e alteration of the level of d-pgj and linoleic acid in plasma samples of rats using gc-ms. asterisk denotes uuo + aa vs uuo, **p < . . f the plasma level of d-pgj in mice treated with aa. *p < . , **p < . natural ligand d-pgj in uuo rats; ( ) d-pgj attenuates interstitial fibrosis in uuo mice; and ( ) aa regulates the production of endogenous d-pgj , which may be through srebp- . this study demonstrates for the first time that the administration of the selective ppar-γ antagonist gw abolishes the renoprotective effects observed upon treatment with aa in rats. this result indicates that the renoprotective effects of aa are closely associated with ppar-γ. ppar-γ, which is expressed in many organs, including the kidney [ ] , is a member of the ligand-activated transcription factor superfamily. ppar-γ is involved in the expression of many genes and regulates glucose metabolism, insulin sensitivity, lipid metabolism, fibrosis, inflammation, and oxidative stress [ , ] . it has been reported that the ppar-γ agonist pioglitazone reduces proteinuria and preserves renal structure and function in passive heymann nephritis rats [ ] . in addition, a ppar-γ agonist can inhibit the generation of ros and the oxidative stress response [ , ] , and oxidative stress plays an important role in the pathogenesis of ckd and its complications [ , ] . in our study, aa attenuated oxidative stress and fibrosis via the activation of ppar-γ in uuo rats. ppar-γ expression was upregulated in uuo rats compared to control rats, which is consistent with previous studies of a renal ischemia reperfusion injury model [ , ] . compared with that in uuo rats, the expression of ppar-γ did not change upon treatment with aa, implying that the renoprotective effects afforded by aa may be due to the overproduction of endogenous ppar-γ agonists instead of the upregulation of the protein expression of ppar-γ. to verify whether endogenous ppar-γ ligands are overproduced, we applied untargeted gc-ms-based metabolomics to investigate plasma samples from the control, uuo, and uuo + aa groups. twenty-two plasma metabolites were identified and are shown in table ; we found two ligands of ppar-γ, namely, d-pgj , and linoleic acid. they were significantly increased in aatreated rats compared with uuo rats (fig. e) . endogenous d-pgj produced by the nonenzymatic dehydration of pgd has an affinity for ppar-γ of approximately μm [ ] . this metabolite is the most potent natural ligand of ppar-γ. ppar-γ is also activated by a variety of long-chain polyunsaturated fatty acids. linoleic acid has been reported to bind to and activate ppar-γ with relatively scale bar, µm; magnification, × ). *p < . , **p < . , ***p < . (compared with control group); # p < . , ## p < . (compared with uuo group) low affinity (k d = - μm) [ ] . then, we further validated that aa indeed enhances the plasma level of d-pgj in mice using targeted gc-ms-based metabolomics (fig. f) . moreover, the administration of d-pgj attenuated ( ) renal dysfunction (reduced the increase in scr and bun levels, fig. a, b) ; ( ) interstitial damage (h&e staining, fig. c); ( ) the expression of fibrotic proteins (fig. a, b) ; and ( ) the disorder of tgf-β/smad and wnt/β-catenin signaling pathways (fig. c, d) . overall, d-pgj showed similar therapeutic effects as those of aa in uuo animal model, and their renoprotective effects were abolished by gw . these data indicate that aa ameliorates renal fibrosis, at least in part via enhanced formation of endogenous d-pgj in a uuo animal model. srebps play a central role in cell metabolism by controlling the synthesis of fatty acids, tgs, and cholesterol [ ] . srebp- is one of the most important srebps, which are synthesized as inactive precursors bound to the er [ , ] . when sterols are depleted in the er membrane, srebp- is transported to the golgi, proteolytically released by two proteases and then shuttled to the nucleus to induce the expression of genes involved in fatty d western blots showing protein expression in the tgf-β/smad signaling pathway in kidneys from all groups. quantitative measurements of protein expression in kidneys from each group as indicated. *p < . , **p < . , or ***p < . acid synthesis. a previous study reported that the expression of an activated form of srebp- causes cells to produce ligand(s) of ppar-γ [ ] . however, the ligand that is produced is still unknown. our results show that uuo animals treated with aa exhibit significant increases in the levels of activated srebp- and d-pgj compared to those in the uuo group (figs. e and a) , while the administration of fatostatin in aa-treated mice produces a decrease in nsrebp- and d-pgj levels (fig. c, d) . the data presented here indicate clearly that nsrebp- triggers the generation of endogenous d-pgj . in summary, we demonstrated that aa increases the expression of activated srebp- , upregulates nsrebp- , enhances the level of endogenous d-pgj , which activates ppar-γ, attenuates oxidative stress, rebalances the disorder of the tgf-β/smad and wnt/β-catenin signaling pathways, and ameliorates renal fibrosis (fig. e) . our study, for the first time, reveals, in part, the mechanism by which aa can treat renal fibrosis through regulating endogenous metabolites. this study also provides a paradigm for mechanistic studies of molecules with similar characteristics as those of aa. fig. the protein (a) and mrna expression (b) of srebp- in kidneys from control, uuo, and uuo + aa rats. c western blots showing nsrebp- expressions in kidneys from control, uuo, uuo + aa, and uuo + aa + fatostatin mice. quantitative measurements of protein expression in kidneys from each group as indicated. *p < . , **p < . . d alteration of the plasma level of d-pgj in mice using gc-ms. *p < . , **p < . . e suggested antifibrotic mechanism of asiatic acid team. the authors would like to thank zhe wang for the discussion of the manuscript. zhz, hcc and nht designed the experiments. zhz and jqh collected and analyzed the data. yyz and nht contributed analytical tools. zhz wrote the manuscript. nht revised the paper. prevalence of chronic kidney disease in the united states effects of losartan on renal and cardiovascular outcomes in patients with type diabetes and nephropathy unmet need in renal protection-do we need a more comprehensive approach? inhibition of lps-induced no and pge production by asiatic acid via nf-kappa b inactivation in raw . macrophages: possible involvement of the ikk and mapk pathways antioxidant and cytotoxic activities of centella asiatica (l) urb asiatic acid, a triterpene, induces apoptosis through intracellular ca + release and enhanced expression of p in hepg human hepatoma cells asiatic acid induces apoptosis in sk-mel- human melanoma cells asiatic acid ameliorates tubulointerstitial fibrosis in mice with ureteral obstruction treatment of renal fibrosis by rebalancing tgf-beta/smad signaling with the combination of asiatic acid and naringenin ppar-gamma agonist attenuates renal interstitial fibrosis and inflammation through reduction of tgf-beta renal fibrosis is attenuated by targeted disruption of kca . potassium channels an integrated lipidomics and metabolomics reveal nephroprotective effect and biochemical mechanism of rheum officinale in chronic renal failure metabolomics analysis reveals the association between lipid abnormalities and oxidative stress. inflammation, fibrosis, and nrf dysfunction in aristolochic acid-induced nephropathy biomarkers of obstructive nephropathy using a metabolomics approach in rat matrix metalloproteinase- as a surrogate marker predicts renal wnt/β-catenin activity in ckd peroxisome proliferator-activated receptors (ppars): novel therapeutic targets in renal disease thermodynamics in gliomas: interactions between the canonical wnt/beta-catenin pathway and ppar gamma the role of ppars in lung fibrosis transcriptional regulation of nephrin gene by peroxisome proliferator-activated receptorgamma agonist: molecular mechanism of the antiproteinuric effect of pioglitazone rosiglitazone, a ppar-gamma ligand, protects against burn-induced oxidative injury of remote organs the metabolic effects of troglitazone in patients with diabetes and end-stage renal disease bardoxolone methyl and kidney function in ckd with type diabetes targeting the transcription factor nrf to ameliorate oxidative stress and inflammation in chronic kidney disease study of peroxisome proliferator-activated receptor (ppar)-gamma in renal ischemia-reperfusion injury the selective ppar-gamma antagonist gw reverses the protection of lps in a model of renal ischemia-reperfusion a prostaglandin j metabolite binds peroxisome proliferator-activated receptor gamma and promotes adipocyte differentiation fatty acids, eicosanoids, and hypolipidemic agents identified as ligands of peroxisome proliferator-activated receptors by coactivator-dependent receptor ligand assay protein sensors for membrane sterols srebps: activators of the complete program of cholesterol and fatty acid synthesis in the liver add /srebp activates ppargamma through the production of endogenous ligand this work was financially supported by the national natural science foundation of china (grant ) and the program for jiangsu province innovative research the online version of this article (https://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users.competing interests: the authors declare no competing interests. key: cord- -vt b ukw authors: voth schrag, rachel j.; ravi, kristen e.; robinson, sarah r. title: the role of social support in the link between economic abuse and economic hardship date: - - journal: j fam violence doi: . /s - - - sha: doc_id: cord_uid: vt b ukw more data is needed about the pathways through which intimate partner violence (ipv) impacts the economic well-being of survivors. the current study assesses the moderating influence of social support on the association between economic abuse (ea) and economic hardship. female participants (n = ) were recruited to participate in a web-based survey which included standardized measures of ea, other forms of ipv, domains of social support, and economic hardship. analysis included bivariate and multivariate regression with an investigation into interaction effects.experiencing ea was significantly correlated with economic hardship, even with extent of physical and emotional ipv controlled. both tangible and appraisal support had significant negative association with extent of material hardship. significant interactions between forms of social support and economic abuse were observed. for those at high levels of economic abuse, support had less influence on economic hardship. a mix of direct economic aid, advocacy, education and support could provide a blueprint for addressing the economic hardship experiences of community-dwelling survivors of economic abuse. a comprehensive response to ea requires interventions aimed directly at economically controlling and exploitative tactics, including credit building, individual economic advocacy, and education. interventions that seek to enhance survivors’ access to social support may be necessary but not sufficient to buffer the impacts of violence on survivors’ economic outcomes. intimate partner violence (ipv) is a worldwide epidemic that impacts at least one in four ( . %) women (smith et al. ). the consequences of economic abuse at the hands of an intimate partner are far-reaching and long lasting. the monetary costs of ipv include $ . billion annually in lost productivity, physical, and mental health care costs, along with expenditures for legal and court costs (max et al. ). survivors of ipv lose approximately million days of work per year, the economic equivalent of , full-time jobs (ncipc ) . ipv also creates serious barriers to establishing or maintaining economic independence for survivors. these barriers are all the more challenging because women cite economic dependence on their abusive partner as a primary limiting factor in establishing safety outside of an abusive relationship (adams et al. ; tolman and rosen ) . while the link between ipv and economic insecurity is well established, the pathways that feed this association are less explored. the current study seeks to expand the knowledge in this area by assessing the moderating influence of social support on the association between economic forms of abuse and economic hardship in a community-based sample of women. scholars have identified specific batterer behaviors aimed at sabotaging economic efforts and maintaining economic power and control as a distinct type of ipv (adams et al. ; stylianou a) . evidence of unique consequences and specific patterns of behavior have led scholars to call for economic abuse (ea) to be considered as a separate form of abuse deserving specific attention to its dynamics, patterns, and impacts (adams et al. ; postmus et al. postmus et al. , stylianou et al. ) . ea is comprised of tactics that hinder economic self-sufficiency and harm economic self-efficacy through financial exploitation, economic control, and employment sabotage (adams et al. ; postmus et al. ; weaver et al. ). these include preventing or limiting work or school hours, stealing income or cash gifts, harassment at work or school, damaging credit history, and dominating family finances by demanding receipts, preventing access to money, or making unilateral decisions (adams et al. ; stylianou a; moe and bell ; sanders ; voth schrag et al. ; weaver et al. ). these behaviors are linked to employment and housing instability, increased use of public assistance, greater material hardship, and increased economic dependence on abusive partners for financial stability (adams et al. ; goodman et al. ; voth schrag ) . economic abuse has also been shown to impact the health and mental health of victims of ipv. ea has been linked with negative health outcomes such as gastrointestinal syndromes, psychosomatic symptoms, pelvic problems, and psychological problems, as well as mental health impacts including increased risk of depression (stockl and penhale ; stylianou b; voth schrag ) . data also show that historical experiences of ea can reverberate through survivors' lives for years due to on-going issues with debt, credit, employment, and economic self-sufficiency (toews and bermea ; ulmestig and eriksson ) . there is growing evidence for the prevalence of ea. in clinical populations of ipv survivors, high levels of ea have been documented by adams et al. ( ) and postmus et al. ( ) among others. these studies found that % of sheltered ipv survivors and % of ipv service seeking women reported economic abuse as an aspect of their abusive relationship. additionally, a recent study found lifetime prevalence rates of economic abuse among women at . % in an australian sample (kutin et al. ) . however, in the united states few studies have examined the dynamics of ea outside of ipv service settings, and those that have often have used non-standardized measure of ea (postmus et al. ). a substantial literature links ipv to decreased economic wellbeing (adams et al. ) . national estimates from rennison and welchans ( ) found seven times more ipv among those in the lowest / th of the income distribution. similarly, tolman and wang ( ) found a % reduction in work hours over years for survivors. the link between ipv and poverty is particularly concerning because economic hardship can further the cycle of violence, deepening women's dependence on an abusive partner for basic needs and security for themselves and their children (adams et al. ; brush ; kutin et al. ; power ) . conversely, economic well-being can provide a buffer against on-going dependence on an abusive partner and may be an important pathway to safety from long-term abuse at the hands of a current or future partner (adams et al. ). however, differences in economic impact by type of ipv experiences (physical, emotional, economic, etc.) is less understood. scholars have identified tactics of economic abuse to include stealing income and limiting access to bank accounts or joint property. thus, measures of economic well-being based on household income or individual income may miss economic dynamics faced by survivors. one strategy for operationalizing economic hardship is through experiences of material hardships, such as utility disconnections, housing instability, food scarcity, and difficulty accessing needed medical care (beverly ) . material hardships are a clear indicators of the extent to which survivors and their families are able to meet basic needs, and the extent to which they face serious economic insecurity (voth schrag ). social support is the availability of individuals in a survivor's social network who provide emotional comfort, helpful advice, tangible assistance, and positive social interactions (coker et al. ) . wright ( ) argues that bfriends, family, or acquaintances may provide instrumental or expressive support to victimized women, which may help her to leave the relationship or cope with the victimization^(p. ). higher levels of social support have demonstrated to be related to increased help-seeking and decreased negative outcomes for survivors of ipv (coker et al. ; dougé et al. ; kamimura et al. ; van wyk et al. ) . however, research concerning the relationship between social support and economic hardship is limited. several studies have attempted to look at the moderating effects of social support on the relationship between economic hardship and psychological distress but have not found significant results (kingston ; manuel et al. ; ayala-nunes et al. ) . alternatively, simmons et al. ( ) found social support was a strong indicator of economic well-being in rural, lowincome, single mothers. further, in a qualitative study exploring economic abuse and unemployment, survivors identified that one impact of job loss was the loss of emotional support and recognition from colleagues (ulmestig and eriksson ) . however, the role of social support in the lives of survivors of ea and the particular impact of social support in addressing the economic impact of ea remains unclear. coercive control theory (cct) views physical violence as one tactic of ipv, rather than the end in itself, and highlights the range of abusive tactics, including ea, that are used to establish power and control. (arnold ). dutton and goodman ( ) argue that a coercive threat must involve both a demand and a credible threat associated with not carrying out the demand. when an ipv survivor believes that the threat or consequence will be carried out, the abusive partner holds both the power of threat and the power of reward (dutton and goodman ; stark ) . importantly for the current study, cct posits that ipv survivors will have lower levels of economic stability compared to others, as an abusive partner uses various tactics (including physical and emotional violence and their resulting mental and physical health consequences, as well as tactics of ea) to increase economic dependence in order to enhance their control over all aspects of life (stark ; postmus et al. ) . the current study seeks to expand our knowledge related to the dynamics of economic hardship within the context of ipv by assessing the association between eawith physical and emotional ipv, and economic hardship. it also seeks to examine the moderating impact of receiving social support on survivors' experiences of hardship. it asks the following research questions: ) is there an association between experiencing ea and experiencing economic hardship in a communitycollege based sample of women? ) is there an association between extent of social support and ea in a community-college based sample of women? ) does the extent of social support moderate the relationship between ea and economic hardship? participants were women who were attending one of four community college campuses within a single community college system in a large midwestern metropolitan area during the fall semester. after study procedures were approved by the irbs of the sponsoring university and the community colleges, the study team was provided with a complete roster of currently enrolled students. from this roster, a simple random sample of participants were recruited via their college e-mail accounts to participate in a web-based survey exploring factors influencing their quality of life and educational outcomes. of female students randomly selected for recruitment, % (n = ) opened a recruitment e-mail. fifty-six percent of those then opened the survey link, and ( % of those who opened the survey) consented to participate. overall, % of females who opened the survey e-mail consented to participate, or % of females contacted. of those who consented, were screened out for reporting that they identified as male, nine were screened out for being younger than or not reporting an age to verify their eligibility, and were screened out for not having been in an intimate relationship in the past months. these participants, along with those who failed to complete the demographic questioner (n = ), were removed from further analysis, leaving a final sample of respondents. to understand the extent to which the sample reflects the broader population of community college students, the demographics of study participants were compared with data for all students enrolled in the community college system published by the national center for education statistics (nces ) . no significant differences were identified between the study sample and the demographics of the four campuses overall on any of the observed variables (see table ). participants were an average age of . years (sd = . ) and racially diverse ( % white, % african american, . % other) (see table ). forty-eight percent had at least one child at home. nearly % ( . %) were currently working for pay, and almost % were currently living with their intimate partner. among those currently working, they averaged h of work per week. participants received a $ gift card for completing the survey which took between and min. the web-based survey included demographic measures and validated scales assessing economic and social indicators along with measures of violence exposure. economic hardship economic hardship was measured via the economic hardship index (ehi), which has been previously used with a version of the scale of economic abuse to look at relationships between ea and material hardship among service seeking ipv survivors (adams et al. ) . the ehi is a checklist measure of forms of material hardship, including difficulty finding stable housing, eviction, food insecurity, and utility disconnection. respondents are asked to report if they have experienced various forms of hardship in the past year, and a total summed score is obtained. adams and colleagues (adams et al. ) found the ehi to have a reliability coefficient of . in a sample of ipv survivors. in the current sample of community college women, the scale alpha was . . economic abuse economic abuse was measured via the scale of economic abuse (sea- ) (stylianou et al. ) . it uses a five-point likert scale ( = never to = very often) to assess the frequency of specific behaviors over the past months including items such as bmade financial decisions without you^, bkept financial information from you^, band bbuild up debt under your name.^the -item version of the sea has demonstrated strong validity and good internal and test-retest reliability (adams et al. (adams et al. , . in the current sample of community college women, the overall sea- scale alpha was . . physical & emotional abuse physical and emotional abuse were measured using the revised abusive behavior inventory (abi-r) (postmus et al. ) . subscales for physical violence ( items) and emotional abuse ( items) were used. responses were on a five-point likert scale ( = never to = very often) and assessed the frequency of specific abusive behaviors over the past months. the abi-r has been used previously along with the sea- to evaluate multiple forms of ipv together (postmus et al. ) . in the current sample, the subscale alphas were . for physical ipv and . for emotional ipv. social support social support was measured via the interpersonal support evaluation list (short) (isel-s) (payne et al. ) . the current study used two isel-s subscales to evaluate extent of social support: appraisal support (advice/encouragement) and tangible support (provision of physical help or needed items) (payne et al. ) . the isel-s has demonstrated reliability coefficients clustered around . for the full scale and all subscales (payne et al. ) . a four-point likert scale ( = definitely false to = definitely true) is used to assess the extent to which participants view the statements as true for them. in the current sample of community college women, subscale alphas were . for appraisal support and . for tangible support. data analysis included descriptive analyses of univariate statistics to describe the extent of exposure to economic abuse and economic hardship, as well as levels of appraisal and tangible support. due to univariate skewness in the economic abuse exposure variable, spearman correlations were conducted to understand the bivariate relationships between key study variables. last, multiple regression analyses were run using the hayes moderation macro (hayes ) to test the moderating effect of different types of social support on the relationship between economic abuse and economic hardship. missing data was low across all items, with tables and indicating the number of observations included for each variable of interest. a power analysis in the study's design phase determined that the sample size was sufficient for the intended moderation analyses. the overall average for the sea- was low (m = . , s. d. = . , range = - . ), however % of respondents indicated having experienced at least one economically abusive behavior in the past months ( table for descriptive statistics of key study variables. bivariate correlations between abuse exposure, key study variables, and demographic characteristics were analyzed (see table table presents the results for linear regression models that assess the impact of the interaction between appraisal and tangible social support and economic abuse on participants' level of material hardship experience. both models control for covariates including physical and emotional ipvexperiences, monthly income, number of children in the home, age, and respondent race. as shown in model , a significant association is observed between appraisal support and extent of economic hardship. as survivors' experience higher levels of economic hardship, they report decreased appraisal support (β = −. , p = . ). similarly, higher levels of ea are associated with higher levels of economic hardship (β = . , p < . ). finally, a significant interaction was observed between appraisal support and ea (β = . , p = . ). for survivors reporting higher levels of ea, the protective influence of appraisal support on extent of economic hardship is less. neither physical (β = . , p = . ) nor emotional (β = . , p = . ) ipv were significantly associated with extent of economic hardship in model . participant income (β = . , p = . ) and race (β = − . , p < . ) remained significantly associated with economic hardship experiences in this multivariate model. table model describes the relationships between ea, tangible support, and economic hardship. in this multivariate model, the relationship between ea and economic hardship remained statistically significant (β = . , p < . ). a significant relationship between tangible support and economic hardship was observed, with increased tangible support being associated with decreased economic hardship (β = −. , p < . ). finally, a significant interaction was observed between tangible support and ea (β = . , p = . ). at higher levels of ea, the protective influence of tangible support on extent economic hardship is reduced. neither physical (β = . , p = . ) nor emotional (β = . , p = . ) ipv were significantly associated with extent of economic hardship in model . participant income (β = . , p < . ) and race (β = − . , p < . ) were still significantly associated with economic hardship experiences. in line with coercive control theory, this study found a significant association between ea and recent experiences of economic hardship in a sample of women attending community college in a large midwestern metropolitan area. the quantitative data identified a strong association between ea and increased economic hardship in a non-service seeking population, and at lower levels of ea severity than previously observed (adams et al. ; postmus et al. ) . previous studies have been in service seeking samples, presumably with survivors of long-term or severe ipv who may be more financially entrenched with their partners. comparatively, women in this sample have a wide a range of relationship stages and types, including many individuals in healthy intimate relationships. of particular importance is that these tactics were associated with increased economic hardship, even when monthly income and other forms of ipv experiences were statistically controlled. this suggests that increasing individual income or reducing physical violence without also addressing economic coercion is not enough to prevent experiences like utility disconnections or housing instability for those living with ea. even those with higher incomes are at risk of economic hardship in the face of ipv. these findings suggest that interventions aimed specifically at addressing the tactics of ea, such as credit sabotage, economic control, and economic exploitation, will be critical in supporting women's efforts to build economic security and long-term financial safety. the results of the moderation analysis provide unique insight into how different forms of social support impact the economic hardship experiences of survivors of ea and other forms of ipv. for participants who report low levels of economically abusive tactics in their current or most recent relationship, high levels of social support are associated with fewer experiences of material hardship. in other words, social support may be protective against material hardship for these individuals. however, for participants who reported high levels of economically abusive tactics, the association between extent of social support and material hardship was weaker. participants who had access to high levels of social support did not have the same experience of protection from material hardship experiences in the face of ea. in bivariate analysis, higher levels of physical and emotional abuse were also linked to increased economic hardship for survivors of intimate partner violence. however, in the multivariate models, when the three forms of ipv were considered together, only ea was significantly linked with economic hardship experiences. with this perspective, experiences of ea appear to be especially confounding, as they seem to be uniquely associated with economic hardship experiences. being entrenched in social networks that have access to material resources can be protective against economic hardship for survivors of ipv. however, previous studies have demonstrated that the social isolation that accompanies ipv can be a threat to these networks, breaking down access to resources and potentially disrupting the protective effect of social support (lanier and maume ; sylaska and edwards ) . for service providers, these data suggest the importance of supporting survivors in identifying strategies for safely maintaining and building social supports and for developing programs that can provide some of the sorts of tangible resources that can decrease survivors' experiences of material hardship. these data also remind us that services which provide advice or knowledge may be necessary but not sufficient to address the material hardship experiences of ipv survivors, especially those for whom ea is a major component of their ipv experience. financial empowerment programs like the moving ahead through financial management curriculum have proven effective in support of the economic self-efficacy of survivors, which could enhance their ability to address the economic consequences of ea (hetling et al. ) . the current findings suggest that it could be useful to compliment these programs with efforts specifically aimed at increasing survivors' safe and secure access to resources which can disrupt material hardship experiences. one such intervention is economic advocacy, which attempts to account for survivors' material and financial needs in the context of planning for safety from abuse (vondelinde ). it requires advocates to understand the complexities of credit, debt, banking, taxation, and saving, along with how to connect survivors with resources, make change at systemic levels, and explore unconventional routes for enhancing economic well-being (vondelinde ). some of these routes could include evidence based interventions such as individual development accounts, housing first programs, and utility assistance. these programs can complement financial education and advocacy and increase survivors' access to tangible resources in order to buffer the impact of ea on their overall economic stability and well-being (baker et al. ; sanders ; sanders and schnabel ) . the literature on survivors' experiences with ea provides an important caution for practitioners seeking to develop such programs. interviews and surveys with survivors reveal a wide array of economically abusive tactics used by abusive partners, including stealing or controlling access to cash and inkind material assistance (postmus et al. ; sanders ) . service providers can address this challenge to survivors' economic power by using a survivor defined advocacy framework, which takes an individualized approach to work with each survivor, considering their own unique combination of risks and opportunities (davies and lyon ) . survivor centered economic advocacy (scea) provides a helpful framework for the integration of tangible supports into an individualized and survivor specific safety plan. as defined by vondelinde ( ), scea is: an alliance between survivors and advocates that addresses both the physical safety and economic safety needs of survivors through reviewing and developing creative strategies on the survivor's current, past, and future priorities. scea builds on the survivor's strengths and uses the advocates knowledge and experience to enhance the survivor's comprehensive safety plan. ( , p. ) limitations a number of methodological limitations should be considered when weighing the voracity of the findings of the current study. first, a major limitation of this work is that the data are cross-sectional and thus unable to measure change over time (kumar ) . theoretically informed assumptions about causality were built into the specification of the moderation model, but with cross-sectional data it is impossible to control for reciprocal relationships between constructs. thus, the current study should be seen as a preliminary assessment of the role of social support in buffering the impact of ea on economic hardship, and serve as justification for further longitudinal evaluation. the study also fails to capture a number of potentially important vectors influencing the relationship between abuse and material hardship. previous work has demonstrated that issues such as the role of abuse in mental and physical health, employment and academic outcomes may all influence survivor's economic outcomes. future studies should consider including these factors as they seek to understand the key pathways for prevention and intervention. the current study response rate is within-but on the lower end of-the range of published campus based studies of victimization (e.g., busch-armendariz et al. ; cantor et al. ) . although low response rates are expected for web-based surveys compared to other forms of measurement, the response rate for the current study threatens the generalizability of the findings. this concern is somewhat ameliorated by the fact that, when demographics of survey respondents are compared with the study population on age, part vs. full time status, and race, the survey respondents are similar to the overall student body of the four campuses. however, there is still a chance that study participants vary in systematic ways from study nonparticipants on un-measured dimensions. for example, the use of a web-based survey means that potential respondents are more likely to participate if they are comfortable with the electronic interface and frequently check their school e-mail. a final set of limitations are related to measurement. the scale of economic abuse has not been used up to this point outside of ipv service seeking populations, and thus the sea- 's validity for this population is not established. future studies should evaluate the psychometric properties of the sea- in other non-service seeking populations. additionally, the tangible social support measure, while validated and demonstrating strong reliability in other samples, has a coefficient alpha of . in this sample, suggesting potential issues with its reliability (payne et al. ) . finally, the measure of economic hardship did not differentiate between joint hardships (i.e. hardships experienced indirectly by living with family members) and individual hardships. future research should separate the types of economic hardships to increase the understanding of the hardships experienced by the survivor. developing effective strategies for addressing the economic consequences of abuse is central to disrupting cycles of victimization. the clear association the current study observed between experiencing abusive tactics and women's extent of economic hardship is particularly concerning because, bfor women, the consequences of poverty include not only hardships such as homelessness and hunger but also additional vulnerability to being trapped in relationships with abusive men^ (brush , p. ) . for survivors faced with violence and economic insecurity, poverty compounds and extends the consequences of ipv. it increases survivors' dependence on their partners for the basic necessities of life, and limits their access to available resources that they might otherwise be able to mobilize in the face of violence and coercion (brush ; raphael ) . future work should recognize that social support may be necessary but not sufficient to buffer the impacts of 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of intimate partner violence development and preliminary psychometric evaluation of the domestic violence-related financial issues scale (dv-fi) the relationship between social support and intimate partner violence in neighborhood context key: cord- -kkxdmzk authors: smirnova, s. s.; pisareva, m. m.; smirnova, t. d.; sivak, k. v.; vorobiev, k. v. title: long-term maintenance of the functional changes induced by influenza a virus and/or lps in human endothelial ecv- cell sublines date: - - journal: cell tissue biol doi: . /s x sha: doc_id: cord_uid: kkxdmzk influenza a virus and secondary bacterial infection may have remote effects in the form of cardiovascular complications or fibrosis in different organs. however, the mechanisms governing the development of complications remain poorly studied. the present work reports the comparative assessment of the functional changes which take place in human ecv- endothelial cell sublines obtained previously by the long-term culturing of cells after exposure to varying infectious doses (ids) of influenza a virus, and/or bacterial lipopolysaccharide (lps). it has been demonstrated that, in the course of long-term culturing (six passages) after exposure to pathogenic agents (influenza virus and/or lps), endothelial cells maintain changes in their migratory activity, permeability, and expression of mrna for cytokines tnfα and tgfβ (along with the changes in their proliferation activity, which has been demonstrated earlier). the pattern of changes depended on the type of the agent (agents) to which the cells were exposed. the differences in migratory activity (which was at its maximum h after wounding) between the cell sublines at the sixth passage correlated with the differences in their proliferation activity at the first passage (proliferation data were obtained previously). in particular, an increase in migration and proliferation was observed in the sublines exposed to low virus doses (ecv- id), as well as exposed to lps (ecv-lps), while the suppression of migration and proliferation was observed in the subline exposed to high virus doses (ecv- id). in the ecv- id, ecv-lps, and most notably in ecv- id + lps sublines, we detected an increase in the expression of mrna for cytokines tnfα and tgfβ, which, however, didn’t lead to the induction of apoptosis. we have also demonstrated an increase in cell permeability in the analyzed sublines, which was indicated by a decrease in the expression of the mrnas for the genes encoding occludin and zo- , the tight junctions proteins . this paper also reports an evaluation of the effects of the antiviral preparations rimantadine and alpisarin on the functional state of cell sublines. as a result, it has been demonstrated that these drugs may be able to prevent the development of the pathological changes caused by influenza a virus and/or lps in endothelial cells. the results obtained in the present work may be of use when studying the mechanisms of development of the influenza a virus and secondary bacterial infection complications. endothelial cells form a semipermeable barrier between circulating liquid and the surrounding tissues. changes in the endothelial barrier function contribute significantly to the development of different pathological states including inflammation and impaired wound sealing. moreover, they may contribute to the development of acute encephalopathies and organ dysfunctions (as a result of cardiovascular system involvement), organ fibrosis, increased angiogenesis, and oncogenesis (di pietro, ; you and stallcup, ) . disruption of the endothelial barrier in the lungs (in particular, as a result of influenza virus or bacterial infection) may cause liquid to penetrate into alveoli, resulting in pulmonary edema and development of acute respiratory distress syndrome (ards) (kwok et al., ) . impaired migration of endotheliocytes and changes in their proliferation and apoptotic activity, as well as altered cell permeability, are critical for the development of pathological states. abbreviations: ros-reactive oxygen species, id-infective dose, lps-lipopolysaccharide, moi-multiplicity of infection, fbs-fetal bovine serum, aj-adherence junctions, cldn -claudin- , ocld-occludin, tgfβ-transforming growth factor beta, tj-tight junctions, tnfα-tumor necrosis factor alpha, vegf-vascular endothelial growth factor. the ability of endothelial cells to regenerate a damaged intravascular layer relies on the activation of migration of the cells most closely located to the wound, as well as on strengthening of their capacity to proliferate. the process of cell migration includes cell polarization, formation of protrusions in the direction of movement, and detachment and contraction of the posterior cell margin, which are associated with dynamic reorganization of the actin cytoskeleton and microtubules involving signaling molecules from the rho family of small gtpases (kasa et al., ) . key physiological regulators of intracellular signaling pathways in endothelial cells are represented by reactive oxygen and nitrogen species (breton-romero, lamas, ) . endogenous hydrogen peroxide (h o ) accumulates in actively migrating endothelial cells at the site of injury caused by the inflammation inducers of different type, in particular, of bacterial or viral nature. the main source of reactive oxygen species (ros) are nox nad(p)h oxidases. endothelial cells are rich in nox and its homolog nox (basuroy et al., ) . a particularly important role in the stimulation of the signaling systems associated with endothelial cell migration is played by the vascular endothelium growth factor (vegf) which interacts with ros produced not only in the cytosol by nox and nox , but also in mitochondria (kim et al., ) . endothelial barrier is controlled by the locally produced vasoactive agents, such as histamine, prostaglandin, thrombin, and vegf. they modulate cell membrane permeability by promoting dynamic changes in adherens (aj) and tight junctions (tj), and regulate the centripetal cell tension as a result of the cortical actin layer reorganization and formation of actomyosin stress fibers (dejana et al., ; kasa et al., ) . an increase in endothelial cell migration in the presence of bacterial lipopolysaccharide (lps) is used as an indicator of the conversion of endothelial cells into activated fibroblasts via the endothelial to mesenchymal transition (sarmiento et al., ) . similarly to lps, influenza virus is also able to trigger ros production in epithelial and endothelial cells by means of nox and nox induction, which may also facilitate the reproduction of the virus itself (armatore et al., ) . it has been demonstrated that the infection of endothelial cells with influenza virus leads to the disruption of intercellular contacts and a decrease in the content of claudin- and claudin- (cldn ) proteins that form part of tjs (armstrong et al., ; short et al., ) . inflammatory response in the cells, induction of apoptosis, increase in cell permeability, and reorganization of cytoskeleton proteins are all performed by influenza a virus through the same cellular mechanisms and signaling pathways (rho/rock, p , and jnk), which form part of the mechanism of action of lps (zhang et al., ; . the data presented above were obtained for in vivo acute viral infection or during the early period ( - h) after the exposure to lps or high infectious doses (ids) of influenza a virus in vitro. however, we haven't found any data on the effects of simultaneous exposure of cell cultures to influenza virus and lps. earlier, we have for the first time demonstrated an increase in proliferation of the influenza virusinfected human ecv- endothelial cells, which are nonpermissive for the influenza virus, with the decrease in the virus id for - days after the infection, which was accompanied by an increase in cell apoptosis. normal reproduction of influenza virus is impaired in nonpermissive cells, and the presence of virus is detected only at the level of the viral surface protein mrna during no more than one or two passages (danilenko et al., ) . the change in the physiological state of the cells at the first passage ( - days) was the first stage in the changes induced in the cells by influenza virus, with further cell culturing (passages to ) resulting in the stably increased proliferation at low apoptosis levels (smirnova et al., ) . the aim of the current work was to find a link between the primary exposure of ecv- cells to the agents (high and low influenza virus ids and/or lps) and the pattern of remote functional changes in the ecv- cell sublines at the sixth passage after the exposure. we have analyzed the changes in the migratory activity of the cells and gene expression at mrna level, including the genes encoding tumor necrosis factor alpha (tnfα), transforming growth factor beta (tgfβ), and nox , as well as the proteins forming part of tj and controlling the permeability of cell membranes, cldn , occludin (ocld), and zo- . additionally, we assessed the correlation between the functional changes and the proliferation activity and apoptosis in cells of sublines that were analyzed previously (smirnova et al., ) , as well as evaluating the ability of rimantadine and alpisarin antiviral preparations to restore the normal functioning of endothelial cells. sublines of the passaged human endothelium ecv- cell line obtained after the infection with the influenza a virus brisbane/ / (h n ) were used in the work. cells were infected with the low virus dose ( id) corresponding to the multiplicity of infection (moi) equal to . or high virus dose ( id, moi = . ) and additionally (or exclusively) exposed to ng/ml of the escherichia coli lps (sigma, united states). cell were passaged on the sixth to seventh day after infection by splitting them in a ratio of : using the alpha-mem medium (biolot, russia) containing % fetal bovine serum (fbs) (hyclone, united states) without antibiotics. to detach cells from the surface, a solution containing vercene and chymopsin was used. both control (intact) and experimental cells were at the sixth passage after the exposure to the agents (smirnova et al., ) . depending on the agent (virus and corresponding id, lps, or virus and lps together), the cell sublines were given the following names: ecv- id, ecv- id, ecv-lps, ecv- id + lps, and ecv- id + lps. assessment of migratory activity. cell suspensions of the corresponding sublines were inoculated into the wells of six-well plates (nunc, denmark) in a concentration of × cells/ml ( ml per well) in the alpha-mem medium with % fbs, and the plates were placed into the co incubator ( °c and % co ). the next day, the cell monolayer was scratched with a -μl tip, cells were washed with serum-free medium, and alpha-mem medium with % fbs was added to the cells. an amount of . ml ( / medium volume) of tenfold solutions of each of the two analyzed antiviral preparation were applied into the wells. immediately after the application of the antiviral preparations (point zero) and then , , , and h after the application, the selected region of the wound surface was imaged using the axio vert a microscope (carl zeiss, germany) with × magnification. with the aid of the axiovision rel. . software, the wound surface area was calculated in the obtained images, and the change in the surface area with time, i.e., wound sealing, was determined. the end result was expressed as a percentage relative to the change in the wound surface area in the control samples (which were not subjected to infection and treated with antiviral drugs) at each time point. analysis of gene expression. on the fourth day after wounding (when the wound was almost completely sealed), cells were removed from the well surface, counted, washed with the serum-free medium, centrifuged, and used to assess the changes in the expression of the genes encoding cytokines and other cellular factors by real-time pcr. rna was isolated from the analyzed samples using the amplipraim ribo-prep kit (central research institute of epidemiology, russia) according to the manufacturer's instructions. to assess gene expression, cdna was synthesized on the rna template in reverse transcription reaction using the reverta-l kit (central research institute of epidemiology, russia). amplification reaction was carried out in the rotor-gene thermal cycler (corbett research, australia) according to the following program: denaturation at °c for min, cycles including °c for s, then °c for s, and °c for s, each. the reaction mixture contained μl of dna, an amount of μl of ready-made . × pcr buffer (sintol, russia), . μl of syntaq dna polymerase ( u/μl), and . μl of each primer and probe ( pmol/μl) (dna-sintez, russia) in μl. primer for the actin gene (actb) were used as an internal standard. the nucleotide sequences of the oligonucleotide primers and probes for tnfα, tgfβ, nox , cldn , ocld, zo- , and actb are provided in table . expression levels were determined for two or three times, and the end result was expressed in relative units, indicating a change in gene expression relative to the control, and calculated according to the for- table . nucleotide sequences of primers and taqman probes used to analyze mrna expression for the genes encoding cytokines, tight junction proteins, and other cellular factors forward primer '- ' probe '- ' reverse primer '- ' mula -ΔΔct , where c t is the cycle threshold value for the analyzed sample (wong and medrano, ) . plants, russia) solutions were prepared using serumfree alpha-mem medium and were used in final concentrations of μg/ml and μg/ml, respectively. at each time point, two wells were analyzed. experiments were performed not less than in triplicates. statistical data analysis was performed using the mann-whitney u test for the comparison of the two groups of nonparametric samplings. differences were considered significant with p < . . analysis of cell migration was performed using the previously obtained ecv- cell sublines (smirnova et al., ) at the sixth passage after the exposure to influenza virus and/or lps treatment. migratory activity was assessed by the change in wound surface area with time (wound sealing). as a result, we have demonstrated that the maximum migratory activity is observed in all cell sublines (compared to the control intact cells) during the first h (here and in what follows, time is given in hours after wounding). cell migration grew slower by the th- th hours and remained almost at the -h and control levels up to the th hours (fig. a) . the comparison of different sublines showed that highest migratory activity was exhibited by the endothelial cells after the contact with the minimum infective dose of influenza a virus (ecv- id subline, migratory activity % of the control activity h after wounding). the migration exhibited by the cells infected with the high virus dose (ecv- id) was the minimum ( % of the control h after wounding). the cells after the contact with lps (both in the case of individual exposure and together with the virus) showed slightly increased migratory activity during the first hours after wounding ( - h), while later migration remained at the control level (fig. a) . to assess the effects of the antiviral preparations (rimantadine and alpisarin) on the migratory activity of the cells, the preparations were applied to the wells containing cells immediately after wounding. as a result, it was demonstrated that they significantly affect cell migration. rimantadine decreased the migratory activity of the control cells by % h after wounding, while alpisarin decreased it by %; however, migration was restored to the control level h after wounding. rimantadine had almost no effect on the migration of the ecv- id subline cells ( % in h), while the level of ecv- id subline cell migration in the presence of this drug grew higher than the control level (up to % in h). alpisarin caused even higher increase in the migratory activity of these subline cells in h (up to % in ecv- id subline and up to % in the ecv- id subline). the highest increase in cell migration was caused by both preparations in the sublines treated with lps, namely, ecv-lps (up to % in the case of rimantadine and % in the case of alpisarin), ecv- id + lps ( and %, respectively), and ecv- id + lps ( and %, respectively) . in all the sublines, maximum migration level in the presence of the antivirals was also observed h after wounding, together with a decrease in the level of migration by eighth hour; however, in this case, migratory activity remained higher than in the control (fig. a) . we have also revealed that the pattern of differences in the migratory activity between the cell sublines at the sixth passage at the maximum activity point ( h after wounding) correlated with the pattern of changes in proliferation activity of the cells at the first passage (proliferation data were obtained previously (smirnova et al., ) ). in particular, the enhancement of cell migration and proliferation was observed in the ecv- id and ecv-lps sublines, while enhancement of suppression was observed in the ecv- id subline (figs. b, c) . comparison of the migration and proliferation patterns for the cells at the sixth passage showed that the similarity was retained for most sublines, except for the ecv-lps subline, in which proliferation activity decreased at the sixth passage, and the ecv- id subline, in which, by contrast, an increase in proliferation was detected (figs. b, c) . therefore, the differences in the migratory activity of the cells in the analyzed sublines at the sixth passage are determined by the initial (at the first passage) effects of the agents (high or low doses of the virus and/or lps). on the fourth day after wounding (when the wound was almost completely sealed), the analyzed subline cells were detached from the surface of the six-well plates and used to determine the expression levels of mrnas for cytokines (tnfα and tgfβ), nox oxidase, and tj proteins (ocld, zo- , and cldn ) (ecv- id and ecv- id + lps sublines were not used). pcr has revealed an increased expression of mrnas for the cytokines tnfα and tgfβ in the cells that were exposed to the virus and/or lps, with the highest level of expression for these cytokines having been observed in the ecv- id + lps subline cells ( and times higher, respectively, than in the control). treatment with the antiviral preparations rimantadine and alpisarin considerably decreased the expression of the mrnas for these cytokines enhanced by the exposure to virus and/or lps, although, rimantadine on its own stimulated the expression of tnfα mrna ( times higher than in the control) (fig. ) . a slight increase in the expression level of nox mrna was detected only in the ecv- id subline ( . times higher than in the control), while antiviral preparations reversed it to the control levels. the expression of the mrna for the cldn gene encoding the tj protein was at the control level in all cell sublines except for ecv- id + lps, in which cldn mrna expression was . times higher than in the control. the treatment with antiviral preparations increased mrna expression level for the gene encoding this protein in all sublines, and especially so in the case of alpisarin (with the maximum increase-by . times-being in the ecv-lps subline), which may indicate that cell permeability returns to its normal level (fig. ) . in a separate experiment, we assessed the expression levels of mrnas for the genes encoding other proteins within the tj family, namely, ocld and zo- . the obtained results revealed that the expression level of mrnas for these genes gradually decreased in ecv- cells from the first passage to the fifth passage after a single exposure to different doses of influenza a virus and/or lps, this effect being clearer in the case of ocld than in the case of zo- (fig. ) . these data provide evidence that the exposure to influenza virus and/or lps leads to an increase in the permeability of cell membranes, which is further maintained in the analyzed cell sublines for a long period of time. angiogenesis plays an important role in many physiological and pathological processes, while apoptosis serves as one of the regulatory factors of angiogenesis. the ability of the highly virulent avian influ- enza virus strains to cause a lethal disease in chicken as a result of apoptosis of the endothelial cells in different organs is well-known (swayne, ) . however, the pathological mechanisms of influenza infection, in particular, the link between the infection and apoptosis and barrier function impairment, are not always clear. it has been demonstrated in vitro that human influenza a viruses are able to trigger tnf-induced apoptosis of endothelial cells (sumikoshi et al., ) . at the same time, increased cell permeability in human microvascular endothelium (hmvec) and association with apoptosis has been demonstrated only for live (infectious) influenza a virus; uv-inactivated avirulent influenza virus also caused an increase in cell permeability, but in this case not as a result of apoptosis, but rather as a result of the degradation of the tj protein cldn- (armstrong et al., ) . the role of the cytokines tnfα and tgfβ in both apoptosis induction and cell barrier function impairment is well-known, although these processes often develop independently of each other (petrache et al., ; meeteren and ten dijke, ). in our previous work, we have demonstrated very low levels of apoptosis, just as in control cells, in all the obtained endothelium cell sublines cultured during more than three passages (six passages, in particular). at the same time, the antiviral preparations rimanta-dine and alpisarin reduced the increased proliferation of the ecv- id subline cells at the sixth passage and simultaneously stimulated apoptosis in all the analyzed sublines (smirnova et al., ) . the comparative study of the human endothelial ecv- cell sublines carried out in the present work and in our previous work (smirnova et al., ) has demonstrated that the infection of nonpermissive cells with influenza a virus (both in high and in very low doses) and exposure to lps can change migratory, proliferation, and apoptotic activity of cells and impair cell barrier function. we have shown for the first time that these functional changes may be observed during a long period of cell culturing after the exposure. in the analyzed cell sublines, the expression of mrna for ocld and zo- encoding the proteins that form part of the tj complex gradually decreased with an increase in the passage number, which indicated an increase in cell permeability (kasa et al., ) . the results obtained in the present work, as well as in the previous one (smirnova et al., ) , show that all sublines of endothelial cells acquire resistance to apoptosis in the process of culturing (after the third passage), notwithstanding the high level of expression of mrna for both tnfα and tgfβ, especially in the ecv- id + lps subline. this indicates a link between the cytokines tnfα and tgfβ and the increase in cell permeability, but not the induction of apoptosis in this case. this conclusion is supported also by the fact that, in the presence of the antivirals rimantadine and alpisarin, expression of tnfα and tgfβ genes is inhibited and cell permeability is restored (due to the increase in the cldr mrna expression) with a simultaneous increase in the apoptosis level, which also indicates that these drugs are capable of restoring the functional state of the cells. it is characteristic that the level of nox mrna expression appeared to be only slightly increased and only in a single subline, which was obtained after the infection with the low dose of influenza virus (ecv- id) and differed from all others in its increased proliferation level. this allows it to be suggested that the dysfunction of cells in the analyzed sublines that develops over the period of their long-term culturing may be caused by the increase in ros content not only in the cytosol (produced by nox and nox oxidases), but, apparently, to a greater extent in mitochondria (harel et al., ) . the multifunctional cytokine tgfβ can enhance cell proliferation and apoptosis resistance through the alk smad / /nf-κb signaling pathway, which induces the transformation of endothelial cells into miofibroblasts (van meeteren and ten dijke, ; montorfano et al., ) . tgfβ is considered to be the key cytokine participating in fibrosis. reduced sensitivity of lung fibroblasts to fas-induced apoptosis (resistance to apoptosis) with increased alveolar epithelial cell apoptosis level is a characteristic feature of pulmonary fibrosis, in which the regions of a disrupted epithelial cell layer with reduced reparation capacity are filled with apoptosis-resistant fibroblasts (maher et al., ; liu and desai, ) . our findings (increased expression level for tgfβ mrna along with the development of apoptosis resistance) provide support for the idea that there is a link between influenza virus infection and, especially, lps treatment with myofibroblast formation and the possibility of pulmonary fibrosis development. thus, the addition of lps at the time of ecv- cell infection with influenza a virus leads to a more serious dysfunction of endothelial cells, in the same way as a secondary bacterial infection, even in the case of mild influenza infection leads to serious and, in many cases, complications that occur much later (mccullers, ) . our data also indicate that endothelial cells infected only with influenza a virus may become atypical (demonstrate increased proliferation activity and an extremely low apoptosis level), which may facilitate the expansion of these cells and hinder the reparation of damaged blood vessel regions with normal endothelial cells. this may lead to enhanced angiogenesis, which in turn promotes oncogenesis, and may possibly cause intravascular lesions of unknown origin with the characteristics of pseudotumor hyperplasia (diaz-flores et al., ) . to summarize, the results of the present work have revealed that under our experimental conditions (the use of varying virus doses and/or lps with subsequent long cell culturing), changes in endothelial cell functioning can be observed for a long period of time after the exposure. we have also demonstrated that there is a connection between the agent used (virus and/or lps) and the direction of functional changes (increase or decrease) in migratory and proliferation activity and cell permeability. the antiviral preparations rimantadine and alpisarin have a substantial effect on all the analyzed live processes in the cell sublines, which indicates that these preparations may pre- the authors declare that they have no conflict of interest. this article does not contain any studies involving animals or human participants performed by any of the authors. influenza virus replication in lung epithelial cells depends on redox-sensitive pathways activated by nox -derived ros influenza infects lung microvascular endothelium leading to microvascular leak: role of apoptosis and claudin- nox nadph oxidase mediates oxidative stress and apoptosis caused by tnf-a in cerebral vascular endothelial cells hydrogen peroxide signaling in vascular endothelial cells human lung carcinoma (a- ) continuing cell line and human endothelial (ecv- ) continuing cell line responses to the influenza virus at different multiplicities of infection the role of adherens junctions and ve-cadherin in the control of vascular permeability angiogenesis and wound repair: when enough is enough morphofunctional basis of the different types of angiogenesis and formation of angiogenesis-related secondary structures nox , nox , and mitochondrial-derived reactive oxygen species contribute to angiopoietin- signaling 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receptor potential melastatin calcium channel activity change in the functional state of cells of the human endothelial cell line ecv- under the influence of influenza a virus, lipopolysaccharide from e. coli, and some drugs human influenza virus infection and apoptosis induction in human vascular endothelial cells understanding the complex pathobiology of high pathogenicity avian influenza viruses in birds real-time pcr for mrna quantitation location of vegf to vascular ecm is an important aspect of tumor angiogenesis, cancers p mapk, rho/rock and pkc pathway are involved in influenza-induced cytoskeletal rearrangement and hyperpermeability in pmvec via phosphorylating erm key: cord- -w esk authors: moreno, gerard; rodríguez, alejandro; reyes, luis f.; gomez, josep; sole-violan, jordi; díaz, emili; bodí, maría; trefler, sandra; guardiola, juan; yébenes, juan c.; soriano, alex; garnacho-montero, josé; socias, lorenzo; del valle ortíz, maría; correig, eudald; marín-corral, judith; vallverdú-vidal, montserrat; restrepo, marcos i.; torres, antoni; martín-loeches, ignacio title: corticosteroid treatment in critically ill patients with severe influenza pneumonia: a propensity score matching study date: - - journal: intensive care med doi: . /s - - - sha: doc_id: cord_uid: w esk purpose: to determine clinical predictors associated with corticosteroid administration and its association with icu mortality in critically ill patients with severe influenza pneumonia. methods: secondary analysis of a prospective cohort study of critically ill patients with confirmed influenza pneumonia admitted to icus in spain between june and april . patients who received corticosteroid treatment for causes other than viral pneumonia (e.g., refractory septic shock and asthma or chronic obstructive pulmonary disease [copd] exacerbation) were excluded. patients with corticosteroid therapy were compared with those without corticosteroid therapy. we use a propensity score (ps) matching analysis to reduce confounding factors. the primary outcome was icu mortality. cox proportional hazards and competing risks analysis was performed to assess the impact of corticosteroids on icu mortality. results: a total of patients with primary influenza pneumonia were enrolled. corticosteroids were administered in ( . %) patients, with methylprednisolone the most frequently used corticosteroid ( / [ . %]). the median daily dose was equivalent to mg of methylprednisolone (iqr – ) for a median duration of days (iqr – ). asthma, copd, hematological disease, and the need for mechanical ventilation were independently associated with corticosteroid use. crude icu mortality was higher in patients who received corticosteroids ( . %) than in patients who did not receive corticosteroids ( . %, p < . ). after ps matching, corticosteroid use was associated with icu mortality in the cox (hr = . [ % ci . – . ], p < . ) and competing risks analysis (shr = . [ % ci . – . ], p = . ). conclusion: administration of corticosteroids in patients with severe influenza pneumonia is associated with increased icu mortality, and these agents should not be used as co-adjuvant therapy. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. pneumonia caused by the influenza a(h n )pdm virus infection may lead to life-threatening acute respiratory failure (arf) and acute respiratory distress syndrome (ards). antiviral therapy is the cornerstone of treatment for influenza pneumonia [ ] [ ] [ ] ; in addition, intravenous corticosteroids have been used as co-adjuvant therapy in patients with arf/ards to modulate lung inflammation and improve clinical outcomes [ ] [ ] [ ] [ ] [ ] . however, no randomized clinical trials have investigated the potential benefit or harm of corticosteroid therapy for arf/ards due to acute influenza pneumonia. during the h n pandemic, corticosteroids were widely used despite contradictory [ , ] , unfavorable [ , [ ] [ ] [ ] , or inconclusive [ , ] available data. a recent cochrane review [ ] concluded that co-adjuvant corticosteroid therapy was associated with increased mortality in patients with influenza pneumonia. however, the data were derived from observational studies of very low quality and with several methodological limitations, including other clinical indications of corticosteroids as a major potential concern. thus, it is impossible to be sure that patients who were treated with corticosteroids did not have other corticosteroid indications or were not more severely ill in the first place. we have previously reported that corticosteroid therapy does not improve survival in patients with primary viral pneumonia [ ] . however, in that observational study, we assessed the effects of corticosteroid therapy on survival between patients who were and were not treated, but we did not apply a statistical method that would have balanced all the variables between the two groups. therefore, the aim of the present study was to identify the factors associated with corticosteroid use and its impact on intensive care unit (icu) mortality using propensity score (ps) matching analysis in icu patients with influenza pneumonia. preliminary results of this analysis were presented in the th international symposium on intensive care and emergency medicine [ ] . this was a secondary analysis of prospective and observational cohorts of critically ill subjects admitted to icus in spain (which represents approximately % of the country's icus) between june and april . data were obtained from a voluntary registry created by semicyuc (sociedad española de medicina intensiva, crítica y unidades coronarias). all consecutive cases admitted to the icu were collected. the study was approved by the joan xxiii university hospital ethics committee (irb# ). patient identity remained anonymous, and the requirement for informed consent was waived due to the observational nature of the study, as reported elsewhere [ , [ ] [ ] [ ] [ ] [ ] . inclusion criteria participants included patients admitted with fever (> °c); respiratory symptoms consistent with cough, sore throat, myalgia, or influenza-like illness; acute respiratory failure requiring icu admission; and microbiological confirmation of viral a, b, or c infection identified by reverse transcription polymerase chain reaction (rt-pcr) at icu admission. data were reported by the attending physician reviewing medical charts and radiological and laboratory records. the attending physician ordered all tests and procedures related to patient care. exclusion criteria patients receiving corticosteroids as rescue therapy (due to shock) or due to chronic obstructive pulmonary disease (copd)/asthma exacerbation were excluded (see definition below). children < years old were not enrolled in the study. patients with non-pulmonary influenza infection and those with healthcareassociated pneumonia were also excluded. the following variables were recorded at icu admission: demographic data, comorbidities, time from illness onset to hospital admission, time to first dose of antiviral delivery, microbiological findings, and laboratory and chest radiological findings at icu admission (all the collected variables are reported in e- table of the supplementary material). to determine illness severity, the acute physiology and chronic health evaluation (apache) ii score [ ] was estimated for all patients within h of icu admission. organ failure was assessed using the sequential organ failure assessment (sofa) scoring system [ ] , also at icu admission. the indication of corticosteroid treatment was clearly reported in the case report form and was confirmed by the medical records. community-acquired pneumonia (cap) was defined in accordance with current american thoracic society and infectious diseases society of america guidelines (ats/ idsa) [ ] . the rt-pcr test for influenza was carried out in accordance with the guidelines of the centers for disease control and prevention (cdc) [ ] . primary viral pneumonia was defined as acute respiratory failure and unequivocal alveolar opacities involving two or more lobes, with negative respiratory and blood bacterial cultures during the acute phase of influenza virus infection at icu admission [ ] . systemic corticosteroids have been widely used as co-adjuvant therapy in patients arf/ards due to influenza pneumonia to modulate lung inflammation, despite controversy on clinical outcomes. our findings provide solid evidence to support the association of corticosteroids administration with increased icu mortality in critically ill patients with influenza pneumonia. copd "exacerbation" was defined according to copd exacerbation guidelines of the european respiratory society/ats [ ] as increased respiratory symptoms, particularly dyspnea, cough, and increased sputum purulence without pulmonary infiltrates in chest x-ray. copd patients with pulmonary infiltrates in chest x-ray were considered as cap and were included in the present analysis. asthma exacerbation was defined as acute or subacute episodes characterized by a progressive increase in one or more typical asthmatic symptoms (dyspnea, coughing, wheezing, and tightness of the chest [ ] without infiltrates in the chest x-ray. asthmatic patients with pulmonary infiltrates in chest x-ray were considered as cap and were included in the present analysis. community-acquired respiratory co-infection (carc) was considered in patients with confirmation of influenza virus infection showing recurrence of fever, increase in cough and production of purulent sputum plus positive bacterial/fungal respiratory or blood cultures at icu admission [ , ] . refractory septic shock was defined in accordance with the surviving sepsis campaign guidelines [ ] ; that is, patients in whom adequate fluid resuscitation and vasopressor therapy are unable to restore hemodynamic stability. ventilator-associated pneumonia was defined according to the new ats/idsa guidelines [ ] among icu patients who developed a new pneumonic event while mechanically ventilated for at least h after clinical presentation. corticosteroid treatment: we considered the primary indication recorded by the treating physician as coadjuvant treatment for viral pneumonia. corticosteroid therapy was defined as corticosteroid administration at icu admission (within the first h). patients receiving corticosteroids as rescue therapy (due to shock) or due to copd/asthma exacerbation were excluded (see exclusion criteria). obese patients were defined as those with a body mass index (bmi) of > kg/m . the icu admission criteria and treatment decisions for all patients, including the decision to intubate and type of antibiotic, antiviral, or corticosteroid therapy administered, were not standardized between centers and were left to the discretion of the attending physician, according to the spanish society of intensive care recommendations [ ] . primary to determine whether corticosteroid use was associated with icu mortality. in addition, the primary outcome was examined in eight pre-specified subgroups defined according to the following baseline characteristics: ( ) severity of illness (apache score < vs. ≥ ), ( ) intensity of organ dysfunction (sofa < vs. ≥ ), ( ) presence of shock upon icu (yes vs. no), ( ) need for mechanical ventilation (mv) upon icu admission (yes vs. no), ( ) inflammatory response to c-reactive protein (crp < vs. ≥ mg/dl), ( ) presence of bacterial coinfection (yes vs. no), ( ) chronic lung disease such as copd (yes vs. no), and ( ) asthma (yes vs. no). the cutoff for continuous variables was determined according to our population median value. secondary to determine risk factors associated with corticosteroid use. icu length of stay (los) and mv days were also examined in survivors between groups receiving and not receiving corticosteroid therapy. discrete variables were expressed as counts (percentage) and continuous variables as means with standard deviation (sd) or medians and interquartile range - % (iqr). for patient demographics and clinical characteristics, differences between groups were assessed using the chi-squared test and fisher's exact test for categorical variables, and the student t test or the mann-whitney u test for continuous variables. to investigate the association between baseline (icu admission) variables and corticosteroid use, a multivariate analysis (binary logistic regression) was performed. the multivariate model comprised factors of clinical interest and all significant covariates in the univariate analysis. the results are presented as odds ratios (or) and % confidence intervals (ci). model integrity was examined using standard diagnostic statistics and plots and goodness of fit for each model for all outcomes, and was assessed with the hosmer-lemeshow test. after this first approach, we generated a full-matching ps analysis in order to minimize the effect of a corticosteroid treatment selection bias and to control for potential confounding factors (additional information about the ps full-matching analysis can be found in the electronic supplementary material) [ ] . this allowed us to study two comparable (almost identical) cohorts: ( ) the corticosteroid-treated group and ( ) the control group, comprising patients who did not receive corticosteroid treatment. ps matching analysis attempts to compare outcomes between patients who have a similar distribution of all the covariates measured. an attractive feature of this approach is that it uses the entire sample. using the ps methodology, all patients were assigned a weight between and ; this propensity-matched cohort was generated by choosing the best weight balance. this method optimizes the post-weighting balance of covariates between groups and, in this way, approximates the conditions of random site-of-treatment assignment. to assess our ps adjustment, we checked for adequate overlap in propensity scores between groups with a crossvalidation model. to do so, we divided the patients in the database into two subsets: (a) a "training set" with patients ( %), and (b) a "validation set" with patients ( %). after the matching, a kaplan-meier survival plot was generated to track icu mortality over time for corticosteroid-treated and untreated patients. in addition, cox proportional hazards regression models were fitted to assess the impact of corticosteroids on icu mortality. the results are presented as hazard ratios (hr) and % ci and adjusted survival plots. because cox hazard survival analysis is not satisfactory for describing icu patient mortality over time [ ] , we performed a competing risks analysis to confirm our results. first, we computed the cumulative incidence function (cif) of death over time. at time t, the cif defines the probability of dying in the icu by that time t when the population can be discharged alive. the cif was estimated from the data using the cmprsk package developed by gray [ ] . we used the fine and gray model [ ] , which extends the cox model to competing risks data by considering the sub-distribution hazard (for instance, the hazard function associated with the cif). the strength of the association between each variable and the outcome was assessed using the sub-hazard ratio (shr), which is the ratio of hazards associated with the cif in the presence of and in the absence of a prognostic factor. in order to avoid spurious associations, the variables that we entered in the regression models were those with a relationship in the univariate analysis (p < . ) or a plausible relationship with the dependent variable. data analysis was performed using spss for windows version . (ibm corp., armonk, ny, usa). mixed-effects models were performed with r (cran.r-project.org). a total of patients with confirmed influenza pneumonia were enrolled at icus in spain during the study period ( ) ( ) ( ) ( ) ( ) ( ) . of these, ( . %) met the inclusion criteria and were included in the study (fig. ) . among patients with corticosteroid therapy, ( . %) received methylprednisolone, ( . %) prednisolone, and three ( . %) dexamethasone. for all patients who received therapy with corticosteroids due to pneumonia, this was initiated within the first h of icu admission. patients received a median (interquartile range [iqr]) daily dose equivalent to ( - ) mg of methylprednisolone, and the median duration of corticosteroid treatment was ( - ) days. the frequency of corticosteroid treatment by study period was . % in , . % in , % in , and . % in . considering the period as baseline, we observed that only in was there a significant reduction (p = . ) in the indication of corticosteroid treatment as co-adjuvant therapy for pneumonia. no differences in the rate of ventilator-associated pneumonia were observed between patients with (n = , . %) and without (n = , . %) corticosteroid therapy. clinical characteristics of patients and their distribution in the two groups are shown in table fig. flowchart of all excluded and included patients who received corticosteroid therapy were sicker according to the apache ii score, more obese, and more likely to have asthma, copd, and hematological diseases than those who did not receive treatment. mv use, serum procalcitonin concentrations, and icu mortality rate were higher in patients who received corticosteroids. there were no significant differences between groups regarding icu los or mv days. no other differences were found between the groups. overall mortality was . % ( / ). to determine factors associated with corticosteroid use, a stepwise logistic regression model was performed. apache ii score, asthma, copd, obesity, hematological disease, and mv were the independent variables included in the model. as shown in table , mv (or = . ), asthma (or = . ), copd (or = . ), and hematological disease (or = . ) were independently associated with corticosteroid use. table , supplementary material). ps matching was applied, and control and treated patients were matched. the summaries of balance for unmatched and matched data are shown in table (and e- fig. in the supplementary material). the apache ii score, sofa score, delay at icu admission, number of quadrants infiltrated in chest x-ray, serum lactate dehydrogenase (ldh), white blood cell (wbc) count, continuous renal replacement therapy (crrt), serum crp, mv, shock, chronic heart disease, human immunodeficiency virus (hiv/aids), primary viral pneumonia, bacterial co-infection, and corticosteroid use were the variables included in the logistic regression analysis of the ps model. the discriminatory power of the model (e- fig. , supplementary material) was good, with an area under the receiver operating characteristic curve of . ( % ci . - . , p < . ). the accuracy of the predictive model (training set) with respect to the validation set was . . e- figure (supplementary material) shows the kaplan-meier estimates of the mortality rate during icu admission, differentiating between patients with and without corticosteroid use. the cumulative survival was lower in patients with corticosteroid therapy than in untreated patients (log-rank test . , p < . ). when we excluded patients with carc, the results were similar (log-rank test . , p = . ; supplementary material) . however, in patients with carc, only a trend towards higher mortality related to corticosteroid treatment was observed p = . ; supplementary material) . finally, to determine the impact of corticosteroid use on icu mortality, a cox regression analysis adjusted for apache ii and potential confounding factors (see e- fig. in supplementary material) was performed. the survival plot (fig. ) showed that the use of corticosteroids was significantly associated with a higher icu mortality rate (hr . [ % ci . - . ], p < . ). when a multivariate fine and gray regression model was used (fig. and e- table in the supplementary material), corticosteroid use remained as a factor associated with mortality (shr = . [ % ci . - . ], p < . ). our results strongly suggest that administration of corticosteroids as co-adjuvant therapy to standard antiviral treatment in critically ill patients with severe influenza pneumonia is associated with increased icu mortality. this negative effect was evident in all subgroups considered and after careful adjustments, including a ps matching analysis. to assess the potential effects of corticosteroids on these severely ill patients, we limited our analysis to a well-defined cohort of icu patients with severe influenza pneumonia, and excluded those with other indications for corticosteroid use. the effect analysis of corticosteroids was restricted to early administration (within the first h of icu admission) in order to avoid the inclusion of patients receiving rescue therapy and to reduce the effects of time-dependent confounders. we found that mv, asthma, copd, and hematological disease were independently associated with corticosteroid use. severe acute lung injury following influenza infection is characterized by uncontrolled local and systemic inflammation [ ] [ ] [ ] . this damage is caused by an excessive host innate response with exaggerated migration of macrophages, neutrophils, and pro-inflammatory cytokines, leading to classic exudative diffuse alveolar damage, severe necrotizing bronchiolitis with predominantly neutrophilic inflammation, and intense alveolar hemorrhage [ ] . corticosteroids have several anti-inflammatory, immunomodulatory, and vascular properties, including inhibition of pro-inflammatory cytokines, reduction of leukocyte trafficking, stimulation of apoptosis in t-lymphocytes, and maintenance of endothelial integrity and vascular permeability. therefore, they may represent an option for adjunctive therapy; however, although they are frequently prescribed in critically ill patients with influenza pneumonia, their potential benefits and harms are controversial [ , , , , ] . three recent systematic reviews and meta-analyses [ ] [ ] [ ] concluded that corticosteroid therapy is significantly associated with mortality, even in the subgroup of patients with influenza hospitalized in or outside the icu. these systematic reviews recognize similar limitations such as the heterogeneity of the studies, lack of sufficient data on indication for corticosteroids, dosage, therapy timing, type of corticosteroid use, and severity of illness. a recent cochrane review [ ] reported an association between corticosteroid therapy and increased mortality. however, all studies included were observational (only seven studies included patients admitted to the icu) and of very low quality due to confounding by indication. therefore, it was impossible to determine whether additional corticosteroid therapy is indeed harmful in patients with influenza infection. several observational studies have evaluated the impact of corticosteroids on mortality in patients with influenza infection [ - , , , , - ] , and have offered conflicting perspectives. observational studies are potentially susceptible to bias and do not provide robust results. despite these weaknesses, however, observational data are representative of current clinical practice, and applying modern methods such as ps matching may help in evaluating the effects of certain interventions in clinical settings and may help to guide decision-making. to the best of our knowledge, only one study has used an analysis similar to ours in patients with influenza infection. in critically ill patients, kim et al. [ ] analyzed the effect of corticosteroid treatment on -day mortality with a similar methodology to ours, applying multivariate adjustment (controlling for variables that differed between the two groups and incorporating the ps) and ps matching ( : ). sixty-five pairs were generated, and the -day mortality rate was higher in the corticosteroid group ( % vs. %, p = . ). these data are in concordance with our results; however, the mortality rate in our patients was substantially lower. this discrepancy might be due to several factors, including differences in severity of illness, endpoint observational period (icu mortality vs. -day mortality), and early recognition vs. standard of care. interestingly, kim et al. reported that half of the patients treated with corticosteroids received hydrocortisone, a non-standard co-adjuvant treatment of pneumonia. the authors did not report the treatment indication for corticosteroid therapy; thus many patients in this cohort may have received corticosteroids for a reason other than influenza-induced acute lung injury. in contrast, our population comprised only patients treated with corticosteroids as an co-adjuvant therapy for severe viral pneumonia, excluding patients with other indications for corticosteroids (such as shock). therefore, with a homogeneous group of critically ill patients, and after carefully controlling for important confounders through a ps matching analysis and competing risks analysis, we provide robust evidence to support the association between corticosteroid administration and increased mortality. interestingly, the subgroup analysis showed that, in contrast to patients with asthma, copd patients treated with corticosteroids had a higher risk of icu mortality than those without corticosteroid therapy. we are not able to explain this finding using our database because we did not collect data on the degree of copd severity. copd patients may be at an advanced stage of disease. this condition, and other uncontrolled confounding factors, may explain the higher mortality among copd patients even after excluding patients with copd exacerbation. the main strengths of this study are the homogeneous and uniform population, the high number of critically ill patients included in our multicenter study, data regarding the kind/indication of corticosteroid treatment, and the carefully executed analysis to resolve confounding factors including the presence of competing risks. however, we recognize some limitations. first, our results were obtained in a homogeneous population of patients with influenza pneumonia and cannot be extrapolated to other populations. second, we did not review the duration of viral shedding or appearance of drug-resistant virus in either group. third, ps matching analysis may also be a limitation, because this method may not reflect the possible biases in observational studies, and some residual confounding may persist. however, as ps matching analysis can balance the population and reduces observational bias, it is the best evidence available for physicians. fourth, data on mv of patients were not recorded. lung-protective ventilation is the standard of care for patients with acute lung injury/ards because of the evidence that it decreases mortality. although we did not provide data regarding ventilation of patients, it is broadly accepted in this country that applying protective ventilation improves results and is one of the national quality indicators. finally, we did not record data about muscle weakness or metabolic alterations related to corticosteroid treatment. in a homogeneous group of critically ill patients with severe influenza pneumonia, after adequate adjustment by ps matching and competing risks, co-adjuvant corticosteroid therapy was significantly associated with increased icu mortality. our data strongly suggest that corticosteroids should not be used as co-adjuvant therapy in patients with influenza pneumonia. the online 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low-tomoderate-dose corticosteroids on mortality of hospitalized adolescents and adults with influenza a (h n ) pdm viral pneumonia the authors are grateful to michael maudsley for language editing and eudald correig for the statistical analysis.getgag (grupo español de trabajo gripe a grave) study group investigators. andalucía: pedro cobo (hospital punta de europa, algeciras); javier martins (hospital santa ana motril, granada); cecilia carbayo (hospital torrecardenas, almería); emilio robles-musso, antonio cárdenas, javier fierro author contributions gm, ar, jsv, iml, ed and at conceived and designed the study. all authors, apart from mr, lfr, jg, and as, contributed to the acquisition and local preparation of the constituent database. gm, ar, ec, st, iml, ed, jgm, ls, and jcy contributed to database creation and standardization, design of statistical analyses, and data analysis. gm, ar, lfr, jg, jsv, ed, mb, st, jg, jcy, as, jgm, ls, mvo, jmc, mvv, mir, at, and iml made important intellectual contributions and actively participated in the interpretation of the data and wrote the paper. all authors contributed to critical examination of the paper for important intellectual content and approval of the final manuscript. this study was supported in part by grants from semicyuc (spanish society of critical care) and the ricardo barri casanovas foundation. the study sponsors had no role in the study design, data collection, data analysis, data interpretation, or writing of the report. the corresponding authors (ar/gm) had full access to all the data in the study and final responsibility for the decision to submit for publication. all named authors declare that they have no conflicting interests. the institutional review board of joan xxiii hospital approved the original study (irbref# ). key: cord- - fpx pxs authors: corless, inge b.; nardi, deena; milstead, jeri a.; larson, elaine; nokes, kathleen m.; orsega, susan; kurth, ann e.; kirksey, kenn m.; woith, wendy title: expanding nursing's role in responding to global pandemics / / date: - - journal: nurs outlook doi: . /j.outlook. . . sha: doc_id: cord_uid: fpx pxs nan inge b. corless, phd, rn, fnap, faan a, *, deena nardi, phd, pmhcns-bc, faan a , jeri a. milstead, phd, rn, nea-bc, faan a , elaine larson, phd, rn, faan, cic b , kathleen m. nokes, phd, rn, aphn-bc, faan a , susan orsega, msn, fnp-bc, faanp, faan a , ann e. kurth, phd, rn, cnm, mph, faan a we know by now that the world will see another pandemic in the not-too-distant future; that random mutations occur often enough in microbes that help them survive and adapt; that new pathogens will inevitably find a way to break through our defenses; and that there is the increased potential for intentional or accidental release of a synthesized agent. every expert commentary and every analysis in recent years tells us that the costs of inaction are immense. and yet, as the havoc caused by the last outbreak turns into a fading memory, we become complacent and relegate the case for investing in preparedness on a back burner, only to bring it to the forefront when the next outbreak occurs. the result is that the world remains scarily vulnerable.-world bank, the world is interdependent, not only in terms of the flow of human beings but also the spread of new, emerging, and re-emerging pathogens (eidr). the number of outbreaks per year has tripled in the last several decades (smith et al., ) and the human and economic impact of human immunodeficiency virus, severe acute respiratory syndrome, ebola, and other diseases, has been staggering. yet only a handful of countries have carried out pandemic preparedness exercises (world bank, ) . furthermore, funding for pandemic preparedness (e.g., the coalition for epidemic preparedness innovations and the bloomberg foundation), has been led by nongovernmental donors more than by governments. global preparedness is critical and must include strengthening global surveillance in public and other health care delivery systems. communities must be engaged as active partners in primary and secondary infectious disease prevention efforts. as the largest cadre of the health workforce in every country, as well as a profession that is dedicated to prevention and alleviation of suffering, nurses must be integrally involved with interprofessional teams, communities, and across sectors for global pandemic preparedness. infectious diseases constitute the third leading cause of death worldwide (who, ) . international mobility contributes to the promulgation of new and reemerging pathogens that frequently are resistant to current form of treatment (morens & fauci, ) . this mobility may result in disease outbreaks that have dire consequences. emerging global pandemics pose high risks for individuals and communities. the unpredictability of pandemic outbreaks (agents, time, and place) is a given in communicable diseases. the gap in the incorporation of nursing knowledge and skills related to screening, disease identification, rapid response, community involvement, inter and intraagency communication, governmental notification, and coordination need not remain challenges to adequate and timely responses. the failure to develop a coordinated system of health care workers who understand the importance of detecting and forwarding information about the identification of an illness is a major gap in our ability to contain new infections. a key issue is the time delay in the early identification of infections that pose a threat to potential epidemics/pandemics. to mitigate the delay, it is essential that the health care professional who is the initial point of contact with the infected person, likely the community health worker in many global settings, communicate with the appropriate provider or agency to initiate the next steps including identification of the pathogen, initiation of appropriate treatment, and prevention of further dissemination. in addition, recognition of a pattern, alerting others as to the emerging disease, and preventive services will aid in preventing further diffusion of pathogens from isolated cases. a delay in such identification may lead to the development of epidemics/pandemics and be an impediment to the prompt initiation of treatment for the infected individual, appropriate interventions and protective devices, and efforts to curtail the spread of the epidemic. recent events, such as the ebola outbreak, demonstrate how lack of preparation and gaps in communication systems during the alert phase when a pathogen has been identified contribute to a delayed response . national and global organizations have evaluated the adequacy of the timeliness and programmatic response to potential outbreaks, and have examined what remains to be accomplished (sands, mundaca-shaw, & dzau, ) . the world health organization department of pandemic and epidemic diseases (ped) develops strategies, initiatives, and mechanisms to address emerging and re-emerging epidemic diseases to reduce impact on affected populations and limit international spread (world health organization, n.d.). although the ped brochures note that their work is implemented through a multidisciplinary team that includes disease-specific and public health experts with field experience responding to outbreaks and emergencies under the international health regulation, it is not clear how nurse input is provided and communicated with the team. the u.s. department of health and human services office of pandemics and emerging threats engages with multiple governmental and nongovernmental partners and agencies to set policy and support initiatives to prevent, detect, and respond to health threats and provides leadership and coordination of international activities through policy analysis, program development and implementation, coordination, and strategic planning (u.s. department of health and human services, n.d.). again, it is not clear how nurse input is provided or utilized during the early stages of the health challenge. the global advisory panel on the u.s. department of health and human services ( ) organized by sigma theta tau international met with key nurse stakeholders throughout the world and released a - summary report (the global advisory panel on the future of nursing & midwifery (gapfon)) (u.s. department of health and human services, ). using accumulated rankings across all geographic regions, communicable disease was one of the top five health priorities identified by nursing leaders (www.gapfon.org). the contribution of nurses from all types and levels of practice to an improved response to global pandemics also has been noted (edmonson, mccarthy, trent-adams, mccain, & marshall, ) . the u.s. centers for disease control and prevention (cdc) identified several time intervals important to organizing an effective response to an emerging pandemic. the first is recognition interval: recognition of increased potential for ongoing transmission, described as the time when increasing numbers of human cases or clusters are identified anywhere in the world (cdc, ) . although the organizational responses noted above contribute to earlier and more rapid identification of communicable diseases, the contributions of nursing are missing. the aim of this academy policy is to fill this gap by identifying the essential role of nurses and community health workers during the time just prior to the confirmation of a potential epidemic and focuses on the early identification of infectious pathogens and prevention of further transmission. for this to occur, nursing leaders must develop a grounds-up approach to the formulation of solutions to local problems of national and international significance. the best response to a potential global pandemic is prevention. early recognition of novel infections will be enhanced by the development of linkages between community and clinic nurses and the initial point of contact with the infected individual. this will require the education and empowerment of community health workers who have regular and on-going contact with the inhabitants of villages, towns, or regions, and are likely to be the first to recognize symptomatic individuals and consequently serve as the first line of defense through the early and prompt identification of emerging or re-emerging infectious diseases. a policy that requires the development of a network incorporating the community health workers into an early warning system will facilitate the prompt identification of potentially communicable diseases. the early identification of suspicious illnesses requires that these individuals are educated regarding symptoms, modes of transmission, information about ways to interrupt the chain of infection, and who and how to make contacts for rapid follow-up. providing a cell phone or other communication device to community health workers or others likely to be an initial point of contact may be an efficient, cost-effective approach to facilitating the early identification of communicable diseases. this will also serve as community recognition of the importance of their role. collection of a specimen from the sick individual, if feasible, and restriction of contact with that individual, to the degree possible, will also minimize transmission of the infection. the use of technology will increase the efficiency of early identification of prospective epidemics. transport of a potentially infected individual, as appropriate, to a primary care clinic or district hospital may be essential to obtaining a differential and confirmatory diagnosis and for treatment. prompt identification and treatment of the infected individuals, and prevention of further transmission, requires the rapid deployment of appropriate treatment regimens and protective gear to the caregivers in the affected areas. in addition to preparing frontline community workers to promptly identify potential emerging or re-emerging infection, appropriate national nursing councils and public health entities could be charged with the responsibility of developing and sharing early reporting networks. engaging international and national agencies (e.g., world health organization, international council of nurses, american nurses association, cdc) in such networks would further facilitate rapid mobilization of responses. these recommended actions are consistent with cdc's national strategic plan for public health preparedness and response to protect people's health and secure the nation's public health, (cdc, ) directly addressing the need for "a change in approach and longterm visioning across the various u.s. agencies involved in global health…with an emphasis on integration and partnership" (national academies of sciences, engineering, and medicine, ). the american academy of nursing affirms the importance of nursing leadership for the early recognition of communicable diseases and therefore calls on nursing leaders in each country to develop a coordinated network for response to emerging or re-emerging infections that is based on a grounds-up approach incorporating frontline individuals and communities likely to be the first to recognize symptomatic individuals as first responders. this policy aligns with the academy's strategic plan - , goal , objective : "support policies and practice design that promotes nurses as clinicians and leaders in care coordination and primary care" (american academy of nursing, ). it is also consistent with the academy policy, increasing the capacity of public health nursing to strengthen the public health infrastructure and to promote and protect the health of communities and populations (kub, kulbok, miner, & merrill, ) . global pandemics are a threat to every nation's health and therefore improvement in recognizing, containing, and controlling infectious diseases must be a global health priority. the american academy of nursing asserts that nurses are prepared for the leadership roles in policy decisions of health systems and government agencies, and can prepare for, identify, respond to, and direct recovery efforts from global pandemics that require an informed, internationally coordinated response (american academy of nursing, ). the policy recommendations provided below will strengthen national health security through the enhanced recognition and expansion of the individuals who are initial points of contact in the community as well as the role of nurses and nursing organizations in responding to and preventing potential global pandemics. have developed a national grounds-up nursecoordinated network, advocate for funding from appropriate sources (e.g., the gates foundation, the world bank) for the education of community health workers and for the distribution of cell phones or other devices to personnel likely to be first points of contact with infected individuals to facilitate rapid communication with next level providers. . encourage the icn, the national league for nursing, the centers for disease control and prevention, and community health worker representatives, to develop a curriculum for community health workers regarding the identification and reporting of infectious diseases. . urge global leaders (icn, who, cdc) to develop a strategic plan for local distribution of resources (pharmaceuticals, lab equipment, and other treatmentrelated materials) in case of emerging epidemics. academy of nursing policy manager and staff liaison - strategic plan a national strategic plan for public health preparedness and response beyond the ebola battle-winning the war against future epidemics emerging global health issues: a nurse's role. ojin: the online journal of issues in nursing emerging infectious disease repository american academy of nursing policy: increasing the capacity of public health nursing to strengthen the public health infrastructure and to promote and protect the health of communities and population emerging infectious diseases: threats to human health and global stability the neglected dimension of global security-a framework for countering infectious disease crises global rise in human infectious disease outbreaks department of health and human services (n.d.) office of global affairs, office of pandemics and emerging threats (pet) the global advisory panel on the future of nursing & midwifery (gapfon®) report. retrieved from the top causes of death. media centre from panic and neglect to investing in health security: financing pandemic preparedness at a national level (english) key: cord- -ijrghpco authors: bein, thomas; karagiannidis, christian; quintel, michael title: climate change, global warming, and intensive care date: - - journal: intensive care med doi: . /s - - - sha: doc_id: cord_uid: ijrghpco nan in the last five decades, human activities have resulted in the release of increasing quantities of carbon dioxide and other greenhouse gases, thus contributing to global climate change by additional heating of the atmosphere. the world has warmed up by approximately . °c. in particular, the last decade has been the warmest since [ ] , and the frequency and intensity of natural disasters (such as earthquakes, devastating storms, forest fires, prolonged heat waves, droughts, and floods) have increased manifold. between and , climaterelated and geophysical disasters killed . million people and left a further . billion injured, homeless, displaced, or in need of emergency assistance [ ] . climate change scenarios include a change in the spread of infectious diseases with warming and changes in outbreaks associated with extreme weather events after floods or as a result of water heating. furthermore, warmer climates provide more favourable conditions for the survival and completion of the life cycle of the vector that transmits pathogens [ ] . natural disasters and extreme events lead to traumatic deaths and injuries, mental illnesses, and infections, while global warming per se promotes heat-associated illnesses (cardiovascular strain, pulmonary diseases, exsiccosis, mental disorder [ ] ). the world health organisation (who) expects approximately , additional deaths per year between and from extreme heat, natural disasters, and changing patterns of infections, mostly in people at risk (people living in coastal regions or mega cities, children, the elderly, people with multiple and/or severe comorbidities, and-last but not least-people living in regions with weak healthcare infrastructures) [ ] . the impact of global warming on diseases requiring intensive care has been extrapolated from some existing data regarding a change in the spread of infectious diseases [ ] , an (further) alteration of the function of the respiratory system-especially in patients suffering from chronic lung diseases-an expected increase in kidney diseases, an expansion of cognitive disorders due to heat waves, particularly in the elderly, and some adverse effects on the cardiovascular system. an overview of health-related changes requiring intensive care is given in fig. . according to statistical models to estimate the global burden of infectious diseases due to climate change, in , there could be a % increase in diarrhoeal diseases, primarily in young children, and the population at risk for malaria could increase by - % [ ] . of late, in germany, the first incidence of meningitis caused by mosquito-borne west nile virus infection was reported in a man who never had travelled abroad [ ] . in consequence, in future, the european intensivist-who already should be aware of uncommon vector-borne diseases due to global tourism-must be further sensitized to presently uncommon or tropical mosquito-borne diseases such as malaria and dengue, or other vector-borne viral, fungal, or bacterial transmissions like coccidioidomycosis or avian influenza. what should we expect regarding the impact of climate change on respiratory health? lungs in a warming world [ ] become more vulnerable, especially those that are yet damaged. in patients with serious lung diseases like asthma or chronic obstructive lung disease (copd), an aggravation of severity or increased mortality has been described as a consequence of global warming. increasing concentration of greenhouse gases, air pollution, forest fires, prolonged heat waves, droughts, and floods contributes to increased respiratory morbidity and mortality. during the heat wave in portugal, for each °c increase copd morbidity increased by . %, especially in women and in persons aged over years [ ] . furthermore, a rapid spread of viral respiratory infections (e.g. hantavirus, respiratory syncytial virus) as well as fungal respiratory infections (coccidioidomycosis, aspergillosis) can be expected [ ] . dehydration and volume loss could be the other consequences of climate-related extreme heat exposure, leading to chronic kidney diseases or acute renal failure [ ] apart from that caused by diabetes, hypertension, or glomerulonephropathy. a recent study [ ] has recognized an epidemic of chronic kidney disease of unknown aetiology in central america ('mesoamerican nephropathy'), which has been attributed to recurrent dehydration due to heat stress. several studies have addressed potential cardiopulmonary health effects associated with climate change parameters [overview in ] , and these effects are expected to further increase in the coming decades. it seems that a rise in mortality, emergency room visits, and (intensive care) hospitalization due to cardiopulmonary diseases attendant on heat stress, extreme weather conditions, or air pollution are more certain than worst-case horror scenarios. but what is about climate change and intensive care medicine? do we have enough awareness (and answers) in the face of all these challenges for the healthcare system? in a recent survey on climate change and health on the members of the american thoracic society [ ] , the majority of the respondents stated that climate change is a fact that it is relevant for patient care. they confirm that physicians and physician organizations should play an active role in educating patients, the public, and policy-makers about the effects of climate change on human health. do we play such an active role yet? are we prepared to confront the increased frequency of critical illnesses about which, in some cases, we have to learn first? [ ] to our knowledge, there is presently no specific activity in intensive care medicine to face current and future challenges in the context of global warming and climate change, although it is expected that intensive care medicine will need more specialized capacities, better knowledge on the part of the intensivists, and better preparation for worst-case scenarios (heat stroke waves or infectious outbreaks) to manage the consequences of climate change adequately. it has been shown that acute and intensive care services vary substantially across economic regions, in highincome as well as in low-income countries [ ] , and it is debatable whether the current structure of intensive care is yet armed for needs related to climate change. icu bed capacities represent by far not the only challenge; changes in knowledge and organization are required to be prepared ( table ). the intensive care staff will in the near future need specific knowledge about 'uncommon' diseases and heat stroke management (especially in the elderly), and the icu must be organized so as to be able to effectively take care of larger numbers of rapidly incoming critically ill patients in the context of natural disasters. especially in megacities and coastal regions, the number of icu beds should be checked so that they are sufficient in times of natural disasters. a functionally adequate capacity for isolating patients with critical infections is indispensable for successful infection control management. expert support from psychiatrists or psychologists must be available in situations of devastating floods, storms, or heat waves. preparing intensive care medicine for the consequences of climate change does not mean neglecting the basic principle of medicine-the best cure is prevention. therefore, supporting efforts that reduce or ideally stop global warming are the need of the hour. increasing number of critically ill patients due to heat waves, natural disasters, air pollution or forest fire increase capacity of icu beds, especially in coastal regions or megacities mass casualties of critically ill patients due to rapid weather changes, floods, heat attacks implement a 'reserve' of staff and icu beds, which can be easily activated increasing incidence of 'uncommon' infectious or non-infectious diseases provide sufficient capacities of patient isolation instruct the icu staff in the diagnosis and management of 'uncommon' diseases increasing number of nephropathy during heat waves provide sufficient machines for renal replacement mitigation of climate change. contribution of working group iii to the fifth assessment report of the intergovernmental panel on climate change united nations office for disaster risk reduction, centre for research on the epidemiology of disasters ( ) economic losses, poverty, and disasters global warming and its health impact medical aspects of global warming global climate change and infectious diseases climate change and infectious diseases: what can we expect? lungs in a warming world: climate change and respiratory health excess mortality and morbidity during the july heat wave in porto climate change and respiratory infections climate change and the emergent epidemic of ckd from heat stress in rural communities: the case for heat stress nephropathy ckd of unknown origin in central america: the case for a mesoamerican nephropathy environment, global climate change, and cardiopulmonary health survey of international members of the american thoracic society on climate change and health prioritizing health in a changing climate international forum of acute care trialists. access to urban acute care services in high-vs middle-income countries: an analysis of seven cities key: cord- -a oy sz authors: yang, shenshu; lian, gaojian title: ros and diseases: role in metabolism and energy supply date: - - journal: mol cell biochem doi: . /s - - - sha: doc_id: cord_uid: a oy sz researches dedicated to reactive oxygen species (ros) had been performed for decades, yet the outcomes remain controversial. with the relentless effort of studies, researchers have explored the role of ros in biosystem and various diseases. ros are beneficial for biosystem presenting as signalling molecules and enhancing immunologic defence. however, they also have harmful effects such as causing tissue and organ damages. the results are controversial in studies focusing on ros and ros-related diseases by regulating ros with inhibitors or promotors. these competing results hindered the process for further investigation of the specific mechanisms lying behind. the opinions presented in this review interpret the researches of ros from a different dimension that might explain the competing results of ros introduced so far from a broader perspective. this review brings a different thinking to researchers, with the neglected features and potentials of ros, to relate their works with ros and to explore the mechanisms between their subject and ros. ros are a set of unstable molecules including hydrogen peroxide (h o ), hydroxyl radical (oh − ), singlet oxygen ( o ) and superoxide (o − ) that are produced by all kinds of cells [ ] . the comprehensive distribution of ros may grant them with a fundamental role in biosystem. although ros play an important role in pathogen resistance and cellular signalling, they are also broadly recognized as harmful reactive particles to cell as they damage intracellular proteins, lipids and nucleic acids. it usually appears in pathological processes when they are not scavenged on time [ ] . the essence that ros are produced in energy demanding conditions where vigorous metabolism is in demand shall not be neglected. the pathogenic role of ros in self-damage and the beneficial role in the immune system may be due to the requirement of energy supply. in these conditions, excessively produced ros bring about oxidative damage to body and pathogens. ros are widely involved in basic mechanisms and pathways. they not only impair cells and tissues with oxidative damage, but also play an important role in many homeostasis processes involving metabolism, immunity, growth and differentiation [ ] . researchers have been regarding ros as byproducts and exploring their effects on organisms, but the fundamental features of ros might illuminate their role in pathologies and biomechanisms. mitochondrial respiratory chain is one of the major sources of cellular ros. atp synthesis produces ros during normal oxygen metabolism. thus, ros are regarded as byproducts during energy perfusion to cell activities in most cases. the primary function of nadph oxidase (nox) enzymes is the generation of ros [ ] . belonging to the nox family, activated nox could promote ros production through ryanodine receptors and thus trigger ca + sparks [ ] . the involvement of nadph and nadh in repiratory chain and cellular metabolisms makes ros produce in all kinds of cells. toll-like receptors (tlr) tlr , tlr and tlr can enhance ros production by recruiting mitochondria to macrophage phagosomes and translocating tumour necrosis factor receptor-associated factor (traf ) to mitochondria to engage in evolutionarily conserved signalling intermediate in toll pathways (ecsit) [ ] . although ros are mostly produced in mitochondria, the detailed mechanisms of the production are still not fully understood. however, the major factors responsible for ros production are respiratory chain complexes (fig. ). nadh-ubiquinone oxidoreductase (complex i) is the major source of mitochondrial ros production in varying diseases. the components responsible for ros production of complex i include ubisemiquinone, flavin mononucleotide, fe-s cluster and nad [ ] . however, ros production by complex i in healthy state is humble presenting little oxidative damage. the major production of ros in this state comes mainly from complex ii through tca cycle. nadh gene mutation which causes deficiency in respiratory complex i could end up in the overproduction of ros and enhance metastasis of tumour cells [ ] . succinate dehydrogenase (sdh) or succinate-coenzyme q reductase (sqr) is composed of sdha, sdhb, sdhc and sdhd. sdha and sdhb are hydrophilic proteins. sdhc and sdhd are hydrophobic proteins that bind to ubiquinone. this oxidoreductase is also known as complex ii that plays an important role in tca cycle and respiratory chain. succinate is the intermediate of tca cycle and also a metabolic signature of ischaemia-reperfusion. it is responsible for ros generation when accumulated from fumarate overproduction and malate/aspartate shuttle during reperfusion. ischaemia injury can be ameliorated by the inhibition of succinate or ros. complex ii turns succinate into fumarate through oxidation in mitochondria with reduced ubiquinone in the membrane [ ] , and succinate could be re-oxidized by sdh, thus increasing ros generation through reverse electron transport in mitochondria. [ ] . ubiquinol-cytochrome c oxidoreductase (complex iii) is encoded by uqcrc (ubiquinol-cytochrome c reductase core protein ) gene and could receive reducing equivalents from complex i and complex ii. the received reducing equivalents are proceeded with ubiquinol and produces semiquinone for further proton transfer. p shc (src homologous-collagen homologue adaptor protein) generates mitochondrial ros as apoptosis signal through oxidation of cytochrome c in mitochondrial electron transfer chain. p mutants could lose the ability to generate ros and induce mitochondrial apoptosis [ ] , but genetic mutation may also contribute to increased generation of ros. isp- and nuo- encode complex iii subunit rieske and complex i subunit ndufb (nadh dehydrogenase [ubiquinone] beta subcomplex subunit ), respectively. mutants in isp- and nuo- are all related with enhanced ros level that leads to lengthened lifespan. ros promotor treatments can lengthen the wild-type lifespan while having no effect on those longevity mutants. and the enhanced ros induces apoptosis pathway fig. generation of ros in mitochondria triggered by ced- that changed the gene expression to protect mitochondrial dysfunction [ ] . tumour necrosis factor alpha (tnfα ) could regulate cell proliferation and death, and the inhibition of nuclear factor kappa-b (nf-κb) makes tnfα bias to cell death. tnfα-induced ros could support c-jun n-terminal kinase (jnk) activation during nf-κb inhibition. the sustained jnk activation enables cytochrome c release and leads to necrotic cell death [ ] . the homeostasis of ros plays an important role in reducing oxidative damage and fulfil energy demand. ros present as signalling molecules in multiple pathways and mechanisms. thus, they are inevitably influenced by proteins and genes involved within. apart from that, other environmental complexes and antioxidants could contribute to ros production according to their redox potential. it is also noted that different mtdna haplotypes may have distinct respiration capacity triggered by varying production of ros [ ] ( table ) . relatively high levels of ros may cause oxidative damage or induce apoptosis during immunological defences or pathological conditions. the mechanisms to survive under such environment are essential for body cells or tumour cells and bacteria. hypoxia inducible factor- alpha (hif- α ) encoded by endothelial pas domain protein (epas ) gene could control ros level in mitochondria through antioxidant enzymes and maintain ros homeostasis [ ] . pparγ coactivator α (pgc- α) is required for antioxidative enzymes including glutathione peroxidase (gpx ) and superoxide dismutase (sod ) [ ] . ros level also could be controlled through degradation of nox on endoplasmic reticulum by protein negative regulator of ros (nrros). this reduces tissue damage and maintains its function upon immunological defence [ , ] . however, ros themselves could activate extracellular signal-regulated kinase (erk) by targeting proteins gαi and gα and protect cardiac cell from oxidative damage [ ] . these proteins present protective effect on body cells and redox balance. it is also noted that the opened potassium channels may reduce ros level [ ] . ros tolerance may be partly involved in the mechanisms behind the tumour cells avoiding immunological defence. cancer cells could produce enough nadph to support vigorous proliferation while maintaining ros homeostasis through gsh (glutathione). enhanced ros in lung cancer cells could inhibit glycolytic enzyme pyruvate kinase m (pkm ). this also allows them to survive under acute oxidative stress and still supports their proliferation [ ] . nuclear factor erythroid- -related factor (nrf ) transcription is increased in tumour cells to suppress ros generation by nrf -keap (kelch-like echassociated protein ) interaction. oncogenic alleles of k-ras, b-raf and myc could increase nrf antioxidant activity and reduce ros level [ ] . researches also indicate that gene ucp (uncoupling protein ) could limit ros production and inflammation in macrophage [ ] . apart from that, antioxidants also include organics like vitamin e, vitamin c and complexes like fhc (ferritin heavy chain). they reduce apoptosis induced by tnfα and jnk activity through suppression of ros accumulation and iron sequestration as a downstream product of nf-κb pathway [ , ] . change cells from proliferation into differentiation ros are important particles involved in immunological defence. overexpressed tnf induces ros in mitochondria through rip -rip -dependent (receptor-interacting protein kinase) pathways. the increased ros leads to both enhanced macrophages killing and necroptosis. this necroptosis relies on mitochondrial cyclophilin d and ceramide [ ] . tlr / / could enhance ros by recruiting mitochondria to macrophage phagosomes and translocating traf to mitochondria to engage ecsit. this further increases the bacterial killing [ ] . ros induces oxidative damage and apoptosis which may contribute to the control of lifespan. p shc generates mitochondrial ros as apoptosis signal through oxidation of cytochrome c in mitochondrial electron transfer chain [ ] . other factors such as matrix metalloproteinase- (mmp- ) could increase cellular ros and stimulate transcription factor snail and epithelial-mesenchymal transition (emt). this process causes dna oxidative damage and genomic instability in breast cancer and turns normal cells into cancer cells [ ] . heart cell stretch could activate nox to produce ros through ryanodine receptors and trigger ca + sparks [ ] . the deficiency of nox inhibitor nrros could lead to elevated oxidative damage. [ ] erythroblastosis virus transcription factor- (ets- ) requires ros to regulate p phox expression. however, this also could contribute to nadph oxidase and ros generation and become an ets-ros positive feedback [ ] . the ros-induced ros-release circle could lead to elevated ros generation as well [ ] . transcription factor upbeat could regulate the balance between cellular proliferation and differentiation through ros. vigorous changes in metabolism may occur during the shift from cell elongation to differentiation to fulfil metabolic demands. upbeat enhances ros level through the repression of peroxidases which could change the pattern of cell from proliferation into differentiation. [ ] . researchers have been trying to elucidate the mechanisms and the role that ros plays in diseases since they were identified. ros influences diseases basically with its function as signalling molecules and oxidants that influence cell survival and oxidative damage. ros could also drive immunity through immunological defence and maintain metabolic balance or heat dissolving. the multiple functions of ros in biosystem may influence each pathema from different aspects ( table ). abnormal cell proliferation and metastasis are common features of cancer. vigorous proliferation demands substantial nadph to produce energy. this process also abundantly increases ros. high levels of ros could induce apoptosis of tumour cells. however, they also protect cells from oxidative damage by suppressing glycolytic enzyme pkm through gsh in cancer [ ] . tumour cells exhibit enhanced nrf transcription. nrf present as antioxidants that control ros level in cancer. the inhibition of nrf could impair tumourigenesis with increased ros level [ ] . oncogenic alleles k-ras, b-raf and myc could contribute to nrf -keap interaction. ros also regulate tumour suppressor protein p and mediate apoptosis in cancer [ ] . stem cells tend to contain lower ros than regularly differentiated cells. cancer stem cells also maintain low levels of ros to avoid apoptosis induced by ros. it also makes them suffer less dna damage from radiation with enhanced ros scavenging systems [ ] . cancer cells resistant to braf and mek inhibitors develop vulnerability to high levels of ros [ ] . thus, the strategy to enhance ros level may seem to present as an important way for cancer chemotherapy. however, researchers also indicated that tumour cells with high metastasis contain nadh gene mutation. the mutation causes deficiency in respiratory complex i and ended up in overproduction of ros, and the metastatic activity could be suppressed with ros scavenger [ ] . increased ros generation could trigger enhanced epidermal growth factor receptor (egfr) signalling and promote tumour progression [ ] . snail and emt stimulated by mmp- could increase ros generation. the elevated ros level could turn normal cells into cancer cells with dna damage and genomic variation. [ ] . ros could also mediate the tumour microenvironment through epithelial-mesenchymal transition that contributes to radioresistance and therapeutic failure [ ] . although suppressing ros signalling to inhibit tumour growth with ros scavenger is not ideal, the process of inhibition impairs ros-mediated oxidative damage and apoptosis [ ] . the uninhibited ros generation and uncontrolled ros level could also promote cancer cell metastasis and the process of canceration. these controversial results bring about hindered exploration of ros-mediated treatments against cancer. rather than focusing on symptoms, the fundamental role of ros and its comprehensive distribution among biosystem may explain these competing results from a broader perspective. ros are highly related with energy production rather than just byproducts. cancer development and tumour metastasis demand larger amounts of energy than normal cells. this energy-acquiring process also produces high levels of ros. the enhancement of ros may also increase energy production to facilitate tumourigenesis. rather than a regulator of the cancer pathology, ros are more likely the representative of energy consumption. it is easy to induce cancer cells death in vitro with oxidative damage. however, the failure to apply oxidative damage to cancer cells clinically seems to be the result of ros homeostasis system in vivo. being part of the mechanisms involved in innate immunity, inflammation eliminates pathogenic factors while causing tissue damage. ros play a similar role in immunity by enhancing immunological defence and causing oxidative damage. nlr family, pyrin domain-containing (nlrp ) inflammasome could enhance inflammation by activating caspase and promoting secretion of il- β and il- . ros are crucial for nlrp activation [ ] . drosophila multipotent haematopoietic progenitors present relatively high levels of ros in in vivo physiological conditions and become low during differentiation. the enhanced ros could promote the differentiation through jnk and the forkhead box o (foxo) pathway, but ros inhibition disabled its differentiation [ ] . although the differentiation prefers low levels of ros, they are still essential for the process. different t-cell subsets also have distinct sensitivity to ros level that may influence their development and function. th cells are involved in autoimmune diseases and inflammatory diseases. experimental autoimmune encephalomyelitis (eae) is a th -mediated autoimmune disease. regulating th cell differentiation by interfering ros level through glutathione metabolism could prevent eae development [ ] . influencing chromatin structure with gls inhibition also enhances ros level and prevents th differentiation [ ] . discovery of brand new t-cell subsets also endows deeper understanding on immune system and provides aspects for the exploration of t-cellregulated autoimmune diseases [ ] . ros could support immune system, but they become cytotoxic while overload [ ] . ros play a role in both activation-induced t-cell death and activated t-cell autonomous death [ ] . oxidative damage leads to cellular damage on dna, protein and lipids. the damage-induced apoptosis plays an important role in inflammatory bowel diseases [ ] . ros also stimulate parasite growth and cause tissue damage to host's organs [ ] . the expression of nadph oxidase is elevated in phagocytic leukocytes upon stimuli. [ ] the ros-mediated autophagy could promote periodontitis and tendinopathy as well [ ] . apart from oxidative damage, ros also serve as signalling molecules and play an important role in homeostasis, metabolism, growth and differentiation [ ] . ucp could limit ros production and inflammation in macrophage and reduce parasitic cysts [ ] . however, ucp relies on ros level required for heat dissipation through thermogenic respiration in brown adipose tissues. the depletion of ros inhibits ucp and heat generation [ ] . cigarette smoking induces oxidative stress in bronchitis and emphysema. inflammation also occurs in these chronic obstructive pulmonary diseases [ ] . the role of ros in bacterial killing appears to be inconsistent among different studies. some research state that increased ros level in bacteria can enhance the killing ability of antibiotics and oxidants [ ] . enhanced ros by excess tnf through rip -rip -dependent pathways in mitochondria lead to both enhanced macrophages killing and necroptosis that relies on mitochondrial cyclophilin d and ceramide [ ] . tnf-α is indicated to contribute to ros production in rheumatoid arthritis [ ] . however, other studies indicate that ros response during bacterial antibiotic killing is dispensable [ ] . ros scavenger and hydroxyl radical inhibitor could suppress antibiotic bacterial killing. antibiotic bacterial killing does not strictly depend on ros [ ] . it is also noted that the level of ros does not influence antibiotics' activity on killing bacteria at all [ ] . antibiotic killing of escherichia coli does not rely on ros [ ] . the enhanced bacterial killing with increased ros level may due to increased metabolism and energy supply that support oxidation and immunity system. however, it applies little effect when they reaches saturation. but moderated metabolism with lower levels of ros surely decreases the ability of bacterial killing. neurons are important cells that control sensory organs and muscle system. the injury of these cells may lead to neuropathy and movement disorder. the relatively low antioxidant activity makes them vulnerable to oxidative damage. the defects in mitochondria may enhance ros generation and thus promote jnk and sterol-regulatory element binding proteins (srebp) activation in neurons that results in neurodegeneration through the accumulation of lipid droplets [ ] . the adipogenesis could also be influenced by ros via signal transducers and activators of transcription (stat ) [ ] . however, antioxidants could rescue the apoptosis [ ] . fhc could suppress ros accumulation and jnk activity through iron sequestration that inhibits tnf-α-dependent apoptosis [ ] . pgc- α could protect neural cells from oxidative damage by reducing ros level via antioxidative enzymes gpx and sod [ ] . methylmercury and manganese could induce neurotoxicity with enhanced ros level [ , ] . and the increased level of ros in the substantia nigra pars compacta leads to neuronal apoptosis of dopaminergic neurons in down syndrome and parkinson's disease. this process may ultimately lead to retardation [ ] . nrros could protect central nervous system from eae by reducing oxidative damage through nox degradation on endoplasmic reticulum [ ] . nevertheless, ros still play an important role in neuronal development and are essential for synaptic plasticity and memory formation with its fundamental role in energy perfusion. the essence that neurons are differentiated cells that lack the potential to proliferate explained these competing results of antioxidative strategies. they maintain a relatively low demand in energy and metabolism. in the heart, angiotensin ii, norepinephrine and tnf-α mediated ros are related with cardiac hypertrophy, myocardial infarction and heart failure. myocardial ischaemia is the most common cause of heart failure. the ischaemia-reperfusion injury leads to apoptosis of cardiomyocytes that is associated with high levels of ros [ ] . the shortage of atp during ischaemia impairs ion pump and causes calcium accumulation. calcium overload and increased ros could rupture plasma membrane and lead to cell death [ ] . cardiac hypertrophy is a compensating process that enables heart to maintain sufficient function. the increased ros during the process is responsive to energy demand caused by insufficient heart function. thioredoxin could reduce cardiac hypertrophy through heat shock protein and class ii histone deacetylases, the latter being a master negative regulator of cardiac hypertrophy [ ] . and it is also noted that ros increased via d-amino acid oxidase in the hearts of rats could directly lead to systolic heart failure without cardiac hypertrophy [ ] . the oxidative damage-mediated apoptosis is the major cause of heart failure as well. the method to fulfil energy demand by using nox to protect heart from failure with improved myocardial energetics via fatty acid oxidation is also proved to be successful [ ] . to reduce oxidative damage, ubia prenyltransferase domain-containing protein presents cardiovascular protective function via antioxidant coenzyme q [ ] . however, the inability to recover from cardiac damage and pathology is also critical for heart failure. postnatal cardiomyocyte cell-cycle arrest is mediated by ros through dna damage response [ ] . heart cell stretch could cause arrhythmogenic ca + sparks based on microtubules [ ] . although the oxidative damage caused by ros is the major reason for heart failure, the role of ros in energy supply is rather important that protect heart from an even sudden failure of insufficient function. ros regulate vascular cell proliferation and apoptosis with their fundamental role in metabolism. oxidative stress could lead to hypertension and promote its pathological process. however, ros are also needed for the relaxation of cerebral arteries [ ] . ets- and angiotensin ii-generated ros play an important role in vascular changes and injury, and no could regulate blood flow homeostasis in blood vessels [ ] . these outcomes seem to be confusing to tell whether ros are beneficial or harmful. the role of ros in biosystem is rather neutral that they mainly respond to energy demand. nox family could influence the neovascularity of tumour and physiological vascular processes [ ] . similar results also presented in ros-mediated wound repair [ ] . ischaemia-reperfusion (ir) causes oxidative damage with increased generation of ros in mitochondria. succinate is the metabolic signature of ischaemia and responsible for ros generation during reperfusion. the reperfusion injury also leads to retinal dysfunction-associated ros production when the blood pressure is low [ ] . succinate accumulates during reperfusion from fumarate overproduction and malate/aspartate shuttle and then re-oxidized by succinate dehydrogenase. this process increases ros generation through reverse electron transport in mitochondria. the inhibition of succinate or ros could ameliorate ir injury [ ] . pre-conditioning protocols could reduce ischaemia-reperfusion injury by regulating ros level [ ] . atherosclerosis could be regulated by ros interacting with transcription factors related with lipid peroxidation and macrophage [ ] . ros induces dna damage and lipid peroxidation in pneumoconiosis and carcinogenesis as well [ ] . increased ros promote thrombus formation in artery and influence other cardiovascular diseases as well [ , ] . the pulmonary vascular lesions and inflammation are broadly recognized pathological changes in acute respiratory distress syndrome (ards) caused by oxidative damage [ ] . taken together, researchers revealed the position of ros in metabolism and energy supply. ros are needed for basic energy demand and vigorous metabolism rather than simply affecting cellular signalling and organism damages. the continuous oxidative damage applied on cell and tissue may lead to severe organic injuries and eventually cause organ failure. the ros level leading to organ failures far exceeds the extent to maintain basic metabolism and thus the balance between energy supply and oxidative damage is tilted. increasing ros grants little beneficial effect in this situation. inhibition of ros could reduce tnf-α-mediated fulminant liver failure. tnfα regulates cell proliferation and death and the inhibition of nf-κb makes tnfα bias to cell death. tnfα-induced ros supports jnk activation during nf-κb inhibition. sustained jnk activation enables cytochrome c release and leads to necrotic cell death [ ] . fhc is a downstream product of nf-κb. they could reduce apoptosis induced by tnfα through suppression of ros accumulation and jnk activity. the suppression of ros is achieved by iron sequestration [ ] . ros are produced by glomerular cells as autacoids [ ] . ros-mediated glomerular basement membrane degradation and altered cell function may contribute to ischaemic renal failure as well [ ] . and the inhibition of ros could decrease caox stone in kidney [ ] . hypoxia-induced requirement of energy supply and metabolism could lead to increased ros response through ca + influx pathway. this mechanism results in physiological, biochemical and molecular changes. the hypoxia-induced ros production is important for respiratory plasticity and sensory plasticity. ros-mediated apoptosis and cellular dysfunction are associated with heart failure [ ] . the arrhythmias caused by elevated ros and altered mitochondrial function may lead to sudden cardiac death [ ] . the comprehensive distribution of ros intrigues researchers to explore the relationship between their subject and ros. the reduced ros level could lower insulin resistance and improve insulin sensitivity in diabetes ii [ ] , and the glucose-stimulated insulin relies on ros signalling [ ] . however, the cellular death owing to ros-mediated oxidative damage also brings about diabetic complications [ ] . mmp activity and transcription factor-β (tgf-β )-induced excessive deposition of extracellular matrix mediated by ros could lead to renal fibrosis [ ] . the process of ageing caused by oxidative damage and muscle dysfunction could lead to sarcopenia. however, ros are also essential for muscle cell development as signalling molecules [ ] . generation of ros in skeletal muscle is enhanced during contractile activity [ ] . ros are increased in the early stage of muscular dystrophy development [ ] . the elevated ros may reduce muscle mass and bring about frailty [ ] . however, ros also plays an important role in muscle remodelling as signalling molecules [ ] . overproduced ros released through mitochondrial permeability transition pore will damage dna and accelerate ageing by reducing cellular nad [ ] . apart from suppressing tumour, p also plays a role in premature ageing by causing reactive damage to dna [ ] . mushroom-contained antioxidants may protect against oxidative damage and ageing [ ] . ros overproduction may contribute to reproductivity issue and infertility through oxidative damage and disturbed hormone balance. the excessive ros may damage spermatogenesis, sperm lipid/protein layer and dna structure. the ros scavenging system to reduce ovarian toxicity is important for follicular development [ ] . the role of ros seems to be similar in different diseases that they are essential for cellular metabolism and become pathogenic while overload. researches of ros have been carried out since last century, the cognition of which varies along elapse of time. ros promote macrophage bacterial killing through oxidative damage and apoptosis. they also engage in multiple cellular pathways as signalling molecules. however, they also have negative effects like inflammation and cytotoxicity. ros can function as intermediates in varying pathways, but they are also widely regarded as etiologic factors for diseases including cancer, inflammation and organ injuries. evidence suggests that the scavenging ros in pathological condition may reduce cell damage and control the pathological process. however, other researches also indicate the positive side of enhancing ros in diseases. thus, the mechanisms of how ros influences diseases remain obscure. europeans have dedicated to the study of ros and made a comprehensive exploration [ ] . yet, it remains controversial in results of ros-targeted strategies applied to clinical research. with the advancement in technique, ros can be measured in living cells of its transient generation with y . eu . vo nanoparticles by illuminating under oxidative conditions [ ] . specific chemical probes and low-temperature electron paramagnetic resonance (epr) technique could monitor ros level of tumour cells in vitro and in vivo [ ] . other environmental factors like ph and ion concentration are also suggested in ros regulation and generation [ ] . also magnetic fields could influence cellular ros level according to its intensity, frequency and exposure time [ ] . the fundamental understandings of ros remain basically the same in the past years. ros are generally regarded as signalling molecules and harmful particles. researchers always focus on ros levels and their results and analyses them from a single perspective. they should be illuminated from different perspectives and with extensive sight. based upon the characteristics of ros discovered by previous researches, i provide a hypothesis here that may explain the competing results so far. ros thrives in conditions that abundant energy is in demand for vigorous metabolism, either in cancer cells' proliferation and inflammatory necrosis, or immunological defence required immune system functioning. thus, ros do not just act as signalling molecules. they may present as basic energy particles like other acknowledged basic nutrient particles including proteins and carbohydrates. and they provide a much more fundamental impact on cellular metabolism. it is more likely that ros respond to elevated metabolism to fulfil energy demand, rather than directly bending itself to oxidative stress, which interprets why different researches demonstrate controversial outcome in regulating ros. the treatment of both inhibition and enhancement of ros in cancer in vitro may due to exhausted energy supply for metabolism and overload of energy supply-induced oxidative damage, rather than just the regulation of a byproduct. of course, it is easier to suppress ros generation with antioxidants or genetic depletion in experimental animal models. but the inability of clinically gene modulation in vivo and the multiple functions of ros in metabolism may lead to limited potrential of direct ros modulation in diseases with ros and metabolic imbalance. the fundamental role of ros grants them with the potential in metabolic regulation. from another aspect, the essence that ros are common particles with comprehensive distribution endows them with more fundamental mechanisms to be explored. the nox family of ros-generating nadph oxidases: 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tnfalpha-induced apoptosis by suppressing reactive oxygen species tnf dually mediates resistance and susceptibility to mycobacteria via mitochondrial reactive oxygen species bissell mj ( ) rac b and reactive oxygen species mediate mmp- -induced emt and genomic instability transcription factor ets- and reactive oxygen species: role in vascular and renal injury mitochondrial rosinduced ros release: an update and review transcriptional regulation of ros controls transition from proliferation to differentiation in the root ros and p : a versatile partnership association of reactive oxygen species levels and radioresistance in cancer stem cells lethally high ros levels thwart resistance the interplay of reactive oxygen species and the epidermal growth factor receptor in tumor progression and drug resistance reactive oxygen species-mediated tumor microenvironment transformation: the mechanism of radioresistant gastric cancer targeting cancer cells by ros-mediated mechanisms: a radical therapeutic approach? nlrp inflammasome activation: the convergence of multiple signalling pathways on ros production? reactive oxygen species prime drosophila haematopoietic progenitors for differentiation glutathione de novo synthesis but not recycling process coordinates with glutamine catabolism to control redox homeostasis and directs murine t cell differentiation distinct regulation of th and th cell differentiation by glutaminase-dependent metabolism a cd (+) t cell population expanded in lupus blood provides b cell help through interleukin- and succinate t cell apoptosis and reactive oxygen species reactive oxygen species and antioxidant defense in human gastrointestinal diseases ros and trypanosoma cruzi: fuel to infection, poison to the heart reactive oxygen species in phagocytic leukocytes sudden death risk in older athletes: increasing the denominator mitochondrial ros regulate thermogenic energy expenditure and sulfenylation of ucp reactive oxygen species in chronic obstructive pulmonary disease potentiating antibacterial activity by predictably enhancing endogenous microbial ros production the role of reactive oxygen species in immunopathogenesis of rheumatoid arthritis fe-s cluster biosynthesis controls uptake of aminoglycosides in a ros-less death pathway antibiotic and ros linkage questioned killing by bactericidal antibiotics does not depend on reactive oygen species cell death from antibiotics without the involvement of reactive oxygen species glial lipid droplets and ros induced by mitochondrial defects promote neurodegeneration mitochondrial stat and reactive oxygen species: a fulcrum of adipogenesis? jak-stat apoptosis and increased generation of reactive oxygen species in down's syndrome neurons in vitro involvement of glutamate and reactive oxygen species in methylmercury neurotoxicity manganese neurotoxicity and the role of reactive oxygen species nitric oxide and reactive oxygen species in parkinson's disease reactive oxygen species, mitochondria, and nad(p)h oxidases in the development and progression of heart failure a redox-dependent pathway for regulating class ii hdacs and cardiac hypertrophy chemogenetic generation of hydrogen peroxide in the heart induces severe cardiac dysfunction cardiac-targeted nadph oxidase in the adaptive cardiac remodelling of the murine heart the oxygen-rich postnatal environment induces cardiomyocyte cellcycle arrest through dna damage response mechanisms underlying acute protection from cardiac ischemia-reperfusion injury ubiad is an antioxidant enzyme that regulates enos activity by coq synthesis reactive oxygen species: influence on cerebral vascular tone sources of vascular nitric oxide and reactive oxygen species and their regulation nadph oxidase(s): new source(s) of reactive oxygen species in the vascular system? reactive oxygen species and nox enzymes are emerging as key players in cutaneous wound repair reactive oxygen species, oxidative stress, glaucoma and hyperbaric 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oxidative stress and nrf in pancreatic beta-cell function the role of reactive oxygen species in apoptosis of the diabetic kidney reactive oxygen species and matrix remodeling in diabetic kidney reactive oxygen species as mediators of cellular senescence reactive oxygen species and redox-regulation of skeletal muscle adaptations to exercise absence of dystrophin disrupts skeletal muscle signaling: roles of ca + , reactive oxygen species, and nitric oxide in the development of muscular dystrophy control of reactive oxygen species production in contracting skeletal muscle reactive oxygen species are signalling molecules for skeletal muscle adaptation role of reactive oxygen species-mediated signaling in aging ros and senescence in the control of aging reactive oxygen species and antioxidant properties from mushrooms reactive oxygen species and sperm cells single europium-doped nanoparticles measure temporal pattern of reactive oxygen species production inside cells teaching the basics of reactive oxygen species and their relevance to cancer biology: mitochondrial reactive oxygen species detection, redox signaling, and targeted therapies extra-cellular but extraordinarily important for cells: apoplastic reactive oxygen species metabolism magnetic fields and reactive oxygen species publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors declare that they have no conflict of interest. key: cord- -ot es zd authors: li, yating; liu, chenxia; xiao, wei; song, tiantian; wang, shuhui title: incidence, risk factors, and outcomes of ventilator-associated pneumonia in traumatic brain injury: a meta-analysis date: - - journal: neurocrit care doi: . /s - - -w sha: doc_id: cord_uid: ot es zd ventilator-associated pneumonia (vap) is one of the most severe complications in patients with traumatic brain injury (tbi) and is considered a risk factor for poor outcomes. however, the incidence of vap among patients with tbi reported in studies varies widely. what is more, the risk factors and outcomes of vap are controversial. this study estimates the incidence, risk factors, and outcomes of vap in patients with tbi and provides evidence for prevention and treatment. pubmed, embase, cochrane library, and web of science databases were searched from the earliest records to may . data involving the incidence, risk factors, and outcomes were extracted for meta-analysis. the results showed that the incidence of vap was % ( % confidence interval (ci) – %); risk factors analyses showed that smoking [odds ratio (or) . ; % ci . – . ], tracheostomy (or . ; % ci . – . ), blood transfusion on admission (or . ; % ci . – . ), barbiturate infusion (or . ; % ci . – . ), injury severity score (or . ; % ci . – . ), and head abbreviated injury scale (or . ; % ci . – . ) were related to the occurrence of vap. when patients developed vap, mechanical ventilation time (or . ; % ci . – . ), icu length of stay (or . ; % ci . – . ), and hospital length of stay (or . ; % ci . – . ) were significantly increased. however, vap was not associated with an increased risk of mortality (or . ; % ci . – . ). vap is common in patients with tbi. it is affected by a series of factors and has a poor prognosis. electronic supplementary material: the online version of this article ( . /s - - -w) contains supplementary material, which is available to authorized users. traumatic brain injury (tbi) is brain damage caused by external force. it is the main cause of death and disability among trauma patients [ ] . it is estimated that , people die each year from tbi in the united states and more than . million americans are currently disabled for tbi [ ] . patients who have undergone tbi often suffer from profound suppression of the cellular immune system and impaired consciousness [ ] , and they usually require tracheal intubation and ventilator support [ ] , both of which increased the incidence of ventilator-associated pneumonia (vap). vap is a type of nosocomial pneumonia developing h or more after receiving mechanical ventilation. studies have shown that vap can lead to increased mortality and morbidity [ ] . in addition, when patients suffered from vap, the medical expenses increase [ ] . the incidence of vap among patients with tbi reported in studies varies widely, ranging from to % [ ] [ ] [ ] . in addition, previous studies have demonstrated that the risk factors for vap among patients with tbi include smoking, higher injury severity score (iss), tracheostomy, diabetes, and so on [ ] [ ] [ ] [ ] [ ] . however, these studies did not provide consistent evidence for prevention and treatment due to many reasons, such as small sample sizes, different population sources, and the use of different study designs [ , ] . what is more, some studies indicated that vap increased the risk of mortality [ , ] , but other studies showed that vap was not associated with mortality [ , ] . identification of risk factors and outcomes associated with vap could permit clinical staff to provide close infection surveillance and timely care for tbi patients at high risk for pneumonia. therefore, a systematic summary is urgently needed. in the present study, we performed a meta-analysis of the incidence, risk factors, and outcomes of vap in patients with tbi, so as to provide evidence for the prevention and treatment of vap among patients with tbi. a literature search was performed using pubmed, embase, cochrane library, and web of science databases up to may . we used medical subject headings and keywords as follows: (traumatic brain injury or brain injuries, traumatic or brain injury or tbi or craniocerebral trauma or craniocerebral injury or head injury or head trauma) and (ventilator-associated pneumonia or vap or pneumonia, ventilator associated or ventilator associated pneumonia). the specific searching strategy is described in table s . in the initial selection phase, two reviewers (y.l. and c.l.) screened the title and abstract of studies independently. studies that obviously did not meet the inclusion criteria were excluded, and those included articles were read in full text to confirm their eligibility. any disagreement was resolved by discussion with a third reviewer (s.w.). reference lists of relevant studies were also manually searched for further studies. the publication language of studies was not limited. our search strategy was in accordance with the preferred reporting items for systematic reviews and meta-analysis guidelines [ ] , the prisma checklist is described in table s . the inclusion criteria for our study were: ( ) all enrolled patients had undergone tbi and had mechanical ventilation for more than h. ( ) a clear definition and diagnosis of vap. ( ) articles must provide one or more index for the incidence, risk factors, or outcomes of vap. ( ) cohort study or case-control study. ( ) full text available. ( ) when multiple publications contain duplicate results, the study with the largest population was included. two people extract data from eligible articles independently, including study characteristics (first author, publication time, country, number of patients, number of vap, gender, age, study design, the definition of vap, the severity of tbi), incidence, risk factors, and indexes representing clinical outcomes (mortality, mechanical ventilation time, intensive care unit (icu) length of stay, hospital length of stay), so as to provide a comprehensive description of vap among patients with tbi. some studies were presenting continuous data (ventilator days, icu length of stay, hospital length of stay, iss [ ] ) using the mean and standard deviation or using the median and the first and third quartiles. to be able to use these data in our meta-analysis, we converted the median and the first and third quartiles to the mean and standard deviation using the method described by wan et al. [ ] . the quality of the studies was evaluated by two independent reviewers (y.l. and c.l.) using the newcastle-ottawa scale (nos) [ ] , which was used for cohort designs and case-control designs. the quality of the studies was evaluated based on three perspectives: selection (four items), comparability (one item), and exposure or outcome (three items). the nos rating system with a score ranging from to stars. high-quality studies should reach seven stars or more, - stars are of medium quality, and less than four stars are of poor quality. the incidence of vap among patients with tbi was calculated using stata . (stata corp, college station, tx); subgroup analyses in terms of study design, region, the definition of vap, and the severity of tbi were also performed. risk factors and outcomes of vap in patients with tbi were analyzed using the review manager software (revman . ; cochrane collaboration, oxford, uk). for measurement data, the weighted mean difference and % confidence intervals (cis) were calculated to compare the mean difference. for count data, odds ratios (ors) and % cis were used. they were considered significantly associated with vap when p < . . heterogeneity was calculated using i statistic. heterogeneity was considered to be present if i > % and random effects models were used; otherwise, a fixed effects model was used. publication bias was performed by begg's and egger's test using stata . [ , ] ; a p value > . indicating the lack of publication bias. a total of records were identified from the databases (pubmed: , embase: , cochrane library: , web of science ) and manually searched. after duplicates were removed, records were retained. after initial screening of article titles and abstracts, records were excluded. then, records were retained for further screening, among which, there were four reviews and three case reports; four studies were not having a useful index for the description of vap, and five studies were not cohort studies or case-control studies. not all the patients enrolled in seven studies were with tbi. finally, articles were included in this meta-analysis (fig. ) . a total of articles were included in this meta-analysis. the characteristics of the included studies are summarized in table . six studies were cohort studies, and nine studies were case-control studies. according to the nos, two articles got eight stars, five articles got seven stars, five articles got six stars, and three articles got five stars ( table ) . the pooled incidence of vap was % ( % ci - %) among patients with tbi, as shown in fig. a . we performed a subgroup analysis by study design (prospective and retrospective) to explore its impact on vap incidence (fig. b) ; the forest plot illustrated that the incidence of vap in prospective studies ( %) was relatively higher than their counterparts in retrospective studies ( %). stratified analysis based on variation in the region was applied because of its potential effect on the incidence of vap (fig. c) , and the results showed that the incidence of vap in north america ( %) and europe ( %) was relatively higher than asia ( %). we also performed a subgroup analysis by definition of vap (fig. d) , and the results showed the incidence of vap in centers for disease control (cdc) criteria [ ] , american thoracic society criteria [ ] , chinese respiratory society criteria [ ] with %, %, %, respectively. considering the impact of tbi severity on the incidence of vap, we performed a subgroup analysis based on the severity of tbi (fig. e) , and the result showed that the incidences of vap in severe tbi patients (defined according to the standards used in the original study, such as glasgow fig. flow diagram of the study selection process coma score (gcs) ≤ , the presence of clinical or radiographic herniation, etc.) and all tbi patients were similar, with % and %, respectively. in addition to the overall incidence of vap, some studies divided vap into earlyonset ventilator-associated pneumonia (eovap) and late-onset ventilator-associated pneumonia (lovap). cdc centers for disease control and prevention, eovap early-onset ventilator-associated pneumonia, lovap late-onset ventilator-associated pneumonia, tbi traumatic brain injury, vap ventilator-associated pneumonia *values are represented as the mean ± standard derivation; † values are represented as the median (interquartile range); a age distribution of patients in both groups (vap and non-vap); b gender distribution of patients in both groups (vap and non-vap) country number of patients the results showed that the incidences of eovap (occurring in the first - days of mechanical ventilation) and lovap (occurring after - days of mechanical ventilation) were % and %, respectively ( fig. f, g) . there are potential risk factors (reported in at least two studies) summarized in this meta-analysis. we found a significant relationship between vap and the following risk factors: smoking, tracheostomy, blood transfusion on admission, barbiturate infusion, iss, head abbreviated injury scale (ais), as noted in case-control studies trauma; therefore, these factors were determined not to be risk factors for vap. the forest plots of risk factors are described in figure s . after data integration of ten studies, pooled results showed that vap was not associated with mortality (or . ; % ci . - . ; p = . ), as shown in fig. a . however, vap increased the mechanical ventilation time (or . ; % ci . - . ; p< . ), as shown in fig. b . in addition, our pooled results also showed that icu length of stay was increased significantly in patients infected with vap (or . ; % ci . - . ; p< . ), as shown in fig. c . patients who developed vap also had a longer duration of hospital length of stay (or . ; % ci . - . ; p< . ), as shown in fig. d . there was no significant publication bias in the analysis of the incidence of vap by egger's test (p = . ) and begg's test (p = . ). the results of publication bias for risk factor analysis are shown in table to our best knowledge, this study was the first metaanalysis exploring the incidence, risk factors, and outcomes of vap in patients with tbi. this meta-analysis showed that the incidence of vap in patients with tbi was % ( % ci - %) by pooling data from the included studies. however, the spectacular disparity in the incidence of vap was shown in the previous studies. bronchard et al. [ ] and jovanovic et al. [ ] reported that the incidence of vap was %, while rincón-ferrari et al. [ ] found that the incidence of vap was about %. differences in the study design, region, definition of vap, and the severity of tbi may explain this wide range, leading to subgroup analyses based on these factors. the result demonstrated that the incidence of vap in prospective studies was significantly higher than retrospective studies ( % vs. %). a study performed by j tobias et al. showed that data obtained retrospectively from the medical records were less complete and accurate compared to data obtained from the patients prospectively [ ] . retrospective information is fraught with the problems of missing data, conflicting data, and illegibility [ ] . we recommend that more prospective studies be carried out in the future so as to obtain more rigorous results about vap. in addition, there was a significant difference in the incidence of vap between north america, europe and asia ( % vs. % vs. %) by subgroup analysis; this difference may be due to the lack of active infection monitoring systems in asian countries compared to european and north american countries, which led to the inadequate feedback of hospital infection surveillance data and underestimate of the incidence of vap [ ] [ ] [ ] . what is more, the result showed that the incidences of vap in severe tbi patients and all tbi patients were similar, with % and %, respectively. in this meta-analysis, three studies included all tbi patients, rather than only those with severe tbi [ , , ] . most of the patients in these three studies had severe neurologic injury. the mean gcs score of patients was . in the study conducted by kallel et al. [ ] , and . in the study conducted by bronchard et al. [ ] . the median gcs score of patients was in the study conducted by leone et al. [ ] . this may explain why the incidence of vap in these three studies is similar to the studies including patients with severe tbi. additionally, as for eovap and lovap, the incidence of eovap and lovap is related to the cutoff point used for definition [ ] . however, the cutoff point in the literature is usually inconsistent [ ] . unifying eovap and lovap cutoff point is of great significance to the treatment of patients and clinical research. further research is needed to accurately assess the cutoff point. identification of risk factors associated with vap could permit clinical staff to provide close infection surveillance and timely care for tbi patients at high risk for pneumonia so as to optimize clinical outcomes. in the present meta-analysis, we integrated all eligible studies of vap in patients with tbi. by integrating the results of previous studies and expanding the sample size, we found a significant relationship between vap and the following risk factors: smoking, tracheostomy, blood transfusion on admission, barbiturate infusion, iss, and head ais. we identified that smoking increased the risk for vap, probably because smoking impairs mucociliary clearance, making it easier for pathogens to colonize in the respiratory tract [ ] . smoking remains an important contributor to pneumonia, and all patients with tbi should be encouraged to quit. additionally, tracheotomy increased the risk for vap; tracheotomy bypasses normal respiratory defense mechanisms, such as oropharynx and cilia, which contribute to the occurrence of vap [ ] . it has been reported that use of suction-above-the-cuff tracheotomy tubes reduces the incidence of vap [ ] . more attention in the details of tracheotomy care such as the use of suction-above-thecuff tracheotomy tubes [ ] may be helpful in decreasing the associated risk when tracheotomy is performed. furthermore, blood transfusion on admission heightens the risk for vap, which may be because the early period of acute severe trauma is associated with an immunosuppressive inflammatory response, and this response was increased in magnitude when patients received a transfusion of blood products [ , ] . in addition, our results showed that barbiturate use increased the risk of vap in tbi patients, which may be related to the immunosuppressive effect of barbiturates given they; promote reversible bone marrow suppression in patients with tbi [ ] and increased susceptibility to develop vap [ ] . although some treatments such as the use of barbiturates or blood transfusions are frequently indicated, they should be administered appropriately. moreover, the result of this meta-analysis demonstrated that patients with higher iss or head ais represent a specific target population for the prevention of vap. the ais provides a method for ranking and comparing injuries by severity in body regions [ ] . the iss is derived from the ais and provides a description of the overall severity of injury for patients with trauma [ ] . a study by teixeira lopes et al. [ ] analyzed the risk factors associated with post-traumatic complications in hospitalized trauma patients. the study revealed that the higher the severity of trauma, the greater the number of hospitalization complications. furthermore, the main complications presented by trauma patients were infections, especially vap and sepsis [ , ] . the results suggest that iss and ais should be considered in therapeutic decisions to help healthcare providers properly identify patients at high risk for vap and modify patient care to minimize the risk of vap. vap should be closely monitored to detect infections in the target population early and to take measures to achieve optimal results. there is much discussion about whether to use prophylactic antibiotics for tbi patients. our meta-analysis showed that there was no association between prophylactic antibiotics and the occurrence of vap. a study by reizine et al. [ ] showed that prophylactic antibiotics decreased the incidence of eovap but had no influence on lovap; additionally, they found that prophylactic antibiotics delayed the occurrence of vap. nevertheless, they suggested that antibiotic prophylaxis should not be routinely used in clinical practice because antibiotic prophylaxis has no effect on length of stay and mortality. in addition, a study by hoth et al. [ ] showed that trauma patients receiving antibiotic prophylaxis showed an increase in multi-drug-resistant bacteria. for the reasons mentioned above, we do not recommend prophylactic antibiotics in patients with tbi. the main finding of this study was that vap did not increase the mortality in patients with tbi. this was consistent with the results of bregeon's [ ] study, which ultimately showed that vap did not seem to be an independent risk factor for death. josephson's [ ] study also showed that vap does not lead to increased mortality in patients with neurovascular diseases, and he pointed out that death in patients with vap was due mainly to neurologic injury and withdrawal of care. additionally, vap increased the mechanical ventilation time and prolonged the icu length of stay and hospital length of stay. this is in agreement with the findings of many studies [ , ] , indicating that vap imposed a great burden on patients [ ] . this meta-analysis has many advantages. first of all, we conducted a systematic literature search. by integrating the results of previous studies, expanding the sample size and performing quantitative analysis, the accurate incidence of vap in patients with tbi was provided from a global perspective. at the same time, subgroup analyses were performed to identify potential factors responsible for inconsistencies in previous studies, and we found that the incidence of vap in north america ( %) and europe ( %) was relatively higher than asia ( %) and the incidence of vap in prospective studies ( %) was relatively higher than that in retrospective studies ( %). in addition, we analyzed many aspects of vap in patients with tbi including the incidence, risk factors, and outcomes so as to provide a comprehensive understanding of vap. however, some limitations of this meta-analysis should be mentioned. first, the criteria used to diagnose vap in the included studies were inconsistent and the analyzed studies used the following methods for vap ascertainment: three studies used the cdc criteria [ ] , two studies used the american thoracic society criteria [ ] , one study used the chinese respiratory society criteria [ ] , nine studies were based on clinical, radiographical and microbiological criteria to provide references for doctors' diagnosis. in addition, the cutoff points used to define eovap and lovap in the included studies were inconsistent. for example, some studies defined that vap occurring in the first days after mechanical ventilation was eovap [ ] , while some studies defined that vap occurring in the first days after mechanical ventilation was eovap [ ] . we recommend that more research about eovap and lovap be conducted in the future, so as to get more accurate knowledge about it. there are still many uncertainties in the diagnosis of vap, which needs further research. in the present study, we integrated all eligible studies of vap in patients with tbi, demonstrating that patients with tbi were at very high risk of developing vap, with a pooled incidence of %. we also found that vap does not increase the mortality in patients with tbi, but vap increases the mechanical ventilation time, icu length of stay, and hospital length of stay. moreover, this meta-analysis demonstrated that smoking, tracheostomy, blood transfusion on admission, barbiturate infusion, iss, and head ais are risk factors for vap, providing specific target population for the prevention of vap. traumatic brain injury and cognition economic evaluations in the diagnosis and management of traumatic brain injury: a systematic review and analysis of quality the injured brain: tbi, mtbi, the immune system, and infection: connecting the dots mechanical ventilation and the injured brain prevention of ventilator-associated pneumonia, mortality and all intensive care unit acquired infections by topically applied antimicrobial or antiseptic agents: a metaanalysis of randomized controlled trials in intensive care units 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head trauma patients ventilator-associated pneumonia in severe traumatic brain injury preferred reporting items for systematic reviews and meta-analyses: the prisma statement the injury severity score: a method for describing patients with multiple injuries and evaluating emergency care estimating the sample mean and standard deviation from the sample size, median, range and/or interquartile range critical evaluation of the newcastle-ottawa scale for the assessment of the quality of nonrandomized studies in meta-analyses bias in meta-analysis detected by a simple, graphical test operating characteristics of a rank correlation test for publication bias tracheal colonisation within h of intubation in patients with head trauma: risk factor for developing early-onset ventilator-associated pneumonia early onset pneumonia. anesthesiology ventilator-associated pneumonia following severe craniocerebral trauma and its clinical characteristics retrospective analysis of the risk factors and pathogens associated with early-onset ventilator-associated pneumonia in surgical-icu head-trauma patients the incidence and risk factors of ventilator-associated pneumonia in patients with severe traumatic brain injury ventilator-associated pneumonia in pediatric traumatic brain injury cdc definitions for nosocomial infections american thoracic society and the infectious diseases society of america guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcareassociated pneumonia chinese society of respiratory medicine. guidelines for the diagnosis and treatment of hospital acquired pneumonia (draft) the accuracy and completeness of data collected by prospective and retrospective methods basics of research (part ): archival data research healthcare-associated infection in developing countries: simple solutions to meet complex challenges impact of availability of guidelines and active surveillance in reducing the incidence of ventilator-associated pneumonia in europe and worldwide epidemiological approach to nosocomial infection surveillance data: the japanese nosocomial infection surveillance system early-and late-onset ventilatorassociated pneumonia acquired in the intensive care unit: comparison of risk factors respective impact of implementation of prevention strategies, colonization with multiresistant bacteria and antimicrobial use on the risk of early-and late-onset vap: an analysis of the outcomerea network risk factors for the development of chest infections in acute stroke: a systematic review an overview of mechanical ventilation in the intensive care unit tracheotomy tubes with suction above the cuff reduce the rate of ventilatorassociated pneumonia in intensive care unit patients association between gene expression biomarkers of immunosuppression and blood transfusion in severely injured polytrauma patients analysis of blood trace elements and biochemical indexes levels in severe craniocerebral trauma adults with glasgow coma scale and injury severity score barbiturate coma may promote reversible bone marrow suppression in patients with severe isolated traumatic brain injury usefulness of the abbreviated injury score and the injury severity score in comparison to the glasgow coma scale in predicting outcome after traumatic brain injury in-hospital complications in trauma patients according to injury severity risk factors for complications of traumatic injuries effects of antibiotic prophylaxis on ventilator-associated pneumonia in severe traumatic brain injury. a post hoc analysis of two trials prophylactic antibiotics adversely affect nosocomial pneumonia in trauma patients is ventilator-associated pneumonia an independent risk factor for death? ventilator-associated pneumonia in a neurologic intensive care unit does not lead to increased mortality ventilator-associated pneumonia in a tertiary paediatric intensive care unit: a -year prospective observational study outcome and attributable cost of ventilator-associated pneumonia among intensive care unit patients in a suburban medical center the authors declare that they have no conflicts of interest. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.published online: july key: cord- - dmp c authors: stillman, michael; gustafson, kristin; fried, guy w.; fried, karen; williams, steve r. title: communication with general practitioners: a survey of spinal cord injury physicians’ perspectives date: - - journal: spinal cord ser cases doi: . /s - - - sha: doc_id: cord_uid: dmp c study design: an online questionnaire. objectives: to gauge spinal cord injury (sci) specialists’ assessment of their communications with general practitioners (gps). to determine whether economic or health-care system-related factors enhance or inhibit such communication. setting: a collaboration of co-authors from a health-care system. methods: an online survey interrogating a number of aspects of communication between sci specialists and gps was developed, distributed, and made available for months. responses were analyzed for the entire cohort then according to descriptions of participants’ home nations’ economies and the type of health-care delivery systems in which they work. results: a total of responses were submitted. the majority ( %) were from nations with developed economies, a plurality ( . %) were from countries that offer universal health coverage, and half used a combination of paper and electronic health records. a majority of respondents ( . %) reported routinely communicating with their patients’ gps, but most ( . %) rated those communications as only “fair”. the most commonly listed barriers to communication with gps were lack of time ( . %) and a perceived lack of receptivity by gps ( . %). nearly all respondents ( . %) believed that the care they provide would be enhanced by improved communication with gps. participants who used electronic means of communication were more likely to communicate with gps and to describe those interactions as “positive”. conclusions: although there are a number of barriers to communication between sci specialists and gps, most sci specialists are eager for such inter-physician communication and believe it would enhance their care they deliver. many people living with spinal cord injury (sci) receive care from both sci specialists and general practitioners (gps). in stillman et al.'s survey of individuals with sci, a plurality of respondents had made between and visits to both an sci specialist and a gp within the past year [ ] . in donnelly et al.'s study of how people with sci in the united states, canada, and the united kingdom utilize physician services [ ] , % of respondents had a gp, % had an sci specialist, and % had both. of note, there was substantial overlap between the medical concerns participants would bring to their sci specialists and to their gps. although people with sci may turn to their gps for care, many generalists may be inadequately prepared to assist them. first, gps' offices are frequently inaccessible to people with sci. in stillman et al. [ ] , % of subjects with sci reported having faced an accessibility barrier while seeking primary care and the majority were encountered inside examination rooms. in the study by donnelly et al. [ ] , only % of respondents were able to use all the equipment in their gp's office. second, many gps lack specific training in the care of people with sci and other complex disabilities. in premo et al.'s [ ] survey of physicians in california, only % reported having received education pertaining to the care of such individuals, whereas in holder et al.'s [ ] study of medical students and attending physicians, the majority felt unprepared to treat their patients with disabilities. this oversight in medical training has real clinical consequences. among stillman et al.'s respondents, only % believed that their gps understood their disability-specific concerns "well or very well" [ ] . given persistent challenges surrounding gps' office accessibility and medical training, it is not surprising that some people with sci turn to sci specialists for attention to their general and preventative health concerns. in johnston et al.'s [ ] survey of men with sci, % considered their physiatrist to be their "main doctor type", and in donnelly et al. [ ] , % of respondents had approached their sci specialist for an annual physical examination. despite this phenomenon, however, some sci specialists feel uncomfortable providing primary care. in the only study to examine physiatrists' attitudes toward serving as gps [ ] , only % believed that they ought to be gps for people with disabilities or that they had the requisite knowledge to do so. with gps being sub-optimally prepared to care for people with sci and with sci specialists being reluctant to serve as gps, communication between the two seems essential. however, this specific form of inter-professional collaboration has remained essentially uninvestigated. in this study, we distributed a survey assessing sci specialists' interest in and assessment of their communication with gps, and whether these communications are influenced by the economies and the health-care systems in which they work. although our findings are preliminary, we hope they will serve as the basis for further study into how to offer more comprehensive and seamless care to individuals living with sci. our online survey was developed and distributed (from november of through february of ) to an international cohort of physicians who care for individuals with sci. the survey was developed by all five authors (four sci specialists and one gp) based on their expertise in the field then administered electronically (surveymonkey inc, san mateo, ca, www.surveymonkey.com) to their colleagues in the field of sci medicine and to members of the international spinal cord society (iscos) via its online newsletter. no identifying information was collected, and consent was implied by participants' completing the survey. no ethics approvals were obtained or considered necessary for this project. descriptive analyses were performed using chi square tests. responses were analyzed for the entire subject pool, then separated according to participants' descriptions of their home nations' economies ("developed", "transitional", or "developing"), health-care systems ("universal/ government funded", "tier-based system, basic universal healthcare with available private coverage", or "no universal health care; system of private/public coverage"), and methods of medical record keeping (paper records, electronic records, or a mix of the two). as fewer responses were submitted from participants living in countries with developing (n = ) or transitional (n = ) economies than from participants working in developed economies (n = ), the first two were combined into the category "developing nation". to bring clarity to other analyses, certain groups and answers were combined, and this is noted in the results. eighty-eight physicians responded to the survey. the majority ( %) were from nations with developed economies, . % were from countries with universal health coverage ( . % from tiered systems with basic universal coverage and . % from systems without universal coverage), and % used a mix of paper and electronic health records (ehr) in their practices ( . % used exclusively electronic records and . % used paper only). just over % reported that their patients with sci also see gps ( . % replied "no" and % that they did not know), a plurality ( . %) felt comfortable serving as a gp for their patients with sci ( . % replied "no" and . % replied "not sure"), and whereas . % routinely communicated with their patients' gps, the majority of them ( . %) rated those communications as only "fair" ( . % "excellent", . % "good", and . % "poor"). nearly all respondents ( . %) felt that the care they provided to their patients with sci would be enhanced by improved communication with gps. among the participants who routinely communicated with their patients' gps (n = ), . % reported primarily corresponding by mail or fax and . % by phone ( . % through ehr, . % in person, and . % through other secure messaging systems). when these same respondents were asked how their patients' gps communicate with them, . % replied "by phone", % "by mail or fax", and . % that their gp colleagues do not communicate with them. when asked to identify the strongest barrier to improved communication with gps, . % selected "lack of time", . % the "administrative burden", and . % the difficulty of accessing secure platforms. however, over one-quarter ( . %) felt that a perceived lack of receptivity by gps was the primary obstacle to enhanced correspondence. participants' home nations' economies did not determine the type of health-care system in which they practiced. respondents from developed nations were no more likely than those from developing nations to work in a system with universal coverage ( . % vs . %; ns). neither participants' home nations' economic status nor the type of health-care system in which they worked influenced whether their patients also saw a gp, their comfort with serving as a gp for individuals with sci, whether they routinely communicated with gps, or their means of communicating with gps. in grouping certain answers for analysis, several significant associations emerged. when we combined two possible responses to type of medical records used (paper only and mix of paper and electronic records), we found that respondents from developed nations were more likely than those from developing nations to exclusively use ehrs ( . % vs . %; p = . ). when we combined responses to satisfaction with communication with gps into "positive" ("excellent" plus "good") and "negative" ("fair" plus "poor"), we found that participants using electronic means of communication were more likely than those using phone or fax to routinely communicate with gps ( % vs . %) and to rate those communications as positive ( . % vs . %; p = . ). to our knowledge, this is the first study to evaluate sci specialists' perspectives on their communication with gps and to begin to determine which factors promote or hinder those communications. although our sample size was small, limiting our analyses and ability to draw conclusions, several findings are notable. first, our results are in line with those of prior studies showing that most individuals with sci see both an sci specialist and a gp. in the study by donnelly et al. [ ] , respondents from countries with universal health coverage were more likely to have a gp than were those from the united states ( % in the united kingdom, % in canada, and % in the united states). in this study, neither the state of economic development nor the system of healthcare influenced whether individuals with sci were co-managed by gps. our data provide a simple reiteration of the fact that many people with sci knit together a complex network of care, and that like other people with chronic medical conditions, turn to a variety of physicians and health-care providers for assistance. second, our results show that sci specialists have an unequivocal interest in improved communication with their patients' gps, but that nearly % of them perceive that interest is not reciprocated. people living with acute and chronic sci have a high risk of hospitalization [ , ] and of several potentially life-threatening secondary effects of paralysis [ ] [ ] [ ] . although communication between sci specialists and gps has never been formally studied nor shown to improve clinical outcomes, it seems evident that it would. future studies may investigate how to better engage gps in the multi-specialty care of their patients with sci and whether that engagement would result in a reduction in re-hospitalization and sci-related complications. third, our findings suggest that clinical practices and health-care systems must continue to explore technologies that facilitate inter-physician communication. many researchers have shown that adoption of ehrs shifts the amount of time traditionally spent in a variety of aspects of patient care [ ] [ ] [ ] , an early manuscript demonstrated that emergency room physicians were eager to use electronic modalities to communicate with community-based colleagues [ ] , and several authors have demonstrated that use of an ehr can improve the quality and efficiency of discharges from inpatient services and of communications between physicians, nurses, and pharmacists [ ] [ ] [ ] . however, only one study to our knowledge has shown that adoption of electronic technologies-specifically, a computerized referral platform-enhances quality or frequency of inter-physician communications [ ] , and our data may support that notion. future work ought to explore whether this association holds, and if it does, how to make these technologies more available and affordable to health-care providers working in less developed nations. this work has several limitations. first, our sample size was small, potentially obscuring findings that would emerge from a larger data set. second, we only invited physicians to take our study, which excluded the insights and experiences of clinicians such as nurse practitioners and physician assistants who often provide front-line care to individuals with sci. future iterations of this study will certainly be more inclusive. finally, participants from less-developed nations were under-represented in this study. moving forward, we must focus our energies on improving representation of colleagues from a variety of nations. this being said, this study is the first to investigate sci specialists' perspectives on their communications with gps and to attempt to determine which factors facilitate and retard those communications. it may serve as stimulus for more-detailed work addressing these questions. health care utilization and barriers experienced by individuals with spinal cord injury utilization, access and satisfaction with primary care among people with spinal cord injuries: a comparison of three countries providing primary health care for people with physical disabilities: a survey of california physicians. center for disability issues and the health profession preparing health professionals to provide care to individuals with disabilities preventive services and health behaviors among people with spinal cord injury physiatry as a primary care specialty etiology and incidence of rehospitalization after traumatic spinal cord injury: a multicenter analysis rehospitalization in the first year of traumatic spinal cord injury after discharge from medical rehabilitation secondary conditions in a community sample of people with spinal cord damage longitudinal outcomes in spinal cord injury: aging, secondary conditions, and well-being incidence of secondary complications in spinal cord injury allocation of physician time in ambulatory practice: a time and motion study in specialties time spent on dedicated patient care and documentation tasks before and after the introduction of a structured and standardized electronic health record electronic health record logs indicate that physicians split time evenly between seeing patients and desktop medicine maintaining continuity of care: a look at the quality of communication between ontario emergency departments and community physicians assessing the impact of the introduction of an electronic hospital discharge system on the completeness and timeliness of discharge communication: a before and after study using the electronic medical record to enhance physician-nurse communication regarding patients' discharge status the effect of electronic medical record system use on communication between pharmacists and prescribers improving referral communication using a referral tool within an electronic medical record conflict of interest the data sets generated and analyzed during the current study are available from the corresponding author on reasonable request. the authors declare that they have no conflict of interest.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - qa e xt authors: lim, su-ho; gu, won mo; cha, soon cheol title: comparison of the retinal nerve fiber layer and ganglion cell complex thickness in korean patients with unilateral exfoliation syndrome and healthy subjects date: - - journal: eye (lond) doi: . /s - - - sha: doc_id: cord_uid: qa e xt purpose: to compare retinal nerve fiber layer (rnfl) and ganglion cell complex (gcc) thickness in patients with unilateral exfoliation syndrome (xfs) and age-matched controls using spectral domain-optical coherence tomography (sd-oct). materials and methods: this prospective case–control study included eyes (the xfs-affected and the fellow eyes) of unilateral xfs patients and eyes of age-matched control subjects. the rnfl and gcc thicknesses were measured using sd-oct (rt-vue , optovue, fremont, ca) after pupillary dilation. rnfl and gcc thicknesses were compared between case and control groups. results: the mean age of xfs patients was . years and that of age-matched controls was . years. both groups demonstrated a male preponderance. superior rnfl thickness of xfs-affected eyes were significantly thinner than those of the healthy age-matched controls (p = . by anova). there were no statistically significant differences in the rnfl thickness between both eyes of unilateral xfs patients. moreover, superior gcc thickness of both eyes in unilateral xfs patients were thinner than those in controls (p = . by anova). conclusions: thinner rnfl and gcc thicknesses were observed in unilateral xfs patients without visual field defects. these findings imply that xfs itself might be a risk factor for development of glaucomatous optic disc and rnfl damage. exfoliative glaucoma (xfg) is a common sight-threatening form of glaucoma which develops as a result of exfoliation syndrome (xfs) [ ] [ ] [ ] [ ] [ ] . blockage of the trabecular meshwork by exogenous or endogenous exfoliative materials (xfm), dysfunction of the trabecular meshwork, and concomitant primary open-angle glaucoma (poag) could cause glaucomatous damage [ , ] . xfg is associated with a greater mean intraocular pressure (iop) and more advanced visual field defects at diagnosis than poag. consequently, xfs itself is thought to be associated with glaucoma. xfs is also associated with numerous systemic diseases, including cardiovascular disease, cerebrovascular disease, sensorineural hearing loss, and alzheimer's dementia [ ] [ ] [ ] . in particular, xfs seems to widely affect various ocular vasculatures, including the ophthalmic artery, ciliary circulation, iris vessels, and central retinal vessels [ , ] . previous studies using color doppler imaging have reported reduced blood-flow velocities and increased resistivity in the ophthalmic artery, central retinal artery, and short posterior ciliary artery in xfs [ , ] . besides ocular vasculature, some studies have reported reduced retrobulbar blood flow and ipsilateral carotid blood flow in the xfsaffected eye [ , ] . these hemodynamic changes may result in retinal nerve fiber thinning and ganglion cell layer changes, even in eyes without glaucomatous damage [ ] . assessment of the macula allows investigation of the clinical impact of the diagnosis of early glaucomatous change [ ] . about % of retinal ganglion cells are located in the macula [ ] . in this context, evaluation of both the ganglion cell complex (gcc, a combination of the nerve fiber layer, ganglion cell layer, and inner plexiform layer) and circumpapillary retinal nerve fiber layer (cprnfl) is important because glaucomatous retinal atrophy mostly affects the rnfl and gcc. however, few studies have compared the rnfl and gcc thicknesses in xfs patients without glaucomatous damage and healthy controls [ , ] , and no studies in an asian population have been reported to date. thus, the purpose of this study was to evaluate automated measurements of cprnfl and gcc thickness, using the rt-vue (optovue, fremont, ca) sd-oct system, in patients with unilateral xfs without glaucomatous damage. this prospective cross-sectional study was approved by the institutional review board of daegu veterans health service medical center. all participants gave informed consent to participate. this study was performed in accordance with the tenets of the declaration of helsinki. the subjects were enrolled and investigated between january and december at daegu veterans health service medical center in south korea. a total of eyes of unilateral xfs patients (the affected and the fellow eyes) and eyes of age-matched control subjects (the right eye) were included in the study. unilateral xfs was defined as having one eye with xfs, diagnosed using slit-lamp biomicroscopy, tonometry, and dilated fundus examination, and a fellow eye without glaucomatous damage or retinal diseases. xfs was defined as follows [ ] : ( ) presence of characteristic fibrillar exfoliation materials at the pupillary border or on the anterior capsule after pupillary dilation, ( ) iop < mmhg, without antiglaucoma treatment, ( ) no glaucomatous optic nerve head (onh) changes confirmed by dilated fundus exam and sd-oct, ( ) open-angle status on gonioscopic examination, without visual field defects confirmed by automated perimetry in all xfs patients. the control group was defined as those without a glaucomatous onh or rnfl defect, visual field defect, or other retinal diseases in either eye. the exclusion criteria were as follows: ( ) patients older than years or younger than years; ( ) eyes with a refractive error of less than − diopters or more than + diopters; ( ) glaucomatous changes including glaucomatous onh changes and glaucomatous visual field defects; ( ) a history of antiglaucoma treatment, including medication and laser treatment; ( ) media opacity that reduced the image quality (as described in the following section); ( ) previous ocular surgery or ocular trauma; ( ) the presence of neurological diseases that might affect the rnfl thickness, such as alzheimer's disease, vascular dementia, or parkinsonism. all subjects underwent complete ophthalmic examinations, including a best-corrected visual acuity measurement, goldmann applanation tonometry, slit-lamp biomicroscopy, and gonioscopy using goldmann three-mirror lens, a dilated fundus examination, and rnfl and gcc thickness measurements using sd-oct. the margins of the optic cup were defined as the point of maximal inflection of vessels crossing the neuroretinal rim. the vertical cup diameter was measured as the vertical distance between the points of maximum centrifugal extension of the cup between and o'clock and and o'clock. disc hemorrhage, notching of the neuroretinal rim, and thinning of the rnfl were also documented. a dilated fundus examination was performed by a single glaucoma specialist (s.h.l). when glaucomatous changes were suspected on fundus examination in control group, automated perimetry was performed using a humphrey visual field analyzer i (carl zeiss meditec inc, dublin, ca) and the - swedish interactive threshold algorithm. visual field defects were defined as follows [ ] : ( ) glaucomatous visual field defects corresponding to onh or rnfl changes; ( ) glaucoma hemifield test results outside of the normal limits; and ( ) a cluster of three or more nonedge, contiguous points, not crossing the horizontal meridian, with p values < . as compared with the age-matched normal on the pattern deviation plot, one of which must have a p value < . . the rnfl and gcc thicknesses were measured using a sd-oct device (rt-vue , rt vue, fremont, ca) after pupillary dilation. an internal nasal fixation light was used to center the onh in the rectangle image mode, and cprnfl thickness was measured. to improve the scan quality, scans with signal strength intensity < were excluded from the analyses. average rnfl thickness and rnfl thickness in eight peripapillary sectors (superior nasal, superior temporal, temporal upper, temporal lower, inferior temporal, inferior nasal, nasal upper, and nasal lower sector) were calculated automatically. thereafter, we grouped eight peripapillary sectors into four sectors. superior rnfl thickness indicates the average of the superior nasal and superior temporal sectors. temporal rnfl thickness indicates the average of the temporal upper and temporal lower sectors. inferior rnfl thickness indicates the average of the inferior temporal and inferior nasal sectors. nasal rnfl thickness indicates the average of the nasal upper and nasal lower sectors. gcc thickness was also measured in two areas (superior and inferior sector). all oct images were evaluated by the same observer (s.h.l). statistical analyses were performed using spss for windows software version . (spss inc, chicago, il). independent t-tests and chi-square tests were used to compare baseline characteristics of unilateral xfs patients and controls. one-way analysis of variance (anova) was used to compare rnfl and gcc thickness among three groups (xfs-affected eye, fellow eye, and control group). in posthoc analysis, tukey's test was used to compare two groups. after bonferroni's correction of multigroup comparisons, a p value of . was considered statistically significant, at a significance level of . . table compares the baseline characteristics of the xfs and control groups. the mean age was . ± . for xfs patients and . ± . for healthy controls (p = . ). there was no difference in the numbers of males and females comprising either group (p = . ). the mean iop of xfs-affected eyes ( . ± . mmhg) was higher than that of fellow eyes ( . ± . mmhg) and healthy controls ( . ± . mmhg) (p = . ). a total of pseudophakic eyes ( xfs-affected eyes, fellow eyes and controls) were included, but, there was no significant difference between groups in terms of lens status (p = . ) or cup to disc ratio (p = . ), and there were no patients with abnormal myopic optic disc features such as severe disc tilt and peripapillary atrophy in any groups. rnfl parameters are summarized in table . although there was borderline statistical significance (p = . by one-way anova), post-hoc analysis revealed average rnfl thickness of xfs-affected eyes was slightly thinner than that of controls (p = . ). in particular, the superior quadrant rnfl thickness of xfs-affected eyes ( . ± . μm) was thinner than that of controls ( . ± . μm) (p = . by anova; p = . by tukey's post-hoc test). gcc parameters are summarized in table . there was a significant difference in the average and superior gcc parameters between groups by one-way anova (p = . , p = . , respectively). in particular, the superior gcc thickness decreased in both xfs-affected eyes and fellow eyes ( . ± . μm, . ± . μm, respectively) as compared with healthy controls ( . ± . μm) (p = . , p = . , respectively, by tukey's test). however, the inferior quadrant gcc thickness did not show a statistically significant difference between three groups (p = . ). rnfl and gcc thickness measurements provide clinically important information about early glaucomatous changes [ ] . however, few studies have compared the rnfl and gcc thicknesses between patients with xfs without glaucomatous damage and healthy controls [ , ] . in the present study, patients with unilateral xfs without glaucomatous optic nerve changes or visual field defects showed thinner rnfl and gcc thicknesses than those of control individuals. our results were similar to those of previous individual studies and meta-analysis [ , , ] . our study demonstrated reduced rnfl and gcc thicknesses in superior quadrants. similarly, aydin et al. [ ] found that rnfl thickness in the superior quadrant was thinner in xfs patients than in healthy subjects using cirrus hd oct- . they reported the gcc thickness was thinner in xfs patients than in healthy subjects, except in the inferior and inferonasal quadrants. in addition, eltutar et al. [ ] reported that in eyes of xfs, the superior and total macular nfl (nerve fiber layer) thickness; superior and total gcl + ipl (ganglion cell layer + inner plexiform layer) thickness were significantly thinner than those of control subject. however, controversies regarding location of thinning still abound. previous meta-analysis showed that rnfl thickness was significantly lower in all quadrants in xfs patients compared with control group [ ] . in their study, the weighted mean difference was − . in superior, − . in inferior, − . in temporal, and − . in nasal quadrants [ ] . subgroup analysis demonstrated no significant differences in the nasal quadrant regardless of oct type in their study [ ] . moreover, publication bias was found in average rnfl thickness and inferior rnfl thickness [ ] . thus, their results seem similar to our own. these variations in measurements may be attributable to the small sample size, different ethnicities, and possible systemic diseases of the study participants, and this was the first study comparing the rnfl thickness using optovue sd-oct the reasons for superior thinning of rnfl thickness may be attributable to vascular factors besides iop. recent octa studies have shown that superficial capillary density is significantly lower in the superior quadrant, but not in the inferior quadrant [ ] . these vascular differences were supported by some studies that report reduced rnfl thickness in xfs patients with normal iop [ , ] . it has, therefore, been suggested that rnfl changes in xfs patients are not only caused by high iop but also by other factors, such as insufficient blood flow to the onh and peripapillary retina [ , ] . in particular, xfs is greatly affected by genetic and environmental factors. many epidemiological studies have found significant variability in the prevalence of xfs among different ethnic groups ( . - . %) [ ] [ ] [ ] [ ] . similarly, the risk allele of loxl rs is reversed in the asian (japanese, chinese, and korean) population, and rs is not associated with xfg risk in greek, indian, mexican, and polish populations [ ] . given the conflicting genetic association results and the variable prevalence, genetic, environmental, and racial differences should be kept in mind. to the best of our knowledge, the rnfl and gcc thicknesses have not yet been compared between xfs patients and controls in an asian population. our results raise the question about the reason for thin rnfl and gcc thickness in xfs patients and the clinical relevance of these changes. first of all, the mean iop in xfs-affected and fellow eyes appears to be higher than reported by the korean national health and nutrition survey (knhanes) [ ] ; it was . ± . mmhg in those more than years of age. therefore, mean iop of xfsaffected and fellow eyes were higher than what was seen in that population; this could be a reason for the rnfl loss. besides the impact of iop, we postulate that the reasons for the thin rnfl and gcc thickness in the present study are related to ( ) systemic, or ( ) local vascular factors, and data are presented as mean ± sd (range); p value was calculated by one-way anova anova analysis of variance, rnfl retinal nerve fiber layer, xfs exfoliation syndrome a although the p value by anova demonstrated statistical borderline significance, post-hoc analysis was performed using tukey's test. the p value for comparison of xfs-affected eyes and fellow eyes (p ) was . ; the p value for comparison of xfs-affected eyes and control (p ) eyes was . ; and the p value for comparison of fellow eyes and control eyes (p ) was . b post-hoc analysis was performed using tukey's method: p = . ; p = . , and p = . [ , ] . these retinal ischemia can lead to ganglion cell death and onh atrophy [ ] , and it has been suggested that diminished ocular blood flow contributes to the development of rnfl damage in xfs patients. second, local vascular factors should also be considered. xfm accumulation in short posterior ciliary arteries, central retinal vessels, the optic nerve sheath, and vortex veins could cause vascular alterations, ischemic changes and subsequent rnfl and gcc thinning [ ] . ocakoglu et al. [ ] reported that microvascular blood flow in the optic nerve and peripapillary retina was decreased, based on heidelberg retinal flowmeter measurements. their study supported our hypothesis. besides compromised vasculature, the effect of mechanical changes of the onh should be considered. a thin lamina cribrosa of xfs was reported by some studies [ , ] . the decreased stiffness of the lamina cribrosa and peripapillary scleral tissues caused by xfs may reflect an inherent tissue weakness, especially in older patients [ ] . this may in turn render these eyes more vulnerable to iop fluctuations and optic nerve damage. in addition, jonas [ ] reported that the obstruction of the venous outflow system could be caused by mechanical distortion of the lamina cribrosa. structural alterations of the lamina cribrosa may also influence hemodynamic changes, such as increased venous outflow resistance or turbulence. in this study, thin rnfl and gcc thicknesses were also observed in the fellow eyes of patients with unilateral xfs, as compared with that of healthy control subjects. these findings suggest that both eyes of patients with unilateral xfs could have structural changes or vulnerability to the development of glaucoma, considering that the rnfl and gcc thicknesses were found to be thinner in glaucoma patients than in healthy control subjects in previous studies [ , ] . in particular, xfs is considered an asymmetric bilateral disease. xfs often occurs unilaterally, but within years, - % of cases may convert to the bilateral form [ , ] . iris vasculopathies are also detected in both eyes of patients with unilateral xfs [ ] . in this context, our study shows thin rnfl and gcc thicknesses in both eyes of patients with unilateral xfs. our study had some limitations. first, the sample sizes were relatively small; this was especially apparent in our subgroup analysis. second, blood pressure and diurnal iop fluctuations were not considered in this study. however, all patients were enrolled at a similar time of the morning, minimizing the effect of diurnal variation. third, a total of pseudophakic eyes were included because elderly patients were enrolled in our study. iop could be influenced by pseudophakia, but there was no significant difference between groups in terms of lens status. fourth, we could not exclude the effect of iop on rnfl and gcc thickness. the mean iop of xfs patients was slightly higher than that of controls. however, the mean iop and iop fluctuation of the xfs patients were higher than controls levels in other studies [ , ] . these results are compatible with those of other studies [ ] . in addition, the difference in iop was only about . mmhg in our study; thus, iop might not affect the thickness of gcc and rnfl markedly. in conclusion, rnfl and gcc thicknesses were significantly thinner in asian patients with xfs without glaucomatous damage compared with healthy controls. systemic or local vascular changes and mechanical alterations could play a role. thus, physicians should consider that diagnosis of xfs itself might predict a thin retinal change and may be a risk factor for development of a glaucomatous optic disc and rnfl damage. what was known before why is glaucoma associated with exfoliation syndrome? exfoliation syndrome a possible link between the pseudoexfoliation syndrome and coronary artery disease exfoliation syndrome and systemic cardiovascular diseases ocular pseudoexfoliation syndrome and vascular disease: a systematic review and metaanalysis early changes in iris blood vessels in exfoliation syndrome retinal vascular occlusions occur more frequently in the more affected eye in exfoliation syndrome color doppler imaging of the ophthalmic artery blood flow spectra of patients who have had a transient ischemic attack: correlations with generalized iris transluminance and pseudoexfoliation syndrome ocular hemodynamics in pseudoexfoliation syndrome and pseudoexfoliation glaucoma monolateral 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parameters in exfoliation glaucoma compared to primary open-angle glaucoma comparison of retinal nerve fiber layer thickness in patients having pseudo exfoliation syndrome with healthy adults analysis of retinal nerve fiber layer thickness in patients with pseudoexfoliation syndrome using optical coherence tomography. ophthalmologica hemodynamic evaluation of the posterior ciliary circulation in exfoliation syndrome and exfoliation glaucoma orbital blood flow parameters in unilateral pseudoexfoliation syndrome epidemiology of pseudoexfoliation in the island of crete (greece) epidemiology of the pseudo-exfoliation syndrome, a review the prevalence of pseudoexfoliation syndrome in chinese people prevalence of pseudoexfoliation syndrome in an isolated island population of korea: the woodo study loxl gene polymorphism with exfoliation syndrome/exfoliation glaucoma: a meta-analysis the distribution of intraocular pressure and associated systemic factors in a korean population: the korea national health and nutrition examination survey blood flow in glaucoma microvascular blood flow of the optic nerve head and peripapillary retina in unilateral exfoliation syndrome evaluation of lamina cribrosa in pseudoexfoliation syndrome using spectral-domain optical coherence tomography enhanced depth imaging evaluation of lamina cribrosa and choroid in nonglaucomatous patients with pseudoexfoliation syndrome using spectral-domain optical coherence tomography schlötzer-schrehardt u. evaluation of lamina cribrosa and peripapillary sclera stiffness in pseudoexfoliation and normal eyes by atomic force microscopy central retinal artery and vein collapse pressure in eyes with chronic open angle glaucoma pseudoexfoliation of the lens capsule ii. development of the exfoliation syndrome unilateral exfoliation syndrome: conversion to bilateral exfoliation and to glaucoma: a prospective -year follow-up study iris indocyanine green angiography in pseudoexfoliation syndrome and capsular glaucoma interocular differences in optic disc configuration in the unilateral exfoliation syndrome optic nerve head topography in nonglaucomatous, normotensive patients with unilateral exfoliation syndrome. graefe's arch clin exp ophthalmol conflict of interest the authors declare that they have no conflict of interest.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -c xj asg authors: ortega, cesar; cañas‐lopez, leticia; irgang, rute; fajardo, raúl; poblete‐morales, matías; valladares-carranza, benjamin; tapia‐cammas, diana; avendaño‐herrera, ruben title: first detection of spring viraemia of carp virus in common carp (cyprinus carpio l.) affected by a septicaemic disease in mexico date: - - journal: j fish dis doi: . /jfd. sha: doc_id: cord_uid: c xj asg spring viraemia of carp (svc) is an infectious disease responsible for severe economic losses for various cyprinid species, particularly common carp (cyprinus carpio carpio). the causative agent is the svc virus (svcv), a member of the sprivivirus genus, rhabdoviridae family, and a list pathogen notifiable by the world organization for animal health. this study describes the diagnosis of an svcv pathogen isolated in october from wild common carp inhabiting a natural lagoon in central mexico. while neither an epidemic nor fish mortalities were reported, the collected killed specimens exhibited clinical signs of disease (e.g., exopthalmia, moderate abdominal distension and haemorrhaging, as well as internal haemorrhages and adhesions). histological results of injuries were consistent with the pathology caused by svcv. this finding was supported by the isolation of a virus in epc and bf‐ cells and subsequent rt‐pcr confirmation of svcv. the phylogenetic analyses of partial svcv glycoprotein gene sequences classified the isolates into the ia genogroup. these findings make this the first report of svcv detection in mexico, extending the southern geographical range of svcv within north america. however, since this pathogen was detected in fish inhabiting a natural body of water without tributaries or effluents, it is difficult to estimate the risk of svcv for other wild/feral cohabitating cyprinid species in the lagoon. the status of this virus is also unknown for other bodies of water within this region. the common carp (cyprinus carpio l) is a freshwater and brackish fish originated in asia and has been farmed in china since at least bc. due to its ability to tolerate eutrophic waters and adverse environmental conditions, carp are farmed across a wide range of geographical and climatic areas worldwide (fao, ) . the common carp was introduced to mexico from asia in (wakida-kusunoki & amador-del-Ángel, ) and is now distributed across the country, with the purpose of providing animal protein to rural populations and additional work opportunities to the community (wakida-kusunoki & amador-del-Ángel, ) . in , carp production reached , tons, reflecting an annual estimated growth of . % (conapesca, ) . despite resilience to a wide range of environmental, climatic and geographical conditions, carp are susceptible to a variety of infectious diseases. primary among these diseases is the spring viraemia of carp virus (svcv), a notifiable pathogen according to the world organization for animal health (oie, a) and a virus responsible for mortalities associated with acute haemorrhaging. to date, svcv has been reported in common carp and other freshwater cyprinids in asia and europe, while detection on the american continent has been reported in the united states, canada (ahne et al., ; ashraf et al., ; garver et al., ; goodwin, ) and brazil (alexandrino, ranzani-paiva, & romano, ; oie, a) . nevertheless, svcv has not yet been detected in mexico. svcv, the type species of the sprivivirus genus and member of the rhabdoviridae family (afonso et al., ; ictv, ; oie, a) , exhibits a genome structure that includes five genes encoded in the order -n-p-m-g-l- , representing the nucleocapsid, phosphoprotein, matrix protein, glycoprotein and polymerase protein genes, respectively. svcv infects different carp species, as well as other cyprinids and ictalurids (ashraf et al., ) , and poses sanitary concerns in the global aquaculture industry. other aquatic rhabdoviruses are also a threat to wild and farmed fish species. these include the viral haemorrhagic septicaemia virus (vhsv) and the infectious haematopoietic necrosis virus (ihnv) of the novirhabdovirus genus, both of which are also listed as notifiable aquatic animal diseases by the oie ( a). in mexico, sanitary control measures associated with diseases in farmed carp are scarce, especially in relation to viral agents. lópez-jiménez ( ) reported on the occurrence of non-published cases of infectious ascites attributed to bacterial infections of aeromonas spp. and pseudomonas spp., whereas the occurrence of viral cases was largely ruled out, excepting one case in which coronavirus-like viral particles were microscopically identified. in turn, vélez-hernández, constantino-casas, garcía-márquez, and osorio-sarabia ( ) all specimens were collected from tecocomulco, a natural freshwater lagoon in the state of hidalgo that is approximately , hectares in size and without tributaries or effluents (rico-sánchez, rodríguez-romero, lópez-lópez, & sedeño-díaz, ) . the lagoon is formed by run-off and springs from the surrounding mountains, has an average length of . km, is between . and . km wide and has a depth, depending on periods of drought, between cm and m (novelo, montejano, cantoral, & tavera, ) . no disease outbreaks and/or disease-related mortalities have been reported for the lagoon for any of the different fish populations. common carp (≈ g) were captured using a net that traps the fish through the gills. many of the collected fish presented signs of septicaemic disease, characterized by moderate exophthalmia, abdominal distension and haemorrhaging in the ventral and lateral areas, as well as at the base of the fins. all fish were killed via anaesthetic overdose ( mg/l tricaine methanesulfonate ; sigma) and immediately subjected to postmortem examination. initial external observations were conducted, and the lesions of each specimen were recorded. examinations were then conducted for the presence of external parasites, and scrapings were obtained from gills and skin for microscopic observation at × and × magnifications. in addition, the microscope slides were gram-stained. for histological analyses, internal samples were taken from each fish, fixed in vials containing % buffered formalin, dehydrated and embedded in paraffin wax following standard procedures. each tissue was sectioned at μm and stained with haematoxylin and eosin to describe histopathological alterations, as in ortega et al. ( ) . sections were observed at different magnifications under an olympus bh- light microscope. the aspect, colour and contents of the body cavity were also reviewed. samples for bacterial isolation were aseptically taken from external skin lesions, the kidney, spleen, liver and brain of each specimen and streaked onto tryptone soy agar (tsa; oxoid) and columbia sheep blood agar (aes laboratoire, france). all plates were aerobically incubated at °c and examined every hr for days. if no colony growth was observed after a week, the cultures were considered negative. the clinical signs presented by the collected carp led to the presumption that an agent similar to svcv could be responsible. to blind-passed at least once before being classified as negative. electron microscopy was carried out on epc monolayers grown in -well tissue culture plates. the cells were inoculated with supernatants from cpe-positive cultures of carp- and carp- . at hr post-inoculation, the cell culture medium was removed. the monolayers were harvested by scraping, centrifuged for hr at g, fixed in glutaraldehyde ( . % in . m sodium cacodylate buffer, ph . ) and post-fixed in % osmium tetroxide for hr. cells were dehydrated with ascending concentrations of acetone and then embedded in epoxy resin (epon ; electron microscopy sciences) and acetone. semi-fine slices ( μm) were obtained using an ultramicrotome and were mounted on slides contrasted with toluidine blue (electron microscopy sciences). once the area of interest was identified, fine slices ( μm) were obtained and mounted on copper grids contrasted with uranyl acetate and lead citrate (electron microscopy sciences). finally, samples were observed using a jeol electron transmission microscope at kv. cell cultures were collected for virus identification when cpe ap- the amplified product was then assessed through semi-nested pcr designed to amplify a -bp product (primers: svcv-f and svcv-r ). the amplification procedure for both protocols adhered to those described by stone et al. ( ) . all pcr amplifications were carried out in a g-storm thermal cycler t gradients and were separated by electrophoresis on . % (w/v) agarose gel, visualized with / , gelred nucleic acid gel staining (biotium) and photographed under uv light. a generuler bp plus dna ladder (thermo scientific) was employed as a molecular mass marker. a sample was considered positive if the expected sizes of the primary and secondary pcr products were and bp, respectively. this report describes an svcv rhabdovirus as the cause of an infective outbreak among wild common carp in mexico. while svcv can replicate in fish, bird and mammalian cell cultures, optimal cell systems are those derived from cyprinid fish, such as epc (fijan et al., ) , which grows at - °c. the cpe of svcv is characterized by a margination of nuclear chromatin followed by a rounding up, detachment and lysis of cells. the presently obtained results are consistent with these descriptions. however, given that svcv is closely related to the pike fry rhabdovirus, an svcv-specific rt-pcr was used to obtain a more precise identification (stone et al., ) . the second round of the semi-nested pcr protocol amplified a prominent -bp product corresponding to nucleotides - of the svcv glycoprotein. genetic clustering and geographical origin are closely linked for svcv strains, which can be divided into the asian and european clades (miller et al., ; stone et al., ) . phylogenetic analysis of the partial glycoprotein genes obtained for the svcv isolates (carp- and carp- ) for mortalities in adult rainbow trout. likewise, tilapia, zebrafish and even trout could act as carriers of this viral agent (emmenegger et al., ; oie, b) . leads to the perivasculitis, degeneration and, ultimately, necrosis of blood cells, which manifests as disseminated haemorrhages f i g u r e histological injuries in common carp affected by an svcv pathogen. (a) liver with perivascular haemorrhaging (asterisk), hepatic degeneration represented by karyolysis (arrow) and pyknosis (arrowhead); (b) lymphocytic pancreatitis (arrows), necrosis (arrowhead) and periacinar lymphocytic infiltration (inset); (c) kidney with haemorrhagic (asterisk) and interstitial mononuclear (arrow) nephritis, as well as tubular dilation with proteinuria (arrowhead); (d) splenic congestion (asterisk) and reticuloendothelial hyperplasia (arrowhead), as well as multifocal haemosiderosis (arrow); (e) interstitial enteritis (lamina propria; asterisk) and necrotic cryptitis (arrow) with epithelial necrosis; and (f) epithelial scaling and villous atrophy (arrow), as well as granulomatous ulcerative colitis (asterisk) [colour figure can be viewed at wileyonlinelibrary.com] et al., ; gaafar et al., ; negele, ) . surprisingly, however, no signs of disease or fish mortalities have been previously described for the tecocomulco lagoon, a situation that is difficult to explain or justify. it is important to note that while this lagoon is rurally located, it is visited by carp fishermen and tourists or weekend visitors. the tecocomulco lagoon is also host to several other species, including fish, such as charal (chirostoma sp.), carps (cyprinus spp., ctenopharyngodon idella, hypophthalmichthys molitrix, amblycephala megalobrema, mylopharyngodon piceus) and goldfish; amphibians, such as axolotl (ambystoma velasci) and anurans like rana moctezumae; and reptiles, such as the aquatic snake (thamnophis eques). although carp- and carp- were isolated, the capacity and extent to which these isolates could cause mortality to carps and other fish species within the tecocomulco lagoon are unknown. (novelo et al., ; quiroz-flores, ramírez-garcía, & lot-helgueras, ) , macroinvertebrate populations (rico-sánchez et al., ) and pesticide presence/levels (aguilar-martínez, ) . therefore, the impact of the isolated svcv pathogen on fish populations in the tecocomulco lagoon remains unclear, and information regarding mortalities associated with this or prior cases is unavailable. this information gap could be due to the large size of the lagoon ( , ha; rico-sánchez et al., ) or, perhaps, due to the asymptomatic nature of affected fish, which act as svcv carriers (ahne et al., ) . furthermore, the lack of an active health-monitoring programme in mexico (ortega, ) and scarce knowledge of aquatic environments have conditioned local residents and fishermen to view low mortality rates as "normal," without concerns for identifying the cause. in the present case, the responsible laboratory only received samples and notified the health authority of suspected svcv. in fact, only five of the sampled fish evidenced classical clinical signs consistent with an svcv infection, which is a notable finding to consider for future health and research assessments. the obtained results are similar to those described by garver et al. ( ) , who isolated svcv from asymptomatic common carp in lake ontario & macías-garcía, ) , which are species susceptible to or that act as carriers for svcv (ahne et al., ; ashraf et al., ) . in any case, the occurrence of this case should alert health authorities to be on guard for other cases. for example, after the first svcv outbreak at a koi farm in north america in , eight subsequent svcv detections or outbreaks have been recorded (emmenegger et al., ) . this exotic virus could, therefore, represent a potential threat to native and culture fish populations in mexico. further contributing to this potential threat is that one of the factors related to svcv appearance is the temperature range in which fish live, with - °c being optimum (ahne et al., ) . in europe, the appearance of svcv is related to temperature increases after winter. in the case of the tecocomulco lagoon, the water temperature fluctuates yearly between . °c and . °c (delgadillo, ; novelo et al., ; quiroz-flores et al., ) ; during the sampling period, the temperature range was closer to . - . °c (delgadillo, ) . identificación de viremia primaveral de la carpa (vpc) carassius auratus en san pablo spring viraemia of carp virus: recent advances comisión nacional de acuicultura y pesca, secretaría de agricultura ganadería desarrollo rural pesca y alimentación determinación de parámetros fisicoquímicos, estado eutrófico y metales pesados de la laguna de tecocomulco, hidalgo; identificación de compuestos quelantes de hydrocotyle ranunculoides l.f. phd dissertation experimental infection of six north american fish species with the north carolina strain of spring viremia of carp virus cultured aquatic species information programme some properties of the epithelioma papulosum cyprini (epc) cell line from carp cyprinus carpio histopathological and ultrastructural study of experimental spring viraemia of carp (svc) infection of common carp with comparison between different immunohistodignostic techniques efficacy first detection and confirmation of spring viraemia of carp virus in common carp first report of spring viremia of carp virus (svcv) in north america the first report of spring viraemia of carp in some rainbow trout propagation and breeding by pathology and molecular techniques in iran genus: sprivivirus mega : molecular evolutionary genetics analysis version . for bigger datasets enfermedades más frecuentes de las carpas cultivadas en méxico phylogenetic analysis of spring viremia of carp virus reveals distinct subgroups with common origins for recent isolates in north america and the uk el uso de helmintos parásitos como bioindicadores en la evaluación de la calidad del agua: lago de tecocomulco vs histopathological changes in some organs of experimentally infected carp fingerlings with rhabdovirus carpio las algas de la laguna de tecocomulco spring viraemia of carp manual of diagnostic tests for aquatic animals. retrieved from www.oie.int/international-standard-setting/aquatic-manual veterinary medical education and veterinary involvement in aquatic animal health and aquaculture in mexico first identification of francisella noatunensis subsp. orientalis causing mortality in mexican tilapia oreochromis spp variación anual de la biomasa de nymphoides fallax ornduff (menyanthaceae) en la laguna de tecocomulco patrones de variación espacial y temporal de los macroinvertebrados acuáticos en la laguna de tecocomulco, hidalgo (méxico) isolation and identification of aeromonas bestiarum in cultured common carp nucleotide sequence analysis of the glycoprotein gene of putative spring viraemia of carp virus and pike fry rhabdovirus isolates reveals four genogroups from aquaculture goal to real social and ecological impacts: carp introduction in rural central mexico gill lesions in common carp, cyprinus carpio l., in mexico due to the metacercariae of centrocestus formosanus first record of the common carp cyprinus carpio var. communis (linnaeus, ) and the mirror carp cyprinus carpio var. specularis (lacepède, ) in tabasco. southern gulf of mexico impact of introduced fish for aqua-culture in mexican freshwater systems key: cord- -sl z i authors: marty, francisco m; chemaly, roy f; mullane, kathleen m; lee, dong-gun; hirsch, hans h; small, catherine b; bergeron, anne; shoham, shmuel; ljungman, per; waghmare, alpana; blanchard, elodie; kim, yae-jean; mckevitt, matt; porter, danielle p; jordan, robert; guo, ying; german, polina; boeckh, michael; watkins, timothy r; chien, jason w; dadwal, sanjeet s title: a phase b, randomized, double-blind, placebo-controlled multicenter study evaluating antiviral effects, pharmacokinetics, safety, and tolerability of presatovir in hematopoietic cell transplant recipients with respiratory syncytial virus (rsv) infection of the lower respiratory tract date: - - journal: clin infect dis doi: . /cid/ciz sha: doc_id: cord_uid: sl z i background: presatovir significantly reduced nasal viral load, signs, and symptoms of respiratory syncytial virus (rsv) infection in a human challenge study. we evaluated presatovir in hematopoietic-cell transplant (hct) recipients with rsv lower respiratory tract infection (lrti). methods: patients with confirmed rsv in upper and lower respiratory tract and new chest x-ray abnormalities were randomized ( : ), stratified by supplemental oxygen and ribavirin use, to receive oral presatovir mg or placebo every days for doses. the primary endpoint was time-weighted average change in nasal rsv viral load through day . secondary endpoints included supplemental oxygen-free days, incident respiratory failure requiring mechanical ventilation, and all-cause mortality. results: from january , , to march , , patients from centers were randomized ( presatovir, placebo); received study treatment ( allogeneic, autologous hct). in the efficacy population ( presatovir, placebo), presatovir treatment did not significantly reduce time-weighted average change in viral load (− . vs − . log( ) copies/ml; treatment difference − . log( ) copies/ml, % confidence interval: −. , . ; p = . ), median supplemental oxygen-free days ( vs days, p = . ), incident respiratory failure ( . vs . %, p = . ), or all-cause mortality ( vs . %, p = . ) versus placebo. adverse events were similar between arms (presatovir %, placebo %). resistance-associated substitutions in rsv fusion protein emerged in / presatovir-treated patients. conclusions: presatovir treatment was well tolerated in hct patients with rsv lrti but did not improve virologic or clinical outcomes versus placebo. clinical trials registration: www.clinicaltrials.gov, nct ; eudract, # - - from to , no patients with radiographic abnormalities consistent with lrti but with rsv detected in upper respiratory tract samples only ("possible" rsv lrti) died, but -day mortality was % in hct recipients with probable or proven rsv lrti [ ] . despite rsv disease burden in hct recipients and other high-risk adults, options for rsv prophylaxis or treatment in adults are limited. aerosolized ribavirin is not indicated for rsv treatment in adults and is associated with concerns regarding difficulty of administration, adverse effects, and high cost [ , ] . although some centers report using aerosolized or oral ribavirin to treat rsv lrti in adult hct recipients, efficacy has not been demonstrated in a randomized controlled clinical trial [ , ] . palivizumab is used for prevention of severe rsv disease in high-risk children ≤ months of age but is not effective as treatment for established infection in children or adult hct recipients [ ] [ ] [ ] . thus, there is an unmet need for specific treatment for adults at risk for severe rsv infection. presatovir is a novel, orally available rsv fusion inhibitor under investigation for treatment of rsv [ ] . presatovir has a favorable safety profile in adult volunteers, and presatovir treatment reduced viral load and respiratory symptoms in healthy adults challenged with rsv [ ] [ ] [ ] . here the safety, tolerability, and efficacy of presatovir in naturally infected hct recipients with rsv lrti were evaluated. this phase , randomized, double-blind, placebo-controlled, -group parallel study recruited hct recipients - years of age from centers in countries (supplemental material, appendix). patients presenting any time post-hct with upper and lower respiratory tract rsv infection documented ≤ days before start of study treatment and evidence of new abnormalities on chest x-ray obtained ≤ hours from screening were eligible for inclusion. lower respiratory tract involvement could be documented from induced sputum, bronchoalveolar lavage, or lung biopsy, but not spontaneous sputum. patients with documented concurrent lrti with other respiratory viruses were excluded. full eligibility criteria are provided in supplemental methods. the study followed the international conference on harmonisation good clinical practice guidelines and the principles of the declaration of helsinki and was approved by local ethics committees. written informed consent was obtained from patients or legally responsible representatives. data monitoring committee activities and changes to the study protocol are described in supplemental methods. this trial was registered with clinicaltrials.gov (nct ) and eudract ( - - ) before enrollment began. patients were randomly assigned ( : ) to receive presatovir or placebo, stratified centrally by supplemental oxygen use (none to ≤ l/min vs > l/min) and ribavirin use (prescribed at randomization, any route of administration) during the current rsv infection. the randomization schedule used permuted blocks of . allocation was concealed by use of presatovir and placebo tablets with identical appearance. study treatment assignment information was provided by an interactive web response system (bracket global, wayne, pa, usa). patients, all study staff, and sponsor were masked to study treatment. patients received presatovir mg ( × mg tablets) or placebo orally or via nasogastric tube every days (± hours) during study visits on days , , , , and , and were followed through study day . patients rsv-positive by local molecular testing on day could participate in an optional extended weekly follow-up through day . a detailed schedule of study assessments and procedures is provided in supplemental table . for virology assessments, bilateral intranasal samples were obtained using midturbinate adult flocked swabs (copan diagnostics, murrieta, ca, usa) at each study visit [ , ] . samples were analyzed using reverse transcription quantitative polymerase chain reaction (rt-qpcr) to determine rsv viral load, rsv sequencing of the f gene to evaluate development of resistance, and a multiplex assay to identify coinfections. all nasal samples were analyzed at central laboratories; further details are provided in supplemental methods. antibody titer and pharmacokinetic methods are described in supplemental methods. clinical assessments included vital signs, weight, and oxygen saturation by pulse oximetry; laboratory safety assessments included complete blood counts and serum electrolyte and liver enzyme measurements. patients were observed without oxygen supplementation at each study visit, and the lowest oxygen saturation during observation was recorded. cardiac safety was assessed via local electrocardiograms and troponin testing on days , , and . additional safety assessments included evaluation of adverse events (aes) and documentation of all concomitant medications, hospitalizations, rehospitalizations, intensive care unit care, invasive and noninvasive mechanical ventilation, and supplemental oxygen use (≥ l/min). the primary endpoint was time-weighted average change in nasal rsv viral load measured by rt-qpcr (log copies/ml) from day to day . key secondary endpoints were number of supplemental oxygen-free days [ ] , proportion of patients developing respiratory failure requiring invasive or noninvasive mechanical ventilation, and all-cause mortality through day . prespecified exploratory endpoints are described in supplemental methods. safety was assessed from aes and clinical and laboratory parameters. assuming time-weighted average change (standard deviation) in rsv log viral load from day to day of - . ( . ) log copies/ml in placebo-treated patients, patients per treatment group were planned to provide approximately % power to detect a ≥ . log decrease in the primary endpoint in patients receiving presatovir relative to placebo using a -sided α of . . we estimated % of patients would be evaluable and planned to enroll patients. the safety population included patients who received ≥ dose of study drug. the efficacy population included safety population patients with quantifiable rsv viral load on day . primary and secondary efficacy endpoints were analyzed in the efficacy population and post hoc in subgroups defined by supplemental oxygen use, ribavirin use, duration of rsv symptoms, graft-vshost disease (gvhd), lymphocyte count, and time from hct to rsv infection on day . the primary analysis tested superiority of presatovir vs placebo using parametric analysis of covariance using baseline viral load and randomization stratification factors as covariates with a -sided α of . (supplemental methods). number of supplemental oxygen-free days was analyzed using a negative binomial model with stratification factors as covariates and an offset parameter to account for on-study duration. patients who died prior to day or received supplemental oxygen on all days of the study period were assigned a value of supplemental oxygen-free days. the proportion of patients developing respiratory failure of any cause requiring invasive or noninvasive mechanical ventilation through day and allcause mortality through day were analyzed using cochran-mantel-haenszel tests adjusting for the stratification factors at the -sided . -level, with -sided % exact confidence interval (ci) based on the clopper-pearson method for each treatment group. where number of events was small, fisher exact test was used. a sequential testing procedure was used to control the type i error rate of . across the primary and secondary endpoints [ ] . from january , , to march , , patients were screened for eligibility and were excluded, mostly due to lack of new radiographical abnormalities or inability to confirm lower respiratory tract rsv infection ( figure ). sixty patients were randomized, of whom were assigned to presatovir and to placebo; patient randomized to presatovir withdrew consent before receiving study drug. notable protocol deviations are described in supplemental results. patient demographics and baseline characteristics were generally balanced between study groups (table ) . overall, the majority of patients ( / ; . %) underwent allogeneic hct and had chronic or acute gvhd ( / , . %). at start of study treatment, ( . %) patients were hospitalized for a median of days (range, - days). twenty-one ( . %) patients required > l/min of oxygen supplementation, and ( . %) patients were prescribed ribavirin (any formulation). rsv lrti was confirmed from induced sputum in ( . %) patients and by bronchoalveolar lavage in ( . %) patients. median time from onset of rsv infection symptoms to start of study treatment was days (range, - days). infection was due to rsv a in ( . %) patients and rsv b in ( . %) patients; ( . %) patients ( presatovir, placebo) had missing day rsv viral load data and were excluded from the efficacy population (n = ). median intranasal rsv viral load on day was . log copies/ml (range, . - . log copies/ml). nine patients ( presatovir, placebo) prematurely discontinued study treatment, and patients discontinued study participation before day ( presatovir, placebo) ( figure ). twenty-seven ( . %) of patients in the presatovir group and / ( . %) patients in the placebo group completed treatment to day ( figure ). figure a -b shows median absolute rsv viral load and change from baseline at each study visit. despite adequate plasma concentrations (supplemental results and supplemental table ), presatovir treatment did not significantly reduce time-weighted average change in log rsv viral load from day to day (− . [ . ] log copies/ml versus − . [ . ] log copies/ ml; treatment difference, − . log copies/ml; % ci, −. , . ; p = . ) compared with placebo ( table ) . during the -day study period, / ( . %) presatovirtreated patients and / ( . %) placebo-treated patients required supplemental oxygen. median (range) number of supplemental oxygen-free days was similar between presatovirtreated ( [ - ] days) and placebo-treated ( [ - ] days) patients (p = . ) ( table ). three presatovir-treated patients ( . %) and placebo-treated patients ( . %) developed respiratory failure requiring mechanical ventilation through study day (p = . ). no presatovir-treated patients and placebotreated patients ( . %) died through day (p = . ) ( table ) ; death was due to respiratory failure. exploratory efficacy outcomes are described in supplemental results. primary and secondary efficacy endpoints did not differ appreciably between patients treated with presatovir relative to placebo in subgroups defined by absolute lymphocyte count on day , presence of gvhd, time from onset of rsv symptoms to study treatment, and timing of rsv infection after hct (supplemental tables - ). optional extended viral monitoring and serologic responses to rsv infection are presented in the supplemental results. sequencing of rsv f gene detected postbaseline amino acid substitutions at resistance-associated positions in / ( . %) of presatovir-treated patients and / placebo-treated patients. these substitutions were detected a median of (range, - ) days after start of treatment (supplemental table ). twenty-four presatovir-treated patients ( . %) and placebo-treated patients ( . %) experienced ≥ ae, whereas presatovir-treated patients ( . %) and placebo-treated patients ( . %) experienced serious aes (saes). adverse events ≥grade occurred in presatovir-treated patients ( . %) and placebo-treated patients ( . %). individual aes occurred in ≤ % of presatovir-treated patients (table ) . numerically more frequent aes in patients treated with presatovir versus placebo were pneumonia, increased alanine aminotransferase, hypokalemia, nausea, acute sinusitis, and epistaxis ( patients each, %), and increased aspartate aminotransferase, dry mouth, and increased alkaline phosphatase ( patients each, . %; table ). except for sae pneumonia in presatovirtreated patients ( %), grade or aes and saes occurred in patient each and were numerically less frequent overall in patients treated with presatovir versus placebo (supplemental tables - ). there were no significant imbalances in electrocardiogram and troponin results during the study. no patients treated with presatovir and patients treated with placebo ( . %) died during the -day study period; death was due to respiratory failure and to progressive acute leukemia. another patients ( . %) who received placebo died after day , of respiratory failure, and of invasive fusariosis. this is the first placebo-controlled clinical trial, to our knowledge, evaluating treatment of rsv lrti with a new antiviral agent in hct recipients. presatovir had a favorable safety profile and was well tolerated but did not decrease timeweighted average change in nasal rsv viral load from day to day , number of days with supplemental oxygen use, or frequency of respiratory failure or mortality relative to placebo. in contrast, presatovir treatment significantly reduced viral load, clinical signs, and symptoms of experimental rsv infection in healthy volunteers treated upon detection of rsv replication [ ] . potential explanations for this discrepancy have important implications for design of clinical trials evaluating antiviral treatments for rsv infection in hct recipients and other patient populations. in the past years, treatment with a fusion inhibitor or nucleoside polymerase inhibitor significantly reduced rsv viral load, signs, and symptoms in challenge studies in healthy human volunteers [ , , ] . however, clinical trials of presatovir conducted in multiple different patient populations, including this study and a companion urti study (chemaly et al [ ] , this issue), indicate difficulties remain in translating challenge study results to successful clinical trials in patients with natural infection [ , ] . one partial explanation is the challenge model's inconsistent representation of the natural infection setting. challenge study volunteers were inoculated intranasally with rsv, then monitored for nasal rsv replication with twice-daily nasal washes that were immediately evaluated with molecular assays for rsv [ , , ] . antiviral treatment was initiated - hours after rsv detection, generally several days before peak viral load and prior to manifestation of significant clinical signs and symptoms [ , , ] . in the present study, patients with naturally acquired rsv lrti received presatovir later in the disease course compared with challenge study subjects (median [range], days after symptom onset); delay was also observed in the urti trial (median [range], [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] days) and other studies of presatovir in natural rsv infection [ , ] . because clinical signs and symptoms tend to correlate with nasal viral load [ , ] , these patients presumably presented near or more likely after peak nasal viral load, potentially beyond the therapeutic window for presatovir even in immunocompromised patients. host immune-mediated clearance of the virus at this stage may also mask treatment-induced reduction in viral load. thus, treatment delay may explain lack of presatovir efficacy in the current study. the mechanism of action of presatovir may also have limited efficacy in this study. because rsv is capable of cell-to-cell spread, inhibition of viral fusion may not halt propagation of established infection along the respiratory tract. therefore, fusion inhibitors, such as presatovir, may need to be administered, whereas virus-cell fusion still represents the main mode of viral spread to appreciably reduce nasal viral load. emergence of f gene protein amino acid substitutions associated with fusion inhibitor resistance was also relatively frequent ( %) in this immunocompromised population. polymerase inhibitors can terminate intracellular rsv replication and may have wider data are n (%) unless otherwise specified. abbreviations: cmv, cytomegalovirus; gvhd, graft-vs-host disease; hct, hematopoietic cell transplant; lrt, lower respiratory tract; max, maximum; min, minimum; rsv, respiratory syncytial virus. a on the day of the first dose of study drug, patients receiving presatovir and patients receiving placebo were being treated with ribavirin. current or intended use of ribavirin on day of randomization was used to stratify randomization. b these patients were excluded from the efficacy population. c for these values, n = for the presatovir arm and n = for the placebo arm. [ ] . however, no polymerase inhibitor has demonstrated clinical efficacy in a natural infection setting to date. furthermore, nasal viral load is questionable as a primary endpoint for proofof-concept studies in naturally infected patients with lrti because upper respiratory tract samples, although more convenient to obtain, may not reflect viral activity in the lower respiratory tract, particularly in immunocompromised patients. alternative approaches are needed for noninvasive measurement of viral disease dynamics and antiviral activity in lower airway and alveolar tissue. the findings of this trial call into question whether appearance of new radiological opacities with documentation of rsv in the upper or lower respiratory tract accurately classifies patients with rsv infection. this approach may be satisfactory for retrospective studies but inadequate as an enrollment criterion for prospective clinical trials. radiographic findings in adults with rsv lrti confirmed from a lower respiratory tract specimen are not well characterized, and radiographic abnormalities in these patients may be caused by other viruses, bacteria, or fungi-particularly in immunocompromised patients-or even noninfectious processes. as rsv chiefly affects airway epithelium [ ] , rsv lrti could manifest without radiographic findings, and present only as lower airway symptoms (eg, wheezing or obstructive spirometry pattern). furthermore, the degree of lung injury in patients with rsv lrti may not be reversible by antiviral treatment alone. these issues will need to be considered in future clinical trials. the lack of clinical benefit in this study may also relate to the selected clinical endpoints, which occurred at lower-thananticipated rates that decreased power to detect a treatment effect. although all subjects enrolled in the current study met waghmare et al's criteria for proven or probable lrti, median number of supplemental oxygen-free days through day was much higher ( and days for presatovir-treated and placebo-treated patients, respectively, vs days), and fewer patients required > l/min supplemental oxygen at baseline ( . % versus %) relative to patients who presented with or developed lrti in the waghmare study [ ] . furthermore, lymphopenia is a major risk factor for rsv infection and subsequent poor outcomes in hct recipients [ , , ] , but only / patients with available data in the current study were lymphopenic (< cells/mm ) at baseline, possibly because rsv infection occurred relatively late after hct (median, vs days in the waghmare study). these differences could be due to limited enrollment of patients perceived as fragile because of the requirement for lower respiratory sampling to confirm rsv lrti. enrichment of the study population for immunosuppressed patients should be considered in future clinical trials of therapies for rsv infection in hct recipients. in summary, presatovir treatment was generally well tolerated in hct recipients with naturally acquired rsv lrti but did not achieve virologic or clinical endpoints. the tendency of adults with naturally acquired rsv infection to seek treatment only after several days of symptoms, when the treatment window may have closed for fusion inhibitors in particular, is a challenge for clinical trials of rsv-specific antiviral therapies. the numerically lower rate of pulmonary complications in the presatovir urti trial suggests that early treatment, before lrti develops, is key for success in future studies. acute kidney injury ( . ) atrial fibrillation ( . ) alkaline phosphatase increased ( . ) dizziness ( . ) fatigue ( . ) neutropenia ( . ) data are shown as n (%). respiratory syncytial virus infection in adults respiratory syncytial virus in hematopoietic cell transplant recipients: factors determining progression to lower respiratory tract disease supplemental oxygen-free days in hematopoietic cell transplant recipients with respiratory syncytial virus impact of aerosolized ribavirin on mortality in allogeneic haematopoietic stem cell transplant recipients with respiratory syncytial virus infections community respiratory virus infections among hospitalized adult bone marrow transplant recipients combination therapy with aerosolized ribavirin and intravenous immunoglobulin for respiratory syncytial virus disease in adult bone marrow transplant recipients management of rsv infections in adult recipients of hematopoietic stem cell transplantation virazole®(ribavirin for inhalation solution, usp) aerosolized ribavirin: the most expensive drug for pneumonia randomized controlled multicenter trial of aerosolized ribavirin for respiratory syncytial virus upper respiratory tract infection in hematopoietic cell transplant recipients how i treat respiratory viral infections in the setting of intensive chemotherapy or hematopoietic cell transplantation synagis® (palivizumab) injection, for intramuscular use updated guidance for palivizumab prophylaxis among infants and young children at increased risk of hospitalization for respiratory syncytial virus infection palivizumab treatment of respiratory syncytial virus infection after allogeneic hematopoietic stem cell transplantation discovery of an oral respiratory syncytial virus (rsv) fusion inhibitor (gs- ) and clinical proof of concept in a human rsv challenge study oral gs- activity in a respiratory syncytial virus challenge study phase first-in-human, single-and multipleascending dose, and food effect studies to assess the safety, tolerability, and pharmacokinetics of presatovir for the treatment of respiratory syncytial virus infection the drug-drug interaction profile of presatovir accuracy and discomfort of different types of intranasal specimen collection methods for molecular influenza testing in emergency department patients development and evaluation of a flocked nasal midturbinate swab for self-collection in respiratory virus infection diagnostic testing gatekeeping strategies for clinical trials that do not require all primary effects to be significant activity of oral als- in a respiratory syncytial virus challenge study safety and efficacy of oral rv in a human respiratory syncytial virus (rsv) phase a challenge study a phase , randomized, double-blind, placebo-controlled trial of presatovir for the treatment of respiratory syncytial virus upper respiratory tract infection in hematopoietic-cell transplant recipients a phase b, randomized controlled trial of presatovir, an oral rsv fusion inhibitor, for the treatment of respiratory syncytial virus (rsv) in lung transplant (lt) recipients a phase b, randomized, double-blind, placebo-controlled trial of presatovir (gs- ), a novel oral rsv fusion inhibitor, for the treatment of respiratory syncytial virus (rsv) in hospitalized adults late therapeutic intervention with a respiratory syncytial virus l-protein polymerase inhibitor, pc , on respiratory syncytial virus infection in human airway epithelium severe human lower respiratory tract illness caused by respiratory syncytial virus and influenza virus is characterized by the absence of pulmonary cytotoxic lymphocyte responses respiratory viral infections in adults with hematologic malignancies and human stem cell transplantation recipients: a retrospective study at a major cancer center financial support. this work was supported by gilead sciences, inc. potential conflicts of interest. f. m. m. reports research grants paid to his institution and consulting fees for clinical trial design from gilead sciences, inc., during the conduct of the study; and research support paid to his institution and consulting fees for clinical trial design from chimerix and glaxosmithkline, research grant support paid to his institution from ansun, and personal fees from roche molecular diagnostics and visterra outside the current study. r. f. c. reports research grants paid to his institution and personal fees from gilead sciences, inc., during the conduct of the study; research grants paid to his institution from ablynx, aicuris, ansun, chimerix, merck, oxford immunotec, novartis, pulmotec, shire, and xenex; and consultant fees/honoraria from ablynx, achaogen, adma biologics, ansun, astellas, chimerix, clinigen, janssen, oxford immunotec, merck/ merck sharp and dohme (msd), pulmotect, shionogi, shire, and xenex outside the current study. k. m. m. reports grants as a clinical trial investigator from gilead sciences, inc., during the conduct of the study; clinical research grants from ansun, astellas, merck, rebiotix, scynexis, and shire; and consultant fees/honoraria from chimerix, glaxosmithkline, merck, and scynexis outside the current study. d. g. l. reports research grants and consultant fees or speaker honoraria from astellas, msd, and pfizer outside the current study. h. h. h. reports nothing to disclose. c. b. s. reports grants paid to her institution from gilead sciences, inc., during the conduct of the study and from abbott, ablynx, chimerix, glaxosmithkline, merck, schering, shire, and viiv outside the current study. a. b. reports funding to her institution from gilead sciences, inc., during the conduct of the study; and research grants from pfizer and sos oxygène and consultant/speaker fees from ablynx; gilead sciences, inc.; merck; pfizer; shire; therakos; and zambon outside the current study. s. s. reports grants paid to his institution from gilead sciences, inc., during the conduct of the study; grants paid to his institution from astellas, ansun, cidara, emergent biosolutions, merck, scynexis, shiongi, shire, and t microsystems outside the current study; and direct payments as a member of a data safety monitoring board from merck outside the current study. p. l. reports research grants or fees/ honoraria paid to his institution from astellas; gilead sciences, inc.; merck; oxford immunotec; and shire; and consultant fees/honoraria from ablynx and aicuris outside the current study. a. w. reports clinical trial support from gilead sciences, inc., during the conduct of the study. e. b. reports speaker fees from boehringer ingelheim; gilead sciences, inc.; novartis; pfizer; and roche outside the current study. y. j. k. reports grants from gilead sciences, inc., during the conduct of the study. m. m., d. p. p., y. g., p. g., and t. r. w. are employees of gilead sciences, inc., and may hold stock. r. j. is a former employee of and holds stock in gilead sciences, inc. m. b. reports clinical trial support and consulting fees from gilead sciences, inc., during the conduct of the study; and grants and personal fees from ablynx; ansun; chimerix; glaxosmithkline; merck; shire; and vir bio; and personal fees from bavarian nordic; humabs, inc.; janssen; modema therapeutics; pulmocide; and pulmotect outside the current study. j. w. c. is an employee and stockholder of janssen biopharma and a former employee and current stockholder of gilead sciences, inc. s. s. d. reports grants paid to his institution from gilead sciences, inc., during the conduct of the study; and grants to his institution and personal fees for consulting, advisory board, and speaker bureau participation from merck; personal fees for advisory board participation from clinigen and janssen; and grants paid to his institution from ablynx, aicuris, ansun, glaxosmithkline, oxford immunotec, and shire outside the current study. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. supplementary materials are available at clinical infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. key: cord- -ec rkxdn authors: chun, audrey; levy, isaiah; yang, ajax; delgado, andrew; tsai, chung-ying; leung, eric; taylor, kristell; kolakowsky-hayner, stephanie; huang, vincent; escalon, miguel; bryce, thomas n. title: treatment of at-level spinal cord injury pain with botulinum toxin a date: - - journal: spinal cord ser cases doi: . /s - - - sha: doc_id: cord_uid: ec rkxdn study design: randomized, double-blinded, placebo-controlled, cross-over study. objective: to explore whether botulinum toxin a (bonta) could be effective for treating at-level spinal cord injury (sci) pain. setting: outpatient sci clinic, new york, usa. methods: participants were randomized to receive subcutaneous injections of either placebo or bonta with follow-up (office visit, telephone, or e-mail) at , , , and weeks to assess the magnitude of pain relief post injection. crossover of participants was then performed. those who received placebo received bonta, and vice versa, with follow-up at , , , and weeks. results: eight participants completed at least one of the two crossover study arms. four completed both arms. the median age of the eight participants was years (range – years) and % were male. all had traumatic, t -l level, complete sci. although our data did not meet statistical significance, we noted a higher proportion of participants reporting a marked change in average pain intensity from baseline to and weeks post-bonta vs. post-placebo ( % vs. %). at and weeks post-bonta, almost all participants reported some degree of reduced pain, while the same was not seen post-placebo ( % vs. %). conclusion: the subcutaneous injection of bonta may be a feasible approach for the control of at-level sci pain and is worthy of further study. sponsorship: the onabotulinumtoxina (botox) used in this study was provided by allergan (irvine, ca). at-level spinal cord injury (sci) pain is neuropathic pain perceived at the neurological level of injury (nli) or within three levels below the nli and is thought to be a result of damage to the spinal cord or its nerve roots [ ] . it has been reported to occur in approximately one third of individuals with sci [ , ] . various pharmacological agents used for treating at-level sci pain have been shown to have only limited efficacy at best [ , ] . therefore, a significant need for the investigation of potential new treatments for at-level sci pain remains. botulinum toxin a (bonta) is a neurotoxin protein produced by the bacterium clostridium botulinum. it acts at the neuromuscular junction and its onset of action occurs within - h after administration, with peak clinical effect occurring - weeks after administration, then - months of effect duration [ ] . at the level of peripheral nerves, bonta may inhibit the synaptic release of local neuropeptides associated with pain transmission, such as glutamate, substance p, and calcitonin gene-related peptide [ ] . at the level of the central nervous system, retrograde effects of bonta on the spinal cord via axonal transport have been proposed [ , ] . bonta therapy has so far been shown to have some effectiveness in treating various peripherally mediated neuropathic pains, but the literature on bonta remains limited for treating centrally mediated pain [ ] . to date, one randomized, double-blinded, placebo-controlled study has successfully been able to demonstrate that subcutaneous bonta may reduce neuropathic pain in persons with sci. this study by han et al. evaluated patients with neuropathic pain associated with sci, both at-level (n = ) and below-level (n = ), and found participants demonstrated significantly reduced pain at and weeks after bonta injections compared with after placebo with saline. they also showed a marginal trend towards improvements on the physical health domain of the world health organization quality of life instrument (whoqol-bref abbreviated form) at weeks post-bonta vs. post-placebo [ ] . bonta has been shown to have a favorable safety and tolerability profile across a broad spectrum of therapeutic uses [ , ] . adverse effects (aes) associated with bonta are generally related to the mechanism of action of the toxin (e.g., dose-dependent focal weakness when injected intramuscularly). other local side effects include temporary injection associated pain, edema, erythema, ecchymosis, headache, and short-term hyperesthesia. systemic side effects include nausea, fatigue, malaise, rash, and flu-like symptoms [ ] . in summary, a significant percentage of persons with sci report at-level sci pain but there is a lack of effective treatment to date, and bonta has shown preliminary evidence of effectiveness for neuropathic pain after sci in one clinical trial while having a favorable safety and tolerability profile. our study sought to further explore whether the subcutaneous injection of bonta could be a feasible approach for the control of at-level sci pain. this study was designed as a randomized, double-blinded, placebo-controlled, crossover study. institutional review board approval (irb # - ) was obtained prior to conducting any study related procedures and informed consent was obtained from each participant. participants received a subcutaneous injection of either normal saline (placebo) or bonta with follow-up (via office visit, telephone, or e-mail) at , , , and weeks post injection (phase , p ). crossover of participants was then performed. those who received placebo received bonta, and vice versa, with follow-up at , , , and weeks (phase , p ). twelve weeks were determined as being the minimum required wash-out period based on the pharmacokinetics of bonta [ ] . participants were given the choice to start the p course at the time of -week follow-up for p , or to defer the p course until a later time point. all study activities took place in a large tertiary care hospital in the outpatient sci rehabilitation clinic setting. portions of this study have been accepted for poster presentation at the american congress of rehabilitation medicine annual conference [ ] . participants were recruited via newsletter advertisements, physician recommendations, and clinicaltrials.gov (nct # ) between and . participants were included if they reported chronic traumatic sci (duration > months) and pain that has been: ( ) present continuously for ≥ month, ( ) of at least moderate average intensity over the prior week (numeric pain rating scale [nprs] score ≥ / ) and ( ) diagnosed by an sci provider as being at-level sci pain with a high degree of certainty [ ] . they were excluded if they were: ( ) < years old, ( ) pregnant, ( ) with any contraindications to bonta (e.g., neuromuscular junction disease), ( ) with a history of intolerance, hypersensitivity, or allergy to bonta, its preservatives, or the ethyl chloride used as an analgesic during injections, ( ) with a history of bonta injections within the past months, ( ) with a history of coagulation disorder or current infection, ( ) with insufficient command of english, and ( ) unable to provide informed consent. consented participants underwent a pre-procedure evaluation, consisting of a focused history and physical exam, by a physician blinded to whether they would be bonta or placebo recipients. each participant was asked to localize his or her area of worst pain, then to describe and rate the intensity of his or her pain before examination of the indicated painful area. participants were then screened for the presence of neuropathic pain based on the douleur neuropathique and sci pain instrument questionnaires to supplement the focused history and exam [ ] . the area of pain was marked using a skin marker and a plastic cut-out template for injection sites separated from each other by a cm radius (fig. ) . to marked areas, participants received subcutaneous injections of either bonta or placebo. each unit vial of bonta was reconstituted with ml preservative-free sterile . % normal saline solution, for a concentration of units of bonta per . ml injectate, as per manufacturer recommendations. placebo consisted of . ml of the same preservative-free sterile . % normal saline. syringes were prefilled by an unblinded physician and research assistant in a separate location from where injections and assessments occurred. the physician administering the injection was blinded to syringe contents. the randomized treatment allocation for each patient was kept in a sealed envelope by another research assistant, separate from those preparing and administering injections. the area was sterilized with alcohol then localized topical anesthesia was provided with sterile ethyl chloride. each injection was given using a gauge needle and administrators were allowed to provide up to injections of units each for a total maximum delivery of units, per participant, per phase injection session. this maximum dose and injection number was determined based on the study team's review of the bonta manufacturer recommendations and the bonta literature to date at the time of protocol formation [ ] [ ] [ ] . following the injection procedure, participants were monitored for ≥ min for immediate aes. they were reeducated on potential aes of bonta (e.g., local hyperesthesia, weakness, erythema), including all serious ones for which they should seek emergency care (e.g., respiratory distress, systemic illness), and provided with contact information for any questions or concerns that they may develop afterwards. neither participants nor their insurance providers were charged for the bonta and participants did not receive any monetary reimbursement for participation. the bonta was stored in a locked refrigerator ( - °c), labeled in a location separate from the outpatient sci clinic. our primary outcome measure was patient reported average pain intensity over the prior week in their area of worst pain as indicated above. this measure is part of the international sci pain basic data set (iscipbds), whose validity and utility in individuals with sci have been demonstrated, and it is among the measures recommended for use in sci clinical trials [ ] [ ] [ ] . participants were asked to rate their average pain using the - nprs from ("no pain") to ("worst pain imaginable"). secondary outcome measures included other iscipbds items, such as the degree to which pain interfered with: ( ) day to day activities, ( ) mood, and ( ) sleep, all on scales from ("no interference") to ("extreme interference"). we defined "marked improvement" as a ≥ -point difference based on the minimal clinical important difference (mcid) for the nprs being ≥ points [ ] . at each follow-up time point, participants were asked to quantify the change in quality of life (qol) appreciated after injections on a -point scale based on the patient global impression of change scale [ ] . lastly, participants were asked about and monitored for aes as described above. all collected data were stored in a password-protected redcap database [ ] and all hard copy forms were kept in locked files in the offices of research staff. statistical analyses were performed using spss version . . power analyses were conducted based on the primary outcome of change in pain intensity from baseline. we determined that mcid on the nprs would be ≥ points, that % of participants in the placebo group would have positive response to treatment, and that % more participants (i.e., %) would respond in the treatment group [ , , ] . assuming a two-sided alpha of . , a sample size of would provide % power. assuming a % drop out rate, a sample size of participants was deemed necessary for sufficient power to detect treatment effect. our study failed to meet target sample size therefore it was determined that our findings would be best presented as a descriptive case series and descriptive statistics were used for participant characteristics and all outcomes. among enrolled and consented participants, did not proceed to injections because they were deemed ineligible for the study during the pre-procedure history and physical or they declined participation in the study at this point. among the eight participants who proceeded, five started the study with bonta. all participants finished the p arm of the study. a single bonta recipient withdrew from the fig. template used to administer botulinum toxin a (bonta) and placebo injections study prior to starting the p arm, reporting increased pain after completing p (participant was blinded to whether bonta or placebo was given). one participant starting the study with bonta and two starting with placebo were lost to follow-up during p . a total of four participants finished both p and p arms (fig. ). participants had a median age of years (range - years) and were predominantly male ( %). all had traumatic, thoracic t to lumbar l level, complete sci (american spinal injury association impairment scale grade a). median duration of injury was years (range - years). table shows detailed participant demographics and injury characteristics. participant c reported some pain reduction at weeks post-bonta, marked reduction at , , and weeks, but not post-placebo. participant d reported some pain reduction at and weeks post-bonta but at the week follow-up, withdrew from the study prior to receiving placebo, reporting increased pain after finishing p . participant e reported some pain reduction at , , and weeks post-bonta, but received placebo for p some months after -week follow-up for p then was lost to follow-up. participant f reported marked pain reduction at and weeks post-bonta but not post-placebo. participant g reported no pain reduction post-placebo, received bonta for p concurrently with -week follow-up for p , then was lost to follow-up. participant h reported no pain reduction post-placebo, received bonta for p some months after -week follow-up for p , then was lost to follow-up (table ) . participants a and c reported no marked improvements compared with baseline in day to day activities, mood or (table ) . participant a reported his qol was "moderately better, with a slight but noticeable change" at and weeks post-bonta. participant b reported his qol was "somewhat better" at and weeks post-bonta. participant c reported "somewhat" better qol at weeks and "moderately" better qol at weeks post-bonta (item assessment missed at weeks). participant d reported qol that was "moderately better, with definite improvement" at weeks post-bonta, but progressively reduced qol at , , and weeks. participant e reported his qol was "somewhat" better weeks post-bonta but reported minimal improvements at , , and weeks. participant f rated his qol as "moderately better, with definite improvement" at and weeks post-bonta, and "somewhat better" at weeks post-bonta (table ). one participant attributed an ae related to bonta injection itself: participant d, who initially reported some improvements in pain, activities, mood, sleep, and qol post-bonta, reported that overall his pain worsened at weeks and withdrew from the study (tables - ) . two participants reported an ae during the course of the study crossover -week follow-up intensity reported using numeric pain rating scale, from ("no pain") to ("worst pain imaginable") cross-over -week follow-up degree of interference reported using a numeric scale with endpoints of ("no interference") to ("extreme interference") (participant a reported "thrombosis of a preexisting inferior vena cava filter" and participant h reported "a fever from a cold") but both denied feeling the events were related to the study. our study sought to explore whether the subcutaneous injection of bonta is a feasible approach for the control of at-level sci pain. this study was designed as a randomized, double-blinded, placebo-controlled, crossover study. however, we failed to meet target sample size and determined our findings would be best presented as a descriptive case series. our data did not meet statistical significance and we were not statistically powered to, but we did note various patterns of improved pain, activities, mood, and sleep among participants after bonta. more participants reported a marked change in average pain intensity from baseline to and weeks post-bonta vs. post-placebo ( % vs. %). at and weeks post-bonta, almost all participants reported some degree of reduced pain; the same was not seen post-placebo ( % vs. %) ( table ). this is consistent with han et al.'s findings that, at and weeks after bonta injections, visual analog scale scores for pain were significantly reduced by . ± . and . ± . , but only reduced by . ± . and . ± . after placebo injections [ ] . more participants reported markedly reduced pain interference with day to day activities at and weeks post-bonta vs. post-placebo ( % vs. %). more participants reported markedly reduced pain interference with mood post-bonta vs. post-placebo at ( % vs. %), ( % vs. %), and ( % vs. %) weeks. more participants reported markedly reduced pain interference with sleep post-bonta vs. post-placebo at ( % vs. %), ( % vs. %), ( % vs. %) and ( % vs. %) weeks (table ) . lastly, more participants reported at least moderate improvements in qol post-bonta vs. post-placebo at ( % vs. %) and ( % vs. %), ( % vs. %), and ( % vs. %) weeks (table ). this too supports han et al.'s trend towards improvements at weeks post-bonta (p = . ) on the physical health domain of the whoqol-bref, which includes facets like activities of daily living and sleep [ ] . our data was statistically insufficient to draw conclusions regarding the effects of bonta on at-level sci pain. despite this, we managed to demonstrate several interesting patterns within our participants' reports on how they felt post-bonta. participants also denied any significant aes = "almost the same, hardly any change at all" = "a little better, but no noticeable change" = "somewhat better, but the change has not made any real difference" = "moderately better, a slight but noticeable change" = "moderately better, a definite improvement that has made a real worthwhile difference" = "a great deal better, a considerable improvement that has made all the difference" a item data could not be obtained for participant c at weeks follow-up for phase (p ) from receiving bonta injections, consistent with the drug's favorable safety and tolerability profile demonstrated in the literature to date [ , ] . one withdrew from the study reporting worsened pain following bonta injections, but it was later noted this participant had initially reported improvements in pain, activities, mood, sleep, and qol post-bonta. the largest limitation to our study was our failure to meet target sample size due to low recruitment and retention rates. bonta injections were offered for free but the time, planning, and costs required of participants to coordinate injection visits were inadequately accounted for. coordinating follow-ups also proved to be difficult once participants received injections, possibly because patients lost incentives to continue participating. these issues could be better addressed with more resources for both improved recruitment and retention in the future. based on our findings, we encourage the field to continue considering bonta as a potential option for treating at-level sci pain. we further hope that sharing our limitations may facilitate additional investigations into this treatment method throughout the field. several encouraging patterns among participants with respect to self-reported average pain levels; pain interference with day to day activities, mood, and sleep; as well as overall qol as related to pain were observed post-bonta injections. these findings are consistent with the literature to date and incite us to continue investigating subcutaneous bonta as a feasible approach for the control of at-level sci pain. survey data are available from the corresponding author upon reasonable request. international spinal cord injury pain (iscip) classification: part . initial validation using vignettes a prospective study of pain and psychological functioning following traumatic spinal cord injury a longitudinal study of the prevalence and characteristics of pain in the first years following spinal cord injury pharmacotherapy for neuropathic pain in adults: a systematic review and meta-analysis the canpain sci clinical practice guidelines for rehabilitation management of neuropathic pain after spinal cord: recommendations for treatment physical medicine and rehabilitation board review botulinum toxin type a for neuropathic pain in patients with spinal cord injury central origin of the antinociceptive action of botulinum toxin type a botulinum toxin a, brain and pain botulinum toxin treatment of pain syndromes-an evidence based review complications of botulinum neurotoxin safety of botulinum toxin type a: a systematic review and meta-analysis treatment of at-level spinal cord injury pain with botulinum toxin a. [presentation] american congress of rehabilitation medicine th annual conference cut point determination in the measurement of pain and its relationship to psychosocial and functional measures after traumatic spinal cord injury: a retrospective model spinal cord injury system analysis screening for neuropathic pain after spinal cord injury with the spinal cord injury pain instrument (scipi): a preliminary validation study botulinum toxin for diabetic neuropathic pain: a randomized double-blind crossover trial subcutaneous injection of botulinum toxin a is beneficial in postherpetic neuralgia botulinum toxin type a for the treatment of trigeminal neuralgia: results from a randomized, double-blind, placebo-controlled trial pain after spinal cord injury: an evidence-based review for clinical practice and research. report of the national institute on disability and rehabilitation research spinal cord injury measures meeting reliability and validity of the international spinal cord injury basic pain data set items as selfreport measures the international spinal cord injury pain basic data set clinically significant change in pain intensity ratings in persons with spinal cord injury or amputation research electronic data capture (redcap)-a metadata-driven methodology and workflow process for providing translational research informatics support the powerful placebo acknowledgements we thank dr. tiffany wong (department of rehabilitation and human performance, ismms, new york, ny) for her assistance with injections during her sci fellowship.funding the onabotulinumtoxina (botox) used for this study was provided by allergan (irvine, ca). funding source personnel had no involvement in the study design; collection, analysis and interpretation of the data; or the writing and submission of this paper for publication.author contributions all listed authors have met all of the following authorship criteria: ( ) conceived and/or designed the work that led to the submission, acquired data, and/or played an important role in interpreting the results. ( ) drafted or revised the paper and approved the final version. ( ) agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. conflict of interest the authors declare no conflict of interest.ethical approval we certify that all applicable institutional and governmental regulations concerning the ethical use of human volunteers were followed during the course of this research.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -m kxc x authors: croop, sarah e. w.; thoyre, suzanne m.; aliaga, sofia; mccaffrey, martin j.; peter-wohl, sigal title: the golden hour: a quality improvement initiative for extremely premature infants in the neonatal intensive care unit date: - - journal: j perinatol doi: . /s - - - sha: doc_id: cord_uid: m kxc x background: following delivery, extremely premature infants are vulnerable to rapid development of hypothermia and hypoglycemia. to reduce local rates of these morbidities, a multidisciplinary team developed a protocol standardizing evidence-based care practices during the first hour after birth. methods: using quality improvement methodology, the golden hour protocol was implemented for all inborn infants < weeks’ gestation. data were collected ( – ) over three phases; pre-protocol (n = ), phase i (n = ), and phase ii (n = ). results: there were no significant differences in infant characteristics. improvements in hypothermia ( % vs % vs %; p = . ), hypoglycemia ( % vs % vs %; p = . ), and minutes to completion of stabilization [median (q ,q ) ( , ) vs ( , ) vs ( , ); p = . ] were observed. conclusions: implementation of an evidence-based, golden hour protocol is an effective intervention for reducing hypothermia and hypoglycemia in extremely premature infants. prematurity is the leading cause of neonatal death and contributes to~ % of childhood disabilities in the united states [ , ] . the highest risks of morbidity and mortality are seen with the most premature infants [ ] . extremely premature (< weeks' gestation), extremely low birth weight (< g) (ep-elbw) infants' mortality rates are - % [ ] . survivors face substantial risk of morbidities including intraventricular hemorrhage (ivh), chronic lung disease (cld), and retinopathy of prematurity (rop) [ ] . these morbidities are predictors of long-term neurodevelopmental, sensorial, and psychiatric disabilities, which affect quality of life and can negatively impact the family [ , [ ] [ ] [ ] . following birth, ep-elbw infants are susceptible to rapid development of hypothermia, hypoglycemia, hypotension, and respiratory failure [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . resuscitation in the delivery room (dr) and stabilization during admission to the neonatal intensive care unit (nicu) involves a series of interdependent tasks and procedures. these interventions must be performed quickly, proficiently, and systematically to minimize the short-term sequelae of prematurity, which contribute to the risk of long-term morbidity and mortality [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the hour immediately following the birth of an ep-elbw infant is referred to as the "golden hour" (gh) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this term denotes the period of time in which medical care to prevent irreversible damage is most effective and represents the inverse relationship between elapsing minutes and likelihood of survival. due to the complexity of the care required at birth, protocols that bundle evidence-based practices and standardize their application to the first few minutes after birth have been used to improve the quality and consistency of care for ep-elbw infants during the gh [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in , vermont oxford network database reports for the nicu at the university of north carolina (unc) children's hospital revealed admission temperatures for ep-elbw infants fell below the unit's goal range ( . - . °c), and the network's reported ranges for similar type nicus. in our unit, % of infants < weeks' gestation and % of infants - weeks' gestation were euthermic compared to and % of similar type nicus in vermont oxford network. a propensity for early hypoglycemia (glucose < mg/ dl) was also noted. at baseline, % of ep-elbw infants were hypoglycemic on admission. in addition, retrospective chart review demonstrated the time from birth to obtaining intravascular (iv) access, subsequent administration of iv fluids and antibiotics, and overall time to completion of admission stabilization was prolonged, increasing the risk for hypothermia and hypoglycemia. in a review of the literature, nine quality improvement (qi) studies were identified presenting outcomes following implementation of a gh protocol in the nicu [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . admission temperature was reported as a short-term outcome measure and improvements in temperatures to goal range were described in eight of the studies [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . one study provided statistical analysis of improvement in admission serum glucose concentration to > mg/dl following protocol implementation [ ] . another described "stable" glucoses throughout the qi project [ ] . the only other study that discussed glycemic control reported an increase in the rate of hypoglycemia which was attributed to increased physiologic stress [ ] . although long-term outcomes were not consistently evaluated in the studies, several authors described improvements in the incidence of ivh, rop, cld, late-onset sepsis, and/or length of stay in their units [ , , , , ] . our goal was to design and implement a systematic, evidencebased approach to the care of ep-elbw infants during the gh. the primary aims were to increase the percentage of temperatures within euthermic range, reduce the incidence of hypoglycemia, and decrease the time to completion of admission stabilization to less than min after birth. a retrospective-prospective study design was used for comparison of data from a preprotocol cohort with postprotocol cohorts. retrospective data were collected january, through march, from electronic medical records. prospective data collection began with project implementation in april, and continued through february, for phase i and march, through march, for phase ii ( table ). the effectiveness of interventions was evaluated using plan-do-study-act (pdsa) cycles in accordance with institutional use of lean six sigma qi methodology [ , ] . this single-center, qi study was conducted in the -bed level iv nicu at unc children's hospital, in chapel hill, nc. the nicu is part of a large academic medical center and serves as a regional referral center for the state. nearly two-thirds of the - infants admitted to the unit each year are inborn. the study population included all inborn infants < weeks' gestation who survived resuscitation in the dr and were admitted to the nicu. exclusion criteria were major congenital anomalies resulting in death within the first h after birth and/or parental decision to provide palliative care on admission. a multidisciplinary team of nicu providers including neonatologists, neonatal nurse practitioners, neonatal fellows, nurses, and respiratory therapists designed the evidence-based bundles included in the protocol. representatives from each of these groups of key stakeholders were chosen to lead the proposed culture change. prior to implementation, presentations demonstrating baseline variation in practices were given to increase awareness of the need for improvement. disciplinary-specific education sessions were provided with each phase. to promote sustainability, gh care was incorporated into the nurses' yearly competencies and new fellows' bootcamp. updates were provided throughout the project to maintain stakeholder engagement. to minimize heat loss, the dr temperature was increased ( - °f) prior to delivery and the radiant warmer was prepared in collaboration with the obstetrical team. in the nicu, the isolette and all supplies were warmed. radiant heat, plastic wrap, and stocking-knit caps were used at delivery in the baseline cohort. with project implementation, a warming mattress was added and the technique for wrapping the infant in plastic was modified to improve integrity during resuscitation. one corner of the plastic was folded over the infant's head and secured with a stocking-knit cap, as plastic head-coverings have been shown to be more effective at preventing heat loss than use of stocking-knit caps alone [ ] . in phase ii, the to achieve early initiation of glucose infusion, peripheral iv access was established in the dr with a maximum of two attempts at placement. on admission to the nicu, a % dextrose infusion was administered peripherally while central umbilical access was being established. following radiographic confirmation of placement, the peripheral infusion was discontinued, and central infusions were initiated using % dextrose, . % amino acid solution via the umbilical venous catheter and . % isotonic amino acid solution via the umbilical arterial catheter. a nicu-specific system for ordering umbilical line placement films was developed in collaboration with the radiology department to reduce time to confirmation of central placement. establishing vascular access was also vital to treating potential sepsis. when indicated, antibiotics were started on admission to the nicu. all lab work including the blood culture was collected as soon as blood return was obtained during central umbilical line placement. once the blood culture was collected, antibiotics could be administered peripherally before central line placement had been completed or confirmed. collaboration with the pediatric pharmacy department improved timing of delivery of antibiotics to the bedside. a respiratory management plan for dr resuscitation was implemented, based on gestational age and risk factors for respiratory compromise (fig. ). this plan was used throughout the project in complement with the neonatal resuscitation program (nrp) guidelines. following publication of the th edition nrp guidelines, cardiorespiratory monitoring in the dr was implemented and initiation of supplemental oxygen was reduced from . to . (table ) [ ] . administration of intramuscular vitamin a and intravenous caffeine was routine for the first two study cohorts. however, there were some inconsistencies with the timing of initiation. in an effort to improve outcomes, the initiation of these medications was standardized (table ) [ , ] . during phase ii, both medications were added to the admission order set in the emr and ordered within the first hour after birth for all ep-elbw infants. many of the interventions implemented with phase ii of the project were focused on improving teamwork and communication. a detailed process flowchart was developed to guide the team through the gh (fig. ) . at baseline, respiratory therapy presence at ep-elbw deliveries was inconsistent. for implementation of phase ii, a respiratory therapist was assigned to attend every gh delivery. an additional, experienced nurse, and an additional, experienced provider were also added to the team. previously, one provider assumed responsibility for establishing the airway and leading the team. with implementation of phase ii, two providers, physician or neonatal nurse practitioner, attended the delivery and divided these roles. the leader was responsible for supervising the resuscitation and stabilization of the infant from birth to completion of the gh as well as communicating with the obstetrical team and the family. the second provider focused on establishing an airway in the dr and inserting umbilical lines in the nicu. at this time, each team member was also assigned specific tasks and positions around the radiant warmer in the dr to improve team dynamics and communication [ ] . just-in-time training simulations were employed to solidify these interventions. when delivery of an infant < weeks' gestation was imminent, the team members assigned to attend reviewed their roles, responsibilities, and positions around the radiant warmer and performed a quick, just-in-time simulation. consistent exposure to each team member's role created a shared mental model of what needed to be accomplished and who was responsible for each task throughout the gh. consequently, the team was able to assume and divide additional responsibilities on the rare occasion one role was unfulfilled. initially, a basic handout reminded providers of the goals of the gh and served as the data collection tool. subsequently, a detailed process flowchart with corresponding questions for data collection replaced the initial handout (fig. ) . all information gathered from the data collection tools was confirmed in the medical record prior to analysis. each phase of the project corresponded to changes in the bundles of interventions (table ) . changes in interventions were determined using pdsa cycles, taking into consideration trends in outcomes, stakeholder feedback, and availability of new products and evidence-based practices. evaluation of short-term outcomes and other process fig. golden hour algorithm initial airway management was based on the infant's gestational age and assessment of risk factors for respiratory failure. choices included intubation with surfactant administration (in/in), intubation for surfactant administration with immediate extubation to cpap (in/out), or cpap without surfactant administration. aa amino acid, cbc w/diff complete blood count with differential, cpap continuous positive airway pressure, cr cardiorespiratory, d %w dextrose % in water; ett endotracheal tube, fio fraction of inspired oxygen, huc hospital unit coordinator; nccc newborn critical care center, nnp neonatal nurse practitioner, ob obstetrician, pal peripheral arterial line, peep positive end expiratory pressure, pip positive inspiratory pressure, piv peripheral intravenous line, poc point-of-care, rn registered nurse, rt respiratory therapist, stat immediately, uac/uvc umbilical arterial and venous catheters measures including time to achieving various gh goals provided benchmarks for determining the effectiveness of interventions. to ensure internal validity, implementation fidelity was evaluated based on the consistency of adherence to protocol interventions. primary, short-term outcome measures were those obtained during admission stabilization. axillary temperatures were measured on admission to the nicu (goal range . - . °c ). serum glucose concentration was obtained during central line placement and measured using point-of-care testing (goal ≥ mg/dl). completion of admission stabilization included dr resuscitation, initiation of iv fluids and antibiotics (when indicated), establishing central access, closing the top of the isolette, initiating humidity, and providing decreased environmental stimulation to approximate the intrauterine environment. process measures that directly contributed to thermoregulation, glycemic control, and time to completion of gh stabilization were tracked. these measures included the percentage of time plastic wrap or thermoregulation suit, warming mattresses, and peripheral iv insertion were utilized in the dr and minutes from birth to admission temperature, initiation of iv fluids, initiation of antibiotics, central line confirmation, and closure of the isolette. long-term outcomes including severe ivh (grade iii or iv hemorrhages), severe cld defined as the need for supplemental oxygen ≥ % or positive pressure at weeks' post menstrual age (pma) or discharge [ ] , rop requiring treatment, pma on the day of discharge home, and mortality served as balancing measures to ensure changes in interventions were not having unintended, negative impacts. statistical process control charts were created in qi macros for excel. outcomes were compared with chi-square or fisher's exact test for categorical data and one-way anova or kruskal-wallis for continuous data using stata statistical software. median values with interquartile ranges are presented due to non-normal distribution of data. statistical significance was set at p values of ≤ . . the study included inborn infants < weeks' gestation. three infants were excluded due to severe congenital anomalies resulting in death shortly after admission. data were collected over three phases; pre-protocol (n = ), phase i (n = ), and phase ii (n = ) ( table ). there were no significant differences in infant characteristics (table ) . adherence to the intended interventions were as follows: the use of plastic wrap or thermoregulation suit was % and % in phases i and ii respectively and the use of warming mattresses was % in both phases. the number of attempts for iv insertion in the dr was limited (max of two) and overall, an iv was placed in the dr for % of infants in phase i and % in phase ii. significant improvements were observed in all three primary outcome measures including the incidence of temperatures outside goal range ( % vs % vs %; p = . ), the incidence of hypoglycemia ( % vs % vs %; p = . ), and median time in minutes to gh stabilization ( vs vs min; p = . ) ( table ). evaluation of long-term morbidity and mortality as balancing measures to stabilize our metrics did not reveal any unintended negative effects (table ) . statistical process control charts were used to analyze process performance. changes in the center line and control limits were based on process shifts that occurred with continuous evaluation and implementation of interventions throughout the project. at baseline, both processes were stable. a few instances of a single measure above the upper control limit or below the lower control limit were noted on both control charts. these special cause variations were related to deviation from or noncompliance with the process. new control limits were calculated for both charts when clinically relevant special cause variation was identified ( fig. a, b) . the limits were recalculated for admission temperature two months after project implementation resulting in a new center line with a euthermic temperature . °c (fig. a) . the limits were recalculated for time to completion during phase ii resulting in a new center line at min (fig. b) . both processes remained stable around the new center line. implementation of an evidence-based gh protocol was effective at significantly improving euglycemia, euthermia, and time to completion of admission stabilization for ep-elbw infants in our nicu. following project implementation, admission temperatures in our unit for infants < weeks' gestation consistently fell within the vermont oxford network's reported ranges for similar type nicus. although we did not consistently meet our goal of completion within the first min after birth, we made significant improvements in time from baseline. our gh protocol was implemented in a more premature population of infants than those of the studies in our review. only one of the review studies focused on a population of infants < weeks' gestation, while inclusion criteria for the remaining studies ranged from < to < weeks' gestation [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, despite a more vulnerable population, the improvements in admission glucoses and temperatures demonstrated in our study were consistent with the results reported by the studies in our review. initially, we noted a slight increase in time which we contributed to the learning curve associated with project implementation. the significant decrease in time to gh stabilization during phase ii was associated with efforts to improve team dynamics and implementation of simulation training. a potential confounder was the increase in the resuscitation and survival of periviable infants ( - weeks' gestation) during the course of our interventions, a phenomenon which was not isolated to our unit [ , ] . we noted that the percent of periviable infants remained stable across the first two cohorts and nearly doubled in the third cohort ( %, %, and % respectively). the inverse relationship between gestational age and survival without morbidities is well-recognized and confers a higher degree of vulnerability to those infants delivered at the "cusp of viability" [ , ] . periviable infants are more susceptible to hypothermia and hypoglycemia and less resilient from the ensuing morbidities [ ] . despite an increasing percentage of more immature infants, the rates of hypothermia and hypoglycemia improved without significant differences in balancing measures. this finding remained when balancing measures were evaluated separately for and week infants. we speculate that implementation of the interventions in our gh protocol may offer a protective effect which may be even more beneficial at lower gestational ages. one limitation to the study was the transfer of stable infants to lower-level facilities. there was a loss of % of the data for cld and % of the data related to rop for survivors transferred prior to weeks' pma. sample size was also a limiting factor. the sample size for phase i of the project was approximately % that of the baseline and phase ii cohorts. although no power analysis was performed, it is likely that the sample size of this study was not adequate to assess for differences in long-term outcomes which were used as balancing measures. the -year span of this project and the retrospective/ prospective cohort design introduce the risk of historical threat to the long-term outcomes which served as balancing measures. although we saw no significant differences in the rates of mortality, ivh, cld, or rop we recognize that these disease processes are multifactorial in etiology. changes in practice over time and other confounding variables, not related to the interventions of the gh might contribute to variations in outcomes and in some instances, provide an alternative explanation of the study findings. other nicus may not have been exposed to the same changes in practice or confounding factors thus limiting the generalizability of the study. the study was a single-center qi project and the generalizability of the protocol is also limited as a result of potential differences in resources, staffing, and physical space. however, transferability is comparatively high as the evidencebased practice bundles that make up the protocol can be modified and adapted to fit the needs of any nicu providing care to ep-elbw infants. gh protocols prioritize maintaining the stability of the most vulnerable population of infants in the nicu through the first, tenuous minutes after birth. these protocols promote efficient care delivery and provide a structure for effective teamwork and communication across multidisciplinary groups. the findings of our study and their consistency with the findings of previous studies support implementation of gh protocols in the nicu to decrease admission hypothermia and hypoglycemia in ep-elbw infants. our findings also fill a gap in the literature related to the care of the periviable infant, suggesting gh protocols may be even more beneficial in this subpopulation. health resources and 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published maps and institutional affiliations. key: cord- -m nlsutd authors: song, zhiquan; xie, ying; guo, zongpei; han, yang; guan, hua; liu, xiaodan; ma, teng; zhou, ping-kun title: genome-wide identification of dna-pkcs-associated rnas by rip-seq date: - - journal: signal transduct target ther doi: . /s - - - sha: doc_id: cord_uid: m nlsutd nan highest binding potential. motif analysis showed that dna-pkcs preferentially binds the agga sequence, which was in accordance with previous findings (fig. c) . then, the docking between dna-pkcs rna-binding sites deduced from the web server pridictor and the rna motif agga was performed on the frodock webserver, and the docking structure was analyzed using pymol software (fig. c) . after analysis with a stringent cutoff,~ rnas were precipitated by dna-pkcs. to categorize the rnas bound by dna-pkcs, we performed kegg analysis. this analysis showed a number of signatures involved in the focal adhesion and receptor-ecm interaction pathways (fig. d) . then, the itga , itga , itgav, sdc , and cd rnas were selected for validation of the rip-seq results. the rip-qpcr results showed different fold enrichment values for these five rnas, which are involved in cell adhesion (fig. e) . regulation of rna alternative splicing is a crucial process in rna-binding proteins function, and aberrant splicing is often associated with various human diseases including cancers; therefore, to discern how dna-pkcs modulates bound rnas, we sought to determine whether dna-pkcs could affect cd alternative splicing. specific primers to amplify the cd standard sequence and variants were designed, and qpcr was performed to examine the expression of different variants after u os cells were treated with nu and nu , which target dna-pkcs. the results showed that v , v , and v increased. next, western blotting further verified that after dna-pk inhibition, expression of cd variants increased (fig. f) . in summary, our findings strongly support a model wherein the dna-pkcs protein controls a variety of biological processes, including alternative splicing, through its rna-binding activity. further work will elucidate the accessory factors of dna-pkcs in regulating alternative splicing and how alternative splicing may contribute to the dna damage response mediated by dna-pkcs. geometry of a complex formed by double strand break repair proteins at a single dna end: recruitment of dna-pkcs induces inward translocation of ku protein autophosphorylation of the dna-dependent protein kinase catalytic subunit is required for rejoining of dna double-strand breaks beyond dna repair: dna-pk function in cancer human ku / interacts directly with htr, the rna component of human telomerase the human telomerase rna component, htr, activates the dna-dependent protein kinase to phosphorylate heterogeneous nuclear ribonucleoprotein a long noncoding rna linp regulates repair of dna double-strand breaks in triple-negative breast cancer dna-dependent protein kinase (dna-pk) phosphorylates nuclear dna helicase ii/rna helicase a and hnrnp proteins in an rna-dependent manner therapeutic targeting of splicing in cancer open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/ . /. key: cord- - ezrjuq authors: li, hongqiang; zhou, runv; wang, chunmei; li, yusheng; zheng, guizhen; jiang, sen; dong, tiancao; bai, jianwen; xu, shumin title: t follicular regulatory cells infiltrate the human airways during the onset of acute respiratory distress syndrome and regulate the development of b regulatory cells date: - - journal: immunol res doi: . /s - - - sha: doc_id: cord_uid: ezrjuq t follicular regulatory (tfr) cell is a cxcr (+)foxp (+) subset of t regulatory (treg) cell with critical roles in regulating germinal center responses and modulating the immune environment in the lymph nodes. studies have shown that the proportion of tfr cells may increase during acute inflammation. in this study, we investigated the role of tfr cells in acute respiratory distress syndrome (ards). we found that tfr cells were significantly enriched in peripheral blood and in mini-bronchoalveolar lavage (bal) during the onset of ards. notably, tfr cells represented the majority of treg cells in the mini-bal samples. tfr cells also showed ctla- , il- , and tgf-β expression, but compared to the non-tfr treg cells, the ctla- and il- expression by tfr cells were slightly reduced. both tfr cells and non-tfr treg cells suppressed the proliferation of autologous cd (+)cd (−) t cells; however, the tfr cells displayed slightly reduced suppression capacity. subsequently, b cells were co-incubated with autologous tfr cells or non-tfr treg cells. interestingly, we found that the frequency of il- (+) breg cells was significantly higher following incubation with tfr cells than with non-tfr treg cells, which suggested that tfr cells were more potent at inducing il- (+) breg cells. together, these results demonstrated that tfr cells were a similar but distinctive subset of treg cells. given that tfr cells were strongly enriched in ards patients, especially in the lung infiltrates, they may exert critical ameliorating effects in ards. t follicular regulatory (tfr) cell is a recently discovered t regulatory (treg) cell subset with critical roles in regulating germinal center responses and modulating the immune environment in the lymph nodes [ ] . tfr cells present features of both t follicular helper (tfh) cells and treg cells, including cxcr , pd- , icos, ctla- , and foxp expression [ ] . currently, it is thought that tfr cells differentiate from treg cells that gain access to the b cell follicles in the spleen, lymph nodes, and peyer's patches [ , ] . following interaction with cognate b cells in the germinal center and the b cell follicles, these treg cells gain fully differentiated tfr phenotype with pd- and icos expression, and may stay in the b cell zones, exit the lymphoid tissues and enter circulation, or infiltrate the peripheral tissues [ , ] . representing approximately % of the total cd + t cells, and % of total treg cells in draining lymph nodes, tfr cells are a small but critical population in the development of antibody responses. in mice, tfr cell depletion resulted in elevated numbers of gc b cells, antigen-specific b cells, and plasma cells, and increased serum antibody titer [ , , ] . sorted human tfr cells (cd + cxcr + cd + cd − t cells) were shown to suppress antibody production in vitro [ ] . mechanistically, tfr cells can suppress tfh cell expansion and cytokine production in a ctla- -dependent fashion [ ] . interestingly, ctla- -deleted tfr cells promoted the production of low-affinity antibodies [ ] , suggesting that tfr cells likely improved the quality of the antibody response by hongqiang li and runv zhou contributed equally to this work. preventing overactive tfh-mediated antibody secretion. recently, studies demonstrated that tfr cells were dynamically regulated in acute and chronic virus infections. during influenza infection or lcmv infection, tfr cells in the lymphoid organs presented a drop in frequency, and it is possible that this drop was required for the generation of virus-specific antibodies [ ] . in contrast, tfr frequency appeared to increase in chronic hiv infections, as well as in chronic hepatitis b and hepatitis c patients [ , ] . the frequency and function of tfr cells in other settings have not been extensively investigated. acute respiratory distress syndrome (ards) is characterized by rapid-onset alveolar damage, hypoemia, interstitial and alveolar edema, fibrin deposition, and fibrocyte recruitment and proliferation in the lung [ ] . despite years of research, ards remains a life-threatening condition with only palliative treatments available [ ] . activation of effector immune cells and secretion of proinflammatory cytokines are found in the lung of ards patients, both of which can increase alveolar membrane permeability and coagulation in a perpetuating positive feedback loop [ ] . on the other hand, treg cells in humans and murine acute lung injury (ali) models can disrupt the proinflammatory process, reduce disease severity, promote injury resolution, and accelerate healing [ ] [ ] [ ] [ ] . in addition, we previously showed that early recruitment of il- -producing breg cells and elevated il- secretion could suppress the production of proinflammatory cytokines and predicted better survival in ards patients [ , ] . the role of tfr cells, on the other hand, has not been elucidated in ards patients. in this study, the tfr cells in ards patients were examined. all procedures of sample collection and experiment were approved by the shanghai east hospital ethics committee. peripheral blood and mini-bronchoalveolar lavage (bal) samples were collected from ten ards patients who provided written informed consent. diagnosis and treatment were performed at shanghai east hospital. all patients satisfied the american-european consensus for ards, including acute onset, ratio of partial pressure of arterial oxygen to fraction of inspired oxygen less or equal to , bilateral infiltrates on frontal chest radiograph, pulmonary artery wedge pressure less or equal than mmhg, and no evidence of left atrial hypertension [ ] . ten age, sex, and bmi-matched healthy controls without prior history of pneumonia, ards, or other respiratory diseases donated peripheral blood samples. peripheral blood mononuclear cells (pbmcs) or mini-bal lymphocytes were harvested using standard ficoll-paque (ge healthcare) centrifugation method. a portion of fresh pbmcs and all mini-bal samples were used directly ex vivo. the remaining pbmcs were stored at − °c until further use. for sorting, pbmcs or mini-bal lymphocytes were incubated with anti-human cd (hit a), cd (a a ), cxcr (j d ), and cd (bc ; biolegend) monoclonal antibodies for min on ice. cells were then washed twice in sterile pbs supplemented with % fbs (gibco), and sorted in facsaria system (bd biosciences). for frequency analysis, pbmcs or mini-bal lymphocytes were incubated with anti-human cd , cd , cxcr , and cd monoclonal antibodies for min on ice, and washed twice. the foxp /transcription factor staining kit (ebioscience) and anti-human foxp monoclonal antibody (clone pch ; ebioscience) was then used according to the manufacturer's protocols. samples were analyzed in the lsr system (bd biosciences). greater than × events were acquired for pbmc samples, and greater than × events were acquired for mini-bal samples. following sorting, tfr and non-tfr treg cells were lysed and treated with the rneasy mini kit (qiagen) to collect total rna. cdna synthesis was then performed using high-capacity cdna reverse transcription kit (thermo fisher scientific) following protocol from the manufacturer. qpcr was performed in the abi prism system (applied biosystems) with pre-packaged taqman gene expression assays (thermo fisher scientific) for human ctla- (hs _m ), il- (hs _m ), tgf-β (hs _m ), and β microglobulin (hs _m ). three independent replicates were performed for each assay. tfr/treg suppression assay cd + cd − t cells were sorted from pbmcs and plated in well round-bottom plates at × cells per well. sorted tfr or non-tfr treg cells were then added to each well at numbers specified per experiment. cells were stimulated with t activator beads (thermo fisher scientific). after h incubation, cells were pulsed with . μci/ml tritiated thymidine (amersham biosciences) for h, and harvested. the amount of incorporated radioactive thymidine was examined using a beta counter. three independent replicates were performed for each assay. tfr/treg-b cell coculture b cells from ards pbmcs were sorted using human b cell enrichment kit (stemcell), and were incubated with sorted tfr cells or non-tfr treg cells at / b/t ratio in the presence of μg/ml seb (sigma). after h, cells were incubated with anti-human cd (biolegend), fixed and permeabilized using cytofix/cytoperm buffer (bd biosciences), and incubated with anti-human il- (biolegend). excess antibodies were removed by washing, and the samples were analyzed in the lsr system. data between two samples were compared using unpaired t test with welch's correction. data between multiple samples were compared using regular or repeated-measures (rm) oneway or two-way anova, as specified per experiment. twotailed p values smaller than . were considered significant. to investigate tfr cells, pbmcs from ten ards patients at day of disease onset, and from age, sex, and bmimatched healthy control volunteers were collected. the frequency of foxp + treg cells and foxp + cxcr + tfr cells was determined by flow cytometry (fig. a) . in healthy controls, the frequency of foxp + treg cells in cd + t cells was . % ± . % (mean ± s.d. for all results), and the frequency of foxp + cxcr + tfr cells in cd + t cells was . % ± . % (fig. b) . in ards patients, the frequency of foxp + treg cells in cd + t cells was . % ± . %, and the frequency of foxp + cxcr + tfr cells in cd + t cells was . % ± . %, both of which were significantly higher compared to that in healthy controls (p < . ). the frequency of non-tfr treg cells, calculated by the frequency of treg cells minus the frequency of tfr cells, was also slightly higher in ards patients than in healthy controls (p < . ). the expression level of foxp was not significantly different between tfr cells and non-tfr treg cells, and not significantly different between healthy controls and ards patients (p > . for all comparisons; fig. c ). in order to investigate the infiltration of treg and tfr cells in the affected lung tissue, mini-bal was performed on day , day , and day after ards onset. the frequency of treg cells and tfr cells was examined in the mini-bal samples by flow cytometry (fig. a) . first, we observed that the foxp expression level in mini-bal samples was not significantly different between tfr cells and non-tfr treg cells (fig. b) . also, the foxp expression by tfr cells from the pbmcs was not significantly different from the foxp expression by tfr cells from mini-bal samples (p > . for all comparisons). in most ards subjects, the frequencies of treg and tfr cells presented an increasing trend (fig. c) . for treg cells, the frequency in mini-bal increased from . % ± . % on day , . % ± . % on day , to . % ± . % on day . for tfr cells, the frequency in mini-bal increased from . % ± . % on day , . % ± . % on day , to . % ± . % on day . the non-tfr treg cells, on the other hand, were not different between day and day , but were elevated between day and day . notably, the tfr cells represented a minor subset in treg cells from pbmcs, but in mini-bal, tfr cells represented the major treg subtype. tfr cells expressed inhibitory molecules at similar or moderately reduced levels compared to non-tfr treg cells next, we sought to determine the function of tfr cells. both tfr (foxp + cxcr + cd + ) cells and non-tfr treg (foxp + cxcr − cd + ) cells presented high cd expression compared to foxp − cd + t cells (fig. a) . we sorted tfr cells as cd + cd + cxcr + t cells, and non-tfr treg cells as cd + cd + cxcr − t cells from pbmcs of healthy controls ards patients, and ards mini-bal samples were sorted using fluorescence activated cell sorting (facs). the cells were then lysed for the collection of mrna. the expression levels of ctla- , il- , and tgf-β were analyzed by rt-pcr. the expression of ctla- in non-tfr treg cells and tfr cells from ards pbmcs and ards mini-bal was significantly higher than the expression of ctla- in non-tfr treg cells and tfr cells from healthy control pbmcs (fig. b) . the ctla- expression in non-tfr treg cells and tfr cells from ards mini-bal was further increased compared to the ctla- expression in non-tfr treg cells and tfr cells from ards pbmcs. tfr cells from ards mini-bal presented lower ctla- expression than non-tfr treg cells. the il- expression by non-tfr treg cells and tfr cells was lower in healthy controls and significantly higher in ards pbmcs and ards mini-bal (fig. c) . non-tfr treg cells and tfr cells from ards mini-bal presented significantly higher il- expression than cells from ards pbmcs. in both ards pbmcs and ards mini-bal, the il- expression by tfr cells was lower than the il- expression by non-tfr treg cells. non-tfr treg cells and tfr cells from healthy controls presented significantly lower tgf-β expression than the non-tfr treg cells and tfr cells from ards patients (fig. d) . the non-tfr treg cells and tfr cells from ards mini-bal presented significantly higher tgf-β expression than the non-tfr treg cells and tfr cells from ards pbmcs. no significant differences between non-tfr treg cells and tfr cells in terms of tgf-β expression were observed. to further analyze tfr and non-tfr treg function, we sorted tfr cells as cd + cd + cxcr + t cells and non-tfr treg cells as cd + cd + cxcr − t cells from ards pbmcs. these cells were then incubated with autologous cd + cd − t cells in the presence of tcr (anti-cd /cd ) stimulation for h, and pulsed with tritiated thymidine for h. the capacity to suppress cd + cd − t cell proliferation was compared between tfr cells and non-tfr treg cells. both non-tfr treg cells and tfr cells were capable of suppressing the proliferation of autologous cd + cd − t cells (fig. ) . the tfr cells were less effective than non-tfr treg cells at high ( / ) regulatory-to-effector ratio. subsequently, the interactions between tfr cells, non-tfr treg cells, and b cells were examined. b cells from ards pbmcs were co-incubated with tfr (cd + cd + cxcr + ) cells or non-tfr treg (cd + cd + cxcr − ) cells in the presence of seb for h. the frequency of il- + breg cells was evaluated before and after stimulation by flow cytometry. the frequency of il- + b cells was significantly increased following incubation with both non-tfr treg cells and tfr cells (fig. ) . interestingly, tfr cells were significantly more potent at increasing il- + breg cell frequency than non-tfr treg cells. the current consensus suggests that treg cells promote ards resolution and recovery and may suppress fibroblast recruitment and proliferation [ , ] . hence, treg cells are considered a beneficial cell type in ards. in this study, we focused on a newly characterized subset of treg cells, the foxp + cxcr + tfr cells, and investigated their frequency and function. three major discoveries were made: first, both the treg cells and the tfr cells were significantly enriched in ards patients compared to in healthy controls; second, compared to the non-tfr treg cells, the tfr cells were slightly less effective in some, but not all, aspects of suppression; and third, compared to non-tfr treg cells, the tfr cells were more effective at promoting il- expression in b cells. overall, these results demonstrated that tfr cells presented similar, but different functions compared to non-treg cells. this study examined the tfr frequency in two distinctive samples, the circulating blood and the local lung infiltrates, represented by the mini-bal samples. a notable feature is that in mini-bal, the vast majority of treg cells were, in fact, tfr cells. in addition, tfr infiltration in the lung increased with the duration of disease onset. hence, the functional difference between non-tfr treg cells and tfr cells may become critical fig. the frequency of treg cells and tfr cells in the pbmcs from ards patients and healthy controls. a fresh pbmcs from ards patients and healthy controls were collected and examined using flow cytometry directly ex vivo. figures shown were pre-gated on cd + t cells from one representative ards patient and one representative healthy control. treg cells were gated as total foxp + cd + t cells, and tfr cells were gated as foxp + cxcr + cd + t cells. b the frequencies of treg cells, tfr cells, and non-tfr treg cells in the pbmcs from ten healthy controls and ten ards patients. c the mean fluorescence intensity (mfi) of foxp , compared between tfr cells and non-tfr treg cells in healthy controls and ards patients. unpaired t test with welch's correction. ***p < . . ns not significant in the determination of disease outcome. a number of mysteries remain. first, since there was no mini-bal sample from controls, it is yet unclear whether tfr cells also infiltrate healthy lungs. second, tfr frequency in mice increased significantly in the lung draining lymph nodes and in blood following influenza infection, which was another disease that involved significant inflammation in the respiratory tract and the lung [ ] . whether tfr upregulation is a general feature of lung inflammation requires further analysis. in order to investigate tfr function, we examined and compared the expression of inhibitory molecules in three samples, including the healthy pbmcs, the ards pbmcs, and the ards mini-bal. we found that the expressions of ctla- , il- , and tgf-β by healthy tfr and non-tfr treg cells were much lower than those by ards tfr and non-tfr treg cells. this unlikely indicated that the tfr or non-tfr treg cells from healthy individuals were defective, but rather, it was likely that tfr and non-tfr treg cells in ards samples were more activated and expressed more effector molecules. the higher il- and tgf-β in ards mini-bal compared to autologous pbmcs likely indicated that the tfr and non-tfr treg cells in the lung infiltrates were further activated. interestingly, although the expression of ctla- and il- , as well as the capacity to suppress cd + cd − t cell proliferation, were slightly reduced in tfr cells compared to non-tfr treg cells, the tfr cells presented higher capacity in inducing il- + breg cells than non-tfr treg cells. possibly, the common cxcr expression by tfr cells and b cells allowed both to localize close to each other, thus increasing interaction. whether tfr cells may represent a specialized breg-helper cell type requires further investigation. of note, the experiments were performed in vitro, where tfr cells, non-tfr treg cells and b cells were placed in the same tissue culture. in vivo, the requirement for common cxcr expression might be much higher for colocalization of t cells and b cells to occur. in chronic hepatitis b and hepatitis c patients, the frequencies of il- + breg cells and tfr cells were both upregulated [ ] . whether a common pathway upregulated both cell types, or one cell type promoted the upregulation of the other, still require further analysis. a number of limitations were present in this study. first, the samples, especially those from mini-bal, were limited in availability. as a result, the suppression studies were performed using tfr cells from pbmcs. since the tfr cells from fig. the frequency of treg cells and tfr cells in the mini-bal from ards patients at day , day , and day after disease onset. a fresh lymphocytes from mini-bal were collected and examined using flow cytometry immediately following isolation. figures shown were pre-gated on cd + t cells from day , day , and day of the same ards patient. treg cells were gated as total foxp + cd + t cells, and tfr cells were gated as foxp + cxcr + cd + t cells. b the mfi of foxp in tfr cells and non-tfr treg cells from the mini-bal. unpaired t test with welch's correction. c the frequencies of treg cells, tfr cells, and non-tfr treg cells in the mini-bal from ten ards patients. rm -way anova followed by tukey's test. *p < . . **p < . . ***p < . . ns not significant fig. the expression of inhibitory molecules by non-tfr treg cells and tfr cells from healthy controls and ards patients. a fresh pbmcs from healthy controls, pbmcs from ards patients, and fresh day mini-bal lymphocytes from ards patients were examined by flow cytometry directly ex vivo. the expression levels of cd by tfr (foxp + cxcr + cd + ) cells (solid black line), non-tfr-treg (foxp + cxcr − cd + ) cells (dotted black line), and foxp − cd + t cells (gray area) in one representative from each of the three samples were shown. b the tfr cells were sorted from each frozen-and-thawed sample as cd + cd + cxcr + t cells, and the non-tfr treg cells were sorted from each sample as cd + cd + cxcr − t cells. the expression levels of ctla- , il- , and tgf-β from each cell type from ten healthy control pbmcs, ten ards pbmcs, and ten ards day mini-bal are represented as ratios over β microglobulin (β m). two-way anova followed by sidak's test. *p < . . **p < . . ***p < . . ns not significant fig. tfr and non-tfr treg-mediated promotion of il- expression in b cells. from frozen-and-thawed ards pbmcs, the tfr cells were sorted as cd + cd + cxcr + t cells, and the non-tfr treg cells were sorted as cd + cd + cxcr − t cells. b cells were enriched using negative selection, and were incubated with tfr cells or non-tfr treg cells at / b/t ratio. after h incubation with seb, the frequency of il- + b cells was examined by flow cytometry. rm two-way anova followed by sidak's test. ***p < . . ns not significant. symbols directly above the datasets indicate the difference between the -h experiment and the -h control fig. tfr and non-tfr treg-mediated suppression of cd + cd − t cells. from frozen-and-thawed ards pbmcs, the tfr cells were sorted as cd + cd + cxcr + t cells, and the non-tfr treg cells were sorted as cd + cd + cxcr − t cells. the cd + cd − t cells (effector) were also sorted and incubated with tfr or non-tfr treg (regulatory) cells at the indicated ratios. after h in the presence of t activator beads (anti-cd / cd ), cells were pulsed for h with tritiated thymidine and harvested, and the amount of radioactive thymidine incorporation was examined. rm two-way anova followed by sidak's test. *p < . . ***p < . . ns not significant. symbols directly above the datasets indicate the difference between the labeled dataset and the no regulatory cell control ( / ) dataset mini-bal samples tended to present higher expressions of inhibitory molecules, it is possible that the tfr cells from mini-bal samples had more potent capacity to mediate suppression. also, our in vitro experiments need to be verified in animal models of ards to prove that tfr cell-mediated effects could ameliorate or prevent tissue damage mediated by inflammation. in addition, specific gene knockout experiments are necessary to investigate which inhibitory molecules are required for tfr cell-mediated suppression in vivo. conflict of interest the authors declare that they have no conflict of interest. ethical approval all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the helsinki declaration and its later amendments or comparable ethical standards. informed consent informed consent was obtained from all individual participants included in the study. t follicular regulatory cells t follicular regulatory cells in the regulation of b cell responses circulating t follicular regulatory and helper cells have memorylike properties the receptor pd- controls follicular regulatory t cells in the lymph nodes and blood association between chemotherapy-response assays and subsets of tumor-infiltrating lymphocytes in gastric cancer: a pilot study regulation of the germinal center reaction by foxp + follicular regulatory t cells human t-follicular helper and t-follicular regulatory cell maintenance is independent of germinal centers the coinhibitory receptor ctla- controls b cell responses by modulating t follicular helper, t follicular regulatory, and t regulatory cells hiv-infected spleens present altered follicular helper t cell (tfh) subsets and skewed b cell maturation increased numbers of cd +cd +cd dhighil- + bregs, cd +foxp + tregs, cd + cxcr +foxp + follicular regulatory t (tfr) cells in chb or chc patients acute respiratory distress syndrome: a clinical review epidemiology, patterns of care, and mortality for patients with acute respiratory distress syndrome in intensive care units in countries acute lung injury and the acute respiratory distress syndrome: four decades of inquiry into pathogenesis and rational management regulatory t cells reduce acute lung injury fibroproliferation by decreasing fibrocyte recruitment regulatory t cell dna methyltransferase inhibition accelerates resolution of lung inflammation cd +cd +foxp + tregs resolve experimental lung injury in mice and are present in humans with acute lung injury regulatory t cells contribute to the recovery of acute lung injury by upregulating tim- upregulation of cd +cd hicd hi regulatory b cells is associated with a reduced risk of acute lung injury in elderly pneumonia patients early recruitment of il- -producing b cells into alveoli improved the resolution of acute lung injury definitions, mechanisms, relevant outcomes, and clinical trial coordination key: cord- -dyk mr q authors: zheng, yong; deng, yan; gao, jian-mei; lv, chun; lang, ling-hu; shi, jing-shan; yu, chang-yin; gong, qi-hai title: icariside ii inhibits lipopolysaccharide-induced inflammation and amyloid production in rat astrocytes by regulating ikk/iκb/nf-κb/bace signaling pathway date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: dyk mr q β-amyloid (aβ) is one of the inducing factors of astrocytes activation and neuroinflammation, and it is also a crucial factor for the development of alzheimer’s disease (ad). icariside ii (ics ii) is an active component isolated from a traditional chinese herb epimedium, which has shown to attnuate lipopolysaccharide (lps)-induced neuroinflammation through regulation of nf-κb signaling pathway. in this study we investigated the effects of ics ii on lps-induced astrocytes activation and aβ accumulation. primary rat astrocytes were pretreated with ics ii ( , , and μm) or dexamethasone (dxms, μm) for h, thereafter, treated with lps for another h. we found that ics ii pretreatment dose dependently mitigated the levels of tumor necrosis factor-alpha (tnf-α), interleukin- beta (il- β), inducible nitric oxide synthase (inos), cyclooxygenase- (cox- ) in the astrocytes. moreover, ics ii not only exerted the inhibitory effect on lps-induced iκb-α degradation and nf-κb activation, but also decreased the levels of aβ( – ), aβ( – ), amyloid precursor protein (app) and beta secretase (bace ) in the astrocytes. interestingly, molecular docking revealed that ics ii might directly bind to bace . it is concluded that ics ii has potential value as a new therapeutic agent to treat neuroinflammation-related diseases, such as ad. alzheimer's disease (ad) is a progressive neurodegenerative disease characterized by beta-amyloid (aβ) peptide fibrils, which are extracellular depositions of a particular protein and are accompanied by extensive neuroinflammation [ ] [ ] [ ] . a number of studies have reported that inflammation, which may precede amyloid deposition, exerts vital effects in the pathogenesis of ad [ , ] . moreover, inflammatory mediators increase the expression of amyloid precursor protein (app) and the formation of aβ and upregulate beta-secretase activity [ ] . therefore, antiinflammatory drugs may prevent or treat ad by inhibiting neuroinflammation, thereby reducing the production or deposition of aβ [ ] [ ] [ ] [ ] . however, there are still no ideal antiinflammatory drugs to prevent or treat ad. astrocytes and microglia are important components of homeostasis in the brain [ ] . when the brain is exposed to undesirable environmental conditions, both astrocytes and microglia, which are crucial perpetrators of inflammation and potential neuronal dysfunction [ , ] , acquire special "response" or "activation" phenotypes [ , ] . in ad, the interaction between microglia and astrocytes may be of great significance for the development of neurodegenerative disease. in particular, astrocytes represent the most plentiful cell type in the brain and play an imperative role in maintaining the homeostasis of the central nervous system [ ] [ ] [ ] . under physiological conditions, astrocytes play a role in supporting and separating nerve cells [ ] [ ] [ ] [ ] . nevertheless, under neuroinflammatory conditions, the excessive activation of astrocytes is involved in the inflammatory response through its ability to release multiple molecules and further lead to neurodegenerative diseases [ ] [ ] [ ] . epimedium is a traditional chinese herb used for the treatment of cardiovascular diseases, osteoporosis, and sexual and neurological disorders [ ] . icariside ii (ics ii) is known as one of the major active pharmaceutical ingredients of epimedium, and it has been indicated to have an extensive range of pharmacological effects, including antiinflammatory [ , ] , anticancer [ , ] , antioxidative [ , ] , and antiaging activities [ ] . our previous study found that ics ii attenuates streptozotocin-or aβ - -induced cognitive deficits in rats by increasing the number of surviving neurons in the hippocampus [ ] or inhibiting neuronal apoptosis and reducing pde protein expression [ ] . in our previous in vivo studies, we proved that ics ii exerts beneficial effects on lipopolysaccharide (lps)-induced neuroinflammation by regulating the tlr /myd /nf-κb signaling pathway in rats and inhibiting lps-induced astrocyte overactivation [ ] . however, whether ics ii can suppress neuroinflammatory responses in vitro remains unclear. thus, in the current study, we investigated the effects of ics ii on lipopolysaccharide (lps)-induced neuroinflammation in primary astrocytes and the underlying molecular mechanism. ics ii (purity ≥ %) was obtained from nanjing zelang medical technology corporation ltd (nanjing, china). dmem/f was obtained from hyclone (logan, ut, usa), and fetal bovine serum (fbs) was purchased from gibco (thermo fisher scientific, ma, usa). dexamethasone (dxms) and lps (l ) (escherichia coli o :b ) were purchased from sigma-aldrich (st louis, mo, usa). dxms and lps were dissolved in normal saline at concentrations of mm and mg/ml, respectively, and ics ii was dissolved in dimethyl sulfoxide (dmso) at a concentration of mm. anticyclooxygenase- (cox- ) (#ab ), anti-nitric oxide synthase (inos) (#ab ), antinuclear factor-κb (nf-κb) (p ) (#ab ), anti-p-nf-κb (p ) (#ab ), anti-iκb-α (#ab ), anti-ikk-α (#ab ), anti-p-ikk-α (#ab ), anti-ikk-β (#ab ), anti-p-ikk-β (#ab ), and anti-β-site app cleaving enzyme (bace ) (#ab ) antibodies were purchased from abcam (cambridge, uk). anti-app (#ab b) was purchased from bbi life sciences corporation (shanghai, china). tumor necrosis factor-alpha (tnf-α) enzyme-linked immunosorbent assay (elisa) kits (an interleukin- beta (il- β) elisa kit, an aβ - elisa assay kit (jl ), and an aβ - elisa kit (jl )) were purchased from shanghai jianglai biotechnology (shanghai, china). a nitric oxide (no) detection kit (s ) was purchased from beyotime biotechnology (shanghai, china). astrocyte culture and drug treatment sprague-dawley rats ( ± g) were housed under a -h-light/ dark cycle at a humidity of % ± % and a temperature of °c. all animals were given free access to water and food. animal experiments were performed in compliance with the state committee of science and technology of the people's republic of china order no. of november , , and the protocol was approved by the experimental animal ethics committee of the zunyi medical university. primary astrocytes were extracted from the safe concentration range of ics ii within h (n = ). c the safe concentration range of ics ii within h (n = ). d the safe concentration range of ics ii within h (n = ). * p < . , ** p < . versus control group neonatal rat brains as previously described [ ] . primary astrocytes were isolated from the cerebral cortex of -h-old neonatal rats. briefly, suckling rats were repeatedly disinfected with % alcohol three times, and then the brains were removed, and the brain tissues were separated. then, the cerebral cortex was collected under low temperatures, and the meninges and blood vessels were slightly removed. the cerebral cortex was fully dissociated by the addition of ml of trypsin, the flask was treated with polylysine, and the suspension was spread in a culture flask at a cell density of × cells/ml. the differential adherence method was used to remove other types of cells in the tissue, and then astrocytes were cultured in dmem/f containing % fbs. the dmem/f was changed every days until the th day. the primary cells were shaken at °c at a constant temperature to remove other glial cells, such as microglia and oligodendrocytes. thereafter, the astrocytes were fixed with % paraformaldehyde, incubated with an anti-gfap antibody ( : , abcam), and visualized using an alexa fluor-conjugated secondary antibody ( : , abcam). more than % of the cultured astrocytes were identified by gfap staining, and the astrocytes were pretreated with different concentrations of ics ii or dxms ( μm) for h. thereafter, they were treated with lps for another h. since dxms is an effective and classical antiinflammatory drug, it was selected as a positive control drug to evaluate the antiinflammatory effect of ics ii, and μm was chosen as the desired concentration of dxms according to a previous study [ ] . cell viability was assessed using the -[ , -dimethylthiazol- -yl]- , -diphenyl tetrazolium bromide (mtt) assay as described in our previous study [ ] . in brief, astrocytes were seeded in a -well plate at × cells/well and treated as described above. then, μl of mtt ( mg/ml) was added to fbs-free medium and cultured for another h. the mtt was removed, and the cells were lysed with μl of dmso in each well. the optical density was measured at nm using a microplate reader. the results of the treatments were expressed as a percentage of the control. in brief, astrocytes were inoculated into -well plates at a density of × cells/well and pretreated with ics ii ( , , μm) for h. lps ( mg/ml) and ics ii were added to the plates for h. thereafter, the culture medium was collected and centrifuged for min at × g. the supernatants were then collected and used to measure tnf-α, il- β, aβ - , and aβ - levels using elisa kits. all data were obtained from at least three independent experiments. measurement of nitric oxide (no) astrocytes were inoculated in -well plates at a density of × cells/well and pretreated with ics ii ( , , μm) for h. lps ( mg/ml) and ics ii were added to the plates for h. the accumulation of nitrite in the supernatant was evaluated using the griess reaction. each μl of supernatant was reacted with an equal volume of griess reagent and cultured for min at room temperature. the absorbance was detected in a microplate absorbance reader at a wavelength of nm, and a series of known concentrations of sodium nitrite was utilized as a standard. western blot analysis western blot analysis was performed as described in our previous study [ ] . briefly, astrocytes were treated as mentioned above, homogenized with protein extraction solution, and lysed for min on ice. the lysate was centrifuged for min at × g. ) . then, the blots were subjected to the corresponding horseradish peroxidase-conjugated anti-rabbit or mouse immunoglobulin g. thereafter, immunoreactive proteins were measured using an enhanced chemiluminescence western blotting detection system. statistical analysis all data were analyzed by spss . statistics software and are expressed as the mean ± sd. the data were analyzed using oneway anova followed by the post hoc least significant difference. all data were obtained from at least three independent experiments. p < . was considered significant. we extracted the cultured cells and identified them by staining for gfap, which is a specific marker of astrocytes [ ] . the results showed that astrocytes made up % of the cultured cells (fig. a) . furthermore, the results showed that ics ii ( . , . , . , . , , or μm) for , , or h had no effect on the astrocytes; however, ics ii ( , , or μm) for , , or h was cytotoxic to the astrocytes. since the concentration of dmso used to dissolve ics ii in the experiment did not exceed . %, it was assumed that the concentration of dmso itself was not toxic to the cells and that the toxicity to the cells therefore resulted from the high concentration of ics ii. thus, a concentration of ics below μm was considered to be the safe concentration range (fig. b-d) . thereafter, , , and μm ics ii were used in subsequent experiments, and dxms ( μm) was used as a positive control agent. the effect of ics ii on the lps-induced production of tnf-α and il- β in astrocytes was determined using an elisa assay. the results showed that ics ii ( , , or μm) slightly mitigated the production of tnf-α (fig. a) and il- β (fig. b) . following lps stimulation, the production of tnf-α and il- β was substantially elevated compared with that in the control group, while ics ii markedly alleviated lps-induced tnf-α and il- β overproduction in a concentration-dependent manner. the effect of ics ii on lps-induced no production and inos and cox- expression in astrocytes was determined using the griess reaction and western blot analysis, respectively. the results showed that ics ii decreased lps-induced no production in astrocytes (fig. c) . moreover, inos and cox- expression levels were dramatically increased after lps insult. however, ics ii ( , , and μm) induced a concentration-dependent decrease in the expression of inos and cox- in astrocytes compared with that in the lps group ( fig. d-f ). the results suggested that ics ii not only suppressed the lpsinduced phosphorylation of p (fig. a, b) but also prevented the nuclear translocation of p (fig. c-f ). furthermore, ics ii also suppressed the lps-induced degradation of iκb-α (fig. a, b) , ikk-α, and ikk-β (fig. c, d) . these results indicate that ics ii mitigates the lps-induced activation of nf-κb via inhibiting iκb-α phosphorylation and the translocation of p to the nucleus. the effect of inflammation on amyloid formation in vitro was also investigated because neuroinflammation can cause amyloid production, whereas the aberrant activation of astrocytes is a major source of neuroinflammation. astrocytes provide both mechanical and metabolic support to neurons, regulating the environment in which they function. to determine the relationship between neuroinflammation and amyloidogenesis, we investigated whether the antiinflammatory effect of ics ii can result in antiamyloidogenesis. as shown in fig. a , b, when the cells were unstimulated, they expressed low protein levels of app and bace , whereas the protein expression of app and bace increased in response to lps after h. in addition, ics ii also decreased lps-induced aβ - and aβ - secretion into the culture media of astrocytes (fig. c, d) . in astrocytes, we also found that ics ii inhibited the lps-induced expression of app and bace , as well as aβ - and aβ - levels in a concentration-dependent manner. these results further indicate that the amyloidogenic pathway can be promoted by neuroinflammatory stimulation and that the antiinflammatory effect of ics ii can result in antiamyloidogenesis. interestingly, mock molecular docking was used to verify whether ics ii binds to bace protein, and the results showed that the binding energy of ics ii and bace was − . kcal/mol, which confirmed that ics ii can bind to bace , as the standard threshold for a molecule to bind to a protein is thought to be greater than or equal to − kcal/mol (fig. a, b) . we further investigated the supposed binding modes and interactions within the amino acid pocket, including gly , gly , gly , thr , tyr , gly , lys , asp , and phe (fig. c, d) . taken together, these findings show that ics ii may directly affect bace and thus exert neuroprotective effects. the present study revealed that ( ) ics ii protects against lpsinduced inflammation in primary-cultured astrocytes; ( ) the inhibitory effect of ics ii is due to regulation of the ikk/iκb/nf-κb signaling pathway; and ( ) ics ii decreases aβ - and aβ - levels by downregulating app and bace expression (fig. ) . lps is an effective component of gram-negative bacilli endotoxin, which can cause a series of inflammatory reactions in the body and is widely applied to establish animal or cellular inflammatory models. moreover, astrocytes represent the most abundant cell type in the central nervous system, and they exert a variety of physiological functions through close association with neurons and other brain structures. similar to microglia, the immune and inflammatory properties of astrocytes, which can promote the secretion of various neuroinflammatory factors, such as tnf-α and il- β, are activated by lps. accumulating evidence has demonstrated that the production of multiple neuroinflammatory factors, including tnf-α, il- β, and inos, can activate the nf-κb pathway, resulting in neuroinflammation [ ] . moreover, the excessive production of neuroinflammatory factors leads to neuronal damage through the activation of glial cells. notably, astrocytes play a key role in central nervous system inflammation by producing cytokines, such as tnf-α, il- β, and no, leading to neuronal injury [ ] . therefore, the suppression of astrocyte activation may be an effective treatment for neuroinflammation-related diseases [ ] . our findings showed that lps induces an increase in proinflammatory factors, which is consistent with the results of previous studies [ , ] . ics ii significantly inhibited the lps-induced accumulation of tnf-α, il- β, no, inos, and cox- in primary culture astrocytes. in addition, these effects were not due to the cytotoxic effect of ics ii, as evidenced by the observation that there was no effect on astrocytes at concentrations up to μm. moreover, ics ii also inhibited inos and cox- protein notably, nf-κb not only regulates a variety of inflammatory factors, such as il- β and tnf-α but also plays a key role in mediating cox- and inos expression [ ] . nf-κb (p /p ) heterodimers exist in the cytoplasm of resting cells. however, under stimulation by lps, the phosphorylation of ikk was induced, which subsequently phosphorylates the iκb protein, resulting in the release of nf-κb (p /p ) heterodimers. then, nf-κb-p translocates from the cytoplasm to the nucleus, thereby promoting the release of proinflammatory factors, including tnf-α and il- [ , ] . consistent with these studies, the present study showed that lps induces the phosphorylation of nf-κb-p , iκb, and ikk. however, ics ii abolished these changes, suggesting that ics ii exerts potent antiinflammatory effects, at least partly through modulating the ikk/iκb/nf-κb pathway. app is a membrane-intrinsic protein expressed in a variety of tissues and is closely related to ad. app can be cleaved by α, β, and γ proteases, and the continuous action of β-protease and γprotease can cause app to be cleaved to produce aβ [ ] . however, aβ can cause the formation of senile plaques in the brain and the apoptosis of neuronal cells, thereby causing ad. most importantly, mounting evidence has shown that neuroinflammation is related to the accumulation of aβ in the brains of ad patients [ , ] . in addition, app is first cleaved by β-secretase at its β-cleavage site and is then proteolyzed by the second enzyme, γ-secretase, to produce aβ - and aβ - . in particular, the aβ - and aβ - peptides are involved in the amyloidogenic pathway; aβ - is the most plentiful species, and aβ - shows the strongest neurotoxicity [ , ] . of note, bace is an important rate-limiting enzyme in the app-aβ pathway and plays an important role in the production of aβ. bace was overexpressed in the brains of animal models of ad [ ] . in contrast, in the brains of bace knockout mice, the levels of aβ and sappβ are decreased [ ] . in addition, activated astrocytes that release mediators can induce app and bace expression, thereby stimulating aβ production [ ] [ ] [ ] [ ] . notably, nf-κb mediates the accumulation of app and the expression of bace to increase aβ production [ , ] . in this study, lps not only augmented the production of aβ - and aβ - but also upregulated app and bace , and these findings are consistent with those of previous studies [ , ] . however, ics ii reversed these changes, revealing that ics ii exerts inhibitory effects on neuroinflammation via suppressing the nf-κb/bace signaling pathway, and this was also evidenced by molecular docking. in conclusion, the current study revealed that ics ii exerts inhibitory effects on lps-induced inflammation in astrocytes through the ikk/iκb/nf-κb/bace signaling pathway, and thus ics ii may be a promising therapeutic agent for neuroinflammatory diseases, including ad. elevated cns inflammation in patients with preclinical alzheimer's disease is 'friendly fire' in the brain provoking alzheimer's disease? modulation of inflammation in transgenic models of alzheimer's disease review: systemic inflammation and alzheimer's disease rodent models of neuroinflammation for alzheimer's disease inflammation combined 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processing of amyloid precursor protein without effect on neuregulin- inflammation in alzheimer disease: driving force, bystander or beneficial response large-scale production of soluble recombinant amyloid-beta peptide - using cold-inducible expression system regional and sub-regional differences in hippocampal gabaergic neuronal vulnerability in the tgcrnd mouse model of alzheimer's disease chitosan oligosaccharides inhibit/ disaggregate fibrils and attenuate amyloid beta-mediated neurotoxicity gene structure and organization of the human beta-secretase (bace) promoter increased nf-kappab signalling up-regulates bace expression and its therapeutic potential in alzheimer's disease neuro-inflammation induced by lipopolysaccharide causes cognitive impairment through enhancement of beta-amyloid generation qhg and jss designed the experimental protocols. yz carried out all the studies except the western blot analysis, which was performed by cl, lhl, and yd. yz wrote the manuscript with help from qhg, cyy, and jmg. competing interests: the authors declare no competing interests. key: cord- - w weox authors: fredman, gabriella; kolpen, mette; hertz, frederik boetius; petersen, pelle trier; jensen, andreas vestergaard; baunbæk‐egelund, gertrud; ravn, pernille; jensen, peter Østrup; faurholt‐jepsen, daniel title: the inflamed sputum in lower respiratory tract infection: l‐lactate levels are correlated to neutrophil accumulation date: - - journal: apmis doi: . /apm. sha: doc_id: cord_uid: w weox lower respiratory tract infections (lrti) are common, but little is known about the response of biomarkers of inflammation in the lungs. therefore, our primary aim was to compare the concentration of l‐lactate to the concentration of neutrophils in sputum and systemic markers of infection. because it is difficult to differentiate viral and bacterial infection on the basis of clinical presentation in lrti, our secondary aim was to evaluate if l‐ and d‐lactate may serve as markers of local inflammation as representatives of neutrophils and bacteria, respectively. methods: patients with acute lrti were prospectively recruited. sputum samples were collected and analysed for neutrophil count, l‐lactate and d‐lactate. we had data on pathogens from sputum cultures and polymerase chain reaction (pcr) (atypical bacteria, virus) and c‐reactive protein (crp) from blood. results: in sputum samples from patients, the median (interquartile range (iqr)) sputum neutrophil granulocyte count was . × ( ) cells/ml ( . – . ). the sputum neutrophil granulocyte count was associated with sputum l‐lactate (p = . ) and crp (p = . ), but not with d‐lactate (p = . ). there was a strong association between sputum d‐lactate and l‐lactate (p < . ). conclusion: as l‐lactate in sputum is closely correlated to sequestration of neutrophils in the lungs, l‐lactate is a marker for local inflammation in lrti and a potential biomarker in clinical management of lrti. on expectorated sputum, d‐lactate had no clinical relevance. lower respiratory tract infections (lrti) are common. the response of biomarkers of inflammation in the lungs is, however, far from fully understood. markers of inflammation and pulmonary infection have been studied in patients with chronic obstructive pulmonary disease (copd) and cystic fibrosis (cf) ( ) ( ) ( ) . cf-related chronic airway inflammation and tissue remodelling are thought to be caused by neutrophils that are present in large numbers in the airways of cf patients ( ) . in a study of sputum from cf patients with pulmonary exacerbation, there was an association between sputum neutrophil count and sputum lactate ( ) . however, this study did not differentiate between the two isomers of lactate. increased markers of neutrophilic inflammation in sputum have been found to be associated with bacterial infection in copd exacerbations ( ) , potentially leading to increased levels of both l-lactate and d-lactate. in lrti, the differentiation between virus and bacteria, based on the clinical presentation in lrti, is difficult. in hospital settings, a causative pathogen is not detected in nearly half of pneumonia cases and often it is a mix of pathogens ( ) ( ) ( ) ( ) . to reduce unnecessary prescription of antibiotics in viral lrti, there is a need for markers to differentiate viral from bacterial infections when detection fails. in nature, lactate exists in two isoforms, l-lactate and d-lactate, produced from pyruvate by the isomeric-specific enzymes, l-lactate dehydrogenase (l-ldh) and d-lactate dehydrogenase (d-ldh) ( ) . l-lactate is produced in human tissue from pyruvate during anaerobic metabolism of carbohydrate and is the primary form of lactate in humans ( ) . d-lactate is only produced by lower organisms equipped with d-ldh such as bacteria ( ) . d-lactate synthesis is possible in mammals via the methylglyoxal pathway, but this pathway only generates moderate amounts of d-lactate in the bloodstream ( ) . blood d-lactate level in healthy humans is often undetected by appropriate laboratory analysis, because it is usually so low ( ) . in contrast, little is known about the response of lactate isomers towards bacteria and virus at the site of infection, namely in the lungs. studies on the clinical utility of d-lactate as an indicator of ongoing bacterial infection in various compartments indicate that d-lactate may prove useful and be a supplement in assessing the effect of antimicrobial therapy ( , ) . in bacterially infected pleural fluid, the sensitivity of d-lactate as an indicator for ongoing bacterial infection was % and the specificity was % ( ) . therefore, our secondary aim was to assess the utility of sputum d-lactate as marker of pulmonary bacterial invasion. markers of inflammation and pulmonary infection have been studied in patients with copd and cf ( - ). cf-related chronic airway inflammation and tissue remodelling are thought to be caused by neutrophils that are present in large numbers in the airways of cf patients ( ) . in a study of sputum from cf patients with pulmonary exacerbation, there was an association between sputum neutrophil count and sputum lactate ( ) . however, this study did not differentiate between the two isomers of lactate. increased markers of neutrophilic inflammation in sputum have been found to be associated with bacterial infection in copd exacerbations ( ), potentially leading to increased levels of both l-lactate and d-lactate. altogether, we aimed to assess the utility of sputum l-lactate and sputum d-lactate as markers of pulmonary neutrophilic inflammation and bacterial invasion in the lungs of patients admitted with acute lrti. the study was a prospective observational study involving patients admitted to the emergency ward with acute infection. patients were enrolled prospectively soon after admission based on clinical suspicion of pneumonia or any other cause of lrti if patients had new purulent expectoration. as participants at enrolment did not comply with the criteria for community-acquired pneumonia on admission x-would not be available to all eligible patients, the lrti was defined as the working diagnosis based on clinical judgement involving purulent expectoration as well as minimum of one of the following: cough, fever, chest pain or (when available) an infiltrate on x-ray. only patients who were ≥ years old and able to expectorate could be enrolled. participants were recruited consecutively during workdays (monday to friday) either during their stay in the emergency ward or after transfer to the department of lung and infectious diseases at university hospital nordsjaelland between november and march . patients not able to expectorate, not willing to participate or unlikely to survive the following h were excluded. the study was approved by the regional scientific ethical committees (vek h- ) and was in accordance with the declaration of helsinki. all study subjects gave written informed consent for their inclusion in the study. data on demography and co-morbidities were obtained from a questionnaire given to the participants on the day of inclusion. same day, paraclinical parameters (e.g. blood leucocyte count and crp) were obtained from medical records. data on prior antibiotic treatment and corticosteroid use were obtained from both the questionnaire and the medical records. research samples were prioritized secondarily to routine samples for microbiological assessment. sputum was collected during admission only on enrolment and repeated if the participant was able to produce a purulent expectorated sample on a subsequent day. sputum samples were diluted ( ) and homogenized in phosphate-buffered saline (pbs), ph . (substrate department, panum institute, denmark) and stored at À °c until analysis. if the sputum was not already sent for routine analysis, the samples were divided into two aliquots, of which one was sent for routine analysis to the department of clinical microbiology, herlev university hospital. results were reported as the presence and identity of the pathogens detected in sputum. respiratory pathogens were identified from culture using biochemical methods and/or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof ms). atypical bacteria (mycoplasma pneumoniae, chlamydophila pneumoniae, chlamydophila psittaci and legionella pneumophila) were identified using real-time pcr, and respiratory viruses were identified using pcr (e.g. metapneumovirus, rhinovirus, influenza a, influenza b, parainfluenza - and respiratory syncytial virus (rsv)). the concentration of neutrophils in the sputum is shown as the exact numbers and was determined by adding ll of diluted sputum to a trucount tube (bd ; becton dickinson). in addition, ll of each of the following mouse anti-human monoclonal antibodies (bd biosciences, la jolla, ca, usa) was added to the samples: peridinin chlorophyll a protein-labelled cd percp (bd ), fluorescein isothiocyanate (fitc)labelled cd (bd ) and allophycocyanin (apc)labelled cd (bd ). the samples were incubated for min and fixated with ml of fluorescence-activated cell sorter (facs) lysis solution (bd ) and stored overnight in dark at °c before analysis by flow cytometry using a facscanto (bd biosciences) equipped with a mv argon-ion laser tuned at nm, and a red laser-emitting diode at nm for excitation. light-scatter and exponentially amplified fluorescence parameters were recorded from at least events. leucocytes were identified by gating on cd and the polymorphonuclear leukocytes (pmn) were discriminated by gating on cd and cd as previously described ( ) . the instrument was calibrated with cs&t beads (bd biosciences, franklin lakes, nj, usa). the concentration of d-lactate in sputum was measured in samples using a d-lactate colorimetric assay kit (mak , sigma-aldrich, missouri, mo, usa) according to the manufacturer's recommendations. samples were diluted ( ) if the useful concentration range of . - mm d-lactate was exceeded. frozen samples with diluted sputum were thawed and transferred to a -well microtitre plate. d-lactate concentration was estimated by the addition of a reaction mix of d-lactate buffer, d-lactate enzyme mix and d-lactate substrate for min in dark at room temperature, whereby the d-lactate was quantitated by the optical density at nm measured with an enzyme-linked immunosorbent assay (elisa) plate reader (thermo scientific multiskan ex; thermo fisher scientific inc., bioimage, søborg, denmark). the d-lactate concentrations were determined by the interpolation of values generated from a standard curve. the concentration of l-lactate in the sputum was measured in samples using a l-lactate assay kit (mak , sigma-aldrich) according to the manufacturer's recommendations. samples were diluted ( x) if the useful concentration range of - mm l-lactate was exceeded. frozen samples with diluted sputum were thawed and transferred to a -well microtiter plate. the l-lactate concentration was estimated by the addition of a master reaction mix of lactate assay buffer, lactate enzyme mix and lactate probe for min in dark at room temperature, whereby the l-lactate was quantitated by the optical density at nm measured with an elisa plate reader (thermo scientific multiskan ex). the l-lactate concentrations were determined by the interpolation of values generated from a standard curve. normally distributed data are expressed as mean (standard deviation) and non-normally distributed expressed as median with - th interquartile range (iqr). categorical variables are expressed as number and percentage. continuous variables not adhering to the normal distribution were analysed with non-parametric statistics. correlation analyses were based on linear regression models presented as beta (b) coefficient and % confidence interval ( % ci). regression analyses were performed to investigate the association between neutrophil granulocyte count in sputum with l-lactate and d-lactate. we used multilevel mixed models to adjust for clustering of individuals with more than one sputum sample to account for intra-individual correlation. post-hoc q-q plots over standardized residuals were performed to verify the assumption of normally distributed data. outliers were identified by plotting the data, but were kept in the final models, as the outliers did not alter the correlations reported (data not shown). a p-value ≤ . was considered statistically significant. descriptive statistics and linear regression including q-q plots were performed in stata (statacorp lp, college station, tx, usa), and graphs were made with graphpad prism . (graphpad software, la jolla, ca, usa). thirty-two participants met the criteria and participated in the study. sixteen ( %) were females, and the median iqr age was years ( - ) ( table ) . data on pre-existing pulmonary disease were available for ( . %) participants, with ( %) having copd, eight ( %) having asthma and two ( . %) having pulmonary fibrosis. a total of sputum samples were collected from participants. although admitted to the emergency ward, participants were clinically stable with a mean (range) respiratory rate of per minute ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , heart rate of beats per minute ( - ), systolic blood pressure mmhg ( - ), peripheral oxygen saturation of % ( - ) and temperature of . c ( . - . ). antibiotic treatment had been initiated in all ( %) participants when research samples were collected. among ( . %) participants with data on corticosteroid treatment, ( . %) used oral prednisolone, ( . %) used inhaled corticosteroid and five ( . %) with mixed use. the neutrophil count was assessed in sputum samples. the median neutrophil count was . cells/ml (iqr . - . ). levels of measured parameters are shown in table . the measurement of l-lactate was performed in sputum samples with a median of . (fig. c) , but the neutrophil count was not associated with the level of d-lactate in sputum (b = . , % ci [À . - . ], p = . ) (fig. b) . we also found an association between sputum l-lactate and sputum d-lactate (b = . , % ci [ . - . ], p < . ) (fig. d) there was no difference in the level of blood crp in participants with a negative sputum culture, compared to those with verified bacteria (b = . , % ci [À . - . ] n = , p = . ). there was no difference in the level of blood crp in participants with a verified viral lrti, compared to those without (b = À . , % ci [À . - . ] n = , p = . ). we measured the neutrophil count and the two isoforms of lactate (land d-lactate) in sputum in patients admitted with acute lrti and found an association between the sputum neutrophil count and l-lactate, but not d-lactate. we saw an includes two samples of mixed bacterial and viral aetiology and includes three sputum samples with more than one bacterial pathogen. includes two samples of mixed bacterial and viral aetiology. association between both l-lactate and d-lactate with viral infection, but not between l-lactate and d-lactate with bacterial infection. there was no difference in sputum neutrophil count among participants with and without verified pathogens. earlier studies of sputum neutrophil count in copd and cf patients have demonstrated that the augmented neutrophil count during exacerbation decreases to baseline values after eradication of the infection ( , ) . it is reasonable to believe that elevated sputum neutrophil count in acute lrti declines when treated appropriately. the low detection rate of bacteria in the clinical laboratory may explain why we did not see an association between d-lactate and bacteria as we had expected. during bacterial infection, neutrophil granulocytes are recruited from the bloodstream to the focus of infection ( ) . in pneumonia, this is expected to result in increased neutrophil count in the lungs and sputum. however, the inaccessibility of spontaneously expectorated sputum from non-inflamed persons without infection prevents the inclusion of sputum samples from healthy persons as appropriate control. our finding of neutrophil count in the lrti sputum being unaffected by the detection of virus or bacteria is in line with copd patients during exacerbation ( ). this indicates a strong contribution of viral infection to the lung inflammation in lrti patients. earlier studies demonstrated that the lungs are capable of releasing lactate by metabolizing glucose, although the net balance of lactate is close to zero in healthy lungs ( ) ( ) ( ) . in the human neutrophil metabolism, lactate is produced in glycolysis by the cleavage from ldh ( ) . in disease states such as acute lung injuries, the net production of pulmonary lactate increases and correlates with the severity of the lung injury ( , ) . this lactate production is thought to be due to augmented cytokine effects on pulmonary and inflammatory cells ( , ) . in addition, viral infection of human cells may induce the warburg effect ( ) ( ) ( ) leading to production of l-lactate ( ) . the elevated levels of l-lactate in sputum and the association between sputum l-lactate and sputum neutrophil count strengthened our theory that l-lactate is a metabolite from human neutrophils and that raised levels of l-lactate may indicate ongoing airway inflammation in lrti. previous studies of lactate production and increase in acute lung injuries have not differentiated between the two isoforms, l-and d-lactate ( , ) . in mammals, the amount of l-ldh exceeds the amount of d-ldh by far, and therefore l-lactate is the primary isoform of lactate in humans ( ) . the metabolic activity in bacteria halts immediately during the early phase of antibiotic treatment, and d-lactate has shown to be a potentially useful biomarker for successful therapy ( ) . measuring d-lactate during antibiotic treatment in various body compartments has been promising as an indicator of decrease in bacterial load ( ) . in our study, d-lactate was detected in sputum, but we could not demonstrate an association between the sputum neutrophil count and sputum d-lactate, indicating that d-lactate does not originate from neutrophils. instead, as we have assumed in this study, bacteria may be the source of d-lactate. however, this assumption is challenged by the lack of association between bacteria and d-lactate in our study. we speculate that the antibiotic treatment may have confounded the distribution of d-lactate in the sputum samples by reducing the bacterial production of d-lactate. virus and bacteria were detected in . % and %, respectively, in sputum samples from patients with acute lrti in our study, which is in line with a previous study ( ) . however, we only detected % with a mixed bacterial/viral lrti. this is in contrast with previous studies' finding of - % with a mixed infection ( , , ) . this lack of detected microbes may have been prevented by using bronchoalveolar lavage, which is a very useful tool for diagnosing lrti ( ) . despite that antibiotics had been initiated in all participants, the rate of positive cultures was relatively high in our study. one could consider if antibiotics, when eradicating sensitive bacteria leading to less diversity, allow overgrowth of other bacteria not involved in infection. that could explain why there is no association between bacterial infection and d-lactate in our study, but between viral infection and d-lactate. it has already been suggested that virus may disrupt the respiratory mucosal epithelium and impair the immune system as seen in influenza, where there is often a superinfection with staphylococcus aureus ( , , ) . that is supported by the strong association between sputum d-lactate and l-lactate. from the present study, d-lactate does not qualify as a good marker to differentiate bacterial from viral infection, while l-lactate seems to be strongly associated with severity of local pulmonary inflammation. the research sampling was done at the first encounter with the participants already admitted and after initiation of antibiotics. sampling should preferably have been done directly at admission before initiation of antibiotic treatment. we could not prioritize research samples over clinical samples, as this could impair detection rates, but unfortunately almost a fifth of samples for microbiology were contaminated or lost, which reduced the number of specimens detected. sampling should preferably have been done before initiation of antibiotic treatment, as culture of common pathogens in lrti (e.g. streptococcus pneumoniae) is difficult after initiation. thus, it is likely that some of the cultured samples from infected participants contained non-viable bacteria that could not be identified in the clinical laboratory and which potentially could have been found if a different pcr had been used. further, culture on expectorated samples treated with antibiotics (e.g. penicillin) may cause opportunistic growth of mainly gram-negative bacteria (e.g. escherichia coli), which are rarely a lrti pathogen, but this was not seen. our study has shown that l-lactate in sputum is associated with the neutrophil invasion in the lungs mixed bacterial/viral lrti n = ( . %). thirteen positive sputum samples from patients. in acute lrti. although d-lactate in sputum was a marker of specific interest, since it is unique to bacterial metabolism, it had no clinical relevance in the current study. both sputum l-lactate and d-lactate were associated with viral lrti but not bacterial, suggesting a greater inflammatory response in viral or mixed viral/bacterial than bacterial lrti. this suggestion may be further verified by comparing to blood-derived biomarkers proposed to discriminate between bacterial and viral or non-bacterial lrti such as procalcitonin ( ) . as l-lactate is widely available to measure in laboratory settings, we recommend further studies to focus on the usefulness of this marker, and how it represents the invasion of leucocytes into the lungs. furthermore, l-lactate should be evaluated as a marker to monitor treatment response directly in the lung, which may be more sensitive than systemic markers such as crp. finally, as patients admitted with lrti often stop producing sputum early in the course of infection, l-lactate should be tested in patients who are intubated or having a tracheostomy, where routine tracheal suctions are performed. the study was supported by the nordsjaellands hospital. alveolar inflammation in cystic fibrosis lactate in cystic fibrosis sputum infections and airway inflammation in chronic obstructive pulmonary disease severe exacerbations community-acquired pneumonia etiology of community-acquired pneumonia: increased microbiological yield with new diagnostic methods incidence and characteristics of viral community-acquired pneumonia in adults penicillin treatment for patients with community-acquired pneumonia in denmark: a retrospective cohort study d-lactic acid production as a monitor of the effectiveness of antimicrobial agents d-lactic acid measurements in the diagnosis of bacterial infections polymorphonuclear leucocytes consume oxygen in sputum from chronic pseudomonas aeruginosa pneumonia in cystic fibrosis neutrophil left shift and white blood cell count as markers of bacterial infection utilization of blood sugar and formation of lactic acid by the lungs lactate, pyruvate, glucose, and free fatty acid in mixed venous and arterial blood the fate of circulating lactic acid in the human lung energy metabolism of human neutrophils during phagocytosis lactate production by the lungs in acute lung injury pulmonary lactate release in patients with sepsis and the adult respiratory distress syndrome bench-to-bedside review: lactate and the lung induction of the warburg effect by kaposi's sarcoma herpesvirus is required for the maintenance of latently infected endothelial cells global metabolic profiling of infection by an oncogenic virus: kshv induces and requires lipogenesis for survival of latent infection targeting epstein-barr virus oncoprotein lmp -mediated glycolysis sensitizes nasopharyngeal carcinoma to radiation therapy -methoxyestradiol reverses the procarcinogenic effect of l-lactate in osteosarcoma b cells comprehensive molecular testing for respiratory pathogens in community-acquired pneumonia viral-bacterial coinfection affects the presentation and alters the prognosis of severe community-acquired pneumonia bronchoalveolar lavage as a diagnostic tool viral potentiation of bacterial superinfection of the respiratory tract insights into the interaction between influenza virus and pneumococcus procalcitonin to initiate or discontinue antibiotics in acute respiratory tract infections none. key: cord- -bc fj h authors: pekmezaris, renee; kozikowski, andrzej; pascarelli, briana; handrakis, john p.; chory, ashley; griffin, doug; bloom, ona title: participant-reported priorities and preferences for developing a home-based physical activity telemonitoring program for persons with tetraplegia: a qualitative analysis date: - - journal: spinal cord ser cases doi: . /s - - - sha: doc_id: cord_uid: bc fj h study design: focus group. objectives: the purpose of this qualitative study was to explore perceptions and priorities of persons with spinal cord injury (sci) for physical activity and to incorporate their feedback to inform future development of a physical activity program delivered via a telemonitoring platform. setting: new york. methods: qualitative data were collected from a purposive sample of adults with tetraplegia (n = ). two investigators led an audio-recorded focus group using a moderator’s guide. data were analyzed using a six-phase thematic analysis approach. results: the discussion focused on two major areas, which resulted in multiple derived themes and subthemes. the first theme centered on the daily life of persons with tetraplegia, including changes after sci, gain of function prioritization, and identification of psychosocial support systems that facilitate community reintegration after injury. the second theme centered on participant perceptions and recommendations for a physical activity program delivered via a telemonitoring platform. desired design features included variations in schedule, diverse activities, or exercises included in each class, and optional two-way video to enable social interactions with classmates. conclusions: participants favorably viewed the concept of a physical activity program delivered via a telemonitoring platform and contributed program design ideas. although this was a small sample size, challenges to obtaining physical activity expressed by participants were consistent with those identified previously in larger studies of persons with tetraplegia. therefore, we expect these concepts and their recommendations to be relevant to the greater sci community. approximately , persons in the us are living with traumatic spinal cord injury (sci) [ , ] . due to reduced mobility, persons with sci are at increased risk for developing obesity, muscle atrophy, osteoporosis, accelerated atherogenesis, type ii diabetes mellitus, and other medical consequences that increase the risk of stroke and coronary heart disease [ ] [ ] [ ] . this reduced mobility often has deleterious psychosocial effects that impact quality of life, including increased social isolation, reduced social participation, reduced exercise self-efficacy, and depression [ , ] . thus, there is a critical need for therapeutic strategies that reduce the risk of multiple medical and psychosocial consequences of sci. physical activity is a recommended therapeutic strategy to reduce risks of common medical consequences across diverse clinical populations [ , ] . physical activity reduces risks of coronary heart disease and diabetes, increases immunity and blood circulation, and decreases inflammation, fat, anxiety, pain, and improves mood and sleep [ ] [ ] [ ] [ ] . the american college of sports medicine recommends that able-bodied adults perform min of moderate-intensity aerobic exercise and participate in two or more days of muscle-strengthening exercise weekly [ ] . the latest physical activity guidelines for adults with sci recommend, "at least min of moderate to vigorousintensity aerobic exercise two times per week and three sets of strength exercise for each functioning muscle group, at moderate to vigorous intensity, two times per week" [ , ] . for cardiometabolic health benefits, it is recommended that adults with sci engage in at least min of moderate to vigorous-intensity aerobic exercise three times per week [ , ] . persons with sci and other disabilities are less likely to engage in regular physical activity, due to many modifiable barriers. these include: lack of knowledge about existing programs/safe exercises, insufficient programming, lack of transportation, cost, and scheduling issues [ ] . there are also other barriers, such as feeling too hot or cold outdoors or distance from an adaptive sports facility [ ] . in the general population, telemonitoring approaches to delivering physical activity are part of a highly successful commercial fitness industry. consumers are offered the ability to choose a program to engage in at home, with recorded or live classes, that can be delivered to a tv, tablet, phone, or computer via a commercial internet provider. compared to a gym membership, telemonitoring is convenient, scalable, and relatively low cost. regardless of the modality, telemonitoring physical activity programs often require minimal exercise equipment and are delivered at home on a personalized schedule. in addition to the physical health benefits, such as increased muscle strength and improved cardiovascular fitness, many physical activity instructors also engage actively in motivational strategies, to promote adherence and increase exercise self-efficacy [ ] . increasingly, telemonitoring enables a participant to experience self-efficacy in the following ways: ( ) mastery of experiences, the strongest predictor of self-efficacy, relate to actual performance when successfully meeting a challenging task. participants performing daily health behaviors and seeing progress, experience mastery. ( ) vicarious modeling (seeing others facing similar challenges and reaching their goals) will be achieved by viewing other participants of similar abilities attaining activity goals. ( ) social persuasion (verbal encouragement) is provided by the instructor. ( ) physiological factors, such as anxiety and distress, can be experienced by participants when they fail to meet activity goals; the instructor can interpret this as situational and not associated with overall success [ , ] . home-based physical activity delivered via telemonitoring may be a particularly useful option for persons with sci as a way to modify common environmental barriers to achieve the benefits of regular physical activity [ ] . to address these and other barriers, telehealth approaches are being increasingly studied in the context of sci [ ] . sweet and colleagues are starting an rct of an -week tele-rehab program for persons with paraplegia to measure changes in psychosocial variables related to exercise participation and quality of life [ ] . another study measured the effects of a home-based exercise program in persons with chronic sci, including outcome measures of metabolism, body composition, physical activity, energy intake, measures of health and wellbeing, resting metabolic rate, heart rate, and blood pressure, aerobic capacity, immune function, and adipose gene expression [ ] . encouraging results using telemonitoring have been obtained across physical health measures (i.e., wound care), as well as psychological health [ ] . there is a need to establish novel methods to facilitate regular physical activity for persons with sci [ ] . here, we report the results of a qualitative study of priorities and preferences for developing a home-based physical activity telemonitoring program for persons with tetraplegia. we consider this to be a first step towards optimizing feasibility and acceptability in a physical activity program for persons with sci [ ] . this is a qualitative study of adults with chronic (at least year from injury) tetraplegia who were recruited from the ny metropolitan area. the rationale for including only persons with tetraplegia was because, in general, this group has fewer opportunities for achieving physical activity in their daily life, lower reference values of cardiovascular fitness (relative vo peak), higher risk factors for cardiovascular disease, and lower life expectancy than persons with paraplegia. a short demonstration video developed by the study team was presented to participants to show the concept of a telemonitoring physical activity program led by a physical therapist for persons with tetraplegia. moderators explained that they envisioned that participants would join the class via a tablet with a split screen that showed themselves, the instructor, and classmates conducting exercises. moderators described that an instructor would monitor vital sign data (heart rate and blood oxygenation) of participants in real time via a pulse oximeter. before engaging in exercise, participants would be trained on proper equipment use. for safety, participants would be asked by the instructor every min during the intervention, to describe any symptoms of discomfort, including pain (musculoskeletal or other), fatigue, shortness of breath, or dizziness. frequency, duration, and type of proposed activities are based on the most recent guidelines on physical activity for persons with sci [ ] . the intervention presented was proposed to be delivered three times/week for min, with ≥ min of activity. the circuit training program proposed was based on evidence of strength and cardiorespiratory benefit in persons with sci [ ] . stretching, cardiovascular, and strengthening exercises would be tailored to participants' functional abilities. theraband, with open handgrips (loops), would be used to provide resistance for strength training [ ] . moderators explained that the program would consist of three repetitions of: (a) warm-up: a series of active (nonresisted) movements: shoulder lateral raises, flies, shoulder rolls, wide biceps curls, shoulder shrugs, triceps extensions to rear; (b) circuit exercise program: resistance followed by aerobic (arm spinning) exercises with rest periods as needed (~ s). resisted movements would include: set : seated rows, horizontal shoulder abduction, arm spinning/circles (aerobic exercise), set : shoulder internal rotation, shoulder external rotation, aerobic exercise, set : straight arm pulldowns, chest press, aerobic exercise [ ] . a -h focus group was conducted in january , led by two moderators previously unknown to participants. moderators used a moderator guide with open-ended questions and probes, related to a range of relevant topics including experiences with and priorities for benefits of physical activity before and after their injuries, technology use, and perceptions of important features that should be incorporated into a telemonitored physical activity program. the discussion was digitally recorded (using two recorders in case of technical failure), stored on an internal password protected server to ensure security, and transcribed professionally. transcripts were checked against the original recordings for accuracy. to optimize credibility, transferability, and dependability of results, we utilized analyst triangulation, peer debriefing, and conducted an audit trail of decisions made during the analysis and rationale. the transcript was analyzed by two researchers (a k and b p), to achieve triangulation to gain a more complex understanding of the data. a six-phase thematic analysis approach was utilized [ , ] . in the first phase, transcripts were reviewed independently multiple times to become familiar with the data. researchers documented initial theoretical and reflective thoughts, and potential codes and themes. in the second phase, researchers focused on data patterns and generated a comprehensive set of codes through inductive and deductive coding. two researchers documented their reasoning for coding blocks of text from the transcript of the focus group to explain how the data were perceived and examined. the third phase consisted of searching for themes after coding and codes were collated. in the fourth phase, themes were reviewed and refined. criteria for retaining themes were that they needed to be specific enough to be concrete, while broad enough to capture ideas. themes with sparse data were eliminated and those with large amounts of data were further divided into separate themes. in the fifth phase, team members met and discussed the finalization of theme names. in the sixth phase, the report was generated. participants were persons with tetraplegia (n = : males and females) who were wheelchair users for community mobility. the discussion explored challenges of living with tetraplegia, gain of function prioritization, social networks, and design recommendations for a telemonitored physical activity program. participants were asked to rank their gain of function prioritization on a seven-point scale, with one being most, and seven being least important, in the following areas: arm/ hand function, upper body/trunk strength and balance, bladder/bowel function, lived experiences of sexual function, elimination of chronic pain, sensation and mobility ("mobility could be anything that gets your body around in space") [ ] . most participants ranked either arm/hand function, sensation, or improvement of mobility as the most important. the next gain of function priorities ranked was upper body/trunk strength and balance, elimination of chronic pain, and sexual function. two major discussion themes emerged from a six-phase thematic analysis approach to the transcript. theme one: daily challenges pain several participants described challenges of performing activities of daily living (adls) while experiencing constant pain. the locations of pain symptoms varied by individual, including the back, neck, shoulders, and feet. multiple participants reported that pain symptoms were worse in the morning and did not resolve completely throughout the day. "right now, i feel like someone's kicking me in the back but that's normal for me, so it's just one of those things you kind of deal with…" "…i have chronic back pain that just will not go away. it's probablyif i say on a scale of one to ten, it's probably around a good eight most of the day…" "i'm in pain every day when i get home. i'm in bed by : because i can't even function." participants also discussed how pain impacted feelings of fatigue and strategies to cope with interruptions in sleep. "and you talk about getting exhausted during the day. i want to sleep every day by : . but i'm at work, so i can go in my office for a little while just to try to rest for a minute." multiple participants reported being athletic and active prior to their sci, had careers in physically enduring professions or participation in active sports including swimming, motocross, running, cycling, and skiing. "yeah, motor cross. yeah. so i used to ride-a lot of cycling, a lot of swimming. i had a home gym that i worked out in all the time. running-i was a terrible runner because my knees weren't that great. so i would run a little bit, but not that much. mostly cycling. i loved cycling. anything with wheels, i was there." "i was very free spirited, i'd say. we'll put it that way. but yeah, i sort of was very spontaneous and enjoyed flying by the seat of my pants and all that. it's like losing a little piece of you." as expected, the intensity and type of physical activity changed for most participants after injury. most focus group participants were not engaged in regular physical activity, outside of the exercises prescribed during physical therapy. for participants who were active, post-injury activities include using a stationary bike, thera-bands for resistance training, and free weights. "when i first came home, i was doing them every day. and then little by little, you're slacking off. but like i said, every day, once i get into bed, that's when i do the most thera-bands or weights or anything because i'll put a wrist [adaptor] on my arm. i'll go on my side and i'll do the left arm. then i get turned the other way, i'll do the right arm." "...everything from thera-bands like you were talking, to cuff weights. i use cuff weights as well that-most people use them on their ankles when they're running or exercising, but they also work great for quads around your arm. it's like a velcro weight. the rickshaw [wheelchair rehab exercise machine]…. it's a great machine for people in wheelchairs. and they have another machine there which is called the upper tone…it's kind of a home gym-type looking machine that's specifically designed for people in wheelchairs and people with limited hand function." variation in physical therapy and interactions with physical therapy personnel were discussed and perceived as impacting the post-injury rehabilitation process. "i've been to great therapists and i've been to not-sogreat therapists, and what they did clinically was not that different from each other. the difference was the therapists' behavior, the interaction." "i mean, it felt like it's a total package there. you get a lot of focused attention. youand theystart on the dime and they give you every second of that hour." participants discussed the critical role of social networks (family and friends) in community reintegration after injury. in addition, participants were motivated and inspired by interacting with peers with sci who demonstrated resilience. "…when you see people getting better, it helps. it makes you believe you can do the same thing too." the importance of self-efficacy to obtaining functional gains was also discussed, including the importance of maintaining both physical and emotional health. the feelings of well-being obtained from exercise were reported to reinforce the desire to continue exercising. "when i'm in a good mood, i feel i can conquer the world. but when i'm in a lousy moodlike today is not a great mood for meis i don't feel good about anything, and i don't want to do anything because i'm miserable. but then tomorrow i'll feel great and say i can pretty much take on the world and do anything i want and just let me do what i got to do." "yeah, inspirational. yeah, it would raise inspiration, want me to build more muscle on my end to want to feel better and know that i'm healthier and to keep going for whatever reason, whether it's for walking or not." "and i can tell you personally that i should probably be further along physically than i am… i think i plateaued and then went the other direction because of my own inability to push those things out of my mind…like if your head's not there, like in anything in life, but especially with sci rehab, it's hard enough knowing that this happened." participants discussed the importance of recognizing that goals and priorities may vary by individual and that each person will begin the program looking for a different outcome. for some, success may be defined as an improvement in mobility, whereas for others success may be defined as increased social interactions, motivation, or health maintenance. "i think everybody's priorities and everybody's goals are different." "so i mean, so i don't want to lose those [functions] any worse than they've been getting over the years because it's like almost limited to what i can kind of do…" "well, i guess it depends on somebody's lifestyle and age has a lot to do with it. so i would say some people are just looking to maintain themselves and stay healthy to be able to continue to do the activities that they currently do." "yeah, and just feeling better as well in daily activities." interaction with other classmates a strong recommendation was made to foster potential interactions among classmates in order to motivate and inspire one another. an additional recommendation was to include a feature to extend class times to allow for social interactions among classmates before or after exercise. some participants suggested that two-way viewing among classmates should be optional, so as to not discourage those who might feel uncomfortable. "i think it would be key to interact with not only the therapist, but with other patients. so i see jack on the one screen and he's struggling, i'm like come on. do one more. do one more. and we're all telling himme, alex-jack, come on. do one more. and he pulls through, so it gives that mental back." "so you said a -minute designed program, but maybe its min and we all log on min before. we could all-oh, maria*, how's that going, or chris*, how's that? so the social muscle to it instead of just working on arms and then logging off, like good old talk." "but to have the ability to [see others] should certainly be an option… but they should at least have the option to turn it off if they want i think, right?" participants suggested that it would be helpful for someone to orient a class member, assist with equipment needs and demonstrate specific exercises included in the program prior to session initiation. participants suggested that including a variety of exercises within each class would be desirable, in order to meet personal preferences and to address varying physical abilities. multiple participants suggested that three times per week would be the preferred frequency of classes, held at a variety of days and times to accommodate different schedules (e.g., weekday, weekend, and evening sessions). the goal of this focus group was to discuss experiences with physical activity and gather input from persons with tetraplegia to inform future design of a physical activity program delivered via telemonitoring that would be feasible, acceptable, and consistent with exercise guidelines for those with sci. a minor aspect of the discussion revealed that, in general, priorities for improvement included: arm/hand function, sensation, and improvement of mobility as being most important. in addition, upper body/trunk strength and balance, elimination of chronic pain, and improving lived experiences of sexual function were also ranked as important. data demonstrate that different modalities of exercise and physical activity have indeed been shown to improve aspects of physical capacity, health, and abilities to perform activities of daily living (e.g., functional wheelchair maneuvers and transfers) in persons with sci [ ] . participants perceived a home-based physical activity program as needed and important. intensity and type of physical activity performed before and after injury were discussed. participants identified family support, psychological state, and having a peer network (e.g., others with sci) as important factors for their overall recovery. participants regarded their input and feedback as critical for ensuring usability and feasibility, including the ability to make choices regarding whether a participant can be seen by classmates, types of exercises in a class, and timing of class delivery to suit multiple schedules. participants generally expressed enthusiasm for interacting with classmates, a desire for help from a caregiver or professional in initial set up, and comfort with a frequency of three times/week for classes and a duration of - min per class. participants perceived multiple potential benefits of a physical activity for persons with sci delivered via telemonitoring. participants had several practical suggestions to optimize design and delivery of such a program. clearly, a pilot study in this population testing this kind of intervention is needed. it is important that future studies incorporate feedback from participants on the design and implementation of a physical activity program. data generated during the focus group are not publicly available in order to protect privacy of participants. deidentified data can be made available upon request to the corresponding author. publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/ . /. annual statistical report for the spinal cord injury model systems public version traumatic spinal injury: global epidemiology and worldwide volume facts and figures at a glance facilitators and barriers to social and community participation following spinal cord injury social and community participation following spinal cord injury: a critical review position statement. part two: maintaining immune health position statement. part one: immune function and exercise reduction in trunk fat predicts cardiovascular exercise training-related reductions in c-reactive protein exercise and respiratory tract viral infections cardiovascular exercise training extends influenza vaccine seroprotection in sedentary older adults: the immune function intervention trial exercise, inflammation, and innate 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international classification of functioning, disability and health as reference framework participation in physical activity in persons with spinal cord injury: a comprehensive perspective and insights into gender differences participation in sport in persons with spinal cord injury in switzerland telehealth for people with spinal cord injury: a narrative review circuit training provides cardiorespiratory and strength benefits in persons with paraplegia a comparison of circuit exercise training techniques for eliciting matched metabolic responses in persons with paraplegia using thematic analysis in psychology causes of death during the first years after spinal cord injury targeting recovery: priorities of the spinal cordinjured population exercise and health-related risks of physical deconditioning after spinal cord injury acknowledgements the authors appreciate the time, effort, and opinions of the focus group participants.funding a grant from the new york state spinal cord injury research board (to ob) and institutional funds supported this work. these funds were used to support sci-related research at our institution and did not influence the specific study in any way.authors' contributions rp, ak, jh, dg, and ob designed the study. rp and ak led development of the moderator guide, to which all authors made contributions. jh and dg developed the proposed physical activity program. rp, ak, jh, dg, and ob created the demo video of the proposed physical activity program. all the authors (rp, ak, jh, dg, ac, bp, and ob) were present for the focus group. rp and ak moderated the focus group. ak and bp analyzed the transcript and wrote the report. all the authors (rp, ak, jh, dg, ac, bp, and ob) contributed to interpreting the data and writing the manuscript. conflict of interest the authors declare that they have no conflict of interest.ethics study activities were deemed not human subject research by the local institutional irb research and therefore did not require irb review. key: cord- -ph eji authors: mostajo, nelly f; lataretu, marie; krautwurst, sebastian; mock, florian; desirò, daniel; lamkiewicz, kevin; collatz, maximilian; schoen, andreas; weber, friedemann; marz, manja; hölzer, martin title: a comprehensive annotation and differential expression analysis of short and long non-coding rnas in bat genomes date: - - journal: nar genom bioinform doi: . /nargab/lqz sha: doc_id: cord_uid: ph eji although bats are increasingly becoming the focus of scientific studies due to their unique properties, these exceptional animals are still among the least studied mammals. assembly quality and completeness of bat genomes vary a lot and especially non-coding rna (ncrna) annotations are incomplete or simply missing. accordingly, standard bioinformatics pipelines for gene expression analysis often ignore ncrnas such as micrornas or long antisense rnas. the main cause of this problem is the use of incomplete genome annotations. we present a complete screening for ncrnas within bat genomes. ncrnas affect a remarkable variety of vital biological functions, including gene expression regulation, rna processing, rna interference and, as recently described, regulatory processes in viral infections. within all investigated bat assemblies, we annotated ncrna families including snornas and mirnas as well as rrnas, trnas, several snrnas and lncrnas, and other structural ncrna elements. we validated our ncrna candidates by six rna-seq data sets and show significant expression patterns that have never been described before in a bat species on such a large scale. our annotations will be usable as a resource (rna.uni-jena.de/supplements/bats) for deeper studying of bat evolution, ncrnas repertoire, gene expression and regulation, ecology and important host–virus interactions. bats (chiroptera) are the most abundant, ecologically diverse and globally distributed animals within all vertebrates ( ), but representative genome arrangements and corresponding coding and non-coding gene annotations are still incomplete ( ) . except for the extreme polar regions, bats can be found throughout the globe, feeding on diverse sources such as insects, blood, nectar, fruits and pollen ( ) . their origin has been dated in the cretaceous period, with a diversification explosion process dating back to the eocene ( ). the bat families known to date are classified into the suborders yinpterochiroptera and yangochiroptera ( , ) ( figure ). although they account for > % of the total living mammalian diversity ( ) , the genomes of only bat species of the estimated > species ( ) have been sequenced with adequate coverage to date and are publicly available ( figure ) . bats have developed a variety of unique biological features that are the rarest among all mammalian, including laryngeal echolocation ( , ) , vocal learning ( ) and the ability to fly ( ) . they occupy a broad range of different ecological niches ( ) , have an exceptional longevity ( - ) and a natural and unique resilience against various pathogenic viruses ( , ) . for example, bats are the suspected reservoirs for some of the deadliest viral diseases such as ebola and sars ( ) ( ) ( ) , but appear to be asymptomatic and survive the infection. possibly, the solution to better understand and fight these pathogens lies in the uniquely developed immune system of bats ( , ) . studying bats and their genomes is likely to have high impacts on various sci- figure . we used available genomes of bat species from eight out of families for non-coding rna annotation in this study. the tree shows their phylogenetic relationship and is based on a molecular consensus on family relationships of bats ( ), further adapted and extended from ( ) . bat families and species with published genomes currently available in the ncbi are shown (details see table ). bat families still lacking a published genome assembly are written in gray color. rna-seq data sets were selected from species marked with an asterisk and additionally obtained from a myotis daubentonii cell line (see table ). bat silhouettes were adapted from artworks created by fiona reid. entific fields, including healthy ageing, immune and ecosystem functioning, the evolution of sensory perception and human communication, and mammalian genome architecture (see the recent bat k review for further details ( ) ). despite the unique biological characteristics of these flying mammals and their important role as natural reservoirs for viruses, bats are one of the least studied taxa of all mammalian ( ) . accordingly, there is little knowledge about the non-protein-coding transcriptome of bats, which plays a crucial role in an extensive number of cellular and regular functions and comprises a very diverse family of untranslated rna molecules ( , ) . in addition, it is believed that due to the early evolutionary radiation of bats (compared to other mammals) their innate and acquired immune responses have a different set of molecules ( ) . genome assemblies and annotations are essential starting points for many molecular-driven and comparative studies ( ) . especially, studies of non-model organisms play important roles in many investigations ( ) . in most cases, however, these organisms lack well-annotated genomes ( ) , which severely limit our ability to gain a deeper understanding of these species and may further impede biomedical research ( ) . in this study, we comprehensively annotated non-coding rnas in available bat genome assemblies (table ) . for each bat species, we provide final annotations that are compatible with current ensembl and ncbi (national center for biotechnology information) standards (gtf format) and that can be directly used in other studies, for example for differential gene expression analysis. we compare our new annotations with the currently available annotations for bats and show that a large number of non-coding genes are simply not annotated and are therefore overlooked by other studies. we used six rna-seq data sets comprising different conditions (table ) to validate our annotations and to determine the expression levels of our newly annotated ncrnas. exemplarily, we show that our novel annotations can be used to identify ncrnas that are significantly differential expressed during viral infections and were missed by previous studies. we downloaded the last recent genome versions for bat species in september from the ncbi genome database (table ) . within the order of yinpterochiroptera, nine genomic sequences were obtained covering the bat families pteropodidae, hipposideridae, rhinolophidae and megadermatidae whereas for the order of yangochiroptera another seven genome assemblies were available for the bat families phyllostomidae, mormoopidae, vespertilionidae and miniopteridae ( figure ). we introduced a unique three-letter abbreviation code (table ) to easily distinguish between the bat species in the manuscript and intermediate annotation files provided in the electronic supplement. we used quast (v . . ) ( ) to calculate several assembly statistics for all genomes, shown in supplementary table s . at the end of , two new bat genomes were presented by the bat k project (http://bat k.ucd.ie) ( ), comprising a newer version of the greater horseshoe bat genome (rhinolophus ferrumequinum; rhinolophidae) and the genome of the pale spear-nose bat (phyllostomus discolor; phyllostomidae). however, these two bat genomes were not included in our present study due to the data use policy of the bat k consortium and to support a fair and productive use of these data. to validate our novel ncrna predictions, we selected six rna-seq data sets ( , ( ) ( ) ( ) ( ) comprising all together samples gathered from four different bat species. we have labeled each published rna-seq data set based on the first authors last name and the year of data set publication ( table and supplementary table s ). all samples were quality trimmed using trimmomatic ( ) (v . ) with a nt-step sliding-window approach (q ) and a minimum remaining read length of nt. for the field- data set ( ) , we additionally removed the three leading ' nucleotides from the reads of each sample because of a generally low quality observed by fastqc (www.bioinformatics.babraham.ac.uk/projects/ table . we have annotated ncrnas within bat genomes of different assembly quality. we introduced three-letter abbreviations for each bat species used throughout the manuscript and in supplemental files and annotations. genome sizes were estimated (est.) by using c-values (dna content per pg) from the animal genome size database (http://genomesize.com) and by applying the following formula: genome size = ( . · ) · c. if multiple entries for one species were available, an average over all c-values was calculated and used to estimate the genome size. if one species could not be found, an average c-value for the corresponding genus was used. supplementary table s provides additional assembly statistics calculated by quast (v . . ) ( ) . ncbi acc. -genbank assembly accession without the prefix gca fastqc/) (v . . ). the remaining reads of all processed samples were individually mapped to all bat assemblies using hisat ( ) (v . . ) and transcript abundances were subsequently calculated from all resulting mappings by featurecounts ( ) (v . . ). if suitable, the appropriate strand-specific counting mode was applied for each data set (see table for information about the strand specificity). for each bat genome assembly, the merged annotation of already known (ncbi) and newly identified (this study) ncr-nas was used (supplementary files s ). due to the size of the annotations and the huge amount of overlaps, long ncr-nas were counted and analyzed for differential expression separately. to enable a better investigation of small rnas (sr-nas), we included a data set of the targeted sequencing of srnas (especially mirnas) from m. daubentonii cells (weber- ). to obtain this srna sequencing data, total rna of samples, which was obtained using the same procedure like explained for the rrna-depleted m. daubentonii data ( ) , was preprocessed using the illumina truseq smallrna protocol, sequenced on one hiseq lane, and finally uploaded in the course of this study under geo accession gse . the reads of these srna samples were additionally preprocessed by removing potential adapter sequences with cutadapt ( ) (v . . ) followed by a quality (q ) trimming using again a window-size of and a minimum length of nt by prinseq ( ) (v . . ). the processed srna samples were either individually mapped to the bat genomes for differential expression analysis or combined and mapped on each bat genome to predict known and novel mirnas with mirdeep ( ). only uniquely mapped reads were counted and used for the differential gene expression analyses with deseq ( ) (v . . ). annotated rrna genes were removed prior de-seq and tpm (transcripts per million) analysis. all raw read counts from samples of one data set were combined and normalized together using the built-in functionality of deseq , followed by pairwise comparisons to detect significant (adjusted p-value < . ; absolute log fold change > ) differential expressed ncrnas. besides the deseq normalization, we calculated tpm values for each ncrna in each sample as previously described ( ) : where c i is the raw read count of ncrna i, l i is the length of ncrna i (and the cumulative exon length in the case of lncrnas) and n is the number of all ncrnas in the given annotation. to this end, we obtained for each rna-seq sample, each bat annotation, and each ncrna one tpm value representing the normalized expression level of this ncrna. if available, we calculated all tpm values in relation to the expression of all already known coding and non-coding genes and not only based on our novel ncrna annotation. although we performed mappings, read countings, and normalization for all samples, bat genome assemblies and all six data sets ( table ; overall mappings), we only selected one comparison per data set to exemplarily show novel and significantly differential expressed ncrnas (supplementary files s . -s . ; divided by data set and input annotation). for each data set, we chose the bat species that was also used in the corresponding study. for the hölzer- and weber- data sets, we used the closely related m. lucifugus genome assembly and annotation as a refer- table . six rna-seq data sets comprising all together samples derived from four different bat species were used to evaluate our novel ncrna annotations. all samples were quality trimmed and individually mapped to all bat assemblies using hisat ( ) and transcript abundances were subsequently calculated from all mappings by featurecounts ( ) . we labeled each rna-seq data set based on the first authors last name and the year of data set publication. raw read data of the enriched sequencing of small rnas (especially mirnas) of a m. daubentonii cell line (accompanying gse ( ) ) have been uploaded in the course of this publication under geo accession gse (weber- ). polya+ -library preparation with mrna selection; rrna--library preparation with rrna depletion and size selection (> nt); srna -library preparation with size selection (< nt); se/pe -single-/paired-end sequencing; ss/not-ss -strand-specific/unstranded sequencing; ss s /ss a -strand-specific in sense orientation/in antisense orientation figure . please note that for m. daubentonii currently no genome assembly is available, so the genome assembly of m. lucifugus was used as a close relative. all of our annotations follow the general transfer format (gtf) as described and defined in the ensembl ( ) database (https://ensembl.org/info/website/upload/gff. html). therefore, each row of each annotation file is either defined as a gene, transcript, or exon (by the feature column) and strictly following a hierarchical structure, even if only one exon (as for most ncrnas) is reported. by adhering to this annotation format, our novel annotations can be easily merged with existing ones as derived from ensembl or ncbi and are directly usable as input for computational tools such as hisat for mapping or featurecounts for transcript abundance estimation. we defined gene, transcript, and exon ids following the ensembl pattern: <species><feature><ncrna>< -digit-number>. in general, we used specialized computational tools for the annotation of specific ncrna classes (supplementary tables s -s ). if not otherwise stated, the main ncrna-discovery is based on homology searches against the rfam database ( - ) (v . ). we used the gorap pipeline (https://github.com/koriege/gorap), a specially developed software suite for genome-wide ncrna screening. gorap screens genomic sequences for all ncrnas present in the rfam database using a generalized strategy by applying multiple filters and specialized software tools. to this end, gorap takes huge advantage of infernal ( , ) (v . . ) to annotate ncrnas based on input alignment files conserved in sequence and secondary structure (so-called stockholm alignment files; stk). all resulting alignment files were automatically pre-filtered by gorap based on different ncrna class-specific parameters including taxonomy, secondary structure and primary sequence comparisons. due to repeats, pseudogenes, undiscriminable un-/functional genes and overlapping results from the different assembly methods, we defined a ncrna set per species for annotation that includes filtered sequences, but allows for variants and multiple copies. this final annotation set is defined by hand-curating the resulting stk alignments of gorap with the help of emacs ralee mode ( ) . due to the removal of sequences in the stockholm alignments, the remaining sequences were extracted and again aligned into stockholm format using cmalign --noprob from infernal. the rfam-derived ncrna alignments were further split into snornas (supplementary table s ), mirnas (supplementary table s ) and other ncrnas including snrnas, lncrnas and other structural rnas (supplementary table s ). in general, our annotation results give an overview about the amount of different ncrnas in bat species and intentionally can include false positive hits and duplicates. all ncrna hits are placed as stk (if available), gtf and fasta-files in the electronic supplement and osf (doi. org/ . /osf.io/ cmdn). rrnas. we used the prediction tool rnammer ( ) (v . ) to identify . s, s and s rrna genes using hidden markov models. the tools output was transformed into regular gtf file format. all output files can be found in supplementary table s . trnas. for the annotation of trnas, we applied trnascan-se ( ) (v . . ) to the bat contigs using default parameters. the results were filtered by removing any trnas of type 'undet' or 'pseudo' and the tabular output was transformed into the gtf file format. additional information about the anticodon and the coverage score were added to the description column. we provide the raw trnascan-se files in supplementary table s . snornas. we annotated snornas based on available alignments from the rfam database using gorap and additionally marked and classified them into box c/d and box h/aca when intersecting with the set of snornas from http://www.bioinf.uni-leipzig.de/publications/supplements/ - ( ) (supplementary table s ). mirnas. additionally to the rfam-screening (supplementary table s ), mirnas were predicted by the mird-eep pipeline ( ) (v . . . ) using default parameters and the combined smallrna-seq data set (weber- ; samples) mapped to each individual bat assembly as an input for mirdeep (supplementary table s ). to validate the accuracy of our approach, we compared our mirdeep annotations of m. lucifugus/p. alecto (based on the transcriptomic data derived from m. daubentonii; weber- ) with annotations of mirnas for transcriptomic data of myotis myotis ( ) and p. alecto ( ) . for reference mapping, huang et al. also used the m. lucifugus genome, so we were theoretically able to directly compare our annotations with the annotations of both studies. unfortunately, no positional information (annotation file) of the identified mirnas derived from the transcriptomic data of m. myotis were given in the manuscript or supplement ( ) . therefore, we blasted the precursor mirna sequences identified with the help of the m. myotis transcriptome against the m. lucifugus genome and retained only hits with a sequence identity of %. the so obtained positional information was further used to calculate the overlap between our predicted mirnas in m. lucifugus. we used the same approach for the p. alecto comparison. if the annotated location of an mirna and one of our identified mirna locations in m. lucifugus/p.alecto were overlapping by at least %, we counted this location as a common prediction. lncrnas. long ncrnas were annotated using a high confidence data set h from the lncipedia ( ) (v . ) database comprising transcripts of potential human lncrnas. the transcripts were used as input for a blastn ( . . +, e − ) search against each of the bat assemblies (compiled as blast databases). the blastn result for each bat assembly was further processed to group single hits into potential transcripts as follows: first, for each query sequence q ∈ h, hits of q found on the same contig c and strand s were selected (hits c, s, q ) and the longest one, q , was chosen as a starting point so that trscp c, s, q = (q ). second, all hits q i ∈ hits c, s, q with q i ∈trscp c, s, q , which overlap neither in the query q nor in the target sequence and do not exceed a maximum range of nt from the most upstream to the most down-stream target sequence position of all q j ∈ trscp c, s, q ∪q i , were added iteratively to trscp c, s, q . to this end, we introduced a simple model of exon-intron structures, naturally occurring when using transcript sequences as queries against a target genome assembly. we defined the nt search range based on an estimation of lncrna gene sizes derived from the human ensembl ( ) annotation. if the sum of the lengths of all q i ∈ trscp c, s, q covers the query transcript length length q at least for %, trscp c, s, q was considered as a transcript and its elements q i as exons, otherwise all q i ∈ trscp c, s, q were withdrawn. this procedure is repeated until all hits ∈ hits c, s, q were used or withdrawn. therefore, each so-defined group of non-overlapping hits derived from the same query sequence and found on the same contig and strand should represent a lncrna transcript with its (rough) exon structure. the defined transcripts were saved as blast-like output and transformed into gtf file format. to follow the gtf annotation format and to harmonize our lncrna annotations with the other ncrna annotations, each lncrna transcript was also saved as a gene annotation and consists of at least one exon. as we observed a lot of different sequences from lncipedia aligning to the same positions in the genomes, we decided to condense exons at the same sequence positions, considering transcripts with one or multiple exons separately. for each contig and strand, starting from the ' end, exons with a minimum overlap of nt were grouped together. in the case of multiple exons, groups of exons were merged, if they shared any transcript origin. if all exons in the group originated from the same lncipedia gene, the group was considered as one gene with several transcripts and its associated exon(s). otherwise, we defined a lncrna hot spot on gene level with several transcripts and their associated exon(s). the lncipedia names of the gathered transcripts of a lncrna hot spot, as well as start and end positions of all exons, are listed in the gtf gene attribute field (supplementary table s ). the scripts used for the identification of lncrnas can be found at https://github.com/rnajena/bats ncrna. table ) . for the other six species we used blastn ( . . +, e − ) with the mlu and pva mitochondrial genomes as queries against the remaining bat genomes. for ehe, we found a possible mtdna contig in full-length ( nt; awhc ) in the genome assembly. due to the circularization of mtdna, we rearranged the sequence of this contig to start with the gene coding for the phenylalanin trna and to match the gene order of the other mitochondrial genomes. only for esp, rsi, mly, efu and mna, we were not able to detect any possible contigs of mtdna (table ). all mitochondrial genomes were annotated with mitos ( ). the ncrna results were filtered by e-value (threshold . ), thus one of two small rrnas in mbr and rfe and one of two large rrnas in ehe were discarded as false positive hits (supplementary table s ). for the five bat assemblies directly including mtdnas (table ) , the mitos annotations were added to the final merged ncrna annotation. all other mtdnas and annotations can be found in supplementary table s . as we annotated all bat assemblies by using different tools, we needed to merge the resulting gtf files to resolve overlapping annotations and to receive a final annotation of ncrnas for each bat species. furthermore, we extended the available ncbi annotations (including protein-and noncoding genes) by integrating our novel ncrna annotations. the scripts used to merge the different annotation files and to calculate overlaps between annotations can be found at https://github.com/rnajena/bats ncrna. due to their size, we have not included the lncrna annotations based on lncipedia. these can be downloaded and used separately (supplementary table s ). of novel non-coding annotations. for each bat species, we merged the ncrna annotations (except for lncrnas) using a custom script (merge gtf global ids.py). after reading in all features and asserting correct file structure, overlaps were resolved in the following manner: (i) exons are considered overlapping if > % of the shorter one is covered by table . mitochondrial bat genomes (mtdna) publicly available and used for annotation with mitos ( ). for out of the bat species investigated in this study, mtdna could be found in the ncbi. for four bat species, the mtdna is also part of the genome assembly as determined using blastn. for e. helvum, no mtdna could be found in the ncbi, but we were able to identify a single contig that is part of the genome assembly as mtdna using blastn and the mitochondrial genomes of the other bats as query. the contig was rearranged to match the gene order of the other mtdnas. r -found via blastn and rearranged merge of ncbi and novel annotations. we first converted and filtered the ncbi annotations to a compatible format with a custom script (format ncbi.py) and then combined the results with our merged novel ncrna annotations using the same strategy to resolve overlaps as above, but imposing less strict format rules (merge gtf ncbi. py). at best, a genome assembly represents the full genetic content of a species at chromosome level. whereas the first complex eukaryotic genomes were generated using sanger chemistry, today's technologies such as illumina short-read sequencing and pacbio or oxford nanopore long-read approaches are increasingly used ( ) . the currently available bat genomes vary widely regarding their assembly quality and completeness (table and figure ; supplementary table s ) and were predominantly assembled by using short illumina-derived reads and low (∼ x) ( ) up to moderate/higher coverage ( - x) approaches ( ) ( ) ( ) ( ) ( ) ) . a new assembly of the cave nectar bat (eonycteris spelaea) ( ) was exclusively generated from long-read data derived from the pacbio platform, and the genome of the egyptian fruit bat (rousettus aegyptiacus) ( ) was assembled by using a hybrid-approach of illumina and pacbio table and supplementary table s ) supplementary table s . data. these two genomes from the pteropodidae family are of a generally higher quality (figure and supplementary table s ). regardless of their assembly quality, these genomes need to be annotated to identify regions of interest, for example, encoding for protein-and non-coding genes or other regulatory elements. current genome annotations, mostly generated by automatic annotation pipelines provided by databases such as the ncbi ( ) or ensembl ( ), are predominantly focusing on protein-coding genes and well-studied ncrnas such as trnas and rrnas. accordingly, the available bat genome annotations vary a lot regarding their quality, ranging from more comprehensive annotations for longstanding bat genomes such as m. lucifugus or p. vampyrus to annotations on region level, completely missing any coding or non-coding gene annotations at the current ncbi version ( figure and table ). furthermore, by using strandspecific rna-seq data, we could show that some genes (e.g. ifna /ifnw in the ensembl annotation of m. lucifugus ( ) ) are annotated on the wrong strand and are therefore entirely missed by differential expression studies when relying on a strand-specific read quantification. for all publicly available bat genomes, ncrnas are generally annotated on low levels and are highly incomplete, mostly only comprising some trnas, rrnas, snrnas, snornas and lncrnas ( figure and table ). therefore, many ncrnas, especially mirnas, are simply overlooked by current molecular studies, for example from rna-seq studies that aim to call differential expressed genes based on such in-complete genome annotation files. studies that have made additional effort on annotating ncrnas in bats ( , , , ( ) ( ) are not reporting their results on a level that can be directly used for further computational assessment (e.g. as a direct input for rna-seq abundance estimation). currently, in the ncbi database, five bat assemblies are entirely lacking any coding/non-coding annotations and mirnas are not annotated at all ( table ). the rfam database ( ) contains mainly for m. lucifugus and p. vampyrus ncrna families. other ncrnas are currently unknown from bat genomes or not well documented. the genome assembly status of different bat species varies widely: ranging for example from contigs and an n of nt (d. rotundus) to contigs and an n of only nt (m. lyra), see table and supplementary table s . accordingly, the annotation status also varies a lot (table ) . within this work, we give an overview of potential ncrna annotations in bats. however, the precise number of ncrnas remains unclear, because of ncrnas being present several times in the assemblies, and others still remaining undiscovered. to give a better estimation of transcribed and potentially functional ncrnas, we used six illumina short-read rna-seq data sets derived from four bat species (table ) to estimate the expression levels of our novel annotations. note that we refer throughout this paper to an rna-seq data set by the first author's name and the year of the respective data set publication. the only included data set derived from a species of the yinpterochiroptera suborder (r. aegyptiacus) was obtained from a study dealing with the differential transcriptional responses of ebola and marburg virus infections in human and bat cells ( ) (data set: hölzer- ) . in this study, total rna of nine samples of r e-j cells, either challenged by the ebola or marburg virus or left un-infected, were harvested at , or h post infection (poi) and sequenced. unfortunately, no biological replicates could be generated for this study. therefore, we did not use this data set for the differential ex-pression analysis, but also calculated normalized expression values (tpm; transcripts per million) as done for all rna-seq data sets. the other five data sets comprise yangochiroptera species of the vespertilionidae (m. lucifugus, m. daubentonii) and miniopteridae (m. natalensis) families (table ). field et al. conducted two transcriptomic studies ( , ) (field- , field- using wing tissue of the hibernating little brown myotis bat. they were especially interested in transcriptional changes between un-infected wing tissue and adjacent tissue infected with pseudogymnoascus destructans, the fungal pathogen that causes the white-nose syndrome. two other data sets were obtained from virus-(rvfv clone ) and interferon (ifn) alphainduced transcriptomes of a myotis daubentonii kidney cell line (mydauni/ c). rna of mock, ifn and clone samples were gathered at two time points, and h poi ( ) . from the same samples, rrna-depleted (hölzer- ) and smallrna-concentrated (weber- ; see methods section for details) libraries were generated and sequenced. finally, we used data of the long-fingered bat m. natalensis initially obtained to characterize the developing bat wing ( ) . here, total rna was extracted from paired forelimbs and hindlimbs from three individuals at three developmental stage. we have mapped each sample to each bat genome, regardless of the origin of the rna. expectedly, the mapping rate decreases in bat species that are evolutionary more far away from the original species from which the rna was sequenced. however, we were interested to find out which ncrnas are consistently transcribed in all investigated bat species or only in certain bat families and sub-groups. over all bat assemblies, we annotated ncrna families for in total trnas, rrnas, mirnas ( predicted by mirdeep ), snornas, mitochondrial (mt-)trnas and mt-rrnas as well as other ncrnas additionally derived from the rfam database ( ) (selected ncrnas are shown in table ). with a broad approach, we have identified potential lncrnas and defined between (m. lyra) and (d. rotundus) lncrna hot spots. all annotations, separated for each ncrna class and summarized for each bat species, can be found in the open science framework (doi.org/ . /osf.io/ cmdn) and in our electronic supplement (rna.uni-jena. de/supplements/bats) and are compatible with the genome assembly versions listed in table . thus, our annotations together with the bat genome assemblies obtained from the ncbi can be directly used for subsequent analysis such as differential gene expression detection. we detected s and s rrna for the majority of investigated bat species (supplementary table s ). the varying number of rrnas is in line with all currently available metazoan genomes, lacking the correct composition of rrnas due to misassemblies. however, the number of . s rrna varies a lot between for p. vampyrus and for m. natalensis (table and supplementary table s ). interestingly, of pteropodidae show a higher number of . s rrnas compared to the other species (∼ - fold). only the p. alecto assembly is in line with the other bat assemblies in regard to the amount of . s rrna copies. for all bat species, we observed various numbers of trnas (table and supplementary table s ). we could detect full sets of trnas for e. fuscus and m. davidii, whereas between one and four trnas are missing from the assemblies of h. armiger, m. brandtii, m. lucifugus, m. lyra, m. natalensis, p. parnellii, r. ferrumequinum, r. sinicus and d. rotundus, and between nine and twelve are missing for e. helvum, p. alecto, p. vampyrus, r. aegyptiacus and e. spelaea (supplementary table s ). interestingly, we identified a large number of trnas ( ) for m. lyra in comparison to all other bat species (supplementary table s ), likely a result of the low assembly quality (figure and supplementary table s ). the lowest amount of trnas was annotated for p. parnellii and d. rotundus with only and copies, respectively. in all other bat genomes, we found between and trna genes (supplementary table s ). the trna encoding for valine (val) with the anticodon structure tac had high copy numbers (over ) in all genome assemblies of the pteropodidae family (table ) . similar copy numbers were achieved by the r. ferrumequinum and r. sinicus assemblies regarding the trna encoding for isoleucine (ile) with the anticodon aat (supplementary table s ). for trna(ile) and the anticodon gat, we also observed high copy numbers in h. armiger. interestingly, all species with high trna(val) and trna(ile) copy numbers had rather low counts (between and ) of trna(sec) with the anticodon tca, while this trna was found with higher copy numbers in p. parnelli ( ) and in d. rotundus ( ) and with even higher counts (between and ) in all other bat species (supplementary table s ). however, high copy numbers might be also occur due to assembly quality and false positive predictions of trnascan-se. in supplementary table s we list all detected snornas, divided into box c/d and box h/aca types. overall, we found snorna families within the investigated bat species, comprising box c/d, box h/aca and unclassified snornas. many snornas were found with exactly one copy present in each bat genome assembly (e.g. scarna ), whereas others were found in multiple copies for each bat species (e.g. snord ) or completely absent from certain bat families (e.g. aca ), see table . exactly one copy of the small nucleolar rna aca was found within the genomes of the pteropodidae family and multiple copies for d. rotundus, p. parnellii, m. natalensis and members of the vespertilionidae family; however, this snorna seems to be completely absent from bat species of the megadermatidae and rhinolophidae families ( table ). the h/aca box snorna aca is predicted to guide the pseudouridylation of s rrna u ( ) . interestingly and as another example, snora was found in higher copy numbers in the genomes of the vespertilionidae family. among others, this h/aca box snorna was described to be commonly altered in human disease ( ) . over all bat assemblies, we detected mirna families based on rfam alignments (supplementary table s ) and predicted between (e. helvum) and (m. davidii) mirnas based on the combined small rna-seq data sets (weber- ) using mirdeep ( ) (supplementary table s ). the higher number of mirnas predicted for myotis species can be explained because the small rna-seq data set is derived from a myotis daubentonii cell line. similar to other ncrna classes, we observe various differences in mirna copy numbers between the bat families. for example, mir- and mir- are absent in all vespertilionidae and m. natalensis (table ) , whereas mir- is present in all yangochiroptera (except d. rotundus) but absent from all pteropodidae (supplementary table s ). the mirna is absent from all yangochiroptera except d. rotundus. there are many other examples of absent/present mirnas in certain bat species/families such as mir- (absent from pteropodidae), mir- (absent from yinpterochiroptera) and mir- (absent from rhinolophidae and megaderma lyra), see supplementary table s . with mirdeep , we detected hundreds of potential mirnas for all investigated bat species (supplementary table s ). generally, all bat species can be divided into two groups. for the majority of the bat assemblies ( out of ) about mirnas were predicted. for the other species of the vespertilionidae family about times as many mir-nas could be found. this is in concordance with the small rna-seq data used for the prediction, that was obtained from myotis daubentonii kidney cells (see methods). nevertheless, ∼ conserved mirnas can be predicted in the evolutionary more distant bat species. table . general genome information for each of the investigated bat assemblies and selected ncrna examples annotated in this study. we selected ncrnas with interesting copy number distributions among the investigated bat species. for snora , pvt and hottip and we additionally found sophisticated differential expression patterns in at least one of the used rna-seq data sets (absolute log fold-change greater , tpm > ). full tables and detailed information for each ncrna class (fasta, stk, gtf files) can be found in the electronic supplement online (supplementary tables s -s ) lncrnas for the annotation of lncrnas, we have deliberately chosen a broad blast-based approach, using , transcripts of potential human lncrnas obtained from the lncipedia database ( ) . we have consciously chosen this approach, because lncr-nas have diverse genomic contexts, reveal various functions and act in different biological mechanisms ( ) ( ) ( ) . from lncipedia transcripts, we annotated genes and lncrna hot spots. we defined regions in a genome as a lncrna hot spot, if different lncipedia transcripts derived from different genes map to the same region (see methods section for detailed description). overall, we found between and potential lncrna genes in m. lucifugus and r. sinicus, respectively, and between and lncrna hot spots in m. lucifugus and d. rotundus, respectively. we annotated the previously described lncrnas tbx -as and hottip ( ) in all bat genomes, except tbx -as in m. lucifugus, presumably due to the lower genome assembly quality (table ) . besides evolutionarily explainable differences in the presence of lncrnas, we have observed that the number of lncrnas and lncrna hot spots increases with increasing assembly quality (e.g. with a higher n ; see supplementary figure s ). we have not observed such a clear correlation between assembly quality and annotation results for short ncrnas. based on the rfam alignments, we were able to detect other ncrna families in addition to the rrnas, trnas, snornas and mirnas described before (supplementary table s ). overlaps with annotated lncrnas (supplementary table s ) are intentional, because the rfam includes only highly structured parts of long ncrnas. the highest number of ncrna copies ( ) was detected for the u spliceosomal rna in m. brandtii, already known to have a lot of pseudogenes ( ) . for ncrnas such as caesar (rf ), g-csf slide (rf ), nron (rf ), tusc (rf ), xist exon (rf ), linc (rf ), and hammerhead hh and hh (rf , rf ), we found exactly one copy in each investigated bat genome assembly (supplementary table s ). again, we also observed ncrna families that are lost for some species or entire families, for example the ribozyme cotc (rf ) that seems to be absent from all vespertilionidae members and m. natalensis. for each investigated bat species except e. spelaea, r. sinicus, m. lyra, e.fuscus and m. natalensis (where no mitochondrial contigs could be identified; table ) mitochondrial protein-coding genes and ncrnas were annotated (see methods). in total, mitochondrial genes comprising trnas, two rrnas ( s and s) and protein-coding genes were detected for each bat species as known for other metazoans ( ) (supplementary table s ). the mitochondrial genome lengths range from nt in e. helvum to nt in m. davidii. for the five bat species, where the mitochondrial genome could be identified as a part of the ncbi genome assembly, we appended the mtdna annotation to the final annotation of ncrnas. exemplarily, we investigated known and novel differentially expressed (de) ncrnas found in the genome of m. lucifugus in more detail. to this end, we used the rna-seq data sets field- , field- , hölzer- and weber- (table ) as a basis to identify de ncrnas that were newly discovered in this study and were not part of the current ncbi or ensembl genome annotations for this bat species. more detailed de results can be found in supplementary file s . we filtered for novel m. lucifugus de ncrnas by (i) an absolute log fold change (fc) > , (ii) an adjusted pvalue < . , and (iii) a tpm > . we further manually investigated the expression patterns with the igv ( ) and discarded de ncrnas overlapping with the current ncbi (myoluc . annotation release ) or ensembl (myoluc . . ) annotations. based on the small rna-seq comparison of mock and virus-infected (clone ) samples h post infection (weber- ), we found several mirnas (rfam-and mirdeep -based) and snornas to be differentially expressed ( figure and details in supplementary files s . ). in general, replicates of virus-infected and ifn-treated samples h post infection tend to cluster together only based on the expression profiles of small ncrnas (mainly mirnas) ( figure a and b) . most differences can be observed between the h virus-infected and all other samples, which seem to show no clearly distinguishable expression pattern. interestingly, at h post infection, we see replicates clustering together regardless of their treatment (mock, ifn, clone ). thus, after only h, few mirnas are differentially expressed and therefore the samples of each replicate (mock, ifn, clone ) cluster together, because they have the same passaging history but the passaging history in between the replicates differ ( ) . after h, more and more mirnas are significantly differentially expressed and the samples can be better distinguished based on their treatment ( figure a and b). we observed that, in general, mirnas tend to be down-regulated ( figure b ; upper half), while snornas tend to be up-regulated (lower half) after h of clone infection compared to mock. for example, we found a novel mirna (mlugd in our annotation; predicted by mirdeep ; supplementary table s ) located in an intron of the protein-coding gene sema g, significantly down-regulated (log fc = - . ) during clone infection ( figure c and d) . based on rfam alignments we further found a histone '-utr stemloop (rf ), an rna element involved in nucleocytoplasmic transport of the histone mrnas, significantly down-regulated during infection. for the same comparison of mock and virus-infected samples at h, the rrna-depleted data set (hölzer- ) revealed several de lncrnas. for example, we found a lncrna potentially transcribed in an intron of mx (mlugl ) up-regulated (log fc = . ) during infection. another lncrna (mlugl ), we found potentially transcribed as a part of two exons of the plat protein-coding gene and down-regulated (log fc = − . ) during viral infection (see details in supplementary files s . ). interestingly, based on the field- rna-seq data, we found two internal ribosomal entry site (ires) in the genes vegfa and odc with rfam ids rf and rf , respectively, to be -fold up-regulated during p. destructans infection. in this study, we comprehensively annotated ncrnas in readily available bat genomes obtained from the ncbi database (table ) . we provide novel annotations in the common gtf format, following a hierarchical structure of gene, transcript and exon features to allow direct integration of our annotations into already available ones (supplementary tables s -s ). finally, we provide for each bat genome assembly an extended annotation file merged with the protein-and non-coding gene annotations that were already available by the ncbi database (supplementary files s ; leaving out potential lncrnas that can be downloaded separately, see supplementary table s ) . we used six rna-seq data sets derived from the transcriptomic sequencing of four bat species (table ) to calculate normalized expression values for our newly annotated ncrnas and exemplarily show significantly differential expressed ncrnas (figure ) , which were never before described on such a large scale for any bat species. in addition to the evolutionarily explainable differences in the pure existence and the amount of annotated ncrnas in bats, we have observed that the assembly quality can also influence the annotation results. while the effects on the annotation of short ncrnas seem to be small (with some exceptions), the number of identified lncrnas and lncrna hot spots increases with increasing assembly quality (e.g. with a higher n ; see supplementary figure s ). however, this observation may be true for our data and analyses, but it also depends strongly on the annotation method used. recently, the bat k project (http://bat k.ucd.ie/) was announced as a global effort to sequence, assemble and annotate high-quality genomes of all living bat species ( ) . we aim to extend our annotation of ncrnas regularly and whenever new bat genomes become publicly available. in mid january , new low-coverage bat genomes of nine families were submitted by the broad institute to the ncbi genome database. unfortunately, our timeconsuming and computationally extensive analyses were already completed at this time point. we want to further automate our ncrna annotation workflow, to easily include these and any new bat (or other mammalian) genomes that will be sequenced and assembled in the future. our current identification of ncrnas in bat species will be usable as a resource (electronic supplement) for deeper studying of bat evolution, ncrnas repertoire, gene expression and regulation, ecology, and important host-virus interactions. detailed information about the bat genomes used in this study, their assembly quality and all ncrna candidates (in fasta, stk and gtf format) can be found in the electronic supplement (rna.uni-jena.de/supplements/ bats). the final extended annotations for each investigated bat species can be found in supplementary files s and the lncrna annotations in supplementary table s . to allow full reproducibility of our study, all final and intermediate data files (such as used genome files and mapping files in bam format) were uploaded to the open science framework under accession doi.org/ . /osf.io/ cmdn. python scripts used to filter and merge our annotations were deposited at github (github.com/rnajena/ bats ncrna). the virus-infected and ifn-stimulated small rna-seq data of the m. daubentonii kidney cell line was uploaded to geo (gse ). bats: important reservoir hosts of emerging viruses bat biology, genomes, and the bat k project: to generate chromosome-level genomes for all living bat species an eocene big bang for bats phylogeny, genes, and hearing: implications for the evolution of echolocation in bats mammal species of the world. a taxonomic and geographic reference a molecular phylogeny for bats illuminates biogeography and the fossil record crowd vocal learning induces vocal 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not out: the role of micrornas in hibernating bats a computational screen for mammalian pseudouridylation guide h/aca rnas small rnas with big implications: new insights into h/aca snorna function and their role in human disease long noncoding rnas: past, present, and future incredible rna: dual functions of coding and noncoding cncrnas: bi-functional rnas with protein coding and non-coding functions evolution of spliceosomal snrna genes in metazoan animals animal mitochondrial genomes integrative genomics viewer (igv): high-performance genomics data visualization and exploration icarus: visualizer for de novo assembly evaluation we thank ivonne görlich key: cord- -vbq izw authors: coban, cevayir; lee, michelle sue jann; ishii, ken j. title: tissue-specific immunopathology during malaria infection date: - - journal: nat rev immunol doi: . /nri. . sha: doc_id: cord_uid: vbq izw systemic inflammation mediated by plasmodium parasites is central to malaria disease and its complications. plasmodium parasites reside in erythrocytes and can theoretically reach all host tissues via the circulation. however, actual interactions between parasitized erythrocytes and host tissues, along with the consequent damage and pathological changes, are limited locally to specific tissue sites. such tissue specificity of the parasite can alter the outcome of malaria disease, determining whether acute or chronic complications occur. here, we give an overview of the recent progress that has been made in understanding tissue-specific immunopathology during plasmodium infection. as knowledge on tissue-specific host–parasite interactions accumulates, better treatment modalities and targets may emerge for intervention in malaria disease. supplementary information: the online version of this article (doi: . /nri. . ) contains supplementary material, which is available to authorized users. activation-induced cytidine deaminase (aid) . an enzyme that is required for two crucial b cell events in the germinal centre: somatic hypermutation and class-switch recombination. by incomplete immunity to malaria causes long-term hidden pathologies . one such pathology is burkitt lymphoma, a cancer that develops in both childhood and adulthood in africa and that is closely related to p. falciparum and epstein-barr virus co-endemicicity . the robust and long-lasting expansion of germinal centre b cell populations that occurs during plasmodium infection may induce increased activation-induced cytidine deaminase (aid) expression, eventually leading to chromosomal translocations [ ] [ ] [ ] that predispose to the development of lymphoma in immune tissues. an increasing recognition of the association between malaria infection and physical growth retardation in children in africa, regardless of nutritional status , , suggests a detrimental effect of chronic malaria infection on growth, possibly via an effect on the bone tissue environ ment . nevertheless, any plasmodium spp. causing infection in humans result in varying degrees of complications, which can not only be severe and life threatening but can also have a long-term impact on patients' quality of life even after recovery. in this review, we focus on how systemic infection by plasmodium parasites causes local but tissue-specific immunopathologies during the blood stage of infection, mainly through the manipulation of inter-tissue interactions between the blood and other host tissues that often result in dysfunction of certain, but not all, organs. acute systemic immune activation, such as pro-inflammatory cytokine production, lymphocyte activation and vessel congestion, is evident; however, how these pathological events affect each tissue or organ is not understood well. current interventions for malaria are based on the administration of anti malarial drugs aimed at killing parasites and on supportive care to reduce systemic symptoms, such as coma, high fever, severe anaemia and acidosis. in this review, we emphasize the need to focus on host interactions with plasmodium parasites at various tissue levels and the importance of targeting local and specific organ failure and/or pathologies during, as well as long after, infection. these pathologies might be critical for determining diagnostic as well as therapeutic targets for the development of novel adjunct therapies to be used in combination with current antimalarials. we describe new evidence and new ways of targeting infected tissues of a few, but not all, unique organs such as the brain, gut and bones. we initially summarize how the blood tissue plays a central role in the initial pathogenesis caused by plasmodium parasites, following which we explore the interaction of infected blood tissue with specific organs composed of multiple, unique interacting tissue layers. largely owing to limitations of space, topics regarding the sporozoite stages of infection in the liver and those on the innate immune recognition of plasmodium para sites , are out of the scope of this review. in fact, plasmodium parasites do possess several ligands used to manipulate host cell invasion that could potentially be exploited as host factor targets for anti-malaria therapy , , a notion close to the topic of this review, but we exclude and leave this to an excellent recent article focusing on this topic . pathology within the blood tissue blood is a tissue (box ) that is formed by the plasma and blood cells (that is, erythrocytes, platelets and leukocytes) and is primarily located in the blood vessels. the infection of erythrocytes by plasmodium parasites results in extensive erythrocyte remodelling and dysfunction followed by cell lysis, which contribute to the pathology of severe anaemia. anaemia. plasmodium parasites (a merozoite form) are released into the bloodstream from liver hepatocytes within specialized vesicles called merosomes and continue a repetitive erythrocyte-invasion cycle in the blood tissue. merozoites released from the liver enter into erythro cytes through a very dynamic and complex process , (fig. ) . once erythrocytic infection is established, continuous destruction of erythrocytes due to schizont rupture contributes to anaemia. a moderate degree of anaemia is caused simply by the naturally occurring life cycle of para sites in erythrocytes. however, studies in both humans and mice have clearly indicated an increased removal of infected and uninfected erythrocytes that plays an essential and major role in severe malarial anaemia [ ] [ ] [ ] . clinical observations and mathematical modelling have suggested that for each infected erythrocyte, approximately uninfected rbcs are removed during malaria . chronic anaemia may also be mediated by the generation of self-reactive anti-phosphatidylserine antibodies . phosphatidylserine on infected erythrocytes is thought to be pathologically exposed to the immune system during malaria, leading to the generation of anti-phosphatidylserine antibodies. under usual circumstances, uninfected erythrocytes, mainly young cells called reticulocytes, express high levels of cd , a 'do not eat me' signal , , which allows them to escape from phagocytosis. however, young erythrocytes generated during malaria expose phosphatidylserine earlier, thus leading anti-phosphatidylserine antibodies to bind to uninfected erythrocytes and facilitate their clearance, contributing to chronic anaemia. one of the outcomes of the destruction of high numbers of erythrocytes during malaria is an increase in intravascular haem release , , which is an important factor for neutrophil activation but in turn could be the reason for increased oxidative damage and thus decreased macrophage function and the neutrophil exhaustion observed during malaria. this implies that not only erythrocytes but also blood tissue components including various leukocytes collectively react to the presence of plasmodium parasites (covered previously by seminal review articles , , ) and their related products in the blood circulation and tissues (fig. ) . erythrocytes undergo extensive deformation and remodelling after invasion by plasmodium parasites. while remodelling allows nutrient acquisition and parasite growth, it leads to changes in erythrocyte structure, with an increase in rigidity and rosetting . hence, the mature schizont stages easily sequester in the host microvasculature, mostly during p. falciparum and p. knowlesi infection and to a lesser extent during p. vivax infection , whereas immature ring stages freely circulate in the vessels. plasmodium berghei anka infection in mice shows a similar sequestration phenotype [ ] [ ] [ ] [ ] , and in this instance, sequestration via cd may be beneficial for the survival of the parasite . it is believed that infected erythrocytes, mostly those in the schizont stages of parasite infection, adhere along with activated leukocytes (mainly cd + t cells) to the endothelial cells of small blood vessels in the brain via binding to cd , intercellular adhesion molecule (icam ), endothelial protein c receptor (epcr) and platelet endothelial cell adhesion molecule (pecam ) [ ] [ ] [ ] [ ] . however, endothelial cell acti vation can occur without direct adhesion of leukocytes to the endothelial cells, possibly as a result of metabolites produced by leukocytes or parasites . indeed, there are unidentified soluble factors released by irbcs that cause endothelial cell pathology . in line with this, endothelial cells, irbcs, platelets, leukocytes and monocytes were shown to increase their release of extracellular vesicles during plasmodium infection, and this contributed to inflammation and correlated with disease severity . extracellular vesicles are mostly released from irbcs during schizogony and contain rbc components, parasite proteins and various rnas that can activate innate immune cells . in addition, endothelial cells have been shown to act as antigen-presenting cells (apcs) through the phagocytosis of merozoites and the presentation of malarial antigens to cd + t cells, which leads to ifnγmediated and perforin-mediated disruption of the blood-brain barrier (bbb) . most of these models of malaria-induced pathology are still under debate, and more evidence from both humans and animal models is clearly needed. overall, while the process of sequestration is not completely understood, it is known to cause obstruction of blood flow in small capillaries and post-capillary venules (pcvs), endothelial cell activation and inflammation and severe pathology in many organs including lung, adipose tissue, spleen and brain , , , (fig. ) . plasmodium-infected erythrocytes, is transported to organs via blood vessels. between large arteries and veins, various smaller sized vessels such as arterioles, capillaries and pcvs are present and have a role in controlling blood pressure and velocity in organs (fig. a) . the speed of blood flow substantially decreases as the blood enters arterioles, drops dramatically again in the capillaries and then slightly increases in the venules and veins (fig. a) . depending on the size and tissue environment, blood vessel structures vary. generally, the smaller the diameter of the vessel, the less smooth muscle it contains. capillaries, which play a major role in exchanging materials in blood, are the smallest vessels and are composed of endothelial cells with firm tight junctions and a basement membrane and do not include a smooth muscle layer (fig. b) . unlike capillaries, pcvs may have a few thin smooth muscle a tissue operates as an orchestration of large numbers of similar cells for a specific function. mammals have four basic types of tissues: epithelial tissue, muscle tissue, nerve tissue and connective tissue. two or more different tissues form and function as an organ. in general, the cells in their related tissue environment and organ are found in harmony, but during infection and inflammation, this harmony is likely to be disrupted. depending on the organ that the tissue or tissues interact with and support, multiple specific layers are present, which gives tissue specificity to healthy and diseased conditions. connective tissue deserves special attention because it includes various subcategories such as blood tissue, which is a major target of plasmodium parasites. this tissue, which mainly connects and supports various other tissues, is composed of various subtypes in addition to blood tissue, such as adipose tissue, areolar tissue, cartilage and bone tissue, lymphatic tissue and fibrous tissue. the main characteristics of connective tissues are that they contain an extracellular matrix composed of various collagen and/or elastin fibres, they contain interstitial fluid and they are vascularized with blood vessels. importantly, blood vessels are lined by endothelial cells, a component of epithelial tissue, and covered by smooth muscles (a muscle tissue); they carry blood cells, infected erythrocytes and various materials to the tissues and organs of the entire body, suggesting that even at the basic blood vessel level, many complex tissues specifically interact with each other. because tissue specificity occurs within an organization of different interacting tissues, it is reasonable to hypothesize that plasmodium parasites will reach different tissues and organ environments and develop unique and specific interactions with other tissues via blood tissue. also known as virchow-robin spaces. spaces located between brain-penetrating pial vessels and the brain tissue grey matter. (isf). the solution present extracellularly, that fills the spaces between cells and tissues. (csf). the solution surrounding the brain and spinal cord, which mainly serves to protect these two important organs. layers (fig. c) . importantly, depending on the organ, capillaries and pcvs interact with additional cells in tissues (fig. d) . for instance, brain capillaries and pcvs are additionally surrounded by pericytes, astrocyte end-feet and microglia (fig. d) . in particular, pcvs differ from capillaries in that they have leakier tight junctions and thus, may create perivascular spaces that apcs can easily home to and that allow for the infiltration of inflammatory cells during inflammatory conditions . moreover, the capillary beds in various organs are mostly in close proximity to lymphatic capillaries, into which drain interstitial fluid (isf; mainly leukocytes) and materials coming from blood vessels. the isf drained into the lymphatic vessels reaches the lymph nodes and finally returns to the final destination veins (fig. a) . therefore, it is reasonable to think that the type and diameter of a blood vessel in a specific organ in addition to the specific connective tissue and smooth muscle that is present to support vessel integrity may determine the degree of sequestration of plasmodium parasites and the outcome of disease. the walls of the smallest capillaries and pcvs are presumably more susceptible to internal changes occurring in the vessels. therefore, in the following sections, we detail the blood vessels within a few organs, such as the brain, gut and bones, and highlight their unique involvement with irbcs during malaria. tissue pathology within the brain the brain is severely affected by p. falciparum infection and to a lesser extent by p. knowlesi and p. vivax infections. this unique brain pathology, known as cerebral malaria, involves convulsions, coma and high fever and develops with the presence of mostly ring-stage infected erythrocytes in the periphery (suggesting a sequestration of late-stage parasites in the organs) [ ] [ ] [ ] . disruption of bbb integrity is an established outcome in cerebral malaria and may cause brain swelling and death in both humans and animals , . the activation of blood tissue components and their effect on brain immune cells (microglia, astrocytes) have been addressed , - , but how an obligate erythrocyte-resident pathogen can cause bbb disruption is not well understood. although the brain is considered an immune-privileged organ that is protected by the presence of the tight and selective bbb and by the lack of lymphatic drainage (a notion that has been disputed recently , ), the loss of bbb integrity due to the direct invasion and inflammation of meninges by various extracellular pathogens is well known . to understand why and how brain tissue is affected during malaria, we should consider recent advances in the neuroscience field, which could help to expand our understanding of cerebral malaria pathogenesis and development. the central nervous system (cns) is composed of the brain and spinal cord, which are protected by the meninges (formed by three layers: dura mater, arachnoid mater and pia mater) and are associated with two types of fluids: cerebrospinal fluid (csf) running through the subarachnoid space and isf in the brain and spinal cord parenchyma (box ) . csf is derived from plasma but has far fewer proteins, no erythrocytes, very nature reviews | immunology cells neutrophils, monocytes, platelets, t and b cells etc. brain, lung, kidney, intestine, bone etc. following the release of plasmodium merozoites from infected hepatocytes into the bloodstream, repetitive erythrocyte-invasion cycles occur in the blood. this leads to the release of several parasite by-products, such as haemozoin, and the parasites themselves into the bloodstream. the blood-stage cycle of plasmodium infection causes various pathologies, such as anaemia, toxic haem release, immune cell activation (modulation of platelets, neutrophils, monocytes, macrophages, t cells and b cells) and can cause a cytokine and chemokine storm. in blood tissue, infected red blood cells (irbcs) and their products may interact with other infected and uninfected rbcs (causing rosetting), or they may interact with immune cell populations (causing a cytokine storm) or with the endothelial cells of blood vessels (causing rbc sequestration and microhaemorrhage). these cell-cell interactions have organ specificity and thus take place in specific tissue environments, resulting in specific immunopathologies. an organ located on the cribriform plate, which functions in smell. the bulb surface is surrounded by complex olfactory nerve structures that originate from the nasal cavity and project to the brain. small trabecular capillary structures are the main vessel structures inside the bulb. few leukocytes and higher sodium, chloride and magnesium contents. isf fills the basement membranes of brain capillaries, where most of the exchange between blood and the cns occurs. the selective bbb inside the brain blood vessels, especially capillaries, is composed of uniquely specialized endothelial cells with intracellular tight junctions, pericytes, astrocyte end-feet and microglia in addition to the basement membrane, suggesting that blood vessel structures in the brain differ from those in other organs of the body, with a unique involvement of brain cell astrocytes and microglia in disease processes (fig. d) . advancing our understanding of brain physiology (box ) clearly has important implications for neurological diseases, such as alzheimer disease and multiple sclerosis , , as well as for cerebral malaria. indeed, recent preclinical studies using cutting-edge imaging technologies, such as ultra-high-field . t magnetic resonance imaging (mri) and multi photon live imaging microscopy, in experimental cerebral malaria models have expanded our understanding of how cerebral malaria develops. below, we consider the roles of the retina, olfactory bulb and the perivascular spaces of the brain during cerebral malaria. arteries supplying blood to the brain divide into smaller arteries on figure | interaction of plasmodium-infected red blood cells with various blood vessels and lymphatics. a | infected red blood cells (irbcs) circulate in blood vessels, which vary in size from large arteries to veins, with blood flow running from arteries, arterioles, capillaries and post-capillary venules (pcvs) to venules and veins, providing controlled blood pressure and velocity in organs. the speed of blood flow is lowest in capillaries and pcvs. in various organs, these capillary beds are mostly near lymphatic vessels, through which interstitial fluid and materials coming from blood vessels drain the lymph nodes and finally return to veins. irbcs and irbc-mediated immune responses, therefore, may have profound effects on these smaller vascular beds in each organ. b | capillaries have only an endothelial cell wall and no smooth muscle; therefore, they are in close contact with irbcs. c | two capillaries fuse and form pcvs, which may have some smooth muscle and where leukocyte rolling can occur. d | capillaries and pcvs in the brain are additionally surrounded by pericytes, astrocyte end-feet and microglia; irbc-related events in capillaries are sensed immediately. a small, perforated bone structure that lies on top of the nasal cavity and supports the olfactory bulb. olfactory nerves running from the nasal cavity to the olfactory bulb pass thorough cribriform plate. the pial surface and penetrate into the brain parenchyma before further dividing into arterioles. arterioles (and venules) are surrounded by perivascular space which is filled with isf drained from the brain parenchyma (fig. a) . a newly defined 'glymphatic system' has been proposed in which the glia limitans, a barrier comprising astrocyte end-feet that surrounds small capillaries and veins, creates perivascular spaces via aquaporin (aqp ) channels. the aqp channels facilitate the flux of isf into perivascular spaces to be mixed with csf , , and this eventually contributes to the clearance of particles from the csf. interestingly, dilatation at perivascular spaces and astroglia and reduced expression of aqp protein have been reported during cerebral malaria in animal models , . furthermore, in the absence of aqp , mice with cerebral malaria succumbed to death more quickly , suggesting aqp has protective roles during cerebral malaria, perhaps in controlling liquid exchange, oedema and cell transmigration at the bbb. apcs with phagocytic properties such as dendritic cells and macrophages located in the pial surfaces and perivascular spaces have been shown to present antigens to t cells in various cns diseases . as visualized by electron microscopy and intravital two-photon microscopy, it seems that leukocytes and cd + t cells, and probably irbcs or their products, increasingly accumulate in perivascular spaces in very deep and branching vessels during cerebral malaria [ ] [ ] [ ] [ ] . these phagocytic apcs and activated endothelial cells in vessels have the capacity to present malarial antigens , . furthermore, all these events that are mediated by ifnγ could be reversed by blocking the adhesion molecules box | the steady state brain drainage system owing to the presence of the highly selective blood-brain-barrier (bbb), the brain has long been considered to have no classical lymphatic system and thus be an immune-privileged organ (see the figure) . in homeostatic conditions, the cerebrospinal fluid (csf) is continuously produced by the choroid plexus and drained into the bloodstream via arachnoid granulations in the venous sinuses or nasal lymphatics under the cribriform plate next to either the olfactory nerves or the spinal and cranial nerve sheaths. immune surveillance via selective epithelial cells and homeostatic leukocyte trafficking in csf have been acknowledged ; however, recent studies by louveau et al. and aspelund et al. have challenged previous knowledge by demonstrating the presence of a classical lymphatic drainage system located in the brain meninges. the authors clearly showed that this lymphatic network works alongside the classical known csf drainage system, with the additional capacity to drain other constituents such as toxic macromolecules and immune cells into the deep cervical lymph nodes, providing evidence of a connection between the csf, the brain and the periphery. essentially, these newly identified brain lymphatic vessels lay in the dura mater and collect csf and interstitial fluid (isf) together with macromolecules such as amyloids and immune cells from perivascular spaces (pvss) of the brain parenchyma. similar lymphatics are also present around another immune-privileged organ, the eye (along with the optic nerve) , and over the olfactory bulb, aligned throughout meninges as simple narrow vessels that become wider in the transverse sinuses, ultimately reaching the cerebellum. all lymphatic vessels finally drain into the deep cervical lymph nodes. it is of note that similar to blood vessels, these lymphatic vessels are different in size depending on their anatomical location in the central nervous system, and owing to that, their interaction with local blood vessels or astrocyte end-feet may be different during homeostasis than in diseased conditions. pcv, post-capillary venule. figure | infected red blood cells in close proximity with brain components. a | small arteries on the pial surface of the brain penetrate into the brain parenchyma and further divide into arterioles; these are surrounded by perivascular space (pvs) which is filled with interstitial fluid (isf) that has drained from the brain parenchyma. arterioles become capillaries, venules then veins, which finally drain into dural venous sinuses, and each of these areas potentially contain abundant infected red blood cells (irbcs), as well as lymphocytes such as cd + t cells. lymphatic vessels located in the dura carry immune cells and cerebrospinal fluid (csf) into deep cervical lymph nodes. b | the retina is rich in photoreceptors (cones, bipolar cells and ganglions), nerve cells and complex blood vessels, especially capillary structures surrounded by müller cells, a retina-specific astrocyte-like cell. the optic nerve serves as a connector between the retinal nervous tissue and the brain, where meningeal membranes, dura lymphatics and csf cover the optic nerve up to the retina border. this area may contain abundant irbcs. c | the olfactory bulb is surrounded by meninges of brain dura and arachnoid and pial layers. csf similarly runs throughout the bulb and reaches the cribriform plate area. olfactory nerves originate from the nasal mucosa and terminate in the olfactory bulb through the cribriform plate, which is also the route of the blood vessels surrounding the olfactory nerves. these olfactory nerves project any signal for smell to the olfactory bulb and the brain. inside the olfactory bulb, very dense blood capillaries are oriented in different directions (radially and tangentially), with thin astrocyte end-feet surrounding the vessels, and sense irbc-related events and secrete several cytokines and/or chemokines including cc-chemokine ligand (ccl ). blood capillaries easily bleed during cerebral malaria. pcv, post-capillary venule. a mechanism explaining the relationship between neuronal activity and the cerebral blood vessels and the blood flow. very late antigen (vla ) and lymphocyte function-associated antigen (lfa ) (ligands for vascular endothelial adhesion protein (vcam ) and icam , respectively), which are expressed on activated cd + t cells during cerebral malaria . one of the important features of inflammation in perivascular spaces is that the parasite and inflammatory products may directly activate astrocytes , , microglia and neurons , and may cause an increase in the levels of protein s b (a biomarker for astrocytes) and in the levels of the microtubule-associated protein tau (a biomarker for degenerated axons) in csf . nevertheless, future studies are needed to provide direct evidence of this. it would also be interesting to investigate in the future whether there are other types of perivascular spaces found at different locations in the brain , , because pia mater also covers the cerebellum and other cns areas, such as the spinal cord. therefore, the interaction of perivascular spaces with small blood vessels may vary depending on the anatomical location and unique architecture of the tissue, for example, in the brain cortex, spinal cord, cerebellum or olfactory bulb. the back of the eye ball is lined by the retina, with the optic nerve in the middle and going through to the brain. the optic nerve serves as a connector between the nervous tissue of the retina and the brain, where brain meninges, dura mater lymphatics and csf cover up the optic nerve up until the retina border. the retina is rich in photo receptors, nerve cells and complex blood vessels, especially capillary structures (fig. b) . retinal changes (such as haemorrhages, multiple peripheral and macular patchy whitening areas and papilloedema) have been commonly observed in children and in adults who develop sudden comatose cerebral malaria symptoms after a few days of fever [ ] [ ] [ ] . these retinal changes occur in areas that show high sequestration of parasites accompanied by thrombi (composed of fibrin and platelets) , , which cause impaired capillary perfusion , and are well correlated with the severity of disease . interestingly, other areas in the eye, such as the optic nerve head, ciliary body and iris, are similarly affected by irbcs , indicating that specific blood vessel similarities may occur within other parts of the brain. retinal homeostasis is maintained through neurovascular coupling involving astrocytes, müller cells (a specific type of glial cell found in the retina) and resident retinal microglia, with müller cells, pericytes and astrocytes mainly ensheathing the retinal blood capillaries . the observation that the accumulation of fluid between processes of müller cells and photoreceptor cells in the macular area produces intra-retinal spaces in human cerebral malaria suggests that these areas represent perivascular-space-like areas in the retina that support immunological events similar to those that occur in the perivascular spaces in deep brain areas. furthermore, high-field mri studies have identified damage to the optic and trigeminal nerves during experimental cerebral malaria even before the development of oedema, which may cause loss of visual acuity . the olfactory bulb and cerebral malaria. the olfactory bulb is situated on the cribriform plate, located on top of the nasal sinus. although the olfactory bulb has several characteristics that distinguish it from the brain, it is covered by similar meningeal structures and by the csf and is thus included in the cns. however, in contrast to other parts of the brain, olfactory nerves initiate from the nasal mucosa and terminate in the olfactory bulb through the cribriform plate, which is also the route of the blood vessels surrounding olfactory nerves. these olfactory nerves project any signals for odour to the olfactory bulb and to the brain. inside the olfactory bulb, very dense blood capillaries are oriented in different directions (radially and tangentially) with thin astrocyte end-feet surrounding the vessels (fig. c) . owing to complex structural interactions of nerves through the cribriform plate, capillaries, microglia and astrocyte endfeet in this region, the olfactory organ is considered to be the gateway of the otherwise well-protected brain to the outside world. it is known that several molecules, cells and even pathogens can gain access to the brain parenchyma through this route . by use of . t mri in mice, it was discovered that the olfactory bulb is the first region during experimental cerebral malaria to show vascular leakage and bleeding , a phenomenon possibly related to the unique small capillary trabecular structure of the olfactory bulb. this was also directly visualized and confirmed by intravital two-photon microscopy during cerebral malaria . these dense and directionally structured olfactory blood capillaries , which are thought to be leakier than those in the brain cortex or pons , are a suitable scaffold for irbcs . sequestration of irbcs in the olfactory bulb capillaries results in astrocyte and microglia activation , , and in the loss of tight junction integrity, and these parasite-mediated events lead to olfactory dysfunction and loss of smell, which could be a valuable early diagnostic marker for cerebral malaria. moreover, astrocytes surrounding small trabecular capillaries in the olfactory bulb were found to sense changes in the vessels and secrete cytokines and chemokines, such as cc-chemokine ligand (ccl ), which are involved in the accumulation of pathological cxcr + cd + t cells in the olfactory bulb . expression of cc-chemokine receptor (ccr ), which is the canonical receptor for ccl and ccl , was shown to be essential for antigen cross-presentation by cd α + dendritic cells and for the activation of cxcr + cd + t cells in the spleen during cerebral malaria . by contrast, ccl secreted from astrocytes played a role in the recruitment of pathological cxcr + cd + t cells in the olfactory bulb via its non-canonical receptor cxcr (ref. ) . a pathological role for several chemokines and chemokine receptors (including cxcchemokine ligand (cxcl ), cxcl and cxcr ) is well recognized in experimental cerebral malaria , but the study discussed above highlights how the specific role for chemokines in cerebral malaria might differ depending on the tissue and organ environment. extended mri studies using sensitive contrast reagents have further confirmed these findings and have shown that micro-haemorrhages originate in the olfactory lacteals small lymphatic capillaries located in the villi of the small intestine. vessels with a structure similar to capillaries but that have larger diameters and porous endothelial cells, which allow for permeability and exchange of materials; they are located in bone marrow, liver and spleen. bulb together with oedema, but that the oedema further spreads along the rostral migratory stream, similar to the migration route of immune cells and neuroblasts , and may reach the brainstem . moreover, the glymphatic system may play a key role in the rapid inflammatory spread of cerebral malaria from the olfactory bulb and rostral migratory stream axis to deeper brain areas such as perivascular spaces . it is also possible that these events that occur in the olfactory bulb during cerebral malaria may block the drainage of csf into nasal lymphatics and aggravate brain oedema (fig. c) . clearly, the role of the olfactory bulb in cerebral malaria in humans warrants further research. tissue pathology within the gut gastrointestinal symptoms such as abdominal pain, vomiting and diarrhoea are common symptoms during malaria infection. autopsies of human patients who died from severe malaria have shown that sequestration of irbcs occurs in the gastrointestinal tract (including in the colon, jejunum, ileum and stomach) , suggesting that gastrointestinal bleeding occurs during malaria. the presence of plasmodium dna in faeces additionally supports the occurrence of intestinal bleeding during infection ; however, how parasites interact with other cells in gut tissue is a research area that has only recently gained attention. recent studies have confirmed that pathological changes (including detachment of epithelia and shortening of villi and the colon) occur in the intestinal tract during malaria ; such changes may cause increased intestinal permeability, ruptures and leakage of infected erythrocytes into the lumen, causing dysbiosis of the intestinal microbiota, which may also contribute to disease severity . moreover, there is a close correlation between malaria infection and increased rates of infection with other gut pathogens (for example, nontyphoidal salmonella and helminths), possibly owing to an impairment of intestinal barrier function during malaria [ ] [ ] [ ] . several factors may have a role in this, such as increased histamine release from mast cells in the gut or the modulation of intestinal inflammation and immune responses via an increase in the number of exhausted neutrophils or an increase in the levels of il- family cytokines, including il- and il- , in the intestinal tissues during malaria infection, as these factors may ameliorate responses to secondary bacterial infections [ ] [ ] [ ] . however, it should be kept in mind that the composition of the gut microbiota has a clear impact on the severity of malaria disease [ ] [ ] [ ] , suggesting that there is a bidirectional relation between the gut and plasmodium parasites that is not well understood and requires further investigation. nevertheless, a close look at the intestinal tissues may give some clues for understanding gut-plasmodium interactions (fig. a) . the blood capillaries carrying irbcs reach the lamina propria of the intestinal mucosa and possibly come into close proximity with lacteals, which are specialized lymphatic vessels of intestinal villi that play a key role in nutrient absorption and in gut immune responses, such as the transport of microbial antigens and apcs to the mesenteric lymph nodes, where antigen presentation by apcs promotes the induction of t cells with gut-homing properties . there is a gut-vascular barrier comprising endothelial cells, enteric pericytes and enteric glial cells that is very similar to the barriers found in other parts of the body (such as the bbb) and that actively blocks bacterial dissemination from the gut epithelium to the blood . therefore, it is reasonable to hypothesize that intestinal bleeding may occur as a result of malaria-mediated immune responses that directly affect lacteals. it is possible that lacteals with disturbed remodelling have impaired survival and villi length , and this may have knock-on effects on immune cell composition and on the microbiota of the gut tissue. tissue pathology within bone bone tissue is a mineralized connective tissue supporting the whole body and it houses the bone marrow, which comprises specialized niches that maintain and regulate haematopoiesis, erythropoiesis and bone remodelling (fig. b) . arteries in the bone marrow unite and become specialized vessels called bone marrow sinusoids, which allow cells to pass between the circulation and bone marrow . without any cyto adherence, plasmodium parasites can invade and multiply in nucleated erythroblasts located in extravascular spaces of the bone marrow [ ] [ ] [ ] . the parasites can survive and hide in these erythroid precursors and develop into gametocyte stages [ ] [ ] [ ] , likely assisting in the continuous success of malaria transmission to mosquitoes. however, it is still not known why plasmodium parasites preferentially differentiate into gametocytes in the bone marrow and whether a specific bone marrow niche is required to support the development of gametocytes. unlike haematopoietic niches, which are located adjacent to sinusoids, erythroid niches are found throughout the bone marrow and are generally located away from the sinusoids, although they move closer to the sinusoids as they mature . this may suggest a major role for the erythroid niche in plasmodium gametocyte development. from an immunological point of view, it has recently been shown that cd + t cells produce ifnγ in response to infected mhc class i-expressing erythroblasts residing in the bone marrow; by contrast, mature erythrocytes residing in the blood circulation lack any mhc molecules and cannot present antigens to cd + t cells , . activated cd + t cells promote the exposure of phosphatidylserine on infected erythro blasts, and this enhances the susceptibility of the infected cells to phagocytosis by macrophages , suggesting a protective role of cd + t cells against malaria. however, whether bone marrow erythroid cells contribute to the expansion of pathological populations of cd + t cells that can drive cerebral malaria has not been investigated. despite the growing body of information on how bone marrow niches are manipulated by plasmodium parasites, very little is known about the potential outcomes of interactions between plasmodium parasites and bone tissue. in bone marrow, haematopoietic stem cells give rise to blood cells and bone-resident osteoclasts, whereas mesenchymal stem cells give rise to osteoblasts, adipocytes and stromal cells. a very recent study has suggested that during plasmodium infection, parasites and their products -mainly haemozoin -continuously accumulate in bone marrow and cause acute as well as chronic bone loss . although it is not precisely known how plasmodium products are retained long term in the bone marrow, it is possible that engulfment by macrophages or other phagocytic cells, such as bone-resident osteoclasts, or trapping by extracellular matrix in the bone marrow contributes to this phenomenon . chronic bone loss during malaria infection is mediated by over-activation of osteoclast resorption activity. the osteoclasts are activated by the key osteoclastogenic cytokine rankl (also known as tnfsf ), which is upregulated in osteoblasts through myd -dependent signalling triggered by the persistence of parasite products in the bone marrow . these findings highlight how, not only during acute malaria infection but also after recovery from infection, plasmodium products continue to interact with tissueresident cells, including bone cells, osteoclasts and osteoblasts, and how they can cause long-term effects in the host, such as bone loss and growth problems. the molecular and immunological pathways underlying the interaction between plasmodium spp. and the niches of various bone marrow cells and the effect of these interactions on the immune system , need to be addressed in the future. malaria is a serious disease with acute life-threatening and long-term complications, all of which can be attributed to local but specific organs in which plasmodium figure | infected red blood cells in gut and bone marrow niches. a | the intestinal tract is made up of several tissue layers. the villous mucosa is covered by mucus that is secreted by the goblet cells of the epithelium. it lies on a connective tissue, the lamina propria, which is an environment rich in blood vessels, lymphatics, nerves and immune cells. it also contains peyer's patches and is lined by smooth muscle tissue (muscularis mucosa). blood capillaries that are carrying infected red blood cells (irbcs) reach the lamina propria of the intestinal tract and can come into close proximity with lacteals at this location. b | bone tissue supports the whole body and is home to bone cells, blood vessels, nerves and endothelium, as well as containing the bone marrow niches. the bone marrow is composed of haematopoetic and mesenchymal stem cells that are involved in both haematopoiesis and bone remodelling, such as osteoclasts and osteoblasts. cells and irbcs pass through the circulation and bone marrow via specialized venules called bone marrow sinusoids. plasmodium parasites can invade erythroblasts, the erythrocyte precursors, and preferentially form gametocytes in the bone marrow erythroblastic niche. released plasmodium products including haemozoin can be engulfed by osteoclasts and activate osteoblasts and osteoclast precursors. hsc, haematopoietic stem cell; msc, mesenchymal stem cell; osb, osteoblast. haemozoin a unique by-product of plasmodium parasites that accumulates in several organs, including spleen, liver and bone marrow, even after the infection resolves. parasites and their products or irbcs interact with the tissue, causing an imbalance in the specific tissue environ ment. here, we have attempted to summarize the recent progress of research focusing on the tissue environment in a few example organs, such as the brain, gut and bone, where plasmodium parasites reach the tissue via the blood. however, the lungs , kidneys and placenta represent other organs that are seriously affected during malaria infection, and these tissues should also be examined more closely in future studies. here, our aim has been to show that the influence of malarial parasites reaches beyond erythrocytes and may vary depending on, and be tightly regulated by, the vessel-specific and/or tissue-specific microenvironment. on the basis of the available data, we suggest that the brain pathology caused by plasmodium parasites is not homogeneously controlled by parasite sequestration but is rather controlled by the local, specific and unique anatomical structure of vessels, parenchyma and lymphatics, which closely interact with each other during both homeostasis and infection. this concept is in accordance with the recent understanding that the vascular and epithelial barriers do cooperate in an organ-specific manner in homeostatic and diseased conditions . we additionally emphasize that parasite-derived products can continue to stay in the body, interact with tissues and cause chronic pathologies even long after recovery from infection, particularly in the case of bone tissue. the gut pathology caused by malaria, which has recently been recognized, was summarized, and the possible anatomical interaction between blood tissues and special gut lymphatics has been introduced. there are multiple benefits of understanding malaria-related pathologies in various tissues. for instance, understanding the reaction of microglia and astrocytes to irbcs will be beneficial for generating intervention strategies that block these interactions to prevent cerebral malaria and/or related long-term sequelae. supporting the gut microbiota during and after plasmodium infection might help to protect against and promote recovery from secondary infections. similarly, additional therapies like vitamin d supplementation to support the bone environment affected during and after malaria might help the growth of children or support bone health in elderly people. the search for a full understanding of malaria-related pathologies is important for ensuring that malaria is one day a curable and/or preventable disease. towards this goal, novel approaches should be investigated to analyse unknown or overlooked anatomical -as well as immunological -host-parasite interactions at various tissue levels of each organ, not only during infection but also long after clearance of the initial infection by the parasite. by doing so, we will be able to prevent the disease, diagnose and treat patients, predict prognoses and allow patients to receive appropriate medical care through the use of effective vaccines, diagnostic tools, adjunct therapies 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doi: . /ilar/ily sha: doc_id: cord_uid: j yaga failure to reproduce results from some scientific studies has raised awareness of the critical need for reproducibility in translational studies. macroscopic and microscopic examination is a common approach to determine changes in tissues, but text descriptions and visual images have limitations for group comparisons. semiquantitative scoring is a way of transforming qualitative tissue data into numerical data that allow more robust group comparisons. semiquantitative scoring has broad uses in preclinical and clinical studies for evaluation of tissue lesions. reproducibility can be improved by constraining bias through appropriate experimental design, randomization of tissues, effective use of multidisciplinary collaborations, and valid masking procedures. scoring can be applied to tissue lesions (eg, size, distribution, characteristics) and also to tissues through evaluation of staining distribution and intensity. semiquantitative scores should be validated to demonstrate relevance to biological data and to demonstrate observer reproducibility. statistical analysis should make use of appropriate tests to give robust confidence in the results and interpretations. following key principles of semiquantitative scoring will not only enhance descriptive tissue evaluation but also improve quality, reproducibility, and rigor of tissue studies. tissue evaluation is a common research tool used in basic science and - toxicological - and clinical studies. [ ] [ ] [ ] [ ] scoring of tissues changes or lesions can aid in assessing model phenotypes, disease pathogenesis, toxicities, and efficacy of therapies. , , [ ] [ ] [ ] [ ] morphological examination of tissues produces text descriptions and visual images that can be valuable to define initial group-specific differences; however, these observations are qualitative in nature and have limitations for rigorous group comparisons. in general, quantitative and semiquantitative approaches can be applied to tissues to produce scores that enhance the rigor of data. "quantitative" scores are derived from measuring tissue parameters often using manual techniques or by using specialized software to analyze digital images , , and yield a discrete numeric value on a continuous scale (eg, . , . , . , etc.) . in contrast, "semiquantitative" scores are assigned by an observer based on predefined morphologic criteria, and these whole number scores are, by definition, less precise than quantitative scores because they approximate relative changes. semiquantitative scoring can be applied to macroscopic and microscopic tissue changes, allowing generation of robust data that are amenable to statistical analysis and evaluation of experimental groups. the goals of this paper are to introduce investigators to key ideas in reproducible semiquantitative scoring of tissues and guide them in finding additional resources for more detailed discussions and examples. for the remainder of this paper, "scores" and "scoring" will refer, unless otherwise specified, to semiquantitative methods. integration of semiquantitative scoring in translational research can be useful in several situations. , , , first, semiquantitative scoring data are relatively inexpensive, because no software or computational tools are necessarily needed. second, it can be a quick screening method to produce pilot data for grant applications or guide future research studies. third, semiquantitative data can enhance the rigor of descriptive text. while annotated images and descriptive text may show apparent differences between groups, semiquantitative scores can provide a comprehensive overview of tissue changes for group comparisons. lastly, semiquantitative data can be used to guide, corroborate, and validate observations or data obtained from other assays. semiquantitative scoring can be used to acquire data in several scientific areas, and fundamentally the core concepts are similar. [ ] [ ] [ ] [ ] [ ] [ ] in the preclinical area, which utilizes models (eg, animal, tissue/cell cultures, etc.) of human diseases/conditions, semiquantitative scoring is regularly used to compare experimental groups. , , , in the clinical area, semiquantitative scoring of human tissues (eg, cancers, tissue/cell cultures, etc.) is often used to help define disease diagnosis, pathogenesis, biomarkers, and clinical prognosis. [ ] [ ] [ ] semiquantitative scoring is also a key component of nonclinical toxicology studies, , which are performed to support regulatory agency submissions and thus have an inherently different purpose than preclinical investigative studies. here, the goal is to evaluate the safety of the material being tested (ie, hazard identification and risk assessment) rather than to assess potential treatment efficacy. to support future clinical trials, all toxicity studies must be performed according to guidance documents from various regulatory agencies, such as the food and drug administration. additionally, the usage of consistent diagnostic terminology for each organ system in rodents and large animals is strongly recommended. , collaboration with experienced toxicologists and toxicological pathologists is highly encouraged before investigators plan these types of studies to ensure the current regulatory guidelines are followed. unless specified, the remainder of the paper will focus on foundational concepts for semiquantitative scoring emphasizing nontoxicologic translational studies (table ). statistician george box once stated, "all models are wrong, but some are useful." to apply this quote in the context of translational research, modeling in itself (eg, animal models) is never fully identical to the condition being modeled (eg, human disease). due to several factors (genetic diversity, comorbidities, etc.), even small cohorts of humans do not fully "model" the human condition. this is, in part, why large and multiple clinical trials are often required to test for efficacy and adverse effects of new therapeutics in humans. in research, studies that model the human condition should be constructed to be as useful and reproducible as possible; one way to do this is to guard against factors that are known to cause bias. in science, bias is a term applied to areas of subjectivity (from overt to subconscious) that can skew data and contribute to lack of scientific reproducibility, an unfortunate reality that has been increasingly recognized. [ ] [ ] [ ] there are several ways to constrain bias when scoring tissues, and by using these precepts investigators can acquire more objective data. a critical step for reproducible science is to establish a strong foundation in sound experimental design. , , [ ] [ ] [ ] [ ] constraining bias early, at the experimental design stage, avoids downstream "junk in, junk out" problems and issues of "regret" that can lead to adverse and unexpected influences in the quality and analyses of tissues. , considerations to address during the experimental planning stage include selection of the appropriate model (eg, species or strain), consideration of the appropriate controls (eg, matching with respect to age, sex, or litter), and calculation of the sufficient sample size needed for statistical significance. it can be helpful to revisit proper techniques for tissue collection as well as the different options available for fixation and storage because tissue handling variables can influence staining quality. , , , staining techniques can also vary in consistency as a function of stain choice and by staining protocol. for example, the planning phase for a hypothetical experiment involving viral-induced inflammation in the lungs of a mouse should address whether there is sufficient tissue for multiple tests (eg, bronchoalveolar lavage, paraffin, and oct embedded tissues, pcr, microarray, protein quantification, and viral culture). novice investigators might make several invalid assumptions (eg, homogenous virus distribution in lungs, bronchoalveolar lavage collection does not affect other analyses, murine lung size will allow for ample tissue sampling, etc.) that can lead to incomplete and/or skewed data. early consultation with all key collaborators (especially pathologists) at the time of experimental design will ensure all needs are accounted for (eg, appropriate amount and type of tissue allocations) to prevent oversights. randomization ("heterogenization") is an important tool to prevent the introduction of treatment bias that arises from overly homogenized groups; this situation has been variably coined as litter effect, cage effect, or batch effect. [ ] [ ] [ ] the introduction of such bias can sometimes happen in innocuous ways. for example, tissue harvest from a large cohort of animals will likely produce a wide range of times from onset of the experimental day until necropsy. if animal in one treatment group were necropsied early, before starting on the other group, tissue parameters such as liver glycogen stores (especially in fasted animals) could be affected and create artifactual group-specific bias. randomization of all the groups (animals and their tissues) can mitigate bias introduced by the experimental procedures. other examples of variables that could render a study nonrandomized include differential housing of subjects (single vs group) or subject/sample processing order. any variable that is not randomized across treatment groups has the potential to confound the data. bias may also be introduced into translational research in studies conducted without the support of expertise-specific collaborators to help plan, execute, and appropriately interpret the study. , specifically, statistical and pathological analyses are common components in translational studies, but trained statisticians and board-certified pathologists are often omitted from these multidisciplinary teams, leading to data interpretations that are more prone to errors. , , for tissue scoring, a designated "observer" must thoroughly examine samples and ascribe scores. various biomedical personnel (including principal investigators, postdocs, and even students) have been assigned the role of observer to score tissues. this approach, which lacks the expertise of a board-certified pathologist trained in tissue interpretation, has been labeled as do-ityourself pathology, a practice that has been associated with numerous publications with erroneous interpretations. , , [ ] [ ] [ ] [ ] while observations made by biomedical personnel may be biologically accurate in some cases, it is important to note that tissue examination by nonpathologists (even those who are "scientific experts" for a particular disease) is not recommended. nonpathologist observers are more prone to making type i errors (ie, "false positives" often from inadequate consideration of other morphologically similar tissue changes) and type ii errors (ie, "false negatives" often from not recognizing unexpected tissue changes). inclusion of experienced and board-certified pathologists, who are specially trained to examine and interpret tissue changes as part of the multidisciplinary team, can greatly enhance the quality of tissue evaluation and scoring. semiquantitative scoring depends on the judgment of an "observer," exposing the evaluation to some level of bias. masking (also known as blinding) is a method to keep the observer from knowing the treatment groups when assigning tissue scores. experts at every level (even pathologists!) are at risk of having their judgment subliminally influenced by information cues from the study. masking significantly reduces this possibility. there are several methods to mask observers to the experimental groups, each with advantages and disadvantages that have been previously reviewed. , , briefly, comprehensive masking prevents the observer from knowing any details about the study design, treatments, or grouping of samples at initial examination. this approach may seem unbiased and even useful upon first glance, but in reality can easily lead to false negatives and skewed interpretations. an alternative approach to comprehensive masking is group masking. here, the study design, treatments, and goals are all transparent to the observer; however, the samples are each assigned into de-identified groups, so that the observer does not know which group had specific treatments. a final example is that of postexamination masking. in this approach, full transparency and access are allowed to all study-related information and slides. this is an important step, especially in new or poorly characterized models, to avoid missing subtle or unexpected treatment-related changes. once the decision is made to score the tissues, the slides are masked to the observer and scores assigned. masking should be a standard component that is defined in the methodology of all studies that use semiquantitative scoring. for each of these approaches, the observer should evaluate the scores and tissues after scoring in a nonmasked fashion to give confidence in the scoring system and interpretation of the results. one of the major benefits of semiquantitative scoring is the transformation of descriptive (qualitative) observations into numerical data so as to allow statistical group comparisons and enrich data quality. a widely accepted premise for tissue scoring is the exhibition of at least three characteristics: it should be definable, reproducible, and produce meaningful results. in translational studies, scoring is typically performed on tissues to detect treatment group differences. there are major types of tissue changes that are targeted when scoring tissues: lesions and stains (or other labeling techniques). some studies have used a merged scoring (ie, an average or sum of scores) approach in which multiple parameters are combined to form one final "composite" score, but if this approach is used it should have biological relevance. , , lesions a tissue lesion can be defined as an observed morphologic change that differs from control or normal tissue architecture. lesions can be scored in many ways, such as size, shape, distribution, presence/absence, etc., depending on the expected disease-specific findings or tissue observations. considerations for selecting the appropriate scoring parameter include a thorough examination of all tissues that catalogs the lesions seen; identification of lesion parameters (size, shaped, etc.) that appear to have chronological or group specific differences; and biological relevance to the pathophysiology of the model. another common approach is to score histochemically or immunohistochemically stained tissues or cells. , here, the observer can assess either the distribution (eg, percent of stained cells) or intensity (eg, weak to robust) of the labeled cells. similar to considerations described for "lesions," selection of a scoring parameter may be dependent on the staining presentation as well as the biology of the model. for example, a virus infection of the lung might warrant evaluation of the distribution of staining, whereas a tp marker might require staining intensity as a gauge of activation in benign vs malignant tumors. several methods of semiquantitative scoring have been discussed in recent reviews, and readers are encouraged to use these for more specific details. , , , , [ ] [ ] [ ] while several types of semiquantitative scoring tests are available, ordinal scoring is by far the most common in translational research and will be further discussed here. ordinal systems produce hierarchal or progressive numeral scores (also known as "grades" or "tiers") that are reflective of the extent and/or severity of change. a mock example of this is an ordinal scoring method composed of whole numbers from to representing distribution of tissue necrosis in which is normal, is < % necrosis, is % to % necrosis, is % to % necrosis, and is > % necrosis. ordinal scoring systems should follow several key principles for enhanced reproducibility. first, the range of levels is recommended to be about to ; fewer than this decreases sensitivity to detect group differences and more than this reduces repeatability , , , second, each progressive level should have welldefined descriptors (such as the percentage of tissue affected, as in the example above). descriptors that are vague and subjective, such as is normal, is mild, is moderate, and is severe, should be avoided or include additional information to clearly discern each level. score descriptors in an ordinal system can be defined by multiple lesion parameters (eg, inflammation, proliferation, necrosis), but in these situations reproducibility can sometimes be limited. therefore, separating each lesion parameter into its own ordinal scoring system is often preferred. third, ordinal scores are inherently discontinuous data that are not normally distributed (bell-shaped) and require nonparametric statistical analyses. data that are normally distributed should be analyzed with parametric analysis (eg, paired or unpaired t tests). many statistics software packages include tests for normality for determining whether a given statistical test will be valid for the dataset. it is not appropriate to use parametric analysis to analyze data derived from ordinal scoring systems. , , evaluation for semiquantitative scoring to have purpose and relevance, it should have validation with biologically relevant data. in this evaluation, semiquantitative scores are tested for a correlation with biologically relevant data in the model. , , if a significantly positive or negative correlation exists, then this confirms that the scoring system is relevant to the model. conversely, if no correlation exists, then one has to question the use and utility of the scoring system for the model. another form of validation is that of repeatability by the observer, both intra-observer (same person scoring the data) and inter-observer (different people scoring the data). , , validation of repeatability gives confidence in the scoring system descriptors as it relates to the model and also gives confidence in its repeatable use by other laboratories. once the semiquantitative tissue scores are collected, appropriate statistical tests can be applied; these have been reviewed. , , , , as mentioned above, appropriate expertise such as a statistician collaborator would be advantageous to guide proper statistical analyses of the data. awareness of the type of data produced by semiquantitative scoring is very important because it guides the type of statistical tests used to give the most compelling interpretations of the study. as alluded to above, ordinal scoring is not parametric in nature, and thus selection of nonparametric tests should be considered. semiquantitative scoring is a simple and relatively inexpensive approach to enhance descriptive/qualitative tissue data. understanding common applications of semiquantitative scoring and the key concepts for repeatability will enhance scientific studies in translational research. the use of mouse models of breast cancer and quantitative image analysis to evaluate hormone receptor antigenicity after microwave-assisted formalin fixation mouseadapted mers coronavirus causes lethal lung disease in human dpp knockin mice principles and approaches for reproducible scoring of tissue stains in research approaches to evaluate lung inflammation in translational research best practices guideline: toxicologic histopathology reporting of toxicologic histopathology: contrasting approaches in 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assurance in histopathology and cytopathology the measurement of observer agreement for categorical data statistical analysis of histopathological endpoints design and statistical methods in studies using animal models of development guidelines for the design and statistical analysis of experiments using laboratory animals the design and statistical analysis of animal experiments key: cord- -tjotro h authors: herrmann, helena a.; dyson, beth c.; vass, lucy; johnson, giles n.; schwartz, jean-marc title: flux sampling is a powerful tool to study metabolism under changing environmental conditions date: - - journal: npj syst biol appl doi: . /s - - - sha: doc_id: cord_uid: tjotro h the development of high-throughput ‘omic techniques has sparked a rising interest in genome-scale metabolic models, with applications ranging from disease diagnostics to crop adaptation. efficient and accurate methods are required to analyze large metabolic networks. flux sampling can be used to explore the feasible flux solutions in metabolic networks by generating probability distributions of steady-state reaction fluxes. unlike other methods, flux sampling can be used without assuming a particular cellular objective. we have undertaken a rigorous comparison of several sampling algorithms and concluded that the coordinate hit-and-run with rounding (chrr) algorithm is the most efficient based on both run-time and multiple convergence diagnostics. we demonstrate the power of chrr by using it to study the metabolic changes that underlie photosynthetic acclimation to cold of arabidopsis thaliana plant leaves. in combination with experimental measurements, we show how the regulated interplay between diurnal starch and organic acid accumulation defines the plant acclimation process. we confirm fumarate accumulation as a requirement for cold acclimation and further predict γ–aminobutyric acid to have a key role in metabolic signaling under cold conditions. these results demonstrate how flux sampling can be used to analyze the feasible flux solutions across changing environmental conditions, whereas eliminating the need to make assumptions which introduce observer bias. high-throughput technologies have resulted in a rapid increase in available 'omic data sets. large-scale metabolic networks constructed using these data integrate known and predicted metabolic pathways. these large-scale networks can be constrained using experimental data and the system behavior can be analyzed using metabolic modeling. metabolism describes a cellular phenotype under given conditions, and changes in metabolite concentrations and reaction fluxes can be used to assess a cellular response to changing environmental conditions. with the existence of large-scale metabolic networks comes the need to have appropriate modeling techniques available for their analysis. the majority of techniques for analyzing large-scale metabolic networks fall within the paradigm of constraint-based modeling (cbm). cbm imposes stoichiometric constraints on the metabolic reactions and analyzes the possible flux solutions at steady state. because the system is assumed to be at steady state, even genome-scale models can be solved at little computational expense. two of the most widely used forms of cbm are flux balance analysis (fba) and flux variability analysis (fva). the key feature of fba and fva is that they compute the steady state of a model using an objective function. the objective function defines a reaction that is to be maximized or minimized when solving the system under the set constraints. a typical objective is "maximum biomass production", whereby essential macromolecules such as proteins and lipids are defined at known ratios or quantities in an outgoing reaction of the metabolic system. , fba computes single steady-state solutions, which satisfy the objective. however, often multiple solutions exist and their range can be computed using fva. fva provides no indication as to whether all singlepoint solutions within the range are feasible and which solutions are the most likely. in order to reduce the number of feasible solutions of fba and to further constrain the feasible flux ranges returned by fva, it is common practice to introduce multiple objective functions. however, defining one or multiple objective function(s) intrinsically introduces an observer bias as to what the main "goal" of the cell is, in the context of the analysis. although biomass production as estimated by fba was seen to match experimental data in escherichia coli, this may not be an appropriate objective when studying short-term environmental changes. in the green algae chlamydomonas reinhardtii, a cmetabolic flux analysis was shown to contradict the assumptions of a prior fba analysis by demonstrating that maximum biomass and maximum atp production cannot stand alone as cellular objectives. optimal growth conditions, to which most objective functions are tailored, are an exception in natural environments. evolutionarily, metabolism is most likely optimized for overall robustness across many conditions, rather than a single conditionspecific objective. bacillus subtilis mutants, which outperform the wild-type in terms of biomass production in control conditions have been found experimentally; the wild-type, however, is more robust to both environmental and genetic perturbations and therefore holds an evolutionary advantage. understanding the optimization process that allows an organism to tolerate changing environmental conditions is of particular interest in crop sciences, where increasing yields will need to be achieved, despite rapidly changing environmental conditions. owing to their sessile nature, plants are exposed to frequent and sometimes extreme environmental fluctuations. plant metabolism can therefore be presumed to hold an inherent robustness to changing environmental conditions. furthermore, plant metabolism is highly intricate as it includes multi-cellular autotrophic and heterotrophic tissues with complex cellular compartmentalization. analyses of plant metabolism and of plant metabolic strategies can therefore be considered a comprehensive case study of metabolism in general. genetic modifications do not always produce a desired effect owing to network robustness: , in crop sciences, optimization for increased yield in control conditions rather than in changing environments is a likely explanation for discrepancies between laboratory and in field results. , if we wish to use cbm techniques to assess network robustness and phenotypic plasticity, we must be able to capture all alternative solutions and the probability with which they occur (the solutions space) of a metabolic network across different conditions. to calculate the exact properties of the solution space, we can use mathematical techniques such as convex analysis and vertex enumeration; however, owing to their computational intensiveness, these methods are only efficient when applied to small, simple networks or genome-scale models with one or more objective function. when wanting to analyze genomescale metabolic networks without the use of an objective function, such approaches cannot practically be applied. sampling of feasible flux solutions provides a realistic alternative. flux sampling generates a sequence of feasible solutions (called a chain) that satisfy the network constraints, until the entire solution space is analyzed. enough samples need to be generated for the samples to provide an accurate representation of the feasible solution space. a chain of samples is said to have converged once it contains enough samples to give an accurate representation of the solution space. flux sampling provides information both on the range of feasible flux solutions (similar to fva) but also on their probability. importantly, unlike fba, flux sampling does not require (but also does not exclude the option for) an objective function to be specified. therefore, flux sampling methods hold great potential for analyzing optimization strategies that are not defined by clear objectives such as a simple biomass reactions. when plants are exposed to a change in environmental conditions, such as temperature, which last only for a few days, optimizing biomass production during this period is arguably of secondary importance; sustaining metabolic function with minimal cost may be a higher priority to plants. for example, the allocation of carbon into different transient storage compounds accumulated to maintain cellular processes, has been shown to change when plants are exposed to environmental stresses. [ ] [ ] [ ] [ ] [ ] starch, malate, and fumarate are the three major carbon storage compounds which accumulate during the day in leaves of the model plant arabidopsis thaliana. , , increased cytosolic fumarate accumulation is a known cold response of a. thaliana leaves and has been linked to an increased photosynthetic capacity that sustains metabolism in cooler temperatures. evidently, tight regulation of carbon partitioning is required for successful cold acclimation. given the large number of reactions and pathways involved in linking primary carbon assimilation to its downstream storage products starch, malate, and fumarate, cbm seems appropriate. of the cbm methods available, flux sampling allows us to gain a detailed understanding of the solution space and the interdependence of the different carbon stores under different temperature conditions, without imposing the constraint of an objective function. multiple large-scale metabolic networks of the model plant a. thaliana have been constructed. [ ] [ ] [ ] [ ] [ ] here, we used three of them to formally assess for the efficiency of existing and easily accessible flux sampling algorithms: the coordinate hit-and-run with rounding (chrr), the artificially centered hit-and-run (achr) and the optimized general parallel (optgp) algorithms. we identified the most efficient sampling method based on runtime and convergence, and applied it to study plant acclimation to cold. we experimentally measured diurnal co uptake and organic carbon accumulation of a. thaliana in control and cold conditions. by constraining a leaf metabolic model to the two conditions and using an appropriate flux sampling algorithm, we were able to explore inherent metabolic robustness to temperature and predict the metabolic changes required to support a photosynthetic acclimation response to cold. although flux sampling has previously been applied as a technique for studying the solution space of metabolic networks, , , this will, to our knowledge, be the first time that the available algorithms are formally compared with one another in the context of metabolic modeling, and that flux sampling is applied to study network robustness across changing environmental conditions. both run-time and convergence are fastest when using chrr in matlab we compared the run-time and convergence of the chrr, achr, and optgp algorithms using three metabolic models of a. thaliana. we tested the run-times of , , , , , and , , samples (s), of which were stored and the rest were discarded with constant measures of thinning. for s = , , , the chrr algorithm was . times faster than the optgp and . times faster than the achr for the arnold model (fig. ) . this difference in speed increases with model complexity, such that, for the poolman model, the chrr was . times faster than the optgp and . times faster than the achr (fig. ) . the optgp was run in two parallel processes; however, even when running it as a single process it is faster than the achr. although we cannot exclude the fact that matlab may have a faster connection to gurobi than python, flux sampling is fastest when using the chrr setup as available in the cobra toolbox for matlab. the number of reactions that did not satisfied the convergence criteria were assessed for each chain ( table ). the longer runtimes of achr and optgp implementations in python are not outweighed by faster convergence. in fact, as the three pilot chains with varying thinnings show, the chrr algorithm, across all model reactions, converges the fastest, with the lowest number of samples required for convergence, the least amount of autocorrelation, and the lowest discrepancy between chains (table ) . we confirmed this difference in convergence by inspecting trace and auto-correlations plots of individual reactions, such as those for the biomass reaction of the arnold model shown in fig. (c). chrr shows little dependence between consecutive samples even with a thinning of t = , whereas optgp and achr show low levels of autocorrelation only when t = , . our results show differences in the outcomes when convergence is reached according to the different convergence diagnostics. all convergence diagnostics agree that the chrr performs best (table ). according to the raftery & lewis and the ipsrf diagnostics, all of the flux samples of reactions in the arnold and poolman models converge in < samples with a thinning constant of , when using chrr. the fact that such large numbers of samples are required for model convergence shows that, owing to the irregular solution shape of genome-scale metabolic networks, autocorrelation in chains is a common problem and must be overcome and tested for using appropriate convergence diagnostics. analyzing samples that have not achieved convergence can lead to incorrect conclusions about the metabolic fluxes under study. currently, many applications of the achr, optgp, and chrr algorithms to biological networks do not report whether convergence has actually been achieved. , [ ] [ ] [ ] previous comparisons between the achr and chrr algorithms have been made using different metabolic models but were based only on a single convergence diagnostic, the potential scale reduction factor (psrf). notably, different convergence diagnostics test different features and may not always be in good agreement. therefore, more than one diagnostic should be used to confirm that the sampling chain is likely to have reached convergence. , the achr and optgp algorithms have been compared using five genome-scale metabolic models and three different convergence diagnostics, including the psrf. however, the psrf assumes a normal distribution of solutions, which is questionable given that sampling is most needed when the distribution of flux estimates is non-normal, as is often the case in metabolic modeling. chrr-based flux sampling generates verifiable hypotheses concerning plant acclimation to cold when plants of a. thaliana are transferred from °c to °c, photosynthesis is inhibited. metabolism slows down in cooler temperatures and, in order to sustain normal metabolic functions, plants need to acclimate their co uptake, altering the concentrations of metabolic enzymes to achieve a new steady state. , , after days of cold, we observe that the a. thaliana wild-type col- is able to achieve the same level of photosynthesis as measured in control conditions (fig. ) . the allocation of carbon to the three main carbon storage compounds, malate, fumarate, and starch, shifts in the cold as part of the metabolic acclimation response. after days of acclimation both photosynthesis and transient carbon accumulation attain a new cold acclimated state. most notably, after days of cold treatment a larger proportion of carbon is partitioned into fumarate (fig. ). using the experimental data shown in fig. to constrain the co input and the malate, fumarate, and starch accumulation reactions using the arnold model (please see methods for further details), we were able to compute converged flux sampling distributions for all reactions. we did so for both control and cold conditions, which allowed us to overlay the sampling distributions of reaction fluxes and to assess changes required in plant metabolic behavior for acclimation (fig. ) . in order to demonstrate how the application of an objective function for an fba analysis can lead to vastly different conclusions, we have overlaid fba results for maximum biomass production (under the same model constraints as applied for the sampling) over the flux sampling distributions (fig. ) . this further emphasizes how an objective function, if inappropriate for the analyses under consideration, can be misleading. both sucrose export to other tissues and cytosolic pyruvate production as a precursor for the tricarboxylic acid (tca) cycle are predicted to be unchanged. the model suggests that, given equal carbon assimilation in control and cold conditions, cellular maintenance and export functions are supported equally in both conditions on the time scale considered. although sucrose export is difficult to measure experimentally, we observed the rate of respiration in the cold to be the same as in control conditions (fig. ) . given that, for both conditions, the model shows equal fluxes from cytosolic pyruvate into the tca cycle (fig. ) , which feeds directly into respiration, this model prediction is in agreement with our experimental data. for fumarate (and other metabolites not shown) the model predicts a shift in cellular compartmentalization with temperature. model results show an increase in fumarate export from the mitochondrion into the cytosol in the cold. the reverse is shown for fumarate export from the chloroplast, where it is produced via the breakdown of arginosuccinate (fig. ) . leaves developed in the cold have increased cytoplasmic and decreased vacuolar volumes; a reshuffling of metabolites across cellular compartments has therefore previously been proposed as an important temperature acclimation response. model results suggest an increase in flux from cytosolic malate to fumarate via cytosolic fumarase (fum ) (fig. ) . this is consistent with previous experimental results from mutant studies this reaction is thus essential for photosynthetic acclimation to cold. flux sampling distributions suggest a link between carbon and nitrogen metabolism to support cold acclimation the sampling distributions suggest a trade off between increased carbon compound accumulation and decreased amino-acid production (fig. ) , linking nitrogen and carbon metabolism. synthesis of γ-aminobutyric acid (gaba), however, is predicted to increase in the cold. gaba has previously been reported to accumulate in response to environmental stresses, including cold treatment. gaba has been suggested as a signaling molecule of the carbon to nitrogen status in plant leaves and evidence for its role in regulating nitrate uptake exists for both rapeseed and a. thaliana. [ ] [ ] [ ] a. thaliana plants in the cold show increased nitrogen assimilation compared with those in control conditions. if gaba is indeed involved in carbon:nitrogen signaling, it may be counteracted by the increased accumulation of malate. increased levels of malate have previously been shown to suppress nitrate reductase expression and activity in tobacco leaves. malate levels in col- may be kept below a certain threshold, by redirecting carbon to fumarate, thereby supporting adequate nitrogen assimilation and an increased photosynthetic capacity. this hypothesis is supported by the observation that a. thaliana mutants, which show increased levels of malate and decreased levels of fumarate, grow significantly less well in highnitrogen conditions than col- . fumarate has few known metabolic functions in a. thaliana leaves, and may thus serve the purpose of a carbon storage buffer in changing environmental conditions. based on run-time and platform, chrr is faster than optgp, which is itself faster than achr. chrr also converges faster than both optgp and achr. users with unrestricted access to matlab are therefore recommended to use chrr. for those who wish to work using an open-source platform, optgp is recommended over achr; in general, optgp converges faster than achr, has a shorter run-time and allows for parallel processes. when running sampling algorithms, sets of flux samples are produced for each reaction in the model. here, we have tested convergence for all reactions of the models using three different sample chains. we have highlighted the importance of checking for convergence using different diagnostics when analyzing an irregular solution space of large networks. if only a subset of reaction fluxes are of interest, only those distributions will have to be checked for convergence. flux sampling provides a powerful tool for exploratory analyses assessing metabolic differences across different environmental conditions. our results further confirm the notion that it is not possible to fully automate convergence analyses using a single diagnostic and that results should confirmed via manual inspections of trace and autocorrelation plots. flux sampling is currently an under-utilized technique in the metabolic modeling of large-scale networks. using cold acclimation of the model plant a. thaliana, we demonstrate how flux sampling can be used effectively to analyze alternative feasible solutions across multiple conditions whilst eliminating the need to make assumptions that introduce observer bias. given short-term environmental changes, adequately sustaining basic metabolic functions with minimal resource investment may be a more-likely cellular objective on that time scale than, for example, maximizing growth. we therefore did not set an objective function for flux sampling but used four experimentally measured flux values (co input and fumarate, malate and starch accumulation) to constrain a leaf metabolic model. we further demonstrated how these flux sampling results can lead to different conclusion than traditional fba analyses. by constraining the model to both cold and control conditions we were able to select reactions that show different flux distributions across the two conditions. our model highlights reactions that are essential to change with temperature (i.e., the flux distribution of the two temperature conditions do not overlap) such as the production of cytosolic fumarate via malate. the model further demonstrates the properties of the flux distributions of gaba to differ in cold and control conditions, highlighting gaba as a plausible signaling molecule for supporting a shift in the nitrogen and carbon balance, required to sustain photosynthesis in the cold. thus, through flux sampling, we were able to generate novel hypotheses about the roles of gaba, fumarate and malate in cold acclimation, which would have been unfeasible to detect using fba and fva methods. by overlaying different fba solutions onto flux sampling distributions obtained under condition-specific model constraints, fba in combination with flux sampling, could, in future work, be used to determine plausible objective functions and help generate predictions about how cellular objectives might be changing in response to environmental changes. constraint-based reconstruction and analysis (cobra) methods for genome-scale metabolic networks are integrated in the cobra toolbox , for the matlab programming language and the cobrapy package for the open-source python programming language. three algorithms for flux sampling exist across the two platforms: chrr (matlab), achr (matlab and python), and optgp (python). further flux-sampling algorithms exist; , - however, as they are not currently available in the cobra packages, they are here not considered for comparison. , . the artificially centered hit-and-run (achr) sampler estimates the center of the solution space in a "warm-up" phase. this estimate is then continuously revised with further sampling. the center estimate is used to inform the direction of further sampling such that the full solution space is covered in fewer steps than in traditional hit-and-run sampling (where the direction of the next sample is chosen at random). although the markovian nature of hit-and-run (i.e., the fact that each future sampling state is dependent only on the current sampling state) is lost in the achr, it overcomes the edge-trapping limitation of the standard hit-andrun algorithm (i.e., it no longer gets stuck at the bounds of a solution space if these are of an elongated shape, a frequent feature in metabolic models). . the optimized general parallel sampler (optgp) is argued to be an improvement on the achr, because from the warm-up point it generates multiple short chains from the estimated center and only considers the last point in the chain as a sample. it thereby increases the randomness and efficiency with which the total solution space is explored. furthermore, it allows for parallel sampling. larger samples can thus be generated in shorter runtimes. . the coordinate hit-and-run with rounding (chrr) algorithm starts with a pre-processing step that rounds the solution space to a more regular, convex shape, and therefore a markov chain can be used to explore the rounded solution space without the limitation of edgetrapping. after sampling, the solutions are back-transformed to match the original solution space in order to obtain the true value of the sampled points. , assessing convergence the aim of flux sampling is to generate enough consecutive samples (a long enough chain) in order to get an accurate depiction of the solution space. a sample chain is considered to have converged once it can be assumed that the sampled subset of solutions represent the properties of h.a. herrmann et al. the true solutions obtained from an infinite amount of samples (i.e., when the shape of the flux distribution no longer changes with more samples). in flux sampling, an algorithm's efficiency is defined by the run-time and the number of samples required for apparent convergence to the true flux distribution. here, we apply three different diagnostics in order to test for convergence: . raftery & lewis diagnostic: this diagnostic estimates the total number of samples, n max , required for a set of samples to achieve convergence, based on a given subset of samples (pilot chain). we estimated n max required for convergence using the coda package in r. the diagnostic further returns a dependence factor, i, which is indicative of autocorrelation (the degree of dependence between consecutive samples). chains with i > are here considered to be problematic as this value suggests high dependence between samples or influential starting values (i.e., the chain was not run long enough). . interval-based potential scale reduction factor (ipsrf): adapted from the original gelman-rubin diagnostic, psrf, the ipsrf is based on comparing the differences between consecutive samples (within sequence interval length) with the total differences observed between all samples (between sequence interval length). because interval length, rather than variance of the samples (on which the original version is based), is considered, a normal distribution of the samples is no longer a requirement. as with psrf, the ipsrf fig. summary of the general influx and outflux of the arnold model, as set up in our analysis. differences in pathways linking carbon assimilation and diurnal carbon storage were compared across the two temperatures ( °c and °c) in order to predict metabolic changes required for cold acclimation of a. thaliana the linear programming solver used was gurobi, version . . . the samplers were used in accordance with cobra documentation using default parameter settings unless otherwise specified. although an achr implementation is available both in the cobra toolbox and in cobrapy, the comparisons made here are based on the python implementation of the achr sampler because it is open-source and because the optgp, which is available only in python, directly builds on the general parallel sampler (gp) of the achr. we did collect preliminary convergence and run-time data of the achr algorithm in matlab; however, because its convergence was evidently slower than chrr, we have chosen to omit its matlab implementation from further analyses. optgp and chrr algorithms were run using two parallel processes; there is currently no option to run achr as multiple processes. to compare the performance of the samplers, three published genomescale a. thaliana metabolic models were obtained in sbml format and are from here on referred to by their first author: the poolman, arnold, and dal'molin models. the poolman model is based on the aracyc database and describes a non-compartmentalized heterotrophic culture using reactions. the arnold model describes a compartmentalized, photoautotrophic system and is based on manually curated a. thaliana specific reactions. the dal'molin model is a compartmentalized network reconstruction of reactions, applicable to both photosynthetic and non-photosynthetic tissues of plant metabolism. the original model constraints , , were used when comparing sampler performance across these models, such that the arnold, poolman, and dal'molin model had , , and degrees of freedom respectively. samples that are close together within a sample chain can be autocorrelated (i.e., be similar to one another due to the way in which the algorithm works). in order to avoid the effects of autocorrelation and to ensure convergence, a large number of samples should be run and a technique called thinning can be applied. sample chains that are thinned store only every k th sample in the chain, where t = k is called the thinning constant. in order to compare autocorrelation of the three algorithms, we applied three different thinning constants (t = , , , ) storing samples for each chain. three replicate chains were run for each value of t. convergence diagnostics were calculated for sample chains produced for each of the model reactions. run-times were obtained on a personal laptop ( . gb ram, i - u cpu processor, cores, . ghz capacity, ubuntu . . os). carbon assimilation and respiration (co influx and outflux) by the a. thaliana wild-type columbia- (col- ) was measured using infrared gas analysis at μmol m − s − light under control conditions ( °c) and after week of cold treatment ( °c) as described previously. fumarate, malate and starch concentrations at the onset and at the end of the photoperiod were measured for both control and cold conditions using enzyme assays. averages of - replicated were calculated. measurements taken across the different conditions were tested for significant differences using an unpaired t test (p < . ) assuming unequal variances, as implemented in r. given that previously published data confirm an approximately constant rate of accumulation of transient carbon storage products during the day, , we subtracted the beginning of day metabolite concentrations from the end of day concentrations for each of the products in order to obtain a flux value for carbon storage over one photoperiod. these flux values were used to constrain the arnold model (fig. ) . this reduced the degrees of freedom of the model to . measurements were taken at control conditions (red) and after days of cold treatment (blue), at the beginning of day (bod) and at the end of day (eod), using infrared gas analysis and enzyme assays. the s.e.m. of the - replicates for each measurement is shown as error bars. the above data (excluding carbon outflux) were converted to mmol gfwÁday and used to constrain the arnold model as outlined in the method. significant differences between measurements across the two temperature conditions are indicated by an asterisk given that the arnold model is leaf specific and manually curated, it suits the purpose of studying photosynthetic plant acclimation. in order to do so we constrained the model with our experimental diurnal flux data for malate, fumarate, and starch accumulation as well as co influx. we set the cytosolic fumarase reaction, which produces cytosolic fumarate from malate, to be reversible. outgoing fumarate, malate, and starch reactions were added to the model in order to simulate diurnal carbon storage (fig. ). diurnal accumulation of the metabolites was calculated by subtracting average beginning of day concentrations from average end of day concentration values. metabolite accumulation and co influx were converted to mmol (gfw) − day − . the resulting values were applied as model constraints. upper and lower bounds were applied according to the calculated standard errors of three to four replicates as shown in fig. . to ensure convergence of all flux sampling distributions, , flux samples with a thinning of , were generated using the chrr algorithm in the cobra toolbox. a kruskal-wallis test, as implemented in the scipy python package, version . . , was used to assess whether flux samples generated using either the cold or the control constrained model stemmed from the same distribution. reporting summary further information on research design is available in the nature research reporting summary linked to this article. the authors declare that all data supporting the findings of this study are available within the paper and online or can be generated using the publicly available source code (https://github.com/haherrmann/fluxsamplingplantmodels; https://doi.org/ . /zenodo. ). flux sampling distributions of key reactions linking photosynthetic input (co ) to transient carbon storage for control (red) and cold (blue) conditions. condition-specific carbon assimilation and fumarate (fum), malate (mal), and starch accumulation were constrained according to experimentally measured results. resulting fluxes of reactions including triose phosphate (tp), fructose , bisphosphate (f p), glyceraldehyde -phosphate (g p), pyruvate (pyr), arginosuccinate (as), arginine (arg), phosphoenolpyruvate carboxylase (pep), oxaloacetate (oaa), γ-aminobutyric acid (gaba), acetyl coenzyme a (acoa), and sucrose (suc) are shown. we overlaid fba results for maximum biomass production (under the same model constraints as applied for the sampling) as vertical blue and red bars over the flux sampling distributions. reactions for which the two distributions are significantly different (p < . ; kruskal-wallis) are marked with an asterisk in the top right corner multi-omics and metabolic modelling pipelines: challenges and tools for systems microbiology aragem, a genome-scale reconstruction of the primary metabolic network in arabidopsis constraint-based models predict metabolic and associated cellular functions what is flux balance analysis? the biomass objective function flux balance analysis of plant metabolism: the effect of biomass composition and model 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international workshop on algorithms and models for the web-graph, waw: algorithms and models for the web graph use of ranks in one-criterion variance analysis publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/ . /. key: cord- -xuztu a authors: davis, samuel; fard, nasser title: theoretical bounds and approximation of the probability mass function of future hospital bed demand date: - - journal: health care manag sci doi: . /s - - - sha: doc_id: cord_uid: xuztu a failing to match the supply of resources to the demand for resources in a hospital can cause non-clinical transfers, diversions, safety risks, and expensive under-utilized resource capacity. forecasting bed demand helps achieve appropriate safety standards and cost management by proactively adjusting staffing levels and patient flow protocols. this paper defines the theoretical bounds on optimal bed demand prediction accuracy and develops a flexible statistical model to approximate the probability mass function of future bed demand. a case study validates the model using blinded data from a mid-sized massachusetts community hospital. this approach expands upon similar work by forecasting multiple days in advance instead of a single day, providing a probability mass function of demand instead of a point estimate, using the exact surgery schedule instead of assuming a cyclic schedule, and using patient-level duration-varying length-of-stay distributions instead of assuming patient homogeneity and exponential length of stay distributions. the primary results of this work are an accurate and lengthy forecast, which provides managers better information and more time to optimize short-term staffing adaptations to stochastic bed demand, and a derivation of the minimum mean absolute error of an ideal forecast. significant cost and safety issues occur when the demand for hospital resources is not matched by the supply of these resources [ ] . when demand exceeds supply, unsafe conditions arise due to less availability of resources, which can cause increased mortality and the rate of medical errors [ , ] . conversely, when supply exceeds demand, excess clinician staffing can produce significant cost with no benefit to patient care quality. hospital management must actively manage the supply of fixed resources, such as beds and specialized equipment, and variable resources, such as nurses, technicians, and providers, that affect care quality and ensure patient safety. matching supply to demand is difficult because the future patient resource demands are random and unknown, while the resource supply is often set far in advance and is difficult to adjust in the short term significantly. uncertainty in patient resource demand is caused by several stochastic processes, including the number and timing of arrivals and discharges, length of stay (los), unit transfers, health improvement and deterioration, surgical complications, and same-day cancellations for outpatient and surgical appointments. variability arises from disease seasonality, holidays, and the numerous types of specialized staff, physical resources, ailments, procedures, allergies, comorbidities, and surgical techniques. additional variability is created when surgeries are scheduled without anticipating and managing the aggregate downstream resource requirements, such as the time needed in a post-anesthesia care unit (pacu) or intensive care unit (icu). the aggregate patient resource demand several weeks in the future, which is the time period for which clinician shifts are being scheduled, is often significantly different from the current demand. short-term staffing adaptations are made to manage the mismatch of resource supply and demand, such as incorporating on-call and per diem staff and canceling unnecessary shifts. effective proactive adaptive staffing requires highconfidence forecasts of short-term resource demand to ensure the appropriate quantity and timing of staffing adjustments. proactive is harder than reactive adaptation, in which the mismatch of supply and demand already exists, which is relatively simple to measure and align. the cost of a staffing adaptation is typically dependent upon its magnitude and timing, and thus requires a forecast that is both accurate and lengthy to be cost-effective. developing and applying an accurate model to forecast patient resource demand for multiple time periods into the future improves both the cost and safety of providing care when coupled with an adaptive staffing strategy. predicting and managing the demand for healthcare resources, such as beds, staff, and specialized equipment, is an extensive research topic. settings have included emergency departments (eds) [ ] [ ] [ ] [ ] [ ] [ ] , surgical suites and recovery beds [ ] [ ] [ ] , medical and surgical inpatient beds [ ] [ ] [ ] [ ] , icus [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , outpatient care [ ] [ ] [ ] and long-term care [ , ] . methods have forecasted aggregate demand either by applying tools like regression [ , ] , time series analysis [ , , , ] , and neural networks [ , ] , or aggregating individual forecasts by applying tools like probability models [ , , [ ] [ ] [ ] , queueing theory [ , , ] , simulation [ , , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , and expert opinion [ , ] . the wealth of literature on predicting the demand for hospital resources demonstrates the strong need to make accurate forecasts. most models make simplifying assumptions to manage tractability and fit data availability, including assuming stationary or cyclic demand patterns [ , , ] , exponential-based inter-arrival times and los distributions [ , , , ] , patient homogeneity [ , , ] , singleday forecasts [ ] , and point estimates instead of probability mass functions (pmfs) [ , ] . the model derived herein provides a generalized approach to forecasting bed demand that does not require these simplifying assumptions. the goal of this study is to forecast an accurate approximation for the multi-period pmf of bed demand using the exact surgical schedule at the time of the forecast, non-stationary inter-arrival times, and patient-level duration-varying los distributions. required to construct this model is an analysis of scheduled patient los distributions, including the probability of being an inpatient given information known at the time of the forecast. the rest of this paper is organized as follows: first, the forecasting model is derived for each patient group. these sub-forecasts are then aggregated, and their aggregate distribution is approximated, producing both a total patient pmf forecast and an inventory of the necessary parameters to construct the model. the theoretical minimum expected mean absolute error (mae) for the forecast is then derived, which helps assess the quality of the model. a case study using blinded real hospital data demonstrates the construction and assessment of the model, followed by a discussion of results, limitations, and future work. the forecasting model uses discrete time measured in periods and is run at period d. all patients currently in the hospital at that time have their discrete los incremented by one period. let t represent the length of the complete forecast, and t be the specific period being forecasted, such that d + ≤ t ≤ d + t. these time periods could correspond to days, non-overlapping shifts, or smaller increments of time. the model may be used to forecast a single unit or collection of hospital units. for instance, a forecast could be created for just an icu, as well as the complete set of beds in the hospital. the first forecast would focus on icu demand, while the second would provide information about the capacity of the hospital as a whole. at any future period t, each patient in a hospital will either be known at current period d or unknown. at period d, all known patients existing at period t will either already be in the hospital or scheduled to arrive on or before period t. all remaining patients in the hospital at period t that were not known of at period d are either emergency arrivals, or patients who have not yet been added to the schedule, but will be scheduled to arrive on or before period t. figure shows these patient groups and their hierarchy. let γ group d t ð Þ represent the forecasted random variable of demand at period t made from period d for a given patient group, where group ∈ { curr, sch, emer, nysched}, representing current patients, scheduled patients, emergency patients, and not-yet-scheduled patients, respectively. forecasts must be made for each of these patient groups individually and then aggregated to produce the overall patient demand forecast. all patient arrivals and los probabilities are assumed to be independent of one another. each current patient i ∈ m, where m represents the number of current patient types indexed by i, currently in the hospital at period d, either is or is not still in the hospital at future period t, and can thus be represented by a bernoulli random variable where success is equal to the probability of having a remaining los from strictly greater than t − d. let f i (h) represent the probability of patient i staying exactly h periods from d, meaning the pmf of their remaining los is f i (h). let f i (h) be the cumulative distribution function (cdf) of f i (h), and r i (h) = − f i (h). the probability of patient i still being present at period t when forecasted from period d is represented by c d, i (t), as shown in eq. . the convolution of all individual patient probabilities shown in eq. , represented by γ curr d t ð Þ, is the random variable representing the number of current patients at period d still present at period t. if these patients all have the same probability of being present at period t, then γ curr d t ð Þ is a binomial random variable. however, if these probabilities are non-homogeneous, then their convolution follows a poisson binomial (pb) distribution [ ] . thus γ curr d t ð Þ, which represents the random variable representing all current patients at period d being present at period t, follows a pb distribution, with mean and variance as shown in eqs. and , respectively. var γ curr a patient j ∈ n, where n represents the number of scheduled patient types indexed by j, still being present at the hospital at period t can be represented by a bernoulli random variable, similar to current patients. let s j represent the future period on which the patient is scheduled to arrive, such that d + ≤ s j ≤ t. let f j (h) represent the pmf of staying exactly h periods, f j (h) be the cdf of f j (h), and r j (h) = − f j (h). then, as represented in eq. , the probability of patient j, scheduled for period s j , being present at period t, when forecasted from period d, is represented by s d, j (t). note that h may equal . similar to current patients, γ sch d t ð Þ is the convolution of the individual patient distributions shown in eq. , which is the sum of non-homogeneous bernoulli random variables, and thus pb. the mean and variance of this random variable are represented by eqs. and , respectively. emergency patients arrive at the hospital without ever being scheduled in advance, and are traditionally modeled as poisson random variables in the literature [ ] , with a time-varying arrival rate. the arrival rate should be determined for each period, and should thus vary across time. let the expected number of arrivals on period s, where d + ≤ s ≤ t, of patient type u ∈ u, where u represents the number of emergency patient types indexed by u, be represented by λ u (s), and the probability of staying exactly h nights be represented by not-yetscheduled variable γ emer d t ð Þ, has a mean equal to its variance, which is shown in eq. . patients booked at period b to arrive on period s, such that d are not-yet-scheduled, but may be present at period t. these patients are unknown, and can thus not be modeled with bernoulli random variables. there is little research on this topic, though linear regression has been shown to reasonably predict the number of not-yet-scheduled patients [ ] . suppose the number of patients of type v ∈ v, where v represents the number of emergency patient types indexed by v, scheduled on a given booking period b for scheduled period s is poisson distributed and represented by λ v (b, s). the probability of patients of type v staying exactly h nights is represented by the random variable representing not-yet-scheduled patients present at period t, with mean and variance as shown in eq. , is thus represented by γ nysch determining the distribution of total demand at period t from forecast period d, represented by Γ d (t), requires a convolution of known and unknown patients present at period t. the pmf of current and scheduled patients are pb distributions, meaning the distribution of known patients is also pb, as the sum of two sums of heterogeneous bernoulli random variables is also a sum of heterogeneous bernoulli random variables. the pmf of emergency and not-yet-scheduled patients are poisson, meaning the distribution of unknown patients is also poisson. calculating the full convolution of total patient demand Γ d (t), which is the sum of a pb random variable representing known patients, and a poisson random variable representing unknown patients, is computationally expensive to calculate [ ] . this is partly because it has an infinite domain and the number of unique combinations of the pb distribution is equal to (m + n) , where m + n is the number of known patients, which exceeds one trillion with just known patients. instead of calculating the complete convolution of Γ d (t), an approximation using the normal distribution is obtained using the mean and variance of forecasted patient demand. the mean, as shown in eq. , is the sum of eqs. , , , and , and the variance, as shown in eq. , is the sum of eqs. , , , and . this normal distribution, as well as the underlying convolution that includes a poisson distribution, both have infinite domains. these distributions represent the demand for beds, and can thus exceed the hospital unit capacity. when calculating the bed demand, this normal distribution should be truncated at zero, and when predicting the bed occupancy, this normal distribution should be truncated at both zero and the hospital unit capacity. let the error of the forecast for period t made from period d be represented by e d, t . if a t is the actual census for period t, then eq. shows the calculation for e d, t . the z-score of the error, represented in eq. by z d, t , equals the number of standard deviations away from the forecasted mean and the standard deviation is equal to the square root of the forecast variance. the case where all los distributions are deterministic, meaning no uncertainty in los, such that all variance comes from unknown future patients, is the theoretical lowestvariance forecast. in this case, the mean and variance of the pb random variable would become an integer constant and zero, respectively. thus, the distribution of the forecasted pmf with the lowest variance is a translated poisson random variable that is translated by the amount equal to the expected value of the pb random variable. if a forecast is made equal to the mean of this translated poisson random variable, then the expected mae of the forecasted pmf with the lowest variance can be obtained as shown in eq. , where λ + c and λ represent the mean and variance of the translated poisson (tp) random variable, respectively. the proof is shown in the appendix. note that ⌊λ⌋ represents the largest integer less than or equal to λ. translating the poisson random variable does not affect the expected mae, and the result in eq. can be further reduced, as shown in eq. , where f p represents the pmf of a standard poisson random variable with a mean of λ. to calculate the theoretical best expected value of a multiperiod forecast with non-homogenous poisson random variables, the value in eq. must be averaged for all periods, using the actual number of arrivals to estimate λ. figure shows the expected mae for a single poisson random variable as a function of λ. populating all the necessary parameters for a forecast of t total periods requires significant parameterization and calculation. table shows the six groups of parameters that must be developed, along with their corresponding indices. these parameters can either be determined from the empirical distribution of the dataset or produced using more powerful methods, such as machine learning. for instance, historical data could populate a logistic regression or decision tree model to estimate the probability of staying any number of nights. several other works have thoroughly analyzed los predictions, and are good resources for this important aspect of forecasting bed demand [ , , ] . unlike for the other patient groups, f j ( ) for scheduled patients must be determined. blinded de-identified data were obtained for all patients scheduled to arrive or having stayed between / / and / / at a mid-sized massachusetts community hospital. the next three subsections describe the raw data, develop the model parameters, and produce and analyze the forecast results. this particular application of the model used days as periods to determine the midnight census, and combined all hospital units into a single entity. to assess the performance of the proposed model, a -period moving average (ma) model was also developed. included in the hospital dataset were about , patients, % of whom were scheduled to arrive, versus % of whom were never scheduled. of the scheduled patients, % had a los of at least one night, meaning the dataset included about scheduled inpatient surgeries. the dataset included fields for arrival date, departure date, if the patient was scheduled or not, and for all scheduled patients, fields for booking the nightly midnight census is set equal to the sum of new arrivals that day and the current patients that morning, minus the departed patients. figure shows the mean arrivals, current patients, and departures by dow in waterfall form, where the bottom of the third bar represents the mean midnight census for that particular dow. about one-third of all inpatient surgeries occur on mondays, and taper across the week, with the highest mean of surgical census occurring on wednesday. mean emergency patient arrivals also peak on monday, with the census peaking on wednesday. few scheduled inpatient surgeries occur on weekends, whereas the mean number of emergency arrivals is roughly constant by dow. construction of the forecasting model required calculation of the flow probabilities and arrival rates in table . in this case study, seven primary patient types were differentiated: emergency patients and six surgical patients grouped by orthopedic, general surgery, urology, gynecology, neurosurgery, and all other specialties combined. the los distribution for emergency patients was set equal to the empirical los distribution for all emergency patients. patients not spending a night in the hospital were excluded from the analysis due to not being part of the midnight inpatient bed census. the los distributions for each surgical group were calculated assuming an inpatient stay of at least one night. the probability of a scheduled surgery becoming an inpatient stay was based on the proportion of patients having that particular procedure being an inpatient to the number scheduled. hence, the los distribution was conditioned on staying at least one night, based purely on the procedure, then determined based on the surgical group. figure shows a bar chart containing the los distributions for each patient type, conditioned on staying at least one night, and truncated after seven nights. due to data availability, the los distributions were not updated at each subsequent time period for each patient. the arrival rates for emergency patients were set equal to the mean number of daily arrivals by dow. figure contains box plots showing the arrival distributions by dow. the bottom and top whiskers represent the minimum and maximum values, respectively, the centerlines represent the medians, and the bottom and top of the boxes represent the th and th percentiles. arrival rates for not-yet-scheduled patients were set to the empirical distribution of scheduled patients by surgical group, appointment dow, and the number of days between the booking date and the appointment date. from each date between / / and / / , the patient volume was forecasted days into the future, representing forecasts for each of the lengths. figure a shows the forecasted -day pmf of demand from / / to / / , based on the discrete normal approximation using the mean and variance of demand. figure b shows an example of a single -day forecast starting from / / versus the actual values, and fig. c shows fourteen consecutive -day forecasts starting from / / versus the actual values. the lower control limits (lcl) and upper control limits (ucl) are drawn two standard deviations below and above the means in both figures, respectively, where the table , in addition to the theoretical minimum expected mae as determined by eq. . the theoretical mae is calculated by setting λ equal to the actual number of unknown daily arrivals and taking the mean of the daily expected mae. the simple -period ma model had a mae of . for a period forecast, representing a % increase in error compared to the proposed model. two-sided z-tests were applied to each forecast length to compare the means of the forecast error z-scores to zero. they all passed with α = . , meaning none of the means of the zscore errors were statistically different than zero. also using two-sided z-tests, the means of the squared values of the zscores were compared to one for each forecast length. the difference between these values and one were not found to be statistically significant for any of the forecasted lengths. given that the standard deviation of a random variable is equal were not statistically different from one. this finding supports the assumption of patient bed demand following a normal distribution. in addition to testing the means and standard deviations of the z-scores of the error, each of the forecast lengths was tested for normality using the kolmogorov-smirnov test, in which the null hypothesis was that the forecast distribution was from a normal distribution with mean and variance equal to the forecasted mean and variance. if the test statistic exceeds the critical value, then the null hypothesis is rejected. otherwise, if the null hypothesis is embraced, then statistical support exists for a normal distribution being a good fit for the true distribution. for all fourteen forecasts lengths, the null hypothesis was embraced at the % significance level, which supports the use of normal approximations for future bed demand. to visually assess the overall fit of the normal approximation, q-q plots were created to identify outliers. q-q plots are used to compare the empirical distribution percentiles to the theoretical distribution percentiles, as shown in fig. . the xaxis in each plot corresponds to the theoretical normal distribution percentiles, and the y-axis in each plot corresponds to the empirical distribution of the z-scores of the forecast errors. a -degree line is drawn in each chart to show the ideal fit of the two sets of quantiles. this forecasting model assumes that all arrival rates and los distributions are independent, and thus does not take into account system effects like congestion and negative feedback loops. a chi-squared test for independence was prepared to test if the -day forecast errors were independent of the census at the time of the forecast. five bins were created for both the error and the census at the time of the forecast, centered on their respective means, creating a total of bins in the joint distribution. the middle bins represented the mean plus or minus half the standard deviation. the next bin on each side also had a width of a single standard deviation. values more than . standard deviations above or below the mean were placed in the highest and lowest bins, respectively. the chisquared test statistic was equal to . , which with degrees of freedom and α = . , was not found to be statistically significant, and hence, there was insufficient evidence to demonstrate non-independence. the forecast residuals versus the census at the forecast times are shown in fig. , the distribution of observed values is shown in table , and the distribution of expected values is shown in table . the total population of patients is a combination of known and unknown patients, represented by a pb and poisson random variable, respectively. figure shows the average ratio of known patients to all patients from each forecast length, representing the proportion of the forecast that could theoretically be known with close to certainty if los distribution predictions improve and approach a smaller variance. as t → ∞, when all known patients will have departed, and only unknown patients remain, the forecast converges to a poisson random variable. on the first day of the forecast, nearly % of the mean of bed demand was from known patients, compared to just % on the seventh day. this paper demonstrates a novel and accurate method to produce a multi-day forecast of the pmf of hospital bed demand. the case study results are promising, including an mae close to the theoretical minimum expected mae, means of error zscores close to zero, standard deviations of error z-scores close to one, and successful kolmogorov-smirnov tests for all forecasted lengths. in addition, the chi-squared test for independence demonstrated that forecast residuals were independent of the bed demand at the time of the forecasting, confirming that the assumption of independent patient flow variables was reasonable. predictably, the forecasts increase in mae as their lengths increase, though the difference between the prediction and the theoretical minimum expected mae decreases. this suggests that although there is room for improvement in predicting the arrival rates for emergency patients and not-yet-scheduled patients, the more significant forecast improvement opportunity lies in predicting (i.e., reducing the variance of the pmf of) the los distributions for current and scheduled patients. improving los distributions could be accomplished via real-time updating from electronic medical records, including data on age, gender, medical history, and list of orders, to drive personalized classification and regression models. furthermore, as natural language processing improves, valuable information from clinician notes may become available for predictive models to improve personalization. only % of the inpatient stays at this hospital were scheduled, representing only % of total nights spent. this relatively low proportion of scheduled patients significantly increases the variance of the theoretical best quality forecast due to the dominance of the poisson random variable for unknown patients relative to the pb random variable for known patients. applying the forecasting model to a hospital that predominantly sees scheduled patients relative to emergency patients could significantly reduce the mae of the predictions. in addition, because the curve in fig. is concave down, the rate of expected mae increase is slower than the rate of the increase of the mean of unknown patients, meaning this forecasting model should produce a smaller ratio of mae to mean census at larger hospitals with similar los distributions. the q-q plots in fig. demonstrate that a normal distribution with the forecasted mean and variance is a suitable estimation of the full probability convolution. the estimations hold well within the range of about two standard deviations, indicated by little deviation from the -degree line. however, the -day high outliers are above the -degree line, indicating that the upper tail of the normal distribution estimate may be too thin to fit the real data. this makes sense given that poisson distributions have positive skew and most of the forecast is driven by the underlying poisson random variable for unknown patients. the q-q plots for other forecast lengths tend to have high outliers below the -degree line, suggesting that consistent multi-day volume increases may be mitigated over several days via reduced los or fewer scheduled surgeries. a prominent outlier occurs on the night of june th, , when the -day forecast from the previous day had an error zscore of − . , and a -day forecast z-score of − . . the following day, june th, , also clearly shows up on the -day q-q plot. although an explanation for this outlier likely exists, tracing its root-cause was beyond the scope of the study. a potential limitation of this work is the assumption that all random variables are independent of each other, as it is possible that some processes adjust when the census is extreme. for instance, a full hospital may prohibit non-critical short-term scheduling, or demonstrate a greater urgency to discharge current patients to make room for new ones. although the chisquared test demonstrated independence between the census at the time of the forecast and the next day's forecast residual, incorporating system-level adjustments in future forecast model expansion work may enhance the quality of the predictions in extreme cases. another limitation is that the parameterization could benefit from the application of machine learning algorithms and additional data, especially data that is updated during the patient visit. for instance, learning that a patient has moved to the icu or is on a ventilator would significantly increase the expected remaining los. incorporating natural language processing to read the clinician notes would further improve the accuracy of the patient-level parameters. the difference in accuracy between the case study accuracy and theoretical best accuracy demonstrates the value in improving these parameters. as these parameter errors compound, the gap between the theoretical best accuracy and the model accuracy widens. furthermore, error can exist within the input parameters themselves, in addition to the inherent error of any probability not equaling zero or one. this model assumes that probabilistic input parameters have been accurately determined, and an opportunity for future work would be to analyze the effect of poor parameter estimation on the aggregate demand prediction. future work could improve this model by incorporating unit-level flows and a joint distribution of demand across these units by addressing unit-level transfers, as well as including resource demand ratios to produce a pmf of resource demand. for instance, beyond predicting the demand for beds, which is a one-to-one relationship to patients, the model could be expanded to predict the need for nurses and technicians in specific units, who can cover multiple patients based on severity and needs. the ultimate intent is to apply a stochastic optimization model that sets base level staffing and adjusts it based on a pool of shared resources based on a joint probability distribution of demand across all units. in addition to expanding the detail of the model structure and parameters, the full convolution of demand could be integrated to improve the predicted pmf accuracy. an accurate forecasting model has been derived and implemented that generalizes previous models in the literature by allowing multi-period forecasts, pmfs of demand instead of point estimates, non-cyclic surgery schedules, time-varying los distributions, and patient heterogeneity. in a case study, the forecasts held up to statistical tests of independence and normality. this forecasting method allows bed managers significantly more information, both in accuracy and timeliness, to adapt staffing levels to stochastic demand, potentially leading to cost reduction and safety improvements. most significantly, this work demonstrates a theoretical minimum error in a single-unit bed demand forecast, which can be used to assess the accuracy of future work in this domain. to err is human nurse staffing and inpatient hospital mortality nurse staffing models, nursing hours, and patient safety outcomes developing nonlinear queuing regressions to increase emergency department patient safety: approximating reneging with balking measuring and forecasting emergency department crowding in real time a simulation study to improve quality of care in the emergency department of a community hospital predicting emergency department inpatient admissions to improve same-day patient flow time series modelling and forecasting of emergency department overcrowding discrete event simulation of emergency department activity: a platform for system-level operations research a simulation model for 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hospital patient flow optimization of hospital ward resources with patient relocation using markov chain modeling development, implementation and evaluation of a tool for forecasting short term demand for beds in an intensive care unit strategies for cutting hospital beds: the impact on patient service on patient flow in hospitals: a data-based queueing-science perspective modelling variability in hospital bed occupancy a generic simulation model for planning critical care resource requirements modeling the impact of changing patient flow processes in an emergency department: insights from a computer simulation study uncovering effective process improvement strategies in an emergency department using discrete event simulation an operating room scheduling strategy to maximize the use of operating room block time: computer simulation of patient scheduling and survey of patients' preferences for surgical waiting time the use of simulation to evaluate hospital operations between the emergency department and a medical telemetry unit discrete event simulation for performance modelling in health care: a review of the literature addressing the variation of post-surgical inpatient census with computer simulation forecasting emergency department crowding : a discrete event simulation using real-time demand capacity management to improve hospitalwide patient flow length of stay-based patient flow models: recent developments and future directions length of stay and imminent discharge probability distributions from multistage models: variation by diagnosis, severity of illness, and hospital on the distribution of the number of success in independent trials stabilisation of inpatient bed occupancy through control of admissions prediction and optimization techniques to streamline surgical scheduling on computing the distribution function for the poisson binomial distribution calculation of expected mae of a translated poisson random variable given its mean (λ + c) and variance (λ).split the sums along λ + c and k.combine the sums without the extra k in the sum.add and subtract identical sums to get (k − c) in the sums that used to have just k.adjust sums by c. key: cord- -omz kibf authors: dixit, shivani; shrivastava, pankaj; kumawat, r. k.; kaitholia, kamlesh; dash, hirak ranjan; sharma, harsh; choubey, gyaneshwer title: forensic genetic analysis of population of madhya pradesh with powerplex fusion c(™) multiplex system date: - - journal: int j legal med doi: . /s - - - sha: doc_id: cord_uid: omz kibf performance of powerplex fusion c kit (pp f c) was assessed in unrelated individuals belonging to madhya pradesh, an indian state. the study evaluated the forensic parameters for the loci included in pp f c multiplex system. the combined discrimination power (cpd) and combined exclusion power (cpe) were and . , respectively, for all autosomal str loci. se showed the greatest power of discrimination ( . ) in the studied population, whereas tpox showed the lowest ( . ). the availability of three y-str loci in the multiplex system is suitable for assessing male contribution and amelogenin deletion in a single multiplex pcr simultaneously. the study also presents the first global report on polymorphism in the indian population on se autosomal str loci and pp fusion c multiplex system. the results revealed that the studied str multiplex system is highly polymorphic and suitable for forensic purposes. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. description present capillary electrophoresis and pcr-based str analysis using expanded combined dna index system (codis) markers are state of art in forensic dna typing [ ] . for performing the identification conclusively and precisely with a significant probability value, an increase in the number of loci is recommended, as the discrimination power of any multiplex system is based on a combination of all the independent loci tested. madhya pradesh is the second largest state in india by area. the population of madhya pradesh is , , comprising of , , males and , , females, contributing % to india's total population as per the census. a few studies have been performed to characterize the population of madhya pradesh on autosomal strs (described in table s ) but these studies are based on str markers either using identifiler/identifiler plus (applied biosystem, usa) or powerplex / hs (promega corporation, madison, usa). based on six dyes, pp f c system (promega corporation, madison, usa) is a -locus multiplex that ensures simultaneous detection of autosomal loci in the expanded codis (csf po, fga, th , tpox, vwa, d s , d s , d s , d s , d s , d s , d s , d s , d s , d s , d s , d s , d s , d s , and d s ), three more loci for better discrimination (penta d, penta e and se ), amelogenin for sex determination and dys and two rapidly mutating y-str loci, dys and dys for further confirmation of gender. a multi-laboratory evaluation of the pp f c system has already been published [ ] following swgdam guidelines [ ] . this study is the first global attempt to work out the efficacy of pp sagar, madhya pradesh, india. the samples included in this study have not been typed by any other multiplex system and this is the first study from india detailing the forensic efficacy of a -marker multi-utility str multiplex system. following the code of ethics of the world medical association (i.e., declaration of helsinki) for experiments involving humans, peripheral blood samples of these unrelated individuals were collected. all individuals provided prior written informed consent. no minor was involved in the study. after dna extraction and pcr, analysis was performed on xl genetic analyzer, and statistical evaluation was done as described before [ ] . the peak height threshold was rfu for heterozygous and rfu for homozygous alleles. internal laboratory control standards and kit controls were used at all steps of analysis. the authors have passed the proficiency test of the gitad, spain (http://gitad.ugr.es/principal.htm). this article follows the population data publication guidelines formulated by the journal. the allele frequencies and results of the forensic efficiency parameters for the autosomal str loci under study are given in table s . the frequency values observed in this study were compared with the previously published data (table s ) on autosomal str markers from india and with recently published data on autosomal str markers [ ] . a total of alleles were observed in the population of madhya pradesh, with se possessing the highest ( ) number of alleles and tpox with lowest ( ) number of alleles (table s and fig. ) . of the samples tested, one sample showed amelogenin deletion, with x-profile on amelogenin marker and deletion on dys and dys marker with amplification on dys marker (fig. s ). the studied population followed the hardy-weinberg equilibrium for all loci except for d s , d s , tpox, d s , d s , d s , and fga after the bonferroni correction. the observed and expected heterozygosity in the studied multiplex system ranged from . to . and . to . , respectively. the most polymorphic and discriminatory str loci in the studied population were se with values of . and . , respectively. all the loci included in the studied multiplex system were found to be polymorphic (pic value = < . to . ). the power of exclusion (pe) ranged from . (d s ) to . (se ) with a value of greater than . for the combined power of exclusion (cpe). the power of discrimination (pd) ranged from . (tpox) to . (se ) with a value of one for the combined power of discrimination (cpd). the combined probability of the match (cp m ) and combined paternity index (cpi) for all autosomal str loci were found to be . × − and . × , respectively. therefore, the autosomal str loci exhibited high efficacy and informativeness for forensic investigation and individualization purposes. the data obtained in this study were compared with published indian population data (table s ) related to common autosomal str loci. pairwise fst matrix between the studied population (marked as madhya pradesh population) and reference populations at the common loci have been shown in table s . nj tree and pca plot are given in fig. s and fig. , respectively. the distribution pattern of populations demonstrated that the genetic affinity has existed within ethnic origins and geographically close populations. the studied population was also compared with previously published data of indian population [ ] and guangdong population of china [ ] at common loci and nj tree was constructed using poptree software. the studied population (marked as madhya pradesh population) made a pool with previously published indian population reflecting the same origin and guangdong population of china was found an outlier (fig. ) . pca plot drawn with the same populations showed that all the compared populations are genetically distant. indian population [ ] and the studied population have the same origin but during evolution, they became genetically different which is reflected in pca plot. thus, the nj tree and pca analyses, both were consistent with our observations in the studied population. for the y-linked loci dys , dys , and dys , estimated heterozygosity was observed as . , . , and . respectively (fig. s ). mean genetic diversity and mean genetic distance between individuals based on the cumulative values observed by the analysis of three y-strs included in pp f c were found to be . and . , respectively. the study will add to the data bank of various studies conducted on the indian population. with respect to the distribution of alleles at each str locus, the loci were found to be substantially polymorphic in this population indicating good informativeness of all studied autosomal str markers. in addition, the multiplex system is found suitable in deciphering the amelogenin deletion to avoid misinterpretation and/or to assess presence of male dna (if the dna is male mixed). the interested researchers can request the data from the authors. selection and implementation of expanded codis core loci in the united states developmental validation of the powerplex® fusion c system validation guidelines for dna analysis methods genetic polymorphism study at autosomal locus in central indian population population genetic analyses and evaluation of autosomal strs in indian populations analysis of genetic polymorphisms and mutations at autosomal str loci in guangdong han the authors gratefully acknowledge the support of director, state forensic science laboratory, sagar (mp) for providing laboratory facility and promega (india) for providing the powerplex fusion c for testing its efficacy on the indian population. conflict of interest the authors declare that they have no competing interest.open access this article is distributed under the terms of the creative comm ons attribution . international license (http:// creativecommons.org/licenses/by/ . /), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -bz iwfan authors: mcgovern, ruth; gilvarry, eilish; addison, michelle; alderson, hayley; geijer-simpson, emma; lingam, raghu; smart, debbie; kaner, eileen title: the association between adverse child health, psychological, educational and social outcomes, and nondependent parental substance: a rapid evidence assessment date: - - journal: trauma violence abuse doi: . / sha: doc_id: cord_uid: bz iwfan background: between % and % of children in high-income countries live with a substance misusing parent, the majority of which is below dependent levels. however, little is understood about the impact of nondependent parental substance misuse upon children. methods: we searched the international literature using rigorous systematic methods to identify studies examining parental substance misuse and adverse outcomes in children. the inclusion criteria were cross-sectional, longitudinal, case-control, and cohort studies; of children aged – years whose parents are high-risk substance misusers; reporting on their health, psychological, substance use, educational, and social outcomes. results: we identified papers (from unique studies), most of which were assessed as being of medium to high methodological quality (n= ). parental nondependent substance misuse was found to be associated with adversity in children, with strong evidence of an association with externalizing difficulties (n = papers, all finding an association) and substance use (n = papers, all finding an association) in adolescents and some evidence of adverse health outcomes in early childhood (n = / papers finding an association). there is less evidence of an association between parental substance misuse and adverse educational and social outcomes. the body of evidence was largest for parental alcohol misuse, with research examining the impact of parental illicit drug use being limited. conclusion: methodological limitations restrict our ability to make causal inference. nonetheless, the prevalence of adverse outcomes in children whose parents are nondependent substance misusers highlights the need for practitioners to intervene with this population before a parent has developed substance dependency. , affecting the quality of the relationship between the parent and the child (cleaver et al., ) , lower levels of parent-child supervision (kandel, ) , harsh parenting (kelley, lawreence, milletich, hollis, & henson, ) , higher prevalence of domestic violence and other traumatic events (sprang, staton-tindall, & clark, ) , and family deprivation (holland et al., ) . many factors have been highlighted as possible mechanisms that impact upon the child, these include direct exposure to alcohol and/or drug use and to other users (advisory council on the misuse of drugs, ) , ineffective parenting practices and a reduction in parenting capacity brought about by the intoxicating effect of the substance and/or withdrawal from it (kandel, ; miller, smyth, & mudar, ) , and a lack of parental emotional availability and warmth (suchman, rounsaville, decoste, & luthar, ) as well as greater likelihood of experiencing trauma such as abuse or neglect as a child (dube et al., ) . due to the potential negative impact on the child, parental substance dependence is often identified as a risk factor in child welfare and child protection assessments. in england, % of all children in need assessments identify parental drug misuse and % identify parental alcohol misuse (department for education, ) . in the united states, parental substance misuse has been associated with up to two thirds of all child maltreatment cases (traube, ) . it has been estimated that , children ( %) in england may live with a dependent opiate-using parent (department for work and pensions, ) and between , ( %) and , ( %) children who live with an alcohol-dependent parent (pryce et al., ) . a far larger number of children are likely to live with substance-misusing parents whose use does not meet the diagnostic criteria for dependence. research estimates that between % and % of children in european countries live with at least one parent who misuses substances (european monitoring centre for drugs and drug addiction [emcdda] , ), . % of children in the united states, and % of children in australia (dawe et al., ) . in the united kingdom, % of children aged under years live with at least one parent who misuses alcohol and % with a parent who misuses illicit drugs (manning, best, faulkner, & titherington, ) . moreover, % of uk infants (aged under year) are exposed to parental problem, drinking, or illicit drug misuse (manning, ) , while u.s. research estimates % of mothers are misusing alcohol year after delivery (liu, mumford, & petras, ) . despite the high prevalence of parental substance misuse that is below the diagnostic criteria for dependence, little is understood about the impact of such patterns of parental substance misuse upon children. as the number of children living with a substance-misusing parent is higher than those living with a substance-dependent parent, the greatest burden of harm on a population level is likely to be experienced by these children. the furthering of knowledge in this area is fundamental to enable effective and early responses to address the needs of the families affected. this rapid evidence assessment reviews published evidence reporting on adverse health, psychological, substance use, educational, and social outcomes of children of nondependent substance-misusing parents. we limit our review to high-risk patterns of parental substance misuse. the international literature was searched in november using electronic databases medline (ovid), psychoinfo (ovid), cinahl (ebsco), scopus, applied social science index and abstract (proquest), international bibliography of social science (proquest), proquest criminal justice (proquest), proquest social science journals (proquest), proquest sociology (proquest), social service abstracts (pro-quest), and sociological abstracts (proquest). due to population flux and changes in economic conditions, we restricted our search for evidence to publications from onward. a search strategy using mesh terms, thesaurus headings, boolean, and proximity operators was adapted for each database and implemented. two researchers independently screened all titles and abstracts using specified inclusion and exclusion criteria, retrieving full articles for all potentially eligible studies and evaluating in full text. discrepancies at each stage were resolved by discussion or by consulting a third researcher if consensus could not be reached. studies adopting a cross-sectional, longitudinal, case-control, and cohort design were included if the sample consisted of children aged - years whose parents were high-risk substance misusers. to be included, studies must report on parental substance misuse that meets one of the following criteria: a pattern of alcohol consumption that leads to the presence of physical or psychological problems (typically over units per week for women and over units per week for men); frequent illicit drug misuse (more than once per month as defined by the crime survey for england and wales); and alcohol or illicit drug abuse defined as a maladaptive pattern of drinking/drug use, leading to clinically significant impairment or distress, as manifested by at least one related problem in a -month period (failure to fulfill major role obligations, use in situations in which it is physically hazardous, alcohol or drug-related legal problems, having persistent or recurrent social or interpersonal problems caused or exacerbated by the effects of alcohol or drugs; american psychiatric association, ). further, studies were required to include comparison samples of children whose parents were not substance misusers. the condition of interest was adversity experienced by the child defined as any negative health, psychological, child substance use, educational, and social effect. a health harm includes direct impact (e.g., brought about by accidental ingestion by the child or exposure to the substance or contaminated environments) or indirect impacts (e.g., child physical injury, health service usage, fatality); psychological harm such as internalizing (e.g., emotional or behavioral problems that are focused inward such as depression, anxiety, dissociative disorder, and eating disorder) and externalizing problems (e.g., behaviors that are directed toward the external environment including physical aggression, disobeying rules, and antisocial and offending behaviors); substance use by the child includes early onset of alcohol and/or drug use, frequent use, and experience of alcohol and/or drug problems; educational impact includes school attainment, punctuality, truancy, or suspension; and social impact includes parent-child relationship quality, family functioning and home environment, parent supervision, and experience of abuse. papers were excluded if % of the parent sample were dependent upon substances other than tobacco (defined as those with a diagnosis of dependence, in receipt of agonist prescribing or attendance at treatment services); insufficient detail is reported for the review team to confidently assess the criteria for high-risk substance misuse levels; were reporting on a qualitative study or were not published in english. the methodological quality of each study included was assessed according to the criteria presented in the quality assessment tool for systematic reviews of observational studies (wong, cheung, & hart, ) . this scale is based on a cumulative score across items: external validity, reporting ( items), bias, and confounding factors. studies achieving % or more in scoring were regarded as high quality, - % medium, and less than % low quality. we have standardized the language used within the review when referring to all studies meeting the criteria for high-risk parental substance misuse. we use the term parental substance misuse when referring to studies that report on parents who misuse alcohol and/or drugs. when the source study examines only alcohol or drug misuse, we use the term parental alcohol misuse or parental drug misuse, respectively. within the tables and figures, however, we will include further clarification relating to the specific levels reported upon within the source studies. we identified papers (reporting on unique studies) that reported upon adverse outcomes of children of nondependent parental substance misusers. the majority (n ¼ ) of the studies were conducted in the united states, in the united kingdom and in other european countries and two other countries worldwide (australia and korea). the sample sizes of the included studies ranged from to , (mean sample , ). we assessed studies as being of high methodological quality, of medium quality, and of low quality. we have divided the adverse child outcomes into physical health, psychological well-being, child substance use, educational, and social. figure provides further details of the flow of the studies identified for the review, and the summary of findings for the included studies is presented in table . table highlights the implications for practitioners working with children at different developmental stages. six unique studies showed a significant positive association between parental substance misuse and negative child health outcomes. baker et al. ( ) and tyrrell, orton, tata, and kendrick ( ) conducted high-quality large uk population-based matched nested case-control studies investigating the association between maternal alcohol misuse and other risk factors for accidental child injury aged - years. children whose mother's medical record showed a history of alcohol misuse were found to have a -fold higher odds of long bone fracture (odds ratio [ medium more frequently and for longer than children whose mothers did not misuse substances. inpatient care episodes per , children were almost double in the group of children with substance misusing mothers to that of the comparison group ( , vs. and , ) with a mean duration of . days and . days, respectively (raitasalo, holmila, autti-ramo, notkola, & tapanainen, ) . this association was most pronounced in mothers who misused both alcohol and drugs. remaining studies were of low quality; therefore, caution should be applied when considering results. these studies reported that poor diet (jeffreys, hirte, rogers, & wilson, ), low weight (below the th percentile; joya et al., ) , and increased rates of dental problems (cornelius et al., , jeffreys et al., ) in children were associated with substance misuse by parents, while studies have found no correlation between parental alcohol misuse and sleep in children (tarokh & carskadon, ; tarokh et al., ) . there is strong evidence of significant positive association between parental alcohol misuse and externalizing problems, with all seven papers (reporting on six unique studies) of medium to high quality found a significant and positive relationship (kendler et al., ; lee & cranford, ; malone, iacono, & mcgue, ; malone, mcgue, & iacono, ; torvik, rognmo, ask, roysamb, & tambs, ) . parental alcohol misuse has been found to be associated with conduct problems (kendler et al., ; malone et al., , malone et al., , most defiant disorders (malone et al., ; malone et al., ) , delinquency (kendler et al., ) , and violence (rossow, pape, & wichstrom, ) . there were mixed findings relating to attention and hyperactivity difficulties in the children of alcohol misusers, with two studies finding a significant association (kendler et al., ; torvik et al., ) , while two papers from one linked study found no association (malone et al., ; malone et al., ) . only one study examined the impact of parental misuse of substances other than alcohol (torvik et al., ) , finding an association of modest effect size upon child attention and conduct problems. studies mostly reported association between both mothers' and fathers' substance misuse and child externalizing difficulties. one paper reported that the relationship was greater when maternal alcohol misuse was present (torvik et al., ) . a further study found gender-modeling associations between the parent and child; maternal alcohol misuse was found to be significantly correlated with rule-breaking and aggressive behavior in girls but not in boys, while paternal alcohol misuse was found to be significantly associated with aggressive behavior in boys but not girls (finan, schulz, gordon, & ohannessian, ) . there was limited evidence of a significant association between maternal or paternal alcohol misuse and internalizing disorders in children. in a medium quality study using child report measures of parental drinking, both paternal and maternal alcohol misuse were related to depression and anxiety for girls but not for boys (ohannessian, ) . in a small, medium-quality cohort study of korean school children aged - years, parental alcohol misuse was found to be early childhood ( - years) greater likelihood of being involved in an accident, self-poisoning incident, and sustaining an injury (baker et al, ; tyrrell et al, ) . requirement for medical attention and admittance to hospital (raitasalo et al, ) . more likely to require inpatient care for a longer period (raitasalo et al, ) . inadequate diet and underweight. children maybe left in places of unknown safety (freishleret al, ). early adolescence ( - years) poor dental hygiene resulting in higher likelihood dental problems, however may not access dental care (corneliuset al, ) . low shyness, hyperactivity, attention difficulties, and conduct problems (kendler et al, ) . early onset alcohol use, cigarette use, and illicit drug use (cranford et al, ; jeffreys et al, ; kendler et al, ; kerr et al, ) . externalizing and internalizing difficulties may begin to emerge (lee and cranford, ; malone et al, ; rossow et al, ; torvik et al, ) . middle adolescence ( - years) externalising difficulties including conduct problems, delinquent behaviour, rule breaking, aggressive behaviour, attention difficulties (finan et al, ; lee and cranford, ; malone et al, ; rossow et al, ; torvik et al, ) . internalising difficulties including depression and anxiety lee and cranford, ; ohannessian, ) . regular substance misuse include frequent intoxication, illicit drug use and the development of substance misuse problems ( positively and significantly associated with internalizing disorders (lee & cranford, ) . associations in a high-quality study however did not reach significance (malone et al., ; malone et al., ) . there is a large volume of evidence that parental substance misuse influenced their child's own substance use, with all included papers (reporting on unique studies) finding a significant association. this evidence is mostly of medium to high quality with only two studies being of low quality (jeffreys et al., ; yule, wilens, martelon, msimon, & biederman, ) . parental alcohol and/or drug misuse was significantly associated with early onset adolescent drinking (kerr, capaldi, pears, & owen, ; vermeulen-smit et al., ) , alcohol consumption (cranford, zucker, jester, puttler, & fitzgerald, ; haugland, holmen, ravndal, & bratberg, ; jennison, ; keeley, mongwa, & corcoran, ; shorey et al., ; vermeulen-smit et al., ) , alcohol intoxication (haugland, holmen, krokstad, sund, & bratberg, ; haugland et al., ; haugland, strandheim, & bratberg, ; keeley et al., ; rossow et al., ) , and the development of alcohol problems (kendler et al., ; lieb et al., ) as well as adolescent illicit drug use (delaney-black et al., ; haugland et al., ; hofler et al., ; hopfer, stallings, hewitt, & crowley, ; jeffreys et al., ; keeley et al., ; malone et al., ; malone et al., ; shorey et al., ) . having two parents who misuse substances was highlighted as being particularly predictive of adolescent substance use (keeley et al., ; swaim, beauvais, walker, & silk-walker, ) , with regular alcohol use being almost times as likely (or ¼ . , ci [ . , . ], p < . ) and past year illicit drug use almost times as likely (or ¼ . , ci [ . , . ], p < . ) as adolescents with two nonsubstance misusing parents (keeley et al., ) . there were mixed findings as to whether the mothers' or fathers' substance misuse had the strongest association with child alcohol and/or drug use. six studies found that both maternal and paternal substance misuse correlated with adolescents' alcohol (finan et al., ; haugland et al., ; keeley et al., ; van der zwaluw et al., ) and drug use (finan et al., ; keeley et al., ) to a similar size of effect. studies found that only fathers' and not mothers' substance misuse was positively associated with adolescent drinking (cranford et al., ; ohannessian, ) and the range of substances used in adolescent children (shorey et al., ) . further studies, however, reported only maternal alcohol misuse (kerr et al., ) and maternal drug misuse (yule et al., ) to be significantly associated with development of alcohol or drug misuse in children. when also considering child gender, both boys and girls have been found to be significantly more likely to engage with substance using behaviors if their parents misused alcohol, with boys in particular being found to experience a negative effect (haugland et al., ) . three papers of mixed quality reported on parental alcohol misuse and its association with negative child education outcomes. using a large cohort of over , swedish individuals, berg, back, vinnerljung, and hjern ( ) conducted a high-quality study finding that alcohol-related hospital admissions in parents were significantly associated with lower school performance in adolescents aged - years (berg, back, vinnerljung, & hjern, ) . school behavioral problems were associated with paternal alcohol misuse in early childhood, with a -fold risk of truancy, absenteeism, suspensions, and conduct problems in a medium quality study (jennison, ) . family dysfunction, conflict, and ineffectual parenting were found to be strongly correlated to adverse school outcomes for children in families with an alcohol misusing father. the low levels of attachment and bonding to biological fathers found to be associated with an increase in school-related behavioral problems of the children were notable. a small low-quality study in australia also found children of substance misusers were more often absent or late for school (jeffreys et al., ) . the literature on the social outcomes of children whose parents misuse substances was mixed. four studies of medium quality considered the association between alcohol misusing parents and the parent-child relationship. while one study showed no significant impact upon the support provided to the child from the parent (van der zwaluw et al., ) , a further study reported that children of alcohol misusing parents were significantly less likely to feel emotionally close to their father, either due to the impact of alcohol misuse upon the father's behavior, conflict within the home, abdication of family responsibilities, or estrangement (jennison, ) . parental bonding and the parent-child relationship were negatively related to both mothers' and fathers' alcohol misuse in other research (shorey et al., ) . there is conflicting evidence that parental monitoring is negatively associated with parental alcohol misuse. one study reported a significant association between parental alcohol misuse and lower levels of parental monitoring (shorey et al., ) , while a further study reported that parental alcohol misuse was unrelated to a number of measures of neglectful parenting practices (freisthler, johnson-motoyama, & kepple, ) . a significant association with a particularly large effect was shown in a high-quality study of the number of children of substance misusing mothers who were placed in care (raitasalo et al., ) . children of alcohol misusing mothers were times more likely to be placed in care by their seventh birthday than those raised by nonalcohol misusing parents. those born to drug misusing mothers were over times more likely to be in care by the age of , while children whose mothers misused both alcohol and drugs faced a -fold increased risk. these relationships persisted after controlling for the child's gender and mothers' socioeconomic status. once in care, children of substance misusing mothers were discharged % faster than those children whose mothers did not misuse substances (hussey & guo, ) . this somewhat counterintuitive finding is most likely to relate to an accelerated decision to place the children in permanent care however rather than reunification of the family. further, the low quality of this study means that findings should be interpreted with caution. a cohort study of children taken into care also reported on the range of abuse children may experience while living within substance misusing homes (jeffreys et al., ) . due to the small sample size in this study, no statistical testing could be conducted. as such, the existence of a correlation between parental substance use and abuse is unknown. it is clear from this evidence that nondependent parental substance misuse is associated with adversity in children. this evidence is more pronounced when both parents misuse alcohol and/or drugs or when one or more parents misuse a combination of alcohol and drugs. in early childhood ( - years), the evidence from high-quality studies suggests an increased likelihood of experiencing an injury or health concern, resulting in the need for medical care. the methodological quality of studies examining an association with diet, child weight, and dental health is however of low quality and should be interpreted with caution. maternal alcohol misuse in particular is highlighted as a key predictor for poorer child health. this may in part relate to the greater role mothers tend to play in the child's early years. however, there was a paucity of research considering substances other than alcohol and relating to fathers' substance misuse that may result in a misleading focus on mothers. the literature suggests that parental alcohol misuse increased the likelihood of externalizing problems in children, with a significant association being reported in all studies being of medium or high quality. there was less evidence for an association between parental substance misuse and internalizing difficulties such as depression and anxiety. those showing an association were of medium quality and involved children exposed to parental intoxication or where family and/or parental factors are present, while a high-quality study did not find a significant correlation. there was a large and methodologically robust evidence base consisting of papers, all finding a significant and positive relationship between parental alcohol and/or drug misuse and the child's own use. the evidence appeared particularly strong in families where both parents misused substances or when the child was directly exposed to the substance misuse. social learning theory explains that we learn behavior from observing, imitating, and modeling those around us (bandura, ) . it is possible that when children observe their parents consuming alcohol and/or drugs, it encourages the development of normative views about substance use. further, the availability of alcohol or other substances within the home may increase the likelihood of adolescent use (peeters, koning, monshouwer, vollebergh, & wiers, ) . there is emerging evidence that parental alcohol misuse is predictive of educational challenges including truancy, school-related behavioral problems, and lower educational attainment, although the methodological quality of these studies varied. the involvement of child welfare services as well as out-ofhome placements for children was also higher in children whose parents misuse substances. the evidence for an association with other social and relational difficulties is however mixed. there was some suggestion that parental alcohol use was associated with lower levels of parent-child bonding, communication, and overall relationship quality. however, evidence of neglectful parenting or inadequate parental supervision was limited and at times contradictory and as such caution should be exercised when drawing conclusions regarding an association between parental substance misuse and the quality of parental supervision. methodological issues further limit the evidence. social workers often interact with families experiencing a wide range of difficulties. while social workers within children's welfare services consider it legitimate within their role to ask parents about their drug and alcohol use, they experience difficulty in identifying parents who are substance misusers (galvani, hutchinson, & dance, ; loughran, honhman, & finnegan, ) . typically, they rely upon observations of the parent's physical presentation and behavior, which is unlikely to detect levels of misuse below the diagnostic criteria for dependence (galvani et al., ) . furthermore, those parents who are identified as being substance misusers often do not receive an intervention, with parents expressing reluctance to engage with specialist drug and alcohol treatment providers as they did not perceive themselves as having a substance misuse problem (forrester & harwin, ) . this suggests that early, opportunistic intervention delivered by a nontreatment specialist may be more appropriate. there is a large amount of high-quality evidence that has accumulated to support the effectiveness of alcohol screening and brief interventions with adults who have an alcohol use disorder with nondependent populations in primary care setting (kaner et al., ; o'donnell et al., ) . however, there are no studies examining the effectiveness of screening and brief interventions with substance misusing parents including those whose children are involved with child welfare services. this represents a missed opportunity to intervene with this population before a parent has developed substance dependency. such intervention has the potential to prevent the development of more problematic patterns of use and prevent harm to children. social workers should engage in conversations with parents, which promote the parent's ability to link their substance misuse with adverse experiences and risk of negative outcomes for their child. such an interaction may replicate the "teachable moment" found to be conducive of behavior change following the delivery of brief interventions within other settings (babor & grant, ) and improving outcomes for children. the evidence examined within this review provides support for an association between parental substance misuse and a number of adverse child outcomes at different stages of development. there are however some notable gaps. in early childhood ( - years), the literature focuses upon the relationship between mothers' substance misuse and child physical health, with a paucity of research examining behavioral problems, parent-child bonding, or preparation for school as well as the impact of fathers' substance misuse. in early adolescence ( - years), there is a lack of research into child education outcomes, and in each stage of adolescence ( - years), there is a lack of research into parental substance misuse and child health. there is limited research that considers parental illicit drug misuse throughout the child's development, with the majority of research examining the difficulties in children of alcohol misusing parents. due to the inclusion of cross-sectional studies, causal relationships cannot be determined. such naive comparisons of exposed and unexposed groups cannot adequately account for the many potential confounders nor precisely account for the measurement of effect (fewell, davey smith, & sterne, ) . for example, genetic predisposition (agrawal & lynskey, ) and the interaction between genes and the environment (cleveland & wiebe, ) may result in intergeneration transmission of substance misuse. alcohol permissive parenting (ennett et al., ; hung, chang, luh, wu, & yen, ) , adolescent monitoring (kerr, stattin, & burk, ), lower parent-child relationship quality (donaldson, handren, & crano, ; el-sheikh & buckhalt, ; shorey et al., ) , and greater family conflict (el-sheikh & flanagan, ; kelly et al., ) have been associated with increased child substance misuse. furthermore, the direction of a reported relationship cannot be determined. for example, while children's conduct difficulties could be a result of parental alcohol misuse, a parent whose child has conduct difficulties may struggle to cope with their child's behavior and their alcohol consumption may increase in response. while longitudinal studies can highlight the temporal associations between variables and may offer greater insight into causation, such inferences are reliant upon the timing of the behavior in relation to the outcome. for example, studies examining the impact of parental substance misuse upon internalizing difficulties in children may assess children prior to the emergence of any symptoms or early childhood exposure to parental substance misuse may be undetected due to behavior change that predates study inclusion (katikireddi, green, taylor, smith, & munafo, ) . further research into the impact of parental substance misuse upon the child is needed to address the gaps in the evidence. specifically, research examining the various impacts of both alcohol and illicit drug misuse throughout the stages of the child's development. future research should include both fathers and mothers and be sufficiently powered to enable analysis of the impact of mothers' versus fathers' use upon male and female children and utilize longitudinal design, with regular follow-up throughout the child's development. this would offer opportunity for causal inferences and also enable age-related and temporal associations to emerge. there is also a need for more high-quality research examining the health, educational, and social impact of parental substance misuse. the significant variation in how substance misuse patterns are described within research has presented a great challenge to this review and ultimately in the further of knowledge in this area. there is a need for consistency in the use of terminology describing levels of parental substance misuse in future research. in considering the evidence for the impact of nondependent parental substance misuse on children, focus invariably is on risks. there is also a need to consider the protective factors that may be present, which may enhance child resilience to harm. this review has highlighted evidence that an association between parental substance misuse is greater when both parents are substance misusers. put another way, the presence of one nonsubstance misusing parent offers some protection. using the language of protection, rather than risk, affords an opportunity to view such protective factors as a possible intervention mechanism to enhance resilience. given the evidence identified that factors such as maternal closeness, attachment, and parent-child relationship quality are moderators of adversity (shorey et al., ) , future research should include a range of mediators and, importantly, moderators of harm, which may inform intervention development. the findings of this review suggest that the vulnerability to adverse outcomes is not restricted to children living with substance-dependent parents. rather, children may be affected by a wider continuum of harmful parental substance misuse; children who are likely to be less visible to practitioners. while practitioners may find it challenging to identify parents whose use is not within the dependent range (galvani et al., ) , intervening early in parental risk factors including alcohol and drug misuse to safeguard children has been highlighted in guidance for health, social care, and third sector partners (department of health, ; hm government, ; munro, ) . as the number of children living with a nondependent substance misusing parent is likely to be greater than those living with a substance-dependent parent, intervening with nondependent parents is likely to bring about most benefit to children on a population level. working to promote resilience and to enhance the child's protective factors is also important. parents who do not misuse substances are a resource to this end. moreover, intervening before a parent has developed a dependency has the potential to prevent the development of more problematic patterns of use and prevent harm to children. the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. the author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: the authors received funding from public health england to undertake this rapid evidence assessment. https://orcid.org/ - - - ruth mcgovern is a senior research associate and nihr post doctoral fellow at the institute of health & society, newcastle university. she is also a qualified and registered social worker. her research is concerned with drug and alcohol misuse, with a particular focus upon parental misuse and the impact upon the family. eilish gilvarry is deputy medical director for appraisal and revalidation at northumberland, tyne & wear nhs foundation trust, consultant psychiatrist in addictions at newcastle addictions service and honorary professor of addiction psychiatry at newcastle university. her research concerns all aspects of drug and alcohol misuse, with a particular focus on pharmacological and psychological interventions. michelle addison is a research associate at the institute of health & society, newcastle university. she is a social scientist and her research is concerned with the relational properties of health and social inequalities impacting on socially marginalised people. hayley alderson is a research associate at the institute of health & society, newcastle university. her research is concerned with developing and evaluating interventions to reduce drug and alcohol use. emma geijer-simpson is a phd student at the institute of health & society, newcastle university. her doctoral research is concerned with developing family-involved interventions to prevent risky alcohol use and co-existing mental health difficulties in young people. raghu lingam is a professor of paediatric population health at the university of new south wales and a community paediatrician within the sydney children's hospital network. his is a clinical academic research interests are in child health services research, mental health and risk taking behaviours and in child development and disability. debbie smart is a research assistant at the institute of health & society, newcastle university. her research interests are concerned with children and families with a particular focus upon young carers and parental risk factors. eileen kaner is a professor in public health at the institute of health & society, newcastle university. she is a behavioural scientist and her research programme aims to improve health by promoting the use of 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- journal: eye (lond) doi: . /s - - - sha: doc_id: cord_uid: d sbfibq purpose: soluble cytokine receptors are potential biomarkers for immune activation and have a promising potential as immunotherapeutic agents. we investigated the levels of soluble cytokine receptors in aqueous humour (ah) samples from patients with specific autoimmune uveitic entities. methods: patients with active uveitis associated with behçet’s disease (bd) (n = ), sarcoidosis (n = ), hla-b -related inflammation (n = ), vogt–koyanagi–harada (vkh) disease (n = ) and control subjects (n = ) were included. ah samples were analyzed with the use of multiplex assays for the proinflammatory cytokine tumour necrosis factor (tnf)-α and the soluble cytokine receptors scd , scd , sgp , sil- receptor-α (sil- r), stnfri and stnfrii. results: tnf-α and soluble cytokine receptor ah levels were significantly higher in uveitis patients (n = ) compared with controls (n = ). when nongranulomatous uveitis (bd and hla-b -associated uveitis) was compared with granulomatous uveitis (sarcoidosis and vkh disease), the levels of scd and stnfri/tnf-α and stnfrii/tnf-α ratios were significantly enhanced in granulomatous uveitis. finally, when comparing the profile in the specific uveitis entities, scd levels were highest in patients with vkh disease. sgp , scd , sil- r, stnfri and stnfrii did not differ significantly between the four different clinical uveitic subgroups. conclusions: soluble cytokine receptors are significantly upregulated in autoimmune uveitis. cd (+) t cells might contribute to the inflammatory process in granulomatous uveitis, particularly in vkh disease. granulomatous uveitis is also characterized by significantly higher stnfrs/tnf-α ratios than nongranulomatous uveitis. patients with autoimmune uveitis present with heterogeneous clinical manifestations of intraocular inflammation and are at risk for severe visual impairment. the patients often also suffer from systemic diseases, such as behçet's disease (bd), sarcoidosis, human leucocyte antigen (hla)-b -associated inflammation and vogt-koyanagi-harada (vkh) disease, which present with different clinical phenotypes. these specific uveitic entities presumably arise as a consequence of different underlying mechanisms, each involving distinct immune molecules [ ] [ ] [ ] [ ] . cytokines are key regulators of inflammation and are thought to play important roles in the pathophysiology of various autoimmune uveitis entities [ ] [ ] [ ] [ ] [ ] . previous studies demonstrated upregulation of proinflammatory cytokines, such as tumour necrosis factor-α (tnf-α), interferon-γ (ifn-γ), interleukin (il)- , il- and il- in the aqueous humour (ah) samples from autoimmune uveitis patients [ ] [ ] [ ] [ ] [ ] . selective blockade of proinflammatory cytokines, therefore, may represent a possible way for clinical intervention. this is exemplified by the introduction and considerable clinical benefits of novel biologicals, such as anti-tnf-α agents and anti-il- receptor (il- r) antibody for the treatment of autoimmune uveitis resistant to conventional treatment [ ] [ ] [ ] . soluble cytokine receptors have been thoroughly studied in experimental and clinical studies as potential biomarkers of immune activation and/or disease activity in many clinical conditions. furthermore, soluble cytokine receptors have a promising potential as immunotherapeutic agents for several autoimmune diseases [ ] . cluster of differentiation (cd ), belonging to the tnf-nerve growth factor receptor superfamily, was first characterized as a cell surface antigen on hodgkin's and reed-sternberg cells [ , ] . subsequently, cd was also found on activated cd ro + memory t helper (th) cells and cd + t cells, but is absent on naive and resting t cells [ ] . in addition, it was demonstrated that t cells isolated from peripheral blood of healthy individuals expressing cd produced more cytokines (ifn-γ or il- ) than cd negative t cells [ ] . activated th cells expressing cd exhibited enhanced helper activity for b cell immunoglobulin production [ ] . a soluble form of cd is released by cd + cells in vitro and in vivo by proteolytic cleavage. the level of scd was demonstrated to correlate well with surface expression of cd and can, hence, be applied as a marker for cd expression [ , ] . cd is a member of the cysteine-rich scavenger receptor family class b and is expressed exclusively by the monocyte-macrophage cell lineage. as an endocytic receptor for haemoglobin-haptoglobin complexes cd is proposed to capture free circulating haemoglobin. cd may have anti-inflammatory and immunoregulatory properties, since it decreases in vitro activation and proliferation of t lymphocytes. the proteolytic cleavage of the membrane-bound cd by matrix metalloproteinases in response to inflammatory stimuli releases scd . thus, scd may be viewed as a biomarker for macrophage activation in several inflammatory disorders [ , ] . dysregulated expression of il- is implicated in the development of various chronic inflammatory autoimmune diseases [ ] [ ] [ ] . the biological activities of il- are mediated by a receptor complex comprising the il- binding transmembrane glycoprotein il- r-α and the transmembrane signal transducer protein gp that is shared by additional cytokines of the il- family [ ] . the high-affinity complex of il- and il- r interacts with two gp chains and activates the underlying signal transduction pathways. gp is broadly expressed, whereas il- r is expressed restrictively in liver cells and certain leucocyte subtypes [ ] . soluble gp (sgp ) isoforms are generated by alternative splicing. the soluble il- r (sil- r) also arises through alternative splicing, in addition to ectodomain shedding. sil- r and intact membrane-bound il- r bind il- with comparable affinity. contrary to the soluble receptors of tnf-α, which inhibit the activity of tnf-α, the il- /sil- r complex can via interaction with gp activate target cells, in the absence of the classical membrane-embedded il- r. such cells cannot be activated by il- alone when sil- r is not available. this latter process is referred to as il- trans-signalling, as opposed to classical signalling via surface il- r. it is increasingly apparent that many of the activities assigned to il- are mediated via sil- r [ , ] . since sgp binds the complex of il- /sil- r, sgp acts as a natural inhibitor of il- trans-signalling in vivo, without interfering with classical signalling [ , ] . tnf-α recognizes two distinct cell surface receptors, namely tnf-α receptor type i (tnfri) and type ii (tnfrii). in addition to membrane-bound forms, both tnfri and tnfrii also exist as soluble forms produced by proteolytic cleavage [ , ] . the soluble receptors stnfri and stnfrii retain their ligand binding capacity and can act as natural inhibitors by sequestering tnf-α and avoiding activation of its membrane-embedded signalling receptors. as tnf-α itself is a principal inducer of stnfrs shedding and expression, determination of stnfri and stnfrii levels could provide indirect evidence of tnf-α production and reflect the activation state of the tnf-α/ tnfr system [ , ] . we hypothesized that different immunopathogenic mechanisms are involved in each clinical subtype of autoimmune uveitis. through analysis of the soluble cytokine receptor profile in ah from patients with specific clinical entities of autoimmune uveitis, a better understanding of the underlying immune mechanisms may be obtained. in addition, it may also be of clinical importance for disease diagnosis and identification of potential targets for selective therapy. for these reasons, we analyzed the ah from patients with active uveitis associated with four systemic inflammatory diseases (sarcoidosis, vkh disease, bd and hla-b -related inflammation) for the presence of scd , scd , sgp , sil- r, stnfri and stnfrii. furthermore, the levels of soluble receptors were correlated with the levels of tnf-α, a major proinflammatory cytokine, and with clinical disease activity. all patients included in the study were seen at the outpatient clinic of king abdulaziz university hospital and all gave informed and written consent. as controls for patients with active uveitis (n = ; n = for bd, n = for hla-b associated uveitis; n = for sarcoidosis; n = for vkh disease), patients who had undergone cataract extraction with no prior history of uveitis (n = ) were included. diagnosis was made as described before following established clinical criteria, with supporting laboratory evidence as needed [ , , ] . in each patient, the uveitis activity was graded according to the criteria of the standardization of the uveitis nomenclature working group grading scheme [ ] . none of the patients was on topical or systemic therapy on presentation. ah sampling was done before the start of therapy. by means of limbic paracentesis - µl of ah was aspirated and processed as described [ ] [ ] [ ] [ ] . the demographic and clinical characteristics of the included patients are described in detail in a previous report in table ; duplicated with permission from [ ] . all procedures were performed according to the tenets of the declaration of helsinki and the protocol of this study was approved by the research center, college of medicine, king saud university. we determined the profile of tnf-α and the soluble cytokine receptor levels in the ah samples using two multiplex assays. the first multiplex contained detection reagents for the proinflammatory cytokine tnf-α (bio-plex catnr ak mr , biorad, hercules, ca, usa), the second multiplex assessed the soluble cytokine receptors scd , scd , sgp , sil- r, stnfri and stnfrii (bio-plex catnr al m, biorad). the two analyses were performed according to the manufacturer's guidelines in separate plates on consecutive days on the same ah samples. in each array, µl of ah was used after dilution with µl of assay buffer (biorad). the data were obtained . ± . . ± . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . ± . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . ± . . ± . vkh vogt-koyanagi-harada when considering the whole group of patients, tnf-α and soluble cytokine receptor levels were significantly enhanced in ah of patients compared with controls ( table ) . compared with controls, tnf-α, scd , sgp , sil- r, stnfri and stnfrii levels were significantly higher in bd and hla-b- -associated uveitis. the levels of tnf-α, scd , scd , sgp , sil- r, stnfri and stnfrii were significantly elevated in sarcoidosis. the levels of scd , scd , sil- r, stnfri and stnfrii were significantly higher in vkh disease ( table ) . next, the kruskal-wallis test was applied to compare the distribution of tnf-α and soluble cytokine receptor levels among the four disease groups. among the cytokine and soluble cytokine receptors analyzed, tnf-α and scd differed significantly between patients with bd, sarcoidosis, hla-b -associated uveitis and vkh disease (p = . ; p = . , respectively) (fig. a) . tnf-α levels were significantly increased in hla-b -associated uveitis compared with vkh disease (mann-whitney test; because the biological activity of tnf-α is regulated by stnfri and stnfrii in vivo, the ratios of tnf-α to its soluble receptors were calculated for the ah samples. when considering the whole patient group, the stnfri/ tnf-α ratio was significantly higher in the ah of controls than in uveitis patients. on the other hand, the stnfrii/ tnf-α ratio was increased in uveitis patients compared with controls, but significance was not reached (table ). in the four disease groups, the stnfri/tnf-α ratio was significantly higher in controls than in bd and hla-b associated uveitis. however, the stnfri/tnf-α ratio did not differ significantly between controls and sarcoidosis or between controls and vkh disease. the stnfrii/tnf-α ratio did not differ significantly between controls and individual disease groups (table ) . next, we evaluated the distribution of stnfri/tnf-α and stnfrii/tnf-α ratios among the four disease groups (kruskal-wallis test). no statistically significant differences were observed. when analysis was done according to subdivision of the patients into those with granulomatous uveitis (sarcoidosis and vkh disease) (n = ) and those with nongranulomatous uveitis (bd and hla-b -associated uveitis) (n = ), scd levels and stnfri/tnf-α and stnfrii/tnf-α ratios in granulomatous uveitis were significantly higher than those in nongranulomatous uveitis. on the other hand, tnf-α levels in nongranulomatous uveitis were significantly higher than those in granulomatous uveitis. levels of scd , sgp , sil- r, stnfri and stnfrii were similar in granulomatous and nongranulomatous uveitic diseases (table and fig. b) . in the whole group of patients, the levels of tnf-α correlated significantly with the levels of stnfri (r = . ; p < . ) and stnfrii (r = . ; p < . ). significant positive correlations were found between ah levels of tnf-α (r = . ; p < . ), sil- r (r = . ; p = . ), stnfri (r = . ; p = . ) and stnfrii (r = . ; p = . ) and clinical disease activity. on the other hand, there was a significant negative correlation between stnfri/tnf-α ratio and clinical disease activity (r = − . ; p = . ). there were no significant correlations between clinical disease activity and scd , scd , sgp and the stnfrii/tnf-α ratio. in the present study of patients, elevated levels of scd in the ah samples from patients with autoimmune uveitis might reflect a recruitment of cd + t cells into the ocular inflammatory microenvironment of patients with uveitis. our subgroup analysis showed that scd levels were significantly higher in patients with vkh disease than in patients with bd and patients with hla-b -associated uveitis. among the four disease groups, the levels of scd were elevated . -fold, . -fold, . -fold and . -fold in patients with vkh disease, sarcoidosis, bd and hla-b associated uveitis, respectively, compared with controls. in addition, scd levels were significantly higher in patients with granulomatous uveitis associated with sarcoidosis and vkh disease than in patients with nongranulomatous uveitis associated with bd and hla-b -associated uveitis. our findings suggest that cd + t cells contribute to the inflammatory process in patients with granulomatous uveitis, particularly in vkh disease. similarly, previous studies demonstrated that cd + t cells are involved in the pathogenesis of several granulomatous inflammatory diseases [ ] [ ] [ ] . recently, shinoda et al. [ ] demonstrated that cd knockout mice showed milder symptoms in a model of experimental autoimmune encephalomyelitis and less antigen-specific th and th cells were induced. these findings suggest that cd expression on cd + t cells is implicated in the pathogenesis of autoimmune diseases of the central nervous system. previous reports demonstrated high serum levels of scd in both th -and th -dominated disorders [ , ] . pellegrini et al. [ ] reported that cd may be an essential costimulatory molecule and marker for an important immunoregulatory subpopulation of t cells which controls the th /th balance in immune responses. increased levels of scd have been reported in several autoimmune diseases [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in addition, elevated scd serum concentrations correlated with clinical and laboratory parameters of disease activity [ , [ ] [ ] [ ] . macrophages activated by classical pathways (m ) have proinflammatory properties. conversely, alternatively activated m macrophages which express cd have antiinflammatory, antioxidant, tissue repair, proangiogenic and fibrosing roles. therefore, scd may represent a marker for alternatively activated macrophages [ , ] . the value of scd as a marker of macrophage activation has been confirmed in several inflammatory conditions [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the present study is the first to demonstrate high levels of scd in the ocular microenvironment of patients with autoimmune uveitis, scd levels being elevated about -fold, compared with controls. these data suggest that active autoimmune uveitis may be associated with increased macrophage activation and are consistent with previous reports that macrophages are involved in the pathogenesis of autoimmune uveitis [ ] . the detected rise in scd may be due to upregulation and/or augmented shedding of cd . increased shedding might be correlated to elevated levels of cd -cleaving proteinases in the ocular inflammatory microenvironment of these patients. several studies demonstrated that il- trans-signalling via the sil- r is critically involved in the initiation and maintenance of several inflammatory and autoimmune diseases [ , ] . elevated sil- r levels have been documented in synovial fluids of patients with rheumatoid arthritis [ , ] . in addition, elevated levels of il- and sil- r correlated with the degree of joint destruction in rheumatoid arthritis [ ] . in a previous study, we demonstrated upregulation of the proinflammatory cytokine il- in ah samples from patients with autoimmune uveitis [ ] . in the present study, sil- r levels were elevated . -fold compared with controls, whereas levels of the inhibitory sgp were elevated only . -fold. these findings suggest that the regulation of il- trans-signalling may be distorted in autoimmune uveitis. interestingly, selective targeting of sil- r-mediated events may represent a novel avenue for therapeutic intervention [ , ] . in animal models of arthritis, specific inhibition of sil- r-mediated signalling by intra-articular injection of sgp effectively suppressed all parameters of disease severity [ ] . similarly, in animal models of colitis, application of a gp -fc fusion protein specifically neutralizating sil- r suppressed colitis activity and induced apoptosis of lamina propria t cells. these results suggest that a t cell activation pathway driven by the il- /sil- r complex supports chronic intestinal inflammation [ ] . collectively, these findings suggest that specific therapeutic targeting of sil- r may be explored as a novel strategy for the treatment of autoimmune uveitis. indeed, neutralizing antibodies against il- r have proven to be effective in uveitis [ ] . in the present study, the levels of stnfri and stnfrii were elevated . -fold and . -fold, respectively, compared with controls. our results might reflect the cellular attempt to antagonize the effect of tnf-α by cleavage of the surface receptors. similarly, several studies reported increased levels of stnfri and stnfrii in inflammatory and autoimmune diseases [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . moreover, stnfri and stnfrii levels correlated with disease activity [ , [ ] [ ] [ ] [ ] [ ] . our findings also suggest the preferential shedding of stnfrii in patients with autoimmune uveitis. these results are in agreement with previous studies that demonstrated higher levels of stnfrii than stnfri levels in the synovial fluid of patients with rheumatoid arthritis [ , ] . cellular responses to tnf-α are influenced by, amongst other factors, the extracellular concentrations of stnfri and stnfrii [ , ] . indeed, elevated concentrations of stnfrs have been demonstrated to inhibit the activity of tnf-α both in vitro and in vivo [ ] . the relative proportion of tnf-α to its soluble receptors in biological fluids has been proven to be important in a number of clinical conditions [ ] [ ] [ ] [ ] [ ] . diminished ratios of stnfri/tnf-α and stnfrii/tnf-α were observed in children with meningococcal septicaemia with a fatal outcome compared with survivors [ ] . increased joint destruction in polyarticular juvenile chronic arthritis may be related to the lower stnfri/tnf-α and stnfrii/tnf-α ratios [ ] . considering the inhibitory effects of the soluble receptors for tnf-α, we wondered whether the ratios of stnfrs/ tnf-α would be different among the disease groups, and be differentially distributed compared with the receptor levels. indeed, the ratios were significantly higher in patients with granulomatous uveitis associated with sarcoidosis and vkh disease than in patients with acute nongranulomatous uveitis associated with bd and hla-b -associated uveitis. these findings suggest that tnf-α would exert more destructive effects in the ocular inflammatory microenvironment of patients with acute nongranulomatous uveitis when compared with granulomatous uveitis and provides an explanation for the different clinical courses of these uveitis subgroups. this may also reflect different pathogenic mechanisms in the two disease groups. this study has several limitations regarding experimental methods and designs. firstly, our control group consisted of patients who had undergone elective cataract extraction with no prior history of uveitis. accordingly, the control patients were older than patients with uveitis. the relationship between age and human ah cytokine levels was recently investigated. increased concentration of proinflammatory and proangiogenic cytokines with age was demonstrated [ ] . although the control patients might have a proinflammatory state, the statistical analysis gave clear differences in comparisons with patients with uveitis. younger controls might have yielded differences with higher statistical significance. furthermore, analysis of ah samples was performed at one-time point during the course of disease. therefore, it was not possible to evaluate the effect of the interval between onset of inflammation and ah sampling and intensity of inflammation on the levels of the studied soluble cytokine receptors. animal models are needed to explore the effect of uveitis activity and to investigate the expression of these soluble cytokine receptors at different time points. nevertheless, with the use of a multiplex assay, we analyzed ah samples from patients with several specific clinical entities of autoimmune uveitis in parallel allowing measurements of these soluble cytokine receptors simultaneously in the same samples. in conclusion, our findings suggest that cd + t cells might be involved in the regulation of inflammation in granulomatous uveitis, particularly vkh disease and that active autoimmune uveitis might be associated with increased macrophage activation, since we observed elevated scd levels. granulomatous uveitis is characterized by significantly higher stnfrs/tnf-α ratios than acute nongranulomatous uveitis. finally, specific therapeutic targeting of sil- r-mediated signalling may represent a novel strategy for the treatment of autoimmune uveitis. to further investigate the relative importance of these soluble cytokine receptors, intervention functional studies in animal models are necessary. what was known before what this study adds • cd + t cells might be involved in the pathogenesis of granulomatous uveitis, particularly vogt-koyanagi-harada disease. • active autoimmune uveitis might be associated with increased macrophage activation, since we observed elevated scd levels. • granulomatous uveitis is characterized by significantly higher stnfrs/tnf-α ratios than acute nongranulomatous uveitis. the cytokine interleukin- and the chemokines ccl and cxcl are novel biomarkers of specific endogenous uveitic entities distinct cytokine and chemokine profiles in the aqueous of patients with uveitis and cystoid macular edema cytokine profiles in aqueous humor of patients with different clinical entities of endogenous uveitis differential cxc and cx c chemokine expression profiles in aqueous humor of patients with specific endogenous uveitic entities intraocular cytokine environment in active behçet uveitis expert panel recommendations for the use of anti-tumor necrosis factor biologic agents in patients with ocular inflammatory disorders longterm clinical outcomes in patients with refractory uveitis associated with behçet disease treated with infliximab targeting interleukin- in autoimmune uveitis soluble cytokine 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with il- and cxcl in patients with long-standing rheumatoid arthritis soluble macrophagederived cd is a marker of disease activity and progression in early rheumatoid arthritis proteolytic shedding of the macrophage scavenger receptor cd in multiple sclerosis soluble cd as a marker of macrophage activity in newly diagnosed patients with multiple sclerosis increased serum levels of soluble cd in patients with scleroderma serum levels of soluble cd in patients with systemic sclerosis the diagnostic significance of soluble cd and soluble interleukin- receptor alpha-chain in macrophage activation syndrome and untreated new-onset systemic juvenile idiopathic arthritis association of cd + macrophages and local production of soluble cd with decreased lymphocyte activation in spondylarthropathy synovitis soluble cd , a specific macrophage activation marker, is decreased by anti-tnf-α antibody treatment in active inflammatory bowel disease bosch-morell f. macrophages and uveitis in experimental animal models interleukin- and soluble interleukin- receptors in the synovial fluids from rheumatoid arthritis patients are responsible for osteoclast-like cell formation concentrations and origins of soluble interleukin receptor-alpha in serum and synovial fluid soluble il- receptor governs il- activity in experimental arthritis: blockade of arthritis severity by soluble glycoprotein blockade of interleukin trans signaling suppresses t-cell resistance against apoptosis in chronic intestinal inflammation: evidence in crohn's disease and experimental colitis in vivo circulating tnfri and tnfrii levels correlated with the disease activity score (das ) in rheumatoid arthritis circulating tumour necrosis factor alpha and soluble tumour necrosis factor receptors in patients with different patterns of rheumatoid synovitis increased levels of soluble tumor necrosis factor receptors in the sera and synovial fluid of patients with rheumatic diseases soluble tumor necrosis factor receptors in human inflammatory synovial fluids soluble tumour necrosis factor receptors stnfr and stnfr are produced at sites of inflammation and are markers of arthritis activity in behçet's disease circulating levels of tumor necrosis factor soluble receptors in systemic lupus erythematosus are significantly higher than in other rheumatic diseases and correlate with disease activity serum p and p tumour necrosis factor receptors as markers of disease activity in juvenile chronic arthritis differences in the profiles of circulating levels of soluble tumor necrosis factor receptors and interleukin receptor antagonist reflect the heterogeneity of the subgroups of juvenile rheumatoid arthritis tumour necrosis factor alpha and its soluble receptors in juvenile chronic arthritis tumor necrosis factor soluble receptors circulate during experimental and clinical inflammation and can protect against excessive tumor necrosis factor alpha in vitro and in vivo the j study group, et al. imbalance between tumour necrosis factoralpha and soluble tnf receptor concentrations in severe meningococcaemia imbalances between tumor necrosis factor-alpha and its soluble receptor forms, and interleukin- beta and interleukin- receptor antagonist in bal fluid of cavitary pulmonary tuberculosis increased synovial fluid levels of interleukin- , scd and stnf-rii/stnf-ri ratio delineate a cytokine pattern characteristic of immune arthropathies age-related proinflammatory and pro-angiogenic changes in human aqueous humor conflict of interest the authors declare that they have no conflict of interest.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -q qh kas authors: sadeghi, behnam; ersmark, bo; moretti, gianluca; mattsson, jonas; ringdén, olle title: treatment of radiculomyelopathy in two patients with placenta-derived decidua stromal cells date: - - journal: int j hematol doi: . /s - - -w sha: doc_id: cord_uid: q qh kas mesenchymal stromal cells may reverse acute inflammatory disorders. the placenta is important in feto-maternal tolerance. we have used placenta-derived decidua stromal cells (dscs) to treat graft-versus-host disease and found an immunomodulatory and anti-inflammatory effect. we here report the use of dscs in two patients with radiculomyelopathy. the first patient was a -year old man treated with parotidectomy and irradiation for lymphoma of the neck. following a yersinia infection, he developed a radiculomyelopathy in c /c and could not elevate his arms. the second patient was a -year old woman who was admitted months after allogeneic hematopoietic stem cell transplantation due to hemolysis, impaired sensorium below arcus, and difficulty in ambulation. following intravenous infusion of dscs ( × ( )/kg/infusion), the first patient could elevate his arms to the facial level. he experienced recurrent paralysis after months, and the efficacy of four additional dsc infusions, at subsequent occasions, were limited and transient. the second patient was treated with two doses of dscs ( × ( )/kg/infusion). after cell infusion, she was able to stand on one leg, sensation in the belly normalized, and she was discharged. these two cases suggest that dscs may be useful in the treatment of neuroinflammatory disorders. mesenchymal stromal cells (mscs) have strong immunomodulatory effects and have been widely used [ ] [ ] [ ] [ ] . we were first to use mscs for acute graft-versus-host disease (gvhd) [ , ] . mscs were used for gvhd, inflammatory disorders, encephalopathies, acute respiratory distress syndrome (ards), arthritis and hemorrhage cystitis [ ] [ ] [ ] [ ] [ ] [ ] [ ] . we have used placenta-derived decidua stromal cells (dscs) for acute and chronic gvhd, ards and hemorrhagic cystitis [ ] [ ] [ ] [ ] . the decidua plays an important role in feto-maternal tolerance [ ] . dscs have stronger immunomodulatory effect than bm-mscs and are easier to obtain than bm-mscs [ , ] . here we report two patients, treated with dscs for radiculomyelopathy. patient was a -years old german man who in october , due to extranodal, stage a, b cell lymphoma, on the left side of neck, was given gy irradiation in gy fractions. following yersinia enterocolitis in august , he got paresis in the shoulders and arms. in october , laboratory tests showed igg positivity and iga anti-yersinia antibodies. spinal fluid and serum showed monoclonal igg gamma chain antibodies against yersinia. it is not known from the patient's charts if yersinia growth was found in stool. peripheral electroneurography was normal. diagnosis was transverse myelitis. ivig and corticosteroids had no effect. plasma exchange was not performed. mri showed atrophy in c /c . he could not raise his arms. electrophysiological investigations showed severe neuropathy of the lower motor neurons from the spinal cord segment c /c to c /th . he had little sensitivity in both arms. reflexes for biceps, triceps, and brachioradialis were negative in both arms. reflexes and sensitivity were normal in the lower extremities. the diagnosis was atypical transverse myelitis. differential diagnosis including multiple sclerosis, myelin oligodendrocyte glycoprotein (mog) antibody-related disease, collagen diseases, antiphospholytic syndrome, vasculitis, sarcoidosis, paraneoplastic neurological syndrome could not be excluded. however, clinical manifestations and the physicians' judgment suggested atypical transverse myelitis. in december , patient was given cyclosporine, . mg/kg twice daily for weeks; to prevent immunization against dscs. cyclosporine had no effect on the paresis. dscs were manufactured as previously described in detail [ , ] . with ethical permissions ( / - - / , / - / , / - / ). he was given two doses, × dscs/kg i.v. week apart. on the day after, he could lift his arms to the level of his face for the first time in almost a year. after the second dose, he went home and improvement continued. afterwards, he could go shoping with his wife and carry the bags. electrophysiological investigation following dscs infusion showed some improvement with more frequent motor unit activity, especially in the right arm. following a α-streptococcal infection in january , he had recurrence of paresis in the arms. we speculate that the occurrence of transverse myelitis was due to infectioninduced inflammation in the site at c /c . he got treatment with two doses of dscs in may. after the first dose, he could elevate his left arm to arcus and his right arm up to the face. his strength was about % normal. he could open doors and hold a glass without assistance. one and weeks later, he was given additional infusions of dscs, but with no further improvement. from then on, there was an unchanged routine neurophysiological status. mri shows decreased volume at levels c and c . the neurophysiological examination showed loss of motor units, most pronounced on the left side and there were signs of re-innervation in the remaining motor units. the patient had no strength and could not carry bags. four months later, he could only raise his hands towards the waist. in september , he received an additional dose of dscs. after this, he has shown improvement and could lift his arms up to the waist. mri was unchanged. following further infusions of dscs on three occasions he had transient improvements, but the effect was not long lasting. patient was a -year old woman who underwent allogeneic hematopoietic stem cell transplantation (hsct) from an unrelated donor in july due to acute myeloid leukemia (aml) , . eight months after hsct, the patient had fever and a dat-positive hemolysis with hemoglobin of g/l, lactate dehydrogenase (ld) of . μkat/l (normal value < . for the age > ) and . % schistocytes in the blood streak. this may exclude thrombotic microangiopathy(tma). she had mild chronic gvhd with sicca. she had a subacute onset of ascending paresthesia starting in the feet and progressing proximally to the navel, including the perineum area. she developed an unsteady gate, weakness of the legs and reduced sensation in the anal canal. she had decreased sensibility for touch below the navel, all the way caudally to feet. the tendon reflexes were normal in the arms, but brisk in the legs (left patellar and achilles +, right patellar and achilles +). the muscle strength was normal in the arms, but slightly reduced in the legs (proximally and distally +). the toes were flexor on babinski's test. she could walk straight in the tandem gait test. romberg´s test was pathological. she needed a walker to walk > m. the cranial nerve examination was unrevealing. mri of the brain and entire spinal cord were normal and showed no gadolinium contrast enhancement in the spinal cord or the brain. the lumbar puncture showed only mononuclear cells/μl cerebrospinal fluid (csf), normal albumin and igg levels and no oligoclonal bands. serum and csf did not show evidence of epstein-barr virus, parvovirus, varicella zoster virus, hhv , cytomegalovirus, herpes simplex and , borrelia, enterovirus, jc virus, htlv or mycoplasma infection. csf had no leukemic cells. six days after onset of neurological symptoms, peripheral nerve electroneurography (eneg) was normal, but increased latency of motor evoked potentials in the lower legs upon cortical transcranial magnetic stimulation (tms), with normal response evoked in hand muscles. lumbosacral root tms stimulation elicited normal motor evoked potentials in the lower legs. somatosensory evoked potentials (sep) showed a clear increased latency of cortical sensory potentials upon stimulation of the tibial nerves, but normal responses upon stimulation of the median nerves. the results of the sep indicated a functional disorder of the spinal cord below c . she got prednisolone ( mg/day) and a dose of intravenous immunoglobulin (ivig, g) for the hemolysis. the neurological symptoms persisted on march , i.e. days after onset. she got dscs ( × cells/kg) with no complications days after neurological symptoms. the day after the dsc infusion, she had normal sensation on neurological examination and subjectively in the lower abdomen and legs. she could walk more steadily, had a normal romberg test and could keep balance standing on one leg. she was discharged and a week later got the second dsc infusion ( . × cells/kg). the patient had some residual paresthesia in the toes and subjective balance impairment but made long walks without aid. the tms showed a slight improvement in the latencies of motor evoked potentials in the legs upon cortical stimulation compared to the previous exam. four months after dscs therapy walking and sensation were normal. she resumed working months after treatment with dsc. the patient was examined with tms and seps every - months up to a year after the onset of neurological symptoms. seps were completely normal. tms, although significantly improved, showed some residual delay in the latency of motor evoked potentials to the legs. due to hemolysis, the patient continued with steroids, which were tapered after months. one year after infusion, prednisolone was discontinued and she has no more hemolysis. at the last follow-up, the patient is doing well with no neurological symptoms. in patient we speculated that the symptoms may be due to a previous irradiation and infection-induced inflammation. the investigation showed a clear pathological electromyelography pointing to radiculomyelopathy. the subjective symptomatology in patient indicated a thoracic myelitis. the objective pathological findings in the investigation were clear and confined to evoked potentials. both patients had a very dramatic response day after dsc infusion. patient could elevate his arms. patient could suddenly walk steadily. in patient the myelopathy re-occurred after months, and the effects of dsc were only transient. in patient , the effects were long-lasting. some differences between the two patients may explain the differences in outcome. most importantly, patient had myelopathy for a year, whereas patient had myelopathy for week. this is also experienced with gvhd, where dsc's effectively reverse acute gvhd [ ] , whereas in chronic gvhd only limited and transient effects were seen [ ] . although dscs appear to have a potent anti-inflammatory effect, it was not known whether the cells would be effective for myelopathy [ ] [ ] [ ] ] . the anti-inflammatory and immunomodulatory mechanisms by dscs in not fully understood. however, in vitro in mixed lymphocyte cultures direct contact with dscs and immune cells are needed for immunosuppression and an increase of regulatory t cells [ ] . interferon gamma, prostaglandin e , indoleamine dioxygenase and programmed death ligand- are probably involved in the mechanism because blocking of these structures using antibodies abrogated immunosuppression by dscs [ ] . infusion of allogeneic cells may cause immunization. a boy who was treated with dscs for epidermolysis bullosa developed multispecific anti-hla antibodies [ ] . anti-hla antibodies have not found in hsct patients treated with dscs [ ] . studies in mice have also shown that stromal cells induce immunization [ ] . to prevent immunization, the first patient was given cya. patient developed a subacute myelopathy at the thoracic spinal level. at this time, she also had a dat-positive hemolytic anemia and chronic gvhd with sicca reaction. the neurological examination and results of the electrophysiological testing with tms and sep indicated a clear functional disorder of the spinal cord despite normal results of imaging and csf analysis. this was presumed to have an autoimmune (chronic gvhd) basis. this is most probably the reason although paraneoplastic syndrome or tma cannot be definitely excluded. the occurrence of gvhd in the cns is debated, but the affection of the brain, and also the spinal cord (myelopathy), has been described [ , ] . causes other than gvhdinduced myelopathy (myelitis) were excluded by a thorough workup of possible viral and infectious factors in blood and csf, and also by ruling outside effects of drugs. based on the outcome in these two patients with paresis, further studies using dscs are warranted. dscs may also be investigated as a therapy in autoimmune and inflammatory neurological disorders such as myasthenia gravis and guillain-barré disease. cell therapy using allogeneic bone marrow mesenchymal stem cells prevents tissue damage in collagen-induced arthritis treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells therapy with stem cells in inflammatory bowel disease mesenchymal stem cells for treatment of therapy-resistant graft-versus-host disease tissue repair using allogeneic mesenchymal stem cells for hemorrhagic cystitis, pneumomediastinum and perforated colon mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing t-cell anergy treatment of acute respiratory distress syndrome with allogeneic adiposederived mesenchymal stem cells: a randomized, placebo-controlled pilot study treatment of severe chronic graft-versus-host disease with decidual stromal cells and tracing with ( )indium radiolabeling fetal membrane cells for treatment of steroid-refractory acute graft-versus-host disease successful reversal of acute lung injury using placenta-derived decidual stromal cells placenta-derived decidua stromal cells for treatment of severe acute graft-versus-host disease mother's little helpers: mechanisms of maternal-fetal tolerance stromal cells from term fetal membrane are highly suppressive in allogeneic settings in vitro placenta-derived decidua stromal cells for hemorrhagic cystitis after stem cell transplantation decidual stromal cells promote regulatory t cells and suppress alloreactivity in a cell contact-dependent manner immunogenicity of decidual stromal cells in an epidermolysis bullosa patient and in allogeneic hematopoietic stem cell transplantation patients immune-mediated myelopathy following allogeneic stem cell transplantation immune-mediated myelopathy after allogeneic marrow transplantation acknowledgements we thank gunilla tillinger for preparation of this manuscript. this work was supported by grants to or from the swedish cancer society (can / ), the swedish research council (k - x- - - ), the cancer society in stockholm ( ). or was the recipient of a distinguished professor award from the karolinska institutet. conflict of interest the author declares that they have no conflict of interest to report. key: cord- -hcd bwy authors: konstantinidis, charalampos; karafotias, achileas; eleftheropoulos, ioannis; delakas, dimitrios title: chronic prostatitis effectively managed by transurethral prostatectomy (turp) in a spinal cord injury male date: - - journal: spinal cord ser cases doi: . /s - - - sha: doc_id: cord_uid: hcd bwy introduction: spinal cord injury (sci), specifically suprasacral sci, results in high intravesical pressures, elevated post-void residual and urinary incontinence which are all risk factors for urinary tract infections (utis). the management of utis usually is conservative medical antibiotic treatment. however, recurrent utis in the sci patient population warrant further investigation. the method of urinary drainage (intermittent or indwelling urinary catheters, urinary diversion) and untreated complications of nlutd (vesicoureteral reflux, stone formation, chronic incomplete emptying of the bladder) are risk factors for recurrent utis (rutis). removal of these uti risk factors and improving urinary drainage are goals of urologic management; however, when conservative interventions do not succeed, surgery may be a viable solution in select cases of rutis. case presentation: we present a case of complicated persisting rutis and associated urethral discharge in a middle-aged sci male who manages his bladder with intermittent catheterization (ic). we detail the evaluation and management approach that leads to an eventual transurethral prostatectomy (turp) as a final solution for his rutis. fortunately, the surgical intervention was successful, and the patient is free of utis after years of follow-up. discussion: in sci male patients with rutis and suspected chronic prostatitis, turp may be a valuable treatment option once all predisposing factors have been remediated. neurogenic lower urinary tract (lut) dysfunction can be caused by injuries of the spinal cord (sci). high intravesical pressure, post-void residual and incontinence are the main consequences of this dysfunction and all of these conditions can cause urinary tract infections (utis). in addition, the potential complications of neurogenic urinary disorders (reflux, stone formation, incomplete emptying of the bladder), and the methods of urine drainage (intermittent or indwelling catheters, urinary diversion), contribute even more to utis. in persons with scis, all utis are considered as complicated ones and there is different microbiology as compared to the general population [ ] [ ] [ ] . recurrent infections of the urinary tract (ruti) usually are treated conservatively. when these methods fail, surgical treatment is an option. in order to proceed in surgery, a constant source of microorganisms and/or appropriate conditions for their proliferation should be present. sources of infections include calculi, fistulae or abscesses. favorable conditions for bacterial proliferation can occur under malignancies, foreign bodies or high post-void residual volumes [ ] . there are four clinical types of prostatitis: acute bacterial prostatitis, chronic bacterial prostatitis, chronic nonbacterial prostatitis/pelvic pain syndrome, and asymptomatic inflammatory prostatitis [ ] . common treatment means are antibiotics, alpha-blockers, hormonal treatment, antiinflammatory drugs and phytotherapeutic agents (plant extracts) [ ] . transurethral prostatectomy (turp) is suggested as a potential treatment option when other therapeutic approaches fail, but it is not widely used as such [ , ] . this report analyses the sequence of diagnosis and treatment for a case of complicated rutis in a middle-aged man with paraplegia and neurogenic lut dysfunction. the final therapeutic step for the patient was a turp, which resulted in the patient being free from rutis at his -year follow-up. this case involves a -year-old male with t ais a sci secondary to a car accident in [ , ] . his bladder management consisted of intermittent catheterization (ic) - times per day in addition to using a condom catheter for concomitant urinary incontinence, especially during his wheelchair basketball sports activity. the patient had a -year history of - escherichia coli rutis per year accompanied by high fever with chills and associated temporary urethral secretion. with the onset of the uti symptoms, his urinary incontinence ceased temporarily and the following day he would notice a thick, purulent urethral discharge. repeated renal ultrasound evaluation did not reveal stones, renal dilatation or parenchymal abnormalities. due to the urethral discharge, a periurethral source of infection/abscess was suspected. a urethral swab/secretion culture revealed > , e. coli cfu/hpf. transrectal prostate ultrasound (trus) and lower abdominal magnetic resonance imaging (mri) were conducted and both tests were without any significant findings. urodynamics in the absence of uti demonstrated a stable bladder pressure system with neurogenic detrusor underactivity accompanied by stress urinary incontinence due to urethral sphincter deficiency. bladder compliance was normal at . cystourethroscopy demonstrated a normal urethra and bladder; however, there was an incidental hypertrophic verum montanum (fig. ) . given the limited findings yet rutis, it was hypothesized that the hypertrophic verum montanum was acting as a source of temporary obstruction during a prostatitis inflammation process. after thorough discussion, the individual underwent endoscopic removal of verum montanum (fig. ) . urodynamic studies were repeated post verum montanum resection demonstrating similar neurogenic detrusor underactivity and urethral sphincter deficiency with an abdominal leak point pressure (alpp) at cmh o. clinical recommendations for management consisted of continuing self-ic - times per day and avoidance of valsalva/straining maneuvers for bladder emptying. our goal to avoid rutis was to maintain a low-pressure reservoir that was emptied completely via ic and to avoid valsalva/strain voiding to minimize pressure in the prostatic area. due to the failure of appropriate culture-sensitive antibiotics to eradicate the rutis and the new findings of prostatic calcifications (fig. ) , a turp was recommended. an uneventful turp was performed months after the last ruti, and pathology revealed chronic inflammatory elements and small abscesses in the prostate gland (fig. ) . the individual has been followed for the past years and he has been free of uti symptoms and urethral discharge after the turp. urodynamic testing was repeated year after the turp and demonstrated stable bladder pressures with neurogenic detrusor underactivity and stress urinary incontinence with urethral sphincter deficiency but now with a lower alpp of cmh o. the lower alpp can be explained by the decreasing of "urethral resistance" after turp. the individual continues to manage his bladder by ic and avoid straining. to manage his stress incontinence due to sphincter deficiency, he continues to use a condom catheter. the turp did not have any adverse effects on his bowel or sexual function. the individual also uses intracavernosal alprostadil injections for his neurogenic erectile dysfunction with good results. to our knowledge, this is the first reported case of successful turp treatment of chronic prostatitis in an individual with sci. turp has been reported as a therapeutic approach to prostatitis in six case studies from to [ ] . one-hundred and ten men were included in the series and none of them had scis. in the series, the mean age at diagnosis was years ( - years) and symptom duration ranged from to months, for patients. prior to turp, patients were treated with conservative antibiotic therapy and alpha-blockers. out of patients, ( %) were referred as cured, ( %) as improved and ( %) with no change. only one patient's symptoms were quantified under the international prostate symptom score (ipss), in which improvement suggested, with a score from downgraded to postoperatively [ ] . chronic bacterial prostatitis is a relatively rare male condition that presents with vague recurrent uti symptoms. the prevalence of prostatitis-like symptoms is only %. due to the high relapse rate, it can have a significant negative impact on a man's quality of life. while the treatment of acute bacterial prostatitis should definitely rely on antibiotics, there are no clear recommendations for the management of chronic prostatitis. our case report documents a role for surgical intervention when conservative medical therapy for chronic prostatitis is ineffective. surgical turp, in this case, resolved the source of infection and also lowered voiding pressures. by "radical" turp (almost complete removal of the prostate adenoma), the microabscesses that were acting as a "nest" for the microbes were eradicated [ ] [ ] [ ] . additionally, the turp assisted with low-pressure conditions with limited urethral resistance [ ] . in neurogenic male patients with ruti symptoms felt to be due to chronic prostatitis, turp may be a valuable treatment option. all predisposing factors such as foreign body, urinary tract stones, altered bladder pressures and high residual urine must first be remediated. once these efforts fail, turp may be a valuable treatment option. conflict of interest the authors declare that they have no conflict of interest. microbiology of urinary tract infections-microbial agents and predisposing factors adult neurogenic lower urinary tract dysfunction and intermittent catheterisation in a community setting: risk factors model for urinary tract infections eau guidelines on urological infections. european association of urology liatsikos e. α-blockers for the treatment of chronic prostatitis in combination with antibiotics pathogenesis of urinary tract infections. host defenses surgical management of recurrent urinary tract infections: a review transurethral resection of the prostate in recurrent acute bacterial prostatitis american spinal injury association. international standards for neurological classifications of spinal cord injury international standards for neurological and functional classification of spinal cord injury surgical therapy of prostatitis: a systematic review transurethral resection of the prostate for chronic bacterial prostatitis the role of transurethral prostatectomy in chronic prostatitis endosurgical alternatives in the treatment of the chronic prostatitis syndrome: part ii. the transurethral procedures patients with prostatic inflammation undergoing transurethral prostatic resection have a larger early improvement of storage symptoms informed consent written informed consent was obtained from the patient. the patient consented of the publication of the data.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -cg i bae authors: stoots, sarah abramson; lief, lindsay; erkan, doruk title: clinical insights into diffuse alveolar hemorrhage in antiphospholipid syndrome date: - - journal: curr rheumatol rep doi: . /s - - - sha: doc_id: cord_uid: cg i bae purpose of review: diffuse alveolar hemorrhage (dah) is a rare but devastating manifestation of antiphospholipid syndrome (aps) patients with or without other systemic autoimmune diseases. data regarding diagnosis and treatment are limited to case series. we review diagnostic and therapeutic strategies employed in aps patients with dah and discuss our experience in managing these complex patients. recent findings: pulmonary capillaritis likely contributes to the pathogenesis, however is only observed in half of the biopsies. corticosteroids induce remission in the majority of patients, however almost half recur and require a steroid-sparing immunosuppressive to maintain remission. cyclophosphamide- or rituximab-based regimens achieve the highest remission rates ( %); other strategies include intravenous immunoglobulin, plasmapheresis, mycophenolate mofetil, and/or azathioprine. summary: given the rarity of dah in aps, treatment is guided by interdisciplinary experience. why certain patients achieve full remission with corticosteroids while others require immunosuppressive agents is unknown; future research should focus on the pathophysiology and optimal management. antiphospholipid syndrome (aps) is a systemic autoimmune disease characterized by thrombosis and obstetric morbidity in patients with persistently elevated antiphospholipid antibodies (apl) (lupus anticoagulant test [la] , anticardiolipin antibodies [acl], or anti-β -glycoprotein-i antibodies [aβ gpi]) [ ] . thrombosis in aps can be grouped as venous, arterial, or microvascular. diffuse alveolar hemorrhage (dah) is a rare and potentially fatal microvascular manifestation of apl in which red blood cells (rbcs) leak from the alveolar capillaries into the intra-alveolar space [ ••] . patients present with diffuse pulmonary infiltrates, hypoxemic respiratory failure, hemoptysis, and anemia. diffuse alveolar hemorrhage in aps is a major therapeutic challenge, particularly in this patient population, who are hypercoagulable and often on life-long anticoagulation. thus, the goal of this review is to offer insights into the diagnosis as well as the management of apl-positive patients with dah. how common is diffuse alveolar hemorrhage in antiphospholipid syndrome? (ards) , and pulmonary artery thrombosis had a combined prevalence of only . % [ ] . one institution reported a % frequency of apl-associated dah among primary aps patients followed over years [ ••] . in catastrophic aps (caps), dah is identified in % of patients [ ] . the nonspecific clinical and radiographic findings and inconsistent hemoptysis in dah, as well as the requirement for an invasive diagnostic procedure, i.e., bronchoscopy, contribute to dah being underdiagnosed [ ] . thus, the true frequency is likely higher than what is reported in the literature [ , ] . what is the etiopathogenesis of diffuse alveolar hemorrhage in antiphospholipid syndrome? the defining feature of dah is injury to the lung's microcirculation: the capillaries, arterioles, and venules lining the alveoli. three major histologic descriptions of dah, not specific for aps, are ( ) pulmonary capillaritis, ( ) bland alveolar hemorrhage, and ( ) diffuse alveolar damage [ ••] . pulmonary capillaritis is defined by neutrophilic infiltration of the perivascular interstitium (alveolar septae) of the capillaries. a vicious cycle of neutrophilic apoptosis, proteolytic enzyme disbursement, fibrin deposition, and recruitment of inflammatory cells culminates in fibrinoid necrosis of the alveolar and capillary walls. once a critical mass of neutrophils and aforementioned debris disrupt the alveolar-capillary basement membrane, rbcs extravagate into the alveoli and interstitium. hemosiderin-filled macrophages begin to accumulate - h after the initial vessel injury [ ••, , ] . bland alveolar hemorrhage is characterized by intra-alveolar rbcs without adjacent capillary inflammation or destruction. diffuse alveolar damage, the primary lesion in ards, is defined by intra-alveolar edema and hyaline membranes filled with debris lining the alveoli with sparse inflammation [ ••, , ] . the specific mechanism behind dah in aps is not well defined. the most frequently proposed theory is antibody-mediated pulmonary capillaritis, possibly caused by apl-induced endothelial cell activation and upregulation of adhesion molecules [ ••] . another postulated mechanism is in situ microvascular thrombosis generating alveoli hemorrhage [ ] . the detailed discussion of mechanisms of apl-mediated dah and micro-thrombosis can be found elsewhere [ , ••, ] ; however, recent data suggest (a) apl activation of the mammalian target of rapamycin (mtor) kinase leads to endothelium proliferation and consequent vasculopathy, which is evident in apl-associated nephropathy, and (b) complement activation is an important contributor to apl-mediated thrombosis; however, complement deposition is rarely seen on lung biopsy [ ] [ ] [ ] . patients with dah present with hypoxemia, hemoptysis, pleuritic chest pain, and cough. dyspnea is the most common symptom, and fever may be present. severity ranges from asymptomatic radiographic abnormalities to respiratory failure. hemoptysis may be initially absent in a third of patients, because the total alveolar capacity can hold a significant pool of blood in distal airways [ ••] . acute respiratory distress syndrome may develop but is more common in caps [ ] [ ] [ ] . recurrent bleeding episodes can lead to mild to moderate (rarely severe) interstitial fibrosis and restrictive lung disease [ , , ] . development of lung hyperinflation and airflow obstruction is rare but has been reported [ , ] . although pulmonary hypertension in aps is primarily a result of chronic pulmonary thromboembolism and vasculopathy, dah without embolic disease can also elicit pulmonary hypertension [ , , ] . laboratory findings often demonstrate anemia, leukocytosis, and elevated erythrocyte sedimentation rate as well as creactive protein; however, the absence of these findings does not exclude dah. imaging may show diffuse or patchy alveolar opacities. bilateral ground glass opacities on chest computed tomography (ct) are classically seen. the differential diagnosis for dah includes infections, medications, systemic vasculitis, heart failure, uremia, and coagulopathies; a detailed review of differential diagnosis can be found elsewhere [ ••] . infection is particularly important to exclude. an opportunistic infection may present identically to an autoimmunemediated pulmonary disease but the two therapies counteract one another-the former requires a robust immune system and the latter necessitates immunosuppression [ ] . pneumonia inciting a dah flare requires concurrent treatment [ ] . the white blood count may be normal or elevated in both infectious and apl-related hemorrhage. over-the-counter medications, herbal supplements, illicit drugs, toxins, and prescribed medications have been found to induce pulmonary hemorrhage. culpable medications, (though rarely reported) include propylthiouracil, diphenylhydantoin, amiodarone, mitomycin, d-penicillamine, sirolimus, methotrexate, haloperidol, nitrofurantoin gold, all-transretinoic acid, bleomycin, montelukast, zafirlukast, inhaled cocaine, hydralazine, and infliximab [ ••, ] . thus, it is important to review patients' medications for offending agents that may accelerate dah in apl-positive patients. during an initial presentation of dah, a thorough work up to evaluate for other connective tissue disorders and systemic vasculitis should be undertaken. cytoplasmic and perinuclear anti-neutrophil cytoplasmic antigens (anca) (anti-proteinase [pr ] and anti-myeloperoxidase [mpo]) can help evaluate for granulomatosis with polyangiitis, microscopic polyangiitis, and eosinophilic granulomatosis with polyangiitis. antinuclear antibodies (ana), double-stranded dna (dsdna), complement and levels, urinalysis, creatinine, and anti-glomerular basement membrane antibodies (anti-gbm) can help evaluate for sle and goodpasture's syndrome [ ] ; however, at times, lupus pneumonitis may be indistinguishable from dah [ ] . cardiac echocardiogram should be obtained to exclude heart failure given the dyspnea and bilateral ground glass opacities resemble pulmonary edema, and to assess for valvular pathology. additional imaging to exclude pulmonary embolism is often performed. pulmonary function tests (pfts) are rarely performed due to the difficulty patients with dyspnea have in completing the necessary maneuvers. however, if done, pfts may show restriction; and the diffusing capacity for carbon monoxide (dlco) may be increased due to blood in the alveolar spaces, which can absorb additional carbon dioxide [ ••, ] . bronchoscopy with bronchoalveolar lavage (bal) is critical in patients with aps who present with respiratory failure, dyspnea, or pulmonary infiltrates to diagnose or exclude dah and infection. persistent and increasing bloody returns on three sequential bal sample aliquots support a diagnosis of dah. neutrophilic predominance and high numbers (greater than %) of hemosiderin-laden macrophages are present on bal cytology. of note, increasing bloody returns may precede the hemosiderin-laden macrophages during acute hemorrhage. yet these macrophages may persist in bal fluid weeks to months after alveolar erythrocytes have vanished [ ] . lavage specimens should be sent for bacterial, mycobacterial, and fungal stains and cultures, fungal markers, and viral polymerase chain reaction (pcr) tests. an extensive list of infectious pathogens is reviewed elsewhere, but includes pneumocystis jiroveci, legionella, mycoplasma, nocardia, leptospirosis, influenza a and b, respiratory syncytial virus, and adenovirus [ ] . transbronchial biopsies during bronchoscopy are not usually recommended [ ••] . surgical lung biopsy (thoracoscopic or open) is not routinely performed given the risk-benefit ratio; however, surgical biopsy may be indicated in certain circumstances when there is persistent diagnostic uncertainty. pathologic results of lung biopsy may reveal either pulmonary capillaritis, bland hemorrhage, or diffuse alveolar damage. pulmonary capillaritis is particularly associated with autoimmune or inflammatory diseases; in a review of lung biopsies of diffuse pulmonary hemorrhage patients (vasculitis-and connective disease-associated dah in and four cases, respectively), capillaritis was present in % of the specimens; however, none of these patients had aps [ ] . causes of bland alveolar hemorrhage include coagulopathies, mitral stenosis and regurgitation, congestive heart failure, and drug-induced; also, it has been reported in aps patients with dah [ ••, , ] . diffuse alveolar damage is the classic histology observed in ards but also evident in infections such as legionella, mycoplasma, and pneumocystis jiroveci, lymphangioleiomyomatosis, radiation therapy, bone marrow transplant complications, and aps-associated dah [ ••] . what is the optimal management of diffuse alveolar hemorrhage in antiphospholipid syndrome? in addition to supportive care, patients are treated with highdose corticosteroids to induce remission during the acute phase of hemorrhage. anticoagulation has not been shown to be beneficial in preventing or treating dah in these patients. anticoagulants are usually temporarily discontinued during the initial bleeding episode and cautiously restarted depending on the patient's clinical stability. many patients require a steroid-sparing agent to achieve remission. cyclophosphamide, rituximab, mycophenolate mofetil, azathioprine, plasmapheresis, and intravenous immunoglobulin (ivig) have been used with varying degrees of success (further discussed in the "literature review" section). our management approach is also described in greater detail in the "our experience" section. we identified aps cases with dah (primary or associated with another systemic autoimmune disease, with or without catastrophic aps) through a literature review (pubmed medline search; english only) of publications between january and december (eight case series and case reports). pubmed search keywords included: "antiphospholipid," "alveolar hemorrhage," "pulmonary hemorrhage," "catastrophic antiphospholipid syndrome," "dah," and "pulmonary capillaritis." references of prominent articles were also reviewed. catastrophic aps was present in cases; we only included their biopsy results in the assessment of pathology findings but otherwise excluded those patients from the analysis (as this review is not focused on caps management) [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . a case series of patients did not report individual treatment and remission data, and thus, these patients were excluded from the outcome analysis; however, we reported their outcomes separately [ ] . while assessing the treatment response in the remaining cases, we defined "remission" or "response" as no or less than mg prednisone daily (or equivalent corticosteroid regimen) to maintain remission. for patients who received multiple immunosuppressive medications started non-simultaneously, we included each medication (excluding corticosteroids) in our analysis individually (positive response versus no response). for a small number of patients who received multiple immunosuppressive medications started simultaneously, we included the combination in our analysis (positive response versus no response, excluding corticosteroids). because we used the most current and final information provided to gauge remission or treatment response, deceased patients were not included as treatment responders. when the data was incomplete, we exercised clinical judgment to best categorize the cases. seventy-nine apl-positive patients with dah (excluding caps patients) were mostly males (n = ; %), often caucasian in their fourth or fifth decade of life (mean age + ) , suggesting the possibility of a genetic disposition. eleven patients ( %) had sle; however, some case series excluded patients with sle [ , , ••, , ] . eleven of of patients in a small case series were current or former smokers [ ••] . in a retrospective study of aps patients, compared to aps patients without dah, patients with dah were more likely to have mitral valve disease, pulmonary hypertension, skin involvement (livedo reticularis/racemosa or chronic ulcers), obstetric morbidity, and central nervous system disease [ ] . diffuse alveolar hemorrhage was the first presentation of aps in / ( %) patients [ ••, , - ] ; three out of patients in one case series were diagnosed with aps only after they presented with dah [ ••] . yet, two other reviews of and patients noted a median dah onset of . and . years, respectively, after the diagnosis of aps [ , ] . furthermore, many patients have a delay in diagnosis [ , ] , e.g., a -year-old female visited her primary care doctor and emergency room for multiple episodes of hemoptysis throughout year before the eventual diagnosis of aps [ ] . viral upper respiratory tract infections or bacterial pneumonia were both reported to incite episodes of alveolar hemorrhage [ ••, ] . while periods of supratherapeutic international normalized ratio (inr) could pre-dispose patients to alveolar hemorrhage, some patients had alveolar hemorrhage while off anticoagulation, and the vast majority of published cases revealed sub-therapeutic or therapeutic inr on presentation. four patients did develop recurrent dah with a supratherapeutic inr and one patient's first episode of dah began weeks after starting warfarin [ ••, , , ] . half of patients ( %) were on anticoagulation (typically warfarin) either before or after the diagnosis of dah, but true prevalence of anticoagulation use is likely higher than reported given the limitations of these studies. pulmonary capillaritis was seen more frequently than microvascular thrombosis. across the cases included in our outcomes analysis, patients ( %) ultimately achieved remission, / ( %) with corticosteroids or supportive care alone (fig. ) , and / ( %) with immunosuppressive agent(s). at one institution, ( %) of patients achieved complete and sustained remission; interestingly, four of these patients had a mild form of dah and reached remission without even corticosteroid treatment (details not reported). in the same case series, three ( %) patients relapsed within a median time of months [ ••]. another large case series (n = ) demonstrated poorer remission rates: % achieved complete remission and % were alive but remained on high-dose corticosteroids after to years follow up; % of all patients experienced recurrent disease [ ••] . based on our review, of patients ( %) experienced recurrent disease, which may underestimate the true relapse rate, since many groups did not report long-term follow up (mean follow up was months; however follow up data was not provided for patients). symptoms often reappeared during corticosteroid tapering, and many patients were never able to discontinue prednisone. we found the mortality rate among all cases as %. this was similar to published rates in patients with dah secondary to numerous etiologies: % during an acute episode and % among those who survived their initial hospitalization [ ] . reported mortality in sle-associated dah has ranged widely from % in two studies up to %, %, and % in others [ ] [ ] [ ] [ ] [ ] table illustrates major therapeutic interventions utilized a n d s u b s e q u e n t c l i n i c a l r e s p o n s e . i n g e n e r a l , cyclophosphamide-or rituximab-based regimens achieved the highest remission rates (approximately %), which aligns with the largest case series reported to date [ ••] . intravenous immunoglobulin (ivig) and plasma exchange together with other immunosuppressive agents demonstrated similar treatment responses (approximately %), whereas mycophenolate mofetil and azathioprine use resulted in lower remission rates (approximately %). given the efficacy of cyclophosphamide in granulomatosis with polyangiitis and other pulmonary vasculitides, this medication was generally utilized initially in the management of apl-associated dah [ ] . patients unable to achieve remission with cyclophosphamide ultimately died, remained on high-dose corticosteroids, or responded to rituximab or ivig [ ••, , , ••, ] . recent papers have shown success with rituximab, an anti-cd monoclonal antibody that targets a surface protein expressed on b-lymphocytes, the precursor of aplproducing plasma cells. for instance, the first patient with primary aps treated with rituximab was a -year-old male who had been hospitalized times over months for recurrent dah; after unsuccessful treatment with azathioprine, ivig, cyclophosphamide, and plasmapheresis, two doses of rituximab ( g each weeks apart) led to a sustained remission over years of follow up [ ] . others have reported similar success with follow up ranging from to years [ ••, ] . several authors have achieved remission using ivig, both acutely to control an active disease flare and long-term with monthly maintenance infusions. for instance, janowiak et al. fig. outcomes in diffuse alveolar hemorrhage. includes two patients whom initially achieved remission but then flared without further remission. includes three patients whom achieved full or partial remission with cyclophosphamide but later died of ( ) sepsis, ( ) bone marrow transplantation complications, and ( ) diffuse alveolar hemorrhage flare when cyclophosphamide was discontinued [ ••, ] , ] ; however, the combination of rituximab and mycophenolate mofetil resulted in complete remission in two patients [ ••] . none of the six patients on monotherapy azathioprine in the largest case series attained remission [ ••] , and others reported similar outcomes [ , , , ] . hydroxychloroquine was used as monotherapy in only two cases (one resulted in remission and the other treatment failure) and demonstrated intermittent success when utilized as an adjunct agent [ ••, ••, , , ] . in patients with clinically significant apl profiles (persistently positive la test and/or moderate-to-high titer acl/aβ gpi igg/m), when imaging studies are suggestive of dah, bronchoscopy is important during initial or ambiguous presentations. during the initial diagnostic assessment, we also obtain chest ct with contrast as pulmonary embolism can present with hemoptysis. in patients with aps and recurrent, welldocumented dah and without suspicion for additional underlying disease or infection, we generally do not perform repeat bronchoscopy. we do not routinely recommend lung biopsy due to a s s o c ia t e d r is k s i n p a ti e n t s w h o a r e g e n e r a l l y anticoagulated and at high risk for thrombosis. lung biopsy is considered if the etiology of the dah is unclear from the serologic work up and an accurate diagnosis cannot otherwise be made. if performed, the pathologist should perform immunofluorescent staining to assess for immune-complex and complement deposition, and tissue cultures should be sent as reviewed above. of note, if the clinical scenario and laboratory tests suggest aps, the absence of capillaritis or microthrombosis on biopsy should not preclude the aps diagnosis. minor hemoptysis presents a diagnostic dilemma when it develops in a patient with aps on warfarin. is the warfarin to blame? we urge providers to consider dah on the differential to avoid missed and delayed diagnoses. and in those with documented aps, high clinical suspicion for dah is necessary, as documented cases of hemoptysis incorrectly attributed to warfarin overdose rather than a first occurrence of alveolar hemorrhage have been reported [ ] . in addition, when patients do not yet carry the dah diagnosis, symptoms may be falsely attributed to respiratory infections. given the rarity of the disease, lack of controlled studies, limited literature regarding treatment, and heterogeneity, there is no "one size fits all" approach to treatment. in our experience, two different presentations of apl-positive patients with dah exist: (a) acute-with moderate to severe pulmonary hemorrhage (with varying degrees of respiratory failure) requiring hospitalization and (b) chronic-with mild hemoptysis, dyspnea, and/or positive imaging findings, usually seen as an outpatient. patients in both settings often require steroids for disease control and a steroid-sparing immunosuppressive for maintenance therapy, as we discuss below. in hospitalized patients, supportive care is a mainstay of acute management, ranging from supplemental oxygen to mechanical ventilation, along with as-needed transfusions of blood products. empiric antibiotic therapy is often coadministered with corticosteroids at initial presentation and subsequently if a patient clinically decompensates. of note, immunosuppressed aps patients with dah are especially susceptible to pulmonary infections (typically bacterial), which can manifest with hemoptysis. we do not routinely treat with antiviral or antifungal antibiotics unless laboratory or culture data suggest a particular pathogen. high-dose corticosteroids (intravenous methylprednisolone - mg daily for days, followed by oral prednisone mg/kg daily in divided doses (maximum mg day)) are generally the primary treatment during the acute phase. due to high mortality in these patients, corticosteroids are rapidly initiated even when diagnostic tests are still pending. for patients with life threatening or ventilator-dependent dah, usually in the setting of caps, we also use plasma exchange and/or intravenous immunoglobulin [ ] . anticoagulation therapy is temporarily discontinued during the acute severe bleeding episode; however, the risks and benefits of stopping anticoagulation should be carefully calculated in apl-positive patients with/without history of thrombosis. when stopped, we recommend restarting anticoagulation as soon as the acute bleeding is controlled. in cases with concurrent pulmonary embolism and hemorrhage, starting anticoagulation and corticosteroids simultaneously may be required [ ] . a multidisciplinary team approach should appraise the risks and benefits, i.e., timing and dosage of safe anticoagulation [ ] . in apl-positive patients without a history of thrombosis presenting with dah, anticoagulation does not treat or prevent dah and we do not recommend anticoagulation for treatment purposes. however, we recommend continuing prophylactic dose anticoagulation to prevent thrombosis in hospitalized patients. in an outpatient setting with mild dah in the absence of respiratory compromise, we recommend initiating oral prednisone (or increasing the dose to) mg/kg daily to a maximum of mg daily. as discussed above, in patients with suspected new-onset dah, urgent referral to a pulmonary team for confirmatory bronchoscopy is required. for patients with known dah and recurrent albeit mild symptoms, oral corticosteroids with close follow up is often sufficient. both in an inpatient and outpatient setting, in addition to steroids, we recommend starting steroid-sparing immunosuppressive agents as, based on our experience, the risk of recurrence is relatively high during corticosteroid tapering. our first-line therapy is generally mycophenolate mofetil (quickly accelerating the dose to g/day) with or without rituximab ( mg twice weeks apart), once infectious etiologies have been excluded. we add rituximab to mycophenolate mofetil especially in apl-positive patients with hematologic and/ or other microthrombotic manifestations, e.g., thrombocytopenia or livedoid vasculopathy, and also in patients hospitalized with relatively severe hemorrhage. after the dah is controlled, we decrease the prednisone dose with close monitoring, usually by - mg per month. we do not use cyclophosphamide as first-line therapy due to the high incidence of treatment-associated toxicity. in cases of recurrent dah on mycophenolate mofetil, we add rituximab depending on the other apl-manifestations discussed above. no data exist on the long-term management of rituximab in aps patients with dah; we generally do not continue rituximab if there is full recovery. if patients have no response or recurrence of dah despite the combination of mycophenolate mofetil and rituximab, we next add ivig. we have treated several patients who achieved remission with the addition of ivig g/kg over - days, every - months, to mycophenolate mofetil (with or without a history of rituximab use) (unpublished data). in all apl-positive patients with dah, despite the lack of clinical data, we add hydroxychloroquine ( - mg daily) and a statin (e.g., atorvastatin - mg/day) to the treatment regimen. hydroxychloroquine reduces the risk of thrombosis in experimental apl/aps models and lupus patients; similarly, statins ameliorate the proinflammatory and prothrombotic markers in apl-positive patients [ ] [ ] [ ] [ ] . we do not understand why certain patients experience a single episode that clears with corticosteroids, while others require long-term immunosuppressive agents. based on our experience, patients may continue to have abnormalities on imaging despite resolution of symptoms. some of these patients, whose symptoms of dah have resolved, but have persistent opacities on imaging, may have recurrence of mild hemoptysis during upper respiratory tract infections or when their inr is supratherapeutic. in asymptomatic patients with persistent imaging findings, we continue the immunosuppressive agents while tapering and ultimately discontinuing prednisone as discussed above. monitoring for recurrence can be challenging, particularly when patients are asymptomatic with normal laboratory findings. for patients on chronic immunosuppression for prior hemorrhagic episodes, we recommend obtaining chest radiograph and pulmonary function tests including dlco yearly or with changes in respiratory symptoms. in our experience, patients with dah (without caps) have lower mortality rates than those reported in the literature; we attribute this to publication bias for this rare condition. diffuse alveolar hemorrhage is a rare yet potentially fatal complication of aps, in which red blood cells leak from the alveolar capillaries into the intra-alveolar space. pulmonary capillaritis, along with antibody-mediated endothelial proliferation, likely contributes to its pathogenesis, yet is not always observed pathologically. diffuse alveolar hemorrhage typically presents with hypoxemia and subsequent respiratory failure, anemia, hemoptysis, and diffuse radiographic pulmonary infiltrates. given the rarity of pulmonary hemorrhage in aps, no randomized trials exist. treatment is guided by case reports, case series, and inter-disciplinary experience. while anticoagulation is standard of care for the treatment and prevention of thrombotic complications of aps, its use is contraindicated during acute dah episodes and is not efficacious in preventing dah. in addition to supportive care, high-dose corticosteroids are the initial treatment for dah in aps. the majority of patients require an immunosuppressive steroid sparing agent; we generally use mycophenolate mofetil with or without rituximab as a first-line treatment strategy, and add ivig, plasmapheresis, and cyclophosphamide in patients with limited response. international consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (aps) diagnosis and management of the antiphospholipid syndrome intraalveolar haemorrhage in the anticardiolipin antibody syndrome antiphospholipid syndrome: clinical and immunologic manifestations and patterns of disease expression in a cohort of , patients one of the two largest and recent case series with a thorough overview of dah presentation catastrophic antiphospholipid syndrome (caps): descriptive analysis of patients from the international caps registry nonthrombotic manifestations of antiphospholipid syndrome: away from thrombosis? catastrophic antiphospholipid syndrome. clinical and laboratory features of patients unusual manifestations of the antiphospholipid 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systemic lupus erythematosus predictors of mortality in diffuse alveolar haemorrhage associated with systemic lupus erythematosus alveolar hemorrhage in systemic lupus erythematosus diffuse alveolar hemorrhage and systemic lupus erythematosus. clinical presentation, histology, survival, and outcome possible primary antiphospholipid syndrome" with concurrent diffuse alveolar hemorrhaging and libman-sacks endocarditis mimicking catastrophic antiphospholipid syndrome rituximab induces resolution of recurrent diffuse alveolar hemorrhage in a patient with primary antiphospholipid antibody syndrome mortality in the catastrophic antiphospholipid syndrome: causes of death and prognostic factors in a series of patients a -year-old man with hypoxemia and bilateral upper-lobe predominant ground-glass infiltrates on chest imaging mcmaster rare-bestpractices clinical practice guideline on diagnosis and management of the catastrophic antiphospholipid syndrome primary antiphospholipid syndrome associated with diffuse alveolar hemorrhage and pulmonary thromboembolism the challenge of bleeding in antiphospholipid antibody-positive patients hydroxychloroquine reverses thrombogenic properties of antiphospholipid antibodies in mice global effects of fluvastatin on the prothrombotic status of patients with antiphospholipid syndrome effect of antimalarials on thrombosis and survival in patients with systemic lupus erythematosus antithrombotic effects of hydroxychloroquine in primary antiphospholipid syndrome patients publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -qqrv npv authors: grodzinsky, ewa; sund levander, märta title: history of the thermometer date: - - journal: understanding fever and body temperature doi: . / - - - - _ sha: doc_id: cord_uid: qqrv npv the temperature of the human body has been used as a diagnostic sign since the earliest days of clinical medicine. the earliest thermal instruments were developed during the sixteenth and seventeenth centuries. in , it was suggested that the melting point of ice and the boiling point of water should be the standard. the most common scales today are the fahrenheit, centigrade, and the kelvin scales. since the earliest days of medicine, physicians have recognized that the human body can exhibit an abnormal rise in temperature, usually defined as fever, as an obvious symptom of illnesses. in , wunderlich established that the temperature in a healthy person is constant and that variation of temperature occurs in disease. the allbutt thermometer was the first practical device to become commercially available. the technology has then improved to provide highly accurate devices, for example, thermal imaging; its use is still growing in medicine. the earliest thermal instruments were developed during the sixteenth and seventeenth centuries. these simple instruments were constructed so as to trap air in glass tubes with the open end of the tube submersed in a reservoir of water. these open thermometers were termed thermoscopes. in , galileo used wine instead of water and was one of the first to use an alcohol thermometer. it was, of course, found that when carrying such a device up a mountain to a different altitude that the level in the tube was affected by the changing atmospheric pressure. these devices illustrated changes in sensible heat, before the concept of temperature had been recognized. while it is sometimes claimed that galileo was the inventor of the thermometer, what he actually produced was a thermoscope. he did discover that glass spheres filled with aqueous alcohol of different densities would rise and fall with changing temperature. today, this is the principle of the galilean thermometer, which is calibrated with a temperature scale. the first illustration of a thermoscope showing a scale, which therefore can be described as a thermometer, was by robert fludd in . however, around , santorio santorio calibrated the tube and went on to attempt to measure human temperature with his thermoscope. at the end of the sealed tube, he had a bulb blown of the optimal size to be inserted in the mouth. the open end was submersed in fluid. as the air expanded due to the oral temperature, fluid was expelled from the tube. after a fixed period of time, the bulb was removed, the air cooled, causing the fluid to rise in the calibrated tube ( fig. . ) [ ] . the thermometer in , ferdinand ii de' medici, grand duke of tuscany, produced sealed tubes with a bulb and stem that were partly filled with alcohol. this was the first thermometer to depend on the expansion and contraction of a liquid, which was independent of barometric pressure. many variants of this concept appeared, each unique as there was no standard scale. christian huygens in suggested using the melting point of ice and the boiling point of water as standards. the danish astronomer ole rømer in copenhagen used these upper and lower limits for a thermometer that he used to record the weather. there was still uncertainty about how well these parameters would work at different geographical latitudes. in , carlo renaldini suggested that the ice and boiling water limits should be adopted as a universal scale. in england, isaac newton proposed in that a scale of °c could be used between melting ice and body temperature! in , a german instrument-maker named gabriel fahrenheit produced a temperature scale that now bears his name. he manufactured high-quality thermometers with mercury (which has a high coefficient of expansion) with an inscribed scale with greater reproducibility. it was this that led to their general adoption. fahrenheit first calibrated his thermometer with ice and sea salt as zero. salt water has a much lower freezing point than ordinary water, so he chose the freezing point as °f. the temperature inside the healthy human mouth was °f, and he established the boiling point of water at °f. he later adjusted his freezing point to °f, so he established °f between boiling and freezing which he measured at sea level [ ] . in uppsala, sweden, anders celsius ( - ) had been involved in meteorological observations as an astronomy student. there were at that time a large number of different thermometers, all with different scales. he may have already at that early stage in his career realized that there was a need for a common international scale. he was appointed as professor of astronomy at uppsala (as his father had been before him) and was involved in meteorological surveys. celsius was the first to perform and publish careful experiments leading to the establishment of an international temperature scale based on scientific data. (he was for many years secretary of the royal society of sciences at uppsala.) his paper 'observations of two persistent degrees on a thermometer' described his detailed experiments to check that the freezing point is independent of latitude and atmospheric pressure. he also determined the dependence of boiling water on atmospheric pressure and gave a rule for the determination of the boiling point if the barometric pressure deviates from a standard pressure [ ] . why would a student of astronomy be interested in scales of temperature measurement? the position of zero was much discussed. the scale used by ole rømer placed zero at the lower temperature. celsius had also used a thermometer created by the french astronomer joseph-nicolas delisle with zero at the boiling point, thus giving a reversed scale with increasing numbers for decreasing temperatures, which avoided having negative values. the reversal of this centigrade scale, placing zero at the freezing point, was inevitable and occurred a few years after celsius's death. various names are associated with this change. while linnaeus is often credited, the history of thermometers in the proceedings of the royal swedish academy of sciences for mentions celsius, his successor strømer, and the instrument-maker ekström in connection with the direct scale. no single person was given credit. a century later, carl august wunderlich stated in the english translation of his treatise on 'temperature in diseases' that he preferred to retain all his measurements in the centigrade scale, because the convenience of this scale will probably shortly lead to its general adoption by all scientific men. celsius is now internationally recognized for his major contribution through his careful experiments and in using fixed points for calibration. this was recognized by the adoption in by an international conference on weights and measures of the preferred scale for temperature now referred to as degree celsius (°c). imagine the setting for scientific discussions and the dissemination of new knowledge at the time of linnaeus and celsius. in scotland in , lord kelvin realized in his study of heat that a much greater range of temperature could be considered, far beyond the centigrade scale. absolute zero, the level at which all molecular motion stops, gives the lowest conceivable temperature that can be found. this he determined to be − . degrees on the centigrade scale and − . degrees on the fahrenheit scale. therefore, the lowest temperature on the kelvin scale is , and the units are the same as the centigrade (celsius) scale. while this scale is not used in clinical medicine, it may sometimes be used to define a temperature calibration source or similar scientific system. thomas seebeck, who was born in estonia in , is the person most closely associated with the thermocouple as a temperature-measuring device. in , when at the berlin academy of sciences, he had studied the magnetic influence of an electrical current. a year later, he announced his discovery that two different metals forming a closed circuit will display magnetic properties when there is a difference of temperature between the two points of contact. this, the seebeck effect, is the basis of all thermoelectricity and led to the development of thermocouples for contact temperature measurement. in recent years, this technology has been improved to provide highly accurate heat-measuring devices capable of measurement from a few degrees above absolute zero to high temperatures over °c ( °f). their main applications generally fall outside the temperature range of the human body, but some patient-monitoring devices used in critical care employ thermocouples taped to the skin for continuous measurements over time. thermocouples and thermistors are also used in sealed catheters for internal body temperature measurements [ ] . the first non-contact radiometer designed to measure body temperature in the inner ear canal was invented in by theodor benzinger. when doing research on human temperature regulation at the us naval medical research institute in bethesda, benzinger developed a small radiometer to measure as close as possible to the brain. this was intended to be a non-invasive procedure, to avoid attaching electrodes to the hypothalamus [ ] . the first systems were produced in the united states, europe, and japan in the early s and have been increasingly adopted as a routine instrument for clinical thermometry (fig. . ). since the earliest days of medicine, physicians have recognized that the human body can exhibit an abnormal rise in temperature, usually defined as fever, as an obvious symptom of certain illnesses. for example, the bible has early references to fever in the book of job, and there are descriptions of 'burning bones' in the book of psalms. physicians were aware of the use of their hand as a standard means for estimating temperature. hippocrates noted that the temperature of the body was important and insisted that physicians should be able to recognize the signs of abnormal temperature. he taught that steps should be taken to raise the temperature where it is depressed and lower it when raised. galen (ad - ) described fever as calor praeter naturam or preternatural heat. as already noted, the first attempts to measure the temperature of a human body seem to have occurred in the sixteenth and seventeenth centuries and then first in italy. giovanni borelli, who had the support of queen christina of sweden, was a pioneer of biomechanics and studied movement in animals. he is reputed to have tried many different measurements of the inner organs of live animals long before anaesthetics were available [ ] . santorio santorio made an elaborate form of oral thermoscope to study human body temperature, although probably only with limited success. herman boerhaave ( - ) and his pupils gerard van swieten and anton de haen noted the value of fahrenheit's thermometer after it became available in . van swieten became a professor of medicine at the university of vienna and recommended that fever should be measured with a thermometer rather than with the hand. he applied the mercury thermometer to both the mouth and axilla as recommended by fahrenheit. anton de haen taught clinical practice at the vienna general hospital and emphasized to all his students of the importance of measuring body temperature in fever. he pointed out that a physician's touch was inadequate, especially when a shivering patient complained of extreme coolness while registering a temperature that was three or more degrees above normal. unfortunately, his studies were scattered throughout his volumes of publications, ratio medendi ( - ). these included observations on temperature related to diurnal fluctuations, in the elderly, and on the action of certain drugs. de haen's detailed observations, just part of his extensive work, went largely unheeded [ ] . excellent work on the temperature of healthy people and animals had been published by george martine ( - ), a physician who had studied in edinburgh and leiden. he theorized that animal heat was the result of the velocity of blood moving through the vessels. his work inspired many others including john lining in on temperature in those suffering from malaria, and john hunter ( - ) one of the great surgeons and pioneers of the circulatory system. hunter subsequently disagreed with martine, claiming that 'warmth depends on a different principle, which is intimately connected with life itself, and is a power which maintains and regulates the machine, independent alike of the circulation, the will, and of sensation' [ ] . many of the early thermometers were of doubtful accuracy, and frequently they were inconveniently large. however, by becquerel and breschet were able to establish the mean temperature of a healthy adult to be °c ( . °f). by the s, the use of the thermometer had become more common, and the physiological significance of body temperature was becoming clearer. by , john davy had noted the variations in temperature resulting from exercise, the intake of food and drink, the influence of external temperature, and the differences in body processes in children. by this time, it was recognized that in many situations, temperature was a better clinical indicator than the pulse, because it was not affected by nervous activity or excitement. reflect on the statement that body temperature is not affected by nervous activity or excitement. during this period of increasing interest in thermometry, carl reinhold wunderlich ( - ) published his major work on temperature in diseases when at leipzig in . this was published in an english translation in [ ] . his treatise was based on regular temperature measurements made on all his patients over the course of years, some as many as four to six times daily. after some , observations, wunderlich showed that when the temperatures were plotted on charts, the disease could be shown to follow certain laws, which could be characterized by the trend in temperature. overall, he studied some , specific cases. this was clearly a significant contribution to the subject and places wunderlich at the forefront of discovery in this aspect of clinical observation. he had established that the temperature in a healthy person is constant and that variation of temperature occurs in disease. from this, wunderlich laid down a code based on principles that he had derived from his large set of observations. by this time it was considered that 'a physician who carried on his profession without the thermometer was like a blind man endeavouring to distinguish colours by feeling'. in the first chapter of his book, wunderlich lists precepts of human body temperature, most of which remain unchallenged in modern medicine. here are some examples: the temperature of healthy person is almost constantly the same, although not absolutely. so, indeed there are spontaneous variations in the course of every twenty-four hours, but these seldom exceed half a degree of the centigrade scale. a normal temperature does not necessarily indicate health, but all those whose temperature either exceeds or falls short of the normal range are unhealthy. the range of temperature in the most severe diseases is between °c ( °f) and . °c ( . °f), and it is very seldom that it exceeds °c ( . °f) or sinks below °c ( . °f). alterations of temperature may be confined to special regions of the body, which are the seat of disease actions (local inflammation), while the general temperature remains more or less normal. a rapid increase in temperature of the body from a chill, or in the normal warmth of the hands, feet, nose, or forehead, is commonly associated with strong feelings of chilliness and convulsive movements ('cold shivers', rigors, 'fever-frost'). a more or less permanent and noticeable rise in temperature amounting to . °c ( . °f) or more, is generally accompanied with subjective feelings of heat, and lassitude, as well as with thirst and headache…and rapidity of the pulse …('feverishness', pyrexia, fever). when there are extremes of temperature, we know that there is great danger. high fever is indicated by temperatures above . °c ( . °f) in the morning, and above . °c ( . °f) in the evening. temperatures in every known disease except relapsing fever, in all probability, indicate a fatal termination ( °c [ . °f] or more-hyperpyretic temperatures). abnormally low temperatures may seriously disturb the various functions of the body; and when the fall is very considerable, it may render the continuation of life impossible [ ] . these extracts are abridged from the very detailed description of differing types of fevers that were accepted in nineteenth-century medicine. in the full text of temperature in diseases, wunderlich provides a most comprehensive list of investigators, mainly german and european, who had studied the role of thermometry in man and animals. he also discusses the various sites on the human body where thermometry may be applied. of the many potential areas, he showed that in the hand or between fingers and toes was too unreliable. rectal and vaginal sites were also criticized, the former being affected by masses of faeces, and the latter lacking in clinical evidence of reliability. the axilla and the mouth were advocated, with warnings of the effects of ingestion of food and drink, and or oral breathing when suffering from nasal congestion. much of this work by wunderlich and others was performed with large, slow thermometers sometimes requiring minutes to fully register. the need for a narrow temperature range of clinical thermometer was obvious. this should also be a maximum registering thermometer, small in size, and able to be fitted into a protective case. in this way a physician could carry a stethoscope and a thermometer in his personal kit, thus increasing the use of temperature measurement in diagnosis. while different names have been associated with the arrival of the 'clinical thermometer', the allbutt clinical thermometer was the first practical device to become commercially available. sir thomas clifford allbutt ( - ) was a celebrated british physician. he spent years working in leeds during which time he devised the small clinical thermometer. a local company, harvey and reynolds, first manufactured this special thermometer in , followed by thackeray in london. allbutt made the design of his thermometer freely available to others, and it was quickly taken up by british physicians. it was notable in that the instrument, centimetres long, had a constriction in the capillary tube that held the mercury at its reading after use, until shaken down to the lower limit of calibration ( fig. . ) . the temperature reading was available in minutes and initially was calibrated to - degrees on the fahrenheit scale ( - . °c). later clinical thermometers were marked with the centigrade scale. thomas allbutt made several significant contributions to medicine, including the ophthalmoscope. he received royal recognition in england, being awarded a knighthood in , and he was made the president of the british medical association in [ , ] . although william herschel in britain had identified the existence of infrared radiation in , it took many years for remote heat sensing to be developed. throughout the s and s, this technology came into practical use, accelerated by the needs of the military during the second world war. in the late s, once infrared technology had been declassified, thermal imaging became available to medicine and industry. although the early systems were slow scanners, it became clear that it was possible to record the temperature distribution of a human subject or an object. an important conference in at the new york academy of sciences revealed the true potential of this technology in the study of human body temperature [ ] . also, in the german physician dr theodore benzinger, who had moved to the united states, developed a small radiometric device to measure the temperature of the inner ear (tympanic membrane). in contrast to the very expensive early thermal imaging systems, this device promised a low cost and reliable means of measuring temperature close to the brain, but without the invasive contact of thermocouples. initially only used for military and space technology, the tympanic radiometer came into medicine some years later. this was undoubtedly stimulated by the concerns about the use of mercury on thermometers and its subsequent banning. the radiometer was further developed in the united states for measuring temperature over the temporal artery and was also used to measure forehead temperature. the latter application is not always successful, as the forehead may be a site of profuse sweating, either from physical exertion or from fever. after years of ever-improving and cheaper thermal imaging, its use is still growing in medicine [ ] . a significant chain of events during the severe acute respiratory syndrome (sars) outbreak and subsequent pandemic threats of the hemagglutinin and newaminidase (hn) viruses has resulted in trials using thermal imaging of the face for airport screening of the travelling public. this has led to the international standards organization publishing documents to highlight the essential requirements of thermal imaging cameras and their optimal use in this application. from this work, it is now established that a close-up thermogram of the subject's frontal face can be used to measure the temperature of the inner canthus of the eye and so to detect fever by remote sensing (see chap. ) [ , ] . thus, the study of human body temperature continues to evolve, and the technology applied to it is still being developed [ ] . many pioneers in medicine, physiology, and the physical sciences have contributed to this story, which, inevitably, cannot be said to be over. our knowledge of the science of the human body will doubtless continue to grow, yet the long centuries of battles against human disease have not yet come to an end. medical thermometry -a short history swedish astronomers - acta universitatis upsaliensis the early history of the thermocouple tympanic thermometry in anaesthesia and surgery physicists and physicians ratio mendendi in nosaocomio practico vindobonensi. vienna: kruckten medical thermometry and human temperature medical thermometry medical thermometry thermography and its clinical applications infrared thermal imaging in medicine. topical review medical electrical equipment_part - : particular requirements for basic safety and essential performance of clinical thermometers for body temperature measurement. switzerland: iso/tc /sc lung ventilators and related equipment medical electrical equipment: deployment, implementation and operational guidelines for identifying febrile humans using a screening thermography radiometric temperature measurement key: cord- - w qxs w authors: cui, dongjin; ai, zhengtao; mak, cheuk-ming; kwok, kenny; xue, peng title: the influence of envelope features on interunit dispersion around a naturally ventilated multi-story building date: - - journal: build simul doi: . /s - - -x sha: doc_id: cord_uid: w qxs w this study examines the influence of building envelope features on interunit dispersion around multi-story buildings, when the presence of an upstream interfering building is also considered. validated cfd methods in the steady-state rans framework are employed. in general, the reentry ratios of pollutant from a source unit to adjacent units are mostly in the order of . %, but there are still many cases being in the order of %. the influence of envelope features is dependent strongly on the interaction between local wind direction and envelope feature. in a downward dominated near-facade flow field, the presence of vertical envelope features forms dispersion channels to intensify the unidirectional spread. horizontal envelope features help induce the dilution of pollutant to the main stream and weakens largely the vertical interunit dispersion. the large influences caused by the presence of envelope features extend the existing understanding of interunit dispersion based on flat-facade buildings. natural ventilation has been recognized as an effective way to achieve low-energy building design and an economic way to provide a healthy indoor environment (homod and sahari ; schulze and eicker ; chau et al. ; wu et al. ) . however, during the natural ventilation process, outdoor pollutants could penetrate indoors through open windows and deteriorate the indoor air quality. among others, the transport of human exhaled bio-aerosols between adjacent units in a building, namely interunit dispersion, is a valid route, which was observed in the outbreak of sars in hong kong ). since being identified, a large of number studies have been conducted to explore the interunit dispersion characteristics and mechanisms as well as to quantify its infectious risks (e.g., li et al. ; niu and tung ; gao et al. ; liu et al. ; ai et al. ; mak , ; cui et al. cui et al. , . li et al. ( ) analyzed the sars transmission characteristics in the most affected housing estate in hong kong and found that airflow has been involved in the transport of sars viruses. later, niu and tung ( ) performed on-site measurements to quantify the interunit dispersion between two vertically adjacent units and reported that the buoyancy-induced upward transmission from a source unit to the unit immediately above it can reach up to %. according to niu and tung ( ) , for an indoor/outdoor temperature difference of - °c, the turbulence effect of a wind speed over . m/s can overwhelm the thermally driven force. wind tunnel experiments conducted by liu et al. ( ) indicated that the vertical interunit dispersion could occur not only along an upward direction but also along a downward direction. ai and mak ( ) further extended the findings on the interunit dispersion route, who reported that wind-induced interunit dispersion could occur along any direction, namely upward, downward, and horizontally lateral directions. the reentry ratios could reach up to . %, which is much higher than that due to buoyancy effect. one important finding obtained from previous studies is that the interunit dispersion along building facades is strongly determined by the near-facade flow patterns. among others, it is obvious that the near-facade flow patterns around a building are largely influenced by the envelope configurations and the surrounding buildings. among all the surrounding interfering buildings, an upstream building has a significant influence when the downstream building is located in the leeward-side recirculation zone (hajra et al. ) . common knowledge tends to support that the presence of an upstream building would lower external wind speeds and then reduce the indoor ventilation performance of its downstream building. although our earlier study (cui et al. ) revealed the significant influence of the presence of an upstream building on interunit dispersion, it is, however, limited to the building models with flat facades. in practice, buildings are attached with various types of envelope features on their facades, such as balconies, bay windows, wing walls and overhangs (see fig. ) and wind catchers (jomehzadeh et al. ; hughes et al. ; pak et al. ) . these envelope features are designed for architectural and/or functional purposes, some of which were proved being able to mitigate noise, moderate the transport of heat and air and reduce transmission of daylight from outside to indoor environments (ai et al. ; mak et al. mak et al. , niu ). since , in new residential projects in hong kong, it has been permitted to exclude envelope features such as balconies from calculations of the gross floor area and/or site coverage (joint practice note no. ). ai et al. ( ) revealed the significant influence of the presence of balconies on interunit dispersion, which however, is limited to isolated building models. overall, previous studies on interunit dispersion focused mostly on isolated buildings with flat facades. this study intends to explore the influence of upstream buildings and envelope features on interunit dispersion in a multistory building. two heights of an upstream building are considered, namely one with the same height of the downstream building and another with the double height of the downstream building. in addition, two typical envelope features are investigated, namely vertical and horizontal envelope features. computational fluid dynamics (cfd) method is used, and the widely used rans turbulence model, namely, renormalization group (rng) k-ε model, is employed to predict the airflow field in and around buildings. cfd method in the rans framework is the most widely used method to investigate flow and dispersion in and around buildings (blocken ) . tracer gas is used to simulate human exhaled flows. owing to the similar aerodynamic characteristics between gas and fine aerosols, tracer gas technique was widely used in past studies, including numerical simulations (gao et al. ; ai et al. ; mak , ) and experiments (liu et al. ; niu and tung ) . after the establishment of flow field, tracer gas is released from a certain unit and its concentrations in all units are monitored to observe interunit dispersion pattern and to quantify infectious risks. the findings from this study are expected to extend the understanding of interunit dispersion in naturally ventilated buildings. franke et al. ( ) , a computational domain with an upstream distance of d z , downstream distance of d z , lateral distance of d z , and height of d z is chosen for compromising accuracy and numerical cost. table shows the different configurations designed based on the -story building with and without vertical or horizontal envelope features and the upstream buildings. for each configuration, there are cases with the source released from the different units. therefore, totally cases were simulated. a grid with approximately . million cells is used after a grid independence test based on the building configurations without envelope features (see also cui et al. ) . three types of grids, namely coarse, medium and fine, are created, which contain . , . and . million cells, respectively. the average ach value of the units in the target building is used as an indicator to evaluate the independence of the grid. for the three types of grid, the average ach values are . , . , . , respectively, which suggests that the medium grid is suitable to be used for further simulations. the commercial cfd software, ansys fluent . , was used to compute the flow and turbulence fields in and around the buildings. the rng k-ε model (orszag et al. ; yakhot and orszag ) was adopted to deal with the turbulence effect. considering the roughness of the ground, the velocity inlet was specified according to the widely used power law profile: where v z is the wind speed at the height z, v ref is the reference velocity at the building height d z , α is the roughness coefficient that was specified as . to represent a typical urban ground roughness. the turbulence at the inlet boundary was characterized by turbulence intensity and length scale, which were % and m, respectively. zero normal gradients and zero background pressure were applied to the domain outlet. on the domain ground and building surfaces, nonslip boundary conditions combined with standard wall functions were used. a tracer gas, co (carbon dioxide), was used to represent fine droplet nuclei in the human exhaled flows. it was released continuously at the center of a source room at a flow rate of mg/s. the species transport model (fluent ) was employed to predict the concentration of the tracer gas. the dimensionless number, sct, could has a large influence on the solution of the species equation (tominaga and stathopoulos ) . ai et al. ( ) used rans model to investigate interunit dispersion and found that sc t equal to . is a suitable setting. therefore, all simulations in this study were conducted with the sc t equal to . . the governing equations were discretized to algebraic equations on a staggered grid system based on the finite volume method (fvm). the pressure and momentum equations were coupled using the simplec algorithm. all the discretization schemes had second-order accuracy. convergences were reached when the solutions of the velocity and concentration at important locations in the computational domain become stable for dozens of iterations. the parameter reentry ratio (rk) was used to evaluate and quantify the interunit dispersion, which is defined as (niu and tung ; ai et al. ) : the reentry ratio indicates the fraction of air leaving from a source unit reenters an affected unit. in the equation, c j and c i are the concentration at an affected unit and a source unit, respectively; (ach) j and (ach) i are the air change rate of the affected unit and the source unit, respectively. ach of a specific unit was calculated using an integral method: where v x is the normalto-opening velocity component, a the area of the opening, and vol r the volume of the unit. validations of the cfd methods described in sections . - . against experiments were reported in our earlier papers (cui et al. ; ai et al. ; ai and mak ) . the experiments used for validation include the influence of an interfering building on the pressure distributions on the building facades of another adjacent building (zhang and gu ) and the flow field and ventilation rate of a naturally ventilated building (jiang et al. ) . zhang and gu ( ) investigated the wind-induced surface pressure on the facades of a building under the effect of a staggered interfering building in an atmospheric wind tunnel. they measured the pressure coefficient along the vertical center line of walls. cfd simulations were employed to reproduce the experiments (cui et al. ). using rng model, the simulated pressure coefficients had a good agreement with the measured results, with an average deviation of . %. this justifies the use of the aforementioned cfd methods to study the influence of an upstream building on the flow field around its downstream building. jiang et al. ( ) conducted a wind tunnel experiment to investigate airflow inside and around a cubic building model with single-sided natural ventilation. they measured the mean air velocities along ten vertical lines on the vertical centerplane of the building using laser doppler anemometer. cfd simulations were performed and the velocities along these vertical lines were predicted (ai et al. ; ai and mak ) . despite the deficiencies of the rng model in predicting the vortex-shedding effects in the wake of a bluff body, the simulated results generally agree well with the measurements in most areas in and around the building. it was concluded that the application of rng model was reliable to establish the flow field in and around a naturally ventilated building. figure presents the streamlines and velocity contours on the vertical centerplane across the buildings when there is no envelope features (see also cui et al. ). the airflow patterns around a building, which have a significant influence on the interunit dispersion, have been widely examined in the field of wind engineering. when encountering an isolated bluff body, such as a flat-roofed building, the urban wind will separate at the windward facade, which includes vertically downward and upward flows as well as horizontal flows. due to the airflow impingement, the windward facade is exposed to a relatively high pressure flow field, where the maximum pressure appears at the stagnation zone at approximately / height of the building (oke ). such a general airflow pattern around an isolated bluff body is similar for both a single-story building and a multi-story building (ai and mak ) . when comparing figs. (a), (b) and (c), it is obvious to find that the presence of an upstream building has a significant influence on the airflow field around its downstream building. as shown in fig. (b) , the presence of a low upstream building makes the windward area of the downstream building to be a downward dominated flow field, where the upward flow around the building top (see fig. (a) ) disappears. such a change in airflow pattern would directly alter the interunit dispersion routes. for example, when pollutants are released from the fourth floor, their dispersion route will change from upward to downward. the presence of the high upstream building also modifies the near-facade airflow pattern of the downstream building in both windward and leeward sides (see fig. (c) ). in addition, a very obvious change caused by the presence of an upstream building is that it increases the velocity magnitude in the vicinity and the inside of the downstream building, which potentially improves the indoor ventilation performance. figures and show the influence of envelope features on the airflow pattern in and around an isolated building. the general external flow fields around a building are similar with those revealed in previous studies on bluff bodies (santos et al. ; ashrae ; martinuzzi and tropea ) . however, the presence of protrusive envelope features introduces more resistances to the near-facade sweeping airflows and intensifies the turbulent fluctuations by the enhanced interaction between approaching wind and envelope features (both vertical and horizontal). as shown in fig. (b) and fig. (b) , small vortices appear in the corners of the envelope features, which indicates that the presence of the envelope features create a rougher and more dynamic near-facade flow field when compared with that formed near flat building facades. according to haghighat et al. ( ) , such near-facade wind fluctuations around building surfaces are the continuous driver of the indoor and outdoor air exchange. figures and present the flow fields on the vertical centerplane of the building when there is an upstream building. again, the presence of envelope features disturbs the near-facade airflow patterns and generates many small vortexes. such a change in near-facade airflow patterns caused by the presence of envelope features is the same no matter there is an upstream building or not. here, an important finding is that the direction of external flows entering the units is different when there is a low upstream building when compared to that when there is a high upstream building. this difference caused by the different heights of an upstream building is not changed by the presence of envelope features. in addition, with the presence of an upstream building, the addition of envelope features mostly forms as a barrier to prevent the indoor and outdoor air exchange, especially when the upstream building has the same height with the target building. figure presents the average ach coefficients of all configurations. ach coefficient is defined as the ratio of the average ach value of the four windward units or the four leeward units of a specific configuration (configurations - ) to the average ach value of the four windward units of configuration . for an isolated building (configurations - ), compared to the configuration with no envelope features (configuration ), the presence of vertical envelope features increases the average ach values considerably on both windward (increased by %) and leeward side (increased by %). while, the presence of horizontal features slightly improves the average ach values for both windward and leeward side of the downstream building. after the inclusion of a low upstream building (configurations - ), both types of envelope features have negative influences on the natural ventilation performance. in particular, compared to the configuration with no envelope features (configuration ), the ach values at windward and leeward sides are decreased by % and % after the presence of horizontal features (configuration ). when the upstream building is increased to double height (configurations - ), the presence of vertical envelope features increases the ach value on windward side by %, whereas horizontal features result in a decrease of ach by % and % on windward and leeward side, respectively. for wind-induced natural ventilation of a multi-story building, pollutant released from one unit may re-enter into other units of the building. in addition, many buildings in cities like hong kong are high-rise cylinder-like buildings, which pose a risk of pollutant transport between windward units and leeward units. the interunit dispersion characteristics of the configurations are discussed in this section. figures - show the pollutant reentry ratios r k from a source unit to other units. to focus on analyzing the major dispersion routes, those reentry ratios smaller than . % are not presented. figure presents the reentry ratios of tracer gas from the source unit w to other units on both windward and ② with a low upstream building; ③ with a high upstream building leeward sides of the building. when a pollutant is released from the ground floor unit w , the r k to all units are less than . % in the case of an isolated building with flat-facade (see fig. (a) ). these relatively small reentry ratios are formed basically because that the ground floor is close to the downward recirculation zone in front of the building, where the tracer gas is restricted in this region and diluted effectively into the lateral flow vortices. however, the presence of an upstream building, especially a low upstream building, enhances obviously the interunit dispersion. the presence of envelope features, especially the vertical features, first extends the affected scope in terms of the number of affected units and second increases the reentry ratios not only on the windward side but also on the leeward side. the reentry ratios can reach up to . % and . % on windward and leeward side, respectively. figure presents the reentry ratios of tracer gas from the source unit w to other units in the building. obviously, the unit just below the source unit on the windward side is the most affected one. the unit w is close to the strong downward flow region in front of the building, which, therefore, leads to a significant downward dispersion to the unit w . this downward flow is formed because of the high pressure difference between the stagnation zone at the / building height and the low-pressure recirculation zone around the ground. the presence of an upstream building, especially the low upstream building, enhances considerably the downward dispersion, where the r k increases from . % to . %. the presence of the vertical envelope features forms a vertical dispersion channel, which restricts further the dispersion direction and reduces tracer gas dilution. as a consequence, the presence of the vertical envelope features results in the increase of reentry ratio in most units. however, the presence of horizontal features performs in a different way, where the reentry ratios to most units are decreased significantly. this can be attributed to that the horizontal features form a barrier to the vertical transport of tracer gas and induce the dilution of the tracer gas to the main flow stream. figure presents the reentry ratios of tracer gas from the source unit w to other units. as described in section . , the presence of a low upstream building modifies the airflow pattern near the windward side of its downstream building, where no stagnation exists and the downward flow becomes the dominated flow pattern. when the pollutant source of an isolated building is located in the stagnation area (w ), the pollutant released from the source unit flows both upward and downward on the windward side, which means that both the unit w and w are affected. however, when there is a low upstream building, almost only the lower floors are affected. for the case of an isolated building, all units with flatfacade have relatively small r k (below . %). the presence of an upstream building, especially a low upstream building, intensifies the interunit dispersion for most units. however, the unit w is an exception when there are vertical features, where it suffers from the highest reentry ( . %) when there is no an upstream building. in general, the enhancing effect of the interunit dispersion due to the presence of vertical features still exists; the reentry ratios of the lower units, especially unit w are significantly increased. however, the presence of horizontal features tends to lower the reentry ratio level. overall, the most affected units are w and w , which are located below the source unit. figure presents the reentry ratios of tracer gas from the source unit w to other units. the basic influences of the presence of an upstream building and envelope features on the interunit dispersion are the same with those analyzed in previous section. overall, the lower units are most affected. the presence of a low upstream building and the presence of vertical envelope features enhance significantly the interunit dispersion. with the combined effect of the upstream building and vertical envelope features, the reentry ratios can reach up to . %. the presence of horizontal features, however, helps to reduce the reentry ratios. this study examines the influence of the presence of an upstream building and envelope features on the natural ventilation performance and interunit dispersion of a multistory building using cfd method. based on the findings, the following conclusions can be obtained. the presence of upstream building changes the nearfacade flow patterns around its downstream building, and the presence of envelope features create a rough and dynamic near-facade flow field and forms a barrier to the vertical or horizontal development of the near-facade flow. on average, for an isolated building, the presence of vertical features increases the ach value by %, but the presence of horizontal features improves only slightly the ach value. with the presence of a low upstream building, both types of envelope features have negative influences on natural ventilation performance; particularly the ach value is decreased by % due to the presence of horizontal features. with the presence of a high upstream building, the addition of vertical features improves the natural ventilation performance on windward side by %, but the addition of horizontal features results in a decrease by %. the presence of an upstream building, especially a low upstream building, significantly enhances the interunit dispersion for most cases. such an enhancement can be up to one order of magnitude. it suggests that findings from previous studies on interunit dispersion based on an isolated building underestimate the dispersion risks. the presence of vertical envelope features forms vertical channels to restrict the dilution of pollutant, which therefore intensifies the interunit dispersion and increases significantly the reentry ratios for most cases. the presence of horizontal features forms barriers that induce the dilution of pollutant to the main flow stream, which therefore weakens largely the vertical interunit dispersion. the large influences caused by the presence of envelope features extend the findings from previous studies based on flat-facade buildings. numerical investigation of wind-induced airflow and interunit dispersion characteristics in multistory residential buildings a study of interunit dispersion around multistory buildings with single-sided ventilation under different wind directions comparison of single-sided ventilation characteristics between single-storey and multi-storey buildings due to wind effect large eddy simulation of wind-induced interunit dispersion around multistory buildings ashrae handbook, hvac applications (si), section . years of computational wind engineering: past, present and future valuing the health benefits of improving indoor air quality in residences effect of balconies and upper-lower vents on ventilation and indoor air quality in a wind-induced, naturally ventilated building cfd simulation of the effect of an upstream building on the inter-unit dispersion in a multi-story building in two wind directions ansys fluent . user's guide, modeling turbulence best practice guideline for the cfd simulation of flows in the urban environment the airborne transmission of infection between flats in high-rise residential buildings: tracer gas simulation the influence of turbulent wind on air changes rates-a modeling approach the effect of upstream buildings on near-field pollutant dispersion in the built environment energy savings by smart utilization of mechanical and natural ventilation for hybrid residential building model in passive climate the development of commercial wind towers for natural ventilation: a review natural ventilation in buildings: measurement in a wind tunnel and numerical simulation with large-eddy simulation building department, lands department and planning department a review on windcatcher for passive cooling and natural ventilation in buildings, part : indoor air quality and thermal comfort assessment multi-zone modeling of probable sars virus transmission by airflow between flats in block e investigation of indoor air pollutant dispersion and cross-contamination around a typical high-rise residential building: wind tunnel tests the application of computational fluid dynamics to the assessment of of green features in buildings: part : wing walls a numerical simulation of wing walls using computational fluid dynamics the flow around surface-mounted, prismatic obstacles placed in a fully developed channel flow some significant environmental issues in high-rise residential building design in urban areas on-site quantification of re-entry ratio of ventilation exhausts in multi-family residential buildings and implications renormalization group modeling and turbulence simulations developing a passive house with a double-skin envelope based on energy and airflow performance experiment investigation of outdoor and indoor mean concentrations and concentration fluctuations of pollutants controlled natural ventilation for energy efficient buildings turbulent schmidt numbers for cfd analysis with various types of flowfield air infiltration induced inter-unit dispersion and infectious risk assessment in a high-rise residential building renormalization group analysis of turbulence: . basic theory evidence of airborne transmission of the severe acute respiratory syndrome virus wind tunnel tests and numerical simulations of wind pressures on buildings in staggered arrangement the work described in this paper was supported by a grant from natural science foundation of guangdong province, china. foundation no: a . key: cord- -yz gynn authors: soliman, yasser; alshaikh, belal; alawad, essa; akierman, albert; elsharkawy, adel; yusuf, kamran title: respiratory outcomes of late preterm infants of mothers with early and late onset preeclampsia date: - - journal: j perinatol doi: . /s - - - sha: doc_id: cord_uid: yz gynn objective: to study the effect of early and late onset preeclampsia (eope, lope, respectively) on outcomes of late preterm infants. study design: cohort study of late preterm infants admitted to a tertiary care nicu from january –july . outcomes of late preterm infants of eope mothers were compared with the next late preterm infant of a lope mother and the next two late preterm infants of normotensive non-pe mothers. primary outcome comprised use of continuous positive airway pressure, mechanical ventilation and/or surfactant in the h after birth. results: compared to normotensives (n = ), adjusted odds ratio (aors) of the primary outcome was higher in the eope (n = ) and lope (n = ) groups but reached statistical significance only in the eope group, aors . , % ci . – and . , % ci . – . , respectively. conclusions: compared to late preterm infants of normotensive and lope mothers, infants of mothers with eope have significantly higher respiratory morbidity. late preterm infants, defined as births between + and + weeks gestation, constitute the largest proportion of preterm births, being % in canada [ ] [ ] [ ] . there is significant morbidity associated with late preterm birth with respiratory distress syndrome (rds) and transient tachypnea of the newborn (ttnb) the commonest [ , ] . compared with term infants, late preterm infants are nine times more likely to be placed on continuous positive airway pressure (cpap), five times more likely to be placed on mechanical ventilation and forty two times more likely to need surfactant replacement [ ] . the cost associated with the care of late preterm infants is substantial, corresponding to more than million dollars annually in canada [ ] . initial studies on morbidity in late preterm births compared outcomes to term births without taking into account underlying maternal medical conditions that may lead to preterm birth. this is now being questioned with the realization that in addition to prematurity per se, maternal conditions also contribute to morbidity in late preterm infants [ , ] . one such condition is preeclampsia. preeclampsia is a pregnancy specific disorder characterized by hypertension and proteinuria manifesting at or after twenty weeks of gestation and is a major cause of maternal and neonatal mortality and morbidity worldwide [ ] . although placental dysfunction is a hallmark of preeclampsia, the disorder is heterogeneous [ ] . based on gestational age at onset of disease, preeclampsia is classified as early, occurring before weeks gestation and late occurring at or after weeks of gestation [ , ] . both have similarities and distinct features. placental changes are more marked in early onset preeclampsia while maternal factors such as increased body mass index (bmi) and metabolic syndrome play a greater role in late onset disease [ ] . early onset preeclampsia is also associated with a greater adverse impact on maternal and fetal health [ ] . although early onset disease manifests before weeks gestation, a third of women deliver at or after weeks gestation [ , ] . surprisingly, reports of the effects preeclampsia on outcomes of late preterm infants have not differentiated between early and late onset preeclampsia, despite significant differences between the two. the objective of our study was to investigate the effects of early and late onset preeclampsia on the outcomes of late preterm infants, with the primary objective being respiratory outcomes. the neonatal intensive care unit (nicu) in calgary maintains a prospectively collected electronic database of all infants admitted to the nicu. late preterm infants born between + and + gestation to a mother with early onset preeclampsia between january and july were included in the study. their outcomes were compared with the next late preterm infant born to a mother with late onset preeclampsia and the next two late preterm infants born to normotensive mothers. a prestructured data form was completed for all infants. in case of missing data, the infant's medical record charts were reviewed. the conjoint health research ethics board of the university of calgary approved the study. the primary outcome comprised the use of cpap or mechanical ventilation and/or surfactant use in the first h after birth. we chose this primary outcome for several reasons. amongst all the morbidities faced by late preterm infants, respiratory morbidity is the most common. the two most common diagnosis for respiratory distress in late preterm period, rds and ttnb, can sometimes be difficult to distinguish based on clinical manifestations and chest x-ray [ ] . importantly, similar definition of respiratory morbidity has been used in other large studies [ , ] . secondary outcomes included (i) rds-diagnosed based on signs of respiratory distress, a typical chest x-ray and/or need for surfactant [ ] . (ii) ttnb-diagnosed on a chest x-ray showing good volume with increased vascularity and the presence of a transverse fissure [ ] . (iii) use of supplemental oxygen by nasal cannula. (iv) small for gestational age (sga)-defined as birthweight below the th centile using the fenton growth charts [ ] . preeclampsia definition was based on society of obstetrics and gynecology canada recommendations and was defined as systolic blood pressure≥ mmhg or a diastolic level of (korotkoff ) ≥ mmhg on two or more occasions at least min apart after weeks gestation in a woman with previously normal blood pressure. proteinuria was defined as ≥ . g protein in a h urine sample. when a h urinary sample was not feasible, or ≥ mg/mmol urinary creatinine in a spot urine sample or ≥ + on a urinary dipstick test-strip was used as criteria for proteinuria [ ] . early onset preeclampsia was onset of symptoms at < weeks gestation and late onset preeclampsia onset of symptoms ≥ weeks gestation. histological chorioamnionitis was defined as infiltration of polymorphonuclear leukocytes in the fetal membranes and chorionic plate, and funisitis as the presence of these cells in the umbilical cord blood vessel walls and wharton's jelly [ ] . antenatal steroids were considered a course if more than twelve hours had elapsed after the first dose [ ] . diabetes included both gestational and pregestational forms of the disease. gestational age was based on a first trimester ultrasound and date of embryo transfer in cases of in vitro fertilization. surfactant was administered when oxygen requirements were persistently > %. exclusion criteria included infants born with any congenital malformations or chromosomal anomalies, infants of mothers with chronic hypertension (onset before weeks of gestation), maternal renal, cardiovascular, endocrine or autoimmune disease, substance abuse, and torch infections. women who did not have a first trimester ultrasound were also excluded. as the distribution of the relevant variables was not normal, we chose conservative nonparametric analysis for continuous variables, using the kruskal-wallis test. categorical variables were compared using the χ or fisher's exact test as appropriate. bonferroni correction was used post hoc for multiple comparisons. to identify risk factors for development of the primary outcome, multivariable ordered logistic regression with backward elimination approach was performed. any risk factors that demonstrated associations, whether statistically significant or judged to be clinically significant, with both preeclampsia and the primary outcome but were not intermediate variables, were included in the modeling process as possible confounders [ ] . the least significant variables were then removed until all remaining variables were significant at p value of . . the p value of . was set conservatively as an entry for variables to proceed to the next step in the analysis [ ] . the adjusted odds ratio (or) and their % confidence interval (ci) are reported. a p < . was considered significant. data were analyzed using stata v. (college station, texas, usa). during the study period, there were admissions to the nicu. amongst the women with early onset preeclampsia, four women with chronic hypertension and two with a history of substance abuse were excluded. there were three sets of twins in the group resulting in infants eligible for the study. of the women with late onset preeclampsia, two women with substance abuse and one with chronic hypertension were excluded. there were no twins in the group resulting in infants eligible for the study. one hundred and thirty three normotensive women who delivered late preterm infants between and weeks gestation were included as controls. on chart review, four of these women had a history of substance abuse, two had chronic hypertension and one each had systemic lupus erythematosus and antiphospholipid syndrome. they were excluded from the study. there were six sets of twins in the group resulting in infants eligible for the study (fig. ) . table shows the maternal and neonatal demographic variables between the three groups. there was no difference in the maternal age, diabetes, twins, chorioamnionitis, smoking, number of male infants and apgar scores < at min between the three groups. compared to the normotensive group, the number of primigravida mothers was late onset preeclampsia vs. normotensive c early onset preeclampsia vs. late onset preeclampsia higher in the two preeclamptic groups but the difference did not reach statistical significance. compared with the other two groups, gestational age was significantly lower in the early onset preeclampsia group. birthweight was significantly lower and sga rates significantly higher in the two preeclampsia groups compared with the normotensive group (p < . ). antenatal steroid use, c-section rates, and apgar scores < at min were significantly higher in the early onset preeclampsia group. table a shows the univariate analysis of the primary outcome and its components in the three groups. all infants who received surfactant were also placed on mechanical ventilation. the composite outcome as well as the individual components were significantly higher in the early onset preeclampsia group (p = . for surfactant and < . for the other variables). table b shows the results of multivariable ordered logistic regression as the odds ratio (ors) of the primary outcome in early and late onset preeclampsia with the normotensive group as the reference group. the final model included gestation, mode of delivery, sex, antenatal steroids, and small for gestational age. the odds of the primary outcome-cpap use, mechanical ventilation and/or surfactant use-was greater in the two preeclampsia group with the early onset preeclampsia group having the higher odds. however, the higher odds in the late onset group did not reach statistical significance (or . % ci . - , p = < . for early onset preeclampsia and or . % ci . - . , p = . ). table shows the secondary outcomes between the three groups. compared to the normotensive group, ttnb, nasal cannula oxygen, hypoglycemia, sga infants and use of phototherapy, duration of hospital stay was higher in the two preeclampsia groups. rds was significantly higher in the early onset group. during the study period, there were no deaths in any of the groups. although infants were placed on anti-biotics for suspected sepsis, none of the infants had a positive blood or cerebrospinal fluid culture. our results show that late preterm infants of mothers with preeclampsia have worse respiratory outcomes compared to late preterm infants of normotensive mothers. the outcome is significantly worse in infants of mothers with early onset preeclampsia and persisted after controlling for potential confounders. we also demonstrate that late preterm infants of mothers with preeclampsia also have significantly more non-respiratory morbidity. this is the first study to differentiate between outcomes of late preterm infants of early and late onset preeclampsia. studies on the outcomes of late preterm infants of preeclamptic mothers show divergent results with worse outcomes and no difference when compared to late preterm infants of normotensive mothers [ , [ ] [ ] [ ] . these conflicting results are, we believe, due to investigators not differentiating between late preterm infants of early and late onset preeclampsia. gouyon et al., using a large cohort from france, reported higher risk of severe respiratory morbidity, in late preterm infants of mothers with hypertensive disorder of pregnancy [ ] . as in our study, severe respiratory morbidity was defined as the need for mechanical ventilation and/or cpap. habli et al., in a secondary analysis of [ ] . respiratory morbidity, defined by the need for oxygen, cpap or mechanical ventilation was higher at each gestational age in infants of hypertensive mothers but reached statistical significance only at weeks. lagenveld et al. compared outcomes of late preterm infants of mothers with preeclampsia, gestational hypertension and normotensive pregnancies in a large cohort from the netherlands [ ] . they found higher rates of hypoglycemia and sga in the preeclampsia group, results similar to our study. the odds of rds were lower in the preeclampsia group with no difference in ttnb and need for oxygen. however, the study did not have data on antenatal corticosteroid use, systolic blood pressure was not used in the definition of preeclampsia and % of the cohort was excluded because of missing data. masoura et al. reported increased rates of hypoglycemia and rds and lower apgar scores in late preterm infants of preeclamptic mothers [ ] . in a study from australia, of infants - weeks gestation, ventilator support was required significantly more in infants of mothers with preeclampsia [ ] . as in other studies, no distinction was made between early and late onset preeclampsia. our results demonstrate increased ttnb in late preterm infants of both early and late onset preeclampsia and increased rds in early onset preeclampsia. in addition, the use of surfactant cpap and mechanical ventilation was higher in late preterm infants of mothers with preeclampsia, with higher rates in infants of mothers with early onset preeclampsia. as primary cpap is associated with better respiratory outcomes compared with elective intubation, nine of the fifteen infants placed on mechanical ventilation were initially tried on cpap but had to be intubated because of worsening respiratory status and increasing oxygen requirements [ ] . of these nine patients, six were from the early onset, two from the late onset and one from the normotensive group. increased respiratory morbidity in preterm infants of mothers with preeclampsia can potentially be explained by the antiangiogenic state in preeclampsia. several studies have demonstrated higher levels of antiangiogenic factors, soluble vascular endothelial growth factor receptor- (sflt- ) and soluble endoglin and lower levels of angiogenic factors, vascular endothelial growth factor (vegf), and placental growth factor in preeclampsia [ , [ ] [ ] [ ] [ ] . vegf plays a key role in lung vascular development, which promotes alveolar growth, and also enhances surfactant protein production [ , ] . in animal models, vegf prevents rds and higher levels of sflt and lower levels of vegf are described in more severe rds [ ] [ ] [ ] . importantly, this antiangiogenic state is much more pronounced in early onset preeclampsia with levels of sflt much higher and levels of vegf much lower in early onset preeclampsia as compared with late onset preeclampsia [ , ] . in addition, in early onset preeclampsia the fetus is exposed to the hostile intrauterine antiangiogenic environment for a longer period. these factors may be responsible for the worse respiratory outcomes in late preterm infants of mothers with early onset preelampsia as compared with infants of mothers with late onset preeclamsia and normotensive mothers. a negative correlation between sflt levels and birthweight is reported in preeclampsia, with higher sflt levels associated with lower birthweight [ , ] . in our cohort, the lower birthweight in infants of preeclamptic mothers, more marked in the early onset group, can conceivably be attributed to this negative correlation. the composite outcome of cpap and/or mechanical ventilation use in the normotensive group in our cohort was %, similar to the reports by habli et al. and gouyon where it was . % and %, respectively [ , ] . the rate of cpap and/or mechanical ventilation use was higher in the preeclamptic group in our cohort compared to the study by habli et al. ( vs. %) . however, their cohort included both preeclampsia and gestational hypertension which is a less severe disease associated with less respiratory morbidity, diluting the number of infants needing cpap or mechanical ventilation. antenatal steroid use was % in our normotensive cohort and % ( / ) in the entire cohort. these mothers had received steroids for threatened preterm labor before weeks gestation. although no study has reported on the antenatal steroid use in late preterm infants of early and late onset preeclamptic mothers, suga et al. and gymanfi-bannerman et al. reported rates of . % and . %, respectively, in late preterm infants, not dissimilar to our cohort [ , ] . the increased respiratory morbidity in infants of mothers with early onset preeclampsia in our cohort was despite the increased use of antenatal steroids. in addition to being the first study to report on the outcomes of late preterm infants of mothers with early and late onset preeclampsia as distinct groups, there are other strengths to our study. we had a well-defined cohort from a recent era with little or no missing data and detailed demographic characteristics that affect outcomes, especially respiratory outcomes in late preterm infants. this included antenatal steroid use and chorioamnionitis, data that is missing from most studies. importantly, our gestation was based on first trimester ultrasound or the date of embryo transfer in cases of ivf, largely limiting misclassification of gestation. in addition, health care in canada is universal, negating the effect of socioeconomic status and differing antenatal and postnatal care on outcomes. however, there are limitations of our study. we did not investigate outcomes like intraventricular hemorrhage, bronchopulmonary dysplasia, or retinopathy of prematurity, which some studies have reported. these outcomes are, however, extremely uncommon in late preterm infants and an extremely large cohort would be needed to study them. our population was also from single center making generalization of our results difficult. although not the focus of the study, we did not measure any angiogenic or antiangiogenic factors in our population. in summary we report, worse outcomes in late preterm infants of mothers with preeclampsia, which are considerably worse in infants of mothers with early onset preeclampsia as compared with late onset preeclampsia. there is uncertainty between planned immediate delivery or expectant management in women with preeclampsia between and weeks gestation [ ] . our data can be used to counsel mothers with preeclampsia in the late preterm period and help in identifying mothers who may benefit from delivery at facilities with higher levels of neonatal intensive care. our results, however, need validation in a larger cohort. how often are late preterm births the result of non-evidence based practices: analysis from a retrospective cohort study at two tertiary referral centres in a nationalised healthcare system canadian perinatal health report. public health agency of canada the problem of late-preterm (near-term) births: a workshop summary respiratory morbidity in late preterm births a systematic review of severe morbidity in infants born late preterm clinical issues in the management of late preterm infants the economic burden of prematurity in canada effect of late-preterm birth and maternal medical conditions on newborn morbidity risk neonatal outcome associated with singleton birth at - weeks of gestation preeclampsia part : current understanding of its pathophysiology subclassification of preeclampsia incidence of pre-eclampsia: risk factors and outcomes associated with early-versus late-onset disease maternal morbidity associated with early-onset and late-onset preeclampsia clinical differences between early-and late-onset severe preeclampsia and analysis of predictors for perinatal outcome diagnosis of neonatal transient tachypnea and its differentiation from respiratory distress syndrome using lung ultrasound core concepts: respiratory distress syndrome respiratory distress in the term and near-term infant a systematic review and meta-analysis to revise the fenton growth chart for preterm infants canadian hypertensive disorders of pregnancy working group diagnosis, evaluation, and management of the hypertensive disorders of pregnancy chorioaminionitis and funisitis: their implications for the neonate antenatal steroids: are incomplete courses beneficial risk factors, confounding, and the illusion of statistical control backward, forward and stepwise automated subset selection algorithms: frequency of obtaining authentic and noise variables neonatal outcomes in pregnancies with preeclampsia or gestational hypertension and in normotensive pregnancies that delivered at , , or weeks of gestation neonatal outcome of pregnancies complicated by hypertensive disorders between and weeks of gestation: a year retrospective analysis of a national registry neonatal outcomes of late preterm deliveries with pre-eclampsia predicting the need for ventilatory support in neonates - weeks' gestational age strategies for the prevention of continuous positive airway pressure failure circulating angiogenic factors and their association with birth outcomes in preeclampsia circulating concentrations of sflt (soluble fms-like tyrosine kinase ) in fetal and maternal serum during pre-eclampsia circulating concentrations of soluble endoglin (cd ) in fetal and maternal serum and in amniotic fluid in preeclampsia change in amniotic fluid levels of multiple antiangiogenic proteins before development of preeclampsia and intrauterine growth restriction angiogenesis in lung development, injury and repair: implications for chronic lung disease of prematurity high-dose vascular endothelial growth factor increases surfactant protein gene expressions in preterm rat lung loss of hif- alpha and inhibition of vegf impair fetal lung maturation, whereas treatment with vegf prevents fatal respiratory distress in premature mice the role of vegf and its soluble receptor vegfr- in preterm newborns of preeclamptic mothers with rds circulating anti-angiogenic factors during hypertensive pregnancy and increased risk of respiratory distress syndrome in preterm neonates the relationship of angiogenic factors to maternal and neonatal manifestations of early-onset and late-onset preeclampsia the course of angiogenic factors in early-vs. late-onset preeclampsia and hellp syndrome maternal, gestational and neonatal characteristics and maternal angiogenic factors in normotensive pregnancies risk factors associated with respiratory disorders in late preterm infants effect of antenatal corticosteroids on respiratory morbidity in singletons after late-preterm birth is early induction or expectant management more beneficial in women with late preterm preeclampsia? acknowledgements the authors are grateful to the alberta children's hospital research institute for providing the funding for the study. conflict of interest the authors declare that they have no conflict of interest.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -upjhmfca authors: tshilenge mfumu, jean-claude; mercier, annabelle; occello, michel; verdier, christine title: a multiagent-based model for epidemic disease monitoring in dr congo date: - - journal: biomedical engineering systems and technologies doi: . / - - - - _ sha: doc_id: cord_uid: upjhmfca any infectious diseases have been reported in sub-saharan countries over the past decade due to the inefficiency of health structures to anticipate outbreaks. in a poorly-infrastructure country such as the democratic republic of congo (drc), with inadequate health staff and laboratories, it is difficult to respond rapidly to an epidemic, especially in rural areas. as the drc’s health system has three levels (peripheral, regional and national), from the production of health data at the peripheral level to the national level that makes the decision, meantime the disease can spread to many people. lack of communication between health centres of the same health zone and health zones of the same health provincial division does not contribute to the regional response. this article, an extended version of [ ], proposes a well elaborated solution track to deal with this problem by using an agent-centric approach to study by simulation how to improve the process. a new experiment is described by arranging twenty-eight health zones of kinshasa to show how their collaboration can provide unique health data source for all stakeholders and help reducing disease propagation. it concerns also health centres, medical laboratory, provincial health division and rapid riposte teams. the simulation data, provided by provincial health division of kinshasa, concerned cholera outbreak from january to december . the interaction between these agents demonstrated that health zone agent can automatically alert his neighbours whenever he encountered a confirmed case of an outbreak. this action can reduce disease propagation as population will be provided with prevention measures. these interactions between agents have provided models to propose to the current system in order to find out the best that can help reducing decision time. introduction experiencing serious difficulties in improving the living conditions of its population, particularly in the field of basic health care. life expectancy at birth is and respectively for men and women [ ] . the country is currently divided into city-province of kinshasa and other provinces. the provinces are subdivided into territories which are divided into sectors. to facilitate the supervision of health structures, drc health system is divided into three levels [ ] : central, intermediate and peripheral. the nearest level to population is the peripheral area composed of health zones (hz) that coordinate the actions of health facilities. a health zone is divided into health area (ha) containing one or more health centers (hc) and a general referral hospital (grh). the central (national) level defines the policies, strategies and resources of the sector. it enforces strategies and policies at the peripheral level through the intermediate level called the provincial health division (phd), which coordinates primary health care and technical support activities for health zones in a province. provincial health minister (phm) is the political authority of the province. each health zone has a health information bureau (hib) which retrieves aggregated data from all its supervised health area to send to national level for decision measures. hib has a health zone executive team (hzet) that organizes weekly meetings to discuss about suspicious cases to report to hierarchy if needed. health zone executive team manages health facilities (hf) that includes health center and grh. as who member country, drc benefits from the technical and financial support of the partners to respond to epidemics under the conditions stipulated in the international health regulations (ihr) [ ] . all cases of these four diseases must be automatically notified to who: smallpox, poliomyelitis due to wild-type poliovirus, severe acute respiratory syndrome (sars) and cases of human influenza caused by a new subtype. on the ever-changing list of diseases provided by ihr, each country is free to add other diseases, with epidemic potential or not, which constitute a public health problem. access to basic care is difficult for a large part of population. people visit the health facilities in case of extreme emergency. this is more evident in rural areas where the diminishing resources of farmers do not allow them to consult medical services regularly. most of time health care is provided during free medical workers' campaigns. the most usual ways to collect data about cases that must be sent to the hierarchy for decision-making are described below [ ] : -pharmacies must report when same medicines are increasingly purchased by population or when they detect some recurrent treatments; -schools reporting unusual rates of pupil absences due to strange signs and symptoms; figure presents an exhaustive list of actors used in providing health data. despite great efforts to improve disease surveillance and response, drc faces big challenges in identifying, diagnosing and reporting infectious diseases properly due to the remoteness of communities, the inadequate transport and communication infrastructures, and the lack of qualified health staff and laboratory facilities to ensure accurate diagnosis. the challenge, in this paper, is to find new solutions based on real population life and situation to improve health services organization and data sharing in order to detect infectious disease very quickly, organize the response and prevent the spread of disease. in this paper, we present a part of this challenge. we propose a multi-agent system to simulate the interactions between actors working together to organize an optimal response to epidemic detection. when a new case of infectious disease is suspicious in health center, actors will collaborate to report it to provincial health division through health zone executive team. the approach will be based on the current drc heath system processes to extract relevant actors' tasks. the identification of these actors and their tasks will provide the opportunity to simulate a new system that distributes the entire competences of the old heath system to those actors in order to improve their collaboration and eventually shorten the making-decision time response. work-sharing protocols will be proposed to simplify the complexity of the data sources. this paper focuses precisely on improving the process of reporting health data from the peripheral level to the hierarchy for rapid decision-making and anticipate as much as possible the medical response using multi-agent systems (mas). hierarchical dependency between three levels forbids peripheral to directly transmit health data to national level for quick decision. as information must pass through intermediate level (provincial health division), with defective means of communications, it drastically hampers the fight against a propagation of a disease. since decision-making is pushed back to the central level, it can intervene belatedly at the risk of witnessing an alarming spread of an epidemic with high epidemic potential. the next section shows the related work in healthcare and multiagent domains. section describes the healthcare system and problematic in drc. the methodological approach and agent's models are explained in sect. . the model is validated by a simulation presented in sect. . future research directions and conclusion are developed in sect. . information and communication technology is a powerful solution for health care in developing countries [ ] . it made possible the improvement of remote patient follow-up [ ] , controlling the progression of malaria [ ] , improving the uptake of information from health systems [ ] . two main ways of research can be studied in this paper: the use of mobile phone as a relevant medium to rapidly transfer medical data and the multi-agent system that is powerful to simulate organizational skills to anticipate diseases spreading. mobile phone coverage in africa grew from % in , % in to more than % in [ ] . this technology is used to cover numeric fraction. to improve drug adherence and suppression of plasma hiv- rna load in kenyan, mobile phone communication between health-care workers and patients starting antiretroviral therapy was set up [ ] text-message reminders sent to health workers' mobile phones improved and maintained their adherence to treatment guidelines for outpatient pediatric malaria [ ] . phone traces are powerful tools to estimate population migration while investigating an outbreak. these techniques were used to demonstrate the feasibility of rapid estimates and to identify areas at potentially increased risk of outbreaks in haiti. they produced reports on sim card movements from a cholera outbreak area at its immediate onset and within h of receiving data. results suggest that estimates of population movements during disasters and outbreaks can be delivered rapidly and with potentially high validity in areas with high mobile phone. a trial of mobile phone text messaging for diabetes management in an eight-month period to transmit data such as blood glucose levels and body weight to a server that automatically answered with a monthly calculated glycosylated hemoglobin result. the trial results suggest that sms may provide a simple, fast and efficient adjunct to the management of diabetes. in developed countries sms messages have been widely used to remind patients of scheduled appointments car and all, ). similarly, more complex mobile phone applications have shown significant improvement in the follow-up of malaria patients in thailand. the same approaches have been tested in africa as part of the sms reminder package to improve patients' adherence to antimalarial treatment schedules in .six sub saharan countries [ ] . even if text-messaging is the simplest and the most widely easy to use technology function in reporting periodic data from the health system peripheral to control managers, it however needs to be experienced in interventions targeting individual patients, for whom a high facility workload or illiteracy may present a barrier. a multi-agent system is a set of agents situated in a common environment, which interact and try to reach a set of goals. through these interactions, a global behavior, more intelligent than the sum of the local intelligence of multiagent system components, can emerge. by 'agent' we mean a software entity evolving in an environment that it can perceive and within which it reacts. there are several kinds of agents. a reactive agent reacts to the stimuli of the environment. cognitive agents have a more developed intelligence: they manage knowledge and make decisions according to their internal states and according to their perception of the environment. it is provided with autonomous behaviors and some objectives. autonomy is the main concept in the agent issue: it is the ability of agents to control their actions and their internal states. the agents' autonomy implies no centralized control [ ] . when a mas integrates agents on the fly dynamically during the execution, the system is qualified as an opened one. otherwise, it is a closed one. one of the advantages of mas is to model systems where a global description is not possible at any given moment. multi-agent conception is well suitable to model actors described in fig. . simulation based on mas approach has wide potential applications in healthcare. first case, mas approaches are suitable to applications where a complete control is unreachable, the high number of entities or the complexity of entities' behavior make hard to represent the overall system. the second case is related to the monitoring of the epidemics. in a general way, the model of each agents going into action in the system is designed in microscopic level. the environment, the agents' interactions and the social organizations are defined at the macroscopic level. in mas domain, there are numerous methods to approach the analysis and the design of the software application. the methods come from various domains such as object oriented design, knowledge engineering or reproduction of behavior or natural phenomena. the first methods were aaii [ ] which uses an external view (roles services, organization) and internal view (agent design bases on belief desire intention architecture). cassiopée [ ] is a bottom-up approach based on natural behavior and desire [ ] is based on knowledge engineering. the method vowels [ ] allows obtaining modularity at the level of the multi-agents models by decomposing the problem into four elementary facets. other method like gaia [ ] extends the concepts used in classical object engineering and provides a microscopic and a macroscopic view of the software system. there are also complete approaches for developing multi-agents systems from the analysis to the deployment by using mase [ ] or prometheus [ ] . moreover some models al-low the designer to develop the agent like agr [ ] based on agent/group/role or bdi [ ] based on belief/desir/intention. the main concept of all those approaches is the agent and the communication between agents to lead to the main objective of the software. the general classification is clinical, operational, managerial and educational simulation. managerial and operational simulations are closely interrelated. both are the core components for healthcare process management. some challenges and trends of simulation models in healthcare in the past two decades have been developed the design of a web-based clinical decision support system that guides patients with low back pain in making suitable choices on self-referral has been experienced in netherlands. mas is used to describe an approach to the analysis and development of telemedicine systems [ ] , to manage communications in wireless sensor networks [ ] , the epidemiological decision support system [ ] , the care of seniors at home [ ] , decision-making for monitoring and prevention of epidemics [ ] , evaluation of disaster response system [ ] , medical sensor modules in conjunction with wireless communication technology supporting a wide range of services including mobile telemedicine, patient monitoring, emergency management and information sharing between patients and doctors or among the healthcare workers [ ] . mas can be considered as a suitable technology for the realization of applications providing healthcare and social services where the use of data and remote collaborations among users are often the most requirements [ ] . cooperation in agent technology can provide better healthcare than the traditional medical system [ ] . real programs built on the multiagent paradigm are still evolving towards a complete maturity. the variety and complexity of the e-health scenario make it one of the most interesting application fields, able to check the advantages of their use to condition their evolution [ ] . mas was used to monitor a generic medical contact center for chronic disease environment, detect important cases, and inform the healthcare and administrative personnel via alert messages, recommendations, and reports, prompting them to action [ ] . developed mas applications in healthcare can provide a reasonable way to mitigate the cost due to increased demand for services [ ] . an agent-based model (abm) with geospatial and medical details was used to evaluate the efficiency of disaster responders to rescue victims in a mass casualty incident situation in south korea [ ] . abm can cooperate to share tasks between sensors observing a phenomena [ ] , to manage diabetes treatment between caregivers and patients. the usability evaluation of a collaborative information system for dementia assessment built using a usercentered design approach was experienced in norway [ ] . but from several research papers we have reviewed we didn't find a paper addressing abm in sharing tasks from multisource health information to organize a rapid response to a high epidemic potential disease. the patient health care and the reporting of suspicious cases are managed at the peripheral level by health facilities (hf): health centers plus the general referral hospitals. health data collected by the health facilities are transmitted to health zone executive team for consolidation, analyzes and decision-making. aggregated data from entire health zone are also transmitted to provincial health division. each week this intermediate level structure convenes meetings to analyze data from each health zone, decide on actions to take. provincial health division produces consolidated data for its province. provincial health division must transmit the health data from its province to the central level for a second analysis and national consolidation. if suspicious cases reported by health zone require deeper investigation, laboratory tests or kits intervention, provincial health division will ask for technical and financial supports from central level. disease control direction (dcd) is a central respondent at central level. it also organizes weekly meetings to analyze health data from all provinces. it often provides advice and recommendations to provincial health division to monitor suspicious cases in accordance with the national policy of the sector. whenever provincial health division asks for a help, dcd can approach government authorities, special programs, partners and even the international community, to fill up needs. difficulties encountered by health facilities to better report information and structures dependency are well expressed at next section. the first challenge in managing epidemics begins with the multi-sources data processing at the health zone level. national policy has provided list of groups of individuals who can retrieve information from suspicious cases. this information transmitting by phone calls or narrative is not exhaustive. hence, the interest in diversifying the mode of communication by adding text and voice sms, tweets and phone calls on green lines can enhance data completeness. a second difficulty in an accurate identification of suspicious cases is the insufficient number of qualified health staff [ ] . despite training courses organized by health zone executive team for community relays and staff of health facilities, there are gaps in the implementation of the information brought to their attention. for example, the provincial health division can conduct a thorough investigation with qualified staff as soon as the number of suspicious cases reaches the threshold for the pathology. lack of information on the list of the nearest laboratories delays response time to confirm cases and ensure accuracy of diagnosis. hierarchical dependences do not favor communication between structures of the same level such as health areas of a same health zone or of contiguous health areas but belonging to different provincial health division. this lack of dialogue can lead to the non-detection of an epidemic for the simple reason that the number of suspicious cases to organize investigation is not reached in an health zone. however, by combining number of suspicious case found in contiguous health areas, we could detect the pathology at the intersection of the provinces. structures authorized to report information relating to suspicious cases to health zone executive team are health facilities. but, community relays can also report their observations that need to be considered by health facilities. reports concern pathologies described at international sanitary regulations (smallpox, poliomyelitis due to wild polio virus, human influenza and severe acute respiratory syndrome (sars)) and local list of diseases with epidemic eradication measures or elimination and other chronic diseases provided by authorities. as soon as it appears, suspicious cases must be transmitted to health zone executive team by all data providers indicated on fig. . when number of suspicious cases in health zone equals to the threshold of observed pathology, a rapid riposte team (rrt) has to investigate some health center and the population of the concerned health area to make sure the allegation was correct. the investigation of rapid ripost team could result to laboratory tests of some samples. in case of riposte many hierarchical structures such as provincial health division or national level would intervene to provide technical and financial supports. the process used to organize riposte simulation is heavily based upon computer science, mathematics, probability theory and statistics: yet the process of simulation modeling and experimentation remains very much an intuitive art. simulation is a very general and somewhat ill-defined subject. for the purpose of this paper, we will define simulation as «the process of designing a computerized model of a system and conducting experiments for the purpose of understanding the behavior of the system and/or of evaluating various strategies for the operation of the system. thus we will understand the process of simulation to include both the construction of the model and the analytical use of the model for studying a problem shortly described in fig. . health zone executive team analyze the report of surveillance to determine if the number of suspicious case has reached the threshold to order an investigation. rapid ripost team will research new cases at health area according to the clinical definition of case. it will find out new determinants of the outbreak to report to provincial health division in other to realize the response. a final evaluation of outbreak response presented as a report of the process can be shared with other health zone and health facilities. this type of system is well suited to mas using an aeio representation. real system is analyzed with four elements: agent, environment, organizations and interactions between agents. this model will be detailed in the next part. the hierarchical organization of the healthcare system in drc is a good candidate for a multiagent model because there are several kinds of agents with personal goals sharing the same global achievement. in the process described in fig. , the agents use some knowledge and tasks to perform a main goal together: collecting data in order to respond with efficiency to epidemic. at the stage of this research, the simulation's objective is to understand how the drc's healthcare system reacts in epidemic diseases. it is a preliminary work before (i) determining metrics to analyze process simulations and (ii) developing modules in embedded systems -phones or tablets-to assist the end-user in the data collection, coupled with the multiagent system. multiagent-based-simulation (mabs) allows explicitly modeling the behavior of each individual and viewing the emergent system from the interactions between the individuals. morvan in [ ] proposes a survey on mabs and presents several multiagents platforms. in these existing platforms, we have not found solutions which can act as both a simulation system and a tool to end-users on embedded systems. diamond method and its associated simulator mash -multiagent software hardware simulation -developed in lcis laboratory provided the capacity of testing a method in a tool of simulation [ , ] . diamond -decentralized iterative multiagent open network design -is a method that guides the designer from the requirements to the implementation. it is based on a-e-i-o decomposition for agent, environment, interactions and organization of the system. the agent dimension concerns the model of the reactive agent represented by a simple automaton, the cognitive agent manipulating information or a more complex agent based on a knowledge system. the environment is the context in which the agent reacts, its geographic location. the interaction dimension specifies the way the agents communicate; it mainly consists of protocols of interaction or the type of communication. the organization dimension reflects the structural relations between the agents (group, hierarchy, market). these four key concepts are considered under a global angle (the society) and a local one (agent view). the diamond method proposes an analysis phase of the problem in four steps: the situation phase helps to find the society's circumference to be represented by defining the limits of the system, agents and environment; the individual phase concerns the internal functioning of the agents (behavior and the knowledge); the social phase defines the relations between agents, particularly by integrating into its knowledge communication protocols and information structures to understand the society organization and -the phase of socialization consists of integrating the individual agents into the society by adding the social influences into its behavior (possible answers in the requests from the outside, the launch of interaction protocols and the choice to be made according to its position in the organization). we decide to use this method because it allows explicitly designing the behavior of each individual. however, coupled with the mash simulator platform, it is useful to view the emergent system from the interactions between the individuals. the process in fig. can be modelized by agents able to be simulated in the mash simulator. the mash simulation platform was used to simulate systems with embedded and software agents. it is suitable to our problem because we plan to provide a tool for collecting data with phones or tablets applications. to have an individual-centered vision of the process is one of the advantages of this simulation. afterwards, we will be able to contribute to the improvement of the process with an exterior view by proposing changes and ideas to reduce the response time for example. this section shows the steps to break down multiagent system's elements. to start the analysis, each individual agent's behavior is studied. it is a way of seeing things at a micro level. the phenomenon at macro level does not change and the process remains the same even if observer's level changes. the objective is to be able to adjust the behavior of each individual agent and probably to add some skills to certain nodes or node types. the first step of this approach is to find out what to model as agents from information of the process. in our study context, agents are: health centers, general referral hospital, health areas, provincial health division, any national health entity related to the administrative structures, health zone executive team and rapid ripost team for human team working group. figure shows for instance the internal behavior of one agent (rapid ripost team). for each agent, we have to list all its skills, what information will be required to store or to handle and how agent acquires this information. this information should be acquired directly by perception (e.g. the user grasps something) or on demand by asking other nodes (higher hierarchy or same level nodes). in this step, we obtain for each agent a vision of the relevant knowledge to perform its individual tasks. that information is required by agents working in the same environment. the result is a set of tasks that an agent can perform. these tasks correspond to the skills of each node. some skills are executed by one agent without the need of other agents. but to achieve a goal, an agent should have partial information and should ask to other agents to complete their goal. however, this will result to a cooperative behavior in place of an individual one which is entirely internal to agent. this kind of social behavior reflects an interaction between several agents: either to gain information or to share tasks. social behavior. in this second step, we will have to create interactions between nodes for example to back up information (health center to health zone executive team) or to receive orders (health zone executive team from provincial health division). these interactions should intervene between different partner groups such as health areas. in the implementation, we define very simple interaction protocols for data exchange such as receiving information, answers/queries or order to perform a task. for some tasks, such as health alert surrounding areas, it is no longer just a request for information but cooperative behavior brings into play several kinds of agents. to communicate with others, agent uses interaction protocols. we will there-fore express how this behavior will be realized by defining a more sophisticated interaction protocol than query/response. various protocols are available for negotiating, giving orders, waiting for answers. the interaction patterns that will govern this cooperative behavior will be organized between the agents. interaction protocols. the protocol is a part of the agents' knowledge. in our simulation, agents have a list of protocols they should initiate to answer others. for the moment, we use a simple protocol with two states as represented in fig. : "inform" and "inform back". for example, agent a launches an instance of protocol p , with the state s . the agent a receives a call from a with the performative "information" in the state s . a knows the protocol and searches the next transition; it slips into state s and sends an acknowledgment to agent a . a treats the message and the conversation finishes. the acl fipa compliant performatives are used: • rdcmessage.acl_query_ref for queries/answers, • rdcmessage.acl_request for an order to perform a task and, • rdcmessage.acl_inform for inform/acknowledgment. position in the organization. the last step is to consider an agent's position in organization when he initiates interactions with others. an organization should be a hierarchy or a simple group. for example, to alert neighboring health zone, health center agents must know the surrounding areas of health zone, which is a group or an organization in the multiagent system, and make a decision based on its position in this group. the agent's position in the organization is integrated in the decision loop. agent's internal decision making loop. the previous steps showed agent's skills, agent's complex behavior (internal and social) and the knowledge of interaction protocols. on the hypothesis that each agent gets this information, we can now build the agent decision loop. on one hand purely individual behavior runs only with an agent's context information and does not need other agents to complete the agent goal. on other hand, social behavior involves relationships between agents. an individualcentered approach defines agent at micro level so that interactions with other agents have to be merged with the internal behavior in the agent's decision loop. the individual and cooperative behaviors are both integrated into the decision loop. in the individual behavior, a set of tasks is launched in the internal decision loop. in its decision loop, the agent should have to respond to the message from others, which are part of interaction protocols initiated by other agents or parts of the agent interaction patterns. these tasks have to be synchronized with the messages received from others agents. as an external view, huge decision loops, which are decentralized in the several kinds of agents, seem to be synchronized at the system level. but in fact, each agent decides in what state to pass according to its knowledge and the state of its interactions. agents would collaborate to achieve some objectives. to investigate on health area, rapid ripost team must wait for an order from health zone executive team. the later receives health data every week from health center and checks if the threshold of the followed pathology has been reached. the same collaboration is needed between rapid ripost team and health area, rapid ripost team and laboratory. the sequence diagram (fig. ) gives a snapshot of the kind of collaboration found in agents concerned with an outbreak investigation. message format for interaction. the messages exchanged between agents contain sender and receiver agents, protocol information and data to manage like [sender; receiver; conversation; perform; protocol; inst_prot; state_prot; data]. the data follow a format according to the performative. to interact through a message sent by another agent, a simple protocol is established. for instance when rapid ripost team asks a laboratory to perform exams, he has to first check its state to be convinced that it can answer his request. a simple protocol with acknowledgment is used. the agent changes state when he asks for information and when he receives answers to his request (fig. ) . in a future simulation, a negotiation protocol with a call for proposal to several laboratories will be tested. however, the agent launching the conversation should negotiate among laboratories which one is available, near or powerful. the analyze of the cycle of outbreak response presented on . isolated some individuals playing different roles to achieve the objectives assigned to the riposte process. a simple class diagram (fig. ) gives a quick overview of main actors. health center as agent can exchange message with agent 'health area' to get information about rivers crossing the area of its location. since this knowledge is crucial to anticipate cholera epidemic in raining season, this collaboration is very important. rapid riposte team (rrtagent) investigate on suspicious case in a health area which means visiting population and health centers found in it if the disease requires laboratory analyzes to confirm the case rrtagent must take samples and transmit it to laboratoryagent available. reported worldwide due to fear of economic consequences, often insufficient surveillance systems and a lack of standardized terminology to define a case of cholera. the disease results from the absorption by the mouth of water or food contaminated by the cholera vibrio. after a few hours to a few days of incubation, violent diarrhea and vomiting occur, without fever. in the absence of treatment, death occurs in to days, by cardiovascular collapse (fall in blood pressure) in to % of cases. mortality is higher among children, the elderly and vulnerable individuals. the treatment consists essentially of compensating the digestive losses of water and electrolytes. rehydration is given orally or intravenously, depending on the degree of dehydration. the improvement is noticeable after a few hours and healing, without sequelae, is obtained in a few days. antibiotic therapy is recommended by who only for severe dehydration. in dlm is a disease control direction located in kinshasa which collects the national data for disease monitoring. it provided us with ten years data of cholera outbreak from to november . we extracted the data for kinshasa grouped on its health zones to compare with provincial health division's data. provincial health division's data contained more details about health centers that reported the suspicious cases and theirs health area. we analyze the data from first january to december th . we reported cumulated data of each health zone to a map, as shown in fig. , to find their neighboring and try to suggest the best way to establish collaboration between them in order to stop disease propagation. we focus on two groups of health zones. in the first group we have binza-météo, mont-ngafula, kokolo and kintambo. the second contains limete and kingabwa. our hypothesis was: if actors from each heath zone could exchange disease information with their neighbors as soon as an outbreak happens, it would be possible to reduce the propagation. for example, epidemic began at kintambo on january . as the communication and sensitization weren't establish with its neighbors, some weeks after disease was reported from kokolo and binza-météo. to test and evaluate our approach, we adapted mash simulator developed by lcis lab for a wireless sensor multiagent system [ ] . we focus our simulation to these health zones: binza-météo, mont-ngafula, kokolo, kintambo, limete and kingabwa. the simulation concerns precisely kintambo and limete that register more suspicious case of cholera outbreak during and from them other neighboring health zones were affected. the main idea is to see how the future system would react if each actor of health system could perform its own task with autonomy. these experiences could result to many scenarios and the best of them will be proposed to drc's health system to reduce decision time as each actor can execute his talks according to the knowledge of the environment and the outbreak determinants he will be provided with. we chose two health-zones of kinshasa provincial health division for the simulation. kintambo and limete are health zones that register respectively and suspicious cases with death in . the epidemic began from them and the propagation of disease affected their neighbors with an important amount of suspicious cases. kokolo counted cases while binza-météo registered suspicious cases at the same period. to respond to an outbreak noticed at a health center, health staff of the concerning health center must refer to health zone executive team. in their turn, health zone executive team must refer to provincial health division and provincial health division to central level. this chain of hierarchical contacts can enlarge time decision. in our simulation, we worked with these hypotheses: (i) each health center actor must contact immediately its health executive team as soon as it encounted a suspicious case; (ii) health zone executive team cumulate suspicious case and create an rrt when the disease's threshold is reached; (iii) rrt can contact the nearest laboagent able to answer to his request or to use his information to make decision. we considered twenty-eight health zone executive team in yellow or red, forty seven health center in green or blue, rrt in grey one provincial health division and one medical test laboratories (laboagent). the first suspicious case was detected in health center # in green. figure illustrates those actors working as agents. the below map represents health zones in north of kinshasa. the simulation trace file (fig. ) shows the communication between agents. as they are autonomous they perform their own tasks like "cumulate new cases", "conduct the investigation on health zone # " and manage messages like "realize analyzes from health center", "receive sample to_ analyze" or "report suspicious case detected". hzet agents (# and # ) are waiting for a suspicious case message from any hc. whenever health zone executive team agent receives such a message he computes cases and compare with the threshold of disease monitoring to decide the necessity of building a rrt agent able to investigate in health centers around suspicious case in health area. the visited health center will turn from green to blue color. rrt agent could send samples to laboagent while searching for new cases in health areas. the organization of outbreak riposte could depend on the results from laboagent and investigation report from rrt agent. the agents operate independently: hcagent transmits data to health zone executive teamagent, rrtagent completes a full investigation, laboagent conducts medical testing and transmits results to hzetagent and phdagent which manages all information from health zone executive team under its supervision. message synchronization between kinds of agent is done in the agent's decision loop. a protocol with two states is used and implements kqml-like performatives. the four numbers in the message are " " for inform (give information), " " for query information (ask for an information) and " " for request (ask for a task to be done). the agents communicate and achieve their goal by reacting to messages from others or executing their inner task as response to a query. with positive results from laboagent, health zone executive team sends warning and preventive measures to his all health centers. phdagent can also alert the surrounding health zone executive teams. in this paper, we have showed how multi-agent system can improve the organizations' tasks in order to decrease the time to response when an epidemic disease is suspected or detected. a main research result can be highlighted: the use of a multi-agent method previously dedicated to wireless sensors and applied to human organizations. we have proved that the method was generic enough and gave good results with real data and a hierarchical and complex health eco-system. mas is often used in health domain and our paper's result complete the panel of applications with real data and under eco-system constraints. some limits must be underlined: (i) in order to adapt the method, we have defined hypothesis that strongly constraint the models; (ii) the stakeholders have been introduced in the method only with their job characteristics and adding their experience in the simulation can probably enhance the results. nevertheless the multiagent method can cover only one part of the global problem. we discussed about an organizational method useful to enlarge all aspects of the problem laid down. we propose in the future works to widen the approach to take into account different and complementary aspects: health data collection and transmission, health data quality, improvement of the complete riposte process, improvement of the health system organization. in order to, we propose a three-phases innovation method named chicken useful to supervise and improve the epidemic disease riposte: -phase : define the life cycle of the epidemic disease to watch over. -phase : health data monitoring -phase : riposte and feed-back each phase of this innovation method is build with models, methods' fragments, tools coming from different sciences domains and proposes a road map to improve the riposte and the health data quality. then the multiagent method becomes a method's fragment in the wider method chicken. towards an agent-based model to monitor epidemics and chronic diseases in dr congo programme des nations unies pour le développement: rapport sur le développement humain organisation mondiale de la santé rdc: guide technique pour la surveillance intégrée de la maladie et riposte icts for poverty 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technology and future roadmap a multiagent tool to simulate hybrid real/virtual embedded agent societies usability evaluation of a collaborative health information system -lessons from a user-centred design process multi-level agent-based modeling -a literature survey key: cord- -zid e z authors: forkpa, hawa; rupp, angela h; shulman, stanford t; patel, sameer j; gray, elizabeth l; zheng, xiaotian; bovee, maria; kociolek, larry k title: association between children’s hospital visitor restrictions and healthcare-associated viral respiratory infections: a quasi-experimental study date: - - journal: j pediatric infect dis soc doi: . /jpids/piz sha: doc_id: cord_uid: zid e z we investigated the effect of annual winter visitor restrictions on hospital respiratory virus transmission. the healthcare-associated (ha) viral respiratory infection (vri) transmission index (number of ha vris per inpatient community-associated vris) was % lower during the months in which visitor restrictions were implemented. these data prompt consideration for instituting year-round visitor restrictions. healthcare-associated (ha) viral respiratory infections (vris) are diagnosed frequently in children's hospitals [ ] and are associated with increased morbidity, mortality rates, and healthcare costs [ ] . nearly % of children's hospitals implement visitor restriction policies and practices (vrpps) [ ] , such as restricting the number of visitors, visitation by other children, and visitation by people with vri symptoms. despite the pervasiveness of vrpps in children's hospitals, the effectiveness of visitor restriction is poorly understood. our objective was to understand the effect of annual winter hospital vrpps on the transmission of respiratory viruses in our children's hospital. to assess the effectiveness of annual winter vrpps, we performed a retrospective quasi-experimental study at lurie children's hospital of chicago, an academic freestanding tertiary care children's hospital with private rooms, including the neonatal intensive care unit. no changes in the hospital's physical structure occurred during the study period. our hospital first implemented visitor restrictions in late january . our winter vrpps include limiting the number of visitors ( in icu rooms, in acute care rooms, including parents/guardians) and restricting visitation by all nonsibling children < years old, siblings < years old, and any person with vri symptoms. the timing of annual winter vrpp implementation varied slightly each year depending on community vri incidence, particularly that of respiratory syncytial virus (rsv) and influenza. during vrpp periods, visitors were screened by the concierge staff in the lobby of the children's hospital for symptoms of viral respiratory and gastrointestinal illness before being granted access to patient care areas and screened again by bedside nurses in patient rooms. suggested scripting was provided to the concierge and nursing staff. nonparent adult visitors with viral respiratory or gastrointestinal symptoms were restricted from visiting. parents with viral respiratory symptoms were allowed to visit but encouraged to wear a surgical mask and practice hand hygiene and respiratory etiquette. bedside nurses were expected to document visitor illness symptoms in the electronic medical record. compliance with vrpps was not monitored. all clinical and nonclinical hospital employees were required to receive an annual influenza vaccine throughout the study period. hospitalized patients were considered to have a vri if their result from a respiratory viral polymerase chain reaction (pcr) test was positive. throughout the study period, we used the filmarray respiratory panel (biomérieux, marcy-l'Étoile, france), which detects viral species (table ) . of note, this assay cannot distinguish between rhinoviruses and enteroviruses. thus, a positive result for either of these viruses is reported as rhinovirus/enterovirus. we also used various combined influenza/rsv pcr assays during winter months; influenza/rsv pcr testing was recommended primarily for outpatients. ha vris were diagnosed on or after hospital day . infection control personnel confirmed vri symptom onset on or after hospital day in all patients with ha vri. community-associated (ca) vris were diagnosed in inpatients before hospital day . we measured monthly ha-vri incidence over four -to -month vrpp seasons (february to april ) and compared the incidence of ha vri in vrpp months with that in non-vrpp months. incidence was expressed as ha-vri incidence density (number of ha vris per patient-days) and vri transmission index (number of ha vris per inpatient ca vris). because seasonal ca-vri incidence varies substantially between vrpp and non-vrpp periods, and because ha-vri transmission depends on inpatient ca-vri burden, we normalized ha-vri incidence to ca-vri burden by calculating the vri transmission index, as previously described [ ] . hand hygiene and personal protective equipment compliance, which were monitored monthly by direct observation in all inpatient units throughout the study period, were also compared between the vrpp and non-vrpp periods. transmission indices, hand hygiene compliance, and personal protective equipment compliance were compared using the pearson χ test with yates continuity correction (r . . [r foundation, vienna, austria]). any -sided p value of <. was considered statistically significant. the lurie children's hospital of chicago institutional review board exempted this study from review. there were ha vris during the study period; and ha vris occurred during vrpp and non-vrpp months, respectively ( table ). the median length of stay at the time of ha-vri diagnosis was days (interquartile range, - days). the incidence density of ha vris was similar between vrpp and non-vrpp months ( . vs . ha vris/ patient-days, respectively). however, when we normalized the incidence data for inpatient burden of ca vris, the hospital transmission index of vris was significantly lower in vrpp months. in more specific terms, the aggregate vri transmission index was % lower in vrpp periods than in non-vrpp periods ( . vs . ha vris/ inpatient ca vris, respectively; p < . ) ( healthcare provider infection prevention practice compliance was high during both the vrpp and non-vrpp months. hand hygiene compliance was observed in ( . %) of and ( . %) of observations during the vrpp and non-vrpp months, respectively (p = . ). personal protective equipment-donning compliance was observed in ( . %) of and ( . %) of observations during the vrpp and non-vrpp months, respectively (p = . ). personal protective equipment-doffing compliance was observed in ( . %) of and ( . %) of observations during the vrpp and non-vrpp months, respectively (p = . ). although nearly % of children's hospitals implement vrpps [ ] , the effectiveness of these practices was characterized only recently in multiple single-center studies [ , ] . our data indicate that vrpps implemented in the winter at a freestanding children's hospital are associated with reduced hospital transmission of respiratory viruses compared with that in nonwinter months when vrpps are not in place. our findings are consistent with those of recently published studies at other large freestanding children's hospitals, which reported reduced ha-vri incidence density after winter vrpp implementation [ , ] . the specific vrpps implemented vary substantially among children's hospitals [ ] ; optimal vrpps have not been defined yet. however, a bundled approach for ha-vri prevention that includes winter visitation limitations, hand hygiene, visitor symptom screening, and close monitoring of routine infection-prevention practices has been shown to effectively reduce ha-vri incidence density by % [ ] . despite ha-vri reduction with vrpps in our study and others, ha vris remain common in winter months, which suggests that additional strategies for reducing ha vris are needed. although vrpps are often limited to the winter months when community incidence of rsv and influenza rises, our data also suggest that seasonal vrpps can result in unacceptably frequent hospital transmission of respiratory viruses, particularly rhinovirus/enterovirus, during nonwinter months. a similar trend has been found in other pediatric studies [ , , ] , which demonstrates the high burden of ha-rhinovirus infections. rhinoviruses and enteroviruses are often benign in otherwise healthy children. however, certain enteroviruses are associated with severe respiratory disease (eg, enterovirus d ) [ ] and severe neurologic sequelae, such as acute flaccid paralysis (eg, enteroviruses d [ ] and a [ ] ), even in otherwise healthy children. furthermore, rhinoviruses are associated with death in highly immunocompromised hosts [ ] . in patients after stem cell transplantation, rhinoviruses cause a rate of pulmonary complications similar to that caused by for rsv, parainfluenza, and human metapneumovirus [ ] . frequent hospital transmission of rhinoviruses/enteroviruses in units that house immunocompromised children and other children at high risk for infection supports consideration of implementing yearround vrpps for preventing rsv/influenza. in contrast to other recent studies of ha-vri epidemiology [ , ] , our study was strengthened by normalizing ha-vri incidence data to inpatient ca-vri burden. by using vri transmission indices, rather than incidence density, as was done in a previous study of influenza and rsv [ ] , we limited bias related to profound differences in seasonal and year-to-year community incidences of many respiratory viruses. however, our study also had several limitations. as a quasi-experimental study, we cannot rule out the effect of other factors that might have differed between the vrpp and non-vrpp months. however, many other factors that can affect hospital transmission of respiratory viruses did not change during the study period. for example, our hospital included all private rooms throughout the figure . aggregate monthly viral respiratory infection transmission indices during visitor restriction policy and practice (vrpp) (shaded) and non-vrpp (nonshaded) periods. the horizontal line and corresponding numeric label in each period represent the overall transmission index for that particular period. we found . fewer healthcare-associated (ha) viral respiratory infections per inpatient community-associated (ca) viral respiratory infections during the vrpp periods, which represents a % decrease compared to that in the non-vrpp periods. study period, and no physical changes were made to the hospital environment. there were no identified clusters of ha vri during the study period that could have led to real-time vrpp modifications. annual influenza vaccination was mandated for employees throughout the study period. furthermore, we found no significant differences in observed hand hygiene or personal protective equipment use between the vrpp and non-vrpp months. although we noted a statistically significant difference in hand hygiene compliance between the vrpp and non-vrpp months ( . % vs . %, respectively; p = . ), we consider it clinically insignificant. furthermore, even if clinically significant, hand hygiene compliance was higher during the non-vrpp months, which would bias our ha-vri transmission data toward the null. compliance with symptom-based isolation at the time of hospital admission is monitored for all patients by infection preventionists year-round, and these practices do not have seasonal variation. we acknowledge that not all patients with vri symptoms had necessarily been tested and their data captured by electronic laboratory surveillance, and we cannot confirm that all patients with a ca vri had respiratory symptoms. however, at our institution, vri testing is discouraged for patients without a compatible illness, and our clinical observations suggest that the multiplex pcr panel is used routinely year-round in inpatients with a compatible illness. in addition, the number of ca-vri cases identified in nonwinter months is consistent with the expected seasonal variation of individual viruses, which suggests that case ascertainment based on seasonal differences in testing decisions was not a source of significant bias. because of the exceedingly low influenza/rsv incidence during nonwinter months, this study was not adequately powered to detect differences in influenza/ rsv transmission or aggregate non-rhinovirus/enterovirus vri transmission index. on the basis of our incidence and transmission data, vrpp seasons would be required to detect a statistically significant difference in the aggregate non-rhinovirus/ enterovirus vri transmission index. we were limited to vrpp seasons because vrpps were not implemented before . in summary, our data indicate that winter vrpps are associated with reduced hospital transmission of respiratory viruses, particularly rhinoviruses/enteroviruses, which were the most commonly transmitted viruses in our study. because of the high burden of ha rhinovirus/enterovirus, particularly in hospital units that house immunocompromised children and other children at high risk for infection, strong consideration should be given to implementing vrpps year-round. additional work is needed to understand how best to limit visitors with communicable respiratory illnesses while maintaining patient-and family-centered care. financial support. l. k. k. is supported by the national institute of allergy and infectious diseases, national institutes of health (grant k ai ). research reported in this publication was supported in part by the national institutes of health, national center for advancing translational sciences (grant ul tr ). disclaimer. the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health. potential conflicts of interest. all authors: no reported conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. burden of healthcare-associated viral respiratory infections in children's hospitals hospital-acquired respiratory viral infections: incidence, morbidity, and mortality in pediatric and adult patients visitor restriction policies and practices in children's hospitals in north america: results of an emerging infections network survey the impact of infection control upon hospital-acquired influenza and respiratory syncytial virus development of a novel prevention bundle for pediatric healthcare-associated viral infections association of visitation policy and health care-acquired respiratory viral infections in hospitalized children severe respiratory illness associated with enterovirus d -missouri and illinois a novel outbreak enterovirus d strain associated with acute flaccid myelitis cases in the usa ( - ): a retrospective cohort study notes from the field: enterovirus a neurologic disease in children-colorado human rhinovirus detection in the lower respiratory tract of hematopoietic cell transplant recipients: association with mortality a multicenter consortium to define the epidemiology and outcomes of inpatient respiratory viral infections in pediatric hematopoietic stem cell transplant recipients key: cord- -l c x tb authors: klomp, richard w.; jones, laurie; watanabe, emi; thompson, william w. title: cdc’s multiple approaches to safeguard the health, safety, and resilience of ebola responders date: - - journal: prehosp disaster med doi: . /s x sha: doc_id: cord_uid: l c x tb over , people were sickened by ebola and over , people died between march of and june of . the us centers for disease control and prevention (cdc; atlanta, georgia usa) was one of many public health organizations that sought to stop this outbreak. this agency deployed almost , individuals to west africa during that timeframe. deployment to these countries exposed these individuals to a wide variety of dangers, stressors, and risks. being concerned about the at-risk populations in africa, and also the well-being of its professionals who willingly deployed, the cdc did several things to help safeguard the health, safety, and resilience of these team members before, during, and after deployment. the accompanying special report highlights innovative pre-deployment training initiatives, customized screening processes, and post-deployment outreach efforts intended to protect and support the public health professionals fighting ebola. before deploying, the cdc team members were expected to participate in both internally-created and externally-provided trainings. these ranged from pre-deployment briefings, to preparing for work overseas (pfwo) and public health readiness certificate program (phrcp) courses, to incident command system (ics) , , and courses. a small subset of non-clinical deployers also participated in a three-day training designed in collaboration with the center for the study of traumatic stress (csts; bethesda, maryland usa) to train individuals to assess and address the well-being and resilience of themselves and their teammates in the field during a deployment. participants in this unique training were immersed in a virtual reality environment (vre) that simulated deployment to one of seven different types of emergencies. the cdc leadership also requested a pre-deployment screening process that helped professionals in the cdc’s occupational health clinic (ohc) determine whether or not individuals were at an increased risk of negative outcomes by participating in a rigorous deployment at that time. when deployers returned from the field, they received personalized invitations to participate in a voluntary, confidential, post-deployment operational debriefing one-on-one or in a group. implementing these approaches provided more information to clinical decision makers about the readiness of deployers. it provided deployers with a greater awareness of the kinds of challenges they were likely to face in the field. the post-deployment outreach efforts reminded staff that their contributions were appreciated and there were resources available if they needed help processing any of the potentially-traumatizing things they may have experienced. the ebola outbreak in guinea, sierra leone, and liberia sickened over , people between march of and june of , and resulted in over , confirmed deaths. it attracted the attention of people all around the world and required the dedicated efforts of countless caring individuals from diverse organizations and numerous countries. almost , public health professionals at the united states centers for disease control and prevention (cdc; atlanta, georgia usa) deployed to west africa. they tested patient samples, communicated health messages, educated health care workers, advised travelers, trained officials, and interviewed people who might have been in contact with ebola patents. approximately , additional cdc personnel contributed by working around the clock in cdc's emergency operations center (eoc) in atlanta, georgia. senior cdc leadership tasked their office of safety, security, and asset management's (ossam) worklife wellness office (wwo) to set up a pre-deployment screening process to reduce the likelihood of deploying someone at-risk of negative mental health outcomes in an emergency response. the cdc added this new screening to pre-deployment safety and resilience briefings, classroom resilience training, and post-deployment outreach initiatives that have been a part of cdc deployments since . the cdc leadership prioritized providing rapid, stigma-free assistance for all individuals who might desire it, who had worked in inhospitable and potentially dangerous environments. the wwo resilience program submitted a request for project determination and approval to the cdc institutional review board (irb). the irb provided a waiver for the report of this resilience-related project. in this report, resilience is defined as "the ability to withstand, recover, and grow in the face of stressors and changing demands." the cdc strongly recommended a variety of trainings focused on topics such as travel safety, personal security, and the incident command system (ics; ics , , and ) for cdc staff preparing to deploy to the field. multiple cdc offices had developed deployment preparation courses and asked wwo to conduct resilience-enhancing segments of those classes. the cdc's three-day "preparing for work overseas" (pfwo) class, which addressed learning objectives from the us department of state (washington, dc usa), included a one-hour section on "physical and mental resiliency while traveling." in that section, instructors shared resilience basics along with diaphragmatic breathing and muscle relaxation techniques designed to combat the well-known fight-or-flight response. the cdc also hosted a four-day training titled: "public health readiness certificate program" (phrcp). this training included a one-hour segment on "deployment resiliency." almost , cdc staff completed these kinds of resilience-related trainings. records show that , staff participated in other wellness offerings, such as on-site support for eoc staff, weekly physical activity sessions, and stress management classes during the ebola response timeframe. pre-deployment trainings also provided valuable information, presented a variety of common responses to unusual situations, and encouraged potential deployers to prepare mentally and emotionally for a deployment. however, for many years, the cdc lacked personnel specifically trained to provide resilienceenhancing support services in-country to deployed cdc staff. several years ago, cdc resilience experts conducted an environmental scan to see what other federal agencies did to protect workers placed in inhospitable or potentially dangerous environments. the cdc's resilience team reached out to the bureau of alcohol, tobacco, firearms, and explosives (washington, dc usa); us border patrol (washington, dc usa); us coast guard (washington, dc usa); drug enforcement agency (springfield, virginia usa); environmental protection agency (washington, dc usa); federal bureau of investigation (washington, dc usa); national aeronautics and space administration (washington, dc usa); and the department of defense (virginia usa). the scan revealed no standardized inter-agency processes. it did appear upon preliminary review that each agency based their approach on some type of psychological model combined with a peer-support component. to develop an impactful intervention, the cdc collaborated with the center for the study of traumatic stress (csts; bethesda, maryland usa) at the uniformed services university of the health sciences. , leaders at csts already had conducted key informant interviews at the cdc in . they had recommended then that the cdc develop training to enhance cdc emergency responder resilience. the csts also recommended that the cdc consider using psychological first aid (pfa) as the foundation for its resilience-supporting training initiative; pfa could be described as a pragmatic, evidence-informed, public health or population-based framework designed to help non-clinicians organize a response to trauma at the individual or community level. it was developed by the national center for post-traumatic stress disorder (washington, dc usa) to assist people immediately after a disaster to reduce initial distress and foster both short-and long-term adaptive functioning. the wwo worked closely with csts psychiatrists to develop a three-day course titled: "deployment safety resiliency team" (dsrt) training that incorporated pfa. the training included two days of pfa principles including: peer support, coping skills, stress management, triage, and proper referral processes ( figure ) . a third day highlighted the basics of disaster site safety including: blood borne pathogens, personal protective equipment, respiratory protection, radiation basics, and fatigue mitigation. the wwo excerpted relevant safety information from a well-established disaster site safety course and a collateral duty course. the focus was on the kinds of safety risks that were likely to be encountered in the field during a deployment. five experienced trainers also shared relevant insights about public health deployments they had gained during prior emergency responses. this innovative, highly-interactive, educational approach also incorporated small group analysis of three realistic, deploymentbased scenarios. the culmination of class included immersion in one, -minute virtual reality environment (vre). the vre options included a simulated deployment to: a rural african village, a city devastated by a hurricane, a town rocked by an earthquake, a community hit by a radiological dispersal device, a location dealing with a pandemic, a deliberate release of a toxic substance, or a food-borne infectious disease outbreak. during the -minute immersive experience, which introduced potentially stressful scenarios, up to trainees could see, hear, and "interact" with characters on a large screen in a darkened training room. the vre's pre-recorded characters addressed typical health, safety, and resilience challenges in a particular scenario. training participants, in three-person teams, used hand-held electronic devices to test their knowledge of relevant course content. they made decisions within their teams about how to assess available assets and threats, and then address these realistic challenges. they also had the opportunity to apply several of the principles they studied during the course in a safe, virtual environment. the wwo resilience team reviewed among other things the military's total force fitness framework to understand if or how it might be adapted to the cdc's workforce. the framework uses the connection between mind, body, spirit, environment, and relationships to holistically build and maintain health, readiness, and optimal performance of the us armed forces. it also assesses soldiers' resilience before and after deployment. the wwo's review included the popular press and media focused on resilience-related concepts and processes. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the goal was to conceptualize, create, and implement a screening process to help safeguard the health, safety, and resilience of staff being considered for deployment. the wwo resilience team convened an internal, -person expert panel to consider ways to address dozens of obstacles the cdc faced as it prepared to implement an assessment process for civilians. the panel was composed of psychometricians, an ethicist, mental health professionals, attorneys, epidemiologists, and experienced deployers. panel members reviewed numerous assessment tools designed to create a current snapshot of an individual's resilience. they eventually agreed upon a focused battery of brief and relatively unobtrusive assessment instruments. the panel report included these recommendations to cdc leadership: • adopt three specific assessment instruments; • develop standard operating procedures (sops) to ensure professional, confidential, stigma-free collection of pre-deployment assessments; and • encourage collaboration with the cdc occupational health clinic's (ohc) medical professionals to make a team-based recommendation to inform deployment decisions. the panel recommended that cdc implement a pre-deployment assessment battery comprised of: the expert panel determined that using these kinds of tools would screen for thoughts and behaviors that might indicate staff would be at elevated risk of a negative mental health outcome during deployment to potentially dangerous environments. the tools were brief enough to encourage completion while avoiding assessment fatigue. the panel also considered the battery broad enough to identify individuals who were struggling with issues that might jeopardize work productivity and personal well-being in the field. between november , and december , , there were , deployments by cdc staff in response to the ebola outbreak in west africa. records indicate that almost of the total deployments were by repeat deployers. the assessment scores of approximately different deployers were outside the norms for the externally validated assessments that were used. in accordance with the sops that the wwo resilience team developed, a licensed mental health professional within the cdc's resilience assessment and maintenance program (ramp) held a confidential conversation with those individuals about factors that might be negatively impacting their assessment scores at that time. during most of those confidential conversations, an exchange of pertinent information made it clear to both parties whether or not a deployment at that time was in the best interests of the individual and the organization. for example, if a person's pregnant spouse was only a couple of weeks from their due date, a deployment probably would be contraindicated. or if a person had just lost their father, it might not be advantageous to the cdc or in the individual's best interests to deploy right away. there were a few occasions when it was advisable for a ramp mental health professional to consult with a cdc ohc medical professional who had completed a physical assessment of the potential deployer. during those consultations, the mental health and medical professionals determined deployment eligibility jointly. the ramp clinicians ensured that all information related to assessments, concerns, and conversations remained secure and confidential in the ohc's electronic medical record system. they also referred several individuals to the cdc's employee assistance program (eap) for support with temporary challenges they faced. the cdc subject matter experts provided pre-deployment briefings for everyone who participated in a deployment managed by the eoc in atlanta. briefings ranged from minutes to minutes, depending on the nature of the response (eg, outbreak, humancaused, or natural disaster) and types of environments and challenges eoc leaders anticipated the deployers might encounter. for several years, eoc leadership requested that the resilience team provide an overview of basic resilience-related principles in those briefings. the resilience briefer highlighted physiological, cognitive, and behavioral symptoms of stress and emphasized the importance of self-care and social support. in virtually every briefing, the resilience briefer made this norm-setting statement: "emergency response is much more like a marathon than a sprint, so it's important to pace yourself while you're deployed." the briefing coordinator shared with briefing participants additional written material and references to supplemental resources, including contact information for eap professionals. during the past dozen years, cdc mental health professionals have reached out to deployers who had worked on challenging and stressful assignments to see how they were doing. for example, the resilience team contacted hundreds of cdc professionals when they returned from deployment to the marburg hemorrhagic individual level-the cdc sought to ensure that returning responders had multiple opportunities to speak with caring colleagues who could provide a non-judgmental listening ear. responders also received access to relevant information and, if desired, additional support after an especially challenging deployment. also, ramp developed a plan and implemented a process for team members to send personalized emails to returned ebola responders. the ramp team member conducting the outreach obtained from the cdc's eoc a list of staff getting ready to return from the field. they sent those individuals a standardized email inviting them by name to participate in a voluntary, confidential, non-clinical conversation about what they experienced in the field. between september , and june , , ramp team members emailed invitations to , different individuals who had deployed as part of the cdc's ebola response. of that group, individuals ( %) chose to participate in a conversation, which typically lasted - minutes (appendix ; available online only). the ramp team members informed responders that relevant information they shared would be aggregated, summarized, de-identified, and included in a report shared regularly with internal stakeholders at the cdc. recipients included the division of emergency operations (deo), incident management leadership, and the ossam. the deo and ossam had the opportunity to use insights and recommendations in these reports to identify gaps or redundancies in the deployment process, improve procedures, and fine-tune the deployment experience to increase the health, safety, and resilience of future deployers. as a result, the cdc provided additional information to potential deployers about proper clothing to wear, hotel conditions, computer and technology issues in the field, and types of personal equipment to bring. because of feedback, the cdc drafted and shared additional guidance about what to do if a deployed team member became ill. group level-many people were willing to share positive and negative feedback about processes and programs in a oneon-one format. the ramp team members believed that others might be more comfortable, and consequently more likely, to ask pertinent questions or share helpful process improvement information in a group setting. voluntary post-deployment group conversations were offered as part of the cdc's eoc activation for ebola from to . approximately individuals participated in one of these weekly or bi-weekly group meetings that typically lasted between and minutes. the ramp team members designed the group sessions to be facilitated conversations. to protect their anonymity, the group session leader asked participants not to share their names. he asked them to share their insights and observations in response to a handful of general questions about their deployment experience (appendix ; available online only). the group session leader informed responders that relevant information they shared would be aggregated, summarized, de-identified, and included in a report shared regularly with internal stakeholders at the cdc to identify gaps or redundancies in the deployment process, improve processes, and fine-tune the deployment experience for future deployers. for example, in response to this input from returned deployers, presenters updated information in security briefings. emergency response leaders tried to reduce the volume of email sent to deployed staff. the practice of having an in-country technology specialist become more standardized. after returning from the field, more deployers had access to help completing complicated travel vouchers and reimbursement paperwork. the cdc granted usb drive exceptions for computers in the field when deemed helpful. feedback collected and shared by resilience team members also supported development of improved checklists of steps in the deployment process and helpful packing lists. approximately individuals completed the three-day dsrt training during the ebola response. as part of this course conducted at the cdc since , the ramp team administered assessments to provide the opportunity for the cdc to evaluate training effectiveness. more specifically, ramp administered pre-and post-training assessments to participants in the following areas: . knowledge of resilience-enhancing principles and processes ( figure ); . knowledge of basic disaster site safety principles and processes ( figure ); . sense of self-efficacy as measured by a -item general self-efficacy scale ( figure ); . overview of course content; and . general effectiveness of the training via a standard training assessment form. the -item dsrt course content survey was intended to assess mastery of constructs related to suicidal ideation, pfa, compassion fatigue, dsrt principles, resilience, support, and referral recommendations (appendix ; available online only). the -item self-efficacy survey was an externally-developed, self-assessment of how an individual thought they could manage challenging situations (appendix ; available online only). to assess the effectiveness of the training, ramp assessed the change in the total score for the dsrt course content survey. they observed a statistically significant improvement in the total mean scores. at baseline, participants, on average, scored a . on the -item test. at post-test, they scored an average of . , an increase of . points ( % ci, . - . ). analysis of the individual items demonstrated that the responses to a large majority of the items improved between baseline and the post-test. for example, it was found for the two items "something you might do if you suspected a person was having thoughts of suicide" and "core principles of psychological first aid (pfa)," there were statistically significant increases in the percent correct (p < . ). for the items "which of the following is part of the 'five steps to getting support?'" and "which of the following can be influenced by compassion fatigue," there were not statistically significant improvements. in the future, ramp will consider revising or removing items that did not demonstrate a statistically significant improvement. the self-efficacy survey total score showed a significant improvement in overall self-efficacy. the individual items were likert scales that ranged from one (do not agree) to four (completely agree). at baseline, on average, individuals scored a . across all items. at post-test, individuals' scores improved to . , which was a statistically significant improvement (change = . points; % ci, . - . ). all individual items demonstrated a statistically significant improvement over the baseline responses. this suggests participants gained useful knowledge of resilience principles and strategies from the course content. the cdc has implemented several evidence-informed approaches to safeguard the health, safety, and resilience of its responders. these included additional pre-deployment trainings, a new pre-deployment assessment process, expansion of training to provide support in the field during a deployment, and multiple postdeployment outreach initiatives. these efforts were in addition to the three-day dsrt training, which the cdc had implemented several years earlier. since , over individuals have completed this unique resilience-focused training, which has created a cadre of individuals who can assist fellow deployers in real time. leadership at ramp trained these primarily non-clinician participants in the basics of assessing and addressing their own and their colleagues' resilience during a field deployment. in addition, ramp helped prepare participants to accomplish these tasks by: • clarifying expectations about their roles in the field; • providing information about core actions and core principles of pfa; • encouraging small groups to analyze and apply their experience and what they learned in class to address challenges presented in three different realistic scenarios; and • sharing an overview of relevant concepts and potentially helpful questions from five valid and reliable assessment tools. also, ramp made presentations highlighting this resilienceenhancing training developed in collaboration with csts at conferences in san diego, california; atlanta, georgia; little rock, arkansas; washington, dc; charleston, south carolina; san juan, puerto rico; mexico city, mexico; and tel aviv, israel. additionally, ramp shared a two-day version of this training with public health agency of canada colleagues in ottawa, canada, and a one-day version with national institute of occupational safety and health (niosh) colleagues in cincinnati, ohio and morgantown, west virginia. introducing a pre-deployment assessment process for the cdc's responders improved the quality and quantity of relevant information to which cdc's clinic staff had access. it also addressed management's directive that a screening be implemented to mitigate the risk of deploying someone who might neither contribute to, nor benefit from, a deployment. access to timely and relevant data allowed better planning and allocation of resources for staff. in addition, ramp gave deployers additional points of contact from whom they could receive support or share input and feedback about emergency planning and coordination. it expanded both the depth and breadth of information available to cdc's clinical staff to help consider the variables that affect medical clearance. it also gave potential deployers the opportunity to proactively explore and prepare for some unintended consequences in the field. this includes fatigue or distress that might negatively impact their work and family dynamics while deployed. the intent was to improve their professional and personal success and happiness in the field. the post-deployment outreach initiative provided on-going, process-improvement data to the cdc's eoc and deployment coordination team. an added benefit of the outreach was that it served as a consistent, unobtrusive vehicle through which the cdc could emphasize the organization's gratitude for personal and professional sacrifices and contributions made during the ebola response. it reminded them about the meaningfulness of their professional contributions in the field and provided an additional opportunity for deployers to connect with supportive resources, if needed. the abbreviated resilience-related training provided during pre-deployment briefings and participation in more general trainings helped increase participant awareness of challenges and opportunities in field deployments. analysis of pre-and post-training assessments of graduates of the cdc's three-day dsrt course indicated, with statistical significance, that they acquired relevant knowledge and that their self-efficacy was increased. finally, ramp determined that the assessment data supported their operating assumptions that it made sense to take multiple approaches to klomp © prehospital and disaster medicine safeguard the health, safety, and resilience of individuals deployed to inhospitable and potentially dangerous environments on an emergency response. to view supplementary material for this article, please visit https:// doi.org/ . /s x ebola outbreak in west africa control strategies, and lessons learned in the cdc response to the - ebola epidemic virtual classroom immersion training: safeguarding the health, safety, and resiliency of emergency responders chairman of the joint chiefs of staff instruction intervention and resilience after mass trauma center for the study of traumatic stress psychological first aid (pfa) field operations guide: nd edition total force fitness army master resilience training course provides valued instruction stronger: develop the resilience you need to succeed the power of resilience: achieving balance, confidence, and personal strength in your life the resilience handbook: approaches to stress and trauma the resilience factor learned optimism: how to change your mind and your life the survivor personality: why some people are stronger, smarter, and more skillful at handling life's difficulties : : : and how you can be too /a-ready-and-resilient-workforcefor-the-department-of-homeland-security-protecting-americas-front-line.aspx key: cord- -b yywn f authors: hasan, ashfaq; praveen, sai haranath; tarke, chandrakant; abdullah, fahad title: clinical aspects and principles of management of tuberculosis date: - - journal: mycobacterium tuberculosis: molecular infection biology, pathogenesis, diagnostics and new interventions doi: . / - - - - _ sha: doc_id: cord_uid: b yywn f tuberculosis over the ages, has killed more people than any other infection has. notwithstanding the advances in modern science, clinical diagnosis sometimes remains elusive, owing principally to the frequent paucibacillary occurrence of the disease and the slow doubling time of the organism; empiric treatment is often fraught with risks in the era of increasing drug resistance. this chapter attempts to provide an overview of the disease, beginning with the pathogenesis and its protean clinical presentations. it also discusses the recent evolution of molecular methods that have lately provided an impetus to early diagnosis with a clear opportunity to unmask drug resistance before initiating “blind”, potentially ineffective, and sometimes harmful treatment with standard therapy. the chapter also provides insight into tuberculosis in special situations, and discusses briefly the treatments in uncomplicated cases as well as in special situations, and in instances of drug resistance. preventive methods including current and upcoming vaccines are mentioned. finally, a short discussion of the sequelae of tuberculosis—which have the potential to be confused with active disease—is presented. tb can involve all organs except the nails, hair and teeth [ekaterina kulchavenya. ther adv infect dis. apr; ( ): - ]. pulmonary tb is the most common form of tb. the symptomatology of tb has to do with its pathogenesis, the specific organ system involved and the duration of the disease. primary tb, which occurs in the mycobacterium-naive individual, usually presents with a frequently occult primary focus in the peripheral lung parenchyma (stead's focus) accompanied by a pleural effusion; or by a primary focus-frequently inapparent-in the lower part of the upper lobe or the upper part of the lower lobe (ghon's focus) accompanied by lymphadenopathy. other manifestations of primary tb such as erythema nodosum or phlyctenular conjunctivitis may be present. in young children with immature cellular immunity, or in immunocompromised individuals, the process may evolve to progressive primary disease. symptoms and signs relate to the compressive effect of enlarged lymph nodes on adjacent structures (collapse or hyperinflation of a lobe or segment), or rupture of caseating lymph nodes into a bronchus, with bronchiectasis later supervening in such a lobe. post-primary disease, the typical adult form of tb, usually occurs by reactivation of a dormant focus-or less frequently, by reinfection; the better aerated lung apices are preferentially involved. the classical triad of the constitutional symptoms of tb (fever, night sweats, weight loss)-most usually of several weeks' duration-is present in a high proportion of patients (heemskerk et al. ) . the extent of disease at the time of first diagnosis may be variable, from patchy consolidation to a large cavity. this not only reflects the protean pulmonary forms of the process but also has to do with host immunity, the virulence of the strain and, most of all, with the time elapsed between disease onset and diagnosis. cavitation is an important milestone in the timeline of the post-primary pulmonary tuberculosis. with ingress of o -rich air into a fresh cavity (caused by the coughing out of a caseous collection), the aerobic mycobacterium is presented with an opportunity to multiply manifold in an aerobic environment: the infectiousness of the subject (and the possibility of a positive diagnosis on sputum testing) commensurately increases (golub et al. ) . cavitation also increases the propensity to haemoptysis (which may sometimes be massive) and that of cavitary colonization with a fungal ball (mycetoma) later on in healed disease. one third of all individuals affected with post-primary tb die within a few months ("galloping consumption") (reported tuberculosis in the united states, ). the remainder either go on to develop a chronic pattern with a more gradual progressive decline or undergo spontaneous remission of the process and restoration to health. with chronicity, fibrosis is common. disseminated tb, which often casts "miliary" shadows in the lungs, occurs due to haematogenous dissemination following either primary or post-primary tb. the name derives from the resemblance of the widespread granulomatous lesions to millet seeds (seen in pathological specimens as - mm yellow granules). symptoms may be nonspecific and depend on the site and degree of organ involvement. hepatosplenomegaly, lymphadenopathy and pleural effusions may be present. fundus examination, often neglected, will frequently show choroid tubercles, which are virtually diagnostic of the condition in a likely setting. diagnosis often hinges on microbiology from broncho-alveolar lavage (bal) or on biopsy of involved organs (lewinsohn et al. ) though it may occasionally be made on sputum (sponaneously expectorated or induced). disseminated tb disseminated tb should always raise suspicion for an accompanying immune deficiency such as hiv disease. sometimes an occult disseminated form of tb ("cryptic miliary tb") presents as a "fever of unknown origin", usually in the elderly or immunocompromised patients. in difficult cases (as with the classic miliary form), bone marrow or liver biopsy with mycobacterial cultures may be diagnostic. unlike the post-primary form of disease, untreated miliary tb is predictably fatal. extra-pulmonary tuberculosis has been increasingly seen since the advent of the hiv epidemic. tb can involve any organ of the body (other than nails, hair and teeth). organ systems typically involved include pleura (pleural effusions), lymph nodes (generally cervical or mediastinal), airway (endobronchial tuberculosis), skeletal (joint or vertebral involvement, sometimes with a psoas abscess), gastrointestinal, genitourinary, meningeal and pericardial involvement. symptoms pertain to the system involved, but diagnosis may be difficult unless a high degree of suspicion is maintained. tissue or fluid samples from the involved organs with staining, mycobacterial cultures or molecular probe assays are often diagnostic although radiological patterns in specific cases may be very suggestive of the process (e.g. vertebral involvement with a paraspinal abscess) (lewinsohn et al. ) . the diagnosis of tuberculosis, especially in the developed nations, rests on the keystone of suspicion. physical examination is generally unrewarding. auscultation of the lung typically underestimates the lung problem relative to that seen on imaging. with a pleural effusion, breath entry is typically decreased, with a flat note over the effusion. cavernous bronchial breathing may be detected over a cavity, and tubular bronchial breathing over an area of consolidation or dense fibrosis. the radiologic features in the various forms of pulmonary tb have already been discussed above under the relevant heads. microbiological isolation of the mycobacterium tuberculosis on culture has been the only definitive test for tb thus far, though it could be argued that molecular probes for specific mycobacterial genes and other technologies (discussed below and also elsewhere in this book) offer viable and more attractive options for diagnosis. presumptive diagnosis is typically achieved by microscopy (usually of sputum, but also of fluid obtained by bal and other means) (pai et al. ). mycobacteria, not being stainable by "conventional" stains, require a modified acid stain (the ziehl-neelsen method) in order to be visualized; led microscopy though relatively expensive, by making the bacilli easy to see under low power, enables more fields to be scanned in a shorter period of time, and thus increases the sensitivity of microscopy several-fold (bhalla et al. ) . conventional culture methods are slow ( - weeks, less by radiometric assays), and frequently there is a compelling need to start appropriate anti-tubercular therapy as early as possible. rapid culture techniques are now in universal use. they use liquid rather than solid media. the mycobacteria growth indicator tube (mgit) method takes days rather than weeks and facilitates drug sensitivity testing. since relapses (see below) occur in approximately % of patients after months of standard therapy, it is a moot point whether establishment of minimum inhibitory concentrations (mics) in the presence of a "critical concentration" of drug is relevant in selected cases (colangeli et al. ) . automated nucleic acid amplification technology (naat) has the potential of amplifying even a fragment of mycobacterial dna rapidly (in h) and has revolutionized tb diagnosis. in its currently available form, the genexpert test is intended specifically to diagnose mycobacterium tuberculosis (and thereby differentiate it from other mycobacteria such as environmental mycobacteria), and also presumptively diagnose rifampicin resistance. though not invariable, rifampicin resistance also implies inh resistance. together, rifampicin and inh resistance fulfil the diagnosis of multiple drugresistant tb (mdr-tb). thus a positive genexpert test makes a presumptive diagnosis of mdr-tb (steingart et al. ). the test is more sensitive than direct microscopy and extremely specific (steingart et al. ). the test is not infallible, however, and any result that is discordant with the clinical picture should either be repeated or further information sought using another modality. false-positive naat results can occur due to laboratory contamination. importantly, naat can detect nucleic acid from dead non-viable bacilli, and so a positive test after completion of the course of treatment has no relevance in that situation (boyles et al. ) nor is it of relevance in monitoring response to treatment (friedrich et al. ) . it is important to realize that naat is not a substitute for afb smear and culture testing; culture is essential for species identification and for drug susceptibility testing (cheng et al. ) . a more sensitive version of xpert has been developed. the line probe assays (lpa) can permit rapid identification of specific gene markers associated with rifampicin resistance alone or in combination with isoniazid, and provide clinically relevant information about the level of inh resistance (low level associated with the inh-a gene; versus high level associated with the kat-g gene) (who treatment guidance for drug resistant tuberculosis ). definitive diagnosis has traditionally hinged on the culture characteristics of mtb: species discrimination can be achieved on the basis of colony morphology as well as on the basis of biochemical tests performed on the culture. conventional culture (lowenstein-jensen medium and its variants) is capable of detecting as few as ten bacteria per millilitre with the sensitivity and specificity of sputum culture approximating and %, respectively (morgan et al. ) . following initial culture, species identification is done by any of the following techniques: biochemical methods (testing, nucleic acid hybridization probes, high-pressure liquid chromatography or mass spectrophotometry). drug sensitivity testing is subsequently carried out, usually at a reference laboratory (shinnick and good ) to first-line anti-tubercular agents, but sometimes to second-line agents as well when resistance to components of first-line anti-tubercular agents has been documented. in individuals living with hiv who have cd counts < cells/mm , or those who are immunocompromised, mycobacterial cultures of blood and urine should complement conventional testing. point-of-care urinary detection of lipoarabinomannan, a component of the mycobacterial cell wall (urine lam test) may be useful for hiv-infected individuals, and its use may possibly confer a mortality benefit (reported tuberculosis in the united states, ). the future of point-of-care devices is next-generation sequencing, and, perhaps, innovations like detection of volatile organic compounds in exhaled breath (garcia-basteiro et al. ) serological tests (tests for antibodies against tb) have poor sensitivity and specificity; false-positive tests are ubiquitous, and serologic testing for the diagnosis of tb is discouraged by the who. a pleural fluid adenosine deaminase (ada) > u/l has been reported to be % sensitive and % specific for the diagnosis of pleural tb in lymphocytic exudates (garcia-zamalloa and taboada-gomez ). tb infection can rapidly progress to disease in infants and young children (progressive primary tb). mortality is high in early childhood because of the severe forms of tb (such as miliary tb and meningeal tb) that occur more frequently in young children. progressive primary tb is a serious consequence of primary tuberculosis. consolidation or bronchopneumonia is more common than cavitary disease in children. haematogenous dissemination leading to miliary tb is also more common in infants and young children. tubercular pleural effusion, however, is less common in children below years of age. central nervous tb is the most serious form of childhood tb (prevention ). the presentation is usually with nonspecific symptoms such as fever, headache, drowsiness and irritability, but in advanced cases, vomiting, neck rigidity, seizures, focal neurologic deficit and coma may supervene. since sputum samples for diagnosis are difficult to obtain in young children (children tend to swallow sputum rather than expectorate), gastric washings can rather be sent for microbiological examination. a ppd test that is greater than mm in bcg-unvaccinated children and > mm in bcg-vaccinated children is considered positive. (targeted tuberculin testing and treatment of latent tuberculosis infection. this official statement of the american thoracic society was adopted by the ats board of directors, july . this is a joint statement of the american thoracic society (ats) and the centers for disease control and prevention (cdc). this statement was endorsed by the council of the infectious diseases society of america. (idsa), september , and the sections of this statement .) the principles of treatment of tuberculosis in children are similar to that of adults. paediatric tb patients should be monitored carefully for ethambutol-induced ocular toxicity, owing to the fact that children are less likely to report visual impairment than adults. in a prospective study of children on multidrug-resistant tuberculosis, children had lower serum concentrations in spite of higher dosing of moxifloxacin, a fact that was ascribed to increased drug elimination in children. quinolones have also been known to rarely cause arthropathy and tendon rupture. more recent studies have shown that most quinolones are quite well tolerated but very specific and narrow indications are present for quinolone use in children (patel and goldman ) . tuberculosis in pregnancy has important implications for the mother and child (loto and awowole ) . the diagnosis of tuberculosis in pregnancy can be challenging, since many of the symptoms of early tb may be confounded by pregnancy; for instance, the normal weight gain in pregnancy may temporarily mask the associated weight loss. chest radiograph is not an absolute contraindication and when necessary may be carried out with "abdominal shielding" of the fetus. the complications of tb in pregnancy include early spontaneous abortion, intrauterine growth retardation, preterm delivery, low birth weight and increased neonatal mortality. congenital tb is rare, but can be associated with high perinatal mortality. first-line oral drugs, i.e. isoniazid, rifampicin, ethambutol and pyrazinamide, can all be used during pregnancy safely. streptomycin and other aminoglycosides have been proven to be potentially teratogenic in pregnancy. they are also capable of causing eighth nerve paralysis, with deficits ranging from mild hearing loss to bilateral deafness (loto and awowole ) . concerns have been raised about the use of fluoroquinolones in pregnancy even though the weight of the evidence seems to point toward safety (acar et al. ) tb is considered an aids-defining disease [centers for disease control and prevention. appendix a: aids-defining conditions. mmwr. ; (rr ): ]. the risk of developing tb is estimated to be between and times greater in people living with hiv than among those without hiv infection. hiv and tb have a salutary impact upon each other and expedite each other's progression. the risk of progressing from latent to active tb is estimated to be between and times greater in people living with hiv than among those without hiv infection (global tb report ). against the backdrop of hiv infection, the rate of progression of tb from the acquisition of infection to full-blown disease may take weeks, rather than years-as is the case in hiv-negative individuals (mayer and dukes hamilton ) . it is important to realize that tb can occur at all "stages" of hiv disease. the clinical presentation of hiv patients who have relatively high cd counts is no different to that which occurs in individuals without hiv. such individuals manifest with the classical manifestations of post-primary tuberculosis such as upper lobe infiltrates. on the other hand, lower lobe involvement, pleural effusions and intrathoracic lymphadenopathy are common in individuals with low cd counts (typically < /μl). indeed the chest x-ray may sometimes only show vague interstitial infiltrates or even be normal (mayer and dukes hamilton ) . taken together, the fact that in advanced hiv disease (a) the sputum is less likely to be positive for afb (b) the ppd is usually non-reactive (c) the x-ray abnormalities are likely to represent other opportunistic infections or even non-infective conditions associated with hiv disease and (d) histopathology of involved tissues is characterized by lack of typical granulomata; these considerations make the diagnosis extremely challenging in this setting. in addition, there is increased frequency of extra-pulmonary tb among hiv-positive individuals. frequently, a positive naat (genexpert) test is the justification for starting treatment. however it must be cautioned that even a negative naat test cannot convincingly rule out disease, and, given that delays in treatment may be fatal, the decision to initiate treatment is sometimes taken on clinical grounds. the treatment of tuberculosis differs from the treatment of most other infections in certain respects. multiple drugs are required in a regimen since spontaneous mutations can lead to resistance to the action of one or more drugs. also, because the bacterium is relatively slow growing, it takes longer to achieve bacteriologic sterility. multidrug therapy, the norm for tb, has been very effective, with a modest rate of relapse (colangeli et al. ) . the standard four-drug regime ("short course") comprises a -month intensive phase with the four front-line drugs (rifampicin, isoniazid, ethambutol and pyrazinamide), followed by a continuation phase with the drugs (minus pyrazinamide and possibly ethambutol) for a further months. the four front-line drugs have been chosen based on their efficacy in rapidly reducing the number of organisms initially (during the initial phase), thus reducing infectivity; and on their sterilizing potential (the ability to eradicate viable bacteria completely and so prevent relapses); and their relative lack of side effects (see table . ). these drugs are combined into regimens. the six classes of second-line agents (fluoroquinolones, the aminoglycosides, capreomycin, ethionamide and prothionamide, para-amino salicylic acid (pas), cycloserine and terizidone) have a rather lower efficacy and a higher propensity for side effect and drug intolerance. several other antibiotics of uncertain efficacy have also been categorized as anti-tubercular agents and have a role in treating multidrug-resistant tb (mdr-tb) (see table . ). two new agents, bedaquiline (a diaryl-quinolone) and delamanid (a nitroimidazole), are now approved for the treatment of mdr-tb. the who recommends using a daily regimen and fixed-dose combinations (guidelines for treatment of drug-susceptible tuberculosis and patient care ). administration of the drugs on a daily basis both during the intensive and during the continuation phase is preferable although daily administration during the initial phase and intermittent (thrice weekly) administration during the continuation phase is also acceptable (guidelines for treatment of drug-susceptible tuberculosis and patient care ). intermittent administration of drugs (especially in the initial phase) is not offered in the presence of hiv disease. if at the end of the intensive phase the bacteria are sensitive to the three most important agents (isoniazid, rifampicin and pyrazinamide), ethambutol may be discontinued (nahid et al. ). however, drug testing for three of the four frontline drugs for each patient at the end of the intensive phase is impractical, and limited testing (genexpert) is usually performed initially in those patients whose sputum remains positive. there is evidence to show that extending the continuation phase by months (making up a total duration of treatment of months) helps in preventing relapse in those patients who have cavitation on initial chest x-rays; and also in those who show positive cultures at the end of the initial phase of treatment (benator et al. ) ; as well as in those whose medication given during the initial phase did not include pyrazinamide. anti-tubercular drugs require to be supplemented with carefully monitored steroid therapy in two circumstances: tubercular meningitis (a short course of dexamethasone or prednisolone is typically given, tapered over to weeks) and tuberculous pericarditis (guidelines for treatment of drug-susceptible tuberculosis and patient care ). since many patients cannot be relied upon to regularly take their drugs, and irregular intake of drugs has the potential to result in drug resistance (see treatment failure and relapse; and drug resistant tb, below), it is imperative to ensure adherence to the regimen. directly observed therapy, short-course (dots) is the direct observation by another person, of the patient taking the medication (chaudhuri ) . the use of dots has improved adherence in subgroups of patients such as tb with hiv or if dots was at the site of dots centre or provider location. self-administered therapy (sat) modified with some form of monitoring incorporated into the process shows some promise. although dots could be administered by a family member, the preferred person is a trained lay provider or healthcare worker (guidelines for treatment of drug-susceptible tuberculosis and patient care ). the treatment of tb in hiv disease merits special consideration. antiretroviral therapy (art) should be started irrespective of the clinical stage of hiv disease and cd count. anti-tubercular therapy (att) should precede the commencement of art. art should then be commenced as soon as it is evident that att is being tolerated (typically between weeks and months). the immune reconstitution inflammatory syndrome (iris) is sometimes seen with the returning immune competence that follows art; an increased immune response to tubercle bacilli or antigens occurs. iris usually occurs between and months of the commencement of art and is associated with a decrease in viral load and an increase in cd + t cell count. there is a worsening in the clinical picture weeks or months into treatment, with an increase in the size of lymph nodes, development of pleural effusions and features of noncommunicating hydrocephalus in a patient with tb meningitis. although most patients with iris have mild to moderate symptoms, iris, especially when related to tb meningitis, can be life-threatening. an "unmasking iris" may occur in patients with unsuspected tb (raviglione ) . treatment of iris includes addition of steroids (which probably work by decreasing the levels of pro-inflammatory cytokines) and the continuation of att and art. with both att and art on board, drug-drug interactions especially involving rifampicin (by induction of the cytochrome p system) may occur. rifamycins induce the metabolism of non-nucleoside reverse transcriptase inhibitors and protease inhibitors. rifampicin is a more potent inducer of the cytochrome p system than rifapentine, which in turn is more potent than rifabutin. despite potential drug interactions, rifamycins should nevertheless be included in tb regimens, with dosage adjustment if necessary. rifabutin is the preferred rifamycin in hiv-positive individuals. host-directed therapy to modulate the immune response to tb is an active area of research (kaufmann et al. ) . the use of an anti-diabetic drug-metformin, for example-is being investigated. also, studies of autologous mesenchymal stromal cell infusions is being investigated in mdr and xdr-tb (skrahin et al. ). an approach using high-altitude sanatoria was also explored (murray ) in a throwback to the pre-anti-tubercular chemotherapy era. study using atypical mycobacterial injection in tuberculous did not show any major outcome differences from placebo although steroids did decrease the subsequent development of constrictive pericarditis (mayosi et al. ) . given the role that vitamin d plays at the level of the antigen-presenting cell, the role of vitamin d deficiency as a possible player in tb predisposition has recently come into focus; indeed a recent meta-analysis has revealed that vitamin d supplementation in fact did not affect time to sputum culture conversion overall, though it did accelerate sputum culture conversion in patients with multidrug-resistant pulmonary tuberculosis (jolliffe et al. ). patients with tb may be admitted to an icu for a variety of reasons, including respiratory failure, multiorgan failure, decreased consciousness associated with tubercular meningitis, pneumothorax, cardiogenic shock (due to massive pericardial effusion) and liver or kidney failure due to a drug reaction induced by att. confluent tuberculous bronchopneumonia and tb ards are less common causes of respiratory failure (hagan and nathani ) . tuberculosis treatment is especially challenging in critically ill patients due to potential for poor gastric absorption and the high rates of organ dysfunction and drug toxicity in this subset of patients. deciding the optimal regimen and dosing becomes challenging in the presence of concomitant hepatic and renal failure. in a recent study done on tb patients requiring icu admission, mortality was . % (tatar et al. ). treatment failure is suspected when the sputum remains positive for afb at the end of the first months of treatment. it occurs due to factors that may involve incorrect regimens or dosing or indeed due to malabsorption or poor-quality drugs (lambregts-van weezenbeek and veen ) . patients are regarded as treatment failures when a microbiologic indicator like sputum smear/culture for tb remains positive after - months of starting treatment or comes back positive after a period of remaining negative (fall and rise phenomenon) (prasad et al. ) . patients may respond for a short time after starting treatment or do not respond to treatment from the inception. in any case the implication of treatment failure is drug-resistant tb (see below), and strenuous efforts must be made to look for microbiological resistance to first-and, in the proper context, to second-line agents-by the available tools. relapse, on the other hand, is the recurrence of tb after completion of treatment that has been successful, with documented culture negativity. relapse may be due either to a reinfection or reactivation of the dormant bacillus. drugresistant tb is to be considered in these settings. the implications of relapse are not as grave, the isolates generally being drug sensitive. in either case, the exact regimen chosen should be based upon the drug sensitivity pattern. specific recommendations upon the construction of such a regimen are available at who treatment guidelines for drug-resistant tuberculosis update (http://www.who.int). drug-resistant tb is a major global problem and is a threat to the success of the end tb strategy. mdr-tb or multidrug-resistant tb (mdr-tb) is defined by resistance of m. tuberculosis against to at least rifampicin and isoniazid. since these two drugs are the most effective drugs available against tb, resistance to both considerably shortens the odds of complete cure. xdr-tb or extensively drug-resistant tb is defined as resistance to rifampicin and inh plus resistance to at least one fluoroquinolone and one second-line injectable agent (amikacin, kanamycin or capreomycin). some of the reasons that lead to drug resistance have been mentioned in the section above. however the most important of these are failure to comply with the treatment regimen on the part of the patient and the addition of a single drug ("monotherapy") to a failing regimen; or a situation of unrecognized primary (that which is present at the start of therapy) or secondary (that which develops during treatment) drug resistance. inh mono-resistance of itself probably does not impact the outcome of therapy provided that ethambutol and possibly pyrazinamide are used for the entire duration of therapy. consideration should be given in this case to extending the duration of treatment to months from the standard (sadanand ). for reasons already discussed, rifampicin-resistant individuals should be managed as mdr-tb patients. in such patients it is not unusual to encounter resistance to additional drugs (e.g. ethambutol). it has been shown that clinical cure is possible in patients with mdr and even xdr-tb (who drug resistant tb ). however, owing to a variety of reasons not the least of which is drug intolerance, only about half of mdr-tb and a third of xdr-tb patients ever complete therapy. the who estimates there are over , mdr-tb or rifampicin-resistant cases annually (global tb report, who update ; wwww.who.int). the treatment of mdr-tb and xdr-tb is complex and requires specialized knowledge and close monitoring to ensure rational and safe therapy (lange et al. ) . the choice of drugs for treatment of mdr-tb is frequently difficult. formulation of drugs into regimens is facilitated by the organization of drugs into groups: group a ¼ levofloxacin/moxifloxacin, bedaquiline, linezolid. group b ¼ clofazimine, cycloserine/terizidone. group c ¼ ethambutol, delamanid, pyrazinamide, imipenem-cilastatin, meropenem, amikacin (streptomycin) , ethionamide/prothionamide, p-aminosalicylic acid. the who makes specific recommendations for short and long mdr-tb regimens. in the who's update (who treatment guidelines for multidrug-and rifampicin-resistant tuberculosis ), for those mdr-tb patients "who have not been previously treated for more than one month with second-line medicines used in the shorter mdr regimen or in whom resistance to fluoroquinolones and second-line injectable agents has been excluded," the who permits a shorter mdr-tb regimen of - months. for mdr/rr (rr¼ rifampicin resistant) tb patients on longer regimens, the who recommends that "all three group-a agents and at least one group-b agent (should) be included to ensure that treatment starts with at least tb agents likely to be effective, and that at least three agents (are) included for the rest of treatment after bedaquiline is stopped. if only one or two group a agents are used, both group b agents are to be included. if the regimen cannot be composed with agents from groups a and b alone, group c agents are added to complete it. kanamycin and capreomycin are not to be included in the treatment of mdr/rr-tb patients on longer regimens" (who treatment guidelines for multidrug-and rifampicin-resistant tuberculosis ). a detailed discussion about the treatment of mdr-tb is available at the foregoing resource. novel anti-tubercular agents (e.g. bedaquiline and delamanid) have been discussed elsewhere in this text. the remodelling of the lung that follows tb can significantly contribute to morbidity and mortality. haemoptysis is frequently one of the remote sequelae and can on occasion be life-threatening. an unresolved cavity is frequently the cause, and bleeding from an unsupported blood vessel in the cavity wall or in a post tubercular bronchiectatic segment can be a source of troublesome bleed. cavities colonized by fungi (aspergillomas), broncholiths eroding into the bronchial wall and rarely expansion and subsequent rupture of an unsupported blood vessel in the cavity wall (rasmussen's aneurysm) can cause massive haemoptysis (van den heuvel and van rensburg ) . a scar carcinoma sometimes develops in an area of fibrosis and may sometimes be difficult to distinguish from a dense fibrotic infiltrate (gao et al. ) . post-tubercular bronchiectasis is predisposed to when caseation necrosis and inflammation with retention of secretions leads to destruction of bronchial walls with ensuing permanent dilatation. sometimes compression of bronchial lumen by enlarged lymph node can produce bronchiectasis by a different mechanism. postprimary tb involves the upper lobes of the lung, and so the gravitationally advantaged upper lobes do not usually undergo a process of chronic and recurrent infection unlike the lower lobes that are involved by bronchiectasis of other aetiologies. mycetoma (fungal ball) is a mass of fungal hyphal material that grows within a cavity. aspergillus fumigatus is the most common aetiological agent, but other fungi such as mucor or fusarium can also cause a fungal ball to develop (rippon ) . such a mycetoma is usually asymptomatic but can sometimes lead to haemoptysis. no treatment required in asymptomatic cases, but surgical treatment needs to be considered in patients with massive or recurrent haemoptysis. a list of adverse effects associated with anti-tubercular agents appears in table . . side effects are possible with any of the anti-tubercular agents, and on rare occasions, they may be serious and even fatal. the risk of liver injury with anti-tb therapy is always a concern. among the four first-line agents inh, rifampicin and pyrazinamide are all capable of causing hepatotoxicity. isoniazid is capable of causing asymptomatic mild elevation in transaminases in - % as well as fatal severe acute hepatitis in . - %. jaundice with liver injury can occur in . - %. peripheral neuropathy due to pyridoxine deficiency can occur with inh therapy in up to . % of elderly individuals (ruan et al. ); pyridoxine supplementation is required for all patients taking inh (john ). factors increasing the chances of inh hepatotoxicity include age, female gender, alcohol use, prior hepatitis, concurrent use of cytochrome-inducing agents and slow acetylator status (sharma et al. ) . acute inh toxicity can present as seizures due to decreased gaba levels. inh is metabolized to several metabolites including the hepatotoxic metabolite hydrazine, toxic-free radicals and mono-methylhydrazine. degradation by acetylation occurs via n-acetyltransferase- gene (nat ); a deficiency of the nat enzyme can lead to accumulation of hepatotoxic metabolite. ten percent of patients with anti-tubercular drug-induced hepatotoxicity show progression to acute liver failure; the rest are self-limited (badrinath and john ) . if the serum bilirubin rises to ! mg/dl or serum transaminases exceed more than five times the upper limit of normal (or exceed three times the upper limit of normal in a symptomatic patient), all drugs should be stopped (nahid et al. ). the decision on what regimen to use on restarting should be individualized. the regimen, even if retailed, might then still include one or more potentially hepatotoxic drugs, and these should be restarted one at a time, only when liver function tests return to their baseline (e.g. the transaminases fall to less than twice normal). in general, a cholestatic type of derangement would prompt addition of inh first; with a non-cholestatic picture, rifampicin could first be added (sterling) . needless to say, careful monitoring with frequent testing of serum liver function is required. most physicians would omit pyrazinamide when restarting att except in the mildest of cases or with other compelling reasons (sterling) . patients receiving ethambutol (emb) should be questioned regarding their vision at each monthly visit. monthly visual acuity and colour vision discrimination testing is recommended in those patients in whom emb doses of greater than - mg/kg are being used, those who have been taking ethambutol for longer than months and those with renal insufficiency in whom the levels of drug may be expected to rise (blumberg et al. ) . for patients who require a regimen with no hepatotoxic agents, potential agents include ethambutol, levofloxacin or moxifloxacin, an injectable agent and other second-line oral drugs. the optimal choice of agents and duration of treatment (at least - months) is uncertain. (see "antituberculous drugs: an overview", section on "second-line agents".) in the end, efforts to finally eradicate tb will need to hinge upon preventative aspects as much as upon its treatment. these strategies would include currently established practices, as well as novel strategies. the former include the following. tuberculosis is now the primary cause of death from infectious diseases exceeding hiv, aids and malaria. the who estimates . million new cases and . million deaths every year (who global tb report ). of these, . million are hiv patients who die mostly in lmic (low-and middle-income countries) (who global tb report ; www.who.int). within the next years, the goal is to reduce tb incidence by % but the progress has been painfully slow. the most effective way to reduce the burden of tb in the community, to date, has been to identify patients affected with tb as early as possible and to treat them effectively with antitubercular therapy. the role of vaccines in this regard, though potentially vital, has been disappointing. of all vaccine tested to date, the bacillus calmette-guerin (bcg)-a live attenuated vaccine that was made in after an incredible years and sub-cultures (hasan )-is still the most effective vaccine we have today. yet its effect has been inconsistent, ranging from % to zero protection; several factors including geographical differences appear to play a role (british adolescents had better responses than south indian participants perhaps due to background exposure to mtb or atypical mycobacteria). the efficacy of bcg has been most manifested in its protection of infants and young children from the dangerous forms of tb (such as disseminated "miliary" tb and tb meningitis). also, its effects appear to wane with time (hasan ) . the bcg is not a single vaccine. several strains-all derived from the original bgc strain-are in use, which also partly explains its variable efficacy. in , the sanger centre in cambridge, britain and paris's pasteur institute in france cracked the genetic code for the old h rv strain of tubercle bacillus, and then, the genome of highly virulent cdc (the "oshkosh" strain) was mapped at the tigr (fine ). these developments have led to the possibility of exciting new candidate vaccines, several of which are in fact in the pipeline. it has been estimated that one-third of the world has been infected with tb bacteria. a relatively small number of immunocompetent individuals affected with ltbi will ever progress to active disease. reactivation of tb occurs when the immune system is compromised by external factors such as steroids or other immunosuppressants or by immune deficiency syndromes, which enables the latent (dormant) bacteria to "awaken" causing active disease. there is unfortunately no test that can currently predict which of these individuals will progress to active disease immunocompromised individuals have a high possibility of doing so. given that perspective, treatment for latent tb infection is most relevant for children under years, the elderly and individuals with hiv infection. to eliminate tb from the planet, a multipronged strategy incorporating early and effective treatment, vaccination and elimination of dormant reservoir of latent tb will be essential. ltbi treatment with inh ("chemoprophylaxis") is still the mainstay for treating latent tb infection but given that treatment extends to months, and that there is one death from hepatotoxicity for every , - , cases with such treatment, alternate strategies including rifamycin-based regimens are being explored (rangaka et al. ) . pregnancy outcomes following quinolone and fluoroquinolone exposure during pregnancy: a systematic review and meta-analysis rifapentine and isoniazid once a week versus rifampicin and isoniazid twice a week for treatment of drug-susceptible pulmonary tuberculosis in hiv-negative patients: a randomised clinical trial performance of light-emitting diode fluorescence microscope for diagnosis of tuberculosis infectious diseases society of america: treatment of tuberculosis false-positive xpert (r) mtb/rif assays in previously treated patients: need for caution in interpreting results recent changes in technical and operational guidelines for tuberculosis control programme in india - : a paradigm shift in tuberculosis control molecular diagnostics in tuberculosis bacterial factors that predict relapse after tuberculosis therapy the bcg story: lessons from the past and implications for the future five-year follow-up of a controlled trial of five -month regimens of chemotherapy for pulmonary tuberculosis assessment of the sensitivity and specificity of xpert mtb/rif assay as an early sputum biomarker of response to tuberculosis treatment ct features of lung scar cancer point of care diagnostics for tuberculosis diagnostic accuracy of adenosine deaminase and lymphocyte proportion in pleural fluid for tuberculous pleurisy in different prevalence scenarios delayed tuberculosis diagnosis and tuberculosis transmission guidelines for treatment of drug-susceptible tuberculosis and patient care ( ) world health organization clinical review: tuberculosis on the intensive care unit adjunctive vitamin d in tuberculosis treatment: meta-analysis of individual participant data progress in tuberculosis vaccine development and host-directed therapies -a state of the art review control of drug-resistant tuberculosis drug-resistant tuberculosis: an update on disease burden, diagnosis and treatment infectious diseases society of america/centers for disease control and prevention clinical practice guidelines: diagnosis of tuberculosis in adults and children tuberculosis in pregnancy: a review synergistic pandemics: confronting the global hiv and tuberculosis epidemics prednisolone and mycobacterium indicus pranii in tuberculous pericarditis comparison of a radiometric method (bactec) and conventional culture media for recovery of mycobacteria from smearnegative specimens worth a try in extensively drug-resistant tuberculosis? official american thoracic society/ centers for disease control and prevention/infectious diseases society of america clinical practice guidelines: treatment of drug-susceptible tuberculosis tuberculosis diagnostics: state of the art and future directions safety concerns surrounding quinolone use in children multidrug-resistant tuberculosis/rifampicin-resistant tuberculosis: principles of management reported tuberculosis in the united states controlling the seedbeds of tuberculosis: diagnosis and treatment of tuberculosis infection (ed) harrison's infectious diseases, th edn. mcgraw-hill education medical mycology: the pathogenic fungi and the pathogenic actinomycetes isoniazid-induced hepatotoxicity and neurotoxicity in rats investigated by ( )h nmr based metabolomics approach harrison's infectious diseases miliary tuberculosis: a new look at an old foe diagnostic mycobacteriology laboratory practices autologous mesenchymal stromal cell infusion as adjunct treatment in patients with multidrug and extensively drug-resistant tuberculosis: an open-label phase safety trial xpert(r) mtb/rif assay for pulmonary tuberculosis and rifampicin resistance in adults treatment of drug-susceptible pulmonary tuberculosis in hiv-uninfected adults this official statement of the american thoracic society was adopted by the ats board of directors contributing factors to mortality rates of pulmonary tuberculosis in intensive care units who treatment guidance for drug resistant tuberculosis ( ) who treatment guidelines for multidrug-and rifampicin-resistant tuberculosis fluoroquinolones for treating tuberculosis key: cord- - qznqp authors: denstaedt, scott j.; singer, benjamin h. title: hemophagocytic lymphohistiocytosis and other culture negative sepsis-like syndromes in the icu date: - - journal: evidence-based critical care doi: . / - - - - _ sha: doc_id: cord_uid: qznqp there are many sepsis-like inflammatory syndromes that may be encountered by critical care practitioners. clinically, these syndromes may imitate sepsis and are often identified after an extensive, but unrevealing evaluation for infection. in some instances, these syndromes are anticipated complications of advanced therapies for malignancy. it is vitally important to identify these disorders and treat them with specific chemotherapeutic or immunomodulating therapies. this chapter will focus on hemophagocytic lympho-histiocytosis (hlh), a rare disorder of pathologic immune system activation that presents as a sepsis-like illness in the critically ill. while treatment of hlh with chemotherapy and immunosuppression should be guided by an expert hematologist, the diagnosis is often made by the critical care medicine practitioner. we present a case illustrating the challenges of defining this syndrome in a patient with recurrent critical illness, and review the evidence underlying diagnostic and prognostic criteria for this protean syndrome. we also review several of the more common sepsis-like inflammatory syndromes that are encountered in the critical care unit along with their specific treatments. a year old man with history of idiopathic panniculitis on long-term steroid treatment presented to the hospital with a month history of dyspnea, malaise, nausea and vomiting. he was hypotensive and tachycardic with an acute kidney injury. prior to this presentation, he had been hospitalized for weeks at a nearby hospital with similar symptoms and had been found to have multifocal ground glass opacities on ct of the chest. his condition after admission improved with fluid resuscitation, stress-dose steroids, and treatment for presumed sepsis. infectious and rheumatologic workups were negative. he was noted to have bilateral pulmonary infiltrates ( fig. . a, b) . one week after discharge, however, he presented again with systemic inflammatory response syndrome (sirs). a bronchoscopy revealed only acute and chronic organizing pneumonia, with no evidence of infection. a bone marrow biopsy was performed, which demonstrated a hypercellular marrow. pulse dose steroids were initiated for organizing pneumonia and he improved significantly over the next week with continuation of empiric antimicrobials and tapering of steroids. after week of this regimen, however, he again decompensated with hypoxia and hypotension. worsening infiltrates were noted on his chest radiograph (fig. . c) . ferritin was elevated at ng/ml, and sil- r was elevated at u/ ml. trigylcerides were normal and fibrinogen was elevated. given his unremarkable bone marrow biopsy days before and elevated fibrinogen, the possibility of hemophagocytic lymphohistiocytosis (hlh) was dismissed as his hyperferritinemia and elevated sil- r were attributed to history of blood transfusion and occult infection. he continued to improve with empiric antimicrobials and steroids, and was discharged from the hospital days after admission to the icu to complete a course of levofloxacin on mg of prednisone daily. he presented to the hospital for the third time days after discharge with hypotension and hypoxia requiring endotracheal intubation and vasopressors, and antibiotics and steroids were restarted. he was admitted to the icu and his pulmonary status rapidly improved. after days, however, he again decompensated with hypotension and an elevated lactate. he received intravenous fluids and intravenous steroids. given the unclear association between changes in his antimicrobial regimen, steroid dosing, and clinical condition, his steroid dose was decreased from mg prednisone daily by mg each day. a repeat bone marrow biopsy was performed, which showed rare hemophagocytic macrophages but no evidence of malignancy. the next day, in the setting of reducing his steroid dose, he again had an episode of hypoxia and hypotension. ferritin was measured at that time and was ng/ml, increased from ng/ml the day before ( fig. . ). sil -r was measured and was elevated at u/ml. a diagnosis of hlh was made, and he was started on chemotherapy with etoposide and high dose dexamethasone. eight weeks had elapsed since his initial presentation. the patient was initiated on etoposide and dexamethasone without cyclosporine per the hlh- protocol. he rapidly improved and was moved to the hematology/oncology ward. his course was complicated by recurrent fevers and rising ferritin with the addition of filgrastim to his regimen after weeks of therapy. this was discontinued and he improved uniformly from then on. after month he was transferred to the physical medicine service for rehabilitation, and was subsequently discharged to home. he continued to do well months after his diagnosis, and was preparing for allogeneic bone marrow transplant at that time. hlh is a group of disorders characterized by aberrant immune activation leading to uncontrolled inflammation and, in many cases, critical illness. the underlying causes of hlh have been divided into primary causes, which encompass genetic defects in immune system regulation and often present in the neonatal period or early childhood, and secondary causes, which may be related to autoimmune disease, viral infection, or malignancy [ , ] . however, there is growing appreciation that many adolescent and adult patients develop hlh due to the interaction of an underlying genetic defect with an environmental trigger, such as viral infection with epstein-barr virus or cytomegalovirus [ ] . in adults, hlh often presents with dramatic, but nonspecific, features of including fever, organomegaly, cytopenias, kidney injury, and altered mental status [ , ] . as many as out of patients with hlh will present with severe coagulation disorders including disseminated intravascular coagulation and hypofibrinoginemia, many with associated severe bleeding [ ] . as illustrated in the case above, in our experience rapid fluctuating hemodynamic instability and hypoxic respiratory failure are also common features resulting from the abnormal inflammatory response. these presenting symptoms and laboratory findings overlap significantly with severe sepsis. it is not uncommon for adult patients with hlh to have recently been hospitalized for presumed sepsis, and hlh is entertained as a diagnosis after one or more unexplained relapses. the most important step in recognizing hlh in the critically ill patient, therefore, is to consider it in the differential diagnosis alongside more common causes of shock and respiratory failure. while primary hlh is a genetic disorder and may be diagnosed by molecular testing with a suggestive family history and clinical presentation, secondary hlh is a clinical syndrome with diverse causes. diagnostic criteria for hlh are currently based on the selection criteria for the hlh- trial, which enrolled pediatric patients (table . [ ] ). the presence of fever, organomegaly, cytopenia, and hypertriglyceridemia or hypofibrinogenemia, while nonspe-cific, are widely accepted as helpful criteria in defining the syndrome. the presence of hemophagocytosis, despite lending hlh its name, is also nonspecific [ ] . the search for appropriate biomarkers, then, remains an area of active and important investigation, as detailed below. in practice, the most critical information leading to the diagnosis of hlh is often derived from following the patient over several days, observing the response, or lack thereof, to treatment for sepsis or other common conditions, and the relationship of biomarkers to the patient's evolving clinical condition. the largest trial of hlh treatment are the hlh- and hlh- trials, which investigated the treatment of hlh with etoposide, dexamethasone, and cyclosporine in children and adolescents [ , ] . long-term follow-up of the hlh- trial suggested that intensified cyclosporine regimens and the addition of intrathecal steroids with reduced time to hsct did not improve outcome significantly in the pediatric population [ ] . etoposide and dexamethasone have become the mainstay of treatment in adult patients with hlh. antithymocyte globulin has also been used in combination with steroids, cyclosporine and intrathecal chemotherapy [ ] . a recent consensus review on malignancy-associated hlh suggests tailoring treatment to the underlying trigger, performance status, organ function and additional therapies the patient is receiving [ ] . early consultation with a hematologist and center experienced in the treatment of hlh is critical, both because immunomodulatory therapy for hlh is an area of active investigation, and because bone marrow transplantation is considered the curative therapy for patient with a known or suspected genetic cause of hlh. early therapies for hlh may be limited by end-organ damage present at the time of diagnosis, especially liver and kidney injury. as highlighted in the case presented here, treatment with high dose steroids may provide adequate temporizing therapy, allowing the patient to stabilize and ultimately receive definitive therapy. outcomes as the case above highlights, adult patients in the icu are often diagnosed with hlh after several episodes of apparent improvement followed by worsening, with periods of critical illness. the most comprehensive longitudinally studied cohort of patients with hlh was enrolled in the hlh- study, which exclusively studied children under age [ ] . in this cohort, cumulative -year mortality was %, with % mortality within weeks of diagnosis. survival was % among those patients who underwent hsct. hlh in adults is far more likely to be secondary than hlh in the pediatric population, though late presentations of primary hlh are possible [ ] . among critically ill patients who receive etoposide for treatment of hlh, a near % survival rate has been reported [ ] . a multi-center seven-year series of adult hlh cases described a median overall survival of months [ ] . while survival to discharge in many adult cohorts mirrors the pediatric population, long-term mortality in secondary hlh may be more reflective of underlying malignancy [ , ] . short-term mortality from hlh depends strongly on local treatment resources and supportive care, and varies widely among single-center studies from to nearly % prior to discharge [ , ] . across centers, however, hlh related to malignancy is found to have the highest mortality, followed by autoimmune and then infectious causes. in a single center descriptive study, the worst prognosis was found in patients with malignancy associated hlh and extreme hyperferritinemia with a median survival of months [ ] . in a north american tertiary care center, patients with hlh secondary to causes other than malignancy had a median survival of months, compared to less than months in cases associated with malignancy [ ] . severity of illness at the time of diagnosis of hlh also appears to bear on outcome despite treatment, with thrombocytopenia, marked hyperferritinemia, and shock at icu admission associated with mortality [ , ] . in a single center study of patients with hlh, in multivariate analyses age, lymphoma, decreasing platelet count and not having etoposide administered as part of treatment were all associated with increased mortality [ ] . as highlighted in the case presentation, diagnosis of hlh is often difficult and delayed. multiple conditions share at least several of the diagnostic criteria of hlh. genetic testing has been proposed as a means for improving diagnosis and therefore treatment of hlh. using a high-throughput sequencing strategy of genes linked to hlh researchers developed a screening test with . % sensitivity in detecting known genetic mutations for hlh [ ] . however, when utilized in a prospective cohort of adult hlh patients, only % of patients had an identifiable mutation, highlighting the lack of clear genetic cause in adult hlh. specific biomarkers for hlh would likely shorten time to diagnosis and therapy. bone marrow hemophagocytosis, while lending its name to hlh, is neither sensitive nor specific for the disorder [ ] . nk cell activity and sil- r levels are part of the hlh diagnostic criteria, but these laboratory studies are available in only a few reference laboratories. ideally, biomarker testing would rely on commonly and rapidly available assays, such as those for determining ferritin levels. in the pediatric population, hyperferritinemia (> , mcg/l) has a greater than % specificity for hlh [ ] . however, a recent retrospective study demonstrated that hlh is diagnosed in only . % of adults and . % of children with ferritin > , mcg/l suggesting this biomarker is less specific than previously thought [ ] . in patients diagnosed with hlh, ferritin levels reflect disease course and activity, and in children a marked decrease in ferritin (> %) portends decreased short term mortality during hlh treatment [ ] . in critically ill adults, however, the increased prevalence of conditions which elevate ferritin, such as infection, malignancy, autoimmune disease, liver injury and chronic blood transfusion make hyperferritinemia a nonspecific finding for hlh [ , ] . only % of hospitalized patients with elevated ferritin levels (> ng/ml) will have a diagnosis of hlh; patients are more likely to have liver injury or infection with elevated ferritin alone [ ] . in addition, particular causes of secondary hlh may be associated with variable biomarker patterns. for example, hlh due to lymphoma may present with a high ratio of sil- r to ferritin compared to other causes [ ] . given that hlh is a disorder of immune dysregulation, efforts have been made to differentiate hlh from infection based on cytokine patterns in the pediatric population [ ] . whether this approach will be fruitful in adults requires further study. there are no randomized controlled trials for the treatment of adult hlh. treatment regimens have been adapted from the hlh- and hlh- studies that primarily included children. a trial of salvage therapy of doxorubicin-etoposidemethylprednisolone in adults with mostly non-familial refractory hlh, demonstrated an overall response rate of %, with of patients surviving to undergo disease specific therapy [ ] . a recent case series reported the use of the interleukin antagonist, anakinra, with steroids and ivig in eight critically ill patients with hlh and demonstrated a hospital survival rate of % [ ] . additional research on nonetoposide based treatment regimens may therefore be warranted for critically ill patients with multiple organ failure. alemtuzumab, a humanized monoclonal antibody targeting cd , has been used as a salvage therapy for refractory adult hlh [ ] and is being evaluated as part of an ongoing clinical trial for primary therapy in combination with etoposide (clinicaltrials.gov identifier nct ). other biologics and small molecules are currently being evaluated for various forms of hlh (ruloxitnib nct , tocilizumab nct , emapalumab nct ). while hlh is associated with a wide variety of primary and secondary etiologies, several other sepsis-like syndromes have been identified in specific clinical contexts. as mentioned earlier, secondary hlh (shlh) occurs in the setting of other medical conditions. mas is a form of shlh associated with autoimmune disease [ ] . in children, it is a well-recognized complication of systemic juvenile idiopathic arthritis (sjia) [ ] . in adults, mas is commonly seen with rheumatologic diseases such as in systemic lupus erythematosus and adult onset still's disease (aosd), the latter being characterized clinically by quotidian fevers, arthralgia, salmon-pink skin rash, sore throat, lymphadenopathy and organomegaly [ ] . nearly half of all mas/shlh patients are critically ill requiring mechanical ventilator or vasoactive support [ ] . clinically, mas/shlh is difficult to diagnose, though should be suspected in critically ill, persistently febrile patients. while there are suggested diagnostic criteria for mas complicating sjia [ ] , there are no such criteria for mas in adult rheumatologic disease. recently, a variable scoring system (hscore) has been developed for use in identifying adults with shlh (table . ) [ ] . an hscore of ≥ corresponds to a sensitivity of % and specificity of % for identifying patients with shlh. treatment should be performed in consultation with hematologic experts as there is no validated approach to treatment of adult patients with mas/shlh. the foundation of treatment is high dose steroids ( g methylprednisolone daily for - days), although many patients may require additional therapy [ ] . use of the il- receptor antagonist, anakinra, has shown benefit in mas associated with sjia [ ] , and may be useful in adult mas/shlh. additional therapies include ivig, rituximab (in ebv associated disease), and cyclosporine [ ] . a macrophage activation-like syndrome (mals) has been described in patients with sepsis who have an hscore of > and/or had evidence of disseminate intravascular coagulation and hepatobiliary dysfunction [ ] . presence of mals was associated with an increased risk of death within the first days of sepsis. a ferritin level of > ng/ml was associated with a % specificity for mals diagnosis in these patients. interestingly, sepsis patients with mas features including hbd/dic have a mortality benefit in the phase iii clinical trial using anakinra [ ] . however, its utility in treating patients with sepsis with mas features requires validation in future clinical trials. chimeric antigen receptor (car) t cells are now being used in treatment of leukemia and lymphoma. while effective in inducing bone marrow remission [ ] [ ] [ ] , car-t cells may be associated with the development of cytokine release syndrome (crs) which frequently requires treatment in the icu. this syndrome is characterized by fevers, hypotension, capillary leak, neurotoxicity and death [ ] . neurologic side effects can be particularly devastating and include encephalopathy, aphasia, obtundation and seizures [ ] . rates of crs are variable among studies ranging from % to %, with about in patients developing severe symptoms [ ] . development of crs is associated with higher tumor burden (> % bone marrow blasts) and higher doses of car-t cells [ ] . diagnostic criteria for crs due to car-t cells have been developed (table . ) [ ] , but generally this syndrome should be suspected in patients receiving car-t therapy who develop evidence of fever or end organ damage. given the common nature of this side effect, grading scales for symptom severity and treatment algorithms have been proposed. at our institution, we consider icu admission for grade toxicity and require icu admission for grade and toxicities (table . ) . after initial studies demonstrated efficacy of interleukin- receptor blocking monoclonal antibody, tocilizumab, in severe crs related to car-t cell therapy [ ] , it has been commonly used as a first line agent in patients with symptoms that include progressive hypoxia and hypotension. management is usually performed in the context of established protocols at the institution administering the car-t therapy. ips is a complication of allogeneic hematopoietic cell transplant (hct) that commonly leads to respiratory failure requiring management in the intensive care unit. it is defined as an idiopathic pneumonia-like syndrome with evidence of widespread alveolar injury in the absence of other common etiologies of respiratory failure including infection [ ] . the pathophysiology of ips is unclear, though it has been suggested that direct toxic effects of hct conditioning regimens, occult infection, and certain inflammatory cytokines including tnfα may play a role. clinically, patients may present with symptoms of pneumonia, hypoxic respiratory failure and multi-lobar infiltrates on chest imaging. ips usually presents in the first months after hematopoietic cell transplant (hct), though later presentations more than days post-hct have also been reported [ ] . the historical incidence is - % with traditional myeloablative conditioning and lower with modern nonmyeloablative regimens [ ] . in one retrospective cohort study, median time to onset was days (iqr - days) from hct and two-year mortality was % [ ] . there is no pathognomonic pattern of histologic alveolar injury and patients are rarely able to tolerate lung biopsy. therefore, diagnosis relies on exclusion of other etiologies that may cause multi-lobar infiltrates and respiratory failure. bronchoalveolar lavage (bal) should be performed and tested for common bacterial (including acid-fast bacilli, nocardia, legionella) and non-bacterial (cmv, rsv, pcp, hsv, vzv, influenza, parainfluenza, human metapneumovirus, rhinovirus, coronavirus, hhv , chlamydia, mycoplasma and aspergillus) organisms [ ] . the importance of this testing is emphasized by the fact that % of bal specimens from ips patients had detectable bacterial or viral pathogens using modern techniques [ ] . thus, lower respiratory tract sampling may be one of the most important diagnostics an intensivist can offer. to reach a diagnosis of ips, patients must also have an absence of cardiac dysfunction, acute renal failure or iatrogenic fluid overload contributing to their pulmonary dysfunction. current treatment for ips includes supportive care for hypoxic respiratory failure and systemic corticosteroids. response rates in recent clinical trials were % for high life-threatening symptoms with requirement for mechanical ventilatory support or grade organ toxicity (except transaminitis) grade death grade ii-iv refers to common terminology criteria for adverse events (ctcae v . ) adapted from [ ] dose steroids, with a median time to response of days [ ] . steroid treatment is typically with mg/kg/day methylprednisolone equivalents continued intravenous for the three days with subsequent oral dosing and then tapering after day . the tnfα binding protein etanercept has been used in combination with steroids, demonstrating improvements in response to therapy and early survival in small single center studies [ ] [ ] [ ] . in a phase iii clinical trial of adult ips, etanercept did not provide additional benefit over steroids alone, though enrollment in this trial was prematurely ended and definitive conclusions regarding use of etanercept in ips cannot be made from this trial alone. notably, etanercept use was not associated with increased risk of opportunistic infection or malignancy relapse compared with placebo controls. veno-venous extracorporeal membrane oxygenation use has been reported in both early and late ips, though its efficacy and impact on outcomes is unclear [ , ] . for patients who do not respond to steroids and anti-tnfα therapies the treatment is also unclear. il- is elevated in the plasma of patients with ips [ ] and it has been suggested that the il- inhibitor, tocilizumab, may have some benefit in treating or preventing ips [ ] . several important clinical syndromes, including hlh, mas, ips, and crs can mimic sepsis and cause critical illness through immune dysregulation in the absence of infection. identification of these syndromes requires a high index of suspicion, awareness of the clinical context and risk factors, as well as careful exclusion of infection and attention to response to empiric therapies. while prognosis in these syndromes is often uncertain, recent evidence indicates that patient survival in the presence of excellent supportive care is strongly driven by the underlying disease rather than the sepsis-like syndrome itself. hemophagocytic syndromes -an update how i treat hemophagocytic lymphohistiocytosis genetic features of late onset primary hemophagocytic lymphohistiocytosis in adolescence or adulthood hemophagocytic lymphohistiocytosis: 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activation-like syndrome: an immunological entity associated with rapid progression to death in sepsis interleukin- receptor blockade is associated with reduced mortality in sepsis patients with features of macrophage activation syndrome: reanalysis of a prior phase iii trial * chimeric antigen receptor t cells for sustained remissions in leukemia efficacy and toxicity management of - z car t cell therapy in b cell acute lymphoblastic leukemia cytokine release syndrome with chimeric antigen receptor t cell therapy toxicities of chimeric antigen receptor t cells: recognition and management cd car -t cells of defined cd + : cd + composition in adult b cell all patients chimeric antigen receptor-modified t cells for acute lymphoid leukemia current concepts in the diagnosis and management of cytokine release syndrome an official american thoracic society research statement: noninfectious lung injury after hematopoietic stem cell transplantation: idiopathic pneumonia syndrome etanercept and corticosteroid therapy for the treatment of late-onset idiopathic pneumonia syndrome the association between platelet transfusion and idiopathic pneumonia syndrome is unaffected by platelet product type idiopathic pneumonia syndrome after hematopoietic cell transplantation: evidence of occult infectious etiologies double-blind, placebo-controlled trial of soluble tumor necrosis factor receptor: enbrel (etanercept) for the treatment of idiopathic pneumonia syndrome after allogeneic stem cell transplantation: blood and marrow transplant clinical trials network protocol the impact of soluble tumor necrosis factor receptor etanercept on the treatment of idiopathic pneumonia syndrome after allogeneic hematopoietic stem cell transplantation etanercept (enbrel) administration for idiopathic pneumonia syndrome after allogeneic hematopoietic stem cell transplantation high-dose corticosteroids with or without etanercept for the treatment of idiopathic pneumonia syndrome after allo-sct concurrent treatment with a tumor necrosis factor-alpha inhibitor and veno-venous extracorporeal membrane oxygenation in a post-hematopoietic stem cell transplant patient with idiopathic pneumonia syndrome: a case report successful use of extracorporeal membrane oxygenation in a hematopoietic stem cell transplant patient with idiopathic pneumonia syndrome tnf-receptor inhibitor therapy for the treatment of children with idiopathic pneumonia syndrome. a joint pediatric blood and marrow transplant consortium and children's oncology group study (asct ) the primacy of il- in ips? key: cord- -i z tdrk authors: dangi, mehak; kumari, rinku; singh, bharat; chhillar, anil kumar title: advanced in silico tools for designing of antigenic epitope as potential vaccine candidates against coronavirus date: - - journal: bioinformatics: sequences, structures, phylogeny doi: . / - - - - _ sha: doc_id: cord_uid: i z tdrk vaccines are the most economical and potent substitute of available medicines to cure various bacterial and viral diseases. earlier, killed or attenuated pathogens were employed for vaccine development. but in present era, the peptide vaccines are in much trend and are favoured over whole vaccines because of their superiority over conventional vaccines. these vaccines are either based on single proteins or on synthetic peptides including several b-cell and t-cell epitopes. however, the overall mechanism of action remains the same and works by prompting the immune system to activate the specific b-cell- and t-cell-mediated responses against the pathogen. rino rappuoli and others have contributed in this field by plotting the design of the most potent and fully computational approach for discovery of potential vaccine candidates which is popular as reverse vaccinology. this is quite an unambiguous advance for vaccine evolution where one begins with the genome information of the pathogen and ends up with the list of certain epitopes after application of multiple bioinformatics tools. this book chapter is an effort to bring this approach of reverse vaccinology into notice of readers using example of coronavirus. it compelled us to apply the well-known reverse vaccinology (rv) approach on available proteome of coronavirus. rv approach has been successfully applied on many prokaryotes, but there are very few known applications on eukaryotes and viruses. so, it is worthwhile to explore the potential of this approach to identify potential vaccine candidates for coronavirus. rv basically does the in silico examination of the viral proteome to hunt antigenic and surface-exposed proteins. this approach was initially applied successfully to neisseria meningitidis serogroup b (kelly and rappuoli ) against which none of the prevailing techniques could develop a vaccine. the present book chapter is intended to explore the potential of rv approach to select the probable vaccine candidates against coronavirus and validate the results using docking studies. undoubtedly, the traditional approaches for vaccine development are fortunate enough to efficiently resist the alarming pathogenic diseases of its time. however, the traditional approach suffers from certain limitations like it is very timeconsuming, the pathogens which can't be cultivated in the lab conditions are out of reach, and certain non-abundant proteins are not accessible using this approach (rappuoli ) . consequently, a number of pathogenic diseases are left without any vaccine against them. all these limitations are conquered by reverse vaccinology approach utilizing genome sequence information which ultimately is translated into proteins. hence all the proteins expressed by the genome are accessible irrespective of their abundance, conditions in which they expressed. the credit of fame of reverse vaccinology should go to the advancements in the sequencing strategies worldwide. accordingly, improvement in the sequencing technologies has flooded the genome databases with huge amount of data which can be computationally undertaken to reveal the various crucial aspects of the virulence factors of the concerned pathogen. reverse vaccinology is based on same approach of computationally analysing the genome of pathogen and proceeds step by step to ultimately identify the highly antigenic, secreted proteins with high epitope densities. the best epitopes are selected as potential vaccine candidates (pizza et al. ) . this approach has brought the unapproachable pathogens of interest in spotlight and is evolving as the most reassuring tool for precise selection of vaccine candidates and brought the use of peptide vaccines in trend (sette and rappuoli ; kanampalliwar et al. ). bexsero is the first universal serogroup b meningococcal vaccine developed using rv, and it has currently earned positive judgement from the european medicines agency (gabutti ) . whether it is discovery of pili in gram-positive pathogens which were thought to not have any pili or the sighting of factor g-binding protein in meningococcus (alessandro and rino ), the reverse vaccinology steals all the credits from other conventional approaches. most of the applications of rv are against prokaryotes and very few against eukaryotes and viruses because of complexity of their genome. corynebacterium urealyticum (guimarães et al. ) , mycobacterium tuberculosis (monterrubio-lópez et al. ) , h. pylori (naz et al. ) , acinetobacter baumannii (chiang et al. ) , rickettsia prowazekii (caro-gomez et al. ) , neospora caninum (goodswen et al. ) and brucella melitensis (vishnu et al. ) are the examples of some pathogens that are recently approached using this in silico technique in order to spot some epitopes having potential of being a vaccine candidate. herpesviridae (bruno et al. ) and hepatitis c virus (hcv) (kolesanova et al. ) are the examples of the viruses that are addressed using this approach. (altschul et al. ; okonechnikov et al. ; golosova et al. ) . multiple sequence alignment (msa) was done via clustalw, and the phylogenetic tree was constructed using nj method from unipro ugene . . bioinformatics toolkit (okonechnikov et al. ). analysis of secondary structure of the proteins of seed genome was done by means of expasy portal. the aim is to forecast the solvent accessibility, instability index, theoretical pi, molecular weight, grand average of hydropathicity (gravy), aliphatic index, number of charged residues, extinction coefficient etc. (http://web. expasy.org/protparam/; gasteiger et al. ) . virus-mploc was used to identify the localization of proteins of virus in the infected cells of host (http://www.csbio.sjtu.edu.cn/bioinf/virus-multi/; hong-bin shen and kuo-chin chou ) . this information is important to understand the destructive role and mechanism of the viral proteins in causing the disease. in total six different subcellular locations, namely, host cytoplasm, viral capsid, host plasma membrane, host nucleus, host endoplasmic reticulum and secreted proteins, were covered. these predictions could help in formulation of better therapeutic options against the virus. as per the protocol of rv, secreted and membrane proteins are of special interest, therefore, filtered for further analysis. to predict the number of transmembrane helices tmhmm server v. . (http://www.cbs.dtu.dk/services/tmhmm/; krogh et al. ) was used. signal peptides are known to impact the immune responses and possess high epitope densities. moreover, most of the known vaccine candidates also possess signal peptides. hence, it is worthwhile to predict signal peptides in proteins prior to epitope predictions. signal-blast web server is used to predict the signal peptides without any false predictions (http://sigpep.services.came.sbg.ac.at/signalblast.html; frank and sippl ) . the prediction options include best sensitivity, balanced prediction, best specificity and detect cleavage site only. we choose to make the predictions using each option, and the proteins predicted as signal peptide by all the four options were preferred for further investigation. the most appropriate targets as vaccine candidates are those which possess the adhesion-like properties because they not only mediate the adhesion of pathogen's proteins with cells of host but also facilitate transmission of virus. adhesions are known to be crucial for virulence and are located on surface which makes them promptly approachable to antibodies. the stand-alone spaan with a sensitivity of % and specificity of % was used to carry out the adhesion probability predictions, and the proteins with having adhesion probabilities higher than or equal to . were selected (sachdeva et al. ). betawrap motifs are dominant in virulence factors of the pathogens. if the proteins are predicted to possess such motifs, then they are appropriate to be taken under reverse vaccinology studies. betawrap server is the only online web server to make such predictions. the proteins having p-value lower than . were anticipated to contain betawraps (http://groups.csail.mit.edu/cb/betawrap/betawrap.html; bradley et al. ). for added identification of the antigenic likely of the proteins, they were subjected to vaxijen server version . . it is basically an empirical method to hunt antigenic proteins. so, if the proteins are not found antigenic using other sequence-based methods, then they can be identified using this method. this step confirms the antigenicity of proteins selected using above-mentioned steps (http://www.ddgpharmfac.net/vaxijen/vaxijen/vaxijen.html; doytchinova and flower ). for being a probable vaccine candidate, the protein should not exhibit the characteristics of an allergen as they trigger the type- hypersensitivity reactions causing allergy. therefore, to escape out such possibilities, the proteins were also subjected to allergenicity predictions using allertop (http://www.pharmfac.net/ allertop; dimitrov et al. ) and algpred tools (http://www.imtech.res.in/ raghava/algpred/submission.html; saha and raghava a, b). to check whether the filtered proteins possess any similarity to host proteins or not, the standard blastp (http://blast.ncbi.nlm.nih.gov/blast) searches were performed. in case of sequence similarity, there is a feasibility of generation of immune responses against own cells. predicting the epitopes binding to mhc class i is the main decisive phase of the rv to carry out valid vaccine predictions. the predicted epitopes were docked with receptor that is hla-a* using cluspro (http://cluspro.bu.edu/login.php; kozakov et al. ) that is an automated protein-protein docking web server. the literature searches provided the information of conserved residues of the receptor site. the default parameters were used for docking (comeau et al. a, b; kozakov et al. ). a total of different sequenced strains of coronavirus are available at ncbi. among them strains are pathogenic to humans. various information regarding source, host and collection of these strains are presented in table . and . . this information can be obtained from ncbi's genome database, the virus pathogen database and analysis resource and genomes online database (liolios et al. ; pickett et al. ) . the mers strain is taken as seed genome as it is the most prevalent and disastrous strain among others. its proteome consists of total proteins as shown in table . . the results of sequence similarity to reveal orthologs using blastp are shown in table . . the sequences with greater than % identity score are considered as homologs. the phylogenetic tree is depicted in fig. . and the mers-cov, taken as seed genome, found clustered with different bat coronaviruses. the results of analysis of secondary structure of the proteome using expasy tools are shown in the table . . from the analysis of charge on the residues and ph values, it is concluded that six of the proteins are basic and positively charged unlike allergens which are acidic in nature. however, five proteins are acidic and show negative charge. the negative gravy score of five proteins justify them to be of hydrophilic nature with majority of the residues positioned towards the surface. for the rest of six proteins, the gravy score is positive; it means that these are the accession number and identity of orthologs obtained in different strains is shown in the table hydrophobic proteins. the proteins with less than value of instability index are quite stable than those with higher values. all the proteins are having the molecular weight less than kda except (yp_ . , yp_ . and yp_ . ). this exhibits the effectiveness of lightweight proteins as targets as they can be easily purified because of their low molecular weights. the protein yp_ . is reported as a spike glycoprotein. it is acidic with prominent negative charge, with negative gravy score which suggests its hydrophilicity and figure . depicts the subcellular localization of proteins of the seed genome, i.e. mers-cov. only one protein was predicted to be localized in host cytoplasm, four in host membrane, two in both host cell membrane and endoplasmic reticulum (er) while two in only er, and two are left unrecognized. the known spike protein is predicted to be localized in host er. from these results we decided to pick the proteins which are located in host membrane or were predicted to be localized in both host membrane and er. the two are known envelop protein and membrane protein from bibliographic studies, and along with that, the known spike protein was also included in the filtered results. out of the filtered proteins, only two (yp_ . and yp_ . ) contain more than two transmembrane helices, therefore filtered out. the results of transmembrane helices prediction are tabulated in table . . figure . depicts the subcellular localization of proteins of all the four selected genomes using virus-mploc prediction tool. the proteins that are predicted to possess the signal peptides by signal-blast web server are yp_ . and yp_ . . the results of signal-blast web server are tabulated in the table . . this step takes into account the concept of adhesion-based virulence. adhesions cause pathogen recognition and initiation of inflammatory responses by the host. spaan predicted (yp_ . and yp_ . ) out of proteins of mers strain as adhesive (table . ). only one protein (yp_ . ) was predicted to contain betawrap motifs within it (table . ). hence, it is considered virulent and might be responsible for initializing the infection in the host. a total of out of proteins of mers strain were predicted antigenic (prediction values greater than . ). the protein with accession number yp_ . and yp_ . were among the filtered proteins, however, not predicted antigenic, therefore filtered out. as a result, only four proteins (yp_ . , yp_ . , yp_ . and yp_ . ) were kept for further analyses. none of the proteins of mers-cov possessed any clue of allergenicity as per prediction results from algpred and allertop tools; it means that no vigorous immune responses will be mounted if the epitopes from these proteins will be adopted as vaccine candidates. none of the protein of mers strain shows similarity with the proteins of host that demonstrates that the epitopes from these proteins can safely elicit the required immune response without the hazard of autoimmunity. in total different -mer epitopes with potential to bind to receptors of both b-cell and t-cell were predicted. the list of the predicted epitopes can be found in the table . and are specific for mers-cov strain. all these epitopes displayed no conservancy with proteins of other human and non-human pathogenic strains. docking permits to reveal the binding energy or potency of connection among epitopes and the receptor in appropriate orientation. the cluspro docking server was used to dock the predicted epitopes against hla-a* . the structure of the receptor was available from pdb and was optimized before docking to free it from the complexed self-peptide ( u y, resolution . Å, bouvier et al. ). pepstr (peptide tertiary structure prediction server; kaur et al. ) was used to derive the tertiary structure of the predicted peptides. figure . depicts the quaternary structure of the receptor hla-a* with its conserved active site known to form complex with the peptides (bouvier et al. ). the binding energy results obtained after performing docking analysis are listed in table . . the -mer epitope vvcaitllv at site of protein yp_ . docked to the receptor with smallest amount of binding energy (À . ) and hydrogen bonds. the next epitope in the list was also from the same protein yp_ . at site , i.e. tllvcmafl. the predicted structure of the top potent epitopes on the basis of docking energy and the snapshots of docking results are displayed in figs. . , . , . , . and . . the most chief restriction for developing a safe and sound vaccine against any of the virus is to identify the protective antigens. the present study is an effort of application of reverse vaccinology approach to investigate a choice of coronavirus proteomes to identify possible vaccine targets. this technique has demonstrated to be a competent way to forecast different epitopes from the selected seed genome. these epitopes are from spike glycoprotein, ns protein, ns b protein and envelope protein. unfortunately none of the epitope is found conserved in other strains, and all are specific to mers-cov. the docking analysis studies revealed perfect binding between hla-a* receptor and epitopes. the conserved residues of the receptor site are also involved in h-bonding with epitope residues. further, the selected antigenic epitopes must be validated using in vitro and in vivo studies to confirm their potential as vaccine candidates. review: reverse vaccinology: developing vaccines in the era of genomics basic local alignment search tool the middle east respiratory syndrome coronavirus -a continuing risk to global health security crystal structures of hla-a* complexed with antigenic peptides with either the amino-or carboxyl-terminal group substituted by a methyl group betawrap: successful prediction of parallel beta -helices from primary sequence reveals an association with many microbial pathogens geminiviridae. in: virus taxonomy-ninth report of the international committee on taxonomy of viruses lessons from reverse vaccinology for viral vaccine design discovery of novel cross-protective rickettsia prowazekii t-cell antigens using a combined reverse vaccinology and in vivo screening approach identification of novel vaccine candidates against acinetobacter baumannii using reverse vaccinology cluspro: a fully automated algorithm for protein-protein docking cluspro: an automated docking and discrimination method for the prediction of protein complexes middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group allertop v. -a server for in silico prediction of allergens vaxijen: a server for prediction of protective antigens, tumour antigens and subunit vaccines high performance signal peptide prediction based on sequence alignment techniques meningococcus b: control of two outbreaks by vaccination protein identification and analysis tools on the expasy server unipro ugene ngs pipelines and components for variant calling, rna-seq and chip-seq data analyses discovering a vaccine against neosporosis using computers: is it feasible? genome informatics and vaccine targets in corynebacterium urealyticum using two whole genomes, comparative genomics, and reverse vaccinology reverse vaccinology: basics and applications pepstr: a de novo method for tertiary structure prediction of small bioactive peptides reverse vaccinology and vaccines for serogroup b neisseria meningitidis way to the peptide vaccine against hepatitis c piper: an fft-based protein docking program with pair wise potentials the cluspro web server for protein-protein docking predicting transmembrane protein topology with a hidden markov model: application to complete genomes an integrative approach to ctl epitope prediction, a combined algorithm integrating mhc-i binding, tap transport efficiency, and proteasomal cleavage prediction improved method for predicting linear b-cell epitopes the genomes on line database (gold) v. : a monitor of genome projects worldwide identification of novel potential vaccine candidates against tuberculosis based on reverse vaccinology identification of putative vaccine candidates against helicobacter pylori exploiting exoproteome and secretome: a reverse vaccinology based approach database resources of the national center for biotechnology information unipro ugene: a unified bioinformatics toolkit scheme for ranking potential hla-a binding peptides based on independent binding of individual peptide side-chains virus pathogen database and analysis resource (vipr): a comprehensive bioinformatics database and analysis resource for the coronavirus research community identification of vaccine candidates against serogroup b meningococcus by whole-genome sequencing reverse vaccinology spaan: a software program for prediction of adhesins and adhesin-like proteins using neural networks algpred: prediction of allergenic proteins and mapping of ige epitopes prediction of continuous b-cell epitopes in an antigen using recurrent neural network reverse vaccinology: developing vaccines in the era of genomics virus-mploc: a fusion classifier for viral protein subcellular location prediction by incorporating multiple sites propred : prediction of promiscuous mhc class-i binding sites identification of potential antigens from non-classically secreted proteins and designing novel multitope peptide vaccine candidate against brucella melitensis through reverse vaccinology and immunoinformatics approach key: cord- -r quizqn authors: kim, won ho; yoon, hyun-kyu; lee, ho-jin title: intensivist coverage and patient outcomes date: - - journal: j anesth doi: . /s - - - sha: doc_id: cord_uid: r quizqn nan with interest, we read the study by oh et al. [ ] , which retrospectively analyzed a large cohort of patients admitted to the surgical intensive care unit. although results are encouraging in the quest to find evidence that supports the relevance of intensivist in the management of post-surgical patients, two questions have come up in the interpretations of the results. firstly, parameters of intraoperative management which may affect the postoperative incidence of acute kidney injury (aki) were not sufficiently included in the contributors of propensity score. just like the patient management variables during postoperative days, intraoperative parameters including hemodynamics, vasopressor infusion, hydroxyethyl starch use, nephrotoxic agent use such as diuretics, and transfusion are equally or more important for the development of aki especially during immediate postoperative period [ ] . secondly, in the sensitivity analysis of aki according to admission time, there were only two time-points where the associations were significant and there seems no dose-response relationship between the duration of nonintensivist coverage and the incidence of aki. to suggest causality in the retrospective analysis, dose-response relationship should be demonstrated in addition to consistency, biological plausibility and temporal relationship [ ] . further evidence are required to substantiate their hypothesis. conflict of interest no competing interest declared. admission to the surgical intensive care unit during intensivist coverage is associated with lower incidence of postoperative acute kidney injury and shorter ventilator time prediction and prevention of acute kidney injury after cardiac surgery the environment and disease: association or causation? key: cord- -g gesvzt authors: heitzer, andrew m.; piercy, jamie c.; peters, brittany n.; mattes, allyssa m.; klarr, judith m.; batton, beau; ofen, noa; raz, sarah title: cumulative antenatal risk and kindergarten readiness in preterm-born preschoolers date: - - journal: j abnorm child psychol doi: . /s - - - sha: doc_id: cord_uid: g gesvzt a suboptimal intrauterine environment is thought to increase the probability of deviation from the typical neurodevelopmental trajectory, potentially contributing to the etiology of learning disorders. yet the cumulative influence of individual antenatal risk factors on emergent learning skills has not been sufficiently examined. we sought to determine whether antenatal complications, in aggregate, are a source of variability in preschoolers’ kindergarten readiness, and whether specific classes of antenatal risk play a prominent role. we recruited preschoolers ( girls; ages – years), born ≤ ( )/( ) weeks’ gestation, and reviewed their hospitalization records. kindergarten readiness skills were assessed with standardized intellectual, oral-language, prewriting, and prenumeracy tasks. cumulative antenatal risk was operationalized as the sum of complications identified out of nine common risks. these were also grouped into four classes in follow-up analyses: complications associated with intra-amniotic infection, placental insufficiency, endocrine dysfunction, and uteroplacental bleeding. linear mixed model analyses, adjusting for sociodemographic and medical background characteristics (socioeconomic status, sex, gestational age, and sum of perinatal complications) revealed an inverse relationship between the sum of antenatal complications and performance in three domains: intelligence, language, and prenumeracy (p = . , . , . , respectively). each of the four classes of antenatal risk accounted for little variance, yet together they explained . %, . %, and . % of the variance in the cognitive, literacy, and numeracy readiness domains, respectively. we conclude that an increase in the co-occurrence of antenatal complications is moderately linked to poorer kindergarten readiness skills even after statistical adjustment for perinatal risk. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. born preschoolers and their term-born peers yielded poorer scores in the former group, regardless of gestational age. group differences have been documented in expressive and receptive language abilities as well as in visuomotor or graphomotor (preprinting) skills in both very preterm (foster-cohen et al. ; caravale et al. ; torrioli et al. ) and late-preterm (baron et al. ; baron et al. ) cohorts. establishing the nature of early biomedical risk factors that forecast deficits in critical academic precursor skills is essential for identification of preschoolers at risk for deviation from the typical neurodevelopmental trajectory. findings from a recent quantitative integration of the literature suggest that both preterm birth and adverse antenatal factors are important antecedents of intellectual disability diagnosed between and years of age (huang et al. ). yet few preschool outcome studies included within-group examination of the links between complications associated with preterm-birth and performance on neuropsychological tasks that tap domain-specific, literacy or numeracy, precursor skills. foster- cohen et al. ( ) , focusing on the impact of peri-, and neonatal, but not antenatal, complications, reported no significant associations between several early risk factors (including the sum of perinatal complications) and language delay within a cohort of four-year-old preschoolers born very preterm. in a similar sample of preschoolers born < weeks gestation torrioli et al. ( ) were able to document a significant relationship between a single major antenatal complication, intrauterine growth restriction, and intelligence (but not visual-motor integration). consistent with the prevailing fetal programming framework (barker ) , conditions or risks originating in-utero have the unique capacity to modify long-term physical health and behavioral outcome. though the exact mechanisms leading to disease or cognitive-behavioral deficits have yet to be specified, it has been suggested that fetal adaptation to environmental stress may involve vascular, metabolic or endocrine changes that permanently alter bodily structure or function (hocher et al. ) . antenatal perturbations are likely transmitted to the infant through their effects on placental function. the latter, in turn, adversely influences fetal and postnatal brain development and cognitive-behavioral outcome (zeltser and leibel ; buss et al. ; davis and sandman ) . within the high-risk group of preterm-born children, both the variability in the base-rates of various antenatal complications associated with prematurity and the sheer number of medical risk factors that require consideration often impede exploration of developmental outcome effects of early biological adversity. additionally, the developmental impact of discrete medical complications is probably cumulative (shalev et al. ) . cumulative risk indices may show increased stability across developmental periods, accounting for more outcome variability between individuals than specific complications (wade et al. ) . aggregate scores that reflect cumulative risk associated with a distinct early risk epoch may be increasingly sensitive compared to discrete complications that are likely linked to small effects that are difficult to detect (whitehouse et al. ) . although the influence of cumulative perinatal risk on developmental outcome of preterm children has received some consideration (e.g., carmody et al. ; foster-cohen et al. ) , the effects of cumulative antenatal risk in this vulnerable group remain essentially unexplored. hence, our chief objective was to examine the combined contribution of common antenatal complications to explaining individual differences in academic (kindergarten) readiness within a preterm-born cohort. in addition to gauging intellectual abilities, we evaluated language skills as precursors of reading attainment, visual-motor integration skills as antecedents of writing-skill development, and early number concepts as the preschool forerunners of math achievement. we predicted that degree of antenatal risk in preterm-born preschoolers will be inversely related to both general cognitive, and domain-specific, neuropsychological skills that provide the basis for scholastic achievement. preterm-born children (< weeks' gestation) were recruited for the study between and years of age and evaluated between may -july . the children were born at william beaumont hospital (wbh), royal oak, mi, in mi, in - , and treated in the neonatal intensive care unit (nicu). at wbh-nicu resuscitation is attempted for all infants with an estimated gestational age ≥ / weeks (batton et al. ) . children with congenital anomalies, or who required mechanical ventilation after discharge, were excluded from the retrospectively identified nicu cohort matching our inclusion criteria. the families of . % of cases could still be reached for recruitment attempt based on contact information provided at the time of birth (see online resource ). of these, families of . % cases were not interested, for multiple reasons. in accord with wbh human investigation committee guidelines nonparticipating families were not specifically queried, yet amongst common reasons spontaneously provided by families for nonparticipation were being "too busy", residing too far away or not wanting to travel from suburban areas to the city. families of additional cases ( . % of those successfully contacted) "no-showed" for the schedule assessment twice and were not rescheduled. the available participants constituted . % of the pool of cases whose families could be contacted between and years of age. of evaluated cases ten were excluded from data analyses, five with suspected antenatal substance exposure and five with missing background medical information pertinent to this investigation. altogether cases were available for this study (see table for sociodemographic characteristics). as the table shows, our sample included participants from a very broad socioeconomic range. nonetheless, the relatively high mean ses ( . of possible points; sd = . ) reflects the composition of the catchment area of wbh. this region includes primarily middle social strata, thereby minimizing the possibility of confounding the effects of antenatal risk with other adverse environmental factors associated with socioeconomic status. correspondingly, about % of nicu admissions were covered by private medical insurance and only % by medicaid. additionally, . % of the mothers and . % of the fathers attained a college degree. the final sample included ( . %) boys and ( . %) participants of african-american descent. the proportion of males to females in our sample ( . %: . %) was not significantly different from the proportion observed in the remaining portion of the total relevant nicu cohort from which we attempted to recruit ( . %: . %; χ [ ] = . , p = . ). the proportion of singletons to multiples in our sample ( . %: . %) was also not significantly different from the proportion observed in the remainder of the total cohort ( . %: . %; χ [ ] = . , p = . ). information about racial distribution was available for two thirds of the total relevant cohort. as noted above, our sample included . % african americans, a somewhat smaller proportion than that observed in the remainder of the relevant nicu cohort ( . %;χ [ ] = . , p = . ). the mean gestational age ( . ± . weeks) and birth weight ( , ± g) in our sample approximated the mean gestational age ( . ± . weeks) and birth weight ( , . ± g) available for the total relevant cohort. likewise, the length of stay was also similar for our sample ( . ± . days) and the total cohort ( . ± . days), and the proportion of children requiring ventilation in our sample was not statistically different from that observed in the remaining portion of the total cohort ( . % vs. . %; χ [ ] = . , p = . ). according to parental report, none of the children had sustained a severe head injury with loss of consciousness. postnatal seizure history was reported for cases, yet only three required anticonvulsant medications. whereas the intellectual abilities of two of these three children fell well within the average range, the remaining case manifested severe intellectual deficits. as table shows, ninety participants were singletons and seventy were products of multiple pregnancy. sixty-four multiples were co-members of twinships or triplets and therefore shared antenatal risk. descriptive statistics regarding pregnancy and perinatal risk in our sample were based on data obtained retrospectively from hospital records ( table ). additional information regarding intervention procedures is provided in online resource . gestational age in our sample ranged from / - / weeks and was determined by maternal dates and confirmed by early prenatal ultrasound in > % of cases. three children with cp diagnosis (spastic diplegia) were included in the sample. additionally, there were three cases with perinatal diagnoses of grade iii intraventricular hemorrhage, one with grade iv, one with periventricular leukomalacia (pvl), one with both pvl and subsequent cerebral palsy (spastic diplegia), and one with grade iv hemorrhage coupled with hydrocephalus (requiring reservoir placement). adding the aforementioned single seizure case with severe intellectual impairment, the "significant brain injury" subgroup included cases. importantly, statistical analyses were performed both with and without these cases. general assessment considerations evaluations were conducted between and by clinical psychology graduate students who had been extensively trained in developmental neuropsychological assessment. they were kept uninformed about participants' medical background data, with the single exception of being aware that the children were nicu graduates. all testing and other data collection procedures were conducted in compliance with ethical standards of the helsinki declaration and its later amendments, as well kramer et al. ( ) i summary score for the nine above listed antenatal complications with sample frequency > % j includes any atypical presentations (breech, transverse lie, footling, etc.) k arterial cord ph examined for participants, arterial ph below . was recorded (n = ). when only venous cord ph was available (n = ), venous ph below . was recorded (n = ). for four cases, initial capillary blood below . was recorded (n = ), whereas acid-base information was unavailable for five cases l as determined by obstetrician; > % of cases were corroborated by antenatal ultrasound. distribution: cases ≤ / weeks ( . %) + cases ≤ / weeks ( . %) + cases < / weeks ( . %), + cases ≤ / weeks ( . %) m initial hematocrit < % n established by positive blood culture o chronic lung disease: supplemental oxygen required at weeks gestation or discharge for infants < weeks gestation. no cases were observed in this sample with gestational age ≥ weeks p based on a chest roentgenogram and clinical evaluation q peak bilirubin ≥ mg/dl r diagnosed at least once during nicu stay s documented on the basis of cranial ultrasound. (mild = bleed grade & ; severe = grade & using grading criteria by volpe, ) . routine cranial ultrasound given to all infants with gestational age ≤ weeks, and when clinically indicated to infants with gestational age > weeks. there were twenty cases ( . %) with mild brain bleeds (sixteen grade i and four grade ii) and seven ( . %) with severe intracranial pathology, including three grade iii intraventricular hemorrhage, two grade iv (one requiring shunt), and two cases with periventricular leukomalacia t infants discharged on the ventilator were not included in the current study u diagnosed by clinical manifestations and echocardiographic information v summary score for the nine above listed perinatal complications with sample frequency > % w the rate of severe retinopathy of prematurity (> stage ) in the total sample was . %, below our inclusion cutoff (one cases with stage iii, four cases with stage iii+, and two cases with stage iv) and there were no cases with grade iv rop after exclusion of eleven cases with significant neurological background. all cases with stage iii+ and stage iv had received laser treatment (wechsler ) . prereading skills language skills were assessed using the core language (cl) score from the clinical evaluation of language fundamentals-preschool (celf-p ; wiig et al. ). the cl stability coefficient for ages : - : is high (r = . ), whereas internal consistency and split-half reliability are excellent (r xx = . and . , respectively). the cl scale is comprised of three subtests: sentence structure, word structure, and expressive vocabulary. prenumeracy skills quantitative knowledge and reasoning were measured using the applied problems (ap) subtest from the woodcock johnson (wj-iii) tests of achievement (woodcock et al. ) . at the prekindergarten level, ap assesses emergent counting, addition, and subtraction skills. the split-half reliability for ages and is excellent (r = . and . , respectively), and test-retest correlations range from r = . -. for ages - (woodcock et al. ) . quantitative concepts (qc), another wj-iii task that may be administered at three years of age does not possess an adequate floor (alfonso and flanagan ) , and was therefore not included. preprinting skills eye-hand coordination skills were assessed using the visual-motor integration (vmi) subtest from the peabody developmental motor scales (pdms- ; folio and fewell ) . the vmi includes items that require reaching and grasping, building with blocks, or copying designs. internal consistency reliability for - months of age is excellent (r = . ), whereas the overall test-retest (r = . ) and inter-scorer (r = . ) reliability coefficients are also exceptionally high (folio and fewell ) . descriptive information for performance measures the mean fsiq, cl, and vmi scores (±sd) of our participants fell well within the average range on three of the four readiness measures ( . ± . , . ± . , and . ± . , respectively), whereas mean ap score fell above average ( . ± . ). these favorable results are likely attributable to the preponderantly middle-class background of our sample. notably, participants' scores spanned a broad range (with similar ranges and sd's for ap and cl) and it was our goal to explore the contribution of antenatal risk to explaining this variability. all standard scores were corrected for prematurity. the recalculation of the preterm preschooler's age-at-testing as time elapsed since the expected date of delivery allows one to derive standard scores based on age-reference norms of typical children who are similar in biological maturity. additional descriptive data for the total-, and subsample without significant brain injury may be found in online resource . cumulative antenatal risk consistent with the fetal programming hypothesis, our chief variable of interest was cumulative antenatal risk, operationalized as an index comprised of equally weighted complications. we included nine major complications with sample frequency > % (see table ) and with well documented relationships to unfavorable neonatal outcome or neurobehavioral sequelae. hence, for each participant we summed the identified complications, out of the nine relatively common antenatal risks, with the following sample distribution: ( %), ( . %), ( . %), ( . %) and ( . %). no cases were observed with frequency ≥ complications. the correlations between pairs of antenatal complications were moderate at best, suggesting that the information provided by discrete complications was not redundant. the strongest relationship was observed between diagnosis of histological chorioamnionitis and pprom latency duration (time between membrane rupture and birth) > h (r = . ; p < . ). cumulative perinatal risk a cumulative perinatal risk score was also computed and was included as a covariate. nine major perinatal complications with sample frequency > % provided the basis for the perinatal summary score (see table ). abnormal presentation was not included because this complication is thought to exert little influence on longterm outcome (eide et al. ) and since the potentially unfavorable outcome effects are believed to be the result of confounding with the effects of prematurity or gestational age (ismail et al. ). the latter variable was taken into account as a separate risk factor in this study. background medical information was obtained retrospectively from the mother's labor and delivery hospitalization as well as the nicu records. intra-class correlations (icc- ; shrout and fleiss ) for the antenatal and the perinatal complications composite scores were excellent, owing in part to the ease and efficiency of searching electronic records. based on two independent and trained medical records reviewers, icc equaled or approached unity for ten cases (icc [ ] = . , and . , for antenatal and perinatal composites, respectively). construct validity of the two summary scores was established in the singletons subsample (n = ) by examination of the associations between the cumulative antenatal risk score and birth weight, an index of fetal growth and well-being, and between the cumulative perinatal risk score and both length of nicu stay and days on the ventilator, two indexes of need for medical intervention. in addition to the singletons and seven single-members of twin pairs, our sample included sets of co-twins and three sets of co-triplets. to capture the correlation between participants within sets of multiples, we used spss . mixed (maximum likelihood) to fit separate linear mixed effects models for each outcome variable, with multiplicity (cases from the same birth mother) as a random effect. thus, the individual multiples nested within each set were conceptualized as replications and were given a random block number that was unique, but identical for set members. in contrast, singleton children or multiples without an evaluated co-multiple had no replicates and were considered random block with size . this model enabled both co-multiples and singletons to be used in the same analysis without either violating independence assumptions or, alternatively, discarding important information by including only a single member of each set. the variable of interest, antenatal risk (the sum of antenatal complications), was entered together with sociodemographic covariates, sex and ses (hollingshead ), into the model. the hollingshead index is a composite score reflecting family social status. the score is based on four factors: parental marital status, employment status, educational attainment and occupational prestige. two additional covariates were entered to capture the influence of perinatal risk: gestational age and the perinatal complications summary score (see table ). birth weight was highly correlated with gestational age and was therefore not included (r = . ; p < . ). hence, the effect of antenatal risk on readiness was statistically adjusted for influence of four covariates, with all five predictors entered simultaneously into the model. information about bivariate correlations among the five predictors may be found in online resource . one should note here that a fifth covariate, iq test edition, was used in analyses of cognitive outcome. the predicted variables were the cognitive (prorated fsiq) and pre-academic (celf-p cl score, wj-iii ap subtest score, and pdms- vmi score) measures of kindergarten readiness corrected for degree of prematurity. information about the correlations between the five predictors and four outcome measures is presented in online resource . three ( . %) of the participants were unable to complete any of the tasks included in this evaluation (two cases with cp and a single case without significant neurological findings who required > . months on the respirator at birth). of the children without significant brain injury who completed at least one task, a minority of cases failed to complete the tasks needed to obtain a score on one or more of the four preacademic performance indices (n = [ . %], [ . %], [ . %], and [ . %] cases for fsiq, cl, ap, and vmi, respectively). due to the children's young age, it was difficult, if not impossible, to ascertain the causes of failure to attempt or follow instructions for specific subtests. examination of the correlates of the sum of incomplete subtests per participant r e v e a l e d s i g n i f i c a n t a s s o c i a t i o n s n e i t h e r w i t h sociodemographic variables (r[ ] = −. and − . for ses and sex; p = . and . , respectively) nor with preschool attendance (r[ ] = . ; p = . ). in contrast, a significant inverse relationship between the number of incomplete subtests and the fsiq was observed (r[ ] = −. , p < . ). to avoid bias resulting from potential influence of the missing subtest scores, we applied multiple imputation to replace these performance data. the results of data analyses are reported both before and after exclusion of cases with significant brain injury. prior to data analyses, interactions between sex and the remaining predictors were examined for all outcome measures. as none of the interactions were significant (all p values > . ), the reduced models were used. follow-up analyses were conducted to explore whether any observed associations between antenatal risk and kindergarten readiness were attributable to a particular class of complications. hence we grouped the nine antenatal complications comprising the summary score (table ) into four categories. these four groupings were based on shared etiological pathways, or shared antepartum symptoms coupled with at least some shared antecedents. specifically, complications associated with either ( ). intra-amniotic infection and inflammation (histologic chorioamnionitis and membranes ruptured > h; e.g., stock et al. ); ( ). placental insufficiency (hypertension in pregnancy, hellp syndrome, and iugr; e.g., stepan et al. ) ; ( ). maternal endocrine dysfunction (hypothyroid and diabetes; e.g., haddow et al. ) or ( ). uteroplacental bleeding (abruption and previa; e.g., berhan ; getahun et al. ) . category scores were then assigned to participants based on the sum of complications accumulated within each class of antenatal risk. the data were then reanalyzed using the same sociodemographic and medical variables as covariates; however, we substituted the four sums of risk scores for the single composite sum of nine antenatal complications. table shows analyses of performance for the total sample and for the subsample without significant brain injury cases. as shown, the antenatal complications score was inversely related to the fsiq, both before (p = . ) and after (p = . ) exclusion of brain injury cases. analyses of preacademic performance revealed similar associations between antenatal risk and both cl and ap scores, before (p = . and p = . , respectively) and after (p = . and p = . , respectively) excluding brain injury cases. no significant relationships were observed between cumulative antenatal risk and the vmi, yet the vmi was linked to gestational age (p = . and . ). added variable plots, depicting relationships between residualized antenatal risk scores and outcome of the remaining cases, two failed to obtain a score on the fsiq (one with brain injury), nine on cl ( with brain injury), seventeen on ap (three with brain injury), six on the vmi. f of the "non-neurological" cases completing tasks required for a score on at least one of the four outcome measures, one (. %) could not obtain a score on the fsiq, six ( . %) on cl, fourteen ( . %) on ap, six ( . %) on the vmi. g computation of Δr based on snijders and bosker ( , pp. - ) . h antenatal complications score is a composite of presence (= ) vs. absence (= ) of nine complications: placental abruption, placenta previa, chorioamnionitis, diabetes, hypertension in pregnancy, hellp syndrome (hemolysis, elevated liver enzymes, low platelet count), hypothyroidism, preterm premature rupture of the membranes (pprom) > h, and intrauterine growth restriction. i due to the paucity of cases with four antenatal complications, analyses were repeated with three and four complications combined into a single category ( table shows the relative contribution of each of the four categories of antenatal risk to the four performance measures; the proportion of variance explained (Δr ) is provided for statistical effects with p < . . as the table reveals, intraamniotic infection risk was significantly related to the fsiq (p = . ), cl (p = . ), and ap (p = . ), whereas conditions associated with placental insufficiency were significantly related to cl (p = . ), with trends for associations with the fsiq and ap (p = . and . , respectively). maternal endocrine dysfunction was associated with cl (p = . ), and disorders associated with uteroplacental bleeding were significantly related to the fsiq (p = . ). following statistical adjustment for sociodemographic and perinatal confounds, the sum of nine, relatively common, antenatal complications remained a significant source of variability in preterm-born preschoolers' cognitive and academic performance. exploration of the relative outcome contribution of four classes of antenatal risk revealed that complications associated with intra-amniotic infection, placental insufficiency and uteroplacental bleeding accounted for . %, . % and . % of iq variance, respectively, altogether . % of variability in kindergarten cognitive readiness. similarly, complications associated with maternal endocrine dysfunction, intraamniotic infection, and placental insufficiency accounted for . %, . % and . %, of the variance in language skills, respectively, altogether . % of variability in literacy readiness. complications associated with intra-amniotic infection risk and placental insufficiency contributed . % and . % of the variance in early number concepts, respectively, a combined share of . % to variability in numeracy readiness. hence, when considered separately, each of the four risk categories accounts for a small slice of outcome variance in one or more preacademic domains (except visual-motor integration). yet in aggregate they accounted for . - . % of the variance in kindergarten readiness of preschoolers free of major handicaps, consistent with effect sizes of moderate magnitude (cohen ) . interestingly, amongst the four categories of antenatal risk examined here, intra-amniotic infection was the most consistent contributor to kindergarten readiness. these findings are compatible with recent evidence that inflammation, including chorioamnionitis, contributes to preterm cns injury and is also an independent risk factor for brain injury in the term infant (yellowhair et al. ) . these results are also consistent with reports of marked increase in the probability of unexplained cerebral palsy in the presence of antenatal inflammation-infection (horvath et al. ) . in this context, one should note that a sizeable proportion of the mothers in our sample received antibiotics prophylaxis for prevention of early onset neonatal infection (see online resource ). in a recent integrative review of the literature, braye et al. ( ) highlighted the observed decrease in incidence of early onset infection since the introduction of intrapartum antibiotic prophylaxis, as well as findings of randomized controlled studies documenting effectiveness. at the same time, however, braye and colleagues emphasized that the longer-term health implications of prophylaxis for early onset infection are unknown. it is difficult, therefore, if not impossible, to draw conclusions regarding the potential implications of antibiotic prophylaxis on kindergarten readiness until such data become available. nonetheless, our findings revealed that each of the four classes of antenatal risk studied here contributed significantly to explaining performance variance on one or more kindergarten readiness domains. the putative influence of each antenatal risk category on brain maturation trajectory and cognitivebehavioral development may be conceptualized within the broad framework of fetal programming of disease (buss et al. ; zeltser and leibel ; myatt ; andersen et al. ; miller et al. ; godfrey ) . however, the biological mechanisms mediating the relationship between cumulative antenatal risk and kindergarten readiness require specification. antenatal stressors lead to placental adaptive responses to the variations in the maternal-fetal environment (myatt ) . these responses, in turn, are followed by fetal adaptations expressed via vascular, metabolic or endocrine changes that permanently modify bodily structure or function (hocher et al. ). the precise nature and sequence of the biological changes mediating deficits in cognitive-behavioral functioning have yet to be elucidated. to accomplish this goal, future investigations should incorporate measures of potential sequential mediators, including indexes of placental size or function, intrauterine cerebral development, and postnatal brain structure or function. our findings are consistent with zeltser and leibel's ( ) observations that dissimilar intrauterine stress factors may nonetheless lead to similar fetal outcomes because they activate related mechanisms of placental adaptation (fowden et al. ) which, in turn, shape the trajectory of fetal brain development (buss et al. ) . the thesis that diverse insults converge on similar unfavorable outcomes (a relationship mediated by fetal brain programming) is compatible with the notion that the amalgamation of antenatal complications into a single index or several classes of risk may offer an important tool for the study of individual differences among pretermborn children in the severity of neurocognitive deficits. antenatal complications contributed less than ses to explaining outcome variance, although effect magnitude was typically moderate for both variables. girls outperformed boys on all measures, consistent with other reports of female outcome advantage following preterm birth (lauterbach et al. ). similar to foster- cohen et al. ( ) , we did not find significant associations between the sum of perinatal complications and kindergarten readiness in this young age group. however, gestational age, often considered a proxy for perinatal risk, was found to be linked to development of visualmotor integration skills even after we excluded cases with evidence of significant brain injury from the analyses. additionally, there was a weak trend (p < . ; table ) for a relationship between gestational age and global intellectual skills. the absence of the oft-reported significant relationships between the degree of prematurity and the remaining outcome measures (e.g., heuvelman et al. ; a recent epidemiological investigation) was somewhat unexpected. it is possible that within a restricted gestational age range, where values ≥ weeks were truncated, the relationships between gestational age and preschool performance are more difficult to demonstrate whereas the adverse influence of other risk scales- . f perinatal complications score is a composite score reflecting presence (= ) vs. absence (= ) of nine complications: anemia, bronchopulmonary dysplasia, bacterial infection, hyaline membrane disease, hyperbilirubinemia, hypoglycemia, intracranial pathology, patent ductus arteriosus, and supplemental oxygen requirement following discharge. g intra-amniotic infection risk score is a composite of two complications believed to share etiological pathways: preterm premature rupture of membranes (pprom) > h and histological chorioamnionitis. h placental insufficiency score is a composite of three complications thought to share etiological pathways: maternal hypertension, hellp syndrome (hemolysis, elevated liver enzymes, low platelet count), and intrauterine growth restriction. i maternal endocrine dysfunction score is a composite of two complications found to share some etiological pathways: maternal diabetes and hypothyroidism. j uteroplacental bleeding risk score is a composite of two complications sharing antenatal symptomatology: placental abruption and placenta previa factors (e.g., antenatal complications) becomes more apparent. interestingly, the absence of gestational age effects on several performance measures employed here seems consistent with findings from a recent meta-analysis of the long-term cognitive and academic performance of children born with various degrees of prematurity (allotey et al. ). the quantitative integration revealed a miniscule, if any, effect of lower gestational age. children born between and weeks performed almost as poorly as those born < weeks. as noted earlier, our findings of an association between cumulative antenatal risk and kindergarten readiness are consistent with the fetal programming hypothesis. researchers of fetal programming have typically studied the role of the prenatal environment without taking into consideration the perinatal, or postnatal environment (grant et al. ). in the current study, however, we statistically adjusted cumulative antenatal risk for perinatal complications and for socioeconomic status in effort to account for confounding influences occurring after birth. limitations of this study include the retrospective nature of the participant identification and recruitment component. medical data were also collected retrospectively, yet interrater reliability for information obtained from medical records was excellent. the difficulty of a small number of participants to complete various tasks is not surprising, given the combination of young age and increased biomedical risk in our sample. the percentage of missing outcome data for the children without significant neurological injury completing ≥ one task ranged from negligible ( . %) for cognitive readiness (fsiq) to moderate ( . %) for numerical readiness (ap). the amount of missing data for three of the four outcome measures was nonetheless small (< . %). although our sample included children with a wide range of socioeconomic circumstances, the mean ses and educational characteristics of the sample revealed a greater representation of middle-class strata, thereby reducing the potentially confounding effects of socioeconomic adversity on kindergarten readiness. nonetheless, because to some extent generalizability to lower strata was traded-off for improved internal validity, our findings may have underestimated statistical effects in samples with greater representation of the lower end of the socioeconomic scale. the cross-sectional nature of this investigation precluded examination of the generalizability of the findings to elementary school readiness and beyond. in the current investigation antenatal risk accounted for up to . % of the variance in kindergarten readiness. antenatal risk estimation was based on a simple frequency count of common complications. a more sensitive measure of risk may be developed to take the severity of discrete complications within each of the four antenatal risk categories examined here into account. increased sensitivity, in turn, may serve to enhance the magnitude of the statistical effects observed here between antenatal risk and emergent academic skills. as noted above, we statistically adjusted for degree of gestational maturity and for the presence of nine common perinatal risk factors (table ) in order to estimate the unique portion of outcome variance that is attributable to cumulative antenatal risk. we further examined the data with and without 'neurological' cases, based on both ultrasound evidence obtained in the nicu and subsequent evidence of cerebral palsy. nonetheless, one may argue that the full effect of confounding perinatal risk factors such as chronic lung disease or germinal matrix hemorrhage may become evident in the longer term only, when academic performance demands are increased. a longitudinal study of educational attainment is best suited to address this issue. the significance of early academic skills as the foundation of scholastic achievement has been established in multiple follow-up investigations of typical kindergartners to the early school years (e.g., pagani et al. ; romano et al. ; grissmer et al. ) . moreover, developmental continuity has been demonstrated between preschool quantitative and oral-language skills and elementary school attainment in math and reading, in a variety of student populations (e.g., davison et al. ; manfra et al. ; manfra et al. ; nguyen et al. ) . the existence of robust developmental continuity between pre-academic and academic skill levels supports the notion that the same factors that explain variability in emergent academic skills also account for variability in mastery of both the reading process and basic arithmetic in the early elementary-school years. based on our findings, it is therefore likely that differences in scholastic achievement among graduates of the nicu are partly explained by cumulative antenatal risk. a follow-up study of preterm-born preschoolers, extending to early 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placental pathology prenatal, perinatal, and neonatal risk factors for specific language impairment: a prospective pregnancy cohort study clinical evaluation of language fundamentals: preschool- woodcock-johnson tests of achievement preclinical chorioamnionitis dysregulates cxcl /cxcr signaling throughout the placentalfetal-brain axis roles of the placenta in fetal brain development acknowledgements the authors thank beth kring and tammy swails for their help in data collection. evaluations and testing materials were funded in part by the merrill-palmer skillman institute. none of the authors has a known conflict of interest concerning this manuscript.funding this work was supported in part by funding from the merrill palmer skillman institute, wayne state university, east ferry, detroit, mi . key: cord- -pz q i authors: zhang, jie title: chai jing: the power of vulnerability date: - - journal: female celebrities in contemporary chinese society doi: . / - - - - _ sha: doc_id: cord_uid: pz q i in the past seventeen years chai jing has risen from china’s official media to become a recognized investigative journalist, public intellectual, author, and more recently, an independent filmmaker and environmental activist. her experience and work reflect how china’s news apparatus has reformed to adapt to the drastic societal changes with emotion being used to open up new ways of news communication. her documentary under the dome further shows how the internet has transformed the ecology of media and provided innovative platforms for social engagement. chai’s embracing her own feelings of vulnerability, which dominated the beginning of her career, and using it to channel public feelings and drive news reporting has made her a distinctively controversial media personality. her leaving the cctv can be viewed as a self-marginalization that helps her sustain that vulnerability, through which she gains resilience and critical power. the use of maternal voice in under the dome exemplifies her use of the power of vulnerability in its most mature form. the controversiality about that voice signals that post-socialist china remains a space where environmental and gender discourses are contested and negotiated. . courtesy of guangxi normal university press some of china's best-known media people, further made chai known as an influential writer. in , chai released under the dome (qiongding zhixia 穹嵿之下), an independently produced, -minute documentary that brought environmental issues to the frontline of china's public debate again. chai declared the film "a personal war against the smog" and its release energized china's burgeoning environmental movement. the film received million online clicks from february to march , after which the government banned it. chai's engagement with the nuanced transition of china's official media, the rise of investigative journalism, and the development of environmental discourses have made it essential to examine her media and literary work within these correlated contexts. this chapter continues existing inquiries about chai's roles in these contexts, with a special focus on how gender plays an indispensable part in forging her news reporting and commentating style on television, her self-positioning in insight, and her "ecofeminist" voice in under the dome. the unapologetically maternal voice that has caused much controversality in the reception of the film is not a radical shift from her established career; instead, femininity is a constant in chai's self-awareness and it has taken different forms in different stages of her career, sometimes making possible her agency and breakthroughs, while at other times making her subject to reckless critiques that mark the limitations of china's gender equality. when chai started working at the news probe department at cctv in , she was a twenty-four-year-old "literary and artistic youth" (wenyi qingnian 文藝青年) whose inspiration predominantly came from "romance fiction and popular songs," as she would reflect more than a decade later. "literary and artistic youth" in the chinese context refers to people in their twenties to forties who love literature, travel, photography, and arts and who speak sentimentally and tend to lose touch with reality. chai admitted self-mockingly that these qualities made her sail through her earlier job at a hunan radio station, where she read listeners' letters on the air for an evening program called "gentle night" (yese wenrou 夜色溫柔). throughout chai's career, she has never hesitated to self-criticize this unpractical aspect of her identity, underlining how it clashes with her journalistic mission of exposing some of the most hidden and complex realities in china. however, noticeably chai has never lost her sentimentality, neither have her audiences and readers failed to recognize it. when chai's insight came out in , some of her disapproving readers denominated her chai huiyin 柴徽因, alluding to lin huiyin 林徽因 ( - , one of china's celebrity intellectuals known for her literary talent and romantic relations with several high-achieving men. the denomination poignantly recognizes chai's literary talent while also reprehends her self-promotion in the book's much publicized press release. some other audiences and readers have also faulted chai for her connections with powerful men. "why are your friends all old men?" asked one of chai "friendlier" audiences in the book release conference. hinting unfairly that chai's success had to do with gender-based favoritism rather than her capability, such questionings nevertheless drive home the fact that china's news corresponding had been male-dominated when chai started her career at cctv. chen meng 陳虻 ( - ), chai's recruiter to cctv, was a widely respected reformer in china's news apparatus, radically envisioning and implementing many groundbreaking changes in the cctv news since the early s. similarly, chai's partner bai yansong 白岩松 (b. ) was then a newly established media personality appreciated for his candid, sharp, and thoughtful approach to news reporting. bai created, produced, and anchored "horizon connection," through which chai had her cctv debut. chai described in the first chapter of insight the many mistakes she made at this onset of her cctv career, highlighting, not without literary dramatization, how her colleagues' challenges and mentoring made possible her professional growth. significantly, all of these nineteen colleague-mentors were men. although chai also mentioned two additional women colleagues in passing, they did not contribute to her professional development. a woman called sister lei 雷姐 attempted to be a matchmaker, arranging chai with another colleague. chai also heard that some cctv executive remarked that she looked like jing yidan 敬一丹 (b. ), an award-winning hostess of economy-related programs. chai's colleagues interpreted this remark as the executive's approval of chai's hire. the deep irony here is that to some of her seniors and her colleagues alike chai's look seemed to matter more than her quality. chai's lack of female role models was further complicated by the unprecedented changes in the cctv news itself. since the early s, a new documentary movement had begun shaping in china. filmmakers and television professionals took advantage of the spread of video technology and hoped to "open up new public spaces for discussion of social problems and dilemmas in the post-socialist era" (berry et al. ) . consciously distancing themselves from the official propagandistic news and culture production mode, filmmakers and media professionals both inside and outside the state-owned film studios and television stations started experimenting with "a more spontaneous mode of documentary" (berry et al. ) that focused on the experiences of people marginalized by china's modernization. they used hand-held cameras to record unrehearsed and unscripted pieces of realities-"my camera doesn't lie" was one of their slogans -and later went as far as completely democratizing video making itself and letting the voiceless record themselves. in news reporting, oriental horizon (dongfang shikong 東方時空, est. ) combined investigative journalism and news-documentary realism and quickly became a hit. chai's seniors chen and bai were both deeply involved in the formation of oriental horizon. bai anchored the news magazine and became its spokesperson. chen famously verbalized the catchphrase-"telling the common folks' own stories" (jiangshu laobaixing ziji de gushi 講述老百姓自己的故事)-for life space (shenghuo kongjian 生活空間), the hallmark segment of oriental horizon. the path-breaking nature of the news programs that chai joined meant that she had no models to follow and must come to an understanding of what news meant to her and her audiences through both soul searching and hand-on experiences. all these uncertainties made chai feel unprepared and vulnerable. apparently, she felt like an impersonator, a syndrome many professional women share in an unsupportive working environment. as she wrote in insight, she kept dreaming about days of her cheating in a vision test in fourth grade by memorizing the last line on the test poster. she also tried to hide herself in the crowd when the station anchors gathered for a group photo. when simultaneously interviewing three guests on the phone from the newsroom-with images of the guests to be edited in later due to technological restrictions-chai felt disconnected and instead became highly conscious of her own gestures. she was too anxious to breathe and had to deep breathe in the restroom. she also found herself resorting to clichéd phrases such as "let's hope for the prompt arrival of a democratic and law-governing society" to safely conclude some of her news corresponding so that she could run away from it. naturally she found strength through withdrawing to literature, where she felt most at home. clarice starling, the twenty-four-year-old f.b.i. trainee in thomas harris's novel the silence of the lambs, became her alter ego. in the novel, starling starts with the need to prove herself in the male-dominated bureau but eventually transforms into a confident agent resolved to serve nobody but the silent women victims of cannibalism. it was the single thought that "nothing can make starling afraid" which kept chai sitting at her newsroom in spite of the perceived failures at the beginning of her cctv career, as she confessed with hindsight in insight. rising from disaster reporting when chai was interviewed for her cctv position, she was asked, "what concerns you the most when you do news?" she answered, "the human in the news." it was this constant concern for the human when suddenly confronting disastrously inhuman situations in , which helped chai breakthrough from her earlier frustration and develop her own news reporting and commentating style. within the two months from february to april , chai found herself doing on-the-spot reporting from two of china's most distinctive locations, the remote uygur area devastated by a . magnitude earthquake that caused more than two hundred deaths, and the political and cultural capital beijing plagued by an outbreak of severe acute respiratory syndrome (sars) epidemic, with more than nine hundred confirmed cases and more than two thousand probable cases by mid and late april. chai would later call these experiences a turning point of her career. "all started with uncertainty. you don't have time to deliberate on what questions you want to ask. only from uncertainty can one forget all about oneself. it was in this situation that i found what i really wanted" (li) . as disasters are unpredictable and destructive, disaster reporting involves a larger extent of on-the-spot improvisation and spontaneity. this gave chai much-needed agency to explore her media persona. in what follows, i analyze chai's sars coverage from four angles, focusing on how her disaster reporting credited her as a journalist. disaster reporting is innately one of the most dramatic genres of the news discourse. disasters are unnegotiable and the stakes are high, with humans stuck in liminality and caught in intensive emotion. this sets up a great background for great storytelling, the core of great news reporting. as a cctv reporter, chai had privileged access to this stage. she was on her way to uygur half an hour after the earthquake and was able to get to its center by taking a military transporter (chai ) . her prompt first-hand experience filled in the need for the latest information to keep her audiences updated and to facilitate coordinated disaster-relief. because the transmission of this information itself can mean the difference between life and death for victims, as media visibility may expedite relief action, this type of reports generally tends to be treated as more valuable. moreover, in an age when social media did not yet exist, television news were the predominant means by which people were informed about the disasters. all these factors made it possible for chai's coverage to get the immediate attention of the largest possible number of audiences. chai's sars report was well-received also because it was perceived to be a significant step toward promoting governmental transparency and civilians' "right of knowing" (zhiqingquan 知情權). precisely because disasters have great potential of causing social instability, the chinese government tended to execute strict control of disaster news release and sometimes opted for a cover-up. just months before the sars outbreak, western media warned how china's "catastrophic mismanagement of its aids crisis" (kristof) , which was largely caused by illegal blood collecting in henan province, will result in unprecedented deaths if the official cover-up continued. the sars outbreak seemed to have been treated in a similar fashion in the beginning, with the chinese media instructed to propagandize that the situation was under control and there was no need to be "panic" (konghuang 恐慌). but an unexpected turn-around took place on april , when china's health minister zhang wenkang 張文康 (b. ) and beijing mayor meng xuenong 孟學農 (b. ) were fired for "mishandling the matter." the firing may have been necessitated by the governmental need to fully publicize the seriousness of the epidemic in order to discourage mass travels anticipated for each may. it created the impression that government officials were held accountable and the people's "right of knowing" was reinstated. chai's reporting on sars, which began three days before the firing and aired several days thereafter, coincidentally provided a timely outlet for people to exercise this newly vindicated "right of knowing." more than million people watched the -minute special report in which chai, covered by white insulating clothing and gauze masks, entered the quarantined sections of beijing hospitals where sars patients were treated and, for the first time since the outbreak, interviewed. chai became the face of sars reporting not only because of her strong work ethics-"if i were to have a child in the future and he asked me, 'mom, what were you doing when sars broke out?' i cannot just say, 'i was watching tv.' i really cannot just say that!" chai reflected later (chai )-but also because that the ways that she conducted the interviews pioneered a profound transformation of chinese reporters from a detached information transmitter into an empathetic individual capable of co-experiencing what the interviewees were experiencing. amid the life-threatening public health crisis, everyone was equally vulnerable; some of the first causalities of the epidemic were health professionals. chai's awareness of her own vulnerability and her refusal to succumb to it made her not only a credible reporter but also a relatable and likable individual. the high risk that she took highlighted the worthiness of the "right of knowing," and the idea of a slender and young woman confronting deadly viruses for the sake of public understanding was immediately heart-warming and guilt-provoking. in fact, in the first interview when chai's team had to let her enter the quarantined area alone with a hidden audio recorder-the cameramen were banned because there was no way to insulate and sanitize their heavy equipment-the left-behind camera lingered on her back and the glass door that she entered through for an extended time. this camera's gaze is meta-narratorial in its recognition of its own limits and its concern for chai. that chai did not stop at where the camera had to stop helped construct her as a credible reporter seeking nothing but truth, truth that she deemed more important than her life. in this process, chai has also transformed from the insecure girl disconnected with the news that she was commentating into a dedicated reporter fully immersed in the news in its making. at the same time, her own sense of insecurity has also been subliminally transcended. the epidemic reveals that being vulnerable is an integral and universal part of human experience and embracing it rather than denying it is the only way to survive or to thrive. through the sars reporting, chai developed her own distinctive style of news probing. in the succeeding hospital visits, she treated her interviewees as fellow human beings and encouraged them to share their feelings. when talking to a patient, she said, "understandably there are currently a lot of fear about the disease. can you tell us from the perspective of a patient what it feels like to have this disease?" by discussing fear with the patient, the target of the fear, chai was including rather than excluding the patient in the construction of sars discourses and also insisting that the humanity of the patient rather than the virality of the patient should be emphasized. the patient's subsequent description, which highlighted that this disease was not so different from other more common diseases in its identifiable pattern of development, serves to demystify the virus. in fact, this patient was so comfortable with sharing her feelings with chai that she expressed that she felt for the nurses and doctors who took care of her. she said, "they wore thirty layers of gauzed masks but they still got infected. they take care of us but then they cannot go home. this is really hard on them!" that a patient infected with a deadly virus can feel empathy for her caregivers only shows how humans are deeply connected when facing their vulnerability. throughout the report, chai effortlessly created contexts for her interviewees to talk about their feelings. "can you go home after work?" she asked a nurse this seemingly obvious question. as the interview went on, the audience learned what going home actually meant for the nurse: she and her fiancé had to postpone their wedding ceremony and had not seen each other for more than a month. this episode reminded people how life had been interrupted by the epidemic but it also emphasized how the epidemic had made people care even more for others. in another occasion, chai asked a doctor, "you knew the risk of working here, but why did you volunteer to stay?" visibly getting emotional in spite of the cover of insulating clothes and masks, the doctor recognized her own vulnerability but insisted that the virus only makes humans more connected. "i could be one of them," she said plainly. this message about shared burden is all the more powerful because it comes from a quarantined hospital room, a space designated to separate people. at the end of the report, chai asked a patient who almost died of the infection but was on her way to recovery, "have you ever thought what you want to do the most when you are discharged?" the patient seemed shocked to face this question and stuttered, "ai-ya…i've thought too much [about this]…life is really, really…i have a new understanding of life now." chai's ability to arouse feelings made her sars report a heart-warming piece about humanity, community, and connectivity. she also exemplified how a reporter's personality can memorably shape the ways that stories unfold. chai's success in reporting the sars epidemic helped consolidate a change in china's news production, which shifted from a scriptwriterand director-centered model to a reporter-centered model. such a shift in the early s reflected the nuancedly transformed relationship between the state and the media apparatus. while the chinese media remained an integral part of state propaganda, since the s the state had cautiously allowed some reform so that the media can be less financially dependent upon the government. this meant that the media, while still subject to top-down censorship, must also take its mass appeal more seriously than ever. in other words, while political correctness remained a central concern, creative storytelling and audience rating (shoushilű 收視率) also became essential. at the same time, the new documentary movement had provided an effective narrative model to engage audiences, through focusing on the underprivileged and using hand-held camera, long shots and long takes, and voiceover, etc. both of these changes made possible a democratic shift in their emphasis on the agency of the conventionally neglected majority. both also pushed for a similar democratic shift in news reporting, from script-based, newsroomsituated monologues to reporter-anchored, on-site heteroglossia that oftentimes resist mainstream outlooks. this shift necessarily gives reporters new importance as the very process of news discovery rather than the official interpretation of the events has become the focal point of news stories. in september , cctv's "news probe" pioneered the reportercentered model, giving reporters unprecedented power to veto a proposed topic and to direct the actual on-site shooting of the news. "the more central a reporter becomes, the more appealing the news becomes," "news probe" director zhang jie 張洁 stated, admitting that chai's success had given him the confidence to implement this overdue reform (li) . the reform gave chai and her peers much agency in exploring their own media person and eventually made it possible for them to become public intellectuals who relentlessly unpack the complexity of post-socialist china and engage audiences in public debates. on november , china's fourth journalists' day, eight journalists including chai were selected to be that year's "journalists of the time" (zhongguo jizhe fengyun renwu 中國記者風雲人物). collectively, their reports covered police violence, monopolized taxi market, controversial private rights, fatal side effects of traditional chinese medicine, a cover-up of a coalmining disaster, a scandal of a drug rehabilitation center, and the iraq war, showing how journalists have become increasingly recognized as a force to push for pluralistic discourses about social transparency and justice. chai's remarkable capability of co-experiencing with her intervieweesvillagers whose lands were taken, abused women who killed their husbands, left-behind children, voiceless gays, earthquake survivors, mentally disabled women forced into prostitution, et al.-have made her news reports pivotal to raise awareness about neglected and misunderstood social-political-cultural issues. through her interview of "crazy english" (fengkuang yingyu 瘋狂英語) founder li yang 李阳 (b. ) and his wife kim lee 李金 (m. - ), chai revealed not only the excruciating pain that the wife experienced as a result of her husband's domestic violence but also how his inability to love and connect (a result of his own problematic childhood) and his over-emphasis on his professional success (a way to escape the fear to connect with his own family through running into "a crowd of strangers," as his wife puts it) have made him insensitive to his family's needs and feelings . through her interview of gays, activists, and their families, chai demystified gay people and normalized their sexuality and their feelings, calling for a more accepting environment. in her report on teen suicides in a northeastern town, she traced how a girl's suicide tipped into a chain of five teen suicides, with each revealing how the confusion of individual boundaries, the anxiety of growing up, and the lack of meaningful contact with parents and teachers have intensified the stress of coming-of-age and made suicide a contagious outlet. these reports have earned chai the reputation of a rising public intellectual (gongzhi 公知) decisively contributing to newly emerging and rapidly shifting public discourses about the underrepresented in the chinese society. chai's approach has also caused much controversiality due to the centrality of emotionality in it. when interviewing the cousin of the girl who committed suicide, chai asked, "have you talked to her [since her death]?" the boy, having felt very close to the girl, answered firmly, "yes." "what did you say to her?" chai pursued. breaking into tears, the boy muttered, "… how are you?" in a moment of heart-felt empathy, chai raised one of her hands to wipe the tears on the boy's face. this moment was eventually aired and became extremely controversial. some of chai's colleagues, embracing the idea that "a female reporter should never be aware of her gender except when she uses the restroom," pointed out that this style of what they called "crying interview" (qisheng caifang 泣声采访) could make her unqualified to pursue "hardcore news" (ying xinwen 硬新闻) (chai ). in fact, one of her colleagues confronted her, "why did you wipe his tears?" "what would you have done?" chai countered. "nothing! that is what makes me a reporter!" the colleague hollered (chai ) . some audiences also challenged chai's ingenuity, wondering whether she cared more about her own image than the news itself and calling her style "performative news anchoring" (biaoyan xing zhuchi 表演性主持) (chai ). still, others questioned whether she was manipulative in triggering these vulnerable moments of her interviewees, making their emotion the selling points (maidian 卖点) of her own news storytelling. this uneasiness with emotion, which is perceived to be opposite to journalistic objectivity, as well as the questioning of chai's sincerity, which is an innate paradox of the new documentary movement itself (some questioned whether the filmmakers are using the stories of the marginalized people for their own identity politics), provides a lens into the media consumption habits of the chinese public in the first two decades of the twenty-first century. for decades prior to these, the emotions allowed on television news were mostly orchestrated, ranging from patriotic pride to nationalistic anger. it was rare for individual emotions to erupt on the screen; any occasion when such moments occurred could be interpreted as a subtler way of propaganda, due to audiences' consensual distrust in the media in general. in fact, since cctv's talk show artistic life (yishu rensheng 艺术人生) first began in , its host zhu jun 朱军 (b. ) has also been heavily criticized for "arousing and manipulating feelings" (shanqing 煽情) among his interviewees. paradoxically, chai's news reporting diversified the emotional dimensions of china's television news, but the more people felt the new possibility of connection the more they felt the need to be reassured about the authenticity of that connection, particularly given the prevalence of artificially mediated feelings on television before this. chai recognized that excessive sentimentality can harm the discovery of truth, but she also believed that emotional affect can be an advantage rather than a liability for truth finding. in , cctv news reformed to focus more on "revealing what is behind the scenes" (kaijue neimu 开掘内幕), leaning heavily toward investigative journalism. chai's reports began to involve more confrontational situations. she covered some of china's darkest sides: bribery in a village election, shady deals in real estates, fraud in international trade, money laundering, and antique trafficking. chai and her partners found themselves having to use hidden cameras, face threats and bribery, cultivate informants, and outsmart deceiving interviewees more frequently than ever. but her deep understanding of humanity quickly made her an example of investigative journalism. chai's interviews are characterized by their persistent demand of truth, and she particularly excelled in belying the hyperbolism and illogicality of political formula that many of her deeply flawed interviewees used for their self-defense. when investigating why an entrepreneur was sentenced to three years in prison just because he refused to obey the mayor, chai asked the judge why the essential piece of evidence was a photocopy. she dispelled the judge's excuse one after another and eventually cornered him to admit that his sentencing was purely based on the photocopy. when the judge, obviously exasperated, cried out "i still believe he is guilty," resorting to his personal belief and thus betraying his lack of legal proof, and threatened chai, "don't you become other people's tool!" chai calmly pursued his hypocrisy further, "in the court debate, the defendant's lawyer stated that the law should not become the government's tool. what do you think of this?" (chai - ). chai's ability to feel for the vulnerable goes hand in hand with her perseverance to question how the system works and whether it is fair. from to , chai covered the annual "two conferences" (lianghui 兩會), or the combined national people's congress (zhonghua renmin gongheguo quanguo renmin daibiao dahui 中華人民共和國全國人民 代表大會) and the chinese people's political consultative conference (zhongguo renmin zhengzhi xieshang huiyi 中國人民政治協商會議), and pushed for capturing the heated discussion of topics essential for people's livelihood, such as governmental control of business, environmental pollution caused by economic development, skyrocketing medical expenses, and the lack of senior care, etc. chai's coverage subverted the conventional "two conferences" reports that tended to feature highly scripted and heavily edited discussion among the representatives in order to deliver a unified voice. "what others experience, i must experience," she wrote after an earlier interview of abused women. this insistence on co-experiencing has made chai's demand for truth almost personal and her commitment to truth all the more earnest, urgent, and powerful. chai's understanding of how a reporter should handle emotions has evolved throughout her cctv career, reflecting how china's news discourses have incorporated emotions in the first two decades of the twentyfirst century. earlier on, the news advocated for the right, particularly of those who were marginalized, for feelings, feelings that challenge ways that things have always been done. these expressions of feelings served to dramatize the news storytelling, drive home the social issues being explored, and call for resolutions. at the same time, because feelings are universal, they also served as an equalizer-wrongdoers are also entitled to express their feelings-and as a result news coverages became more inquisitive and comprehensive. a prominent example of this use of emotion was chai's interview of a woman who stomped a cat to death and a man who videotaped this scene for an online platform. the woman used this abusive action to vent out her frustration as a single mother and the man recorded it to compensate on his financial woes. in her report, chai suspended her own judgments, refrained from anchoring the interviewees' feelings, and instead retreated to quietly observe as the complexity of humanity emerged. as chai matured as a reporter, she began to be more self-reflexive of the constructed nature of emotion. she reflected how the limited scope of her own experience can make her own feelings biased, uninspiring, and even dangerously propagandistic. she also pondered how feelings can be performative and manipulated to resist deeper inquiries of the truth. she reminded herself that she must not be overwhelmed or "hijacked" (xiepo 脅迫) by anything, including the feelings of the majority (minyi 民意) (chai ). as a result she attempted to be more attentive to unsmooth and paradoxical elements in stories that resist a holistic interpretation. she also became more willing to leave her stories open-ended, allowing her audiences to experience and reach their own conclusion. "a society that does not care about truth is hopeless and immoral" (chai ) , she wrote in , clarifying that a reporter's job is "not to express, but to serve" (要服務,不要表達) (chai ) . chai left the cctv in and returned to the public sphere in with her documentary under the dome, which uses a ted talk format to combine personal testimonials, graphs and data, animation, and interviews to investigate the causes of china's air pollution. because the film was taken down from online shortly after its release, many assumed an antagonistic relation between chai and the official censorship. but such an assumption is reductive and unhelpful in explaining chai's dynamic engagement with authorities. just as chai had used the cctv as a platform for her investigative journalism, she had also used the network and resources developed from her cctv career to gather insiders' information and win interview opportunities for this film. many also assumed that chai had left cctv to develop an independent voice-chai's fans often asked how she could have, for so long, "held fast to" (jianshou 堅守) her ideals while in cctv-but i would like to argue that it was the ongoing changes in cctv that made possible and sustained chai's rise. chai's book insight clearly shows how her career is about negotiating with media conventions and creatively testing the boundaries of governmental control. "many things exist only because there were people who believed in them," chai wrote in , showing how she perceived her work to be a force to push for the realization of ideals through navigating through the system, rather than a force to attack and invalidate the system itself . the production and circulation of under the dome demonstrates that chai has been consistent with her active negotiation with powers rather than suddenly shifting her selfpositioning to subvert those powers. chai's purpose was to develop a niche to discuss china's smog hazard in the hope to propel an effective solution. earlier on, she had covered environmental topics such as sandstorms, polluted rivers, and problems caused by trash burning and coking plants, during her cctv career. the beginning of under the dome particularly referenced her earlier coverage of how the coal mining and coking plants in her hometown shanxi province 山西 had become a public health hazard. one of the most memorable moments of this segment was when a six-year-old local girl confessed that she had seen neither a blue sky nor a piece of white cloud in her whole life. inspired by al gore's inconvenient truth, chai aimed to unpack the information that she discovered and made them accessible and memorable. throughout the documentary, she kept reassuring her audiences that she, just as everyone else, was also ignorant of the concepts, data, graphs, and discourses on the smog. the film is simultaneously a piece of scientific popularization (kepu 科普) and public relation (gongguan 公關), intending to dispel myth about the smog and call for some collective pressure upon the government for reform. specifically, the timing of the documentary's online release ( february ) corresponded with the annual "two conferences" ( and march ) , which chai had earlier reported, in a particularly important year when discussions at the conferences were anticipated to shape china's key policymaking in the many years to come. clearly chai knew how the system works and attempted to expand what an insider-turned-independent journalist can do within the limitation of that system. chai adopted a maternal voice to engage her audiences emotionally from the very beginning. her use of the ted talk format, the best examples of which all summon emotion powerfully (gallo) , allows her to reference her own personal experience, focusing on the emotional turmoil she felt when she found out that her newborn daughter had a lung tumor. she then engaged her audiences visually, another powerful tool of ted talks (gallo) , showing a picture of her toddler confined at home peeking at the heavy smog outside the window (image . ). "i panic when my daughter smiles to me [because of the smog she is breathing in]," chai stated, turning a mother-daughter bonding moment into a nightmarish battle that she has no way to win. chai embraced her vulnerability in the most resolutely desperate form here: she was a helpless mother that did not know who her enemy was and how she could fight back. notably chai is drawing on a biological discourse (her being a mother) to justify her exploration of the environmental discourse, making it possible to interpret the documentary through the lens of ecofeminism. for instance, earlier ecofeminists defended animal rights as a biological calling, indicating that women have more compassion for animals (ropers-huilman ix). the maternal voice intended to address the universal ground-that is, parental concern for their children-of the environmental awareness that chai hoped to instill; however, it has proven to be a "soft spot easily challenged by both discursive rhetoric and the virtual internet" image . chai presenting her daughter, who was confined at home and gazing at the smog through a window, in under the dome ( , produced by chai) (cui ) . considering reason and nature as being separated and incompatible, some audiences stated that chai lacked scientific proof to link her baby's tumor to the smog. one of china's most rebellious artists, ai weiwei 艾未未 (b. ), went as far as calling chai a "brain-damaged mother" and further insulted that "those who comprehend the notion of smog only through chai's womb must be brain-damaged, too" (cui ) . it is ironic that the iconic artist eagerly volunteered to defend scientific data, only betraying that however politically idiosyncratic he may appear, his gendered biases remain deeply entrenched. ai's criticism of chai is based on the belief that the brain and the womb are antagonistic to each other and a woman's thoughts and feelings all have to do with her womb. ai's reductive reception of the documentary, however, demonstrates how gendered biases can drive people to criticize chai's work based on their own deeply flawed perceptions unhelpful to understand the work itself. chai may have used the maternal voice to mitigate potential offenses to the chinese government-she hoped to propel a policy level change after all-but her being attacked for embracing that voice becomes a lens into how gendered biases remain fully charged in the social and cultural norms that she attempted to change. in fact, chai's self-funded film represents the most comprehensive and in-depth investigation of the causes of china's air quality problems and fills in an urgent information gap through remarkably engaging storytelling and solid reasoning. chai reveals how china's excessive coal consumptions, ineffectively regulated automobile emission, inferior oil quality, flawed policymaking dominated by petroleum companies, and powerless environmental agency have collectively contributed to the smog hazard. she not only explored what caused the problems but also explained how they caused the problems, situating the environmental problems into larger historical contexts. for instance, she revealed how china's energy industries have inherited policymaking privileges from the state-regulated economy era and conveniently translated this political status quo into economic gains in the market-driven economy. she also revealed many gray areas in automobile and industrial emission controls, showing how state and local environmental agencies have been marginalized by recent state-level policies that heavily prioritize economic development. chai also referenced the lessons of london and los angeles in their tough recovery from air pollution, driving home her points about the worrying long-term damages caused by short-term economic gains. chai's interviewees are all insiders of the government, the law reinforcements, the industries, and the academia. each of them is not only dissatisfied with the situation but also remarkably critical of the existing systems. chai provided a platform for them to candidly express themselves and to contradict each other's claims, shedding light on how unclearly defined governmental functions, intertwined with prevalent self-interests and lack in transparency, have collectively led to non-action and dangerous numbness to the urgency of this problem. chai did not claim originality for these information, but she firmly pushed the publicity of these information in a systematic manner so that reform could become possible. the banning of under the dome should be situated in the context of how the chinese government has become increasingly responsive to public discourses. when facing environmental issues, the state actually also has a high stake. environmental issues pose a challenge to the sustainability of china's economic model and can potentially mobilize people and escalate into a threat to the "harmonious society" (hexie shehui 和諧社會) that the chinese government hopes to build. the government therefore must take them seriously. in this context, chai's film could be useful for the state to vent out people's frustration and to test the possibility of reform. with frequent, unprecedented changes happening in all aspects of chinese society within a short period of time, it is not uncommon for practices to precede regulation in post-socialist china, as new changes demand new regulations. the rise and fall of chai's documentary apparently follows a familiar pattern: as something independently produced, the documnentary rose from the margin of china's media apparatus, suddenly became viral and mainstream (the documentary received million views within hours), and eventually faced state's regulation because of its popularity. in this case, the state stepped in to shut down the online circulation of under the dome in order to contain potential damages, for the film could make people interpret the smog as a "human-caused catastrophe" and finger point at the state, whose legitimacy has largely depended on aggressive economic development that devastated the environment. but pulling the documentary offline does not mean that chai's work does not have any meaningful impact upon the government. notably the chinese government finally began incorporating environmental factors into the evaluation of state and local officials' work in . the various attacks of under the dome provide a useful lens into how the construction of environmental discourses in china is radically contested and unavoidably linked to political and economic discourses. firstly, some audiences criticized chai for prioritizing environmental issues over economic development. they further claimed that this tension reflected the conflicted interests between the "well-off middle-class elites," to whom chai belongs, and the poor who presumably could benefit from aggressive economic development. when there are many people struggling with basic food and housing issues, they declared, it is insensitive and pretentious for chai to discuss clean air as a human right. this criticism of chai is driven by an anxiety about modernization, which the state has effectively used to justify its economic ambition, and will be a continuous burden of china's environmental movement. secondly, some specifically attacked chai's elitist status, showing how the chinese society has been stratified and how the tensions among different social classes have intensified as a result of china's uneven development. that chai's daughter was born in america seemed to fuel this criticism, triggering heated debate about whether she was hypocritical to give birth to an american citizen while also claiming she deeply cared about china. some, justifiably concerned about the urban and rural gap, also argued that chai's campaign against smog may divert limited sources from more serious environmental issues, such as polluted lands and water in the countryside, that have plagued powerless farmers. thirdly, chai's film has also suffered nationalistic interpretations fueled by conspiracy theory. critics stated that chai's self-declared "personal war against the smog" was actually a western smear of china's economic miracle. presumably the film received funding from western powers-while in reality chai used the royalty of her book to self-produce the film-in order to sabotage china's economic development through portraying the smog as a negative result of that development. under the dome triggered an intensive, renewed interest in chai's interview of ding zhongli 丁仲禮 (b. ), a geologist and fellow of the chinese academy of science. clearly, the interview demonstrates how chai's neoliberal perspectiveshe emphasized universal standards, co-existence, and shared environmental burden across the world-clashes with ding's china-centered perspective. ding articulated with powerful elocution that the right for emission (paifangquan 排放權) equals the right for economic development (fazhanquan 發展權) and that each chinese people should be allowed to have a fairly calculated amount of emission when compared to their western counterpart. ding's openly admitted patriotism gave the impression that chai lacked it in her emphasis on the shared burden of the international community and her insistence that environmental issues transcend international politics, making under the dome even more politically suspicious for some of its chinese audiences. in the past seventeen years, chai jing has risen from china's official media to become a recognized investigative journalist, public intellectual, author, and more recently, an independent filmmaker and environmental activist. her experience and work reflect how china's news apparatus has reformed to adapt to the drastic societal changes with emotion being used to open up new ways of news communication. her documentary under the dome further shows how the internet has transformed the ecology of media and provided innovative platforms for social engagement. chai's embracing her own feelings of vulnerability, which dominated the beginning of her career, and using it to channel public feelings and drive news reporting has made her a distinctively controversial media personality. her leaving the cctv can be viewed as a self-marginalization that helps her sustain that vulnerability, through which she gains resilience and critical power. the use of maternal voice in under the dome exemplifies her use of the power of vulnerability in its most mature form. the controversiality about that voice signals that post-socialist china remains a space where environmental and gender discourses are contested and negotiated. notes . the abbreviation is "wenqing" 文青. some also translated the term as "the cultured youth." the use of the term has a clearly negative undertone. when one uses this to refer to oneself it often intends to humorously self-disparage. a stereotypical female wenqing usually wears cotton clothes and shoes, reads literary works by eileen chang 張愛玲 and haruki murakami (b. ) , watches arthouse movies, listens to non-mainstream music, carries professional cameras, drinks coffee, and loves to travel. she also embraces romantic fantasies and indulges in sentimental self-reflection. a stereotypical male wenqing tends to be quiet and mature, capable of commanding language and echoing others' feelings, and leans toward spirituality and perfectionism. whose independent documentary bumming in beijing: the last dreamers 流浪北京 (liulang beijing, ) was widely considered the inaugurating piece of new documentary movement in china to commemorate the th anniversary of china youth news reporters' association (zhongguo qingnian xinwen jizhe xiehui 中國青年新聞記 者協會, est. , renamed all-china journalists association 中華全國 新聞工作者協會 in ) according to goldman ( - ), there were no laws to protect public intellectuals in chinese history and some of the intellectuals that mao persecuted and that were later rehabilitated in the s were the earliest public intellectuals in post-mao china. although contemporary chinese public intellectuals have been silenced or purged continuously, they have been able to spread their ideas through private publishing, the internet, and working out contracts with hong kong and foreign media since the reform he became a target of vehement criticism after cui yongyuan 崔永元 (b. ), one of china's best known media people, mentioned in an interview that one of his cctv colleagues had some very disrespectful comments on the crying interviewees' of a talk show; many linked cui's remarks to zhu's show and accused zhu of this presumed wrongdoing at the end of an eight-minute interview about whether gore intended to run the president race. chai noted that stahl's question intended to expose how a politician cannot provide a straight answer even to the simplest question. chai concluded her own interview of an official who tried to brush away the pollution issue by asking him cui shuqin was the first critic to propose an ecofeminist reading of chai's documentary introduction the power of vulnerability guilin: guangxi shifan daxue chubanshe chai jing's under the dome: a multimedia documentary in the digital age what i learned watching hours of ted talks repression of china's public intellectuals in the post-mao era dir. my camera doesn't lie china's deadly cover-up chai jing: a protagonist of news drama editorial introduction: ecofeminism translated by cathryn clayton comparative study on the appraisal resources of china daily's disaster news the urban generation: chinese cinema and society at the turn of the twenty-first century key: cord- -o ai r authors: wang, wei; xiong, liang; wang, pengyun; wang, fubing; ma, qingfeng title: major vault protein plays important roles in viral infection date: - - journal: iubmb life doi: . /iub. sha: doc_id: cord_uid: o ai r viral replication and related protein expression inside the host cells, and host antiviral immune responses can lead to the occurrence of diverse diseases. with the outbreak of viral infection, a large number of newly diagnosed and died patients infected with various viruses are still reported every year. viral infection has already been one of the major global public health issues and lead to huge economic and social burdens. studying of viral pathogenesis is a very important way to find methods for prevention, diagnosis, and cure of viral infection; more evidence has confirmed that major vault protein (mvp) is closely associated with viral infection and pathogenesis, and this review is intended to provide a broad relationship between viruses and mvp to stimulate the interest of related researchers. viruses are acellular that cannot naturally reproduce outside of the living host cells and only assemble themselves depending on the host cellular metabolism. virion, known as the complete viral particle, consists of nucleic acid surrounded by capsid, which is enveloped with lipids in some viruses. virion is less than nm in diameter, and its self-assembly is very fast, viral replication inside of the host cells may manipulate and damage the host cells, and the antiviral immune response of the host can damage tissue simultaneously. under the effort of viral toxicity and host immunity, the host is prone to get many kinds of diseases. hepatitis b virus (hbv) and hepatitis c virus (hcv) can cause chronic infection, which can lead to liver cirrhosis and subsequently develop hepatocarcinoma, the patients with viral hepatitis serve as reservoirs of infectious virus. some viruses, including hepatitis a virus (hav), human enterovirus, ebola virus, sars virus, and avian influenza, can cause an outbreak of epidemic infection. [ ] [ ] [ ] [ ] the typical antibiotics are not effective of antiviral infection, antigenic drift of viruses can make effective treatments ineffective, and treatment of viral infection is still one of challenges for humanity. abbreviations: aids, acquired immunodeficiency syndrome; atf, activating transcription factors; c/ebpβ, ccaat-enhancer-binding protein β; egf, endothelial growth factor; eif a, eukaryotic initiation factor a; erk, extracellular signal-related kinase; irf , interferon regulatory factor ; mapk, mitogen-activated protein kinase; mda , melanoma differentiation-associated protein ; mdm, monocytederived macrophages; mvp, major vault protein; myd , myeloid differentiation primary response ; nf-kb, nuclear factor kappa-lightchain-enhancer of activated b cells; pbmc, peripheral blood mononuclear cells; pkm , pyruvate kinase isozyme m ; prrs, pattern recognition receptors; pten, phosphatase and tensin homolog deleted on chromosome ; srsfs, serine/arginine-rich splicing factors; stat- , signal transducer and activator of transcription- . recent studies have shown that many host-encoded proteins are associated with viruses: heat shock protein is incorporated into the virions of human immunodeficiency virus type (hiv- ) ; serine/arginine-rich splicing factors (srsfs) are related to viral replication, srsf promotes anogenital tumorigenesis by maintaining the stability of e e mrnas of human papillomavirus (hpv ), which is the pathogen of anogenital cancer; hiv- replication is increased by srsf , srsf , and srsf within the host cells ; host-encoded proteins are presented in influenza virions ; mvp is involved in antiviral immune response ; and the study of hostencoded proteins in relation to viruses contributes to finding novel targets for antiviral drugs. vaults, the large ribonucleoprotein particles, are composed with mvp, poly (adp-ribose) polymerase, telomerase-associated protein- (tep ), and one or more noncoding rna. , the human mvp, encoded by mvp gene that is located in chromosome p . , is highly conserved during evolution , and predominant component of vaults. [ ] [ ] [ ] [ ] the expression of mvp is very strong and widespread, the mvp is mainly located in the cytoplasm and associated with the cytoskeleton, and a small amount is localized at or around the nuclear membrane and the nuclear pore complex. , current studies have confirmed that mvps are associated with multidrug resistance in treatment of non-small lung cancer, human colon cancer, and mesial temporal lobe epilepsy with hippocampal sclerosis. mvp/vaults play important roles in several signal transduction pathways, suppress c-jun-mediated ap- transactivation by associating with cop , participate the phosphoinositide -kinase pathway by interacting with endogenous phosphatase and tensin homolog deleted on chromosome (pten) with the help of ca + modulation, act as a signaling scaffold protein of extracellular signal-related kinase (erk)/mitogen-activated protein kinase (mapk) pathway by interacting with src in response to endothelial growth factor (egf), and affect the jak-stat signaling pathway by responding and interfering the interferon (ifn)-gamma-mediated stat signals. growing evidences also confirmed that mvp is closely associated with other multiple cellular processes, such as nuclear-cytoplasmic transport, malignant transformation, senescence/ aging, autophagy, and innate immunity. interestingly, mvp has been linked to several types of viral infectious diseases as well as to virus-mediated immune responses. , here, we focus on the roles of mvp in the intracellular viral replication and host immune responses. the innate immune response, including the production of ifn- , is the first barrier of eliminating invaded pathogens early. in host cells, tlrs, rig- (rig-i-like receptor dsrna helicase enzyme), and mda (melanoma differentiation-associated protein ) act as pattern recognition receptors (prrs), ifn-stimulated proteins, and sensors for viral infection. [ ] [ ] [ ] the interferon regulatory factor (irf ) plays the master transcriptional role in viral infection-induced ifn production and immune responses, [ ] [ ] [ ] activates ifn-β production mediated by myd (myeloid differentiation primary response )independent rig- /mda pathway, also activates ifn-α production mediated by the myd -dependent tlrs pathway. [ ] [ ] [ ] the ifn- inhibits viral replication (including hcv, influenza a virus [iav], and hiv) by the production of ifn-stimulated effective proteins. , , after host cells or tissues are infected by hcv, prrs of host cells recognize stimulation signals of products of hcv processing, the interaction between prrs and stimulation signals activates ikba kinase to phosphorylate ikbα, which is associated with nf-kb protein complex in the cytoplasm, phosphorylated ikbα is released from nf-kb complex and degraded by ubiquitin-proteasome pathway, free nf-kb complex translocates to the nucleus, and subsequently activates mvp transcription under coactivators including hcv protein ns a. hcv infection also induces mvp expression through the sp signal pathways, and the infection of vesicular stomatitis virus (vsv), iav, and enterovirus (ev ) has the same effect with hcv infection. inducible mvp is helpful for the nuclear translocation of irf and nf-kb, and performs antiviral activity by promoting endogenous ifn- production and expression of the ifn-stimulated genes. the production of ifn is the critical step in an innate immune response, and mvp plays strong antiviral activity in an ifn- -dependent manner. with the advent of effectively prophylactic vaccines and antiviral drugs, hbv infection remains a global public health problem, , an estimated million people with chronic hbv infection are hbv carriers, deadly complications of hbv chronic infection (including cirrhosis and hepatocellular carcinoma) result in approximately , deaths per year, and hbv infection brings heavy economic pressure for individuals and heavy social burden for the world. as a type of pathogen, hbv causes host cells to produce ifns to increase protective defense of host immune system, ifns play important roles of antivirus by regulating the host immune system, and have been used to treat some cancers and hbv infection. , hbv virus infection leads to the production of type ifns by two main pathways. toll-like receptors / (tlr / ) recognize viral nucleotides and glycolipids and recruit the adaptor protein trif (tir-domaincontaining adapter-inducing ifn-β), trif interacts with traf (tumor necrosis factor [tnf] receptor-associated factor ) to activate nf-kb (nuclear factor kappa-lightchain enhancer of activated b cells), and activated nf-kb provokes ifnb production. , another pathway is triggered by tlr / and tlr , tlrs recognized viral nucleotides in the endosome recruit myd , in turn recruit irak / (interleukin- receptor-associated kinase / ) to the complex and interact with traf (tnf receptorassociated factor ), , and then activate irf / (ifn regulatory factor / ) to induce ifnα expression. mvp is a virus-induced protein, and the level of mvp in peripheral blood mononuclear cells (pbmcs), sera, and liver tissue derived from patients with chronic hepatitis b (chb) is higher than healthy individuals; mvp expression is also increased in hbv stable expression cell lines (hepg . . and huh . ) and hbv-infected hepatocarcinoma cell lines (hepg and huh ). during hbv infection, tlrs recruit and activate myd , which interacts with irak / , irf / , and traf to form a complex, the middle domain (aa - ) of mvp can interact with myd , high expressed mvp joins the myd -mediated complex by interacting with myd to promote ifn- production through translocation of irf and nf-kb from the cytoplasm to the nucleus. , however, hbsag and hbeag competitively bind the myd -binding region of mvp and suppress the ifn- production by disrupting mvp/myd interaction; the ifn- increment effect induced by mvp is counterattacked through hbeag and hbsag binding to mvp (figure ). evidence suggests that hbv has other strategies to suppress the host immune response. hbv polymerase (pol) may inhibit ifn-ɑ-induced myd induction, hbeag suppresses tlr-induced ifn-β, hbsag can block the irf- mediated ifn-ɑ production pathway, and multiple mechanisms lead to hbv immune escape. when the host is attacked by harmful pathogens including viral infection, one of protective immune response is inflammation to eliminate damage. ifn to interfere viral replication, interleukin (il- ) acted as a proinflammatory cytokine, and interleukin (il- ) served as a chemokine for neutrophils and monocytes are important mediators of immune response, and activation of il- and il- gene expression is regulated by transcription factors. , activator protein (ap- ), composed of proteins belonging to c-fos, c-jun, activating transcription factors (atf) and maf families, is a heterodimeric complex and acts as a transcription factor. the function of ap- complex is heavily dependent on the c-fos and c-jun subunits, ap- complex binds dna at ap- specific sites at the promoter and enhancer regions of target genes and increases target gene expression, , and researchers had confirmed that the ap- complex is involved in il- and il- regulation. , ccaatenhancer-binding protein β (c/ebpβ) is a member of the c/ebp transcription factor family, the gene of c/ebpβ can be translated into three polypeptides: the kda and kda liver-enriched transcriptional activating proteins (laps), and the -kda liver-enriched transcriptional inhibitory protein (lip). c/ebp proteins interact with certain gene promoters containing ccaat box motif, then recruit co-activators to promote gene expression. the promoters of il- and il- consists of the ccaat box motif region, wherein c/ebpβ can bind and affect il- and il- expression. f i g u r e hbsag and hbeag weaken the effect of mvp on promoting ifn production in order to restrict the spread of infected virus, some activated transcription factors contribute to the production of inflammatory-related cytokines and chemokines. iav, as a kind of negative single-stranded rna viruses (ssrna), produces replicative intermediate double-stranded rna (dsrna) in the infected cells, dsrna and the synthetic dsrna analog polyinosinic-polycytidylic acid (poly[i:c]) are recognized by tlr , , then activate the tlr -ifn production pathway to robustly express type i ifns. , mvp, as a regulator in the proinflammatory response and an effector in ifn signaling pathway, increases to against viral replication during viral infection. mvp has been proven to be a nuclear-cytoplasmic transport protein and interacts with c-fos of the ap- complex components and c/ebpβ-laps. the interaction promotes the ap- complex and c/ebpβ-laps translocation from the cytoplasm to nucleus and follows to activate the il- and il- expression by the ap- complex and c/ebpβ-laps binding to the il and il promoters, and mvp plays a synergistic role in the expression of il- and il- . the expression of mvp, il- , and il- increases simultaneously in iavinfected a or dsrna-stimulated pbmcs, and the expression of il- and il- is impaired in mvp knockdown cells and knockout mice ; mvp plays a pivotal role in virus-triggered proinflammatory response by mediating the ap- and c/ebpβ signaling pathways. the model of mvp functions for proinflammatory response is summarized in figure . hepatitis e virus (hev), belonged to the genus hepevirus, is classified as a positive-strand rna virus ([+] ssrna virus), and hev infection is an important public health problem. hev is mostly transmitted via the fecal-oral route in developing countries under poor sanitary conditions, , and often spread in many countries by food borne, blood transfusion, , and zoonotic origin. hev can cause chronic infection in immunosuppressed patients, pegylated ifn-alpha- b is used in the treatments for chronic hepatitis e (che) virus infection in liver transplant patients, pegylated ifn-alpha- a is used in the treatments for che virus infection in a hemodialysis patient, and ribavirin as monotherapy may be effective in the treatment for che virus infection in solid-organ transplant patients. silvestrol is a natural cyclopenta(b)benzofuran and acts as an inhibitor of the eukaryotic initiation factor a (eif a) via hindering translation initiation from the capped and -utr of mrnas. the hev is a (+) ssrna virus containing -cap and -utr structure, the released hev particles from persistently hev-infected a cells treated with silvestrol are robustly reduced, which are caused by the decrease of the intracellular hev capsid protein. silvestrol also affect the expression and localization of antiviral host factor mvp, the mvp amount of the cytoplasm is reduced after treating with silvestrol in hev-infected cells, and the mvp transfers from the cytoplasm to the perinuclear area that affects mvp-mediated ifn production. the translation of mvp is highly activated to play an antiviral role by hev infection; however, the change of translation and cytoplasmic localization affected by the silvestrol treatment counteracts part of antiviral effect, mvp plays a complex interplay between the anti-hev replication and the effect of treating with silvestrol for hev infection. the infection of hiv is the pathogenesis of acquired immunodeficiency syndrome (aids) and one of major global public health issues. hiv infected immune cells, including monocytes, lymphocytes, and macrophages, act as stable rival reservoirs, and are main barrier to f i g u r e mvp plays a pivotal role in the proinflammatory response caused by (−) ssrna viral infection eradicate virus by antiviral therapy. the level of cystatin b, a cysteine protease inhibitor, is higher in blood monocyte-derived macrophages (mdm) than in placental macrophages, which are more resistant to hiv- infection than mdm. , the expression of cystatin b is upregulated in hiv- -infected mdm, and cystatin b promotes hiv- replication by interacting with pyruvate kinase isozyme m (pkm ), which is associated with the cocaine enhancement of hiv- replication. in hiv-infected mdm, upregulated cystatin b interacts with mvp and signal transducer and activator of transcription- (stat- ). mvp, as an ifn-responsive protein, directly inhibits tyrosine phosphorylation of stat- to weaken ifn-induced antiviral response by interfering the jak/stat signal pathway, then promote hiv replication. cystatin b directly interacts and decreases tyrosine phosphorylation of stat- , and inhibits ifn-β response and stat- translocation from the cytoplasm to nucleus to reduce jak/stat signal pathway activity, and ultimately promote hiv replication. under the cooperation of the cystatin b and mvp, hiv replication is activated by the damage of jak/stat signal pathway activity mediated by the low tyrosine phosphorylation of stat- . mvp is involved in the diversely cellular processes, including multiresistant cancers, - signal transmission pathways, [ ] [ ] [ ] [ ] and immune response associated with viral infection and treatment. , , , , viruses with divergent virulence and spreadways can cause diverse human diseases with different types and degrees of damage, as a response of viral infection, studies have confirmed that mvp is enhanced in diverse viral infection, including hbv, hcv, hiv, iav and vsv, and so on. the infection of (−) ssrna viruses (including hcv, vsv, iav, and ev ) or dsrna stimulation activates proinflammatory response by inducing the expression of mvp, il- , and il- , enhanced mvp further increase the expression of il- and il- by translocating transregulatory elements (ap- protein complex and c/ebpβ-laps) to the nucleus, and lipopolysaccharide synthesized during viral replication also activates the tlr signaling pathway to induce cytokines, chemokines, and ifn- against iav replication ; however, the value of mvp in the diagnosis, treatment, and prognosis of viral infection remains unclear and additional studies are still required. hbsag and hbeag compete to bind with mvp, facilitate hbv replication and survival by attenuating the effect of mvp-induced ifn, and ifn and nucleotide analogs (nas) are used for the treatment of patients infected with hbv, the stage of liver diseases is important in guiding antiviral therapy ; however, the effect of mvp on the severity of liver disease and the efficacy of different treatments is unclear. silvestrol, as a potent antiviral compound, inhibits hev assembly by interfering hev capsid protein translation, but deactivates the antiviral effect of mvp by translocating mvp to the perinuclear membrane ; cystatin b, as a cysteine protease inhibitor, increases hiv replication by interacting with mvp and pkm to inhibit ifn response and tyrosine phosphorylation of stat- . mvp plays an opposite role in hiv infection by comparing with iva and hbv infection, weakens the antiviral efficacy of silvestrol in the treatment of hev infection, and additional studies are necessary to clarify the role of mvp more clearly in viral infection. i would like to thank my collaborators for their kind help to organize the thoughts and concepts. synthetic viruses: a new opportunity to understand and prevent viral disease significance of hbv dna by pcr over serological markers of hbv in acute and chronic patients. 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authors declare no potential conflict of interest.orcid pengyun wang https://orcid.org/ - - - fubing wang https://orcid.org/ - - - qingfeng ma https://orcid.org/ - - - key: cord- - k xk hs authors: summo, carmine; palasciano, marino; de angelis, davide; paradiso, vito m; caponio, francesco; pasqualone, antonella title: evaluation of the chemical and nutritional characteristics of almonds (prunus dulcis (mill). d.a. webb) as influenced by harvest time and cultivar date: - - journal: j sci food agric doi: . /jsfa. sha: doc_id: cord_uid: k xk hs background: several workers have studied the effect of harvest time on chemical and nutritional composition of almonds, but the results are partly conflicting, probably due to differences in the cultivars considered and to different agronomic and climatic conditions in the growing areas. in this paper, the influence of harvest time and cultivar on the chemical and nutritional composition of almonds (prunus dulcis (mill). d.a. webb) were evaluated. ten cultivars were considered, grown in the same orchard and subjected to the same agronomical regime. almonds were collected at two different harvest times: (i) when the fruits were unripe, but already edible, and showed green and moist hull; and (ii) when the fruits were ripe, with dry brown hull. the analyses of proximate composition, fatty acid profile, total phenolic compounds, and antioxidant activity were carried out. results: lipid content increased (p < . ) during ripening, while both protein and carbohydrate content decreased (p < . ). fatty acid composition showed a not univocal behavior during ripening and was highly influenced by cultivar. total phenolic compounds and antioxidant activity varied among cultivars but increased during ripening with the exception of cv marcona. the ‘genco’ and ‘francolì’ cultivars were found to be phenolic rich. conclusion: harvest time and cultivar significantly influenced the chemical and nutritional composition of almonds. genotype strongly influenced fatty acid composition and total phenolic compounds. the changes of bioactive compounds and antioxidant activity suggest that the synthesis of antioxidants also occurs in the last stage of ripening. unripe almonds, a valuable niche product, showed interesting nutritional value. © society of chemical industry almond (prunus dulcis (mill.) d.a. webb) is one of the oldest cultivated nut trees in the world and a major nut tree crop in hot-arid countries of the mediterranean basin, , including southern italy and, particularly, the apulia region. almond trees produce nutrient-dense nuts appreciated for their favorable lipid profile, and for high contents of vitamin e and polyphenols. almonds are mostly consumed without removing the skin, but may also be blanched and peeled, then milled and processed to nondairy beverages, or confectionery delicacies. the importance of almonds from an agronomical and nutritional point of view explains the presence of numerous studies related in particular to the characterization of the chemical composition of fruit, with particular emphasis on the lipid fraction. the latter is characterized by the predominance of unsaturated fatty acids, such as oleic and linoleic acid, and low amounts of saturated fatty acids, as well as by the presence of antioxidant compounds. previous studies showed the influence of several factors, such as genotype and harvest year, on the antioxidant compounds of almonds. in particular, bolling et al. reported that the synthesis of the individual polyphenols was related only to the cultivar. flavonoids, antioxidant activity, and total polyphenols instead showed a significant interaction between genotype and environmental conditions. furthermore, almond genotype is the main cause of variability of fatty acid composition, oleic/linoleic acid ratio, and content of minor compounds (squalene and -tocopherol). , at the same time, several studies , reported that both lipid content and fatty acid composition were affected by growing region, pointing out the significant interaction between genotype and environment. finally, yada et al. showed that moisture, total lipid, monounsaturated fatty acids, dietary fiber, and ash content of almonds were significantly affected by the harvest year, although this aspect was not univocally demonstrated in literature. moreover, many other studies were carried out in order to investigate the use of almonds to produce processed food, such as almond milk, almond fermented milk, and almond paste for desserts, all pointing out the health benefits of almonds and related products. [ ] [ ] [ ] [ ] at the same time, to take advantage of the high phenolic content of almond skins, several studies evaluated the potential use of this by-product of almond blanching as a new ingredient, with the aim of improving the health value of foods. , the composition of almonds is also strongly influenced by harvest time; that is, it varies at increasing ripening degree, as reported by several studies. nanos et al. carried out a study on two cultivars ('texas' and 'ferragnès') studying the effect of both harvest time and irrigation strategies on lipid content, lipid quality and sugar content. cherif et al. studied three cultivars ('achaak', 'perlees', and 'mazzetto') and reported an increase of lipid content during ripening, with significant changes in fatty acid composition. hawker and buttrose considered two cultivars ('chellaston' and 'johnston prolific') and studied the anatomy and the chemical composition of different parts of the fruit, reporting the evolution of lipid, protein, and sugar contents during ripening. soler et al. reported the changes of carbohydrates, proteins, and free amino acids during almond fruit developing, considering a single cultivar. egea et al. investigated the changes of both carbohydrate and protein content during fruit development in a single almond cultivar ('marta'), under deficit irrigation conditions. however, these studies focused on very few cultivars, and their results were partly conflicting. this could be due to a difference in the cultivars considered as well as to different agronomic and climatic conditions in the growing areas. in this framework, the aim of this study was to determine the influence of harvest time and cultivar on the chemical composition of almond fruit by considering ten different cultivars, all grown in the same orchard. two different harvest times were considered. the first corresponded to an early stage of almond maturity, when the fruit was still unripe but could be already consumed as fresh product, whereas the second harvest time corresponded to ripe fruits with dry hull. the research was carried out on adult almond trees ( years old) belonging to the germplasm collection of the department of soil, plant and food science (disspa) of the university of bari, grown in valenzano, near bari (apulia region, southeastern italy). ten commercially important cultivars were studied, of which eight were selected among the most widespread in the top producing countries (australia, california, italy, and spain), [ ] [ ] [ ] [ ] and two were new cultivars obtained in recent breeding programs. , in detail, the cultivars examined were the australian 'johnston prolific' (jp cv ), the californian 'texas' (te cv ) and 'thompson' (th cv ), the italian 'filippo ceo' (fc cv ), 'genco' (ge cv ) and 'tuono' (tu cv ), the spanish 'desmajo largueta' (dl cv ), and 'marcona' (ma cv ), as well as 'francolì' (fr cv ) and 'ferragnès' (fe cv ), the latter two being the new ones, recently grown in italian and spanish new plantations. , among these cultivars, the italian 'tuono', is also grown in greece, libya, tunisia and in spain (where it is known as 'guara'). all almond cultivars were grafted, on sweet almond cv don carlo, by t-budding in the fall onto almond seedling rootstocks, a common grafting technique used for almond orchards located in the mediterranean region and already used in other studies. , almond cultivars were grown under rainfed conditions and with a tree spacing of . m × . m. all trees had the same age, and standard cultural practices were performed. fruits were randomly collected from four different trees for each cultivar at two different harvest times, as follows: (i) t ( july ) corresponding to stage 'j' -that is, an early stage of almond maturity, when the fruit was still unripe, with green and moist hull, but with developed cotyledons, easily separable from almond skin; (ii) t (between the second half of august and the beginning of september ), corresponding to stage 'l' -that is, ripe fruits with dry brown hull. whole fruits collected at t and t were quickly stored at − ∘ c until analysis. then, the hull and shell were removed to obtain the kernel. finally, almonds were finely milled by an electric grinder (b , imetec, azzano s. paolo, bergamo, italy) and analyzed with three replicates. samples at t were lyophilized (de mori, milan, italy) prior to milling. weight was determined by an analytical scale on fruits. width, length, and thickness were measured by a caliper on fruits. all parameters were measured on fruits collected at t and t . protein (total nitrogen × . ), ash, and moisture content were determined according to the aacc methods - a, , and - a respectively. fat content was determined by means of a soxhlet apparatus using diethyl ether (sigma aldrich, milan, italy) as extracting solvent. total carbohydrates were calculated by difference. energy value was expressed as kilocalories per kilogram and was calculated using atwater's coefficients. the fatty acid composition was determined by gas chromatographic analysis of fatty acid methyl esters according to aocs method ch - . the gas chromatography system used consisted of a a gas chromatograph (agilent technologies, palo alto, usa) equipped with a flame ionization detector and an sp fused-silica capillary column m × . mm × . m film thickness (supelco park, bellefonte, pa, usa). the gas chromatography conditions used were the same as those reported in our previous study. in particular, the temperature of the split injector was ∘ c, with a splitting ratio of : ; the detector temperature was ∘ c. the oven temperature was programmed from to ∘ c, with increments of ∘ c min − , then to ∘ c with increments of ∘ c min − , and a final isothermal of min. helium was utilized as carrier gas at a constant flow rate of ml min − . the identification of each fatty acid was carried out by comparing the retention time with that of the corresponding methyl ester standard (sigma aldrich, milan, italy). the results were expressed as grams per kilogram. radical , -diphenyl- -picrylhydrazyl (dpph) scavenging activity and content of total phenolic compounds were measured on the methanol extract prepared as follows: . g of sample powder was mixed with ml of aqueous methanol ( % v/v) and stirred for h. after centrifugation at rpm for min, the supernatant was utilized for the determination of the antioxidant activity, as reported in cosmai et al. with some modifications. in particular, l of extract were diluted ten times wileyonlinelibrary.com/jsfa and added at l of mol l − dpph methanol solution (sigma aldrich, milan, italy). samples were stored in dark condition for h, then the absorbance at nm was read for each sample with a cary uv-vis spectrometer (agilent technologies, santa clara, ca, usa). a calibration curve was prepared with (±)- -hydroxy- , , , -tetramethylchromane- -carboxylic acid (trolox, sigma aldrich, milan, italy) in order to express antioxidant activity in equivalents of this compound. total phenolic compounds were determined using the folin-ciocalteu method, previously reported in singleton and rossi with some variations. basically, l of extract was added to l of folin and ciocalteu's phenol reagent (sigma aldrich, milan, italy) and to l of sodium carbonate ( % w/v). the mixture was kept stirring in dark condition for h, then was centrifuged at rpm for min. finally, the absorbance at nm was read. total phenolic compounds were expressed as milligrams per kilogram of gallic acid, previously used to obtain the calibration curve. to analyze the differences among samples, one-way analysis of variance (anova), followed by fisher's test (least significant difference) for multiple comparisons at p < . , was carried out on the experimental data by means of the xlstat software (addinsoft sarl, new york, ny, usa). principal component analysis was applied to define the influence of cultivar and harvest time. table reports the mean values of weight and size of kernels collected at two different harvest times: t , corresponding to unripe fruits with green and moist hull, traditionally consumed fresh, and t , corresponding to drier fruits collected at full ripening time. almond size's indices at t were characterized by a great variability; in particular, kernel weight ranged from . to . g. kernel weight remarkably decreased at t , due to moisture loss, with a lower variability than at t (range . - . g). jp cv showed the highest kernel weight both at t and t and was affected by the highest weight loss (exceeding %) during ripening. the same cultivar showed the longest kernels. the cultivars under investigation were characterized by different kernel shape, varying from round (ma cv , with . and . width/length ratio at t and at t respectively) to elongated (jp cv , with . and . width/length ratio at t and at t respectively). table reports the chemical composition of almond cultivars at the two different harvest times considered. all almond cultivars showed a remarkable variability for the chemical parameters evaluated. t almonds were characterized by a high moisture content ( . g kg − as mean value, reaching . g kg − in te cv ), which dramatically decreased to . g kg − in t almonds. the lipid content at t was between . g kg − (in te cv ) and . g kg − (in dl cv ) on fresh matter, and considerably increased at t , reaching . g kg − (in fc cv ). the latter cultivar also showed the lowest protein content at both t and t ( . g kg − and . g kg − respectively). in this regard, other authors reported that the highest lipid content was coupled with the lowest protein content. ma cv showed the highest protein content at t ( . g kg − ), whereas jp cv showed the highest protein content at t ( . g kg − ). carbohydrates mean content accounted for . g kg − and . g kg − at t and t respectively. ash content was below . g kg − at t , rising to a range of . - . g kg − at t . owing to the strong difference in moisture content (much lower at t than at t ), the total energy value (expressed on fresh matter) of almonds collected at t was dramatically higher ( . kcal kg − ) than at t ( . kcal kg − ). these observations could induce modern consumers, searching for less energetic food, to prefer fresh unripe almonds over ripe fruits. the differences between t and t are highlighted in fig. , which reports the mean value and the results of statistical analysis (one-way anova) of proteins, lipids, carbohydrates, and ash content expressed on dry matter. harvest time significantly influenced the lipid content, making it strongly increase during ripening (p < . ). this trend was due to the incomplete biosynthesis of triacylglycerols at t . cherif et al. egea et al., and piscopo et al. previously reported similar results, whereas nanos et al. reported no significant differences in lipid content during ripening, considering two cultivars (te cv and fe cv ) under two different irrigation strategies. protein content exhibited a significant decrease (p < . ) in almonds harvested at t , ranging from . to . g kg − . a previous study carried out over years in cv marta reported a sharp decrease of protein content immediately after fruit dehydration, but just in year of production. thus, protein content could be influenced by several factors in addition to cultivar, such as climatic conditions during kernel filling stage. changes in protein content during ripening were previously studied by soler et al. who reported a protein increase as ripening proceeded. this trend was not found in our samples. paralleling the same trend of protein content, a drop of total carbohydrates on dry matter was observed, from an average of . g kg − (t ) to . g kg − (t ). carbohydrates could be used as substrate for the biosynthesis of other chemical compounds during ripening. finally, ash content remained constant during almond ripening (p = . ). the changes that occurred in chemical composition could also be explained by considering the differences in kernel weight at the two harvest times (table ) . considering the chemical composition on a dry basis (data not shown), a significant and negative correlation was found between kernel weight and lipid content (r = − . ), whereas a positive correlation occurred with carbohydrate content (r = . ). thickness, length and width were not significantly correlated with chemical composition. greater variations in kernel weight during ripening (such as in fc cv and jp cv ) corresponded to stronger changes in the chemical composition. considering the data obtained in our investigation, unripe almonds (t ) revealed interesting nutritional characteristics compared with fully ripe almonds (t ). as a matter of fact, almonds have much lower lipid content and higher protein content at t than at t . moreover, owing to their high moisture content, unripe almonds have a lower energy value than fully ripe fruits. however, such a high moisture level determines a very low shelf-life, limiting the consumption of unripe almonds, as fresh product, to a short period of the year. frozen storage could be effective to extend the shelf-life of this nutritionally valuable product. table reports the fatty acid composition and the results of statistical analysis of almond kernels collected at t and t , expressed as grams per kilogram. all the cultivars showed a predominance of wileyonlinelibrary.com/jsfa oleic acid, ranging from . g kg − (te cv at t ) to . g kg − (ge cv at t ). linoleic acid was the second most abundant fatty acid, with the lowest content in jp cv at t ( . g kg − ) and the highest content in dl cv at t ( . g kg − ). at the same time, dl cv showed the highest content of palmitic acid at both t and t , with values of . g kg − and . g kg − respectively. the lowest contents of palmitic acid were found in ge cv and fc cv at both harvest times. therefore, almonds showed a well-balanced and healthy fatty acid composition, even at the earliest stages of ripeness. the results of the statistical analysis (one-way anova) revealed that fatty acid composition was influenced by harvest time, pointing out significant differences among seven out of ten cultivars under investigation. the evolution of fatty acid composition during ripening did not exhibit univocal behavior. in particular, ge cv and te cv showed a significant increase of oleic acid content during ripening and, oppositely, fc cv , fe cv , and jp cv revealed a significant decrease of the same fatty acid. in a previous study, nanos et al. examined te cv and fe cv and reported a higher oleic content in early-harvested almonds than in late-harvested ones. on the other hand, both cherif et al. and piscopo et al. reported an increase of oleic acid during ripening. in fc cv , fe cv , and jp cv , the decrease of oleic acid corresponded to a significant increase of linoleic acid during ripening, whereas ge cv and te cv showed a decrease. palmitoleic acid significantly decreased in fe cv , ge cv , and ma cv . the saturated lipid fraction was represented mostly by palmitic acid, which also exhibited divergent trends among cultivars. its content significantly decreased in te cv and tu cv . stearic acid increased in jp cv and tu cv , but significantly decreased in te cv . to better point out the influence of cultivar in our samples, fatty acid compositional data were submitted to principal component analysis, setting the cultivar as supplementary variable (fig. ) . as expected, the distribution of samples was strongly influenced by the variable 'cultivar', regardless of harvest time. however, we found an irregular behavior in sample distribution among the two principal components. pc explained over the % of total variability and was influenced by minor and saturated fatty acids. in this case, some cultivars, such as ge cv , jp cv , te cv , and dl cv , exhibited a great variability on this axis. pc , instead, was influenced by major fatty acids (oleic and linoleic acids), and both jp cv and te cv showed a large variability related to harvest time. on the whole, our results agreed with existing literature , , , , , and highlighted that the evolution of fatty acid composition during ripening was related to varietal factors. the positive effects of almond consumption on health are widely reported in the literature. these properties are mainly related to almond fatty acid composition, which contributes to enrich the diet in monounsaturated fatty acids. the latter have a more favorable effect on health than polyunsaturated fatty acids, , besides the obvious positive effect over saturated fatty acids. , a moderate and regular consumption of almonds and nuts (∼ g daily) is associated with health-promoting effects, and the use of almonds as a nutraceutical tool is conceivable in metabolic diseases because they reduce low-density lipoprotein and total cholesterol and improve glycemic control. , [ ] [ ] [ ] thus, the consumption of almonds is gaining interest both locally, in the producing areas, and worldwide. the total phenolic compounds and the antioxidant activity of almond cultivars at both t and t are reported in table . the total phenolic compounds showed a great variability among cultivars, ranging from . (jp cv ) to . mg kg − gallic acid (fr cv ) on wileyonlinelibrary.com/jsfa table . mean value (n = ), plus/minus standard deviation, and results of statistical analysis of the antioxidant activity ( mol trolox equivalents g − on dry matter) and of the total phenolic compounds (mg kg − gallic acid on dry matter) of almonds collected at two different harvest times total phenolic compounds it should be underlined that almond phenolic compounds have positive health effects, such as the reduction of oxidative stress and inflammation. , in particular, the most abundant class of polyphenols in almonds is represented by proanthocyanidins, recognized as strong contributors of the stability of intestinal microbiota, improving the immune response. , therefore, the variations observed among cultivars could have consequences on the health benefits associated with almond consumption. , , moreover, some phenolic-rich cultivars, in particular ge cv and fr cv , could be used in the formulation of food products in order to extend their shelf-life, by reducing the lipid oxidation and preventing the formation of off-flavors. these cultivars will be the object of further studies of shelf-life assessment. considering the effect of harvest time, eight out of ten cultivars showed significant differences between t and t . in particular, six cultivars showed a significant increase during ripening, probably as a consequence of the incomplete biosynthesis of phenolic compounds in unripe green almonds. two cultivars (ma cv and jp cv ), on the contrary, showed a significant decrease of the total phenolic compounds during ripening, while for dl cv and te cv the variation observed during ripening was not significant. the antioxidant activity was significantly correlated with the total phenolic content (r = . and . at t and t respectively). also, the antioxidant activity varied greatly among cultivars, in particular at t , when the lowest value accounted for . mol trolox equivalents per gram for ma cv and the highest value, observed in fr cv and ge cv , was five times higher. with the exception of ma cv , jp cv , and te cv , the antioxidant activity at t was significantly higher than at t . even for this parameter, ma cv showed a peculiar behavior, with a significant decrease during ripening. the differences in the content of total phenolic compounds and antioxidant activity observed among cultivars could be due to several factors, such as genetic influence and harvest year, as reported by numerous workers. , , furthermore, total phenolic compounds and antioxidant activity were influenced by harvest time. in this study, the influence of harvest time and cultivar on the chemical composition of almonds was evaluated considering ten cultivars grown in the same orchard. harvest time significantly influenced the chemical composition of almonds, showing an increase of the lipid content, and, at the same time, a decrease in carbohydrates and protein content. ash content remained constant during ripening. the fatty acid composition was also affected by harvest time, showing no univocal behavior among the cultivars and then pointing out a strong varietal influence. a great variability of antioxidant activity and content of total phenolic compounds was found among the ten cultivars considered, pointing out the strong influence of the genotype. these parameters tended to increase with harvest time, suggesting that the synthesis of antioxidant compounds also occurred in the last stage of ripening. data variability, also considering the influence of kernel weight, suggests that each cultivar has a particular attitude to different purposes. lower lipid content and higher levels of phenolic compounds could positively influence the shelf-life, by limiting the oxidative process during almond storage. moreover, in this study we also carried out a nutritional characterization of unripe almonds. owing to lower lipid content and higher moisture content, they show a markedly lower energy value than fully ripe fruits. unripe almonds are a valuable niche product. owing to their very low shelf-life, unripe almonds are traditionally consumed fresh in the producing area during a very short period of the year when they are naturally available. however, unripe almonds could be stored frozen and then marketed all the year round and far beyond the area of production, fulfilling the expectation of consumers aware of the relation between healthy diet and well-being. irrigation and harvest time affect almond kernel quality and composition effect of harvest time on kernel quality of several almond varieties (prunus dulcis (mill.) da webb) evaluation of almond (prunus amygdalus batsch) cultivars from the apulia region in southern italy almond polyphenols: methods of analysis, contribution to food quality, and health promotion a review of composition studies of cultivated almonds: macronutrients and micronutrients antioxidant constituents of almond (prunus dulcis (mill.) da webb) hulls polyphenol content and antioxidant activity of california almonds depend on cultivar and harvest year chemometric characterization of almond germplasm: compositional aspects involved in quality and breeding variability in almond oil chemical traits from traditional cultivars and native genetic resources from argentina oil content and fatty acid composition of almond kernels from different genotypes and california production regions protein content and oil composition of almond from moroccan seedlings: genetic diversity, oil quality and geographical origin natural variability in the nutrient composition of california-grown almonds almond milk preparation process and products obtained almond milk fermented with different potentially probiotic bacteria improves iron uptake by intestinal epithelial (caco- ) cells almond nut paste for beverages and desserts the almond milk: a new approach to the management of cow-milk allergy/intolerance in infants in-depth proteomic analysis 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their distribution in almonds key: cord- -lez dcao authors: de oliveira, marcelo m.; alves, sidiney g.; ferreira, silvio c. title: dynamical correlations and pairwise theory for the symbiotic contact process on networks date: - - journal: nan doi: . /physreve. . sha: doc_id: cord_uid: lez dcao the two-species symbiotic contact process ( scp) is a stochastic process in which each vertex of a graph may be vacant or host at most one individual of each species. vertices with both species have a reduced death rate, representing a symbiotic interaction, while the dynamics evolves according to the standard (single species) contact process rules otherwise. we investigate the role of dynamical correlations on the scp on homogeneous and heterogeneous networks using pairwise mean-field theory. this approach is compared with the ordinary one-site theory and stochastic simulations. we show that our approach significantly outperforms the one-site theory. in particular, the stationary state of the scp model on random regular networks is very accurately reproduced by the pairwise mean-field, even for relatively small values of vertex degree, where expressive deviations of the standard mean-field are observed. the pairwise approach is also able to capture the transition points accurately for heterogeneous networks and provides rich phase diagrams with transitions not predicted by the one-site method. our theoretical results are corroborated by extensive numerical simulations. discontinuous transitions [ ] , observed in distinct classes of cooperative systems, have led to a renewed burst of interest within different contexts, such as social interactions [ , ] , coinfections [ ] [ ] [ ] , synchronization [ , ] , and percolation [ , ] , to cite only a few fundamental processes. many of these investigations have focused on phenomena occurring at the top of complex networks [ ] , which constitute basic substrates for describing interacting patterns of complex systems [ ] [ ] [ ] . while in a continuous transition the order parameter varies continuously from zero, in the discontinuous case it suddenly jumps to a finite value at the transition point, involving a macroscopic portion of the system, and this transition is commonly referred to as catastrophic or abrupt [ ] . coinfection epidemics [ ] , i.e., when a host can be infected simultaneously by two distinct diseases, can result in coexisting thresholds when competitive interactions are considered [ , ] . nevertheless, cooperative or synergistic interactions result in richer phase diagrams, which may include discontinuous phase transitions, and they have been a topic of intense research [ , , [ ] [ ] [ ] [ ] [ ] . cooperation and competition have been investigated in multispecies interacting models in distinct ecological contexts [ ] [ ] [ ] [ ] [ ] [ ] . in particular, symbiotic interactions were recently investigated in a two-species contact process ( scp) [ ] . in the standard, * mmdeoliveira@ufsj.edu.br † sidiney@ufsj.edu.br ‡ silviojr@ufv.br single-species contact process (cp) [ ] , individuals lay on the vertices of a graph, which was originally a lattice, but the process was later extended to networks [ , ] . in the standard cp model, each individual can reproduce asexually at a rate λ, with its offspring occupying one empty nearest neighbor randomly chosen, or it can die at a rate μ (which can be taken as μ = without loss of generality). in the scp [ ] , two species, a and b, inhabit the same graph. the symbiotic interaction is modeled via a reduced death rate, μ < , at sites doubly occupied (by one individual of each species). with the exception of this interaction, the two populations evolve independently according to the standard cp. apart from its interest as an elementary model of symbiosis, the scp is fundamentally interesting for the study of nonequilibrium phase transitions. extinction represents an absorbing state, i.e., a frozen state without fluctuations of populations [ ] . on regular lattices, it was found that the scp exhibits a continuous phase transition in one and two dimensions [ ] . however, the transition becomes discontinuous in the regime of strong symbiosis if diffusion is introduced [ ] . the scp was recently investigated in complete graphs and random regular networks [ ] , and it was conjectured that the nature of its transition changes at the upper critical dimension, from continuous to discontinuous. the phase diagram determining the regions of the scp space parameter μ versus λ was determined in the ordinary, onesite mean-field level [ , , ] , which neglects all dynamical correlations with the assumption of statistical independence among the states of individuals even if they are nearest neighbors. this assumption is a strong approximation that can make the theory inaccurate even for high-dimensional systems such as complex networks [ ] , where the smallworld property [ ] resembles a mean-field (fully connected) regime. dynamical correlations can be introduced using pairwise approximations [ , ] where the mean-field equations for pairs of connected vertices are considered. despite being inaccurate in low dimensions [ ] , pairwise correlations greatly improve the theoretical results in the case of dynamical processes on complex networks [ , [ ] [ ] [ ] [ ] . in the present work, we investigate the role of dynamical correlations in the scp using a homogeneous pairwise meanfield (pmf) theory. the theoretical predictions are compared with stochastic simulations on different random networks, including homogeneous, poissonian, and scale-free degree distributions [ ] . we observe that the pmf theory substantially improves the ordinary mean-field in all investigated cases, being very accurate for determining the transition points and phase diagrams. moreover, the pmf phase diagrams are more complex, showing discontinuous transitions with either symbiosis parameter μ or rate infection λ fixed in contrast with the ordinary mean-field where only the former one can happen. the remainder of this paper is organized as follows. in sec. ii we present the definitions of the model and networks used as substrates for simulations. the mean-field theories are developed in sec. iii and appendix while the simulation methods used in the present work are described in sec. iv. section v is devoted to a comparison between mean-field theories and simulation outcomes. finally, our conclusions and prospects are drawn in sec. vi. we investigate the symmetric scp [ ] , in which the involved rates are the same for both species. the model is defined for a networked substrate as follows. each vertex of the network can hold at most one individual of each species a and b. so, the state s of a vertex i can be empty, represented by s i = ×, occupied by only one individual of type a (s i = •) or b (s i = •), or occupied by one individual of each species, represented by s i = ••. the reproduction of new individuals of a given species happens independently of each other according to the standard cp rules [ , ] . a vertex i with one individual of type a, s i = • or s i = ••, creates an offspring of type a at a randomly selected nearest neighbor j at a rate λ if j does not have an individual of type a (i.e., if s j = × or s j = •). same rules and rates are used for the replication of a type b. any individual in a vertex i spontaneously dies with rate if they are alone (s i = • or •) or with rate μ otherwise (s i = ••). if μ = , the two processes evolve independently, but for μ < they interact symbiotically since the annihilation rates are reduced at vertices where both species are present. the stationary scp can evolve asymptotically to a fully active state, where both species coexist, or to an inactive (absorbing) phase in which both species are extinct. there are also two partly active states, where only one of species a or b is extinct, that can be reached for specific initial conditions (the dynamics in these partly active states is beyond our interest since it is that of a standard single-species cp). in this work, we use random graph models as substrates on which the scp takes place. the number of neighbors of a vertex i, the vertex degree, is denoted by k i . we consider both homogeneous and heterogeneous networks. in random regular (rr) networks [ ] , all vertices have the same degree, k i = k, and connections are performed at random following the configuration model [ ] avoiding both multiple and selfconnections. in the erdös-renyi (er) model [ ] , each pair of vertices is connected with probability p. when the size of the graph n → ∞, its degree distribution is a poissonian with a finite mean k = pn. both rr and er models belong to the class of homogeneous degree networks, where large deviations from the average value do not occur [ ] , whereas only rr has a strictly homogeneous degree distribution. the third substrate is the barabási-albert (ba) model [ ] , in which the networks are generated through a preferential attachment mechanism [ ] starting from a fully connected set with m + vertices and vertices with m edges being added one at a time. we used m = m such that the average degree k = m can be fixed accordingly for comparison with the homogeneous case. the ba degree distribution follows asymptotically a power law (pl), p k ∼ k −γ , with γ = and is representative of a highly heterogeneous network possessing a heavy-tailed degree distribution. we consider a homogeneous mean-field approximation where all vertices are assumed to have the same degree k i = k. to apply this theory to the heterogeneous cases of er and ba, we use the same equations of the homogeneous theory replacing k by k . this strategy has been used to compare the transition points of the cp obtained in a homogeneous pmf theory, given by λ c = k/(k − ) [ ] , with the simulations on heterogeneous networks using λ c = k /( k − ) [ , ] . let [s] be the probability that a vertex is in state s, and let [s, s ] be the joint probability that a vertex is in the state s and its nearest neighbor is in the state s . symmetries in the rates with respect to distinct species imply that following this approach, one identifies three independent one-site variables, [•], [••], and [×], related by the closure relation the dynamical equations for these variables are readily derived as and equations ( )-( ) are exact but not closed since the evolution of the one-site probabilities depends on pairs. cutting the correlations at a vertex level with the approximation [s, s ] = [s][s ], we obtain the ordinary mean-field equations [ ] and note that if one species is extinct, the above system reduces to the mean-field theory for the one-species cp, with a transition point at λ = . the stationary solution of eqs. ( )- ( ) is given by [ ] [ and for μ / , [•] grows continuously from zero at λ = , marking the latter value as the transition point. the activity grows linearly, for μ < / , however, the expression is already positive for λ = √ μ( − μ) < , and there is a discontinuous transition at this point. further improvement in including dynamical correlations is given by a pmf theory, in which the system is described in terms of pairwise variables [s, s ]. the dynamical equations of pairs will depend on triplets [s, s , s ] in which a pair s, s of nearest neighbors is connected to a vertex in the state s through the vertex in the state s . here, we neglect that s and s can also be connected, i.e., we assume the network does not form triangles having thus a negligible clustering coefficient [ ] we now proceed with the standard pairwise approximation [ , ] [s, and apply the generic closure relation to obtain a set of seven dynamical equations for the independent pairwise variables, given by eqs. (a )-(a ) in appendix. these equations, in addition to the one-site dynamical equations ( )-( ), build a closed system. it is worth mentioning that pairwise approximations have been presented for one-dimensional chains and square lattices in ref. [ ] while our approach is valid for generic homogeneous graphs (conditioned to have a very low clustering coefficient). our simulations of the scp on networks were implemented using an optimized gillespie algorithm [ ] , in which we maintain two lists, one of singly and another of doubly occupied vertices. let n and n denote, respectively, the numbers of such nodes. the total rate of (attempted) transitions is λn + λn + n + μn ≡ ( t ) − , where t is the average time increment associated with a given step of the simulation. at each time step, we randomly choose among the events: (i) creation attempt by an isolated individual, with probability λn t; (ii) creation attempt by an individual at a doubly occupied node, with probability λn t; (iii) death of an isolated individual, with probability n t; (iv) death of an individual at a doubly occupied node, with probability μn . once the event type is selected, a vertex i is chosen at random from the respective list. the creation attempt occurs at a vertex j randomly selected among the nearest neighbors of i. if j is already occupied by an individual of the species to be created, no change of state is implemented, and the simulation continues to the next step. if node i is doubly occupied, the species of its offspring in a creation event is chosen to be a or b with equal probability. in the same way, in an annihilation event at a doubly occupied node, we choose the species to be removed at random. time is incremented by t. in the simulations, we sample the quasistationary (qs) distribution of active nodes employing the qs simulation method [ ] . this scheme consists in replacing the absorbing state, every time the system attempts to visit it, with an active configuration randomly taken from the history of the simulation. this procedure optimizes the numerical simulations restricting the dynamics of the process to active states, and it is a powerful tool in analyzing continuous absorbing state phase transitions in networks [ , , [ ] [ ] [ ] . for a recent analysis on such simulation methods applied to networks, see ref. [ ] . in this work, we performed qs simulations for systems of sizes up to n = nodes, with each run lasting at least time units. averages are taken in the qs regime, after discarding an initial transient which depends on the system size and symbiosis strength used. finally, mean-field analysis was performed via a numerical integration of the respective dynamical systems using the fourth-order runge-kutta method with a time step t = − . the steady state is computed after a relaxation of t > . the quasistationary densities of sites occupied by a single ρ = ρ • + ρ • and by two species ρ = ρ •• are compared with mean-field approximations in fig. for a rr network with connectivity degree k = , using distinct values of the symbiotic strength parameter μ. this dynamics can exhibit bistability with both active and absorbing stable stationary states [ ] . so, we first analyze an initial condition having all vertices in a doubly occupied state ρ = such that this threshold represents the loss of global stability of the absorbing state, marking the lower spinodal point. we observe that the ordinary mean-field theory, although qualitatively predicting the discontinuous or continuous nature of the transition, is not quantitatively accurate as indicated by the dashed curves in fig. . however, a far better result is obtained with the pmf theory, represented by the solid curves, presenting an excellent agreement between theory and simulations, for both near and above the transition point regimes irrespective of the μ values. increasing the connectivity k reduces the transition value of λ as can be seen in table i . actually, the threshold will converge to the one-site mean-field value for k since this limit corresponds to the fully connected graph for which the one-site theory is exact in the thermodynamical limit. figure compares the results from simulations and meanfield theories for er networks with k = using the substitution of k by the mean connectivity k . as observed in rr networks, the one-site approximation reproduces qualitatively well the nature of transition but underestimates the transition point. the pmf accurately matches the transition point and density of singly occupied vertices in the active phase of simulations but underestimates the density of doubly occupied vertices and consequently the overall density of active vertices ρ = ρ + ρ . the role of heterogeneity is further investigated in fig. where the results on ba networks with k = are shown. the pmf theory outperforms the ordinary one but still underestimates the location of the phase transition for small values of μ, where the phase transition is discontinuous while yields a very good agreement for the transition point for the continuous cases with μ > . despite the high heterogeneity of the ba networks, where hubs are expected to play some important role. this finding in the continuous case is in agreement with one-species cp on scale-free networks [ , ] . the transition points for the investigated networks (rr, er, and ba) with different average degrees are presented in table i . we observe that the pmf theory exhibits a higher accuracy for large average connectivity, as expected, since in the limit k it corresponds to the fully connected graph where mean-field theories become exact. another important observation is that the pmf performs worst for ba networks when μ is small while the accuracy tends to be reduced for larger μ in rr networks. in ba networks, where hubs are present, small μ will enhance activity localized in the neighborhood of hubs, which, in turn, are not sufficiently mixed for the regime of scale-free networks with γ = [ ] . more precisely, the star subgraph containing a hub and its nearest neighbors can stay active in isolation for long periods through a feedback mechanism where the hub activates its neighbors, which in turn reactivate the hub recurrently [ , , ] . since localization is opposed to the homogeneous mixing hypothesis of the mean-field methods, larger deviations from the theory are indeed expected in this regime. another intriguing feature of the data of table i is that the pmf thresholds are nearer to the simulations for the slightly heterogeneous er than from the homogeneous rr networks, which becomes more evident for lower k and higher μ. a similar phenomenon was also observed for the one-species cp on networks [ ] and associated with the approximation k ≈ k , which would not be observed in a degree-based mean-field theory [ ] . up to this point, we have addressed the global loss of the stability of the absorbing state that, in the case of discontinuous transitions, involves the transition from an absorbing to a bistable stationary state where both the absorbing phase and active phases can be stable depending on the initial condition. we now also consider the loss of global stability of the active phase, i.e., the transition from bistable to active phases. for this aim, we consider an initial condition very close to the absorbing state with a very low density of doubly occupied vertices (ρ = and ρ •• ) in conjunction with the fully occupied state ρ •• = used previously. we can therefore compute hysteresis curves where the phase coexistence can active fig. . hysteresis analysis for scp on rr networks with k = and μ = . . solid curves: pmf theory (arrows indicate the hysteresis flow). dashed curves: ordinary mean-field theory. symbols: results from simulations for the rr network with n = vertices using different initial conditions. be derived as in fig. where one sees absorbing, bistable, and active regions. phase diagrams in the parameter space (λ, μ) obtained via pmf for different connectivities are shown in fig. . the diagrams exhibit three connected regions corresponding to globally inactive (dotted region), globally active (empty region), and bistable (hashed region). an interesting difference with respect to the ordinary mean-field is a transition from a globally stable active phase to a bistable dynamics obtained either by fixing the infection rate λ and varying the symbiotic parameter μ or using fixed μ and changing λ, whereas in an ordinary mean-field theory such a transition occurs only for fixed μ; see, for example, fig. of ref. [ ] , which resembles very much the limit of large degree shown in fig. (c) . indeed, the region with double transitions (inactive to bistable and bistable to active) at fixed λ, occurring in the small μ region, shrinks as the connectivity increases toward a fully connected graph limit as shown in figs. (b) and (c). triple points shown in the phase diagrams depend on connectivity, departing from (λ t , μ t ) = ( , / ) for the complete graph (one-site mean-field) [ ] to λ t = k k − and μ t = . ( ), . ( ), and . ( ) for k = , , and , respectively. it is worthwhile to comment that phase diagrams with qualitatively similar spinoidals found in scp with our pmf theory have been reported for other models with abrupt transitions on networks [ ] [ ] [ ] , with the difference that they were obtained by a first-order mean-field theory, whereas in the scp model we needed to go up to a second-order pairwise theory. figure (a) also presents the phase diagram obtained via simulations on different graphs with average degree k = . one observes a remarkably good match between simulations and theory for rr and er (homogeneous) networks. the quantitative agreement is less satisfactory for the ba model, but it is still qualitatively correct. the dynamical correlations in these infinite dimensional systems have proven to lead not just to a quantitatively better theory but also to new behaviors not predicted by the ordinary mean-field theory. stochastic interacting systems with cooperative or synergistic couplings can be used for modeling different phenomena, such as coinfections of pathological agents, social relations, and ecological interactions, among others. one key issue of these models involves the nonequilibrium phase transitions among active and absorbing phases that can emerge in some systems, especially the abrupt ones, where macroscopic order parameters change discontinuously. moreover, understanding the behavior of these transitions taking place at the top of complex networks has become very relevant since most of these dynamical processes indeed involve interactions mediated by networked systems. in the present work, we investigate a simple model in which two species lying on networks interact symbiotically, i.e., the symbiotic contact process [ ] . we used both stochastic simulations and mean-field pairwise approximations, the latter reckoning dynamical correlations not considered in previous analytical (one-site mean-field) studies of this model. the pmf theory outperforms the ordinary one and, more importantly, predicts features not captured by the latter, in agreement with the simulations on distinct types of networks with homogeneous, poissonian, and scale-free degree distributions. in the case of homogeneous networks, the pmf theory is accurate at describing very well both the dynamics near and above the transitions (supercritical region). although it disregards the heterogeneity of the networks, our theory also gives very good estimates of the transition points in the case of poissonian and scale-free networks, but it cannot quantitatively capture the prevalence in the highly active regime. we also investigated the phase diagrams exhibiting globally absorbing, bistable, and globally active regions. the pmf theory, beyond being quantitatively more accurate than the ordinary one, provides a much richer phase diagram where discontinuous transitions, involving bistable phases can be obtained by either varying the infection rate λ or the symbiosis parameter μ independently, whereas this transition happens only at fixed μ in the one-site approach. these pmf results are confirmed by extensive numerical simulations. our findings provide an important example of the role played by dynamical correlations in cooperative processes on networked substrates, which is commonly not considered in other theoretical approaches. pair approximations such as the one developed in this work can be used to improve the accuracy of phase diagrams observed in other related works [ , ] where first-order mean-field theory captures the qualitative behavior but analytical spinoidals deviate from simulations when the average connectivity is reduced. we expect that our work will stimulate future analysis of dynamical correlations on other cooperative processes. the theories presented in this paper do not include the network heterogeneity and, especially, the degree distribution. as a prospect for forthcoming analysis, one could use degree-based [ ] or individual-based 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regular and complex networks universal scaling of distances in complex networks contact processes on random graphs with power law degree distributions have critical value nature of the epidemic threshold for the susceptible-infected-susceptible dynamics in networks spontaneous recovery in dynamical networks critical behaviors in contagion dynamics failurerecovery model with competition between failures in complex networks: a dynamical approach failure and recovery in dynamical networks epidemic phase transition of the sis type in networks the authors acknowledge the financial support of conselho nacional de desenvolvimento científico e tecnológico (cnpq) and fundação de amparo à pesquisa do estado de minas gerais (fapemig). in the pair approximation, the seven two-site variables to be considered are [ . all other combinations are equivalent to some of them, due to symmetry. considering that every vertex has k nearest-neighbors, after some algebra we obtain the following set of dynamical equations in eqs. (a ) to (a ), and find that they can be approximated as key: cord- -tf dpo authors: enes, sara rolandsson; uriarte, juan j.; pouliot, robert a.; weiss, daniel j. title: clinical application of stem/stromal cells in copd date: - - journal: stem cell-based therapy for lung disease doi: . / - - - - _ sha: doc_id: cord_uid: tf dpo chronic obstructive pulmonary disease (copd) is a progressive life-threatening disease that is significantly increasing in prevalence and is predicted to become the third leading cause of death worldwide by . at present, there are no true curative treatments that can stop the progression of the disease, and new therapeutic strategies are desperately needed. advances in cell-based therapies provide a platform for the development of new therapeutic approaches in severe lung diseases such as copd. at present, a lot of focus is on mesenchymal stem (stromal) cell (msc)-based therapies, mainly due to their immunomodulatory properties. despite increasing number of preclinical studies demonstrating that systemic msc administration can prevent or treat experimental copd and emphysema, clinical studies have not been able to reproduce the preclinical results and to date no efficacy or significantly improved lung function or quality of life has been observed in copd patients. importantly, the completed appropriately conducted clinical trials uniformly demonstrate that msc treatment in copd patients is well tolerated and no toxicities have been observed. all clinical trials performed so far, have been phase i/ii studies, underpowered for the detection of potential efficacy. there are several challenges ahead for this field such as standardized isolation and culture procedures to obtain a cell product with high quality and reproducibility, administration strategies, improvement of methods to measure outcomes, and development of potency assays. moreover, copd is a complex pathology with a diverse spectrum of clinical phenotypes, and therefore it is essential to develop methods to select the subpopulation of patients that is most likely to potentially respond to msc administration. in this chapter, we will discuss the current state of the art of msc-based cell therapy for copd and the hurdles that need to be overcome. chronic obstructive pulmonary disease (copd) is a progressive life-threatening disease that is significantly increasing in prevalence. the world health organization (who) predicts that copd will become the third leading cause of death worldwide by [ ] [ ] [ ] . there is currently no cure for this disease, and smoking cessation remains the most prominent intervention [ ] . because of the lack of effective curative pharmaceutical options and the increase in prevalence, extensive efforts have been devoted to the development of new strategies for cell replacement and tissue remodeling in copd. so far, most focus has been on mesenchymal stromal cell (msc) therapy. msc are theoretically ideal candidates for cell therapeutic approaches because of their low or absent constitutive hla class i and ii expression, allowing allogeneic administration of mscs obtained from normal healthy volunteers, and their immunosuppressive and antibacterial properties [ , ] . in this chapter, we will examine in detail the biological rationale for use of mscs in copd, clinical trials, and the current challenges for implementing this approach as a potential therapy for copd. copd is a progressive lower respiratory condition, which has a massive impact on public health worldwide. increasing in prevalence, copd is currently responsible for over , us deaths annually and is expected to become the third leading cause of death globally in the next few years [ ] . copd is most often associated with longterm smokers over the age of and is thought to be driven by abnormal tissue response(s) to inhaled toxic particles over time. the life expectancy of continuous cigarette smokers is at least years shorter than nonsmokers and the absolute risk of developing copd among this population has been estimated to be - % [ ] ; however, there is evidence for significant underdiagnosis [ , ] . the most common symptoms of copd are chronic bronchitis (persistent cough with chronic mucus production), dyspnea (shortness-of-breath), wheezing, and chest tightness. as a progressive disease, these symptoms get worse over time. current treatments, most importantly smoking cessation, are part of a delay strategy to slow down the physiological disease progression. these physiologic changes all contribute to the impairment of efficient breathing and include: the gradual loss-of-elasticity of the lung tissue leading to collapse of airway and alveolar sacs, weakening-to-rupture of alveolar septal walls, enlargement of segmented airspace, loss of gasexchange surface area, increased mucus production, airway plugging, and airway narrowing driven by swelling and fibrosis ( fig. . ). copd is a complex pathology with a diverse spectrum of clinical phenotypes, comorbidities, and treatment profiles [ , ] . the gold criteria have been widely utilized to help standardize the copd definitions and treatment guidelines; however, they do not fully encompass the diversity of copd phenotypes [ , ] . the treatments available to patients diagnosed with copd are not curative and cannot completely stop disease progression; however, indicate alveolar space (side-to-side alveolar wall distance). scale bars at × magnification represent μm and at × magnification μm they are key to slowing disease progression and importantly to improve quality of life. the most important intervention at any stage is the cessation of smoking and/or limitation of exposure to other identified environmental risk factors. symptomatic treatment throughout disease progression often relies on bronchodilators, which are inhaled beta-agonists or muscarinic antagonists. early-stage individuals will most often be treated with short-acting bronchodilator therapies (saba/sama); however, as the disease progresses treatment will need to incorporate long-acting drugs that affect these receptors (laba/lama). unfortunately, bronchodilators can only partially resolve lung hyperinflation in emphysema [ ] , becomingly increasingly less effective as the disease progresses. inhaled corticosteroids (ics), often used to treat acute respiratory exacerbations, work by interfering with the transcription pathways of key inflammation genes; however, this treatment does not always work and unfortunately can have little to no longterm benefits [ ] . in addition to direct toxic effects of cigarette smoke on lung epithelial cells, there is increasing appreciation that altered or aberrant immune cell signaling significantly contributes to much of the irreparable tissue damage. smokers with undiagnosed copd normally experience lowlevel infiltration of inflammatory cells into the large airways and peripheral lung parenchyma and have what is increasingly recognized as early disease. in individuals with diagnosed copd, the inflammatory process is amplified and prolonged leading to many of the tissue-remodeling events associated with chronic bronchitis and emphysema; hallmarks of copd [ ] . for example, in smoking-induced emphysema, chronically activated macrophages have been found to express upregulated levels of several proteinases and matrix metalloproteinases (mmps) in both human smokers and in mouse models of cigarette exposure [ ] . macrophages also play a crucial role in triggering the initial immune response in responding to smoking induced inflammation. alveolar macrophages are usually in a quiescent state and actually work to suppress the adaptive immune system in the healthy lung; however, in chronic inflammatory situations alveolar macrophages are the main source of proinflammatory amplification and play a significant role in causing an influx of other immune cells [ ] . ultimately, the disease progresses to a point where gas exchange is limited by the tissue damage and extent of hyperinflation. in many cases invasive surgical interventions are the only option; these include endobronchial valve insertion, bullectomy, lung volume reduction surgery, and lung transplantation [ ] . lung volume reduction surgeries can successfully address some issues with hyperinflation in selected patients by returning some of the mechanical advantage of normal breathing. however, invasive surgeries are associated with high morbidity and operative mortality [ ] [ ] [ ] , especially in late-stage copd patients who are often poor targets for surgical intervention. for some patients with end-stage copd, lung transplantation is the only option. however, this approach offers its own unique challenges including rejection risk, requirement for immunosuppression, and the limited supply of donor lungs. while transplanted lungs can certainly facilitate better gas exchange than severe copd lungs, the benefits are balanced by the risks, as the years survival of transplant recipients is only around % [ ] [ ] [ ] [ ] . at present, there are no true curative treatments that can stop the progression of copd, thus new therapeutic strategies are needed. advances in cell-based therapies provide a platform for development of new therapeutic approaches in copd. at this moment, much focus has been given to msc cell-based therapies, mainly because of their immunomodulatory properties. the promising results in animal models have translated into clinical trials for treatment of copd and emphysema. searching on the clinicaltrials.gov database for trials listed through november , using the keywords "copd" and "stromal cell"; "copd" and "mesenchymal stromal cell"; "copd" and "mesenchymal stem cell"; "emphysema" and "stromal cell"; "emphysema" and "mesenchymal stromal cell"; and "emphysema" and "mesenchymal stem cell", identified studies of human clinical trials. so far, four of the studies have been completed and had their results published in the pubmed database, four are still in the process of recruiting patients, three of them are active but not recruiting patients, three have an unknown status, and four of them have been withdrawn [ ] . this section will be focusing on the clinical studies that have been completed and for which results have been published (table . ). in , ribeiro-paes et al. conducted the first clinical investigation evaluating the safety of using bone marrow-derived mononuclear cells (bmmc) in four patients with advanced-stage copd (nct ). autologous bmmc were collected after days of granulocyte colony stimulating factor (g-csf) stimulation, and bmmc were isolated using ficoll-hypaque premium™. the cells were further resuspended in albumin saline solution (ass) at a final concentration of × mononuclear cells/ml, and intravenously administered directly to the patients without freezing or in vitro culture procedures. the patients were evaluated by several pulmonary function tests, including forced vital capacity (fvc), forced expiratory volume in s (fev ), and partial pressure of carbon dioxide (paco ). [ ] importantly, due to the small size of this study, lack of controls, and the lack of statistical analysis no clear conclusions can be drawn from these results. furthermore, the cells used in this study were heterogeneous mononuclear cells isolated from bone marrow aspirates, and not mscs, and therefore this study cannot be considered as the first msc study for treatment of copd patients. in , weiss et al. performed a prospective, randomized, double-blind, placebo-controlled industry-sponsored trial evaluating the safety and the efficacy of intravenous allogeneic mscs (nct ). the study enrolled patients ( - years of age), from six different centers, with moderate-to-severe copd (gold ii or iii). the patients were randomized into two groups, where the first group received non-hlamatched allogeneic mscs and the second group the infusions were well tolerated and no severe or fatal adverse events were observed during the msc or vehicle administration. no significant differences in fev , fvc, and total lung capacity were seen between the groups. nor were differences in -min walk test or dyspnea assessment observed between the two groups. for most of the circulating inflammatory cytokines no significant differences were seen between the msctreated patients and the vehicle group. however, a decrease in the crp level in patients treated with msc compared to their baseline crp levels was observed. the most important finding in this study was that msc administration was safe in an older population of patients with moderate-tosevere copd [ ] . stolk et al. performed a phase i, prospective, open-label study (nct ) where they aimed to assess the safety and feasibility of intravenously infused bone marrow-derived mscs for ten patients with severe emphysema that had serial lung volume reduction surgeries (lvrs). during the first lvrs bone marrow was aspirated. mscs were isolated from the bone marrow aspirates and expanded in vitro (passage - ) followed by cryopreservation. at three and four weeks prior to the second lvrs, mscs were intravenously administered to the patients at two different occasions. spirometry, gas transfer, lung volumes, and lung densitometry were evaluated at baseline and at the months follow-up. seven patients completed the full protocol. three patients were withdrawn from the study due to problem aspirating bone marrow, no msc growth, or persistent air leak after the first lvrs. no toxicity after the msc infusions was observed and the patients did not report any symptoms that were considered related to the treatment. at months follow-up, a significant increase in fev and body weight was observed compared to baseline levels. however, if changes in fev and body weight was due to msc administration or to the surgeries remain unknown, since this study protocol did not include a control group. importantly, no signs of increased pulmonary fibrosis were observed when lung tissue was evaluated by both histology and ct-derived lung density [ ] . de oliveira et al. combined msc administration with one-way endobronchial valve (ebv) insertion [ ] . this study was a prospective, patient-blinded, placebo (vehicle)-controlled, phase i study on ten patients with advanced heterogeneous emphysema (nct ). de oliveira et al. aimed to investigate the safety of combining ebv insertion with intrabronchial msc administration. the authors hypothesized that combining intrabronchial msc administration with ebv would reduce the inflammation, a common side effect of ebv placement. this study, however, was not designed to investigate msc as a treatment for copd, but rather specifically to investigate if msc treatment would enhance ebv placement by reducing the underlying inflammation. therefore, the secondary aim was to investigate if msc administration reduced the systemic inflammation. mononuclear cells (mncs) were isolated from ml bone marrow aspirate collected from the iliac crest of a single healthy donor using density-gradient centrifugation. mncs were cultured at a density of × cells per cm in iscove's modified dulbecco's medium supplemented with % fetal bovine serum, penicillin, and streptomycin at °c, % co for generation of mscs. mscs were immunophenotyped and samples were taken for microbiological and cytogenetic testing. mscs were harvested at passage three or four, diluted in saline solution, and placed in infusion bags. right before ebv insertion mscs (in ml saline) were administered to five of ten patients using a video bronchoscope with a . -mm instrument channel. the patients in the vehicle group received saline. in both groups, the infusions were performed in the region where the ebvs were supposed to be placed (the segmental or subsegmental bronchus of all branches of the target lobe). immediately after the msc administration or vehicle administration and ebv insertion, a chest radiograph was performed to confirm the ebv placement. for the following days, the patients were evaluated for body temperature, blood pressure, oxygen saturation, heart, and respiratory rates. arterial blood gas, complete blood count, urea, creatinine, glucose, and electrolytes were evaluated at day , , , , and . chest ct scans were performed at day , , and . circulating levels of inflammatory cytokines were assessed in serial blood samples obtained throughout the study period. efficacy was evaluated as improvement from baseline in fev , fvc, fev /fvc, total lung capacity, single-breath carbon monoxide diffusing capacity, the body mass index, airway obstruction, dyspnea, exercise index, and health-related quality of life (st. george's respiratory questionnaire). all ten patients completed the full protocol. the msc administration was well tolerated and all patients tolerated the ebv insertion but one, who developed pneumonia, pneumothorax, empyema, and respiratory failure. no severe adverse events were seen in the group receiving msc, but % in the msc group and % in the placebo group experienced adverse events during the study period, and importantly none of the adverse events was reported to be related to the msc administration. no difference in toxicological or lung function parameters such as fev , fvc, and total lung capacity were observed between the groups. in accordance with data reported by weiss et al. [ ] the msc treated group had significantly reduced levels in crp at day and post administration. patients receiving msc infusions were reported to have a significant decrease in the st. george's respiratory questionnaire scores compared to the placebo group at day post administration. the authors concluded that intrabronchial msc administration in combination with ebv insertion appears to be safe in patients with severe heterogeneous emphysema. furthermore, in this study msc administration tended towards decreased circulating crp levels; however, due to the low number of recruited patients and the limited follow-up period it was not possible to evaluate if msc treatment altered the efficiency of the ebv placement or the subsequent clinical copd course. [ ] . finally, armitage et al. recently published a single site, phase i study that was not listed at the nih clinicaltrials.gov database, rather only in the australian clinical trials registry (number ), which aimed to investigate the distribution of intravenously infused mscs into copd patients. nine patients with mild-tosevere copd (gold i-iv) received infusion of low passage allogenic bone marrow-derived mscs radiolabeled with indium- , followed by a second infusion of unlabeled mscs one week post the first administration. in similarity with the other clinical trials, all patients tolerated the msc infusions well and no infusional or short-term adverse effects were reported. following the first infusion, labeled mscs were detected in the lungs within min by computed tomography (ct) scan, and remained detectible h after the infusion. after h, indium- was detected in spleen, liver, and bone marrow up to days after infusion. moreover, h after the first infusion the patients were assessed by single-photon emission computed tomography (spect) to evaluate msc localization within the lungs. furthermore, the amount of indium- positively correlated with the baseline fev and the diffusing capacity of the lung for carbon monoxide. in addition, this study further aimed to investigate systemic inflammation following the msc infusion. the authors were not able to detect il- beta, il- , il- p , or il- a; however, increased circulating levels of crp were detected at h and up to days after msc administration. interestingly, this study suggests that msc infusion shifted the balance towards a more anti-inflammatory profile, as the number of circulating regulatory t-cells were increased days after msc administration and the proportion of dendritic cells were altered, favoring plasmacytoid dendritic cells [ ] . current clinical trials that aimed to evaluate the effect of msc administration in copd patients differ in a wide range of factors such as routes of administration, number of msc administered, number of administrations, use of fresh mscs or culture-expanded mscs. furthermore, all the investigations discussed above, were phase i-ii studies that were underpowered in order to detect potential efficacy and no improved pulmonary function or respiratory quality of life was observed. although the primary end-point was safety and all studies reported that msc administration was well tolerated and no toxicity was observed, further studies, both clinical and preclinical, are needed to better understand potential therapeutic efficacy of mscs in copd. despite increasing number of preclinical studies demonstrating that msc administration could prevent or treat experimental copd and emphysema [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , clinical studies have not been able to reproduce the preclinical results, and to date no efficacy or significantly improved pulmonary function in copd patients have been observed. in this section, we will be discussing some of the challenges in the field and the hurdles that need to be overcome in order to improve the efficacy of msc therapy in copd [ ] . mscs are known to be a heterogeneous cell population [ , ] , containing subpopulations that have been demonstrated to be functionally different from each other [ , ] . many of the phenotypic and functional differences depend on differences in culture conditions, individual donors, the harvest site, and the tissue source [ ] [ ] [ ] [ ] . this makes it difficult to compare results between different studies, both preclinical and clinical, and importantly it hinders progression in the field. efforts should therefore be concentrated on developing standardized msc isolation methods and culture conditions. in , the international society for cellular therapy published a position paper in order to address this issue. in this article, they defined minimal suggested criteria for cultured human mscs [ ] . since this position paper by dominici et al. was published it has been updated once in [ ] , but the msc field has advanced and today mscs are isolated from different organs and tissues and therefore these minimal criteria urgently need to be modified and updated. to date, bone marrow-derived mscs are the most widely investigated, but preclinical studies have demonstrated that mscs with immuneregulatory and regenerative properties can be isolated from other tissues such as adipose tissue, umbilical cord, and lung [ , [ ] [ ] [ ] [ ] [ ] [ ] . a large body of data demonstrates that mscs execute their therapeutic effects through a spectrum of paracrine activities, and interestingly preclinical data suggest that mscs isolated from different tissues have different secretome profiles [ , ] . it is also important to realize that primary mscs change phenotype when they are isolated from their native tissue and plated on a plastic culture dish [ ] . the bona-fide msc, which are thought to be small and quiescent, noncycling cells in vivo, changes phenotype into a spindle-shaped and active proliferating and secretory cell in culture [ , ] . at early passages, mscs have a high proliferation rate but as their time in culture progresses their proliferation rate declines and they finally enter a senescence stage [ ] [ ] [ ] . also the morphology is changed during culture expansion, mscs in early passages have a thin spindle-shaped morphology, but at higher passages mscs tend to become larger and more flattened cells with an irregular shape [ , ] . moreover, mscs have been reported to accumulate dna damage during in vitro expansion, which could potentially lead to tumorigenesis upon implantation [ , ] . importantly, tumor development was not observed in any of the clinical studies using msc as treatment for copd and/or emphysema patients, although longer follow-up is necessary [ ] [ ] [ ] [ ] . furthermore, the biological properties of mscs can also be strongly influenced by the cell culture medium. cell culture media are often supplemented with serum, and most often fetal bovine serum (fbs) is used. the use of fbs has several disadvantages, especially in clinical settings. first, the possibility of contamination with pathogens such as prions and viruses and the potential immune reaction to bovine proteins. second, lot-to-lot variation between different fbs batches might induce differences in msc behavior such as proliferation rates and differentiation potential, and make it difficult to standardize methods and reproducibility of results [ , , ] . human platelet lysate (hpl) is as an alternative to fbs in clinical settings. hpl has the advantages of containing non-animal products and therefore no risk of xenogeneic infections and immune rejection. on the other hand, hpl is a human product and has the potential to transmit human diseases such as hepatitis b and c, and human immune deficiency virus (hiv). in similarity with fbs, hpl also brings the disadvantage of having lot-to-lot variation [ , ] . a third option would be to use serum-free cell or synthetic culture media. these media are highly promising, but more studies are needed in order to have evaluate their utility for producing clinical grade-mscs. recently, lensch et al. demonstrated that mscs had a higher proliferation rate when growing in xeno-free medium which resulted in a greater viable cell yield compared to standard fbs containing culture medium [ ] . nevertheless, further studies are needed in order to evaluate if the in vivo biological properties of msc are altered when expanded in the in vitro setting. another factor that may influence the biological function of mscs is the freezing and thawing of cells before administrated to patients. the current model for allogeneic msc use is to expand cells on plastic culture dishes following harvest and isolation from bone marrow or other source and cryopreserve the cells until usage. when it is time for administration, cells are thawed, washed, and directly administered to the patients most commonly through intravenous infusions [ ] . however, a number of studies have demonstrated that mscs that have been freeze-thawed have impaired functional properties. francois et al. reported that cryopreserved mscs had impaired immunosuppressive properties [ ] . in accordance with these results, moll et al. published an article where they demonstrated that freezethawed mscs had a reduced responsiveness to proinflammatory stimuli and an impaired production of anti-inflammatory mediators [ ] . minor effects on gene expression of freeze-thawed mscs compared to continuously cultured mscs have been observed. however, the alterations in gene expression between different donors were larger than the effects of cryopreservation [ ] . although there is a practical need for expanding and cryo-banking cells for therapeutic use [ ] , most preclinical studies have been performed using log phase of growth msc. there are studies, some of them discussed above, which demonstrated that freeze-thawing procedure alters the biological properties of msc. francois et al. found that during the thawing process a heat-shock stress response was initiated that was associated with the impaired immunosuppressive properties of msc. interestingly, this response was reversible and cells were recovered after h of culture [ ] . these results imply that cryopreservation and banking of cells might be possible, as long as the cells are allowed to recover in culture before use. the study by cruz et al. further supports the potential of using freeze-thawed cells for clinical trials. in this study, the authors compared the therapeutic effect of continuously cultured versus freshly thawed bone marrow-derived mscs in an aspergillus hyphal extract (ahe) exposed asthma mouse model, and found no difference in therapeutic effect between the two groups [ ] . utilizing plastic culture dishes are by far the most traditional way of cultivating and expanding msc; however, alternative culture systems have been developed that might mimic the in vivo situation more compared to the more traditional d cultivation on plastic. the use of alternative threedimensional cell culture systems can hopefully contribute to narrowing the gap between preclinical and clinical research. different groups have studied the possibility to grow mscs on plastic culture dishes coated with extracellular matrix molecules (ecm) such as collagen and fibronectin [ , ] . ecm is a three-dimensional network composed of noncellular structures that play an important role within the lung, not only by providing structural support and adding stability but also as a bioactive environment that can influence cellular responses [ ] . engler et al. demonstrated that changing the elasticity of the ecm that mscs were grown on significantly affected the msc phenotype. mscs grown on a stiffer ecm differentiated towards the osteoblast lineage, whereas mscs grown on a softer ecm differentiated towards the adipocyte lineage [ ] . the msc differentiation potential could also be altered by changing the cross-linking of the collagen fibers [ ] . in addition, modifications of the geometric shape, cell density, and cell size have been implicated in the differentiation potential of msc [ , ] . interestingly, mcmurray et al. developed a nanoscale surface that maintained the phenotype and multilineage potential of longterm cultured mscs [ ] . how the ecm environment affects the msc therapeutic behavior, especially in a fibrotic or emphysematous copd lung, is currently a largely untouched area that will most likely play a pivotal role in the development of successful msc-based therapies. a different approach of the three-dimensional cultures is the usage of the hanging drop model. in conformity with primary mscs, culturing mscs using the hanging drop method resulted in nondividing cells [ ] , but an increased potential to differentiate towards osteoblast and adipocyte lineages was also demonstrated [ ] . another strategy that has been used for msc expansion relies on culturing mscs in d scaffolds (decellularized lung tissue or synthetic scaffolds) [ ] [ ] [ ] . in this system, cultivation on a plastic surface could be avoided, but a perfusion-based bioreactor system is required [ ] . studies have shown that mscs cultured in lung ecm hydrogels have enhanced viability and increased expression of sox and oct compared to cells grown on plastic [ ] . furthermore, changes in secretion of cytokines including il- ra, vegf, g-csf, fgf, and hgf have been demonstrated in mscs grown in d culture compared to d [ , ] . taken together, the traditional way of cultivating mscs as monolayer on a plastic surface may result in mscs with a different phenotype compared to mscs expanded in three-dimensional culture systems. however, whether cultivating mscs on ecm coating, in scaffolds, or in hanging drops increases the beneficial effects when used for clinical settings remains to be evaluated and further studies are needed. it is well known that oxygen levels can affect cell functions, such as differentiation, cytokine production, and proliferation [ ] [ ] [ ] [ ] . furthermore, it is also known that different adult tissues experience a wide range of oxygen levels [ ] and that severe pathological inflammation can cause hypoxia, reduced ph, and oxidative stress [ , ] . nevertheless, mscs tend to be cultured at atmospheric oxygen levels ( - % o ) which do not reflect the microenvironment they normally reside in, or the microenvironment they will encounter when administered into the diseased lung [ ] . culturing mscs at oxygen levels that more closely represent their in vivo situation have a huge impact on msc behaviors. lennon et al. observed that mscs grown at lower oxygen levels had a greater number of colony-forming cells and proliferated at a higher rate compared to mscs grown at higher oxygen levels. also, lennon et al. demonstrated that mscs cultured at % oxygen formed more bone structures in vivo, compared to mscs grown in % oxygen [ ] . moreover, adipose-derived mscs grown at low oxygen levels, secreted higher levels of cytokines such as vegf and fgf compared to cells cultured at % oxygen [ ] . combining the low oxygen condition with growing the mscs in d cultures has been shown to increase the expression of pluripotent genes such as oct- , sox- , nanog, and rex- compared to control [ , ] . beegle et al. reported that mscs pretreated with hypoxia before administration enhanced survival rate and cell retention compared to cell grown at % oxygen. taken together, these studies emphasize the importance of understanding the effects of differences in protocols, culture conditions, and oxygen levels in the context of culturing mscs for clinical trials for copd where you have gasexchange impairment, active immune response, and inflammation. despite an enormous interest in using mscs for clinical settings, the exact in vivo function is not understood, especially not within the lung. a compelling amount of data now points towards that mscs act by paracrine mechanisms rather than through engraftment [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . tracking studies of intravenous injected mscs reveal that most of the injected cells disappear after h [ , , , ] , and since mscs do not engraft it is unlikely that mscs can remodel injured tissue by differentiating into other cell types. the mechanisms by which mscs are the most likely to be involved in copd and emphysema are discussed below. immunomodulation through paracrine actions is one of the main mechanisms of actions of mscs and involves both the innate and the adaptive immune system [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (fig. . ) . these effects include inhibition of t-cell [ , ] and b-cell proliferation [ ] , macrophage polarization [ ] , and differentiation of t-cells towards t-regulatory cells [ ] [ ] [ ] . the paracrine actions have been associated with several mediators such as hepatocyte growth factor (hgf), transforming growth factor beta (tgfβ), prostaglandin (pge ), il- , ifn-gamma, tnf-stimulated gene (tsg ), and indoleamine , -dioxygenase (ido) [ , , , , ] . in addition to the paracrine immunomodulatory effects, mscs might activate the immune system by recognition of the immune cells. as mentioned, mscs are rapidly cleared from the lung after infusion, which was recently demonstrated to be mainly through phagocytosis by monocytes [ ] . the recognition of mscs by monocytes results in a polarization of monocytes/macrophages towards an immuno- suppressive phenotype that results in an immunomodulatory response [ , ] . similar results have also been demonstrated with heatinactivated mscs, suggesting that mscs also can act in a passive immunomodulatory manner [ ] . however the potency of apoptotic mscs are controversial, apoptotic mscs have been demonstrated to be completely ineffective when injected intravenously in mice [ ] . msc are also known to secrete antimicrobial proteins and polypeptides that are molecules responsible for bacterial killing. mscs secrete the antimicrobial peptide, ll- , following eschericia coli. stimulation, which was subsequently found to be responsible for the antimicrobial activity in a model of e. coli pneumonia [ ] . in addition to its antimicrobial activities, ll- can also play an important role in inflammatory and immune modulatory actions [ , ] . a growing body of data suggests that mscs can form links with other cells, and that they have the potential to transfer components such as mitochondria [ ] [ ] [ ] [ ] . through mitochondrial transfer msc have been demonstrated to be able to rescue epithelial cells with defective mitochondria [ ] . the mitochondria transfer is thought to be via direct transfer by microtubules and tunneling nanotubes (tnt) [ , ] . mscs can also transfer mitochondria to macrophages resulting in an increased phagocytic activity [ ] . mitochondrial biogenesis is regulated by extracellular stimuli [ ] and several lung diseases are associated with impaired mitochondrial biogenesis and dysfunctional mitochondria [ , ] . however, beyond the mitochondria-derived reactive oxygen species (ros), the contribution of mitochondria in the development of copd is still under investigation [ ] . in addition to mitochondria transfer through microtubules and tnt, mitochondria can also be transported via extracellular vesicles (ev) [ , [ ] [ ] [ ] . it is also becoming increasingly clear that msc-derived evs can influence the behavior of surrounding inflammatory and structural cells. for example, evs released from mscs can stimulate bronchial epithelial cells and alveolar cells to secrete proinflammatory cytokines [ , ] . furthermore, msc-derived evs suppress the potential of lung fibroblasts to differentiate towards myofibroblasts [ ] . it is not only mitochondria that could be transferred by msc-derived evs, also other components such as microrna, proteins, lipids, dna, and mrna [ , ] . evs are taken up by other cells, and evs derived from mscs have been demonstrated to impact immune cells. evs isolated from il-beta pretreated mscs induced macrophage polarization towards the anti-inflammatory phenotype (m ) [ ] . mscderived evs have also been associated with inhibition of t-cell proliferation, inducing apoptosis of activated t-cells and promotion of regulatory t-cells [ ] . msc-derived evs have been tested in experimental copd models, but further studies are needed [ ] . it is now widely accepted that, following in vivo delivery, culture-derived mscs respond to the microenvironment they encounter, which in copd and emphysema could encompass everything from massive inflammatory environment to emphysematous tissue destruction. therefore, it is important to consider several important aspects of the msc preparation and administration used today. the route by which mscs are delivered into the patients most likely plays an important role in the msc potential function. despite the fact that several clinical trials has been performed using mscs for severe lung disorders [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , the best route of administration have not been determined. in preclinical studies, two main administration routes have been evaluated: systemic administration [ , , , , , , , , ] and local administration [ , - , , ] . copd is a systemic disease and therefore systemic administration might be better suitable for these patients. in addition, systemic administration is less invasive and has less contamination risks compared to local administration [ ] . not only has the route of administration been different in the different studies conducted to date but also the number of cells administered with each injection and whether single or multiple injections were administered during the trial. according to antunes et al. a wide range of msc doses in preclinical settings have been used, from up to × [ ] . so far, bone marrow-derived mscs are the most frequently used cell source for msc-treatments, especially when used in human clinical trials. however, mscs derived from other sources such as adipose-derived, umbilical cord-derived, lungderived, and amniotic fluid-derived mscs have been evaluated for treatment of copd/emphysema models [ , , , ] . since it is known that the environment affects msc function and viability, several preconditioning strategies have been tested. some researchers have been focusing on the effect of the inflammatory environment and the cytokines that may be encountered in the diseased lung [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . krampera et al. reported that mscs stimulated with ifn-gamma, increased the levels of ido produced and secreted by mscs, leading to an increased suppressive effect on t-lymphocyte proliferation. moreover, the authors were able to demonstrate that the inhibitory effects of mscs on t-lymphocyte proliferation were completely abolished when adding an ifn-gamma blocking antibody to the culture system [ ] . in an ifngamma knock out mouse model, polchert et al. were able to demonstrate that endogenous ifngamma was required to initiate msc efficacy. however, after pretreatment of mscs with high doses of ifn-gamma they immediately became active [ ] . also pre-stimulating mscs with a combination of inflammatory cytokines has been explored [ , ] . another interesting approach to mimic the microenvironment is to utilize patient samples such as serum and bronchoalveolar lavage (bal) fluid from patients and pre-stimulate cells with such prior to the administration [ , ] . moreover, attempts to improve the beneficial effects of mscs have utilized treatment with the toll-like receptor- ligand (poly(i:c)). the authors found that mscs pretreated with poly(i:c) had improved immunosuppressive properties, an effect that was inhibited by addition of the microrna mir- [ ] . in addition to the inflammatory environ-ment, others have studied the effect of pretreating mscs with hypoxia and nutrient deficiency. during culture under hypoxic conditions, mscs have been shown to have decreased expression of senescence-associated beta-galactosidase and an increase in the expression of anti-apoptotic proteins such as bcl- and bcl-xl [ , ] . by exposing mscs to hypoxia the hypothesis is that the cells will adapt to the ischemic environment with oxidative stress, an environment they likely will encounter in the copd lung. this might potentially enhance the time that mscs can survive and exert their therapeutic paracrine actions in the recipient lung. a different way of increasing the therapeutic effect by mscs is to genetically manipulate the cells prior to administration [ ] [ ] [ ] . for example, jiang et al. demonstrated that after co-overexpressing the genes ang- and akt in mscs, an increased cell survival and improved angiomyogenesis was observed in an experimental model of acute myocardial infarction [ ] . in lung, mscs overexpressing ang- have been demonstrated to more potently decrease lpsinduced pulmonary inflammation and proinflammatory cytokine release into the bal fluid [ ] . in another study by mcginley et al., overexpression of heat shock protein (hsp ) in mscs led to decreased apoptosis and improved cardiac function [ ] . overexpression of manganese superoxide dismutase in adipose-derived mscs, a gene strongly upregulated during hypoxia, increased the time that the mscs were detectable in a matrigel plug implanted into a mouse model [ ] . moreover, he et al. transduced mscs with angiotensin-converting enzyme (ace ), an enzyme that degrades angiotensin ii and had previously been demonstrated to have a protective role against acute lung injury. the ace transduced mscs were demonstrated to reduce pulmonary vascular permeability, normalize the expression of enos, and improve the endothelial barrier integrity, when infused into an ali-mouse model. furthermore, the ace overexpressing mscs also displayed an improvement in the suppression of the inflammatory response [ ] . combination of different treatments could be another approach to enhance msc efficacy. this approach was used in two of the clinical trials discussed above. stolk et al. combined msc treatment with lung volume reduction surgery and de oliveira et al. with a one-way endobronchial valve insertion [ , ] . an alternative could be to pretreat the recipient tissue with pharmacological drugs in order to make the recipient site more accessible to the infused cells [ , [ ] [ ] [ ] . in a cardiac disease model, pharmacological pretreatment of a vasodilator drug in the recipient site of transplantation resulted in an enhanced delivery of mscs [ ] . in a clinical trial using mscs for treatment of chronic heart failure, the administration site was treated with a shock wave prior to the administration of the cells. in the group receiving both the shock wave pretreatment and the msc infusion, the overall occurrence of major adverse cardiac events were significantly decreased compared to the control groups [ ] .these are all important observations for potential cell-based therapies for lung diseases and should be investigated further. the lack of translating the encouraging preclinical data into clinically relevant effects in patients with copd and emphysema brings up the following question: is copd the most suitable pulmonary disease for msc-based treatment? the animal models of copd and emphysema used in the preclinical studies were optimized to detect the maximum therapeutic effects [ ] , and might therefore not reflect the in vivo situation that mscs encounter when infused into patients with copd and/or emphysema. copd is characterized by tissue damage, structural changes, and inflammation, and as mentioned it is a heterogeneous disease with different degrees of fibrosis and emphysema [ ] . copd patients with different phenotypes might respond differently to msc administration [ ] , and choosing patients that are more likely to respond to the treatment could be one way to improve the clinical outcome. another possible way to improve the outcome could be the timing of the treatment. in animal studies, mscs are frequently administered to the animals in close proximity to the induction of the disease [ , , , ] or even at the same time or prior to the disease induction [ , ] . based on these preclinical findings, mscs might be more beneficial earlier in the disease than in later stages of the disease. however, copd patients tend not to seek medical attention in early stage of the disease [ ] . one way to foresee which patients would most likely respond to the treatment could be to develop in vitro potency assays. even if it is widely accepted that the therapeutic effect of mscs is mainly mediated by paracrine effects, the exact mechanism of action is not determined. this makes it difficult to develop one single analytic or biological assay, and most likely, a combination of evaluating different mechanisms would be needed [ , ] . another potential way would be to develop biomarkers to indicate which patients have an active disease and therefore might benefit more from a msc-based therapy. to date, several potential biomarkers, including circulating fragments of ecm proteins, have been shown to be increased in copd patients with an active disease e.g. in relation to acute exacerbations [ ] [ ] [ ] . finally, broekman et al. suggested that in addition to the optimization of msc-treatment and potency assays, challenges such as improved outcome parameters needs to be addressed [ ] . in parallel with the growing interest of cellbased therapies for copd and other lung diseases, an increased market for commercial stem cell therapies has developed both in the usa and globally [ ] . this very unfortunate and problematic outcome might partly be due to an increased visibility to desperate patients through the internet and open social media channels [ ] . these unproven and often unsafe stem cell treatments can create a situation in which desperate patients easily can be misled into participating in very expensive treatments, which are not covered by insurance. furthermore, the providers at the stem cell clinics often fail to prove safety and efficacy of their treatments failing to fulfill recognized biological and medical standards, exposing the patients to unnecessary risks and leaving the patient and their family with dashed hopes [ , ] . these stem cell clinics have the potential to harm even more patients and their families, as well as bring the field into disrepute and hamper the progression of safe and effective msc-based therapies. therefore, organizations such as the international society for stem cell research (isscr) and the international society for cell and gene therapies (isct) have taken stances against these unethical cell-therapy clinics. also, the food and drug administration (fda) is beginning to take actions against the stem cell tourism [ , ] . in a review by dominici et al., the authors discuss the importance of having proper communication between different players such as medical doctors, industry, patient organizations, and patients, in order to enhance credibility and patient welfare [ ] . in an attempt to begin proactively addressing this issue, the american thoracic society (ats) respiratory cell and molecular biology assembly stem cell working group posted a statement online and several other related publications [ , [ ] [ ] [ ] [ ] . this statement will help to translate new scientific findings into patient education in an unbiased way and to make the public aware of the limitations and potential risks associated with such therapeutic approaches [ , ] . however, it is not only the patients that need education, many pulmonologists are also not familiar with the stem cell field, and the ats respiratory cell and molecular biology assembly stem cell working group has developed educational resources for this audience also [ ] . msc-based therapy for treatment of copd and emphysema has demonstrated promising results in animal models; however, this has not translated into clinically relevant effects in patients to date. current clinical trials have failed to demonstrate efficacy and improved lung function, but importantly they have uniformly demonstrated the msc administration to be safe. the challenges ahead for this field are to standardize the isolation and culture conditions in order to have a cell product with high quality and reproducibility, to select the proper subpopulation of patients that is most likely to respond to the cell treatment, to develop appropriate potency assays, and to improve or develop new methods to measure outcomes. furthermore, the usage of cell-free products such as evs and conditioned medium, or pretreating mscs prior to administration has demonstrated promising results. however, there is still a long way to go and many challenges are ahead before we have an optimal msc-based 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inhibit neutrophil apoptosis: a model for neutrophil preservation in the bone marrow niche a new mesenchymal stem cell (msc) paradigm: polarization into a pro-inflammatory msc or an immunosuppressive msc phenotype mesenchymal-stem-cell-induced immunoregulation involves fas-ligand-/fas-mediated t cell apoptosis interaction of human mesenchymal stem cells with cells involved in alloantigen-specific immune response favors the differentiation of cd + t-cell subsets expressing a regulatory/suppressive phenotype cell contact, prostaglandin e( ) and transforming growth factor beta play non-redundant roles in human mesenchymal stem cell induction of cd +cd (high) forkhead box p + regulatory t cells bone marrow stromal cells attenuate sepsis via prostaglandin e( )-dependent reprogramming of host macrophages to increase their interleukin- production anti-inflammatory protein tsg- secreted by activated mscs attenuates zymosan-induced mouse peritonitis by decreasing tlr /nf-kappab signaling in resident macrophages immunomodulation by therapeutic mesenchymal stromal cells (msc) is triggered through phagocytosis of msc by monocytic cells mesenchymal stem cells induce suppressive macrophages through phagocytosis in a mouse model of asthma inactivated mesenchymal stem cells maintain immunomodulatory capacity mesenchymal stromal cells: clinical challenges and therapeutic opportunities antibacterial effect of human mesenchymal stem cells is mediated in part from secretion of the antimicrobial peptide ll- antimicrobial cathelicidin peptide ll- inhibits the lps/atp-induced pyroptosis of macrophages by dual mechanism stem cells, cell therapies, and bioengineering in lung biology and diseases. comprehensive review of the recent literature - mitochondrial transfer between cells can rescue aerobic respiration cell-tocell cross-talk between mesenchymal stem cells and cardiomyocytes in co-culture mechanisms of mesenchymal stem/stromal cell function characterization of intercellular communication and mitochondrial donation by mesenchymal stromal cells derived from the human lung mitochondrial transfer via tunneling nanotubes is an important mechanism by which mesenchymal stem cells enhance macrophage phagocytosis in the in vitro and in vivo models of ards mitochondria in mesenchymal stem cell biology and cell therapy: from cellular differentiation to mitochondrial transfer mitochondrial dysfunction increases allergic airway inflammation association between mitochondrial dysfunction and severity and outcome of septic shock mitochondria in lung diseases intra-and intercellular quality control mechanisms of mitochondria mitochondrial transfer from bone-marrow-derived stromal cells to pulmonary alveoli protects against acute lung injury mesenchymal stem cells use extracellular vesicles to outsource mitophagy and shuttle micrornas monocyte/macrophage-derived microparticles up-regulate inflammatory mediator synthesis by human airway epithelial cells cd -mediated adhesion is required for the induction of a proinflammatory phenotype in lung epithelial cells by mononuclear cell-derived extracellular vesicles thy- dependent uptake of mesenchymal stem cell-derived extracellular vesicles blocks myofibroblastic differentiation extracellular vesicle-shuttled mrna in mesenchymal stem cell communication biological properties of extracellular vesicles and their physiological functions exosomal mir- a contributes to the enhanced therapeutic efficacy of interleukin- beta-primed mesenchymal stem cells against sepsis microvesicles derived from mesenchymal stem cells: potent organelles for induction of tolerogenic signaling adipose stem cell-derived nanovesicles inhibit emphysema primarily via an fgf -dependent pathway treatment with allogeneic mesenchymal stromal cells for moderate to severe acute respiratory distress syndrome (start study): a randomised phase a safety trial a prospective, non-randomized, no placebo-controlled, phase ib clinical trial to 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interleukin (il)- and il- rn levels serum from asthmatic mice potentiates the therapeutic effects of mesenchymal stromal cells in experimental allergic asthma the toll-like receptor ligand, poly(i:c), improves immunosuppressive function and therapeutic effect of mesenchymal stem cells on sepsis via inhibiting mir- transplantation of hypoxia-preconditioned mesenchymal stem cells improves infarcted heart function via enhanced survival of implanted cells and angiogenesis reduced oxygen tension attenuates differentiation capacity of human mesenchymal stem cells and prolongs their lifespan mesenchymal stem cells modified with akt prevent remodeling and restore performance of infarcted hearts hepatocyte growth factor-modified mesenchymal stem cells improve ischemia/reperfusioninduced acute lung injury in rats transfection of mesenchymal stem cells with the fgf- gene improves their survival under hypoxic conditions supportive interaction between cell survival signaling and angiocompetent factors enhances donor cell survival and promotes angiomyogenesis for cardiac repair prevention of lps-induced acute lung injury in mice by mesenchymal stem cells overexpressing angiopoietin mesenchymal stem cell survival in the infarcted heart is enhanced by lentivirus vector-mediated heat shock protein expression promotion of survival and engraftment of transplanted adipose tissue-derived stromal and vascular cells by overexpression of manganese superoxide dismutase mesenchymal stem cells overexpressing angiotensin-converting enzyme rescue lipopolysaccharide-induced lung injury prostacyclin improves transcoronary myocardial delivery of adipose tissue-derived stromal cells effect of shock wave-facilitated intracoronary cell therapy on lvef in patients with chronic heart failure: the cellwave randomized clinical trial angiogenic pretreatment improves the efficacy of cellular cardiomyoplasty performed with fetal cardiomyocyte implantation mesenchymal stromal cells: a novel therapy for the treatment of chronic obstructive pulmonary disease? thorax extracellular matrix remodelling in copd understanding copd: a vision on phenotypes, comorbidities and treatment approach copd: early diagnosis and treatment to slow disease progression international society for cellular therapy perspective on immune functional assays for mesenchymal stromal cells as potency release criterion for advanced phase clinical trials the challenge of defining mesenchymal stromal cell potency assays and their potential use as release criteria characterization of serological neo-epitope biomarkers reflecting collagen remodeling in clinically stable chronic obstructive pulmonary disease high levels of biomarkers of collagen remodeling are associated with increased mortality in copd -results from the eclipse study accelerated extracellular matrix turnover during exacerbations of copd positioning a scientific community on unproven cellular therapies: the international society for cellular therapy perspective science, ethics and communication remain essential for the success of cell-based therapies unproven stem cell treatments for lung disease-an emerging public health problem balancing safety and innovation for cell-based regenerative medicine statement on unproven stem cell interventions for lung diseases medical societies, patient education initiatives, public debate and marketing of unproven stem cell interventions co-opting of clinicaltrials. gov by patient-funded studies the global emergence of unregulated stem cell treatments for respiratory diseases. professional societies need to act key: cord- -ms t fx authors: pietzka, sebastian; kämmerer, peer w.; pietzka, silke; schramm, alexander; lampl, lorenz; lefering, rolf; bieler, dan; kulla, martin title: maxillofacial injuries in severely injured patients after road traffic accidents—a retrospective evaluation of the traumaregister dgu® – date: - - journal: clin oral investig doi: . /s - - - sha: doc_id: cord_uid: ms t fx objectives: it was the aim of the study to analyse the prevalence of maxillofacial trauma (mft) in severely injured patients after road traffic accidence (rta) and to investigate associated factors. materials and methods: in a retrospective study, data from patients after rta by the traumaregister dgu® from to were evaluated for demographical and injury characteristics. the predictor variable was mechanism of injury and the outcome variables were type of injury, severity and hospital resources utilization. results: during the investigation period, n = , patients were enclosed with a prevalence of maxillofacial injuries of . % (mft positive). the injury severity score of mft-positive patients was higher than in the mtf-negative subgroup ( ± . vs. . ± . ). if mft positive, . % show minor, . % moderate, . % serious and . % severe maxillofacial injuries. injuries of the midface occurred in . % of mtf-positive patients. a relevant blood loss (> % of total blood volume) occurred in . %. mft-positive patients had a higher coincidence with cervical spine fractures ( . % vs. . %) and traumatic brain injuries ( . % vs. . %) than mft-negative patients. there was a noticeable decrease in the incidence of facial injuries in car/truck drivers during the study period. conclusions: every th patient after rta shows a mft and the whole trauma team must be aware that this indicates a high prevalence of traumatic brain and cervical spine injuries. clinical relevance: even if sole injuries of the face are seldom life threatening, maxillofacial expertise in interdisciplinary trauma centres is strongly recommended. epidemiology of facial trauma was reported to vary across the world and depends on social and economic differences [ , ] . traffic accidents contribute to the worldwide deaths in a significant way also leading to moderate as well as serious injuries requiring hospitalization [ ] . even if the introduction of seat belts and the improvement of safety systems led to a significant reduction of injury severity and death after traffic accidence, they are still one of the major causes for maxillofacial injuries in adults [ , ] . those injuries may result in a loss of function, disfiguration, psychological problems or even disability and death [ ] . the assessment of facial injuries is often perceived as difficult by the ambulance. in particular, this is due to the fact that injuries in the maxillofacial area offer special features and that there is often a lack of subject-specific competence [ ] . nevertheless, esmer et al. found evidence that about % of all severely injured patients are suffering from maxillofacial injuries as well [ ] . these injuries can be less severe, such as injuries to soft tissue or teeth, or life-threatening, such as bleedings from major arteries [ ] . even in primarily isolated maxillofacial injuries, energy may inevitably be transmitted to other anatomical structures of the head and neck, which can also be involved in a variety of pathological lesions such as spine injury [ ] . this impact which is focused toward the body resulting in multiple injuries which varies in severity may be increased in cases of polytraumatised patients after traffic accidents. in order to develop and implement effective interventions to reduce the medical burden, a better understanding of maxillofacial injuries in severely injured patients after traffic accidents is needed. though, as the causes of maxillofacial injuries have been shown to vary not only from one country to another but even within the same country depending on the prevailing socioeconomic, cultural, and environmental factors [ ] , studies with large case numbers are needed in order to allow general conclusions. therefore, the aims of this study on a large cohort of affected patients were to describe the patterns of the respective injuries and to identify those factors associated with road traffic accidents. as secondary parameter, changes within the patterns of injury within a -year period were examined. in this retrospective study, data provided by the traumaregister dgu® were analysed following the strobe recommendations [ ] . the study has received a positive vote from the ethics committee of the medical school of the university of ulm, germany (from th of december ), and was performed in compliance with the helsinki declaration (october ). the traumaregister dgu® of the german trauma society (tr-dgu) was founded in . the aim of this multi-centre database is the pseudonymised and standardised documentation of care for severely injured patients. data are collected prospectively from the site of the accident until discharge from hospital. included are patients who are admitted to hospital via emergency room and subsequently receive intensive or intermediate care and patients who arrive at hospital with vital signs and die before admission to the intensive care unit. the infrastructure for documentation, data management and data analysis is provided by the academy for trauma surgery, which is affiliated to the german trauma society. scientific data analysis is approved according to a peer review procedure established by the committee on emergency medicine, intensive care and trauma management (sektion nis) of the german trauma society. the participating hospitals are mainly located in germany. currently, approximately , cases from more than hospitals are entered into the database per year. for hospitals associated with traumanetzwerk dgu®, the entry of at least a basic data set is obligatory for reasons of quality assurance [ ] . the present study is in line with the publication guidelines of the traumaregister dgu® and is registered as tr-dgu project id - . during the study period from to , patients who underwent primary treatment at a local (level iii), regional (level ii) or supra-regional trauma centres (level ) (as defined by the dgu) were included [ ] . the study collective comprised all patients who were involved into a road traffic accident (rtc) and were admitted to the trauma room with at least one "serious injury" (abbreviated injury scale (ais) or higher). patients from hospitals other than germany were excluded due to their potentially other emergency medical services (ems) systems. patients who did not undergo immediate surgery or were not admitted to intensive care unit (icu) were also excluded. revised injury severity classification ii (risc ii) scores were calculated in order to predict mortality and thus to assess the probability of survival at the time of hospital admission [ ] . an exclusion on variable level was carried out when values were not available [ ] . next to demographic details, the type of transportation (car/truck, motorcycle, bike, pedestrian) before trauma was assessed. patients with an injury to the face (ais region ), including orbita, nose, ears, jaw, (inner) mouth, were assigned to the maxillofacial trauma group (mft pos). patient without maxillofacial injuries belonged to the mft neg group. in mft pos, the respective facial injury pattern was analysed. furthermore, the different injury combinations in comparison of mft pos and mft neg were investigated. in addition, the association between injury pattern and type of trauma centre was examined. finally, changes over time have also been analysed. results are presented in a descriptive manner. continuous variables are given as mean and standard deviation (sd). categorical variables were presented with frequency and percentages. ninety-five percent confidence intervals ( % ci) were calculated for selected results in order to demonstrate the degree of uncertainty. in order to give a better insight into the distribution of the data, median values were given in addition where appropriate. there was no formal testing (mft pos vs. mft neg) for statistical significance due to the large number of cases. even small differences without clinical relevance were likely to be statistically significant. for this reason, differences of medical importance were termed "relevant". data were analysed using spss statistical software (version , ibm, armonk, usa). demographic and clinical details of all included patients and noticeable differences between mft pos and mft neg during the investigation period, the tr-dgu recorded n = , patients having suffered from a traffic accident with at least one serious injury. the mean age of all casualties was years (median ). a total of . % of the casualties were men and . % were women (table ) . a total of , patients ( . %) had a maxillofacial injury and , patients ( . %) had not. the mean injury severity score (iss) was . points (median ). the iss was relevantly higher in mft pos ( . points/median ) than in mft neg ( . points/median ). there was also a remarkable higher number of unconscious patients in mft pos ( . % vs . %). in hospital, ( . %) of the patients died from the consequences of the accident and the resulting total inhospital mortality in the collective at hand was . %. the predicted mortality according to risc ii was higher in mft pos ( . %) than in mft neg ( . %) just as the observed hospital mortality (mft pos . % vs. mft neg . %). the highest inhospital mortality was among pedestrians in both groups between and % (tables , , and ). cyclists showed a mean age of . (median ) years and pedestrians of . (median ) years. those were relevant older when compared to car/truck inmates (mean . /median years) and motorcyclists (mean . /median years). a more detailed analysis regarding the type of road accident of patients without and with maxillofacial trauma is displayed in table and table , respectively. in mft pos, ( . %) patients showed minor (ais = ), ( . %) moderate (ais = ), ( . %) serious (ais = ) and ( . %) severe (ais = ) injuries in region . most commonly, injuries of the midface were seen ( patients ( . %)). lower jaw injuries were diagnosed in ( . %) and nasal injuries in patients ( . %). injuries of the eyes ( . %), the mouth ( . %) and the ear ( . %) were less common (fig. ) . critical or massive bleeding (more than % blood volume loss is due to the facial injury) was documented in only patients ( . %). in mft pos, . % of , cases additionally showed a relevant injury of the neurocranium (fig. ) . in contrast, mft neg had relevant less lesions in this body region ( . % of , cases). in addition, cervical spine injuries were seen in mft neg in only . % of , cases and mft pos had relevant more respective lesions in this region ( . % of , cases; fig. ). when comparing cervical spine trauma with ais = and ais = , higher incidences were seen in mft pos as well ( . vs. . % and . vs. . %) than in the mft neg group (figs. and ). nevertheless, in mft neg, a relevant increased combination of thoracic, abdominal as well as limb injuries was found. a total of , patients ( . %) were primarily admitted to a trauma centre. the remaining . % were secondary transfers from one hospital to another. of these primary admitted patients in the database, in total, , patients ( . %) were admitted to a level i trauma centre, , ( . %) in a level ii trauma centre and in ( . %) of the patients in a level iii trauma centre. a total of , ( . %) of these patients were diagnosed with facial injuries. a total of of those ( . %) were treated in a level i, ( . %) in a level ii and ( %) in a level iii trauma centre. in contrast, patients without facial injuries were treated relevant less in a level i trauma centre ( . %) and relevant more often in level ii as well as level iii trauma centres ( . % & . %) (fig. ). an accompanying intracranial trauma (ais head ≥ ) represents another predictor for admission to a level i trauma centre ( table ). in the mft neg group, in . % ( , ) intubation of conscious patients (glasgow coma scale (gcs) > ) was not performed. a total of . % ( ) was intubated which might depend on preventive or sedation reasons. in contrast, within the mft pos group, . % (of ) conscious patients were primarily intubated. in unconscious patients (gcs ≤ ), the intubation rate increased from . % (mft neg) to . % (mft pos) only. accordingly, similar results could be found in patients with critical or massive bleeding in the facial region. if such a bleeding occurred, the intubation rate in conscious patients (glasgow there was a noticeable decrease in the incidence of facial injuries in car/truck drivers over the years ( there were no relevant changes in the incidences of injuries in the eye, ear and nose region or in the findings of critical bleedings (fig. a, b ). over a period of more than years, this study covers all traffic accidents resulting in severely injured patients recorded in the traumaregister dgu® and represents the largest predominance of injuries of the midface was described before whereas the mandible, even if an exposed location, was affected less commonly [ ] . in accordance, rashid et al. concluded that most mandibular fractures were associated with interpersonal violence and falls whereas the incidence of fractures associated with traffic accidents became low also reflecting improvements in car safety. schneider et al. came to the conclusion that only % of maxillofacial fractures were result of traffic accidents [ , , ] . nevertheless, this seems to apply for germany/central europe only as other countries reported different major causes for maxillofacial injuries [ ] . the given prevalence is similar if not slightly higher to those reported by others [ , ] whereas a recent publication showed concomitant injuries to the facial skull in about % of cases [ ] . the injury pattern of severely injured patients after traffic accidents has changed for car/truck accidents only. for all other types of accident, the combinations of injury patterns have largely remained the same over the years. some authors reported that accidents involving motorcycles were the most prevalent. for example, in malaysia, motorcycle-related fatalities are reported to be three times higher than car fatalities, six times higher than pedestrian and times higher than bus passenger fatalities [ ] . nevertheless, others analysed in that accidents involving automobiles were the most frequent cause [ , ] . in the developed countries, greater prevalence of facial trauma among victims of automobile accidents than among victims of motorcycle accidents may be due to the use of airbags, which, despite reducing the incidence and severity of injuries in general, may contribute to facial injuries. in contrast to this, showing a lack of established cultural patterns [ ] . the mean age of years in the study at hand is similar to others [ ] whereas in less recent studies, the peak was reported to be between and years [ , ] . in accordance, a shift towards older patients may be assumed. maxillofacial injuries are rarely the leading diagnosis as stabilization and warming of patients on the trauma unit is of paramount importance so facial injuries are often diagnosed later [ ] . nevertheless, they are frequently associated with major trauma of the cervical spine and the neurocranium/head. analogue to this, a correlation between facial injuries and other injuries of the head has been described to exist in - % of cases [ , , ] . tsang and whitfield stated that in % of cases with craniofacial fractures, additional fractures in the base of the skull were seen [ ] . this could be attributed to transfer of force from the facial skeleton to the cranium. however, some investigations have suggested that the facial skeleton may actually transmit forces directly to the neurocranium, resulting in serious brain injury. an association between injury to the upper and midfacial skeleton and varying severity of brain injury had been suggested [ , ] . in accordance, at the latest in the trauma room, the entire patient must be re-evaluated in a priority-oriented manner in order not to endanger him or her due to initially unrecognised or underestimated injuries [ ] . though, especially in polytraumatised patients, a delay in correct diagnosis of lesions is evident in - % of cases [ ] . for example, the unsuspected comorbidity of the cervical spine as an infrequent but serious risk after maxillofacial injury was illustrated by a recent publication by reich et al. underlining the results of the study at hand. from a traumatological/orthopaedic point of view, patients with cervical spine injuries present in - % of cases with significant maxillofacial trauma [ , , ] and the probability of a blunt spine injury seem to rise with an increased complexity of the maxillofacial injury (fig. ) [ ] . sargent and rogers formulated that early restoration with anatomic reduction and fixation of the midfacial fractures led to a reduction in oedema formation as well as a better recontouring of facial soft parts [ ] . scholz et al. found the least rate of complications for surgical reduction of midface fractures between the nd and the th day [ ] . in contrary, other authors did not see an increase in complications when using a rather delayed approach for maxillo-facial fractures [ , ] . rothweiler et al. even concluded that a delayed approach (> h) led to less complications [ ] . of course, events such as unstable bleeding, exposed cartilage to the nose and ear as well as intra-orbital or intra-canalicular damage to the visual pathway make a therapeutic action necessary within a few hours. in accordance, basic maxillofacial competence is recommended in all trauma centres, for example in order to immediately detect a retrobulbal hematoma and shift the patient to a centre with maxillo-facial-and/or ophthalmologic expertise as soon as possible. here, early decompression of the orbita is one major aspect to prevent a permanent loss of function of the optical nerve with otherwise might lead to a consecutive reduced vision or even blindness [ ] . another rare but lifethreatening complication of midfacial fractures is a traumatic bleeding of the maxillary artery. this can occur directly after the trauma or delayed after rupture of a traumatic pseudoaneurysm or caused by movement of bony fragments [ ] . therefore, all emergency physicians and trauma surgeons should be aware of this complication and should be trained in stopping the bleeding by using packings, belocq tamponades or expandable local catheters until definitive open surgical ligature or interventional radiological catheter-based embolization can be performed. a safer and less complication-associated solution might be sufficient availability of maxillofacial surgeons, as well as ophthalmologists and ent specialists on demand in each trauma centre. on one hand, it appears reasonable that this lack of experts in local and regional trauma centres (level ii and iii) is due to economic reasons. on the other hand, there will be no second chance for primary reconstruction in some cases. the costs for secondary functional rehabilitation of these patients can be higher if rehabilitation is still possible at all. in addition, facial injuries often have a disfiguring and extremely stressful effect. baecher et al. were able to prove a dependence on injury region and post-traumatic stress disorder. the risk to be taken ill by post-traumatic stress disorder (ptsd) significantly increased if injuries of the skull and face region are prevalent compared to other body regions. this secondary data analysis is limited by its retrospective nature. although efforts are undertaken by the tr-dgu to increase data completeness and data correctness as well as to include all severely injured patients (e.g. regular reports of data completeness, audits of all trauma centres regarding data correctness), up to % of all cases likely go unreported [ ] . in addition, it must be mentioned that the number of participating trauma centres increased from in the year to more than in . furthermore, in the beginning of the tr-dgu, also, maxillofacial injuries might be overseen if they were not obvious. in accordance, ct scan of the midface was not always conducted in the past. though, as several authors showed a positive integration of whole-body ct in the diagnostic assessment of trauma room patients, an increase of the respective detection over the years can be assumed [ , ] . a further limitation of the study is the absence of details of maxillofacial injuries. even documentation and especially coding has shown to be not reliable [ ] and it can be assumed that this especially applies for maxillofacial injuries if they are primarily documented and coded by trauma surgeons. moreover, it should be stressed that all data are limited to severely injured patients who arrived alive the emergency department and were treated by a multidisciplinary trauma team. data from the study at hand indicate that patients with sustained multiple injuries (including maxillofacial injuries) may benefit from an early multidisciplinary management in a specialised trauma centre wherein close collaboration between trauma-, neuro-, ophthalmology-, ent-and maxillofacial surgeons can be ensured. on one hand, every th patient after road traffic accident needs a maxillofacial expertise and the trauma team must be aware, that maxillofacial trauma indicates a high suspicious of traumatic brain and cervical spine injuries. on the other hand, injuries of the midface are seldom the leading diagnosis of this patient collective-especially a relevant bleeding is rare. acknowledgements the authors state that parts of this article are based on a doctoral dissertation that will be submitted to the medical school of the university of ulm by silke pietzka. conflict of interest sebastian pietzka declares that he has no conflict of interest. peer w. kämmerer declares that he has no conflict of interest. silke pietzka declares that she has no conflict of interest. alexander schramm declares that he has no conflict of interest. lorenz lampl declares that he has no conflict of interest. rolf lefering declares that he has no conflict of interest. dan bieler declares that he has no conflict of interest. martin kulla declares that he has no conflict of interest. ethical approval the present study has been approved by the ethic committee of the medical school of the university of ulm (positive vote th december ). all procedures were in accordance 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maxillofacial and cervical spine fractures the prevalence of cervical spine injury, head injury, or both with isolated and multiple craniomaxillofacial fractures nasoethmoid orbital fractures: diagnosis and management delayed primary management of midfacial fractures retrobulbar hematoma: a systematic review of factors related to outcomes traumatic pseudoaneurysm of the internal maxillary artery: arare lifethreatening hemorrhage as a complication of maxillofacial fractures numbers of severely injured patients in germany. a retrospective analysis from the dgu (german society for trauma surgery) trauma registry effect of whole-body ct during trauma resuscitation on survival: a retrospective, multicentre study changes in trauma management following the implementation of the whole-body computed tomography: a retrospective multi-centre study based on the trauma registry of the german trauma society (traumaregister dgu((r))) abbreviated injury scale: not a reliable basis for summation of injury severity in trauma facilities publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- - vd mdu authors: nan title: abstracts from the th european society for animal cell technology meeting: cell technologies for innovative therapies: lausanne, switzerland. - may date: - - journal: bmc proc doi: . /s - - -x sha: doc_id: cord_uid: vd mdu nan . a schematic representation of crispr based synthetic transcription factor technology. b mrna expression levels of protein transport related genes (napg, rab a and arpc b). background an increasing number of biologics are entering the development pipelines of pharmaceutical companies [ ] . today, the preferred production host for therapeutic proteins is the cho cell line. however one of the major hurdles, especially for the production of non-antibody glycoproteins, is host cell-related proteolytic degradation which can drastically impact developability and timelines of pipeline projects. material and methods spike-in: cho cells were cultivated in a chemically defined culture medium at . °c/ % co in shake-flasks. when the cells reached their maximum viable density, they were removed by centrifugation and the conditioned medium was collected. a model mab was spiked into the conditioned medium and incubated at °c ± protease inhibitors. the amount of proteolytic degradation was analysed by western blot and lc-ms. transcriptomics: total rna was extracted after days of cell cultivation. rna sequencing libraries were constructed and processed on the hiseq platform from illumina. generation of matriptase knockout: cho-k cells were transfected with mrna encoding "transcription activator-like effector nucleases" or "zinc finger nucleases" targeting matriptase exon . the transfected cells were subsequently sorted into single cells and analysed for frameshift mutations in both alleles via sanger sequencing. cell cultivation: fed batch cultivation was performed in -ml miniaturized bioreactors (ambr ). approximately proteases are known in rodents. to reduce the number of candidate proteases we showed first that a model mab (prone to proteolytic degradation) incubated in conditioned medium of cho-k cells resulted in clipping of the mab, demonstrating the involvement of secreted/shedded proteases (fig. a) . broad spectrum inhibitors of the different protease classes revealed that only serine protease inhibitors prevented clipping. serine protease inhibitors of higher specificity highlighted the group of "s a trypsin-like proteases" (fig. a) . comparison of the proteolytic degradation profile of several therapeutic proteins between cho-k with another cho cell line (cho-a) revealed less degradation in cho-a. therefore expression of the involved protease(s) is likely lower in cho-a. gene expression profile analysis of both cell lines showed five secreted/shedded "s a trypsin-like serine proteases" more than . fold lower expressed in cho-cho-a versus cho-k (fig. b) . surprisingly, sirna knockdown experiments of these five candidates identified "matriptase" as the major protease involved in degradation of recombinant proteins expressed in cho-k cells ( fig. c upper panel) . next, we generated a cho-k matriptase knockout (ko) cell line. no proteolytic degradation product was detected when the model mab was spiked into conditioned medium of the ko cell line (fig. c lower panel) . also, stable expression of the model mab in the ko cell line resulted in no/significantly less clipping (fig. e) . the protein titer and the cell growth behaviour of the matriptase ko cells were similar to the corresponding wildtype (wt) cells (fig. d) as shown by comparative cultivation in ambr system. conclusions one major challenge for the production of recombinant proteins is cho host cell mediated proteolytic degradation which can negatively impact or even result in termination of projects [ ; ] . using a variety of techniques such as applying protease inhibitors, transcriptomics and sirna mediated knock-down we were able to identify "matriptase" as the major protease involved in degradation of recombinant proteins expressed in cho-k cells. subsequently we generated a matriptase deficient cho cell line. protein candidates of diverse formats, severely degraded in wt cho-k cell line, were not or significantly less cleaved in the matriptase ko cell line. furthermore cell growth, viability and productivity levels were comparable between the wt and the matriptase ko cell line. in summary, we have generated a superior platform-compatible cho production host cell line with the same favourable productivity properties as the parental host cell line [ ; ] , allowing expression of complex glycoproteins prone to clipping. background cho cell lines are common hosts for the production of biopharmaceutical proteins. so far, considerable progress has been made increasing productivity of cell culture to meet the rapidly growing demand for antibody biopharmaceuticals through increased cell densities and longer culture times. the downside is the increase of the process related impurities, bringing new challenges for process and harvest development. among the process related impurities such as host cell proteins (hcps) or dna the potential impact of lipids production and release during cell culture is still poorly understood due to the complex nature and diversity of this class of molecules. thanks to recent advances in analytical tools especially mass spectrometry, the advent of lipidomics offers now the feasibility to study several thousands of lipid species thus unraveling the possibility to understand and potentially control the interactions between high performance bioreactor processes, harvest conditions and purification. in order to analyze and quantify lipids, we developed a three steps method. in a first step, lipids were extracted with methyl tert-butyl ether (mtbe) according to matyash method [ ] . lipids were then separated by liquid chromatography using either hilic of reverse phase column prior to detection and quantification by mass spectrometry. all lipid classes were detected by esi-ms/ms excepted cholesterol (apci-ms/ms). finally we applied this method to analyze the lipid content of different cell lines each expressing a different recombinant protein, during a days fed batch process. lipid from cho cells were successfully extracted with a yield between % and % depending on the different lipid classes. stable isotope labeled lipids were used as internal standard in order to have comparable results between batches. the obtained results (fig. ) show that for a given cell line, lipid distribution is changing over the process. moreover, this distribution may vary significantly depending on the cell line: cl- and in a lower extend, cl- , show an accumulation of triglycerides from day to the end of the process, while cl- doesn't seems to follow this trend. conclusion interestingly, in some cell lines/experimental conditions, we highlighted an overproduction of triglycerides and cholesterol leading to the accumulation of lipid droplets known as energy storage sink. at the metabolic level, these findings suggest a relative overflow of the carbon metabolism. from a process development perspective these findings can be considered on the one hand as a resource waste since the stored energy is not used for protein/biomass biosynthesis and on the second hand as the root cause of additional process challenges especially during the harvest and the first capture steps given the hydrophobic nature of these molecules. implementation of lipidomics analysis enables us to highlight a new type of process variability and to anticipate potential problems for the downstream steps. the application of this methodology on our platform has helped us to design tailor made solutions (pretreatment selection, filter selection,…) at the clarification step which are now implemented in our harvest development platform approach. . matriptase knock-out in cho cells prevents clipping of recombinant proteins. a serine protease inhibitors protect model mab from proteolytic degradation in cho-k cell derived conditioned medium. the model mab was incubated in conditioned medium for h or h at °c, subsequently samples were analyzed by western blot. broad spectrum serine protease inhibitors (aprotinin, leupetin) were added during incubation. aprotinin and leupetin are inhibiting proteolytic degradation. the intact mab (upper band) and the clipped mab (lower band) are indicated by arrows. b gene expression profiling of cho-k versus cho-a by ngs. shown is the gene expression profile of "secreted/shedded members of the s a trypsin-like serine protease family" for cho-k and cho-a cell lines using next generation sequencing. the gene expression analysis highlights that five proteases were more than . fold higher expressed in cho-k cells (labelled with a red asterix). the y-axis shows the transcript abundance as rpkm (reads per kilobase of exon model per million mapped reads). c sirna knock-down identifies matriptase as major clipping protease and cho matriptase ko clone shows no detectable clipping activity. upper figure: sirnas directed against the five protease genes and scrambled (scr.) sirna were transfected and conditioned medium was collected three days after transfection. the model mab was incubated in fresh medium as control (first lane) and conditioned medium from the sirna transfected cells. samples were analyzed by western blot. only sirna targeting matriptase (st ) showed reduced proteolytic degradation. the intact mab (upper band) and the clipped mab (lower band) are indicated by arrows. lower figure: the model mab was incubated for h in conditioned medium collected from wt cho-k as well as the matriptase knockout clone. samples were analyzed by western blot. the intact mab (upper band) and the clipped mab (lower band) are indicated by arrows. no proteolytic degradation could be detected in the samples originating from the matriptase ko clone. d cell growth, viability and productivity in ambr (fed batch with temperature shift). cell growth, viability and volumetric productivity profiles of wt cho-k (red circles, n= ) and matriptase ko clone (blue squares, n= ) cultivated in -ml ambr. no significant differences were seen between wt and matriptase ko clone regarding cell growth and viability. comparable or slightly higher productivity was detected for the matriptase ko clone compared to the wt. e significant reduced proteolytic clipping applying matriptase ko clone. the model mab was stable expressed in cho-k (wt) as well as the cho-k matriptase ko clone. samples were analyzed by western blot. the intact mab (upper band) and the clipped mab (lower band) are indicated by arrows. significant reduced proteolytic degradation could be detected in the samples originating from the matriptase ko clone ( samples each is shown for wt and ko cells) the glycosylation of therapeutic proteins is a critical quality attribute (cqa) and needs to be analyzed during cell line and bioprocess development. the current methods for analyzing glycosylation are mainly based on the enzymatic release of glycans. they are tedious and offer only limited throughput, which makes them unsuitable for cell line development work. in this study we evaluated a novel paia assay for measuring intact glycoproteins with capture beads and fluorescence labeled plant lectins to analyze glycans in a high throughput -well plate format. material and methods analytes: erbitux © , mabthera © , arzerra © and avastin © . two glycoengineered variants of one igg were kindly provided by merck (vevey, switzerland). all analytes were spiked into cho-k cell culture supernatant or buffer, diluted : with a denaturation solution and incubated at or °c for minutes to expose the fc glycans. erbitux samples were analyzed under denaturing conditions to detect fab-and fc-glycosylation and in native conditions for fab glycosylation only. μl of pretreated sample was added to each well of the special -well paiaplate, containing labeled lectin and capture beads. the microplate was incubated for minutes at rpm on an orbital shaker at room temperature and spun down at xg. the read-out was done on a fluorescence microscope (synentec, elmshorn, germany) in less than five minutes. results figure a : lectin binding profiles of different iggs. the analysis of different igg results in lectin binding profiles which show the different degrees in glycosylation. high abundance of sugars leads to high binding rates of the lectin for the respective sugar. avastin has a very low degree of galactosylation and high mannose species compared to mabthera and arzerra (fig. a) . only arzerra is carrying glycans with - linked sialic acids. these findings are in line with results from literature [ ] . figure b : distinction between fc and fab glycosylation in erbitux. without denaturation only the fab glycans are detectable in erbitux. denaturation leads to additional exposure of the fc glycans and thus higher lectin binding rates compared to native erbitux. gna and npl only bind to denatured erbitux indicating that the high mannose glycans are only present on the fc part. the equal sna binding rates for both conditions confirm that the - linked sialic acids are almost exclusively found on the fab part. this is in agreement with published data [ ] . figure c : lectin binding rates correlate with the levels of galactosylation and fucosylation. increasing degrees of glycosylation in the mixtures of the glycan variants from merck lead to higher lectin binding rates for all galactose and fucose markers proving that quantitative analysis can be performed with these assays. the cona lectin which binds to the common core mannose glycan motive remains at the same level, suggesting that the fc glycans were similarly exposed in all samples. the results demonstrate that paia assays are capable of quickly detecting differences in glycan patterns of different antibodies. in addition it was shown that glycan variants of the same igg can be analyzed quantitatively. and finally we could confirm the differences in fab and fc glycosylation in erbitux. we believe that bead-based assays with lectins have a great potential for monitoring product quality early in the development process. background gene-and cell therapy-based medicines are experiencing resurgence due to the introduction of "next generation" transfer viral vectors, which have demonstrated improved safety and efficacy. adeno associated viruses (aav) and lentiviruses are very commonly used in therapeutics and often produced by transient gene expression, using pei-mediated transient transfection in hek- or hek- t cells [ ] . the critical raw materials needed for cgmp vector production must be sourced from approved suppliers and should have gone through a rigorous testing program to reduce the risk of introducing adventitious agents into the production process. correspondingly, the pei transfection reagent must also be sourced from a qualified supplier, and have gone through rigorous testing to ensure reliable transfection efficiencies, and hence reproducible virus production yields. here, we present peipro® and peipro®-hq, the unique pei-based transfection reagents suitable for use in process development and in cgmp biomanufacturing, respectively. unlike commercially available peis, peipro® benefits from extensive research and development in polymer chemistry and formulation for mammalian cell transfection. we further demonstrate that peipro® and peipro®-hq are the reagents of choice for virus production runs in most cell culture systems, hence facilitating the transition from initial optimization during process development up to large-scale therapeutic viral vector production in adherent or suspension cells. manufacturing process of peipro® and peipro®-hq reagents. peipro® and peipro®-hq are fully synthetic reagents, free of any animal-origin components. in comparison to peipro®, a more extensive number of quality controls are performed on peipro®-hq to enable its use as a qualified raw material in gmp processes for the manufacturing of clinical batches of therapeutic products. lentivirus and aav production. irrespective of the cell culture vessel type, transfection using peipro® was performed following our recommandations. as an example, hek- t (lentivirus) and hek- (aav) cells were thawed directly into each medium and passaged every to days before going into a liter benchtop bioreactor. cells were resuspended and cultured for days before transfection with peipro®. hek- t cells were transfected with a third-generation system (four plasmids) for lentivirus production. hek- cells were co-transfected with three plasmids for aav production. lentiviral and aav titers were measured and hours post-transfection (data kindly provided by généthon). peipro® is the reagent of choice for virus production runs in most adherent and suspension cell culture systems from process development up to large scale clinical-grade virus production. irrespective of the cell culture-based system and production scale, peipro® and peipro®-hq have led to efficient viral vector yields in standard laboratory cell systems, such as in flasks, cell factories, and roller bottles, as well as in multilayers flasks or fixed-bed culture systems that take into account time and space concerns for the scaling-up process (table ) . for example, high viral vector yields superior to ig/ml and - vg/ml were obtained respectively for lentiviruses and aavs in suspension hek- t and hek- cells cultured in one of the commercially available synthetic cell culture medium balancd® hek (irvine scientific®). conclusion peipro® and its higher quality grade peipro®-hq are the unique pei suitable for efficient and reproducible production of therapeutic viral vectors. efficient viral vector production yields can be achieved in most cell culture systems, irrespective of the production scale. with appropriate and advanced quality controls, the highest quality grade peipro®-hq is commercially available to accompany academics and biopharmaceutical companies in terms of qualified raw material for their gmp-grade viral vector production needs. b distinction between fc and fab glycosylation in erbitux. erbitux was diluted to a concentration of μg/ml in tris buffer and measured in native and denatured conditions to distinguish fab glycosylation (native erbitux) from fab and fc glycosylation (denatured erbitux). it could be confirmed that sialic acids are almost exclusively present on the fab part of erbitux and that the high mannose glycans are only found in the fc part. c lectin binding rates correlate with the levels of galactosylation and fucosylation. two glycan variants samples of the same igg from merck were mixed in different ratios to yield glycosylation rates of to % in terminal β-galactose and to % in core-fucose, based on data from -ab uplc analysis. the mixtures all contained . μg igg per well. the measured lectin binding rates for all galactose and fucose markers correlate very well with the respective degree of glycosylation in the mixtures. all measurements were performed in triplicates (irvine scientific®) . hek- t (lentivirus) and hek- (aav) cells were thawed directly into each medium and passaged every to days before going into a liter benchtop bioreactor. cells were seeded and cultured for days before being transfected by peipro® (polyplus). for transfection, four plasmids were used for lentivirus and three plasmids were used for aav. lentiviral and aav titer were measured and hours post transfection (data kindly provided by généthon) table (abstract p- ). peipro®, the reagent of choice for virus production runs in most cell culture systems in both adherent and suspension cells. irrespective of the cell culture-based system and production scale, peipro® and peipro®-hq have led to efficient viral vector yields superior to ig/ml and vg/ml, respectively for lentiviruses and aavs background continuous perfusion process is making a comeback as a competing upstream manufacturing technology for the production of biopharmaceuticals compared to the standard fed batch processes. this is primarily because of cost advantages such as reduced capital cost and increased product yield. the change in status of perfusion process from older perfusion to the new-age perfusion is due to availability of better cell retention devices leading to more efficient processes, improved cell lines, cell culture medium capable of supporting high cell densities and better bioreactor control strategies. in this work, we present the advantages, limitations and challenges of fed batch and perfusion type of processes through case studies. table results the perfusion run yielded -fold higher titer compared to fed batch run (fig. a) . considering the number of runs that could be executed in a manufacturing facility within the same calendar days, about fold increase in product output can be achieved with the perfusion process (fig. a) . this difference is attributed to higher ivcc, higher pcd and longevity of cells because of decreased level of toxic metabolite concentrations such as lactate and ammonia. case : understanding product retention in perfusion process the new-age perfusion processes utilize hollow fiber filters. this has been observed to cause retention of product within the bioreactor especially towards the end of the production run. two types of experiments were conducted to study the factors contributing to product retention: -spiking studies: -role of product titer: product was spiked into chemically defined media -role of different cell viability: different broths with varying viability spiked with same product titer -evaluation of different hollow fiber membrane (m , m and m ) on product retention. from spiking studies, it was evident that cell debris and poor quality cell broth (lower viability) were the major factors contributing to product retention (fig. c) . from the different membranes experiments, it was identified that at pilot scale, m showed much higher retention from the first perfusion cycle itself and it increased to more than % towards the end of the batch. however, with m membrane, product retention started only late (after % of batch duration) and it remained low (~ - %). on the contrary, this difference was not observed at l scale due to the usage of membranes with larger filter area ( - folds higher compared to pilot scale). when the filter area per unit volume of perfusate was decreased by half (m _batch ) for the pilot scale, even m showed retention profile similar to m (fig. d ). we presented data to show that perfusion process has -fold increase in product yield on a per-batch basis and a -fold increase when facility throughput is considered. product retention is a technical challenge that requires optimization (perfusion rates and filter membrane types). we believe it is imperative that labs that develop processes for biologics can now consider both perfusion and fedbatch based processes as both these technologies can now closely compete with each other. the choice of the process format going forward should now solely be dependent on the requirement for the biologic rather than the earlier perception that fed-batch is the preferred choice because of its simplicity. background chinese hamster ovary (cho) cell culture has been widely used for production of monoclonal antibodies in the pharmaceutical industry. previous studies have shown that the cell specific productivity in cho cells can be increased by glucose limitation [ ] . introducing a productivity enhancing effect it is possible that this also affects the quality of the product such as glycosylation or other posttranslational modifications. in this work, we are focusing on the impact of glucose limitation and increased productivity on the product quality of a monoclonal antibody produced in a fed-batch cultivation of cho cells. materials and methods cho cells were cultivated both under limiting and non-limiting nutrient conditions in fed-batch. for fed-batch cultivation the reduced range for glucose concentration was chosen between . and . g/ l. reference cultivation was performed between . and . g/l. both cultures were fed with similar volumes of a complex nutrient supplement. all cultivations were performed in chemically-defined, animalcomponent free cho growth media (xell ag). viable cell density and viability were determined using the automated cell counting system cedex (roche diagnostics), glucose and lactate concentrations were detected via ysi (ysi life sciences). amino acid were quantified using hplc-fld, vitamins were quantified using reversed phase chromatography coupled to a triple quadrupol mass spectrometer (varian , selected reaction monitoring). amounts of igg were quantified via protein a hplc, mab purified from another cho cell clone was used as a standard. the analysis of product quality was performed by intact mass analysis using reversed phase chromatography coupled to a microotof-q ii mass spectrometer (bruker daltonik). the cho cell culture cultivated under low nutrient conditions reached a % higher viable cell density than the reference culture (fig. a) . the product titer was even increased by % (fig. b) . the spent media analysis shows that some amino acids and vitamins were present at presumably limiting concentrations after day / , mostly in the low nutrient level culture (down to to μm for tyr, gln, arg, and asn, below μm for pyridoxine, data not shown). the product quality showed significant changes for the changed feeding strategy ( fig. c and d) . as expected, the glycation level decreased from % to % compared to the reference culture. the truncation level of c-terminal lysine at the heavy chain of the mab increased from % to %. the glycosylation was also significantly influenced by the low nutrient level (fig. e) : the nonfucosylated variants increased from % to % (fig. f) , the degree of galactosylation increased from % to % (fig. g) . cultivation under low nutrient level led to % higher viable cell density and a product titer increased by % when compared to reference culture grown under non-limiting nutrient conditions. the analysis of product quality reveals % less glycation of light chain for cho cells grown under low nutrient conditions ( . % vs . % in reference culture). the truncation of c-terminal lysine decreased by % (from % to %), the degree of galactosylation increased by % (from % to %, also observed by takuma et al. [ ] ) and non-fucosylated glycans increased by % (from . % to . %) under low nutrient conditions. the product quality analysis by intact mass proved to be highly robust (average cv for four replicates = %). in summary, cultivation with alternative feed led to higher igg product titer and better product quality (glycation unwanted, higher amount of non-fucosylated glycans leads to higher antibody-dependent cellmediated cytotoxicity (adcc), higher amount of galactosylation to higher complement-dependent cytotoxicity (cdc) and adcc [ , ] ). background we developed an automated, multiwell plate (mwp) based screening system for suspension cell cultures (fig. a) which is now routinely used in cell culture process development. it is characterized by a fully automated workflow with integrated analytical instrumentation. it uses shaken - well plates as bioreactors which can be run in batch and fed-batch mode with a capacity of up to reactors in parallel [ ] [ ] [ ] . a wide ranging analytical portfolio to monitor cell culture processes and also a cooperation with internal high throughput (ht) analytic groups to characterize product quality are available. in addition the use and the benefits of spectroscopic methods for cell culture automation were shown in the past [ , ] . automated cell culture systems enable broader screening within a shorter time frame for many applications in upstream process development. the higher degree of parallelization and automation helps to screen for most promising parameters in a shorter time. the use of broad doe screening design allows in addition the identification of parameters that support high titers while keeping high product quality (multiple factors at the same time). the illustration (fig. b) shows an example how this combination can speed up process development steps. main applications of the cell culture automation are for example the identification of product quality levers and media or feed optimization. the application of the cell culture automation is shown for two examples. the goal in the first application was to identify levers to reduce trisulfides. by a screening of conditions in parallel (in -fold replication, wells in sum) the reduction of trisulfides by . % (normalized to start level) was possible. in addition the levers for trisulfide reduction were identified. the best and start conditions were verified in bioreactor scale (fig. c) . the goal in the second application was to increase product concentration without an impact on product quality. by a screening of conditions in parallel (in -fold replication, wells in sum) the increase of titer from . g/l to . g/l (> factor ) was possible by media platform change and media optimization. an impact on product quality could not been shown. the best conditions were also verified in bioreactor scale (fig. d) . the benefits of using cell culture automation in late stage process development were shown based on two examples of current applications. for this purpose the experimental results of the development work of two late state projects using the in-house developed automated cell culture system were shown. the first example shows the capability of the automated cell culture system by reducing trisulfides significantly in just one experiment. for the other project the final product concentration could be increased by factor . by a media screening and changing to the in-house media platform. these two examples show the potential of cell culture automation as a routine tool in process development. the cell line development process has become faster and is simultaneously generating more clone-and product-related analytical data. in order to select the best producer cell line, extremely heterogeneous data types need to be systematically compared. the timely availability of all data needed to decide which cell line to pursue has become a bottleneck in the cell line development workflow. to ensure sound decision making, new integrated workflow support and data analysis methods are needed. we have developed a new end-to-end platform for bioprocess development, which includes a cell line development workflow system supporting seeding, selection, passaging, analyzing, cryo-conservation, and processing in (micro-) bioreactors. this platform, genedata bioprocess™, enables partially or fully automated cell line selection and assessment processes, and it increases process efficiency and quality. the system tracks the full history of all clones -from initial transfection all the way to their evaluation in bioreactor runs -and combines this information with analytics data on molecules, clones, and product quality. it can directly integrate with all instruments, such as pipetting robots, bioreactors, and bioanalyzers. the system is designed for a wide range of . a schematic illustration of the automated cell culture system. only the core system is shown with a robotic plate handler as key device connecting cultivation, processing and analytical parts. b illustration of an example how cell culture automation can speed up process development steps. c application in the identification of product quality levers. d application in titer optimization biologic molecules, including antibodies (iggs, novel formats) and other therapeutic proteins (e.g., fusion proteins). highlighted use cases describe the identification of top producer cell lines, decision making support, bioreactor data management, and full clone history report documentation (fig. ). genedata bioprocess, which was developed in collaboration with top pharmaceutical companies, can flexibly support various (non-linear) workflows and structure the collected information in a way that fosters collaboration across an organization. while increasing throughput is crucial to ensure the timely availability of optimal producer cell lines, high-throughput is only possible when automated processes in the laboratory and the resulting data collection and aggregation can be streamlined. genedata bioprocess helps to establish more productive processes by offering support and integration for automation stations and measurement devices. thanks to the comprehensive workflow support and the possibility to integrate results from cell line stability experiments, product quality assessment, and bioreactor suitability tests, genedata bioprocess provides a unique way to evaluate cell lines. comprehensive analysis of all data collected in the process helps to ensure the highest possible quality and minimize the time and resources needed for data analysis and management. integration of bioreactor data analysis and visualization with other parameters measured in cell line development, streamlines clone evaluation in micro-bioreactors and supports highthroughput operations. genedata bioprocess comprehensively tracks the full clone history from the origin of the host cell line to the generation of the validated monoclonal producer cell line. for promising clones, the clone history report can be generated with one click. besides supporting cell line development, genedata bioprocess is a comprehensive platform capable of tracking the complete bioprocess development process. in , . million people suffered from cancer [ ] making it to a major concern of our society. since common cancer treatment is limited and not effective for late stage carcinoma, alternative methods are needed to reduce the high mortality rate of cancer patients. one alternative approach is the application of the oncolytic measles virus (omv), because omv has a natural affinity against cancer cells. the major drawbacks of omv is to produce the extremely high amount of at least tcid ( % tissue culture infective dose) per dose [ ] which is needed. to solve this problem, a high titer process must be established including an efficient downstream processing (dsp). we developed an appropriate upstream processing and are able to produce - tcid ml - in a bioreactor with . l working volume [ ] . now, we focus on the dsp part. the following study tested the application of charged depth filters for the omv clarification. in contrast to common dsp schemes, a depletion of virus particles or a loss of infectivity is not desired. the aim is a reduction of protein content and dna with minimal loss of infective omv. further, we investigated the influence of the cell culture medium on the depth filtration process. to explore the influence of the surrounding cell culture medium on the depth filtration performance, omv was either produced in serum-free medium (vp-sfm) or serum-containing medium (dmem + % fcs). the production was done in a str with a working volume of . l as described in [ ] . cells and carriers were separated with an opticap xl -module (polygard-cr; μm; merck). for the depth filtration millistak+ ce filters (merck) were used. the filter material was autoclaved and rinsed with ml of mm tris-hcl (ph= . ). the virus suspension was filtered with a load of l m - using a peristaltic pump (ism c; ismatec) applying a flux of l m - h - (fig. ). samples were collected at the beginning and end of a filtration run. the omv titer (tcid ml - ) of the samples was determined according to kärber and reed [ , ] . protein content was measured with the pierce bca protein assay kit (thermofisher scientific) according to the manufacturer's instructions. dna was measured by a microtiter assay using quant-it picogreen dsdna reagent (thermofisher scientific) according to the manufacturer's instructions. we found that positively charged depth filters were suitable to clarify omv suspensions. the cell culture medium, in which the omv was produced, influenced the outcome of the depth filtration. a log reduction value (lrv) of . was determined for omv present in serum-containing medium (scm), whereas the titer of omv in serumfree medium (sfm) was reduced . log levels. this indicates that without serum in the surrounding liquid, omv will adsorb to the filter material. however, we must evaluate if the missing serum or other components present in sfm are responsible for this effect. total protein was not relevantly reduced by the clarification using charged depth filters. for omv present in scm, the residual protein content was slightly less compared to omv present in sfm (table ). in contrast, host cell dna (hcdna) was bound to the filter material. we achieved a % reduction of hcdna for an omv suspension in sfm. after clarifying an omv suspension in sfm, the remaining hcdna content was even lower being only %. conclusions charged depth filters are suitable for the first clarification step of omv downstream processing. residual protein could pass the depth scheme of the complete cell line development workflow support in genedata bioprocess. showcasing integration of data from diverse measurement instruments, data visualization for decision making support as well as, tracking of full clone history filter almost unhindered, whereas the hcdna content was already reduced to % at maximum. however, the omv titer was also reduced by the depth filtration. this undesired effect was stronger for the omv present in sfm. because the agencies require avoiding serum in clinical-grade production processes, this is disadvantageous. nonetheless, because sfm will be soon standard for omv production, further experiments have to be done preventing the omv reduction during clarification. one option can be to reduce the adsorption strength of the virus to the filter material by the addition of salt. moreover, it is important to establish a standardized protocol for the upstream processing. we determined batch-to-batch variations within the clarification indicating a strong impact of upstream processing (usp) on the outcome of the dsp. therefore, further studies must investigate the influence of usp parameter e.g. time of harvest and ph of the harvest solution on the omv. fed-batch culture is commonly employed to maximize cell and product concentrations in upstream mammalian cell culture processes. typical standard platform processes rely on fixed-volume bolus feeding of concentrated feed supplements at regular intervals. however, such static approaches might result in over-or underfeeding. to mimic more closely the dynamics of a fed-batch culture, we developed a dynamic feeding strategy responsive to the actual nutrient needs of a mab-producing recombinant cho cell line. results and discussion improvements made at different steps during fed-batch development are shown in fig. . at step , all eight cell boost supplements were added to cdm ns according to a doe approach, and batch cultures were performed. this evaluation allowed us to select only those cell boost supplements that were beneficial to the overall culture performance. non-performing cell boost supplements were removed and not considered further. at step , the selected cell boost supplements were added daily to the cultures at different ratios according to a doe approach, and fedbatch cultures were conducted. as expected, daily feed additions to replenish consumed nutrients substantially improved mab and peak cell concentrations as well as viable cumulative cell days (vccd) compared to batch cultivation. further, the results enabled us to fine-tune the feed ratio of selected cell boost supplements. at step , we further optimized the best performing feed ratio by investigation of static and dynamic feed protocols. most fed-batch protocols rely on constant feed additions on distinct days. however, these approaches often lead to substantial over-or underfeeding during bioprocessing. to improve such "static" protocols, we investigated three different "dynamic" approaches as shown in table by applying the selected cell boost supplements with the optimized feed ratio. this investigation allowed us to further improve the bioprocess performance. the best performing approaches, constant and retrospective feed, were further investigated in fully automated bioreactors under controlled conditions. in general, constant cultivation parameters in the bioreactor slightly enhanced mab titers compared to shake flask cultivation. the retrospective feed strategy yielded % higher titers than the constant strategy. overall, the established methodology for fed-batch development allowed us to obtain . × higher mab titers (batch mean: . g/l vs. fed-batch . g/l) in a short time and three simple steps. in addition, the product quality was investigated. compared to the legacy fedbatch process, fed-batches that were conducted with the newly selected basal and feed media altered the distribution of charge and glycan variants. the amount of aggregated product was not altered. the established methodology for fed-batch development is a rapid protocol to select well-performing feed supplements and optimize their ratio to the culture requirements. in three steps, mab titers were boosted . x from . g/l to . g/l. product glycosylation and charge variants could be influenced by the newly selected basal and feed media compared to a legacy fed-batch process. the amount of aggregated product was not altered. the present study investigates the beneficial effect of spiking hyclone™ actipro™ basal medium with hyclone cell boost™ a and cell boost b feed supplements on growth and productivity of a recombinant cho cell line. to evaluate the impact of feed-spiking compared with cultivation in basal medium only, the cell line was grown in bioreactors under controlled conditions to determine cellspecific metabolic rates, nutrient consumption, and byproduct accumulation over the process time. transcriptome analysis of the cultivated cells, using microarrays on four consecutive days to investigate differential gene expression, revealed the beneficial effect of feed-spiking compared with cells grown in basal medium. model cell line was a mab-expressing cho dg (licensed from cellca gmbh) cultivated either in actipro basal medium only (ge healthcare) or in actipro basal medium feed-spiked with additional supplementation with % cell boost a and . % cell boost b (ge healthcare). both cultures were grown in batch mode using dasgip™ cellferm-pro™ stirred-tank bioreactors (eppendorf). the beneficial effect of feed-spiking was analysed by transcriptome analysis using microarray technology ( × k design, agilent). both basal and feed-spiked processes lasted for seven days with viabilities above % until day . on day seven, a sharp decline in viability indicated the end of the batch process (fig. a) . in feed-spiked medium, cells initially grew slower but reached almost twice as high peak cell concentrations ( . × c/ml) than in basal medium only ( . × c/ml). remarkably, the integral of the viable cell concentration over the total process time (viable cumulative cell days [vccd] ) was similar between both process strategies (fig. c) . while mab production plateaued after day in basal medium only (final titer . g/l), a continuous increase to three-fold higher final titers ( . g/l) was observed in feed-spiked medium (fig. b) . the higher titers could be attributed to generally higher cell-specific productivities (qp), which remained rather constant (~ pg/cell/day) in feedspiked cultures. in basal medium, the qp continuously dropped by % (day to ), % (day ), and > % (day to ) from to pg/cell/day in basal medium cultures. in average, the qp was % higher in feed-spiked cultures (fig. d ). transcriptome analysis of differentially expressed genes between cells grown in basal medium or feed-spiked medium were used to identify relevant go terms that indicated a more active proliferative state for feed-spiked cultures (data not shown). the top go terms significantly related to cell cycle and primary metabolism, cellular division, as well as nucleobase formation or regulation. furthermore, gsea revealed several significantly enriched set of genes related to gene transcription, dna replication and repair, cell growth and proliferation, as well as inhibition of apoptosis in feed-spiked cultures. thus, feed-spiking increased the proliferative activity of cultivated cells. several of the identified genes appear as promising targets for cell line engineering, but have not yet been described in relation to high-producing recombinant cell lines and will need to be evaluated in future studies. feed-spiking of basal medium is a convenient and easy way to considerably increase product concentrations in a simple batch culture. differential gene expression revealed genes that appear important for high cell-specific production rates, and this knowledge can be leveraged into cell line engineering approaches or the design of high producing cho cell media. in the latter case, a maximized supply of high biosimilarity must be demonstrated by physicochemical and functional characterization for approval requirements of phase i and phase iii studies in terms of efficacy, safety and immunogenicity. in this study, rounds of upstream and downstream processes were run to reach the cqa limits of the originator molecule. after conducting many different development strategies, the mirror plot images of the intact deconvoluted mass were found to be identical corresponding to similar levels of glycoforms. the uv chromatogram of reversed phase ultraperformance liquid chromatography (rp-uplc) of tryptic peptide mapping demonstrated that the primary structure of tur is identical to the originator as shown in fig. a . post-translational modifications (ptms) such as oxidation, deamidation, n-terminal pyroglutamic acid, c-terminal lysine truncation levels were also comparable for two products. the glycosylation site (hc-asn ) was confirmed by peptide mapping analysis and % glycan site occupancy was proven for tur and originator. the glycosylation pattern for two products were highly similar in terms of major glycans (g f, g f, g f-gn and etc.). man level was lower in tur compared to the originator product which may not have any clinical effect on the molecule. the secondary structure was determined by atr-ftir spectroscopy. absorption bands (amide i and amide ii) were overlapped completely and amounts of α-helix and β-sheet structures were comparable. furthermore; size-exclusion chromatography (sec) analysis revealed that both products have the same level of purity (> %) and aggregate (< %) levels. the level of impurities were determined as below % by ce-sds. the capillary isoelectric focusing (cief) experiments showed that the charge variant profiles of two products are indistinguishable and the isoelectric point of main peak is observed at . for both products. the association/dissociation rate constants and binding affinity for both tur and originator were highly similar and similarity score was calculated greater than %, as shown in fig. b . in this study, state-of-art analytical techniques were used to assess the biosimilarity of tur to the originator adalimumab. head-tohead comparison data clearly demonstrated that tur is highly similar to the originator adalimumab in terms of physicochemical and functional characteristics. based on the analytical similarities, we . process performance of basal medium (black) and feed-spiked (red) bioreactor batch cultures: a cell concentrations and viability, b viable cumulative cell days and specific growth rate, and c antibody concentrations and cell-specific productivity. error bars indicate standard deviation from three independent experiments. the black arrows on day indicate the beginning of decreasing cell-specific productivities and lower cell-specific growth rates in basal medium cultures believe that tur will have comparable pk/pd, potency, and efficacy results to the originator adalimumab. the expanded interest in intensified continuous bioprocessing has highlighted the need to develop a small scale model for perfusion cell culture. the direction in the industry has been to increase target cell densities to ≥ x vc/ml and decrease perfusion rates to ≤ vvd. in order to increase the throughput of our perfusion media development capabilities we sought to develop a small scale model of perfusion using the ambr® instrument (sartorius, germany). we used a modified cell settling model from the previously published by kreye et. al. to achieve the cell retention necessary to reach perfusion relevant viable cell concentrations [ ] . in this work, we will show the application of this small scale model for: ( ) identification of specific productivity performance over a steady-state for tested media, ( ) identification of cspr min for a specific cell line and medium combination, and ( ) confirmation of consistent product quality profiles between the small scale model and benchtop perfusion (data not shown). a chozn® cell line producing an igg was evaluated in several proprietary chemically defined media prototypes generated during the development of the catalog excell® advanced hd perfusion medium: "fed batch medium", "early prototype", "mid prototype", "intermediate prototype" and "late prototype" [ ] . small scale simulation of perfusion experiments were run in ambr® . media exchange was performed times per day in equal amounts. agitation, gassing, and liquid handling were stopped for an optimized period of time to allow cells to settle to the bottom. spent media was removed in an amount proportional to / rd daily exchange volume. agitation, gassing, and liquid handling were resumed and fresh media was added back to the vessels. for benchtop perfusion, cells were inoculated in l applikon bioreactors (applikon, netherlands). at a concentration of~ . x vc/ml, perfusion was initiated using the atf (repligen, massachusetts). perfusion rate was limited at . vvd during steady-state. using the cell settling method described above we have been able to achieve ≥ % cell retention efficiency. all media tested in this work were able to reach and maintain the x vc/ml target cell density at vvd (fig. ) . performance of each media formulation was ranked based on specific productivity (table ) . using "intermediate prototype", minimum steady-state cspr was determined to be . pl/c/d for this cell line. n-glycan analysis of ambr® and bioreactor samples via intact mass spectrometry displayed only slight differences in product quality profile (data not shown). our work has shown a clear distinction between various prototype perfusion media and demonstrated a % increase in specific productivity over "fed batch medium" used in perfusion. additionally, we have shown the application to further characterize the process using this model to determine cspr min for a given medium and cell line. the transient process has been successfully operated at l in a sartorius biostat single use bioreactor (sub), yielding . kg of crude product from a two-week expression culture (table ) . successful scale up of the process to l creates the potential to supply transiently expressed products to support toxicology studies or even early gmp clinical supply, enabling accelerated biopharmaceutical development project timelines. the scale up from rocking bioreactors (rbr) to sub scale identified some scalability issues. lower specific productivity due to increased cell growth and decreased titres were observed in the sub ( fig. iii & iv). to improve the predictability of scale up, a new process was developed and evaluated in the sub vessels utilising a modified transfection method, which resulted in comparable expression levels and specific productivity between rbr and sub scales. two sets of expression vectors comprising heavy chain and light chain plasmids expressing a human igg kappa mab, as previously described [ , ] were used in the process optimisation study. the cell line used for transient expression and the pei mediated transfection method has been described previously [ ] . transfected cultures were run under fed batch conditions for days in l ge healthcare wave bioreactors (rbr), hyclone sub using l and l hyclone bioreactor bags (thermo scientific). the transfection process was modified to address the reduced titres and higher viable cell density (vcd) seen in the sub cultures. shake flask cultures were used to assess the standard (a) and modified transfection processes (b and c) (fig. , i & ii). process c was identified as the process to be studied at sub scale, offering the potential to mitigate the high viable cell densities (vcd) observed. scaling up process c to l and l sub resulted in cultures producing titres exceeding g/l with desired cell growth profiles. scale up of process a into sub vessels resulted in decreased productivity compared to the rbr scale. after optimisation, the sub process c yielded increased specific productivities and expression titres comparable to those seen at rbr scale (table ) . medimmune has successfully completed the first known successful cho transient culture at l scale producing > mg/l of mab at harvest. process optimisation has subsequently demonstrated reproducible titres at l to l scale exceeding g/l with comparable glycosylation profiles between sub and rbr cultures across scales. comprehensive analysis of the impact of trace elements in media on clone dependent process performance and product quality background state-of-the-art biopharmaceutical processes are accounting concomitantly for process performance and product quality. even though high yielding, robust processes are the cornerstones of any process development, product quality parameters such as structural integrity, charge variances and post-translational modifications are progressively becoming the focus of the developmental work. in conjunction with host cell line selection and process performance parameters, media components are crucial for the continued progress in rational modulation of product quality attributes affecting biological activity, immunogenicity, half-life or stability. among media components, trace elements (te) are of particular interest as they play a pivotal role in various cell metabolism pathways. based on a comprehensive doe approach, extensive process performance-and product quality evaluation combined with metabolic flux analysis, the impact of several trace elements on the biopharmaceutical process is assessed. in a comprehensive i-optimal doe approach ( fig. ) , the effect of six te in various concentration levels and combinations in serum-free media was studied for four different cho-k cells lines in an ambr® setup. a scrutiny of the process performance parameters such as cellular growth, productivity, amino acids and vitamins consumptions rates for each of the conditions was performed. the process performance evaluation was accompanied by extensive product quality analysis including size and charge variants, glycosylation patterns, oxidation and methylation. furthermore, a metabolic flux analysis was performed based on the nitrogen balance. based on extensive analytical data, the obtained response surface model provides a clear insight into the impact of particular te and their combinations on process performance and product quality. the high model quality enables discriminations between clone dependent and clone independent effects. with an elevation in titer up to % in the best condition of the cell lines clearly show, that even state-of-the-art media can be outperformed by trace element rebalancing. analyzing specific rates in combination with metabolic flux analysis improves the understanding of metabolic restructuring of the cell lines under distinct te levels and combinations. modulation of trace elements levels had a tremendous effect on the charge heterogeneity and glycosylation structure of the different proteins. this provides a toolbox for the fine tuning and control of product quality parameters. taken together, the data further paves the way to the rational fine tuning of process performance and product quality attributes. background due to regulatory concerns and economic impact, ensuring product quality and consistency is now one of the main challenge faced by the biopharmaceutical industry. for monoclonal antibodies (mab), glycosylation is one of the most important quality attributes as it impacts on mab structure integrity, and ultimately on both clinical efficacy and safety. many factors affect mab glycosylation and its inherent heterogeneity, including the host cell, the culture medium, the mode of operation and the operating conditions. in this context, the capacity to monitor and control on-line the antibody glycosylation, from early-to late-stage process development, would be of salient interest to reduce the time and cost to market. in order to address this unmet need, we have designed an improved spr biosensor assay to measure the kinetics of interaction between a mab and the extracellular domain of the fcγriiia receptor bound at the biosensor surface [ , ] . of salient interest, we also demonstrated that various binding kinetic signatures, especially different dissociation kinetics could be correlated with distinct mab glycosylation patterns and with therapeutic efficacies, as deduced from mass spectrometry and a surrogate adcc assay, respectively. in parallel, we have also harnessed a spr biosensor directly to a bioreactor, which permitted the at-line determination of the concentration of antibodies by hybridoma cells during a bioreactor culture. we now plan on combining both approaches to determine on-line the glycosylation profile of the produced mabs. our ultimate goal is to design a unique and highly innovative bioprocess control tool that can be readily applied in an industrial bio-manufacturing setting. reducing timelines and costs are key factors for bio-pharmaceutical industries to accelerate process development and drug delivery to patients. enhancing throughput of bioprocess development has become increasingly important for the screening and optimization of cell culture processes. this challenge requires high throughput tools. in a previous study [ ] , we showed that ambr® , a robotically driven mini-bioreactor system developed by tap-sartorius, could be advantageous to accelerate process development. the use of ambr® system allows us to test a large number of experimental conditions in a single experiment. therefore, the large amount of production samples to be characterized for product quality attributes (pqa) increases as well: the bottleneck has moved from the generation of samples at the production bioreactor step to in-process analysis. for product quality attribute analysis at lab scale, protein purification is generally carried out on > ml columns which is incompatible with the size of ambr® bioreactors. moreover, the applied methods are relatively low throughput. the development of a high binding capacity resin (up to mg/ml) [ ] , combined with high performing new cell lines which are able to produce up to g/l of recombinant monoclonal antibodies, allow require the development of an efficient and high throughput (hts) purification method robot. the use of robotic equipment for small scale purification purposes is a great opportunity for us to tackle this bottleneck, by enabling highthroughput sample purification at smaller scale ( μl). recombinant monoclonal antibodies were produced by a genetically engineered dihydrofolate reductase (dhfr)-/-dg chinese hamster ovary (cho) cell line. clarified cell culture fluid (cccf) was obtained from and k liter bioreactors after three filtration steps. minipurifications were performed on tecan freedom evo® robot with predictor robocol-umns® containing μl mabselect sure® resin. larger scale purification were executed using an aktaxpress using hitrap column prota. to assess monoclonal antibody purification at small scale, we first tested the repeatability of the minipurification, purifying the samples times on the same columns and using different columns, focusing on the yield of the purification and the impact on product quality attributes, especially the hmws. then, we compared those results to those obtained with the aktaxpress at larger scale purification, comparing the yield of the purification and the pqa of the protein-a eluates obtained with both purification systems. finally, we assessed the capability of the robot to perform hts of buffer and purification conditions, evaluating three different buffers at different concentrations and ph values, and also testing different loading column capacities. in this study we established that the tecan can be used as a robust platform for purification at small scale. we observed similar purification yields, intra and inter run. the analysis of the pqa a level showed there is also very high reproducibility. and the ph of the eluate showed as well strong comparability as well. table shows the coefficient of variation (cv) of the yield, hmws and eluate ph are low, demonstrating the good reproducibility of the purification. the strong reproducibility obtained between the different purifications showed that the tecan and the aktaxpress are similar in terms of purification performance and pqa (fig. a, b) . the tecan is a versatile automated liquid handler allowing the screening of huge purification conditions (fig. c) , the possibility to purify large quantities of samples, while the samples amount is limited. the tecan has the potential to purify more than samples/day, reducing timelines and allowing us to deliver faster to the patients. viable cell density monitoring in bioreactor with lensless imaging geoffrey monitoring cell density and viability of mammalian cell culture bioreactors is a necessary task that presents today a number of remaining challenges. the traditional measurement for bioreactor cell count and viability rely on using the trypan blue exclusion method once a day. while automatic cell counters have reduced the statistical manual error, sampling the bioreactor remains a contamination risk and is prohibiting process control as the sampled volume becomes siginficant. lensless imaging technology is a new method for accurately determining cell concentration and viability without staining. this technique directly acquires the light diffraction properties of each individual cells through their holograms images without any objective, lens or focus settings. living and dead cells have significant holographic patterns that can be distinguished and precisely counted. lensless imaging technique directly acquires the light diffraction properties of each individual cells through their holograms images without any objective, lens or focus settings. living and dead cells have significant holographic patterns that can be distinguished and precisely counted. we compare cell counts and viability between the reference method and our lensless imaging device, the cytonote counter. measures are performed once a day on samples from bioreactors, from the inoculation to the end of the culture. we also assessed the repeatability of our method. another lensless imaging prototype is setup as a measurement chamber directly connected to a perfusion bioreactor, for continuously receiving the bioreactor broth, and therefore reproducing an in situ measure. with a concentration range up to x cells/ml ( fig. ) and viability range at - %, we obtained a correlation factor of . between the two compared methods. the large field of view allows the analyze of several thousand cells within a single image, keeping the statistical variability of the measure as low as %. our measurement chamber prototype has demonstarted its capability for continuous viable cell density and viability monitoring. we are now working at designing a steam strerilizable probe, and we envision lensless imaging to become the future method of choice for on-line monitoring of suspension cells cultures. lensless imaging technology is capable of accurately and precisely monitoring viable cell density and viability with a combination of significant advantages starting from low sample volume use, label free detection, quick measure, simple device, to high number of cell analyzed which let us think that it is a good candidate for very small- comparison between both purification systems and the ability of the system to be used as a high throughput tools for buffers screening. a purification yield (%). b pqa .a (normalized). c impact of the ph and buffer concentration on the pqa .a scale bioreactor and high-throughput measures. its high repeatability is also a key parameters in the effort to narrow batch to batch deviations. in addition we demonstrate that this technique is potentially powerful for in-line and continuous monitoring of a lab bioreactor. we envision lensless imaging to become the future method of choice for on-line and in-situ monitoring of suspension cells and a perfect tool for process control in fed-batch or perfusion mode in single-use bioreactors or traditional steam sterilized vessels. it can certainly become the first vcd measurement technique to work from cell line engineering, to process development, pilot scale, and up to manufacturing scale. time-dependent product heterogeneity in mammalian cell fermentation processes background a consistent product quality is a major goal in the production of biotherapeutics, especially recombinant glycoproteins. whereas it is unlikely that the polypeptide chain changes during a production process, posttranslational modifications and protein folding are sensitive to fluctuations in parameters and conditions. here we focus on protein glycosylation as one important indication for product quality [ ] . during a batch process conditions change continuously. at the beginning, the supply situation for the cell is excellent, but the secreted material stays a long time in the culture fluid. later during cultivation substrate provision decreases, whereas the exposition time of the protein to the culture fluid is much shorter. altogether this leads to product heterogeneity of the secreted protein during a batch culture. four different cell lines, two of human origin and two cho clones, producing four different recombinant glycoproteins were investigated in this study. together with their respective parental cell line the clones were cultured in three replicates in shakers. supernatant from the cultures were harvested at four time points. the removed culture volume was replaced by culture supernatant of the identically cultured corresponding parental cell line. the product was isolated from the supernatant and the glycans were released. one part of the released glycans was labeled with -ab and separated by hilic-fld. the other part of the glycans was permethylated and analyzed by maldi-tof mass spectrometry (fig. a) . the investigated proteins were antibody, antithrombin iii from cho clones and α -antitrypsin, c -inhibitor from human clones. the antennarity of the glycans is quite stable in all production phases. the degree of core fucosylation is very high in all products. a low fucosylation degree of antibodies may be favorable for a higher adcc performance [ ] . some of the products showed an antennary fucosylation, which seemed to change not very much in different cultivation phases. nevertheless, this might be an issue due to an antigenic impact in the patient. the antennary galactosylation changes noticeable for the antibody and α -antitrypsin. in both cases the degree is highest in the first phase. an incomplete galactosylation leads to truncated glycans. this leads inevitable to undersialylated antennas to be seen for α -antitrypsin. the sialylation is the highest in the early phases and decreases during cultivation time. sialylation of a therapeutical protein is important for the half-life in the patient. therefore highly sialylated products are desired [ ] . in further studies the consistency of the galactosylation and the sialylation will investigated for fed batch and long term continuous cultures in comparison to batch cultures. due to the feed solution or the fresh media being present during such processes, the supply situation should be excellent for the whole cultivation time. the differences between the maldi-tof and hilic-fld data originate from complex and unresolved chromatograms (fig. b , chromatograms not shown). for that reason coupling of hilic-fld and ms is very much recommended. background novel biologics are often selected from a large library of lead candidates in the initial stage of preclinical and clinical developments. for this selection, there is a demand for high-throughput production of recombinant proteins of high quality and in sufficient quantity. transient gene expression offers a rapid approach to the production of numerous recombinant proteins for the initial-stage developments of biologics. mammalian cells are major host cells for transient gene expression, but they have the disadvantages of complicated operations and high cost of culture. insect cells are easy to handle and can be grown to a high cell density in suspension with a serum-free medium. insect cells can also produce large amount of recombinant proteins through post-translational processing and modifications of higher eukaryotes. hence, insect cells have been recognized as an excellent platform for the production of functional recombinant proteins [ , ] . in the present study, the production of an antibody fab fragment through transient gene expression in lepidopteran insect cells was examined. the dna fragments encoding the heavy chain (hc) and light chain (lc) genes of an fab fragment of mouse anti-bovine rnasea [ ] were respectively cloned into the plasmid vector pihaneo, which contained the bombyx mori actin promoter downstream of the b. mori nucleopolyhedrovirus (bmnpv) ie- transactivator and the bmnpv hr enhancer for high-level expression [ ] . trichoplusia ni bti-tn- b - (high five) cells were co-transfected with the resultant plasmid vectors using linear polyethyleneimine (pei; mw , ). before transfection, the plasmids and pei were prepared in mm nacl, ph . and incubated at room temperature for min. when the transfection efficiency was checked, a plasmid vector encoding the enhanced green fluorescent protein (egfp) gene was also co-transfected. transfected cells were incubated with a serum-free medium in a static or shake-flask culture. culture supernatants were analysed by western blotting and enzyme-linked immunosorbent assay (elisa). the numbers of green fluorescent cells and total cells in culture broth was determined using a flow cytometer. western blot analysis and elisa of culture supernatants showed that transfected high five cells secreted the fab fragment with antigenbinding activity. in static cultures, transfection and culture conditions, such as hc:lc gene ratio, a serum-free medium, dna:pei ratio, and dna amount per cell, were successfully optimized by flow cytometry of egfp expression in transfected cells and the yield of the secreted fab fragment measured by elisa. the effects of culture temperature and initial cell density were also examined by comparing the cell growth and the production of fab fragments in shake-flask cultures. under optimal conditions (medium, psfm-j (wako pure chemical industries, japan); hc:lc gene ratio, : ; dna, μg/( cells); pei, μg/( cells); initial cell density, x cells/cm ; temperature, °c), the yield of more than mg/l of fab fragment was achieved in days in a shake-flask culture ( fig. ) . transfection did not significantly affect the growth of high five cells as compared with untransfected cells. transient gene expression using insect cells may offer a promising approach to high-throughput production of candidate proteins for the development of biologics. the increasing demand for biopharmaceuticals produced in mammalian cells has led industries to increase volumetric productivity of bioprocesses through different strategies [ , , ] . in this context, fedbatch and perfusion cultures have attracted more interest than conventional batch processes. the efficient application of such alternative processes requires the availability of reliable on-line measuring tools for cell density and cell metabolic activity estimation [ ] . the comparison of different culture strategies for hek cell line producing ifn-γ are presented below: batch, fortified batch and fed-batch. in this context, a new robust feeding strategy based on the monitoring of alkali buffer addition was applied for the estimation of nutrient requirements. this method allows to increase cell density and product titer compared with the other strategies assessed. three different culture strategies were carried out in -litre biostat b-dcu ii bioreactor. first, a reference batch and a batch using fortified medium (nutrient enriched medium) were run and assessed in terms of viable cell density (vcd) and product titer, and set as initial references. then, a fed-batch was performed applying a feeding strategy based on the nutrient requirements estimation by monitoring the alkali buffer addition used for the control of ph. results vcd and product titer achieved for the different culture strategies assessed (batch, fortified-batch and fed-batch) are presented in table . in fortified batch an increase in vcd of % and also % in product titer were obtained compared with batch. in the fed-batch culture carried out (fig. ) , we observed that alkali buffer addition profile matched the vcd evolution trend. thus, the monitoring of alkali buffer addition was used for estimating the nutrients requirements (i.e. the volume of feeding medium) at any time during the fed batch phase. the feeding strategy based on alkali buffer addition enabled to maintain glucose concentration set point therein a narrow range during fed-batch phase (around mm). as a result, higher vcd ( . · cells/ml) was obtained when compared with both batch references: vcd was enhanced to % and % and an increase up to % and % in product titer in respect to batch and fortified batch respectively. the results prove that fed-batch strategy based on the alkali buffer addition is a robust on-line monitoring method that enables to optimize the feeding strategy in a fed-batch cultures. three different culture strategies have been tested in bioreactor with a hek cell line producing ifn-γ. results show as the higher vcd is reached, the higher product concentration is achieved. therefore, from bioprocess development point of view, it is very interesting to implement strategies with higher vcd outcome, such as fed-batch operation mode. in this context, a new robust method for vcd estimation in fed-batch was applied. the alkali buffer addition necessary for maintaining the ph set-point is an on-line reliable and easy measuring variable that provides information about by-products formation (mainly lactic acid). the monitoring of this variable can provide information about the cell concentration, activity and metabolism, to detect changes in culture. besides that, a relationship between alkali buffer addition and vcd can be established since the first is strongly correlated with cell growth and metabolites consumption/formation. the application of alkali buffer addition measure to implement an optimal feeding strategy in fed-batch permits to enhance vcd and product titer when comparing with batch strategies. a novel approach to high throughput screening for perfusion background perfusion systems for suspended mammalian cells raise growing interest in the biomanufacturing industry. continuous manufacturing is growing in the field and is encouraged by health authorities [ , , ] . this work addresses scale down limitations inherent to continuous media exchange and cell retention by using a semi-continuous system. data was generated with a set of different clones that were previously studied in fed-batch mode [ ] . materials and methods cho-k cell lines expressing the same monoclonal antibody (mab) and issued from the same transfection were used as models. . l bioreactors (sartorius) were used for fed-batch and perfusion production runs. the perfusion bioreactors were run using an alternating tangential flow filtration device (repligen, xcell™ atf system). the cell biomass was controlled by removing cells through a bleed line and was controlled using a biocapacitance probe (hamilton, incyte). the perfusion rate (d) was fixed to one vessel volume a day (vvd - ). the semi-continuous runs were made in ml shake tube (tpp, tubespin® bioreactor ). once a day, the tubes were centrifuged ( min, g), the supernatant removed (to mimic a perfusion rate of vvd - ), replaced with fresh media and cells were re-suspended. the clone's growth potential were preserved across the systems (fig. ). clone # always reached the highest viable cell density (vcd), followed by clone # . clone # and # showed similar growth characteristics. it is interesting to note that in the perfusion bioreactor different patterns in terms of vcd were observed although the cell biomass signals were similar for all runs. this reflects the fact that the capacitance measures the biomass and not the absolute cell count [ ] . to estimate the minimum cell specific perfusion rate (cspr min ) in the semi-continuous experiment, the perfusion rate was divided by the maximum viable cell density (vcd max ). this value was compared to the cspr obtained during the th set-point (sp ) of the perfusion runs. as expected, the bleed fraction decreased when the capacitance set-point was increased and went down to % or less of the total perfusion rate (data not shown). since the bleed removes the excess biomass, it is an indication of how close to a limitation the system is. therefore, the cspr calculated at sp was considered as the minimum cspr. the cspr min obtained in both systems were very close ( table ). the semi-continuous system can therefore be used to identify the cspr min before running a continuous bioreactor, it therefore facilitates the decision making early in the development (to define the target cell density for a defined perfusion rate). the specific productivity (q p ) of the clones was quantified at the maximum vcd (semi-continuous) or at sp (perfusion). absolute values are not representative since the cell environment is so different in both systems. nevertheless, a relative ranking proved to be indicative of the respective performances ( table ). the maximum cell growth in fed-batch, semi-continuous and perfusion were also compared, their ranking was always preserved. both indications can be used to assess a performance ranking for different clones. the performance of clones was studied in different cultivation systems: fed-batch/perfusion bioreactors and semi-continuous shake tube. the semi-continuous system was able to precisely predict the cspr min , an important process parameter for perfusion. specific productivity and maximum cell density ranking was preserved across the systems, therefore the scale down experiment can be used to assess a performance ranking for perfusion clone screening. modulating antibody galactosylation through cell culture medium for improved function and product quality jenny y. bang, james-kevin y. tan the production of therapeutic antibodies (abs) requires high titers and excellent product quality to ensure efficient manufacturing and potent drug efficacy. glycosylation, or the attachment of sugars to organic molecules, is a critical quality aspect that can significantly alter ab binding, function, and therapeutic effect [ ] . galactose is a key sugar of interest due to its significant impact on ab function and the ability to control galactosylation through cell culture medium. herein, irvine scientific assessed the ability of media components to modulate galactose levels on a model therapeutic ab. various media compositions were able to modulate galactosylation levels without compromising cell growth and ab titers. in addition, an in vitro assay was utilized to evaluate the functional ability of abs to bind and activate complement-dependent cytotoxicity (cdc). differences in galactosylation significantly altered the abs' ability to induce cell cytotoxicity. furthermore, design of experiment analysis determined the optimal ratio of supplements to maximize galactosylation. this "optimized supplement" was verified and evaluated against other suppliers' galactosylation supplements in terms of growth, titer, glycan analysis, and ab function. the optimized supplement outperformed all other suppliers' supplements and resulted in the best overall cell growth, titer, galactosylation, and ab function. ab against cd were grown in balancd® cho growth a and were fed with balancd® cho feed on days - of the cultures. viable cell density and cell viability were assessed by a beckman coulter vi-cell xr, ab titer was assessed by a pall fortébio qk e , and glycan analysis was assessed by a perkinelmer labchip gxii. for the functional cdc assay, abs were incubated with daudi b lymphoblast cells and normal human complement serum. cell cytotoxicity was assessed with a promega cytotox-glo kit. various supplements were evaluated in fed-batch cultures and resulted in - % ab galactosylation without compromising cell growth and ab titers. design of experiment analysis determined an optimal composition, deemed "optimized supplement," which was evaluated against a panel of galactose-modulating supplements from other suppliers. the optimized supplement resulted in a similar viable cell density (vcd) and cell viability compared to the fed-batch culture control which had no supplements (fig. a) . supplements from supplier resulted in similar to half the vcd of the control while supplements from supplier resulted in very low vcd and percent viability. all of the supplements except those from supplier resulted in ab titers similar to the control (fig. b) . due to the poor growth and subsequently low titer from supplier 's supplement, supplier was not further evaluated. the glycan profiles were analyzed and are presented in (fig. c ). all the evaluated supplements were able to raise galactosylation; however, only the optimized supplement and the x supplier supplement resulted in over % galactosylation. the function of the abs was further evaluated in a cdc assay (fig. d) . abs from the optimized supplement were more effective than the control abs and had a significantly lower half-maximal effective concentration (ec , . μg/ml) than the control ( . μg/ml). abs from the x supplier supplement had a similar ec to the control which may be due to the higher man % of the abs. an optimized supplement was produced through fed-batch evaluation and design of experiment analysis. the optimized supplement outperformed all other supplenments from other suppliers and resulted in the best overall cell growth, glycan profile, and functional ab activity (table ) . industry practice for mammalian cell culture media and feed development typically employs a high-throughput screening (hts) platform along with large sets of experiments [ ] . modern hts systems often include robotic liquid handlers to replace labor intensive steps. to align with advancements in the field, a semi-automated hts platform was developed to facilitate in-house media and feed development for early stage biologics projects. selecting appropriate instruments and integrating them into a seamless system are the keys to a hts platform. the developed hts platform uses deep well plates (dwps) for culture vessels, the liquid handler of the advance microscale bioreactor (ambr ) for media/ feed formulation preparation in an aseptic environment, nyone cell imager for viability and cell growth analysis, tecan freedom evo's liquid handler for activity assay sample preparation, and cedex bioht for high-throughput metabolite analysis. dwps offer comparable cell growth to shake flasks and compatible layout to ambr , which makes the dwp an ideal candidate for the platform. in addition, the user friendly design of experiments (does) interface and liquid handler function of the ambr expediates the formulation preparation of varying doe conditions [ ] . a macro program was written and developed in excel to enable the easy import of does design from major statistics software packages, such as jmp and simca, into ambr . performance qualification of each component were performed prior to implementing the hts platform. comparable cell growth profile and productivity were achived between shake flasks and dwps (fig. a ), indicating compatable cell culture environment for the cells. cell counts using nyone gave identical cell growth ranking as the traditional count from vi-cell xr (fig. b) . freedom evo's liquid handler was optimized to produce comparable activity results to manual operation while expediting the sample preparation with improved consistency (fig. c) . finally, implementing the liquid handler function of ambr to support media and feed formulation significantly reduced the labor for each experiment. summary of the capability comparison between the hts platform and the traditional method are listed in table . a case study of a complex feed screening with definitive screening design was completed using the semi-automatic hts platform. this experiment, containing more than feed formulations in duplicates, was handled by one operator and delivered a % improvement in productivity within a week period (data not shown). in addition, implementing the hts platform for this study also resulted into~ % reduction in labor while improving the traceability of formulation preparation. a semi-automated hts platform was developed to support media and feeds screening and development for early stage biologics projects. the platform utilizes dwps, nyone cell imager, ambr , freedom evo liquid handler system, and bioht metabolic analyzer to accelerate the screening process. this screening platform not only improves process throughput, operational precision, and traceability of formulation preparation, but also reduces the labor for the media and feed formulation preparation. background a perfusion medium requires high concentrations of specific nutrients while balancing other components to support intensified perfusion processes. using a combination of design of experiment (doe), multivariate analysis (mva), and spent media analysis, we developed a catalog "de novo" perfusion medium by working with multiple cho cell lines and proteins. the optimization of the medium in bioreactors using alternating tangential flow (atf) cell retention devices reduced the minimum cspr from over pl/cell/d to under pl/cell/d for most cell lines while increasing specific productivity during day steady states with stable growth rates, viability, volumetric productivity and product quality. high throughput screening (hts) was performed with seven cell lines, while four were used in bioreactors: cho-s, dg , and two chozn® gs lines, each producing different monoclonal antibodies and include a fusion protein. for hts experiments, cells were inoculated at . x vc/ml with a ml working volume in ml tpp® tubes and cultured for days in a multitron shaken at rpm, °c, % rh, and % co . for benchtop perfusion, cells were inoculated at . - . x vc/ml in l applikon bioreactors (applikon, netherlands) with a l working volume. bioreactors were operated at rpm, °c, % do, and a ph of . or . ± . depending on the cell line. oxygen was supplied through an l-sparger or microsparger as needed, and excell® antifoam (milliporesigma, germany) was added at a maximum rate of . % v/v to control foam. at a cell concentration of~ . x vc/ml, perfusion was initiated using the atf (repligen, massachusetts), with a bleed set to maintain cell concentrations at or * vc/ml. two "de novo" prototype media were developed using doe and mva in hts with tpps and an ambr® [ ] and one was chosen for further development after comparing to a basal medium enriched with feed in bioreactors. eleven components were identified as significant effectors of critical parameters for perfusion processes across evaluated cell lines. doe central composite experiments were run and component concentrations were optimized in the selected prototype. in parallel, amino acid specific consumption rates were calculated from bioreactor spent media samples and used to adjust the concentration of amino acids to target a reduced cspr. increasing specific amino acids concentrations resulted in a significant reduction of the minimum cspr across all tested cell lines -for example the cspr of cho-s was reduced from to pl/cell/day (table ). however, even at the lower cspr, spent media analysis revealed excess concentration of some amino acids, so specific accumulating amino acids were reduced and components were streamlined for the final medium: excell® advanced hd perfusion medium. using this medium, a cho-s and a chozn® gs cell line producing a fusion protein were cultured at a cspr of less than pl/cell/day with a vcd of * vc/ml. metabolic profile, productivity, and product quality were constant over the day steady state. the chozn® gs cell line was also tested at * vc/ml with a cspr of pl/cell/day (fig. ). we have developed a catalog perfusion medium from first principles, ensuring broadness of application by using seven cell lines in scaleddown systems and four in perfusion bioreactors. the final catalog medium showed significant improvements in productivity across all cell lines, with reduced csprs when compared to enriched fed-batch medium or initial prototypes (table ) . there is a rising demand for accelerated process development, increased efficiency and economics for biopharmaceutical production processes. furthermore, increased process understanding have evolved from the process analytical tool initiative (pat) and the quality by design (qbd) methodology. in contrast to one-factor-at-atime methods, statistical design of experiment (doe) methods are widely used to develop biopharmaceutical processes. even if highthroughput systems can handle these numbers of experiments in parallel, the heuristic restriction of boundaries and the high number of factors results in stepwise iterations with multiple runs. therefore, the combination of model-based simulations with doe methods (mdoe) for the development of sophisticated cell culture processes is a novel tool for process development [ ] . it is used to reduce the number of experiments during doe and the time needed for the development of more knowledge-based cell culture processes. this concept was applied to the optimization of the initial glutamine and glucose concentrations of a cho batch process. a mechanistic model was adapted and modified from [ ] and used to describe the dynamics of cell metabolism and antibody production of an il- antibody producing cho cell line (see abbreviation of fig. for cultivation details). experiments were simulated and compared to a fully experimental doe. as can be seen from table , user defined constraints were chosen to get a stable and reproducible process with the aim of maximizing the cell density but decreased lactate and ammonia production. at first, the experimental space was estimated by simulating the responses for broad concentration ranges and calculating the multiple response desirability function (fig. a) . this results in a small area (turquoise) suggested as experimental space. experiments were planned within these boundaries and responses were either simulated ( fig. b, cultivations for fitting the model) or compared with the purely experimental responses (fig. c, cultivations). optimal concentrations for glutamine and glucose with respect to the constraints are in the lower right corner and similar for both methods (red frame, fig. ). compared with the fully experimental design, mdoe results in a reduction of % in the number of experiments ( experiments for modelling vs. experiments in experimental doe). the method is intended to optimize cultivation strategies for mammalian cell lines and evaluated these before experiments have to be performed in laboratory scale. this results in a significant time and cost reduction during process development and process establishment. the strategy is especially intended for the use in multi-single-use-devices to speed up process development. . at a target cell density of * vc/ml, volumetric productivity was stable for a day steady state with excell® advanced hd perfusion medium. shorter steady states were tested at * vc/ml background for the large-scale production of therapeutic glycoproteins, fedbatch culture has been widely used for its operational simplicity and high titer. however, repeated feeding of medium concentrates and/ or addition of a base to maintain optimal ph during fed-batch culture lead to increase in osmolality. the hyperosmolality affects glycosylation in a protein-specific manner. however, the mechanism behind such osmolality-dependent variations in glycosylation in recombinant chinese hamster ovary (rcho) cells remains unclear. in this study, to better understand the effect of hyperosmolality on the glycosylation of a protein produced from rcho cells, we investigated n-glycosylation-related gene expression and n-linked glycan structure in fc-fusion protein-producing rcho cells exposed to hyperosmotic conditions. furthermore, to validate the effect of hyperosmolality on protein glycosylation, we performed hyperosmotic culture supplemented with betaine, an osmoprotectant, and then analyzed the n-linked glycan structure and mrna levels of n-glycan branching/antennary genes. after three days of hyperosmotic culture, nine genes (ugp, slc a , slc d , gcs , manea, mgat , mgat b, b galt , and b galt ) were differentially expressed over . -fold of the control, and all these genes were down-regulated. n-linked glycan analysis by anion exchange and hydrophilic interaction hplc showed that the proportion of highly sialylated (di-, tri-, tetra-) and tetra-antennary n-linked glycans was significantly decreased upon hyperosmotic culture. addition of betaine, an osmoprotectant, to the hyperosmotic culture significantly increased the proportion of highly sialylated and tetra-antennary n-linked glycans (p ≤ . ), while it increased the expression of the n-glycan branching/antennary genes (mgat and mgat b). thus, decreased expression of the genes with roles in the n-glycan biosynthesis pathway correlated with reduced sialic acid content of fc-fusion protein caused by hyperosmolar conditions. conclusions taken together, the results obtained in this study provide a better understanding of the detrimental effects of hyperosmolality on n-glycosylation, especially sialylation, in rcho cells. the identified genes, particularly mgat and mgat b, are potential targets for engineering in cho cells to overcome the impact of hyperosmolality on glycoprotein sialylation. disruptive cost-effective antibody manufacturing platform based on cutting-edge purification process v. medvedev, m. duyck, t. albano, j. castillo univercells sa, gosselies, belgium correspondence: v. medvedev (v.medvedev@univercells.com) bmc proceedings , (suppl ):p- background demand for high-quality monoclonal antibodies is growing exponentially, calling for new production capacities. overcoming current limitations of conventional manufacturing strategies, namely the high capital investment and production cost, can only be achieved through innovative process designs based on the latest technologies. this study presents a process design combining batch-fed technology with continuous multi-column capture. an advanced cell culture clarification method was introduced to simplify downstream operations and increase overall cost-effectiveness of the process, for an optimized production of recombinant proteins. this study was performed with cho cells expressing a monoclonal antibody targeted against the coronoavirus responsible for the middle east respiratory syndrome (mers), developed by organic vaccines tm and the nih, kindly provided to univercells. upstream process: -fed-batch, days culture at l scale with cd-cho chemically defined media and feeds. harvest treatment: -precipitation of impurities in the production bioreactor using organic compounds (< % v/v) and flocculation by electropositive organics (< . % w/v). -acidic ph and physiological conductivity. upstream processing and harvest treatment: culture reached . g/ l ( x cells/ml; % viability), harvest treatment was found to be very effective in terms of impurities clearance. capture: capture strategies were evaluated from the point of view of simplification of downstream operations, with hcp impurities content monitored as a key performance indicator. -protein a affinity chromatography: advanced harvest clarification enabled major improvements in affinity capture, in terms of eluate purity and reduction of host cell impurities (< ppm in all conditions tested). (fig. ). -cation exchange chromatography: cex allows higher capacities (> g/l) than protein a, whilst being more affordable (from -to -fold cheaper). low residual hcp (< ppm) was observed with all cex resins tested. without harvest treatment and clarification preceding the capture studies (either affinity or cex), results showed a lower binding capacity of the resin, a higher content of hcp in the eluate (up to ppm), a higher content of hmw species in the elution fraction (up to -fold higher) and a significant turbidity of the neutralized eluate. -continuous multicolumn chromatography: further options to increase cost efficiency include using a continuous multicolumn setup (table ) . two models were assessed based on two different static binding capacities (sbc), demonstrating that to columns of ml were able to process a l production in less than h. this method provides a great opportunity for designing simplified and low footprint mabs dsp processes, while maintaining similar or achieving superior quality profile compared to standard approaches: -harvests treatments followed by depth filtration proved to be a cost-efficient way to obtain pretreated feed and minimize the burden on downstream operations. -protein a resins exhibited advantages of extracting key contaminants during harvest treatment, while caex confirmed to be a competitive capture strategy. -switching from batch to continuous multicolumn mode allowed to process a complete batch in less than hours, requiring lower media and resins volumes. followed by a single polishing step, such process set-up strongly supports the reduction of operations required to deliver a high-quality product. analyses of product quality of complex polymeric igm produced by cho cells background immunoglobulin m (igm) antibodies are secreted by b cells as the first defense against invading pathogens during primary immune response. some igm antibodies already gained the orphan drug status, which shows their unique capability in therapy of rare diseases. potential fields for applications are discovered with increasing knowledge about these molecules. it seems that the most active forms are pentameric and hexameric igms. unfortunately, recombinant production of igms is rather difficult as secretion and correct polymer formation results in low expression yields and mixtures of polymers. we established stable producing chinese hamster ovary (cho) dg cell lines to analyze cellular and extracellular factors that influence quantity and quality of the produced recombinant polymeric igm in future studies [ ] . one quality parameter is polymer distribution, which can be measured directly in cell culture supernatant using densitometric analyses [ ] . additionally, we developed a very efficient single-step-affinity purification strategy using the poros captureselect igm affinity matrix to analyze pure igms. for more precise measurements of the igm isoform distribution we separated the purified polymers by high performance liquid size exclusion chromatography (sec hplc). our cho dg cell lines grow to peak cell concentration of . x cells/ml in erlenmeyer flasks and . x cells/ml in bioreactors. similar productivity of approximately mg/l was observed for cells cultivated in both cultivation vessels in a nonoptimized batch culture using chemically defined media. analysing how cultivation conditions affect the fraction of polymers may offer clues about the assembly of polymers and the challenges of igm production. we quantified polymeric distribution of igm directly in the supernatant using a densitometric method [ ] . cultivated under standard conditions ( °c, ph ) igm is produced as % pentamers, whereas igm _gl only consists of approximately % pentamers. the purified igm _gl was analysed with sec-hplc and contained % pentamer and % dimer, which is comparable to the results achieved with densitometry. the purification of the igm antibodies was quite challenging as the manufacturer recommend acidic elution, which led to aggregation and inefficient elution of our model igms. therefore, we screened for different elution buffers that prevent denaturation and aggregation. by combining high salt concentrations with moderate ph reduction we optimized elution conditions to - % igm recovery, which corresponded to a five to six fold improvement compared to the manufacturers' conditions. sds-page analysis and sec-hplc showed that our elution strategy resulted in a very pure product after a single chromatographic step. the purification strategy was verified with the igm , igm and igm . our model igms were produced in a ratio of approximately : pentameric to dimeric igm, measured concordantly with both analytical methods. process development on igm purification using the poros capture select human igm affinity matrix enabled the recovery of highly pure fractions. through optimization, by combining mild ph and high salt concentrations, the relatively low elution yields were increased by a factor of - . applying densitometry and sec-hplc we will investigate how culture conditions influence polymer formation in future. currently, no small scale (< . l) cell culture system is commercially available for high cell density perfusion cultivations to use in high throughput screening studies. to increase throughput for process characterization activities at janssen vaccines and prevention, a shaker flask-based scale down model was developed. though, the control possibilities of shaker flask cultures are technically very limited and different compared to a bioreactor controlled process. in addition, the sensitivity of the shaker flask model should allow the detection of the effects of process parameters on critical quality attributes (cqas) of the vaccine produced at large scale. iterative experiments were performed in shake flasks to evaluate the influence of cultivation parameters such as shaking speed, working volume, co % in the incubator and daily base additions on cultivation parameters (as cell growth, ph and do). in addition, a medium exchange was tested to mimic the perfusion mode used in the bioreactor process. the presens shake flask reader was implemented to allow for ph and do monitoring. the conditions for which the performance as reflected in specific virus titer showed the best fit were selected. at these conditions, a series of parallel shaker flask infections were conducted to demonstrate statistical equivalence of performance parameter and cqas (as cell specific iu titer and vp/iu ratio) between the production scale and reduced scale processes and thus to qualify the shake flask as a scale-down model. a daily medium exchange by centrifugation was implemented and cultivation parameters for shake flasks were identified. based on performance parameter (cell specific vp titer) and the cqas of the vaccine (cell specific iu titer and vp/iu ratio), equivalence between the production-scale and scale down systems was confirmed. the scale down model data fall into the % prediction intervals calculated on manufacturing data whereas scale down model data from batch mode experiments (using non optimized cultivation conditions) do not. the shaker flask as a scale down model for the l bioreactor perfusion process was qualified. this model is a tool to screen a subset of process parameters at a higher throughput, thereby reducing process characterization timelines. background until today, the market for therapeutic proteins, especially monoclonal antibodies, is gaining more and more importance in the pharmaceutical field. to meet the increasing demand for these products, the industry made tremendous efforts to generate highly efficient production systems. one of the pharmaceutical industry's research focuses is the improvement of the secretion process in eukaryotic cells. in mammalian cells, the efficiency of protein transportation strongly depends on the translocation of a nascent protein into the er, which is mostly conducted by the signal peptide (sp) coupled to the nterminus. through the interchangeability of signal peptides between products and even species, a large variety can be used to enhance protein expression in already existing production systems materials and methods at first the influence of four different natural sps (sp ( ), ( ), ( ) and ( )) was compared on the secreted amount of an igg model antibody (product a) in fed batches using a cho dg host cell line. in the second part, one promising sp-candidate showing improved secretion (sp ( )) was identified and the influence of this sp on four additional antibody products, which varied in their expressability from good to mid/bad, was investigated. in both approaches, the standard sp was implemented for comparative reasons. in the first approach, four signal peptides sp ( ), sp( ), sp( ) and sp( ) were screened for their potential to improve the product secretion of cho dg cells expressing a model antibody (product a). the results revealed a . -fold increase in average final fed-batch antibody titer of sp( ) when compared to the standard sp approach (standard sp = . g/l; sp( ) = . g/l). in the second approach, the enhancing capacity of sp( ) on secretion of four other igg products (named product b to e, table ) was further evaluated. an improved performance was observed for all products when comparing sp( ) and the standard sp in a fed batch process (fig. ) . with an increase in average final fed-batch titers ranging from to % and up to % in cell-specific productivities. taken together, with a positive influence on the final concentrations of all tested products, the results obtained with sp( ) contribute to -signal peptide sp( ) was identified as a promising candidate with an average . -fold titer increase during screening of four signal peptides. -sp( ) was able to improve production titers up to % compared to standard sp. -sp( ) was able to improve cell-specific productivities up to % compared to standard sp. -future usage of sp ( ) contributes to the further optimization of sartorius stedim cellca's standard cell line development process. new platform for the integrated analysis of bioreactor online and offline data lukasz gricman , milan ganguly , amanda fitzgerald , hans peter more and more experiments are used to assess bioreactor suitability and stability of clones, to evaluate media composition and other process parameters, and to start upscaling campaigns. this has resulted in a major bottleneck due to the increase in data capturing, processing, aggregation, visualization, and statistical analysis. in addition, the association of the data with the experimental context (e.g., fermentation protocols, media recipes, bioreactor control parameters) is not easily accomplished in high throughput. the data generated in the process must not only be analyzed, but also managed and stored to enable easy tracking and relating to historical records. furthermore, the processes are often developed by global teams interacting in complex enterprise it ecosystems. therefore, new and high performing systems for data capture, processing, and analysis need to be integrated in order to enable storage and correlation of experimental context information and various types of time course analytics data. we have developed genedata bioprocess™, a new enterprise platform for bioprocess development. the platform enables automatic capture and visualization of all online and offline data (e.g., ph, o , metabolic data), auto-calculations and aggregations (e.g., ivcd, qp, consumption rates) and multi-parametric assessment of any type of time-series bioreactor data in the context of experimental protocol data (e.g., process parameters, feeds). genedata bioprocess comes with dedicated interfaces for integrating with relevant laboratory instruments, control systems, statistical analysis software packages and custom enterprise solutions. it enables the modeling and tracking of complex nonlinear workflows and supports decision making in bioprocess development. the data can be analyzed in the context of upstream process development, and also be correlated to other unit operations. automation support assists the ever increasing throughput of bioprocess development operations, and the analysis of experimental data and process parameters across unit operations or even different projects. this overall integration enhances process development workflows. highlighted use cases describe the selection of the best producer clones (fig. a) , the identification of optimized media feeding strategies (fig. b) , and the comparison of clone performance across different fermentation scales (fig. c) . a special focus is on the analysis of data from micro-and bench-top bioreactors (such as the ambr ™ and dasgip™ systems) operated in parallel. these bioreactors allow for increased throughput of clone selection and process optimization studies, which in turn leads to an increase in data generation. genedata bioprocess supports integration with such systems and enables a comparison of data regardless of the instrument provider or scale. automated bioreactor data analysis allows development groups to take advantage of even richer datasets and, as data management is built-in to the system, the data can be easily tracked and associated to historical records. another focus is on cross-reactor scale comparisons. data coming from different bioreactor scales can be easily imported into the platform and analyzed to establish the best conditions for upscaling. genedata bioprocess enables the correlation of process parameters (e.g., fermentation protocols, media recipes, bioreactor control parameters), with key performance indicators of the processes (e.g., titer, qp) and the product quality attributes (e.g., aggregation, glycosylation profiles). finally, bioreactor time course data can be tracked together with clone analytics and product quality parameters, which makes the platform uniquely able to support end-to-end biopharma development. upstream bioprocesses are at particular risk of contamination from adventitious agents. the typical . μm filters used at this step protect bioreactors from bacteria and mycoplasma but offer no protection from viral contaminations. a new polyethersulfone (pes) upstream virus filter, viresolve® barrier, has demonstrated high levels of microorganism retention -full retention for bacteria and mycoplasma (> . lrv -log reduction value) and~ lrv for small viruses, such as parvoviruses. it also has improved flow and capacity as compared to virus removal filters designed for monoclonal antibody purification. given the small pore size of virus retentive filters, implementing a virus filter upstream of the bioreactor raises the question of whether critical cell culture media components are removed. therefore, it is important to evaluate the cell culture performance and protein quality attributes using virus-filtered media to ensure that filtration does not negatively impact the process. materials and methods ex-cell® cho media and corresponding feeds were processed through either viresolve® barrier filters or . μm filters (control). media composition post-filtration was evaluated by high performance liquid chromatography (hplc), inductively coupled plasma/ optical emission spectrometry (icp-oes), and nuclear magnetic resonance (nmr). recombinant cho cells were cultured in fed batch culture. cell density and viability were measured by vi-cell tm cell viability analyzer while metabolites were analyzed by bioprofile® flex analyzer. shake flasks and bioreactors were utilized to verify that surfactants, such as poloxamer, (which are essential for shear protection in stirred tank bioreactors and can be difficult to filter) have not been removed during filtration. monoclonal antibody titer was quantitated by protein a hplc. characterization of the antibody product quality was assessed via weak cation-exchange chromatography (charge heterogeneity), size exclusion chromatography (aggregate profile), and -ab fluorescent labeling with np-uplc (glycan species). media and feed compositions were unaffected by filtration through the virus barrier filter. no significant differences in concentrations were observed with icp-oes (trace metals) or hplc (amino acids and water soluble vitamins). nmr showed no change in the organic composition of the media including poloxamer. the aromatic region with vitamin and amino acid signals is shown (fig. a) . cell cultures showed no differences in cell growth or titer, in either shake flasks or bioreactors (fig. b) . cell viability was unaffected, metabolite levels were within limits, and titer was consistent. the protein quality of the secreted antibodies showed no differences in the glycosylation pattern (fig. c) , amount of aggregates or charge variants. the risk of virus contamination in the bioreactor remains a concern for biotherapeutic manufacturers as there is no universal technology that provides a reliable, cost effective solution for virus removal that can be applied to all components of cell culture media. this study evaluated the viresolve® barrier filter that provides an efficient and easy way to protect bioprocesses from adventitious virus contamination. study results demonstrated that media and feed compositions, cell culture performance, and product quality were unaffected by filtration through the viresolve® barrier filter. implementation of vire-solve® barrier filters provides efficient filtration performance, high virus retention, and minimal cell culture impact and offers a viable option to improve the overall virus risk mitigation strategy for the manufacture of biotherapeutics. b tracking of process conditions together with online and offline performance analytics. the system allows to flexibly define tracked parameters and select optimal process conditions. c comparison of process performance across different reactor scales. the open architecture makes genedata bioprocess a provider agnostic system which allows to aggregate and compare data regardless of provider background bi-and multi-specific antibodies, antibody-cytokine fusion proteins, nonimmunoglobulin scaffolds, chimeric antigen receptors (cars), engineered t-cell receptors (tcrs) and tcr-based bispecific constructs can provide significant advantages for use in cancer immunotherapy. however, as highly engineered molecules they pose new challenges in design, engineering, cloning, expression, purification, and analytics. we have thus implemented an infrastructure that addresses these challenges and enables the industrialization of these various novel therapeutic platforms. in close collaboration with leading biopharmaceutical companies, we implemented a workflow, data management and analysis support system, genedata biologics™, enabling the automated design, screening, and expression of large panels of therapeutic candidates using these novel technologies. we have also built tools for developability and manufacturability assessments of these complex molecules. we have ensured that there is a seamless integration of all data generated and that functionalities such as bulk protein and vector generation using our in silico cloning engine, configurable library of template vectors and cloning strategies, fully annotated in silico protein molecules and dna constructs, and dna synthesis verification support, can be used for the newest protein formats and molecule topologies. we implemented data structures and data handling systems, which mirror how these complex next-generation biologics molecules and cell lines are being designed, screened, and analyzed. the result successfully addresses workflows for tcr optimization and engineering. we exemplified this with the generation and evaluation of a panel of engineered tcrs with an alpha chain cdr randomization and successfully supported the analysis and selection of beneficial mutations. the system also successfully supported workflows for the design and generation of a panel of tcr-based bispecifics (tcr coupled with anti-cd ) using automated molecule registration and in silico cloning tools and subsequent capture of expression, purification, and functional and analytical characterization data. on the car-t cell front, the system is able to provide traceability of the work from antibody generation, optimization, car engineering (e.g., attachment to the scfv with cd -zeta and co-stimulatory domains to mimic the natural tcr complex) to the engineering of the t-cell. the genedata biologics platform successfully enabled automation, increased data integrity and traceability during research and development work, and will contribute towards the industrialization of these very exciting novel approaches for cancer immunotherapy. optimal selection of therapeutic antibodies and production cell lines by assessment of critical quality attributes and developability background the increasing cost of bringing a new drug to the market has put significant pressure on biopharma organizations. to increase efficiency in r&d processes and reduce costs, organizations need to evaluate potential drug candidates earlier in the r&d process, eliminate those with undesirable characteristics, and focus on the most promising candidates. after designing and thorough testing of successful candidates, efficient production of new biological entities in mammalian cell lines is necessary. the main goal here is to find a suitable cell line and optimal upstream and downstream processing conditions that not only lead to a satisfactory product yield, but also to a product with the desired biochemical properties. the evaluation of production cell lines, processes, and product quality attributes is performed earlier and in higher throughput for an increasing number of drug candidates. in addition, new methods in molecular and cell biology (e.g., novel genome engineering approaches such as crispr/cas ), in analytics [e.g., process analytical techniques (pats)], in process miniaturization, and in automation promise to make process development more efficient. however, the management and analysis of the increasing amount of experimental data during candidate selection and cell line and process development has become a bottleneck. in addition, quality-compromising steps in biopharma organizations can negatively impact the cost of goods and substantially prolong the drug candidate's time to market. therefore, systems for integrated management and analysis of wellstructured and curated data that comprehensively integrate molecule and sample information, manufacturing process parameters, and process and product quality attributes are needed. critical quality attribute (cqa) assessment should be enabled along the whole bioprocess development workflow, including cell line development, upstream and downstream process development, as well as analytical and formulation development. we have developed a comprehensive platform, genedata bioprocess™, which supports drug candidate developability and manufacturability assessment and bioprocess development. the platform captures and structures the cell line and process parameters together with analytical data for cell lines, processes, and protein products. the protein analytical data being tracked include biological data (such as bioactivity, immunogenicity), and physicochemical properties. these properties include glycosylation, chemical liabilities (such as deamidation and oxidation), aggregation, stability under different conditions (low ph, low and high temperature), solubility, and impurities. genedata bioprocess™ simplifies and streamlines laborious, manual process and supports tools for molecule, clone and process selection. furthermore, the platform allows for seamless integration with laboratory instruments, statistical software packages, and custom solutions. here, we present use cases showing how to identify and annotate liability sites prone to chemical modifications (fig. a) and how to monitor cqas of molecules allowing to assess developability more efficiently. we show how the analytical data generated in the course of a developability assessment are compiled to select the best drug candidate (fig. b) . implemented traffic-light systems indicate where molecules harbor issues such as in case of the antibody tpp- , which is compromised by low temperature and repeated freeze-thaw operations. the same assessment views can also be applied on batches and cell lines. the underlying data can be visualized graphically. as an example, we show glycan types of products obtained from different cell line clones generated in a cell line development campaign for the molecule tpp- (fig. c) . even though the selected clone cli- meets the glycosylation criteria (e. g., < % afucosylation, < % galactosylation, < % sialylation), the produced next-generation biologics molecules are composed from a number of specific subdomains. each type of molecule is composed of a specific set of domains, which must be mirrored in the registration and further research and development workflow. molecule registration and hit-selection using data from a number of assays is shown here using the example of car-t cells. the image is a screenshot from the genedata biologics™ software molecule harbors some stability issues as mentioned above. therefore, more attempts would be needed either in formulation or in reengineering of the complimentarity determining regions (cdrs) in order to provide a developable ttp- -like drug candidate. background environmental process variables are often used as tools to optimize the performance of mammalian cell cultures to achieve higher cell densities and high productivities of r-proteins (q p ). the manipulation of culture temperature in the range of mild hypothermia (mh) ( - °c) [ , ] , as well as different glucose availability scenarios [ , ] , has been shown to improve productivity in different cell lines. however, the manipulation of these variables individually or together has a concomitant effect on the rate at which cells grow, masking the net response exhibited by the cells. in order to identify the effects of these variables, we have taken advantage of the use of the chemostat culture. chemostat cultures were performed at two dilution rate (d)( . or . (h- )), two temperatures ( or °c) and three feed glucose concentrations ( , or mm). the response was analysed considering r-protein production, cell growth and key metabolites. r-tpa protein concentration was determined by immunoassay (trinilize tpa kit); cells were counted using a hemocytometer and cell viability was determined by the method of exclusion using trypan blue (t , sigma, usa); glucose, lactate and glutamate were determined by enzymatic assay using a biochemical analyser ysi (yellow spring instruments). statistical analysis of the results was performed by anova (design-expert for windows). a decrease in cell density was observed in response to an increase of glucose feeding concentration, regardless of temperature or specific growth rate (in this case μ=d) evaluated. the maximum cell densities were reached at mm, achieving . and . x cells/ml at / °c and . (h - ); and . and . x cells/ml at / °c and . (h - ) respectively (fig. a) . the increase in glucose concentration from to mm resulted in an q p increase of and . fold at °c/ . (h - ) and °c/ . (h - ) respectively. a lower increase of . and . fold was reached at °c/ . (h - ) and °c/ . (h - ) respectively (fig. b) . the highest q p s were reached at °c and . (h - ). however, a positive effect of mh was not observed, in contrast to that observed in batch culture [ , , ] . this behaviour suggests that low μ is a main factor on increased r-protein production in batch cultures exposed at mh condition. the specific consumption rate of glucose was significantly increased by the glucose increase from to mm and reduced by mh (fig. c) . at . (h - ) the specific production rate of lactate (q lac ) was increased by glucose increase, independent of the culture temperature used. while at °c/ . (h - ) the q lac decreased with increasing glucose concentration and at °c/ . (h - ) a maximum consumption was observed at mm glucose (fig. d) . the lactate-glucose yield ( fig. e ) not showed relevant changes at . (h - ), while at . (h - ) this yield showed a more efficient utilization of glucose, as glucose concentration was increased. however, this last behaviour was not reflected in an increase of r-protein production. the concentration of glucose has the greatest impact on the behaviour of the culture, and its increase affects positively the protein productivity. the mh did not improve proteins productivity of cho cells producing tpa under the different conditions evaluated; low dilution rate and at high glucose concentration impact positively the protein productivity and the metabolism exhibited by the cells. background mammalian cell cultures are the most commonly used bioprocess for the production of therapeutic recombinant proteins such as monoclonal antibodies (mabs). facing to the increasing demand of these biopharmaceuticals, the fda has initiated the process analytical technology (pat) framework in order to encourage pharmaceutical industries to use innovative technologies to monitor in real time the critical process parameters (cpps), and to ensure the final product quality [ ] . one of the most important cpps for cell culture bioprocesses is the specific growth rate (μ), which is a direct indicator of cellular physiological state. indeed, μ is sensible to culture conditions and its value decreases when cells are in the unfavourable environment for growth [ ] , which may greatly influence mab production and quality. however, until this day, the online monitoring of μ remains a great challenge for mammalian cell culture bioprocesses. igg-producing cho cells were cultured in l stirred bioreactors equipped with an in situ dielectric spectroscopy (hamilton). operating conditions were fixed at rpm, % of air saturation, ph . and °c. permittivity of cell culture was measured every min, which allowed to calculate in real time the vcd by using a previously established linear correlation. then, a model of online estimation of μ was developed based on vcd prediction and cell mass balance equations. several signal noise filters and various calculation methods were evaluated to reach better model stability. cell cultures were performed in both batch and feed-harvest modes. feed-harvest cultures consisted of sequential renewals of / volume of the culture medium by following different strategies. this study proposed an innovative methodology based on dielectric spectroscopy to monitor in real time the cellular physiological state, by online estimating the specific growth rate (μ) of cells. model of online estimation of μ was developed from cultures in batch mode, and was validated by comparing online estimated μ with the experimental ones calculated at the end of the culture. with this model, the moment when μ started to decrease significantly, which indicated that cells were no longer in the exponential growth phase, was identified as the critical moment. to demonstrate the interest of online estimation of μ, the developed model was applied to a feedharvest culture, where the medium renewals were performed at the critical moments indicated by the model. this culture was then compared with the traditional feed-harvest culture where medium renewals were performed by following offline measurements of glucose and glutamine. we found that the online strategy allowed to maintain the value of μ by renewing the medium at the right time, while the values of μ varied a lot when using offline strategy. moreover, by using the online estimation of μ, the glycosylation of igg was kept at a high level (about %) throughout the whole culture. however, for the culture using offline strategy, the glycosylation level decreased progressively and was only about % at the end of the culture (fig. ) . model of online estimation of μ was developed by using dielectric spectroscopy, which allowed to monitor the physiological state of cells in cell culture bioprocesses. implementation of this model in feed-harvest cell culture led to better mab glycosylation, which demonstrates clearly the potential of this methodology in mab production bioprocesses. background monoclonal antibodies are normally synthesised from transfected mammalian cells as heterogeneous mixtures of glycoforms [ ] . however, clinical efficacy may depend upon single glycoforms which have been difficult to isolate [ ] . we have now developed an efficient method for generating single glycoforms by solid phase re-modelling which is superior to previous methods because it allows a sequential series of enzymatic changes without the need for intermediate purification of the antibody. solidphase binding exposes the antibody glycans to enable easier access of the transforming glycosylation enzyme. the antibodies subjected to modification were a chimeric human/ camelid monoclonal antibody (eg ), a humanized monoclonal antibody (il ), a full size chimeric antibody (cetuximab) and polyclonal antibodies obtained from pooled human serum. the antibodies were bound to a protein a column using conditions typical of mab purification (fig. ). after washing out non-bound impurities by a neutral ph buffer, each antibody was subjected to enzymatic modification directed to a targeted glycan profile ( table ). the antibodies were then eluted with a low ph buffer and neutralized. the glycan profiles were analysed following glycan removal with pngase f, labelling with -aminobenzamide and separation on a hilic-hplc column [ ] . prior to enzymatic modification glycan analysis of all antibodies showed variable galactosylation and sialylation typical of human abs. this included a distribution of fg , fg , fg , fs and fs with galactosylation indices ranging from . for il to . for eg . there was minimal sialylation in il but up to % in eg . glycan modifications were made as each antibody was held on a protein a column in accordance with procedures shown in table . agalactosylated glycans were enriched by treatment with the single addition of galactosidase and neuraminidase. this resulted in - % of agalactosylated structures in the mabs and % in the polyclonal antibody. galactosylated antibodies (> % yield) were produced by a single stage reaction involving sialidase and by galactosyltransferase with udp-gal. breakdown of the glycans to a trimannosyl core was accomplished by treatment of the agalactosylated structures with hexosaminidase. this produced a yield of - % of the fm structure with a small remainder of fa . sialylated antibodies (> %) were produced by a stage reaction involving sialidase, galactosyltransferase and finally treatment with , sialyltransferase in the presence of cmp-nana. the latter reaction produced equimolar quantities of monosialylated and disialylated cetuximab and polyclonal antibodies. the results suggest that for human antibodies ( kda) there may be a limitation for sialylation given the steric constraints between the two ch domains of the dimeric structure. the ability to sialylate the smaller camelid antibody ( kda) was greater resulting in a high (> %) level of disialylated glycans. this suggests that the steric constraints for glycosylation may be lower. these sialylated antibodies have significant potential clinical importance for their ant-inflammatory activities. we have modified the glycans of antibodies following immobilization on an affinity ligand column. this allows enzymatic transformation in a solid state that has a distinct advantage over the equivalent transformation in solution because the enzymes and buffers can be washed out on completion of the modification leaving the antibody still attached to the affinity ligand. this enables repeated rounds of an enzymatic reaction or sequential reaction steps without the need for intermediate antibody purification. the antibody can be removed eventually from the column by application of an elution buffer once all desired glycan modification have been made. since affinity ligand purification of antibodies is performed routinely as an initial step of purification after cell culture, the glycan modification can easily be incorporated into this process. the enrichment of the resulting antibody for a targeted glycoform can enhance the potential therapeutic efficacy as it is known that specific glycoforms are required for certain biological effects. [ , ] . this is mainly because microvesicles can be enriched/deprived for specific proteins, based on their functional purpose and their cellular origin. recently, microvesicles purified from the supernatant of t bladder cancer cells were reported to be enriched for bcl- and cyclin d (anti-apoptotic proteins), but deprived for bax and caspase- proteins (pro-apoptotic proteins) contributing towards immunity against programmed cell-death [ ] . however, impact of microvesicles on cho-based bioprocess has not been evaluated yet. therefore, in this investigation, we aimed to evaluate their impact on cell growth and recombinant protein production from cho cells. materials and methods cho-k cells were grown in chemically-defined protein-free culture medium (life technologies- ) in shake flask (gx- p). the different fractions of spent-media (microvesicles and microvesicle-free spent media) were collected using ultracentrifugation method [ , ] . quality of different fractions was ensured using western blotting for exosomal marker, cd (sc- ) and coomassie stained gel for loading control (fig. a) . to evaluate impact on cell growth, cells were seeded with microvesicles and microvesicle-free fraction collected from log-phase of culture and cell counts were performed by vicell using trypan-blue dye exclusion method. for impact on productivity, cell-free supernatant, collected from microvesicle-treated human igg secreting cho culture from stationary-phase of culture with respective control, was evaluated using elisa (ab ). microvesicles collected from % of media (by volume) from routine maintenance cultures compared to working volume for microvesiclesupplementation were used in each experiment. the growth of microvesicle-supplemented cultures had shorter lag-phase and achieved . fold higher maximum cell density ( . x viable cells/ml) compared to untreated standard culture ( . x viable cells/ml) and maintained higher for the remaining period of batch culture (fig. b) . however, microvesicle-free fraction did not had significant impact on growth. the viability of microvesicle-supplemented cultures, similar to microvesicle-free media supplemented, was also higher compared to standard culture suggesting potential use of microvesicles for regulating cho growth in production cultures. this could be possibily because microvesicles have already been reported to be enriched with cell growth/death-regulating proteins and hence facilitating cell growth [ , ] . we have also observed abundance of cell cycle regulators including cyclin d in microvesicle-fraction compared to microvesicle-free spent-media in our laboratory (data not shown); however, further investigation are required to prove the hypothesis. the overall productivity of human igg secreting cho cells was also observed to increase bỹ fold following supplementation of microvesicles to the culture without significantly affecting per-cell productivity. since microvesicle-supplementation facilitates cell growth, increased number of viable producer cells in the culture could be expected to be the basis of observed increase in the overall productivity of the culture [ , ] . the further work is ongoing to in-depth explore the potential of microvesicles for improving recombinant protein production from cho cells. the data indicate that microvesicles secreted from cho cells can improve cell growth and hence recombinat protein production in culture. therefore, strategies need to be developed for sterile isolation of cho microvesicles from routine maintenance cultures and their supplementation into the production culture for improving the performance of cho-based production process. the glycosylation profile of a recombinant protein is one of the most important attributes when defining product quality. producing a protein with desired characteristics requires the ability to modify and target specific glycosylation profiles. traditionally the approach to modify the glycosylation profile of a protein involves supplementing a culture with components that can improve galactosylation. experimentation using this supplemental approach resulted in a dramatic increase in terminal galactosylation, but lacked the ability to easily and repeatedly target specific glycosylation profiles. using novel and proprietary technology, we have developed a feed (glycantune™) and a unique feeding process that will maximize growth and titer while being able to modulate glycan profiles. this new feed can be added as a standalone process that can result in a significant shift from g f to g f and g f (maximum galactosylation). using a unique fed-batch process, glycantune can also be used with a standard feed to dial in targeted glycosylation profiles. through process development, we created a method where a transition point is used to switch from a standard feed to a glycan modulating feed. the timing of the transition point will determine the specificity of the glycan profile. ) . n-linked glycans were digested with pngase f and quantified using pmole maltohexose/maltopentose internal standards labeled with -aminopyrene- , , -trisulfonic acid (atps) as described by laroy et al [ ] or the user guide for the glycan labeling and analysis kit (glycanassure™ user guide, thermo fisher scientific). all ce separations were performed using the applied biosystems™ xl. the timing of transition from efc+ to gtc+ made it possible to target specific glycosylation profiles. modulating g f from % down to %, while increasing g f and increasing g f (fig. ) . transitioning to gtc+ early in culture resulted in a greater shift from g f to g f and g f. transitioning midway or late in culture resulted in a greater proportion of g f compared to g f and g f. supplementation based approaches using glycosylation modulating media components to modify and target specific glycosylation profiles proved to be difficult. these approaches were able to increase terminal galactosylation (g f and g f), but lacked the ability to fine tune glycan profiles. this could result in numerous rounds of titration experiments to target specific glycan profiles that would likely remain inconsistent between cell lines, culture media and feeds, and process scale. the development of a unique process made it possible to predictably target specific glycosylation profiles. transition from standard feeding to glycantune allowed for precise targeting of glycan profiles. transition to glycantune early in culture resulted in an increased shift from g f to g f and g f. a transition late in culture resulted in increased g f and decreased g f and g f. growth performance during precultures and batch curves in plain shaking flasks did not show any differences among tested surfactants or lots thereof, and cell densities reached - · cells/ ml ( fig. a and b) . experiments with hek -f cells at elevated power input in baffled shaking flasks revealed distinct differences between pluronic® f- , f- and kolliphor® p , with f- showing the best performance. peak viable cell densities reached with lots a and b of pluronic® f- and f- were comparable to those in plain shaking flasks, while those for kolliphor® p and lots c and d of pluronic® f- were significantly lower. peak viale cell densities were of - · cells/ml (fig. c) . similar transient transfection efficiency and mean fluorescence of transfected cells independent of applied surfactant and lot thereof indicated no major impact of respective poloxamer (fig. d) . interestingly, experiments using fluorescein-labelled pluronic® showed a time-dependent uptake into hek cells. visual tracking revealed an endocytic uptake of poloxamers by the cells (> fold increase in signal after h) and its co-localisation with cell membrane and lysosomes. sec (fig. e) analyses showed differences between the tested poloxamers. especially tested lots of pluronic® f- revealed notable deviations in the low molecular weight fraction (peak , fig. e ), compared to the other poloxamers. cultures subjected to varying levels of shear stress showed distinct growth differences depending on used poloxamer. while experiments in plain shake flasks did not show any differences in growth, cultivations under elevated shear stress in baffled shake flasks resulted in lower peak viable cell densities with kolliphor® p and some pluronic® f- lots. it remaines unclear whether this can be explained by different membrane protective activities alone, or if other mechanisms, occuring during and after cellular uptake, contribute to this effect. especially for the tested lots of pluronic® f- , sec of surfactants showed differences in the low molecular weight fraction. this fraction mainly represents polyethylen oxide (peo) (revealed by nmr), which is likely to be a remnant from synthesis. these observations indicate that the use of different poloxamers and lots thereof should be carefully evaluated, especially under elevated shear stress. further experiments will focus on investigating distinct sec fractions of poloxamers. overcoming (fig. ) . aurintricarboxylic acid (endonuclease inhibitor; enhancer used in e.g. salivary gland transfection) and polyvinylpyrrolidone (polymer; beneficial in electroporation) were both found to negatively impact peimediated transfection of cho cells, while another tested polymer enhanced growth as well as transfection efficiency. the use of a strong chelator led to a high transfection efficiency, but impaired cell growth. based on the results of the independent substance testings, the medium formulation was modified by the addition of a weak chelator and further components including vitamins. different osmolalities between mosmol/kg and mosmol/kg were tested for the final formulation, but no major impact was seen neither on transfection efficiency nor on viability days post-transfection. the final cho tf medium formulation supported high cell growth of finally tested cho cell lines and with peak viable cell densities above ⋅ cells/ml in batch cultivations with an overall cultivation time of - days (fig. ) . further improvements of the process might be achieved by adapting the protocol, as the results shown are based on a simple precomplexing of dna-pei. moreover, product yields could potentially be increased by using feeds, temperature shifts or commonly used enhancers (e.g. valproic acids). scaling of a cell culture process is an essential part in its development. in a typical approach scaling [ ] is performed by keeping a (critical) process parameter constant throughout the complete bioreactor range. this can lead to non-beneficial results either on the high or the low end of the range. for instance, the specific power input [p/v] of w/m might result in a good agitation in production scale whereas it leads to a nonturbulent mixing behavior in process development scale. to overcome this issue a new approach for an easy scaling procedure was developed. this "utility function" approach for agitation scaling is based on individual functions with a value-based mapping independent of bioreactor scale. process insight information (established either from doe process investigation or existing experience with a process platform) is directly formalized into a set of mappings which transform bioprocess values into perceived benefits ( to ). at each bioreactor scale, parameters (e.g. stirring and gassing) are then chosen to maximize the product of resultant utility functions. the model cho fed-batch process in this trial comprised a cho dg cell line that was transfected to produce a humanized antibody igg . a chemically defined media system was used. the process, including cell line, medium and feeding strategy was designed and developed by sartorius stedim cellca. the aim of the gassing scale-up was to achieve similar cell densities when the addition of pure oxygen starts. for all flexsafe str® bags oxygen was sparged via the micro sparger part of the combi sparger. all other systems used a ring sparger with holes face up. the initial air flow rate was set to an oxygen transfer rate (k l a) of /h at the corresponding agitation rate and volume. all process engineering characterization parameters were determined according to dechema guidelines [ ] . with the use of the utility functions the discrete agitation rate was determined (table ) . the utility functions led to discrete agitation rates where not only homogeneous mixing but also a turbulent flow pattern and a suitable specific power input was guaranteed. the initial gassing rate of air supplied enough oxygen for x cells/ml in all bioreactors. due to the used scaling methods the growth patterns in all bioreactor scales were comparable. peak viable cell densities (vcd) of - x cells/ml were achieved and viability at the point of harvest was above % in all scales. the final product concentration was in an acceptable range of . - . g/l. product quality attributes show comparability over the complete bioreactor range (fig. ) . the harvest criteria of days gave a combination of viability and product concentration that made it easy to process the cell broth during cell removal and other downstream steps. the process implementation of the cho production system -expressing mab was successfully performed with the use of utility functions. cell growth, productivity and product quality is comparable over the complete bioreactor range. background endoplasmic reticulum (er), the central part of the secretory pathways in eukaryotic cells, is responsible for controlling the quality of secreted and resident proteins through the regulation of protein translocation, protein folding, and early post-translational modifications [ ] . a number of physiological conditions such as oxidative stress, hypoglycemia, acidosis, and thermal instability can disturb the er functions, which triggers er stress [ ] . prolonged er stress induces apoptotic cell death [ ] . oxidative stress that naturally accumulates in the er as a result of mitochondrial energy metabolism and protein synthesis can disturb the er function [ ] . because er has a responsibility on the protein synthesis and quality control of the secreted proteins, er homeostasis has to be well maintained. when h o , an oxidative stress inducer, was added to recombinant chinese hamster ovary (rcho) cell cultures, it reduced cell growth, monoclonal antibody (mab) production, and galactosylated form of mab in a dose-dependent manner. antioxidants can reduce the oxidative stress level and suppress the apoptotic cell death by scavenging oxygen free radicals, inhibiting chain reaction of oxidation, and detoxifying peroxide [ ] . however, despite the importance of mass production of mabs, studies on the effect of antioxidants on the production and quality of mabs in rcho cell cultures have not been fully substantiated. to find a more effective antioxidant in rcho cell cultures, six different antioxidants including baicalein, which have used widely in mammalian cell cultures, were evaluated as chemical supplements with two different rcho cell lines producing the same mab in -well plates. then, batch and fed-batch cultures were performed in shake flasks with the supplementation of baicalein, which showed the best effect on culture performance among the antioxidants. the reactive oxygen species (ros) and er stress levels were measured to study the effect of baicalein on mab production and quality. among these antioxidants, baicalein showed the best mab production performance. addition of baicalein significantly reduced the expression level of bip and chop along with reduced ros level, suggesting oxidative stress accumulated in the cells can be relieved using baicalein. as a result, addition of baicalein in batch cultures resulted in . - . -fold increase in the maximum mab concentration (mmc), while maintaining the galactosylation of mab ( fig. and table ). likewise, addition of baicalein in fed-batch culture resulted in . -fold increase in the mmc while maintaining the galactosylation of mab. oxidative stress negatively affected the production and galactosylation of mab in rcho cell cultures. among the various antioxidants tested in this study, baicalein showed the best mab production performance in both batch and fed-batch cultures of rcho cells. baicalein addition significantly enhanced mab production while maintaining galactosylated forms of mab. thus, baicalein is an effective antioxidant for use in rcho cell cultures for improved mab production. background the production of many biopharmaceuticals (e.g. antibodies & proteins for diagnostic and therapeutic purposes) requires the cultivation of mammalian cell lines, which is demanding with respect to various aspects such as complex cell metabolism, variabilities in cell behavior, scale dependencies, influences of changes in cultivation conditions, medium composition etc. although an increasing number of measurement parameters is available, only a part of them is routinely utilized in industrial cell culture processes and their corresponding seed trains. nevertheless, the data base grows, statistical investigation of data gains importance and process data are more easily accessible in the context of industry . . cell cultivation has to consider these complex requirements, e.g. for fed-batch control and seed train design. furthermore, cultivation strategies have to be adapted to new products, cell lines and clones as well as to different production plants when transferring processes. one approach to encounter the variabilities and to include actual information from the process and from data analysis is adaptive model-assisted control [ ] . two software tools enabling adaptive model-assisted control applying unstructured, unsegregated models have been developed and implemented using matlab © , winers and fortran, one tool for fedbatch control and another one for seed train simulation and optimization. one key element of adaptive model-assisted control is the underlying process model. in order to provide an adaptive character, model parameters should be easily identifiable from routine cultivation data, which is available during seed train and fed-batch without additional sophisticated measurements. therefore, the usage of unstructured, unsegregated models is recommended. a) example of an unstructured, unsegregated cell culture model (for adaptive model-assisted control) one example, describing cell growth, cell death, uptake of substrates and production of metabolites via a first order system of ordinary differential equations and monod-type kinetics, is shown in table . this mathematical model includes cell specific model parameters [ ] . ii) b) open-loop control sequence for seed train simulation and optimization [ ] : using model, a priori identified model parameters and starting concentration values, the temporal concentration courses can be predicted for the first scale. subsequently, points in time for passaging and starting values for the next scale can be computed by adding a passaging strategy, seed train conditions and medium concentrations. prediction for the following scales can be obtained iteratively. integrating feedback from the process in terms of cultivation data enables increasing prediction accuracy and responding to possible changes in cell behaviour. process design and optimization, e.g. regarding seed train and fed-batch, is realized by adaptive model-assisted software tools using unstructured, unsegregated models. they enable feedback from the process via routine cultivation data and allow adaptation to diverse circumstances such as different cell lines, products, cultivation conditions, plant configurations etc. ) in polyelectrolyte capsules. significant advantages, such as great mechanical stability, good biocompatibility and good mass transfer properties characterized these capsules based on sodium cellulose sulfate/poly(diallyldimethyl) ammonium chloride (scs/pdadmac) [ , ] . here, we present the possibility to cultivate human t cells, freshly isolated from blood, to high densities in similar semipermeable polyelectrolyte microcapsules within less than days. cells were encapsulated in semipermeable scs/pdadmac polyelectrolyte microcapsules or confined in . % alginate/poly-l-lysine (pll) beads, a standard approach for cell immobilization. the permeability of the microcapsules was estimated using dextran-based molecular weight standards ( and kda) and vitamin b ( . kda). gentle digestion with endocellulase allows an easy release of the cells out of the capsules. cell growth, cytokines production and phenotype were measured in non-encapsulated and encapsulated cells grown under standard culture conditions. moreover, we analyzed the interplay between the secreted cytokines and the scs within the capsules and its putative influence on cell growth. cells mixed in the cellulose sulfate solution under physiological conditions can be safely trapped within a liquid core during capsule formation. encapsulated cells can reached cell densities ≤ x cells ml capsule - , whereas cells confined in alginate/pll beads and non-encapsulated ones reached . x cells ml bead - and . x cells ml, respectively. one major advantage of these polyelectrolyte microcapsules (< mm) is the low mwco (< kda) (fig. a-b) . this restricted permeability allows for a conditioning of the capsule core by autocrine factors, which in turn permits the use of basal cell culture medium instead of expensive t cell specialized media, hence does not necessitate high amounts of rhil- and reduces the cultivation costs. moreover, co-encapsulation of rhil- had a beneficial effect on the growth kinetics in most cases (fig. c) . some evidence is presented that the scs used to form the polyelectrolyte microcapsules, specifically adsorbs il- (table ) -a cytokine which provides an essential signal for t-cell proliferation and differentiation [ ] . therefore, we postulate that the scs used for encapsulation has biomimetic properties, creating an artificial extracellular matrix mimicking heparin sulfate which in turn positively affect t cell proliferation via trans-presentation of il- (fig. d) [ ] . primary t lymphocytes can be expanded under appropriate conditions outside the body. in the latter, t cells grow/expand in specific environments where the cells are tightly packed, leading to multiple cell-cell contacts and manifold interactions with the extracellular matrix. ex vivo suspension cultures of diluted cells cannot provide such a microenvironment. in the microcapsulesbased cultivation system presented, the cells are suspended in a viscous scs-solution. the low molecular weight cut off of the surrounding polyelectrolyte membrane assures that typical signaling molecules produced by the cells are retained thus facilitates the "conditioning" of the cellular microenvironment, while nutrients and metabolites can pass. expensive additives, such as interleukin- (il- ), can be co-encapsulated. expansion then no longer requires specialized t-cell media. moreover, the scs seems to have biomimetic properties, representing an artificial extracellular matrix mimicking heparin sulfate. we consider that the described method may be an appropriate alternative to expand t cells while creating a local microenvironment mimicking in vivo conditions. - ) . equations of balances and kinetics of an employed process model including x v viable cell density, x t total cell density, μ cell-specific growth rate, μ d cell-specific death rate, t time, k s and k monod kinetic constant and monod constant for uptake, k lys cell lysis constant, q cell-specific uptake rate or production rate, respectively, y kinetic production constant, c concentration, glc glucose, gln glutamine, lac lactate, amm ammonia, f feed rate, v volume balances with fed-batch terms kinetics ;uptake if c glc ≥ . mm : q lac,uptake = if c glc < . mm : q lac,uptake = q lac,uptake,max q amm = y amm/gln • q gln background digital manufacturing (dm) is heightening the productivity and robustness of existing processes and facilities. it also enables the efficient development of previously unmanageable products or processes and provided the basis for a wave of innovations. dm is a resident and on-line source of continuous optimization of process performance. it relies upon the comprehensive, real-time interfacing of both human and machine sourced information through one centralized system. more than legacy distributed control system (dcs) and supervisory control and data acquisition (scada), it is an integral interconnection of real-time access to divergent sources of information. as such, it can promise deep analysis and predictions leading to shortened product cycle and advanced process control. this comprehensive analysis is extending beyond operations performance data from the production floor to data driving such activities as raw materials security of supply (sos) and business continuity management systems (bcms). digital biomanufacturing (db) can be viewed as yet another, larger, embodiment of digital biotechnology. db is similar to digital manufacturing in that it promotes innovations in the manufacturing of biologicals by using such things as computer aided design, manufacture, verification and deep process analysis using software sensors (fig. ) . however, the fact that there are living components (cells) involved in the processes puts a distinctly different flavor to the systems employed. it is desirable to use a distinct term here to distinguish it because, as in the terms bioproduction and biopharmacology, db addresses many unique aspects of biologically-based activities. the reasons why the biotech and biopharma industry lags behind other sectors such as the automotive regarding the transformation to digital manufacturing are (i) the complexity and dissipative nature of biological systems, (ii) distributed heterogeneous data and (iii) limited at-line or on-line data sources. however, the costs of genomic sequencing, omics data generation, and computing resources are decreasing rapidly, and at the same time process analytical technologies, computational power and predictive modeling as well as data management infrastructures are greatly improving (table ) . by removing roadblocks that used to limit approaches, these changes have paved the way to transforming the bioeconomy into an industry that is based on digital knowledge. such new and optimized manufacturing technologies as continuous biomanufacturing and d bioprinting can actually demand the interfacing of many sources of information, deep data prior to elisa, the various proteins were incubated at °c in scs prepared as for encapsulation. as control, the scs was replaced by pbs. shown are mean values ± sd, n = analysis including software sensors for metabolic fluxes, and model-based predictions of digital biomanufacturing. the application of predictive models for bioprocess optimization greatly improves established platforms and finally leads to a massively increased mechanistic process understanding. four essential benefits result from the increased bioprocess understanding, development, and control of db. first, personnel are relieved of many manual and repetitive tasks. second, strategic planning and operational efficiency are improved. third, we see real-time optimization of end-to-end manufacturing based on such high-value criteria as projected product quality and profitability. fourth, it enables previously unmanageable operations and creates innovative solutions. monitoring between-batch behavior of real-time adjusted cellculture parameters xavier lories, jean-françois michiels arlenda, mont-saint-guibert, , belgium correspondence: xavier lories (xavier.lories@arlenda.com) bmc proceedings , (suppl ):p- background cell-culture parameters (ccp), such as ph, may be continuously measured online and subject to real-time automated adjustment (e.g. automated addition of a base to prevent the ph to drop too low). this is an efficient method to maintain the parameter within specified limits. this type of control constraint the variability within the predefined limits and does not provide any information on the between-batch variability of the process. online measurements of ccp provide time-dependent curves presenting one or more transitions. different types of transition can be observed: -the process can shift from a state in which adjustment is needed to keep the ccp in range to a state in which it is not. typically, the ccp drifts away from a limit. -the process shifts from a state in which adjustment is not needed to one in which it is. for instance, a drifting ccp reaches the lower or upper limit of the accepted range. the timepoints at which those transitions take place are here called changepoints. those are aspects of the process and, as such, should be controlled. in the multiple changepoints cases, the approach allows the early termination of runs showing very early or very late first changepoint. the identification of the changepoints position is based on simple rules rather than complex statistical modeling to keep the identification methodology simple. once the changepoint are identified, a multivariate bayesian model is adjusted on the appropriately transformed data. prediction regions are obtained and used as control limits [ ] . results obtained for a -changepoint case are shown on fig. . points on the right-hand graph represent new batches. the red triangle represents a failed batch. it appears that the control strategy fails to identify the failed batch. two reasons can be considered: -the limits of the prediction region have been established based on points, such a small sample size is likely to be insufficient for the definition of such a control chart. -the tested batches were produced out of set point. a control chart should be used on a stable process, ran in the same conditions, in order to be really relevant. this work was based on available historical data, which is never an ideal situation. the suggested strategy offers a simple approach to the monitoring of between-batch behavior for cell-culture. once the limits have been defined, the approach is quite straightforward and usable by nonstatistician. however, such strategy, as any other of this type, must be based on a sufficient number of batches for the definition of the control limits in order to have a good estimation of the batch-to batch variability. fig. (abstract p- ) . intelligent software applications support digital biomanufacturing process development and control. • databases using data collected online, at-line, and offline from bioprocesses operating worldwide. • process data are used to generate metabolic network models that represent a specific host cell line in a bioprocess. • modelbased computational simulations improve process understanding and reduce experimental efforts for media design, clone selection, and metabolic engineering. • automated data import and processing allow for a streamlined and standardized metabolic process analysis. • identification of critical metabolic parameters is used for proactive steering and control of production processes background rabies is a zoonotic viral disease with a mortality close to % [ ] . as there is not an efficacious treatment available, post-exposure vaccination is recommended for individuals in contact with the virus. on the other hand, the most common source of virus transmission is saliva of infected animals, mostly dogs, whereby mass vaccination of pets is the most cost-effective way to reduce human infections. in this context, availability of both human and veterinary vaccines is critical [ , ] . our group had previously developed an effective vlp-based rabies vaccine candidate produced in high density hek cell cultures with serum free medium (sfm) [ , ] . one of the aims in vaccine production process is the achievement of a good productivity with a low cost per dose, mainly in the case of vaccines for animal use in which case the sfm is one of the principal expenses. in this work, we show the adaptation of the producer clone to a non-expensive in-house developed culture medium, in order to reduce the global cost of the process and therefore the price per dose. experimental approach first, we compared a direct and a sequential adaptation protocol of our hek rv-vlps producer clone, from % of the commercial sfm (ex-cell , safc) to a new formulation with only % of the sfm and a minimum essential medium (p g), developed in our laboratory specifically for rv-vlps production. this new formulation was called rvpm (rabies vaccine production medium). the specific productivity of rv-vlps in culture supernatants was measured by sandwich elisa, using the th international standard for rabies vaccine that quantify the glycoprotein content (nibsc, expressed in elisa units per ml). further, we evaluated both media for the production of the rabies vaccine, using stirred tank bioreactors operated in continuous mode (biostat qplus, sartorius). the production of the rv-vlps was daily evaluated by elisa and the obtained harvests analysed by the nih potency test for rabies vaccine. after the adaptation process, suspension cultures without aggregates or clumps were obtained, with the same specific growth rate. a lower maximum cell density with the rvpm was reached, achieving x cells.ml - , compared with the sfm that reach cell densities between and x cells.ml - in batch mode. the specific rv-vlps productivity per cell was maintained, obtaining values of . and . eu. cells - .day - for the clone being cultured in sfm and rvpm, respectively. taking into account that this producer clone can be changed directly from one medium to the other without lag phase or cell damage, and that in rvpm the maximum cell density reached was lower, this medium was proposed to be analysed in high cell density in perfusion mode for a continuous culture in bioreactor. therefore, we performed two cultures in parallel to compare the efficacy of each media formulation in perfusion. as shown in fig. , we obtained very similar culture performances in both bioreactors; . eu.ml - and . eu.ml - of rv-vlps for the commercial sfm and rvpm, respectively. after that, the harvests were evaluated by the nih potency test obtaining a rabies vaccine potency of . iu.ml - for both cultures (being iu.ml - the minimum potency required for animal vaccine). thus, the results obtained represent an interesting advance in the optimization of this vaccine production process since the use of this new medium formulation represents a reduction of % of the total cost which will be reflected in a considerable reduction of the price of the vaccine dose. background vaccines are one of the most powerful and effective health inventions ever developedproviding tremendous economic and societal value; yet several factors hinder comprehensive immunization coverage. traditional methods of biologics production, based on stainless steel bioreactors, allow pharmaceutical companies to achieve economy of scale, but are limited by high capital expenditures. such approaches stifle manufacturing innovation and lack long-term cost-effectiveness and sustainability. current innovations can cut biologics' production costs to revolutionize the mainstream use of biologic treatments, focusing on developing fast, potent and cost-effective vaccine production. univercells' mission to make biologics affordable to all initiated a paradigm shift, targeting an innovative single-use manufacturing platform incorporating bioprocess into continuous operations. univercells employs process intensification, using high volumetric productivity bioreactors; and unit steps integration, coupling usp and dsp into continuous operations. the objective is a down-scaled high-productivity process for a cost-effective manufacturing solution. the resulting micro-facilities are easily-deployable in developing countries, breaking entry barriers to biomanufacturing (fig. ) . manufacturing and distribution advancements, from centralized to distributed, foresee affordable treatments' obtainability via supplying local populations with local production units. -bench-scale fixed-bed bioreactor; -carriers made of % pure non-woven hydrophilized pet fibers; -vero cells grown in serum-free and serum containing media; -attenuated polio strains; -cell nuclei on carriers counted by the crystal violet method; -polio virus production estimated by elisa assay (d-antigen content). cultivation of vero cells in medium with serum and in serum-free medium, was carried out in bench-scale compact fixed-bed bioreactors, to determine which culture conditions result in the highest growth rate, the highest cell biomass by carriers and virus production. cells were inoculated at . x cells/cm and infected during the mid-exponential phase, following a complete media exchange. viral infection took place in serum-free media. in-line clarification and purification is targeted to be performed in only a few steps (maximum one of two) without intermediary diafiltration. in such configuration, we measured that vero cells can reach a cell density of - x cells/cm with pdl/day of . - . in serum-containing media. this new facility is expected to manufacture any type of viral vaccine at a very low cost and could be deployed at the site of the manufacturer in emerging countries, killing the two birds of cost of manufacturing and distribution with one stone. the presentation will feature the description of the engineering development, but also the preliminary results of cell growth, infections, and product quality, as well as a description of the cogs calculation. univercells developed a disruptive polio vaccine manufacturing technology exceeding expectations when compared to traditional methodsachieving a superior result via its all-in-one solution of a simple, scalable, and fully-disposable vaccine production platform resulting in long-term cost-effectiveness, flexibility and sustainability: -all upstream, downstream and inactivation steps take place within a closed system with all the equipment contained in a low footprint isolatorcreating a confined area for polio virus handling that facilitates the deployment of micro-facilities. -this leads to a dramatic reduction in capital investment, time required for development and increases production capacity. -in conclusion, this is a simple and elegant solution for the industrial production of human vaccines at a low cost in micro-facilities, making polio vaccines available to all. comparison of media formulations for the vaccine production in l stirred tank bioreactors operated in continuous mode. both cultures were performed in parallel using the corresponding medium for the perfusion. a feeding was performed with the commercial sfm. b the first two days of perfusion feeding was performed with sfm until the cell density reach cells.ml - and, after that, the bioreactor was fed using the rvpm formulation. (↓) on day number , % of the reactor volume was punctually bled maintaining the working volume background vectored vaccines based on modified vaccinia virus ankara (mva) are reported to stably maintain large transgenes, and to be safe, immunogenic and tolerant to pre-existing immunity. mva is usually produced on primary chicken embryo fibroblasts but continuous cell lines are being investigated as more versatile substrates. we have previously reported development of a continuous suspension cell line (cr.pix) derived from the muscovy duck and efficient production process for mva in chemically defined media [ , ] . this process allowed isolation of an hitherto undescribed genotype (mva-cr ) that induced fewer syncytia in adherent cultures and replicated to higher infectious titers in the extracellular volume of suspension cultures [ ] . replication of mva-cr remained restricted predominantely to avian cells, an important property of mva vectors. homologous recombination in cr.pix cells was used to generate viruses with various expression cassettes in deletion site iii [ ] and combinations of the differentiating point mutations of mva-cr in a backbone of wildtype virus. all recombinant viruses were plaquepurified. successful introduction of the mutations was confirmed by sequencing and specifically designed restriction fragment length polymorphisms (rflps). viruses were analyzed by serial passaging, diagnostic pcrs accross deletion sites [ ] , replication kinetics, plaque phenotype and electron microscopy. the genome was further investigated by anchored pcr and long pcr. efficiency of spread of recombinant viruses (fig. a) could be mapped to a point mutation in one of the genes, a r. however, although mva-cr carries mutations in three structural proteins we detected no obvious differences to wildtype by electron microscopy (fig. b) . the replacement of the left viral telomere by the right counterpart was the most surprising result of our new study (fig. c) . this extensive rearrangement affects % of the viral genome and has also increased the area of complementarity between the two telomeres. the recombination site was precisely located and shown via analysis of earlier and subsequent passages to be a stable property of mva-cr . various viruses, including those with larger dual (dsred and gfp) expression cassettes, were serially passaged at least -fold. although the genotype of mva-cr is advantageous for replication, all genomic and genetic markers of wildtype and mva-cr were stably maintained in all passages of the recombinant viruses, independent of wildtype or mva-cr backbone. we confirmed our previous results that suggested that mva-cr replicates efficiently in single-cell suspensions and were able to connect this property with the d y mutation in a , a structural protein on the surface of the virions. mva-cr was also found to differ from wildtype mva by a recombination between left and right viral telomere. due to this event, several genes encoded at the left terminus have been deleted whereas the gene dosis of those originally encoded only at the right terminus may have increased. we do not currently know how much the various point mutations and changes in genomic structure combine to explain the improved replication of mva-cr . as several of the affected genes have been reported in the literature to impact interaction of mva with the host we would expect that in vivo studies may reveal additional novel properties of mva-cr . an extremely important distinction between our earlier study [ ] and this one concerns the source of the viruses. here, we investigated plaque-purified viruses and confirm the high genetic and genomic stability of mva. different expression cassettes inserted into deletion site iii, all diagnostic rflps and pcrs over various sites of the genome and within the viral telomeres remained unchanged throughout at least serial passages -independent of whether recombinant viruses with wildtype or cr -derived backbones were characterized. fig. (abstract p- ) . a one hallmark of mva-cr is a significantly reduced tendency to induce syncytia and an increased dispersion of plaques in cr.pix cell monolayers. this property appears to be supported by the mutation in a r. b electron microscopy reveals no obvious differences between novel genotype and wildtype. background transient gene expression systems using polyethylenimine (pei) are considered to be fast, flexible and cost-efficient for recombinant protein production [ ] . transfection efficiency depends on different factors; one of them is the type of media. production media support cell growth and protein production but not high transfection efficiency (te) mediated by pei [ ] . therefore, media were selected for transfection followed by feeding of production media [ ] to improve te and protein production. two different transfection strategies are compared: conventional transfection by preparing polyplex of a plasmid (pdna) and pei interaction before transfection and insitu transfection by direct addition both of them to the cell suspension and the polyplex formed spontaneously [ ] . cells were seeded hr in chomacs cd media before transfection. at transfection time point an equal amount of cells were resuspended in each media type. transfection was applied either insitu or conventional (polyplex prepared in μl of mm nacl and incubated for min.), media addition was performed hours post-transfection (hpt). media type and transfection condition were illustrated in table . media screen result exhibits the highest transfection efficiency of around % transfected cells by opti-mem medium coming along with low cell growth and viability. to improve the transfection efficiency, basic parameters including cell density, pdna, and pei concentrations were varied and higher transfection efficiency was reached by reducing media or accordingly increasing cell density, pei and pdna concentration for transfection. further optimization results show that the transfection of cho-k cells in opti-mem (transfection medium) for hours followed by addition of cho-macs cd (production medium) for further enhancing the transfection, cell count, and cell viability. the transfection efficiency (te) increased up to ± . % coincide with increases in viable cell concentration (vcc) in comparsion to transfection and cultivation in opti-mem media alone fig. a . both conventional and insitu methods are successfully transfected cho-k to the same similar high te as shown in fluorescence microscope images of fig. b . insitu transfection shows super-priority for suspension cell transfection concerning the reduction of handling steps (one step) compared to the conventional way (two steps). the insitu transfection avoiding the optimization step required for the incubation period to prepare transfection polyplex but require a higher amount of pdna and pei than conventional way as shown in table . in order to deal with the growing demand of large quantities of therapeutic proteins in a timely fashion, expression systems are being optimized to reduce the time of generation of stable clones as well as to increase the levels of protein secretion. this can be achieved by a combination of expression cassette optimization, cell engineering and selection process. we have previously developed the cumate gene-switch, which is a very efficient expression system for protein production [ ] . we have shown that the cumate-inducible promoter (cr ) was the strongest promoter we had tested so far in chinese hamster ovary (cho) cells. with this promoter, we were able to generate stable cho pools capable of producing high levels of a fc fusion protein ( mg/l), outperforming by to fold those generated with cmv and hybrid ef α-htlv constitutive promoters. besides the strength of the cr promoter, we demonstrated that the ability to control both the time and the level of expression during pool generation and maintenance gave a real advantage to the inducible expression system. indeed, we observed that keeping the expression off during selection enabled the generation of pools with superior productivity compared with the pools whose expression was maintained on. moreover, preliminary results suggest that keeping recombinant protein expression down increases the frequency of high producer clones [ ] . knowing that one of the main bottlenecks of the successful bioprocessing of recombinant proteins using cho cells is the rapid isolation of a high producer, our data suggest that the cumate gene-switch system could be a valuable platform for the generation of stable clones. the main regulatory authorities and organizations demand proof of monoclonality for biotechnological producer cells. with increasing pressure to shorten timelines and to improve drug safety, technologically advanced methods have to be established to ensure that production cell lines are derived from a single progenitor cell. sartorius stedim cellca's single cell cloning approach is based on one round of fluorescence-activated cell sorting (facs) using becton dickinson (bd) facsariatm fusion cell sorter combined with photodocumentation by synentec cellavista microscopic imaging system. for the approach, critical process parameters such as different cell lines, viability and cell aggregation levels were investigated separately to assess their contribution to the probability of monoclonality. immediately after single cell cloning into -well plates ( cell/ well) the plates were centrifuged followed by imaging using the cellavista (day ). further cellavista images are taken on day , day and on one day between day and . outgrowth was defined at day . cell lines expressing different recombinant products were investigated to calculate probability of having ≥ cells/well after facs sorting p(d), the apparent probability p(i) of having ghostcells (cells that are out-of-focus and, thus, are not visible during initial microscopic imaging), and the apparent probability p(k) of having ghostcells that outgrow the -well stage (fig. ). using these results, the probability of obtaining a monoclonal cell by using sartorius stedim cellca's single cell cloning approach was determined (table ) by conservative examination: p(monoclonal, conservative) = -(p(d) x p(i)) realistic examination: p(monoclonal, realistic) = -(p(d) x p(k)) cell pools with low viability can theoretically impact the probability of monoclonality by e.g. diminishing microscopic imaging quality (cell debris). therefore, pool cell line with very low viability (≥ %) was used to demonstrate, that the probability of monoclonality is still . % in case of low viability on day of sorting: p(monoclonal, conservative) = p(d) x p(i) = . % p(monoclonal, realistic) = p(d) x p(k) = . % furthermore, cell pools with high aggregation levels can theoretically impact the probability of monoclonality by sticking together during facs sorting and therefore increase the probability p(d) of having ≥ cells/droplet. therefore, pool cell line with high aggregation levels (≥ . %) was used to demonstrate, that the probability of monoclonality is still ≥ . % in case of highly aggregated cell pools on day of sorting: p(monoclonal, conservative) = p(d) x p(i) = > . % p(monoclonal, realistic) = p(d) x p(k) = > . % conclusions in summary, there is no obvious correlation between protein product type and the determined probabilities for monoclonality. furthermore, pools with a viability as low as % and pools with an aggregation level as high as . % can be used for scc resulting in acceptable probabilities of monoclonality. background ich guidance [ ] requires that any cell line used to produce biopharmaceuticals originates from a single progenitor cell. recently, there has been increased scrutiny of the method(s) used to achieve this requirement. here, we review the suitability of the legacy capillary aided cell cloning (cacc) method in light of this changing landscape of expectations. the cacc method is based on the 'spotting' technique [ ] and relies on independent visual conformation by two scientists of the presence of a single cell in a μl droplet. this method achieves a high probability of monoclonality in one cloning round. although the method has since been replaced by facs single cell deposition for routine use, it remains a viable cloning method. -performed by trained scientists -dilute culture to ± cells/ml with ≤ % doublets -draw cell suspension into pipette tip by capillary action; tap tip against the centre of the base of each well of a well plate. -size of resulting droplet =~ μl (fig. a ) -two scientists independently view all wells using a microscope (initially use x magnification with the entire rim of the droplet visible within the field-of-view. next, examine particles using x or x magnification to confirm they are cells) and individually record the number of cells present in each well's droplet (fig. b to d) . -exclude droplet from further analysis if full visualisation is hindered (fig. e to h) . -add growth medium, and incubate plates. record all wells containing colonies; only progress colonies from wells that both scientists agree contains only one cell. -data analysis: -each scientist's observations categorised as: cells, cell or > cell -observed outcome for each well: growth or no growth -probability of monoclonality estimated from data using a statistical model cloning (ldc) increased accuracy of p(monoclonality) with cacc -ldc weakness: no visualisation after seeding (to check both well seeding and subsequent growth of colonies is well described by the poisson distribution), potentially overestimating p(monoclonality) -addressed by cacc: visual examination with colonies arising from wells seeded with cell distinguished from those seeded with > cell -visualisation step further strengthened by: using controls for exclusion of wells; measuring errors based on the presence or absence of colonies in wells where two scientists independently reported cells; and formally analysing the data using a suitable statistical model decreased time and resource requirements with cacc -high p(monoclonality) possible in single round as each well examined individually with only those containing a single cell progressed, and because the error rate for incorrect scoring is considered to be low two scientists miss a cell one cell sitting on top of another and the two thus appearing as one an experiment was performed to estimate error frequency [ ] . conclusion -scientists miss a cell infrequently (in the range . % to . %, [ ] ) -error frequency does not invalidate use of direct observation methods for cell cloning -single cell seen by both scientists is highly likely to be monoclonal -during method development, strategies established to control potential sources of error ( table ) use of a contemporaneous visualisation approach, a strict control strategy, and a suitable statistical model (which takes into account potential errors) results in: -the cacc method being at least as robust as the ldc method -the cacc method being a reliable, single-step method for cloning to achieve a high p(monoclonality) background vector design is a key step in cell line development for the expression of therapeutic biologics. it is essential that the vector design results in high, stable expression of the encoded protein. other considerations include ease of cloning, stability for propagation in e. coli as well as in the mammalian host cell line, and ease of sequence amplification for verification of vector construction and for detection of insertion site and copy number in stably expressing cells. for these reasons, use of the same promoters and polya tails in dual cassette vectors, as is common for expression of the heavy and light chains of monoclonal antibodies, can be problematic. in order to minimize sequence similarities between the two expression cassettes, we have modified the promoters, introns, and polya tails of the light chain and heavy chain expression cassettes in the dual expression vector commonly used for the expression of therapeutic antibodies in the chozn® gs -/cell line development platform. gene synthesis and vector construction of igg and fluorophore-expressing vectors was done by atum. vectors were transfected into chozn® gs -/cells via electroporation. analysis of gfp and rfp expression was achieved using a macsquant instrument. selection and generation of stable pools and single cell clones from transfections with igg -encoding vectors was performed as described in the chozn® platform technical bulletin. titer analysis was performed in static ( well plate), in a day tpp assay and in a day fed batch assay using a qk fortebio. initial screening experiments identified a lead vector, # , and a vector, # , which produced very low titers and relatively few minipools expressing detectable levels of igg . analysis of gfp and rfp expression from the modified vectors indicated relatively high expression from the rfp/hc expression cassette of vector # . a stronger promoter resulting in overabundance of hc, known to be toxic to cells, provides a possible explanation for the poor results with this vector. interestingly, swapping the positions of the lc and hc in # resulted in a vector, # , that outperformed the initially identified lead vector (fig. ). this same change was made to vector # without any resulting improvement in titers (vector # , fig. ). interestingly, vector # had a smaller difference in relative promoter strengths, based on mean channel fluorescence ratio of gfp to rfp, suggesting that overabundance of hc was not an impediment to igg expression from # . poor titers were also seen with a modified version of vector # (vector # , fig. ) in which the glutamine synthethase selection cassette was in the reverse orientation. this second screen identified vector # as the lead vector design (fig. ) . a full comparative study of vector # and the control vector was performed, cumulating in the generation and comparison of single-cell clones from each. these studies have demonstrated the equivalence of these vectors in terms of igg titer. this work has resulted in the identification and characterization of a dual expression vector with minimized similarity between the two expression cassettes, easing the cloning, propagation and analysis of vector integration in stable cell lines while maintaining the high, stable expression of the encoded protein of the original vector design. background traditional cell line engineering strategies mainly include an antibiotics resistance selection. in this process, cells are transfected with the goi (gene of interest) together with an antibiotics resistance gene and those cells are selected that survive treatment with the respective antibiotic [ ] . although the gene responsible for the survival of the cell is transfected together with the goi, resistance is not necessarily linked to high goi expression. thus, a significant proportion of resistant cells may not express the goi at all, necessitating the search for alternative, more closely linked selection systems. sirnas (silencing inducing rnas) are short, noncoding rnas that can bind to complementary mrna and inhibit their translation. this function has been used in many approaches to silence the expression of certain genes [ ] . with their short length, sirnas can be hidden in introns (non-translating regions) of genes, making it possible to couple the expression of a sirna to a gene. this way a cell produces a correlating amount of sirna when transcribing the gene, without adding any further translational burden on the cell. the co-expression of the sirna can be used as a selective marker by one of the following methods: ( ) knock-down of a suicide gene to enable a cell's survival after suicide gene mrna transfection, ( ) down-regulation of a surface marker which is used in macs (magnetic cell separation) to filter out wanted or unwanted cells, and ( ) inhibition of a fluorophore marker for selection using facs without product specific antibodies. for sirna based cell selection systems, sirnas replace the commonly used antibiotics resistance as a marker. cells that produce goi will also produce the sirna that protects the cell from a suicide gene. the selection protein (suicide genes, fluorophores, surface markers, etc.) is transfected as mrna and is only expressed during selection. the general process is outlined in fig. . (a) the traditional antibiotics resistance marker is replaced by an sirna, which is cotranscribed with the goi. unlike in antibiotic resistance, the marker here is not a protein, reducing the translational burden and providing more resources for goi production [ ] . transfection with the suicide gene proved to be % lethal within days, with no outgrowth over two weeks. protection by expression of the sirna was shown to be efficient. currently a comparison of stable cell line development programs based on sirna selection and neomycin selection is ongoing. conclusions the novel selection system should speed up cell line development, as the system kills rapidly and directly selects for cells transcribing the product gene on a high level. we expect to see more high producers earlier in the process, which will allow for an easier and faster selection in the following steps. sirna based selection offers great opportunities. by directly selecting based on goi transcription and not a proxy marker, we expect more relevant cells on a pool level. in addition, the elimination of an antibiotics resistance allows more cellular resources for goi production. the system offers multiple ways of application, either by enriching wanted, or depleting unwanted cells. background single-cell cloning is an essential step used in the upstream development of transformed cell lines for therapeutic protein production. while single-cell clones are typically used to ensure product consistency, such low cell density cultures present a survival challenge; cells grow more slowly or may even not survive at low densities in protein-free media, costing the industry time and money and limiting the pool of candidate colonies for choice of production clones [ , ] . to address this problem, we aimed to develop a highly efficient serum-free medium suitable for optimising single-cell cloning efficiency by studying a range of conditioned media (cm) samples isolated from different chinese hamster ovary (cho) cell lines. materials and methods cho-s, dg and cho-k were adapted to cho-s sfm-ii (gibco) medium for a minimum of three passages. conditioned media was then collected when the cultures reached a cell density of x cells/ml (typically day -day depending on the growth profiles of each cell line and whether they grew in suspension or attached conditions). samples were then centrifuged twice to remove cell pellet/debris and stored at - °c. the ability of conditioned media to support cho colony formation was then assayed using -well plates, seeding the cells at low cell density ( - cells/well) by diluting down cho cultures in media/conditioned media. after incubation at °c for days, cloning efficiency was assayed using a standard xtt assay. initial screening of the nine cm samples was performed using cho-k cells due to their widespread use in industrial antibody production. successful media candidates were subsequently screened using additional cho cell lines. table ) . the k -sfmii-cm product improved cell cloning efficiency for dg cells (avg. increase> . -fold) and cho-s cells (avg. increase> -fold) ( fig. ) and also the adherent cho-k cell line growing in atcc + %fbs. the ability of conditioned media to support cho growth in limiting-dilution conditions ( , and cells/ml) was investigated. from a range of nine conditioned media samples; four compelling products have been identified which improve low-cell density growth of cho-k cells, compared to sfm-ii control media. we feel that these early-stage conditioned media products may increase cloning efficiencies during upstream cho cell line development, resulting in financial savings for industry and increasing the possibilities of identifying particularly highperforming transformed clones. ( ): - . the main rate-limiting step in the upstream stages of protein biomanufacture is the isolation of stable, high producing cell clones. ubiquitous chromatin opening elements (ucoe®s) consist of at least one promoter region with associated methylation-free cpg island from housekeeping genes; they possess a dominant chromatin opening capability and thus confer stable transgene expression. ucoe®-viral promoter (e.g. cmv) based plasmid vectors markedly reduce the time it takes to isolate high, stably producing cell clones. although some ucoe®-viral promoter combinations have been tested, they have not been thoroughly evaluated in chinese hamster ovary (cho) cells. plasmid vectors containing combinations of either the human hnrpa b -cbx ucoe® (a ucoe®) or murine rps ucoe® linked to different viral promoters (hcmv, gpcmv, sffv) driving expression of an egfp reporter gene were functionally analysed by stable transfection into cho-k cells and expression analysed by flow cytometry and qpcr to determine vector copy number. the results at days post-transfection and selection clearly indicate that the rps ucoe®-gpcmv and -hcmv combinations give the highest transgene expression as shown in fig. . the a ucoe®-hcmv/gpcmv constructs were the next efficacious but -fold lower than the rps ucoe® vectors. the sffv promoter linked with either of the two ucoe®s was the least effective with expression levels -fold lower than the rps -cmv constructs. the rps ucoe®-gpcmv/hcmv constructs are now being further modified to include elements that will provide optimal post-transcriptional pre-mrna processing (splicing, polyadenylation, transcription termination, mrna stability) thereby maximising stable cytoplasmic transgene mrna levels and protein production. in the last years, growing number of innovator biologics and biosimilars have formed a competitive environment, where speed and efficiency of generating robust and highly productive cell lines needs to be improved continuously. through various advances, especially in media development and process optimization, product titers as high as g/l were achieved in the pharmaceutical industry (kim et al., ) for standard products such as monoclonal antibodies. nevertheless, other proteins e.g. bispecific antibodies, fc-fusion proteins or fab-related products are difficult-to-express (dte) in chinese hamster ovary (cho) and may result in delays or even in termination of the cell line development process. we developed a new robust pool generation approach (cld . ) addressing both, easy-and difficult-to-express molecules, while reducing timelines down to months (cld standard = months), improving reliability of cell line development as well as clearly increasing obtained titers. in order to create stable cell lines, we transfected our cho dg host cells by electroporation. cells processed using the standard approach were cultivated in selective medium or medium containing additional . nm methotrexate (mtx) for three weeks. after an amplification step with nm mtx for three weeks, stable individual cell pools were expanded and clones were generated by facs-sorting. clones were analyzed for growth performance and product concentration in fed-batch studies. in our new cld . approach, we increased mtx concentrations ( . nm, nm and nm mtx) during the first selection phase of three weeks. afterwards we omitted the nm mtx amplification step. thereby, pool generation finished four weeks earlier than in the standard approach. to evaluate the stability of cell clones derived from mini pools (mps) generated according to the cld . approach, stability studies were performed for eight weeks, including stability fed batches at t= weeks and t= weeks. altogether three different proteins of interest with six cell clones each were tested. we adapted our cell line development process by increasing the initial selection during the first selection phase, thereby allowing the omission of the nm mtx amplification step. we observed that the capacity of amplifiability varied for different products. cell lines with a protein titer ranging from > g/l to . g/l (dte) in shake flask fed-batch showed to be more susceptible to increased initial mtx levels and were thus not amplifiable with nm mtx. in contrast, cell lines with high protein titer > . g/l were observed to adapt to nm mtx easily and were amplifiable. finale shake flask fed-batch data with cld . clones of highexpressing products showed comparable titers to clones from the standard approach. cld . clone titers for dte proteins revealed in average a . -fold increase compared to clones generated in the standard approach. titers of top producing clones were in a range of . g/l to to . g/l (fig. ) . furthermore, stability data of cld . cell clones from different dte products showed a stable specific productivity in a range of +/- % over eight weeks cultivation. fed-batch titer from t= weeks and t= weeks were in a normal range of +/- % of the standard nm projects. our results demonstrate that cld . is a robust and reliable process for standard products (mab) and dte proteins. with our new process, we were able to increase titer of difficult-to-express proteins up to %. by omitting the amplification step ( nm mtx) % of generated clones were stable over eight weeks cultivation time. additionally using the cld . approach, the time line from dna to rcb was reduced to months. background cho cells have become the most popular platform for production of therapeutic proteins [ ] . however, the generation of high-producer cells is a time-consuming and labor-intensive process that requires the screening of large amount of cells to get a clone of high titer and stability. since the expression titer and stability of clone is highly dependent on the site of integration, we demonstrated a new cell line development strategy by using ngs to identify the integration site and using crispr/cas to generate the target integrated high producing cell lines [ , ] . to identify the high expression sites in the cho cells, we employed ngs to analyze the integration sites of a high producing cell line (titer > g/l). the pair-end reads with one read mapped to the vector and the other read mapped to the cho reference genome are extracted to identify the integration sites. to test the expression activity of the integration sites, we employed crispr/cas to specifically integrate the antibody gene into cho genome for expression. our data showed integration sites are in the high producing cell line. among the integration site, is integration site was tested by crispr/ cas for target integration of antibody gene for expression. the is target integrated cell pool present higher expression titer than cell pool generated by target integration into other integration sites (fig. a) . the single cell clones derived from is -target integrated cell pool had low copy number of goi (fig. b) . after normalization with copy numbers, the single cell clones derived from is -target integrated cell pool showed high titer per copy ( ~ mg/l/copy) (fig. c) . this study demonstrated the generation of high-producing cell lines by crispr/cas mediated target integration. this approach will cost less time and labor than traditional method. the active integration site will serve as a platform like a cassette player for therapeutic antibody production. background cho, hek and sp / are the dominant host cells for biologics drug production. achieving high level of recombinant protein production by these cell lines still remains a challenge. in order to understand the potential roles of lipids in protein production, secretion, vesicular transport and energy metabolism, we coupled high-throughput transcriptomics and lipidomics technologies. quantitative lipidomics is an emerging 'omics technology which can help us understand the physiological limitations of each cell line. the two types of major lipid groups in cells are non-polar and polar lipids. polar lipids such as glycerophospholipids (pls) include phosphatidylethanolamine (pe), phosphatidylcholine (pc), phosphatidylinositol (pi), phosphatidylserine (ps), phosphatidylglycerol (pg), and phosphatidic acid (pa). in this study; we integrated two dimensional high performance thin layer chromatography ( d-hptlc) and mass spectrometry (ms) lipid analysis of sp / , cho, and hek cell lines to understand the major differences in the lipid content of these hosts. bligh-dyer method was used to extract the lipids and extracts were analyzed by hp-tlc and ms. the polar lipids were separated into different categories by -d hp-tlc using a chcl -meoh-h o ( : : . , v/v/v) solvent system in the first dimension and a chcl -meoh-acetic acid-h o ( : : : , v/v/v/v) solvent system in the second dimension. non-polar lipids were separated by -d hptlc using hexane-diethyl ether-acetic acid. , -dichlorofluorescein dye was used to visualize both polar and non-polar lipids. further detailed analysis was performed on a qqq mass spectrometer (thermo tsq vantage, san jose, ca) using negative-ion and positive-ion esi modes as well as negative-ion esi mode in the presence of lithium hydroxide. in this study, quantitative lipidomics was coupled with transcriptomics to further understand the physiological pathways of hek, cho-m and sp / cells. initial hp-tlc analysis indicated that major lipids in these industrial cell lines were pe and pc. other polar lipids such as pi, ps, pg, pa, and sm were lower compared to pc and pe in exponential and stationary phases of each cell line. figure represents d hp-tlc results of hek with the relative quantitation of polar lipids. in order to investigate the lipid subgroups, shotgun ms analysis was conducted for both exponential and stationary growth phases of the three cell lines. ms analysis indicated that lysophosphatidylethanolamine (lpe) and lyso-phosphatidylcholine (lpc) amounts were - fold and - fold higher in hek cells compared to sp / and cho cell lines. sphingomyelin (sm) was another lipid subgroup that was shown to have a major difference between sp / and other mammalian cell lines. sm was - fold lower in sp / cell line compared to cho and hek. to understand these metabolic differences, transcriptomics analysis using illumina highseq and gene expression omnibus was conducted on these mammalian cells. the kyoto encyclopedia of genes and genomes (kegg) database was used to map the transcriptomics data to the lipid synthetic pathways. transcriptomics data mapping to kegg pathways demonstrated that differences in lpe and lpc pathways correlate with the expression profiles of secretory phospholipase a (spla ), lysophospholipid acyltransferase (lpeat), lysophosphatidylcholine acyltransferase (lpcat), and lysophospholipase (lypla) [ ] . the hp-tlc and lc/ms findings demonstrated that high levels of lpe and lpc existed in the hek cell line and low levels of sm were observed in the sp / cell line. coupling lipidomics with transcriptomics provides us with an improved understanding of the physiological differences across sp / , cho, and hek cell lines that could be used to guide cell engineering efforts with the goal of increasing the recombinant protein expression capabilities of these three cell lines. biopharmaceuticals are a class of biological macromolecules that include antibodies and antibody derivatives, generally produced from cultured mammalian cell lines via secretion directly into the media. manufacturing at medimmune requires the generation of chinese hamster ovary (cho) clonal cell lines capable of producing the biopharmaceutical product at commercially relevant quantities with optimal product quality. the isolation of cell clones based on random single cell deposition via fluorescence activated cell sorting (facs) provides a heterogeneous panel of expressers. we hypothesize that the application of facs to provide an additional sorting step based on desirable cell attributes that correlate with productivity, product quality or cell growth attributes could lead to the isolation of higher producing cell lines with enhanced product quality attributes. a panel of cell lines expressing a model recombinant monoclonal antibody were characterised in terms of growth, productivity, intracellular recombinant protein and mrna amounts. assays were also developed to investigate cell attributes using the commercially available imagestream instrument, an imaging flow cytometer, which enables the investigation of cellular characteristics that correlate with cell productivity at the single cell level. characterisation revealed the cell lines exhibited a range of values for productivity, growth, and intracellular (ic) antibody mrna and protein expression, ideal for further imagestream characterisation. western blot and qrt-pcr analysis demonstrated that final titre correlated with both ic heavy chain (hc) protein and mrna amounts (pearson correlation coefficient (pcc) = . and pcc = . , respectively). to assess productivity at the single cell level, assays multiplexing ic hc protein and mrna with cell attributes were therefore developed. initial assay development focusing on hc mrna and protein amounts has revealed interesting results; four cell lines displayed two distinct populations, one producing the antibody and another nonexpressing population. the ratio of these populations varied amongst the cell lines. images obtained from the imagestream have shown the cellular localization and expression of hc and lc message and protein (fig. ) . for both message and protein, hc and lc colocalize in the cell. whether there is any relationship between ic hc protein and cell attributes at the single cell level was then also investigated, as well as correlations with cell culture parameters at the population level. at the population level, correlations were found between titre and ic hc protein and mrna (pcc = . and pcc = . , respectively) confirming the data obtained by western blot and qrt-pcr analysis. a panel of cell lines has been characterised at the population level and show a wide range of antibody expression profiles at both the mrna and protein level. in parallel, assays have been developed for the imagestream to measure hc and lc message and protein amounts at the single cell level. protein and message quantification with the imagestream are consistent with more traditional approaches, such as western blots and qrt-pcr, that operate at the population level. the developed assays are now being used to investigate single cell productivity attributes and for the isolation of more productive clones. background productivity and stability are key factors for the selection of cell line in protein drugs production. large amount of target gene integrated in cell genome could lead to the instability of production. therefore, cells with low copies of target gene integrated in high yield sites could be an ideal production cells for manufacturing. it has been known that the transposon system can control the integrated copy number of target gene and can generate high yield producing cells, it could be a great approach to generate stable high yield producing cell lines carrying low copies of target gene through transposon system. we intended to develop a platform to generate high yield producing cell lines carrying - copy of the integrated target gene using transposon system. two cho cell lines, cho-s cells and dxb cells, have been applied. cells were co-transfected with transposon and target gene expression plasmids. after drug selection, the cell pool with highest productivity per target gene copy was applied to single cell cloning. the productivity and copy number of cell clones were determined, and the stability of cell clones was analysed after culture of about generations. in the stable pools of cho-s and dxb cells, the productivities per integrated target gene copy were about - mg/l/copy and - mg/l/copy in a batch culture, respectively. after single cell cloning, the integrated copy numbers in most cell clones were less than three copies per cell. in cho-s and dxb cell clones, the productivities per integrated target gene copy were - mg/l/copy and - mg/l/copy in a batch culture, respectively. the productivity per integrated target gene in cell clones developed by the transposon system was much higher than that in cell clones developed by random integration (fig. a and b) . to evaluate the productivity stability of cell clones developed by the transposon system, ten cell clones at generation , , , and were applied in the analysis. of interest, about % of cell clones were stable at generation , but lost the productivity at generation (fig. c) , implying the most cell clones could maintain the stability within months. using the optimized conditions of the transposon system to develop the stable gene expression cells, the productivity per integrated target gene was higher than random integration. these results suggested that our platform is capable to develop high yield producing cells with - copy of integrated target antibody gene and can be applied to identify high yield integration sites. background mammalian cells show an inefficient metabolism characterized by high glucose uptake and the production of high amounts of lactate, a widely known growth inhibition by-product [ ] . recently, we have observed a different glucose-lactate metabolism in some cell lines. while some cell lines are unable to metabolize lactate, others can co-metabolize simultaneously glucose and lactate under certain culture conditions, even during the exponential growth phase [ ] . these metabolic differences between different mammalian cell lines (cho, hek and hybridoma) have been studied by means of flux balance analysis (fba). three different cell lines were cultured in a -liter bioreactor: cho-s, hek sf and hybridoma kb- . . for the fba, two adapted genome-scale metabolic models were used: a reconstruction of mus musculus for cho and hybridoma [ ] , and a reconstruction of human metabolic model (recon ) for hek [ ] . in cultures where ph was not controlled, two different metabolic phases were observed for cho and hek cells. during the first phase both cell lines produced large amounts of lactate as a consequence of the high glucose consumption rates. interestingly, when ph dropped below . , due to acid lactic secretion and accumulation, a second metabolic phase was identified, in which concomitant consumption of glucose and lactate was observed even during the exponential growth phase. conversely, hybridoma cells were unable to co-consume lactate and glucose simultaneously even under noncontrolled ph conditions. therefore, the hybridoma physiological data used for the fba corresponded to only phase of phcontrolled cultures. a summary of the main cell growth and metabolic parameters obtained from the different experiments performed is presented in table . fba shows ( fig. for hek cell culture) that lactate is produced in phase because pyruvate has to be converted to lactate to fulfill the nadh regeneration in the cytoplasm and only a small amount of pyruvate can be transported into tca through acetyl-coa. cell metabolism in phase is highly inefficient, as the majority of the carbon source is not used for the generation of energy nor biomass. in phase , in which mitochondrial ldh was considered, tca fluxes could be maintained as in phase at the maximal rate encountered; hence, the energy available for cells to grow was similar in both phases, obtaining similar growth rate. two different glucose and lactate metabolism behaviors have been observed in cho and hek cultures depending on the culture conditions: phase ) glucose consumption and lactate production, and phase ) glucose and lactate simultaneous consumption. in contrast, only phase was observed in hybridoma cultures even when ph was non-controlled. fba showed that tca fluxes in phase and phase were similar, obtaining similar cell growth rate, but glucose uptake rate was much lower in phase due to the lactate co-consumption. some authors hypothesize that cells metabolize extracellular lactate as a strategy for ph detoxification [ ] . glucose and lactate co-metabolization resulted in a better-balanced cell metabolism, as can be seen from the metabolic fluxes calculated, with minor effects on cell growth. the observation of glucose and lactate co-consumption metabolic behavior and its deeper study and characterization could open the door of novel culturing strategies with the aim of increasing bioprocesses productivity. background transient protein expression in mammalian cell lines has gained increasing relevance as it enables fast and flexible production of high-quality eukaryotic protein. considerable efforts have thus been made to overcome existing limiting aspects of transient gene expression systems, in terms of cell lines, cell culture-based systems, and protein production in a cost-effective manner. milligram amounts of protein per liter can be produced within several days, allowing a significant shortening of the bioproduction process in comparison to protein production from stable clones. to ensure the robustness of the process, it is essential to have a reliable and easy-to-use transfection method. to palliate for the need of a reliable transfection reagent, we developed peipro®, the only commercially available pei optimised for mid to large-scale transient protein production during process development. peipro® is a non-polydiperse and fully-characterised polymer that has become the gold pei standard due to its reliability, reproducibility in high dna delivery efficiency and in ensuing high protein production yields. here, we present experimental data showing the benefits of using peipro® for protein production in comparison to other peis. we further demonstrate compatibility of using peipro® for recombinant protein production in most commonly used chemicallydefined media. materiel and methods suspension hek- and cho cells were cultured in shaker flasks in various synthetic media, as listed in table . hek- and cho cells were resuspended at × cells/ml of serum-free medium, on the day before transfection. cells were transfected with . - mg of plasmid dna encoding for the luciferase gene reporter using peipro®, pei "max" and l-pei kda (polysciences, warrington, pa) resuspended at mg/ml according to the manufacturer's recommendation. protein expression of the luciferase reporter gene was assayed hours post-transfection by affinity chromatography using protein g (hplc). comparison of peipro® to other commercially available peis was achieved by transfecting suspension hek- and cho cells with plasmid dna encoding for the luciferase gene reporter. luciferase production yields obtained in hek- and cho cells were at least respectively -fold and -fold higher when using a similar amount of peipro in comparison to the other peis (fig. ) . furthermore, peipro® was the only pei that led to similar luciferase production yields when decreasing the amount of plasmid dna per liter of cell culture. conversely, at least mg of plasmid dna and -fold more of pei "max" and l-pei kda were needed to obtain a similar luciferase expression range in both hek- and cho cells. we further assessed the compatibility and versatility of peipro® by measuring protein production yields obtained in most commonly used animal-free synthetic media. as shown in fig. , peipro® leads to high protein production yieds in several commercially avaialble media formulations for hek- anc cho cell lines. peipro® is the only fully characterised pei transfection reagent that is suitable for reliable and reproducible recombinant protein production, irrespective of the scale of production and of the type of adherent and suspension cell culture system. fig. (abstract p- ) . peipro® requires less reagent and similar to lower dna amount compared to other peis. suspension hek- and cho cells were seeded at × cells/ml in serum free medium and transfected with peipro®, pei "max" and l-pei kda (polysciences, warrington, pa) resuspended at mg/ml. luciferase expression was assayed h after transfection using a conventional luciferase assay fig. (abstract p- ) . peipro® is optimized for transfection of hek- and cho cells in several specific synthetic culture media. suspension hek- and cho cells were seeded following the recommended protocol in serum-free media and transfected with peipro® using the standard conditions. igg -fc production was assayed h after transfection using protein g affinity quantification (hplc) monoclonal antibodies (mabs), which are widely used in anticancer therapies, are mainly produced by mammalian cell lines. mab conjugation to biological molecules for enhancing their antitumor activity offers a new powerful tool for anticancer therapies. we have assessed the production of commercially approved anti-her therapeutic antibody trastuzumab (tzmb) [ ] and also its fusion with interferon-α b (ifnα b). two cloning strategies consisting in transfecting cho-s and hek cell lines with two bicistronic or with a single tricistronic plasmids have been assessed. the in vitro efficacy of both antibodies has been tested and compared side by side. tzmb heavy and light chains were cloned in two bicistronic plasmids (pirespuro and piresneo , clontech) and in a tricistronic plasmid derived from pirespuro . ifnα b was spliced to tzmb heavy chain by overlap extension pcr and the resulting tzmb-ifnα b fusion protein was also cloned in the expression vectors in the same way than non-modified tzmb. selected cell pools were cultured in ml shake flasks containing sfmtransfx supplemented with % v/v of cell boost (hyclone), mm of glutamax (gibco) and μg/ml of puromycin and also with μg/ml neomycin in the case of the cells transfected with pires-neo . cells were cultivated in the same conditions as described elsewhere [ ] . purified products (using protein a chromatography (hitrap mabselect sure, Äkta avant )) were quantified by both elisa and sds-page. antigen binding test was performed in sk-br- breast cancer cell line by means of flow cytometry analysis. the biological activity of the different candidates was tested with mtt assay. both tzmb and the fusion protein tzmb-ifnα b have been successfully expressed in cho-s and hek , which use for heterologous protein expression have previously been optimized in prior works [ ] . the tricistronic strategy resulted in the most efficient, showing a . fold increase in terms of productivity with respect to the bicistronic double-transfection for tzmb in cho-s cells and a -fold increase in hek cells (fig. a) . in the case of tzmb-ifnα b, the tricistronic strategy also allowed to achieve higher productivities than the bicistronic one (fig. b) . regarding the differences of specific productivity between both cell lines tested, hek emerged as the best production host candidate, for the two tested strategies (tricistronic and bicistronic) and for the two produced proteins, showing a . -fold increase in terms of productivity with respect to cho-s cells for tzmb using the tricistronic strategy. tzmb and tzmb-ifnα b were analysed in terms of their antigen binding capacity, and both were find to efficiently bind to her + skbr- cells (fig. c) . thus, the antibody affinity to her antigen has not been affected when fused to inf-α b. finally, antiproliferative activity of tzmb and tzmb-ifnα b were assessed on the same sk-br- cells. at a concentration of nm of tzmb, and after a -hour incubation, sk-br- cells presented a % growth with respect to the untreated control. however, no antiproliferative effect was observed for tzmb-ifnα b (fig. d) . the tricistronic strategy provides higher productivity yields in hek and cho-s cell lines for both recombinant proteins (trastuzumab and tzmb-ifnα b). regarding which cell line is the best production host candidate, hek achieved higher productivity than cho-s cells for the two proteins tested. all constructions performed preserved the binding affinity to its antigen, trastuzumab and tzmb-ifnα b bind efficiently to the her antigen present in skbr- cells. finally, tzmb-ifnα b does not present an improved antiproliferative effect with respect to trastuzumab when compared by means of an in vitro assay. the genetic engineering of patient-specific t cells with lentiviral vectors (lvv) expressing chimeric antigen receptors (car) for late phase clinical trials requires the large-scale manufacture of high-titer vector stocks. the state-of-the-art production of lvv is based on -to layer cell factories transiently transfected in the presence of serum. this manufacturing process is extremely limited by its labor intensity, open-system handling operations, its requirements for significant incubator space plus costs and patience risk due to presence of serum. to circumvent these limitations, this study aims to develop a stable and serum-free process to produce lvv with pei-mediated transfection. in addition, this study also focuses on the development of a a c b d fig. (abstract p- ) . expression of trastuzumab (a) and trastuzumab-ifnα b (b) from bicistronic strategy (bc) and tricistronic strategy (tri) with cho-s and hek cells. relative specific productivity units are used for comparing the different strategies. c antigen binding analysis of trastuzumab and trastuzumab-ifnα b. d antiproliferative activity of trastuzumab and trastuzumab-ifnα b on sk-br- cells production system not only using a gfp marker but also a therapeutically relevant transgene (cd -car) [ ] . therefore, three different cell lines (hek , t, ft) were investigated concerning their productivity of lvv and their growing behavior in the in-house serum-free medium transmacs. as part of this, design of experiment was used to investigate the optimal conditions for pei/ dna-transfection. furthermore, this statistical approach was used focusing an ideal ratio between the rd generation plasmids (transfer plasmid cd -car or gfp, envelope plasmid, packaging plasmids). in addition, different enhancers (sodium butyrate, lithium acetate, caffeine, trichostatin a, cholesterol, hydroxyurea, valproic acid) were investigated concerning their effects on productivity comparing hek cultures producing lvv encoding for gfp-marker or cd -car. concerning productivity and growing behavior, hek t was the favored cell line for our serum-free lv manufacturing process. in addition, an additive screen revealed that sodium butyrate alone had the most promising effect on both gfp-lvv and cd -car-lvv production. after pei/dna titration, we finally could increase lvv productivity by lowering pei/dna amount at higher cell densities referred to our standard transfection protocol. furthermore, the titration for the optimal plasmid ration revealed, that for large transfer constructs higher amounts of transfer plasmid are required than for smaller constructs to achieve a high productivity (fig. ) . the outcome of these experiments enabled the development of a robust hek t based process to produce clinical relevant lvv under serumfree conditions. furthermore, it provides an insight how therapeutic genes and the expression of its transgene can influence cell productivity. led to a vast increase in productivity, cho cells yield less than other expression systems like yeast or bacteria [ ] . to improve yields and find beneficial bioprocess phenotypes, genetic engineering plays an essential role in recent research. the mir- cluster with its genomic paralogues (mir- a and mir- b) was first identified as differentially expressed during temperature shift, suggesting its role in proliferation and productivity [ ] . the common approach to deplete mirnas is the use of a sponge decoy which, requires the introduction of reporter genes. as an alternative this work aims to knockdown mirna expression using the recently developed crispr/cas system which does not require a reporter transcript. this system consists of two main components: the single guide rna (sgrna) and an endonuclease (cas ) which induces double strand breaks (dsbs). these dsbs can result in insertion or deletion (indels) of base pairs which can disrupt mirna function and processing [ ] . a cho-k cell line stably expressing an igg was used for knockdown experiments. sgrnas were designed to target the seed region of each mirna member and stable mixed populations were generated (fig. a) . total rna form each mixed population was reverse transcribed into cdna using mirna specific stemloop primers. the expression was quantified by rt-qpcr. to further analyse the range of indels the mir- a and mir- b clusters were amplified by a standard high-fidelity pcr. amplicons were cloned into pcr tm -topo® vector and positive clones were analysed by sanger sequencing. cell growth was monitored using viacount tm viability stain on a guava tm benchtop flow cytometer. productivity was assessed by elisa. students t-test was used for statistical analysis. it was shown that mirna expression was significantly reduced in mixed populations. a knockdown up to % was achieved for mir- a, mir- b and mir- . the knockdown in mir- a and mir- b expression was considerably less -between - % (n= , * p ≤ . , ** p ≤ . , *** p ≤ . ) (fig. b) . furthermore, it was shown that various sizes of indels were generated by targeting the seed region. smaller indels (+ / + /- /- bps) seemed to be more common but larger deletions were detected as well (fig. c) . mir- a, mir- b and mir- b showed increased viability in late stages of the culture. depletion of mir- a reduced growth significantly whereas knockdown of mir- showed increased proliferation as well as boosting igg titers (table ) . in this work, we have shown that crispr/cas can be successfully applied as a tool to knockdown mirna expression in cho cells. the data was generated using mixed pools and it remains to be established if both alleles can be successfully targeted e.g. using nextgeneration sequencing of individual clones. background chinese hamster ovary (cho) cells are the most widely used host cell line for the production of therapeutic antibodies. pre-and posttranslational modifications and optimization of culture methods contributed to increase the productivity, resulting in a very high titre [ , ] . however, it has been pointed out that the intracellular secretion process is a bottleneck in the production of therapeutic antibodies [ ] . in addition, the details of the process of secretion of humanized recombinant antibodies from cho cells have not been well investigated. in this study, we thus analysed the detailed process of secretion of therapeutic antibodies using cho cell lines, which have already been established as high producers, with the aim of obtaining information for the more rational and efficient establishment of high-producer cells. we performed ) chase assay, ) immunofluorescent microscopy observation, and ) size exclusion chromatography (sec) analysis to investigate the duration of secretion, bottleneck position, and formation of recombinant igg, respectively. high-producer cho cells expressing humanized igg [ ] and igg were used. for the chase assay, cells were cultivated in shake flasks with serum-free medium containing μg/ml cycloheximide (chx) to stop nascent peptide synthesis. the amounts of igg both remaining in the cell and secreted into the medium at each time point were measured by quantitative western blotting. for immunofluorescent microscopy observation, cells were cultivated on coverslips with chx for h. immunofluorescent staining against the recombinant igg, endoplasmic reticulum (er), and golgi apparatus was performed after chemical fixation. for sec, cells cultured with chx were re-suspended in a buffer containing tritonx- and injected into a column. the amount of igg in each fraction was measured by quantitative western blotting. the amount of igg in the supernatant increased until - h after the inhibition of protein synthesis by chx; however, it hardly changed thereafter (fig. , upper panel) . at this point in time, however, around % of igg still remained in the cells (fig. , lower panel) , meaning that all of the synthesized igg could not be secreted into the medium and remained in the cells for several hours. this result was almost the same as that of studies using igg -expressing cells [ , ] . the localization of igg in the cells was checked before and after the addition of chx, with the results showing that igg remained in the er and was hardly seen in the golgi apparatus [ ] [ ] [ ] ; igg did not seem to be efficiently transported to the golgi apparatus. the sec experiment showed that most of the igg remaining in the cell seemed to form full-sized antibodies [ , ] , but it could not be secreted despite this. the high-producer cells could not secrete all of the synthesized igg, and around % of igg remained in the cells for several hours. this incomplete secretion is a common phenomenon among cho cells producing different types of recombinant igg. the igg could not be transported from the er to the golgi despite its formation of fullsized antibodies. solving this bottleneck in the transportation of igg from the er to the golgi and/or achieving more efficient glycosylation of igg after the formation of full-sized antibodies might be the next target to improve productivity. background humanized monoclonal antibodies (mabs) are among the most promising drugs, but defined strategies for their modification are still not available. our work deals with humanization of murine mab / h . the superhumanization approach leads to a loss of binding affinity which was partially restored by a single human-to-mouse backmutation (t hr). [ ] this residue was selected by synergistic combination of sequence analyses of antibody framework regions and structural information using novel in silico simulations. for structural stabilization, a conglomeration of tyrosine residues surrounding t hr was identified, the so called "tyrosine cage". [ ] analysis of the "tyrosine cage" was done by alanine scanning mutations with a double mutation variant t hr + y ha (bm ) and a triple mutation variant t hr + y ha + y ha (bm ). in a recent series of experiments we tried to enhance binding affinity by three new variants with backmutations in the variable light chain (vl). originating from t hr, residues in the vl were selected based on their spatial proximity to the cdr loop of the variable heavy chain. affinity improvement of t hr was evaluated by vl-double backmutation variants t hr + f ll (su ) and t hr + q ls (su ) and a triple backmutation variant t hr + f ll + q ls (su ). all five variants were expressed transiently in hek - e cells and binding affinities were investigated in two individual settings with bio-layer interferometry. in the first approach concentrated cell culture supernatants were directly applied and mabs were captured on protein a tips, blocked with d scfv-fc and the association and dissociation of f igg was measured. for the second approach, the culture supernatants were purified and the affinity was determined with streptavidin biosensors. first, biotinylated f igg was bound and then the association/dissociation of the purified h variants was measured. affinity evaluation of concentrated culture supernatants with protein a sensor tips showed a decrease of binding affinity of bm and a loss of binding of bm . the protein a measurement showed an increased binding strength of su , su and su compared to su h and bm . su and su result in a higher binding affinity compared to su . these results can be confirmed with purified variants by the streptavidin bio-assay (fig. ) . alanine scanning of the tyrosine cage demonstrated a reduction of binding affinity (bm ) and a severe loss of binding (bm ), concluding that the tyrosine cage plays an important role for supporting a correct cdr loop conformation. further affinity improvement of the single mutation variant t hr could have been reached via mutations in the vl. it demonstrates the underestimated role of the vl for the interaction with its binding partner. although cho cells are a major expression system for production of recombinant biopharmaceuticals, the molecular and cellular background characterizing a high producer is largely unknown. it has been observed that in producer cell lines important signaling pathways like the akt-signaling are altered in characteristical ways. thus analyzing according signaling events should lead to identification of key elements characterizing high producer cells. to investigate this, our emphasis lies on the phosphorylation status of involved proteins as reversible switches in all signaling pathways. we aimed to establish a workflow for cho-specific phosphoproteomics and focused on igf signaling, as cell culture media often are supplemented with this growth factor. two producer cell lines and the according parental cells were cultivated in a stable isotope labeling with amino acids in cell culture (silac) experiment, followed by quantitative ms phosphoproteomic analysis including chospecific data evaluation. the chosen cho cell lines were cultivated in triplicates in silac media containing isotopically-labeled lysine/arginine (hlys/harg) and in parallel in identical standard media (llys/larg, tcx d, xell). cell density, viability, metabolism and cell cycle distribution were monitored during ml batch culture for - days. at day . igf was added into hlys/harg cultures. min later a part of the cells was harvested. for ms analysis igf-treated (hlys/harg cultures) and control cultures (llys/larg cultures) were combined. the following ms sample preparation workflow included digestion of whole protein lysate and phosphopeptide enrichment via tio beads. nanolc-esi-orbitrap ms (q exactive plus, thermo fisher scientific) of phosphopeptides was excecuted with subsequent identification and quantification in maxquant [ ] . in addition to silac quantification of h/l ratios for investigation of igf effects, aquired data was also used to perform label-free quantification (lfq) in maxquant [ ] for comparison of cell lines. statistical significance was calculated via t-test (p< . ) or anova (permutation-based fdr< . ) in perseus [ ] . results igf effects on growth and production the igf treatment resulted in a prolonged viability for all cell lines. however, an increased vcd was only observed for producer cell line , yielding in an enhanced integral of vcd (ivcd). for the parental cells growth was inhibited by igf, although s-phase cells were enriched at least temporary (fig. a) . regarding antibody production igf led to a decreased qp and product titer, concomitantly with an increase in s-phase cells (fig. a) . this inverse correlation of proliferation and cell specific productivity is known from different productivity enhancing molecules, like butyrate [ ] . ms investigation of signaling events the phosphoproteomic experiment resulted in the identification of . class i-phosphorylation sites. statistical evaluation of phosphopeptide abundances in perseus showed up significant differences between the cell lines and led to producer vs. parental classifications (fig. b) . the quantitative evaluation via silac yielded in about . quantifiable phosphosites in at least biological replicates. rapid phosphorylation changes after growth factor treatment indicated signaling towards protein synthesis, cell cycle and regulation of actin cytoskeleton amongst others. for phosphosites significantly different h/l ratios were calculated between the two groups parental vs. producer, four of them are listed (table ) . the workflow to study phosphorylation states revealed differences in the related cell lines and gave insights into signal transduction as a response on igf. on the one hand, igf-treatment resulted in a fast and widespread upregulation of phosphorylation sites within aktand mapk-signaling. on the other hand, a different phosphorylation status for producer compared to parental cell lines uncovered distinctions in biological processes like rna-and dna-binding and regulation of cytoskeleton. in sum, our sucessfully established phosphoproteomic approach allows to detect important signaling key players in cho cells that subsequently can be targeted through cell engineering or small molecule treatment. to improve antibody production in the cho cell expression system, it seems to be useful to up-or downregulate gene expression including antibody folding, secretion, and cell metabolism. many cell engineering approaches, including gene introduction, knockout and knockdown, have been employed to enhance recombinant antibody production [ ] . however, identifying production enhancer genes is the rate-limiting step for cho cell engineering, because the conventional method requires a series of experiments including genomic integration of the tested genes, selection of stable cell clones and cell culture experiments of several clones. in this study, we propose an approach for rapid evaluation of production enhancer genes based on an episomal expression system. plasmid vector carrying the epstein-barr virus (ebv) encoded nuclear antigen (ebna ) was transfected into cho cell line producing igg antibody. after g selection and single colony isolation, ebna expression was checked with capillary electrophoresis system wes (proteinsimple). ebv ebna -antibody ( eb ) was used for detection as the primary antibody. the expression vector for the gene of interest was prepared by inserting bp of an orip dna sequence into a plasmid vector carrying cag promoter, resulting in the potc vector. pei max (polysciences, inc.) and balancd transfectory cho (irvine scientific) were used for the transfection. the number of viable cells and gfp-positive cells were counted using countess ii fl automated cell counter (thermo fisher scientific). the transfected cells were cultured in cellstar cellreactor tubes. the tubes were incubated in a climo-shaker isf -x (kuhner). antibody production was measured using biolayer interferometry with an octet qk system (fortebio). we constructed four cho cell lines stably expressing ebna , termed igg -eb to eb . in capillary electrophoresis analysis, we observed a clear peak corresponding to the ebna expression in all four cell lines. we tested the transfection efficiency by potc-gfp plasmids. in the best transfection condition, pei/dna ratio of / , igg -eb cell showed the highest gfp-positive cell number ( . × cell/ml) and transfection efficiency ( %) among the four cell lines. therefore, igg -eb cell lines were selected for further study. after the transfection, the number of gfp-positive cells continued to increase even after the passage (fig. ) , suggesting that the potc-gfp plasmid was stably retained and replicated by ebna /orip system in igg -eb cell lines. in preliminary experiments, we introduced three genes, mdh , gss and gclm, into igg -eb cell lines. cotransfection of these three genes led to an increase in igg production from ± mg/l (control) to ± mg/l at day (p< . , t-test, n= ). this result suggests that these three genes work as production enhancer genes. conventional methods based on stable cells take up to months to determine whether the gene of interest is beneficial for recombinant igg production. in contrast, identification of production enhancer genes is achievable within days by our proposed method based on ebna /orip system. the proposed method makes it possible to evaluate production enhancer genes in a rapid manner. the proposed method is a promising approach to identify genes enhancing recombinant antibody production. background g unic™ ( gun) technology comprises a set of protected genetic elements that improve protein production by acting on transcription as well as on translation. the elements can either be inserted into existing (platform) vectors or be provided as complete ready-to-use vectors. the technology can be used in stable and in transient transfection to boost protein production for product development and is being applied in cld for pharmaceutical proteins. in combination with antibiotic selection or dhfr selection, gun technology routinely results in - fold increase in expression of client antibodies or fusion proteins, both in pools and after clonal selection. previously, we have successfully combined gun technology with glutamine synthetase (gs) selection and the cho gs null cells of horizon discovery, resulting in clonal cell lines producing > g/l of a biosimilar mab in fed-batch assay. here we present data on the successful application of the gun technology for the enhanced expression of a large (> kda) human heterotrimeric glycoprotein, a renowned difficult-to-express (dte) protein. all expression vectors comprised a hcmv promoter and bgh polyadenylation sequence in the expression cassettes for the gene of interest, and a selection marker gene with sv promoter and sv polyadenylation sequence. g unic™ vectors also contained genetic elements ( g unic™ technology, proteonic). cho gs null cells (horizon discovery) were transfected in duplicate with reference or gun expression vectors and selected in media lacking glutamine and containing the appropriate antibiotics. the bulk pools were seeded at equal viable cell density after obtaining maximum viability and cultured for days without feeding (batch). expression of the target protein in cell culture supernatants of stable bulk pools was measured by elisa. the three protein subunit genes were expressed from vectors with different selection markers. in the reference constructs (without gun), the α, β, and γ chains were expressed from vectors with marker genes for zeocin, blasticidin, and gs, respectively. a similar vector combination was also generated with gun elements integrated in each vector. in addition, vectors with subunits (γ-α and α-γ), each with a separate gun element, promoter and polyadenylation signal, were generated with a gs marker gene. cho gs -/cells were transfected with the appropriate vector combinations in equimolar ratios and selected in bulk in medium lacking glutamine and or antibiotics. the -vector transfected cell pools recovered first, due to the presence of only antibiotic in the medium (fig. a) . the pools transfected with three gun vectors recovered to maximum viability just a few days after the -vector gun pools. recovery of the reference pools took up to a week longer than the gun pools. production of each pool was assessed in a batch production run in shaker flasks. all -vector gun pools which recovered first produced titers around . g/l, which is almost -fold higher as compared to the production by reference pools (fig. b) . the highest titers of . g/l were obtained in the -vector gun pools. these data show that the g unic™ genetic elements can be successfully used to obtain a significant increase in the titer of difficult-to-express proteins. similar results have been obtained with other dte proteins, including fc-fusion proteins and bi-specific antibodies (not shown). the expression of a large, glycosylated multimeric difficult to express protein can be increased more than ten-fold in cho gs pools by application of g unic™ genetic elements. the highest expression of is obtained using a separate vector for each subunit. characterization of antibody-producing cho cells with chromosome aneuploidy noriko yamano , , sho tanaka background chinese hamster ovary (cho) cells are commonly used as host cells to produce biopharmaceuticals. however, the number of chromosomes in cho cells varies. previously, dg -sc and dg -sc cell lines with modal chromosome numbers of and were isolated from parental cho-dg cells, from which igg -expressing cell lines named igg -sc and igg -sc were established, respectively. the igg -sc cell pool showed a higher specific igg production rate than the igg -sc cell pool [ ] . even though all of the igg -sc clones and half of the igg -sc clones contained the same number of vector integration sites (single integration site), igg-sc cell clones produced more igg following the culture of single-cell clones than any of the igg -sc clones [ ] . in this study, we performed transcriptome analysis to investigate the characteristics of high-producer cells with chromosome aneuploidy. transcriptome analyses using amplified fragment length polymorphism (aflp)-based high-coverage expression profiling (hicep) and de novo mrna-seq were performed on dg -sc , dg -sc , igg -sc and igg -sc . to compare cell lines with different numbers of chromosomes, transcriptome data from mrna-seq were adjusted for cell number using rna reference materials (nmij crm -a; national institute of advanced industrial science and technology) mixed at equal amounts per cell. pathways related to differentially expressed genes were searched using keymolnet (km data). high-chromosome-number cho cells showed larger cell diameters, as determined by vi-cell (beckman coulter) measurement. the predicted volume ratios, based on these diameters, are . (dg -sc :dg -sc ) and . (igg -sc :igg -sc ). the levels of β-actin and the products of most other genes that were detected by mrna-seq differed by approximately % in the comparison between sc and sc (sc > sc ). based on the analysis of gene expression levels per cell volume, approximately % of detected genes showed lower expression in both dg -sc and igg -sc compared with the levels in dg -sc and igg -sc , respectively. in addition, the number of genes whose expression level was decreased in igg -sc compared with that in dg -sc was larger than those showing the opposite pattern. the results of the comparisons between igg -sc and igg -sc indicate that differentially expressed genes were mainly related to cell growth (e.g. myc, smad), apoptosis (e.g. caspase), lipid metabolism (e.g. srebp, pparγ) and epigenetic histone modification (e.g. brca, hat) pathways. the mrna levels of myc, smad, caspase, brca and hat related genes were lower in igg -sc , while those of srebp and pparγ related genes were higher in igg -sc . the effects of these pathways on antibody production should be examined in future. in this study, we found that high-chromosome-number cho cells have lower amounts of mrna relative to their volume. a reduction per unit volume in the expression of genes that are required for survival might generate additional energy for recombinant protein production in high-chromosome-number cells. from an evolutionary perspective, an increased set of chromosomes underlies rapid evolutionary adaptation. although there are issues to be considered, such as stability, there may also be advantages to using high-chromosome-number aneuploid cho cells as a production host cells of recombinant proteins. background human growth factors have an enormous therapeutic potential. among them, the bone morphogenetic protein- (bmp- ) can induce de novo bone formation endowing the protein a high therapeutic potential. however, finding a suitable recombinant production system for such a protein still remains a challenge. recombinant expression of hbmp was investigated in transiently transfected hek- cells and in stable clones established in cho-k cells cultivated in excell and pro-cho medium, respectively. protein stability and interaction of the hbmp with the producer cells were investigated in vitro using commercially available rhbmp . in addition, we investigated a cell-free protein synthesis system harboring translocationally active microsomal structures, hence having the potential to perform post-translational modifications, as an alternative production method. we showed that growth rates and viabilities of the rhbmp producing cells were similar to those of the parent cell line, while entry into the death phase was delayed in case of the recombinant cells. the maximum rhbmp concentration detected in the culture supernatant was low for stable clones but can be greatly improved combining the hek- cells transient expression system and batch reactor cultivation which reflects a better compatibility of the codon usage in the human cells (table ) . hbmp protein is sensitive to slightly acidic ph and to a lesser extend to proteases (fig. a ) and binds to both producers cell lines (fig. b) -all this could incidentally contribute to the low product titers. cell-free protein synthesis has been proposed as alternative for "difficult-to-express" proteins. since native hbmp is glycosylated, a cell-free system based on eukaryotic cell lysates is required for its production. cho cell lysates were chosen, since they had previously been established as the most productive eukaryotic system in our hands [ ] , while concomitantly enabling a direct comparison to the production of hbmp in stable clones established in cho-k . the ability to perform post-translational modifications is a major advantage of eukaryotic systems. the cho lysates prepared by the protocol used here have previously been shown to contain significant amounts of endogenous microsomes derived from the endoplasmatic reticulum during lysis [ ] . to enforce translocation of the target protein into the microsomal structures, a melittin signal peptide was fused to the hbmp cdna. the glycosylation of the protein was assessed by enzymatic treatment (pngase, endoh) and confirmed using c-mannose for the de novo protein synthesis. upon cell-free protein synthesis, the hbmp yield was -fold higher than the best one in the hek- cells. the difference becomes even more dramatic, when productivities are considered (table ) , i.e. the fact that maximum product titers are reached within h in the cellfree system compared to h in the cell-based ones. this demonstrates that the cell-free expression system is most suitable compared to mammalian cell expression method for the production of glycosylated human bmp (table ) [ ] . human growth factors are complex molecules, which make their production in mammalian cells desirable. however, low product titers caused by a variety of both cell and process related effects may hinder the development of highly productive processes. in such cases, cell-free protein production using cho cell lysates containing endogenous microsomes for posttranslational processing, may eventually present an attractive alternative. in particular since these lysates can be used under tightly controlled conditions assuring a higher degree of reproducibility, than, e.g. transient transfection systems. cell-free systems are known to circumvent typical bottlenecks of cellbased ones, e.g. metabolic regulation and cell maintenance mechanisms. in consequence, the production of a recombinant protein is neither inhibited by its accumulation nor by any interaction with the cells, e.g. through the activation of inhibitory signaling pathways. core. preliminary studies showed that the corresponding polyplexes, but also some of the cells that came into contact with them, became magnetic and were manageable by magnetic fields [ ] [ ] [ ] . here, we present a characterization of the influence of structure and composition on the function of these polymers using a library of highly homogeneous, paramagnetic nano-stars with varied arm lengths and densities [ ] . the paramagnetic nano-stars library was synthesized by coating maghemite nanoparticles (γ-fe o ) with a thin silica-shell functionalized with an atomic transfer radical polymerization (atrp) initiator. pdmaema arms were grown from the core particles via atrp. in one case, the pdmaema arms was end-capped with pdegma blocks produced during a second atrp step. all nanostars were characterized by size exclusion chromatography and thermogravimetry to calculate number and length of the pdmaema arms. the core diameter was determined by transmission electron microscopy and dynamic light scattering (dls). the different variants (table ) were analyzed for their ability to complex pdna (pegfp-n ) using various physicochemical methods (dls, zeta sizer). transfection efficiency/cytotoxicity in cho-k cells were determined by flow cytometry. transfected cells were placed in a magnetic field and the influence of the polymer architecture on the magnetic separation was investigated. nonparametric spearman analysis was used to correlate between arm length/arm densities, magnetic properties of the cells and transfection efficiency. based on the hydrodynamic radii of the polyplexes, the investigated nano-stars could be divided into three subgroups (table ) . middle, but also high arm density nano-stars formed smaller polyplexes with hydrodynamic radii ≤ nm, a size that is considered suitable for endocytosis and transfection. transfection efficiencies and cytotoxicities varied systematically with the nano-stars architecture, with viability showing a more pronounced dependency on the characteristics of the transfection agent than the transfection efficiency itself. the arm density was particularly important, with values of approximately . arms/ nm yielding the best results (fig. a) . the end-capping the polycation arms with pdegma significantly improved the serum compatibility (fig. b) . the gene delivery potential of a given nano-star and its ability to render the cells magnetic did not correlate. although, compared to the non-separated cells, egfp-expressing cells were consistently more frequent in the magnetic cell fraction, while the non-magnetic fraction was slightly depleted. when the egfp-expressing cells were further divided into low, middle and high producers, a statistically significant shift towards the high producers was observed in the magnetic cell fraction (fig. c) . a nonparametric spearman correlation analysis was used to statistically evaluate possible links between the molecular characteristics of the nano-stars, the physicochemical properties of the corresponding polyplexes, the transfection conditions, and the cellular reactions. the resulting correlogram is shown in fig. d . transfection agents with magnetic properties enlarge the toolbox for studying non-viral gene delivery, since cellular magnetism is added as a new parameter. this allows, inter alia, a distinction between mere cellular interaction and actual uptake, which is otherwise difficult. viability showed a much more pronounced dependency on the characteristics of the transfection agent/polyplex than the transfection efficiency itself, which should be taken into account during method optimization. end-capping the polycationic pdmaema-arms with pdegma-blocks improved the compatibility of the polycationic nano-stars with serum components. in future optimized, blood-compatible, nano-stars, which can be retained/directed by magnetic fields, could become options for non-viral gene delivery in vivo. the increasing demand for monoclonal antibodies has necessitated the need to increase the productivity of current industrial cell lines. in our earlier study [ ] , we had shown that treatment with er-stress inducer, tunicamycin significantly increased the titers and productivity in recombinant cho cell lines with a simultaneous upregulation of many genes from the unfolded protein response pathway (upr). however the loss in cell viability prevented a sustained increase in titers. in the current study we explore the effect of varying concentrations of tunicamycin and treatment times, such as to modulate the increase in protein folding capacity while preventing induction of apoptosis. anti-rhesus igg-secreting cho cells [ ] were cultured in sf-cdm in ml shake flasks. the cells were treated with varying concentrations ( - ng/ml) of tunicamycin in a batch culture. further, the effect of treatment with tunicamycin for short periods of time ( hrs) was also evaluated. igg titers and mrna expression levels were quantified using elisa and qrt-pcr (illumina), respectively. results cho cells were treated with different concentrations of tunicamycin and cultured in a batch for days (referred as continuous treatment/cte). figure a presents the maximum vcd and % drop in viability under treatment. a dose-dependent inhibitory effect is observed on growth and viability of cells in cte-cultures, with minimal inhibition as lower concentrations. contrastingly, igg titers (fig. b) were higher in treated cultures w.r.t. control in initial phase of the cultures at all the concentrations of tunicamycin. the per-cell productivity (fig. c) also showed a significant increase w.r.t control at all the concentrations of tunicamycin. however, the increased productivity due to tunicamycin was not sustained and levels become similar to control after day (data not shown). to prevent loss of viability due to tunicamycin, the effect of short-term treatment (ste) with tunicamycin was explored. cells treated with tunicamycin for hours were harvested (corresponding to day of cte cultures) and inoculated in fresh media. the ste-cultures showed improved viability and higher maximum vcd as compared to cte-cultures (fig. a) . the fold increase in igg titers was not sustained beyond day - in stecultures ( fig. d ) but significant increase in productivity was seen in the initial phase (fig. e) . further, the cells were adapted over continuous generations under ng/ml tunicamycin. the adapted cells had overall . -fold higher productivity, as compared to control (fig. f) , in a batch culture. to understand the molecular basis of increase in productivity, mrna expression level of key genes was determined. xbp s is a transcription factor involved in activation of chaperones (like grp , calreticulin) and apoptotic genes (such as chop). significant increase in the levels of calreticulin was seen on treatment with tunicamycin (fig. g) . both xbp s and grp were marginally induced when treated with ng/ml of tunicamycin in both cte-and ste-cultures (fig. h) , and significantly up-regulated when treated with ng/ml of tunicamycin. the chop mrna levels also increase with increasing tunicamycin concentrations, with levels in ste-cultures lower than cte-cultures (fig. h) . the results suggest that upr induction may be important to increase productivity in these cte/ste-cultures. note that, tunicamycin had no effect on the expression levels of igg heavy-chain, thus eliminating the involvement of igghc-mrna in increasing productivity (fig. i) . tunicamycin induced er-stress increased productivity in the initial phase of the culture and enhanced upr-mediated folding capacity can be attributed as one of the reasons for it. at lower concentrations of tunicamycin, a fine balance between optimum upr induction and apoptosis can be achieved, as seen in ng/ml tunicamycin ste-cultures. in summary, this study demonstrates an alternate approach to enhance productivity of current industrial cell lines. background chinese hamster ovary (cho) cells have been widely used for the large-scale production of biopharmaceuticals [ ] . to construct antibody-producing cho cells, exogenous genes encoding antibodies are usually integrated into unspecified regions of chromosomes (random integration). however, the chromatin structure differs depending on the location of the chromosomal region, which affects the expression level of the gene of interest [ ] . recently, gene-targeting methods that enable site-specific integration of expression vectors have been developed. however, the regions that are most efficient for exogenous gene expression have not been clarified. we previously constructed a cho genomic bacterial artificial chromosome (bac) library generated from the recombinant cho-dg cell line. it was expected to cover the entire cho genome five times. the chromosomes in cho-dg cells were aligned in decreasing order of size and assigned letters from a to t [ ] . three hundred and four bac clones were mapped to every chromosome of cho-dg . among the karyotypes of cho-dg , cho-k and primary chinese hamster cells, chromosomes a and b are considered as the sole paired chromosomes corresponding to chromosome in primary chinese hamster cells. hence, chromosomes a and b are considered to be stable [ ] . in this study, we constructed antibody-producing cells by using a gene-targeting method, which focused on the stable chromosomes. a gene map of chromosome was constructed by combining the bac-fluorescence in situ hybridization (fish)-based chromosome physical map and sequence data of mapped bac clones. the sequences of bac clones were searched by blast with ncbi and chogenome.org databases. three different regions on chromosomes a and b were selected based on cho genomic bac library sequences as target sites. cho-k cells were stably transfected by lipofection. the target sequences were broken using the clustered regularly interspaced short palindromic repeats (crispr)/crispr-associated protein (cas ) system and humanized igg genes were integrated by non-homologous end joining recombination. transfection without using the crispr/ cas system was also performed. these cell pools were cultivated for six days with serum-supplemented medium, and their levels of antibody productivity were evaluated by elisa. copy number analysis was also performed using real-time pcr. results and discussion construction of gene map of chromosome : eighty-three bac clones were mapped onto chromosomes a and b (each clone contained - kb of the cho genome sequence). as a result of annotations of bac clone sequences, genes were mapped on chromosome . investigation of the differences of productivity among antibodyproducing cells that were constructed by chromosome targeting and/or random integration: cell growth was not affected by the gene targeting site. the specific production rates of antibodyproducing cell pools constructed by gene targeting of chromosome were higher than those of the cell pool constructed by random integration. all cell pools constructed by gene targeting showed lower copy numbers of heavy chain and light chain in genomic dna than those in the cell pool constructed by random integration, despite showing high productivity. our results indicate that high productivity of the cells constructed by gene targeting of chromosome does not depend on the increase of the antibody copy number, and that the environments around these target regions are suitable for exogenous gene expression. the approach of using gene targeting to chromosome may be promising for constructing antibodyproducing cells. retroviral vectors have been widely used as gene delivery tools in various biotechnology fields. however, the random integration feature of retroviral vectors seems to cause problems such as insertional mutagenesis and gene silencing. we previously demonstrated cre-mediated retroviral transgene insertion into a pre-determined site of the founder cells using integrasedefective retroviral vectors (idrvs), where a cre expression plasmid was transfected into the cells prior to retroviral transduction [ ] . recently, we reported novel hybrid idrvs (cre-idrvs) incorporating bioactive cre recombinase protein, and validated site-specific gene integration of an scfv-fc antibody expression unit into the chinese hamster ovary (cho) cell genome [ ] . we also developed an accumulative site-specific gene integration system, which enables repeated integration of multiple transgenes into a pre-determined locus of the cell genome [ ] . here, we attempted repeated integration of transgenes using cre-idrvs. a viral vector plasmid (pqmscv/hd[scfv-fc]) encoding reporter genes and an scfv-fc expression unit flanked with wild-type and mutant loxps was constructed for the production of idrvs. cre-idrvs were produced described previously [ ] . results and discussion figure a shows a schematic drawing of each round of targeted transgene integration using cre-idrvs harboring an scfv-fc expression unit. (fig. b) . genomic dna extracted from the cells were subjected to pcr using specific primer pairs α and β, and γ and δ to confirm site-specific integration. dna fragments with expected sizes were amplified in each cell clone (fig. c) . these results indicate that site-specific repeated integration was achieved using cre-idrvs. in contrast, scfv-fc productivity in cho/hd[scfv-fc]× cells was slightly decreased compared with that of cho/ne[scfv-fc]× (data not shown). although the reason remains unclear, repeat-induced gene silencing might occur due to tandem repeat structure of expression units. we reported improved recombinant antibody production using a production enhancer element [ ] . such a cis-regulatory element might be a feasible approach to enhance the productivity. we demonstrated site-specific repeated transgene integration into a pre-determined chromosomal locus using cre-idrvs for the production of an scfv-fc antibody. if lipids role in the cell have been reduced for a long time to cell membrane formation, it is now understood that lipids plays also a role into energy metabolism, vesicular transport, membrane structure, dynamics and signaling. however, the exact mechanism of how compositional complexity affects cell homeostasis remains unclear. thanks to recent advances in mass spectrometry, it is now possible to study a wide range of lipids, providing a better understanding of lipid homeostasis in high performance cell culture processes. the purpose of this work was to develop a robust lipidomics method applied to mammalian cell cultures in a three step method: extraction, separation and detection (fig. ). both matyash [ ] and folch [ ] extraction method were performed on our cells to reach the highest yield. two separation techniques were also tested: hydrophilic interaction liquid chromatography (hilic) and reverse phase chromatography. finally lipid classes' identification was achieved by tandem mass spectrometry analysis thanks to structure-specific fragmentation ions. the yield obtained with matyash extraction method was higher than with folch method for each lipid class tested. besides, matyash method presents also the advantage to be less toxic and suitable for high throughput analysis since the organic layer is above the aqueous layer. lipids separation by hilic is based on their polar head. since lipid classes are defined by polar head, the lipids are eluted class by class, making their identification easier. the separation of lipids by reverse phase was correct but the method is longer and we observed a massive carryover of triglycerides on the column. finally each lipid class was screened in ms/ms parent ion mode. target daughter ion was set according to the lipid class structure and fragmentation pattern. this detection technique enabled the identification of different lipids. to ensure the absolute quantification of the detected lipids and to guarantee comparable results between batches labeled internal standard were added prior to extraction. this method was optimized in a stepwise process to ensure a sensitive and selective measurement of the lipids. lipids were extracted by matyash method, separated by hilic and detected by tandem mass spectrometry. this method is suitable for both in process sample lipid analysis providing information on the cell lipid content, and for harvest samples, enabling to follow the lipid release during the different harvest steps. this non-targeted lipidomic quantitation method will enable us to better control lipid synthesis during biopharmaceutical fed batch production through clone selection, metabolomics studies and harvest development. background human mesenchymal stem/stromal cells (hmsc) can easily be isolated from e.g. bone marrow, fat tissue or umbilical cord blood and are therefore a central player in regenerative medicine, gene therapy and cell therapy [ ] [ ] [ ] . the necessary gene shuttle is mainly provided by viruses associated with diseases, like retrovirus or adenovirus [ ] [ ] [ ] [ ] . these possible pathogen viruses demand for high safety standards. also, they are prone to genomic alterations and there is the possibility of virus inactivation, triggered due to pre-existing immunity in the patient [ ] [ ] [ ] . in this context, the autographa californica multicapsid nucleopolyhedrovirus (acmnpv) is a safe alternative. the virus replication is hostspecific for insects [ ] , but it is known since the mid- s, that a temporary transduction of mammalian cells is possible [ ] . some modifications of the virus increased the applicability in stem cells. pseudotyping the virus with the vesicular stomatitis glycoprotein (vsv-g) led to an expansion of the transducable cell [ , ] and the integration of the woodchuck hepatitis virus post-transcriptional regulatory element (wpre) prolonged the recombinant protein expression [ , ] . for achieving a baculovirus-induced differentiation of hmscs, the promotor and the expression strength of the recombinant protein are crucial factors. still, there are still few comparative promotor studies [ , ] . however, a successful virus uptake is the prerequisite for a successful protein expression. we therefore investigated factors significantly influencing the transduction process by applying design of experiments (s. fig. a ). the experimental design comprises a two level factorial screening, set-up using design expert v .for the transduction , c/cm were seeded in -well plates with dmem + % fcs and incubated overnight at °c, % co and humidified atmosphere. the recombinant baculovirus using an integrated ef α promoter to control gfp expression, described elsewhere [ ] , was diluted to the respective concentrations in the different surrounding fluids. after discarding the cultivation medium of the hmsc-tert, ml of virus containing solution was added to the cells. the following incubation was varied in duration before replacing the virus solution with growth medium and an incubation overnight. h post transduction (hpt) the cells were washed with pbs, trypsinized with μl trypsin/edta and incubated for min at °c. trypsination was then stopped applying μl soybean trypsin inhibitor and the cells were analyzed using flow cytometry. as shown in fig. a , the virus concentration and incubation time exert the highest influence on the transduction efficiency. obviously, a higher concentration of viral particles and longer incubation of cells with virus increases the probability for hits between cells and virus particles. additionally, the surrounding fluid can have a negative impact on the transduction. this is due to the interaction of medium components with the baculovirus. therefore, pbs containing ca + & mg + is recommended as surrounding fluid for transduction experiments. in fig. b , the transduction conditions resulting in the highest percentage of gfp+ cells are displayed: virus particles per cell (ppc) and an incubation time of h with hmsc-tert. the experiments show, that especially the virus concentration and the incubation time of cells with virus influence the transduction efficiency. based on the results of the screening, further optimization of the transduction conditions will be done using a face centered central composite design with pbs containing ca + & mg + as surrounding fluid and at an incubation temperature of °c. background breast cancer is the second main cause of cancer related deaths for women worldwide and among them the triple negative subtype (tnbc) represents a clinical challenge by being associated with high mortality and having no effective therapies against it [ ] , [ ] . accordingly, there is an urgent need to design new and more effective drugs to treat breast cancer. notch signaling is an evolutionary conserved cell-to-cell communication pathway crucial during embryonic and breast development and tissue homeostasis. this pathway is often hyper-activated by overexpression of notch receptors and/or its ligands in several types of cancers, such as breast cancer (tnbc included), where it contributes to its development, progression and drug resistance [ ] , [ ] , [ ] . our aim is to generate a function blocking antibody against the notch delta-like- (dll ) ligand with therapeutic efficacy against breast cancer. materials and methods dna of human dll full length extracellular domain (dll -ecd) and a truncated version, containing the minimal binding region to the notch receptor (dll -egf ), were cloned into pfuse-fc -igg , and expressed in hek e cells. recombinant proteins were purified from culture media by protein-a affinity and size exclusion chromatography. the human scfv phage display tomlinson i+j library was used to select specific scfv against peptides targeting dll binding regions to notch. the binding ability and specificity of the selected scfv clones was evaluated by scfv-on-phage elisa. our strategy allowed us to obtain mg of pure (> %) and stable dll -ecd-fc as confirmed by sds page and thermofluor assay. dll -egf -fc yield was very low and buffer screenings are ongoing to optimize protein stability. functional studies performed in human breast cancer mcf cells showed that both ligands are biologically active as they increased the expression of the notch-dependent genes hes- , hey-l and hey- . recombinant dll and peptides were used to select for monoclonal antibodies by phage display. after three rounds of panning with dll peptides we identified scfv positive clones, of which presented high affinity to dll -ecd-fc. currently we are performing more phage display selections to increase the number of positive clones. scfv with higher affinities will be reformatted into iggs and their ability to inhibit the notch pathway will be evaluated. the anti-oncogenic effects of anti-dll iggs will be assessed in breast cancer cells in viability/apoptosis, proliferation, migration, and invasion assays. an anti-dll igg with therapeutic efficacy against breast cancer will demonstrate that targeting dll could be one of the key factors for successfully targeting breast cancer. recombinant adeno-associated virus (raav) approaches have an outstanding reputation in gene therapy and are evaluated for cancer therapy [ ] . advantages include long-term gene expression, targeting of dividing and non-dividing cells, and low immunogenicity. established raav production utilizes triple transfection of adherent hek cells, which hardly meets product yield requirements for clinical applications. we transferred the aav production system to hek -f suspension cells. this process is scalable and uses serum-free media streamlining downstream procedures. after optimization of transfection efficiencies and shaker cultivations, we produced titers of × viral genomes per cell in a l bioreactor. the suspension adapted hek-freestyle -f cell line was used for the experiments in chemically defined animal component free media (hek-tf, hek-gm (xell ag), freestyle f (thermo fisher scientific)). samples for viable cell density and viabilities were taken daily and analyzed using an automated cell counting system (cedex, roche diagnostics). transient transfection of × cells/ml was carried out with polyethylenimine max in a : dna-pei ratio (w/w) with μg dna. three plasmids (pgoi, prepcap, phelper) were applied in a molar : : ratio (fig. a) . pretests were performed in orbital shaking tube spin bioreactors. for scale-up, batch processes were carried out in ml shake flasks as well as in l stirred bioreactors at % air saturation and ph . . transfection efficiencies and raav production were quantified by flow cytometry using a goi coding for a fluorescent protein and qpcr of genomic copies, respectively. by optimizing the dna amount for transfection of -f cells more than % of the cells were reproducibly transfected. batch cultivations in shaker flasks revealed that raav were produced in the first - h after transfection. figure b shows viable cell densities and viabilities in relation to the genomic titer. genomic titers were determined from raw cell extracts and up to copies/ml were repetitively achievable. a decrease in viability marked the decline in genomic copies per ml showing that a prolongation of the process e.g. by addition of a feed would probably not increase yield. in a first scale-up, the raav production was transferred to a l bioreactor (fig. c) . transfection efficiencies in bioreactors of up to % were comparable to that obtained in a simultaneous shaker flask experiment. transfection efficiencies were lower compared to prior experiments due to controlled conditions in the bioreactor. nonetheless the titer with up to × genomic copies per cell was elevated compared to that of shaker flasks. first experiments with -f cells in hek tf medium showed promising results of transferring raav production from the adherent system to suspension. after improvement of transfections by the adjustment of dna amounts in small scale experiments, aav production was analyzed in shaker flasks. the batch process showed an expected increase in cell density with low variability between biological replicates (fig. b) . the genomic titer increased according to the viable cell density until day four where a sudden drop started. this observation was made for aav productions in hek-tf, hek-gm and freestyle f medium. for optimal yields, we assume that a slight decrease in viability marks the point in time for harvest. from optimized protocols, a batch process in a l bioreactor was carried out. interestingly the bioreactor cultivation resulted in lower overall viable cell densities but in higher genomic copies per cell compared to shaker flasks (fig. c) . these results are comparable to already published data for suspension cells [ ] . subsequent optimization of the bioreactor protocol will lead to further increase in raav yield. genethon and pall have collaborated to assess pall's single-use icellis fixed-bed bioreactor for viral vector production. clinical use of gene therapies to treat formerly incurable genetic diseases is advancing rapidly. viral vectors are an important tool for introducing genes into target cells. many gene therapies have been developed using adherent cells in -dimensional flatware or roller bottles but using these technologies to reach commercial-scale production represents a significant challenge. the icellis bioreactor enables large-scale viral vector production by providing a -dimensional matrix for cell growth in a compact configuration (fig. ). up to m of surface area is available in a compact bioreactor measuring mm in diameter in a total volume of l with ph, do and temperature control. a key feature of the icellis bioreactor is that it scales by increasing the diameter of the fixed-bed while keeping the height constant with no change in aspect ratios. the height of the fixed-bed can be varied ( , and cm) as well as density of carrier packing ( gm/l or gm/l). the icellis system comes in two formats, the icellis nano bioreactor ( . - . m ) and the icellis bioreactor ( - m ). processes developed in the bench top icellis nano bioreactor can be directly transferred to the corresponding icellis system. the icellis nano bioreactor enables an efficient platform for process optimization. the genethon raav- process was transferred to an icellis nano bioreactor . m ( cm bed height, gm/l density) bioreactor using freestyle media. the initial icellis nano process was established as ( ) seed on day , ( ) transfect at day , ( ) harvest at day and yielded < x vp/cm (n= ). media exchange, cell density at transfection, pdna/cell ratio, and lysis method were then changed to determine the effect on productivity. the modified process was then scaled from . m to . m ( cm bed height, gm/l density) icellis nano bioreactor. -media: a media exchange at hours post transfection with dmem substituted for freestyle medium resulted in an x increase in specific productivity. (abstract p- ) . a schematic overview of raav production in hek cells with triple-transfection system. b viable cell densities (vcd), viabilities and genomic copies per ml (gc) of a raav production with -f batch cultivations in shaker flasks. genomic copies per ml refer to the titer determined in ml culture volume. error bars represent biological and technical duplicate measurements of samples. c viable cell densities and genomic copies per cell of a raav production with -f batch cultivation in a l bioreactor. for reasons of comparability between shaker and bioreactor data genomic copies are given per cell. error bars represent technical duplicate measurements of samples -cell density at transfection: cells were seeded at , cells/cm and reached , cells/cm at day which was determined to be the optimal cell density for transfection. -pdna/cell ratio: reducing pdna by % had no significant effect on productivity. -lysis: use of trion x- at . % with mm nacl at ph resulted in > % virus recovery compared to sampled carriers. -scaling: specific productivity was maintained as the system was scaled from . m to . m . -overall, an average yield of x vg/m was achieved. the icellis technology is being adopted widely for viral vector production. transferring a process to the icellis nano bioreactor can be easily achieved and once in place can be optimized to provide significant productivity increases and cost savings such as reduced pdna. the icellis nano bioreactor is an efficient bench-top system the results of which can be readily scaled to the icellis system. background tissuse multi-organ-chip (moc) platform contributes to the ongoing advancement in systemic substance testing in vitro. current in vitro and animal tests for drug development are failing to emulate the systemic organ complexity of the human body and, therefore, often do not accurately predict drug toxicity. especially, cardiotoxicity is one of the main reasons why new compounds are failing in clinical trials. therefore, we aimed to establish an autologous dynamic multiorgan-device integrating cardiomyocytes for substance testing. generic d monolayer and d suspension ipsc derived cardiomyocytes differentiation protocols were established. beating cardiomyocytes were first seen on day in monolayer as well as in spheroid culture. cardiomyocytes show up to % cardiac troponin t positive cells and % myosin heavy chain positive cells by flow cytometry (fig. g, h) . myosin ii heavy chain, α-actinin, myosin / , myosin and caldesmon expression was shown by immunohistochemistry ( fig. a-d) . due to the exclusion of a lactate enrichment of cardiomyocytes, cardiac fibroblasts are also expressed in the spheroids shown by vimentin staining. those cardiac fibroblasts lead to a physiological heterologous cell population similar to the human heart. beating spheroids were cultivated for days under dynamic culture conditions in the multi-organ-chip. the integrated on-chip micropump provides physiological-like pulsatile circulation at a microliter scale and leads to better nutrition and oxygen supply. the next significant step is to combine multiple autologous d organ equivalents in our multi-organ-chip using ipsc differentiation technology. differentiating all cell types from one ipsc donor is crucial to overcome source and rejection problems. combining our multi-organ-chip platform with ipsc differentiation technology will eventually lead to a personalized system for drug and substance testing. lab as a service -automated cell-based assays lena schober, moriz walter, andrea traube laboratory automation and biomanufacturing engineering, fraunhofer ipa, stuttgart, germany correspondence: lena schober (lena.schober@ipa.fraunhofer.de) bmc proceedings , (suppl ):p- background the use of cell-based assays in pharmaceutical industry and academic research is a growing trend that is a driving force to reduce costs for drug development. academic research is gaining information about intracellular targets or functional mechanisms through the variety of different assays. these benefits can be used in preclinical studies and furthermore costly late-stage drug failures may be reduced by the use of cell-based assays. the use of automated systems is also in great demand and will change the testing of substances and research activities. nevertheless, there are a lot of barriers at the moment limiting the successful application of automated systems in this field. by the lack of flexibility and the demand for skilled computer scientists & engineers just the two main aspects stated by experts shall be mentioned. our strong background on automated cell culture technologies and expertise, gained in several projects, let us rethink the overall process chain and overcome established principles. a new service orientated platform for the execution of cell-based assays that are commonly used will be introduced. the main idea is to give access to automated infrastructure for academic research or spin-offs which cannot afford the special infrastructure. nowadays it is known that the development of inhibitory antibodies by hemophiliac patients is closely related with immunogenic epitopes present in the coagulation factors. these proteins are produced in hamsters cells [ - ] which insert a different posttranslational modification profile when compared with the human profile. patients with high-titer/high-responding inhibitors must be treated with bypassing agents that can achieve hemostasis. activated factor vii (fviia) is an attractive candidate for hemostasis, independent of fviii/fix, making this coagulation factor an alternative for hemophilia patients with inhibitory antibodies. however recombinant factor vii is produced in bhk- cells (baby hamster kidney cells) and as well as the others coagulation factors, it may contain immunogenic epitopes [ - ] . in this context, becomes extremely important to produce recombinant proteins with complex posttranslational modifications in a cell line not yet used [ - ] . we have been using the sk-hep- human cell line for the production of recombinant fvii. to generate the recombinant cell line we have used a bicistronic lentiviral vector, -gfp, containing a fvii gene and the gfp selection marker gene. a master cell bank and a work cell bank were generated in gmp conditions. the rfvii analyses were made by elisa assay, western blot, gene expression quantification and biological activity using the prothrombin time (pt) assay. rfvii purification by affinity chromatography using viiselect (ge) column. after purification the rfvii was formulated and dry froze to be used in in vivo experiments. in static conditions sk-hep- cells showed, for a period of months, a stable fvii production with an average of , iu/ml of fvii, % of cell viability and % of cells expressing the gfp gene. after purification with viiselect column it was possible observe a recover of % of the purified protein with % degree of purity (fig. ) . this recombinant purified fvii is being used in in vivo experiments to determine the pharmacokinetics parameters and to evaluate the posttranslation modifications profile. in conclusion, this study reports the use of sk-hep- cell line for high-level production of recombinant factor vii. these cells have proven to be effective in the production of recombinant protein and can be used as a new platform for the production of recombinant proteins. fig. (abstract p- ) . a determination of protein yield of egfr (epidermal growth factor receptor) synthesized in a cho cecf system. analysis of egfr protein yield obtained in a various batches of cecf formatted reaction. cecf synthesis was performed in the presence of c leucine for radio labeling of target proteins. radio labeled proteins were precipitated using tca followed by scintillation measurement. b detection of radio labeled egfr by autoradiography. a no template control (ntc) was prepared containing no egfr dna template background emergence of stem cell-based regenerative medicine recently leaded to the necessity to reach a sustained production of such cells [ ] . hence, new bioreactors and carriers were designed for cell expansion. however, to meet this increasing demand, improvement of both quality and quantity of stem cells remains necessary. soft biocompatible microcarriers mimicking extracellular matrix in term of structure and stiffness should be of valuable utility as substrate stiffness strongly influence in vitro stem cell fate and differentiation [ , ] . our expertise in the field of microbeads design using jetcutting technology [ ] enabled us to engineer +/- μm alginate beads of various g/m monomer ratio. we used jetcutter (genialab gmbh) with μm nozzle at max speed rpm. alginate solutions with concentrations % to % were gelifyed in % cacl etoh % solution. alginates with estimated viscosity (@ %) from to mpa were tested. a further surface treatment with gelatine ( , %, %) and poly-l-lysine ( , %) was carried out to reach an optimal cell anchoring of human adiposederived mesenchymal stem cells (atcc-psc- - ) in mesempro rs medium (gibco). jetcutter technology allowed us to obtain alginate microcarriers with a good homogeneity in size around μm and sphericity comparable to commercial carriers (table ) . best adhesion of human adipose-derived mesenchymal stem cells was obtained on , % gelatine coated alginate carriers (fig. ) . we observed limited apoptosis and human adipose-derived mesenchymal cells stemness was conserved after days in culture (data not shown). cellular bioassays developed with functionally immortalized cell lines aileen bleisch , aleksandra velkova , tom wahlicht , dagmar wirth , tobias may inscreenex gmbh, braunschweig, germany; msys, helmholtz centre for infection research, braunschweig, germany; greiner bio-one gmbh, frickenhausen, germany bmc proceedings , (suppl ):p- background a major challenge of current research is the limited availability of physiologically relevant cells [ ] . thus the development of relevant cellular bioassays that are robust, reproducible and scalable is hindered. to overcome current limitations we developed an immortalization strategy allowing the efficient and reproducible establishment of novel cell lines showing an in vivo-like phenotype. the main feature of our ci-screen technology is the ability to combine the advantage of cell linesthe unlimited cell supplywith the advantage of primary cellsthe physiological relevance. using this technology we have immortalized, amongst others, a human osteoblast cell line (ci-huob) [ ] . in the present study, the in vivo-like phenotype and functionality of the novel ci-huob was examined. therefore, ci-huob cells were used to develop a d cell culture model by using the magnetic d bioprinting technology (nano d biosciences, houston, tx, usa) [ ] . the ci-huob cell line was recently described and cultivated in huob maintenance medium (inscreenex, germany). for spheroid creation ci-huobs were grown in a monolayer, magnetized by adding a magnetic nanoparticle assembly (nanoshuttle, ns, nano d biosciences, houston, tx, usa) at a concentration of μl ns/cm growth area. after an overnight incubation magnetized ci-huob were detached and seeded into cellstar® cell-repellent -well plates (greiner bio-one, frickenhausen, germany). with the help of mild magnetic forces cells were printed into spheroids within h. these consist of . - . cells and were cultured for a period of up to days. the cell viability was analyzed by a propidium iodide (pi) and calcein am staining. to improve spheroid functionality spheroids were cultivated with huob differentiation medium (inscreenex, germany). "mini bone" tissue functionality and thus mineralization was analyzed by an alkaline phosphatase (alkaline phosphatase activity) and an alizarin red s staining (ca + deposits). the combination of ci-huob cells with the magnetic d bioprinting technology enabled the establishment of reproducible and consistent d spheroids. single spheroids per well were formed independent of the amount of cells ( . - . cells) (fig. a) . formed spheroids were stable for a culture period of up to days (fig. b) . neither cell death nor cell proliferation were observed in the bioprinted spheroids which is indicated by the stable size of the spheroids throughout the cultivation (fig. c) . after treatment with a differentiation stimulus the d bioprinted spheroids became fully functional "mini bones". this was highlighted by the alkaline phosphatase activity and the ca + deposits within the d bioprinted spheroids (fig. d,e) . taken together, these results demonstrated that the functional immortalization technology provides physiologically relevant cells in sufficient numbers and that the magnetic d bioprinting technology enabled a fast, consistent cell aggregation and the formation of stable uniform spheroids. importantly, these immortalized cells are capable to differentiate when a suitable stimulus is provided. for differentiation into mini bones, d spheroid cultivation and additional stimulation by small molecules are required. the combination of physiologically relevant cell systems with three dimensional culturing will help to generate in vitro test systems which closely resemble the in vivo physiology and thereby supporting future drug discovery approaches. fig. (abstract p- ) . characterization of spheroid "mini bones". a different number ( . - . cells) of ci-huob cells were printed into spheroids. b . ci-huobs were printed into spheroids and cultivated for indicated time points. c for analyzing spheroid sizes, pictures were taken and quantified by imagej. (d/e) . ci-huob cells were printed into spheroids and cultivated with (huob differentiation medium) or without a differentiation stimulus for two weeks. afterwards, bioprinted spheroids were sectioned by a cryo microtome and d stained for ca + deposits (alizarin red s) or e stained for alkaline phosphatase activity crispr/cas , a novel genomic tool to knock down microrna in vitro and in vivo degrontagged dcas /cpf effectors for multi-directional drug-inducible control of synthetic gene regulation assessing the variability of an innovator molecule n-glycan profile correct primary structure assessment and extensive glyco-profiling of cetuximab by a combination of intact, middle-up, middle-down and bottom-up esi and maldi mass spectrometry techniques d-dige screening of high productive cho cells under glucoselimitation -basic changes in the proteome equipment and hints for epigenetic effects dependence on glucose limitation of the pco influences on cho cell growth, metabolism and igg 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th annual and international meeting of the japanese association for animal cell technology humanization strategies for an anti-idiotypic antibody mimicking hiv-i gp antibody humanization by molecular dynamics simulations-in-silico guided selection of critical backmutations maxquant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification the perseus computational platform for comprehensive analysis of (prote)omics data hoffrogge: label-free protein quantification of sodium butyrate treated cho cells by esi-uhr-tof-ms the art of cho cell engineering: a comprehensive retrospect and future perspectives gs system for increased expression of difficult-to-express proteins the netherlands correspondence: maurice van der heijden (heijden@proteonic.nl) bmc proceedings increased recombinant protein production owing to expanded opportunities for vector integration in high chromosome number chinese hamster ovary cells ires-mediated translation of membrane proteins and glycoproteins in eukaryotic cell-free systems cell-free protein expression based on extracts from cho cells comparison of cell-based vs. cell-free mammalian systems for the production of a recombinant human bone morphogenic growth factor. eng dual-responsive magnetic core-shell nanoparticles for nonviral gene delivery and cell separation pdmaema-grafted core-shell-corona particles for nonviral gene delivery and magnetic cell separation influence of polyplex formation on the performance of starshaped polycationic transfection agents for mammalian cells systematic study of a library of pdmaema-based, superparamagnetic nano-stars for the transfection of cho-k cells systems biology of unfolded protein response in recombinant cho cells dynamics of unfolded protein response in recombinant cho cells cells were transfected with np@(pdmaema ) (n/p ), separated h post transfection (t = ) by magnetically-assisted cell sorting and placed into separated cultures. the bars represent the overall transfection efficiency. distribution of low (light green), middle (green), and high (dark green) producers within the egfp-expressing cell fraction. data represent one experiment carried out in duplicate, with random experimental error shown. d correlogram between the molecular characteristics of the nano-stars (core diameter, arm density, arm length, number of monomeric units per nano-star), the physicochemical properties of the corresponding polyplexes (hydrodynamic radius, zeta potential), the transfection conditions (n/p ratio, amount of polymer), and the cellular reactions (transfection efficiency, magnetism, viability) production of recombinant protein therapeutics in cultivated mammalian cells position effects on eukaryotic gene expression bacterial artificial chromosome library for genome-wide analysis of chinese hamster ovary cells construction of bac-based physical map and analysis of chromosome rearrangement in chinese 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cells and continuous harvest of vector from the culture media for gmp fix and flt clinical vector development of a cost-efficient scalable production process for raav- based gene therapy by transfection of hek- cells simon arias , mustapha hohoud , roel lievrouw , fabien moncaubeig b- brussels, belgium; généthon, rue de l'internationale cell-free systems based on cho cell lysates: optimization strategies, synthesis of "difficult-to-express" proteins and future perspectives cell-free protein expression based on extracts from cho cells comparison of cell-based vs. cell-free mammalian systems for the production of a recombinant human bone morphogenic growth factor ires-mediated translation of membrane proteins and glycoproteins in production of recombinant factor vii in sk-hep- human cell line zip - , brazil; department of clinical, toxicological and food science analysis, faculty of pharmaceutical sciences of ribeirão preto human cell lines for the production of recombinant proteins: on the horizon production of recombinant protein therapeutics in cultivated mammalian cells recombinant protein therapeutics from cho cells - years and counting establishment of a cell line expressing recombinant factor vii and its subsequent conversion to active form fviia through hepsin by genetic engineering method expression and fast preparation of biologically active recombinant human coagulation factor vii in cho-k cells implications of the presence of n-glycolylneuraminic acid in recombinant therapeutic glycoproteins uniquely human evolution of sialic acid genetics and biology production platforms for biotherapeutic glycoproteins. occurrence, impact, and challenges of non-human sialylation human cells: new platform for recombinant therapeutic protein production therapeutic glycoprotein production in mammalian cells masthering industrialization of cell therapy products tissue cells feel and respond to the stiffness of their substrate matrix elasticity directs stem cell lineage specification continuous cider fermentation with co-immobilized yeast and leuconostoc oenos cells eternity and functionality -rational access to physiologically relevant cell lines generation and characterization of two immortalized human osteoblastic cell lines useful for epigenetic studies biocompatibility of nanoshuttletm and the magnetic field in magnetic d bioprinting publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal • we provide round the clock customer support • convenient online submission • thorough peer review • inclusion in pubmed and all major indexing services • maximum visibility for your research submit your manuscript at www submit your next manuscript to biomed central and we will help you at every step authors thankfully acknowledge the biotechnology and biological sciences research council for funding this research work. sns thanks esact for providing her with the opportunity to present her work at the meeting. we would like to thank moritz frei for his support for the generation of the ngs transcriptomics data. many thanks to valentine chevallier for her precious advices, to stefanos grammatikos for his support and to the whole upstream process sciences team. we thank david bruehlmann and thomas vuillemin from merck (vevey, switzerland) for providing the igg glycan variants and the -ab-uplc glycan data. polyplus-transfection would like to thank généthon for their kindly provided data.acknowledgements asmita mukerji, reetesh pm, sasi kumar k, pilot plant team acknowledgment to cedric allier from cea leti, grenoble, france. we would like to thank a. schemel and a. ehrlich for technical assistance. the authors would like to mention that this research was supported by the fi-dgr ( ) from spanish government and the project was led by prof. jordi joan cairó badillo. the authors want to thank the biotech process sciences team at merck in corsier-sur-vevey for their support and also the members of the morbidelli group at eth zürich for their input and collaboration. authors would like to thank dr. benjamin youn in manufacturing science and technology (msat) at biomarin for his help on coding the excel macro program for ambr , and dr. donald l. traul from tap biosystems (now part of the sartorius stedim biotech group) for his assistance on ambr operations. thanks to the bioreactor team of dustin davis, amer al-lozi, and jana mahadevan. we organic vaccines tm and the nih, who kindly provided to univercells. we thank polymun scientific immunbiologische forschung gmbh for providing the antibodies igm , igm and igm as a kind gift. this allison kurz and gian andrea signorell, genedata ag, basel, switzerland allison kurz, gian andrea signorell, genedata, basel, switzerland. allison kurz, gian andrea signorell, genedata ag high glucose concentration and low specific cell growth rate improve specific r-tpa productivity in chemostat culture of cho acknowledgements r. boraston for photographs in fig. . we acknowledge atum for their contributions to vector design and construction. austrian bmwfw, bmvit, sfg, standortagentur tirol, government of lower austria and business agency vienna through the austrian ffg-comet-k . this research is supported by sfi grant /ia/ . financial support of the austrian science fund (fwf; grant number p ) is gratefully acknowledged. we would like to thank the australian institute for bioengineering and nanotechnology, university of queensland-brisbane, australia (aibn) for providing the cho clones. this research is partially supported by the developing key technologies for discovering and manufacturing pharmaceuticals used for next-generation treatments and diagnoses both from meti and amed, japan. this work was supported in part by grants for developing key technologies for discovering and manufacturing pharmaceuticals used for next-generation treatment and diagnoses, both from the ministry of economy, trade and industry (meti), japan and from the japan agency for medical research and developments (amed). many thanks stefanos grammatikos for his support and to the whole upstream process sciences team. the authors thank the hessen state ministry of higher education, research and the arts within the hessen initiative for scientific and economic excellence (loewe program) for the financial support. the authors thank xell ag, bielefeld, for providing hek serum-free media (hek gm and hek tf) and for fruitful discussions. the authors would like to thank dana wenzel for cho lysate preparation (fraunhofer izi, potsdam-golm, germany). this work is supported by the european regional development fund (efre) and the german ministry of education and research (bmbf, no. b a). the authors acknowledge são paulo research foundation -fapesp ( / - ), centro de pesquisa, inovação e difusão (cepid), and national institute of science and technology in stem cell and cell therapy -inctc for financial support. this work was supported by grants from the niedersächsisches ministerium für wissenschaft und kultur ( ) and the german ministry for economic affairs and energy (igf n). the infrastructure which was modularly built up, consists of automated liquid handling robots, plate and tube handling robots as well as incubators, refrigerator and analysis systems as for example an imaging system. the aim is to address the need on reproducibility and reliability of results and to offer access to a maximal controlled and automated environment. with the help of a web-based configurator assay selection as well as parameterization of the assays can be done in an easy way. after the order process, test items can be shipped to the lab. assays will be executed on the fully automated platform. by capturing in process data as well as environmental conditions, a real complete data set is leading to comprehensively results. as soon as results are available during the process, the view and analysing can be done in a secure cloud. the service can be used for single experiments in low throughput applications and is therefore a benefit for labs which cannot afford automated infrastructure or the staff for the maintenance for such platforms. extensive monitoring and data capturing during the run leads to a gapless data trail and the possibility of detailed result analysis. due to automated processing the reproducibility is increased associated with direct reduction of costs and time. the centralized service paired with specific know-how allows up-scaling of processes at any time. the web-based interface provides a flexible guidance for the user and the online order gives / access on the infrastructure, leading to a fast reliable result generation. furthermore the secure interaction with additional services e.g. other specific data analysis tool is possible. this dynamic access to automation offers high flexibility for low throughput experiments and will push high quality research and drug development in early stage. development of alternative animal cell technology platforms: cho based cell-free protein synthesis systems for the production of "difficult-to-express" proteins lena thoring , background nowadays, animal cell technologies are commonly used for a broad range of medical and pharmaceutical applications. one main topic of these technologies is the production of proteins used for therapeutical purposes. these in vivo production processes are often time consuming and limited in production of so called "difficult-to-express" proteins including the pharmaceutical relevant class of membrane proteins. to overcome these issues, novel cell-free protein synthesis platforms were developed based on the industrial working horse cho cells [ ] . cell lysates provide a basis for this technology by including all components of the translational machinery and enabling protein production within a few hours. microsomal structures present in cho cell lysates enable posttranslational modification of target proteins and insertion of membrane proteins into lipid bilayer. in this study a cell-free protein synthesis platform was developed based on a combination of cho cell lysates and a continuous exchange reaction format. the continuous exchange reactor consists of a twochamber system, a reaction and a feeding chamber, separated by a semipermeable membrane. due to concentration gradients, energy components can diffuse to the reaction chamber, while inhibitory byproducts are continuously removed. different classes of proteins were selected to evaluate the quality of the cho cecf system including a transmembrane receptor, a single chain variable fragment and an ion channel. cell-free protein synthesis was performed in the presence of c leucine for radio labeling of synthesized proteins. protein yield was quantified by tca precipitation of radio labeled proteins followed by scintillation measurement and molecular mass was detected by autoradiography. posttranslational modifications and activities of proteins were estimated by kinase assays, elisa, endoglycosidase treatment and electrophysiological measurements. the demonstrated results showed a protein production of up to around g/l while detecting correct molecular weights by autoradiography. analysis of the productivity using different lysate batches by the production of the membrane protein egfr revealed only minimal batch-to-batch variations (fig. a) . posttranslational modifications of proteins, including phosphorylation and glycosylation, were detected using western blot and autoradiography (fig. b) . evaluation of localization of membrane embedded eyfp fusion proteins by confocal laser scanning microscopy resulted in the detection of proteins in the microsomal fraction of cho cell lysate. produced single chain variable fragments showed binding specificity in elisa experiments. the activity of synthesized ion channels was underlined by electrophysiological measurements and detected single channel activities. a cell-free system based on cho cell lysates for high yield production of proteins was developed that provides a platform for efficient production of "difficult-to-express" proteins. the combination of a cho lysate based cell-free system and a continuous exchange cell-free system leads to be a highly efficient production system for various classes of "difficult-to-express" proteins. this approach opens up a fast and cost-effective process pipeline for the production of "difficult-to-express" proteins and shows a high potential for industrial applications including screening technologies, protein structure determination and just-in-time protein production processes. key: cord- -dzl afq authors: stoclin, a.; rotolo, f.; hicheri, y.; mons, m.; chachaty, e.; gachot, b.; pignon, j.-p.; wartelle, m.; blot, f. title: ventilator-associated pneumonia and bloodstream infections in intensive care unit cancer patients: a retrospective -year study on prospectively monitored patients date: - - journal: support care cancer doi: . /s - - - sha: doc_id: cord_uid: dzl afq purpose: some publications suggest high rates of healthcare-associated infections (hais) and of nosocomial pneumonia portending a poor prognosis in icu cancer patients. a better understanding of the epidemiology of hais in these patients is needed. methods: a retrospective analysis of all the patients hospitalized for ≥ h during a -year period in the -bed icu of the gustave roussy hospital, monitored prospectively for ventilator-associated pneumonia (vap) and bloodstream infection (bsi) and for use of medical devices. results: during first stays in the icu, cases of vap and primary, secondary, and catheter-related bsis were recorded. the vap rate was . / ventilator days ( % confidence interval [ci] . – . ); the catheter-related bsi rate was . / catheter days ( % ci . – . ). the cumulative incidence during the first days of exposure was . % ( % ci . – . %) for vap, . % ( % ci, . – . %) for primary, . % ( % ci . – . %) for secondary and . % ( % ci . – . %) for catheter-related bsis. vap or bsis were not associated with a higher risk of icu mortality. conclusions: this is the first study to report hai rates in a large cohort of critically ill cancer patients. although both the incidence of vap and the rate of bsi are higher than in general icu populations, this does not impact patient outcomes. the occurrence of device-associated infections is essentially due to severe medical conditions in patients and to the characteristics of malignancy. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. intensive care unit (icu) patients develop life-threatening healthcare-associated infections (hais) more frequently than other patients due to their acute illness and invasive procedures. infection surveillance networks provide comparative hai data that can be adjusted, at least partially, for intrinsic and extrinsic risk factors in patients [ ] . the hai rates differ electronic supplementary material the online version of this article (https://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users. according to the icu type and patient mix [ ] . among the hais, ventilator-associated pneumonia (vap), clinical sepsis and bloodstream infections (bsis) are associated with a poorer prognosis [ ] . a growing number of cancer patients are admitted to icus and regular improvement of their prognosis has been observed [ ] . the rare publications available show high hai rates [ ] and the poor prognosis of nosocomial pneumonia in critically ill cancer patients [ ] . there is a need for a better understanding of the epidemiology, risk factors, and outcomes concerning hais in this population. our main objective was to report the incidence of vaps and bsis in critically ill cancer patients based on a -year prospective cohort in our oncology icu. our secondary objectives were to describe pathogen distribution and assess the risk factors for hais and their potential influence on icu mortality. the gustave roussy cancer centre is a tertiary care hospital treating exclusively patients with solid or hematological malignancies. the average annual admission volume in the -bed medical surgical icu is to patients. a dedicated infection control team is in place since . we collected data from the hospital activity and associated expenditure database (programme de médicalisation des systèmes d'information, pmsi) and from the icu case report forms for stays ≥ h. the pmsi national database contains information on admission categories, patient demographics, disease characteristics, eastern cooperative oncology group performance status (ecog-ps [ ] ), and simplified acute physiology score (saps ii [ ] ). the icu registry is based on a questionnaire, filled out by the same two physicians since . the case report forms include information on the following: invasive devices (mechanical ventilation [mv] and central venous catheters [cvcs]), hais (vap, primary bsis, catheter-related bsis, and secondary bsis), neutropenia (white blood cell [wbc] count < /mm or acute leukemia) before admission (duration and nadir), and outcomes at discharge from icu (infections [date of diagnosis, pathogen] and death. mv by intubation or tracheotomy and cvcs (including totally implanted ports and hemodialysis lines) were studied. as the majority of long-term cvcs were used throughout the icu stay, we did not differentiate between long-term and temporary central lines. all patients were prospectively monitored for infections from admission to h after discharge. all the icu stays between january , , and december , , were considered. during this period, the main change in routine practice was the use of a sedation scale for mechanically ventilated patients since . the data are strictly confidential and available only to authorized clinicians and staff. (medical procedures are described in the supplementary material) diagnostic techniques and infection criteria remained unchanged during the study period. all cases of pneumonia and bsis were audited by two authors (as, fb) in - , using the microbiology data and medical records. all clinically suspected vap were confirmed using a quantitative culture of distal respiratory tract secretions, bblindly,^or via a fiberoptic bronchoscope (or sometimes a semi-quantitative culture of sputum after extubation). for details, see web supplements. bacteremia or fungemia were defined as at least one positive blood culture (except for skin commensals). bsis were classified as primary, secondary, or catheter-related bsi (cr-bsi) (see supplementary material). we retrospectively described the use of invasive devices in terms of the number of devices, median placement time, and inter-quartile ranges (iqrs). device usage rates (ratio of the duration of device use to the duration of the stays) were calculated separately for the first (per patient) icu stays and for the remaining stays. we computed the rates of hais as times the ratio of the total number of infections to the total number of icu days. hai rates were calculated as ( ×) the ratio of the total number of infections (vap and cr-bsis) to the total duration of the device. for patients with several stays exceeding h, only the first stay was included in the prognostic analyses. analogously, only the first hai was considered. to verify whether the infection risk was constant over time, we compared the exponential estimation of the cumulative incidence to the % confidence bands of the kaplan-meier estimate. we identified factors associated with infection using logistic models adjusted for the exposure time. we computed univariate models, then multivariate models via stepwise selection based on likelihood ratio tests (α in = . , α out = . ). the methods used for sensitivity analyses and factors associated with mortality are shown in the supplementary material. all analyses were performed using sas . and r . . the number of icu stays was , of which lasted < h (flowchart: figure s ). the number of first stays ≥ h was to per year until , then it increased. the median length of the icu stay was constantly about - days ( figure s ). table describes patient characteristics. among the stays ≥ h, were first stays (median duration days). most of the patients ( ; %) were admitted for medical reasons; ( %) had solid tumors, and ( %) had metastatic disease. at icu admission, patients ( %) had experienced leukopenia, for more than days in ( %) cases. the icu mortality rate was constantly %. among the first stays (n = ), patients ( %) experienced one or more episodes of mv (median duration days) and patients ( %) underwent at least one cvc placement (median dwell time days; le s ). the total cvc dwell time ( , days) exceeded the total duration of stays ( , days) because most patients had several catheters, including cvcs (preexisting and implanted in the icu). the icu device usage rate was . % for mv and . % for cvcs. figure shows the yearly number of stays ≥ h with devices and median device duration. during the first stays (table ) the most common secondary bsis were of abdominal origin ( . %, / ). thirty-four bsis were due to vap and to urinary tract infections. figure shows the number of stays with at least one hai of each type. among the patients with infections, % ( ) experienced only one type of infection, two types, three, and all four types. the most common pathogens (table s ) were gram-negative aerobes bacilli ( isolates, . %) and gram-positive cocci ( isolates, . %). candida species and other fungi accounted for isolates ( . %). polymicrobial infections were recorded in / ( . %) vaps (first episodes) and / ( . %) bsis: / ( . %) primary bsis, / ( . %) secondary bsis, and / ( . %) cr-bsis. almost . % of bloodstream isolates (n = ) were candida species, % (n = ) of which were candida albicans. figure shows the cumulative incidence rates of vap and primary and secondary bsis and cr-bsis. the results of multivariate prognostic analyses are summarized in table , and details are provided in the supplementary material (tables s -s ) . among the patients with mv, were excluded from the prognostic analyses ( had vap) due to missing values among risk factors. vaps were recorded for ( %) of the fig. ). the duration of mv, older age, scheduled surgery, and solid tumors were vap-specific risk factors (table s ) . among the first stays, were excluded from the prognostic analyses for the risk of bsi ( had primary and secondary bsis) because of missing values among risk factors. primary bsis were recorded for ( %) of the remaining stays. the cumulative risk of primary bsi after a -day icu stay was . % ( % ci . - . %; fig. ). the length of stay, a high saps ii score, scheduled surgery, ecog-ps > , absence of metastases, and recent leukopenia were significant risk factors (table s ) . secondary bsis were recorded for / stays ( %). the cumulative risk of secondary bsis after a -day icu stay was . % ( % ci . - . %; fig. ). the length of stay, surgery, and leukopenia were significant risk factors for secondary bsi (table s ) . among the patients with a cvc, were excluded ( had cr-bsi) because of missing risk factors. cr-bsis were recorded for ( %) of the remaining stays. the cumulative risk of cr-bsis after a -day cvc dwell time was . % ( % ci . - . %; fig. ). no significant risk factors were associated with the risk of cr-bsi, probably due to a lack of power (the low number of cr-bsis; table s ). sensitivity analyses (tables s -s ) confirmed the robustness of these results. the occurrence of a vap episode or bsi was not associated with a higher risk of icu mortality in the univariate or multivariate analyses (table s ) . a significantly higher risk of death was observed for high saps ii and ecog-ps values, medical admission, and the presence of metastases. we presented the results of a retrospective study on prospectively collected data, with drastic quality control measures which eliminated some inconsistencies. the stability of the admission categories, severity scores, and length of stay suggest that the case mix has not changed over time. to our knowledge, this study is the largest prospective series ( ; years) on hais in cancer patients (see supplementary material). the study began after the introduction of alcoholbased hand gels. the main change in routine practice was the use of a sedation scale for mechanically ventilated patients since . specific comments on device usage rates, slightly different from those reported previously, are given in the online supplementary material. briefly, the rate of catheter use is higher (almost all of our patients have a long-term intravascular device) and the mv rate is lower [ ] but has been increasing over time. as we focused on avoidable device-associated infections only, the overall incidence cannot be compared with other non-cancer populations. we observed a higher vap rate ( . / ventilator days) than in other studies ( / at the end of the s to < / during the past decade [ , ] ), but lower than the vap rate in the only study in cancer patients ( / [ ] ). the incidence of vap may have been underestimated given that microbiological samples were postponed when palliative care was decided, and some patients may have died with untreated pneumonia (or, similarly, with undiagnosed bsi…). immunodeficiency due to malignancies and anticancer therapies can explain the high rate of vap in cancer patients. however, neutropenia did not appear to be a risk factor for vap, which is consistent with other studies [ , ] . we observed a higher bsi rate ( . / icu days) than that reported in a mixed population of french icu patients ( . / ) [ ] and by the french national surveillance network [ ] ( . / ). our cr-bsi rate ( . / catheter days) is higher than those reported in the usa [ ] . mixing long-term and short-term cvcs in our study makes interpretation difficult: the reacat study excluded long-term and pre-inserted cvcs. interestingly, when cr-bsi secondary to long-term and short-term cvcs were examined in a post hoc analysis, a very similar infection rate was observed for catheters previously inserted when in the operating room (including totally implanted ports) and those inserted during the icu stay. unlike the steadily declining device-associated infection rates often reported [ , ] , our incidence of cr-bsi increased between and and then decreased to below / cvc days ( figure s , right) . we focused on cr-bsis because they are more sensitive to preventive interventions than central line-associated bsis and more relevant for comparisons between icus [ ] . the distribution of germs is relatively close to that of general icu populations, except for yeasts and anaerobic organisms. candida species represented . % (n = , including cases of candidemia during leukopenia) of the isolated blood culture organisms, a rate that is similar to [ ] or higher than [ ] that of previous reports. indeed, our population exhibited many recognized risk factors for candidemia [ ] . our high rate of anaerobic germs ( %) is mainly due to the numerous heavy abdominal surgery cases admitted in our center. scheduled surgery and a solid tumor were vapspecific risk factors, mainly due to hyperthermic intraoperative peritoneal chemotherapy (hipec) surgeries and esophagectomies, with regular complications requiring mv. surgery (scheduled or not) and leukopenia were risk factors for secondary bsi, which could be due to hipec, often complicated by leukopenia and peritonitis. nevertheless, after excluding scheduled surgery patients (table s ) , unscheduled surgery and leukopenia remained significant risk factors. mucosal barrier injury (bsi), due to neutropenic enterocolitis, is not preventable and is classified as a secondary bsi in our study: in this case, leukopenia is not only a risk factor but also the fig. number of first stays in the intensive care unit (total = ) with at least one of each type of infection. vap ventilator-associated pneumonia, pbsi primary bloodstream infections, sbsi secondary bloodstream infections, cr-bsi catheter-related bloodstream infections cause of the bsi. the fact that icu mortality was not influenced by the occurrence of vaps or bsis means that nosocomial infections mainly reflect the severity of the underlying disease or of the patient's condition. nevertheless, we had no information on the adequacy of initial antimicrobial treatment, a key point in mortality attributable to vap [ ] . finally, the design of our study does not allow a detailed analysis of the real prognostic burden of each hai on mortality; aggregation of data into broad categories, such as vaps and bsis, lessens the actual impact of some types of hai on the prognosis. thus, the occurrence of a deep fungal infection in a neutropenic patient obviously has a greater impact on the prognosis than a catheter-related fungemia, for example. identifying the mortality indeed attributable to each hai (rather to underlying conditions) would require further analysis and studies. the occurrence of device-associated infections is essentially due to severe medical conditions in patients and to the characteristics of the malignancy, but these infections do not influence the outcome of icu cancer patients. given the data were obtained in an oncology icu in a specialized cancer center, extrapolation from these findings should be made very cautiously. however, these data may be useful for comparative studies with other oncology icus and for developing quality improvement activities. they could be also useful for comparison with hai rates in the era of innovative treatments such as immunotherapy. estimated probabilities (pr) with their % confidence interval ( % ci) from univariate and multivariate analyses, adjusted for the icu length of stay. for each category, the probability is computed for a mean profile of the other factors vap ventilator associated pneumonia, pbsi primary bloodstream infection, sbsi secondary bloodstream infection, saps simplified acute physiology score, ecog ps eastern cooperative oncology group performance status and fr had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. author contributions as and fb contributed to conception and design. as, mw, and mm were involved in the data acquisition. fr and jpp planned and performed the statistical analyses. all the authors were involved in the interpretation of the results, read, and approved the final manuscript. conflict of interest the authors declare that they have no conflict of interest. national nosocomial infections surveillance system ( ) nosocomial infection rates in adult and pediatric intensive care units in the united states. national nosocomial infections surveillance system the prevalence of nosocomial infection in intensive care units in europe. results of the european prevalence of infection in intensive care (epic) study. epic international advisory committee intensive care of the cancer patient: recent achievements and remaining challenges nosocomial infections in an oncology intensive care unit nosocomial pneumonia in haematological malignancies in the medical intensive care unit toxicity and response criteria of the eastern cooperative oncology group a new simplified acute physiology score (saps ii) based on a european/north american multicenter study a report from the nnis system effect of an education program aimed at reducing the occurrence of ventilator-associated pneumonia consider saying yes prognosis of neutropenic patients admitted to the intensive care unit outcomes of primary and catheter-related bacteremia. a cohort and case-control study in critically ill patients saint-maurice : institut de veille sanitaire sustaining reductions in catheter related bloodstream infections in michigan intensive care units: observational study national healthcare safety network (nhsn) report, data summary for , deviceassociated module catheter-related vs. catheter-associated blood stream infections in the intensive care unit: incidence, microbiology, and implications bacteremia and severe sepsis in adults: a multicenter prospective survey in icus and wards of hospitals. french bacteremia-sepsis study group epidemiology of candidemia: a oneyear prospective observational study in the west of france attributable mortality of ventilator-associated pneumonia: a metaanalysis publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments we thank monique monhonval for data entry, pascale jan for technical support, and lorna saint ange for editing. as key: cord- -uiabpbxd authors: gebrekidan, hagos; perera, piyumali k.; ghafar, abdul; abbas, tariq; gasser, robin b.; jabbar, abdul title: an appraisal of oriental theileriosis and the theileria orientalis complex, with an emphasis on diagnosis and genetic characterisation date: - - journal: parasitol res doi: . /s - - - sha: doc_id: cord_uid: uiabpbxd oriental theileriosis, a tick-borne disease of bovids caused by members of the theileria orientalis complex, has a worldwide distribution. globally, at least distinct genotypes of t. orientalis complex, including type (chitose), type (ikeda), type (buffeli), types to , and n –n , have been described based on the sequence of the major piroplasm surface protein (mpsp) gene. of these genotypes, mainly ikeda and chitose are known to be pathogenic and cause considerable morbidity (including high fever, anaemia, jaundice and abortion), production losses and/or mortality in cattle. mixed infections with two or more genotypes of t. orientalis is common, but do not always lead to a clinical disease, posing challenges in the diagnosis of asymptomatic or subclinical forms of oriental theileriosis. the diagnosis of oriental theileriosis is usually based on clinical signs, the detection of piroplasms of t. orientalis in blood smears, and/or the use of serological or molecular techniques. this paper reviews current methods used for the diagnosis of t. orientalis infections and the genetic characterisation of members of the t. orientalis complex, and proposes that advanced genomic tools should be established for investigations of these and related haemoparasites. ticks and tick-borne diseases (tbds) affect the productivity of bovines in tropical and subtropical regions of the world, leading to a significant socioeconomic impact on the livelihood of resource-poor farming communities (makala et al. ) . globally, four main tbds, namely anaplasmosis (primarily caused by anaplasma marginale and a. centrale), babesiosis (babesia bovis, b. bigemina and b. divergens), theileriosis (theileria annulata, t. parva, t. orientalis complex and others) and heartwater/cowdriosis (ehrlichia ruminantium) affect bovines, and cause substantial economic losses to cattle industry, particularly in the tropics and subtropics (makala et al. ; jabbar et al. ) . the annual global costs of ticks and tbds to cattle industry are estimated at us$ to billion worldwide (de castro ) . this impact is higher in developing countries where the costs of tick control and treatment strategies are almost unaffordable for lowincome smallholder farmers (minjauw and mcleod ; rajput et al. ) , particularly in many countries of sub-saharan africa, asia and latin america (delgado et al. ) . bovine theilerioses are caused by intracellular protistan parasites of the genus theileria (apicomplexa: piroplasmida; theileriidae) and are considered as one of the most economically important diseases of bovines globally (uilenberg ) . the geographical distribution of theileria spp. is usually restricted to tropical and subtropical regions where suitable tick vectors occur. theileria spp. infect primarily domestic and wild ruminants, and cause economically significant diseases in cattle, sheep and goats. for instance, t. annulata and t. parva (the causative agents of tropical or mediterranean and east coast hagos gebrekidan and piyumali k. perera contributed equally to this work. section editor: panagiotis karanis fever, respectively) are known to be the most pathogenic species in bovines, whereas other species, such as t. mutans, t. taurotragi and members of the t. orientalis complex, often cause asymptomatic infections (cf. jabbar et al. ) . depending on species of theileria, a number of hard ticks of the genera amblyomma, haemaphysalis, hyalomma and rhipicephalus can transmit theilerioses. traditionally, theileriosis caused by t. orientalis complex was considered to be benign and asymptomatic, but clinical cases had been reported from australia (rogers and callow ) , japan (shimizu et al. ) and new zealand (james et al. ) . however, since , a number of outbreaks of oriental theileriosis have occurred in australia and new zealand, leading to significant economic losses in dairy (perera et al. ) and beef (lane et al. ) cattle. currently, genotypes of t. orientalis are recognised, and only ikeda and chitose are known to be pathogenic, causing considerable morbidity and mortality in cattle. the diagnosis of oriental theileriosis can be made using approaches including clinical signs, the detection of piroplasms of t. orientalis in blood smears and/or the use of serological or molecular techniques. this article provides an account of oriental theileriosis and then reviews the current methods employed for the diagnosis of oriental theileriosis and the genetic characterisation of t. orientalis. it also highlights the need for advanced genomic tools for diagnostic and analytical applications. the theileria orientalis complex the taxonomy and nomenclature of t. orientalis is still unresolved as different names are being used to describe similar parasites from different geographical locations, including t. sergenti from japan, t. buffeli from australia and t. orientalis from europe and elsewhere (watts et al. ) . recently, based on serological and morphological characteristics, uilenberg ( ) proposed that t. orientalis represented a single species but suggested that it be called a complex. different molecular markers, including the small subunit (ssu) of nuclear ribosomal rna ( s rrna), the first and second internal transcribed spacers (its- and its- = its) of nuclear ribosomal dna, the cytochrome c oxidase iii, -kda piroplasm membrane protein (p ) and major piroplasm surface protein (mpsp) genes have been used to study and characterise members of the t. orientalis complex (ota et al. ; altangerel et al. ; kamau et al. a, b; yokoyama et al. yokoyama et al. , perera et al. a, b, c) . of these markers, the mpsp gene is the most commonly used marker; based on this gene's sequence, at least distinct genotypes of t. orientalis (types - and n -n ) from cattle, water buffaloes, sheep and ticks have been reported to date, worldwide (sivakumar et al. ). oriental theileriosis caused by one or more genotypes of t. orientalis has become an important tbd of bovines, particularly in the asia-pacific region (sivakumar et al. ) . recently, the first case of clinical oriental theileriosis was reported in cattle from virginia, usa (oakes et al. ) . theileria orientalis appears to be widely distributed globally, but in most countries, the genotypic identity of t. orientalis complex and the clinical relevance of the distinct genotypes is unclear. to date, t. orientalis has been recorded in bovines, sheep, ticks and other blood-feeding insects in more than countries (sivakumar et al. ). however, most reports originate from japan, followed by australia, china, korea and new zealand. the life cycles of theileria spp. are indirect, involving ticks as vectors. significant variation occurs in the life cycles of theileria spp.; some species induce cell transformation and proliferation (e.g., t. parva and t. annulata), whereas others do not (e.g., t. orientalis complex). generally, theileria parasites have three distinct stages: (i) schizogony (asexual reproduction), which takes place in vertebrate hosts; (ii) gametogony (sexual reproduction)-the development and union of gametes inside the intestinal cells of tick vectors; and (iii) sporogony (asexual reproduction), which takes place in the salivary glands of tick vectors (mehlhorn and schein ; nene et al. ) . sporozoites (the infective stage of the parasite) are inoculated with the saliva of a tick when it takes a blood meal from a vertebrate host. these sporozoites invade lymphoid cells and develop into a multinucleate schizont (e.g., in t. annulata)~ h after inoculation. following merogony/ schizogony, lymphocytes rupture, releasing merozoites which then enter erythrocytes. when a tick feeds on blood from the infected host, infected erythrocytes are ingested. following ingestion, gametes are released into the gut lumen of the tick, where fertilisation occurs. fertilisation gives rise to a spherical zygote that invades the gut epithelium of the tick. this zygote develops into motile ookinetes, which then invade the salivary gland of the tick, develop into sporoblasts and multiply asexually, producing thousands of sporozoites. sporozoites are the infective stage and are injected when infected ticks feed on blood from another vertebrate host, thereby continuing the life cycle (norval et al. ; shaw ). an ixodid tick, haemaphysalis longicornis is the main vector of t. orientalis (see watts et al. ). in addition, h. bancrofti and h. humerosa are also believed to be potential vectors in australia (stewart et al. a, b; watts et al. ) . additionally, other blood-sucking insects, such as march flies (tabanids), mosquitoes or sucking lice are also suggested as potential vectors of t. orientalis (see hammer et al. hammer et al. , watts et al. ) . furthermore, t. orientalis dna has been detected in other tick species, including amblyomma variegatum, a. cohaerens, rhipicephalus decoloratus, r. evertsi and r. praetextatus in ethiopia (hornok et al. ; kumsa et al. ) , as well as from r. annulatus ticks in italy (toma et al. ) . however, the competence of these vectors has not been established, as these ticks were collected from cattle, not from pastures, and no experimental infection with t. orientalis from these ticks has been demonstrated to date. for many years, t. orientalis was recognised as a benign parasite of bovines (watts et al. ) , as its schizonts do not induce cell transformation or lymphoproliferation, unlike t. annulata and t. parva. although the pathogenesis of t. orientalis is not yet completely understood (watts et al. ), a rapid rate of division of both macroschizonts and microschizonts has been observed in calves infected with t. orientalis (uilenberg et al. ) , which highlights the potential of members of t. orientalis to be categorised as celltransforming pathogens, but this hypothesis requires further testing. irrespective of the non-lymphoproliferative nature of t. orientalis complex, in the last decade, numerous outbreaks of oriental theileriosis have been recorded in both dairy and beef cattle in the asia-pacific region, mainly in australia and new zealand (izzo et al. ; aparna et al. ; islam et al. ; kamau et al. a, b; mcfadden et al. ; perera et al. perera et al. , perera et al. , c and, recently, in the usa (oakes et al. ) . based on the characterisation of t. orientalis genotypes from cattle affected by clinical oriental theileriosis, only two of the currently known genotypes, namely chitose and ikeda, are known to be pathogenic, and these two genotypes have been found to be associated with hundreds of recent oriental theileriosis outbreaks in australasia and the usa (izzo et al. ; islam et al. ; mcfadden et al. ; perera et al. perera et al. , c . most oriental theileriosis outbreaks occurred when cows had been stressed and immunosuppressed due to calving or late pregnancy and/or other associated farm management, nutritional and environmental challenges (reviewed by watts et al. ) . the main economic losses due to oriental theileriosis relate to abortions, significant reduction in milk production (quantity and quality) in dairy cattle, and severe morbidity and mortality rates in affected cattle herds (perera et al. ; lane et al. ) . various methods have been used for the detection, identification, quantitation and characterisation of t. orientalis complex and closely related haemoparasites that infect bovines. the diagnostic methods used for t. orientalis can be broadly categorised into traditional and molecular methods. the traditional diagnostic methods used for t. orientalis are mainly based on the clinical signs observed in infected cattle, as well as findings of postmortem, microscopic and serological evaluations. although traditional diagnostic methods might lack sensitivity and specificity, they are still widely used and useful, to support clinical and epidemiological investigations. clinical diagnosis clinical and postmortem examinations assist the diagnosis of oriental theileriosis. table provides details of the major clinical signs of this disease. the main clinical signs include anorexia, lethargy, pyrexia, haemolytic anaemia, jaundice, pale mucous membranes, haemoglobinuria, increased heart and respiratory rates, nasal discharge, swollen lymph nodes, depression, abortions, reduced production, still birth and death in severe cases (islam et al. ; izzo et al. ; aparna et al. ; mcfadden et al. ; eamens et al. a, b; perera et al. ) . as a diagnostic method, this approach can provide some information about the disease occurrence in affected herds. similarly, postmortem examination of suspected cases can also assist in diagnosis. gross pathological changes observed in oriental theileriosis include jaundice, pale, rounded and enlarged liver, kidney and spleen, haemorrhagic duodenitis, ulcers in the abomasal mucosa, pulmonary oedema and enteritis (izzo et al. ; aparna et al. ; mcfadden et al. ; gebrekidan et al. gebrekidan et al. , a . however, both clinical signs and postmortem findings are often subjective, and are not specific to oriental theileriosis, as diseases caused by other blood parasites can cause similar clinical signs or lesions in bovines (gharbi et al. ; magona et al. ). microscopic detection of t. orientalis the microscopic examination involves the identification of the parasite (piroplasm) in the red blood cells on giemsa-stained blood smears ( fig. ) (aktas et al. a; khattak et al. ; perera et al. ; chauhan et al. ) . microscopy can also be used to estimate the parasite burden (parasitaemia), particularly in clinical cases when a large number of erythrocytes are infected with t. orientalis (see rogers and callow ; izzo et al. ). however, blood smears are not useful when the parasitaemia is low. furthermore, it does not allow the discrimination of t. orientalis genotypes or other theileria spp. infecting bovines based on their morphological characteristics. many theileria spp. infecting bovines are very similar in their morphology at the piroplasm and schizont stages, except for t. velifera and t. taurotragi which have a unique veil and/or bar-like structures in the infected red blood cells. the main limitations of microscopic examination for t. orientalis include lower specificity, sensitivity, inability to identify t. orientalis to the genotypic level, and the time required to screen blood smears (aktas et al. b; izzo et al. ; aparna et al. ; nayel et al. ; perera et al. ; charaya et al. ). the immunofluorescence antibody test (ifat), enzyme-linked immunosorbent assay (elisa) and latex agglutination test have been employed for the detection of anti-t. orientalis serum antibodies in bovines (table ). all these serological tests are targeted at the surface proteins of piroplasms of the parasite(s). of all available serological methods, elisa is a relatively sensitive technique to study the prevalence of t. orientalis infections in cattle populations (not on an individual animal basis). serological methods are reported to be more sensitive compared to other traditional diagnostic methods (clinical signs, postmortem and microscopic examinations) used for t. orientalis. however, these techniques also have limitations. for example, they are unable to discriminate among different genotypes of t. orientalis or differentiate between current and past t. orientalis infections, and cross-reactivity with other theileria spp. is common in serological techniques (mans et al. ) . hence, none of the traditional methods used for t. orientalis detection allows the differentiation of known genotypes, or yields genetic information about the parasite(s) present. thus, molecular methods are crucial to identify and distinguish pathogenic and nonpathogenic genotypes of t. orientalis. molecular diagnostic methods can overcome many of the limitations of traditional methods (conraths and schares ) . to date, a number of molecular assays have been utilised to detect, characterise, differentiate and quantitate members of the t. orientalis complex, including conventional polymerase chain reaction (pcr), nested-pcr, reverse line blot hybridisation assay (rlb), loop-mediated isothermal amplification (lamp), real-time/quantitative pcr (qpcr) using hydrolysis probes and multiplexed tandem pcr (mt-pcr) assays (table ) . cpcr this technique is widely used to detect and characterise t. orientalis. as compared to traditional diagnostic methods used for t. orientalis, conventional polymerase chain reaction (cpcr) has been shown to achieve higher diagnostic sensitivity (dse) and diagnostic specificity (dsp) using different genetic markers (perera et al. ; gebrekidan et al. gebrekidan et al. , a mans et al. ) . for this assay, markers including the ssu, mpsp, p , -kda piroplasm membrane protein (p ), -kda piroplasm membrane protein (p ), -kda piroplasm membrane protein (p ), cox-iii, β-tubulin genes, or the its have been used to detect, differentiate and characterise t. orientalis from different countries (reviewed by mans et al. ) . of these markers, the mpsp gene is the most commonly applied to the t. orientalis complex. the cpcr using mpsp offers the advantage of amplifying relatively long dna regions from t. orientalis, particularly to characterise novel species/ genotypes for the first time compared with established qpcr assays, which usually only amplify short dna fragments ( - base pairs). limitations of cpcr include its (i) inability to differentiate genotypes in the case of mixed infections with t. orientalis and, (ii) potentially, pcr inhibition due to the presence of different blood constitutes such as haemoglobin and lactoferrin, and other chemicals and reagents used for dna extraction (perera et al. a) . furthermore, compared with established qpcr assays used for the detection of t. orientalis (perera et al. a) , cpcr is less sensitive. in addition, in the cpcr assay, post-pcr steps like gel preparation, gel electrophoresis and purification of pcr products for sequencing are costly, laborious and time-consuming. based on the molecular size of the pcr products on regular agarose gels, it is difficult to discriminate among genotypes of t. orientalis complex. to circumvent this issue, mutation scanning technique, such as singlestrand conformation polymorphism (sscp), has been used to discriminate complex and closely related sequences (from a distinct parasite) even that differ only by a single nucleotide over < bp (cf. gasser et al. ). sscp has also been used to identify sequence variation within and among genotypes of t. orientalis (see cufos et al. ; perera et al. ). the sensitivity of detection of nucleotide differences depends on the size of pcr products and sscp conditions (gasser et al. ) . npcr members of the theileria orientalis complex have been detected in cattle blood samples in brazil, iran, south africa, uganda and the usa using semi-nested or nested pcr (npcr) assay employing the ssu or its loci (chae et al. ; oura et al. ; ghaemi et al. ; chaisi et al. ; silveira et al. ). the principle of npcr is similar to that of cpcr, except npcr requires two rounds of amplification of the marker(s) using outer and inner primers. the inner primers bind to sequences in the target dna that are within the dna fragment amplified by the outer primers. the advantage of this technique is that, if the outer primers bind and amplify non-target regions of the dna sequence, it is rare that the inner primers will bind within the incorrect regions of the dna (odongo et al. ). uilenberg et al. ( ) , papadopoulos et al. ( ) enzyme-linked immunosorbent assay (elisa) using piroplasm antigens shimizu et al. ( ) latex agglutination test using p antigens jeong et al. ( ) indirect elisa using p antigens wang et al. ( a) molecular reverse line blot hybridisation (rlb) using s rrna gene gubbels et al. ( ) pan-fret real-time pcr using cox gene chaisi et al. ( ) conventional pcr using markers mpsp, p , p , p and/or p , s rrna (ssu) gene tanaka et al. ( ) , kawazu et al. ( ) , kubota et al. ( ), govaerts et al. ( , sarataphan et al. ( sarataphan et al. ( , , liu et al. ( liu et al. ( , , ota et al. ( ), islam et al. ( semi-nested pcr for s rrna ghaemi et al. ( ) nested pcr for its- - . s-its- region aktas et al. ( ) loop-mediated isothermal amplification (lamp) for p and its - . s-its region wang et al. ( b) , liu et al. ( ) quantitative pcr using its- and its- regions kamau et al. ( b) multiplexed tandem pcr using mpsp, its- and p perera et al. ( a) hydrolysis probe-based quantitative pcr using mpsp bogema et al. ( ) multiplexed tandem pcr using mpsp gebrekidan et al. ( ) a limitation of npcr can be the higher probability of contamination as the amplicons of the first round of pcr are diluted for the second pcr, and the chance to contaminate the reaction is high which may lead to false-positive results (cox-singh et al. ) . furthermore, npcr requires more time (due to the two rounds of amplification) than cpcr (janardhanan et al. ) . the estimation of dse and dsp of npcr assay is difficult. however, some studies have compared the estimated dse and dsp of npcr and with those of cpcr assays, and found that the dse of the npcr was higher than that of cpcr. however, due to the higher probability of false-positive results, dsp of npcr can be lower than that of cpcr (kim et al. (kim et al. , rigotto et al. ) . rlb hybridisation rlb hybridisation allows the detection of multiple molecular targets in a sample using one multiplex pcr reaction, followed by probe hybridisation on a nylon membrane. this technique has been used to screen and differentiate several pathogens of veterinary and medical importance (e.g. rijpkema et al. ; kamerbeek et al. ; gubbels et al. ) . the development and validation of a novel rlb hybridisation overcomes most of the limitations of cpcr, which lacks the simultaneous detection of pathogens in the same sample, and it is much more sensitive and cheaper than cpcr. in this assay, multiple pathogens can be detected using multiple probes for simultaneous detection. rlb hybridisation was originally developed for the identification of streptococcus serotypes (kaufhold et al. ). subsequently, this assay was also used for the detection and differentiation of pathogens in ticks (e.g. borrelia spirochetes) (rijpkema et al. ) , and then rapidly became a standard molecular diagnostic tool for the detection and differentiation of known theileria and babesia spp. infecting bovines. for instance, it has been used for the characterisation of babesia divergens in a human case, and for the discovery of novel theileria and babesia spp. (gubbels et al. ; centeno-lima et al. ; nijhof et al. ; schnittger et al. ; oura et al. ; brigido et al. ; tomassone et al. ) . gubbels et al. ( ) validated an rlb hybridisation assay for the simultaneous detection and differentiation of theileria and babesia spp. infecting bovines, and it was able to differentiate six and three theileria and babesia spp., respectively. the main limitations of rlb assay include a time-consuming protocol with many steps and its low specificity (mans et al. ) . lamp lamp is a simple, sensitive, rapid and cost-effective technique that amplifies different templates, including genomic dna, heat-treated blood and blood dried on filter papers under isothermal conditions (notomi et al. ; kuboki et al. ; poon et al. ; thekisoe et al. ). this technique is a useful diagnostic method to be employed in laboratories with limited resources. it does not require expensive laboratory equipment, such as pcr thermocyclers and fridges/ refrigerators, and the reagents are stable at room temperature ( to °c), which makes this method appropriate for use in field conditions (thekisoe et al. ). many studies have reported the utility of the lamp technique for the rapid detection of both theileria and babesia spp. infecting bovines (iseki et al. ; salih et al. ; he et al. ; thekisoe et al. ; liu et al. ). however, only two studies have reported the use of lamp for the diagnosis of t. orientalis infection in china (wang et al. ; liu et al. ). one of the main limitations of lamp for t. orientalis is the challenge of designing reliable primers for closely related genotypes (torres et al. ) . qpcr this technique was first described in the mid- s, and its popularity and usage grew in the field of molecular microbiology, as it allowed sensitive, accurate and reproducible detection of pathogens in a variety of samples (bretagne et al. ; nicolas et al. ; bossolasco et al. ; mary et al. ; vitale et al. ) . it is a reliable assay to measure gene expression, to quantitate parasite intensity and to undertake single nucleotide polymorphism (snp) analysis. as compared with other commonly used molecular diagnostic tools, such as cpcr, npcr, rlb hybridisation and lamp assays, qpcr is more sensitive and less time-consuming, and, once critically standardised and validated, does not require post-pcr analyses, such as gel electrophoresis and dna sequencing (francino et al. ; abda et al. ) . another advantage of this assay is that highresolution melt (hrm) curve analysis can be used to infer variation in pcr products. high-resolution melt curve analysis is a rapid, practical and inexpensive technique to discriminate parasites to the species-level based on the melting characteristics of amplicons (morick et al. ; mehta et al. ) . many studies have evaluated qpcr assay for the detection, identification and quantitation of theileria spp. infecting bovines globally (sibeko et al. ; papli et al. ; ros-garcía et al. ; chaisi et al. ) . recently, taqman qpcr assays were developed for t. orientalis genotypes, targeting the mpsp gene for use in australia (bogema et al. ) and in new zealand (pulford et al. ) . these two assays performed similarly, in terms of specificity and sensitivity, but these assays are not able to detect all currently known genotypes of t. orientalis or novel genotypes in australia and new zealand. moreover, initial costs linked to the setting up and standardisation of qpcr can be relatively high (e.g., mackay et al. ) . due to the high analytical sensitivity of qpcr assays, sound experimental design, the inclusion of suitable internal controls and data normalisation are crucial to achieving reliable results (wong and medrano ) . mt-pcr various duplex and/or multiplex qpcr assays have been developed for the simultaneous detection of different pathogen species, including theileria spp. of bovines (criado-fornelio et al. ; peleg et al. ; bilgiç et al. ; junlong et al. ). an mt-pcr assay was developed for the simultaneous detection of the four common genotypes (buffeli, chitose, ikeda and type ) of the t. orientalis complex known to occur in australasia (perera et al. a ). this assay used multiple markers, including the mpsp gene for genotypes chitose and type , the p gene for buffeli and its- for ikeda (perera et al. a ). however, subsequently, gebrekidan et al. ( d detected cross-amplification of p gene and its- from both genotypes buffeli and ikeda. consequently, the mt-pcr assay was modified to detect the four common genotypes (buffeli, chitose, ikeda and type ) of t. orientalis by using only one marker (i.e. mpsp) in the assay (gebrekidan et al. ) . this technique requires the easy-plex platform (ausdiagnostics pty. ltd., australia), which includes a well-variant of the easy-plex processor, a well-easy-plex analyser, personal computer with easy-plex software, vortexer, -well plates, dilution plate, plate sealing films and a liquid handling robot. the assay involves primary amplification using primers designed to each of the genotype of t. orientalis targeted, and secondary amplification for quantification using nested primers to internal regions of specific markers used. samples tested in this assay are recorded as testpositive or test-negative using the auto-call function in the easy-plex software. similarly, the intensity of infection (dna-copy numbers) for each genotype in each sample is determined by comparison with the cycle threshold data predetermined for an internal spike-control containing a known number of dna copies ( , ) provided by the ausdiagnostics pty. ltd., australia (cf. perera et al. a) . although this assay is a time-and cost-effective and sensitive method to simultaneously detect multiple genotypes of t. orientalis, it requires specific reagents and equipment, and, thus, can be relatively costly to set up initially. although the currently available advanced molecular diagnostic tools used for t. orientalis allow for a rapid and accurate diagnosis (mainly for the two pathogenic genotypes chitose and ikeda), some assays can be expensive for routine use due to individual testing of blood samples, particularly when outbreaks of oriental theileriosis occur in cattle herds. however, herd-level prevalence and intensity of infection with t. orientalis can be readily determined at a considerably low cost by testing pooled blood samples from herds. several studies have evaluated and reported the usefulness and costeffectiveness of pooled sampling technique for distinct applications. for example, pooled testing has been utilised for the screening of blood samples for malaria from blood donors, the detection of bovine leukaemia and viral diarrhoea pathogens in cattle and salmonella infections in poultry (muñoz-zanzi et al. ; singer et al. ; bharti et al. ; hsiang et al. ) . recently, the performance and cost-effectiveness of mt-pcr were assessed for the diagnosis of the two pathogenic genotypes (ikeda and chitose) of t. orientalis using pooled blood samples from cattle (gebrekidan et al. a, b, c, d) . although shown to be useful in this proof of concept study, further experimental optimisation is required before this assay can be routinely used in the monitoring of t. orientalis infection(s) in bovine herds. recent outbreaks of oriental theileriosis in cattle in the asia-pacific region and the usa have challenged the dogma that oriental theileriosis is a benign infection. evidence shows that the clinical form of oriental theileriosis can lead to significant economic losses in dairy and beef cattle. furthermore, the two genotypes (ikeda and chitose) of t. orientalis are mainly linked to the clinical form of oriental theileriosis. the taxonomy and nomenclature of members of the t. orientalis complex are still unresolved, and a number of molecular markers have been utilised to characterise t. orientalis from cattle. various aspects of oriental theileriosis such as transmission, pathogenesis, epidemiology and control remain to be fully explored. a critical appraisal of the literature shows that the currently available traditional diagnostic methods used for the detection of t. orientalis can have limitations, particularly regarding diagnostic and analytical sensitivity and specificity. however, most of the limitations associated with the traditional diagnostic methods used for detection can be overcome by using molecular diagnostic tools. of the currently available molecular diagnostic tools, cpcr employing the mpsp gene is the most commonly used technique for the characterisation of t. orientalis genotypes globally, although other molecular markers, such as s rrna, its and p gene, have also been employed. a few quantitative pcr assays employing different markers have also been developed and validated in australia and new zealand to detect, differentiate and quantitate pathogenic genotypes (ikeda and chitose) of t. orientalis (e.g. bogema et al. ; perera et al. a; gebrekidan et al. ) . currently available, advanced molecular tools for the diagnosis and characterisation of t. orientalis are unable to simultaneously reliably detect/distinguish all of the currently known and/or novel genotypes of t. orientalis. therefore, validation of other molecular tools that can simultaneously detect all currently known and/or novel genotypes of t. orientalis is required. it is expected that next-generation sequencing (ngs) of pcr amplicons using the illumina platform will be a suitable method that could allow both infection intensity and genetic diversity within t. orientalis populations to be measured directly. recently, chaudhry et al. 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orientalis in tick vectors detected in hokkaido and okinawa publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations cufos n, jabbar a, de carvalho lm, gasser rb ( ) key: cord- -gpl zbvd authors: barry, maura; chandra, sunandana; hymes, kenneth b. title: cytopenias in transplant patients date: - - journal: principles and practice of transplant infectious diseases doi: . / - - - - _ sha: doc_id: cord_uid: gpl zbvd anemia, leukopenia, thrombocytopenia, as well as pancytopenias can be seen following solid organ transplant. varying patterns of cytopenia can be seen based on the drugs used in the posttransplant period, infections encountered by the individual, as well as the individual’s immune response and bone marrow function. the chapter discusses the main causes of anemia, leukopenia/neutropenia, and thrombocytopenia. the differential diagnosis for anemia after solid organ transplant includes hemolysis, drug toxicities, iron deficiency, infection, posttransplant lymphoproliferative disorder, graft-vs.-host disease, and hemophagocytic syndrome. etiologies for leukopenia and neutropenia include drug toxicities and infection, and etiologies for thrombocytopenia include drug toxicities, infections, autoimmune events such as immune thrombocytopenic purpura, and underlying causes such as persistent portal hypertension and splenomegaly. mismatched abo solid organ transplantation is often employed due to the shortage of transplantable organs. three different groups of abo incompatibility can be found in transplantation: minor, major, and bidirectional. complications arising from minor abo-mismatched solid organ transplants include the passenger lymphocyte syndrome (pls) [ ] . recipients of minor abo-incompatible transplantation express abo antigens that are not expressed in the donor and may result in a graft-versus-host (gvh) reaction, including delayed hemolysis of recipient red blood cells [ ] . passenger lymphocyte syndrome occurs when antibodies that are produced by the donor b-lymphocytes result in a primary or secondary immune response against the recipient's abo and rh antigens. the severity of hemolysis depends on the level of red cell isoagglutinins in the donor tissue that are passively transferred with the organ and the subsequent rise in antibodies in the transplant recipient that occurs - weeks posttransplant and usually resolves within months posttransplant [ ] . in rare instances, pls can occur due to non-abo/rh antibodies if the organ had been previously sensitized to other red cell antigens in the setting of pregnancy or transfusion [ ] [ ] [ ] [ ] . pls occurs more frequently in the heart and lung transplants and less frequently in liver and kidney transplants [ ] . there are numerous drugs that are often used in the solid organ transplant setting that can cause myelosuppression, including anemia, through a variety of pathophysiologic mechanisms. a number of immunosuppressants with various pharmacologic mechanisms of action are used to prolong graft and recipient survival. the immunosuppressants mycophenolate mofetil and tacrolimus have been shown to cause anemia in renal transplant recipients [ ] . one-year posttransplant, renal transplant patients with anemia who are on mycophenolate mofetil have a lower rates of survival and higher rates of cardiovascular death [ ] . sirolimus, another immunosuppressant, may result in greater myelosuppression compared to mycophenolate mofetil [ ] . sirolimus and calcineurin inhibitors such as tacrolimus and cyclosporine have been shown in renal and lung transplant recipients to cause hemolytic anemia, thrombotic thrombocytopenic purpura, and atypical hemolytic uremic syndrome [ ] [ ] [ ] . the calcineurin inhibitors have been shown to cause anemia ranging from % to % in european trials to - % in us trial [ ] . the antimetabolite azathioprine, a purine-analog drug, can also cause cytopenias. mycophenolate mofetil, tacrolimus, azathioprine, and anti-thymocyte globulin have all shown to cause pure red cell aplasia [ , ] . primaquine and dapsone are used for pcp treatment, and both can result in hemolysis in glucose- -phosphatedehydrogenase-deficient patients, which is not restricted to solid organ transplant settings. in patients with low body weight or renal failure, dapsone may induce a hemolytic anemia and produce methemoglobinemia even if the g pd levels are normal [ ] [ ] [ ] . ribavirin and interferon can cause bone marrow suppression in liver transplant recipients who are being treated with recurrent hepatitis c virus [ ] . ribavirin is used in treating respiratory syncytial virus after transplant in both the oral and inhaled formulations, both of which can cause bone marrow suppression [ ] . in patients who are co-infected with human deficiency virus (hiv) and hepatitis c virus, myelosuppression can be seen with the anti-retroviral medication, azt, and the anemia can be exacerbated with the coadministration of ribavirin. the antibiotic trimethoprimsulfamethoxazole can also cause myelosuppression including anemia. valganciclovir has been reported to cause bone marrow failure in renal transplant patients who received this antiviral as prophylaxis [ ] . newer immunosuppressants have been developed which allow for the sparing of steroids and calcineurin inhibitors, the latter of which can cause chronic nephropathy. these newer agents include alemtuzumab, a human anti-cd antibody that depletes t-and b-cells, daclizumab, a human anti-cd antibody that targets the il- alpha subunit, and anti-thymocyte globulin (atg). alemtuzumab has been reported to be associated with red cell aplasia, autoimmune hemolytic anemia, and idiopathic thrombocytopenia purpura in pancreas transplant patients [ ] . iron deficiency is often overlooked in transplant patients. in renal transplant patients, those with a hematocrit of less than % have iron studies checked only % of the time [ ] . perioperative bleeding and frequent phlebotomies for labo-ratory studies can contribute to iron deficiency anemia. anemia of chronic disease is also frequently seen in the transplant population due to chronic inflammation, abnormal erythropoietin production due to allograft nephropathy after renal transplant. drugs such as ace inhibitors that are often used in chronic kidney disease are also associated postkidney transplant anemia [ ] . numerous infectious etiologies that can occur during the posttransplant immunosuppressed period have been shown to cause myelosuppression including anemia. parvovirus b , a single-stranded dna virus, has been known to cause red cell aplasia with anemia, reticulocytopenia, and erythroid maturation arrest [ ] . elevated parvoviral b titers have been found by pcr in lung transplant recipients who had anemia with other etiologies that were ruled out [ ] [ ] [ ] . cytomegalovirus (cmv) infection as well as its first-line therapies, ganciclovir and valganciclovir, can be associated with bone marrow suppression. also tuberculosis, histoplasmosis, epstein-barr virus (ebv), human herpes virus- , and human herpes virus- infections can be associated with bone marrow suppression and pancytopenia [ ] . posttransplant lymphoproliferative disorder that can be seen with immunosuppressive therapy can also be associated with pancytopenia. posttransplant lymphoproliferative disorder (ptld) which includes the spectrum of infectious mononucleosis, ebvdriven polyclonal lymphocyte proliferation, and non-hodgkin's lymphoma can be seen with solid organ transplantation [ ] . ptld is due to the impaired ebvspecific cytotoxic t-cell activity that allows for recipient b cells that have latent ebv infection to expand. ptld can result in bone marrow infiltration and pancytopenia, as well as cause autoimmune hemolytic anemia. the severity of ptld depends on the level of immunosuppression and usually occurs within the st year after transplant. graft-versus-host disease (gvhd) is rarely seen after sot and is due to the engraftment and proliferation of allograftassociated lymphocytes in the immunosuppressed transplant recipient causing an immune-mediated response toward hla-unmatched host tissue. risk factors for the development of gvhd includes the volume of lymphoid tissue that is transplanted and therefore is seen more with small bowel and liver transplants, in those over years of age and with hla mismatch between donor and recipient [ , ] . gvhd, in contrast to the development of ptld, occurs early after sot, on the order of weeks to months depending on the type of solid organ transplant. the clinical presentation usually includes fever, rash, diarrhea, and cytopenias, and diagnosis is made by histologic confirmation of affected tissue. hemophagocytic syndrome is a systemic inflammatory disease that can include the symptoms of fever, hepatosplenomegaly, lymphadenopathy, pancytopenia, rash, jaundice, cough, dyspnea, cachexia, and neurologic dysfunction and can often occur in response to a precipitant, such as infection [ ] . this syndrome is a result of aberrant immune response of abnormal t-cell activation leading to hemophagocytosis by activated, nonmalignant macrophages that secrete numerous cytokines including interleukin (il)- , il- , il- , and tumor necrosis factor-alpha in the bone marrow, liver, lymph nodes, and spleen, resulting in a "cytokine storm" [ ] . acquired hemophagocytic syndrome has been documented in the renal, liver, heart, and pancreaskidney solid organ transplants. there have been cases of hemophagocytic syndrome due to disseminated histoplasmosis in renal transplant recipients which were diagnosed by bone marrow biopsy [ ] . leukopenia can be defined as having a white blood cell (wbc) count of less than - cells/μl, with neutropenia defined as an absolute neutrophil count (anc) < / mm by the infectious diseases society of america [ ] . leukopenia is commonly seen after solid organ transplantation and can be caused by noninfectious and infectious etiologies. it can signal an underlying infection or disease process, such as posttransplant lymphoproliferative disorder (ptld). it also increases the risk of developing further complications such as opportunistic infection and can require reduction of immunosuppression, increasing the risk of graft rejection. while there is no data to suggest a clear independent relationship between leukopenia and graft rejection, the complications of leukopenia mentioned above provide ample reason to investigate the etiology of the decreased white cell count. solid organ transplant recipients are at risk for developing infections due to their medically induced immunodeficiency following transplant, required to prevent rejection of the transplanted organ. noninfectious causes of leukopenia include drugs that are often used in transplant settings. numerous immunosuppressants can cause leukopenia, but given their use in combination, it is difficult to elucidate each agent's individual role in incidence and management. in one retrospective study of adult kidney and pancreas transplantations, the incidence of either leukopenia or neutropenia was %, with the first episode occurring at a mean of days posttransplant [ ] . one of the most common immunosuppressants, azathioprine, is a purine analog that causes an antimetabolite effect. azathioprine may result in leukopenia in a dose-dependent manner, as well as based on the duration of treatment. the leukopenia that results from azathioprine is usually reversible upon dose-reduction or drug discontinuation. the leukopenia, often occurring late in the course of therapy, can be related to low or absent levels of s-methyl-transferase (tpmt) activity, which metabolizes -mercaptopurine, and can result in increased myelotoxicity [ ] . drugs that result in the depletion of t cells, such as thymoglobulin and alemtuzumab, can also lead to leukopenia in - % of patients [ ] . the immunosuppressant, mycophenolate mofetil (mmf), reversibly and noncompetitively inhibits the enzyme, inosine monophosphate dehydrogenase, the rate-limiting enzyme for de novo purine synthesis during lymphocyte proliferation [ ] . mmf can result in leukopenia in - % of patients. the myelosuppression of mmf is dose-dependent and is related to the trough level of the active metabolite, mycophenolic acid; however, brief discontinuations of the drug can lead to organ rejection, especially in the era of steroid-sparing regimens [ , ] . the calcineurin inhibitors such as cyclosporine, tacrolimus, and sirolimus can also lead to cytopenias, including leukopenia. some of these agents can cause leukopenia as one of many symptoms of infection. for example, leukopenia (and often thrombocytopenia as well) have been observed as a sign/ symptoms of infection with pathogens such as adenovirus, coronavirus, lymphocytic choriomeningitis virus (lcmv), parainfluenza, ehrlichiosis, and measles [ ] [ ] [ ] . in areas endemic for the disease, dengue infection also causes both leukopenia and thrombocytopenia in patients after solid organ transplant [ ] . fungal infections such as histoplasmosis can cause disseminated organ infiltration, with the bone marrow being a common area of involvement, resulting in decreased hematopoiesis and cytopenias [ ] . parvovirus b , much better known for its role in causing both acute and chronic anemia in solid organ transplants, is also reported to cause acute and chronic leukopenia in approximately . % of solid organ as well as hematopoietic stem cell transplant recipients who develop the infection [ , ] . an acute infection with hhv- can present with fever, splenomegaly, and leukopenia (as part of a pancytopenia), with bone marrow biopsy revealing hypocellularity, plasma cell infiltration, and evidence of viral infection by immunohistochemical staining [ ] . a retrospective analysis of liver and kidney transplant recipients was performed to assess the relationship between leukopenia and positive hepatitis b and c serologies. the investigators found that there was no significant correlation between leukopenia and hepatitis c infection, but that the incidence of leukopenia in those with active hepatitis b infection was . %. they posited that, similar to other viruses, infection with hepatitis b virus could lead to "decreased or ineffective leukocyte production in the bone marrow…shifts of cells from the circulation to the marginal blood pools… [and] also produce peripheral destruction of white blood cells due to immune and nonimmune processes" [ ] . cytomegalovirus infection is the most well-known transplant-related infection to cause cytopenias, with leukopenia found in approximately % of infected transplant recipients and with most of the data and research conducted in kidney transplant populations [ ] . infection with cmv has direct effects on the bone marrow, inhibiting hematopoiesis by affecting both the bone marrow stroma and the stem cells and hematopoietic precursors [ , ] . cmv disease (acute symptomatic infection) is most often seen in the first months, particularly during the first months posttransplant, and presents with constitutional complaints such as fever, abdominal pain, diarrhea, and respiratory symptoms along with cytopenias [ , , ] . however, in heart transplant patients, a subclinical infection during the st year where infected individuals are asymptomatic has also been associated with leukopenia, with the most significant reductions occurring in the neutrophil and monocyte populations and preservation of the lymphocyte counts [ ] . an added challenge when addressing cmv infection and leukopenia results from the frequent finding that the treatments for the disease can result in further leukopenia (discussed in "noninfectious etiologies of leukopenia" section). additional diagnoses to consider when assessing the etiologies of leukopenia, as well as pancytopenia, with regard to infection are hemophagocytic syndrome (hps) which is associated with cmv, ebv, hhv- , hhv- , and histoplasmosis, as well as ebv-associated ptld [ , ] . as in all cases of thrombocytopenia, when evaluating a finding of low platelets in a patient after sot, it must be determined whether the primary problem is one of impaired production in the bone marrow or if it is a matter of consumption or sequestration outside the marrow. infections and drugs are known to suppress megakaryocyte production in the marrow, such as cytomegalovirus and trimethoprimsulfamethoxazole (tmp-smx). additionally, both infections and medications as well as auto-and alloimmune processes can lead to destruction of platelets despite adequate production of megakaryocytes in the bone marrow. solid organ transplant recipients are at risk for developing infections due to their immunosuppression, and viral infections in particular are a potential contributor to the development of thrombocytopenia following solid organ transplant. detailed discussions of these infections are found in other chapters of this book, but their involvement in thrombocytopenia is discussed below. in addition to the viral infections that contribute to thrombocytopenia, it is important to remember that thrombocytopenia can be a sign of bacterial infection and sepsis most often in the context of disseminated intravascular coagulation (dic). appropriate workup to rule out infection is among the first steps in examining thrombocytopenia in a solid organ recipient. the virus of particular concern in regard to platelet count in transplant patients specifically is cytomegalovirus, though thrombocytopenia due to other viruses has also been described, often among a constellation of systemic symptoms. cytomegalovirus can cause thrombocytopenia both by decreasing production of and through destruction of platelets. studies have shown that cmv can impair megakaryocyte production in its early stages by infection of stromal cells, which interferes with growth factor production, as well as by directly infecting myeloid cells [ ] , similar to cmvrelated leukopenia. the other reported etiology of thrombocytopenia from cmv is due to intravascular destruction of platelets by cmv-associated thrombotic microangiopathy (tma), with a clinical picture resembling that of thrombotic thrombocytopenic purpura (ttp)/atypical hemolytic uremic syndrome (ahus), consisting of varying degrees of coombs-negative hemolytic anemia, thrombocytopenia, acute kidney injury, fever, and neurological findings. while this etiology is more often identified as a drug-related phenomenon, particularly due to the immunosuppressants required to prevent organ rejection (see next section), there have been multiple case reports associating cmv infection as a trigger of tma in the posttransplant setting [ , ] . this has been noted with particular frequency in the renal transplant literature, where both de novo and recurrent forms of ahus were associated with cmv infection in renal transplant recipients. however, particularly in the patients with "de novo" disease, it is possible that cmv may be directly driving the thrombotic microangiopathy, rather than solely by the complementmediated events of ahus. mechanisms thought to be underlying cmv's endothelial effects include activation of cmv-specific cytotoxic immune responses and induction of primitive endothelial dysfunction as well as direct infection of endothelial cells by cmv [ , ] . however, some investigators question how significant a contributor cmv actually is to thrombotic microangiopathy in transplant patients. in a review of tma among lung transplant recipients by hachem and colleagues, an analysis of patients who were diagnosed with tma following lung transplantation revealed that only patients had evidence of cmv infection, and additionally that there were incidences of cmv viremia among the lung transplant patients who did not develop tma. additionally, in their univariate and multivariate analyses, neither cmv viremia nor serologic status was identified as a risk factor for tma in the study population [ ] . infection with epstein-barr virus often results in conditions associated with pancytopenia, such as ptld and hemophagocytic syndrome, both of which are described in greater detail in previous sections of this chapter (see "leukopenia and anemia" sections). ebv should be considered as a possible infectious etiology during the workup of thrombocytopenia, particularly if other systemic signs or symptoms are present. other infectious etiologies that have thrombocytopenia among the constellation of presenting symptoms that have been described in organ transplant recipients include coronavirus, particularly sars, lymphocytic choriomeningitis virus (lcmv), and hhv- , though the thrombocytopenia is unlikely to be the primary issue at presentation [ ] . parvovirus b- and polyoma bk virus infection have also been associated with development of ahus [ , ] . it is also important to note that chronic infection with hepatitis c can be an etiology for thrombocytopenia both in and outside the context of solid organ transplantation. the etiology of thrombocytopenia in the setting of hepatitis c infection can be due to hepatocellular damage including fibrosis and/or cirrhosis affecting thrombopoietin (tpo) production, hypersplenism due to portal hypertension, bone marrow suppression, immune dysfunction, and development of platelet autoantibodies [ ] . additionally, treatment for hepatitis c with interferon is known to cause thrombocytopenia. there are numerous noninfectious etiologies of thrombocytopenia that have been identified in sot patients. many pharmacologic agents have been implicated in the development of thrombocytopenia following sot through varying mechanisms, such as tma, decreased megakaryocyte production, and auto-and allo-immune mechanisms of platelet destruction. the drugs most strongly associated with decreased platelet counts due to thrombotic microangiopathy are the calcineurin inhibitors (ci) cyclosporine and tacrolimus. calcineurin inhibitor induced tma often occurs within weeks following sot, and the cis are thought to cause direct endothelial injury and platelet aggregation, although the specific mechanism has not been identified. when this is identified, numerous case studies in multiple different organ systems (lung, liver, kidney solid organ transplant) have reported that changing from one ci to another (tacrolimus to cyclosporine or vice versa) or to another class of medication such as sirolimus or mycophenolate mofetil can prevent further episodes of tma from occurring [ ] [ ] [ ] [ ] . however, the addition of the mtor inhibitor sirolimus to a calcineurin inhibitor also increases the chance of developing tma [ , ] . ganciclovir and valganciclovir are used in prophylaxis and treatment of cmv. both are known to have myelosuppressive effects, particularly on granulocytes and platelets, but generally there is rapid recovery of counts following withdrawal of the medication. one of linezolid's most well-known adverse effects is thrombocytopenia, with the package insert reporting a rate of % in adults. other studies have reported rates of grades iii-iv thrombocytopenia of approximately . % [ ] . no mechanism has been identified for linezolid-related thrombocytopenia, though some evidence suggests that it is an immune-mediated phenomenon [ ] . the medication is frequently used in the treatment of vancomycin-resistant enterococcus (vre), which has been an infection seen in transplant patients, as well as non-transplant patients, with increasing frequency. a multicenter compassionate use trial published in showed that it was an effective drug in treating vre, which was identified as having a mortality rate of up to %, with the authors reporting a % survival rate after treatment with linezolid. thrombocytopenia was the main adverse effect of treatment, seen in . %, but did not necessitate the cessation of therapy [ ] . a second study in liver transplant patients treated with linezolid for vre infec-tion showed a similar treatment efficacy and again reported no cases ( / patients) requiring cessation of therapy due to severe thrombocytopenia, and furthermore found no correlation between treatment duration and platelet counts [ ] though other articles advise caution when using linezolid for extended time periods [ ] . thus, while it may or may not require any intervention or change in treatment plan, it should be considered as part of the differential diagnosis when assessing thrombocytopenia. heparin-induced thrombocytopenia is an additional drugrelated event that can occur in the setting of solid organ transplant. assessment of this as a possible etiology for thrombocytopenia follows the same algorithm as it would for any patient receiving heparin. the probability of the thrombocytopenia being related to heparin use would be based on the ts whether the timing (> days following start of heparin use or sooner if heparin was used previously), degree of fall (> % decrease from baseline), presence of thrombosis, and lack of alternate explanations for the thrombocytopenia suggest that heparin could be the causative agent [ ] . studies reveal that hit is an uncommon occurrence in liver transplant recipients, and that thrombotic events and hit antibody positivity were not well correlated [ , ] . case studies in renal transplant patients have reported some incidences of hit posttransplant and graft-failure related to hit, in part related to previous exposure to heparin in hemodialysis [ , ] . hit antibody immunoassays are often sent if a patient develops thrombocytopenia and has received heparin at any time during the hospitalization. however, the high sensitivity but low specificity of the test results in overdiagnosis of heparin-induced thrombocytopenia exposes patients to unnecessary risks associated with therapeutic anticoagulation. chaturvedi and colleagues examined this phenomenon at cleveland clinic and found that utilizing the ts algorithm to first rule out patients at low risk for hit was a safe, reliable, and cost-effective [ ] . therefore, we recommend that immunoassays for hit antibodies be utilized only in those patients whose t scores suggest intermediate or high probability of heparin-induced thrombocytopenia. if a hit antibody immunoassay is sent once a patient is determined to be of intermediate/high risk for hit, it is important to understand how this test is interpreted. the immunoassay detects the presence of antiplatelet factor (pf ) antibodies in patient serum and is interpreted by optical density (od). a higher reported od correlates to a higher titer of the antibody and is more strongly suggestive of a diagnosis of hit. as mentioned previously, elisas for hit have a high sensitivity (meaning a negative test can rule out the diagnosis) but a low specificity, underscoring the need to first confirm a high pretest probability. immune thrombocytopenic purpura (itp) is characterized by very low platelet counts, petechiae and bruising, as well as mucosal bleeding, due to opsonization of platelets in the circulation. occurrence of itp following solid organ transplant has been documented particularly in the liver transplant literature, with the cases attributed to either autoimmune itp, at times precipitated by an identified infectious etiology such as tuberculosis [ ] or alloimmune etiologies. the literature reports that chronic renal disease and renal transplant in patients with itp are noted to be very rare [ ] , and thus most of our knowledge of itp as an etiology following transplant is from the liver transplant literature. one study reported a case series of eight patients who developed itp following orthotopic liver transplantation (olt), with a mean time of presentation of itp since olt of . years [ ] . these cases were all felt to be autoimmune cases, as there was no history of itp in the donors. this case series also presented a review of the previous literature on itp after olt, and they noted two distinct time patterns of itp presentation, early (within months) or late (> months). the authors note that it has been proposed that the earlyonset presentation may be due to passive transfer of antibodies from the donor to the recipient. those that developed late-onset itp were felt to have developed the antibodies independently of their donors [ ] . additionally, studies have reported on development of alloimmune thrombocytopenic purpura, with antibodies introduced from donors with a history of itp [ , ] . one case study described a case where a donor liver was obtained from a donor who had died after a cerebral hemorrhage secondary to itp. the recipient developed itp within days of transplant and subsequently expired after developing portal vein thrombosis. the authors attributed the death to itp in that they were unable to anticoagulate but were providing blood products that may have resulted in increased likelihood of thrombosis. it is also possible, however, that the donor was producing procoagulant antibodies, as approximately - % of patients with itp also have antiphospholipid antibodies [ , ] . based on this event, the authors recommended excluding cadaveric transplants from donors whose death is attributed to itp [ ] . particularly in liver transplant patients, thrombocytopenia is often seen prior to transplant, and generally approximately % of transplant recipients develop worsening thrombocytopenia within weeks following transplant. this acute decrease generally resolves within the st month after trans-plant, and if thrombocytopenia persists, another etiology should be sought [ , , ] . thrombocytopenia following liver transplant can also be attributed to residual portal hypertension or hypersplenism, if either of these conditions persist following transplant. however, it is important to remain vigilant to other causes particularly drug-related and infectious etiologies that could cause a drop in the platelet count. additionally, while case reports exist of tma with low adamts levels attributed to inhibitors present in the blood [ ] , this is not a common phenomenon, and etiologies of tma mentioned previously (infectious and drug-related) would be much more likely. in most cases, treatment of the underlying etiology of the thrombocytopenia will result in improvement in platelet counts. that may require antivirals, adjustment of the immunosuppressant regimen, withdrawal of other pharmacologic agents such as heparin, or supportive care. platelet transfusions may be necessary if bleeding events occur or if additional procedures are necessary, but we do not recommend prophylactic transfusions for maintenance of the platelet count above a specific threshold. tpo receptor agonists, romiplostim and eltrombopag, have been used in management of chronic thrombocytopenia due to itp and liver disease and are being studied as a supportive medication in stem cell transplantation [ ] . there is a case report in the pediatric transplant literature where romiplostim was used in the peri-transplant setting, which resulted in a platelet-transfusion-free liver transplant [ ] . however at this time, there is no data to support use of tpo agonists outside of their approved indications following solid organ transplant. prevalence and management of anemia in renal transplant recipients: a european survey late post-transplant anemia in adult renal transplant recipients anemia and erythrocytosis after kidney transplantation: a year graft function and survival analysis anemia is a predictor of outcome in heart transplant recipients the effects of pre-and post-transplant anemia on -year survival after cardiac transplantation immune hemolysis following abomismatched stem cell or solid organ transplantation passenger lymphocyte syndrome and liver transplantation a 'dangerous' group o donor: severe hemolysis in all recipients of organs from a donor with multiple red cell alloantibodies donor derived alloantibodies and passenger lymphocyte syndrome in two of four patients who received different organs from the same donor donor-derived antibodies and hemolysis after abocompatible but nonidentical heart-lung and lung transplantation severe hemolytic anemia due to passenger lymphocytes after living related bowel transplant pharmacoepidemiology of anemia in kidney transplant recipients posttransplantation anemia at months in kidney recipients treated with mycophenolate mofetil: risk factors and implications for mortality comparative effects of sirolimus and mycophenolate mofetil on erythropoiesis in kidney transplant patients post-renal transplant hemolytic uremic syndrome following combination therapy with tacrolimus and everolimus the spectrum of thrombotic thrombocytopenic purpura: a clinicopathologic demonstration of tacrolimus-induced thrombotic thrombocytopenic 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transplant recipients: report of a multicenter compassionate-use trial efficacy and safety of linezolid in liver transplant patients thrombocytopenia associated with linezolid therapy evaluation of pretest clinical score ( t's) for the diagnosis of heparin-induced thrombocytopenia in two clinical settings incidence of heparin-induced thrombocytopenia type ii and postoperative recovery of platelet count in liver graft recipients: a retrospective cohort analysis heparin-induced thrombocytopenia after liver transplantation heparin-induced thrombocytopenia type ii: a serious hazard in preemptive renal transplantation: a case report successful preemptive renal retransplantation in a patient with previous acute graft loss secondary to hit type ii: a case report and review of literature over-testing for heparin induced thrombocytopenia in hospitalized patients immune thrombocytopenic purpura induced by intestinal tuberculosis in a liver transplant recipient renal transplantation and 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schuhmacher, alexander; von zedtwitz, max; reepmeyer, gerrit title: the internationalization challenge: where to access innovation date: - - journal: leading pharmaceutical innovation doi: . / - - - - _ sha: doc_id: cord_uid: cegevjw pharma innovation is becoming increasingly global, partly due to the lure of serving new markets, partly because of the need to access early new technology and talent wherever it emerges. apart from the established centers of innovation in the united states, europe and japan, india, china and singapore are rising attractors for global life science r&d. china as a pharma market and host of pharma r&d is highlighted as a case study, both from the angle of foreign r&d investors and from the perspective of indigenous chinese players. given the significant cost pressures in mature markets, reverse innovation in healthcare has drawn considerable attention by local governments. in the u.s., takeda in japan, and bayer in germany had large home markets and a substantial domestic r&d base, and hence had less pressure to internationalize their r&d activities. only in recent years, starting in the mid- s, increased competition from within and outside their industries forced companies from large countries to source technological knowledge on a global scale. global mergers ensued, expanding the research of r&d organizations quickly into new territories. the rise of china as an emerging market for health care and pharmaceuticals has attracted much pharma r&d investment in the mid- s. as a result, the top pharmaceutical companies today have r&d locations in all major markets, not just for the coordination of local clinical development projects but also for discovery research. the management of cross-border r&d is characterized by a significantly higher degree of complexity than local, single-site r&d management. the extra costs of international coordination must be balanced by synergy effects such as decreased time-to-market, improved effectiveness, and enhanced learning capabilities. top corporate managers are confronted with the task of finding the optimal r&d organization based on the type of r&d activities, the present geographic dispersion of subsequent value-adding activities such as production and marketing, and the coordination between a multitude of contributors to the r&d process. the case for r&d internationalization is not unchallenged. besides the ubiquitous cost argument, foreign r&d units are more difficult to manage, and control, and may be less efficient due to missed scale effects. table . summarizes some of the most cited arguments against international r&d. what drives r&d internationalization? most factors are due to either science & technology-related issues or sales & output efficiency (table . ). science and technology-related factors are concerned with r&d personnel qualification, knowhow sourcing and regional infrastructure. these factors are largely outside the direct influence of r&d but necessary for its fundamental operations, such as the proximity to universities or the r&d environment. proximity to markets and customers, improvements of image and collaborations are notable sales & output efficiencyrelated factors. efficiency-related criteria focus on the costs of running and the critical mass of r&d units, as well as efficient hand-over processes between r&d and other corporate functions. direct cost advantages (such as the often-publicized labor costs) rarely influence the internationalization of r&d in the long run, but other efficiency-oriented factors such as costs of coordination and transfer, and critical laboratory size do have an even bigger impact on international r&d organization. direct costs may become more important in the coming years as the other factors improve in low labor cost countries. political and socio-cultural factors such as local content rules, technology acceptance and public approval times, all play an important role in locating r&d abroad. protectionist, legal and cultural constraints imposed by national governments, however, often require a company to establish local r&d units. r&d-external forces such as a business unit's striving for autonomy and the build-up of local competence alters the original mission of a local r&d unit. this evolution may take place without hq's knowledge, particularly in strongly decentralized companies. in the pharmaceutical industry, mergers and acquisitions have significantly contributed to the internationalization of r&d, particularly with recent cross-border mergers. even though small size is a driver for internationalization, the global marketing reach of large firms facilitates the leverage of global r&d and innovation. for instance, when pfizer acquired wyeth and merck acquired schering-plough in , on paper the two merged firms would have accounted for % of the total u.s. pharmaceutical r&d spending and % of total world-wide spending (comanor and scherer ). subsequent cost reduction programs eliminated r&d units in the combined companies, but since both domestic and foreign units were closed down, this had little effect on their new extent of r&d internationalization. rather, we ( ) . trends in r&d internationalization observed a centralization of r&d in certain internationally leading regions of innovation (centers of excellence). however, mergers are rarely driven by scientific or technological reasons. access to new markets and economies-of-scale effects are primary drivers for mergers. nevertheless, the resulting r&d conglomerate must somehow come to terms with a new and more international organization. the development of local products requires the early involvement of market and customer application know-how, which is more likely to be found in regional business units. companies with local r&d exhibit an inclination towards overemphasizing different local market specification in order to support local autonomy and independence from the parent company. host country restrictions, such as local content requirements, tolls, import quota, and fulfillment of standards, can attract r&d into key market countries (pull regulations): both the u.s. and china have plenty of such regulations attracting inbound r&d investments from overseas. on the other hand, home country restrictions may induce companies to move r&d abroad (push regulations): european regulations caused biotechnology r&d to be transferred to the u.s., and lack of homegrown talent forced swiss pharma companies to set up r&d centers abroad. a study of r&d sites across various industries (including automotive, engineering, electrical, it, software, food, chemical and pharmaceutical companies) produced the following overall results concerning international r&d locations (see also von zedtwitz and gassmann ): • r&d is concentrated in the triad regions of europe, the united states, japan, as well as major regional centers in south korea, singapore and other emerging economies in asia-pacific, such as india and china. china has seen a particularly strong increase of inbound r&d investments in recent years, but domestic companies are also conducting more and more r&d. compared to china and india, the other frequently mentioned bric countries-brazil, russia, and sometimes also south africa-are far behind. they lack economic and institutional attractiveness, and their domestic high-tech ecosystem is still underdeveloped, comparatively speaking. • over % of all research sites are in the five regions of the northeastern usa (new jersey, new york, massachusetts), california, the united kingdom, western continental europe (in particular germany), and the far east (japan, south korea). china (especially shanghai and beijing) and india (especially mumbai and bangalore) are catching up as locations for research, too. the trend of research concentration is even more apparent when only foreign research locations are considered: almost % are in the triad regions of the u.s., europe, and japan. • although the main regional centers for development largely coincide with the regional centers for research, development is more evenly distributed among european countries and the northeastern united states, and extends into southeast asia, australia, africa, and south america. only slightly more than half of all development sites are located in the eight most development-intensive countries. moreover, research and development sites of the same company are not necessarily co-located. this is especially true in the pharmaceutical industry, where development mandates differ so much from research. since the late s many large companies have made multiple efforts to consolidate their activities in order to realize synergy and coordination potential in international r&d. transnational r&d projects are managed more easily if the r&d network consists of competence centers (pharmaceutical firms tend to be ahead in reorganizing themselves around competence centers), given that complementary competencies are provided locally. with increasing complementarities of resources, competencies, and knowledge bases, as well as the division of labor and specialization of work, synergy potentials in r&d projects can be exploited. figure . shows a subset of pharmaceutical r&d centers of the r&d locations. it presents the r&d locations of pharmaceutical companies, including abbott, astrazeneca, boehringer ingelheim, j&j, eli lilly, eisai, gsk, pfizer, merck, novartis, novo nordisk, pfizer, roche, sanofi, sinopharm, takeda, or teva. the distribution of these r&d sites shows a similar pattern to global r&d dispersion across all industries but differs in the following observations: • there are very few r&d centers outside the globally most attractive countries (the triad countries, as well as china). • in the us, r&d is focused more on the north-east, in particular the tristate area, north carolina, massachusetts, and a few centers in the mid-west. relatively speaking, there is less r&d in california, but the bay area, san diego and los angeles are catching up also as hosts for pharma and biotech r&d. • in europe, r&d is concentrated in the u.k., france, germany and switzerland, mirroring the relative strength of these countries as homes of big pharma (novartis and roche are from switzerland, sanofi and servier are french, astrazeneca and gsk are from the u.k., and bayer and merck kg are german). • despite what some call japan's lost two decades, there is a strong representation of r&d in japan, led by the many japanese pharmaceutical firms, although most of them are smaller compared to their western competitors (e.g., takeda or eisai). • emerging new centers of pharma r&d are shanghai, beijing and singapore, but also india (mumbai and bangalore), and israel. given this data, the pharmaceutical industry is one of the most internationalized in terms of r&d locations. pharmaceutical companies also seem to internationalize research as fast as development (albeit for different reasons). most other industries tend to keep research at home and localize development. although not obvious from location data alone, pharmaceutical companies also tend to organize r&d as competence-based networks, as opposed to r&d hubs (e.g., automotive and chemicals), polycentric networks (e.g., local market-dependent companies such as royal dutch/shell) or centralized r&d (e.g., dominant design industries). in competence-based r&d networks, each r&d node has a clearly defined competence-and responsibility!-which it brings into the network of other r&d centers. the coordination and management of such r&d networks is more demanding and costly than rather centralized and directive r&d hubs, or the laissez-faire style of polycentric r&d networks. as a consequence, pharmaceutical companies (and many electrical and it companies, who also favor this type of r&d organization) try to coordinate r&d activities across multiple levels, including the deployment of transnational project teams, platform management, informal as well as formal network techniques. the history of novartis has been documented in detail by zeller ( ). we will focus here only on some of the more recent developments as they are representative of big pharma. in , about scientists worked in research centers worldwide in several therapeutic areas. only of them were employed in research centers in switzerland, along with a comparable number in pre-clinical and clinical development: more than half of the r&d workforce was located outside switzerland. novartis had drug candidates in clinical development at that time. in may , novartis announced it would move its global research headquarters to cambridge, massachusetts. over the following years, the cambridge-based center has grown by people in r&d alone and now occupies multiple sites. in , it opened a research center focused on dengue and other tropical diseases (the nitd institute for tropical diseases) in singapore. it closed its palo alto-based systemix r&d and moved it to east hanover, new jersey. in , novartis opened a r&d center in shanghai, being one of the first of the big pharma to do so. over the next few years, novartis would continue to consolidate r&d in its preferred research hubs. in , novartis closed its horsham, u.k., respiratory research center, its center for topical dermatology treatments in vienna, and a biotherapeutics development unit in la jolla. it also relocated oncology r&d from emeryville in california to its cambridge r&d center. in , novartis closed its schlieren, switzerland-based esbatech r&d center, and moved its institute for tropical diseases from singapore to emeryville, although singapore remained a major r&d hub for novartis in asia-pacific. it also shut down its biologics group in shanghai. however, it opened a brand-new us$ billion research center in shanghai, to become its third largest research center behind cambridge and basel. as of , novartis employed scientists in its global r&d organization, almost half of them in cambridge alone. novartis's headquarters remained in basel, switzerland. chugai is one of japan's pharmaceutical pioneers. it was acquired by roche in but continues to operate independently. founded in in tokyo, it had established r&d centers in ukima, kanagawa, and shizuoka. with new ambitious research targets in the early s, it started to establish overseas offices in the mid- s, and to invest or acquire u.s.-based biotech firms. in it set up its first international research center in korea, and expanded its london office into a fully-ledged pharma center in . in the same year, it founded a research center in ibaraki, and years later one in tsukuba and in singapore. with the alliance with roche in , global r&d was reorganized as well. the tsukuba and the takada centers were both closed, but the forerunner pharma research company formed, as was a new clinical research center organization, which eventually also opened a base in singapore in . an interesting case of internationalization is ferring pharmaceuticals. these examples are typical for the changes in global r&d in the pharmaceutical industry (see fig. . for takeda). we see three major trends: . consolidation in hubs with abundance of talent and technology. . acquisitions of smaller competitors and biotech firms expand the geographic dispersion of r&d. . increased presence in asia-pacific, especially china. the last trend is also observed for global r&d in other industries, which nevertheless requires an explanation, as china is not known to be a place in which r&d is conducted easily, nor is it a country in which intellectual property-the main result of r&d work-is well protected. the healthcare context in china china is no longer a mystique country closed off to foreigners, but it is still very much misunderstood. under almost constant reform mode, the china's economy was opened up in the s, picked up speed in the s when it became a major destination for manufacturing investments, excelled at growth rates beyond % and % in the s, when it also became attractive for r&d, but in the wake of the economic crisis of it slowed down to what is now called a "new normal" economic growth of - %. it passed japan as the second largest economy in the world in , and in its gdp was us$ trillion, behind the u.s. at about us$ trillion. despite a growing middle class (whose definition is ambiguous in china), most of china is still a developing country, which translates into great tensions about healthcare services needed and wanted, and what role the pharmaceutical industry can play in this context. improved living conditions further mean that china's population is aging fast, with the share of the population aged or older to double to about % by . china's one-child policy, although relaxed to a two-child policy in , led to wealth being distributed among fewer children and family members, which means that more earnings are disposable for life-style and healthrelated products. furthermore, as china westernizes, its disease profile will become more and more comparable with the western world, including afflictions such as hypertension, cancer, diabetes, etc., for which multinational companies already have developed drugs and therapies. with a population of more than . billion, china presents a huge market opportunity as it is. a rapidly aging population (in , an estimated million people are aged or over), coupled with new afflictions resulting from air and water pollution, pushes the emerging healthcare system to the brink, and the solutions required will need to concerted effort of government, pharmaceutical industry, and healthcare institutions. administrative hurdles, low healthcare spending, the lack of intellectual property protection and the poor distribution network infrastructure remain the biggest challenges. major pharmaceutical companies pursue aggressive growth strategies and try to benefit from the chinese market in the longer term. they invest in the sourcing of active ingredients, research and development, and the production and selling of generic and proprietary drugs. in , china was still a low-tiered market of about us$ billion in total. pharmaceutical sales grew at a cagr of % between and , exceeding overall growth in gdp and overall healthcare expenditure (deloitte ). it passed us$ billion in and is estimated to grow to us$ billion in . china has the world's fastest-growing over-the-counter (otc) drug market and is already the second largest pharmaceutical market worldwide. pharmaceutical sales amounted to % of total health expenditures in , or us$ per person. generics accounted for % of total sales, patented drugs only % (ita ). the generics market is dominated by low cost domestic producers. webber ( ) put their number at approximately chinese pharmaceutical companies, of which produced medicines (small-molecule generics and biotech products, i.e. not including herbal medicines or traditional chinese medicines) and the remainder were involved in pharmaceutical-related activities such as packaging and equipment supply. a large share of them are merely producers of raw materials (wang and von zedtwitz ). by , this market was still highly fragmented, with domestic companies in operation, of which the top accounting for only one third of the total market (ita ). government and chinese fda (cfda) policies have been expected to weed out those not in compliance with gmp (good manufacturing process) practices, but have made only limited progress. the strategic emerging industries initiative plans to create national champions, and policies are being put in place that seem to favor domestic over foreign companies (prud'homme ; ita ) . chinese manufacturers are very strong in their ability to copy foreign drugs, sometimes selling them under foreign labels. the vast majority of the - pharmaceutical products manufactured in china are copies of foreign products, either legal generics or illegal counterfeits. they serve a worldwide counterfeit market estimated to exceed us$ billion in alone (philipp ), with % of counterfeit pharmaceuticals seized by u.s. authorities in coming from china. the situation is complex to control: in addition to the almost chinese companies involved in medicine manufacturing, there are , retail pharmacy shops, and , firms involved in shipping medical products (philipp ). china lacks effective regulatory control over the manufacture and distribution of active pharmaceutical ingredients (apis). chemical manufacturers only need to register with the cfda if their product is intended for medical use. the cfda has no authority to regulate them if they declare otherwise, and the cfda does not monitor or inspect apis intended for export (ita ) . for many chinese companies, the term 'r&d' refers mainly to the production of additional generic products particularly for china-in terms of strengths, dosage forms and even specific compounds (webber ). as of , what constitutes a "new drug" was still of unclear definition and subject to policy debates (ita ). in , tu youyou won the first nobel prize awarded to a chinese in physiology or medicine for her discovery of a novel malaria therapy, artemisinin. while traditional chinese medicine (tcm) has been a staple in china for a long time, it put the spotlight on the uneasy relationship between western and chinese approaches to medicine (many chinese doctors prescribe tcm side-by-side with western ethical drugs). more than (including different dosage forms) traditional chinese medicines (tcm) are manufactured and sold. with few market entry barriers, there were over tcm companies in china. tcm accounts for more than % of sales and is particularly strong in rural areas. tcm drugs make up % of the essential drug list (edl) and % of the national drug reimbursement list (ndrl) (export ). in the chinese view, tcm has already proved itself to be effective in curing many kinds of diseases, having "gone through thousands of years of clinical trials" (guo , quoting wang guangji, a deputy to the national people's congress (npc) and former vice president of china pharmaceutical university who specialized in western medicine): "but important terms like yin (substance) and yang (energy), whose balance are essential for harmonious operation of the body, according to traditional chinese medicine, would hardly be accepted by foreigners." western pharmaceutical firms shy away from tcm r&d, as it is difficult to isolate single, scientifically testable compounds (waldmeir ). according to ye yang, deputy director of shanghai institute of materia medica, "to get a single compound out of a single plant, like in the case of artemisinin, took so many years. in traditional herbal medicine [you might have] - compounds working together. we don't have the technology to follow all of them to see how they are working individually and together." the chinese government promotes tcm, and several government departments are guiding the tcm industry's attempts to modernize. according to webber ( ) , the chinese authorities have identified two tracks for developing tcm r&d. the first is through purification and standardization to meet global standards and remove impurities such as pesticides and heavy metals. the second track is to utilize tcm as a starting point for producing novel medicines. this may be through the identification and purification of the active element(s) (often complex molecules) or the discovery and development of small molecules which mimic the activity of the original tcm. liu and xiao ( ) found that about new drugs have originated directly or indirectly from chinese medicinal plants by means of modern scientific methods. the chinese government has also set clear focus on certain areas of biotech research, especially genomics research. in , the ministry of science and technology established the chinese national human genome centre based in beijing and shanghai, and the beijing institute of genomics, as centers of excellence for genome sequencing and analysis, thus enabling china to join the international human genome sequencing consortium in , in which china played a significant part. china now is on par with major international research leaders in areas such as gene mapping, transgenic technology for animals and plants, gene therapy technology, stem cell research, gene chips and gene research of some major diseases (webber ) . the country has several world-class scientific biomedical institutions-the north and south genome centers, the institute of materia medica, tsinghua and beijing universities, for example. china also has some research institutes for biotechnology and more than of the key state laboratories in biopharmaceutical-related areas. in china, the domestic pharmaceutical research and development environment is still dominated by universities and scientific institutes rather than pharmaceutical enterprises. the industrialization of pharmaceutical research and biotechnology still lags the western world, but is catching up quickly as well (shi et al. ) . foreign pharmaceutical companies have conducted r&d in chins since the late s, although in the earlier years the type of research done was very low key and part of larger studies that were rooted elsewhere. for instance, roche had an r&d office in shanghai long before it opened a dedicated r&d center in . it was not until the mid- s that global pharma companies discovered chinas as a harbor for strategic r&d operations, perhaps a decade after global electronics, it and automotive firms started to invest in r&d in china. there are several reasons for this relative late arrival in china: • uncertain ip protection: china had reintroduced patent law only as recently as , and neither the general public nor the scientific ecosystem was used to developing, protecting, and honoring intellectual property. the courts, the legal system, and the police had little-if any-experience in dealing with ip cases. chinese employees had little sensitivity for personal safety or safety as a public good. tacit knowledge and knowledge that was pre-patent was not easy to secure and appropriate. of course, these conditions affected also other industries, but in pharma-with its extremely long lead times-such unwanted exposure of technical knowledge is very harmful. • fragmented nature and complexity of china's market: domestic companies accounted for % of annual sales, of which % operated in the low value generics market (dierks et al. ) . most needs are met by local generics, with only between % and % being patent-protected innovative drugs. • price pressure: % of china's healthcare budget was being spent on medicine compared with - % in western countries, leading to considerable political pressure to reduce prices (knowledgewharton ). on the other hand, there were also several strong china-specific reasons to consider r&d in china: • quick development of a middle-income market demanding medicines for their evolving needs. these needs stem from rising life expectancy, pollution, and enhanced abilities to detect and diagnose diseases. • understanding the uniqueness of the genomics and metabolomics of the chinese population gives competitive leverage to domestic r&d entrants. • access to natural products in china as sources for new chemical entities (nces). newman et al. ( ) calculated that about % of nearly nces could be traced back to natural products. one famous example is lipitor, a statin first discovered in natural sources with sales exceeding us$ billion annually. • access and research on tcm: china-based innovations include artemisinin, invented in china using sweet wormwood and hailed as a miracle malaria drug, sobuzoxan, an anti-tumor drug, and huperzine a (hupa), a novel alkaloid isolated from a chinese medicinal herb, which improves memory deficiencies in alzheimer patients. testing the waters for captive local r&d operations often begins with r&d collaborations. glaxosmithkline (gsk), for instance, invested over us$ million in cooperative r&d with chinese research institutions since the mid- s. novartis started a collaboration with the shanghai institute of materia medica (simm), with the objective of isolating compounds from chinese medicinal plants for novartis to further screen and identify lead compounds. after an initial phase and us$ million in funding, training and equipment, by simm had isolated more than compounds from natural herbs covering immunology, oncology, diabetes and the central nervous system. other braved the uncertain conditions and started dedicated r&d centers early. in , novozymes opened an r&d center in the zhongguancun science park in beijing. costing million €, the facility focused on the customized development of enzymes and processes for the chinese market. roche opened a new r&d center for chemists in zhangjiang high-tech park and established r&d alliances with the state-owned genomics centers in shanghai and beijing, to conduct research in the genetic predisposition to diseases such as diabetes and alzheimer's. local r&d enabled these foreign pharma companies to enter more credibly into a dialogue with authorities and opinion leaders in the country, which ultimately is good for business in china. by the mid- s, the ip situation in china was better understood, other foreign multinationals had been successful with captive r&d in china, and some of pharma's r&d pioneers seemed to succeed with r&d in china as well. as a result, pharma companies started to invest more strategically in r&d in china, in the expectation to access the natural resource base in china, and to tap into an increasingly large body of medical researchers and pharmaceutical scientists. also, china is also an attractive base for clinical research, given that there were more than , hospitals and healthcare facilities. table . presents an overview of this early stage of r&d setup in china. in july , glaxosmithkline china set up an otc (over-the-counter) medicines r&d center at tianjin smith kline & french laboratories, a joint venture funded by glaxosmithkline. its aim: to excel in creating innovative science-based products to meet consumer needs and support the joint venture's vision of becoming the premier otc company in china. pfizer set up an r&d center in shanghai focusing on developing trial protocol design and assessment of trial results, and novartis built a us$ million drug discovery research center in shanghai in . the research facility, which expanded to scientists by , focused initially on discovering medicines to treat cancers caused by infections, which make up a considerable proportion of the cancer cases diagnosed in china. some smaller biotech firms also started local r&d in china, for instance bicoll in shanghai. while pharma r&d focused initially on shanghai and beijing, other industries explored locations in second-tier cities and interior china, a trend that pharma r&d is following only slowly. even globally, pharma r&d tends to be more concentrated in few hubs, and there is no reason to assume this should be different in china (fig. . ) . in the years after the global financial crisis china benefited from its domestic stimulus program, but as of , china's gdp growth has marked slowed down. this is especially true for the already well developed provinces along the pacific shoreline, while many of the central provinces continue to enjoy high growth rates, albeit from a lower base level (according to the national bureau of statistics in china). cities in those provinces-e.g., chengdu, chongqing, and wuhan-are expected to become more attractive as hosts for pharma r&d. at the same time, foreign pharma companies continued to consolidate and expand r&d in their original r&d locations. roche, which has occupied the same location in shanghai since , poured in rmb . billion (about us$ million) to expand to buildings covering , square meters and employing professionals. shanghai became its third most important strategic r&d site worldwide, with major projects being run from china globally. construction is under way for the roche innovation campus shanghai, as is a diagnostic manufacturing facility in suzhou; these projects cost another us$ million. novartis, which started with a -square meter facility in , invested us$ million to build a dedicated r&d facility for scientists in . in , it opened a us$ billion r&d center in shanghai, banking on the expectation that the chinese government's own increased spending on biomedical research and training will deliver talented and highly educated researchers. a drug for liver fibrosis was fully discovered in china and brought into the late clinical stages, with hopes running high that it will be a high-performance global drug soon. astrazeneca doubled down on its start in clinical research in china and in added a us$ million investment in its new astrazeneca innovation center in shanghai. the center's initial research mandate was "in china, for china," but it has assumed a broader mission as a full-fledged discovery center focusing on diseases more prevalent in asia. in it made available us$ as an initial investment in a widened collaboration with wuxi apptec (so renamed after the merger of wuxi pharmatech with apptec laboratory services) to produce innovative biological medicines in china, with several hundred million us$ earmarked for further r&d investments in china across the board. it is important to note that pharma r&d in china is still very much in its early stages, and it is far from certain that the upfront multibillion-dollar investment will indeed deliver the expected payoffs. research by grimes and miozzo ( ) showed that only . % of the patents granted by the uspto between and by the top big pharma companies had chinese citations and only . % had chinese inventors. of the latter, roche accounted for % and novartis for %. china's share of biotechnology patents was also at around %, which is the same level as its overall uspto share. while china is often credited with speed and rapid development, many of the problems that kept pharma investment away in the s are perhaps still present, or at least inadequately addressed (see table . ). astrazeneca's the question is whether economic growth coupled with the shaping of a positive environment will encourage increasing investment into innovative pharmaceutical r&d in china. the global pharma companies are well advised to explore the china option carefully and systematically, given the dangers involved, but certainly they are advised to take this opportunity seriously. reverse innovation in healthcare to the nobel prize for medicine to be awarded to a chinese researcher, tu youyou. innovations from emerging markets carry the stigma of being 'low cost' or 'geared towards the needs of under-developed civilzations' (for a summary of roadblocks against reverse innovation, see hadengue et al. ) and often face steep adoption and acceptance difficulties (depasse and lee ). western policymakers and governmental institutions have therefore launched several initiatives at making the reverse transfer of emerging market solutions (from so-called lic low-income countries) more predictable and more systematic: • prize competitions: snowdon et al. ( ) reported the results of an open competition for healthcare solutions from developing or emerging countries, with the winner to be awarded us$ , for further financing their project. a committee evaluated all submitted projects on the basis of seven weighted criteria. apart from the winning innovation being supported through the prize money, the competing entries also benefited from project-specific feedback, and through media exposure the public was made aware of the potential of reverse innovation in health care. • crossover identifications: depasse and lee ( ) described a four-step model to transfer reverse innovations that includes a common problem identification in a lic, a dissemination analysis in that country, a crossover proposal, and subsequent dissemination in a high-income country (hic). the trick is not only to execute that crossover effectively, but also to identify suitable reverse innovation candidates and to support disseminiation in the home country. • scoring assessments: battacharaya et al. ( ) developed a two-step scoring system with eight assessment criteria, mostly for use by policymakers, public institutions or funding agencies. the purpose of this assessment is to identify potential reverse innovations and qualify them for their suitability to be transferred and applicability to an unmet need in the home country. in conclusion, the potential of reverse innovation in pharmaceuticals is still difficult to estimate, but the healthcare sector has taken a strong interest in considering any emerging solution from emerging market countries due to the mounting cost pressures in industrialized nations. pharmaceutical multinational firms that already have r&d centers in those emerging markets are well positioned to leverage their reach into different population groups not only as patient pools but also as sources of innovation, and ultimately may play an important role in acting as conduits of reverse innovation in their home countries. r&d in multinational companies has developed from centralized and geographically confined towards distributed and open structures. the pharmaceutical industry is one of the most advanced in terms of r&d internationalization, and one of the most it captures not only innovations first introduced in such markets but also those developed there (von zedtwitz et al. ), and it also includes truly indigenous sources of innovation rather than primarily foreign-invested subsidiaries of technology-intensive multinations in those regions the list of examples illustrating the potential that reverse innovation can have on healthcare in general and the pharmaceutical industry in particular is growing. among the first instances of reverse innovation mentioned ever are medical devices: ge's mac electrocardiogram from india, or ge's portable ultrasound devices developed in china. but medicines are also increasingly coming from emerging market countries: vicks honey cough drops were developed by p&g in their caracas r&d center, initially for their latin american market, only to see it do well with large populations in the u.s. and europe as well reverse innovation in healthcare specific when it comes to regulation and significance of science and technology. still, maintaining a well-balanced locally responsive and globally efficient r&d network is one of the great challenges of multinational organizations. rapidly evolving biotech firms and new pharma entrants (whether from emerging economies or in the • introduction of local culture of innovation and know-how • challenging projects coupled with bottom-up creativity • personal interactions more important in decentralized r&d • synchronization of international drug development by means of transnational project management in order to shorten r&d cycles • worldwide integrated r&d data management • acquisition of external ideas and projects as important as internal r&d • international teams require new organizations • manage platforms, not individual r&d projects • better inclusion and leverage of local university and national research programs • effective management of local open innovation partners • foster networking and collaboration in conclusion, transnational r&d management is a key ingredient to success, and the high stakes of the drug approval and medical safety have made the pharmaceutical innovation pipeline one of the best understood r&d engines (gassmann and von zedtwitz b). however, there is still untapped potential to improve this engine with new technologies, new managerial approaches, and new scientific talent drawn from countries around the world. key: cord- -r hzazp authors: stowe, julia; andrews, nick; miller, elizabeth title: do vaccines trigger neurological diseases? epidemiological evaluation of vaccination and neurological diseases using examples of multiple sclerosis, guillain–barré syndrome and narcolepsy date: - - journal: cns drugs doi: . /s - - -y sha: doc_id: cord_uid: r hzazp this article evaluates the epidemiological evidence for a relationship between vaccination and neurological disease, specifically multiple sclerosis, guillain–barré syndrome and narcolepsy. the statistical methods used to test vaccine safety hypotheses are described and the merits of different study designs evaluated; these include the cohort, case-control, case-coverage and the self-controlled case-series methods. for multiple sclerosis, the evidence does not support the hypothesized relationship with hepatitis b vaccine. for guillain−barré syndrome, the evidence suggests a small elevated risk after influenza vaccines, though considerably lower than after natural influenza infection, with no elevated risk after human papilloma virus vaccine. for narcolepsy, there is strong evidence of a causal association with one adjuvanted vaccine used in the / influenza pandemic. rapid investigation of vaccine safety concerns, however biologically implausible, is essential to maintain public and professional confidence in vaccination programmes. vaccination is one of the most effective public health interventions successfully controlling many serious infectious diseases and saving millions of lives globally each year [ ] . however, as with any medical treatment or drug, vaccination can never be entirely risk free in terms of unwanted side effects. an important feature of vaccination is that unlike most therapeutic drugs, vaccines are given prophylactically to healthy individuals, often young children. when an event occurs shortly after vaccination in an otherwise healthy individual without an obvious cause, it is tempting to attribute its occurrence to the preceding vaccination. the assumption of a causal association with a vaccine from purely a temporal association is often incorrect as unrelated events will occur by chance irrespective of vaccination. it can be hard to disentangle these temporal associations when there is a strong perception that a temporal association is necessarily evidence of a causal association and the onset of the condition is insidious and its timing relies on patient or parental recall [ ] . even if only based on a temporal sequence of events, it is important that such safety concerns are rapidly investigated with robust epidemiological studies to allow mitigation procedures to be put in place if an association is confirmed or, if unfounded, to have the necessary evidence to sustain public confidence in the vaccination programme without which coverage drops and disease control is lost. in this article, which focusses on the evaluation of the relationship between vaccination and neurological diseases, the statistical approaches to causality assessment are first discussed and their relative merits evaluated, followed by an overview of a selection of vaccine safety studies involving neurological disease with differing conclusions; some of the included studies have shown a small elevated risk, others none, two lack evidence to draw any definitive conclusion and one provides robust evidence of causal association. to establish whether the signal seen is associated with the vaccine and to quantify the risk, a formal epidemiological study is usually needed. this requires a pre-specified protocol detailing the population under study, the period after vaccination for which an elevated risk is suspected, and the methods for case identification and statistical analysis. most importantly, the ascertainment of the condition of interest must be unbiased with respect to vaccination history [ ] . the following statistical methods have been used most commonly to address vaccine safety questions and to control for the inherent biases in the population and data under study. although these methods aim to address confounding, it can be difficult to fully control for this in an observational study. an assessment of the likelihood of residual confounding/ bias and its potential extent is an important consideration when weighing up the strength of a study and drawing a conclusion with regard to causality. in a cohort study, the risk of developing the condition is compared in the vaccinated and unvaccinated individuals in the study population. cohort studies need to be very large to detect rare vaccine adverse events and this often makes them impractical for a prospective study. retrospective cohort designs can use routinely collected data and cases identified by clinical coding but this study design may be disadvantaged by the need to collect a large number of confounding variables. factors such as underlying illnesses, sociodemographic characteristics, and propensity to consult may differ between unvaccinated and vaccinated individuals and would therefore need to be adjusted for in the analysis as they can independently determine the likelihood of the adverse event under study. the advantage is that an entire population is studied and relative and absolute incidence estimates can be reported. in addition, once the cohort is defined, several outcomes can be assessed within the same study design. when studying a vaccine that is given as part of a national schedule and high coverage is achieved, the small unvaccinated group may differ from the vaccinated group in ways that are difficult to capture and control for in an adjusted analyses. additionally, care must be taken to ensure unvaccinated cases are indeed unvaccinated and the data are not missing. this can occur when regional vaccine datasets are used and the transfer and sharing of data are not comprehensive. cohort studies are feasible for vaccine safety studies when data from a whole country or region can be used. an example of this is in denmark where danish residents contribute to a large linked dataset consisting of demographic factors that are linked to health information including potential confounding variables [ ] . the self-controlled case-series method (sccs) was designed for rapid unbiased assessment in vaccine safety studies using available disease surveillance data that may not be amenable to cohort analysis. the method only requires information on the timing of cases during a defined observation period and their vaccination status [ ] . the cases act as their own controls as the incidence of the event in pre-defined risk periods following vaccination is compared to the incidence outside the risk period generating a relative incidence (ri) measure ( fig. ) . a significant advantage of the method is that confounding factors that do not vary over the observation period, such as co-morbidities or sociodemographic status, are automatically controlled for. adjustment for timevarying confounders such as age is also possible by dividing up the observation period further into age categories. it has been demonstrated that the power of the sccs method is nearly as good as a cohort study when uptake is high and risk intervals are short, and it is superior to that of a casecontrol study [ ] . the self-controlled case-series method has been used by public health england to address many pertinent vaccine safety concerns [ ] [ ] [ ] [ ] . this design has been chosen both because of its simplicity and ability to control for individual level confounding and also because a national cohort of cases cannot be easily defined using the national hospital data as no national immunisation register is available. unlike a cohort study, the sccs method does not provide absolute risk estimates. however, if the number of doses given to the population from which the cases are derived is known and if ascertainment is complete, then absolute risks can be estimated and the cases attributable to vaccination estimated from the magnitude of the ri. a case-control study requires smaller numbers than a cohort study but the same confounding and bias can occur and it also has the added difficulty of selecting the correct controls for comparison. for vaccinations given in the short age range in the first and second year of life or during a short calendar period to target ages, close matching of the controls on date of birth is required. prior vaccination status is then compared between cases and controls using the date of onset in cases as a reference date. to obtain enough power to assess the required risk, multiple controls per case are often needed and defining appropriate criteria for the selection of controls can be problematic. while it is important to ensure that controls are similar to cases on characteristics such as age and geographical location that can independently affect vaccination status, over matching is a risk if too many extraneous variables are included in the matching, resulting in loss of efficiency and potentially introducing bias. a case-control study does not provide absolute risk estimates, rather it measures the odds of vaccination in cases compared to controls. however, as with the sccs method, if the number of doses given to the population from which the cases are derived is known and if ascertainment is complete, then absolute risks can be estimated and the cases attributable to vaccination estimated from the magnitude of the odds ratio. the case-control design has been used where controls can be selected from the same population as cases and can be readily matched on the relevant variables. as a case-control approach is more efficient than the cohort approach, it is often used on large databases that could be used for a cohort analysis. examples include the vaccine safety datalink in the usa, which accesses complete patient records from health maintenance organisations, or studies using hospital admission data bases linked to national immunisation registers such as the australian childhood immunisation register [ ] [ ] [ ] . the case-coverage design has recently been used in vaccine safety studies [ , ] . it is similar to the screening method, which until recently has been primarily used for vaccine effectiveness assessment [ ] , although it is more limited in terms of adjustment for possible confounders than the sccs method. each case is matched to a population coverage estimate and this is then used to see if the number of cases vaccinated is greater than expected. the method uses logistic regression on the odds of vaccination with an offset for the log-odds of the matched population coverage, thus it is similar to a case-control study with thousands of controls per individual. this design has been used by public health england to assess the risk between as adjuvanted h n pandemic vaccine pandemrix™ and narcolepsy. because pandemrix™ was rolled out over a short period of time in the winter season of / targeting children of different ages according to whether they had certain co-morbid conditions, it was necessary to have detailed information on dates of vaccination and dates of birth to estimate the population coverage for each narcolepsy case by age and time period. this was available from a representative subset of general practices in england, which also provided information on co-morbidities, the only other variable considered as a potential confounder [ ] . in the first study assessing the risk of narcolepsy in children [ ] , both the sccs method and the case-coverage design was used. the results from the sccs method were found not to be clear as this method requires the incidence in a pre-specified risk period after vaccination relative to the baseline incidence to be compared. because the duration of the risk period had not been defined at the time, the postvaccination interval was found to be too short and resulted in the inclusion in the baseline period of four patients with symptoms more than months after vaccination. the choice of study design to answer a vaccine safety question will depend on the hypothesis to be tested, the available data sources and the extent to which confounding variables are likely to bias the results. the sccs method has now become the gold standard design in vaccine safety studies, owing to the benefits highlighted above, but for each study question the methods should be adapted and potential biases considered in the context of the population under study, the dataset being utilised and the hypothesis being tested. it will inevitably be a trade-off between the ideal and the practical and the best designs will vary according to setting. when many studies are performed to answer the same question, the key to demonstrating causality is consistency in the results from well-designed studies [ ] . neurological conditions have a long history of causal associations with vaccination being inferred from temporally related onsets. an example is the damage that was made to the uk whole-cell pertussis vaccination programme in the late s when neurological damage was wrongly attributed to the vaccine based on case reports of infants with onset of encephalopathy shortly after vaccination. these reports of permanent brain damage following vaccination attracted intense and sustained professional and media interest causing vaccination rates to fall from % in to % in . following this, three national epidemics of pertussis occurred with an estimated hospital admissions, cases of pneumonia, cases of convulsions and deaths [ , ] . neurological vaccine safety concerns can be broadly assigned to either being biologically plausible or unsubstantiated and unexpected. the biologically plausible group are often a direct effect from a component of the vaccine. for example, in the case of a live attenuated vaccine, the adverse reaction could mimic, at a lower frequency, what the non-attenuated wild virus would do. this is demonstrated in the rare risk of acute flaccid paralysis following the oral polio vaccine after a reversion to virulence or with the risk of aseptic meningitis after the attenuated urabe mumps strain in the measles-mumps-rubella vaccine due to retention of some neurovirulent characteristics [ , ] . the unsubstantiated and unexpected group occurs usually because of the timing of the vaccine, which coincides with the diagnosis of the condition and has no immediate biologically plausible explanation. examples of this are measles-mumps-rubella and autism [ ] , gait disturbance and measles-mumps-rubella [ ] , and thiomersal and developmental delay [ ] . although a signal may not have a clear biological basis for its causation, it still needs to be fully investigated using robust epidemiological methods. neurological diseases for which a causal association with vaccination has been suspected have some common features. first, they are often serious conditions that are rare, second, their aetiology and pathophysiology are poorly understood, and third, immune stimulation is thought to play a role in the pathogenesis of the condition. because vaccines provoke an immune response, albeit targeted to a specific antigen, it can be tempting to invoke a superficially plausible causal pathway when adverse events with a suspected immune aetiology arise shortly after vaccination. universal hepatitis b vaccine was recommended by the world health organization in the early s to protect against the hepatitis b virus, which can cause chronic liver damage and cancer. following this recommendation, france carried out a mass vaccine campaign in . shortly after, reports of cases of multiple sclerosis (ms) with onset or relapse after vaccination were reported, leading to the hypothesis that the vaccine could cause an acute autoimmune reaction in susceptible persons soon after administration. with a lack of adequate background rates of ms in the vaccinated population to put the reported cases into perspective, mistrust in the vaccine soon grew and the vaccine programme was subsequently suspended. a systematic review and meta-analysis by mouchet et al. published in that included studies with a control group found no evidence of an increased risk. the overall adjusted risk ratios for ms was . ( % confidence interval [ci] . - . ) and for central demyelination was . ( % ci . - . ) [ ] . within the systematic review, there was one study that found a significant association using a primary care database from england [ ] . this study was unable to adjust for all risk factors and additionally no routine hepatitis b vaccination programme was in place at the time with most of the vaccine delivered via occupational health departments whose records may not be routinely transferred to primary care databases. france continues to have suboptimal vaccine coverage [ , ] and has the lowest level of confidence in vaccine safety in europe [ , ] . this demonstrates the need to have robust methods in place to rapidly respond to such scares because once confidence is lost in a vaccine it is difficult to restore and may generate a more general lack of confidence in vaccine safety. guillain-barré syndrome (gbs) is the most common cause of acute neuromuscular paralysis in the developed world resulting in muscle weakness and sometimes paralysis, which can lead to respiratory failure and a death rate in up to % [ ] . the strongest evidence of a causal link with a vaccine was obtained during the us swine influenza vaccine programme in military personnel, which was found to be associated with a risk of one case per , and resulted in the suspension of the vaccine programme [ ] . since then, gbs has been a potential vaccine-associated adverse event of interest particularly for vaccines given in adolescence, an age coinciding with the age at which autoimmune diseases are often diagnosed. . - . ) . the overall relative risk for gbs after seasonal vaccine was marginally increased at . ( % ci . - . ), with a somewhat larger relative risk of . ( . - . ) for the h n pandemic vaccine but this was not significantly higher than the relative risk for seasonal vaccine [ ] . the authors did not find any statistically significant differences by geographical region nor between adjuvanted and unadjuvanted vaccines. an earlier meta-analysis of studies using the sccs method also found a small elevated risk of gbs after the monovalent h n pandemic vaccine, with an ri of . ( % ci . - . ) in the days following vaccination [ ] . similarly, salmon et al. found an ri of . ( % ci . - . ) in a large study in the usa [ ] . in contrast, a strong association between gbs and a preceding influenza-like-illness was shown in a study in england using primary care data and the sccs method. no association was seen with influenza vaccine in the - days after administration (ri . [ % ci . - . ]) but a significantly increased risk was found in the days after influenza-like illness (ri . [ % ci . - . ]) [ ] . these studies show that a small overall risk of gbs after influenza vaccine probably does exist with a slightly larger risk after the monovalent pandemic vaccine. the mechanism may be multi-factored with the risk varying with the vaccine used, co-circulation of other infections and the inherent susceptibility to developing gbs. however, the small risk that exists does not outweigh the risk of developing gbs after influenza itself. human papilloma virus vaccine is given at an age when autoimmune disorders are often diagnosed. following a french study reporting a signal for gbs after human papilloma virus vaccination, a study was conducted in england identifying gbs cases in a national hospital discharge database (hospital episode statistics) [ ] . primary care practitioners were then contacted for the vaccination history and asked to confirm gbs diagnosis and provide an onset date and send supporting documentation. in a selfcontrolled case-series analysis of cases with a record of human papilloma virus vaccination, there were episodes in the -to -day risk period after any dose with no significant increased risk, ri . ( % ci . - . ). the analysis was also stratified by manufacturer (of either the quadrivalent or bivalent product); there was no difference in the ri between products and no significant increased risk for either manufacturer. the pandemic influenza vaccine, pandemrix™, was the most widely used vaccine in europe during the pandemic. it was a monovalent h n pdm vaccine containing as , a powerful oil-in-water adjuvant. uptake of the vaccine varied between countries with high coverage of % in children in finland [ ] and lower coverage in england where children in a risk group eligible for the seasonal influenza vaccine and later all children under years of age were targeted, with uptake being % and %, respectively. in england, pandemrix™ was also used in the influenza season / because of a shortage of seasonal influenza vaccine. in august , concerns were raised in finland and sweden, where vaccine coverage was high, about a possible association between narcolepsy and pandemrix ™ when a large increase in cases of narcolepsy in vaccinated cases was reported by sleep centres [ , ] . a subsequent cohort study in finland reported a -fold increased risk of narcolepsy following pandemrix™ in children aged - years, the majority of whom had onset within months of vaccination and almost all within months [ , ] . narcolepsy was a totally unexpected adverse event and the early reports were met with initial scepticism in the global vaccine community. the world health organization global advisory committee in vaccine safety issued a statement in april stating "no excess of narcolepsy has been reported from several other european states where pandemrix was used" and "it seems likely that some as yet unidentified additional factor was operating in sweden and finland". however, it was unlikely that narcolepsy would be identified by passive surveillance systems in other countries where pandemrix™ coverage was low given the low background incidence of the condition and the complexity and frequent delays in diagnosis. to assess this risk identified in finland, the health protection agency (now public health england) performed a study in sleep centres in england where the majority of children with sleep disorders are seen. this study identified a -fold increased risk in those vaccinated with pandem-rix™ [ ] with the attributable risk estimated to be . per , doses. this demonstrated that even in a country were vaccine coverage was low, the association can be demonstrated using robust epidemiological methods. the study of the relationship between narcolepsy and pandemrix™ has been an epidemiological challenge in terms of identifying the cases and their vaccine histories in a non-biased manner. not only can the diagnosis be lengthy and complex, but admitted patient care databases, which are widely used for a non-biased ascertainment of cases in vaccine safety studies, are incomplete as patients experiencing narcolepsy may not be admitted and if they are admitted, the admission date is not an accurate reflection of the onset of the narcolepsy symptoms leading to misclassification bias. an important consideration when selecting cases is the awareness of the hypothesised association. this awareness may lead to an increased reporting of cases known to be vaccinated and has two aspects; public awareness and professional awareness. first, this heightened awareness may lead to vaccinated individuals presenting to healthcare institutions and being diagnosed earlier than unvaccinated cases leading to ascertainment bias. if a condition has an insidious onset making the recall of the first symptom difficult to determine, media attention may lead to a differential recall of the symptom-onset date in the vaccinated cases. using source documents, which were created prior to any media attention in the country of study, can address this potential recall bias. professional awareness is likely to occur even if media attention is low, as health professionals in the specialty will be aware of current topics of interest through professional bodies and literature. differential misclassification bias will occur if cases known to have been vaccinated are more likely to be assigned a diagnosis of narcolepsy than unvaccinated cases. in the study from england, public awareness of the association was assessed by analysing google searches for "narcolepsy" in the period of interest and found there was little activity in the uk compared to sweden (fig. ) [ ] . even with these practical challenges, there has now been a consistent strong association demonstrated in countries that used pandemrix™ but no association has been seen with other pandemic or seasonal vaccines [ ] . as with all vaccine safety studies, but particularly in the case of narcolepsy and pandemrix™ where the association was completely unexpected, the key to demonstrating causality was consistency of results from well-designed studies in different settings. the answer to the question of whether vaccination can cause neurological disease is multifaceted. the evidence does not support an association between ms and the hepatitis vaccine, while for gbs and influenza vaccines the evidence suggests a small increased risk though it is much smaller than the risk from a natural influenza virus infection. the now established association between narcolepsy and pan-demrix™ should act as a lesson for the vaccine safety community that sometimes unexpected but serious conditions can arise and need to be investigated rapidly however biologically implausible. the neurological vaccine safety issues outlined here demonstrate that rapid assessments of safety signals are needed to ensure that public confidence is maintained in national immunisation programmes. the confirmation of a signal and estimation of the magnitude of vaccine-attributable risk will require consistent results from a number of well-designed epidemiological studies, preferably conducted in different settings. as the experience with narcolepsy has shown, not all vaccine safety concerns can be anticipated on the basis of biologically plausible and thus predictable effects. as new vaccines are introduced, the basis of discussions on vaccine safety should be the acceptance that vaccination can carry a small risk but that this risk needs to be balanced against the enormous individual and public health benefits. funding public health england, national infection service, immunisation and countermeasures division has provided vaccine manufacturers with post-marketing surveillance reports, which the marketing authorisation holders are required to submit to the uk licensing authority in compliance with their risk management strategy. a cost recovery charge is made for these reports. world health organization. the power of vaccines: still not fully utilized recall bias, mmr, and autism vaccine safety surveillance postlicensure epidemiology of childhood vaccination: the danish experience control without separate controls: evaluation of vaccine safety using case-only methods statistical assessment of the association between vaccination and rare adverse events post-licensure autism and measles, mumps, and rubella vaccine: no epidemiological evidence for a causal association guillain-barre syndrome and h n ( ) pandemic influenza vaccination using an as adjuvanted vaccine in the united kingdom: self-controlled case series idiopathic thrombocytopenic purpura and mmr vaccine the risk of intussusception following monovalent rotavirus vaccination in england: a self-controlled case-series evaluation population-based study of rotavirus vaccination and intussusception intussusception risk and disease prevention associated with rotavirus vaccines in australia's national immunization program mmr vaccine and idiopathic thrombocytopaenic purpura risk of narcolepsy in children and young people receiving as adjuvanted pandemic a/h n influenza vaccine: retrospective analysis risk of narcolepsy after as adjuvanted pandemic a/ h n influenza vaccine in adults: a case-coverage study in england estimation of vaccine effectiveness using the screening method incidence of narcolepsy after h n influenza and vaccinations: systematic review and meta-analysis pertussis immunisation and control in england and wales, to : a historical review the pertussis vaccine controversy in great britain risk of aseptic meningitis after measles, mumps, and rubella vaccine in uk children risks of convulsion and aseptic meningitis following measlesmumps-rubella vaccination in the united kingdom autism and mmr vaccination in north london; no causal relationship no evidence of an association between mmr vaccine and gait disturbance thiomerosal exposure in infants and developmental disorders: a retrospective cohort study in the united kingdom does not support a causal association hepatitis b vaccination and the putative risk of central demyelinating diseases: a systematic review and metaanalysis. vaccine recombinant hepatitis b vaccine and the risk of multiple sclerosis: a prospective study estimates of national immunization coverage european centre for disease prevention and control. measles vaccination coverage (second dose the state of vaccine confidence : global insights through a -country survey vaccine hesitancy among general practitioners and its determinants during controversies: a national cross-sectional survey in france guillain-barre syndrome guillain-barre syndrome following vaccination in the national influenza immunization program guillain-barre syndrome and influenza vaccines: a meta-analysis international collaboration to assess the risk of guillain barre syndrome following influenza a (h n ) monovalent vaccines association between guillain-barre syndrome and influenza a (h n ) monovalent inactivated vaccines in the usa: a meta-analysis investigation of the temporal association of guillain-barre syndrome with influenza vaccine and influenzalike illness using the united kingdom general practice research database no increased risk of guillain-barre syndrome after human papilloma virus vaccine: a self-controlled case-series study in england increased incidence and clinical picture of childhood narcolepsy following the h n pandemic vaccination campaign in finland risks of neurological and immune-related diseases, including narcolepsy, after vaccination with pandemrix: a population-and registry-based cohort study with over years of follow-up as adjuvanted ah n vaccine associated with an abrupt increase in the incidence of childhood narcolepsy in finland key: cord- - g c r authors: kim, yeunbae; cha, jaehyuk title: artificial intelligence technology and social problem solving date: - - journal: evolutionary computing and artificial intelligence doi: . / - - - - _ sha: doc_id: cord_uid: g c r modern societal issues occur in a broad spectrum with very high levels of complexity and challenges, many of which are becoming increasingly difficult to address without the aid of cutting-edge technology. to alleviate these social problems, the korean government recently announced the implementation of mega-projects to solve low employment, population aging, low birth rate and social safety net problems by utilizing ai and icbm (iot, cloud computing, big data, mobile) technologies. in this letter, we will present the views on how ai and ict technologies can be applied to ease or solve social problems by sharing examples of research results from studies of social anxiety, environmental noise, mobility of the disabled, and problems in social safety. we will also describe how all these technologies, big data, methodologies and knowledge can be combined onto an open social informatics platform. a string of breakthroughs in artificial intelligence has placed ai in increasingly visible positions in society, heralding its emergence as a viable, practical, and revolutionary technology. in recent years, we have witnessed ibm's watson win first place in the american quiz show jeopardy! and google's alphago beat the go world champion, and in the very near future, self-driving cars are expected to become a common sight on every street. such promising developments spur optimism for an exciting future produced by the integration of ai technology and human creativity. ai technology has grown remarkably over the past decade. countries around the world have invested heavily in ai technology research and development. major corporations are also applying ai technology to social problem solving; notably, ibm is actively working on their science for social good initiative. the initiative will build on the success of the company's noted ai program, watson, which has helped address healthcare, education, and environmental challenges since its development. one particularly successful project used machine learning models to better understand the spread of the zika virus. using complex data, the team developed a predictive model that identified which primate species should be targeted for zika virus surveillance and management. the results of the project are now leading new testing in the field to help prevent the spread of the disease [ ] . on the other hand, investments in technology are generally mostly used for industrial and service growth, while investments for positive social impact appear to be relatively small and passive. this passive attitude seems to reflect the influence of a given nation's politics and policies rather than the absence of technology. for example, in , only . % of the total budget of the korean government's r&d of ict (information and communication technology) was used for social problem solving, but this investment will be increased to % within the next five years as the improvement of korean people's livelihoods and social problems are selected as important issues by the present government [ ] . in addition, new categories within ict, including ai, are required as a key means of improving quality of life and achieving population growth in this country. in this letter, i introduce research on the informatics platform for social problem solving, specifically based on spatio-temporal data, conducted by hanyang university and cooperating institutions. this research ultimately intends to develop informatics and convergent scientific methodologies that can explain, predict and deal with diverse social problems through a transdisciplinary convergence of social sciences, data science and ai. the research focuses on social problems that involve spatio-temporal information, and applies social scientific approaches and data-analytic methods on a pilot basis to explore basic research issues and the validity of the approaches. furthermore, ( ) open-source informatics using convergent-scientific methodology and models, and ( ) the spatio-temporal data sets that are to be acquired in the midst of exploring social problems for potential resolution are developed. in order to examine the applicability of the models and informatics platform in addressing a variety of social problems in the public as well as in private sectors, the following social problems are identified and chosen: . analysis of individual characteristics with suicidal impulse . study on the mobility of the disabled using gps data . visualization of the distribution of anxiety using social network services . big data-based analysis of noise environment and exploration of technical and institutional solutions for its improvement . analysis of the response governance regarding the middle eastern respiratory syndrome (mers) the research issues in the above social problems are explored, and the validity of the convergent-scientific methodologies are tested. the feasibility for the potential resolution of the problems are also examined. the relevant data and information are stored in a knowledge base (kb), and at the same time research methods that are used in data extraction, collection, analysis and visualization are also developed. furthermore, the kb and the method database are merged into an open informatics platform in order to be used in various research projects, business activities, and policy debates. while suicide rates in oecd countries are declining, only south korea has increasing suicide rates; moreover, korea currently has the highest suicide rate among oecd countries as shown in fig. . its high suicide rate is one of korea's biggest social problems, entailing the establishment of effective suicide prevention measures by understanding the causes of suicide. the goals of the research are to: ( ) understand suicidal impulse by analyzing the characteristics of members of society according to suicidal impulse experience; ( ) predict the likelihood of attempting suicide and analyzing the spatio-temporal quality of life; and ( ) to establish a policy to help prevent suicide. the korean social survey and survey of youth health status data are used for the analysis of suicide risk groups through data mining techniques, using a predictive model based on cell propagation to overcome the limitations of existing statistics methods such as characterization or classification. in the case of the characterization technique, results indicate that there are too many features related to suicide, and that there are variables including many categorical values, making it difficult to identify the variables that affect suicide. on the other hand, the classification technique had difficulties identifying the variables that affect suicide because the number of members attempting suicide was too small. correlations between suicide impulses and individual attributes of members of society and the trends of the correlations by year are obtained. the concepts of support, confidence and density are introduced to identify risk groups of suicide attempts, and computational performance problems caused by excessive numbers of risk groups are solved by applying a convex growing method. the y social survey including personal and household information of members of the society are used for analysis. the attributes include gender, age, education, marital status, level of satisfaction, disability status, occupation status, housing, and household income. the high-risk suicide cluster was identified using a small number of convexes. a convex is a set of cells, with one cell being the smallest unit of the cluster for the analysis, and a density is the ratio of the number of non-empty cells to the total number of cells in convex c [ ] . figure shows that the highest suicidal risk group c is composed of members with low income and education level. it was identified that level of satisfaction with life has the highest impact on suicidal impulse, followed in order of impact by disability, marital status, housing, household income, occupation status, gender, age and level of education. the results showed that women and young people tend to have more suicidal impulse. new prediction models with other machine learning methods and the establishment of mitigation policies are still in development. subjective analyses of change of wellbeing, social exclusion, and characteristics of spatio-temporal analysis will also be explored in the future. mobility rights are closely related to quality of life as a part of social rights. therefore social efforts are needed to guarantee mobility rights to both the physically and mentally disabled. the goal of the study is to suggest a policy for the extension of mobility rights of the disabled. in order to achieve this, travel patterns and sociodemographic characteristics of the physically impaired with low levels of mobility are studied. the study focused on individuals with physical impairments as the initial test group as a means to eventually gain insight into the mobility of the wider disabled population. conventional studies on mobility measurement obtained data from travel diaries, interviews, and questionnaire surveys. a few studies used geo-location tracking gps data. gps data is collected via mobile device and used to analyze the mobility patterns (distance, speed, frequency of outings) by using regression analysis, and to search for methods to extend mobility. a new metrics for mobility with a new indicator (travel range) was developed, and the way mobility impacts the quality of life of the disabled has been verified [ ] . about people with physical disabilities participated and collected more than , geo-location data over a month using an open mobile application called traccar. their trajectories are visualized based on the gps data as shown in fig. . the use of location data explained mobility status better than the conventional questionnaire survey method. the questionnaire surveyed mainly the frequency of outings over a certain period and number of complaints about these outings. gps data enabled researchers to conduct empirical observations on distance and range of travel. it was found that the disabled preferred bus routes that visit diverse locations over the shortest route. age and monthly income are negatively associated with a disabled individual's mobility. based on the research results, the following has been suggested: ( ) development of new bus routes for the disabled and ( ) recommendation of a new location for the current welfare center that would enable a greater range of travel. further study on travel patterns by using indoor positioning technology and cctv image data will be deployed. many social issues including political polarization, competition in private education, increases in suicide rate, youth unemployment, low birth rate, and hate crime have anxiety as their background. the increase of social anxiety can intensify competition and conflict, which can interfere with social solidarity and cause a decrease in social trust. existing social science research mainly focused on grasping public opinion through questionnaires, and ignored the role of emotions. the internet and social media were used to access emotional traits since they provide a platform not only for the active exchange of information, but also for the sharing and diffusion of emotional responses. if such emotional responses on the internet and geo-locations can be captured in realtime through machine learning, their spatio-temporal distribution could be visualized in order to observe their current status and changes by geographical region. a visualization system was built to map the regional and temporal distribution of anxiety psychology by combining spatio-temporal information using sns (twitter) with sentiment analysis. a twitter message collecting crawler was also developed to build a dictionary and tweet corpus. based on these, an automatic classification system of anxiety-related messages was developed for the first time in korea by applying machine learning to visualize the nationwide distribution of anxiety (see fig. ) [ ] . an average of , tweets with place_id are collected using open api twitter j. to date, about , units of data have been collected. a naïve bayes classifier was used for anxiety identification. an accuracy of . % was obtained by using , and , anxiety and non-anxiety tweets as training data respectively, and and , anxiety and non-anxiety tweets as testing data, respectively. the system indicated the existence of regional disparities in anxiety emotions. it was found that twitter users who reside in politicized regions have a lower degree of disclosure about their residing areas. this can be interpreted as the act of avoiding situations where the individual and the political position of the region coincide. as anxiety is not a permanent characteristic of an individual, it can change depending on the time and situation, making it difficult to measure by questionnaire survey at any given time. the twitter-based system can compensate for the limitations of such a survey method because it can continuously classify accumulated tweet text data and provide a temporal visualization of anxiety distribution at a given time within a desired visual scale (by ward, city, province and nationwide) as shown in fig. . environmental issues are a major social concern in our age, and interest has been increasing not only in the consequences of pollution but also in the effects of general environmental aesthetics on quality of life. there is much active effort to improve the visual environment, but not nearly as much interest has been given to improve the auditory environment. until now, policies on the auditory environment have remained passive countermeasures to simply quantified acoustic qualities (e.g., volume in db) in specific places such as construction sites, railroads, highways, and residential areas. they lack a comprehensive study of contextual correlations, such as the physical properties of sound, the environmental factors in time and space, and the human emotional response of noise perception. the goal of this study is to provide a cognitive-based, human-friendly solution to improve noise problems. in order to achieve this, the study aimed to ( ) develop a tool for collecting sound data and converting into a sound database, and ( ) build spatiotemporal features and a management platform for indoor and outdoor noise sources. first, pilot experiments were conducted to predict the indicators that measure emotional reactions by developing a handheld device application for data collection. three separate free-walking experiments and in-depth interviews were conducted with subjects at international airport lobbies and outdoor environments. through the experiment, the behavior patterns of the subjects in various acoustic environments were analyzed, and indicators of emotional reactions were identified. it was determined that the psychological state and the personal environment of the subject are important indicators of the perception of the auditory environment. in order to take into account both the psychological state of the subject and the physical properties of the external sound stimulus, an omnidirectional microphone is used to record the entire acoustic environment. subjects with smartphones with the built-in application walked for an hour in downtown seoul for data collection. on the app, after entering the prerequisite information, subjects pressed 'good' or 'bad' whenever they heard a sound that caught their attention. pressing the button would record the sound for s, and subjects were additionally asked to answer a series of questions about the physical characteristics of the specific location and the characteristics of the auditory environment. during the one-hour experiment, about sound environment reports were accumulated, with one subject reporting the sound characteristics from an average of different places. unlike previous studies, the subjects' paths were not pre-determined, and the position, sound and emotional response of the subject are collected simultaneously. the paths can be displayed to analyze the relations of the soundscapes to the paths (fig. ) . the study helped to build a positive auditory environment for specific places, to provide policy data for noise regulation and positive auditory environments, to identify the contexts and areas that are alienated from the auditory environment, and to extend the social meaning of "noise" within the study of sound. respiratory syndrome (mers) the development and spread of new infectious diseases are increasing due to the expansion of international exchange. as can be seen from the mers outbreak in korea in , epidemics have profound social and economic impacts. it is imperative to establish an effective shelter and rapid response system (rrs) for infectious diseases control. the goal of the study is to compare the official response system with the actual response system in order to understand the institutional mechanism of the epidemic response system, and to find effective policy alternatives through the collaboration of policy scholars and data scientists. web-based newspaper articles were analyzed to compare the official crisis response system designed to operate in outbreaks to the actual crisis response. an automatic news article crawling tool was developed, and , mers-related articles were collected, clustered and stored in the database (fig. ) . in order to manage and search for news articles related to mers from the article database, a curation tool was developed. this tool is able to extract information into triplet graphs (subjects/verbs/objects) from the articles by applying natural language processing techniques. a basic dictionary for the analysis of the infectious disease response system was created based on the extracted triplet information. the information extracted by the curation tool is massive and complex, which limits the ability to correctly understand and interpret information. a tool for visualizing information at a specific time with a network graph was developed and utilized to facilitate analysis and visualization of the networks (fig. ). all tools are integrated into a single platform to maximize the efficiency of the process. as for the official crisis response manual in case of an infectious disease, social network analysis indicated that while the national security bureau (nsb) and public health centers play as large a role as the center for disease control (cdc) in crisis management, the analysis of the news articles showed that the nsb was in fact rarely mentioned. it was found that the cdc and central disaster response headquarters, the official government organizations that deal with infectious diseases, as well as the central mers management countermeasures & support headquarters, a temporarily established organization, were not playing an important role in response to the mers outbreak. on the other hand, the ministry of health and welfare, medical institutions, and local governments all have played a central role in responding to mers. this means that the structure and characteristics of the command & control and communication in the official response system seems to have a decisive influence on the cooperative response in a real crisis response. these results provided concrete information on the role of each respondent and the communication system that previous studies based on interviews and surveys have not found. much research based on machine learning has been criticized for giving more importance on method itself from the start rather than focusing on data reliability. this study is based on a kb in which policy researchers manually analyze news articles and prepare basic data by tagging them. this way, it provides a basis for improving the reliability of results when executing text mining work through machine learning. by using text mining techniques and social network analysis, it is possible to get a comprehensive view of social problems such as the occurrence of infectious diseases by examining the structure and characteristics of the response system from a holistic perspective of the entire system. with the results of this study, new policies for infectious disease control are suggested in the following directions: ( ) strengthen cooperation networks in early response systems of infectious diseases; ( ) develop new, effective and efficient management plans of cooperative networks; and ( ) create new research to cover other diseases such as avian influenza and sars [ ] . an ever-present obstacle in the traditional social sciences when addressing social issues are the difficulties of obtaining evidences from massive data for hypothesis and theory verification. data science and ai can ease such difficulties and support social science by discovering hidden contexts and new patterns of social phenomena via low-cost analyses of large data. on the other hand, knowledge and patterns derived by machine learning from a large data set with noise often lack validity. although data-driven inductive methods are effective for finding patterns and correlations, there is a clear limitation to discovering causal relationships. social science can help data science and ai by interpreting social phenomena through humanistic literacy and social-scientific thought to verify theoretical validity, and identifying causal relationships through deductive and qualitative approaches. this is why we need convergent-scientific approaches for social problem solving. convergent approaches offer the new possibility of building an informatics platform that can interpret, predict and solve various social problems through the combination of social science and data science. in all pilot studies, the convergent-scientific approaches are found valid and sound. most of the research agendas involved the real-time collection and development of spatio-temporal databases in a real-time manner, and analytic visualization of the results. such visualization promises new possibilities in data interpretation. the data sets and tools for data collection, analysis and visualization are integrated onto an informatics platform so that they can be used in future research projects and policy debates. the research was the first transdisciplinary attempt to converge social sciences and data sciences in korea. this approach will offer a breakthrough in predicting, preventing and addressing future social problems. the research methodology, as a trailblazer, will offer new ground for a research field of a transdisciplinary nature converging data sciences, ai and social sciences. the data, information, knowledge, methodologies, and techniques will all be combined onto an open informatics platform. the platform will be maintained on an open-source basis so that it can be used as a hub for various academic research projects, business activities, and policy debates (see fig. ). the open informatics platform is planned to be expanded to incorporate citizen sensing, in which people's observations and views are shared via mobile devices and internet services in the future [ ] . in the area of social problem solving, fundamental problems have complex political, social and economic aspects that have their roots in human nature. both technical and social approaches are essential for tackling social problem solving. in fact, it is the fig. . structure of informatics platform integrated, orchestrated marriage between the two that would bring us closer to effective social problem management. we need to first study and carefully define the indicators specific to a given social problem or domain. there are many qualitative indicators that cannot be directly and explicitly measured such as social emotions, basic human needs and rights, and life fulfillment [ ] . if the results of machine learning are difficult to measure or include combinations of results that are difficult to define, that particular social problem may not be suitable for machine learning. therefore, there is a need for new social methods and algorithms that can accurately collect and identify the measurable indicators from opinions of social demanders. recently, mit has developed a device to quantitatively measure social signals. the small, lightweight wearable device contains sensors that record the people's behaviors (physical activity, gestures, and the amount of variation in speech prosody, etc.) [ ] . machine learning technologies working on already existing data sets are relatively inexpensive compared to conventional million-dollar social programs since machine learning tools can be easily extended. however, they can introduce bias and errors depending on the data content used to train machine learning models or can also be misinterpreted. human experts are always needed to recognize and correct erroneous outputs and interpretations in order to prevent prejudices [ ] . in the development of ai applications, a great amount of time and resources are required to sort, identify and refine data to provide massive data for training. for instance, machine learning models need to learn millions of photos to recognize specific animals or faces, but human intelligence is able to recognize visual cues by looking at only a few photos. perhaps it is time to develop a new ai framework which can infer and recognize objects based on small amounts of data, such as transfer learning [ ] , generate lacking data (gan), or integrate traditional ai technologies, such as symbolic ai and statistical machine learning into new frameworks. machine learning is excellent in predicting, but many social problem solutions do not depend on predictions. the organic ways solutions to specific problems actually unfold according to new policies and programs can be more practical and worth studying than building a cure-all machine learning algorithm. while the evolution of ai is progressing at a stunning rate, there are still challenges to solving social problems. further research on the integration of social science and ai is required. a world in which artificial intelligence actually makes policy decisions is still hard to imagine. considering the current limitations and capabilities of ai, ai should primarily be used as a decision aid. ibm: science for social good -applying ai, cloud and deep science toward new societal challenges analyzing suicide-ideation survey to identify high-risk groups: a data mining approach mobility among people with physical impairment: a study using geo-location tracking data sns data visualization for analyzing spatial-temporal distribution of social anxiety application of network analysis into emergency response: focusing on the outbreak of the middle-eastern respiratory syndrome in korea a platform for citizen sensing in sentient cities chapter : the sociology of social indicators to signal is human a guide to solving social problems with machine learning how transferable are features in deep neural networks? in: advances in neural information processing systems (nips ). nips foundation acknowledgements. this work was supported by the national research foundation of korea (nrf) grant funded by the korean government (msit ) (no. r a a ). key: cord- -zyjd rmp authors: peixoto, tiago p. title: network reconstruction and community detection from dynamics date: - - journal: nan doi: . /physrevlett. . sha: doc_id: cord_uid: zyjd rmp we present a scalable nonparametric bayesian method to perform network reconstruction from observed functional behavior that at the same time infers the communities present in the network. we show that the joint reconstruction with community detection has a synergistic effect, where the edge correlations used to inform the existence of communities are also inherently used to improve the accuracy of the reconstruction which, in turn, can better inform the uncovering of communities. we illustrate the use of our method with observations arising from epidemic models and the ising model, both on synthetic and empirical networks, as well as on data containing only functional information. the observed functional behavior of a wide variety largescale system is often the result of a network of pairwise interactions. however, in many cases, these interactions are hidden from us, either because they are impossible to measure directly, or because their measurement can be done only at significant experimental cost. examples include the mechanisms of gene and metabolic regulation [ ] , brain connectivity [ ] , the spread of epidemics [ ] , systemic risk in financial institutions [ ] , and influence in social media [ ] . in such situations, we are required to infer the network of interactions from the observed functional behavior. researchers have approached this reconstruction task from a variety of angles, resulting in many different methods, including thresholding the correlation between time series [ ] , inversion of deterministic dynamics [ ] [ ] [ ] , statistical inference of graphical models [ ] [ ] [ ] [ ] [ ] and of models of epidemic spreading [ ] [ ] [ ] [ ] [ ] [ ] , as well as approaches that avoid explicit modeling, such as those based on transfer entropy [ ] , granger causality [ ] , compressed sensing [ ] [ ] [ ] , generalized linearization [ ] , and matching of pairwise correlations [ , ] . in this letter, we approach the problem of network reconstruction in a manner that is different from the aforementioned methods in two important ways. first, we employ a nonparametric bayesian formulation of the problem, which yields a full posterior distribution of possible networks that are compatible with the observed dynamical behavior. second, we perform network reconstruction jointly with community detection [ ] , where, at the same time as we infer the edges of the underlying network, we also infer its modular structure [ ] . as we will show, while network reconstruction and community detection are desirable goals on their own, joining these two tasks has a synergistic effect, whereby the detection of communities significantly increases the accuracy of the reconstruction, which in turn improves the discovery of the communities, when compared to performing these tasks in isolation. some other approaches combine community detection with functional observation. berthet et al. [ ] derived necessary conditions for the exact recovery of group assignments for dense weighted networks generated with community structure given observed microstates of an ising model. hoffmann et al. [ ] proposed a method to infer community structure from time-series data that bypasses network reconstruction by employing a direct modeling of the dynamics given the group assignments, instead. however, neither of these approaches attempt to perform network reconstruction together with community detection. furthermore, they are tied down to one particular inverse problem, and as we will show, our general approach can be easily extended to an open-ended variety of functional models. bayesian network reconstruction.-we approach the network reconstruction task similarly to the situation where the network edges are measured directly, but via an uncertain process [ , ] : if d is the measurement of some process that takes place on a network, we can define a posterior distribution for the underlying adjacency matrix a via bayes' rule where pðdjaÞ is an arbitrary forward model for the dynamics given the network, pðaÞ is the prior information on the network structure, and pðdÞ ¼ p a pðdjaÞpðaÞ is a normalization constant comprising the total evidence for the data d. we can unite reconstruction with community detection via an, at first, seemingly minor, but ultimately consequential modification of the above equation where we introduce a structured prior pðajbÞ where b represents the partition of the network in communities, i.e., b ¼ fb i g, where b i ∈ f ; …; bg is group membership of node i. this partition is unknown, and is inferred together with the network itself, via the joint posterior distribution the prior pðajbÞ is an assumed generative model for the network structure. in our work, we will use the degreecorrected stochastic block model (dc-sbm) [ ] , which assumes that, besides differences in degree, nodes belonging to the same group have statistically equivalent connection patterns, according to the joint probability with λ rs determining the average number of edges between groups r and s and κ i the average degree of node i. the marginal prior is obtained by integrating over all remaining parameters weighted by their respective prior distributions, which can be computed exactly for standard prior choices, although it can be modified to include hierarchical priors that have an improved explanatory power [ ] (see supplemental material [ ] for a concise summary.). the use of the dc-sbm as a prior probability in eq. ( ) is motivated by its ability to inform link prediction in networks where some fraction of edges have not been observed or have been observed erroneously [ , ] . the latent conditional probabilities of edges existing between groups of nodes is learned by the collective observation of many similar edges, and these correlations are leveraged to extrapolate the existence of missing or spurious ones. the same mechanism is expected to aid the reconstruction task, where edges are not observed directly, but the observed functional behavior yields a posterior distribution on them, allowing the same kind of correlations to be used as an additional source of evidence for the reconstruction, going beyond what the dynamics alone says. our reconstruction approach is finalized by defining an appropriate model for the functional behavior, determining pðdjaÞ. here, we will consider two kinds of indirect data. the first comes from a susceptible-infected-susceptible (sis) epidemic spreading model [ ] , where σ i ðtÞ ¼ means node i is infected at time t, , otherwise. the likelihood for this model is where is the transition probability for node i at time t, with fðp; σÞ ¼ ð − pÞ σ p −σ , and where m i ðtÞ ¼ p j a ij lnð − τ ij Þσ j ðtÞ is the contribution from all neighbors of node i to its infection probability at time t. in the equations above, the value τ ij is the probability of an infection via an existing edge ði; jÞ, and γ is the → recovery probability. with these additional parameters, the full posterior distribution for the reconstruction becomes since τ ij ∈ ½ ; , we use the uniform prior pðτÞ ¼ . note, also, that the recovery probability γ plays no role on the reconstruction algorithm, since its term in the likelihood does not involve a [and, hence, gets cancelled out in the denominator pðσjγÞ ¼ pðγjσÞpðσÞ=pðγÞ]. this means that the above posterior only depends on the infection events → and, thus, is also valid without any modifications to all epidemic variants susceptible-infected (si), susceptibleinfected-recovered (sir), susceptible-exposed-infectedrecovered (seir), etc., [ ] , since the infection events occur with the same probability for all these models. the second functional model we consider is the ising model, where spin variables on the nodes s ∈ f− ; g n are sampled according to the joint distribution where β is the inverse temperature, j ij is the coupling on edge ði; jÞ, h i is a local field on node i, and zða; β; j; hÞ ¼ p s expðβ p i<j j ij a ij s i s j þ p i h i s i Þ is the partition function. note that this is not a dynamical model as each microstate s is sampled independently according to the above distribution. unlike the sis model considered before, this distribution cannot be written in closed form since zða; β; j; hÞ cannot be computed exactly, rendering the reconstruction problem intractable. therefore, instead, we make use of the pseudolikelihood approximation [ ] , which is very accurate for the purpose at hand [ ] , where we approximate eq. ( ) as a product of (properly normalized) conditional probabilities for each spin variable s i pðsja; β; j; hÞ ¼ in the above, we use uniform priors pðjjaÞ ¼ q ij ½− = < j ij < = a ij , thus, forcing, without loss of generality, the values of j ij to lie in the shifted unit interval ½− = ; = . for the remaining parameters, we use uniform priors, pðhÞ ∝ and pðβÞ ∝ , for β ∈ ½−∞; ∞ and h ∈ ½−∞; ∞ n . for any of the above posterior distributions, we perform sampling using a markov chain monte carlo procedure: for each proposal a → a , it is accepted with the metropolis-hastings probability [ , ] and likewise, for the node partition b → b , and any of the remaining parameters θ → θ . note that the acceptance probability does not require the intractable normalization constant pðdÞ to be computed. for both functional models considered, a whole sweep over e entries of the adjacency matrix and n nodes is done in time oðem þ nhkiÞ, where m is the number of data samples per node, allowing the method to be applied for large systems. we summarize and give more details about the technical aspects of the algorithm in the supplemental material [ ] . synthetic networks.-we begin by investigating the reconstruction performance of networks sampled from the planted partition (pp) model, i.e., a dc-sbm with κ i ¼ , λ rs ¼ λ in δ rs þ λ out ð − δ rs Þ, with λ in ¼ hki½ þ ϵðb − Þ=n and λ out ¼ hkið − ϵÞ=n, where ϵ ¼ nðλ in − λ out Þ=hkib controls the strength of the modular structure. the detectability threshold for this model is given by , below which it is impossible to recover the planted community structure [ ] . given a network a à from this model, we sample m independent ising microstates s according to eq. ( ) using j ij ¼ , h i ¼ , and β ¼ β à being the critical inverse temperature for the particular network. we compare two inference approaches: in the first, we sample both the reconstructed network as well as its community structure form the joint posterior of eq. ( ). in the second approach, we perform reconstruction and community detection separately, by first performing reconstruction in isolation, by replacing the dc-sbm prior pðajbÞ by the likelihood of an erdős-rényi model. we evaluate the quality of the reconstruction via the posterior similarity s ∈ ½ ; , defined as where a à is the true network and π is the marginal posterior probability for each edge, i.e., π ij ¼ p a;b;θ a ij pða; b; θjdÞ. a value s ¼ means perfect reconstruction. we then perform community detection a posteriori by obtaining the maximum marginal point estimate and then sampling from the posterior pðbjÂÞ. figure contains the comparison between both approaches for networks sampled from the pp model, which shows how sampling from the joint posterior improves both the reconstruction as well as community detection. for the latter, the joint inference allows the detection all the way down to the detectability threshold, for the examples considered, which, otherwise, is not possible with the separate method. real networks with synthetic dynamics.-now, we investigate the reconstruction of networks not generated by the dc-sbm. we take two empirical networks, the with e ¼ edges, and a food web from little rock lake [ ] , containing n ¼ nodes and e ¼ edges, and we sample from the sis (mimicking the spread of a pandemic) and ising model (representing simplified interspecies interactions) on them, respectively, and evaluate the reconstruction obtained via the joint and separate inference with community detection, with results shown in fig. . as is also the case for synthetic networks, the reconstruction quality is significantly improved by performing joint community detection [ ] . the quality of the reconstruction peaks at the critical threshold for each model, at which the sensitivity to perturbations is the largest. as the number of observed samples increases, so does the quality of the reconstruction, and the relative advantage of the joint reconstruction diminishes, as the data eventually "washes out" the contribution from the prior. for the ising model, we compare the results of our method with the mean-field inverse correlations method [ ] , i.e., βa ij j ij ¼ ½c − ij , where c ij ¼ hσ i σ j i − hσ i ihσ j i is the connected correlation matrix. as seen in fig. , this simpler reconstruction method can be just as accurate as our separate reconstruction approach, but only close to the critical point. for higher inverse temperatures, the reconstruction deteriorates rapidly and breaks down completely as the system becomes locally magnetized, with whole rows and columns of the matrix c being equal to zero, causing it to be singular [ ] . in such situations, this kind of approach requires explicit regularization techniques [ ] , which become unnecessary with our bayesian method. the joint inference with community structure improves the reconstruction even further, beyond what is obtainable with typical inverse ising methods, since it incorporates a different source of evidence. in fig. , we show a comparison of the reconstruction of the food web network from a simulated ising model, using different approaches. optimal thresholding corresponds to the naive approach of imputing the existence of an edge to the connected correlation between two nodes exceeding a threshold c à , i.e., π ij ¼ f if c ij > c à ; ; otherwiseg. the value of c à was chosen to maximize the posterior similarity, which represents the best possible reconstruction achievable with this method. nevertheless, the network thus obtained is severely distorted. the inverse correlation method comes much closer to the true network, but is superseded by the joint inference with community detection. empirical dynamics.-we turn to the reconstruction from observed empirical dynamics with unknown underlying interactions. the first example is the sequence of m ¼ votes of n ¼ deputies in the to session of the lower chamber of the brazilian congress. each deputy voted yes, no, or abstained for each legislation, which we represent as f ; − ; g, respectively. since the temporal ordering of the voting sessions is likely to be of secondary importance to the voting outcomes, we assume the votes are sampled from an ising model [the addition of zero-valued spins changes eq. ( ) only slightly by replacing coshðxÞ → þ coshðxÞ]. figure shows the result of the reconstruction, where the division of the nodes uncovers a cohesive government and a split opposition, as well as a marginal center group, which correlates very well with the known party memberships and can be used to predict unseen voting behavior (see supplemental material [ ] for more details). in fig. , we show the result of the reconstruction of the directed network of influence between n ¼ twitter users from retweets [ ] using a si epidemic model (the act of "retweeting" is modeled as an infection event, using eqs. ( ) and ( ) with γ ¼ ) and the nested dc-sbm. the reconstruction uncovers isolated groups with varying propensities to retweet, as well as groups that tend to influence a large fraction of users. by inspecting the geolocation metadata on the users, we see that the inferred groups amount, to a large extent, to different countries, although clear subdivisions indicate that this is not the only factor governing the influence among users (see supplemental material [ ] for more details). conclusion.-we have presented a scalable bayesian method to reconstruct networks from functional observations that uses the sbm as a structured prior and, hence, performs community detection together with reconstruction. the method is nonparametric and, hence, requires no prior stipulation of aspects of the network and size of the model, such as number of groups. by leveraging inferred correlations between edges, the sbm includes an additional source of evidence and, thereby, improves the reconstruction accuracy, which in turn also increases the accuracy of the inferred communities. the overall approach is general, requiring only appropriate functional model specifications, and can be coupled with an open ended variety of such models other than those considered here. [ , ] for details on the layout algorithm), and the edge colors indicate the infection probabilities τ ij as shown in the legend. the text labels show the dominating country membership for the users in each group. inferring gene regulatory networks from multiple microarray datasets dynamic models of large-scale brain activity estimating spatial coupling in epidemiological systems: a mechanistic approach bootstrapping topological properties and systemic risk of complex networks using the fitness model the role of social networks in information diffusion network inference with confidence from multivariate time series revealing network connectivity from response dynamics inferring network topology from complex dynamics revealing physical interaction networks from statistics of collective dynamics learning factor graphs in polynomial time and sample complexity reconstruction of markov random fields from samples: some observations and algorithms, in approximation, randomization and 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authors: bhattacharyya, sankar title: inflammation during virus infection: swings and roundabouts date: - - journal: dynamics of immune activation in viral diseases doi: . / - - - - _ sha: doc_id: cord_uid: yigz qiz inflammation constitutes a concerted series of cellular and molecular responses that follow disturbance of systemic homeostasis, by either toxins or infectious organisms. leukocytes modulate inflammation through production of secretory mediators, like cytokines and chemokines, which work in an autocrine and/or paracrine manner. these mediators can either promote or attenuate the inflammatory response and depending on differential temporal and spatial expression play a crucial role in the outcome of infection. even though the objective is clearance of the pathogen with minimum damage to host, the pathogenesis of multiple human pathogenic viruses has been suggested to emanate from a dysregulation of the inflammatory response, sometimes with fatal consequences. this review discusses the nature and the outcome of inflammatory response, which is triggered in the human host subsequent to infection by single-sense plus-strand rna viruses. in view of such harmful effects of a dysregulated inflammatory response, an exogenous regulation of these reactions by either interference or supplementation of critical regulators has been suggested. currently multiple such factors are being tested for their beneficial and adverse effects. a successful use of such an approach in diseases of viral etiology can potentially protect the affected individual without directly affecting the virus life cycle. further, such approaches whenever applicable would be useful in mitigating death and/or debility that is caused by the infection of those viruses which have proven particularly difficult to control by either prophylactic vaccines and/or therapeutic strategies using specific antiviral drugs. the mammalian immune system has evolved arsenal and strategies to make a distinction between microbes that are either beneficial or benign or bad, an integral part of which is differential treatment of "self" and "non-self." whereas recognition of "self" as "non-self" can cause autoimmunity, the converse results in microbial colonization. in fact the human gut does harbor multiple variety of microbes as natural part of the biological ecosystem (scarpellini et al. ). the recognized non-self are counteracted by adaptive and innate effectors of the immune system, using dedicated cells and biochemicals, which attempt to restrict the growth and impede colonization by the pathogen. the innate response is nonspecific, while the secondary adaptive response is specific for the pathogen or closely related species. the cellular component includes innate immune cells like the monocytes/macrophages, neutrophils, and natural killer (nk) cells and adaptive immune cells like b-and t-lymphocytes, which coordinate for an effective response. cytokines are a dedicated group of biochemicals involved in this coordination and include interferons (ifns), interleukins (ils), and chemokines that are responsible for synchronizing the initiation, regulation, and termination of an immune response. a group (~ ) of small polypeptides (< kda) produced predominantly although not exclusively by immune cells like macrophages and lymphocytes, cytokines are secreted out exerting their function by engaging respective cell-surface receptors and depending on biological function are labeled as either pro-inflammatory (pic) or antiinflammatory (aic) cytokines (turner et al. ). on the one hand, several cytokines are functionally redundant, and on the other hand, some cells can express receptors for multiple cytokines. viruses with positive-sense single-stranded rna as genome can either be enveloped (togaviridae, flaviviridae, and coronaviridae) or non-enveloped (astroviridae, caliciviridae, and picornaviridae), and several from either group cause severe human pathology (fields et al. ) . entry into human host can be by diverse means including mucosal contact (gut in enteroviruses) or vectorial inoculation (e.g., in dengue and jev) or parenteral blood transfer (e.g., hepatitis c virus). immobilization by interaction with extracellular matrix components like glycosaminoglycan is followed by tropism determinant cognate receptor-mediated entry (chen et al. ; olenina et al. ; tan et al. ) . in enveloped viruses, the envelope fuses with the endosomal membrane, while non-enveloped viruses breach the membrane of either the cell or the endosome using specific cofactors, ultimately releasing viral genome into the host cytosol (kumar et al. ; plemper ) . a culmination of the following steps results in direct translation of the genomic rna to produce a polyprotein, which is cleaved by virus-derived and host-origin proteases to yield the multiple structural and nonstructural proteins (fields et al. although viruses can replicate in multiple types of cells, the pathological outcome manifests in only one or a few specific cell/tissue types. independent of organismal entry site, the likeliest primary encounter of a virus is with mononuclear-phagocytic cells like monocytes, macrophages (mϕ), and dendritic cells (dcs). mϕ and dcs are tissue-localized cells constituting the first line of cellular defense, which when infected can undertake antiviral steps in addition to "informing" the other effectors of the innate and adaptive immune system (pohl et al. ; ginhoux and jung ) . activated dcs shift to lymph nodes, process viral antigen, and "present" or display it for clonal expansion of cognate lymphocytes population. mϕ, which can be either tissue-resident or differentiated from afferent monocytes postinfection, play a more regulatory role and are important determinants of the outcome of the inflammatory response (ginhoux and jung ; mercer and greber ; hou et al. ; schulz et al. ) . tissue-resident mϕ , which are a distinct population from monocyte-derived ones, include microglial cells in cns, liver kupffer cells, skin langerhans cells, etc. (davies et al. ) monocytes/mϕ (and many other cell types) express molecular detector proteins called pattern recognition receptors (prrs), specialized for interacting with signature motifs on microbe-derived molecules, termed as pathogen-associated molecular pattern (pamp). viral pamps include double-stranded (dsrna) rna (replication-intermediate formed during replication) and ′-ppp (uncapped genomic rna polymerized by de novo replication). cellular prrs specific for these include toll-like receptors (tlrs) like tlr (dsrna) and rig-i-like receptors (rlrs) like rig-i, mda (dsrna, ′-ppp end on rna) (jensen and thomsen ) . nod-like receptors or nlrs form another class of cytosolic prrs that can detect virus infection, albeit in an indirect manner (takeuchi and akira ; ichinohe et al. ) . physical engagement with pamps activates the respective prrs, stimulating alterations in conformation of these sensors that allow them to interact with adapter molecules mediating the assembly of multi-protein complexes called inflammosome, in parallel to activating the expression of cytokine genes coding for type- interferons (ifns) and nfκb target genes (kawai et al. ; pichlmair and reis e sousa ; chen and ichinohe ; seth et al. ) . secreted type-i ifns attach specific receptors, in a paracrine or autocrine manner, thereby activating the expression of many interferon-sensitive genes (isgs) with diverse functions that confer antiviral property to their activity (schneider et al. ; schoggins and rice ) . isgs include prr-coding genes producing a feed-forward loop and aggravating inflammation. in parallel, nfκb enhances expression of pro-inflammatory genes like tnf-α, il- β, cox , il , il- p , or il- besides components of nlrp (tak and firestein ; bauernfeind et al. ). upon assembly the nlrp inflammosome catalyzes caspase- activation, a protease which slices the precursor form of pleiotropic pro-inflammatory cytokines like il- β and il- generating their active secreted forms (garlanda et al. ; biet et al. ) . il- β potentiates the antiviral response by multiple ways in addition to inducing expression of type-i ifns and isgs in dcs (ben-sasson et al. ; aarreberg et al. ) . chemokines (chemotactic cytokines) flag/point to the site of infection by a concentration gradient, attracting leukocytes like neutrophils, monocytes, and lymphocytes, subsequently activating them to release more cytokines thereby amplifying the inflammatory response (sokol and luster ; ley ) . among these il- and il- (produced predominantly by dcs) have crucial immunomodulatory functions. il- attracts cd + t-helper (th) cells influencing their differentiation into ifn-γ secreting th cells in addition to augmenting the cytotoxic activity of cd + t cells and nk cells (athie- morales et al. ; henry et al. ). il- on the other hand increases nk-cell sensitivity to il- by receptor upregulation (wang et al. ) . ifn-γ which in contrast to type-i ifns is produced exclusively by immune cells (t and nk cells) has pleiotropic antiviral effect including the capacity to polarize existing or newly recruited mϕ to m phenotype (hu and ivashkiv ; verreck et al. ). mϕ either resident or monocyte-derived can acquire either an m or an m phenotype differing in ontology, phenotype, and secretome, with unidirectional plasticity from m to m (halstead et al. ; guiducci et al. ; smith et al. ). m -mϕ promotes a th immune response which is necessary for resolution of infection, while the m -mϕ endorses tissue repair following inflammation, suggesting that a premature skew in abundance of m -mϕ at the expense of m -mϕ would limit viral clearance leading to chronic infection and prolonged inflammatory response (klenerman and hill ). an emerging concept in modulation of inflammation involves the role of bacterial surface components like lipopolysaccharide on concurrent viral infection (smith et al. ; wilks and golovkina ) . alterations in gut microbiome have been reported and potential influences this might have on disease outcome have been suggested (preveden et al. ; banks et al. ) . though it is difficult to ascertain the number of asymptomatic infections for any given virus, the percentage of symptomatic infection vis-à-vis asymptomatic ones is often a multivariate variable, being known for only a few. for example, only among individuals infected with denv shows febrile symptoms. this suggests a success for the antiviral immune mechanisms in the majority of individuals. animal studies using gene knockout models have given evidence of this efficacy for many viruses (suthar et al. ; samuel and diamond ; lazear et al. ; deonarain et al. ; burdeinick-kerr et al. ). in case of humans, these information are complicated by differential efficacy of these pathways, protecting or predisposing individuals under the influence of genotype, environment, etc. (paalani et al. ; mitchell and aneshensel ; liu and taioli ) besides, there are few studies that indicate potential influence of medication or noninfectious ailments or societal stress on the outcome of infection through an influence on the immune system (mehrbod et al. ; gilbert et al. ; htun et al. ; jean et al. ). hcv and denv infection can cause liver damage through a chronic and acute infection regime, respectively (samanta and sharma ; axley et al. ) . liver as an organ is characterized by a high capacity to regenerate; however, chronic injury/scarring can lead to fibrosis, steatosis, or even hepatocellular carcinoma resulting in liver failure (forbes and newsome ). hepatocytes constitute twothirds of all liver cells and are associated with all major liver functions besides playing a crucial role in innate immune signaling (kmiec ; zhou et al. ) . hepatocytes are permissible to both hcv and denv, the latter being reported to additionally infect kupffer cells (chang et al. ; zehender et al. ; boisvert et al. ; caussin-schwemling et al. ; goutagny et al. ; marianneau et al. ; de macedo et al. ; huerre et al. ) . in acute infection, the major damage is through apoptosis following direct infection of these cells, whereas establishment of a chronic infection usually causes a sustained inflammation leading to infiltration of polymorphonuclear cells and lymphocytes (huerre et al. ; lim et al. ; masalova et al. ; deng et al. ; bala et al. ; sung et al. the central nervous system (cns) is physiologically isolated from the rest of the body by a specialized selectively permeable barricade called as the blood-brain barrier (bbb), which allows passage to selected metabolites, respiratory gases, and an extremely limited repertoire of circulatory tissue cells. this isolation is necessary for protection of low regeneration capacity neuronal cells from systemic inflammation, which can also upset the structural and functional plasticity of neurons that is dependent on cytokine signaling (arnett et al. ; gougeon et al. ; mason et al. ; fischer et al. ; brissoni et al. ) . the cns can have either neuronal or non-neuronal glial cells; the latter provide vital functional support and include microglia (macrophage-like immune cells), oligodendrocytes (which provide insulation for neurons), and astrocytes (responsible for repair of damaged neuronal tissue). microglial cells have immunomodulatory function in suppressing a pathogenic inflammation (seitz et al. ) . multiple viruses in the +ve-ssrna genome group, including coronavirus, picornavirus, flaviviridae, and togaviridae, cause opportunistic infection of cns (bergmann et al. ; koyuncu et al. ; fletcher and mckeating ) . in the absence of a direct admission route, these viruses undergo limited replication in peripheral tissue, before entering through either peripheral nerves or bbb microvasculature or cns infiltrating leukocytes (functioning as the proverbial "trojan horse") (koyuncu et al. ; jeha et al. ) . a feature common here is a breach of the vascular endothelial barrier at varying locations, e.g., bbb for jev/wnv, blood retinal barrier for zikv, and endothelial barriers in lungs/ peritoneum for denv. breach in bbb is more common for some viruses (e.g., wnv, jev, zikav, poliovirus) correlating with fatality. interestingly, wnv and jev have been suggested to cause bbb disruption from inside the cns (li et al. ; verma et al. ). still other reports suggest infected endothelial cells to secrete pics that disrupt the bbb (chen et al. ; chang et al. ; roach and alcendor ) . the tissue damage is caused from a combination of either direct neuronal infection which activates intrinsic apoptosis or a hyperactive inflammatory response mediated by pics or cd + cytotoxic t cells (ctls) (wang et al. ; samuel et al. ) . infected neurons secrete chemokines that attract leukocytes like monocytes and lymphocytes (klein et al. ; shrestha and diamond ; glass et al. ; kelley et al. ; lim et al. ; bardina et al. ; durrant et al. ; shrestha et al. ). the relation between a "good" and "bad" immune response is, however, very tricky when it comes to the cns. migration of ctls expressing receptors for chemokines like ccl , ccl , ccl , ccl , cxcl - , as well as its timing with respect to establishment of infection, seems to play a crucial role in virus eradication and survival (wang et al. ; shrestha and diamond ; diamond et al. ; sussman et al. ; getts et al. ; nansen et al. ; chen et al. ; liu et al. ; zink et al. ; winter et al. ) . the ctls exert their antiviral role by inducing cell death through either a perforin-dependent or fas-fasl-mediated mechanism (rossi et al. ; shrestha and diamond ) . in addition to ctls, other pics might also induce direct cell death in neurons (dhanwani et al. ; olmo et al. ; baer et al. ; kumar et al. ). dengue virus causes a febrile illness with can turn fatal after a subsidence of the fever. the severity emanates from leakage of fluid from the blood vessels by a breach of the vascular endothelium. circulating in four serotypes, severe disease is mostly associated with secondary infection by a serotype different from the one causing primary infection. neutralizing antibodies generated during primary infection incompletely neutralize the secondary infection virus and instead promote their uptake by monocytes, by a phenomenon called antibody-dependent enhancement or ade (katzelnick et al. ; dejnirattisai et al. ). notwithstanding a primary or secondary infection, the pathological symptoms are considered to be the result of an unbridled host immune response (basu and chaturvedi ; rothman ). (wu et al. ; jessie et al. ; tolfvenstam et al. ). different studies have reported a positive association of dhf/dss development with extraordinarily augmented levels of different pics that include macrophage migration inhibitory factor (mif), ifn-α, tnf-α (green et al. ; kurane et al. ; huang et al. ; chen et al. ) . although multiple reports have suggested correlation between specific pic level and plasma leakage, the mechanism is still elusive and limited to association studies (priyadarshini et al. ; her et al. ; sehrawat et al. ; malavige et al. ). interestingly however, multiple similar association studies have suggested a positive association between levels of il (an aic) and severe/critical symptoms related to dengue infection (malavige et al. ; tsai et al. ; flores-mendoza et al. ). il , produced by multiple immune cells, suppresses immune response through upregulation of socs (suppressor of cytokine signaling) function and downregulation of ifn activity, the result being decreased t-cell cytotoxicity (halstead et al. ; katzelnick et al. ; tsai et al. ; azeredo et al. ; brasier et al. ). the augmentation of il level has been suggested to emanate from monocytes infected by the ade route with additional influence from high viremia (tsai et al. ) . il is a dominant regulator of the immune system that can prolong pathogen clearance through a subversion of the immune response (couper et al. ). coronavirus infections are usually benign causing self-limiting mild flu-like symptoms. however, recent outbreaks involving, e.g., severe acute respiratory syndrome coronavirus (sars-cov), which jumped species barrier through acquisition of minor genome mutations, have projected them as potentially severe human pathogens (guan et al. ) . spread through aerosols, sars-cov primarily infect lung cells triggering an often fatal inflammatory response clinically called acute respiratory distress syndrome (ards) that starts with severe hypoxia, pulmonary edema progressing to systemic inflammation, and failure of multiple organs, culminating in high rate of mortality (peiris et al. ; lew et al. ; tsushima et al. ; farcas et al. ) . although evidence suggests that sars-cov can infect multiple cell types, lung type-ii pneumocytes and ciliated epithelial cells constitute primary sites of virus replication, consequent to which these cells undergo apoptotic and/or necrotic death attracting innate immune cells and activating them to secrete pics (sims et al. ; chow et al. ; nicholls et al. ) . the nature of inflammation following sars-cov infection is characterized by a prompt production of pics through immediate nfκb activation and a delayed expression of type-i ifn genes (shi et al. ; kong et al. ; wong et al. ). severity of symptoms correlates positively with il- levels while exhibiting negative correlation with that of il- and tgfβ (zhang et al. ) . as observed with many other viral pathogenesis models, macrophage polarization culminating in preferential enrichment of m -macrophages has been suggested to be responsible for sars-cov pathogenesis (page et al. ) . sars-cov infection is also associated with hemophagocytosis or engulfment of different types of blood cells by histiocytes (a class within macrophages), which is a clinical marker of immune system hyper-activation (usmani et al. ). traditionally prophylactic or therapeutic strategies for combating viral pathogenesis are designed using vaccines or directly acting antivirals (daa), respectively. but for many viruses there is no clinically approved product to serve in either approach. since the etiology of critical pathogenic symptoms is often associated with an unbridled host inflammatory response, there have been suggestions and attempts to control the harmful effects through modulation of key inflammatory signaling (d'elia et al. ). however, a holistic approach to complete "cure" should probably involve investigations to provide support to both approaches simultaneously. only ribavirin or the same combined with pegylated ifn-α was the therapeutic strategy for controlling hcv infection, before the advent of high efficacy daas. similarly ifn-λ and glucocorticoids, both of which can consolidate the bbb, have been suggested as therapeutics for combating viral diseases that disrupt this barrier (rhen and cidlowski ; daniels et al. ; lazear et al. ; wang et al. ; blecharz et al. ; fabene et al. ) . likewise, administration of pics like ccl and il a has shown efficacy in increasing survival of mice experimentally infected with wnv ( bardina et al. ; acharya et al. ) . in dengue patients, however, meddling with either promoter or inhibitor of inflammation has been suggested as possible approaches (tsai et al. ; goh et al. ; callaway et al. ; ji et al. ; dinarello ) . small molecules that can influence the function of the nlrp inflammosome have also been projected as potential therapies for chikv and can be tested against dengue as well (chen et al. ; coll et al. ; hottz et al. ) . alternative approaches using pharmaceuticals that indirectly mitigate the pathological effect without interfering with inflammation have also been discussed (olmo et al. ; grip and janciauskiene ; reynolds and miller ; thomas and grossberg ; giguere and tremblay ; raemer et al. ). an ability to suppress innate immunity pathways is common among viruses that cause severe human diseases. nonetheless modulating inflammation needs extreme caution, in order to reduce potential cytotoxicity of the administered therapeutic. therefore, there is a need to go beyond association studies to generate a clearer picture of the exact role that inflammation plays in viral pathology, which can then assist in developing therapeutic strategies that tinker with inflammation. the author declares that they have no competing interests. dengue virus infects human endothelial cells and induces il- and il- production liver histopathology and biological correlates in five cases of fatal dengue 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serum cytokines in patients with severe acute respiratory syndrome hepatocytes: a key cell type for innate immunity increased macrophage chemoattractant protein- in cerebrospinal fluid precedes and predicts simian immunodeficiency virus encephalitis key: cord- -sstpidvk authors: younan, duraid; delozier, sarah j.; adamski, john; loudon, andrew; violette, aisha; ustin, jeffrey; tinkoff, glen; moorman, matthew l.; mcquay, nathaniel title: factors predictive of ventilator-associated pneumonia in critically ill trauma patients date: - - journal: world j surg doi: . /s - - - sha: doc_id: cord_uid: sstpidvk background: ventilator-associated pneumonia (vap) is a serious complication of mechanical ventilation. we sought to investigate factors associated with the development of vap in critically ill trauma patients. methods: we conducted a retrospective review of trauma patients admitted to our trauma intensive care unit between and . patients with ventilator-associated pneumonia were identified from the trauma database. data collected from the trauma database included demographics (age, gender and race), mechanism of injury (blunt, penetrating), injury severity (injury severity score “iss”), the presence of vap, transfused blood products and presenting vital signs. results: a total of patients were admitted to the trauma intensive care unit (ticu) during the study period; of these, had ventilator-associated pneumonia. patients with vap were older (p = . ), and they had a higher incidence of massive transfusion (p = . ) and received more packed cells in the first h of admission (p = . ). they had a higher incidence of face injury (p = . ), injury to sternum (p = . ) and injury to spine (p = . ). patients with vap also had a higher incidence of acute kidney injury (aki) (p < . ) and had a longer icu (p < . ) and hospital length of stay (p < . ). multiple logistic regression models controlling for age and injury severity (iss) showed massive transfusion (p = . ), aki (p < . ), injury to face (p < . ), injury to sternum (p = . ), injury to spine (p = . ) and icu length of stay (p < . ) to be independent predictors of vap. conclusions: among critically ill trauma patients, acute kidney injury, injury to the spine, face or sternum, massive transfusion and intensive care unit length of stay were associated with vap. ventilator-associated pneumonia (vap) is the most common nosocomial infection among trauma patients requiring prolonged mechanical ventilation [ ] ; it is associated with prolonged mechanical ventilation, intensive care unit stay and hospital length of stay [ ] . because of reported mortality from vap reaching as high as - % [ ] , preventive measures like the ventilator bundle have been implemented. despite the fact that hospitals have reported prolonged periods of decreased vap rates and periods free of vap [ , ] , cook et al. in a prospective cohort study involving icus and mechanically ventilated patients in canada found the incidence of ventilator-associated pneumonia was . % [ ] , and other authors reported an incidence of % among trauma patients admitted to the icu [ ] . chastre et al., in a review of the literature, found vap to be associated with increased hospital length of stay, intensive care unit stay and cost [ ] , and other authors found similar results in different patient populations [ ] [ ] [ ] . because of this increased morbidity, hyde et al. in a retrospective review of trauma patients with vap and found early tracheostomy (mean hospital day ) significantly decreased both pulmonary morbidity and critical care resource utilization [ ] . vap is associated with increased mortality. magnotti et al. reported an associated mortality of % with vap being an independent predictor [ ] , and cavalcanti et al., in a case-control study, reported a mortality of % in patients with vap who did not respond to antimicrobial treatment [ ] . because of the associated morbidity and mortality of vap, implementation of the ventilator bundle gained popularity in intensive care units with some authors reporting up to % decrease in incidence of vap [ ] [ ] [ ] . known risk factors for vap are existing such as lung disease, aspiration, emergency endotracheal intubation, prolonged mechanical ventilation [ , , ] and coagulopathy [ ] . we hypothesized that other factors exist that are associated with development of vap in critically ill trauma patients. we performed a retrospective review of trauma patients admitted to our intensive care unit between january and october after institutional review board (irb) approval was obtained. patients with ventilator-associated pneumonia (vap) were identified from the trauma database. our hospital is a level academic trauma center and has beds. the trauma intensive care unit has beds and is staffed by surgical intensivists. there are trauma intensive care specialists in our division who alternate between managing patients in the intensive care unit patients and regular floor. the study included all adult trauma patients admitted to the intensive care unit. variables collected from the patients' medical records and trauma database included age, race, gender (demographics), presenting vital signs, injury type and severity, the presence of ventilator-associated pneumonia, hospital and intensive care unit length of stay and survival data. the attendings at our institution follow the center for disease control (cdc) criteria for diagnosing ventilatorassociated pneumonia: ''the presence of a new or progressive radiographic infiltrate plus at least two of three clinical features (fever greater than c, leukocytosis or leukopenia and purulent secretions) represent the combination of criteria for starting empiric antibiotic therapy'' [ ] . the massive transfusion protocol (mtp) at our institution is initiated by the attending trauma surgeon on-call; the first pack includes units of packed red blood cells (prbcs), units of fresh frozen plasma (ffp) and unit equivalent of platelets. the second pack includes units of prbcs, units of ffp, unit equivalent of platelets and unit equivalent of cryoprecipitate. massive transfusion in this study was defined as massive transfusion protocol ''initiated'' and the patients receiving units or more of packed red blood cells (prbcs) in the first h. demographics and clinical characteristics were summarized using descriptive statistics (mean and standard deviation for continuous variables; frequency and percentage for categorical variables). t-test and chi-square tests were used to detect differences between those with vap and those without vap (for continuous and categorical variables, respectively). mann-whitney u test was used to compare the medians of iss, ed sbp, ed hr and ed rr. multiple logistic regression models were used to assess association between vap and variables of interest to detect associations, and each individual model examined the influence of a single predictor while controlling for injury severity (iss) and age. analysis was performed using spss software version , ibm company, armonk, new york, , . one thousand four hundred and three patients were admitted to the trauma intensive care unit (ticu) during the study period; of these, had ventilator-associated pneumonia (vap). the average age was . ± . years, and the median (iqr) for iss was ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . there were ( . %) men, ( . %) were white, and ( %) had blunt trauma. mean icu stay was . ± . days, and mean total hospital length of stay (los) was . ± . days. thirty-one patients received massive transfusion ( . %), incidence of acute kidney injury (aki) was . % and ( . %) died. patients with vap were older (p = . ) and had an average bmi of . ± . . their co-morbidities included cvas (stroke) in ( . %), hypertension in ( . %), ckd (chronic kidney disease) in ( . %), smoking in ( . %), copd (chronic obstructive pulmonary disease) in ( . %), diabetes in ( %) and coronary artery disease in ( . %). these patients also had higher iss (p = . ), and they had a higher incidence of massive transfusion (p = . ) and received more packed cells in the first h of admission (p = . ). among those with massive transfusion, the average number of packed red blood cells received in the first h was ± . , while the average number of fresh frozen plasma units was . ± . . vap patients also had a higher incidence of face injury (p = . ), injury to sternum (p = . ) and injury to spine (p = . ). patients with vap also had a higher incidence of acute kidney injury (aki) (p \ . ) and had a longer icu (p \ . ) and hospital length of stay (p \ . ). there were deaths among patients with vap; were vap-associated (table ) . multiple logistic regression models controlling for age and injury severity (iss) showed massive transfusion (p = . ), aki (p \ . ), injury to face (p \ . ), injury to sternum (p = . ), injury to spine (p = . ) and icu length of stay (p \ . ) to be independent predictors of vap (table ). we found that, among critically ill trauma patients admitted to the intensive care unit, injury to the spine, face or sternum, massive transfusion, acute kidney injury and intensive care unit length of stay were associated with ventilator-associated pneumonia (vap). these findings have significant consequences on the care of these critically ill patients. published risk factors for vap include head and neck trauma, hypotension, craniotomy, pre-existing pulmonary disease, witnessed aspiration, emergency intubation, prolonged mechanical ventilation [ , , ] and more recently coagulopathy [ ] . we identified other associative factors in this study including icu length of stay and hospital length of stay, injury severity score and acute kidney injury. it is not entirely clear why acute kidney injury predicts vap, and it might be a measure of the severity of the injury and illness. in this study, certain injuries were associated with development of vap in these patients, including face fractures, sternum fracture and injury to the spine. while chest trauma was found to be predictive of vap in previous studies [ ] , and some authors advocated early broncho-alveolar lavage to identify it in patients with chest trauma [ ] , sternal fractures by themselves-although often associated with significant pain and underlying pulmonary contusions-have not. chest wall trauma (without sternum) in this study was not associated with development of vap. spine trauma was associated with vap in this study, and multiple previous studies have described this association. aarabi et al. indicated a significant relationship between pulmonary complications and spinal cord injury [ ] , and other authors even advocated performing early tracheostomy in patients with spinal cord injury [ ] . our data are in agreement with published literature. while gursel et al. and others noted a high incidence of acute renal failure during episodes of ventilator-associated pneumonia in the intensive care unit and showed an association with vap [ , ] , in this study, acute kidney injury (aki) was an independent predictor of vap on multivariable analysis. the higher incidence of aki in patients with vap is a marker of the high injury severity these patients sustained. we also found the length of intensive care unit stay to be associated with vap. while other authors similarly found vap to be associated with icu stay [ ] , tsakiridou et al., in a prospective observation study performed at a tertiary care hospital, found that delayed ([ h) icu admission was an independent risk factor for vap [ ] . we also found massive transfusion to be associated with development of vap in this study, and others have noted that transfusion was associated with augmentation of the inflammatory response with resultant increased susceptibility to nosocomial infections [ ] . our study has limitations. first, the study is retrospective and performed at a single center. second, the clinical diagnosis of vap in an intensive care unit setting is not an easy one to make and will differ between providers depending on experience and practice patterns. it is important to note that differences in physician practice patterns may result in differences in the observed incidence of vap. third, we did not have data on the existing pulmonary disease or witnessed aspiration, and these have been identified as predictive factors for vap in the past and would have helped shed more light on the associations and predictive factors of vap. among critically ill trauma patients admitted to the intensive care unit, acute kidney injury, injury to the spine, face or sternum, massive transfusion and intensive care unit length of stay were associated with vap. further studies are needed to confirm these findings. conflict of interest the authors declare that they have no conflict interests. informed consent informed consent was waived by the institution review board. protein c depletion early after trauma increases the risk of ventilator-associated pneumonia attributable mortality of ventilator-associated pneumonia: a meta-analysis of individual patient data from randomised prevention studies prevention of ventilator-associated pneumonia ventilator bundle'' approach to prevention of ventilator-associated pneumonia implementing a ventilator bundle in a community hospital incidence of and risk factors for ventilatorassociated pneumonia in critically ill patients is ventilator-associated pneumonia in trauma patients an epiphenomenon or a cause of death? ventilator-associated pneumonia toward improved surveillance: the impact of ventilator-associated complications on length of stay and antibiotic use in patients in intensive care units the clinical impact and preventability of ventilator-associated conditions in critically ill patients who are mechanically ventilated review article: ventilator-associated pneumonia in major burns early tracheostomy in trauma patients saves time and money risk and prognostic factors of ventilator-associated pneumonia in trauma patients prevention of ventilator-associated pneumonia: the multimodal approach of the spanish icu ''pneumonia zero'' program sustained reduction of ventilator-associated pneumonia rates using real-time course correction with a ventilator bundle compliance dashboard preventing ventilator-associated infections ventilator-associated pneumonia is more common and of less consequence in trauma patients compared with other critically ill patients early trauma-induced coagulopathy is associated with increased ventilator-associated pneumonia in spinal cord injury patients risk factors for ventilator-associated pneumonia in trauma patients: a descriptive analysis early bronchoalveolar lavage for intubated trauma patients with tbi or chest trauma predictors of pulmonary complications in blunt traumatic spinal cord injury early tracheostomy in patients with traumatic cervical spinal cord injury appears safe and may improve outcomes incidence and risk factors for the development of acute renal failure in patients with ventilatorassociated pneumonia fluid overload and kidney injury score as a predictor for ventilator-associated events pre-intensive care unit intubation and subsequent delayed intensive care unit admission is independently associated with increased occurrence of ventilator-associated pneumonia association between gene expression biomarkers of immunosuppression and blood transfusion in severely injured polytrauma patients key: cord- - zrbo w authors: wardhan, rashmi; mudgal, padmshree title: membrane transport date: - - journal: textbook of membrane biology doi: . / - - - - _ sha: doc_id: cord_uid: zrbo w every living cell has to exchange molecules across the membrane for cellular functions. the hydrophobic or lipophilic molecules do not require energy for crossing the membrane. they can diffuse freely from higher to lower concentration till equilibrium is established. this process is called passive transport or diffusion. every living cell has to exchange molecules across the membrane for cellular functions. the hydrophobic or lipophilic molecules do not require energy for crossing the membrane. they can diffuse freely from higher to lower concentration till equilibrium is established. this process is called passive transport or diffusion. this is not true for polar or lipophobic molecules because of the nonpolar nature of membrane lipid bilayer. for polar molecules, the cell recruits mediator or transporter proteins in the membrane to facilitate their movement. if the direction of transport is from higher to lower concentration region, the transporter proteins do not use external energy and the process is called facilitated transport, because the energy needed for transport is dispensed by molecule's own concentration gradient (unequal distribution across the membrane) in the favorable direction. the free energy is minimal in unequal distribution of polar molecules. if polar molecule is transported from lower to higher concentration region, against the concentration gradient, the energy has to be provided by external source to mediator protein to make the process spontaneous in the cell, and this process is called active transport. in the cell, the energy source is mostly adenosine triphosphate (atp). the facilitated transport unlike diffusion has shown speed and specificity, saturation kinetics, susceptibility to competitive inhibition, and susceptibility to chemical inactivation. the active transport of polar molecules against concentration gradient requires energy for transport. a large group of transporters that hydrolyze atp to transport molecules across the membrane are known as atpases. these carrier atpases are classified into five subclasses according to their structure and physiological functions as p-type atpases, v-type atpases, f-type atpases, ecto-type atpases, and a-type atpases. p-type atpase transporters are multidomain integral membrane proteins involved in the transport of cation and lipids. phylogenetically according to their function, these p-type atpases are divided into five major types and various subclasses. the vacuolar atpases are atp-dependent oligomeric protein proton pump, which regulate acidic ph in organelle compartment like phagosome and endosome for the separation of ligand from their receptors transport proton (h + ) across the plasma membrane. the secondary transport, also known as ion-coupled transport involves the use of electrochemical potential generated by an ion transport for the co-transport of another ion against the gradient across the membrane. atp-binding cassette (abc) transporters, importers, and exporters, the newly identified integral proteins, comprise a large superfamily of integral membrane proteins with diverse functions. there are three major transporters which are known as flippases, floppases, and scramblases. the flippases are also known as p -atpases, which transport phospholipids to inner leaflet of membrane and have substrate specificity to phosphatidyl serine and phosphatidyl choline. aquaporins (aqps), the inte-gral proteins of membrane, are small, hydrophobic, and homotetramer proteins, which are involved in bidirectional transport. the bacteria take nutrients from their surroundings by various modes of transport but the transport of sugars and its derivatives like sugar alcohols, amino sugars, glucuronic acids, and disaccharides is transported by group translocation with histidine heat resistance protein hpr as high-energy phosphate donor protein. the transporting sugar molecule is also phosphorylated during transport, which makes sugar negatively charged. the various organisms have specialized proteins of rhodopsin family to seize light energy for various physiological functions. the rhodopsin protein part, opsin, is covalently linked to retinal chromophore, which may be in trans-or cis-configuration. the animal rhodopsin (also known as type ii) are cell g-coupled receptors, while microbial rhodopsin (type i) may act as ion pump, ion channels, sensors, photosensory receptors and regulator for gene expression and kinases. pore-forming toxins (pfts), the bacterial virulence factor, single major family of proteins, are secreted by gram-positive and gram-negative bacteria. pfts may be present either in cytoplasm or in the membrane. the conformational change in cytoplasmic pfts may translocate them to membrane in need of hour. ionophores are natural and synthetic organic molecules of diverse type, which can transport cations by forming lipid ion-soluble complex. each ionophore possesses a unique property of changing transmembrane ion gradient and electrical potential, synthesized by bacteria, and acts as protector against competing microbes. the bacterial and eukaryotic membranes are found to have special proteins in their outer membrane for transport, which are known as porins and form channels for various small size solutes, ions, and other nutrients. the protein ion channels are passive transporters, are made up of transmembrane, are selective for their substrate ions, and have much faster rate of transport than pumps. these channel proteins do not interact with transporting ions and have two conformations, closed and open, to regulate flow of ions. the trimeric atp-activated channels are permeable to cations like sodium, potassium, and calcium. there are seven subclasses of the group, which are involved in various physiological functions like muscle contraction, neurotransmitter release and immune responses regulation. the gaba and glycine receptors are anion channels but inhibitory in nature. gaba receptors have two subdivisions as gaba receptors family of ligand-gated channel (ionotropic) gaba a and g receptor protein family (gaba b ). the n-methyl-d-aspartate (nmda) receptors are known as glutamate-gated cation channel for calcium permeation. the tetrameric ionotropic receptors include nmda, a-amino- -hydroxy- -methyl- -isoxazolepropionic acid (ampa), and kainate receptor as per their synthetic ligand binding. the voltage-gated ion channels open or close in response to change in voltage and the unit of membrane potential. in excitable cells like nerve and muscle, the chemical or electrical signaling is initiated through voltage-dependent sodium or calcium influx. the k p channels or voltage insensitive channels formerly referred to as leaky or resting channels, are ubiquitously found in all organisms. the functionally active k p channel has two subunits, and each subunit is made up of two pore regions (p and p ), four transmembrane domains (m -m ), and two characteristic extracellular helices cap c and c . this chapter explains all the mechanisms of membrane transport. while osmosis is the movement of solution from high concentration to low concentration without transporters across the lipid bilayer, the diffusion is a type of passive transport where small lipophilic or hydrophobic solute moves from higher to lower concentration. the membrane is made up of lipid bilayer to have hydrophobic interior in membrane, which is selectively permeable to small hydrophobic molecules but not to large polar ones. the passive diffusion of charged species across the membrane depends upon the concentration and also on charge of particle z and the electrical potential difference across the membrane dw. the diffusion of particle across the membrane is determined by fick's law of diffusion and affected by magnitude of concentration gradient, molecular weight of solute, distance, permeability, and surface area of membrane. the diffusion coefficient of any solute molecule of fick's law is proportionality factor, which can be defined as mass of solute diffusing through a unit area in a unit time (unit is square meter per second). the diffusion coefficient depends on the size, temperature, charge on the molecule because of difference between proton and electron and polarity (uneven distribution of charge in the molecule) ( fig. . ). according to fick's law, for membrane area a, thickness x, and concentration gradient c (higher) and c (lower) across the membrane, the diffusion rate will be proportional to probability coefficient and can be written as follows: the diffusion rate dx dt ¼ pa c Àc ð Þ ð : Þ the rate of diffusion is also proportional to partition coefficient k. so, where d is diffusion coefficient. from eqs. ( . ) and ( . ), equation ( . ) explains that the rate of diffusion is directly proportional to both partition coefficient and diffusion coefficient and inversely proportional to thickness of the membrane x; the thickness is constant factor for membrane; that's why diffusion largely depends on partition coefficient. the more hydrophobic molecule will move much faster ( fig. . ). as depicted in fig. . , the hydrophobic nonpolar molecule diffuses much faster than polar molecule. among the polar molecules, anionic molecules have higher permeability than cationic molecules. the gases like o and co are nonpolar and hydrophobic, so they easily diffuse. glycerol also diffuses across the membrane, but if this is phosphorylated, then this becomes polar. the urea (nh conh ) is less hydrophobic than diethyl urea (c h nhconhc h ), so urea diffuses slower than diethyl urea. the diffusion of molecules and gases from mother to fetus has been observed, and another molecule sodium thiopental is observed to diffuse through blood-brain barrier. facilitated transport is mediated by transporter protein, which is found in the membrane without using external source of energy. the transport occurs from higher concentration to lower concentration. in mammal's erythrocytes, the . passive diffusion glucose is transported by facilitated diffusion by glucose transporter protein. the facilitated transport unlike diffusion has shown speed and specificity, saturation kinetics, susceptibility to competitive inhibition, and susceptibility to chemical inactivation ( fig. . ). the permeability coefficient of the facilitated transport of glucose is much higher than permeability coefficient ( .  − cm/s) of diffusion. the glucose transporter proteins are divided into two major families like na + /glucose cotransporter (sglts) and facilitative glucose transporter (gluts). the sglt is found in specialized epithelial cells of intestine, proximal tubule of kidney, trachea, heart, brain, testis, and prostate. the na + /glucose cotransporters (sglts) are involved in secondary transport of glucose in intestine and nephrons. the members of sglt class are identified; out of them sglt , sglt , sglt , and sglt function as sugar transporter and sglt acts as glucose sensor. sglt is present in the kidney, brain, liver, thyroid, muscle, and heart. the facilitative glucose transporter (gluts) has fig. . diffusion of polar and nonpolar molecules in the artificial phospholipid bilayer. the small nonpolar molecules move much faster than polar and large molecules fig. . cellular uptake of glucose mediated by glut protein exhibits simple enzyme kinetics and greatly exceeds the calculated rate of glucose entry by simple diffusion members, and out of transport sugars. glut is present in erythrocytes, fetus placenta, brain, and kidney but low in liver and muscles. glut expressed in liver maintains glucose homeostasis. glut is neuron-specific glucose transporter with km of around mm for glucose. glut , a high-affinity, insulinresponsive transporter with approximately mm k m , is expressed in adipose tissue and muscle tissue. in the process of insulinstimulated glut translocation, the various proteins of signal pathway also participate. the high-affinity enzyme glut has km for glucose around - mm lower than blood glucose level ( - mm), hence transports glucose at significant level even in hypoglycemic conditions. the human erythrocyte glucose transporter (glut ) is a type iii transporter, having -residue glycoprotein with transmembrane (m -m ) spanning a helices (mr- , ). the n-terminal and c-terminal are located in cytoplasm. amino acids in the extra cellular loop between m and m , have n-linked glycosylation sites. the large hydrophobic amino acids are present between m and m . the , , , , and amphipathic helices are involved to form hydrophilic channel for glucose transport ( fig. . ) . the glucose transporter accounts for % of erythrocyte membrane proteins and runs as band . in sds-page gels of erythrocyte membranes. it is generally not visible on the gel because the heterogeneity of its oligosaccharides makes the protein band to diffuse (fig. . ). the glucose concentration is maintained at mm for all metabolic purposes inside the cell. the process of glucose transport can be compared to an enzymatic reaction, where the glucose concentration outside the cell is the substrate (s out ), glucose concentration inside the cell is the product (s in ), transporter t is the enzyme, initial velocity is (v ) and (kt) is the transport constant like michaelis constant. the graph between velocity of transport and extracellular glucose concentration [s] out shows saturation kinetics. v max and kt can be calculated from this double reciprocal plot between /v and /s out when [s] = kt, and the rate of uptake is / of v max . the rate equations for this process can be derived like enzyme-catalyzed reactions from michaelis-menten equation: the glucose molecule does not undergo any chemical change during transport across the fig. . structure of glut with transmembranes, and the alpha-helices , , , , and are involved to form hydrophilic channel for glucose . facilitated transport of glucose membrane making this a reversible process and helps to achieve equilibrium much faster in the presence of transporter glut transporter. the transporter protein undergoes a change in conformation on binding to glucose. it has a kt of . mm for d-glucose ( fig. . ). the carbon dioxide formed by cellular respiration of tissues is transported to lung as bicarbonate through erythrocytes and plasma. the hydration of co by carbonic anhydrase in erythrocytes forms bicarbonate, which is exchanged with chloride. the anion transporter involved in facilitated transport across the erythrocyte membrane is high-capacity transporter as the diffusion rate of chloride/bicarbonate is never rate limiting for co transport to lungs. this anion transporter constitutes % of erythrocyte membrane. the two identified families of anion exchanger proteins are known as slc/ a and slc a. the chloride-bicarbonate anion transporter belongs to slc a family. slc/ a has na + /bicarbonate cotransporter. the anion exchanger can transport bicarbonate in both directions. these transporters regulate ph of the cell, volume, and acid-base secretion. the carbonic anhydrase enzyme (pka of . , close to cellular ph) along with co /hco equilibrium acts as a buffer and maintains ph in the cell (fig. . ) . the human chloride-bicarbonate anion exchanger protein has residues with n-terminal domain, transmembrane, and c-terminal domain. the n-terminal and c-terminal are cytosolic. this comprises % of rbc protein. there are - transmembrane domains involved in transport. the protein is glycosylated at asparagine residue of extracellular domain four. the c-terminal domains of amino acids interact with carbonic anhydrase enzyme (transport metabolon). in erythrocyte, carbonic anhydrase reaction forms bicarbonate. during mitochondrial respiration, co is diffused in erythrocytes via capillaries, where it is converted into bicarbonate reaction as shown here. the bicarbonate formed in tissue erythrocytes is released in plasma for lungs. the process is reversed in lungs; the bicarbonate is transported back to lung erythrocytes, which is converted there into co by carbonic anhydrase ii isoform of lung and co is exhaled from lung by diffusion. in the exchange of one bicarbonate ion, chloride ion also moves in opposite direction; that's why this anion transporter is called electroneutral transport ( fig. . ). this anion transporter increases blood exchange capacity for co and provides flexibility of erythrocytes by interacting with rbc cytoskeleton. this transporter may be dimer or tetramer in erythrocytes. the n-terminal interacts with ankyrin, b-spectrin, . and . band proteins of rbc. the mutation in rbc anion exchanger results in defective interaction with cytoskeletal proteins to make rbc osmotic fragile and causes disease such as hereditary spherocytosis. in kidney, the anion exchanger protein controls acid-base homeostasis, electrolyte, and fluid concentration. the carrier protein as discussed earlier can be involved in active or passive transport. the active transport of polar molecules against concentration gradient requires energy for transport. a large group of transporters that hydrolyze atp to transport molecules across the membrane are known as atpases. these carrier atpases are classified into five subclasses according to their structure and physiological functions as p-type atpases, v-type atpases, f-type atpases, ecto-type atpaeses, and a-type atpases. . p-type atpases (e -e atpases): these atpases transport various cations and lipids across the cellular and organelle membrane to regulate cell functions. the various cations may be h + , na + , k + , ca + , cu + , cd + , hg + , zn + , ni, and pb + . the phylogenetic study has divided this group into five major types and more than subtypes. . e-type atpases: these are cell surface transporters involved in extracellular transport, which can use nucleoside diphosphates and nucleosides triphosphates including extracellular atp. these transporters depend upon ca + and mg + and are insensitive to specific inhibitor of p-type. examples are ecto-atpase and animal ecto-apyrase. the e-type atpases participate in termination of purinergic receptor-mediated responses. they are also involved in cell adhesion processes and are important for the maintenance of hemostasis in the cardio vasculature. . f-type atpases (f -f atpases): these atpases are involved in atp synthesis and localized on bacterial membrane, thylakoid membranes of chloroplasts, and inner membranes of mitochondria. the detail of these atpases is discussed in chap. . . v-type atpases (v -v atpases): these atpases act as proton pump and are found on various organelle membranes to regulate acidic ph in organelles. . a-type atpases (a -a atpases): these atpases are present in archaeal bacteria and in some other prokaryotes. they functionally resemble to f-type atpases and structurally to v-type atpases. . p-type (e -e ) atpases p-type atpase transporters are multidomain integral membrane proteins involved in transport of cation and lipids. phylogenetically according to their function, these p-type atpases are divided into five major types and various subclasses ( fig. . ). the p-type atpase or pump is a - kd protein having approximately around amino acids. p-type atpases may have maximum hydrophobic transmembranes from m -m with their n-terminal and c-terminal of proteins in cytoplasm. the cytoplasmic domain is highly conserved and inserted between m and m and m -m . these proteins have characteristic conserved amino acids dktgtlt, where aspartic acid (d) is reversibly phosphorylated during catalytic cycle. the five types of p-type atpases are divided as type i, type ii, type iii, type iv, and type v. type i p-type atpases are bacterial ion pump like escherichia coli-kdp atpases, which are made up of four different subunits with six transmembrane domains namely, kdp f, a, b, and c. the kdp b is the catalytic and ion-transporting subunit of the pump. type ib atpases of single peptide with eight transmembranes like znta and copa physiologically detoxify bacterial cell by throwing out toxic metals. type ii p-type atpases are majorly involved in membrane potential regulation in the cell. they are present ubiquitously in all the cells like na + /k + atpases and h + /k + atpases. type iia is most studied and diversified class that includes sarcoplasmic reticulum ca + atpase pump (serca). this calcium atpase transports two calcium ions from muscle to lumen of endoplasmic reticulum at the expanse of one atp and in exchange of two or three hydrogen ions. the muscle contraction is controlled by ca + atpases because the sudden calcium increase in muscle cell results in muscle contraction. the type ii a calcium atpases is inhibited by phospholamban, which is active in dephosphorylated form and blocks calcium entry by interfering in m transmembrane movement. the calcium atpases are inhibited by thapsigargin. type iib includes plasma membrane calcium atpases or pump (pmca). the type iib fig. . phylogenetic data of the p-type atpase. subfamily division according to their function and ion binding specificity type ia, bacterial kdp-like k + -atpases; type ib, soft-transition-metal translocating atpases; type iia, sarcoplasmic reticulum (sr) ca + -atpases; type iib, plasma membrane ca + -atpases; type iic, na + /k + -atpases and h + /k + -atpases; type iid, eukaryotic na + -atpases; type iiia, h + -atpases; type iiib, bacterial mg + -atpases; type iv, "lipid flippases"; and type v, eukaryotic p-type atpases of unknown substrate specificity. colour coded by species: green, genes from arabidopsis thaliana; orange, c. elegans; grey, e. coli; dark blue, homo sapiens; light blue, methanobacterium thermoautotrophicum; yellow, methanococcus jannaschii; purple, synechocystis pcc ; and red, saccharomyces cerevisiae atpases are regulated by their c-terminal or n-terminal calmodulin-binding domains. the type iic atpases are heteroligomers with aand b-subunits. the sodium-potassium atpases (na + /k + atpase) belong to this class. the a-subunit with maximum ten transmembranes (m -m ) is catalytic unit with autophosphorylation site and ion binding site, while b-subunit, glycosylated single transmembrane of kd, is required for insertion and assembly. the renal na + /k + atpase has additional c-subunit with fxyd motif (fig. . ) . the fxyd proteins act as regulator and stabilizer. the fxyd motifs provide stability to pump. the sodium-potassium pump is inhibited by digitalis glycosides and ouabain, and h + /k + atpases are inhibited by omeprazole, an antiulcer drug, so these pumps are the site of drug action also. the na + /k + atpases transport three na + ions out of the cell and two k + ions inside the cell per catalytic cycle. the type iid atpases are eukaryotic na + /k + atpases. the type iii atpases are prominently found in fungi and plants, where these pumps maintain intracellular ph of . against extracellular ph of . and maintain membrane potential in the similar way of animal na + /k + pump. the h + atpases are regulated by c-terminal autoinhibitory amino acids. fusicoccin is a fungal toxin, which activates h + atpases irreversibly. type iv and type v atpases' role is not very clear but structurally they resemble to type i and are found in eukaryotes. they are also known as flippases, which transport phospholipids from outer leaflet to inner leaflet and maintain lipid asymmetry of the membrane. the erythrocyte membrane has mg + -dependent flippases. these ion pumps may work in correlation with other lipid transport proteins to maintain asymmetry of membrane. . . structure of p-type atpases x-ray crystallography, nmr spectroscopy and electron cryo-microscopy (em) studies on various types of p-type atpases especially, calcium atpases and sodium-potassium atpases revealed that p-type atpases have four domains with conserved sequences, namely, actuator a, membrane m, nucleotide-binding domain nbd, and phosphorylation p-domain. the variation of amino acids has been observed only in extracytoplasmic loop. the most conserved phosphorylation domain is the catalytic domain with dktgtlt signature motif, where aspartic acid (d) is phosphorylated and dephosphorylated during catalytic cycle. it has rossmann fold (characteristics of many nucleotides proteins) made up of central seven-stranded b-sheet flanked by a-helices, including the cytoplasmic end of m . the nucleotide-binding domain inserts into p-domain through conserved hinge region of two antiparallel peptides. seven strands of b-sheet in central have lysine for nucleotide binding. in na + /k + atpases-n-domain phenylalanine clustering is observed whereas acidic residues are rich in calcium atpases. this might be site for regulator interaction, as observed with em. in these atpases are composed of two subunits (a and b). the a-subunit ( kd) binds atp and ions (na + and k + ) and contains an autophosphorylation site (p) but some isoforms like renal sodium-potassium have extra c subunit with fxyd the nucleotide binding site, adenosine base and phenylalanine interaction forms hydrophobic stacking and three phosphate group bulge out in solution so that c-phosphate could reach to p-domain aspartic acid for phosphorylation. the n-terminal actuator domain is highly conserved. similarly like p-domain, a-domain has two unequal divisions by m and m helices hairpin structure. the larger part has b-sheet secondary structure with conserved tge sequence. the tge sequence interacts with phosphorylation site during transport cycle. some of atpases like fungal h + -atpases have extra-long amino-terminal amino acids fused with actuator domain, which might play role in substrate sensing or in regulation. the membrane domain is the largest domain of approximately amino acids with ten transmembrane segments to enclose ion binding site. this domain is less conserved and is directly connected to catalytic core through m -m and m -m polar ionic side chains. as observed in calcium atpases, the m aspartic acid and m glutamate acidic side coordinate ca + ; the other amino acids such as valine , alanine , and isoleucine of m also participate in ion binding. m is the flexible region, which is arranged and rearranged during transport cycle for accommodating different sizes and charge ions and for catalysis ( fig. . ). the catalytic cycle of atp binding, phosphorylation, changes in conformation by arrangements of m-domain helices, hydrolysis of atp for energy are same in all p-type transporters. that's why p-, n-, and a-domains have conserved residues but m-domain with variable residues regions with ion binding site may change to accommodate various ions. the cycle starts with loading of ion (x + ) to high-affinity binding site and releasing of extracellular ion (y + ) in cytoplasm through cytoplasmic access channel. the interaction of ion (x + ) with polar group of amino acids near binding site at p-domain twists m , m , and m helices to move in e state. mg + /atp binding to nbd domain brings this close to p-domain through slight movement of b-strands. these movements result in correct positioning of c phosphate group of atp to p site, and e state of atpase is changed to e *p state. in e *p state, ion (x + ) is trapped in protein, which can be visible from any side of membrane. when ion (x + ) binding is complete, the phosphorylation takes place on aspartic acid residue of p-domain because the energy released by ion binding to e atpase favors the inclination of p-domain to bring more closure c phosphate group to aspartic acid for reaction ( fig. . ). the reaction may be explained as nucleophilic attack on aspartic acid in p-domain by c phosphate group. this may be the reason that sodium atpase needs three na + ions in comparison with two ca + ions in ca + atpases for p-domain movement. a-domain rotates parallel to membrane, and tge loop becomes closure to phosphorylation site. the e *p state changes to e *p state. simultaneously, m , m , and m helices of a-domain because of their rearrangement induced by phosphorylation may close polar ion access channel from cytoplasmic site. this might be understood that the inclination of p-domain induces the movement of helices in such a way that e *p state has low affinity to ion (x + ) and leaves this to extracellular site or into lumen of er. the e *p state has high affinity for ion (y + ), and binding to this ion may induce the hydrolysis of phosphorylated aspartic acid. the tge loop interaction with mg + ion will leave phosphate group unprotected, and this may result in hydrolysis by water. after this hydrolysis, e transforms into e state with open binding site in cytoplasm for next cycle of transport. the vacuolar atpases are atp-dependent oligomeric protein proton pump, which regulate acidic ph in organelle compartment like phagosome and endosome for the separation of ligand from their receptors and transport proton (h + ) across the plasma membrane. the v type atpases activity can be regulated by reversible disulphide bond formation between conserved cysteine residues at catalytic site. glutamate uptake in presynaptic vesicle is found to be dependent on v-type atpases. the v-type atpases regulate plasma ph by exporting proton through renal intercalated cells of kidney. the mutation in v-type atpases may cause renal tubular acidosis. the vacuolar atpases also regulates acidic ph in osteoclasts for bone degradation, and the genetic defect in v-type atpases may cause osteoporosis. v-type atpases are present at the surface of neutrophil and macrophage for maintaining ph. the sperm maturation and storage also need v-type atpases in epididymis of testis. the cancer cells are also found to manipulate v-type atpases. the v-type atpases are potential target for drug in various diseases. the prerenin receptors' study has indicated the role of renin/angiotensin signaling in v-type atpases' regulation. the v-type atpases are oligomeric protein complex with two different domains, which are known as peripheral (v ) of eight subunits and integral (v ) of six subunits. while peripheral proteins (v ) carry out atp hydrolysis, the v -domain of integral proteins transports proton ions. the peripheral proteins v are designated in bold alphabet like v a, b, c, d, e, f, g, and h, and integral membrane proteins are known as a, b, c, c", d, and v e. the various isoforms of these subunits are found in various tissues and organs. the peripheral proteins are present in stoichiometry concentration of a b c d e f g h - . the catalytic domain of vacuolar atpases contains heterohexamer of alternate a-and b-subunits with the atp binding site at the interface of a-and b-subunits. three subunits of a contribute majorly to three catalytic sites, and b-subunits are regulatory in functions. the remaining peripheral proteins form central and peripheral stalk, which is linked to v -and v domains. c-, e-, g-, and h-subunits form the peripheral stalk. the v -subunits are present in the ratio of a d e n c - c and c (n is variable number of e). the subunits c, c , and c have fig. . transport cycle of p-type atpases. in the e conformation, ion (x + ) binds to its high-affinity site in the membrane (m)-domain through cytoplasmic side. the binding of x + ion initiate phosphorylation of aspartic residue in e state (p-domain by atp). phosphorylated e p state transporter still is in same state. during transition of e to e change in conformation (regulated step), the p-domain rearranged itself for bringing tge loop near to phosphorylation site, which stabilizes phosphoryl group binding and adp dissociates. the a-domain m and m segments movement closes access to cytoplasmic ion access channel (light gray). the p-domain rotation disturbs the high-affinity x + binding site through its attachment to helices m -m (dark gray). x + is delivered to the outside (extracellular/lumenal side) through an exit channel. the ion binding site of e p now has a high affinity for second ion (y + ), which may bind from the outside. the aspartic acid is dephosphorylated leaving enzyme into e conformation. mg + and inorganic phosphate (pi) are dissociated, and the enzyme reverts back to the e state, in which y + is released into the cell. the atpases are ready for new cycle hydrophobic residues in high concentration and have one buried carboxyl group in one of the transmembranes as tm (for c and c ) and tm (for c ) for proton translocation. these buried carboxyl groups may be reversibly protonated during proton translocation. these hydrophobic subunits with single copy of c and c and four copies of c form proteolipid ring. the hydrophilic subunit d at the cytoplasmic side interacts with v subunits (d and f) of the central stalk complex, and this whole complex is referred as rotatory complex (fig. . ). the subunit a of v with eight transmembrane domains has buried arginine amino acid in seventh transmembrane domain, which participates in proton translocation by forming two hemichannels (one toward cytoplasmic side and other in opposite side). the cytoplasmic proton enters through cytoplasmic hemichannel of subunit a, which may protonate buried carboxyl group of v -subunit. the atp hydrolysis by a-subunit induces conformational change in heterohexamer of ab resulting in rotation of central motor with d-subunit as axle of the motor. f is regulatory unit. v d-subunit couples this rotation to ion-transporting proteolipid ring of band csubunits. the b-, c-, and a-subunits form channel for proton transport. the proton moves with rotation of b-and c-subunits. the v-type atpases function through rotatory mechanism, where v -induced atp hydrolysis results in rotation of central rotatory domain with ring of hydrophobic subunits (c, c , and c ) in relation to other subunits, which causes ion transport. this proton remains to interact with carboxyl group because of only polar group in the hydrophobic surroundings of v -subunits. when atp hydrolysis induces conformation change by rotation, the protonated carboxyl group is exposed to luminal hemichannel. these carboxyl groups interact with buried arginine residue of a-subunit and stabilize carboxyl group as carbonate ion, so proton gets disassociated and released into the luminal hemichannel. the continued movement of charged carboxyl group because of changes in conformation by rotation of subunit a places it once again in contact with the cytoplasmic hemichannel, where it is again available for protonation. regulation of v-type atpases can be observed through the following different ways either induced or natural. the v-type atpases may be regulated by ( ) reversible dissociation of v and v -subunit, ( ) by changing or uncoupling of proton transport and atp hydrolysis, ( ) by reversible disulfide bond formation between cysteine and cysteine of catalytic subunit a, ( ) by modulation proton pump density as in apical membrane of renal intercalated cell of kidney proton pump activity is regulated by reversible fusion of the pump with apical cells induced by bicarbonate-sensitive adenylate cyclase. the net ph of a compartment is the result of active proton transport and passive proton leakage. if passive proton ion diffusion is controlled, proton pump automatically will be regulated ( fig. . ). f-type atpases are discussed in detail in chap. . the secondary transport is also known as ion-coupled transport because the electrochemical potential generated free energy of an ion transport which is used by transporters for other substrate transports across the membrane. these transporters ubiquitously found in all cells constitute a major facilitator superfamily (mfs) or solute carrier group of transporters (slc). more than of these transporters are involved in lipid, amino acids, sugar transport, and antibiotics. the important examples may include na + /glucose (sglt ) transporter present in kidney proximal tubule and epithelial cells of intestine. oligonucleotide/proton transporter (facilitated proton transport and uphill peptide transport), proton/neurotransmitter transports in synaptic vesicle membrane of neuron (neurotransmitter transport against concentration gradient by using electrochemical energy generated by proton transfer), lactose permease lacy in bacteria, sodium taurocholate cotransporting peptide (oatp), apical sodium-dependent bile acids (apsbt). the major facilitator superfamily transporters have - transmembrane segments and fig. . v-type atpases may be regulated by a reversible disulfide bond formation between conserved cysteine residues of catalytic a-subunit, b controlling proton ion pump density through fusion with membrane, c regulation of other transporters for cl − or h + , d reversible dissociation of v and v domains and e uncoupling atp hydrolysis and transporting pump subunits. source cipriano et al. ( ) transport substrate by alternating conformations. the bacterial lactose secondary transporters (lacy) and sodium glucose transporters (sglt ) in human intestines are two well-explored transporters to understand secondary transport mechanism, which is discussed here. lactose permease (lacy) transporter of e. coli bacteria has selective affinity for disaccharides containing a d-galactopyranosyl ring, as well as d-galactose and symport d-galactopyranoside and hydrogen ion together in cell (symport). lacy protein is an integral protein ( amino acids) of transmembrane helices with a connecting loop between two pseudo-symmetrical six a-helice ( - and - amino acids) bundles. these a-helices form a central hydrophomation. the other conformation with open cavity toward periplasmic side is called outward-facing conformation. this alternate conformation of transporter (involved helices ii, iv, v in n-terminal and vii, x, xi in c-terminal) exposes bound sugar and proton to cytosol and periplasmic side alternatively; the basic principle of this type secondary transport is the conformation change in transporter by binding with substrate. the sugar-interacting residues are present in n-terminal region and the proton-interacting amino acids are present in c-terminal side ( fig. . ). the mutagenesis study has identified the role e of helix iv, r and w of helix v in substrate recognition and binding and r of helix ix, e and h of helix x in proton translocation. e is required for sugar identification and proton translocation. lacy has shown affinity toward numbers of dgalactopyranosyl disaccharide and with d-galactose also but not to d-glucose. the -hydroxyl group is found to have role in discrimination. the x-ray crystallography of lacy transporter has explained the mechanism. the transport cycle starts with inward open conformation. in the absence of sugar, lactose permease transporter is closed from periplasmic side. the binding of d-galactosyl pyranoside to lacy transporter induces changes in protein to open periplasmic side by closing cytosolic inward open cavity. the deprotonation of e causes closing of inward cavity and opening of periplasmic cavity. the membrane potential has no effect on substrate binding. the five residues r , w , e , n , and h bind the substrate binding and dissociation from transporter promote transition between inwardand outward-facing conformations. the lactose/proton symport has same mechanism in active or facilitative transport for galactose and proton but with the difference in rate limiting steps. the deprotonation step is rate limiting in favorable concentration gradient downhill transports, but in uphill transport, conformation changed induced protonation is rate limiting. the lactose permease (lacy) transfer potential energy, preserved in proton gradient facilitates galactose transport down the concentration gradient. hence, the transport is typically a thermodynamically driven and kinetically regulated secondary transport. there are various ways of glucose transporters as discussed earlier. the sodium/glucose (sglt ) transporter is present in epithelial cells of human intestine and also found on incretin (intestinal hormone, which enhances insulin secretion)-secreting vesicles and also in s and s segments of in kidney proximal renal tubule. sglt has transmembranes ( fig. . ). sglt has specificity for glucose and galactose. in intestine, the sodium/glucose transporters translocate one molecule of glucose per two sodium ions across the epithelial membrane of intestine by using electrochemical gradient of another ion (sodium/potassium). the electrochemical potential is generated by another sodium/potassium pump located in basolateral membrane of these polarized cells of intestine. the another sodium/potassium atpase pump is present in basolateral membrane to generate electrochemical gradient along with an independent glucose transporter, which also participates in translocation of glucose molecule from intestine to blood vessels. the detail mechanism of secondary transport is discussed above. the sodium-potassium atpase pump at basolateral membrane transports two potassiums inside the cell and three sodium ions out in blood in atp-dependent manner. the electrochemical gradient energy generated by this transport is used by sglt transporter in epithelial cell of intestine to import glucose against concentration gradient. when glucose concentration is accumulated in cell, the other facilitator transporter of summary of glucose transport in human intestine and then to blood vessels by secondary transporter sodium/glucose (sglt ). the mechanism is discussed in text glucose transports accumulated glucose from cell to blood (fig. . ). atp-binding cassette (abc) transporters, importers, and exporters, the newly identified integral proteins, comprise a large superfamily of integral membrane proteins with diverse functions. a total of transporters are identified in human. in bacteria % and in archaeabacteria % of the genome codes for abc transporters. they might transport various substrates like amino acids and peptides, monosaccharide, polysaccharides, nucleosides, oligonucleotides, vitamins, coenzymes, and different types of lipid. the abc transporter proteins are classified on the basis of highly conserved approximately amino acid residues with three motifs like walker a/p-loop ( amino acids), a signature motif/c-loop (lsggq), and the walker b motif (five amino acids), which participate in atp hydrolysis. abc exporters are physiologically involved in multidrug resistance and detoxification, while importers play role in nutrient uptake and also are used by some prokaryotic pathogens to show pathogenicity and virulence in plants, prokaryotes, and archaea. the abc exporters are also known as multidrug resistance transporters because of their role in detoxification. the other distinct classes of energy-coupling factor (ecf) transporters are also as class iii abc importers. the abc exporters and importers are further classified into subclasses as type i, ii, and iii. functionally, these all transporters may be divided into two major domains known as nucleotide-binding domain (nbd) and transmembrane domain (tmd). the nbd and tmd can be further divided into nbd and nbd and tmd into tmd and tmd . these domains may be separate, dimer, or fused peptides. in eukaryotes, the abc transporters are mostly multifunctional with single peptide with four domains but the assemble to form dimers or heteromers. in bacterial abc transporters nbd and tmd may be separate, dimer or fused. in some bacteria an extra subunit of - kd is found for binding to substrate and delivery to tmd. the bacterial abc transporters may be c the "half-transporter" with six transmembrane domains and one atp-binding region. the atp-binding camay be on the carboxy-terminal (c) or n-terminal localized in periplasm like gram-negative bacteria or may be membrane bound like gram-positive bacteria ( fig. . ). as mentioned above, the characteristic abc transporters conserved signature motif like walker a (gkt/s for phosphate binding domain), lsggq, and walker b motif in nbd are involved in nucleotide-induced conformation change in nbd region. the highly conserved aspartate/glutamate of the walker b motif forms hydrogen bonds with the threonine/serine of the gkt/s box and with a bound water molecule and facilitates nucleotide hydrolysis (fig. . ). abc transporters transport substrates against a chemical gradient, a process that requires atp hydrolysis as a driving force. under physiological conditions, abc transporters operate in a single direction (either import or export) except drug efflux pump lmra having reversible transport under acidic conditions. the transmembrane domain may face outside or inside by changing conformation. the energy for the conformational change in tmd in inward and out ward face is provided by the binding of substrate and mg + atp, followed by atp hydrolysis and substrate release. abc transporters have similarity to secondary transport in mechanism ( fig. . ) . the abc importers bind to substrate through mediator protein, while exporters directly interact with substrate through their tmd. the nbd forms catalytic domain with walker a or p-loop, walker b, q-loop and h motif (switch region) and conserved sequence lsggq in alpha-helical region. the relative orientation of catalytic sequence to alpha-helical region is decided by atp binding. usually, in active abc transporters, its subunits are arranged in head to tail in fig. . a abc transporters' various domain assemblies. the red half-circles and dashed lines indicate nbd interface, p-loops and conserved signature motifs. in all abc transporters, coupling helices transmit conformational changes between the nbds and the tmds. b in single nbd, various domains like p-loop, a-loop, catalytic glutamate, lsggq signature sequence is shown. c linear subunit presentation with conserved sequence opposite direction to each other. tmd and tmd heterogeneous helices are further divided into three separate fold as type abc transporter, type ii abc transporter, and abc exporters' folds. in outward positions, the tmd helices are in extended wing because of tm and tm from one subunit and tm and tm from other subunit. tmd primarily interacts with variable q-loop with conserved glutamine in a-helical domain. any change in q-loop is directly coupled to atp hydrolysis and conformation change of tmd. walker b-loop participates in binding. magnesium interaction with atp requires d of walker b. abc transporters utilize two atps per one molecule of substrate transport. in addition to substrate binding, two sets of amino acids of d-, q-, and h-loops are required for catalysis. one set acts as general base to activate water molecule to attack on c-phosphate group of atp, and other set of amino acids stabilizes phosphate oxygen. the amino acid e near walker b, q of q-loop and h in h motif essentially contributes to catalysis. atp molecule is bound at interface between tmd and nbd, and terminal c-phosphate is positioned between p-loop and lsggq signature sequence. the most important factor in catalysis is the relative position between nbd and tmd domain because of atp, adp, and substrate binding. the transport cycles move in order of substrate binding, hydrolysis, substrate release, which induces tmd conformation change from close form with bound atp to open form without atp. the change in conformation stimulates substrate translocation also. abc transporters are ubiquitously found in lung, brain, muscle, spleen, endosome, and rod photoreceptors in mammals. these transporters are classified into various classes as abca, b, c, d, e, f. the class abc a transporters of members are basically involved in transport of phospholipids and cholesterol to hdl. in lung, they protect cell surface (table . ). these are multidrug transporter and efflux n retinyl-diester-phosphatidyl ethanol amine. the abc b class of members are found in kidney, brain, er, liver, and mitochondria. these are involved in multidrug resistance. they are involved in peptide transport to er. the c class of members, present in all tissues, is involved in drug resistance, organic anion, nucleoside, chloride ion transport. abc d of four members are identified in peroxisomes. abc e class has only one member. the abc f is also present in all tissues. the abc g of eight members regulates transport of cholesterol and other sterols. the plasma membrane has to maintain lipid asymmetry for biological functions and requires constant and regulated transport of membrane components for the purpose. the nonpolar molecules can translocate easily from inner to outer in other direction but lipids with polar group are transported by transporters in atp-dependent manner. there are three major transporters which are known as flippases, floppases, and scramblases. the flippases are also known as p atpases, which transport phospholipids to inner leaflet of membrane and have substrate specificity to phosphatidyl serine and phosphatidyl choline. the flippases' transporters may cause change in shape of membrane like bending of the membrane for vesicle formation or vesicle budding. in erythrocyte, amino phospholipid flippases are identified for ps and pc transportation to inner membrane. atp b flippase is a phosphatidylserine translocase in human. the other enzyme, floppases, belongs to abc transporters' large family, which transport polar lipids to outer membrane in energy-dependent mode for transport. p-glycoprotein floppase is a multidrug resistance protein in human. the scramblases can transfer lipid in both the directions without requiring energy. these are activated by calcium during membrane injury, apoptosis, or coagulation and transport ps and pe to outer leaflet for destroying lipid symmetry. phospholipid scramblases (plscrs) are palmitoylated proteins of lipid raft, which is under regulation by phosphorylation and water is the main component of life, and its exchange is characteristic of any living being. first water channel proteins, known as aquaporins were first isolated from rbc and later from renal proximal tubule membrane peter agre group in . aquaporins (aqps), the integral proteins of membrane, are small, hydrophobic, and homotetramer proteins, which are involved in bidirectional transport. these proteins are present in all living beings. the aquaporins may facilitate the transport of urea, nitrate, no, hydrogen peroxide, carbon dioxide (co ), and ammonia (nh ) along with water. numerous identified isoforms are differentially expressed and modified by posttranslational processes for tissue-specific osmoregulation in various organisms. in mammal, aquaporins are involved in multiple physiological processes including urine concentration by kidney tubules, nerve transmission, metabolism of lipids, fluid secretion, tissue swelling, cell migration, and salivary gland secretion along with skin hydration. the genetic defects in aquaporins may lead to disorders like kidney dysfunction, loss of vision, and brain edema. according to their substrate specificity and localization, the aquaporins are divided into two subgroups: . classical aquaporins are exclusively water channels. . aquaglyceroporins are the proteins with broader specificity for nonpolar molecules like polyols, glycerol, ammonia, co , no, urea along with water and may participate in nutrient uptake and osmoregulation but bacterial glycerol facilitator glpf only allows the movement of polyols. the unusual aquaporin acts as anion channel. the aquaporins are mostly found in plasma membrane but may be localized inside the cell organelle membrane. the movement of water and other solutes through aquaglyceroporins (aqps) supports bidirectional downhill movement through pore or channel formed by aquaporins. the number of genes identified for coding aquaporins in various organisms is variable with one or two genes in bacteria and yeasts, around in mammals ( are for renal tubules to concentrate urine) and more than genes in plants. aquaporins are highly selective and efficient for the transport of water and glycerol but do not allow hydroxide, hydronium ion, and proton movement. this selective transport protects the membrane electrochemical gradient (figs. . and . ). the active form of aqps are homo-tetramers. each monomer of - kd contains amino acids with six transmembrane helices (tm - ) and five connecting loops (a-e). the primary structure of aquaporin oligomer is made of symmetrical two halves, hemipore- (transmembrane - ) and hemipore- (transmembrane - ), as shown in fig. . . the transmembranes are arranged into two halves of , , and , , , connected by loops to form channel. the four monomers together form a central pore or channel for ions and gases to move but water is not allowed through this central channel. the water diffuses through individual monomers of aquaporins. the three connecting loops a, c, e are externally located, while b and d face cytosolic side. the two loops, one from each side b and e, have signature motif of aquaporin family (major intrinsic family) asparagine, proline, and alanine (npa). there are two conserved sequences of npa in aquaporin channel with two asparagine side chains toward pore at the end of hemipore. all transmembranes have their n-terminal and c-terminal in cytoplasm. the b-loop entering from extracellular side and e from other cytosolic side create seventh alpha-helical domain to form a part of surface for water channel. if water is moving from outside to inside, it has to interact with side chain carboxylic group of amino acid at the surface of aquaporin. the presence of arginine and aromatic amino acids at the narrowest part of the pore is the final selection for any molecule to enter. the overlapping of asparagine, proline, and alanine (npa) in the center of pore reduces the size of pore for movement, which is important for water molecule interaction for selectivity. the water-specific aquaporin is observed with pore size of . Å, so bigger molecule or proton will not pass. after interaction with surface protein side carboxylic group, the water molecule will be selected by polar or nonpolar amino acid residues of pore wall proteins, which will provide channel specificity of molecule and will determine the rate of transport. figure . shows the various important amino acids found at pore site. the b and e short helices from opposite side of the pore with positive-charged amino acids with their side group projecting toward center play an important at the constricted site of npa with conserved aromatic and arginine amino acids, water ionic bonds become weak. the aromatic amino acids may vary in various aquaporins. in bovine aquaporin (aqp ), phenylalanine and arginine form constriction site. histidine at surface of pore provides ionic interaction to water molecule, while phenylalanine repels water. after clearing aro/r constriction site, the water molecule interacts with two conserved npa sequences and its neighboring amino acids, where protruding valine pulls water molecule to interact with asparagine and asparagine in npa segment. in the pore, the electrostatic interaction of the npa repeats and the aromatic/arginine (ar/r) constriction results in the exclusion of protons. these amino acids' segment of npa is strong lipid anchor for changing water orientation, which is also supported by strong dipole of b and e helices to change water dipole in opposite side to previous one. the npa amino acids are preceeded and followed by hydrophobic residues (isoleucine and phenyl alanine before and glycine , bovine aquaporin (aqp ) detail structure of pore region with conserved npa provides interaction mechanism of channel with water. half helices dipoles and the hydrophilic and hydrophobic residues lining the pore are shown in red and yellow, respectively; the aromatic/r and npa selective filters are shown as well alanine and valine after npa sequence). these hydrophobic residues stop water interaction with other amino acids of wall proteins for faster movement. tyrosine of d-loop is found responsible for closing of channel because of its insertion into npa site. the phosphorylation at serine of aquaporin induces change in conformation, which may open aquaporin channels, because serine is present in b-loop, which is directly connected to tyrosine . these channels are very fast to have permeability of water  mol/s. the aquaporin channels may be regulated by concentration gradient of water, phosphorylation and dephosphorylation, temperature, and ph. this has been observed in plants that decrease in ph may close the channel because of histidine protonation in d-loop. the mercury chloride binding to cysteine at npa site also closes aquaporins channel. vasopressin in kidney may activate channels through camp-induced phosphorylation. the genetic defect in human aquaporins aqp leads to diabetes insipidus. ca + is found to regulate translocation of aqp to the plasma membrane. aqp overexpression because of heart failure leads to water retention. aqps role in homeostasis of the cerebrospinal fluid (csf) and neuron excitability is well-established ( localized specifically to organelle membrane of endosome phosphorylated during transport, which makes sugar negatively charged. the phosphorylation of sugar does not change intracellular concentration gradient and also check sugar leakage from cell. the seven families of phosphotransferases have been identified in various groups of bacteria with mostly conserved sequences and dissimilarity in substrate specificity. during this transport, the sugar is chemically modified across the membrane and that's why this transport is also called as group translocation transport. the phosphortransferases complex has cytoplasmic, peripheral, and integral proteins. the cytoplasmic proteins are constitutive and nonspecific, which are involved in group translocation of various other proteins also. the cytoplasmic proteins comprise of heat-resistant histidine protein hpr and enzyme e . e protein has histidine at n-terminal, which participates in reversible phosphorylation of hpr, and its c-terminal domain is required for protein autophosphorylation by pep. hpr is small monomeric protein, which is phosphorylated at n-terminal histidine by e*p. the cytoplasmic pts proteins or membrane-associated hydrophilic pts domains undergo transient phosphorylation during transport of sugars. the integral membrane proteins are known as enzyme ii (eii), which participate in substrate translocation and its phosphorylation. the eii enzymes are inducible and substrate-specific proteins, which include eiia, cytoplasmic protein, a peripheral protein associated with inner membrane eiib, and carrier integral protein eiic. eiia and eiib are sugar-specific proteins. eiia for glucose is phosphorylated at histidine . the eii component may be separate or fused in different organisms. eiia is conserved; eiib is hydrophobic which is phosphorylated at conserved cysteine residue by eiia. eiic has - transmembrane regions with one conserved gxxe motif in hydrophobic loop. protein eiic has three periplasmic ( , , loops) and two cytoplasmic ( and % loops). in bacillus subtilis, oligo-b-glucoside-specific pts has three separate enzymes, while in the same organism, mannitol pts has eiib fused with eiic. the reaction cycle starts with transfer of phosphate group from pep to a histidine residue on enzyme i. the enzyme ei transfers this phosphate to hpr protein on histidine . in the next step, this phosphate group is transferred from hpr to eiia protein again on histidine residue. the specific enzyme eiia for sugar transfers phosphate to the cysteine residue of enzyme iib. finally, enzyme eiib transfers phosphate group to eiic bound sugar, which is phosphorylated and translocated to cytoplasm (fig. . ) . the majority of sugars are transported in phosphorylated form but fucosyl-a- , -n-acetylglucosamine in lactobacillus casei is translocated in unphosphorylated form exceptionally. some sugars are immediately dephosphorylated after translocation in cytoplasm like mannose in enterobacter faecalis. the glucose uptake is enhanced by the availability of nitrogen in bacteria through these phosphotransferases. the various organisms have specialized proteins of rhodopsin family to seize light energy for various physiological functions. the rhodopsin protein part, opsin, is covalently linked to retinal chromophore, which may be in trans-or cisconfiguration. the animal rhodopsin (also known as type ii) are cell g-coupled receptors, while microbial rhodopsin (type i) may act as ion pump, ion channel, sensor, photosensory receptor, regulator for gene expression or a kinase. halobacterium salinarum has normal respiration in the presence of o and high nutrients, but in the absence of nutrients, it survives by using light energy, which is identified with purple patch on the bacterial membrane because of retinal base. the three microbial pumps are well-characterized in their structure and function known as the bacterio rhodopsin h + proton pump, channel rhodopsin for chloride ion. the halobacterium salinarum has sensory rhodopsin i and sensory rhodopsin ii, which activate transducers for senses ( fig. . ) . the animal and microbial rhodopsins are transmembrane of a-helices, covalently linked to retinal through lysine residue and form protonated schiff base with it. the retinal chromophore may be in cis-or transconfiguration. the ground state of microbial and animal rhodopsins has all trans-and -cis-retinal configuration ( fig. . ) . thermodynamically, retinal is preferred in all transconfigurations in nonpolar environment with absorption maxima at nm wavelength but the protein interaction with retinal shifts this absorption maxima to broader range in visible light range from to nm. the light absorption by retinal stimulates isomerization from trans-cis to cis-trans in less than a fraction of second. the energy released by this transition isomerization induces change in opsin protein conformation for various activities. the sequences of animal and microbial proteins are quite dissimilar in spite of both having seven salinarum contains four rhodopsins, bacteriorhodopsin (br), halorhodopsin (hr), sensory rhodopsin i (sri), and sensory rhodopsin ii (srii, also called phoborhodopsin, pr). br and hr work as a light-driven transmembranes. bacteriorhodopsin translocates one proton inside to outside in cyclic reaction and generates membrane potential across the membrane for atp synthesis. the channel rhodopsin of h. salirium transports chloride ion from outside to inside of cell for atp synthesis and for osmotic balance. the sensory rhodopsin i (sri) acts as stimulator of positive phototaxis, while srii works oppositely. the bacterial movement toward particular wavelength light is positive phototaxis and moving away is negative. sri ( nm k max ) and srii ( k max ) perform through their transducers and cotransducers. the cotransducers are named as htri for sri and htrii for (srii). sr proteins with their cotransducers are localized in cell membrane in : ratio to form tetramer. the signal induced by retinal trans-cis isomerization by light is propagated to cytoplasmic transducers chea and chey resulting in phosphorylation and dephosphorylation for flagellar movement in desired direction ( fig. . ) . the crystallographic studies and electron density of catalytic side have explained the photocycle of ion transport. the seven transmembranes of opsin protein may be designated as a b c d e f g, which are arranged to cover centrally linked protonated retinal schiff base to g-helix by lysine . basically, the light absorption by retinal redistributes its own electrons in protonated base resulting in isomerization, proton transfer between various amino acids at catalytic side, water relocalization, and finally change in protein conformation for physiological function. the water molecule at position in catalytic site accepts proton from retinal and fig. . synthesis of retinal chromophore in microorganism and animal from b carotene. the ground state of microbial and animal rhodopsins possesses all-trans-and -cis-retinal as its chromophore, respectively, bound to a lys residue via a schiff base, which is normally protonated and exists in the -anti configuration. it should be noted that microbial rhodopsins depend exclusively on all trans-retinal, while some animal rhodopsins possess vitamin a (c =c double bond for fish visual pigments) and hydroxyl (c -oh for insect visual pigments) forms of -cis-retinal. usually, photoactivation isomerizes microbial rhodopsin selectively at the c =c double bond and animal rhodopsin at the c =c double bond. post-content transports it to aspartic acid and asp . the protonation of asp and retinal deprotonation induce proton movement to outside of membrane, and proton is released. cytoplasmic proton migration protonates retinal schiff base to its original position to complete light-induced cycle. the conserved residues involved in proton transfer are shown in figs. . and . . at the ground state of stable active site, the positively charged retinal schiff base interaction with three water molecules , , and aspartic acid (d) , d stabilize separate noninduced light changes fat active site. the h-bond interaction of aspartic acid d with threonine t asp d , tyrosine y , y , and water molecule keeps asp d in anionic form. the aspartic acid d and water also interact with positively charged arginine r . the transition of retinal base from trans to cis by light will dramatically destabilize this fig. . bacteriorhodopsin molecule is purple and is most efficient at absorbing green light (wavelength - nm, with the absorption maximum at nm). bacteriorhodopsin has a broad excitation spectrum fig. . a structure of bacteriorhodopsin (br), with conserved aromatic residues highlighted (pdb id: qm ). tyr , trp , and trp are strongly conserved among microbial rhodopsins (orange). aromatic amino acids are strongly conserved at the position of tyr , trp , and phe (yellow). in br, trp , trp , tyr , and trp constitute the chromophore binding pocket for all transretinal configuration (gray). b crystallographically observed internal water molecules of br (shown as green spheres) ground state. the hydrogen bond between these amino acids and water will be disturbed and rearranged, which will cause to disassociation of proton from schiff base. the asp d is first proton acceptor which is centrally located. water actively participate in hydrogen bond rearrangement because of its position its h + and oh − easily may protonate and deprotonate neighboring residues. the extracellular side of retinal linked to arginine r interacts with three-dimensional hydrogen bond mesh contributed by water molecule , , , and and amino acids. the retinal molecule is linearly connected to arginine through h bonds starting from water -d -water, -arg r , and other water -d -w -water -arg r nh /nh . the whole mesh is interconnected. the proton release complex with water , , , glu e , glu e is separated by isoleucine i and leul from hydrophilic environment. at the c-terminal of d helices transmembrane, a unpaired buried arginine r interacts with salt bridge to glutamate e and interacts with carboxylic group of amino acids at , , and positions. the transmembrane g at cytoplasmic side gets kink because of p bulging at alanine a , which results in peptide bending between alanine a and lysine k . this bending stimulates local rearrangement of h bonding between water and amino acids ( fig. . ) . the new hydrogen bonding between molecules and new local arrangements around g transmembrane helices slightly tilt this from canter, resulting in c-terminal displacement to outer side, and induce change in conformation. the water molecule at stabilizes this change through interactions with amino acids and carboxylic group of retinal schiff base. the rearrangement of g helices and water molecule amino acids interactions decrease pka of aspartic acid d , which donates proton in photocycle and control reprotonation of aspartic by isomerization of retinal schiff base from cis to all trans ( fig. . ). pore-forming toxins (pfts), the bacterial virulence factor, single major family of proteins, are secreted by gram-positive and gram-negative bacteria. pfts may be present either in in bacteriorhodopsin helices g at cytoplasmic side, a kink between alanine a and lysk is produced because of alanine p-bulging. the local hydrogen bonds between water and amino acids are rearranged to release proton cytoplasm or in the membrane. the conformational change in cytoplasmic pfts may translocate them to membrane in need of hour. the numbers of bacteria like staphylococcus aureus, e. faecalis, e. coli, and vibrio cholera had shown the presence of these proteins during pathogenesis. the size of pore may be small ( . - nm) or large (more than nm). mostly, pfts are water-soluble. binding of pore-forming toxin to host cell receptor through sugar, lipids, or protein leads to change in conformation for pore formation. the strength of pore formation is directly proportional to their virulence power. as per transmembrane structure of pfts in host cell membrane, the pfts are divided into two groups as a-pfts because of a-helical pore and b-pfts because of b-barrel pore (fig. . ) . the various molecules in the host membrane may act as receptor molecules like glycosyl phosphatidyl inositol (gpi), chemokine receptor (ccr or cd ) of white blood cells, disintegrin and metallo-protease (adam)-type proteins, cholesterol, and other lipids. pore-forming toxins may change their conformation to adjust in host environment. the pore formed by pfts may be continuous barrel layer of proteins or may be in toroidal form because of lipid protein interaction. the major pathogens producing different pfts proteins are shown in table . . the psts act first by binding to receptors, which are followed by oligomerization, change in conformation, and insertion into host membrane to form pore. till date, six families of pore-forming toxins, including three a-pfts and three b-pfts, are known. a-pore-forming toxins are heterogeneous group with various pfts like cytolysin of e. coli, exotoxin of pseudomonas aeruginosa, and diphtheria toxin from corynebacterium diphtheria. the various pfts may form various types of pores with different characteristics. the e. coli produced a-pfts colicins family produces pore in other bacterial inner membranes. the isolated colicin a and e pft, according to their function, may be divided into three domains for receptor binding, translocation, and pore formation. the pore-forming domain of colicin has ten alpha-helices. two of these hydrophobic helices are surrounded by eight amphipathic alpha-helices. the hydrophobic a-helices are inserted into inner membrane to make nonspecific voltage-gated ion channel. this pore destabilizes membrane potential of host cell. the other apft such as insecticidal cry toxin from bacillus thuringiensis, diphtheria toxin translocation domain, and eukaryotic protein bak, has shown similar structure like colicin for pore formation. the colicin pft of this group forms the pore by binding to receptor, translocates to periplasm, and causes cell death by forming pore in inner membrane. bcl- completes pore formation in mitochondria by releasing cytochrome c to activate caspases for apoptosis. a member of apft cytolysin family, cytolysin a(cly), hemolysin e(nhe) enterotoxin of salmonella enterica and shigella flexneri and e. coli forms pore of - nm diameter for cation in macrophage leading to apoptosis. cytolysin a is found to be a-helical protein in elongated shape with short hydrophobic b-strands in tongue shape (btongue). during formation after assembly, the b-strands become disassociated from the protein for insertion into lipid bilayer; this event causes the rearrangement of n-terminal region of amphipathic helices at the pore site. the n-terminal domain arrangement may vary in length but this variation cause is not clear till now. the conformational change by the n-terminal amphipathic alpha-helices catalyzes a very organized step-by-step oligomerization of monomers. there are various ways of protein-protein or lipid-protein interaction for pore formation as shown in fig. . . when toxin protein interacts with host lipid for pore formation, lipid may provide a curved nonbilayer shape at pore periphery. in this pore, the protein regulates stress caused by distortion of membrane. as seen in eukaryotic b-barrel actinoporin pft from sea anemones, in which cation selective pore is formed by interaction with sphingomyelin of host membrane. usually, the toxins recognize and interact with specific lipid domain of sphingomyelin clustering or cholesterol-sphingomyelin complexes, which support the accumulation of pfts locally to ease their function of pore formation. this may result in lamellar structure destabilization by lipid transmovement across the membrane (fig. . ) . the b-pore-forming toxins include aerolysin and cholesterol-dependent cytolysin family (cdc), and these are secreted majorly by gram-positive bacteria and minor by gram-negative bacteria. gram-negative aeromonas saps produces aerolysin as protoxin. the c-terminal cleavage converts protoxin into toxin protein, which is elongated multidomain protein. the aerolysin protein may be divided into four domains; the domains , , and are together, and domain is separated. the domain three has multiple short a helices with b-sheet. the domain four ( ) interacts with cholesterol for binding by inserting several loops. the domain four ( ) has conserved domain of tryptophan residues with three short hydrophobic regions known as l , l , and l . the domain has two transmembrane a-helices in soluble form, which are converted to b-hairpin transmembrane during pore formation. while a-pfts start pore formation by the insertion of hydrophobic helices, the b-barrel formation occurs in concerted mode (fig. . ) . . actinoporin and colicin family pfts pore formation by monomer alpha-helical hydrophobic segment extraction and insertion into host membrane, umbrella formation, oligomerisation either into two or more peptides as mentioned above, the other major family of b-pore-forming toxins is cholesterol-dependent cytolysins (cdc), which include perfringolysin o (pfo) of c. perfringens, streptolysin o (slo) of streptococcus pyogenes, pneumolysin of s. pneumoniae, and listeriolysin o (llo) of listeria monocytogenes belong to cholesteroldependent cytolysin family. these toxins are produced mostly by gram-positive bacteria and by few gram-negative bacteria. the pore formation is dependent on cholesterol for pore formation. in cholesterol-dependent cytolysin toxin, pore may contain from to proteins subunits, and each subunit donates b-hairpin for pore unlike other b-pfts. cytolysins are produced as monomer or dimer and have some similar features like eukaryotic membrane attack complex (mac). cytolysin also has four domains like aerolysin in elongated structure and makes well-defined prepore in the membrane. the cdc, unlike some other toxins, which rearranges their structure for pore formation, goes under transition from alpha-helices to their b hair pin secondary structure at membrane surface (prepore). the transmembrane interacts with lipid cholesterol and forms arc-like structure to create gap (semi pore) in the membrane. at this stage, oligomerization cytolysin monomers occur. the oligomerization transforms the surrounding arrangements of molecules to form functional pore. the conserved tryptophan in toxin and cholesterol play an important role in pore formation (fig. . ) . a-hemolysin (hla) of cholesterol-dependent cytolysin (cdc) family anchors through n-linked glycan proteins of hosts. aerolysin binds through glycosylphosphatidylinositol (gpi) anchors. colicins f a-pft family interact with lipopolysaccharides (lps) in the outer fig. . depiction of pore formation pathway of pore-forming toxins (pfts). soluble pfts are recruited to the host membrane by protein receptors and/or specific interactions with lipids (e.g., sphingomyelin for actinoporins or sterols for cholesterol-dependent cytolysins (cdcs)). upon membrane binding, the toxins concentrate and start the oligomerization process, which usually follows one of two pathways. in the pathway followed by most b-pfts, oligomerization occurs at the membrane surface, producing an intermediate structure known as a prepore (mechanism ), which eventually undergoes conformational rearrangements that lead to concerted membrane insertion. in the pathway followed by most a-pfts, pft insertion into the membrane occurs concomitantly with a sequential oligomerization mechanism, which can lead to the formation of either a partially formed, but active, pore (mechanism ) bacterial membrane. a-pft fragaceatoxin c (frac) binds to sphingomyelin clustering. the eukaryotic cell also has pore-forming toxins as the mechanism of their defense, which is known as membrane attack complex (mac) or performin toxins. the t cell secretes them to destroy pathogen. these toxins like cdc have two transmembrane domains. the hydrolysin from cnidaria (phylum of aquatic invertebrate animals), enterolobin toxin from enterolobium contortisiliquum, and human c are the examples of eukaryotic toxins. the various defense mechanisms against these virulent pore-forming toxins exit in host like human. the binding of toxin to cell membrane may also activate p mitogen-activated phosphokinase (mapk), c-jun n-terminal kinase like mapk-kgb pathway. the detail of receptor signaling is discussed in chap. . pfts pore formation may result sometimes in increase in extracellular potassium as a defense mechanism. the potassium efflux activates nucleotide-binding oligomerization domain (nod)-like receptors (nlrs), which have variable n-terminal domains such as caspase recruitment domain (card) and pyrin domain (pyd), which activate apoptosis and inflammation pathways. these receptors are found on immune cells like lymphocytes, macrophages, dendritic cells, and also in nonimmune cells and play a role in innate immunity, phagocytosis, and killing of pathogens. the caspase induces sterol regulatory element binding protein (srebps), the regulator of membrane lipid synthesis. the extracellular potassium increases in response to toxin interaction and also activates caspase for inducing apoptosis. the activation of p and extracellular response-activated kinase (erk) brings back potassium level to normal. a-defensin proteins are also detected in host to act against pfts (fig. . ) . calcium efflux in response to pfts activates the release of lysosomal enzymes such as sphingomyelinase and hydrolase to degrade pathogenic molecules endocytosis is another defense used by host cell. pfts also interact with cellular signaling, and molecules such as arthritis were caused by c-hemolysin in human. the hemolysin induced the expression of interleukin in osteoclasts. the downregulation of interleukin and nitric oxide synthetase is observed in macrophage by cytolysin toxin. ionophores are natural and synthetic organic molecules of diverse type, which can transport cations by forming lipid ion-soluble complex. each ionophore possesses a unique property of changing transmembrane ion gradient and electrical potential, synthesized by bacteria, and acts as protector against competing microbes. they disrupt membrane's ionic gradient required for proper transport; therefore, they have got antibiotic properties. ionophores increase the permeability of biological membranes to certain ions and antibiotics; that's why they are used as growth enhancers as additives in certain animal feed. the various ionophore antibiotics are macrolides. the two classes of ionophores are identified as carriers and channels. the carriers bind to specific ion, shield it from hydrophobic environment, leave the ion on other side of membrane by diffusing across the membrane, and come back without ion to original place. the carrier polyether ionophores include approximately more than types having antibiotic properties against bacteria, fungi, and virus. some of them are found to have antitumor activity also. the antibiotic property of these polyether ionophores structure specifies their cation metal binding and is directly correlated to their antibiotic property. the carrier ionophores are not found to be effective in gram-negative bacteria because of their cell wall but the opposite is true for gram-positive bacteria. the gram-positive bacterial membrane is permeable for these small molecules. because of this reason, e. coli is found to be important for food and human health. the carrier ionophore examples include valinomycin, calcimycin, salinomycin, monensin. valinomycin is produced by various strains of streptomyces species as s. tsusimaensis and s. fulvissimus. the valinomycin is highly selective for potassium and rubidium (rb + ) ion and does not allow sodium or lithium transport ( fig. . ) . the binding and affinity constant for the potassium-valinomycin complex are ten times higher than sodium. valinomycin was observed to downregulate the lymphocytes multiplication induced by phytohemagglutinins in human. valinomycin-metal ion interaction essentially requires dehydrating form of ions, and complex is formed in energy-dependent process. the amount of energy required is the deciding factor for forming valinomycin-potassium and rubidium complex but not with sodium (table . ). as shown in table . , because of unequal distribution of ions across the membrane, the electrical potential across the transmembrane is generated, which initiates binding of metal ion to ionophore for diffusion. the movement of ions may decrease or balance electrical potential of membrane. the ionophores may reduce membrane electrical potential through ions movement, which is the basis of their antibiotic property. these metal ion ionophores act as uncouplers like the cell's own uncouplers in mitochondrial respiration. the synthesis of atp becomes disassociated from electron transport chain. valinomycin acting as antibiotic may kill the cell by disturbing electrical gradient in transport and energy-generating process. in s. aureus, listeria innocua, l. monocytogenes, b. subtilis, and bacillus cereus, gram-positive bacteria the valinomycin activity is ph dependent. recently, the valinomycin is reported to inhibit severe acute respiratory-syndrome corona virus survival at . - lm (table . ). the structural study of carrier ionophore by x-ray has revealed the binding site of ions calcium-magnesium, calcium- hydrogen hydrogen > cesium (cs + ) % rubidium (rb) > potassium (k + ) > sodium (na + ) > lithium (li + ) nystatin monovalent cation and anion through oxygen atoms in the center of ionophore. ionophores are neutral complexes with monovalent cations (monensin, salinomycin) and with divalent metal cations (lasalocid, calcimycin). the carboxylic group is deprotonated at physiological ph, so more of neutral complexes are found in the cell. the pseudocyclic structure formed by cation may be stabilized by intramolecular hydrogen bonds of carboxylic group and hydroxyl groups. the hydrophobic residues of ionophore encircle the ionic interactions for the protection. the polyether ionophore anion (i-coo − ) binds the metal (m + ) cation or proton (h + ) and forms either (i-coo − m + ) or (i-cooh) depending on ph. this interaction changes na + /k + atpases activity to change in gradient of the cell. resultantly, this may cause the death of the bacteria cell because of increase in osmatic pressure, swelling and bursting of cell by disturbed atpases. the coordinated geometry between ionophores and ions is important for its function. the other examples of carrier ionophore, isolated from streptomyces chartreuses, are calcimycin (a ) and ionomycin calcium ionophore. ionomycin hydrolyzes phosphatidyl inositides in the t-cell membrane to activate phosphokinase c (pkc) through autophosphorylation by phosphorylating t helper cell (cd ) and t suppressor (cd ) cells. the hemisodium, synthetic ionophore, transports sodium ions. the calcium ionophore calcimycin, oxidative phosphorylation, and electron transport chain uncoupler are often used for enhancing thyroid and insulin secretion. calcimycin, because of fluorescent property, may be used as biomarker in membranes for transport study (fig. . ) . calcium ionophores promote three types of signal in calcium-resistant cell for cytosolic calcium increase: ( ) activate receptor-mediated calcium transport into cytosol by native ca + channels, ( ) phospholipase c-dependent calcium mobilization from intracellular stores like er, ( ) at low concentration of ca + , ionomycin in endothelial cell, like histamine, increases cytosolic calcium, while in fibroblast, only intracellular calcium from organelle store releases out. beauvericin ionophore has both antibiotic and insecticidal property. this has phenylalanine and hydroxyl-iso-valine amino acids alternatively to form cyclic hexodepsipeptide. it transports alkaline and alkali metals across the cell membrane. beauvericin, found in beauveria bassiana, wheat, barley, and corn, causes apoptosis in mammals additionally. the other class of enniatins ionophores are found in fusarium fungi. usually, proteins have the characteristic of forming channels in the membrane for transport of various molecules, but some of the ionophore also transport by forming a channel in the membrane. these ionophores have a few unusual amino acids normally not found in proteins. gramicidin, isolated from bacillus brevis, has amino acids, linearly arranged in d and l alternate fashion with amino-terminal formaldehyde and carboxyl terminal ethanol amine. the hydrophobic amino acids in gramicidin support its function in channel formation. these channels have outer lining of hydrophobic amino acids to interact with lipid bilayer of membrane with hydrophilic interior for ion interaction. these channels allow very fast diffusion of ions through them in very short period unlike carrier ions. their size is larger than fig. . crystal structure of : complex of calcimycin with the magnesium cation. https://www.hindawi.com/ journals/bmri/ / /fig / protein channel. gramicidin channels allow protons and other cation movements but not of divalent cations like ca , which block them. gramicidin has antibiotic property against gram-negative bacteria, gram-positive bacteria, and pathogenic fungi but sometimes its hemolytic activity may limit its use. in various solvents, gramicidin has stable amphiphilic antiparallel b-sheet structure with a polar and a nonpolar surface as a-helix is not possible because of alternate d-and l-amino acid residues. gramicidin has . residues per turn in b-sheet structure with channel diameter approximately . nm. the ethanol residue is projected on the surface and formyl group inserted in membrane. the nonpolar amino acids play a role in microbial specificity, and disrupted beta-sheet structure adds to its antimicrobial activity ( fig. . ) . the natural mixture of gramicidin, known as gramicidin a, is observed fluorescent because of tryptophan amino acid. its antimicrobial activity may be through gene regulation as e. coli rna polymerase was found to be inhibited by gramicidin ( fig. . ) . gramicidin is not synthesized by ribosomes but by cellular enzymes, and at position, it has tryptophan which is replaced by phenylalanine in gramicidin b and tyrosine in gramicidin c, respectively. the interior of the channel is formed by the polar amino acids as discussed above. gramicidin has affinity for ions in the following order as maximum for cesium and minimum for lithium cs + > rb + > k + > na + > li + . gramicidin is also used to stimulate the production of cytokines interferon. the property of ionophore to induce apoptosis in cancer cells is being used for drug targeting especially sodium and calcium, because ionophore has capacity to disrupt lysosomal function and can inhibit proteasome function for apoptosis ( fig. . ). the bacterial and eukaryotic membranes are found to have special proteins in their outer membrane for transport, known as porins which form channels for various small size solutes, ions, and other nutrients. the porins found in mitochondria are also known as voltage-dependent anion channel (vdac). the mitochondrial conserved porin proteins are tissue specific, but in bacteria, these porins constitute a diverse group because of bacterial variable environment. the porins are made of , , , or b-barrel strands in slightly tilted form. the porins may be monomer but mostly are trimers. based on their structure and function, porins are broadly classified as -stranded nonspecific b-barrel such as omp , oprp, and phoe, -stranded specific b-barrel (lamb from e. coli), b-strands and monomeric such as ompg and multidomain like cyma from bacteria. t. thermophilus which is p porin protein with n-terminal peptidoglycan binding domain and a coiled coil domain with large pore size. nmr and crystallographic studies have revealed mitochondrial vdac pore size of . nm height and nm diameter. the vdac has b-strands with n-terminal helix buried in pore. the sugar-specific porins (fig. . ) may allow solute up to kd. the bacterial porins are oval in shape with pore size - . and nm height. the porin first and last b-strands are joined in antiparallel mode to form complete pore. these b-strands are connected to each other from cytoplasmic side by - long loops and - small turns from periplasmic side. the loop from the cytosolic side in all porins is folded in side in barrel as shown in fig. . and regulates pore size. usually, loop is found between and color -blue (n terminus) , red (c terminus) with approximate molecular dimensions. the first and last b-strands (b and b ) are paired in a parallel way (yellow arrows). the upper view highlights n-terminal a-helix localized inside the barrel strands of barrel. this has mostly polar amino acids aspartic acid and glutamic acids. the l , l , and l interact with each other monomer to form trimer. l , l , and l are present on the surface of porin. the loop l being folded into interior contributes in channel opening toward extracellular side (fig. . ) . the aromatic amino acids like tyrosine and phenylalanine are predominantly present in inner side and outer side of pore. the tyrosine is more toward outer side and phenylalanine to cytosolic side. all porins are found to have phenylalanine at their c-terminal which is required for proper folding and import. the alge porin from p. aeruginosa ( b-strands) has positive amino acids like arginine inside the pore to translocate negatively charged alginate polymer. the mitochondrial porins have three isoforms and are of round shape. the first and last b-strands of strands pair parallel teach other unlike bacterial porins. all strands are connected through short loops, little elongated to cytoplasmic side ( - number) (fig. . ) . the mitochondrial porins are imported by tom protein and inserted by sorting and assembly machinery (sam complex) bacterial porins are inserted by b-barrel assembly machinery (bam) complex. in -stranded b-barrel porins of nonspecific class, the transport depends on molecular mass and rate of transport is directly proportional to concentration gradient. the specific b-barrel porins like lamb maltoporin of e. coli have specific binding site for substrate inside channel and shows michaelis-menten kinetics of transport. many prokaryotic porins are closed at high voltage of - mv. vdac also has voltage dependence and can be easily gated by change in ph, and their permeability is controlled by membrane potential. they are involved in transport of atp and adp; that's why a large number of positively charged amino acids are present in interior wall of pore. the protein ion channels are passive transporters, are made up of transmembrane, are selective for their substrate ions, and have much faster rate of transport than pumps. these channel proteins do not interact with transporting ions and have two conformations closed and open, to regulate flow of ions. the channels may be classified into ligand-gated channel and voltage-gated channel. ligand-gated channel opening and closing depends on the interaction with positive or negative ligand. the binding of ligand induces conformational change in protein for opening or closing. on structural basis, ligand channel (lgic) is classified into three classes. (nicotinic ach) superfamily, which has pentamer cysteine loop such as nicotine acetyl receptor hydroxyl tryptamine, zn-activated channel cation transport, gama-aminobutyrate (gaba a ), strychnine-sensitive glycine receptors for anions, glutamate-gated chloride channel of c. elegans. which has four subunits divided into three subclasses like n-methyl d-aspartate (ndma) receptor, a-amino- -hydroxy- -methyl- isoxazolepropionic acid (ampa) and kainate receptors. . p x receptor family, which has three subunits of cysteine loop. these are cation permeable-gated channels and opened or closed by atp interaction (fig. . ). the trimeric atp-activated channels are permeable to cations like sodium, potassium, and calcium. there are seven subclasses of the group, which are involved in various physiological functions like muscle contraction, neurotransmitter release, immune responses' regulation. p x receptors are present only in eukaryotes and ubiquitously expressed. p x are trimers of any three from seven variable subunits (p x - ) and exist as homomer or heteromer. each of the subunit may have two transmembranes. the detail is discussed in chap. . in nerve transmission, the neurotransmitter is released in synaptic cleft by presynaptic vesicles. these neurotransmitters have to be internalized by postsynaptic membrane for the propagation of impulses. for internalization, these neurotransmitters bind to receptors on postsynaptic membrane and induce conformational change in receptor to open channel for translocation. the gaba and glycine receptors are anion channels but inhibitory in nature. gaba receptors have two subdivisions as gaba receptors family of ligand-gated channel (ionotropic) gaba a and g receptor protein family (gaba b ). the g receptors are discussed in chaps. and . the ligand for gaba a receptor is c-aminobutyrate. the cysteine loop family in eukaryotes has conserved motif of two cysteine separated by other amino acids. the gaba a receptors are multisubunits with major isoform of gaba a. receptors are a b and c . the various isoforms of these proteins are found in human as asubunits with six isoforms, b-subunits three, c-subunits three, q-subunits three, and -, d-, h-, and p-subunits one copy. the single subunit of gaba a receptor has around amino acids with both c-terminal and n-terminal that are extracellular. its half hydrophilic n-terminal has signature motif of cysteine, and other c-terminal has four transmembrane helices from m to m . the m transmembrane forms lining of channel. the large intracellular loop between m and m constitutes modulation site for phosphorylation. the m and m domains of receptor may interact with other regulatory proteins. the functional receptors are made up of five subunits as homopentamer or heteropentamer. the -and d-subunits enhance polymeric association of monomer ( fig. . ). ( ) the pentameric cysteine loop superfamily receptor superfamily. ( ) the tetrameric ionotropic glutamate receptors are subdivided into n-methyl-d-aspartate (nmda), a-amino- -hydroxy- -methyl- -isoxazolepropionic acid (ampa), and kainate receptor subfamilies. the highly schematic topography of each receptor category indicates the locations of the extracellular and intracellular termini, the number of transmembrane spans (large colored cylinders), and cysteine residues participating in disulfide bond formation (yellow circles). red cylinders indicate a-helical regions participating in ion conduction/selectivity the density of these receptors varies from cell to cell. the gabaa are found in nerve cells, muscle, and liver and on immune cells. the gaba receptors initially were thought to be only on postsynaptic membrane for opening of gaba chloride channel for hyperpolarization of a depolarized membrane but recent studies have confirmed their presence on dendrites, and other tissues also for the regulation of synapses. the gaba a receptors are gated chloride anion channels and occasionally may translocate bicarbonate anions. gaba binds to two binding sites at the interface of a-/b-subunit of receptor. the important region for ion translocation is a-subunit segment (f loop), which bulges out from ligand binding site toward the ion channel, and in c-subunit, this segment modulates channel activity. the second segment, a lysine residue of the short loop between m and m , is important for the formation of a salt bridge during the process of gating. the third segment resides within the b -b linker of the b-subunit. gaba binding to binding sites at the interface of a-/ b-subunit stimulates conformational change around binding site which passes through open and many closed states (flipped states) for final gate opening. glutamate receptor channels n-methyl-d-aspartate (nmda) the n-methyl-d-aspartate (nmda) receptors are known as glutamate-gated cation channel for calcium permeation. the tetrameric ionotropic receptors include nmda, a-amino- -hydroxy- methyl- -isoxazolepropionic acid (ampa), and kainate receptor as per their synthetic ligand binding. these are excitatory neurotransmitters. in excitatory transmission, glutamate released from presynaptic membrane binds to ndma or ampa receptor of postsynaptic membrane. the ndma receptor family have three different subunits known as glu n - . the crystallographic and functional studies have revealed detail structure and mechanism of channel opening. active nmda receptors are tetramer with two glun and two glun . the monomer subunit may be divided into four distinct domains as extracellular n-terminal domain, ligand binding domain, pore-forming membrane domain, and c-terminal intracellular. the extracellular n-terminal domain is linked to ligand binding domain through small linker, which is also attached to pore-forming transmembrane ( fig. . ). the transmembrane is liked to c-terminal domain. the channel opening starts in sequential order of ligand binding ! change in conformation ! channel opening. two separate ligands are required for nmda receptors activation. the ligand binding domain of subunit has two subdomains s and s . the s domain of two neighboring subunits, at their interface, creates ligand binding site and is rigid in structure but lower one s domain is mobile without any interaction. in typical ndma receptor, glun and glun will interact with two molecules of glutamate and two molecules of glycine. the receptor with glu n and glu n will require only glycine for the activation. the nmda receptors have high affinity to glutamate, high calcium (ca + ) permeability, which may be blocked by magnesium ion in voltage-dependent manner. the nmda differs from ampa receptor of the same family in its function. the n-methyl-d-aspartate (nmda) receptors unlike a-amino- -hydroxy- -methyl- -isoxazolepropionic acid (ampa, activation time ms) are slow in activation (app. - ms) and deactivation (app. - ms). ampa receptor initiates rapid polarization in response to neurotransmitter release during synaptic transmission but nmda receptors only care for time span of synaptic currents. the glycine is coagonist for nmda receptor. two molecules of glycine and two molecules of glutamate bind on separate binding site in receptor for the activation and opening of channel, which results in influx of calcium and sodium with efflux of potassium. the electrical synapses are converted into chemical synapses through nmda receptor because influx of a the arrangement of nmdar subunits with four hydrophobic domains (m through m ), two ligand binding domains (s and s ), and amino and carboxyl terminal domains (ntd, ctd). b monomer subunit with n-terminal signal peptide on extracellular side, transmembrane helices m , m , and m , m , a cytoplasmic reentrant hairpin loop a link between m and m and ligand binding domains s and s , c twodimensional membrane folding model of glycine binding nr and glutamate binding nr subunits . tetrameric ionotropic glutamate receptor channels … calcium in postsynaptic membrane activates calcium-dependent signaling pathway for the modulation of synaptic structure, plasticity, and connectivity. the signaling is discussed in chap. . the independent binding of two ligands, glycine and glutamate, collectively induces change in conformation to open channel. the closing of channel may result in glutamic acid release and desensitization of receptors. it has been observed that the nmda receptors may be in desensitized form with bound glutamate for long period. the other function of nmda receptor in learning and memory can be inhibited by magnesium ion in voltage-dependent mode. in close state of receptor, the magnesium is bound to receptor in inhibition mode, which can be released only by sufficient depolarization of the membrane. this kind of interaction empowers nmda receptor to act as coincidence detector for pre-and postsynaptic changes. the slow kinetics of nmdar-mediated excitatory postsynaptic current (epsc) supports temporal summation and decreases dendritic filtering of synaptic input. the receptors provide strength to synapses through long-term potentiation (ltp) and weakness by long-term depression (ltd) for learning and memory. through calcium influx, the overstimulation of nmda receptors may cause alzheimer, parkinson, huntington, and amyotrophic lateral sclerosis (als). physiologically, nmda receptors may be desensitized by unfavorable interaction between glycine and glutamate to cause close conformation of receptor or calcium influx activate calmodulin to remove actin from receptor to keep receptor in desensitized mode. the receptors activity may be decreased by zinc, polyamines, and proton. the phosphorylation of receptor protein enhances open form of channel by removing magnesium and inducing favorable conformation. the voltage-gated ion channels open or close in response to change in voltage and the unit of membrane potential. in excitable cells like nerve and muscle, the chemical or electrical signaling is initiated through voltage-dependent sodium or calcium influx. the sodium channel protein in human is composed of large a-subunits ( kd with amino acids) and small b-subunits ( - kd); the small b-subunit has two isoforms b- and b- . the sodium channel b-subunits have n-terminal extracellular domain into immunoglobulin-like fold, a single transmembrane segment, and a short intracellular segment. these units may form heterodimer or heterotrimer. the a-subunits are linked noncovalently to b- and through disulfide linkage to b- . the voltage-gated sodium ion channels are also known as nav channels. in human, a-subunit has various isoforms like nav . , nav . , nav . , nav . , nav . , nav . , nav . , and nav . . the nav . , nav . , and nav . are found in peripheral nervous system and nav . , nav . , nav . , and nav . in central nervous system, and the nav . resides in heart muscle and nav . in skeletal muscle. the a-subunit is coded by ten different genes in human but one of these codes for nonchannel a-protein is known as sensing helix. b-subunits have four isoforms as b- , b- , b- , and b- . the b- and b- associate with a-subunit noncovalently and b- and b- through disulfide linkage. navb-subunits resemble cell adhesion molecules in structure and may have role in localization. the three-dimensional structure of voltage-gated sodium channel has revealed structural detail, mechanism and catalytic amino acids involved, drug target site, and regulatory site. the bacterial sodium channel (navab) is seen to have central pore surrounded by four pore-forming helix segments s and s segments and the intervening pore loop. the four voltage-sensing elements made up of s -s segments are symmetrically associated with the peripheral pore elements. the structural analysis of bacterial sodium channel (navab) has shown the swapping of functional domain of one subunit to other like all individual voltage-sensing elements are found in close associated with the pore-forming element to neighbor as shown in fig. . in dark colors. this type of arrangement and association is also observed in potassium channel (fig. . ). the navab sodium channel are found to have large outer entrance with narrow selectivity filter, a large central cavity filled with water and layered by the s segments and an intracellular activation gate at the crossing of the s segments and intracellular surface of the membrane. sodium and potassium channel has similar type of structure but differs significantly in permeation mechanism. the potassium ion channels discriminate k + by direct interaction with a series of four ion coordination sites created by the carboxyl group of ion selectivity filter amino acid residues in the zone. water is excluded and does not interact with selection filter, while the navab ion selectivity filter has a high-field-strength site for ions at its extracellular end because of the side chains participation of four highly conserved and key determinants glutamate residues. the sodium ion with two planar water molecules for hydration could fit in this high-field-strength site. on outer side of the field, two carboxylic groups of peptide create coordination site for sodium binding with four planar waters of hydration. these negatively charged group interaction with sodium removes water of hydration partially for permeating sodium as a hydrated ion, which interact on the way with the pore residues through its inner shell of bound waters. the local anesthetics, antiarrhythmic and antiepileptic drugs may inhibit this channel by binding to inner side of segment (drug receptor site) but binding to this site navab channels is solely controlled by the side chain of phe , single amino acid residue. the sodium channels open in response to depolarization and within fraction of second are closed also for repetitive firing of action potential in synapse. the short intracellular loop connecting a-subunit iii and iv domain acts as inactivation gate in channel inactivation. this acts as an intracellular blocking particle by folding into the channel structure and blocks the pore during inactivation. the nmr study of channel inactivation gate has shown the presence of a rigid a-helix followed by two loops of protein with ifm motif and a neighboring threonine residue on their surface for interaction participate in closing. the inactivation gate bends at conserved pair of glycine residues to fold into the intracellular mouth of the pore. the fast inactivation gate is not found in homotetrameric bacterial sodium channels. the voltage sensing is executed by the s transmembrane segments of sodium channels, which has four to eight repeated motifs of a positively charged amino acid residue (usually arginine), followed by two hydrophobic residues, and shifts the gating charges of sodium channels in the sliding helix in voltage sensing. the gating charges are stabilized in their transmembrane position by ionic interacting pairing with neighboring negatively charged residues, which can exchange their ion partners during outward movement. fig. . subunit primary structures of the voltage-gated sodium channels y, sites of probable n-linked glycosylation; p in red circles, sites of demonstrated protein phosphorylation by pka (circles) and pkc (diamonds); green, pore-lining segments; white circles, the outer (eeee) and inner (deka) rings of amino residues that form the ion selectivity filter and the drug binding site; yellow, s voltage sensors; h in blue circle, inactivation particle in the inactivation gate loop; blue circles, sites implicated in forming the inactivation gate receptor the formerly referred leaky channels, resting, back ground, or constitutive channels are now referred as k p or voltage insensitive channels. the k p channels are ubiquitously found in all organisms. the functionally active k p channel has two subunits, and each subunit is made up of two pore regions (p and p ), four transmembrane domains (m -m ), and two characteristic extracellular helices cap c and c . the yeast tok , kp channel with eight transmembrane helices (m -m ). the tok ( p/ tm) is exceptionally outward rectifier. these channels are highly regulated and functionally important in nervous system, muscles, blood vessels, endocrine system, and kidney. according to sequence homology, eukaryotic k p channels are classified into six groups; ( ) the twik clade is weak inward rectifying channels, ( ) trek clade is mechanosensitive and also regulated by lipids, ( ) task clade channels are regulated by acid ph, ( ) talk clade is alkaline ph-sensitive, ( ) thik clade is inhibited by halothane, and ( ) tresk clade is present in spinal cord. unlike voltage-dependent channels (nav, kv, cav), these k p channel do not have voltage-sensing domain and have two pores. the k p are not inactivated during depolarization except twik- . the voltage-dependent channels have one pore made by four transmembranes (fig. . ) . k p-channels in the cell membrane can be regulated at the transcriptional level. the presence of cap helices c and c is unique to these channels, which vertically extends * Å on the extracellular side of membrane to cover entrance with two lateral portals for k + ions. the pore-lining helices, m and m , traverse obliquely through the membrane and m and m , and the outer helices are more vertically oriented. the transmembrane segments of the channel have lateral openings between m and m facing toward lipid environment. the selectivity filter and the pore helices have fourfold symmetry with inner wide open cavity. the extracellular cap, the central cavity, and the inner opening of channel are found in twofold symmetry unlike other potassium channels. the two subunits were found to be assembled in a simple antiparallel manner. the c -c linkers are linked via a disulfide bridge. the two opposite pore helices and the corresponding pore loops contribute to forming the selectivity filter. the m -helix of one subunit is adjacent to the end to m -helix of the other subunit. the c-helix is amphipathic with charged residues facing the cytoplasmic side. the human k p channel cytosolic end of m has two to four charged residues and forms an amphipathic helix. the cytosolic part of the pore participates in gating and permeation. the crystallized k p-channel has been found to have lateral opening at the interface between the m -helix of one subunit and the m -helix of the other subunit to connect the inner cavity with the hydrophobic core of the cell membrane. the lateral cavity, occupied with the hydrophobic tails of membrane phospholipids, may be important in channel gating because of lipid interaction with m and m domains and alkyl chains of membrane lipids into the cavity through the lateral opening influences the conductance and the pharmacological properties. like all voltage-dependent channels, the glycine residues are conserved m and the m helices of all k p-channels for creating kink in m and the m domain around the hinge of g for moving m -helix from a "down" to an "up" configuration for transport, where lateral fenestration is closed and ions are passed through the selectivity filter. in down conformation, the central cavity m -helix crosses the membrane in such a way that lipids of the central cavity blocks the permeation. k p-channels are lined up with hydrophobic residues in layer. the opening of lower activation gate promotes the opening of upper inactivation gate, and c-type inactivation is promoted unlike voltage-dependent gate. during depolarization, all k p-channels have shown immediate current component and following time-dependent component of outward current. this instantaneous component supports all k p channels to have open probability at negative potentials, and the time-dependent current changes support the regulation of k p-channels by voltage changes. the steady-state current-voltage relation under physiological conditions (with - mm k + extracellular concentration) of k p channel, studied at depolarized potentials, is much larger than the current flowing through k p channel at resting membrane potential to support role of k p channel in action potential configuration. the anesthetics enhance the leakage of ions in neural tissue and decrease excitability. in various tissues, k p channels are present to perform various functions and may also play role in apoptosis subjected to further analysis (figs. . and . ). structure and regulation of the vacuolar atpases assembling the puzzle: oligomerization of alpha pore forming protens in membrane structure of a bacterial multidrug abc transporter the bacterial phosphoenolpyruvate: carbohydrate phosphotransferase system: regulation by protein phosphorylation and phosphorylation-dependent -protein interactions aquaporins are multifunctional water and solute transporters highly divergent in living organisms understanding poly specificity of multidrug abc transporters: closing in on the gaps in abcb structure of an abc transporter in complex with its binding protein structural mechanism for light-driven transport by a new type of chloride ion pump, non labens marinus rhodopsin- the gramicidin ion channel: a model membrane protein gating of two pore domain potassium channels a structural overview of the plasma membrane na + , k + -atpase and h + -atpase ion pumps lactose permease h -lactose symporter: mechanical switch or brownian ratchet? p-type atpases phosphoenolpyruvate: carbohydrate phosphotransferase systems of bacteria abc transporters: the power to change much more than a leak: structure and function of k p-channels the mechanism of pore formation by bacterial toxins aquaporins: important but elusive drug targets structure, function, and pharmacology of nmda receptor channels biology of human sodium glucose transporters the role of mitochondrial porins and the permeability transition pore in learning and synaptic plasticity structure and mechanism of abc transporters structure and evolution of mitochondrial outer membrane proteins of b-barrel topology key: cord- -pnxjrtgo authors: zhang, pingping title: application of upt-poct in anti-bioterrorism and biosecurity date: - - journal: principles and applications of up-converting phosphor technology doi: . / - - - - _ sha: doc_id: cord_uid: pnxjrtgo with the exception of toxins, bioterrorism agents are mainly microorganisms, many of which cause serious infectious diseases. up-converting phosphor technology-based point-of-care testing (upt-poct) can detect bioterrorism agents from various samples with high sensitivity and specificity, in particular it shows robust performance for complicated samples, such as food, powder, viscera and grains. the tolerance of upt-poct to sample is based on the physical and luminescence stability of ucnps, the stable covalent interaction between ucnps and antibody, as well as the strong buffering capacity of the detection system. reliable results can be obtained in a short time period using a portable biosensor by nonprofessionals owing to the simple nature of upt-poct operation and sample pre-treatment. based on pathogenicity, bioterrorism agents can be categorized into lethal and incapacitating agents. lethal bioterrorism agents have high mortality, for example, the mortality of septicemic plague and pulmonary tularemia can reach % and %, respectively, in the absence of antibiotic treatment. y. pestis, b. anthracis, f. tularensis type a, b. pseudomallei, yellow fever virus, smallpox virus, rickettsia rickettsii, chlamydia psittaci, and botulinum toxin are all lethal bioterrorism agents. incapacitating bioterrorism agents can make people defenseless, examples include brucella spp. and coxiella burnetii. bioterrorism agents can be transmitted by air, food, and water, and some can be transmitted person-to-person, such as y. pestis (begier et al. ) , b. anthracis, and ebola virus. the bioterrorism agents disseminated through air can be made into aerosols and threaten public health on a large-scale, examples include rickettsia rickettsii, y. pestis (agar et al. ), and b. anthracis (estill ). some zoonotic pathogens can infect people through contact with infectious animals during slaughter and leather treatment, and animal husbandry, as well as contact between people (begier et al. ) . transmission through food includes consumption of the meat or milk of infected animals, for instance, people can be infected by v. cholerae through contaminated seafood (finelli et al. ) . water is also an important transmission medium and is the main transmission route for v. cholerae (hill et al. ). in addition, some agents, for example, b. anthracis spore can survive in the silt at the bottom of a riverbed for decades, and f. tularensis can survive in the cold water of a river for months (chitadze et al. ), seeking the chance for outbreak. transmission media arthropods such as mosquitos, flies, lice, mites, and ticks, are widespread in nature. the transmission media of y. pestis, f. tularensis, yellow fever virus, and rickettsia przewalskii are flea, tick, mosquito and pediculus humanus corporis, respectively. the suspicious incidents caused by bioterrorism agents can easily lead to public panic because of their perniciousness. the misdiagnosis of the diseases caused by bioterrorism agents, rapid deteriorations for acute and serious infectious diseases, and the limitations of therapeutic means, all highlight the importance of preventing the release of bioterrorism agents. low pathogenic dose, diversity of pathogenic types, high mortality or disability rate, and the potential for widespread dissemination, are all characteristics of bioterrorism agents, as well as strong adaptability to the environment in the exposed zone resulting in long-term threat to public health. several microorganisms can multiply rapidly in vivo, causing serious diseases, for instance, the infectious dose of f. tularensis type a is less than ten live cells. bioterrorism agents attack the respiratory tract, digestive tract, skin, blood, and glands. many of the agents cannot be handled by the immune system, and y. pestis can even survive, proliferate and spread in macrophages (lukaszewski et al. ) . acute and serious diseases occur after a short incubation period, often only several days. for instance, the symptoms of pneumonic plague include high fever, cold shivers, cough, chest pain, hemoptysis, and dyspnea, followed by serious poisoning symptoms and death in two to four days. the mortality of pneumonic and generalized plague is up to ~ %. brucella spp. with anti-phagocytic capsules can proliferate in the lymphatic system and then enter into the blood resulting in toxemia, and its ability to evade elimination by the body results in long-term joint pain, fatigue, and disability, dramatically decreasing the quality of life of the patient. the most catastrophic potential outcome is a worldwide epidemic. three historic large-scale plague epidemics caused million deaths (prentice and rahalison ) , seven historic cholera epidemics involved countries, and brucellosis is prevalent in countries. diagnosis can be based on epidemiological history, clinical syndromes, etiology, and serology detection. epidemiological history includes residency in the epidemic areas or entrance into these areas in the last two weeks before morbidity, bites by arthropods, and contact with or consumption of infected products or water. clinical syndromes of some bioterrorism agents appear in isolation, however agents with many infection routes can cause various clinical syndromes. for example, the main clinical syndrome of cholera is diarrhea, however at least six syndromes have been found for tularaemia, including bubonic, pneumonic, gastrointestinal, and systemic (typhoidal and septicemic) tularaemia. the similarity of clinical syndromes between diseases caused by bioterrorism agents and by other factors, makes accurate pathogenesis diagnosis more difficult for doctors when the etiology is unknown. for example, patients with fever caused by f. tularensis are easily misdiagnosed with influenza (simsek et al. ) , while there are no differences the in clinical syndromes between pneumonia caused by f. tularensis or that arising from other causes (stralin et al. ) . misdiagnosis and delayed treatment are responsible for lack of safeguard implementation and the subsequent dissemination of bioterrorism agents in medical institutions. therefore, etiology detection is critical for determining and preventing infectious disease. however, the low numbers of pathogens in the initial stage of a disease are difficult to find by etiology detection, fortunately early diagnosis based on the detection of antibodies in blood is a plausible approach in practice. because of the great threat of bioterrorism agents to the lives of patients, essential therapy must be administered in time to avoid death and poor prognosis. for instance, water and electrolytes must be administered to patients infected by v. cholerae in time because severe dehydration and failure of microcirculation caused by diarrhea often occur during the progression of cholera. the prognosis is usually poor owing to the limited therapeutic means. antibiotics are often excessively applied for saving the lives of patients infected by bacterial bioterrorism agents, such as streptomycin specific for plague and many antibiotics that are effective for tularaemia, however the side effects of this therapy method are osteoporosis and joint injury. eliminating infection sources, cutting-off transmission routes, and protecting susceptible patients, are all methods for prevention and control of the spread of bioterrorism agents. quarantine is essential for infectious bioterrorism agents, including isolation of prime areas of disease outbreak, quarantine of patients and people who have gone to countries with epidemics, conflagration and deep interment of cadavers, use of disinfectant, and correct treatment of material from patients with high-temperature and high-pressure. individual and environmental defenses should be enhanced when nursing and treating patients or infected animals, as well in the resulting treatment of cultures in scientific research. pre-inoculation of vaccines is a good prevention method for protection of people in natural foci, doctors and scientific staff, for example, vaccines or attenuate strains of y. pestis, f. tularensis, and b. anthracis can be inoculated by cutaneous scarification. however, no effective vaccine has been obtained for some bioterrorism agents, such as chlamydia psittaci. there are some effective virus vaccines, such as vaccinia vaccine (against smallpox virus), rabies vaccines, hepatitis b vaccines, and hantavirus vaccine. however, mutated viruses often emerge as rna viruses, especially for retroviruses such as human immunodeficiency virus, leading to some vaccine failure and the necessity for preparation of new vaccines against the mutated virus. owing to the high pathogenicity of bioterrorism agents, as well as the high transmission capacity of microorganisms, essential isolation and protection measures must be carried out to ensure biosecurity during the process of detection. prompt and accurate testing favors the detection of suspicious substances, so that an emergency signal can be issued or a false alarm can be revealed. in addition, multiplexed detections can be applied to unknown pathogens to improve efficiency and reduce environmental contamination caused by excessive operation. protective measures during the detection process are essential to ensure individual and environmental security and prevent the spread of bioterrorism agents. specialized laboratories for pathogenic microorganisms, or detection vehicles on site equipped with such laboratories are necessary for lethal bioterrorism agents. for detection on site, operators should wear rubber gloves, surgical masks, hats, and protective clothes. isolation for patients and infected animals, and blockades of the contaminated region are essential. the screened suspicious matter should be sent to the laboratory for further identification in a biohazard marked container that is waterproof, breakage-proof, and high temperature and pressure resistant. according to the national standard for laboratory security, the defense levels of bio-laboratories are from to . experiments involving the most infectious bioterrorism agents must be performed by professionals using a biosafety cabinet in a level or laboratory. the measures for infection prevention in the laboratory include pre-inoculation with vaccine, wearing protective clothes, using biosafety cabinets for culture and infective material, high-pressure treatment for growth medium and contaminated gloves, as well as sprinkling disinfectant in the biosafety cabinet. the detection limit, specificity, and tolerance of detection methods determine their accuracy. the detection limit for bioterrorism agents must be very low, ideally single cell, because of their high pathogenicity in low dose and the high capacity for proliferation of some microorganism bioterrorism agents. pre-incubation of microorganism bioterrorism agents to increase detection rates is not permitted. specificity is particularly critical for a detection method because there are incalculable microorganisms and other organisms in nature. no specific reaction should occur for substances or strains that share structural similarity, close genetic relatedness or similar transmission routes with the targets. a detection method must be available for different specimens within appropriate operational-error to ensure the stability. various fresh or decomposed animal tissues obtained in natural foci and the white powders used by terrorists for concealment, such as flour, milk powder, and putty powder, are representative of the complicated specimens that the detection method will be confronted with. the errors brought by non-professional operation are also considered to ensure the detection accuracy in practice. in addition to safety and accuracy, detection time should also be shortened. a short detection time is beneficial for therapy, cutting-off transmission routes, preventing the dissemination of contamination, and promptly eliminating negative social effects. multiplex detection has been developed to improve detection efficiency, and reduce sample volumes and operational handling. compared with the detection of individual targets, multiplex detection reduces the workload and shortens detection times. in addition, multiplex detection can give results for many targets at once, therefore smaller sample sizes are sufficient, which is especially important for precious low-volume samples. less operational handling minimizes the possibility of contamination of the inspector and environment with bioterrorism agents. because of difficulties arising from multiple reactions in one system and simultaneous multiple signal extraction, multiplex detection still shows unsatisfactory performance in stability, anti-interference, and reproducibility. despite these shortcomings, multiplex detection is urgently required for identification of unknown pathogens and simultaneous detection of multiple pathogens in one sample because multiple bioterrorism agents can be released in one sample, for example, coxiella burnetii, chlamydia psittaci and influenza virus have been combined into aerosols and applied for biowarfare. bio-threats are becoming increasingly serious with developing technology because of reductions in cost, and improved transmission and operation. many bioterrorism agents can be easily obtained from nature, such as zoonotic microorganisms from natural hosts, and ricin and abrin, which are easily prepared from plants. modern fermentation technology promotes mass-production of microorganism bioterrorism agents (yang ), while many toxins can be synthetized using chemical methods. water, air conditioning systems, food, and letters have been used as the vectors for bioterrorism agents in bio-attack incidents, and the agents can appear in the form of powders and aerosols, amongst others. the detection of bioterrorism agents includes surveillance in natural foci and treatment for public health emergencies. surveillance, especially for common infectious disease, is an efficient measure for preventing disease outbreaks. the objects of the monitoring are different depending on the sort of disease. for example, rodents and fleas are natural reservoirs for y. pestis (prentice and rahalison ) ; water and plankton are the natural environment and reservoir of v. cholerea respectively (huq et al. ) ; and b. pseudomallei can survive in tropical and subtropical natural foci (draper et al. ) . public health emergencies, such as bioterrorism attacks, laboratory releases, collective food poisoning, and concentrated outbreaks of cases, require prompt responses based on the detection results. according to the definition specified by the committee of poct, chinese association of medical equipment, point of care testing (poct) is a detection method implemented on site, and it reports results in a short time period using portable analytical instruments and associated reagents. to satisfy the strict requirements of poct, a detection method must be sufficiently rapid, sensitive, and specific. screening detection on site mainly relies on poct methods. the short response time for surveillance in natural foci and during public health emergencies has provided powerful support for the prevention of disease outbreaks. the minimal operation procedures that are a feature of poct methods make poct applicable for detecting bioterrorism agents to reduce the release of the agents. immunochromatography is well known as a poct method for onsite screening of bioterrorism agents because it can give results from simple sample loading and the used strip can be directly disposed of after treatment with high temperature and pressure. the sensitivity and specificity of traditional immunochromatography methods are often too low because the physical interaction between antibodies and gold particles is fragile, and the results are analyzed by the naked eye. up-converting phosphor technologybased point-of-care testing (upt-poct) as described below are based on the covalent conjugation of upconversion nanoparticles (ucnps) and antibodies, and then the emission signal at nm resulting from excitation at nm can be transmitted into readable electrical signals by biosensors. therefore, the upt-lf assay can detect various samples with high sensitivity and specificity, in particular it shows robust performance for complicated samples. the detection mode of upt-poct for bioterrorism agents hinges on the molecular size of the detection target. double-antibody sandwich mode is used for detection of macromolecules, while competition mode is used for micro-molecules. in addition, the principle of the double-antigen sandwich mode is in parallel with that of the double-antibody sandwich mode for utilization of macromolecules, mainly antibodies. upt-poct based on the double-antibody sandwich mode is mainly applied for the detection of bacterial antigens or protein toxins, such as y. pestis (yan et al. ) , brucella spp. (qu et al. ), b. anthracis spore (li et al. ) , f. tularensis (hua et al. b) , b. pseudomallei (hua et al. a) , v. cholerae (hao et al. ), e. coli o (wang et al. ), ricin , and abrin . while upt-poct based on the double-antigen sandwich mode is applied for the detection of antibodies against bioterrorism agents, such as antibodies against y. pestis (hong et al. ) and hepatitis b surface antibody (li et al. ). for example, in the detection of y. pestis ( fig. . ) ; mouse anti-y. pestis antibody is immobilized on the nitrocellulose membrane as the test (t) line, while mouse anti-y. pestis antibody is combined with ucnps covalently. for positive detection, y. pestis in samples is captured by ucnps-mouse antibody conjugates, and then flows forward and is captured by mouse antibody at the t line of the nitrocellulose membrane, forming t line-mouse antibody -y. pestis-mouse antibody -ucnps conjugates. ucnps can emit visible light signals after excitation by infrared light, and the intensity of the signals is in direct proportion to the concentration of y. pestis. whether there are y. pestis present or not, goat anti-mouse antibody-mouse antibody -ucnps conjugate will be formed on the control (c) line on the nitrocellulose membrane for quality control. the signal ratio between the t and c lines, the t/c ratio, is defined as the detection result, and t/c values increase with the increase in quantity of y. pestis in the sample. in the double-antigen sandwich mode, bioterrorism antigens, such as f antigen of y. pestis, are dispensed in the t line and combined with ucnps respectively, and then the corresponding antibody is detected. competitive mode is used to detect micro-molecular matter that is too small to be detected by double-antibody sandwich mode. upt-poct based on competitive mode is applied for the detection of mycotoxin, such as aflatoxin b (afb ) , aflatoxin m and t- toxin. using the detection of aflatoxin b (afb ) as an example (fig. . ) , afb -bsa cross-linking agent is immobilized on the nitrocellulose membrane as the t line, while mouse anti-afb antibody is combined with ucnps covalently. for positive detection, ucnps-anti-afb antibody is combined with afb in samples and cannot be further captured by afb -bsa crosslinking agent on the nitrocellulose membrane, leading to the p. zhang the upt-poct assay for multiplex detection is based on a -channel upt-lf disc or multiple t lines for one strip. the upt-lf disc is composed of ten one-target strips, therefore ten targets can be detected from the loading of one sample. for instance, a upt-lf disc prepared with ten proteins of y. pestis based on the doubleantigen sandwich mode has been realized for the detection of antibodies against y. pestis, providing a clue for seeking diagnosis biomarker of y. pestis (hong et al. ) . a strip with multiple t lines can detect multiple targets, such as the upt-lf assay developed for simultaneous detection of v. cholerae serogroup o and o (hao et al. ). sensitivity and specificity are crucial to the performance evaluation for a detection method, and the evaluations of upt-poct for detection of different bioterrorism agents are shown in table . . the sensitivity of upt-poct for bacterial bioterrorism agents can reach cfu/ml (namely cfu for each test based on ten-fold sample dilution and µl of sample loading volume), and that for toxin can reach . ng/ml. the quantitative range covers four to five orders of magnitude, even six for detection of b. anthracis spore (zhang et al. ). the coefficients of variation for detection are all below %. upt-poct shows excellent specificity to the bioterrorism agents that share genetic relatedness, similar transmission routes, or similar structure with the targets. the tolerance to biochemical agents (table . ) and operation error of upt-poct for the detection of five bacterial bioterrorism agents has been evaluated (zhang et al. ; hua et al. a, b) . the high concentration of agent that the detection method can tolerate is defined as the tolerance limit. the tolerance limits of upt-poct for ph, ionic strength, viscosity, and bio-macromolecule concentration can reach ph - , ≥ mol/l of nacl and kcl solution, ≤ mg/ml peg , ≥ % glycerol, ≥ mg/ml of bsa and ≥ mg/ml of casein, respectively. at some tolerance limits, the sensitivity could be improved by one order of magnitude. the operation error, including the volume variation of the sample (from − to %), sample treating buffer (from − to %), and loading mixture (from − to %), has little influence on the sensitivity and specificity of upt-poct. upt-poct shows excellent performance for detection of bacterial bioterrorism agents in simulated samples, such as food, powder, and viscera (zhang et al. ; hua et al. a, b) (table . ), and it can also effectively detect abrin and afaltoxin b in food and grains zhao et al. ) (table . ). the highest tolerance limit of upt-poct to simulated samples could reach mg/ml. for detection of v. cholerae in field water samples obtained from sample collection sites in guangzhou city (china), upt-poct is more sensitive than the isolation-culture method and colloidal gold immnochromatography assay, and its sensitivity could match that of real-time fluorescent pcr with fewer false positive results (hao et al. ) . the sample pre-treatment for detection by upt-poct is merely grinding, or supernatant extraction by centrifugation after grinding and shaking on a vortex shaker for min (or ultrasonicating for min), and then the sample can be directly mixed with sample-treating buffer for detection. the tolerance of upt-poct to sample is based on the physical and luminescence stability of ucnps, the stable covalent interaction between ucnps and antibody, as well as the strong buffering capacity of the detection system. detection methods for bioterrorism agents include the isolation-culture or animal inoculation method, biochemical method, nucleic acid method, and immunization method. ≥ ** ≥ ** the sensitivity of upt-lf strip improved by one order of magnitude at that tolerance limit microorganism bioterrorism agents can be identified by the isolation-culture method or inoculation of animals, and toxin can also be injected into animals for species identification. selective culture medium, as well as inoculation and dissection of susceptible animals, are both common experiments. the culture methods for the various bioterrorism agents are different. ( ) bacteria can be identified by selection in selective medium, for example, alkaline peptone water is the medium for selective culture of v. cholerea, while y. pestis can be identified by culturing with bacteriophage lysis. ( ) isolation of viruses by cell culture is the main method for virus detection because viruses can only survive in live cells. after virus infection the mutated cell can be directly detected by microscopy, or observed through the change in ph of the medium, hemadsorption or hemagglutination. culture in chick embryo is also common for viruses such as influenza virus. animal incubations are better than cell incubation for some viruses, for example, inoculation of mice is the best culture method for rabies virus. ( ) rickettsia bioterrorism-associated agents are cultured and isolated by guinea pig and chick embryo. ( ) the types of toxin can be identified by lethality or the animal response after inoculation of susceptible animals or cells, such as vomiting and diarrhea caused by s. aureus enterotoxin b. the susceptible animals for toxins are different, for instance, mouse and cat are sensitive for botulinum toxin and s. aureus enterotoxin, respectively. the isolation-culture method is the basic detection method-even gold standard detection method-for bacterial bioterrorism agents. however, it must be conducted by professionals in particular institutes equipped with biosafety facilities, and bioterrorism agents can be easily transmitted owing to improper operation or defense. because of its low sensitivity, the isolation-culture method is often combined with other methods to improve the accuracy of detection, such as the bacteriophage lysis method (zhao et al. ) . for example, the bacteriophage lysis method for identification of y. pestis is realized through adding bacteriophage into cultured bacteria at - °c. the low sensitivity and specificity of the animal injection method for detection of toxins are caused by the diversity of toxins and individual differences between animals. the biochemical method is based on the properties or metabolism characteristics of the microorganism or toxin, and is in fact a detection system because the species cannot be determined by one property, such as the systemic biochemical detection according to diagnostic criteria for cholera (ws - , healthy industry standards of the people's republic of china). the poor sensitivity and specificity of the biochemical method, as well as the complicated operation, are a result of the property similarities between microorganisms or biological substances. the nucleic acid method is based on the principle of dna replication in vitro, such as the polymerase chain reaction (pcr) and loop-mediated isothermal amplification (lamp). pcr is a laboratory detection method that parallels the dna replication process in vivo. the single strand dna template is formed at °c, and then it can match with a primer at annealing temperature (about °c) based on their complementary sequences, subsequently a new complementary dna strand can be obtained using dntp as a reactive material catalyzed by taq dna polymerase. these strands can be further used as a template for the next cycle, therefore the target dna can be multiplied millions of times by dozens of cycles. the products can be determined by dna electrophoresis for common pcr, while for real-time quantitative pcr the amplification process can be monitored by the change of fluorescence signals. in addition to dna as a template, rna could also be used for amplification, and this is realized using the reverse transcriptional pcr (rt-pcr) method. the target genes of pcr for y. pestis include the caf gene that encodes f antigen, ymt gene that encodes murine toxin, pla gene that encodes plasminogen activator, hms gene that is related to pigmentation, and specific segment a in chromosome (qu et al. ) . segment a is the main target gene because it is not as easily lost as the genes of plasmid such as pla and caf . the target genes of pcr for f. tularensis include fopa gene (ay ) that encodes outer membrane protein, and the akr gene (am , - ) in chromosomes that encodes aldo/ketoreductase. the genes at two specific toxin plasmid for b. anthracis, including cya, lef, paga at plasmid pxo and capa, capb, capc at plasmid pxo , are often used for species identification (koehler ), while further identification by the detection of the genes in chromosomes, such as gs sequences, is necessary because of the inaccuracy of detection results caused by the deletion or change of plasmids. the pcr method has higher sensitivity than other current methods, but its application on site as a poct method is limited by its dependency on expensive equipment, sample pre-treatment (particularly difficult for dna extraction from complicated samples) and professional operation, as well as its high false positive results. for detection of bioterrorism agents, the special biosecurity facilities for dna extraction (even in an equipped laboratory the infection and contamination in the dna extraction procedure must be paid particular attention) were the major obstacle for utilization of pcr on site. currently, an instrument based on pcr, called a filmarray (seiner et al. ) , demonstrates a promising prospect for application of pct in the field by integration of the sample treatment, amplification, and result analysis in airtight system. however, the complexity of pre-treatment of complicated samples in routine surveillance and public health emergencies is still a significant limitation of application of pcr on site. loop-mediated isothermal amplification (lamp) is a new method for gene diagnosis invented by a japanese professor, in which a dna strand that is complementary to template dna can be synthesized at a determined temperature through strand displacement reaction. using four primers designed according the six segments of the target dna, lamp detection can be realized through one procedure following mixing of the template, primer, strand displacement type dna polymerase and other substrates. the pyrophosphate isolated from dntps in the dna synthesis process can react with mg + ions, resulting in the formation of a white precipitate. lamp has been developed for detection of some bioterrorism agents, such as y. pestis (feng et al. ) and b. anthracis (qiao et al. ) , and it is very suitable for rapid detection on site because only a thermostat is needed and the results can be observed by the naked eye. however, it is not suitable for long strand target dna, because sequences longer than bp are difficult to amplify based on the strand replacement reaction. because lamp is an amplification reaction similar to pcr, a high frequency of false positive results are often generated by lamp because of contamination. the immunization method is based on the reaction between antigens and antibodies, such as enzyme linked immunosorbent assay (elisa), immunochromatography, immunodiffusion, and immunoprecipitation. immunodiffusion and immunoprecipitation were developed in the s, and are not widely used at present owing to poor sensitivity. elisa is a routine laboratory method with excellent sensitivity and specificity owing to signal amplification by enzymes and several rinse procedures. however, a high number of rinse procedures increases the complexity of the operation and the operation error, as well as the potential for spread of bioterrorismassociated agents. immunochromatography was described in sect. . . . many upt-lf assays have been developed for detection of several bioterrorismassociated agents, including detection of bacteria (y. pestis, brucella, b. anthracis spore, f. tularensis, b. pseudomallei, v. cholorae, e. coli o :h , antibody against y. pestis), viruses (hepatitis b surface antibody) and toxins (abrin, ricin, aflatoxin b , aflatoxin m and t- toxin). upt-poct has been provided at many centers for disease control and prevention, as well as entry-exit inspection and quarantine bureaus, and it also provides technology support for biosecurity at large events, such as the games of the olympiad in beijing, shanghai world exposition, and asia games in guangzhou. in , upt-poct was integrated as a mobile biological rapid detection instrument in the stand for construction of city fire station ( - , issued by the ministry of housing and urban-rural development of china and the national development and reform commission of china). upt-poct is suitable for detection of bioterrorism agents on site as a poct method because of the integration of the benefits of immunochromatography, upconverting phosphor particles, and portable biosensing. first, upt-poct can give reliable detection results. given the extremely low pathogenic dose and high social perniciousness of bioterrorism agents, particularly the high transmission capacity of microorganism agents, detection sensitivity and reliability are important for detection methods. compared with the isolation-culture method, biochemical method, and colloidal gold method, upt-poct can realize sensitive and quantitative detection based on immunological interactions, and shows excellent performance that is comparable with that of real-time quantitative pcr in some applications (hao et al. ) , resulting from the merits of the immunological recognition mode, up-converting phosphor, and biosensor. first, immunological recognition between antigens and antibodies is highly sensitive and specific. in addition, the infrared excitation light for up-converting phosphor particles avoids the interference from other biomaterial in the samples, resulting in a more efficient signal isolation rate than other luminous detection methods. the efficient signal extraction and quantitation calculation by the biosensors also facilitate the recognition of the weak positive signal, which is superior to observation with the naked eye for colloidal-gold immunochromatography assays. upt-poct can be tolerant to a great diversity of complex samples in the bioterrorism and biosecurity fields (such as meat, decomposed viscera, and flour). many detection methods are limited by the complicated pre-treatment of samples. for example, repeated selection and identification is required for the isolation-culture method, and the complex composition of samples can easily influence the result of biochemical detection. the pretreatment of complex samples is also a significant challenge for laboratory methods, such as the extraction of dna in complicated samples in pcr that could seriously influence the detection rate. in detection of many bioterrorism agents, upt-poct shows robust performance for various samples with simple pretreatments, such as grinding, or supernatant extraction by centrifugation after grinding and shaking (ultrasonic). this robust performance derives from the physical stability, luminous stability, and up-converting capacity of ucnps, the solid covalent combination between the ucnps and antibody, and the excellent buffering capacity of the detection system. upt-poct is also safer than other detection methods. there are some operations that are unfavorable for the control of bioterrorism agents during detection using other methods, such as repeated proliferation for the isolation-cultured method, various rinses for elisa, complex pre-treatment of sample for the pcr method, and multiple tests for bio-chemical detection. compared with these methods, the simple sampletreatment process based on its high tolerance, and the simple sample-loading manner of upt-poct, reduces the potential for the spread of bioterrorism agents in the detection process. the short acquisition time for upt-poct facilitates rapid response to disease outbreaks in surveillance and public health emergencies, and is derived from the min reaction process of the immunochromatography detection mode and the simple sample pretreatment. as a quantitative detection method, the detection time needed for pcr is more than that for upt-poct owing to the time required for dna extraction. portability is the main obstacle to many laboratory detection methods for application on site, for example the expensive and cumbersome equipment for the pcr method. the portability of upt-poct derives from the small size of the strips and biosensors. in addition, the biosensor could work with a standard mains electricity source or battery. for upt-poct, reliable results can be obtained by nonprofessionals owing to the simple nature of upt-poct operation and sample pre-treatment, making it possible to treat incidents rapidly for surveillance and in public health emergencies. characterization of the rat pneumonic plague model: infection kinetics following aerosolization of yersinia pestis co pneumonic plague cluster water-borne outbreak of oropharyngeal and glandular tularemia in georgia: investigation and follow-up association of the melioidosis agent burkholderia pseudomallei with water parameters in rural water supplies in northern australia recovery efficiency and limit of detection of aerosolized bacillus anthracis sterne from environmental surface samples yersinia pestis detection by loop-mediated isothermal amplification combined with magnetic bead capture of dna outbreak of cholera associated with crab brought from an area with epidemic disease the far-reaching impact of bioterrorism: what the european union is doing regarding deliberate releases of biological/chemical agents based on the events in the united states development and evaluation of an up-converting phosphor technologybased lateral flow assay for the rapid, simultaneous detection of vibrio cholerae serogroups o and o toxigenic vibrio cholerae o in water and seafood development of an up-converting phosphor technology-based -channel lateral flow assay for profiling antibodies against yersinia pestis development and systematical evaluation of an up-converting phosphor technology based lateral flow assay for quantitative detection of burkholderia pseudomallei development and evaluation of an up-converting phosphor technology-based lateral flow assay for rapid detection of francisella tularensis coexistence of vibrio cholerae o and o bengal in plankton in bangladesh bacillus anthracis physiology and genetics development of up-converting phosphor technology-based lateral-flow assay for rapidly quantitative detection of hepatitis b surface antibody development of up-converting phosphor immunochromatography for fast and quantitive detection of bacillus anthracis spore rapid detection of aflatoxin m in milk powder and milk based on up-converting phosphor technology rapid detection of abrin in foods with an up-converting phosphor technology-based lateral flow assay pathogenesis of yersinia pestis infection in balb/c mice: effects on host macrophages and neutrophils biological and chemical bioterrorism agents loop-mediated isothermal amplification for rapid detection of bacillus anthracis spores rapid and quantitative detection of brucella by up-converting phosphor technology-based lateral-flow assay ambient stable quantitative pcr reagents for the detection of yersinia pestis evaluation of the filmarray(r) system for detection of bacillus anthracis, francisella tularensis and yersinia pestis identification of francisella tularensis by both culture and real-time taqman pcr methods from environmental water specimens in outbreak areas where tularemia cases were not previously reported an outbreak of primary pneumonic tularemia development of ucp-immunochromatography test for rapid detection of e. coli o toxin warfare agents: recognition molecules and drugs for control preparation of monoclonal antibodies against ricin toxin and development of up-converting phosphor technology-based lateral flow assay for its quantitative detection rapid quantitative detection of yersinia pestis by lateral-flow immunoassay and up-converting phosphor technology-based biosensor evaluation of up-converting phosphor technology-based lateral flow strips for rapid detection of bacillus anthracis spore, brucella spp., and yersinia pestis outer membrane proteins ail and ompf of yersinia pestis are involved in the adsorption of t -related bacteriophage yep-phi development and evaluation of an up-converting phosphor technologybased lateral flow assay for rapid and quantitative detection of aflatoxin b in crops key: cord- -o e na authors: nan title: scientific session of the th world congress of endoscopic surgery, jointly hosted by society of american gastrointestinal and endoscopic surgeons (sages) & canadian association of general surgeons (cags), seattle, washington, usa, – april : poster abstracts date: - - journal: surg endosc doi: . /s - - - sha: doc_id: cord_uid: o e na nan purpose: to evaluate the efficacy of single-incision laparoscopic surgery for totally extraperitoneal repair (sils-tep) of incarcerated inguinal hernia. patients and methods: clinical setting a retrospective analysis of patients undergoing sils-tep for incarcerated hernia from may to august at kinki central hospital was performed. exclusion criteria sils-tep was contraindicated for the following conditions in our hospital: a history of radical prostatectomy; a small indirect inguinal hernia in a young patient; and unsuitable for general anesthesia. surgical procedure laparoscopic abdominal exploration through a single, . -cm, intraumbilical incision was performed. the incarcerated hernia content was gently retracted from the hernia sac into the abdominal cavity. in some cases, simultaneous manual compression on the incarcerated hernia from the body surface was required. if no bowel resection was needed, a standard sils-tep using mesh was performed following laparoscopic abdominal exploration and incarcerated hernia reduction. if bowel resection was required, inguinal hernia repair using mesh was not performed to avoid postoperative mesh infection, and two-stage sils-tep was performed - months after the bowel resection. results: fourteen patients ( men, women) with irreducible inguinal hernias, including with unilateral hernias and with bilateral hernias, underwent surgery. the patients' median age was years (range, - years), and median bmi was . kg/m (range, . - . kg/m ). of the patients, had acute incarceration, and had a chronic irreducible hernia. seven patients with acute incarcerated hernias underwent emergency surgery, and two of the seven patients needed singleincision laparoscopic partial resection of the ileum, followed by two-stage sils-tep. twelve patients, excluding two patients who required single-incision laparoscopic partial resection of the ileum, underwent laparoscopic exploration with hernia reduction followed by sils-tep. one case of chronic incarceration out of the twelve patients who underwent sils-tep after hernia reduction required conversion to kugel patch repair. the median operative times were min (range - min) for unilateral hernias and min (range - min) for bilateral hernias. the median blood loss was minimal (range - ml). the median postoperative hospital stay was day (range - days). the median follow-up period was months (range - months). a seroma developed in % ( / ) of patients and was managed conservatively. no other major complications or hernia recurrence were noted during the follow-up period. conclusions: sils-tep, which offers good cosmetic results, could be safely performed for incarcerated inguinal hernia. objective: introduction of mis in pediatric age group has been proved feasible and safe. there is considerable evolution with introduction of a number of invovation in mis pediatric inguinal hernia repair. high ligation of sac is the basic premise of surgical repair in pediatric inguinal hernias. there are different mis techniques broadly grouped into intracorporeal or intracorporeal with extracorporeal component namely the suturing. every techniques has its own complications. the main objective of our study was to focus on different anatomical pointers which can lead inadverent complications mainly bleeding and recurrence. methods and procedures: prospective review of hernias ( male and female) ( months- years) performed laparoscopically between september and june . under laparoscopic guidance, the internal ring was encircled extraperitoneally using a - non-absorbable suture and knotted extraperitoneally. data analyzed included operating time, ease of procedure, occult patent processus vaginalis (ppv), contralateral inguinal hernia, complications, cosmesis and recurrence. results: sixteen right ( %), left ( %) and bilateral hernia ( %) were repaired. five unilateral hernias ( . %), all left, had a contralateral ppv that was repaired (p= . ). mean operative time for a unilateral and bilateral repair were . ( - ) and . min ( - min) respectively. one hernia repair still recurred ( . %) even with all precautions and another had a post operative hydrocoele ( . %). one case ( . %) needed an additional port placement due to inability to reduce the contents of hernia completely. because of our techinique we could not find any adverent peroperative bleeding. there were no stitch abscess/granulomas, obvious spermatic cord injuries, testicular atrophy, or nerve injuries. conclusion: the results confirm safety, efficacy and cost effectiveness of laparoscopic inguinal hernia repair. during our per-operative analysis we focus to address the anatomical landmark to minimize future recurrence and peroperative surgical complications. we identified and named a point as j. point at the tip of triangle of "doom". that is most important point to address peroperatively. there is high chance of recurrence if that point is not encircled well or inadequately circled because of fear of iliac vessels injury. we aslo concluded that 'water dissection technique' is effective techniques in un-experienced hand and in early stages of laparoscopic hernia repair to prevent inadvertent iliac vessels injury. medstar georgetown university hospital, georgetown university school of medicine, introduction: incisional hernias following abdominal surgery can be associated with significant morbidity leading to decreased quality of life, increase in health care spending and need for repeat operations. patients undergoing gastrointestinal and hepatobiliary surgery for malignant disease may be at higher risk for developing incisional hernias. identifying these risk factors for incisional hernia development can help decrease occurrence. this will be the largest multi-institutional study looking at incidence of symptomatic hernia rates for major abdominal operations including colectomy, hepatectomy, pancreatectomy, and gastrectomy. methods and procedures: an irb-approved retrospective study within the medstar hospital database was conducted, incorporating all isolated colectomy, hepatectomy, pancreatectomy, and gastrectomy procedures performed across hospitals between the years of to . all patients were identified using icd- and icd- codes for relevant procedures and then subdivided into either having benign or malignant disease. exclusion criteria comprised of patients who had concomitant organ resection, or those undergoing organ transplant. data validation was performed to verify the accuracy of the data set. the rate of symptomatic incisional hernia rates (ihrs) were determined for each cohort based on subsequent hernia procedural codes identified and repairs performed. descriptive statistics and chi squared test were used to report ihrs in each group. results: during this -year span, a total of , major abdominal operations were performed at all institutions, comprising of , colectomies, , hepatectomies, , pancreatectomies, and gastrectomies. malignancy was the indication for surgery in , ( . %) colectomies, ( . %) hepatectomies, ( . %) pancreatectomies, and ( . %) gastrectomies. ihr in each cohort for benign vs malignant etiologies, respectively, are as follows: ( . %) vs ( . %) in colectomy (p= . ), ( . %) vs ( . %) in hepatectomy (p= . ), ( . %) vs ( . %) in pancreatectomy (p= . ), and ( . %) vs ( . %) in gastrectomy (p= . ) patients. conclusion: symptomatic incisional hernia rates following major gastrointestinal and hepatobiliary surgery ranges from . to . %. there was no significant increase in hernia rates in patients undergoing surgery for malignancy. patients undergoing colectomy for benign disease had a high incidence of symptomatic ihrs. introduction: prosthetic infections, although relatively uncommon, are a major source of cost and morbidity. the study aimed to evaluate the influence of mesh structure including the polymer type and mean pore size on bacterial adherence in a mouse model. methods: three commercially available hernia meshes were included in the study. for each mesh type, a cm square was surgically placed intraabdominally in mice. one mouse served as a control while an enterotomy was made in the subsequent mice to introduce a bacterial load onto the mesh. after hours the meshes were harvested. the inoculated meshes were then plated on agar plates and bacterial counts were counted after hours. the bacterial counts were compared between the various mesh types. results: the mean bacterial adherence was increased in the large pore mesh was colonies, for the small pore mesh was colonies, and in the biologic mesh group it was colonies. conclusions: through the use of a mouse model, the influence of mesh type and pore size on bacterial adherence was evaluated. meshes that have larger pores with a lower prosthetic load and the biologic mesh interestingly had lower early bacterial colonization after hours following an enterotomy. further evaluation with a longer incubation time could be helpful to determine the effect of bacterial colonization of mesh. hrishikesh salgaonkar, raquel maia, lynette loo, wee boon tan, sujith wijerathne, davide lomanto; national university hospital, singapore laparoscopic repair of groin hernias is widely accepted approach over open due to lesser pain, faster recovery, better cosmesis and decreased morbidity. however, there is still debate on its use in large inguino-scrotal hernias, recurrent hernias and history of lower abdominal surgery anticipating adhesions and difficulty in dissecting extensive hernia sac. retrospective analysis of prospectively collected data was done of patients undergoing laparoscopic repair of large inguino-scrotal, incarcerated groin hernia, recurrent cases after open or laparoscopic repair and history of previous lower abdominal surgery. between january to july , patients with large inguino-scrotal hernias, recurrent hernia, history of lower abdominal surgery, incarcerated femoral hernia underwent laparoscopic inguinal hernia repair. patient characteristics, operating time, surgical technique, conversion rate, complications and recurrence up to months recorded. patients had large inguino-scrotal hernia, recurrent hernia ( previous open, previous lap) , history of lower abdominal surgery ( lscs, appendectomy, prostatectomy, midline laparotomy), incarcerated femoral hernia, meshoma removal. patients underwent total extraperitoneal (tep) repair, transabdominal pre-peritoneal (tapp), needed conversion to open. mean operation time was min for unilateral and min for bilateral hernia. seroma formation seen in patients, minor wound infections treated conservatively. we conclude that the laparoscopic approach can be safely employed for the treatment of complex groin hernias; surgical experience in laparoscopic hernia repair is mandatory with tailored technique in order to minimize morbidity and achieve good clinical outcomes with acceptable recurrence rates. mesh fixation in ventral incisional hernia is a topic of ongoing debate. permanent and absorbable tacks are acceptable and widely used methods for mesh fixation. the purpose of this study was to compare outcomes of permanent tack fixation versus absorbable when used alone or with suture fixation in laparoscopic incisional hernia repairs. a retrospective review of all patients undergoing laparoscopic ventral hernia using tack fixation (absorbable/permanent) alone or in conjunction with suture fixation was queried from the ahsqc database. outcome measures included hernia recurrence rate, pain, quality of life, wound related issues, and hospital length of stay. propensity match scoring was performed to compare patients undergoing tack only fixation versus tack and suture fixation with a p-value of . considered significant. a total of patients were identified after propensity match scoring with who underwent repair with permanent tacks alone or with sutures and who underwent repair with absorbable tacks alone or with sutures. following matching there were no differences in bmi, age, hernia width/length, or baseline pain/ quality of life. there were no significant differences found in outcome measures including recurrence rates, pain and quality of life outcomes at days, months, and year, surgical site infection (ssi), and postoperative length of stay (p[ . ). there was a significant increase in any post op complication in the permanent tack fixation group compared to the absorbable tack fixation group ( % vs %, p. ) which is likely due to the increase in surgical site occurrences noted in the permanent tack fixation group ( % vs. %, p. ). based on this large data set, there are no significant differences in postoperative outcomes in permanent versus absorbable fixation in laparoscopic hernia repair except in surgical site occurrences. further study is needed to evaluate but at the present time, there is no convincing evidence that one type of fixation is superior to another in laparoscopic ventral hernia repair. introduction: inguinal hernia repair is the most common procedure in general and visceral surgery worldwide. laparoscopic transabdominal preperitoneal mesh hernioplasty (tapp) has been also popular surgical method in japan. single incision laparoscopic surgery is one of the newest branches of advanced laparoscopy, and its indication has been spread to not only simple surgery such as cholecystectomy, but also complex surgery. we report our experience with single incision laparoscopic tapp (s-tapp) for japanese patients with inguinal hernia. case description: a consecutive series of patients ( male, female) who underwent s-tapp during june to september in a single institution. twenty eight of the patients had bilateral inguinal hernia. the mean follow-up was days. the average age of the patients was . ± . years. establishment of the ports: a -mm vertical intra-umbilical incision is made for port access. one -mm optical port and two -mm ports were placed side-by-side through the umbilical scar. surgical procedure: the procedure was carried out in the conventional fashion with a wide incision in the peritoneum to achieve broad and clear access to the preperitoneal space, and an appropriate placement of polypropylene mesh ( dmaxtm light, bard) with fixation using the tacking device (absorbatack®, covidien). the hernia sac is usually reduced by blunt dissection, or is ligated and transected with ultrasound activated device. the peritoneal flap is closed by one suture with - pds and the - tacks using absorbatack®. discussion: in one patient, we encountered a large sliding hernia on the right side having sigmoid colon as content of the sac, which required conversion to the conventional laparoscopic procedure. there were nine recurrence cases after surgery of laparoscopic or anterior approach, and two cases after prostatectomy. there was no intra-operative complication. the mean operative time was . ± . min, and blood loss was minimum in all cases. the average postoperative stay was . ± . days. there was one recurrence case ( . %) months after the surgery. there was no severe complication after the surgery, but there were seromas ( . %) and one hematoma ( . %). two patients had blunt tactile sense in the area of the lateral femoral cutaneous nerve ( . %), which improved in two months. conclusion: our results suggest that s-tapp is a safe and feasible method without additional risk. moreover, cosmetic benefit is clear. however, further evaluation for postoperative pain and longterm complications compared to standard laparoscopic tapp mesh hernioplasty should be required. manuel garcia, md, daniel srikureja, md, marcos j michelotti, md, facs; loma linda university health introduction: prosthetic mesh use has become standard practice during ventral hernia repair to reduce the risk of recurrence. the ideal mesh is macro-porous which favors rapid cellular ingrowth and tissue integration, has limited tissue reactivity, low profile and weight, and has high tensile strength to add resilience to the repair. additionally, the material is expected to have good handling characteristics. currently, there is a wide variety of options for mesh. biosynthetic material (poliglycolic acid/trimethylene carbonate -pga/tmc) has been shown to behave well in terms of early vascularization and ingrowth as well as adequate long term tissue generation. gore® synecor® biomaterial is a composite mesh including two layers of absorbable biosynthetic material (pga/tmc) with one tridimensional non-absorbable macro-porous knit of dense ptfe mesh. it has shown good vascularization and ingrowth at days in animal examination. however, there is still no evidence of long term behavior of this mesh in human tissue. we present the first histologic analysis of this mesh year after placement in a human. objective: to perform a histologic analysis of the gore® synecor® biomaterial one year after placement in the human body. methods: after incidentally finding incorporated gore® synecor® mesh in a patient with prior ventral hernia repair year ago, during open bilateral inguinal hernia repair, a sample of mesh was taken and sent to pathology lab for analysis. tissue healing, vascularization, and ingrowth of the composite mesh were analyzed. results: histologic findings significant for a biomaterial consistent with a knitted ptfe material surrounded by mature fibrovascular tissue and foreign body inflammation consistent with expected healing response for this time frame. no evidence of any other biomaterial (pga/tmc) or evidence of infection. conclusion: gore® synecor® biomaterial has shown to be well integrated into appropriately healed tissue, with pronounced vascularization and ingrowth. the pga/tmc layers have been seen to be completely absorbed and replaced by collagen. these findings, in a human months sample, replicate what had been shown in animal specimens. method: from to , patients came to hospital with renal paratransplant hernia. they were evaluated for this study. the following data were collected from their records: age, gender, weight, age at graft rejection, surgical complications, treatment method and the treatment results with composite ptfe mesh. results: for laparoscopic repair of incisional hernia after renal transplant, the median interval between kidney transplantation and developing of incisional hernia was (range to ) days. predisposing factors were obesity, age over fifty years, and female gender. in six patients, hernia was large, and the repair was performed with using composite ptfe mesh. one patient had developed serous collection in surgical site, which was managed successfully with multiple punctures. hernia recurrence or infection was not noted in these patients during to months follow-up periods. conclusion: incisional hernia is not a rare entity after kidney transplantation. predisposing factors, such as obesity, age over years, and female gender have a role in its development. repeated surgeries in kidney recipients can increase the risk of incisional hernia. managing this complication by laparoscopic approach is a safe and effective method. sujith wijerathne, raquel maia, hrishikesh salgaonkar, wee boon tan, lynette loo, davide lomanto; national university hospital, singapore introduction: a femoral hernia is a less common type of hernia. it is estimated to account for less than % of all abdominal wall hernias. only about in every groin hernias are femoral hernias. they are found more commonly in females due to wider shape of pelvis. laparoscopy by offering magnification and better vision provides us the opportunity for clear visualization of the myopectineal orifice. laparoscopy seems to be a safe and feasible approach for femoral hernia repair in an asian population. case description: between and , consecutive patients with femoral hernia who underwent laparoscopic hernia repair were prospectively studied. patient demographics, hernia characteristics, operating time, conversion rate, intraoperative, postoperative complications and recurrence were measured. discussion: total of femoral hernias were repaired, on right and on left groin. this included patients with bilateral and unilateral hernia. concomitant obturator hernia were found. there were male and female patient. no conversion was reported. one patient had injury to bowel at the mm port entry site, without contamination, identified and managed immediately. patients developed seroma, all were managed conservatively except one who needed aspiration. peri-port bruising was noticed in patients and patients had hematoma. one patient with hematoma underwent excision of the organised hematoma. of the hematoma patient was on aspirin pre-operatively. no wound infection, chronic groin pain or recurrence was documented during follow up till date. conclusion: laparoscopic repair offers accurate diagnosis and simultaneous treatment of both inguinal and femoral hernia with minimum morbidity and good clinical outcomes. better visualisation and magnification gives us an opportunity to identify occult hernias which can be repaired during the same setting, thereby reducing the chance of recurrence and possible need for second surgery. laparoscopic repair has become the procedure of choice for the treatment of the majority of groin hernia at our institution. introduction: totally extraperitoneal (tep) repair that does not require peritoneal incisions is a good procedure that involves minimal visceral damage. however, balloon-or camera-assisted blunt dissections that are performed in a haphazard manner do not follow precise dissection of the fascia layer. furthermore, they have a disadvantage in that they are difficult to understand anatomically. we therefore developed a novel preperitoneal approach to resolve this issue. methods: a -mm trocar is inserted into the rectus abdominis sheath cavity after a small incision is made below the umbilicus and the posterior rectus sheath is exposed. a -mm trocar is inserted cm towards the pubic bone from the umbilicus. using forceps from this position, narrow branches that enter the posterior rectus sheath from the inferior epigastric vessels are dissected, thereby broadly exposing the anterior surface of the posterior rectus sheath. the third mm-trocar is inserted near the lateral margin of the rectus abdominis. on the outside, local anesthetic is injected beneath the posterior rectus sheath and the preperitoneal cavity is separated in fluid so that the peritoneum is not injured during posterior rectus sheath incision. a small incision is made to the posterior rectus sheath or attenuated posterior rectus sheath at one finger width higher than the expected upper margin of the prosthetic mesh. due to the effects of local injection, a sharp incision to the fascia can be made with an electric scalpel. utilizing this mechanism, the posterior rectus sheath aponeurosis and the lining transverse fascia and superficial preperitoneal layer are individually identified. once the preperitoneal cavity is reached, the peritoneal margin is determined in the lateral direction, and the peritoneum that is pulled due to pneumoperitoneum is separated from the preperitoneal fascia on the outside from the cranial side towards the deep inguinal ring. on the inside, the pneumoperitoneum pressure pushes the peritoneum inferiorly, leading to enlargement and increased visibility of the posterior rectus sheath deep fascia, which is dissected one layer at a time from the outside. the umbilical prevesical fascia is dropped inferiorly, and the dissection of the preperitoneal cavity necessary for mesh deployment is performed. results: by individually dissecting each fascia using emphysema through pneumoperitoneum and enlargement through local injection, the method for reaching the preperitoneal cavity could be successfully completed by following the dissection of the fascia layer without proceeding with the operation blindly, thereby resulting in the elimination of intraoperative bleeding and postoperative hematoma. introduction: in the field of abdominal wall reconstruction, the utility of drain placement is of debatable value. we present outcomes evaluating drain placement vs no drain placement at the time of robotic transversus abdominis release (rtar) technique with placement of mesh in the retromuscular position, a currently understudied subject. methods: retrospective review of a prospectively maintained hernia patient database was conducted identifying individuals who received either drain placement or no drain placement during abdominal wall reconstruction via the rtar technique from august to june at a single high volume hernia center. perioperative data and postoperative outcomes between the two groups are presented with statistical analysis for comparison and quality of life (qol) measures assessed using the carolina comfort scale. results: thirty-five patients were identified for this study, of which had drains placed intraoperatively in the retromuscular position at the conclusion of rtar (drn) and underwent rtar without the placement of draining devices (nd). the drn cohort had a mean bmi, defect area, mesh area, and operative time of . , cm , cm and minutes, respectively, compared to . , cm , cm , and minutes in the nd group. all cases utilized medium weight macroporous polypropylene synthetic implantable mesh materials in both the drn and nd subgroups. there were no reported postoperative complications, including no development of hematoma, seroma, or surgical site infections in either group. hernia recurrence was not identified in either the drn or nd cohorts through a mean follow up of days ( . months). there were no statistically significant differences in postoperative qol outcomes. conclusion: our series review suggests that the use of intraoperative drains may not afford any benefits with the rtar technique when mesh is placed in the retromuscular position. additional postoperative management associated with drain care may be unnecessary. surg endosc ( ) :s -s background: appendectomy is one of the most common operations performed during emergency surgery. although laparoscopic appendectomy (la) has become the treatment of choice, there is still a debate regarding the use of la for treating complicated appendicitis. in this retrospective analysis, we aimed to clinically compare la and open appendectomy (oa) for treating complicated appendicitis. methods: we retrospectively identified patients who underwent an operation for complicated appendicitis at our hospital; these patients were operated on between and july . [editor ] in total, patients underwent conventional appendectomy and patients were laparoscopically treated. outcomes included operation time, blood loss, length of hospital stay, and postoperative complications. logistic regression analysis was performed to analyze the concurrent effects of various factors on the rate of postoperative complications. objective: small bowel perforation has conventionally been dealt with open exploration, which frequently leads to many wound-related complications. wound infection is the major reason for increasing morbidity in these patients and delay recovery. laparoscopic surgery has various benefits over open surgery like, smaller wound, lesser pain and faster recovery. the aim of this study was to relay the advantages of minimally invasive surgery (mis) to patients with small bowel perforation to decrease postoperative wound complications and duration of hospital stay. methods: it is a retrospective study, including patients with small bowel perforation from to . of these , had traumatic etiology, had typhoid-related perforation and the remaining had a duodenal perforation. of them were male, and the average age was . years. only patients who presented within hours of perforation were included in the study. laparoscopic exploration was done on introducing camera from -mm infraumbilical port after intraperitoneal carbon dioxide insufflation. the remaining two -mm working ports were then introduced depending on the site of perforation once identified. the perforations were then repaired using intracorporeal single-layer suturing using polydioxanone - suture. the peritoneal cavity was given thorough lavage and abdominal drain placed in the pouch of douglas. fecal contamination was found in all the patients. a total of patients underwent conversion to open surgery due to inability to find the site of perforation laparoscopically. of the operated patients, patients developed port-site infection, and there were no major postoperative complications in the -week follow up period. conclusion: we conclude from our study that laparoscopic intervention in early small bowel perforation is a safe approach with favorable outcomes, especially with regards to wound complications, that are a major factor in increasing the morbidity in such patients postoperatively. laparoscopic approach leads to early discharge and recovery postoperatively. with the emerging era of laparoscopic surgery, leading to its easy accessibility, more patients can advantage from this technique when they arrive in emergency with intestinal perforation. s surg endosc ( ) :s -s introduction: pneumatosis intestinalis (pi), or gas in the bowel wall, can be seen on various imaging modalities. the pathophysiology behind pi is unclear. one theory proposes a mechanical cause (e.g. small bowel obstruction) while another proposes a bacterial etiology. management of pi in adults is difficult as often there is a benign clinical course. however, when paired with specific clinical features such as hepatic portal venous gas (hpvg) on imaging, the course of management changes as the suspicion of bowel ischemia increases. hpvg alone has been associated with a high mortality rate and a poor prognosis. management in this case becomes surgical. case presentation: we present a case of -year-old latino male who presented to the emergency room with abdominal pain and altered mental status. focused physical examination revealed a non-rigid abdomen, no rebound tenderness, no guarding, and diffuse tenderness only to deep palpation. ct scan of the abdomen and pelvis demonstrated moderate portal venous gas in the right and left hepatic lobes, an upper midline dilated small bowel loop with pneumatosis intestinalis, and a moderately distended stomach with gas and fluid. laboratory studies revealed metabolic acidosis and a lactic acid level of . mmol/l. due to these findings, bowel ischemia was suspected, and the patient was taken to the operating room for a diagnostic laparoscopy. the laparoscopy was converted to an exploratory laparotomy due to extensive adhesions. intraoperatively, there was no small bowel compromise and no identifiable transition point. extensive lysis of adhesions and repair of iatrogenic enterotomy were performed. patient tolerated the procedure well, clinically improved, and was discharged from the hospital. discussion: this case illustrates the difficulty in management of a patient with pneumatosis intestinalis and, specifically, hepatic portal vein gas seen on ct imaging. hpvg has traditionally been a harbinger of morbidity and mortality, but exploratory laparotomy revealed only diffuse abdominal adhesions and the absence of bowel ischemia despite high clinical suspicion. background: ventral hernia repair is one of the most common surgical procedures facing the general surgeon. there is little consensus as to the best surgical technique for complex scenarios. often these patients have complicating co-morbid conditions such as radiation therapy, that has an inevitable effect in the abdominal wall structures, which can lead to non-traditional repairs. case report: we present a case of a year-old female who underwent a tah/bso and right hemicolectomy which was complicated by wound dehiscence. she underwent primary repair and adjuvant whole pelvis radiation for her squamous cell carcinoma. subsequently, the patient developed acute obstructive symptoms do to a stricture within her small bowel and a large ventral hernia measuring cm with non-reducible abdominal contents below the level of the fascia more prominent in the suprapubic area. the patient's bmi was . . various considerations are important in planning a surgical repair in a previously irradiated field with loss of domain which include, minimal dissection, and the use of an atraumatic surgical techniqueque with either external oblique release or transversus abdominis muscle release (tar). we chose a a tar, as it provides wider myofascial release and dissection below the arcuate line towards the space of retzius and bogros allowing for a larger sublay mesh placement. also it avoids the need of skin flaps reducing the risk for wound complications in under-perfused tissue. the tar was performed successfully and there were no intraoperative and postoperative complications. her follow-up at months revealed no wound complications or hernia recurrence. conclusion: for patients with compromised tissue and loss of domain a tar technique may be useful when reconstructing complex abdominal wall hernias. it provides the core principals of hernia repair such as primary fascial closure, wide mesh overlap, and finally it provides a reliable approach for the under-perfused tissue without need of skin and soft tissue flap creation. outcomes in the management of cholecystectomy patients in the setting of a new acute care surgery service model: impact on hospital course larsa al-omaishi, bs, william s richardson, md; ochsner medical clinic foundation introduction: the acute care surgery (acs) model, defined as a dedicated team of surgeons to address all emergency department, inpatient, and transfer consultations, is quickly evolving within hospitals across the united states due to demonstrated improved patient outcomes in the non-trauma setting. the traditional model of call scheduling consisted of one senior attending and one senior resident on call per -hour shift. attendings were responsible for consults, previously scheduled operations, as well as clinic time. multiple recent studies have shown statistically significant improvements in several parameters of patient care by using acs including but not limited to . time from emergency department to surgical evaluation . time from surgical evaluation to operating room . operative time . percent laparoscopic . length of hospital stay . intra-operative complications (blood loss, perforation rates) . post-operative complications (fever, infection, redo) . cost. one study demonstrated a statistically significant cost savings for the acute care surgery model with respect to appendectomies, but not cholecystectomies. study design: a retrospective analysis of patients who underwent cholecystectomy in the setting of non-traumatic emergent cholecystitis was performed to compare data from two cohorts: the traditional model and the acs between january , and dec , at ochsner medical center, a -bed acute care center in new orleans. parameters gathered included . time from emergency department to surgical evaluation . time from surgical evaluation to operating room . operative time . percent laparoscopic . length of hospital stay . intra-operative complications (blood loss, perforation rates, conversion to open) . post-operative complications (fever, infection, redo). demographics were also collected including age, weight, height, ethnicity, asa, etc. inclusion criteria included: age[ and having undergone cholecystectomy between jan , and december , . exclusion criteria included choledocholithiasis, gallstone pancreatitis, ascending cholangitis, gangrenous cholecystitis, septic complications precipitating further procedures and delays, or researcher discretion. results: patients were initially identified as having undergone cholecystectomy within the allotted time period [ - , - , - , - ] . were excluded due to one of the reasons above. median patient age was years old and the average patient encounter was . days. conclusion: the acs model is better suited to manage emergent non-traumatic cholecystectomies than the traditional call service at our institution, as evidenced by several parameters. s surg endosc ( ) :s -s he nailed it background: nail guns are powerful tools and are widely used. injuries with these devices may be devastating due to the significant force they can deploy. patients and methods: we herein report a first case of a self inflicted abdominal injury with a nail gun. results: a year old male with history of coronary artery disease, type dm and early signs of dementia attempted to refill a nail gun. he lodged the device against his right abdomen while the air hose was still attached and then accidently fired nails into his abdomen. after he unsuccessfully tried to pull the nails out he drove himself minutes to our emergency room. he was hemodynamically stable on arrival; pain control was achieved, antibiotics were given and he received tetanus immunization. ct-scan showed the two foreign bodies penetrating from the ruq with one reaching the transverse colon. on emergency laparoscopy, the nails were found to have penetrated the thick omentum and the puncture site of one nail into the colon was identified. the omentum was resected off the colon and the right colon was completely mobilized. no additional injuries were found. the entrance area of the nails was then used to create a loop colostomy. the postoperative course was initially uneventful but the patient developed a severe posttraumatic inflammatory reaction of the fat tissue in the right upper quadrant and had to be readmitted for pain control and antibiotics were again administered. he recovered and was discharged with a plan for laparoscopically assisted colostomy closure after weeks. discussion: to the best of our knowledge this is the first reported isolated colonic injury by a nail gun. given the tremendous force of the device with unknown collateral damage to the surrounding tissue it was decided to manage the accident with a laparoscopic assisted colostomy using the entrance point of the nails for fecal diversion. introduction: it is difficult to diagnose obturator hernias by routine physical examination. obturator hernias are frequently complicated by ileus and the diagnosis is often first made from abdominal ct. obturator hernias are difficult to reduce, and often necessitate emergency surgery. they are common in elderly people, and they often had bad general condition. so it was high in the death rate. at our hospital, we first attempt to reduce the hernia from the body surface under ultrasonographic guidance. after relieving the strangulation, we perform radical operation electively in patients who are for possible for surgery under the general anesthesia. we perform laparoscopic repair for obturator hernias. obturator hernias are often complicated by other types of hernia. in these cases, we perform total repair. herein, we present a review of the patients who underwent surgery for obturator hernia at our hospital. methods: we review the data of cases of obturator hernia encountered by us from february to december . we performed total repair in three of the cases. however, it is difficult to procure a mesh that would be adequate for all the defects (inner inguinal ring, femoral ring, obturator). no single mesh can fit, because the inguinal and pelvic curves present opposing curves near the obturator. therefore, we placed two pieces of mesh available at our hospital ( d max [bard] and onlay sheet of kugel patch [bard] ) together in the patientswe could successfully cover all the defects using these two pieces of mesh and could fit the mesh to the pelvic shape by devising an appropriate connection between the meshes. results: we reviewed a total of operated cases for obturator hernia. the hernia was bilateral in cases, and complicated by other hernias in cases. we first determined the appropriate approach for the repair. we performed total repair in cases. they were no complications and no cases of recurrence. conclusion: our approach to the repair of obturator hernias was very useful. we can use the exact area and shape of the mesh needed in individual patients by this method. we show the method of shaping the mesh to fit the pelvic form. demin aleksandr, do, ajit singh, do, noman khan, do; flushing hospital introduction: internal hernias are known complications that are well documented to involve peterson's defect. in bariatric patient's post gastric bypass there is a high index of suspicion for internal hernias as well as a low threshold to operate. there have been some debates around the closure of the potential peterson's space with several studies advocating closure versus some which show that there is no difference in the rate of symptomatic internal hernias. we present a case of an unusual cause of small bowel obstruction due to internal hernia caused by a cecal volvulus. it is an atypical presentation however the patient was triaged and brought to the or within hours of admission. although it is rare there have been reports of internal hernias caused by other structures like congenital bands or natural potential spaces. there have been reports of unusual presentations of the cecum herniating through the foramen of winslow. the anatomical rearrangements after bypass create potential areas where an internal hernia can occur. in this case a bowel resection was undertaken due to the anatomical variation of the cecal bascule and cecal volvulus due to high rate of recurrence of this cecal pathology. majority of internal hernias do not require bowel resection especially when detected earlier and prompt surgical exploration is undertaken. mortality as direct consequence internal hernia is extremely rare. however late diagnosis of internal hernias can lead to catastrophic gut loss and may require lifelong tpn and/or visceral transplantation or autologous reconstruction. conclusion: careful history and physical of our bariatric patient can elicit the signs and symptoms of internal hernias and prevent the morbidity and mortality that can come with the complications of this condition. unusual presentations and causes are reason for prompt diagnosis and complete exploration. shingo ishida , naotsugu yamashiro , satoshi taga , koichi yano ; shinkomonji hospital, shinmizumaki hospital symptomatic cholelithiasis is common disease performed with laparoscopic cholecystectomy (lc). we will hesitate to operate if the patient is pregnant in the third trimester. pregnant patients undergoing laparoscopic surgery have been reported increasingly. however, most case reports are confined to patients in the first and second trimester. we report a patient who underwent lc in the third trimester and review the relevant literature. a -year-old woman in the third trimester ( w d) of pregnancy was seen in the emergency department of our hospital with a history of upper abdominal pain. there was no problem in the course of pregnancy. the result of the examination proved to be attack of gallstone colic. she was hospitalized the same day and underwent lc the next day. the base of pregnancy uterus was cm above the navel. we needed to consider the surgical approach, for example inserting the first trocar under left hypochondrium. operative duration was minutes. she complained abdominal distension at postoperative day (pod) and but there was no abnormality in the fetus. she was discharged on pod . after that she gave birth to a healthy baby. lc in third trimester of pregnancy was safely performed with obstetrics back up. weekday or weekend hospital discharge: does it matter for acute care surgery? ibrahim albabtain , roaa alsuhaibani , sami almalki , hassan arishi , hatim alsulaim ; kamc, background: hospitals usually reduce staffing levels over weekend. this raises the question of whether patients discharged over a weekend may be inadequately prepared and possibly at higher risk for adverse events post-discharge. the aim of this study was to assess the outcomes of common acute care surgery procedures for patients discharged over weekend, and identify the key predictors of early readmission. methods: this retrospective cohort study was conducted at a tertiary care hospital between january and december . surgical procedures included were cholecystectomy, appendectomy, and hernia repairs. patients' demographic, co-morbidities, complications, readmission and follow-up details were collected from the electronic medical records. predictors and post-operative outcomes associated with weekend discharge were identified by multivariable analysis using univariable and multivariable logistic regression models controlling for potential confounders. results: a total of patients were included. overall median age was years (iqr: , ). the majority of patients were female (n= , . %). patients ( . %) underwent a cholecystectomy, ( . %) an appendectomy, and ( . %) hernia repairs. weekend discharge was . % vs. . % of weekday discharge. patients discharged during weekend were younger ( . vs. , p-value. , mean) . post-discharge -day follow-up visits were significantly lower in the weekend discharge subgroup ( . % vs. . %, p-value . ). overall, -day readmission rate was . % (n= ), and did not differ between those of weekend and weekday discharge (or= . , % ci . - . ). conclusions: patients discharged on weekends tended to be younger in age and less likely to have chronic diseases. patients discharged over the weekend were less likely to follow up compared to weekday discharge patients. however, the readmissions rate did not differ between the two groups. intrauterine device (iud) migration out of the uterine cavity is a serious complication. its incidence in the us has been reported to be about . % annually. previously published systematic review supports the use of laparoscopic surgery for elective removal of migrated iucds from the peritoneal cavity. we present the safety and efficacy of the laparoscopic approach to this complication in the acute care setting. depicted is an otherwise healthy year old female with no previous surgical history who presented to the ed with worsening abdominal pain for one week with no associated symptoms. on physical exam, patient was non toxic. abdomen was moderately distended with guarding and rebound tenderness to palpation, no rigid. patient had been seen shorlty prior to ed admission by her obgyn and recent work up with abdominal/pelvic x-ray and ultrasound has revealed a misplaced iud in the transverse position (side ways). pregnancy test was negative. based on patient clinical presentation and recent radiologic findings, we decided to proceed with diagnostic laparoscopy. after systematic review of cavity, the foreign body was found to be incorporated within the greater omentum. we procceded, laparoscopically with omentectomy+foreign body removal. there were no perioperative complications, patiet was discharged on the following day. the use of laparoscopy in elective iud retrieval within in the abdominal cavity has been considered standard of care in surgical management to date. this poster demonstrates its use as an effective approach for safe removal of intra-abdominal foreign bodies also in the acute setting. symptomatic inguinal and umbilical hernias in the emergency department: opportunity lost? andrew t bates, md, jie yang, phd, maria altieri, chencan zhu, bs, salvatore docimo, jr., do, konstantinos spaniolas, md, aurora pryor, md; stony brook university hospital introduction: patients with symptomatic inguinal and umbilical hernias often present to the emergency department (ed) when their symptoms change or increase, usually not requiring emergent surgery. however, little is known about how often these patients present prior to eventual repair and whether they undergo surgery at the initial presenting institution. the aim of this study was to assess the clinical flow of patients presenting in the ed for inguinal and umbilical hernia. methods: all patients presenting to eds in new york state from to with symptomatic inguinal and umbilical hernias were identified using the new york state longitudinal hospital claims database (sparcs). patients were followed for records of hernia repair and subsequent inpatient and outpatient visits up to . results: , patients presenting to the ed for symptomatic inguinal hernia were identified. . % ( , ) of ed presentations resulted in inpatient admissions. , ( . %) had repair later and their average time from ed presentation to inguinal hernia repair was (± ) days. . % of patients who did not have subsequent surgery had only one ed visit. of those that underwent interval repair, . % had only one ed visit prior to surgery. for those patients with only one ed visit before repair, . % had repair at a different hospital, as opposed to . % if multiple ed visits were made. , umbilical hernia patients presenting to the ed were identified. . % ( , ) resulted in inpatient admission. , ( . %) had interval repair, with the average time from ed presentation to umbilical hernia repair being (± . ) days. % of patients who did not record of later repair presented to the ed once. of those patients who underwent repair, . % did so after one ed visit. for those patients with only one ed visit before repair, . % had repair at a different hospital, as opposed to . % if multiple ed visits were made. conclusion: a majority of patients with symptomatic inguinal and umbilical hernias that present to the ed do so once with no subsequent follow-up or repair. for those patients that undergo interval repair, a significant portion willnopt for surgery at other hospitals. a significant proportion of patients with acutely symptomatic inguinal/umbilical hernias who undergo interval repair after a previous ed visit, will opt for definitive surgery at another hospital facility. this represents a missed opportunity for continuity of care for providers and healthcare systems. nikhil gupta, dr, himanshu agrawal, dr, arun k gupta, dr, dipankar naskar, dr, c k durga, dr; pgimer dr rml hospital, delhi introduction: peritonitis is the inflammation of the serous membrane that lines the abdominal cavity and the organ contained therein and is one of the most common infections, and an important problem that a surgeon has to face. reproducible scoring system that allows a surgeon to determine the severity of intra-abdominal infections are essential to prognosticate the patient. this study was done to compare apache ii scoring and mpi score to assess prognosis in perforation peritonitis. methods: all patients admitted with hollow viscus perforation from st november till st march was included in the study. it was a cross sectional observational study. apache ii and mannheim peritonitis index (mpi) scoring systems were calculated in all the patients in order to assess their individual risk of morbidity and mortality. the outcome variables were studied postoperatively -post-operative wound infection, wound dehiscence, anastomotic leak, respiratory complications, duration of hospital stay, need of ventilator support and mortality. the inferences were drawn with the use of appropriate tests of significance. results: the study comprised of patients. neither apache ii nor mpi could predict postoperative wound infection. the mean apache ii score of subjects included in the study was . ± . with range of to and the mean mpi score of subjects included in the study was . ± . with range of to . apache ii was able to predict postoperative respiratory complications, post-operative need for ventilatory support, hospital stay duration and mortality while mpi was able to predict post-operative wound dehiscence, post-operative respiratory complications, post-operative need for ventilatory support and mortality. neither apache ii nor mpi could predict postoperative anastomotic leak and postoperative wound infection. conclusion: mannheim peritonitis index is a useful and simple method to determine outcome in patients with peritonitis. mpi is comparable to apache ii in assessing the prognosis in perforation peritonitis and can well be used in emergency setting in place of apache ii scoring when time is a definite constraint. microrna- and the prognosis of human carcinomas: a systematic review and meta-analysis chengzhi huang, mengya yu; guangdong general hospital (guangdong academy of medical science) muhammad nadeem , julian ambrus, md , steven schwaitzberg, md , john butsch, md ; university at buffalo, introduction: mitochondria is a small energy producing structure of a cell. mitochondrial myopathy (mm) is mixed disorder clinically, which can affect various systems besides skeletal muscle. mm starts with muscle weakness or exercise weakness. mm patients have decreased skeletal muscle mitochondrial function than the healthy person, because of weakened intrinsic mitochondrial function and decreased mitochondrial volume density. no one has studied the mm role in gerd and constipation so far. this study is aimed to see effects of mm on the gastrointestinal system specifically gastroesophageal reflux disease (gerd), gall bladder issues, and constipation. methods: between may and june , mm diagnosed patients at buffalo general hospital were included in this retrospective study. we assessed their demeester score for gerd and wexner's constipation questionnaire for constipation. demeester score[ and constipation score[ were set points for gerd and constipation respectively. data was analyzed by using spss version . mitochondrial enzymes were assessed by using their muscle biopsy report. results: out of ( . % female, . % male) mitochondrial myopathy patients, . % and . % were suffering from gerd and constipation respectively. . %, . % and . % patients had gall bladder issues, obstructive sleep apnea (osa) and fatigue respectively. mm gerd patients ( . % female, . male) had mean demeester score . (sd: . ) more than normal although . % patients were on gerd medications and . % patients had nadh cytochrome c reductase, cytochrome c oxidase and citrate synthase abnormal mitochondrial enzyme in mm associated gerd but . % mm patients had abnormal cytochrome c oxidase enzyme only. mm along with constipation had mean wexner's constipation score . (sd: . ) more than the normal although . % were taking enema, medications or digital assistance. % patients had cytochrome c oxidase and nadh cytochrome c reductase enzymes were abnormal in those patients. . % mm associated gall bladder issues patients had cytochrome c oxidase abnormal. . % mm associated gerd and constipation patients had gall bladder issues. conclusion: in this present study, we found that mm had effects on gastrointestinal system causing gerd, constipation and gall bladder issues. gerd, constipation and gall bladder problems are common in mm patients even patients are taking medications for gerd and constipation. cytochrome c oxidase, citrate synthase and nadh cytochrome c reductase are the most commonly impaired mitochondrial enzyme in mm patients and mm associated gerd, constipation and gall bladder issues patients. objectives: gulf war illness (gwi) is a chronic, multisymptom illness marked by cognitive and mood dysfunction and disrupted neuroendocrine-immune homeostasis affecting % of gw veterans. after + years, useful treatments are lacking and its cause is poorly understood, although exposures to pyridostigmine bromide and pesticides are consistently identified among the strongest risk factors. previous work in our laboratory using an established rat model of gwi identified persistent elevation of microrna- (mir- ) levels in the hippocampus whose gene targets are involved in cognition-associated pathways and neuroendocrine function, suggesting that mir- inhibition is a promising therapeutic approach to improve the complex symptoms exhibited by gwi. the purpose of this study was to identify broad effects of mir- inhibition in the brain by profiling the expression of genes known to play a critical role in synaptic plasticity, glucocorticoid signaling, and neurogenesis in gwi rats administered a mir- antisense oligonucleotide (mir- inhibitor). methods and procedures: nine months after completion of a -day exposure regimen involving gw-relevant chemicals and stress, rats underwent intracerebroventricular infusion of mir- inhibitor (n= ) or scrambled negative control oligonucleotide (n= ) and were implanted with -day osmotic pumps delivering . nmol/day. intranasal delivery of oligonucleotides was performed on additional rats (n= per group; daily for days) to determine whether mir- inhibition is achievable using a noninvasive procedure. hippocampi were harvested and quantitative pcr arrays were used to profile the expression of focused panels of genes important for ) synaptic alterations during learning and memory, ) signaling initiated by the glucocorticoid receptor (known mir- target), and ) neurogenesis. hippocampi were also analyzed by quantitative pcr to examine expression levels of endogenous mir- . results: upregulation ([ . fold change, p. ) of synaptic plasticity genes, glucocorticoid signaling genes, and neurogenesis genes was observed in the hippocampus of gwi rats infused with mir- inhibitor compared to scrambled control, consistent with a significant reduction (p\ . ) in mir- levels detected in rats receiving mir- inhibitor. altered gene expression and a reduction in mir- levels were not observed in rats after intranasal delivery. conclusion: mir- antagonism in the hippocampus upregulates the expression of several downstream targets involved in synaptic plasticity, glucocorticoid signaling, and neurogenesis and is a promising therapeutic approach to improve cognition, emotion regulation, and neuroendocrine dysfunction in gwi. further testing is being pursued to discover the optimal dose for intranasal administration to test viability of this option for ill gw veterans. nikhil gupta, dr, ananya deori, dr, arun k gupta, dr, dipankar naskar, dr, c k durga, dr; pgimer dr rml hospital, delhi background: the ultrasonic dissector, commonly known as the harmonic scalpel, has been in use for achieving haemostasis in surgery for almost yrs. its advantages in breast surgery, especially in the dissection of axilla, have been a matter of debate as previous studies have shown inconsistent results. this study compares the outcomes of the ultrasonic dissector in axillary dissection with that of the conventional electrocautery. methods: patients who were undergoing mrm and bcs with axillary dissection from november till march were included in the study. patients were randomized into two groups, group a undergoing axillary dissection with ultrasonic dissector and group b with electrocautery. the operative time, intra-op bleeding, post-op pain, post op drain volume, hospital stay and any other complications were noted in the two groups. results: the numbers of patients in both groups were each. group a had a significantly shorter operative time, both for axillary dissection ( . min vs. . min, p. ) and the total duration ( . vs. . min, p= . ). the blood loss was significantly less in group a, as measured by the mop count. there was significant reduction in the total post-op drainage volume, which resulted in fewer days of drain in-situ and the total number days stayed in the hospital. there was no significant change in the post-op complications such as haematoma, seroma, flap necrosis, oedema, etc. conclusion: with the use of ultrasonic dissector, the operative time, blood loss and the axillary drainage was significantly reduced. the axillary drainage in turn, reduced the hospital stay. there was no significant difference in terms of complications like haematoma formation, seroma formation, skin flap necrosis or oedema. for the statistical analysis, χ or fisher's exact tests to compare proportions and the nonparametric mann-whitney u test for analysis of values with abnormal distribution were used. discussion: the study included patients. all preoperative laboratory indicators were elevated. the laboratory tests do not demonstrate any statistical significance between these two groups. the group of the patients without stones in the cbd diagnosed by ioc was also divided in patients with diameters. ?mm and with diameters≥ . ?mm of the cbd. also in these two groups, the statistical analysis of the laboratory tests does not demonstrate significant difference. all patients underwent ioc. ioc showed stones in / patients ( . %) . a comparison of patients with and without stones at ioc showed similar mean times from hospitalization to surgery ( . background: housed in a high volume tertiary referral center, our division receives a large amount of transfers and referrals from outside institutions for patients who require completion cholecystectomies. in this study "completion cholecystectomy" refers to patients that meet one of three criteria: . previous subtotal cholecystectomy, . previously aborted cholecystectomy, or . previous cholecystectomy with incidental finding of cancer on pathology. traditionally, exploration of a reoperative field in the right-upper quadrant mandates an open approach due to dense adhesions and inflammation. over the past few years, we have found that robotic-assisted surgery has allowed us to perform these completion cholecystectomies in a minimally invasive fashion. methods: case logs and operating room billing logs were reviewed from to to identify all robotic-assisted cholecystectomies performed at our institution. review of all reports identified completion cholecystectomies. all additional variables including demographics, operative variables, and postoperative outcomes were determined from manual chart review of all consultation notes, operative reports, anesthesia records, progress notes, discharge summaries, and postoperative office visits. results: of the identified robotic-assisted completion cholecystectomies, patients had a previous subtotal cholecystectomy, patients had an aborted cholecystectomy, and patients had an incidental finding of t gallbladder carcinoma on pathology. fifteen patients ( %) underwent preoperative ercp either for choledocolithiasis or to determine biliary anatomy. average time from original procedure was months with . % of previous procedures performed in an open approach. average or time was . minutes, average ebl was . cc, and average length of stay was . days. one patient ( . %) was readmitted within days for nausea that resolved with antiemetics. three patients ( . %) had minor postoperative complications (clavien-dindo grade or ) which resolved with pharmacologic therapy. no patients suffered a -day mortality. all cases were completed in minimally invasive fashion without a conversion to an open procedure. conclusions: although rare, completion cholecystectomies present a challenging surgical scenario. although traditionally performed in an open approach, we have had success in recent years at our institution with a robotic-assisted approach to completion cholecystectomy. we feel that the robotic approach offers certain advantages in a hostile, reoperative field which allows us to perform these procedures in a minimally invasive fashion with no conversions to an open procedure to date. previously limited to case reports, this report of procedures represents the largest case series of robot-assisted completion cholecystectomies to our knowledge. s surg endosc ( ) :s -s background: percutaneous cholecystostomy tube (pct) has been used as a bridge treatment for grade ii-iii moderate to severe acute cholecystitis (ac) to "cool" the gallbladder down over several weeks and allow the inflammation to resolve prior to performing interval cholecystectomy (ic) and removal of the pct, often laparoscopically. the aim of this study was to assess the impact of timing of ic after pct on operative success and outcomes. methods: a retrospective review of electronic medical records of patients who were treated for ac with a pct, and subsequently underwent ic at our institution between january to december was performed. the patients were divided into three groups (n= each), based on the duration of the pct prior to ic, and these groups were comparatively analyzed. a comparative sub-analysis of clinical outcomes between patients who underwent surgery within the first week vs. third week or later after pct was also performed. results: a total of patients met the study criteria. each group had patients. there were no statistically significant differences between the groups in regards to age, gender, bmi, imaging findings, and indications for cholecystostomy tube placement. overall, there was no statistically significant difference in outcomes between performing ic within the first weeks, - weeks and [ weeks after pct placement. the length of stay, overall morbidity, clavien-dindo grade of complications and mortality were similar between the time intervals. however, a sub-analysis showed that patients who underwent ic within the first week of pct placement had statistically significant higher mortality rate (p= . ) compared to those who underwent ic[ weeks of pct placement. the two patients who died in our sample had ic within a week after pct placement. even though there was a statistically significantly higher morbidity rate in those who had ic[ weeks after pct, the clavien-dindo grade of these complications was lower than. conclusion: delaying ic to [ weeks after pct placement for ac is not associated with any improvement in patient morbidity, length of stay or rate of conversion from laparoscopic to open cholecystectomy. cholecystectomy within the first week of pct placement is associated with higher mortality rate than after weeks likely due to associated sepsis. introduction: the effect of intraoperative bile spillage during laparoscopic cholecystectomy (lc) on operative time (or time), length of stay (los), postoperative complication rates, and day readmission rates was analyzed. laparoscopic cholecystectomy is the gold standard operation for gallbladder disease in the united states. number of studies have shown that same day discharge in elective laparoscopic cholecystectomy is feasible and safe. bile spillage during this procedure can be a common occurrence in teaching institutions, however, data on the effects of operative outcomes is lacking. methods: this is a retrospective study analyzing all of the laparoscopic cholecystectomies performed at the brooklyn hospital center (tbhc), both emergent and elective, from to . patient data was collected on demographics, comorbidities, bile spillage, operative findings, complications, los, and day readmission rates. statistical analysis was performed using imb spss statistics v. . covaried analysis of variance (ancova) was performed on continues variables and significance levels were calculated. pearson's chi square significance level was calculated for all binomial variables. results: of the patients who underwent lc during this time period, intraoperative bile spillage was encountered in patients. interestingly, bile spillage was significantly more likely to be seen in elective cases over acute cases ( . % vs . %, p. ). there was a statistically significant increase in or time in cases where intraoperative bile spillage was encountered vs. cases where no bile spillage was encountered ( vs. min, p= . ). there was a significant increase in rate of conversion to open procedure when bile spillage was encountered ( . % vs. . %, p. ). drain placement rates increased, not surprisingly, when bile spillage was encountered ( . % vs. . %, p. ). there was no statistically significant difference in los between cases with bile spillage and cases without ( . days vs. . days). there was no significant increase in complication rate or day readmission rates. conclusions: intraoperative bile spillage significantly increases or time, conversion to open procedure, and drain placement. however, there was no significant effect observed of intraoperative bile spillage on length of stay, complication, and day readmission rates. thus, intraoperative bile spillage appears to have little clinical significance on surgical outcomes. however it may have an impact on overall healthcare costs. larger prospective studies evaluating the effect of intraoperative bile spillage on los, or time, complication rates, and day readmission rates are needed to analyze these effects further. tariq nawaz, md; rawalpindi medical university study design: prospective and observational study. place and duration: from january, to july . surgical unit ll, holy family hospital, rawalpindi. patients and methods: thousand patients with a diagnosis of cholithiasis were included. exclusion criteria are patient younger than year and older than year. calot's triangle dissection was done meticulously. cystic artery and hepatic artery anomalies and variations were observed and analyzed on spss . results: the age varies from to years. on the basis of distributional variation the cystic artery was single in % cases, branched in % cases and absent in % cases. on positional variations the cystic artery was superomedial to the cystic duct in % cases, anterior in % cases, and posterior in % cases and low lying in % of the cases. on the basis of length variation results showed that ( %) cases had a normal cystic artery. a short cystic artery was found in ( %) cases and a long cystic artery was present in ( %) cases. other arterial variations are of hepatic artery i.e moynihan's hump ( %) and and right hepatic artery present in calots triangle in % conclusions: for the safety of laparoscopic cholecystectomy one should be well aware of the anatomical variations of the cystic and hepatic artery. keywords: cholelithiasis, cholecystitis, laparoscopic cholecystectomy. as small as it gets: micro-invasive laparoscopic cholecystectomy using only two mm trocars and a needle grasper background: the majority of surgeons use four ports including for laparoscopic cholecystectomy (lc). multiple efforts have been made to reduce number and size of ports. left upper quadrant (luq). patients and methods: of lcs performed from / - / , ( %) were done using three instruments including cases in which trocars and the teleflex needle grasper were used. in cases only two mm trocars were (left upper quadrant (luq) and umbilicus) with the minigrasper being placed between the two. the gallbladder (gb) serosa was incised on both sides and a window was created behind the gb midportion and widened towards fundus and infundibulum. cystic artery (ca) and cystic duct (cd) were dissected out obtaining the critical view and after the last fundus adhesion was cut, ca and cd were secured with clips or endoloop. results: median age of women and men was . (range . - . ) years. lc was done for acute cholecystitis (n= ), chronic cholecystitis (n= ), biliary dyskinesia (n= ), choledocholithiasis (n= ). three patients had an ercp with bile duct clearance prior to the lc. in one case a keith needle was used to suspend the gb fundus for better exposure. twelve patients had additional procedures together with their lc (wedge liver biopsy ( ), lysis of adhesions ( ) , umbilical hernia repair ( ) , mesenteric/lymphnode biopsies ( ) . median or time was (range - ) minutes. the specimen was removed through the luq port site in patients. there were no vascular or bile duct injuries in this series. % of cases were done as outpatient procedures, % of patients required hours observation only three patients were hospitalized for medical reasons. conclusion: in selected cases with either small stones or biliary dyskinesia, lc with only two mm ports and a needle grasper is possible. the teleflex minigrasper can completely replace a port based grasper. introduction: the standard treatment for lithiasic acute cholecystitis remains the laparoscopic cholecystectomy despite the timing of surgery is still controversial. the aim of this prospective study is to evaluate the advantages and limitations of early laparoscopic cholecystectomy in a district hospital. methods and procedure: all patients undergoing laparoscopic cholecystectomy at the surgical department of "carlo urbani" hospital in jesi (italy) from may to september were consecutively enrolled. clinical data such as gender, age, bmi, comorbidity, previous abdominal surgery, previous acute cholecystitis were collected. subsequently, the patients were arranged in two groups according to the timing of intervention (early versus elective surgery). for each group, we compared data concerning surgery, such as operative time, intraoperative and postoperative complications, length of hospital stay and cost analysis. results: this study is a part of an ongoing research. so far, we collected laparoscopic cholecystectomies. ten ( %) of them were admitted with acute cholecystitis and were operated during the hospital stay (group a). group b included patients scheduled for elective surgery (n= ; %). the two groups were comparable with respect to clinical data. conversion to open approach was performed in cases, all of them in group b. mean surgical time was . ± . minutes in group a and . ± . minutes in group b (p= . ). no significant differences in intraoperative and postoperative complications rates were seen in the two groups, just a few in both of them. mean overall length of hospitalization was . ± . days in group a and ± . days in group b (p= . ), whereas the difference in length of postoperative hospitalization was not statistically significant. due to the extended hospitalization for group a, the cost increase as compared to group b was statistically significant, too. conclusions: early laparoscopy is comparable to delayed laparoscopy in terms of postoperative hospitalization and complications in the management of acute cholecystitis. a longer hospital stay among patients scheduled for immediate surgery may be associated with a more time-consuming diagnostic work-up before surgery. however, in future research we expect to enhance our cost analysis with more data regarding the costs incurred in the first hospitalization reserved to nonoperative treatment of group b inpatients with acute cholecystitis. s surg endosc ( ) introduction: with improvements in healthcare access and technology, admissions of octogenarian population with acute cholangitis (ac) are increasing. octogenarians are vulnerable to inferior outcomes. there is no study to evaluate factors predicting outcomes of ac in octogenarians. the aim of our study is identify factors predicting outcomes, and to evaluate the quick sequential organ failure assessment (qsofa) score and tokyo guidelines (tg ) severity grading for octogenarian patients with ac. methods: a retrospective review of octogenarian patients admitted with ac from january to december was performed. demographic profile, clinical presentation and discharge outcomes were studied. systemic inflammatory response syndrome (sirs), qsofa and tg severity grading scores were calculated. mortality is defined as death within days of admission or in hospital mortality. statistical analysis was performed using spss version . results: there were a total of patients admitted for ac, of which ( %) were octogenarians. majority (n= , %) were female, with a mean age of (range - ) years. majority were secondary to gallstones (n= , %), and ( %) were due to malignancies. ( %) and ( %) patients fulfilled sirs and qsofa criteria of severity respectively. ( %) and ( %) of patients had a tg severity grading of moderate and severe respectively. nine ( %) patients required inotropic support in the emergency department (ed) and ( %) patients were admitted to critical care unit (ccu). ( %) patients underwent endoscopic retrograde cholangiopancreatography (ercp) and ( %) underwent percutaneous transhepatic biliary drainage (ptbd) for biliary decompression. patients underwent index cholecystectomy. length of stay was . (range - ) days and -day mortality of %. multivariate analysis performed showed that an abnormal glasgow coma score (p= . ) and malignancy (p. ) predicted -day mortality. the use of ed inotropic support predicted ccu admission (p= ). a positive blood culture (p= . ), presence of malignancy (p. ), use of ed inotropes (p= . ), and index cholecystectomy (p= . ) predicted a longer length of stay. qsofa (p. ) and tg severity grading (p= . ) were predictive of -day mortality. sirs criteria did not predict -day mortality. conclusion: reduced consciousness and malignancy predicted -day mortality in octogenarian patients with ac. qsofa and tg severity grading system is superior to sirs criteria in predicting mortality of octogenerians with ac. our group has performed needlescopic grasper assisted silc (nsilc) to overcome these problems. we evaluate the technical feasibility, safety and benefit of nsilc versus three-port laparoscopic cholecystectomy (tplc). methods and procedures: this prospective randomized control study was conducted to compare the advantages if any between the nsilc and tplc. one hundred and forty eight patient were randomized into two groups, with one group underwent n slic ( patients) and a control group underwent tplc ( patients). basic information about the patient and diagnosis was collected. the surgical outcome that was composed with critical view of safety (cvs) time, major procedure time and total operation time, and the comparison of postoperative complication was made. result: nsilc group was consisted of male ( . %) and female ( . %), and tplc group was consisted of male ( . %) and female ( . %) (p= . ). the average age of nsilc group was . ± . years old, and tplc group was . ± . years old (p= . ). cvs time of tplc group was shorter than silc group (nsilc: . ± . min, tplc: . ± . min, p= . ), major procedure time (skin incision to gb removal from liver bed) of tplc group was shorter than nsilc group (silc group: . ± . min, tplc: . ± . min, p= . ). however, there was no significant difference in postoperative complication (nsilc: , tlc: , p= . ). conclusion: although cvs time, major procedure time, and operation time of silc were longer than tplc, overall clinical results were similar. nsilc is feasible and safe surgical procedure in patient with benign gallbladder disease. introduction: management of malignant biliary obstruction not amenable to surgery is usually by means of ercp or pthc. however, on occasions, these routes are not accessible and the alternate decompressive technique of percutaneous cholecystostomy (pc) has to be adopted. the aim of this study was to evaluate the efficacy and outcomes of pc in a highly selected series at a tertiary referral center. methods: we retrospectively reviewed all patients that had undergone pc from to . data collected included baseline demographics, comorbidities, details of pc placement and management, etiology of mbo, and post-procedure outcomes. the charlson comorbidity index (cci) was calculated for all patients at the time of pc. results: four hundred and eight patients underwent pc placement of which patients including ( %) males and ( %) females, with malignant biliary obstruction. the mean age at the time of pc placement was . ± . years of age, and the mean cci was . ± . for all patients. of mbo in all patients was due to pancreatic malignancies (n= ), cholangiocarcinoma (n= ), primary hepatic malignancies (n= ), secondary hepatic tumors (n= ), and ampullary carcinoma (n= ). pc tube complications were reported in ( %) patients. mean number of tube exchanges was . ± . . mean duration from pc tube placement to death was ± . days. total deaths were recorded. conclusion: pc placement appears to be a viable option in mbo in elderly and frail patients. in this cohort, pc may be a potential definitive management to improve quality of life. melanie boyle, daivyd palencia, philip leggett; houston northwest medical center background: there are very few studies assessing the relationship between gastroesophageal reflux and biliary disease. this is surprising as they share presenting symptoms as well as risk factors, particularly obesity. our group previously produced a review of patients in our practice who had undergone some type of reflux procedure. conclusions showed that the prevalence of gallbladder disease in our severe reflux population is much higher compared to that found in the general population. our goal of this study is to expand on that data to include a larger sample size to investigate the incidence of biliary disease in our reflux population and decide if this should influence our pre-operative algorithm for anti-reflux surgery patients. methods: we expanded on our previously performed retrospective review of patients that underwent laparoscopic fundoplication for reflux disease. we previously reviewed data from to . we are now looking at data from to . our expected sample size will include approximately patients, of which have currently been reviewed. our previous study included only . the surgery preformed was either a toupet or nissen fundoplication, and one underwent a dor. demographic data, imaging studies, and pathology results were reviewed. results: we looked at whether each patient who underwent antireflux surgery had a prior cholecystectomy either remotely or recently, underwent concomitant cholecystectomy, or had no biliary disease in their workup. the groups had similar age and were predominantly women. we once again demonstrated that the prevalence of gallbladder disease in our severe reflux population is much higher than the general population. when approaching a patient with gastroesophageal reflux disease, attention should be paid to gallbladder symptomatology as well. we recommend that it may be beneficial to include gallbladder ultrasound in pre-operative workup for antireflux surgery so that concomitant cholecystectomy can be performed if indicated. steven schulberg, do, jonathan gumer, do, matt goldstein, vadim meytes, do, george ferzli, md; nyu langone hospital -brooklyn introduction: acute cholecystitis is a common surgical disease with roughly , cholecystectomies performed in the us annually. the current dogma revolves around the " hour rule" advocating early cholecystectomy if within the window, and if beyond hours, conservative treatment and interval operation. in patients beyond the hour window, as well as with multiple comorbidities, advanced age, and other complicating factors, cholecystostomy has become an acceptable treatment as a bridge to interval cholecystectomy. while this has become an appropriate treatment modality, it does not come without its own set of complications. we aim to evaluate the rate of complications in our institution. methods: this is a retrospective review of all patients at our institution who underwent cholecystostomy placement between and . we evaluate the comorbidities, readmission rate, overall rate of complication associated with cholecystostomy tubes, and eventual definitive cholecystectomy. results: our cohort includes patients, % of whom were male, with a mean age of . we had an overall complication rate of . %, including tube dislodgements, leaking tubes, and misplaced tubes. all cause readmission rate was % and only % of patients who had cholecystostomy drains underwent interval cholecystectomy. conclusion: there has been much interest in treatment of acute cholecystitis in patients with multiple comorbidities. in review of our data, a surprisingly large number of patients had mechanical complications involving the cholecystostomy drain. in an era focused on decreasing readmission rates and their associated costs, drains carry a high risk of malfunction which will in turn, lead to increases in these two metrics. while there is more work to be done in the evaluation of early cholecystectomy versus cholecystostomy in this subgroup of patients, we suspect that early cholecystectomy in the medically optimized patient will lead to reduced length of stay and hospital costs as well as increased patient satisfaction. does selective use of hepatobiliary scintigraphy (hida) scan for diagnosis of acute cholecystitis, following equivocal nondiagnostic gallbladder ultrasonography, affect outcomes fahad ali, ba, amir aryaie, md, eneko larumbe, phd, mark williams, md, edwin onkendi, md; texas tech university health sciences center introduction: acute cholecystitis (ac) is diagnosed by characteristic gallbladder ultrasonographic findings (high specificity, low sensitivity). hepatobiliary scintigraphy (hida) may be needed to confirm ac (higher sensitivity and specificity). the aim of this study was to assess the impact of the current selective use of hida scan for sonographically equivocal cases of ac on outcomes. methods: a retrospective chart review of patients treated for ac at our institution ( / to / ) was performed. patients were divided into groups: the ultrasound only group (us-only) and the ultrasound-hida group (us-hida). timing of us and hida, and intervention for ac since presentation to emergency room (er), and their impact on outcomes were analyzed. ac severity was graded per the tg -tokyo guidelines. results: a total of patients were analyzed. the groups were statistically similar with regards to age, body mass index, asa class ii, iii and iv, extent of leukocytosis at presentation and liver functions test levels at presentation. in the us-only group, diagnostic ultrasound was obtained sooner, [median of (interquartile range, iqr . - . ) hours] from presentation to the er compared to the us-hida group, ) hours], p= . . hida was obtained after a median delay of . (iqr . - ) hours from a nondiagnostic ultrasound. majority of patients ( %) in the us-only group had mild (tg grade i) to moderate (tg grade ii) ac, while % of the us-hida group had moderate (tg grade ii) to severe (tg grade iii) ac (p= . ). despite this, more patients in the us-hida group ( %) had a "normal" non-diagnostic ultrasound compared to the us-only group ( . %), p. . seven patients in the us-hida group had no intervention due to normal hida scan ( ) , ac misdiagnosis due to liver cirrhosis ( ) , and severe medical comorbidities ( ) . more patients ( %) in the us-only group underwent laparoscopic cholecystectomy, compared to % in the us-hida group (p= . ). between the two groups, there was no significant differences in -day morbidity, mortality and reoperations. however, the length of stay was longer by a median of . days in the us-hida group (p= . ). conclusion: patients with moderate to severe ac are more likely to need hida scan due to a "normal" non-diagnostic ultrasound, have a delay in diagnosis, not have intervention for ac due to severe medical comorbidities and have lower chance of laparoscopic cholecystectomy. the length of hospital stay is significantly longer for these patient by a median of . days. introduction: benign gallbladder disease is commonly treated with laparoscopic cholecystectomy (lc). gallbladder cancer (gbc) is a rare malignancy characterized by high invasiveness and poor survival. in our institution, all gallbladder specimens are routinely sent to pathology, to rule out gbc. the purpose of our study was to assess the efficacy for routine histopathology of gallbladder specimens after cholecystectomy (cly) for all gallbladder disease. methods and procedures: after obtaining approval from our institutional review board, a retrospective review was conducted on all patients who underwent cly from june of to may were included in the study. the data obtained include gender, age, american society of anesthesiologist score (asa), body mass index (bmi), comorbidities, length of stay (los), radiological imaging and pathology results. independent t and chi-square tests were performed using ibm® spss® software. results: there were cly performed at our institution, of which ( %) were lc. females composed of ( %) patients and the median age was . ( %) gallbladder specimens were found to be cancerous. ( %) gallbladder specimens were benign. majority ( %) were chronic cholecystitis, ( %) were acute cholecystitis and ( %) were gangrenous cholecystitis. ( %) were found to be acalculus cholecystitis and ( %) were cholelithiasis. ( %) were found to be adenomyositis, and other. conclusion: in our institution, less than % ( ) of all gallbladder specimens were found to be cancerous. it would decrease cost and work load if gallbladder specimens are selectively sent to pathology. emanuel a shapera, md we sought to determine clinical factors associated with recurrent cholangitis in two las vegas community hospitals to aid providers in management of this disease. methods and procedures: retrospective, multi-center study. over ercps were analyzed between and . patients were identified as having multiple ( ) admissions for cholangitis per tokyo criteria. univariate and multivariate analysis was conducted. results: patients with a significantly (p. ) higher albumin level on admission ( . ) were discharged home more often than patients discharged to a facility or hospice ( . ). on multivariate analysis, non-home discharge was associated with lower albumin level at admission (p= . ) and greater maximum temperature prior to decompression (p= . ). increased hospital stay was associated with lower albumin level at admission (p= . ). a majority ( / ) of recurrent episodes involved stent placement, exchange or removal. patients ( %) had either biliary malignancy, gallbladder or both. blood cultures were drawn in % of all episodes and positive in %, e coli being the most common pathogen isolated. all patients had low hdl levels ( - , mean ) . conclusions: high fevers and poor nutritional status was associated with increased length of hospital stay and fewer home discharges. tumors, gallbladders and malfunctioning stents contribute substantially to morbidity. close follow up for indicated gallbladder removal, stent management and nutritional optimization is critical to reduce the burden of this disease. we compared the surgical method in neonate choledochal cyst between oec and lec. the perioperative and surgical outcomes that were reviewed included age, operative time, postoperative hospital stay, time to diet, and surgical complications. the patients were followed up for months (range, - months) . results: there was no difference in range of bile duct excision and manner of roux-en-y hepaticojejunostomy between oec and lec groups. there was no intraoperative complication in both groups and no open conversion in the lec group except one case which was ruptured choledochal cyst. the median age of oec and lec groups were days (range, - ) and . days (range, and median body weight at the time of operation were . kg (range, . - . ) and . kg (range, . - . ) , respectively. the median operative time was minutes (range, - ) in oec and . minutes (range, in lec groups and there was no significant difference between oec and lec groups (p= . ). intraoperative bleeding was minimal in both groups. the postoperative hospital-stay, time to start diet, and time to return to full feeding had no significant differences in both groups. after discharge, of ( %) oec patients experienced readmission due to cholangitis and ileus, while there were none in the lec group. conclusions: this study revealed that lec had better prognosis compared to oec. lec provided an excellent cosmetic result. so we suggest lec could be the treatment of choice for neonatal choledochal cyst. this is a small series, therefore future studies will have to include a larger number of patients and evaluate long-term follow-up. keywords: choledochal cyst, laparoscopy, neonate. laparoscopic narrow band imaging for intraoperative diagnosis of tumor invasiveness in gallbladder carcinoma: a preliminary study yukio iwashita, hiroki uchida, teijiro hirashita, yuichi endo, kazuhiro tada, kunihiro saga, hiroomi takayama, masayuki ohta, masafumi inomata; oita university faculty of medicine introduction: determining tumor invasiveness before operation is one of the most important unsolved issues in the management of gallbladder cancer. we hypothesized that the assessment of irregular vessels on the gallbladder wall may be useful for detecting subserosal infiltration. we present an initial report on the clinical usefulness of laparoscopic narrow band imaging (nbi) for the intraoperative diagnosis of tumor invasiveness in gallbladder carcinoma. methods: thirteen patients with gallbladder cancer were included in this study. patients with tumors located in the liver bed and those with definitive invasion observed on computed tomography findings were excluded from this study. gallbladders were observed using nbi and the microvasculature was evaluated. according to previous reports of endoscopic nbi, we defined four findings as positive: vessel dilatation, tortuousness, interruption, and heterogeneity. the nbi findings were compared with postoperative pathological findings. the study protocol was approved by the institutional review board of the oita university. results: the serosal surface of the tumor site and its microvasculature were successfully observed in all patients. laparoscopic nbi detected at least one abnormal finding in seven patients, and postoperative pathology showed subserosal infiltration accompanied by vessel invasion. on the contrary, six patients with no positive nbi findings showed mild or no subserosal infiltration and no vessel invasion. conclusions: our study indicated that laparoscopic nbi may be useful for diagnosing subserosal infiltration accompanied by a vessel invasion. shuichi iwahashi, mitsuo shimada, satoru imura, yuji morine, tetsuya ikemoto, yu saito, hiroki teraoku; department of surgery, tokushima university introduction: laparoscopic cholecystectomy (lap-c) is the standard operation for the benign diseases. we have reported reduced port lap-c (rpl-c) was safely and comparable method to sils-c and conventional lap-c (sages ) . in this time, we examined the utility of rpl-c containing the post-operative adverse event. procedures: the adjustment is the benign illness including the cholecystolithiasis, and advanced obesity and the cases of the inflammation remaining have been excluded. the incision is put and cut open the abdomen to the umbilical region, and camera port was inserted. we used mm flexible scope. mm forceps for holding of the gallbladder bottom and left hand of operator were inserted directly with no port. methods: rpl-c has been introduced in this department since july, . we performed cases of lap-c, containing sils-c and american style conventional lap-c, and we performed rpl-c has been performed already cases. we compared the patient background and the operation factor between rpl-c, sils-c, conventional lap-c. operators were young surgeons, they were not specialists of gastroenterological surgery or endoscopic surgery. results: the difference was not admitted in the age, gender, the physique, and the disease, and the difference was not admitted in hospital stay after the operation (rpl-c:sils-c:conventional lap-c= . ± . days: . ± . days: . ± . days) and the amount of blood loss (rpl-c:sils-c:conventional lap-c= . ± . ml: . ± . ml: . ± . ml) and operation time (rpl-c:sils-c:conventional lap-c= ± min: ± min: ± min). and surgical wound after rpl-c was cosmetically acceptable. regarding as the post-operative adverse event, there were no patients of bile duct injury. conclusion: in the patients on reduced port lap-c, there were no bile duct injuries of postoperative adverse event. reduced port lap-c is safely for young surgeons and comparable method. introduction: acute cholangitis is an ascending infection of the biliary tree secondary to obstruction and can be severe if proper intervention and treatment are not performed in a timely fashion. the most common management of cholangitis with ductal obstruction due to choledocholithiasis is intravenous hydration, empiric antibiotic therapy, endoscopic retrograde cholangiopancreatogram (ercp) with sphincterotomy and stone extraction with or without stent placement, followed by a delayed laparoscopic cholecystectomy. we present the case of a patient with blood clot obstruction of a common bile duct (cbd) stent after ercp with sphincterotomy and stone extraction. case presentation: a year old male presented to the emergency department with jaundice, right upper quadrant abdominal pain, truncal pruritis, nausea, vomiting, and fever. biochemical analyses and liver profile demonstrated an elevated white blood cell count, hyperbilirubinemia, and elevated liver enzymes consistent with cholestasis. biliary ultrasound demonstrated multiple gallstones and dilation of the cbd with a distal obstructing calculus. he proceeded to ercp where biliary cannulation was achieved, sphincterotomy performed, and a large amount of sludge and pus was drained. an mm stone was removed from the cbd by balloon sweep with completion cholangiogram demonstrating no filling defects. a stent was then placed in the cbd with adequate flow. following the procedure, the patient continued to have increasing hyperbilirubinemia. a repeat ercp revealed a large blood clot and continued bleeding at the previous sphincterotomy that resolved with epinephrine injection. the former stent was visualized in the proper position, removed with a snare, and found to be fully occluded with blood clots. after retrieval of additional clots, a new stent was placed with adequate return of bile. the patient recovered with resolution of his symptoms and hyperbilirubinemia with laparoscopic cholecystectomy. discussion: cholangitis is characterized by charcot's triad of right upper quadrant abdominal pain, fever, and jaundice due to an ascending bacterial infection of the biliary tree coinciding with obstruction of biliary flow most commonly from gallstones. cholangiography via ercp with associated sphincterotomy, stone extraction, and stenting is both diagnostic and therapeutic. while debated by endoscopists, stent placement has shown to reduce recurrent biliary complications, decrease length of hospital stay, and lessen morbidity. although pancreatitis is the most common cause of hyperbilirubinemia post-ercp, stent occlusion secondary to stones or blood clots should be considered to effectively treat patients. proper hemostasis is important in any procedure and close patient follow-up should be performed to prevent further complications. sarrath sutthipong, md, panot yimcharoen, md, poschong suesat, md; bhumibol adulyadej hospital background: choledochal cyst (cc) is a rare disease, characterized by dilatations of the extra-or/ and intrahepatic bile ducts. ccs occur most frequently in asian and female populations. cc is associated with biliary lithiasis and considered at risk of malignant transformation. todani's classification dividing cc into types is the most useful in clinical practice. the current standard treatment is complete cyst excision with roux-en-y hepaticojejunostomy and cholecystectomy for the extrahepatic disease (todani type i and iv). in this report we present our experience using a total laparoscopic technique to treat adult patients with cc in -year period. methods: a retrospective review of the records of the patients above years who underwent laparoscopic cyst excision and roux-en-y hepaticojejunostomy in our hospital between january and may was carried out. the data included the clinical presentation, investigation, perioperative details and complication. the type of cc was classified according to todani's classification. results: seven cases of cc were reviewed, females and male with mean age years (range - years). these included cases of todani type ib and cases of type a. the predominant symptoms were chronic abdominal pain and jaundice. a case of both pancreatitis and cholangitis were also seen. investigations included ultrasound with mrcp in cases and ercp in case. the mean operative time was hours and minutes ( hours minutes to hours range) with mean intraoperative blood loss ml (range - ml). all the resected specimens showed chronic inflammation. malignancy was not seen in any patients. the early postoperative complications included bile leakage with intra-abdominal collection in patients, which were managed conservatively (evidenced by clinical status and imaging study), re-operation was not required. the median duration of hospital stay was days (range - days). there was no perioperative mortality. all patients were followed up at , , and months postoperatively, late complication were not detected during each visit. conclusion: in our opinion, laparoscopic cyst excision and hepaticojejunostomy could offer more feasible and safe methods of treatment for ccs in adult patients with potentially less postoperative morbidity, a shortened length of stay and a lower blood loss when compared to the preferred open approach. however, we would need to study this on a larger sample of patients to report the efficacy and safety of laparoscopic approach. endoscopic trans-papillary gallbladder drainage (etgbd) in acute cholecystitis: a single center experience arun kritsanasakul, chotirot angkurawaranon, jerasak wannapraset, thawee rattanachu-ek, kannikar laohavichitra; rajavithi hospital background: surgery is the mainstay of treatment for cholecystitis, however, it may not be safe or feasible in some circumstances such as severe cholecystitis or cholecystitis in extremely high-risk patients. gallbladder drainage may be an appropriate alternative or a bridging option prior to cholecystectomy. endoscopic trans-papillary gallbladder drainage (etgbd) has been proposed as a modality that is feasible and effective in cholecystitis. objective: the primary outcome of this study is to evaluate the effectiveness of etgbd. the secondary outcome is to evaluate the safety, early experience outcomes, and complications of this procedure. methods: retrospective medical records review between january -december from a single tertiary referral hospital center, rajavithi hospital, bangkok, thailand. a total of patients who was diagnosed with cholecystitis and underwent etgbd. the procedure was performed at the endoscopic suite under light sedation via total intravenous anesthesia. the patient demographic data and procedures were collected. the technical success of etgbd was defined as decompression of the gallbladder by successful cystic duct stent placement. the clinical success was defined as resolution of symptoms and/or improved laboratory data or ultra-sonographic findings. results: a total of patients underwent etgbd. among these patients, were high risk for surgery due to age or comorbidity, had concomitant jaundice and was failure of medical treatment. both technical and clinical success of etgbd was achieved in of cases ( %). the two patients that did not achieve technical success were due to failure to cannulate guidewire through cystic duct and the other had trans-cystic guidewire perforation that needed surgical intervention. there were two intra-operative complications ( %). one was the patient who had trans-cystic guidewire perforation and another had anesthesia-related complication (hypoventilation requiring endotracheal intubation). there were no -day mortality. conclusion: endoscopic trans-papillary gallbladder drainage is an alternative treatment modality for patients with cholecystitis who are at high-risk for surgery and or those who are unsuitable for percutaneous gallbladder drainage. the technique is feasible, however, careful case selection and high endoscopic skill is needed. julia f kohn, bs , alexander trenk, md , woody denham, md , john linn, md , stephen haggerty, md , ray joehl, md , michael ujiki, md ; university of illinois at chicago; northshore university healthsystem, northshore university healthsystem introduction: subtotal cholecystectomy, where the infundibulum of the gallbladder is transected to avoid dissecting within a heavily inflamed triangle of calot, has been suggested as a method to conclude laparoscopic cholecystectomy while avoiding common bile duct injury. however, some case reports have suggested the possibility of recurrent symptoms from the remnant gallbladder. this retrospective case series reports a minimum of two-year follow-up on patients who underwent subtotal cholecystectomy within one four-hospital system. methods: a retrospective chart review database containing randomly selected cholecystectomies, all of which occurred between and , was reviewed to identify all instances of subtotal cholecystectomy. charts for these patients were reviewed through / , including any documentation from other providers, including primary care. results: six patients who underwent subtotal cholecystectomy with a remnant of infundibulum left following surgery were identified. surgical approach and the choice to perform subtotal cholecystectomy were dependent on the attending surgeon; all decisions were made intraoperatively. there was an average of months of follow-up for these patients within our institution. discussion: this case series adds six cases to the literature surrounding long-term outcomes in patients who underwent subtotal cholecystectomy. although one patient was lost to follow-up, no patient had recurrent biliary colic or other complications arising from the remnant gallbladder. this may be encouraging to surgeons who feel that subtotal cholecystectomy with an infundibular remnant is the safest way to proceed with cholecystectomy in patients with severe inflammation. objective: this study aims to evaluate the utility and efficiency of icg as an alternative to routine intraoperative cholangiogram in patients undergoing cholecystectomy. introduction: common bile duct injury is an uncommon, but serious complication associated with laparoscopic cholecystectomy. current guidelines state that when used routinely intraoperative cholangiogram (ioc) can decrease biliary injury, however it is not routinely used due to increased time of operation, and inaccessibility of equipment. indocyanine green (icg) has been found to be effective for identification of biliary anatomy during cholecystectomy, however has not yet been widely adopted. we aim to assess if icg is able to overcome the obstacles of ioc, while still effectively assessing biliary anatomy. methods: we performed a retrospective analysis of laparoscopic cholecystectomies performed in a single institution from january to september . elective and emergent cases were included. we stratified patients into icg and non-icg groups. patients who had concomitant procedures performed were excluded. we analyzed patient demographic information, as well as bmi, asa classification and comorbidities in both groups. our primary outcome was operation time (skin to skin), and laparotomy conversion rate. secondary outcomes were effectiveness of icg in visualizing biliary anatomy, and cost. results: patients were included in our study, in the non-icg arm and in the icg arm. both groups were similar in background. there were no statistical differences in patient demographics, asa classification, bmi, or comorbidities. there was no statistical difference in operation time ( . vs . minutes; p. ) or conversion rate ( . vs %; p. ). icg was able to delineate biliary anatomy in % of the patients. the cost of a mg/vial kit of icg is approximately $ . conclusion: the use of icg does not increase operating time during laparoscopic cholecystectomy. icg is an inexpensive and effective tool used to delineate biliary anatomy without the inherent burden and limitations of ioc. benefsha mohammad, md , michele richard, md , steve brandwein, md , keith zuccala, md ; danbury hospital, danbury hospital department of gastroenterology, introduction: obesity is a prevalent issue in today's society, which has increased the number of gastric weight loss surgeries. this presents an anatomical challenge to biliary disease requiring endoscopic retrograde cholangiopancreatography (ercp). in gastric bypass patients, traditional ercp via the mouth in these patients is technically more challenging, requiring a longer endoscope with a reported success rate of less than %. a solution is laparoscopic assisted ercp (la-ercp) via gastrostomy. this minimally invasive technique has become increasingly more prevalent and safe. we present our experience with la-ercp at our teaching community hospital in a large cohort of patients. methods and procedures: retrospective chart review was performed on all patients with a history of prior laparoscopic gastric bypass surgery who underwent la-ercp from april to april . the procedure was performed by two different general surgeons and one gastroenterologist. a pursestring suture and transfacial stay sutures were used to bring the gastric remnant to the abdominal wall. a gastrostomy was then created and accessed by the duodenoscope to perform the ercp. biliary sphincterotomy, papillary or biliary dilation, lithotripsy, stent placement, and/or stone removal were performed as indicated. we observed the incidence of postoperative outcomes, including acute pancreatitis, reoperation, post-procedure infection, pain control, hospital re-admission and bile leak. results: thirty-two patients met inclusion criteria. six patients were male and twenty-six were female, with mean ages of (std dev ) and years (std dev ), respectively. indications for la-ercp included suspected choledocholithiasis ( / ), cholangitis with choledocholithiasis ( / ), acute pancreatitis ( / ), abdominal pain with abnormal lft ( / ), cholangitis with cholecystitis ( / ), and bile leak ( / ). la-ercp was successfully performed in all thirty-two patients. biliary cannulation, sphincterotomy and stone extraction were performed on / patients, and one patient underwent sphincterotomy and stent placement for bile leak after recent laparoscopic cholecystectomy. one patient developed acute pancreatitis with elevated pancreatic enzymes which resolved after conservative treatment. one patient required a second la-ercp for stent replacement due to a persistent bile leak. the median length of stay was days (range - days). conclusions: la-ercp is a safe and feasible alternative to open surgery, and can be safely implemented at community hospitals with adequately trained providers. obesity is a growing burden on society, increasing the incidence of weight loss surgery. our large study proves that in this minimally invasive era, la-ercp provides gastric bypass patients a safe alternative with less pain and increased satisfaction. ahmed elgeidie, elsayed adel; gastrointestinal surgery center background: endoscopic sphincterotomy (es) is an effective therapeutic procedure for common bile duct (cbd) stone clearance but it carries a substantial risk of recurrent stones at long-term outcome. aim of the study: to evaluate the rate of cbd stones recurrence after primary complete endoscopic clearance, and to identify the risk factors of recurrence. methods: between january and december , patients with cbd stones who underwent successful es and complete stone clearance were studied retrospectively. recurrent cbd stone, was defined by the confirmation of the presence of cbd stone at least months after previous complete cbd stone clearance by es. the risk factors for recurrent cbd stones and mean time interval between initial es and stone recurrence were analyzed. results: in total, patients we included. the median follow up period was months. recurrent cbd stones appeared in / ( . %) patients after a median time interval of ( - ) months following es. stone recurrences were observed on multiple occasions in patients ( . %). on the univariate analysis, the significant risk factors related to recurrent cbd stone were male sex (p= . ), previous history of cholecystectomy (p= . ) multiple cbd stones (p= . ), large cbd stone (p= . ) the presence of periampulary diverticulum (p= . ) and stone crushing using mechanical lithotripsy (p= . ) conclusion: recurrence of cbd stones is an identified long-term risk after es and stone clearance. background: laparoscopic cholecystectomy during advanced pregnancy is challenging due to the limited intraabdominal space. patients may be at increased risk for developing trocar site hernia. case report: a year old hispanic female in her th week of pregnancy came to the er with acute right upper quadrant pain. due to lack of accessibility she had poor prenatal care. she had mildly elevated amylase but normal lfts and ultrasound showed some gallbladder wall thickening suggestive for acute cholecystitis and no dilated biliary duct. fetal ultrasound was normal. she was admitted to the hospital and started on antibiotics, obstetrics was consulted. her amylase peaked at [ u/l but then normalized and indication for laparoscopic cholecystectomy was made. mrcp and ercp were not performed as it was assumed that the patient had passed a stone. five mm trocars were placed in the luq and the umbilicus and a teleflex minigrasper between the tow. the uterus was found at the umbilical level. the gb was pulled out and the serosa was incised on both sides and a window was created behind the gb midportion and widened towards infundibulum and fundus. there was gb wall thickening and edema. the critical view was obtained and the cystic artery and duct were clipped and divided. the common bile duct appeared normal and no ioc was done. the specimen was retrieved through the luq port site using a mm endobag after dilatation to . cm due to the presence of two large stones. the port site fascia was closed using a suture passer. the postoperative course was uneventful and both mother and baby were well at the two weeks follow up. discussion: in case of biliary pancreatitis during pregnancy, lc should be performed and if ultrasound shows a normal biliary system and amylase/lipase normalize, mrcp/ercp and ioc may be avoidable to protect the baby. lc with two ports is feasible during pregnancy. removal of the specimen through a lateral abdominal wall site may help prevent an umbilical port site hernia in this patient population. s surg endosc ( ) :s -s introduction: splenic abscess is a rare, potentially lethal condition, with autopsy studies showing incidence rates between . - . %. mortality rates ranging from to % making early diagnosis and prompt intervention vital. several case reports have documented post surgical splenic abscess, most notably after laparoscopic sleeve gastrectomy. to the best of our knowledge, there has not been any reported cases of splenic abscess arising after laparoscopic cholecystectomy. it is important to remember this disease process for expeditious targeted treatment in future cases. case presentation: a year-old female with past medical history significant for cholilithiasis, hypertension, and hyperlipidemia presented to the emergency department (ed) with a chief complaint of abdominal pain for two days. labs and imaging were obtained which confirmed the diagnosis of choledocholithiasis and pancreatitis. ercp was performed which showed a . cm stone causing obstruction, with several other smaller filling defects. the stones were removed after sphincterotomy. post procedurally, the patient underwent an uncomplicated laparoscopic cholecystectomy on hospital day (hd) # . post operatively, the patient had persistent leukocytosis peaking at . thousand on postoperative day (pod) # . a ct scan was performed which showed a rim-enhancing splenic collection measuring . . cm suggestive of an abscess. interventional radiology was consulted and aspirated ml of purulent fluid. cultures grew out klebsiella pneumoniae and enterobacter cloacae complex, and the patient was discharged home on zosyn. discussion: laparoscopic cholecystectomy has become the cornerstone in treatment of symptomatic biliary colic and acute cholecystitis. of the many recognized complications of laparoscopic cholecystectomy, splenic abscess has not yet been reported in current literature. the nonspecific signs and symptoms of splenic abscess make clinical diagnosis difficult. the classic triad of fever, palpable spleen and left upper quadrant pain are only seen in about two-thirds of patients. ct scan has been shown to be the most sensitive imaging modality for diagnosis of splenic abscess. current treatment options for splenic abscess are broken down into two subsets: percutaneous and surgical intervention. percutaneous treatment includes image guided aspiration with or without placement of drainage catheter. surgical intervention can be either laparoscopic or open and includes drainage of abscess with splenectomy or splenic conservation. the best treatment option remains unclear, and there is lacking prospective data demonstrating which modality is superior. introduction: laparoscopic subtotal cholecystectomy is widely accepted as a safe alternative to the conventional laparoscopic cholecystectomy in case of acute cholecystitis with frozen calot's triangle. the remnant stump of the gallbladder may be either sutured or looped. however, there are limited studies comparing the outcomes of the two techniques. the present study is aimed at comparing loop and suture closure of the gall bladder stump. methods: a retrospective analysis of our prospectively maintained database revealed that between january and december . patients underwent laparoscopic subtotal cholecystectomy for acute cholecystitis, chronic cholecystitis or empyema gallbladder with frozen calot's triangle. the decision to use endoloop or sutures for stump closure was made intra-operatively after dividing the gallbladder through the infundibulum. a no. sized drain was kept in all the cases. the patients were discharged with drain in situ, and were reviewed on post-operative day during which an ultrasound was done and drain removed if the progress was satisfactory. the intra-operative and post-operative data between the two groups were recorded and analyzed. results: endoloop closure was performed in patients and suture closure using . ethibond was done in patients. three patients from the sutured group had post operative bile leak among which one patient underwent endobiliary stenting. the other were managed conservatively while the drain had to be retained for weeks. two patients in the endoloop group were detected to have retained stone in the remnant gallbladder cuff among which one had recurrent cholecystitis requiring laparoscopic completion cholecystectomy. none of the patients had bile duct injury or surgical site infection. mean post operative stay was . + . days, did not significantly vary between the groups. suturing needed more surgical expertise and had prolonged operative time than endoloop ( + min versus + min, p= . ). conclusion: suture or loop closure of the remnant gallbladder after subtotal cholecystectomy are equally effective. suturing the stump may be associated with increased incidence of biliary leak while endoloop may have higher incidence of retained gallstones. the choice between the two may be made intra-operatively based on the surgeon's expertise and preference. background and aim: in recent years, due to the spread of laparoscopic cholecystectomy, bile duct injury as its complication has been reported at a certain frequency. current surgical treatments include ) suturing and closing the injured part laparoscopically during surgery, ) transitioning to laparotomy and closing the suture, ) inserting a tube such as t-tube under the laparotomy, ) bile duct-intestinal anastomosis under the laparotomy, etc. are taken into consideration. regardless of which treatment method, it is not a definite ideal treatment. we have developed a bioabsorbable material (caprolactone: lactic acid ( : ) polymer reinforced with polyglycolic acid fiber and designed to be absorbed in about weeks). at this conference, we would like to talk about the current state and problems of development of minimally invasive therapy for biliary damaged area using bioabsorbable materials we developed. method: in order to overcome the problem of the current bile duct injury cure method, we have been developed, a) a method of closing a perforation part endoscopically from the luminal side of a bile duct (a covered stent using a bioabsorbable material in the damaged part), b) develop a method of closing the biliary duct injury under the laparoscope from the outside of the bile duct (adhering the bioabsorbable sheet to the bile duct perforation using a biocompatible adhesive). results: experimental results of suturing the bioabsorbable material in the biliary duct in surgery of laparotomy were able to regenerate the bile duct without stenosis in the damaged area. however, various adhesives were tried to bond the sheet of this bioabsorbable material and the native bile duct under the endoscope, but at the moment, there is no glue that will allow the sheet to be adhered readily and reliably where there is moisture to a certain extent. a tool for delivering the sheet from the bile duct into the injured part is under development and good results are obtained at present. conclusion: it is possible to regenerate the bile duct without constriction using a bioabsorbable material. it is difficult to laparoscopically adhere to the injured part of the bile duct, but we hope that it will be possible in the near future to develop further adhesives. s surg endosc ( ) , - kg/m (c) and more than kg/m (d). we made a . -cm longitudinal skin incision within the umbilicus. a wound retractor and a surgical glove were applied at that incision. we used the three -mm ports technique. after retracting the gallbladder upward, the cystic duct and artery were divided and identified using pre-bending forceps through the flexible port and laparoscopic coagulating shears (lcs). the cystic artery was dissected using the lcs and the cystic duct was also dissected after clipping. the gallbladder was freed from the liver bed using the lcs, and the specimen was retrieved from the umbilical wound. results: there were conversions to open laparotomy in cases ( . %) and requirement of additional ports in ( . %). the mean age (years), operation time (min), blood loss (ml) and postoperative hospital stay (days) in group a, b, c and d were . , . , . and . (p= . [), . , . , . and . (p= . ), . , . , . and . (p= . ) , and . , . , . , and . (p= . ), respectively. there was a significant difference in age only. the complications were bile duct injury in one case ( . %) and pneumothorax in two ( . %). conclusion: obesity had no influence of surgical outcomes for performing silc. introduction: recent studies have reported mixed outcomes when comparing surgeon case volume and laparoscopic cholecystectomy (lc) outcomes. formal minimally invasive surgical training (mist) has been shown to be associated with shorter post-operative length of stay (los), but no difference in major adverse events such as bile leak, bile duct injury, intra-abdominal abscess formation, and death. we aim to determine -day rates of major adverse events after lc in a university hospital setting, to identify significant associated risk factors, and to determine if mist or surgeon volume are associated with differences in los and major adverse events. methods: we conducted a single-center retrospective review of , cholecystectomies performed over a seven-year period ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . characteristics and outcomes were compared using chi squared or rank sum tests. multivariable regression modeling was used to determine independent associations with the two main outcomes, major adverse events and los. results: we identified , adults who underwent lc during the study period, with a median age of , and % women. about % (n= ) of patients had a los[ day and . % (n= ) were re-admitted within the first days after surgery for any reason. within days of lc, . % (n= ) of patients suffered from one or more major adverse events. this includes . % (n= ) of patients with bile duct injury, . % (n= ) of patients with bile leak, . % (n= ) of patients with intra-abdominal abscess, and . % (n= ) of patients died for reasons related to their procedure or post-operative recovery. table shows the characteristics of the patients and procedures with a comparison of the patients with an adverse event versus those without one. in univariate analysis, high annual surgical volume ( + cases/year) and procedure urgency were found to be significant predictors of adverse events and los, however, mist was not. in multivariable analysis, controlling for significant univariate predictors, urgent or emergent cases were associated with a -fold increase in odds of an adverse event (or= . introduction: laparoscopic cholecystectomy is an extremely common procedure in the united states, with over , cases performed annually. despite the procedure's overall safety, there has been some evidence that tobacco use is associated with increased risk of wound infection after lc. this retrospective chart review sought to examine whether tobacco use is associated with increased complications following laparoscopic cholecystectomy within a high-volume healthcare system. methods: after irb approval, of approximately , cholecystectomies performed within one four-hospital system between and were randomly selected, and patient charts were retrospectively reviewed. pre-, intra-, and postoperative data were collected, including all complications within days. tobacco use cohorts were defined as follows: never, former (any historical tobacco use), and current (active tobacco use within year of surgery) per the acs nsqip surgical risk guidelines. following preliminary data analysis, multivariable logistic regression models were generated to identify whether tobacco use was predictive of outcomes of interest. of the cases analyzed, patients ( . %) were never smokers; . % were former smokers, and . % were current tobacco users or had quit less than months prior to surgery. there were surgical site infections, one wound dehiscence, one port site hernia, three common bile duct injuries, and medical complications requiring prolonged hospitalization or readmission within days. current tobacco users were significantly more likely to undergo urgent surgery (following emergency admission or direct admission to the hospital) than former or nonsmokers. however, there was no difference between cohorts for prolonged duration of surgery, conversion to an open procedure, surgical site infection, wound dehiscence or hernia, common bile duct injury, or other medical complication. there was no significant difference between cohorts when all postoperative complications were pooled. conclusions: there does not appear to be a significant difference in -day surgical outcomes or complications in active tobacco users vs. former or non-users. although studies in other surgical settings have indicated a possible reduction in complications if patients abstained from smoking prior to surgery, this may not be beneficial in laparoscopic cholecystectomy. moreover, as current tobacco use appears to be associated with higher rates of urgent surgery, these patients may not be able to stop smoking prior to an elective procedure. prospective studies to further clarify whether there is any benefit towards tobacco cessation prior to lc may be valuable. , [ , [ respectively ( - ) , cyfra were . , . , . respectively ( - . ) . afp and cea were negative. as for this patient, he is of high risk of hepatobiliary system diseases. introduction: thymoma is one of the rare tumor entity benign or malignant arsisng from the epithelial cells of thymus gland, frequently associated with neuromuscular disorder myasthenia gravis. so, we are presenting this rare case of thymoma with myasthenia gravis in our institute. methods: we operated a single patient of thymoma in a case of myasthenia gravis by video assissted thoracoscopic approach. results: operative time- min, intraoperative blood loss − ml, post operative analgesia requirement in form of nsaids is for days, no ventilatory support required post operatively, with follow up reduction in achr ab from nmol/l to nmol/l and reduction in symptoms in form of reduced ptosis. conclusion: thoracoscopic thymectomy is feasible and safe in terms less operative time, less post operative pain and analgesia requirement and no post operative ventilatory support requirement. carter c lebares, md, stanley j rogers, md; ucsf background: duodenal fistulas are uncommon but morbid complications of acute necrotizing pancreatitis. if percutaneous drainage fails, surgical correction via roux-en-y diversion or pancreaticoduodenectomy can be required. while self-expanding metal stents have been tried, complications like migration and perforation have limited such use. endoscopic transmural stents have successfully treated fistulas of the stomach, particularly post-sleeve gastrectomy. here we present a case of endoscopic transmural stents used to treat a non-resolving duodenal fistula following acute necrotizing pancreatitis. methods: under general anesthesia, using a standard adult gastroscope, the fistula was identified in the second portion of the duodenum (fig. ) . a flexible-tipped guide wire was used to identify the fistula tract and two fr cm double pigtail biliary stents were deployed ( fig. ) with positioning verified under fluoroscopy. two weeks later these were removed and a single stent deployed into the visibly smaller tract (fig. ). two weeks after that, the single stent was removed and contrast medium was injected under fluoroscopic visualization, demonstrating resolution of the fistula (fig. ) . case: this patient is a year old woman with hypertension and congenital hearing loss who underwent a cholecystectomy for biliary colic and subsequent ercp with sphincterotomy for retained stone. this was complicated by acute pancreatitis which progressed to severe necrotizing pancreatitis with infected retroperitoneal necrosis. percutaneous drainage yielded initial improvement but a persistent moderate collection ( cc per day) lead to the identification of a fistula in the second part of the duodenum. repositioning and exchange of percutaneous drains over weeks did not hasten resolution. endoscopic transmural pigtail stents were tried after visualization of a large ( - mm diameter) fistula tract. stents were utilized as described in methods, with a total of three endoscopic interventions, at week intervals, resulting in resolution of the fistula as evidenced by contrast injection into the duodenum under fluoroscopy and subsequent ct scan with oral contrast. the patient's symptoms resolved and she was tolerating a normal diet. she remained thus at month follow-up. conclusion: this case demonstrates the benefit of endoscopic transmural stents for the resolution of duodenal fistulas, expanding the utility of this technique to address leaks and fistulas of the upper gastrointestinal tract. further study is warranted to clarify the timing and adjuncts to optimize the use of this promising approach. totally laparoscopic alpps combined with the microwave ablation for a patient with a huge hcc hua zhang; department of hepatopancreatobiliary surgery, west china hospital, sichuan university introduction: associating liver partition and portal vein ligation for staged hepatectomy (alpps) is a novel technique for resecting hepatic tumors that were previously considered unresectable due to the insufficient future liver remnant (flr) which may result in postoperative liver failure (plf). the procedure has been accepted and modified in many medical centers worldwide. but reports about the laparoscopic alpps were rare. this study aimed to report a totally alpps combined with microwave ablation for a patient with huge hcc and confirm the feasibility of laparoscopic alpps. methods: a -year-old man had complained of -year history of right upper abdominal pain, and the syndrome was worsened in recent month. abdominal enhanced computed tomography (ct) imaging revealed a cm solid mass in right lobe of liver with non-uniform and unclear boundary, the right posterior branch of the portal vein was invaded. in addition, a small lesion was simultaneous found in left lateral lobe of liver. the tumor was evaluated as unresectable due to the flr was only ml ( %). we decided to perform the laparoscopic alpps procedure. first stage including microwave ablation of the lesion in left lobe, cholecystectomy, ligation of the portal vein and transection of liver parenchyma. the second stage was done days later and consisted of laparoscopic right hemihepatectomy. results: the two stages were underwent by laparoscopy successfully. the operation duration was and minutes, respectively. estimated blood loss was and ml. the hospitalization time in intensive care unit was and days. there was no need for transfusion in both stages. the patient was discharged days after the second stage and the total hospitalization time was days. recovery of the patient was uneventful in addition to the incision infection after the second stage which recovered with conservative management. the patient did not show any signs of liver failure. the ct scan before the second stage showed an enlargement of left lobe, the flr was ml ( . %). there was no signs of residual liver disease in the ct scan days after the operation. the patient showed no signs of recurrence or liver failure in the following up period of six months. conclusion: totally laparoscopic alpps combined with microwave ablation is safe and feasible for the multiple hcc which was not resectable. the hypertrophy of remaining liver was fast and can achieve an adequate volume in a short time. introduction: chronic pancreatitis is a benign, irreversible inflammatory disorder characterized by the conversion of the pancreatic parenchyma into fibrous tissue. initial management should be conservative, surgery is applied in case of failure of medical treatment. the development of minimally invasive techniques has made it possible to perform these highly technical procedures in a laparoscopic manner. materials and method: we have the history of patients with and years with chronic pancreatitis and pancreatic lithiasis of difficult handling but intractable pain to those who decided to surgical management. we performed the procedure under general anesthesia, epidural analgesia catheter was placed. neumoperitoneum technique of cali, at mmhg and approach using a mm umbilical port, working ports of and a of mm port,. the pancreas was exposed by a section of the gastrocolic ligament with a mm ultrasonic scalpel, with cephalic retraction of the stomach, opening of a smaller sac and approaching the transpavity of omentum. the ventral surface of the pancreas was exposed from the neck. an incision was made in a pancreas body with a monopolar hook. primary pancreatic duct lumen was identified and the incision was extended longitudinally from the neck to the tail of the pancreas ( cm). roux's y loop was prepared cm from the treitz ligament, with a jejunum section with a mm stapler, roux's loop was transmecoscopically retrocollic, closing the gap of the mesocolon with monocryl. a -cm jejunum-jejunal anastomosis was performed with endo-gia stapler and closure of enterotomy with - polypropylene intracorporeal suture. jejunal (roux) isoperistaltic loop was placed longitudinally at the opening of the main pancreatic duct, and enterotomy was performed with monopolar in antimesenteric segment. the intracorporeal pancreatico and jejunum anastomosis was performed using a lower and an upper plane, with single points of total thickness with ethnobond - . closed drains were placed towards each anastomosis. this procedure was performed in the patients reported. operative time - min complications none operative time - days minimal bleeding drains no retired in both cases at days year follow-up of patients improved pain\ conclusions: minimally invasive surgery is a fundamental tool for the approach and management of patients with biliopancreatic pathologies. the establishment of multidisciplinary groups, offer an excellent alteranativa in the integral management of the patients. surg endosc ( ) gallbladder anatomy is highly variable, and surgeons must be prepared to identify anomalies of form, number, and position. variants include gallbladder agenesis, diverticulum, duplication, bilobed, multiseptate, phrygian cap, ectopic, and hourglass gallbladder. the hourglass gallbladder has been described from the earliest days of cholecystectomy, as morton described a congenital case in , and else thoroughly described the acquired and congenital strictures leading to the hourglass deformity in . we describe a case of an hourglass gallbladder found during one-step endoscopic retrograde cholangiopancreatography (ercp) and laparoscopic cholecystectomy. this year old male presented to an outside hospital with one day of nausea, and constant, severe, epigastric pain that radiated to his back. he endorsed a history of similar pain several times in the past. his abdomen was soft, nontender, and without murphy sign. laboratory evaluation revealed total bilirubin . mg/dl, alkaline phosphatase u/l, ast u/l, alt u/l, and no leukocytosis. ct abdomen and pelvis revealed cholelithiasis, distal choledocholithiasis, intra-and extra-hepatic ductal dilation, and a . centimeter left liver hemangioma. he was transferred for management of choledocholithiasis, and an abdominal ultrasound revealed cholelithiasis, without gallbladder wall thickening or pericholecystic fluid, and a . millimeter common bile duct without choledocholithiasis. he was taken to the operating room for a one-step ercp and laparoscopic cholecystectomy. upon laparoscopy, dense adhesions to the gallbladder were found. after initially attempting to obtain the critical view of safety, we then embarked on the retrograde "top down" dissection. this isolated a spherical structure measuring . . centimeters. two very thin tubular structures were identified, clipped, and transected after we found they were too small to place a cholangiocatheter. the common bile duct appeared to be pulled anteriorly by surrounding inflammation, though this was later found to be the proximal segment of gallbladder. the intra-operative ercp identified a remnant gallbladder with cholelithiasis and no extravasation of contrast. given the unusual anatomy, we completed the operation, ordered a post-operative ct liver and mrcp, and consulted a hepatopancreatobiliary surgeon. a small remnant gallbladder was identified on ct liver, though not on mrcp. completion laparoscopic cholecystectomy with intraoperative cholangiogram and ultrasound was performed on hospital day . this hourglass gallbladder variant likely occurred secondary to chronic fibrosis from cholecystitis, leading to a proximal and distal gallbladder lumen. in anatomic uncertainty, the "top down" dissection, intraoperative cholangiography, ct liver, and expert consultation are safe methods to avoid iatrogenic injury. introduction: endoscopic entero-enteral bypass could change our approach to small bowel obstruction in patients with prohibitively high operative risk. magnetic compression anastomoses have been well-vetted in animal studies, but remain infrequent in humans. isolated cases of successful use in humans include treatment of biliary strictures and esophageal atresia. while endoscopic gastro-enteric magnetic anastomoses have been described, the associated multicenter cohort study was terminated due to serious adverse events. since then, the technology has evolved and recently our own institution reported results of the first in-human trial of magnetic compression anastomosis (magnamosis), deployed through an open approach. here we present the first case of endoscopic delivery of the magnamosis device and the successful creation of an enteroenteral anastomosis for chronic small bowel obstruction in a patient with prohibitively high operative risk. methods: the magnamosis device has previously been approved by the food and drug administration (fda) for use in clinical trial. our institutional review board approved emergency compassionate endoscopic use of the device in this patient due to a non-resolving small bowel resection and prohibitively high operative risk. case: this is a year old man with advanced liver disease, chronic obstructive pulmonary disease, and history of emergent right colectomy with end ileostomy for cecal perforation. he presented with multiple acute on chronic episodes of small bowel obstruction with a stable transition point in the distal ileum, radiographically estimated at centimeters proximal to the ileostomy. endoscopic evaluation through the ileostomy revealed a traversable obstruction with proximally dilated small bowel. the magnets were delivered via endoscopic snare under fluoroscopic guidance and positioned in adjacent loops of bowel on either side of the obstruction (image ). by days post-procedure, healthy villi were visible through the central portion of the mated magnetic rings (image ). by days the magnetic rings were mobile and the anastomosis was widely patent allowing easy passage of the gastroscope (image ), and the patient's symptoms were completely resolved. the rings passed through the ileostomy days post-procedure. at month follow up, the anastomosis was unchanged (image ). conclusion: this case demonstrates the benefit of an endoscopically created magnetic compression anastomosis in a patient with small bowel obstruction and high operative risk. further studies are indicated to evaluate the use of this technique in similar patients or those with malignant obstruct, ion. desiree raygor, md, ruchir puri, md; university of florida health jacksonville cholecystectomy is one of the commonest operations in general surgery [ ] . occasionally chronic cholecystitis can lead to a small contracted gallbladder. this diagnosis can be misleading as it may represent congenital agenesis of the gallbladder [ ] . a -year-old female with a past history of pancreatitis presented with a three day history of right upper quadrant pain associated with nausea and vomiting. upon exam she exhibited tenderness in the right upper quadrant. her leukocyte count and liver function tests were within normal limits. ultrasound revealed a poorly visualized, contracted gallbladder without stones and a dilated common bile duct (cbd). cholescintigraphy revealed non visualization of the gallbladder after two hours, which was suggestive of acute cholecystitis. decision was made to proceed with a laparoscopic cholecystectomy. the abdomen was entered by an open hasson technique and standard trocar placement for a cholecystectomy was performed. on initial inspection, the gallbladder was not readily visible. a structure appearing to be the cbd was present and was mobilized circumferentially (fig. ) . a gauge butterfly cannula was utilized and multiple cholangiographic images were obtained (fig. ). no cystic duct or gallbladder was identified which was suggestive of congenital agenesis of the gallbladder. the patient did well postoperatively, and was discharged home on postoperative day two. the patient's symptoms resolved and she continues to be pain free one month postoperatively. congenital agenesis of the gall bladder is a rare disorder. a high index of suspicion is required especially in the setting of a small contracted gall bladder. if preoperative imaging is inconclusive then diagnostic laparoscopy should be the next step. cholangiogram should be performed routinely to confirm the diagnosis and to rule out an ectopic gall bladder. conversion to open does not offer any distinct advantage, and laparotomy should be avoided if possible given its associated morbidity. there are many reports upper abdominal major arterial aneurysms. however, an aneurysm of left inferior phrenic artery had never been reported. a -year-old woman with liver cirrhosis associated with hepatitis b viral infection was referred to department of surgery for treatment of aneurysm of left inferior phrenic artery. she underwent trans-arterial chemoembolization (tace) for treatment of hepatocellular carcinoma three times, previously. on months after last tace, mm sized highly enhancing nodular lesion of gastric fundus was found on follow-up abdomenpelvis computed tomography (a-p ct). one year later, the size of this lesion increased to mm, and an aneurysm was diagnosed. she underwent angiography and attempted embolization with an aneurysm of the left inferior phrenic artery, but access failed. we performed a laparoscopic vessel ligation. she recovered with no complication and discharged on the th postoperative day. s surg endosc ( ) :s -s yousef almuhanna, vatsal trivedi, fady balaa; university of ottawa a years old female, g and weeks pregnant, was brought to the hospital by ems, after being found on the floor in her toilette surrounded by vomitus and urine. mother-inlaw, who happens to be at the house that time, have heard severe retching followed by a loud bang sound. firefighters have found no pulse and therefore started cpr. return of spontaneous circulation was achieved, yet unfortunately, she had arrested again minutes prior to arrival to er. pocus assessment showed large rvot, and therefore tpa was started on the assumption of pulmonary embolism. upon arrival of blood work, it was found that her hemoglobin had dropped from to . fast was repeated showing moderate to severe amount of free fluid in the morrison's pouch and pelvis. she was then taken to the operating theatre, had undergone laparotomy showing liver segment ii injury. pringle's maneuver and aortic clamping did not control the bleed, therefore finger fracture and venous clips were used to temporary minimize the bleed, and head to interventional radiology suite. after multiple attempts to control the bleed, and the massive transfusion, she vital signs were not maintained, and had arrested afterwards. sarrath sutthipong, md, chumpunut chuthanan, md, chinnavat sutthivana, md, petch kasetsuwan, md; bhumibol adulyadej hospital, bangkok, thailand background: mesenteric panniculitis (mp) is a rare, benign and chronic fibrosing inflammatory disease that affects the adipose tissue of the mesentery of the small bowel and colon. the specific etiology is unknown and no clear information about the incidence. the diagnosis is suggested by ct and is usually confirmed by surgical biopsy. treatment is based on some selected drugs. surgical resection is sometimes attempted for definitive therapy, although the surgical approach is often limited. we reported a case of the mp diagnosed with ct and surgical biopsy by laparoscopic approach. case report: -year-old woman with months history of chronic abdominal pain, mainly localized in the sub-epigastrium, intermittent and mild. she had anorexia but no weight loss or change in bowel habits. no history of medical illness or surgery. the physical examination was unremarkable, except for palpation of ill-defined mass about cm at mid-abdomen, firm, smooth surface with mild tenderness. the laboratory profile and tumor marker were normal. ct of the abdomen, which showed focal heterogeneous enhancement of the mesenteric fat with stranding ( . . cm) with multiple internal subcentimeter lns in the supraumbilical area, which was probably inflammatory in origin and suggestive of mp. f-fdg pet/ct showed faint fdg uptake in multiple mesenteric lns. the patient was subsequently underwent diagnostic laparoscopy with biopsy. intra-operative finding showed a fat-like surface of yellowish mass at mesentery of jejunal segment, incisional biopsy was performed laparoscopically. the histology showed adipose tissue with areas of fat necrosis, fibrosis, foamy macrophages infiltration and predominant chronic inflammation, no evidence of malignancy. ihc studies (including cd , s- , cd and cd ) were performed and the result was compatible with reactive process. treatment was started with mg prednisone once daily and planned for follow-up with repeated ct scan. discussion: mp involves the small bowel mesentery in over % of cases. the diagnosis is made by pathologic findings: fibrosis, chronic inflammation and fatty infiltration. the differential diagnosis is broad and has been associated with malignancies such as lymphoma, well-differentiated liposarcoma and melanoma. the imaging appearance varies depending on the predominant tissue component. a definitive diagnosis is biopsy but open biopsy is not always necessary. no data of laparoscopic biopsy, which has been reported previously. treatment has been reserved for symptomatic cases with a variety of drugs. our case was started on oral corticosteroid treatment and waited for responsive evaluation. background: laparoscopic appendectomy is the gold standard for treatment of acute appendicitis. stapled closure of the appendiceal stump is often performed and has been shown to have several advantages. few prior cases have been reported demonstrating complications from free staples left within the abdominal cavity after the laparoscopic stapler has been fired. case report: a previously healthy year old female initially underwent laparoscopic appendectomy for acute uncomplicated appendicitis during which the appendix and mesoappendix were divided using laparoscopic gastrointestinal anastomosis (gia) staplers. her initial postoperative recovery was uncomplicated and she was discharged home the same day. the patient returned to the emergency department on postoperative day with one day of sharp mid-abdominal pain, obstipation, and emesis. her abdomen was distended and mildly tender but not peritoneal. she was afebrile but was found to have a leukocytosis of . . ct demonstrated twisted loops of dilated small bowel in the right lower quadrant with two transition points, suggestive of internal hernia with closed loop bowel obstruction. diagnostic laparoscopy was performed through the three prior appendectomy incisions. an adhesion was noted between the veil of treves and the mesentery of a more proximal loop of ileum caused by a solitary free closed staple, remote from the staple lines, resulting in an internal hernia containing several loops of ileum ( fig. ). the hernia was reduced, and the small bowel was noted to have early ischemic discoloration. the adhesion was lysed by removing the staple from both structures to prevent recurrence. through the remainder of the procedure, the compromised loops of bowel began to peristalse and the color normalized. the procedure was concluded without resection. the patient recovered on a surgical floor and was discharged home on postoperative day one. conclusion: gastrointestinal staplers are commonly used secondary to ease of use and low complication rate. it is not uncommon to leave free staples in the abdomen during laparoscopy as retrieval can often be more difficult and time consuming. our case is only the second in the literature reporting an internal hernia with closed loop bowel obstruction as a complication of retained staple. choosing the most appropriate size staple load, to reduce the number of extra staples after the fire, and removing as many free staples as possible can prevent potentially devastating complications. video-assisted thoracoscopic pulmonary wedge resection in a patient with hemopytsis and intralobar sequestration: a case report mary k lindemuth, md, subrato j deb, md; the university of oklahoma health science center case report: a -year-old male with history of noonan's syndrome, bronchitis, and asthma presented with acute hemoptysis. while chest x-ray was unremarkable, a computed tomography angiogram of his chest was significant for intralobar pulmonary sequestration in the right lower lobe. the aberrant pulmonary artery originated from the abdominal aorta, immediately proximal to the celiac axis, and coursed through the hiatus in the retroperitoneum. flexible, fiberoptic bronchoscopy revealed blood within the right lower lobe bronchus with no appreciable source. a right video-assisted thoracoscopic approach was taken for wedge resection of the sequestration. twoportal technique was utilized with the patient on single lung ventilation. the sequestration was easily identified; the anomalous pulmonary artery coursed directly to a large, focal area of hemorrhage noted within the lower lobe pulmonary parenchyma, as seen in image [rectangle marking the aberrant artery and oval marking the sequestration]. pathologically, the specimen was noted to be benign lung parenchyma with bronchiectasis and abundant, acute hemorrhage. discussion: pulmonary sequestration (ps) is a rare, congenital bronchopulmonary foregut malformation. literature describes the incidence of ps to be only . - . % of all pulmonary malformations. as ps is most frequently diagnosed during childhood, the occurrence of diagnosis during adulthood is estimated to be less than per , adults. two types (intra-and extralobar) are described, with intralobar sequestration most common and contained within the normal visceral pleura. both types have aberrant systemic arterial blood supply, most frequently from the thoracic aorta. likewise, both types are nonfunctioning lung tissue, as there is no direct communication with the bronchopulmonary tree. the most common presentation is pneumonia, and often patients will have had recurrent symptoms before diagnosis. it is rare to present with hemoptysis, which is understood to be secondary to elevated capillary pressure within the sequestration and then communication through the pores of kohn. while endovascular embolization of the aberrant pulmonary artery has been described as a safe alterative for surgical intervention, the subjects of these studies have primarily been children and long-term outcomes are unknown. the definitive treatment of ps continues to be surgical intervention. the surgeon should strive to leave as much normal lung parenchyma as possible. video-assisted thoracoscopic resection is well tolerated by patients when compared to thoracotomy. however, it is vital for the surgeon to be aware of the potential risk of life-threatening hemorrhage secondary to the sequestration having systemic blood supply that must be controlled and ligated. case report: a years-old female patient with history of an increased mass and weight loss of kilograms in months, associated with vomiting and nausea for eight months. abdominal ultrasound showed an irregular cyst, without solid projections and without signs of flow in doppler, measuring cm. investigation continued with ct scan that showed a large homogeneous cystic lesion with no septum in the abdominopelvic region, possibly mesenteric, measuring . . cm. a laparoscopic approach for resection of the cyst was then performed. the surgery was performed with a patient in the dorsal decubitus, using three trocars: one in the umbilical region ( -mm) for the camera, and where the pneumoperitoneum was created by the hasson open technique under direct vision; and another two located in the epigastrium ( -mm) and in the right upper quadrant ( -mm) . in addition to the mesenteric cyst, a simple cyst in the right ovary and a solid nodule with a lipomatous characteristic of approximately cm in the abdominal cavity were visualized. total resection of the mesenteric cyst with periprancreatic fibrous tissue was performed. the cyst was punctured and its contents fully aspirated. resection of the right ovarian cyst was also performed. at the end of the procedure the mesenteric and ovarian cysts, the nodule, part of the omentum, and the peripancreatic tissue were removed through the -mm trocar at the umbilicus. patient had no further complications, being discharged four days after the procedure. histopathologic result showed a serous cyst in the right ovary, serous cyst in peripancreatic mesentery with chronic inflammatory process and signs of calcification; no signs of malignancy were observed in any specimen. we aimed to present the succesul therapeutic approach utilizing laparoscopy for safely removing a gastrointestinal stromal tumor. depicted is a year old jehova's witness female who presented to the emergency department for evaluation of bitemporal headache and dizziness and found with profound anemia with hemoglobin . and hematocrit . upon arrival to ed. the patient refused blood transfusion as her religious beliefs, jehovah's witness, preclude her from taking blood products. as part of her work up, endoscopy was performed and revealed a large, approximatelly cm, prolapsed, ulcerated, nodular lesion with active bleeding in the cardia of the stomach. this was temporized but the friable tissue, with no single identifiable lesion for clip placement, left the patient at high risk for re-bleeding. she was taken to the operating room and laparoscopic partial gastrectomy with intraoperative esophagogastroduodenoscopy were succefully perfomed, with minimall blood loss and no intra operative complications. patient was discharged on post op day . we present the case of a -year-old male with a history of morbid obesity with an initial bmi of . , who underwent an elective laparoscopic single anastomosis duodenal-ileal bypass with sleeve gastrectomy (sadi-s). postoperatively he developed an anastomotic leak at the duodeno-ileal anastomosis that would not resolve despite reoperation. he was then converted to a roux-en-y gastric bypass (rygb). postoperative imaging failed to reveal any signs of anastomotic leak and the patient was discharged tolerating an oral diet. he returned to the emergency department days later with a cm sub-hepatic collection arising from the duodenal stump from the surgical conversion. interventional radiology percutaneously drained the collection and found a connection between the cavity and the duodenum. using this connection, a percutaneous decompressive duodenostomy drain was successfully inserted into the duodenum using a guidewire through the abscess cavity along with an extra-enteric drain placed within this cavity. the collection was obliterated and the duodenal leak was controlled successfully with percutaneous drainage, bowel rest with parenteral nutrition and broad-spectrum intravenous (iv) antibiotics. the patient was reintroduced to a bariatric clear diet after a week of bowel rest and the abscess drain was then discontinued during the same hospital admission. the patient was discharged with the percutaneous duodenostomy tube which was removed in clinic days later, after the patient tolerated capping trials and imaging failed to reveal any further collections, oral contrast extravasation or distal obstruction. in this article we analyze notable imaging from the case and review current literature on the different management options for a duodenal stump blowout. we also discuss the basics of the sadi-s procedure and conversion of a sadi-s procedure to a rygb. keywords: anastomotic leak, duodenal stump blowout, sadi-s, duodenostomy tube. pancreatopic heterotopia is often an incidental finding on autopsy, but in some cases can lead to abdominal pain, obstruction, or intussusception. we present a case of pancreatic herterotopia mimicking an internal hernia on radiologic imaging. a year old female with seven month history of chronic abdominal pain treated for low back pain and recurrent urinary tract infections. she was found to have a computed tomography (ct) scan concerning for internal hernia and labs consistent with acidosis. she was taken for a laparotomy and did not have an internal hernia, but an exophytic mass in the proximal jejunum. the mass was resected and a stapled side to side jejunojejunostomy was created. on pathologic review, the specimen was found to be pancreatic heterotopia. her post operative course was complicated by an ileus, but was discharged post op day three. at her two week follow up she had minimal incisional pain and at one year follow-up she had resolution of her left upper quadrant abdominal pain. prior to this report, pancreatic heterotopia has never been described as presenting on ct scan as an internal hernia. although uncommon it should remain in the differential when evaluating a patient presenting with abdominal pain and radiologic evidence of obstruction or internal hernia. case report: a -year-old male patient who was diagnosed with high blood pressure at years-old and presented tetraparesis and intense asthenia for six months. blood tests showed hypokalemia, hypernatremia, and suppressed renin activity. ultrasound of the urinary tract was normal. ct scan of the abdomen showed a hypodense nodule with regular margins, measuring . . cm with a density of hu in the non-contrast phase and heterogeneous uptake after the injection of the contrast in the left adrenal gland. thus, the diagnosis of hyperaldosteronism secondary to the left adrenal nodule was confirmed, and surgical resection was indicated. the procedure was performed with the patient in the right lateral decubitus. two -mm and one -mm trocars were used on the left flank, as well as the -mm portal for the camera in the lower right quadrant under direct vision. the pneumoperitoneum was created by the hasson open technique in the transumbilical incision. the procedure consisted of the dissection, isolation and electrocautery of the left renal capsule and the left adrenal region with ultrasonic device, as well as the periadrenal vessels, adjacent lymph nodes and periadrenal and adrenal fat tissue. the surgery was uneventful and the patient had no further complications, being discharged the next day. histopathologic result showed a completely excised adrenocortical adenoma. conclusions: the hybrid minimally invasive approach proved to be safe and effective for this procedure, and the known advantages of minilaparoscopy such as less trauma, better visualization, better dexterity, better aesthetics, and reduced hospital stay were observed. s surg endosc ( ) background: coccidioidomycosis is a fungal infection endemic to the southwestern united states, central america and south america. coccidioides is ubiquitous in many of these endemic regions, with near % seroconversion in some communities. two-thirds of these mycotic infections may be asymptomatic. the most common presentation of coccidioidomycosis consists of "flu-like" symptoms or pneumonia. less than five percent of symptomatic cases progress to disseminated coccidioidomycosis which may involve any organ system. very rarely infection may include the peritoneum. we report a case of coccidioidomycosis with peritoneal involvement in an immunocompetent individual. case: a -year-old male presented to the emergency department with progressive abdominal pain. he was seen and treated for pneumonia in the emergency department one week prior. the patient worked outdoors in arizona and was otherwise healthy with a family history of malignancy and blood disorders. fever, leukocytosis and ascites on computed tomography scan prompted a diagnostic laparoscopy which revealed peritoneal granulomas positive for coccidioides. the patient was treated outpatient with fluconazole. discussion: since this is the th reported case of peritoneal coccidioidomycosis to our knowledge. the patient described in this case report was an otherwise healthy -year-old male; this is incongruent with many of the previously recorded cases which involved disseminated disease in immunocompromised patients. the patient's family history of malignancy and blood disorders suggests a potential underlying genetic predisposition that could account for this abdominal presentation. possible mutations include genes coding for the interleukin- β receptor and the signal transducer and activator of transcription which have been implicated in increased coccidioidomycosis susceptibility. peritoneal infection presents a unique challenge in diagnosis. in these cases coccidioidomycosis may not be suspected due to nonspecific symptoms and imaging, the infrequency of this extra-pulmonary manifestation and clinical characteristics that mimic the presentation of tuberculosis and malignancy. abdominal infections have been misdiagnosed as appendicular abscesses, iliopsoas abscesses, adnexal abscesses and pancreatic masses. consequently, the diagnosis of peritoneal coccidioidomycosis is often made after laparoscopic exploration of the abdomen and histopathology, as it was in this case report. conclusions: coccidioidomycosis incidence is on the rise in endemic areas and it often falls on the surgeon to make the diagnosis in extra-pulmonary cases. the peritoneal subset of coccidioidomycosis should be considered in endemic areas when a young, otherwise healthy patient presents with abdominal pain. failure to recognize the possibility of coccidioidomycosis may lead to unnecessary treatments and procedures. indocyanine green cholangiography to detect anomalous biliary anatomy steven d schwaitzberg, md, gabrielle yee, ms; university at buffalo jacobs school of medicine introduction: common bile duct injury is the most feared complication of cholecystectomy. imaging with indocyanine green (icg) is a safe and effective technique to detect biliary anatomy in open, laparoscopic and robotic surgery. several studies report detecting aberrant biliary anatomy with the use of icg in laparoscopic cholecystectomy with high success rates. by identifying the cystic duct-common hepatic duct confluence before dissecting calot's triangle, icg allows surgeons to perform "virtual" cholangiography at the start of procedures to identify either normal anatomy or possible anatomic variants. it is clear that icg use is an effective tool to achieve the critical view of safety. however, no reports have suggested icg cholangiography as the last operative step in cholecystectomy to identify hidden biliary anomalies and avoid postoperative bile leak complications. case report: we report a novel use of icg cholangiography in visualizing anomalous biliary anatomy prior to closing, thus avoiding potential bile duct leakage. in our case, icg cholangiography was used to fluoresce the common hepatic duct, common bile duct and cystic duct. the cystic duct was transected, and the gallbladder was removed using electrosurgery. at the completion of the gallbladder removal, the liver was elevated to inspect the clips on the cystic duct and artery. at this point, near infrared imaging was reinitiated, and a small mm structure was noted to fluoresce next to the cystic artery. this structure was identified using white light and subsequently clipped. discussion: the use of icg in this context after the completion of the cholecystectomy facilitated the identification of a small hepatocystic or aberrant duct, which would have likely leaked bile sometime in the postoperative period. based on our experience, we recommend one additional routine near infrared viewing to identify small structures or potential leaks at the completion of cholecystectomy. improved visualization of the extrahepatic biliary anatomy by icg has the potential to translate into improved clinical outcomes. solitary fibrous tumors (sft) are uncommon fibroblastic mesenchymal neoplasms that display a wide range of histologic behaviors. these tumors, which are estimated to account for % of all soft tissue neoplasms, typically follow a benign clinical course. however, it is estimated that - % of sfts are malignant and demonstrate aggressive behavior with local recurrence and metastasis up to several years after surgical resection. we report a case of sft arising from the stomach, which is an exceptionally rare finding and has been reported only six times in the literature. additionally, this tumor was associated with dedifferentiation into undifferentiated pleomorphic sarcoma. to our knowledge, there are no documented cases of a malignant sft arising from the stomach to demonstrate dedifferentiation into an undifferentiated pleomorphic sarcoma. a -year-old male presented to the emergency department with vague complaints of right-sided flank pain. the patient had a history of nephrolithiasis and underwent a ct abdomen. this scan revealed a large heterogeneous mass in the left upper quadrant. the patient underwent endoscopic ultrasonography with fine needle aspiration of the mass, which stained strongly for cd . gastrointestinal stromal tumor (gist) was the favored diagnosis as it is by far the most common mesenchymal neoplasm of the stomach, especially cd positive spindle cell neoplasm. accordingly, the patient began treatment with imatinib; however, after four weeks of therapy, there was no significant radiologic regression. a second biopsy was performed and the specimen was sent for stat immunohistochemistry, which revealed diffuse strong nuclear positivity. a diagnosis of solitary fibrous tumor was provided. surgical resection of the tumor was performed, which measured . cm. the patient was to undergo surveillance imaging every to months post-operatively. surveillance scan showed solitary metastatic disease in the left lateral segment of the liver. he underwent left lateral segmentectomy with an uneventful recovery. our case was complicated by diagnostic dilemma with gist, highlighting the challenges of diagnosing and characterizing sfts. dedifferentiation, or the abrupt transition from a classic sft into a high-grade sarcoma, is a particularly concerning finding in our case, as it is associated with a worse prognosis than classic malignant sft. the stat marker by immunohistochemistry is very specific for sft and may have aided in the diagnosis earlier. therefore, it is imperative to keep solitary fibrous tumor, albeit exceedingly rare, in the differential diagnosis of mesenchymal neoplasms of the stomach. appendiceal diverticulitits is an uncommon pathology that can clinically mimic acute appendicitis. some radiographic distinctions have been reported, but final pathologic examination of the surgical specimen is required to confirm the diagnosis. symptoms are often more mild, which can lead to a delayed diagnosis, and increases the risk of severe complications such as perforation. a year old female presented with a three day history of right lower quadrant pain. she described the pain as constant and radiating to the left lower quadrant. associated symptoms included nausea and vomiting, and decreased appetite; she denied fevers or diarrhea. the patient had no significant past medical history, and surgical history was significant for a total nephrectomy for living donor kidney transplant to her mother. on physical exam she was tender in the right lower quadrant with rebound and a positive rosving's sign. all laboratory results were unremarkable, and she was hemodynamically stable. ct scan was performed and demonstrated a dilated fluid filled appendix with surrounding inflammatory change without abscess or free intra-peritoneal air. she was subsequently admitted to the hospital, made npo, started on iv antibiotics, and was taken to the operating room where she underwent an uncomplicated laparoscopic appendectomy. post-operatively, her hospital course was unremarkable. pathology revealed acute suppurative appendicitis secondary to an acutely inflamed appendiceal diverticula, consistent with a final diagnosis of acute appendiceal diverticulitis. appendiceal diverticulitis should be considered in patients presenting with acute right lower quadrant abdominal pain. although some consider appendiceal diverticulitis a variant of acute appendicitis, it is important to distinguish between the two diagnoses. appendiceal diverticulitis has a higher rate of complications, including perforation, and is associated with a higher risk of neoplasm, particularly mucinous adenomas and carcinoid tumors. appendectomy should be performed in all cases in order to obtain appropriate pathological examination and rule out coexistent neoplasms. laparoscopic appendectomy is a safe and appropriate approach to treatment of appendiceal diverticulitis. upper gi endoscopy and biopsy showed a gastrointestinal stromal tumor (gist) in the stomach. a videolaparoscopic partial gastrectomy was then proposed. the surgery was performed with the patient in the right lateral decubitus. two -mm minilaparoscopic trocars, a -mm conventional trocar for an ultrasonic instrument and a -mm trocar in the umbilical region for the camera were used. pneumoperitoneum was created using the hasson open technique under direct vision. trans-operatory endoscopy was perfomed to identify the tumor easily. initially, the ultrasonic device released the large omentum, and, then, the tumor was resected in the body of the stomach. the gastric wall was manually sutured with a - vicryl, and the tumor was removed in an endobag through the -mm incision in the umbilicus. the surgery was uneventful, with a total time of minutes. the patient had no further complications, being discharged two days after the procedure with good clinical conditions. histopathological result showed a free margins gist. conclusion: the minimally invasive approach proved to be safe and effective for this procedure. the known advantages of video-surgery such as less trauma, better visualization, increased dexterity, better esthetics, and less postoperative recovery time were confirmed. the upper gi endoscopy contributed to improve the safety and efficacy of the procedure, allowing a more precise resection of the gist, as well as the intragastric review of the suture line at the end of the surgery. background: portal vein thrombosis (pvt) is a rare post-operative complication, which has been associated with a wide range of precipitating factors. most commonly described associated conditions include; cirrhosis, bacteremia, myeloproliferative disorders and hypercoagulable states. pvt most frequently occurs as a complication after hepatobiliary surgery, and although possible, very few cases have been documented occurring after laparoscopic surgery of the gastrointestinal tract. herein, we describe a case of pvt in a patient who underwent elective laparoscopic right hemicolectomy and was treated successfully at our center. case: a year-old female with past medical history of depression, migraines and endometriosis underwent an uncomplicated laparoscopic right hemicolectomy at our facility, for recurrent rightsided diverticulitis. she had suffered previous episodes of diverticulitis and desired definitive surgical treatment. her hospital course was uneventful and she was discharged to home on postoperative day . on post-operative day , she presented to the emergency department complaining of severe abdominal pain, back pain and nausea. computed tomography of abdomen and pelvis revealed pvt. she was initiated on therapeutic anticoagulation with heparin. hematology was consulted for hypercoagulable workup. further investigation revealed that she had a family history of a brother who had had a lower extremity deep venous thrombosis, with negative hypercoagulable workup. she had also previously been taking leuprolide and conjugated estrogen and medroxyprogesterone for her endometriosis. she was ultimately found to have a heterozygous prothrombin g a gene mutation. her anticoagulation was bridged to coumadin and she was discharged home. she has recovered as expected, without any further complications. discussion: although more common in patients with cirrhosis after hepatobiliary surgery, pvt is a rare complication that can occur after virtually all types laparoscopic surgeries, including elective right hemicolectomy. patients may be completely asymptomatic, or present with a broad spectrum of symptoms including; severe abdominal pain, fever, diarrhea, or gastrointestinal bleeding. physicians should be aware of this possible complication, since early diagnosis and treatment is imperative to prevent life-threatening complications, such as intestinal ischemia and perforation. a detailed medical and family history is imperative, and all patients with post-operative pvt should undergo complete hypercoagulability workup. this is a case of a year old male with a previous history of a redo-hiatal hernia years prior who presented with two episodes of upper gastrointestinal bleeding with no identifiable source noted on both endoscopy and angiography. during his second admission, initial hemoglobin was . g/dl and endoscopy performed showed massive amount of blood in the stomach. continuous oozing was seen originating in the fundus area but no clear source could be identified. empiric epinephrine was injected to the area but failed to achieve hemostasis. angiography was also negative. repeat endoscopy performed showed no active bleeding, however, distention of the wrap into the gastric cavity was observed. the patient re-bled and was taken to the operating room emergently after failed attempt at endoscopic control. the patient underwent proximal gastrectomy after intra-operative gastrostomy and exploration was unable to identify a bleeding source. the patient was left with an open abdomen and in discontinuity while resuscitation was performed in the surgical intensive care unit. he subsequently underwent a roux-en-y reconstruction and gastrostomy tube placement via the distal gastric remnant. upper gastrointestinal series performed demonstrated absence of leak, and the patient was started on a liquid diet supplemented with tube feeding. his recovery was uneventful and he was discharged home in stable condition. pathology revealed gastric ischemia at the base of the wrap making it impossible to visualize through endoscopy. on reviewing the literature, gastric ulcers and ischemia have been previously described. incidence was up to % and their onset of presentation ranged from the early post-operative period up to years. most were located in the lesser curvature. the exact pathophysiology for its occurrence is not completely understood. factors hypothesized include technical aspect of the fundoplication causing inappropriate tension, vessel disruption and ischemia, and injury to the vagus nerve affecting gastric emptying which was thought to increase gastrin secretion. treatment includes medical management with proton pump inhibitors; however, few cases describe antrectomy with inclusion of the bleeding ulcer. our case presents failed medical and endoscopic management. we recommend take down of the fundoplication in hemodynamically stable patients to completely evaluate the gastric mucosa, identify, and address the source of bleeding. otherwise emergent cases will require staged gastrectomy including the wrap followed by roux-en-y reconstruction. acalculous cholecystitis associated with a large periampullary duodenal diverticulum: a case report peng yu, md, phd, austin iovoli, aaron hoffman, md; department of surgery, suny buffalo, kaleida health system, buffalo, ny introduction: periampullary diverticulum (pad) could compress common bile duct (cbd), and consequently cause obstructive jaundice and cholangitis as few publications have documented. here we first report an acalculous cholecystitis associated with a pad-related cbd obstruction. case: the patient was a -year-old female with a past surgical history of laparoscopic sleeve gastrectomy who presented at the emergency room with upper abdominal pain and vomiting for one day, associated with leukocytosis and left shift. serum total bilirubin raised up to . mg/dl on hospital day (hd) . ct, ultrasound, and mrcp images confirmed a distended, wall-thickening gallbladder with pericholecystic fluid, and a significantly dilated cbd at . cm of diameter ( fig. ) , without cholelithiasis or choledocholithiasis. ercp was unable to be completed due to the post-gastrectomy anatomy and the failure in cannulation into the ampulla which embedded in a large foodimpacted pad (fig. ). on hd , the patient underwent a diagnostic laparoscopy and an intra-operative cholangiogram which confirmed a mildly inflamed edematous gallbladder, and a . . cm large pad with a narrow neck that was distorting the distal cbd (fig. ). since the patient's bilirubin level had been improving, we decided to only do a laparoscopic cholecystectomy. intraoperatively an anatomic variation of the cystic artery encircling the cystic duct ( fig. ) was also identified. postoperatively the patient recovered well during the thereafter inpatient course and at the postoperative -week outpatient follow-up. the pathology of the excised gallbladder confirmed cholecystitis without cholelithiasis. discussion: lemmel's syndrome is defined, in the absence of cholelithiasis or other detectable obstacle, by obstructive jaundice due to pad. since lemmel described this duodenal-diverticulum-obstructive jaundice in , there still have been very few cases reported or investigated. to date there is no report describing the association of acalculous cholecystitis with lemmel's syndrome. this patient's mild acalculous cholecystitis probably attributed to the biliary obstruction and consequent gallbladder hydrops. her symptoms could be from either acalculous cholecystitis or intermittently worsening biliary obstruction. in this case, the contribution of the anatomic variation of the cystic artery is unclear. in the future, if this patient's symptoms recur, the treatment plans for her will be sphincterotomy, removal of the impacted food in the pad, or diverticulectomy. accidental fish bone ingestion masquerading as acute abdomen aim: to report a case of fish bone ingestion masquerading as acute abdomen. case report: a years old female patient presented with complaints of severe abdominal pain since days. there was no history of associated nausea or vomiting, fever or altered in bowel habits. on examination patient had tenderness and guarding localized to the right iliac fossa. blood investigations revealed raised inflammatory markers. ultrasound whole abdomen and contrast enhanced computed tomography (cect) were normal. patient was managed conservatively but in view of persistence of symptoms a triple puncture diagnostic laparoscopy was performed on day of admission. omental inflammation with soapy appendix was found and appendicectomy was performed. on further assessment a foreign body was also found in the ileum which was removed and identified as a fish bone. patient had a satisfactory post operative recovery and was discharged in stable condition. discussion: acute abdomen due to fish bone ingestion is not a very common occurrence. unfortunately the history is often non-specific and these people can be misdiagnosed with acute appendicitis & other pathologies. ct scans can be useful to aid diagnostics. it is however not fully sensitive in detecting complications arising from fishbone ingestion. conclusion: any patient with acute abdomen, with non-specific history and normal imaging may still benefit from a diagnostic laparoscopy. discussion: this patient presented with a bowel obstruction, partial cecal necrosis and neuroendocrine carcinoma. literature suggests that cecal necrosis in the majority of cases is caused by a vascular event, occlusive or non-occlusive. the patient had atherosclerosis and an underlying malignancy which can be associated with prothrombotic states and contributes to an overall risk of thrombosis. the cecum can sustain ischemic ischemic injury in the presence of severe or prolonged hypotension. most frequent causes being decompensated heart failure, hemorrhage, arrhythmia or severe dehydration, only of which was present in this patient. the midgut neuroendocrine tumor is generally located in the terminal ileum, as a fibrotic submucosal tumor cm or less. mesenteric metastases are often larger than the primary tumor and associated with fibrosis which may entrap loops of the small intestine and cause bowel obstruction. this may eventually encase the mesenteric vessels with resulting venous stasis and ischemia in segments of the intestine as seen in this patient. conclusion: cecal necrosis is a rare entity, but its incidence increases with age. isolated cecal necrosis may manifest as a ct-negative appendicitis or a small bowel obstruction in the absence of past surgical history. s surg endosc ( ) laparoscopic transection of the falciform and triangular ligament successfully released the entrapped loop with successful reperfusion by the end of the surgery. in the absence of any prothrombotic comorbidity, the patients were discharged asymptomatic without further anticoagulation. to date only few similar cases have been reported, and most of them described in neonates and pediatric patients. to our knowledge, this cases reporteds in the elderlys. in this patients laparoscopic approach was both diagnostic and therapeutic with the transection the ligament. roberto javier rueda esteban , andres mauricio garcia sierra , felipe perdomo ; universidad de los andes, fundacion santa fe this is a patient´s rare case of spontaneous splenic rupture associated to chronic myeloid leukemia as an uncommon complication. the case report and review of the relevant literature on symptomatology and clinical management is presented. emphasis is made about the importance of including splenic rupture as differential diagnosis for acute abdominal pain, especially in a patient with neoplastic hematopathology, since early treatment increases patient survival and prognosis. esophagectomy is a complex operation associated with serious immediate complications and long term chronic complications. gastric ulcers are a common chronic complication after esophagectomy with gastric conduit reconstruction. these are rarely complicated by significant bleeding or perforation. we report a case of delayed diagnosis of a fistula forming between a gastric conduit and right bronchial tree years after esophagectomy. this was successfully treated using multiple therapeutic approaches including endoscopic localization and resection through a right thoractomy. to the best of our knowledge, our patient is the only survivor from a chronic gastric conduit bronchial fistula. a year old male with type diabetes mellitus, dyslipidemia, asthma and smoking history presented years after an ivory-lewis esophagectomy for a gastrointestinal stromal tumor (gist) with a chronic cough starting years after his esophagectomy followed by multiple episodes of hematoptysis over the next years. the patient was known to have ulcers in his gastric conduit with a massive bleed year after his esophagectomy. repeat endoscopy revealed two large chronic ulcers that had increased in size based on comparison of pictures from endoscopies to years after his esophagectomy despite maximal medical management. the patient presented to numerous specialists at tertiary care centers in canada and the united states. ultimately, in a clinic the patient was observed to cough immediately after the ingestion of water, but not solids leading to a provisional diagnosis of a gastrobronchial fistula. a barium swallow failed to show a fistula (fig. ). however at endoscopy, instillation of saline directed at an ulcer immediately induced a cough, but this was not reproduced when the saline was directed away from the ulcer. the fistula was ultimately demonstrated by placing a wire through the ulcer and visualizing it bronchoscopically in the right superior segmental bronchus . in an effort to pursue a minimally invasive approach two attempts were made to close the fistula with over-the-scope clips (otsc). unfortunately, the patient's symptoms persisted. a wire was placed through the fistula and delivered through the patient's mouth and endotracheal tube. a right thoracotomy allowed access to the conduit, which was opened and the fistula localized using the wire. the fistula was resected and the bronchus closed. at twelve month follow up the patient did not have a recurrent cough or hemoptysis while tolerating a full diet. introduction: roux en-y gastric bypass (rygb) is one of the initial and most studied weight reduction procedures and remains the gold standard for comparison in bariatric surgery clinical outcomes. although rygb is an effective procedure for weight loss, it has been less popular over last several years because of increased morbidity compared to the more utilized vertical sleeve gastrectomy (vsg). early complications of rygb include bleeding, perforation, or leakage. late complications include internal hernias, small bowel obstruction, anastomotic stenosis, marginal ulcers, and gastrogastric fistulas. case report: a -year old female with a past medical history of morbid obesity, diabetes mellitus type , hypertension, gerd, peptic ulcer disease, cholelithiasis, liver dysfunction with ascites, asthma, and a past surgical history of rygb ( years ago) presented to our institution with acute on chronic abdominal pain associated with nausea, vomiting, dysphagia, inability to eat and maintain hydration, and an additional weight loss of about lbs. over the last year. in addition, the patient was a chronic opioid and nsaid user, had an extensive smoking history, and had not followed with her surgeon for years. at the time of presentation, the patient weighed lbs (bmi: . ), had normal vital signs, and appeared cachectic. an upper gastrointestinal study followed by an upper endoscopic examination demonstrated complete obliteration of the gastrojejunal anastomosis and revealed a -cm long gastrogastric fistula originating from the distal end of the gastric pouch to the lesser curvature of the excluded stomach. after conservative measures were initiated to hydrate and metabolically stabilize the patient, the decision was made to proceed with diagnostic laparoscopy and surgical placement of a gastrostomy tube to the gastric remnant. the patient was discharged after tolerating a full liquid diet and gastrostomy tube feedings, for plan of future revision of gastrojejunostomy when optimal nutritional status is achieved. conclusions: late complications of rygb occur at a rate of - %. major risk factors for anastomotic complications include non-compliance, smoking, and opiate and nsaid abuse. though abdominal pain, anastomotic stenosis, marginal ulcers, and fistulas are relatively common late complications of rygb, complete obliteration of the gastrojejunal anastomosis has not been well described in the literature. this case demonstrates the importance of long term follow up post rygb for early diagnosis of late complications and brings attention to this rare, but possible sequele that can arise in patients after rygb. contrast radiograms and upper endoscopic photographs will be presented. introduction: retroperitoneal sarcoma represents approximately - % of all sarcomas and less than . % of all neoplasia. radiotherapy and chemotherapy still do not represent valid therapeutic alternatives; therefore complete surgical resection is the only potential curative treatment modality for retroperitoneal sarcomas. the ability of complete resection of a retroperitoneal sarcoma with tumor grading remains the most important predictor of local recurrence and disease-specific survival. in a patient with a large fibrosarcoma and associated hypoglycemia, assays for insulin-like activity (ila) were found to be high in the extract of tumor tissue, while insulin was not detected in significant concentration neither in the same extract nor in his serum. laparoscopic surgery represents an alternative technique for radical resection of such tumors as a minimally invasive rather than traditional surgery. only few cases were reported in the literature. introduction: roux-en-y gastric bypass (rygb) is a frequently performed bariatric procedure, of which internal hernia (ih) is a known complication. we discuss a rare finding of occult gastric remnant perforation as a result of an obstructed ih in a post bypass patient. methods: we present a case report of a single bariatric surgeon's experience at a tertiary care hospital. literature review of pubmed confirms the unique presentation and operative findings in our patient, as few similar cases have been published. a -year-old male s/p rygb years ago presented to the ed with right upper quadrant pain, nausea, vomiting, and a leukocytosis of , . bmi was . ; weight was lbs. workup included an abdominal ultrasound showing gallbladder distention without signs of cholecystitis. liver function tests were normal. further imaging included a ct scan, remarkable for a paraesophageal hernia (peh) containing the gastric pouch, and an elevated left hemidiaphragm. the scan showed no evidence of ih or bowel obstruction. an upper gi series was additionally obtained, which was also negative for small bowel obstruction. due to unclear etiology for this patient's symptoms or source of leukocytosis, diagnostic laparoscopy was planned. results: intraoperative findings were significant for ih containing dilated small bowel with twisted and incarcerated omentum through the jejunojenunostomy site, as well as a distended gallbladder without acute inflammation. ih was reduced and closed without bowel resection. cholecystectomy was completed. subsequent inspection of the diaphragmatic hiatus revealed uncomplicated herniation of the gastric pouch. in attempts to dissect the left diaphragmatic crus, a large pocket of purulent material was encountered below the left diaphragm in the region of the remnant stomach fundus. methylene blue test and intraoperative endoscopy did not demonstrate any connection to gastric pouch. the purulence was attributed to an occult remnant stomach perforation related to distal obstructed ih. a drain was left in the abscess and the peh was not surgically addressed. patient was discharged on postoperative day . he has not suffered any further complications or recurrent complaints. conclusion: gastric perforation following rygb is an uncommon complication resulting from ih. this diagnosis was missed by preoperative imaging and was only found after thorough laparoscopic investigation. surgeons should maintain a high clinical suspicion of ih in post rygb patients with otherwise unexplained abdominal symptoms, fever, and leukocytosis, even in the absence of confirmatory diagnostic testing. threshold for operative exploration in this clinical setting should remain low. alejandro garza, md, robert alleyn, md, jose almeda, md, ricardo martinez, md; utrgv obesity is an epidemic condition worldwide carrying significant morbidity and mortality. surgical therapy is the only proven effective method to sustain weight loss. among the different surgical procedures gastric bypass is the most effective. during this surgery, most of the stomach is excluded from the upper gastrointestinal tract which makes future evaluation of the same very challenging. this could potentially lead to delay in diagnosis of any pathology in the bypass stomach. gastric cancer is the th most common cause of cancer and cause of cancer death in the united states. we present a case report of a patient who underwent a roux-en-y gastric bypass and went on to developed adenocarcinoma in the gastric remnant year after her surgery. she underwent an exploratory laparotomy, extended antrectomy, subtotal gastrectomy including the gastro-colic ligament, and incidental appendectomy. pathology showed grade undifferentiated adenocarcinoma that penetrated the visceral peritoneum with clear margins. there was angiolymphatic invasion and perineural invasion along with metastatic carcinoma in out of lymph nodes. introduction: polyarteritis nodosa (pan) is a systemic transmural inflammatory vasculitis that affects medium-sized arteries. inflammation of the vessel wall and intimal proliferation creates luminal narrowing which can lead to stenosis and insufficiency. the same inflammatory process causes disruption of the elastic lamina leading to aneurysm formation and possible spontaneous rupture with life-threatening bleeding. multifocal segments of stenosis and aneurysm formation are characteristically identified as a "rosary sign" or "beads on a string". unlike other vasculitides, pan does not involve small arteries or veins, and is not associated with anti-neutrophil cytoplasmic antibodies. we present the case of a year old female with a significant intra-abdominal bleed that was explored and repaired primarily. she was subsequently found on angiogram and postmortem pathology to have findings consistent with pan. case presentation: year old female who presented to the emergency department with abdominal pain followed by hemorrhagic shock and found to have a ruptured left hepatic artery aneurysm during exploratory laparotomy. this aneurysm was suture ligated with a successful outcome. a mesenteric arteriogram was performed the following day and demonstrated lesions consistent with pan including aneurysms of the left gastric branches, right and left hepatic arteries, and beaded appearance of the iliac artery. however, days after hospital discharge she developed massive pulmonary embolism from which she did not recover. postmortem examination confirmed rupture of the left hepatic artery aneurysm in addition to gross anatomical and histological findings consistent with pan. discussion: polyarteritis nodosa is a systemic inflammatory vasculitis that causes intimal proliferation and elastic lamina disruption. this multifocal disruption of the vessel results in aneurysm formation alternating with stenosis creating a characteristic "rosary sign" on imaging. spontaneous rupture of these aneurysms is rare and almost always fatal due to life-threatening hemorrhage. with acutely ruptured aneurysms, prompt diagnosis, aggressive resuscitation, and hemostasis through transarterial embolization or surgery is paramount for patient survival. while acute rupture of an aneurysm as the result of pan is exceedingly rare, it must be considered as a differential diagnosis in the setting of acute abdominal pain and hemodynamic instability. in a patient known to have a medical history of pan and aneurysm formation, routine monitoring and disease progression should be followed. introduction: , surgeries are done annually in the us for small bowel obstruction, which is most commonly caused by intraabdominal adhesions, malignancy, and hernias. . to . % of small bowel obstructions are due to paraduodenal hernias. paraduodenal hernias carry a % lifetime risk of incarceration with a mortality of to %. case report: the patient is a year old male who presented with severe upper abdominal pain for one day. he was passing flatus and had had a bowel movement the previous day. on examination, the patient was tender over the upper abdomen. computed tomography (ct) scan with iv contrast showed a mesenteric swirl sign. the decision was made to perform diagnostic laparoscopy with possible small bowel resection. intraoperatively, a mesenteric defect was noted posterior and to the right of the duodenum, through which bowel was herniating. the herniated bowel and its mesentery were edematous. the defect was sutured closed, taking seromuscular and mesenteric bites through the stomach, jejunum, and mesentery. the patient had an uneventful recovery postoperatively and was discharged on postoperative day . he returned on postoperative day with periumbilical pain which resolved with conservative management. he was followed up weeks postoperatively and was doing well. discussion: paraduodenal hernias are the most common internal hernias. they are seen more often in males. they are caused by failure of the counterclockwise rotation of the prearterial segment of the embryonic midgut in weeks to of embryonic development. paraduodenal hernias usually present with chronic intermittent abdominal pain, weight loss, nausea, and vomiting. they may present acutely with symptoms of bowel obstruction. peritoneal signs are often not appreciated due to retroperitoneal position of the hernia. ct scan of the abdomen often shows clustering of bowel loops, which cannot be displaced on repositioning the patient. if imaging is equivocal, diagnostic laparoscopy may be undertaken. surgical correction consists of reducing the bowel, resecting nonviable segments, and either closing the defect or opening the sac laterally into the general peritoneal cavity. in summary, paraduodenal hernias are a rare cause of bowel obstruction and as such present a challenge in diagnosis and early intervention. diverticulosis of the appendix is a rare disease found in . - . % of appendectomies, first described in . the clinical presentation may be acute inflammatory with or without appendicitis or it may be an incidental finding in an uninflamed appendix. the congenital type is rare and it has all the bowel wall layers. it most frequently represents as pseudo diverticulum which lacks the muscularis layer. the pathogenesis of appendiceal diverticula is not completely elucidated. its symptoms are similar to and often misdiagnosed for that early acute or chronic appendicitis. while appendectomy is curative for both entities, it is important to distinguish diverticulum of the appendix from appendicitis as it is four times more likely to perforate and may be a sign of an underlying neoplasm. we reported a very rare giant pseudo diverticulum of the appendix in a -year-old male presenting with chronic abdominal discomfort for months. abdominal x-ray showed abnormal gaseous finding. physical exam was significant for a soft rubbery mass in the periumbilical region. blood work revealed slight elevation of c-reactive protein. preoperative ct and mri showed a -centimeter-large cavity composed of thin wall, located at the tip of the appendix with peri appendicular fat stranding. in the concern of pending obstructive symptom and chronic abdominal pain, we decided to perform the resection laparoscopic. the soft mass arose from the tip of the appendix. there were dense adhesions between the appendix, mesentery, and sigmoid colon. after adhesiohedlysis, laparoscopic appendectomy was performed with endogia. the specimen was extracted through a small incision without spillage. hospital course was uneventful and the patient was discharged on post-operative day . the pathological finding was consistent with a pseudo diverticulum of the appendix which lacked muscularis layer and the inner wall of the cavity was lined with a scattered cubital epithelial layer in the continuity with the appendiceal mucosal membrane. here we report a successful laparoscopic resection of an extremely rare giant chronic pseudo diverticulum of the appendix. yvette farran, ms, jorge a miranda, ms, benjamin clapp, md, elizabeth de la rosa, md; texas tech university health sciences center introduction: sigmoid colon intussusception is rarely encountered and given its vague symptomatology diagnosis and management can be difficult. the treatment of an intussusception in adults is different than in children. lipomas as the causative etiology for intussusception are encountered up to . % of the times and up to %- % of the patients require surgical resection for treatment. methods: this is a case report about a year old male that presented with two weeks of worsening abdominal pain and distention. physical exam was only pertinent for abdominal pain on light palpation, guarding and moderate distress. ct scan of abdomen and pelvis demonstrated a lipomatous mass causing complete obstruction of the sigmoid colon with intussusception. this was managed with laparoscopic sigmoidectomy. the patient had an uncomplicated post-operative period and was discharged on post-operative day . pathology of the lipomatous mass confirmed a benign lipoma. discussion: intussusception is rarely encountered in clinical practice in adults and constitutes % of all cases. lipoma induced sigmoid intussusception with complete obstruction is rare. symptoms can be non-specific as in this case. this case report highlights the importance of timely diagnosis and treatment of an intussusception in adult patients. ct scan is the gold standard for diagnosis and often shows a "target sign". other imaging techniques like ultrasound have shown adequate results but remain less effective than ct scan. the treatment in adults is not a reduction by enema like in pediatrics but rather resection of the lead point. this can be appropriately done with a laparoscopic technique in most cases. conclusion: colonic intussusception is rare. surgery is the only treatment for an intussusception in adults since the lead point needs to be removed, and can be attempted safely with a laparoscopic approach. surg endosc ( ) :s -s joshua smith, md, kern brittany, md, amie hop, md, amy banks-venegoni, md; spectrum health case report: year-old female with no significant past medical history presents with a -year history of nocturnal cough that had worsened over the past months and had associated regurgitation. she underwent esophagogastroduodenoscopy (egd) that showed a tortuous esophagus and tight lower esophageal sphincter that required dilation. she received an upper gastrointestinal (ugi) contrast study that showed a dilated, tortuous esophagus with 'bird's beak' tapering, consistent with achalasia, as well as a large epiphrenic diverticulum measuring cm. esophageal manometry confirmed "pan-esophageal pressurization" consistent with type ii achalasia. given her symptoms in the presence of these findings, she elected to proceed with surgery. she underwent laparoscopic, trans-hiatal epiphrenic diverticulectomy, heller myotomy and dorr fundoplication. extensive dissection allowed for approximately cm of retraction down from the chest and we were able to come across it with a single blue load of a mm linear cutting stapler. post-operatively, she tolerated the procedure well with immediate improvement in her symptoms. her ugi on post-operative day showed no evidence of leak, she tolerated a soft diet and was discharged home. she was seen at -week and -year follow-up appointments with complete resolution of symptoms. discussion: epiphrenic diverticula in the presence of achalasia has an occurrence rate of %. large diverticula ([ cm), are even more rare with only a handful of case reports in the literature. historically, thoracotomy or, more recently, thoracoscopic approaches are required for resection. however, thoracic approaches are associated with a % increase in morbidity, namely due to staple line leak and the resulting pulmonary complications. only a single case report exists on our review of the literature that demonstrates successful trans-hiatal laparoscopic resection without post-operative complications of a diverticulum of this size. the shortest documented length of hospital stay postoperatively for similar cases is days, while the average is - days or longer for those with complications. our patient was able to go home on post-operative day after a normal ugi and was tolerating a soft diet. not only does this case show that a large epiphrenic diverticulm can be successfully resected via the trans-abdominal laparoscopic approach, this case makes the argument that patients undergoing any minimally-invasive epiphrenic diverticulectomy and myotomy, with or without fundoplication, may be successfully managed with early post-operative contrast studies and dietary advancement, thus decreasing their length of hospitalization and overall cost of treatment. kazuma sato, shunji kinuta, koichi takiguchi, naoyuki hanari, naoki koshiishi; takeda general hospital background: situs inversus totalis (sit) is a rare congenital condition in which the abdominal and thoracic organs are located opposite to their normal positions. few cases of laparoscopic surgery for gastric cancer with sit have been reported. we report a case of laparoscopic distal gastrectomy with d lymph node dissection performed for gastric cancer in a patient with sit. case description: an -year-old woman was admitted to our hospital for treatment of gastric cancer that was diagnosed by esophagogastroduodenoscopy (egd) at a local clinic after she experienced anemia and nausea. egd identified an irregularly shaped gastric ulcer located at the anterior side of the lesser curvature of the antrum. a biopsy revealed a moderately differentiated adenocarcinoma. she was then diagnosed with sit by chest radiography and abdominal computed tomography (ct). the abdominal ct showed that all organs were inversely positioned and that the wall of the antrum had thickened; it also showed the lymph nodes in the lesser curvature of the stomach, without distant metastasis or an abnormal course of vascularity. the patient was clinically diagnosed with t n m stage iiia gastric cancer according to the japanese classification of gastric carcinoma. a laparoscopic distal gastrectomy with d lymph node dissection in accordance with the japanese gastric cancer treatment guidelines as well as a roux-en-y anastomosis due to an esophageal hiatal hernia were performed. the surgery was safely and successfully performed, although it required more time than usual because the inverted anatomic structures were repeatedly examined during the surgery. the postoperative course was positive, and the patient was discharged on postoperative day without any complications. the final stage of this case was pt bn m stage ia. currently, the patient is doing well without recurrent gastric cancer. conclusion: gastric cancer with sit is an extremely rare occurrence. we experienced a case of laparoscopic distal gastrectomy with d lymph node dissection performed for gastric cancer in a patient with sit. we simulated the operation for sit by viewing left-right reversed ordinary surgical videos. the abdominal ct angiography with a three-dimensional reconstruction helped reveal any variation and confirmed the structures and locations of vessels before the surgery. the operation could safely be performed following the standardized surgical technique by reversing the surgeon standing position and trocar position. sternum or chest wall resection is performed for a variety of conditions such as primary and secondary tumors of the chest wall or the sternum. sternum reconstruction has been a complex problem in the past due to intraoperative technical difficulties, surgical complications, and respiratory failure caused by the chest wall instability and paradoxical respiratory movements. advances in the fields of surgery and anesthesia result in more aggressive resections. nowadays neither the size nor the position of the chest wall defect limits surgical management, because resection and reconstruction are performed in a single operation that provides immediate chest wall stability. chest wall resection involves resection of the ribs, sternum, costal cartilages and the accompanying soft tissues and the reconstruction strategy depends on the site and extent of the resected chest wall defect. here i'll present, the youngest ever case reported, years old girl with rhabdomyosarcoma involving the sternum. i will present the management challenges and the reconstruction options. introduction: neuroendrocrine malignancies constitute . % of all cancers. the gastrointestinal tract is the commonest site, followed by the lung. the last decade has seen a steady increase in their incidence. this is a case series of twenty five such tumours and their clinicopathological characteristics. materials and methods: twenty five patients with neuroendocrine tumours of the gastrointestinal tract were studied with reference to their demographic and clinicopathological characteristics. apart from routine pathological examination, these tumours were also checked for e cadherin expression as an independent marker of aggressive disease. results: the age of our patients ranged from to years. we had female and male patients, contradicting a female preponderance in literature. the vast majority of the tumours we encountered were from the stomach and duodenum, with and patients, respectively. two tumours were at the gastroduodenal junction, two from the appendix, small intestine and pancreas, each, and one each from the rectum and gall bladder. this is in contrast to literature that shows that neuroendocrine tumours of the git most commonly arise from the appendix and small bowel, followed by the rectum, stomach and duodenum. two of these tumours were functional. the diagnosis was confirmed by immunohistochemistry staining for chromogranin a and synaptophysin. grading was done using who criteria that takes into account the mitotic count, ki index and necrosis. of our cases were grade i. further, immunohistochemistry for e cadherin showed that absence of expression correlated with more aggressive clinical behavior. out of twenty five patients were operable at presentation and standard resections depending on the organ of origin with adjuvant therapies were given as required. could only be given palliative care. the functional tumours were treated with radiolabelled somatostatin analogues following uptake studies. conclusion: as neuroendocrine tumours are relatively rare, information about them is not as abundant as with other malignancies. absence of e cadherin expression is associated with more aggressive disease. more studies are required that document the pathological characteristics and clinical behavior in order to offer well rounded treatment protocols that treat not only the primary, but also the generalized effects of the secretions produced by them. targeted chemotherapy is gaining prominence, but more specific drugs directed at the plethora of receptors these tumours express, could potentially revolutionize treatment. ( ) . unfortunately there are no publications from denmark. we would like to present first to our knowledge reported case of double gallbladder in denmark. double gallbladder is a rare anomaly with a prevalence of : in autopsy studies, described first by boyden in ( ) . there are several classifications of double gallbladder that are based on relation between gallbladder, cystic duct and common bile duct ( , ) . non-specific symptoms and inadequate imaging are possible causes of lack of awareness of the condition. removal of all gallbladders, preferably laparoscopic with special attention to the biliary anatomy, is recommended ( ). method: case report with review of the literature. a -year-old female patient of polish origin was hospitalized due to upper right quadrant pain. on admission clinical manifestations and paraclinical abnormalities of pancreatitis were present. ultrasound scanning of the abdomen showed bile stones, ultrasonic manifestations of acute cholecystitis and normal intra-and extrahepatic bile ducts. because of elevated liver enzymes mrcp was performed and showed double gallbladder, double cystic duct and signs of pancreas anulare. scheduled ercp confirmed bile stones in cbd, double gallbladder with double cystic duct, h-type according to harlaftis classification ( ) . because of minor retroperitoneal perforation second ercp was needed for removal of all stones. the patient was then scheduled to laparoscopic cholecystectomy with perioperativ cholangiography. conclusion: anatomical variations of the gallbladder such as double gallbladder are rare and often remain unnoticed. they are most often identified because of clinical manifestations symptoms, diverse imaging studies, during surgery or autopsy. as most of them are not expected, they can contribute to complications during surgery. careful preoperative imaging is very important to prevent accidental bile duct injury. looking at the number of case reports, double gallbladder seems to be slightly more common than expected. the interesting question is whether a gallbladder discovered during an unrelated radiological investigation in a patient that previously underwent a cholecystectomy can represent undetected case of double gallbladder. we would like to present a review of the literature as well as images from mrcp, ercp and laparoscopy. michael jaroncyzk, md, courtney e collins, md, ms, vladimir p daoud, md, ms, ibrahim daoud, md; st. francis hospital; hartford ct introduction: several decades ago, surgical training was saturated with procedures to treated peptic ulcer disease. since the introduction of histamine- blockers and proton pump inhibitors, these procedures have dwindled significantly. however, there are still instances where patients require surgical intervention for peptic ulcer disease. perforation is one of the indications for surgery. the surgical options to treat a perforated peptic ulcer are numerous. one of the most common options is a graham patch. we are presenting a case of a patient with a perforated ulcer that did not have available omentum for the repair. methods and procedures: recently, a -year-old female with a past history of an open total abdominal hysterectomy and bilateral salpingo-oophorectomy presented as an outpatient with chronic lower abdominal pain. she underwent a work-up and imaging that did not reveal any pathology. at diagnostic laparoscopy, she had diffuse lower abdominal adhesions, which were lysed. she was discharged on the same day, but presented to the emergency department two days later with severe abdominal pain and fevers. the work-up revealed tachycardia, diffuse abdominal tenderness with peritoneal signs, leukocytosis and a large amount of free air on imaging. she was emergently brought to the operating room for a diagnostic laparoscopy. during laparoscopic exploration, the lower abdominal cavity appeared normal for a recent lysis of adhesions. attention was turned to the upper cavity to find the pathology. bile-stained free fluid and peri-gastric exudates were identified, but no perforation was visualized. intra-operative endoscopy revealed the site of perforation in the antrum on the lesser curvature. a biopsy was performed and the decision was made to perform a graham patch. however, the omentum was already densely involved with the lower abdominal cavity from the enterolysis. due to the close proximity of the falciform ligament, it was mobilized laparoscopically and the pedicle was used as a graham patch. the patient recovered without any additional issues. the biopsy was reported as a chronic gastric ulcer. conclusion: surgical history has given us many options to treat peptic ulcer disease that are not nearly as common as they were decades ago. perforated ulcers can be managed laparoscopically and graham patches are a common choice for repair. however, the lack of the omentum for a proper pedicle flap can pose a problem in some patients. we have shown in this patient that a falciform pedicle flap can be successfully used as a substitution. laparoscopic management of boerhaave's syndrome after a late presentation: a case report and literature review tahir yunus, hager aref, obadah alhallaq; imc background: boerhaave's syndrome involves an abrupt elevation in the intraluminal pressure of the oesophagus, causing a transmural perforation. it is associated with high morbidity and mortality. having a nonspecific presentation may contribute to a delay in diagnosis and results in poor outcomes. treatment is challenging, yet early surgical intervention is the most important prognostic factor. case presentation: we present a case of a thirty-two-year-old male with a long medical history of dysphagia due to benign oesophagal stricture. he presented with acute onset of epigastric pain after severe emesis. based on computed tomography scan, he was diagnosed with boerhaave's syndrome. presenting with signs of shock, mandated immediate surgical exploration. for which he was taken for laparoscopic primary repair with uneventful postoperative recovery. the golden period of the first hours of insult still applies for cases of oesophagal perforation. the rarity of these cases makes a comparison between the various treatment methods difficult. our data support that the use of laparoscopic operative intervention with primary repair as the mainstay of treatment for the management of oesophageal perforation. lipomas of the gastrointestinal tract are rare benign soft tissue tumors that are often discovered incidentally. these lesions are often asymptomatic, but have occasionally been reported to have clinical significance as will be described in this case report. a year old male initially presented to his primary care physician's office with a three week history of vague intermittent abdominal pain. his pain was located in the mid epigastrium and was associated with mild nausea. past medical history was significant for hyperlipidemia and a right-sided goiter, and he denied any previous surgeries. outpatient work up revealed a microcytic anemia, intermittent melena and hemoccult positive stools. the patient was referred to hematology and gastroenterology. endoscopies revealed gastritis, and small internal and external hemorrhoids. he underwent an outpatient ct scan which demonstrated a . . cm mass within the lumen of the jejunum causing long segment non-obstucting intussusception. subsequently, the patient was referred to surgery and underwent a diagnostic laparoscopy. at the time of surgery, an approximately twelve centimeter segment of proximal jejunum was identified intussuscepting into a distal limb. this segment was attempted to be reduced laparoscopically, however there was significant mesentery within in the intussusceptum and the segment could not be safely reduced. therefore, the section of bowel was delivered through a small periumbilical incision. the intussusceptum was then able to be manually reduced from the intussusception. at this point a large mass was palpated inside the lumen of the jejunum. a small bowel side to side, functional end to end resection and anastomosis was preformed. the bowel was returned to the abdomen and the abdomen was re-insufflated. the remainder of the small bowel was run and no additional lesions were identified. final pathology revealed a . . . cm submucosal partially obstructing lipoma with ulceration at the tip. the patient recovered uneventfully and was discharged home on the second post operative day. this case report describes a submucosal jejunal lipoma that was acting as a lead point for intermittent non-obstructing small bowel intussusception, while simultaneously causing a microcytic anemia due to ulceration at the tip of the lipoma. laparoscopic assisted reduction and small bowel resection is a safe and effective treatment for gastrointestinal tract lipomas that are unable to removed endoscopically. percutaneous endoscopic gastrostomy (peg) is an alternative to laparotomy for open gastrostomy tube placement to provide enteral nutrition for those who are unable to pass nutrition orally. despite being less invasive, the procedure is not without its complications, one of which includes the formation of a gastrocolocutaneous fistula. the case describes a year old female who presented with a peg placed months prior with reports of leakage of tube feeds from the gastrostomy site. as there was concern for possible ileus or obstruction, an upper gi series was completed which seemed to indicate dislodgement of the g-tube. the g-tube was replaced and a follow-up gastrograffin study was repeated which now indicated that the g-tube was within the lumen of the colon. soon thereafter fecal matter was noted to be draining around the g-tube site; however, patient was without clinical signs of peritonitis. the patient was managed non-surgically as she was a poor surgical candidate with multiple prohibitive co-morbidities. the g-tube was removed bedside by cutting it flush at the skin level with the anticipation that the remainder of the tube would be excreted with bowel movements. the decision was then made to attempt closure of the gastric fistula endoscopically which was accomplished with hemoclips. a follow up upper gi study hours later showed no extravasation of contrast through the gastric fistula. the colocutaneous fistula had self-resolved over the next couple days as well. placement of the peg tube through the transverse colon can present with varying ill effects including diarrhea, pneumoperitoneum, peritonitis, gram negative pulmonary infection or feculent vomiting with the formation of a gastrocutaneous fistula. treatment historically for a gastrocolocutaneous fistula has been exploration and excision of the fistula tract with resection of the involved colonic segment. however, there currently is no gold standard for the management of, and really ranges from conservative management to surgical and is dependent on the presenting symptoms. if the peg becomes dislodged with resultant spillage from the colon with resultant peritonitis, surgical exploration is needed with removal of the g-tube and repair of the stomach and colon. on the other hand, non-surgical management has been suggested in management of a well-established fistula. fistula closure may be spontaneous; however, can be inhibited due to delayed gastric emptying or leakage of gastric secretions through the fistula. endoscopic clipping of the fistula tract employing the hemoclips is a treatment option. median arcuate ligament syndrome (mals) is a rare etiology of abdominal pain caused by narrowing of the celiac artery at its origin by the median arcuate ligament with relative hypoperfusion downstream. patients suffer from post-prandial abdominal pain, abdominal pain associated with exercise, nausea, and unintentional weight loss. diagnosis is historically made by demonstrating elevated celiac artery velocities and respiratory variation on dynamic vascular studies. standard of care for mals patients is laparoscopic celiac artery dissection with release of the median arcuate ligament. at our institution, we have encountered fourteen patients (eleven female, three male) diagnosed by elevated peak velocity in the celiac artery by duplex ultrasound in conjunction with ct angiogram, mr angiogram, arteriogram, or multiple modalities. all but one patient had multiple diagnostic imaging modalities, with the most common being ct angiogram; eight patients had invasive imaging. the mean age at presentation was . years in men and . years in women. on average, male patients presented with a longer duration of symptoms, . years (range - years), as compared to women, . years (range - years). symptoms were fairly consistent between genders and included nausea, emesis, abnormal bowel habits, early satiety, post-prandial pain, and weight loss. all male patients reported at least two symptoms, most commonly nausea and post-prandial pain. in female patients, % reported having three or more symptoms. notably, post-prandial pain was universal among men and women, while weight loss was exclusive to female patients as reported by %. pre-operative peak velocities were recorded in all but one patient, with mean values more elevated in female patients as opposed to male patients, cm/s versus cm/s. post-operative duplexes were obtained in seven patients; pooled data show a mean change of negative cm/s for an average of cm/s after decompression. in all cases, the celiac artery trifurcation was visualized and noted to have a distinct change in artery caliber after division of the ligament. in total, % of patients reported significant improvement with return to normal diet and healthy weight gain post-operatively. of the three without complete resolution, two were diagnosed with motility disorders and one was lost to follow-up. our experience demonstrates that laparoscopic release of the median arcuate ligament in patients with significant flow limitation of the celiac artery on dynamic and anatomic imaging can be a successful treatment option for patients with recalcitrant pain and gastrointestinal dysfunction with no alternative diagnosis. matthew a goldstein, ma, kirill zakharov, do, sharique nazir, md; nyu langone brooklyn adhesions are fibrotic bands that form between and among abdominal organs. the most common cause of abdominal adhesions is previous surgery in the area as well as radiation, infection and frequently occurring with unknown etiology. these bands occur among abdominal organs, commonly the small bowel, and can lead to obstruction or remain asymptomatic, akin to the patient discussed here. congenital abdominal adhesions are rare and have received little attention in research and field of study. the patient described in this case is a -year-old female with a past medical history of morbid obesity, bmi of , hypertension and no past abdominal surgical procedures. the patient presented in august for bariatric surgical consultation and was ultimately taken for an attempted laparoscopic sleeve gastrectomy. upon entering the abdomen, significant adhesions were encountered and an additional attending was called to assist in identifying the stomach. the splenic flexure was found to be plastered to the diaphragm and the descending and transverse colon were adhered to the anterior surface of the stomach. additionally, small bowel adhesions encased the area between the right and left hepatic lobes as well as the caudate lobe. after extensive enterolysis, the pylorus remained the only identifiable portion of the stomach. the patient also demonstrated significant hepatomegaly and a wedge resection was performed. the amount of adhesion and matting of the small and large bowel obscured the view of the stomach and the procedure was deemed too dangerous and terminated. this case represents the uncommon scenario in which an abdomen with no prior surgical history presents with extensive, obscuring adhesions. one such recent study describes the influence of cytokines and proinflammatory states as contributors to obstruction and malrotation in children, but this patient demonstrated no significant history. further investigation is needed to determine potential etiologies of symptomatic and non-symptomatic congenital adhesions among bariatric patients who fail conservative treatment. today the patient is doing well and the surgical team will attempt to complete the procedure in the coming months. laparoscopic spenulectomy: an interesting case report riva das, md , daniel a ringold, md , thai q vu, md ; orlando health, abington jefferson health introduction: spenules, or accessory spleens, are a rare disease entity. most often, they are asymptomatic, and found incidentally during radiographic workup for an unrelated problem. torsion can cause a splenule to not only become symptomatic, but also confound the results of usual diagnostic studies. case description: a -year-old female patient with history of uncomplicated hypertension, hyperlipidemia, hysterectomy, cholecystectomy, spinal surgery, and partial left nephrectomy, presented to the hospital with a two-week history of intermittent left upper quadrant abdominal pain. she denied any similar episodes in the past, or any associated symptoms. further investigation with a ct scan of the abdomen and pelvis showed an acute inflammatory process in the left upper quadrant in same location as some colonic diverticulosis, as well as a . cm soft tissue mass. this indeterminate soft tissue mass was described as having decreased attenuation compared with the spleen. differential diagnosis for this mass included malignancy, an atypical splenule, or an infectious/inflammatory mass. an mri was recommended for further evaluation, but did not reveal any additional significant findings. nuclear medicine liver/spleen scintigraphy was performed, which showed no focal activity associated with the indeterminate left upper quadrant mass, therefore making it unlikely to reflect a splenule, and making malignancy the diagnosis of exclusion. following a period of observation with analgesia, intravenous antibiotics, and bowel rest, her abdominal pain did not resolve, and the decision was made to proceed with operative exploration. diagnostic laparoscopy revealed an approximately cm spherical mass in the left upper quadrant located just below the inferior aspect of the spleen. the superior aspect of the mass gave rise to a vascular pedicle, which upon tracing, seemed to originate from the splenic hilum. this pedicle was easily ligated, and the mass removed. pathology revealed an extensive infarcted hemorrhagic nodule with organizing thrombus and attached thrombosed artery, consistent with an infarcted splenule due to torsion along its own axis. the patient had an uncomplicated postoperative course. discussion: this case report demonstrates the unusual presentation and workup of a patient that was ultimately diagnosed with an infarcted splenule, despite imaging findings that did not correlate, and may even have confused her diagnosis. scintigraphy, which is normally the gold standard for diagnosing and localizing accessory splenic tissue, was in this case unrevealing, due to inability of the tracer to traverse the torsed vascular pedicle. operative exploration was both diagnostic and therapeutic. patients which was treated with antibiotics suggested by culture and sensitivity report and local wound care. one patient died due to sepsis at presentation. conclusion: chikungunya virus was found circulating in rodents in pakistan as early as . duodenal ulcer perforation which is a common surgical emergency in our part of the world usually presents with pinpoint perforation in ant wall of first part of duodenum unlike in already diagnosed cases of chikungunya disease where a slit like duodenal perforation is noted in the anterior wall of first part of duodenum. literature and consensus relate this perforation with the excessive use of nsaids due to usual presentation of arthritis in chikungunya disease but the unusual presentation is still to be answered. introduction: bouveret's syndrome is a rare form of gallstone ileus in which an impaction of a gallstone in the duodenum results in a gastric outlet obstruction. gallstone ileus accounts for approximately - % of all cases of small bowel obstruction. the terminal ileum is the most common location for a calculus to cause obstruction followed by the proximal ileum, jejunum and duodenum/stomach respectively. open and laparoscopic surgery has previously been the mainstay of treatment for bouveret's syndrome, however with the advent of new endoscopic techniques and instruments there has been increasing success in endoscopic management. this case report looks at a patient with a gastric outlet obstruction from a gallstone, and discusses the current literature regarding diagnosis and management. case: year old male presented with several day history of epigastric abdominal pain and multiple episodes of nonbloody, nonbilious emesis. he had previously been diagnosed with cholelithiasis, however had refused surgery at that time. on admission the patient was found to have a leukocytosis of . . an ultrasound was performed in which the images were limited due to pneumobilia. a subsequent ct scan revealed pneumobilia, and a large cm gallstone impacted in the first portion of the duodenum causing a gastric outlet obstruction. the patient underwent failed endoscopic attempts at removal and ultimately required a laparotomy, enerotomy with stone extraction. discussion: bouveret's syndrome is a rare variant of gallstone ileus. with newer endoscopic techniques and electrohydraulic lithotripsy, there has been increasing success with endoscopic retrieval of the impacted gallstones. there is some controversy in regards to the need for definitive operative management. stone extraction, without cholecystectomy and fistula repair, has been shown to have less postoperative complications as well as lower mortality rates compared to when a cholecystectomy and fistula repair has been performed. total mesorectal excision (tme) with neoadjuvant chemoradiotherapy (nacrt) is standard treatment for rectal cancer, which has resulted in a decrease in local recurrence. however, nacrt has shown no significant overall survival and some adverse effects mainly caused by radiation therapy. recently, the usefulness of neoadjuvant chemotherapy (nac) has been reported. we retrospectively assessed the efficacy and safety of the neoadjuvant mfolfoxiri compared with nacrt followed by laparoscopic surgery. a total of patients undergoing laparoscopic surgery for lower rectal cancer (clinical stage: ii or iii) from july to february in our department were retrospectively evaluated. patients underwent nac, and patients underwent nacrt. the following data were collected: pathological complete response (pcr), histological grade, down staging, radial margin (rm) and postoperative complications. histological grade was defined as follows: tumor cell necrosis or degeneration is present in less than one third of the tumor area (grade a), between one and two thirds (grade b), more than two thirds but viable cells remain (grade ), and complete response (grade ). these two groups were demographically comparable. down staging did not differ between the two groups. histological grade (?grade b) and pcr were significantly higher in the nacrt than in the nac group (p. ). rm had no significant difference in both groups, but tended to be able to secure negative rm in the nac group ( % vs. . %, p= . aims: increasing evidence suggest that cme may improve overall and disease free survival in colon cancer. our aims were to investigate the safety and efficacy of single incision laparoscopic cme colectomy (silcc) compared to multiport cme laparoscopic colectomy (mpclc) providing the first meta-analytical evidence. methods: pubmed, scopus and cochrane library were searched. studies comparing the silcc to mpclc in adults with colon adenocarcinoma were included. the studies were critically appraised using the newcastle ottawa scale. statistical heterogeneity was assessed with x and i . the symmetry of funnel plots was examined for publication bias. results: one randomized and four case control trials were included ( silcc vs sl introduction: obesity has been associated with increased morbidity following total proctocolectomies with ilealpouch anal anastomosis (tpc-ipaa). however, the incremental added risk of increasing obesity class is not known. the aim of this study was to evaluate the additional morbidity of increasing obesity class for tpc-ipaa. methods: after ethics board approval, the acs-nsqip database ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) was accessed to identify patients who underwent elective tpc-ipaa. body mass index (bmi, kg/m ) was classified as normal ( . - . ) , overweight ( . - . ), obesity class-i ( - . ), obesity class-ii ( - . ) and obesity class-iii (≥ ). primary outcomes were overall surgical site infection (ssi) and organ-space infection (osi). secondary outcomes were -day major morbidity and length of hospital stay (los aim: in curatively intended resection of sigmoid and rectal cancer, many surgeons prefer to perform ligation of the root of the inferior mesenteric artery (ima), high tie, because of oncological reasons. however, ligation of the ima has been known to decrease blood flow to the anastomosis. there are few reports of patients undergoing the reduced port laparoscopic approach (rps) including single-incision laparoscopic approach (sils) even among those undergoing laparoscopic lymph node dissection around the ima with preservation of the left colic artery (lca). our objective was to evaluate the quality of this procedure regarding application of rps for the treatment of sigmoid and rectal cancer. methods: the feasibility of this procedure was evaluated in consecutive cases of rps for sigmoid and rectal cancer. a lap protector (lp) was inserted through a . cm transumbilical incision, and an ez-access was mounted to lp and three -mm ports were placed. almost all procedures were performed with standard laparoscopic instruments using a flexible scope (sils). a mm port was inserted in right lower quadrant mainly in rectal cancer surgery (sils + ). our method involves peeling off the vascular sheath from the ima and dissection of the ln around the ima together with the sheath. results: lymph nodes around the ima were dissected with preservation of the lca in cases (group a). the ima was ligated at its root in cases (high tie, group b). in group a, patients were treated with sils and patients were treated with sils+ . in group b, patients were treated with sils and patients were treated with sils+ . median operative time was . , and . min for group a, and b, respectively. the operative time was significantly longer in group a. estimated blood loss was . and . g, and mean numbers of harvested ln were . , and . . none of the other operative results of groups a and b were different statistically. in this series, there was only one anastomotic leakage in group b. conclusion: our method allows equivalent laparoscopic lymph node dissection to the high tie technique. the operative time tends to be longer, however this procedure has a possibility to reduce an anastomotic leakage. introduction: the routine mobilization of the left colonic flexure in colorectal surgery is still a matter of debate. we present our surgical approach with data. this technique may increases the surgical expertise/confidence when the surgical maneuver is necessary. up to % of all splenectomies are for surgery-related injuries; % of those splenic injuries are treated by splenectomy. the iatrogenic splenic injury rate during colorectal surgery is . %. iatrogenic splenic injuries create: increased risk of mortality/morbidity, extended operative time/patient in-hospital stay and increased healthcare costs. risk factors for iatrogenic splenic injury are: advanced age, adhesions, underlying pathology. obesity is not a risk factor. it is debated if the left colonic flexure mobilization is a risk factor for splenic injury. the ligament over-traction is the most frequent damage mechanism. the most dangerous surgical manuever is the spleno-colic ligament surgical dissection. moreover, laparoscopy descreases by almost , times the splenic injury risk. some surgeons are reluctant to routinely take down the splenic flexure. materials and procedures: robotic left colonic/rectal cases with routine splenic flexure mobilization technique have been performed: left colectomy (n= ), rectal surgery (n= ), transverse-colectomy (n= ) and pancolectomy (n = ). conversion rate , %, ebl\ ml, postop-leak ( . %) and % iatrogenic splenic injuries. results: in our approach, there are pathways that need to be mastered for the splenic flexure mobilization:a) medial to lateral dissection (underneath the inferior mesenteric vein); b) lateral to medial (from the lateral peritoneal reflection); c) access to the lesser sac with omental detachment from the transverse colon; d) access to the lesser sac with the gastrocolic opening, following the inferior border of the pancreas. the dissection should be closer to the colon rather than to the spleen. in our experience the routine mobilization of the splenic flexure may have some advantages: a) better (without tension) distal anastomosis formation; b) better perfusion of the proxiaml stump; c) wider oncological dissection; d) no need of going back to the flexure when the proximal stump is too short; e) mastering a surgical manuver useful in other procedures (e.g. distal pancreasectomy). the theoretical drawbacks of routine splenic flexure mobilization can be:a) longer operative time, which is on average increased by minutes; b) risk of splenic injuries, in our experience, no splenic injuries have been registered. conclusions: technical accuracy with cautious dissection/visualization can reduce iatrogenic splenic damages rate. laparoscopy decreases splenic injury rate. robotic surgery may have the potential to further reduce this complications. our data suggest that the routine mobilization of the splenic flexure, has more advantages than drawbacks and it can reduce the iatrogenic splenic injury rate. more trials are needed in order confirm our findings. introduction: the robotic stapler with the endowrist™ technology (intuitive surgical, inc.) includes a larger range of motion and articulation compared to the laparoscopic device, and may provide some benefits in difficult areas like the pelvis. to date, few studies have been published on the application of robotic endowristed stapling. we present our preliminary experience using the robotic stapler in low anterior rectal resection (larr) with total mesorectal excision (tme) for rectal cancer. methods and procedures: between march and september , patients underwent elective robotic larr with tme and primary colorectal anastomosis within the eras program. patient demographic, intra-operative data and post-operative outcomes were compared between the endowrist™ robotic stapler group (rs group) and the laparoscopic stapler group (ls group). results: the two groups were homogeneous in terms of demographic and clinical characteristics. thirteen ( males) and patients ( males) were included in rs and in ls group, respectively. seven patients received preoperative chemoradiation in rs group, in ls group. there was no difference in intra-operative blood loss and total operative time. the median number of stapler fires for patients in rs group and in ls group was (range, - ) and (range, - ), respectively. loop-ileostomy was fashioned in patients in rs group ( . %) and patients in ls group ( . %). the days mortality was nil. two cases of anastomotic leaks have been detected in rs group ( . %), cases ( . %), occurred in ls group, all treated conservatively. the mean length of postoperative stay was . ± . days in rs group, . ± . days in ls group. conclusions: in our preliminary experience the application of robotic stapler during larr with tme has shown to be safe and feasible with acceptable morbidity. even if our case series is pretty small, fewer stapler fires were required in the rsg compared to lsg. we believe that the robotic stapler might lead to a more precise firing during pelvic surgery: it can explain the trend toward a decreased number of fires, that has been well documented in literature to be related to a lower risk of anastomotic leak. further high quality studies are required to confirm these findings. background and objectives: the present study was aimed at investigating the safety and feasibility of laparoscopic ultra-low anterior resection (l-ular) with total mesorectal excision (tme) and transanal specimen extraction for rectal cancer located at lower one-third rectum, and specifically understanding the oncological outcome of the operation. patients and method: a prospective designed database of a consecutive series of patients undergoing laparoscopic ultra-low anterior resection for rectal malignancy with various tumornode-metastasis (tnm) classifications from to at the texas endosurgery institute was analyzed. in this study ultra-low anterior resection is defined as low anterior resection for the malignant lesion at distal / of rectum. results: ultralow anterior resections were completed laparoscopically with tme and transanal specimen extraction. the operating time for the surgery was . ± . minutes, and estimated blood loss during the procedure was . ± . ml. the length of the lesion from the anal verge measured with intraoperative colonoscopy ranged from . cm to . cm, and shortest distance of colorectal anastomosis from the anal verge is cm. since diverting ileostomy was routinely installed after l-ular, none was found to have anastomotic leakage, however patients developed anal stenosis within -month follow-up. therefore the overall rate of postoperative complication is . %. moreover patients were reported to have local recurrence in -year followup with the rate of . %. conclusions: l-ular is safe and effective procedure for the rectal cancer at distal / rectum with comparable local recurrence and postoperative complication rates, thereby suggesting l-ular can be considered as a procedure of choice for rectal cancer at very low location in the rectum. for rectal cancer, however, local full-thickness excisions are fraught with high local recurrence rates -even if limited to early and best selected lesions. this corroborated observation is likely caused by a combination of missed nodal disease and direct implantation of tumor cells into the mesorectum, which upstages even early t lesions to at least a t lesion. the treatment of choice for invasive adenocarcinoma consists of an oncological total mesorectal resection, possibly with other modalities. rectal tumors of uncertain behavior can present a treatment dilemma between over-treatment vs under-treatment. concept: if the nature of a lesion is not certain or if contradictory results have been obtained, we propose a superficial local excision as a mucosal excisional biopsy to establish the diagnosis while avoiding interference with subsequent definitive treatment modalities by preserving the integrity of the external rectal wall and mesorectum. a benign final pathology concludes the treatment, whereas a detection of invasive cancer will be managed with a subsequent oncological resection. methods: this is a case report of a -year-old woman found to have a . cm villous lesion in the mid to distal rectum without proven or disproven invasive cancer. a tems-guided mucosal resection of the rectal mass at cm above the anal verge was performed whereby the lesion was dissected off the underlying muscularis. results: with preoperative discrepant erus and mri staging ut - vs ct lesion, a technically successful mucosal resection of the large rectal mass was carried out. pathology revealed a tubulovillous adenoma without high grade dysplasia or malignancy and a complete resection. conclusion: tems mucosal excisional biopsy of rectal tumors of uncertain behavior allows for a less invasive diagnostic approach that may (a) be definitive treatment if the lesion is proven benign, or (b) confirm the need for more aggressive treatment without having burned any treatment bridges or upstaged an early tumor by violating the mesorectal plane. an oncologic resection with appropriate (neo-)adjuvant chemotherapy can be carried out while preventing the potential for tumor seeding at initial operation. background: adequate visualization of the entire lumen of the large bowel is essential in detecting pathology and establishing diagnoses during colonoscopies. patients are provided dietary instructions and medications in order to achieve adequate bowel preparation. given the extensive amount of preparation required, some patients may be unable to adhere to the prescribed routine, resulting in rescheduling or repeat procedures and misallocation of limited resources. a number of previous quality-improvement efforts have been implemented to ensure adequate preparation prior to colonoscopy. objective: the objective of this study was to develop and assess the feasibility of a novel smart phone application in the delivery of bowel preparation instructions. methods: a novel smart phone application was developed to deliver bowel preparation instructions to patients undergoing colonoscopy for the first time. patients were included in the pilot phase of this project if they were undergoing a colonoscopy for the first time. we included patients who had access to a smart phone, had not previously had a bowel preparation for any reason. we excluded patients with a previous diagnosis of inflammatory bowel disease or colorectal cancer. patient surveys were administered at the time of colonoscopy. patients were questioned regarding the completeness of bowel preparation and adherence to bowel preparation instructions. patient questionnaires were completed to ascertain the ease of use of the smart phone application and any concerns that arose. quality of bowel preparation was assessed by the colonoscopist using the validated ottawa bowel preparation score. this is the pilot study results for the "coloprep" trial (nct ). results: a total of patients were enrolled in the pilot phase of this study. patient satisfaction, adherence to instructions and ease of use of the smart phone application were ascertained. bowel preparation, as assessed by the colonoscopist, was reported. conclusions: this study assessed the feasibility of using a novel smart phone application for delivery of bowel preparation instruction. this pilot study is the initial phase of a randomized controlled trial to compare smart phone application vs. written instructions in the delivery of bowel preparation instructions. the . median follow-up was months. there were no statistically significant differences found in clinical features and laboratory findings between the two groups. no statistically significant difference was found regarding the overall success rates and the complication rates between the conservative and the surgical arms (success rates: . % and . % (p= . ) and complication rates: . % and . % (p= . ), respectively). however, surgical treatment was better than conservative treatment in preventing recurrent diverticulitis (recurrence rates: % and . % (p= . ), respectively). conclusion: conservative management with bowel rest and antibiotics is a safe and effective treatment for right-sided colonic uncomplicated diverticulitis and may be considered as the initial option. on the other hand, laparoscopic diverticulectomy is also safe, effective and adequate. surgery is advocated to decrease the recurrence rate. introduction: it has been hypothesized that the structural and functional changes that develop in the defunctioned segment of bowel may contribute to the development of postoperative ileus (poi) after loop ileostomy closure (lic). as such, longer intersurgery interval between ileostomy creation and lic may increase poi. methods and procedures: after institutional review board approval, all patients who underwent lic at a single institution between - were identified. the primary endpoint, primary poi, was defined as either a) being kept nil-per-os on or after postoperative day for symptoms of nausea/vomiting, distension, and/or obstipation or b) having a nasogastric tube (ngt) inserted, without postoperative obstruction or sepsis. secondary endpoints included length of hospital stay (los) and non-poi related morbidity. patients who left the operating room with a ngt, had a planned laparotomy with a concomitant procedure at the time of lic, had a total proctocolectomy as their index operation, or had secondary poi, were excluded. patients were then divided into two groups based on timing from the index operation to lic (\ months vs. objective: fecal incontinence can be a debilitating problem significantly diminishing productivity and quality of life. sacral neuromodulation has emerged as a first line surgical option treatment in patients with fecal incontinence. though its efficacy has been rigorously evaluated in adult populations there is scant data available for its use in the pediatric pateints with fecal incontinence. this case study discusses the management of fecal incontinence in a pediatric patient with a history of hirschsprung's disease utilizing sacral nerve stimulation. methods: our patient is a -year-old female with a history of hirshsprung's diagnosed in infancy and treated surgically with coloanal pull through at the age of who presented with complaints of fecal incontinence. the patient was wearing pads daily, noting frequent uncontrolled bowel movements as well as having frequent missed days of school due to these symptoms. despite maximal medical management and pelvic floor physical therapy the patient continued to have - episodes of fecal incontinence daily. a ct scan with rectal contrast was used to establish her postoperative anatomy. anal manometry showed low rest/squeeze pressures, absent resting anal inhibitory reflex, and abnormal sensation. furthermore, during balloon expulsion testing the patient failed to pass device. the patient was deemed a candidate for stage testing with sacral nerve neuromodulation. during follow-up, the patient was noted to have resolution of her episodes of fecal incontinence and the second stage was completed. the patient continues to note % continence and dramatic improvement in her quality of life. conclusion: in this patient with a history of severe fecal incontinence due to hirschsprung's disease, sacral neuromodulation has had a significant impact on her quality of life. post-operatively she continues to have marked improvement in her symptoms with - bowel movements a day with no recurrence of fecal incontinence. the use of sacral neuromodulation is a promising treatment for fecal incontinence in the pediatric population. future research investigating the longterm efficacy of this treatment modality in the pediatric population is needed. cases of bowel obstruction caused by colorectal cancer recurrence and progression were excluded. surgical cases ( . %) were considered to be early bowel obstruction and ( . %) were classified as late bowel obstruction. left hemicolectomy (n= , . %) was a significantly more frequent procedure in early bowel obstruction, and abdominoperineal resection (n= , . %) was significantly more common in late bowel obstruction (p. ). both early and late bowel obstruction included adhesive small bowel obstruction (n= ), internal hernia (n= ), and strangulation obstruction (n= ). internal hernia (n= ) and strangulation obstruction (n= ) occurred after left hemicolectomy and abdominoperineal resection, respectively. there is no apparent relationship between surgical procedures and adhesion regions (abdominal wall, intestinal tract, and pelvic cavity). the incidence rate of postoperative small bowel obstruction remained low, and laparoscopic colectomy had been safely performed. however, countermeasures are needed because of the high frequency of both early and late bowel obstruction which occurred after left hemicolectomy and abdominoperineal resection, respectively. improved utilization of resources as an improvement introduction: nowadays, treatment decisions about patients with rectal cancer are increasingly made within the context of a multi-disciplinary team (mdt) meeting. the outcomes of rectal cancer patients before and after the era of multi-disciplinary team was analyzed and compared in this paper. the purpose of the present study is to evaluate the value of discussing rectal cancer patients in a multi-disciplinary team. methods and procedures: in our health institute, weekly mdt conferences were initiated in january . meetings were attended by surgeons, radiologists, radiation and medical oncologists and key nursing personnel. all rectal cancer patients diagnosed and treated in - in the general surgery division of the "carlo urbani" hospital in jesi (an, italy) were included. then, the data from rectal cancer patients in were evaluated, before the adoption of mdt and in year , after the adoption of meetings. datasets regarding demographics, tumor stage, treatment, and outcomes based on pathology after operation were obtained. during an mdt discussion patient history, clinical and psychological condition, co-morbidity, modes of work-up, clinical staging, and optimal treatment strategies were discussed. a database was created to include each patient's workup, treatments to date and recommendations by each specialty. ''demographic variables'' consisted of age at diagnosis, sex, body mass index, comorbidities, american society of anesthesiologists physical status classification system, clinical stage and pathological stage. other analyzed variables included baseline carcinoembryonic antigen (cea), the type of imaging, use of neoadjuvant chemo-radiation, restaging following neoadjuvant therapy, distance from the anal verge, operation type and use of adjuvant chemo-radiation. ''outcome variables'' consisted in a comparison for each group between clinical and pathological stage. results: sixty-five patients were included in this study: thirty patients in (pre-mdt) and thirty-five patients in . demographic variables did not differ significantly between groups. preoperative clinical stages with baseline preoperative cea and postoperative pathological stage were analysed, too. thanks to the mdt and the increased use of the neoadjuvant therapy, a statistically significant difference in reduction of the stage between the clinical and pathological stage in the patients of the mdt group was verified. conclusions: the vast majority of rectal mdt decisions were implemented and when decisions changed, it mostly related to patient factors that had not been taken into account prior to the adoption of multi-disciplinary team. analysis of the implementation of team decisions is an informative process in order to monitor the quality of mdt decision-making. purpose: in japan, lateral pelvic node dissection (lpnd) is the standard treatment for locally advanced lower rectal cancer. there are few reports of patients undergoing single-incision plus one port laparoscopic (sils+ ) lpnd even among those undergoing laparoscopic lpnd. the aim of this study is to describe our initial experience and assess the feasibility and safety of sils+ lpnd for patients with advanced lower rectal cancer. methods: a lap protector (lp) was inserted through a . cm transumbilical incision, and an ezaccess was mounted to lp and three -mm ports were placed. a mm port was inserted in right lower quadrant. a single institutional experience of sils+ lplnd for rectal cancer are presented. inclusion criteria was indications for lld were lower rectal cancer with t - , or t - rectal cancer with metastasis of lateral lymph node, as described by the japanese society for cancer of the colon and rectum (jsccr) guidelines for the treatment of colorectal cancer. perioperative outcomes including operative time, operative blood loss, length of stay, postoperative complications, and histopathological data were collected prospectively. introduction: endoscopic stenting with a self-expandable metallic stent (sems) is widely accepted procedure for malignant colorectal obstruction. we assessed the safety and efficacy of insertion of a sems followed by elective surgery as 'bridge to surgery (bts)' in our institute. methods: this study was a retrospective study in our institute. the data was collected from medical charts from january to june . results: a total of consecutive patients underwent radical surgery for colorectal malignancy during this period. in this series, patients ( . %) were diagnosed malignant colorectal obstruction and intended to a bts. the stent was successfully placed in patients and all the patients were planned to undergo radical surgery. the failed patients underwent stoma creation ( patients) and hartmann's procedure. the technical success rate was % and the clinical success rate was %. the median time from sems to surgery was days ( - days) . open and laparoscopic surgery was performed in and patients, respectively, except for one patient refused radical surgery because of a great age. the tumor could be resected in patients (bts patients) with primary anastomosis. however, diverting stoma creation was needed in patients and decompression rectal tube was placed in patient. the entire patient laparoscopically was no conversion to open surgery. there was no anastomotic leakage in bts patients. the median duration of postoperative hospital stay was days ( - days). the overall postoperative complication was % ( / ) including bowel obstruction and anastomotic stricture. the median follow-up period was days. during the follow-up period, patients were relapsed peritoneal dissemination, ovarian metastasis, and liver and pulmonary metastases, respectively. former patients were diagnosed stage iva at the time of primary surgery. one patient died from sudden death. conclusions: our data suggested that routine use of sems insertion was safe and effective procedure for malignant colorectal obstruction as a bts. moreover, laparoscopic procedure was useful procedure in bts patient. the short-and long-term surgical outcomes were also acceptable. introduction: serpin e , also known as plasminogen activator inhibitor- (pai- ) is an inhibitor of urokinase type plasminogen activator (upa) and tissue-type plasminogen activators (tpa ). pai- plays a role in the regulation of angiogenesis, wound healing, and tumor cell invasion; over expression has been noted in breast, esophageal, and colorectal cancer (crc). pai- is also a potent regulator of endothelial cell (ec) proliferation and migration in vitro and of angiogenesis and tumor growth in vivo. the plasminogen/plasmin system plays a key role in cancer progression by mediating extracellular matrix degradation and tumor cell migration. surgery's impact on plasma pai- levels is unknown. this study's purpose was to measure plasma pai- levels before and during the first month after minimally invasive colorectal resection (micr) for crc. objectives: retroflexion in the rectum at the end of a colonoscopy is a requirement for a complete endoscopic evaluation. retroflexion helps to visualize and detect polyps which would be missed otherwise. currently new endoscopes are available which can do retroflexion in the caecum. aim: our study aims to compare the rate of polyp detection rate in cecum and ascending colon with and without retroflexion in cecum. methods: this is a single center, single operator, retrospective study. a total of two hundred patients were involved. a single center irb waiver was obtained. patients were divided into two groups based on the presence/absence of retroflexion in caecum during their colonoscopy. the data was obtained from records. group a (n= ) had colonoscopy without retroflexion in caecum group b (n= ) had colonoscopy with retroflexion in caecum inclusion criteria: patients undergoing screening colonoscopy between the age of and . results: group a: total of patients were screened. a total of polyps were detected in group a. number of cecal polyps were ( . % of total polyp count). number of ascending colon polyp were ( % of total polyp). on analyzing the pathology % of the cecal polyps were tubular adenoma, % hyperplastic polyps % and % lymphoid aggregate. number of ascending colon polyps were , of which % were tubular adenoma, % tubular adenoma and % tubulovillous adenoma group b: total of patients were screened. a total of polyps were detected. number of cecal polyps detected were ( . % of total polyp count). number of ascending of ascending colon polyps were ( %). on analyzing pathology, % cecal polyps were tubular adenoma and % were sessile serrated. out of the ascending colon polyps % were tubular adenoma, % sessile serrated, % tubulovillous and % hyperplastic polyp. side events: two mass lesions were noted in both group a and b. there was incomplete colonoscopy in group a and b. conclusion: this retrospective analysis reveals a small increase in polyp detection in the cecum with retroflexion, especially in detecting sessile polyps which have more malignant potential. however, a large multicenter analysis will be required to validate the above observation. background: while uncommon, rectal prolapse is a disabling condition affecting older females. in a small subset of patients, concomitant organ prolapses with or without incarceration can lead to significant morbidity. as the field of laparoscopy has evolved, minimally invasive surgical options for rectal prolapse have led to improved quality and reduced morbidity for patients suffering this debilitating disease. methods: the - acs-nsqip databases was queried for patients undergoing a traditional or minimally invasive rectopexy based on cpt codes ( , , , and ) . emergent cases and patients with preoperative infections or inflammatory states were excluded. the primary outcome of interest was a -day postoperative composite morbidity score. statistical analysis incorporated multivariate analysis and binomial logistic regression with p. holding significance. results: these inclusion and exclusion criteria identified patients undergoing traditional ( ) and minimally invasive ( ) rectopexy for prolapse between and . patients undergoing traditional rectopexy were older (p. ), had a higher body mass index (p= . ), more comorbid conditions (diabetes, copd, hypertension) and less functional independence (p= . ). patients undergoing a traditional rectopexy had a higher composite morbidity incidence of . % vs. % for minimally invasive rectopexy (p. ). specifically, minimally invasive rectopexy patients had a . % reduction in wound complications (p= . ) and a shorter hospital stay ( . days vs. . days, p . ) compared to a traditional rectopexy. readmission rates were also . % lower in the minimally invasive group (p= . ). after controlling for the differences in the cohorts, a minimally invasive approach was a significant protective factor against the incidence of -day postoperative morbidity (or . , p. ). conclusion: a minimally invasive rectopexy has improved -day postoperative morbidity compared to a traditional rectopexy and should be strongly considered for the treatment of rectal prolapse. objectives: the short-term safety and efficacy of a self-expandable metallic stent (sems) placement followed by elective surgery, "bridge to surgery (bts)", for malignant large-bowel obstruction (mlbo) have been well described. the aim of this study was to investigate the risk factors for postoperative complications and optimal interval between sems placement and surgery in patients with mlbo. methods: retrospective examination of patient records revealed that the bts strategy was attempted in patients with mlbo from january to march in our institution. two of these patients were excluded because they had undergone emergency surgery for sems migration; thus, patients with mlbo who had undergone sems placement followed by elective surgery were included. of these patients, eight had developed postoperative complications (clavien-dindo grading≥ii) (postoperative complication: poc group) whereas patients had no such complications (no poc group). results: univariate analyses showed that the factors of asa score, number of lymph nodes resected, interval between sems and surgery, and preoperative albumin concentration were associated with postoperative complications. multivariate analysis identified only the interval between sems and surgery as an independent risk factor. furthermore, a cut-off value of days for interval between sems and surgery was identified by roc curve analysis. conclusions: an interval of ≥ days from sems placement to surgery is an independent predictive factor for postoperative complications in patients undergoing elective surgery in a bts setting. thus, an interval of over days is recommended for minimizing postoperative complications. haseeb kothar, ronan cahill; mater misericordiae university hospital current clinical advances in operative near-infrared visualisation of cells, tissues and structures are predicated on the use of commercial available near-infrared cameras to excite and visualise emission energy from non-selective, approved compounds (predominantly indocyanine green (icg)). it is expected that new generation compounds wholly selective for specific cellular components are now needed for further advance and a variety of molecular targets have been proposed and are being developed primarily for oncological imaging purposes. recent publications have however suggested icg itself is retained within malignant tissue differently to its uptake and clearance from surrounding non-malignant tissue which is important for two reasons. firstly, it exploits and makes visual the increased vascular permeability and disordered clearance associated with carcinogenesis which is a common endpoint of a variety of mediators including but not limited to vegf. this raises the useful option of targeting downstream effects of cancer compounds on a metabolic basis as opposed to tagging individual cell or antigen components. this means that a single agent could be used to target a variety of cancers rather then needing a specific one for each specific sub-type as well as obviating the issue of cancer cells heterogeneity even in a single cancer deposit. second, it is very likely that some or all of the "localisation" effect of proposed selective compounds may well be due to a similar phenomenum rather then cell-specific binding and may make distinction from other areas of similar metabolic behaviour (ie inflammatory regions) difficult. the crucial step-advance for such agent development so may well relate to timing of compound delivery and "visualisation window" at the region of interest rather then highly selective oncocellular-targeting. to illustrate this in more detail, we have been examining the tissue-specific effects and actions of near-infrared excitation in patients (n= ) with localised malignant colorectal primaries receiving an aliquot of icg before such examination at the time of resection. icg can be selectively apparent in the colorectal primary minutes after its systemic administration likely due to altered vascular dynamics. additional dose-related work has shown that early administration ( - minutes before examination) does not give useful information related to tumour fluorescence. interestingly none of these patients had fluorescence seen within their regional lymphatics but none also had malignant lymph nodes associated with their large primaries on pathological examination. however, this procedure is not usually performed in laparoscopic apr for its technique difficulty, which may lead to increased rates of complications ( fig. ) . here, we compared the feasibility and peri-operative outcomes of the laparoscopic apr with and without pelvic peritoneum closure (ppc) for lower rectal cancer. introduction: there are reports of increased operative duration, blood loss and postoperative morbidity, caused by difficulties in obtaining good visualization and in controlling bleeding when laparoscopic resection is performed in obese patients with colon cancer. purpose: the aim of this study was to investigate the impact of obesity on perioperative outcomes after laparoscopic colorectal resection performed by various operative methods in our department. patients and methods: we conducted a retrospective analysis of patients with colorectal cancer who underwent laparoscopic surgery between january to december . right colectomy was performed in patients, sigmoidectomy in patients, and low anterior resection in patients. the surgical outcomes were compared between non-obese (body mass index [bmi]\ kg/m ) and obese (bmi ? kg/m ) patients. results: right colectomy cases: the amount of blood loss was significantly increased in the obese group compared with the non-obese group, but operation time did not differ significantly between the groups. there were no significant differences between the two groups in the rate of postoperative complications and duration of post-operative hospitalization. sigmoidectomy cases: there were no significant differences between the two groups in operation time and amount of blood loss. even though the preoperative asa score and the rate of postoperative complications were higher in the obese group, the mean postoperative hospital stay did not differ significantly between the two groups. low anterior resection cases: there were no significant differences between the obese group and the non-obese groups in operation time, amount of blood loss, rate of postoperative complications, and duration of post-operative hospitalization. discussion: although there are some reports of increased operative times in obese patients, the operative procedure was not extended in any of the present study patients. the amount of blood loss was significantly increased in the obese group compared with the non-obese group when right colectomy was performed. among the patients undergoing sigmoidectomy, the postoperative rate of complications was higher in the obese group; however, the preoperative asa status was also higher in the obese group than non-obese group, indicating that factors other than obesity may be involved. conclusion: we concluded that laparoscopic colorectal resection appeared to be safe and feasible in both obese patients and non-obese patients. however, bmi may not accurately reflect the amount of visceral fat present. background: for the complete rectal prolapse (basically longer than cm), we thought sling rectopexy was most reasonable to hang up and fix the rectum, which drooped down and prolapsed due to the relaxation of supporting tissue. we considered ripstein method had enough fixed power of rectum to sacrum. however, complications of rectal stenosis, constipation, mesh infection and mesh penetration were reported. therefore, we modified ripstein method to conquer such complications. aim: a prospective study beyond the randomized control trial (rct) between our modified ( introduction: the results of the japan clinical oncology group (jcog) study suggested that total mesorectal excision (tme) and lateral lymph node dissection (llnd) could become the standard treatment for lower rectal carcinoma. however, llnd must also be performed laparoscopically if surgery for lower rectal carcinoma is to be carried out as a completely laparoscopic procedure. transanal tme (tatme) is expected to provide better results than the conventional tme, both oncologically and in terms of pelvic function, and its use has recently been spreading in japan. we started performing laparoscopic tatme+llnd in our department in july and here report the short-term outcomes. subjects and methods: we used laparoscopic tatme+llnd to treat men and women with ct or deeper rectal carcinoma in whom the inferior margin of the tumor was on the anal side of the peritoneal reflection. this was a retrospective study of short-term postoperative outcomes. surgical procedure: laparoscopic surgery was started simultaneously by two teams, one working transabdominally and the other working transanally. the transabdominal team performed the standard proximal llnd and mobilization of the splenic flexure via five ports. they then dissected the bilateral lateral lymph nodes, mainly in the obturator (# ) and internal iliac (# ) groups. during this time, the transanal team performed laparoscopic tatme. finally, both dissection layers were connected and the cancer was excised. results: six patients had clinical stage ii and two had clinical stage iii lower rectal carcinoma. all the patients underwent preoperative chemotherapy with s- +l-ohp. five underwent a sphincterpreserving surgery, and three underwent rectal amputation. the mean operating time was minutes (range, - minutes), and the mean amount of hemorrhage was g ( - g). the mean number of lymph nodes dissected was , and r resection was performed in all the cases. the mean length of hospital stay was days, and a postoperative complication of clavien-dindo grade iii or higher occurred in one patient (anastomotic failure). conclusions: laparoscopic tatme+llnd performed by two teams simultaneously is an extremely useful procedure that not only reduces operating time, but also is less invasive than laparoscopic surgery. it may also be effective for improving curative nature, nerve preservation, and anal function. objective: in laparoscopic appendectomy, the base of the appendix is usually secured by applying a roeders knot. the aim of this study was to compare the advantages of using staplers and hem-olocks for securing the base of the appendix. method: the study included patients between age of to years with acute appendicitis randomly divided into two groups. in the first group, the base of the appendix was secured using roeders knot. in the second group, mesoappendix was not dissected and was included in the endostapler jaws. the primary outcome was overall morbidity. secondary outcomes were total duration of surgery, total length of stay and ease in difficult cases. result: no morbidity was recorded in any group. the time of the operative procedure was significantly longer in the cases with roeders knot than in the stapler group (p. ) as mesoappendix was not dissected in the later. cases with unhealthy base were progressed to laparoscopic quadricolectomy. apart from the ease of applying a stapler, cases of second group with gangrenous base were easily tackled using endostapler, avoiding the need of a hemicolectomy. conclusion: all forms of closure of the appendix base are acceptable, but endostapler technique apart from providing a secure base, reduces operative time and is an essential tool in cases of gangrenous base. introduction: accurate staging is essential to estimate the prognosis of patients with colorectal cancer (crc) and lymph node evaluation is key to determine it. in non-metastatic crc, the number of harvested lymph nodes is the strongest prognostic factor for outcome and survival. additionally, it is thought that a higher lymph node yield may be representative of a higher quality of surgical care. due to the importance of the association between lymph node evaluation and outcome in crc, it is necessary to evaluate factors which may affect lymph node harvest. introduction: hatmann's procedure is commonly done in treating complicated diverticulitis, negleccted rectal trauma with sepsis and sometimes malignancy. the traditional techniques to restore the intestinal continuity after hartmann's procedure were for many years the standard of care in these operations, but in fact they carry many morbidity and even mortality and failure. laparoscopic techniques is not only carry the advantage of minimal invasive surgery, but also of better visualizationn and magnification. the aim is evaluating the outcome of using the laparoscope in reversal of hartmann's procedure as regard feasibility and safety. patients and method: forty patients were subjected to laparoscopic reversal of hatmann's procedure in tanta university hospital, there ages ranged between to years, the time elapsed after the original operation ranged from months to years, excluding advanced malignany. conversin occurred in cases due to extensive adhesions and bleeding. results: no mortality, or major morbidity in our study and only single leak treated by covering ilestomy. conclusion; laparoscopic hartmann's procedure is feasible, promising tehnique with minimal morbidity. background: minimal invasive surgery has been well established in the elective colorectal surgery and it has been proven better clinical outcome compared with open surgery. in the emergent setting, laparoscope is used mostly in the colecystectomy, appendectomy but laparoscopic emergent colorectal surgery is limited for it's complexity and difficulity. the aim of this study was to envaluate the feasibility of laparoscopic emergent colorectal surgery. methods: this study is prospective collected, observational single center study of patients undergoing laparoscopic emergent colorectal surgery from to . the patient demographics, surgery indication and detail, complication, clinical outcome and hospital stay were collected and analyzed. results: there are total emergent colorectal operations and patients were managed with minimal invasive method. among these laparoscopic emergent surgery, there are male patients and female patients. mean age of the patients was . years (range - years). the main indication for operation: perforation . % ( / ), leakage after elective colorectal surgery . % ( / ), obstruction . % ( / ), ischemia colitis . % ( / ,), bleeding . % ( / ). there are cases in asa , cases in asa , cases in asa . the qsofa score for sepsis: cases was , cases was , cases was , case was . there are cases undergoing laparoscopic lavage with diverting stomy, cases were hartmann procedure, cases were anterior resection, cases were right hemicolectomy, cases were perforation repair, cases were redo anastomosis. there are cases coversion to open method including cases were due to bowel adhesion, cases were due to bowel distension, case was due to severe shock status. mean operative time is . minutes. the overall mortality rate was . % and major complication rate (clavien-dindo grade above ) was . %. re-operation rate was . %. the mean hospital stay was . days. conclusions: this study presents evidence of an initially clinical outcome in emergent laparoscopic colorectal suregry. in the absence of large case series, the benefits of a laparoscopic approach should befall to at least a minority of these patients. confocal laser endomicroscopy (cle) can provide real-time observation of the cell structure and tissue morphology. in our study, we aim to assess the situation of anastomotic perfusion using cle. method: the experimental rabbits were separated into two groups: group a (good anastomotic perfusion, n= ), group b (poor anastomotic perfusion, n= ). the partial colectomy and anastomosis was performed for group a and b. then detection for anastomotic perfusion using cle was carried out after the surgery. during the continuous scanning, we counted the number of blood cells that cross over the certain point of anastomotic stoma in the same period. results: assistant with fluorescein sodium, the blood vessels are highlighted. we can see significant difference of imaging effect between group a and group b. the average number of blood cells are . /min of group a and . /min of group b (p. ), which has significant difference. conclusion: cle can allow real-time observation of the blood flow of anastomotic stoma in vivo. therefore, it is feasible to assess the anastomotic perfusion using cle in colorectal surgery. cigdem benlice, ahmet rencuzogullari, james church, gokhan ozuner, david liska, scott steele, emre gorgun; cleveland clinic background: intraoperative colonoscopy (ioc) is an adjunct in colorectal surgery (crs) especially in patients with malignancies in order to detect location of the primary or synchronous lesions as well as assessing anastomotic integrity. however, effects of intraoperative colonoscopy on short term outcomes during crs is a concern. this study aims to evaluate safety and feasibility and post-operative outcomes of intraoperative colonoscopy in left-sided colectomy patients for colorectal cancer patients by using the nationwide database. patients and methods: patients undergoing elective left-sided colectomy with low pelvic anastomosis without any proximal diversion for colorectal cancer were reviewed from the american college of surgeons national surgical quality improvement program (acs-nsqip) proceduretargeted database ( ) ( ) ( ) according to their primary procedure current procedural terminology (cpt) code. subsequently, patients who underwent intraoperative colonoscopy were identified from concurrent cpt codes and divided into two groups based on the simultaneous intraoperative colonoscopy. demographics, comorbidities, -day postoperative complications were evaluated and compared between the groups. multivariate logistic regression was conducted adjusting for significant factors between the groups. results: a total of patients were identified and ioc was performed for ( . %) patients. objective: laparoscopic ileostomy commonly performed for the patients with colorectal obstruction due to cancer, peritonitis with perforation of colon or the other reason. reduced port surgery is a novel technique that may be performed when considering minimally invasive surgery and desiring a cosmetic benefit. the aim of this study was to evaluate safety and feasibility of reduced port laparoscopic ileostomy for the patients with advanced colorectal cancer before chemotherapy. methods: between july and august , patients who underwent reduced port laparoscopic ileostomy were included ( male and female, age: years old. the outcomes were evaluated in terms of operation time, intraoperative blood loss and perioperative complications. sugical procedures: the patients were placed in the supine position and the operator stood left side. an access device with the wound-protector (ez access, hakko, nagono, japan) was inserted on the future ileostomy site in the right lower abdomen, inserting two of -mm trocars, maintaining pneumoperitoneum at mmhg with carbon dioxide. a -mm trocar was inserted in the left lower abdomen. a -mm flexible laparoscope was inserted from access device port. after exploring abdominal cavity, ileum end was identified. then the marking using dye was put on the ileum of cm proximal from the ileum end. the ileum marked by dye was grasped, and extracted through the access devise. then a blooke ileostomy was created. results: reduced port laparoscopic ileostomy was performed for patients with colorectal obstruction due to cancer before chemotherapy. the mean operative time was minutes, the mean blood loss was . ml. three patient received one additional port. there were no intraoperative complications. five patients ( . %) experienced postoperative complications (two of deep surgical site infection, one of pneumonia, one of outlet obstruction and one of renal dysfunction). there were no other intraoperative or postoperative complications. conclusion: reduced port laparoscopic ileostomy is a safe and feasible procedure for the patients with advanced colorectal cancer before chemotherapy. methods: we performed elective lcr on patients for primary colorectal cancers between june and june . seventy-two patients were excluded in this study following reasons: patients underwent multiple organ resection, and colorectal cancer was diagnosed with stage iv in patients. accordingly, patients were eligible for comparative analysis, with in group po (post operation) and in group c (control). in group po, past operative procedures were as follows: appendectomy ( %), digestive tract ( %), hepato-billiary-pancreatic ( %), gynecologic ( %), urologic surgery ( %), and others ( %). results: there were no significant differences between two groups in asa (grade≤ : vs. %, p= . ), bmi ( introduction: the treatment of rectal cancer requires highly skilled practice by the entire multidisciplinary team. important aims of treatment are: to reduce the risk of residual disease in the pelvis, with lower morbidity and to preserve good sphincter function. the tata procedure is transanal transabdominal radical proctosigmoidectomy with coloanal anastomosis. this technique was first developed in by dr. gerald marks to avoid a permanent colostomy for low-lying rectal cancer. this study reports the long-term results of tata procedure for low rectal cancer. methods and procedures: a prospective study was on patients with low rectal cancer between april and july in a tertiary referral university-affiliated center specializing in laparoscopic surgery. all resections were carried out by a team of dedicated colorectal surgery and standard protocol was used for all pre-and-post-operative care. all the patients underwent total mesorectal excision. results: consecutive patients ( male, female, mean age ) underwent tata procedure, of them ( , %) after neoadjuvant radiochemotherapy. the mean operation time was min (range - ) and the mean estimated blood loss was ml (range - ). the overall incidence of morbidity was , % ( / ) and the mean hospital stay was , days. the mean follow-up period was , (range, - ) months with a recurrence rate of , % ( / ), overall estimated -year survival , % and the disease-free survival rate , %. conclusion: laparoscopic total mesorectal excision with tata procedure is safe with excellent local recurrence and disease-free survival rate. jacek piatkowski, md, phd, marek jackowski, prof; clinic of general, gastroenterological and oncological surgery introduction: more than years ago, laparoscopic technique was considered to be a fully accepted surgical method for treatment of rectal cancer. the following years are a further search for a new surgical method that reduces invasiveness and improves treatment outcomes. it seems that such a method is transanal total mesorectal excision. the aim of this study was to evaluate the new method of rectal cancer surgery (tatme) after years of its use. methods: radicality of treatment (r resection, local recurrence), outcome of surgical treatment and quality of life of patients after surgery were evaluated. results: in the period from . . . - . . . patients ( men, women) were operated in the clinic. in cases the indication for surgery was lower and middle rectal cancer and in cases high grade dysplasia. all patients underwent laparoscopic rectal proctectomy with transanal access (tatme). in all cases, complete oncological radicalization (resection r ) was obtained. the average operation time was minutes. we had used two teams approach (cecil approach) with laparoscopic sets -abdominal and perineal starting at the same time. in the postoperative course, patients had signs of anastomosis leak ( of them required reoperation). the follow-up period is - months. none of the patients had any recurrence of cancer. conclusions: . transanal tme for rectal cancer surgery is an alternative method to conventional laparoscopic surgery. . in a large proportion of patients with lower and middle tumors, the rectum can avoid abdomino-perineal resection with permanent colostomy. background: the double stapling technique (dst) has widely spread colorectal anastomosis especially for anastomosis after low anterior resection. as for the colorectal cancer treatment, heald reported total mesorectal excision (tme) in , and has been accepted as the standard technique for rectal resection due to the decreased local recurrence rate and improved functional results. with advent of dst, there is a background that it has become possible to preserve anus, even in the case with the lesion at lower rectum. laparoscopic surgery for colon cancer was introduced in the s, and has had promising results including long-term outcomes. according to the spread of laparoscopic surgery, laparoscopic surgery had been applied to the rectal resection, with technical difficulty. one of the reasons for the difficulty is that the high rate of anastomotic leakage, a critical adverse effect of low anterior resection (lar). thus, risk factors for anastomotic leakage were widely discussed, including technical factors such as pre-compression and number or firing. the decisive difference in conventional lar and laparoscopic lar in dst, is the stapler used for transection of the rectum. the laparoscopic staplers which are currently available are thought to be not ideal, and there is little evidence of specific specifications of stapler for laparoscopic surgery. materials and methods: all method described in this study was approved by the institutional ethical review committee. we reviewed the colon and rectal wall thickness according to histological examination using h&e staining of distal margin of resected specimen of the patients who conclusions: rstc for severe acute uc is at least as safe as the laparoscopic approach. although the robotic cohort had more comorbidities, major postoperative complications, readmissions, and reoperation rates were less when compared to lstc. rstc was also associated with an earlier return of bowel function and shorter length of stay. a prospective study with larger numbers is needed to see if the superiority of robotic versus laparoscopic approaches is reproducible. s surg endosc ( ) introduction: complete mesocolic excision (cme) has been advocated based on oncologic superiority, but is not commonly performed in north america. furthermore, many data are limited to case series with few comparative studies. therefore the objective was to systematically review studies comparing the short-and long-term outcomes between cme and non-cme colectomy for colon cancer. methods: a systematic review was performed according to prisma guidelines of medline, embase, healthstar, web of science, and cochrane library. studies were only included if they compared conventional resection (non-cme) to cme for colon cancer. quality was assessed using the methodological index for non-randomized studies (minors). the main outcome measures were short-term morbidity and oncologic outcomes. study eligibility, data extraction and quality assessment was performed by two independent reviewers, and disagreements resolved by consensus. weighted pooled means and proportions with %ci were calculated using a randomeffects model when appropriate. results: out of citations, studies underwent full-text review and met the inclusion criteria, of which were unique series. mean minors score was . (range - ). the mean sample size in the cme group was (range - ) and (range - ) in the non-cme group. in the unique studies, included only right-sided resection, and . % ( % ci . - . ) of the remaining were right-sided colectomies. of the studies that reported surgical approach, . % ( %ci . - . ) of cme were performed laparoscopically. there were papers reporting plane of dissection, with cme plane achieved in . % ( . - . ). mean or time in cme group was minutes (range - ) and in non-cme group minutes (range - ). perioperative morbidity was reported in studies, with pooled overall complications of . % ( %ci . - . ) for cme and . ( %ci . - . ) for non-cme resections. anastomotic leak occurred in . % ( %ci . - . ) of cme versus . % ( %ci . - . ) in non-cme colectomies. cme surgery consistently resulted in more lymph nodes retrieved, longer distance to high tie, and specimen length. there were studies that compared -or -year overall or disease-free survival, or local recurrence. only studies reported statistically significant higher disease-free or overall survival in favour of cme. local recurrence was lower after cme in of reported studies. conclusions: the quality of the current evidence is limited and does not consistently support the superiority of cme. more rigorous data are needed before cme can be recommended as the standard of care for colon cancer resections. gilberto lozano dubernard, md, facs, ramon gil-ortiz, md, gustavo cruz-santiago, md, bernardo rueda-torres, md, javier lopez-gutierrez, md, facs; hospital angeles del pedregal introduction: to assess the feasibility of a single-stage colorectal laparoscopic re intervention without ostomy. colonic laparoscopic interventions on patients that previously underwent a minimally invasive procedure, constitutes the current boundary in the management of the acute colorectal pathology. that includes, patients with fecal peritonitis due to diverting procedures already treated surgically. the outcome of our patients could significantly improve if the surgical procedure is performed in one time, with no stoma. method and procedures: from september to june , one hundred thirty-two patients underwent colorectal laparoscopic surgery. five of these patients developed complications: three perforations due to colonoscopy and two due to dehiscence of the anastomosis. these five patients underwent a second laparoscopic procedure that included resection and anastomosis. no stoma required. results: all five patients underwent a second laparoscopic procedure due to an anastomosis leak. no stoma was required. the procedure consisted on resection of the previous anastomosis, re anastomosis, abdominal lavage, aspiration and drains placement. all of them supported with parenteral nutrition. there were no surgical complications. only one patient developed pneumonic symptoms that were solved. conclusion: the reported results, regarding no conversion rate, nor mortality, on our series of patients, suggest that single stage laparoscopic re intervention is feasible, despite fecal peritonitis. introduction: total mesorectal excision is known to be a gold standard surgical procedure for the rectal cancer. subsequently complete mesocolic excision (cme) is recognized as an essential surgical procedure for the colon cancer. the transverse colon is relatively minor location for colon cancer. variety of vessels and mobilization of splenic flexure and dissection close to pancreas make operations for the transverse colon cancer complicated. laparoscopic transverse mesocolic excision in our hospital is presented. method: laparoscopic surgery is conducted with five trocars under the lithotomy position. inferior mesenteric vein is cut after dissection of the descending colon with medial approach. the lower edge of pancreas is exposed near the inferior mesenteric vein and is dissected along toward the tail of pancreas. the splenic flexure is mobilized with lateral approach and the dissection between transverse mesocolon and the lower edge of pancreas is continued in the direction to the pancreas head. coming to the exposure of superior mesenteric artery and vein, the origin of middle colic artery and vein are cut. the transverse mesocolon is separated from the pancreas head and the duodenum with preserving the gastrocolic trunk of henle and the right gastroepiploic vein. the hepatic flexure is mobilized and cme for the transverse colon is finished. this method, the 'tail to head of pancreas' approach, we called, was performed from september . this method is well performed with one series of surgical view, and seems to be a simple procedure as cme with central vascular ligation for the transverse colonic cancer. there were no intraoperative complications, and one postoperative pancreatitis with grade ? of clavien-dindo classification of surgical complications. conclusion: our method, the 'tail to head of pancreas' approach, with transverse mesocoloc excision is simple, safe and feasible. the introduction: anastomotic complication after stapled anastomosis in colorectal cancer surgery is a considerable problem. there are various types of anastomotic complication and they have different severity. this study was aimed to evaluate the impact of intraoperative colonoscopy on detection of anastomotic complication, and its effectiveness in treatment of anastomotic complications after anterior resection (ar) and low anterior resection (lar) for colorectal cancer intraoperatively. methods: from dec. to jul. , a total of patients who underwent anastomosis between sigmoid colon and rectum after colorectal resection were reviewed retrospectively. intraoperative colonoscopy was performed routinely since december in our hospital after anterior resection and low anterior resection. to identify effectiveness of intraoperative colonoscopy, we compared postoperative complications with non-intraoperative colonoscopy group during previous months. intraoperative colonoscopy was performed after anastomosis to visualize the anastomosis line and to perform an air leakage test. if anastomotic defect and moderate bleeding were found in intraoperative colonoscopy, it was managed by means of reinforcement suture or transanal suture repair. we used logistic regression to analyze anastomotic complication between two groups with or without intraoperative colonoscopy. results: of the patients who were performed intraoperative colonoscopy after ar (n= ) and lar (n= ), abnormal findings including bleeding and air leak were found in patients ( . %). among those, cases were observed without any procedure, additional procedures were performed in patients ( . %, transanal suture ( ), lembert suture ( )). postoperative complication was developed in patients; patients had anastomosis bleeding ( . %), patients had ileus ( . %), patient had pneumonia ( . %), patients had minor complication ( . %, acute urinary retention, chylous drainage, laparoscopic port site bleeding). among patients who had anastomosis bleeding, patients were treated by endoscopic clipping, patients were cured by conservative treatment. there was no postoperative anastomotic leakage. the cases of ar and lar were and in non-intraoperative colonoscopy group, there was no significant difference between two group (p= . ). the proportion of laparoscopic surgery was . % and . % on intraoperative colonoscopy and non-intraoperative colonoscopy group, respectively, there was significant difference statistically (p= . ). however, there was no significant difference in anastomotic complication rate between two groups. (rr= . , % ci, . - . ). conclusions: although there was no significant difference in postoperative anastomotic complication rate between two groups, intraoperative colonoscopy may be valuable method for decreasing postoperative complication by visualizing anastomosis line and performing additional procedure. conclusion: it was suggested that lymph node dissection of both middle and left colic regions is necessary for splenic flexure colon cancer, because lymph node metastasis was recognized in both region. surg endosc ( ) :s -s the aims: laparoscopic right hemicolectomy became the standard of care for treating cecum, ascending and proximal transverse colon cancer in many centers. most centers use laparoscopic colectomy with extracorporeal resection and anastomosis (lc). single-incision laparoscopic colectomy with intracorporeal resection and extracorporeal (sc) remains controversial. the aim of the present study is to compare these two techniques using propensity score matching analysis. methods: we analysed the data of patients who underwent laparoscopic right hemicolectomy with lc or sc between december and december . the propensity score was calculated from age, gender, body mass index, the american society of anesthesiologists score, previous abdominal surgery and d lymphnode dissection. short-term outcomes were recorded. postoperative pain was evaluated using a visual analogue scale (vas) and postoperative analgesic use as outcome measure. results: the length of skin incision in the sc group was significantly shorter than in the lc group: median (range) ( . - ) cm verses ( - ) cm (p= . ). the vas score on day and day after surgery was significantly less in the sc group than in the lc group: median (range) ( - ) verses ( - ) on day (p= . ) and median (range) ( - ) verses ( - ) on day (p= . ). significantly fewer the number of requiring analgesia in the sc group on day and day after surgery: median (range) ( - ) times verses ( - ) times on day (p= . ) and ( - ) times verses ( - ) times on day (p= . ). there were no significant differences in operative time, intraoperative blood loss, the number of lymph nodes removed and postoperative courses between the groups. conclusions: sc for right colon cancer is safe and technically feasible. sc reduces the length of skin incision and postoperative pain compared with conventional lc. patients were divided into the following groups: cephalo-medial-to-lateral approach group (cml group, n= ) and medial-to-lateral approach group (ml group, n= introduction: laparoscopic technique has been widely used in the treatment of colorectal cancer, while playing its minimally invasive advantages, but also achieved a good effect of radical oncology. however, t colorectal cancer is not recommended laparoscopic surgery. methods: retrospectively collected pt colorectal cancer data from to in guangdong general hospital, all cases were undergoing radical surgery. results: a total of cases were enrolled in the pt group, including cases of laparoscopic group, cases of open group, conversion rate was . %. there was no difference in baseline data (age, sex, bmi, asa, etc.)(p. ). there was a significant difference between the two groups (p. ) in blood loss, postoperative complications and postoperative recovery index. in the pathologic t a/b, combined-organ resection, postoperative recurrence, the laparotomy group had more cases, and there was a statistically significant difference between the two groups (p\ . ). the -and -year overall survival rates were . % and . % for the lap group and . % and . % for the open group (p= . ). meanwhile, the -and -years disease-(p= . ). iiic stage, lymph node status, ca - and adjuvant chemotherapy were independent prognostic factors affecting overall survival. the age, pt a/b, iiic stage, ca - and adjuvant chemotherapy were independent influencing factors of disease-free survival. conclusions: laparoscopic surgery for pt colorectal cancer surgery, it is not only in the play of its minimally invasive but also obtained with the similar long-term effect. but we need more multicenter, prospective, and large sample clinical studies to validate our findings. introduction: lymph node (ln) retrieval after surgery is important. in the present study we evaluated the efficacy of the fat dissolution technique using fluid containing collagenase and lipase to avoid staging migration after laparoscopic colorectal surgery. methods: seventeen patients who underwent laparoscopic ln dissection for colorectal cancer were evaluated. first, unfixed lns within the resected mesentery were explored by visual inspection and palpation immediately after the operation by the surgeon, which is the most common practice in japan. subsequently, the fat dissolution technique was used on remnant fat tissue, and the lns were evaluated again. the primary endpoint was whether the second assessment increased the number of lns evaluated. results: the median number of lns identified at the first and second assessments was and , respectively, resulting in a significant increase in the total number of lns evaluated ( vs. , p\ . , paired t-test). one positive node was identified among all the additional lns identified ( . %; / ). although staging was not altered in any patient, the second assessment resulted in an increase in the originally insufficient number of lns evaluated (\ for stage ii) in three patients, whose treatment may be altered. tumor cells detected after the fat dissolution technique were stained with carcinoembryonic antigen and cytokeratin- . conclusion: using the fat dissolution liquid on remnant fat tissue of the mesentery of the colon and rectum enabled identification of additional lns. this method should be considered when the number of lns identified is not sufficient after conventional ln retrieval, and may avoid stage migration. aim: the aim of this study is to evaluate the pathological resection margin after laparoscopic intersphincteric resection for low rectal cancer. method: from to , there were eight laparoscopic intersphincteric resection cases for low rectal cancer. we evaluated the clinicopathological findings and the positivity of pathological resection margin. results: the median distance from the anal verge to the tumor was mm (range, - ), and the median diameter of the tumor was mm (range, - ). there was no case with neoadjuvant therapy. the estimated tumor depth were ct in cases ( . %) and ct in cases ( . %), and the actual tumor depth were ptis in cases ( . %) and pt in cases ( . %) and pt in cases ( . %). the median distal resection margin was mm (range, - ). pathological resection margin, such as the proximal, distal and circumferential margin was negative in all cases ( %). there was no mortality, but morbidity occurred in two cases (one case of anastomotic leakage and one case of small bowel obstruction). no recurrence nor distant metastasis was observed in the follow up period. conclusion: there was no positive resection margin case in the series. our patient selection, indication and the technique were considered to be precise and appropriate. introduction: the fistulas of the intestine to the vagina or the bladder include a highly morbid entity, with several functional limitation and loss of the quality of life, its diagnosis is complex and more than its treatment, which include a wide range of possibilities that go from the simple derivative colostomy in search of the spontaneous closure of the fistula, under the complete correction of the pathology with resections, anastomosis and mini-vasive reconstructions. give to know our experience in the minimally invasive treatment of whole vaginal and whole vesicial fistules by laparoscopic via, for the last years. results: a total of patients were operated in this period, women and men, all those by laparoscopic via, with intestinal resection, in thick intestine cases, in one small intestine and in another case with the commitment of the two, everyone restriction and intestinal anastomosis and in no matter were colostomy, primary closures of the fistula in patients were required, conversion to open surgery in a case and there was no recurrence, patients had prolonged hospitalization for localized infections, a requirement reintervencion for revision. a patient suffried a umbilical eventration for the extraction site, which was corrected one year after laparoscopy. conclusion: minimally invasive surgery in patients with this type of pathology becomes an excellent strategy for the integral management of these patients. group work guarantees good results. robbie sparks, dr, ronan cahill; mater misericordiae university hospital background: precise preoperative localisation of colonic cancer is a prerequisite for correct oncological resection. effective endoscopic lesional tattoo is vital for small, radiologically unseen tumors planned for laparoscopic resection but its practice may be imperfect. methods: retrospective review of consecutive patients with preoperative endoscopic lesional tattoo who underwent laparoscopic colonic resection identified from our prospectively-maintained cancer database with supplementary clinical chart and radiological, histological, endoscopic and theatre database/logbook interrogation. results: patients ( males, mean age years, median bmi . kg/m , left sided lesions, screen detected, benign polyps, % conversion rate). in operations ( %) tattoo visibility was documented with tattoo absence noted in ( . %) although tattoo was identifiable in the pathological specimen in four. in those with "missing tattoos", six of the lesions were radiologically occult and in three the tumor was found in a different colonic segment then had been judged at colonoscopy. four patients had on-table colonoscopy and five were converted to laparotomy ( % conversion rate, p. ). mean postoperative length of stay was . (range - ) days. one patient's segmental resection contained only benign pathology requiring a second operation to remove the cancer. on univariate analysis, time between endoscopy and surgery (but not patient age, gender, bmi, endoscopist or surgeon seniority, tumor size or location) was significantly associated with absence of tattoo intraoperatively (p= . ). conclusion: recording related to tattoo is variable but definite lack of gross tattoo visualisation significantly impacts the procedure. the mechanism of tattoo absence is multifactorial needing careful consideration but solvable. the aim of the present study was to perform a systematic review of the literature to determine the role of antibiotics in the management of acute uncomplicated diverticulitis (aud). diverticular disease is the most common disease of the large bowel and poses a significant burden on healthcare resources. in the united states alone, the cost of diverticular disease has been estimated to be over $ billion making it the fifth most important gastrointestinal disease economically. the use of antibiotics in the management of aud, however, is primarily based on expert opinion as current high-quality evidence is lacking. recent studies have not only questioned the optimal type and duration of antibiotic regimens, but whether antibiotics provide any benefit in the treatment of aud. conclusions: antibiotic use in patients with acute uncomplicated diverticulitis is not associated with a reduction in major complications, readmissions, treatment failure, progression to complicated diverticulitis, or need for elective and emergent surgery. however, it increases the length of hospital stay. given the risk of selection bias in included studies, further randomized trials are needed to clarify the need for antibiotics in uncomplicated diverticulitis. laparoscopic para-aortic lymph node resection for colorectal cancer aim: we want to highlight the feasibility of a sigmoidectomy using total laparoscopic with a transanal extraction of the specimen. methods: it is a -year-old female patient, obese (bmi= kg/m ) to the antecedents of laparoscopic cholecystectomy and chronic constipation. she was treated three months ago for a sigmoidal diverticulitis complicated with a pelvic abscess. the evolution has been favorable under antibiotic therapy and percutaneous drainage of the abscess. the colonoscopy showed a multiple diverticula located between and cm from the anal verge. prophylactic sigmoidectomy was performed laparoscopically using trocars ( mm supra ombilical, mm fid and mm right flank). the specimen was extracted transanally, thus avoiding a pubic incision. the steps of the intervention were: -mobilisation of left colon -closing of distal left colon stump -rectal stump lavage -opening on the rectum -transanal introduction of the anvil -specimen transanal extraction -closing og rectal stump -colonic positioning of the anvil -coloractal anastomosis. results: the intervention was minutes. no perioperative incidents. the liquid regime was authorized on the night of the intervention. the operating procedures were favorable with an exit to j post operative. the anapath examination of the surgical specimen confirmed the presence of sigmoidal diverticula. conclusion: laparoscopic sigmoidectomy with transanal extraction of the specimen for benign desease is a seductive technique with satisfactory results. it avoids a pubic incision with its parietal and aesthetic complications. chengzhi huang; guangdong general hospital (guangdong academy of medical science) background: colorectal cancer (crc) is one of the most common malignant diseases over the world. of the causes of the death of crc, metastasis to liver or lung are the major factors. however, there is still lack of precise tumor biomarker that precisely predict the clinical outcome of crc. the salt-inducible kinase (sik ) encodes a serine kinase of amp-activated protein kinase (ampk) family, which may play critical roles in tumorigenesis and tumor progression. this study aimed the study the expression and clinical significance of sik and crc patients. methods: the expression of sik protein was measured by western-blot and analysis of immunohistochemistry. sik mrna expression in cancerous tissue was measured by rt-pcr. results: the expression level of sik was correlated with the following factors: tumor invasion (t stages), lymph node metastasis, clinical stages (tnm) and tumor location. the down-regulated sik implies poor clinical outcome measured by kaplan-meier analysis (p-value. ), and may act as an independent risk factor of crc patients. background: surgical specimens for resected colon cancer vary in quality and there remains no universally accepted technique to guide resection margins. a minimum of lymph nodes provides some quality assurance, however this remains a crude marker of optimal oncological surgery. a tool to precisely identify lymphatic drainage within the mesentery could improve the oncologic quality of resection and better guide adjuvant treatment through more optimal mesenteric lymphadenectomy. while fluorescence imaging (fi) has been described to identify nodal disease in several other cancers, feasibility and best practices have not been established in colon cancer. we describe a novel technique of fi using indocyanine green (icg) to identify lymphatic spread and potentially guide optimal mesenteric lymphadenectomy in colon cancer. methods: three consecutive patients with colon cancer undergoing a laparoscopic resection had peritumoral subserosal injection of icg for fi after extracorporealization of the mobilized specimen. three concentrations of icg were injected − mg/ ml, mg/ ml, and mg/ ml. a total of ml was given for each patient. using a modified laparoscopic camera, the icg was excited by light in the near-infrared (nir) spectrum, for real-time visualization of the lymphatic drainage. the main outcome measure was identification of lymphatic drainage. results: three patients with right-sided primary colon cancer were evaluated. all three patients had successful identification of the lymphatic drainage pattern along the mesentery. the most successful protocol was ml (concentration mg/ ml) subserosal injection at points within close proximity ( cm) of the tumor with a -gauge needle, then waiting minutes for complete mapping. no intraoperative or injection-related adverse effects occurred with -day follow-up. the median lymph node yield was . all specimens had tumor-free margins. conclusion: from this small series, fluorescence imaging with icg is a potentially safe and feasible technique for identifying mesocolic lymphatic drainage patterns. this proof of concept and protocol will lead to future studies to examine the utility of fluoresence imaging to guide more precise surgery in colon cancer. introduction: anastomotic leakage in colon/rectal surgery is a dangerous event with an occurance rate ranging from to %. the associated mortality rate is between - %. the white-light intraoperative subjective surgical assessment (the most frequently used approach) underestimates the actual anastomotic leakage rate. intraoperative tissue perfusion assessment by indocyanine green (icg)-enhanced fluorescence has been reported in multiple clinical scenarios in laparoscopic/ robotic surgery, as well as for for bowel perfusion assessment. this technology can detect microvascular impairment, potentially preventing anastomotic leakage. we reviewed the literature and present our data to evaluate the feasibility and usefulness of icg-enhanced ?uorescence in the intraoperative assessment of vascular peri-anastomotic tissue perfusion in colorectal surgery. methods and procedures: a pubmed literature narrative review has been performed. moreover, out of a total of robotic colorectal cases, we retrospectively analyzed icg-enhanced fluorescence robotic colorectal resections ( left colectomies- rectal resections- right- transverse- pancolectomy). results: after icg-technology use, the biggest (n[ ) case-series showed a rate of . - % of cases in which they changed the level of resection based on icg. icg technology may variably reduce the anastomotic leak rate from to %. however, the threshold values to define the actual sub-optimal perfusion are still under investigation. in our experience, out of icg cases performed: the conversion, intraoperative complication, dye allergic reactionand mortality rates were all %. post-op surgical complications: case of leak ( , %) and sbo for incarcerated hernia ( . %). in cases, with normal white-light assessment, the level of the anastomosis was changed after icg showed ischemic tissues. despite the application of icg, anastomotic leak has been registered. conclusions: icg-enhanced ?uorescence may intraoperatively change the white-light assessed resection/anastomotic level, potentially decreasing the anastomotic leakage rate. our data shows that this technology is safe, feasibile and may prevent anastomotic leakage. however, the decision making is still too subjective and not data driven. at this stage icg, beside being a promising technique, doesn't have high level of evidence (most of the reports are retrospective). some randomized prospective trials with an adequate statistical power are needed. a precise injection dose and timing standardization is required. the main challange is to develop a method to objectively obtain a real-time intensity assessement. this may provide objective metric tresholds for an intraoperative evidence/data-based surgical decision making. introduction: according to the world health organization, colorectal cancer is the rd most commonly diagnosed cancer in the world. one of the main risk factors for the development of colorectal cancer is obesity. obesity is seen to increase the risk of colorectal cancer by % in women per kg/m and % in men per kg/m . bariatric surgery is one of the treatments that is considered to achieve and sustain a significant amount of intentional weight loss in patients. considering that fact that bariatric surgery decreases obesity, this intentional weight loss would seem to provide a favorable outcome in terms of diagnosis and prognosis of colorectal cancer. a systemic review of the literature was conducted via pubmed to identify relevant studies from january through may . the main outcome for this study is to assess whether patients who underwent bariatric surgery (restrictive and malabsorptive procedures) had an increased or decreased risk of colorectal cancer. all studies included in this meta-analysis are retrospective cohort studies. results were expressed as standard difference in means with standard error. statistical analysis was done using fixed-effects meta-analysis to compare the mean value of the two groups between bariatric surgery and non-surgery in patients with colorectal cancer. (comprehensive meta-analysis version . . software; biostat inc., englewood, nj). results: four out of studies were quantitatively assessed and included for meta-analysis. among the four studies, , underwent bariatric surgery and , did not undergo bariatric surgery. there is a significant decrease ( . ± . ; p= . ) in the risk in patients developing colorectal cancer in patients who underwent bariatric surgery compared to those who didn't get surgery. conclusion: bariatric surgery patients appear to have a decreased risk of colorectal cancer compared to patients who did not have bariatric surgery. guh jung seo, hyung-suk cho; department of colorectal surgery, dae han surgical clinic, gwangju, south korea introduction: the incidence of rectal carcinoid tumors is increasing due to the widespread use of screening colonoscopy. endoscopic mucosal resection (emr) is a useful method for small rectal carcinoid tumors (≤ mm) because of its simplicity, quick procedure and low complication rates. we aimed to describe our experience and evaluate the outcomes of emr for rectal carcinoid tumors. the patients enrolled in this study were patients with small rectal carcinoid tumors who underwent emr using a submucosal injection technique of epinephrinesaline mixture between august and october . all medical records, including characteristics of the patients and tumors, complications, were retrospectively reviewed. results: the patients were men and women, with a mean age of . years (range, - years). en block resection was performed by emr in all cases. the endoscopic mean size of tumors was . mm (range, - mm). the pathologically measured mean size of the resected specimens was . mm (range, - mm). the mean size of resected carcinoid tumors was . mm (range, . - mm). the tumor shape was submucosal tumor in and polyp in . histological examination revealed that cases had resection margin positive of tumor and case had undetermined resection margin of tumor. of the patients, patients underwent endoscopic treatment and patients underwent transanal excision. no residual tumor was found in additionally removed tissue. there were cases with emr-related complications: early postprocedural bleeding and postpolypectomy syndrome. there was no significant bleeding requiring blood transfusion or perforations. conclusion: endoscopic mucosal resection is considered to be a relatively safe and useful method for treatment of small rectal carcinoids in selected patients. background: disturbance of sexual function after an operation for rectal cancer has often occurred. the relationship between autonomic nerves and arteries in pelvis was examined. methods: clinical studies of male patients with resected rectal cancer were performed using snap gauge method, penile-brachial index and evoked bulvo-cavernous reflex. in canine experiments, pelvic splanchnic nerve (psn) electric stimulation, arterial flow measurement, corpus cavernosum pressure measurement and muscle strip study using drugs were evaluated. results: in clinical studies of male patients, transection of the hypogastric nerve (hgn) and the sympathetic trunk did not affect the erectile function in the postoperative course. in animal experiments transection of these nerves did not affect the increase in inner pressure of the penis cavernosum. in postoperative cases in which only one side of the lower grade branches of the psn (s ) were preserved, the erectile function was preserved. in animal experiments in which the psn of one side was disturbed, the ipa flow of the same side decreased, while the flow of the other side increased. we have evaluated the role of adrenergic components in the psn on the erectile function in the dog. the effect of norepinephrine hydrochloride on canine vascular smooth muscle was examined in vitro. vascular smooth muscle strips from the ipa relaxed longitudinally. electrical stimulation of the psn increased blood flow in the ipa and also elevated the cavernous pressure. these increases were blocked in part by phentolamine, but not by propranolol or atropine. the effects of cholinergic and adrenergic agonists and antagonists on mechanical responses were also examined in muscle strips obtained from various arteries in the intra-pelvic region including the ipa. norepinephrine induced contraction in the iliac artery and relaxation in the ipa, and both the contraction and relaxation responses were blocked by phentolamine but not by propranolol. these findings suggest that in the dog, α-adrenergic components projected through the psn may contribute to penile erection. conclusion: blood flow in the ipa was controlled significantly by the same side psn, but compensatory by the other side psn. it is also conceivable that the erectile function through the psn is controlled by the sympathetic nerve, not by the parasympathetic nerve. in postoperative cases in which only one side of the lower grade branches of the psn (s ) were preserved, the erectile function was preserved. introduction: currently, neoadjuvant chemo-radiotherapy (ncrt) followed by low anterior resection or abdominoperineal resection are the standard treatments for locally advanced rectal cancer. ncrt can improve resecability, achieve better sphincter preservation and reduce local recurrence. although total mesorectal excision is the standard treatment for advanced rectal cancer, recent trends in minimally invasive treatments led to an increase in local excision or "watch and wait" in patients with an excellent response to ncrt. the purpose of this study, part of an ongoing research, is critically evaluating the feasibility of "non-operative treatment" for rectal cancer in a district hospital. methods and procedures: a total of patients with rectal cancer, who where treated with ncrt from january to august at "carlo urbani" district hospital in jesi (italy), were retrospectively reviewed. all patients had histologically-confirmed primary adenocarcinoma of the rectum located within cm from the anal verge. the involved patients completed ncrt and had no recurrence disease, distant metastasis, synchronous malignancies. they were classified according to the mandard's tumor regression grade (trg) into two clusters: group a (trg - ) and b . results: the average age of people is . and were male. five patients underwent abdominoperineal resection and % fell within group a. six patients had lymph nodes involved. four patients suffered relevant complications, such as wound complication, anastomotic leak, operative reintervention and death. univariate analysis showed that the main predictors of tumor regression were the absence of lymph-nodes involvement from initial imaging (p. ), normal initial carcinoembryonic antigen level (p. ) and tumor downstaging in imaging (p. ). in addition, most relevant complications occurred to elderly patients although they observed a good clinical response. besides, % of patients were found to be complete pathologic responders upon examination of the surgical specimen. conclusions: the oncologic feasibility of non-operative management for the patients with complete clinical response after ncrt has been growing, but some studies have suggested lack of oncologic safety in these patients. the patients with a complete clinical response expect good survival, but they may still harbor residual disease. no consensus on "watch and wait" policy in the field of rectal cancer was obtained, yet. our data did not entirely support this policy although it might be the best strategy, based on the predictors of tumor regression, to avoid the complications associated with surgery in elderly patients with significant medical comorbidities and fear of a permanent stoma. introduction: conventional incision laparoscopic surgery procedure for rectal cancer is widely accepted as a successful alternative to laparotomy now, bestowing specific advantages without causing detriment to oncological outcome. evolving from this, single-incision laparoscopic surgery (sils) has been successfully utilized for the removal of colonic tumors, but the literature lacks sufficient data analyzing the suitability of sils for rectal cancer especially for total resection mesorectal excision (tme), particularlyon oncological outcome. we report the short-term clinical and oncological outcomes from a large cases retrospective analysis of observational study of sils for tme procedure of rectal cancer. methods: rectal cancer patients who underwent transumbilical single incision laparoscopic tme surgery were recruited in the current study. short-term perioperative clinical parameters and oncological outcomes were observed and all patients were followed up after surgery. then summarize the preliminary application results. results: operations were accomplished successfully with single incision laparoscopy, patients were converted to multiport approach, and was converted to laparotomy, no diverting ileostomy was performed. the average operative time was ( . ± . ) min, with an average blood loss of ( . ± . ) ml, the median postoperative hospital stay was ( . ± . ) days. all patients received a r resection and the surgical margin were conformed negative in all cases, the median number of harvested lymph node is ( . ± . ), the specimens met the requirement of tme. there were postoperational complications, no operation-related mortality or postoperative anastomotic leakage was observed. no patient appeared recurrent in a median follow up of months. conclusions: total mesorectal excision surgery for rectal cancer can be safely performed using transumbilical single incision laparoscopic technique, with acceptable short-term clinical and oncological outcome. surg endosc ( ) background: any surgical trauma induces an inflammatory response, which is considered as a negative factor in the general immune response, specially in malignant disease. the c-reactive protein (crp) is an acute phase protein often used as a marker of surgical trauma. stent treatment has been used as a treatment option for colonic obstruction in palliative cases for many years, and also as a bridge to surgery in selected cases. in a pilot study we compared the inflammatory response after acute stent treatment or surgery for malignant colonic obstruction. method: we compared two consecutive series of treatment of acute malignant colonic obstruction, stent treatment or emergency surgery during - . all patients were admitted with acute colonic obstruction due to colorectal cancer. choice of treatment was based on attending senior colorectal surgeons' preference, patient comorbidities and disseminated disease was considered. patient age, crp, time to first defecation and length of stay was recorded. results: a total of patients were identified in a retrospective analysis. patients had acute stent treatment and had acute surgical treatment for colonic obstruction, all due to colorectal cancer. median age was y ( - ) with no difference between the groups. there was no difference in metastatic disease between the groups. median time until first defecation after treatment was significantly shorter for the stented patients ( h ( - )) compared with those operated ( h ( - )) (p, ). median hospital stay was also shorter in the stent group, days ( - ), versus days ( - ) in the surgical group (p= , ). crp did not differ between the groups before treatment. both treatments resulted in increased crp levels at postoperative days and , but the crp levels were significantly higher in the surgical group than in the stent group at both time points (pod p= , , pod p, ) conclusion: acute stent treatment in colonic malignant obstruction seems to induce a less pronounced inflammatory response compared with surgery, as shown by a significantly reduced increase in postoperative crp resulting in shorter time to first defecation and a shorter hospital stay. introduction: meckel's diverticulum is the most common congenital abnormality in newborns, present in about - % of them. diagnostic of meckel's diverticulum requires a high index of suspicion, and even with the use of modern imaging technologies, they are often diagnosed intraoperatively. what to do when an asymptomatic diverticulum is found incidentally during surgery for other causes is a matter of discussion. objective: the aim of this article is to report symptomatic and asymptomatic incidentally found cases seen in a fourth-level hospital in colombia. the reports of the histopathologic examinations carried out in the hospital in the last years were reviewed searching for those containing meckel's diverticulum in their diagnosis. patients were divided in asymptomatic and symptomatic groups. the asymptomatic group was defined as patients who were operated for a different indication and a meckel's diverticulum was found incidentally. morbidity was divided in early and late complications after the initial surgery. results: from january to june , a total of pathology reports included the diagnosis meckel's diverticulum. a total of adult patients were retrieved. all of those patients with meckel's diverticulum a total of patients were symptomatic, being sbo the most common complication and required the surgical remove incidentally. conclusion: the correct approach of the patients with diverticular pathology allows the early identification and the appropriate management of the surgical complications that can be presented. robert j czuprynski, md, grace montenegro, md; saint louis university hospital presacral masses are a rare entity, with an incidence of . % and can be classified in several categories, including inflammatory, neurogenic, congenital, osseous and miscellaneous. in this case, a neuroendocrine tumor was identified with concern for iliac chain lymphatic and gluteal metastasis. the patient underwent abdominoperineal resection, excision of presacral mass, lymph node biopsy and omental flap. final pathology returned as a grade ii neuroendocrine tumor arising from a tailgut cyst. a year old female with a ten year history of recurrent perianal, ischiorectal and deep postanal abscesses presents with a presacral mass biopsy proven well-differentiated neuroendocrine tumor. octreotide scan demonstrated avidity for presacral mas as well as left intergluteal lymph node and two internal iliac lymph nodes. chromogranin a, neuron-specific enolase and serotonin markers were all negative. the patient was taken to the operating room and underwent abdominoperineal resection, resection of presacral mass and internal iliac nodes with an omental flap. neuroendocrine tumors arising from tailgut cysts of the presacral space are rare in nature. in a retrospective study from great britain, four of thirty one tailgut cysts had malignant transformation, so it is generally recommended to resect the cysts. in this case, the patient's tumor was a moderately differentiated, grade ii with extensive lymphovascular and perineural invasion. there are no prospective studies showing neoadjuvant therapies in neuroendocrine tumors of the presacral space. according nccn guidelines, patient is currently asymptomatic with low tumor burden. recommended treatment at this time is observation with surveillance tumor markers every - months or octreotide. anastomotic leakage has been commonly regarded as one of the toughing postoperative complications in laparoscopic mid/low rectal cancer surgery, attenuating the short-term clinical benefits. the left colic artery (lca) has been routinely central-ligated in dissection process to guarantee the oncological effects, which may potentially attribute to the postoperative ischemia-induced anastomotic leakage in the patients with left-colic vessel variation, e.g. bypass or absent of riolan arch. however, no specific study focuses on the surgical benefits of lca preservation compares to conventional ones. herein, we conduct a single center randomized controlled trial, demonstrating that lca-preserving technique shows significant reduction rate of postoperative leakage as well as overall complications comparing to the traditional central-ligation group. no difference in survival rate and recurrence in short term is found between the two groups. the lca-preserving strategy is proven to be repeatedly safe and feasible, potentially reduce the risk of anastomotic leakage with comparable short-term outcomes. further investigation is required for both the oncological safety and long-term prognosis for this innovative technique. background: three-photon imaging (tpi), which was based on the field of nonlinear optics and femtosecond lasers, has been proved to be able to provide the -dimensional ( d) morphological feature of living tissues without the administration of exogenous contrast agents. the purpose of this study is to investigate whether tpi could make a real-time histological d diagnosis for colorectal cancer compared with the gold standard hematoxylin-eosin (h-e). methods: this study was conducted between january and august . a total of patients diagnosed as colon or rectum carcinoma by preoperative colonoscopy were included. all patients received radical surgery. the fresh, unfixed and unstained full-thickness cancerous and the corresponding normal specimens in the same patient, were immediately prepared to receive tpi after surgery. for d visualization, the z-stacks were reconstructed. all tissue went through routine histological procedures. tpi images were compared with h-e by the same attending pathologist. results: the schematic diagram of tpi is shown in fig. a . peak tpi signal intensity excited at nm was detected in living tissues. the field of view (fov) was µm and the imaging deep was µm in each specimen. in normal specimens, glands lined regularly and characterized as a typical foveolar, which was comparable to h-e images ( fig. b and d ). in cancerous specimens, irregular tissue architecture and shape were identified by tpi, which was also validated by corresponding h-e images ( fig. c and e ). tpi images can be acquired with a view of d visualization. based on rates of correlation with pathological diagnosis, the accuracy, sensitivity, specificity, positive predictive value, negative predictive value were %, %, %, %, . %, respectively. conclusions: it is feasible to use tpi to make a real-time d optical diagnosis for colorectal cancer. with the miniaturization and integration of colonoscopy, tpi has the potential to make a real-time histological d diagnosis for colorectal cancer in the future, especially in low rectal cancer. erica pettke , abhinit shah , vesna cekic , daniel feingold , tracey arnell , nipa gandhi , carl winkler, md , richard whelan ; mount sinai west, columbia university introduction: alvimopan (alvim) is a peripherally acting µ-opioid receptor antagonist used to accelerate gastrointestinal functional recovery postoperatively (postop) after bowel resection. the purpose of this retrospective study was to compare the time to first flatus and bowel movement (bm) as well as length of stay (los) following elective minimally invasive colorectal resection (crr) in a group of patients (pts) who received alvimopan perioperatively (periop) vs a group that did not get this agent. methods: a data review from to from irb approved databases was carried out. operative, hospital and office charts were reviewed. routine use of alvim for elective crr cases was stared in . besides gi data, preoperative comorbidities and day postop complication rates were assessed. the results with periop alvim were compared to a no-alvim group. the students t and chi-square tests were used. results: a total of pts underwent elective crr. alvim was administered periop to pts ( %). the breakdown of indications between groups were similar. alvim pts were younger ( . vs. . years old, p= . ) and, as regards comorbidities, less likely to have heart disease (cad . % vs . %, other heart disease . % vs . %) but were otherwise similar. the rate of laparoscopic-assisted (alvim, . %; no alvim, %) and hand assisted or hybrid operations (alvim, . %; no alvim, %) were similar. alvim pts had significantly earlier return of flatus ( . vs . days) and first bm ( . vs . , p. for both) than the no alvim group. there was also a trend toward a shorter los ( . vs . days, p= . ) for the alvim group. overall complication rates were similar, however, alvim pts had lower rates of post-operative ileus ( . % vs . %, p. ), sssi's ( . vs %, p= . ), and blood transfusion ( . vs . %, p= . ) than the no alvim group. conclusion: the two groups compared were largely similar (most co-morbidities, indications, crr type) with the differences in age and cardiac issues noted. the impact of the higher rates of sssi's, blood transfusion, and mi in the no alvim group on gi function is unclear. pts who received alvim periop had an accelerated return of bowel function, decreased postoperative ileus and shorter length of stay. these results suggest that alvim is effective in reducing the postoperative ileus but further study is warranted. background: laparoscopic total proctocolectomy (tpc) is selected for minimally invasive surgical treatment of familial adenomatous polyposis (fap) and ulcerative colitis (uc). our policy of tpc is no diverting ileostomy for fap and creating ileostomy for ibd because most of the patients received steroid therapy. objective: we examined the outcome of laparoscopic tpc according to disease of fap and ibd (uc and crohn's disease). methods: twenty-three consecutive patients who underwent laparoscopic tpc between april and march were examined. the patients were divided into fap group and ibd group. results: seven patients of fap and patients of ibd (uc , crohn's disease ) underwent laparoscopic tpc or total colectomy. among them, patients (fap , ibd ) were cancerassociated cases. the procedures of the fap group was tpc with iaca in patients and hals total colectomy with ira in patient. the procedures of ibd group were tpc with iaca in patients, tpc with iaa in patients, total colectomy with ira in patients, of which hals cases. the mean operative time and blood loss were minutes, . g in the fap group and minutes, . g in the ibd group, respectively. diverting ileostomy was constructed in patients of only uc group. early complications of fap group were observed in cases (postoperative ileus , anastomotic leak with conservative treatment ), and those of ibd were observed in cases (ileus , anastomotic leak with conservative treatment , abdominal abscess , wound infection ). the median postoperative hospital stay was days in the fap group and days in the ibd group. complications requiring reoperation were cases (fap : intestinal obstruction, ibd : inflammation of stoma-closure site). no cancer recurrence and mortality were observed. one case of fap underwent additional transanal mucosal resection due to new lesion of adenoma. conclusions: laparoscopic total proctocolectomy for fap and ibd was performed safely, especially less complications occurred in fap patients without diverting ileostomy. in addition, followup of remaining mucosa is important in iaca and ira patients. treatment of complex anal fistula has always been a nightmare for surgeonsby conventional means. even the lowest and simple looking fistula at times comes out to be a complex one with high incidence of recurrence above %. most of the availability diagnostic including mri is nit conclusive and many a times the surgeon remains in a state of confusion as to what is going to come at the operation table. the conventional treatment modalities also usually leave the patient wounded needing almost to weeks to heal with a risk of sphincter damage and a high risk of recurrence. we would be presenting the technical details and results of our series of cases of complex anal fistula treated by video assisted endoscopic therapy. jun higashijima, phd, mitsuo shimada, professor, kozo yoshikawa, phd, takuya tokunaga, phd, masaaki nishi, phd, hideya kashihara, phd, chie takasu, phd, daichi ishikawa, phd; department of surgery, the university of tokushima background: one of the important causes for anastomotic leakage (al) in anterior resection is an insufficient blood flow of the stump. the hems (hyper eye medical system) and spies (laparoscopic icg system) can detect the blood flow of fresh organ intraoperatively by injection of indocyanine green (icg). and thermography also can evaluate the bloodflow less invasively. the aim of this study is to evaluate the usefulness of icg system and thermography in laparoscopic anterior resection. patients and methods: this study retrospectively included patients who underwent laparoscopic anterior resection for colon cancer with double stapling anastomosis procedure. blood flow evaluation of oral stumps was performed with measurement of fluorescence time (ft) using hems and spies. and bloodflow was also evaluated by thermography. result: evaluation by icg system: in all cases, the al rate was . % ( / cases). over ft cases, the al rate was %, higher than that of under s cases and these patinets need additional management, covering stoma or additional resection. and in border cases, ft * sec, al rate is . %, higher than under s cases. in these borderline cases, if covering stoma was performed in patinets with more than three well known risk factors, the al rate reduced to . % and false positive was . %. and under s cases, they need no additional management. evaluation by thermography: in residual intestine, the temperature was siginificantly higher than resected intestine ( . vs . ?, p. ). and the temperature in ft under s cases was significantly higher than over ft over s cases ( . vs . ?). the temperatue and ft was tended to be oppositely correlated (r = . ). conclusion: both icg system and thermography may be useful to avoid anastomotic leakage. introduction: some patients who undergo neoadjuvant chemoradiation therapy (crt) for rectal cancer achieve a pathologic complete response (pcr) in which no tumor cells are discovered during pathologic analysis of the resection specimen. achievement of pcr is correlated to improved prognoses relative to non-pcr counterparts. such correlations are not well established in the context of a community-based hospital. the study sought to examine response rates, recurrences, and survivals in locally advanced rectal cancer patients and compare patient outcomes to those achieved at major academic institutions. methods and procedures: a single-center retrospective chart review was performed at a local, community-based hospital. study population consisted of patients with locally advanced rectal cancer treated with neoadjuvant crt followed by surgical resection. patients with a history of metastasis, inflammatory bowel disease (ibd), hereditary cancer syndromes, concurrent or prior malignancy, and emergent surgery were excluded. results: patients ( . %) achieved pcr in the test population. across both groups, mean age (p =. ), gender (p=. ), and ethnicity (p=. ) were found to be comparable. mean interval between crt and or (p=. ), pre-op stage (p=. ), number of nodes (p=. ), radiation dose (p=. ), tumor location (p=. ), and days of follow-up (p=. ) presented statistically insignificant differences between groups. at years, non-pcr patients ( . %) had a recurrence with zero recurrences in the pcr group. -year mortality presented non-pcr patients ( . %) compared to pcr patient ( . %). conclusion: a multidisciplinary approach to rectal cancer consisting of standardized preoperative treatment and surgical resection can achieve patient outcomes and survival similar to those of larger academic institutions, even in the context of a community-based hospital. objective: the aim of this study was to assess safety and feasibility of total mesorectum excision (tme) within the holy plane based on embryology for rectal cancer. methods: prospectively collected data of consecutive patients with rectal cancer who underwent tatme from november to august were enrolled. surgical outcomes including tme completeness, operative time for tme completion, blood loss, complications, pathological findings and length of hospital stay were assessed. surgical procedure: after performing ractal lavage, self-retaining anal retractor was set, and anal dilators were used for an atraumatic introduction of the transanal access devise (gelpoint path). three of -mm trocars and one of -mm trocar were inserted through the gelpoint path in a quadrant shape. then the gelpoint path was introduced through the anal to rectum. after rectosigmoid colon was temporally clamped using an atraumatic endo bulldog clip, pneumoperitoneum was maintained at mmhg with carbon dioxide via an air seal platform. a purse-string suture using a polypropylen with -mm rounded needle was performed clock-wise to tightly occlude the rectum with a cm margin distal to the tumor. after irrigation with saline and marking dissection line with tattooing the rectal mucosa distal to the mucosal folds, a mucosal transection of rectum was initiated. then a full-thickness rectal transection was performed circumferentially. after dissection of rectococcygeal muscle at o'clock and rectourethral muscle in the anterior wall, circumferential sharp dissection within the holy plane was performed. dissection proceeded between the endopelvic fascia and the prehypogastric nerve fascia in the posterior plane, between the denonvilliers's fascia and the anterior mesorectum in the anterior plane, and between pelvic nerve and the mesorectum with recognition of the neurovascular bandle in the lateral plane. then the dissection connected to the abdominal plane via laparoscopic team with working together until tme completed. results: tme completion performed in ( . %) patients. thirty five ( . %) patients had negative of circumferential resection margin. mean of tme completion time and blood loss were min and g, respectively. one ( . %) patient had an intraoperative complication and ( . %) patients had postoperative complications. no other complications occurred. the length of hospital stay was days. conclusions: tatme within the holy plane on based on embryology is a safe and feasible procedure for rectal cancer. abstract: acromegaly is a debilitating condition marked by excessive production of growth hormone. this leads to disfiguration, cardiopulmonary complications, and increased risk for cancer. with up to a two-fold increased risk of developing colon cancer and worse prognosis for diagnosed patients, earlier and more frequent screening has been recommended. we present a case of a -year-old hispanic male with acromegaly who presented to our hospital with hematochezia and weight loss. a near-obstructing rectal adenocarcinoma with metastasis to the liver was discovered. after completing neoadjuvant chemoradiotherapy, he underwent laparoscopic low-anterior colon resection and simultaneous open hepatic trisegmentectomy. in this case report, we review the literature and current guidelines in screening this high-risk group of patients. introduction: in this study, we discovered that in cme for laparoscopic right hemi-colectomy starting at the ileocolic vessel and proceeds along the superior mesenteric artery (sma) achieved a better oncologic outcome compared with the conventional ones proceeding along the superior mesenteric vein (smv). methods and procedures: patients admitted to a shanghai minimally invasive surgical center were included from september to january and were randomly divided into two groups: study group (n = ) and conventional group (n = ). operation time, blood loss during surgery, liquid intake time, postoperative hospital stay, postoperative complications within days after surgery, specimen length, and number of lymph nodes harvested as well as the positive lymph node rate were observed and studied. results: there was no statistical difference between the two groups with the exception of number of lymph node dissected and the positive lymph node rate for stage iii colon cancer. the study group had more lymph node retrieved and also a higher positive rate compared with the conventional group. the mean number of lymph node retrieved of study group was . ± . , while the conventional group was . ± . (p. ). and the positive lymph node rate for study group was . %, the conventional group was . %. conclusion: when performing the laparoscopic right hemi-colectomy, dissecting the lymph node along with the left side of sma could be achievable and there were no differences of surgical outcomes compared with the conventional ways, while there was a higher number of lymph nodes dissected and positive rate probably leading to a better oncologic outcome. aims: we describe laparoscopic surgery for rectal cancer using needlescopic instruments performed at our department. methods: from to , cases of rectal cancer underwent surgery using needlescopic instruments: cases at rectosigmoid colon, at upper rectum, and at lower rectum. an umbilical camera port ( -mm) and two needlescopic instruments (endorelieftm) were directly punctured into the assistant surgical site. we started with port sites. in low rectum cancer cases, we kept the good pelvic visualization to lifting the peritoneum of the bladder onto the ventral side using the lone star retractor staystm. results: the median age was years ( - years), with males and females, and body mass index was . kg/ m ( - kg/m ). anterior resection was performed in cases, low anterior resection in cases, intersphincteric resection in cases, abdominoperineal resection in cases, hartmann's procedure in cases, and lateral lymph node dissection in case. in addition, one case of t b (bladder) was converted from laparoscopic to open surgery. however, there were no cases in which needlescopic instruments were replaced with conventional forceps. moreover, intraoperative complications related to the forceps were not observed. conclusions: in rectum cancer surgery, needlescopic instruments leave a small postoperative wound; healing is rapid and the cosmetic result is excellent. surgical safety is comparable to that using conventional forceps. there is no problem with the rigidity of needlescopic instruments. however, where the shaft is curved, operative control requires attention to mobility and directionality. in low rectum surgery, use of needlescopic instruments is limited due to the curvature of the shaft during the dissection of the anterior rectum wall, but it is possible to maintain a good field of view by using auxiliary equipment. therefore, more cases could be considered for surgeries using needlescopic instruments with the help of auxiliary equipment. introduction: anastomotic leaks are devastating complications of colorectal operations that lead to significant morbidity and potential mortality. inadequate tissue perfusion is considered a key contributor to anastomotic failure following colorectal operations. currently, clinical judgment is the most commonly used method for evaluating adequate blood supply to an anastomosis. more recently intraoperative laser angiography using indocyanine green (icg) has been utilized to assess tissue viability, particularly in reconstructive plastic surgery. this technology provides a real-time evaluation of tissue perfusion and is a helpful tool for intra-operative decisions, particularly in deciding to revise an intended colorectal anastomosis. our study aimed to determine if there is a statistical significance in colorectal anastomotic leak or abscess rate using icg compared to common clinical practice. methods and procedures: patients undergoing left-sided colorectal operations, between march and february , were retrospectively reviewed. patients' colorectal anastomoses were evaluated using icg angiography (icga) to qualitatively assess tissue perfusion (icg group). peri-operative and post-operative outcomes, including anastomotic leak and abscess rates, were compared to patients who had colorectal operations without icga (control group). the primary outcomes of intra-abdominal leak rate and intra-abdominal abscess rate were compared using exact chi-square tests. the secondary outcomes of -days or return, mortality, and readmission rate were compared using chi-square tests. all statistical analyses were performed using sas software. results: two leading indications for surgery included malignancy (n = ) and diverticulitis (n = ). the majority of patients either had a low anterior resection (n = ) or sigmoidectomy (n = ). all operations were primarily minimally invasive. no statistically significant difference was seen between the two groups in regards to patient demographics, rate of proximal diversion (p = . ), and splenic flexure mobilization (p = . ). patients in the icga group were more likely to have high ima ligation than in the control group ( . % vs. . %, p-value. ). of the icga group, of the patients underwent additional colonic resection while of the did not undergo additional colonic resection. there was no statistically significant difference in primary or secondary outcomes between the two groups. conclusion: icg angiography has become a helpful adjunct in determining adequate perfusion to an intended colorectal anastomosis. this data is unable to support any difference in patient outcome utilizing this technology over surgeons' visual and clinical assessment. our results may contribute to larger studies to determine if there is a true difference in anastomotic leak or abscess rate using this technology. objective: to investigate the feasibility and surgical strategy of complete mesocolic excision (cme) with completely medial access by "page-turning" approach (cmapa) for the laparoscopic right hemi-colectomy. the cmapa is a modified medial approach of cme, which focus on the exploration of surgical plane instead of the recognition of vessels. surgical procedures: ( ) start point: the anatomy projection of ileocolic vessel; ( ) expose the whole trunk of smv to the level of inferior edge of pancreas before ligating any branches, for the purpose of high tie and verifying their location; ( ) enter the intermesenteric space (ims) and right retrocolic space (rrcs) with cranial and right extension through transverse retrocolic space (trcs); ( ) complete mobilize the mesocolon and remove the tumor en-bloc. see figure ? . clinical outcome: from september to march , there were patients underwent cmapa in shanghai ruijin hospital. the average operation time was . ± . minutes, average blood loss was . ± . ml, number of lymph node was . ± . , average specimen length was . ± . cm, flatus time was . ± . days, fluid intake time was . ± . days and average hospital stay was . ± . days. the overall complications rate was . % ( / ). compared to traditional medial approach of cme performed in our center, the blood loss, operation time and hospital stay were significantly reduced by performing cmapa for laparoscopic right hemi-colectomy. conclusion: the advantage of the cmapa ( ) to avoid the laparoscopic "leverage effect" and "tunnel effect". ( ) to make the branches of superior mesenteric vessels more easily recognized. ( ) to offer surgeons an alternative route entering the trcs, ims and rrcs. ( ) to avoid repetitive flipping of the colon complying with the "no touch" principle, and to lower the requirements of assistants. figure : anatomy and surgical planes concerning cmapa. aim: we have reported a possibility of "one-stop shop" simulation for liver surgery by mri using gadoliniumethoxybenzyl-diethylenetriamine pentaacetic acid (eob-mri) (emerging technology, sages )., which is characterized by ( ) one-time examination, ( ) no-radiation exposure, ( ) demonstration of liver vasculatures including biliary tract, ( ) diagnosis of tumors, ( ) volumetry and ( ) estimation of liver functional reserve in each segment. the aim of this study is to investigate usefulness of "one-stop shop" simulation for liver surgery using eob-mri. methods: accuracy of liver vasculatures: d-reconstruction of dynamic eob-mri imaging was done by synapse vincent software (fujifilm medical co., ltd., japan), using a manual tracing method. visualization of hepatic vessels in eob-mri was compared with that in dynamic ct in patients. assessment of liver functional reserve: the standardized signal intensity (si) of each segment was calculated by si of each segment divided by si of the right erector spine muscle. the standardized total liver functional volume (tlfv) was calculated by ∑ [k= to ] (standardized si of segment (k) volume of segment (k)) divided by body surface area. the following formula of resection limit was established using normal liver cases ( % of the liver is resectable) and unresectable cirrhotic patients such as recipients of liver transplantation ( % of the liver is resectable). the estimated resection limit (%)= % (the standardized tlfv of the patient - )/ , . this formula was validated using other patients who underwent hepatectomy. results: accuracy of liver vasculatures: the liver simulation by eob-mri succeeded in demonstrating hepatic vasculatures including biliary tract, diagnosis of hepatic tumors, and volumetry without any radiation exposure. regarding the vessel anatomy at hilar area, biliary tract was more clearly visualized in eob-mri. regarding the hepatic artery, right and left hepatic arteries were well visualized in all cases, however, small-sized middle hepatic artery was visualized in only one out of patients. assessment of liver functional reserve: as a result of validation of the patients, one patient having resection volume with over the resection limit died of liver failure, however, the other cases within their resection limits did not suffer from liver failure. conclusion: "one-stop shop" liver surgery simulation could contribute to safety of liver surgery such as laparoscopic hepatectomy, because of no radiation exposure, accurate assessment of anatomical variations especially biliary tract, and helping decision making of resection volume. showing key steps of the procedure to be viewed. the in-studio program was hosted by an education specialist from the science center and a surgical resident from our institution, with laparoscopic instruments available for manipulation by participants. participants then viewed a video highlighting the roles of all healthcare providers involved in the specialty to be featured, including nurses, physicians, dietitians, psychologists, technologists, etc. live questions and answers were then encouraged between students and surgeons during the surgery broadcast. the program also expanded from high schools to vocational-technical colleges and nursing schools. results: during the - academic year there were sessions presented to schools, with student participants. by the - year this increased to sessions presented to schools, with participants. in sum, throughout the first years of the program, there were schools attending, with a total of , participants. of polled high school participants, % of responders acknowledged considering a career in healthcare after this experience. conclusion: over years, our program has grown steadily in popularity such that schools from several counties attend and regularly return, and we have been asked to expand the program to create a surgical summer camp for students interested in science and technology. live broadcast surgery in an elective, minimally invasive format provides unique visibility and access to surgical procedures for student audiences and promotes future interest in healthcare careers. surg endosc ( ) :s -s p improving trainees' self-assessment through gaze guidance introduction: effective learning to become competent in surgery depends on a trainee's ability to accurately recognize their strengths and weaknesses. however, a surgical trainee's self-assessment is poorly correlated with expert assessment. this study aimed to improve self-assessment by the visual gaze guidance provided through telestration in laparoscopic training. we hypothesized that visual conveyance of where to look or perform actions on the laparoscopic video enhances the trainees' awareness of the gaps in their skills and knowledge. methods and procedures: a lab-developed telestration system that enables the trainer to point or draw a free hand sketch over a laparoscopic video was used in the study (fig. ). seven surgical trainees ( surgical fellow, research fellow, pyg- and pyg- ) participated in a counterbalanced, within subjects controlled experiment, comparing standard guidance with telestration-supplemented guidance. the trainees performed four laparoscopic cholecystectomy tasks -mobilizing cystic duct and artery, clipping the duct, clipping the artery, and cutting the duct and artery, on a laparoscopic simulation. performance assessment, adapted from the global rating scale (grs) instrument, was completed by the trainers and trainees at the end of each task. the mean self-assessment scores were compared with the trainers' scores by the linear mixed model, where the trainees' performance indicated by the trainers' scores was control. the assessment alignment was evaluated by spearman's rho. results: the trainers' scores were significantly lower than the self-assessment scores in the standard guidance, while the scores of the trainers and trainees were much more similar (fig. ) . the correlation between the trainers' and trainees' assessment in telestration guidance was high (r= . , p. ), compared to the standard guidance (r= . , p= . ). the correlation comparison for each grs criterion shows a significant increase (p= . ) in the assessment alignment for depth perception in telestration guidance (r= . , p. ), compared to the standard guidance (r= . , p= . ) (fig. ) . the visual gaze guidance improved the alignment of assessment between the trainer and trainees, especially for the assessment alignment in depth perception. for visual gaze guidance to become an integrated part of the training, further work needs to be conducted to understand how gaze guidance change the nature of the training process. applying to surgical residency: what makes the best candidates? yann beaulieu, beng, louis guertin, md, frcsc, ariane p smith, md, margeret henri, md, frcsc, facs; university of montreal objective: while quotas for canadian surgical residency programs are at their lowest point in ten years, the number of canadian graduating medical students is at an apogee. this year, only spots in surgical residency programs were available for students applying to carms. undergraduate medical students individually collect anecdotal information regarding what influences admission to their surgical subspecialties of interest, as scarce literature covers the topic. we thus surveyed surgeons and residents to analyze the relative importance of modifiable factors and innate attributes in the selection of new surgical residents. methods: an electronic survey was sent to all surgeons and surgical residents affiliated with the university of montreal. participants were asked to specify their surgical subspecialty, their status, their level of experience and whether they were an active member of a residency selection committee. the subjective importance of predefined application elements and candidate qualities was assessed using -point likert-type items. results: of the surgeons and residents to whom the survey was sent, ( . %) and ( . %) completed the survey. evaluations of elective rotations and evaluations of core rotations were considered very important by . % and . % of responders respectively. regarding letters of recommendation, the content was rated very important ( . %) more often than the notoriety of the author ( . %). networking with key surgeons was considered the least important element to prioritize with % of negative assessments. with regards to the fundamental qualities of surgical candidates, the extremes were "clinical judgement" with . % and "innate technical ability" with . % of responders rating them very important. no significant differences in responses were observed between staffs and residents, between members and non-members of selection committees, between different levels of surgical experience and between surgical subspecialties. conclusion: clinical judgement and performance in core and elective rotations along with strong personalized letters of recommendation should be prioritized by medical students aiming for a surgical career. kazuhiko shinohara, phd, md; school of health science, tokyo university of technology background and objective: many types of training devices had been proposed since the early days of endoscopic surgery. however, they are too expensive for daily training of novices. we developed a simple and economical training device made of frozen fruit and agar. material and methods: to make this device, g of agar powder was added to ml of boiling water and boiled for min. the solution was then poured into a stainless steel tray containing frozen blueberries and lychees and refrigerated for h. basic maneuvers required during endoscopic dissection and resection of a tumor with laparoscopic forceps and electrosurgical devices were then performed using this agar model in a conventional laparoscopic training box. results: using this model, endoscopic dissection and enucleation of a tumor with an electrosurgical device could be practiced repeatedly with minimal expense and preparation. background: situs inversus totalis (sit) is a rare congenital anatomy and a challenging condition for laparoscopic surgeries because standardized strategy to overcome such anatomical difficulties. mirror-reversed video images of laparoscopic surgeries for patients with normal anatomy could help to develop surgical strategies for patients with sit. we had a chance to evaluate this idea with a treatment of a patient of early gastric cancer, and describe the surgical results of the case. patient and methods: seventy-two-year-old women with a history of sit was referred to our department for the treatment of early gastric cancer, and laparoscopic distal gastrectomy with d + lymphadenectomy was scheduled. a video record of the same surgery for a patient with similar physical attribute performed before then was retrieved, and was edited with a computer into full length, totally mirror-reversed images of the surgery. designated operator and assistant simulated the operation using the video several times before surgery. results: laparoscopic distal gastrectomy was performed with d + lymphadenectomy while the operator was on the left side of the patient and the assistant on the other side, being opposite positions as usual. laparoscopic b- reconstruction was followed using "delta anastomosis" technique reported by kanaya et al. total laparoscopic procedures were completed with the operation time of minutes and the blood loss below measurable limits. no appreciable complications were observed after surgery and the patient was discharged on postoperative day . no recurrence of the disease was detected until years after surgery, conclusion: although further validation is unlikely because of a rare incidence of this anatomy, the same technique would be recommended for one of the preoperative preparations for similar cases. background: surgical simulation is thought to provide a basis for improvement of resident surgical skill training, in the safety of a simulation setting. it is unclear whether surgical skills learned in a simulation curriculum actually contribute to the improvement of surgical skills when transferred to the or. methods: a ten question online survey was sent to attending surgeons and residents. the questionnaire focused on domains: confidence, independence, transferable skills, improvement of skills/knowledge and time spent on the simulation curriculum. evaluation data was collected and anonymously analyzed. background: minimally invasive surgery poses a unique learning curve due to the requirement for non-intuitive psychomotor skills. programmes such as the fundamentals of laparoscopic surgery (fls) provide mandatory training and certification for many residents. however, predictors of fls performance and retention remain to be described. this single-centre observational study aimed to assess for factors predicting the acquisition and retention of fls performance amongst a surgically naïve cohort. methods: laparoscopically naïve individuals were recruited consecutively from preclinical years of a medical university. participants completed five visuospatial and psychomotor tests followed by a questionnaire surveying demographics, extracurricular experiences and personality traits. individuals completed a baseline assessment of the five fls tasks evaluated by fls standards. subsequently, participants attended a -minute training-course over week one and two on inanimate box trainers. a post-training assessment was performed in week three to evaluate skill acquisition. participants were withdrawn from laparoscopic exposure and retested at four onemonth intervals to assess skill retention. introduction: bipolar energy can cause thermal injury to adjacent organs when used improperly. sages fuse curriculum provides didactic knowledge on principles and best practices for safety, but there is no hands-on component to practice these skills. the objective of this study is to compare the effectiveness of the vest™ bipolar training module in addition to the fuse curriculum. methods and procedures: the study was a mixed design with two groups, control and simulation. after a pre-test that assessed their baseline knowledge, the subjects were randomized to two groups. both groups were given a min presentation, reading materials from the fuse manual and an online didactic module on bipolar energy. the simulation group also practiced on the simulator for one session that consisted of five trials on the effect of activation time on thermal damage and the importance of providing a margin of safety by sealing short gastric vessels. after one week the performance of both groups was assessed using a post-questionnaire. one week after the post-test both groups performed sealing of vessels on an explanted porcine mesentery with vessels perfused. their performance was videotaped and their activation times were recorded. a total safety score was calculated by assessing the proximity of the location of activation to the intestine by two independent raters. wilcoxon -signed rank and mann-whitney u tests were used to assess difference within and between groups. results: a total of residents ( in each group) participated in this irb approved study. median test scores for both groups increased (simulation, p= . and control, p= . ). no difference was found between the two groups in their pre-test (p= . ) and post-test (p= . ) scores indicating learning. the median total activation time for control group was higher ( . s) compared to simulation ( . s) but was not statistically significant (p= . ). there was a moderate agreement between two raters for margin of safety (kappa= . , p. ). total safety scores showed no difference between the two groups (p= . ). conclusions: subjects with simulation training had lower activation time compared to control. training for margin of safety requires more simulation refinement. small sample size and variations in the explanted models contributed to variability in data but even with small sample size, simulation training along with the fuse curriculum trended towards being more beneficial than the fuse curriculum alone. the general, that aims to build educational infrastructure and standardize training and education in laparoscopy throughout mexico. ilap participants engage in didactic and hands-on modules in educational theory, laparoscopic techniques, and simulation based education (sbe), and then develop and implement a -day sbe course for local trainees. the purposes of this study were to understand the existing educational environment at a single institution in mexico and measure the changes in perceptions, attitudes, and engagement in surgical education after an intensive training course. methods and procedures: all faculty and of general surgery resident participants completed a survey that contained items designed to assess the existing educational environment at a large, public hospital in mexico. using a -point likert scale, residents self-rated the quality of faculty feedback and the learning environment within their institution ( =strongly disagree, = neutral, =strongly agree). faculty rated their perceptions of the same educational themes. upon completion of a faculty-lead simulation course, residents rated the educational environment during the course. faculty provided additional qualitative feedback. descriptive analyses were performed. irb-exemption was obtained through lurie children's hospital. results: discordance existed in perceptions of the existing educational environment. the greatest disparity between resident and faculty perceptions included "faculty provide sufficient feedback in the operating room" ( % vs. %), "faculty promote an active learning environment" ( % vs. %), and "residents may ask questions without fear of negative evaluation" ( % vs. %). faculty and residents agreed with "residents are sometimes afraid to speak up in the operating room for fear of retaliation" ( % each). post-course evaluations (n= ) revealed universal improvement in all educational themes during the simulation course. qualitative feedback revealed most faculty plan to incorporate open communication and safe learning into their practice. residents were equally positive, with % optimistic that they will see changes within the educational environment. conclusions: significant discordance exists in resident and faculty perceptions of the educational environment at a large teaching hospital in guadalajara, mexico. after participation in the ilap course, residents noted demonstrable change in the faculty approach to education and feedback, and both faculty and residents expressed optimism for increased engagement in education. the immediate successes of the ilap initiative should be followed over time, as the ultimate measure of success is sustainability and scalability throughout mexico. background: laparoscopic anterior resection is technically challenging and the learning curve is long. well-designed formative assessments can provide trainees effective and constructive feedback, an important element in efficient learning. previously reported assessments for laparoscopic colorectal procedures were developed for summative assessment. we aimed to develop a formative assessment tool to evaluate competence and provide trainees with effective feedback in laparoscopic anterior resection. methods: the assessment tool was developed by an expert panel from mcgill university affiliated hospitals. the procedure was deconstructed into a series of sequential steps including general domains, surgical principles, injury prevention and technical skills specific to laparoscopic anterior resection. the tool contains discrete items with global rating scales for each step of the operation; each domain was scored using a -point likert scale, with anchors for scores of , and . each operation was assessed through direct observation in the operating-room by the attending, a trained observer, and trainees themselves. intraclass correlation coefficients (iccs) were calculated to estimate interrater reliability for ( ) attending surgeon and trained observer, ( ) attending surgeon and self-assessment, and ( ) trained observer and self-assessment. internal consistency was measured using cronbach's alpha. comparison between training levels was done using mann-whitney u-test. the global operative assessment of laparoscopic skills (goals) was also used to assess trainees' general laproscopic skills. spearman's correlation was used to determine association between goals and this procedure-specific tool. overall usefulness of this tool was evaluated using a cm visual analog scale. results: in this pilot study, fourteen operations, performed by experienced surgeons and trainees were assessed. the icc between ( ) attending surgeon and observer was . ( % ci . to . ) ( ) observer and self-assessment was . ( % ci . to . ), and ( ) attending surgeon and self-assessment was . ( % ci - . to . ). the internal consistency of the items was excellent (cronbach's α= . ). there was a significant difference in median total score between experienced surgeons and trainees ( . ± . vs. . ± . ; p= . ). there was strong correlation (r= . ) between goals and this procedure-specific score. overall usefulness of this assessment tool was rated as . ± . . all assessments were completed in about minutes. conclusions: we present a new procedure-specific formative assessment tool for laparoscopic anterior resection and provide preliminary evidence of its reliability and validity. this formative assessment tool could be used for constructive feedback and tracking performance in competencybased surgical training. cullen introduction: one of the key challenges to the proliferation of endoscopic submucosal dissection (esd) in the west has been a lack of training platforms. therefore, the virtual endoluminal surgery simulator (vess) is being developed as a training tool for esd. the aim of our study is to inform the design of vess using cognitive task analysis (cta), which is a human factors engineering framework to describe practitioners' mental models and cognitive processes and incorporate insights into the simulator's design. methods and procedures: cta-based interview questions were developed to probe the cognitive challenges and strategies employed at each stage of the esd procedure. six esd practitioners were interviewed for varying lengths of time. two of these interviews were conducted simultaneously during an observation of a training workshop where the cta participants were instructors (total observation time was five hours, and interview time was * minutes). another interview was conducted during observation of esd procedures (total observation time was hours, and interview time was * minutes). participants had varying levels of experience in esd, with of them being 'super-experts' (exclusively esd exponents), an 'expert' and a fellow. a cta of the data is currently being conducted to systematically inform design of functionalities in the simulator. results: analysis of our data highlights a few prominent themes at each stage of esd: goals, challenges (e.g., avoiding perforation of muscularis); points of decision-making (e.g., partial or full incision for boundary demarcation); skills involved (e.g., dissection); and ambiguity (e.g., unclear lesion boundaries). participants also described risks associated with each stage of esd and strategies to prevent or overcome the same. conclusions: qualitative data for a cta were collected through observations and interviews of esd practitioners. preliminary analysis has indicated prominent themes to consider in the design of the training simulator. the next step in the study is to conduct a full-scale cta of esd based on the current data. the ultimate benefit of the cta would be to incorporate the results into informing the design of vess in a way that is compatible with the mental models of esd trainees, thus enhancing the fidelity and effectiveness of the simulator. background: colonoscopy is an important diagnostic and therapeutic procedure in the management of colonic disease; achieving competence during residency is an integral part of performing high-quality colonoscopy in-practice, regardless of specialty. there is debate and controversy however, regarding what, if any, number of procedures achieves said proficiency. furthermore, there is significant heterogeneity in the current guidelines and studies published to-date on the definition of competence in colonoscopy. objective: to determine individualized learning curves as an alternative to 'number of procedures' for assessing colonoscopy competence. methods and procedures: this is a multi-institutional prospective cohort study involving eleven surgical trainees (novice endoscopists). the main outcome, colonoscopy competence, was assessed by determining the independent colonoscopy completion rate (iccr), the number of procedures required to reach % independent colonoscopy completion and polyp detection rate. individual and overall iccr were calculated using moving average analysis. conclusions: while a benchmark for a minimum number of procedures may be necessary to allow supervisors to adequately assess performance, it is difficult to determine what number is optimal. there appears to be significant heterogeneity in both overall number of colonoscopies completed by each resident, as well as the mean iccr and the number of procedures required to reach the current benchmark for competency. the use of learning curves allows real-time tracking of progress and training tailored to the individual, as we move forward in the era of competency-based medical education. background: with the growing popularity of robotic-assisted surgery, new methods for evaluation of technical skill are necessary to determine when a surgeon is qualified to perform an operation independently. current evaluation methods are limited to point likert scales which require a degree of subjective scoring. surgeons in training need an objective method of evaluation to view progress and target areas for improvement. one method of objectively evaluating surgical performance is a cumulative sum control chart (cusum). by plotting consecutive operative outcomes on a cusum chart, surgeons can view their learning curve for a given task. another method of objective evaluation is the dv logger®, or "black box," which records objective measurements directly from the da vinci® system. methods: we followed two hpb fellows during dry lab simulation of robotic-assisted hepaticojejunostomy reconstructions using biotissues to model a portion of a whipple procedure. we simultaneously recorded objective measurements of dexterity from the da vinci® system and performed cusum analyses for each procedural step. we modeled each variable using machine learning (a self-correcting and autoregressive modeling tool) to reflect the fellows' learning curves for each task. statistically significant objective variables were then combined into a single formula to create an operative robotic index (ori). results: variables that significantly improved over the course of the simulation included completion time (p= . ), economy of motion in arm (p= . ), number of times head was removed from the console (p= . ), total time left master manipulator was active (p= . ), total time right master manipulator was active (p. ), and total time that any arm was active (p\ . ). the inflection points of our cusum charts and plots of objective variables both showed improvement in technical performance beginning between trials and [ fig. and fig. ]. the operative robotic index showed a strong fit to our observed data and improved with additional trials (r = . ). [ figure ]. conclusions: in this study we identified objective variables recorded by the da vinci® system which correlated with the technical dexterity of fellows during a robotics dry lab. we broke a complex procedure down in stepwise fashion with cusum analyses to determine targets for improvement. using variables which correlated with the improved performance of the fellows, we effectively modeled the learning curve with the creation of an operative robotics index (ori). this study successfully models the learning curve of novice robotic surgeons using a novel combination of objective measures. georg wiese, md, paula veldhuis, steve eubanks, md, facs, scott w bloom, md, frcsc, facs; florida hospital institute for surgical advancement introduction: robotic surgery is a specialized skill which requires time and resources to master. in a general surgery residency program that seeks to train competent surgeons in both open, laparoscopic and endoscopic techniques it is difficult to see where adding robotic training will be of benefit and at what cost this will be to the remaining surgical skills. we therefore sought to ascertain robotic surgery's current role in the training of new general surgeons by soliciting the opinions of current general surgery program directors on the role of robotic surgery at their respective institutions. methods: an irb approved survey was created and sent to general surgery program directors across the country to assess how robotic surgery training is being integrated into current surgical training. the survey was sent via email to publicly available email addresses from the acgme website of program directors. it was voluntary in nature and consisted of questions regarding current status of robotic training in residency as well as future goals. results: overall response from our pd survey were at % of the surgical programs with addresses available via acgme, though responses continue to be submitted at the time of this abstract. approximately % of all respondents are from independent, university based programs. % felt that robotics was an emerging skillset important for residents to master versus % feeling that it was more appropriate for fellowship. all respondents noted that robotic surgeons were present at their institution, % within the core faculty, and % indicated that they were actively recruiting robotically trained surgeons. additionally, % of programs indicated that residents were exposed to robotic surgery, % of these on core general surgery rotations. % of respondents indicated that they had a formal robotic training curriculum with % of programs taking measures to integrate robotics into the future curriculum though % lacked specific milestones for such training. finally, opinion was evenly divided among respondents as to whether one could sign off on residents to perform robotic assisted cases upon completion of pgy year with % agreeing with that statement and the remainder indicating some additional training would be necessary. conclusions: our study highlights the emerging field of robotic assisted mis surgery and its increasing role in residency training. it is evident from the data, that robotic surgery is a growing part of residency experience. importantly, however, milestones were significantly lacking for determining resident progress in robotic training. introduction: in chile, medical students have the opportunity to undertake a month-long medicine elective (me) in a community hospital, primary care center or emergency department within the country at the end of their first clinical year. due to the lack of opportunities to practice suturing in the first years, students usually do not have an optimal performance in this type of medical procedure during the me. simulation training programs in suturing improve technical skills, selfconfidence and patient safety in the medical internship. the objective of this study is to evaluate the impact of implementing a simulated suture training program earlier in the medical curriculum, before the me. methods: we conducted a prospective, randomized controlled trial with medical students at the end of their first clinical year. they were randomized into two equal groups. the intervention group received an intensive suture training program consisting in one theory class, four practical sessions and effective feedback from an expert surgeon. the control group did not receive training, remaining with the classic opportunistic learning approach during the me. after the me, all students undertook an electronic survey. statistical analysis was performed on the answers of both groups. per protocol analysis was applied. results: there were no statistical differences between groups in terms of age and sex. four students did not complete the training program. one student in the control group did not reply to the survey. higher self-confidence with regards to suturing was reported in the intervention group in comparison with the control group [ / ( %) vs / ( %), p, ]. also, a greater student desire to carry out suture-related procedures was reported in the intervention group than the control group [ / ( %) vs / ( %), p, ]. in addition, a lower rate of overseeing physician intervention was reported in the intervention group [ / ( %) vs / ( %), p, ] ( table ) . a greater number of patients requiring sutures were treated by the intervention group than the control group, with a median of patients ( - ) against ( ) ( ) ( ) ( ) . the intervention group performed a higher number of sutures with a median of ( - ) vs ( - ), with a statistically significant difference (p, ) in both cases (fig. ) . conclusion: a simulated suture training program prior to the me generates a positive impact on medical students by improving self-confidence and desire to attend patients that require sutures. this leads to a higher rate of both exposure to suture techniques and suture execution. introduction: measuring performance in the operating room (or) is challenging. performance is a multifaceted construct a complex interaction of many behaviors and actions that reflect an individual's knowledge and skill. no assessment tool to date provides an expertise-based, comprehensive evaluation of the various aptitudes necessary to excel in the or, especially with respect to advanced cognitive skills. using qualitative methodologies, we previously defined behavioral themes that guide surgeons' behaviors, decisions, and actions, within a universal framework of domains that reflect intra-operative performance. the purpose of this pilot study was to use this framework to derive a comprehensive assessment tool and to obtain evidence for its validity as a measure of intra-operative performance. methods: an assessment tool was developed by a panel of surgeons and surgical trainees based on the five-domain model of intra-operative performance: ) psychomotor skills; ) declarative knowledge; ) interpersonal skills (two items); ) personal resourcefulness, and ) advanced cognitive skills (ten items). all items were rated on an ordinal scale of (inadequate) to (expert) and equally weighted. surgical residents and surgeons from a single academic center were evaluated on their performance during standard general surgery operations, for example, open inguinal hernia repair and laparoscopic cholecystectomy. for residents, there were evaluators -the attending surgeon and an observing surgeon. attending surgeons evaluated their own performances and were also assessed by observing surgeons. internal consistency, inter-rater reliability, and correlation of total scores with training level (junior residents, senior residents, staff surgeons) were calculated. likert scale questionnaires were administered to evaluate the tool's usability, feasibility, and educational value. results: fifteen subjects ( junior residents, senior residents, surgeons) participated. the total score on the assessment demonstrated significant differences between training levels ( figure) . inter-rater reliability was high (interclass correlation coefficient= . ), as were internal consistency between each domain score (cronbach's alpha= . ), internal consistency amongst items in the advanced cognitive skill domain (cronbach's alpha= . ), and internal consistency amongst items in the interpersonal skills domain (cronbach's alpha= . ). all assessments required less than five minutes to complete. overall, evaluators agreed that the assessment tool was easy to use, was comprehensive, and should be used routinely throughout training to track performance and provide formative feedback. conclusion: in this pilot study, we developed a comprehensive assessment tool for intra-operative performance and provide preliminary validity evidence for the score. surg endosc ( ) introduction: the purpose of this study was to evaluate the validity of our developed system for assessing suturing skills in laparoscopic surgery (fig. ) . we have updated numbers of participants and a comparison method compared with the last year report. methods and procedures: fig. shows our developed computerized system for objective assessment of suturing skills by using a laparoscopic intestinal suturing model, e-lap. the system includes a new artificial intestinal model that mimics living tissue and pressure-measuring and image-processing devices. each examinee performs a specific skill using the artificial model, which is linked to a suture simulator instruction evaluation unit. the model uses internal air pressure measurements and image processing to evaluate suturing skills. five criteria, scored on a five-grade scale, were used to evaluate participants' skills ( fig. ) . the volume of air pressure leak was determined by the volume of air inside the sutured artificial intestine. for example, for the criterion "air pressure leakage", the approximate midpoint of the acceptable range was grade . values lower than the minimum acceptable value received lower grades and those above the midpoint of the acceptable range higher grades. we enrolled surgeons who participated a simulator competition event at the th annual meeting of the japan society for endoscopic surgery (jses houston methodist hosptial, baylor college of medicine introduction: the sages flexible endoscopy course for minimally-invasive surgery (mis) fellows has been shown to improve confidence and skills in performing gi endoscopy. this study evaluated the long-term retention of these confidence levels and investigated how fellows have changed practices within their fellowships as a result of the course. methods: participating mis fellows completed surveys six months after the course. respondents rated their confidence to independently perform sixteen endoscopic procedures ( =not at all; =very). while the pre-and post-course surveys identified anticipated endoscopy uses and barriers to use, the -month follow-up survey evaluated actual usage and barriers to use in each fellow's practice. respondents also noted participation in additional skills courses and status of fundamentals of endoscopic surgery (fes) certification. comparison of responses from the immediate postcourse survey to the -month follow-up survey were examined. mcnemar and paired t-tests were used for analyses. results: twenty-three of ( %) course participants returned the -month survey. % had passed the fes skills examination and % had attended another flexible endoscopy course. no major barriers to endoscopy use were identified. in fact, fellows reported less competition with gi providers as a barrier to practice compared to their original post-course expectations ( % versus %, p. ). in addition, confidence was maintained in performing the majority of the endoscopic procedures, although fellows reported significant decreases in confidence in independently performing snare polypectomy (− %; p. ), control of variceal bleeding (− %; p. ), colonic stenting (− %; p. ), barrx (− %; p. ), and tif (− %; p. ). fewer fellows used the gi suite to manage surgical problems than was anticipated post course ( % versus %, p. ). fellows without fes certification reported loss in confidence to independently perform barrx (− %; p. ) and colonic stenting (− %; p. ), and also a % decrease in the use of gi suite to manage surgical problems (p. ) fellows who passed fes noted no significant loss of independence, changes in use, or barriers to use. % of fellows made additional partnerships with industry after the course. % stated flexible endoscopy has influenced their post-fellowship job choice. % would recommend the course to other fellows. the sages flexible endoscopy course for mis fellows results in long-term practice changes with participating fellows maintaining confidence to perform the majority of taught endoscopic procedures six months later, and over % reporting that flexible endoscopy influenced their career choice. additionally, fellows experienced no major barriers to implementing endoscopy into practice. the materials and methods: at our center, we formulated a laparoscopic mentorship program where a senior consultant was paired with a particular trainee resident for a period of weeks. consultants & residents were a part of the study. the or schedules were rearranged to accommodate these pairs. an evaluation of the residents' views was performed prior to the study and once at its completion, using a simple questionnaire with each parameter scored between & . results and discussion: continuous, consistent evaluation by a consultant over an extended period of time allowed them to assess their assigned resident's laparoscopic skill set. all pairs observed an increased frequency of errors being noticed & improved upon. the consultants stressed upon shedding undesirable operative habits. there was a significant improvement in residents' scores at the end of the short study. conclusion: we found that the short-term mentorship program was easy to incorporate within our or schedule and was well received by the participants. continuous short rotations under senior consultants appear to allow residents to not only fully observe and imbibe correct operative techniques, but also helps shed unfavorable habits. we are currently amid the second cycle of our study & looking forward to the results at the end of this academic year. introduction: colorectal cancer is one of the most common cancers in the united states. endoscopic submucosal dissection (esd) is an emerging minimally invasive technique that allows complete en-bloc resection and a much lower recurrence rate at long-term follow-ups. however, performing colorectal esd is technically demanding since the colorectal wall is thin and constantly moving, and potentially higher rates of complications (e.g., bleeding and perforations). hence, an adequate training for colorectal esd is needed to acquire basic proficiency with minimum complications. objectives: a virtual reality (vr)-based simulator with visual and haptic feedback for training in colorectal esd is being developed, which the aim to allow trainees to attain competence in a controlled environment with no risk to patients. in this work, a newly developed application of the virtual simulator that promotes the endoscopists to perform and assess technical skills in esd is developed. training tasks are built based on physics-based computational models of human anatomy with tumors. methods: the main modules of the vr-based simulator for colorectal esd involve: ( ) rendering; ( ) haptic interface; ( ) physics-based simulation; and ( ) performance recording and assessment metrics. the rendering engine allows surgical tasks to be performed in the three-dimensional virtual environment. haptic feedback mechanisms allow users to physically feel the interaction forces. physics-based simulation technologies are employed to enable the complicated simulation for performing virtual surgical tool-tissue interactions. the simulator can also collect learners' performance data to offer feedback based on the built-in metrics. results: four training tasks involving marking, injection solution, circumferential cutting, and submucosal dissection are designed to practice skills with different surgical tools. the marking task aims to identify the lesion. the injection solution task minimizes the risk of bleeding and perforation to protect the muscularis. in the circumferential cutting task, the objective is initial incision of the lesion with the surgical tools. the objective of the dissection task is to remove the tumor from the connective tissue of the submucosa under the lesion. conclusions: the vr-based simulator enables realistic esd tasks to provide a possibility for developing, validating and objectively evaluating the performance metrics in colorectal esd training, and offers an opportunity to rise up the learning curve before application to patients. background: the virtual translumenal endoscopic surgery trainer (vtest) simulator is a virtual reality system that was designed to train the hybrid-notes technique. transfer of skill acquired while training on the vtest was measured in a near-real cholecystectomy procedure staged in the easie-r model. methods: sixteen medical students were divided randomly and evenly into groups: control, training. all subjects performed the cholecystectomy procedure on the vtest simulator to establish a baseline (pre-test). the training group received training sessions, over a period of consecutive weeks, consisting of trials per session or as many trials as can be accomplished in one hour, whichever was achieved first. at the end of the training period, all subjects performed one trial on the vtest simulator (post-test), and again to weeks later (retention test). two months after that, subjects performed the hybrid-notes cholecystectomy procedure on an easie-r model. performance with the easie-r simulator was video-recorded, and three tasks within the cholecystectomy procedure were isolated for evaluation: clipping, cutting, and dissecting the gallbladder. objective performance measures, such as time and error, were extracted from the videos by two independent reviewers, while subjective performance was scored by four expert surgeons who were blinded to the training conditions. expert reviewers used a modified version of the operative performance rating system by the american board of surgery and the objective structured assessment of technical skills (osats) tool. results: there was no difference in task completion time between the control and training groups, (t( )= . , p =. ) in the cutting and clipping tasks. however, there was a significant difference in the number of errors, t( )=- . , p=. . there was no difference in subjective performance between the training groups for the clipping and cutting tasks. in the gallbladder dissection task, however, there was a statistical significance in "instrument handling" based on one of the surgeons' ratings (t( )= . , p=. ), and a statistical significance in "time and motion" based on another surgeon's rating (t( )= . , p=. ). conclusions: results indicate that weeks of training on the vtest simulator did not allow the subjects to transfer their learned skills equally to the near-real environment, even though they retained the skills when tested for retention. this new insight suggests that modification of the training method for different types of surgical skills may be warranted to optimize their transfer to the real environment. examining conclusions: this study provides evidence to suggest that for bariatric surgeons, experience and skills acquired in performing non-bariatric surgery may not translate to improved outcomes in bariatric surgery. as seen in this study, improvement in bariatric surgical outcomes is likely more dependent on experience specifically performing bariatric procedures. as there may be no benefit acquired from performing surrogate procedures, this may have implications in the design of subspecialty training programs and for accreditation purposes. . a universally adjustable cellphone holder was used where smartphones could be placed inside the fls box in order to capture the task from a similar angle as the onboard camera. residents were able to use their own smartphones to record their performance on each of the five fls tasks in high definition (hd) quality. after each practicing session, they would upload their videos to a designated folder on a password-protected computer in the simulation lab. this folder was linked to a cloud-based storage system that fls instructor had exclusive access. the faculty was able to review each video in the next hours and provide immediate feedback to the residents via email, over the phone or in-person. the video library of performance also allowed the instructor to track the progress of the residents and whether they reached proficiency level in all five tasks to take the fls examination. this program was offered to all surgical trainees. results: utilization of simulation lab to practice fls tasks increased significantly across all postgraduate years after implementation of this model. six residents took the fls examination. the passing rate of the residents remained the same ( % before and after) but their scores in fls manual skills improved significantly compared to the group prior to implementation. the residents evaluated this change positively and reported that the use of videos and immediate feedback by faculty was a valuable intervention in their learning experience. conclusions: the smartphone cameras are readily available and can be used for telementoring. incorporation of telementoring in standard proficiency based fls training can promote self-directed learning and improve the access to experts for immediate feedback as a crucial element of effective training in acquisition of laparoscopic skills. background: it is important that making individual procedures a language, and an objective qualitative evaluation for the laproscopic training. recently, task training and the sham operation using the virtual simulator are carried out for medical students as the basic laparoscopic maneuver training, but there are few reports of objective qualitative evaluation for the training. in this study, we investigated rubric evaluation as the qualitative evaluation for laparoscopic training. materials and methods: one hundred and six students in th grade of tokushima univ. were participated. basic laparoscopic task training (gummy band ligation, beads transfer, delivery of beads, gauze excision) with training box and sham laparoscopic cholecystectomy with virtual simulator were performed. task execution time and rubric evaluation which includes the evaluation standard that became a language for each maneuver were performed before and after basic task training and sham operation. the group who are bad at laparoscopic maneuver was decided by time exceeded in tasks more than two from before practice. relationship between the group who are bad at laparoscopic maneuver and the group which self-evaluation was higher in a rubric evaluation was investigated. results: in basic task training, average task execution time in all students was shortened after practice compared with before practice, but investigated individual, students exceeded in more than two tasks. rubric evaluation in basic task training showed no difference between self-evaluation and evaluation by tutor before and after practice. in sham laparoscopic cholecystectomy, all students and tutor showed high score by rubric evaluation after practice compared with before practice. some students showed higher score than tutor, especially in part of extension of operation field by elevation of the gall bladder, exposure of triangle of calot, and exposure of cystic duct. students who showed high score by self-evaluation in many maneuver of sham laparoscopic cholecystectomy also exceeded in more than two basic tasks. conclusions: as rubric evaluation showed the point of the maneuver is made a language definitely, it was useful for an objective qualitative evaluation for laparoscopic training. pre introduction: bariatric surgery candidates have the opportunity to research bariatric surgeons and hospitals prior to scheduling their elective surgery. pre-operative information sessions are important tools for bariatric surgeons to provide patient education while increasing their patient population. online education is becoming increasingly popular, but its utility over in-person education is uncertain. our objective was to compare patients attending the two most commonly used educational formats: online (webinars) and in-person (seminars) and determine which were more likely to undergo bariatric surgery. methods: we conducted a retrospective cohort study of , patients who attended pre-operative information sessions from january to december by reviewing data maintained by the obesity, prevention, policy and management (oppm) database from our institution. the patients were divided into two groups: those who attended an in-person session (n= ) and those who attended an online session (n= , ). the proportion of patients who went on to have bariatric surgery was compared between the two groups. to categorize the study sample, patient demographics, surgeon providing the information session, and procedure performed were compared between groups. multivariate logistic regression model was applied to compare the effectiveness of in-person session and online session. results: of , patients analyzed, % attended online information sessions ( % female, mean age ). the remaining % attended in-person information sessions ( % female, mean age ). analysis found that . % of patients who attended online information sessions went on to have a bariatric surgical procedure, while . % of patients who attended in-person sessions went on to have a bariatric surgical procedure. after controlling for differences in age and gender, results of multivariate logistic regression analysis indicate that patients who attended inperson sessions were % more likely to have a bariatric surgical procedure than patients who attended an online session ( introduction: knot security is the ability of knots to resist slippage as force is applied, and the optimal number of throws to ensure a secure knot improves efficiency and outcome. the literature on the accepted number of throws per type of suture material has been largely anecdotal, often referring to throws for silk, for polyglactin (vicryl), five for polydioxanone (pds), and six for polyproprolene (prolene). we report a pilot knot-tying study of four suture types to determine optimal numbers of throws. materials and methods: four senior general surgery residents (pgy- and above) and four attending surgeons participated. participants viewed a standardized instructional video and a one-handed knot-tying tutorial. they were instructed to tie one-handed knots, beginning each knot with two throws in the same direction, and square the third and subsequent throws in the opposite direction. each surgeon tied knots, using differenttypes of - suture material: silk, polyglactin, polydioxanone, and polyproprolene. suture types were evaluated using , , , or throws. the participants were randomized to both suture type and order of throw numbers. the knots were then tested on the f.a.s. t knot tester (sawbones, vashon island, wa) for slippage (insecure knot) or breakage (secure knot). generalized estimating equation (gee) analysis was used to determine optimal throw number. results: knots were individually tested on the knot tester for slippage and recorded as % slipped (see table) . the percentage of slipped knots varied by participant and ranged from to %. generalized estimating equation analysis suggested that the only significant variable when determining knot security was number of throws (p= . ), not suture type or participant training level. the optimal number of throws for - silk, polydioxanone, and polypropylene was five, whereas six throws was optimal for polyglactin. conclusion: knot security is dependent on the number of throws placed, and these optimal numbers were higher in our study than the commonly accepted number of throws. evaluation of take introduction: laparoscopic skills can be learned using portable simulators and these skills are transferrable to the operating room. several training regions within the uk have therefore developed and delivered home-based laparoscopic training programmes for junior surgical trainees. although performance improved in some, overall engagement has been poor. similar results have been observed in north america. the aim of our study was to uncover the reasons for poor engagement with home-based simulation with a view to developing a future, more successful, programme. methods: this was a qualitative study utilising focus groups. interviews were undertaken with key stakeholders involved in various laparoscopic home-based simulation programmes through the uk. training equipment comprised the eosim portable simulator paired with online training tasks. the tasks were similar to those used in the fundamentals of laparoscopic surgery programme (fls). basic metric feedback was provided (eg time to complete task). a total of individuals were interviewed, including surgical trainees, consultant trainers, training directors and programme faculty. this generated approximately hours of data which was coded using nvivo software. a basic thematic analysis was performed. results: trainees cited multiple competing professional commitments as a barrier to engaging with home-based simulation. they tended to focus on scoring 'points' which contributed toward career progression rather than tasks which were interesting, or associated with personal development. this approach is perpetuated by the surgical training system, which rewards trainees with points for publications and exams, but not for operative skill. this leads to conflict between trainers and trainees, the former expecting trainees to instead focus upon developing their technical abilities. trainees were unsatisfied with metric feedback and wanted individual feedback from consultant trainers (attending equivalent). trainees generally perceived consultants as lacking interest toward the programmes and training in general. however, some consultants were in fact unaware of the programmes being delivered and others felt lacking in confidence to deliver necessary training to trainees. conclusions: our findings are widely generalizable and have implications for any institution delivering a similar programme. as a means of improving engagement, the the inception of scheduled simulation study days, providing trainees with the opportunity for personalised feedback from consultants, has been suggested. equipping trainers with the necessary competencies to deliver training can be achieved by ensuring attendance at the necessary professional development courses. tackling the 'box ticking' culture is more challenging and may involve a move toward restructuring the current surgical training scheme. introduction: to provide evidence for the face and content validity of a hybrid active-shooter team training simulation and the impact of a hybrid curricular model on learner's engagement and performance. the following study was conducted because hospitals are increasingly threatened by active-shooter incidents, and no active and noticeable training is currently available to train hospital staff members. methods: thirty-five volunteers (medical students, residents and other allied health providers) from the university of minnesota affiliated medical centers were randomly selected and divided into control and experimental groups. the control group (n= ) was given a traditional lecture-style presentation. the experimental group (n= ) participated in the hybrid curriculum which included augmented reality, kinesthetic simulation, and debriefing components. following both curriculum styles, nasa task load index (tlx) surveys were completed by each group member. a final active shooter simulation experience was presented and evaluated by active-shooter trained raters using a checklist of critical actions from the department of defense. a post-simulation nasa tlx survey and post-test were provided. to assess face and content validation of a hybrid team-training simulation exercise to prepare healthcare personnel in the event of a hospital-related active-shooter crisis, a -point likert-scale survey determined the realism, utility, and applicability of this type of training while engagement and performance during the simulation were measured using a nasa-tlx survey and contrasted with the rater's evaluation. our study provided evidence to support the face and content validation of an active-shooter simulation team training curriculum as a useful adjunct to health care institutional safety planning. we demonstrated that this type of training requires an optimal level of cognitive activation to increases learner's engagement and performance. we concluded that the hybrid design of our curriculum was successful in delivering these optimal levels of cognitive stimuli by producing engaging team training simulation experience capable of motivating our learners to acquire the tactical skills and life-preserving behaviors consistent with better survival opportunities during a hospital related active-shooter crisis. the introduction: the virtual electrosurgical skill trainer (vest) provides surgeons and trainees with a hands-on approach to learning the best practices in electrosurgery. it is comprised of five modules covering tissue effects, stray currents, bipolar tools, monopolar tools and or fire safety. the module in this study teaches the origins of stray currents and shows the learner how they can cause damage to non-target tissues via direct and capacitive coupling. the aim of this study was to assess learning using the vest system. methods: the irb approved study followed a single group pretest-posttest design and was conducted at the sages learning center. thirty-eight subjects participated and out of these, % were attending surgeons while the rest were medical students, residents and fellows. % of subjects had prior fuse exposure, while the remaining had none. subjects were asked to complete a five-question multiple choice questionnaire before and after using the simulator. it assessed their knowledge in topics such as direct coupling, capacitive coupling and insulation failure. participants then used the simulator to complete three tasks. first, the subject used direct coupling to seal a vessel and observed the desired effects and potential pitfalls. in the second task the subject was immersed inside the peritoneal cavity and was directed to use the active electrode to observe how the activation of energy can cause capacitive coupling. in the third task the subject practiced evaluating the insulation of electrosurgical tools for defects. wilcoxon's signed rank test was used to differentiate between pre-and post-test scores, and the mann-whitney u test was used to differentiate between the groups of subjects as a function of fuse experience. results: the median score on the pre-simulator assessment was % and the post-simulator median score was % (p = . ). there was no statistically significant difference in pre-assessment scores between attending surgeons and the others (p= . ). subjects with prior fuse exposure scored significantly higher on the pre-module assessment compared to those that had no prior fuse exposure ( % vs %, p= . ). in the post-assessment their median scores were % and %, respectively (p= . ). conclusions: the vest simulator module successfully increased the overall participants' knowledge of coupling in electrosurgery regardless of level of surgical experience. participants with prior exposure to the fuse curriculum had increased knowledge on this topic at baseline as compared to participants without any fuse exposure. introduction: the objective of this study was to assess the reliability of a modified notechs rating scale for the evaluation of medical students' non-technical (nt) skills. the importance of physician nt skills for the safe care of patients is receiving increasing attention in the literature. tools to assess nt skills such as notechs that addresses communication, situation awareness, cooperation, leadership, and decision-making have been shown to be valid and reliable. despite its importance, the assessment of nt skills of medical students, our future physicians, has received little attention. methods and procedures: twenty-seven medical students participated in of acute care simulated scenarios, each approximately minutes long. video recordings of student performance were reviewed and assessed using a modified notechs rating tool adapted for these scenarios with input from a team of clinicians, nurses, and human factors specialists. the rating scale ranged from to , representing very problematic behavior (e.g., not vocalizing concerns or decision process) and representing model behavior (e.g., identifies future problems and remains calm to unexpected events). two reviewers rated all videos independently on the notechs domains and specific subscales. student scores in each nt skill domain and interrater reliability were assessed. results: a summary of the scores of each notechs domain is shown in table . the highest overall average score of a participant was . while the lowest was . . the intra-class correlation (icc; two-way random model) was . , and the cronbach's α coefficient was [ . . the lowest icc agreement was in the situation awareness domain ( . ) while the highest agreement was in leadership ( . ). conclusion: medical student nt skills during acute care simulated scenarios vary significantly using a modified notechs assessment. this newly developed tool provides a framework for educators to evaluate medical students' nt skills during simulation training. it further identified domains where students scored lower, such as situation awareness, and could be targeted for education. the moderate icc, between the . - . range, shows that further refinement of the tool is needed to reliably assess the constructs. future steps to obtain validity evidence include additional raters and applying the tool in non-simulated settings. introduction: a general misperception of the real concept of robotic surgery seems to be revealed in our clinical practice. despite its introduction almost years ago, robotic surgery is still related to many myths and beliefs. before designing a trial to see if these false awareness could impact on outcome, we measured this misperception by a survey. moreover we tested if medical school is able today to give to the future doctors a necessary knowledge about robotic surgery. with the same survey we explore the feelings about the introduction of the artificial intelligence in medicine and the perception of the consequences of a larger use of technology in medicine. methods and procedures: a multiple choice survey was designed and anonymously administered via the platform surveymonkey (http://www.surveymonkey.com). a total of questions were selected from the research team and included in the survey. the questionnaire was divided in three parts: the first was to get information on participants' population; the second asked specific questions about robotic surgery; the third focused on technology use in medical education. results: we received and analyzed questionnaires, of which totally filled. many undergraduates consider robotic surgery as "experimental", will prefer open surgery on themselves and see a risk for robotic surgery in damaging the patient-surgeon relationship. this situation is better for medical students, but still a great diffidence were encountered. % of ug consider robotic surgery as "experimental" vs only . % of ms (q ). most thought robotic surgery had been used for only years or less (q ). . % of ug and . % of ms gave the right answer (p=. ). almost % of ug see robotic surgery as a risk in damaging the patient-surgeon relationship. this is not seen among ms (q ) (p=. ). % of ug are fearful of robots used to operate them. this fear is significantly reduced among medical students (p=. ). ug were less familiar with the indications and uses for robotics. ms gave a correct response more frequently (q , . % vs . %, p= . ). conclusions: our results indicates that nowadays, the robotic surgery is related a lot of misperceptions and a generally low level of information. this general picture is partially mitigated during the medical school, but the level of knowledge is still low. a big effort seems mandatory in clarify every technical aspect and an ethic debate about robotics, technology and ai as part of medical curriculum is advisable. background: learning theory states that a certain level of physiological stress or cognitive activation is required to achieve optimal task engagement and performance by the learners. our study will seek to determine if a hybrid team training curriculum inclusive of a task-oriented interactive virtual environment could help achieve the optimal level of cognitive activation required to result in a higher task engagement and performance. methods: a total of thirty-five medical professionals from the university of minnesota participated in several team training simulations. participants were randomly selected to an experimental and control groups. the experimental group (n= ) was exposed to a hybrid team training module, consisting of a task-oriented augmented reality phase followed by a second and third phase consisting of a kinesthetic simulation scenario and debriefing, respectively. the augmented reality phase presented the trainees to an interactive -degree image of the same clinical room where the simulation would take place allowing for ''situated-learning'' to take place. during the learning phase, trainees were encouraged to interact and communicate with each other while completing the tasks allowing for ''social-learning'' to effect. the control group (n= ), educational component consisted of a traditional audiovisual lecture-style introductory presentation, a simulation, and debriefing. after completing their respective educational components, each group completed a nasa task load index survey to assess the cognitive load experience of the individual educational models. subjects were then exposed to a final simulation (test simulation) similar in content and structure to the initial simulation. this was followed by a second nasa tlx survey. raters evaluated both group level of engagement and performance using a validated checklist of critical actions. results: the experimental groups showed higher weighted overall nasa cognitive load index scores than the control group (p= . ) prior to the test simulation. the weighted nasa score remained elevated in the experimental participant groups following the test simulation, whereas in the control group the post-simulation nasa assessment revealed a decrease in cognitive load (p= . ). expert raters using a validated checklist determined that . ± . % of the experimental (hybrid curriculum) group and . ± . % of the control group appeared to be more engaged and performed better during the simulation. conclusions: pre-simulation task-oriented augmented reality learning environments designed to incorporate situated, and social learning virtual experiences can provide the optimal level of cognitive boost that can result in a higher participant engagement and performance during team training simulation scenarios. introduction: despite the huge importance of laparoscopy, medical students have a brief contact with this surgical specialty during medical school in brazil. usually, they get in touch with this specialty during the surgery clerkship in the last years of medical school. therefore, few students perform clinical research or develop interest for this area during graduation. objective: to awaken the interest in laparoscopy of medical students early in medical school, improving the development of clinical research projects, and to prepare new generations of minimally invasive surgeons. discussion: the academic league of videolaparoscopy was created in under the guidance of dr. gustavo carvalho from the university of pernambuco, brazil. an academic league is a group of medical students who are guided by a tutor to develop three areas: research, teaching, and clinical practice. every year new students join the league after being selected with a multiple question test and an analysis of the curriculum vitae. the students are stimulated to participate in laparoscopic procedures as observers, learning about the techniques and instruments. moreover, there are minimally invasive surgery lectures and courses during the year. general surgery residents can also be part of the program as tutors. they are encouraged to present lectures, and to assist with research projects. medical students participated of this program in years. % pursued a surgical specialty after graduation. % did minimally invasive surgery as a fellowship. conclusions: the students who participate in several activities provided by the league have an increased interest in pursuing the path to become a laparoscopic surgeon. background: surgical education is an active and adaptive process of developing knowledge, technical and non-technical skills. the rise of social media has created a paradigm shift in surgical education, with online learning platforms offering exposure to real-time content, expert instruction, and global collaboration. while these disruptive technologies evolve, their influence on surgical education has not been investigated. our goal was to evaluate the growth and impact of an online surgical education model-the advances in surgery (ais) channel. our hypothesis was that utilization and engagement with the platform continues to grow, providing novel methods of measuring successful education. methods: assessment of the platform's membership demographic, user activity, and engagement was performed from inception in to quarter . the ais channel uniquely provides free, high quality, innovative content from elite surgeons in scheduled and continuously available formats across colorectal, bariatric and endocrine surgery service lines. users login to access content, with demographics, time spent, and content accessed recorded as measures of active account utilization and engagement. the main outcome measures were overall membership trends, utilization patterns by region, content type, and surgical specialty for the platform. results: users were predominately male ( . %), surgeons ( . %), and ranged in age from to years ( . %). the main surgical subspecialty represented was colorectal ( . %). active account usage/weekly recurrence was . % ( % industry benchmark), with users engaged for a mean minutes/session (excluding live events). since inception, steady exponential growth was seen across several dimensions. registered users and unique ip addresses increased from over , and , in to over , and . million in , respectively. the number of countries represented increased to reach across continents. at present, over live surgeries and live congresses have been broadcast from countries, with over , surgical videos available on demand to facilitate surgical education. the greatest engagement is seen with live surgical broadcasts. conclusion: our analysis demonstrated proof of concept for a unique, online surgical education model to provide effective surgical education. success was validated through the increase in overall users, sustained active account usage, and global penetration. user preferences for live surgical broadcasts were seen. knowing the utilization and preference patterns, the platform can continue to evolve and enhance the learners' experience. with this growth and penetrance, there is the potential to globally improve patient outcomes and the quality of care provided. background: a realistic simulator for transabdominal preperitoneal (tapp) inguinal hernia repair would enhance the surgeons' training experience before they enter the operating theater. the purpose of this study was to evaluate the efficacy of d-printed tapp simulator in evaluating preoperative skill before entering operative theater. methods: surgeons in our institution were enrolled in this study. they performed simulation tapp and the performance score was measured using tapp check list. the tapp simulator allows for the performance of all procedures required in tapp. the correlation between post -graduate years (pgys), age, experienced a number of laparoscopic surgery (more than , less than ), experienced number of tapp and the performance score was evaluated. results: strong correlation between experienced member of tapp inguinal hernia repair and the performance score was evaluated in this study (r= . ). however, the correlation between pgy, age and score was weak ( introduction: as the field of laparoscopic surgery grows, the need for standard measures of complex laparoscopic surgical skills is apparent. fundamentals of laparoscopic skills (fls) testing is required to complete general surgery residency, but there is no standard metric to convey expertise in advanced laparoscopic procedures. in an effort to develop a standardized assessment of laparoscopic suturing expertise, a group of experts was surveyed using delphi methodology to reach consensus on observed laparoscopic suturing skills reflective of performing at an expert level. methods: expert laparoscopic surgeons participated in serial surveys via redcap (research electronic data capture). experts included surgeons who perform[ /year laparoscopic procedures that involve intra-corporeal suturing, obtained from the authors' personal and professions networks. using a point likert scale, participants were asked to agree/disagree if different observed laparoscopic suturing skills indicate performing at an expert level. these skills were chosen from prior assessment instruments in the literature and the authors' previously published work. tasks were considered to meet criteria for consensus and eliminated from the next round of the survey after reaching % consensus as "strongly agree." results of the previous round of surveys were shared with participants at the start of the next round. the predefined endpoint for the delphi was set as maximum of rounds, reaching % consensus on each skill, or if[ % of initial respondents fail to return for subsequent surveys. results: after the first round of the delphi survey, respondents met inclusion criteria. preliminary data demonstrated skills that reached consensus ([ % of respondents chose "strongly agree"): forehand suturing, avoiding tissue trauma, having a technically acceptable final product (ie. tight closure), and tying a secure knot at the end of suturing. items did not approach consensus (\ % of respondents chose "strongly agree" or "agree"): alternating hands for each throw while tying, never missing a target when grabbing needle/suture, alternating direction of throws when tying, and backhand suturing. data from all four rounds of surveys as well as the final draft of the assessment instrument will be available at time of presentation. conclusion: preliminary data of this delphi study allowed us to reach consensus amongst a group of expert laparoscopic surgeons on the characteristics of expert laparoscopic suturing, which will allow creation of a comprehensive assessment tool for this domain. validation of such a tool will help advance the surgical field towards true competency-based credentialing and promotion. the study was designed to assess the knowledge of scp among european surgeons (specialists and residents). additionally, surgeons' opinion on usefulness of each of the rules of scp was gathered. the data were analyzed in terms of differences between residents and specialists. this is to set ground for and an educational program and increase the safety of elective laparoscopic cholecystectomy by minimizing the occurrence of cbdi. methods: the data on the knowledge of scp and opinion on usefulness of its rules were gathered in form of an anonymous questionnaire distributed among participants of several surgical conferences in poland. the questionnaire then asked about the surgeon's experience in terms of cholecystectomies performed and the number of complications in form of cbdi. it then listed the scp rules and asked the surgeon about their opinion on usefulness of each of the rules on a -point scale. gathered data were subject to statistical analysis and a comparison between specialists and residents was performed. the study has been registered in the clinicaltrials.gov-nct . , although these numbers are still low. significant differences in the mean usefulness score between residents and specialists were observed in regard to two rules: rule was found more useful by residents (mean score , vs. , , p= . ), whereas rule was found more useful by specialists (mean . vs. . , p= . ). the awareness of the sages safe cholecystectomy program in poland is still low and needs to be promoted. both surgical residents and specialists consider the rules of scp to be useful during surgery, although there are slight differences in the usefulness scores between the groups. an educational program to promote and further implement the scp should be established. introduction: transanal total mesorectal excision (tatme) has attracted substantial interest amongst colorectal surgeons throughout the world. technical challenges of the technique however have been acknowledged by early adopters and this may underpin the early reports of visceral injuries which occurred during the perineal phase. evidence from previous surgical training programs suggest that a structured proctorship programme can shorten the learning curve, operative time and most importantly reduce major complications. the aim of this study was to report on the first national pilot training initiative which was developed in the uk to ensure safe introduction of this technique. methods: a pilot training programme for the uk has been established in partnership with the healthcare industry, and supported by the association of coloproctology of great britain and ireland. the programme consists of three phases: (i) development of a consensus process on the optimum training curriculum of tatme from all relevant stakeholders, including experts, early adopters, and potential learners, to guide the training of this technique (ii) piloting of this training curriculum and (iii) assessment and quality assurance mechanisms to monitor training and measure outcomes. results: a cohesive multi-modal training curriculum has been developed providing clear guidance on case selection, supporting multi-disciplinary and multimodal training including online modules, dry-lab, purse-string simulators, cadaveric training and formal clinical proctoring programme. the uk pilot programme opened for applications in may and, after a rigorous selection process, the initiative was launched in september with trainers mentoring consultant colorectal surgeons from five centres. the selection of learners was based on suitable case volume and prior experience in laparoscopic rectal surgery. objective assessment tools were applied to an unedited video of a laparoscopic rectal surgery case for each applicant. for the selected centres, access to the ilapp tatme app was provided to access educational content including operative video footage, prior to attending a bespoke cadaveric workshop. each learner will then benefit from a structured, centrally organised and funded proctorship programme at their own institutions. a global assessment score form has been specifically designed to monitor training and a formal accreditation process will be used to sign off each learner using competency assessment tool. data on the cadaveric workshop and initial outcomes of the clinical mentorship will be presented at the conference. conclusion: a competency-based pilot training programme for transanal total mesorectal excision has been launched in the uk to support safe introduction of this technique. practicing on a fls trainer box is effective but requires large amount of consumables and is scored subjectively. the purpose of this study is to evaluate the face validity of the intracorporeal suturing task on a virtual fundamentals of laparoscopic surgery simulator (virtual fls). we hypothesize that the virtual fls will demonstrate face validity. methods and procedures: after a video demonstration and a practice period, twenty-three medical students and residents completed an evaluation of the simulator. the participants were asked to perform the standard intracorporeal suturing task on each of the virtual fls and the traditional fls box trainer. the presentation order of the devices was balanced. the performance scores on each device were calculated based on time (seconds), deviations to the black dots (mm), and incision gap (mm). the participants were then asked to finish a -question questionnaire regarding the face validity of the simulator. participants answered questions with ratings from (not realistic/useful) to (very realistic/ useful). a wilcoxon signed ranks test was performed to identify differences in performance on the virtual fls compared to the traditional fls box trainer. results: responses to of the questions ( . %) averaged above a . out of . those questions that rated the highest were the degree of realism of the target objects in the virtual fls compared to the fls ( . ) presently, most training methods for thoracoscopic esophagectomy use live porcines; this presents several problems including cost, long preparation times, and ethical issues. these problems further prevent frequent training. currently, no alternative models for thoracocopic esophagectomy training. we report, for the first time, the development and use of a non-biomaterial training model for thoracoscopic esophagectomy. methods: we collaborated with sunarrow co., ltd. (tokyo, japan) to develop the training model. we created organ models for esophagus, trachea, bronchus, aorta, vagus nerve, recurrent nerve, bronchial artery, lymph node, vertebrae, azygos vein, and thoracic duct, and filled the models with a polyvinyl alcohol hydrogel. the gaps between organs were filled with a filler material mimicking connective tissue. we chose a synthetic resin that closely mimics the characteristics (rigidity or elasticity) of each organ. after each organ was fixed, the model was covered with a filler to create a pleural membrane to allow training in peeling operations. in addition, because a patient plate was attached to the rear of the training model, excision with an energy device was possible and more closely simulated surgical conditions. results: using the training model resulted in a highly satisfactory level of experience in three trainees. the trainees were able to learn anatomical positions and sequence of surgical procedures, including endoscope handling. centre for rural health, aberdeen university introduction: as doctors become expert in a complex procedure, they develop automatic nuances of performance that are difficult to explain to a peer or a trainee (so called 'unconscious competence'). traditional methods which aim to allow sharing of expertise have limitations: concurrent reporting alters the flow of the task at hand while retrospective reporting is subject to bias and often incomplete. iview expert is a technique validated in the aerospace domain which externalises an expert's cognitive processes, without disrupting the task at hand. the aim of this project is to assess the feasibility of adapting the technique to medical training. methods: this was an observational case study in which an expert endoscopist wore a head mounted camera to capture a complex procedure (colonoscopy). captured video was reviewed during a facilitated debrief which externalised the expert's cognitive processes. the debrief was recorded and formed an audio commentary. the video and accompanying audio commentary formed a learning package which was watched by a specialty trainee. the technique differs from standard procedural videos in that it provides a more detailed insight into cognitive processes of the expert. this is achieved through the debrief, which encourages reflection upon kinaesthetic (head movement) as well as auditory and visual cues, resulting in a higher level of experiential immersion. questionnaires examined acceptability and educational value of the technique using likert scales and free text answers. quantitative data were presented using basic descriptions in terms of agreement with statements. qualitative data from free text responses were coded in order to identify key themes. results: the expert agreed that wearing the camera was acceptable and did not interfere with the procedure, nor usual decision making processes. qualitative analysis revealed the debrief process to be associated with a high level of experiential immersion: "as if they were there". both the expert and the trainee strongly agreed that the process was educationally valuable and that they learned something new. qualitative analysis demonstrated that the technique revealed useful and unique nuances of the procedure. the intervention could represent a powerful adjunct to existing training methods, especially amongst more experienced practitioners. we are currently undertaking a larger study involving a greater range of procedures with more learners. introduction: endoscopy is an important skill for general surgeons to possess. however, there is lack of training within surgery residency programs. we implemented a one-day endoscopic surgery course with the aim of improving the confidence of surgical residents in performing endoscopic procedures. we also aimed to examine the effect of the exposure to this course on self-reported confidence in performing endoscopic procedures. methods and procedures: the fundamental of endoscopic surgery course at texas tech university health science center is a one-day course consisting of both didactic training and lab training. the didactic part of the course is taught by attending physicians and focuses on the basics of endoscopy, management of upper and lower gastrointestinal (gi) bleed, and techniques to perform a variety of gi endoscopic procedures on swine esophagus and stomach explant. the lab portion of the course allows residents to perform different endoscopic surgical procedures with the attending physicians providing guidance. residents from pgy- to pgy- participated in the course. a -item questionnaire that measured the self-reported confidence in performing several endoscopic procedures on a - likert scale was administered before and after the course. results: twenty-two participants successfully completed the training and the questionnaires. a significant improvement was observed in the overall confidence in performing a variety of endoscopic procedures ( . ± . , p. ). the improvements remained significant even after controlling for the years of postgraduate surgical training (p. ). conclusion: the one-day fundamental of endoscopic surgery course enabled residents to be more confident with endoscopic procedures. overall, the residents felt that the course was helpful and would like to attend more than one session per year. this course should be held, at least, annually to allow the general surgery residents to become even more confident with this important skill. by being more confident in their surgical endoscopy skills, they will ultimately be able to provide better care for patients. introduction: a course evaluation study on the effectiveness of improving laparoscopic skills of surgical residents using swine models was evaluated through a self-report questionnaire administered before and after course completion. the purpose of the training is to provide surgical residents opportunities to practice and advance their laparoscopic proficiencies. methods and procedures: participating residents in all post-graduate year levels (pgy through pgy , n= ) were provided anesthetized pigs with which to perform a variety of simple to complex laparoscopic cases. prior to training, residents were given a questionnaire composed of eleven questions requiring the subjects to rate their confidence in performing various laparoscopic procedures on a - likert scale. after completion of the course, an identical questionnaire was distributed with two additional questions relating to the overall impact of the course. all statistical analyses were conducted using r statistical software (version . conclusion: overall, one-day hands-on training using swine models improved resident's skills, confidence, and understanding of laparoscopic surgery. the information acquired through the questionnaire emphasized the importance of providing a laparoscopic training course as a standard requirement at all medical institutions. allowing opportunities for surgical residents to practice their laparoscopic skillset will not only help in their individual academic advancements, it will allow them to provide optimum care for their patients. background: learning laparoscopy is difficult and many educational tools including simulation training are required. feedback plays a crucial role for motor skill training but require expert tutors and its time consuming. e-learning increases knowledge acquisition through a more interacting multimedia experience and reduces de costs of learning. in the last decade multiple applications (apps) have been developed for mobile medical training. a new ios app was developed using specially designed educational videos that explain the main technical aspects in advanced laparoscopy through simulation training. the aim of this study is to present the first results of its incorporation in a surgical simulation lab as a complement of effective feedback. methods: twenty-five consecutive residents were trained in our simulation lab through a session validated training program for the acquisition of advanced laparoscopic skills needed for the performance of a laparoscopic hand-sewn jejuno-jejunostomy. every session had written instructions and a basic tutorial video. the app consist two main sections, the first one explains the essential techniques needed for intracorporeal suturing and the second is a complete walkthrough of the validated training program. the trainees were divided in two groups, the first was trained without using the app (napp) and the second group was trained using the app (yapp). both groups of trainees could ask for feedback anytime they needed. trainees were assessed before and after the training program using validated rating scales and the number of necessary tutor-feedback sessions were registered. finally the yapp group answered a survey about the strengths and weaknesses of the app for learning advanced laparoscopic skills. results: twenty-five residents completed the training program; yapp and napp. both groups finalized their training with no statistical significant differences in their scores (p: . ). the number of tutor-feedback needed to complete the training in the yapp vs napp was of [ ( - ) vs ( - ) (p. )] respectively. in the questionnaire all participants considered that the app was effective for learning advanced laparoscopy. over downloads have been registered since the app was published in the apple app store in . we present a novel smartphone app that guides laparoscopic training using simulation-based educational videos with very good results. the use of app guided learning reduces de need of expert tutor feedback reducing the costs of simulated training. jemin choi, young-il choi; kosin university gospel hospital purpose: laparoscopic appendectomy (la) has been widely performed for acute appendicitis. in addition, minimally invasive surgery such as la is common surgical technique to the surgical residents. however, single incision laparoscopic surgery (sils) is a challenge to inexperienced surgical residents. we described our initial experience in teaching sils procedure for appendectomy in our medical center. methods: twenty nine cases of single incision laparoscopic appendectomy (sila) were performed by single surgical resident and cases of la were performed by surgical residents and boardcertified surgeons. a study was reviewed retrospectively. ( ) clinical stressors (i.e., vitals of patient coding). we developed a stress simulator testbed by integrating an fls box trainer with a linux computer, running custom c++ code. the code generated various stressor conditions, while recording sensor data from the trainer and human operator. we tested groups of participants in an irb approved trial including: novices (non-medical students), intermediates (medical students), and experts (pgy residents and fellows). the study consisted of subjects performing the peg transfer and the pattern cut six times (baseline, four randomized stressors, posttest). after each task, the nasa-tlx survey was administered to determine the overall workload of that stressor condition. an analysis of variance was conducted to identify significant trends in terms of stressor type. results: when compared to baseline nasa-tlx scores, the intermediate group had the greatest changes in overall workload than novices and experts (p= . ). additionally, the change between baseline and post-test workload was significantly lower than for the environmental, negative evaluative, and clinical stressors (p= . ). for pattern cutting, subjects reported a significantly lower perception of failure (p= . ) in both the positive evaluative (mean= . ) and post-test conditions (mean= . ), yet, though not statistically significant (p= . ), the measured accuracy in the task during the positive evaluative condition was actually worse ( . %), second only to the pre-test accuracy ( . %). the best accuracy for pattern cutting across all expertise levels was % for the post-test followed by . % in the negative evaluative condition. these results are interesting as they show that despite perceived improvements in performance with a positive feedback condition, performance actually degrades and is better in the negative feedback condition, which is perceived to be more difficult. these results were not found in the peg transfer task, which is arguably an easier task. conclusion: from the evidence gathered in the study, it is clear that there is a correlation between distractors and performance. further analysis is needed to identify the relationship between the type of stressor, and inherent difficulty of the tasks, in terms of which type of stressor best improves learning and outcomes. surg endosc ( ) each received credentials to perform diagnostic and therapeutic ercp from their respective hospitals in nevada, minnesota, and idaho. one continues to teach ercp to general surgery residents, and another taught the skill to fellows in an advanced endoscopy fellowship. all three continue to use ercp in their practice ( to times per month), as they each specialized in a field that utilizes ercp routinely. choledocholothiaisis is the most frequent indication, though ercp is also performed for iatrogenic biliary duct leaks, traumatic biliary or pancreatic duct leaks, chronic pancreatitis, and malignancy. conclusions: training in esophagogastroduodenoscopy and colonoscopy is required for general surgery residents, but the addition of ercp to select residents' training enables them to completely manage their patients' surgical disease. the training of select general surgery residents in this skill has been successful, evidenced by the continued use of ercp in the practices of three residents who completed this training program at our institution. the decision to train residents in this skill should be left to individual program directors and department chairs. we recommend that residents selected for this additional training should plan to practice in specialties where ercp can be implemented. conclusion: same-day discharge after nissen fundoplication and hiatal hernia repair is feasible for select patients. one major challenge for same day discharge is the current insurance provisions required for hospital reimbursement. within the parameters of this study, bmi and asa score did not differ between discharged and admitted patients, while older age and increased procedure duration were associated with need for admission. premkumar anandan, ms, facs; bangalore medical college and research institute introduction: minimal access surgery is an imperative element of enhanced recovery program and has significantly improved the outcomes. enhanced recovery program (erp) synonym "fast track" surgery "was first conceived by dr henrich kelhet. largely described for colorectal surgery and reported to be feasible and useful for maintaining physiological function and smooth the progress of recovery. most of the patients who present for surgical emergency are not adequately prepared and many are not in normal physiological state. the feasibility of enhanced recovery programs protocol in such emergency minimal access surgery remains indistinct. this study was designed to validate an enhanced recovery program in patients who undergo emergency minimal access surgery. introduction: pathways for enhanced recovery after surgery (eras) have been shown to improve length of stay and postoperative complication rates across various surgical fields, however there is a relative lack of evidence-based studies in bariatric surgery. the objective of the current study was to determine if starting a bariatric full liquid diet on postoperative day (pod) zero was associated with shorter length of stay (los) for patients who underwent laparoscopic sleeve gastrectomy (lsg) or roux-en-y gastric bypass (rygb). methods: retrospective review of a prospectively collected dataset was conducted at a single institution before and after implementation of a new diet protocol for lsg and rygb. postoperative diet orders were changed from full liquid diet on pod to pod . length of stay and -day readmissions were reviewed from june to august . independent samples t-tests were used to compare continuous variables and chi-squared tests for categorical variables before and after diet change was implemented. patients were excluded if they were undergoing revision surgery, were discharged directly from pacu, or had significant intraoperative complications or required reoperation within the same admission. introduction: data suggests value in using tap (transversus abdominis plane) neural blockade in abdominal surgical procedures. we deploy tap blockade using liposomal bupivacaine via ultrasound (us) as part of a narcotic sparing pain management pathway for patients undergoing abdominal surgery in our rural community setting. our goal was to evaluate adequacy of postoperative discomfort and the success in avoiding narcotic usage. methods and procedures: records of patients undergoing abdominal surgical procedures performed by one surgeon over an month period were reviewed under irb approval. patients taking narcotics prior to the procedure (except for discomfort due to the condition being surgically treated) were excluded from analysis, as were those admitted to the hospital for postoperative treatment. us guided lateral tap blocks were performed by the surgeon using mg of liposomal bupivacaine and mg of bupivacaine in the or prior to the incision. unilateral block was performed for unilateral procedures (e.g. inguinal hernia) and bilateral for laparoscopic or midline procedures. incisional sites were treated with a field block of mg of bupivacaine. prescriptions for medications included , mg of acetaminophen qid and mg of naproxen sodium tid for days. a prescription for tramadol ( to mg prn up to times daily; tablets with no refill) was given. patients were seen in followup two weeks postoperatively. data (following standard scales/metrics) for patient-reported-outcomes e.g. pain, nausea-vomiting, & fatigue will be analyzed with the above data and the analysis with conclusions will be presented & discussed. federico sertic, md, ashwin gojanur, dr, ahmed hammad, md; guy's and st thomas' hospital introduction: the aim of this project is to assess the quality of post-operative pain relief in colorectal surgery and identify patients in whom pain management has not been effective, in order to improve the quality of post-operative care. effective management of post-operative pain has long been recognised as important in improving the post-operative experience, reducing complications and promoting early discharge from hospital. standards: all patients should be pain free at rest, % of elective patients should be told about what analgesia they will have post-operatively, % of patients should be satisfied with their pain management and % of patients should feel staff did everything they could to control their pain. methods and procedures: questionnaires were given to patients on the day prior to discharge. questions about pre-operative and post-operative pain experience were asked. data regarding post-operative analgesia were collected from medication charts and medical notes. data were collected over a period of two months (august/september ). range of procedures: elective laparoscopic abdomino-perineal-excision-of-rectum with igap flaps, elective laparoscopic right hemicholectomy, laparotomy+bowel resection/stoma formation ( elective, emergency), elective repair of parastomal hernia, appendicectomy ( laparoscopic elective, laparoscopic emergency, laparotomy emergency) and elective reversal of ileosomy. pain scores ( - ): immediately post-operative pain, day post-operative pain, post-operative pain after day and pain on moving/coughing/straining. results: mean immediate post-operative pain score was . ( % of patients with score +), mean day post-operative pain score was . , mean post-operative pain score after day was . , mean pain score on moving was . ( % of patients with score +), mean pain score on coughing/ straining was . ( % of patients with score +). % of patients were satisfied with their post-operative pain management and felt that the staff had done everything they could to manage their pain. % of patients were not aware of their post-operative analgesia regimen and % did not know how regularly they could request analgesia. conclusions: effective management of post-operative pain is a key part of post-operative care and an important component of enhanced recovery programmes. patient satisfaction with pain management has been found to correlate with received pre-operative information. increasing ward nurses' and acute pain teams' knowledge is important in improving patients' pain experiences. interestingly, those patients who had a background of long-term opioid requirements reported that they were satisfied with their pain management. methods and procedures: a patient undergoing a standard ultrasound guided ql block by an anesthesiologist established the baseline anticipated response, and procedure time. the procedure, performed under sedation preoperatively, required over minutes. for this study, patients undergoing laparoscopic colorectal surgery were administered a lateral ql block (modified ql ) under ultrasound guidance by the operating surgeon. ml of a mixture ( ml injectable liposomal bupivacaine suspension, ml . % bupivacaine hydrochloride and ml normal saline) was injected bilaterally, after induction, skin preparation, draping, and prior to the operation. postoperative narcotic use and pain vas scores were documented. results: six patients were administered a bi-lateral ql block intraoperatively. procedures were: laparoscopic sigmoid colectomies, one end ileostomy reversal, laparoscopic completion proctectomy with ileal pouch anal anastomosis, and a laparoscopic descending colectomy. of the narcotic naïve patients, mean pain vas on post op days , and were . , . and . respectively within a multimodality pain management/enhanced recovery program, where standing orders prompting narcotic administration by nursing staff is pain vas . all were discharged on pod or without narcotic prescriptions. two of the patients were chronic narcotic users, and they were discharged on their baseline narcotics, i.e. without additional narcotics. all intraoperative blocks were performed in less than minutes. conclusion: a novel, surgeon-administered lateral ql block under ultrasound guidance, is feasible and provides post-operative pain control. patients are discharged home on no/baseline narcotics. a randomized controlled trial is being constructed based on these striking findings. keywords: lc-laparoscopic cholecystectomy, ga-general anaesthesia, sa-spinal anaesthesia. nikhil gupta, rachan kathpal, dr, arun k gupta, dr, dipankar naskar, dr, c k durga, dr; pgimer dr rml hospital, delhi introduction: cholecystectomy have shown some advantages when done under spinal anaesthesia (sa) and associated with less intra operative and post -operative morbidity and mortality. laparoscopic cholecystectomy (lc) under regional anaesthesia alone included patients with coexisting pulmonary disease, who are deemed high risk for ga. the aim of the present study is to assess the efficacy and safety of laparoscopic cholecystectomy under sa. materials: this prospective, interventional study was conducted on patients with chronic calculous cholecystitis attending general surgery out-patient department of our institution. results: in our study, intraoperative complications recorded were hypotension, bradycardia, intra op shoulder tip pain, bleeding from the liver bed, bile spillage, post-op pain and vomiting. % patients had intraoperative pain, % had shoulder tip pain, . % had bradycardia, . % had hypotension, . % had bile spillage and . % had bleeding. laparoscopic cholecystectomy under spinal anaesthesia should be promoted more even in developing countries but we need to establish well evaluated safety guidelines that could be followed faithfully for minimizing the risk of complication. background: the "opioid crisis" has taken over headlines with increasing public attention brought to the drastically increasing rates of addiction to prescription narcotics. in , the american society of addiction medicine reported million americans with an addiction to prescription pain relievers and a four-fold increase in overdose related deaths. in a medical setting, increased opiate use is associated with increased rates of delirium, ileus, urinary retention, and respiratory depression. these risks are increased in the obese/bariatric population. transversus abdominis plane (tap) block is a safe and effective approach to achieve optimum pain control. it reduces the use of opiates in patients undergoing major abdominal surgery. however, there is currently no data in the literature examining its use in the bariatric population. our study examines the use of liposomal bupivacaine for tap block in patients undergoing laparoscopic sleeve gastrectomy (lsg). methods: sixteen patients undergoing lsg with tap block were compared with historical cohort of sixteen patients undergoing lsg without tap block (standard group). the primary outcome measured was post-operative in-hospital opiate use (morphine equivalents). statistical analysis was performed using student's t test for continuous variables and fisher's exact test for categorical variables. results: both groups were well matched in regards to bmi, age, and asa class. there was a significant decrease in the post-operative use of opiates with the use of the tap block ( . mg in the tap block group vs. mg in the standard group; p . ). there was no difference in the mean length of stay between the two groups. there was an increase in the mean operative time with use of the tap block ( minutes in the tap block group vs. minutes in the standard group; p. ) conclusions: the use liposomal bupivacaine for tap block provides substantial analgesia, allowing for significant reduction in post-operative opiate use in our bariatric patients. this can be an important adjunct in pain control for the bariatric population and aid in post-operative complication risk reduction. introduction: the objective of this study was to identify variation in weight and demographics in the distribution of pre-operative clinical characteristics between super obese females compared with males who were about to undergo bpd/ds surgery. as the american obesity epidemic increases, morbidly obese patients have become integral to every surgical practice; they are no longer limited to bariatric surgeons. every clinical insight helps the surgeon to optimize outcomes when operating on and managing these medically fragile individuals. in this context, however, clinically and statistically significant differences in demographics, body mass, and in the distribution of weight-related medical problems between super-obese women and men are unknown. introduction: a transversus abdominis plane (tap) block is an ultrasound-guided injection of local anesthetic in the plane between the internal oblique and transversus abdominis muscles to interrupt innervation to the abdominal skin, muscles, and parietal peritoneum. currently there are incongruent findings on the benefit of this regional anesthetic to surgical patients, particularly the obese population. we hypothesized the addition of a tap block in an enhanced recovery pathway (eras) for bariatric patients would decrease opioid use and shorten hospital length of stay. methods: a retrospective review of all patients who underwent bariatric surgery at a single institution from january to december was performed. patients were identified as: no tap block (no tap), tap blocks that were performed after induction either pre-surgery (pre-tap) or post-surgery (post-tap). the primary outcome was time to first opioid (min) and total morphine (mg) equivalents in pacu. objective: prolonged postoperative ileus increases hospital length of stay and therefore impacts healthcare costs. although many surgeons recommend ambulation in the postoperative period to hasten return of bowel function, little evidence exists to support this practice. our hypothesis is that early ambulation does reduce the time to return of bowel function after intestinal surgery. methods: a subset of patients undergoing intestinal surgery from an ongoing, prospective trial evaluating perioperative physical activity was analyzed. preoperatively, patients wore an activity tracker for a minimum of three days to establish a baseline activity level, measured by daily steps. postoperatively, steps were recorded for days. patients were included in this study if they underwent an operation on the small bowel, colon, or rectum. resolution of postoperative ileus was defined as the postoperative day when patients were noted to meet all of the following criteria on review of nursing documentation: passing flatus, stooling or having ostomy output, and tolerating a regular diet without intravenous fluids. "early" postoperative activity was defined as the average number of daily steps during the first two postoperative days. discussion: these results suggest the patients who received an intraoperative block laparoscopically were more likely to be able to spend less time in the post anesthesia care unit and be discharged home the same day. based on these results, additional process improvement ideas will be implemented in an attempt to improve outcomes. riley d stewart, md, msc, frcsc, james ellsmere, md, msc, frcsc; dalhousie university division of general surgery introduction: oropharyngeal and gastrointestinal (gi) perforations from bbq brush bristles are being reported in the literature with increasing frequency. media attention to this problem has increased awareness by the public. most commonly, bbq bristles lodged in the gi tract can be removed endoscopically or pass without complication. rarely, surgical intervention is required for removal of the bristle or drainage of an associated abscess. we report a case of gastric perforation by a bbq bristle leading to a pancreatic abscess. case report: a -year-old male presented to a regional center with epigastric pain and malaise. his medical history included: hypertension, dyslipidemia, gerd, and smoking. his surgical history included: a tonsillectomy, excision of bronchial cleft cyst, and an umbilical hernia repair. on presentation, his laboratory investigations where unremarkable aside from an elevated white blood cell count. investigations including an abdominal x-rays and an abdominal ultrasound were unremarkable. he was initially treated with a proton pump inhibitor for presumed peptic ulcer disease. he returned to the local emergency room, no better than before. a ct scan was arranged which demonstrated a foreign body at the pylorus consistent with a bbq bristle and a peripancreatic fluid collection (figs. & ) . a gastroscopy failed to identify the bristle. he was admitted, placed on iv antibiotics and referred to our center. despite several days of antibiotics prior to arrival, the collection size on repeat ct scan had increased and the patient had ongoing pain. we repeated the endoscopy with a side viewing endoscope. the perforation was identified posteriorly at the pylorus. the bristle had migrated into the peripancreatic space. the perforation was cannulated with a jagtome. fluoroscopy was used to confirm the position of a wire in the fluid collection (figs. & ) . pus was drained from the collection into the stomach by placement of a french pigtail catheter (fig. ) . the patient was discharged pain free the following day. the patient was asymptomatic at weeks' follow-up. a repeat ct scan showed resolution of the abscess and safe migration of the bristle and stent out of the gi tract (fig. ) conclusion: to our knowledge, this is the first reported transgastric endoscopic drainage of a peripancreatic abscess caused by a bbq bristle gastric perforation. this case is a demonstration of the ever-expanding role of therapeutic endoscopy in a surgical practice. andrew w white, md, carl westcott, md; wake forest baptist medical center introduction: endoscopic balloon dilation of the gastroesophageal junction (gej) is generally limited to mm in diameter. in many stenotic or spastic disorders of the gej mm is just not big enough. larger balloon sizes are available ( and mm), although these are deployed under fluoroscopy without endoscopy. thus, these larger dilations are often not feasible at the time of the diagnostic endoscopy because different facilities and/or equipment are needed. also, fluoroscopic mm balloon dilations are associated with a percent perforation rate. to address these shortcomings we present an experience with a retroflexed "against the scope" balloon dilation of the gej. in detail, the gej is visualized while retroflexed and a balloon is then placed through the scope. the gej is cannulated next to the scope and deployed. please see the attached image for example. methods and procedures: a retrospective chart review was performed for a single surgeon during the past five years. we identified those who had retrograde dilations and evaluated the indications, repeat dilations, complications and symptomatic response. results: a total of retrograde dilations were performed on patients with gej related dysphagia. the average age was . years. of dilations were with a mm balloon while other dilations used as small as a mm balloon. dilations were performed for persistent dysphagia after cardiomyotomy between and days after surgery. other indications for dilation were dysphagia after fundoplication ( / ), dysphagia after paraesophageal hernia repair ( / ) and achalasia during pregnancy ( / ). patients required a total of repeat retrograde dilations at an average time of days after previous dilation. there were instances reported where the dilation did not improve symptoms. there was mucosal breakdown noted in instances although there were no perforations. bleeding was noted in instances although this was always minimal and selfresolving. conclusions: retrograde endoscopic dilation is safe and effective in this small series. the mm balloon against a mm scope gives a mm diameter, but a different shape and a decreased total circumference. there is a possible added safety advantage given that the balloon is inflated under visualization. it can be inflated in steps or stopped if it appears too aggressive. in addition these larger dilations were provided at the time of the initial diagnostic egd without extra equipment. more studies are needed to compare retrograde endoscopic dilation to other methods of management of gej stenosis. introduction: robot-assisted surgery allows surgeons to perform many types of complex laparoscopic surgical procedures. more and more patients are treated with this sophisticated system. however, all the instruments used in the currently available surgical robot system is rigid. therefore, there exists a limitation in the extent of reach to the deeper surgical fields. in order to overcome this difficulty, we are developing a novel flexible endoscopic surgery system (fess) which has flexible single port platform of cm in diameter, independently controlled endoscope and instruments, open architecture that is compatible with existing flexible devices and a magnified d hd camera that has sensors of both rgb and infrared. furthermore, the system is smaller and would be more cost-effective than existing robotic surgical system. a preliminary experiment was performed in surgical procedures using porcine model to evaluate effectiveness and feasibility of fess. methods and procedures: experimental protocols were approved by the animal research committees of our institution. we used a female swine of kg. an assistant forcep lifted up the fundus of gallbladder to create good visualization of surgical field. the cystic duct was ligated by laparoscopic clip device from assistant port. blunt dissection was performed by pushing the forceps and sharp dissection by monopolar electrocoagulation. results: the fess accomplished the dissection of the gallbladder from the liver bed successfully. two mm forceps had enough grasping and dissecting force and dexterity. the gallbladder was removed from single port site easily. conclusions: this experiment showed that it is feasible to intuitively operate single-site cholecystectomy with fess. in order to realize a pure fess procedure, an additional novel device to create good visualization of the surgical field is necessary for the fess platform. a prototype has already been developed for evaluation in securing the surgical field. the optimal working range, or "sweet spot" of fess is not relatively large. in addressing this issue, the feature of easy setup is being improved to enable more efficient positioning and shifting of the sweet spot for the surgical field. this mechanism could enhance the expansion of procedures suitable for fess. the target procedures of fess are those specifically suitable for single port surgery, such as transanal surgeries and transcervical mediastinoscopic surgeries. intraluminal procedures and natural orifice translumenal surgery (notes), which are not considered suitable for rigid surgical robot, are also good applications of fess. regression of anal and scrotal squamous cell carcinoma (hpv related) with imiquimod index patient is a year old hiv positive homosexual man with anal-scrotal condylomas (ain) initially resected in , then treated with radiation in for recurrence. recurred in with changes severe enough to ''…consider diagnosis of invasive squamous cell carcinoma…''. patient elected trial of imiquimod % cream three times per week to defer recommendation of abdominoperineal resection. imiquimod has no antiviral effect but stimulates interferon and cytokines to suppress hpv subtypes and , among other immune effects. no data exists as to systemic effects of imiquimod. after three months of therapy, lesions had largely regressed with only one specimen showing ''…concern for squamous cell carcinoma in situ…''. patient has elected to continue treatment pending further biopsy. this report is typical of a number of other reports of small numbers of cases of neoplasia regression with imiquimod % cream to include melanoma-in-situ, basal cell cancer of skin and other cutaneous malignancies as well as vin. a second female patient, years old, hiv+ with hpv lesions (ain ) including urethral lesions, is being treated with vulvar application of imiquimod to determine if urethral lesions will regress. there is no fda-approved indication for mucosal application of imiquimod. biopsies are pending at completion of six month trial of imiquiimod. surg endosc ( ) introduction: training in flexible endoscopy remains a critical skill for surgeons, as therapeutic endoscopy procedures continue to evolve and to supplant standard surgical operations. the role of endoscopy across surgical subspecialties is shifting, as endolumenal procedures (like per-oral endoscopic myotomy and endolumenal bariatric interventions) have become commonplace. while surgical residency minimum case volumes are mandated, little is known about the volume of endoscopic procedures surgical fellows participate in. we aimed to characterize the volume of flexible endoscopy cases logged by surgical subspecialty fellows as a measure of endoscopic platform use by surgeons. methods: operative case logs for fellows enrolled in post-graduate training programs participating in the fellowship council were de-identified (no patient or program specific information) and provided for analysis. the case log is an online, mandatory, self-reported collection of all surgeries, procedures and endoscopies performed during fellowship year. all cases listed within the category of "gi endoscopy" in which the fellow designated their role as "primary" surgeon for the procedure were further sorted based on subcategory and linked to the year of fellowship graduation. rigid endoscopy, trans-anal endoscopic procedures, and those in which the fellows roll was "first assistant" were excluded. introduction: complex pancreatic and duodenal injuries due to trauma continue to present a formidable challenge to the trauma surgeon with a described mortality of - % and morbidity of - %. duodenal fistula formation subsequent to failure of attempted primary repair is associated with significant morbidity and mortality. we present the first reported series of four patients with complex trauma-related duodenal injuries who had failure of primary repair which were managed with duodenal stenting. we compared outcomes to a matched case control cohort of patients with trauma related duodenal injuries. the aim of this study is to document our experience with enteral stents in patients with complex duodenopancreatic traumatic injuries. methods: a retrospective review at a level i trauma center identified patients who underwent endoscopically placed indwelling covered metal stents after failure of primary duodenal repair in the form of high output duodenal fistulas. a matched case control cohort was identified including patients with duodenal fistulas who were not treated with stents. drainage volumes were collected and classified according to source and phase of intervention (i.e. admission to fistula diagnosis, to stent insertion, after removal, and until discharge). results: there was a decrease in the mean combined drain output of ml/day (p= . ) after stent placement. when comparing the sum of all output sources, there was a statistically significant difference across phases (p= . ) and "after removal" was significantly less when compared to the reference phase (p= . ). there was also a change in the directionality of the slope for the sum of all drain outputs with an increase of ml/day prior to stent placement compared to a decrease of ml/day (p= . ) after stent placement. the stenting group demonstrated a decrease in mean drain output ( ml/day vs ml/day, p= . ) and increase in distal gastrointestinal output ( ml/day vs ml/day, p= . ). one patient in the stent group required later operative repair. all other patients in the stenting and control group had resolution of their fistulas over time. there were late mortalities in the control group. the stent treated patients demonstrated diversion of approximately ml/day of enteral contents distally. while all patients eventually healed their fistulas, the stent treated patients demonstrated an accelerated abatement of drain outputs when compared to the control cohort, but did not reach statistical significance. indwelling enteral-coated stents appear to be an effective rescue method for an otherwise inaccessible duodenal fistula after failure of primary repair. kevin l chow, md, hassan mashbari, md, mohannad hemdi, md, eduardo smith-singares, md; university of illinois at chicago introduction: esophageal trauma represents an uncommon but potentially catastrophic injury with a reported overall mortality of up to %. the management of iatrogenic and spontaneous perforations have been previously described with well-established guidelines which have been mirrored in the trauma setting. esophageal leaks are the most feared complication after primary surgical management and present a challenge to salvage. there has been increasing reports in the literature supporting the use of removable covered metal stents to treat esophageal perforations and leaks in the non-trauma setting. we present the first reported case series of four patients presenting with external penetrating trauma induced esophageal injuries, complicated by failure of initial primary surgical repair and leak development, successfully managed with the use of esophageal stents. materials and methods: a retrospective review was performed at a level i trauma center identifying four patients who underwent endoscopically placed removable covered metal stents, either by a surgical endoscopist or an interventional gastroenterologist, after failure of primary surgical repair of esophageal traumatic injuries. demographic information, hospital stay, additional interventions, complications, imaging studies, iss scores, and outcomes were collected. results: our cohort consisted of patients with penetrating injuries to the chest and neck with esophageal injuries ( thoracic and cervical esophageal injuries) managed with esophageal stenting after leaks were diagnosed following primary surgical repair. their initial esophageal injuries included grades , and . leaks were diagnosed on average post-operative day . two patients underwent an additional attempted surgical repair and subsequent leak development. esophageal stents were placed under endoscopic and fluoroscopic guidance within days of leak diagnosis. there was resolution of their esophageal fistulas with all patients resuming oral intake (averaging days after stent placement). three patients ( %) required further endoscopic interventions to adjust the stent due to migration or for dilations due to strictures. mortality was %, all patients survived to be discharged from the hospital with average icu length of stay of days. conclusion: the use of esophageal stenting has progressed over the last few years, with successful management of both post-operative upper gastrointestinal leaks as well as benign, spontaneous, or iatrogenic esophageal perforations. while the mainstay of external penetrating traumatic esophageal injuries remains surgical exploration, debridement, and repair with perivisceral drainage; our case series illustrates that the use of esophageal stents is an attractive adjunct that can be effective in the management of post-operative leaks in the trauma patient. results of the ovesco-over-overstitch technique for managing bariatric surgical complications introduction: since , the preferred method of enteral access has been the percutaneous endoscopic gastrostomy tube (peg). accidental removal is a common complication associated with excessive cost and possible significant morbidity. removal prior to days is considered ''early removal.'' early removal has more significant risk associated with it, and can necessitate emergent operation to prevent peritonitis and sepsis. some patients, who do not exhibit signs of peritonitis, may be simply observed. for these patients, peg replacement would typically be delayed - days to ensure closure. this delay results in prolonged npo status and worsened nutritional status. presented below is a case of early accidental removal followed by endoscopic clip closure, and immediate peg replacement. case report: a -year-old male presented after a large left middle cerebral artery infarct. a peg placement was completed without complication. eleven hours after the procedure the patient had pulled the peg tube out of the abdominal wall. at this time the patient appeared to have no abdominal pain and no signs of peritonitis. twelve hours following the accidental removal of his peg tube, the patient was taken back to the endoscopy suite, and an egd was performed. the previous peg site was identified and appeared closed and ulcerated. the mucosal defect was closed with two endoscopic metallic clips. a peg tube was then placed at an adjacent site. the following day, the patient was restarted on trickle feeds and advanced to regular tube feeding over a period of hours. since that time, his peg has been functioning well. discussion: we propose that in the case of early accidental peg removal, the patient should be examined first for evidence of peritonitis. if initial physical exam and radiographic investigation do not reveal peritonitis or significant pneumoperitoneum, the patient should undergo urgent repeat endoscopy. at this time, the gastrotomy can be closed endoscopically via metallic clips and peg can be replaced immediately. tube feeds can be initiated after a - hour period of dependent drainage with serial abdominal exams. introduction: since its inception in , poem has become a viable procedure for the treatment of achalasia and esophageal dysmotility disorders. however many institutions are in the beginning stages of implementing the procedure into their programs. in view of training, we report the successful ability to dissect and identify common landmarks during a poem procedure performed by trainees under supervision in a high volume poem center. methods: posterior poem procedures performed by trainees with experienced proctor guidance during the period between february to july were evaluated for the frequency of identifying the perforating vessels, the presence of sling fibers, and position on the lesser curvature of stomach evaluated by double scoping method during the creation of the tunnel and myotomy for procedure. results: all poem procedures were successfully completed by trainees (gi and surgery fellows). the average length of procedure was minutes. indication for procedure included patients with type achalasia ( %), with type achalasia ( %) and des ( %). average length of myotomy for all procedures was . cm. during these procedures or perforator vessels were identified in ( %) of patients, sling muscle was identified in patients ( %) of patients. myotomy extended to anterior lesser curvature of stomach on double scope exam in % of patients. no patient had a serious complication requiring intervention. conclusion: trainees performing a posterior poem procedure were able to correctly dissect and identify the sling muscle and/or perforating vessels in approximately % and % respectively of procedures. however the myotomy position was correctly placed in all procedures. this indicates that while ideally the sling fibers and perforating vessels should be identified, a correctly positioned myotomy can still be successfully performed by trainees without identification of these landmarks. introduction: gastroparesis is a rapidly increasing problem with sometimes devestating patient consequenses. surgical treatments, particularly laparoscopic pyloroplasty, have recently gained popularity but require general anesthesia, advanced skills and create risk of leaks. peroral pyloromyotomy (pop) is a less invasive alternative but is technically demanding and not widely available. we propose an hybrid laparo-endoscopic collaborative approach using a novel gastric access device to allow a endoluminal stapled pyloroplasty as an alternative treatment option for functional gastric outlet obstruction. methods and procedures: under general anesthesia six female pigs (mean weight kg) had endoscopic placement of or mm intragastric ports (taggs, kansas, usa) using a technique similar to percutaneous endoscopic gastrostomy. a mm laparoscope was used for visualization. endoflip (crospon, inc., galway, ireland) was used to measure cross sectional area (csa) and compliance of the pylorus before intervention, immediately after and at week survival. pyloroplasty was performed using a mm articulating laparoscopic stapler (dextera microcutter). after removing the taggs ports, the gastrotomies were closed by either endoscopic clip, endoscopic suture or suture under laparoscopic vision. the animals were survived for week. after - days, a second laparo-endoscopic procedure was performed to verify healing of the pyloroplasty as well as intraluminal dimensions. at the end of the protocol, animals were euthanized. results: six endoluminal linear stapled pyloroplasty were performed. the mean operative time was min. in all cases, this technique was effective in achieving optimal pyloric dilatation. median pyloric diameter (d) and median cross-sectional area (csa) pre-pyloroplasty were mm ( . - . mm) and . mm ( - mm ). after the procedure, these values were increased to . mm ( . - . mm) and . mm ( - mm ) respectively (p= . the quality of endoscopic examination depends on the quality of endoscopic equipment, experience of the endoscopist and preparation of the patient. contemporarily electronic endoscopes make feasible to transfer image directly to external device which is subsequently linked to computer network and can be transferred further. dynamic image viewed in real time is more accurately interpreted by a physician than a static one. the possibility of simultaneous voice contact makes teleconsultation sterling. the aim of this study was to present our own experience regarding endoscopic teleconsultations. materials and methods: analysis enrolled examinations performed in endoscopic centers located in lesser poland district and in denmark. consultations took place in real time, consulting physicians had more than years of experience in endoscopic procedures and over colonoscopies and therapeutic procedures performed. there were teleconsultations via standard internet connection mb/s. endoscopic centers were equipped with olympus and series linked to video card. each card had its own ip address, and the image was accessible through internet login from anywhere. consulting physicians used computers connected to internet for tracing the image synchronously and giving advice. results: teleconsultations were undertaken in . % of all endoscopic procedures. teleconsultations concerned difficulties in endoscopic image interpretation in cases and decisions regarding further treatment in cases. the consulting physician solved all problems concerning proper endoscopic image interpretation. in cases the elective procedure was rejected. the elective treatment was continued in remaining cases. patients had a complication of polypectomy that was endoscopically treated. conclusions: the opinion of independent consulting physician in difficult clinical cases regarding endoscopic procedures helps to understand the endoscopic image in real time and implicates a decrease in complications after endoscopic procedures. michelle ganyo, md, robert lawson, md; naval medical center san diego introduction: a presacral phlegmon is a contained collection of infected fluid and inflammation within the bony pelvis, posterior to the rectum and anterior to the sacrum, that usually arises as a complication of surgery, malignancy, inflammatory bowel disease, ischemic colitis or perforated viscous. symptoms include low-back pain, pelvic pain and fevers. antibiotics and supportive therapy are the mainstay of treatment. however, if abscess develops, drainage is required usually by trans-gluteal percutaneous and/or surgical methods, both of which are associated with significant morbidity and mortality. endoscopic ultrasound (eus) -guided drainage of perirectal and presacral abscesses is a well described minimally-invasive approach that permits clear definition of anatomy, real-time access to the abscess and creation of an internalized fistula through placement of one or more transluminal stents. however, to date there is no published report describing endoscopic treatment of the more complicated, clinically challenging presacral phlegmon. here we present a case of a symptomatic presacral phlegmon recalcitrant to medical management that was successfully treated with an endoscopically placed retrievable, transmural, lumen-apposing metal stent. case report: this is a case-report of a -year-old, post-partum female who presented with fevers and recurrent lower back pain radiating to her rectum and vagina. her spontaneous vaginal delivery was notable for a second-degree laceration that was primarily repaired at the time of delivery months prior to presentation. her past medical history was otherwise unremarkable. radiographic imaging revealed several perirectal and presacral abscesses that were considered too small for percutaneous drainage. iv antibiotics were started and the largest abscess was targeted for eusguided aspiration. unfortunately, her pain became constant and progressed in severity. a follow-up mri a week later revealed a -cm presacral phlegmon. results: colonoscopy revealed a luminal bulge in the rectum but was otherwise normal. to permit drainage and multiple sessions of endoscopic necrosectomy, a mm lumen-apposing metal stent (lams) was placed transrectally under eus-guidance into the presacral phlegmon. endoscopic debridement with forceps and copious irrigation was performed. over the following weeks the patient reported purulent rectal drainage and resolution of her fevers and pain. repeat endoscopy revealed a normal rectum and no sign of the stent. a follow up mri showed a -cm area of heterogenous tissue in the presacral area. conclusions: although not previously described for management of a presacral phlegmon, lams appears to be a safe and effective, minimally-invasive treatment option. introduction: flexible endoscopy has evolved to include multiple endoluminal procedures such as anti-reflux procedures, pyloromyotomy, and mucosal and submucosal tumor resections. however, these remain technically demanding procedures as they are hindered by the state of flexible technology which has difficult imaging, limited energy devices, no staplers, and cumbersome suturing abilities. an alternative approach is transgastric laparoscopy, which for almost decades has been shown to be a good procedure for pancreatic pseudocyst drainage and full-thickness and mucosal resection of various lesions. we propose to expand the indications of transgastric laparoscopy by using novel endoscopically placed transgastric laparoscopy ports (taggs, kansa, usa) to replicate endoscopic procedures such as endoluminal antireflux surgery. methods and procedures: under general anesthesia female pigs (mean weight . kg) had endoscopic placement of mm-intragastric ports (taggs, kansas, usa) using a technique similar to percutaneous endoscopic gastrostomy. a mm laparoscope was used for visualization. endoflip, (crospon, inc., galway, ireland) was used to measure cross sectional area (csa) and compliance of the gastroesophageal junction (gej) before and after intervention. laparoendoscopic-assited suture plication of the gej was performed using - sutures (polysorb®). once the taggs ports were removed, the gastrotomies were closed by using endoscopic clip. at the end of the protocol, animals were euthanized. results: five laparoendoscopic-assited sewing plication were performed. the mean operative time was , min (endoscopic evaluation: . min, tagss insertion: min, endoflip evaluation+ gej plication: , min, gastric wall closure: min). in all cases, this technique was effective in achieving adequate gej plication. median gej diameter (d) and median cross-sectional area (csa) pre-plication were . mm ( . - . mm) and . mm ( - mm ). after the procedure, these values were decreased to . mm ( . - . mm) and . mm ( - mm ) respectively (p= , ). median distensibility (d) and median compliance (c) pre-plication were . mm /mmhg ( . - . mm /mmhg) and . mm /mmhg ( , - , mm /mmhg). after the procedure, these values were decreased to , mm /mmhg ( . - . mm /mmhg) and . mm /mmhg ( . - . mm /mmhg) respectively (p= , ). no intraoperative events were observed. conclusion: a hybrid laparoendoscopic approach is a feasible alternative for performing intragastric procedures with the assistance of conventional laparoscopic instruments; especially in cases where the location of the intervention limits the access of standard endoscopy or where endoscopic technology is inadequate. further evaluation is planned in survival models and clinical trials. introduction: due to previous manipulation or submucosal invasion, colonic lesions referred for endoscopic mucosal resection (emr) frequently have flat areas of visible tissue that cannot be snared. current methods for treating residual tissue may lead to incomplete eradication or not allow complete tissue sampling for histologic evaluation. our aim is to describe dissection-enabled scaffold assisted resection (descar): a new technique combining circumferential esd with emr for removal of superficial non-lifting or residual "islands" with suspected submucosal involvement/fibrosis. methods: from to , lesions referred for emr were retrospectively reviewed. cases were identified where lifting and/or snaring of the lesion was incomplete and the descar technique was undertaken. cases were reviewed for location, prior manipulation, rates of successful hybrid resection and adverse events. results: lesions underwent descar due to non-lifting or residual "islands" of tissue. patients were % m, % f, and average age (sd ± . yrs). lesions were located in the cecum (n= ), right colon (n= ), left colon (n= ) and rectum (n= ). average size was mm (sd ± . mm). previous manipulation occurred in / cases ( % biopsy, % resection attempt, % tattoo). the technical success rate for resection of non-lifting lesions was %. there was one delayed bleeding episode but no other adverse events. approximately % of patients have been followed up endoscopically to date with no evidence of residual adenoma. conclusions: descar is a feasible and safe alternative to argon plasma coagulation and avulsion for the endoscopic management of non-lifting or residual colonic lesions, providing en-bloc resection of tissue for histologic review. further studies are needed to demonstrate long-term eradication and for comparison with other methods. results: patients underwent fully covered stent placement procedures. indications for stent placement were leak in patients ( sleeve; bypass) and stricture in patients ( bypass, sleeve). five patients had stent migration. three required surgical removal, one patient endoscopic repositioning and one passed the stent per rectum. all eight patients with enteric leak successfully underwent stent placement in conjunction with diagnostic laparoscopy and drainage. all but one of these patients developed an enteric leak perioperative to index procedure. the average duration of stent treatment in these patients was days ( - days). of the patients treated for a stricture, patients ( sleeve, bypass) failed treatment and required subsequent definitive operative revision. average length of time of stent treatment in these patients was days (range, - days) and five had severe intolerance. conclusions: endoscopic stent placement of leak may require multiple procedures and carries the risk of migration; however, this therapy seems to be an effective treatment. failure rates are higher with strictures and are not as tolerated by patients. background: colonoscopy is the most commonly performed endoscopic examination worldwide and is considered the gold standard for colorectal cancer screening. the quality of examination and endoscopic treatment is affected by a number of factors that are verified by recognized parameters such as cecal intubation rate and time (cir, cit), withdrawal time, adenoma detection rate (adr) and polyp detection rate (pdr). advanced endoscopic imaging improves accurate recognition of the nature and variety of pathologic lesions, while the endoscope tips, third eye retroscope and wide-angle endoscopy allow detection of lesions located on the proximal side of the intestinal folds. the aim of the study was to assess the suitability of wide-angle colonoscopy for the detection of colorectal lesions and to analyze the functionality of a special endoscope series regarding cir, cit and withdrawal time. introduction: leak is an uncommon but serious complication of gastrointestinal surgery. when identified post-operatively, percutaneous drains are used to manage abscesses and prevent further peritoneal contamination. if drain position is suboptimal, however, the consequences of persistent leak may necessitate a formal surgical intervention in a hostile abdomen. in select situations, we have utilized natural orifice transluminal endoscopic surgery (notes) methods to enter the abdominal cavity and place/reposition drains under direct endoscopic visualization a part of our comprehensive endoscopic management algorithm for leaks. methods and procedures: a prospectively collected database was queried for patients who had undergone transluminal endoscopic drain repositioning (tedr) as part of multimodal endolumenal therapy for leak (including interventions like defect closure, enteral feeding access, or endolumenal stent placement). inadequate drainage was identified pre-procedurally by undrained fluid collections in conjunction with clinical signs of sepsis. translumenal access was obtained via the leak site and carbon dioxide insufflation was used in all cases. the peritoneal cavity was surveilled and cleared of gross debris by irrigation and suction. intraabdominal drains were located endoscopically and fluoroscopically, grasped with an endoscopic snare or grasper and repositioned adjacent to the leak site to ensure better drainage. results: four patients ( female), average age (range - ), average body mass index (range - ) were managed with tedr as a component of endoscopic treatment of full-thickness gastrointestinal leak. two patients developed leak following revisional bariatric surgery. one patient had an acutely dislodged gastrostomy tube with intraperitoneal leak after multiple laparotomies recently closed with a granulating vicryl mesh. one patient developed a leak at an esophagojejunostomy following total gastrectomy. three patients had adequate drainage after the initial tedr, while one patient required tedr on two occasions. all patients had improved drainage demonstrated by resolution of clinical signs of sepsis and resolution of fluid collections. drains were removed as clinically indicated. conclusion: intraabdominal drains are an essential element in the management of full-thickness gastrointestinal leaks, but are not always able to be adequately positioned percutaneously. transluminal endoscopic drain repositioning via a gastrointestinal defect is a viable option to avoid surgical intervention in an otherwise hostile field and is a novel practical notes application. background: epiphrenic diverticula (ed) arise from increased intraluminal pressures, often secondary to achalasia or another underlying esophageal motility disorder which causes "pulsion" physiology. ed are traditionally thought to contribute to patients' symptoms of regurgitation and dysphagia, and are frequently resected at time of heller myotomy and fundoplication done for treatment of the primary motility disorder. ed excision carries significant risks (staple line leak, pulmonary complications, mortality), and little is known regarding patients with ed and esophageal motility disorder who undergo surgical myotomy without ed resection. the goal of this study was to compare outcomes of patients with ed and esophageal motility disorder who did and did not undergo diverticulectomy at time of myotomy and fundoplication. methods: retrospective analysis of prospectively collected database from to was performed. patients with diagnosis of ed undergoing surgical treatment of symptomatic esophageal motility disorder were included. all patients underwent laparoscopic heller myotomy with toupet fundoplication by a single surgeon at a tertiary referral hospital. patients were stratified according to whether ed was excised or not excised at time of primary surgery. patient-reported symptoms were obtained from pre/post-operative clinic evaluations and mailed surveys during the follow-up period. independent samples t-test and fisher's exact test were used to compare continuous and categorical variables respectively. results: ed was identified in patients prior to surgery. primary diagnoses included achalasia (n = ), nutcracker esophagus (n= ), and diffuse esophageal spasm (n= ). ed was excised in five patients ( . %) and not excised in ten patients ( . %), with no significant difference in frequency of preoperative dysphagia ( % vs. %, p= . ) or regurgitation ( % vs. %, p= . ) between groups respectively. reasons for non-resection included ed was too proximal (n= ), patient/surgeon preference (n= ), and small ed size (n= ). the resection group did not experience any leaks and there were no mortalities in either cohort during the follow-up period. at mean clinic follow-up of days, there was no difference in frequency of residual dysphagia in patients who did or did not undergo ed resection ( % vs. %, p= . ) and neither cohort reported residual regurgitation symptoms. conclusions: this study suggests that leaving ed in place during surgical treatment of an esophageal motility disorder may achieve similar rates of postoperative symptom control. while ed excision in this study did not cause significant excess morbidity, ed resection introduces risk of leak and requires more extensive surgery that may not provide significant benefit to patients. introduction: median arcuate ligament syndrome (mals) has been described in the literature as presenting with a constellation of symptoms including nausea, vomiting, weight loss, and post-prandial epigastric pain. while many of these symptoms are consistent with foregut pathology, a cohort of patients with mals presenting with delayed gastric emptying has not been described in the literature. in this study we report on the possible association of mals with delayed gastric emptying. methods: cases of mal release were collected between and . eight patients were identified who presented with mals and underwent subsequent mal release. all patients underwent laparoscopic or robotic surgery. patients were compiled into a retrospective database and their demographic, symptomatic, imaging, and outcomes data were analyzed. background: laparoscopic fundoplication (lf) is often performed to treat paraesophageal hernia and/or gerd. care is taken to select the right patients for the operation. some patients may not improve, and others experience dysphagia or bloating after surgery. factors associated with patient satisfaction after fundoplication would be helpful during the patient selection process. methods: a retrospective review of a prospectively collected database was performed. queried patients underwent lf from to . non-elective operations and fundoplications after heller myotomy were excluded. of this cohort, patients were included only if they responded to a two-year postoperative quality of life survey. surveys were distributed preoperatively, at three weeks, at one year, and at two years. the surveys include the reflux severity index, gerd-hrql, and dysphagia score. the gerd-hrql asks about patient satisfaction with their current state ( = dissatisfied, = somewhat satisfied, = very satisfied). the cohort was divided according to their answer to this question at two years. demographics and preoperative factors were compared between the groups with kruskal-wallis and fisher's exact tests. univariable and multivariable ordinal logistic regression was performed to identify preoperative symptoms associated with satisfaction at two years. scores on the surveys over time were were also analyzed. results: a total of patients were included in the analysis (dissatisfied = , somewhat satisfied = , very satisfied = ). the only significant demographic or preoperative difference was a high number of paraesophageal hernias in the 'very satisfied' cohort (p = . ). on univariable regression, younger age and paraesophageal hernia predicted satisfaction. several variables negatively predicted satisfaction with an or \ . multivariable regression, controlled for age and hernia type, identified throat clearing, post-nasal drip, and globus sensation as preoperative symptoms less likely to result in patient satisfaction (p = . , . , and . , respectively). subgroup analysis of patients with paraesophageal hernias revealed that patients with bloating preoperatively are less likely to be satisfied at two years. survey scores over time showed all groups improving over three weeks, but while satisfied patients continued to improved, dissatisfied patients symptomatically worsened over time. conclusion: this study confirms previous reports stating atypical symptoms of gerd are less likely to improve after lf. it also shows individuals with paraesophageal hernia tend to do quite well, unless they report bloating preoperatively. patient-centered analysis such as this can be useful when discussing postoperative expectations with patients, and may reveal opportunities to individualize operative approach. objective: the study was performed to assess whether sutured crural closure or mesh reinforcement for hiatal closure yields better results with regards to symptom resolution and recurrence post-operatively. material and methods: a prospective randomized controlled trial was carried out at grant medical college and sir j. j. group of hospitals, mumbai, india. patients were randomized to receive either sutured repair or mesh reinforcement of hiatal closure. outcomes of interest were symptom resolution, quality of life scores and recurrence in the postoperative period. results: patients were recruited for the trial ( -sutured repair, -mesh reinforcement). the two groups were comparable in terms of demographic profiles, symptom severity and findings at esophagogastroscopy and manometry in the pre-operative period as well as size of the hiatal defect measured intra-operatively. post-operatively the mesh repair group had significantly better symptom resolution in terms of early satiety, chest pain and regurgitation (p\ . ) while with respect to heartburn, dysphagia and post-prandial pain there was no significant difference between the improvements demonstrated. improvement in quality of life scores after either procedure was not significantly different. recurrence was higher in the suture repair group ( vs , p. ). recurrence lead to poorer symptom severity scores as well as quality of life scores and one patient underwent re-operation. the change in the symptom severity score from baseline after the procedure at months in the subgroup population. conclusion: mesh reinforcement results in a reduced rate of recurrence and offers excellent symptom control in the short-term without a rise in complications when compared to sutured repair for the closure of hiatal defects in laparoscopic hiatal hernia repairs. material and methods: in a period from to , patients underwent laparoscopic resection ( -gastric resection, -duodenal resection), using different techniques. all patients were investigated with upper gi endoscopy, eus and abdominal contrast-ct, which allows us to get the complete evaluation of tumor, including size, location, type of growth and the gi layer. based on the findings the decision on the type of resection was made. the majority of resections were wedge or partial resections, performed using endoscopic steplers or using ultrasound scissors followed by double-suturing of gatro/duodenotomy. in the cases of tumor location on the posterior gastric wall we mobilized the the greater curvature to get a direct approach to the tumor with extraluminal growth. in the cases with intraluminal growth we used transgastric approach with small , cm incision on the anterior gastric wall for endoscopic stepler. technically the most complex procedures were in the cases of tumor location close to anatomically narrow places and muscle sphincters (gastroesophageal junction, pylorus, duodenal bulb, duodenal flexure), with high risk of stenosis and dysfunction of anatomical sphincters. in such cases we used «lifting-technique» in which we dissect serous and muscle layers circumferentially around the tumor making partial enucleation of lesion followed by total resection preserving almost all normal tissue with minimal suturing and deformity at the site of surgery. ( : ), mean age was . years (sd ± . ), patients ( %) had mis. the type of reconstruction was predominantly with a "pull-up" technique (n = , . %) followed by the kirschner-akiyama procedure (n = , . %), stapled gastroplasty was performed in patients. all the anastomosis were performed at the level of the neck and only one of the patients had a stapled anastomosis, mean operative time was min (sd ± min) including resection of the specimen. primary neoplasms were predominantly hypopharynx (n = , . %), distal esophagus (n = , %), cervical esophagus (n = , . %) and thoracic esophagus (n = , . %). histologic types were mainly squamous cell carcinoma (n = , . %) and adenocarcinoma (n = , . %). mean of hospitalization days was . (sd ± . ). no complications were observed in patients and major complications (dindo-clavien ≥iiib) were found in patients. anastomotic leak was present in patients ( . %) and perioperative mortality ( days) was . %. progressive shift to laparoscopic surgery was evidenced through the years ( - : . %, - : . % and - : . %; p = . ) and reduction in major complications (p = , ) was observed. anastomotic leaks (p = , ) and perioperative mortality (p = . ) did not show significant differences in the present study. conclusions: results in our center show that major complications decrease with time after application of minimally-invasive surgery and no differences in anastomotic leaks and mortality were seen. current data has lead us to abandon open total esophagectomy as a first-choice procedure. introduction: minimal invasive three-fields esophagectomy for minimal invasion is the surgical standard for oncological procedures and benign diseases. cervical dissection has a risk of to % in some series, of, lesion or paralysis of the rnl, but the standard in mckeon approach is %. a high level of suspicion is needed because this type of lesion has an impact on postoperative evolution and the hospital stay. main: to describe three cases of rnl post esophagectomy paralysis in three planes by least invasion. methods: in a period of years, january to june , esophagectomies for bening disease were performed. three patients ( males female) with diagnosis of terminal achalasia and stenosis secondary to caustic ingestion consulted at the minimal invasion service fundcacion valle del lili. they were schedualed for minimal invasive three fields esophagectomy, one patient without complications and early discharge ( postoperative day) but occasional dysphagia, the other two required early reintubation after de surgery with ards, patient requiered tracheostomy, the second patient could be extubated after days but with occasional dysphagia. all three had mild hoarseness after surgery. the patient who required tracheostomy was decannulated at days without complication. results: the three patients underwent endoscopy without complication in the cervical anastomosis stenosis or disorder in the emptying of the gastric tube, swallowing study without alteration and laryngoscopy with paralysis of the left vocal cord. these patients went to speech therapy with total paralysis recovery at months corroborated with laryngoscopy, without dysphagia or hoarseness. conclusion: rnl innervates the larynx and upper esophageal sphincter, therefore lesion or paresis causes symptoms such as hoarseness, dysphagia, difficulty swallowing, aspiration, difficulty in coughing, pneumonia and ards. injury has a predecessor factor in pulmonary complications and prolongation of the hospital stay. % of these patients may require some surgical procedure to restore the function of rnl. noninvasive monitoring of the laryngeal nerve decreases the risk of injury. philip case report: multiple esophageal diverticula associated with achalasia introduction: achalasia is well defined disorder of increased lower esophageal sphincter tone ( ). epiphrenic esophageal diverticulum are a rare disorder believed to result from increased intraesophageal pressure often in conjunction with a motility disorder causing functional outflow obstruction. they are a pulsion-type pseudo-diverticulum with mucosal bulging most frequently from the right posterior esophageal wall ( ) . we present a very rare case of achalasia associated with multiple esophageal diverticula successfully treated with laparoscopic heller myotomy with dor fundoplication. case presentation: a year old woman presented with years of dysphagia, chest discomfort, regurgitation, and weight loss. esophagoscopy showed a patulous esophagus with multiple esophageal diverticula (figure ). barium esophogram demonstrated esophageal diverticula in the distal esophagus and delayed clearance of esophageal contrast (figure ). high resolution monometry revealed a hypertensive mean les, an aperistaltic body on of wet swallows, and panesophageal pressurization in of wet swallows -consistent with type ii achalasia by chicago classification ( ). we performed a laparoscopic heller myotomy with dor fundoplication. the myotomy was extended cm above the gasgtroesophageal junction and cm onto the gastric cardia. an anterior diaphragmatic defect with a moderate type hiatal hernia was repaired with two sutures, ensuring to not impinge the esophagus (figure ). at weeks post operatively the patient reports excellent results. her dysphagia and chest discomfort have entirely resolved. her eckhardt score improved from seven preoperatively to one post operatively. discussion: type ii achalasia is successfully treated in the majority of cases with laparoscopic heller myotomy and partial fundoplication ( ). however, esophageal diverticula typically require both myotomy as well as diverticulectomy for successful treatment ( ) . there is little experience with the surgical management of multiple esophageal diverticula. we propose a two stage surgical approach for these patients. we reason that the risk of esophageal leak or stenosis in the case of multiple esophageal diverticulectomies out weighs the proposed benefit. indeed epidemiologic studies indicate that the majority of esophageal diverticula are asymptomatic ( ) . in the event the patient remains symptomatic after myotomy a second stage operation with diverticulectomies would be possible. this single experience suggests that diverticulectomy may not be necessary in the case of multiple diverticula associated with achalasia. instead, treatment may be directed at relieving the functional obstruction responsible for the symptoms by performing laparoscopic heller myotomy with dor fundoplication. takahiro kinoshita, md, facs, masanori tokunaga, md, akio kaito, md, masahiro watanabe, md, shizuki sugita, md; national cancer center hospital east, japan objective: the optimal surgical approach for siwert type ii cancer is still controversial due to the anatomical complexity of the region. potential advantages of laparoscopic transhiatal approach have not been fully investigated. methods and procedures: we retrospectively analyzed consecutive patients with siewert type ii cancer who underwent laparoscopic transhiatal resection. indication of surgery is patients with siewert type ii cancer with less than cm esophageal invasion. regarding the extent of resection, basically proximal gastrectomy with the lower esophageal resection was selected, aiming at preservation of gastric reservoir function. in terms of reconstruction after proximal gastrectomy, double-tract method was performed. intraoperative peroral endoscopy was routinely employed for determination of the appropriate resection level of the stomach. esophagojejunostomy was employed by overlap method using a mm linear stapler. in order to obtain a wider operative field in the lower mediastinum, the diaphragmatic crus was dissected to widen the esophageal hiatus. results: in patients ( males and females), median operation time was minutes, and estimated blood loss was g. the rate of surgical morbidity was %, and that of anastomotic leakage was %. there was no mortality. the mean length of proximal margin was mm, and no positive margin was recorded. the -and -year overall survival rate was . % and %, respectively. conclusions: laparoscopic transhiatal resection for siewert type ii cancer is technically challenging, but appears feasible and safe when performed by an experienced surgical team. a largescale prospective study is necessary for final conclusion. introduction: mesh use for reinforcement of primary crural closure is controversial. synthetic mesh use poses a risk of erosion but there is no evidence that non-synthetic mesh is useful to minimize the risk of hernia recurrence. we evaluated a fully bioresorbable mesh made from poly- -hydroxybutyrate (p hb) for crural reinforcement after para-esophageal hernia (peh) repair. the aim of this study was to evaluate the safety and efficacy of p hb mesh at the hiatus in patients undergoing peh repair. this was a review of prospectively collected data on consecutive patients that had repair of a peh with reinforcement of the crural closure with p hb mesh. to be considered a peh at least % of the stomach was herniated into the chest. a collis gastroplasty or crural relaxing incision was added for short esophagus or crural tension when necessary. routine follow-up consisted of esophagogastroduodenoscopy (egd) at months for patients that had a collis gastroplasty and a barium upper gi study (ugi), high resolution manometry (hrm) and ph test in all patients at months. a hernia of any size identified during objective follow-up testing was considered a recurrence. overall, there was a significant difference in mean measured tension between the three subjective suture ratings by the surgeons. however, there was substantial variability and overlap amongst the surgeon's ratings (figure) . the tension necessary to approximate the crura during peh repair can be objectively measured and as expected increases progressively with anterior movement up the hiatus. while there was some correlation between a surgeon's subjective assessment of the tension necessary to bring the crura together and actual measured tension, there was wide variability and imprecision from one stitch to another. objective tension measurement may provide a more reliable assessment of when excessive force is being used to re-approximate the crura and potentially improve peh recurrence rates. ahmed introduction: paraesophageal hernia repairs are increasing in prevalence, and unfortunately carry a high recurrence rate. consequently, reoperation is expected to increase in frequency. published data on the outcomes of recurrent paraesophageal hernia (rpeh) repair is very limited. because of the technical difficulties of revisional surgery, we hypothesize that laparoscopic revisional paraesophageal hernia repairs are associated with high perioperative morbidity and poor patient outcomes. methods: all rpeh repairs performed by the foregut surgical service at our institution from to were reviewed. patients were included if their index operation was a true pehr (initial type hiatal hernia repairs were excluded, as well as multiply recurrent hernias). demographics, medical and surgical history, and operative notes from the index surgery were reviewed. details from standardized pre-operative symptom assessment, objective testing and operative details for the revisional surgery were collected. patients were routinely offered month post-operative upper gastrointestinal contrast evaluation. postoperative outcomes included a standardized symptom assessment and results of objective testing at any time after surgery. results: twenty six patients were identified who underwent repair of rpeh. demographic, operative and perioperative data was available for all patients (table ) . twenty four patients underwent followup symptom evaluation (two were lost to follow-up after the initial hospitalization). sixteen patients underwent follow-up objective testing by radiographic evaluation with contrast, endoscopy or both. these subgroups were used to calculate symptomatic and objective outcomes (table ) . conclusion: reoperative laparoscopic surgery for recurrent paraesophageal hernias is technically challenging as evidenced by long operative times. despite this, perioperative outcomes at a high volume center are good, with low morbidity and no mortality. importantly, symptomatic outcomes for this difficult problem are excellent. introduction: hypotension of the lower esophageal sphincter (hles) and the presence of hiatal hernia (hh) have both been associated with gastroesophageal reflux disease (gerd). the exact likelihood with which a hles or a hiatal hernia predict gerd continues to be defined. we hypothesize a synergistic interaction in those with hles and hh in predicting gerd as defined by a positive ph study. methods and procedures: between and , consecutive patients presenting to a surgical practice with symptoms most concerning for gerd, without prior antireflux surgery were evaluated by high resolution manometry (hrm), esophagogastroduodenoscopy (egd), videoesophagography (veg) and an ambulatory ph study. hles was defined as residual les pressure of\ mmhg, hh was defined as having been noted and measured by the radiologist, these were further categorized into any hh, - cm, [ - cm background: while clinical outcomes have been reported for antireflux surgery, there is limited data on postoperative outpatient encounters and their associated costs. the aim of this study is to evaluate the utilization of healthcare and its associated costs during the -day postoperative period following antireflux surgery. methods: we analyzed data from the truven health marketscan® research databases. patients ≥ years with an icd- procedure code or cpt code for antireflux surgery and a primary diagnosis of gerd during - were selected. only patients with continuous enrollment six months prior to the date of surgery and -days after surgery were analyzed. patients with a diagnosis of esophageal cancer or achalasia during the six-month period prior to antireflux surgery, a length of stay [ days following index procedure, a capitated plan, or patients who underwent emergency surgery were excluded. outpatient endoscopy was defined using icd- and cpt codes, and related readmission was defined by clinical classification software. introduction: the development of postsurgical gastroparesis following nissen fundoplication is poorly understood. in this study, we analyze the development of gastroparesis requiring intervention and other subsequent procedures following fundoplication and paraesophageal hernia (peh) repair procedures in the state of new york. methods: using a comprehensive state-wide administrative database (sparcs), we examined all in-patient and outpatient records for adult patients who underwent fundoplication or peh repair as a primary procedure for the treatment of gerd between the years of - . patients with an initial gastroparesis diagnosis were excluded from the analysis. through the use of a unique identifier, each patient was followed until for the subsequent diagnosis of gastroparesis or reoperation. surgical procedures for the treatment of gastroparesis included pyloroplasty, pyloromyotomy, or gastroenterostomy procedures. multivariable logistic regression models were used to identify independent predictors for having subsequent reoperation. results: a total of , patients were analyzed. this included , fundoplication patients ( . %) and , ( . %) with peh repair. in the fundoplication group, ( . %) patients had a follow-up diagnosis of gastroparesis or secondary procedure. ( . %) of the patients who underwent a primary peh repair procedure had a follow-up procedure or gastroparesis diagnosis (table ) . mean time to follow-up procedure or diagnosis was . years for the fundoplication group and . years for the peh repair group. the majority of the follow-up procedures in the fundoplication group were revisional procedures (fundoplication or peh repair) (n = , . %), while ( . %) patients were newly diagnosed with gastroparesis and/or underwent a secondary procedure for its treatment. conclusion: fundoplication and peh repair procedures have a relatively low post-operative incidence of gastroparesis following initial procedure for treatment of gerd. secondary fundoplication or peh repair was more commonly performed compared to any of the surgical procedures for gastroparesis for both procedures. further analysis of association with subsequent procedures is needed. during this procedure, gastro-esophageal reflux was evaluated and assigned to severe, moderate and slight category. if the reflux was observed slightly up to cervical esophagus, the case was assigned to moderate category. if the reflux was observed intensely up to cervical esophagus, the position was returned to head high position for the safety and the case was assigned to severe category. the anti-reflux surgery was considered in the moderate and severe categories. results: we have performed laparoscopic nissen procedure in cases. the mean operation time was min. the outcome was assessed by reflux test performed on - postoperative day, and the results showed the reflux was disappeared in every cases. median follow-up period of this study was months ( - months). in cases ( . %) ppi was restarted before months after the anti-reflux surgery. in cases ( . %) ppi was restarted after the anti-reflux surgery during the whole follow-up period of this study. the bmi of the patients had no relationship to the needed restart of ppi. to evaluate the degree of esophagitis objectively before and after the anti-reflux surgery we designed "the esophagitis score". in this scoring method, a number from - was assigned according to the degree of esophagitis along with the la classification. the results of the study have shown that the reflux esophagitis was improved obviously after the anti-reflux surgery even in the ppi restarted group (p. ). discussion: the number of gerd patients who needed anti-reflux surgery seems to be so high. to extract the patients who needed it remarkably is important. the anti-reflux surgery is most effective for the patients who really have the obvious reflux. reflux test is feasible because of its convenience and visual effects for the patients. the results of the laparoscopic nissen fundoplication were good and satisfied by the patients mostly. surg endosc ( ) :s -s introduction: fundoplication at the time of giant paraesophageal hernia repair is controversial. the proposed advantages are better reflux control and lower recurrence. disadvantages include fundoplication specific complications, might be unnecessary and may not decrease recurrence. we retrospectively reviewed giant paraesophageal hernia repairs (peh) with two point gastropexy in the fundus and body, and no antireflux procedure. data collected is postoperative gerd symptoms, postoperative proton pump inhibitors (ppis) therapy and recurrence. methods: a retrospective review of patients who underwent repair of giant peh from to december of . giant was defined as a hernia with % or more of the stomach above the diaphragm. follow up consisted of upper gi (ugi) study one year postoperatively and reflux symptom questionnaire. patients were followed every months in the surgery clinic and a ppi wean was initiated at the second postoperative visit. the primary outcome we evaluated was discontinuation of ppis. in addition, we utilized a standardized reflux scale and recurrence rates collected. chi-squared was used for statistical analysis. background: gastroesophageal reflux disease (gerd) is a highly prevalent disorder with a multitude of treatment options ranging from lifestyle modifications and medical management to surgical options. despite the numerous treatments available, there is still debate over which approach is most appropriate and effective for patients. this study aims to examine the effect of robotic hiatal hernia repair (rhhr) with the novel addition of esophagopexy in patients with gerd. methods: a single institution, single surgeon, prospectively maintained database was used to identify patients who underwent rhhr with a partial fundoplication and concomitant esophagopexy for gerd from november to july . patient characteristics, operative details and postoperative outcomes were analyzed. primary endpoint was resolution of subjective gerd symptoms and discontinuation of proton pump inhibitor (ppi). recurrence of hiatal hernia was a secondary endpoint. results: eleven patients were identified meeting the inclusion criteria (rhhr + esophagopexy) with a mean follow-up of . weeks ± . weeks. in regards to the rhhr, % underwent a partial fundoplication and the additional % underwent a re-do wrap. this patient cohort was . % female with a mean age of . ± . years. preoperative esophagogastroduodenoscopy (egd) was performed in % of patients with the study showing a hiatal hernia in . %, gastritis in . % and esophagitis in . % of patients. manometry was performed in . % of the patients showing % of these patients with esophageal dysmotility. esophagograms and ph studies were performed preoperatively in . % and . % of patients respectively. preoperatively, % of patients had a documented diagnosis of gerd and were taking a ppi and/or h blocker. after rhhr with esophagopexy, . % of patients had resolution of their gerd symptoms while . % (n = ) remained symptomatic. however, one of two patients reported a subjective decrease in symptom severity following the procedure. despite resolution of symptoms, . % remained on ppis. another % switched to h blockers and one patient discontinued all antisecretory therapy. none of the patients experienced recurrence of their hiatal hernia. conclusion: based on our data, rhhr with esophagopexy results in resolution gerd symptoms in over % of symptomatic patient. in patients with hiatal hernias and gerd, rhhr with esophagopexy does lead to resolution of symptoms, however, the majority of patients remained on ppis. long-term follow up is needed to investigate whether these patients are able to discontinue ppis and remain symptom free. chaya shwaartz, nadav zilka, mustapha siddiq, yuri goldes, md; sheba medical center, israel background: d gastrectomy for gastric carcinoma is a well-established procedure in patients undergoing surgery for gastric cancer and is the standard of care in our institution. reduced pain, early ambulation, and better cosmetics are some of the benefits of minimally invasive surgery for early gastric cancer. we aimed to describe our experience in laparoscopic d gastrectomies undertaken by a single surgeon in our institution. methods: this is a single-center retrospective review of prospectively collected d gastrectomies performed by a single surgeon. between november and february , laparoscopic subtotal/total gastrectomies were performed at sheba medical center, a tertiary center for forgut cancer. clinicopathological characteristics of the patients, surgical performance, postoperative outcomes and pathological data were collected. results: forty-five patients underwent laparoscopic gastrectomy. of these, had subtotal gastrectomy and had total gastrectomy. the median age in our series ( - ). most of the patients in our series had early gastric cancer (t - ) ( %). the mean average of dissected lymph nodes was ± . the mean operative time was ± . the postoperative complications, classified using the clavien-dindo classification. severe complications ([ cd iiia) rate was %. conclusions: laparoscopic d gastrectomy for invasive gastric cancer is safe and feasible when carried out in high-volume centers by an experienced surgeon as part of a multidisciplinary team with careful case selection and appropriate high-quality postoperative support. minimally invasive management of diaphragmatic hernias after esophagectomy: a case report introduction: esophagectomy is a common treatment for both benign and malignant pathologies of the foregut. hiatial paraconduit hernias are rare complications following esophagectomy. in this study, we review our experience with these rare diaphragmatic hernias. methods: a retrospective analysis of all patients presenting with hiatial hernia after esophageal resection at the university of oklahoma health science center between and was performed. data was abstracted from the medical record for evaluation and included demographics, symptoms, repair techniques and outcomes. no patients were excluded. results: a total of ten patients were identified to have paraconduit hernias. during this time interval, there were a total of esophageal resections performed. all patients had esophagectomy for malignant disease. seven of the patients have undergone surgery. two patients are asymptomatic and are being followed at their request, and one patient is pending elective correction. of the seven patients who underwent surgery, the median age was , with males and two females. six of the seven patients underwent minimally invasive ivor lewis esophagectomy and one had an open mckeown procedure. the median time from esophagectomy to hernia repair was months, with range from month to months. the most common presenting complaint was abdominal pain and nausea. one patient was noted to have a paraconduit hernia on postoperative day and taken to surgery for repair during the hospitalization. there was one death in a patient who presented with necrosis of the small bowel. the remaining patients all had laparoscopic approach. one patient required a hand port to reduce incarcerated colon and one patient was noted to have a cecal perforation during port closure requiring repair. all patients had herniated colon, with small intestine or pancreas herniation noted in three. repair was performed by reducing the viscera, a left phrenic relaxing incision, closure of the hiatus around the conduit and then closure of the diaphragmatic defect with mesh. at median follow up of months, there are no recurrences. conclusion: hiatal paraconduit hernias are becoming a frequent finding among survivors of esophageal cancer surgery. our study demonstrates that there is a propensity for patients who undergo minimally invasive esophagectomy to develop these hernias. the vast majority of patients can undergo laparoscopic repair. our recommendation is to perform a diaphragmatic relaxing incision and liberal use of mesh. early results appear to be favorable regarding recurrence. aim: there have been several reports illustrating the safety and efficacy of various surgical techniques in performing laparoscopic esophagojejunostomy (ej). this study aims to compare two established methods of ej anastomosis -circular stapling with purse-string suture ("lap-jack") and linear stapling technique -in laparoscopic total gastrectomy. methods: patients diagnosed with gastric cancer underwent intracorporeal ej anastomosis in laparoscopic total gastrectomy from january, to october, . cases used the circular stapler with purse-string "lap-jack" method, and patients used the linear stapling method for ej anastomosis. were matched using propensity scores, and retrospective data for patient characteristics, surgical outcome, and post-operative complications was reviewed. the two groups showed no significant difference in age, bmi, or other clinicopathological characteristics, and there was no conversion to an open procedure. after propensity score matching analysis, the linear group had significantly shorter operating time ( . ± . vs . ± . , p≤ . ) and more sufficient proximal margin ( . ± . vs . ± . , p = . ). no significant difference was found in estimated blood loss, retrieved lymph node, hospital stay, and time for first flatus. there was no postoperative mortality. early postoperative complication of the circular and linear group occurred in ( . %) and ( . %, p = . ) patients respectively. ej leakage occurred in ( . %) cases from each groups, with ( %) case from both group needing radiologic or surgical intervention. no other significant difference in early complication was found. late complication was observed in ( . %) cases (circular = linear = , p = . ) with ej anastomosis stricture in the linear group, but there was no statistical significance. conclusion: both circular stapling and linear stapling techniques are feasible and safe in performing intracorporeal ej anastomosis during laparoscopic total gastrectomy. linear-stapling technique had more sufficient proximal margin and shorter operating time. there was no significant difference in anastomosis related complication between the two groups. masahiro watanabe, masanori tokunaga, akio kaito, shizuki sugita, takahiro kinoshita; national cancer center hospital east, gastric surgery division background: although the current standard treatment for advanced gastric cancer (agc) is open gastrectomy, laparoscopic gastrectomy (lg) is increasingly performed, especially in the east. however, it is a technically demanding procedure, and the feasibility remains unclear. the aim of the present study was to clarify the feasibility of lg for agc. patients and methods: the present study included patients who underwent lg for agc between and . the indication of lg has gradually expanded in our institute, and is currently any stage gastric cancer except for gastric cancer obviously invading adjacent organs or gastric stump carcinoma. we retrospectively reviewed short-and long-term surgical outcomes of the patients. results: male/female ratio was : , and median age (range) was ( - ) years. distal gastrectomy was most frequently performed ( %), followed by total gastrectomy ( %). median operation time and intraoperative blood loss was ( - ) minutes and ( - ) g, respectively. clavien-dindo grade iii or more complication rate was . %. with a median followup period of months, the -year recurrence free survival rates of pstage ii and iii patients were % and %, respectively. conclusion: the outcomes of lg for agc are satisfactory, provided that an experienced team performs the surgery. introduction: the present study aims to evaluate the predictive value of indocyanine green (icg) for the detection and prevention of anastomotic leak following esophagectomy. anastomotic leak is a highly morbid and potentially fatal complication of esophagectomy. ensuring adequate perfusion of the gastric conduit can minimize the risk of postoperative leak. intraoperative evaluation with fluorescence angiography using icg offers a dynamic assessment of gastric conduit perfusion, and can guide anastomotic site selection. methods: a search of electronic databases medline, embase, scopus, web of science and the cochrane library using the search terms "indocyanine/fluorescence" and esophagectomy was completed to include all english articles published between and august . articles were selected by two independent reviewers based on the following major inclusion criteria: ( ) esophagectomy with gastric conduit reconstruction; ( ) use of fluorescence angiography with indocyanine green to assess perfusion; ( ) age ≥ years; ( ) sufficient outcome data for the calculation of leak rates and ( ) sample size ≥ . the quality of included studies was assessed using the quality assessment of diagnostic accuracy studies- . results: our literature search yielded potential studies, of which studies were included for meta-analysis after screening and exclusions. there were eleven prospective and three retrospective studies. the pooled anastomotic leak rate when icg was used was found to be %. pooled sensitivity and specificity for leak detection were . ( . - . ) and . ( . - . ), respectively. when studies involving intraoperative modifications were removed, pooled sensitivity and specificity were only marginally changed to . ( . - . ) and . ( . - . ), respectively. the diagnostic odds ratio was found to be . ( . - . ) across all studies and . ( . - . ) when intraoperative interventions were excluded. only three trials included a control group, giving a sample size of . in studies with a comparator group, icg was associated with an % reduction in the risk of anastomotic leak [or: . ( . - . )]. conclusions: in non-randomized trials, the use of icg as an intraoperative tool for visualizing vascular perfusion and conduit site selection, is promising. however, poor data quality and heterogeneity in reported variables limits cross-study comparisons and generalizability of findings. randomized, multi-center trials are needed to account for independent risk factors for leak rates and to better elucidate the impact of icg in predicting and preventing anastomotic leaks. objective: robotic assistance for bariatric surgery represents a novel application of a rapidly emerging technology. its safety and efficacy remains primarily characterized by smaller, singleinstitution studies. in this investigation, the influence of robotic assistance on short-term perioperative outcomes is contrasted with the more established primary multi-port laparoscopic approach for patients undergoing roux-en-y gastric bypass (rygb), using data from a national bariatric database. methods: a retrospective analysis of , robotic-assist and , laparoscopic rygb patients from the metabolic and bariatric surgery accreditation and quality improvement program national database were reviewed for differences in patient characteristics and short-term outcomes. on bivariate analysis, variables associated with primary outcomes of -day reoperation, readmission and reintervention were imputed into multivariate analyses to determine independent significance. results: robotic-assist bypass patients were older (p\. ), had a higher prevalence of comorbidities and had concomitant operations more frequently performed during surgery (p\. ). on bivariate analysis, robotic-assist patients had a higher rate of readmission than laparoscopic patients ( . % vs. . %; p=. ), but no differences in -day reoperation ( conclusion: robotic-assistance does not confer an increased rate of morbidity and mortality after rygb, and represents a feasible surgical modality for the surgeon willing to adopt the technology and accept its limitations. alicia m bonanno, md, brandon tieu, md, farah husain, md; oregon health and science university introduction: marginal ulcer is a common complication following roux-en-y gastric bypass with incidence rates between and %. most marginal ulcers resolve with medical management and lifestyle changes, but in the rare case of a non-healing marginal ulcer there are few treatment options. revision of the gastrojejunal (gj) anastomosis carries significant morbidity and mortality with complication rates ranging from to %. thoracoscopic truncal vagotomy (ttv) may be a safer alternative with decreased operative times. the purpose of this study is to evaluate the safety and effectiveness of ttv in comparison to gj revision for treatment of recalcitrant marginal ulcers. methods and procedures: a retrospective chart review of patients who required surgical intervention for non-healing marginal ulcers was performed from st september to st september . all underwent medical therapy along with lifestyle changes prior to intervention and had preoperative egd that demonstrated a recalcitrant marginal ulcer. revision of the gj anastomosis or ttv was performed. data collected included operative time, ulcer recurrence, morbidity rate, and mortality rate. statistical analysis was performed using t-test and fischer's exact test. results: a total of fifteen patients were identified who underwent either gj revision (n= ) or ttv (n= ). there were no -day mortalities in either group. mean operative time was significantly lower in the ttv group in comparison to gj revision ( . ± vs. . ± minutes respectively, p= . ). recurrence of the ulcer was not significant between groups and occurred following gj revisions and ttv. overall complication rate was not significantly different with % in the gj revision group and % in the ttv group. complications included anastomotic leak ( gj), anastomotic stricture ( gj), aspiration ( ttv), dysphagia ( gj and ttv), and dumping syndrome ( gj). conclusions: our results demonstrate that thoracoscopic vagotomy may be a better alternative with decreased operative times and similar effectiveness. however, further prospective observational studies with a larger patient population would be beneficial to evaluate complication rates and ulcer recurrence rates between groups. we present a case of a -year-old female with a history of thyroid cancer who initially presented to an outside hospital complaining of reflux, abdominal pain, early satiety, and -pound unintentional weight loss. endoscopy demonstrated a cm pre-pyloric mass; with initial biopsies of the mass demonstrating only gastric mucosa. endoscopic ultrasound and fna of the lesion also failed to elucidate its pathology. due to the pyloric location of the mass and inability to rule out invasive malignancy, we recommended a robotic-assisted transgastric submucosal resection with possible distal gastrectomy. intraoperatively we found a -degree circumferential pre-pyloric exophytic sessile tumor. frozen sections suggested a benign papillary tumor therefore we proceeded with submucosal resection. the resulting mucosal defect and gastrotomy were closed primarily with absorbable suture. final pathology showed the tumor to be a tubulovillous adenoma with high grade dysplasia arising against a background of intestinal metaplasia. the resection margins were negative for dysplasia. the postoperative course was complicated by a minor leak which did not require operative intervention and subsequent gastric outlet narrowing which required endoscopic dilation and feeding tube placement. however, the patient has recovered well and has advanced to diet as tolerated. gastric adenoma has a prevalence of . - . % in the western hemisphere. the risk of carcinomatous transformation in gastric adenomas is related to size, degree of dysplasia, and villosity. gastric adenomas are considered precancerous lesions. pre-operative pathologic diagnosis of dysplasia is often elusive as biopsies will often miss or under-grade the lesion. guidelines advocate for complete resection with either endoscopic submucosal dissection or surgical resection depending on surgeon preference and local expertise. endoscopic resection has been shown to be safe and efficacious in the removal of adenomas with good long-term outcomes. in this case the pathology of the lesion was unclear after multiple unsuccessful biopsies and required a surgical diagnosis to rule out invasive malignancy. management of gastric adenomas, while rare, may require a multidisciplinary approach between surgical endoscopy, minimally invasive surgery, and surgical oncology to achieve local control in an oncologically sound manner. we show that transgastric submucosal resection can be achieved in a minimally invasive fashion using robotic assistance. objective: parahiatal hernia is a rare type of diaphragmatic hernia with incidence of . - . %. para-hiatal hernias arises lateral to the left crural musculature adjacent to but separate from the oesophageal diaphragmatic hiatus. in view of its rare occurance and little clinical suspicion, it is almost never diagnosed clinically. the current case report is intended to depict the clinical profile of an intraoperatively diagnosed para-hiatal hernia and feasibility of laparoscopic repair of parahiatal hernias. method: laparoscopic fundoplication is frequently performed at grant medical college and sir j. j. group of hospitals, india. during one such case intraoperatively para-hiatal hernia was diagnosed. discussion: primary or true parahiatal hernias occur as a result of a congenital weakness and secondary defects follow hiatal surgery. the primary treatment of para-hiatal hernia is mesh-plasty. this is coupled with fundoplication in cases of large hernia and those symptomatic for gastroesophageal reflux disease. laparoscopic repair of these uncommon hernias is safe, effective and provides all of the benefits of minimally invasive surgery. conclusion: due to its rare occurrence, knowledge about this condition among laparoscopic surgeons is important to avoid diagnostic dilemma. knowledge about its management aids intraoperatvely to avoid performing incomplete procedure. introduction: extended indications of endoscopic resection for early gastric cancer (egc) have been widely accepted. according to current japanese guidelines, additional gastrectomy with lymph node dissection (lnd) is recommended for patients proven to have potential risks of lymph node metastasis (lnm) on histopathological findings. on the other hand, the frequency of lnm in these patients is exteremely low. the aim of this study was to elucidate the accurate risk of lnm based on the number of risk factors (rf) for possible lnm, and to compare the stratified risk of lnm with predicted risk from additional radical resection. methods and procedures: we enrolled egc patients who did not meet absolute or extended indications of endoscopic resection, and investigated the risk stratification of lnm according to the total number of lnm rfs described below; ( ) sm , ( ) lymphatic vessels invasion, ( ) undifferentiated adenocarinoma and [ mm in diameter, and ( ) [ mm in diameter and ulcer formation. we compared the stratification risk to the surgical risk that was calculated based on the japanese national clinical database (ncd) risk calculator in patients with additional gastrectomy after esd. results: the total number of lnm rfs and frequency of lnm were significantly correlated ( / rf; . %, rfs; . %, rfs, . %, rfs, . %; p. , fischer exact test). the estimated frequency of lnm was found to be lower than the predicted value of in-hospital mortality rate based on ncd in . % of / rf-patients who underwent additional gastrectomy with lnd after esd. the present study suggested that some patients must be over-indicated for additional gastrectomy with lnd, and no additional surgical treatment or less invasive surgery, such as local lnd (sentinel node navigation surgery or lymphatic basin resection), might be indicated for some patients with low number ( / rf) of lnm risk factors after esd. aims: laparoscopic proximal gastrectomy has been applied for early gastric cancer in upper third. we previously reported outcomes of laparoscopic total gastrectomy in managing this condition. in this study, we applied this modified technique for upper third early gastric cancer with double tract reconstruction. it is expected that our technique could be useful for treating these cases. methods: from april of to june of , consecutive patients with upper third early gastric cancer were assigned to undergo surgical treatment with proximal gastrectory at our hospital. we had cases of total gastrectory for upper third early gastric cancer in the same study period. background: laparoscopic total gastrectomy for remnant gastric cancer is much more difficult than common laparoscopic total gastrectomy due to severe adhesions to adjacent organs, displacement of anatomical structure. purpose: the aim was to analyze cases of laparoscopic total gastrectomy for remnant gastric cancer at the department of surgery of juntendo university urayasu hospital between november and april . method: we analyzed outcome and feasibility of laparoscopic total gastrectomy surgery for remnant gastric cancer. and we compared with laparoscopic total remnant gastrectomy ( cases) versus laparoscopic total gastrectomy ( cases) in our hospital. results: in the previous laparoscopic surgeries. we performed laparoscopic distal gastrectomy in cases, laparoscopic proximal gastrectomy in pcases, and open distal gastrectomy in cases. all cases were performed laparoscopic total gastrectomy with r-y reconstruction. case of them had been converted to open surgery due to severe adhesions. the mean operative time was min and the mean blood loss was ml. there were no intraoperative complications, and there were postoperative complications as a pancreatic fistula and a bowel obstruction. however, there were no intra-operative complications more than grade according to the clavien-dindo classification. the mean postoperative hospital stay was . days. all cases were without recurrence. thus, there were no significant differences in operative time, bleeding volumes, intra and postoperative complications and hospital stay compared with laparoscopic total gastrectomy. conclusions: laparoscopic total remnant gastrectomy can be performed with similar short-term outcomes to laparoscopic total gastrectomy, and may be feasible and safe procedure, and can become an option of therapeutic strategy. although this study was not powered to show lower recurrence rates with synthetic absorbable as compared to biologic, the . % recurrence rate is consistent with other series utilizing this mesh. it is interesting to note the difference in time to recurrence. these results suggest that while synthetic absorbable mesh may result in lower recurrence rates, recurrence seems to occur earlier. the results also suggest that deconditioning (lower bmi), and difficult cases and/or recovery may predispose to recurrence. these findings can help inform lf mesh selection and predict which patients are at higher risk of recurrence. introduction: little discussion of gastroparesis (gp) following laparoscopic paraesophageal hernia repair (lphr) has been reported in the literature. we wished to examine the incidence in our institution, and identify potential risk factors for development of gastroparesis following lphr. methods and procedures: a single institution retrospective chart review was preformed using cpt codes corresponding to paraesophageal hernia repair and fundoplication to identify patients undergoing laparoscopic paraesophageal hernia repair over a five year period ( / / - / / ) by three surgeons. emergency procedures and reoperations were excluded. in total, patients undergoing non-emergent first time lphrs were identified. size of the hiatal defect was identified when able, via either measurement between the diaphragmatic crura on ct or by medical record documentation. data obtained included sex, age, hernia type, mesh usage, and existence of specific comorbidities associated with gastroparesis. presence of gastroparesis was identified either by documentation of diagnosis via clinical judgment, or by results of gastric emptying nuclear medicine studies, with timing being no longer than months from date of surgery. independent students t-test and fisher exact test were used to determine statistical differences between the groups. results: patients undergoing non-emergent first time lphrs were identified. of these, we were able to obtain the size of the hiatal defect in patients. patients overall were diagnosed with gastroparesis, with an overall incidence of . %. when comparing all patients who developed gastroparesis to those who did not, only females comprised the group which did develop gastroparesis ( males/ females with gp, males/ females without gp, p= . ). age was also found to be greater in the group which developed gastroparesis. for patients in which the size of the hernia defect was identified, the average age was years older in the group diagnosed with gastroparesis ( step under laparoscopic view, left part of the lesser omentum was cut with preserving the hepatic branch of vagus nerve. the right crus of the diaphragma has been dissected free from the soft tissue around the stomach and abdominal esophagus. in this step the fascia of the right crus should be preserved and the soft tissue should not been damaged to avoid bleeding. after cutting the peritoneum just inside the right crus, the soft tissue was dissected bluntly to left side. then the inside margin of the left crus of the diaphragma was recognized from the right side. in this part of the procedure, laparoscope uses trocar (a), the assistant uses trocar (b) to pull the stomach to left lower side and the operator's right hand uses trocar (c). step the branches of left gastroepiploic vessels and the short gastric vessels were divided with ultrasonic coagulation and dissection device. the left crus of the diaphragma was exposed and the window at the posterior side of the abdominal esophagus was widely opened. in this part of the procedure, laparoscope uses trocar (a) at the beginning of dividing left gastroepiploic vessels, trocar (b) when dividing short gastric vessels. step the right and left crus are sutured with interrupted stitches to reduce the hiatus. from the right side, the fundus of the stomach is grasped through the widely opened window behind the abdominal esophagus. then the fundus of the stomach is pulled to obtain a degree ''stomach-wrap'' around the abdominal esophagus (fundoplication). using - non-absorbable braided suture, stitches are placed between both gastric flaps. purpose: laparoscopic gastrecomy has been widely adopted as the treatment of choice by many countries and institutions. internal hernia is a well-known complication after rouxen-y gastric bypass in the field of bariatric surgery. however, there were only a few reports of internal hernia after gastrectomy in gastric cancer patients. the purpose of this study was to analyze the incidence and clinical features of internal hernia after gastric cancer surgery in a high-volume center. method: , gastric cancer patients who underwent curative gastrectomy at seoul national university bundang hospital between january and december were retrospectively reviewed in this study. internal hernia was classified into two types, mesenteric hernia and petersen's hernia. result: patients who underwent distal gastrectomy (dg) with reconstruction by billroth ii, rouxen-y gastrojejunostomy and uncut rouxen-y gastrojejunostomy, total gastrectomy (tg) with esophagojejunostomy, and proximal gastrectomy with double tract reconstruction (pg dtr) with esophagojejunostomy and gastrojejunostomy had potential space for internal hernia. among these patients, ( . %) were determined as internal hernia by computed tomography and patients ( . %) underwent surgical treatment of internal herniation. two patients were conservatively managed. all patients suffered from abdominal pain and / ( %) patients showed nausea and vomiting. the median interval between the initial gastrectomy and surgery for internal hernia was days. mesenteric hernia was observed in cases and petersen's hernia in cases. since we started closing the mesenteric and petersen's defects from may of , there were only cases ( %) observed afterwards but there were cases ( %) before closure of the defects. conclusion: internal hernia after gastrectomy is likely underreported. although we analyzed patients with internal hernia, there might be more patients with mild symptoms who were managed conservatively by their own. a high degree of suspiciousness for internal hernia should be maintained in patients presenting symptoms like nausea, vomiting and abdominal pain after gastrectomy with potential space for internal hernia. with our experience, closure of the mesenteric and petersen's defect is helpful in reducing internal hernia. however, due to low incidence, a multicenter retrospective study is necessary. introduction: the increased incidence of anemia in patients with a hiatal hernia (hh) has been clearly demonstrated, as has resolution of anemia after hh repair in these patients. despite this, the implications of preoperative anemia on postoperative outcomes have not been well described. in this study, we aimed to identify the incidence of preoperative anemia in patients undergoing hh repair at our institution and sought to determine whether preoperative anemia had an impact on postoperative outcomes. methods and procedures: using our irb-approved institutional hh database, we retrospectively identified patients undergoing hh repair between january and april at our institution. we identified all patients with anemia, defined as serum hemoglobin levels less than mg/dl in men and mg/dl in women, measured within two weeks prior to surgery, and compared this cohort to those that had normal hemoglobin values preoperatively. specific perioperative outcomes analyzed included: estimated blood loss (ebl), operative time, need for blood transfusion, failure to extubate postoperatively, intensive care unit (icu) admission, postoperative complications, length of stay (los), and -day readmission. results: we identified patients undergoing hh repair, of which had preoperative bloodwork available for review. the average age was years and the majority of patients were female ( %, n= ). most were treated electively ( %, n= ) and with a minimally invasive approach ( %, n= ). patients ( . %) had preoperative anemia. compared to patients without anemia, patients with anemia had increased rates of failed extubation postoperatively ( . % vs. . %, p= . ), increased icu admissions ( . % vs. . %, p= . ), increased need for perioperative blood transfusions ( . % vs %, p= . ), and increased rates of postoperative complications ( . % vs. . %, p. ). although mean los ( . days vs. . days, p . ), mean operating time ( mins vs. mins, p= . ), and ebl ( ml vs ml, p= . ) were greater in the anemic group, they did not reach statistical significance, and there was no significant difference in -day readmission rate ( . % vs . %, p= . ). conclusions: anemia diagnosed on preoperative bloodwork appears to be associated with increased failure to extubate postoperatively, need for icu admissions, need for perioperative blood transfusion, and increased overall complication rate after hh repair. however, we found no significant difference in los or -day readmissions between anemic and non-anemic patients. since the majority of patients in this analysis underwent elective repairs, these results would support the preoperative treatment of anemia in patients undergoing hh repair. few studies have compared the procedures' long-term effectiveness with none looking beyond years. this study sought to characterize the efficacy of laparoscopic toupet versus nissen fundoplication for types iii and iv hiatal hernia using a telephone survey. methods and procedures: with irb approval, a review of all laparoscopic hiatal hernia repairs with mesh reinforcement performed over seven years at a single center by one surgeon was conducted. patient demographics and perioperative characteristics were recorded. hiatal hernia was classified per published sages guidelines as type iii or iv using operative reports and preoperative imaging. patients with type i or ii or recurrent hiatal hernia and patients receiving concomitant procedures were excluded. the gerd-health related quality of life survey was administered by telephone no earlier than months postoperatively. patients responded to items concerning symptom severity using a -point scale ( =no symptoms to =symptoms are incapacitating to do daily activities). symptoms surveyed included heartburn ( items), difficulty swallowing ( item) and regurgitation ( items introduction: as the thoracic esophageal carcinoma has a high metastatic rate of upper mediastinal lymph nodes, especially along the recurrent laryngeal nerve (rln), it is crucial to perform complete lymph node dissection along the rln without complications. although intraoperative neural monitoring (ionm) during thyroid and parathyroid surgery has gained widespread acceptance as the useful tool of visual nerve identification, the utilization of ionm during esophageal surgery has not become common. here, we describe our procedures focusing on a lymphadenectomy along the rln utilizing the ionm. methods and procedures: we first dissect ventral and dorsal side of the esophagus preserving the membranous structure (meso-esophagus), which contains tracheoesophageal artery, rln and lymph nodes. we next identify the location of the rln which runs in the meso-esophagus using ionm before visual contact. after that, we perform lymphadenectomy around the rln preserving the nerve. this technique was evaluated in consecutive cases (neural monitoring group; nm) of esophagectomy in prone positioning, and compared with our historical cases (conventional method group; cm background: laparoscopic hiatal hernia repair, particularly large type and type hernias, is associated with high recurrence rates. various use of overlay mesh reinforcement have been described in an attempt to improve outcomes. unfortunately, overlay use of biologic mesh continues to result in high recurrence rates, and more effective repairs employing permanent mesh raise serious erosion concerns and are therefore rarely used. we theorize that employing an interlay technique with permanent mesh (positioned between both crura) will help enhance crural closure and improve rates of hiatal hernia recurrences with minimal risk of erosion. methods: we reviewed all patients who underwent a laparoscopic hiatal hernia repair from april to august by a single surgeon from a prospectively maintained database at a tertiary care referral center (n= ). patients who underwent surgery for achalasia with concurrent hiatal repair were excluded. during this time frame, a new interlay technique of polypropylene mesh was employed upon suture closure of the crura. outcomes of repair were retrospectively reviewed. recurrence of hernia was identified by positive work up of patient's symptoms (new onset dysphagia, gerd, pain). results: a total of consecutive laparoscopic hiatal hernia repair were reported in a period of months. interlay polypropylene mesh was utilized in all repairs. patients were majority females ( . %), had a median age of and had a mean bmi of . . eleven ( . %) patients were redo repairs. majority of patients received a nissen fundoplication (n= , . %) followed by a toupet fundoplication (n= , . %). median length of stay after surgery was day. median follow up was days (range: - days). there were zero reported recurrences. conclusion: laparoscopic hiatal hernia repair with interlay polypropylene mesh appears in the short term to be a safe and durable technique to reduce the incidence of hiatal hernia recurrences. further studies are needed to assess more long term outcomes of this novel technique. zia kanani , melissa helm , max schumm , jon c gould, md ; introduction: laparoscopic fundoplication remains the current gold standard surgical intervention for medically refractory gastroesophageal reflux disease. studies suggest that on average - % of patients undergo reoperative surgery due to recurrent, persistent, or new symptoms. the primary objective of this study was to characterize the long-term symptomatic outcomes of primary and reoperative fundoplications in a clinical series of patients who have undergone one or more fundoplications. methods: patients who underwent laparoscopic primary or reoperative fundoplication between and by a single surgeon were retrospectively identified using a prospectively maintained database. patients undergoing takedown of a failed fundoplication and conversion to roux-en y gastric bypass (for morbid obesity, severe gastroparesis, or or more prior failed attempts) were excluded from the current analysis. all procedures were performed laparoscopically. patients were asked to complete the validated gerd-health related quality of life (gerd-hrql) survey prior to surgery and postoperatively at standard intervals to assess long-term symptomatic outcomes and quality of life. gerd-hrql composite scores range from (highest disease-related quality of life) to (lowest diseaserelated quality of life, most severe symptoms conclusions: patients who need to undergo reoperative fundoplication have more severe gerd-related symptoms at years post-op compared to patients undergoing primary fundoplication. however, good outcomes and morbidity rates of laparoscopic reoperation that approximate that of a primary fundoplication are possible in the hands of an experienced surgeon. adenocarcinoma of duodenum: surgical or endoscopic treatment? introduction: it is well known that the adenocarcinoma of the duodenum (adc) is a quite rare lesion infact represents % of cancer of the small bowel and % of these are localized in the periampullary area: % affect the sub-papillary tract and only % the supra-papillary segment of the duodenum. the adc may arise from duodenal polyps (familial polyposis, or gardner's syndrom or be associated with coeliac disease). until now the treatment was the pancreatoduodenectomy (for anatomo-surgical reasons and for the possibility of regional lymphonode resection). infact in my series of of such procedures, where performed for duodenal cancer. in this last years patients with adc of supra-papillary segment of the duodenum underwent endoscopic submucosal dissection (esd). the purpose of this study were to check the feasibility of the esd in treating such cases. in our experience this kind of endoscopic operation was feasible with high complication rate; perforation in cases ( . %); and bleeding occurred in case ( . %). all the complications were successfully treated endoscopically and the long-term outcomes was favorable. consitering the high rate of complications, the difficult and long procedure, the compliance of patients (c ), the general anesthesia, a very very skilled endoscopist is needed. conclusions: the esd represent a new endoscopic approach enstablished in clinical practice: end is performed following the intraluminal path ( rd space) wich, unlike the others, remain virtual and has to be created by dissecting and expanding the tissues layer between the mucosa and the muscolaris propria allowing the endoscope to gain access. the benefit of esd for treating the adc of the supra-papillary segment of the duodenum, according to our experience, must be validate in the future; a pre-operative pet-tac scan examination must be performed in order to demostred the lesion of the duodenum and if there is any limphatic involvement and no infiltration of the head of the pancreas. yoontaek lee, md, sa-hong min, md, young suk park, md, sang-hoon ahn, md, do joong park, md, phd; seoul national university bundang hospital purpose: this study summarizes the single institution experience of laparoscopic gastrectomy in advanced gastric cancer and evaluates the postoperative morbidities and long-term oncologic outcomes. methods: a total of , laparoscopic gastrectomy for advanced gastric cancer were performed at seoul national university bundang hospital between may and may . the characteristics of patients, surgical techniques, postoperative morbidities, and long-term oncologic outcomes were retrospectively reviewed using electronic medical records. results: patients required conversion to open surgery. the reasons of conversion to open surgery were advanced stage (n= ), intraoperative bleeding (n= ), adhesion due to previous abdominal operation (n= ), small abdominal cavity (n= ), associated disease (n= ), and intraoperative pleural injury (n= ). the mean hospital stay was . days for distal gastrectomy, . days for total gastrectomy, . days for proximal gastrectomy, and . days for pylorus preserving gastrectomy. the mean number of collected lymph nodes was . for distal gastrectomy, . for total gastrectomy, . for proximal gastrectomy, and . for pylorus preserving gastrectomy. the rates of postoperative complications of grade ii or more were . %. there was one case of postoperative mortality due to delayed bleeding after discharge. old age was the only independent predictor of surgical morbidities. background: intrathoracic gastric volvulus is a life-threatening condition of paraesophageal hernia. the therapeutic is a challenge because in acute volvulus it may lead to gastric strangulation and necrosis. most patients are elderly and with a significant associated medical illness which has higher morbidity and mortality of major surgery. we present a laparoscopic surgery is safe in paraesophageal hernia with acute intrathoracic gastric volvulus in a high-risk patient. case presentation: an -year-old woman with underlying of diabetes mellitus and hypertension was transferred from an outlying hospital with anemia, dysphagia, urinary tract infection and aspiration pneumonia. she had severe recurrent emesis after admission. ct scan of the chest and abdomen revealed a large esophageal hiatal hernia, and most of the stomach was in the inferior mediastinum with organoaxial gastric volvulus. endoscopy revealed flat pigmented spot gastric ulcer which compatible with cameron lesion and twisting of gastric folds without evidence of ischemia. the endoscopic reduction was unsuccessful. a laparoscopic surgery was performed and the herniated stomach was successfully reduced. the hernial sac was excised. the crura were approximated and reinforced with composite mesh. nissen fundoplication was performed along with gastropexy of the greater curve of the stomach to the abdominal wall. there was no perioperative complication. she tolerated enteral diet on a postoperative day . she had an uneventful recovery and discharged in weeks after treatment of her associated medical illnesses. she had no relapse of previous symptoms at her six-month follow-up assessment. discussion: endoscopic reduction of acute gastric volvulus may be the first option in a patient with severe comorbidities. however, if there is evidence of ischemia or failure of endoscopic reduction, surgical treatment should be considered. laparoscopic reduction and gastropexy may be a lessinvasive and viable alternative to the more aggressive surgical procedure but definitive surgery with repair hiatal hernia can be done in a selected patient. conclusion: minimally invasive treatments of acute gastric volvulus with paraesophageal hernia, either endoscopic or laparoscopic offer the option for reducing morbidity and mortality in elderly with significant comorbidities. the definitive laparoscopic surgery can be accomplished successfully and safely when it is performed with meticulous attention to the surgical technique and perioperative care. reid fletcher, md, mph, emily ramirez, rn, alfonso torquati, md, philip omotosho, md; rush university medical center introduction: the objective of this study was to evaluate the impact of an enhanced recovery after surgery (eras) program on post-operative length of stay following laparoscopic sleeve gastrectomy. eras programs have been demonstrated to improve outcomes and decrease length of stay in multiple surgical disciplines however relatively little has been published regarding the impact of eras programs in bariatric surgery. methods: an eras program for all patients undergoing bariatric surgery was implemented in february at a single institution. we retrospectively reviewed all patients undergoing laparoscopic sleeve gastrectomy between february and august . as a pre-eras historical control, we also reviewed all patients undergoing laparoscopic sleeve gastrectomy between january and december . baseline patient characteristics, additional concomitant operative procedures as well as -day readmission and complication rates were reviewed. logistic regression analysis was used in univariate and multivariate models to identify factors that predicted early post-operative discharge. data analysis was completed using stata se software (statacorp lp; college station, tx). results: eighty-five patients underwent laparoscopic sleeve gastrectomy after implementation of the eras program while patients were included in the pre-eras control group. there were no statistically significant differences in the baseline characteristics between the two groups and there were no differences in the rate of concomitant procedures performed. there was a statistically significant decrease in post-operative length of stay following implementation of the eras program from . it has been reported that laparoscopic redo surgery is effective for recurrent gerd and/or hiatal hernia after surgery. however, there has been very few reports from japan. we report an initial experience of laparoscopic surgery for japanese patients with recurrent gerd and/or hiatal hernia. among patients who had undergone laparoscopic fundoplication in our hospital from to , patients with recurrent gerd/hiatal hernia underwent redo surgery. preoperative work-up included upper gi series, endoscopy, ct, h ph-impedance and manometry. the patients consisted of women and men with a mean age of . years. the interval from the initial surgery was . months ( days- months). the types of initial fundoplication were nissen: , toupet: , anterior: . the types of recurrence were sliding hernia: and paraesophageal hernia: . one patient with recurrent sliding hernia had poor gastric motility. laparoscopic redo surgery was performed on patients. redo surgery included crural repair with mesh reinforcement: , refundoplication: (nissen-nissen: , nissen-toupet: , toupet-toupet: , toupet-lateral: ) and reduction of the incarcerated paraesophageal hernia: . additional procedure included mesh reinforcement: and pyloroplasty: . open partial gastrectomy was performed for one patient with incarcerated and strangulated hernia. operation time was min. patients was converted to open surgery. oral intake was started on the st pod and postoperative stay was . days. two patients recurred after redo surgery, one of whom underwent re-redo surgery. during the surgery, ivc was injured but rescued by open surgery. eleven patients had good outcome and patients required ppi after redo surgery. our morphological fundoplication score significantly improved after redo surgery. symptom score and acid exposure time were also significantly improved after redo surgery. laparoscopic redo surgery for recurrent gerd and/or hiatal hernia after surgery is safe and effective, although attention should be paid during surgery to avoid injury of the adjacent organs. surg endosc ( ) introduction: cameron ulcers (cu) are linear erosions or ulcerations in the gastric mucosa at the level of the diaphragmatic hiatus in patients with a hiatal hernia (hh) and are frequently associated with anemia. perioperative outcomes of patients with cu undergoing hh repair are not well described. we sought to identify the incidence of cu in patients undergoing hh repair at our institution and determine whether the presence of cu impacted postoperative outcomes. methods and procedures: using our irb-approved institutional hh database, we retrospectively identified patients undergoing repair between january and april . we identified all patients with cu found on preoperative esophagogastroduodenoscopy (egd). we compared patients with and without cu to determine if they differed in terms of preoperative anemia (defined as hemoglobin levels less than mg/dl in men and mg/dl in women). lastly, we compared outcomes between the cu group and the non-cu group, focusing on need for perioperative blood transfusion, failure to extubate postoperatively, intensive care unit (icu) admission, postoperative complications, length of stay (los), and -day readmission. conclusions: the presence of cu on preoperative egd is associated with increased rate of preoperative anemia, increased los, and increased icu admission after hh repair. although the cause of anemia in patients with hh is commonly attributed to cu, only % of cu patients were anemic, indicating that differences in outcomes may not only be attributed to a higher incidence of anemia in cu patients. the implications of cu in patients undergoing hh repair need to be further elucidated. laparoscopic heller myotomy as treatment for achalasia objective: aim of this stud was to review our experience with laparoscopic heller dor myotomy. disphagia constitutes the main symptom. diagnosis is performed by means of esophageal manometry. materials and method: over a period of years, patients were treated with heller myotomy plus dor fundoplication laparoscopically. all patients had lost weight, and there was a prevalence of females with an average age of . twenty five patients had chagas disease. they were all assessed with serial x-rays, endoscopy, esophageal manometry, and their symptoms were assessed with a - score, being the most severe. results: there was no conversion or mortality. in patients the mucosa was perforated during myotomy. the mucosa was sutured without altering the result of the treatment. average hospital stay was hours. one patient had to be reoperate because of esophageal perforation with peritonitis. sixty patients were followed up with manometric control and ph-probe testing, and only % of those had pathologic reflux. conclusions: laparoscopic treatment of achalasia is possible and reproducible, while reducing the morbility of laparotomy with relieve of patients symptoms. introduction: stent treatment in the gastrointestinal tract is emerging as a standard therapy for overcoming strictures and sealing perforations. we have started to treat patients with perforated duodenal ulcers using a partially covered stent and external drainage achieving good clinical results. stent migration is a serious complication that may require surgery. pyloric physiology during stent-treatment has not been studied and mechanisms for migration are unknown. the aims of this study were to investigate the pyloric response to distention mimicking stent-treatment, using the endoflip, investigating changes in motility patterns due to distention at baseline, after a pro-kinetic drug and after food ingestion. methods: a non-survival study in five pigs was carried out, followed by a pilot study in one human volunteer. a gastroscopy was performed in anaesthetized pigs and the endoflip was placed through the scope straddling the pylorus. baseline distensibility readings were performed at stepwise balloon distention to ml, ml, ml and ml, measuring pyloric cross sectional area and pyloric pressure. measurements were repeated after administration of a pro-kinetic drug (neostigmin) and after instillation of a liquid meal. in the human study readings were performed in conscious sedation at baseline and after stimulation with metoclopramide. results: during baseline readings the pylorus was shown to open more with increasing distention, together with higher amplitude motility waves. reaching maximum distention-volume ( ml), pyloric pressure increased significantly (p= . ) and motility waves disappeared. after prokinetic stimulation pyloric pressure decreased and motility waves increased in frequency and amplitude at , and ml distentions. after food stimulation pyloric pressure stayed low and motility waves showed increase in amplitude at distentions of , and ml. during both tests the pylorus showed higher pressure and lack of motility waves at maximum probe distention of ml. similar results were found in the human study. the pylorus seems to acts as a sphincter at low distention but when further dilated starts acting as a peristaltic pump. when fully distended, pyloric motility waves almost disappeared and the pressure remained high, leaving the pylorus open and inactive. stent placement in the pylorus results in pyloric distention, possibly changing motility. this study indicates that a duodenal stent placed over the pylorus should have a high radial force in the pyloric part in order to dilate the pylorus and diminish the contraction waves, this might reduce stent migration. introduction: cutting-edge technology in the field of minimal invasive surgery allows the application of singleincision laparoscopic surgery on gastric cancer. however, single-incision distal gastrectomy (sidg) is still technically difficult due to limited range of motion and unstable field of view-even in the hands of an experienced scopist. solo surgery using a passive scope holder may be the key in allowing sidg to be safer and efficient. we report our initial experience of consecutive cases of solo sidg. methods: prospectively collected database of patients clinically diagnosed as early gastric cancer who underwent solo sidg from october until july were analyzed. all the operations were held by a single surgeon and a scrub nurse. a passive laparoscopic scope holder was controlled by the surgeon to fix the field of view. results: the mean operation time (sd) was . (± . ) min, and the average estimated blood loss was . ± . ml. average body mass index was . ± . kg/m . the median hospital stay (range) was ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) days, and the mean number of retrieved lymph nodes was . ± . . there was no conversion to multiport or open surgery. early postoperative complication occurred on % with three delayed gastric emptying, two postoperative pneumonia, one pancreatitis, and one wound complication. conclusion: solo sidg using a passive scope holder allows sidg to become more feasible by providing a stable field of view. there were no peri-operative deaths in either group. in the elective group, age was not an independent risk factor for complications (or . , % ci . - . ). conclusions: the incidence of major complications and mortality in this series were much lower than those previously reported for elective lpehr, while morbidity after emergency repair remains high. the paradigm of watchful waiting for elderly and/or minimally symptomatic patients with giant peh should be revisited. the impact of vagal nerve integrity testing in the surgical kamthorn yolsuriyanwong, md, eric marcotte, md, mukund venu, bipan chand, md; loyola university chicago, stritch school of medicine background: thoracic and gastric operations can cause vagal nerve injury, either accidentally or intended. the most common procedure, which can lead to such an injury, includes fundoplication, lung or heart transplantation and esophageal or gastric surgery. patients may present with minimal symptoms or some degree of gastroparesis. gastroparetic symptoms of include nausea, vomiting, early satiety, bloating and abdominal pain. if these symptoms occur and persist, the clinician should have a high suspicion of a possible vagal injury. investigative studies include endoscopy, esophageal motility, contrast imaging and often nuclear medicine gastric emptying studies (ges). however, ges in the post-surgical patient have limited sensitivity and specificity. if a vagal nerve injury is encountered, subsequent secondary operations must be planned accordingly. methods: from january to august , patients who had a previous surgical history of a foregut operation, with the potential risk of a vagal nerve injury, had vagal nerve integrity (vni) test results reviewed. vni test was measured indirectly by the response of plasma pancreatic polypeptide to sham feeding. the data collected and analyzed included age, gender, previous surgical procedures, clinical presentation, results of vni testing and the secondary procedure planned or performed. vni testing was compared to other testing modalities to determine if outcomes would have changed. results: eight patients ( females) were included. the age ranged from to years. two patients had prior lung transplantation and six patients had prior hiatal hernia repair with fundoplication. seven patients presented with reflux and delayed gastric emptying symptoms. one lung transplantation patient had no symptoms but his lung biopsy pathology showed chronic micro-aspiration with rejection. the vni testing results were compatible with vagal nerve injury in patients. according to these abnormal results, the plans for nissen fundoplication in patients were modified by an additional pyloroplasty and the plans for redo-nissen fundoplication in patients were changed to redo-nissen fundoplication plus pyloroplasty in patient and partial gastrectomy with roux-en-y reconstruction in patients. the operative plans in patients with a normal vni test were not altered. all patients that had secondary surgery had improvement in symptoms and or improvement in objective tests (ie signs of rejection). conclusion: the addition of vni testing in patients with previous potential risks of vagal nerve injury may help the surgeon select the appropriate secondary procedure. . we present a single-center experience with a "myotomy first" approach for all patients, regardless of diverticular size. the hypothesis is that cardiomyotomy alone will provide satisfactory symptom abatement in some patients. and mis cardiomyotomy causes minimal scarring, so a staged mis diverticulectomy is feasible at a later date if diverticular retention/stasis continues. in order to discuss this treatment algorithm we present our experience with cardiomyotomy alone for patients with epiphrenic diverticula. methods: the electronic medical record was queried for patients with esophageal diverticula who were managed with cardiomyotomy and dor fundoplication alone. pre and post-operative reflux/dysphagia questionnaires were gathered; imaging studies, operative data, complications and follow up were reviewed. results: from march of until the present, patients with esophageal diverticula were treated using the "myotomy first" approach. intraoperative esophagoscopy was done to internally visualize the elimination of the inciting spastic esophageal muscle. preoperatively, all patients complained of regurgitation, followed by dysphagia in ( %) and weight loss ( %). postoperatively, dysphagia and weight loss resolved in all subjects. regurgitation symptoms resolved in ( %) patients. the average size of the diverticula was . cm , the range was - cm . post operative esophagream's showed persistent diverticual, however most had decreased in size. there were no perioperative complications, average length of stay was . days and there were no icu admissions or returns to the or. the average length of follow up for these patients was days where all patients reported being satisfied with their results and none of them have yet desired to pursue diverticulectomy. discussion: a "myotomy first" approach resulted in excellent short term symptomatic control. none of the have retained or re-experienced symptoms of diverticular retention worthy of surgical intervention. in the age of laparoscopic surgery, an esophageal epiphrenic diverteculectomy should be staged. this step wise approach seeks to assure surgical necessity for a morbid endeavor. surg endosc ( ) :s -s the background: the two-stage oesophagectomy (ivor-lewis procedure) remains the mainstay of curative surgery for oesophageal cancers in the uk. gastro-oesophageal anastomotic leak is a potentially devastating complication of this procedure affecting perioperative morbidity and mortality. although the leak rates have improved over the years, it still remains widely variable. intraoperative reinforcement of gastro-oesophageal anastomosis with an 'omental wrap' has been proposed as a measure to reduce anastomotic leak rates. there is some data to suggest that this additional technique reduces anastomotic leak. we reviewed our single institution data to assess if the omental wrap indeed had a 'cocoon' effect in maturing the anastomosis and reducing leak rates. methods: data for all cancer oesophagectomies (ilog) performed in our institute since april - was retrospectively analysed from a prospectively maintained database. the patients were categorised into two groups. masafumi ohira; department of gastroenterological surgery, hokkaido university graduate school of medicine background: in laparoscopic surgery, both surgical technique and adequate support and traction by an assistant are highly important. this study assessed the impact of the first assistant on shortterm outcomes of laparoscopic distal gastrectomy (ldg) and laparoscope-assisted distal gastrectomy (ladg). methods: patients who underwent ldg or ladg for gastric cancer at our hospital, between november and august , were included. ldg and ladg cases of billroth i reconstruction, performed by a single surgeon accredited in endoscopic procedures, were analyzed. the cases were categorized into the following groups according to the first assistant's postgraduate years (pgy) of experience: group a, - years; group b, - years; group c, - years; and group d, [ years. short-term outcomes were compared between the groups. results: we examined cases. operative time was significantly longer in group a than in group b (p= . ). no significant differences in operative time were found between groups b, c, and d. the cases were recategorized into groups as follows: group a, the young assistant group (group y, n= ), and groups b, c, and d, the senior assistant group (group s, n= ). significant differences in operative time and method of anastomosis (circular stapler or delta anastomosis) were observed between the groups (p= . and p= . , respectively), but no significant differences in complication rates were found (p= . ). the unadjusted analysis revealed that the group, method of anastomosis, and body mass index (bmi) were significant factors associated with longer operative time. multivariate linear regression analysis with stepwise model selection using akaike's information criterion (aic) revealed that bmi and group were significant factors associated with longer operative time (p= . and p= . , respectively). multivariate analysis using these variables and the method of anastomosis confirmed the significance of bmi and group for longer operative time, but no significance was found in the method of anastomosis (p= . , p= . , and p= . , respectively). conclusions: our study showed that operative time tended to be longer when the first assistant had experience of less than pgy, but the morbidity did not increase. as with the operator, the first assistant needs adequate training to ensure a smooth operation. steven g leeds, md, marc ward, md, brittany buckmaster, pa, estrellita ontiveros, ms; baylor university medical center at dallas background: gastric contents can reach beyond the esophagus into the larynx and pharynx causing an increasingly prevalent disease called laryngopharyngeal reflux (lpr). magnetic sphincter augmentation (msa) has been used as an alternative treatment for gerd with good success, but there is no data to support its use in lpr. methods: forty-five patients with msa implants for symptomatic relief with both gerd and lpr symptoms were examined. all patients experienced at least one typical gerd symptom as well as at least one extra-esophageal symptom. this was assessed using the gerd-hrql which is questions graded - on each question, and reflux symptom index (rsi) which is questions graded - on each question. patients filled out questionnaires preoperatively, one month postoperatively (early follow up), and at months to year postoperatively (late follow up). the responses on the gerd-hrql were clustered into questions inquiring about heartburn ( ), dysphagia ( ), and regurgitation ( ) like all surgical fields there is a push towards standardization of the post operative course while maintaining safe practices. other surgical fields have streamlined recovery processes in an effort to standardize care and minimize costs. laparoscopic hiatal hernia repair is a complex procedure, but with experience and a team approach, this operation can become a streamline process. methods: a retrospective review was done for over laparoscopic hiatal hernia repairs at a single institution. aspects of post operative care such hospital floor, nursing ratio utilized, pain medication, diet advancement, use of foley catheters and length of hospital stay were tracked. statistical analysis was done to compare utilization of resources as years went on along with complications and readmissions. results: a total of hiatal hernias were performed between and . improvements were noted in nearly every field over time, including faster foley removal, decreased length of hospital stay, decreased use of patient controlled analgesics (pcas) and faster advancement of diet. furthermore these patients are now treated on a surgical floor rather than the intensive care unit or step down with a higher nurse to patient ratio, decreasing hospital cost. there were no changes in complications, reoperations or readmissions over the course of the study. conclusions: cost, length of stay and so called "advanced recovery pathways" are all the rage in the surgical literature. anytime a procedure and its post operative course can become less of a "major undertaking" and more routine, the more streamline it becomes. this comes from making a standard protocol that deescalates treatment based on what is actually needed. nearly every aspect of post operative care was simplified; length of stay and cost to the hospital was decreased while no additional complications or readmissions were accrued. the foundation of a formalized advanced recovery pathway will be implemented from these factors which were studied. background: the obesity epidemic continues to worsen. bariatric surgery remains the most effective way to achieve weight loss and resolution of comorbidities. laparoscopic sleeve gastrectomy has become the most common bariatric operation due to excellent efficacy and low morbidity and mortality. the most common complication of sleeve gastrectomy is gastroesophageal reflux disease (gerd), which can adversely impact the quality of life and lead to additional esophageal complications. recently, esophageal magnetic sphincter augmentation (linx®) has become an acceptable alternative to fundoplication for certain patients with gerd. the use of linx® in patients who previously underwent laparoscopic sleeve gastrectomy was described in a case series in . the known complications of these devices include dysphagia, need for endoscopic dilation, and device erosion. the complication profile of linx® in the setting of sleeve gastrectomy has not been reported heretofore. methods: we present a case of a patient with prior sleeve gastrectomy who received a linx® device one year after her bariatric operation due to severe gerd refractory to medical management. initial evaluation demonstrated a hypotensive lower esophageal sphincter and hiatal hernia, but no evidence of stricture or twisting. soon after linx® implantation, the patient developed progressive dysphagia and worsened reflux. repeat evaluation showed esophagitis, a moderate stricture with angulation at the incisura, and a large amount of retained food. discussion: the patient was recommended conversion to roux-en-y gastric bypass, but was deemed to be a poor candidate due to heavy smoking. thus, laparoscopic removal of the linx® device was performed with hiatal hernia repair and gastric stricturoplasty. post-operative fluoroscopic evaluation revealed improvement in the stricture, but persistent gastroesophageal reflux. the patient experienced a significant improvement in her symptoms of dysphagia, nausea, and vomiting. however, once smoking cessation is achieved, she may still need a conversion to roux-en-y gastric bypass in order to address persistent gerd. conclusion: conversion to roux-en-y gastric bypass remains the standard approach to treatment of gerd post sleeve gastrectomy. new approaches to this problem, including placement of linx®, are promising but have not been evaluated for long-term safety and efficacy in the setting of prior bariatric surgery. careful diagnostic evaluation prior to placement of magnetic sphincter augmentation device should be routinely undertaken. postoperatively, close long-term follow up is imperative, particularly in patients with prior sleeve gastrectomy. presence of linx® in a patient with prior bariatric surgery may lead to worsening symptoms if complications of initial operation are present. kazuto tsuboi, md , nobuo omura, md , fumiaki yano, md , masato hoshino, md , se-ryung yamamoto , shunsuke akimoto, md , takahiro masuda , hideyuki kashiwagi, md , norio mitsumori, md , katsuhiko yanaga, md ; fuji city general hospital, shizuoka, japan, nishisaitama-chuo national hospital, saitama, japan, the jikei university school of medicine, tokyo, japan background: esophageal achalasia is one of the primary esophageal motility disorders, and the patients suffer from dysphagia, vomiting and chest pain. timed barium esophagogram (tbe) is a convenient method to assess esophageal clearance, which we usually performed before and after surgery. meanwhile, laparoscopic heller-dor operation (lhd) has been considered worldwide as a gold standard for the surgical management of esophageal achalasia. the aim of this study is to examine the effect of preoperative clearance rate at the lower part of the esophagus on surgical outcomes in patients with esophageal achalasia. patients and method: between august and april , patients who underwent lhd at our institution were extracted from the database. out of patients, patients met our inclusion criteria; such as the patients who underwent lhd as an initial operation with complete evaluation with preoperative esophageal clearance by tbe. these patients were divided into three groups by the degree of esophageal clearance (group a: clearance rate \ %, group b: %? clearance rate \ %, and group c: %? clearance rate). patients' background, pre-and post-operative symptom scores, and surgical results were compared. before and after surgery, the standardized questionnaire was used to assess the degree of frequency and severity of symptoms (dysphagia, vomiting, chest pain and heartburn). moreover, satisfaction with operation was evaluated using the standardized questionnaire. statistical analysis was performed by using krasukal-wallis test or chi-square test, and p-value less than . was defined as statistically different. results: their mean age was . years and of them were male ( . %). one hundred and sixty-eight patients ( . %) were in group a, ( . %) in group b, and ( . %) in group c. the maximum width of the esophagus in group c was smaller than that in other groups (p= . ). as to the pre-operative symptom score, the frequency score of dysphagia was significantly lower in group c (p= . ), whereas the severity score of chest pain was significantly higher in group c (p= . ). surgical outcomes including the incidence of mucosal injury were not different among the groups. moreover, the patient satisfaction with lhd was excellent regardless of preoperative esophageal clearance. conclusion: preoperative clearance rate at the lower part of the esophagus in patients with esophageal achalasia did not affect the surgical outcomes of lhd, but the characteristics of preoperative symptoms in patients with poor esophageal clearance was low dysphagia and high chest pain. surg endosc ( ) ( . cm . cm) was made by dissecting between submucosal and muscular layers at the anterior remnant gastric wall. after creation of the double flap, the posterior esophageal wall ( cm from the edge) and the anterior gastric wall (superior edge of the mucosal window) were sutured for fixation, and . cm from the inferior edge of the mucosal window was opened, and the wall of the esophageal edge and the opening of the remnant gastric mucosa were sutured continuously. the anastomosis was fully covered by the seromuscular flaps with suturing. in latg, roux-en-y reconstruction was performed through a small incision using a circular stapler. introduction: the purpose of this study was to clarify the long-term and short-term outcomes of consecutive patients who underwent thoracoscopic esophagectomy in the prone position using a preceding anterior approach for the resection of esophageal cancer at a single institution. this method was established to make an esophagectomy easier to perform and to achieve better outcomes in terms of safety and curativity. methods and procedures: we retrospectively reviewed a database of patients with thoracic esophageal cancer who had undergone a thoracoscopic esophagectomy (te, patients) or an esophagectomy through thoracotomy (oe, patients) between january and august . to compare the long-term outcomes of te and oe, we used a propensity score matching analysis and a kaplan-meier survival analysis. to analyze the short-term outcomes of te, patients were chronologically divided into three groups: a first period group ( patients), a second period group ( patients), and a third period group ( patients). as for thoracoscopic procedure, the esophagus was mobilized from the anterior structure during the first step and from the posterior structure during the second step. the lymph nodes around the esophagus were also dissected anteriorly and posteriorly. the intraoperative factors, the number of dissected lymph nodes, and the incidence of adverse events were compared among the three period groups using a one-way anova or chi-square test. results: one hundred and twenty-three patients from each group, for a total of patients, were completely selected and paired. background: it is also difficult to anastomose using circular stapler in the narrow neck field. to overcome the problem we modified circular stapling for anastomosis. gastric juice reflux is frequently observed at the esophagogastric anastomosis. we develop and report trapezoidal tunnel method to reduce the incidence reflux. ( ) patients one hundred thirteen cases ( in left lateral and in prone position), with esophageal carcinomas underwent vats-e, respectively. esophago-gastric anastomosis is performed for cases by modified circular stapling and cases by trapezoidal tunnel method. ( ) methods at first the patients are fixed at semi-prone position and esophagectomy is performed in prone position that can be set by rotating and ports are used at the intercostal space (ics). esophagectomy and the l.n. dissection are performed with pneumothorax by maintaining co insufflation. esophago-gastric anastomosis is performed as following, i) trapezoidal tunnel method sero-muscular layer of anterior wall in the near top of gastric conduit is peeled from submucosal layer after parallel horizontal incision of sero-muscular layer, and then trapezoidal tunnel of sero-muscular layer is created. the edge of the proximal esophagus is drawn into the tunnel and esophago-gastric submucosa anastomosis is performed. to wrap anastomosis distal side of parallel line is closed. ii) modified circular stapling at first the circular stapler is introduced into the gastric conduit and joined to an anvil, and close a little. and then a joined anvil is placed into the proximal esophagus and secured by means of a pursestring suture. the gastric conduit opening is closed by a linear stapler. purpose: mesh utilization and its impact on postoperative hernia recurrence following paraesophageal hernia repair remains a polarizing topic. this analysis evaluates the recent trends in laparoscopic paraesophageal hernia repairs and analyzes the impact of operative time on postoperative morbidity. methods: the - acs-nsqip database was queried for primary cpt code for laparoscopic paraesophageal hernia repair with and without mesh ( / ). only elective cases performed by a general surgeon were included. operative time was grouped into quartiles ( - , - , - , - min) and statistical analysis was performed using anova univariate with post-hoc testing and multivariate regression modeling controlling for age, diabetes, renal disease and weight loss. this analysis was powered to detect a greater than % difference in outcomes based on mesh utilization. the outcomes of interest were composite morbidity scores and readmission rates within days of surgery. results: the database identified a cohort of , laparoscopic paraesophageal hernia repairs performed between and . average patient age was years and average patient body mass index was . mesh was utilized in % of cases per year and did not change over the study period (p= . ) however mesh utilization was %, %, %, and % within operative time quartiles - respectively (p. ). postoperative morbidity and readmission rates for each operative time quartile were . %, . %, . %, and . % (p. ) and . %, %, . %, and . % (p= . ), respectively. post-hoc testing indicated statistically significant differences in postoperative morbidity and readmission rates between quartiles and / . multivariate regression analysis documented operative time as a risk factor for postoperative morbidities and readmission, even after controlling for covariates. mesh utilization was only significant for a reduction in the rate of venous thromboembolic complications (or . , p= . ) but did not impact other morbidities or readmission rates. conclusion: this analysis suggests that patients with higher operative times have increased postoperative morbidity and readmission while mesh utilization does not impact postoperative outcomes, after accounting for the longer operative time of a paraesophageal hernia repair with mesh. introduction: gastroparesis is a chronic gastric motility disorder defined by delayed gastric emptying and symptoms such as nausea, vomiting, bloating and abdominal pain. surgical options for refractory gastroparesis include pyloroplasty, gastric stimulator insertion, and gastrectomy. the palliation from a pyloroplasty and gastric stimulator may be synergistic, however concerns remain regarding the possibility of stimulator infection when performing both procedures simultaneously. we present our initial experience of combined laparoscopic pyloroplasty and insertion of gastric stimulator. methods: gastroparesis patients diagnosed by solid gastric scintigraphy or endoscopic evidence of retained food after prolonged npo status who underwent combined laparoscopic heineke-mikulicz pyloroplasty and gastric stimulator insertion between july and july were reviewed. patient demographics, pre-and post-operative symptom scores and outcomes were collected. results were analyzed using statistical tests as appropriate. p value . were considered significant. results: seven patients underwent the simultaneous pyloroplasty and gastric stimulator insertion. six patients ( %) were idiopathic and one patient ( %) was diabetic. one patient was male and six patients were female. charleen yeo, enming yong, danson yeo, kaushal sanghvi, aaryan koura, jaideepraj rao, myint oo aung; tan tock seng hospital introduction: gastric cancer is one of the most common cancers in the asian population, with recent literature supporting the laparoscopic approach in early disease. however, the minimally invasive approach in advanced disease is still controversial. the outcomes of laparoscopic gastrectomy in the elderly have also not been extensively studied. we aim to evaluate our institution's short term outcomes of laparoscopic versus open gastrectomy for gastric cancer-with particular focus on advanced disease and elderly patients. methodology: we prospectively collected the data of all patients who underwent gastrectomies for stomach cancer from to . all patients underwent a partial or total gastrectomy with d lymphadenectomy. the decision for open or laparoscopic approach was decided between surgeon and patient. we excluded patients who underwent palliative resection. all patients were followed up for at least one year post-operatively. introduction: it was an eye-opener when the lancet brought the attention about global surgery. it is estimated that the deaths due to lack of access to surgery is far greater than deaths due to malaria, tuberculosis and hiv/aids put together. there is greater need to stress the importance in developing countries. there is a responsibility at the medical schools to enlighten students about this necessity and arouse interest in concept of global surgery. the students or surgical residents in the future are a great resource to solve this major problem. the first step would be to educate surgical residents. we need to assess the existing awareness about global surgery problem among surgical residents. we can plan a program to train the next generation surgeons. methods and procedure: all the surgical residents in our institution (victoria hospital, bangalore, india) were enrolled for this study. a total of residents were enrolled. a multiple-choice questionnaire regarding global surgery was designed. the received questionnaire was analyzed to assess the depth of knowledge about global surgery. there were multiple choice questions (mcq) and an option was provided at the end for feedback and suggestion to improve the global surgery in our country. each question carried one mark. score more than was considered the cutoff for pass and those students were termed 'informed'. results: ( . %) students cleared the cut off score of and were termed 'informed'. among this group ( %) residents scored marks. ( . %) students did not cross the cut off and were termed 'non-informed'. among these ( . %) students scored marks and did not know anything on the topic. students provided relevant suggestions and opinions to improve global surgery issue. conclusion: there is a great lacuna in knowledge about global surgery among surgical residents. we need to plan a program integrating global surgery in the syllabus of surgical training. the awareness among residents would arouse interest and participation in the future. introduction: minimally invasive surgical techniques (mists) could have tremendous applications and benefits in resource poor environment. these include but are not limited to short hospital stay, reduced cost of care, and reduced morbidity, especially related to post operative infections. there is growing interest in mists in most low and middle income countries (lmic) but its adoption has remained limited largely due to high cost of initial set-up, lack of technological backup and limited access to training among others. one of the most limiting factors is the maintenance of the vision system. an affordable laparoscopic set-up as an example will therefore go a long way in improving access to mists. methods and procedures: a common zero-degrees mm scope is attached on the camera of a low price smartphone (samsung galaxy j , samsung®, seoul, south korea). two elastic bands are used to fix the scope right in front of the main camera on the smartphone. the device is covered with sterile transparent drapes (tegaderm®, m corporate, st. paul, mn, usa). a light source is connected with a fiber optic cable for endoscopic use. the image can be seen in real time on a common tv screen through an hdmi connection to the smartphone, with a sterile drape. holding the vision system through the scope would guarantee to keep the camera in place without issues. to operate in full screen the vision was digitally zoomed at . , without losing quality (that is more related to the intensity of the light). as a collateral project we built a low cost simulator training box with the same camera to train the surgeon, obtaining a high fidelity and affordable simulation setting. results: we were able to perform the tasks of the fundamentals of laparoscopic surgery curriculum using our vision system with proficiency. in a pig model, we performed a tubal ligation to simulate an appendectomy and we were able to perform basic laparoscopic suturing. no major issue were encountered and small adjustment only were required to have an acceptable, stable and clear view. conclusion: there is growing interest in minimally invasive surgeries among surgeons in lmic, but its adoption has remained limited due to reasons such as high cost of initial set-up, lack of technological backup and limited access to training among others. an affordable laparoscopic camera system will therefore go a long way in improving access to mis in such settings. open. there were no deaths or bile duct injuries in our series. two patients undergoing laparoscopic approach were converted to open ( . %). complications, los, and gender were similar between the two groups. the laparoscopic group were significantly younger and had a significantly longer operative duration (table) . long term outcomes were not available for analysis. laparoscopic and open cholecystectomy appear safe in the setting of short term surgical missions. neither group suffered major complications. both had similar immediate outcomes. los for both groups was surprisingly similar and shorter than larger series which may possibly due to patient selection. given similar immediate outcomes and large burden of disease, the open approach should be considered. however, this cost may be extracted in terms of greater pain or longer recovery time for patients, which may outweigh the benefits. further data is needed to study pain, long term outcomes, and return to work. introduction: minimally invasive surgery relies on optimal camera control for the successful execution of operations. one disadvantage of laparoscopic surgery is that camera control is dependent on a surgical assistant's interpretation of visual cues and ability to predict the next field of focus in addition to verbal commands from the operating physician to provide the optimal view. robot-assisted minimally invasive surgery provides the operating surgeon the advantage of dictating their field of view. this study aims to utilize a video processing algorithm to determine the incidence of improperly centered field of view in laparoscopic vs. robot-assisted surgery. methods: in this study, recordings of minimally invasive resection of rectal cancer ( laparoscopic and robot-assisted surgery) were evaluated. recordings were input into matlab® video processing to generate single frames at each second interval. a single reviewer would indicate the pixel which best determined where the camera should be centered based on positioning of instruments, current action (dissection/hemostasis/traction) depicted in the frame, and previous review of recordings. pixel locations were recorded for subsequent analysis. centered views were determined as those with the identified centered position pixel lying within the center quadrant when frames were split into a uniform grid. in addition, distance of each point to the absolute center of the frame was calculated based on the pixel's x and y positions. results: individual operation data was analyzed for percent of centered pixel locations and pixel distance from the center pixel of the frame. robot-assisted surgery demonstrated higher percentage of centered views over laparoscopic surgery ( . ± . vs. . ± . ; p. ). robot-assisted surgery also demonstrated shorter distances to frame center than laparoscopic surgery ( . ± . vs. . ± . ; p. ). conclusion: robot-assisted surgery aims to resolve conflicts of cooperation that occur between surgeon and assistant in laparoscopic surgery by enabling manual visual control of the operative field by the operating surgeon. this study demonstrates that by eliminating such conflicts, optimal surgical view is more frequently obtained. surg endosc ( ) background/objective: valveless laparoscopic insufflator systems are marketed for ability to prevent loss of abdominal collapse and desufflation during laparoscopy. however, community surgeons raised concern for possible entrainment of room air, including oxygen ( ), with these systems. this study seeks to quantify o and non-medical air entrainment by a laparoscopic valveless cannula system to understand the risk of intraoperative air embolism. a communityuniversity collaborative was created to design a model and test this hypothesis. methods: an artificial abdomen was developed and calibrated to equivalent compliance and intraoperative volume of an average adult abdomen. it was connected to a flow meter, oxygen concentration sensor, and commercially available laparoscopic valveless cannula system. background: further advance of near-infrared (nir) imaging capability into greater clinical usefulness will be helped by the development of new targetable agents. to avoid issues related to dose timing and contamination, compounds that become fluorescent only at the site being targeted would be a significant advance. here we build on earlier laboratory work to show step-wise advance of the agent towards clinical trialling. methods: a novel agent (nir-aza) was tested in ex vivo colorectal specimens using two commercially available systems to determine characteristics in biological tissue. it was then trialled in a large animal cohort (n= ) to determine its performance for both intestinal perfusion assessment and lymph node mapping (both stomach and colon) using again a commercially available optical imaging system and including a direct comparison with indocyanine green. results: the novel agent was easily detectable in biological tissue in the near infrared wavelength relevant to commercial instrumentation both as a local depot tattoo and as a lymphatic tracing agent. porcine model trialling again showing excellent detection and tracking characteristics both in the circulation and in gastrointestinal tissue with clear tracking to relevant lymph nodes within minutes evident with the latter. while these studies were non-survival, there was no evidence of local tissue or systemic system toxicity in any case. direct qualitative and quantificative comparison between in situ nir-aza and icg at both intestinal and lymph basin regions showed similar levels of fluorescence. conclusion: the trial compound underwent successful testing indicating proof of earlier projected potential. this is encouraging for further work to advance to first in human testing. introduction: enhanced imaging systems have been developed to alter laparoscopic camera output to facilitate visualization during laparoscopic surgery using several novel imaging modes: clara mode reduces overexposure and reflections while brightening darker areas of the image; chroma mode intensifies color contrast to more clearly delineate blood vessels; and a combined chroma-clara mode. the ies also allows the surgeon to change imaging modes throughout the procedure as needed to facilitate different portions of the operation. we hypothesized that this technology would enhance visualization of critical structures during laparoscopic cholecystectomy (lc) compared to standard laparoscopic imaging. methods: videos and still images from an ies (karl storz endoscopy) were assessed in patients undergoing lc using the four imaging modalities. three time points were assessed: ) after adhesions were taken down but before any other dissection; ) after partial dissection of the hepatocystic triangle; and ) after establishment of the critical view of safety (cvs). seven surgeons blinded to the imaging modalities ranked each modality from (best) to (worst) for each of time points ( dissection points for cases). structures identified on achievement of the cvs were also analyzed. all statistics were performed using spss. rank data was analyzed with the friedman and wilcoxon signed rank tests. results: the median ranks of the chroma and chroma-clara imaging modalities (median [iqr] [ ] [ ] [ ] vs ( - ), p= . ) were not significantly different from each other, but both ranked significantly higher than the clara and standard modalities (median rank [iqr] [ ] [ ] and [ ] [ ] , respectively, p. ). individual surgeon preferences varied; four surgeons preferred chroma-clara, two preferred chroma, one preferred clara, and none preferred the standard mode. in addition, the cystic artery and cystic duct were visible in all cases after achieving the cvs, but the common bile duct was visible in only % of cases. conclusion: enhanced imaging system technology provides modalities that were significantly preferred over standard laparoscopic imaging on retrospective review of still and video images during lc. enhanced imaging modalities should be evaluated further to assess their impact on outcomes of lc and other laparoscopic procedures. introduction: cholangiocarcinoma is often diagnosed at an unresectable stage. endoscopic stent placement is generally performed to release the tumor-induced biliary obstruction. however, stents misplacement and migration, tumor tissue ingrowth and cholangitis are relatively frequent complications. energy-based techniques (radiofrequency ablation and photodynamic therapy) have been proposed as alternatives or in addition to the stent placement, showing controversial results. the use of laser sources in the ablation of the biliary wall has not been investigated so far. this study aims at the evaluation of the optimal power and exposure time to achieve a controlled circumferential intraluminal laser ablation of the common bile duct (cbd). methods: through a laparotomy access, the cbd of pigs was exposed and a small choledocotomy was made. a confocal endomicroscopy (ce) scanning (cellvizio) was performed through the choledocotomy, after injection of ml of sodium fluorescein. the . mm diameter circumferentiallyemitting diode laser probe ( nm wavelength) was introduced in the cbd. laser ablation was performed at w during s (n= ) or s (n= ). the power setting was predetermined on preliminary ex-vivo tests on porcine liver specimen. local temperature control was monitored through a fiber bragg grating, embedded in the laser probe. ce scanning was then repeated. the extent of the ablation was measured on hematoxilin-eosin and nadh stained slides. results: the diameter of the probe was too small to enable a single-shot circumferential ablation. there were no full-thickness perforations. after s from turning laser on, the temperature at the application site reached a plateau with minimal oscillations, and remained at mean values of . ± . °c during both and min. histology revealed that the mucosa ablation, at the contact areas, induced a consistent cellular necrosis (nadh-). ce scanning provided real-time images with a specific aspect of the post-ablation mucosa, including an alteration of the normal glandular structure and a general lack of enhanced imaging. the local application of a circumferential laser source induced a precise and safe mucosa ablation with a long-standing increase in temperature in the cbd, in this experimental trial. however, there is a need of an adapted probe, better fitting the diameter of the cbd to enable a single-shot circumferential treatment. goutaro katsuno, md, phd , yasuhiko nakata, md, phd , nobuyuki kubota, md, phd , teruo kaiga, md, phd , takao mamiya, md , masahiro yan, md , naoaki shimamoto, md , shuichi sakamoto, md, phd ; department of gastrointestinal and minimally invasive surgery, mitsuwadai general hospital, introduction: recently major developments in video imaging have been achieved for performing complete mesocolic excisions (cme) or total mesorectum excisions (tme). indocyanine green (icg) fluorescence imaging is already contributing greatly to making intraoperative decisions for keeping an intact visceral fascial layer, making suitable mesentery division lines and identifying anastomotic perfusions. the aim of this study is to present our experience with laparoscopic procedures for colo-rectal cancers using icg fluorescence imaging (lap icg-fi). patients and methods: we usually use the near-infrared (nir) laparoscopy (stryker corporation, michigan, usa) for lap icg-fi. [indocyanine green fluorescent imaging] visualization of lymph flow: icg ( . mg/ . ml) was injected into the submucosal layer around the tumor at points with a -gauge localized injection before the lymph node dissection. visualization of blood flow: after complete colorectal mobilization, the mesocolon was completely divided at the planned proximal or distal transection line. indocyanine green was injected intravenously and the transection location(s) and/or distal rectal stump, if applicable, were re-assessed in fluorescent imaging mode. results: we experienced lap icg-fi cases with colo-rectal cancer patients. tumor was located at the rectum in of them, at the sigmoid colon in , at the transverse colon in , at the descending colon in , at the ascending colon in , and at the cecum in . tnm stage was -i in patients, ii in , iii in , and iv in . the median (range) age of the patients was ( - ) years with a median (range) bmi of . ( - . ) kg/m . the lymph flow was visualized in patients ( %) intraoperatively. however, a high-quality intraoperative icg lymphangiogram was achieved in patients ( %). in high-quality lymphangiogram, the lymphatic ducts and lymph nodes were clearly visualized in real time, and this proved useful in keeping an intact visceral fascial layer as well as in making a suitable mesentery division line even in the bmi[ patients. a high-quality intraoperative icg angiogram was achieved in all patients. anastomotic perfusion was satisfactory in all cases. in patients ( . %), the use of nir+icg resulted in revision of the proximal colonic transection point before formation of the anastomosis. there were no postoperative anastomotic leakages. no injection-related adverse effects were reported. conclusion: lap icg-fi is a simple, safe and useful tool to help us complete lap cme or tme and check real-time anastomotic tissue perfusion. introduction: recently, the spread of laparoscopic surgery as a standard treatment and the development of information & communication technology have yielded abundant video data of laparoscopic procedures. these data have been accumulated and we can access them anytime, anywhere. however, the direction of how to use the abundant video data are still unclear. conventionally, surgical procedures have been performed based on surgeon's subjective decisions and skills, so called "tacit knowledge". for the purpose of objective analysis of laparoscopic procedures in video data, automatic recognition of surgical tools and understanding of surgical workflow must be the first critical step. we used convolutional neural network (cnn) which is the current trend in machine learning and computer vision tasks. methods: using video database of laparoscopic sigmoid colectomy in our institute, we performed annotation of tools and phases in every frame of the operating videos. for the tool detection, we annotated bounding boxes for both left and right tools in the videos. furthermore, phase annotation was performed by watching the videos in consultation with laparoscopic surgeons. the laparoscopic sigmoid colectomy operation passes through phases; -placement of ports and preparation, -dissection of retrorectal space, -medial approach to ima, -isolation and division of ima, -medial-to-lateral retromesenteric dissection, -lateral mobilization of left colon, -rectosigmoid mobilization, -division of mesorectum, -rectosigmoid resection and anastomosis, -finishing. we used cnn architecture to perform surgical tool detection and workflow recognition. results: we totally labeled tools used in the procedures of laparoscopic sigmoid colectomy and successfully developed tool detection system by cnn. as for surgical workflow, average times of phase - were . , . , . , . , . , . , . , . , . , . min, respectively. workflow recognition system using cnn was also successfully developed, while we needed to extract pure operating scenes in advance for efficient recognition outcomes. we've developed tool detection and phase recognition systems using cnn. we need more datasets to improve the detecting ability for future clinical uses. introduction: surgical environments require special aseptic conditions for direct interaction with the preoperative images and surgical equipment, which hampers the use of traditional input devices. we presented the feasibility of using a natural user interface (nui) for gesture control combined with voice control to directly interact in a more intuitive and sterile manner with the preoperative images and the integrated operating room (or) functionalities during laparoscopic surgery. in this study, efficiency and face validity of using this nui for medical image navigation and remote control during the performance of a set of basic tasks in the or will be assessed. methods and procedures: twenty experienced laparoscopic surgeons participated in this study. they performed basic tasks in the or focused on the interaction with a medical image viewer (osirix; pixmeo) and with the functionalities of the integrated or (or ; karl storz). these tasks were carried out by means of traditional manual interaction, using a computer keyboard and mouse and a touching screen, and using a gesture control sensor (myo armband) in combination with voice commands. this nui is controlled by the tedcube system (tedcas medical systems). time required to complete the tasks using each interaction method was recorded. at the end of the tasks, participants completed a questionnaire for face validation and usability assessment. results: the use of the nui required significantly less time than conventional manual control to show preoperative studies and information for surgical support. however, the interaction with the medical image viewer was significantly faster using the traditional input devices. participants evaluated the nui as an intuitive, simple and versatile tool that improves sterility during surgical activity. seventy-five percent of the participants would choose the gesture control system as a method of interaction with the patient's preoperative information during surgery. conclusions: the presented gesture control system allows surgeons to directly interact with preoperative imaging studies and the functionalities of an integrated or during surgery maintaining the aseptic conditions. for the traditional manual interaction, it is necessary to take into account the possible reaction time and displacement time of the technician to execute the surgeon's requests. a more personalized medical image viewer is required and with higher integration with the capabilities of the presented gesture control system. emma k gibson, bs, jacqueline j blank, md, timothy j ridolfi, introduction: following a generous left hemicolectomy an anastomosis between the transverse colon and rectum may be required. extensive mobilization and retroileal routing is sometimes necessary to create a tension-free anastomosis. retroileal routing is a technique in which a window is created in the ilieocolic mesentery. the colon is routed through this window, beneath the ileum, prior to entering the pelvis. retroileal routing is uncommon and there is no data on this technique when performed in using a hand-assisted laparoscopic technique. the aim of this study was to review our experience with hand-assisted laparoscopic left sided colon resections including retroileal routing of the proximal colon to the rectum. methods and procedures: we performed a retrospective review of a single surgeon's experience with hand-assisted laparoscopic left sided resections over a seven-year period from - . indication for operation, basic demographics, bmi, procedure time, short-and long-term morbidity, and mortality were recorded. results: a total of patients underwent a hand-assisted laparoscopic left sided resection with a colorectal or coloanal anastomosis. of these, underwent hand-assisted laparoscopic procedures with retroileal routing of the proximal colon. in each case, operations included a midline hand port incision and two mm ports in the lower abdomen. the indications for operation were diverticular disease and neoplasm in nine and four patients respectively. procedures took an average of . ( - ) minutes to complete. postoperative morbidity included intubation for co retention in one patient and a rll effusion in another patient. there were no anastomotic leaks and there were no -day or -day mortalities. conclusion: retroileal routing of the colon following left hemicolectomy occurs infrequently. a hand-assisted laparoscopic approach appears to be a safe and efficient in these technically challenging cases. objective: approximation of the diaphragmatic crus pillars is a key step in hiatal hernia repair. the dogma of successful hernia repair requires tension free approximation of tissue. there are no techniques described to measure tension across the crus closure. aim of this study is to describe a novel technique for measuring the tension exerted on crural sutures and report initial findings. methods: data was collected at institutions by the same surgeon. after hiatus dissection was complete the crus defect was measured both anterio-posterior and transverse dimension. the crus closure sutures were placed posterior and then lateral to the esophagus. the initial suture is started posteriorly with a figure of eight fashion (# ). with each subsequent stitch placed anteriorly (# and # ) or laterally (l , l ) till adequate hiatus closure is achieved. we measured tension on each suture placed as follows. conclusions: the autolap system provides improved image stability, staff interactions, and enhanced ergonomic comfort for the surgical team. it also offers cost-savings from decreased staffing requirements for hospitals that routinely use staff camera holders. the system set up of - min was less variable after cases, representing the learning curve. in addition, our approach identified problems with the system that require improvement by the manufacturer. notably, we identified significant ergonomic problems for human camera holders, which has been previously described and can be addressed by this device. background: gastric leaks continue to be a troubling predicament for physicians and patients alike. they are especially concerning after bariatric surgery. electrolyte abnormalities and dehydration continuously pose a life threatening problem in these patients. methods: this is an irb approved retrospective review of our experience with a biologic tissue mesh plug closure of gastric leaks. our interventional radiology colleagues percutaneously accessed the perigastric collection with a wire and a straight catheter was guided through the gastric wall defect and advanced over the wire until it was intraluminal. the surgeon then placed an endoscope down to the level of the gastric defect. the wire was then retrieved by the endoscope achieving percutaneo-oral wire access. the biologic tissue matrix was then measured and cut to a square and inverted into a cone like structure with a flat straight piece on the open end. the cone patch was then secured to the wire with braided polyglactin suture loop. the wire was then withdrawn back through the gastric defect pulling the plug and patch into position and placement was confirmed by endoscopy. results: we attempted closure of a gastric leak arising after bariatric surgery in six patients. five underwent successful deployment while one had premature disconnection of the plug from the wire and could not be deployed. the five who had successful deployment had immediate success and within days resumed enteral intake of liquids and resolution of the leak. two of the six patients additionally underwent covered stent placement to stent a stenotic area at the incisura angularis from the esophagus to the antrum. this stent was typically removed - weeks later. there were no complications related to the procedure or the plug. only one patient has undergone repeat endoscopy to evaluate the status of the plug. in that patient an ulcer at the plug site was visualized one month after the procedure. three months later endoscopy showed the clean ulcer had shrunk to half of the original ulcer size. conclusion: this novel minimally invasive technique utilizing ir and endoscopic placement of a biologic mesh plug into gastric leaks after bariatric surgery has been highly successful in treating chronic and subacute gastric leaks. we recommend that these endoscopic techniques be used to close gastric defects prior to operative intervention. introduction: laparoscopic surgery has spread worldwide and become a standard procedure among many abdominal surgical fields. the incidence of postoperative adhesion, which is a typical postoperative complication, is considered low compared with that after laparotomy, but once complications develop, such as adhesion-induced intestinal obstruction and chronic abdominal pain, the low-invasiveness of laparoscopic surgery may decrease markedly. while we have previously used a sheet-type absorbable barrier to prevent adhesion, it requires a technique in many cases when it is applied in the abdominal cavity. in this study, we used a spray-type absorbable barrier, which is considered simple to apply, as an adhesion-preventing absorbable barrier following laparoscopic surgery. subjects and methods: a spray-type absorbable barrier for prevention of adhesion (ad spray type l®) was applied to the dissected surface, port region, and beneath the small incised wound in patients who underwent laparoscopic surgery of the large intestine after february . the nozzle is long ( mm in length) and the angle of the tip is adjustable to some extent, so that the spray could be applied easily to the target region, even in areas in which it would be difficult to secure a work space, by rotating the shaft and finely adjusting the angle of the tip. in order for the barrier to remain in the target region, this preparation must remain viscous after application. discussion: approaches for the insertion and affixing of a conventional sheet-type absorbable barrier for the prevention of adhesion has been reported previously by various researchers. the adhesion-preventing absorbable barrier used in this study was a spray type with a long nozzle, which may have been useful because it made the laparoscopic application easy. however, its application requires some experience and time for preparation compared with the use of the sheet type, which could be disadvantageous. further accumulation of cases, including evaluation of prevention of adhesion after use of the adhesion-preventing absorbable barrier may be necessary. christopher g yheulon, md, priya rajdev, md, s. scott davis, md; introduction: evidence has demonstrated that biosynthetic glue for laparoscopic inguinal hernia repair results in decreased pain. however, the two glue sub-types (biologic-fibrin based; synthetic -cyanoacrylate based) have never been compared. this study aims to assess the outcomes of those subtypes. method and procedures: a systematic review of the medline database was undertaken. randomized trials assessing the outcomes of laparoscopic inguinal hernia repair with penetrating and glue fixation methods were considered for inclusion and data analysis. thirteen trials involving patients were identified with eight trials utilizing fibrin and five trials utilizing cyanoacrylate. results: there were no differences in recurrence or wound infection between the glue subtypes when compared individually to penetrating fixation alone or indirectly to each other. there was a significant reduction in urinary retention with fibrin glue when compared to penetrating fixation (or . , % c.i. . - . ). no studies utilizing cyanoacrylate analyzed urinary retention as an outcome. there were non-significant trends in reduction of hematoma and seroma for both glue subtypes when compared to penetrating fixation (or . , % confidence interval . - . ). conclusions: glue fixation in laparoscopic inguinal hernia repair reduces the incidence of urinary retention and may reduce the rate of hematoma or seroma formation. as there are no differences in outcomes when comparing fibrin or cyanoacrylate glue, surgeons should choose the glue that is available at the lowest cost at their respective institution. however, improvement of the optical system is necessary to further utilize this advantage. we are developing an optical lens system covering the range from macroscopic to microscopic. methods: we developed a handheld prototype created by combining the objective lens system of an optical microscope and a telescope lens. a feasibility study using a porcine model was conducted. macroscopic observation was done at a distance followed by microscopic observation in contact with tissue. first, we observed the operative field macroscopically. we then observed the serosa of the small intestine microscopically, and effects of blood flow occlusion were studied. results: ( fig. and fig. ) the same visual field as ordinary laparoscopy was achieved during macroscopic observation, while using microscopic observation it was possible to observe the complex peristaltic movements of the intestine. the minute blood vessels of the visceral peritoneum and larger, deeper blood vessels were also observed. when the mesenteric vessels were occluded, changes in peristaltic movement were seen directly. congestion in blood vessels in the deep layers of the serosa was observed. improvement in peristalsis and congestion were confirmed by restoring blood flow. this system enables direct visual observations not possible with conventional optics. this system can be utilized in both laparoscopic and open surgery. the microscopic visual information obtained by this system may help with intra-operative decision making and serve to facilitate safe and precise surgery. introduction: accurate, real-time visualization is critical for efficient, effective and safe surgery. although optical imaging using near-infrared (nir) fluorescence has been used for visualization of anatomic structures and physiologic functions in open and minimally invasive surgeries, its efficacy and adoption remain suboptimal due to the lack of specificity and sensitivity. herein, we report a novel class of compounds, which are exclusively metabolized in liver or kidney, rapidly excreted into to biliary or urinary systems, and emitted two different nir fluorescence spectrums. methods: novel, water-soluble heptamethine cyanines; compound x (biliary) and compound y (urinary), unreactive towards gluthathione and the cellular proteome were synthesized, and visualized using real-time, dual-color nir imaging device. sprague-dawley rats (n= ) and yorkshire pigs (n= ) were used to demonstrate and validate its usefulness, distributed into a control group (icg; rat n= , irdye cw rat n= ), a biliary group (compound x; rat n= , pig n= ), a urinary group (compound y; rat n= , pig n= ), and dual-labeling group (compound x&y; rat n= , pig n= ). each rat and pig received one or two of the compounds at optimized dose of . -mg/kg intravenously, fluorescence signals and bio-distributions were monitored and recorded over time. the target to background ratio (tbr) was calculated in each target systems and compared to assess sensitivity and specificity. results: compound x was rapidly cleared from liver within min after intravenous injection while the fluorescence signals in biliary system lasted up to h both in rats and pigs. compound y showed significant renal excretion up to h and the urinary signals remained up to h. they were both highly specific to target organs with tbr values of . (biliary), . (urinary) and . (cf. icg) at peak signals. these new compounds have approximately - times higher quantum yields than icg and . - . times higher specificity to kidney and liver than irdye cw. one-way anova showed significant differences between control, biliary, and urinary group (p. .) dual-labeling results also showed a complete separation of these two metabolic systems (p= . ) and a real-time display of these two systems were clearly visualized with pseudo-colored labeling inside the animal body. conclusion: we report a new generation of organ-specific, real-time fluorescent markers for intraoperative visualization, navigation and potential geo-fencing. these new compounds have significantly higher quantum yields and higher specificity to visualize kidney and/or liver than any currently available reagents. background: porcine models have been widely accepted for gastrointestinal surgery studies, due to their similarities to human anatomy, histology and physiology. devices such as laparoscopic staplers have been widely used in bariatrics and are currently the cornerstone of bariatric. there are currently few published articles regarding surgical stapler testing in porcine models by means of a survival design. the purpose of this study is to present a new model for stapler testing in porcines. we present the following study in which we asses a novel stapler's feasibility and safety, and its compatibility to currently used stapler reloads. this novel stapler, the aeon™ endoscopic linear stapler (lexington medical inc., billerica, ma. pending fda approval), has been previously tested in-vitro and in-vivo by the lexington medical engineering department in matters of mechanical function, staple line bursting pressure, staple formation and hemostasis. duffy et al. used this instrument for small bowel anastomoses in a two-week survival study in porcine models. methods and procedures: four porcine animal model was used under iacuc protocol for a -day survival study held at the fiu (doral, fl, u.s.a) research facility. all animals underwent sleeve gastrectomy using the novel stapler handle, combined with the endo gia™ (medtronic, mansfield, ma) mm-staple reloads in two of the animals and aeon™ mm-staple reloads in the remaining two. no reinforcements or oversewing of the staple line was done. these procedures were performed by two bariatric surgeons. animals were monitored perioperatively by the facility staff as per protocol. the animals were euthanized at day . post-mortem assessments were done blindly. gross evaluation and comparison of the gastric tube and their staple lines was done, as well as patency, strictures, and staple line integrity. results: stapler function was equivalent with both reload brands, no technical issues were encountered. - firings were used per animal. no intraoperative complications related to stapler function ensued. no postoperative complications were encountered. all animals survived the full length of the study- days. all sleeves were patent, no strictures or bowel obstruction were present. conclusions: in an animal survival study, a follow-up period of weeks appears to be a good benchmark for stapler testing. the use of the novel stapler for gastric resections appears feasible and safe. further studies such as microscopic examination of the staple lines, might help confirm equivalence, safety and feasibility of these products for the sleeve gastrectomy procedure. jason m samuels, md , peter einersen, md , krzysztof j wikiel, md , heather carmichael , douglas m overby , john t moore , carlton c barnett , thomas n robinson, md , teresa s jones , edward l jones, md ; university of colorado denver, denver va medical center introduction: the purpose of our study was to evaluate the impact of smoke evacuation devices on operating room fires caused by surgical skin preps. surgical fires are rare but preventable events that cause devastating injuries. alcohol-based surgical skin prep serves as the fuel for a fire ignited by electrosurgical instruments. we hypothesized that increasing air exchanges near the tip of the active electrode will reduce the concentration of alcohol thus reducing the incidence of surgical fires. methods: a standardized, ex vivo model was created with a cm section of clipped, porcine skin. surgical skin preparations tested: % isopropyl alcohol with % chlorhexidine gluconate (chg-ipa) and % isopropyl alcohol with . % iodine povacrylex (iodine-ipa). based upon previous studies, a high-risk situation was replicated with immediate energy activation in the presence of pooled alcohol-based prep. the site was draped to simulate a small surgical procedure with approximately square cm exposed. (figure ) a standard and smoke evacuating electrosurgical pencil was activated for s on w coagulation mode in the presence of % oxygen. a standard wall suction was also tested with the tip held cm from the tip of the electrosurgical pencil. a chi-square test was used to compare differences between groups. results: surgical fires were created in % ( / ) of the tests with the chg-ipa and % ( / ; p= . ) of the tests with iodine-ipa. continuous wall suction did not change the incidence of fire. the smoke evacuation electrosurgical pencil significantly decreased the incidence of fire when compared to the standard pencil and continuous wall suction for both preparations (table ) . with chg-ipa, the smoke evacuation electrosurgical pencil decreased the frequency of fire by % (figure , p. ). similarly, when using iodine-ipa, the electrosurgical pencil with integrated smoke evacuation demonstrated a % decrease in fires (figure , p. ). conclusion: alcohol-based skin preps fuel surgical fires. the use of a smoke evacuator electrosurgical pencil reduces the occurrence of surgical fires. elimination of alcohol-based preps and the use of smoke evacuation devices decrease the risk of operating room fires. brian bassiri-tehrani, md, netanel alper, md, jeffrey s aronoff, md, yaniv larish, md; lenox hill hospital ureteral stents have historically been used in pelvic surgery when anatomical or clinical considerations warrant urological expertise to aid in identifying the ureters. in the colorectal and gynecologic surgery literature, prophylactic ureteral stents appear to increase the ability to detect ureteral injuries while not being shown to prevent such injuries. with the increasingly widespread use of laparoscopy and the robotic platform in complex colorectal and pelvic surgery, the utility of stents remains unclear. one of the limiting factors regarding the use of ureteral stents in minimally invasive surgery is the lack of tactile feedback; the inability of the surgeon to directly palpate the stents. one proposed method to overcome this deficiency has been the use of lighted ureteral stents. increased operating time, increased cost, and need for specialized equipment are potential drawbacks of lighted stents. an alternative to using lighted stents in minimally-invasive surgery is to directly inject indocyanine green (icg) into the ureters after cystoscopy-guided placement of ureteral stents. intraoperative visualization of the ureters is acheived by using either the pinpoint endoscopic fluorescence imaging system in laparoscopy, or firefly integrated with the robotic platform. it is hoped that the risk of inadvertent ureteral injuries during colorectal and pelvic operations will be minimized using this technique, due to improved visualization of the ureters throughout the procedure. in this case presentation, we describe a novel use of icg in a patient undergoing a laparoscopic surgery for resection of a . . . cm pelvic mass abutting the bladder, sigmoid colon and left ureter. preoperatively, there was concern that the mass would be intimately adherent to, or even invading, the bilateral ureters based on ct scan findings. after ureteral injection of icg, visualization of both ureters was easily achieved at the time of operation, and the procedure proceeded with careful and safe dissection of the mass with visualization of the ureters at all times. though there is a paucity of studies evaluating the use of icg in the laparoscopic modality, this technique was safe, easy to employ, inexpensive and very useful to visualize the ureters intraoperatively. indeed, larger studies with appropriate sample sizes would help to further validate this novel use of icg. university of colorado -denver, va eastern colorado healthcare system introduction: operating room fires are "never events" that expose the patient to the risk of devastating complications. our group has previously demonstrated that alcohol-based surgical skin preparations fuel operating room fires. manufacturer guidelines recommend a three-minute delay after application of alcohol-based preps to decrease the risk of prep pooling and surgical fires. the purpose of this study was to evaluate the efficacy of the three-minute dry time in reducing the incidence of surgical fires. methods and procedures: a standardized, ex vivo model was used with a cm section of clipped, porcine skin. alcohol-based surgical skin preparations tested were % isopropyl alcohol (ipa) with % chlorhexidine gluconate (chg) and % ipa with . % iodine povacrylex (iodine-ipa). nonalcohol-based solutions included % chlorhexidine gluconate and % povidone-iodine "paint." an electrosurgical ''bovie'' pencil was activated for seconds on watts coagulation mode in % oxygen, both immediately and minutes after skin preparation application, with and without solution pooling. results: no fires occurred with immediate testing of nonalcohol-based preparations ( / ). alcohol-based preps created flames on immediate testing in % ( / ) of cases when pooling was present. without pooling, flames occurred in % ( / ) of cases on immediate testing. after a -minute delay, there was no difference in the incidence of fire when pooling was present ( / vs. / , p [ ) . similarly, there was no difference when pooling was not present ( / vs. / , p= ). (table ) conclusions: alcohol-based surgical skin preparations fuel surgical fires. waiting minutes for drying of the surgical skin prep did not change the incidence of surgical fire (regardless of whether there was pooling of the prep solution). the use of nonalcohol-based skin preps eliminated the risk of fire. introduction: laparoscopic port sites are associated with a significant incidence of long-term hernia formation. in addition, closure with closed loop suture may lead to increased post operative pain thereby limiting patient mobility. the development of novel trocar closure systems could offer a pathway towards quality improvement and warrants investigation. we performed a randomized controlled trial comparing a novel anchor based system (neoclose®) versus standard suture closure. methods: a prospective randomized controlled trial of patients undergoing port site closure following robotic assisted laparoscopic sleeve gastrectomy or gastric bypass was completed ( with neoclose® device and with standard laparoscopic suture closure). each patient had both the camera port and stapling port closed ( port sites in each group). primary outcome measures included the incidence of hernia ( week ultrasound), time for port site closure, and depth of needle penetration. secondary outcome measures were analog pain scoring at post op day , week and week . results: physical exam as well as ultrasound evaluation showed no hernias in either group at weeks. when compared to suture closure, the neoclose® device was associated with shorter closure times ( . ± . versus . ± . s, p. ) and needle depth penetration ( . ± . versus . ± . cm, p\ . ). the neoclose® device was associated with decreased pain at week after the operation (analog pain score . ± . versus . ± . , p. ). no difference in pain scoring was observed on post operative day or at week . conclusions: trocar site closure with the neoclose® device is associated with decreased closure times and needle depth penetration. no difference in the incidence of hernias was identified very early after operation. the neoclose® device led to decreased pain week after trocar closure which is potentially secondary to decreased tension when compared to closure with closed loop suture. long term hernia data ( year) is pending with patients scheduled for follow up physical exams and ultrasounds. federico gheza, md, mario a masrur, md, simone crivellaro, md; uic introduction: robotic instruments provides a better ergonomics during suturing compared to standard laparoscopy. minimally invasive procedures with limited need of few suture may benefit from an economically affordable device able to overcome some limitations of laparoscopic suturing. flexdex surgical recently obtained the fda approval for human use of its articulated laparoscopic needle driver. the official training provided by the company (available at https://flexdex.com/register-for-training) is a h basic dry lab. the training curriculum as well as the accreditation process is not well structured. no literature is available today on this matter. our goal was building a dedicated training, to allow a safe and predictable early use in humans. methods and procedures: the training module design and implementation was done in our minimally invasive laboratory. in the preliminary phase we define with a small group of residents and research specialists a short list of mandatory concepts to detail showing the instrument. a simple suturing task was then performed by the same group with the new device, laparoscopically and with the robot, available in our lab for training only. a more complex task, based on a dedicated self-designed high-fidelity model of urethral anastomosis was then proposed, exploring different options (one flexdex only vs two flexdex, surgeon vs assistant holding the camera). lastly, we applied the new device in animals to evaluate the usefulness of including simple tasks or entire procedures in the training curriculum. results: we were able to define a multilevel, adaptable training module including a basic information session, a dry lab with inanimate low-and highfidelity models and a pig lab. subjects with different level of expertise (medical student, resident, fellow, expert and very expert surgeon) were involved to have an extensive feedback. however, our main focus was to design a training module for laparoscopic and robotic surgeons, to safely introduce the flexdex in their practice. the only outcome for this preliminary work was collected through a "post exposure" survey. the expert surgeon that did the entire training was able to give feedback after his first application of the device in humans as well. conclusions: flexdex is a promising device, available in the united states in approved facilities only. a minimally invasive lab with high laparoscopic and robotic training experience is the ideal setting to build a curriculum. a first adaptable, multilevel, original, high-fidelity training is proposed to be validated with further studies and could be implementable for accreditation purposes. surg endosc ( ) :s -s augmenting spatial awareness in laparoscopic surgery by immersive holographic mixed reality navigation using hololens objectives: endoscopic minimally invasive surgery provides a limited field of view, thus requiring a high degree of spatial awareness and orientation. because of a d field of endoscopic view, a surgeon's spatial awareness is diminished. this study aims to evaluate the efficacy of our novel surgical navigation system of immersive holographic mixed reality (mr) using a head-mounted smart glass display hololens to enhance spatial awareness of the operating field in laparoscopic surgery. the authors describe a method of registering and overlaying the preoperative mdct imaging localization of tumors, vessels, and organs onto the real world in the operating theatre through holographic smartglasses in augmented reality (ar). methods: in this study we included laparoscopic gi, hpb, urology, and gynecologic surgeries using this system. we developed a ct-based patient-specific holographic mr surgical navigating application using hololens, that is a pair of see-through monitors built-in head-mounted display. by reconstructing the patient-specific d surface polygons of tumors, vessels, and organs out of the patient's mdct, mr anatomy was displayed on the see-through grasses three-dimensionally during actual surgery. the hololens features an inertial measurement unit which includes an accelerometer, gyroscope, and a magnetometer for environment understanding sensors, an energy-efficient depth camera, a photographic video camera, and an ambient light sensor. results: the accurate surgical anatomy of size, position, and depth of the tumors, surrounding organs, and vessels during surgeries could be measured using build-in dual infrared light sensors. the exact location between surgical devices and patient's anatomy could be traced on the pair of mr smart-glasses by satellite tracking. the gesture controlled manipulation by surgeons' hands with surgical groves was useful for intraoperative anatomical references of tumors and vascular position under sterilized environment. it allowed the user to manipulate the spatial attributes of the virtual and real anatomies. this system reduced the length of the operation and discussion time. this could support complex procedures with the help of pre-and intra-operative imaging with better visualization of the surgical anatomy and spatial awareness with visualization of surgical instruments in relation to anatomical landmarks. conclusions: the immersive holographic mr system provides a real-time d interactive perspective of the inside of the patient, accurately guiding the surgeon. this helps spatial awareness of the surgeons in the operating field and has illustrative benefits in surgical planning, simulation, education, and navigation. enhancing scene visualization is a feasible strategy for augmenting spatial awareness in laparoscopic surgery. francisco miguel sánchez margallo, phd , juan a. sánchez-margallo, phd , andreas skiadopoulos, phd , konstantinos gianikellis, phd ; minimally invasive surgery centre, cáceres, spain, university of nebraska at omaha, university of extremadura, spain introduction: new handheld devices have been developed in order to address the technical limitations and ergonomic issues present in laparoscopic surgery. the aim of this study is to analyze the surgeon's performance and ergonomics using the radius r drive instruments (tubingen scientific medical, germany) during the execution of laparoscopic cutting and suturing tasks. methods and procedures: three experienced laparoscopic surgeons performed both an intracorporeal suturing task and a cutting task on a box trainer. both tasks were repeated three times. a maryland dissector and a pair of scissors were used for the cutting task. for the suturing task, a maryland dissector and needle holder were used. conventional laparoscopic instruments and their equivalent r drive instruments were used. the order in the use of the type of instruments was randomized. execution time and surgeon's ergonomics were assessed. for the latter, surface electromyography (trapezius, deltoid and paravertebral muscles) and the nasa-tlx index were analyzed. for the cutting task, the percentage of the area of deviation from the cutting pattern (% of error) was assessed. the suturing performance was assessed by means of a task-specific validated checklist. results: surgeons required more time to perform both laparoscopic tasks using the r drive instruments. the use of both instruments had a similar percentage of deviation from the exterior part of the cutting pattern. however, the deviation from the inner part was significantly higher using the r drive instruments (conv: . ± . % vs r drive: . ± . %; p\. ). needle driving was scored lower using the r drive instruments, but quality of knot tying was similar to conventional instruments. the use of r drive increased the muscle activity of the trapezius muscles bilaterally for both laparoscopic tasks. this muscle activity also increased for the left deltoid muscle during the cutting task. surgeons stated that the use of r drive instruments leads to a higher mental and physical workload when compared to traditional laparoscopic instruments. conclusions: despite the novel and ergonomic design of the r drive laparoscopic instruments, the results of this study suggest that an improvement in surgical performance and physical workload is required prior their use in an actual surgical setting. further studies should be done to analyze the use of these instruments during other laparoscopic tasks and procedures. we believe that surgeons need a longer and comprehensive training period with these laparoscopic instruments to reach their full potential in laparoscopic practice. background/objectives: d printing has been shown to be a useful tool for preoperative planning in various surgical disciplines. however, there are only several single case reports in the field of liver surgery. this is because of problematic visualization of anatomy, difficulties in methodology and-most importantly-high costs limiting implementation of d printing. the goal of this study is to evaluate the utility of personalized d-printed liver models as routinely used tools in planning and guidance of laparoscopic liver resections. materials and methods: contrast-enhanced computed tomography images of consecutive patients who underwent laparoscopic liver resections in a single centre were acquired and processed. proper segmentation algorithms were used to obtain virtual models of anatomical structures, including vessels, tumor, gallbladder and liver parenchyma in stl (stereolithography) format. after processing files, models in parts were subsequently printed with desktop ultimaker + (ultimaker, netherlands) d printer, using polylactic acid filaments as printing material. all parts were matched together to create a mold, which was later casted with transparent silicone. models were delivered to surgical teams prior to the surgery as well as used in patients' education. results: up to now, six full-sized, transparent, personalized liver models were created before laparoscopic liver resections and used as a tool for preoperative planning and intraoperative guidance. usefulness of these models has been evaluated qualitatively with surgeons. operative data was obtained for each patient and it will be used for quantitative analysis in further study phases. costs of one model varied between $ and $ and whole process of development took approximately days in every case. conclusions: d-printed models allow precise planning in complex cases of minimally invasive liver surgery by providing high-quality visualization of patient-specific anatomy. implementation of this technology might potentially lead to clinical benefits, such as reduction of operative time or improvement of short-term outcomes. having said that, more data is needed to decisively prove these hypotheses. introduction: modern laparoscopic graspers may risk inadvertent injury to tissues, and have been shown to produce crush and puncture injuries. in addition, the force transmitted to the tissues by grasper handles can be highly variable, dependent on the orientation and amount of tissue engaged by the grasper. we have developed a novel vacuum-based laparoscopic grasper designed to reduce tissue injury from grasping. the aim of this study is to compare the incidence and severity of tissue trauma caused by vacuum-based graspers versus standard compressive graspers while manipulating tissue. we performed an in vivo surgical porcine study to assess gross and histologic tissue injury after grasping trials. grasping trials were divided equally between two adult porcine models; samples of small bowel were grasped with a standard atraumatic laparoscopic grasper (aesculap double-action atraumatic wave grasper) and were grasped with our novel vacuum grasper with varying vacuum head designs ( for head a, each for heads b and c). following grasping, the porcine model was allowed to dwell for hours prior to harvest. gross injury was graded as follows: ) no injury, ) ecchymosis only, ) serosal injury, ) seromuscular injury, and ) perforation. histologic injury was graded as follows: ) serositis, ) partial-thickness injury to the muscularis propria (mp), ) full-thickness mp injury, and ) full-thickness mp and mucosal injury. mann-whitney u test was performed to compare both gross and histologic injury scores between the groups. results: on gross assessment, no samples were noted to have injury more severe than ecchymoses following grasping. the vacuum grasper was found to cause more ecchymosis (median= ) than the compressive laparoscopic grasper (med.= , u= , p. ). on histologic assessment, the compressive grasper caused significantly more severe injury (med.= ) compared to the vacuum grasper (med.= , u= , p= . ). subgroup analysis showed that heads a (med.= , u= . , p= . ) and b (med.= , u= , p= . ) caused significantly less injury compared to the compressive grasper. head c (med.= , u= . , p= . ) also showed less injury but did not reach statistical significance. conclusion: this study demonstrates that our novel laparoscopic vacuum grasper produces less tissue trauma than standard compressive graspers. vacuum-based grasping is a viable alterative for reducing inadvertent tissue injury in laparoscopy. minimally invasive surgery centre, cáceres, spain, university of nebraska at omaha, university of extremadura, spain introduction: the aim of this study is to analyze the surgeon's performance, workload and ergonomics using an ergonomically designed handheld robotic needle holder during laparoscopic urethrovesical anastomosis in an animal model, and comparing it with the use of a conventional laparoscopic needle holder. methods and procedures: six experienced surgeons performed an urethrovesical anastomosis in a porcine model using a handheld robotic needle holder and a conventional laparoscopic axialhandled needle holder (karl storz gmbh). the robotic instrument (dex®, dextérité surgical) has an ergonomic handle and a flexible tip with unlimited rotation, providing seven degrees of freedom. the use of the surgical instrument was randomized. for each procedure, an expert surgeon evaluated the surgical performance in a blinded fashion using the global operative assessment of laparoscopic skills rating scale. besides, the quality of the intracorporeal suture was assess by a validated suturing-specific checklist. the surgeon's posture was recorded and analyzed using the xsens mvn biomech system based on inertial measurement units. the surgeon's workload was evaluated by means of the nasa task load index, a subjective, multidimensional assessment tool. the patency of each anastomosis was assessed using methylene blue. results: all urethrovesical anastomoses were completed without complications. only one anastomosis with the robotic device failed the patency test. surgeons showed similar surgical skills with both instruments, although they presented greater autonomy with the conventional instruments (p =. ). for the suturing performance, the use of the robotic device led to an increase in the number of movements during the needle driving and lower tendency to follow its curvature during the withdrawal maneuver (p=. ). the level of workload increased with the robotic device. however, the surgeon's satisfaction with the surgical outcome did not differ using both instruments. the use of the robotic instrument led to similar posture of the shoulder and wrist and better posture of the right elbow (p=. ) when compared to the conventional instrument. conclusions: the use of the robotized needle holder obtained similar results for the surgical performance and surgical outcome of the urethrovesical anastomosis when compared to the conventional instrument. we consider that aspects such as the surgeon's autonomy, dexterity in driving the needle and workload could be improved with a comprehensive training with the new device. inertial sensors can be an alternative for actual and crowded surgical environments. surgeons acquired a better body posture using the novel robotic needle holder. surg endosc ( ) :s -s introduction: temporal and spatial tissue temperature profile in electrosurgical devises, such as ultrasonic scissors and bipolar vessel sealing system, was experimentally measured, and the incidence of postoperative complications after thoracoscopic esophagectomy was assessed according to the electrosurgical devises used. methods and procedures: experiment of thermal spread: sonicision (sonic) was used for ultrasonic scissors and ligasure (ls) was used for bipolar vessel sealing system. each device was activated in order to cut porcine muscle at room temperature. temperatures of both the device blade and porcine tissues beside the device were measured using a temperature probe. each experiment was performed at least three times. room temperature was degrees. clinical analysis: the patients who underwent thoracoscopic esophagectomy with -field lymph node dissection in the prone position were selected in the study. incidence of postoperative complications after thoracoscopic esophagectomy was compared according to electrosurgical devises. bronchoscopy was used for diagnosis of recurrent laryngeal nerve paralysis (rlnp). sonic and ls was employed in and patients, respectively. material: we compared consecutive cases using d laparoscopic surgery versus cases of d conventional laparoscopic surgery from january to june . all surgical procedures were performed by experienced laparoscopic surgeons using d (einsteinvision system) and hd conventional laparoscopic optic. d-laparoscopic surgery offers the depth perception of the surgical field that is lost with the conventional ( d) laparoscopic surgery, and in many series is reported to be better in terms of surgical performance. outcome measures was operation time, surgical performance, blood looses, complications and surgeon satisfaction with the procedure. results: cholecystectomy was the most frequent surgery performed with cases ( %); hernia surgery cases ( %); fundoplication cases ( %), appendectomy cases ( %), left colon excison with colo-rectal anastomosis cases( %), and other cases ( %) wich included ovarian cyst excision, liver biopsy, prostatectomy and pediatric surgery. we compared each d procedure with a standard laparoscopy case performed by the same surgeon during the time of the study. d vs d surgical procedures outcome measures are shown in table . we found better results in operation time, surgical performance and less blood looses in favor of three-dimensional laparoscopy (. ). conclusion: d laparoscopy reduces operation time related to better performance during the procedure. depth perception facilitates dissection, intracorporeal knotting, mesh placement and colo-rectal anastomosis. surgeons reported better surgical performance and comfort during d laparoscopy; there were any reported side effects such as headache or dizziness. background: social media (some) uniquely allows international collaboration, with immediacy and ease of access and communication. in areas where surgical management is contentious, this could be a valuable tool to frame the current state, propose best practices, and possibly guide management in a rapid, cost-effective, global scale. our goal was to determine the ability to use twitter-a some platform-as an alternative surgical research tool. methods: twitter was used to host an online poll on a pre-selected controversial topic with no current consensus guidelines-pathological complete response in rectal cancer. an influential colorectal surgeon published the survey "t n rectal cancer undergoes a complete response" on two separate occasions. both polls were open for duration of three days. two methodologies were tested to increase exposure and direct towards relevant participants: first, tagging several worldwide experts, then using the well-established hashtag #colorectalsurgery and publishing during an international surgical conference. the main outcome measure was the feasibility, validity, reproducibility, and methods to further participation of a twitter survey. results: the tweet polls were posted three weeks apart. there was no cost and the time required for the process was three minutes, demonstrating the feasibility. providing three closed options to select from facilitated validity. the poll's anonymity limited knowledge of the participant's qualifications, but public comments and "retweets" came from surgeons with experience ranging from trainee to department chair. a robust volume of respondents was observed. the st post received votes, "likes", "retweets", and comments from a diverse international group ( countries). all tagged members participated in the forum. the nd received votes, "likes", "retweets", and comments. the results were reproducible, with the majority favoring option on both occasions ( % and %, respectively; p= . ). treatment recommendations, their rationale, and open questions were identified in the thread. conclusions: some can be used as a research tool, with valid, reproducible, and representative survey results. while exposure was comparable across the two methods, tagging specific members guided experts to provide more opinions than using conference and specialty hashtags. this could expand awareness, education, and possibly affect management in a transparent, cost-effective method. the anonymous nature of respondents limited the ability to make conclusions, but interest and opinion leaders for further study can be easily identified. this demonstrates the potential for some to facilitate international collaborative research. background: despite the technological advancement of a minimally invasive approach to pylorus -preserving pancreaticoduodenectomy (pppd), the morbidity is still high. among the many complications, postoperative pancreatic fistula (popf) is reported in high incidence rate, which varies from researcher to researcher, and a fistula risk score (frs) has been developed to predict the popf. the aim of this study is validate the fistula risk score in minimally invasive approach of pppd and find the other meaningful parameter for prediction of popf. method and materials: from january to august , laparoscopy attempted right-sided pancreas resection was performed on patients including robotic reconstruction in the division of hepatobiliary and pancreas at yonsei university health system. among them, patients were excluded due to total pancreatectomy (n= ), open conversion (n= ), pancreaticogastrostomy and hybrid manual anastomosis (n= ), non-measurable drain and missing datas (n= conclusions: fistula risk score is significant prediction factor of popf including biochemical leaks. in addition to the previously known frs variables, our data showed that bmi is an important predictor of popf with clinical relavancy in a minimally invasive approach of pppd. laparoscopic hemi-hepatectomy for liver tumor satoru imura, hiroki teraoku, yuji saito, shuichi iwahashi, tetsuya ikemoto, yuji morine, mitsuo shimada; tokushima university introduction: with progress of surgical technique and devices, laparoscopic liver resection became a realizable option for patients with liver tumor. major liver resection such as anatomical left or right hemi-hepatectomy has also been introduced in many centers. herein, we evaluate surgical results of laparoscopic hemi-hepatectomy for liver tumor. patients and methods: until march , consecutive patients who underwent laparoscopic or laparoscope-assisted hemi-hepatectomy (left: , right: ) were reviewed and the surgical data such as operation time, blood loss, postoperative complications were analyzed retrospectively. results: of the patients underwent left hemi-hepatectomy, cases were primary liver cancer, cases were metastatic tumor, and cases were benign tumor. pure laparoscopic surgery was performed in cases. the mean blood loss was ( - ) ml, mean operating time was ( - ) minutes and mean postoperative hospital stay was ( - ) days. the rate of postoperative complications was . % (wound infection; n= ). all right hemi-hepatectomy was performed by laparoscope-assisted method. of the patients underwent right hemi-hepatectomy, cases were primary liver cancer, cases were metastatic tumor, and cases were benign tumor. the mean blood loss was ( - ) ml, mean operating time was ( - ) minutes and mean postoperative hospital stay was ( - ) days. the rate of postoperative complications was . % (biliary stenosis; n= ). the patients with hepatocellular carcinoma were followed up for a median of ( - ) months. recurrence occurred in cases and none of them had died at the time of follow-up. conclusion: laparoscopic hemi-hepatectomy is a safe and effective procedure for the treatment of benign and malignant liver tumors. ibrahim a salama, professor; department of hepatobiliary surgery, national liver institute, menoufia university abstract background: iatrogenic biliary injuries are considered as the most serious complications during cholecystectomy. better outcome of such injuries have been shown in cases managed in a specialized center. objective: evaluatation of biliary injuries management in major referral hepatobiliary center. patients and methods: four hundred seventy two consecutive patients with post-cholecystectomy biliary injuries were managed with multidisciplinary team (hepatobiliary surgeon, gastroenterologist and radiologist) at major hepatobiliary center in egypt over years period using endoscopy in patients, percutaneous techniques in patients and surgery in patients. results: endoscopy was very successful initial treatment of patients ( %) with mild/moderate biliary leakage ( %) and biliary stricture ( %) with increased success by addition of percutaneous (rendezvous technique) in patients ( . %). however, surgery was needed in ( %) for major duct transection, ligation, major leakage and massive stricture. surgery was urgently in patients and electively in patients. hepaticojejunostomy was done in most of cases with transanastomatic stents. one mortality after surgery due to biliary sepsis and postoperative stricture was in cases ( . %) treated with percutaneous dilation and stenting. conclusion: management of biliary injuries was much better with multidisciplinary care team with initial minimal invasive technique to major surgery in major complex injury encouraging for early referral to highly specialized hepatobiliary center. introduction: simple liver cyst is the solitary non parasitic cystic lesion of the liver. teatment of symptomatic liver cyst varies from simple aspiration to hepatic resection. each treatment has its own merits and associatied complications. laparoscopic unroofing (fenestration) offers the best balance between efficacy and safety. polycystic liver disese (pcld) treatment by this method are less clear because of high failure rate. liver resection though more effective carries higher risks. treatment of hydatid disease are controversial. materials and method: simple cyst may be asymptomatic and picked up as incidental findings on ultrasound examination for other abdominal complaints. few cyst have symptoms of mass effect or with complication effect due to haemorrage, rupture, infection. on examination liver is palpable. compression over bile duct give rise to jaundice. the commonest symptoms are pain, early satiety, nausea and vomiting. simple cyst are more common in female after years of age. the cyst located antriorly inferiorly and laterally are the ideal case. investigation like ultrasonography is important. it will helps us to detect the cyst nature, will help to differentiate bet ween simple cyst from poly cystic liver disease, from neoplastic liver. in endemic area of hydatid liver disease serological test is mandatory. ct scan is important regarding details information about to localise the cyst, to identify the liver tissue arroud the cyst, relationship of cyst with the nearby vital structures, number of cyst, calcification and carcinomatous changes in its wall. aspiration of cystfluid, biological and cytological examination to rule out the presence of infection, biliary communication and malignancy. recently, ca - estimation is helpful for the differentiating the simple cyst from the cystadenoma or carcinoma. for jaundice patient ercp is impotant to locate the intraductal polyp causing the biliary obstruction or cyst causes the compression of the biliary tree. for bleeding in cyst mri is helpful. carcinoma at epithelial lining may occur. result: laparoscopic de-roofing (fenestration) less radical procedure ensures adequate drainage of cyst content into the peritoneal cavity. the cyst wall can be removed using harmonic scalpel so smoked produced and fogging of lens can be minimized. the interior surface inspected with care to exclude neoplastic growth and biliary communication. whole operative procedure, duration of postoperative recovery, hospital stay is much shorter in this procedure. large cevron incision can be avoided. no recurrence in two years follow up period. liver resection and total cystectomy theoretically minimizes the recurrence risk but invoke the a real risk of postoperative complications and death. conclusion: careful case selection and meticulous surgical skills are the two major determinants of the outcome. in the llr group, the first port was placed with an alexis® wound retractor (applied medical, usa) and free access® (top corporation, japan) at the abdominal defect made by previous sc. an additional or trocars were placed as needed. results: all patients in the llr group were treated using the laparoscopic approach. there were no other significant differences in patient background and characteristics. operative duration was similar for these groups. blood loss, complication rate, and hospital stay in the llr group were significantly decreased compared with the olr group. conclusion: in concurrent liver resection and sc, the open approach may require multiple large incisions, but the laparoscopic approach can complete procedures with a stoma wound and a few port wounds. additionally, use of a platform on the wound for sc enhances safety and efficacy for dissection of intraabdominal adhesions and a clear operative view. primary hepatic lymphoma: the importance of liver biopsy diego t enjuto , carlos ortiz , laura casanova , jose luis castro , pablo sánchez , jaime vázquez , norberto herrera , benjamín tallon , carmen jimenez ; hospital severo ochoa, hospital san rafael, hospital henares primary hepatic lymphoma (phl) is a very uncommon lymphoproliferative malignancy. it accounts for only . % of all extranodal non-hodgkin lymphoma and . % of all cases of non-hodgkin disease. the diagnosis is made when there is only liver involvement or when there is minimal non-liver disease. bone marrow, spleen, or hematologic affection should be excluded to confirm the diagnosis. we present our experience with two phl's that were correctly diagnosed thanks to laparoscopic liver biopsy. -year-old male admitted because of a -month history of right upper quadrant pain and nonmeasured weight loss. liver function tests and cholestasic enzymes showed normal values. serologic tests showed negative results for both hbv (hepatitis b virus) and hcv (hepatitis c virus). ct (computed tomography) scan showed three intrahepatic lesions in segments v, vi, and vii. ct-guided fine needle did not reach the diagnosis so a laparoscopic hepatic biopsy was performed. the final diagnosis was burkitt-like lymphoma. chemotherapy with r-chop (rutiximab, cyclophosphamide, adriamycin, vincristine, and prednisone) modality was started and completed after cycles. it is currently years since the patient was diagnosed and there are no clinical or radiological signs of recurrence. -year-old male who complained of diarrhoea and abdominal pain. chronic hb infection with no viral charge was detected. ultrasound showed heterogeneity of the whole left hepatic lobe and an mri was performed. a ten by segen centimeters lesion occupying the left hepatic lobe enhanced in arterial phase was seen suggesting adenoma. laparoscopic hepatic biopsy was completed to reach a definitive diagnosis. non-hodgkin lymphoma follicular type has just been confirmed with the histology and immuno-histochemistry. chemotherapy with r-chop should be started in the following weeks. phl's diagnosis is hard to achieve. fine needle biopsies are frequently negative because of the large area of necrosis. surgical biopsies are sometimes indispensable to get enough tissue to reach the diagnosis. phls are sometimes misdiagnosed as hepatocellular carcinoma because of its relation to hcv meaning a major hepatic resection. that is the reason why we consider that all diagnostic measures should be undertaken to rule out a different type of tumor. surgical resection is normally not needed in phls; as they are chemosensitive lesions. surgical options usually add unnecessary morbidity and mortality to these patients. chemotherapy standard treatment for phl consists on r-chop combination. pancreatic neoplasm enucleation -when is it safe? case report and review of the literature elaine jayne buckley , k molik , j mellinger ; siu-som, hshs pediatric surgery introduction: solid pseudopapillary tumors are rare neoplasms accounting for - % of pancreatic malignancies with a low risk of recurrence and metastasis. pancreatic malignancies are less common in pediatric populations, though small case series have identified that pseudopapillary tumors comprise between and % of pediatric pancreatic neoplasms. as these tumors have a low risk of metastasis, the mainstay of treatment has remained surgical excision. several surgical approaches have been described from extensive resections such as pancreaticoduodenectomy to local enucleation. we present a case of enucleation of a large pseudopapillary tumor from the pancreatic head complicated by pancreatic fistula. a literature review was performed given the rarity of this tumor to review surgical approaches, to compare complications and long-term outcomes, and to identify specific strategies to decrease the risk of pancreatic fistula. case description: a year-old female presented with months of abdominal pain. computed tomography identified a right upper quadrant mass felt to be consistent with a lipoma. follow up ct at months suggested the mass was more likely a gastrointestinal stromal tumor (gist), and surgical resection was recommended. enucleation of the mass was chosen in view of a wellcircumscribed appearance, clear operative tissue planes, and concern for long-term morbidity of a more extensive resection given the patient's young age. pathology demonstrated an . cm pseudopapillary tumor with negative margins. her post-operative course was complicated by a grade b pancreatic fistula, managed with nutritional support, external drain maintenance, and endoscopic stenting. the patient achieved healing of the pancreatic fistula after four months. results: our literature review demonstrates no difference in recurrence, mortality or morbidity between types of surgery. pancreatic fistula contributed to the majority of postoperative morbidity in all cases. recommendations for enucleation include small ( - cm) tumors with between and mm margin from the main pancreatic duct. techniques identified to minimized post-operative pancreatic fistula include preoperative imaging of the duct anatomy, preoperative pancreatic stent placement, and intraoperative ultrasound to identify the pancreatic duct. some literature supports preservation of pancreatic parenchyma, particularly in younger patients, to reduce endocrine and exocrine dysfunction given the low rates of recurrence and metastasis with this rare neoplasm. conclusion: our case demonstrates complications of enucleation of a large pseudopapillary tumor with successful multidisciplinary post-operative management. with the risk reduction strategies identified, we suggest that enucleation may be considered for pseudopapillary tumors in younger patients to preserve pancreatic parenchyma and long-term pancreatic function. introduction: recent advancements in minimally invasive techniques led to increased effort and interest in laparoscopic pancreatic surgery. laparoscopic distal pancreatectomy is a widely accepted procedure for left-sided pancreatic lesions. in other cases, the adoption of laparoscopic pancreaticoduodenectomy has been hindered by the technical complexity of laparoscopic reconstruction. hybrid laparoscopy-assisted pancreaticoduodenectomy (hlapd) in which pancreaticoduodenal resection is performed laparoscopically, while reconstruction is completed via a small upper midline minilaparotomy, is combines the efficacy of open approach, and the benefits of laparoscopic approach. the purpose of this study is to report our experience of hlapd and to define the learning curves. methods: patients with benign and malignant periampullary lesion underwent hlapd by a single surgeon between july and may were retrospectively reviewed. the clinicopathologic variables were prospectively collected and analyzed. the learning curve for hlapd was assessed using cumulative sum (cusum) and risk-adjusted cusum (ra-cusum) methods. results: the most common histopathology was pancreatic ductal adenocarcinoma (n= , . %), followed by intraductal papillary mucinous neoplasms (n= , . %), ampulla of vater cancer (n= , . %), and common bile duct cancer (n= , . %). the median operation time was min (range, - min) and the median estimated blood loss was ml. based on the cusum and the ra-cusum analyses, the learning curve for hlapd was grouped into four phases: phase i was the initial learning period (cases - ), phase ii was the technical stabilizing period (cases - ), phase iii was the second learning period (cases - ) and phase iv represented the second stabilizing period (cases - ). there was a statistical difference in terms of surgical indication between phase ii and iii (p= . ). conclusions: hlapd is a technically feasible and safe procedure in selected patients. this procedure has benefits of both open and minimally invasive procedure, and could be a stepping-stone for transition from open to purely minimally invasive pancreaticoduodenectomy. in silico investigation of the background: wilson's disease is a rare autosomal recessive genetic disorder of copper metabolism, which is characterized by hepatic and neurological disease. the gene atp b (on chromosome ) leads to wilson's disease is highly expressed in the liver, kidney, and placenta and encodes a transmembrane protein atpase (atp b), which functions as a copper-dependent p-type atpase. methods: here, the rare codons of atp b gene and their location in the structure of atp b protein was studied with rare codon calculator (racc) (http://nihserver.mbi.ucla.edu/racc/), atgme (http://atgme.org/), latcom (http://structure.biol.ucy.ac.cy/latcom.html) and sherlocc program (http://bcb.med.usherbrooke.ca/sherlocc.php). racc server identified arg, leu, ile, and pro codons as rare codons. results: results showed that cyp a gene have single rare codons of arg. additionally, racc detected two rare codons of leu, single rare codons of ile and rare codon of pro. atp b gene analysis in minmax and sliding_window algorithm resulted in identification of and rare codon clusters, which shows the difference features of these algorithms in detection of rcc. analyzing the d model of atp b protein show that arg residue constitute hydrogen bonds with glu and glu that with mutation of this residue to ser this hydrogen bonds were disrupted and may interfere in the proper folding of this protein. moreover, the side chain of arg don't forms any bond with others residues that with mutation to thr form new hydrogen bond with the side chain of arg . these addition and deletion of hydrogen bonds effects on the folding mechanism of atp b protein and interfere with the proper function of the atp b position. his forms the hydrogen bonds with the his and it seems that this hydrogen bond close together two region of this protein and it seems that has a critical role in the final folding of atp b protein. conclusions: computational study of diseases such as wilson's disease and involved genes (atp b) help us in understanding of disease's physiopathology and finding new approaches for detection and treatment. pancreatic stump leak and fistula formation are significant causes of morbidity in patients undergoing distal pancreatectomy (dp), with incidence of % to as high as % in a large systematic review. we present a case of a year old female, four months status post distal pancreatectomy and splenectomy for pseudopapillary neoplasm of pancreatic tail. patient presented to our institution with day history of left upper quadrant pain and general malaise. differential diagnosis on admission was abdominal wall abscess vs incarcerated incisional hernia. physical exam was positive for severe tenderness to palpation over a * cm cm non reducible mass in left upper quadrant with surrounding skin erythema. patient underwent a diagnostic laparoscopy and intraoperative findings revealed extensive adhesions to the anterior abdominal wall and a loop of small bowel was found adhered to the previous incision site in left upper quadrant. upon further dissection we entered a large cm cavity with saponified caseous material. the saponified material and thick tan fluid were evacuated into an endocatch bag and two large bore jackson pratt drains were left within the cavity. further examination showed that the small intestine was normal with no signs of obstruction or ischemia. fluid studies and cultures were sent and showed yeast like organisms and negative for acid fast bacillus. we report an unusual presentation of a distal pancreatectomy stump leak in the formation of an intra-abdominal saponified fluid collection four months after the primary procedure. given the high incidence of pancreatic stump leak and fistula formation after distal pancreatectomy, much effort has been made to identify factors associated with higher incidence of leaks and their usual and unusual presentations, which will be reviewed in this report. initial concerns regarding healthy donor's safety and graft integrity, need for acquiring surgical expertise in both laparoscopic liver surgery and living donor transplantation (ldlt) have delayed the development of laparoscopic donor hepatectomy in adult-to-adult ldlt. however, decreased blood loss, less postoperative pain, shorter length of stay in hospital, and excellent cosmetic outcome have well been validated as the advantage of laparoscopic hepatectomy. hence, the safety and feasibility for laparoscopic donor should be further investigated. we present initial experiences and safety for totally laparoscopic living donor right hepatectomy. in cases who received elective living donor right hepatectomy for adult-to-adult ldlt, totally laparoscopic approach was applied from may up to august . the anatomical variation of portal vein was not considered as an exclusion criteria, but all donors were with type i portal vein variation. the bile duct anomaly was preoperatively evaluated with magnetic resonance cholangiopancreatography (mrcp) and was never excluded for totally laparoscopic approach. d conventional rigid º rigid laparoscopic system was used in cases and the remaining cases used d flexible laparoscopic system. in about %, hepatic duct anomalies (type , a, b) were identified. the operation time was from hours to hours. and the time for the graft removal was within minutes. the hepatic duct transection was performed under operative cholangiography via a cystic duct and the patency of left hepatic duct was also confirmed by operative cholangiography. however, during postoperative period, bile leakage was identified in only case and resolved after the biliary stent insertion by ercp. during operation, there was no transfusion and the inflow control like pringle maneuver was not used at all. v or v were reconstructed in cases and large right inferior hepatic vein was prepared for anastomosis in cases. all grafts were removed through the suprapubic transverse incision. most donors were discharged at days after hepatectomy. during the short-term follow-up period in the donors except this case, complications were not identified. conclusively, totally laparoscopic right donor hepatectomy in elective adult-to-adult ldlt can be initially attempted after enough experiences of laparoscopic hepatectomy and ldlt. however, the true benefits of totally laparoscopic living donor right hepatectomy should be fully assessed through various experiences from multi-institutes. background: the role of neoadjuvent chemotherapy on the treatment of pancreatic cancer remains widely controversial. studies have evaluated its effect on resectability and survival; however, few have studied the consequence of neoadjuvent therapy on surgical outcomes and complications. methods and procedures: a retrospective analysis was performed utilizing the targeted pancreas module of the national surgical quality improvement project (nsqip) for patients undergoing pancreaticoduodenectomy. neoadjuvent therapy was defined by chemotherapy and/or radiation in the -days before surgery. patient demographics, operative characteristics, and -day outcomes were compared amongst patients undergoing neoadjuvent chemotherapy, radiation, chemoradiation, and no neoadjuvent therapy. both univariable and multivariable analysis were completed. results: pancreaticoduodenectomy was completed in , patients. , patients had no neoadjuvent therapy; underwent both chemotherapy and radiation; underwent chemotherapy alone, and underwent radiation alone. there were no differences in demographics or comorbidities. no difference in -day mortality was found; however pancreatic fistula formation was affected by neoadjuvent therapy. neoadjuvant radiation increased fistula formation (or: . , % ci: . - . ) while neoadjuvent chemotherapy (or: . , % ci: . - . ) was protective. conclusion: neoadjuvent therapy significantly impacts surgical outcomes following pancreaticoduodenectomy. given that pancreatic fistula formation can delay post-operative chemotherapy, it may be reasonable to refrain from neoadjuvent radiation therapy for patients with resectable and borderlineresectable disease. the influence of thickest background: the use of stapling devices for distal pancreatectomy remains controversial, due to concerns about the development of postoperative pancreatic ?stula (popf). pancreas thickness might be associated with popf, but suitable thickness of stapler remains also inconclusive in view of reducing popf. methods: we routinely use thickest endo gia™ reloads with tri-staple™ (covidien, north haven, ct) for pancreas closure during laparoscopic left side pancreatectomy (lp) since . we compared short term surgical results of the consecutive ten patients underwent lp using new stapler (ns) and patients with lp using other type of stapler (os) focusing on popf. results: no patients developed clinically relavent (cr)-popf in ns group and two patients ( . %) with os group experienced cr-popf. however, there was no difference of cr-popf between two groups. pancreas thickness on stapling point were not different between two groups ( . mm vs . mm, p= . ). in ns group, patients ( . %) developed a popf, whereas in os group, patients ( . %) developed a popf. there was also no difference of popf between groups. conclusion: the gia™ reloads with the thickest tri-staple™ allows effective prevention of cr-popf after distal pancreatectomy. however, there was no advantage over thinner stapler for lp. introduction: single-incision laparoscopic hepatectomy (silh) has been showed feasible and safe in experienced hands for selected patients with benign or malignant liver diseases. there were only small series reported and most of the procedures were minor liver resections. we herein present our experience of silh during a period of months. methods and procedures: consecutive patients underwent silh which were performed by two experienced laparoscopic surgeons with straight instruments. patient characteristics and surgical outcomes were analyzed by reviewing the medical charts. results: the patient age was . ± . ( - ) years with male predominance ( patients, . %). six patients ( . %) had liver cirrhosis proved by pathologic examinations. nine procedures ( . %) were indicated for malignancy. four major hepatectomies (over two segments) and nine minor ones were performed including seven anatomical resections. the abdominal incisions were para-or trans-umbilical except one which was along the old operative scar at lower midline, while most of them (n= , . %) was within cm in length. inflow control was carried out by either individual hilar dissection or extraglissonian approach instead of pringle maneuver. the operations were all accomplished successfully without additional ports or open conversion. the operative time was . ± . ( - ) min and the estimated blood loss was . ± . ( - ) ml. five ( . %) patients encountered complications and four of them were classified as clavien-dindo grade i. the postoperative length of hospital stay was . ± . ( - ) days. there was no mortality. conclusion: silh can be performed safely and efficaciously for selected patients with benign and malignant liver diseases including cirrhosis. not only minor but also major liver resections are feasible. this innovative procedure provides low postoperative pain and fast recovery. before adopting this demanding technique, surgeons should be familiar with both single-incision laparoscopic surgery and laparoscopic hepatectomy. better outcomes after the learning curve could be anticipated. background: laparoscopic distal panreatectomy (ldp) has been replacing the open procedure for benign or malignant diseases of the pancreas. however, it is often difficult to apply ldp for pancreatic ductal adenocarcinoma (pdac) because its aggressive invasion to adjacent organs or major vessels. objectives: the objective of this study was to report our experiences for laparoscopic extended pancreatectomy with en-bloc resection of adjacent organs or major vessels for left-sided pdac. methods: we reviewed data for all consecutive patients undergoing ldp for left-sided pdac at asan medical center (seoul, south korea) between april and december . the patients who underwent laparoscopic extended panreatectomy with en-bloc resection of adjacent organs or major vessels were included in analyses. results: of total patients, underwent laparoscopic extended pancreatectomy. there were male and female patients with a median age of . years. resected adjacent organs or vessels were as following: stomach in , duodenum in , colon in , kidney in , superior mesenteric vein in , and celiac axis in . median operative duration was minutes, and median length of hospital stay was days. pathological reports revealed the following: a median tumor size of . cm, the tumor differentiation (well differentiated in , moderately differentiated in , and poorly differentiated in ), t stages (t in , t in , and t in ) , and n stages (n in and n in ). r resection was achieved in patients, and most r resection were tangential retroperitoneal margins. postoperatively, clinically relevant postoperative pancreatic fistula was occured in patients, and there was no -day mortality. median overall survival was . months and year survival rate was . %. conclusions: although laparoscopic surgery has limitations in treating extensive diseases, some selected patients can be applicable for laparoscopic extended pancreatectomy with acceptable complication and survival rates. who underwent hepatic resection was included. these patients were divided into llr or olr. demographics, tumor characteristics, recurrence rates and over-all survival were compared between the groups. results: patients were included and grouped into llr (n= ) and olr (n= ). the average tumor number was ± for both groups, while the mean tumor size was . cm and . cm for the llr and olr group, respectively. when compared with olr, llr had lower post-operative complication rates ( . % vs . %, p= . ) and shorter hospital stay ( vs days, p= . ), although the difference was not statistically significant. overall, recurrence-free and disease-free survival was comparable between llr and olr. introduction: single port surgery has been described since with cholecystectomy, colectomy, gastrectomy, and others. nevertheless, few cases are still reported in field of hbp surgery. herein, we report single port pancreatic surgery developed from our previous experience. we had started single port surgery in , since then we have done more than cases of single port surgery using surgical glove port including cholecystectomy, appendectomy, and colectomy. because we consider this experience should develop to pancreatic surgery, cases of single port staging laparoscopy for potentially resectable and borderline resectable pancreatic cancer and cases of single port plus one port distal pancreatectomy (spop-dp) have been done in our institution. single port staging laparoscopy for pancreatic cancer. resectability was proved in ( %) out of patients while patents had unresectale factor such as small liver and peritoneal metastases that was not able to detect pre-operatively. the length of hospital days were . ± . days and the days to chemotherapy were . ± . days. single port plus one port distal pancreatectomy (spop-dp) spop-dp starts with . cm skin incision on umbilicus. subsequently, a wound retractor is installed at umbilical wound. then, a non-powdered surgical glove ( . inches) is put on the wound retractor through which three -mm slim trocars and one -mm trocar are inserted via each finger tips. a semi-flexible laparoscopic camera is inserted via the middle finger port. -mm port is used when laparoscopic us, mechanical stapler, endo intestinal clip or retrieval bag were needed. an additional -mm port is inserted at left subcostal lesion mainly used for surgeon's right hand instrument. gastric posterior wall is fixed to abdominal wall by suture instead of manual retraction. pre-compression before transection of the pancreas was done using endo intestinal clip before firing. discussion: as we have seen in these two decades, surgery has dramatically been changed by laparoscopic surgery or robotic surgery. nevertheless, because of technical difficulty and relatively high post-operative complication rate, introduction of reduced port surgery to hbp surgery has just started. spop-dp using endo intestinal clip, glove port and gastric wall hanging method is feasible. but its advantage is not clear so far, multicenter rct is highly desired to clear the benefit of reduced port surgery for pancreas. introduction: scoring systems (ss) are an essential pillar of care in acute pancreatitis (ap) management. we compared six ss (acute physiology and chronic health examination (apache-ii), bedside index for severity in ap (bisap), glasgow score, harmless ap score (haps), ranson's score and sequential organ failure assessment (sofa) score) for their utility in predicting severity, intensive care unit (icu) admission and mortality. methods: ap patients treated between july and september were studied retrospectively. demographic profile, clinical presentation and discharge outcomes were recorded. predictive accuracy of six ss was assessed using areas under receiver-operative curve (auc) with pairwise comparisons. results: patients were treated for ap. twenty-two ( . %) patients were excluded for insufficient data. / ( . %) were male and mean age was . ( - ) years. most common aetiology was gallstones ( . %). mean length of stay was . ( - ) days. ( . %) patients had severe ap, ( . %) required icu admission and ( . %) died. table below shows positive predictive value (ppv), negative predictive value (npv) and auc of six ss in predicting outcomes. pairwise comparisons revealed ranson's (p. ) and sofa (p. ) scores were superior than other ss in predicting all three outcomes. auc of sofa was greater than ranson's score in predicting severity (p. ), but similar in predicting icu admission (p= . ) and mortality (p = . ). conclusion: sofa score is superior to classical ss in predicting severity, icu admission, and mortality in ap. introduction: necrotizing pancreatitis is often a devastating sequelae of acute pancreatitis. historically several approaches have been described with variable outcome. open necrosectomy is associated with higher morbidity ( %) and mortality ( %). endoscopic necrosectomy often is tolerated well but associated with stent migration and multiple procedures. video-assisted retroperitoneal debridement is tolerated well but associated with severe bleeding if adjacent blood vessels are injured during the procedure leading to severe complications. methods: in our series, we perform a step up approach by involvement of a multidisciplinary group consisting of general surgeons, gastroenterologists, infectious disease physicians, critical care internalist, interventional radiologist and nutritional services to formulate a management plan. the necrotized pancreas is initially drained with an ir guided drain, fluid cultures sent for microbiology and treatment with appropriate antibiotics if deemed necessary. the drain is gradually upsized to a fr sized drain to form a well-defined tract for surgical debridement; a preoperative ct scan of the abdomen with iv contrast to access the location and proximity of the vasculature around the necrotized pancreas. a collaboration with the interventional radiologist to discuss possible ir embolization of splenic artery prior to surgical debridement. the patient would then undergo video assisted retroperitoneal pancreatic necrosectomy and a sump drain left in-situ at the pancreatic fossa. post-operative management in the surgical icu would be lead by the critical care internalist. results: three patients were managed by this multidisciplinary approach with excellent outcomes. one patient underwent preoperative ir embolization followed by surgical debridement; second patient underwent embolization immediately following debridement; one patient did not require any embolization but had ir on standby if needed to intervene. post-operatively all three patients recovered well. they all were tolerating good oral intake and were discharged to rehabilitation facilities. conclusion: our preliminary experience demonstrates that an early multidisciplinary plan by various subspecialties can result in a pragmatic and successful approach to this potentially catastrophic condition. introduction: liver resection with preservation of as much liver parenchyma as possible is called parenchymal sparing hepatectomy (psh). psh has been shown to improve overall survival by increasing the re-resection rate in patients with colorectal liver metastases (crlm) and recurrence. the caudal-cranial perspective in laparoscopy makes the cranial segments ( , a, , and ) more difficult to access. the objective of this systematic review is to analyze feasibility, safety, morbidity, and oncologic outcomes of laparoscopic psh. methods: a systematic review of the literature was performed. medline/pubmed, scopus, and cochrane databases were searched. a search strategy was published with the prospero registry. a systematic review was conducted on all cases reported, they were categorized by area of resection and quantitative meta-analysis of operative time, blood loss, length of hospital stay, complications, and r resection was performed. results: of the studies screened for relevance, studies were selected. because interventions or endpoints were noncontributory or reporting incomplete, were excluded. only publications remained, reporting data from patients who underwent laparoscopic psh. the highest oxford evidence level was b and selective reporting bias was common due to single center and noncontrolled reports. among them, ( . %) resections were in the cranial segments ( . %), a ( . %), ( %), and ( . %), which previously would have required laparoscopic hemi-hepatectomies or sectorectomies. the most common tumor type was crlm ( %) and the second most common tumor type was hepatocellular carcinoma ( %). feasibility of laparoscopic psh was %, conversion rate was %, and complications were seen in % of cases. no perioperative mortality was reported. no standardized reporting format for complications was used across studies. meta-analysis revealed a weighted average operating time of minutes, estimated blood loss of cc, and length of stay of days. r resections were achieved in % of cases. conclusion: laparoscopic psh of difficult to reach liver tumors are feasible with acceptable conversion and complication rate, but relatively long operating times and relatively high blood loss. in future studies, data on long term survival and specific tumor type recurrence should be reported and bias reduced. yangseok koh , eun-kyu park , hee-joon kim , young-hoe hur , chol-kyoon cho ; chonnam national university hwasun hospital, chonnam national university hospital purpose: laparoscopic surgery has become the mainstream surgical operation due to its stability and feasibility. even for liver surgery, the laparoscopic approach has become an integral procedure. according to the recent international consensus meeting on laparoscopic liver surgery, laparoscopic left lateral sectionectomy ( conclusion: this study showed that laparoscopic lls is safe and feasible, because it involves less blood loss and a shorter hospital stay. for left lateral lesions, laparoscopic lls might be the first option to be considered. keywords: laparoscopy, left lateral sectionectomy. outcome analysis of pure laparoscopic hepatectomy for hcc and cirrhosis by icg immunofluorescence in.-a propensity score analysis introduction: in laparoscopic hepatectomy, the surgeon cannot use their hand to palpate the liver lesion and estimate margin of resection. the use of icg immunofluorescence technique can show up the liver tumour and has the potential to facilitate a throughout assessment during the operation. method: between and , there were patients undergone pure laparoscopic liver resection for hcc in our hospital. patients had undergone surgery by the conventional laparoscopic approach. patients had laparoscopic hepatectomy with additional icg immunofluorescence augmented technique. the surgical outcome was compared with propensity score analysis in a ratio of : . result: patients had icg immunofluorescence assisted laparoscopic hepatectomy (group ). patients using conventional laparoscopic liver resection with propensity-matched were selected for comparison (group ). the median operation time was minutes vs minutes p= . , the median blood loss was ml vs ml (p= . ). additional tumours were identified by icg technique. patients had suspicious lesion picked up by icg technique but proven to be benign pathology on frozen section examination. the sensitivity of tumour detection by group was %. % r resection was achieved in group and group respectively. hospital stay was days vs days (p= . ), post-operative complication was ( %) vs ( . %) (p= . ) none of the patient developed icg related complication. conclusion: in the current study, the new technique showed equally good short-term outcome when compared with conventional laparoscopic hepatectomy. icg immunofluorescence augmented reality is a promising technique that might facilitate easier identification tumour during laparoscopic hepatectomy. surg endosc ( ) :s -s taking the training wheels off: transitioning from robotic assisted to total laparoscopic whipple introduction: there is a substantial learning curve to performing minimally invasive pancreatoduodenectomy (mis-pd) for surgeons who are trained in open pd. the learning curve to transition from robotic assisted pd (rapd) to total laparoscopic pd (tlpd) is not well established. methods: mis-pds performed between january and june performed by sc as a surgeon or co-surgeon were included for analysis. mis-pds were performed using a robotic assisted technique prior to august , and tlpds were performed subsequently. rapds performed prior to were excluded to limit the comparison to rapds after the initial learning curve. demographics, clinical and pathologic outcomes, operative and post-operative outcomes were compared. results: a total of rapds and tlpds were scheduled during the study period. there was no statistically significant difference in age, body mass index, or prior abdominal surgery. median time from initial clinic consultation to surgery was days for the rapd group versus days in the tlpd group (p= . ). conversion to laparotomy was required in of patients ( there were no operative complications or mortality. the mean hospital stay was ± . hours. there was no postoperative jaundice, bile leak, intra-abdominal collections or mortality. conclusion: when surgery is indicated for difficult acute calculous cholecystitis, laparoscopic subtotal cholecystectomy with control of the cystic duct is safe with excellent outcomes. however, if the critical view of safety can't be achieved due to obscured anatomy at calot's triangle, conversion to open surgery or cholecystostomy must be performed to prevent bile duct injury. scott revell, md , joshua parreco , rishi rattan, md , alvaro castillo, md ; u. miami -jfk gme consortium, university of miami, miller school of medicine introduction: over the last two decades the increasing incidence of benign liver tumors has led to the expanded need for clinicians to make therapeutic decisions regarding the utilization of open, minimally invasive and ablative techniques. the purpose of this study was to compare outcomes of the management of benign liver disease based on operative approach and pathology. methods: patients aged years or older who underwent liver surgery for benign liver tumors from to were identified in the nationwide readmissions database. patients were compared based on liver pathology, resection versus ablation, and an open versus laparoscopic/robotic approach. the outcomes of interest were in-hospital mortality, prolonged length of stay (los) [ days, and readmission within -days. univariable analysis was performed for these outcomes and multivariable logistic regression was performed using the variables with a p-value . on univariable analysis. results were weighted for national estimates. results: there were , patients undergoing surgery for benign hepatic tumors in the us during the study period. the most common pathology was benign neoplasm ( . %) followed by hemangioma ( . %), and congenital cystic disease ( . %). resection alone was performed in . %, ablation alone in . %, and resection with ablation in . %. a laparoscopic/robotic approach was used in . % of cases. the overall mortality rate was . %, a prolonged los was found in . %, and readmission within days occurred in . %. an increased risk for mortality was found with hemangioma (or . , p= . ) and congenital cystic disease (or . , p= . ). resection with ablation was associated with an increased risk of prolonged los (or . , p. ), while a laparoscopic/robotic approach was a protective factor for prolonged los (or . , p. ). patients treated with ablation alone were at decreased risk for readmission (or . , p. ). omar m ghanem, md , desmond huynh, md , tomasz rogula, md ; mosaic life care, cedars sinai, introduction: laparoscopic sleeve gastrectomy is the most commonly weight loss procedures performed worldwide. as such, there is great diversity in the techniques utilized. this study aims to identify and categorize the differences in techniques and assess the need for guidelines in this field. case description: surgeons were surveyed on the techniques they employ on biweekly basis using the international bariatric club facebook group. the survey included sleeve staple line reinforcement, preoperative work up, intraoperative hiatal dissection, bougie size, distance from pylorus to distal staple line, and intraoperative leak testing. surveys were conducted between may and july . each survey was active for weeks after which data was collected. participants were required to select a single answer per question. discussion: when surveyed on staple line reinforcement (n= ), surgeons used no reinforcement, over-sewed, buttressed, clipped as necessary, over-sewed as necessary. for preoperative work up (n= ), utilized routine endoscopy, routinely obtained upper gi series, routinely obtained both endoscopy and upper gi, and employed endoscopy or upper gi series only in patients who were symptomatic. for hiatal dissection (n= ), surgeons dissected the hiatus routinely, dissected only when obvious hernias intraoperatively, dissected only if the hernia was detected on preoperative work up, and dissected in the setting of gerd symptoms. for sleeve caliber sizing (n= ), bougie \ f was used by surgeon, bougie size f, f, f were utilized by , bougie size f and f were utilized by , bougie[ f were used by , and gastroscopes ( f) were used by . with regards to distance from pylorus to where the sleeve staple line was initiated (n= ), participants started \ cm away from pylorus, between and cm, and started [ cm from pylorus. finally, for preferred intraoperative leak test during sleeve (n= ), methylene blue was used by surgeons, air leak test by , used both, and opted for none. conclusion: this study characterizes the wide varieties in the techniques used during sleeve gastrectomy. a great number of variations exist in every parameter surveyed; however, there is little evidence comparing the effectiveness and safety of these variations. in this setting, further randomized controlled trials are necessary and should be used to construct guidelines to best optimize outcomes in this extremely common and necessary operation. yen-yi juo, md, mph, yas sanaiha, md, yijun chen, md, erik dutson, md; ucla introduction: bariatric surgeries are commonly performed in accredited centers of excellence, but no consensus exists regarding the optimal readmission destination when complications occurred. our study aims to examine the impact of care fragmentation on post-operative outcome and evaluate its causes and consequences among patients undergoing -day readmission after bariatric surgery. methods: the metabolic and bariatric surgery accreditation and quality improvement program (mbsaqip) database was used to identify patients who experienced -day unplanned readmission following bariatric surgery. non-index readmission was defined as any readmission occurring at a hospital other than the one where initial surgery was performed. primary outcome was -day mortality after surgery. logistic regressions were used to identify risk factors for nonindex readmission and to adjust for confounders in the association between non-index readmission and -day mortality. results: a total of , patients were identified as experiencing -day unplanned readmission following bariatric surgery, among whom ( . %) were non-index readmissions. occurrence of postoperative complication during initial hospitalization was the most significant risk factor for non-index readmission (or . , % ci . - . , p= . ) in our multivariate logistic regression. the three most common reasons for readmission were similar within the two comparison groups, including nausea/vomit, abdominal pain and anastomotic leakage. similar proportion of patients underwent reoperation among the two comparison groups ( . vs . %, p= . ). even after adjusting for occurrence of complications, being readmitted to a non-index facility was still associated with a . -fold odds of -day mortality ( % ci . - . , p\. ). conclusion: non-index readmission significantly increases the risk of -day mortality following bariatric surgery. patients were more likely to visit a non-index facility if complications occurred during their initial hospitalization. further patient education is required to re-inforce the importance of continuity-of-care during management of bariatric complications and guide patient's decision making in choosing readmission destinations. introduction: sleeve gastrectomy has become the most performed bariatric surgery. removing part of the stomach causes weight loss by restricting food intake and regulating the production of incretins, particularly ghrelin. however, prognostic factors to weight loss after sleeve gastrectomy have been difficult to find. the goal of this research was to study the correlation between the volume of resected stomach and weight loss. methods and procedures: volume of resected stomach of patients undergoing sleeve gastrectomy was measured. a standard laparoscopic technique was used. calibration was performed tightly around a fr bougie, and stapling started - cm from the pylorus. the standardized technique for measurement involved insufflation with a g catheter with saline solution to a pressure of cm h o immediately after removal of the specimen. resected stomach's volume, gender, age, bmi, height and % of total weight loss (%twl) at months and year were prospectively recorded. correlation between variables was analyzed with pearson's test and linear regression models. conclusion: removed stomach was larger on men than women and its size slightly correlated to height. however, volume of resected stomach did not seem to have an incidence on short termweight loss. gastric size should not be considered as a prognostic factor for weight loss in patients undergoing sleeve gastrectomy. revisional bariatric surgery after initial laparoscopic sleeve gastrectomy: what to choose salman alsabah, eliana al haddad, ahmad almulla, khaled alenezi, shehab akrouf, waleed buhamid, mohannad alhaddad, saud al-subaie; amiri hospital introduction: bariatric surgery has been shown to produce the most predictable weight loss results, with laparoscopic sleeve gastrectomy (lsg) being the most performed procedure as of . however, inadequate weight-loss may present the need for a revisional procedure. the aim of this study is to compare the efficacy of laparoscopic re-sleeve gastrectomy (lrsg), laparoscopic roux-en-y gastric bypass (lrygb) and gastric mini-bypass surgery (mgbp) in attaining successful weight loss following initial lsg. methods: a retrospective analysis was performed on all patients who underwent lsg at amiri and royale hayat hospital, kuwait from to . a list was obtained of those who underwent revisional bariatric surgery after initial lsg, and their demographics were analyzed. introduction: the aim of this study is to identify potential risk factors or early indicators, specifically related to perioperative blood pressure, and its association with perioperative hemorrhage in the bariatric population. laparoscopic bariatric surgery in the united states has been steadily increasing over the past several years. between and , the annual number of cases has increased by %. although rare, hemorrhagic complications (hc) occur at a rate of - % and can lead to significant morbidity and mortality. by identifying factors which may place a patient at higher chance of hc, surgeons can potentially mitigate those risks. these modifications could reduce morbidity and limit the requirement of transfusions or reoperations. methods and procedures: a retrospective case-control series was performed to include all patients who underwent either laparoscopic sleeve gastrectomy (sg) or laparoscopic roux-en-y gastric bypass (gb) in at a single bariatric center of excellence. a total of patients were identified with perioperative hc. each patient was matched : for procedure, body mass index, and medical comorbidities. peak systolic, diastolic, and mean arterial pressures were compared between groups at time of admission, intraoperative, and during remainder of initial hospital stay. welch's t-tests were used for comparison between groups. results: a total of procedures were performed with de novo sg, and de novo gb. revisional bariatric cases were excluded from the study. hc occurred in ( . %) total patients, gs and gb. four patients required operative treatment for hc, were treated laparoscopically and required laparotomy. the mean diastolic pressures at time of arrival on day of surgery was higher in patients who develop hc (p= . ) and mean peak diastolic pressure intraoperatively was lower in patients who develop hc (p= . ). there was no statistical difference in peak systolic or mean arterial pressures throughout the hospital stay. conclusions: bariatric surgical patients with elevated preoperative diastolic blood pressures are at an increased risk of postoperative hc. additionally, decreased peak diastolic blood pressures may be an early indication of an hc in bariatric patients. introduction: bariatric surgery in the adult population is recognized as one of the most effective treatments for obesity and its comorbidities. nonetheless, the safety, efficacy, and substantial outcomes of bariatric surgery in young adults are still not well documented. the aim of our study is to evaluate the safety and efficacy of laparoscopic sleeve gastrectomy (lsg) in young adults (\ years old) versus older adults (≥ years old). methods: we retrospectively reviewed all patients who underwent bariatric surgery at our institution from to. propensity score matching was used in order to balance covariates, matching for common demographics and comorbidities between the younger patient population (\ years old) and the control group ([ years old). all tests were two-tailed and performed at a significant level of . . statistical software r, version . . ( - - ) was used for all analyses. results: of patients, . % (n= ) met our inclusion criteria after matching. we found . % (n= ) patients under years old and . % (n= ) patients greater or equal to years old (control group). we observed that our younger population distribution was predominantly caucasian and female, . % (n= ) and . % (n= ) respectively. the mean age was . ± . years with a preoperative body mass index (bmi) of . ± . kg/m in the younger group compared to . ± . years and a bmi of . ± . kg/m in the control group. diagnosis of diabetes and hypertension were present in . % (n= ) and . % (n= ) of our younger group, respectively. no statistical significance was found when assessing the percentage of bmi loss (% ebmil) at and months follow-up as shown in table . when comparing the % ebmil at months follow-up, the younger group had . % more ebmil than the control group (p= . ). when assessing post-operative complications we observed no statistical significance. conclusions: bariatric surgery is equally effective and safe in young adult population demonstrating a significant better %ebmil at months following bariatric surgery. following prospective studies are needed to elucidate the resolution and behavior of comorbidities in a younger bariatric population. minimally invasive conversion of sleeve gastrectomy to rouxen-y gastric bypass for intractable gastroesophageal reflux disease: short term outcome background: surgical management recommendations for intractable gastroesophageal reflux disease (gerd) after sleeve gastrectomy (sg) remain controversial. this case series demonstrates our experience with treatment of post-operative intractable gerd using minimally invasive conversion of sg to roux-en-y gastric bypass (rygb). patients and methods: this is a retrospective review of a prospective data registry (mbsaqip) from jan through sept . eleven patients, female and male, were evaluated. of the surgeries, were laparoscopic, assisted with xi da vinci robot, and assisted with si da vinci robot. all patients presented with intractable reflux on high dose ppi. three had a history of aspiration pneumonia. ± . %, respectively. one was omitted due to pending results. conclusion: several solutions exist for operative management of intractable gerd after sg including redo-sleeve gastrectomy, combined gastrectomy with fundoplication, conversion to gastric bypass or anti-reflux procedures such as linx. reports remain small in series and require further study to evaluate the consistency of results. we found minimally invasive conversion of sg to rygb is a highly effective and safe option for treatment of intractable gerd. setthasiri pantanakul, chotirot angkurawaranon, ratchamon pinyoteppratarn, poochong timrattana; rajavithi hospital background: obesity is an important health problem affecting more than million people worldwide. esophageal dysmotility is a gastrointestinal pathology associated with obesity; however, its prevalence and characteristics remain unclear. esophageal dysmotilities have a high prevalence among obese patients regardless of gastrointestinal symptoms. objective: to identify the prevalence of esophageal motility disorder in asymptomatic obese patient. materials and methods: prospective study was performed between june and march . a total of of morbid obese patients who visited the bariatric and metabolic clinic at rajavithi hospital (bangkok, thailand) underwent preoperative evaluation with high resolution esophageal manometric test with manoscantm eso (smith medical). tracings were retrospective analysis and reviewed according to chicago classification criteria for esophageal motility disorders. results: among asymptomatic obese participants, twenty five of them were female. the mean age was . ( - ) years old. most of the participants were classified as class three obesity or over. the mean bmi was . kg/m . no hiatal hernia was found and the anatomy of esophagus was normal in all patients. the mean irp was . mmhg. twenty-one patients ( . %) demonstrated high irp over normal limit ([ mmhg) . four patients demonstrated premature contraction (dl\ . second). hypercontractile esophagus was identified in patients and ineffective motility disorder was found in patients. two patients were diagnosed as distal esophageal spasm (des). two patients were compatible with type achalasia and patients ( . %) have esophageal outflow obstruction. none of the patient demonstrate incomplete bolus clearance even high irp or abnormal motility. conclusion: this study reveals a high prevalence of esophageal dysmotility in asymptomatic thai obese patients. the most common abnormality were esophageal outflow obstruction and ineffective motility. the chicago classification of esophageal motility disorder may not suitable among obese population. sitembile lee, ms , chike okolocha , aliu sanni, mdfacs ; philadelphia college of osteopathic medicine ga campus, eastside bariatric and general surgery introduction: roux-en-y gastric bypass (rygb) is the most popular bariatric procedure performed worldwide, accounting for % of all bariatric procedures. however, in patients with a body mass index (bmi) ≥ kg/m (super-super obese) the rygb procedure can be technically challenging. this has led to the adoption of a single-stage treatment such as one anastomosis (mini) gastric bypass (oagb/mgb) in the super-super obese patients. proponents of the oagb/mgb claim the clinical outcomes are comparable to the rygb. the aim of this study is to compare the outcomes of the two procedures by examining the literature. methods: a systematic review was conducted through pubmed to identify relevant studies from to with comparative data on rygb versus oagb/mgb on super-super obese populations. the primary outcome was the percentage excess weight loss (%ewl). other outcomes include operative times, complication rates and length of hospital stay. results were expressed as standard difference in means with standard error. statistical analysis was done using randomeffects meta-analysis to compare the mean value of the two groups (comprehensive meta analysis version . . software; biostat inc., englewood, nj introduction: obesity is becoming more prevalent in patients with inflammatory bowel disease (ibd). the obese body habitus increases the complexity of surgeries that are often needed to treat ibd. some surgeons may delay definitive surgical treatment because of obesity. little data exists on bariatric surgery in the obese patient with ibd. methods: we retrospectively identified patients who had known diagnosis of ibd who underwent bariatric surgery from to . demographics and post-operative outcomes were assessed. results: patients were identified: with ulcerative colitis (uc) and with crohn's disease (cd). of the uc patients, none of the patients had surgery for uc and only one was on a biologic. of the uc patients, had adjustable gastric band (agb), had gastric bypass and had sleeve gastrectomy. one patient with agb had it replaced for slip and subsequently removed for dysphagia. uc preoperative bmi average was . . postoperative bmi was . with excess weight loss (ewl) of %. average follow up was months. of the cd patients, patients had ileocolic resections and one had total proctocolectomy with end ileostomy. one was on remicade and one on mp. of the cd patients, had agb, had gastric bypass and had sleeve gastrectomy. one agb patient had conversion to gastric bypass because of dysphagia and poor weight loss. a second abg patient had band removal because of dysphagia. cd patients' preoperative bmi average was . . postoperative bmi was . with average ewl of %. average follow up was months. overall, agb patients had % ewl, sleeves % and gastric bypass %. two uc patients had post-operative flares, one immediately post op and one month post-operative. four of the band patients had dysphagia, with one replacement, two removals and one conversion to bypass. there were no leaks, intraabdominal infections, fistulas or wound infections. conclusions: uc patients appear to have higher excess weight loss compared to crohn's patients; ewl % compared to % but was not statistically significant. agb had poor results in both uc and cd patients. sleeve gastrectomy and gastric bypass results in effective weight loss for obese patients with ibd. gastric bypass in ibd patient is controversial, but may be appropriate in the right clinical setting. introduction: previous studies suggest that modest preoperative weight loss is associated with improved weight loss following bariatric surgery. however, there remains a need to investigate factors which may successfully predict preoperative weight loss among bariatric patients. methods and procedures: this analysis included patients who underwent laparoscopic roux-en-y gastric bypass (rygb), sleeve gastrectomy, or gastric banding at an academic medical center in california. data were measured at patients' consult and preoperative clinical visits. preoperative weight loss outcomes were categorized as follows: no weight loss, lost weight, or gained weight. associations between categorical sociodemographic and surgical characteristics and preoperative weight loss outcomes were assessed using the chi-square test of association. associations between continuous measures and preoperative weight loss outcomes were assessed using anova. a sub-group analysis was completed among participants who lost weight prior to bariatric surgery. wilcoxon-rank-sum and kruskal-wallis tests were used to evaluate associations between patient characteristics and the number of pounds lost. results: patients (n= , ) were predominately ages - ( %), female ( %), white ( %), and privately insured ( %). patient race was significantly associated with weight loss outcomes (p = . ): whereas % of white patients lost weight prior to surgery, only % of black patients lost preoperative weight. among privately insured patients, % lost weight. in contrast, % of patients insured by medi-cal/medicaid lost weight (p= . ). on average, lower baseline excess body weight was associated with no weight loss. patients who lost preoperative weight (n= , ) were included in the sub-group analysis. male sex (p\. ), black race (p. ), undergoing laparoscopic rygb (p= . ), no previous abdominal surgeries (p= . ), upper tertile baseline weight (p. ), waist circumference (p\. ), percent body fat (p\. ), bmi (p. ), excess body weight (p. ), and systolic blood pressure (p= . ) were associated with more pounds lost. conclusions: this study demonstrates various associations between sociodemographic and clinical patient characteristics and preoperative weight loss. given previous literature indicating the positive relationship between preoperative and postoperative weight loss following bariatric surgery, the results of this study suggest an opportunity to improve preoperative weight loss among specific groups. yen-yi juo, md, mph , usah khrucharoen, md , yijun chen, md , yas sanaiha, md , peyman benharash, md , erik dutson, md ; background: besides rate and extent of weight loss, little is known regarding factors predicting interval cholecystectomy following bariatric surgery, which are important factors in a surgeon's consideration during decision-making regarding whether to perform prophylactic cholecystectomy. in addition, no previous studies have quantified the incremental costs associated with ic. we aim to identify risk factors predicting interval cholecystectomy (ic) following bariatric surgery and quantify its costs. methods: a retrospective cohort study was performed using the national readmission database - . cox proportional hazard analyses were used to identify risk factors for ic. linear regression models were constructed to examine associations between cholecystectomy timing and cumulative hospitalization costs. background: patient-reported outcomes after bariatric surgery are important in understanding the longitudinal effects of surgery. the impact of hospital practices and surgical outcomes on followup rates remains unexplored. objective: to assess the effect of hospital-level practices and -day complication rates on -year follow-up rates of a standardized patient-reported outcomes survey. methods: bariatric surgery program coordinators in a statewide quality improvement collaborative were surveyed in june about their practices for obtaining patient-reported outcomes data one year after surgery. hospitals were ranked based on their follow-up rates between and (accounting for overall performance and improvement). univariate analysis was used to identify hospital practices associated with higher follow-up rates. multivariable regression was used to identify independent associations between -day outcomes and follow-up rates after adjusting for patient factors. results: overall, follow-up rates improved from ( . %± . ) to ( . %± . ) though there was wide variability between hospitals ( . % vs . % in ) . coordinator survey response rate was %. sixty-one percent of all surveyed coordinators perceived that surgeons prioritize high follow-up rates. when asked how long were their patients followed for, % of coordinators noted their programs provided lifelong follow-up. patient reminders about the -year survey were used by % of programs, mostly during clinic visits ( %). most programs ( %) had implemented strategies to improve follow-up rates, such as handing out the survey ( %) during clinic visits. follow-up providers included surgeons ( %), nurse practitioners ( %), and/or registered dietitians ( %). patient disinterest ( %), loss to follow-up ( %), survey length ( %), and lack of staff/ resources ( %) were the factors most commonly perceived as barriers to high follow-up rates. when compared to programs in the bottom quartile of follow-up rates, those in the top quartile were more likely to hand out the survey to patients during clinic visits ( % vs . %; p= . ) and had lower rates of risk-adjusted severe complications ( . % vs . %; p= . ), readmissions ( . % vs . %; p= . ), and reoperations ( . % vs . %; p= . ). conclusions: hospitals vary considerably in their -year follow-up rates when seeking patientreported outcomes data after bariatric surgery. there were also significant differences in programspecific practices for obtaining these data. hospitals with higher -year follow-up rates were more likely to physically hand surveys to patients during a clinic visit and had lower -day severe complication, readmission, and reoperation rates. improved -year patient-reported outcomes follow-up after bariatric surgery may be a proxy for higher quality perioperative care. david merkle , kazim mohommed , danielle r rioux , dilendra weerasinghe, md, facs ; nova southeastern university, herbert wertheim college of medicine, bariatric surgery is gaining popularity not only for its weight loss benefits, but also for its metabolic effects. we present a -year-old female patient with symptoms of neuroglycopenia, occurring -years post roux-en-y gastric bypass surgery. during one of her syncopal episodes, her blood sugar was noted to be mg/dl. continuous glucose monitoring demonstrated post prandial hypoglycemia, averaging episodes per day, with a maximum of episodes in one day. upon further evaluation, the lab results of the hba c, chromogranin a, somatostatin, and urinary sulfonylurea levels were all normal, with the c-peptide level within the upper limit of normal. ct scan of the abdomen and pelvis did not show any obvious masses in the pancreas, and since the chromogranin a level was normal, it lead to the empiric diagnosis of nesidioblastosis by exclusion. we placed the patient initially on medical management which included a carbohydrate restricted diet of g per meal, eating - small meals per day, and taking mg of acarbose three times per day. overall, symptoms have improved, and she has - episodes per month, compared with about episodes per day. we will also present the data with regards to other invasive treatment options, which are available when medical treatment options have failed, such as gastric bypass reversal versus distal gastrectomy. vertical banded gastroplasties (vbgs) were a common bariatric procedure in the s but have largely fallen out of favor due to unsatisfactory weight loss and a relatively high incidence of longterm complications such as dysphagia and severe gastroesophageal reflux disease (gerd). one of the ways to address these undesirable effects is to convert to a roux-en-y gastric bypass (rygb). the aim of this study was to assess the safety and efficacy of vbg-to-rygb conversion. outcomes of vbg revisions performed at an academic center between and were reviewed. of the vbg revisions, gastrogastrostomies were created in two patients, two underwent a planned -stage conversion, and vbgs were converted to rygbs. patients were operated on an average of years after their initial vbg. presenting symptoms were weight regain (n= , . %), dysphagia (n= , . %), or severe gerd (n= , . %). fourteen patients ( %) had a gastric staple line dehiscence. of the vbg to rygb conversions, were laparoscopic, were converted to open, were open, and were robotic-assisted. average operative time and length of hospital stay were . minutes and . days, respectively. within the first months post-operatively, twelve ( %) patients required readmission directly related to surgery, while eight ( %) visited the emergency department. eight patients ( %) required at least one unplanned operation due to complication(s) during the entire follow-up: small bowel obstruction (n= , at -week, -months, and -months), necrosis/leak of remnant stomach requiring remnant gastrectomy (n= ), tracheostomy for prolonged respiratory failure (n= ), bleeding (n= ), anastomotic leak (n= ), and hemothorax requiring vats (n= ). four patients ( %) had a contained perforation that was medically managed and five ( %) developed a gastrojejunal anastomosis stricture requiring endoscopic intervention. one patient ( . %) developed pulmonary embolism. there was no mortality directly related to surgery. complete resolution or improvement of gerd/dysphagia was appreciated in all patients in the short term follow-up. patients who presented with weight regain had a mean bmi loss of . ± . points in the median follow-up time of . months up to a year after conversion to rygb. in summary, reoperative bariatric surgeries after vbgs are complex, requiring longer operative times and length of stay. our study found % risk of severe complications requiring reoperations, compared to the previously cited % in short and long-term complications. conversion of vbg to rygb provides excellent relief of severe gerd and dysphagia and is a viable option for significant weight reduction. introduction: bariatric surgery is a safe and effective treatment for severe obesity and its comorbidities. however, concomitant splenectomy is sometimes required due to uncontrolled bleeding during the surgery. limited literature exists regarding the effects of concurrent splenectomy on outcomes of bariatric surgery. this study aimed to determine these outcomes. methods: adult patients with obesity who underwent primary, elective laparoscopic roux-en-y gastric bypass (lrygb) or laparoscopic sleeve gastrectomy (lsg) with concomitant splenectomy were identified from the metabolic and bariatric surgery accreditation and quality improvement program (mbsaqip, ) and national surgical quality improvement program (nsqip, (nsqip, - datasets. using propensity scores (based on baseline variables), patients who underwent primary bariatric surgery were matched : to a control group (primary lrygb/lsg without concomitant splenectomy) and thirty-day postoperative outcomes were compared. continuous variables and categorical variables were categorized as medians with interquartile range (iqr) and counts with percentages, respectively. background: several previous studies have suggested a correlation between weight loss and age after bariatric surgery. objective: the aim of our study is to further address age as a preoperative factor to determine the amount of weight loss after bariatric surgery. materials and methods: we performed a retrospective analysis of outcomes of a prospectively maintained database of , obese patients who underwent either sleeve gastrectomy (sg) or roux-en-y gastric bypass surgery (rygb) at our hospital between and . we analyzed the -month, -month, and -year postoperative percent total body weight loss (%tbwl) of obese patients who underwent bariatric surgery based on their preoperative age. results: the average age of patients included in the study was years old with a range of - years. an inverse relationship between preoperative age and postoperative weight loss was observed. younger patients achieved a higher % tbwl than older patients at the -month, -month, and -year postoperative follow-up. the average %tbwl for all patients at the -month, -month, and -year postoperative follow-up periods were . %, . %, and . %, respectively. at the -year follow-up, for every decade increase in age (above the average age of ), patients lost % less tbwl. conclusion: in our study, younger patients tend to lose a greater amount of %tbwl than older patients after bariatric surgery. results: patients participated in the survey. the median age was yo (iqr: - ) and . % were females. the following responses were encountered when asked about the importance of surgery-related factors: the study population indicated the following responses regarding expectations from magnetic surgery compared to conventional laparoscopy: there was no significant evidence of different responses by demographic groups. additionally, . % of the population indicated that a surgeon performing magnetic surgery should be more skillful than a surgeon performing conventional laparoscopy. conclusion: this study represents the first report of bariatric patient's perception regarding surgery-related factors. notably, nearly % of the cohort indicated that cosmesis after surgery is an important factor, whereas the responses regarding the rest of the factors were indicated as expected. the bariatric population included in this study had a positive perception of magnetic surgery. furthermore, the population perceived that this technique is associated with better outcomes, better cosmetic results, and higher surgeon dexterity. introduction: although much is known regarding medical outcomes of metabolic surgery, less is known regarding quality of life outcomes. we hypothesized that the collection of patient-reported outcomes (pros) could help us understand quality of life in this patient population. we chose to primarily use patient reported outcomes measurement information system (promis) instruments because of their broad applicability, low cost, and ability to use computer-adapted technology to survey. methods: we implemented the routine collection of pros as part of clinical care in december, . patients were offered tablets in clinic, and were asked to complete the surveys at most of their visits. we used computer-adapted technology to decrease the length of time needed to survey. we collected the following promis instruments: depression, pain interference, physical function, and satisfaction with social roles. we also collected the gerd-hrql, a general health question, and a current health visual analog scale (vas). we retrospectively reviewed our results from december through september . results: our response rate was % over the last year of collection. in total, assessments were completed by patients. the mean scores in our total patient population were as follows: vas , gerd-hrql , general health , depression , pain , physical function , and social roles . for promis instruments, the mean for the national population is , with as the standard deviation. for the depression and pain scores a higher score is worse, while a higher score indicates better quality of life for social roles and physical function. conclusions: routine collection of patient reported outcomes can be implemented in a metabolic surgery clinic. health-related quality of life appears to be decreased in this patient population compared to the general public. further work is ongoing to learn about postoperative trends, as well as differential effects of metabolic procedures. the effect of peri-operative antibiotic drug class on the resolution rate of hypertension after roux-en-y gastric bypass and sleeve gastrectomy. results: in total, rygb and sg were included in our analysis. no significant differences were found between cefazolin and clindamycin regarding hypertension resolution rates after sg. there was a significant difference in the resolution of hypertension after rygb with the use of prophylactic clindamycin or cefazolin. as shown in figure , patients who underwent rygb and received clindamycin had a significantly higher rate of hypertension resolution compared to cefazolin. this effect started at weeks post-operatively ( . % vs . % respectively, p= . ) and persisted up to the -year ( . % vs . % respectively, p= . ). we found no significant differences in patient age, sex, number of pre-operative hypertensive medications, pre-operative bmi, or %bmi change after year to account for the significant effect of antibiotic choice on hypertension resolution. conclusion: this study represents the first clinical report to suggest an impact of the type of antibiotic administered at the time of rygb on co-morbidity resolution, specifically hypertension. future studies will be needed to confirm that the mechanism of action for this novel finding is due to the differing modifications of the gastrointestinal microflora population based on the specific peri-operative antibiotic administered. introduction: laparoscopic adjustable gastric band with plication (lagbp) is a novel bariatric procedure which combines the adjustability of the laparoscopic adjustable gastric band (lagb) with the restrictive nature of the vertical sleeve gastrectomy (vsg). the addition of plication of the stomach to lagb should provide better appetite control, more effective weight loss, and greater weight loss potential. objective: the purpose of the study was to analyze the outcomes of lagbp at months. setting: this is a retrospective analysis from one surgeon at a single private institution. methods: data from all patients who underwent a primary laparoscopic lagbp procedure from december to june were retrospectively analyzed. data collected from each patient included age, gender, weight, body mass index (bmi), and excess weight loss (ewl). results: sixty-six patients underwent lagbp. the mean age and bmi was . ± . years and . ± . kg/m, respectively. all patients were beyond the -month postoperative mark. no patient was lost to follow-up. the patients lost an average of % and . % excess weight loss (ewl) at months ( . % follow-up) and months ( . % follow-up), respectively. also, the patients lost a mean bmi of . kg/m and . kg/m at months and months, respectively. the total number of fills during the study period was , and the mean fill volume was . ± cc. dysphagia was the most common long-term complication. the mortality rate was %. conclusions: lagbp is a relatively safe and effective bariatric procedure. in light of recent studies demonstrating poor outcomes following lagb, lagbp may prove to be the future for patients desiring a bariatric procedure without resection of the stomach. the median interval between (lrygb) and reoperation is months in group a and months in group b. the median percentage of excess weight loss (%ewl) is % vs %, respectively (p= . ). patients % ( in group a) were admitted in an emergency with an acute abdomen pain. ct scan was performed in patients % and has shown signs of occlusion in all cases. the most common symptoms were abdominal pain and vomiting. the surgery was performed by laparoscopy in patients % and by laparotomy or conversion in patients %. in all cases internal hernia was reduced and closed all defects. in only one patient in (group a) small bowel at jja was resected. there was no mortality and one patient had pneumonia with acute respiratory distress which was treated medically. conclusions: the closure of mesenteric defects at (lrygb) by tight non-absorbable continued sutures is recommended because it is associated with a significant reduction in the incidence of internal hernia. introduction: laparoscopic roux-en-y gastric bypass (rygb) is a common and effective form of bariatric weight loss surgery. however, a subset of patients will fail to achieve the expected total body weight loss (tbwl) greater than % after months or experience significant weight regain despite dietary, psychiatric, and behavioral counseling. although alternative procedural interventions exist for operative revision after suboptimal rygb weight loss, laparoscopic adjustable gastric banding (lagb) provides an option with short operative time, low morbidity, and effective results. we have previously demonstrated that short-term ( -month), and mid-term ( -month) weight loss is achievable with lagb for failed rygb. the objective of this study is to report the long term year outcomes of lagb after rygb failure. methods and procedures: a retrospective review of prospectively collected data before and after rygb when available, and before and after revision with lagb was performed. background: saline filled intragastic balloons have become a common outpatient procedure for the treatment of obesity. acute dilation, ischemia and necrosis of the stomach has been described in the medical literature. gastric necrosis from acute gastric dilation is a rare but life-threatening condition, which requires timely diagnosis and management. we present a case of partial gastric ischemia with necrosis hours following placement of a saline filled intragastric balloon. postoperative complaints of bloating, nausea and vomiting are common complaints following placement of saline filled intragastric balloons and can lead to a delay in diagnosis. early diagnosis and management is essential in avoiding this life threatening complication. case report: a year old woman, bmi , comorbid conditions of diabetes mellitus underwent uncomplicated placement of a saline filled intragastric balloon for treatment of obesity. hours after placement the patient complained of cramping and bloating. hours following placement the patient developed vomiting and presented to an emergency room for evaluation. she was found to have blood glucose exceeding and a severely dilated stomach with pneumotosis on ct evaluation. ng tube decompression and icu management of the severe hyperglycemia was initiated. removal of the intragastric balloon was delayed - hours until an appropriate endoscopic retrieval kit could be obtained. endoscopic retrieval was performed without incident and near complete necrosis of the gastric mucosa was noted. the antrum was the only area spared. hours after retrieval a laparoscopic evaluation of the stomach revealed full thickness necross of the entire fundus and greater curve. indocyanine green (icg) fluorescent dye was used to assess vascular integrity of the remaining stomach and to define lines of resection. resection of the greater curvature was performed using icg florescent dye to ensure that the angle of hiss was viable and well perfused. the patient had a full recovery and subtotal gastrectomy was avoided. conclusions: spontaneous gastric distension exacerbated by gastric outlet obstruction following placement of a saline filled intragastric balloon can occur. unrecognized this condition can lead to ischemia, necrosis and perforation of the stomach. appropriate evaluation of patients following placement of intragastric balloons is essential. recognition of this condition can be delayed due to the complaints of cramping, bloating and vomiting which are typical following placement of saline filled intragastric balloons. untreated, gastric ischemia and necrosis can lead to early perforation which is associated with a high mortality rate. introduction: morbid obesity has become a growing health risk in the united states with up to % of americans suffering with obesity. bariatric surgery remains the best treatment for morbid obesity. the recent use of laparoscopic sleeve gastrectomy (lsg) as a single stage procedure has met with great success because of its quick learning curve and minimal postoperative complication rates. however, there are concerns if the lsg is an effective procedure for long-term weight loss. although criticized at first, the mini-gastric bypass (mgb) surgery has become a great option for morbidly obese patients because of the ability to lose weight with minimal post-op complications. the aim of this review is to assess the outcomes of lsg as it compares to mgb for the management of morbid obesity. introduction: we hypothesize that a jejunoileal anastomosis and partial diversion using magnamosis, a novel magnetic compression device, is technically feasible and will improve insulin resistance and metabolic syndrome similarly to patients who underwent bariatric surgery. metabolic surgery has demonstrated improvements in various parameters including insulin resistance, triglyceride levels, and cholesterol. it may be technically feasible to perform a less-invasive operation through partial diversion, and thereby stimulate an increase in incretins from the l-cells of the ileum to glean these benefits. methods and procedures: we performed a laparotomy and jejunoileal partial diversion using magnamosis in five rhesus macaques with induced insulin resistance through dietary modifications. after surgery, weight was monitored and a metabolic laboratory evaluation was performed weekly. timed tests were performed at baseline and again at and weeks postoperatively for triglyceride levels, glp- , insulin, glucose, and bile acids. the primates were followed for weeks prior to euthanasia. results are represented as mean±sem and all p-values were calculated using a two-sample students' t-test. introduction: many studies concerning individuals seeking bariatric surgery indicate a higher prevalence of psychiatric disorder in this population, both before and after surgery, however results are not conclusive. the aim of this study was to investigate changes in psychiatric health after gastric bypass surgery. methods: patients within the catchment area of the department of psychiatry of the south alvsborg hospital, operated with gastric bypass surgery during - were identified through the scandinavian quality registry (soreg). patients files were examined and psychiatric diagnoses and alcohol/drug abuse were recorded preoperatively and with a follow up time of years. results: a total of operated patients were identified. of these patients had been in contact with the psychiatric department before or after surgery. patients had attempted suicide preoperatively, but no attempts were made postoperatively, all women. patients attempted suicide postoperatively without a previous history of suicidal attempts, men woman. four patients with a preoperative history of alcohol abuse were identified, all women. these individuals did not seem to abuse alcohol/drugs postoperatively. postoperatively patients with an alcohol/drug abuse were identified, men, women. none of them had a former history of abuse. of the patient performing suicidal attempts postoperatively, men woman, had a postoperatively emerging alcohol/drug abuse. conclusion: preoperatively known alcohol/drug abuse or suicidal attempts do not seem to predispose for postoperative abusive problems or suicidal behavior. preoperative identification of individuals prone to alcohol/drug abuse or suicidal attempts seems difficult. introduction: in the past, our group has popularized models for gastric bypass, sleeve and gastric imbrication. there are currently no models to predict weight loss following single anastomosis duodenal switch. surgeons who offer this procedure are left to guess based on their limited experience how their patients will do following surgery we have developed a simple office based algorithm to predict weight loss following this procedure. method: patients met the criteria for this study. these patients underwent surgery at a single institution from june to december . non-linear regression analysis was performed to interpolate weight loss at one year. a multilinear regression was run to determine the significant variables. a model was then constructed to predict weight loss after single anastomosis duodenal switch. results: bmi, htn, gender, and the interaction between htn and dm were found to affect weight loss. the model achieved a r value of . and the average error of prediction in the model was . %ewl. conclusion: today too many surgical practices offer procedures tailored to surgeon instead of the needs of the patient. using our models predicting postoperative weight loss can be a straightforward process using easily gathered data. all surgeons should be doing this currently in their own practice to allow patient to choose targeted healthcare interventions based on patient's personal goals. surg endosc ( ) introduction: there is a long-standing practice of testing anastamosis both in upper and lower gi surgery. post-operative leaks in bariatric surgery are an uncommon but serious compilation increasing morbidity and risk of mortality. the present study looks at the practice of performing an intra-operative leak test during roux-en-y gastric bypass (rygb) and sleeve gastrectomy (sg). methods and procedures: the study was divided in two independent phases of six months and months. data was collected from all patients undergoing sg, rygb or revision rygb within those two periods. to confirm the integrity of the staple line all patients underwent a methylene blue and air test intra-operatively. this was followed by a gastrograffin swallow the morning post procedure. results: total number of patients in the study was . there were four positive intraoperative tests. one patient was a primary rygb and three patients were undergoing revision rygb. all were reinforced and subsequent recovery and gastrograffin swallow showed no leak. one revision rygb had an undetected small bowel injury distal to jejuno-jejunostomy that was not identified on intraoperative or next day imaging. we used multivariate statistical analysis to study our population sample and classified the impact of each factor or their combination with the use of principal component analysis. we used systematic clustering to identify subpopulations that have significant differences in statistical distribution. result: the main determinant of total operative time was the surgeon and the level of his assistant. prior surgeries, bmi and smoking history had a statistically significant impact on the laparoscopic time (p value. ). removing the impact of various surgeons, we detected four clusters of patients based on more than patient characteristics. we noticed total or time had two different clusters: one with a standard-deviation of - min while the other had over min. conclusion: this study may have practical implications on improving scheduling. the different comorbidities of these bariatric patients helped to stratify patients into these main cluster groups. better predictability on length of operative procedure can lead to more efficient use of or time and staff, thus ultimately leading to savings for the hospital. in addition, we used automated noninvasive tracking methods to identify phases of bariatric procedures that will allow more accurate estimated or time to efficiently schedule cases. the smart or, which is equipped with multiple noninvasive sensors, allows for error free tracking and monitoring without human interference. objectives: successful outcomes after bariatric surgery (bs) require a comprehensive educational program (cep) focused on post-surgical dietary and lifestyle changes. at our institution, patients must comply with a -week life-after-surgery program prior to surgery. since many patients are not able to participate in-person, an online cep was created to improve accessibility. to evaluate comprehension, a -question test is administered at the last preoperative visit to participants of both classes. the primary objective of this study is to evaluate the effectiveness of online versus inperson cep in terms of comprehension and post-operative weight loss. methods: patients who underwent bs from august -may were retrospectively reviewed at a single institution. all patients who underwent the in-person or online cep, completed the -question test, and had post-operative follow-up for at least months were included. baseline demographic, operative, and weight data were obtained using the electronic medical record. background: body weight loss after bariatric surgery is affected by several factors. diabetes status or preoperative body mass index (bmi) would affect the body weight loss after surgery. age and sexuality may also be the predictor. furthermore, the malabsorptive procedure is considered more effective for body weight loss than the restrictive procedure alone. we investigated the contribution of preoperative background data and procedures to the body weight loss after surgery. methods: this was a multicenter, retrospective study to validate the efficacy of bariatric surgery for morbidly obese patients in japan. patients underwent sleeve gastrectomy (lsg) or lsg with duodenal-jejunal bypass (lsg/djb) in each institution from january to december , and whose bmi was kg/m or more at the first visit were included in this study. we investigated the percent excess body weight loss (%ewl) at months after surgery. univariate and multivariate analyses were done to evaluate the predictive factors of body weight loss. we defined that %ewl more than % as well response (wr background: despite its known safety and efficacy, bariatric surgery is an underutilized treatment for morbid obesity in the united states. objective: our goal was to identify factors associated with failing to proceed with surgery despite being considered an eligible candidate by a bariatric surgery program. methods: this is a retrospective study that includes all patients (n= ) who attended a bariatric surgery informational session (bis) at a single center academic institution in . eligible candidates were identified after clinical evaluation and multidisciplinary candidacy review (mcr). we compared patients who underwent surgery to those who did not (i.e. dropped out) by evaluating patient-specific, insurance-specific, and bariatric surgery program-specific variables. univariate analysis and multivariable regression were performed to identify risk factors associated with failing to undergo surgery among eligible candidates. introduction: the elderly are a special subset of the population due to their limited physiological reserve with aging. revisional bariatric surgery is becoming more common with increase in primary bariatric procedures. data on safety, weight loss, and metabolic effects of revisional bariatric surgery in elderly is limited. the aim of this study was to assess the safety and efficacy of revisional bariatric surgery in the elderly. methods: clinical data of all elderly patients ( years and above) who underwent elective revisional bariatric surgery at an academic institute between and were reviewed. demographic data, perioperative variables, and postoperative outcomes were studied. results: a total of patients were identified with a female predominance ( : ). mean age was ± . years. mean bmi at the time of revisional surgery was . ± . kg/m . the primary indication for revisional surgery included management of postoperative adverse events (n= , . %) and weight recidivism (n= , . %). in patients with postoperative complications, the most common indications for revisional surgery were dysphagia (n= , . %), marginal ulcer (n= , . %), gastric outlet obstruction (n= , . %), and fistula formation (n= , . %). the most common type of revisions included conversion of vertical banded gastroplasty to roux-en-y gastric bypass (rygb, n= ), revision of rygb (n= ), conversion of adjustable gastric banding to sleeve gastrectomy (sg, n= ), and sg to rygb (n= ). two out of seven ( . %) patients with -day postoperative readmissions had serious complications that required reoperation. one of them underwent small bowel resection for ischemia and the other had thoracotomy for hemothorax evacuation developing secondary to a gastropleural fistula. while there was no mortality over the first days postoperatively, two patients died months after surgery due to infectious complications. in the median follow-up time of (interquartile range, - ) months, mean weight and bmi changes of − . kg and − . kg/m were observed. twenty-three ( . %) patients had diabetes at time of revisional surgery. a mean reduction of . mg/dl in fasting blood glucose and . % in glycated hemoglobin were noted between baseline and last follow-up. conclusion: revisional bariatric surgery in elderly is associated with high complication rates. our data indicate that revisional bariatric surgery can potentially alleviate symptoms and resolve complications of primary bariatric surgery. elderly patients should have their risk stratified and weighed against the benefits of surgery. anne-marie carpenter, bs, alexander l ayzengart, md, mph; university of florida introduction: bariatric surgery is the most effective treatment for morbid obesity. of all available procedures, laparoscopic sleeve gastrectomy (lsg) is now the most popular worldwide. common complications of lsg include gastroesophageal reflux, stricture, and staple-line leak. although rare, portomesenteric venous thrombosis (pmvt) and liver retractor-induced injuries are increasingly reported. we present a case of isolated left portal vein thrombus after routine lsg that was likely caused by prolonged compression of left liver lobe by the nathanson retractor. case presentation: a -year-old female with a bmi of and biliary colic due to cholelithiasis underwent lsg with hiatal hernia repair and cholecystectomy. she tolerated the procedure without complication and was discharged home on the following day. on postoperative day , she presented to the emergency department with fever and epigastric pain. contrast ct revealed an isolated filling defect within the proximal left portal vein; abdominal doppler demonstrated an acute thrombus occluding the left portal vein with normal flow in the main and right portal veins. the patient was treated with a -month course of therapeutic anticoagulation with lovenox. a complete hematologic workup did not uncover any hypercoagulable conditions. the patient recovered well and remained asymptomatic at her follow-up visit weeks after operation. discussion: pmvt is a rare surgical complication with multifactorial etiology. in bariatric surgery, evidence suggests lsg elicits more frequent pmvt compared with roux-en-y gastric bypass. a systematic review cited the incidence rate of pmvt as . - % after lsg. the mechanisms are thought to be due to pneumoperitoneum, procoagulant obese state, manipulation of portomesenteric venous system during division of the gastrocolic ligament, and postoperative dehydration. liver retraction is paramount during laparoscopic bariatric surgery to provide adequate visualization of the upper stomach and diaphragmatic hiatus. most methods of liver retraction produce significant pressure on the liver parenchyma by compressing it against the diaphragm. three types of liver injury have been documented in literature: minor congestion, traumatic parenchymal rupture, and delayed liver necrosis. uniquely, we propose an additional type of injury-left portal vein thrombosis due to compression of left liver lobe with the nathanson retractor. conclusion: the case described herein represents the first documented report of isolated left portal vein thrombosis after lsg. this is a unique presentation of retraction-related liver injury causing pmvt by mechanical compression of liver parenchyma. as surgical procedures increase in duration, intermittent release of liver retraction should be performed at regular intervals. introduction: up to % of patients experience internal hernia (ih) after laparoscopic roux-en-y gastric bypass (rygb). studies have shown that antecolic roux limb orientation, and closure of the mesenteric defect reduce, but do not eliminate, the incidence of ih. we hypothesize that despite operative differences, ih occur more frequently in patients who experience significant weight loss. this study aims to determine whether those patients who present with ih following rygb experience greater than % excess body weight loss (ebwl). methods: a retrospective chart of all patients who underwent ih repair following rygb at our institution between sept and sept was performed. all applicable cpt codes to encompass ih repair were reviewed (n= ). patients with ih repair after rygb were identified. results: of the patients, were female. the mean pre-rygb weight was lbs (sd± . ), bmi . kg/m (sd± . ). all procedures but one were performed in an antecolic configuration; the other retrocolic-antegastric. fifteen cases were laparoscopic and two were open; nine had the jejunal mesenteric defect closed, eight did not. the average weight loss from the time of rygbp to ih presentation was . lbs (sd± . ) and %ebwl from rygb to the nadir weight was % (sd± ). when evaluated by t-test, there was no statistical difference in bmi at the time of program initiation, rygb, or ih presentation, as well as number of pounds lost, %ebwl, or time to ih presentation, when comparing patients for whom the mesenteric defect was closed or not. average time from rygb to ih presentation was . years (range - days) . conclusion: in our limited cohort of patients who have presented with internal hernia after rygb, there was an average of % ebwl. this is greater than the average expected %ebwl at our institution and others, suggesting that ih may occur in patients with greater weight loss at a higher frequency. mesenteric defect closure did not appear to have any influence in this limited cohort, suggesting that weight loss is a stronger factor in ih development. we plan a more extensive evaluation in a larger cohort of patients to determine if greater %ebwl is a predictor of ih formation in patients undergoing rygb. introduction: introduction of enhanced recovery after surgery (eras) pathways has led to early recovery and shorter hospital stay after laparoscopic roux-en-y gastric bypass (lrygb) and laparoscopic sleeve gastrectomy (lsg). this study aims to assess feasibility and outcomes of postoperative day (pod) discharge after lrygb and lsg from a national database. methods: patients who underwent elective primary lrygb and lsg and were discharged on pod and were extracted from metabolic and bariatric surgery accreditation and quality improvement program (mbsaqip) dataset. a : propensity score matching was performed between cases with pod vs pod discharge, and the -day outcomes of the propensity-matched cohorts were compared. high risk patients were excluded from the analysis. purpose: the aim of this study was to evaluate a large volume, multi surgeon bariatric surgery center producing the largest sample size to date proving efficacy (% weight loss) and safety of sleeve gastrectomy following band removal in one or two step procedures. methods: all patients undergoing conversion of lagb to lrygb ( ) and lsg ( ) regardless of one step vs two step conversion from january to january were included. a retrospective analysis of our prospectively maintained database was performed to compare outcomes in patients undergoing conversion to lrybg vs lsg after lagb to identify the outcomes. introduction: the purpose of the study was to describe the use of intraoperative indocyanine green (icg) fluorescence angiography to identify the blood supply patterns of the stomach and gastroesophageal junction (gej). we hypothesized that identifying these vascular patterns may help modifying the surgical technique to prevent ischemia-related postoperative leaks. methods: patients underwent laparoscopic sg and were examined intraoperatively with icg fluorescence angiography at an academic center from january to september . prior to the construction of the sg, ml of icg was injected intravenously and pinpoint® technology was used to identify the blood supply of the stomach. afterwards, the sg was created with attention to preserving the identified blood supply to the gej and gastric tube. finally, ml of icg were injected and pinpoint® technology was used again to ensure that all the pertinent blood vessels were preserved. results: patients successfully underwent the procedure with no complications. the following blood supply patterns to the gej were found: the incidence of overall accessory blood supply to the right-side dominant pattern was more common than expected. in about half of the cases where an accessory vessel was found in the gastrohepatic ligament, the blood flow was toward the stomach (and not the liver). furthermore, the incidence of accessory blood supply from the left side was found in % of the cases. % of patients had both the left side accessory and accessory gastric artery pattern. in these particular patients, if a concurrent hiatal hernia repair is performed, these accessory blood supplies are at risk of being injured if care is not taken to preserve them, rendering the gej relatively ischemic. conclusion: icg fluorescence angiography allows determining the major blood supply to the proximal stomach prior to any dissection during sleeve gastrectomy so that an effort can be made to avoid unnecessary injury to these vessels. background: morbid obesity, a common medical concern with significant health risks, has a prevalence of . % among u.s. adults. bariatric surgery provides effective weight loss for morbidly obese patients with improvement in their comorbid conditions. traditionally, routine intraoperative drain placement (idp) and postoperative esophagram (ugis) were thought to identify early postoperative complications. recently, these interventions have been scrutinized for their effectiveness. we hypothesized that idp and postoperative ugis do not alter outcomes in bariatric surgery and only increase hospital length of stay (los). methods: two cohorts, each consisting of patients from either or were analyzed from our institution. in the cohort, all patients had idp and an ugis on postoperative day , prior to starting a clear liquid diet. in the cohort, no patients had idp or ugis, but instead were started on a clear liquid diet on postoperative day , in the absence of vomiting. all patients in each cohort underwent either a laparoscopic sleeve gastrectomy or a roux-en-y gastric bypass. a retrospective study was performed to analyze whether there was a significant difference in postoperative complications, length of stay, and operating room time between these two cohorts. those who underwent t dm remission were less likely to be vdd at all time points. the rates of vdd appear to be slightly higher in rygb at each time points. the rates of macrocytic anemia, microcytic anemia and hypoalbuminemia were low and varied depending on surgical procedure, with no relevant increase following surgery (see figure ). conclusions: vitamin d deficiency is prevalent among diabetic patients with obesity presenting for bariatric surgery. the postoperative management was successful in addressing vdd following surgery; those who experienced t dm remission after surgery were less likely to be vdd. further prospective studies are needed to explore this relationship. surg endosc ( ) :s -s introduction: it is well known that morbid obesity is strongly associated with high blood pressure. cardiovascular risk reduction is a well studied and described result of bariatric surgery. the objective of this study is to quantify hypertension resolution in patients who underwent bariatric surgery at our institution. methods: we retrospectively reviewed all the patients who underwent either laparoscopic sleeve gastrectomy (lsg) or laparoscopic roux en y gastric bypass (lrygb) at our institution between and . we selected those patients who were on antihypertensive medical treatment and had a -month follow-up. hypertension resolution was defined as the interruption of any blood pressure medications within the follow-up period. we compared the patients who had resolution of hypertension (group ) with patients who did not (group ), based on demographics, comorbidities, and outcomes. chi-square and student t-test were used for categorical and continuous variables respectively. results: out of patients, ( . %) patients met the inclusion criteria, out of which, ( . %) had a complete resolution of hypertension within months. the patient population included in group was predominantly female n= patients ( . %), diabetic (n= , %), with a mean bmi of . ± . kg/m , a mean age of . ± . years, and a preoperative systolic blood pressure mean of ± . mmhg. the most common procedure performed was lsg with n= ( %). comparison between group and group based on age, gender, bmi, and diabetes showed no statistically significant difference. estimated bmi loss % at months, type of procedure and % ebmil showed no statistically significant difference between the groups. conclusions: rapid weight loss is associated with a drastic reduction of blood pressure. besides weight loss, we did not identify a clear correlation between risk factors when we compared patients who had resolution of hypertension with patients without resolution. further prospective studies should be done for better understand these findings. the mount sinai hospital, university of chicago introduction: for many patients, hiv has transformed from a life-threatening illness into a manageable chronic disease. reflecting trends in the general population, obesity is increasingly prevalent among hiv-positive patients. surgical intervention has shown the greatest effectiveness in treating obesity. it is unknown, however, whether physician attitudes reflect the changing trends in obesity care for hiv-positive patients. methods and procedures: medical students from the first, second, and fourth years of training were invited to participate in an irb-approved survey, handed out during didactic sessions, which was designed to assess their knowledge and attitudes regarding bariatric surgery in hiv-positive patients. self-reported demographic information of respondents was also collected. the outcome of interest was the proportion of correct responses. univariate and multivariate regression analyses were performed. results: surveys were completed by medical students. demographic covariates included the following: age, sex, race, bmi, and year of training. age, sex, race, and bmi were not statistically significant in the multivariate model. however, in both univariate and multivariate models, each additional year of training was associated with a significant increase in the proportion of correct responses (multivariate model beta coefficient= . , p. ). conclusions: obese and hiv-positive patients suffer from well-documented stigma in health care. these findings suggest that medical training corrects common misperceptions of obese and hivpositive patients, and may lead to a better understanding of the appropriateness of bariatric surgery for hiv patients. whether these attitudes are predictive of referral practices remains to be seen. introduction: obesity is a common problem worldwide with numerous associated comorbidities and is associated with an increased risk of developing some cancers. despite bariatric surgery being associated with a risk reduction for cancer development, some will develop cancer after surgery and little is known about complications which might arise during multimodality cancer treatment. here we report the case of a year-old female who developed an unusual giant marginal ulcer (mu) post laparoscopic roux-en-y-gastric bypass (lrygb) while receiving systemic chemotherapy for an early stage breast cancer. case report: in summary, a year-old female with a preoperative bmi of kg/m had an uncomplicated lrygb one year prior to her presentation. she was a non-smoker, was abstinent of alcohol and did not use nsaids, steroids or other ulcerogenic medications. eight months post procedure with a bmi of . kg/m she was diagnosed and treated with bcs plus slnb for a pt n m er/pr +ve her −ve breast cancer. one week following her third cycle of docetaxel and cyclophosphamide, she presented with two days of melena, small volume hematemesis and abdominal discomfort. the patient was resuscitated with prbc, started on a ppi infusion and had free air ruled out on a cxr. upper endoscopy was complete showing a giant mu at the gastrojejunal anastomosis, biopsies ruled out malignancy and h. pylori. subsequent ct abdomen/pelvis identified contrast extravasation from the anastomosis confirming a free perforation. broad spectrum antibiotics were started and a diagnostic laparoscopy complete. a graham patch repair utilizing omentum and abdominal washout were complete with placement of surgical drains. the patient was supported with parenteral nutrition while npo. diet was advanced after an upper gi series on post operative day showed no ongoing leak. the patient was discharged on post operative day , recovered and although further chemotherapy was discontinued she completed whole breast radiotherapy. conclusion: leaks and hemorrhage are early postoperative complications that are not seen intraoperatively in our experience. furthermore, endoscopy significantly increases mean operative time. routine use should be left to the discretion of the surgeon but should not be considered an essential step of the sleeve gastrectomy. the objective of the study: surgical site infection (ssi) following bariatric surgery contributes to patient morbidity and additional use of health care resources. we investigated whether a ssi quality control initiative in the form of a refined preoperativeantimicrobial protocol affected the rate of ssi following laparoscopic roux-en-y gastric bypass (lrygb). we reviewed all lrygb procedures performed between june and december at a single bariatric surgery centre of excellence. two preoperative antimicrobial protocols were compared. patients undergoing surgery prior to february received g of cefazolin whereas patients undergoing surgery after february , received a new antimicrobial protocol consisting of g cefazolin, mg metronidazole and ml oralchlorhexidine rinse. the primary outcome was day ssi including superficial ssi, deep incisional ssi and organ/space infection as defined by the centre for disease control. clinic charts and provincial electronic medical records were reviewed for emergency department visits, microbiology investigations and physician dictations diagnosing ssi. outcomes were assessed using a students t-test. results: two hundred seventy six patients underwent lrygb of which received the refined antimicrobial protocol and received cefazolin. the refined antimicrobial protocol significantly decreased the rate of deep incisional ssi compared to cefazolin (n= , . % vs n= , . %; p\ . ). the refined antimicrobial protocol resulted in an insignificant overall reduction in the rate of superficial ssi (n= , . % vs n= , . %; p[ . ) and organ/space infection (n= , . % vs n= , . %; p[ . ) respectively. conclusions: a preoperative antimicrobial protocol using cefazolin, metronidazole and chlorhexidine oral rinse appears to reduce the rate of ssi following lrygb. this protocol may be most effective to prevent deep incisional ssi. additional patient cases or alternative study design including a randomized control trial is required to better understand the efficacy of this protocol. background: for many years, the roux-en-y gastric bypass (rygb) was considered a good balance of complications and weight loss. according to a several short-term studies single anastomosis duodenal switch or stomach intestinal pylorus sparing surgery (sips) offers similar to weight loss to rygb with fewer complications and better diabetes resolution. however, no one has substantiated complication and nutritional differences between these two procedures over the midterm. this paper seeks to substantiate previous studies and compare complication and nutritional outcomes between rygb and sips. methods: a retrospective analysis of patients who either had sips or rygb from to . complications were gathered for each patient. nutritional outcomes were measured for each group at , , and years. regression analysis was applied to interpolate each patient's weight at , , , , , , and months. these were then compared with t tests, fisher exact tests, and chi squared tests. results: rygb and sips have statistically similar weight loss at , , , , and months. they statistically differ at and months. at months, there is a trend for weight loss difference. there were only statistical differences in nutritional outcomes between the two procedures with calcium at and years and vitamin d at year. there were statistically significantly more long term major complications, minor complications, reoperations, ulcers, small bowel obstructions, nausea, and vomiting with the rygb than sips. conclusion: with comparable weight loss and nutritional outcomes, sips has fewer short and long-term complications than rygb and better type diabetes resolution rates. introduction: the purpose of this study is to determine the risk factors that contributed to increased postoperative complications, as noted in prior studies within the publicly funded insurance population undergoing bariatric surgery. methods and procedures: data was collected via a retrospective review of the medical records of patients who underwent laparoscopic roux en y gastric bypass or laparoscopic sleeve gastrectomy from to at a single institution. for each patient, data was collected in the following categories: baseline demographics, insurance status, medical comorbidities, immediate complications, re-admissions and associated complications, and follow up out to years. results: a total of patient charts were reviewed, patients were categorized as private insurance and patients were categorized as public insurance. there was no statistically significant difference in mean patient age (private . years vs public years), sex (male:female %: % for both groups), or bmi ( vs ). there was a statistical significance in relationship status in the categories of single ( % vs %), married ( % vs %) or living with a partner ( % vs %), as well as employment status ( % vs %). when comparing comorbid conditions preoperatively there was no difference except for diabetes which was less common in the private insurance group % vs %. readmission rates for complications were significantly different as well at % vs % with public insurance patients having increased complication rates and readmissions. there was no difference in follow up percentages at each time point for the two groups. interestingly postoperative bmi was significantly different in the two groups until year out ( vs ) when the difference disappears. conclusions: our current data set confirms prior research that documented higher complication rates in public insurance patient populations without differences in long term results in regards to weight loss. it also shows that the public insurance group is possibly at higher risk for complications and readmissions postoperatively due to the lack of social support at home given that a much higher percentage of them are single or divorced, and lack employment. it is likely that this lack of support at home prompts more frequent readmissions and associated complications. introduction: gastric bypass has been an acceptable treatment for the morbidly obese patient, with proven efficacy on weight loss and remission of co morbidities, especially diabetes (t dm). laparoscopic sleeve gastrectomy (lsg) is gaining momentum as an alternative procedure for the morbidly obese patient. the aim of this study is to assess the resolution of t dm by examining hba c, bmi, fat %, and % excess weight loss in t dm patients in our lsg patients. methods: we performed a retrospective chart review of t dm patients before and after lsg, analyzing hga c, bmi, % weight loss, fat %, and diabetic medications. data was analyzed by using spss version . paired t-test was applied to see the significance of bmi, weight, fat % and hba c before and after the procedure. introduction: gastroesophageal reflux disease (gerd) is a known risk following laparoscopic sleeve gastrectomy (lsg), with up to % of patients affected by the disease postoperatively. of these patients, an unknown number progress to medically refractory gerd. due to their postsurgical anatomy, these patients have limited options for intervention. while endoluminal therapies are available, surgical revision to roux-en-y gastric bypass (lrygb) has become an accepted revisional treatment. despite this therapeutic option, many payors deny coverage for this treatment. in this study, we report outcomes of revision of lsg to lrygb and difficulties in obtaining insurance approval for the operation. methods: we conducted a retrospective review of all patients who underwent a revisional bariatric operation at a single institution between january and august . we analyzed all patients who underwent conversion of lsg to lrygb. we collected data on -day mortality and morbidities, pre-and postoperative antacid use, and the insurance approval process. results: within the study period, we identified patients undergoing revisional bariatric surgery. seventeen patients had undergone conversion of lsg to lrygb. all of these patients underwent revision due gerd refractory to maximal medical therapy. the average body mass index was kg/m , and our average operative time was minutes. one patient required laparoscopic cholecystectomy within days due to acute cholecystitis, and another patient required reoperation for control of staple line bleeding. there were otherwise no -day morbidities or readmissions. fifty nine percent stopped all antacid medication by six months, and % stopped by months. of the % percent of patient still on proton pump inhibitor therapy, none of those patients complained of reflux symptoms. of non-medicare patients, % were initially denied insurance coverage for revision. only one plan accounted for all initial approvals. twenty five percent of denied patients eventually paid out of pocket, and the remaining % ultimately secured coverage after an appeal process. with no significant differences in mortality or hospital stay. significantly shorter operative times were observed in the adolescent group ( . ± vs . ± , p. ). in univariate analysis blood transfusions and vte rates were significantly lower in the adolescent group but there was no difference after risk-adjusted logistic regression analysis. analysis of readmission data showed lower rates in adolescents compared to young adults ( . % vs . % p= . ). however, adolescents are more frequently readmitted secondary to gallstone disease ( . % vs . %, p. ). the most common reason for readmissions in both groups was nausea and vomiting with fluid/electrolyte depletion, followed by abdominal pain. conclusion: adolescent bariatric surgery is feasible and safe, with outcomes similar to that of young adults. lsg is currently the most common bariatric procedure performed in adolescents which is reasonable given the relative lack of co-morbid conditions within this group. nausea and vomiting are the most common reason of readmission in both groups, but gallstone disease is significantly higher in adolescents, suggesting that this population should be carefully screened for gallbladder disease preoperatively. further studies regarding long-term results are needed to elucidate long-term outcomes, such as the durability of comorbidity resolutions in adolescent patients. introduction: revision bariatric surgery is always considered to be associated with higher complication rates. there is currently controversy in the literature regarding one stage and two stage revisions. methods: the present study is ongoing longitudinal prospective analysis of data of revision surgery in a single unit. the revision surgery was offered after initial failed or complicated gastric band, sleeve gastrectomy and roux-en-y gastric bypass (rygb). results: there were forty-two individuals who had revision bariatric surgery. the age of the cohort of patients ranged from twenty-six to seventy-five years. thirty-three were females and nine males. all patients who were hypertensive or diabetic at the time of their initial bariatric operation had a relapse of their co-morbidity prior to their revision surgery. the two stage revisions patients had their band removed at another facility, had a compilation from the band itself or did not wish for revision surgery initially. of the two failed bypasses one had a large pouch and very short limbs. the other had a gastro-gastric fistula and ultra short limbs. there were no deaths in this study. one patient who underwent one stage revision of a gastric band to bypass had an iatrogenic small bowel injury that required a second operation. amelioration of diabetes and hypertension was seen in all who had relapsed. weight loss was good in all patients except for the those undergoing revision from short limbed to long limbed bypass. conclusion: there is enough evidence that revision surgery is feasible, and can ameliorate metabolic co-morbidities after failed band and sleeve. two staged surgery is not necessarily safer compared to one stage revision. in the present study an inadvertent iatrogenic injury occurred in one stage revision group but is not true reflection of increased complications. the association between preoperative endoscopic esophagitis and post operative gerd in sleeve gastrectomy patients samer elkassem, md; medicine hat regional hospital introduction: gerd is a common complication after sleeve gastrectomy (sg). the purpose of this study is assess the relationship between pre-operative findings of endoscopic esophagitis and postoperative gerd in sg patients. the hypothesis of this study is that patients with pre-op esophagitis are more likely to have gerd post-op than patients with no esophagitis pre-op. methods: a retrospective review of sg patients who had pre-operative endoscopy and followed prospectively for at least one year was preformed. patients were divided into two groups based on pre-op endoscopic findings: those with no findings of esophagitis (ne), and those with endoscopic esophagitis, including barretts (ee). patients were followed for at least one year, and assessed for usage of a proton pump inhibitor (ppi) usage. the two groups were compared using both student t-test and chi square test. results: a total of patients did not have any findings of esophagitis on pre-op endoscopy (ne group), and patients had findings of endoscopic esophigitis (ee). there was no difference in preoperative demographics and post-op weight loss at one year (table i) . follow-up ranged from one to years post-op. the dependency on ppi usage and de novo reflux are shown in table ii . introduction: patients with "super-super obesity", defined as a bmi≥ , are at higher risk of weight-related health problems and might benefit more than others from metabolic and bariatric surgery. however, these benefits need to be weighed against the potential for increased operative and perioperative risks. accurate data regarding these patients is critical to guide procedure choice and informed, shared decision-making. the metabolic and bariatric surgery accreditation and quality improvement program (mbsa-qip) is a national accreditation and quality improvement program, which captures clinically-rich specialty-specific data for the majority of all bariatric operations in the united states. this is the first analysis of the mbsaqip participant use file (puf) focusing on this at-risk subpopulation. introduction: sleeve gastrectomy represents one of the most common surgical procedure used in bariatric surgery. the most feared complication following laparoscopic sleeve gastrectomy is the leak that occurs at the staple line. one method to reduce the risk of leak is the use of reinforcement material at the suture line. in this study, the efficacy of sutures and fibrin glue in the prevention of staple leak has been compared retrospectively. materials and methods: a total of patients undergoing lsg between october and august at the medical faculty of firat university were retrospectively assessed using the hospital database system records. results: there were males ( %) and ( %) females, with a mean age of years (range: - y), and body mass index of kg/m . while no reinforcement material was used in patients ( %) at the suture line, reinforcement sutures or fibrin glue were used in ( %) and ( %) patients, respectively. postoperative leak occurred in patients ( . %), and ( . %) of these had no use of reinforcment material for leak prevention, while additional sutures or fibrin glue had been used in patients, one in each group ( . %). one patient died due to leak and the consequent development of sepsis ( . %). discussion: lsg is increasingly more frequently used in bariatric surgery practice. however, an increase also occurs in the rate of complications. a discrepancy exists in the published literature regarding the benefit of reinforcment the suture line on the risk of leak risk. in our patient series, patients without the use of additional material in the staple line had a significantly increased risk of leak. conclusion: despite some controversy, strong evidence exists on the effectiveness of fibrin glue in the prevention of leaks in patients undergoing laparoscopic sleeve gastrectomy. background: laparoscopic bariatric surgery has been performed safely since . in a persistent search for fewer and smaller scars, single port and acuscopic surgery or even notes have been implemented. the goal of this study is to analyze the safety and feasibility of using a low cost incisionless liver retraction compared to a standard laparoscopic retractor for sleeve gastrectomy. methods and procedures: candidates for sleeve gastrectomy that fulfilled nih criteria for bariatric surgery were selected. those younger than and/or with prior upper-left quadrant surgery were excluded. all patients signed written consent. patients were randomized : to either a standard port technique with a fan-type liver retractor through a mm port (group a); or a port technique with the liver retracted by a polypropylene suture passed through the right crura and retrieved at the epigastrium with the use of a fascia closure needle (group b). all surgeries were performed by the same surgeon. surgery length from insertion of first port to withdrawal of the last was the primary endpoint. anthropometric data, % of pre-surgical total weight loss (%ptwl), visualization of the surgical field, complications inherent to liver retraction and postoperative morbidity were recorded. background: comprehensive web and hospital based preparative patient education allow the morbidly obese patients to understand weight loss surgery, its benefits, the necessity of follow up and the risk of weight regain. while the inhouse seminars provide a face-to-face interaction with the bariatric program staff, the online seminars are easily accessible and more cost effective. the primary objective of this study is to compare demographics and weight loss surgery outcomes between patients who participated in the online vs in-house preparative seminars. methods: after obtaining institutional review board approval, a retrospective chart review was performed involving patients who underwent bariatric surgery between january and december at a tertiary care center. the patients were divided into two groups based on their choice of educational seminar, online or in-house, prior to their initial consult with a surgeon. data was collected on age, type of insurance, length of stay (los), longest follow up and change in bmi to assess weight loss. results: one hundred and eighteen patients were included in this study. eighty patients attended in-house seminar while completed online seminar. the various types of surgery (laparoscopic gastric bypass, sleeve gastrectomy, and band) were similarly represented between the two groups. there was no difference in the type of insurance policy between the groups. patients who elected to take the in-house seminar were on average years older than those who chose the online course, which was statistically significant (p. ). there were no differences in los, longest follow up after surgery, and weight loss at months between the groups. conclusions: based on mbsaqip registry data, patients age or over did not have higher odds of a -day readmission compared to younger patients after lsg or lrygb. rates of -day readmission, reoperation, and death were similar, but rates of complications (e.g. pneumonias, unplanned intubations) were higher in the older group. bariatric surgery in the elderly should therefore be performed only after careful and patient-centered selection processes. introduction: revisional bariatric surgery has become more common in recent years. it is to address short and long-term complications of primary bariatric surgery as well as the issue of weight regain. the aim of this study was to retrospectively analyze the indications for reoperation and short-term outcomes in our institution. methods and procedures: between and , patients who underwent bariatric surgery in our center were included in a prospectively collected database. demographic data, primary and revisional bariatric procedures, reasons for revisions and outcomes were recorded and reviewed retrospectively. results: a total of patients underwent bariatric surgery at our institution and % of these (n= ) were revisional bariatric surgery. we identified groups of patients according to their primary procedures: adjustable gastric band (agb), roux-en-y gastric bypass (rygbp), vertical band gastroplasty (vbg), and sleeve gastrectomy (sg). of the patients, ( %) had abg as primary procedure. of those, % had their band removed due to food intolerance and severe dysphagia and % had a conversion to either rygbp or sleeve gastrectomy (sg) due to weight recidivism. in the rygpbp group (n= ), % of the patients presented with late complications. of these, % had an acute presentation (small bowel obstruction, internal hernia, or perforated marginal ulcer) requiring emergency surgery. only % patients needed gastric bypass takedown due to severe hypoglycemia. weight recidivism was noted in % of the patients that necessitated either revising the anastomosis, trimming of the gastric pouch or gastrogastric fistula takedown. in the vbg group (n= ), % of the patients experienced weight recidivism that required conversion to rygb and % of the patients required the vbg to be taken down due to obstructive symptoms. in the sg group (n= ), % of the patients experienced early complications needing a second procedure. weight recidivism was found as the most common reason for conversion ( %) to rygbp. twenty nine percent of the patients in this group underwent conversion to a rygbp due to severe de novo gerd. introduction: our aim was to systematically review the literature to compare weight loss outcomes and safety of secondary surgery after sleeve gastrectomy (sg), particularly between roux-en-y gastric bypass (rygb) and biliopancreatic diversion with duodenal switch (bpd-ds). sg was originally developed as the first part of a two-stage procedure for bpd-ds. however, it is now the most common standalone bariatric surgery performed in the united states. the majority of sg are done as the sole bariatric operation but in %, a second operation is necessary, due to insufficient weight loss, weight regain or reflux. the most common second-stage operations are rygb at % and bpd-ds at %. there are a few small case series comparing rygb to bpd-ds as a secondary surgery after sg. these studies suggest that after failed sg, bpd-ds results in greater weight loss but higher early complication rates than rygb. we had one mortality, related in part to supra-therapeutic anticoagulation perioperatively. one patient underwent successful heart transplantation and additional patients were reactivated on the transplant list. conclusion: laparoscopic sleeve gastrectomy is effective in advanced heart failure patients for meaningful weight loss, reactivation to the transplant wait list, and ultimately cardiac transplantation. however, this complex population carries a high perioperative risk and close multidisciplinary collaboration is required. more data is needed to best optimize perioperative management of these patients. the introduction: bariatric surgery is a highly effective treatment for severe obesity. while its effect on improvement of the metabolic syndrome is well described, its effect on intrinsic bone fragility and fracture propagation is unclear. therefore, the aims of this systematic review of the literature were to examine ( ) the incidence of fracture following bariatric surgery, ( ) the association of fracture with the specific bariatric surgical procedure ( ) conclusion: it appears that the overall risk of sustaining a fracture of any type after undergoing bariatric surgery is approximately percent after an average follow up of . years. the greatest risk of fractures is associated with the bpd, with the rygb being the most favorable. fractures following bariatric surgeries tend to follow osteoporotic and fragility patterns. post-operative supplementation of vitamin d, calcium and weight bearing exercises need to be optimized, and long term follow-up studies will be needed to confirm that these interventions will indeed reduce fracture risk following bariatric surgery. background: the effect of sleeve gastrectomy on gastroesophageal reflux (gerd) remains controversial. it is currently common practice to perform a hiatal hernia repair (hhr) at the time of the sleeve gastrectomy, however, there are few data on the outcomes of gerd symptoms in these patients. the aim of this study was to evaluate the effect of performing an esophagopexy hiatal hernia repair on gerd symptoms in morbidly obese patients undergoing robotic sleeve gastrectomy (rsg). methods: a single institution, single surgeon, prospectively maintained database was used to identify patients who underwent rsg and concomitant esophagopexy for hiatal hernia repair from november to july . patient characteristics, operative details and postoperative outcomes were analyzed. primary endpoint was subjective gerd symptoms and recurrence of hiatal hernia. results: thirty-seven patients were identified meeting the inclusion criteria (rsg+hhr+esophagopexy) with a mean follow-up of . over the past years there have been several bariatric surgeries cancelled secondarily to abnormal pre-operative test results within eastern health. these surgeries are often cancelled the day before their scheduled surgery, which does not provide sufficient time to book other patients. the end result is that the or gets underutilized and the bariatric surgery waitlist grows. prior to any major surgery patients are often subjected to a routine screening process, which includes a history and physical along with diagnostic screening tests and screening blood work. a preliminary analysis was done of the first patients through the bariatric surgery program at eastern health assessing the coagulation study results and outcomes. analysis showed that out of the first patients % were found to have a history of bleeding, % were using anticoagulants preoperatively, another % were noted to have a family history of bleeding. in the preoperative blood work that was done, % were found to have an elevated ptt/ inr for which hematology ended up being consulted in % of the patients. overall this did not change the preoperative management of these patients and they went on to have their surgery. intraoperatively patient was noted to have excessive bleeding and this was found not be associated with any preoperative elevation in their coagulation studies or family history of bleeding disorders. post operatively there was bleeding in patient which required transfusion, however this too was found not to be associated with any preoperative elevation in their coagulation studies or family history of bleeding disorders. overall this initial analysis showed no difference in operative management or delay in surgery secondarily to abnormal preoperative assessment findings. further analysis of a larger population of the bariatric surgery program patients is needed in order to determine whether any changes should be made to the preoperative assessment protocol. introduction: patients undergoing bariatric surgery frequently present with various obesity-related psychiatric comorbidities, including depression. furthermore, previous literature has demonstrated a positive association between depression and cardiovascular disease, and obesity serves as an independent risk factor for cardiovascular disease. however, the relationship between preoperative depression and cardio-metabolic risk factors following bariatric surgery remains unknown. methods and procedures: this retrospective analysis utilized data obtained from patients (n= , ) who underwent bariatric surgery at a single academic medical center in california. patients underwent either laparoscopic roux-en-y gastric bypass or sleeve gastrectomy. using medical record data, patients were preoperatively categorized as follows: not depressed, history of depression but not currently on anti-depressive medication, and history of depression and presently taking anti-depressive medication. patient demographic characteristics were obtained preoperatively. clinical and biochemical risk factors for cardiovascular disease were evaluated preoperatively and and months following bariatric surgery. anova, kruskal-wallis, and chisquare tests were applied where appropriate. results: in this sample, % of patients were not depressed, % had a history of depression but were not taking anti-depressive medication preoperatively, and % had a history of depression and were taking anti-depressive medication preoperatively. at baseline, depressive history was positively associated with female sex (p\. ), older age (p\. ), white race (p\. ), medicare insurance (p\. ), previous abdominal surgery (p\. ), length of stay (p\. ), requiring an inferior vena cava filter (p=. ), total cholesterol (p\. ), and triglycerides (p =. ). on average, patients with a history of depression taking anti-depressive medication weighed less than patients with a history of depression not on medication and patients without depression preoperatively (p=. ) and (p=. ) and (p=. ) months after surgery. after six months of follow-up, preoperative depressive history was positively associated with total cholesterol (p=. ), triglycerides (p\. ), hba c (p=. ), and fasting serum concentrations of insulin (p=. ). after months of follow-up, preoperative depressive history was positively associated with higher levels of total cholesterol (p=. ), ldl cholesterol (p=. ), and triglycerides (p= . ). conclusion: a history of depression prior to surgery was associated with higher levels of total cholesterol and triglycerides at baseline and and months postoperatively. after months, preoperative depressive history was also associated with higher levels of ldl cholesterol. this study suggests that, on average, bariatric patients with comorbid depression have worse lipid profiles prior to-and up to one year after-bariatric surgery relative to counterparts without depression. yen-yi juo, md, mph, yas sanaiha, md, erik dutson, md, yijun chen, md; ucla introduction: anastomotic leak is one of the most morbid complications of roux-en-y gastric bypass (rygb), yet its risk factors are ill-defined due to the rarity of the complication. we aim to identify both patient-and operative-level risk factors for anastomotic leak after rygb using a national clinical database. methods: a retrospective cohort study was performed using the metabolic and bariatric surgery accreditation and quality improvement program (mbsaqip) database. all adult patients who underwent laparoscopic or open rygb were included. multivariate logistic regression models were used to identify patient-and operative-level variables associated with development of anastomotic leakage. clinically relevant anastomotic leakage is defined as those that required readmission, intervention, or reoperation. introduction: hyperammonemia secondary to ornithine transcarbamylase (otc) deficiency is a rare and potentially lethal disorder. the prevalence of otc deficiency is reported to be : , to : , in the general population. otc deficiency has been reported in patients presenting with neurological symptoms after roux-en-y gastric bypass (rygb), and less than cases have been reported in the literature. the aims of this study are to examine the apparent incidence of this uncommon disorder in patients after bariatric surgery and to examine potential predictors of mortality. methods and procedures: this is a single center, retrospective study in a large, urban teaching hospital of postbariatric surgery patients who developed hyperammonemia from january to august . elevated plasma ammonia with an elevated urinary orotic acid level is accepted as consistent with a diagnosis of otc deficiency. all patients in our program are instructed on a post-operative diet containing grams/day of protein. descriptive and correlative statistics are calculated for all variables. results: between january and august , bariatric surgical procedures were performed at this single medical center. seven women with neurological symptoms had plasma ammonia levels above the upper limit of normal range. their average bmi is kg/m . two patients underwent vertical sleeve gastrectomy (vsg), underwent vsg with duodenal switch, and underwent rygb. all patients were hospitalized. the mean peak plasma ammonia level is umol/l (range: - ). the mean urinary orotic acid level is . mmol/mol creatinine (range: . - . ). there were patients with no orotic acid level checked, secondary to demise. no patient had clinical features or findings of progressive hepatic failure. there are four mortalities ( . %). serum folate and peak lactic acid levels are predictors of mortality with p-values of . and . respectively. the apparent incidence of otc deficiency is : in post-operative patients. conclusions: in our post-operative population, hyperammonemia results in a high mortality. its apparent incidence, secondary to otc deficiency, amongst bariatric surgery patients is higher than that reported in the general population. since otc deficiency is identified after multiple bariatric surgical procedures, further investigation will be important to examine potential mechanisms for its development which may include a genetic predisposition (possibly triggered by nutritional deficiencies), upper gut bacterial overgrowth (supported by elevated serum folate levels), or preexisting, subclinical hepatic dysfunction. introduction: the use of closed suction drains is associated with poor outcomes in many anastomotic operations and routine use is not recommended. in this context, intraoperative drain placement for primary bariatric surgery remains controversial. recent studies demonstrate that drains confer no benefit to patients; however, data are limited to descriptive single center experiences with low sample size. in order to characterize this practice gap, and implement evidence based recommendations, we sought to evaluate the use of closed suction drain and outcomes following primary bariatric cases using the mbsaqip registry. methods: we used data from the metabolic and bariatric surgery accreditation and quality improvement program (mbsaqip) public use file for patients who underwent a non-revisional laparoscopic roux-en-y gastric bypass (rygb), laparoscopic sleeve gastrectomy (lsg), or laparoscopic adjustable gastric banding (lagb). we excluded patients with asa status greater than or conversion to an open procedure. we analyzed demographics, preoperative comorbidities, procedure type for patients who did and did not undergo drain placement. adjusted rates of postoperative complications and mortality were then compared based on receipt of postoperative drain placement. results: of the , included patients who underwent laparoscopic bariatric surgery, , ( . %) underwent intraoperative drain placement. drains were more often placed in patients who underwent lrygb, were older, had higher preoperative bmi, had higher preoperative asa status, and had more comorbid conditions. after patient level risk adjustment, there was no difference in rates of leaks requiring intervention ( . % versus . %, p= . ) or mortality ( . % versus . %, p= . ) for patients with and without drains. in patients who underwent drain placement, there were higher rates of transfusion ( . % versus . %, p. ), reoperations for bleeding ( . % versus . %, p= . ), all reoperations ( . % versus . %, p. ), and surgical site infections (ssi) ( . % versus . %, p. ). conclusion: our analysis demonstrates that nearly one quarter of all laparoscopic bariatric surgery patients undergo drain placement. we found that drain placement is more common in preoperatively higher risk patients and following higher complexity procedures as suggested by associated increased rates of transfusion and reoperations for bleeding. we found no benefit to drain placement in terms of interventions for clinically significant leaks or mortality. finally, patients who underwent drain placement were more likely to develop ssi suggesting routine placement is not without risk. although further prospective studies are warranted, our analysis demonstrates that drains have the potential for harm with minimal protective benefit for patients after primary bariatric surgery. sleeve gastrectomy ( % n= ) and laparoscopic roux-en-y gastric bypass ( % n= ) were the two types of surgeries done in our population. the risk of developing atrial fibrillation was calculated preoperatively and found a -fold higher risk in females and -fold greater risk in males when compared with the ideal risk for each category. at months follow-up the preoperative risk was . ± . % with an absolute risk reduction of . % corresponding to a relative risk reduction of . % with males having a more significant change at months follow-up. these findings and the electrocardiographic changes at months follow-up are better described in background: the sleeve gastrectomy (lsg) is the most popular procedure worldwide to treat obesity. among those that are obese, gerd has a prevalence of . percent. many surgeons do not perform lsg in these patients because only . percent of symptomatic patients showed resolution of gerd-like symptoms after concomitant sleeve gastrectomy with hiatal hernia repair. many surgeons perform the gastric bypass on gerd patients with hiatal hernias because they believe its superior for the resolution of gerd. when they do this they overlook the many long term complication associated with gastric bypass. also, many patients do not want the gastric bypass under any circumstances. surgeons need to be open to finding better way to reduce the high recurrent rates of gerd after lsg. materials and methods: this is a single institution, multi-surgeon, retrospective study involving morbidly obese patients in a prospectively kept data base from january of through july of . these patients all had gerd with preoperatively identified hiatal hernias on egd. all patients were dependent on anti-reflux medications. there were ( . %) males and ( . %) females. bmi ranged from to . hiatal hernias measured from cm to cm. all lsg patients received a primary crural closure, with or without gore bio a mesh placement, at least weeks prior to the sleeve gastrectomy. post-operatively, patients were interviewed for gerd symptomatology and anti-reflux medication dependency. results: of the patients, ( . %) patients had resolution of gerd-like symptoms and off all anti-reflux medications after the staged hiatal hernia repair and sleeve gastrectomy. patients ( . %) had improvement of gerd but still dependent on anti-reflux medication. patients ( . %) had no resolution or improvement of gerd. there was one post-operative complication of laryngospasm with pulmonary edema status post extubation. there were no mortalities in the series. conclusions: in this study, staged hiatal hernia repair, at least weeks prior to sleeve gastrectomy, doubled the published rate of gerd resolution from % to %. % showed improvement in symptoms at one year. this rate is comparable to gerd resolution after gastric bypass. this may be an alternative approach to hiatal hernias in the morbidly obese patient with gastroesophageal reflux disease who do not want a gastric bypass. background: bariatric surgery is a common procedure in general surgery. gastric bypass has been performed laparoscopically for over two decades and multiple techniques are described. the circular stapled anastomosis, one of the earliest methods for gastrojejunostomy, is performed in two ways: a transoral method to introduce the anvil and a transabdominal approach developed later. the former technique requires passing the anvil of the circular stapler through the mouth, down the esophagus, and into the gastric pouch. in the latter method, a gastrotomy is made, the anvil is introduced, and the gastrotomy is stapled off, creating the gastric pouch. this study aims to objectively compare the two methods of circular stapled gastrojejunostomy in terms of surgical site infection (ssi) rate. methods: a retrospective chart review of patients undergoing laparoscopic roux-en-y gastric bypass with one of two surgeons at a bariatric center of excellence in an academic hospital from january introduction: laparoscopic sleeve gastrectomy (lsg) has become the most commonly performed procedure in the treatment of morbid obesity, but there is significant variability in its performance. from national database analysis, more restrictive sleeve construction, based on smaller bougie size, has not correlated with greater weight loss. we hypothesize that bougie size is not reflective of actual restriction, or that sleeve restriction does not correlate with weight loss. we performed qualitative and volumetric analysis of immediate post-sleeve contrast studies to determine the association of sleeve restriction with post-operative weight loss and complications. methods: between and , patients underwent immediate post-sleeve contrast studies. based on standardized vertebral body height assessment by preoperative chest radiograph, sleeve diameter at intervals (including the narrowest point) was measured in mm, and the volume above the narrowest point of the sleeve was calculated. sleeve shape was assumed as dual-tiered or simple truncated cone based on morphology. sleeve restriction, morphology and volumetric analysis were associated with clinical outcomes including complications, post-op symptoms, and weight loss at months. background: variability in surgical technique resulting in narrowing at the incisura angularis, twisting along the staple line, and retention of the gastric fundus has been implicated in increased gastroesophageal reflux disease (gerd) following laparoscopic sleeve gastrectomy (lsg). standardizing creation of the sleeve based on anatomic landmarks may help produce more consistent sleeve anatomy and improve outcomes. methods: a retrospective review of all patients undergoing lsg from january to november at a single institution specializing in bariatric surgery was performed (n= ). patients underwent either traditional lsg with use of a f suction bougie to guide creation of the sleeve (n = ) or anatomy-based sleeve gastrectomy (abs, n= ). abs was performed using a gastric clamp to maintain predetermined distances from key landmarks ( cm from gastroesophageal junction, cm from incisura angularis, cm from pylorus) during stapling. patient demographics, perioperative characteristics, and post-operative outcomes were compared using chi-square and student's t-tests as required. helicobacter pylori (hp) is prevalent in up to % of the population worldwide with increased rates observed in the bariatric population. bariatric surgery has seen a rapid expansion over the last years with the growing rates of severe obesity. higher hp rates are thought to be associated with increased rates of postoperative complications including increased marginal ulceration and leak rates. accordingly, some bariatric centers have adopted routine pre-operative screening and hp eradication programs. yet, while hp correlation with gastritis and malignancy has now been well defined, its impact on patients undergoing bariatric surgery remains unclear. background: the risk of developing a hiatal hernia in the obese population is . fold compared to patients with a bmi \ . most hiatal hernias after bariatric surgery are asymptomatic and when symptoms are present they may be difficult to differentiate from overeating or maladaptive eating habits. the aim of this study was to define the risk and symptoms associated with a hiatal hernia in the post-bariatric surgery cohort. methods: a retrospective review of prospectively collected data for patients who underwent laparoscopic hiatal hernia repair who previously had primary roux-en-y gastric bypass (rygb) or sleeve gastrectomy (sg). data collection spanned a five-year interval ( / - / ). preoperative and follow up data were collected from medical records and questionnaires in the clinic or by telephone. variables obtained include age, gender, psychiatric history, pre-index procedure bmi, pre-hiatal hernia repair bmi, post-hernia repair bmi, pre and post operative symptoms, and associated morbidity. all hiatal hernia repairs were done laparoscopically, with posterior cruroplasty after circumferential hiatal dissection. results: we identified patients with a symptomatic hiatal hernia who had previously (range: - years) underwent bariatric surgery. fourteen rygb patients presented at a mean of . years compared to sg patients who presented at a mean of . years after index procedure. diagnosis was by a combination of ugi ( %), ct scan ( %) and egd ( %). mean follow up was . months (range: - months). laparoscopic hiatal hernia repair was successfully performed in all patients with % mortality. dysphagia and regurgitative symptoms markedly improved in [ % of patients however, nausea, vomiting and abdominal pain were not changed in - % of patients ( figure) . conclusion: hiatal hernia following bariatric surgery is a rare but important cause of bloating manifested as nausea and vomiting, abdominal pain, regurgitation or reflux, and food intolerance or dysphagia (barf)-and should be further evaluated with imaging or endoscopy when present. laparoscopic repair of hiatal hernia is warranted and results in resolution of symptoms in the majority of symptomatic patients. mid-term outcomes of sleeve introduction: obese patients suffer from multiple organ comorbidities which contribute to a shortened lifespan. one of the effects of obesity is thought to be pseudotumor cerebri, which is secondary to increase in intracranial pressure (icp) in the absence of an obstruction. over the past two years, we have measured icp after insufflating with a laparoscopy device. we found that icp increases dramatically and it correlates with the amount of insufflation in the abdomen. over the years, there have been studies in obese patients and intra-abdominal pressure. these studies have shown that some obese patients have an intra-abdominal pressure of - mmhg. increasing intraabdominal pressure is thought to escalate intracranial pressure (icp). the objective of this pilot study was to observed change in icp after the raising intra-abdominal pressure. method: in this retrospective chart review preliminary study, pressure in each of the patients either normal pressure hydrocephalous or high pressure hydrocephalous receiving a ventricle shunt were measured by manometer. once the shunt was placed into the ventricle, we attached a manometer to measure the opening pressure. after we accessed the abdominal cavity using the standardoptiview technique, we created a pneumoperitoneum. after achieving an intraabominal pressure of mmhg, were measured the icp using the manometer. spss software version was used for data analysis. paired t-test was applied on icp before and after the procedure. introduction: postoperative bleeding represents an infrequent, yet serious complication after bariatric surgery. differences in the rate of postoperative bleeding reported for the two most common weight loss procedures-laparoscopic roux-en-y gastric bypass (lrygb) and laparoscopic sleeve gastrectomy (lsg)-are ostensibly confounded by patient and surgeon specific preoperative, intraoperative and postoperative factors, in particular, by the utilization of staple line reinforcement or oversewing. with this understanding, we aim to use a large national database to definitively characterize differences in bleeding rates between lsg and lrygb. conclusions: after appropriate risk-matching, lsg patients have a reduced likelihood of a postoperative bleeding event compared to those undergoing lrygb. this difference is likely more pronounced with intraoperative securing of the staple line via oversew, buttress or an alternative method. these findings from a large national database represent an important consideration for surgeons and patients alike when evaluating the appropriate bariatric operation. background: bariatric surgery has shown to be the most effective treatment, with documented improvement in obesity-related comorbidities. the type of health insurance coverage plays an important role in the access to bariatric surgery, but might also affect postoperative outcomes. the objective of this study is to determine whether there is a difference in outcomes based on the type of insurance months after bariatric surgery. methods: we retrospectively reviewed all the patients that underwent bariatric surgery at our institution from to . we divided the patients into two groups, based on the type of insurance, private (group one), and public (group two). we compared demographics and months outcomes between the groups, using t-test for continuous variables and chi-square for categorical variables. we also compared months estimated bmi loss between different private insurances using anova. introduction: bariatric surgeons are now performing primary and revisional procedures on the extremes of age. there is controversy surrounding the safety and effectiveness of bariatric surgery among older age groups compared to younger age groups. to address this knowledge gap, we designed a study assessing short-term bariatric surgery outcomes among various age groupings across a large national database. methods and procedures: de-identified patient data across from the mbsaqip registry was used. age groupings were organized into young, middle-aged, and older adults (in years) as follows: \ , - , and [ , respectively. the following -day outcomes were evaluated between all possible pairwise age groupings: mortality, surgical site infection (ssi), and readmission; logistic regression was used to compare outcomes between age groupings controlling for primary vs. revisional index operation, patient factors, and procedure factors. a p value of . was deemed statistically significant. results: a total of , patients were identified (age range: to [ ); % (n= , ) underwent primary bariatric operations while % (n= , ) underwent revisional cases. older adults had significantly worse outcomes than middle-aged and younger adults, respectively, for over comparisons across all outcomes; in contrast, younger adults had significantly worse outcomes than middle-aged adults for only comparisons across ssi and readmission. for primary bariatric cases, older adults had significantly higher mortality rates than middle-aged and younger adults, respectively, in the following categories: asa , laparoscopic sleeve gastrectomy (lsg), or laparoscopic roux-en-y gastric bypass (lrygb). for revisional cases, older adults had significantly higher mortality rates than middle-aged and younger adults, respectively, in the setting of female gender, caucasian race, or asa . regarding ssi, older adults undergoing primary lrygb had significantly higher organ space infections compared to younger adults. in addition, older adults who had revisional lrygb had significantly higher deep surgical site infections compared to middle-aged adults. following primary bariatric cases, older adults had significantly higher readmission rates compared to younger adults in the presence of male gender, caucasian race, asa , copd, or after lsg. following revisional cases, older adults had significantly higher readmission rates than middle-aged and younger adults, respectively, in the setting of pre-operative chronic steroid use. conclusions: overall, older adults had worse short-term outcomes compared to their younger counterparts following primary and revisional cases. further research is required to investigate these findings with the goal of targeting interventions to improve outcomes among bariatric surgical patients. background: the obesity epidemic in the united states has been accompanied by surge in bariatric surgery. nearly , bariatric procedures were performed in the us in , % of which involved roux-en-y gastric bypass (rnygb). while rnygb has proven an effective tool in combating obesity, it also alters a patient's anatomy in a way that makes traditional ercp a difficult, if not impossible option for interrogating the common bile duct. one way to approach the post-rnygb patient with obstructive jaundice is to access the peritoneal cavity via a laparoscopic/ robotic approach followed by direct cannulation of the gastric remnant with a laparoscopic port, allowing passage of an endoscope. the aim of this study was to evaluate our single center experience with minimally-invasive transgastric ercp (tg-ercp) from to . methods: we compiled a list of all patients who underwent laparoscopically or robotically assisted tg-ercp at our institution from - . we then examined patient demographics, procedural details, postoperative outcomes, and success rate, with success defined as cannulation of the ampulla, clearance of obstruction if present (stones/sludge/stenotic ampulla), and completion imaging of the biliary and pancreatic ducts. results: patients were included in the study. cases were performed robotically ( %), and laparoscopically ( %). ercp was successful in cases ( %). all unsuccessful attempts were aborted when the endoscopist was unable to pass the scope through a tight pylorus. median time of operation was minutes ( minutes if concomitant cholecystectomy was performed, minutes if not). median length of stay after operation was days (range - days). median estimated blood loss (ebl) was ml. post ercp pancreatitis occurred in patients ( . %), and was mild and self limited in all cases. patients had postoperative bleeding requiring transfusion. both of these had concomitant cholecystectomy. discussion: in patients with biliary obstruction and anatomy not suitable for traditional ercp, tg-ercp is a viable option. it can be performed with in a minimally invasive fashion (either laparoscopically or robotically) with a high success rate and low morbidity. as the population of patients who have undergone rnygb continues to grow, so does the likelihood of encountering one with obstructive jaundice. tg-ercp, therefore, should be thought of as an essential tool in the armamentarium of the general surgeon. introduction: primary palmar hyperhidrosis (ph) is a pathological condition of over perspiration caused by body produces an excessive amount of sweat. this disorder affects to decrease quality of life of patients. thoracoscopic sympathectomy is minimally invasive and an effective procedure to treat hyperhidrosis. different of level of sympathectomy has been debate for the best outcomes. many researchers studied about short term outcomes but no empirical research evidences long term outcomes of thoracoscopic sympathectomy in thailand. this study purposed to evaluate and compare the long term clinical outcomes between patients who underwent t and t thoracoscopic sympathectomy for ph with particular attention to patient satisfaction and quality of life. methods and procedures: sixty patients with ph underwent thoracoscopic sympathectomy. patients were divided into two groups by the level of thoracoscopic sympathectomy as t group and t group. they were investigated the improvement of sweating, compensatory sweating, satisfaction and quality of life. the long-term investigation was designed to examine clinical outcomes at before surgery, six months after surgery, year after surgery, years after surgery, and last follow up days were compared within group and between of t and t group. they were subjected to telephone interview using multiple questionnaires to investigate surgery outcomes, degree of satisfaction, and quality of life improvement. results: sixty patients responded to the telephone interview. patients demographic data and also recurrence rate of ph between t and t group was not significant different (p= . ). both groups improved severity of sweating without any statistical significant. but the t thoracoscopic sympathectomy led to significantly lower incidence of compensatory hyperhidrosis when compared with t group at back and trunk sites. the t group had higher overall satisfaction than t group with was not significantly different. long term result are followed after years. conclusions: there was no difference in decreasing severity of sweating between t and t level of thoracoscopic sympathectomy. both group equally archived patient satisfaction. but the t level of thoracoscopic significantly had lower severity of ch and better quality of life in long term period. introduction: acute pancreatitis due trauma is commonest cause of pseudocyst in pediatric age. due to limited literature available and under diagnosis by pediatricians, the true incidence of pseudocyst in - age group is not known. material and methods: retrospective analysis of pediatric age ( - years) patients who underwent laparoscopic cystogastrostomy at distric teaching hospital was done. patients data, presentation, investigations, opetation done and post operative course was studied. result: total of patients ( males & females) had mean age of . years, mean weight of kg. etiollogies included blunt abdominal trauma ( ), idiopathic ( ), gallstones ( ) . average cyst diameter was . cm. laparoscopic cystogastrostomy by transgastric approach was successfully possible in cases with no conversion. cystogastrostomy was performed using sutures in patients and ultrasonic energy device in patients. gastrotomy was closed with sutures in all cases. mean operative time was minutes. post operative imaging at months revealed no persistence or recurrence of cyst. conclusion: minimally invasive laparoscopic approach for chronic pancreatic pseudocyst in pediatric age group is safe and effective strategy and should be adopted as primary modality of treatment. introduction: videoscopic neck surgery is developing despite the fact that only potential spaces exist in the neck. gagner first described the endoscopic subtotal parathyroidectomy with constant co gas insufflations for hyperparathyroidism in . the cervical approach utilizes small incisions in the neck thus making it cosmetically unacceptable and cannot be used for lesions greater than cm. the axillary approach makes it difficult to visualize the opposite lobe. the anterior chest wall approach utilizes port access at various positions on the anterior chest wall depending on the surgeon. this technique also allows bilateral neck exploration. hence we have been able to perform total thyroidectomies with central compartment clearance for papillary carcinoma and near-total thyroidectomies for large multinodular goiters, materials and methods: three incisions subplatysmal plane pneumoinsufflation with carbon dioxide (co ) ports creating a subplatysmal palne dissection begins at the inferior pole posterior dissection clipping superior thyroid vessels specimen freed up thyroid lobectomy was performed in the twenty cases. the average blood loss was ml mean operative time was min there were no complications and no cases were converted to open. there were no cases of recurrent laryngeal nerve injury or postoperative tetany. no subcutaneous emphysema, ecchymosis or hypercarbia was observed in any patient. all patients were discharged on the second postoperative day except the first on the fifth day. in conclusion this approach seems to be safe in case of unilateral lobectomy but early to say it is superior to conventional thyroidectomy especially in total thyroidectomy. introduction: laparoscopic sleeve gastrectomy (lsg) is one of the most commonly performed weight loss surgeries. prolonged hospital admissions are associated with both increased morbidity and mortality and increased strain on the health care system; studies are now investigating the safety and feasibility of outpatient lsg. this study examined a single surgeon's postoperative admission trends for patients who underwent lsg. the patients were divided into two cohorts based on the date of surgery, and we hypothesize institutional experience has a significant impact on postoperative stay and hospital readmission rate. methods: this is a retrospective study on lsgs performed by a single surgeon in a tertiary center from - . inclusion criteria: patients [ years old, bmi [ with comorbidities or bmi [ , and patient approval by the bariatric surgical program in victoria, british columbia. patients with prior weight-loss surgery were excluded. patients were discharged home on a care plan involving: nurse and surgeon telephone follow-ups within one week post-surgery. patients were divided into two cohorts: cohort a (procedures between - inclusive) and cohort b (procedures between - inclusive). results: patients were included in this study: females ( . %) and males ( . %). the mean preoperative age was . ± . years, and the mean preoperative bmi was . ± . kg/m . the average postoperative discharge day for the population was day . ± . and the average or time was . ± . minutes. one patient in cohort b was re-admitted pod with a diagnosis of postoperative edema managed conservatively and is included in the analysis as pod . a second patient in cohort b returned to hospital (pod ) for abdominal pain and was managed conservatively as outpatient. conclusion: there was a significant difference in the average postoperative discharge day between patients in cohort a and cohort b who underwent lsg with patients in cohort b requiring a shorter average admission time. this study suggests that with increasing institutional experience and a postoperative discharge plan, patients undergoing lsg may be discharged on postoperative day one safely. surg endosc ( ) introduction: minimally invasive techniques have revolutionized the art of the surgical practice. the laparoscopic approach to cholecystectomy has become the gold standard and is the most common laparoscopic general surgery procedure worldwide. in an effort to further enhance the advantages of laparoscopic surgery, even less invasive methods have been attempted, including smaller and fewer incisions. the objective of this study was describing our results of years of needlescopic cholecystectomy. methods: since march all patients that underwent to needlescopic cholecystectomy micro-laparoscopic procedure with instruments of mm were included in this study in a prospective database and the information was analyzed. results: between march and september , needlescopic cholecystectomies have been done at texas endosurgery institute in san antonio, texas by a single surgeon. % of the patients were female. the average age was . (range of - years old). average operating time was . minutes (range of - minutes). the minute operation required laparoscopic cbd exploration, accounting for the extended time. average estimated blood loss (ebl) was cc (range of - cc). % of cases required conversion to standard mm cholecystectomy and was completed without incidents. all patients were followed up at weeks, weeks, and months after the procedure. only patient presented with a hernia at the umbilical site. otherwise no wound, bile duct, bile leak, bleeding or thermal injury complications were identified. conclusions: micro-laparoscopic procedures with mm instruments in this specific procedure of needlescopic cholecystectomy is safe and feasible, and is a cosmetic alternative to the standard laparoscopic cholecystectomy. there's still less report about thyroid cancer cases in toetva. this study reviews all cases of thyroid cancer which surgery were performed. there were cases of toetva in thyroid cancer and cases of opened thyroidectomy. objective: to review and report in terms of surgical outcome, complication, post-surgical treatment and recurrence in all cases of thyroid cancer surgery, especially in toetva technique. material and methods: from march -july in police general hospital, a total of patients underwent toetva with cases of toetva in thyroid cancer and cases of opened thyroid surgery in thyroid cancer. all patients were recorded in multiple parameters. results: this study have total of thyroid cancer cases which cases ( %) were male and cases ( %) were female, with an average age of . most clinical presentation was thyroid mass or nodule which was at cases ( . %), case ( . %) was non-toxic goiter and case ( . %) was grave disease. the clinical presentation mean time was . years ( weeks- years). there were cases ( . %) with a mass at right lobe, cases ( . %) with a mass at left lobe, and cases ( . %) with mass at both lobes. the size of thyroid mass was . ± . centimeters ( - centimeters). there were cases ( . %) had euthyroid, case ( . %) had subclinical hyperthyroid, cases ( . %) had subclinical hypothyroid, and cases ( . %) had hyperthyroid. for type of surgery, there were cases ( . %) of toetva surgery and cases ( . %) of opened total thyroidectomy. most patients at cases ( . %) didn't have any post-operative complication. and there were hypothyroid cases ( . %), transient hypocalcemia with no symptom cases ( . %), and transient hoarseness cases ( . %). after toetva surgery performed, cases ( . %) were redo completion thyroidectomy, cases ( . %) were transaxillary completion thyroidectomy, cases ( . %) were redo toetva, and case ( . %) deny for reoperation. and cases ( %) didn't have any complication after redo surgery, cases ( . %) were hypothyroid, cases ( . %) were hypocalcemia and hypoparathyroid, and case ( . %) was transient hoarseness. after did thyroidectomy, ultrasound neck shown that cases had no residual or recurrence thyroid mass, cases had residual thyroid tissue. all cases received radioactive iodine ablation. radionuclide total body scan showed no evidence of distant functioning metastasis. conclusion: three-year short-term followed up toetva in thyroid cancer has shown less complication and no recurrence cancer. objective of the study: sentinel node navigation surgery (snns) in gastric cancer has been investigated for almost two decades in an effort to reduce operative morbidity. indocyanine green (icg) with enhanced infrared visualization is one technique with increasing evidence for clinical use. we are the first to systematically review and perform metaanalysis to assess the diagnostic utility of icg and infrared electronic endoscopy (iree) or near infrared fluorescent imaging (nifi) for snns exclusively in gastric cancer. methods and procedures: a search of electronic databases medline, embase, scopus, web of science and the cochrane library using search terms "gastric/stomach" and "tumor/carcinoma/cancer/neoplasm/adenocarcinoma/malignancy" and "indocyanine green" was completed in may . all human, english language randomized control trials, non-randomized studies, and case series were evaluated. articles were selected by two independent reviewers based on the following major inclusion criteria: ( ) diagnostic accuracy study design; ( ) indocyanine green was injected at tumor site; ( ) iree or nifi was used for intraoperative visualization. the primary outcomes of interest were identification rate, sensitivity and specificity. titles or abstracts were screened after removing duplicates. the quality of all included studies was assessed using the quality assessment of diagnostic accuracy studies- . results: ten full text studies were selected for meta-analysis. a total of patients were identified with the majority of patients possessing t tumors ( . %). pooled identification rate, diagnostic odds ratio, sensitivity and specificity was . ( . - . ), . ( . - ), . ( . - . ) and . ( . - . ) respectively. the summary receiver operator characteristic for icg+iree/nifi demonstrated a test accuracy of . %. subgroup analysis found improved test performance for studies with low risk quadas- scores, studies published after and submucosal icg injection. iree had improved diagnostic odds ratio, sensitivity and identification rate compared to nifi. heterogeneity among studies ranged from low (i \ %) to high (i [ %). conclusions: the idea of snns in gastric cancer is intriguing because of the potential to limit operative morbidity. we found encouraging results regarding the accuracy, diagnostic odds ratio and specificity of the test. the sensitivity was not optimal but may be improved by a carefully planned and strict protocol to augment the technique. given the limited number and heterogeneity of studies, our results must be viewed with caution. objective: to evaluate the feasibility, cost effectiveness and safety of single incision laparoscopic surgery using routine laparoscopy instruments. method: cases of acute appendicitis and cases of symptomatic gallstone disease were included in study. cases were enrolled in study and prospective observational study was performed. ruptured appendicitis/abscess formation were excluded from study. similarly empyema gallbladder/gallbladder perforation were also excluded. results: total cases included; cases of appendicitis and cases of symptomatic cholelithiasis. mean age of appendectomy group was . ± . years and mean age of cholecystectomy group was . ± . years. in our study, mean operative time for sil appendectomy was . ± . min. post-operative fever was noted in cases ( . %). mean post-operative pain as per vas score taken after hours, on pod was . . average post op stay in hospital was . days, port site infection occurred in one case ( . %). patient satisfaction score obtained on the scale of - on one month follow up was . , while scar cosmesis score was . . in our study, cases underwent sil cholecystectomy, of which were male ( . %) and were females ( . %), and mean age of patients was . yrs. mean operative time in our study was . min, mean post-operative pain taken on pod as per vas score was . , mean post-operative hospital stay was . days, port site infections occurred in cases. post-op fever was noted in cases, post-operative patient satisfaction score obtained at month follow up was . and scar score of . on the scale of - . no case required drain placement and conversion. conclusion: sils can be performed using conventional laparoscopic instruments especially in a government setup where per capita economic burden to patient will be less. though it has more operative time, it has comparably less post-operative hospital stay, causes less pain, and has significantly more patient satisfaction regarding post-operative scar and cosmesis. since sils has more patient acceptance and satisfaction, it can be offered to all patients undergoing laparoscopic surgery. it is very useful in government setup where lower economic class of patients will also benefit, irrespective of unavailability of special instruments and financial constraints, as it can be performed using routine laparoscopic instruments. in the year we started to practice the pericardic window by laparoscopy to diagnostic of head injury hidden in precausal trauma, although lucketally for our society, this type of injury has decreased considerably, we have achieved an important number of patients and in the last year we have performed the procedure for another type of pathologies and also diversified the approach route according to the case. objective: sharing accumulated experience in years in the pericardic window practice by laparoscopy or thoracoscopy. material and methods description of cases results: during this period, we have accomplished cases of laparoscopic pericardal window with two unique ports for the diagnosis of head injury in trauma precordial, additionally there were practiced windows through traumatic trauma of which have been derived in treatment of cardiac injury on this way, without performing open approach. in another scenario, we have performed pericardial spill treatments for different causes by minimally invasive via. no complication or mortality associated with the procedure has been presented. conclusions: the pericardic window performed by a minimally invasive surgery is an effective, replicable strategy for the management of diagnosis and the medical and traumatic treatment of this pathology. patient selection is key and work in multidisciplinary groups guarantees good results. introduction: for the transabdominal preperitoneal repair (tapp) for groin hernia, single port surgery (sps) has been reported to reduce the abdominal wall damages. to reduce the length of the umbilical scar and to keep the view of triangulation, we use one needle forceps plus sps. patients and methods: from may to july , consecutive tapp patients were retrospectively investigated. there were male and female. we use two mm ports ( for the scope and for the operator's right hand forceps) through an umbilical multi-channel port and additional mm needle instrument is pierced above the pubic bone. a mm flexible scope allowed us to keep the triangular formation easily. we studied the safety and usefulness of this method from the viewpoints of operation time and the complications. results: median operation time of single side hernia ( cases) was min ( - ) and the bilateral case ( cases) was min ( - ). five cases needed one or two additional mm ports, and one case with severe preperitoneal adhesion due to the previous prostate cancer surgery was converted to open method because of the venous bleeding. other complications were spermatic cord injury and postoperative seroma that required the percutaneous puncture. umbilical scars and the pierced needle instrument scars became gradually invisible within or months. there were no incisional hernia nor wound infections in our series. these data was comparable to the conventional laparoscopic hernia repairs. conclusions: operation scars of this method had better cosmesis than the conventional tapp or sps tapp, and there were no differences between our sps-tapp with one needle foerceps and conventional method in operation time and the complication rate. our method was demonstrated as a less invasive approach for laparoscopic groin hernia repair. clinical application: fj clip is a stainless steel that can be used to hold organs in the abdominal cavity. it is available in two sizes: mm and mm. the device is short, it has a strong grasp, and it causes no or only negligible organ damage. we have used fj clip in the performance of local gastric excision (n= ), colectomy (n= ), and cholecystectomy (n= ) with no resulting difficulty. f loop plus is a g stainless steel loop-like device into which we can insert φ . mm nt alloy thread, which we draw out extracorporeally via simple puncture. laparoscopic total and proximal gastrectomy. we made a small incision at the umbilicus and inserted a -mm camera port and -mm metal cannula. we placed two (left and right) epigastric ports. retraction of the left hepatic lobe was easy with use of the -mm fj clip and a -mm penrose drain. for # lymph node dissection, we used the fj clip to grasp the upper part of the stomach, inserted the f loop plus from the upper right abdomen. for # dissection, we grasped the pyloric vestibule and pulled it leftward. for dissection of the upper edge of the pancreas, we grasped the left gastric arteriovenous pedicle and pulled it toward the abdomen. the fj clip's grasp and traction exerted on the stomach wall were strong and effective, and there was little organ damage. reconstruction (roux-y) or double tract were performed within the abdominal cavity by hand-sewn purse string suture of the esophageal stump, insertion of an anvil, and use of an automated anastomosis device. we have experienced total and proximally cases to date, but there have been no complications, and both intraoperative bleeding and operation time were within normal limits. conclusion: we believe the fj clip and f loop plus will replace conventional forceps for various tasks in reduced port gastrectomy. introduction: pulmonary anatomical resection is considered as standard treatment for early staged lung cancer. uniportal video-assisted thoracoscopic surgery (uvats) has recently showed favorable surgical outcomes, but remains technically demanding, especially in a complex procedure such as anatomic segmentectomy. needlescopic instruments facilitates complex laparoscopic surgeries with nearly painless and scarless postoperative outcomes, however, its utilization of thoracoscopic surgery were mostly for minor procedures such as bullectomy and sympathectomy. we presented our initial experience of lung cancer surgery performed by uniportal vats and additional needlescopic instruments, and we also compare the operative results with conventional uniportal vats. methods: from december to august , consecutive patients with lung cancer undergoing anatomical lung resections including lobectomies and segmentectomies were reviewed retrospectively. of these patients, patients received conventional uniportal vats (uvats), and patients received needlescopic-assisted uniportal vats (na-uvats). we compared the peri-and post-operative outcomes in these groups. results: there was no significant difference in demographic, anesthetic, or operative characteristics in two groups except for age. the mean operation time was statistically less in the na-uvats group ( . ± . min vs . ± . min, p= . ). the intraoperative blood loss was significantly less in the na-uvats group ( . ± . ml vs . ± . ml, p= . ). there were two major pulmonary arterial bleeding events and one conversion to thoracotomy in the uvats group. the hospital stay, duration of chest tube drainage and post-operative pain scale were comparative in the two groups. conclusion: under the assistance of additional needlescopic instruments, uniportal vats can be performed more efficiently and safely without compromising its benefit in less postoperative pain and early recovery. purpose: we applicated the v-loc into abdominal wall closure in single incision laparoscopic appendectomy (sila) from . the aim of our study is to present our experience of abdominal wall wound closure technique using barbed suture in sila and comparision of perioperative outcomes with conventional method of layer by layer abdominal wall closure after sila. methods: from august to june , sila was performed on patients with acute appendicitis at the department of surgery, hallym sacred heart hospital. under approval of institutional review board, data concerning demographic characteristics, operative outcomes, postoperative complications were compared between both v-loc closure group and conventional layer by layer closure procedures. in v-loc closure group, after removing the appendix, divided linear alba was closed using unidirectional absorbable barbed suture v-loc - with continous running fashon. begins at the end of incision, and coming back with reinforced running. subcutaneous closure was also done using same thread, and the subcuticular suture along incision line was performed with remaning portion of v-loc. results: the demographic data of patients's characteristics were similar between the two groups. the use of barbed suture significantly reduced the suturing time for abdominal wall closure (p= . ) compared with conventional suture. the postoperative incision length was significantly shorter in v-loc group than conventional group (p= . ). the rate of surgical site infection were similar in both group. no incisional hernia were noted in both group with median follow up periods of . months. the total costs of the procedure were comparable in both group under korean drg system. the use of barbed suture in abdominal wall closure in single port laparoscopic appendectomy is safe, and feasible method, reduces the suturing time, thereby decreasing the total operation time, and incision length with cosmetic effect. angela m kao, md, michael r arnold, md, julia e marx, paul d colavita, md, b todd heniford; carolinas medical center introduction: morgagni hernia is an anteromedial congenital diaphragmatic hernia seen in approximately in live births and rarely identified in adulthood. patients may be asymptomatic, have intermittent symptoms, or present acutely with incarceration/obstruction. given this, surgical repair is recommended, but a standardized technique has not yet been described. methods: a prospectively collected hernia-specific database was queried for all adult morgagni hernias performed at a tertiary hernia center. demographics and peri-operative data were compared. ( ) repair. the most common ( . %) method of repair included suturing mesh to the diaphragmatic portion of the defect and securing the anterior-inferior edge to anterior abdominal wall with transfascial sutures and/or tacks. four patients ( . %) underwent primary repair. average defect and mesh size was . cm and . cm , respectively. three patients ( %) underwent a concomitant paraesophageal hernia repair. mean ebl and length of stay was ml (range - ml) and . days (range - days). postoperative morbidity included transient postoperative hypoxemia ( patients) and pleural effusion ( ) . there was no mortality, mesh complications or recurrences with a mean follow-up of months. conclusions: morgagni hernias patients were more often older, obese, and women. these hernias remained unrepaired in % of patients despite their having had previous abdominal surgery. a laparoscopic or robotic approach offers an effective hernia repair with minimal complications, short hospital stay, and excellent long-term results for both elective and acute operations. mesh repair, sutured to the diaphragm and sutured/tacked to the abdominal wall, appears to be a very successful means to repair larger defects. introduction: hydatidosis is a zoonotic disease caused by echinococcus granulosus. it is endemic in the mediterranean, south america and middle east. it is a systemic disease wherein lungs are the second most common organ involved, after liver. radio-imaging plays an important role in diagnosing and determining the extent of the disease. surgical enucleation of cyst has been the classical treatment for this disease. bilateral lung involvement has been traditionally treated by median sternotomy or a bilateral thoracotomy. video assisted thoracoscopic surgery (vats) is an effective surgical approach in such settings. materials and methods: at our center, we have operated cases of pulmonary hydatidosis thoracoscopically over the past years. in all cases, area around the cyst was cordoned off with . % cetrimide soaked gauze pieces. a pericystotomy is performed with ultrasonic shears & the germinal membrane is delivered en masse into an endo-bag. an air leak test after saline instillation into the cavity, is a standard part of the procedure. for those cases with cysto-bronchiolar communications, the defect was sealed by either suturing or glue application. traditionally, bilateral cases & cysts larger than cm in size were tackled by an open approach. but, in our experience, cyst size, bilaterality & presence of complications are not contraindications for vats. all cases are administered perioperative albendazole ( mg twice a day, administered for three cycles of days each, with a gap of days in between) which helps in preventing recurrence and also takes care of any inadvertent intra-operative spillage. introduction: minimally invasive surgery (mis) is the standard approach for most of the surgical procedures performed by general surgeons. traditionally the majority of operations for trauma are performed open due to the complexity of the cases, however, trauma surgeons are expanding their armamentarium to include mis in a variety of acute procedures. we report our experience with the application of laparoscopy in a variety of trauma cases. methods: a retrospective review of trauma cases performed between / - / . during that time laparoscopic cases were performed after traumatic injury. patient demographics, injury severity (iss), injury mechanisms, the types of procedures and outcomes will be described. means and standard deviations were calculated and t test were performed. a p value of . was statistically significant. results: demographics-a total of trauma cases were performed laparoscopically during the study period. the majority were male, n= and the age was sd . obesity was documented in %, hypertension or cad was in %, and substance abuse was in %. blunt trauma was in % and penetrating %. the iss was sd . surgical procedures-the majority, %, of the procedures were completed laparoscopically. non-therapeutic laparoscopy was performed in %. repair of diaphragmatic or traumatic abdominal wall hernias were %. hematoma evacuation and control of bleeding was %. control of solid organ bleeding and repair was performed in %. intestinal repair occurred in %. for the cases that required open conversion iss was sd vs. laparoscopic cases iss was sd , p= . . outcomes: the overall length of stay was days sd . there was n= late death in a poly-trauma patient that required open conversion for complex solid organ and intestinal injuries. there was n= case of a community acquired pneumonia, and n= case of a recurrent pneumothorax. conclusions: a descriptive series of trauma operations approached with mis techniques is described. this cohort had high injury severity and a predominance of comorbid conditions. laparoscopy was successfully applied in the majority of cases for a variety of therapeutic procedures and mortality and morbidity was low. mis is safe and is gaining momentum for application in traumatic injury. objectives: laparoscopic distal gastrectomy for early gastric cancer is a standard treatment in japan described in guidelines. the surgical procedure has been shifting from laparoscopic assisted to complete laparoscopic surgery. in this study, we evaluated the outcomes and safety of the laparoscopic assisted distal gastrectomy. methods: for the marking of the oral side transecting line, the clipping at oral side of cancer lesion was performed by gastro-endoscopy before surgery. the lymph node dissection (d +/d ) is performed laparoscopically. as the dissection of the pancreatic superior region, the assistant hold the left gastric artery and keep the good view by retracting the pancreas. the common hepatic artery and proximal side of splenic artery are exposed. both sides of the left gastric artery and vein are exposed. left gastric vein and left gastric artery are cut after clipping and sealing. lymph node dissention of hepato-duodenal ligament is done and right gastric artery is cut after clipping and sealing. minor curvature of upper gastric wall is exposed (no , dissection). billroth i reconstruction by the circular stapler (cdh) is performed. through the upper median incision with cm, operator pulls out the stomach and transects the oral side of stomach with linear stapler after palpating the clips. duodenum is transected after purse string suture. gastroduodenal anastomosis is performed by cdh. results: two hundred cases were analyzed. the operation time, blood loss and the conversion to open surgery rate were minutes, ml, and . %, respectively. as postoperative complications, anastomotic failure, pancreatic fistula and postoperative bleeding were %, . % and %, respectively. the reoperation rate was %. one surgical death due to cerebral infarction was experienced. there were no patients with ppm (pathological proximal margin) positive and too much pm distance. frequency of abdominal wall incisional hernia and ileus were % and %, respectively. conclusion: although there is the disadvantage that small laparotomy can be made in the upper abdomen, laparoscopic assisted distal gastrectomy with billroth i reconstruction in our procedure is enough good from the viewpoint of the precision of proximal margin, and the incidence of serious complications. introduction: minilaparoscopy (mini) is a modality of minimally invasive surgery that attempts to produce less surgical trauma to the abdominal wall by reducing the diameter of surgical instruments to mm. searching for better outcomes in inguinal hernia repair, surgeons have looked for new and less invasive alternatives such as single-incision surgery, single-port surgery and mini. minilaparoscopic transabdominal preperitoneal hernia repair (mini-tapp) demonstrates some of the known advantages of mini general surgery procedures such as enhanced visualization, improved dexterity and great cosmetic outcome. it is safe and reproducible since it does not differ from standard laparoscopy. introduction: the celiac plexus is a structure located in the retroperitoneum, at the level of the lumbar vertebra, which is located in the prevertebral region and has sympathetic fibers. patients with advanced gastrointestinal cancer and associated pain, one of the management strategies is pain control. neurolysis of the celiac pleural by laparoscopy was first reported in humans in in patients with advanced pancreatic adenocarcinoma with excellent results. experience will be shown in the simplification of the technique for the procedure. method: neurolysis of the celiac pleura was performed in patients with advanced gastrointestinal cancer, stomach %, pancreas % liver % other %, no complications associated with the procedure, pain improvement was achieved in % of patients after process. the standardization of the technique by laparoscopy and its simplification, has made this procedure that is replicable and safe. description of the technique: patient in french position, technique of trocars, umbilical trocar mm and trocars of mm paraumbilical, staging laparoscopy is performed and sampling if necessary, is identified in the region of the lowercurvature of the stomach, the celiac trunk and the emergence of the left gastric artery are identified and cc of % alcohol diluted to the medium in the lateral fatty bearing are instilled through a pericranial under direct vision, verifying the non-arterial instillation of the alcohol. there were no complications related to the procedure. results: we report the experience of one group who underwent celiac pleura neurolysis in patients with advanced gastrointestinal cancer, gastric cancer %, pancreatic cancer %, liver cancer % and another %. the most frequent pathology report was adenocarcinoma, % of the patients were managed at hours with sustained effects, up to months of follow-up. with a significant decrease in pain medication. only patient required new laparoscopic neurolysis because of difficult-to-manage pain. the operative time of this procedure was minutes. the standardization of the technique, the use of low cost inputs, makes this type of procedure easily replicable with goodresults in pain management in cancerpatients. conclusions: mis is offered as one of the fundamental tools for the management of palliative procedures in gastrointestinal cancer. neurolysis of the celiac pleura with standardization of the technique, use of low cost elements, and the surgeon's skills make this procedure an option of management and control of pain in patients with advanced gastrointestinal cancer, is easily replicable, economical and insurance. background: the non-absorbable polymer clip offers a solution to the disadvantage of traditional metallic clip. due to its metallic property, it is not only expensive but also causes artifacts on imaging studies and often migrates into cbd. this study compares the traditional standard metallic clip with hem-o-lock used in laparoscopic cholecystectomy (lc) in regard of the safety and efficacy?. material and methods: this study includes patients who underwent lc implementing metallic clip (mc) and patients implementing hem-o-lock clips (h )?. both clips were applied to cystic duct and artery, then the gallbladder was dissected from the liver bed by diathermy. the intraoperative and postoperative parameters were collected including duration of the operation and complications?. results: the median operative time was not statistically different between the mc and the hc group ( . vs . minutes, respectively; p= . ) with no significantly less incidence of bile spillage ( vs. , p= . ) . no statistically significant difference was found in the incidence of postoperative complications between both groups ( vs. , p= . ). no postoperative bile leakage was encountered in both groups. conclusion: hem-o-lock clip provides a complete hemobiliary stasis and a secure cystic duct and artery control. its cost effectiveness is also attractive while provides efficacy equivalent to that of the standard metallic clip. introduction: most of the blunt thoracoabdominal injury patients always have multiple organ injuries. plan of definite treatment depends on the preoperative diagnosis. in isolating diaphragmatic traumatic injury without others organ injury laparoscopic approach is helpful, decrease a length of hospital stay as well as decrease a wound complication. authors describe the laparoscopic treatment of the patient who had rupture of a diaphragm from blunt trauma in an emergency setting. methods and procedures: a years old man presented with motor vehicle accident and mechanism of injury was blunt thoracoabdominal injury. he complains about chest tightness and tachycardia. complete evaluation and ct scan ware performed. stomach was herniated to the left chest and diaphragmatic ruptured was found neither others great vessels nor solid organs injury. the laparoscopic approach was desired and left diaphragm was repair by non-absorbable sutured without intraoperative complication. results: the patient has been discharged days post-operative with full recovery. chest x-ray was taken before discharge, in out-patient department weeks as well as months after discharge which shown no diaphragmatic herniation. conclusion(s): laparoscopic approach in isolated traumatic ruptured diaphragm patients is safe and should be considered. short-term outcome of laparoscopy-assisted distal gastrectomy with roux-en-y reconstruction through mini-laparotomy for gastric cancer since , we have introduced laparoscopy-assisted distal gastrectomy (ladg) with b-i reconstruction through mini-laparotomy. regarding to reconstruction, roux-en y reconstruction are also one of the choice in ladg, however, the technical feasibility has not been well documented so far. the purpose of this study was to compare the short-term outcome of ladg with roux-en-y reconstruction through mini-laparotomy compared to that of ladg with b-i anastomosis. between and , patients who underwent ladg for gastric cancer in oita university were enrolled in this retrospective study. since , the roux-en-y reconstruction has been performed as a standard method in our department. these patients were divided two groups based on anatstomosis; roux-en-y (r-y) group (n= ) and billroth i (b-i) group (n= ). baseline characteristics, operative results (including complications) and pathological results were evaluated. there were a considerably greater number of patients with advanced clinical stage and having ≥t invasion in the r-y group. estimated blood loss was lower in r-y than in b-i (p. ) and operative time was longer in r-y than in b-i (p. ). there were no significant differences in all grade intra-operative complications (p= . ). in addition, there were no significant differences in all grade post-operative complications between the two groups except internal hernia. hospital mortality was % in each group. ladg with r-y reconstruction through mini-laparotomy was technically feasible as well as ladg with b-i anastomosis. utilization of laparoscopy associated with blunt abdominal trauma: the nationwide inpatient sample - kenneth w bueltmann , marek rudnicki ; advocate illinois masonic medical center, chicago, il, university of illinois introduction: the incidence of trauma and its heavy burden upon the healthcare system remain strong. paradigm shifts in the management of these cases has, however, improved the mortality in such cases. it can be expected that improvements in management, when combined with the benefits of laparoscopy, will demonstrate positive impacts upon treatment outcomes. methods: the nationwide inpatient sample was referenced for inpatient stays for the years to . abdominal trauma cases were selected and identified as hollow (ho) or solid organ (so) type, and as blunt or penetrating. the trauma subset was then scanned for the presence of discrete laparoscopic procedures, laparotomy, and converted cases, and flagged accordingly. conclusion: utilization of laparoscopy in treatment of intraabdominal solid and hollow organs injury increases over time. although current analysis based on available hcup nis data include any procedures done during post-traumatic hospitalization, its results can lead to conclusion that minimally invasive technique is being utilized in increased fashion. introduction: single incision laparoscopic (sil) surgery is a laparoscopic procedure which leaves a single small incision in navel, and has been reported to be less invasive than and as safe and efficient as the conventional multiport laparoscopic (mpl) surgery. the long-term rate of incisional hernia after sils colectomy is unknown, and the risk factors of incisional hernia formation is not fully elucidated. methods and procedures: this is a retrospective from a prospectively collected database. the investigation took place in a high-volume multidisciplinary tertiary private hospital in japan. introduction: laparoscopic approach in the acute surgical care setting continues to be underutilized. we aim to report the successful diagnostic and therapeutic use of laparoscopy in the management of a nontoxic patient presenting with acute abdomen and to highlight the benefits of a minimally invasive approach without added morbidity. case report: presented is a -year-old male with history of cad s/p cabgx two years prior and no abdominal surgical history who presented to the ed with sudden onset severe, diffuse, abdominal pain of six-hour duration with n/v. there was no trauma to the abdomen. he had mildmoderate hypertension, but was otherwise hemodynamically stable. on examination, the patient was in severe distress and writhing in pain. fast exam was unable to be performed secondary to pain. cta of the abdomen revealed mesenteric abnormalities with associated small bowel edema in the rlq suspicious for small bowel ischemia. he was taken to the or for diagnostic laparoscopy. he was found to have an omental adhesive band to the abdominal wall with herniation of the small bowel through the small opening. approximately cm of ischemic, nonviable small bowel was resected and anastomosed intracorporeally. he tolerated the procedure well and was discharged home on post-operative day . discussion: primary omental related internal herniation of small bowel is exceedingly rare. there have been only few cases reported in the literature ( , , , ) . two were diagnosed on exploratory laparotomy, one on diagnostic laparoscopy and one at autopsy. the one who underwent diagnostic laparoscopy did not require bowel resection. in presenting this case, we hope to illustrate the role of laparoscopy in the management of acute abdominal pain due to bowel compromise. introduction: morgagni hernias are a rare finding in the adult population, and represent - % of all congenital diaphragmatic hernias. multiple approaches to these rare hernias have been described in the literature. here we present a novel technique of laparoscopic trans-abdominal repair using a combination of the endo-close device (medtronic, minneapolis, mn) and the ti-knot (lsi solutions, victor, ny.) methods: in a patient with a large left anterior diaphragmatic defect we performed trans-abdominal suturing utilizing the endo-close to perform primary closure of the defect, using the ti-knot to secure the pledged sutures along the anterior fascia. due to the size of the defect ( cm) this primary repair was buttressed with polyester mesh. in a second patient with a smaller ( cm) classic right-sided anterior diaphragmatic defect we similarly performed laparoscopic trans-abdominal suturing using the endo-close to traverse both the anterior and posterior fascia and the ti-knot to secure the sutures in order to perform a primary repair of the hernia. both patients presented had an uneventful postoperative course and no indication of recurrence at months. conclusions: morgagni hernias present unique technical challenges. in our experience the combined use of trans-abdominal suture with laparoscopic knot replacement device allowed for completion of both cases laparoscopically with minimal tension on the repairs. feasibility of concomitant laparoscopic splenectomy and cholecystectomy in situs inversus totalis: first case report worldwide ibrahim a salama, md, phd; department of hepatobiliary surgery, national liver institute, menoufia university introduction: situs inversus totalis is a rare anomaly characterized by transposition of organs to the opposite site of the body. combined laparoscopic splenectomy and cholecystectomy in those patients is technically more demanding and needs reorientation of visual-motor skills. presentation of case: herein, we report a year old girl presented with yellowish discoloration and left hypochondrium and epigastric pain diagnosed as hereditary spherocytosis (hs). the patient had not been diagnosed as situs inversus totalis before. the patient exhibit a left sided "murphy's sign" and spleen palpable in right hypochondruim. diagnosis of situs inversus totalis was confirmed with ultrasound, computerized tomography (ct) and magnetic resonant image (mri) with enlarged right sided spleen and presence of multiplegall bladder stones with no intra or extrabiliary duct dilatation. the patient underwent combined laparoscopic splenectomy and cholecystectomy as treatment of hereditary spherocytosis (hs). discussion: feasibility and technical difficulty in diagnosis and treatment of such case pose challenge problem due to the contra lateral disposition of the viscera. difficulty is the laparoscopic technique encountered in skelatonizing the structures in calot's triangle, which consume extra time than normally located gall bladder with right sided standing surgeon and the position changed to left sided standing surgeon during splenectomy. in review up to date medical literature this is the first case reported worldwide. conclusion: provided that the technique is performed by an experienced surgical team, concomitant laparoscopic splenectomy and cholecystectomy in situs inversus totalis is a safe and feasible procedure and may be considered for coexisting spleen and gallbladder disease as in hereditary spherocytosis (hs) as changes in anatomical disposition of organ not only influence the localization of symptoms and signs arising from a diseased organ but also imposes special demands on the diagnosis and surgical skills of the surgeon. objective: to identify the preference among medical students of the following surgical approaches: open surgery, conventional laparoscopy, minilaparoscopy (mini), single incision laparoscopic surgery (sils), natural orifice transluminal endoscopic surgery (notes), and robotic surgery. methods: an online google questionnaire was filled by medical students of different years in medical school. before answering the questionnaire, they watched an online video showing the different techniques, its advantages and disadvantages. the questionnaire consisted of questions about the hypothetical situation where the participants were going to be submitted to an elective cholecystectomy and they could decide which technique they would prefer. all statistical analysis was performed using the r software program, version . . . the chi-squared test was performed for categorical variables where appropriate. a p value . was statistically significant. results: one hundred and eleven medical students answered the survey. ( . %) were female and men. most of the students were between to years old ( . %). they were in the first four years of medical school. when asked if they would consider notes or single incision even if they know that they are new procedures and with not completely established security standards, . % ( ) answered that they wouldn´t consider with no difference between gender (p= . ). when asked if only conventional laparoscopy, robotics or mini were offered, which one they would choose: % of women and . % men chose mini first (p= . ). about the factors that they would consider the most important when choosing the surgical technique, they answered safety first ( . %), followed by the surgeon´s experience with the procedure ( . %), with no statistically significant result between genders (p= . ). when asked if they would consider an open technique even with the other techniques available and compared according to their year in medical school, students closer to finishing medical school would not consider it, with a statistically significant result (p= . ). regarding the most important factors they would consider and compared by year in medical school, safety and experience of the surgeon performed best, with a statistically significant result (p. ). conclusion: among the available surgical approaches, minilaparoscopy tends to be the preference among women medical students who considered safety the most important aspect. the closer they get to the end to medical school, the less they consider the open technique. background: extension of the single incision for the purpose of specimen removal in singleincision plus one additional port laparoscopic surgery (sils+ ) can undermine the merits of sils + , either by increasing wound-related morbidity or by destroying cosmesis. methods: we retrospectively analyzed the clinical outcomes of patients underwent elective sils + anterior resection, either with transanal specimen extraction (tase, n= ) or transumbilical specimen extraction (tuse, n= ), for colorectal cancer from january to june . this study included patients with a tumor diameter less than cm, measured by preoperative computer tomography. results: both groups were similar in patient's basic information and oncologic condition. most surgical data and postoperative clinical variables were comparable between tase and tuse group, except for increasing operative time in tase ( . + . vs. ± . min, p= . ) and reducing wound complications in tase ( % vs . %, p= . ). dosage requirement of narcotic analgesics was not inferior in tase group compare to tuse group. no significant differences were observed in conversion rate, perioperative and overall morbidity between the two groups. conclusion: although sils+ with tase prolonged operative time compare to with tuse, implement of tase is expected to provide benefit of reduced wound-related morbidity in patients with a tumor diameter less than cm. medhat ibrahim, md; al-azhar university, naser city, cairo, egypt purpose: morgagni hernia (mh) is a rare condition. mh is less than % of surgically treated diaphragmatic hernias in infants. there is no specific symptom for the maorgagni hernia. open surgical repair was the golden stander before the introduction of the laparoscopic surgery in the children and infant. there are many different laparoscopic techniques for mh repair have been reported. i report laparoscopic repair of mh in five infants using primary sutures closure with inrta-corporeal knot tying and ethicon secure strap device. this study is an evaluation of the safety and efficacy of this new laparoscopic technique of mh repair in infants with it is short-term outcomes follow up. patients and methods: five infants with mhs underwent laparoscopic repair by hernia sac excision then two primary sutures, non-absorbable proline through the full thickness of the anterior abdominal wall and the posterior rim of the defect, intra corporeal sutures knot tying, ethicon secure strap device which was used to complete the colures of the defect. there was no insertion of chest tube or drain. results: five infants with mh were operated upon. there were males and female. all cases were left side mh, male-female ratio was : . intraoperative and postoperative analgesia requirement was minimal (paracetamole mg/kg/rectal suppository/ hours for the first hours). ceftriaxone mg/kg single dose at the anesthesia induction. all operations were completed laparoscopic. all infants started and tolerated oral regular feeding with in hours from surgery. none of the patients developed intraoperative or postoperative complications. the maximum follow-up was months (mean, months). all patients are in good health without recurrence or port site compilation. conclusion: this easy save technique of mh repair is reducing the operative time and postoperative hospital stay. it is minims the need of postoperative analgesia, anti biotic. the early oral feeding is also a good benefit. the introduction: transumbilical single port laparoscopic appendectomy (tspla) is the most popularized single port surgery in the world. it provides more cosmetic benefits than conventional laparoscopic surgery. however, single port appendectomy requires longer operation time and advanced surgical skills. we aimed to investigate the learning curve for tspla. material and methods: data were collected from patients who underwent tspla by single surgeon between march and february . the learning curve were analyzed using a cumulative sum control chart (cusum) for operation time and complication. results: a total of patients were included in this study. mean operation time is . ± . minutes. there was no open or multi-port conversion. based on cusum for operation time, learning curve were cases. conclusions: tspla is a safe and effective alternative procedure. the learning curve could be overcome safely without major complications. our results suggest that the cases are sufficient to achieve surgical skills for tspla. introduction: anastomotic leakage (al) is a life threatening complication after minimally invasive ivor lewis esophagectomy (tmie ile) and has diverse treatment strategies such as conservative treatment, endoscopic treatment and surgery. however, there is no consensus on which treatment strategy is best. the aim of this study was to analyse various therapeutic strategies for al and their outcomes. methods and procedures: this retrospective multicentre study was performed in three highvolume hospitals. all patients that developed al after tmie ile in the period of january -july were included. the different endoscopic (stenting, clipping and suction-drainage) and surgical treatments and their success-rate were described; success was defined as clinical improvement after primary treatment. primary endpoint was the time until oral feeding was resumed. secondary endpoints were hospital stay and the total amount of surgical, endoscopic and radiologic interventions. results: in total patients that developed al were identified; four patients received antibiotics only. in the remaining patient, endoscopic treatment was performed as primary treatment in %; % received primary surgical treatment. basic variables were similar in these groups. median postoperative day of diagnosis of al was day in the endoscopic-group and day in the surgical-group (p= . ). admission to the icu as a result of the leakage was necessary in % in the endoscopic-group versus % in the surgical-group (p. ). however, median icu-stay was significantly shorter in the endoscopic-group ( days versus days, p= . ). success-rate of the primary treatment was similar; % and % respectively (p= . ). primary and secondary endpoints were comparable for both the endoscopic-and surgical-group; median time until oral feeding was resumed was days and days respectively (p= . ), median total hospital stay days and days respectively (p= . ) and the median number of interventions was in both groups (p= . ). conclusion: endoscopic treatment appears to be a safe and efficient therapy for al after tmie ile. a patient-tailored approach based on the condition of the patient and the morphology of the leak can be adapted to avoid surgery in a selection of patients. this may prevent surgical reoperations and reduce icu admissions. background: lymph node (ln) dissection around recurrent laryngeal nerve (rln) is one of the most important and difficult procedure in esophageal cancer surgery because of high rate of ln metastasis and risk of rln palsy. especially around left rln, the surgical area is far and narrow by thoracic approach which tends to results in insufficient ln dissection. therefore, we tried to remove this ln by imaging lymphatic chain to dissect sufficient ln. surgical procedure: we perform thoracoscopic esophagectomy by semi-prone position using - mmhg thoracic air pressure. after dissection of right rln ln, middle and lower esophagus, encircle the esophagus at the level of bifurcation of bronchus and pull toward right side by tape to dissect the dorsal and left side of upper esophagus. dissect the tissue including left rln ln from trachea by pulling esophagus up to dorsal side and try to move this tissue toward dorsal side of left rln so that this rln ln tissue can recognize as the "lymphatic chain". to increase the mobility of esophagus, cut the esophagus at the level of aortic arch and pull further up this upper esophagus to dorsal side. cut the esophageal branch of rln and separate this lymphatic chain from rln. at the end of thoracic procedure, this lymphatic chain is attached to upper esophagus. after the upper esophagus has pulled out from cervical site, lymphatic chain can easily recognize at the esophageal wall. result: we performed this lymphatic chain procedure in cases. to evaluate this procedure, cases of conventional method by same prone positioned esophagectomy was used for control. there was no statistical difference between these two groups in amount of blood loss (lymphatic chain: conventional= ml: ml, p= . ), rate of rln palsy ( . %: . %, p= . ). although the thoracic operation time was extended in some degree ( min: min, p= . ), number of dissected ln was increased ( . : . , p= . ) and recurrence along left rln has been relatively fewer by this method ( . %: . % p= . ). conclusion: ln dissection around left rln would be easy and sufficient by imaging lymphatic chain. further improvement is needed to secure this procedure and further evaluation should be done to support this data. introduction: to evaluate the role of robotic assisted surgery as part of an appropriate patient work-up and treatment of ipmn and its consistency in terms of perioperative and long term results. few reports described singular minimally invasive procedures for ipmn. this study aims to describe a comprehensive, oncologically adequate treatment of ipmn in a minimally invasive unit with an extremely high robotic penetrance. methods and procedures: we retrospectively analyze our database of resected ipmn between and . this case series includes consecutive, unselected patients: all candidates with a preoperative diagnosis of ipmn were approached robotically. results: among robot assisted pancreatic resections, we identified patients with ipmn. one was excluded for having less than months follow-up, so patients were included and analyzed. they underwent duodenopancreatectomy in cases, distal pancreatectomy in cases and central pancratectomy in . all but one indications followed the most updated available guidelines (sendai from to and fukoka from to ; american gastroenterology association guidelines were used for comparison only). one patient was operated even if the guidelines were suggesting to follow up, because of a strong familiar cancer history. the final pathology for this patient was high grade dysplasia. in another patient we were inside fukoka's recommendations, but outside aga guidelines and the final pathology was adenoma in chronic pancreatitis. postoperative morbidity was . ( low grade complications, one grade a pancreatic fistula, now considered a biochemical leakage only) and mortality was zero. one conversions to open surgery occurred only: a dp in jehowah's witness with a bulky mass behind the portal vein. the mean follow up was months (range: - ), with only one loss to follow up after months for a high grade dysplasia. conclusion: in hepatobiliary pancreatic minimally invasive centers the treatment of ipmn can be grant following the same principles of major cancer centers, with comparable results. large unbiased studies are needed to evaluate if a minimally invasive approach could modify the ratio between operated and surveilled patients. reducing the use of catheters, tubes and imaging after hiatal hernia surgery significantly reduces length of hospital stay sophia s oswald, candice l wilshire, md, brian e louie, md, ralph w aye, md, alexander s farivar, md; swedish medical center introduction: historically, standard post-operative management of patients undergoing laparoscopic hiatal hernia surgery has been placement of a foley catheter and nasogastric tube (ngt) at the time of surgery with removal early on postoperative day (pod) one, at which time an upper-gastrointestinal series study (ugi) would be performed. we initiated a quality improvement project, seeking to assess if we could safely forego placement of foley and ngt along with the ugi, unless clinically indicated. our aim was to determine if this decreased overall length of stay (los), and how often and which demographic of patients needed placement of foley or ngt postoperatively. methods and procedures: we reviewed patients who had undergone laparoscopic hiatal hernia surgery between and under a single thoracic surgeon. patients were excluded for poor esophageal motility (peristalsis \ %), previous esophageal surgery, and presence of a paraesophageal hernia (peh) with over % of the stomach contained in the chest. eligible patients were further stratified into two groups: fast track and non-fast track. fast track was defined as patients who left the operating room (or) with no foley or ngt, and did not receive a routine ugi on pod one. non-fast track was defined as patients who left the or with a foley and ngt and received a routine ugi on pod one. los was measured in hours from the start of surgery to the time of discharge. results: of the patients included, were categorized as fast track and as non-fast track. the two groups were similar in terms of age, gender, bmi and asa; however, the fast track group had fewer paraesophageal hernias and shorter surgery times [table] . the hospital los, however, was significantly shorter in the fast track group, even though there were more postoperative urinary catheters utilized. no patients in fast track group needed an ngt placed or ugi ordered during initial stay. conclusion: in more straightforward laparoscopic hiatal hernia surgery, surgeons can safely forego ngt and foley placement, as well as ugi evaluation the following morning. these initiatives may translate to a quicker discharge from the ward, and may allow safe transition to performing these cases in hour ambulatory outpatient setting. further evaluation of additional interventions and patient education to decrease los are underway. the conclusion: laparoscopic surgery seems to be a safe and feasible option, with long-term benefit for primary tumor resection with metastatic colorectal cancer, but optimal treatment has yet to be defined. the canadian association of gastroenterology (cag) has implemented the colonoscopy skills improvement (csi) program across canada with a goal of improving colonoscopy quality. the programs' efficacy has not yet been formally assessed. this retrospective cohort study was performed on fourteen endoscopists practicing in a tertiary referral center who have undergone csi training between october and december . procedural data were collected before and after csi training. data were extracted from the electronic medical record (emr) and entered into spss version . for analysis. student's t-test was used to compare groups for continuous data; chi-squared tests were used for categorical data. data were collected for a total of procedures; were done before csi training and procedures since csi training. our sample size provided % power to detect a mean difference in adr improvement of %. the most common indication for colonoscopy was family history of colorectal cancer in ( . %) patients. while age ( . yrs v. . yrs, p. ) and gender ( . % male v. . % male, p= . ) were similar, they were statistically different between groups. groups were comparable in terms of indication, and completion rate ( . % v. . %). adr improved significantly after completing the course ( . % v. %, p. ). an improvement was also noted in both polyp detection ( . % v. . %, p. ) and polyp removal ( . % v. . %, p\ . ). we have seen a significant increase in adr at out institution since implementing the csi program. gastric stomach cancer is a rapid major cause of cancer-related death globally, have higher incidence in men and it is noticeable by its heterogeneity. a lot of studies have expressed out the molecular basis of this cancer, include pathogenesis, invasion and metastasis. the invention of new technologies has help to bring out several novel biomarkers that have diagnostic and prognostic value. therefore, this review centers on biomarkers for the early diagnosis, treatment and prognosis of gastric cancer, elaborate the clinical important of serum tumor markers in a patient with this cancer as well as checking the growths, prognosis together with epigenetic changes and genetic polymorphisms. a deep and rigorous search was carried out in pub med/medline using specific words; "gastric cancer", with "tumor marker". our search yielded important reports about related topic from books and articles that were published before the end of september . conclusively, scientists are utilizing time and resource to salvage this nemesis which is of global burden. classical and novel biomarkers are important for treatment as well as pre-post diagnosis of gc. major causes for this disease are cigarette smoking, infection by helicobacter pylori, atrophic gastritis, male sex, and high salt intake. the treatment of which early diagnoses is of important to the management, after pathological diagnoses by stage prognosis and metastatic setting, although the outcome proved not so good includes chemotherapy, and oral medication are oxaliplatin, capecitabine, cisplatin and -fluorouracil ( -fu). introduction: emergent appendectomy is the standard of care in usa based on tradition rooted in theory that delaying surgery allows for progression of disease and poorer outcomes. antibiotic treatment alone has been shown feasible in the treatment of uncomplicated appendicitis. in clinical practice surgical treatment can be delayed due to a multitude of medical and logistical reasons. this study evaluates the relation between timing of surgery to outcomes. methods and procedures: consecutive adult patients undergoing appendectomy in a teaching community hospital were risk stratified using the acs risk calculator. time from imaging to incision defined early and delayed groups. statistical analysis was used to determine association between risk level, timing of surgery and outcomes. results: % of patients in this study were considered high risk. average time to incision was . hours. shorter time to incision was associated with a statistically significant lower length of stay (p. ). for every hours in surgery delay, one day was added to the length of stay. no statistical difference was found between time to incision and other outcome variables of clinical complications, conversion to open appendectomy or frequency of complicated appendicitis. length of stay was longer than predicted by acs risk calculator in both high and low risk groups. a multidisciplinary, obesity-focused approach improves diagnosis of obesity-related illnesses: a new paradigm for the care of patients with obesity roderick olivas, aaron brown, md, racquel s bueno, md, cedric s lorenzo, md; university of hawaii -department of surgery introduction: patients suffering from the burden of obesity are at significant risk for medical problems that lead to premature death and disability. we hypothesize that a multidisciplinary bariatric team will be better equipped to recognize and diagnose these conditions. this study hopes to quantify that a patient focused approach leads to increased recognition of obesity-associated comorbidities, thus improving quality of care and surgical outcomes. methods and procedure: a retrospective medical chart review of patients who underwent bariatric surgery from / / to / / was performed comparing patient problem lists obtained from their primary care providers upon entry into the bariatric program, and the final problem list generated after evaluation by the program's multidisciplinary team. the total number and specific comorbidities identified before and after multidisciplinary team evaluation was analyzed with a paired t-test and manova, respectively. comparison of the number of comorbidities identified against specific patient demographics was conducted using paired t-test. results: a total of patient charts were selected and met inclusion criteria. the sample consisted of % women and % men; the mean age was . ; the mean bmi was . ; % were morbidly obese (bmi ) and % were obese . the total number of comorbidities identified after evaluation by a multidisciplinary team was significantly greater (p=. ), with the average number of comorbidities diagnosed before and after being . and . , respectively. a significant increase (p. ) in the identification of comorbidities before and after evaluation were noted for all demographics, and no disparities regarding gender, age, marital status, employment status, bmi, or ethnicity where identified. conclusion: patients with obesity unknowingly suffer from many obesity-associated comorbidities simply because their health care providers have failed to recognize the existence of these conditions. surprisingly, this include diseases that are highly associated with obesity, such as osa and t dm, for which obese patients should be screened. although the root of this dereliction is yet to be determined, insufficient obesity-focused education and inherent weight bias among providers must be considered. assessment by a multidisciplinary bariatric team resulted in the identification and treatment of an increased number of comorbidities in this patient population. increased recognition of obesity-related comorbidities improves quality of care, which can translate into improved surgical outcomes. introduction: it is known that surgical residents suffer from sleep deprivation. no recent study evaluated the type and number of calls received at night. lately, burn out, depression and suicide have been the subject of interest in studies and media because of the higher rate among the residents compared to general population. the objective of our study is to evaluate junior resident's level of fatigue and the quantity and quality of calls received during on-call nights in general surgery at chus. methods and procedure: transversal study conducted on junior residents that were on-call in general surgery at the chus between april and august , . the participants detailed all the calls received between pm and am on an database created on the application handbase and completed a daily calendar of their on-call night noting all the tasks they did every half hour (surgery/consultation/sleep). the level of fatigue was evaluated at the end of the night at am with a visual analog of sleep scale on a score over points. results: the level of fatigue / (tired) or / (exhausted) was reached in closed to % of the oncall nights. the median number of calls by night was and the median duration of sleep was only . hours. the median lenght of uninterrupted sleep was . hours by night. among the total nights and calls analyzed, % were ''not pertinent'' and % were ''reportable in the morning''. more than % of the nights had at least one call ''not pertinent'' or ''reportable in the morning'' that have interrupted the junior resident's sleep. the level of fatigue was significantly correlated to the number of calls received during the night (spearman's rho=+ . , p. ) and to the number of uninterrupted hours of sleep (spearman's rho=− . , p. ). conclusion: the level of fatigue is very high among the junior residents in general surgery. many of the calls received during the night are not pertinent or could have been delayed to the morning. our results lead us to the conclusion that interventions and recommendations should be made to raise nurses and resident's awareness about the situation to reduce the unnecessary calls and the level of fatigue of the residents. we hope that on-call resident sleep will be better preserved and that will result in fewer health issues for them (burn out, depression, suicide). without interruptions: does twitter level the playing field? heather j logghe, md , laurel milam, ma , natalie tully, bs , arghavan salles, md, phd ; thomas jefferson university, washington university, introduction: frequent interruption of women in conversation has long been noted anecdotally, and studies confirm that women are interrupted more often than men. such interruptions can diminish perceptions of authority and compromise women's self-confidence. on twitter, users cannot be interrupted in the same way they can be in live conversation. thus the platform may provide a means for women to overcome this obstacle. to determine the degree to which women surgeon leaders utilize twitter compared to their male colleagues, we examined the twitter accounts and activity of the leaders of three national surgical societies. methods and procedures: lists of surgeons holding leadership positions in three surgical societies; the american college of surgeons, the academic association of surgery, and the society of american gastrointestinal and endoscopic surgeons, were obtained and duplicate names were deleted. table details the organizations and leadership positions included. the twitter accounts of these leaders were then identified and confirmed by reviewing the accounts for surgical content. account duration was calculated from the join date. the number of tweets, accounts following, followers, and likes were recorded for each account. outliers were defined as two standard deviations from the mean. results: one hundred sixty-eight men and women surgeon leaders were identified. forty-nine percent of the men and % of the women were found to have twitter accounts. mean account durations for men and women were similar, . years and . years, respectively. outliers for total tweets ( men, women), accounts following ( men), followers ( men), and likes ( men) were excluded from analyses. almost all positive outliers were men. there were no negative outliers. overall, excluding the outliers, there were no significant differences between men and women in any metric. conclusion: among leaders in the surgical organizations analyzed, a higher percentage of women than men have twitter accounts. those with the greatest number of tweets, accounts following, followers, and likes, however, are overwhelmingly male. thus, although women in this sample were more likely than the men to have twitter accounts, men were more likely to gain influence through their accounts. increasing women's influence in this public forum may position them as much-needed role models for the current and next generations. surgical societies may help reduce the disparity in women's representation in surgical fields through education of their members on how to use social media. introduction: the aim of this study was to report the perioperative morbidity and short-term outcomes of a case series of robotic-assisted laparoscopic transabdominal preperitoneal (tapp) inguinal hernia repairs. methods and procedures: a retrospective review (january through december ) of patients who underwent either unilateral or bilateral robotic-assisted laparoscopic tapp inguinal herniorrhaphy by two attending surgeons was performed. patient demographics, perioperative morbidity, operative time, and follow-up data were analyzed. results: patient demographics are summarized in table . mean operative times for unilateral and bilateral inguinal herniorrhaphy were . ± . and . ± . minutes, respectively. mean robot console times for unilateral and bilateral inguinal herniorrhaphy were . ± . and . ± . minutes, respectively. postoperative complications included urinary retention ( . %), conversion to open repair ( %), and delayed reoperation ( . %). no major bleeding, surgical site infection (ssi), or mortality was observed. at first follow-up visit ( ± days), symptoms/signs included groin/scrotal swelling ( %), seroma ( %), groin pain ( %), burning ( %), numbness ( %), and persistent urinary retention ( %). % of patients required a second follow-up visit. two patients underwent reoperation for suspected recurrence but instead a cord lipoma was found without a hernia defect. conclusions: robotic-assisted tapp inguinal herniorrhaphy can be performed with operative times and short-term outcomes similar to those published for open technique. the robotic-assisted tapp inguinal herniorrhaphy is a safe and an efficient minimally invasive surgical option with lower ssi risk and better cosmetic results. gunnar nelson, nathan lau, phd; virginia polytechnic institute & state university introduction: the fundamentals of robotic surgery (frs) and fundamentals skills of robotic surgery (fsrs) are universal curriculums covering a range of topics to assure a high level of surgical skills for optimal patient outcomes. this assurance of skills should include management and response to adverse events. thus, we reviewed frs and fsrs to identify any gaps in educational contents pertaining to how surgical teams are trained to handle adverse events in robotic surgery. methods and procedures: we conducted a literature search through google scholar, journal of robotic surgery, and plos one on frs and fsrs from to . we reviewed articles on preparing medical professionals in handling adverse events during robotic surgeries. besides the two curriculums, we also surveyed the literature on the characteristics of the adverse events and responses of the medical team. this literature survey provided a basis for recommending additional education contents to frs and fsrs. results: in our review, the frs contains modules consisting of an introduction to robotic surgery, with cognitive, psychomotor, and team training/communication skills. meanwhile, the fsrs contains different tasks, half of which on human-machine interaction and another half on operative interaction. both curriculums appear to lack contents on managing adverse events in robotic surgery. according to fda data, , adverse events were reported per , surgeries, of which (i) % relates to broken pieces of surgical instruments falling into patients, (ii) . % pertains to burning holes in tissue from electric arching, and (iii) . % relates to unexpected operations of the instrument such as power outage and issues with electrosurgical units. thus, medical professionals should be trained to manage common adverse events in robotic surgery. for frs, augmenting the five current scenarios in the communication section with common adverse events (i.e., broken pieces falling into patients) would minimize complications under abnormal circumstances. for fsrs, the most logical method would be augmenting the operative interaction tasks with adverse events to train medical professionals. conclusion: we discovered universal curriculums on robotic surgery lack education contents for training medical professionals to manage adverse events and out of the , procedures, ( . %) pertained to device malfunction. to protect the patient's health, universal curriculums must incorporate contents preparing medical professionals in responding to adverse events, particularly device malfunctions, during robotic surgeries. introduction: this retrospective study was performed to evaluate the safety and feasibility of the new senhance robotic system (transenterix) for laparoscopic cholecystectomies. we report the first single-institutional experience utilizing this new robotic platform. methods: approximately robotic cholecystectomies were performed using the senhance robotic system. the senhance surgical system is a new robotic platform that consists of a cockpit, manipulator arm and a connection node ( figure ). this new system provides robotic surgery with numerous advantages including eye-tracking camera control system, haptic feedback, reusable endoscopic instruments, and a high configuration versatility due to total independency of the manipulator arms. patients were between and years of age, eligible for a laparoscopic procedure with general anesthesia, had no life-threatening disease with a life-expectancy of less than month and a bmi\ . a retrospective review of a variety of prospectively collected pre-, peri-and postoperative data including but not limited to patient demographics, intraoperative as well as postoperative complications was performed. cholecystectomies were performed by expert level laparoscopic surgeons. results: the standard laparoscopic technique and setup was easily applicable to the senhance robotic system for this particular surgery. operative time and perioperative complications were comparable to reports of standard laparoscopic cholecystectomies. there was no significant learning curve detected in our case series. conclusion: we report the first experience with laparoscopic cholecystectomies using the new senhance robotic system. there were no major perioperative complications and operative time was comparable to standard laparoscopic cholecystectomies well reported in the literature. this case series suggests that the senhance robotic system can be safely and easily used for laparoscopic cholecystectomies by experienced laparoscopic surgeons. background: the ergonomic benefits or robotic surgery for the health of the surgeon are widely touted as benefits of this technique, though concern remains over a perception of increased risk of injury to patients, particularly in the novice robotic surgeon. injury to the bedside surgeon and assistants due to robotic movement can also occur, though not previously reported. we describe a finger fracture to the bedside surgeon due to entrapment between robotic arms and discuss potential risks to the surgeon in robotic procedures. procedure: a distal pancreatectomy and splenectomy was performed utilizing the davinci si system (intuitive surgical, inc., sunnyvale, ca). during the operation, hemorrhage was encountered which required an instrument exchange that was delayed by self-testing failures. after the instrument was validated and advanced into the field by the bedside surgeon, the operator abruptly took control of the device to reposition. the external portion of the active arm was then rapidly and forcefully propelled laterally toward a stationary retracting arm. the bedside surgeon's hand was still engaged on the instrument being inserted and became trapped between the two arms, leading to a right middle finger crush injury. results: the bedside surgeon sustained a fracture to the distal phalanx at the insertion of the flexor tendon with significant hyperextension of the joint. there was temporary paresthesia of the fingertip. while flexor tendon function was preserved and surgery was not required, the surgeon was required to maintain continuous splinting and was unable to return to full duty for a total of weeks. the surgeon has mild residual hyperextension. conclusions: while complications to the patient have previously been attributed to the robotic platform, this case demonstrates that there are other inherent hazards to members of the operative team. as is natural with all indirect visual surgical techniques, the operator becomes intensely focused on the internal view and instruments in the field. this spatial separation is accentuated on the robotic platform as the isolated console provides a complete visual field immersion, no tactile feedback, and a disconnect between the rapid, sizeable outward arm motions need to produce small internal movements. given the need for maximum dexterity internally, the device doesn't have external proximity sensors to prevent arm-arm or arm-operator collisions. while many bedside operators report anecdotes of collisions with the device, this case reveals the forces involved at the human-machine interface can lead to more significant injuries. robtic approach to non-midline abdominal wall hernias: a single institution experience from a high volume center emily benzer, do, j. stephen scott, md, facs; university of missouri introduction: the objective of our study was to evaluate our experience with robotically repaired non-midline abdominal wall hernias at a high-volume robotic surgery program. we also will discuss the technical advantages of the use of robotic technology in repair of these unusual hernias which have typically had higher recurrence rates then midline hernias. laparoscopic approach for lateral ventral abdominal wall hernia (spigelian) and lumbar hernia has been described, however the success of robotic assisted repair for these hernias has yet to be determined. methods: a retrospective case analysis of all robotic abdominal hernia cases between june and june at an academic institution with a single high volume robotic surgeon was performed. the operative details of robotic repair of non-midline abdominal hernias, patient demographics, length of stay and smoking status were recorded and analyzed. the technical advantages of the use of robotic technology for example circumferential fixation of the mesh, ease of intracorporeal suturing, and the use of wristed instruments to gain better angles for posterior fascial release were evaluated. results: a total of cases were identified. the average age of the patients was . years (range - years) and patients were predominantly female ( %). spigelian hernias represented % (n = ) and lumbar hernias % (n= ). all patients had primary closure of their defect and patients ( %) had a posterior myofascial release performed. mesh types placed included polypropylene uncoated (n= ), polypropylene coated (n= ), and biologic (n= ). with uncoated polypropylene mesh placed had peritoneum closed over the mesh. the average length of stat was . days (range - days). there were no recurrences identified over a mean follow up period of . months (range . - . months). conclusion: robotic assisted repair of non-midline abdominal wall hernias is a viable option in the elective setting with no recurrences noted in this case series. the technical advantages of using robotic technology were identified and discussed in detail. these advantages theoretically improve outcomes in these patients however further analysis on long-term outcome and costs will have to be determined in future studies. the inguinal hernia repair has seen several critical improvements in recent times due to the implementation of new techniques, including laparoscopic repair, as well as robotic repair. with over , inguinal hernia repairs performed annually, it is important to identify the safest and most patient-friendly method. for surgeons, robotic assisted laparoscopic surgery is gaining in popularity for its dexterity and d visualization. but despite the growing interest in robotic hernia repairs, there is a scarcity of literature to support its superiority over open inguinal hernia repair. this study hypothesizes that patients who undergo robot assisted laparoscopic inguinal hernia repair will have decreased immediate post-operative pain, shorter recovery room stays, decreased narcotic requirement, and overall decreased pain at follow up compared to open inguinal hernia repair. in this study, we performed a retrospective analysis of patients who underwent either an open or robotic assisted laparoscopic inguinal hernia repair at stamford hospital, from july -july . the following characteristics were analyzed for both subsets of patients: gender, bmi, type of repair, operative time, recovery room time, immediate post-operative pain, and post-operative pain at follow up. our study demonstrated longer average operative time for patients undergoing robotic hernia repair compared to open repair, which was statistically significant (p value=. ). patients who underwent robotic inguinal hernia repair spent less time in the recovery room compared to patient who underwent open repair. in addition, patients in the robotic hernia group required less narcotics in the recovery room compared to patients who underwent open repair (p value = . ). there was no statistically significant difference between lengths of hospital stay between the two groups. this study highlights several possible advantages of robotic inguinal hernia repair, including lower post-operative pain scores, less narcotic usage required in the post-operative period, as well as shorter recovery room time. the results from this study should increase interest in investigating the superiority of robotic inguinal hernia repair. future plans for study involve comparing robotic to laparoscopic repair. in addition, we plan to continue to follow the study patients to look at additional qualitative metrics, including time to return to work and time to return to daily activities. introduction: buccal mucosal grafts (bmg) are traditionally used in urethral reconstruction. there may be insufficient bmg for applications requiring large amounts of graft, such as urethral stricture after gender affirming phalloplasty. rectal mucosa is an alternative with less post-operative pain, no impairment in eating and speaking, and larger graft dimension. laparoscopic transanal minimally invasive surgery (tamis) has been described by our group. due to the technical challenges of harvesting a sizable graft within a confined space, we adopted a new approach using the intuitive da vinci xi® system. we demonstrate the feasibility and safety of a novel technique of robotic tamis (r-tamis) in the harvest of rectal mucosa for the purpose of onlay graft urethroplasty. methods and procedures: irb approval was obtained. three female-to-male transgender adults (age range: - years) presenting with post-phalloplasty urethral strictures underwent robotic rectal mucosal harvest. the procedure was first rehearsed on an inanimate model using bovine colon. the surgery was performed under general anesthesia with the patient in lithotomy position. the gelpoint path transanal access platform was used. the rectal mucosa was harvested by the robotic instruments after submucosal hydrodissection. specimen size harvested correlated with clinical surface area needed for urethral reconstruction. following specimen retrieval, flexible sigmoidoscopy was used to ensure hemostasis. the rectal mucosa graft was placed as an onlay for urethroplasty. results: there were no intraoperative or postoperative complications. average graft size was cm (range: - cm). every case had excellent graft take for reconstruction. all patients recovered without morbidity or mortality. they reported minimal postoperative pain and all regained bowel function on the first postoperative day. all reported significantly less postoperative pain and greater quality of life in comparison to prior bmg harvests. the procedure has been refined to increase efficiency and decrease operative time by maintaining adequate insufflation, retraction of the mucosal graft, and maintaining graft integrity. conclusions: to our knowledge, this is the first use of r-tamis for harvest of rectal mucosal graft. our preliminary series indicates the robotic approach is feasible and safe. it constitutes a promising minimally-invasive technique to employ in urethral reconstruction. demonstrated feasibility and avoidance of the challenging recovery associated with bmg harvest warrants further application and long-term evaluation of this procedure. prospective studies evaluating graft success, donor site morbidity and long-term outcomes are needed. introduction: the proportion of robotic minimally invasive procedures that are being performed annually is growing rapidly, specifically in the field of general surgery. a robotic approach to minimally invasive procedures potentially confers a number of benefits ranging from a magnified viewing field to greater attenuation and translation of hand movements leading to improved stability and maneuverability. it is paramount that a robust curriculum is designed for training surgical residents in robotic techniques. the aim of this project is to assess the current state of robotic surgery training at the ohio state university, with specific regard to whether it is currently temporally effective in addition to establishing a baseline against which the robotic surgery curriculum can be compared. methods and procedures: data were obtained for cases performed at the ohio state university hospital east, between january and september of . case time, date, type, and attending surgeon were recorded and tracked for review. of the cases, were cholecystectomies, were unilateral inguinal hernia repairs, and were bilateral inguinal hernia repairs-for a total of procedures included in the analysis. chief residents were trained in two-month blocks, beginning in january of . mean console operative times for the first and second months were compared for cholecystectomies as well as unilateral and bilateral inguinal hernia repairs. results: mean console time decreased for cholecystectomies (− . %; n= ), bilateral (− . %; n= ) and unilateral (− . %; n= ) inguinal hernia repairs from month one to month two. there was a large amount of variance across training blocks, but there was a systematic improvement in operative time across the training period. average operation length was shortest for cholecystectomies (m= . min), followed by unilateral inguinal hernia repairs (m= . min), and finally bilateral inguinal hernia repairs (m= . min). discussion: this preliminary data suggests that residents are able to decrease their robotic operation time over the course of the two-month rotation. although sample sizes were relatively small for each block, the consistency of the trend supports this conclusion. further data collection will allow for more precise estimates in the future, and stronger conclusions to be drawn. these results show that rapid improvement is possible and provide motivation to establish robotic surgery curricula for general surgery residents nationally. robotic pancreas-sparing treatment of pancreatic neuroendocrine tumors: three case reports and review of the literature alessandra marano, giorgio giraudo, stefano giaccardi, desiree cianflocca, diego sasia, felice borghi; santa croce e carle hospital introduction: pancreas-sparing resections would be the ideal procedure in case of small pancreatic neuroendocrine tumors (p-nets) reducing the risk of exocrine and endocrine insufficiency. compared to standard resection, this type of surgery is safe and feasible without increasing the risk of postoperative complications except the overall rate of clinical pancreatic fistula (pf), which did not result in higher mortality or overall morbidity. robotic surgery for pnets enucleation has been rarely described but initials experiences have shown that this approach is associated with favorable outcomes. the aim of this study is to describe three cases of dv®si™ pancreatic enucleation for p-nets located in the uncinate process, in the body and in the posterior aspect of the tail of the pancreas, respectively. a brief review of the literature regarding the application of robotics for pnets enucleation is also included. methods and procedures: this study includes patients undergoing dv®si™ enucleation for pnets with a maximum diameter no more than cm and a distance between tumour and main pancreatic duct (mpd) greater than mm. at surgery, exposure of the pancreas was achieved by separation and traction of the gastrocolic and gastropancreatic ligaments. the pancreas was explored: an intraoperative ultrasound was used ensuring negative margins and leaving the mpd intact. thus, a cross-stitch through the tumour was made routinely in order to pull the tumour. enucleoresection was carried out with monopolar scissors and bipolar forceps. the tumour was placed into a specimen bag and removed from the trocar port. a drain was always left. results: median total operative time was min. no conversion neither intraoperative complications occurred. median length of stay was . days. two patients presented a pf grade a (classification isgpf) while a pf grade b occurred in case of pancreatic tail net enucleation. final pathology revealed two insulinomas and one non-functioning net of the pancreatic body. at a median follow-up of months no pancreatic insufficiency, reoperation or tumour reoccurrence was observed in all cases. the robotic approach for the treatment of p-nets is safe and feasible and, in selected cases, it may extend the indications of minimally invasive pancreatic-sparing surgery. in particular, the robotic approach provides a more precise dissection and may ensure negative margins and the mpd intact. these preliminary results are consistent with literature data about over robotic pancreatic enucleations for p-nets that shows favourable surgical outcomes, especially if compared with those of open surgery. introduction: rectal cancer continues to be a surgical challenge. new technologies must be incorporated into practice and, at the same time, oncologic surgery and overall outcomes must be improved. the use of da vinci robotic surgery systems has spread rapidly in the field of rectal cancer treatment showing several technical advantages and favorable outcome compared to laparoscopy. since the introduction of the robotic platform in our institution in , we have adopted a single-docking robotic technique for rectal resection. the aim of this study is to present our standardized technique and to analyse the clinical outcomes of the first robotic rectal procedures. methods and procedures: prospectively collected data reviewed from consecutive patients who underwent single docking totally robotic (da vinci® si™) dissection for rectal cancer resection between june and august under eras program. robotic rectal surgery was performed without changing the position of the robotic cart but only the robotic arms are repositioned between two phases: ) vascular ligation, and sigmoid colon to splenic flexure mobilization; and ) pelvic tme. results: there were men ( %) and the median age was years (range- - ). thirty-five patients had neo-adjuvant chemoradiotherapy whilst patients had bmi [ . procedures performed included anterior resection (n= ) and abdominoperineal resection (n= ). protective ileostomy was performed in patients. the median operating time was min (range- - ). there was one conversion and two intra-operative complications (one bladder lesion and one ureteral lesion, respectively). median length of stay was . days (range, , and readmission rate was %. thirty-day mortality was zero. anastomotic leak rate was %, and all patients except by one were managed conservatively. the mean lymph node harvested was (sd± . ). radial margin was negative in all patients. at median follow-up of months, there were no local recurrences. the single docking robotic technique is a safe and feasible approach for rectal surgery: in our study it has demonstrated favourable clinical outcomes and the adoption of a standardized stepwise approach was useful especially during the initial learning phase. to the best of our knowledge, this is the largest series from italy to report this standardized approach and the short-term clinical and oncological outcomes. in the complex laparoscopic surgical procedure, there is a problem such as that the laparoscope and the surgical instruments interfere with each other because multiple instruments is concentrated in one place. this problem is significantly appear in the laparoendoscopic single site surgery. therefore we suggested multi degrees of freedom (dof) manipulator with mantle tube for assisting laparoendoscopic surgery, which manipulator has two flexion and one telescopic mechanisms actuated by wire. it is possible to insert any thin surgical instruments such an endoscope the mantle tube of the multi dof manipulator, which the manipulator can let those surgical instruments access the operative field from different axis with other instruments. the use of this manipulator has two advantages, one of which is avoidance of fighting between instruments and laparoscope. the other is that become possible to ensure a satisfactory field of vision in the operative field. in this report, we assumed that this multi-dof manipulator is used as laparoendscope. in order to evaluate the performance of this manipulator, the operation time of the test in the abdominal cavity simulator (fasotec inc.) was measured. the test is a contact test to multiple-targets, which is a test that bring a forceps contact multiple-targets in the abdominal cavity simulator according to the defined pattern. as a general comparison and evaluation target for this measurement result, it is compared with the case using the same access method as the conventional rigid endoscope. in this test, the number of contacts between forceps and laparoendoscope were recorded by using electrical device. subjects (n= ) are adult men who trained the peg transfer in the above simulator. it was compared of total operating times of the test and the field of vision obtaining each device. from these results, using the suggested manipulator device rather than using rigid laparoscope a satisfactory field of vision is obtained, and it is possible to short the operating time approximately seconds, and to small the number of contacts significantly. therefore it was shown that the effectiveness using the suggested manipulator device. for this reason, use of this device is expected to facilitate the complex surgical operation. additionally, it is performed para ablative operation of swine liver tissue in the abdominal cavity simulator, as previous step of clinical test. the operative field in this test was surveyed, the refinements of this manipulator for improvement performance were described in this report. yoshiyuki usui, md, phd, ichiro akiyama, md, phd, hironori kunisue, md, phd, hideaki mori, md, phd, tetsuya ota, md, phd; okayama medical center background and methods: we have performed approximately cases of gasless endoscopic thyroid surgery since for years. this surgery was performed through a small subclavian incision and using a wire traction and inserting an endoscope. we have modified and improved our surgical techniques by inventing various surgical instruments. here we introduce four newly invented surgical instruments, chronologically. results: we made u-retractor ( ), u-trocar ( ), u-kelly forceps ( ), and u-suction tube retractor ( ). all surgical instruments were modified from conventional surgical instruments. the u-retractor was a piercing retractor, each end of which had a sharp tip and a retractor. this retractor was inserted from the -cm working port outside the body and retracted the muscles effectively. the u-trocar was reversely set from inside to outside to make the working space wider. the u-kelly forceps which had a special ratchet were made to dissect loose connective tissue around the thyroid gland avoiding injury of the recurrent laryngeal nerve. the u-suction tube retractor facilitated a wider working port and eliminated the mist created by the ultrasonically activated scalpel effectively. recent data showed no difference of operative time, hoarseness, blood loss and hospital stay between conventional thyroid lobectomy and gasless endoscopic lobectomy. conclusion: gasless endoscopic thyroid surgery has been improved in the last years. this procedure made the excision of not only benign thyroid tumors but also small thyroid carcinomas. this operation is still cost effective, because almost all surgical instruments are reusable and is a satisfactory experience to both the patients and surgeons. objective: to put forward the importance of complete (r ) resection for the treatment of retroperitoneal tumors increasing overall survey. methods: in this study; patients having the diagnosis of retroperitoneal tumors with different histopathological subtypes whom were hospitalized in emergency surgery department of istanbul medical faculty between the years of and were evaluated retrospectively. the database of the department was analyzed. operational backgrounds, histopathological results, radiological evaluations, and assesments about relapses, and overall survey were obtained from the medical archieve. results: the average follow-up time was , years. all of the patients included into the study were undergone operations. the average time of hospital stay was calculated as days. of the patients were found to have positive surgical margins in their histopathological evaluations. overall mortality rate of the study was % ( / ). we have observed a direct correlation between complete (r ) resection and disease-free survival. patients having relapses had worse prognosis in terms of overall survey ( % mortality rate). after having done the statistical evaluation, surgery was found to be the main determining factor for the assesment of overall survey. conclusion: reference to an experienced and multidisciplinary surgical center after an early diagnosis has upmost importance for the treatment of retroperitoneal tumors. surgical approach constitutes the main element in the management. overall survey is directly correlated with complete (r ) resection. novel fluorescent dyes for real-time, intraoperative, organ-specific visualization of biliary and urinary systems using dual-color near-infrared imaging ; children's national health system, nih/nci p multidisciplinary approach for management of necrotizing pancreatitis: a case series prabhu senthil-kumar university of alberta, centre for the advancement of minimally invasive surgery introduction: the objective of this study was to systematically review the bariatric surgery literature to understand how weight loss is reported. the incidence of obesity has increased globally. according to the world health organization more than million were obese in . in the last decade, bariatric surgery has been increasingly utilized as an effective treatment option for severely obese patients. currently, bariatric surgeries are among the most commonly performed operations. the primary outcome of such procedures is weight loss which has been shown to vary according to the type of surgery. however, there are different methods used to report weight loss which makes it difficult to directly compare outcomes between studies. a previous review by dixon et al. in revealed a wide heterogeneity in weight loss reporting. however, there have been no recent reviews on the reporting of weight loss in bariatric surgery. methods: a search of the medline electronic database was performed for studies published in using search terms gastric bypass/sleeve gastrectomy, weight, human, and english. articles were selected by two independent reviewers based on the following inclusion criteria: ( ) adult participant ≥ years predictive factors for excess body weight loss after bariatric surgery in japanese obese patients takeshi naitoh hypertension resolution after rapid weight loss: a single institution experience cristian milla matute reoperative bariatric surgery: analysis of indications and outcomes: a single center experience iman ghaderi objective: to observe the effects of duodenal-jejunal transit on glucose tolerance and diabetes remission in gastric bypass rat model. method: in order to verify the effect of duodenal-jejunal transit on glucose tolerance and diabetes remission in gastric bypass, twenty-two type- diabetes sprague-dawley rat model established through high fat diet and low dose streptozotocin (stz) administered intraperitoneally were assigned to one of three groups: gastric bypass with duodenal-jejunal transit (gb-djt n= ), gastric bypass without duodenal-jejunal transit (rygb n= ) and sham (n= ). body weight, food intake, blood glucose, as well as meal-stimulated insulin, and incretin hormones responses were assess to ascertain the effect of surgery in all groups. oral glucose tolerance test (ogtt) and insulin tolerance test (itt) were conducted three and seven weeks after surgery. results: comparing our gb-djt to the rygb group, we saw no differences in the mean decline in bodyweight, food intake, and blood glucose -weeks after surgery. gb-djt group exhibited immediate and sustained glucose control throughout the study outcomes with sham operation did not differ from preoperative level. conclusion: preserving duodenal-jejunal transit does not impede glucose tolerance and diabetes remission after gastric bypass in type- diabetes sprague-dawley rat model is bariatric surgery effective for comorbidity resolution in super obese patients? methods: a retrospective analysis of outcomes of a prospectively maintained database was done on obese patients with a diagnosis of at least one or more of the following comorbidities-t dm, htn, osa, or hld-at the time of initial visit who had undergone either a sleeve gastrectomy (sg) or a roux-en-y gastric bypass (rygb) at our hospital between and . the patients were stratified based on their preoperative body mass index (bmi) class: bmi methods: we retrospectively reviewed all patients that underwent laparoscopic sleeve gastrectomy (lsg) at our institution from - . common demographics and comorbidities were collected as well as creatinine, preoperatively and up to hours after surgery. the renal function was calculated using the ckd-epi formula, derived and validated by levey et al. acute kidney injury was defined as an increase in serum creatinine by ≥ . mg/dl within hours after surgery. all tests were two-tailed and performed at a significant level of . . statistical software r, version . . ( - - ) was used for all analyses. results: of the patients reviewed conclusion: the impact of laparoscopic sleeve gastrectomy in renal function is evident within the first hours after surgery. patients undergoing lsg, especially patients with baseline chronic kidney disease stage ≥ are at increased risk of developing acute kidney injury in the perioperative setting the body mass index (bmi), fasting plasma glucose (fpg), glycosylated hemoglobin (hba c), serum triglyceride, serum cholesterol and blood pressure of all patients were measured before and at months after surgery. the results were collected and analyzed. results: patients suffered from metabolic disease undertook lsg surgery successfully (a mean age of years), were male and were female. all of patients suffered from obesity and the mean bmi of them was . ± . kg/m before surgery. among them, patients had type diabetes mellitus (t dm), patients had hypertriglyceridemia (htg), patients had hypercholesterolemia (hc) and patients had hypertension. the mean bmi of patients at months after surgery was . ± . kg/m and decreased significantly (p. ). the mean excess weight loss (ewl%) of patients was . %± . %( %* %) at months after surgery. the average levels fpg, hba c of t dm patients at months after surgery were . ± . mmol/l, . %± . % methods: we retrospectively reviewed all patients who underwent bariatric surgery from to . we assessed kidney function using the chronic kidney disease epidemiology collaboration (ckd-epi) and cardiovascular risk using framingham risk score (frs) equation pre-operatively and at and months of follow-up. our population was divided into two groups: patients with ckd stage ≥ (gfr\ ml/min) and patients with normal gfr. significance. results: of the , patients reviewed, . % (n= ) met the criteria for ckd-epi glomerular filtration rate (gfr) and framingham risk score (frs) calculations. after matching, patients ( . %) were left to analyze, % (n= ) of which had a laparoscopic sleeve gastrectomy. eighty-six patients ( %) had an impaired kidney function (ckd≥ ) (group ) and patients ( %) had a normal gfr (group ). common demographics and comorbidities after matching are described in table . the mean creatinine in group was . ± . mg/dl versus . ± . mg/ dl in group (p
). glomerular filtration rate was . ± . ml/min in group and ± . ml/min in group . furthermore, when the frs was calculated at months follow-up, patients with impaired kidney function had an absolute risk reduction of . % corresponding to a relative risk reduction (rrr) of group . the percentage of estimated bmi loss was found to be similar in both groups ( . ± . and . ± . respectively p= . ). conclusions: bariatric surgery, especially lsg, has a positive impact on kidney function particularly in patients with chronic kidney disease stage or greater. despite these patients having a higher preoperative cardiovascular risk, they showed similar risk reduction when compared to patients with normal kidney function at months of follow-up the impact of socioeconomic factors and indigenous status jerry t dang only ( . %) patients underwent urgent conversion for management of complications after sg. three patients had intraoperative complications necessitating blood transfusion. fourteen ( . %) patients required readmission within days postoperatively. six patients ( . %) required surgical interventions including for gastrointestinal leak, for hemodynamic instability, for a cecal perforation, and for a small bowel obstruction. there were no mortalities within the first year of revisional surgery. in patients with bmi[ kg/m at the time of revisional surgery, at the median postoperative follow-up of (interquartile range, - ) months, a median (interquartile range, - ) kg/m reduction in bmi was observed. overall, ( . %) patients had persistent type diabetes at time of revisional surgery. improvement of diabetes was observed in patients ( . %) after conversion of sg to rygb. among patients with gerd symptoms, subjective symptomatic relief was reported at the last follow-up. conclusion: weight recidivism is the most common indication for revision of sg objective: to evaluate laparoscopic mini-gastric bypass in the treatment of morbid obesity. method: three hundred patients with a mean bmi of . . kg/m underwent a laparoscopic mini-gastric bypass between to . a laparoscopic approach with five trocar incisions was used to create a long narrow gastric tube; this was then anastomosed ante-colically to a loop of jejunum cm. distal to the ligament of treitz peri-operative and short-term follow-up results up to does age or preoperative bmi influence weight loss after bariatric surgery? one-way anova or the kruskal-wallis test was used to compare continuous data across all groups. subsequent analysis of categorical data was achieved by chi-square or fisher's exact test. statistical significance was accepted as p. . results: a total of patients ( % male) were analyzed. average age and preoperative bmi were . ( . ) years and . ( . ) kg/m , respectively. preoperative comorbidities included: diabetes ( . %), hypertension ( . %), hyperlipidemia ( . %), previous myocardial infarction ( . %), obstructive sleep apnea ( . %), chronic obstructive pulmonary disease ( . %), gastroesophageal reflux ( . %), tobacco use ( . %). the asa classes of patients undergoing sg were ii ( . %), iii ( . %), and iv ( . %). the follow up rate at , and months was . %, . %, and . %, respectively. the -day mortality and readmission rate were % and . %, respectively. the %ewl was not different among age groups at , or months for the total, male, or female cohorts. among preoperative bmi groups, %ewl was not different in any cohort at or months, but was different at months for the total cohort (p. ) and female cohort (p\ . ), and trended toward significance in the male cohort (p= . ). the highest %ewl was found to be in patients with preoperative bmi of - . there was no difference in -day mortality or readmissions among groups a crp≥ mg/dl had a sensitivity for a complication of % and a specificity of %. primary bariatric surgery patients with a post-operative complication had higher crp levels compared to those who did not ( . ± . mg/dl vs . ± . mg/dl; p= . ). there was no difference in crp levels for patients with a -day reoperation or readmission. there were no mortalities. conclusions: bariatric surgery patients with elevated post-operative crp levels are at increased risk for -day complications. the low sensitivity of a crp≥ mg/dl suggests that a normal crp methods and procedures: the patients, who formed the previously published cohort, were contacted and their charts were reviewed. follow-up visits, symptom severity scores, and any subsequent medical or surgical interventions were collected. symptoms were assessed using the symptom severity score (sss) and the gastroparesis cardinal symptom index (gcsi) questionnaires. success was defined as a sss of or less. results: out of original patients, patients ( males, females) were available for follow-up ( patients declined participation, were lost to follow-up, patient was deceased, and was excluded after undergoing esophagectomy for unrelated indication) mbbs ; grant government medical college and sir jj government hospitals methods and procedures: twenty-six nh patients with dm were prospectively randomized to undergo either lrygb or lsg. patients were followed for -years with primary end points consisting of total weight loss (twl), percent excess body weight loss (%ebw) and impact on dm as measured by fasting blood glucose (fbs) and hba c. in addition, baseline, week, and , , , , and months post-operative levels of glucagon-like peptide (glp- ), peptide yy (pyy), leptin, and ghrelin were collected. results: a total of / patients completed follow-up. the %ebw at year for lrygb and lsg were % and %, respectively. resolution of dm occurred in / patients, the remaining three subjects were in the lgs arm. pre-operative fbs in lrgyb and lsg groups, were and , respectively. pre-operative hba c in the lrygb and lsg groups, were . and . , respectively. fbs at year for lrygb and lsg were and , while hba c for lrygb and lsg were . and . , respectively. a consistent post-operative decrease in fbs was only seen in lrygb. lrygb ghrelin percentages increased at , , and months, while levels decreased in lsg. leptin percentages decreased in both groups. the ppy levels remained relatively unchanged in both groups. lrygb glp- levels increased at week, , , and months. lsg glp- trends were similar except at months where glp- levels decreased. conclusion: lrygb and lsg resulted in equivalent post-surgical weight loss and resolution of dm in the nh population video assisted thoracoscopic thymectomy (vats) has emerged as a minimally invasive alternative to the standard transsternal approach. we present herewith the surgical and neurological outcomes after vats their operative time, blood loss, conversion rate and post operative parameters like intensive care unit (icu) stay, inter-costal drainage (icd) indwelling time, hospital stay were recorded. neurological outcomes were assessed based on myasthenia gravis foundation of america (mgfa) post intervention status classification. statistical analysis was done using stata software. results: ninety patients underwent thoracoscopic thymectomy during the study period. vats was done through right approach in ( . %), left approach in ( %) bilateral approach in patients ( %) and subxiphoid approach in ( . %). there was conversion to open approach in ( . %) patients due to dense adhesions at westchina hospital of sichuan university were included. all of the operations were performed by a single skilled surgeon. we divided our patients into two groups based on whether isao was used. of them, patients received isao for lps and patients received lps without isao. surgical skills and safety were evaluated. results: there were no significant differences in preoperative patients characteristics of the two groups. significantly less intraoperative blood loss( . ± . ml vs . ± . ml; t=− . , p= . ) were observed in group of isao conclusions: isao is technically feasible, safe surgical skills for patients reveived lps, and its represents an effective method to decreased intraoperative blood loss. p modular laser-based endoluminal ablation of early cancers: in-vivo dose-effect evaluation and predictive numerical modelling giuseppe endoscopic submucosal dissection enables en-bloc removal of early gastrointestinal neoplasms. however, it is technically demanding and time-consuming. laser-based ablation (la) techniques, are limited by the lack of depth penetration control and thermal damage (td) prediction. our aim was to evaluate a predictive numerical modelling (pnm) of the td to preoperatively select the optimal power and exposure time enabling a controlled ablation down to the submucosa (sm). additionally, the ability of confocal endomicroscopy (ce) to provide information on the td was assessed at the histology, there was an increased damage depth per higher j applications. the r value at . j was . ± . , and was significantly lower when compared to energies from j (r= . ± . ; p. ) up to j ( . ± . ; p\ . ). safe m and sm ablations were achieved applying lower p settings ( . and w), at different t values, leading to an mp impairment only in and % of the cases, respectively. ce provided relevant images of the td, consisting in architecture's distortion and disappearance of the gland's contours. the predicted damage depth we also analyzed early gastric cancer patients who received lpg-ip with cm jejunal interposition. anastomosis procedure was overlap method for eshophagojejunostomy and gastrojejunostomy, feea for jejuno-jejunostomy. results: the comparison between otg/opg-ip shows no significant difference in perioperative complications and qol scores, significant smaller body weight loss in opg-ip group. lpg-ip group also shows good result in short term outcomes. consideration: as comparison in open surgery implies superiority in jejunal interposition, we have introduced lpg-ip. esophagogastrostomy after proximal gastrectomy is simple but has a risk for sever gerd symptoms, no optimal procedure for reconstruction after proximal gastrectomy has yet been established. although laparoscopic jejunal interposition is relatively complicated in procedure, we can safely perform in combination with common anastomosis techniques. conclusion: body weight loss in otg-ip group is smaller compared to otg group consecutive patients with early gastric cancer underwent solo spdg (n= ) and mldg (n= ) performed by same surgical team. solo spdg can be defined as practice in which a surgeon operates alone using camera holder. mldg usually requires two or three surgical assistants. the inclusion criteria in this study were (i) pathologic proven stage i-ii gastric cancer (ii) no other malignancy (iii) more than d lymph node dissection (iv) r surgery. one-to-two propensity score matching was performed to compensate for the differences between two groups. results: after the propensity score matching, solo spdg (n= ) and mldg (n= ) patients were selected. mean operation time ( ± . vs ± . mins, p= . ) and estimated blood loss (ebl) ( . ± . vs . ± . ml, p= . ) were significantly lower in the solo spdg group than in the mldg group. the hospital stay and the use of pain control were similar between the two groups. although the initiation of semi fluid diet was similar, the time to first flatus was earlier in the solo spdg adhesional omental hernia: a case report an unexpected cause of small intestinal obstruction in crohn's disease strangulation inguinal hernia due to an omental band adhesion within the hernia sac: a case report omental adhesion, intestinal herniation, and unexpected death in the elderly small bowel obstruction secondary to greater omental encircling band-unusual case report the median operative time was min. the median postoperative hospital stay was . d. histological examination of the tumors revealed carcinomas, adenomas, and carcinoid. complications occurred in ( %) patients, viz., ssi (two patients), pancreatic fistula (two patients), bleeding (two patients), passing failure (one patient), and cholangitis (one patient). however, no severe postoperative complications (clavien-dindo classification grade or higher) were reported in these cases. conclusion: our cases showed that duodenal tumor resection using lecs enables curability through a minimally this study aimed to compare the outcomes of tltg with those of latg by using a meta-analysis. methods: we searched pubmed, embase, and cochrane library in may, to locate prospective or retrospective studies on surgical outcomes of tltg versus latg. the outcome measures were postoperative complications such as anastomosis leakage and anastomosis stenosis, operation time, blood loss, time to flatus, time to first oral intake, and postoperative hospital stay endoscopic thyroid lobectomy: our early experience at tertiary care hospitals of lahore univariate analysis was performed followed by logistic regression to identify independent predictors for the primary outcome. results: forty-six out of ( %) patients referred for gp required jt insertion to treat malnutrition. etiology of gp included: % idiopathic, % diabetic, % post-surgical. thirty-six patients ( %) reported severe daily symptoms. twenty-five patients ( %) had successful return to oral intake while ( %) required prolonged feeding access, reinsertion of a jt or tpn initiation. on multivariate analysis patients who had a pyloroplasty (p= . , or . ) and those who were married (p= . , or . ) were found to be independent predictors of successful discontinuation of tube feedings. on subgroup analysis -hour gastric emptying time normalized after pyloroplasty (p= . ) in patients which had a successful re-initiation of oral intake while persistent gastric emptying refractory to pyloroplasty was associated with failure. the group of patients who underwent pyloroplasty did not differ in terms of demographics, marital status (p= . ) and preoperative gastric emptying (p= . ) from those who did not. gp etiology (p= . ) psychiatric conditions (p= . ) and substance abuse laparoscopic transabdominal repair of morgagni hernia rebekah macfie average procedure length was . minutes. average hospital length of stay was . days, with all patients tolerating a regular diet prior to discharge. our -day readmission rate was / ( . %). / ( . %) patients required repeat egd evaluation for either recurrence of symptoms or impacted food bolus. at week follow-up, / patients ( %) complained of dysphagia and / patients ( %) had eliminated ppi from their daily medication regimen. at month follow-up, / patients ( %) complained of dysphagia and / patients ( %) had eliminated ppis. at year follow-up, / patients ( %) complained of dysphagia and / patients ( %) had eliminated ppis. conclusion: as a recently introduced surgical option, no long-term data exists detailing the linx procedures ultimate success rates and complication profile mini-laparoscopic vs traditional laparoscopic cholecystectomy: preliminary report deniz atasoy since the introduction of minilaparoscopic cholecystectomy (mlc) in , it gained little interest that could be attributed to decreased durability of the reduced size instruments, poorer optical resolution and smaller jaws of the instrument tips. our aim was to compare the outcomes of mlc with traditional laparoscopic cholecystectomy (tlc) one developed choledocholithiasis on postoperative day one and after ercp the course was uneventful. the other patient developed choledocholithiasis and acute pancreatitis on the sixth postoperative day and was treated conservatively. the stone in the ampulla had fallen by itself without a need for ercp single-incision plus one additional port laparoscopic surgery for colorectal cancer with transanal specimen extraction: a comparative study two patients had a previous attempt of hernia repair, one with mesh. one patient did not have any immunosuppression due to hiv infection, whereas the other were on cyclosporine, tacrolimus and/or mycophenolate mofetil. there were two laparoscopic and two open cases, mean operative time was . minutes ( - ), mean blood loss was ml ( - ). mesh used were biological porcine dermis in one case, polypropylene with absorbable hydrogel barrier in three cases. mean mesh length and width were cm ( - ) and . cm ( - ) respectively. one patient underwent a component separation, though none of the patients had the fascial defect closed. there were no intra-operative complications. three patients were readmitted for hyperkalemia, abdominal pain, and seroma respectively. neither recurrences nor reoperations were reported. mean follow-up was . days ( - ) conclusion: post liver transplant incisional hernia repair is feasible either laparoscopic or in an open fashion. because of the size and location of the defect, fascial closure is unlikely achievable. the use of standard techniques and materials give a similar result of the non-transplant population. p technique of esophagojejunostomy using orvil after laparoscopy assisted total gastrectomy for gastric cancer shinichi sakuramoto there was a significant difference in mortality between the two time-periods, / patients died during - and / died during - (p= . ). those who died were significantly older ( years ( - )) than the survivors ( y ( - )) (p= . ). five of the patients who died in the previous group died without any intervention. / of those who had an acute open necrosectomy died. surgical necrosectomy correlated significantly with mortality (p= . ). the only patient who died in the recent group died without any intervention. none of the patients receiving minimal invasive drainage in this group died until now only cases in adults and fewer than cases in children have been reported in world literature, with surgical management being the only option. an innovative, minimally invasive laparoscopic excision of the abdominal sac was performed and the scrotal component was managed by jaboulay's procedure. this is probably the first case report in world literature describing laparoscopic management of hydrocele-en-bissac. case report: a year old male presented with complaints of bilateral hydrocele and swelling in right lower abdomen since one year. computed tomography of the abdomen revealed an encysted hypodense lesion with enhancing walls along the right side of pelvis, anterior to the psoas muscle and extending through the internal ring into the right inguinal region upto the scrotal sac; measuring . cm . cm suggestive of an encysted hydrocele of cord associated with hydrocele of both scrotal sacs excessive gastric resection may result in postoperative deformity of the stomach, with consequent gastric stasis in food uptake. to minimize the resection of stomach tissue, especially for lesions close to the esophagogastric junction or pyloric ring, we have developed laparoscopic wedge resection (lwr) with the serosal and muscular layers incision technique (samit) for gastric gastrointestinal stromal tumors. this samit is simple and does not require special devices. purpose: the purpose of this study was to clarify whether lwr with samit for gastric gists is technically feasible in term of short-term outcome methods: all patients who went through lsg in our department between / to / have been evaluated for bleeding complications, after implementation of anti-bleeding policy: blood pressure was controlled to mmhg during stomach resection and staple line was reinforced throughout it's length with a running - absorbable v-lock suture. drains were used selectively. results: out of patients who went through the procedure ( . %) suffered hemorrhagic complications: patients had? hb[ gr%. patients received - red blood pc's. no patients were re-operated for bleeding. patients were readmitted for infected hematoma and had ct guided drainage. one patient ( . %) suffered from leak. conclusion: implementation of anti-bleeding policy in lsg is very effective. there is no need to use expensive buttress material to achieve these results. drains can be used selectively. the impact of this policy on leak rate needs to be fifty procedures immediately prior to, immediately after, and eight months after completion of training were included for each endoscopist. data were extracted from the electronic medical record and entered into spss for analysis. student's t-test was used to compare groups for continuous data, and chi-squared tests were used for categorical data. data were collected for procedures. patient groups pre, post, and eight months after csi training were comparable in terms of age ( . yrs, . yrs, and . yrs), sex ( it's in the bag; can stoma output predict acute kidney injury in new ostomates? robert fearn colostomy output stabilised rapidly, whilst ileostomy output increased progressively throughout the first postoperative days as can be seen in chart . twelve patients ( %) developed aki during index admission. length of stay was significantly greater in the aki group at ( % ci - ) days vs ( - ) days. highest daily stoma output was non significantly higher in the aki group ml ( % ci - , ml) vs , ( - , ml) as was mean daily stoma output at ml ( - , ml) vs ml ( - ml) (chart ). seventeen patients ( %) were readmitted for any reason, ( %) specifically for aki. in total patients ( %) developed aki within three months of their stoma surgery only of whom had developed aki during their index admission. all patients who developed aki following their index admission were ileostomy patients. conclusion: acute kidney injury in new stoma patients is associated with prolonged hospital stay and readmissions with associated morbidity and healthcare costs consecutive laparoscopic bariatric operations were performed, including primary roux-en-y gastric bypasses (lrygb), primary adjustable gastric bands (lagb), primary sleeve gastrectomies (lsg) and secondary bariatric surgeries and revisions. all bariatric procedures were approached laparoscopically ( procedures were stapled and were nonstapled). the mean patient age was years ( - ), females represented % and mean bmi was . kg/m ( - ). there were no perioperative mortalities, no conversions to open surgery and no intraoperative blood transfusions. there we two major intraoperative complications (hypopharyngeal perforation- , malignant hyperthermia- ). mean hospital stay was . days ( - days). eleven patients ( . %, in gastric bypass group and one in lsg group) required -day reoperations for postoperative complications (staple line gastrointestinal bleeding- , anastomotic leak- , strangulated port site hernia- , unexplained severe abdominal pain- , intestinal obstruction- , and intraabdominal abscess- ). there were no long term ( -year) mortalities in patients that required reoperation. there was one transfer to another institution. the dynamics of further improving safety was such that there was no complication on the recent consecutive stapled procedures and the mean hospital stay was . days ( - days). detailed subgroup analyses will be provided. conclusions: with well-controlled and structured pre-, intra-, and post-operative care, laparoscopic bariatric surgery can be performed with minimal reoperations and zero mortality in a teaching institution does concomitant placement of a feeding jejunostomy tube during esophagectomy affect quality outcomes? md, facs; icahn school of medicine at mount sinai background: placement of a feeding jejunostomy tube (fj) is often performed during esophagectomy. few studies, however, have sought to determine whether concomitant placement affects postoperative outcomes of esophagectomy of these, ldg was performed patients and odg was performed . we compared elderly patients (aged years or more) with younger patients in each operative procedure. (ldg: elderly , younger ; odg: elderly , younger ) preoperative comorbidity and surgical results were analyzed. multivariate analysis was performed to detect predictive factors for postoperative complications. results: in both ldg and odg groups, the operative time and amount of blood loss did not differ, while comorbidity was more common in elderly patients than in the nonelderly, and there were fewer retrieved lymph nodes in elderly patients. the incidence of all postoperative complications did not differ between both groups in each procedure, and there were no significant differences in the time to first flatus or postoperative hospital stay. however, in terms of specific postoperative complications, respiratory complications were more frequently observed in eldery group with odg significantly (p= . ), while not with ldg group. in multivariable analysis, age was not independent predictor of postoperative complications. conclusion: odg for eldery patients requires attention particularly in postoperative respiratory complications. ldg is a safe and less invasive treatment for gastric cancer in elderly patients who have greater comorbidity. p examining the role of preoperative ineffective esophageal motility in laparoscopic fundoplication outcomes tyler hall there were no significant differences in complications or recurrence rates. preoperative quality of life measures did not vary between the cohorts nor did postoperative scores at three weeks or six months. patients with % ineffective clearance exhibited worse gerd-hrql scores one and two years postoperatively conclusion: preoperative ineffective esophageal motility was shown to result in comparable short-term quality of life following ars. however, gerd-hrql scores at one and two yearsshowed worse outcomes in patients with preoperative iem robotic surgery as part of oncologically adequate ipmn treatment: indications, short and long term results federico gheza eligible patients who had minimally invasive surgery were stratified in multiport laparoscopic and robotic cohorts, and included if they had poi/sbo after surgery. comparative analysis assessed the demographic, perioperative, and postoperative outcomes. the main outcome measures were the incidence rate, associated variables, and time to ileus/ sbo across the mis platforms. results: during the study period total patients were reviewed- laparoscopic and robotic. postoperatively, ( . %) laparoscopic and ( . %) robotic patients suffered from poi/sbo laparoscopic sbo occur significantly later after the index procedure than robotic sbo ( conclusions: the rate of poi/ sbo is considerable and comparable across laparoscopic and robotic approaches. however, there are distinct differences in the severity, time to occurrence, and impact on quality measures, such as los and readmissions between laparoscopic and robotics. this information could be an important factor in which approach the surgeon choses laparoscopic surgical procedure was standard with using laparoscopic linear stapler. responses to surgery were evaluated a month after the operation based upon the american society of hematology evidence-based practice guidelines for itp. results: there was no open conversion in this study. the mean operation time and blood loss were min and g, respectively. there was no case using blood transfusion during and after operation. with regard to complications, one patient ( %) had a postoperative pancreatic fistula that did not require percutaneous drainage. positive responses, including the complete and partial remissions, were achieved in % ( / ). the mean follow-up duration was months, and the -, -, and -year relapse-free survival rates were % for all three time points. conclusions: the present study demonstrated that ls for itp can provide good long-term outcomes two cases of conversion from sp-c to open surgery were excluded. all procedures were followed postoperatively for a minimum of months, and wound complications such as bleeding, fat lysis, infection, or hernia were recorded. patients were classified as having a wound complication or not. results: pure transumbilical sp-c was completed . %, additional trocars were used in . %, and the rate of conversion to open surgery was . %. after a median follow-up of . (range, - ) months few cases performed with hand assist, notes, or single-incision. utilization of robotics was highest for bpd/ds ( of , cases, . %). the greatest number of robotic-assisted cases were sleeve gastrectomy ( , of , , . %) and gastric bypass ( , of , cases, . %). relatively few operations were converted to a different approach (see table). operative time was longer when using robotic approaches for both sleeve ( . vs . minutes, p. ) and bypass ( . vs . , p\ . ). postoperative los was no shorter when using robotic-assistance (see table). unadjusted -day outcomes revealed slightly higher rates of readmission for both operations when using robotic-assistance (see table), and slightly higher rates of complications after robotic sleeve gastrectomy p comparision of perioperative and survival outcomes of laparoscopic versus open gastrectomy after preoperative chemotherapy: a propensity score-matched analysis adjustment for potential selection bias in the surgical approach was made with propensity score-matched (psm) analysis. perioperative and survival outcomes were compared between the lag and og groups. results: in total, patients were identified from the database. after psm analysis, patients who underwent og were one-to-one matched to patients who underwent lag in the setting of nact. these two groups had similar outcomes in terms of intra-and postoperative complications and -year overall survival. however, the lag group had a longer operation time (p= . ) and lower estimated blood loss (p= . ). moreover, compared with patients in the og group, those in the lag group had fewer days until first ambulation conclusion: the present study indicates that lag performed by well-qualified surgeons for treatment of locally advanced gastric cancer after preoperative chemotherapy is as acceptable as og in terms of oncological outcomes. p outcomes of laparoscopic antireflux surgery for gastroesophageal reflux disease: effectiveness and economic benefits kyung won seo, phd; kosin university college of medicine purpose: laparoscopic antireflux surgery (ars) is an alternative treatment option for gastroesophageal reflux disease (gerd) in the world. however, the effectiveness and economic feasibility of ars versus medical treatment is unknown. this study was performed to evaluate the effectiveness and economic benefits of ars. methods: nine patients with gerd were treated using laparoscopic ars between and . surgical results and total cost for surgery were reviewed. results: seven men and women were enrolled. preoperatively, typical symptoms were present in patients, while atypical symptoms were present in patients. one patient underwent partial fundoplication due to absent peristalsis and the other underwent nissen fundoplication. postoperatively, typical symptoms were controlled in of patients, while atypical symptoms were controlled in of patients. overall, at months after surgery, reported partial resolution of gerd symptoms, with achieving complete control. the average cost of ars for nine patients was usd. conclusion: laparoscopic ars is effective for controlling typical and atypical gerd symptoms. the cost of ars may be more economical over the long term compared to medical treatment since laparoscopic surgery is reported to affect respiration and circulation, we should take indication of lag for elderly patients into consideration carefully. indication of lag for elderly patients, however, is still controversial. the aim of this study is to assess the safety and validity of lag for elderly patients. method: medical records were retrospectively reviewed for patients who underwent lag for gastric cancer between and . in this study, patients over years of age were defined as elderly patients. patients were divided into two groups according to age; group a (age ≥ , n= ), group b (age \ , n= ). preoperative characteristics and postoperative outcomes were analyzed. two-tailed student's test and/or pearson's chi-square test were used for statistical analysis. results: there were no significant differences in male/female ratio and body mass index between two groups. number of patients whose asa physical status was ≥ , and/or performance status was ≥ did not differ total gastrectomy ( . vs . %, p= . ), proximal gastrectomy ( vs . %, p= . ). intra-operative blood loss, operating time, and number of harvested lymph nodes did not differ between the two groups. as for postoperative complications such as intra-abdominal abscess ( . vs . %, p= . ), anastomotic leakage ( vs . %, p= . ), significant difference was not observed between the two groups. in addition, respiratory and cardiovascular complication was not observed in elderly patients. incidence of clavien-dindo classification ≥grade ( . vs . %, p= . ), and postoperative hospital stay ( . vs . days, p= . ) did not differ. conclusion: short-term outcomes of lag in elderly patients were not different from those in young patients the essential role of the transcystic duct tube (c-tube) during laparoscopic common bile duct exploration (lcbde) towakai hospital introduction: laparoscopic common bile duct exploration (lcbde) is a standard surgical procedure for the treatment of common bile duct stones (cbds). however, there are some problems associated with cbd drainage after operations even if performing with the primary closure. therefore, we developed a new drainage tube, c-tube, which contributes to shorter drainage periods and reduces perioperative complications. method: c-tube is a type of bile drainage tube which is fixed to the cystic duct with an elastic band. closing the duct with an elastic band as soon as c-tube is removed prevents bile leakage from the stump of the cystic duct. the essential roles of this tube include: . assisting suturing during operations, . use during intra-and post-operative cholangiograpy, . assisting post-operative endoscopic sphincterotomy when necessary we included patients from -years prior to our intervention and compared this with patients who had follow-up after implementation. we excluded patients having revisions, gastric banding, and patients whose primary surgeon had left during the data collection period. we analyzed demographics and follow-up rates at , , , , and months. chi-square test was used to evaluate for significance, and results were corrected for multiple comparison. results: patients met inclusion criteria in the pre-intervention group, and in the postintervention group. of those, were analyzed for the year follow-up visit. the pre-intervention group had males, females, and an average age of . approximately / of the surgeries performed were sg, / were rygb. the post-intervention group had males, females, average age of . approximately half of the post-intervention cases were sg while the rest were rygb. conclusion: bariatric surgery is a useful tool in aiding weight loss and improving comorbidities. it is essential that patients receive long-term follow-up and monitoring to achieve these goals. our program now uses a system of phone call reminders for scheduled visits, as well as calls and letters for annual visits surgeon's evaluation of an intraoperative microbreaks web-app workload questions were modified nasa task load index (physical demand, mental demand, and complexity) and procedural difficulty on - ( =maximum impact) scales. primary outcomes were the impact of microbreaks on surgeons' physical performance, mental focus, pain/discomfort and fatigue with checkboxes for improved, no change and diminished. secondary outcomes were microbreaks impact on distraction level and workflow disruption using a - ( =maximum impact) scale. descriptive statistics were calculated for median and interquartile ranges (iqr) of these responses. results: seven surgeons ( male, female), with a median (iqr) surgical experience of ( . , ) years, completed ten surgical days with a median (iqr) operative duration of ( , ) minutes/surgical day. the median number of microbreaks/surgical day was . the median (iqr) for mental demand, physical demand, surgical complexity and difficulty are shown in table . following each surgical day, surgeons reported / improved physical performance situs inversus totalis (sit) is inherited in an autosomalrecessive fashion with complete abnormal transposition of thoracic & abdominal viscera. its incidence varies from in to live births. for those undergoing surgery, laparoscopic approach is preferred as it avoids inappropriate incisions. however, due to mirroring of the viscera, the surgeon faces constant visio-spatial disorientation during laparoscopy. p ''how to be a surgeon and not dying trying'' control of basic physiological parameters in perioperative phase second main variable: blood pressure (bp) with manual measurement sleeve. preoperative bp and immediate postoperative bp were measured, we were not able to measure intraoperative bp due to the lack of consent of the surgeons involved for the use of other devices different from the heart rate band. secondary variables: years from graduation, years of practice, age, body mass index (bmi), number of medical co-morbidities, number of jobs, sleeping hours the night before. we took measurements to surgeons during a laparoscopic cholecystectomy. results: the mean preoperative heart rate was . bpm. the mean minimum intraoperative heart rate was bpm. the mean maximum intraoperative heart rate was . bpm ( % with tachycardia at the surgery). the mean immediate postoperative heart rate was . cpm. the mean heart rate minutes after the postoperative phase was . cpm. at the immediate preoperative phase % of surgeons had elevated bp level (usual normotensives) articles were randomly selected and the gender of the first and last authors determined. results: of the bariatric surgery publications reviewed, only % of first authors and . % of last authors were female surgeons. even though the proportion of female authors has increased over time, this is not proportional to the increase in the number of female surgeons or surgery residents (figure ). discussion: female surgeons are under-represented in bariatric surgery research. the number of female surgeons and residents has a continuous up trend over the last few decades our survey also included the validated quick-dash (disabilities of the arm, shoulder, and hand) questionnaire for upper-limb symptoms and the ability to perform certain physical activities. the quickdash is scored into two components: disability/symptom score, and the optional work module, which represent the impact of disability on daily activities and work responsibilities, respectively. both scores range from - , with a higher score indicating greater disability. surgeons were grouped according surgical focus (open, lap, or ra), and comparisons were made between groups. surveys with more than % of responses missing were excluded. statistical analysis were done using spss . , with α= . . results: completed surveys were evaluated (open: n= , lap: n= , ra: n= ). the survey response rate was %. . % of respondents were general surgeons, and mean age was ± . years. surgeons reported an average of ± . cases performed per month ra: . %, p= . ). likewise, there were no differences in the mean disability similarly, there was a positive correlation between mean work scores and reported pain in the upper-limb for lap and ra, both p. . conclusions: this nationwide survey revealed a similar prevalence of pain in the upper-limb among surgeons performing open, laparoscopic and robotic-assisted procedures. likewise, similar disability scores were reported between the three surgical groups. older surgeons performing laparoscopic and robotic-assisted approaches reported a higher impact of upper-limb problems interfering with their daily activities, unlike open surgeons. among all surgeons who reported pain in the upper-limb, laparoscopic and robotic surgeons were more likely to report that this pain interferes with their work activities an analysis of subjective and objective fatigue between laparoscopic and robotic surgical skills practice p d laparoscopic versus robotic gastrectomy for gastric cancer: comparisons of short-term surgical outcomes lin chen, xin guo patients who underwent d-lag (n= ) or rag (n= ) for gastric cancer were enrolled. the clinicopathological factors and short-term surgical outcomes were compared with retrospectively analysis. results: the clinicopathological factors between the two groups were well matched. postoperative recovery factors including the days of first flatus, days of eating liquid diet and hospital stay were similar. the rate of postoperative complications between the two groups were with no statistical differences in the subgroups of patients with total gastrectomy, d-lag had less blood loss and shorter operative time than rag (p= . and p. ), while for distal gastrectomy, blood loss and operative time showed no statistical differences. conclusions: this study suggests that d-lag is a novel and acceptable surgical technology in terms of surgical and oncological outcomes. d-lag is a promising approach for gastric cancer therapy methods: patients underwent robotic surgery between the beginning of to first half of in turkey were included. data were obtained from a prospectively maintained database. patient, surgeon and hospital identifiers were encrypted. parameters were operation type, operation year, robotic system used (s, si, xi), hospital volume and surgeon volume. high volume robotic colorectal hospital and surgeon was defined as the caseload within the forth interquartile ( th- th) based on the median value. results: there were colorectal procedures. surgeons performed robotic colorectal surgery at hospitals. ( . %) and ( . %) procedures were performed with the s-si and xi platforms respectively. hospitals have both of the si and xi platforms. hospitals are the si, hospitals are the xi hospital currently. the number of robotic colorectal operations increased gradually by years (figure ). the median numbers of colorectal procedures were (range - ) and (range - ) per hospital and per surgeon respectively among those hvrcs, the numbers of si and xi users were and respectively. the surgeons who performed more than procedures continued to use robot in their practice except one surgeon who stopped at . only left colectomies and no right colonic resection were performed before introduction of the xi platform first robotic cases and implementation of a robotics curriculum in a general surgery residency domenech asbun armonk ny) and utilized student's t test and chi-square. we also performed a linear regression analysis to determine the effect of or time, robotic surgery, and diagnosis on operating room costs and postoperative length of stay. results: laparoscopic and robotic cholecystectomies were performed. demographic parameters (age, gender, medical comorbidities, preoperative albumin and bmi, surgical history and smoking) were comparable. primary diagnosis was significantly different (chi-square . ), driven by more acute cholecystitis in the laparoscopic group. / robotic cases and / ( . %, p = . ) laparoscopic cases were converted to open ( for adhesions, for failure to progress, and for visualization of anatomy after adjusting for or time and diagnosis, robotic surgery was associated with a $ increase in costs robotic surgery is independently associated with increased or cost, but individual hospital systems must decide if this additional cost outweighs increased robot utilization and training benefits for physicians and staff robotic abdominal wall hernia repairs: technical considerations and lessons learned inguinal hernia repairs (ihrs) comprised the majority ( . %) of cases ( . % male, mean age . , mean bmi . ). there were unilateral ihrs with an average operative time of . ± . min and an average ebl of . ml. there were bilateral ihrs with an average operative time of . ± . min and average ebl of . ml. thirteen ihrs were combined with umbilical hernias and two with incisional hernias. average operative time for combined procedures was . min and average ebl was . ml. fifty-five incisional hernias were repaired robotically ( . % male, mean age . , mean bmi . ), four of which were retrorectus and two of those required transversus abdominis release. median hernia size was cm ( - cm). mean operative time was . ± . min and average ebl was . ml. twenty-three ventral/umbilical hernias were repaired robotically ( . % male, mean age . , mean bmi . , median size . cm ( - cm), mean operative time . ± . min, average ebl . ml). one spigelian hernia (operative time min, ebl ml) and one parastomal hernia (operative time min, ebl ml) were repaired robotically. there were no major complications and only groin seroma requiring percutaneous aspiration. nine patients required conclusion: this study demonstrates improved outcomes of robotic inguinal hernia repair compared to an open or laparoscopic approach. robotic hernia repair showed overall lower -day complication and readmission rates, and shortened los. while open approach had the highest rate of opiate use we retrospectively investigated consecutive overweight gc patients (bmi≥ ) underwent distal gastrectomy with d lymphadenectomy ( for rag and for lag) performed by two surgeons. the clinicopathological and surgical features were compared between groups. the cutoff point for initial phase (phase i) and stable phase (phase ii) were determined by cumulative sum (cusum) curve of operation time. results: generally, the surgical outcomes including postoperative complication rate, duration of postoperative hospital stay and lymph nodes harvest in the overweight patients have comparable results between rag and lag groups. the cutoff determining phase i and ii according to the cusum figure for rag group was and cases for surgeon a and b, respectively. and comparison analysis showed that the operation time of phase ii rag was significantly shorter robotic-assisted transabdominal preperitoneal inguinal herniorrhaphy: a single-center experience including perioperative morbidity and short-term outcomes patient factors, treatment factors, and outcome measures were collected in an attempt to gain insight and to generate ideas to potentially improve outcomes. results: there were no operative complications. six patients ( %) had failed gastric pacemaker placement prior to intervention. nine patients ( %) reported improvement in their symptoms and overall quality of life. four patients ( %) reported no improvement in symptoms and required additional intervention for symptom control and supportive care (one underwent roux-en-y gastric bypass, three underwent laparoscopic jejunostomy feeding tube placement to maintain nutrition). conclusion: robotic-assisted pyloroplasty is a safe option that improves symptoms and quality of life in % of our patients patients were matched into cohorts by procedure type. outcomes were analyzed using unpaired t-test and fisher's exact test. results: cost data was available for patients undergoing ras or la procedures. significant increases in equipment, labor, and overhead costs resulted with ras vs. la. variable-labor and variable-overhead costs were significantly higher in la procedures. higher supply costs and longer procedure time was seen with ras in all cohorts however, total -day costs were not significantly different in any group. conclusion: ras led to significant increases in fixed clinical, operative and pathologic factors were reviewed and analyzed. results: seventy patients underwent robotic surgery for rectal cancer during the study period. the locations of tumor were upper rectum, lower rectum. the procedure were as follow, high anterior resection in , low anterior resection in , isr in , apr in patients. eight patients underwent bilateral lymph nodes dissection (llnd). the procedures were performed successfully in all cases. mean age was . years, and % of the patients were men, and the mean body mass index was . (range, . - . ) kg/m . median operative duration was ( - ) minutes. median blood loss was ( - ) ml. median postoperative stay was ( - ) days. mean harvest lymph node number was . ( - ). surgical margins were negative in all cases. there was one conversion due to bleeding during the llnd and anastomotic leakage occurred in two patients. morbidity was %. there was no mortality postoperatively in this series. conclusion: in early series of the selected patients, this technique appears to be fesible and safe when performed by surgeons skilled in laparoscopic colorectal surgery the inactive electrode was placed touching small bowel to simulate accidental thermal injury. the bowel tissue at the site of temperature change was immediately resected and examined histologically for tissue injury. student t-tests were used for all comparisons with a p-value less than . considered statistically significant. results: comparison of the laparoscopic and robotic techniques are displayed in table . energy transfer was quantified using energy leak (per ma), which in these tests averaged . degree celsius change ( % ci . - . ) at the inactive electrode. surface temperature heated to a maximum of . degrees celsius, more in the robotic system than laparoscopy but still clinically negligible. pathology results from in vivo testing showed only thermal injury to the serosa without deeper mural injury. conclusions: stray energy transfer occurs in both laparoscopic and robotic surgery in amounts that are measurable but without clinical relevance. the average change in tissue temperature is less than degrees celsius laparoscopically and less than degrees robotically. while the robotic surgery appears to transfer more stray energy, no significant bowel injuries were caused in either group. p robot assistance can improve the performance of laparoscopic extensive concomitant adhesiolysis: results from a large observational study federico gheza outcomes compared were operative time, conversion rate, overall complications, gastrointestinal (gi) related complications (wound infection, abdominal abscess, anastomotic leak, ileus and small bowel obstruction), hospital length of stay, and -day re-admission rate. two sample t-test was used and p. was considered statistically significant. results: fifty-five robotic colectomies were matched with laparoscopic counterparts based on type of operation: right colectomy (n= ), sigmoidectomy (n= ), low anterior resection (n= ), proctocolectomy (n= ), transverse colectomy (n= ), abdominoperineal resection (n= ), and total abdominal colectomy (n= ) we assessed if technical obstacles of laparoscopic suturing were decreased and if laparoscopic skills overall were improved. surgical outcomes were compared relative to our historic values; we assessed procedure time and operating room efficiency, including set up and turn-over times. results: overall, the d/flexdex system permitted a greater improvement in working speed, superior optical visualization, and better suture handling compared to standard laparoscopy. all surgeries were completed without any complications. historically, we considered laparoscopic suturing to be complicated and inefficient. we relied on tacking devices for mesh fixation, suturing was previously completed with large cumbersome straight laparascopic devices. however, with flexdex and endoeye flex d, tacking devices have been eliminated and suturing technique improved. the mean total procedure times remained comparable for inguinal and hiatal hernia surgeries, and slightly longer for ventral hernias. operating room efficiency, including mean set up and turn-over times also remained unchanged. the acquisition cost for both the olympus endoeye flex d laparoscopic imaging system we performed a cost analysis which showed an average total cost of $ , for laparoscopic sleeve gastrectomy and an average of $ , for robotic assisted. the total reimbursements were $ , for laparoscopic sleeve gastrectomy and $ , for robot assisted. this translated to an average contribution margin of $ , for laparoscopic vs $ , for robot assisted. we analyzed these differences for bypasses as well. laparoscopic bypasses averaged minutes laparoscopically vs robotically. we found an average cost of laparoscopic $ , vs robot assisted $ , , with a contribution margin of $ , laparoscopic vs $ , robot assisted. conclusions: in our study we noted increased operative times with robot assisted operations, especially bypasses which could be explained by increased use of the robotic system for difficult cases such as revisional bypasses. the impact of cost is especially important in this financial climate, and judicious use of resources becomes important when determining surgical approach average or time for rih was minutes compared to lih which was minutes. average intraoperative cost for rih was $ , compared to lih which was $ . of note, one lih was converted to open, whereas none of the rih required conversion. average los was . hours for rih compared to . hours for lih. postoperative pain at one week follow up was the same between both groups. two postoperative surgical site occurrences (sso) occurred in the lih group ( groin seromas), whereas no ssos occurred in the rih group. eleven ventral hernia repairs were examined, were robotic (rvh) and were laparoscopic (lvh). average or time for rvh was minutes compared to minutes for lvh. average intraoperative cost for rvh was $ , compared to lvh which was $ , . no procedure from either group required conversion to open. average los was . hours for rvh, and . hours for lvh. again, postoperative pain was the same at one week follow up for both groups. there were no postoperative complications noted in either cohort. conclusion: operative time and procedural costs for rvh and rih repairs were shown to be longer and more expensive when compared to their laparoscopic counterparts. however, with increased operative experience using the robotic platform, surgical time did show a decreasing trend does robotic system have advantages over laparoscopic system for distal pancreatectomy? results: a total of consecutive patients underwent minimally invasive distal pancreatectomy (ldp n= ; ra-ldp n= ). most common pathologic finding was pancreatic ductal adenocarcinomas ( cases). there was no in-hospital mortality or cases of conversion to open surgery in this study. spleen-preserving approach was performed more often in the ra-ldp ( %) than in the ldp ( . %) groups (p= . ) both groups showed no significant differences in the total number of lymph nodes, number of positive lymph nodes, tumor differentiation, tumor stage, and resection margins. conclusions: ra-ldp is a safe and feasible approach that has an advantage of performing spleenpreserving distal pancreatectomy, with perioperative and short-term oncologic outcomes comparable to those of ldp. p robot-assisted alpps technique mike fruscione right portal vein embolization was not feasible secondary to the proximity and size of the right hemi-liver tumor burden relative to the right portal vein. the pre-operative planned procedure was a right trisectionectomy and microwave ablation of the segment lesion. results: using the da vinci xi surgical system (intuitive surgical, inc.) the right portal vein was dissected, doubly-ligated, and divided. the liver parenchyma was split from the inferior edge to the dome mm medial to the falciform ligament and down to the middle hepatic vein which was preserved to maintain adequate venous outflow. the patient was discharged home on post-operative day two. on post-operative day six, ct volumetrics demonstrated a flr of %. on post-operative day seven, a second stage alpps procedure was performed where the right hepatic artery, middle and right hepatic veins and right hepatic duct were ligated and divided. segments a/b, , , and were removed. the patient was discharged home on post-operative day five they were asked to answer demographic questions and rate their comfort level ( =not comfortable, =very comfortable) with aspects of robotic surgery. paired t-tests and wilcoxon tests were used to assess whether there were changes in comfort level before and after labs, and chi-square goodness of fit tests were used to assess whether dry lab (using inanimate objects), wet lab (using a porcine model), or simulator modules were thought to be most helpful in obtaining specific robotic skills. results: the survey response rate was % (n= ). ninety-one percent of residents felt that robotic surgery is not intuitive. prior to simulation, % of residents felt inadequately prepared to safely operate on the robotic console. following simulation, % felt better prepared and more confident to participate in robotic surgery for the first patients whom we treated (the first-stage group), we invited a visiting expert from a high-volume center to perform the procedure jointly with our hospital's surgeons by using a dual console. for the subsequent patients (the second-stage group), the procedure was performed by our hospital staff alone. in this report, we describe our experience of introduction of robotassisted colectomy and discuss issues for the future. patients and methods: the operative procedure was sigmoid colectomy, low anterior resection, and intersphincteric resection. the median number of lymph nodes dissected was . . the mean operating time was minutes for the first-stage group and minutes for the second-stage group. the median console time was minutes for the first-stage group and minutes for the second-stage group, with no significant differences between the two groups. the mean operating time other than console time was minutes for the first-stage group and minutes for the second-stage group, significantly longer in the latter group. the mean amount of hemorrhage was . g in the first-stage group and g in the second-stage group. no significant differences were found between the two groups in the mean length of postoperative hospital stay. none of the patients in either group developed a complication of clavien-dindo grade iii or higher. conclusions: the use of dual console system was particularly useful for the introduction of robotassisted surgery in our hospital. for the patients whom we treated, we found almost no difference in console time between the first-and second-stage groups. the high-quality instruction received via the dual console was considered to have had a beneficial effect on the operators' learning curve. however, the operations that were set up other than console time, such as roll-in and docking, took significantly longer in the second-stage group when the proctor was not present select specimens from each trial were immediately resected and evaluated for histologic thermal injury. experiments were repeated times based to detect an expected difference of five degrees. student t-tests were used for all comparisons with significance set at . . results: stray energy transfer was higher in the single incision setup compared to the traditional setup (figure ). stray energy in the assistant grasper caused . ± . °c of temperature change in the standard configuration, and . ± . °c in the single incision configuration (p= . ). doubling energy output to w amplified the same finding robotic single-site cholecystectomy of cases: surgical outcomes and comparing with laparoscopic single-site procedure jae hoon lee incisional hernia occurred one case in each group. rssc is safe and feasible procedures. with accumulating of experience, rssc had more short operative time than sslc. comparing to sslc, rssc is relatively suitable to acute gallbladder disease and high bmi and requires a minimal learning curve to transition from traditional multiport to single-port robotic cholecystectomy. p initial experience using da vinci xi robot in colorectal surgery anna r spivak, do, john marks, md; lankenau medical center introduction: the xi robot has been developed to facilitate multiquadrant abdominal surgery. this report presents initial experience to evaluate feasibility and safety of xi robot in colorectal surgery. methods: all cases performed on xi robot were prospectively entered into a robotic database that was queried for colorectal cases performed from intraoperative complications were encountered in cases ( . %), requiring conversion to laparoscopy. none were converted to open. mean length of largest incision . cm. median ebl ml. there was no mortality. there were ( . %) immediate postoperative morbidities: postoperative abscess, bowel perforation, two postoperative bleeds, two hernias, two hematomas, smv thrombosis, small bowel obstruction. perioperative blood transfusions were required in . % of cases. there was one anastomotic leak. median time from surgery to low residue diet and discharge was days. conclusion: initial experience shows robotic colorectal resection with da vinci xi learning curve for robotic sleeve gastrectomy and roux-en-y gastric bypass: achieving equivalence to laparoscopy residents and fellows participated in an analogous fashion in both arms of the study, and patients undergoing re-operative bariatric surgery were excluded. results: a total of patients undergoing rsg (n= ) or rrygb (n= ) were included. for the overall robotic cohort, median age was (range - ), % were american society of anesthesiologists (asa) score , % were asa score , and mean body mass index (bmi) was ± with no differences between procedures. there were no conversions to open. there was one patient with portal vein thrombosis after rsg which occurred in the th rsg and one patient who underwent re-operation in the immediate post-operative period for hemorrhage at the gastro-jejunal anastomosis in the rrygb group; this occurred in the th rrygb. there were no leaks, strictures, or mortalities in either group. mean length of stay was days± for rsg with no difference based on number of procedures performed. in the rrygb group, los decreased after the first five procedures from days± to days±(p= . ). for both procedures, operative time decreased by number of procedures performed (figure). equivalence to lsg in operative time ( minutes± ) was reached after eight robotic procedures were included. the da-vinci xi® was used for the operations. age, gender, body mass index (bmi), asa score, indication for surgery, urgency of procedure, type of procedure, docking number, operation time, estimated blood loss, complications, short (≤ days) and long term ([ days) complications were evaluated. results: patients ( females) were included. median age was . median bmi was , median asa score was . total and completion rrp-ipaa were performed for and patients respectively. the indications were as follows: medical refractory uc (n= ), cancer/dysplasia (n= ), fulminant colitis (n= ), toxic megacolon (n= ), medical treatment resulting in growth retardation (n= ), medical treatment refractory bleeding (n= ). patient with toxic megacolon had an emergent operation. the median docking number was and for completion and total rrp-ipaa respectively. median operative time was minutes. median blood loss was ml. all patients had a stapled ileal j pouch anal anastomosis. all patients had a diverting loop ileostomy at the time of ipaa creation. no intraoperative complications were observed. no conversion to open surgery was needed. the median time to flatus was day. the median time to oral intake was day. patient had a laparotomy on postoperative day due to intra-abdominal bleeding. patient had a bleeding from ileostomy which was treated endoscopically. superficial surgical site infection was observed in patients. patient had a pouchitis managed with oral antibiotics. patient had an ileus responded to conservative treatment. patient had a per-anal bleeding stopped spontaneously. patient had a urinary tract infection responded to antibiotics. patients had pouchitis, patient had a perianal fistula requiring a loop ileostomy and a parastomal hernia was developed in another patient in long term follow up ) were significantly different between the two groups. , pairs undergoing primary and pairs undergoing revisional procedures were successfully matched. robotic gastric bypass was associated with a significantly longer operation length than laparoscopic gastric bypass for both primary (median difference minutes, p. ) and revisional (median difference minutes, p. ) procedures overall, there were no significant differences in anastomotic/staple line leak, -day readmission, reoperation, re-intervention, total event, and mortality rates between matched cohorts. conclusion: when controlling for patient characteristics, those undergoing primary and revisional lrygb and rrygb had no difference in early morbidity. despite the prolonged operative duration, the robotic approach was not associated with any clinical benefit or increased complications for primary or revisional gastric bypass surgery preoperative risk factors were collected. we focused on perioperative outcomes and in hospital complication rate. results: thirty-three patients underwent robot assisted giant hiatal hernia repair at our institution. patients ( %) were years and older and patients ( %) had a bmi higher then. there were no significant differences in patient characteristics between the groups. no patient underwent conversion to open or standard laparoscopy. no mortality was observed and no transfusions were needed. four patients ( %) had a complication, two of them were older than years old. three of the four patient ( %) that had a complication were obese. there were no statistical differences in mortality % and . % of them were with s-si and xi platforms respectively. the median numbers of procedures were (range - ) and (range - ) cases per hospital and per general surgeon respectively. the high volume surgeons (higher than th percentile) performed ( %) of the cases. the xi platform has been the main tool for colorectal surgery only (figure ). conclusions: while xi platform significantly increased caseload in general surgery by facilitating performance of colorectal surgery, its preference in other general surgical fields is not superior to si laparoscopic inguinal hernia repair (tapp) -first experience with the new senhance robotic system robin schmitz ; intuitive surgical inc, loma linda university medical center introduction: crohn's disease is an incurable inflammatory disorder that can affect the entire gastrointestinal tract. while medical management is considered first-line treatment, approximately % of patients with crohn's disease require surgery within years of their initial diagnosis. traditionally, surgery has been performed via an open approach with poor adoption of minimally invasive technique. the aim of this study is to demonstrate the feasibility of robotic-assisted approach as a minimally invasive option for surgical management of crohn's disease and compare the perioperative outcomes with traditional laparotomy. methods: patients who underwent elective resection of the intestine for crohn's disease by roboticassisted or laparotomy approach from to q were identified using icd- codes from premier healthcare database. all the procedures were performed by either general surgeons or colorectal surgeons. since hospital characteristics were comparable between the two cohorts before propensity-score matching, : matching was performed using patient characteristics such as age, gender, race, charlson index score and year of the surgery to create comparable cohorts. sample selection and creation of analytic variables were performed using instant health data (ihd) platform (bhe methods: we conducted a retrospective analysis of , mis inguinal hernia repairs ( , robotic, , laparoscopic) from through with data collected in the premier hospital database. patient, surgeon, and hospital demographics of robotic and laparoscopic inguinal hernia repairs were compared. the adjusted odds ratio of receiving a robotic procedure was calculated for each of the demographic factors using a multivariable logistic regression model. statistical significance was defined as p. . sas software version . was used for statistical analysis. results: the odds of a procedure being robotic increased from inguinal hernia repair is one of the most common general surgery procedures with over , performed annually in the united states. when compared to traditional open inguinal hernia repair (oihr), laparoscopic inguinal hernia repair (lihr) has been associated with faster postoperative recovery rates and lower postoperative pain. with advances in the robotic platform, robotic inguinal hernia repair (rihr) is an available technique that is currently being explored. this study examines lihr and rihr as described in literature to see if one is superior to the other. study design: search terms: ''inguinal hernia repair surgical complications including hematomas ( . %), seromas ( . %), and trocar site infection ( . %) resolved with antibiotics, with a . % postoperative complication rate. conclusion: rihr repair is a safe alternative to lihr, with fewer postoperative complications and a faster recovery time. however, operative time as well as or room time is significantly longer, which may increase overall cost laparoscopic or robotic approach were chosen on a schedule availability basis. data was collected prospectively and it involved anthropometric data, presence of type diabetes mellitus (t dm), % of preoperative total weight loss (%ptwl), surgical time, postoperative length of stay, -day complications, and need for readmission or reoperation. comparison between groups was carried on with t-test for continuous data and with chi-square test for dichotomous variables. a p lower than . was considered significant. results: overall sagb were performed, laparoscopic and robotic. a long and thin gastric pouch was created calibrated by a fr bougie and a . cm antecolic antegastric gastrojejunal (gj) anastomosis was groups (laparoscopic vs robotic) were comparable regarding age ( vs . years, p= . ), bmi ( . vs kg/m , p= . ), %ptwl ( . vs . %, p= . ) and % with t dm ( vs there were fewer men in the laparoscopic group ( . vs % there were ( . %) major complications in the laparoscopic group: bleedings from the gj anastomosis, one of which required reoperation, severe dumping syndrome, gerd requiring revision and gj stricture that underwent relaparoscopy. the only complication ( %) in the robotic group was an acute pancreatitis. readmission rate was % in both groups and reoperation rate was % for laparoscopic and % for robotic surgeries. conclusions: totally robotic sagb with manual gastro jejunal anastomosis was safe and feasible in this early experience compared to laparoscopic approach multi degrees of freedom manipulator with mantle tube for assisting endoscopic and laparoscopic surgical operations masataka nakabayashi, phd , yuta hoshito, masters student p step by step anatomic mapping during laparoscopic transabdominal adrenalectomy lateral flank approach ranbir singh steps analyzed were: right adrenalectomy: step ) mobilize liver; ) medial dissection; ) adrenal vein isolation; ) inferior dissection; ) adrenal off kidney; ) detachment. left adrenalectomy: step ) division splenorenal ligament; ) develop plane pancreas/kidney; ) mobilization medial/lateral borders adrenal; ) adrenal vein isolatoin; ) dissection adrenal off kidney; ) detachment. structures were identified as yes/no and results expressed as percentage total n of cases seen at each step. results: structures identified at each step are shown (table) incisions were made at the oral vestibule under the inferior lip. a -mm trocar was inserted through the center of the oral vestibule with two -mm trocars above incisors. the subplatysmal space was created down to the sternal notch, and carbon dioxide was insufflated at pressure mmhg to maintain the working space. parathyroidectomy was performed using laparoscopic instruments. intraoperative parathromone levels were measured minutes after excision of gland. primary end-points were the success rate in achieving the cure from hyperparathyroid state and hypocalcemia rate. secondary end-points were operating time, scar length, pain intensity assessed by the visual-analogue scale, analgesia request rate, analgesic consumption, quality of life within postoperative days (sf- ), cosmetic satisfaction, duration of postoperative hospitalization, and cost-effectiveness analysis. result: one patient experienced a transient recurrent laryngeal nerve palsy which was spontaneously resolved within month. no permanent recurrent laryngeal nerve injury was found no mental nerve injury or infection was found. conclusion: with highly sensitive localising sestamibi and ct scans, focussed exploration is the current standard of treatment. among all minimally invasive surgeries, toepva is a feasible, safe, and almost pain-free surgical option when combined with intraoperative parathormone monitoring for patients with hyperparathyroidism indocyanine green is a water soluble nontoxic compound exhibiting near infrared renal function and long-term survival. indocyanine fluorescence helps in assessing vascular flow, tissue perfusion and aberrant anatomy and thereby leads to lower conversion rates in partial nephrectomy. we aim to present our experience in patients who underwent partial nephrectomy over years. materials and methods: of the partial nephrectomies performed at our institution, were done by laparoscopic approach alone and rest by patients who underwent llr for whole hepatoma in our facility, underwent llr for a solitary hepatoma and were divided into "before standardization" (bs; n= ) and "after standardization" (as) groups (n= ). patient background, characteristics, and perioperative outcomes were compared between these groups. procedure: we chose the devices according to phases of liver transection. a soft-coagulation monopolar device was used for marking surface. an ultrasonically activated device was used for transection of the liver surface within a -cm depth. crash and sealing with biclamp were indicated for deep-phase transection. the cavitron ultrasonic surgical aspirator was used if the lesion was close to the major glisson's sheath or the major hepatic vein. results: no significant differences in the patients' background were found between the two groups. the operative durations were min ( - min) and min ( - min) in the as and bs groups, respectively, with a significant difference (p. ). the blood loss volumes were cc ( - cc) and cc ( - cc), respectively (p= . ). the lengths of hospital stay after llr were days (range, - days) and days ( - days), respectively, with a significant difference iwao kitazono, phd , kentaro gejima , hizuru kumemura , akira hiwatashi , yuichiro nasu , fumisato sasaki , akio ido , yutaka imoto ; cardiovascular and gastroenterological surgery, kagoshima university graduate school of medical and dental science, digestive and lifestyle disease, kagoshima university graduate school of medical and dental science introduction: in locally-treatable gastrointestinal tumors, laparoscopic endoscopic cooperative surgery (lecs) is a minimally-invasive technique that can avoid excessive resection of the gastrointestinal tract. objective: to share our therapeutic guidelines and surgical technique of lecs for gastroduodenal tumors. subjects: nineteen patients who underwent lecs for gastroduodenal tumors ( patients with gastric tumor and patients with duodenal tumor).[results] ) gastric tumors ( gist, glomus): . site of lesion was u ( patients), m ( ), or l ( ), . operative procedure was acquired in a stepwise manner from classical lecs ( patients) to inverted lecs ( ) to non-exposed endoscopic wall-inversion surgery: news ( ). . operative outcome revealed no postoperative complications. ) duodenal tumors ( adenoma, m cancer, ectopic pancreas): . site of lesion was bulbus duodeni ( patient), superior part ( ), or descending part ( ); . operative procedure was esd followed by laparoscopic continuous suture in a single seromuscular layer for patients with preoperatively confirmed or suspected cancer, or full-thickness resection followed by albert-lembert suture along the short axis for patients unable to undergo esd. in all cases, c-tube was placed to prevent bleeding and perforation at the site of resection due to exposure to bile; . operative outcome included successful endoscopic hemostasis upon bleeding from exposed vessel on postoperative day in patient and anastomotic leak in patient. the event of anastomotic leak resolved after days of bile drainage through c-tube and conservative therapy. compared with patients who underwent esd alone, those who underwent lecs had significantly larger diameters of resected specimens and tumors (p. ) but no significant difference in the incidence of postoperative bleeding and delayed perforation. conclusion: for gastroduodenal tumors, lecs is a minimally-invasive and safe therapeutic option as it combines advantages of both laparoscopy and endoscopy. in particular, c-tube placement for bile drainage was effective in reducing exposure of the suture site to bile as well as supporting drainage after anastomotic leak. introduction: in japan, transurethral balloon catheters (tuc) are currently inserted in most surgical patients to maintain a urine outflow route and to measure the urine output both intraoperatively and postoperatively. however, tuc insertion not only causes postoperative pain but can also lead to urinary tract infections. temporary suprapubic catheters (spc) are used in the field of obstetrics and gynecology as a method of postoperative management to avoid performing transurethral procedures. in the field of surgery, especially in laparoscopic surgery, spc also considered how it would be a useful way to reduce patient suffering. here we report our prospective study on whether an spc can be safely inserted as a substitute for tuc during laparoscopic-assisted colectomy. subjects and methods: the subjects in this study were patients who underwent laparoscopic surgery for primary colorectal cancer from to , and who would normally have had their urinary balloon catheter removed early after surgery. during surgery, an angiomed cystostomy set was installed for patients who gave their consent to participate in this study as an alternative to a urinary balloon catheter. we prospectively collected patient information including sex and age, in addition to other perioperative data, such as, time required for cystostomy, complications accompanying cystostomy, sense of discomfort or pain associated with the vesical fistula after surgery, the time of the removal of the vesical fistula, the frequency of releasing the vesical fistula, postoperative complications. results: our subjects included cases who gave their informed consent to have an spc inserted. an spc was inserted into the remaining case. the mean surgical duration was min, and the spc insertion was performed at a mean of min after the start of surgery. insertion required a mean duration of . s. the bladder of one case ( . %) was perforated, and hematuria was observed at the time of insertion in two cases ( . %), but surgery completed without any incident. six out of cases ( . %) demonstrated neither urinary urgency nor independent urination on the day the catheter was clamped. however, the clamp was released two to four times, and draining of an average of ml urine, urinary urgency, and independent urination were confirmed - days later. conclusion: spc is a procedure that avoids crossing the urethra and its associated disadvantages. here we were able to demonstrate that the procedure can be safely used in laparoscopic surgery patients.our objective is to devise methods for proper port placement to overcome the ergonomic challenges. procedure: patients with sit were operated laparoscopically in our hospital in the period of may to november , males suffering from cholelithiasis without cholecystitis and female with acute appendicitis. after thorough review of literature and proper planning, the patients were posted for surgery. for laparoscopic appendectomy, a thorough initial diagnostic survey is performed on introducing a scope through the umbilical port and confirming the exact location of the appendix. the two working ports are introduced accordingly, which is usually a mirror image of the standard port sites. the appendix was visualised in the left iliac fossa and after meticulous dissection, the appendix and mesoappendix were divided using an endostapler. the operative time was minutes and there were no intraoperative or postoperative complications.the port placement for laparoscopic cholecystectomy in such a case is trickier as the anatomical variation and the contralateral disposition of the biliary tree demand an accurate dissection and exposure of the biliary structures to avoid iatrogenic injuries. it is important to conform to the principles of triangulation during port placement. the mirror image of -port placement is convenient for left-handed surgeons. whereas, to make the procedure comfortable for right-handed surgeons, the working ports need to be shifted caudally with the surgeon standing between the patient's legs. the mean operative time was minutes and there were no minor or major intraoperative or postoperative complications.conclusion: ergonomic comfort is vital to a smooth procedure. while mirroring ports suffices for appendectomy, all other procedures require forethought for port placement. it should be noted that ambidexterity is a desirable skill in the operating room for a laparoscopic surgeon.priscila r armijo, md, chun-kai huang, phd, gurteshwar rana, md, dmitry oleynikov, md, ka-chun siu, phd; university of nebraska medical center introduction: the aim of this study was to determine how objectively-measured and self-reported fatigue of the upper-limb differ between laparoscopic and robotic surgical training environments. methods: surgeons at the sages conference learning center, and at our institution were enrolled. two surgical skills practical environments were utilized: ) a laparoscopic training-box environment (fls) and ) the mimic® dv-trainer (mimic). two standardized surgical tasks were chosen for both environments: peg transfer, and needle passing. each task was performed twice. objective fatigue was evaluated by muscle activation and fatigue, and comparisons were made between fls and mimic, for each surgical task. muscle activation of the upper trapezius, anterior deltoid, flexor carpi radialis, and extensor digitorum were recorded during practice using surface electromyography (emg; trignotm, delsys, inc., boston, ma). the maximal voluntary contraction (mvc) was obtained to normalize muscle effort as %mvc. the median frequency (mdf) was calculated to assess muscle fatigue. subjective fatigue was self-reported by completing the validated -scale score piper fatigue scale- (pfh- ) before and after practice. statistical analysis was done using spss v . , with α= . . results: this abstract represented the performance of trainees (fls: n= , mimic: n= ) as part of larger cohort of the study. for peg transfer, emg analysis revealed that mimic had a significant increase in mean muscle activation for the upper trapezius and anterior deltoid, both p\ . . conversely, practice with fls led to significantly more muscle fatigue than mimic for the same muscle groups (upper trapezius: p= . , anterior deltoid: p= . ), represented by a significantly lower mdf. similarly, for needle passing, mimic had a significant increase in mean muscle activation for the upper trapezius (p= . ) and anterior deltoid (p= . ), but practice with fls significantly induced more muscle fatigue effort for anterior deltoid (p= . ). survey analysis revealed a significant decrease in self-reported fatigue after performing fls tasks (before: . ± . , after: . ± . , p= . ), but no difference after mimic tasks (before: . ± . , after: . ± . , p= . ). conclusions: although different muscle groups are preferentially required in the performance of fls and mimic, our analysis for both surgical tasks showed practice with mimic required more activation of shoulder muscles, whereas practice with fls could lead more muscle fatigue for the same muscle groups. interestingly, surgeons reported improved or no change in perceived fatigue after the tasks, despite of having an increase in muscular activation and effort. subjective selfreport fatigue might not truly reflect the level of fatigue when trainees practice surgical tasks using fls or mimic. objective: to investigate the prevalence of musculoskeletal (msk) injuries in bariatric surgeons around the world. background: as the popularity of bariatric surgery increases, efforts into improving its patient safety and decreasing its invasiveness have also been on the rise. however, with this shift towards minimal invasiveness, surgeon ergonomic constraints have been imposed, with a recent report showing a - % prevalence of physical complaints in surgeons performing laparoscopic surgeries. methods: a web-based survey was designed and sent out to bariatric surgeons around the world. participants were queried about professional background, primary practice setting, and various issues related to bariatric surgeries and msk injuries. results: there were responses returned from surgeons from countries around the world. . % of the surgeons have had more than years of experience in laparoscopic surgery, . % in open and . % in robotic surgery. % of participants reported that they have experienced some level of discomfort/pain attributed to surgical reasons, causing the case load to decrease in . % of the surgeons. it was seen that the back was the most affected area in those performing open surgery, while shoulders and back were equally as affected in those performing laparoscopic, and the neck for those performing robotic, with . % of the surgeons reporting that this pain has affected their task accuracy/surgical performance. a higher percentage of females than males reported pain in the neck, back and shoulder area when performing laparoscopic procedures. supine positioning of patients evoked more discomfort in the wrists, while the french position caused more discomfort in the back region. only . % sought medical treatment for their msk problem, of which . % had to undergo surgery for their issue, and . % of those felt that the treatment resolved their problem. conclusion: msk injuries and pain are a common occurrence among the population of bariatric surgeons, and has the ability to hinder performance at work. therefore, it is of importance to investigate ways in which to improve ergonomics for these surgeons as to improve quality of life.introduction: the use of robotic technology is rapidly increasing among general surgeons but is not being routinely taught in general surgery residency. we aimed to evaluate our first robotic cases during which time we developed a robotic surgery curriculum incorporating residents. methods: the first robotic cases performed at our institution from - by two surgeons were analyzed. a residency curriculum was developed and instituted after the first months. it consisted of online modules offered by intuitive surgical resulting in certification, simulator training, hands on workshops for cannula placement, docking, instrument exchange, camera clutching and other introductory tasks. patient demographics, type of procedure, resident involvement, total operative and console times, comorbid conditions and complications were evaluated. unpaired t tests were performed for statistical analysis. results: females and males comprised this series with an average age of years ± . the majority of patients, % had comorbidities, with a predominance of hypertension, % and diabetes, %. the bariatric patients had an average bmi of ± . a variety of procedures were performed including hernias, foregut and bariatric. residents participated in % of cases. there were no differences in total operative and console times in cases with residents except bariatric procedures. there were complications in this series; postoperative ileus, gallbladder fossa hematoma and an enterotomy. there was one early conversion to open in a complex foregut case and no deaths in this series.conclusions: we report our initial experience of robotics in a variety of general surgery and complex foregut cases. the implementation of a robotic surgery program and residency curriculum was safe with similar outcomes related to operative times and complications. as mis expands with the application of robotics in general surgery, residency curriculums will need to be revised. further data is needed to determine residency learning curves between robotics and laparoscopy.background: robotic surgery has made a large impact in the fields of urology and gynecology. its use is significantly increasing in the fields of general and bariatric surgery. evidence remains unclear as to the clinical impact on outcomes, and significant questions remain as to the impact of cost. our goal was to evaluate the economic impact of robotic surgeries in general and bariatric surgery at our institution. methods: this study is a retrospective analysis of minimally invasive general and bariatric procedures done at a single institution from january through june . we performed a cost and reimbursement analysis of robotic versus conventional laparoscopic surgery. the cost evaluation included operative time, operating room costs, length of stay and overall hospital expenses. in addition, we looked at reimbursement and the contribution margin per cpt code. results: our study included a total of patients who underwent laparoscopic and robot assisted general and bariatric surgeries. the average time duration for laparoscopic surgeries was minutes vs minutes for robot assisted. we performed a cost analysis which showed an average total cost of $ , for laparoscopic and an average of $ , for robot assisted. the total reimbursements were $ , for laparoscopic and $ , for robot assisted. this translated to an average contribution margin of $ , for laparoscopic vs $ , for robot assisted. for general surgery we found an average cost of laparoscopic $ , vs robot assisted $ , , with a contribution margin of $ , laparoscopic vs $ , robot assisted. for bariatric surgeries we found an average contribution margin of $ , for laparoscopic vs $ , for robot assisted. conclusions: robotic surgery has been associated with higher costs and longer operative times. in this economic climate of increased cost awareness with institutions under increasing financial pressures, judicious use of resources becomes important when determining surgical approach. although cost of robot assisted surgery may decrease with time, other quality factors may be important in patient selection. although there is no clear evidence that institutions lose money with robot assisted surgery, in our experience the contribution margin is lower with robot assisted surgery as compared to conventional laparoscopy.introduction: this retrospective study was performed to evaluate the safety and feasibility of the new senhance robotic system (transenterix) for inguinal hernia repairs using the transabdominal preperitoneal approach. our series is the first experience in the field of general surgery utilizing this new robotic platform. methods: from march to september , inguinal hernia repairs in patients were performed using the senhance robotic system. the senhance surgical system is a new robotic platform that consists of a cockpit, manipulator arm and a connection node (figure ). this new system provides robotic surgery with numerous advantages including eye-tracking camera control system, haptic feedback, reusable endoscopic instruments, and a high configuration versatility due to total independency of the manipulator arms. patients were between and years of age, eligible for a laparoscopic procedure with general anesthesia, had no life-threatening disease with a life-expectancy of less than month and a bmi \ . a retrospective chart review was performed for a variety of pre-, peri-and postoperative data including but not limited to patient demographics, hernia characteristics, intraoperative and postoperative complications. results: male and female patients were included in the study. median age was . years (range - years), and median bmi was . (range . - . kg/m ). median docking time was minutes (range - minutes), and median operative time was minutes (range - minutes). two cases were converted to standard laparoscopic surgery due to robot malfunction and intraoperative bleeding respectively. one patient developed a postop seroma that did not require any further intervention. conclusion: we report the first series of laparoscopic inguinal hernia repairs using the new senhance robotic system. compared to previously published conventional laparoscopic or robotic tapp hernia repairs these data suggest similar outcomes in operative time and perioperative complications. additionally there was no significant learning curve detected due to its intuitive applicability. therefor the senhance robotic system can be safely and easily used for tapp hernia repairs by experienced laparoscopic surgeons. this is a video presentation of years old female, who presented with suprapubic pain and mass to gynecology office. she has a history of robotic hysterectomy and sbladder sling operation years ago. this was complicated with peritonitis and long icu stay, due to what she was called ''bowel injury'' but treated only conservatively with antibiotics and subsequent abscess drainages at that time. she has occasional appearing nodule and pain at the left suprapubic region. ct ordered by gynecology read as abdominal wall hernia with long sigmoid diverticuli in hernia. also there was small amount of subcutaneous air at the tip of herniated diverticuli. after antibiotic treatment and improvement, colonoscopy shows, actually the diverticuli is the limb of the sling going through the simoid and anchored in subcutaneous fat on abdominal wall ahich represents clocutaneous fistula as gets infected. clip was placed on sling and repeat imaging comfirmed that the localion of this sling fits to location of so called ''hernia'' the sling limb was resected robotically and colon was repaired with side stapling of clolonic wall. the abdominal wall defect is repaired with long term absorbable suture. as far as we have found, the presentation and treatment of this complication is unique and could not find a similar case to guide us for the plan. background: robot-assisted surgery using da vinci surgical system (dvss) is thought to have many advantages over conventional laparoscopic surgery. it was reported that the use of the surgical robot might reduce surgery-related complications, then a multi-institutional historically controlled prospective cohort study on the feasibility, safety, effectiveness and economical efficiency of robotic gastrectomy (rg) for resectable gastric cancer was conducted in japan. this study evaluated the safety of rg using dvss xi. methods: this single-center, prospective phase ii study included patients with resectable gastric cancer (umin ). the primary endpoint was the incidence of post-operative complications greater than grade iii according to clavien-dindo classification during one month after surgery. the secondary endpoints included all adverse events and completion rate of robotic surgery. results: from oct to jan , patients were enrolled for this study. the incidence of post-operative complication greater than grade iii was %. the overall incidence of adverse events was . % (grade i; . %, grade ii; . %). no patient required conversion to laparoscopic or open surgery; thus, the rg completion rate was %. conclusion: this study suggested the introduction of rg using dvss xi for gastric cancer seems to be safe and feasible. priscila r armijo, md , dmitry oleynikov, md , sages robotic task force* ; university of nebraska medical center, sages robotic task force introduction: while robotic companies continue to aggressively market and promote the use of robots in general surgery, little is known about how this technology is employed by general surgeons, and what is expected of this technology from both novice and experts in the field. the aim of this study is to evaluate the needs of general surgeons who are new to robotic surgery and the needs of established robotic surgeons. methods: the sages robotic task force survey, a one-page survey, was designed and sent electronically to all sages members. questions regarding fellowship training, area of expertise, robotic simulation and in clinical case use, services offered in the current hospital, mentorship, likelihood of switching to a different approach, and expectations for the robot were included in the survey. two groups were created based on previous use of davinci® system in a clinical scenario, or not. statistical analysis was conducted using ibm spss v. . . , using fischer's exact and pearson's chi-squared tests where appropriate. results: sages members answered the survey. surprisingly, respondents ( %) had used the davinci® in a clinical setting. among these, ( %) had additional fellowship training, compared to ( %) in the non-clinical use group, p= . . of all surgeons with additional fellowship training, the great majority ( %) had specialization in advanced gi, mis and bariatric surgery, followed by colorectal ( %). most surgeons are performing less than cases per month using the robotic system, and with the majority of cases performed using the platform being hernia repairs ( %), followed by foregut-related procedures ( %). interestingly, from all the surgeons who replied the survey, only . % are planning to switch from open procedures to its robot counterpart, whereas . % are planning to adopt robotic-assisted procedures rather than laparoscopy. conclusions: the majority of sages members who responded to the survey have used the davinci® in a clinical setting in the past. surgeons who stated they perform mainly laparoscopic procedures were likely to continue to adopt robotic techniques, whereas those who perform open hernia repair for example were not very likely to switch to robotic approach. while the use of the robot may be enabling surgeons who used to perform mostly open procedures in the urology or gynecology fields, laparoscopic skills predict robotic utilization in general surgery. hernia and foregut appear to be the most common procedures that are being utilized.aim: while conventional multiport laparoscopic splenectomy has become gold standard for some hematological or splenic diseases, reduced-port laparoscopic splenectomy (rpls) including singleincision laparoscopic splenectomy (sils) is regarded as highly challenging. herein, we describe the technical refinements for safe rpls especially for patient with splenomegaly. methods: in all cases, access was achieved via a . -cm mini-laparotomy at the umbilicus into which a sils tm port or e-z access ® with three -mm trocars was placed. a -mm flexible scope, an articulating grasper, and straight instruments were used. our rpls is characterized by the followings: a) early ligation of the splenic artery to shrink the spleen, b) application of our original "tug exposure technique," which provides good exposure of the splenic hilum by retracting (tugging) the spleen with a cloth tape, and c) safe introduction of stapler under the guidance with a flat drain into the splenic hilum. results: rpls patients ( men and women, ± years old) comprised hematological disorder (n= ), splenic disease (n= ), and liver cirrhosis (n= ). in patients ( %), rpls was successfully completed: sils in and sils plus one additional port only in patients. conversion to open surgery was necessary in patients including liver cirrhosis with remarkable collateral varicose veins around the spleen. operation time and blood loss were ± min and ± g, respectively. weight of the extracted spleen was heavier than normal and ± g (maximum g). no intra-or postoperative complication occurred. the postoperative scar was nearly invisible. conclusions: rpls might safely be performed even for splenomegaly (up to , g). however, care should be taken for cirrhotic patient with collateral veins. rpls can be the procedure of choice even in the patients with splenomegaly and who are concerned about postoperative cosmesis. the aim of this feasibility study was to evaluate laparoscopic sn biopsy for laparoscopic snns in early gastric cancer patients. subjects and methods: this study includes patients with ct n m (primary tumor \ cm) gastric cancer who underwent laparoscopic sn biopsy in conjunction with radioisotope and dye methods between jan. and jul. . first, we looked for green-dyed sns after injection of indocyanine green (icg) without near-infrared light system, and then tried to detect the radioactivity of sns using a hand-held gamma probe inserted through a small incision at the umbilical port. after the areas where sns were distributed were resected, a gastrectomy with prophylactic lymphadenectomy was performed according to the gastric cancer treatment guidelines of the japanese gastric cancer association. we looked for undetected sns in the resected specimen at the back table. results: among cases, there were ( %) in which sns were not detected in the resected specimen. there were cases in whom sns were detected in the resected specimen. in both cases, the primary tumors were located in the middle and greater curvature of the stomach. in case , laparoscopic sn biopsy identified the left ( sb) and right ( d) greater-curvature lymph node (lns) as sns, however, lesser-curvature ( ) and infrapyloric ( ) lns remained as sns in the resected specimen. in case , the left ( sb) and right ( d) greater-curvature lns were identified as sns intraoperatively, while the lesser-curvature ( ) ln remained as an sn in the resected specimen. the sns overlooked with laparoscopic sn biopsy method were detected by radioisotope only. no cases had ln metastasis, and the -year relapse-free survival rate of these patients was %. conclusions: our feasibility study of laparoscopic sentinel node biopsy for early gastric cancer showed that we should search for sns of the lesser curvature carefully even if the primary lesion is located at the greater curvature. key: cord- - kwlev authors: maan, sushila; dalal, sangeeta; kumar, aman; dalal, anita; bansal, nitish; chaudhary, deepika; gupta, akhil; maan, narender singh title: novel molecular diagnostics and therapeutic tools for livestock diseases date: - - journal: advances in animal biotechnology and its applications doi: . / - - - - _ sha: doc_id: cord_uid: kwlev recent novelties in diverse diagnostics and therapeutic tools in animal health sector have paved a brighter and clearer way ahead. these are proved to be better in detection, management, control and eradication of animal sufferings caused by various infectious and non-infectious diseases. these innovations have potential impact that extends beyond the animal health and welfare. the advancements have significantly contributed towards improvement in the economy of the country as well as food security. in the present competitive era of evolution, the organisms have inculcated a number of new strategies for survival and spread. therefore, science needs to continuously evolve more sensitive, specific and high-throughput tools to overcome pathogen cleverness to escape from host immune surveillance. for visible or remarkable changes, it is necessary to use full potential of these advanced molecular techniques into current animal health standards and practices. under ‘one health’ concept, the health of animals and humans has to be taken care simultaneously. at present, these advanced molecular diagnostic methods play a significant role in the detection of new and emerging pathogens of livestock. the acquired information also helps to study the interrelationships of pathogens, their hosts and their surroundings. additionally new vaccines bridging human and animal health development may be discovered. latest developments in the field of diagnostics and vaccine design through genomics approach have also laid the foundation to enhance the diagnosis and surveillance and in turn helped in the control of infectious diseases. latest high-throughput dna sequencing platforms are currently being used for identification and detailed analysis of both disease pathogen and host genomes. the high-throughput data generated using these platforms need to be analysed adopting the bioinformatics and computational genomics that have taken a very high pace nowadays. in the context of animal health, the data analysis may provide some key opportunities for the development of better diagnostic and therapeutic tools for emerging or re-emerging diseases. such novel and potent technologies put forward a significantly new scenario of disease knowledge, which enables more accurate predictions leading to faster and greater management responses to combat potentially devastating disease crises. owners. maintenance of good health status of animals by effective control and treatment of diseases lies in the access to various standard diagnostic tests which are rapid, reliable, precise and highly sensitive and therefore helps in confirming early detection of causative agent. the conventional diagnostic methods are too much demanding in terms of time and labour. latest advances in the molecular biology and biotechnology have opened new avenues in disease diagnosis and therapeutics, but these technologies are in budding stage so need to be further explored to their full potential to safeguard the health of human in addition to their companion animals. along with this there is a need to build up qualified, trained and competent manpower for bringing the existing disease diagnostic methods in laboratories to field of action. because of the lack of skilled manpower in different institutions of public undertakings, it is highly desirable that the gained expertise and knowledge be disseminated to users of these techniques directly so benefits can be harnessed more efficiently and effectively. veterinary pathogens have been traditionally diagnosed by detecting the pathogens by culture isolation or serological detection of antigen or antibody such as neutralization tests, enzyme-linked immunosorbent assays, agar gel immunodiffusion and complement fixation tests. in the past diagnostic capabilities have been enhanced by complementing conventional assays with molecular diagnostics. incredible progress in the molecular diagnosis and characterization of avian influenza virus infections in the past two decades is the true example of this. moreover, recent advancements in the field of molecular diagnostics have made the speedy identification of group a influenza and h and h subtype viruses feasible, which is a major global cause for the spread of avian influenza from poultry to humans. the innovative, reliable, rapid and appropriate dna-based diagnostic techniques have the potential to assist international efforts to check the incursion of exotic diseases into new geographic areas. this write-up focuses on recent advancements in molecular diagnostic tools which can help clinicians in promoting human and animal health within 'one health' concept. the use of molecular diagnostics has been rampant in recent years for the detection of veterinary pathogens. these involves detection of pathogen either directly by sensing the presence of dna or rna in the host or indirectly by prior amplification of genome of infectious agent. these techniques have not only helped in earlier disease diagnosis but also animal disease control programmes. further, modifications in pcr-based molecular detection techniques have generated a vast array of fast, reliable and specific assays which have widespread applications in veterinary diagnostics. the sensitivity of any genome detection-based method can be enhanced to a very high degree by manipulating any of the three pillars of the assay, i.e. by amplification of target, signal and probe. the revolutionary, very sensitive and specific pcr method was developed by kary mullis in the s. it has allowed scientists to detect and amplify a unique region of dna multiple folds under strict thermal conditions (mullis ). the specificity of the assay is determined by complementarity of short stretches of synthetic dna oligonucleotides or primers to the target sequence followed by amplification of the sequences between these primers by the thermostable dna-dependent dna polymerase enzyme. it is a faster technique than other methods of detection of pathogens (bacteria, fungi or viruses), which require initial isolation in culture. pcr has been used in veterinary diagnostics for specific genomic detection, e.g. infectious bovine rhinotracheitis virus, foot-and-mouth disease virus, bovine viral diarrhoea virus, buffalopox virus, ephemeral fever virus and many others. mass level detection of avian influenza and newcastle disease poultry pathogens in recent outbreaks in the usa has been made with pcr. the following is the list of the different types of pcr being used in the diagnosis of animal diseases: . reverse transcription pcr (rt-pcr) . semi-quantitative pcr . nested/semi-nested pcr (npcr) . asymmetric pcr . quantitative pcr (qpcr)/real-time pcr . linear after the exponential pcr (late-pcr) . multiplex pcr . other variants of pcr ever since the discovery of pcr, various changes in the pcr protocol have been developed (erlich et al. ) . some of them effectively increased the diagnostic capabilities of pcr and enhanced its uses in the molecular diagnostics. reverse transcription pcr (rt-pcr) is particularly designed for the amplification of rna sequences by converting into complementary dna (cdna). after reverse transcription, cdna is amplified using pcr. the rt-pcr has played a vital role in diseases diagnosis especially rna viral infections, for example, influenza viruses, rotavirus, bluetongue virus, foot-and-mouth disease virus, etc. (erlich et al. ; persing ) . furthermore, in vitro gene expression study can be done by application of rt-pcr as the acquired cdna retains the original complementary rna sequences. the main confrontation in rt-pcr is the difficulties in handling low level of target rna/mrna. moreover, low stability of rna/mrna and its susceptibility to be degraded by ribonucleases and ph change poses many problems to combat. this technique helps in the approximate estimation of nucleic acids present in a given sample. for rna quantitation, firstly, the rna sample is converted into cdna by rt-pcr. the internal controls/markers (e.g. apo a and β-actin) are also amplified simultaneously. amplified products are separated by agarose gel electrophoresis that are photographed on gel documentation system. the amount of ethidium bromide bound visible dna is calculated by measuring its optical density using densitometer. the drawback of this method is the possibility of nonspecific annealing, producing false positive/negative results. the specificity can be enhanced by using highly specific primers. this pcr uses two sets of primer pairs for two rounds of amplification for increased sensitivity and specificity. the product of the first set of outer primer pair is used as template for the second pcr using the second set of inner primer pairs. the latter are specific to the amplified product sequence of the first pcr thereby verifying the specificity of the first round of pcr with the specific product availability of second pcr. nested pcr (npcr) has been used to detect a number of etiological agents of veterinary importance such as canine corona virus, west nile virus and orf virus (pratelli et al. ; bora et al. ) . the drawback of nested pcr is the possibility of contamination of the first amplified product. this problem can be overcome by using the primer pairs annealing at different temperatures. secondly, ultrapure oil or wax can be used to make physical separation between two amplification mixtures as a contamination control measure (erlich et al. ). in a symmetric pcr, there is exponential growth of double-stranded dna, whereas an asymmetric pcr generates a single-stranded dna predominantly as it is governed by the concentration ratio of the primers (i.e. one of the strands of dna by the primer in excess) by linear amplification due to the use of unequal concentrations of two primers in a primer pair and a fraction of dsdna. in the asymmetric pcr, the primer in lower concentration is quantitatively incorporated into dsdna. thus the diagnostic technique provides lower intensity signal in agarose gel leading to lesser sensitivity than symmetric pcr. asymmetric pcr has been used in detection of gene mutations (zhang et al. ). real-time pcr is a modification of conventional pcr to address the need for robust quantification of nucleic acids. during this type of pcr, fluorescent signals arise due to the use of fluorescent dyes. these signals are directly proportional to the number of amplified products produced. the amount of product is measured after each cycle of pcr. the exponential phase data represent reaction yield or quantitative information based on the starting quantity of the target. common real-time pcrs include ( ) sybr green method where the fluorescent dye sybr green binds to random dsdna and can also give nonspecific amplification and ( ) dual dyelabelled probe method which involves the use of sequence-specific dna probes that are labelled with a fluorescent reporter, permitting specific detection after hybridization of the probe with its complementary sequence. there are several advantages of real-time pcr that include: ( ) its ability to monitor the reaction progress in real time. ( ) it is helpful for accurate quantification of samples. ( ) it has a wide dynamic range of detection, and being a single-tube method, post-pcr manipulations and contamination is not a problem. rt-pcr is also extensively used for the genotyping and phylogenetic analysis (relatedness) of veterinary pathogens. real-time rt-pcr is the most sensitive, informative technique yielding rapid results, with the only drawback of high cost of start-up and of reagents. it has been employed for detection of bluetongue virus (de leeuw et al. ; maan et al. ) , foot-and-mouth disease virus and bovine piroplasmids (criado-fornelio ) etc. the quantification of nucleic acids in rt-pcr is performed by two methods, i.e. the relative and absolute quantitation. in absolute quantification, exact numbers of target dna molecules are calculated by using a calibration curve. the calibration curve is built with dna standards. limitation of this method is that sample and standard should have the same amplification efficiency. the relative quantification is depending on fold differences in the expression of target gene as compared to internal reference genes. the result is expressed in different expression levels of mrna as cdna (reverse transcription). it is an easy approach for quantification because there no need of making calibration curves. there are multiple reasons for considering conventional asymmetric pcr inefficient, as there are difficulties in optimization and it tends to promote nonspecific amplification and restricted concentration of one primer lowers the tm (melting temperature) below the reaction annealing temperature. to overcome these limitations and to increase efficiency comparable to symmetric pcrs, linear after the exponential (late)-pcr was developed based on primer pairs purposely designed for use at unequal concentrations to yield specific single-stranded dna products in a robust way (pierce et al. ). in the late-pcr, detection step is distinct from the annealing and extension step, and it improves allele discrimination and increases signal strength significantly as compared to symmetric pcr (sanchez et al. ). late-pcr does not reach the characteristic plateau like conventional pcr but ends in a nearly linear phase (johann et al. ) . late-pcr is highly appropriate for high-throughput field applications, e.g. clinical analysis, biodefense, forensics and dna sequencing. multiplex pcr (mpcr) is a modification of pcr for concurrent amplification of many sequences by inclusion of different sets of genome sequence-specific primer pairs for different targets. mpcr is used for diagnosis of different disease pathogens in a single reaction. it can also be used to identify exonic and intronic sequences in specific genes. in multiplex pcr the design of various primer pairs is crucial so that they complementarily anneal to specific dna sequences at more or less similar temperatures, i.e. annealing temperature should be the same for different primer pairs used in combination. a multiplex pcr assay to detect h n and other human respiratory pathogens (lam et al. ; rheem et al. ) and mastitis in animals has been developed (shome et al. ) . other variant and combination of this technique is multiplex one-step real-time pcr kits that are available with all plus points inherited (gautam et al. ). during the amplification of target dna, sometimes amplification of nontarget sequences also takes place which are nonbeneficial, and their presence in huge amount decreases the amount of desired product that leads to complication in data analysis. for getting higher reaction specificity and yield, 'hot-start pcr' was developed, where the taq polymerase enzyme stays inactivate/blocked by specific antibodies at temperatures below °c, the optimal temperature of primer extension by taq polymerase, thus avoiding non-specific amplification. the blocking antibodies get denatured and removed at an initial step at °c allowing the specific reaction to proceed (kellogg et al. ). in this type of pcr, the annealing temperature (ta) is deliberately brought down during the cycling process. at initial stage, ta is set - °c higher than the tm of the primers, while in the follow-up cycles, ta is slowly decreased, and by the end of the amplification, ta is below - °c from tm (don et al. ) . the high temperature increases the specificity of primer and template binding leading to amplification of only target sequences. pca involves two sets of pcr. in the first set of pcr, overlap primers are used. the product of the first pcr is used as a template for the second pcr, which amplifies final full-length product. pca synthesizes dna from a pool of long oligonucleotides having short overlapping segments and can be used as an alternative for ligation-based assembly (bang and church ) . this pcr is specifically to screen the bacterial colonies. the bacterial colonies are transferred into a pcr master mix aseptically. the cellular dna is released either by incubation at °c (with standard polymerase) or by shortened denaturation step at °c with a recombinant dna polymerase (pavlov et al. ). this technique is used for an established fung's double-tube method for rapid detection and confirmation of clostridium perfringens (ruengwilysup et al. ). the digital pcr performs simultaneous amplification for a large number of samples. these samples are present in an emulsion in a separate droplet, e.g. sequencing platforms and ion torrent technology. suicide pcr is performed where very high specificity of the desired product is required, e.g. palaeogenetics. the target of this pcr is the genomic region, which has never been amplified in the laboratory with any sets of primers. this is to avoid false positive results from contaminating dna from previous pcr reactions (raoult et al. ) . nucleic acid techniques (nats) are sensitive, rapid and reliable diagnostics that are based on amplification of specific regions of pathogen genome. though quantitative in nature, the non-amplification nucleic acid detection methods are not so commonly used because of their lower sensitivity compared to amplification methods. these methods have less specific requirements for performing enzymatic process and also less dependency on good reagents. the property of less sensitivity of these methods is favoured to reduce carryover product contamination. the different methods of nucleic acid amplification have been developed, e.g. rolling circle amplification technique and direct signal amplification system. in human medicine these techniques are presently being used for the detection of cytomegalovirus (cmv) and human immunodeficiency virus (hiv). these methods can be useful in the diagnosis of livestock diseases. to minimize the chances of contamination during amplification, various molecular technologies have been developed. one alternate to enzymatic amplification of target nucleic acid is hybridized probe-based signal amplification. the most common signal amplification technologies are branched dna (bdna) and hybrid capture (hc) assays (datar and joshi ) . in bdna technology, the presence of specific nucleic acid is detected by measuring the signal generated by specific hybridization of many branched labelled dna probes on an immobilized target nucleic acid. one end of bdna binds to the immobilized target dna, and the other end of it has many branches of dna which amplify the detection signals. the amplification of signals is linear and is achieved by sequential or simultaneous hybridization of synthetic oligonucleotides. final detection uses alkaline phosphatase (ap) as it generates chemiluminescence. bdna is a quantitative technology and is used in the determination of viral or pathogen load (cao et al. ; kern et al. ; collins et al. ). this technology is a highly sensitive, specific, less labour-intensive, less contamination prone and reliable tool in the diagnosis of viral and bacterial infections and for monitoring disease status during the course of therapy. this technology has been proved versatile since it helped in the detection of infections by a wide range of microorganisms (tsongalis ). nasba, introduced in , is an isothermal amplification method which is carried out at a constant temperature of °c. it is also called self-sustained sequence replication and transcription-mediated amplification. three enzymes, viz. avian myeloblastosis reverse transcriptase, rnase h and t dna-dependent rna polymerase, are involved in the process (fakruddin et al. ) . it is a sensitive, isothermal, transcription-based amplification system which is specifically designed for the detection of rna targets (van gemen et al. ; deiman et al. ) . the major advantage of nasba is the production of single-stranded rna amplicons. these amplified rna products can be sequenced directly with a dideoxy method using rt and a labelled oligonucleotide primer. additionally, the ssrna amplicons can either be amplified or probed for direct detection. the technique can be easily adopted for the development of various pathogen detection kits (fakruddin et al. ). tas was developed by kwoh and his coworkers in . in tas, rna is the target molecule as well as primary product (kwoh et al. ) . a copy of dna is made from rna, and then transcription produces million copies of rna. the variations of tas are transcription-mediated amplification (tma), nucleic acid sequencebased amplification (nasba) and self-sustaining sequence replication ( sr). in the process, the primer complementary to the sequences that has the binding site for rna polymerase is added to the targeted rna molecule. three enzymes, reverse transcriptase (rt), rnase h and t dna-dependent rna polymerase, are used in the amplification reaction. the primer anneals and then rt enzyme is used to produce cdna copy of target rna. rnase h then degrades the initial rna from dna-rna hybrid. the second primer set binds to newly synthesized cdna which is extended to produce double-stranded cdna. both the strands of this ds cdna can act as a template for rna polymerase for transcription. tas has few advantages over various amplification processes: ( ) tas is an isothermal process, negating the requirement of thermal cycling to drive reactions due to which it helps to minimize contamination risks (guatelli et al. ). apart from direct detection of rna-containing viruses, e.g. hepatitis c virus, this technique can be used for detection of low amount of certain bacterial and fungal pathogens (compton ) . the only demerit of tas is heating step which denatures the enzyme due to which enzyme is needed after every denaturation step. isothermal amplification (lamp) sda, a non-pcr nucleic acid amplification technique, developed in , involves dna polymerase-initiated dna synthesis. it works on a single-stranded nick followed by the displacement of the nicked strand during the process. the displaced strand is used as a substrate for further simultaneous nicking and displacement reactions. sda uses specific primers, a dna polymerase and a restriction endonuclease, to achieve exponential amplification of target. in brief, this method makes copies of the target sequence flanked by nickable restriction sites, allowing the exponential increase of these modified target sequences by recurrent nicking, strand dislocation and further priming of banished strands (spargo et al. ) . sda has two important advantages: (a) sda is isothermal with the exception of the initial denaturation step and (b) it can be applied to both single-and doublestranded dna (kim and easley ) . loop-mediated isothermal amplification (lamp) assay, initially described by notomi et al. , is an important method for disease diagnosis especially in the developing countries (abdullahi et al. ) . lamp is a simple, quick, highly specific and cheap single-tube technique for the amplification of dna. lamp can be used as a simple test in the field or at the point of disease outbreak. in lamp test, four different types of primers, a forward inner primer (fip) and a backward inner primer (bip) set and a forward and backward outer primer set, are used. these primers recognize six distinct binding sites on target dna. lamp uses bst dna polymerase having strand displacement activity. both primers and enzymes work together for lamp cycling initiation and continuation of dna synthesis with auto-strand displacement leading to the accumulation of copies of target in less than an hour. dna synthesis is primed by fip and bip, in combination with initial strand displacement mediated by the extension of forward and backward outer primers, resulting in the successive formation of a dna molecule. this dna molecule is looped out at one end, further by a dumb-bell dna structure, which is rapidly transformed to a stem-loop structure which is the substrate for the elongation and recycling step of lamp. lamp-based commercial detection kits for bacterial and viral pathogens are available. lamp has also been developed for important animal pathogens, e.g. footand-mouth disease virus (yamazaki et al. ) , bluetongue virus (maan et al. ) , capripox viruses (batra et al. ) and peste des petits ruminants virus (zhan et al. ). the process demands the use of multiple primers where a major disadvantage of lamp over pcr lies, that is, the need of frequently updated primer sequences in order to detect the prevalent virus strains with adequate sensitivity and specificity. ligase chain reaction (lcr) is a probe-based amplification technique and was first described by wu and wallace ( ) to detect point mutations. in this technique one probe is formed by ligation of two adjacent probes. the main characteristic of lcr is the second set of primer which is complementary to the first pair, designed with the nucleotide at the ′ end of the upstream primer indicating the sequence difference. when target dna is present, the two adjacent probes are ligated by dna ligase. if ligated product is absent, that means there is at least a single base-pair change in the target sequence. in the following steps, the ligated products can serve as templates and can be amplified by thermal cycling in an exponential manner (wiedmann et al. ) . in lcr contamination risk and variation in copy number of the plasmid containing the target is a problem (umesha and manukumar ). rna aptamers are defined as rna oligonucleotides ( - long) having variable and constant region that bind to a specific target with high affinity and specificity (ellington and szostak ) . the method for isolation of aptamers is called systematic evolution of ligands by exponential enrichment (selex) (tuerk and gold ) . rna aptamers are an important tool in rna nanotechnology as compared to dna and protein aptamers (germer et al. ) . they are easy to synthesize in huge quantity under controlled way having well-defined structure and stereochemistry. similar to antibodies, rna aptamers have low immunogenicity as compared to other macromolecule such as proteins. rna aptamers are more thermodynamically stable and its unique tertiary structure leads to specific binding. it is small in size due to its single-stranded nature so it can easily enter into the cell. rna aptamers can be used in different fields of science, i.e. diagnostic, prognostic and therapeutic (germer et al. ). nowadays whole-genome sequencing techniques are being extensively utilized to study a wide variety of infectious agents. with the advent of high-throughput 'nextgeneration' sequencing technologies, detailed analyses of entire pathogen genomes have been made possible within a few days which previously took many years. different rapid whole-genome sequencing platforms as illumina, and ion torrent have tremendously revolutionized the world of disease diagnostics by enhancing the ability to rapidly screen complex mixtures of genomes. it has become easier to make comparison between diseased and apparently normal/healthy states of patients. no prior knowledge is required about the infectious agent to be sequenced, so it plays an important role in the detection and identification of new and emerging pathogens. the latest sequencing methods hold promises in increasing the present knowledge regarding the evolution of pathogenic microorganisms especially with respect to the development of antimicrobial resistance. expensiveness is always a matter of consideration when any diagnostic assay or kit is developed. at present whole-genome sequencing technologies particularly the next-generation sequencing (ngs) are very costly, but as per the increasing demand and use in different field of scientific research, diagnostics and many others, it can be expected to become cheaper in the future. some of the ngs platforms are briefly described below. the very first next-generation system brought in the market was genome sequencer (gs) instrument based on the principle of pyrosequencing by life science in (margulies et al. ) . traditional sequencing methods required cloning of the gene to be sequenced, but this demerit was removed by gs pyrosequencing system. briefly, the procedure involved initial random shearing of genome (may be enzymatic or physical by ultrasonication), followed by ligation of both the ends of each fragmented dna genome with specific adaptors. these adaptors allowed the dna molecule capturing on the surface of beads. now these ligated fragments were amplified (emulsion pcr) and captured in emulsion droplet. individual bead with amplified fragment arrayed in a well of a fibre-optic slide. the gs followed the same sequencing principle as of most primitive sanger sequencing method. in the method, when a new base was incorporated in the growing chain, a pyrophosphate group was released and detected as emitted light by a ccd imager coupled to the fibre-optic array. the main disadvantage of this system was high error rates as insertion and deletion mutations (indels) were produced because of the false reading of template by misjudging the homopolymers. to increase the read lengths, a new gs flx titanium xl+ has been developed which is able to generate mb of sequence data with . % accuracy. the system involves a relatively high cost and lower throughput than other developed next-generation sequencing methods and therefore has decreased its preferences. ion torrent is known by different synonyms as ion semiconductor sequencing or ph-mediated sequencing or silicon sequencing. it is based on the principle of detection of hydrogen ions by ion-sensitive field-effect transistor (isfet) ion sensor that is released during polymerization reaction of new dna strand synthesis. this is a method of sequencing by synthesis, i.e. sequence data of template is generated, while complementary strand is synthesized. the labelled or modified nucleotides and optical instruments are not employed as done in other sequencing methods. this system also has various limitations like difficult enumeration of long homopolymer repeats. signals obtained from high repeats of different nucleotides create obstacles as differentiation could not be made from that of similar but different number homopolymer repeats. it gives an average read length of nucleotides per read, which is a shorter read length compared to pyrosequencing and sanger sequencing methods. thus this is best suited for small-scale applications as microbial genome/transcriptome sequencing, amplicon sequencing or quality testing of sequencing libraries. illumina ngs technology was developed by canard and sarfati in as a second next-generation sequencing technology, which uses the reversible termination chemistry concept (canard and sarfati ) . this concept allows the identification of single-nucleotide base as it is polymerized into new dna strand, i.e. sequencing by synthesis. it is multipurpose solving as has been used in whole-genome sequencing, transcriptome analysis, metagenomics, small rna and methylation profiling and also helpful in the analysis of protein and nucleic acid interaction. it is also known as solexa, released in as solexa sequencing platform. solexa platform uses sequencing by synthesis (sbs) technology where fluorescently labelled nucleotides are used as terminating base. the latter when removed leave an unblocked ′ terminus making the process of chain termination reversible. the illumina technology involves dna fragmentation, ligation with specific adapters and followed by denaturation. the single-stranded denatured templates are then immobilized on one end on a flow cell surface which is already laid with complementary adapters. immobilization of every fragmented template strand on flow cell surface occurs by hybridization of free end to the complementary adapter. it is followed by amplification, which results up to identical copies of every ssdna template molecule named as dna 'polonies'. during the cycling process of sequencing, as a single fluorescent dye-labelled dntp is incorporated, the fluorescence of dye is imaged by the ccd camera for identification of the base, and then the dye is enzymatically cleaved, which allows incorporation of the subsequent nucleotide. microarray technology is very powerful and high throughput which can be used for expression studies, transcriptome analysis, detection and characterization of genetic variants, dna-protein interaction study and detecting genome methylation. the important feature of this method is immobilization of different molecules (oligonucleotides, proteins, small drug-like compounds) onto a solid and activated surface as matrix (schena et al. ) . this matrix arrangement is called microarray. microarray plate has specific point at which high concentration of immobilized molecules are present to interact with their targets. these are named as microchips, biochips, dna chips or gene chips to detect dna in diverse biological samples. the advantages of microarray technology are the following: the expression of the entire gene content of a genome of interest can be monitored and it also provides data analysis in field conditions because detection is simple and rapid and also provides real-time data analysis (zhou and tompson ) . the term 'nanotechnology' was given for the first time by norio tangiuch in . this technology includes manipulation in atoms and molecules at very small scale that is nano in size (savage et al. ; medina et al. ). till date so many types of nanoparticles are obtained from transition metals, silicon, carbon and metal oxides (torres-sangiao et al. ) . this nanomaterial shows different type of physico-chemical properties, thereby widening its application area. this technology is very helpful in designing molecular diagnostic assays, pen-side test or chip-based diagnostics for medical and veterinary fields. the very small size also increases its use in nanomedicine (jos et al. ). gold nanoparticles (aunps) are very flexible nanostructures and can be used in different field of biomedical science (mirkin et al. ) . aunps show so many useful properties like more flexible structure, defined size, shape, structure and better optical properties. these particles are being used for diseases diagnosis as biosensors (dilbaghi et al. ; mirkin et al. ; shah et al. ). the whole set of proteins expressed by a genome, cell, tissue or organism is termed as 'proteome'. the study and characterization of complete set of proteins is called as 'proteomics' (anderson and anderson ) . proteomics recently in the last two decades has emerged as a field of research and has developed rapidly (ceciliani et al. a, b) . proteomics is a better approach than genomics for studying changes in metabolism under stress conditions. various methods in proteomics, viz. -d gel electrophoresis ( dge), maldi-tof/ms, etc., play a significant role in the analysis of novel proteins and in disease diagnosis. the expression of proteins depends on the status of the body and different environmental factors. this field of study has an important role in novel drug discovery and in the early-stage disease diagnosis (oie ) . proteome maps are being derived from a range of veterinary pathogens (mujer et al. ; rout and field ; yatsuda et al. ) . proteomics-based diagnosis may be used to identify known or unknown disease markers in the future. proteomics may have applications in the diagnosis of disease pattern development in combination with biochip technology, bead technology, mass spectrometry and other separation chromatographic methods. this type of combination of technologies can be useful for identification of pathogens that do not induce predictable serological reactions, i.e. bovine tuberculosis. molecular diagnostic assays are becoming more popular for confirmation of fieldbased diagnosis of livestock diseases on the basis of clinical signs and symptoms. recent advancements of diverse biotechnological tools promise for improvement in the speed and accuracy of diagnostics for both human and veterinary medicine. this can also 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loop-mediated isothermal amplification (lamp) method for the detection of peste des petits ruminants virus the application of asymmetric pcr-sscp in gene mutation detecting microarray technology and applications in environmental microbiology key: cord- -r nv ph authors: pai, vidya v.; kan, peiyi; gould, jeffrey b.; hackel, alvin; lee, henry c. title: clinical deterioration during neonatal transport in california date: - - journal: j perinatol doi: . /s - - - sha: doc_id: cord_uid: r nv ph objective: identify clinical factors, transport characteristics and transport time intervals associated with clinical deterioration during neonatal transport in california. study design: population-based database was used to evaluate , infants transported before days after birth from to . log binomial regression was used to estimate relative risks. results: . % of infants had clinical deterioration. clinical deterioration was associated with prematurity, delivery room resuscitation, severe birth defects, emergent transports, transports by helicopter and requests for delivery room attendance. when evaluating transport time intervals, time required for evaluation by the transport team was associated with increased risk of clinical deterioration. modifiable transport intervals were not associated with increased risk. conclusion: our results suggest that high-risk infants are more likely to be unstable during transport. coordination and timing of neonatal transport in california appears to be effective and does not seem to contribute to clinical deterioration despite variation in the duration of these processes. the inter-facility transport of critically ill newborns is an integral component of regionalized perinatal care in the united states [ ] . treatment of sick neonates in higher-level neonatal intensive care units (nicus) has been shown to be associated with decreased morbidity and mortality when compared to those cared for in lower-level nicus [ ] [ ] [ ] [ ] [ ] . for this reason, mothers who are anticipated to have highrisk deliveries should be transferred to more specialized centers when feasible. unfortunately, not every high-risk delivery can be predicted, and transport is often imperative when neonates are born in hospitals that may not be equipped to deliver higher-level neonatal intensive care. in addition, sick neonates who need more advanced care but are not transported may have higher risk of death [ ] . although transport to higher-level nicus is often necessary for critically ill neonates to receive specialized care, the transport environment is not without risks. compared to inborn neonates or those born after maternal transfer, neonates who require acute postnatal transport have increased risk of morbidities such as hypoxemia, glucose abnormalities, intraventricular hemorrhage and death [ ] [ ] [ ] [ ] . several factors have been associated with adverse neonatal outcomes after transport: the condition of an infant around the time of transport and provision of intensive care during transport have both been linked to increased morbidity and mortality [ , ] . duration of transport may also impact outcomes. studies of acute transports involving older children have shown that remoteness of the referral hospital and longer duration of transport are associated with increased hospital lengths of stay, higher illness acuity scores and increased mortality rates [ ] . a retrospective study in japan of neonatal transports demonstrated that inter-facility transport longer than h was associated with a higher risk of neonatal death compared to transports of shorter duration [ ] . during transport, neonates may be exposed to significant physiologic stressors that can lead to clinical deterioration. in addition, identification and management of patient deterioration may be more difficult while transport is occurring. although it has been demonstrated that certain clinical characteristics are associated with adverse outcomes after transport [ , ] , little is known about factors associated with clinical deterioration during transport. in addition, it is unknown how certain time intervals during the transport process may affect the likelihood to deteriorate during transport. the goal of this study was to identify maternal and neonatal risk factors, transport characteristics and transport time intervals that are associated with increased risk of clinical deterioration during transport. it is crucial to understand which infants are likely to deteriorate during transport so that transport teams can prepare and respond appropriately. in addition, understanding the association of transport time intervals with deterioration during transport provides information on the quality of the neonatal transport process and may lead to opportunities for improvement. in california, the california perinatal quality care collaborative (cpqcc) collects clinical prospective data on infants admitted to nicus in california. standard definitions align with those used by the vermont oxford network. eligibility for the database includes gestational age less than weeks, birthweight between and g, whether transport occurred into or out of a nicu, need for intubated or non-intubated assisted ventilation for greater than h, early sepsis, major surgery, severe hyperbilirubinemia, suspected encephalopathy or active therapeutic hypothermia [ ] . the majority of neonatal transports in california are conducted by members of the california perinatal transport system (cpets), a network of over specialized nicus and transport teams, who conduct~ acute neonatal transports each year. this network serves to facilitate the transport of critically ill infants to nicus offering a higher level of care that are better able to meet their needs [ ] . these transports are extremely heterogeneous with respect to gestational age at birth, postnatal age at transport and clinical status [ ] . the cpets collects comprehensive neonatal transport data that are linked to the cpqcc database. acute transfer makes an infant eligible for data collection for cpqcc, therefore this dataset accounts for all infants who were transported for care to one of the cpqcc nicus. this study was based on data collected on infants born from january to december who were transported within days after birth. this study was approved by the california committee for the protection of human subjects and the stanford university institutional review board. the canadian transport risk index of physiologic stability (trips) score is a physiology-based assessment developed by lee et al. [ ] . the trips score can be used to calculate the risk of death of an infant within seven days of transport and an increase or decrease in trips scores after transport has been shown to be associated with increased or decreased mortality, respectively [ ] . this scoring system has been optimized and validated for the california neonatal population by gould and colleagues (ca-trips) [ ] . the ca-trips score comprises temperature, blood pressure, response to noxious stimuli, respiratory status, use of vasopressors to support blood pressure and use of a ventilator [ ] . ca-trips scores are evaluated at the point at which the transport team arrives at the bedside (pre-transport score) and again when the infant begins care at the receiving nicu (post-transport score). infants with clinical deterioration during transport were defined as those who had a post-transport score greater than pretransport score. this definition was chosen because any increase in score after transport has been shown to be associated with higher mortality [ ] . covariates were selected a priori and evaluated from cpqcc and cpets. we included relevant covariates that would be known about the infant either prior to or shortly after birth. maternal and neonatal clinical covariates included infant sex (male or female), gestational age (weeks), birthweight (grams), maternal age (years), maternal race/ ethnicity (african american, hispanic, white, asian/pacific islander, or other/unknown), prenatal care, multiple birth, birth defect severity, delivery room resuscitation and -min apgar score less than or equal to . birth defect severity was divided into six levels, as is defined in cpqcc: level (no birth defect), level (not severe, i.e. hemangioma, atrial or ventricular septal defects), level (moderately severe, i.e. gastrointestinal atresias, imperforate anus, trisomy ), level (severe, i.e. encephalocele, abdominal wall defect, complete atrio-ventricular canal), level (very severe, i.e. congenital diaphragmatic hernia, hydrops fetalis), level (most severe, i.e. ancencephaly, hypoplastic left heart syndrome, bilateral renal agenesis, trisomy or ). transport characteristics included: age at transport, hour and day of transport, type of transport, mode of transport, indication for transport and team leader composition. transport type was defined as delivery attendance (transport team was requested to attend the delivery), emergent transport (immediate transport was requested), urgent transport (transport requested within h), and scheduled transport (transport was planned for an infant who required eventual transfer but who was currently in stable condition). time (in minutes or hours) between key periods of neonatal transport were calculated from the cpets data and were divided into clinically relevant intervals. these time periods included: time from birth to when the referral call was made, time from the referral call to admission acceptance by the accepting nicu, time from admission acceptance to departure of the transport team, time from departure of the transport team to arrival at the referring nicu, time from start of evaluation by the transport team to admission at the accepting nicu. total time of transport was calculated from time of referral to admission at the accepting nicu. infants who experienced clinical deterioration during transport were compared to those who remained stable or improved during transport. student's t-test compared means and standard deviations of ca-trips scores before and after transport in these infants. infants were compared according to maternal, neonatal clinical, transport and timing factors. relative risks and % confidence intervals for the association between each covariate and clinical deterioration during transport were estimated using log-binomial regression models. models included variables that may influence the practice of transport, including year of transport, mode of transport, and transport type. as the intent of our study was to identify neonates at risk for clinical deterioration, specific neonatal and maternal characteristics were not included in the models. significance was defined as p < . or a confidence interval that excludes . . statistical analyses were computed using sas . (sas institute, cary, north carolina). of , cpqcc-eligible infants born from to , , infants required acute transport with , ( . %) transported within days after birth and ( . %) transported later than days after birth. ( . %) infants were excluded because of missing birthdate, birth time, accepting nicu evaluation date or evaluation time. , infants had valid ca-trips scores documented and were included in the analysis. of these infants, , ( . %) had clinical deterioration during transport. , ( . %) infants remained stable with either no change or an improvement in ca-trips score. pre-transport, infants with clinical deterioration had a ca-trips score of . ± . ; infants who remained stable had a score of . ± . (p = . ). post transport, infants with clinical deterioration had a mean score of . ± . ; those who remained stable had a score of . ± . (p < . ). table displays the association of maternal and neonatal characteristics with risk of clinical deterioration. early gestational age was associated with increased risk of clinical deterioration during transport with the highest risk at the earliest gestational ages. this pattern was similar for birthweight. increased risk of clinical deterioration was also associated with a low apgar score at min, the need for delivery room resuscitation and greater birth defect severity (levels and ). transport characteristics are displayed in table . there was no association between hour of transport or day of the week with risk of deterioration. transports by helicopter, emergent transports and transports requested for delivery attendance were all associated with increased risk. transports with a nurse or nurse practitioner as the team leader were associated with decreased risk. table displays time intervals during the transport process and their association with risk of clinical deterioration. compared to infants who were referred for transport within h after birth, those who were referred later had lower risk of clinical deterioration. transport teams that took more than min to arrive at referring nicus were associated with an increased risk of clinical deterioration. the longer the total time for the transport process and the longer the time period from initial evaluation by the transport team to nicu admission, the greater the risk of clinical deterioration. this is a large population-based study evaluating the relationship between clinical deterioration during transport and various clinical factors, transport characteristics and time intervals during the transport process. our goal was twofold: ( ) identify risk factors associated with clinical deterioration that might aid transport teams in appropriately preparing for transport, and ( ) describe the relationship between time intervals during the transport process and clinical deterioration since particular intervals may be modifiable, providing opportunities to improve transport quality. we found that high-risk infants were most at risk for deterioration during transport; this includes the smallest and most premature infants, infants requiring delivery room resuscitation, and those with more severe birth defects. this finding is not unexpected given that these infants are likely to be more ill. other variables associated with illness severity, such as transports by helicopter and emergent transports, were also associated with increased risk of clinical deterioration. a recent study of neonates in sweden found no significant change in ca-trips scores before and after acute airborne transports [ ] , which contrasts with our findings. this difference may reflect differences in our study population and illness severity, in addition to a larger transport volume. we also found that transports led by a nurse or nurse practitioner were associated with decreased risk of clinical deterioration. a study of transport outcomes in the canadian population, similarly showed that infants traveling with transport teams led by nurses showed the greatest decrease in trips score compared to transport teams lead by emergency medical technicians [ ] ; however, that analysis did not differentiate between teams that contained physicians or advanced transport specialists. we hypothesize that nurses and nurse practitioners may lead transports that are lower acuity where a provider with advanced neonatal resuscitation skills may not be needed. transports requested for delivery attendance were found to be associated with increased risk; this reinforces that mothers who are anticipated to have high-risk deliveries should be transferred to more specialized centers when feasible to avoid infants being born in centers that may not be equipped to deliver advanced care. when feasible, antenatal transfer of high-risk patients has been shown to improve outcomes for both the mother and infant [ ] . despite the association of clinical deterioration with high-risk clinical factors, the mean pre-transport ca-trips score was similar among infants with and without clinical deterioration; in fact, infants without clinical deterioration had a slightly higher baseline ca-trips score. although our results demonstrate a statistically significant difference due to the large sample size, from a clinical perspective, this minor difference in scores is likely not relevant. this contrasts with the study of transport outcomes in the canadian population by eliason et al. that showed that baseline pretransport trips score correlated to the change in trips score [ ] . our data suggest that an infant's history, rather than physical exam, may be more indicative of the likelihood for deterioration during transport. an important finding of this study is that there was no relationship between modifiable transport time intervals and the risk of clinical deterioration. these include the time required for a nicu to accept a patient after a referral call is made and the time required to mobilize a transport team after a patient is accepted. these intervals correspond to the time required for the transport team to be organized. this suggests that the process of facilitating and dispatching a transport team appears to be effective and that despite variation in the duration of these intervals, there was no association with risk of clinical deterioration. our study is in the context of a relatively efficient statewide transport system where in % of transports, the time from initial referral call to acceptance of the transfer occurred in less than min, and the large majority of transports occurred expeditiously in regard to initiation of transport and arrival at the referring nicu (table ) . standards do not exist for appropriate lengths of components of the transport process; however, the time intervals in our study are similar to those in an analysis of neonatal transports in australia that demonstrated a median discussion time after the initial referral call to be (interquartile range - ) min [ ] . we did not find that the time interval of acceptance to transport team departure was associated with clinical deterioration. this time interval could be considered as one that may be most modifiable from the standpoint of the transport team in order to optimize outcomes. a delay in response of the transport team could presumably lead to further clinical deterioration in a sick infant at a facility without appropriate resources. this is likely to be true to some extent, and our finding of no association in time and clinical deterioration may be due to several reasons, first, the response times were generally quick, with % occurring in less than h. second, it may be the case that those response times which occurred later were ones in which the transport team leader appreciated that the clinical scenario and the referring hospital situation was one in which a later departure time would be safe. our findings suggest that in california, the response times of transport teams are generally appropriate. we did find that transports teams that take longer than min to arrive at the referring nicu were associated with increased risk of clinical deterioration. although we were unable to evaluate physical distance and weather-related limitations that may impact transit time, transfer of pediatric patients from more remote healthcare centers has been shown to be associated with adverse outcomes [ ] . in general, the time intervals during transport that were most associated with risk of deterioration are likely related to the condition of the infant being transported, and not related to the transport process itself. we found that the longer it took for the referral call to be made after birth, the lower likelihood of clinical deterioration. this is potentially because infants who are referred later after birth may be less ill. risk of deterioration during transport was associated with longer total transport times; however, when evaluating the individual intervals of the transport process, this risk was associated with longer evaluation periods by the transport team. this is likely to reflect the situation when transport teams may need more time to stabilize sicker infants either prior to or during transport. this is a key finding since the need for intensive care during transport is associated with adverse outcomes [ ] . prior research has similarly found that longer transport times are associated with increased neonatal mortality [ , ] ; however, this study further elucidates which specific components of the transport process may be associated with increased risk of deterioration. one limitation of this study is that there was missing data. of the , infants transported within days after birth, had missing transport information and had missing ca-trips scores. however, in the final analysis of , infants, no more than % had missing information for any given clinical factor or transport timing period. the large sample size is a significant strength of this study. we utilized a population-based dataset encompassing more than , infants who required acute transport in california. the combined cpqcc and cpets network encompass more than % of newborns born in california, which is a strength of this analysis. the size and scope of this database allows the ability to provide a comprehensive analysis of neonatal transport and evaluate how components of the transport process are associated with clinical deterioration during transport in california. in summary, this study provides evidence that the process of organizing and facilitating neonatal transport is effective in california and that there is no increased risk of clinical deterioration despite variation in the duration of these processes. clinical deterioration during transport instead appears to be associated with certain groups of high-risk infants. understanding the clinical factors associated with deterioration is crucial to ensure transport teams are adequately prepared to manage these infants. it is notable that a large number of transports continue to occur for infants in california, which may have a potential impact on both short-term and long-term outcomes of these infants. quality improvement efforts focusing on antenatal maternal transfer when appropriate may reduce the need for neonatal transport. in addition, exploring the impact of the transport process on specific high-risk populations, such as extremely premature and very-low birthweight infants, is a priority for future research. regionalized neonatal emergency transport perinatal outcomes for extremely preterm babies in relation to place of birth in england: the epicure study the effect of birth hospital type on the outcome of very low birth weight infants the effect of level of care on gastroschisis outcomes effect of deregionalized care on mortality in very low-birth-weight infants with necrotizing enterocolitis hospital variation and risk factors for bronchopulmonary dysplasia in a population-based cohort perinatal mortality and morbidity. comparison between maternal transport, neonatal transport and inpatient antenatal treatment effect of place of birth and transport on morbidity and mortality of preterm newborns transport of premature infants is associated with increased risk for intraventricular haemorrhage the effect of transport on the rate of severe intraventricular hemorrhage in very low birth weight infants transport risk index of physiologic stability: a practical system for assessing infant transport care estimating the quality of neonatal transport in california the relationship between remoteness and outcomes in critically ill children duration of inter-facility neonatal transport and neonatal mortality: systematic review and cohort study cpqcc manual of definitions for infants born characteristics of neonatal transports in california critically ill neonates displayed stable vital parameters and reduced metabolic acidosis during neonatal emergency airborne transport in sweden variations in transport outcomes of outborn infants among canadian neonatal intensive care units analysis of the retrieval times of a centralised transport service acknowledgements this work was supported by the stanford child health research institute. conflict of interest the authors declare that they have no conflict of interest.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - wyl h authors: appanna, vasu d. title: dysbiosis, probiotics, and prebiotics: in diseases and health date: - - journal: human microbes - the power within doi: . / - - - - _ sha: doc_id: cord_uid: wyl h the microbiome like any other components of the body undergoes numerous challenges during the life-span of a human being. these complications may involve injuries, aggression by pathogens, pollution, hormonal variations, genetic pre-disposition, unbalanced nutrition and onset of diseases. although the microbial reconfiguration provoked by these stressors are not immediately evident as in the case of an afflicted visible organ where the abnormality is readily observable, the biological perturbations induced manifest themselves in form of various illnesses. the disruption of a working microbiome is referred to as dysbiosis and is a condition whereby the fine balance between the microbial communities and the host is distressed. diseases such as cancer, irritable bowel syndrome, rheumatoid arthritis, acne, gastric ulcers, obesity and hypertension can ensue. the pathogeneses of some pulmonary disorders, digestive complications and neurological abnormalities can be traced to the imbalance in the constituents of the microbiome. however, rebiosis, the re-establishment of the native microbiota is proving to be an excellent remedy against this condition. probiotics, prebiotics, and synbiotics are potent therapeutic tools designed to rectify this situation. probiotics such as lactobacillus spp are more or less like stem cells utilized to replenish and rejuvenate the microbiome while prebiotics like fructose oligosaccharides (fos) are microbiome fertilizers akin to mineral supplements or energy nutrients aimed at promoting the proliferation of select microbes in the invisible organ. synbiotics is a combination of both probiotics and prebiotics in a proper dosage aimed at remedying dysbiosis. the molecular understanding of dysbiosis and rebiosis will offer a very effective non-invasive means in preventing and curing diseases with probiotics and prebiotics. this will have a dramatic impact on our well-being. abnormalities can be traced to the imbalance in the constituents of the microbiome. however, rebiosis, the re-establishment of the native microbiota is proving to be an excellent remedy against this condition. probiotics, prebiotics, and synbiotics are potent therapeutic tools designed to rectify this situation. probiotics such as lactobacillus spp are more or less like stem cells utilized to replenish and rejuvenate the microbiome while prebiotics like fructose oligosaccharides (fos) are microbiome fertilizers akin to mineral supplements or energy nutrients aimed at promoting the proliferation of select microbes in the invisible organ. synbiotics is a combination of both probiotics and prebiotics in a proper dosage aimed at remedying dysbiosis. the molecular understanding of dysbiosis and rebiosis will offer a very effective non-invasive means in preventing and curing diseases with probiotics and prebiotics. this will have a dramatic impact on our well-being. keywords microbial imbalance · antibiotics · cancer · obesity · anti-oxidant · drug activation as our microbiome accomplishes a variety of critical tasks for us, it is clear that we will not be the way we are without the microbes inhabiting in and on our body. without this microscopic support, our anatomical constituents and our diet would have been completely different. the diets of carnivores and herbivores are also dependent on the microbial helpers these animals possess. the cows we see grazing would not be engaging in such an activity if it was not for the microbeladen stomach they have. indeed, humans also have evolved to be reliant on microbes to carry numerous functions they are not able to execute without the assistance of their traditional organs. without the presence of these microbes, human anatomy and physiology would have been entirely different; we would not have been biologically the way we are. thus, it is not surprising that if this harmonious relationship is perturbed, major complications may arise with disastrous consequences to the bodily operation. in fact, the delicate balance among the microbial ecosystems needs to be preserved if the body is to function normally. the metabolically compatible members of this community work in harmony and as they are assigned a specific job that enables the community to function properly. this synergy breeds inter-dependence and a specialization of functions. the community forges a collective work ethic amongst all the stake-holders. constant interspecies communication ensures that their activity is controlled and excessive growth is under check. however, perturbation of this fine microbial-balancing act can result in debilitating impact on the host. the increase in some bacterial species with the concomitant decrease of others triggers abnormal signals that can be the harbingers of diseases. this situation is similar to the operation of our social communities. for instance, if there is an excessive amount of doctors and limited number of nurses and other health professionals, the medical delivery system becomes sick. the disruption in the microbial community is bound to create a shift in the families, genera or species of the resident microbes. this can be fatal or make the business of living unpleasant. numerous diseases are known to be triggered by the disturbance of microbial population within the body. the imbalance of the microbiome is referred to as dysbiosis (fig. . ) . diet, physical stress, exercise, psychological distress, radiation, flying, humidity, geographical location, age and medications including antibiotics have been shown to exert demonstrable change in the microbial landscape in the body. for instance, ampicillin utilization leads to a sharp decrease in the gut microbiome, while the antibiotic, cefoxilin perturbs the fine balance by promoting the proliferation of clostridium difficile at the expense of other microbes. the amount of some lactobacillus species is sharply diminished during flight and may lead to anxiety associated with air travel. the increased levels of catecholamine produced during stress trigger the growth of pathogenic microbes like e.coli and impede the proliferation of good microbes like lactobacillus and bifidobacteria. farm workers who are exposed to pesticides suffer a similar diminution in lactobacillus and bifidobacteria. exposure to metal pollution like cadmium limits the amount of micro-organisms belonging to the family of bacteriodes that in turn is reflected in reduction of metabolites like scfa (shortchain fatty acids), contributors to numerous pivotal functions in the body as we have seen before. these changes lead to various abnormalities ( fig. . ). the skin is the primary contact with the external environment and is constantly exposed to a variety of fluctuating conditions that affect the microbial communities residing on it. humidity, temperature, clothing, cosmetics, soaps, age, and personal hygiene are some of disruptors of the skin microbiome leading to the onset of various abnormalities (box. . ). acne is a common skin complication that afflicts primarily adolescents. during puberty there is a major change in the landscape of the skin with the increase of hair follicles and the maturation of the pilosebaceous glands. these oil-producing vesicles enrich the nutritional content of the skin and trigger the proliferation of oil-loving microbe like the propionibacterium acnes. these microbes secrete lipases and proteins that enable them to gobble oil. unfortunately, these molecular scissors also bruise the tissues adjoining the oily glands. such an unintended assault compels the body to unleash its own guards to defend itself, with devastating outcome. this counter response to the shift in microbial population results in the formation of those unwelcomed blemishes on the face. hormonal imbalance may also aggravate this problem (fig. . ). the dermal microbiota is prone to disruption by a wide variety of factors as it is permanently exposed to the external environment. geography, pollution, ultraviolet radiation (uv-r), occupation and the host biology are major contributors to the skin microbiome. injury, chronic conditions, infection, cosmetics and the use of hygiene products may result in a shift in the bacterial communities. uv-r promotes the release of anti-microbial peptides that may block the body's immune system and creates an environment for the cancer-causing viruses to flourish. chemicals like triclosan, a common ingredient in household products like soap and toothpaste can perturb the fine microbial balance. this is further compounded by the inherent diverse landscape that the skin offers; the face is oily, hairy and exposed to environment while the axilla is occluded, moist and laden with microbial nutrients. the dysbiosis triggered by the host of environmental and microbial-driven features are at the origin of numerous skin diseases. for instance, the atopic dermatitis that is a chronic pruritic inflammatory disorder is characterized by an overwhelming increase in staphylococcus aureus. a decrease in filaggrin, a microbial sensing protein coupled with a decrease in dermicidin, a peptide defending bacterial invasion results in this lack of microbial diversity and the proliferation of a select few microbes. prebiotics like fucosylated oligosaccharides derived from chicory roots and probiotics like staphylococcus epidermis are being introduced in cream to fight dermal dysbiosis. eczema is another common skin ailment that finds its origin in the imbalance of the dermal microbial community. it is a chronic, recurring skin disorder that is more prevalent in children compared to adults. this disease is characterized by a major shift in the microbial population resulting in diminished biodiversity. such a situation promotes the unchecked growth of the microbe, staphylococcus aureus. increased colonization by this organism provides a fertile landscape for the nasty streptococcus to proliferate. the tight-knit functional community is unraveled. the new uncontrolled colonizers secrete toxins that induce the degranulation of the dermal mast cells and force the body's immune system to respond. this change in landscape is partly responsible for the dry red spots that are characteristic of eczema. these patches are sometimes accompanied by local inflammation. in the case of psoriasis, a disease that is prevalent world-wide, similar conditions conspire to create a dramatic shift in the harmonious microbial population. the ensuing diminished biodiversity propelled by the over-representation of the genera staphylococcus belonging to the firmicutes family automatically leads to a reduction in other family members that had bonded into this dermal community and learnt to live in harmony. in this instance, the territorial presence of members belonging to the acetinobacteria family is restrained. thus, begins the initiation of psoriasis. environmental factors like temperature, humidity or presence of cosmetics on our skin may hasten the onset and the severity of this malady. the establishment of the unbalanced microbial community residing in the psoriatic lesions generate antimicrobial peptides and other modulators that hyper-activate the body's immune system. this reaction modifies the life-cycle of the skin cells that respond by growing rapidly, a situation culminating in itchy, dry, red plaques. these become the hub of other uncontrolled microbial activity and can aggravate into psoriatic arthritis. wound healing in diabetic patients is a problem as cuts on the skin of these individuals take unusually longer time to repair. here again the inability to effectively recover from wounds is again due to the uncontrolled proliferation of certain microbes at the expense of others. the sugary environment provided by the diabetic blood and the presence of other nutrients create an ideal ecosystem for the multiplication of staphylococcus. this nutrient-rich landscape on the skin-cut acts as a magnet for some bacteria and disrupts the harmony among the otherwise orderly community members. this microbial imbalance is a recipe for disaster and opens the body for invasion as some of our trusted partners are either too small in numbers to make any difference or are completely wipe-out of our intimate landscape by the opportunistic intruders. and wound healing becomes a very abnormal exercise for the body to partake in the absence of some of the invisible allies. the mouth is another part of the body that lends itself to constant contact with the invisible world. as it is an extension of our environment, the microbial traffic is high. anytime we open our mouth to eat, drink, laugh, smile, yawn or cry, we are enabling millions of microbes to access our body. however, due to the vigilant watch of resident oral microbes and their anti-microbial components, only a select few can contemplate to occupy a piece of the real estate either in our mouth or lungs. the microbial communities composed of diverse constituents that set foot are scrupulously and carefully interrogated by our molecular sentinels. interactions with prospective residents are permitted only if they work for a common purpose. only when a member can contribute to a specific function that the community depends on, the microbial member is accepted and a shelter is made available. the contribution of each of the member is critical for the functioning of the community. however, this situation can rapidly change specially in the mouth where there is a constant flux of diverse exchanges occurring with respect to microorganisms. furthermore the buccal chemical landscape also undergoes tremendous fluctuation due to the regular intake of foods and drinks. this situation can be complicated if one allows any food component to remain in the mouth for too long. carbohydrate-rich nutrients become easy prey on which some opportunistic microbes can thrive. despite the vulnerability of the mouth to possible invisible intruders, the microbial population is kept in check by the watchful guard of other community members including the host. for instance, in the establishment of dental caries, the streptococcus mutans utilizes these sugary goodies stuck on the tooth to produce lactic acid, an event that increases the acidity of the buccal ecosystem. this presents an opportune situation for other microbes like the veillonella and lactobacillus salivarus/acidophilus to colonize this territory and further aggravate the affliction on the tooth (fig. . ) . the fine microbial balance that is perturbed as a result of ensuing chemical change in the mouth is an ongoing concern for people especially children with poor dental hygiene. this disruption and the resulting medical complication can be averted by controlling the chemical landscape in the mouth. halitosis is also caused by microbial disruption that results in the increase in h s, a molecule responsible for smelly breath. this may also be aggravated by the genetic make-up of the host (fig. . ). the perturbation of controlled microbial growth in the oral cavity is known to trigger the gum disease referred to as periodontics. the homeostatic equilibrium in the microbial community for a healthy gum is maintained partly by the body's own immune system. this is upset by the invasion and colonization of porphyromonas gingivalis, a microbe responsible for the destruction of gum tissues. these degraded components create a nutrient-rich environment that becomes a breeding ground for other opportunistic organisms that are usually kept at bay by our oral microbiome and the lack of appropriate nourishments they can thrive on. this feeding frenzy results in the overpopulation of some species compared to others and give rise to a misfit community of microbes. bacteria such as prevotella and desulfobulbus are two culprits that exploit this situation and assist in the task of invading the gum. thus, dysbiosis an uncontrolled microbial proliferation resulting from the temporary nutritional change in the oral ecosystem is at the root cause of numerous complications that ache both the hard and soft components belonging to this part of the body. the lack of microbial biodiversity in the crypts of the soft tissue like the tongue is at the origin of halitosis, an affliction that emanates unpleasant smell that most of us find repulsive (box . ). the mouth has the second most diverse microbiota in the body with over species. the chemical landscape of the saliva plays an important role in the establishment of the microbial communities that reside in the buccal (continued) box . salivary microbiome and its health impact (continued) cavity. nearly microorganisms are found in one milliliter of saliva, a fluid rich in proteins, lipids, carbohydrates and enzymes. the presence of lactoferrin, lysozyme and lactoperoxidase act in concert to dissuade the colonization by fortuitous bacteria. hydrogen peroxide, hypothiocyanite and nitric oxide derived from nitrate in foods are potent antimicrobial and are part of the defense armoury deployed to fend against cariogenic intruders. however, poor hygiene, nutritional habits, smoking and diseases like diabetes tend to weaken this defense. the presence of increased glucose level generates acidic metabolites that drop the ph of the mouth from . to . . this promotes the acid tolerant microbe like staphylococcus mutans and results in an increase in the firmicutes family coupled with a decrease in the bacteriodetes family. the solubilization of calcium and phosphate is preceded by a variety of dental diseases. furthermore, the dysbiosis created by this situation leads to reduction of nitrate reducing bacteria, a situation restricting the output of nitric oxide, no. this has major health impact including cardiovascular complications. the use of chlorhexidine containing mouthwash also has a negative influence on no-producing bacteria. hence, the saliva and its microbial residents are an important contributor to a healthy outcome. the mouth and the nostrils are constantly providing the lungs the air we need to live. without this obligatory and mundane routine we will not be able to produce energy (atp) that keeps our body machine active. this process is hard-wired in our brain that we keep on doing until we are no more alive. thus we are constantly exposed to the microbes and chemicals in the environment where we are studying, sleeping, playing, working or just relaxing. the lungs are permanently bombarded with the myriad of micro-organisms our surroundings have to offer. although the microbial populations in the lungs are well-maintained, these communities can be harassed by medications we ingest and by the chemicals in the air we are surrounded by. farmer workers, employees in factories and other industrial organizations are at high risk in this regard as they are surrounded by chemical constituents that affect the microbial composition of the lungs. the development of asthma is triggered by the decrease in the diversity of the pulmonary microbial ecosystem. the release of nutrient-rich mucus during lung infection aimed at arresting the proliferation of intruders can also inadvertently act as a magnet for some opportunistic microbes that further compound the problem. a shift in the microbial community favouring a group of lactobacillus is observed during the onset of chronic obstructive pulmonary disease (copd). during cystic fibrosis (cf) the viscous sputum allows the rapid growth of pseudomonas aeruginosa, a microbe that is controlled in non-cf lungs. this microbial imbalance generates chemicals like alginate that further aggravates the situation. the nearly unimpeded pathway of the air tract between the mouth, lungs and the environment render these organs an easy target of dysbiosis. these body parts are challenged on an ongoing basis with changing microbial population and environmental factors such as chemicals and gaseous pollutants that make them a fertile ground for microbial disharmony. hence, it is critical that the crevices in the mouth do not have nutrients stuck for long as these are perfect ingredients that can dislodge the well-established functional microbial communities and inflict us with diseases. the mouth and nose gears are a common sight in cities around the world with high level of pollution. in beijing, new-delhi and mexico city, citizens are often seen wearing these protective screens in an effort to limit the intake of the polluted air in order to diminish its influence on the lung microbiome. during outbreaks of microbial diseases such as severe acute respiratory syndrome (sars) or influenza, this phenomenon is observed around the globe in order to ensure that the lungs are not further burdened by air loaded with viruses as these will undoubtedly promote dysbiosis. the mouth and lungs are the first line of defence to combat the growth of opportunistic microbes lurking around in the air. thus, oral hygiene and the quality of air one breathes in will go a long way in helping maintain the proper microbial balance required for a healthy body. however, if these conditions are not met, microbial disruption resulting from the reduction of biodiversity in the pulmonary and oral landscape can be a cause of concern. it is not surprising that the deteriorating quality of the air across the planet has led to sharp rise in asthma and other lung-related illnesses ( fig. . , box . ). rheumatoid arthritis (ra) is a debilitating autoimmune disorder associated with the inflammation of the joints that can lead to bone erosion and deformity. the hyperactive immune response can also damage other parts of the body like the eyes, lungs and skin. this dysregulation of the immune system triggers pro-inflammatory tendencies with an increase in t-lymphocytes and self-reactive antibodies. although genetic factors may contribute to this disorder, a major shift in the constituents of the microbiome is observed in ra patients. there is an overrepresentation in the provetella species including provetella copri, an increase in clostridium spp and a reduction in bacteriodes spp. this dysbiosis evokes the elevated production of homocysteine, hydrogen sulphide and lipopolyssacharide (lps) that all known to promote inflammation, a feature culminating in the attack of one's own body part. probiotics like lactobacillus casei has been shown to mitigate symptoms associated with ra. asthma is another inflammatory disease where the respiratory airways are challenged by the aggressive action of the immune system. there is an overproduction of mucus and an intense remodeling of the airway wall ensues. the presence of dust, pollen and spores tend to aggravate this situation. however, this disease is characterized by a marked change in the lung microbiota. in children there is reduction in such microbes as the faecalcibacteria, veillonella and rothia while in adults an increase in proteobacteria and a decrease in bacteriodetes and firmicutes are common. hence, restoration of the microbiome with probiotics and prebiotics can be an alternative therapy. the digestive tract is the home of most bacteria in the body. although the vast majority is housed in the intestine, some do find refuge in the stomach. despite the inhospitable environment presented by this acidic organ, the microbes belonging to the helicobacter spp have set-up their home here. they have done so by learning how to tame the low ph these environs are immersed in. they cling to the mucus of the cell-wall where the acidity tends to a bit more manageable. by neutralising this unfriendly territory, these microbes are able to develop a friendly habitat where they can reside. even with these territorial adjustments, the stomach is the organ that harbours the least amounts of bacteria. however, the perturbation of this microclimate can quickly change this microbial landscape. the intake of medications like the proton pump inhibitors and antibiotics tend to promote a shift in this microbial population to the upper confines of the gastrointestinal tract. once, this uncontrolled colony is established, it produces ammonia that compels an empty stomach to generate acid. it is critical that this digestive compartment is acidic only when it has food in its midst. any change in the acidity in the absence of food distresses this organ. it is more or less like the stomach is chewing itself. this becomes the genesis of aches and heartburns that result into peptic ulcers. there is a rise in this microbially-induced disease around the world and the disruption of the fine working arrangement struck among the invisible residents may be at play. it is not surprising that the majority of ulcers can be cured by antibiotics and not by drugs aimed at diminishing the acidity of stomach, a finding that has dismayed many pharmaceutical companies as they have invested heavily on the incorrect cause of the disease. the understanding of how the disruption of these gastric microbial communities will pave the way for better remedies ( fig. . ). the majority of the constituents of our microbiome is lodged in the small and large intestines with the latter harbouring the bulk of the invisible partners. once the colonies are established in these locations, the stability of the residents is constantly being questioned due to the passage of foods and other edibles that transit through. this environment is under constant flux due to our daily intake of foods that can come in all shape, size, content, well-done, rare, or raw. the colonizers have to adapt. low carbohydrate, high fat, high protein, dietary fibres, medications, antibiotics, sugary drinks all have major impact on the ecosystem. this constant pounding by these diverse ingredients, roughly around tons during our life span is bound to take its toll on these miniscule residents within. such a situation is akin to us changing our clothes conatantly in response to temperature changes in the environment. how would you feel if you have to face snow, rain, sun and fog on an ongoing basis and keep modifying your outerwear continually ( fig. . ). whether the food is starchy, oily or meaty; the microbes have to adjust. a shift from a high fat/high sugar diet to a regime of low-fat/plant-based polysaccharides can change the gastrointestinal microbial ecosystem in a matter of days. for instance, while bacteriodes spp thrive in people who consume high fat foods, prevotella spp dominates the guts of individuals who eat more carbohydrate-rich products; refined sugar intake on the other hand favours clostridium spp. the lovers of vegetarian diets are host to less pathogenic bacteria while protein-rich nutrients tend to provoke increased activity of enzymes like nitroreductases derived from microbial sources. this situation contributes to the weakening of the inflammatory response and prevents the metabolism of short-chain fatty acids (scfa). remember the oxidation of scfa like butyrate provides the intestines with an important source of energy and perturbation of this process can result in abnormal physiological functions. preserved foods, dehydrated vegetables and alcoholic drinks with their high content in sulphate provide a fertile ecosystem for suphate-reducing bacteria like desulfovibrio spp to thrive and distress the body. these micro-organisms generate metabolites that restrict the growth of probiotics that help us execute numerous tasks. medications like opioids and the popular diabetic drug metformin impair the mobility of the gut. these are also responsible for shifting and deranging the community-like environment that good bacteria nurture. this disruption provides an opportune situation for bad microbe like the clostridium difficile to multiply. even psychological stress that is known to trigger the production of signaling chemicals like catecholamines has an impact on the microbiome. well-established microbes like lactobacilli and bifidobacteria that are known to confer a number of healthy attributes in the body tend to diminish (box . ). box . food additives, dysbiosis and health impact numerous chemicals are introduced into foods to improve taste, to extend shelf-life, to add volume, to enhance appearance and to impart a variety of other properties. some of these are known to interact with the microbial communities in the gut and perturb the fine communal balance. dietary emulsifiers like carboxymethyl cellulose (cmc) is one of the additives that is widely utilized in foods, cosmetics, and hygiene products. ice-cream, cookies, toothpaste and laxatives are some of the products where this polysaccharide provides texture, viscosity and structural integrity. despite being mostly composed of glucose, cmc cannot be digested either by the visible or the invisible organs. however, it creates a major ecological change in the gut by interfering with mucin, a natural barrier lining the gut. mucin is heavily glycosylated and act as a potent fence against any opportunistic pathogens to access the inner walls of the intestine. it inhibits the absorption of dietary flavonoids and also promotes the proliferation of microbial flora desired by the body. these functions are perturbed by cmc as it shares some chemical and physical features with mucin. this disruption results in dysbiosis leading to the multiplication of pathogens and their translocation into the intestinal tissues. there is an increase in proteobacteria spp and inflammation ensues. chronic intestinal complications have been attributed to these additives. hence, it is not surprising that these chemical, environmental and behavioural changes that the gut microbiome has to contend with invariably lead to the disturbance of the delicate balance cementing the microbial communities and the host in an effective functional relationship. this flux in the gut ecosystem creates conditions for the abnormal proliferation of some members at the expense of others. the subsequent activation of enzymes and production of disease-causing factors can contribute to such maladies as irritable bowel syndrome (ibs), colorectal cancer, rheumatoid arthritis (ra), type diabetes, obesity, cardiovascular complications and neurological disorders. some of the metabolic diseases like diabetes, obesity and heart ailments have reached an epidemic proportion globally. a recent report revealed there are more obese people than underweight individuals, a phenomenon that is a first for human history. an average person has become . kg heavier over each decade. thus, if your grand-father was say kg, your dad is kg and you will be kg at the same age of course. this scenario will be true if nothing else changes. the study on body mass index that was done over years in countries is alarming indeed. policies on food and the quality of food we have access to will help to some extent. it is not surprising to see various north american cities are imposing an obesity tax aimed at sugar-laden soft drinks. examining the role of our microbial ecosystem is also pivotal if this issue is to be remedied. the gut microbiome is an important participant in the manner we digest food, extract the maximal energy and nourish the rest of the organs. a disruption in the microbial landscape can trigger metabolic diseases. for instance, during irritable bowel syndrome (ibs) microbes belonging to the firmicutes family decrease while an increase in members of the bacteroidetes family is observed. this disorder in the intricate microbial balance propels a concomitant rise in facultative bacteria like the enterobacteriae spp. the unregulated establishment of the microbial community tends to impair the integrity of the mucus lining of the intestine. this shift perturbs the ability of the resident intestinal microbes to properly communicate with the mucosal barrier; such a situation results in the inability of the body to tolerate the harmless bacteria and neutralize the invading squatters. following this distress imposed on this natural fence, conditions are perfect for the release of signals and chemical shuttles by the microbes to enable their migration across the intestinal defensive wall. this sequence of events forces the host to activate its guards with no clear mandate on the proper target and the result is an unintended inflammation giving rise to crohn's disease and ulcerative colitis. these are the two most prevalent forms of ibs that can be traced to the perturbation of microbes residing in the gut. in fact, these diseases can be mitigated by increasing the presence of firmicutes in the gastro-intestinal tract. it is clear that the conditions promoting the opportunistic microbes and the colonizing factors they produce are at the root cause of these disorders and re-establishing the original microbial communities goes a long in alleviating the pain of individuals suffering from these complications. obesity is a major problem afflicting the world. the rise in overweight individuals has been staggering and this malady attacks regardless of any economic and geographical boundaries. almost all countries surveyed are experiencing the illeffects of this metabolic disease. a variety of elements such as diet rich in simple carbohydrates, low in vegetables and fibres; life-style and genetic factors may be at play. however, the disturbance of the microbial harmony in the body cannot be discounted as a potential trigger responsible for the soaring rate of obesity around the globe. there is a clear distinction in the microbial landscape associated with obese people compared to underweight individuals. lean individuals tend to have higher amounts of microbes belonging to the bacteriodetes family while obese subjects and those suffering from metabolic syndrome are home to more members from the firmicutes family. although the specific species of these two microbial families responsible for the lean and obese traits have yet be catalogued, it is clear that obesity is characterized with increased levels of toxins like lipopolysaccharide (lps) in the blood. symptoms associated with weight gain and decreased sensitivity to insulin have been attributed to the presence of microbial toxins. hence, modulating the nature of the gut microbiome and restoring the proper microbiota have to be included as part of the strategy dedicated to combating these disorders. the food we eat is a major instigator of the establishment and fluctuation of the microbiome we possess. in fact, the adage we are what we eat applies aptly to the microbiota that constitute our body. one can even add that our physiological processes are the way they are because of the microbes we own. these invisible partners that accompany us throughout our live-journey are part of the elaborate communities responsible for our well-being. however, this is not a permanent relationship as it is susceptible to evolution and disruption by what we eat. this intimate relationship that we have forged with our invisible partners and all of our other bodily organs faces lots of twists and turns as we navigate through our lifelong journey. in the case of our microbial ecosystem these changes are non-stop as it is extremely prone to the vagaries of what we put in the mouth. and this is a daily activity that is most relentless and continual and commences the day we are born and ceased only upon our death. indeed it is important to remember that every component of body is in constant flux, we are never exactly with the same body parts that we are born with. these are evolving every second. for instance, we get a new skeletal system every years and this happens on an ongoing basis day by day. otherwise you will be soft like jelly one day and then wake up the next morning with a brand new skeleton. thus, depending on our eating habits, we may be saddled with a microbial ecosystem that can promote or impede our wellness in the same way we have toned body if we exercise or we are stuck with a flabby belly if we are a potato couch. a fat-rich diet together with a combo of a sugary drink is sure a concoction to upset the fine balance of our invisible organ. such a nutritional habit is responsible for the non-alcoholic fatty acid disease (nafld) and cardiovascular complications. these greasy foods are known to promote the proliferation of some bacteria and arrest the growth of others. the establishment of this unbalanced ecosystem is a perfect brewing ground for the genesis of liver and heart diseases. these oilladen nutrients contain copious amounts of choline, a chemical responsible for the yellow colour in the egg yolk. it plays an important role in the development of the brain in children. however, its excessive consumption via the food we intake specifically in adults can be problematic and the unwelcomed microbial residents aggravate our overindulgence in these choline-containing goodies. in reality our gut microbiota transform this into a product known as trimethylamine (tma). this diversion of choline into tma prevents the body from making some good lipids and subsequently leads to the accumulation of the bad lipid known as triglycerides. remember the number associated with triglycerides in the blood of adults. this magic number if higher is a relatively precise predictor of potential coronary abnormalities. these triglycerides have nowhere to go but to accumulate in the liver where they become the precursor of fatty acid disease. this is not all that can come out of this greasy food, it can become worst. if you harbour microbes that can metabolize tma i.e. process it into the oxygenated variety known as trimethylamine oxide (tmao), this can spell danger for your heart. the benign tma falls prey to microbial manipulation in our gut that renders this relatively innocuous agent into a toxin with devastating impact on our well-being. tmao is an important tell-tale sign of cardiovascular diseases. there are other intestinally derived toxins fuelled by microbial activities that can cause havoc to the normal functioning of our body. sulphate derivatives of aromatic chemicals originating from microbial intervention in the intestine can be a major burden on the kidney and may lead to renal abnormalities. controlling the microbes that are in the business of transforming harmless chemicals into noxious ones is critical strategy that need to be pursued in order not to succumb to the microbial imbalance promoted by fatty foods (fig. . ). even though we may not be completely aware there is an important communication corridor between the gut and the brain that is nourished by numerous signals emanating from the microbial activities in the intestine. the gut-brain axis relies on the messengers and/or their precursors generated by the microbes we harbour. they produce short-chain fatty acid (scfa) like butyrate and neurotransmitters like γ-aminobutyric acid (gaba), dopamine and serotonin. whenever you have a rewarding feeling due to some activity like eating that you are engaged in, blame it on dopamine. these instruction-laden chemicals that are also produced by other organs in our body are central to numerous tasks executed by the brain and can stir our behaviour in one direction or the other depending on the concentration of a specific neurotransmitter. hence, factors that modulate the flux of the microbial communities and their propensity to dish out these neuro-active commanders have tremendous influence on our health and well-being. for instance the lack of microbial diversity associated with ageing impedes the ability of the body to generate scfa such as acetate, propionate and butyrate that supply the brain cells with energy. the loss and/or the rationing of the nutrients fuelling the brain result in a reduction of cognitive and memory power. this attribute is a common characteristic reminiscent of ageing. hence, the propensity of people in retirement homes not to remember or retain mundane facts (fig. . ) . the microbiome definitely has its imprint on the functioning of the brain, a feature that has yet to be fully deciphered. for instance, the amino-acid tryptophan is the precursor of numerous neurotransmitters that dictate a variety of our behavioural responses that are usually tailored to the stimuli we receive. this is an essential nutrient that humans cannot make with their own traditional organs and have to acquire it from the food we eat. however, depending on our microbial communities we can easily have ample supply as numerous gut microbes are capable of synthesizing this nutrient; but with caveat that the amounts of tryptophan we have access to need to be modulated if our response is to be commensurate to its presence or its derivatives. any abnormal variation of this commodity in our body can become a harbinger of neurological complications. a shift in microbial communities induced by a myriad of factors can create havoc with blood tryptophan levels. this can then results in depression, mood swings and neurological ailments like parkinson's or alzheimer's diseases. keeping a careful watch on our fig. . dysbiosis, abnormal metabolite production and neurological disorders microbial partners and their ability to generate these brain responsive signals will lead to better health outcomes (box . ). the ability of the microbiome to produce a variety of neuroactive chemicals makes the invisible organ a potent modulator of human behavior and an instigator of neurological diseases. for instance, parkinson's disease is an incurable adult neurodegenerative disorder characterized by abnormal movements due to defective motor control. dopamine production is impeded and there is an increased formation of aggregated neurotoxic protein known as α-synuclein. these two biochemical manifestations may be shaped by the nature of the microbial residents by limiting precursors of dopamine and activating the immune system with the release of pro-inflammatory chemicals like lps. the gut microbiome in parkinson's patients is usually characterized by a decrease in prevotella spp and an increase in enterobacteria spp. a diminution of mucin synthesis increases intestinal permeability and leads to an acute dysbiosis that contributes to the abdominal discomfort, bloating and premature satiety reported in these patients. autism spectrum disorders (asd) are another set of neurological perturbations leading to social and behavioural impairments including repetitive behaviours and nonstandard communications. here again, the microbiome may be a contributing factor. a decrease in veillonella spp coupled with a rise in clostridium spp and campylobacter spp have been reported. microbial products like pcresol has been observed in elevated amounts in autistic children and -ethyl sulfate ( -eps) has been shown to trigger anxiety like behavior. this microbial link is prompting the search of probiotics (e.g. bifidobacterium fragilis) that may help in curing these diseases. cancer is a multi-factorial disease that is a major global concern. almost everyone knows of someone who has been affected by this disease. numerous governments have recognized this disease as a national priority and have decided to provide all the support necessary to fight cancer. although life-style, genetic disposition and environmental factors are important contributors to the spread of this disease, it is becoming amply clear that our microbial landscape and cancer proliferation are interconnected. it is being increasing recognized that the microbiome plays a key role in preventing and promoting cancer. the gut microbial ecology provides an important cover against invading pathogens that contribute to the onset of tumour formation. however, a shift in microbial communities induced by diet and pollution creates a favourable environment for opportunistic microbes to thrive and neutralise the protection offered by the finely tuned original microbial guards. the invading microbes produce virulence elements responsible for unimpeded cellular growth, a key feature of all cancers. they also put in motion processes that result in the instability of the dna and furthering the progress of cancerous cells. understanding the communal work of the intestinal microbes is pivotal if this problem is to be tackled. indeed it has been shown that africans from rural areas exhibit lower risk of colon and rectal cancers due to the increased abundance of the microbes known as bacteroides spp. these are avid producers of short-chain fatty acid like butyrate, an important energy-producer and a promoter of cellular growth. this is a critical signaling molecule that can goad the body to synthesize sentinels that stop cancer cells on their tract. on the other hand, the group of individuals whose colon is home to higher amounts of prevotella spp tend to have higher levels of bile acid and lower amounts of butyrate. this switch in colonic microbial ecology is suspected to be responsible for the colorectal cancers ( fig. . ) . the prevotella-rich individuals are programmed to secrete bile acids in the feces. these compounds are known to promote the proliferation of cancerous cells. the interplay between these two microbial communities and their ability to produce these metabolites afforded wellness to one group and colorectal cancer to the other. hence, the promotion and nurturing of functional microbial communities and limiting the presence of unregulated microbial ecosystem in the gut are pivotal to maintaining our wellbeing. this invisible organ has to be tended in the same manner like any other visible organ; one cannot expect to have working lungs supplying the furthest nooks in the body with oxygen if one indulges in smoking or toiling in occupation with toxic air. this cardinal rule also applies to the microbiome even if its anatomical features do not readily pop out to our eyes. otherwise we run the risk of turning this organ that we almost never see but hear rumblings of once in a while into a cancer-causing entity. the critical attributes of these microbes need to be tapped as cancer preventers and fighters. the microbiome is an essential component of humans if not of all multicellular organisms. this invisible component of the body that quite often makes itself heard with a rumbling sound is involved in a variety of biological tasks that are essential for the proper functioning of any human. although these microbes residing in and on us have their genetic information they are bound by the communal existence they have adopted. the role and behaviour of each member are dictated by the host and other members of the collectivity. this harmony and synchronicity of purpose force everyone to comply with the rules set-up during the establishment of the community. however, these communities evolve in relation to the host and the changing landscape they have to deal with. they accompany us from the very beginning when we are developing till death. it is becoming more apparent that these microbes contribute to our development and eventually mould us the way we are both anatomically and physiologically. they have a say virtually in all aspects of our life and prod us as we go about our daily living. from providing essential nutrients like vitamin k that we are dependent on but incapable to make on our own to protecting us against opportunistic organisms and helping heal our wounds, these microbes partake in a myriad of tasks just as visible organs do. however, their constituents and interactions like all other organs in the body are modulated to large extent by the changes in environment, food, stress, and hormonal fluctuations we are subjected to. the invisibility of this organ and its dispersed anatomy have contributed to the relegation of the microbiome to a low esteem in the hierarchy of our body parts. but this view is being discarded at a rapid pace as intriguing information about its role is being revealed. its intimate link to our existence was only visualized very recently. what you cannot see, you cannot appreciate. this dictum fits perfectly the microbiome. the advent of next generation sequencing (ngs) coupled with bioinformatics tools is laying bare the indispensability of the microbial communities roaming within and on us to our very existence. our life will not be the same without our intricate network of microbial communities. hence, understanding how this invisible communal habitat is constituted, how it evolves and how the various partners communicate amongst themselves is crucial for our being. the host will not exist without the microbiome. it is as crucial as the heart and any other body part. this mission is as critical as our desire to unravel the workings of the brain or to venture in the outer galaxy. its systematic functional identification will be akin to the discovery of a new organ as in occurred in the sixteenth century when human was first beginning to comprehend the workings of various body parts. just imagine the excitement when the italian surgeon, realdo columbo identified how the four valves of the heart permitted the flow of blood in only one direction i.e. from the right ventricle to lungs and back to the left ventricle and on to the aorta. we are on the brink of tantalizing discovery on a part of our body that has long been ignored due to the lack of proper tools to decipher the microbial communities. it is indeed more exciting as this invisible organ has at least , times more genes than our own visible body. the microbiota we possess must play an important role in our development and we are physically and physiologically the way we are because of them. we would be entirely different if it was not for the microbiome and this holds true to most if not all organisms wandering on this planet and most probably beyond. the proper functioning of this community with multiple partners depends on the finely tuned relationship among all the constituents. every member operates in a mode that is beneficial to this cooperative life-style where interdependence is the modus operandi. however, this harmony can be perturbed by a variety interfering influences and can result in a multitude of ailments. the intrusion of opportunistic organisms fuelled by a change in the ecosystem gives rise to dysfunctional communities that are the precursors of debilitating diseases like cancers, ibs, and vaginosis. the latter is a disease arising from the uncontrolled proliferation of microbes such as gardnernella vaginalis, veillonella spp, and bacteriodes spp. that promotes an increase in ph, fatty acids and polyamines (fig. . ) . cadaverine, a polyamine secreted during this dysbiosis is the cause of the malodor characteristic of the disease. once the fine balance involving the constituents and the signals that are responsible for a proper microbial community is uncovered, we will be in a better position to predict what makes us tick and what ails us. this knowledge is just now beginning to emerge and ignoring this important aspect of our body will be to our peril; it would be akin to not wanting to know how the brain functions (fig. . ; fig. . ). the microbial communities that constitute our microbiome are dependent on a myriad of factors such as the genes we inherit, our mother's milk, the environment we live in, our hobbies, the seasonal changes we are subjected to, the food we eat, the life-style we lead and the medications we take. despite the ability of our microbiota to respond and adjust to these situations, the invisible organ can be influenced by either taking in select beneficial microbes with known functional attributes or by consuming foods and plant products that promote the proliferation of specific microorganisms. this microbiome-rearing strategy is akin to feeding our brain with the books we read or the educational programs we watch or the games we play. remember some eager mothers keen on giving their children a head-start in life, read to them or play music even when babies are in the womb. to promote a healthy life-style, seniors are being encouraged to partake in neuron-stimulating games on a regular basis. this brain exercise has becoming a common place in residences for the elderly. thus probiotics, prebiotics and synbiotics can be utilized to guide the microbiome to perform optimally and ameliorate our well-being (fig. . ) . probiotics are live microbes administered as foods or even in capsules that confer health benefits on the body. microbial cultures have been consumed by humans since the dawn of civilization. mongolian women sprayed fermented milk on horsemen and their horses in belief that this provided strength and health on the recipients, while in some civilizations fermented milk was utilized to treat a variety of ailments. dahi, a fermented milk is widely consumed in the indian sub-continent and is known to impart numerous healthy outcomes. however, the first clinic trials on the health claims of these bacteria-rich fermented foods were not performed until the twentieth century. eli metchnikoff who obtained a nobel laureate in medicine was first to correlate the longevity of some bulgarian citizens to the prolific consumption of fermented milk that was rich in lactobacillus bulgaricus and streptococcus thermophilus. in , the japanese scientist shirota isolated microbes with probiotic properties from healthy human subjects. these were later utilized in the development of milk products that were commercialized as yakult. subsequently, a french pediatrician reported the lack of bifidobacteria in stool of infants suffering from diarrhea. in the term probiotics was coined by lilly and stillwell to describe products that stimulate the growth of microorganisms beneficial to the body. it was in the first probiotic species, lactobacillus acidophilus was introduced followed subsequently by bifidobacterium spp. their use in food products is widespread. the significance of probiotics in fortifying the immune system has officially been recognized fig. . nurturing of the microbiome, the brain and the muscle. (activities and nutrients involved in fortifying these organs) fig. . consumption of probiotic-rich foods around the globe by the world health organization (who). currently a wide variety of microorganisms such as e. coli, propionobacterium, enterococcus, streptococcus, leucomostoc, and bacillus cereus are being consumed in order to regulate and adjust the body's microbiome (fig. . , box . ) . box . probiotics: occurrence and uses worldwide humans have been consuming microbe-laden foods unknowingly since the dawn of civilization. fermented milk, vegetables, meat and fish were and are a regular part of the daily diet for most people around the globe. recently probiotics enriched foods like yogurt, yakult, kefir, dahi and cheese have become a regular repertoire of natural products that nutritionists are eager to extoll the health virtues of. lactobacillus spp, bifidobacterium spp and bacillus spp. are the more prominent probiotics even though other microbes are being added to this list. they are also being used in non-consumable items like oral care gel and anti-ageing serum. their ability to produce acid, enzymes, scfa, immune responsive factors and to help establish functional microbial flora have made these probiotics excellent candidates to cure a variety of diseases that are provoked by dysbiosis. while e.coli nissle provides relief to patients suffering from ulcerative colitis, a disease like vaginitis can be remedied with lactobacillus rhamnosus gg. irritable bowel syndrome victims can find comfort with the intake of bifidobacterium animalis and lactobacillus acidophilus. the role lactic-acid microbes like bifidobacterium breve has been recognized in diminishing high blood pressure due to their ability to produce vitamin d, anti-hypertensive factors and interrupting cholesterol absorption. although influence of probiotics in promoting wellness and as therapeutic agents aimed at numerous illnesses are becoming more prominent, their usage will become universal once the dosage and the intake frequency of these microbial supplements have been properly evaluated. probiotics not only help the body to fine-tune the microbiome, they can also perform some very specific functions. depending on the probiotics, these microbes act as a barrier and prevent colonization by opportunistic bacteria. they do so by re-enforcing the protective function of the gut mucosa and by generating signals that arrest the invasion by infectious organisms. they produce antibiotics, antioxidants and improve the body's immune system. probiotics are also known for their ability to synthesize scfa and vitamins, ingredients essential for numerous metabolic activities. they secrete enzymes that help in digestion and in the elimination of wastes. a good probiotic should be able to impart health benefits to the host and be bestowed with no pathogenic properties. its presence in the microbial community should enhance some biological functions and/or curtail any negative influence on an individual's well-being. these can preferably be taken orally and must survive the harsh environment of the digestive system, especially the stomach where the ph can be unforgiving. the low ph of the stomach can be avoided if the probiotics are mixed with food such as milk, dietary fibres and yogurt. they can be designed to generate specific biomolecules that can be of benefit to the host. for example, lactose-intolerant individuals can consume probiotics with the ability to synthesize lactase or galactosidase that helps in metabolizing the milk sugar, lactose. probiotics have virtues almost like stem cells. stem cells i.e. cells that have not yet made up their mind what they will become or which organ they will develop into can be utilized as a therapy to fortify ailing components of the body. for instance, they can be injected in the cornea or the liver with the proper commands that enable them to mature into corneal or hepatic cells that become part of the rejuvenated organs with optimal biological functions. probiotics act in a similar manner in regard to our microbial communities. they can be consumed with the aim of enhancing a specific function like in the case of a select targeted fortification that is desired in the body. individuals suffering from the inability to digest milk are readily relieved of their aversion to milk or milk products by the consumption of lactobacillus. this microbe secretes lactase, an enzyme that can clip the milk sugar lactose into glucose and galactose which are then utilized by the body. probiotics such as aspergillus oryzae found in fermented foods like soy sauce, and sake secretes amylase and lipases that help in the digestion of starchy and oily meals. in instances where the microbiome has been infiltrated by opportunistic microorganisms that prevent the invisible organ from performing its regular task, an intake of the probiotics can shift the microbial balance toward a more fruitful one for the body. one case in point is the colonization of the dental space by streptococcus, a microbe responsible for carving cavities on the tooth. food enriched with various species of lactobacillus can rectify this situation by creating an unfavourable situation for the invading bacteria to survive. among other various biological weapons they have in their armoury, lactobacillus group of probiotics can generate an acidic environment. this situation is known to impede the growth of the occupying bacteria and arrest their assault on the teeth. unlike the stem cells that are mainly involved in regenerating desired tissue or organ, probiotics can tilt the balance of a non-functional microbiome towards a functional one by quashing undesirable elements in the community. they can also be engaged in a targeted task designed to thwart a discomfort an individual is experiencing as in the case of intolerance to lactose or abnormal movement of the digestive system. world-wide people have been consuming fermented products laden with probiotics. these foods have evolved through centuries and each region has its own speciality of delicacies full of microbes. these probiotics have become part of the culinary culture and it will not be surprising if we learn that our palates have evolved accordingly. yogurt, a diary product supplemented with microbes lactobacillus, bifidobacteria and lactococcus is part of the grocery shelves in almost all countries. in india dahi, a fermented milk product rich in lactococcus lactis and lactobacillus acidophilus is taken with nearly all foods and its curative powers are readily touted. in the caucus region, kefir with its bountiful of lactobacillus, lactococcus, leuconostoc and bifidobacteria is a daily staple. this is derived from goat's milk and contains yellowish grains resembling cauliflower. these grains are in fact polysaccharide capsules full of probiotics. in japan the miso soup prepared from barley, rice, beans and rye is king while in africa uji the probiotics charged maize or sorghum or millet is widely cherished. although not currently available probiotics with the ability to metabolize ethanol or cure obesity will be a welcome help to those individuals who are susceptible to adverse reaction upon intake of alcoholic drinks or have difficulty controlling their body mass index (fig. . ). the mouth is literally a continuation of our external environment and is being constantly challenged by the microbes we are exposed to in our surroundings. even under this threat, the soft and hard components of the mouth harbour a good number of microbial communities that allow them to fulfill various essential tasks. the change in the chemical milieu in the mouth triggered by the intake of foods provides a fertile landscape for foreign organisms to set foot, proliferate and disturb the working microbial harmony. this happens at the onset of dental cavities when streptococcus rapidly colonizes the tooth. in this instance, a probiotic like lactobacillus rhamnosus can spring in action and create an acidic environment by the secretion of lactic acid. its ability to produce antimicrobial factors administers another debilitating punch thwarting the streptococcus-driven assault. this probiotic also helps train the immune system to fend the noxious microbe. bile, a product that is produced in the liver has been shown to be involved in a variety of tasks that make the body ticks. its acid derivatives are emerging as important signals that allow us to control our weight, regulate the level of cholesterol in the blood and determine how much fat we are going to store. the key enzyme that facilitates this formation of the bile-derived acids is bile salt hydrolase. a number of probiotics like lactobacillus, bifidobacteria, bacteriodes and enterococcus spp is known to secrete this enzyme. these probiotics can be designed to produce this enzyme that will be an excellent tool to control weight gain. this role of probiotics is akin to the use of lactase-producing lactobacillus. intake of food enriched with lactobacillus is a common therapy given in order to easy the misery of people suffering from the aversion to milk. in fact, lactase derived from probiotics is the most popular form of therapy for this ailment. nearly - % adults in east asia cease to produce lactase as they reach adulthood. although it is totally natural not to have lactase after the weaning years, the omnipresence of dairy products significantly raises the importance of this enzyme beyond our childhood. hence, the remedy provided by lactobacillus-supplemented foods or capsules is a boon to the adult population world-wide who wants to partake in their dairy-laden delicacies. the curing of enzyme deficient diseases or conditions with the aid of probiotics will become a common practice as our understanding of the microbiome becomes clearer (fig. . ) . in diabetic patients a microorganism referred to as akkermansia muciniphilia that shelters in the mucus in the intestine can be an excellent candidate to promote carbohydrate metabolism. these microbes constitute - % of the gut microflora and are known to stimulate glucagonlike peptide (glp), an important modulator of blood sugar. glucagon and insulin are the sugar police in the blood. glucagon becomes active at night during asleep. this is when the blood sugar is low. the sugar sentinel ensures that we wake up healthy by maintaining the right sugar balance. insulin on other the hand, protects against high blood sugar. for instance, when you indulge in a big bar of chocolate, it preserves the proper blood sugar and directs the extra to be stored. hence, people with a hardy dose of a. muciniphilia are less insulin resistance, a major contributing factor to obesity. the abundance of these microbes decreases with age and the intake of high fat diet. the magic of a.muciniphilia is in the production copious amount of mucin, a slimy lining for intestine that promotes a diverse microbial ecosystem and fends off opportunistic microbes. hence, the population of this blood-sugar balancing microbe can achieved by either taking it as a probiotic or by consuming nutritional fertilizer like fibers aimed at stimulating its proliferation. bifidobacteria is another group of microorganisms that dissuade infectious microbes to colonize the gut by blocking the anchoring sites and stimulating the immune system. it is not surprising that they constitute almost % of the cultivable microbes in the stool of infants as opposed to the meagre % found in adults. they contribute to the defense power in babies as they are vulnerable at this tender age (fig. . a) . infants especially those who are born premature can also suffer from damaged intestinal tissues that begin to die. this disease known as necrotizing enterocolitis is characterized by bloating and swollen abdomen responds favourably to probiotics. use of the probiotic lactobacillus is quite effective in mitigating this disease. on the other hand, the intervention with a concoction of bifidobacterium, propionobacterium and lactobacillus significantly reduces the risk of allergy in infants delivered by c-section. in fact these c-section babies are devoid of the microbes that mothers impart on them if they are not born naturally. recent studies involving the sprucing the babies with the mums' microbiota are showing positive results. this kind of probiotic treatment where someone else or one's own microbe is used as probiotics to rectify aberrant biological activities is becoming more prevalent. transfer of microbes from fecal matter, ear wax, skin and other body parts is gaining medical traction and are being implemented in health centers world-wide. microbial transplant undoubtedly impart numerous health benefits (fig. . b ; . c). elderly individuals are another segment of the population who can benefit tremendously from the enrichment of their microbiome with probiotics. ageing is characterized with a sharp decline in microbial diversity and density. this phenomenon is the leading cause of the decrease of metabolic activities and in some cases of physiological abnormalities observed in seniors. the lower abundance of bifidobacteria coupled with the increased presence of enterobacteriaceae is a common predictor of the ageing process and such a shift in microbial population is responsible for the diminished production of enzymes and essential vitamins like vitamin b that are critical in maintaining a constant supply of energy in the body. there is also a build-up of toxic products as the proper microbial communities involved in their decomposition are severely hampered. this situation is analogous to someone not going to the toilet on regular basis. the build-up of noxious elements in the colon can be the cause of a variety of ailments including head aches and digestive discomfort. the advanced age of centenarians is attributed to a diversity of microbial populations that are a hallmark of all individuals living beyond the ripe of age years. the mission of these microbes may be compared to the role stem cells play in rejuvenating the organs and extending their functional life span (fig . ). in this instance the diversity of the microbes observed in centenarians may be allowing these microbiota to supplement some of the functions their visible organs cannot perform or to generate chemical ingredients triggering the proper physiological responses from these ageing organs. hence, harnessing the power of probiotics in reconfiguring the dysfunctional microbiome characterized by the ageing process can be an important gamechanger in the life-style of seniors. this will be critical in seniors who are usually on microbiome-distressing medications. that is why it should not come as a surprise if elderly individuals take more time to heal and are invaded at a rapid rate by opportunistic bacteria. in fact microbial infection is a major concern when elderly patients are admitted to health facilities. they may be in for a hip replacement but unfortunately they run a higher risk of being contaminated by hospital dwelling-microbes. the prevalence of hard to eliminate microbes like clostridium difficile in elderly patients is a problem world-wide. and often infectious outbreaks occur in these places. unbalanced nutrition and erratic eating habits induced by reduced sensation in the olfactory and taste systems add to the dilemma facing seniors. these conditions create a nutritional environment deficient in essential nutrients like calcium and vitamins that further exacerbate the microbial ecosystem. intake of the probiotic bifidobacterium longum stimulates the proliferation of other bifidobacterium spp. administration of lactobacillus acidophilus is known to increase the synthesis of antioxidants like glutathione and oxidant bursting enzymes like catalase. while the regular consumption of bifidobacterium animalis helps reduce the time food transits in the gut, the probiotics lactobacillus rhamnonus and a propionibacterium spp contribute to the improved defecation frequency. hence the implementation of a nutritional regime rich in probiotics will go a long way in easing the discomfort and some of the ailments that are associated with old age. evidence-based research is revealing how probiotics can help reverse ailments caused by the disruption of the microbial communities in our body. irritable bowel syndrome (ibs) is one such disease where a perturbation in microbial population is the main cause of the disease. this shift in the ecosystem of the microbiota is punctuated by a loss in immune tolerance and a rise in inflammatory response. to mitigate this situation and to remedy this ailment, the administration of probiotics like e.coli nissle has been relatively more effective than the use of antibiotics. in this instance, the probiotics appear to deliver a three-pronged attack on the invasive and crafty microbes. they improve the fence lining the intestine, they out-compete the bad bacteria and they secrete molecular soldiers like hydrogen peroxide and lactate that stop invaders right on their tract. the significance of probiotics as therapeutic agents is only now beginning to be appreciated and is slowly challenging the aggressive use of antibiotics in treating these conditions. unfortunately, antibiotic-based remedies tend to seriously interfere with the workings of our microbiome as their action against toxic microbes also eliminates some of our trusted invisible partners. the uncontrolled proliferation of the stomach residing helicobacter pylori is a cause of concern as it is a risk factor for gastroduodenal ulcers and lymphomas. the intake of probiotics such as lactobacillus and streptococcus thermophilus has been demonstrated to correct this imbalance. the ability of these probiotics to secrete scfa like acetate and butyrate is known to be a key factor in impeding the march of the over-reactive h. pylori. wound healing and cancer prevention are other two activities that probiotics are also involved in. lactobacillus acidophilus that is an excellent candidate with the power to heal injuries is known to arrest the colonization driven by the scheming pseudomonas aeruginosa. the acidic environment nurtured by the lactobacillus renders the ability to conquer the tear on the skin almost impossible. the propensity of probiotics to bind to mutagenic compounds and inhibit the production of carcinogens by opportunistic pathogens is central for their anti-tumour activity. the lactic acid producing bacteria such as lactobacillus acidophilus, bifidobacterium bifidum and streptococcus lactis are routinely being recommended for the prevention of cancers. some select tumours like in colorectal cancers are being treated with the aid of these life-microbial concoctions. hence it comes as no surprise that the use of probiotics as a prevention of diseases and a therapy against some specific disorders is on the rise. the commercial value of this industry is into hundreds of billions of dollars, a figure that is further expected to increase as probiotics are safe and well-tolerated in children, premature infants and in the general population as a whole. furthermore, unlike pharmaceutical products, probiotics are very easily administered mostly as components of foods and the lingering effects of these chemicals are quickly becoming a relic of the past. the microbiome can also be nurtured and programmed with the assistance of prebiotics. these are natural products that foster an environment propelling the proliferation of some desirable microbes. in the same manner as we are always eager to give our visible organs a boost when they are not working well or when they are sluggish, we can revitalize our invisible organ. our more traditional visible organs can be invigorated by taking supplements like vitamins or minerals that provide ammunition to various metabolic activities in the muscle, brain, heart or liver. for instance, athletes take creatine, a product responsible for energy stabilization to improve their performance. creatine and creatine kinase duo enables us to engage in physical activities quickly and fast. without the intervention of this dynamic couple, daily chores will happen in a relatively sluggish manner. individuals with excess body mass consume carnitine with the hope of inducing the various body parts in burning fats. well, the constituents of the invisible organ can be strengthened with the intake of prebiotics. these are like fertilizers that create the proper environment for the desired microbial ecosystem to flourish. they tilt the microbial community in a manner favouring some specific tasks to be performed. for instance, a prebiotic promoting the growth of lactase-secreting microbes will help lactose intolerant people while a prebiotic inhibiting the competitors of vitamin k-producing bacteria will be of immense assistance to patients having difficulty with blood coagulation. prebiotics are excellent nutritional supplements involved in modulating and adjusting the microbiome that can undergo disruption due to ageing, infection, climate change and a sleuth of other factors one may be subjected to as one goes through one's daily activities (fig. . ) . what are these magical prebiotics? they are usually plant derived fibers and complex carbohydrates contained in milk that help promote the growth of specific microbial ecology in our body especially in our digestive system. they are mostly resistant to digestion by the enzymes produced by the gut. they are metabolized only by our invisible organ and/or promote proper microbial communities that impart health benefits to the host. the production of short-chain fatty acids (scfa-acetate, butyrate) in response to the intake of prebiotics can contribute to the energy budget of the muscle, can regulate cholesterol biosynthesis and can provide fuel to the colon. stimulation of calcium absorption, reduction in infections and repression of allergic symptoms are some of the other activities prebiotic intake is known confer on the host. these physiological processes are of course a major boon to our well-being as these are like lubricants oiling our body machines. the prebiotics prime the microbiome and this invisible organ is reflective of the prebiotics consumed. as a toned muscle system is indicative of the time spent at the gymnasium, specificity and diversity of the invisible organ is a tell-tale sign of our dietary fiber-eating habit (fig. . ) . prebiotics or dietary fibers have been part of human nutrition since the dawn of civilization. in bc hippocrates had noted the laxative attribute of coarse wheat compared to the refined variety. in the s kellogg sang the praise of bran as a nutrient that increased stool weight, prevented diseases and acted as a laxative. all health gurus extoll the praise of the nutritional quality of fiber-rich foods. after a relative quiet epoch in the health benefits of the dietary fibers, their involvement in mitigating ailments like obesity, cardiovascular disorders and diabetes were widely revived in the s and has now become a staple of balanced nutrition. the main prebiotics are the galacto-oligosaccharides (gos) obtained from milk, inulin associated-fructo-sugar (fos) found in chicory roots and xylooligosaccharides (xos) present in plant products like palm oil and corn plants. they also occur naturally in milk, asparagus, garlic, onion, leeks, wheat, oats and soya beans. they are soluble, resistant to the acidic environment in the stomach, fermentable and stimulate growth or the activity of intestinal microbes responsible for the well-being of the host. prebiotics tend to be relatively selective to microorganisms like bifidobacteria and bacteroides. they are converted into simpler derivatives before they reach the colon. here they act as an anchor for the establishment of a unique ecosystem that is aimed at aiding the host in accomplishing a variety of tasks. they generate energy that fuels the muscles and the colon. they are also involved in repressing allergic symptoms. the reduction of infection and promoting the proliferation of good microbes like akkermansia are some other positive features prebiotics contribute to. they are also known to decrease glucose absorption, activate cholesterol excretion, and promote laxation. one of the most intriguing influence of prebiotics in dictating our microbial ecosystem and promoting our well-being has been observed in infants just immediately after birth. babies who are fed their mothers' milk have much better health outcomes than those fed formulated milk. they are less susceptible to allergic reactions and kids nourished by natural milk tend to be less prone to asthma. the gos, a natural ingredient in the mother's milk cannot be digested by the babies' developing guts. they are designed to promote the growth of microbes that are crucial for a variety of tasks at this critical juncture in their lives. one can safely conclude that mother-nature is making these goodies not for the babies but for the microbes feeding on them. in fact, the presence of a unique ingredient known as sialic acid in the milk of some malawian mothers result in well-nourished infants as opposed to infants who are fed by mothers without this magical tonic in the milk. this prebiotic is responsible for the growth of a unique set of microbes that generates simple sugars utilized by infants for their development. even from such an early stage of human life, the body is programmed to depend on these invisible partners. it is not surprising to learn that various milk products are being formulated with oligosaccaharides in an effort to generate increased microbial population in growing infants. the elderly can also benefit from the intake of prebiotics as their microbiota tend to undergo a drastic change and there is a significant reduction in the diversity and abundance of the microbial ecosystem as age progresses. the fructooligosaccharide (fos)-based prebiotics have an excellent bifidogenic activity while resistant starch like sprouted legumes improves bowel movement by promoting the growth of lactobaccilli and bifidobacteria. these microbial fertilizers can be given to relieve seniors of constipation, a condition quite acute in this segment of the population. prebiotics not only promote a healthy life, their intake can be tailored to remedy a number of ailments that are due to dysbiosis i.e the perturbation of microbiota in the body. their ability to nurture a select group of bacteria that possess a range of tools to combat the opportunistic invisible invaders can be put to good use. obesity is a disease that is characterized by low grade inflammation and is punctuated by a decrease in the diversity of gut microbiome, a situation resulting in the ineffective harvesting of calories from what we eat. the inadequate energy extraction from food intake automatically triggers the hunger hormone, ghrelin which stimulates the desire to eat more food. this vicious circle causes the body to gain weight. the utilization of fos prebiotics like inulin-laced pasta decreases the bacterial lps, suppresses the hunger signal, promotes satiety and increases microbial diversity events aimed at countering obesity. a prebiotics is an effective means in controlling weight gain. cardiovascular diseases do also respond well to prebiotics therapy. water soluble fibers such as pectin found in fruits and guar gum from guar beans are known to decrease the bad cholesterol (ldl low density lipoprotein) without affecting the good cholesterol (hdl high density lipoprotein). they also help lower the blood pressure. the positive influence of these prebiotics on the functioning of the heart has been widely documented and accepted as a means of averting cardiovascular diseases. mitigating constipation and prompting proper bowel mobility are the gold standard treatment fuelled by the intake of prebiotics. cereal fibers like wheat bran are religiously utilized to counter any abnormality associated with fecal bulking. prebiotics can also be a potent remedy against cancers. enhanced micronutrient absorption and stimulation of microbes with cancer fighting enzymes like glutathione transferase are some of the mechanisms that these nutritional elements confer to the body. the ability of prebiotics to enrich the diversity of the microbiome is central to its curative power (fig. . ; fig. . ; fig. . ). prebiotics is almost like a dietary manure that transforms the microbial landscape in the gut. a similar strategy is utilized to help the visible body parts if something goes amiss. during anemia when the level of hemoglobin is low, iron sulfate pills enables the blood system to stimulate the production of this oxygen carrier. in the case of keratoconus, an eye condition where the cornea loses its focusing power, the surgical administration of vitamin b allows the eye to function normally. in this instance, the infusion of vitamin b promotes the crosslinking of collagen, a biological event that fortifies the cornea and enables it to contribute to visual sensation. the same can be said of lipoic acid and calcium supplements. the former helps fortify the workings of the brain while the latter enable the proper functioning of the skeletal system. as these ingredients assist the target organs optimize their biological roles, the intake of prebiotics provides a proper nutritional environment for the right microbial ecosystem to flourish in order to maximize the output of the invisible organ in promoting a healthy body. however, this is a non-invasive exercise and usually corresponds to an accompaniment of the food we consume unlike the surgical intervention needed to feed the cornea with vitamin b . hence, prebiotics are natural fertilizers for the microbiome and can remedy the microbial perturbations that the body has to often undergo due to age, environmental change or intake of medications. one can also utilize a two-pronged approach comprising probiotics and prebiotics to regulate an unbalanced microbial landscape. this is termed as synbiotic and can be equated to a multi-drug regime to cure a disease. a combination of these two stimulators can have a more vigorous impact on the invisible organ and can provide a very effective therapy against ailments mediated by uncontrolled proliferation of some opportunistic microbes. the blending of live microbes and the microbial fertilizers that enable them to thrive is a very powerful tool to goad the constituents of the invisible organ in executing a dedicated task. for instance, muscle wasting and inflammation that characterize cancer cachexia has been shown to be mitigated with a concoction of a synbiotic consisting of lactobacillus reuteri and inulin-based fructans (fos). the intake of this synbiotic reduces the population of enterobacteriacae, replenishes the lactobacillus colonies and prolongs the life of the patients. the therapeutic value of the fos and lactobacilli taken together in combatting hemorrhage triggered by bacterial infection has also been recognized. in this instance, a sharp decrease in e.coli counts coupled with an increase in the population of lactobacillus spp contribute to bringing relief to these patients. quite often a cocktail of drugs is prescribed to combat a viral infection; this can be equated to synbiotic therapy. the aids virus, can only be eliminated from the blood system of infected individuals by the use of anti-proteases and medications inhibiting the replication of the viral genetic code. hence, this double punch aimed at aiding the microbiome naturally is of immense benefit to the body. the human body is made of cells that are in constant flux due a variety of factors including wear and tear as age progresses. these can be repaired or rejuvenated with the aid of medical intervention. one can seek the help of a cosmetic surgeon to remove ageing spots or modify the shape of the nose. in case of the microbiome, any perturbations (dysbiosis) that the invisible organ experiences can be modulated by the programmed intake of probiotics and prebiotics. hence, the microbiome behaves in manner that is comparable to the organs composed of visible cells. it is clear that the body is made up of visible and invisible cells. most visible cells assemble as various organs we can see; there are few exceptions like the blood system where the cells are mobile and interact directly and indirectly with all the organs. just like dysbiosis that robs the microbiome of its natural functions, the organs can also be subjected to a number of challenges over body's life span. the inherent repair machinery cannot remedy the situations the organs are facing in a timely manner, intervention in form of medication or surgery is needed to bring them back as contributing members of the body. for example if some oily clutters are impeding the flow of blood, the arteries are unclogged by surgical procedure. if organs of the body are insulted by overuse of some products or by accidental intake of toxic compounds, antidotes are utilized to bring back these anatomical structures in tip-top shape. for instance, fomepizole can give reprieve to a liver challenged by alcohol abuse while high levels of cholesterol in the blood that contribute to plaque build-up in the arteries can be rectified by statin family of cholesterol-busters (fig. . ). in the same manner any disturbance in the microbial communities constituting the microbiome can be adjusted with the intake of probiotics, prebiotics or synbiotics. these 'therapies' are akin to the medications, the surgery, antidotes or stem cells that are deployed to tweak the visible organs in an effort to bring them to a normal working condition (fig. . ) . the fact that the microbiome is invisible and its molecular operation is still not fully understood compared its visible counterparts like the liver should not preclude it from being an important component of the cellular collectivity responsible for the body's well-being. the microbial system spans a wide surface area within and on the body just like the blood system as it imparts extremely valuable functions. this microbial ecosystem is to some extent responsible for the human anatomy and makes it work the way it does. understanding how the invisible organ operates, how the harmony amongst its cells are disrupted and how the fine-balance is reconstituted with the aid of probiotics and prebiotics are essential if we are to understand the intimate details hence, dysbiosis and rebiosis propelled by the infusion of live seeds (probiotics) and the fertilizers (prebiotics) will go a long way toward promoting wellness and will bestow upon us a healthy life probiotics, prebiotics and colorectal cancer prevention immune system stimulation by probiotic microorganisms interactions between the microbiota and pathogenic bacteria in the gut individual diet has sex-dependent effects on vertebrate gut microbiota microbiota and healthy ageing: observational and nutritional intervention studies a gastroenterologist's guide to probiotics the impact of the gut microbiota on human health: an integrative view probiotics and gastrointestinal disease: successes, problems and future prospects probiotics and prebiotics in ulcerative colitis microbiome-wide association studies link dynamic microbial consortia to disease prebiotics: why definitions matter the oral microbiome -an update for oral healthcare professionals probiotics in prevention and treatment of obesity: a critical view comparison of the immunomodulatory properties of three probiotic strains of lactobacilli using complex culture systems: prediction for in vivo efficacy the microbiome in asthma the skin microbiome: is it affected by uv-induced immune suppression? front microbiol : the role of bile acids in reducing the metabolic complications of obesity after bariatric surgery: a systematic review recent developments in prebiotics to selectively impact beneficial microbes and promote intestinal health regulation of the immune system by biodiversity from the natural environment: an ecosystem service essential to health functions of the skin microbiota in health and disease psychobiotics and the manipulation of bacteria-gutbrain signals cancer-promoting effects of microbial dysbiosis key: cord- -mszlz jk authors: al-subu, awni m.; hacker, timothy a.; eickhoff, jens c.; ofori-amanfo, george; eldridge, marlowe w. title: two-site regional oxygen saturation and capnography monitoring during resuscitation after cardiac arrest in a swine pediatric ventricular fibrillatory arrest model date: - - journal: j clin monit comput doi: . /s - - - sha: doc_id: cord_uid: mszlz jk to investigate the use of two-site regional oxygen saturations (rso( )) and end tidal carbon dioxide (etco( )) to assess the effectiveness of resuscitation and return of spontaneous circulation (rosc). eight mechanically ventilated juvenile swine underwent ventricular fibrillatory arrests with open cardiac massage. cardiac massage was administered to achieve target pulmonary blood flow (pbf) as a percentage of pre-cardiac arrest baseline. non-invasive data, including, etco( ), cerebral rso( ) (c-rso( )) and renal rso( ) (r-rso( )) were collected continuously. our data demonstrate the ability to measure both rso( ) and etco( ) during cpr and after rosc. during resuscitation etco( ) had a strong correlation with goal co with r = . (p < . ) % ci [ . – . ]. both c-rso( ) and r-rso( ) had moderate and statistically significant correlation with co with r = . (p = . ) % ci ( . – . ) and . (p = . ) % ci [ . – . ]. the aucs for sudden increase of etco( ), c-rso( ), and r-rso( ) at rosc were . [ % ci, . – . ], . [ % ci, . – . ], and . [ % ci, . – . ] respectively. measurement of continuous etco( ) and rso( ) may be used during cpr to ensure effective chest compressions. moreover, both rso( ) and etco( ) may be used to detect rosc in a swine pediatric ventricular fibrillatory arrest model. cardiac arrest occurs in - % of children admitted to pediatric intensive care units [ , ] . the overall survival for pediatric patients with in-hospital cardiac arrest remains low at less than % [ , ] . although severe hypoxia and respiratory failure remain the most common cause of pediatric in hospital cardiac arrests, - % have rhythm that requires defibrillation [ ] . the outcome of cardiac arrest is dependent on critical interventions, especially early defibrillation, effective chest compressions and assisted ventilation [ , ] . invasive blood pressure and cardiac output monitoring provide an accurate means of assessing the effectiveness of chest compressions [ ] . however, invasive monitoring at the time of either the arrest or initiation of resuscitation occurs in less than % of in-hospital pediatrics cardiac arrest cases and almost never occurs in out-of-hospital pediatric cardiac arrest cases [ ] . the need for accurate, non-invasive, and real-time monitoring of both oxygenation and circulation during resuscitation is, therefore, paramount to the improvement of resuscitation outcomes. despite its limitations, end-tidal carbon dioxide (etco ) monitoring remains the only noninvasive tool that has been used and advocated during cpr as a marker of effectiveness of resuscitation [ ] . furthermore, etco has been suggested to correlate with cardiac output and cerebral perfusion, and it has also been reported to be a predictor of survival and prognosis in patients after cardiac arrest and resuscitation [ , ] . however, measurement of etco can be particularly unreliable in low-flow states such as during cpr or in the presence of significant pulmonary pathology. moreover, the accuracy of etco measurement depends on proper capnography waveforms that may be difficult to obtain during chest compressions [ , ] . finally, etco does not provide any meaningful information about the quality of organ perfusion and oxygenation during resuscitation [ ] . regional oxygen saturation monitoring with near infrared spectroscopy (nirs) has emerged as a noninvasive surrogate monitor of regional oxygen saturation (rso ) [ , ] . during critical illness, nirs probes are routinely placed on the forehead to measure cerebral rso (c-rso ) and the back to measure renal rso (r-rso ), referred to as two-site rso . two-site rso can provide a real-time indicator of the balance between oxygen supply and demand in the intraoperative and intensive care setting. moreover, two-site rso represent two opposite poles of circulation, and the compensatory response to low cardiac output can be reflected in a decrease in r-rso while c-rso is preserved. this clinical information can help the provider to make treatment decisions and assess the effect of therapeutic interventions in settings of low cardiac output, such as cardiogenic, hemorrhagic and septic shock [ ] . moreover, nirs does not require pulsatile flow, which makes it an ideal monitoring tool during cardiac arrest [ ] . although nirs technology has been validated and used in many clinical scenarios, limited studies have examined the role of two-site rso monitoring during cardiac arrest to assess the effectiveness of chest compressions and detection of rosc without interrupting resuscitation efforts [ ] [ ] [ ] [ ] . all applicable international, national, and institutional guidelines for the care of experimental animals for scientific purposes were applied throughout the study; the experimental protocol was approved by university of wisconsin institutional animal care and use committee, in accordance with the guidelines in the us department of health and human services and the national institutes for health. the choice of domestic pigs was based on the fact that the porcine heart closely resembles the human heart from the point of view of size, physiology and anatomy. this study was performed in eight healthy -to -monthold mixed-breed domestic swine (sus scrofa) with weight of ± kg. animals had free access to water but were fasted overnight. each animal was pre-medicated with intramuscular telazol ( - mg/kg) and xylazine ( mg/ kg). subsequently, a g intravenous catheter was placed into an ear vein for additional anaesthetic agents (propofol, - mg/kg) prior to the procedure. the animal was intubated with cuffed endotracheal tube and mechanically ventilated under general anaesthesia using mixture of room air and titrated isoflurane ( . - . % inspired concentration). volumetric capnography monitor (philips, carlsbad, ca) was connected to the ventilator circuit via a side stream capnostat sensor placed at the proximal end of the endotracheal tube (between the endotracheal tube and the ventilator circuit). then animals were deeply sedated with adequate pain control throughout the experiment using fentanyl ( . - . μg/kg/h iv) as needed. the tidal volume was initially set at - ml/kg and the ventilator rate at breaths per minute; ventilator settings then were adjusted to maintain paco at to mm hg. the femoral artery was accessed by percutaneous methods then a fr sheath was placed and advanced to the abdominal aorta to measure systemic pressure and to withdraw blood for blood gas analysis. a fr sheath was also placed in the femoral vein by percutaneous methods for further administration of medications. a median sternotomy was performed and the heart was exposed. finally, an ultrasonic flow probe (pau series, adinstruments, dunedin, new zealand) was placed around the pulmonary artery. following surgical procedures, adhesive, non-invasive nirs probes were placed on a shaved portion of the left forehead scalp laterally from the midline, and left somatic (flank) region above the kidney. these locations were selected to reflect the locations used for clinical c-rso and r-rso monitoring in children. then probes were connected to a somanetics invos c monitor (oxy-alerttm, somanetics invos c, somanetics corporation, troy, mi, usa). baseline c-rso and r-rso readings were continuously obtained before vf induction and then was continuously monitored and recorded throughout the entirety of the experimental protocol. continuous monitoring included ecg, peripheral oxygen saturation, invasive arterial blood pressure monitoring, cerebral and renal saturation by near infrared spectroscopy (invos® cerebral/somatic oximeter monitor, somanetics, troy, mi, usa), and the respiratory volumes, pressures, etco , volume of carbon dioxide monitoring, and tidal volume and dead space ratio by means of a volumetric capnography monitor. blood gases, lactate, and hemoglobin were analyzed using a blood gas analyzer (phox basics; nova biomedical, waltham, ma). following baseline data acquisition, ventricular fibrillation was induced with current from a v battery applied to the epicardial surface of the right ventricle for s. examination of the ecg tracing, absence of contractility, and the absence of pulses in the arterial pressure waveform was used to confirm vf. one minute following loss of pulse pressure, cpr was initiated by open manual cardiac massage. cardiac massage was administered and was altered every min during resuscitation to achieve target pulmonary blood flow (pbf) as a percentage of pre-cardiac arrest baselines ( %, %, and % of prearrest mean pbf) until rosc or euthanasia. non-invasive and invasive hemodynamic data was continuously monitored and collected through the whole experiment. cardiac massage was at a rate of approximately cycles per minute. epinephrine, lidocaine, and defibrillation was administered per pediatric advanced life support (pals) guidelines (fig. ). if there was no rosc, the cycle was repeated per pals protocol. rosc was defined as restoration of perfusing rhythm, arterial systolic blood pressure of at least mmhg and pulse pressure of mmhg lasting at least min. if there is no rosc after min resuscitation was discontinued. after successful resuscitation, the animals was maintained on mechanical ventilation and continuous monitoring. blood gases were obtained, metabolic acidosis was treated with normal saline and/or sodium bicarbonate and the ventilator was adjusted to achieve normal ventilation (paco = - mmhg). normal saline boluses were administered to treat hypotension. after min of stability, the experiment was repeated in the animal up to four additional times. hemodynamic variables and levels of blood gases were summarized in terms of means ± standard deviations, stratified by event type. the correlations between measured cardiac output and rso and etco parameters were evaluated using the nonparametric spearman's rank correlation analysis. receiver operating characteristics (roc) curve analysis was conducted to evaluate whether cerebral, renal or etco can predict rosc. the strength of the prediction is quantified by calculating the area under the curve (auc) which was reported along with the corresponding % confidence intervals. the youden criterion was used to determine optimal threshold cutoff threshold for predicting rosc [ ] . linear mixed effects modeling with animal-specific random effects was utilized when comparing outcome parameters between goal cardiac outputs. all reported p-values are two-sided and p < . was used to define statistical significance. statistical analysis was conducted using sas software (sas institute inc., cary nc), version . . a total of mechanically ventilated juvenile swine underwent vf arrests with open cardiac massage ( animals underwent vf arrests, underwent vf arrests, and one animal underwent only vf arrests). hemodynamic variables and blood gases before cardiac arrest, during cpr and after rosc are shown in table . with onset of cardiac arrest, there was a sudden (within s) decrease in etco , r-rso and c-rso by . %, . %, and . % respectively. correlations between r-rso , c-rso , and etco before cardiac arrest, and after rosc are shown in table . there was a strong and statistically significant correlation between etco and cardiac output after rosc with r = . (p = . ) % ci [ . - . ]. during resuscitation, etco had a strong correlation with goal co, measured as a percentage of baseline pbf, with correlation coefficient of r = . (p < . ) % ci [ . - . ]. both c-rso and r-rso had moderate and statistically significant correlation with measured co with correlation coefficient of . (p = . ) % ci ( . - . ) and . (p = . ) % ci [ . - . ]. there were no significant differences detected in c-rso , r-rso or etco levels when goal cardiac output fig. receiver operation for sudden increase in a c-rso , b r-rso , and c etco to detect return of spontneous circultion (rosc). auc area under roc the curve was increased from to % (fig. ) . the aucs for sudden increase in etco , c-rso , and r-rso (fig. ) . the optimal threshold for percentage change cutoffs for etco , c-rso , and r-rso were . %, . %, and . % respectively. at these cutoffs, a sudden increase in etco , c-rso , and r-rso had similar sensitivities ( %, %, and %). however, r-rso was the most specific when compared to etco and c-rso ( % vs. % and %). further analysis included only the first vf arrest for each animal showed that aucs for sudden increase in etco , c-rso , and r-rso (fig. ) . the optimal threshold for percentage change cutoffs for etco , c-rso , and r-rso were . %, . %, and . % respectively. at these cutoffs, a sudden increase in etco was more sensitive than c-rso and r-rso ( % vs. % and %). however, c-rso and r-rso were more specific when compared to etco ( % and % vs. %) (fig. ). published guidelines for cpr increased the focus on ways to ensure that high-quality cpr is performed in all resuscitation attempts to improve outcomes from cardiac arrest [ , ] . recent adult studies have assessed the usefulness of etco and c-rso as a guide to resuscitative efforts during cpr [ ] [ ] [ ] , yet there have been no studies that assessed the use of r-rso , nor the role of two-site rso in assessing quality of resuscitation or rosc. we report the potential use of two-site rso , and etco to monitor the effectiveness and quality of cpr, and rosc. this is the first study to evaluate the relationship between changes in c-rso , r-rso , and etco and co during resuscitation and rosc in swine pediatric vf arrest model. in this study we demonstrate that etco and two-site rso can be used to assess and guide effectiveness of chest compressions especially when resuscitation efforts are suboptimal. as etco reflects pbf, and two-site rso reflects the balance between oxygen delivery and extraction at two different vascular beds [ ] , the use of both monitoring devices during resuscitation may provide complementary information on the quality of resuscitation organ perfusion, and tissue oxygenation during cpr. our result is consistent with the findings of koyama et al. who reported that c-rso could reliably assess the quality of chest compressions and improve effectiveness of cpr in adult cardiac arrest patients. however, in that study only c-rso waveform (not the absolute value) was used to assess the quality of chest compression [ ] . in our study the quality of chest compressions was controlled and continuously monitored and the absolute rso was monitored as well. in contrast to our results, kämäräinen et al. found that c-rso remained low during high-quality resuscitation and that improvement in cpr was not significantly reflected in c-rso [ ] . however, the assessment of c-rso was not monitored prior to initiation of cpr and therefore the c-rso before cardiac arrest was not known. moreover, quality of cpr provided was monitored using defibrillator with cpr quality analysis features and no other direct or invasive measurements of cpr quality was measured. in regard to etco , a recent study using a lamb asphyxial cardiac arrest model, chandrasekharan et al. demonstrated a relationship between etco and adequate chest compression during resuscitation, and concluded that a rapid increase in etco with a threshold of > mmhg is sensitive and % specific in predicting rosc [ ] . moreover, a recent multicenter study of adult patients with in-and out hospital ca showed that depth of chest compression was associated with higher etco which suggest that etco monitoring during cpr might be a useful tool to guide effective resuscitation after ca [ ] . our data also suggest that both rso and etco can detect rosc during cpr without interrupting chest compressions. although c-rso , r-rso , and etco were all very sensitive tests to detect rosc, r-rso was more specific than both c-rso and etco . thus adding renal nirs monitoring during resuscitation might not only guide resuscitation, but can be used to detect with more confidence rosc. our results is consistent with pokorna et al. who showed that a sudden increase in etco > mmhg during out-of-hospital cpr could be used as an early indicator of rosc [ ] . similarly, with the findings of singer et al. who reported that during outside hospital ca, both mean c-rso and etco during cpr can be used to predict rosc [ ] . they found that etco was more sensitive and c-rso was more specific at predicting rosc. however, they didn't assess the sensitivity and specificity of sudden increase in both to detect rosc. moreover, a growing number of recent studies demonstrated that a higher mean cerebral rso during cpr was observed in patients who achieved rosc compared to nonsurvivors [ , , , ] . we are not aware of any studies that assessed the ability of two-site rso to detect rosc during cpr. finally, our results demonstrated that both rso and etco rapidly decline after loss of pulse pressure. this is consistent with the findings of reynolds et al. who reported that forelimb rso in a porcine vf arrest model rapidly declined by % in min after loss of pulse pressure. however, they reported that etco did not drop immediately [ ] . similarly, putzer et al. found that c-rso decreased during periods of untreated cardiac arrest in an animal model of hypothermic cardiac arrest [ ] . the recognition of acute sudden drop in rso and etco in a hemodynamically unstable patient could be extremely vital in the recognition of cardiac arrest and early initiation of resuscitation efforts especially in patients who lack invasive lines. our study has obvious limitations. first, our study included animals with open chest without sufficient thoracic pump function, which may have affected venous return and cardiac output. we acknowledge that our model might not resemble the clinical situation of most critically ill patients who suffer cardiac arrest and require cpr, however, it might be clinically relevant in anaesthetized patients or patients who just undergone cardiac surgery and suffer a sudden, cardiac deterioration and arrest. second, the cardiac massage was performed by hand and mean pbf goal was used during resuscitation. also animals received normal saline and/or sodium bicarbonate after rosc which might led to a transient increase in etco without an actual increase in cardiac output during subsequent arrests, however, further analysis of the first vf arrest didn't indicate such effect, in the contrary, etco was more sensitive than c-rso and r-rso to detect rosc during the first vf arrests when compared to subsequent arrests (fig. ) . third, all animals had relatively healthy lungs, which may not reflect common pediatric cardiac arrests. this latter point presents a concern as etco is affected by alveolar dead space, which may be a confounding factor when assessing changes in co. finally, analgosedative drugs were administered, as necessary, to maintain adequate analgesia and anesthesia. the use of these drugs may have affected cardiovascular function and autonomic control during resuscitation. in this swine pediatric vf arrest model, two-site rso obtained by nirs technology, and etco correlates with cardiac output changes during cpr and can be used to guide resuscitations efforts and detect rosc without interrupting resuscitation efforts. further studies are required to explore the use of two-site rso and capnography as a tool for early detection of ca, monitor effectiveness of chest compression, and detection of rosc among critically ill pediatric patients who suffer from cardiac arrest. survival trends in pediatric in-hospital cardiac arrests: an analysis from get with the 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new zealand council on resuscitation, heart and stroke foundation of canada, interamerican heart foundation, resuscitation council of southern africa, resuscitation council of asia); and the american heart association emergency cardiovascular care committee and the council on cardiopulmonary, critical care, perioperative and resuscitation end-tidal co as a predictor of survival in out-of-hospital cardiac arrest correlation of end-tidal co to cerebral perfusion during cpr end-tidal carbon dioxide concentration during cardiopulmonary resuscitation in patients with pre-hospital cardiac arrest capnographic waveforms in the mechanically ventilated patient etco monitoring during low flow states: clinical aims and limits a pilot study examining the role of regional cerebral oxygen saturation monitoring as a marker of return of spontaneous circulation in shockable (vf/vt) and non-shockable (pea/asystole) causes of cardiac arrest near-infrared spectroscopy to monitor cerebral oxygen saturation in single-ventricle physiology monitoring of regional tissue oxygenation with near-infrared spectroscopy during the early postoperative course after superior cavopulmonary anastomosis non invasive monitoring in mechanically ventilated pediatric patients a proposed algorithm for the intraoperative use of cerebral near-infrared spectroscopy detection of rosc in patients with cardiac arrest during chest compression using nirs: a pilot study regional cerebral oximetry during cardiopulmonary resuscitation: useful or useless? monitoring of cerebral oxygen saturation during resuscitation in out-of-hospital cardiac arrest: a feasibility study in a physician staffed emergency medical system cerebral oximetry levels during cpr are associated with return of spontaneous circulation following cardiac arrest: an observational study the interpretation of diagnostic tests characteristics of regional cerebral oxygen saturation levels in patients with out-of-hospital cardiac arrest with or without return of spontaneous circulation: a prospective observational multicentre study european resuscitation council guidelines for resuscitation : sect. . adult basic life support and automated external defibrillation a new method to detect cerebral blood flow waveform in synchrony with chest compression by near-infrared spectroscopy during cpr quality controlled manual chest compressions and cerebral oxygenation during in-hospital cardiac arrest cerebral oximetry versus end tidal co in predicting rosc after cardiac arrest continuous capnography monitoring during resuscitation in a transitional large mammalian model of asphyxial cardiac arrest quantitative relationship between end-tidal carbon dioxide and cpr quality during both in-hospital and out-of-hospital cardiac arrest a sudden increase in partial pressure end-tidal carbon dioxide (p(et)co( )) at the moment of return of spontaneous circulation near infrared spectrophotometry (cerebral oximetry) in predicting the return of spontaneous circulation in out-of-hospital cardiac arrest regional cerebral oxygen saturation during cardiopulmonary resuscitation as a predictor of return of spontaneous circulation and favourable neurological outcome-a review of the current literature tissue oximetry by near-infrared spectroscopy in a porcine model of out-of-hospital cardiac arrest and resuscitation monitoring of brain oxygenation during hypothermic cpr-a prospective porcine study acknowledgements we thank imran sayed, md, arij beshish, md, ph.d. for their diligent work in collecting data during experiments. we also thank allison brodbeck for surgical and administrative support of this project. conflict of interest dr. eldridge received salary support for research from the national heart, lung, and blood institute (nhlbi) of the national institutes of health (r hl - and r hl ) and the u.s. department of defense, navy (n - -c- and n a- -c- ). the remaining authors have disclosed that they do not have any potential conflicts of interest. key: cord- - h eiihp authors: guan, wei-jie title: giants in chest medicine: professor nan-shan zhong, md date: - - journal: chest doi: . /j.chest. . . sha: doc_id: cord_uid: h eiihp nan professor nan-shan zhong, md wei-jie guan, phd professor nan-shan zhong was born in nanjing in . following graduation from high school, he pursued medical studies at peking medical university. due to excellent physical fitness, he became well known for his competitiveness as an athlete. the spirit of pursuing excellence has underlined his mental strengths in achieving countless breakthroughs in his career. in , he became a resident at the first affiliated hospital of guangzhou medical university. despite hardships such as poor working conditions, he quickly made great strides in the accumulation of medical knowledge, which lent him support in pursuing future progress. in , the significant national burden of chronic bronchitis prompted the central government to establish the group for prevention and management of chronic bronchitis, which in became guangzhou institute of respiratory disease. in recognition of their achievement in investigating the effects of a chinese herb on chronic bronchitis, professor zhong and colleagues were awarded the national prize for scientific advances. in and , he pursued further studies at the royal infirmary, university of edinburgh and at st. bartholomew's hospital, university of london as a research fellow. apart from his brave behavior of inhalation of carbon monoxide to achieve high blood carboxyhemoglobin concentrations, his findings on the effects of carbon monoxide on the hemoglobin dissociation curve dramatically reshaped scientists' contemporary understanding. in , he was appointed director of the guangzhou institute of respiratory disease (which is currently called guangzhou institute for respiratory health). the development of a hand-squeezed atomizer for performing bronchial provocation tests allowed the exploration of the epidemiology of asthma and the definition of "asymptomatic asthma" (probable asthma) possible in the s and , respectively. the development of a corrected formula for calculating nutritional energy balance in critically ill patients has significantly improved patient health care in icus. in recognition of these achievements, he became an academician of the chinese academy of engineering in . his brave behavior of admitting the most critically ill patients with severe acute respiratory syndrome (sars) and challenging the authoritative opinions about the pathogens of sars made him famous as the "hero of china." he successfully led his team in minimizing the mortality of sars, resulting in the world's lowest record of the disease. his experience was quickly adopted by the central government, leading to the ultimate success of the fight against sars, "the war without gunfire." dedication, innovation, exploration, and collaboration have become the "nan-shan spirit," which remains the famous motto that inspires medical students and staff today. despite enormous achievement in his earlier career, the spirit of pursuing excellence has relentlessly inspired him to make greater strides. the establishment of the state key laboratory of respiratory disease has transformed his visions into an ideal platform for bridging the gap between basic scientific research and clinical practice. he has frequently stated that scientific research should be pursued for the improvement of people's well-being, which has led to copious landmark translational research that has dramatically changed the way medicine should be practiced. the study finding that carbocisteine effectively reduced the annual acute exacerbation rate of copd was awarded "paper of the year " by the lancet editorial board. recognizing challenges of copd management in china, he and colleagues performed the largest epidemiologic investigation on the national disease burden, highlighting that approximately % of patients with copd remained asymptomatic on presentation, that biomass fuel combustion might be the main risk factor for copd in women (particularly in rural areas), and that simple practical approaches (eg, installation of exhaust fans and replacing biomass with biogas) may significantly ameliorate the rate of lung function decline and reduce the incidence of copd. recently, he designed the first multicenter clinical trial proving that early intervention in copd (eg, with tiotropium inhalation) may effectively ameliorate the decline in lung function. his comments on improving air quality to reduce the burden on chronic respiratory diseases have inspired scientists and clinicians to dedicate themselves to the prevention and management of air pollution in china. despite countless awards and prizes, he never ceased his progress in pursuing further research. as stated in a press release, he remains active "post- ." one is never too old to learn, he explained. his open mind, coupled with a laudable drive and physical fitness, have lent him measurable support to pursue further achievement, which will ultimately benefit people worldwide, making him a true giant in chest medicine. physicians and scientists worldwide can benefit considerably by standing on the shoulders of his work. we encourage all to view the interview of professor zhong's words of wisdom (video ). bronchial hyperresponsiveness in young students in china, relation to respiratory symptoms, diagnosed asthma and risk factors is asymptomatic bronchial hyperresponsiveness an indication of potential asthma? using sirna in prophylactic and therapeutic regimens against sars coronavirus in rhesus macaque treatment of sars with glucosteroids: the guangzhou experience what have we learned from sars epidemics in china? biomass fuels are the probable risk factor for chronic obstructive pulmonary disease in rural south china prevalence of chronic obstructive pulmonary disease in china: a large, population-based survey effect of carbocisteine on acute exacerbation of chronic obstructive pulmonary disease (peace study): a randomised placebo-controlled study community based integrated intervention for prevention and management of chronic obstructive pulmonary disease (copd) in guangdong, china: cluster randomised controlled trial clinical findings in cases of influenza a (h n ) virus infection leukotriene d and methacholine bronchial provocation tests for identifying leukotriene-responsiveness subtypes a prospective, multicenter survey on causes of chronic cough in china twice daily n-acetylcysteine mg for exacerbations of chronic obstructive pulmonary disease (pantheon): a randomised, double-blind placebo-controlled trial lung function and incidence of chronic obstructive pulmonary disease after improved cooking fuels and kitchen ventilation: a -year prospective cohort study human infection with a novel avian influenza a(h n ) virus ceftaroline fosamil versus ceftriaxone for the treatment of asian patients with community-acquired pneumonia: a randomised, controlled, double-blind, phase , non-inferiority with nested superiority trial the bronchiectasis severity index and faced score for bronchiectasis prevention and management of copd in china: successes and major challenges impact of air pollution on the burden of chronic respiratory diseases in china: time for urgent action tiotropium in early-stage chronic obstructive pulmonary disease role of sponsors: the sponsor had no role in the design of the study, the collection and analysis of the data, or the preparation of the manuscript. key: cord- - ff jc authors: noval, s.; henríquez-recine, m. a.; contreras, i.; galdós, m.; zafra, b.; barrio-barrio, j.; carceller, f. title: macular ganglion cell complex thinning in children with visual field defects due to central nervous system pathology date: - - journal: eye (lond) doi: . /s - - - sha: doc_id: cord_uid: ff jc purpose: to study the relationship between macular ganglion cell complex (gcc) thickness and visual field defects (vfd) caused by central nervous system (cns) lesions in children and evaluate the possibility of predicting vfd according to gcc maps. methods: the gcc maps of a group of children with vfd due to cns lesions with respect of the vertical meridian in at least one eye (study group), as well as of children with other neuro-ophthalmological problems and healthy children were presented to two masked evaluators, who were asked to predict the patients’ vfd on the basis of gcc damage: the evaluators classified vfd as normal, hemianopia (homonymous or heteronymous) or diffuse. results: seventeen patients were included in the study group, with a median age of years. fifteen had brain tumours and two epilepsy. the mean md of the affected hemifields was − . db (sd . db) versus − . db (sd . db) for the nonaffected hemifields, p < . . the mean gcc thickness was of . μm (sd . μm) in the affected hemiretinas versus . μm (sd . μm) for the non-affected, p < . . kappa coefficients between vfd and those estimated by the evaluators were . and . (p < . ) for evaluators and . conclusions: gcc thickness can reflect damage to the visual pathway and gcc maps may be useful to identify chiasmal and retrochiasmal lesions, since gcc atrophy in most of these cases respects the vertical meridian. gcc maps might be used as a surrogate marker for visual damage in patients unable to perform perimetry. bilateral heteronymous or homonymous hemianopia can develop due to several diseases of the central nervous system such as tumours, inflammatory or vascular diseases which damage the visual pathway. hemianopia rarely appears in children; however, recognition of visual field defects is important to diagnose and monitor cerebral damage and to evaluate the possible repercussion on the child's development. unfortunately, children often do not recognise or complain of visual loss [ ] . evaluating only visual acuity (va) would underestimate visual damage since it can be normal in the presence of visual field defects [ ] . often an anomalous position of the head, with the face turned towards the side of the hemifield defect may be the only sign in children with homonymous hemianopia [ ] . the location and radiological features of the brain lesions cannot always predict visual damage, particularly in visual pathway glioma. the management of these lesions is difficult and is based on their clinical repercussion, i.e., the extent of visual damage and the potential for visual improvement [ ] . being able to detect early, subtle changes are essential because visual pathway glioma may not require chemotherapy (which may lead to severe side effects) unless visual loss or progression in visual field defects are demonstrated [ ] . automatic perimetry may sometimes be performed by children as young as years-old [ , ] , but often even older children are unable to perform reliable visual fields. damage to the human central nervous system leads to retrograde trans-synaptic degeneration and when the visual pathway is involved, it often leads to axonal loss at the optic nerve and retinal ganglion cell loss at the retina. optical coherence tomography (oct) is capable of detecting axonal loss in the peripapillary area. however, when there is a decreased peripapillary retinal nerve fibre layer (prnfl) thickness, the extent of damage to the visual pathway may preclude visual recovery after treatment [ ] . software developments now also allow oct measurement of the ganglion cell layer in the macular area of the retina, which might be better correlated to visual field loss than rnfl thickness. oct evaluation would be especially helpful in evaluating children who cannot perform visual fields, since it is a very fast test which requires minimum patient collaboration [ ] . the aim of this study was to evaluate the relationship between reduction in macular ganglion cell complex (gcc) thickness as detected with spectral-domain oct and visual field defects caused by central nervous system lesions in children, as well as to determine whether it is possible to predict visual field defects from gcc maps. children with visual field defects due to brain lesions, with respect of the vertical meridian in at least one eye, who had been followed-up in the neuro-ophthalmology department between and , were evaluated for inclusion in the study. to be included, two standard automated perimetry (humphrey field analyser , zeiss/humphrey systems, dublin, calif.) - tests, swedish interactive test algorithm (sita) strategy had to be available with similar results in extension and severity of the visual defect. at least months had to have elapsed between diagnosis of the brain tumour or its treatment and the first of these visual fields; a further months were required between both fields in order to ensure that visual damage was stable. only the second of these visual fields was included in the analysis. visual fields were considered reliable when fixation losses and positive errors were < %. hemianopia was diagnosed following the criteria proposed by thomas et al., when the threshold values showed depression of three or more contiguous points by db or more along the vertical meridian as compared with their mirror image points in one quadrant and depressed threshold values were present in the other quadrant of the same hemifield. alternatively, the diagnosis was made if three points of the pattern deviation probability plot were depressed to % or lower probability level along the vertical meridian and as compared with their mirror image points in one quadrant and depressed threshold values were present in the other quadrant of the same hemifield [ , ] . in order to simplify the evaluation, visual field defects were classified as normal, hemianopia or diffuse; and then as homonymous or heteronymous. the average mean deviation of each hemifield was calculated and recorded. when va was less or equal to counting finger in one eye, the visual field was not tested and md was defined as − db for the purpose of analysis. all patients also underwent an extensive ophthalmic evaluation including at least best-corrected va, internal and external ocular motor examinations and anterior and posterior pole biomicroscopy and funduscopy. optical coherence tomography scanning was performed through dilated pupils using cirrus-hd model (carl zeiss meditec dublin, ca, usa). an internal fixation target was used whenever possible. to evaluate the prnfl thickness and the gcc thickness, the optic disc cube protocol and the macular cube × protocols were used. the gcc analysis algorithm divides the macula into six equal sectors across a horizontal meridian. in normal subjects all segments should have a similar thickness. to establish a thickness measurement for the nasal macula, the superonasal sector and the inferonasal sector were averaged, and similarly the temporal thickness was calculated as the average of the superotemporal and inferotemporal sectors. (fig. ) . the inferior and superior segments were left out of the analysis as they include both crossed and uncrossed fibres [ ] . the gcc maps were presented to two neuroophthalmologists (ic and mg) who were asked to predict the visual field defects the patients had on the basis of gcc damage. maps from the children included in the study (with visual field defects caused by central nervous system lesions) were presented mixed with maps from a control group. this group consisted of subjects: healthy volunteers and subjects diagnosed with other neuroophthalmic diseases such as optic neuritis, optic nerve drusen or intracranial hypertension, of which only ten eyes had visual field defects. the study was approved by our institutional review board and adhered to the tenets of the declaration of helsinki. since this was an exploratory study, no power calculation was performed prior to initiation. statistical analysis was performed using the statistical package for social sciences software (version . ; spss, inc, chicago, il). kappa coefficients were calculated to determine the correlation between the responses of the evaluators and the actual visual field defects of patients. the correlation is considered to be low if the kappa coefficient is < . , good if it is between . and . and excellent if it is > . (fig. ). seventeen patients younger than years at the time visual field and oct examination were performed were included in the study. one patient with a homonymous defect due to ischaemic damage was excluded because criteria for hemianopia were not fulfilled. there were eight girls ( %) and nine boys ( %), with a median age of years (range from to years). brain tumour was the most frequent pathology with cases ( both evaluators predicted there would be a diffuse defect in both eyes when presented with the cgg analysis, due to the diffuse gcc loss. the mean gcc for the right eye is above the cutoff point of µm, which increases the probability of finding an hemianopia instead of a diffuse defect two cases were caused by sequels of disseminated acute encephalitis and shaken baby syndrome with occipital cortex involvement. only five children had a visual acuity of / (snellen equivalent) in both eyes ( %); six had a va of / in one eye with decreased visual acuity in the fellow eye ( %), ranging from amaurosis to / ; two ( %) had a bilateral moderate reduction (between / and / ) and four ( %) had a visual acuity of / or less in both eyes. five children had strabismus; four with reduced visual acuity in at least one eye presented exotropia ( . %) and one child with bilateral normal va, esotropia ( %). two other children had nystagmus ( %). nine patients had a homonymous hemianopia ( %), seven had a temporal defect in one eye and a diffuse defect in the contralateral eye ( %) and one case, an heteronymous hemianopia ( %). mean deviation (md) ranged from − . to − . db, with a mean md for the eyes of − . db (sd . db). good quality scans of the optic nerve could be obtained in all except one eye. mean prnfl thickness for the eyes was . μm (sd . μm), with a range between and μm. in two eyes, the oct software did not accurately segment the macular region and gcc measurements were unreliable. the mean gcc thickness was . μm (sd . μm), with a range between and μm in the remaining eyes. table shows the values for prnfl thickness and gcc, with significant differences in mean md and oct thicknesses between eyes with hemianopia and diffuse defects. a more detailed analysis was performed in eyes with hemianopia ( eyes), in order to compare the affected with the unaffected hemifields. the mean md of the affected hemifields was − . db (sd . db) and − . db (sd . db) for the non-affected hemifields. the difference was statistically significant (p < . , wilcoxon test). the mean gcc thickness of the affected hemiretinas (nasal or temporal macula) was . μm (sd . μm) and . μm (sd . μm) for the non-affected hemiretinas. the difference was statically significant (p < . , wilcoxon test). no significant correlations were found between md and mean gcc thickness in any hemifield. we calculated kappa coefficients to determine the correlation between the responses of the neuro-ophthalmologist evaluators and the actual visual field defects of patients. between evaluator and actual defects the kappa coefficient was . (p < . ); between evaluator and actual defects the kappa coefficient was . (p < . ) and between the two evaluators the kappa coefficient was . (p < . ). when taking into account only cases with chiasmatic or retrochiasmatic lesions, the most frequent mistake was made in the prediction of diffuse defects instead of the real hemianopias: evaluator predicted diffuse defects and evaluator , diffuse defects, when there were only seven diffuse defects, with three hemianopias. overall mean gcc thickness was significantly different in eyes with hemianopia and eyes with a diffuse defect [ . μm (sd . μm) versus . μm (sd . μm), p < . ]. its ability to discriminate hemianopia from diffuse defect was calculated by the area under the curve of the roc curve (roc = . , p < . confidence interval : . - . ). according to the jouden index, a mean gcc thickness of μm would be the best discriminating cut-off point, with a sensitivity of % and a specificity of %. in glaucomatous optic neuropathy, damage to the prnfl can precede future perimetric damage by up to years, and therefore, oct has been recognised as a useful diagnostic tool in detecting early glaucoma [ , ] . however, in compressive optic neuropathy, the relationship between oct measurements and visual damage is more complex. damage to the visual pathway has been shown to lead to transynaptic retrograde degeneration (tsrd), which may be detected with oct evaluation both of the prnfl and gcc [ ] . acute or rapidly progressive chiasmal compression may produce axonal dysfunction with visual field deficits such as bitemporal hemianopias before structural damage can be demonstrated. thus, prnfl values within normal values in patients with visual fields due to chiasmal tumours have been shown to predict visual improvement after treatment. this predictive role has been established in adults with pituitary adenoma [ ] and craniopharingioma [ ] . therefore, perimetry would seem to be better than oct evaluation of the prnfl to detect early compression of the visual pathway in order to achieve visual improvement after treatment, both after the initial diagnosis or after recurrences. most of these tumours cannot be completely resected (especially in the case of childhood craniopharyngiomas) and follow-up is critical to detect visual impairment which is an important factor to decide if further treatment is necessary. but children are often unable to perform reliable visual fields and it is necessary to search for an alternative surrogate marker for visual loss for both diagnosis and follow-up. furthermore, it seems that in chronic compressive neuropathies, ganglion cell loss can take place before visual field deficits [ ] [ ] [ ] . several studies suggest that gcc thinning is more apt to occur than prnfl thinning following damage to the visual pathway and that it may reflect damage earlier than the prnfl. blanch et al. reported seven patients with radiologic compression of the chiasm by a sellar tumour, in whom oct-gcc analysis detected damage without visual field defects apparent on standard automated visual field testing; three of these patients had normal prnfl thicknesses [ ] . tieger et al. also reported six cases of patients with sellar tumours without visual field loss, with more significant gcc than rnfl thinning, as well as cases with binasal gcc loss without rnfl thinning on oct [ ] . persistent gcc thinning with improvement in visual fields was noted in seven of eight patients who underwent surgical decompression (after decompression, % of eyes had nasal gcc loss postoperatively and % experienced vf improvement) [ ] . after decompression of the optic chiasm, physiological conduction block is reversed in axons that were still viable and thus visual function might improve with stable gcc and prnfl. yum et al. evaluated the detection rate of abnormal colour codes on gcc sector maps in patients with pituitary adenoma; an abnormal yellow colour code was found in the inferonasal sector ( . %), followed by the superonasal ( . %) and inferior ( . %) sectors. furthermore, analysis of the gcc sector maps revealed that % of preperimetric eyes in patients with pituitary adenomas had an abnormal yellow or red colour code in the inferonasal sector [ ] . it seems therefore that gcc thickness is affected early in the course of compressive optic neuropathy and that it may be easier to suspect chiasmal and retrochiasmal damage when evaluating gcc maps than when evaluating prnfl as the atrophy respects the vertical meridian in both eyes. conversely, the more complex pattern of prnfl thinning in chiasmal and retrochiasmal pathologies must be carefully analysed to differentiate tsrd from optic atrophy due to other causes [ , ] . therefore, our purpose was to evaluate the relationship between topographic reduction in gcc thickness and visual field defects caused by central nervous system lesions in children, to explore whether the gcc could provide a valid surrogate marker for visual loss when visual fields are unavailable. the evaluation of the colour maps is especially important in children since there is currently no available normality data. brain tumours were the most common cause of hemianopia in this paediatric case series, mainly craniopharyngiomas and optic pathway gliomas. children were included if they presented hemianopia in at least one eye and it is noteworthy that % had a diffuse defect in the fellow eye. however, visual acuity was below / in both eyes in only one out of four cases. this confirms that visual acuity underestimates visual damage when compared with perimetry. as might be expected, visual field and oct variables were significantly lower in the affected hemifield than in the unaffected one, although they did not show a significant correlation. herro et al. studied eyes of nine adults with visual field defects secondary to ischaemic cortical injury with the cirrus sd-oct device. they did not find a statistically significant difference between affected and unaffected prnfl. however, there was a significant difference in gcc layer reduction between the affected ( . μm) and unaffected ( . μm) sides (p = . ) [ ) . goto et al. detected a reduction of the prnfl thickness corresponding to the hemianopic visual field loss due to acquired post-geniculate visual pathway lesions [ ] . the lack of a correlation between visual field damage and oct parameters might be due to the different time elapsed between the damage to the visual pathway and the moment of visual field and oct examination. jindhara et al. found that the speed of rtsd is greatest within the first years, occurring in the prnfl at a rate of . μm/year. clear evidence of rtsd typically becomes apparent by year, and progression may continue over the course of a decade [ ] . as regards studies performed in children, gu et al. studied the relationship between gcc thickness and vision loss in children with optic pathway gliomas. they compared normal eyes with eyes with decreased visual acuity or visual field defects. all the prnfl and gcc thickness measures were significantly different between normal and affected eyes, although the highest area under the curve was obtained for the fifth percentile gcc of the inner macula. the positive predictive value of the centre ( mm) macula demonstrated that . % of those with decreased gcc thickness had visual loss. they concluded that if children with normal vision demonstrate a progressive decline in their gcc thickness, this may be an indication to initiate an early treatment [ ] . thus, it seems that there is enough evidence (from ours and previous studies) to support that gcc thickness may be a valid surrogate marker for visual loss in patients unable to perform visual fields, such as young children. however, the utility of gcc thickness to predict the extent of visual field damage in clinical practice has not been tested yet, to the best of our knowledge. in this study we found that the evaluation of the gcc map by an experienced neuroophthalmologist may be quite sensible for detecting visual damage; in fact the most frequent mistake was anticipating diffuse defects instead of the real hemianopias. if an evaluation of the map was to be combined with an analysis of the mean gcl-ipl thickness, the prediction of visual field damage might improve. therefore, gcc evaluation might be useful both for diagnosis and for follow-up of children with suspected central nervous system pathology who are unable to undergo visual field testing. however, visual field testing should be performed whenever possible, since depending on the clinical situation, both oct and perimetry have advantages and disadvantages for the detection of chiasmal compression [ ] . the main limitation of our study is that it is a small, retrospective series and the results must be considered with caution. the lack of a correlation between gcc thickness and visual field mean deviation might be due to the less number of patients included, i.e., our study might be underpowered. it included children with very different brain lesions and with different time-courses. another limitation is that we included only patients with a stable clinical situation and therefore it is uncertain whether in a clinical setting, gcc analysis would be truly useful to suspect diagnosis of cns or progression of the lesion if visual fields are unavailable. however, it seems clear that gcc can reflect damage to the visual pathway and that it's especially useful to identify chiasmal and retrochiasmal lesions, since gcc atrophy in these cases respects the vertical meridian. thus, it might be used as a surrogate marker to detect visual damage in patients unable to perform visual fields. what was known before • several studies have shown that perimetry would be better than oct evaluation of the peripapillary retinal nerve fibre layer (prnfl) to detect early compression of the visual pathway. • however, children are often unable to perform reliable visual fields and it is necessary to search for an alternative surrogate marker for visual loss for both diagnosis and follow-up. • damage to the visual pathway has been shown to lead to transynaptic retrograde degeneration, which may be detected with oct evaluation both of the prnfl and ganglion cell complex (gcc). • therefore, our purpose was to evaluate the relationship between topographic reduction in gcc thickness and visual field defects caused by central nervous system lesions in children, to explore whether the gcc could provide a valid surrogate marker for visual loss when visual fields are unavailable. conflict of interest the authors declare that they have no conflict of interest. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. pediatric suprasellar tumors ganglion cell layer -inner plexiform layer thickness and vision loss in young children with optic pathway gliomas anomalous head posture with early-onset homonymous hemianopia optic pathway glioma in children: does visual deficit correlate with 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thinning and its association with visual field loss in homonymous hemianopia caused by post-geniculate lesions using spectral-domain optical coherence tomography. graefe's arch clin exp ophthalmol the time course of retrograde transsynaptic degeneration following occipital lobe damage in humans invited commentary: ganglion cell complex measurement in compressive optic neuropathy key: cord- - e krzxe authors: coquillette, madeline; lee, richard s.; pagni, sarah e.; cataltepe, sule; stein, deborah r. title: renal outcomes of neonates with early presentation of posterior urethral valves: a -year single center experience date: - - journal: j perinatol doi: . /s - - - sha: doc_id: cord_uid: e krzxe objective: evaluate renal outcomes and early predictive factors in infants with congenital posterior urethral valves who required catheter or surgical urinary tract decompression within the first days of life. study design: a -year retrospective study at a single hospital. primary outcomes were estimated glomerular filtration rate (egfr) and development of end stage renal disease (esrd). results: of infants, % developed egfr < ml/min/ . m( ) and % progressed to esrd. nadir creatinine, need for invasive ventilation in the newborn period, and need for surgical diversion after catheter diversion were associated with worse outcomes. % of infants requiring invasive ventilation as neonates developed egfr < ml/min/ . m( ) in childhood. conclusions: half of infants with early presentation and intervention developed significant renal insufficiency in childhood, similar to children with later presentation or who had fetal intervention. invasive ventilation in the newborn period and need for surgical urinary diversion are associated with worse outcomes. congenital posterior urethral valves (puv) is the most common cause of lower urinary tract obstruction (luto) in males, accounting for more than half of pregnancies complicated by luto, and results in a spectrum of urologic and renal sequelae [ , ] . overall, infants with puv have poor renal outcomes. the current literature indicates that - % of patients with puv will develop chronic kidney disease (ckd) and - % will progress to end stage renal disease (esrd) during childhood. furthermore, there have been minimal improvements in outcomes over time [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . due to the poor prognosis, significant effort has been put forth to develop interventions for this population, particularly focusing on fetal interventions to relieve the obstruction and allow normal renal and pulmonary development. fetal intervention, primarily by vesico-amniotic shunting or fetal cystoscopic ablation, has been attempted for nearly years. the cumulative literature suggests that while in utero intervention may reduce perinatal mortality, fetal mortality remains high at - %. furthermore, these interventions have not improved postnatal outcomes and are associated with surgical complications [ ] [ ] [ ] [ ] [ ] . although the anatomic anomaly in puv is relatively uniform, the evaluation of potential interventions has been limited by significant heterogeneity in available cohorts. reported outcomes combine patients with perinatal and childhood diagnoses, varying severity of obstruction, and both fetal and postnatal interventions. in addition, patients with poor outcomes, such as early death or need for subsequent interventions, are often excluded from data analysis. such heterogeneity likely also contributes to difficulties in identifying factors predictive of renal outcomes. small studies have inconsistently shown association of antenatal diagnosis, prematurity, oligohydramnios, renal parenchymal changes, nadir creatinine, vesicoureteral reflux, bladder dysfunction, and younger age at diagnosis with worse renal outcomes; however in multivariate analyses only nadir creatinine has been predictive [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . given the challenges associated with the heterogeneity of this patient population, the aim of this study was to isolate a specific cohort of infants with severe luto caused by puv who presented and had intervention in the first week of life, to evaluate renal outcomes and early predictive factors. this study was approved by the institutional review board at boston children's hospital. requirement for consent was waived by the board. study data were collected and managed using redcap electronic data capture tools hosted at the study institution [ ] . eligible subjects for this study were male infants born during a retrospective -year study period from january , to december , who received treatment at a single tertiary children's hospital for severe luto from puv in the first week of life. patients treated at the institution for puv were identified through a data warehouse query using icd- code q . (congenital puv) or cpt procedure code (cystourethroscopy with incision, fulguration, or resection of congenital puv, or congenital obstructive hypertrophic mucosal folds). patients identified using this query were then reviewed for study inclusion by review of electronic medical records. patients were included in data collection and analysis if they were: ( ) confirmed to have puv in medical records and ( ) had catheter or surgical intervention for urinary tract decompression during the first week of life (n = ). the aim was to isolate a homogeneous population of infants with severe luto, therefore presentation by days of life was selected as it is likely that infants with severe luto would develop clinical signs within this time period, and infants with less severe luto would likely remain undetected. patients that were excluded included those who presented after the first week of life or who underwent antenatal urinary tract intervention. although some excluded patients presented during infancy, many presented during childhood. children who transferred care after infancy or came to the study institution for second opinion were also excluded. the primary outcome measure was the estimated glomerular filtration rate (egfr) at last follow-up, and was only used after age years. egfr was calculated using the bedside schwartz equation [ ] . esrd was defined as egfr < ml/min/ . m or requirement for renal replacement therapy. covariates included birth gestational age, race, prenatal, and postnatal ultrasound (us) findings, presence of vesicoureteral reflux, nadir creatinine in first year of life, duration of follow-up, other renal or urologic surgical interventions, and any respiratory support required during the newborn or follow-up period. prenatal us findings included hydroureter, oligohydramnios, renal parenchymal changes, and bladder changes. postnatal us findings included hydroureter and renal parenchymal changes. postnatal us findings were assessed at two time points, at first postnatal us and at year of age. nadir creatinine was categorized as < mg/dl or ≥ mg/dl based on findings in the existing literature, and excluded values after dialysis initiation. descriptive statistics were calculated. differences in egfr at last follow-up between groups by race, birth gestational age, respiratory supports in the newborn period, ultrasound findings, nadir creatinine, vesicoureteral reflux, and other surgical interventions were assessed with either the kruskal-wallis test or mann-whitney u test. post hoc pairwise comparisons following the kruskal-wallis test were performed with dunn's test with the bonferroni correction. a kaplan-meier curve was used to analyze time to development of egfr < ml/min/ . m (fig. ) . stata version . (statacorp llc, college station, tx) and spss version (ibm corp, armonk, ny) were used in the analysis. statistical significance was set at %. of children treated for puv at the study institution between and who did not have antenatal intervention, infants presented and had catheter or surgical urinary tract decompression within the first week of life. the remaining presented after week of age. the demographics and characteristics of participants are shown in table . there were ( %) singleton births and ( %) twin births. one infant died at days of life. this infant was included in patient characteristics, however due to the absence of follow-up, analyses that required follow-up data were completed using data from the surviving infants. mean laboratory follow-up was months, although clinical follow-up was frequently longer. during this time period, ( %) infants developed egfr < ml/min/ . m , including nine ( %) with egfr < ml/min/ . m and five ( %) progressed to esrd (table ) . all five infants who progressed to esrd received renal transplants during the study period. two children included also had other significant medical conditions. one child developed acute lymphoblastic leukemia in infancy, and one child was diagnosed with neurofibromatosis type . ( %) children required invasive mechanical ventilation in the neonatal period. ten ( %) of these children needed high-frequency ventilation. eleven ( %) required surgical urinary diversion by nephrostomy tube, ureterostomy, or vesicostomy after attempt of catheter drainage rather than, or in addition to ablation of valves. among those who survived the newborn period, there were no significant differences in egfr at last follow-up by race, gestational age, prenatal, or postnatal us findings, or presence of vur ( table ). all children with nadir creatinine ≥ mg/dl developed esrd. in addition, all children with nadir creatinine ≥ mg/dl were diagnosed with esrd within the first month of life. there was a significant number of preterm patients included in this study, with % of included patients born before weeks gestation, however there was no difference in overall renal survival between very preterm (< weeks gestation), moderate or late preterm ( - weeks gestation), or term infants ( or greater weeks gestation) (p = . ). figure summarizes overall renal survival of all included patients, defined as last egfr of ml/min/ . m or greater without a renal transplant. infants who required invasive ventilation in the newborn period had lower egfr at the end of the follow-up period ( ml/min/ . m ) compared with infants who did not require invasive ventilation ( ml/min/ . m ) (p = . ). in addition, % of infants who developed esrd required invasive ventilation in the newborn period. infants who required surgical urinary diversion other than valve ablation had lower egfr ( ml/min/ . m ) at last follow-up than those with sufficient diversion by catheter drainage ( ml/min/ . m ). in this cohort, % of infants with puv who presented and required catheter or surgical urinary tract decompression in the first week of life developed significant renal insufficiency or esrd during the study period. these results are concordant with other published cohorts of all children with puv. we found that elevated nadir creatinine, invasive mechanical ventilation, and requirement for surgical urinary diversion other than routine ablation of valves were predictive factors of egfr < and esrd. given the progressive nature of renal disease associated with puv, we would anticipate that egfr would continue to decline over time in this cohort of patients, and further study to trend renal function through childhood and into adulthood is needed, including comparison with children who present at fig. overall renal survival defined as last egfr of ml/ min/ . m or greater without a renal transplant a later age. children who present at a later age may be at a lower risk for ckd and esrd, as their pathology is likely associated with only partial obstruction, and may progress at a different rate. the follow-up duration in this study was similar to published cohorts and further emphasizes the need for extended follow-up in all subpopulations of children with puv. the renal outcomes of our cohort were similar to those reported for patients who have received fetal intervention for puv. efforts to date to improve outcomes by fetal intervention have been relatively unsuccessful and are associated with high fetal and perinatal morbidity and [ ] [ ] [ ] [ ] [ ] . furthermore, the ability to antenatally recognize and prognosticate the luto patients who are at the greatest risk for ckd and esrd is limited. if technological and scientific developments allow for optimization of fetal interventions, the ability to identify those at greatest risk for ckd will be important to select the correct patients who can benefit from these interventions. our cohort contributes to understanding how elements of early care during fetal monitoring and neonatal intensive care could inform outcomes. our cohort contained a significant number of premature infants and infants who required mechanical ventilation in the neonatal period. the need for invasive ventilation was associated with lower egfr. this observation is similar to prior studies that also suggest that need for respiratory support may portend worse renal outcomes. prior studies of patients with puv have found an association of oligohydramnios with need for respiratory support after birth [ ] and an association of early ventilator support with the development of ckd [ ] . although pulmonary hypoplasia of varying severity is associated with other congenital anomalies of the kidney and urinary tract, this has not been typical of puv. in fact, no children in this cohort had signs of respiratory insufficiency during the follow-up period. these findings suggest that while respiratory failure in the neonatal period may be associated with the development of significant ckd in childhood, it does not appear to lead to long-term pulmonary morbidity in children with puv. as in other studies, we hypothesize that the need for pulmonary support in the neonatal period is likely related to a combination of factors including oligohydramnios and prematurity. although there was no association of oligohydramnios with egfr in our cohort, oligohydramnios was significantly associated with need for invasive ventilation (data not shown). there is also existing literature suggesting an association of prematurity with ckd [ ] . further studies are needed to clarify the causes of neonatal respiratory compromise in children with puv. nadir creatinine ≥ mg/dl in the first year was found to be significantly associated with low egfr in this study, supporting previously reported findings. notably, all infants with elevated nadir creatinine in this study were diagnosed with esrd and initiated on dialysis within the first month of life; therefore this variable may have limited use as an early predictive factor for the later development of ckd and esrd. if more sensitive markers of renal function are identified and validated, they may allow an opportunity to more accurately identify changes in renal function sooner than serum creatinine, especially in the neonate. important limitations of this study include the retrospective design, single institution experience, small sample size and short duration of follow-up, although similar to existing literature. prenatal us reports were not universally available and reported prenatal findings relied on documentation in admission and consult notes and likely are underreported. the postnatal us findings were fairly uniformly documented in this institution but results still may be subjective in certain cases. half of infants with puv who presented and had catheter or surgical urinary tract decompression in the first week of life have poor renal outcomes during childhood, however they appear to have similar outcomes compared to published cohorts that include children with later presentation and children after fetal intervention. infants who required invasive ventilation in the newborn period or required surgical urinary diversion beyond valve ablation after catheter drainage had worse renal outcomes. conflict of interest the authors declare that they have no conflict of interest. publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. congenital lower urinary tract obstruction: a population-based epidemiological study fetal lower urinary tract obstruction (luto): a practical review for providers renal outcome in patients with congenital anomalies of the kidney and urinary tract antenatal determinants of longterm kidney outcome in boys with posterior urethral valves posterior urethral valves: risk factors for progression to renal failure ibrahiem el hi. posterior urethral valves: multivariate analysis of factors affecting the final renal outcome risk factors for progression to end-stage renal disease in children with posterior urethral valves risk factors for end stage renal disease in children with posterior urethral valves long-term outcome of prenatally detected posterior urethral valves: single center study of cases managed by primary valve ablation prognostic factors of posterior urethral valves and the role of antenatal detection prognostic factors in prenatally-detected posterior urethral valves: a multivariate analysis fetal bladder outlet obstruction: embryopathology, in utero intervention and outcome prenatal bladder drainage in the management of fetal lower urinary tract obstruction: a systematic review and metaanalysis laser ablation of posterior urethral valves by fetal cystoscopy fetal surgery for posterior urethral valves: long-term postnatal outcomes posterior urethral valve: outcome of antenatal intervention posterior urethral valves: are neonatal imaging findings predictive of renal function during early childhood? renal impairment in children with posterior urethral valves timing of posterior urethral valve diagnosis and its impact on clinical outcome abnormal antenatal sonogram: an indicator of disease severity in children with posterior urethral valves posterior urethral valves: prognostic factors did antenatal diagnosis protect against chronic kidney disease in patients with posterior urethral valves? a multicenter study nadir creatinine in posterior urethral valves: how high is low enough? research electronic data capture (redcap)-a metadata-driven methodology and workflow process for providing translational research informatics support measurement and estimation of gfr in children and adolescents posterior urethral valves: prenatal diagnostic signs and outcome short-term gestation, long-term risk: prematurity and chronic kidney disease key: cord- -bvxbzgx authors: jagadevan, mohanakrishnan; mohanakrishnan, bhanumathy; murugesan, salaja; sharma, deep; agarwal, navin kumar; fletcher, jebaraj; balasubramanian, vengatesan title: progression to ambulation following lower limb fractures in an individual with a spinal cord injury: a case report date: - - journal: spinal cord ser cases doi: . /s - - - sha: doc_id: cord_uid: bvxbzgx introduction: patients with spinal cord injury (sci) and concomitant lower limb fractures are a challenge to rehabilitate. conventionally, postural orientation is an important milestone in the rehabilitative process. we propose an alternative strategy in achieving goals in individuals with an sci with concomitant injuries that preclude weight bearing below the knee. case presentation: a -year-old girl sustained a burst fracture of l in conjunction with bilateral ankle fractures. during rehabilitation, the calcaneal fracture on the left and tibial plafond fracture on the right prevented her progression in conventional rehabilitation. an alternative strategy “k-ing” (kneel standing/kneel walking) was adopted to facilitate truncal activation without loading the ankle joints. this was found to be helpful in obtaining upright posture stability without hampering her recovery of associated ankle injuries. discussion: “k-ing” strategy can be useful and presents a simple alternative in the presence of associated ankle injuries. it also avoids complications associated with bedrest when there is delay in initiation of ambulation. gait training in individuals with an sci is challenging to patients and physical therapists. the possible comorbidities in the progression of rehabilitation in these cases are postural hypotension [ ] , heterotrophic ossification [ ] , and other concomitant injuries like lower-extremity fractures [ , ] . the incidence of lumbar spine injuries in calcaneal fractures has been reported to be % [ ] . in the rehabilitation process, bipedal loading is an integral part [ , ] prior to ambulation training. the associated lower-extremity fractures can be a setback in individuals with an sci, which can always pose a threat in achieving standing balance and gait training [ , ] . the objective of this case report is to present a low-cost rehabilitation strategy that can be performed without loading the fractured lower limbs and help achieve functional goals. a -year-old girl sustained an l fracture and bilateral fractures involving foot and ankle (left calcaneum and right tibial plafond). the clinical presentation was ais a paraplegia with the absence of sensation below t and loss of motor power below t along with loss of bladder and bowel sensation. she was treated surgically with decompression and posterior stabilization with pedicle screws and rods. fracture of the tibial plafond was managed by closed reduction and below-knee plaster-of-paris cast application. fracture of the calcaneus was treated by closed reduction followed by below-knee plaster-of-paris cast application. the patient was transferred to rehabilitation at the end of the first week following surgery. the initial assessment of the neurological level of injury postoperatively remained t ais a [ ] . the manual muscle test (mmt) was grade zero in all the muscles of both the lower limbs. the motor index score was of , while the sensory index score was of . our postoperative spinal stabilization physiotherapy protocol was followed for the first week ( table ). the individual progressed to sitting upright by the end of the second week using a spinal hyperextension support (ash brace). electrical stimulation to the knee extensors and flexors with galvanic currents ( contractions- sets/day) was provided [ , ] . at the end of weeks, a trace of contraction was noted in the left quadriceps with muscle power grade (mmt). although the spinal cord injury rehabilitation evidence guidelines indicate that standing in a tilt table or in the parallel bars is a recommended therapy at the end of - weeks [ ] , this is to be done only if the individual does not have an associated lower limb fracture. since the calcaneal [ ] and tibial plafond fractures [ ] prevented this individual from loading through the feet, physician clearance was obtained to progress the subject to quadruped positioning. the objective was to obtain static balance training to enhance trunk stability with loading through the femurs. medical clearance was also given to progress to quadruped crawling. once quadruped balance was achieved for - min, during weeks through , crawling was initiated. this facilitated the progression to kneel standing with support on a cushioned mat and later in the parallel bars with adequate cushion support in order to avoid skin complications. at months post injury, static kneeling was achieved for - min at months and kneel walking was initiated afterward. initially, kneel walking was introduced gradually with pillows over the knee with height adjusted in the parallel bars to accommodate the lower trunk alignment (fig. ) . the progression started with - min twice a day and increased to - min twice a day as tolerated. at the end of months, kneeling balance with the cushion was achieved for about - min and kneel walking inside the parallel bars for ( - steps) × was achieved. subsequently, loading started in the bilateral lower limb as the fracture union was radiologically confirmed. bilateral knee ankle foot orthoses (kafo) were fabricated to allow standing balance training and ambulation progression. the participant was made to stand inside the parallel bars for - min for a few days and the standing duration was increased gradually to - min. as the standing balance improved, walking was initiated. within a week, walking inside parallel bars was achieved for about ( - steps) × / day (fig. ) . during initial assessment, there was a complete loss of sensation below t and the predischarge examination at months post injury revealed a partial return of sensation from t to s on the right and from t to s on the left. the sensory index score was out of . there was a partial recovery of muscle power in lower limb key muscles [hip flexors (from / to / bilateral) and knee extensors (from / to / bilateral)]. the motor index score was table ). the walking index for spinal cord injury was used to measure her outcome ambulation score. she obtained a score of (ambulates with two crutches, with braces and physical assistance of one person, meters) at the end of months [ ] . this case report describes the k-ing strategy used in early mobilization on a participant with cauda equina injury and bilateral foot and ankle injury. studies indicate that early mobilization reduces postoperative complications and provides physiological and psychological benefits [ , ] . early vertical loading can promote balance and ambulation training in a sci rehabilitation program [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the k-ing strategy can be a safe alternative in promoting vertical orientation [ , ] without loading the leg and foot component and simultaneously preserving the fracture sites. since the kneel standing and kneel walking re-education simulated standing and walking respectively, this might also have been a positive effect on improving motor function. in sci rehabilitation, loading the hip joint is a critical stimulus in the process of re-innervation of muscles concerned with locomotor training [ ] [ ] [ ] . hence, this strategy could be a hip extension [ ] is crucial in achieving standing balance in sci rehabilitation. in this case, k-ing strategy facilitated hip extension by loading through femurs when bipedal stance could not be allowed. bipedal stance and ambulatory training with orthotic support may be achieved by - weeks. in this case, bipedal stance had to be delayed due to the associated lower limb fractures, thereby resulting in a possible delay of another - weeks. introducing this alternative strategy in early mobilization to an upright position may have reduced the length of stay and therefore the cost of hospitalization [ ] . conclusion "k-ing strategy" may be useful for progression to ambulation in sci patients associated with foot and ankle fractures. this technique may also have a positive effect on neural activation and prevent the deleterious effects of prolonged inactivity. further studies are required to validate if "k-ing strategy" ambulation outcomes are similar to or more effective than other conventional interventions in sci patients. conflict of interest the authors declare that they have no conflict of interest. publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. rehabilitation of spinal cord injuries medical rehabilitation of people with spinal cord injury during years of academic physiatric practice morbidity following lower extremity fractures in men with spinal cord injury long-bone fractures in persons with spinal cord injury association of calcaneal and spinal fractures locomotor training: as a treatment of spinal cord injury and in the progression of neurologic rehabilitation locomotor activity in spinal cord-injured persons advantages of delaying the onset of rehabilitative reaching training in rats with incomplete spinal cord injury anatomical changes in human motor cortex and motor pathways following complete thoracic spinal cord injury classifications in brief: american spinal injury association (asia) impairment scale early cyclical neuromuscular electrical stimulation improves strength and trophism by akt pathway signaling in partially paralyzed biceps muscle after spinal cord injury in rats a clinical practice guideline for the management of patients with acute spinal cord injury: recommendations on the type and timing of rehabilitation spinal cord injury rehabilitation evidence: methods of the scire systematic review treatment of displaced intraarticular calcaneal fractures with or without bone grafts: a systematic review of the literature the sequential recovery of health status after tibial plafond fractures ambulation following spinal cord injury and its correlates identifying predictors of resilience at inpatient and -month post-spinal cord injury systematic review describing the effect of early mobilisation after dysvascular major lower limb amputations management of spinal cord injuries trunk extension strength and muscle activity in standing and kneeling postures body positions used by healthy and frail older adults to rise from the floor plasticity of the spinal neural circuitry after injury heiner sell memorial lecture: neuronal plasticity after spinal cord injury: significance for present and future treatments basic concepts of activitybased interventions for improved recovery of motor function after spinal cord injury neuroplasticity after spinal cord injury and training: an emerging paradigm shift in rehabilitation and walking recovery impact of therapy on recovery during rehabilitation in patients with traumatic spinal cord injury key: cord- -h vbmbo authors: takeda, yohei; okuyama, yuko; nakano, hiroto; yaoita, yasunori; machida, koich; ogawa, haruko; imai, kunitoshi title: antiviral activities of hibiscus sabdariffa l. tea extract against human influenza a virus rely largely on acidic ph but partially on a low-ph-independent mechanism date: - - journal: food environ virol doi: . /s - - -x sha: doc_id: cord_uid: h vbmbo influenza a virus (iav) infection is perennially one of the leading causes of death worldwide. effective therapy and vaccination are needed to control viral expansion. however, current anti-iav drugs risk inducing drug-resistant virus emergence. although intranasal administration of whole inactivated virus vaccine can induce efficient protective immunity, formalin and β-propiolactone are the currently used and harmful inactivating agents. here, we analyzed the antiviral activity of hibiscus (hibiscus sabdariffa l.) tea extract against human iav and evaluated its potential as a novel anti-iav drug and a safe inactivating agent for whole inactivated vaccine. the in vitro study revealed that the ph of hibiscus tea extract is acidic, and its rapid and potent antiviral activity relied largely on the acidic ph. furthermore, the mouse study showed that the acidic extract was not effective for either therapeutic or vaccination purposes. however, hibiscus tea extract and protocatechuic acid, one of the major components of the extract, showed not only potent acid-dependent antiviral activity but also weak low-ph-independent activity. the low-ph-independent activity did not affect the conformation of immunodominant hemagglutinin protein. although this low-ph-independent activity is very limited, it may be suitable for the application to medication and vaccination because this activity is not affected by the neutral blood environment and does not lose antigenicity of hemagglutinin. further study of the low-ph-independent antiviral mechanism and attempts to enhance the antiviral activity may establish a novel anti-iav therapy and vaccination strategy. outbreaks of seasonal influenza caused by influenza a virus (iav) or b virus occur all over the world and lead to approximately to million severe cases and , to , deaths every year (world health organization ). moreover, novel iav strains have emerged repeatedly in the past, leading to a large number of deaths by pandemics in the twentieth century (frederick ; saunders and krewski ; world health organization ) . to combat iav, there is a relentless need to develop more effective antiviral therapies and vaccine strategies. the calyces of hibiscus sabdariffa l. is an ingredient in hibiscus tea. hibiscus tea contains abundant bioactive compounds including anthocyanins, polyphenols, organic acids, and flavonoids (da-costa-rocha et al. ) . earlier reports have shown various biological and pharmacological activities of hibiscus tea and hibiscus tea-derived compounds like anti-inflammatory, anti-oxidant, anti-cholesterol, anti-hypertensive, anti-bacterial, and antiviral activities (d'souza et al. ; da-costa-rocha et al. ; hassan et al. ; joshi et al. ) . we previously reported the antiviral activity of hibiscus tea extract against h subtype avian influenza viruses (baatartsogt et al. ) . hibiscus tea extract inactivated the avian influenza viruses, showing activity in min. based on these results, we hypothesized that these potent and rapid antiviral activities could be used to develop a novel anti-iav drug and a safe inactivating agent for the whole inactivated vaccine. a number of anti-iav drugs have been in practice or under development (li et al. ; noshi et al. ) . although these drugs elicit the therapeutic effect, viral strains resistant against these drugs tend to appear after prolonged use (hurt ; li et al. ) , and some of these drugs also exhibit adverse effects. rna polymerase inhibitor favipiravir showed teratogenicity and embryotoxicity in animal experiments. therefore, its clinical use is strictly restricted in japan (nagata et al. ) . in addition, the possibility of insufficient existing drug supply is a concern during pandemics. finding novel anti-iav substances would be a promising approach for treatment to deal with these problems. vaccination is also important to control iav expansion. one of the effective vaccination strategies is intranasal (i.n.) administration of whole inactivated vaccine. whole inactivated vaccine can prime potent protective immunity because it preserves virus particle integrity (soema et al. ) . in addition, i.n. vaccination can induce systemic igg production and mucosal iga production, the latter of which is indispensable to prevent viral infection establishment (rose et al. ; tamura et al. ) . formalin and β-propiolactone (β-pl) are widely used as inactivating agents for whole-virus vaccines (delrue et al. ) . however, removal of these substances is essential due to their chemical toxicity, making the procedure time consuming and expensive. finding a safe anti-iav compound that can be used as a novel inactivating agent for a whole-virus vaccine may solve critical problems. here, we attempted to clarify the mechanism of antiviral activity against h n human iav strain a/puerto rico/ / (pr virus) and identify the antiviral compounds present in hibiscus tea extract. we focused on three compounds including protocatechuic acid (pca), ferulic acid, and ( s, r)-tetrahydro- -hydroxy- -oxo- , -furandicarboxylic acid (hibiscus acid). pca and ferulic acid showed antiviral activity against several virus species (hassan et al. ; joshi et al. ) . hibiscus acid is one of the major organic acids in hibiscus calyces (da-costa-rocha et al. ). in addition, we assessed hibiscus tea extract's potential as a candidate for novel anti-iav drug and as an inactivating agent for whole-virus vaccines. pr virus (atcc® catalog no. vr- ™) was purchased from atcc (manassas, va), and propagated in the allantoic fluid of -day-old embryonated chicken eggs. madin-darby canine kidney (mdck) cells were kindly provided by dr. h. nagano (hokkaido institute of public health, sapporo, japan). for passaging, mdck cells were cultured in dulbecco's modified eagle's minimal essential medium (dmem) (nissui pharmaceutical co., ltd., tokyo, japan) supplemented with % fetal bovine serum, μm l-glutamine (wako pure chemical industries, ltd., osaka, japan), . % nahco (wako pure chemical industries, ltd.), µg/ml amphotericin b (bristol-myers squibb co., new york, ny), and µg/ml kanamycin (meiji seika pharma co., ltd., tokyo, japan). after viral inoculation, mdck cells were cultured in viral growth medium composed of dmem supplemented with . % bovine serum albumin (wako pure chemical industries, ltd.), . % glucose (wako pure chemical industries, ltd.), . % nahco , . mm hepes (wako pure chemical industries, ltd.), and . % trypsin (wako pure chemical industries, ltd.). ten to -week-old female balb/c mice were purchased from clea japan inc. (tokyo, japan) or generated in our laboratory. all animal experiments were approved by the institutional animal care and use committee of obihiro university of agriculture and veterinary medicine and performed in compliance with institutional guidelines. pca was purchased from tokyo chemical industry co., ltd. (tokyo, japan). ferulic acid was purchased from cayman chemical co. (an arbor, mi). formalin and β-pl were purchased from wako pure chemical industries, ltd. hibiscus tea (roselle) was purchased from nakazen corp. (nanjo, japan). to prepare acidic crude hibiscus tea extract (hib[crude]), g of hibiscus tea powder was boiled in ml ultrapure water until the volume reached ml. to isolate the small molecular weight (< kda) fraction (frhibis), hib[crude] was fractionated using vivaspin® ( mwco pes) (sartorius ag., göttingen, germany) (baatartsogt et al. ). hibiscus acid was isolated as previously described (ibnusaud et al. ; ibrahim et al. ) . in brief, hibiscus tea powder was soaked in a sufficient volume of ultrapure water at °c for h, and this process was repeated several times. the combined extract was concentrated under reduced pressure to obtain a viscous syrup. methanol was added to the syrup to precipitate insoluble materials. the precipitate was removed by filtering, and the filtrate was concentrated to obtain a second viscous syrup that was extracted several times with acetone. acetone was removed under reduced pressure, and the extract was further extracted several times with ether. the ether was removed under reduced pressure, and a crude acid was obtained which was recrystallized (ether + chloroform) to obtain pure hibiscus acid (molecular weight: . , . g, %) in the form of colorless crystal. the structure and purity of isolated hibiscus acid was confirmed using previously reported spectroscopic and other physical analyses (ibnusaud et al. ; ibrahim et al. ) . hibiscus acid-na was synthesized as previously described (ibnusaud et al. ; ibrahim et al. ) . in brief, saturated aqueous sodium bicarbonate was added dropwise into an aqueous hibiscus acid solution ( . g, . mmol, in ml water) at °c until the ph of the solution was neutral. after removing water under reduced pressure, the residue was triturated and washed using dry acetone. the product was finally dried under reduced pressure to obtain a colorless solid (molecular weight: . , . g, %). % tissue culture infective dose (tcid )/ml of pr virus propagated in allantoic fluid was mixed with an equal amount of phosphate-buffered saline (pbs), hib[crude], frhibis, pca, ferulic acid, hibiscus acid, or hibiscus acid-na. the effective concentrations of pca, ferulic acid, hibiscus acid, and hibiscus acid-na in the mixture were . mg/ml, mg/ml, . mg/ml, and . mg/ml, respectively. ph of the mixture was adjusted within a range of acidic to around neutral by adding hcl (wako pure chemical industries, ltd.) or naoh (wako pure chemical industries, ltd.). ph of the mixture was determined by measuring the ph of a virus-free mixture as a substitute for the virus-containing mixture. the mixture was placed at °c for min, h, or h. after each reaction time, the mixture was inoculated into mdck cells and a tenfold serial dilution was performed. three days after inoculation, the viral titer was evaluated by observing the cytopathic effect (cpe) on mdck cells. we calculated log tcid /ml using the behrens-kärber method (kärber ) . pr virus propagated in allantoic fluid was mixed with an equal amount of neutral and acidic ph pbs, hib[crude], frhibis, or pca. the effective concentration of pca in the mixture was . mg/ml. the mixture was placed at °c for min or h. a twofold serial dilution was performed after each reaction time. an ha test was conducted using . % chicken blood cells (nippon bio-test laboratories inc., asaka, japan) according to the who manual on animal influenza diagnosis and surveillance (who manual, world health organization ). the ha test was also conducted with formalin-, β-pl-, or acidic hib[crude]-inactivated purified pr virus. mice were intranasally given tcid / μl pr virus propagated in allantoic fluid. eight hours after the viral inoculation, μl of neutral pbs or acidic hib[crude] was administered once orally (p.o.) (day ). from day to , pbs or hib[crude] was administered p.o. twice a day. each group had mice. the change in the body weight of each mouse was monitored daily. mice were euthanized when the body weight decreased to % or weeks after the viral inoculation. to determine the lung viral titer, mice were euthanized at days and and lungs were harvested from the euthanized mice. the lungs were homogenized in % (w/v) pbs supplemented with μg/ml amphotericin b, mg/ml kanamycin, and μg/ml gentamicin (msd k.k., tokyo, japan). the lung homogenate supernatant was added to mdck cells, and a tenfold serial dilution was performed. three days after inoculation, the viral titer (log tcid /g tissue) was evaluated. to prepare formalin-inactivated whole-virus vaccine, mg/ml of purified pr virus was mixed with . % formalin and kept at °c for h. after viral inactivation, . % na s o (wako pure chemical industries, ltd.) was added and the mixture was placed at °c overnight. similarly, to prepare β-pl-inactivated whole-virus vaccine, mg/ ml of purified pr virus was mixed with . % β-pl and placed at °c for h. afterward, formalin and β-pl were removed using membrane dialysis with spectra/por® ( , mwco) (spectrum laboratories inc., los angeles, ca). the concentration of formalin-or β-pl-inactivated pr virus was measured using the micro bca protein assay kit (pierce, rockford, il) and adjusted to μg/ ml. to prepare acidic hib[crude]-inactivated whole-virus vaccine, μg/ml of purified pr virus was mixed with an equal amount of acidic hib[crude] and placed at °c for min. the inactivated virus was directly used to vaccinate mice. viral inactivation was confirmed based on the absence of viral infectivity by egg inoculation followed by an ha test. μl pbs, formalin-, β-pl-, or acidic hib[crude]-inactivated pr virus vaccine was intranasally administered in mice (first vaccination) under light anesthesia with isolflurane (intervet k.k., tokyo, japan). the second vaccination was performed weeks after the first vaccination. there were mice in each group. six weeks after the second vaccination, the mice were intranasally inoculated with tcid / μl of pr virus propagated in allantoic fluid (day ). changes in body weight were monitored daily after pr viral inoculation. the mice were euthanized when the body weight decreased %. all surviving mice were euthanized on day . blood samples were collected on day (pre-inoculation) and day (post-inoculation) to evaluate anti-pr virus antibody (ab) titer using the hemagglutination inhibition (hi) test. the obtained serum samples were treated with receptordestroying enzyme (denka seiken co., ltd., japan) to remove non-specific ha inhibitors. the serum was mixed with chicken blood cells (the final concentration was %), and the mixture was rotated at room temperature for h to remove any non-specific ha factors. the supernatant was collected after centrifugation, and the hi test was conducted in the presence of pr viral antigen according to the who manual. the serum anti-pr virus ab titer was determined as log hi titer. since hib[crude] is acidic, we assessed whether the anti-pr virus activity of hib[crude] depends on its acidic ph. pr virus was mixed with non-ph-adjusted and phadjusted pbs or hib[crude] (ph of the mixture ranged from . to . ), and the titer of the treated virus was determined. the ph of the mixture containing non-phadjusted hib[crude] was around . . after a -min reaction, hib[crude] and pbs of ph ≤ . strongly inactivated the pr virus, but the effect of hib[crude] at ph . seemed to be slightly higher than pbs at ph . . on the other hand, both pbs and hib[crude] of ph ≥ . did not inactivate pr virus. after a -h reaction, pr virus was inactivated using both pbs and hib[crude] at ph ≤ . on the other hand, the viral titer of hib[crude]-treated pr virus was -times lower than pbs-treated pr virus under the conditions of ph ≥ . (fig. a ). the differences were statistically significant (p < . or . ). we also evaluated viral titers of acidic and neutral (ph-adjusted) frhibis-treated pr virus. the ph of the mixture containing non-ph-adjusted frhibis was around . . acidic frhibis inactivated pr virus after a -min reaction, but neutral frhibis did not. after a -h reaction, the viral titer of neutral frhibis-treated pr virus was consistent with neutral hib[crude]-treated pr virus and -times lower (p < . ) than pbs-treated pr virus (fig. b) . these results show that frhibis has two different anti-pr virus activities: one is a rapid and potent activity largely dependent on acid, and the other is a slow and weak low-ph-independent activity. next, we evaluated the anti-pr virus activity of pca, ferulic acid, and hibiscus acid. these compounds are acidic in solution. pr virus was treated with pbs or each of these solutions, and the titer of the virus was measured. both acidic (non-ph-adjusted) and neutral (ph-adjusted) solutions were tested to correlate between acidic ph of the solutions and the anti-pr virus activity. the estimated ph values of the mixtures containing acidic pca, ferulic acid, and hibiscus acid were approximately . , . , and . , respectively. the mixtures containing acidic pbs (ph . , . , and . ) were also tested as acidic solution controls. after a -min reaction, the titers of acidic and neutral pca-treated pr virus were similar to a pbs-treated pr virus. acidic pbs and pca completely inactivated pr virus after a -h reaction. on the other hand, the viral titer of pca-treated pr virus was . -times lower (p < . ) than pbs-treated pr virus under neutral conditions (fig. a) . neither a -min nor -h reaction in acidic or neutral conditions showed anti-pr viral activity for ferulic acid (fig. b) . finally, anti-pr viral activity of hibiscus acid was analyzed. hibiscus acid-na is a disodium salt of hibiscus acid, and its ph was approximately neutral. acidic hibiscus acid inactivated pr virus after a -min or -h reaction, but neutral hibiscus acid and hibiscus acid-na did not do so (fig. c) . these results show that pca has weak low-ph-independent anti-pr virus activity. next, we assessed whether acidic and neutral hibiscus tea extracts affect the ha activity of pr virus. pr virus was mixed with acidic or neutral pbs, hib[crude], or frhibis. ha titer of pr virus was measured after a -min or -h reaction. acidic pbs-, hib[crude]-, and frhibis-treated pr virus lost ha activity in min. it was confirmed earlier that the acidic hib[crude] did not directly affect chicken blood cell agglutination (data not shown). after a -h reaction, the ha titer of neutral hib[crude]-and frhibis-treated pr virus did not decrease compared to pbs-treated pr virus (fig. a) . ha titers of acidic and neutral pca-treated pr virus were also measured. acidic pbs-and pca-treated pr virus lost ha activity after a -h reaction. on the other hand, the ha titer of neutral pca-treated pr virus did not decrease compared to pbs-treated pr virus (fig. b) . these results show that neutral hib[crude], frhibis, and pca does not affect viral ha activity. next, we assessed the therapeutic efficacy of p.o. administration of acidic hibiscus tea extract in pr virus-infected mice. neutral pbs or acidic hib[crude] was administered p.o. to mice twice a day for the first days after pr virus inoculation (fig. a) . a significant loss in body weight was observed in all treated mice (fig. b) . all pbs-treated mice and % ( out of ) hib[crude]-treated mice died within days post-infection. although only one hib[crude]-administered mouse survived and recovered from the disease, there was no significant difference in the survival rate between the pbs-and hib[crude]-treated groups (fig. c) . the viral titer in the lungs was determined on days and , and it was found to be similar to levels in both the pbs and hib[crude] groups (fig. d) . these results show that p.o. administration of acidic hib[crude] does not exert a therapeutic effect in pr virus-infected mice. next, we assessed the efficacy of acidic hibiscus tea extract as an inactivating agent for whole virus. the pr virus inactivated by formalin, β-pl, or acidic hib[crude] was intranasally administered to mice twice before administering a lethal dose of pr virus (fig. a) . non-vaccinated mice receiving pbs served as a control group. non-vaccinated mice rapidly lost body weight after the viral inoculation, and all mice in the group died by day . on the other hand, the body weight in the formalin-and β-pl-treated groups did not change, and all the mice survived until day . the mice lost body weight in the hib[crude]-treated group, but the degree of weight loss was ameliorated compared to the non-vaccinated group. the vaccine efficacy was much lower in the hib[crude]-treated group, but the survival rate was significantly improved (fig. b, c) . the hi-active ab titer in serum was also measured pre-inoculation on day and postinoculation on day . prior to inoculation, the hi titers were below the detection limit in all non-vaccinated mice, but the titers were in the range of to in the formalin and . the ph of each aqueous solution was adjusted to acidic or approximately neutral. the ph shown was an approximate value with a range ± . . the mixture was placed at °c for min or h and mixed with an equal volume of chicken blood cells. ha titer of the mixture was evaluated after a -min reaction. error bars indicate mean ± sd; n = to per group. student's t test with bonferroni's test (a), and student's t test (b) were performed to analyze statistical significance. the results represent more than two independent experiments β-pl groups. in the hib[crude] group, the titers were less than in all mice and below the detection limit in two mice (fig. d) . although the hi titers were low in the hib[crude] group, the hi-positive mice survived until day (data not shown). post-inoculation hi titer did not differ from the preinoculation titer in the formalin and β-pl groups. in the hib[crude] group, the hi titer increased to the same level as the formalin and β-pl groups (fig. d) . these results indicate that formalin-and β-pl-inactivated vaccine completely prevented viral infection, but acidic hib[crude]-inactivated vaccine did not prevent infection. the viral particle structure integrity in the whole inactivated vaccine relates to protective activity against iav infection. we assessed whether the conformation of hemagglutinin (hain) was preserved in each vaccine using the ha assay. ha activity of iav is lost by a change in hain conformation (sriwilaijaroen and suzuki ) . intact (untreated) or inactivated pr virus was mixed with chicken blood cells and the ha titer was measured. the ha titer of acidic hib[crude]-treated pr virus was below the detection limit, but the ha titer of formalin-and β-pl-treated pr virus was similar to the intact pr virus (fig. e) . these results show that formalin and β-pl treatments but not acidic hib[crude] preserve hain conformation. earlier studies indicated that the extract derived from hibiscus sabdariffa l. calyces has antiviral activity against the h subtype of highly pathogenic and low-pathogenic avian influenza viruses, herpes simplex virus- , feline calicivirus, murine norovirus- , hepatitis a virus, and aichi virus (baatartsogt et al. ; d'souza et al. ; hassan et al. ; joshi et al. ) . moreover, the leaves of hibiscus sabdariffa l. also showed anti-measles virus activity (sunday et al. ). however, antiviral activity mechanism against these viruses is still unclear. since many acidic compounds in hibiscus calyces contribute to the low ph, we focused on correlating anti-pr viral activity and the acidic ph of hibiscus tea components. we found that not only hib[crude] but also pbs of ph ≤ . potently inactivated pr virus in a short period of time ( min) (fig. ) . this finding indicates that the anti-pr virus activity of hibiscus tea extract is largely dependent on acid. however, we could not deny a possibility that there are unknown antiviral compounds which show its activity only in the acidic condition. this unknown mechanism might partially contribute to the viral inactivation. we further analyzed the anti-pr virus activity of pca, ferulic acid, and hibiscus acid, which are all components of hibiscus tea extract. in the previous reports, pca showed antiviral activity against feline calicivirus, murine norovirus- , and herpes simplex virus- (hassan et al. ; joshi et al. ) . ferulic acid showed antiviral activity against feline calicivirus and murine norovirus- (joshi et al. ) . here, acidic pca and hibiscus acid showed anti-pr virus activity. non-ph-adjusted ferulic acid solution with a relatively higher ph (ph . ) did not inactivate pr virus. when the iav particle is exposed to acidic ph conditions, viral hain changes its conformation and irreversibly loses binding affinity for sialic acid (quan et al. ; sriwilaijaroen and suzuki ) . here, all pr virus particles treated with acidic pbs-, (fig. ) . it is also known that low-ph treatment of iav induces m protein layer loss and coagulation of ribonucleoprotein complexes (fontana et al. ). these comprehensive structural changes due to acid could have resulted in rapid and potent viral inactivation from acidic hibiscus component treatment. many iav strains are susceptible to acid, but the degree of sensitivity varies between strains (puri et al. ; ruigrok et al. ) . some highly pathogenic strains of avian h influenza virus like h n strain isolated from thailand were resistant to ph . (treated for min at room temperature) treatment (wanaratana et al. ) . although acidic hib[crude] and frhibis inactivated highly pathogenic and low-pathogenic avian h influenza viruses in min in our previous study (baatartsogt et al. ) , we have yet to determine whether all potent and rapid antiviral activity against these h viral strains is completely acid dependent. here, frhibis showed potent acid-dependent antiviral activity and weak low-ph-independent antiviral activity against pr virus (fig. ) . the neutral pca also showed anti-pr virus activity (fig. ) . the anti-pr virus activity by both neutral frhibis and pca did not affect hain activity (fig. ) . since pca accounts for % of hibiscus sabdariffa water extract (da-costa-rocha et al. ), a major part of the anti-pr virus activities of neutral hibiscus extract may be due to pca. in addition, frhibis also contains abundant bioactive compounds such as phenolic compounds (da-costa-rocha et al. ) . several studies reported that polyphenol-rich plant extracts and plant-derived phenolic compounds have anti-iav activities (droebner et al. ; kim et al. ; yamada et al. ). these phenolic compounds in frhibis may also contribute to antiviral activity. an attempt to identify the mechanism of low-ph-independent anti-pr virus activity of pca and to search for other low-ph-independent anti-pr virus constituents is currently ongoing. we assessed the therapeutic efficacy of p.o. administration of acidic hib[crude] in pr virus-infected mice. however, no therapeutic effect was observed (fig. ) . in the case of p.o. administration, hib[crude] is first absorbed from the digestive tract and distributed into systemic blood. subsequently, it reaches the respiratory organs where iav resides. the neutralized hib[crude] in the blood loses potent anti-iav activity due to acid, and the low-ph-independent antiviral activity is inadequate to inactivate virus in vivo. the low content, weak antiviral activity, and short biological half-life of low-ph-independent anti-iav compounds in hib[crude] could contribute to therapeutic failure. to achieve a successful p.o. administration therapy using hibiscus tea components such as pca, it would be necessary to chemically modify them to extend their biological half-life and enhance antiviral activity or use them in combination with a carrier to retain them in blood. in iav vaccination, i.n. vaccine administration induces effective anti-iav immunity. we previously showed that i.n. co-administration of innate immune receptor adjuvants with split vaccine efficiently induced both systemic and mucosal anti-iav immunity (takaki et al. , takeda et al. ). on the other hand, whole inactivated vaccine comprises viral structural components with immune priming activity like immunostimulatory viral rna. we assessed the utility of acidic hib[crude] as a safe inactivating agent for whole-virus vaccine. however, unlike the viruses inactivated by formalin and β-pl, the viruses inactivated by acidic hib[crude] failed to induce efficient protective immunity (fig. ) . as shown in other reports (pawar et al. ) , formalin-and β-pl-treated virus but not acidic hib[crude] showed intact ha activity (fig. e ). hain is an immunodominant protein (kaminski and lee ; liu et al. ; sautto et al. ) . the acidic ph of hib[crude] could cause the loss of antigenicity by inducing a change in hain conformation. these results show that the acidic hibiscus components cannot be used as inactivating agents for vaccine preparation. on the other hand, low-ph-independent activity of frhibis and pca which does not affect hain conformation is suitable for vaccine application. however, these weak virucidal effects have not yet been sufficient for use as an inactivating agent. further study of the low-ph-independent anti-iav mechanism and enhancement of its antiviral activity may contribute to iav vaccine strategy development. in conclusion, we showed the rapid and potent anti-pr virus activity of acidic hibiscus tea extract, which relies largely on acid. this acidic extract is not suitable for use in therapeutics and vaccine development. however, frhibis and pca were found to have weak low-ph-independent anti-pr virus activity, which does not affect hain conformation. hence, further study of low-ph-independent antiviral compounds may enable us to establish a novel anti-iav therapy and vaccination strategy. high antiviral effects of hibiscus tea extract on the h subtypes of low and highly pathogenic avian influenza viruses aqueous extracts of hibiscus sabdariffa calyces to control aichi virus hibiscus sabdariffa l.-a phytochemical and pharmacological review inactivated virus vaccines from chemistry to prophylaxis: merits, risks and challenges. expert review of vaccines cystus , a polyphenol-rich plant extract, exerts antiinfluenza virus activity in mice structural changes in influenza virus at low ph characterized by cryo-electron tomography pandemic influenza in ; 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published maps and institutional affiliations conflict of interest the authors declare that they have no conflict of interest. key: cord- -ymsag iq authors: wong, tiffany k.; alexander, marcalee sipski; new, peter wayne; delgado, andrew d.; bryce, thomas n. title: pulse article: opioid prescription for pain after spinal cord damage (scd), differences from recommended guidelines, and a proposed algorithm for the use of opioids for pain after scd date: - - journal: spinal cord ser cases doi: . /s - - - sha: doc_id: cord_uid: ymsag iq study design: online questionnaire of spinal cord injury (sci) physicians. objectives: the objective of this study is to characterize the approach to opioid prescription for persons with spinal cord damage (scd). setting: an international online questionnaire. methods: a survey was posted online and circulated among international societies within the field of sci medicine from august to november . results: one hundred and twenty-three physicians responded to the survey. of these, ( %) managed pain for persons with scd. most ( %) felt that opioid prescription was appropriate for uncontrolled acute pain, but fewer ( %) felt it was appropriate for chronic pain. of those who felt opioids had a role in the treatment of neuropathic pain, % did not think there should be a specific upper limit of opioid dose. the majority ( %) would continue prescribing high doses ( morphine milligram equivalent (mme) doses/day) if that dose were effective. tramadol was the most common opioid prescribed first line. conclusion: most physicians who responded to this survey prescribe opioids for intractable pain after scd. a significant proportion of respondents believed that there should not be a specific upper limit of opioid dose prescribed if the drug is tolerated; this does not align with current recommendations. most physicians do not feel influenced in their prescribing habits by regulatory bodies. if physicians decide to taper an opioid that is being tolerated well, it is most commonly related to a fear of the patient developing an opioid-use disorder. the authors propose an algorithm that may help align practice patterns with current recommended practice guidelines. four out of every five people with spinal cord damage (scd) report pain is an ongoing problem [ ] . in more than half of individuals with ongoing pain, the pain interferes with activities of daily living and work [ ] . this high prevalence of disabling pain is present in persons who receive care at scd centers throughout the world [ , ] and illustrates that the treatments available, both pharmacological and non-pharmacological, are not all that effective. as such, clinicians sometimes resort to the use of opioids for the treatment of pain after scd as is done for other types of intractable pain. however, similar to that in other causes of non-cancer chronic pain, the evidence for opioid effectiveness in the treatment of pain after scd is sparse [ , ] . nevertheless, over one quarter of persons with scd who have been treated in spinal cord injury (sci) specialty centers take opioids on an ongoing basis to treat chronic pain [ ] . the united states in remains in the midst of an opioid overdose epidemic, which has been exacerbated by the prescription of opioids for pain over the past two decades. in the united states, the rate of drug overdose deaths tripled between and [ ] . drug overdose is now the leading cause of unintentional death in the united states, with unintentional death being the third leading cause of death overall after cancer and heart disease [ ] . there are also signs of emerging opioid epidemics in other countries such as canada and australia [ ] . in , canada had the highest rate of per capita of opioid consumption in the world, at more than morphine milligram equivalent (mme) per capita, with the united states just behind at nearly mme per capita, germany at over mme per capita, and australia having a rate less than mme per capita [ ] . because of the increased awareness of the problem of opioid overdose in the context of limited clinical effectiveness for chronic pain, many countries have developed prescribing guidelines for opioids. representative guidelines include those developed in the united states by the centers for disease control and prevention (cdc) [ ] , in canada by the national pain centre [ ] , in germany by the german pain society [ ] , in great britain by the faculty of pain medicine of the royal college of anaesthetists [ ] , in australia by the royal australian college of general practitioners [ ] , and in south africa by a group of physicians whose guideline was endorsed by multiple professional societies [ ] . it is notable in all guidelines that there are recommendations for restricting the maximum prescribed daily dose of opioids prescribed. the recommended upper limit is of mme per day in the canadian guideline from , with a recommendation to taper the dose for those on higher doses [ ] , mme in both the south african guideline from [ ] , and the us cdc guideline from with a recommendation not to exceed mme per day without careful justification [ ] . the recommended upper limit is mme per day in the australian guideline from [ ] , mme per day in the german guideline from [ ] , and mme per day in the british guideline from [ ] . persons with scd are prescribed opioids for longer durations and at higher mme doses than persons without scd who are also prescribed opioids [ ] . persons with scd are also at increased risk for overdose death as compared with their matched controls due to conditions related to their scd. these conditions include respiratory insufficiency related to the scd, sleep disordered breathing, and polypharmacy from other respiratory depressant medications [ ] . given the risks associated with the prescription of opioids to persons with scd and the lack of convincing evidence for their effectiveness, a survey of physicians working with these patients was planned to look at their prescription of opioids. the aim of this survey was to characterize how physicians throughout the world working with patients who have scd approach the prescribing of opioids for pain, and to compare the results to representative clinical practice guideline recommendations. the survey was developed by thomas bryce with input from marcalee alexander and peter new. the survey was posted online and circulated among international societies within the field of sci medicine including the american spinal injury association, the international spinal cord society, and the australian and new zealand spinal cord society. the survey was available online on redcap from august to november . a total of clinicians responded to the survey, of whom were physicians. not every physician answered every survey question. the data for the five non-physicians were excluded. the majority of physicians worked more than h per week in clinical practice, with only % working h or less. of the physicians, % were involved with pain management of persons with scd as part of clinical practice duties. the physicians who managed pain were mostly from europe ( %), % were from asia, % from oceania, % from north america, and % were from africa. the vast majority of physicians who completed the survey, who managed pain, indicated a primary specialty of physical medicine and rehabilitation ( %), with the remainder indicating a primary specialty of orthopedic surgery ( %), urology ( %), internal medicine ( %), neurosurgery ( %), or other ( %). when respondents were asked about what non-opioid medications they felt would be appropriate to prescribe for the treatment of scd-related neuropathic pain, most felt that medications of the gabapentinoid class ( %) and tricyclic antidepressants ( %) were appropriate. fewer but still more than half of respondents felt that selective serotonin and norepinephrine inhibitors ( %) were appropriate, with other drug classes thought to be appropriate by a third or less of respondents ( fig. ) . when respondents were asked about the treatment of any type of acute pain, defined as pain present for less than months, physicians ( %) felt that opioids were appropriate to be considered for prescription if non-opioid interventions did not provide adequate relief. when they were asked whether there was a maximum length of time they would prescribe an opioid for non-cancer-related acute pain, over half of respondents ( %) believed that opioids should not be prescribed for more than weeks, whereas over three quarters ( %) believed that opioids should not be prescribed for more than month. notably, % of respondents indicated that opioids should be used as long as required (without specific time limit) to control the pain (fig. ) . when asked specifically about the treatment of any type of chronic non-cancer pain with opioids, about two-thirds of physicians ( %) felt opioids were indicated at any time. of those physicians who felt that there were indications for the prescription of opioids for chronic non-cancer pain, the majority ( %) felt opioids could be indicated for the treatment of neuropathic pain due to scd. for these physicians, when given the choice of different potentially available opioids they might prescribe, the opioid chosen first line was tramadol, with nearly two-thirds of responses ( %), with other drugs selected as first line much less often: oxycodone ( %), fentanyl ( %), hydrocodone ( %), buprenorphine ( %), morphine ( %), tapentadol ( %), codeine ( %), and methadone ( %). when physicians who felt that opioids were sometimes appropriate in the treatment of chronic non-cancer neuropathic pain related to scd were asked whether there was a maximum mme dosage that should be routinely prescribed for intractable pain, nearly half ( %) felt that there should be no specific limit on dose, indicating that it depends on an individual's opioid tolerance. of those who felt that there should be a limit on how much opioid should be prescribed for use in a day, one quarter of physicians ( %) thought the maximum should be no more than mme, whereas another % thought the maximum should be no more than mme. respondents were presented with a hypothetical scenario in which they were treating a person with chronic scd with neuropathic pain (not related to cancer and with no treatable cause), which was controlled on a stable dose of mme per day of an opioid. all previous attempts at dose reduction had been unsuccessful, because the pain became uncontrolled when the dose was decreased. all available treatments recommended in a recent clinical practice guideline had been tried and there were no signs of an opioid-use disorder, diversion of drug, or problematic adverse effects such as constipation or sedation. the respondents were asked whether they would continue the same effective dose of opioid. the overwhelming majority ( %) of the physicians who responded said yes. of the respondents ( %) who noted they would do something different, one-third ( %) would taper off the opioid completely, just over one-fifth ( %) would taper to a maximum dose of mme per day and continue to prescribe it, the same proportion ( %) would taper to a maximum dose of mme per day and continue to prescribe it, % would refer the person to an addiction specialist, whereas the same proportion ( %) would do something different-specifically, try a gabapentinoid. the same nine respondents ( %) who indicated that they would do something different, when asked how much influence different factors had on the decision to not continue prescribing the same opioid dose, most ( %) reported that what had the maximal influence on their decision was the fear the patient would develop an opioid-use disorder. however, one-third ( %) reported that level and quality of scientific evidence regarding strong opioids being effective or ineffective for the treatment of scd-related neuropathic pain had the maximal influence on them. the majority felt minimal to no influence of the potential perception by regulatory bodies that one could be identified as an opioid over-prescriber ( %). also, the majority were not troubled by the regulatory burden in prescribing opioids ( %) or by the time needed to monitor for diversion or signs of an opioid-use disorder ( %). respondents were subsequently presented with a second scenario in which they were treating a person with chronic scd-related neuropathic pain (not related to cancer and with no treatable cause), which was controlled on a stable dose of mme per day of an opioid and for whom all the other related conditions were the same as in the previous scenario. they were asked whether they would continue the same effective dose of opioid. the overwhelming majority ( %) of the eligible respondents said yes. of the two respondents ( %) who noted they would not continue the same dose, both responded that they would taper off the opioid completely. finally, when asked if cannabinoids were legally available for prescription, would the physician considering prescribing these for uncontrolled scd-related neuropathic pain not related to cancer, the majority responded affirmatively ( % of responses). the practice patterns related to the prescription of opioids for chronic pain in persons with scd in this international sample of clinicians differs in several key areas from those recommended in clinical practice guidelines published in the united states [ ] , canada [ ] , australia [ ] , great britain [ ] , south africa [ ] , and germany [ ] . one of these areas relates to the maximum dose of opioids that should be prescribed per day. all the guidelines state, in essence, that clinicians should use caution when prescribing opioids at any dosage and should carefully reassess evidence of individual benefits and risks when increasing dosage above a certain daily threshold. the us guideline identifies this threshold to be mme per day citing evidence that holding doses below this level would "likely reduce risk among a large proportion of patients who would experience fatal overdose at higher prescribed dosages," while further noting "in general, that increasing dosages above or more mme day increases overdose risk without necessarily adding benefits for pain control or function" [ ] . it further recommends that when prescribing dosages above the threshold of mme per day, additional precautions should be implemented, such as increasing the frequency of follow-up, and that prescribers should avoid increasing dosages to more than mme per day citing incremental benefits for pain and function relative to harms as dosages approach the mme per day limit [ ] . the dose limit recommendation in the canadian and south african guidelines similarly is mme per day and slightly higher, at mme per day, in the australian guideline and mme per day in both the german and british guidelines. in contrast to all these guidelines, nearly half ( %) the clinicians who prescribe opioids, who completed the survey, felt that there should be no specific limit on dose, indicating that it depends on opioid tolerance how high a dose one may prescribe. this difference between clinical practice and guideline recommendations may indicate there is a lack of awareness on the part of physicians of the reasons made in the guidelines to justify limiting the maximal recommended daily dose, namely a lack of evidence for improved effectiveness with higher doses as well as emerging evidence for the much higher risk of developing an opioid overdose with higher doses. it is very important to note that many persons with scd are also at a greatly increased risk of overdose, for a range of reasons. those who have a cervical or thoracic level of damage and a complete grade can have a reduction of their pulmonary reserve, and there is a high prevalence of polypharmacy, often with agents that are neurologically sedative. there is clearly a need to educate prescribers about the risks associated with higher doses of opioids, especially in the population with scd. most clinicians ( %) who completed the survey were willing to continue opioids indefinitely if the pain was controlled at very high doses ( mme per day) as long as there were no signs of an opioid-use disorder, diversion of drug, or problematic adverse effects such as constipation or sedation. this practice is not consistent, e.g., with the canadian guideline, which recommended that patients who are currently using mme per day or more be tapered to the lowest effective dose, potentially including discontinuation, rather than making no change in opioid therapy [ ] . another area that perhaps was not addressed adequately in the survey, but which was emphasized in the us guideline, is the necessity that: "before starting opioid therapy for chronic pain, clinicians should establish treatment goals with all patients, including realistic goals for pain and function, and should consider how therapy will be discontinued if benefits do not outweigh risks. clinicians should continue opioid therapy only if there is clinically meaningful improvement in pain and function that outweighs risks to patient safety." one fact that needs to be acknowledged when treating pain after scd is that many persons with scd have limited function due to the scd itself, and to require that there be changes in function as a condition of opioid use may be ineffectual. instead, other types of pain interference, such as with sleep or mood, may need to be substituted. this additional outcome of effectiveness, namely a reduction in pain interference, should be included in the decision to initiate or continue opioid prescription, rather than depending solely on reports of decreases in pain intensity. these interference items are recommended as part of the international sci basic pain data set [ ] . a third area of difference in the practice patterns of the clinicians completing the survey with the recommended us guideline relates to how long opioids should be prescribed for acute pain. the us guideline recommend that when opioids are used for acute pain, clinicians should prescribe the lowest effective dose of immediate-release opioids and should prescribe no greater quantity than needed for the expected duration of pain severe enough to require opioids. "three days or less will often be sufficient; more than days will rarely be needed" [ ] . the reason the united states recommended short durations of use is because longterm opioid use usually begins with the treatment of acute pain. in order to start to address these differences between the clinical practice of physicians who treat pain after scd and widely available clinical practice guidelines, the authors propose an algorithm that can be used as a general guide for the prescription of opioids after scd (fig. ) . the algorithm, which was conceived before this survey was conducted, is based on the key principles outlined in the us guideline [ ] , identified primarily to reduce the risk of unintentional death due to prescription opioid overdose. decision points within the algorithm incorporate proximate consensus guideline-recommended opioid prescription threshold doses, which are loosely correlated to the risk of opioid overdose and the development of an opioid-use disorder. in order to incorporate the increased risks of the use of opioids in special populations, such as those with scd and pain, who may be at even higher risk of unintentional death due to prescription opioid overdose because of underlying respiratory compromise, sleep disordered breathing, and the concomitant use of other prescribed and non-prescribed sedating medications (such as are used for spasticity management), these threshold doses were weighted to the more conservative us guideline threshold doses as opposed to other older guidelines with which higher threshold doses are incorporated. the specific mme threshold dose of mme (rather than mme for instance) was chosen for a purely practical reason, as commercially available doses of opioids from around the world often add up to this number. use of this number fig. algorithm for the treatment of pain after sci with opioids [ ] . *low dose = mme per day or less. **moderate dose = > mme but ≤ mme per day. ***high dose = > mme per day; mme = morphine milligram equivalent therefore simplifies the decision-making regarding mme dosing. finally, the algorithm takes into consideration the consensus guideline belief that pain intensity should not be the only pain outcome evaluated in the decision to continue opioid prescription. specifically, as related to persons with scd, the algorithm was designed to be applicable to those who have significant disability as well as pain, acknowledging that these individuals who may have limited function due to their disability may not be able to improve their function if their pain improves due to the continuance of their underlying disability, and that other measures of pain interference rather than function should be considered in these situations. this algorithm, which has yet to be validated in any population and may require further revision, may help guide practitioners in managing acute and chronic pain in patients with scd, while reducing the risk of adverse consequences of opioid use. limitations of this study include the relatively small sample size and the over/under-representation of different regions of the world. it is also acknowledged that each of the different guidelines to which the results of this survey were compared were developed using different methods and were based upon different study questions. however, importantly, the guidelines all included similar common questions regarding the maximal does of opioids. this study is the first to survey clinicians regarding the prescription of opioids to people with pain following scd. although this survey represents an international crosssection of treating physicians and there clearly is an international opiate focus at the present time, the fact remains that for the physicians and their patients with scd, pain remains a significant problem. this article delves into the viewpoints of treating physicians from around the world that are working with these patients and reveals that many are willing to continue to prescribe what has been working for them despite the guidelines, or in ignorance of them. further research with regard to the physicians' willingness to follow the algorithm is indicated, as it is would be helpful to gain more insight into the perspective of the treating physicians and not just their adherence to various clinical practice guidelines. the practice patterns related to the prescription of opioids for chronic pain for persons with scd for this international sample of clinicians differ in several key areas from those recommended in widely disseminated clinical practice guidelines. use of an algorithm adapted for use by persons with scd has the potential for aligning practice patterns with recommended clinical practice guidelines, although this needs further review and study. survey data are available from the corresponding author upon reasonable request. conflict of interest the authors declare that they have no conflict of interest. publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. gender and minority differences in the pain experience of people with spinal cord injury a prospective study of pain and psychological functioning following traumatic spinal cord injury a longitudinal study of the prevalence and characteristics of pain in the first years following spinal cord injury pharmacotherapy for neuropathic pain in adults: a systematic review and meta-analysis the canpain sci clinical practice guidelines for rehabilitation management of neuropathic pain after spinal cord: recommendations for treatment bryce tn unpublished data from sci model systems increases in drug and opioid overdose deaths-united states department of health and human services the opioid epidemic and national guidelines for opioid therapy for chronic noncancer pain: a perspective from different continents opioid consumption data cdc guideline for prescribing opioids for chronic pain−united states guideline for opioid therapy and chronic noncancer pain long-term opioid use in non-cancer pain opioids aware: a resource for patients and healthcare professionals to support prescribing of opioid medicines for pain the royal australian college of general practitioners. prescribing drugs of dependence in general practice south african guideline for the use of chronic opioid therapy for chronic non-cancer pain dose and duration of opioid use in propensity score-matched, privately insured opioid users with and without spinal cord injury opioids should not be prescribed for chronic pain after spinal cord injury the international spinal cord injury pain basic data set (version . ) pain management in persons with spinal cord injury key: cord- -fm rm authors: leja, mārcis; dumpis, uga title: what would the screen-and-treat strategy for helicobacter pylori mean in terms of antibiotic consumption? date: - - journal: dig dis sci doi: . /s - - -z sha: doc_id: cord_uid: fm rm several guidelines recommend the screen-and-treat strategy, i.e. active search for the presence of helicobacter pylori infection and its eradication to prevent the possibility of gastric cancer. it is thought that a relatively short duration antibiotic regimen given once in a lifetime would not significantly increase overall antibiotic consumption. however, this would mean offering antibiotic treatment to the majority of the population in countries with the biggest burden of gastric cancer who would, therefore, have the greatest benefit from such a strategy. so far, no country has implemented an eradication strategy. with an example based on the current situation in latvia, we have estimated the increase in antibiotic consumption if the screen-and-treat strategy was applied. depending on the scenario that might be chosen, clarithromycin consumption would increase up to sixfold, and amoxicillin consumption would double if the recommendations of the current guideline in the local circumstances was applied. it appears that an increase in commonly used antibiotic consumption cannot be justified from the viewpoint of antibiotic stewardship policies. solutions to this problem could be the use of antibiotics that are not required for treating life-threatening diseases or more narrow selection of the target group, e.g. young people before family planning to avoid transmission to offspring. additional costs related to the increase in resistome should be considered for future cost-effectiveness modelling of the screen-and-treat strategy. although declining in incidence, gastric cancer will remain an important healthcare issue for the foreseeable future due to aging and the increase in global population. the group of experts gathered by the international agency for research on cancer (iarc) has suggested implementation of gastric cancer prevention by helicobacter pylori (h. pylori) eradication in well-controlled research settings (ml was part of the working group) [ ] . the annual total number of new cases accounts for ~ one million, responsible for > % of global cancer-related deaths each year [ ] . this number is estimated to remain stable for at least the next years if no prevention measures are implemented [ ] . infection with h. pylori is the key risk factor for this type of cancer, responsible for ~ % of the distal (non-cardia) cancer cases [ , ] . several prevention strategies, including primary and secondary prevention are suggested to decrease the mortality caused by gastric cancer. only about - % of individuals infected with h. pylori are likely to develop gastric cancer in their lifetime [ , ] . ideally, only the bacteria potentially leading to cancer or other diseases should be eradicated; however, risk stratification attempts based on h. pylori virulence factor identification or host susceptibility, e.g. by detecting the relevant polymorphisms of proinflammatory cytokines, have not led to a strategy that could be recommended for routine practice [ ] . vaccine development still does not provide encouraging results suggesting that it is close to a routine practice [ ] . eradication of h. pylori in adults hosting the infection appears to be the most effective prevention strategy. the only reliable approach to eliminate the infection is simultaneous use of at least two different antibiotics in combination with potent acid suppression using a proton pump inhibitor or a potassium-competitive acid blocker (pcab) [ ] . a screen-and-treat strategy would mean active testing for presence of h. pylori in the (mostly healthy) general population, and offering eradication to those testing positive [ ] . it is expected that h. pylori eradication would reduce the risk of gastric cancer in the population for ~ % [ ] . such a strategy should comply with the principles of good governance and organization, and the benefits should be well-balanced against the risks that any potential intervention could create [ ] . several risks have been suggested for population-based h. pylori eradication; however, the possible adverse effects related to increased antibiotic consumption probably are the most significant. resistant h. pylori strains are emerging due to high antibiotic consumption [ ] . however, the most significant risks will probably be related to induction of resistance in other clinically relevant bacteria than h. pylori and increase the pool of resistant genes in the gut and upper respiratory system [ ] [ ] [ ] . the currently recommended choice of antibiotics depends on the local resistance pattern of h. pylori, unless susceptibility-based individual therapy is prescribed [ , ] . clarithromycin and amoxicillin are the most widely prescribed antibiotics, frequently used in both low and high clarithromycin-resistant regions. according to the guidelines, the clarithromycin-containing regimen is the choice where h. pylori is clearly sensitive to clarithromycin (for individualized treatment) or if h. pylori resistance to clarithromycin does not exceed % in the reference population [ ] . although the resistance of h. pylori to clarithromycin correlates to the overall use of this antibiotic in a particular population, the choice of treatment solely depends on h. pylori resistance. furthermore, no differences in the eradication regimen are currently recommended, depending on whether the treatment is given for a clinically evident disease, e.g. for complicated ulcer disease or malt lymphoma from prevention strategies in population-based settings. the author viewpoint is that negative effects upon the gut microbiome would not be a significant concern in the case of treating patients with a clinically significant disease, yet should be considered in preventive interventions. the european maastricht v/florence consensus (ml was part of the working group) recommended the screenand-treat strategy in communities at high risk of gastric cancer and consideration of this approach in communities with intermediate to low risk of gastric cancer [ ] . kyoto global consensus has for the first time categorized h. pylori gastritis as an infectious disease, irrespective of symptoms and complications [ ] , which has been reinforced by the maastricht v [ ] and brazilian consensus [ ] . all h. pylori infected subjects are recommended to undergo eradication therapy according to the kyoto consensus unless there are competing considerations, such as comorbidities, re-infection rates in their communities, competing health priorities of society or financial issues [ ] . the recent guidelines of asean (association southeast asian nations) countries support eradication to prevent gastric cancer by considering this strategy as costeffective, depending on the disease burden in the relevant community [ ] . a similar opinion was formed in the second asia-pacific consensus guidelines for h. pylori infection a decade ago for communities with high incidence of gastric cancer [ ] . at the same time, some of the guidelines and expert group recommendations are less enthusiastic regarding population-based eradication. the american college of gastroenterology does not recommend active search of h. pylori in an asymptomatic population [ ] . an expert group hosted by iarc has suggested the need for interventional strategies to decrease the burden of gastric cancer [ , ] ; however, experts recommended that this be done by the means of well-designed clinical studies evaluating the feasibility, acceptance, costs, effectiveness and adverse consequences. a european expert group (ml was part of the working group) within the eu joint action in cancer control (cancon) has been even more critical-they concluded that, as of today, there is no screening method that can be readily recommended for implementation in the eu member states, although there seems to be a need for one [ ] . this also included the screen-and-treat strategy for h. pylori. there is common agreement [ , , ] that the optimal timing for eradicating h. pylori must come before the development of precancerous lesions, since a proportion of the subjects could be progressing to gastric cancer if eradicated at the stage that precancerous lesions (atrophy, intestinal metaplasia, dysplasia) become evident. in japan, currently eradication therapy is reimbursed for all the individuals with h. pylori infection and active gastritis (in addition to the ongoing screening activities for gastric cancer), which means that endoscopy is required to confirm the condition. thereafter, significant increase in eradication therapies has occurred [ ] , yet this cannot be considered an organized screening strategy. in korea, a high gastric cancer incidence country with a gastric cancer screening program [ ] , h. pylori management guidelines do not address the search for the presence of the bacteria in general population [ ] . the country probably closest to the real implementation of screen-and-treat strategy in an organized way is slovenia, where the professional society has issued guidelines for such a strategy [ ] ; it must be mentioned that this country has considerable experience in implementing other types of cancer screening in an organized manner, including screening for colorectal cancer. fourteen-day duration of eradication therapies, including for bismuth-containing therapies, is currently recommended by maastricht v, unless shorter duration therapies ( days) are proven effective locally [ ] . similarly, the toronto consensus recommends a -day treatment [ ] . it is noteworthy that the recommended duration of treatment has extended with the time to achieve higher effectiveness of these therapies. in , the maastricht iii consensus considered days as a valid duration for treatment [ ] . maastricht iv in extended treatment to - days giving a gain of ~ % in the success rates [ ] . the general who principles for screening have been set more than half a century ago by wilson and jungner [ ] , and they are still used as of today although updated in the new genomic era [ ] . furthermore, principles of good screening organization and governance have been consistently emphasized by the expert groups [ ] . the above principles include implementation of the program only when sufficient scientific evidence (with proven effects on the mortality as the end-point) is available and following thorough cost effectiveness analysis, precise definition of the target population as well as invitation strategy, piloting of a screening system, setting up a robust quality assurance system before the system is launched in full operation. h. pylori infection is highly prevalent, affecting about half of the global population [ ] ; the prevalence of precancerous lesions in the general population is also considerable [ ] . therefore, population-based efforts such as the screen-and-treat strategy should follow the general rules of screening. it must be mentioned that the current h. pylori management guidelines so far are lacking this approach. adverse events related to h. pylori eradication therapy are common, but usually they are mild and of short-term duration; the most common symptoms are diarrhea, nausea and/or vomiting, epigastric pain, and altered taste [ ] . a large systemic review performed in demonstrated adverse effects in % of the subjects receiving eradication therapy for peptic ulcer compared to % in patients on a proton pump inhibitor (ppi) or no treatment (rr . ; % ci: . to . ) [ ] . treatment with oral and parenteral antibiotics results in a rapid and significant alteration of the intestinal microbiota. the most obvious outcome of disruption of normal gut microbiome is clostridium difficile infection (cdi). cdi is a major threat to both outpatients and those hospitalized. although any antimicrobium can predispose a patient to cdi, the risk is especially great when using broad-spectrum antimicrobials, which disrupt normal enteric flora [ ] . prolonged treatment with antimicrobial agents is also associated with an increased risk of cdi by extending the time disruption of normal enteric flora [ ] . although not being a typical adverse event, a number of cases have been published on cdi after eradication therapy of h. pylori [ ] . awareness of the complication is particularly important when both duration and indications for h. pylori eradication therapy have been extended. significant perturbation of the gut microbiome might follow the use of antibiotics; however, in the majority of cases the gut microflora would be expected to return to its initial state within a few months [ , ] . resistome is defined as a collection of all antibiotic resistance genes and their precursors in both pathogenetic and non-pathogenic bacteria [ ] . the gut microbiota is a large reservoir of antibiotic-resistance genes [ ] ; an average number of such genes per sample has been reported [ ] . many earlier and more recent studies have suggested long-lasting persistence of resistant pharyngeal and/or gut bacteria following the use of traditional antibiotics used to eradicate h. pylori. one-week treatment of healthy volunteers with macrolides (azithromycin or clarithromycin) has been associated to a significantly increased proportion of macrolide-resistant streptococci in the pharynx compared to a placebo-treated group; resistant streptococci were present for up to days following treatment [ ] . similar data on macrolide-resistant streptococci persistence in the pharynx for more than year in patients receiving clarithromycin-containing h. pylori eradication regimens have been reported by others [ ] . another study has addressed the presence of resistant staphylococcus, streptococcus, enterococcus and bacteroides spp. in samples from nostrils, throat and feces before, weeks and year following triple h. pylori eradication therapy (clarithromycin, metronidazole and omeprazole for days) in a group with peptic ulcer disease, as well as a control group not receiving this treatment [ ] . resistant isolates, in particular of staphylococci and streptococci were higher after year, but not in the control group. another study based on the same patient data demonstrated that this treatment facilitated the selection of highly resistant enterococci present, even years after treatment [ ] . the same group has also addressed erm(b) gene levels (one of the mechanisms for macrolide resistance) both in throat and fecal samples. these levels increased dramatically by - orders of magnitude immediately after antibiotic treatment. in a proportion of the subjects, erm(b) remained elevated years after treatment [ ] . however, the small sample of subjects in this study should be noted. more recently, larger studies applying s rrna gene and metagenomic sequencing have been reported by yap et al. [ ] who investigated stool samples in -year-old volunteers from malaysia before h. pylori eradication, and at , , months thereafter. despite microbial diversity was similar pre-and post-h. pylori eradication with no significant differences in richness and evenness of bacterial species, changes in the bacterial communities at the phylum and genus levels were noted, e.g., the relative abundance of bacterioidetes decreased and firmicutes increased. an important study primarily addressing metabolic effects of h. pylori eradication in general population in taiwan was recently published [ ] . significant perturbation of gut microbiota in short-term was revealed, and it was significantly greater for concomitant and bismuth quadruple therapies than for a standard triple. bismuth quadruple therapy, on the other hand, was not associated with an increase in resistance in e. coli. the authors also demonstrated that for other therapies the resistance rates of e. coli and k. pneumoniae to certain antibiotics were restored at week and year after therapy [ ] . however, this study focussed only on gut microbiome and effect on resistance rates in gram-negative bacteria. widespread use of amoxicillin and clarithromycin causes concern about resistance rates in gram-positive bacteria such as str. pneumoniae and s. aureus [ , ] . current evolution of sequencing methods will provide significant evidence related to the potential perturbation of microbiota following antibiotic treatment, including h. pylori eradication. several studies comparing the effect of different h. pylori eradication regimens on the gut microbiome or resistome are in progress in different countries, including taiwan, spain and latvia. in most of them, the diversity of microbiota is addressed by means of s rrna gene sequencing, whereas others apply metagenome sequencing. the use of antibiotics is the primary driver for the development of resistance and also leads to other adverse effects ranging from allergic reactions to cdi [ ] . the term "antimicrobial stewardship" is encountered in a growing number and increasingly diverse range of contexts, from antimicrobial stewardship programmes in hospitals and the community [ ] , to veterinary antimicrobial stewardship [ ] , one health antimicrobial stewardship [ ] and the who global stewardship framework [ ] . because of the rapidly increasing use of the term without a sole clear definition, it has evolved differently in different settings, influenced by local interpretations [ ] . in general, antimicrobial stewardship programs have a direct responsibility to ensure prudent antibiotic prescribing. reducing antibiotic exposure should minimize the duration and extent of disruption of the microbiome, thereby reducing collateral damage and improving patient outcomes. prolonged courses of antibiotics also increase the risk of colonization with multidrug resistant organisms. therefore, the chain of transmission increases the risk of horizontally infecting more than one patient. interrupting this chain is as important as preventing the development of resistance. data from developed nations suggest that % or more of antibiotic prescriptions are for outpatients [ ] . therefore, limiting their use of antibiotics is essential in reducing both resistance and adverse events. over the last years, several developed countries introduced nationwide initiatives aimed at reduced antibiotic consumption, achieved a drop of > %. in sweden between and , the number of prescriptions per inhabitants per year in outpatient care, including primary health care, decreased by % from to , whereas among children aged - years it decreased by % from to [ ] . between and , a statistically significant decreasing trend in antibiotic use was also seen in finland, luxembourg and norway [ ] . in march , the white house released the national action plan for combating antibiotic-resistant bacteria, which set a target of reducing inappropriate antibiotic use in the outpatient setting by % by [ ] . in light of these trends, any suggestion for mass treatment of infections that would lead to an increase in antibiotic consumption will be carefully scrutinized by national authorities and experts. amoxicillin and clarithromycin, drugs of choice for treatment of h. pylori infections suggested by current guidelines, have wide application for the treatment of several community-acquired infections and account for large proportion of ambulatory antibiotic prescriptions in many countries. they are part of the suggested first or second line treatment regimens for community acquired pneumonia [ ] [ ] [ ] [ ] . amoxicillin or amoxicillin/clavulanate is often suggested as first-line treatment for otitis media, bacterial rhinosinusitis, dental infection and urinary tract infections. macrolides are often recommended as replacement treatment for patients with penicillin allergy. use of azithromycin has been suggested for mass treatment for chlamydia trachomatis eye infections in developing countries. this has already provoked significant concern on its potential adverse events, even though the prevalence of chlamydia is significantly lower than h. pylori. monitoring of the resistance in multiple organisms has been suggested on this indication [ ] . we conducted an exercise by estimating the expected increase in clarithromycin and amoxicillin consumption in latvia if an h. pylori eradication program would be implemented. latvia is a small country (population ~ two million) in northern europe with a relatively low consumption of antibiotics and relatively high incidence of gastric cancerthe incidence per , inhabitants in both genders is . (asr, world population) [ ] . we used data on the clarithromycin (j fa ), amoxicillin (j cr ), and amoxicillin beta-lactam combination (j cr ) consumption in the country provided by the state agency of medicines and expressed in defined daily doses (ddd) per inhabitants in latvia within the period - . mathematical projection for the next years was made, and population distribution per relevant age groups from the official statistics was used. according to the guidelines [ ] , a -day eradication regimen with g per day amoxicillin and g per day clarithromycin was used in the estimates, considering that latvia still belongs to low h. pylori resistance areas to clarithromycin, and therefore, clarithromycin-based triple therapy could be considered the first choice. the prevalence of the infection was considered %, based on our previous studies [ ] . three different scenarios for a screen-and-treat strategy were evaluated: ( ) eradication is limited every year just to persons reaching adulthood- years of age; for the estimates we considered a % adherence rate, since it was expected that the campaign would result in additional therapies outside the target group; ( ) within a -year period, screen-and-treat would be offered to the risk-group defined as - -year-old individuals; one third of the target group would be covered per year with the % compliance as an assumption; ( ) within a -year period, screen-and-treat would be offered to all adult individuals assuming a similar % compliance rate. for scenarios and , additional eradication of the group reaching adulthood was considered starting from year . the results are given in fig. . scenario would lead to a moderate increase in clarithromycin and amoxicillin consumption, scenario to ~ threefold increase, but scenario to more than a sixfold increase of clarithromycin, with a slightly lower (twofold) increase in amoxicillin consumption. such an increase in antibiotic consumption would move latvia from a group of countries with low antibiotic consumption to a group with average antibiotic consumption (see fig. ; data on antibiotic consumption vaccine (either preventive or therapeutic) would be potentially the best solution of the problem [ , ] ; however, current developments have not been promising. non-antibiotic h. pylori eradication regimens have gained significant clinical interest. the use of natural products, including various plant (even mushroom) and fruit extracts, natural oils, chinese herbs, garlic, ginger, green tea, curcumin, cranberries and pistacia gum have been studied, predominantly under laboratory conditions [ ] [ ] [ ] [ ] . some of these compounds had activity against h. pylori, but the results of clinical evaluations in monotherapies have been less promising; also, the quality of these trials has been criticized [ ] . probiotics as a single agent have been also used without major success [ ] . laboratory and animal experiments show the effectiveness of several non-antibiotic treatment modalities against h. pylori. antimicrobial polypeptides (ph-sensitive, helix-coil conformation transitionable agent with a bactericidal activity) have been developed to target and selectively eradicate h. pylori as a single therapeutic agent, without significant effects on commensal bacteria [ ] . another group developed docosahexaenoic acid-loaded lipid nanoparticles with bactericidal activity against h. pylori [ ] . this bactericidal in latvia compared to other countries in the european union/european economic area, with the hypothetical scenarios of an h. pylori screenand-treat strategy expressed as ddd per inhabitants per day. note: the bars in blue indicate the current consumption of antibacterials (including latvia). the bars in red are consumption of antibacterials in latvia with various scenarios during the first year of a screenand-treat strategy implementation. scenario : eradication limited to anybody reaching years of age. scenario : the age group - years invited for screen-and-treat within a -year period; % compliance. scenario : all adults invited for screen-and-treat within a -year period; % compliance agent could also eradicate h. pylori without affecting the other bacteria (lactobacillus, e. coli, s. epidermidis and s. aureus) [ ] . since the abovementioned approaches are still far from clinical practice, regimens that are expected to interfere less with the microbiota and less likely to be responsible for gut (and other location) resistome development are more attractive for clinical applications in the near future. single antibiotic regimens, in particular those not containing macrolides, could attract interest in this respect. use of high-dose amoxicillin is one case, although more frequent dosing is required due to the pharmacokinetics of this drug. high-dose amoxicillin-based dual therapy for first-line h. pylori eradication has so far been evaluated predominantly in asia [ , ] , although a pilot study has been reported in europe [ ] . more profound reduction of the gastric acidity also could contribute to a higher effectiveness of the therapy. sugimoto and yamaoka [ ] have recently reviewed the effectiveness of pcab in h. pylori eradication therapies compared to the traditional ppi containing therapies, and demonstrated significant advantages of the pcab. therefore, one directions for future therapies could be pcab and high-dose (multiple dose) amoxicillin in combination. another approach would be the use of antimicrobial agents that are not typically used in managing life-threatening disease, as well as those causing less induction of the pool resistant genes. bismuth-based therapies are mainly used to overcome the resistance of h. pylori to commonly used antibiotics in clinical settings [ , , ] . an additional gain with these therapies is avoiding the use of clarithromycin and amoxicillin. however, bismuth is not available in many countries. the concern with bismuth-related adverse events is predominantly related to the fact that in the s, use of high-dose bismuth salts for long periods was associated with neurotoxicity; however, systemic review and meta-analysis on bismuth use for h. pylori eradication did not reveal serious adverse events for such therapy [ ] . a recent meta-analysis by ko et al. [ ] has suggested the superiority of bismuth-containing therapies over nonbismuth regimens; furthermore, adding bismuth to conventional standard eradication regimens provides additional gain in the efficacy of the therapies. another meta-analysis on a single capsule -day bismuth-containing quadruple therapy has suggested ~ % eradication success both in first-and second-line therapy [ ] . short-term dysbiosis restoration to baseline levels within an -week period following -day bismuth quadruple therapy has been reported from taiwan; s rrna gene sequencing of the v -v region and sampling before the treatment, as well as , , and weeks thereafter, was used [ ] . therefore, bismuth-containing quadruple therapy is also related to dysbiosis; other antibacterial agents contained in this treatment, in particular metronidazole, could be responsible for perturbations in the microbiota. there are still insufficient data on the potency of bismuth-based therapies to induce and cause persistent resistome. an alternative to the empiric eradication regimen is a h. pylori susceptibility-based individual therapy. this allows avoiding unnecessary use of antibiotics and increasing the effectiveness of the treatment [ ] . however, this is considering only h. pylori resistance patterns, not the effects upon the other gut microbiome. usually, even after h. pylori resistance testing, several options for eradication therapies are remaining, and lower negative effects upon the gut microbiome should ideally considered to be the choice. finally, narrowing the target group for h. pylori eradication, e.g. by targeting young adults before family planning and, therefore, before the potential transmission of the infection to offspring, could be another approach in decreasing the misuse of antibiotics. in spite of the failures mentioned above, individual risk stratification based on gender, lifestyle factors, host and h. pylori genetics continue to be of interest in risk stratification. several meta-analyses have suggested convincing cost-effectiveness of population-wide h. pylori eradication [ ] [ ] [ ] . the benefit is likely to be highest in communities with a high risk of gastric cancer; in developed countries, such an approach could be either cost-effective or cost-neutral if considered on the positive side in reducing the cost of dyspepsia treatment [ ] . few other cost-effectiveness studies have since been published. in denmark, a low h. pylori prevalence country, a -year follow-up of a randomized study population ( , individuals aged - years at enrollment) failed to achieve either quality-of-life or cost-effectiveness [ ] , whereas a modelling exercise in china proved cost-effective in a population-based screen-and-treat strategy related to gastric cancer, peptic ulcer disease and dyspepsia reduction. furthermore, the highest effectiveness was in the age group of years [ ] . screen-and-treat has also been estimated as cost-effective for employees in japan [ ] . however, these studies have not considered the potential costs associated to the increase in the pool of resistant bacteria. the real costs behind the resistome are difficult to estimate; at least two major issues must be considered: ) cost of the resistance and ) cost-effectiveness of interventions to reduce it [ ] . there is also a huge range in the estimates of additional cost, varying from < $ to > $ , per patient episode [ ] . however, generally the current costings could be an underestimated, and therefore, interventions in using antibiotics on a wide-scale could reflect as highly cost-saving benefit. the hidden costs of antibiotic resistance in the united states have recently been estimated by michaelidis et al. [ ] who considered: ( ) hospitalization costs; ( ) secondline inpatient antibiotic costs; ( ) second-line out-patient antibiotic costs, and ( ) antibiotic stewardship costs. the authors estimated that the total hidden cost attributable to each ambulatory antibiotic prescription was $ (range: $ - ), and each ambulatory antibiotic prescription would increase antibiotic costs by % (range: - %), if the cost of the resistance was incorporated into antibiotic costs paid by patients or payers [ ] . applying this estimate to the cost-effectiveness estimates of h. pylori eradication and modelling the costs of resistome for other countries globally probably would change the very beneficial cost-effectiveness picture of screen-and-treat strategy. the cost-effectiveness estimates should also incorporate those of organizing the activities and governance of the process, exactly as in traditional cancer screening program settings [ ] . finally, compliance rates of the target population, equal participation of both genders and coverage of the lower socioeconomic class members is critical for the success for any preventive strategy, and should be considered in cost-effectiveness modelling. because of the necessity of antibiotic use and potential adverse events, participation rates could be lower for an h. pylori screen-and-treat strategy than for traditional screening approaches. this has been suggested by the participation results in the danish community h. pylori screening trial [ ] and is currently being addressed in the gistar cohort in latvia [ ] . the screen-and-treat strategy clearly increases the consumption of antibiotics on a population level. in avoiding the problems of treating life-threatening diseases due to increased resistome, antibiotics with high potential for resistome induction, and use for treating life-threatening diseases (such as macrolides) should be avoided in populationbased h. pylori eradication regimens for otherwise healthy people. narrowing of the target groups for the screen-andtreat strategy is desirable, e.g. for young people before family planning and potential transmission of h. pylori to their offspring. finally, in implementing any screen-and-treat strategy, this should be done under thorough surveillance corresponding to the general principles of screening governance, including surveillance of the incidence of serious infections and all-cause mortality. fr/book-and-repor t-serie s/iarc-worki ng-group -repor ts/-em-helic obact er-pylor i-em-eradi catio n-as-a-strat egy-for-preve nting -gastr ic-cance r- global cancer statistics : globocan estimates of incidence and mortality worldwide for cancers in countries fr/book-and-repor t-serie s/iarc-worki ng-group -repor ts/-em-helic obact er-pylor i-em-eradi catio n-as-a-strat egy-for-preve nting -gastr ic-cance r- global burden of cancers attributable to infections in : a synthetic analysis global burden of gastric cancer attributable to helicobacter pylori review article: 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antibiotic resistance european centre for disease prevention and control. antimicrobial consumption national action plan for combating antibiotic-resistant bacteria guideline development g. diagnosis and management of community and hospital acquired pneumonia in adults: summary of nice guidance management of community-acquired pneumonia in adults: guideline update from the dutch working party on antibiotic policy (swab) and dutch association of chest physicians swedish guidelines on the management of community-acquired pneumonia in immunocompetent adults-swedish society of infectious diseases infectious diseases society of america/american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults antimicrobial resistance following mass azithromycin distribution for trachoma: a systematic review global cancer observatory: cancer today. lyon: international agency for research on cancer prevalence of helicobacter pylori infection and atrophic gastritis in latvia a systematic review of treating helicobacter pylori infection with traditional chinese medicine antimicrobial activity of natural products against helicobacter pylori: a review review: pharmacological ins and outs of medicinal plants against helicobacter pylori: a review non-pharmacological treatment of helicobacter pylori probiotic monotherapy and helicobacter pylori eradication: a systematic review with pooled-data analysis selective killing of helicobacter pylori with ph-responsive helix-coil conformation transitionable antimicrobial polypeptides docosahexaenoic acid loaded lipid nanoparticles with bactericidal activity against helicobacter pylori lipid nanoparticles to counteract gastric infection without affecting gut microbiota high dose ilaprazole/ amoxicillin as first-line regimen for helicobacter pylori infection in korea high and frequent dose of dexlansoprazole and amoxicillin dual therapy for helicobacter pylori infections: a single arm prospective study high-dose esomeprazole and amoxicillin dual therapy for first-line helicobacter pylori eradication: a proof of concept study role of bismuth in improving helicobacter pylori eradication with triple therapy understanding treatment guidelines with bismuth and non-bismuth quadruple helicobacter pylori eradication therapies adverse events with bismuth salts for helicobacter pylori eradication: systematic review and meta-analysis bismuth supplements as the first-line regimen for helicobacter pylori eradication therapy: systemic review and meta-analysis meta-analysis of threein-one single capsule bismuth-containing quadruple therapy for the eradication of helicobacter pylori helicobacter pylori eradication with bismuth quadruple therapy leads to dysbiosis of gut microbiota with an increased relative abundance of proteobacteria and decreased relative abundances of bacteroidetes and actinobacteria treating helicobacter pylori effectively while minimizing misuse of antibiotics screening for gastric cancer and surveillance of premalignant lesions: a systematic review of cost-effectiveness studies cost-effectiveness of screening and treating helicobacter pylori for gastric cancer prevention helicobacter pylori eradication therapy to prevent gastric cancer in healthy asymptomatic infected individuals: systematic review and metaanalysis of randomised controlled trials the cost effectiveness of helicobacter pylori population screening-economic evaluation alongside a randomised controlled trial with -year follow-up cost-effectiveness analysis of screen-and-treat strategy in asymptomatic chinese for preventing helicobacter pylori-associated diseases cost-effectiveness of helicobacter pylori screening followed by eradication treatment for employees in japan the true cost of antimicrobial resistance the hidden societal cost of antibiotic resistance per antibiotic prescribed in the united states: an exploratory analysis mass eradication of helicobacter pylori to prevent gastric cancer: theoretical and practical considerations effects of community screening for helicobacter pylori: -year follow-up evaluation of a randomized controlled trial multicentric randomised study of helicobacter pylori eradication and pepsinogen testing for prevention of gastric cancer mortality: the gistar study acknowledgments the work was supported by erdf (european regional development fund) in latvia, project id. nr. . . . / /a/ 'optimisation of h. pylori eradication therapy for population-based gastric cancer prevention'. conflict of interest the authors declare that they have no conflict of interest. key: cord- - ai chbu authors: andersen, bjørg marit title: background information: isolation routines date: - - journal: prevention and control of infections in hospitals doi: . / - - - - _ sha: doc_id: cord_uid: ai chbu the isolation of patients with suspected or documented infections—to not spread to others—has been discussed for hundreds of years. guidelines are many, methods are different, attitudes show vide variations, routines and procedures are still changing, regulations by law may be absent, and some healthcare professionals may be afraid of adverse outcomes of isolation [ – ]. microbes that are spread in the environment, on the hands and equipment are invisible. the invisible agent does not call on attention before the infection; clinical disease, hospital infection or nosocomial infection is a factum that can be registered [ , , , – ]. how to stop the transmission is often “to believe and not believe” in infection control. like luster in sogn and glitre in akershus or in separate isolation buildings at the hospital, like epidemic buildings at ullevål hospital. despite academic controversy about "infection or not" from the late s to the s, the isolation treatment was used for presumably "infectious diseases" like typhoid, cholera, scarlet fever, diphtheria, smallpox, plague, etc. patients were isolated in hospital, at home or in own local epidemic houses in the country. chronic carriers of typhoid bacteria (salmonella typhi) were isolated alone at home or at the counties epidemic house by themselves (smitteborg, "infection castle" in skreia, norway, is still a local name). the epidemic houses had access control and were monitored by the healthcare board mayor. hygiene and infection control were very important since they had few curative agents. it was prepared vaccines and carried out partly or complete mass vaccinations against some infections. pulmonary tuberculosis was treated at sanatoriums in the mountains or at coast hospitals. the patient did not come home until he was free from infection. during outbreaks of typhoid fever, diphtheria or scarlet fever, the patients were isolated in separate sick barracks or at home. to be a carrier of an infectious disease could lead to work prohibition and isolation. schools were closed intermittently during outbreaks, and posters were put on houses with infectious disease as a warning. disinfection of houses, clothing and all of its contents was completed after the termination of the isolation period, using chlorine and sulphur smoke. carbolic acid and hydrochloric acid were also used where sulphur fumes treatment failed. bedding, clothing and fixtures that could not be washed or disinfected were often burned, with a financial compensation for this "outlay." patients who broke the isolation routines could be penalized and committed to further isolation. the society's need went first before each individual's needs [ ] . during the years from to , among approximately . million inhabitants in norway, there were a total of , , cases of acute laryngitis and bronchitis, , with acute gastroenteritis, , with "croup pneumonia," , cases of diphtheria, , with scarlet fever, , with rheumatic fever, , with typhoid and paratyphoid fever, with poliomyelitis and with dysentery! [ ] knowledge of infection, sanitation and housing conditions (less overcrowded), clean water, good personal hygiene, vaccinations and nutritional status were factors mitigating epidemic outbreaks beginning of the twentieth century. this demonstrates that most infectious diseases can be prevented and controlled, even without antibiotics. in , before the breakthrough of the penicillin, there were the most important precautions against infectious diseases in norway: [ ] isolation of the source of infection, [ ] disinfection, [ ] vaccination, [ ] quarantine, [ ] food hygiene and [ ] eradication of insects [ ] . airborne infection was estimated as "the most important form of spreading infections" which may be correct even today [ , ] . the last years of isolation treatment carried out in norway have been in separate houses and buildings until ca. when most infectious diseases were integrated into appropriate wards for the underlying diseases. in there was still isolate buildings where all kinds of infections were taken care of and with good routines. today, this type of isolation is gone, with exception of some buildings in preparedness for strict isolation. in each third to fifth hospital bed, there is an infected patient, and each third patient is treated with resistance-driving antibiotics today [ , , , ] . isolation units are therefore of great need. in norway with . million inhabitants, ca. , patients are treated annually in hospitals and about , receive hospital infections, resulting in at least , extra days in hospitals because of infection. hospital infections with staphylococcus aureus occur in % of the patients [ , ] . isolation treatment is laborious and expensive (ca. , nkr or usd per day). out of the , somatic beds in norway in , hospital infections charged ca. % of these, which would generate a need for ca. infection beds. however, the somatic bed capacity in norway is steadily being reduced, which creates several problems in the healthcare. there is an increased need of isolates for patients with infections, especially due to pulmonary tuberculosis, mrsa, vre, clostridium difficile (cd), multiresistant gram-negative bacteria and other "multidrug-resistant organisms" (mdro) [ , , , , [ ] [ ] [ ] [ ] . global incidence of entero-pathogenic bacteria and imported resistant agents increases, and there is an ongoing microbial spread via food, animals, fish, water, supplies and the environment [ , ] . new knowledge of transmission, robustness and ability to survive in the environment may reinforce the methods for isolation. cd, originally defined as contact transmission, may also spread through the air [ ] . cd problem increases, particularly in the united states where half a million patients are infected each year and , of them died in [ ] . a special dangerous cd type (nap ) is detected [ ] . the incidence of new respiratory viruses like human metapneumovirus, bocavirus, human coronavirus, etc. increases. for the first time in more than years, a highly pathogenic virus as ebola has been introduced to norway and to other countries in europe. pandemic-actual viruses such as sars, mers, different types of bird flu, new outbreaks of ebola and other viral haemorrhagic fevers show the need for isolation and emergency preparedness [ ] [ ] [ ] [ ] [ ] . children's diseases, who disappeared with vaccinations, like whooping cough, mumps, rubella and measles, are now increasing with outbreaks in areas with high vaccination rate in america and europe [ ] . from to , europe had more than , measles cases [ ] . by hospitalization, these need airborne infection isolation units. noro-, rota-and sapoviruses and other types of virus gastroenteritis are highly infectious with vomiting and producing of aerosols, i.e. airborne infection. growing global outbreaks of norovirus may cause fast and widely spread of gastroenteritis among patients and personnel in health institutions and cause inactivity and stop of new admissions [ , ] . the closure of wards or hospitals. outbreaks often end with the closure of the ward, as shown in one study, median in days [ ] . geriatric wards are usually closed during outbreaks. the most frequent cause is outbreaks of norovirus, %, p < . and influenza/parainfluenza, . %, p < . [ ] . intensive care units (icu) are often particularly vulnerable to infection. crosscontamination between such patients is normal [ ] . in a -bed icu for adults, patients were followed for patient days and cross-transmission was followed by genetic typing of the microbes [ ] . a total of episodes of cross-transmission was demonstrated and dominated by pseudomonas aeruginosa, other gram-negative bacilli and s aureus. cross-transmission was associated with understaffing, use of nasogastric tube, ventilator and other multiple patient-to-nurse contacts and immune-compromised patients [ ] . patients treated at understaffed icus were three times more likely to be infected; immunosuppressed had four times greater risk, and bronchoscopy-treated patients led five times higher risk [ ] . intensive units often have no isolates, and if they have, they are not so much used because of staff shortages. airborne infection isolation units will quickly be economic by more control over the spread of infection, fewer extra days in hospitals and fewer sick leave for personnel, as was demonstrated during the sars epidemic [ ] . the larger outbreaks of infectious childhood diseases require more isolation for airborne infections [ ] . the same goes for whooping cough which years ago was an unknown phenomenon in norway and now in widespread child and adult disease [ ] . imported infections are increasing, and hospital infections like wound infections, respiratory tract infections and antibiotic-associated diseases are also increasing. in the s, the state board of health in norway recommended that the number of isolation beds should be % of the hospital's total bed numbers and the icu % [ ] . at ullevål hospital, % of the beds were airborne infection isolates, and % contact isolates in [ , , ]. in , a european investigation was done as regards the number of "high-level isolation rooms" (hirs), i.e. airborne infection isolation units with negative pressure (not defined) with at least air changes per hour and sluice (anteroom) [ ] . this is a unit intended for high-risk infections like haemorrhagic viral infection, sars, the starting phase of pandemic influenza, xdr (extended drug-resistant) tuberculosis, anthrax, plague, biological terrorism, etc. [ ] in all, european network countries (eunid) with hospitals and about isolation beds participated. many of these had fewer than air changes per hour [ ] . the total proportion of airborne infection isolates (hir) per country were calculated per million inhabitants. the largest proportion of these isolation units were detected in luxemburg, . per million; followed by finland, [ ] . most countries used hepa filter for air extraction from the isolates ( out of ). among countries, had localized airborne isolates (hir) to own buildings or wards, in was hir in the same ward as other patient rooms, and in both solutions were adopted [ ] . a total of hirs were equipped for intensive care, most in italy ( beds) and in denmark ( portable beds) [ ] . after year , hirs have been used in europe to approximately imported haemorrhagic fever cases and confirmed sars cases. in the united states, there are three "high-level isolation units" (hliu), placed at the same level as laboratory with biosafety level (bsl) and , in separate buildings, with limited and controlled access, negative air pressure and special treatment of sewage and garbage [ ] . isolates rely on "evidence-based design," i.e. knowledge of the construction, design, materials, equipment, furnishing, standards, etc., which are considered as core elements of the infection prevention [ ] . solid evidence of the spread of infection is used to control good environmental measures, disinfection and proper use of the isolates [ ] [ ] [ ] . infection isolation involves admittance of the patient to a separate room with disinfection/bathroom, sluice or anteroom (see fig. . ). it is always an advantage with some negative pressure of infection isolates-even for contact infection-to avoid spread of infections outside the isolate. a contact isolate has sluice, patient room and disinfection/bathroom, about. m . the sluice should be at least m with room for bed or stretcher and wash basin. it shall have a defined clean side, space for clean clothes and unused ppe and a dirty side, for infectious waste and infectious waste bags or containers. patient room should be at least m , with the head end against the wall and about m free zone on both long sides and from the foot end, to avoid too close contact with the patient. disinfection room, combined with bathroom, - m , contains shower, toilet and wash basin, with plenty of space to be able to help the patient as needed. it should also be equipped with decontaminator, preferably a well-sealed, throughput decontaminator from disinfection room to the sluice. in throughput decontaminators (or autoclaves), infected equipment can be put in from the disinfection room and removed decontaminated in the sluice. all throughput systems should be assured with complete sealing against air leakage. it should only be opened from one side at the time, i.e. interlock. avoid throughput cabinets between the sluice and disinfection room. all isolation unit surfaces must be smooth, robust and withstand disinfectants, both liquid and dry gaseous form. avoid tiles, gaps and joints where dirt can form the basis for growth and biofilms of microbes which may prevent effect of disinfection. there should be at least eight air changes per hour (> six air changes [ ] ), which is important to remove the infectious agent in the room air. incoming air must enter on the wall or ceiling; air extraction shall go out by the floor ( cm above), preferably in the disinfection room. the isolate should have separate ventilation system so korridor ute dekont. that the infection does not come across to other patients by setbacks in joint ventilation ducts. intake air should be a certain distance from the air outlet. hepa filters should be set on air outlet, and procedures for maintenance and control should be established. airborne infection isolate is provided and formed as for a contact isolate. the isolate should be organized for direct access from outside. in addition, the controlled and defined negative pressure is graded down from the sluice to the disinfection-bathroom compartment [ ] . pressure is usually, − to − pascal relative to the corridor and the adjacent rooms. in the sluice there is - pascal, in the patient room − to − pascal and disinfection room − pascal. the air will then be drawn from the cleanest room (sluice) to the dirtiest room (disinfection room), if the doors are closed. the most stable, controlled negative pressure may be made by doors opened outwards from the patient room to the sluice and further from the sluice to the corridor (see fig. . ). the vacuum will suck the door even tighter to the door frame to hinder air leakage. interlocked doors should be used so that only one door at a time may be opened into the patient room. the stability is dependent on the number of air changes per hour. the minimum and most stabile is air changes per hour ( ) ( ) ( ) ( ) ( ) ( ) ( ) [ , , ] . each air exchange reduces the pathogenic agents in the air substantially. all air expelled exits at the floor ( cm above) and is gathered into an outgoing ventilation channel from the disinfection compartment. avoid "shortcuts" of the air circulation. as for contact isolates, airborne infection isolates should have separate ventilation systems with no setbacks in ventilation ducts; external intake of air over the roof should be well away from the external air outlet over the roof, and all air from the unit is hepa filtrated. procedures for maintenance, disinfection and control of filters and ducts should be established. cohort isolation of several patients with the same infectious agent may be appropriate. it is a good initiative during pandemics. the sars epidemic released a rapid learning process where the chinese built-within a week-a dedicated sars hospital for patients in beijing [ ] . in a study from brazil, all patients with proven mdros (multidrug-resistant organisms) were transferred to a -bed unit for isolation treatment by a trained personnel, where personnel, patients and family were weekly taught about how to handle mdro's by a special team [ ] . after the intervention, the mdro rate was significantly reduced, specially by the reduction of vre. isolated patients had at least the same good treatment quality as not isolated patients [ ] . in norway, cohort isolation was utilized in trondheim during the first national outbreak of mrsa ( ) ( ) ( ) ( ) ( ) . a total of patients were infected (including personnel), and died ( . %) directly or indirectly from the hospital epidemic [ ] . the control of the outbreak came first when infected patients and personnel were cohort isolated in a separate isolation ward [ ] . barrier care of one infectious patient among other patients without infections on the same bedroom in dormitory is not recommended. a good standard of patient isolates depends on good daily cleaning and a thorough disinfection by termination of the isolation. long-time surviving bacteria, viruses and fungi should not be a risk for the next patient to be isolated, which may happen [ , ] . mrsa, multiresistant acinetobacter baumannii and other mdros are contaminating bedding, bed and environment for months, if not removed [ ] . disinfection of rooms and isolates are often not well enough done [ ] . the terminal disinfection, removal and disinfection of all equipment in the room and disinfecting of all surfaces are important. disinfectants (liquid) that are effective against all microbial agents are chloramine %, household bleach % or peracetic acid for h. treatment with hot water (> °c) for min is a very effective disinfection of equipment and textiles. different types of gas or steam disinfection (hydrogen-peroxide dry gas, chlorine gas, formaldehyde gas) may be effective for equipment and room disinfection [ ] [ ] [ ] [ ] . note that there is yet no gas treatment that is documented to kill mycobacteria [ ] . the isolates are often used incorrectly. in the same ward and at the same time, patients without infection are placed on the isolates while infectious patients are not isolated. although defined isolate treatment is determined and ppe used, it turns out that the patient is walking freely around in the ward, not really isolated. knowledge and practices increases understanding of disease control measures. some patients that are both prone to infection and have serious infections, like cystic fibrosis patients, should be treated in isolates [ ] . depression, anxiety and anger get less visits and information by health professionals; more often fall injuries or other injuries, inferior care, etc. are claimed to be side effects of isolation [ , ] . however, patients previously depressed and anxious at admission suffer no more under an isolation stay [ ] . there is not shown differences between isolated and non-isolated patient, according to the patient's view of treatment, on the contrary [ ] . but the patient satisfaction is the highest among those who are well informed [ ] . most studies show that contact time with the medical doctor is approximately equal for isolated and non-isolated patients and approximately equally frequent [ , ] . although mrsa or vre infected patients, in one study, had more frequent falls and pressure ulcers, this was not reduced by removing the contact isolation, so that it was likely other factors which could contribute to such complications [ ] . outbreaks are very costly and take up significant resources and healthcare for other patients [ ] . a patient infected with mrsa was at ullevål university hospital, norway, estimated to generate an additional charge of , nkr (ca. $ , ), including extended length, spread to a hospital employee, screening tests and antibacterial treatment in [ ] . in , in connection with a mrsa outbreak in neonatal ward at ullevål, the calculated additional costs were , nkr (ca. $ , ) per mrsa-positive patient, including isolation expenditure, disinfection and cleaning, screening and extra days in the hospital [ ] . active screening and isolation of patients with mrsa are significantly and markedly cheaper for the healthcare than uncontrolled transmission which can result in serious infection and death [ ] . anchoring isolation procedures in laws is essential to the implementation of isolation. norway has several laws, regulations and guidance to control infections in healthcare facilities [ ] [ ] [ ] [ ] [ ] [ ] . international studies have laid the basis for the norwegian isolation measures and techniques [ - , - , ] . a number of studies regarding the design and function of isolation units exist [ , , [ ] [ ] [ ] [ ] . simply to place patients in single rooms provides noticeable improvement in the spread of infection [ ] . the implementation of good isolation measures and compliance is often weakened by confusion, lack of written procedures, lack of training, competence and understaffing. a number of other factors also influence the attitude of healthcare professionals with regard to follow the hospital's infection control procedures, not least professional disagreement about the procedures. • in emergency wards in the united states, only % of the wards used contact protection (gloves and gown) upon receipt of patients with diarrhoea or faecal incontinence, % for suspected cd, % for purulent skin infections, % for suspected mrsa and % by multiresistant gram-negative rods [ ] . • it may be a big difference between what staff and others say they do and what happens in reality [ ] . among persons who entered isolates, the compliance was higher by strict isolation infections ( %) than with other infectious conditions [ ] . visitors followed the measures better than personnel ( % versus %) [ ] . the compliance with isolation procedures increased significantly when more time was spent with the patient, being a visitor, and when the patient was on a strict isolation [ ] . the study pointed out the danger of increased demands for doctors and nurses due to savings can result in adverse effects such as reduced compliance with key routines as isolation [ ] . morgan et al. observed visits of a healthcare professional to isolated and non-isolated patients. patients on contact isolation had % fewer visits and % less contact, and there were fewer visitors than non-isolated patients [ ] . however, the staff implemented significantly more frequent handwashing after visit in isolates, %, than by visits with non-isolated patients, % [ ] . • during the mrsa outbreak with ca patients in trondheim, norway, - , kvittingen et al. described problems that may occur during epidemic and endemic conditions as "doctors more or less wholehearted support and scepticism to the measures made by the hygiene committee, discussions concerning the virulence and resistance development, what to do when a local outbreak paralyzes the department completely, information problems, lack of qualified personnel, etc." [ , ] . they stressed that "larger hospitals should dispose well-planned isolation opportunities, both for patients who are infectious spreaders and perhaps equally important, to protect patients with reduced infection defence," and that "personnel in management positions (should) provide effective and loyal support for infection control personnel" [ ] . • this was a very important observation done for more than years ago. it is still causing major problems during outbreaks and endemic situations when there may be a choice between shortcut thoughts about "economy" and infection control. still, impression and experience is that staff working directly with the patients have great respect for contact and airborne routines, at least in norway. isolation regimes and interpretation of these are an almost perpetual discussion which changes with endemic and epidemic conditions, economy and guesswork. although isolation of patients with infectious diseases has been used for hundreds of years, there is little evidence in terms of endemic conditions [ ] [ ] [ ] [ ] . the norwegian isolation regimes are based mostly on guidelines from cdc [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in addition, an increasing number of guidelines are focusing on special isolation factors [ , - , - , ]. cdc's isolation guideline is graded from high evidence, highly recommended as the ia, to no recommendation and unresolved questions or professional disagreement [ ] . category ic is included in the legislation for patients and caregivers [ ] . classification into categories is essential for the most important infection control measures in hospitals. "these recommendations are designed to prevent transmission of infectious agents among patients and healthcare personnel in all settings where healthcare is delivered. as in other cdc/hicpac guidelines, each recommendation is categorized on the basis of existing scientific data, theoretical rationale, applicability and, when possible, economic impact" [ ] . the cdc recommendations are changed over time [ ] [ ] [ ] [ ] [ ] [ ] [ ] . cdc/hicpac system recommendations in categories in correspondence with investigations, theory and economic possibility; quoted [ ] . cdc's isolation guideline refers to the administrative responsibility around isolation treatment [ ] . the hospital's management is responsible for ensuring the implementation of good practical measures and to be the driving force in the hospital's infection control preparedness. this should particularly be done with regard to isolate patients, an extra burden, and also for the economy. enough isolate capacity is to be provided in relation to citizens, patient categories, type of hospital activity and endemic/epidemic conditions. the isolates must be of good quality and design, and it should be enough airborne infection isolation units [ ] . some of the cdc-recommended administrative responsibilities are quoted here: [ ] citation: "healthcare organization administrators should ensure the implementation of recommendations in this section: transmission-based precautions), establish processes to monitor adherence to those performance measures and provide feedback to staff members. category ib" [ ] . citation ended. a good and active hospital management is of major importance to stop the outbreak [ , , , , , , , ] . ransjø et al. attached the importance to the activity of the hospital management during outbreaks of multidrug-resistant microbes in sweden [ ] . in norway, an outbreak of vre by a dialysis department spring , at ullevål university hospital, was expected from the department's critical building condition [ ] . after great effort from the staff, the outbreak stopped within days [ ] . a structural rehabilitation started then, with expansion of bed numbers, assisted by the hospital management. one year later, all patients were vre negative [ ] . a number of outbreaks at ullevål's premature intensive care unit during years have been linked to chronic understaffing, overcrowding, part-time work, lack of infection control practices and isolates, lack of information and a lack of management responsibilities [ , ] . the responsibility of the hospital management to prevent and combat outbreaks of infection should be clearly stated in the hospital's infection control program. cdc defines contact isolation, using gown and gloves when in contact with patients infected with resistant bacteria like mrsa and other mdros (multidrug-resistant organisms), and single rooms are recommended [ ] . direct contact contamination occurs when microbes are transmitted from an infected person to another without the "intermediate stage" in the environment. microbes transferred to the hands can further spread to the own mouth, nose, hair, ears, clothing, etc. [ ] healthcare personnel is likely to be a "source, vector or a victim" of infections like mrsa [ , ] . patients bring their own microbes into the hospital, with often large contact before a known infection [ ] [ ] [ ] [ ] [ ] . therefore, rapid tests are recommended where possible to detect pathogens [ , ] . indirect contact contamination happens when the microbes are transferred to an object in the environment and from there transmitted to other objects or people. infectious patients themselves will have the agent on the body and clothes and transmit it to the environment. the undetected microbes can spread quietly and calmly in many directions via equipment, rooms, surfaces and missing/incorrect procedures [ ] . this is often a large and persistent environmental problem and a threat to other patients [ , , , , , , [ ] [ ] [ ] [ ] [ ] ] . the most typical spread occurs when people with contaminated hands are depositing microbes on equipment, machinery, knobs, door handles, uniforms, other textiles, furniture, toys, etc. [ ] [ ] [ ] fellow-patients on the same room are highly susceptible to infection [ , , , , , , [ ] [ ] [ ] [ ] . infection can be spread further via equipment (bp apparatus, stethoscope, medicine tray, blood sugar test equipment, journal, etc.) and the personnel's uniform. it is often brought to the ward office, to service rooms and to other personnel or patients. a long "domino" chain of infections may follow infectious agents not stopped by simple hygienic measures and isolation [ ] . contact infection may also be an airborne infection from patients coughing and sneezing by certain aerosol procedures, when cleaning the surfaces, by bedding and by strong air currents (e.g. by rapidly opening and closing doors) which dislodges dust from the lamps, shelves, etc. [ , , , , [ ] [ ] [ ] therefore, a daily good housekeeping and good routines for final disinfection in isolates is very important to reduce the burden and risk of pathogenic microbes [ , ] . contact infection prevention (cp)-recommended by cdc [ ] . some citations selected from siegel et al. ; cdc guideline for isolation, where recommendations are in categories where ia is strongest recommendation. selected citations: [ ] " . in acute care hospitals, place patients who require cp in a single-patient room when available. category ib. . when single-patient rooms are in short supply, apply the following principles for making decisions on patient placement: ----place together in the same room (cohort) patients who are infected or colonized with the same pathogen--. category ib. in close proximity to the patient (e.g., medical equipment, bed rails). don gloves upon entry into the room or cubicle. category ib. . wear a gown whenever anticipating that clothing will have direct contact with the patient or potentially contaminated environmental surfaces or equipment in close proximity to the patient. don gown upon entry into the room or cubicle. remove gown and observe hand hygiene before leaving the patient-care environment. category ib. . patient transport. in acute care hospitals and long-term care and other residential settings, limit transport and movement of patients outside of the room to medically-necessary purposes. category ii. . when transport or movement in any healthcare setting is necessary, ensure that infected or colonized areas of the patient's body are contained and covered. category ii. . remove and dispose of contaminated ppe and perform hand hygiene prior to transporting patients on cp. category ii. . don clean ppe to handle the patient at the transport destination. category ii. precautions. category ib/ic. . in acute care hospitals and long-term care and other residential settings, use disposable noncritical patient-care equipment (e.g., blood pressure cuffs) or implement patient-dedicated use of such equipment. if common use of equipment for multiple patients is unavoidable, clean and disinfect such equipment before use on another patient. category ib [ ] ." selected citation ended. spread of pathogenic infectious agents through the air and droplets requires a defined negative pressure ventilation isolate and a system which reduces airborne infection in the patient's room. it is best done by air replacement - times an hour [ , ] . the cause of airborne, pathogenic microbes may include: and the urinary tract. . by rapid removal of the bandage from pus and discharging wounds. . by raise up of infected dust, skin cells and other contaminants by activities that create air currents, often in poorly cleaned rooms [ ] . . re-aerosolized pathogens from used textiles and equipment in the patient room or where these contaminated items are brought, for instance, to the ward's disinfection room or to the laundry. by massive contamination and poor routines for washing equipment and hospital textiles, this may cause a special problem of re-aerosols [ ] . . construction activity of all types without measures against airborne infection is influencing the airborne spread of bacteria, virus and fungi. this must be taken into account when rebuilding and repairing buildings and ventilation ducts etc. [ , ] . if ignored, it may cause large problems like the outbreak of bacillus cereus among patients, most with sepsis, during months of a large construction project next to the hospital [ ] . air samples showed growth of large quantities of bacillus inside the hospital, the units and also isolates, and hospital textiles were heavily contaminated [ ] . on a daily basis, the human inhales , particles or more via airways; many of these are bacteria, viruses and fungi but mostly come up again without causing disease. a solid documentation shows that airborne infection may occur at a variety of infectious diseases [ , , , , , , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . rooms with mrsa-infected cases may have larger numbers of mrsa bacteria in air samples than in samples from surfaces [ , ] . mrsa may even be spread to nearby located rooms, to patients not infected [ , ] . the few existing units for airborne infection isolation mean that most patients, except for pulmonary tuberculosis, most often are admitted to contact isolation or single-patient rooms, with the use of ppe corresponding to airborne infection: gloves, infection-protecting gown, cap and surgical mask and sometimes room-bound shoes [ ] . airborne precautions (ap)-recommended by cdc [ ] . an aiir directly to the outside, the air may be returned to the air-handling system or adjacent spaces if all air is directed through hepa filters. (c) whenever an aiir is in use for a patient on airborne precautions, monitor air pressure daily with visual indicators (e.g., smoke tubes, flutter strips), regardless of the presence of differential pressure sensing devices (e.g., manometers). (d) keep the aiir door closed when not required for entry and exit. . when an aiir is not available, transfer the patient to a facility that has an available aiir. category ii. . in the event of an outbreak or exposure involving large numbers of patients who require airborne precautions: (a) consult infection control professionals before patient placement to determine the safety of alternative room that do not meet engineering requirements for an aiir. (b) place together (cohort) patients who are presumed to have the same infection(based on clinical presentation and diagnosis when known) in areas of the facility that are away from other patients, especially patients who are at increased risk for infection (e.g., immune-compromised patients). (c) use temporary portable solutions (e.g., exhaust fan) to create a negative pressure environment in the converted area of the facility. discharge air directly to the outside, away from people and air intakes, or direct all the air through hepa filters before it is introduced to other air spaces. category ii. . place the patient in an aiir as soon as possible. if an aiir is not available, place a surgical mask on the patient and place him/her in an examination room. once the patient leaves, the room should remain vacant for the appropriate time, generally h, to allow for a full exchange of air. category ib/ic. instruct patients with a known or suspected airborne infection to wear a surgical mask and observe respiratory hygiene/cough etiquette. once in an aiir, the mask may be removed; the mask should remain on if the patient is not in an aiir. category ib/ic. . restrict susceptible healthcare personnel from entering the rooms of patients known or suspected to have measles (rubeola), varicella (chickenpox), disseminated zoster, or smallpox if other immune healthcare personnel are available. category ib. . wear a fit-tested niosh-approved n or higher level respirator for respiratory protection when entering the room or home of a patient when the following diseases are suspected or confirmed: (a) infectious pulmonary or laryngeal tuberculosis or when infectious tuberculosis skin lesions are present and procedures that would aerosolize viable organisms (e.g., irrigation, incision and drainage, whirlpool treatments) are performed. category ib. (b) smallpox (vaccinated and unvaccinated) ---category ii. (c) no recommendation is made regarding the type of personal protective equipment (i.e., surgical mask or respiratory protection with a n or higher respirator) to be worn by susceptible healthcare personnel who must have contact with patients with known or suspected measles, chickenpox or disseminated herpes zoster. unresolved issue. . in acute care hospitals and long-term care and other residential settings, limit transport and movement of patients outside of the room to medically-necessary purposes. category ii. . if transport or movement outside an aiir is necessary, instruct patients to wear a surgical mask, if possible, and observe respiratory hygiene/cough etiquette. category ii. . for patients with skin lesions associated with varicella or smallpox or draining skin lesions caused by m. tuberculosis, cover the affected areas to prevent aerosolization or contact with the infectious agent in skin lesions. category ib. . healthcare personnel transporting patients who are on airborne precautions do not need to wear a mask or respirator during transport if the patient is wearing a mask and infectious skin lesions are covered. category ii. . exposure management immunize or provide the appropriate immune globulin to susceptible persons as soon as possible following unprotected contact (i.e., exposed) to a patient with measles, varicella or smallpox: category ia. (a) administer measles vaccine to exposed susceptible persons within h after the exposure or administer immune globulin within days of the exposure event for high-risk persons in whom vaccine is contraindicated. (b) administer varicella vaccine to exposed susceptible persons within h after the exposure or administer varicella immune globulin (vzig or alternative product), when available, within h for high-risk persons in whom vaccine is contraindicated (e.g., immune compromised patients, pregnant women, newborns whose mother's varicella onset was < days before or within h after delivery. (c) administer smallpox vaccine to exposed susceptible persons within days after exposure." citation ended. "--the environmental recommendations in these guidelines may be applied to patients with other infections that require airborne precautions". selected citation ended. particularly dangerous or unknown infectious agents with high mortality need special treatment in high-risk isolates with separately, controlled ventilation and disinfection of air extraction/waste/water, etc. or in buildings separated from other patients and without common ventilation [ , [ ] [ ] [ ] [ ] [ ] . during the ebola epidemic from march , health professionals and patients were not protected well enough since the who recommended procedures for close contact/droplet infection [ ] . the who excluded risk of airborne transmission of ebola, even though it was documented to be transferred via air to primates [ , ] . the uncertain infection control procedures of who were upgraded from september to october by the who and cdc, from contact/droplet infection metre from the patient to strict isolation, but still without measures against airborne infection [ , ] . the principle of "prevention is better than cure" was not in use. the same happened in connection with sars in where who was recommending a low infection control level, contact/droplets metre from the patient, and had to raise the disease control level up to strict isolation ca. months later on [ ] . high-risk infections are recorded almost daily around the world, like the pneumonic plague in madagascar, outbreaks (partly nosocomial) of multidrug-resistant tuberculosis in all parts of the world and nosocomial outbreaks of crimean-congo haemorrhagic fever in germany after import of ill us soldier from afghanistan [ ] [ ] [ ] . nearly each day, there are reported new cases of avian influenza, mers, various haemorrhagic viruses or other dangerous microbes [ , [ ] [ ] [ ] [ ] . since , there has been no need for strict isolation in norway, with the exception of the imported healthcare professionals with ebola in autumn . risk of infection may occur when patients with greatly reduced infection defence are coming into contact with infected equipment, textiles or other patients, staff or visitors who have infection or are carriers of possible pathogenic microbes [ , , ] . the following conditions are particularly vulnerable to contamination, agranulocytosis, neutropenia; < . in granulocytes or rapidly decreasing number < , severe burns, patients with severely weakened immune system due to the treatment with immunosuppressive, chemotherapy or radiation (transplants, cancer or leukaemia). this heterogeneous group is growing due to a more advanced treatment and more cancer and transplant cases. they should be protected against infection from "outside" by treatment in protective isolation in a clean room with anteroom or sluice and with hepa-filtered air in a positive pressure room [ , , [ ] [ ] [ ] [ ] [ ] [ ] . cdc recommends that by allogeneic haematopoietic stem cell transplantation (hsct), the patients should be treated in protective isolation, especially to protect against fungal infections such as aspergillus; category ib. [ ] "as defined by the american institute of architecture, air quality for hsct patients is improved through a combination of environmental controls that include [ ] hepa filtration of incoming air, [ ] directed room air flow, [ ] positive room air pressure relative to the corridor, [ ] well-sealed rooms (including sealed walls, floors, ceilings, windows, electrical outlets) to prevent flow of air from the outside, [ ] ventilation to provide ≥ air changes per hour, [ ] strategies to minimize dust (e.g., scrub-able surfaces rather than upholstery and carpet, and routinely cleaning crevices and sprinkler heads), and [ ] prohibiting dried and fresh flowers and potted plants in the rooms of hsct patients. the latter is based on molecular typing studies that have found indistinguishable strains of aspergillus terreus in patients with haematologic malignancies and in potted plants in the vicinity of the patients" [ ] . • in protective isolate should intake air be hepa filtered which removes . % of particles larger than or equal . μm in diameter; category ib. [ ] • the room should be at positive pressure relative to the corridor with a pressure difference of . pascal or more; category ib. [ ] • the pressure should be monitored visually every day; category ia [ ] . • the room must be sealed so that no outside air to seep in; category ib. [ ] • there should be a minimum of air changes per hour; category ib. [ ] • use smooth surfaces, avoiding textile furniture and the like, and perform good cleaning; category ii [ ] . • do not use carpeting in corridors or rooms in the area; category ib. [ ] • the patient should be at least possible out of the room, the necessary examinations, etc., to be implemented in the shortest possible time; category ib. [ , , , , ] • if necessary, use respiratory protection if out of isolation; category ii [ , ] . • staff are using adequate infection control equipment if the patient has infection; category ia/ib. [ ] • patients with respiratory infection-at the same time they need protective isolation-should be transferred to a defined airborne isolate. if hepa filters are lacking on the air intake to the airborne infection isolation units, a portable system of the hepa filter may be used in the room to remove fungal spores and bacteria; category ii [ ] . avoid transfers outside the isolate [ , ] . a retrospective, case-control study of immunocompromised patients in switzerland showed that when invasive fungal infection occurred, the mortality was over % [ ] . an outbreak of fungal infection among patients on a haematological department showed that the more often . protective isolation the patient was transferred to other departments/was outside the isolate, the more frequently he was infected with invasive fungi [ ] . with more than five patient transfers outside protective isolates, the risk of fungal infection was sixfold higher than if staying in protective isolation. if the patient was transferred during neutropenia, the risk increased by nearly sevenfold [ ] . building activities. fungal spores are always released during construction activity and may become airborne also in risk areas where severely immunocompromised patients are located [ , , ] . infectious agents may be spread via air, textiles and equipment with deposition of fungal spores and other microbes during the construction period. fungal spores follow air currents far away [ ] . construction activity must therefore be coordinated with appropriate departments and wards so that the patient area is shielded. air from construction places should not leak into patient areas and be taken out directly from the building in such a way that it does not re-enter through the systems for intake of air. leakage of water in buildings. fungal growth in patient room walls is due to neglected maintenance of buildings for many years and usually comes from water leakage from the old pipes, roof leaks or poorly maintained bathrooms. patients should not stay in rooms with suspected fungal growth and decay! control of fungi should be implemented in protective isolates and storages for such isolates once a year or more frequently when suspected water leaks and rot. the requirement is fungal spores per litres of air using the sas-slit sampler. patients must be protected against infections during care and treatment [ , , [ ] [ ] [ ] . cleaning and disinfection of rooms, surfaces and equipment between infection susceptible patients must be extra careful since many-at the same time-have serious infections, including resistant bacteria. despite a good cleaning, abundant amounts of microbes may be detected in the environment and the equipment after cleaning [ , ] . healthcare professionals who treat infectious patients may be highly exposed to infection, although they really should be protected [ , - , , , , ] . at the beginning of the sars epidemic in , % of sars patients were the hospital personnel who had taken care of the first sars patients [ ] . during the ebola outbreak in , healthcare workers were again severely attacked by the disease and death in the first phase, until a more protective personal protective equipment system was in place [ , [ ] [ ] [ ] . there is still a great variety in international guidelines in terms of personal protection equipment (ppe) by ebola virus for healthcare workers [ ] . the who, united kingdom and cdc defined ebola as non-airborne infection in and still does the same in [ ] . none of these recommended respiratory protection for suspected ebola infection in august-september , at least half a year after the start of the epidemic. only the united kingdom recommended respirator mask for confirmed ebola. there was no covering of the head or neck, not either during aerosol-generating procedures! in october , the cdc upgraded the ppe measures to strict isolation measures for proven ebola infection, while the earlier ppe measures were recommended for suspected ebola infection (table . ) [ ] [ ] [ ] . during the pandemic flu in , norwegian health authorities did not allow the staff at hospitals to use respirator masks (p masks) when taking care of the flu patients; they should use surgical masks [ ] . exceptions were for aerosol-generating procedures. the same health authorities went public and said that surgical masks did not have any protective effect! [ ] recurrent problems are that the health authorities are not able to see the importance in protecting health professionals who are-and will always be-the frontline workers, even at the most serious infectious diseases [ ] . governments that are not willing to take care of their healthcare employees create unnecessary fear and reluctance to make a good contribution during outbreaks. if the disease is not as dangerous as sars and ebola, a highly contagious pandemic influenza or a large norovirus outbreak could paralyse the healthcare system when a high number of the staff get sick at the same time. by preventing disease transmission to health professionals, the hospital can be operatively enough to handle properly other serious diseases such as traffic accidents, myocardial infarction, stroke, etc., even at largescale epidemics. in spite of problems concerning isolation and prevention of spread of emerging and new serious infections, new guidelines are now appearing, which works with reduction of infection control in severely affected countries with endemic infections [ , ] . 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pneumonia in surgical intensive-care unit droplet fate in indoor environments, or can we prevent the spread of infection environmental survey to assess viral contamination of air and surfaces in hospital settings international infection control guidelines may not protect against ebola. hospital health care guidelines does not protect against ebola. bit's th annual wold congress of microbes - (wcm ) pneumonic plague outbreak multi drug-resistant tuberculosis outbreak in gaming centres health care response to cchf in us soldier and nosocomial transmission to health care providers incidence of, and risk factors for, nosocomial infections among haematopoietic stem cell transplantation recipients, with impact on procedure-related mortality ventilation systems for hospital rooms devoted two immune compromised and/or infectious patients invasive aspergillosis in severely neutropenic patients over years: impact of intranasal amphotericin b and hepa filtration hospital infection control in haematopoietic stem cell transplant recipients masking of neutropenic patients transport from hospital rooms is associated with a decrease in nosocomial aspergillosis during construction in-hospital transfer is a risk factor for invasive filamentous fungal infection among hospitalized patients with haematological malignancies: a matched case-control study rikshospitalet: daily work in protective isolation rikshospitalet: for you to be a stem cell transplant. guideline for adult patients and their families protective isolation. in: handbook of hygiene and infection control for hospitals. oslo: ullevål university hospital risk factors for acquiring vancomycin-resistant enterococcus and methicillin-resistant staphylococcus aureus on a burn surgery step-down unit residual viral and bacterial contamination of surfaces after cleaning and disinfection protecting the frontline: designing an infection prevention platform for preventing emerging respiratory viral illness in healthcare personnel in: handbook of hygiene and infection control in hospitals. part microbiology and infection control reconsidering contact precautions for endemic methicillin-resistant staphylococcus aureus and vancomycin-resistant enterococcus duration of contact precautions for acute-care settings comparing the recommended ppe equipment by ebola infection in guidelines from the who, united kingdom and cdc in [ ] [ ] [ ] who key: cord- -fm gl b authors: andersen, bjørg marit title: scenarios: serious, infectious diseases date: - - journal: prevention and control of infections in hospitals doi: . / - - - - _ sha: doc_id: cord_uid: fm gl b scenarios for serious, infectious diseases are important procedures used to understand the special microbe’s behaviour (clinical illness, spread of infection, etc.) and how to act most rational during special dangerous outbreaks. furthermore, scenarios describe how to handle patients, personnel and others possibly exposed to infections,- outside and inside the hospital- to stop spread of the infection as soon as possible. today, it is not acceptable to place a patient with a known high-risk, serious infection in the same hospital room as other patients with not the same disease (who). in this chapter, some seldom but realistic scenario is described to better understand how to react and treat patients to stop spread of microbes during the primary phase of dangerous transmittable diseases. • all personnel who are the first in contact with the infected/exposed person, relatives, other contacts and environment. • it is not acceptable to place a patient (with not defined same disease) in the same room with a patient with known high-risk infectious disease (who). the hospital's management provides written plans for how to react in situations where personnel and others may be exposed to known/unknown serious communicable disease and a practical arrangement for how to handle the situation. the infection control officer at the level of where the problem occurs, and at the departments/ward where the infection may spread, is responsible for following local emergency plans. personnel who unprotected have come in a seriously contagious situation and may have been exposed to infectious agents are responsible for contacting the nearest responsible/infectious unit for advice and of following written guideline and practical advice. [ ] • the patient (infected/suspected infected) is usually relatively easy to deal with since there are guidelines for preventing spread of infection and treating the patient for the current disease; see isolation routines. • contacts exposed to infection are often worse to handle since it may be larger numbers of people (travel company, etc.) and because fear of being infected itself creates uncertainty. therefore, some imaginable scenarios are made that deal with infected contacts. • transport by ambulance. all transport of infectious patients from the place of arrival to the hospital should take place in ambulances using the same infection control regime as for the individual infectious disease (contact infection, airborne infection, strict isolation); see isolation regimes; chaps. proper use of protective equipment is used when handling such patients. the ebola outbreak in african countries in showed that almost , were registered ill, more than , died, health professionals became ill and more than half of them died. lack of use of ppe and proper infection control led to escalation of the epidemic. the staff used only m distance from the patient as a zone of infection, as recommended by the who and cdc, and lacked protection for the head, hair and neck when within the m zone and used only ordinary masks [ ] [ ] [ ] [ ] . this occurred despite the fact that ebola is defined as a high-risk, biosafety level infection in which airborne infection could be relevant [ ] [ ] [ ] [ ] . the epidemic declined from september , following the introduction of more proper use of personal protection equipment and infection control routines [ , , ] . contacts are differentiated after infection risk: . high-risk contact: physical contact with vhf, or with blood secretion or excretion. healthcare staff, ambulance staff, laboratory staff, family or others who have treated the patient before admission. . low-risk contact: been in the same room with the patient after the onset of the disease, but not in direct contact with the patient, equipment or others in the room. examine the contacts; measure temperature two times for weeks. . transmission from healthy contacts is considered unlikely. however, everyone who has been in the same place at the same time with a vhf sick patient should be informed and followed up. if probable or verified vhf, inform the contacts-low chance of infection: • the contacts keep calm at home and measure the temperature daily two times for weeks after the last contact with the index patient. no crowding with many people and no use of collective traffic. • if temperature °c or more, or rash/flu symptoms/sickness, contact the infection medical department. • if this cannot be achieved at home, the contact may come to defined outpatient clinic for temperature measurement by appointment or is admitted to hospital. • served with food, etc. brought out from store to door, possibly, while isolated at home. mrsa (methicillin-resistant staphylococcus aureus), vancomycin-resistant mrsa, penicillin-resistant pneumococci, super-resistant gram-negative bacteria (esbl, cre,cp, ndm- bacteria), multidrug-resistant tubercle bacteria (tuberculosis), vancomycin-resistant enterococci (vre), etc. evaluated in collaboration with microbiological laboratory. this was especially observed during the major tsunami disaster in [ ] . serious problems can occur in areas with melioidosis and other highly virulent bacteria. in hospital, not usually many at the same time, but dependent on endemic situation. the patient can be contact or air isolated, depending on the infectious agent. • registering of direct contacts-depending on the infectious agent (name, address), and include where the patient have been earlier (information). • ambulance personnel and other personnel use routines for the relevant infection type according to isolation procedures and in accordance with emergency department's report. in case of doubt, contact infection control personnel. • use respiratory protection, p mask, if suspecting pulmonary tuberculosis, and put a surgical mask or p /p mask without a valve on the patient in case of suspected pulmonary tuberculosis or respiratory tract infection. contacts/carriers-differentiated follow-up-low chance of getting sick: example: three people in a norwegian travel company get voluminous, watery, painless diarrhoea on return from bangladesh, just before landing at oslo airport, gardermoen. due to a loss of fluid, they were transported with an ambulance equipped for "import infection" and admitted directly into contact isolation. municipal infection control doctor is notified and is responsible for reporting, measures and follow-up outside the hospital together with the municipal emergency response group. this is one of the old, major quarantine diseases. only a few get sick (top of the iceberg), i.e. - patients out of infected. there is a low mortality by proper treatment (< %). the transmission risk is relatively low due to good hygiene and good sanitation in today's norway and other developed countries. most patients are shedding bacteria in large amounts and are also carriers without symptoms. patients are isolated with contact isolation regimens. the infection can reach unmanageable heights in disaster areas, by hunger, contaminated water supply and destroyed infrastructure. following the natural disaster in haiti, cholera was introduced with infected helper crew from asia (carriers), and an epidemic started in , which in had increased to , cholera patients, , hospitalized and deaths [ ] . • registering of contacts and remaining passengers in the airplane and from same travel company (name, address, telephone number). • ambulance staff and other personnel use the contact regime. if risk of spills, etc., use also surgical mask, visor and cap in addition to gloves and gown/overall. example: an elderly woman who recently attended a bus trip to moscow became sick days after returning to oslo. she had sore throat, fever, cough and eventually a white, firm-sitting "plaque" in the throat. she was hospitalized after days because of suspected diphtheria. contact persons and other close contacts were contacted for follow-up and treatment with erythromycin (according to resistance pattern). municipal infection control doctor is notified and is responsible for reporting, measures and follow-up outside the hospital together with the municipal emergency response group. diphtheria may still be periodic problems in eastern europe and many places in the world. vaccination status is good in children in most countries but more uncertain in elderly, especially in women. this is a contact and airborne infection, relatively highly infectious. there are probably few cases in an outbreak due to herd immunity. patients are isolated with air and contact isolation regime until free from bacteria (negative culture). a historically serious infectious disease also in norway, with high mortality rates until the middle of the last century [ ] . ullevål hospital, oslo, introduced treatment with the diphtheria serum in and achieved an impressive response-from - % mortality to - % [ ] . • registering: all exposed persons (name, address, telephone number) and followup; see below. • ambulance staff and other personnel use the contact and airborne infection regime when picking up and transporting a patient. use respiratory protection, visor and cap in addition to gloves and gown/overall, and put a surgical mask on the patient. contacts/carriers of diphtheria are differentiated-vaccination protects against disease: . sampling (nasopharynx samples) of all exposed persons (even if vaccinated and are not sick, you may be a carrier). . prophylactic/therapeutic treatment with erythromycin may be initiated rapidly. . if living at home, others should not be exposed to infection/carrier state. . short-time airborne isolation may be relevant for carrier or exposed to infection until the infection state is clarified/effect of antibacterial therapy. . booster vaccine against diphtheria is considered for all contacts. example: a person of a family of five who has stayed in madagascar for a month got sick on his way home to norway. he coughs, has fever and develops skin rashes that resemble big boils, especially in the groin. he was admitted directly to strict isolation in hospital and suspected of serious import infection. the municipal infection control doctor is notified and is responsible for reporting, measures and follow-up outside the hospital together with the municipal emergency response group. • quarantine disease: periodic problem in the east asia, especially india, and ongoing outbreaks in africa, namibia and madagascar. pest may be a warrelated disease (biological warfare). untreated dies more than % of the cases while, with streptomycin treatment, less than %. this is a typical airborne disease, relatively highly infectious when respiratory tract symptoms. there is usually small outbreak with few cases ( - ). air and contact infection regime until free for bacteria. in november , new pest outbreaks were reported in madagascar. about patients got sick, of whom died of bubonic plague [ ] . the infection spread rapidly between humans and "killed quickly" [ ] . multidrug-resistant yersinia pestis is described in this country [ ] . • registering: all infected persons (same travel company, all in the same flight home) are registered (name, address, telephone number) and followed up. • ambulance staff and other personnel use the contact and airborne isolation regime when picking up and transporting a patient. use respiratory protection (p mask), visor and cap in addition to gloves and gown/overall, and put a surgical mask on the patient. all close contacts/carriers are isolated to infection state is clarified-little chance of getting sick: . sampling from all exposed persons . prophylactic/therapeutic treatment, eventually vaccine . short-time airborne isolation of exposed cases until the infection state is clarified/effect of antibacterial therapy . . anthrax after staying in turkey, sick on the plane home . . . patient: strict isolation-air pressure isolate with pressure [ , ] example: two out of six people who have been on family visits in turkey for a week, on farms with goats and skin production, are acutely ill on the plane home with cough, shortness of breath and fever. upon arrival, the emergency outpatient clinic was contacted by the patients who were immediately transferred to intensive care unit for airway symptoms and suspected import infection. at the hospital, anthrax is suspected, and patients are strictly isolated in air isolate and treated. municipal infection control doctor is notified and is responsible for reporting, measures and follow-up outside the hospital together with the municipal emergency response group. • endemic problem in several places (asia, africa, middle east, north and south america), war-related (biological warfare). varying number of cases of anthrax at outbreaks during peacetime, depending on how many people have eaten, for example, infected food, etc. • symptoms: % are cutaneous, % are respiratory, and a few cases are gastrointestinal anthrax. the latter two are two-phasic and almost always fatal. • the bacterium bacillus anthracis is a spore-forming, resistant, gram-positive rod that survives nearly infinity in the environment if not removed. the bacteria are usually penicillin-sensitive. • infection: person-to-person infection is unlikely, but hospital infection is described. air and contact regime is conducted around such patients in hospitals until free of bacteria (ca h treatment); however spores may survive for a long time. • registering: all directly exposed persons are registered (name, address, telephone number) and followed up. • ambulance personnel use contact and airborne regime for patient pickup and transport. use respiratory protection (p mask), visor and cap in addition to gloves and gown/overall, and put a surgical mask on the patient. contacts/carriers-low risk of getting sick: . sampling and antibacterial treatment are offered to all contacts that may have a common source of infection with the index patients (travel company, co-passenger). person-to-person transmission is unlikely. . vaccine may, in addition to antibacterial treatment, be applicable to people with a common source of infection with the index patients-when risk of large outbreaks (note that vaccination should be discussed due to some serious adverse reactions). . while waiting for result of the sampling, close contacts live at home with contact isolation restrictions. . in case of detected anthrax in contact (incubation phase for disease - days), the person is isolated and treated, and vaccination may be assessed for close contacts. this is an endemic problem among wild animals in most countries. person-toperson transmission is unlikely. it is almost never reported more than one case at a time, infected by animal bites or licking but occasionally without known exposure. close contacts/exposed persons are registered. • registering: all exposed persons are registered (name, address, telephone number) and followed up. • ambulance staff and other personnel use the contact and airborne regime when picking up and transporting a patient. use respiratory protection and ppr; put a surgical mask on the patient. contacts/exposed-low chance of getting sick: . close contacts/exposed persons are assessed for vaccine and rabies immunoglobulin. . followed up at the infection outpatient clinic. patients and contacts are treated just like at vhf. patients and contacts are treated mainly like vhf. see also sars and bird flu. new infectious diseases and agents still appear, for example, sars, avian viruses (h n ), htlv and other retroviruses, hpv, sindbis virus, parvovirus, bocavirus, coronavirus, etc., or bacteria like legionella and borrelia, or agents with virulence changes, like group a streptococci, meningococci, etc. • biological terrorism made anthrax, plague, botulism, coxiella, brucella, vhf, poxviruses and a number of other unusual agents more appropriate as biological weapons . • bacteria sensitive to common antibacterial agents will probably not be a major problem. • viruses with no vaccine or treatment against low infection dose and high capacity to survive will be a major problem if associated with incurable disease, disability or death. • it is probable that this will be a problem first in countries with low hygiene standards/high population density. if the infectious agent is unknown, transmission ways are unknown and the situation is uncertain or uncontrollable, this practical measure may be followed: patient and contacts: strict isolation-negative air pressure isolation . serious illness: isolation of index case and all contacts . less severe disease: isolation of index case and close contacts • registering: all exposed persons are registered (name, address, telephone number) and followed up. • ambulance personnel use contact and airborne regime for patient pickup and transport. use respiratory protection (p mask), visor and cap in addition to gloves and gown/overall/shoe covers/dedicated shoes, and put a surgical mask on the patient. • botulism is caused by a bacteria-produced toxin (clostridium botulinum) that causes paresis and is common in soil as spores. the disease can be associated with toxin formation in contaminated and poorly canned foods, shrimp fish, bacon, etc. under anaerobic conditions and randomly affects both healthy people and vulnerable groups, such as infants who have had honey infected with the bacterium, which has happened repeatedly [ ] . the toxin is the most dangerous we know and is on the list of bioterrorism. • brucella bacteria (zoonosis) are particularly related to laboratory outbreaks but are easily transferable outside the laboratory and are considered highly infectious. [ ] • francisella tularensis (zoonosis) is defined as a category a bioterrorism agent, highly infectious and increasing in the society, has low infection dose ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) bacteria) and can be inhaled or infected via food and water. occasionally, such patients are detected in hospitals, even in the operating department [ ] . • mers-middle east respiratory syndrome-newly discovered coronavirus zoonosis (dromedaries, bats, etc.) and acts as sars, also with a tendency for nosocomial spread in hospitals. in there were cases, of which % died [ ] . • polio-like illnesses, especially among children, were discovered in august in different states in the united states. probably caused by enterovirus d [ ] . • hiv-aggressive variants (crf ) have been detected in cuba in and earlier in africa, with a faster course from infection to aids development [ ] . • prion disease-new shy-drager syndrome is rediscovered in ; multiplesystem atrophy (msa) [ ] . • ricin is a plant-derived toxin that is still used for bioterrorism in letters to, among others, president obama [ ]. • fungi and mould: different types that are especially related to floods, water damage, etc. and to pollution of medical products [ ] [ ] [ ] [ ] . in norway, a recent overview of candida in blood cultures showed a stable state for the past years [ ] . • zika virus: newly discovered flavivirus with mild to serious symptoms and teratogenic effect [ ] . ministry of labour and administration. regulations on protection against exposure to biological factors (bacteria, viruses, fungi and more) in the workplace guidelines for environmental infection control in health-care facilities disinfection, sterilization, and control of hospital waste serious, common contagious scenarios. in: handbook in hygiene and infection control for hospitals. olso: ullevål university hospital other serious viral infections-zoonoses. in: handbook in hygiene and infection control for hospitals. part fagbokforlaget lassa, and other haemorrhagic viruses. in: handbook in hygiene and infection control for hospitals. part . fagbokforlaget interim-infection prevention and control guidance for care of patients with suspected or confirmed filovirus haemorrhagic fever in health-care settings, with focus on ebola infection prevention and control recommendations for hospitalized patients with known or suspected ebola haemorrhagic fever in us hospitals department of health. management of hazard group viral haemorrhagic fevers and similar human infectious diseases of high consequences ebola guideline-from the norwegian institute of public health / international infection control guidelines for ebola. hospital healthcare europe. facilities management transmission or ebola virus from pigs to nonhuman primates violent expiratory events: on coughing and sneezing guidance on personal protective equipment to be used by healthcare workers during management of patients with ebola virus disease in us hospitals, including procedures for putting on (donning) and removal multi-resistant infections in repatriated patients after natural disaster: lessons learned from the tsunami for hospital infection control in: handbook in hygiene and infection control for hospitals. part . fagbokforlaget tonsillitis in europe's outskirt-when the diphtheria toxin came to the county of romsdal blood is a very special juice"-introduction of serum therapy for diphtheria in norway preparedness at ullevål university hospital in connection with biological weapons anthrax and emergency routines at ullevål university hospital the threat of biological attack: why concern now? haemorrhagic fever viruses as biological weapons: medical and public health management bioterrorism-related inhalation anthrax: the first cases reported in the united states anthrax as a biological weapon, : updated recommendations for management anthrax of the gastrointestinal tract nosocomial spread of bacillus anthracis bacteria and disease. epidemiology, infections and infection protection. oslo: gyldendal akademisk, gyldendal norsk forlag as can anthrax infect from patient to patient? bacillus anthracis aerosolization associated with a contaminated mail sorting machine secondary aerosolization of viable bacillus anthracis spores in a contaminated us senate office risk assessments of anthrax threat letters. defense research establishment suffield. dres technical report - anthrax inhalation and lethal human infection bioterrorism: crime and opportunity bioterrorism preparedness and response in european public health institutes deliberate releases of biological agents. initial lessons for europe from events in the united states the use of smallpox virus as a biological weapon: the vaccination situation in france are there «new » and «old » ways to track infectious diseases hazards and outbreaks ? infectious disease disaster: bioterrorism, emerging infections, and pandemics. apic text of infection control and epidemiology bioterrorism readiness plan: a template for healthcare facilities an infant with acute paresis hospital-associated transmission of brucella melitensis outside the laboratory potential risk of aerosol-borne francisella tularensis transmission in the operating room acute neurological disease of unknown etiology in children-colorado hiv-cuba: new aggressive variant prion disease updated: novel prion disease-shy-drager syndrome mould-preventing strategies and possible health effects in the aftermath of hurricanes and major floods fungi-human pathogenic. in: handbook in hygiene and infection control for hospitals. part . fagbokforlaget mucormycosis-usa: fatal, premature infant, probiotic supplement, recall, alert twenty-two years of candidaemia surveillance: results from a norwegian national study key: cord- -awbubjy authors: acevedo, edwin; mazzei, michael; zhao, huaqing; lu, xiaoning; edwards, michael a. title: outcomes in conventional laparoscopic versus robotic-assisted revisional bariatric surgery: a retrospective, case–controlled study of the mbsaqip database date: - - journal: surg endosc doi: . /s - - - sha: doc_id: cord_uid: awbubjy introduction: revisional bariatric surgery is being increasingly performed and is associated with higher operative risks. optimal techniques to minimize complications remain controversial. here, we report a retrospective review of the metabolic and bariatric surgery accreditation and quality improvement program (mbsaqip) participant user files (puf) database, comparing outcomes between revision rbs and lbs. methods: the and mbsaqip puf database was retrospectively reviewed. revision cases were identified using the revision/conversion flag. selected cases were further stratified by surgical approach. subgroup analysis of sleeve gastrectomy and gastric bypass cases was performed. case–controlled matching ( : ) was performed of the rbs and lbs cohorts, including gastric bypass and sleeve gastrectomy cohorts separately. cases and controls were match by demographics, asa classification, and preoperative comorbidities. results: , revision cases were identified ( . % lbs, . % rbs). . % were female and % white. mean age and bmi were years and . kg/m( ). matched rbs and lbs cases were identified. rbs was associated with longer operative duration (p < . ), los (p = . ) and a higher rate of icu admissions ( . % vs . %, p = . ). aggregate bleeding and leak rates were higher in the rbs cohort. in both gastric bypass and sleeve gastrectomy cohorts, the robotic-assisted surgery remain associated with longer operative duration (p < . ). in gastric bypass, rates of aggregate leak and bleeding were higher with robotic surgery, while transfusion was higher with laparoscopy. for sleeve gastrectomy cases, reoperation, readmission, intervention, sepsis, organ space ssi, and transfusion were higher with robotic surgery. conclusion: in this matched cohort analysis of revision bariatric surgery, both approaches were overall safe. rbs was associated with longer operative duration and higher rates of some complications. complications were higher in the robotic sleeve cohort. robotic is likely less cost-effective with no clear patient safety benefit, particularly for sleeve gastrectomy cases. all of which contribute to increased healthcare-related costs. this high rate of weight recidivism following bariatric surgery is also consistent with the reported twofold increase in revisional bariatric procedures in recent literature [ , ] . the optimal treatment modality for weight recidivism post-bariatric surgery remains controversial. most practitioners agree that early recognition and intervention for weight recidivism post-bariatric surgery is important in containing obesity-related healthcare costs in this cohort of patients [ , , ] ; however, standardized practice guidelines for managing these patients are lacking. the spectrum of treatment recommendation includes behavior modification [ , ] , medication [ ] , endoscopic bariatric therapy [ ] , and revisional bariatric surgery [ - , , , - ] , with varying results. revisional bariatric surgery is often recommended for those with inadequate weight loss or significant weight regain, as well as persistence comorbid conditions following primary bariatric surgery [ , , ] . other reasons for revisional or conversional bariatric surgery vary and are related to physiologic and anatomic complications associated with the index surgical procedure [ , , , ] . outcomes following revision or conversion bariatric surgery are not similar to outcomes following primary bariatric surgery [ , , , ] . while some small cohorts and meta-analyses have reported no difference in complication rates between primary and revisional bariatric cases [ , , , ] , others have reported that weight loss is less and complication rates are higher in revisional bariatric surgery [ , , ] . the optimal surgical approach also remains a point of controversy. as technical approaches to surgical weight loss continue to evolve, the robotic platform continues to be increasing used; however, the role, safety, and cost-effectiveness of this platform remain unclear for both primary and revisional bariatric surgery. there are limited published studies on revisional or conversional robotic bariatric surgery [ , , ] . most are small retrospective cohorts, limiting our understanding of outcomes following robotic revisional bariatric surgery. we present the largest retrospective cohort analysis of revisional bariatric surgery comparing conventional laparoscopic and the roboticassisted techniques. we performed a retrospective analysis of the and metabolic and bariatric surgery accreditation and quality improvement program participant use file (mbsaqip puf) database for this study. we compared outcomes in revision or conversion metabolic and bariatric surgery performed with conventional laparoscopic or robotic-assisted techniques. the mbsaqip accredits bariatric surgical facilities in the united states, who are then required to report bariatric surgical outcomes to the mbsaqip puf. the mbsa-qip puf serves as a file registry that contains prospective, risk-adjusted data based on preoperative, intraoperative, and post-operative variables specific to bariatric surgery. data is collected by trained metabolic and bariatric surgery (mbs) clinical reviewers at each bariatric center and audited similar by the national surgical quality improvement program (nsqip). de-identified data is reported on patient characteristics, operative details, and intraoperative and perioperative outcomes. as our study utilized deidentified data from a national clinical database, neither institutional review board (irb) approval nor consent was required. there are , bariatric cases in the combined and mbsaqip puf. we first excluded cases without the revision/conversion flag in the database. this excluded all primary mbs procedures. we then excluded cases by surgical approach, including only revision cases performed by either conventional laparoscopic or robotic-assisted techniques. from this cohort, we identified patients who had a revision/conversion bariatric operation using current procedure terminology (cpt) codes for laparoscopic gastric proximal gastric bypass ( , ), laparoscopic distal gastric bypass ( , ), laparoscopic sleeve gastrectomy ( , ), laparoscopic gastric band ( , ), and laparoscopic duodenal switch ( , ). our case selection algorithm resulted in exclusion of primary bariatric cases, all cases not performed by conventional laparoscopic or robotic-assisted techniques, revision cases that were not a revision/conversion to another bariatric procedure, as well as cases in our final study cohort with missing data points. in order to control for possible confounding variables, we performed : case-control matching of the entire cohort. cases and controls were matched by patient demographics (age, gender, race/ethnicity, and body mass index (bmi) closest to surgery), asa classification and preoperative comorbid conditions (history of myocardial infarction (mi), hypertension requiring medication, hyperlipidemia, renal insufficiency, need for dialysis, venous thrombosis requiring therapy, history of pulmonary embolism (pe), ambulation status, functional dependence, diabetes mellitus, steroid and immunosuppressant use, smoking status within year of surgery, obstructive sleep apnea (osa), chronic obstructive pulmonary disease (copd), and oxygen dependence) ( table ) . procedure-specific subgroup analyses were also performed, comparing case-control matched roboticassisted versus conventional laparoscopic sleeve gastrectomy (sg) cases and robotic-assisted versus conventional laparoscopic roux-en-y gastric bypass (rnygb) cases. thirty primary outcomes variables were assessed, including operative time, hospital length of stay, conversion rate, discharge status, -day icu admission, reoperation, readmission, intervention, or mortality, death likely related to bariatric surgery, drain present at -days, renal failure, progressive renal insufficiency, cardiopulmonary resuscitation (cpr), coma > h, stroke, myocardial infarction, venous thrombosis requiring therapy, pulmonary emboli, transfusion, pneumonia, on ventilator > h, unplanned intubation, peripheral nerve injury, urinary tract infection (uti), sepsis, septic shock, superficial soft tissue infection (ssi), deep ssi, and organ space ssi. seven aggregate complications were also assessed, including aggregate leak-as previously described by berger et al. [ ] , bleeding, renal failure, cardiovascular and pulmonary complications, venous thromboembolic events and surgical site infection. aggregate methodology is reported in table . primary and aggregate outcomes were analyzed for the entire unmatched cohort and case-control matched cohorts. pearson's chi squared test for categorical variables (i.e., gender, race, asa class, and preoperative comorbidities) and an independent two sample t test and mann-whitney test for normally and non-normally distributed continuous variables perioperative and aggregate outcomes for the entire unmatched conventional laparoscopic and robotic-assisted cohorts are described in table . operative duration (min) ( . ± . vs. . ± . , p < . ) and hospital length of stay (days) ( . ± . vs. . ± . , p = . ) were significantly longer in the robotic-assisted cohort. -day reoperation ( . % vs. . %, p = . ), readmission ( . % vs. . %, p = . ), and intervention ( . % vs. . %, p = . ) were also significantly higher in the robotic-assisted cohort. perioperative complications were similar between the two cohorts, except for a higher rate of intraoperative or post-operative transfusion ( . % vs. . %, p = . ) in the conventional laparoscopic cohort. aggregate complications were also similar between the cohorts, except for a significantly higher rate of leak ( . % vs. . %, p = . ) in the robotic-assisted cohort. aggregate bleeding trended toward being significantly higher in the conventional laparoscopic cohort (p = . ). there was no mortality difference between the two cohorts ( . % vs. . %, p = . ). perioperative and aggregate outcomes following matched cohort analysis of all included bariatric procedures are described in table . after : case-control matching for patient demographics and preoperative comorbidities (table ) , cases and controls were identified. operative duration (min) ( . ± . vs. . ± . , p < . ) and hospital length of stay (days) ( . ± . vs. . ± . , p = . ) remained significantly longer in the robotic-assisted cohort. -day outcomes were similar between the two cohorts, except for a higher rate of unplanned icu admission in the robotic-assisted cohort ( . % vs. . %, p = . ). all perioperative complications were also similar between the two cohorts, including intraoperative or post-operative transfusion with -h, which was significantly higher for the conventional laparoscopic cohort in the unmatched cohort analysis (p = . vs. . ). aggregate bleeding ( . % vs. . %, p = . ) and leak ( . % vs. . %, p = . ) remained higher in the roboticassisted cohort, trending toward statistical significance. all other aggregate complications were similar between the two cohorts ( table ). subgroup analyses of sg and rnygb cohorts were then performed. perioperative and aggregate outcomes for the unmatched revision sg and rnygb cohorts are detailed in table . in the unmatched rnygb cohort (n = ), . % were performed robotically. in comparison with conventional laparoscopic cases, robotic-assisted cases were associated with significantly longer operative duration ( . ± . min vs. . ± . min, p < . ) and higher rates of conversion ( . % vs. . %, p = . ) and aggregate bleeding ( . % vs. . %, p = . ). in contrast, the conventional laparoscopic cohort had significantly higher rates of transfusion requirement ( . % vs. . %, p = . ), aggregate leak ( . % vs. . %, p = . ), and pulmonary complications ( . % vs. . %, p = . ). mortality, morbidity, -day adverse outcomes, and other complications were not significantly different in the unmatched revision robotic and laparoscopic bypass cohorts. in the unmatched sleeve gastrectomy cohort (n = , ), . % progressive renal insufficiency peri-operative and aggregate outcomes for procedurespecific matched cohorts are detailed in table . following : case-control matching, revisional gastric bypass ( robotic-assisted and conventional laparoscopic) and revisional sleeve gastrectomy ( robotic-assisted and conventional laparoscopic) cases were compared. in the matched revisional gastric bypass cohort, outcomes were preserved and similar to the unmatched analysis. roboticassisted rnygb was associated with longer operative duration ( . ± . vs. . ± . , p < . ) and conventional laparoscopy was associated with fivefold higher rate of transfusion requirement ( . % vs. . %, p = . ). all other outcome measures were similar between the two surgical approaches for gastric bypass cases. in matched sleeve gastrectomy cohort analysis, robotic-assisted surgery remains associated with significantly longer operative duration ( . ± . min vs. . ± . min, p < . ) and a higher rate of post-operative sepsis ( . % vs. %, p = . ). however, post-operative length of stay and outcome measures that were significantly different in unmatched analysis, were similar among the two surgical approaches in matched sleeve gastrectomy cases. as the number of total bariatric procedure performed annually continues to increase, it is expected that a concomitant increase will be seen in the total number of complications, cases with weight recidivism, and other post-operative morbidities that may require the need for revisional/conversional bariatric procedures [ , , , ] . this is a challenging cohort. in a recent systematic review of re-operative bariatric surgery, mortality was estimated to be %, which is significantly higher than the . - . % reported for primary bariatric procedures [ ] . in a case-matched analysis comparing primary and revisional laparoscopic roux-en-y gastric bypass (lrygb), the revisional cohort was found to have significantly longer length of stay ( . vs. . , p = . ), conversion to laparotomy ( . % vs. %, p = . ), and -day morbidity ( % vs. . %, p = . ) [ ] . a meta-analysis comparing bariatric reoperations after adjustable gastric banding (abg) found that conversion to sleeve gastrectomy had the lowest long-term complication rates ( . %), while conversion to rygb had the highest short-term and long-term complication rate at . % and . %, respectively [ ] . the current literature suggests that revisional bariatric surgery is associated with higher rates of mortality and morbidity and outcomes may be related to the primary and re-operative operation performed. however, there have been limited studies evaluating outcomes in revisional bariatric surgery comparing conventional laparoscopic-and robotic-assisted surgical approaches [ , , ] . this study represents the largest case-controlled retrospective review of the mbsaqip puf database comparing perioperative outcomes in laparoscopic-and robotic-assisted revisional/ conversional bariatric surgery. our case-control matched analysis of revisional bariatric cases revealed longer operative duration and hospital length of stay, and higher rates of icu admission, aggregate leak and bleeding complications in the robotic-assisted bariatric surgery compared to conventional laparoscopy. this is in contrast with other studies. buchs et al. performed a comparison of consecutive revisional bariatric procedures performed laparoscopically, open, or robotic-assisted [ ] . they found that while operative duration was significantly longer in the robotic-assisted cohort, there were less complications and a shorter hospital stay when the robotic platform was used. in another small series (n = ) evaluating robotic-assisted revisional roux-en-y gastric bypass, the authors concluded that their complications and perioperative outcomes were similar to the published results on conventional laparoscopic revisional bariatric surgery [ ] . there remained some similarities and differences between the findings in our study and prior studies. in overall and procedure-specific match analysis, robotic-assisted surgery was associated with significantly longer operative duration, which is consistent with the published literature. while outcomes between robotic-assisted and conventional laparoscopic revisional gastric bypass were statistically similar, robotic-assisted surgery was associated with higher rates of aggregate bleeding (fivefold higher) and aggregate leak ( . -fold higher). in our matched analysis of robotic and laparoscopic sleeve gastrectomy, most outcomes were statistically similar, as with the gastric bypass cohorts. however, robotic-assisted revisional sleeve gastrectomy was associated with higher rates of conversion (twofold higher), -day reoperation ( . -fold higher), -day readmission ( . -fold higher), -day intervention ( . -fold higher), anticoagulation for presumed for confirmed vte (twofold higher), transfusion requirement (fourfold higher), organ space ssi (sixfold higher), aggregate leak ( . -fold higher), aggregate venous thromboembolism ( . -fold higher), and aggregate ssi ( . -fold higher). much of the higher complication rates observed in the robotic-assisted cohorts were not statistically different. this may be a reflection of the smaller sample size compared after our procedure-specific case-control matching. while unclear, this suggests that the robotic-assisted platform is associated with higher rates of adverse outcomes in sleeve gastrectomy revisional cases compared to gastric bypass revisional cases. the reasons for our findings remain unclear. our study represents the largest case-controlled matched study comparing these two surgical platforms for revision/ conversion bariatric surgery. we show that while most peri-operative outcomes are similar after controlling for confounders, operative duration remains significantly higher in both robotic-assisted gastric bypass and sleeve gastrectomy. while the robotic platform was overall safe for both revisional gastric bypass and sleeve gastrectomy cases, we also showed that while most complications were statistically similar in matched gastric bypass (robotic vs. laparoscopic) and matched sleeve gastrectomy (robotic vs. laparoscopic) cohorts, robotic revisional metabolic and bariatric surgery was associated with non-significantly higher rates of some complications. these complications rates were overall higher in the sleeve gastrectomy cohort compared to the gastric bypass cohort. giving these findings, the robotic platform seems overall safe, but is likely less cost-effective, and value added for patient safety remains unclear for revisional metabolic and bariatric surgical procedures, and particularly for revisional sleeve gastrectomy cases. our study has a number of limitations that should be highlighted. first, this study is limited to peri-operative outcomes only, so long-term outcomes cannot be assessed. second, the database does not provide the details about the initial bariatric operation performed for cases designated as revision/conversion. as the primary bariatric operation may impact the level of difficulty of a revision/conversion bariatric procedure, the lack of detail about the initial bariatric operation performed is a possible confounding variable our study could not account for. third, the dataset does not provide details about anastomotic techniques and surgeon experience, which are variables previously shown to impact outcomes following metabolic and bariatric surgery [ , ] . the level of surgeon experience is not accounted for in this database, including where surgeons are on the laparoscopic or robotic learning curve. it is also unclear if anastomotic techniques varied by surgical approach. for instance, were more robotic anastomosis hand-sewn and laparoscopic stapled? it is also unclear what primary bariatric procedures were converted to what revisional procedures. were more difficult conversion cases performed using the robotic platform versus conventional laparoscopy. these nuances could not be illicit from the mbsaqip database, and may be confounding variables not accounted for in our study. lastly, this is a retrospective analysis and is therefore susceptible to biases associated with retrospective analyses of clinical databases. taking into consideration the above outlined study limitations, the findings of this case-control matched analysis comparing these two surgical approaches for revision/conversion metabolic and bariatric surgery show that using the robotic platform is overall safe, but is associated with longer operative times and a higher rate of some perioperative outcome measures. it has been shown that prolonged operative duration is associated with increased complications. in a recent meta-analysis, the authors found that the likelihood of complications approximately doubles with operative time thresholds exceeding [ ] . moreover, perioperative complications [ ] , hospital length of stay [ , ] , -day adverse outcomes, such as reoperation, readmission, and intervention [ ] have all been reported to be associated with increased costs. therefore, outcome measures that were higher in the robotic-assisted gastric bypass (operative duration, aggregate bleeding and aggregate leak) and robotic-assisted sleeve gastrectomy cohorts (operative duration and rates of conversion, -day reoperation, -day readmission, -day intervention, anticoagulation for presumed or confirmed vte, transfusion requirement, organ space ssi, aggregate leak, aggregate venous thromboembolism and aggregate ssi), can serve as proxies for higher cost associated with robotic-assisted metabolic and bariatric surgery. while revisional cases have been reported to be a safe and effective way to treat patients who have significant weight recidivism and relapse of comorbid conditions post-bariatric surgery [ , , , ] , there are no clear patient benefits to utilizing robotic assistance for these cases. because of the large initial investment, consumables, annual maintenance, and other reusable equipment also associated with the robotic platforms [ , ] , health systems must be cognizant of the fact that some peri-operative outcomes may favor the use of conventional laparoscopy over the roboticassisted approach for revisional bariatric procedures. these differences can contribute to higher healthcare expenditures with little effect on patient safety outcomes when the robotic platform is utilized in this patient cohort. conventional laparoscopic and robotic-assisted revision/ conversion metabolic and bariatric procedures are both safe and effective surgical approaches. however, we found that robotic-assisted revision/conversion gastric bypass and sleeve gastrectomy is associated with longer operative times. robotic-assisted and conventional laparoscopic gastric bypass were similar in outcomes, except a non-significantly higher rate of aggregate leak and bleeding. outcomes between robotic-assisted and conventional laparoscopic sleeve gastrectomy were also statistically similar; however, the robotic-assisted cohort had numerous -day adverse outcomes, complications, and aggregate complications that were higher. these findings suggest less cost-effectiveness and no clear patient safety benefit with use of the robotic platform, particularly for revisional sleeve gastrectomy cases. larger revisional cohorts are needed to validate our finding, given the limited sample size included in our analysis following our procedure-specific matching. reoperations after bariatric surgery in years of follow-up of the swedish obese subjects study re-operative bariatric surgery: a systematic review of the reasons for surgery, medical and weight loss outcomes, relevant behavioral factors case-matched analysis comparing outcomes of revisional versus primary laparoscopic roux-en-y gastric bypass re-operations after secondary bariatric surgery: a systematic review outcomes of robotassisted roux-en-y gastric bypass as a reoperative bariatric procedure revisional bariatric surgery for unsuccessful weight loss and complications systematic review on reoperative bariatric surgery american society for metabolic and bariatric surgery revision task force predictors of long-term remission and relapse of type diabetes mellitus following gastric bypass in severely obese patients revisional bariatric surgery weight recidivism post-bariatric surgery: a systematic review productivity loss due to overweight and obesity: a systematic review of indirect costs trends in bariatric surgery: procedure selection, revisional surgeries, and readmissions laparoscopic sleeve gastrectomy leads the us utilization of bariatric surgery at academic medical centers should bariatric revisional surgery be avoided secondary to increased morbidity and mortality? emerging technology and procedures committee ( ) endoluminal revision of gastric bypass for weight regain-a systematic review robotic revisional bariatric surgery: a comparative study with laparoscopic and open surgery robotically assisted revision of bariatric surgeries is safe and effective to achieve further weight loss roux-en-y gastric bypass after previous unsuccessful gastric restrictive surgery conversion of failed laparoscopic gastric banding to gastric bypass as safe and effective as primary gastric bypass in morbidly obese patients laparoscopic revisional surgery after roux-en-y gastric bypass and sleeve gastrectomy the impact of different surgical techniques on outcomes in laparoscopic sleeve gastrectomies: the first report from the metabolic and bariatric surgery accreditation and quality improvement program (mbsaqip) estimate of bariatric surgery numbers the results of robotic versus laparoscopic gastric bypass procedures: a single high volume centre experience robotic roux-en-y gastric bypass, is it safer than laparoscopic bypass? prolonged operative duration is associated with complications: a systematic review and meta-analysis predictors of high cost after bariatric surgery: a single institution review cost of bariatric surgery and factors associated with increased cost: an analysis of national inpatient sample cost of bariatric surgery and factors associated with increased cost: an analysis of national inpatient sample healthcare utilization and outcomes after bariatric surgery robot-assisted surgery compared with open surgery and laparoscopic surgery: clinical effectiveness and economic analyses. canadian agency for drugs and technologies in health robotic versus laparoscopic roux-en-y gastric bypass in obese adults ages to years: a systematic review and economic analysis disclosures drs. edwin acevedo, jr., michael mazzei, huaqing zhao, michael a. edwards, and mr. xiaoning lu have no conflicts of interests or financial ties to disclose. key: cord- -f rnhc authors: zhang, xia; yuan, jing; zhan, yong; wu, jingyi; liu, biaohu; zhang, peng; yu, tao; wang, zhen; jiang, xiaogan; lu, weihua title: evaluation of diaphragm ultrasound in predicting extubation outcome in mechanically ventilated patients with copd date: - - journal: ir j med sci doi: . /s - - - sha: doc_id: cord_uid: f rnhc background: to explore the value of the right hemi-diaphragmatic excursion (de) and its variation in predicting extubation outcome in mechanically ventilated patients with copd. methods: all included patients with copd received mechanical ventilation (mv) and were ready to wean from mv. after patients passed the min spontaneous breathing trail (sbt), extubation was considered to be feasible, and the right de measured by ultrasound at min, min, and min of sbt were named as de( ), de( ), and de( ), respectively. results: twenty-five patients succeeded extubation; patients failed. the area under receiver operator characteristic curve (auc(roc)) of de( ) and Δde( − ) (the variation between and min) were . and . ; a cutoff value of de( ) > . cm and Δde( − ) > . cm were associated with a successful extubation with a sensitivity of % and %, a specificity of % and . %, respectively. the predictive probability equation of the de( ) plus ∆de( − ) was p = /[ + e(−(− . + . ×∆de)( − )(+ . ×de)( )())], a cutoff value of p > . was associated with a successful extubation with the auc(roc) of . , a sensitivity of %, and a specificity of . %. conclusion: the combination of de( ) and ∆de( − ) could improve the predictive value and could be used as the predictor of extubation outcome in mechanically ventilated patients with copd. mechanical ventilation (mv) is an important and effective treatment for patients with respiratory failure. more than % patients in intensive care units (icu) require mv as a part of their process of care [ ] . clinicians in icu are often challenged with the decisions regarding when and how to wean critically ill patients from mv. weaning from mv is initiated as early as possible to avoid the complications associated with prolonged ventilation, such as ventilator-induced diaphragmatic dysfunction (vidd). however, clinicians have to balance the benefits of early extubation with the risk of failure extubation and reintubation, while reintubation increases risk of hospital acquired pneumonia by times, death by - times [ ]. copd is characterized by incomplete reversible airflow limitation; the respiratory muscle has high workload for a long time. the diaphragm, as the major respiratory muscle, is responsible for approximately - % of the workload [ ] . as a result, the diaphragmatic function plays a critical role in weaning from mv. consequently, the thinning diaphragm, descending movement, and impaired contractility easily develop vidd in patients with copd due to systemic inflammatory response, oxidative stress, malnutrition, and chronic hypoxia [ ] . in fact, the previous studies have demonstrated a majority of mechanically ventilated patients spent a staggering % of the time devoted to weaning from mv in the icu, % for copd [ ] [ ] [ ] . in general, the evaluation of diaphragmatic function was common in mechanically ventilated patients in the icu. several traditional variables for evaluating diaphragmatic function have been used in clinical practice, such as maximum inspiratory, expiratory pressure, and maximum trans-diaphragmatic pressure. but these variables presented some limitations that were invasive and dependent greatly on patient's cooperation, which make them to be put into difficult practice in the icu. other methods of assessing diaphragmatic function showed also disadvantages, like radiolesion, movement artifacts, and non-continuity, as in the case of chest xrays, or were difficult to be carried out at the bedside, such as computed tomography and magnetic resonance imaging [ ] . ultrasonography has raised great interest as a noninvasive, easily available, and high reproducible method in evaluation of diaphragmatic function. along with the growing use of ultrasonography in the icu in recent years, it could be used to observe the morphology of the diaphragm, thus to evaluate diaphragmatic function and spontaneous breathing ability [ ] [ ] [ ] . the previous studies have showed that the de measured by ultrasound could be used to assess respiratory effort, and while diaphragmatic thickening fraction was shown to be correlated strongly with diaphragm strength and ventilator support level, the right de assessed by ultrasound was more reliable, more feasible, and more repeatable [ ] [ ] [ ] [ ] [ ] [ ] [ ] . therefore, the aim of our study was to explore the value of the right de and its variation assessed by ultrasound in predicting extubation outcome in mechanically ventilated patients with copd. this prospective observation study was performed from january to august in the icu of the first affiliated hospital of wannan medical college. all patients with acute respiratory failure due to copd and necessitating mv were included in the study. the study protocol was approved by the ethics committee of the first affiliated hospital of wannan medical college, and informed consent was obtained from each patient's family. inclusion criteria: ( ) global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease and practical guidelines for mv worked out by chinese society of critical care medicine were used as the diagnostic criteria [ ] [ ] . patients were selected by inclusion criteria and exclusion criteria. clinicians took charge of the patient's treatment including spontaneous breathing trail (sbt) and extubation. after patients passed the -min sbt, extubation was considered to be feasible. during the sbt, diaphragmatic movement was examined and recorded by the ultrasound physician (all images of ultrasound were saved, and measurements were repeated again.). in the whole process of sbt, infusion speed of drugs and other treatment remained unchanged. the patient was monitored by the ecg monitor to record the changes of heart rate (hr), mean arterial pressure (map), respiration rate (rr), and pulse oxygen saturation (s po ), and to observe the change of mental status. the patients were divided into successful extubation group and failure extubation group according to extubation outcome. general information and various weaning parameters of patients were collected prospectively, such as fraction of inspired oxygen (f io ), partial pressure of arterial oxygen (p ao ), and partial pressure of arterial carbon dioxide (p aco ). the efficacy (sensitivity, specificity, etc.) of de and its changes assessed by ultrasound in predicting extubation outcome were calculated and compared. attending clinicians were blind to the ultrasound results. the criteria for successful extubation: successful extubation was defined as the ability to maintain spontaneous breathing for at least h, without any ventilatory support. in contrast, failure extubation was defined as the reconnection to ventilator (invasive or noninvasive) within h due to respiratory failure or other reasons [ , ] . extubation and the reconnection to ventilator were all based on sbt results and physicians' decision. pressure support ventilation with a support pressure of - cmh o and a peep level of ≤ cmh o were employed for min in the process of sbt. patients were assessed daily until the factors which developed respiratory failure were controlled or reversed. the criteria for sbt initiation were as follows: ( ) clear consciousness (aroused, glasgow coma score ≥ ); ( ) without dyspnea, breaths/min (bpm) ≤ rr ≤ bpm; ( ) f io ≤ . , peep ≤ cmh o, p ao /f io ≥ mmhg, ph > . , and p aco reached the level of remission; ( ) hemodynamic stability, and without myocardial ischemia, absence of severe hypotension, in the absence of any vasopressors or small dose (dopamine < ug/(kg min) or norepinephrine < . ug/(kg min); ( ) no apparent acid; ( ) hemoglobin > g/l; ( ) subjective clinical evaluation: clinicians considered that patients have sufficient airway protection and could be weaned [ ]. the criteria for sbt termination were as follows: ( ) rr > or < bpm for min or involving auxiliary respiratory muscles; ( ) s po < %, p ao /f io ≤ mmhg, ph ≤ . ; ( ) hr > bpm or continuous polycardia, faster or slower than basic heart rate by %; ( ) systolic blood pressure > or < mmhg; ( ) change in mental status (such as somnolence, coma); ( ) new myocardial ischemia. when one of the abovementioned criteria was reached, patients were reconnected and evaluated every day until successful sbt [ ]. all patients were evaluated in semi-recumbent position (with elevating the head of the bed to °). first, the diaphragm was visualized by placing the - mhz convex probe to the right subcostal area between the anterior axillary and the midclavicular lines, the probe was directed to the head, with the right lobe of liver as a window, and the optimal images of diaphragm were captured by adjusting the probe direction. the right hemi-diaphragm was observed by twodimensional ultrasonography until its movement was stable and its image was clear. then, m-mode was used to record the movement of the diaphragm during tidal breathing when the sampling line and diaphragm were as vertical as possible (not < °). after the first data was taken, with the position markers fixed on the skin surface, the average of the three measurements was recorded. general information such as patient name, gender, age, acute physical and chronic health score ii (apache ii), and mv time before admission were all recorded. the data was measured from the first respiratory cycle at min after sbt. the de at min, min, and min of sbt was respectively named as de , de , and de . the variation of right de between each time point was named as Δde − and Δde − . the p ao /f io (p/f) and p aco were observed and recorded at min and min after sbt, besides hr, tidal volume (vt), rr, map, rapid shallow breathing index (rsbi), de of each time point after sbt. all statistical analyses were performed using spss . . data were presented as the mean ± standard deviation (χ(−) ± s) or median and interquartile range when appropriate. fisher exact test was used to compare categorical data (gender). student's t test was used to compare differences of continuous variables between the two groups. mann-whitney u tests were used to compare differences of mv time between the two groups. receiver operator characteristic curves (roc curves) analysis and binary logistic regression analysis were used to evaluate the predictive value of diaphragmatic parameters. a p value < . was considered statistically significant. during the study period, patients were excluded from the study because of poor ultrasound measurement images, while patients were included in this study. of the patients, patients ( . %) failed extubation after sbt, as shown in fig. . there was no statistical difference in age, gender, apache ii score, and mv time between the two groups (p > . ), as shown in table . at all time points of sbt ( , , and min), there was no significant difference in hr and map between the two groups (p > . ). the difference of rr, vt, rsbi, p aco , p/f, and de between the two groups was not significant at min time point (p > . ). however, at min and min time points, the rr and rsbi of patients in the successful extubation group were significantly lower, vt and de were significantly higher (p < . ). in addition, at min time point, the p/f of patients was significantly higher in the successful extubation group (p < . ), while the difference of p aco was not significant between the two groups (p > . ), as shown in table . as shown in table , Δde − between the two groups has no significant difference (p > . ). compared with the failure extubation group, Δde − and Δde − were significantly higher in the successful extubation group (p < . ). a roc curve analysis was used to assess the accuracy of diaphragmatic parameters in predicting extubation outcome. as shown in table , a cutoff value of rsbi less than . bpm/l was associated with a successful extubation with the auc roc of . , a sensitivity of %, a specificity of %, a positive predictive value (ppv) of . %, and a negative predictive value (npv) of . %. a cutoff value of de more than . cm was associated with a successful extubation with the auc roc of . , a sensitivity of %, a specificity of %, a ppv of . %, and a npv of %. similarly, the auc roc was . for de , and a de value of > . cm predicted successful extubation with a sensitivity of %, a specificity of %, a ppv of . %, and a npv of % (fig. ) . our study presented the variation of de between and minutes of sbt was named as Δde − ; a cutoff value of Δde − > . cm was associated with a successful extubation with the auc roc of . , a sensitivity of %, a specificity of . %, a ppv of . %, and a npv of . %. likewise, the auc roc , sensitivity, specificity, ppv, and npv of de − were . , %, %, %, and . %, respectively (fig. ) . the ], a cutoff value p > . was associated with a successful extubation with the auc roc of . , a sensitivity of %, a specificity of . %, a ppv of %, and a npv of . % (fig. ) . this study demonstrated that the de and Δde − could be used to predict extubation outcome in patients with copd. the combination of two indicators could improve the predictive value. the diaphragm is the major respiratory muscle; diaphragmatic dysfunction is responsible for difficult weaning from mv and prolonged mv which in turn may promote diaphragmatic dysfunction named as vidd [ ] [ ] [ ] [ ] . mv for - h can result in significant diaphragmatic atrophy or diaphragmatic dysfunction, as showed by animal studies [ , ] . therefore, diaphragmatic dysfunction can interact with mv, and it is time-dependent increase in diaphragmatic dysfunction in patients with the increase of mv time [ , ] . diaphragmatic dysfunction has a relatively higher incidence in patients with copd undergoing mv [ ] . consequently, the prompt and accurate evaluation of the diaphragmatic function is crucial to successful weaning from mv in patients with copd. sbt has been usually employed to predict extubation outcome. even if patients passed sbt, the rate of failure extubation could still reach approximately % [ , , ] . as a result, it will be very valuable to seek an easily available and accurate indicator to predict extubation outcome and improve survival rate of patients in the icu. along with the growing use of ultrasonography in the icu in recent years, both diaphragmatic thickening fraction and diaphragmatic excursion (de) could be used as predictors of weaning from mv [ , , ] . however, recent studies shown that de was closely correlated with the first second exhalation, lung volume, residual volume, minute ventilation, and maximum inspiratory volume [ , ] . moreover, de correlated well with trans-diaphragmatic pressure [ ] . de assessed by ultrasound was more accurate than diaphragmthickening fraction in predicting successful weaning from mv [ ] . accordingly, de assessed by ultrasound could evaluate well-diaphragmatic function. in addition, the right de was usually easier to be observed, while the descending lung, bowel, and gas on the left side often interfered with the evaluation of diaphragmatic function during inspiration. furthermore, descending diaphragm was more susceptible to interference from the surrounding intestine in patients with copd. previous studies suggested ultrasound measured successfully the right de of patients in patients with a % success rate, while the success rate of the left de was only % [ , ] . therefore, the right de and its variation were assessed by ultrasound in this study. the rate of failure extubation was % in our study, slightly higher than previous reports. some reasons maybe ( ) patients who were included all had copd and acute respiratory failure. they were prone to diaphragmatic dysfunction. ( ) the patients in this study were relatively older. the aged patients were susceptible to weaning due to descended thoracic compliance and weak respiratory muscle. as a result, the rate of successful extubation was negatively affected. de is a surrogate of diaphragmatic contractility. normal data of de is still lacking in patients with mv, and it has a great variation in different respiratory status among different studies [ , ] . in this study, the range of de measured by ultrasound was . - . cm; the de was significantly higher in the successful extubation than in the failure extubation at the different time of weaning process (p < . ). moreover, compared with de , de was more accurate (characterized as higher sensitivity and specificity, larger auc roc ). the key to the successful extubation is not only the respiratory muscle contractility but also the endurance of the respiratory muscle contraction. therefore, one measurement often does not sufficiently show the endurance of the respiratory muscle contraction. the variations among each time point for de may more sufficiently reflect the endurance of diaphragmatic contraction. our study indicated that compared with Δde − , the auc roc and sensitivity of Δde − were significantly larger and higher, but specificity was lower. overall, Δde − was considered to be more accurate in predicting extubation outcome in this study. accordingly, our study showed that the positive predictive value was . % when both indicators were positive, in contrast, the negative predictive value of %. when the de plus Δde − was used to predict extubation outcome was considered to be more accurate in predicting extubation outcome(the auc roc and specificity were significantly higher). rsbi reflected effort of all respiratory muscles. so, in presence of diaphragmatic fatigue, accessory muscles could preserve tidal volume for a short time of sbt but not a long time and thus it was prone to failure extubation despite successfully passing the sbt. the limitations of this study are as follows: firstly, cardiac function had not been fully taken into account, which may be responsible for failure extubation. secondly, the diaphragmatic ultrasound was not operated by two observers at the same time. thirdly, the left diaphragm was not assessed by ultrasound. the subjects were older copd patients in this study, and the left hemidiaphragm was frequently obscured by the expanding lung. however, kim et al.'s study showed that unilateral diaphragm dysfunction was more common than bilateral, and the bilateral evaluation of ultrasound can more accurately represent the diaphragm function [ ] . at last, the rr, rsbi, and p/f are also commonly used to predict extubation outcome. when we analyze de combined with these indicators, we found that the predicted value of joint judgment has not been improved significantly (the auc roc has not been increased). perhaps because this study is a singlecenter study, the number of cases is a limitation, 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stroke patients with dysphagia as assessed by m-mode sonography sonographic evaluation of the diaphragm in critically ill patients. technique and clinical applications diaphragm dysfunction assessed by ultrasonography: influence on weaning from mechanical ventilation ventilator-induced diaphragmatic dysfunction ventilatorinduced diaphragmatic dysfunction both high level pressure support ventilation and controlled mechanical ventilation induce diaphragm dysfunction and atrophy rapid onset of specific diaphragm weakness in a healthy murine model of ventilatorinduced diaphragmatic dysfunction ventilator-induced diaphragm dysfunction: cause and effect diaphragm muscle thinning in patients who are mechanically ventilated ultrasound-assessed diaphragmatic impairment is a predictor of outcomes in patients with acute exacerbation of chronic obstructive pulmonary disease undergoing noninvasive ventilation outcomes of extubation failure in medical intensive care unit patients practice variation in spontaneous breathing trial performance and reporting comparison of clinical utility between diaphragm excursion and thickening change using ultrasonography to predict extubation success diaphragmatic mobility: relationship with lung function, respiratory muscle strength, dyspnea, and physical activity in daily life in patients with copd diaphragmatic excursion measurement in emergency patients with acute dyspnea: toward a new diagnostic tool? publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations authors' contributions authors' contributions: x. z. designed this study, supervised the study, and revised the manuscript. j. w., j. y., and y. z. performed the study, contributed to the collection of data, and participated in writing the manuscript. the other authors were involved in the execution of the study and data collection. all the authors participated in writing the manuscript and in reviewing the final manuscript and approving it for publication. the study protocol was approved by the ethics committee of the first affiliated hospital of wannan medical college, and informed consent was obtained from each patient's family. the authors declare that they have no conflict of interest. key: cord- - jezc authors: Özdemir, seray karagöz; iltar, utku; salim, ozan; yücel, orhan kemal; erdem, ramazan; turhan, Özge; undar, levent title: investigation of seasonal frequency and pathogens in febrile neutropenia date: - - journal: memo doi: . /s - - -z sha: doc_id: cord_uid: jezc background: in patients with hematological malignancies, febrile neutropenia (fen) is the most frequent complication and the most important cause of mortality. various risk factors have been identified for severe infection in neutropenic patients. however, to the best of our knowledge, it is not defined whether there is a change in the risk of febrile neutropenia according to seasons. the first aim of study was to determine the difference in frequency of febrile neutropenic episodes (fnes) according to months and seasons. the second aim was to document isolated pathogens, as well as demographical and clinical characteristics of patients. methods: in the study, fnes of patients who have been followed with hematological malignancies between june and may were evaluated retrospectively. results: although the number of fnes increased in autumn, there was no significant difference in frequency of fnes between months (p = . ) and seasons (p = . ). there was no isolated pathogen in . % of fnes. in . % of fnes, pathogens were isolated. of all pathogens, . % were gram negative bacteria, . % were gram positive bacteria, . % were viruses, . % were fungi, and . % were parasites. conclusİons: the frequency of fen does not change according to months or seasons. also, the relative proportions of different pathogens in the cause of fen do not vary according to seasons. febrile neutropenia (fen) is the most common complication requiring hospitalization and causing mortality in patients with hematological cancer. also, febrile episodes prolong the duration of hospitalization in neutropenic patients [ ] . clinically documented infections occur in - % of febrile episodes [ , ] . most of the infections documented in patients with fen are caused by endogenous flora [ ] . among these, circulatory and respiratory system infections are the most common. while gram negative bacteria were the most frequently detected infectious agents up until the s, gram positive bacteria became the most common factors in the following years, due to increased use of vascular catheters, prophylactic antibiotic applications, and toxic intestinal chemotherapy applications. however, in recent years, the rate of gram negative bacteria has increased again [ ] [ ] [ ] [ ] [ ] . commonly seen gram positive bacteria are s. aureus, coagulase negative staphylococcus (cns), and streptococcus species; gram negative bacteria include e. coli, klebsiella species, and p. aeruginosa [ ] . fungal agents, most commonly candida and aspergillus, can also be identified as causes of fen. severe neutropenia, prolonged neutropenia, presence of mucositis, type of cancer (such as acute leukemia, high-risk myelodysplastic syndrome), uncontrolled or progressive cancer, induction therapy or application of intensive chemotherapy such as hematopoietic stem cell transplantation, presence of comorbid disorders requiring hospitalization, use of central venous catheter, use of monoclonal antibody, poor performance status, and advanced age are some of the risk factors defined for fen development [ ] . to the best of our knowledge, seasons or months have not been defined as risk factors for fen in hematological cancer patients. the primary aim of the study was to investigate whether there is a relationship between the frequency of febrile neutropenic episodes (fnes) and seasons in hematological cancer patients. the second aim of the study was to determine the type and frequency of pathogens detected in fnes. in this study, fnes of patients with hematological malignancies who had been followed at a multidisciplinary hospital (akdeniz university school of medicine hospital) between june and may were evaluated. patients with allogeneic stem cell transplants were not included in the study. the available medical files and biological results of all patients were retrospectively reviewed. according to the asco guidelines, fever was defined as a single oral temperature measurement of > . °c ( °f) or a temperature of > . °c ( . °f) sustained over a one hour period. we defined neutropenia as an absolute neutrophil count (anc) < cells/mm or an anc that was expected to decrease to < cells/mm during the next h [ ] . clinical and demographic data of the patients and the months and seasons of fnes were noted. in addition, neutrophil count, c-reactive protein (crp) level, length of hospitalization, culture results of blood and other body specimens, isolated pathogens, detected foci of infection, and antibacterial, antiviral, or antifungal treatments were reviewed. the study protocol was approved by the institutional ethics committee of akdeniz university school of medicine hospital and conducted in accordance with the principles of the declaration of helsinki and the good clinical practice guidelines of the international conference on harmonization. in addition to descriptive statistical methods (mean, median, and standard deviation), the χ test was used to compare qualitative data, and the student's t-test was used for quantitative two-group comparisons of the parameters with normal distribution. the results were evaluated with a confidence interval of % and a significance level of p < . . there were ( . %) male and ( . %) female patients. the median length of hospital stay was . days . demographic characteristics of the cases are presented in table . the fnes occurring during a given period of chemotherapy-induced neutropenia were % of all fnes, and the chemotherapy regimens included different protocols. ten percent of fnes were not related to chemotherapy. the mean number of fnes was . ± . in females, . ± . in males, and . ± . in all patients. there was no difference in the number of fnes according to gender (p = . ). the median neutrophil count was ( - )/mm and the median crp level was mg/dl ( . - ; normal range: - . ). no pathogen was detected in . % of fnes. in of the fnes, only one microorganism was isolated, while in fnes, more than one microorganism was isolated. isolated microorganisms are presented in table . of the isolated bacterial microorganisms, . % were gram negative bacteria and . % were gram positive bacteria. among table . although the number of fnes increased in autumn, there was no significant difference in frequency of fnes between months (p = . ) and seasons (p = . ) (figs. and ) . it was observed that ( . %) fnes were treated with single antibacterial therapy and ( . %) fnes were treated with combined antibacterial treatment. antifungal treatment was added to antibacterial treatment in ( . %) fnes, and antiviral treatment was applied in ( . %) fnes. the management did not differ according to particular seasons. twenty-seven ( . %) of the fnes resulted in death. the main causes of death were sepsis ( . %), intracranial bleeding ( . %), and acute respiratory distress ( . %). original report discussion today, various cytotoxic antineoplastic therapies are widely used in hematological cancers. however, there is an increase in the frequency of infections due to myelosuppression and immunosuppression caused by the disease itself and current treatments. studies show that life-threatening infection occurs in - % of fen patients [ ] . seasonal patterns of infection can be observed for some of the viruses that cause the common cold. in temperate regions of the northern hemisphere, the frequency of respiratory infections increases rapidly in autumn, remains fairly high throughout winter, and decreases again in spring. in tropical areas, most colds arise during the rainy season [ , ] . based on the presence of seasonal viral infection patterns, this study investigated whether there was a similar seasonal change in fnes. it was found that there was no difference in frequency of fnes according to months and seasons. the probable reason why there is no difference according to months and seasons is that the pathogens are largely caused by endogenous flora. the second aim of the study was to determine the type and frequency of pathogens detected in fnes in patients with hematologic cancer. the proportion of gram negative bacteria was significantly higher (approximately twice as much) than the ratio of gram positive bacteria. among all pathogens, e. coli was the most common ( . %). also, these pathogens did not show any change according to seasons. therefore, empirical antimicrobial therapy does not require a change according to the seasons. fen is a medical emergency and empirical treatment should be started as soon as possible. the aim of empirical therapy is to cover the most likely and most virulent pathogens that may rapidly cause life-threatening infections. therefore, clinicians need to be aware of the current microbiology surveillance data from their own institution, which can vary widely from center to center. this allows for better management of the process with selection of more appropriate empirical antimicrobial therapy. the limitation of the study was that the follow-up period of the participants was not sufficient. a study with a longer follow-up period is needed to more thoroughly investigate seasonal frequency in febrile neutropenia. a more wide view of the whole world practice is needed. it would be nice to know the frequencies of admissions for febrile episodes per month and per season also for non-cancer patients in order to evaluate if the seasonal variations of infections which have been reported elsewhere can be also observed in our region. although there is no reported study in our region, it can be said that viral infections are more common in autumn and winter months. if a more comprehensive study is to be carried out, the presence of a noncancer control group will provide clearer information. mortality,morbidity, andcostassociatedwithfebrileneutropeniain adultcancer patients infectious diseases society of americaa. clinical practice guideline for the use of antimicrobial agents in neutropenic patients with cancer: update by the infectious diseases society of america management of fever in patients with cancer and treatment-induced neutropenia significance of hospital acquisition of potential pathogens retrospective analysis of episodes guidelines for the use of antimicrobial agents in neutropenic patients with cancer current trends in the epidemiology of nosocomial bloodstream infections in patients with hematological malignancies and solid neoplasms in hospitals in the united states invasive grampositive bacterial infection in cancer patients treatment of febrile neutropenia: what is new? perspectives for the management of febrile neutropenic patients with cancer in the st century riskfactorsforfebrileneutropenia among patients with cancer receiving chemotherapy: a systematic review antimicrobial prophylaxis and outpatient management of fever and neutropeniain adults treatedfor malignancy: american society of clinical oncology clinical practice guideline prophylaxis with fluoroquinolones for bacterial infections in neutropenic patients: a meta-analysis the common cold. prim care the common cold we are grateful to all treating physicians in our center for collaboration and data collection.conflict of interest s.k. Özdemir, u. iltar, o. salim, o.k. yücel, r. erdem, Ö. turhan, and l. undar declare that they have no competing interests. key: cord- -hgzneooy authors: david, yadin; judd, thomas title: evidence-based impact by clinical engineers on global patients outcomes date: - - journal: health technol (berl) doi: . /s - - - sha: doc_id: cord_uid: hgzneooy the intersection of technological changes and societal evolution has transformed every aspect of human life. technological advancements are transforming how healthcare knowledge is expanding and accelerating the outreach of critical medical services delivery (jamal et al. in health information management journal ( ): – , ). while this transformation facilitates new opportunities simultaneously it also introduces challenges (jacobzone and oxley, ). appropriate health technology (ht) is vital to new and existing global health care programs. therefore, qualified professionals who can safely guide the development, evaluation, installation, integration, performance assurance, and risk mitigation of ht must be in position to lead. trained clinical engineers (ce) and biomedical engineers (be) have been recognized by the world health organization (who) as the essential practitioners to providing this critically needed guidance. over the past four years, a senior professional group participated in an international project that seeks evidence for the hypothesis - that the engagement of ce and be in guiding ht - impacts positively on patient outcomes, while the alternative is that there is no difference. the group collected published data that was subjected to peer review screening; additional data qualification conditions are described in this paper. the project was initiated at the global ce summit during the first international clinical engineering and health technology management congress (icehtmc) in hangzhou, china in october (global clinical engineering summit at the first international clinical engineering and health technology management congress, ). following the adoption of a resolution to investigate ce contributions to the improvement of world health status, an international survey and literature survey were initiated. during the first two years of this project case studies from countries were identified covering the previous ten years. the results of this survey were presented to health leaders at the world health organization (who) world health assembly in . last year, case studies were added including more countries covering the – period. the combined project contains qualified submissions from countries. the conclusion was that engagement of ce and bme is critical for successful investment in ht and for achieving intended patient outcomes. this paper describes the project’s plan, the results of the literature review performed, and the evidence identified during the process. the intersection of technological changes and social evolution has transformed every aspect of human life [ ] . this transformation is expansive and most obvious in the changes that has been occurring over the past fifty years in the provisioning of healthcare services [ ] . the dependence of health, rehabilitation, and wellness programs on technology for the delivery of services has never been greater [ ] . therefore, it is essential that health technology (ht) be strategically guided and optimally managed [ ] . guidance can only be provided by educated and experienced professionals who can safely lead the full life cycle of the technology, starting with innovation and progressing to development, regulatory compliance, evaluation, installation, training, integration, performance assurance, and risk mitigation. however, these professionals must be familiar with the relationship between contributions from ht and their impact on patient outcomes. the understanding of this relationship is a fundamental requirement for achieving optimal return on investment and improvement of outcomes. such practitioners are critical members of the healthcare team and should be in position to facilitate technology-related plans. beyond the ongoing healthcare burdens of population growth, political and economic instability, disease management, disasters, refugees, accidents, terror attacks, and increasing dependence level on technology, our world of healthcare systems is facing enormous challenges to manage its resources in the twenty-first century. the flood of scientific and technological innovation is radically redefining the nature of healthcare in virtually every dimension, from vascular nanoengineered interventions and predictive diagnostic tests to image guided surgery and remote telehealth-based services at the national and global levels. however, most healthcare systems are not adequately staffed to safely and effectively manage these forces of change. most systems are structured around vertically-expert professions (medical doctors, medical physicists, nurses, administrators), but lack the "horizontal expertise" that trained biomedical and clinical engineers (be&ce) provide. for example, the expertise in assessing and managing the integration and the performance of complex smart systems that have varying areas of service, durations of technological lifecycle [ ] , hardware and software platforms, and middleware in support of integrated medical and surgical services. disproving the myth -that there is a lack of evidence to qualify how much the dependency of ht is well-guided by ce expertise and best practice methodologyled to our examination of published literature and formal presentations of case studies in which ces, bes, and those in similar roles have participated. this allowed us to answer the question whether their participation contributed to improvement in overall healthcare outcomes. in the field of ht management and ce, the incentives to publish studies are lower than it should be, resulting in limited volume of resources to develop best practice measures. despite these perceived limitations, our results were recently published [ ] . in this paper, the focus is on the process used for the selection of data sources and the methodology to qualify their inclusion, described in the methodology section of this paper. on the other hand, over the past years, concerns were expressed that there is a lack of knowledge by government agencies and key stakeholders, coupled with limited recognition for those contributions for the practitioners that guide the deployment, creation and safe deployment of health technology. our data answers these concerns. if the knowledge and the expertise of the global ce community does have a critical role in optimal guidance of ht deployment, how can that expertise be best demonstrated. the collection of the case studies (that were later called success stories) from all over the world can facilitate the determination if there is competency unique to ces around the world that leads the development and optimal management of these technology life cycles. having this knowledge can help to reach better understanding of the required strategy to achieve desired patient outcomes when technology is used in care and rehabilitation management. the ebola virus disease crisis [ ] has demonstrated that multidisciplinary team expertise and collaboration are keys to success. low resources countries in particular face a challenge of improving their health services because, in addition to the above stated challenges, they also have scarce availability of professional expertise trained to address technology-related issues [ ] . varied availability and state of infrastructure and human resources place higher demand on adequate management of ht innovation and deployment. the effective health workforce of the twenty-first century consists of more individual practitioners caring for complex health-issues and thus charged with deploying the most optimal benefits from medical technology, such as proper selection, effectiveness, timely access, and affordable. in academia, government, and industry, teams of be&ce translate design innovations and integrate knowledge of science, engineering, standards and regulations with clinical strategy to create new tools that save and improve lives while building more quality into patient outcomes. in hospitals, be&ce practitioners ensure that proper acquisition, installation, integration and operation of devices and systems are safe and efficient. with the increasing role of technology in the delivery health care services, professional competency of the entire span of the technology lifecycleacross systems and sectorsis critical to achieving the full benefits and best outcomes clinically, economically, and operationally. following the resolution adopted at the first international clinical engineering and health technology management congress [ ] that took place in hangzhou, china, in october , senior members from the ce profession from around the world who participated in the global ce summit [ ] initiated the international project seeking evidence to the hypothesis that the engagement of ce and be in guiding ht deployment positively impacts patient outcomes while the null hypothesis was that there is no difference. the group identified the volume of published data that and developed criteria for inclusion pertinent and qualified publications. the rules are shown in fig. below. several conditions were placed on the total volume of publications and formal presentations that were found. only sources that responded positive to the challenge of the criteria were included in the final examination. to begin with, the source must be subjected to peer-review screening. secondly, the source must include care-related outcomes in the body of the manuscript, thirdly the source had to be published in ifmbe [ ] sponsored publication or event (meeting) proceedings, fourthly, the source must describe how ce or bme practices led to the second criterion of outcomes, and the fifth criterion limit the source inclusion to specific window of time. this window was defined as - for the first phase of this examination, and - for the second phase. during the first two years of the project case studies from countries were identified and satisfied the criteria described in fig. . searching through the time span over a period of previous ten years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the results of the initial review were presented to health leaders from member countries at the who world health assembly in . the interest generated in the project helped to sustain its work and as a result of the continuation of data analysis the team identified and qualified additional case studies from a total of countries covering the period of the - . the combined project's two stages examine and qualified over qualified submissions from countries. next, we looked at the methodology of putting together peer recognized clinical engineering experts from around the world, all members of the ifmbe clinical engineering division and issued a call for collections of papers from around the world, that will demonstrate what is the involvement and what is the contribution of individual programs from around the world in the ce arena. within months, in , we were able to collect a vast volume of evidence that was qualified and filter into specific studies from countries. the literature sources and the results of the examination are presented, grouped into six categories of outcomes impact. the resulting qualified volume of sources were categories into groups. the six groups were created to facilitate decision if sufficient evidence has been accumulated to support conclusion of outcomes. while the six categories were reviewed independently the significant overall commonality is that they all address different aspects of ht technology's impact on outcomes. data collected that met the inclusion criteria was grouped into six categories as follows: through provision of new ht solutions, adaptation of existing, or a combination to address several issues. ease in reaching ht-related health services or facilities in terms of location, time, and ease of approach. positive impact from more efficient and effective deployment of ht at national or policy level. ht's positive impact on health services safety or quality outcomes, or through ht human resource development. establishing or improving htm methodology resulting in improved population health or wellness. improvements achieved due to deployment of internet-based ht tools.following phases of the technology life cycle the analysis of the data began with the group that starts the cycle -with the innovation phase. innovation and the provision of new solutions to existing problems. the next group, reasonable progression to health services. where the question to address was the possible existence of evidence to demonstrate that the access to health care services has increased because of technology management programs. or, did the ht management program established methodologies that improve the overall finance and/or wellness of the population. after that, review of data was conducted with regard to overall impact on national or regional systems or multi-hospital health systems. safety and quality services that dependent on complex technological systems is critical for outcomes and therefore identified with its own group of data technology management group was the next category to be reviewed where ce/bme contributions to organized, integrate, manage, and improve safe and efficient sustainable ht. finally, in a way of looking forward the future, the group of e-technology where telemedicine, image guided interventions, informatics and disaster response operation were grouped together. making assumption that with the introduction of complex technological systems improvements in patient care safety and the quality of services receive were evident. with review of data in these six categories this study was able to cover major activities that are technology-dependent in health. successful source (or submission) was defined as satisfying objective measures developed by the investigators: timeliness, cost saving, deployment or adoption by care providers, impact on services, and overall projection for success. each success metric was evaluated using -point scale against a statement representing the success construct ( = strongly disagree; = strongly agree). timeliness refers to whether the project/submission was implemented in timely manner. this was measure by the statement "the submission will impact outcomes on present time." the cost measure was evaluated on whether the submission's overall costs were within budget constraints and reasonable for the conditions in the region. this was assessed by the statement, "the submission cost objectives can be met in the region." the final two metrics were combined into the statements "the submission will be deployed by its intended users" and "the submission will have a positive impact on those who will adopt it." finally, overall submission success was assessed with the statement "all things considered, the submission will be a success." success was determined when the source received overall rating of or above. sources for data collection included the following: ifmbe/ clinical engineering division (ced) health technologies resources [ ] document provided to the world health assembly, who in may , the nd and rd global forums on medical devices [ ] organized by the world health organization ifmbe/ ced's china and brazil icehtmc [ ] october and september respectively, others [ ] ifmbe journals proceedings and published sources from the period - . the results containing criteria inclusive and qualified data were tabulated and categorized into six categories that are described in tables below. the tables provide detailed information about the category of the submission, the region summitted it, the submission title and authors identification. each of submission is accompanied by hot link facilitating further data and evidence details that the reader is welcome to pursue. the data in these tables with the accompanied links demonstrates that evidence exists for benefit registered in each of the six categories from every region around the world. overall this review identified evidence from case studies received from countries where management of medical devices (as main component of health technologies) made a positive difference over the past twelve years. the results of first phase of the literature survey were incorporated into a document that in may of was successfully presented to the world health assembly consisting of member country delegations of ministry of health from around the world to who in geneva switzerland [ ] . data collection and analysis was conducted over three years period employing the same selection factors as shown in fig. . the analysis shows that volume of evidence exists in the literature to demonstrate the important and critical contributions of ce and bme to the initiation of new and improvement of present care outcomes. these contributions are evident on every continent and every day of the year. not just randomly but continuously x x days. after the completion of the two phases of this literature survey publications from countries document evidence and showing the success of clinical engineering competency serving on the healthcare delivery team (tables , , , , and ). the case studies are actually ht success stories demonstrating, in a limited resource environment, that it is desirable to include professional ht expertise, such as clinical engineers, in national decision-making in order to maximize health systems' services. case studies from the links on the following pages demonstrate these benefits: & access: the ministry of health ht unit-led project in albania that doubled access to critical diagnostic services, such as computed tomography scanners, magnetic resonance and angiography imaging, while reducing equipment downtime to zero, and significantly reducing cost. & health systems: improved coordination between multiple stakeholders in the national laboratory and its satellites in colombia, led by the ministry of health and clinical engineers who partner with experts from academia and industry. & quality & safety: a clinical engineer-led -hospital program in the shanghai region that cooperates with officials, industry, and academic entities, resulting in improved device user satisfaction, tracking of emerging technologies, and closer partnerships with industry. & table innovation & table access & table management & table health systems & table e-technology & table quality & safety in all of the above mentioned topics, data collection, review and validation continued throughout the project period as access to ifmbe sponsored events and related publications was secured in phases. during and we added more evidence that was qualified by our criteria. additional stories from additional of countries, were now increasing the overall count to publications from countries. all with evidence, showing the success from ce involvement in the relationship to improving patient outcomes, and the derived benefits from ht creation, management, and deployment. involvement that is documented through services provided over days a year, h a day, days a week. to be included in the project evidence database, shown in the tables above, each entry must comply with conditions for inclusion and with performance parameters described earlier of timeliness, costsaving, extent of deployment or adoption by care providers, impact on overall services and estimated projection for the entry success. the timeliness parameter complied if an impact has been described in the entry as immediate as in present tense. other parameters were similarly considered similarly. all entries included can be viewed through the on-line links provided in the tables. the hot links to all the resources the task force reviewed and qualified were validated. the tables are color coded to facilitate ease readers interest of seeking additional details for a specific technology category. examples of entries from the table above describe details as follow: in the innovation category, for example, anne-louise smith from adelaide, australia, with a team of clinicians identified a need for solution to specific clinical problem related to retrieval to transfusion of fluids of patients who maybe in a shock. no device was able to meet the need of fulfilling the task without external power source. the entry -bme development of non-electric portable blood/fluid warmer for roadside trauma, describe the critical contribution of ce to create solution, test it, identify and resolve usability barriers and bring it to commercialization. transferring of patients in rural areas is now safer and having better patient outcomes. the engineering expertise and the collaboration with physicians were key factors for the success evident in this entry. in the health systems category: bilal beceren, from turkey, affiliated with ministry of health (moh) of turkey practices at the national ht management program, involved public hospitals. prior to there was no moh based program and knowledge of the medical technology assets deployed. they embarked on national project in that built information about medical assets purchasing, commissioning and facilitated better performance support. ce training was initiated, and maintenance support has increased. the outcomes show that medical technology has been acquired under better terms, more efficiently maintained, the uptime of % for covered inventory now was reachable facilitating better patient care. annual audits conducted since show that from unknown level prior to the program in reached coverage of % of the inventory in the country. national health technology management system for public hospitals in turkey improve the performance and cost efficiency of the technology that patient management is dependent upon. in the access category: ledina picari from the moh in albania, a clinical engineer by training identified concern about the access to diagnostic services. diagnostic imaging technology was not properly maintained and equipment up time did not meet patients' need. in a collaborative national project was initiated to examine the state of equipment management and identify opportunities for increasing access to diagnostic services, to increase clinical availability of diagnostic technology at the local level, and to increase efficient and effective use of public funds. the evidence provided shows that in the volume of ct examinations more than doubled from to exams while the equipment downtime was reduced from almost four months a year down to near days. this is important achievement that in addition delivered the benefit of reducing the maintenance costs from about to % before the project was initiated down to % of the purchase price per annum afterwards. diagnostic technology availability significantly improves patient's outcome. a second example in the access to health services category that bridges to e-technology and specifically a telemedicine program was initiated with ce guidance (yadin david) in houston, texas. the project aimed at connecting rural community in central america village in the safety and quality category, li bin, a ce from shanghai, china, identified the need for having better technology quality control as there was not clear measure in the management of the technology in large network of care providers before . network of care providers facilities in community of million population, hospitals above grade two, and about ce & bme in the region. as result of this project in they changed the conditions from lack of quality standards in purchasing and servicing of diagnostic technology they implemented enhanced management program with collaboration of industry. data sharing and benchmarking information led to better cooperation between the parties, improve service personnel training, the initiation of annual quality improvement reporting and to sustain readiness of technology to serve clinical objectives. they now know that there are billion yuan of medical equipment assets, this is about billion dollars usd that due to ce management improved outcomes for both financial investment in technology and clinical services to patients. the e-technology category has another example of how be & ce contributed to better outcomes, specifically during the devastating earthquake in port-au-prince, haiti. in that occasion, article of new england journal of medicine, march , describes how support staff, including ce, arrived at haiti and within two days after the earthquake, established a field hospital that was able to treat patients, performed surgeries, and delivered babies. the first baby born there was named by his mother 'israel'after the group origin that came to establish the field hospital there. finally, in the ht management category, in brazilian rainforest, we found another evidence for how ce expertise has helped to achieve better patient outcomes and improving care. ryan pinto ferreira from university of campinas, optimal transportation method and assembly all the medical devices that clinicians needed. they transport it over the challenge of difficult route, to be placed in a highly humid rainforest environment. they assembled, commissioned, and operated the equipment and provided support for clinical services that those patients needed. at the who, in the health systems category, adriana velasquez have implemented many technology-based patient care programs that have far reach all over the world. her collaborative efforts perhaps best known through assembling networking of international stake holders during the successful series of global forums on medical devices. another successful contribution she achieved has been the development and dissemination of international publication and resources [ ] for addressing ht issues such as creating a resource for global atlas of medical devices, global model for regulatory framework, medical device policies, compendium of new and emerging health technologies, human resources ht is vital to health and the dependence of health, rehabilitation, and wellness programs that rely on ht for the delivery of their services has never been greater. beyond the ongoing healthcare burdens of population growth, political and economic instability, disease management, disasters, the refugee crisis, accidents, and terror attacks, world healthcare technological systems are facing enormous challenges to be innovative and optimally managed. the transition into health programs for the st century requires the employment of trained competent ce professionals. disease prevention, treatment, and rehabilitation is more efficient and effective when health services are provided with appropriate tools. along with world health organization (who) [ ] , the international it is critical, therefore, that with limited availability of resources, ht must be professionally managed and its creation and deployment over its life-cycle be appropriately guided. this paper describes the extensive study of published data on the vast contributions by ce that positively impact patient outcomes. this finding of this study shows that every region of the world including lowresource regions face a challenge of improving health services while facing varied levels of infrastructure and human resources capacity challenges. ces play vital roles in all stages of healthcare technology life-cycle management. from creation to planning, and from commissioning to utilization and integration; technology-based systems must and can be managed for optimal performance. in each of the technology life-cycle stages the requirement for trained and competent ce input makes critical difference as evidence show in the analyzed data reviewed above. it is our hope that government agencies and other interested parties will have better understanding of ces role and thus will support their inclusion in the healthcare team of professionals. the identified and qualified case studies shown in this manuscript support the need to expand the reach of ce community in order to provide competent guide to management of healthcare technologies around the world. case studiesgrouped in categoriescan assist to formulate national strategies and plans on how to improve the creation and deployment of ht while improving quality of care and efficient use of scares funding. in several countries, case studies demonstrated, this has best been achieved by developing a ht unit at the level of ministry of health that engages the ce community. these studies provide evidence that ht is beneficial; however, at times, deployment of such complex systems when it is not effectively guided and managed may not realize intended outcomes for optimal impact. the who wha resolution . urges member states to create national ht management plans in collaboration with clinical and biomedical engineers. who further clarified the definition of these personnel in - as part of a global survey [ ] in coordination with ifmbe/ced. "trained and qualified biomedical engineering professionals are required to design, evaluate, regulate, maintain and manage medical devices, and train on their safe use in health systems around the world. " these occupations have various names in different countries like clinical engineers, medical engineers, … and related professionals and technicians." we encourage the dissemination of survey tools as describe here to better understand the need for and monitoring of progress towards safe, appropriate and optimal quality care outcomes. the authors express gratitude for the intense work invested by members of the project task force and for kallirroi stavrianou for creating and validating all of evidence links on the tables above. conflict of interest the authors declare that they have no conflict of interest. ethical approval this article does not contain any studies with human participants or animals performed by any of the authors. informed consent informed consent was not obtained since there were individual participants included in the study. using technology to advance global health, proceedings of a workshop, forum on public-private partnerships for global health and safety a 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biomedical equipment the benefit of in-hospital clinical engineer services for medical devices maintenance medical equipment maintenance personnel and training in zambia the potential power of sub-saharan africa professional associations for biomedical/clinical engineering professionals status of biomedical engineering education in the asia pacific cost estimate methodology in procurement processes of me study of medical device purchasing cycles through temporal series analysis rios cuartas i, identifying the needs in the integration of disciplines in the hospital infrastructure management in colombia trading barriers in the medical devices industry. are these barriers hindering the development of this sector in cuba hospital based hta -implementation for the czech republic ifmbe/clinical engineering division projects for the advancement of the profession of clinical engineering the potential role of ifmbe in improving the state of medical equipment in developing countries assessing the impact of a cis/pacs technology for a cardiology department using qfd methodology the status of bme programs in latin america medical devices management strategy in the republic of moldova a comprehensive system for healthcare technology management htm how clinical engineers will solve the billion dollar healthcare funding gap indicators for evaluating and measuring the impact of healthcare infrastructure and technology management on investments, service delivery and quality of care becoming of ubiquitous sensors for ubiquitous healthcare study on medical equipment location systems that use rfid technology (eds) international conference on advancements of medicine and health care through technology; th - th integrating an electronic health record graphical user interface into nanoelectronic-based biosensor technology camacua: low cost real time risk alert and location system for healthcare environments assessing risk in the kaiser permanente clinical technology program intelligent system for identification of patients in healthcare design of a web-based medical equipment management system for clinical engineering technological surveillance and integrity monitoring of infusion systems implementation of six sigma on corrective maintenance case study at the directorate of biomedical engineering in the jordanian ministry of health human resources for medical devices, the role of biomedical engineers, who, medical devices the impact of health information technology on the quality of medical and health care: a systematic review healthcare expenditure a future in question + & f o r m = e d g h p t & q s = h s & c v i d = & r e f i g = dd b a c f cf &cc=us&setlang=en-us&plvar= &pc=dcts publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -repm vw authors: ungchusak, kumnuan; heymann, david; pollack, marjorie title: public health surveillance: a vital alert and response function date: - - journal: the palgrave handbook of global health data methods for policy and practice doi: . / - - - - _ sha: doc_id: cord_uid: repm vw ungchusak, heymann and pollack address the critical global issue of public health surveillance. they describe how epidemiologists collect and use surveillance data to detect unusual events or outbreaks and to guide control programmes. drawing on their combined international experience, the authors explain the vital role that data play in alerting authorities to respond to outbreaks such as severe acute respiratory syndrome, ebola, zika virus and avian influenza. they point to the importance of sharing information globally while ensuring equal benefits to providers of data, coordinating surveillance activities across sectors, building capacity for surveillance and coordinating national surveillance activities. the authors emphasise the need for enhanced global cooperation to prepare for future public health emergencies of international concern. a three-month delay in identifying the outbreak of ebola virus in rural guinea in late resulted in its rapid spread to urban areas and to neighbouring liberia and sierra leone [ ] . once local and international responders identified the virus, they took a year to interrupt its widespread transmission. by april , ebola had accounted for more than , cases and over , deaths. people around the world watched with increasing alarm, as this tragic course of events played out, and with concern that air travel could enable the virus to spread across continents. this epidemic highlighted not only the inadequacy of local health systems to recognise and respond but also that international organisations were not ready to provide timely expertise and resources to control the situation and ameliorate the virus's spread through the region. had health officials identified ebola in west africa promptly, they could have minimised its impact on the lives and livelihoods of the populations of west africa by implementing appropriate control procedures. public health officials coined the term surveillance to describe systems they set up to watch out for and control occurrence of health threats. just as police, for example, set up closed-circuit television devices and community watch programmes to detect and prevent crime, public health surveillance systems engage all possible means to detect unwanted health events and prevent them from escalating and damaging population health. while public health surveillance originated to control spread of infectious diseases such as plague and cholera, it has evolved to include some non-communicable diseases, occupational health and injuries as well as surveillance of biological, behavioural and social determinants of these conditions. we start by reviewing the public health need for surveillance and the development by the international community of regulations to control infectious diseases and other public health emergencies of international concern (pheic). we describe how epidemiologists use surveillance data to detect unusual events or outbreaks and to guide control programmes, and we provide guidance about maintaining data quality. we examine networks that contribute to global surveillance systems and highlight the role of social media and information technology in providing data to monitor new events of international importance. we consider challenges facing epidemiologists responsible for surveillance and describe efforts to address them. public health surveillance is vital to the functioning of national and global health systems. policymakers and health administrators need surveillance information to set priorities to address population health problems, allocate resources and monitor progress of prevention and control programmes; they need surveillance systems to alert them immediately of public health threats. emerging infectious diseases, such as avian influenza of different subtypes, severe acute respiratory syndrome (sars) coronavirus, pandemic influenza h n and the zika virus (zikv) have the potential to spread rapidly causing severe loss of life and to impact socio-economic activity, especially trade and travel [ ] . the outbreak of sars in november highlighted the importance of every country having functioning and connected surveillance systems (see box . ). surveillance requires high-level government support, well-trained health workers, strong health information systems, well-functioning laboratories, effective communication systems and operational health facilities. to be effective, surveillance systems also require a strong legal framework to ensure that individual data can be shared while maintaining confidentiality as far as possible. global cooperation between countries, with up-to-date international health agreements to build and maintain these capacities, is essential to decrease risk of international spread of infectious diseases and contain the risk of bio-terrorism. sars originated in wildlife and spread silently among humans as atypical pneumonia in guangdong province, china, two months before officials became aware of it. authorities began surveillance to identify atypical pneumonia cases but this, and the containment response, were too late to stop sars spreading. a chinese urologist who was infected travelled to hong kong and spread sars to another persons. within weeks, sars spread to countries with more than , reported cases ( fig. . ) [ ] . by the end of the epidemic in july , sars had killed people [ ] . although unable to contain the outbreak of sars, the international community was able to bring the epidemic under control within six months-by collaborating across countries to identify and isolate all probable cases. nevertheless, the asian development bank estimated that the economic loss due to sars in affected countries was up to us $ billion with us$ . billion on mainland china (approximately . per cent of its annual gross domestic product (gdp)) and us$ . billion in hong kong (approximately per cent of its annual gdp) [ ] . plague ravaged europe during the fourteenth century and although authorities had no cure, they realised it was important to swiftly identify and isolate cases to prevent and control this lethal condition. authorities understood that international spread of such diseases followed cross-border trade, pilgrimage and war; and so prevention of disease was a national security issue. in the city-state of venice, authorities instigated quarantine measures-keeping arriving ships in the harbour for days before docking, and holding people in isolation for days at land borders to prevent entry of plague [ ] . in the mid-nineteenth century, recognising that quarantine measures were not enough, governments agreed international conventions aimed at stopping spread of plague and cholera-and two other infectious diseases, yellow fever and smallpox. the conventions required each country to report outbreaks of these diseases to all signatories of the convention, and permitted application of certain public health measures at international borders once a country reported of one of the diseases. in the early twentieth century, governments in the americas and in europe set up regional conventions called international sanitary bureaus. in , the newly formed world health organization (who) led establishment of the international sanitary regulations (isr) to foster global cooperation in reporting and acting at international borders to guard against spread of cholera, plague, yellow fever and smallpox. in , the who replaced the isr with the international health regulations (ihr) which required countries to report any cases of cholera, plague, yellow fever and smallpox to who [ ] . if a country reported one of these diseases, other countries could apply pre-established control measures at international borders-such as a requirement of proof of vaccination against yellow fever of any passenger arriving from a country that reported yellow fever to who. some countries reported to who late, or not at all, because of lack of capacity for public health surveillance, or because of fear of stigmatisation and economic repercussions. after hiv spread across international borders before being identified in , the international community realised that infectious diseases could not be stopped at borders. diseases often cross borders while still being incubated in humans, or in non-human hosts-insects, animals, and food and agricultural goods. in , after the sars outbreak, who updated and revised the ihr as a legal framework to include more diseases, and developed real-time evidence-based recommendations for prevention and control of outbreaks. who evaluates each newly identified outbreak for its potential to become a pheic by the country in which it is occurring. the ihr mandate who member countries to report immediately the occurrence of a single case of four diseases (smallpox, poliomyelitis due to wild type poliovirus, human influenza caused by a new subtype, and sars) [ ] . even though the world eradicated smallpox in , the ihr still maintain it on the list to cover the risk of the virus escaping from a laboratory. each country has an additional list of diseases that it requires its health workers to report by law. diseases of greatest public health threat are reportable, meaning that health workers or laboratory technicians must report individual cases as they occur. reportable diseases include those required by ihr and, for example, anthrax, cholera, ebola, legionellosis, plague and the zikv. other conditions are notifiable, meaning that health workers should report the number of cases that have occurred in a given time period. the number, frequency of reporting and breakdown of reportable and notifiable diseases varies by country. diarrheal cases, influenza cases, tuberculosis, aids and other significant endemic diseases are usually required to be notified to local health authorities. in some countries the notifiable list can include non-infectious conditions such as maternal or infant deaths. the ihr require countries to develop core capacities in public health, including surveillance systems and epidemiology services, that can analyse and act on surveillance information to detect and respond to diseases where and when they occur so that their potential to spread internationally is decreased. the purpose of surveillance activities is to: ( ) detect at an early stage, acute public health threats from all hazards-biological, chemical, radiation, natural disaster and deliberate acts-which require rapid investigation and response; and ( ) guide control programmes by measuring disease burden, monitoring trends, describing disease distribution and evaluating public health programme effectiveness (see table . ). the structure of government responsibilities for public health surveillance varies across countries. most often, countries set up dedicated early warning and rapid response surveillance teams that work with or complement surveillance activities of vertical control programmes such as malaria, hiv/aids or tuberculosis. surveillance and response teams detect early stage public health threats while control programmes gather disease (or condition) specific information to plan activities. control programmes share information with surveillance teams as required. a national network of public health laboratories, often linked to international reference laboratories, confirms etiologic agents, genetic strains and antibiotic resistance patterns. surveillance activities are said to be active when health workers pro-actively seek out cases and passive when the system relies on patients to report themselves to a clinic. using standard case definitions, health workers report individual cases of reportable and notifiable diseases to the local or national surveillance centre where staff aggregates reports, and clean and analyse the data. in cleaning the data, staff look for coding and classification errors, and for duplicate reports. epidemiologists analyse the data to determine how many new cases have occurred during the past day or week and their distribution in time, place and by person to see whether the magnitude and pattern of the disease under surveillance is changing. they note any changes in frequency, clustering or distribution and flag them for verification and explanation. box . illustrates how careful data analysis led to malaysia identifying nipah virus in [ ] . reporting of specific information about cases or patients or behaviour of populations under surveillance produces indicator-based data, that is individual or aggregated data derived from patients diagnosed-by syndrome description, clinical or laboratory confirmation-and identified through routine collection or active case search. the surveillance unit will also use eventbased data about outbreaks, unusual events or changes in human exposure [ ] . rather than wait for official reports, the surveillance team gathers information and rumours through the media, internet and unusual events reported by the community, and investigates these reports. the team captures abnormal health events in real-time and confirms potential outbreaks by triangulating these data with indicator-based data. epidemiologists responsible for surveillance use standard epidemiological methods to analyse trends, identify clusters and investigate suspected risk factors (see chap. for an overview of epidemiological methods). for example, high numbers of reported cases of kaposi sarcoma among young men in new york and california during the early s led to an investigation which showed a japanese encephalitis commonly occurs in school-age children of both sexes. there is a seasonal pattern of disease related to the rainy season when transmission and therefore disease occurrence, increases; there is no difference in occurrence between ethnic or religious groups. from september to april , surveillance teams sent reports of cases of febrile encephalitis ( per cent fatal) to the malaysian ministry of health [ ] . initially, the ministry considered japanese encephalitis virus to be the probable etiologic agent for this outbreak, and instituted conventional interventions of vaccination and insecticide to control mosquitoes. when they examined the surveillance data closely, the epidemiological pattern of encephalitis cases was different to what they expected-the disease occurred mostly among male adults of chinese ethnic origin whose occupations related to pig farming. the ministry sought a different cause and found the etiologic agent to be a new paramyxovirus, later named nipah virus. common risk factor of homosexual behaviour and its relationship with hiv/ aids [ ] . using increasingly sophisticated technologies for data capture and analysis, surveillance teams can monitor real-time occurrence, in time and place, of unusual events such as cholera or legionella, or seasonal outbreaks such as malaria (see chap. for an introduction to spatial and spatio-temporal techniques and to chap. which discusses predicting climate-related health outcomes such as malaria). once epidemiologists have concluded their analyses (sometimes in realtime), they prepare reports which can trigger immediate action by a rapid response team to visit the site of the events, investigate the situation and contain the outbreak. the team also sends reports to clinicians in hospitals and to local and national programme managers. many countries publish weekly disease surveillance reports that are also available to the general public: for example, the us centers for disease control and prevention (cdc) publish the morbidity and mortality weekly report (mmwr) [ ] , the european centre for disease control (ecdc) publishes eurosurveillance [ ] , and the who publishes the weekly epidemiological record [ ] . box . shows how epidemiologists associated microcephaly with zikv which led who to declare zikv a pheic [ ] . public health surveillance guides control programmes by undertaking the following functions: in late , zikv spread rapidly through latin america especially in brazil and el salvador. surveillance of birth defects in brazil identified a major increase in microcephaly during the period when zikv transmission increased. this alerted policymakers and epidemiologists to study whether the increase in birth defects was associated with zikv infection during pregnancy. who declared the suspected increase in microcephaly in association with zikv infection of pregnant women a pheic and recommended pregnant women to protect themselves from mosquito bites and to avoid travel to areas with known zikv transmission. the observation that men who travelled to areas with known zikv transmission could sexually transmit zikv to their partners led who to recommend practising safer sex or abstinence for a period of six months for men and women returning from areas of active transmission. its epidemiological patterns disease in humans results from interactions between the human host and causative agents or hazards of all types. the natural and socio-economic environment influences these interactions. diseases usually occur in the same pattern when there is no change in the causative agent (such as mutation), in the human host (such as vaccination) or in the environment (such as climate change). a surveillance system can closely monitor any changes in these dynamic factors and their consequences, as illustrated by the case of nipah virus in malaysia (box . ). public health surveillance must also address risk. for example, surveillance of annual per capita cigarette consumption in the us showed an increased trend from cigarettes in to , cigarettes in . researchers related this trend to advertising and an expansion in the number of cigarette companies. in , after the first studies suggesting cigarette consumption was related to lung cancer, and the us surgeon general issued a warning, the annual per capita consumption decreased to , [ ] . with surveillance information, epidemiologists can forecast an increase in lung cancer without intervention thereby providing evidence for policy to implement effective interventions such as taxation to prevent smoking. evaluating performance of control programmes after they have implemented interventions, health authorities use surveillance data to see if disease incidence declines. for example, when vaccine coverage increases, the number of cases of vaccine preventable diseases is expected to decrease. increasing taxes on cigarettes is one way to reduce consumption. surveillance data can document a correlation between increasing taxes and decreasing trends in cigarette consumption. to achieve these functions, programme managers collect data through patient records, surveys, programme records or informal sources. types of data include determinants of the condition, behaviours or risk factors associated with the condition, morbidity and mortality associated with the condition, programme responses, and abnormal or unusual events associated with the condition. table . provides examples of these types of data for surveillance of an hiv/aids control programme. to ensure surveillance programmes have adequate resources and produce useful information, public health authorities regularly review their surveillance activities. in , the us cdc issued guidelines to evaluate surveillance systems which, with some updating, are still widely used [ ] . these guidelines focus evaluation of public health surveillance on three areas: ( ) the surveillance system itself, describing the system, its structure, diseases under surveillance, sources of data, and how data are processed, analysed and disseminated; ( ) the resources used to operate the system, including funding sources, adequately trained staff and information technology; and ( ) the usefulness and quality of surveillance information, using the following indicators: usefulness of data do the data and information disseminated to data providers and users contain comprehensible facts and findings and useful recommendations to improve control measures and guide programme management? has the system detected outbreaks? how many of the detected outbreaks were investigated and controlled in a timely manner? timeliness of data and other information is data dissemination timely and regular? for example, epidemic prone diseases require weekly summary, while other diseases require only monthly or quarterly summaries. are these requirements met? validity and completeness of data much of the data come from clinical diagnoses that do not have laboratory confirmation. it is useful to conduct studies to determine the accuracy of diagnoses using standard laboratory confirmation testing. this helps in preparing estimates of the proportion of confirmed cases among all reported cases. when undertaking field investigations, investigators can compare the number of actual cases they find with the number of cases reported through the system. this provides an estimate of reporting completeness of the system. global public health surveillance is the collection, analysis and use of standardised information about health threats or their risk factors from more than one country, and usually worldwide. while surveillance mainly focuses on infectious diseases, global systems also seek to identify deliberate use of biological agents or toxins to cause harm. who leads the global public health surveillance system, gathering information from formal and informal sources working through its country and regional offices. who extends its reach through the global outbreak and response network (goarn) [ ] which comprises over national technical institutions that support who to detect public health threats and respond to outbreaks. who uses the information for risk assessment and analysis as part of its routine disease control and prevention programme activities. when requested by countries for support, who works with goarn institutions to recruit suitable experts. goarn includes regional networks of countries that cooperate independently to prevent and control infectious diseases occurring in their regions, for example, the east african integrated disease surveillance network (eaidsnet), [ ] and the mekong basin disease surveillance network (mbds) [ ] . who leads global networks that work to control specific diseases. these networks depend on cooperation of governments, public health workers and scientists to report cases, provide specimens and share information so that specific diseases can be controlled globally. these include: networks to support influenza control through vaccine development the global influenza surveillance and response system (gisrs) consists of national sentinel centres and national and regional laboratories which annually collect , - , nasal swabs from patients presenting with influenza-like illness. their analyses provide information about the distribution of strains circulating each year and enable scientists to recommend the influenza vaccine composition for the following year based on predominant sequences. gisrs also uses flunet, a public web-based data collection and reporting tool that tracks movement of influenza viruses globally and provides epidemiological data about influenza outbreaks [ ] . initiative. clinical health workers and epidemiologists report all cases of acute flaccid paralysis (afp) in children under years of age from whom they have collected stool specimens for isolation and identification of the poliovirus. through its network of national, regional and specialised laboratories, gpln determines whether polio was the cause of the afp, genetically sequences viruses and compares them to a global database to understand their geographic source. if a polio virus is found, gpln informs the national authority and who regional office for appropriate action. project on anti-tuberculosis drug resistance surveillance [ ] is a common surveillance platform to which countries can provide data that are then used to monitor the evolution and spread of multi-drug resistant tuberculosis (mdr-tb) and extensively drug-resistant tuberculosis (xdr-tb). national laboratories provide susceptibility testing of tuberculosis organisms collected from patients, supported by a supranational tuberculosis reference laboratory network. the global project provides understanding of the prevalence and distribution of tuberculosis resistance worldwide. [ ] . its goal is to develop a standardised strategy to collect, analyse and share clinical, laboratory and epidemiological data globally, assess the burden and support local, national and global strategies to control amr. until recently, surveillance systems depended on paper-based reporting, compilation and analysis of data. computers and electronic reporting have made compilation and analysis of data much easier, and the world wide web (www) and the internet improve the comprehensiveness of reporting. digital and internet-based technology can retrieve information from medical records on a daily basis-but this must be done without infringing personal privacy. hospitals, especially private ones, may refuse to provide patient information to the public health sector unless privacy issues are addressed. cell phone technology has extended the scope of informal and event-based surveillance while social media has transformed exploring rumours of new events. some ground-breaking examples of the use of information and communication technology include: electronic reporting of events the programme for monitoring emerging diseases (promed-mail) is a fully moderated internet-based listserv, that receives and publishes reports of public health events in humans, animals, wildlife and plants from its subscribers and other traditional and nontraditional information sources [ ] . promed-mail uses information available on the www and from voluntary listserv reporters who actively search for and report public health events in realtime from the media, internet blogs and other sites. promed-mail editors and expert moderators review, analyse, evaluate and where possible validate reports, and then disseminate them to listserv members and post them on its website. using big data to identify events the subscription-based application global public health intelligence network (gphin) continuously scans the www gathering information from multiple source news aggregators in real-time [ ] . gphin searches in nine languages for key words that could indicate infectious disease outbreaks, or environmental, radioactive and natural disasters. analysts identify new events and inform subscribers-who are governmental and non-governmental agencies with an established public health mandate. every hours, analysts communicate new information to who which validates reports through its network of regional and country offices. who discusses events that it validates in confidence with health departments in the countries involved. mapping events in real-time healthmap, a fully automated application, utilises online informal sources for disease outbreak monitoring and real-time surveillance of emerging public health threats [ ] . healthmap trawls www sources of information (in nine languages) including online eyewitness reports, expert-curated discussions such as promed-mail, validated official reports, for example from who, or the food and agriculture organization of the united nations, and news aggregation services such as google news. using open source software, healthmap displays the events by time, geographic location and aetiology. participatory flu tracking diseases and abnormal events happen all the time in the community. only some patients, especially those presenting with severe disease manifestations, seek medical care. flu near you invites anyone living north america, over years of age, to report if they have an influenza-like illness [ ] . once registered, participants are asked weekly by e-mail to complete a brief survey that seeks information on ten symptoms linked to influ-enza, and other information such as whether or not the registered participant has had an influenza vaccination. other countries, including the uk, have adopted similar participatory influenza surveillance systems, thereby adding a greater understanding of the epidemiology of influenza around the world. participatory onehealth disease detection (podd) chang mai university in thailand, with support from the skool foundation, developed this mobile application which connects volunteers in local governments. when volunteers notice an abnormal event such as poultry dying off or sickness in animals or humans, they use podd to notify local authorities who dispatch a surveillance and rapid response team to investigate and contain the event. after months of implementation, podd has enabled the detection of , abnormal events, including chicken high-mortality outbreaks, four cattle disease outbreaks, three pig disease outbreaks and three fish disease outbreaks, all of which were detected and controlled [ ] . since revision of the ihr in , outbreaks due to infections, including the middle east respiratory syndrome coronavirus and ebola virus, have highlighted continued weaknesses in public health surveillance and response capacities in most countries, with international spread causing disruptions in trade and travel, and negatively impacting economies. we present some challenges and suggest some solutions. most countries have established disease control programmes each with a surveillance component reporting from grassroots through provincial and national levels. national surveillance units may have sufficient staff for each disease control programme, but at lower levels of the health system, the same individuals often manage more than one programme and are heavily burdened by reporting requirements. there is also duplication of effort in reporting between programmes. who supports countries to coordinate surveillance activities across departments, programmes and administrative levels through integrated disease surveillance and response (idsr) [ ] . idsr links surveillance with other health information activities and strengthens overall capacity of countries to maintain public health surveillance. the ihr obligates countries to develop comprehensive disease surveillance, detection and response when and where infectious diseases and other acute public health threats occur. in reality, national surveillance capacity in many countries is still not at expected and necessary levels. this may be, as the ebola epidemic demonstrated in west africa, that health systems are weak and under-funded, or that the surveillance system itself does not function efficiently. regular evaluation of the system, as we describe in sect. . , can identify which components need to be strengthened. an over-riding issue is for the system to deploy and maintain enough professionals throughout the system with the required skills-understanding the nature and limitations of the data they are working with and able to interpret and draw important findings from the analyses of the surveillance data. approximately per cent of newly identified human diseases are zoonotic in origin [ ] and per cent of these diseases have their origins in wildlife [ ] . since the outbreak of h n avian influenza in hong kong, animal surveillance and human surveillance units have begun to share information and alert each other of unusual events. environmental factors are also crucial to disease occurrence, for example, paralytic shellfish poisoning among people who consume shellfish affected by harmful algae growth in the sea [ ] . the one health approach involves sharing information between multiple health sectors and working together to identify and resolve outbreaks [ ] . during the avian influenza outbreak, who requested all affected countries to share the virus isolated from humans for further study and vaccine development. some governments expressed concern about potential negative economic consequences of sharing information and about possible inequities in the benefits of sharing. this led to the jakarta declaration on responsible practices for sharing avian influenza viruses and resulting benefits [ ] . this declaration underlined need for continued open, timely and equitable sharing of information, data and biological specimens related to influenza; it also emphasised need for more equitable sharing of benefits for example in the generation of diagnostics, drugs and vaccines. the jakarta declaration led to the pandemic influenza preparedness framework (pip) under which manufacturers of influenza vaccines, diagnostics and pharmaceuticals that use gisrs information make annual financial contributions to who. who uses approximately per cent of these contributions for pandemic preparedness activities and surveillance, and per cent for pandemic response including purchase of vaccines and antivirals at the time of a pandemic for countries without access to these supplies. in may , the chatham house centre on global health security, after a series of roundtable consultation with experts in public health surveillance, produced a guide on strengthening data sharing for public health surveillance. this guide facilitates both informal and formal data sharing. the guide proposes seven principles: building trust; articulating the value; planning; using quality data; understanding the legal context; coming to agreement; and evaluating. the guidelines help create the right environment for data sharing and to facilitate good practice in addressing technical, political, ethical, economic and legal concerns that may arise. the guidelines aim to ensure, to the greatest extent possible, that any benefits arising from use of the data are shared equitably [ ] . similar to clinical or public health practice, institutions or agencies responsible for public health surveillance need a set of ethical principles to guide their operations. the who guidelines on ethical issues in public health surveillance proposed guidelines [ ] . these guidelines fall into three major groups: first, the mandate and broad responsibility of the agency to undertake surveillance and subject it to ethical scrutiny; second, the obligation to ensure appropriate protection and rights of individuals under surveillance; and third, considerations in making decisions about how to communicate and share surveillance data to pursue common good and equity of population without harm to individual. the west african ebola outbreak provided a costly lesson that policymakers must commit to establishing, maintaining and advancing public health surveillance systems to protect and promote population health. to prepare for the next major outbreak, the world needs to invest in a strong warning and response system led by a global institution with sufficient authority and funding to react swiftly [ ] . who serves this role but is chronically underfunded. similar investment is needed in countries where a fully supported, well-functioning surveillance office or programme must coordinate different components of the surveillance system. surveillance information should be disseminated widely to alert the public and health programmes of outbreaks so that they can contain the disease at source before it spreads internationally. because the world urgently needs reliable 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guidelines on ethical issues in public health surveillance the next epidemic-lessons from ebola key: cord- -rmtyrbg authors: saad, farouk tijjani; sanlidag, tamer; hincal, evren; sayan, murat; baba, isa abdullahi; kaymakamzade, bilgen title: global stability analysis of hiv+ model date: - - journal: th international conference on theory and application of fuzzy systems and soft computing &#x ; icafs- doi: . / - - - - _ sha: doc_id: cord_uid: rmtyrbg we developed and studied a mathematical model of hiv+. two equilibriums points were found, disease free and endemic equilibrium, and basic reproduction ratio [formula: see text] was also calculated by the use of next generation matrix. global stability analysis of the equilibria was carried out by the use of lyapunov function, and it was shown that the stability of the equilibria depends on the magnitude of the basic reproduction ratio. when [formula: see text] , the disease free equilibrium is globally asymptotically stable, and disease dies out. on the other hand if [formula: see text] , the endemic equilibrium is globally asymptotically stable and epidemics occurs. reported cases of hiv- positive were obtained in the year from ministry of health, turkey (moh). this data is used to present the numerical simulations, which supports the analytic result. [formula: see text] was found to be . , which is bigger than , this shows the threat posed by hiv in turkey. historically, infectious diseases posed a real threat to the human population. although they have been in human population all the time to some extent, the effects of epidemics are the most obvious and noticeable. only in the th century europe, around million out of about million individuals died from the black death [ ] . several diseases were discovered in america which included smallpox, measles, influenza, and typhus, whooping cough, the mumps, and diphtheria. infectious disease was the main reason for the demise of the indians [ ] . in the early 's, some homosexual men in the united states were diagnosed with a type of fungal infection and a tumor called candidiasis and kaposi's sarcoma respectively. paris pasteur institute in detected a virus responsible for those diseases and called it the human immunodeficiency virus (hiv) [ ] . hiv is the virus that causes acquired immunodeficiency syndrome (aids). the virus is responsible for attacking and destroying the immune systems mainly the cd + t-lymphocytes or t-cells [ ] . on a normal basis, these cd + t-lymphocytes or t-cells detect foreign and infected cells, and attack, spread and kill them [ ] [ ] [ ] . hiv is able to infect cd + t-lymphocytes and insert its genome to host genome. this integrated hiv genome may exist in states. they can be either transcriptionally active generating new viruses that can infect other cd + t-lymphocytes or in latent state which may become activated later. in transcriptionally active stage, the infected cd + t-lymphocytes die due to cytopathic effect of the virus. as a result, the number of cd + t-lymphocytes, which are able to recognize foreign and infected cells, declines, and this decrement lead stoper manent and lasting damage to the immune system [ ] . the immune system finally loses its ability to fight and kill infections due to the number of cd + t-lymphocytes count which is so small. when an individual reaches this stage, the person is said to have aids [ ] . the time between getting infected with hiv and advancing to aids is, in general, five years but changes due to many factors. if the cd + t-lymphocytes cells count falls below cells/mm , then the person is considered to be in the "aids phase", otherwise the person is said to be hiv infected [ ] . the mode of transmission of hiv includes heterosexual intercourse, homosexual/bisexual intercourse, intravenous drug use, vertical transmission, and unknown reasons [ ] . since its discovery (hiv/aids), the extensive increase and epidemic continues around the globe. the greatest number in any one year was in , where almost five million individuals became newly infected, with a total of million hiv/aids patients, and almost three million people died from aids in the same year [ ] . to continue its record of one of the most destructive epidemics in history, it killed at least million people by . efforts to improve the use of antiretroviral treatment in some part of the world were still not enough to reduce a significant number of deaths, the hiv/aids epidemic claimed . million lives in , of which about were children (unaids/who [ ] ). the region that suffered most is africa, with sub-saharan africa as the home of the epidemic. in , . million people were newly infected, and . million patients died of aids-related causes across the globe. by the end of , around million individuals were victims of hiv/aids [ ] . china stated that about . million died of aids-related causes in , and there were about . million hiv-infected individuals by the end of . in the year , two patients were diagnosed with aids in turkey, since then, the importance of aids started and still continue to be on the forefront. the number of new cases increases every year, with , , and cases in , , and respectively [ ] . in , the ministry of health (moh) published the total number of hiv patients , of which % are between the ages of to years and sexually active. among these patients, about were aids patients. this is an official data from moh, however, these numbers does not reflect the actual figures of hiv infected individual in turkey, due to insufficiency of the registration system, and the lack awareness and phobia of the patients to attend health centers or hospitals [ ] . at the end of , the ministry stated that the total number of hiv/aids infected people was . it also published in that at least individuals were infected with hiv, and the numbers of newly reported cases in , , and first six months of were , , and respectively. according to a report, poor knowledge of sexually transmitted diseases, poor socio-economic conditions, increase in number of unregistered sex workers, increase in number of homosexuals, and intravenous drug use contributed to the spread of hiv infection in turkey. it was reported that, the main way of transmitting hiv in turkey is via heterosexual sex ( %), then men having sex with men (msm at %), and intravenous drug users (idu at %) among the recorded cases. according to positive living association istanbul, personal communication, hiv/aids will become a major public health issue in turkey in the coming years, as such; it must be regarded as a rising disease for turkey [ ] . the main ways in which hiv/aids is transmitted between individuals are now well understood, but the factors that contribute to the disparities in its prevalence and trends among populations remain an area of interest to scientific researchers. to understand these disparities, it is essential to understand the system, its components and its dynamics. mathematical models of hiv/aids transmission dynamics are important research tool in this category [ , ] . primary purpose of any mathematical model of hiv transmission lies in using individual level inputs to project population level outcomes. some of the important outcomes that can be examined with a model are; the incidence of infection, the prevalence of infection, or the doubling time of the epidemic. more important than these however, is simply the likelihood of an epidemic to occur that is whether there is sufficient transmission potential for a chain of infection to be sustained. this outcome is termed by a simple summary statistic: the reproduction number of the infectious process, r . in a susceptible population, r represents the expected number of secondary infections generated by the first infected individual. if r is equal or greater than an epidemic is expected. if r is less than , the infection is expected to die out [ ] . the magnitude of r is used to measure the risk of an epidemic or pandemic in any emerging infectious disease. it was used for understanding the outbreak and danger of sars. r was also used to characterize bovine spongiform encephalitis (bse), foot and mouth disease (fmd), strains of influenza, and west nile virus [ ] [ ] [ ] [ ] . the incidence and spread of dengue, ebola, and scrapie have also been assessed by r [ ] [ ] [ ] . tropical issues such as the risks of indoor airborne infection, bioterrorism, and computer viruses also depend on this important parameter [ ] [ ] [ ] [ ] . in this paper, we shall first introduce the model involving systems of ode, and then discuss the biological meaning of the parameters involved. we shall study the global stabilities of both disease free and endemic equilibria by the use of lyapunov function. by the use of real data obtained from turkey in , we will conduct numerical simulations to support the analytic result. the organization of the paper is as follows: in sect. , the model is presented and the basic reproduction number is obtained. in sect. , stability of the equilibria are investigated. in sect. , results are obtained by numerical simulations of the real data obtained from turkey in . finally sect. is the discussion and conclusion of the research. the system of ordinary differential equations derived is for the whole turkish population. consider the birth k(t) to the susceptible population per unit time. susceptibles individuals are removed through infection or through natural death. let µ be the natural death rate for the whole population. the removal rate of susceptible individuals through infection is the number of new hiv infections per unit time. we use this rate in calculating hiv incidence which by definition is the number of new infected persons in a specified period of time divided by the number of uninfected persons that were exposed for this same time. let each susceptible have c contacts per unit time. assume that a proportion h/n of these contacts are with infectives and at each of these contacts with infectives, a susceptible has a probability b of becoming infected. let a the incidence rate, then the total probability of one susceptible getting hiv infected from any of their contacts per unit time is ah/n. this is the expression for the force of infection. the force of infection is the probability that a susceptible will get an hiv infection per unit time. therefore in a population of s susceptibles, the number of new hiv infections per unit time is given by ahs/n. infectives are recruited through new hiv infections described above and removed through death at rate v and through natural death at rate µ. hence, /v is the duration spent in the infective stage and /µ is the life expectancy of the population. all these rates are assumed constant in the model. removed cases are recruited either through natural death l or through deaths due to hiv at the rate v. with these assumptions, we arrive at the following system of ode. ( ) can be reduced to it follows from ( ) that, thus the feasible region for ( ) is for the simulation we make further assumptions as follows; (i) s( ) is considered to be the whole population in (ii) we considered homogeneous mixing in the population (iii) v ¼ : is considered (that is averagely years is the life span of hiv+ people) equating the equations in (*) to zero and solving simultaneously we find two equilibrium points. disease free and endemic equilibrium points. this is the number of secondary infections caused by a single infective individual in a completely susceptible population. it is denoted by r . using the next generation of matrix (ngm) method we have, the spectral radius, which is the dominant eigenvalue, is as v þ l hence the basic reproduction ration is; in this section stability analysis of the two equilibrium points is obtained by the use of lyapunov function. the conditions for the global stability of the equilibria in each case depends on the magnitude of the basic reproduction ratio r . hence we have the following theorems and their proofs. theorem : the disease free equilibrium is globally asymptotically stable when r . we construct the following lyapunov function by the relation between geometric and arithmetic means and if as v þ l ð Þ. this implies _ v if r . the endemic equilibrium e is globally asymptotically stable when r [ . proof: we construct the following lyapunov function by the relation between arithmetic and geometric mean and if in this section, results are calculated by simulating the model using the real data obtained from turkey in . here we use the real data obtained from moh, in which there were a total of hiv- positive reported cases in the year , in the year to study and predict the dynamics of hiv in turkey using our model. table presents the values of the parameters as calculated based on the data obtained. since r [ , the disease free equilibrium is not stable and the endemic equilibrium is stable. hence, there is going to be epidemics. simulating the above result, fig. shows the epidemic of hiv/aids in turkey. a mathematical model for hiv+ is constructed and analyzed. two equilibrium points (disease free and endemic) are found and stability of each of the equilibrium point was shown to depend on the magnitude of basic reproduction ratio, using lyapunov function. it was shown that if r ¼ as v þ l is less than one, the disease free equilibrium is globally asymptotically stable. also, if the value is greater than or equals to one the endemic equilibrium is globally asymptotically stable. the turkish population in the year is , and the hiv positive population is . the value of the basic reproduction ratio is . , which is bigger than one. this implies one hiv positive individual in turkey can be able to transfer the disease to almost individuals, hence there is going to be hiv epidemic in turkey. numerical simulations were carried out and the results support the analytic findings. the results in the simulation shows that if appropriate measures are not taken, in years to come, there will be more hiv positive individuals in turkey as there are susceptible individuals. the main limitation to our analysis may be that we did not account for the effect of behavioral change arising both from number of hiv cases in the community as well as awareness by governmental and nongovernmental organizations. secondary, the possible effects of extensive use of antiretroviral drugs (arvs) in terms of method of distributing drugs through public or private health institution or a combination of both could determine whether patients on arvs revert back to the infective class. this together with reduced infectiousness due to lower viral loads for those on treatment was not accounted for. despite the limitations mentioned above, there are several implications of our findings to public health. first, the endemic equilibrium should be brought as low as possible especially during the first wave of the epidemic. this model suggests that this can be achieved by prolonging the lifetime of the hiv patients for as long as possible. second, hiv prevalence at low prevalence levels become less sensitive to changes in the dynamics of hiv epidemic because it is overpowered by demographic changes especially the recruitment of susceptibles. at low prevalence levels, there is hence need to track trends in number of persons infected with hiv than tracking hiv prevalence. a preliminary study of the transmission dynamics of the human immunodeficiency virus (hiv), the causative agent of aids stability analysis of an hiv/aids epidemic model with treatment mathematical epidemiology of infectious diseases: model building, analysis and interpretation stability analysis of an hiv/aids epidemic model with screening stability analysis of an hiv/aids dynamics model with drug resistance analysis of the treatment costs of hiv/aids in turkey global analysis of an hiv/aids epidemic model dynamics of hiv/aids in turkey from to aids knowledge and attitudes in a turkish population. an epidemiology study status hiv/aids epidemic in turkey molecular epidemiology of hiv in a cohort of men having sex with men from istanbul mathematical models for hiv transmission dynamics: tools for social and behavioral science research hiv dynamics and behaviour change as determinants of the impact of sexually transmitted disease treatment on hiv transmission in the context of the rakai trial can population differences explain the contrasting results of the mwanza, rakai, and masaka hiv/sexually transmitted disease intervention trials: a modeling study a simple approximate mathematical model to predict the number of severe acute respiratory syndrome cases and deaths understanding the epidemiology of bse the foot -and -mouth epidemic in great britain: pattern of spread and impact of interventions transmissibility of pandemic influenza an epidemiological model for west nile virus: invasion analysis and control applications uncertainties regarding dengue modeling in rio de janeiro malaria and its possible control on the island of principe the basic ratio number of ebola and the effects of public health measures: the cases of congo and uganda a scrapie epidemic in cyprus risk of indoor airborne infection transmission estimated from carbon dioxide concentration emergence response to small pox attack: the case for mass vaccination role of awareness in controlling hiv/aids: a mathematical model key: cord- - vl xb authors: keser, tobias; kofler, mario; katzmayr, mariella; schiefecker, alois j.; rass, verena; ianosi, bogdan a.; lindner, anna; gaasch, maxime; beer, ronny; rhomberg, paul; schmutzhard, erich; pfausler, bettina; helbok, raimund title: risk factors for dysphagia and the impact on outcome after spontaneous subarachnoid hemorrhage date: - - journal: neurocrit care doi: . /s - - - sha: doc_id: cord_uid: vl xb background: despite the tremendous impact of swallowing disorders on outcome following ischemic stroke, little is known about the incidence of dysphagia after subarachnoid hemorrhage (sah) and its contribution to hospital complications, length of intensive care unit stay, and functional outcome. methods: this is a retrospective analysis of an ongoing prospective cohort study. swallowing ability was assessed in consecutive non-traumatic sah patients admitted to our neurological intensive care unit using the bogenhausen dysphagia score (bods). a bods > points indicated dysphagia. functional outcome was assessed months after the sah using the modified rankin scale with a score > defined as poor functional outcome. results: two-hundred and fifty consecutive sah patients comprising all clinical severity grades with a median age of years (interquartile range – ) were eligible for analysis. dysphagia was diagnosed in patients ( . %). factors independently associated with the development of dysphagia were poor clinical grade on admission (hunt & hess grades – ), sah-associated parenchymal hematoma, hydrocephalus, detection of an aneurysm, and prolonged mechanical ventilation (> h). dysphagia was independently associated with a higher rate of pneumonia (or = . , % ci = . – . ), blood stream infection (or = . , % ci = . – . ), longer icu stay [ ( – ) days versus . ( – ) days, p < . ], and poor functional outcome after months (or = . , % ci = . – . ). conclusions: dysphagia is a frequent complication of non-traumatic sah and associated with poor functional outcome, infectious complications, and prolonged stay in the intensive care unit. early identification of high-risk patients is needed to timely stratify individual patients for dysphagia treatment. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. despite the advances in the clinical management, subarachnoid hemorrhage (sah) is still associated with a high mortality rate and substantial morbidity [ ] . patient and disease-specific factors such as initial disease severity, hospital complications as well as the need for prolonged ventilation contribute to longer intensive care unit (icu) stays and poor functional outcome [ ] [ ] [ ] . post-extubation dysphagia occurs in most patients with neurological impairment and is associated with prolonged mechanical ventilation, the development of pneumonia, and longterm morbidity [ ] . although the incidence of swallowing disorders has been extensively studied in ischemic stroke patients, little is known about the true incidence of dysphagia in sah patients and its contribution to the clinical course and outcome. swallowing involves a complex sequence of neuromuscular events. several cortical brain regions, the cerebellum and the brainstem, are involved in the processing of afferent stimuli, initiating the voluntary oral phase of swallowing and coordinating consecutive reflexive mechanisms [ ] [ ] [ ] . all of those regions may incur functional impairment or structural damage through mechanisms of early and secondary brain injury, as well as global cerebral dysfunction following sah. furthermore, brainstem dysfunction without obvious underlying brain pathology has been described in sah patients [ ] . so far, the incidence of dysphagia in sah patients has been reported to range between . and . % [ , ] . several risk factors for the development of dysphagia were identified; however, the impact of impaired swallowing on functional outcome remains poorly described. in ischemic stroke patients, dysphagia is one of the most important determinants of functional outcome and self-sufficient living. in sah, the early identification of risk factors for the development of dysphagia may help to timely allocate resources to individual patients. furthermore, the novel understanding of neuronal plasticity of brain regions involved in the swallowing process has led to new treatment approaches, such as electrical pharyngeal stimulation, which can be applied already early after ictus. this intervention was recently found to be associated with an improved decannulation rate in tracheotomized stroke patients [ ] . in addition, oropharyngeal air-pulse application was associated with increased resting swallowing rates in tube-fed patients with hemispheric stroke [ ] . such treatments could potentially be applied in mechanically ventilated patients as an attempt to early initiate dysphagia therapy in high-risk patients. the main goal of the current study was ( ) to quantify the rate of swallowing disorders after sah by using a simple clinical assessment tool, ( ) to identify early predictors of dysphagia in all severity grades of sah patients, and ( ) to evaluate how dysphagia contributes to hospital complications, length of icu stay, and poor outcome. we hypothesized that dysphagia would be independently associated with higher rates of infectious complications, a longer stay in the icu, and poor functional outcome. two-hundred and seventy consecutive patients with nontraumatic sah admitted to the neurological icu of our tertiary referral center (medical university of innsbruck, austria) between and were screened. the conduct of this study was approved by the local ethics committee (medical university innsbruck, an / . , am - / . ), and informed consent was obtained from all patients according to federal regulations. twenty patients were excluded due to a lack of information on swallowing ability, because of early death or repatriation, leaving patients for the final analysis. standard critical care conformed to current international guidelines [ , ] , with the exception of intravenous instead of oral nimodipine application in poor-grade sah patients. ruptured aneurysms were secured by neurosurgical clipping or endovascular coiling. transcranial color-coded duplex sonography was routinely performed for the detection of vasospasm. catheter cerebral angiography was performed in patients with severe sonographic vasospasm (> - cm/s mean flow velocity or a lindegaard ratio > ), and intraarterial nimodipine treatment was considered. patients who developed severe vasospasm or delayed cerebral ischemia were treated with blood pressure augmentation, ventilator settings to achieve a pco > mmhg and normothermia. clinical disease severity was graded using the hunt & hess (h&h) scale [ ] . h&h grades - were considered as good grade and h&h grades - as poor-grade patients. cerebral computed tomography (ct) scans were analyzed by a neuroradiologist using the modified fisher scale and screening it for the presence of parenchymal hemorrhage [ ] . delayed cerebral ischemia was defined as the occurrence of a new focal neurological deficit, a decrease of ≥ points on the glasgow coma scale or a new infarct on ct or magnetic resonance imaging (mri) scans not attributable to other causes [ ] . infectious complications were diagnosed using the centers for disease control and prevention criteria. the variable prolonged intubation was defined as duration of mechanical ventilation of more than h in order to exclude patients with short-time intubation for aneurysm treatment. functional outcome was prospectively evaluated by a study nurse, blinded to the clinical course of patients, months after sah using the modified rankin scale (mrs) score, and categorized into good (mrs score - ) and poor (mrs score - ) functional outcome. swallowing ability was routinely assessed by our speech therapists and quantified using the bogenhausen dysphagia score (bods), a score grading the ability to swallow saliva (bods- ) and the possibility of oral food intake (bods- ). the overall score (bods- + bods- ) ranges from (no dysphagia) to (most severe dysphagia). the full bods description is available in table e- . a bods ≥ indicates dysphagia [ ] . we considered a patient as dysphagic, if at least one bods ≥ was documented during neuro-icu stay. furthermore, dysphagia was subclassified using the worst documented bods during the icu stay as mild to moderate (bods - ) or severe (bods - ), as proposed by the german society for neurology (https ://www.dgn.org/image s/red_leitl inien /ll_ /archi v/ll k ap_ .pdf ). a total bods of or greater implies that patients either ( ) require predominantly artificial (enteral or parenteral) nutrition, ( ) have a tracheal cannula, or ( ) both. there was no systematic bods evaluation at months follow-up. baseline characteristics, admission variables, interventions, hospital complications, and measures of functional outcome were prospectively collected in our institutional sah database. data on swallowing ability were obtained retrospectively from our electronic patient chart, where documentation of dysphagia using the bods is performed by our speech therapists as a part of clinical routine patient management. continuous variables are reported as median and interquartile range (iqr). categorical variables are reported as counts and proportions (%) in each group. univariate comparisons between patients with and without dysphagia were performed using the chi-square test for categorical variables and the wilcoxon rank-sum test for continuous variables, as they were not normally distributed. in a second step, factors associated with dysphagia in univariate analysis (p < . ) were included in a stepwise backward elimination binary logistic regression model with the dichotomized dysphagia variable as dependent variable to identify factors independently associated with the development of dysphagia. in the same way, factors associated with poor functional outcome were identified in univariate analyses (chi-square test, wilcoxon rank-sum test, as appropriate) and included in a stepwise backward elimination binary logistic regression model with the dichotomized outcome variable as dependent variable. the association between dysphagia and length of icu stay was assessed in a linear logistic regression model. all analyses were performed with ibm-spss (ibm spss statistics, version . . armonk, ny, usa). a p value smaller than . was considered as statistically significant. baseline characteristics, admission variables, hospital complications, and outcomes of patients with nontraumatic sah are shown in table . dysphagia was diagnosed in patients ( . %) of which ( %) had severe dysphagia. overall, the diagnosis was made after a median of . (iqr - ) days and, in patients who were intubated, (iqr - ) day after extubation. percutaneous endoscopic gastrostomy (peg) was placed in patients ( . %), of whom two had mild or moderate and had severe dysphagia, corresponding to a peg rate of . % ( / patients with mild or moderate dysphagia) and . % ( / patients with severe dysphagia). forty patients ( . %) admitted with good clinical grade and patients ( . %) with poor initial clinical grade developed dysphagia. in / patients ( %), no focal brain lesion was detected on any ct or mri scan during the icu stay. of those, ( %) suffered from dysphagia. factors associated with dysphagia in univariate analysis are shown in table . the risk of developing dysphagia did not differ between aneurysm securing methods (p = . ) or aneurysm locations (p = . ). based on the results of the univariate analysis, age, sex, hunt & hess grade, detection of an aneurysm, modified fisher score, parenchymal hemorrhage, delayed cerebral ischemia, hydrocephalus, prolonged intubation, pneumonia, and blood stream infection were the variables included in the multivariable analysis assessing independent associations with the development of dysphagia. those results are presented in table . ninety-eight patients ( %) had a poor functional outcome after months. we found a significant association between dysphagia and poor functional outcome (univariate or . , % ci = . - . , p < . ). the detailed distribution of functional outcomes in patients with and without dysphagia is shown in fig. . furthermore, we identified dysphagia as being independently associated with poor functional outcome (table ). in addition, patients with severe dysphagia had a higher risk of poor functional outcome (adj. or = . , % ci = . - . , p < . ) when compared to sah patients with mild/moderate dysphagia. moreover, dysphagia was associated with a higher rate of pneumonia within the group of dysphagic patients, peg placement (as an indicator of persistent dysphagia) was associated with poor functional outcome (or = . , ci % = . - . , p = . ), adjusted for h&h grade and age. when patients with peg placement were excluded from the analysis, dysphagia was still associated with poor outcome in the overall cohort (or = . , ci % = . - . , p = . ). patients with poor outcome after months had a significantly higher median worst bods compared to patients with good outcome ( [iqr - ] versus [iqr - ], p < . ). there was an association between a higher bods and poor functional outcome (or per bods point = . , % ci = . - . , p < . ), adjusted for h&h grade and age. in this study, we investigated the frequency of dysphagia in all clinical severity grades of sah patients using a simple score for the assessment of swallowing disorders in critical care patients. the main findings were that ( ) dysphagia was diagnosed in every third sah patient, including patients admitted in good clinical grade, that ( ) dysphagia was associated with radiographic and clinical disease severity on admission, and ( ) that dysphagia was strongly associated with hospital complications, prolonged icu stay, and poor functional outcome. bold p-values indicate significant associations (p < . ) ct computed tomography, evd external ventricular drain, icu intensive care unit, iqr interquartile range statistical analysis was performed using the wilcoxon rank-sum test (*), the chi-square test ( †), and the linear-by-linear association test for trend ( ‡) although it is well known that the high incidence of swallowing disorders after ischemic stroke contributes to impaired quality of life and poor functional outcome, only few studies report the incidence of dysphagia following sah [ , ] . in a retrospective cohort study including predominantly good-grade sah patients, the incidence was . %, which is comparable to our findings [ ] . it is important to mention that even patients presenting with good clinical grades on admission are at risk of developing swallowing disorders, especially when prolonged mechanical ventilation is needed. our data may help to identify patients at high risk early in order to allocate specific resources already early after extubation or even during the time of mechanical ventilation. known risk factors for the development of dysphagia after sah include older age, initial disease severity, the amount of cisternal and intraventricular blood, detection of an aneurysm, rebleeding, hydrocephalus, vasospasm, cerebral ischemia, and mechanical ventilation [ , ] . we additionally identified parenchymal hematoma as being independently associated with the development of dysphagia. however, our data and previous studies indicate that swallowing disorders after sah are not only due to structural brain damage, but also occur in patients without focal parenchymal injury ( % in our cohort), possibly owing to global cerebral dysfunction after sah and/or local mechanical irritation during intubation. in our cohort, intubation for more than h was by far the strongest predictor of dysphagia, which certainly needs a differentiated interpretation. dysphagia after intubation is a well-known complication with an incidence exceeding % in most studies, even in non-neurological patients [ ] . one mechanism seems to be mechanical irritation of the pharynx and larynx, as, among others, prolonged intubation, increased operative time, multiple intubations, fig. functional outcome after months (assessed using the modified rankin scale) in patients with and without dysphagia during the intensive care unit stay. the asterisks (***) indicate a significant difference (p < . ) in outcomes between groups (linear-by-linear association test); mrs = modified rankin scale and perioperative transesophageal echocardiography have been identified as risk factors for dysphagia after intubation [ ] . notably, patients suffering from severe sah with poor admission clinical grades, and those with parenchymal hematoma, hydrocephalus, or detection of an aneurysm (data not shown) more frequently required prolonged intubation. therefore, despite being of independent statistical significance, prolonged intubation may simply reflect disease severity in our cohort. another entity associated with a higher incidence of dysphagia is critical illness polyneuropathy and myopathy [ ] . unfortunately, we only performed electroneurographic studies in poor-grade patients with high clinical suspicion, which made it impossible to further elucidate this association. delayed cerebral ischemia was a univariate predictor of swallowing disturbance, but lost its significance in multivariable analysis. this may be due to small ischemic areas and infarction outside of brain regions involved in the swallowing process. here, we established a strong association between dysphagia and poor functional outcome after months. this is in line with findings in ischemic stroke patients, in whom dysphagia was associated with an increased risk of death, a higher rate of disability, longer hospitalization, and consecutive need for institutional care [ , ] . moreover, this study demonstrates that there is a strong association between dysphagia severity and the probability of poor functional outcome, reflected by the association of a higher bods and peg placement with poor outcome. importantly, dysphagia was still associated with poor outcome in the overall cohort when patients with peg placement (an indicator of persistent dysphagia) were excluded from the analysis. this signifies that also transient dysphagia negatively impacts on functional outcome. in this cohort, dysphagia and pneumonia were the only modifiable risk factors contributing to poor functional outcome in the logistic regression model. our findings underline the importance of early diagnosis and treatment of dysphagia in sah patients. conventional treatment options for dysphagic patients include texture-modified diets, speech and language therapy programs, non-oral (enteral) feeding, medication, as well as physical and olfactory stimulation [ ] . recent studies demonstrated experience-dependent neuronal plasticity of brain regions involved in the process of swallowing [ ] , which led to the development of several experimental interventions such as stimulation of the pharynx with air pulses. increased resting swallowing rates in tube-fed stroke patients with dysphagia have been shown in these patients [ ] . moreover, pharyngeal electrical stimulation favorably influenced the decannulation rate in tracheotomized ischemic stroke patients [ ] . these novel approaches may be of special interest in sah patients, as prolonged mechanical ventilation is common in poor admission grade patients rendering an early time window for these interventions. some limitations merit consideration. although every patient was evaluated for swallowing disorders, the time from ictus to assessment largely differed between patients as the bods assessment requires a certain level of vigilance and cooperation, as well as a clinically stable condition, which also explains the comparably long time to diagnosis. furthermore, assessment of swallowing disorders was not available on weekends, which may have led to a delay in diagnosis in individual patients. we only included the worst bods in the final analysis as, due to the retrospective nature of the study, consecutive bods data of sufficient quality were not available in all patients. on the other hand, this approach may illustrate the negative impact on outcome even of temporary dysphagia. importantly, the bods was not assessed in patients with metabolic encephalopathy or delirium. in summary, prospective data with predefined intervals of bods assessment are needed to further elucidate the association between dysphagia and functional outcome in sah. furthermore, we used fiber-endoscopic evaluation of swallowing only in patients with severe swallowing disorders, which diminishes the comparability of our results with studies assessing dysphagia with instrumental methods. the strength of the bods evaluation is its simplicity, and it can be applied in all hospitals independent of available equipment. dysphagia is a frequent complication of non-traumatic subarachnoid hemorrhage and it is associated with poor functional outcome, infectious complications, and prolonged stay at the icu. early identification of high-risk patients may be possible and opens the opportunity for early treatment, maybe even in unconscious and intubated patients. further investigations are needed to prove the efficacy of such interventions. this study argues for comprehensive dysphagia screening in all clinical severity grades of sah patients. the online version of this article (https ://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users. changes in case fatality of aneurysmal subarachnoid haemorrhage over time, according to age, sex, and region: a meta-analysis impact of medical complications on outcome after subarachnoid hemorrhage impact of nosocomial infectious complications after subarachnoid hemorrhage post-extubation dysphagia is associated with longer hospitalization in survivors of critical illness with neurologic impairment cerebral cortical processing of swallowing in older adults explaining oropharyngeal dysphagia after unilateral hemispheric stroke brain stem control of the phases of swallowing subarachnoid haemorrhage: diagnosis, causes and management incidence and risk factors for dysphagia following non-traumatic subarachnoid hemorrhage: a retrospective cohort study characteristics of patients with aneurysmal subarachnoid hemorrhage and risk factors related to dysphagia pharyngeal electrical stimulation for early decannulation in tracheotomised patients with neurogenic dysphagia after stroke (phast-trac): a prospective, single-blinded, randomised trial proof-of-principle pilot study of oropharyngeal air-pulse application in individuals with dysphagia after hemispheric stroke guidelines for the management of aneurysmal subarachnoid hemorrhage: a guideline for healthcare professionals from the american heart association/american stroke association european stroke organization guidelines for the management of intracranial aneurysms and subarachnoid haemorrhage surgical risk as related to time of intervention in the repair of intracranial aneurysms prediction of symptomatic vasospasm after subarachnoid hemorrhage: the modified fisher scale definition of delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage as an outcome event in clinical trials and observational studies: proposal of a multidisciplinary research group grundlagen der funktionellen dysphagietherapie (fdt) the incidence of dysphagia following endotracheal intubation: a systematic review dysphagia-a common, transient symptom in critical illness polyneuropathy: a fiberoptic endoscopic evaluation of swallowing study* dysphagia in acute stroke: incidence, burden and impact on clinical outcome complications and outcome after acute stroke. does dysphagia matter? dysphagia treatment post stroke: a systematic review of randomised controlled trials neuroplasticity and swallowing open access funding provided by university of innsbruck and medical university of innsbruck. tk was involved in data acquisition, data analysis and writing the manuscript. mk and rh were involved study design, data collection, data analysis and writing the manuscript. mka was involved in study design and data collection. as, vr, bi, al and mg were involved in data collection. rb, pr, es and bp were involved in study design. all authors drafted and critically reviewed the manuscript and approved the final version. this study received an unrestricted grant from fresenius kabi, germany. the funding body did not participate in the design of the study, collection, analysis, and interpretation of data or in writing the manuscript. the authors declare that they have no conflict of interest. this study has been approved by the ethics committee of the medical university of innsbruck, austria, and has been performed in accordance with the ethical standards as laid down in the declaration of helsinki and its later amendments. informed consent has been obtained from all patients according to federal regulations. this article is distributed under the terms of the creative commons attribution . international license (http://creat iveco mmons .org/licen ses/by/ . /), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -rxx ash authors: krogh, klaus; halvorsen, annette; pettersen, ann louise; biering-sørensen, fin title: version . of the international spinal cord injury bowel function basic data set date: - - journal: spinal cord ser cases doi: . /s - - -z sha: doc_id: cord_uid: rxx ash nan in the process of translation of the english version into nordic languages (danish, icelandic, norwegian, and swedish) of the international spinal cord injury bowel function basic data set version . [ ] some inconsistencies were recognized which here will be corrected and clarified: . for the variable "surgical procedures on the gastrointestinal tract": in the data-collection form 'hemorrhoidectomy, date performed yyyymmdd' was missing . the variable: "frequency of defecation (within the last weeks)" a. in the description in the syllabus and in the datacollection form should the code 'once or more per day' be changed to 'daily', ' - times per week' be changed to ' - times per week', and 'once per week or less' to be changed to 'less than once per week' to be in accordance with the original neurogenic bowel dysfunction score. b. in the data-collection form 'not applicable' is missing. . the variable: "frequency of fecal incontinence (within the last weeks)" in the data-collection form "less than once per month/never" shall be together in one code, because the variable is describing the frequency 'within the last weeks'which is also the description given in the syllabus. . the variable: "need to wear diaper, pad, or plug (within the last weeks)" a. the description made regarding the change is correct. b. the description made in the syllabus should only have the codes: in the data-collection form 'not applicable' is missing. rectal prolapse is removed from the syllabus as it already was from the data-collection form . the variable: "abdominal pain and discomfort (within the last weeks)" the codes in the syllabus and the data-collection form are now the same: the code 'once per week or less' is changed to 'less than once per week'because otherwise once per week was included in two answers: conflict of interest the authors declare that they have no conflict of interest. publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. biering-sørensen f. international spinal cord injury bowel function basic data set (version . ) key: cord- -m flptcv authors: bossé, ynuk title: the strain on airway smooth muscle during a deep inspiration to total lung capacity date: - - journal: j eng sci med diagn ther doi: . / . sha: doc_id: cord_uid: m flptcv the deep inspiration (di) maneuver entices a great deal of interest because of its ability to temporarily ease the flow of air into the lungs. this salutary effect of a di is proposed to be mediated, at least partially, by momentarily increasing the operating length of airway smooth muscle (asm). concerningly, this premise is largely derived from a growing body of in vitro studies investigating the effect of stretching asm by different magnitudes on its contractility. the relevance of these in vitro findings remains uncertain, as the real range of strains asm undergoes in vivo during a di is somewhat elusive. in order to understand the regulation of asm contractility by a di and to infer on its putative contribution to the bronchodilator effect of a di, it is imperative that in vitro studies incorporate levels of strains that are physiologically relevant. this review summarizes the methods that may be used in vivo in humans to estimate the strain experienced by asm during a di from functional residual capacity (frc) to total lung capacity (tlc). the strengths and limitations of each method, as well as the potential confounders, are also discussed. a rough estimated range of asm strains is provided for the purpose of guiding future in vitro studies that aim at quantifying the regulatory effect of di on asm contractility. however, it is emphasized that, owing to the many limitations and confounders, more studies will be needed to reach conclusive statements. the beneficial effect of a deep inspiration (di) on respiratory mechanics has long been an important topic of discussion among lung physiologists [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, the mechanism accountable for the respiratory relief that is normally afforded by a di is still not clear [ , ] . several mechanisms are likely involved and each mechanism may contribute to various extents in different individuals. however, one mechanism that arguably received the most attention is the transient change of length (i.e., strain or stretch) that airway smooth muscle (asm) undergoes during the di . indeed, the expansion of lung volume during a di modifies the caliber of the airways (table ) ; the word "caliber" herein refers to the cross-sectional area of the airway lumen. the increase in caliber during a di results from increasing radial stress that stems from the tethering force of the lung parenchyma. since asm is embedded within the airway wall in an orientation that is nearly orthogonal to the long axis of the airways [ , ] , an increase in airway caliber necessarily implies that asm is transiently elongated. the question of whether, and to what extent, strain affects the contractility of asm is easier to investigate in vitro. this is because in vitro experiments can specifically control or monitor the length of asm. several types of asm preparations derived from resected or postmortem lung specimens are used to study asm mechanics. they include rings, spirals and segments of bronchi; strips and segments of tracheas; precision cut lung slices; and strips of lung parenchyma. our understanding of asm mechanics evolves quickly due to the use of these in vitro preparations. they can truly address questions that would otherwise be impossible to investigate in vivo. nowadays, the tools to study asm mechanics in vitro can recreate the dynamic lung movements that occur during breathing maneuvers . they thus emulate the in vivo environment within which asm normally resides and operates. this was a steppingstone in the field of asm mechanics. it has provided instrumental insights regarding the response of asm to stress and strain. these in vitro studies demonstrated that perturbing the length of asm greatly affects its contractile capacity. this effect is certainly relevant to the bronchodilator effect of a di. however, the reported in vitro results are also plagued with a lack of consistency. the main concern relates to the size of the effect, i.e., the magnitude by which strain decreases the contractile capacity of asm. however, the discrepancies go as far as being contradictory, as some studies reported that strain increases both airway responsiveness in vivo [ ] [ ] [ ] and the contractile capacity of asm in vitro [ ] . the controversy also extends to studies performed with isolated asm cells, where it was shown that strain can both decrease and increase surrogates for asm contractility [ , [ ] [ ] [ ] [ ] [ ] . all together, these findings are conducive to alternative conclusions regarding the potential contribution of asm strain to the bronchodilator effect of di. the purpose of the present review is not to discuss the variety of factors that may be responsible for the inconsistencies reported in in vitro studies, but rather to focus on one possible source of error, namely the magnitude by which asm is strained during a di. i attempted, based on published studies on human subjects in vivo, to determine the physiological range of strains that asm undergoes in vivo during a di from functional residual capacity (frc) to total lung capacity (tlc). the first section describes the methods that can be used to estimate asm strain. the second section discusses the limitations associated with those methods and to which extent they may affect the accuracy of the reported strain. finally, the third section describes the confounders that affect asm strain and that inevitably contribute to its variability. [ ] v d (using co as the tracer gas) healthy baseline exercise, co inhalation ( - %) or both all . % [ ] v d (using co as the tracer gas) healthy baseline always from tlc, expired to a given lung volume, and then inhaled different allocated tidal volume to subsequently measure v d on the subsequent expiration. all . % [ ] whole-body plethysmography the complexity of the question in hand stems from the difficulty to assess asm strain in vivo. although refining technologies are currently emerging, no direct measurement of asm strain during a di has yet been performed in vivo. however, many technologies provide reliable measures of luminal volume, airway caliber and resistance. these measurements at different lung volumes then allow approximation of the extent to which asm is strained during a di from frc to tlc. this section describes the methods that may be used to assess airway wall strain in vivo in humans. the major strengths of each method are also briefly stated. . dead volume. the measurement of dead volume (v d ) is the more archaic [ ] but still useful way to estimate the luminal volume of the airway tree at any given lung volume. the change in v d from one lung volume to the other then allows estimation of the strain undergone by the airway tree during such an excursion of lung volume ( table ). the measurement relies on the fact that the last part of the inspired air in a breath does not reach the zone of gas-exchanging units. that last part is rather contained in the v d (also called the dead space), which includes the upper airways down to the terminal bronchi. since air in the v d does not mix with the alveolar gas, the composition of gas at the mouth during an expiration suddenly changes when it transitions from the air that was contained in the v d to the air that was contained in the alveolar space. the volume of air that comes out of the lungs before this transition of air composition is v d . because diffusion inevitably occurs at the boundary between v d and alveolar volume, and that the amount of diffusion is time-dependent, appropriate timing during the respiratory maneuver is essential to obtain an accurate measure of v d . fowler was the first to use the single-breath nitrogen (n ) washout to measure v d [ ] . this technique has been adopted and is still used [ ] [ ] [ ] . alternatively, some have used co as the tracer gas to estimate v d [ ] . johns and coworkers have demonstrated that a breath-by-breath analysis of v d is possible using co as the tracer [ ] . by progressively changing the lung volume, one can then obtain a slope of the relationship between v d and end-inspiratory lung volume. this slope has been abbreviated dv d and has been used to assess airway distensibility (i.e., the ease by which the airways dilate in response to increasing lung volume) [ ] . dv d is advantageous because it can be measured quickly in a noninvasive manner. . although x-rays in humans may provide enough resolution to detect abnormal size of the trachea [ ] , it is not sufficient to quantitate the differences that may occur during an excursion of lung volume from frc to tlc. the resolution can be improved by contrasting agents. for example, dionosil oily inserted through the cricothyroid ligament under local anesthesia, has been used successfully to measure the changes in diameter of large and relatively smaller airways during a full inspiration in man [ ] . fluoroscopy is somewhat better, as it allows motion recording. it has been used without contrast agents to monitor changes in airway caliber during breathing maneuvers. it was to assess the degree of collapsibility of the trachea during coughing in controls and in cases of acquired tracheomalacia [ ] . fluoroscopy may be appropriate to diagnose and assign a degree of tracheomalacia, as it was done in that study. however, it does not seem to provide note: aoct-anatomical optical coherence tomography, copd-chronic obstructive pulmonary disease, di-deep inspiration, fev -forced expiratory volume in s, fot-forced oscillation technique, frc-functional residual capacity, hrct-high-resolution computed tomography, laba-long-acting b agonist, mch-methacholine, pc -the provocative concentration of methacholine causing a % decline in fev , raw-airway resistance, rv-residual volume, saba-short-acting b -agonist, sbnw-single breath nitogen washout, tlc-total lung capacity, vc-vital capacity, and v d -dead volume. a it was assumed that asm is arranged orthogonally in relation to the long axis of the airways. b it was assumed that the volume of the airways expands isotropically during an increase in lung volume from frc to tlc. c it was assumed that the changes in resistance are inversely proportional to the changes in airway luminal radius at the fourth power. the resolution to quantitatively evaluate the changes in airway luminal area during a di. whole-body plethysmography. the measurement of airway resistance (raw) by whole-body plethysmography (raw (pleth) ) is routinely performed in laboratories of lung physiology. raw (pleth) truly represents the resistance attributed to the viscous flow of air passing through the airways. the practical and theoretical principles involved were previously expatiated [ ] . briefly, the subject is enclosed in a hermetic box of known internal volume. the subject is then instructed to pant inside the box into a tube connected to an open shutter and a pneumotachograph. the shutter is then suddenly closed while the subject tries to keep panting. the airflow at the mouth and the pressures at the mouth and inside the box are monitored continuously. the variations of pressure inside the box (dpbox) when the shutter is open reflect corresponding but opposite changes in alveolar pressure (dpalv). using the law of boyle-mariotte (v p ¼ p v ), the dpbox are converted into changes in volume (dvbox: often called the shift volume). the dvbox happens to be the mirror image of the changes in thoracic gas volume (tgv) caused by rarefaction and compression of air during the panting, which are required to generate the driving pressure (dpalv) that draws air in-and-out of the lungs. when the shutter is closed, the dvbox can be related to the changes in alveolar pressure (dpalv), since dpalv are then the same as the changes in mouth pressure. once the relationships between both airflow/dvbox (when the shutter is open) and dpalv/dvbox (when the shutter is closed) are known, the dpalv required to accommodate any chosen airflow (usually l/s) can be determined. the ratio of dpalv/airflow is raw (pleth) . conveniently, the same panting maneuver and boyle-mariotte's law also allow the tgv from which the subject is panting to be measured. the panting can be performed at different lung volumes to measure the relationship between raw (pleth) and lung volume. from this relationship, the airway wall strain from frc to tlc can be roughly estimated ( table ). the limitations are discussed below. the greatest advantages of the whole-body plethysmography are that it is noninvasive and nonionizing. deflation. the interrupted deflation can be used to measure pulmonary resistance (r l ) and to estimate airway resistance (or conductance). the subject is instructed to take a di to tlc and then to expire slowly (sometimes at a controlled rate) while the flow is being interrupted at the mouth for a short period at specified intervals. the flow at the mouth and p l need to be measured simultaneously. p l is measured conventionally using an oesophageal balloon to estimate pleural pressure and to subtract it from airway opening pressure. during the measurement, the change in lung volume is being tracked by integrating flow. this is required to relate punctual changes in p l caused by the interruptions to the lung volumes at which they were measured. the increase in p l caused by the interruption is the driving pressure required to accommodate the airflow (and the lung tissue flow) just preceding the interruption. since the resistance attributed to tissue flow (sometimes called tissue viscance) is small [ ] , especially during deflation, the rise in p l caused by the interruption of flow is mainly ascribed to the frictional resistance to airflow within the airway tree. by dividing the increase in p l to the flow at the mouth, airway resistance can then be calculated at every lung volume at which the interruption took place. the change in resistance between frc and tlc can then be converted to estimate asm strain the same way it is done for raw (pleth) (discussed below) ( table ). the advantage of the interrupted deflation in comparison to the measurement of raw (pleth) is that the breathing maneuver performed by the subject is much simpler. technique. the acoustic reflection technique is an especially good tool to diagnose tracheal stenosis and malacia [ ] . the use of this technique to assess large airway caliber has also been validated in glass tube models and in humans [ , ] . changes in large airway caliber caused by changing lung volumes were also estimated ( table ). the acoustic reflection technique consists of a sound wave emitted by a loudspeaker that is traveling along the airways where it is reflected and perceived by a recording microphone located on the external device near the subject's mouth. the time gap between the emitted sound and the reflections is a measure of the distance between the microphone and the points within the airway where those reflections come from. the amplitude of the reflections is a proxy of the change in airway cross-sectional areas at those specified airway locations. the greatest advantages of the acoustic reflection technique are that it is noninvasive, nonionizing, and quick. it is also easy for the subject, as no complicated breathing maneuvers are required. . high-resolution computed tomography. high-resolution computed tomography (hrct) has first been used by wilson and coworkers in to assess airway caliber at different lung volumes [ ] . since then, hrct was used extensively to assess the bronchodilator effect of di ( table ). the breathing protocol used varied widely, but a scan is normally taken at frc and then at tlc and the differences in airway caliber at corresponding airway locations are measured. hrct is still considered by some the gold standard for imaging the airways. this is because it assesses directly the geometry of individual airways. it is also noninvasive and does not require sedation. the tools to analyze ct scan have also evolved to provide an accurate measure of airway wall thickness. another important asset is the fact that many airways of different sizes can be assessed simultaneously. magnetic resonance imaging (mri) is a technology that is currently emerging to image the geometry of airways in vivo in humans. the technology has evolved to offer a good temporal resolution (scanning time of . s), providing images without blurring, even during cough [ ] . although mri is primarily used in the field of pulmonary medicine to image ventilation defects [ ] [ ] [ ] [ ] [ ] , it can now be used to obtain descent three-dimensional reconstruction of the tracheobronchial tree [ ] . however, the resolution does not equate hrct [ ] . despite other limitations that will be discussed in sec. , mri still represents an appealing noninvasive and nonradiating alternative strategy to image airway caliber. further developments are awaited before mri can accurately assess the changes in airway caliber that occur during a breathing maneuver from frc to tlc (table ). probe. the pitot static probe is a device used to measure the velocity of a moving fluid. it has several applications in modern world. a miniature pitot static probe (few centimeters long and mm external diameter) can be inserted into the airway tree to assess airway caliber on the basis of physiological measurements. the probe is positioned at desired heights down the airway tree, which is determined prior the measurements using a bronchoscope. the probe has an opening at the distal end leading to a first catheter coming out at the mouth, which is used to measure impaction pressure (also called total pressure or ptot). the probe also has side holes that merge into a distinct catheter to measure lateral pressure (plat). the difference between impaction pressure and lateral pressure is the drop of pressure due to convective acceleration (i.e., the kinetic energy of the gas passing the cross-sectional area). assuming incompressible air flowing in a tube with a blunt velocity profile, bernoulli equation can then be used to estimate air velocity (ptot ¼ plat þ qv / , where q is air density and v is velocity). the flow at the pitot static probe can also be calculated from the mouth flow and correcting for difference in air pressure at that location (barometric pressure þ plat) using boyle's law. with this local flow and velocity, one can than calculate the cross-sectional area at the location of the pitot static probe ( is flow in l/s and v is velocity in m/s). calculating the cumulative expelled volume by integrating the flow at the mouth allows for changes in airway caliber to be related to changes in lung volume. theoretically, one maneuver would allow the measurement of airway caliber at every lung volume. the greatest advantage of the pitot static probe is thus to follow the kinetics of change in airway caliber. the insertion of an esophageal balloon as a proxy of pleural pressure (ppl) can also provide additional insights. for example, by relating airway caliber to transmural pressure (ptm, which is plat-ppl), airway compliance (caw) can be obtained. the pitot static probe is not often used to assess airway caliber at different lung volumes. in fact, only one study documented the utility of the pitot static probe for that purpose. in that study, the measurement was made during a maximal expiratory flow-volume maneuver (mefv), i.e., the subject was instructed to inhale to tlc and then provide a maximal expiratory effort to rv. several limitations are associated with this method. of special concern is the instability of the probe during the maneuver. the plugging of the end and side holes by mucus and secretions is also common. maximal reproducible efforts are also required, which can be demanding for the subject. these limitations, together with the other limitations discussed in sec. , may have dissuaded many investigators from using this method. technique. this technique consists of forcing the movement of air in or out the respiratory track using different devices and to then calculate impedance from the resulting flow and pressure that are measured at different locations in relation to the subject (reviewed in ref. [ ] ). the most common devices force air directly into the subject's mouth and the resulting flow and pressure within the mouth are used to calculate impedance (input impedance). the motion of air imposed by the device can also take different shapes, but sinusoidal forcing at one or several simultaneous frequencies are often used. during the measurement, the subject is usually instructed to keep breathing normally at tidal volume. the forced oscillation is thus simply superimposed on top of the natural motion of air resulting from breathing. the forced oscillation technique (fot) has gained tremendous momentum in human respiratory research. this is because it provides valuable readouts pertaining to the mechanics of the respiratory system, including respiratory system resistance (rrs) and reactance (xrs) at every tested frequencies. these measurements allow one to infer on phenomena as complex as recruitment-derecruitment and airway caliber heterogeneity. one of the greatest advantages of the fot is its fine time resolution. because of this fine time resolution, it is possible to measure the kinetics of events happening over a short time-scale, e.g., such as the extent and dynamics of airway dilatation during a di. its usefulness is also ascribed to its noninvasiveness, the ease with which it is operated and the very low level of cooperation that is required from the tested subjects. forced oscillation technique can be used to estimate asm strain during a di from frc to tlc because rrs and its inverse, respiratory system conductance (abbreviated grs), represent proper proxies of airway caliber when measured near the resonant frequency ( - hz) [ ] . this is especially true at zero flow, when the resistance is not affected by frictional airway resistance to airflow. many have measured rrs (or grs) to assess the changes in airway caliber during a di (see table ). . anatomic optical coherence tomography. anatomic optical coherence tomography (aoct) is an in vivo imaging technology that broadcasts live the interior of the airways [ ] . aoct uses a fiber-optic probe that passes through a bronchoscope and emits a beam of near-infrared light toward the airway wall. the resulting reflections are detected and analyzed using lowcoherence interferometry, which allows the measurement of the position of the airway wall in relation to the probe. by rotating the probe, one can obtained two-dimensional cross-sectional images of the lumen, and by moving the probe forward or backward, these serial two-dimensional images can provide a full d representation of the airway lumen. although not used extensively so far, aoct is likely to provide insightful information regarding the excursion of airway caliber during a di. i am aware of only one published study that has used aoct for that purpose [ ] (table ) . a new cousin of oct has also recently been developed, called orientation-resolved oct (or-oct) [ ] . or-oct uses the birefringent property of asm to capture its exact localization within the wall of large airways. asm in humans in vivo can be visualized with a sensational level of resolution by this method [ ] . or-oct should ultimately enable the direct measurement of asm strain during a di from frc to tlc. none of the aforementioned methods directly measure asm strain. they rather reported a change in the cross-sectional area of the lumen in individual airways, a change in luminal volume of the entire airway tree or a change in resistance to airflow in the airways or to the whole respiratory system. this obliges the transformation of the original data into radial airway wall strain. for the sake of this review, it was then assumed that the airway wall strain can be used as an appropriate proxy of asm longitudinal strain. this section describes the limitations associated with several methods, as well as the limitations associated with the transformations and assumptions that were made to convert the original data into asm strain. muscle. the strain on the airway wall reported by most methods during the di refers to the change in perimeter at the apical surface of the epithelium (i.e., the size of the lumen). this is true for imaging methods such as bronchography, hrct, mri, and aoct, but also for the methods relying of physiological measurements, such as v d and resistance (plethysmography, interrupted deflation, acoustic reflection technique, pitot static probe, and fot). it is important to understand that the change in luminal geometry does not represent precisely the change in perimeter occurring at the middle of asm. due to the combined thickness of the epithelium, the lamina propria, and the asm, the extent by which asm is stretched during a di is slightly overestimated when the luminal geometry (radius, diameter, perimeter, and cross-sectional area) is used to calculate the change in asm length (fig. ) . the size of this effect is more prominent in smaller airways. in fact, the size of this effect increases as the ratio of the perimeter measured at the middle of asm to the perimeter of the lumen increases. the thickening of the airway wall observed in some respiratory disorders also amplifies the size of this effect (fig. ). smooth muscle alignment. the reported strain (the one estimated in table ) represents more accurately the radial strain on the airway wall than the longitudinal strain on the asm. this is because the orientation of asm within the airway wall varies. here, i assumed that asm is arranged in an orthogonal fashion in relation to the long axis of the airways. this assumption is correct for the trachea and the main stem bronchi. however, asm is oriented differently in smaller airways. the angle of orientation of asm relative to the long axis of the airways is on average deg [ ] . however, this angle is obviously another parameter that is variable from one airway generation to another, as well as from one asm bundle to another. the implication here is that the strain reported is somewhat overestimated compared to the real strain on the long axis of the asm bundle. for example, when one assumes a radial airway wall strain (viz., an increase of airway perimeter) of % with no longitudinal airway strain (viz., no lengthening of the airway) and an asm bundle oriented deg off the long axis of the airway, the strain on the long axis of the asm bundle is . % (fig. ) . the magnitude by which asm strain is overestimated depends on how far off the angle of orientation deviates from deg. the reality, however, is that the airway is also strained on its longitudinal axis (this is discussed below). if both the radial strain and the longitudinal strain of the airway increase by %, the asm are then also strained by % regardless of the orientation of the asm bundle in relation to the long axis of the airway (fig. ). containing airway smooth muscle. although the bundles of asm completely encircle the small airways, it is not the case for the trachea and the main stem bronchi. in fact, asm occupies approximately / and / of the total airway wall circumference in the trachea and the main stem bronchi, respectively. the remaining circumference is made up of cartilage. since the cartilage is stiffer than the asm, the strain is not distributed homogeneously along the entire circumference of the airway. in reality, near % of the change in circumference is taken up by / to / of the airway wall; the portion occupied by asm and free of cartilage. asm strain during a di in the trachea and the main stem bronchi may thus be well beyond the one estimated based on the change in airway caliber. dimensions. the estimation of asm strain during a di from frc to tlc is sometimes accomplished by measuring a change in volume. this is the case for the methods measuring and comparing v d at different lung volumes. to convert a change in volume into radial airway wall strain (or $asm longitudinal strain), an additional step is required. this is because not only the caliber but also the length of the airways fluctuates during breathing maneuvers [ ] . the relative extent by which the length of the airways elongate and shorten during breathing maneuvers compared to the extent by which they dilate and narrow have been poorly studied. one can assume that the percentage change is equal in every directions; so that the luminal airway volume expands or shrinks isotropically without changing shape during both inflation and deflation. some experimental data support this assumption [ , [ ] [ ] [ ] . studies showing that dv d is linearly related to dlung volume also support this assumption [ , ] . however, other evidences suggest that it is not always the case [ ] . in fact, the accuracy of this assumption seems greatly affected by the starting lung volume and the magnitude of the lung volume excursion [ , , ] . the strain in one direction relative to the other directions is also likely to vary between airways from different individuals and between airways within the same individual. regardless, the degree by which the deformation of the airways deviates from isotropy obviously affects the precision with which asm strain can be estimated by these methods. fig. the changes in luminal geometry overestimate the stretch the asm undergoes during a deep inspiration (di). the schematic illustrates a normal (left) and an asthmatic (right) airways at frc and after a di to tlc. the dimensions are zoomed but at scale to an average airway of the ten generation. the springs represent lung recoil. they are stretched at tlc relative to frc. the arrow at approximately o'clock is the radius (in mm) of the airway lumen (r l ). the arrow at approximately o'clock is the radius up to the middle of the airway smooth muscle layer (r m ). the material composing the airway wall was considered inextensible. notice the thinning of the airway wall when the lungs are inflating to tlc. it this schematic it was assumed that the luminal geometry at frc was equal between normal and asthmatic. it was further assumed that the di was increasing luminal radius by % in both normal and asthmatic, so that the luminal geometry at tlc was also equal between normal and asthmatic. the schematic demonstrates that a % increase of luminal perimeter (p l ) only causes a . % increase of the perimeter at the middle of the asm layer (p m ). this effect is further amplified in asthmatic because of a thicker airway wall ( versus . %). the mathematics is developed in the middle. other abbreviations: a aw -area of the airway wall from the lumen to the middle of the asm layer, a l -luminal area, a m -area internal to the middle of the asm layer, and %d-change in percentage. due to the elongation of the airway tree during a di, the relative distance of a specific point within an airway to a recording device at the mouth increases. this is an issue for several methods. this vertical displacement is important to consider in order to allocate a change in caliber from frc to tlc at specific airway locations. otherwise, erroneous changes are caused by comparing a upper (and larger) part of the airway to a lower part due to lung and airway elongation at tlc. for the acoustic reflection technique, coronal radiographs can be acquired. the radiograph allows the identification of the carina and to subsequently measure its distance from the microphone. the other measured areas can then be allocated to specific airway locations. since the carina is moving at different locations during a breathing maneuver, several radiographs for monitoring its location at different lung volumes is ideally required. however, this is not always done [ ] . it is thus likely that a small variability arises due to a vertical displacement of the airways relative to the recording microphone. to limit this problem, the cross-sectional area is usually averaged over a few centimeter-long segment. the vertical displacement is also an issue for the pitot static probe. indeed, the distance of the probe from the mouth is always the same, as the catheter is fixed at the mouth. this means that the probe is actually moving in relation to the airway tree during the forced expiratory maneuver. this is not taken into account with the pitot static probe. the issue of vertical displacement is more easily overcome by hrct. this is because an image of the entire (or a major portion of the) lungs is obtained. landmarks can thus be identified to assess the vertical displacement of the lungs during the di. the strain can be applied in the three dimensions, as well as in two directions (stretch or compress). the focus of this review is on the expansion of airway caliber during a di causing a stretch to asm. however, the elongation of the airways during a di also implies that asm is strained in at least another dimension, namely on its transversal axis. the effect of transversal strain on asm contractility has not been studied sufficiently. one study demonstrated that cyclical lengthening of isolated airways mainly increases their contractile capacity [ ] . the third dimension (thinner versus thicker airway wall) is also important to consider. one may think that an increase in p l should thicken the airway wall, for the same reasons as it increases the length and the perimeter of the airways. however, the materials constituting the airway wall hardly change in volume during a di compared to the air-filled compartments (i.e., the lumen of the airways and the alveoli). increasing the length of an airway and its caliber during a di to tlc should thus exert a tensile stress on the airway wall, which tends to make it thinner. many ct studies actually reported that the airway wall is thinner at tlc, supporting that the airway wall is compressed in this dimension during a di [ , ] . this was also shown in cat lobes frozen at different volumes [ ] , as well as in isolated airways stretched to the letter a represents the airway perimeter and, in this example, is set to mm. the letter b is the airway longitudinal distance covered by the asm bundle going around the full circumference of the airway. finally, the letter c represents the length of the asm bundle and, in this example, is set at an angle deg off the long axis of the airway. using trigonometry, b and c can be determined. now imagine that this airway is stretched radially to increase its perimeter by %. on the flattened airway shown in (a), this would increase the height of the rectangle by % without changing its length (b). compared to (a), the length of a would increase by %, the length of b would remain unchanged and the length of c would increase by . %. the angle of c would also change to . deg. therefore, a radial stretch to the airway increasing its perimeter by % is expected to strain the asm bundle by only . %. now imagine that the airway length is also strained by %. on the flattened airway shown in (b), this would increase the length of the rectangle by % (c). compared to (a), the length of a, b, and c would all increase by %. in contrast to (b), the angle of c would remain unchanged. therefore, when both radial and longitudinal strains are applied simultaneously at the same magnitude on an airway, the strain on the asm is also of this magnitude. elongated lengths [ ] . overall, these suggest that the airway wall is stretched in two dimensions and compressed in one dimension during a di to tlc. the asm should thus be longer and wider but thinner at tlc. this is different from stretching an asm bundle in vitro, where it becomes longer but consequently less wide and less thick. thus, in contrast to in vivo, the asm in vitro is stretched in only one dimension and compressed in two directions. the effects of varying the orientation and the direction of strain on asm contractility have been largely overlooked. only few studies conducted with isolated cells have examined the effect of strain orientation on asm contractility [ , [ ] [ ] [ ] [ ] [ ] . the findings suggest that strain orientation has a major impact on the outcome. to the best of our knowledge, no study has yet investigated the impact of strain orientation and direction, as well as their different combinations, on the contractility of asm tissues. these studies are clearly warranted. airways. the effect of the upper airways (mouth, larynx, and pharynx) cannot always be discounted. whether the upper airways can be considered a mere extension of the lower airways can be justifiably questioned. some evidence suggests that the cross-sectional area of upper airways is also dependent on lung volume [ ] . however, the upper airways also have an intricate geometry. the concepts of fluid mechanics that apply to a single rigid cylindrical tube in which the flow regime is laminar might not be relevant. for example, the flow path during an expiration bends when it reaches the larger space of the mouth. the flow path also encounters many protruding structures and diverticula that can alter its flow regime such as the vocal cords, the uvula, the epiglottis, the tongue, and the alimentary and nasal canals. all these structure are likely to foster turbulent flow. the measurements of resistance by the fot, the interrupted deflation, and the whole-body plethysmography are affected by upper airways. in the case of raw (pleth) , attempts were made to subtract upper airway resistance from raw in order to obtain lower raw [ ] . in these experiments, the pressure difference between two lateral taps, one at the mouth and one inserted midstream in the tracheal lumen via a puncture - cm below the cricoid cartilage, was measured. this difference in pressure over the mouth flow was then used to estimate upper raw and to subtract it from raw (pleth) [ ] . the acoustic reflection technique is one convenient method to assess upper airways. this is because the time at which the signal is collected can be allocated to a specific distance from the mouth. the intensity of the signal is thus dictated only by the structural features that are in place at that distance. however, it does not provide information regarding the structural features altering the signal. therefore, a notch, a dent or a circular decrease in caliber are equally detected. capturing the excursion of small airway caliber during a di is a challenge. indeed, many methods described in sec. are restricted to the assessment of large airways. this is probably the main limitation of the acoustic reflection technique. according to the results obtained with rigid casts of the airway tree, the areas determined acoustically agreed well with the real areas for airway segments extending up to cm past the carina [ ] . therefore, the airways further down the main bronchi cannot be assessed. this obviously restrains the use of the acoustic reflection technique for the assessment of airway wall strain during di. also, only one measurement per distance can be obtained. so beyond the branching point, the signal emanates from the cumulative effect of the two main bronchi and their respective contribution cannot be distinguished. the pitot static probe is also limited to the assessment of the large airways. the lowest position measured in brackel and coworkers' study was at the entrance of the middle lobe [ ] . in this case, the penetration depth of the device is limited by its size in relation to airway caliber. the same constraints apply to aoct. high-resolution computed tomography is probably the best method to assess the small airways. yet, adequate resolutions are restricted to airway size over $ mm of internal diameter. the contribution of the small airways is certainly embodied in measurements such as v d , raw (pleth) , and fot. however, the extent by which the small airways contribute to the overall signal cannot be quantified. fot was initially suggested by some to distinguish the small from the large airways. the rationale lies in the traveling distances of the oscillations at different frequencies. while oscillations at low frequencies travel far down into the lungs, the traveling distance of high frequencies are shorter. therefore, while the low frequencies should probe the entire airway tree, the high frequencies should only probe the large airways. by looking at the frequency-dependence of rrs, or by using a simpler readout such a r - (which is the subtraction of rrs at hz from the rrs at hz), it was suggested that the degree of obstruction of the small airways can be deduced. although this line of reasoning is logical, this is no longer the prevailing belief. first, the penetration depth of the forced oscillations at different frequencies cannot be ascertained. it is thus not sure from how far deep into the lungs the signals used to calculate rrs at different frequencies emanate. second, and most importantly, computational models have clearly demonstrated that the frequencydependence of resistance is particularly sensitive to the pattern of constriction in peripheral airways [ ] [ ] [ ] . on one hand, a homogeneous pattern of small airway constriction increases rrs equally throughout the frequency range. on the other hand, a heterogeneous pattern of small airway constriction increases rrs way more at low than at high frequencies. therefore, the frequencydependence of rrs (or r [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ) is now considered a readout that is more useful to quantify the degree of airway narrowing heterogeneity in the periphery than to assess the degree of small airway obstruction. near total lung capacity. the measurements made at tlc are sometimes difficult and sometimes just not possible. raw (pleth) , for example, cannot be measured at tlc but only near tlc. the measurement requires a certain amount of air to flow in-and-out of the lungs to calculate raw (pleth) . this panting maneuver near tlc is also difficult for the subject. the measurement of r l with the interrupted deflation also required a certain amount of air to evacuate the lungs in order to measure the change of p l caused by the interruption. therefore, the value at tlc cannot be determined. holding the breath at tlc during hrct can also be difficult, especially for older and/or sicker patients. concerning the pitot probe, the airway caliber at different positions were grouped in volume range (e.g., the caliber between % and % of fvc). the total strain experienced by asm may thus have been underestimated because many measurements were not at tlc. notwithstanding this possibility, the error associated with not assessing airway caliber at tlc compared with near-tlc would be small since the airways are stiffer in that range of lung volume. the variation in transmural pressure may disproportionately exceed the strain on asm. in fact, submaximal volumes and/or p l are generally sufficient to achieve maximal dilation [ , ] . other issues are also more common at or near tlc than other lung volumes and can affect the accuracy of the measurement. for example, glottic closure commonly occurs at extreme volumes (rv and tlc). therefore, the changes in airway caliber sometimes need to be assessed within these volumes (i.e., a little above rv and a little below tlc), which may slightly underestimate the full range of airway wall excursion that occurs during a di from frc to tlc. . bronchial tree geometry. the estimation of asm strain during a di from frc to tlc is often accomplished by measuring the changes in resistance at different lung volumes. this is the case for the whole-body plethysmography, the interrupted deflation, and the fot. to convert resistance into asm strain, some arithmetics are needed. the calculation is based on poiseuille's equation. this equation states that within a tube with a perfect cylindrical geometry in which flow is running in a laminar fashion, airway resistance is inversely proportional to the luminal radius at the fourth power. a change in airway resistance at different lung volumes can thus be converted into asm strain, as the percent change in radius is the same as the percent change in airway perimeter (viz., $asm length). as aforementioned, the airways also elongate during a di. according to poiseuille's equation, resistance to airflow is proportional to the length of the airway. so while the increase in caliber at tlc decreases resistance, the elongation increases resistance. as resistance is related to the radius at the fourth power, the effect of changing caliber largely predominates over the effect of changing the length of the airway. however, the estimation of asm strain based on measurements of resistance might be slightly underestimated because the increase in airway length is usually not taken into account. it is understood that the airway tree is way more complicated than a single cylindrical tube with identical size on both ends. it is also understood that the flow is not always laminar. the airway tree branches in different directions, contains numerous bifurcations, and many bronchi are bending and exhibit a tapered end. all these elements obviously disturb flow and increase the degree of turbulence. notwithstanding this complexity, the most accurate computational model of the human lungs is the singlecompartment model [ ] . this model is merely constituted of a single airway with a perfectly cylindrical geometry that runs into a single elastic compartment. the resistance that stems from the viscous flow of air within the airways in a real airway tree, as well as its response to different interventions, thus seem to be predicted quite precisely just by altering the caliber of a single conducting airway in the modeled lungs. it seems that, sometimes, the gestalt behaves as a simple element. there is thus no reason to treat it differently as such in order to extract information that would otherwise be impossible to infer. the presence of chokepoints (sometimes called stenosis) is also important to consider. the excursion of asm length at these chokepoints during breathing maneuvers can be greatly altered [ ] . chokepoints also obstruct the dynamics of fluid. consequently, they severely increase resistance without necessarily affecting the caliber along the entire airway. the presence of chokepoints is thus likely to insert inaccuracies in the estimation of asm strain in methods using measures of resistance. in addition, chokepoints affect the regional transmural pressures, especially during forced maneuvers. while the transmural pressure downstream of the chokepoint increases during expiration (as the luminal pressure increases in the airways and alveoli subtended by the choked airway), it decreases upstream of the chokepoint. the opposite occurs during inspiration. either way, the transmural pressures are altered. the strain asm undergoes is thus attended to be affected in these regions. also, these chokepoints are usually not present at every lung volume. the chokepoint may thus affect resistance at low lung volumes but not at high lung volumes, and thus affect the change measured between two lung volumes. more generally, the entire geometry of the airway tree (not only chokepoints) is likely to change at different lung volumes, which inevitably affects differently the resistance to airflow. . chest wall. the chest wall can also affect some measurements. this is the case for the fot. the estimated change in airway caliber calculated from the change in rrs from frc to tlc needs to be taken with caution. this is because rrs at volumes surrounding frc is compounded by the resistance of the chest wall (rcw), which is not the case at tlc [ ] . consequently, the delta of rrs from frc to tlc overestimates asm strain. this is consistent with brown and coworkers' study, in which the estimated asm strain by fot using rrs was greater than the one predicted based on delta v d assessed by the single-breath nitrogen washout [ ] . r l estimated with the esophageal balloon can remedy this overestimation by discarding the effect of the chest wall. . ionizing radiation. the exposure to ionizing radiation is obviously a major concern. it is probably the most important downside of hrct. repeated measures should absolutely be avoided. unfortunately, the investigators are left with a few snapshots at a few chosen lung volumes to quantify the excursion of airway caliber, which is far from ideal. radiation is also a concern with bronchography, although the doses used are lower. many factors influence the estimated strain asm undergoes during a di from frc to tlc and should thereby be considered as confounders. the confounders can be related to methodological procedures (an intervention or the breathing maneuvers during and preceding the measurement), which can either be controlled or not. alternatively, some confounders are inherent biological factors that cannot be controlled. this section describes the many procedural and biological confounders that are known to affect airway wall strain during a di. the magnitude by which the airways are strained during a di is dictated in great part by the swing in transmural pressure (i.e., the pressure across the airway wall). the swing in transmural pressure during a di depends on the extent by which the lung parenchyma is pulling the airway open, which, in turn, depends on the transpulmonary pressure (p l ). a greater swing in p l and/or lung volume during a di inflicts a greater excursion of airway caliber and, thereby, a greater stretch to asm. p l and the lung volume attained at tlc are variable between individuals. these biological factors thus affect asm strain during a di and should thus be considered as important confounders. as discussed above, many methods that are used to estimate asm strain from frc to tlc rely on tricky breathing maneuvers near tlc (e.g., raw (pleth) ) or a holding maneuver at tlc (e.g., hrct). the level of inspiration that is achieved/maintained by the subjects during such maneuvers relative to their maximum also contributes to variability [ ] . it is also important to mention that considerable regional differences in p l exist within the lungs. during the same breathing maneuvers, the airways in lower lobes are more strained than airways of the same generation in the apical lobes [ , , , ] . the estimated strain asm undergoes is thus highly influenced by the region of the lungs in which the airways are embedded. pressure and volume. the pressure-volume curve of the lungs is not linear, so as the curve defining the relationship between transmural pressure and airway radius. by changing the starting (frc) transpulmonary pressure or lung volume not only the swing in pressure and/or volume from frc to tlc may be affected [ ] but it also implies that the airways are now operating on a different part of the transmural pressure-radius curve. this is important as it indicates that the same swing in pressure now translates to different changes in radius and thus to different degrees of asm strain. this was predicted by computational modeling [ ] and clearly shown in vitro [ , ] . an increase in frc (i.e., hyperinflation) also impairs the ability to generate high p l [ ] . consequently, it reduces the pressure distending the airways during a di. in fact, hyperinflation is a strong predictor of the bronchodilator effect of di [ ] . more severe is the hyperinflation, smaller is the excursion of airway caliber from frc to tlc and less efficient is the bronchodilator effect of a di [ ] . lung volume and p l at frc also vary between individuals. these biological factors are thus important confounders that affect the strain asm undergoes during a di. all the methods used to estimate asm strain during a di rely on measurements made at frc. they are thus influenced by the starting lung pressure and volume. . hysteresis. the pressure-volume curve of the lungs during inflation and deflation do not overlap. in fact, when the two curves are combined they form a loop that rotates clockwise, meaning that the volume at any given pressure is greater during deflation. this is called hysteresis and is due to the resistive component of the lungs, which is chiefly perceived during inflation but dissipated and barely not perceived during deflation. this hysteretic behavior also applies independently to both the airways and the lung parenchyma. therefore, when tracking the transpulmonary pressure and the cross-sectional area of an airway during breathing, a loop is also formed. however, the direction of rotation depends of the relative hysteresis between the airway and the surrounding parenchyma. when the airway is more hysteretic than the parenchyma the loop rotates clockwise. inversely, when the parenchyma is more hysteretic than the airway the loop rotates anticlockwise. in healthy individuals, airway hysteresis dominates and is largely due to asm tone [ , ] . this was also shown in isolated airways [ , ] . hysteresis has important implications. it indicates that the caliber of an airway at a particular lung volume varies according to whether it follows a deflation from a higher lung volume or a inflation from a lower lung volume [ ] . after either a deflation or an inflation, the caliber can be larger or smaller depending on whether airway or parenchymal hysteresis predominates. for example, when airway hysteresis predominates, the caliber of the airways at frc after a di to tlc is greater than the caliber at frc prior to the di. in essence, this is the definition of the bronchodilator effect of a di. hysteresis also implies that the estimated strain asm undergoes during a di is not the same when the measurements are made from frc to tlc than when they are made from tlc to frc. an important procedural confounder is thus related to whether the change in airway caliber is measured during inflationary maneuvers versus deflationary maneuvers. the methods used to estimate asm strain described in sec. involve either types of maneuvers. for example, the measurements of r l at different lung volumes with the interrupted deflation are performed during deflation. the pitot static probe also takes the measurement during a forced deflationary maneuver. contrastingly, the change in crosssectional area of the airway lumen with the mri is measured during inflation. this is because the image has to be captured during the flow of hyperpolarized gas ( he or xe) into the conducting airways in order to achieve a better contrast between the airways and the parenchyma. this also implies that the image is not obtained at fixed volumes but rather during a dynamic change in lung volume. the caliber at which an airway settles after a perturbation, such as a di, also takes time due to hysteresis. the timing at which the measurements are made can thus affect the results and should thereby be considered as a confounder. likewise, some methods used to estimate asm strain require interferences with the normal pattern of breathing. hrct, for example, requires breath-holding to assess airway caliber at different lung volumes. the breathhold usually decreases the bronchodilator effect of a di [ , ] . however, whether the breath-hold overestimates or underestimates airway wall strain depends: -on the relative hysteresis between the airways and the parenchyma; -at which lung volume it is measured; and -whether the lungs were inflating or deflating before the breath-hold. regardless, the longer time that is allowed for the airways to settle at a new lung volume is likely to affect its caliber. consequently, the estimated strain excursion undergone by asm during such a change in lung volume will also be different compared to the one caused by the same change in volume with no breath-hold. all the other methods using measurements made at static (or quasi-static) lung volumes instead of dynamic lung volumes are likely to be affected by this procedural confounder. other procedures that clearly affect the estimation of asm strain include forced breathing maneuvers, such as forced expiratory volume in s (fev ) and coughing. the transitory peak of negative airway transmural pressure achieved during these maneuvers can transiently narrow the airway lumen to a level beyond that observed at the end of the maneuver [ , , , ] . this phenomenon is also known for causing dynamic collapses and expiratory flow limitation. one method used to estimate asm strain that can certainly be affected by forced maneuvers is the pitot static probe. the flow rate (or speed of inspiration) during the di is also important to consider. a di at a high flow rate is more effective to bronchodilate the airways than a di at slow flow rate [ , , ] . this was also demonstrated with isolated airways [ , ] and asm strips stretched at different rates [ ] . an increase in transmural pressure was proposed to account for the greater bronchodilator effect observed at high flow rate [ ] . with regards to the estimation of asm strain, both inspiratory and expiratory forced maneuvers should be considered as procedural confounders. beyond the different breathing maneuvers used during the measurements (inflation versus deflation, static versus dynamic, slow versus forced maneuvers), the sequence of events preceding the measurements should be considered. the most prominent example is the bronchoprotective effect of a di [ ] [ ] [ ] [ ] [ ] [ ] . so a di, or a series of dis, prior to a bronchoconstriction elicited by inhaled methacholine, facilitates bronchodilatation induced by a subsequent di postmethacholine [ ] . the time elapsed between the di premethacholine and the subsequent di under a bronchoconstricted state also affects its bronchodilator effect [ ] . the time spent without di under bronchoconstriction also affects the excursion of airway caliber during a di because it increases narrowing [ , ] . the bronchodilator effect of a di is also not the same after a sequence of dis than after a single di [ ] . all together, these results indicate that the strain asm undergoes during a di is affected by lung history. the geometry of the airways at frc can also be affected if the preceding measurements were performed at rv compared to if they were performed at tlc [ ] . the caliber of the airways at a given lung volume is greater when preceded by measurements done at a higher lung volume [ ] . as explained above, part of it is due to hysteresis. notably, history can affect the measurements in both directions, as it can either increase or decrease the estimated strain asm undergoes during a di. lung history thus represents a serious procedural confounder and should be controlled whenever possible. . posture. the lungs undergo important deformation during changes in posture. this is because it changes the direction of the gravitational forces, which then affects the local p l and the gradient of p l [ ] . a computational model predicted that a change of $ cmh o can occur at specific locations in the lungs during a change in posture from supine to prone [ ] . in turn, this can be expected to change lung volume by approximately twofold, depending on which part of the pressure-volume curve this change in pressure occurs. this is then predicted to strain asm by $ %, assuming that the perimeter of the airways changes proportionally to the changes in the cube root of lung volume. a change in posture also affects the starting (frc) lung volume, engendering the consequences stated above. for example, frc is decreased in supine posture [ ] . the methods used to estimate asm strain involved measurements at different postures. the measurements of airway resistance by fot and plethysmography are usually performed seated upright. contrastingly, hrct and mri are usually performed supine. the changes in gravitational forces due to these different postures are likely to affect asm strain differently in different locations of the lungs. the posture should thus be considered a procedural confounder. all the elements that constitute the airway wall can be organized differently, in addition to vary in absolute amount and relative proportion [ ] . these elements include the cellular and the extracellular components of the airway wall, as well as other structures such as the cartilages. any elements constituting the airway wall should thus be considered as biological confounders. this is because any change in the structural composition of an airway is likely to modify its mechanical properties and thereby affects the strain asm undergoes during a di from frc to tlc. many of these elements vary in healthy subjects according to airway size. small airways, for example, has a structural composition that makes them more compliant [ , , , , , ] . their percentage of dilation during a di is thus greater compared to larger airways [ , ] . interestingly, it was shown in mice that the specific compliance of the small airways are greater than the specific compliance of the lungs [ ] . the p l at which the airway dilation plateaus during an inflation maneuver also varies with airway size. in healthy individuals, the p l at which this plateau occurs increases progressively with increasing airway size [ , ] . this indicates that maximal airway dilation occurs rapidly in small airways during inflation, while the large airways continue to dilate until later on in the maneuver. this is concerning for the methods that use the change in the entire volume of the airway tree in order to estimate the longitudinal strain on asm. this is because these methods assume that all the airways expand to the same extent. as stated above, airways of different size have different compliance. their compliance also varies according to the pressure range across which they are studied [ , ] . in airway generations (trachea) through , the curve that describes the relationship between luminal area and p l in every generation is sigmoidal. in fact, most of the change in luminal area occurs within a narrowed range of p l of about cmh o [ ] . therefore, a limited window exists where changes in p l actually cause changes in airway caliber. this implies that no change in airway caliber occurs at either low and high p l . contrastingly, in the steeper part of the curve, large changes in caliber occur across a very small range of p l . similar to the mouse study stated above [ ] , the specific compliance of the small airways in dogs and sheep are greater than the specific compliance of the lungs in that steeper part of the curve [ , ] . collectively, these last observations suggest that the starting p l , the maximal p l attained during the di, and the curve describing the relationship between p l and the caliber of the airways under scrutiny need to be known in order to allocate an exact amount of strain on asm during a di. as mentioned above, the swing in transmural pressure during a di is a major determinant of asm strain. apart from the transpulmonary pressure, the transmural pressure across the wall of any airway is influenced by the stiffness of the surrounding lung parenchyma and the degree of interdependence between the airway and the parenchyma [ ] . a more compliant parenchyma may dampen the swing in distending pressure generated by lung inflation and thus limit dilation. a loss of connectivity between the airway and the parenchyma, due to alveolar detachments [ ] or geometric decoupling due to thicker adventitia [ ] , also limits the transmission of pressure across the airway wall. the stiffness of the surrounding parenchyma and the level of interdependence between the airways and the parenchyma are thus biological confounders that are important to consider. . age. aging influences the mechanical properties of the lungs and its constituents [ ] . age should thus be considered a biological confounder. for example, the extensibility (i.e., the ease by which it is elongated) of extraparenchymal airways, such as the trachea, decreases with age [ ] . in contrast, intraparenchymal airways and the parenchyma seem to be more distensible [ ] . indeed, loss of lung elastic recoil was reported with aging [ ] . the larger percentage change in caliber of small versus large airways during a change in lung volume also seems to vanish with age, probably due to an increase in lung compliance [ ] . age can thus affect asm strain during a di from frc to tlc due to various reasons. these phenomena may account for the attenuated bronchodilator effect of di with aging [ ] and the lack of bronchoprotective effect of di in infants [ ] . . health status. respiratory disorders can influence to various extent and by several ways the strain asm undergoes during a di. the driving pressure, the mechanical properties of the airways and the lung parenchyma, as well as the forces of interdependence can all be affected by diseases. in fact, changes in the structural composition of the airways, a process called remodeling, are common in many respiratory disorders and can have a major impact on the mechanical properties of the airway wall [ ] . airway distensibility is decreased in asthma [ , , , , , , ] , especially at high [ ] but also at low lung volumes [ ] . this seems to be mainly caused by airway remodeling [ , ] but also inflammation [ , ] . asthmatic patients also tend to exhibit airways of smaller caliber [ , , , ] . the sigmoidal curve that describes the relationship between p l and airway caliber is also shifted to the left in asthmatic patients, at least when a bronchodilator (salbutamol) is administered prior to measurements [ ] . this suggests that the changes in airway caliber due to breathing maneuvers occur at lower p l in asthma [ ] . this may explain why fluctuation of rrs during tidal breathing is increased in asthma [ ] . interestingly, this increased tidal fluctuation of rrs also negatively correlates with the bronchodilator effect of di [ ] . this is somewhat logical. by operating at a steeper part of the sigmoidal curve during tidal breathing, less of the total possible strain is available during a di to tlc. in contrast to asthmatic patients, the lungs of nonasthmatic individuals seem to breath in a range of p l below the one where airway caliber is greatly affected by small changes in p l (based on small tidal fluctuation of rrs [ ] ). however, for the same reason, the total possible strain the airways can undergo is still available and may occur during a di to tlc. the benefit is a greater stretch to asm and, thus, a commensurately greater bronchodilator effect of di. finally, a decline in lung elastic recoil was also reported in longstanding asthma [ ] [ ] [ ] . all these phenomena may account for the attenuated bronchodilator effect of di in asthma [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , as well as its attenuated bronchoprotector effect [ , , ] . airway distensibility also seems to be attenuated in copd patients [ , ] , especially in emphysema-predominant compared to airway-predominant subtypes of copd [ ] . in this case, the lower apparent distensibility seems mainly attributed to a loss of recoil due to parenchymal destruction [ , ] , as well as hyperinflation, which limits inspiratory capacity and the driving p l during the di [ ] . these phenomena may account for the attenuated bronchodilator effect of di in copd [ , , ] . in contradistinction, the distensibility of the airways is increased in patients with cystic fibrosis [ ] . lung volume dependence of tracheal cross-sectional area in these patients was calculated to . cm /l, compared to almost nil in healthy subjects. cystic fibrosis patients also demonstrate increased lung compliance [ ] , which may limit the swing in p l . therefore, the reported increase in tracheal distensibility may be attributable to flaccidity, as previously reported in older patients with cystic fibrosis, as well as postmortem on isolated tracheas from patients with cystic fibrosis [ ] . bronchiectatic bronchi, which are common in cystic fibrosis patients, are also prone to bronchomalacia. it is important to mention though that the incidence of bronchomalacia is high ( %) in patients with bronchiectasis with or without coexistent lung disorders [ ] . even nonrespiratory disorders are sometimes important to consider. allogeneic haematopoietic stem-cell transplantation, for example, increases the changes in airway caliber during a lung excursion from frc to tlc [ ] . this seems to be entirely related to an increase in lung parenchymal stiffness rather than an increase in airway distensibility. obesity is another example that can significantly impact asm strain during a di, mainly by changing lung volumes [ ] . this phenomenon may account for the attenuated bronchodilator effect of di in obesity [ ] [ ] [ ] . most respiratory disorders also affect the airways nonuniformly [ , ] . the amount of strain the asm experiences during a di is thus expected to vary according to this patchy pattern of affection. overall, diseases can exert a huge and variable impact on the strain asm undergoes during a di. they should thus be considered as biological confounders. among all the elements that constitute the airway wall, asm is one that is very special. this tissue is endowed with the ability to quickly adjust the mechanical properties of the airway wall. greater is its level of activation, stiffer are the airways. in turn, stiffer airways are intuitively expected to expand less in response to a di. however, this is not always the case. the change in airway caliber caused by a simulated di is sometimes greater when asm is activated by methacholine than when it is relaxed by atropine [ ] . this is because increasing the level of asm activation may also cause airway constriction, which affects asm strain during a di for at least two reasons. first, it modifies the length of asm before the di, which impacts its contractile capacity. more precisely, shortening the length of asm decreases its contractile capacity [ , ] . second, airway constriction also changes the positions over which the airways are operating on the pressure-radius curve [ ] . following airway narrowing, the di thus takes place on a more compliant part of the pressure-radius curve and thereby increases the strain for any given stress [ ] . therefore, while the maximal distention attained by the airway during a di is certainly limited by the level of asm activation, the strain excursion may not. these have important implications for in vitro studies that attempt to emulate in vivo situations. for example, it may be appropriate to impose an equivalent level of strain on asm during a simulated di with or without spasmogeninduced contraction. however, this simulation only recreates adequately the in vivo situation if the asm is also adjusted to a shorter starting length in the presence of a spasmogen. the intrinsic level of asm activation is tuned by many spasmogens and bronchodilators that are produced endogenously [ ] . the intrinsic level of asm activation should thus be considered a biological confounder. however, some interventions also affect the level of asm activation, which consequently changes the stiffness of the airway wall and the strain caused by a di. on one hand, many methods used to estimate asm strain during a di intentionally manipulate the level of asm activation with bronchoactive substances (bronchoconstrictor such as methacholine or bronchodilator such as salbutamol). some studies are actually designed to dissociate the active contribution of asm in determining airway wall stiffness from the passive structural components of the airway wall [ , , , ] . these studies investigated the response to dis before and after the activation or the inhibition of asm. on the other hand, many methods used to estimate asm strain during a di unintentionally manipulate the level of asm activation by utilizing topical or systemic anesthesia. this is the case for aoct [ ] . for these reasons, the level of asm activation should sometimes be considered a procedural confounder. some patients also take medications with either short or long duration of action. those are likely to affect the stress-strain relationship of the airway wall and thus the distension of the airways during a di. they should thus be considered as confounders. the duration of withholding from these medications prior to measurements is also an important confounder to take into account. apart from medications, some interventional procedures also have the potential to affect asm strain during a di. this is the case for bronchial thermoplasty, which is an alternative treatment for severe refractory asthma. it was demonstrated in dogs that bronchial thermoplasty increases airway caliber at any airway pressure from to cmh o [ ] . the change in airway caliber though from to cmh o was identical pre-versus postbronchial thermoplasty. based on these observations, geometrical considerations need to be taken into account to understand the impact of bronchial thermoplasty on asm strain. airway caliber changes proportionately to the square of airway perimeter (viz., $asm length). the same change in caliber induced by rising the pressure from to cmh o in an airway starting at a larger caliber implies that asm strain was attenuated postbronchial thermoplasty. therefore, the initial (frc) length of asm may be greater after bronchial thermoplasty, but the strain asm undergoes during the di may be attenuated. the goal of this review was to estimate the strain asm undergoes in vivo in humans during a di from frc to tlc. this transient stretch is important to quantify as it was proposed to contribute to the beneficial effect of di by decreasing the contractile capacity of asm. however, it remains unclear to what extent asm is strained during a di. despite this gap in knowledge, the effect of strain on asm contractility has been tested extensively in vitro. a variable range of strains at different frequencies and for different durations in a variety of asm preparations have been tested. we came to learn that the dynamic environment in which asm operates in vivo has the potential to greatly affect its contractile capacity. in general, the in vitro results indicate that the decline in contractile capacity is largely dictated by the amount of strain [ ] [ ] [ ] [ ] [ ] , , [ ] [ ] [ ] , which is consistent with in vivo results in rabbits [ ] and humans [ , , , , , ] , as well as with isolated cells [ ] . together, these studies support the hypothesis that the beneficial effect of di on respiratory mechanics is related to the decrease in the contractility of asm elicited by the stretch. however, the in vitro results are also variable, sometimes contradictory, and it is still uncertain if the strain required to significantly decrease the contractile capacity of asm is physiologically attainable [ , ] . incorporating an appropriate range of strains in these in vitro studies is obviously essential in order to understand the integrated role of asm in respiratory mechanics. as seen in sec. of this review, many former methods and more recent technological advents can be used to estimate asm strain in humans in vivo during a di. on one hand, some methods assess the distensibility of the entire airway tree. these methods have the advantage to assess the effect of all the airways combined, but provide little insights on the localized response in any given airway and miss completely in reporting the spatial heterogeneity of the response. they are also unable to distinguish between radial versus longitudinal airway strain. among those methods, the measurement of v d , rrs near the resonance frequency with the fot and raw by whole-body plethysmography are the most relevant. the fot is especially appealing and increasingly used in clinical settings. combined with imaging technique and computational modeling, fot may eventually be able to document the changes in airway caliber during breathing maneuvers with a tremendous temporal and spatial resolution [ , ] . on the other hand, other methods assess individual airway distensibility. these methods have the advantage to measure directly the localized response, but usually fail to assess the small peripheral airways. the better being hrct, which can provide adequate resolution for airways down to $ mm of internal diameter. the other methods include bronchoscopy, fluoroscopy, acoustic reflection technique, mri, pitot static probe, and aoct. these methods were all very useful as they have clearly demonstrated that asm strain during a di differs according to airway size and location. as seen in sec. of this review, several limitations need to be considered as none of these methods assess asm strain directly. each of these limitations can putatively add inaccuracies that are sometimes not negligible. a few assumptions also need to be made in order to transform the original data into asm strain. the veracity of these assumptions can certainly be argued. owing to these limitations and assumptions, it is understood that the strains reported in this review are nothing more than estimations. as seen in sec. of this review, a single value of asm strain cannot be attributed to a di. this is because many procedural and biological confounders contribute to the variability between individuals and between airways within the same individual, as well as to the temporal variability of any given airway of a given individual [ ] . at this point, it seems more appropriate to provide a rough estimated range of strains that is physiologically relevant. despite the different setbacks of each method and the many confounders, the level of concordance between the methods for predicting the level of asm strain during a di is decent (table ) . this suggests that, although a greater level of temporal and spatial resolution can now be achieved, the new and refined technologies have simply confirmed the estimations made previously with antecedent methods. set apart the stiffer trachea and main stem bronchi, the longitudinal strain asm undergoes during a di from frc to tlc is estimated to reside within the range of - %. these values should assist investigators who attempt to impose a physiologically relevant level of strain to different asm tissues in in vitro settings in order to mimic a di. it is also important to realize that the value of strain that i was chasing in this review is the maximal attainable strain, i.e., the one achieves at tlc. involuntary dis, or should i call them sighs, which occur spontaneously every few minutes [ , , ] , do not reach tlc. provided that an in vitro study is interested to investigate the regulatory effect of these sighs on asm contractility, the range of acceptable strains is quite large. this is because beyond all the factors discussed in this review, the size of the breath during a sigh is obviously variable. the strain asm undergoes during sighs is thus commensurately variable. any intermediate values of strain occurring between frc to another higher lung volume anywhere from end-tidal inspiration to tlc can justifiably be used to simulate these spontaneous respiratory motions of the lungs. i think the one advice to take from this review is to stay within the reported range of strains and, more importantly, to never exceed the limit of what is physiologically attainable. to simulate more accurately particular in vivo situations, more data will be needed. indeed, owing to the number of biological confounders affecting the variability, more studies using different populations of patients and controlling for as many confounders as possible are required. these studies will allow us to adjust the strain in our in vitro settings according to the particular real-life situations that we are trying to model. in turn, applying a correct range of strains in our in vitro settings should: -ameliorate the validity of these in vitro simulations; -justify the incorporation of those in vitro results in 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asthma bronchomotor effect of bronchoconstriction-induced deep inspirations in asthmatics respiratory impedance measurements for assessment of lung mechanics: focus on asthma oscillometry and pulmonary mri measurements of ventilation heterogeneity in obstructive lung disease: relationship to quality of life and disease control spontaneous sigh rates during sedentary activity: watching television vs reading pattern of ventilation in young adults key: cord- - gr lh w authors: khraif, rshood m.; salam, asharaf abdul; nair, p. s.; elsegaey, ibrahim title: migration in saudi arabia: present and prospects date: - - journal: india's low-skilled migration to the middle east doi: . / - - - - _ sha: doc_id: cord_uid: gr lh w as compared with other gulf cooperation council (gcc) countries, saudi arabia has distinct demographic features. this leads the saudi government to take measures to revisit its existing policies and schemes concerned with population. with this brief outline, migration data used in this article are pitched to understand five dimensions: ( ) to look at the saudi citizens’ migration trends within the kingdom, ( ) to analyse the impact of migration on the kingdom’s population growth and also on the neighbouring gcc countries, ( ) to look at the changes taking place annually in the kingdom’s migration trends and in the other gcc countries, ( ) to examine the migrants, looking at their origin place and ( ) to analyse the variation between immigrants and saudi arabia citizens in terms of demographic parameters like population growth, sex ratio, broad age groups and age-sex distribution. various databases available at both national and international levels were taken to address the five dimensions. the findings showed that push and pull factors and infrastructure differentials are taken into consideration while saudi population internally migrate. all the gcc countries, including saudi arabia, experience international migration streams subject to the labour requirements and governmental regulations for both arabs and non-arabs. immigrant demographics are characterized by saudi arabia’s labour demand controlled by adult males from other countries. ; khraif ) . labour force in the kingdom experiences structural changes, with service sector taking the lead in replacing the agricultural sector which perpetuated the heavy need for foreign labour (khraif ; sufian ; united nations ) . expatriate labour force, with their sincere involvement in daily responsibilities, created a total reliance even in private space-direct impact of non-nationals on the lives of citizens (forstenlechner and rutledge ) . migration is of wide concern among nations, especially in the gcc, although the migration load has declined with time (tabutin and schoumaker ) . the kingdom of saudi arabia has the largest land area and population among the gcc members, where expatriates form . per cent of the total population (center for population studies ). that is, one in every four persons in the kingdom is an expatriate, indirectly influencing fertility decisions (khraif a, b; courbage ) . the expert and technical labour force brought in to fulfil the increasing requirements of technological development and innovation affects the locally available native manpower in the fast changing saudi arabia, resulting in an imbalance on regional and urban scales, impacting upon economy, urban planning, transport, housing and employment and, in return, creating an over-urbanization (united nations khraif khraif , makki ). still, from the role played by migrants in the economy, provisions of government services and social contracts, citizens benefit from their unregulated cheap employment at domestic level (forstenlechner and rutledge ) . the labour and immigration policies are undergoing changes in the kingdom, expecting its outcome in the near future-absorption of native saudi youth into the labour force-and skill upgradation-educational, technical and professional. with this scenario, an analysis of migration data is made in this context in order to: ( ) look at the saudi citizens' migration trends within the kingdom; ( ) analyse the impact of migration on kingdom's population growth and also on the neighbouring gcc countries; ( ) look at the changes taking place annually in kingdom's migration trends and in the other gcc countries; ( ) examine the migrants by looking at the origin place; and ( ) analyse the variation between immigrants and saudi citizens in terms of demographic parameters. the saudi arabian national census has started publishing migration data by administrative area (place of birth and place of current residence) since , specifically for the native population. analyses were carried out to calculate the following: • in-migration rate = (number of persons entering an administrative area/population in the area) * . the international database of us census bureau provides migration data-net migrants by year, which has been collected for saudi arabia and the other gcc states from to -to compare the changes and its contribution to the population growth. individual country experiences on migration are plotted. further, the world bank bilateral migration database has been accessed online to trace the migration flow to the gcc states for three periods: , and . all these data were utilized to explain the migration process in the kingdom of saudi arabia. the findings of the analyses are presented under varying sections such as internal migration, international migration and differences between natives and migrants. the capital, al-riyadh, is vibrant in terms of not only population size but also migration flows (table . ). as against an in-migration rate of per , the out-migration rate is only per , showing high levels of attraction or in-migrant flows determined by the administrative requirements, educational and employment prospects and the quality of life. other areas having pull factors are tabouk (the military headquarters) and the eastern region (the industrial hub). makkah al-mokarramah also stands as attractive with high pull factors, whereas al-baha, jazan, hail, al-qaseem, aseer and the northern borders exhibit pushing power. neither the urban infrastructure in al-baha (al-baha, beljarshy), jazan (sbia and abu arish), hail (hail and baqaa), aseer (khamis mushayt and abha) and northern borders (arar and rafha) nor the major governorates in these administrative areas are able to retain the native labour force. makkah al-mokarramah maintains the status without being influenced significantly by the migration flows. madina al-monawarah, the pilgrim location, also records an out-migration, which might explain the decreasing demand for or interest of natives to serve the pilgrims. the replacement of currently existing expatriate health-care staff by the local nationals-untapped women resources-is imperative to meet the complex health needs and rising public health issues (maben et al. ). but the current trend of out-migration from the makkah al-mokarramah leads to positive net migration. promoting local-level human resources development shall help in bringing a workable link between the three tiers of health-care delivery through referral system and continuing medical education ensuring population health (al-yousuf et al. ) . the increasing administrative jobs and industrial job market influence the internal migration in the kingdom, which has reasons other than employment, such as residential mobility-buying or building homes, increase in family size, affordability for better dwelling, rental rates, evacuation by the owner or inadequate services substantiate the migrants' relation with house ownership (helderman et al. ) . however, majority of internal movements are from rural to urban in nature, accelerating the urbanization process, as stated by clarke and murray ( ) , with a successful spatial distribution policy that reduces widespread regional disparity in physical and social infrastructure and uneven settlements scattered at long distances (al-khalifeh ; makki ) . migration intentions to continue living in a city are determined by the years lived in the city, home ownership in the city, parents' place of residence, land ownership in the village and area of origin (khraif ; ) . areas with higher levels of out-migration require increased levels of employment potential in the destinations in both public and private sectors. on the other hand, those areas experiencing substantial levels of inmigration require redistribution policies, both for population and infrastructure development programmes. according to the recent international strategies on industrial location, the production and manufacturing units are relocated to land areas not used hitherto, which currently paves way for a return migration. saudi arabia's efforts in these lines shall create prospects in the near future in population redistribution. the spatial mobility may be regulated through encouraging local-level human resource development and regional development efforts underway in the kingdom such as the creation of industrial, educational and medical townships at various geographic destinations. the kingdom is no way an exception in terms of sex differentials in migration flows. as seen elsewhere, males migrate more than females here also, as observed in the absolute numbers of in-migrants and out-migrants as well as net migrants. there are , males migrated from one area to another, as of . the corresponding figure for females was , . while male migrations are determined largely by employment opportunities, the female mobility is caused generally by marriages and family settlements. area wise, the number of migrants (in-migrants and out-migrants) varies in such a way that al-riyadh, makkah al-mokarramah, al-qaseem, eastern region, aseer, tabouk, jazan and najran have more male inmigrants and out-migrants than females; al-madinah al-monawarah, hail, northern borders, al-baha and al-jouf have less male in-migrants. however, male out-migrants dominate heavily in all areas. the migration rates range from . (jazan in-migrants) to . (al-riyadh in-migrants), giving rise to positive net migration rates for al-riyadh, makkah al-mokarramah, eastern region and tabouk. all the other areas experienced negative net migration rates. in other words, former four areas have more in-migrants than out-migrants; and the latter have more out-migrants than in-migrants. thus, the areas of saudi arabia experience high levels of spatial mobility, with the level of net migration varying largely from − . (al-baha) to . (al-riyadh). a recent demographic survey enlightens this issue by statistics on population classified by previous residence. it reveals a drastic change in the internal migration trend, comparatively. there is a rapid decline in the number of net migrants to a narrow level, of both males and females. all areas, except najran and al-jouf, show a similar trend ( fig. . ). immigrants constitute a considerable share of total population in the kingdom. (salam et al. ) . immigrants seeking jobs prompted by the development of oil fields foster not only the national economy but also immigrants' home countries (roudi-fahmi and kent ) . however, the crowding and mass gathering, resulting from an influx of job seekers, pose public health challenges such as accidents, corona virus infections, viral transmissions and middle east respiratroy syndrome -(mers), (memis and al-rabeeah ) demanding national attention. as seen in fig. . , the positive net migration plays a key role in the kingdom's population growth. this situation may be compared with other gcc states as the contributions of net migration remain more or less equal in all these nations. such a high role of migration on population growth may necessitate a review and reformulation of population and labour-related policies and programmes so that immigrants do not pose any burden on the gcc states, especially saudi arabia, in the near future. the unregulated import of labour played a more decisive role than natural increase in the higher population growth registered during the period - ( , , ) compared with - ( , , ) (forstenlechner and rutledge ; sufian ) . however, as compared with the other gcc countries-bahrain, qatar and united arab emirates (uae)-the effect of migration on population growth is found lesser in the kingdom, despite the push to develop infrastructure-power stations, government ministries and industries (khraif ) . perhaps, the saudization policies that changed labour and immigration laws since have influenced this trend. on the other hand, it is a fact that liberalization and globalization attract professional or skilled labour force from east asia and africa to western countries. with the changing labour visa regulations, a decline in immigration in the kingdom is expected in the future, which will create a population structure and distribution conducive for enabling an equitable treatment, policies and programmes. to achieve a positive relation between population dynamics and socioeconomic development, planning for migration at all levels should be associated with decentralization of decision-making in large urban centres and distribution of economic investment over a wide area, where urban population spreads out in medium and small centres (makki ). according to the us census data, net migrants in the kingdom kept on decreasing annually since , reaching the lowest level in . thereafter, with slight changes, their numbers declined and reached a negative level by , and the same trend continues at present. that is, the immigrants have started returning to their native countries more often (fig. . ). in contrast, the national censuses show a continuously increasing immigrant population in the kingdom but with lower growth rates, during - , as compared to - . in short, the migration data (net migrants) as given by the us census bureau do not tally with the national datasets. a similar trend is visibly noted in all the other gcc states but at different periods. oman started experiencing negative net migration since . similarly, qatar has the highest rate of net migration, followed by bahrain. definitely, these countries will soon follow the trends of saudi arabia, as the labour laws of saudi arabia are usually adopted by the other gcc states. these data also require comparisons with the national censuses for understanding the trend of immigration. saudi arabia has gcc's major population share as well as land share and the largest share of immigrants, during and . (aldossary et al. ) . also, in the professions having no language requirements, non-arabs are employed. higher emigration to other arab countries prevailed in the kingdom during and , which has changed to outside the arab world in , hopefully revealing the higher return migration from the arab countries as well as increased emigration of saudis to europe and america. this has resulted in the kingdom turning into "heterogeneous, multinational, and multi-religious with higher overall sex ratio, especially at prime working ages, creating social, economic, and security-related problems" (center for population studies ; forstenlechner and rutledge ; khraif a, b; fouad and al-badr ; ali ). the immigrant population (non-saudi) in the kingdom has a unique profile in terms of sex ratio, age distribution, residential status and geographic preferences, different from that of the native saudi population. migrants bring different demands and desires (forstenlechner and rutledge ) , showing a unique profile-adult to middle-aged single males with low educational levels, further impacting upon the national socio-demographics (alrouh et al. ) . sex ratio, the basic demographic indicator, varies from ( ) to ( ), with native sex ratio slightly more than ( in , in , in , in and ) but higher than in case of immigrants ( in , in , in , in and in ) . native sex ratios are close to in the childhood ages but favour males in the middle ages (above years), whereas that of immigrants favour males in the working ages and old ages in (table . ), but show marked differences in and . differences of immigrants from natives in terms of broad age groups are clear from fig. . , with a large majority of them being adult and middle-aged ( - years). among the natives, as of , in all, . per cent of population is aged - years, whereas among immigrants, the corresponding figure is . per cent, which is attributed to the immigration policies in the kingdom. there are marked changes observed thereafter in and . foreign-born population in the kingdom is employed on job contract, with the majority of them without a family status. thus, only the working-age people immigrate for employment, a --- , , , and leaving their family in their native countries. immigrants have rights over the occupation, not on their residential status. thus, the labour sector in the kingdom is dominated by males, except in schools and hospitals, thus reducing the opportunities of females in the job market. the sex differentials reflect the job market requirements. of late, the labour scenario of the kingdom of saudi arabia is undergoing changes in terms of labour laws, migration policies and sponsorshipbased employment contracts (forstenlechner and rutledge ) . effective implementation of such reforms is expected to develop a labour market steered by the native workforce. the labour market shall undergo a number of reforms in the near future before it stabilizes to a particular overall involvement of native labour force. as of , immigrants form . per cent of the total population in the kingdom; their proportions are higher in makkah al-mokarramah ( . per cent) and al-riyadh ( . per cent), eastern region and al-madinah al-monawarah (table . ). the number of persons per household, an indicator of population density at household level, shows differences between the natives and immigrants. while native households have more than six persons on an average, immigrants have just four, which could explain the immigrants' status (as forced bachelorship), expanded male adults and age-sex distribution. native households reflect an ideal family setup (father, mother and children), whereas a lesser proportion of immigrants live with their families. most immigrants live as single in bachelor quarters, employee compounds or in the so-called bedspace arrangements. thus, census counting of the immigrant population itself is an arduous task. housing, yet another indicator of social status, brings out certain unique differences of immigrants' living arrangements from that of natives. housing sector is composed of traditional housing, villas, apartments and others: natives live mostly in the first three types, whereas immigrants stay in apartments ( . per cent), traditional houses ( . per cent), and other types (table . ). apartments are available in the newly developed urban centres and upcoming townships, whereas the less urbanized areas have more traditional houses. concrete houses are commonly seen all over the kingdom but vary across areas, depending upon the climatic conditions and livelihoods. areas such as northern borders, eastern region, al-riyadh and al-jouf have more than per cent of their houses concreted, whereas the percentages are lower in jazan. more than per cent of saudi households in northern borders, al-riyadh, al-jouf and eastern house ownership is generally restricted to the natives, as per the kingdom's housing policies; however, there are few immigrants who own their housing as per special sanctions granted by the kingdom. but the rental housing system is more focussed on the immigrants; still, there are natives who also live in rented housings, which may be due to migrations or for the flexibility of labour force. one-third of the immigrants reside in rented houses, while the rest reside in official houses. rental houses remain less than per cent in al-qaseem and al-baha, implying that official houses are higher in proportions. but the proportions of natives living in rental houses are very low in these areas, as compared to immigrants. with the change in the type of housing, the housing infrastructuresources of power, water and sewage-changes, especially along areas. for example, the dependence of power varies from . per cent (hail) to . per cent (eastern region). native-immigrant variations in the utilization of public source of power are minimal- . per cent of natives as against . per cent of immigrants. among the areas, jazan has lesser proportion of native households, depending on public sources ( . per cent) and northern borders has least proportion of immigrant households ( . per cent). at the same time, eastern region has the highest proportion of native public sector power beneficiaries ( . per cent), whereas najran has the highest per cent of immigrant public sector power beneficiaries ( . per cent). but these cannot be considered as a constraint to quality of life as households have alternative sources such as private stations and private generators. in al-riyadh, makkah al-mokarramah and al-madina al-monawarah, public water supply users are less than per cent. it is in the eastern region that there are more than . per cent of households having access to public sector water supply. while the public sector water usage is lower among the natives ( . per cent), its usage is higher among immigrants ( . per cent). this is because of varying dependence on catchment tank water and well water. conversely, water remains as a severe constraint in the desert areas of the kingdom due to non-availability or difficulties of supplying to distant places. hence, some areas have less than per cent of households, depending upon the availability of public water sources (al-baha, najran and aseer). similar situation with the sewage system exists as well; the dependence on public sewage is less than half in the kingdom, ranging from . per cent (al-baha) to . per cent (eastern region). while the major areas have higher proportions, other areas have lesser proportions of households depending upon public sewage. a large proportion of immigrant households depends upon public sewage ( . per cent) as against the natives ( . per cent), indicating the type of housing along areas. native households have different accesses like ditches and private sewages. as pointed out earlier, immigrants are located more in the urban areas as per the preliminary reports of census, that is, . per cent of immigrant population as against . per cent of native population live in urban areas (table . ). immigrant females are concentrated more in the urban areas ( . per cent) as against immigrant males ( . per cent), which reflects the job demands. in comparison, the native females are lesser in urban areas. there are regional differences in this dimension as well. the kingdom of saudi arabia is vibrant with high levels of internal migration flows across the administrative areas. there exits tremendous and varying degrees of pull and push factors determined by socioeconomic development, industrialization and urbanization. the major in-migrating areas are tabouk, al-riyadh and eastern region, while the major outmigrating destinations are al-baha, jazan, hail, al-qaseem, aseer and northern borders. a few areas remain more or less stable without pulling or pushing native population. public sector employment and proximity to metropolis (riyadh, jeddah/makkah or dammam) are major pull factors. makkah al-mokarramah maintains the status without being influenced significantly by the migration flows. again, as seen elsewhere, males migrate more than females, as observed in the absolute numbers of inmigrants and out-migrants as well as net migrants. the regional development-infrastructure development and promoting medium-sized cities-with an aim to create employment encourages native population to continue living at their own birth places. those areas experiencing substantial levels of immigration require redistribution policies, both for population and infrastructure development programmes. the spatial mobility may be regulated by encouraging local-level human resource development and regional development efforts underway in the kingdom such as the creation of industrial, educational and medical townships at various geographic destinations. saudi arabia is the largest country in the gcc in terms of both land area and population, thus having the highest level of international migration within the arab world and beyond. the changing labour laws and saudization efforts shall curb this situation, bringing a balance between the demand and supply in the labour market. the hiked net migration rate ( ) ( ) ( ) ( ) ( ) ( ) has immediately been brought to unity, which will reduce at prevailing emigration trend. expatriates, in a way, complicate the demographics of saudi arabiasex ratio, age-sex distribution, dependency ratio, regional distribution of population, urban share and housing conditions. demographic transition underway in the country is reflected in the native age-sex distribution. the positive net migration plays a key role in the kingdom's population growth. such a high role of migration on population growth may necessitate a review and reformulation of population and labour policies and programmes so that immigrants would not pose any burden in the gcc states, especially saudi arabia, in the near future. out of the total immigrants in the kingdom, three-fifths are non-arabs. of late, the labour scenario of the kingdom of saudi arabia is undergoing changes in terms of labour laws and migration policies. effective implementation of such reforms is expected to develop a labour market steered by the native workforce. the labour market shall undergo a number of reforms in the near future before it stabilizes to a particular overall desired optimum level of expatriate labour force. health care and nursing in saudi arabia labour immigration in the arab gulf states: patterns, trends and problems population spatial distribution policies in saudi arabia demographic and health indicators in gulf cooperation council nations with an emphasis on qatar organization of the saudi health system dynamics of saudi arabian population: analysis through four censuses - , population dynamics of large middle eastern cities declining fertility in the arab peninsula the gcc's "demographic imbalance": perceptions, realities and policy options the composition of population with respect to sex in saudi arabia migration and home ownership permanent versus temporary rural migrants in riyadh, saudi arabia -a logit analysis of their intentions of future mobility residential mobility in the city of riyadh: a study of its directions, reasons and characteristics the labor force in saudi arabia: spatial dimensions and socioeconomic and demographic characteristics fertility in saudi arabia: levels and determinants. xxiv general population conference urbanization and growth of cities in saudi arabia demographic imbalances in gcc: solutions and confrontations fertility behaviours of grazing groups in suman and northern parts of saudi arabia uneven development: comparing the indigenous health care workforce in saudi arabia, bahrain and oman regional and urban population size weights in saudi arabia public health management of mass gatherings: the saudi arabian experience with mers-cov challenges and opportunities -the population of the middle east and north africa components and public health impact of population growth in the arab world the demography of saudi arabia the demography of the arab world and the middle east from the s to the kingdom of saudi arabia-human development report country program document for saudi arabia the use of urban observatories as a tool for localizing urban and social policy. economic and social council for western asia the demographic dilemma of the arab world: the employment aspect key: cord- -excn a authors: han, xiaoqing; shi, huifang; sun, yingying; shang, chao; luan, tao; wang, dake; ba, xueqing; zeng, xianlu title: cxcr expression on granulocyte and macrophage progenitors under tumor conditions contributes to mo-mdsc generation via sap /erk/stat date: - - journal: cell death dis doi: . /s - - - sha: doc_id: cord_uid: excn a myeloid-derived suppressor cells (mdscs) comprise a critical component of the tumor environment and cxcr reportedly plays a key role in the pathophysiology of various inflammatory diseases. here, cxcr expression on granulocyte and macrophage progenitor cells (gmps) was found to participate in myeloid cell differentiation within the tumor environment. in cxcr -deficient tumor-bearing mice, gmps exhibited fewer macrophage and dendritic cell progenitor cells than wild-type tumor-bearing mice, thereby decreasing monocytic mdscs (mo-mdscs) expansion. cxcr deficiency increased sap expression in tumor-bearing mice, which reduced stat phosphorylation through restraining erk / activation. our findings reveal a critical role for cxcr in regulating hematopoietic progenitor cell differentiation under tumor conditions, and sap is a key negative regulator in this process. thus, inhibiting cxcr expression may alter the tumor microenvironment and attenuate tumor progression. myeloid cells constitute the most abundant hematopoietic cells and are important for adaptive immunity . increasing evidence indicates that the phenotype and function of myeloid cells can be altered by the tumor conditions and converted into immunosuppressive cells . myeloid-derived suppressor cells (mdscs) have been identified as an immature myeloid heterogeneous population and markedly expand and accumulate in the tumor environment [ ] [ ] [ ] . moreover, mdscs exhibit a strong suppressive function towards t cell-mediated immune responses and can also facilitate tumor progression (e.g., tumor growth, angiogenesis, and metastasis) [ ] [ ] [ ] . thus, it is necessary to illuminate the molecules and mechanism associated with regulating the abnormal differentiation of hematopoietic progenitor cells into mdscs. in the tumor microenvironment, mdscs are generated from multipotent hematopoietic progenitor cells following stimulation by cytokines and growth factors released by tumor and stromal cells [ ] [ ] [ ] [ ] . our recent study demonstrated a novel role of two chemokines, cxcl and cxcl , in promoting monocytic mdscs (mo-mdscs) generation by favoring the differentiation of bone marrow cells under tumor conditions . however, the precise precursors that cxcl and cxcl act upon and the related signaling pathways remain unknown. cxc chemokine receptor (cxcr ) is the receptor for cxcl and cxcl , . the crucial immune function of cxcr is to regulate neutrophil mobilization from the bone marrow to the blood and promote the migration of the neutrophil into inflammatory sites . moreover, cxcr has been shown to play a role in maintaining the fate of normal hematopoietic stem/progenitor cells, including survival and self-renewal . cxcr also plays a significant role in melanoma growth, corrected: correction angiogenesis, and metastasis . in addition, cxcr deficient mice display the characteristics of decreased gr + tumor-associated granulocytes, f / + tumorassociated macrophages, and cd b + gr + mdscs, as well as delayed tumor progression . collectively, these studies suggest that cxcr might participate in mdscs accumulation. mdscs can be further divided into two subsets: ( ) granulocytic mdscs (g-mdscs, cd b + ly g + ly c low ); and ( ) mo-mdscs (cd b + ly g − ly c hig ) in mice . although g-mdscs account for the majority of mdscs, mo-mdscs have been found to be no less immunosuppressive than g-mdscs on a per cell basis . importantly, neoplastic and tumor-associated stromal cells release multiple tumor-derived soluble factors that can increase mo-mdscs expansion . nevertheless, the relevant mechanism by which cxcr regulates mo-mdscs accumulation in the tumor microenvironment remains unexplored. here, we reported that granulocyte and macrophage progenitor cells (gmps) express cxcr , and cxcr deficiency reduces the ability of gmps to give rise to macrophage and dendritic cell progenitor cells (mdps), leading to the decreased mo-mdscs generation. furthermore, sap expression was found to be upregulated in hematopoietic stem cells and their progenitor cells (hspcs) of cxcr -deficient tumor-bearing mice. the increase of sap expression inhibited the erk/stat signaling pathway, which regulates the differentiation of hspcs into mo-mdscs. thus, these findings reveal a novel role for cxcr through which sap /erk/stat signaling regulates hematopoietic cells differentiation in the tumor microenvironment. tumor progression is accompanied by mo-mdscs accumulation , . first, the changes to mo-mdscs in the bone marrow and spleen were examined in a b f bearing mouse model. the data showed that the percentage of mo-mdscs in the bone marrow was significantly increased in tumor-bearing mice after weeks (fig. a) . the percentage of mo-mdscs in the spleen was also increased with the extension of tumor-bearing time (fig. b) . next, the expression of cxcr in bone marrow cells was analyzed. the results showed that the expression of cxcr on lineage − cd + ly g − bone marrow cells increased with the extension of tumor-bearing time (fig. c) . to investigate whether the expression of cxcr was related to mo-mdscs accumulation under tumor conditions, we analyzed the number of mo-mdscs in the blood, bone marrow, and spleen of wild-type (wt) and cxcr -deficient (cxcr −/−) mice. the results showed that normal myelopoiesis was not affected in cxcr −/− mice (fig. s a-d) , however, the number of mo-mdscs were all significantly reduced in cxcr −/− mice bearing tumors after weeks (fig. d) , indicating that cxcr was required for mo-mdscs expansion under tumor conditions. to determine whether the decreased number of mo-mdscs resulting from a cxcr deficiency contributes to the suppression of tumor progression, b f cells were intravenously injected into wt and cxcr −/− mice. the results showed that the number of lung metastatic nodules was substantially reduced in cxcr -/-tumorbearing mice. however, when the mo-mdscs were supplemented in cxcr −/− tumor-bearing mice, the number of lung metastatic nodules was increased compared with the cxcr −/− tumor-bearing mice (fig. e) . a significant inhibition in melanoma growth was observed in the cxcr −/− mice compared with wt mice after bearing tumors for weeks (fig. f) . these results suggest that a cxcr deficiency impairs the establishment of a tumor-supportive microenvironment by reducing mo-mdscs accumulation. to determine whether cxcr contributes to mo-mdscs expansion, bone marrow progenitor cells from wt or cxcr −/− mice were exposed to the serum isolated from different mice. the results showed that the serum of tumor-bearing mice exhibited a substantially increased proportion of mo-mdscs than that of control mice and the addition of cxcl or cxcl induced a greater proportion of mo-mdscs compared to the serum of only tumor-bearing mice. furthermore, the differentiation of cxcr −/− bone marrow progenitor cells into mo-mdscs was markedly reduced compared with the wt mice in the serum of tumor-bearing mice or tumor-bearing mice supplemented with recombinant cxcl or cxcl (fig. a, b ). there were no differences in the proportion of g-mdscs between the bone marrow progenitor cells of wt and cxcr −/− mice (fig. c) . these results suggest that cxcr could drive mo-mdscs accumulation. next the mo-mdscs were sorted from the groups exposed to the serum of control or tumor-bearing mice, and the level of arginase- (arg- ) and inducible nitric oxide synthase (inos) mrna was detected. the results showed that there were no differences between the level of arg- or inos mrna in mo-mdscs induced from wt and cxcr −/− mice (fig. d) . there was also no significant difference in the proliferation and apoptosis of mo-mdscs in wt and cxcr −/− mice (fig. s a-d) . and mo-mdscs from cxcr −/− mice gave rise to a similar percentage of cd b + cd c + or cd b + f / + cells as the mo-mdscs from wt mice upon appropriate stimulation (fig. s e and f) . taken together, these results indicate that the impaired differentiation of hematopoietic progenitor cells into mo-mdscs is responsible for the decreased mo-mdscs accumulation in cxcr −/− tumor-bearing mice. mdps, the hematopoietic progenitor cells of mo-mdscs, are decreased in cxcr −/− mice hematopoietic stem cells (hscs) undergo a cascade of multiple progenitor cell stages to generate all the blood cell lineages required for an immune response . common myeloid progenitor cells (cmps) give rise to gmps, which generate mdps, and mdps give rise to mo-mdscs within the tumor environment . we next examined whether cxcr deficiency changed the number of progenitor cells in a specific stage required for mo-mdscs differentiation, thereby leading to the decreased mo-mdscs generation. to this end, the number of total bone marrow cells, lks (lin − sca- − c-kit + ), cmps (lk, fcγrii/iii int cd + ), gmps (lk, fcγrii/iii hi cd + ), and mdps (lin -cd + cd + cd + ly c − cd b − ) were examined (fig. a, b) . there was no difference in the number of total bone marrow cells, lks, cmps, and gmps between the wt and cxcr tumor-bearing mice, however, the number of mdps of cxcr −/− tumorbearing mice was remarkably reduced (fig. c) . the results suggest that a cxcr deficiency leads to perturbations in the hspc compartment. next, the proliferative activity of mdps was assessed. the results showed that (see figure on previous page) fig. a lack of cxcr leads to reduced mo-mdscs accumulation and delayed tumor progression. a, b the percentage of mo-mdscs in the bone marrow and spleen of tumor-bearing mice was analyzed by flow cytometry. the samples were analyzed on the second, third, and week th of tumor-bearing. c the cxcr positive cells in bone marrow were analyzed by flow cytometry. the mice were treated as described in a, b. d absolute number of mo-mdscs in the blood, bone marrow, and spleen of control mice or mice bearing tumors after weeks. e the number of metastatic foci was detected in the lungs of wt control mice, cxcr −/− control mice, -week wt, -week cxcr −/−, and adoptively transferred mo-mdscs in two-week cxcr −/− tumor-bearing mice. all mice were sacrificed at weeks after a tail vein injection of b f cells. the transferred mo-mdscs were isolated from the blood of wt tumor-bearing mice. f b f cells were subcutaneously inoculated into wt or cxcr −/− mice, and the size of tumors was measured at the indicated time points. bars represent the mean ± sd of five independent experiments. a one-way anova with repeated measures followed by a dunnett's post hoc test or a two-way anova followed by holm-sidak's post hoc test were used to determine the level of statistical significance (*p < . ; **p < . ; and ***p < . ; ns, not significant) fig. cxcr deficiency impairs the differentiation of myeloid progenitor cells into mo-mdscs. a the percentage of mo-mdscs and g-mdscs induced from wt or cxcr −/− bone marrow cells were detected by flow cytometry. the bone marrow cells were treated with the serum of control mice, tumor-bearing mice (tb), tumor-bearing mice + ng/ml cxcl , or tumor-bearing mice + ng/ml cxcl in the presence of gm-csf for days in vitro. b, c quantitative analyses of mo-mdscs and g-mdscs as shown in a. d the relative level of arg and inos mrna expression in mo-mdscs was analyzed by qpcr. the mo-mdscs were sorted from wt or cxcr −/− bone marrow progenitor cells induced by the serum of control (control) or tumor-bearing mice (tb) for days. the bars represent the mean ± sd of five independent experiments. a one-way anova with repeated measures followed by a dunnett's post hoc test or two-way anova followed by a holm-sidak's post hoc test show the level of statistical significance (*p < . ; **p < . ; and ***p < . ; ns, not significant) the proliferative activity of mdps was similar between wt and cxcr −/− tumor-bearing mice (fig. d) , suggesting that the cxcr deficiency did not impair the proliferative activity of mdps. we next examined the expression of cxcr on cmps, gmps, and mdps. gmps expressed remarkably cxcr , but cmps and mdps did not express cxcr (fig. e) , suggesting that the cxcr deficiency influences the differentiation of gmps into mdps, which leads to a decrease in mo-mdscs. the relative level of hematopoiesis gene (e.g., hoxa , hoxa , evi , and meis , ) mrna expression was decreased in cxcr −/− tumor-bearing mice (fig. f ). and the important transcription factors for the differentiation of monocytes (e.g., pu. and egr ) were expressed at low levels in cxcr −/− tumor-bearing mice (fig. g) . these data suggest that a deficiency in cxcr reduces the hematopoietic activity in tumor conditions. to confirm the role of cxcr on the differentiation of gmps to mdps, cxcr was transfected into d clone three cells to simulate gmps ( fig. s a and b) and recombinant cxcl or cxcl was administered to the treated d clone three cells. the data showed that d clone three cells transfected with cxcr generated more mo-mdscs in the presence of m-csf, cxcl , or cxcl (fig. h ). this suggests that cxcr activated by cxcl or cxcl promotes gmps to generate mdps, which give rise to mo-mdscs. taken together, these results indicate that the lack of cxcr on gmps reduces the generation of mdps, which are the hematopoietic progenitor cells of mo-mdscs under tumor conditions. cxcr deficiency reduces the differentiation of gmps to mo-mdscs through suppression of the erk/stat pathway cytokines and growth factors secreted by tumor and stromal cells regulate the differentiation of myeloid progenitor cells through a variety of transcription factors, of which the stat plays a critical role , . thus, the effect of stat on the differentiation of gmps into mo-mdscs was examined. the results showed that treatment with the stat inhibitor, stattic, resulted in an significant decrease in the inductive effect of cytokines on mo-mdscs accumulation ( fig. a, b) , suggesting that stat is involved in activated cxcr pathways for regulating mo-mdscs accumulation. to specify the role of cxcr associated with the signal transduction in the activation of stat , d clone three cells were transfected with either cxcr or an empty vector and incubated with m-csf, cxcl , or cxcl . the results showed that treatment with m-csf increased the phosphorylation of erk / and stat in the d clone three cells transfected with either cxcr or an empty vector. however, treatment with cxcl or cxcl only activated erk / and stat in the d clone three cells transfected with cxcr (fig. c ). to verify whether cxcr drives the hematopoietic activity of d clone three cells, the level of hoxa , hoxa , evi , and meis mrna was assessed. the results show that hoxa and hoxa were increased in the d clone three cells transfected with cxcr and exposed to m-csf, cxcl , or cxcl (fig. d) . these data suggest a vital role of the erk/stat axis in the cxcr -mediated regulation of mo-mdscs accumulation. the phosphorylation of erk / and stat was detected in hspcs isolated from different mice. the results showed that the phosphorylation of erk / and stat was up-regulated, whereas a lack of cxcr suppressed the activation of erk / and stat in the tumor conditions (fig. e) . treatment with the erk / inhibitor, u , dramatically inhibited erk / phosphorylation, and stat phosphorylation was also decreased (fig. e ). other molecules (e.g., parp , jak , and shp ) related to stat activation [ ] [ ] [ ] were also examined, and the results showed that the signaling molecules were not substantially influenced by a cxcr deficiency (fig. s ) . furthermore, stat phosphorylation was also decreased in the gmps of cxcr -/-tumor-bearing mice (fig. f) . these data further verify that cxcr regulates the differentiation of gmps into mo-mdscs through the erk/stat pathway. (see figure on previous page) fig. lack of cxcr decreases the production of mdps, the hematopoietic progenitors of mo-mdscs. a, b the classification of hspcs was analyzed by flow cytometry. bone marrow cells from control and tumor-bearing mice were stained with antibodies specific to lin-specific antigens, as well as sca- , c-kit (cd ), cd , fcγrii/iii, cd , and cd . c the total number of bone marrow cells and quantitative analyses of hspcs subsets as shown in a, b: the expression of ki in mdps from wt control mice, wt, and cxcr −/− tumor-bearing mice was analyzed by flow cytometry. e the expression of cxcr in gmps, cmps, and mdps was analyzed by flow cytometry in control and tumor-bearing mice. f, g the level of hoxa , hoxa , evi , meis , pu. , and egr mrna expression in the hspcs of wt and cxcr −/− tumor-bearing mice was evaluated by qpcr. h the percentage of mo-mdscs was analyzed by flow cytometry in d clone three cells transfected with an empty vector or cxcr . the treated cells were administered with m-csf, cxcl , or cxcl for days in the presence of gm-csf. the d clone three cells transfected with cxcr were treated with an equal concentration of m-csf as the positive control group. bars represent the mean ± sd of five independent experiments. a one-way anova with repeated measures followed by a dunnett's post hoc test or two-way anova followed by a holm-sidak's post hoc test was used to determine the level of statistical significance (*p < . ; **p < . ; and ***p < . ; ns, not significant) the erk / phosphorylation induced by treatment with isoproterenol was insufficient to increase the erk / phosphorylation and mo-mdscs generation in cxcr −/− tumor-bearing mice as shown in wt tumor-bearing mice ( fig. s a−d) . to thoroughly investigate whether cxcr signaling in hspcs activates other molecules involved in the differentiation of hspcs into mo-mdscs, d clone three cells transfected with cxcr or an empty vector were incubated with cxcl and cxcl , and rna sequencing was performed. a venn diagram analysis of the predicted cxcr targets was performed from the two independent databases and the data revealed two candidate targets of cxcr : sap and gm (fig. a) . since gm is an unprocessed pseudogene, we hypothesized that sap was the candidate target of cxcr . immunoblot analyses showed that sap expression was decreased in d clone three cells transfected with cxcr and incubated with m-csf, cxcl , or cxcl for h (fig. b top) . next, sap expression was analyzed in hspcs. the results showed that sap expression was decreased in the hspcs of wt tumor-bearing mice; however, sap expression was increased in cxcr −/− tumor-bearing mice (fig. b down) . these observations suggest that cxcr could regulates sap expression under tumor conditions. c analysis of the expression of stat , p-stat , erk / , and p-erk / in the d clone three cells transfected with an empty vector or cxcr and stimulated with m-csf, cxcl , or cxcl for h. d the level of hoxa , hoxa , evi , and meis mrna was evaluated by qpcr in the d clone three cells that were treated as described in c. e the expression of erk / , p-erk / , stat , p-stat , and β-actin in hspcs was analyzed by western blot. left: hspcs were sorted from tumor-bearing mice (tb) or control mice; right: hspcs isolated from tumor-bearing mice that were tail vein injected with and without u (inhibitor of erk / ). f the level of phosphorylated stat was analyzed by flow cytometry in the gmps of tumor-bearing or control mice. bars represent the mean ± sd of five independent experiments. a one-way anova with repeated measures followed by dunnett's post hoc test or a two-way anova followed by a holm-sidak's post hoc test was used to determine the level of statistical significance (*p < . ; **p < . ; and ***p < . ; ns, not significant) based on a previous report , lentivirus-based rna interference against sap was intrapleurally injected into cxcr −/− mice bearing tumors for weeks to test the function of sap in the differentiation of hspcs into mo-mdscs. immunoblot analyses showed that the knock down of sap was evident in the hspcs of cxcr −/− tumor-bearing mice (fig. c) . next, the percentage of mo-mdscs in the bone marrow was examined. the results showed that knocking down sap in cxcr −/− tumor-bearing mice increased the percentage of the genes that are consistently . -fold upregulated or two-fold downregulated in d clone three cells transfected with cxcr or the empty vector, and both cells were incubated with cxcl and cxcl for h. the total amount of differentially expressed genes within a given subset is reported as an underlined number. below this number (next to a brace), its composition in terms of upregulated (↑, red) or downregulated (↓, green) genes is indicated. the genes at the intersection that are systematically either upregulated or downregulated are termed "coherent". the number of coherent genes within the intersection was noted, and their identities (gene names) are reported at the right end of the diagrams. b the expression of sap was analyzed by western blot in d clone three cells transfected with cxcr and administrated with m-csf, cxcl , or cxcl for h (top). the expression of sap in hspcs of tumor-bearing or control mice was analyzed by western blot (down). c the expression of sap in hspcs was evaluated in control and tumor-bearing mice. d the percentage of mo-mdscs in the bone marrow was analyzed by flow cytometry. the mice were treated as described in c. e the level of pu. and egr mrna in the hspcs was detected by qpcr. the mice were treated as described in c. f the expression of stat , p-stat , and sap was tested in d clone three cells transfected with cxcr (control) or d clone three cells transfected with cxcr and sap (sap ). all of the cells were treated with m-csf, cxcl , or cxcl for h. g the percentage of mo-mdscs was analyzed by flow cytometry. the cells were treated as described in f. all of the cells were incubated with m-csf, cxcl or cxcl for days in the presence of gm-csf. bars represent the mean ± sd of five independent experiments. a one-way anova with repeated measures followed by a dunnett's post hoc test or a two-way anova followed by a holm-sidak's post hoc test were used to evaluate the level of statistical significance (*p < . ; **p < . ; and ***p < . ; ns, not significant) mo-mdscs (fig. d) , and the level of pu. and egr mrna was also recovered (fig. e) . these data suggest that sap negatively regulates mo-mdscs generation. to verify the effect of sap on mo-mdscs accumulation, d clone three cells transfected with cxcr and over-expressing sap were incubated with m-csf, cxcl , or cxcl . the results showed that sap overexpression in d clone three cells transfected with cxcr inhibited stat phosphorylation and decreased mo-mdscs accumulation (fig. f, g) . moreover, sap overexpression in d clone three cells transfected with cxcr also inhibited the level of pu. , and egr mrna (fig. s ) . these data suggest that activated cxcr on hspcs increases mo-mdscs generation by inhibiting sap expression under tumor conditions. based on the above findings, we assumed that there was a relationship between erk / and sap . to test this hypothesis, wt and cxcr −/− tumor-bearing mice were intravenously injected with u . the results showed that the inhibition of erk / phosphorylation had no effect on sap expression in hspcs ( fig. a and fig. s a ), but decreased the proportion of mo-mdscs (fig. b) . next, d clone three cells transfected with cxcr were treated with u or over-expressed sap . immunoblot analyses showed that sap overexpression inhibited erk / phosphorylation (fig. c and fig. s b ), which exhibited an effect similar to that of the erk / inhibitor on stat phosphorylation and mo-mdscs generation (fig. c, d) . these results indicate that sap is responsible for erk / activation. to specify the role of sap on erk / activation, a lentivirus-based sap overexpression sequence was intrapleurally injected into wt tumor-bearing mice to overexpress sap , and lentivirus-based rna interference against sap was intrapleurally injected into cxcr −/− tumor-bearing mice to knock down sap . immunoblot analyses showed that erk / phosphorylation was markedly decreased in the wt tumor-bearing mice over-expressing sap and increased in cxcr −/− tumor-bearing mice treated with the sap knock down (fig. e and fig. s c) . moreover, the percentage of mo-mdscs in the bone marrow was decreased by up-regulating sap expression and increased by knocking down sap (fig. f, g) . consistently, there were similar changes in the percentage of mo-mdscs in the blood (fig. s a and b) ; and, sap did not influence the percentage of g-mdscs in the blood ( fig. s a and c) . these data suggest that activated cxcr regulates erk-mediated stat activation by restraining sap expression under tumor conditions. activates the erk / signaling cascade, and pi kγ plays a key role in sustaining the function of erk during multiple cellular processes , . thus, we next performed chip-qpcr analyses to investigate the mechanisms by which sap regulates erk / activation. the results showed that treatment with the anti-sap antibodies (abs) enriched the dna fragment at the hras and pi kγ promoters. moreover, enrichment with the anti-sap abs was enhanced in cxcr −/− tumor-bearing mice (fig. h) . since sap has been identified as a subunit of the transcriptional co-repressor, sin a-hdac complex , we next investigated whether msin a binds to the regulatory sequences of hras, kras, nras, or pi kγ. treatment with anti-msin a abs enriched the same dna fragments which had been enriched by the anti-sap abs, and the enrichment by the anti-msin a abs was enhanced when sap was overexpressed in wt tumor-bearing mice (fig. i) . these results indicate that sap binds to the regulatory sequences of hras and pi kγ, which enhances the binding of msin a to the promoter of hras and pi kγ to inhibit the transcription of hras and pi kγ. immunoblot analyses further showed that the knockdown of sap in cxcr −/− tumor-bearing mice resulted in a substantial increase in hras and pi kγ expression. in addition, sap overexpression in wt tumor-bearing mice markedly decreased the expression of hras and pi kγ and the expression of hras and pi kγ was closely connected with erk / activation (fig. j and fig. s d ). these results indicate that sap participates in the activation of the erk signaling pathway by inhibiting hras and pi kγ expression in hspcs. cxcr is typically described as a chemokine receptor expressed on neutrophils and plays a critical role in the regulation of neutrophil homeostasis , . cxcr is also expressed on microvascular endothelial cells and is responsible for mediating angiogenesis . in the central nervous system, the interaction of cxcl and cxcr on oligodendrocyte progenitor cells prevents the apoptosis of these cells , . moreover, cxcr plays a critical role in angiogenesis, tumorigenesis, and metastasis of cancer - . recently, cxcr has been reported to play an important role in hscs maintenance . in the present study, we demonstrated that activated cxcr on hspcs contributes to mo-mdscs generation under tumor conditions. cxcr expressed on gmps (fig. e) and facilitates the differentiation of gmps into mdps (fig. c) , which results in mo-mdscs generation (fig. d) . other than a classical chemokine receptor, we found a novel function of cxcr in regulating the differentiation of hematopoietic progenitor cells under tumor conditions. the role of cxcr in cancer-related mdscs expansion has been already described in preclinical models, which showed that cxcr signaling in the ly g + mdscs promotes pancreatic tumorigenesis and is required for pancreatic cancer metastasis . the results we presented here suggest that cxcr could play a cell-type-specific role in mo-mdscs generation. the expansion of mo-mdscs is a critical step associated with immunosuppression and multiple cxcr antagonists exist. thus, our findings identify cxcr as a potential pharmaceutical target for the inhibition of tumor progression. it has been reported that the stat -mediated upregulation of s a , s a and c/ebpβ in myeloid progenitors promotes mdscs accumulation in cancer . moreover, stat inhibits the differentiation of myeloid cells by downregulating irf expression . while the classic model of mo-mdscs expansion only demonstrates that stat regulates mo-mdscs expansion, we have further verified that gmps are the critical myeloid progenitor cells, of which stat activity was increased during tumorbearing conditions (fig. f) . the identification of gmps is significant to elucidate the mechanisms of mo-mdscs accumulation in the tumor microenvironment. thus, our study contributes to the further understanding of the differentiation of myeloid progenitor cells into mo-mdscs. according to a previous report , the concentration of stat inhibitor, stattic, used in this study was μm, at which stat inhibition is only partial (fig. s ) . the result suggests that other pathways might be involved in mo-mdscs generation. it has been reported that notch signaling and ezh pathway are involved in mdscs generation , , and hmgb facilitates mo-mdscs generation via p /nfκb/erk / pathway . therefore, further research will be needed to thoroughly clarify the mechanism of mo-mdscs generation. our work also reports the novel discovery that sap is an important molecule required for the coupling of cxcr to its downstream signaling molecules to modulate mo-mdscs expansion. it is important to note that sap was originally identified as a subunit of a transcriptional corepressor (the sin a-hdac complex) which mediates the interaction between sin a and dna-bound transcription factors , , . our results showed that sap expression was not affected by a cxcr deficiency in normal mice; however, sap expression was increased when cxcr was deficient under tumor conditions (fig. b) . thus, these data reveal a novel function for sap in limiting mo-mdscs expansion. furthermore, we found that there was a reverse trend of erk-mediated stat activation in response to the alteration of sap expression under tumor conditions. (fig. e) . chip studies indicated the direct binding of sap at the regulatory sequences of hras and pi kγ and sap overexpression enhanced the binding of msin a to the same regulatory sequences (fig. h, i) . these findings indicate that sap inhibits hras and pi kγ expression by enhancing the binding of msin a to these regulatory sequences. it has been previously reported that erk / is activated via the ras-regulated raf-mek / -erk / signaling pathway . pi kγ also sustains the function of erk in multiple cellular processes . our present findings show that sap represents a novel upstream component of the erk / pathway. in summary, we report cxcr as a novel contributor involved in the differentiation of hspcs into mo-mdscs. the increased binds of cxcl or cxcl to cxcr leads to reduced sap expression under tumor conditions, which subsequently activates the erk/stat signaling pathway to promote mo-mdscs accumulation (fig. ) . the identification of the specific signaling pathway activated by cxcr provides a novel regulatory mechanism (see figure on previous page) fig. the transcriptional suppression of pi kγ and hras regulated by sap attenuates erk-mediated stat activation and leads to an inhibition of mo-mdscs accumulation. a the expression of erk / , p-erk / , stat , p-stat , and sap in the hspcs isolated from wt, cxcr −/−, u -treated wt, and u -treated cxcr −/− tumor-bearing mice was determined by western blot. b the mice were treated as described in a and the percentage of mo-mdscs in the bone marrow was analyzed by flow cytometry. c the expression of erk / , p-erk / , stat , p-stat , and sap in the d clone three cells transfected with the empty vector, cxcr , cxcr administrated with u or cxcr combined with sap . all cells were incubated with cxcl and cxcl for h. d the cells were treated as described in c and cultured with cxcl and cxcl for days in the presence of gm-csf. the percentage of mo-mdscs was evaluated from d clone three cells by flow cytometry. e the expression of erk / , p-erk / , stat , p-stat , and sap was analyzed by western blot in the hspcs isolated from wt control mice, cxcr −/− control mice, wt, sap over-expressing wt, u -treated wt, cxcr −/−, and shsap cxcr −/− tumor-bearing mice. f the mice were treated as described in e and the percentage of mo-mdscs was analyzed in the bone marrow by flow cytometry. g quantitative analyses of the data described in f. h chip analysis using anti-sap antibodies in hspcs. hspcs were sorted from both wt and cxcr −/− tumor-bearing mice. i chip analysis using an anti-msina antibody in hspcs. hspcs were sorted from wt and sap over-expressing wt tumor-bearing mice. j the expression of hras, pi kγ, erk / , p-erk / , stat , p-stat , and sap in the hspcs was analyzed by western blot. hspcs were sorted from wt control mice, cxcr −/− control mice, wt, cxcr −/−, sap over-expressing wt and shsap cxcr −/− tumor-bearing mice. quantitative analyses of the protein in fig. were conducted by image j software. sap , hras and pi kγ were normalized over β-actin. p-erk / was normalized over erk / , and p-stat was normalized over stat . bars represent the mean ± sd of five independent experiments. a one-way anova with repeated measures followed by a dunnett's post hoc test or a two-way anova followed by a holm-sidak's post hoc test were used to evaluate the level of statistical significance (*p < . ; **p < . ; and ***p < . ; ns, not significant) of stat activation in myeloid cell differentiation and a potential strategy for reducing immune suppression in the tumor microenvironment. female c bl/ j mice ( - -weeks-old) were purchased from the animal experimental center of jilin university (changchun, china) to be used as wt controls. cxcr deficient mice on a c bl/ j background were provided by dr. hong zhou (department of immunology, nanjing medical university). all mice were housed in a specific pathogen-free environment under protocols approved by the animal care committee of northeast normal university, china, and all experiments related to the mice were performed in accordance with the approved guidelines. b f cells and hek- t cells were maintained in dulbecco's modified eagle's medium (gibco) supplemented with % heat-inactivated fetal bovine serum (corning) and % penicillin/streptomycin. d clone three cells were maintained in rpmi medium supplemented with % heatinactivated fetal bovine serum and % mouse interleukin (il)- ( - , peprotech) culture supplement. bone marrow cells were obtained from the femurs of mice . the cells were ground and filtered through a -μm cell strainer and erythrocytes were eliminated using hypotonic lysis buffer ( mm nh cl, . mm edta, and mm khco ). the remaining cells were cultured in complete medium (the serum from normal mice or tumor-bearing mice) supplemented with gm-csf ( ng/ ml, - , peprotech) for days. in a separate experiment, m-csf ( ng/ml, - , peprotech), cxcl ( ng/ml, - , peprotech), or cxcl ( ng/ml, - , peprotech) was added to the induction system. next, d clone three cells transfected with pegp-n (an empty vector), cxcr , or cxcr and sap were cultured in complete medium supplemented with gm-csf ( ng/ml) for days. m-csf ( ng/ml), cxcl ( ng/ ml), or cxcl ( ng/ml) was respectively added into the induction system. synthesized short hairpin (sh)rnas or overexpression sequences were cloned into the vectors, and the constructed plasmids or shctrl plasmid were transfected into hek- t cells, together with the packaging plasmid pspax and the envelope plasmid, pmd .g (both from addgene) using lipofectamine reagent (invitrogen). to knock down sap or overexpress sap in tumorbearing mice, the collected supernatant was concentrated and intrapleurally injected into three-week tumor-bearing fig. schematic illustration of the proposed model. the level of cxcl and cxcl was very low under normal physiological conditions, but the level was greatly increased under tumor conditions. the increased cxcl and cxcl under tumor conditions binds to its receptor cxcr on gmps. in this case, the gmps expressed low level of sap resulting in decreasing the binding of msin a to the promoter of hras and pi kγ, which enhanced the transcription of hras and pi kγ in the gmps. the increased expression of hras and pi kγ promoted the phosphorylation of erk / and subsequently activated stat signaling pathway to promote mo-mdscs accumulation mice four times every other day. to overexpress cxcr or sap in d clone three cells, pegfp-n -cxcr or pegfp-n -sap was electrotransfected into the cells. the relative sequences that were used are listed in supplementary table . total rna was extracted using trizol reagent (invitrogen) and reverse transcribed into cdna according to the manufacturer's instructions (thermo scientific). quantitative pcr was performed using sybr green master mix (roche, basel, switzerland) and a roche lightcycler (roche, basel, switzerland) real-time rt-pcr system. the level of mrna expression was normalized to the housekeeping gene, β-actin, based on the ΔΔct algorithm. the primer sequences that were used are listed in supplementary table . single-cell suspensions of bone marrow, spleen, and blood samples were prepared and stained as previously described . the bone marrow and spleens were ground and filtered through a -μm cell strainer. to eliminate the erythrocytes, single cell suspensions were treated with a hypotonic lysis buffer. the single cell suspensions were stained for min at °c with appropriate dilutions of various combinations of the following fluorochromeconjugated antibodies: anti-cd b-fitc (clone m / ), anti-cd -pe/cy (clone -f ), anti-ly g-apc/cy (clone a ), anti-ly c-pe (clone al- ), anti-cxcr -alexa® fluor (clone sa g ), anti-lineage-percp-cy . mouse lineage antibody cocktail, anti-cd -bb (clone b ), anti-sca -pe/cy (clone d ), anti-fcγrii/iii-apc/cy (clone . g ), anti-cd -pe (clone a f . ), anti-cd -alexa® fluor (clone raw ), anti-cd bbiotin (clone m / ), anti-cxcr -pe (clone sa g ), anti-cd -pe/cy (clone afs ), and anti-cd -apc (clone afs ), which were all purchased from bd biosciences, ebioscience or biolegend. the cells were further fixed using % formaldehyde (sigma aldrich) for min and permeabilized using . % tritox- (sigma aldrich) for min, then stained for p-stat (clone a , pe/ cy . -conjugated, biolegend) or ki (clone a , fitcconjugated, biolegend). for annexin-v analysis, cells were stained according to the manufacturer's instructions (bd biosciences). the stained cells were acquired on a facscanto ii (bd biosciences) and the data were analyzed using facsdiva software (bd biosciences) and flow jo . . software (treestar). dead cells and doublets were excluded based on the forward and side scatter. the indicated cells were lysed in lysis buffer ( mm nacl, mm tris-hcl [ph . ], % np- , mm edta, . % sodium deoxycholate, . % sodium dodecyl sulfate, mm na vo , mm naf, mm pmsf, and . mg/ml leupeptin/aprotinin) on ice for min, centrifugated at , ×g for min, and the supernatants were collected for western blotting. the primary antibodies were incubated with the membrane overnight at °c. horseradish peroxidase-conjugated goat anti-rabbit-mouse or rabbit igg secondary antibodies (beyotime, haimen, china) were incubated with the nitrocellulose blotting membranes (ge healthcare life science, boston, massachusetts) for h at room temperature in the next morning. images were obtained using a chemiluminescence (ecl) detection system (proteinsimple, san jose, ca). quantified band intensities were normalized using β-actin as housekeeping protein. blots were scanned with a tanon imaging system ( , shanghai, china). the primary antibodies used included anti-stat , anti-erk / , antip-stat , anti-p-erk / , anti-pi kγ, anti-hras, anti-sap , anti-jak, anti-p-jak, anti-shp , anti-p-shp , anti-parp , anti-par, and anti-β-actin, which were all purchased from santa cruz biotechnology and cell signaling technology. to isolate mo-mdscs, tumor-bearing mice were sacrificed by a tail vein injection with % edta. blood was collected, and the erythrocytes were eliminated with a hypotonic lysis buffer. the remaining cells were collected and the mo-mdscs were sorted with a myeloid-derived suppressor cell isolation kit using an automacs sorter (miltenyi biotec, germany) according to the manufacturer's instructions. to isolate hspcs, bone marrow cells were obtained from the femurs of mice and erythrocytes were eliminated using a hypotonic lysis buffer. first, the lineagecells were sorted using a mouse lineage cell depletion kit (miltenyi biotec, germany) using the automacs sorter (miltenyi biotec, germany) according to the manufacturer's instructions. then, lineage -cd + cells were sorted from the lineagecells using a mouse cd microbead kit (miltenyi biotec, germany) using an automacs sorter (miltenyi biotec, germany) according to the manufacturer's instructions. the lineage -cd + cells were hspcs. the purity of the mo-mdscs and hspcs was assessed by flow cytometry and found to be ≥ %. for rna sequencing (rna-seq), d clone three cells transfected with cxcr or an empty vector were both incubated with cxcl and cxcl for h and harvested. the cellular rna was extracted using trizol reagent followed by a genomic dna elimination step. rna purity was assessed using the kaiaok ® spectrophotometer (kaiao, beijing, china). the rna integrity and concentration was assessed using an rna nano assay kit of the bioanalyzer system (agilent technologies, ca, usa). library construction and sequencing on an illumina hiseq instrument was performed at annoroad gene technology corporation (beijing, china). bowtie v . . was used to build the genome index, and clean data was then aligned to the reference genome using hisat v . . . the level of gene expression was quantified using a software package called fpkm (fragments per kilobase millon mapped reads). degseq v . . was used for differential gene expression analysis between two samples with non-biological replicates. genes that were consistently . -fold upregulated or twofold downregulated in rna sequencing are listed in supplementary table . rna sequencing of the raw data and processed expression data for this study have been deposited in the ncbi gene expression omnibus and are accessible through geo series accession number: [geo: gse ]. hspcs were chemically cross-linked with a % formaldehyde solution for min at room temperature with gentle agitation and quenched with . m glycine. the fixed cells were resuspended, lysed, and sonicated to solubilize and shear the crosslinked dna. the samples were ultra-sonicated for min with s ultra-sonication at s intervals. the resulting fragmented chromatin extract was precleared with protein a/g beads (ther-mofisher, beijing, china) and then incubated separately overnight with sap (santa cruz biotechnology) or msin a (santa cruz biotechnology) antibodies followed by washing, elution, and reverse cross-linking. dna was purified using pcr purification kits (qiagen, hilden, germany) and amplified by quantitative pcr using the specific primer sets as listed below for individual genes. chip-qpcr calculations were performed as described previously . in brief, the protein-specific ab chip-ed dna signal intensity value was divided by the intensity value of the igg-chip-ed signal, representing the fold enrichment of the protein on the specific region of genomic dna. the primer sequences used are listed in supplementary table . statistical analysis was performed using prism software (graphpad software, la jolla, ca, usa). data correspond to the mean ± sd. results were analyzed using paired t-test, or a one-or two-way anova (analysis of variance) followed by post hoc tests (see legends). p values were considered statistically significant when p < . 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depend on the c/ebpbeta transcription factor tnf signaling drives myeloid-derived suppressor cell accumulation dysregulated hematopoiesis caused by mammary cancer is associated with epigenetic changes and hox gene expression in hematopoietic cells chemokine (c-x-c motif) ligand and cxcl produced by tumor promote the generation of monocytic myeloid-derived suppressor cells the carboxyl-terminal pdz ligand motif of chemokine receptor cxcr modulates post-endocytic sorting and cellular chemotaxis international union of pharmacology. xxii. nomenclature for chemokine receptors cxcr and cxcr antagonistically regulate neutrophil trafficking from murine bone marrow cxcr and cxcl regulate survival and self-renewal of hematopoietic stem/progenitor cells host cxcr -dependent regulation of melanoma growth, angiogenesis, and experimental lung metastasis host cxcr -dependent regulation of mammary tumor growth and metastasis myeloid-derived suppressor cell heterogeneity and subset definition mir- promotes proliferation and suppresses apoptosis in myeloid cells by targeting cebpa in vitro lack of muc -regulated betacatenin stability results in aberrant expansion of cd b+gr + myeloid-derived suppressor cells from the bone marrow a clonogenic common myeloid progenitor that gives rise to all myeloid lineages hox genes in hematopoiesis and leukemogenesis insertional mutagenesis identifies genes that promote the immortalization of primary bone marrow progenitor cells myeloid-derived suppressor cells: linking inflammation and cancer parp interacts with stat and retains active phosphorylated-stat in nucleus during pathological myocardial hypertrophy a schistosoma japonicum infection promotes the expansion of myeloid-derived suppressor cells by activating the jak/stat pathway the tyrosine phosphatase shp controls tgfbeta-induced stat signaling to regulate fibroblast activation and fibrosis recruited monocytic myeloid-derived suppressor cells promote the arrest of tumor cells in the premetastatic niche through an il- beta-mediated increase in e-selectin expression at the crossroads of neural crest development noncanonical regulation of insulin-mediated erk activation by phosphoinositide -kinase gamma sin a-associated protein, kda, a novel binding partner of trib , regulates mttp expression the functional significance behind expressing two il- receptor types on pmn the c. x. c. chemokine receptor , cxcr , is the putative receptor for elr+ cxc chemokine-induced angiogenic activity alterations in the oligodendrocyte lineage, myelin, and white matter in adult mice lacking the chemokine receptor cxcr cxcr signaling protects oligodendrocyte progenitor cells from ifn-gamma/cxcl -mediated apoptosis a growthrelated oncogene/cxc chemokine receptor autocrine loop contributes to cellular proliferation in esophageal cancer cxcl /il- and cxcl /sdf- alpha co-operatively promote invasiveness and angiogenesis in pancreatic cancer cxcr promotes ovarian cancer growth through dysregulated cell cycle, diminished apoptosis, and enhanced angiogenesis cxcr promotes breast cancer metastasis and chemoresistance via suppression of akt and activation of cox cxcr inhibition profoundly suppresses metastases and augments immunotherapy in pancreatic ductal adenocarcinoma inhibition of dendritic cell differentiation and accumulation of myeloid-derived suppressor cells in cancer is regulated by s a protein myeloid-derived suppressor cell development is regulated by a stat/irf- axis stattic inhibits rankl-mediated osteoclastogenesis by suppressing activation of stat and nf-κb pathways effects of notch signaling on regulation of myeloid cell differentiation in cancer ezh inhibitor gsk suppresses anti-tumor immunity by driving production of myeloid-derived suppressor cells sap promotes kruppel-dependent transcriptional repression by enhancer-specific histone deacetylation the homeobox gene mohawk represses transcription by recruiting the sin a/hdac co-repressor complex erk / inhibitors: new weapons to inhibit the ras-regulated raf-mek / -erk / pathway myeloid derived suppressor cells (mdscs) can induce the generation of th response from naive cd + t cells target dna sequence directly regulates the frequency of activation-induced deaminasedependent mutations we thank dr. hong zhou (department of immunology, nanjing medical university) for the support with c bl/ j cxcr -/-mice. this work was funded by the national nature science foundation of china. the grant numbers are , and . the authors declare that they have no conflict of interest.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.supplementary information accompanies this paper at (https://doi.org/ . /s - - - ). key: cord- - hnaiyc authors: cieslak, theodore j.; herstein, jocelyn j.; kortepeter, mark g. title: communicable diseases and emerging pathogens: the past, present, and future of high-level containment care date: - - journal: bioemergency planning doi: . / - - - - _ sha: doc_id: cord_uid: hnaiyc high-level containment care involves the management of patients with highly hazardous communicable diseases in specialized biocontainment units possessing a unique collection of engineering, administrative, and personnel controls. these controls are more stringent than those found in conventional airborne infection isolation rooms and provide additional safeguards against nosocomial disease transmission. borne amidst a convergence of events in , the employment of hlcc units was validated during the – ebola virus disease outbreak, and the united states (as well as many other nations) is in the process of expanding its hlcc capacity. beyond ebola, however, the specific diseases that might warrant care in a hlcc unit remain unclear. we review here the fascinating history of hlcc and of biocontainment units and make recommendations regarding those highly hazardous communicable diseases that might optimally be managed in these units. the past few decades have witnessed a number of outbreaks of communicable disease caused by novel or emerging pathogens with the potential to produce significant morbidity and mortality in humans. although highly hazardous communicable diseases (hhcds) such as smallpox and plague date to antiquity, the year saw a convergence of events that brought such diseases, and the pathogens that cause them, into the forefront of public consciousness. the publication, that year, of michael crichton's fictional work, the andromeda strain, magnified concerns initially raised by media coverage surrounding the decision to shutter the us offensive biological weapons program. the success of the apollo mission later that year and the possibility that returning astronauts might bring extraterrestrial pathogens back to earth upon their return further heightened these concerns and was partly responsible for the decision to build the first specialized units dedicated to the isolation of patients potentially harboring highly hazardous communicable pathogens [ ] . these units, the lunar receiving laboratory at the johnson manned spaceflight center in houston and the "slammer" at the us army's medical research institute of infectious diseases (usamriid) at fort detrick md, would usher in a new era of "high-level containment care" (hlcc) and foster a reexamination of institutional infection control procedures. nearly simultaneously, an emphasis on improved laboratory safety would be prompted by the discovery of the contagious and deadly viral hemorrhagic fever virus, lassa, by researchers at yale [ ] . after a yale technician died of laboratory-acquired lassa fever, research activities were relocated to a new maximum-security laboratory (the predecessor of today's biosafety-level- laboratories) at the communicable disease center (now the centers of disease control and prevention [cdc]) in atlanta. usamriid's "slammer" was a two-bed unit that employed engineering controls analogous to those seen in bsl- laboratories. designed with the ability to isolate (care for patients infected with hhcds) and quarantine (observation of individuals potentially exposed to these diseases), the unit was utilized for the latter role times over its -year existence [ ] , but was never required to isolate a symptomatic patient, a fact which likely factored into its decommissioning in . in the meantime, partly in response to bioterrorism concerns raised by the anthrax attacks, as well as to the subsequent outbreaks of severe acute respiratory syndrome (sars) and monkeypox, civilian hlcc units were constructed at emory university and at the university of nebraska. these two facilities cared for seven ebola virus disease (evd) patients during the - outbreak, while another two were cared for at the national institutes of health's special clinical studies unit, which had also developed hlcc capability. a tenth evd patient was successfully managed under improvised hlcc conditions at new york's bellevue hospital. while us etcs have, as of this writing, cared for a handful of evd victims and a single patient with lassa fever [ ] , it is expected that such facilities will be ready to manage patients with a number of additional hhcds. in this regard, we envision that four broad categories of patients might be viewed as candidates for admission to a hlcc unit (table . ). we briefly discuss each of these categories, as well as the pathogens contained therein, allowing that the list of such pathogens is designed to be neither under-nor over-inclusive. in this regard, we acknowledge that some institutions may elect to care for patients in a hlcc setting even though the disease they harbor may not be included on this list, and the same patient might not necessarily be managed under hlcc conditions at another institution. similarly, we realize that new diseases may emerge and be cared for under hlcc conditions until a certain level of confidence with their management is gained. finally, we acknowledge that future developments in vaccinology and therapeutics may well lead to the removal of some diseases from the current list. first, patients harboring diseases caused by pathogens that require handling under bsl- conditions in the laboratory would seem to be obvious candidates for clinical management under hlcc conditions. several taxonomic families contain such highly hazardous viruses: of these, we advocate that patients infected with any except the flaviviruses be considered for movement to a hlcc facility. the latter do not require containment because they lack person-to-person (ptp) transmission, with naturally occurring disease being acquired solely through the bite of an arthropod vector. with the exception of variola and the henipaviruses, the remaining bsl- pathogens produce a clinical syndrome of viral hemorrhagic fever (vhf) in humans, and all are transmissible, to varying degrees, from ptp. in addition to the bsl- pathogens, there are several other diseases that cause high morbidity and mortality, which are potentially communicable and thus might prompt management in a hlcc unit. while a lower risk to laboratorians permits the causative agents of these diseases to be handled utilizing bsl- or even bsl- precautions, in many cases, the precise nature of ptp transmission risk is unknown. we thus propose that the optimal management of patients harboring these agents take place under hlcc conditions. included in this category are persons infected with a number of viral pathogens: the sars or mers coronaviruses, highly pathogenic avian influenza (hpai) and other seemingly morbid novel influenza strains, and the orthopoxvirus, monkeypox. in addition, while pneumonic plague, caused by the bacterium yersinia pestis, is treatable with antibiotics, its high degree of contagion, almost universal lethality in the absence of prompt intervention, and narrow window of opportunity for successful postexposure treatment or prophylaxis make its management in a conventional hospital setting dangerous. moreover, its history as a cause of frightening global pandemics and its past association with biological warfare make it a public perceptual and surety risk (see below). finally, while most patients with pulmonary tuberculosis caused by conventional strains of mycobacterium tuberculosis are readily cared for in a negative-pressure isolation room within a conventional medical facility, the recent emergence of strains of extensively drug-resistant tuberculosis (xdr-tb) provides justification of the need for high-level containment. we allow for the certainty that new diseases will continue to emerge and that these new diseases may initially be severe yet insidious, their causative agents unidentified, and optimal means of their control unknown (the andromeda strain problem). in such circumstances, it may, on occasion, be prudent to manage and study victims in a hlcc environment. in this regard, sars, mers, nipah, hendra, and most of the vhfs could have been considered "andromeda strains" at the time of their initial appearance. finally, we acknowledge that newly emerging and highly lethal diseases raise security and surety issues and that these are compounded by fears of biological warfare and terrorism. there may be circumstances, then, when it would be reasonable to manage patients in a hlcc unit in response to political, public assurance, and risk management concerns. the - outbreak of ebola virus disease (evd) in west africa involved at least , cases with , deaths [ ] . the th such outbreak, larger than the previous combined [ ] , resulted in the infection of several western aid workers, led to their repatriation to the united states and europe [ ] , and prompted the current ongoing expansion of hlcc facilities and capabilities. evd is caused by viruses of the genus ebolavirus in the family filoviridae; the viruses derive their name from the ebola river in the democratic republic of the congo (drc, then called zaire), from whence the first cases were described in . while the - outbreak was caused by the same ebola virus (the type species, previously known as ebola zaire), evd is also caused by at least three additional members of the ebolavirus genus, bundibugyo, sudan, and tai forest viruses. a fifth species, reston virus, produces fatal disease in nonhuman primates and has resulted in seroconversions among laboratory workers, but is not known to cause overt disease in humans. to date, all outbreaks of evd have arisen in a small handful of african nations; guinea, liberia, and sierra leone accounted for the vast majority of cases during the - outbreak, while previous outbreaks occurred in the drc, congo, gabon, uganda, and south sudan. while the ecology of evd remains unclear, fruit bats likely serve as a reservoir host for the viruses [ ] ; outbreaks may begin when humans acquire the disease through contact with these bats or with duikers and nonhuman primates (who are also susceptible to evd) consumed as bush meat. evd is then transmitted from ptp through infected blood and body fluids. given the profuse amount of vomitus and diarrhea often seen among victims, along with the high concentration of virus in those fluids, the risk of such transmission is quite high, especially among family caregivers and those involved in funeral preparations. similarly, patients with evd pose an extraordinary risk of nosocomial transmission through exposure to blood and other bodily fluids during medical procedures. finally, ebola virus persists in semen for many months after recovery, raising the possibility of sexual transmission. ebola has an incubation period of - days (mean - days), after which patients develop fever, headache, myalgia, and gastrointestinal symptoms. hemorrhagic manifestations may be mild or profound, with hematemesis and hematochezia, purpura, and ecchymosis sometimes seen. death typically occurs as a result of multi-organ failure rather than hemorrhage per se, and the mortality rate has ranged from % to %, depending on the species. supportive care is the cornerstone of evd management, and meticulous attention needs to be paid to hemodynamics, respiratory status, fluid balance, and electrolyte abnormalities. while no licensed antiviral therapy exists, monoclonal antibodies [ ] and other experimental treatments have demonstrated great promise, and a vaccine candidate has been shown to be % efficacious in a postexposure vaccination trial [ ] . ebola viruses should be handled in the laboratory using bsl- safety measures, and the processing of clinical specimens potentially harboring these viruses should involve at least bsl- precautions, as would be the case with all of the pathogens discussed in this chapter. the corpses of evd victims are teeming with virus and pose an extreme risk to handlers and family members; they should only be handled by trained persons wearing appropriate ppe. patients infected with ebola were managed under hlcc conditions in multiple countries that evacuated infected expatriate healthcare and aid workers from west africa, including germany, switzerland, britain, france, norway, italy, netherlands, spain, as well as the united states. the infection of two nurses who cared for an evd patient in a conventional hospital setting in dallas, texas, and the more favorable mortality rates of patients treated in hlcc conditions ( . % compared to - % in west africa) [ ] underscore the value of hlcc units in evd management. marburg virus was first described as the cause of a lethal outbreak of viral hemorrhagic fever among laboratory workers in marburg, germany, in . the prototype member of the newly described family filoviridae, marburg, caused deaths among the victims ( % mortality rate) managed in modern german medical facilities, of whom represented secondary cases attributable to ptp transmission. only rare sporadic cases of marburg were seen subsequently until , when an outbreak occurred in congo in which of known cases died (mortality rate %). a second large and very lethal outbreak occurred in angola in ( deaths among victims, mortality %). marburg disease typically begins, following a - -day incubation period, with the onset of fever, headache, myalgia, and gastrointestinal symptoms (vomiting and diarrhea). rash, hemorrhage, and thrombocytopenia are common, and uveitis often occurs; death results from multi-organ failure. nosocomial transmission is well documented. management is supportive; there are no licensed therapeutic agents available to treat marburg hemorrhagic fever, although immune plasma has been used [ ] , and experimental therapies have proven efficacious in nonhuman primates [ ] . laboratories should handle marburg virus under bsl- conditions. the management of patients with marburg infection in hlcc settings has occurred in both the netherlands [ ] and germany [ ] . lassa virus is a member of the family arenaviridae and is an important cause of viral hemorrhagic fever in west africa. endemic in the same region affected by the - evd outbreak, lassa causes as many as , - , human infections annually in nigeria, guinea, liberia, sierra leone, and other nations in the region, where it accounts for - % of all hospitalizations [ ] . seroprevalence rates in these countries range as high as %, attesting to the fact that many infections are mild or silent. among patients ill enough to require hospitalization, mortality typically ranges from % to %. while most lassa infections result from exposure (via ingestion or inhalation) to the urine and feces of mastomys rats, ptp transmission is well documented, and the risk of nosocomial spread via exposure to blood and body fluids is high. like ebola, lassa virus can persist in semen for several months, thus promoting sexual transmission. symptomatic lassa fever begins, following a - -day incubation period, as a non-specific flu-like illness. fever, myalgia, sore throat, and cough are followed by gastrointestinal symptoms and, often, by a maculopapular rash. in severe cases, manifestations of vascular leak (edema, ascites, pleural effusion) occur during the second week, as do neurologic symptoms such as seizures and coma. overt hemorrhage occurs in only about % of patients [ ] , and death results from shock and organ failure. an elevated serum aspartate aminotransferase portends a poor prognosis, and levels above iu/liter have been associated with a % mortality rate. it is in this group, however, that ribavirin was initially studied [ ] . when administered intravenously within the first days of illness, it reduced mortality to %. the use of ribavirin is also advocated for postexposure prophylaxis. lassa is a bsl- pathogen and should be handled accordingly in the laboratory. until the west africa evd outbreak, lassa fever had been the most common vhf treated in an hlcc setting. multiple hlcc units in germany, sweden, and the united kingdom have experience managing lassa fever patients, as does emory university hospital's serious communicable diseases unit, which treated a patient with lassa in . while three lassa fever patients treated in the european facilities were medically evacuated from endemic countries, most were imported cases that were locally hospitalized and later transported to an hlcc facility [ , ]. lujo virus, an old world arenavirus closely related to lassa, was first described in as the cause of a single outbreak of viral hemorrhagic fever involving five patients in lusaka, zambia, and johannesburg, south africa (the name, lujo, derives from the two cities) [ ] . four of these patients died (mortality rate %), and there was evidence of spread to medical caregivers. of note, the lone survivor was the only patient to receive ribavirin. no other cases have been reported as of this writing. laboratories should handle specimens containing lujo virus as they would those containing lassa. guanarito, junin, machupo, and sabia are members of the family arenaviridae and the causative agents of venezuelan, argentinian, bolivian, and brazilian hemorrhagic fevers, respectively. the new world arenaviruses all have rodent hosts, and humans are incidentally infected, likely through exposure to aerosolized rodent excreta. only a single case of naturally occurring (and two cases of laboratoryacquired) sabia has been reported, and its potential for ptp and nosocomial transmission is thus unknown. data supporting ptp transmission of guanarito is likewise sparse. the close relationship of these agents to junin and machupo, however, both of which are known to be transmitted from ptp in nosocomial and other settings, prompts us to advocate for the management of patients infected with any of the four viruses under hlcc conditions. the incubation periods of these diseases are generally thought to range from to days (shorter in the case of parenteral exposure such as via needle stick injury), after which time patients develop fever and malaise, accompanied by headache, myalgia, vomiting, and diarrhea. neurologic manifestations may differentiate new world arenaviral infections from other vhfs, where they are far less common. these manifestations may include hyporeflexia, tremor of the tongue and upper extremities, gait abnormalities, seizures, and coma. leukopenia and thrombocytopenia can be profound, and signs of vascular leak, such as proteinuria and large ecchymoses, are often prominent. mortality is approximately - % in the case of junin or machupo [ ] . as with lassa, however, ribavirin appears quite beneficial in the treatment of new world arenaviral infections, as does convalescent plasma [ ] . although not licensed in the united states, a junin virus vaccine has been widely employed in argentina and is thought to be more than % efficacious [ ] . limited animal data supports the possibility that it may protect against machupo infection as well. all four of these viruses warrant the use of bsl- precautions in the laboratory. chapare, another virus closely related to the abovementioned four, was isolated from a single fatal case of hemorrhagic fever in bolivia [ ] , and three fatal hemorrhagic fever cases in california were attributed to another closely related arenavirus, the whitewater arroyo virus [ ] , which is widely distributed among woodrats in the american southwest. whether these two viruses can be transmitted from ptp is unknown, and while many additional arenaviruses have been discovered in rodents, their role in human disease likewise remains unknown. it would seem prudent to manage patients potentially harboring such diseases under hlcc conditions when feasible and to handle their causative viruses in a bsl- laboratory. although reports of a disease consistent with crimean-congo hemorrhagic fever (cchf) date to the twelfth century, the illness derives its name from a large outbreak of hemorrhagic fever in - among soviet troops serving in the crimea and a subsequent outbreak in the belgian congo [ ] . the causative agent of cchf is a nairovirus in the family bunyaviridae, the only member of this taxon requiring bsl- handling. spread largely via the bite of hyalomma ticks, it is endemic throughout much of this vector's range in africa, central asia, the middle east, and the balkans [ ] . nonetheless, it also poses a significant risk of ptp transmission (the only bunyavirus apparently capable of this), with over cases having occurred among healthcare personnel. on a global scale, cchf is the most important tick-borne infection of humans, responsible for over separate outbreaks since isolation of the virus in . while % of human infections are thought to be subclinical, symptomatic cchf presents in similar fashion to most other vhfsfollowing an incubation period of - days after a tick bite (or - days after exposure to the fluids of an infected person), patients typically experience the abrupt onset of fever, myalgia, and headache, often accompanied by neck stiffness and photophobia. gastrointestinal symptoms (nausea, vomiting, diarrhea) are frequently seen, and thrombocytopenia with hemorrhagic manifestations (petechiae, ecchymoses) is often prominent. although cchf has a mortality rate of - % in the absence of therapy, ribavirin may be beneficial in reducing mortality and in the prophylaxis of exposed contacts [ ] . despite a relatively wide geographic distribution, confirmed cases of cchf in western europe are relatively rare. in and , imported cases of cchf were treated in hlcc units in france and the united kingdom, respectively [ , ] . two autochthonous cases of cchf occurred in spain in : the first patient was initially admitted to an icu before being transported to an hlcc facility, and the second patient was a nurse who had cared for the first patient in the conventional icu [ ] . another bunyavirus, rift valley fever (rvf) virus, is a relatively common cause of disease outbreaks throughout africa and the arabian peninsula. although it can cause a hemorrhagic fever syndrome in a small minority of cases, there is no evidence for ptp transmission of rvf and hlcc management is thus unnecessary. nipah virus infection is caused by a paramyxovirus in the genus henipavirus. it was first described in as the cause of an outbreak of respiratory illness and encephalitis in malaysia and singapore; during the initial malaysian outbreak, at least of cases resulted in death [ ] , for a mortality rate of %. subsequent outbreaks have been limited to bangladesh and neighboring areas of india. while virtually all cases of nipah infection in malaysia and singapore are thought to have resulted from close contact with infected pigs, ptp transmission appears to be a factor in the spread of disease during bangladeshi outbreaks [ ] . the malaysian pigs presumably acquire the disease from pteropus bats which roost in the orchards where pigs are permitted to feed on fallen fruit. in bangladesh, a muslim-majority nation in which pigs are seldom raised, disease appears instead to be spread via the consumption of raw date palm sap contaminated with bat excrement. nipah has an incubation period of - days, after which time patients develop fever and headache, followed by drowsiness, confusion, and, in some cases, respiratory distress. permanent neurologic sequelae, including personality changes and seizures, occur frequently. treatment of nipah is generally supportive, although ribavirin appears efficacious in vitro. laboratories should handle nipah virus under bsl- conditions. like nipah, hendra virus infection is caused by a henipavirus in the family paramyxoviridae. the virus was discovered in as the cause of fatal infections among horses in hendra, a suburb of brisbane, australia. similar to the situation with nipah, the horses appear to have contracted the disease through exposure to the secretions of infected pteropus bats. although human infection has been exceedingly rare (only seven cases have been reported as of this writing), mortality is high ( / , %). hendra appears to have an incubation period of - days, and clinical disease involves a severe respiratory illness accompanied by encephalitis. ribavirin has in vitro activity against the virus but has not been studied in vivo. while ptp transmission of hendra has not been documented, we feel that the similarities between this virus and nipah, coupled with scant clinical experience, warrant extreme prudence. we thus advocate for the management of human hendra virus infection in a hlcc setting: laboratory handling should be done under bsl- conditions. the severe acute respiratory syndrome (sars) first appeared as a distinct clinical entity in the guangdong province of china in . the disease was ultimately attributed to a newly described coronavirus, a taxonomic family previously associated with the common cold. while % of the - , cases associated with the epidemic occurred in china, global travel resulted in infections in nations, with a mortality rate of approximately %. toronto, canada, experienced roughly cases, with evidence of local ptp transmission. the disease then disappeared, and there have been no reported cases anywhere in the world since . bats appear to be the reservoir for the sars coronavirus (sars-cov) and are thought to have spread the pathogen to civet cats; early human infections resulted from contact with these cats [ ] . although animal hosts may be asymptomatic, human infection with sars-cov results, after a - -day incubation period, in an initial flu-like illness with fever, cough, sore throat, myalgia, and lethargy. this typically progresses to a severe viral pneumonia, sometimes with secondary bacterial involvement. diarrhea and other gastrointestinal symptoms occur in a significant minority of patients. laboratory findings often include lymphopenia, as well as elevated transaminases, lactate dehydrogenase, and creatine phosphokinase. the treatment of sars is supportive, and bsl- precautions should be employed by laboratories. during the sars outbreak, cases were imported into western europe, and most were treated in hlcc settings in germany, france, italy, sweden, and the united kingdom [ ] . at the epicenter of the outbreak, isolation capabilities in beijing and singapore were exhausted quickly, and hospital complexes with hlcc capability were built for increasing numbers of sars patients [ , ] . in singapore and taiwan, insufficient space led to the temporary designation of sars hospitals, where engineering controls were installed, contamination zones established, and ppe donning and doffing areas designated [ ] . the middle east respiratory syndrome is caused by another coronavirus (mers-cov) closely related to sars-cov and was first described as the cause of an outbreak of severe and often fatal respiratory disease on the arabian peninsula in . while autochthonous cases have been reported only in the middle east, travelassociated cases have occurred in the united states, western europe, and east asia. as of this writing, the outbreak is still ongoing, with well over cases reported; although asymptomatic infection does occur, the mortality rate of mers is - % among symptomatic patients. person-to-person transmission occurs frequently. following an incubation period of - days, mers begins with non-specific flulike symptoms and progresses, much like sars, to a severe viral pneumonia, acute respiratory distress syndrome, and respiratory failure. laboratory findings are similar to those seen in patients with sars, and clinical management is likewise supportive. the world health organization has published comprehensive guidance for the provision of supportive care to patients with mers [ ] . mers-cov isolates, as well as specimens from patients suspected of having mers, should be handled in the laboratory using bsl- precautions. influenza is caused by a number of viruses in the family orthomyxoviridae. human infections are attributed to three genera, influenza viruses a, b, and c. while influenza b and c are known to cause seasonal disease in humans, only influenza a has the potential for causing devastating pandemics. this potential results from the virus's ability to exchange genes among strains, a process resulting in "antigenic shift." the most important of these genes are those coding for two viral proteins, hemagglutinin (h) and neuraminidase (n). viral strains are characterized by these proteins, with h n and h n currently circulating as causes of seasonal influenza among humans. while most viruses are somewhat species specific, both human and avian strains have the ability to infect pigs; simultaneous infection of pigs risks the exchange of genetic material between human and avian strains within the porcine host. such an exchange caused the h n swine variant outbreak and, while this outbreak was milder than some feared initially, the devastating potential of novel influenza strains is best highlighted by the global spanish flu pandemic, which is estimated to have caused - million deaths worldwide ( - % of the world's population) [ ] . even in the absence of a pandemic, seasonal influenza causes a mean of , excess deaths annually in the united states alone, largely among the elderly. although most patients recover from seasonal human influenza, disease caused by avian strains among poultry and aquatic birds can be devastating. highly pathogenic avian influenza (hpai) h or h strains can have mortality rates of - % in chickens, turkeys, and ducks. while most avian influenza viruses are not particularly infectious for humans, there is fear that a novel emerging influenza strain might combine the infectivity for humans with mortality rates seen in birds. seasonal influenza has an incubation period of - days, and patients may be contagious h prior to the onset of symptoms, which poses a challenge to infection control efforts. initial symptoms include fever and respiratory complaints (cough, sore throat, rhinitis), accompanied by myalgia, headache, and malaise. primary viral and secondary bacterial pneumonias occur frequently and are often the cause of death in fatal cases. avian influenza strains, when they do produce symptomatic disease in humans, often result in rapid progression to severe respiratory distress and respiratory (as well as other organ) failure. while supportive care is a mainstay of treatment for all forms of influenza, adamantanes (amantadine, rimantidine) and neuraminidase inhibitors (oseltamivir, zanamivir, peramivir) have been beneficial in some cases. susceptibility to these drugs varies greatly among strains, however, and their use should be considered in view of guidelines put forth annually by the cdc and world health organization. annual influenza vaccination will not protect against novel and avian strains but is useful in lowering the prevalence of seasonal influenza in the community, thereby potentially providing diminished opportunities for viral reassortment events. the sporadic occurrence of a case of influenza caused by a novel, seemingly highly pathogenic strain might merit isolation and care under hlcc conditions. in the face of a pandemic, however, hlcc bed capacity would quickly be overwhelmed, and alternative management strategies would need to be employed. seasonal influenza virus strains, and clinical specimens harboring such strains, can be handled under bsl- conditions. non-contemporary and hpai strains warrant bsl- handling. smallpox ranks as, perhaps, humanity's greatest killer. responsible for the deaths of several hundred million people over the course of history, its eradication, the result of an intense and coordinated global effort, similarly ranks among public health's greatest achievements. naturally occurring smallpox was last seen in somalia in , and worldwide vaccination against the disease was halted in the early s. a case today would likely be the result of a laboratory accident, a bioterror attack, or a reawakening of dormant virus (e.g., from a corpse preserved in permafrost). smallpox is caused by variola virus, a member of the orthopoxvirus genus in the family poxviridae. its control was enabled by the use of a vaccine derived from vaccinia, a related, but far less pathogenic virus in the same genus. unusual among viruses, variola is quite stable ex vivo and can survive (e.g., in crusted scab material) for decades. smallpox is transmissible via both contact (e.g., with scabs) and droplet nuclei. following an incubation period of - days, initial symptoms include fever, malaise, prostration, headache, and myalgia [ ] . these symptoms are followed very closely by a characteristic exanthem and enanthem (lesions can be seen in the mouth and other mucosal surfaces and are present on internal organs). the rash progresses in synchronous fashion from macules to papules to vesicles to pustules to tense painful lesions said to mimic pellets embedded in the skin. the lesions are centrifugal in distribution and involve the palms and soles, differentiating them from those of chickenpox. when the disease was endemic, smallpox had a % mortality rate (from multisystem organ failure), and survivors were left with deep scars from these lesions. historically, supportive care served as the primary means of treatment for smallpox patients, although recently cidofovir [ ] , licensed for the treatment of cytomegalovirus retinitis, has shown promise in treating other orthopoxviruses in animal models and in immunocompromised humans. similarly, tecovirimat has been used under an investigational new drug protocol to treat persons with complications arising from receipt of live vaccinia virus and has also demonstrated efficacy in animal orthopoxvirus models [ ] . as evidenced by its past success, vaccination is quite effective in preventing smallpox, and the us strategic national stockpile contains robust quantities of vaccinia vaccine. administering vaccine promptly postexposure (within days) may prevent or ameliorate disease, an unusual attribute among vaccines. despite these countermeasures, an outbreak of smallpox today, occurring in an immunologically naïve population, would likely pose a significant risk of mortality, and a single case would constitute a grave public health emergency. smallpox stores are held in only two authorized laboratories at the centers for disease control and prevention (cdc) and at the state research center of virology and technology in koltsovo, russia. any handling of clinical materials potentially containing virus should only be done under tight security and bsl- conditions. monkeypox is caused by an orthopoxvirus closely related to variola and was only differentiated from smallpox in during efforts to eradicate the latter [ ] . while its primary host appears to be macaque monkeys, it can infect humans and a number of other animals, notably rodent species. while monkeypox is endemic in the democratic republic of the congo, contact with infected rodents has resulted in cases elsewhere. in , an outbreak of monkeypox in the upper midwestern united states was traced to the importation of infected gambian giant rats. the rats transmitted the disease to prairie dogs; exposure to these rodents resulted in the infection of over people [ ] . in fact, fear among clinicians and refusal to care for infected patients during this outbreak [ ] was one factor leading to the university of nebraska's decision to build its hlcc unit. human monkeypox presents a clinical picture very similar to that of smallpox, albeit with a milder course and lower mortality rate (< %). in addition, monkeypox produces significant generalized lymphadenopathy whereas smallpox does not, perhaps indicative of more effective immune recognition [ ] . it is principally for this reason-the need to rule out smallpox-that we advocate for the management of suspected monkeypox patients under hlcc conditions. monkeypox is transmissible from ptp, and special caution is warranted until such time as smallpox is ruled out. vaccinia vaccine administration appears protective against other orthopoxviruses, including monkeypox, and hlcc unit personnel can thus be protected through immunization. monkeypox should be handled in the laboratory using bsl- precautions. few diseases conjure up images of fear and destruction as vividly as plague. the disease was first described as the cause of a great pandemic that began in egypt in ad. known as the "plague of justinian," it led to the death of - % of the population of europe and is said to have sealed the fate of the eastern roman empire. a second pandemic, known as the "black death," struck europe in and wiped out one-third of the existing population. a third pandemic began in china in and killed at least million. plague, caused by the gram-negative bacillus, yersinia pestis, presents clinically in multiple forms, with bubonic, septicemic, and pneumonic being the most common. bubonic plague is most often contracted by the bite of an infected flea, particularly xenopsylla cheopis, the oriental rat flea; in the united states, prairie dogs often serve as a reservoir. pneumonic plague can be acquired primarily through exposure to infectious droplets (as might be generated by coughing or sneezing) or secondarily following the seeding of the lungs of a septicemic patient. it is this form of the disease that is readily transmissible from ptp. pneumonic plague typically begins following an incubation period of just - days, when patients experience the abrupt onset of high fever, chills, and rapidly developing tachypnea and dyspnea. hemoptysis, a hallmark finding in pneumonic plague, occurs within - h of symptom onset and heralds an almost universal and rapidly impending death. as a bacterial disease, plague is amenable to treatment with antibiotics; aminoglycosides (streptomycin, gentamicin), fluoroquinolones, doxycycline, and chloramphenicol are typically effective but must be started very early in the course of disease. we advocate for the management of patients with pneumonic plague under hlcc conditions due to plague's extreme infectivity, short incubation period, very rapid progression from the onset of symptoms to death, and futility of treatment once patients have become symptomatic. preexposure and postexposure prophylaxis with oral doxycycline or ciprofloxacin may be useful in protecting healthcare workers caring for a plague victim. a licensed vaccine is currently out of production; while it provided some efficacy against bubonic plague, it was ineffective at protecting against disease acquired via inhalation. bsl- controls should be employed by clinical laboratories handling yersinia pestis or specimens potentially containing the organism. tuberculosis (tb) is caused by infection with the bacterium mycobacterium tuberculosis (and occasionally by m. bovis) and has been a scourge of mankind since antiquity. responsible for the deaths of % of adults in eighteenth-century europe, the disease was brought under control with the discovery of streptomycin in and isoniazid in . most isolates of m. tuberculosis remain susceptible to these drugs today and to others such as rifampin, ethambutol, and pyrazinamide. nonetheless, the recent emergence of multi-drug-resistant tb (mdr-tb) strains, defined as having resistance to both isoniazid and rifampin, is a cause for concern given the limited number of effective tuberculocidal drugs available. even more ominous are strains of extensively drug-resistant tb (xdr-tb), defined as having resistance to isoniazid, rifampin, and fluoroquinolones, plus at least one of three "second-line" drugs (kanamycin, amikacin, and capreomycin, all of which are only effective when administered parenterally). patients harboring such strains pose significant treatment challenges and only - % achieve cure. even in cases where treatment is ultimately successful, the period of contagion may be prolonged, and patients may require airborne isolation for many months. one-third of the world's population has tb, although the vast majority of these persons have latent infection and are asymptomatic (and noninfectious). while tuberculous disease may involve lymph nodes, kidneys, spine, bone, and other organs, it is the pulmonary form of tb which is most common among symptomatic patients, however, and the form which poses the greatest risk of ptp transmission. patients with symptomatic pulmonary tb typically present with chronic cough, night sweats, low-grade fever, and weight loss. radiographic findings vary considerably, but often include hilar and paratracheal lymphadenopathy, as well as pulmonary cavitary lesions and upper lobe atelectasis or infiltrates. most patients with pulmonary tuberculosis caused by susceptible strains are readily managed in a negative-pressure isolation room using airborne precautions. we believe, however, that disease due to mdr-tb should prompt consultation with an expert in tb management. we further advocate that disease due to xdr-tb should be considered for management in a hlcc unit; the agent, and clinical specimens potentially containing it, should be handled under bsl- conditions. the list of pathogens that might warrant care under hlcc conditions is short, and the incidence of disease caused by the majority of these pathogens, at least in developed settings, is low. this is fortunate given the very limited capacity to provide such care. while we foresee that this capacity will increase in the coming years, driven in large part by the collaborative efforts of the national ebola training and education center (netec), we expect that their principal benefit will derive from a reexamination and a strengthening of "conventional" infection control practices throughout the healthcare system. while some might call for a more dramatic expansion in hlcc capacity, further additions to the list of diseases managed in hlcc units, or even a return to bsl- -like care, there is reason to proceed cautiously. hlcc, and especially bsl- -like care, is not without disadvantages. the most obvious of these are economic. it is estimated that the average cost incurred by us ebola treatment centers in acquiring hlcc capabilities during the - evd outbreak exceeded $ . million per hospital [ ] . while some of these are one-time investments (e.g., unit construction and planning), others are ongoing operational costs, such as staff training and the maintenance of supply stocks. moreover, the provision of care under hlcc or bsl- -like conditions creates numerous challenges for caregivers: ppe ensembles can be awkward and clumsy and can lead to claustrophobia. they also limit the time that caregivers can spend performing direct patient care activities, and they decrement auditory and tactile sense. all of these factors risk decreasing, rather than improving, patient and provider safety. while intense training and frequent exercising assist in mitigating against these risks in existing hlcc units, they may be impractical on a larger scale. conversely, the management of these hhcds outside the hlcc environment is fraught with hazard. cases have been successfully treated in conventional hospitals when an hlcc setting was unavailable or there was a delay in diagnosis; a woman with undiagnosed marburg virus infection was successfully managed at a community hospital in colorado [ ] . institutional responses to such exigencies have involved modifying policies, adapting infrastructure, and relying on universal standard precautions. such approaches, however, heighten the exposure risk to healthcare workers and can cause critical delays in treatment and laboratory testing. documented nosocomial transmission of many of the aforementioned diseases and high infection rates among healthcare workers during the sars and evd outbreaks reinforce the importance of engineering controls and highly trained staff to provide safe, quality care to patients harboring highly hazardous contagious pathogens. therefore, while these diseases may be managed safely in a conventional facility if absolutely necessary, in most cases transfer to an hlcc unit is warranted to ensure the safety of healthcare workers, other patients, and the general public. in summary, the hlcc unit incorporates a broad range of infection control measures, engineering modifications, and personnel considerations (detailed in another chapter in this text) that differentiate it from the "conventional" negative-pressure hospital isolation ward. these serve to: . protect patients by providing care in a self-contained unit staffed by selected individuals with expertise in critical care and infectious diseases . protect families by removing difficult decisions about visitation . protect other patients from the threat of contagion . protect laboratory personnel handling specimens containing highly hazardous communicable pathogens . protect the community by offering an additional level of safety, surety, and confidence . protect the healthcare worker against nosocomial transmission this latter protection is especially vital given that at least cases of nosocomial ebola occurred during the - west african outbreak [ ] , a risk to clinical personnel - times that of the general population [ ] . a brief history of biocontainment the invisible enemy: a natural history of viruses managing potential laboratory exposure to ebola virus by using a patient biocontainment care unit emory hospital admits lassa fever patient. the emory wheel ebola outbreak in west africa ebola and marburg haemorrhagic fever clinical management of ebola virus disease in the united states and europe fruit bats as reservoirs of ebola virus multi-national prevail ii study team, et al. a randomized controlled trial of zmapp for ebola virus infection efficacy and effectiveness of an rvsvvectored vaccine in preventing ebola 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centers for disease control and prevention. fatal illness associated with a new world arenavirus-california crimean-congo hemorrhagic fever: history, epidemiology, pathogenesis, clinical syndrome, and genetic diversity crimean-congo hemorrhagic fever health care response to cchf in a us soldier and nosocomial transmission to health care providers first confirmed case of crimean-congo haemorrhagic fever in the uk imported crimean-congo hemorrhagic fever european centre for disease prevention and control. crimean-congo haemorrhagic fever in spain outbreak of hendra-like virus-malaysia and singapore, - nipah virus encephalitis reemergence a review of studies on animal reservoirs of the sars coronavirus a curriculum for training healthcare workers in the management of highly infectious diseases the sars epidemic: treatment; beijing hurries to build hospital complex for increasing number of sars patients epidemiology and control of sars in singapore rapid creation of a temporary isolation ward for patients with severe acute respiratory syndrome in taiwan clinical management of severe acute respiratory infections when novel coronavirus is suspected: what to do and what not to do influenza: the mother of all pandemics smallpox as a biological weapon: medical and public health management cidofovir activity against poxvirus infections tecovirimat for smallpox infections human monkeypox update: multistate outbreak of monkeypox-illinois why were doctors afraid to treat rebecca mclester status of human monkeypox: clinical disease, epidemiology, and research initial costs of ebola treatment centers in the united states imported case of marburg hemorrhagic fever-colorado world health organization. health worker infections in guinea world health organization. ebola health worker infections key: cord- -hrb vt authors: hipgrave, david; mu, yan title: health system in china date: - - journal: health services evaluation doi: . / - - - - _ sha: doc_id: cord_uid: hrb vt the health of china’s population improved dramatically during the first years of the people’s republic, established in . by the mid- s, china was already undergoing the epidemiologic transition, years ahead of other nations of similar economic status, and by , life expectancy ( years) exceeded that of most similarly low-income nations by years. almost years later, china’s health reforms were a response to deep inequity in access to affordable, quality healthcare resulting from three decades of marketization, including de facto privatization of the health sector, along with decentralized accountability and, to a large degree, financing of public health services. the reforms are built on earlier, equity-enhancing initiatives, particularly the reintroduction of social health insurance since , and are planned to continue until , with gradual achievement of overarching objectives on universal and equitable access to health services. the second phase of reform commenced in early . china’s health reforms remain encouragingly specific but not prescriptive on strategy; set in the decentralized governance structure, they avoid the issue of reliance on local government support for the national equity objective, leaving the detailed design of health service financing, human resource distribution and accountability, essential drug lists and application of clinical care pathways, etc. to local health authorities answerable to local government, not the ministry of health. community engagement in government processes, including in provision of healthcare, remains limited. this chapter uses the documentation and literature on health reform in china to provide a comprehensive overview of the current situation of the health sector and its reform in the people’s republic. the health of china's population improved dramatically during the first years of the people 's republic, established in . by the mid- s, china was already undergoing the epidemiologic transition, years ahead of other nations of similar economic status, and by , life expectancy ( years) exceeded that of most similarly low-income nations by years. almost years later, china's health reforms were a response to deep inequity in access to affordable, quality healthcare resulting from three decades of marketization, including de facto privatization of the health sector, along with decentralized accountability and, to a large degree, financing of public health services. the reforms are built on earlier, equity-enhancing initiatives, particularly the reintroduction of social health insurance since , and are planned to continue until , with gradual achievement of overarching objectives on universal and equitable access to health services. the second phase of reform commenced in early . china's health reforms remain encouragingly specific but not prescriptive on strategy; set in the decentralized governance structure, they avoid the issue of reliance on local government support for the national equity objective, leaving the detailed design of health service financing, human resource distribution and accountability, essential drug lists and application of clinical care pathways, etc. to local health authorities answerable to local government, not the ministry of health. community engagement in government processes, including in provision of healthcare, remains limited. this chapter uses the documentation and literature on health reform in china to provide a comprehensive overview of the current situation of the health sector and its reform in the people's republic. most people are familiar with two things about modern china. the first is its physical size and enormous population. in land area, china is the world's third largest nation, theoretically spanning h of time difference from west to east (while officially operating on one time zone). its census revealed a population approaching . billion, the world's largest. china's population grew most rapidly from the late s to the early s, due to the formerly high fecundity of its women alongside a rapid fall in the crude death rate due to communicable disease control (cdc) and basic public health measures. life expectancy also rose rapidly during this period ( fig. ) (hipgrave a ). the second familiar aspect is china's meteoric economic development, with an average annual growth rate of around % for most of the last years, only falling to - % since the global financial crisis. these familiar aspects of china have depended on the health of its population improving dramatically during the first years of the people's republic of china (prc) since its establishment in . by the mid- s, china was already undergoing the epidemiologic transition, years ahead of other nations of similar economic status, and by , life expectancy in low-income china ( years) exceeded that of most similarly low-income nations by years (jamison et al. ) . however, with cdc (hipgrave a), economic development, rapid urbanization, and a dramatically ageing population, china's health system now faces a vastly different range of issues. china will soon become the first large nation to age before achieving developed nation status. noncommunicable diseases (ncds) now account for over % of deaths in china and almost % of its total disease burden (the world bank human development unit ). a world bank analysis of ncds in china (the world bank human development unit ) concluded that "a reduced ratio of healthy workers to sicker, older dependents will certainly increase the odds of a future economic slowdown and pose a significant social challenge in china" (page ). equally challenging is the provision of new services for the prevention and management of chronic illness and the government's averred commitment to equity and universal health coverage. these challenges and commitments were among the stimuli to the major health system reform (hsr) that china commenced in (state council ). china's most recent hsr was a response to deep inequity resulting from three decades of marketization and de facto privatization of the health sector. it was the culmination of many years of debate (tang et al. a ) after acknowledged inaction on the heavy burden of healthcare on household expenditure (blumenthal and hsiao ; huang ; liu ; liu et al. ; tang et al. ) . it comprises initiatives in five main areas: . expanding the coverage and benefit of health insurance schemes in urban and rural areas . establishing a national essential medicines scheme to ensure the availability of affordable medicines and reduce the ability of health providers to profit from the sale of drugs . improving basic service availability and quality while also reducing referrals to specialist care and hospitals . ensuring the availability of basic public health services for all populations . piloting public hospital reform, particularly in order to separate hospital management and clinical service provision the current hsr builds on earlier, equityenhancing initiatives including the reestablishment of rural health insurance (meng et al. ) and subsidized hospital maternity services (feng et al. a) . early progress on the first phase of china's current hsr ( was extensively reviewed, both internally by domestically commissioned teams of international (unpublished) and national experts (wu and yang ; li and chen ) and externally (yip et al. ). the reform is planned to continue to , with gradual achievement of its overarching objectives on universal and equitable access to health services; the second phase ( - ) was announced in early (ministry of health a), and a major additional pronouncement on county hospital reform was made in early (state council ). monitoring and evaluation of the reform is slated to prioritize its different hierarchical elements (figs. and ), although detailed plans for such evaluation have not been released. china's commitment to hsr indicates its ongoing priority for the highest echelons of government (ministry of health a). the four-year plan for phase reiterates the goal of universal access to basic health services and seeks to resolve constraints to the supply of china's increasing and diverse health needs. it again commits to expanding insurance benefits and introduces priority to unifying china's several health insurance schemes; it encourages development of commercial insurance and the introduction of capitation and other payment reforms to separate doctors from the financial management of hospitals; it suggests that the private sector should manage % of health services by ; family general practice is promoted alongside expanding community and public health services, and the drug production, prescription, and pricing will be further consolidated and regulated; performancebased funding of health staff is also mentioned. these individual areas are discussed further below. the plan is encouragingly specific but not prescriptive on strategy and avoids the issue of local accountability for financing various health programs, stipulating only that government spending on health should gradually increase as a proportion of total government expenditure. this vagueness hints at a major problem for china's health sector, the reliance on local government support for the national equity objective . another major problem remains the difficulty of reforming hospital management, effectively undoing the private, for-profit system that evolved over recent decades. as a result, china's hsr has not yet reduced the proportional financial burden of healthcare on households or their risk of catastrophic expenditure on health (meng et al. ). organization of the health system china's former ministry of health (moh) recently merged with the body previously responsible for family planning to form the national health and family planning commission (nhfpc). the commission contains different departments, offices, and bureaux responsible for setting standards and for the planning, administration, oversight, and reporting on china's health sector. however, as with most of china's social sectors, there is a heavy decentralization of responsibility for local planning, financing, and implementation of health services in china (wong ; zhou a) . in china's decentralized system, policies and reform guidelines are set at national level but implementation is delegated to local authorities at provincial and lower levels. a hierarchy of health authorities oversees these issues at province, prefecture, county, and township levels. in china's political economy and governance structure, local health authorities are more responsive to local government than to higher-level cadres within the health sector, meaning that uptake of national policies and recommendations is only guaranteed if there is broad agreement across all sectors of government and at local government level. in the past, when the health sector was of low priority, this severely limited the implementation of national laws relevant to the health sector. for example, the law on control of infectious diseases conferred on local government's responsibility for various forms of reporting and action, but was weakly implemented, culminating in the wake-up call of sars in , redrafting of the law and major reform of cdc (hipgrave a; wang et al. a) . initiatives depending on countrywide uptake such as the national measles vaccination campaign still rely heavily on local funding and prioritization; recent environmental degradation and food and drug safety scandals are further evidence of the lack of cross-sectoral priority given to the health sector in china. the partial rollback of the one-child policy announced at national level in remains subject to interpretation and optional implementation by provincial governments. despite its evident high priority (tang et al. a ), many aspects of the hsr itself are dependent on the same support and follow-up by provincial and even county governments brixi et al. ) . to ensure that hsr would receive adequate local priority despite this structure and accountability, in early the hsr leading group in the state council signed "accountability contracts" with provinces on key reform areas, for subsequent delegation and implementation at lower levels (china news network ). in some provinces, a few key hsr targets such as health insurance coverage were incorporated into subnational officials' performance evaluation criteria, which has been effective in ensuring progress. however, in other, more complicated reform areas, such as strengthening primary healthcare, public hospital reforms, and others, ensuring progress has been more difficult. indeed, the reform of public hospitals suffers from a lack of consensus or clear national guidance on direction, limiting its prioritization and implementation outside pilot areas, particularly at low levels. figure illustrates the ideal accountability relationships among government, healthcare providers, and citizens (society) in the delivery of healthcare. however in china, such relationships have not yet been forged. while there are promising moves to make local government generally more accountable to the public (such as measurement of "green gross domestic product (gdp)" and independent surveys of public opinion on local government performance in some provinces), the main motivation for subnational authorities remains economic development and revenue generation (zhou b) . moreover, while banking, communications, etc. are carefully regulated and monitored from above, like most social sectors, health services are largely organized and monitored at the local level. it is too costly for china's undermanned central government to independently monitor and evaluate subnational health performance (wong ; zhou b) . these circumstances explain the limited ability of national health officials to ensure the hsr is fully pursued at grassroots level. in theory, all government plans represent the will of the people as they are ratified by the national people's congress. however, many congress members are unelected (in the western democratic sense) appointees, and the people's congress generally rubber-stamps the documents presented. however, with the increasing attention of the party and government in china to public comment through social media, albeit increasingly censored (osnos ) , and local protests, there is growing acknowledgment of their answerability to the general public. therefore, while during local planning there is almost no formal process for the public to make input, there are opportunities for the general population to voice concerns through the courts, social media, petitions, protests, etc., especially when issues affect a significant proportion of a community. although the process is usually slow (the hsr took many years to be formalized (tang et al. a )), there is usually gradual recognition and acknowledgment of the need to act. on the other hand, implementation of plans usually requires higher-level pressure on the various lower tiers of government, and this pressure progressively dissipates further down the hierarchy; it may be ignored for issues that don't have high-level and cross-sectoral support and the support of local government. hence, targets for insurance coverage and drug price control are accepted, but controlling the environmental impact of local industry is often ignored (human rights watch ). in this process, public influence is rather indirect and can be ignored if local economic, political, or vested interests override it. patients' concerns in healthcare delivery may be channeled formally through the national people's congress at different levels (although usually only major complaints reach this level) or informally through social media. however, mechanisms to tap the feedback of patients, as the end users of health services, have not been established. there is no ombudsman or independent regulator in china's health system, and senior appointments are normally approved by the ruling party organization. however, since launching the hsr, government is learning that empowering patients and regularly collecting their feedback on key parameters such as service prices and quality strengthens accountability across the government levels and can help achieve the overall goals of the reform (state council ). patient satisfaction and feedback is increasingly incorporated into the performance evaluation framework for hsr implementation (ma ) . however, this practice has not yet been standardized, systematized, and regularized throughout china. an example of the problem china is having in effecting the most difficult aspect of the hsr, the reform of public hospitals, was recently summarized by eminent researchers on china (yip et al. ) , who noted the complex web of relationships that govern this endeavor (fig. yip et al. ) to make progress in this area of reform, although some commentators doubt this will be achieved in the current context (zhang and navarro as part of the government's regular planning, the new npfpc drafts annual national health work plans with annual targets and submits annual budget proposals for approval by the ministry of finance and the ndrc, which approves major construction initiatives such as health infrastructure development. with major events as the hsr, new changes and innovations are often seen in the plans year on year. at subnational levels, healthrelated authorities (not only health bureaux) in provinces, prefectures, and counties submit annual planning and budget proposals in line with health service delivery needs and stewardship to the development planning and finance authorities at the corresponding tier. implementation is financed by local budget supplemented by transfers from higher tiers of government (explained below). local data should be used in formulating plans, but as there is little tradition of regular, independent, or audited data gathering in china, desensitization of administrative and economic data is suspected (cai ; hu et al. ; walter and howie ; kaiman ; anonymous ) . regulation of the health sector follows the accountability structure outlined above and appraises progress and achievement against high-level targets set at national and local levels. performance assessment tends to be quantitative (relating to coverage or throughput of health services), although assessment on more subtle measures such as patient satisfaction, service quality, and disease management has commenced (as outlined in a guidance on performance assessment of basic public health services delivery, jointly promulgated by ministry of health and ministry of finance in january ). at management level, government officials are also increasingly being appraised according to efficiency and innovations in rolling out reform initiatives at local level. with around % of the world's people, population-level changes in china's health status or indeed any globally important indicator have a major influence on corresponding global progress. for example, china's progress toward regional and global achievement of the millennium development goal (mdg) targets will impact any final evaluation of the mdgs in . however, global statistics in any of the biological, physical, and social sciences can only be calculated if china's data is included and considered to be reasonably accurate, and data from china is not always available. many lists of global indicators lack an entry from china, and the accuracy of what is released has been questioned (cai ; mulholland and temple ) . usually, this is simply because china itself does not collect national statistics on the relevant indicators or not in ways comparable with other nations (e.g., see http://www.countdown mnch.org/documents/ report/ / _china.pdf). however, as long ago as , perspectives on china's mortality data were quite positive (banister and hill ) . the overall lack of data from china rouses suspicion. but while china's official statistics often lack breakdowns on key indicators (e.g., until recently, child mortality by gender or cause of death; nutrition status by province) or vary widely from one official source to the next (such as the annual birth cohort (cai ) or number of road deaths (hu et al. ) ), these issues distract from china's efforts to improve the content, frequency, quality, and public availability of official data in recent decades (banister and hill ) . indeed, unicef's "atlas on children in china" publishes a wide range of official and recent data (http://www.unicefchina.org/ en/index.php?m=content&c=index&a=lists& catid= ), and health statistics and other yearbooks are published annually (ministry of health b; national bureau of statistics , ) with a great degree of detail and disaggregation. an increasing number of official and peerreviewed publications on maternal and child health (mch) in china report official government data (wang et al. rudan et al. ; ministry of health a; feng et al. b feng et al. , , and this is contributing to summaries of global progress on the world's health status and mdgs and . china relies on several different sources to provide health administrators, the public and academia with information on the health sector. while it has never conducted a demographic and health survey, and its last multi-indicator cluster survey was in , china's national health services survey has been undertaken with a reasonably consistent methodology on a five-yearly basis since . many publications have used this source to assess progress in aspects of china's health system (meng et al. ) and on its health indicators . as an example of the other sources used, china's official mch management information system (mis) and the china health statistics yearbook (ministry of health b) rely on data from the following: . mch annual reports: administrative reports submitted by~ counties and districts across the nation (ministry of health ). surveillance network, which has been summarized elsewhere (wang et al. ). system, which surveys surveillance sites on a five-yearly basis, most recently in . . the ten-yearly national nutrition survey, a comprehensive, age-stratified, sex-stratified, and geographically stratified survey with a sample size of almost , (last completed in ). information system, a newly computerized administrative system that reports vaccination coverage to the nhfpc. . data gathered on health facilities, human resources, equipment, and services provided to outpatients and inpatients at various subnational levels and collected by the moh center for health statistics and information. reporting system, through which each county reports on notifiable diseases. after sars, this reporting system was massively upgraded to become web-based with reporting in real time (fig. ). . disease surveillance points on births, deaths, and on cases of notifiable diseases at selected points around the nation. . china's vital registration system, which covers around % of the nation's population but is biased toward urban and eastern locations. . national health services survey, which focuses on health status, service uptake, and health financing (meng et al. ) ; it was last conducted in . . national census, last conducted in (national bureau of statistics ), including substantive demographic information. . national one percent (inter-census) household survey, conducted between the ten-yearly national censuses, last conducted in . notwithstanding recent attempts to improve the health mis (hmis), monitoring china's hsr and health status relies largely on outputbased reporting or describes numeric improvements emanating from high-profile national initiatives (meng et al. ) , often lacking denominators (huang ; yip et al. ; ministry of health c). china does not have a tradition of locally representative, populationbased surveys on health outcomes; those which are undertaken are almost never independent. the disaggregated impact of health initiatives and local health status remains unknown except at crude (regional and urban-rural) levels (meng et al. ; ministry of health centre for health statistics and information ). this lack of data reduces the ability of governments to allocate resources according to local demography and disease epidemiology (which are changing rapidly with urbanization). in this context, quality implementation of new hmis initiatives (hipgrave b) will be critical; however, again these are national initiatives reliant on local funding. the hmis is mentioned as a priority for the second phase of the hsr (ministry of health a), but in general the monitoring and evaluation of china's health sector remains weak and non-independent and is not prioritized at subnational level. sources of funding and accountability for its use subnational governments, even at county and township level, are responsible for about % of social sector financing and for the provision of essential services including health (national bureau of statistics ). government expenditure on health depends heavily on local fiscal capacity (yip et al. ; wong ; feltenstein and iwata ) ; this varies widely across china, even after adjusting for formula-based "equalization transfers" from central government (wong ; bloom ). on average, tax revenue sharing and intergovernmental transfers finance up to % of subnational government expenditure (world bank ). this system bestows considerable power on provincial governments but also significant financial stress at the lowest levels of government. each level of government has considerable discretion in transferring resources to successively lower levels. provincial governments are the main recipients of the central government equalization grants and tax sharing and have significant autonomy in what they do with these funds. prefecture governments in turn have similar autonomy. in this system, funding for public service delivery by poorer townships and counties tends to be insufficient (wong ; zhou b) . apart from earmarked transfers from the moh and funds for selected nationwide priorities, local governments may withhold resources for lower levels or favor spending in more populous areas or on issues strategic to their career (zhou a; liu ) . this kind of bias at subnational levels can undermine progress on national development goals (yang ; uchimura and jütting ) . to supplement resources received from the higher levels, subnational governments raise resources from various fees, the sale of land use rights, and taxes on real estate transactions (world bank ). however, poor localities tend to have limited scope for such revenue generation. the imbalance between resources and expenditure responsibilities, particularly in poor jurisdictions, impacts on health service quality (yang ) and on household health expenditure (blumenthal and hsiao ; meng et al. ; world bank ) . moreover, income disparities have widened across localities and population groups within local jurisdictions (xing et al. ; zheng et al. ; undp china and china institute for reform and development ). the national urban-rural ratio of income per capita has risen from . in to . (up to within certain provinces) in ( fig. ) (national bureau of statistics ). at subnational level, only four provinces (sichuan, tibet, xinjiang, and yunnan) bucked this trend due to large subsidies to stimulate economic development and poverty reduction. subsidies for these provinces impact the shape of the line of best fit in fig. , which depicts provincial expenditure on health in relation to provincial gdp, per capita. aligned with policy priorities across sectors and programs. there are four distinct components of the national budget system, two of which impact on social sector spending: the general government budget (which relies on various taxation revenues and allocates funds to publicly funded services and activities) and the social security budget. the first of these allocates funds at the sectoral level; line ministries can then decide on and allocate earmarked transfers to the provinces (wong ; zhou b ). however, subnational government spending also relies on off-budget revenues (such as local taxes) for off-budget programs. monitoring is limited and there is little effort to align subnational budgets or plans with higherlevel priorities. moreover, apart from some individually monitored earmarked transfers, little information is available on whether governments actually spend money according to budgetary allocations or whether government expenditures and programs lead to the desired outputs and expected outcomes. achievement of high-profile input and output hsr targets masks the absence of substantive analysis of outcome-level impact (meng et al. ; yip et al. ) . audits tend to focus on detecting malfeasance, not program performance. additionally, china's budget and expenditure cycles are not synchronous. the fiscal year starts with the calendar year, but the budget is not endorsed by the national people's congress until the end of march. this delay reduces the budget's operational significance for subnational governments and central ministries (world bank ). fragmentation, information limitations, and delays in budget execution limit the ability of national authorities to transform policy priorities into resource allocation and results at the local levels (world bank ). total health expenditure (the) in china was us$ . bn in , at us$ per capita, and . % of gdp (china national health development research centre ). the/gdp is modest compared with industrialized countries, which averaged . % in (oecd ), but is average among low-and middle-income countries (lmic), whose the/gdp ranges from . % to % (e.g., indonesia . %, thailand . %, india . %, russia . %, vietnam . %, south africa . %, and brazil, . %) (see data at http://apps.who.int/nha/data base). health expenditure as a proportion of gdp has increased from~ % to~ % since , but numeric growth has been enormous due to china's rapid economic growth (figs. , , and ) . the sources of the have changed dramatically over time, reflecting changes in the role of government. marketization beginning in the s led to historically high out-of-pocket expenditure in ( %), but this had decreased to~ % in (china national health development research centre ), mostly through public subsidies for primary health programs, for health providers and for the social insurance schemes. in , tax-based government expenditures accounted for . % of the, social health expenditure . %, and out of pocket . % (fig. ) . overall, public expenditure on health as a share of the is similar to that of many other lmic and also to the united states (even higher if the government contribution to social health insurance is considered), but most high-income countries average around % (tangcharoensathien et al. ) . who calculates this figure differently and has china's figure at %; most nations in south and east asia average around % (see http:// apps.who.int/nha/database and hipgrave and hort ). to provide essential health services, reduce inequity, and provide financial protection against catastrophic health expenditure, governments must mobilize sufficient resources via: ( ) collecting revenues, ( ) pooling of risk, and ( ) purchasing goods and services (gottret and schieber ) . globally, three models of basic healthcare financing are practiced: nationalized health services, social insurance, and private insurance. china's total health expenditure % of gdp unit: million renminbi healthcare financing has evolved to a structure dominated by three social insurance schemes with almost universal population coverage: the urban employees basic medical insurance (uebmi) (financed by formal sector employers and employee contributions), the rural cooperative medical (insurance) scheme (rcms), and urban residents' basic medical insurance (urbmi). the latter two receive heavy government subsidization in addition to individual contributions (in a roughly : ratio). government health expenditure stems from tax revenue, as described above. china does not have tax instruments specifically designated to health expenses; the funds are allocated from overall tax revenue. these funds are used to pay the salaries of health workers, purchase equipment, and build infrastructure at various levels and for various specific programs such as public health subsidies or other schemes earmarked by the moh. government also funds a social assistance program (the medical financial assistance scheme), which provides cash for designated poor households to purchase health services. there also remains "free medical treatment" for those on the government payroll and for retired military and party cadres; these arrangements are slated for phasing out. however, government does not as yet contribute substantively to the funding of hospital care, which remains predominantly managed in-house from various sources of revenue (in particular, out-of-pocket payments and insurance) (state council ; barber et al. ). table summarizes the current basic health financing arrangements and benefit provided by the various health insurance schemes in china. it is evident that the major challenge remains fragmentation of the schemes and arrangements and the associated inequity and inefficiency. this is also highlighted in fig. , which depicts the large variation in average numeric benefit and other information about the various schemes. in this context, and given china's highly mobile population and the limited access of migrant populations to urban health services (di martino ), the government is prioritizing integration of the various insurance schemes (ministry of health a), but this is a difficult and complex proposition. before the hsr, to ensure financial accessibility, the chinese government priced primary healthcare services at below cost, but allowed providers to charge high prices for diagnostic tests using high-tech equipment, effectively cross-subsidizing primary services. providers could also levy a % profit on drug sales. under the prevailing fee-for-service payment modality, this created an incentive for providers to maximize profit by ordering tests and overprescription of drugs. cost-effective and efficient primary healthcare services were ignored by providers because they were not profitable; those who could not pay for services often chose to forego them . the recent reforms to provider payment, and those mooted for the future, aim to: ( ) encourage the provision of cost-effective and efficient primary healthcare services, ( ) reduce provider reliance on drug income and curb overprescription, and ( ) curb cost inflation. innovative provider payment methods, such as capitation (for primary heath mostly), gross budget, diagnosis-related groups (for hospitals), as well as performance-based payment for health workers, are being piloted at county and district level. other related policy reforms include a zero markup policy (for essential drugs), implementation of essential drug list, and so on (yang et al. a ). by international standards china's average health infrastructure level has been poor. for example, the number of hospital beds per population in was around , among the lowest in the world (ministry of health b). health infrastructure in china also suffered from a major urban-rural divide in the earlier stages of social and economic development. not only did urban health infrastructure enjoy greater public financial support, it attracted loans and other financial instruments because it was profitable and boosted the local economy. for many years, rural facilities received very limited government subsidy and relied on collective funding among farmers. rural health infrastructure lagged seriously, in terms of both the basic condition of health facilities (buildings, beds, etc.) and the equipment, while big urban hospitals acquired technical equipment of high quality. in , there were . hospital beds per urban residents, but only . in rural townships (ministry of health ). this inequity was recognized by national government, and in the majority of a national bond issue was used to finance a project earmarked for rural health, specifically to finance the rebuilding, renovation, and updating of medical equipment for rural providers, including primary health facilities such as cdc and mch institutions. the ndrc and its local branches approved the funding proposals for physical health infrastructure. more recently, the hsr allocated large sums to further improve physical health sector infrastructure (focusing on rural remote rural areas, but also urban community health centers). progress on this aspect of the reform has been very positive (yip et al. ). for the majority of china's population, access to western and formally regulated traditional chinese medicine (tcm) only commenced with the introduction of china's famed "barefoot doctors" in the mid- s. these cadres numbered . million at their peak (around one per people), but numbers fell rapidly with economic marketization and liberalization of population movement (bien ). moreover, village-level care lost its funding base with the dismantling of the rural cooperatives in the early s, and training and supervision of the quality of care provided fell off. as recently as the late s, many doctors lacked training to the level suggested by their rank and title (youlong et al. ) , and overprescribing of drugs and inappropriate use of parenteral preparations continue to exemplify the low quality of care, especially in rural areas (blumenthal and hsiao ; bloom and xingyuan ; zhan et al. ; pavin et al. ; dong et al. ; chen et al. ) . with economic marketization, medicine at all levels became privatized, physician salaries were paltry, standard consultation fees were fixed below cost (eggleston et al. ) , and over % of doctors' and health facilities' income derived from the sale of drugs (hu ) . as a result, doctors worked where they could be assured of income, patients became disillusioned with the care at rural clinics, self-referral to urban clinics increased, and the distribution of doctors, nurses, and health facilities was heavily biased to urban areas (yip et al. ; undp china and china institute for reform and development ; youlong et al. ; anand et al. ) ( table ) . residents of urban areas in china, particularly in the large eastern cities, enjoy physical access to health services to the same level as in most developed nations. however, like many other asian nations, china has trained more doctors than nurses or midwives, and there are progressively fewer staff with formal health training in progressively poorer rural areas (youlong et al. ; anand et al. ) (table ) . china includes tcm practitioners ( %) in headcounts of health staff (anand et al. ) . china is still paying for the interruption of university education during the cultural revolution of revolution of - , and the paucity of new village doctors trained since the breakup of the village cooperatives in the late s. first, as of , . % of china's doctors and . % of nurses had only completed junior college or secondary technical school level training, and % and % respectively had just high school or lower education (anand et al. ) . the duration and standard of professional education varies widely across the country (youlong et al. ) . village doctors are an ageing cohort, with a likely high attrition rate in the coming decade (xu et al. ). however, with massive increases in the number of formal trainees since , the distribution and quality of personnel are probably bigger problems than the overall number of china's health human resources. indeed, some data suggest an excess of trainees and the likelihood that many health graduates do not take up professional service. nonetheless, inequality and inequity in the distribution of doctors and especially nurses between and particularly within provinces remains extreme and has been linked to key health outcomes including infant mortality (anand et al. ) . authorities in china recognize the prevailing inequity in distribution of health human resources and have initiated training and other schemes to increase the number of qualified personnel and improve their distribution. the th five-year plan for health sector development, released in , sets targets for assistant physicians ( . / population) and nurses ( . ) and lays out plans for increased priority of staffing in rural areas and at community level, of personnel and financial support for poor rural and western health facilities by wealthier urban and eastern facilities, of intensive efforts to fill known human resource gaps among various health and allied health providers, and of tiered registration for doctors that first requires a period of rural service. a focus on community general practice is reiterated in the plan, with a target of , staff newly trained or upgraded personnel to provide such services. in addition, in a "guidance" the state council announced new roles for village doctors, recommending a wide range of tasks (government of china ). by , these cadres should be providing standardized primary care (following new clinical guidelines), implementing public health programs, undertaking disease surveillance, conducting community education, participating in health financing schemes, and maintaining individual e-health dossiers. in theory, it will be possible for the national hmis to monitor their work. the official engagement of village doctors in a national system is positive development and should improve public confidence in their services. however, payment for the planned elevation of village doctors' responsibilities will derive from a complex mix of funding streams (government of china ; ministry of health b) overseen and additionally funded by county-level authorities (government of china ) whose accountability for this national initiative will be to local government (wong ; zhou b) , not health authorities. it is well established that marketization and the de facto privatization of clinical care by salaried doctors working in public facilities had, by , resulted in china having one of the least equitable health systems in the world (the world health organization ), with over % of the being out of pocket (blumenthal and hsiao ; ho and gostin ; wang et al. ). one of the main objectives of china's hsr is to regulate the remuneration of doctors and to separate their income from choices on clinical care. however, while china has reduced the level of out-ofpocket expenditure on health to around % through increases in public funding and insurance initiatives (yip et al. ) , household health expenditure has not decreased either numerically or as a proportion of total household expenditure (meng et al. ) . although there is indirect evidence of increased non-health expenditure by insured households in comparison to before the schemes were introduced (bai and wu ), this objective of the hsr is proving to be the most difficult to achieve. china's the is increasing at around % per year, and a large proportion of the increase is due to payment of health facilities, doctors, and other providers by individuals or insurers. as patient expectations rise but out-ofpocket expenses remain numerically high, an increasing number of assaults of doctors by patients' families are being reported. on the other hand, the scheduled fees payable to doctors for listed services are set below cost, forcing clinicians and facilities to charge for other services, investigations, procedures, and drugs (including those not on the essential drugs list with unregulated prices) (blumenthal and hsiao ; ho and gostin ; wang et al. ; tian et al. ) or through accepting bribes and kickbacks (yang and fan ) . while the government has committed to improving both the quality of care provided by health providers, and is exploring remunerating them through capitation, diagnostic-related groups and performance-based incentives (ministry of health a), separating hospital management from doctors' income is proving to be the most difficult element of the current hsr (yip et al. ). as reviewed elsewhere (hipgrave a), public health services in china suffered badly under the marketization of the s and s. cdc in particular was weak, culminating in the sars epidemic in . public funding for preventive health services fell dramatically and was insufficient to even cover salaries. public health authorities were left to raise their own income through charging fees for services, including vaccination (for which fees were only completely dropped in ) and various inspections and screening. community approaches to disease control were abandoned in favor of vertical programs reliant on national or external funding, and disease surveillance was poor. sars and health authorities' realization of the epidemic of ncds due to ageing, urbanization, and decreasingly active lifestyles has led to major changes to public health programming in china. disease surveillance is now conducted online, in real time, and funding for cdc and preventive health has increased dramatically. new vaccines were introduced in , although globally recommended vaccines against haemophilus influenzae type b, pneumococci, human papilloma viruses, and rotaviruses are only available privately (ironically, through government providers). the largest boost to public health came with the hsr, when government introduced a minimum renminbi (rmb)/capita subsidy for public health/screening activities to be conducted across the nation. this had been pre-dated by various vertical preventive health programs, such as funding of hepatitis b vaccine since (cui et al. ) and national funding of the epi since . the hsr public health funding is provided by a mix of national and local authorities according to their ability to pay (problematic for poor counties in rich provinces) and the rmb was increased to rmb in ; it is much higher in wealthy areas. the funds pay providers to conduct the following services, notionally free of charge: ( ) maintenance of individual electronic health records, ( ) health education, ( ) vaccination, ( ) infectious diseases' prevention and treatment, ( ) screening and management of chronic diseases such as hypertension and diabetes, ( ) mental healthcare, ( ) child healthcare, ( ) pregnancy and maternity care, and ( ) healthcare for the aged. for the elderly and those with chronic diseases, this kind of screening, along with the introduction of zero markup and full reimbursement for drug treatment of ncds (yang et al. a ), has made a huge difference to their care. however, rollout of this initiative is slow, and although most targets are being met (yip et al. ) , monitoring is hampered by the absence of local denominators. moreover, some of the programs, such as management of mental illness, have not been founded upon a training program for staff ill-equipped to provide them. in addition, unpublished evidence gathered by unicef in suggests that some of the funds are being used as salary supplements to support the new responsibilities of village doctors (in public health and other programs) and that the volume of money allocated to some rural localities is actually too high, due to out-migration to cities. meanwhile, the increasing proportion of china's population living in urban areas, including most rural-urban migrants, cannot access such services. another boost to public health came with the moh's program, also introduced in , to prioritize interventions for certain vulnerable populations. these include: ( ) catch-up hepatitis b vaccination for those aged < years; ( ) cervical and breast cancer screening for women in rural areas; ( ) an expansion of the hospital delivery subsidies first introduced in , to cover women in all rural counties; ( ) free cataract surgery for the poor; ( ) free folic acid supplementation for rural women before and during pregnancy; ( ) improved stoves and fuel to reduce fluorosis; and ( ) introduction of eco-friendly toilets. again, targets for introduction of these measures have been set and rollout is proceeding (yip et al. ) . finally, although firm evidence of impact is scant, local authorities in most chinese cities have introduced public education and health literacy programs to enhance awareness on issues like diet, exercise, cigarette smoking, appropriate care of women before and during pregnancy, infants and young children, and the elderly. as usual, implementation of national guidelines on such activities depends on uptake and funding by other sectors and local authorities. the regular occurrence of outbreaks of food (xinhua ) and environmental contamination (human rights watch ) and other scandals with public health implications indicates the difficulty faced by national authorities in china's decentralized context. recent high-profile summaries of china's health system tend to focus on its administration and financing and neglect the considerable improvements in clinical care available to the local population. while standards at all levels of the service hierarchy vary very widely, health authorities have augmented the care available at virtually all public facilities across the nation. moreover, access to services to services has improved for all the population, albeit at high cost to both government and individuals (meng et al. ) . clinical services in china are conducted through a hierarchically arranged network of facilities ranging from tertiary referral centers in the large cities (most having high-quality diagnostic and laboratory equipment) to second-tier hospitals at county and district level. rural townships and urban communities are served by clinics or hospitals with varying capacity for inpatient care and surgery. at village or neighborhood level, public or (mostly) private facilities provide basic outpatient care, usually with an attached dispensary and possibly with links to a laboratory or radiology service. concern about the standard of care provided by local facilities has resulted in many patients self-referring to higher-level facilities and hospitals (table ) . as a result, hospitals in china tend to provide care for all level of illness, resulting in inefficiency and overcrowding. expenditure on hospital-based care as a proportion of the in china far exceeds that in many oecd nations (barber et al. ) , resulting in the high priority given to improving primary care, community general practice, and lower-level facilities in the hsr (yip et al. ; ministry of health a) and to moving outpatient care in particular from hospitals to primary care facilities (barber et al. ) . as would be expected for a nation of this size and variation, clinical services in china vary widely, from the world-class care available to residents in shanghai, beijing, guangzhou, and similar cities to the most basic care in rural clinics in far western china. similarly, models for the care of chronic illness and the use of day-care and hospital in the home vary widely, but in general these options are not yet well developed in china. the average length of inpatient stay is high in china compared to oecd nations (meng et al. ) , particularly in public hospitals, which account for % of total beds and % of hospital admissions (barber et al. ) . clinicians at community level have usually had training in tcm and many practice both western medicine and chinese medicine. however, the preparedness of clinicians in primary care for the wide range of conditions they treat varies widely. for example, china's current hsr acknowledges that the system's clinical focus has been ill-suited to the screening and outpatient care of chronic illness, an increasing priority as rates of noncommunicable diseases rise (the world bank human development unit ) . similarly, the high-volume model of clinical care in china is poorly suited to the management of mental illness (qin et al. ) , aged care and dementia, and prevention of tobacco-related illness and alcohol consumption, all of which are needed in china (the world bank human development unit ; phillips et al. ; yang et al. b; zhou et al. ; chan et al. ) . with respect to quality of care, in the last decade china has moved to standardize many clinical pathways and practices, and the concept of evidence-based medicine is increasing. however, attention to such standards and their influence on clinical care is perceived to be low (yang and fan ) . moreover, funding for and the quality and independence of clinical research, access to information, and the ability of clinicians to practice independent of the profit motive are china's pharmaceutical sector has been one of the most problematic for health authorities over recent decades and the focus of major reform efforts in the last few years. in , . % of china's the was on drugs (hu ), compared to % in developed nations (seiter et al. ) . excessive drug prescription was common in rural china (zhan et al. ; pavin et al. ; dong et al. ; chen et al. ; yu et al. ) , and there is evidence that china's rural health insurance scheme was encouraging overprescription (chen et al. ; sun et al. ). drug sales continue to provide the largest income source for china's county health facilities; doctors have a pecuniary incentive to prescribe more and more expensive drugs (chen et al. ; yu et al. ) . hospitals and doctors profit significantly from the sale of drugs (yu et al. ; the world bank group east asia pacific region ), affecting financial access to healthcare meng et al. ) . weak regulation of drug manufacture and distribution raises safety concerns (yu et al. ; guan et al. ) . previous efforts to improve the pharmaceutical sector had limited effect. the impact of laws, decrees, and separate price reductions over - was constrained by hospital financing/income generation, market influences, and patient preferences (chen et al. ; yu et al. ) . price controls were undermined by manufacturers, wholesalers, and retailers and by hospitals and physicians controlling the prescription of price-controlled drugs (hu ; yu et al. ; chen and schweitzer ) . new drug approvals were issued at astonishing rates (ho and gostin ) and the former head of the national drug administration authority was executed in for accepting bribes. kickbacks and corruption continue to mar the sector (yip et al. ; yang and fan ) . acknowledging these problems, china's hsr included establishment of a national essential medicines scheme (nems) to improve population access to and reduce the cost of essential medicines (state council ), particularly at grassroots (township and village) level. the scheme covers drug production, pricing, distribution, procurement, prescribing, and payment (hu ) and a new national essential drugs list (nedl) for primary healthcare institutions. the nedl comprises western drugs and tcm commodities (increased from western and in ) for storage and use by grassroots facilities. bidding prices for nedl drugs were capped (schatz and nowlin ) , and a "zero markup" (no profit) policy was introduced, although markups remain allowed at county-level and higher facilities. by late january , . % of township hospitals and . % of village clinics had implemented the policy (ministry of health d). in addition, most (urban) districts and (rural) counties had made nedl medicines reimbursable by the various health insurance schemes, with higher reimbursement rates than for nonessential medicines (ministry of health c). finally, to regulate the pharmaceutical market and distribution of essential drugs, the nems introduced province-wise, collective, internet-based public bidding and procurement for nedl medicines. these four elementsthe nedl, zero markup, reimbursement of certain drug costs by insurers, and public procurementwere designed by the government to wrest control of the public pharmaceutical sector from the private sector. however, the official hsr documents encourage local adaptation of the broad design (ho ) , including the nedl (which has indeed been widely augmented (guan et al. ; shi et al. ) ) and strategies to compensate providers for the zero markup policy. few evaluations of the impact of the scheme have emerged. very early indications suggested little change in prescribing practices (yip et al. ) , but a small field evaluation found that while drug procurement has been systematized and the cost of care had declined coincident with reduced drug prices, manufacturers have not uniformly supported the changes, and some drug prices have actually increased. provider compensation for reduced income was mostly ineffective, forcing some to seek alternative sources of income within and outside the health sector. rational drug prescribing had improved in this study. the loss of drug income had forced health facilities to rely more on public financing, and providers complained of higher workload and lower incomes (yang et al. a) . similar issues were found in another study in different locations (xiao et al. ) . the nems particularly impacts small rural health facilities and will again rely on considerable local support for its implementation. meanwhile, provinces are continuing to augment even a revised version of the nedl (tang et al. a) , and zero markup has not yet been applied in county or higher-level facilities. while insurance reimbursement and capitation may help to improve prescribing practices and reduce patient outlays, more control of procurement, manufacturer, and prescriber practices are required. the recently announced reforms of county hospital funding and administration include a major focus on drug procurement, prescription, management, and pricing (state council ). as a consequence of the marketization of china's health sector in the s, provision of health services was opened significantly to private providers. the number of private providers increased rapidly and now comprises a significant proportion of the market. for example, in , private hospitals accounted for only . % of total hospitals, but the share had increased to . % by . in among all , health facilities (hospitals, clinics, and other institutions), % operated as "private" entities. reports indicated that private health providers can offer services at a cheaper price and shorter physical distance and waiting time for patients (deng et al. ) and are highly active in the provision of healthcare in china. however, most private facilities are small and poorly equipped, and collectively they only employed . % of the total labor force, owned . % of total medical beds, and received . % of total patient hospital visits (ministry of health b). compared with public facilities, a large percentage of elderly physicians and new laborers in health market are practicing in private clinics (tang et al. b ). this staffing structure could have negative impact on quality of services. in general, despite rapid development in recent years, private health services are at an early stage of development in china. one major reason is that the evolution and current standing of national policy generally still favors public providers in terms of resource allocation, stewardship (entry and registration control), opportunities for promotion, and social insurance entitlements. this accounts for common challenges in the private sector, i.e., lack of technical capacity, poor infrastructure, and thus compromised service quality. health authorities are now promoting a robust private sector to encourage competition and efficiency within the health sector, aiming for % of beds and services to be privately provided by . however, subsidization of grassroots level public institutions may prevent moves in this direction. while china's progress on major health indicators during the years immediately following the foundation of the prc is unparalleled (jamison et al. ) , marketization and the unaffordability of healthcare for a large proportion of the population stymied progress in the s and s. there are even suggestions that child mortality rates in china actually rose in the s (banister and hill ) , with the breakup of the communebased health cooperatives. moreover, improvement in certain indicators has been slow. for example, urban maternal mortality has been slow to fall, almost certainly because reductions in maternity risk for urban residents have been diluted by the much higher risk of death in pregnancy among urban migrants (fig. ) . geographic disparities also remain great, particularly between eastern and western provinces . in general, the priority given to china's recent hsr acknowledges that progress in its population's health status was less than could have occurred, given the nation's economic growth since the s (yip et al. ) . acknowledgement of this is the government target of a one-year increase in life expectancy by (ministry of health a). the most comprehensive analysis of the causes of death and disability in china, published in mid- , highlighted the dramatic evolution of its demographic transition, with ncds now making up all but two of the top causes of lost life years, and most infectious diseases having fallen precipitously. the report also noted the contribution of air and household pollution to mortality and morbidity and the need for cross-sectoral action to tackle the major causes of ill-health in china (yang et al. b) . nonetheless, in , average life expectancy in china was . years, and in the maternal mortality ratio was . / , live births, infant mortality rate . ‰, and under-five mortality rate . ‰ (china national health and family planning commission ). these figures compare favorably with other developing countries, and china's performance in reducing rural maternal and neonatal mortality has been outstanding (feng et al. b (feng et al. , . china has already achieved all the health targets in mdgs , , and and achieved the target on reducing child underweight in the early s. urban-rural disparity in under-five and particularly maternal mortality has declined since , but remains high for child underweight and stunting and especially for child micronutrient deficiency (unicef china, unpublished data; ). challenges to population health status have been alluded to already and include the rise of ncds, especially smoking-related illness (the world bank human development unit ), illness due to environmental damage and air pollution (the world bank human development unit ; millman et al. ) , urbanization, and the provision of services for newly arrived migrants (gong et al. ) . the prevention of accidents and injury will also play an increasing role in maintaining china's trajectory on reducing preventable death and ill-health (wang et al. b) . as the population ages, private and institutional care of the elderly is another major issue for china's health and other social sectors. china's progress in maternal and child health, urban health, and communicable disease control are very encouraging, but the nation's health system now faces a vastly different range of issues from those it faced before. in addition to health insurance reforms that commenced in , in many ways the comprehensive health system reforms announced in have been highly successful. insurance coverage is almost universal, and the benefit package is gradually expanding, even for outpatient services, although a system for ensuring coverage for the huge population of rural-urban migrants remains under development. introduction of public health screening and management, building of new health infrastructure and expansion of community-based services, measures to control profiteering from the sales of drugs, scale-up training of health personnel, and other measures were both needed and are being implemented. on the other hand, the reform of hospital management and financing remains at the pilot stage, with suggestions but no formal guidance on the model to be followed. china's hsr is encouragingly specific but not prescriptive on strategy. monitoring the reform remains predominantly output-based at macrolevel; no detailed independent assessments have been undertaken, and population-level studies of health outcomes related to the reforms have not been undertaken. moreover, mechanisms to incorporate patient feedback into health service provision have not been established and may be ignored if local economic, political, or vested interests override such input, as has been observed in relation to china's natural environment. public financing of the health sector, although modest by global standards, has improved, particularly in relation to the proportion of the that is out of pocket. but costs are rising faster than government inputs, and poorer constituencies remain least able to fund public services, despite having the greatest needs. as a result, proportional household expenditure on healthcare has not declined. urban residents of china's industrialized eastern provinces enjoy a high quality of healthcare and access to trained personnel. this is not the case for poorer rural residents, particularly in the nation's vast western region. the official engagement of village doctors to provide publicly funded health services in rural areas should improve the standard of and public confidence in their care, but the burden on this ageing cadre of staff is rising and may be untenable; again, accountability for this national initiative will be to local government and health officials unused to the application of treatment algorithms, performance-based assessment, and clinical audit. concern about the care provided by community providers continues to result in many patients self-referring to higherlevel facilities and hospitals. population health in china is threatened by the rise of ncds, especially illness due to diabetes, cardiovascular disease, overweight, tobacco smoking, environmental damage, and air pollution. the prevention of accidents and injury and management of mental illness will also play an increasing role in maintaining china's trajectory on reducing preventable death and ill-health. the required focus of the health sector on chronic illnesses, aged care, and outpatient services requires a dramatic increase in the engagement and stewardship of community providers. this has been a major focus of china's health reforms, now well into their second phase, and it is likely that further major policy and financial inputs will be announced before this phase concludes in . the private sector will play an increasing role in the provision of health services in china, but a higher level of stewardship and the use of financial mechanisms to reign in escalating costs will almost certainly be required, especially for hospital care. to ensure consistency and transferability, this may involve stronger oversight by and involvement of national health policy and financing authorities, notwithstanding the power vested in subnational authorities in china's system of 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children's health development in china. beijing: china ministry of health -moh website now deleted. ministry of health. health statistical monthly reports. beijing: ministry of health; d. ministry of health centre for health statistics and information. an analysis report of the fourth national health services survey in china causes of death in children younger than years in china in china statistical yearbook (in chinese) national bureau of statistics. tabulation of the population census of people's republic of china health at a glance: oecd indicators, oecd publishing china's censored world prescribing practices of rural primary health care physicians in uzbekistan prevalence, treatment, and associated disability of mental disorders in four provinces in china during - : an epidemiological survey prevalence and rates of recognition of depressive disorders in internal medicine outpatient departments of general hospitals in shenyang causes of deaths in children younger than years in 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healthcare reforms health human resource development in rural china pharmaceutical supply chain in china: current issues and implications for health system reform drug prescribing in rural health facilities in china: implications for service quality and cost why hasn't china's high-profile health reform ( - ) delivered? an analysis of its neoliberal roots maternal deaths among rural-urban migrants in china: a case-control study comparative study on structural changes in income disparities in urban households in chongqing municipality, shanghai municipality and sichuan province reforming china's local government governance. in: incentives and governance: china's local governments incentives and governance: china's local governments epidemiology of alcohol use in rural men in two provinces of china key: cord- -dw xblyr authors: norbäck, dan; cai, gui-hong title: microbial agents in the indoor environment: associations with health date: - - journal: indoor environmental quality and health risk toward healthier environment for all doi: . / - - - - _ sha: doc_id: cord_uid: dw xblyr there is international consensus that damp buildings and indoor mould can increase the risk of asthma, rhinitis, bronchitis and respiratory tract infections but we do not know which types of microbial agents that are causing the observed adverse health effects. microbial indoor exposure is a broader concept than microbial growth in buildings. other sources of indoor microbial exposure include the outdoor environment, humans (crowdedness) and furry pet keeping. microbial exposure can have different health effects depending on the dose, different exposure route, genetic disposition and the timing of exposure. microbial stimulation linked to large microbial diversity in early life can protect against disease development, especially for allergic asthma and atopy. protective effects are more often reported for bacterial exposure and adverse health effects are more often linked to mould exposure. there are many studies on health associations for indoor exposure to endotoxin, mainly from homes. the risk of getting atopic asthma may be less if you are exposed to endotoxin in childhood but the risk of non-atopic asthma may increase if exposed to endotoxin especially in adulthood. moreover, genetic disposition modifies health effects of endotoxin. epidemiological studies on muramic acid (from gram-positive bacteria) or ergosterol (from mould) are few. studies on health effects of indoor exposure to beta- - -glucan (from mould) have conflicting results (positive as well as negative associations). epidemiological studies on health effects of indoor exposure to mycotoxins are very few. some studies have reported health associations for mvoc, but it is unclear to what extent mvoc has microbial sources in indoor environments. many studies have reported health associations for fungal dna, especially as a risk factor for childhood asthma at home. since most studies on health effects of indoor exposure to mould, bacteria and microbial agents are cross-sectional, it is difficult to draw conclusions on causality. more prospective studies on indoor microbial exposure are needed and studies should include other indoor environments than homes, such as day care centers, schools, hospitals and offices. keywords mould · bacteria · endotoxin · beta- - -glucan · muramic acid fungal dna · microbial volatile organic compounds (mvoc) · mycotoxins asthma · respiratory symptoms a large proportion of the total biomass in our planet consists of mould and bacteria. thus it is not surprising that some of the microorganisms found in indoor environments have outdoor sources. moreover, mould and bacteria can grow on damp surfaces or in water damaged building materials and spread to the indoor environment [ ] . moreover, free living amoeba can sometimes be found in indoor environments and survive on water damaged building materials [ ] . presence of humans is an important indoor source of bacteria, especially for gram-positive skin bacteria. keeping cats and dog and crowdedness in indoor environments can increase the exposure to endotoxin, a microbial compound from gram-negative bacteria [ ] . moreover, there can be higher levels of mould in settled dust if the concentration of furry pet allergens is higher, suggesting a link between furry pet keeping and mould contamination [ ] . dust on indoor surfaces is an important reservoir of mould, bacteria and microbial compounds which can be re-suspended into the air when people are moving around in the buildings. the world health organization (who) has concluded that damp buildings and mould can increase the risk of asthma, rhinitis, bronchitis and respiratory tract infections, but it is unclear which types of microbial agents that are causing the health effects [ ] . moisture and mould are common in homes. in the european community respiratory health survey (ecrhs), . % of a random sample of adult population reported that they had observed indoor mould in the current home and . % had water damage. inspectors visiting the homes observed mould in . % and moisture stains in . % of the homes [ ] . microorganisms and microbial agents can be measured in building materials, on indoor surfaces, in indoor surface dust (settled particles) and in air samples. the measurements include a sampling procedure, sample preparation and detection and quantification of mould, bacteria or microbial compounds. one major problem with air sampling of mould and bacteria is the short sampling time (usually from minutes up to hours) and the large fluctuation of air concentrations of microorganisms over time in the same building. mould and bacteria can produce different types of microbial components such as cell-wall compounds (endotoxin, muramic acid, beta- - -glucan, ergosterol), allergenic proteins (fungal allergens), volatile organic compounds of microbial origin (mvoc) and microbial secondary metabolites (often called "mycotoxins") [ , ] . a recent hot topic is the role of microbial diversity for human health. epidemiological studies have demonstrated that exposure to high microbial diversity in early life can protect against the development of asthma and allergic disease [ ] . another recent topic is the importance of biological ultrafine particles in indoor dust containing fragments from bacteria [ ] and fungal fragments [ ] . most health effects of indoor exposure to mould and bacteria are not linked to infections by pathogenic species. however, the legionella bacteria can be spread in indoor environments and cause lung inflammation (legionellosis) [ ] . aspergillus mould species can colonise the respiratory tract of immunosuppressed subjects and cause lung aspergillosis [ ] . moreover, there can be indoor airborne transmission of virus and bacteria causing sars, influenza, measles, tuberculosis and middle east respiratory syndrome coronavirus infection (mers) [ ] . this book chapter will describe health associations for mould, bacteria and microbial agents in different indoor environments ( fig. . and table . ). viable airborne mould has been measured in indoor air for many decades but cultivation methods are more and more replaced by molecular methods. one major methodological limitation when measuring viable airborne mould is the large day to day variation of air concentrations [ ] . many samples must be taken in different seasons to get a reliable estimate of the long-term exposure. most indoor mould are non-viable mould and can only be detected by dna based molecular methods or counting methods. when using cultivation methods, cladosporium, alternaria, aspergillus and penicillium are dominating fungal genera in indoor air samples but when identifying indoor mould by metagenomic methods other species are dominating. as an example, cladosporium, alternaria, aspergillus and penicillium genera represented < % of the total dna sequences found in dust from day care centers [ ] . some mould species are normally found indoors but may come mainly from the outdoor environment. other moulds are typical for damp buildings. fungal genera that may grow on damp building materials include: acremonium, aspergillus, aureobasidium, chaetomium, chrysosporium, cladosporium, eurotium, exophiala, fusarium, geomyces, geotrichum, monocillium, mucor, oidiodendrum, paecilomyces, penicillium, phialophora, phoma, rhizopus, scopulariopsis, sphaeropsidales group, stachybotrys, trichoderma, tritirachium, ulocladium, wallemia and yeasts [ ] . some mould species can contain allergenic proteins causing ige mediated allergy. cladosporium sp. and alternaria sp. are important outdoor fungal allergens and penicillium sp. and aspergillus sp. are implicated in allergic diseases as indoor allergens. malassezia sp. are associated with atopic dermatitis but not respiratory allergies [ ] . one review of the epidemiological literature on health associations for different mould species concluded that penicillium, aspergillus, cladosporium and alternaria species pose a respiratory health risk for children and adults, including exacerbation of asthma [ ] . another review on indoor environmental exposure focused specifically on exacerbation of asthma [ ] . endotoxin: a cell-wall compound found in gram-negative bacteria (endotoxin can have different chain length of the -hydroxy acids in the molecule) muramic acid (mua): a cell-wall compound found mainly in gram-positive bacteria ergosterol: a cell-wall compound found in mould (but also in plant materials) beta - glucans: a group of cell-wall compounds in mould (but also in pollen)t fungal dna: dna sequences specific for mould (general or species specific sequences) bacterial dna: dna sequences specific for bacteria (general or species specific sequences) mvoc: volatile organic compounds produced by microorganisms (but can have non-microbial sources as well) secondary microbial metabolites: chemical compounds produced by the secondary metabolism of microorganisms mycotoxins: chemical compounds with toxic properties produced by mould (a subgroup of secondary microbial metabolites) they concluded that there is sufficient evidence of a causal association between outdoor culturable fungal exposure and exacerbation in asthmatics sensitised to fungi. they also concluded that there is limited or suggestive evidence of an association between indoor culturable penicillium exposure and exacerbation in asthmatic children with specific sensitization, any fungal sensitization, or unspecific sensitization. moreover they concluded that there is limited or suggestive evidence of an association between indoor total culturable fungal exposure and exacerbation of asthma in children with any fungal sensitisation [ ] . few studies exist on associations between sick building syndrome (sbs) symptoms and viable airborne mould. one study from newly built dwellings in japan found that rhodotorula sp. were associated with any sbs symptoms and aspergillus sp. was associated with eye symptoms. in contrast, eurotium sp. was negatively associated with skin symptoms (a protective effect) [ ] . human beings is one main source of indoor bacteria, especially gram positive skin bacteria. moreover, bacteria can grow on damp building materials [ ] but few studies exist on viable airborne bacteria in relation to damp buildings. in building investigations in damp buildings in sweden, the genera bacillus, pseudomonas and streptomyces can often be detected in air samples and material samples [ , ] . one study from day care centers in turkey reported that staphylococcus, bacillus, corynebacterium and micrococcus were dominating genera in indoor air [ ] . another study from schools in ethiopia reported that staphylococcus aureus and coagulase-negative staphylococcus species and bacillus species were among the bacterial species in classroom air [ ] . actinobacteria is a large group of grampositive soil bacteria common in indoor environments. some species of sporeforming filamentous actinomycetes have been associated with moisture damage and respiratory disorders [ ] . streptomyces sp. is one type of spore-forming actinomycetes, producing geosmin, a compound with strong soil odour [ ] . few studies exist on respiratory health associations for non-pathogenic viable airborne bacteria. a study in a water damaged office building found that thermophilic actinomycetes in vacuumed dust was associated with lower lung function (fev ) and nontuberculosis mycobacteria level was associated with asthmatic symptoms [ ] . since only a small proportion of indoor mould and bacteria are viable, there is a need for other methods detecting non-viable and non-culturable mould and bacteria in indoor air samples. counting based methods have been used for many decades. they are based on staining of sampled mould and bacteria followed by counting in a microscope. counting methods have limited measurement range, can be disturbed by other particles, are time consuming and need trained laboratory staff. because of these limitations, they are nowadays replaced by molecular methods, e.g. quantitative pcr (qpcr) [ , ] . the camnea method (collection of airborne microorganisms on nucleopore filters, estimation and analysis) is an established counting method used to quantify total mould and total bacteria in air samples and material samples from damp buildings. it is based on staining with acridine orange followed by epifluorescence microscopy [ ] . some studies exist on respiratory health associations for total mould and bacteria by the camnea method. one study from sweden found higher levels of airborne total mould and total bacteria in home of asthmatic adults as compared to levels in homes of non-asthmatics [ ] . in the european hese study (health effects of the school environment), a similar association was found between the concentration of airborne total mould and cough and rhinitis [ ] . a prospective study among young adults in australia found that increasing the total level of mould in air at home increased the risk of developing atopy [ ] . a four-year prospective school study from sweden found an association between total mould concentration in classroom air at baseline and new asthma diagnosis among school children [ ] . another swedish school study found associations between total mould concentration in the classrooms and nasal mucosal swelling and increase of biomarkers of inflammation in nasal lavage from the teachers [ ] . in contrast, the total bacteria concentration in air was negatively associated with nasal mucosal swelling and eosinophilic cationic protein (ecp) concentration in nasal lavage fluid (a protective effect) [ ] . ecp is a marker of eosinophilic (allergic) inflammation. bacteria are classified as gram-negative or gram-positive bacteria. the cell walls of all gram-negative bacteria contain lipopolysaccharide (lps, endotoxin). endotoxin is mostly measured by the biological limulus test [ ] but -hydroxy fatty acid from endotoxin can also be measured by chemical analysis [ , ] the chemical method can classify endotoxin into five different subgroups (c , c , c , c , c ) depending on the length of the -hydoxy fatty acids. different species of gramnegative bacteria produce different length of the -hydroxy fatty acid. katja radon has summarised the health effects of endotoxin with respect to different types of asthma [ ] . endotoxin has immune stimulatory and proinflammatory properties even in small amounts linked to the cd molecule. endotoxin can influence the innate immune system, trigger toll-like receptors (tlr- and tlr- ) and influence cytokine production. the connection between endotoxin exposure and respiratory disease and allergies has been investigated in many studies. the risk of getting atopic asthma may be less if you are exposed to endotoxin in childhood. however, the risk of non-atopic asthma may increase if exposed to endotoxin especially in adulthood [ ] . one office study from the netherlands reported an association between gram-negative bacteria and endotoxin concentrations in indoor air, measured by the limulus method, and sick building syndrome (sbs) [ ] . sbs include eye nose, throat and dermal symptoms, headache and fatigue. a study among school children in malaysia found different health associations for different types of endotoxin in classroom dust, measured by chemical analysis of -hydroxy fatty acids. endotoxin with c -hydroxy fatty acids was associated with rhinitis, c endotoxin was associated with dermal symptoms and c endotoxin was negatively associated with throat symptoms (a protective effect) [ ] . other studies on association between endotoxin and asthmatic symptoms, using chemical analysis of -hydroxy fatty acids, have demonstrated different health associations for different types of endotoxin [ , ] . peptidoglycan is present in all bacteria, but the largest amounts are found in grampositive bacteria. muramic acid (mua) is a chemical compound present in peptidoglycan and is a marker of gram-positive bacteria. muramic acid can trigger toll-like receptors (tlr- ) in the immune system. some epidemiological studies on health associations for mua exist. in a birth cohort from finland, the concentration of mua in household dust was associated with asthma incidence in an inverted u-shape way. the highest risk was found at medium levels and the lowest risk at the highest levels [ ] . among school children in austria, switzerland and germany, children with higher mua concentration in mattress dust had lower prevalence of wheeze (a protective effect) [ ] . in two school environment studies from china, mua concentration in classroom dust was negatively associated with prevalence of asthmatic symptoms [ ] and onset of mucosal symptoms (a protective effect) [ ] . in the european community respiratory health survey (ecrhs) mua concentration in mattress dust was positively associated with asthmatic symptom score [ ] but a case-control study within the ecrhs study found a negative association between mua and asthma [ ] . ergosterol is a cell-wall compound found in all microfungi (mould) and macrofungi (mushrooms). however, ergosterol can be found in plant materials and is not entirely specific for mould. some epidemiological studies on health associations for ergosterol exist. in a school study from malaysia, ergosterol in classroom dust was a risk factor for doctors' diagnosed asthma [ ] but a protective factor for rhinitis [ ] . one study from usa in a water damaged office building found an association between levels of ergosterol in vacuumed dust and current asthma [ ] . another study from usa in two hospital buildings found associations between ergosterol levels in vacuumed dust and work-related asthmatic symptoms among hospital workers [ ] . beta- - -glucans is a group of polysaccharide found in the cell-walls of mould. this compound can have different chain length and configuration. in , the literature on health associations for beta- - -glucans was reviewed. it was concluded that available epidemiology does not permit conclusions on respiratory health associations for beta- - -glucans [ ] . a more recent review on fungal exposure and asthma reported that mounting data supports the view that beta- - -glucans contributes to asthma development and severity but there are several studies that support that beta- - -glucan is protective in asthma development. the review concluded that the conflicting results imply that beta- - -glucans may have different health effects depending on the dose, different exposure route and the timing of exposure [ ] . detection and quantification of various mould species can be done by molecular methods (pcr) that can measure dna from different species of mould in dust or air, no matter whether they are dead or alive. the method is called mould specific quantitative pcr (msqpcr) [ ] . it can detect both general groups of mould and species-specific dna sequences. in the last decades, epidemiological articles have been published on health association for fungal dna, mainly on childhood asthma. data on fungal dna in indoor samples can be analysed in different ways. studies have analysed health associations for the number of fungal dna-sequences per gram vacuumed dust ("concentration in dust") or the number of fungal dna sequences per surface area ("surface contamination"). one swedish study found associations between the concentration of fungal dna in dust from day care centers and fraction of exhaled nitric oxide (feno), highsensitivity c-reactive protein in blood (hscrp) and dyspnea among day care center staff. health associations were found for total fungal dna and dna from aspergillus or penicillium species and aspergillus versicolor. the sequence "total fungal dna" is a sequence common for many hundreds of mould species, but not for all mould. the authors concluded that in day care centers, dna from some species of mould (aspergillus sp., penicillium sp. and aspergillus versicolor) as well as the total load of mould (measured as total fungal dna) can be risk factors for airway inflammation [ ] . school studies in europe, malaysia and china have investigated associations between levels of fungal dna in classroom dust and health among the school children [ , , , [ ] [ ] [ ] [ ] . all school studies found associations between levels of fungal dna in the classrooms and respiratory illness (elevated feno levels, asthmatic symptoms, respiratory infections or lower lung function) as well as sbs symptoms (ocular symptoms, rhinitis, tiredness). sbs symptoms include ocular, nasal throat and dermal symptoms, headache and fatigue [ ] . most consistent associations were found for aspergillus versicolor dna. total fungal dna was associated with respiratory illness in some, but not all studies. some studies have investigated health association for fungal dna in household dust. one case-control study reported that levels of aspergillus versicolor dna were higher in asthmatics homes as compared to controls [ ] . another study found that the dna from the fungal genus volutella was associated with increased asthma severity in atopic children [ ] . vesper et al. has developed a concept called enviromental relative moldiness index (ermi) to quantify the mould burden in homes [ ] . the ermi value is computed from the concentrations of species specific dna sequences from indicator mould species in home dust samples. the mould species are divided in two groups. the first group (group mould) consists of mould species that indicate water damage (table . ). the second group (group mould) consist of sequences from ten group species that can be from outdoor sources and are commonly found indoors even without water damage [ ] (table . ). for each home, the mould burden is computed by taking the sum of log-transformed group mould species concentrations minus the sum of log-transformed group mould species concentrations. the ermi value does not measure the total fungal concentration in the dust or the total fungal exposure. it is used to rank homes with respect to the relative mould burden in homes [ ] [ ] [ ] . ermi has been used in epidemiological studies and higher ermi levels have been found in home dust among children with asthma as compared to controls without asthma [ ] [ ] [ ] . one longitudinal study found that early exposure to molds as measured by ermi at year of age, but not years of age, increased the risk for asthma at years of age [ ] . in addition, one recent study found higher ermi values in school dust from schools with high prevalence of asthma as compared to schools with low asthma prevalence [ ] . finally, one study found lower lung function (fev ) among children who lived in homes with higher ermi-values [ ] . a recent study found associations between ermi values and asthma and asthma and respiratory illness in two low-income hispanic communities in california [ ] a few studies exist on associations between ermi values and adult asthma. one study from scotland (uk) found lower lung function (fev ) among non-smoking adults living in homes with higher ermi-values [ ] . another study from usa found an association between ermi values in household dust and asthma and rhinitis among adults [ ] . only few studies exist on associations between bacterial dna in indoor environments and respiratory health. in one study from the ecrhs cohort, mattress dust from homes was extracted by gel electrophoresis and selected bands were sequenced by qpcr. the clostridium cluster xi band was associated with a lower risk of prevalent adult asthma [ ] . another study among pre-school children in usa found a negative association between streptomyces dna concentration in household dust and feno in asthmatic children (a protective effect) but not in healthy children [ ] . feno is a marker of allergic lower airway inflammation. one home environment study from usa investigated associations between indoor microbial community structure and concentration in household dust and asthma severity in atopic and non-atopic children. increased bacterial richness was associated with increased asthma severity. richness was based on number of operational taxonomic units among sequences per sample [ ] . in a school environment study from malaysia, streptomyces dna concentration in classroom dust was associated with more asthma [ ] but less tiredness among school children [ ] . in the european hese school study, streptomyces dna concentration in classroom dust was associated with lower lung function (fev and fvc) [ ] . dampness can cause chemical degradation of building materials with chemical emissions and odour not linked to microbial growth. one example is emission of -ethyl- -hexanol by alkaline degradation of a phthalate (dehp; di-ethyl-hexyl phthalate) used as plasticiser in pvc floor materials. in addition, alkaline degradation of di-ethyl-hexyl acrylate in water-based floor can produce the same compound [ ] . chlorophenols, including pentachlorophenol (pcp), were commonly used as wood preservative in wooden houses until the 'ies in many countries. in damp conditions, microbes can produce various chloroanisoles by dechlorination and methylation of pcp. some chloroanisols have a strong mould odour [ ] . the metabolism of mould and bacteria can produce certain volatile organic compounds (voc) called microbial volatile organic compounds (mvoc) [ ] . mvocs can be produced by mould as well as bacteria. the production of mvoc can be influenced by the type of species but is mainly linked to environmental conditions and the material where the microorganisms grow. some of these compounds have specific mould odour. the compound -octen- -ol has a specific mushroom odour. the compound geosmin is produced by streptomyces species and has a strong soil odour. mvoc includes alcohols ( -butanol; iso-butanol; -methyl- -butanol; -methyl- -butanol; -methyl- -propanol; -pentanol; -pentanol; -octen- -ol; -octanol), ketones ( -hexanone, -heptanone, -octanone, -octanone; -methyl- -heptene- one), esters (isobutyl acetate; bornylacetate, isobornyl acetate, linalylacetate, butanoic acid, -methylbutyl-ester; ethyl-isobutyrate; ethyl- -methylbutyrate), furans ( -methylfuran; -methylfuran; -pentylfuran), terpenes (beta-myrcene; verbenone; terpinolene; camphene; sabinene; alfa-phellandrene; beta-phellandrene; isoterpinolene; alfa-terpinene; gamma-terpinene; fenchone) and other compounds (camphor; estragol; dimethyl disulphide, geosmin) ( table . ). in the 'ies, researchers in sweden, finland and germany started to measure mvocs in the indoor environment to detect hidden microbial growth in the construction [ ] [ ] [ ] . the concentration of most mvocs in the indoor environment is very low, typically ranging from ng/m to μg/m of air. one early study from germany reported that mvoc levels were higher in homes with mould growth [ ] . a study from homes in three north european cities found higher levels of table . examples of mvoc compounds reported in the literature alcohols: -butanol; iso-butanol; -methyl- -butanol; -methyl- -butanol; -methyl- propanol; -pentanol; -pentanol; -octen- -ol; -octanol ketones: -hexanone, -heptanone, -octanone, -octanone; -methyl- -heptene- -one esters: isobutyl acetate; bornylacetate, isobornyl acetate, linalylacetate, butanoic acid, -methylbutyl-ester; ethyl-isobutyrate; ethyl- -methylbutyrate furans: -methylfuran; -methylfuran; -pentylfuran terpenes: beta-myrcene; verbenone; terpinolene; camphene; sabinene; alfa-phellandrene; beta-phellandrene; isoterpinolene; alfa-terpinene; gamma-terpinene; fenchone other compounds: camphor; estragol; dimethyl disulphide, geosmin -methylfuran and ethyl-isobutyrate in air in damp homes as compared to non-damp homes [ ] . later research has demonstrated that many mvocs are not specific for microbial growth. one study compared air concentration of mvocs in dwellings in germany with and without indoor mould growth. only -methyl- -butanol and -octen- -ol showed a significant association with mould status [ ] . another study measuring mvoc compounds found that mvocs are related to synthetic materials at home and to less extent related to microbial sources [ ] . even if some studies found significantly higher levels of some mvoc in damp and mouldy buildings as compared to normal buildings, the differences in mvoc concentrations are not large. one literature review on mvoc reported that more than compounds have been identified as mvocs in laboratory experiments but the recognition of microbially contaminated indoor environment by mvoc measurements has not been successful with current analytical methods [ ] . another methodological problem is that the indoor concentration of mvocs is influenced by the ventilation flow, which means that it can be difficult to estimate if there is increased emission of mvocs from the construction without taking into consideration the ventilation flow. some epidemiological studies have demonstrated associations between mvoc air concentrations in homes and health. in the german study, children in dwellings with elevated mvoc levels had a non-significant tendency of more asthma, hay fever, wheeze and eye irritation [ ] . one study from france reported an association between a fungal index, based on mvoc measurements in the home, and current asthma and bronchitis [ ] . one study from japan found an association between -octen- -ol concentrations in the home and allergic rhinitis and conjunctivitis [ ] . another article from the same japanese study found an association between -octen- -ol and home-related mucous membrane symptoms [ ] . one swedish school study reported associations between total mvoc levels in the classrooms and nocturnal breathlessness and doctor diagnosed asthma among the school children. wheeze was associated with -octanone, only. doctor diagnosed asthma was associated with -heptanone and -methyl- -butanol. nocturnal attacks of breathlessness were associated with many types of mvoc ( -methylfuran, -methyl- -butanol, dimethyldisulphide, -heptanone, -octen- -ol, -octanone, -methyl- -butanol, -penthylfuran, isobutylacetate and -butanol) [ ] . in the home environment study in homes in three north european cities, there were positive associations between any sbs symptom and -pentanol, -hexaone, -pentylfuran, -octen- -ol. two compounds, -octen- -ol and -methylfuran, were positively associated with mucosal sbs symptoms. two compounds (ethyl isobutyrate and ethyl- -methylbutyrate) were negatively associated with any sbs symptom (a protective effect) [ ] . in the case-control study among pre-school children in sweden by choi et al., there was an association between sum concentration of mvoc and case status, but only in homes with high absolute air humidity. the case definition among the pre-school children was based on wheeze, rhinitis or eczema [ ] . in the literature review on mvoc, toxicological and exposure data was collected for common mvoc. the most obvious health effects of mvoc exposure were eye and upper-airway irritation but the toxicological database was poor for these compounds. the review suggested that there may be more potent compounds and other endpoint not yet evaluated [ ] . microorganisms can produce metabolites in their secondary metabolism, sometimes called microbial toxins. mycotoxins are defined as toxic compounds produced by mould. mycotoxins can grow in food and the main concern about human exposure is linked to food contamination. aflatoxins (aflatoxin b , b , g , g and m ) are classified as human carcinogens (group ) and the mycotoxins fumonisin b , fumonisin b , fusarin c, ochratoxin a and sterigmatocystin are classified as possible human carcinogens (group b) [ ] . the classification of these mycotoxins as human carcinogens is based on dietary intake of contaminated food, not indoor exposure. recent development of chemical analytical methods has made it possible to measure secondary microbial metabolites (and mycotoxins) in damp building materials and in indoor dust. the european hitea school study (health effects of indoor pollutants: integrating microbial, toxicological and epidemiological approaches) is one of the largest studies on exposure to secondary microbial metabolites in indoor environments [ ] . totally secondary metabolites could be detected in european schools, but the article did not include any health evaluation. the most common mycotoxins were emodin, physcion and the enniatins a , b and b . less common mycotoxins were alamethicin, melagrin, griseofulvin, apicidin, beauvericin, trichodermol and verrucarol. about half of the school contained at least one secondary microbial metabolite. the levels were very low, typically . - picogram/m of swabbed area. schools with dampness or moisture damage contained higher levels of microbial secondary metabolites than non-damp schools [ ] . the health significance of indoor exposure to fungal secondary metabolites (mycotoxins) remains unclear because of lack of epidemiological studies. one review article describes possible adverse health effects of indoor mould, including effects of mycotoxins [ ] . certain mycotoxins can have pronounced health effects in experimental animal studies or in vitro tests but it has been unclear if the exposure levels in indoor environments are enough to cause any health effects. one recent review article summarised available data on the low-molecular-weight toxins from fungi common in damp building materials, and exposure levels found in indoor environments. the review conclude that it is possible that toxin doses at levels found in damp buildings could modulate genes that are in the asthma pathways, but next decade of research will illuminate the significance of this information [ ] . in the context of indoor mycotoxin exposure, there has been a special focus on trichothecene mycotoxins from the mould stachybotrys chartarum [ ] . however, existing epidemiology on association between mycotoxins in general, including mycotoxins from stachybotrys chartarum, is sparse. one study from finland measured secondary metabolites in homes of -year old children. a total of different microbial metabolites were detected in the homes. the number of metabolites tended to be higher in homes with dampness and mould. however, the sum of microbial secondary metabolites was negatively associated with current asthma (a protective effect). the authors concluded that there were no evidence indicating that secondary microbial metabolite could explain the well-known association between indoor mould and dampness and asthma [ ] . one school study from malaysia measured mycotoxins and fungal dna in classroom dust in classrooms. stachybotrys chartarum dna was detected in % of the classrooms. three types of mycotoxins were detected. aflatoxin b was detected in %, sterigmatocystin in % and verrucarol in % of the classrooms. verrucarol is produced by stachybotrys chartarum. there were negative associations between the mycotoxin verrucarol and stachybotrys chartarum dna levels on indoor surfaces and daytime attacks of breathlessness among school children (a protective effect) [ ] . in contrast, there were positive associations in the study between verrucarol levels and stachybotrys chartarum dna levels in the classrooms and the prevalence of tiredness among the children [ ] . dampness and indoor microbial growth of mould and bacteria is common in indoor environments in many countries. there is international consensus, based on epidemiological evidence, that damp buildings and indoor mould can increase the risk of asthma, rhinitis, bronchitis and respiratory tract infections. however, there is not enough epidemiological studies to clarify which types of microbial agents that are causing the observed adverse health effects in damp buildings. microbial indoor exposure is a broader concept than microbial growth in buildings. other sources of indoor microbial exposure include air and dust from the outdoor environment, humans (crowdedness), furry pet keeping. microbial exposure can have different health effects depending on the dose, different exposure route, genetic disposition and the timing of exposure. there is now epidemiological evidence that microbial stimulation linked to large microbial diversity in early life can protect against disease development, especially for allergic asthma and atopy. in epidemiological studies, protective effects are more often reported for bacterial exposure and adverse health effects are more linked to mould exposure. however, some studies have reported protective effects of mould and adverse effects of bacterial exposure. there are many epidemiological studies on health associations for indoor exposure to endotoxin, mainly from the home environment. the risk of getting atopic asthma may be less if you are exposed to endotoxin in childhood but the risk of non-atopic asthma may increase if exposed to endotoxin especially in adulthood. moreover, genetic disposition is an important factor influencing health effects of endotoxin exposure. epidemiological studies reporting health associations (positively or negatively) with other cell-wall compounds such as muramic acid and ergosterol are few. studies on health effects of indoor exposure to beta- - -glucan have found positive as well as negative health associations (conflicting results). epidemiological studies on health effects of indoor exposure to mycotoxins are very few. some epidemiological studies have reported health associations for mvoc, but it is unclear to what extent mvoc has microbial sources in indoor environments. there are an increasing number of studies reporting health associations for fungal dna, especially adverse effects in relation to childhood asthma. most of these studies have investigated fungal dna in homes. since most studies on health effects of indoor exposure to mould, bacteria and microbial compounds are crosssectional, it is difficult to draw conclusions on causality. more prospective studies on indoor microbial exposure are needed. moreover, epidemiological studies on microbial exposure should include other indoor environments than homes, such as day care centers, schools, hospitals and offices. indoor fungi: companions and contaminants survival of amoebae on building materials geographic variation and the determinants of domestic endotoxin levels in mattress dust in quantitative pcr analysis of fungal dna in swedish day care centers and comparison with building characteristics and allergen levels world health organization (who) regional office for europe. guidelines for indoor air quality: dampness and mould. copenhagen: who regional office for europe mould and dampness in dwelling places, and onset of asthma: the population-based cohort ecrhs exposure 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-associations with rhinitis and sick building syndrome (sbs) in junior high school students airborne gram-negative bacteria and endotoxin in sick building syndrome. a study in dutch governmental office buildings asthmatic symptoms among pupils in relation to microbial dust exposure in schools in taiyuan quantity and diversity of environmental microbial exposure and development of asthma: a birth cohort study study team alex. microbial exposure of rural school children, as assessed by levels of n-acetyl-muramic acid in mattress dust, and its association with respiratory health a longitudinal study of sick building syndrome among pupils in relation to microbial components in dust in schools in china predictors of microbial agents in dust and respiratory health in the ecrhs microbial characteristics in homes of asthmatic and non-asthmatic adults in the ecrhs cohort hydrophilic fungi and ergosterol associated with respiratory illness in a water-damaged building asthma and respiratory symptoms in hospital workers related to dampness and biological contaminants → )-beta-d-glucans and respiratory health: a review of the scientific evidence comparison of populations of mould species in homes in the uk and usa using mould-specific quantitative pcr (msqpcr) fungal dna in dust in swedish day care centres: associations with respiratory symptoms, fractional exhaled nitric oxide (feno) and c-reactive protein (crp) in serum among day care centre staff fungal dna, allergens, mycotoxins and associations with asthmatic symptoms among pupils in schools in respiratory symptoms and fractional exhaled nitric oxide (feno) among students in penang, malaysia in relation to signs of dampness at school and fungal dna in school dust rhinitis, throat and dermal symptoms, headache and tiredness among students in schools from johor bahru, malaysia: associations with fungal dna and mycotoxins in classroom dust ocular symptoms and tear film break up time (but) among junior high school students in penang, malaysia-associations with fungal dna in school dust an update on sick building syndrome indoor fungal contamination of moisture-damaged and allergic patient housing analyzed using real-time pcr indoor microbial communities: influence on asthma severity in atopic and noatopic children development of an environmental relative moldiness index for us homes correlation between ermi values and other moisture and mold assessments of homes in the american healthy homes survey screening tools to estimate mold burdens in homes geographic distribution of environmental relative moldiness index molds in usa homes quantitative pcr analysis of molds in the dust from homes of asthmatic children in north carolina higher environmental relative moldiness index (ermism) values measured in detroit homes of severely asthmatic children higher environmental relative moldiness index (ermi) values measured in homes of asthmatic children in high environmental relative moldiness index during infancy as a predictor of asthma at years of age mold contamination in schools with either high or low prevalence of asthma decreased pulmonary function measured in children exposed to high environmental relative moldiness index homes asthma risk associated with indoor mold contamination in hispanic communities in eastern cochella valley, california decreased fev % in asthmatic adults in scottish homes with high environmental relative moldiness index values higher environmental relative moldiness index values measured in homes of adults with asthma, rhinitis or both conditions indoor bacteria and asthma in adults: a multicenter case-control study within ecrhsii microbial content of household dust associated with exhaled no in asthmatic children dampness and -ethyl- -hexanol in floor construction of rehabilitation center: health effects in staff chloroanisoles may explain mold odor and represent a major indoor environment problem in sweden microbial volatile organic compounds-what substances can be found in sick buildings? sensory irritation potency of some microbial volatile organic compounds (mvocs) and a mixture of five mvocs relevance of airborne fungi and their secondary metabolites for environmental, occupational and indoor hygiene determination of selected microbial volatile organic compounds by diffusion sampling and dual-column capillary gc-fid-a new feasible approach for the detection of an exposure to indoor mould fungi? airborne molds and bacteria, microbial volatile organic compounds (mvoc), plasticizers and formaldehyde in dwellings in three north european cities in relation to sick building syndrome (sbs) microbial volatile organic compounds in the air of moldy and mold-free environments non-microbial sources of microbial volatile organic compounds microbial volatile organic compounds annesi-maesano i. positive associations between respiratory outcomes and fungal index in rural inhabitants of a representative sample of french dwellings the relationship between exposure to microbial volatile organic compound and allergy prevalence in single-family homes relationship between selected indoor volatile organic compounds, so called microbial voc, and the prevalence of mucous membrane symptoms in single family homes indoor molds, bacteria, microbial volatile organic compounds and plasticizers in schools-associations with asthma and respiratory symptoms in pupils mycotoxins as human carcinogens-the iarc monographs classification microbial secondary metabolites in school buildings inspected for moisture damage in finland, the netherlands and spain adverse health effects of indoor molds fungal secondary metabolites as harmful indoor air contaminants: years on stachybotrys chartarum, trichothecene mycotoxins, and damp building-related illness: new insights into a public enigma microbial secondary metabolites in homes in association with moisture damage and asthma key: cord- -rhpfpku authors: zhong, hui-hai; wang, hui-yuan; li, jian; huang, yong-zhuo title: trail-based gene delivery and therapeutic strategies date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: rhpfpku trail (tumor necrosis factor-related apoptosis-inducing ligand), also known as apo l, belongs to the tumor necrosis factor family. by binding to the death receptor (dr ) or dr , trail induces apoptosis of tumor cells without causing side toxicity in normal tissues. in recent years trail-based therapy has attracted great attention for its promise of serving as a cancer drug candidate. however, the treatment efficacy of trail protein was under expectation in the clinical trials because of the short half-life and the resistance of cancer cells. trail gene transfection can produce a “bystander effect” of tumor cell killing and provide a potential solution to trail-based cancer therapy. in this review we focus on trail gene therapy and various design strategies of trail dna delivery including non-viral vectors and cell-based trail therapy. in order to sensitize the tumor cells to trail-induced apoptosis, combination therapy of trail dna with other drugs by the codelivery methods for yielding a synergistic antitumor efficacy is summarized. the opportunities and challenges of trail-based gene delivery and therapy are discussed. nucleic acid-based therapy has been considered one of the most promising strategies for the treatment of various diseases [ ] . tumor necrosis factor (tnf) plays an important role in the homeostatic regulation of the immune system [ ] . although tnf is potent in causing tumor necrosis, the first two clinical trials of tnflike molecules for cancer therapy failed because of lethal inflammatory shock syndrome and fulminant liver toxicity [ , ] . subsequentlyx, a novel tnf family member, tnf-related apoptosis-inducing ligand (trail), was found [ , ] ; this protein is a type ii transmembrane protein and can be released from the cell surface in soluble form via proteolysis [ ] . soluble trail is nontoxic to normal cells, and in fact, there is a trace amount of endogenous trail (~ pg/ml) in healthy adult plasma [ , ] . the trail protein is expressed in various tissues-predominantly in the spleen, lung, and prostate-and on the surface of cytotoxic t cells and natural killer (nk) cells [ ] . its death receptors (drs), dr and dr , are overexpressed in many types of cancer cells. importantly, trail is capable of killing tumor cells without causing lethal adverse effects [ , ] . apoptosis is an essential function of the maintenance of cellular homeostasis and prevents a number of diseases, including cancer [ ] . tumorigenesis is associated with defects in apoptosis regulation [ ] . there are two major apoptotic pathways: the intrinsic, or mitochondrial, pathway usually induced by chemotherapy [ ] , and the extrinsic, or dr, pathway that mediates extrinsic programs of cell death, such as trail-induced apoptosis. however, these two pathways usually associate with each other downstream via "crosstalk" [ ] . the extrinsic pathway is activated by extracellular proapoptotic stimulators that bind to cell surface receptors [ ] . there are five homologous human receptors for trail: the full-length intracellular death domain (dd)-containing receptors dr [ ] and dr (trail-r ) [ ] [ ] [ ] ; the decoy receptor (dcr or trail-r ), which lacks an intracellular domain; [ , , ] dcr (trail-r ), which contains a truncated dd; [ , ] and the soluble receptor osteoprotegerin (opg). among these receptors, only the binding of trail to dr or dr -because of their integrated intracellular structure-can induce an apoptotic effect; dr has the highest affinity for trail [ ] . however, trail/dcr binding cannot induce downstream signaling, because dcr lacks an intracellular domain, while dcr and opg act as nf-κb ligand receptors, which induce nf-κb activation but not apoptosis. after dr or dr binding to trimeric trail, the intracellular dd structure of the drs is altered, and binding to fas-associated death domain-containing protein (fadd) then occurs. then, fadd binds to procaspase- /- via the death effector domain (ded) on the n-terminus, thereby forming dr /dr -fadd-procaspase- /- , which is called the death-inducing signaling complex (disc). oligomerization and autocatalysis of procaspase- leads to the activation of caspase- , which consequently triggers cleavage of the effector caspases- /- /- to induce apoptosis. furthermore, caspase- promotes the release of cytochrome c, inducing intrinsic apoptosis via the mitochondrial pathway in type ii cells [ ] . the trail-induced apoptosis process is summarized in fig . the ability of the tumor-specific action of trail to induce the apoptosis of cancer cells while sparing normal cells is attractive and renders trail signaling a potential therapeutic target. to date, clinical trials of trail have focused on recombinant trail protein and antibodies against trail-r (table ) . however, clinical trials have shown inadequate treatment outcomes [ ] . the recombinant form of trail and anti-trail-r antibodies, as well as their combination with other components, have not achieved the expected efficacy [ ] . for example, the recombinant form of trail did not exhibit significant antitumor effects, partially due to its short half-life [ , ] , while tas , an antibody targeting dr , showed acute toxicity in a phase i clinical study [ ] . there are three major limitations of trail-based therapy: its short in vivo half-life [ , ] , its poor tumor-targeting efficacy, and resistance to trail monotherapy [ , ] . the emergence of nanotechnology has provided a useful tool to address these problems. trail-based nanotherapies offer the potential to improve the stability of trail and prolong its half-life in the bloodstream, to specifically deliver trail to target sites and to overcome resistance to trail [ ] . compared with direct administration of trail proteins, trail gene therapy also has the unique advantages of delivering trail-encoding dna into tumor cells to locally secrete the trail protein on the membrane or into the tumor microenvironment, thereby overcoming the limitations of recombinant trail protein. notably, combination therapies of trail with other anticancer agents via a codelivery system may solve the problem of trail resistance. trail gene therapy also benefits from the "bystander" effect, by which not only the host cancer cells but also the neighboring cancer cells can be killed by both secreted and membrane-bound trail [ ] . trail shows a unique advantage over other cell deathinducing ligands (e.g., fas ligand, fasl), of which only the membrane-bound form can induce apoptosis, while the intrinsic soluble form cannot [ ] . liposome-bound trail induces even more efficient apoptosis than the soluble form [ ] . trail-based gene therapy has been investigated in various types of tumors, such as hepatocellular carcinoma and cervical cancer. since the completion of the human genome project, the development of gene therapy has accelerated. gene therapy depends on the success of delivering specific nucleic acids to target sites by overcoming a series of biobarriers; in other words, it relies on the efficient delivery of vectors. viral vectors are well known for their high transfection efficiency, which mainly relies on their ability to integrate genes into the genome of host cells. this approach increases the risk of insertion mutations at the integration site, especially if there are hotspots near prooncogenes. thus, there is an urgent need to find a highly efficient vector with low genotoxicity and immunogenicity. nonviral vectors delivering genes typically via a nanosized platform are another option, with obvious advantages of safety and the high packing capacity of nucleic acids. in addition, advances in nanotechnology have provided good insight into rational designs for targeted delivery. although numerous nonviral vectors have been developed, the amount of data from clinical trials has been very limited due to their low transfection efficiency [ ] . thus, there is a pressing need to develop nonviral vectors with increased efficiency. nonviral vectors include cationic lipids, cationic polymers, and inorganic nanoparticles. polyethyleneimine (pei), for example, has high transfection efficiency in various cell lines. for the systemic delivery of gene therapeutics, delivery systems must cross a series of barriers, which is an important consideration in the design of delivery systems. regarding trail-based therapy, viral vectors are prominently used for cell therapy, while nonviral vectors deliver plasmidencoded trail (ptrail) to targets. in this review, we summarize trail-based gene therapeutic strategies and discuss the challenges facing clinical trials of trail. most gene therapy clinical trials involved viral vectors [ ] . modified viruses such as retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses (aavs) are commonly used to deliver genes. viruses are able to transfer genes into many cell types, with highly efficient transfection. griffith et al. constructed a trail-encoded adenovirus and found that rapid expression of the trail protein and apoptosis of tumor cells were triggered by the activation of caspase- [ ] . oncolytic adenoviruses (oads) can selectively replicate in cancer cells while sparing normal cells; thus, these viruses have been used in clinical trials for anticancer therapy. el-shemi et al. applied systemic therapy with ad-Δb/ing (inhibitor of growth ) plus ad-Δb/trail in an orthotopic human hepatocellular carcinoma (hcc) -bearing mouse model. this study found that the combination of these agents elicited potent eradicative effects by inducing apoptosis and immune responses, and suppressing tumor angiogenesis without causing obvious overlapping toxicity [ ] . in addition, the use of viral vectors has been explored for trailbased cell therapy, which will be introduced in section . because of the inherent shortcomings of viral vectors, including their limited dna packaging capacity, complicated production processes, broad tropism, cytotoxicity, immunogenicity, and tumorigenicity, nonviral vectors have also been widely investigated as an option for gene therapy [ ] . in contrast to viral vectors, nonviral vectors have the advantages of low immunogenicity, high delivery capacity, and easy preparation [ , ] . before they reach the target cells, delivery systems need to cross a series of biobarriers. in the physiological environment, positively charged complexes are more prone to bind serum proteins and aggregate and thus be cleared rapidly [ , ] . shielding the positive charge using polyethylene glycol (peg) or anionic materials such as γ-pga can be helpful. another challenge is selective accumulation at the target tissue, which requires specific designs, such as arming targeted ligands. subsequently, fig. pathway of trail-induced apoptosis. trail binds to five receptors, including four membrane-bound receptors (i.e., dr , dr , trail-r , and trail-r ) and one soluble receptor (opg). only binding to dr or dr results in receptor trimerization and recruitment of fadd via the dds of dr or dr . after further recruitment of caspase- , these proteins form a complex named disc, which can activate caspase- . in type i cells, caspase- activates caspase- and triggers apoptosis via the extrinsic pathway. however, in type ii cells, the intrinsic pathway is triggered via caspase- /bid/tbid, and consequently, bax/bak on the mitochondrial membrane is activated to induce the release of cytochrome c, which promotes the formation of the apoptosome with apaf and procaspase- . subsequently, activation of caspase- and caspase- is induced. figure adapted from fig. in ref. [ ] the dna of interest needs to be delivered to the nucleus for transcription [ ] . thus, the design of gene delivery systems should account for these considerations. polymers. cationic polymers are an important class of nonviral vectors. poly(l-lysine) (pll) and polyethyleneimine (pei) were developed for gene delivery in the s. subsequently, numerous cationic polymers have been developed and used, including poly [( -dimethylamino) ethyl methacrylate] (pdmaema), poly(β-amino ester)s, and various carbohydrate-based polymers and dendrimers. pei and its derivatives are the most commonly used polymeric vectors for gene delivery, with the advantage of the "proton sponge" effect, which facilitates the endosomal escape of gene drugs [ ] . however, the major hurdles to overcome for the in vivo use of pei are the substantial cytotoxicity related to its strong positive charge and the issue of its stability in the bloodstream [ ] . to address these issues, modification of pei and the surface coating have been investigated. for example, a γ-pga corona can shield the positive charge of the pei/dna complex, thereby decreasing its toxicity and increasing its stability. it has been reported that the γ-pga-coated branched pei/ptrail complex can efficiently transfect pancreatic stellate cells expressing fibroblast growth factor receptors [ ] . although pei k is the gold standard for polymer transfection, its high molecular weight generally causes high toxicity. therefore, many studies focusing on low-molecular-weight pei have been reported. for instance, a gene delivery system for brain targeting was established by using pei k modified with myristic acid (mc); mc-pei k /dna nanoparticles could interact with the cell membrane via the hydrophobic segment on mc that can be incorporated into the phospholipid bilayer. mc-pei k /porf-htrail nanoparticles were effective against the growth of intracranial tumors [ ] . furthermore, cell-penetrating peptide (cpp)-modified and mannosylated low-molecular-weight pei (termed man-pei k -cpp) was constructed as a vector to deliver ptrail for colorectal cancer treatment. man-pei k -cpp increased the cellular uptake efficiency and improved the efficiency of transfection [ ] . then, a ternary complex system was developed by a γ-pga-based γ-glutamyl transpeptidase (ggt)-targeting and surface camouflage strategy via a layer-bylayer self-assembly method. biodegradable polyanionic γ-pga protected the pei/pdna complexes from interaction with body fluid components and interacted with tumor-overexpressed ggt, which can mediate the endocytosis of nanoparticles for cervical cancer gene therapy (fig. ) [ ] . hyaluronic acid (ha) also acts as a biocompatible polyanionic biomaterial for shielding positive charges. an ha-decorated polyethyleneimine-poly(d, l-lactide-co-glycolide) nanoparticle (pei-plga np) system was established for targeted codelivery of ptrail and gambogic acid (ga) for triple-negative breast cancer (tnbc) therapy [ ] . ga was encapsulated into the hydrophobic core of pei-plga nps, while ptrail was adsorbed onto the positively charged np surface. the ha coating on the pei-plga nps not only functioned as a shell to neutralize the excess positive charge on the nps but also served as a targeting ligand by binding to the cd receptor on tnbc cells. a series of terpolymers were synthesized via the enzyme-catalyzed copolymerization of lactone with dialkyl diester and aminodiol. targeted delivery of ptrail to the tumor by the terpolymers resulted in significant inhibition of tumor growth with minimal toxicity in vivo, and the transfection efficiency was associated with the high molecular weight and increased hydrophobicity [ ] . intriguingly, it was found that preparation via a high concentration the clinical trials can be found at https://www.clinicaltrials.gov trail-based gene delivery and therapeutic strategies hh zhong process (i.e., a small reaction volume) resulted in large pei/dna complexes that had a higher gene transfection efficiency than their small counterparts prepared at a low concentration (fig. ) [ ] . the mechanisms were associated with macropinocytosis and fast dissociation. accordingly, large-sized pei/ptrail complexes exhibited increased anticancer efficacy via local or regional administration in subcutaneous xenograft and peritoneal xenograft mouse models. poly(beta-amino ester)s (pbaes) are a class of cationic synthetic polymers that can be used as nonviral gene carriers. these compounds are easy to synthesize, bind effectively to dna, and are hydrolytically degradable under physiological conditions. tzeng et al. reported the use of pbae nanoparticles containing ptrail for the selective transfection of cancer cells and the induction of apoptosis in several human cancer cells [ ] . additionally, shen et al. developed an ingenious vector comprising quaternary amines carrying n-propionic -acetoxybenzyl ester substituents; the -acetoxybenzyl ester group can undergo rapid intracellular esterase-catalyzed hydrolysis, which subsequently triggers a reversal of the polymer's charge from cationic to zwitterionic [ ] . due to the high cytosolic esterase activity in cancer cells but low activity in fibroblasts, the loaded ptrail can be delivered specifically into cancer cells without damaging fibroblasts, preventing the expression of wnt b (fig. ) [ ] . the same group also synthesized another enzyme-responsive cationic polymer, poly (pqdea), which is rapidly hydrolyzed by intracellular esterases to form anionic poly(acrylic acid) to confer low cytotoxicity and fast release of dna [ ] . pqdea/dna polyplexes were further coated with a lipid layer to generate serum-stable lipidic polyplexes (lpqdea/dnas) for in vivo use. lpqdea/ptrail strongly inhibited tumor formation as effectively as paclitaxel but gave less tumor relapse and longer survival. in addition, some inorganic materials have been modified with polymers for ptrail delivery. for example, a sandwich-type peicoated gold nanocomposite coloaded with ptrail and nucleartargeted dexamethasone (dexa) (termed au-pei/ptrail/pei-dexa) exhibited efficient transfection and significantly inhibited the growth of hep b tumors [ ] . a targeted iron oxide np coated with a chitosan-peg-pei copolymer and chlorotoxin was developed; this np efficiently delivered ptrail into human t g gbm cells and induced the secretion of trail [ ] . further, systemic administration to mice bearing t g-derived flank xenografts resulted in almost imperceptible tumor growth and induced apoptosis in tumor tissue. to overcome treatment limitations of adenoid cystic carcinoma, the fe o -pei-plasmid complex (fpp) was generated, in which iron oxide nps were modified by positively charged pei to enable them to carry pactert-trail [ ] . the efficiency of fpp-mediated transfection was sixfold higher than that of pei alone or of lipo , and fpp-mediated trail gene transfer efficiently inhibited sacc- tumor growth. dendrimers. dendrimers, which are characterized by their welldefined size and low polydispersity index, have been widely used for drug and gene delivery [ ] . jiang et al. reported a gene vector generated by polyamidoamine (pamam) and angiopep- another strategy has been reported in which transferrin (tf) was conjugated to a generation diaminobutyric polypropylenimine (dab) dendrimer; this delivery system harbors plasmids encoding for tnf-α, trail, or il- and leads to therapeutic effects on prostate tumors following intravenous administration [ ] . in addition, lactoferrin (lf)-bearing dab dendriplexes showed similar efficacy [ ] . later, coumarin-anchored low-generation dendrimers (g pamam dendrimers) were established to improve dna binding and gene delivery [ ] . the coumarin moieties endowed these materials with light-responsive drug delivery behaviors, and the drug-loaded nanoparticles exhibited complementary anticancer activity through the codelivery of -fluorouracil and ptrail. a triazine-modified dendrimer, g -dat , was synthesized and used as a vector for ptrail therapy, showing higher transfection efficacy than commercial transfection reagents such as lipo and superfect [ ] . furthermore, in vivo studies demonstrated that g -dat /ptrail efficiently inhibited tumor growth in osteosarcoma-bearing mice. pishavar et al. synthesized a vector composed of pamam dendrimers modified with alkyl-carboxylate chains, peg, and cholesteryl chloroformate for delivering ptrail, and these pamam g -alkyl-peg ( %)-chol ( %)-trail complexes inhibited ct tumor growth in mice [ ] . peptides and proteins. cationic peptides rich in residues such as lysines or arginines are able to condense dna. furthermore, conjugation of peptide ligands to delivery systems allows targeting to specific types of cancer cells [ ] . a biomimetic vector was developed for delivering ptrail to tumor; it contained an adenovirus μ peptide for pdna condensation, a synthetic cyclic peptide for tumor-targeting and intracellular delivery, a phresponsive synthetic fusogenic peptide for endosome escape, and a nuclear localization signal from human immuno-deficiency virus for intranuclear delivery [ ] . up to % of zr- - breast cancer cells were killed after exposure to ptrail in complexation with this vector. a dimerized hiv- tat peptide was used to formulate fig. illustration of the esterase-responsive charge-reversal polymer (erp) and its lipid-coated esterase-responsive polyplexes with trail plasmid for cancer gene therapy. a the erp is a pei whose amines are quaternized with propionic -acetoxybenzyl ester. hydrolysis of the phenolic acetate triggers the elimination of p-hydroxymethylphenol and consequent conversion of the cationic polymer into a zwitterionic form. b erp condenses plasmid dna into the polyplexes, which are easily coated with dc-chol/dope lipids to form lipidic esterase-responsive polyplexes (lerps). after i.p. injection into nude mice bearing hela cell-derived tumors, tumor cells internalize lerps into the cytosol, which is rich in esterases. the lerps disassemble and release the polyplexes to allow the esterases to trigger the charge reversal of the erp and thus plasmid release. these free plasmids enter the nucleus for effective gene expression, inducing apoptosis when delivering the trail gene. in tumor fibroblasts, the low esterase level cannot efficiently induce the charge reversal process, and the trail plasmids will not be expressed, preventing wnt b production. reprinted with permission from [ ] a nanoparticle vector (dtat np) to leverage the efficiency of this cell penetration strategy for tumor-targeted gene delivery [ ] . in cell culture, dtat np was an effective pdna transfection vector with negligible cytotoxicity. gene expression in tumor tissues lasted for > days after intratracheal administration. bolus administration of dtat np-encapsulated ptrail markedly attenuated the growth of tumors derived from lewis lung carcinoma cells. phosphatase and tensin homolog (pten) and trail genes loaded into zein nanoparticles showed antiproliferative activity against hepg cell lines, indicating their potential for gene therapy for the treatment of hcc [ ] . liposomes. cationic lipids have been widely used for nucleic acid delivery and for advances in gene delivery through their molecular design. cationic lipids can spontaneously form specific types of complexes for condensing and encapsulating dna into particles [ ] . huang et al. reported a novel lipid ( , -di-( z-octadecenoyl)- biguanide-propane (dobp)) that was elaborately designed by utilizing biguanide as the cationic head group [ ] . this novel cationic lipid acted as a gene carrier and had a metformin-like antitumor activity via activation of the ampk and inhibiting the mtor pathways. dobp-lpdtrail nps showed potent efficacy against tumor progression. then, these nps were used to transfer a secreted form of trail (strail) to tumor-associated fibroblasts in order to secrete cytotoxic proteins to tumor cells via lipidcoated protamine [ ] . strail triggered apoptosis in tumor cell nests adjacent to tafs. furthermore, strail converted the residual fibroblasts to a quiescent state, thus arresting the tumor growth and remodeling the microenvironment to facilitate the secondwave nanotherapy. the system showed good efficacy in an orthotopic xenograft model of human pancreatic cancer, where the desmoplastic stroma is a major barrier to the delivery of therapeutic nanoparticles. chen et al. developed a tumor-targeted lcpp (lipid/calcium/ phosphate/protamine) np to deliver ptrail into hcc cells in a mouse model of hcc [ ] . ptrail was entrapped in a phresponsive calcium phosphate (cap) core, and protamine was included to direct intranuclear delivery. trail resistance could be reversed by intracellular release of ca + from the cap core that induced dr up-regulation through camkii activation (fig. ) . other vectors. gong et al. developed a well-tailored and versatile "core-shell" ternary system (rrphc) for systemic gene delivery to treat aggressive melanoma [ ] . this system consisted of a core of fluorinated polymers (pfs) that bound to a plasmid (pdna) and a negatively charged multifunctional rrph (rgd-r -peg-ha) shell constructed by grafting a hyaluronan polymer with peg side chains, which were further conjugated with the r -rgd tandem peptide on the distal side, simultaneously targeting the cd receptors and integrin α v β receptors overexpressed on the neovasculature and most malignant tumor cells. systemic injection of the proapoptotic mtrail plasmid by rrphc ternary complexes inhibited the melanoma growth, without noticeable side toxicity. next, this group reported a similar system of an artificial virus core-shell to target cancer stem cell-like cells [ ] . the intravenously injected nanoparticles accumulated at the tumor sites while reducing the exposure to the normal tissues, and efficiently arrest the tumor growth, without obvious systemic toxicity. mesenchymal stem cells (mscs) are a population of fibroblast-like cells originally isolated from bone marrow and other tissues, including adipose tissue, peripheral blood, umbilical cord blood, and wharton's jelly, among others [ , ] . mscs have therapeutic potential in several pathological conditions and unique immunological features [ ] [ ] [ ] . in recent years, the cellular vehicle function of mscs has been used to transfer trail to the tumor parenchyma [ ] [ ] [ ] . lee et al. reported a novel application of magnetic core-shell nanoparticles for the dual purpose of delivering and activating a heat-inducible gene vector that encodes trail in adipose-derived mesenchymal stem cells (ad-mscs) [ ] . this group developed a plasmid harboring the heat shock protein b' (hsp b ) promoter. the magnetic core-shell nanoparticles (mc nps) was composed of znfe o magnetic nanoparticle core and mesoporous silica shell, as well as a surface coating of pei for dna binding. the mc nps facilitated the intracellular delivery of the heat-inducible plasmid into ad-mscs via magnetic guidance, and after systemic injection, the engineered ad-mscs could home to tumors/metastases. trail expression could be specifically activated via the induction of mild magnetic hyperthermia (~ °c). this system enhanced control over the activation of stem cell-based gene therapies. it was reported that human mscs were transduced with trail and the ires-egfp reporter gene under the control of the tetracycline promoter using a lentiviral vector [ ] . the transduced and activated mscs led to the apoptosis and death in various cancer cells in coculture experiments. the in vivo studies demonstrated that the i.v. injected trail-expressing mscs significantly arrested the tumor growth. gao et al. transfected ptrail into mscs with a nonviral vector, pei -cyd, prepared by linking low-molecular-weight polyethyleneimine (pei) and β-cyclodextrin (β-cd) [ ] . the lung tumor homing ability of mscs expressing trail in vivo proved to be efficient for lung metastasis therapy. fig. shows a schematic of the specific process of this cell-based trail therapy. human umbilical cord-derived mesenchymal stem cells (humscs) were transfected by lentiviral vectors coding the strail with the alpha-fetoprotein (afp) promoter, and the treatment efficacy of these engineered humscs on orthotopically implanted hepatocarcinomas in mice was examined [ ] . humscs could migrate to the hepatocarcinoma, where the afp promoter was triggered by the early hepatic differentiation of humscs, and expressed strail at the cancer cells and yielded significant antitumor activity. dominici et al. transduced human ad-mscs with a retroviral vector encoding full-length human trail [ ] . ad-mscs could target various cancer cell lines in vitro, and reverse the trail resistance by coadministration of bortezomib. these ad-mscs targeted to tumors and induced apoptosis, without apparent side toxicity. the engineered mscs with trail expression also induced apoptosis by cell-to-cell contact in the trailresistant ewing sarcoma (ews) that is insensitive to amg , an antibody against the death receptor dr , too, and the treatment effect was confirmed in two orthotopic models of ews [ ] . despite these encouraging results, there is some concern regarding the safety of inoculating both wild-type and genetically modified mscs, especially regarding their possible damaging effects on normal organs, malignant transformation, and promotion of cancer growth [ ] . then, researchers incorporated suicide genes-the herpes simplex virus thymidine kinase (hsv-tk) and cytosine deaminase genes-into mscs to control their fate once infused [ ] . this approach is based on a variant of human caspase- that binds with high affinity to a synthetic, bioinert small molecule (ap ), leading to cell death [ ] . conventionally, mscs have been genetically modified for cancer therapy by using viral vectors that can elicit oncogenicity, thus limiting their use in clinical trials. chen et al. used nonviral agents such as a polylysine-modified polyethyleneimine (pei-pll) copolymer to generate genetically engineered mscs with suicide genes, namely, hsv-tk and trail [ ] . the mscs armed with suicide genes along with prodrug ganciclovir can induce significant antitumor effect to glioblastoma by intratumoral injection both in vitro and in vivo. another study on delivering the trail gene for stem cell-mediated gene therapy was conducted by using nonviral vectors (a less efficient but safer method). na et al. prepared the polyplexes of ptrail and bpei, and photochemical internalization (pci) was applied to improve the polyplex entrapping in hmscs and enhance the transfection efficiency of ptrail; the tumor-homing hmscs could also increase the trail secretion in the tumors [ ] . pci-mediated polyplex loading significantly enhanced trail expression in stem cells, and that homing ability enhanced cancer targeting. exposure of polyplex-loaded hmscs (ptrail/bpei@hmscs) to laser irradiation resulted in a beneficial therapeutic antitumor effect in a xenograft mouse model. in addition to mscs, hu et al. reported a dc cell-based therapy for colon cancer cells [ ] . tyrosine kinase receptor ligand (fl) and trail plasmids were constructed for combination therapy. fl, a hemopoietic growth factor, is important in progenitor cell proliferation and differentiation, which can enhance the proliferative and antitumor effect of dcs. these two plasmids were transfected into dcs by lipo . the combination of fl-carrying b trail expression and secretion were quantified by confocal microscopy and elisa, respectively. c sp -lcpp nps without pdna significantly increased dr expression in a dose-dependent manner in hcc cells. d the camkii inhibitor k a prevented the effects of lcpp nps on dr upregulation. e, f treatment with trail pdna loaded into sp -lcpp nps showed higher cytotoxicity in hcc cells than to control cells. however, these nps exhibited slight cytotoxicity in murine hepatocyte fl b cells. reprinted with permission from [ ] dcs and trail-carrying dcs showed a good level of apoptosis in colon cancer [ ] . although trail-induced apoptosis is an attractive therapeutic target, resistance to trail readily develops during treatment. this therapeutic resistance derives from two sources: intrinsic resistance in some highly malignant tumors [ ] [ ] [ ] and acquired resistance after repeated exposure to trail [ ] . resistance to trail is conferred by multiple receptors and involves a series of signaling pathways and activation of inhibitory molecules [ ] . trail signaling begins with the binding of trail to the death receptors. first, binding to decoy receptors (e.g., trail-r and r ) will not lead to the activation of downstream trail-signaling. second, mutation or downregulation of the functional drs (e.g., dr or dr ) can also result in resistance. for instance, transfection of mutated dr into sw colon cancer cells caused a lower efficacy of cell killing than transfection of the wild-type counterpart [ ] . low expression of dr contributed to trail resistance in anti-dr antibody therapy [ ] . in this case, upregulation of dr is a therapeutic strategy for sensitization to trail treatment [ ] . third, the multifunctionality of downstream trail signaling also induces resistance. the assembly of the disc can be inhibited by apoptosis inhibitors. for example, cflip, with a similar structure to caspase- , can inhibit caspase- activation after binding to fadd [ ] . the ratio of cflip/caspase- is correlated with trail resistance in various tumors, such as hepatocellular carcinoma and burkitt lymphoma [ , ] . in type ii cells, trail-initiating apoptosis is mainly mediated by the mitochondrial pathway (fig. ) . upregulation of the proapoptotic proteins bax and bak promotes cell death, while the antiapoptotic proteins bcl- and bcl-x l determine cell survival [ , ] . furthermore, inhibitors of apoptosis proteins (iaps) can block apoptosis by inhibiting the activity of the effector caspases (e.g., caspase- and caspase- ), and overexpression of iaps can confer trail resistance [ , ] . however, this effect can be reversed by iap antagonists such as smac/diablo, which promotes apoptosis by interacting with iaps [ , ] . the mechanisms are summarized in fig. . in addition, epigenetic changes play a role in trail resistance via the regulation of caspase- gene expression, and dna methylation can restore apoptosis in trail-resistant tumor cells. trail resistance is also related to nf-κb and mitogen-activated protein (map) kinases. however, these signaling pathways showed both proapoptotic and antiapoptotic effects [ ] . in conclusion, dr dysfunction and overexpression of cflip, bcl- , bcl-x l , and iaps contribute to trail resistance, and therefore, regulation of drs and inhibition of antiapoptotic proteins resensitizes cells to trail treatment [ ] . the resistance of cancer cells to trail has encouraged the investigation of combination therapy. in recent years, many multifunctional drug delivery systems, including liposomes, micelles, and polymeric nanoparticles, have been developed for the codelivery of genes and drugs. here, we summarize the delivery and therapeutic strategies of combinations of ptrail and different types of drugs. combination with trail sensitizers the combination of trail with its sensitizers is a promising strategy to overcome trail resistance. it was found that polyether ionophore antibiotics (e.g., monensin) can overcome trail resistance via endoplasmic reticulum stress induction, dr fig. the nonviral vector pei -cyd was used to transduce trail plasmid into mscs. trail-armed mscs showed a lung tumor-homing ability and an antitumor effect on lung metastasis-bearing c bl/ mice after i.v. injection. reprinted with permission from [ ] fig. inhibition of trail signaling results in trail resistance. when the disc assembles, flip (flice-like inhibitory protein) can competitively bind to fadd and limit the recruitment of caspase- . while bcl- and bcl-x l can inhibit bax and bak, tbid plays roles in inhibiting bcl- and bcl-x l and activating bax and bak in type ii cells. iaps can strongly inhibit the activation of effector caspases, as smac/ diablo is needed to interact with iaps to release effector caspases. figure adapted from fig. in ref. [ ] upregulation and cflip downregulation [ ] . huang et al. constructed a biocompatible nanosystem for the codelivery of ptrail and monensin, in which low-molecular-weight pei . k (lmw-pei) was intermolecularly crosslinked via disulfide bonds using sulfhydryl β-cyclodextrin as a linker (fig. ) [ ] . the resulting β-cd-sspei carrier, which can bind efficiently with ptrail, can serve as a carrier for codelivery, while monensin is encapsulated inside the cavities of the β-cyclodextrin molecules. the mo β-cd-sspei ptrail nanocomplex can further be modified by a polyanionic polymer γ-pga, thus protecting the nanocomplex from interaction with serum proteins and consequently extending its half-life in the bloodstream. furthermore, γ-pga/ mo β-cd-sspei ptrail can achieve tumor targeting via specific binding between γ-pga and tumor-overexpressed ggt, which can mediate the endocytosis of the nanocomplex. inside cancer cells, the acidic endosomal environment facilitates the detachment of γ-pga, whose conformation is ph-dependent, and the exposed pei facilitates endosomal escape. importantly, the intermolecular crosslinks of pei can be degraded, and ptrail and monensin can be released. monensin can increase intracellular ros levels and induce apoptosis. moreover, dr expression is upregulated, thus synergizing with trail-based treatment for colon cancer gene therapy. combination with chemotherapeutic drugs trail exhibits improved efficacy in combination with chemotherapy because chemotherapeutic agents can sensitize tumors to trail-induced apoptosis via crosstalk between the intrinsic and extrinsic pathways of cell death [ ] . the chemotherapeutic drugs doxorubicin (dox) and paclitaxel (ptx) were explored to determine their synergistic antitumor effect with ptrail. ebrahimian et al. reported a vector composed of polypropylenimine (ppi) modified with -bromodecanoic acid for the codelivery of ptrail and dox for tumor therapy [ ] . in addition, a host-guest conjugated nanoparticle for the codelivery of dox and ptrail was designed [ ] . the adamantane-conjugated dox (ad-dox, guest component) and the pei-cyclodextrin conjugates (pei-cd, host) were selfassembled into the supramolecular pei-cd/ad-dox, which further bound with trail dna to form the pei-cd/ad-dox/pdna snps. the snps exhibited the enhanced therapeutic efficacy with the significantly increased survival rate of the tumor-bearing mice. jiang et al. investigated the codelivery of the htrail-encoding plasmid open reading frame (porf-htrail) and dox using a tumor-targeting carrier, a peptide haiyprh (t )-conjugated polyethylene glycol-modified polyamidoamine dendrimer (pamam-peg-t ) [ ] . in this system, approximately dox molecules were bound to one porf-htrail molecule, and t served as a ligand targeting tumor cell-overexpressed transferrin receptors. this codelivery system induced apoptosis of tumor cells and efficiently inhibited bel- tumor growth in vivo. furthermore, the combination therapy strategy was further applied to glioma, in which dendrigraft poly-l-lysine (dgl), modified by the t peptide and conjugated with dox via a ph-sensitive hydrazone bond, was used to deliver porf-htrail to glioma tissue [ ] . in addition, other dual targeting systems have also been developed for the codelivery of dox and porf-htrail for glioma treatment [ , ] . regarding the combination of ptx and trail, an angiopep- peptide-modified cationic liposome (ang-clp) for the efficient codelivery of pegfp-htrail and ptx to glioma was reported. angiopep- can target the low-density lipoprotein receptorrelated protein (lrp) overexpressed on the bbb and on glioma cells [ ] . lu et al. developed two kinds of nanoparticles for the delivery of porf-htrail and ptx to glioma tissues [ , ] . porf-htrail was delivered by c(rgdyk)-poly(ethylene glycol)-polyethyleneimine (rgd-peg-pei). rgd can bind to integrin α v β , which is overexpressed in the neovasculature and on u glioblastoma cells. ptx was loaded in a cdx-poly(ethylene glycol)-block-poly(lactic acid) micelle. cdx is a peptide derived from the loop ii region of the snake neurotoxin candoxin, with a high-binding affinity to nicotinic acetylcholine receptors (nachrs). dominici et al. investigated the combination of ptrail and ptx for msc therapy [ ] . ptx restored the sensitivity of pancreatic cancer to msc-delivered trail by reverting its prosurvival gene expression profile. additionally, a combination of cisplatin and trail with high anticancer activity was found [ ] . fig. the process of codelivery of ptrail and monensin nanocomplexes. as a trail sensitizer, monensin upregulates dr and sensitizes tumor cells to trail. reprinted with permission from [ ] trail-based gene delivery and therapeutic strategies hh zhong these results indicate that trail gene therapy in combination with chemotherapy can be promising for ovarian cancer therapy. although it is not fully known how chemotherapy sensitizes cells to trail-induced apoptosis, the effect may be related to a p independent pathway, in addition to changes in the expression of proteins involved in trail signaling, which also play an important role [ ] . combination with intracellular apoptosis-induced agents as trail induces extracellular apoptosis, its combination with agents mediating the intracellular apoptosis pathway can yield a synergistic effect. histone deacetylase inhibitors (hadci) induce cell cycle arrest and apoptosis in tumor cells, and hadci can resensitize trail-resistant cancer cells. codelivery of ptrail and vorinostat (saha) with a reactive oxygen species (ros)-triggered charge reversal polymer (b-pdeaea) produced a good antitumor effect [ ] . the increased transfection efficiency of ptrail and saha-induced ros accumulation caused significant apoptosis of the cancer cells. the mscs-based gene therapy for antiglioma was developed, in which hat-mscs was transfected by pires -egfp-strail [ ] . the engineered mscs effectively inhibited the proliferation of malignant glioma cells and upregulated drs, yielding a potent antiglioma effect in combination with panobinostat. iaps play an important role in cancer cell resistance to trail, and the combination of trail with iap antagonists can help sensitize cells to trail-induced apoptosis. ge et al. demonstrated that an oncolytic adenovirus coexpressing trail and smac, in combination with the cyclin-dependent kinase (cdk) inhibitor sns- , synergistically reinforced their individual anti-pancreatic cancer activities, and sns- enhanced zd -trail-ietd-smac-induced apoptosis [ ] . shi et al. developed an aav-mediated gene therapy that was characterized by coexpression of trail with mir- -zip, which produced a synergistic effect on the enhanced apoptosis induction via the sensitizing effect of mir- -zip by upregulation of pten and downregulation of survivin [ ] . compared with trail monotherapy, combination therapy provides enhanced treatment outcomes. therefore, combinations including trail-mediated therapy are a promising approach for cancer therapy. trail is a promising drug candidate for the treatment of many cancers. the investigation of trail-based therapy in clinical trials focuses on recombinant trail proteins and anti-trail antibodies, but delivery and drug resistance issues are the main hurdles in the successful translation of these results. trail-based gene therapy is another potential treatment approach. importantly, the codelivery systems loaded with ptrail and drugs combined with trail have been actively explored. the development of safe and efficient gene vectors is the central issue for gene therapy. the elegant design of advanced systems provides the tumor-targeted codelivery of both agents, thus achieving synergistic treatment effects. in addition, it is necessary to identify proper biomarkers for maximizing trail-based therapy, which can also provide helpful information for the appropriate selection of drug combinations. however, clinical trials have made little progress. efforts should be directed toward the rational design of appropriate formulations of trail to improve its in vivo pharmacokinetic profile and the exploration of biomarkers specific to trail-based therapy. the future application of trail may benefit from a better understanding of the anticancer mechanisms of trail as well as its combination with trail sensitizers. it is expected that as precision medicine progresses, trail-mediated antitumor functions will be better understood to allow the development of precise and effective trail-based treatments for patients. overcoming cellular barriers for rna therapeutics the cd (apo- /fas) and the trail (apo- l) apoptosis systems cachetin/tnf-alpha in septic shock and septic adult respiratory distress syndrome lethal effect of the anti-fas antibody in mice identification and characterization of a new member of the tnf family that induces apoptosis induction of apoptosis by apo- ligand, a new member of the tumor 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historical perspective dual targeting effect of angiopep- -modified, dna-loaded nanoparticles for glioma therapeutic efficacy of intravenously administered transferrin-conjugated dendriplexes on prostate carcinomas regression of prostate tumors after intravenous administration of lactoferrinbearing polypropylenimine dendriplexes encoding tnf-alpha, trail, and interleukin- a self-assembled coumarin-anchored dendrimer for efficient gene delivery and light-responsive drug delivery triazine-modified dendrimer for efficient trail gene therapy in osteosarcoma modified pamam vehicles for effective trail gene delivery to colon adenocarcinoma: in vitro and in vivo evaluation peptide-guided gene delivery development of a genetically engineered biomimetic vector for targeted gene transfer to breast cancer cells intratracheal administration of a nanoparticle-based therapy with the angiotensin ii type receptor gene attenuates lung cancer growth pten and trail genes loaded zein nanoparticles as 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risks and rewards understanding tumor-stroma interplays for targeted therapies by armed mesenchymal stromal progenitors: the mesenkillers adiposederived mesenchymal stem cells as stable source of tumor necrosis factorrelated apoptosis-inducing ligand delivery for cancer therapy trail delivered by mesenchymal stromal/stem cells counteracts tumor development in orthotopic ewing sarcoma models stem cell-based gene therapy activated using magnetic hyperthermia to enhance the treatment of cancer mesenchymal stem cell delivery of trail can eliminate metastatic cancer mesenchymal stem cells as a novel carrier for targeted delivery of gene in cancer therapy based on nonviral transfection suppression of orthotopically implanted hepatocarcinoma in mice by umbilical cord-derived mesenchymal stem cells with strail gene expression driven by afp promoter an inducible caspase suicide gene to improve the safety of mesenchymal stromal cell therapies inducible caspase -mediated suicide gene for msc-based cancer gene therapy an inducible caspase safety switch for t-cell therapy polylysine-modified polyethylenimine polymer can generate genetically engineered mesenchymal stem cells for combinational suicidal gene therapy in glioblastoma trail-secreting human mesenchymal stem cells engineered by a non-viral vector and photochemical internalization for pancreatic cancer gene therapy the roles of flt in hematopoiesis and leukemia nanoliposomemediated fl/trail double-gene therapy for colon cancer: in vitro and in vivo evaluation bcl-xl protects pancreatic adenocarcinoma cells against cd -and trail-receptormediated apoptosis sensitization for death receptor-or drug-induced apoptosis by re-expression of caspase- through demethylation or gene transfer resistance to tumor necrosis factor-related apoptosis-inducing ligand (trail)-induced apoptosis in neuroblastoma cells correlates with a loss of caspase- expression mechanisms involved in development of resistance to adenovirus-mediated proapoptotic gene therapy in dld human colon cancer cell line molecular determinants of response to trail in killing of normal and cancer cells tumoricidal activity of a novel anti-human dr monoclonal antibody without hepatocyte cytotoxicity flice-inhibitory proteins: regulators of death receptor-mediated apoptosis cellular flice/caspase- -inhibitory protein as a principal regulator of cell death and survival in human hepatocellular carcinoma modulation of caspase- and flice-inhibitory protein expression as a potential mechanism of epstein-barr virus tumorigenesis in burkitt's lymphoma induction of apoptotic program in cell-free extracts: requirement for datp and cytochrome c the release of cytochrome c from mitochondria: a primary site for bcl- regulation of apoptosis synergy is achieved by complementation with apo l/trail and actinomycin d in apo l/trail-mediated apoptosis of prostate cancer cells: role of xiap in resistance x-linked inhibitor of apoptosis (xiap) blocks apo ligand/ 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co-delivery of trail gene enhances the anti-glioblastoma effect of paclitaxel in vitro and in vivo msc-delivered soluble trail and paclitaxel as novel combinatory treatment for pancreatic adenocarcinoma in vitro and in vivo growth inhibition of drug-resistant ovarian carcinoma cells using a combination of cisplatin and a trail-encoding retrovirus is trail the holy grail of cancer therapy? saha (vorinostat) facilitates functional polymer-based gene transfection via upregulation of ros and synergizes with trail gene delivery for cancer therapy histone deacetylase inhibitor panobinostat potentiates the anti-cancer effects of mesenchymal stem cell-based strail gene therapy against malignant glioma synergistic antitumor effects of cdk inhibitor sns and an oncolytic adenovirus coexpressing trail and smac in pancreatic cancer combination of aav-trail with mir- -zip therapeutic strategy overcomes the resistance to trail induced apoptosis in liver cancer competing interests: the authors declare no competing interests.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/ . /. key: cord- -fdn c hx authors: leanza, matthias title: the darkened horizon: two modes of organizing pandemics date: - - journal: how organizations manage the future doi: . / - - - - _ sha: doc_id: cord_uid: fdn c hx this chapter deals with the recent darkening of the future horizon in the global fight against pandemics. since roughly the year , the world health organization has collaborated with a large number of local actors and made a concentrated effort to protect the world’s population against emerging infectious diseases, such as severe acute respiratory syndrome (sars), swine flu, ebola and zika. although efforts have been made so that the spread of future infectious diseases will be contained through early intervention, the actors in charge anticipate that the extant measures will fail to some degree. they believe it is simply impossible to prevent all pandemics from happening. but steps can and should be taken to lessen the impact of an unavoidable pandemic through emergency preparation. this chapter deals with organizations and organizational networks as key actors in these processes of emergency planning. without the capacity of organizations to produce binding decisions for their members, which makes planning for an uncertain future possible, pandemic preparedness would not be feasible—especially not on a global scale. the horizon has darkened. the future no longer seems like an open space full of opportunities and risks. rather, what is in store appears to be deeply threatening. whether one thinks of global warming, terrorism or the continuing instability of the banking and finance sector, our expectations for the future in many areas of public life exemplify what craig calhoun ( , p. ) calls an 'emergency imaginary': 'a discourse of emergencies is now', as he wrote more than years ago in a diagnosis that is even more applicable today, 'central to international affairs. it shapes not only humanitarian assistance, but also military intervention and the pursuit of public health.' due to this emergency imaginary, we feel that our social institutions, our health and well-being, and even, as in the case of global warming, the future of mankind as such are deeply endangered. m. leanza (*) sociology department, university of basel, basel, switzerland this chapter deals with the recent darkening of the future horizon in the global fight against pandemics. around the year , the world health organization (who) started collaborating with a large number of local actors and made a concentrated effort to protect the world's population against emerging infectious diseases such as severe acute respiratory syndrome (sars), swine flu, ebola and zika. although efforts have been made so that the spread of future infectious diseases will be contained through early intervention, the actors in charge expect the extant measures to fail to some degree. they believe it is simply impossible to prevent all pandemics from happening. but steps can and should be taken through emergency preparation to lessen an unavoidable pandemic's impact. as andrew lakoff ( , pp. - ) summarizes: preparedness assumes the disruptive, potentially catastrophic nature of certain events. since the probability and severity of such events cannot be calculated, the only way to avert catastrophes is to have plans to address them already in place and to have exercised for their eventuality-in other words, to maintain an ongoing capability to respond appropriately. in recent years, scholars of security studies, cultural studies and other research areas have paid much attention to these developments in emergency preparedness, which, it is worth noting, are not limited to the domain of public health. this issue has primarily been addressed at two levels: first, by changing global security policies after the / attacks, and, second, by scrutinizing the narratives and rhetorical strategies through which the emergency imaginary is constructed and gains plausibility (e.g. massumi ; aradau and van munster ; horn ) . in this chapter, i will focus on organizations as key actors in these processes of emergency planning. without the capacity of organizations to produce binding decisions for their members, which allows them to plan for an uncertain future, pandemic preparedness would not be feasibleespecially not on a global scale. i will unfold my argument in four steps. with regard to the who, which was established in , i will discuss the question of how supranational coordination and planning for the future is rendered possible by building formal organizations and organizational networks at a global level. i will then highlight some aspects of the attempts undertaken by the who and its partners after the year to fight pandemics on a global scale. my analysis of relevant policy papers, legal norms and manuals shows that two different though complementary strategies are applied: early intervention and emergency planning. these are, as i will discuss more explicitly in the final section, two different kinds of organizing (for) the future or, to put it differently, two modes of how organizations manage pandemics. the overall aim of the empirical analysis offered in this chapter is to reconstruct organizational programmes and rationales rather than to give an account of the actual operations of these systems. the focus lies on public discourses and normative texts and not so much on the 'inside' of these organizations, meaning their day-to-day routines and practices. contagious diseases do not stop at state borders. pathogens circulate without regard for political and administrative spheres of influence. what gilles deleuze and félix guattari ( , introduction) establish for rhizomes in general also applies to infection chains in particular: by growing rampantly, they produce a 'deterritorializing effect'. pathogens connect distant regions and different kinds of people; zoonoses even trespass the boundary between animals and humans. by doing so, communicable diseases create spaces and communalities that did not exist before. this is also the reason why every epidemic requires new maps (e.g. koch ) . even though pathogens do not stop at state borders, sovereignty ends there, and the difficult terrain of diplomacy begins. the international sanitary conferences, which took place between and , made a first step towards creating a global field of public health (howard-jones ; bynum ) . while the first couple of these conferences-there were in all-dealt primarily, though not exclusively, with cholera, further diseases and topics were discussed and negotiated beginning in the s. laborious agreements regarding quarantine, inspection and surveillance measures were worked out and in some cases ratified. but the field of global health diplomacy did not receive a coordination and control unit until with the establishment of the who as a specialized agency of the united nations (zimmer ) . in passing the international health regulations (ihr) of , which superseded the international sanitation regulations of , the who established standards and norms with a legally binding character for its signatory states. the primary goal of these regulations was to provide 'maximum security against the international spread of disease with the minimum interference with world traffic' (who , p. ) . to this end, epidemiological surveillance and alarm systems were installed in signatory states, or already existing structures were expanded. in addition, the who made more specific efforts to combat infectious diseases. one of the first large projects was the global malaria eradication program ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . in order to defeat the dangerous tropical disease, the insecticide ddt (dichloro-diphenyl-trichloroethane) was used liberally and repeatedly in over countries. even though certain regions profited from this measure, the actors in charge had to accept in the end that this goal was, on the whole, too ambitious (zimmer , pp. - ) . more successful, however, was the vaccination programme against smallpox, which was enacted in and intensified in (fenner et al. (fenner et al. , pp. - , . after roughly years, it finally reached its goal. in , the who announced: 'smallpox is dead!' (ibid., p. ). sovereign nation states use the mechanism of formal organization to cooperate in this and further policy areas of international concern. while 'leagues of subjects' within a state 'savour of unlawful design', as thomas hobbes ( , p. ) famously wrote in leviathan, 'leagues between commonwealths, over whom there is no human power established to keep them all in awe, are not only lawful, but also profitable for the time they last'. as well as mutual agreements and legally binding contracts, inter-or supranational organizations are a specific form taken by such leagues today. drawing on niklas luhmann ( ) , organizations can be perceived as a type of social system that is defined by formal membership roles and processes of decision-making. as inter-or supranational organizations demonstrate, not only natural but also legal persons, such as states, can become members of organizations. by entering an organization, sovereign nation states are, in principle, capable of producing collectively binding decisions on a global level, without losing their autonomy to a sovereign world state. today, a wide range of organizations constitute the global field of public health and disease control (youde , part ) . besides the who, they include the world bank, unaids (which was established in ) and governmental and nongovernmental organizations. these organizations are the main action centres within the field of global public health. they deliver expertise, develop policies, launch programmes and mobilize the global community. to achieve their goals, they regularly ally with other organizations and build networks that can be activated when necessary. this especially holds true for the global fight against pandemics. in certain respects, in order to deal with an unfolding threat, organizational networks have to spread as rampantly as the pathogens themselves. otherwise they will be unable to prevent further harm. in , and thus very much in the shadow of the global aids crisis, the who laid the foundation for a new regime in the global fight against pandemics by setting up the global outbreak alert and response network (goarn). since then it has acted in more than cases (mackenzie et al. ). through 'rapid identification, verification and communication of threats' (who , p. ), goarn seeks to contain the spread of infectious diseases, especially highly infectious ones. 'no single institution or country', so the main argument for this international cooperation goes, 'has all of the capacities to respond to international public health emergencies caused by epidemics and by new and emerging infectious diseases' (who n.d.) . in - , the sars pandemic, which resulted in nearly registered deaths, triggered a global health alarm due to goarn, though the communication of this risk kindled by the predicted future potential of the pandemic outstripped, in certain respects, its actual impact (smith ; ong ). the thoroughly redesigned ihr from , which came into force in , further developed and shaped this process. in contrast to the regulations it replaced-the international sanitation and health regulations of and , which, in comparison, were quite static since they only applied to a specific catalogue of communicable diseasethe ihr now includes an early warning system that seeks to detect every potential 'public health emergency of international concern' (pheic) (fidler ) . the focus is on so-called points of entry, especially sea-and airports (who a, pp. - , - ) . the member states of the who are responsible for implementing this global safety net at the local level; they must establish surveillance, contact and coordination units. in germany, for instance, the federal office of civil protection and disaster assistance coordinates and oversees this implementation process in cooperation with the robert koch institute. the robert koch institute, in turn, works with the european centre for disease prevention and control, which is an important partner of goarn. because many different kinds of organizations across a wide range of countries are connected in this network, it is necessary to standardize decision-making. without the 'structural coupling' (maturana and varela , pp. - ) of a shared decision process, cooperation and coordination between the participating organizations would not be feasible. decisions would simply not be able to circulate within the network. instead, they would have to be re-evaluated at every nodal point. for this reason, the ihr (who a, pp. - ; see also who ) stipulates a risk-assessment matrix for signatory countries: after a local surveillance unit has detected an event 'that may constitute a public health emergency of international concern' (who a, p. ) , three yes/no questions regarding its actual and potential impact must be answered. then, it is determined whether the event should be rated as unusual or unexpected. if the answers are all positive, the who must be notified within hours. if they are not all positive, there are two further levels of such yes/no questions, which address the risk of international spread and, in a final step, the possibility that countries or other entities would impose international travel or trade restrictions in response to the outbreak. the answers to these questions then determine whether notifying the who is required or not. this decision-making tool can be understood as an 'attention filter'. since there are now many globally connected surveillance units, mechanisms have to be installed that not only allow and trigger but also suppress communication between them. otherwise, the network would be flooded with more information than it can process. in other words, the elements within this structure would be too closely connected. nonetheless, the goal is to set the attention thresholds as low as possible. even if notifying the who is not required at one point, the event in question has to be kept under surveillance. this, of course, does not prevent the situation from being evaluated incorrectly. the - ebola outbreak, for instance, was declared a pheic relatively late because the actors in charge initially viewed it as only a local problem of a poor region in west africa (lakoff et al. ) . together with the attention thresholds, the reaction times of the relevant public health organizations are also to be lowered. while the decentralized structure of networks improves the alarm function, since attention is widely distributed, a missing or weakly developed action centre has an adverse effect on the intervention function. in defiance of all network rhetoric, the global fight against pandemics cannot proceed without the structural principles of hierarchy and the distribution of tasks. according to the ihr, after being informed of a positive risk assessment by local organizations, the who has to provide them with further information and instructions and send experts to the affected regions (who a, pp. - , - , - ) . the who is the 'obligatory passage point' (callon ) for this process. a combination of the network, hierarchy and the distribution of tasks aims to make rapid intervention possible. even though the who wants, in principle, global traffic to flow without any hindrance, in some cases a temporary interruption of the circulation of goods and people may be considered necessary to protect global public health (stephenson ; opitz ) . the ihr and national regulations therefore stipulate travel restrictions on certain people and allow measures like quarantine and isolation to be imposed. in an age of global flows and a greater awareness of fundamental rights, this specific kind of intervention has to some extent become problematic. as the first principle of the ihr states 'the implementation of these regulations shall be with full respect for the dignity, human rights and fundamental freedoms of persons' (who a, p. ) . similarly, the who ( , p. ) explained in : 'in emergency situations, the enjoyment of individual human rights and civil liberties may have to be limited in the public interest. however, efforts to protect individual rights should be part of any policy. measures that limit individual rights and civil liberties must be necessary, reasonable, proportional, equitable, non-discriminatory and in full compliance with national and international laws.' besides these reservations, the global fight against pandemics cannot proceed without restrictive measures, as the sars pandemic and ebola outbreak have shown. although a concentrated global effort has been made to prevent pandemics via early detection and rapid response, the actors in charge expect them to happen. it is only a matter of time, they believe, until the next health emergency occurs. 'influenza experts agree', the who ( b, pp. vi-vii) warned in , 'that another pandemic is likely to happen but are unable to say when. the specific characteristics of a future pandemic virus cannot be predicted. nobody knows how pathogenic a new virus would be, and which age groups it would affect.' although its exact time of emergence, etiological nature and epidemiological distribution pattern may be unpredictable, it is considered a fact that the next pandemic will occur in the near or not so distant future (see also macphil ). a glossy brochure on pandemic planning by the us department of homeland security ( , p. ) presented a similar way of looking at things. in a quotation in the brochure, the us secretary of health and human services, mike leavitt, states: 'some will say this discussion of the avian flu is an overreaction. some may say, "did we cry the wolf?" the reality is that if the h n virus does not trigger pandemic flu, there will be another virus that will. ' this statement demonstrates that the general trend of thinking about emergencies and accidents as 'normal' has permeated the field of global public health (calhoun ; lakoff ) . in the s, in many areas of public life, the future was already perceived as unsafe and potentially catastrophic, and this view was intensified after the year (aradau and van munster ; horn ) . although the future horizon has darkened with the looming prospect of ecological, political and economic crises, not all hope is lost. the occurrence of (massive) harm might be inevitable, but what can yet be prevented is the worst-case scenario. it is assumed that through emergency planning, the severity of the potential damage can be lessened. this is what 'preparedness' means: acting, deciding and governing under conditions of insecurity (lentzos and rose ; anderson ) . as the who explained in : 'although it is not considered feasible to halt the spread of a pandemic virus, it should be possible to minimize its consequences through advance preparation to meet the challenge' (who c, p. ) . in his address to the nd world health assembly in , the un secretary-general, ban ki-moon, posed the same question: 'how do we build resilience in an age of unpredictability and interconnection?' through emergency planning is his answer. 'this is how we will make the global community more resilient. this is how we ensure that wherever the next threat to health, peace or economic stability may emerge, we will be ready.' of special interest in this regard are critical infrastructures, such as water supply, that might be affected by a severe disease outbreak. local public health emergency centres, which the who ( ) assembled as a global network (eoc-net) in , are responsible for the planning process. as well as taking stock of the available resources and contingents in a country or region, scenario planning and agent-based computer simulations are of fundamental importance ; they enable us to imagine possible scenarios via enactment and visualization without the necessity of making any probability assumptions. it is believed that in order to be prepared for future emergencies, the organizational imagination must to some extent be liberated from restrictions imposed by past experiences. organizations are of crucial importance for the planning process. for instance, the who guidelines, whole-of-society pandemic readiness, aim 'to support integrated planning and preparations for pandemic influenza across all sectors of society, including public and private sector organizations and essential services' (who , p. ) . to strengthen organizational resiliency against the stresses and strains that may result from a pandemic, thorough preparation is required. 'in the absence of early and effective planning, countries may face wider social and economic disruption, significant threats to the continuity of essential services, lower production levels, distribution difficulties, and shortages of supplies' (p. ). emergency planning is furthermore imperative since '[t]he failure of businesses to sustain operations would add to the economic consequences of a pandemic. some business sectors will be especially vulnerable (e.g. those dependent on tourism and travel), and certain groups in society are likely to suffer more than others' (p. ). the 'readiness framework' therefore asks all organizations that provide basic services such as food, water, health, defence, law and order, finance, transportation, telecommunications and energy to prepare for pandemics via simulation exercises and drills based on different scenarios. furthermore, business continuity plans have to be developed. for this purpose, a pandemic coordinator should be assigned to oversee the planning process. all organizations that are crucial for public life are strongly advised to prepare themselves for the next pandemic. given the interdependencies between these organizations, general preparedness is the only way to prevent a complete breakdown. or, as the guidelines put it: 'it is prudent to plan for the worst, while hoping for the best' (p. ). according to lakoff ( ) , today's highly differentiated field of global health is characterized by, among other things, the juxtaposition of two regimes: global health security and humanitarian biomedicine. 'each of these regimes', he elaborates, 'combines normative and technical elements to provide a rationale for managing infectious disease on a global scale. they each envision a form of social life that requires the fulfillment of an innovative technological project. however, the two regimes rest on very different visions of both the social order that is at stake in global health and the most appropriate technical means of achieving it' (p. ). while global health security turns its attention to emerging infectious diseases, 'which are seen to threaten wealthy countries, and which typically (though not always) emanate from asia, sub-saharan africa, latin america', humanitarian biomedicine deals with 'diseases that currently afflict the poorer nations of the world, such as malaria, tuberculosis, and hiv/aids' (p. ). in addition to lakoff's ( lakoff's ( , see also distinction between the two regimes of global health, my analysis highlights two layers that are encompassed by one of these regimes, the global fight against emerging infectious diseases. the two modes of organizing of such pandemics are not organizations themselves. they are programmes that structure the organizational decision-making and the corresponding membership roles. in analysing these programmes, the focus lies not so much on the actual operation of the system-since it is always a creative translation of cognitive and normative schemes into concrete practice-but rather on the intended actions of the system. a first line of defence is defined through early intervention. for this purpose, a wide and ramified organizational network is put in place. it allows pandemics to be detected while they are still emerging, and this makes it possible to limit the potential scope of their spread. because the goal is to prevent a further unfolding of potential threats into actual damages, time is of the essence in detection. the organizations must react quickly while ensuring, at same time, that the information they generate, process and communicate to others is reliable. the strategies they decide to follow also have to be effective. otherwise the primary goal is not achieved: preventing pandemics from happening. in reality, this highly ambitious goal cannot always be met. but the organizations in charge know their limitations and are therefore requested to install a second line of defence: emergency planning. all organizations that are critical for society are asked to have emergency plans in place so that, in the case of a pandemic, they would still be able to react. the goal here shifts from preventing the spread of disease towards securing the 'autopoiesis' (maturana and varela , pp. - ) of the system, meaning its ability to reproduce itself even under enormous environmental pressures. while early intervention requires organizations to be capable of acting quickly, pandemic preparedness aims to produce robust systems that are immune to breakdown. despite operating from different angles, these two modes of organizing pandemics are complementary. early intervention relates to preventable damages. the underlying assumption is that pandemics can be avoided through early detection and rapid response. the future scenario of early intervention is therefore an altogether positive one, in which organizations are capable of doing their job in the face of danger, namely containing infectious diseases. pandemic preparedness, in contrast, works not with one but with two kinds of damages: primary damages, which cannot be prevented, and preventable consequential damages, which pose an existential threat. the aim is still to prevent harm, but preparedness does not focus on the pandemics per se but on the fatal repercussions that they might have for societies. this is a minimal form of prevention, and it is no longer believed that it is possible to escape such a pandemic unscathed. both modes of relating to the future do not exclude but rather complement each other. if early intervention does not work in a specific scenario, there is still a second prevention strategy, which, of course, can only partially contain the effects of the pandemic since (massive) harm will have already occurred. but by strengthening the resilience of organizations and societies, pandemic preparedness aims to preserve existential functions and operations. in his by now classic essay from , charles perrow describes organizations, especially large ones, as a key element of modern societies. according to perrow, fundamental social functions are maintained by private and public organizations. this also holds true for responding to pandemics. in a 'society of organizations' (perrow ) , it is organizations and their professionals who manage pandemics. but two kinds of organizations have to be distinguished which correspond to the two modes or programmes for managing emerging infectious diseases: first, organizations and professionals in the public health sector try to prevent pandemics through early intervention (and further preventative measures, such as vaccination programmes). it is their job to protect the general public from health risks; this is the purpose of these specialized organizations and the goal of their corresponding professional activities. second, and in contrast, all organizations that provide basic services for society are asked to make emergency plans and prepare themselves for the next pandemic. this includes public health organizations but is also addressed to, first and foremost, private and public organizations that provide food, water, defence, law and order, finance, transportation, telecommunications and energy. the second programme is no less ambitious than the first. organizations and professionals in the public health sector may not always succeed in preventing pandemics: as we have seen, they are well aware of this fact, and that is why emergency plans are developed in the first place. but this implies that, in principle, all organizations that provide basic services for society have to professionalize themselves in this specific area. one could describe this as a 'colonization' of non-health organizations through public health imperatives. this is, of course, not a completely new development if one considers, for instance, company doctors or health and safety officers. furthermore, many large organizations have undergone a professionalization in areas that do not traditionally belong to their core activities, such as when they maintain legal, public relations or research departments, or when they offer childcare or psychological counselling to their employees. to some extent, this is a likely consequence of the 'functional differentiation of modern societies' (luhmann ) : even if organizations are typically specialized in providing only one or two services, they have to take further social functions into account. what is new here is the kind of task, that is, preparing for pandemics in order to prevent the worst-case scenario-a complete breakdown of the system that would result from the absence of employees due to illness. in a society of organizations, the autopoiesis of society as whole cannot be separated from the autopoiesis of its organizations. preserving society in a public health emergency depends on keeping organizations functional. for measures of disease assessment and control in germany, see the 'gesetz zur neuordnung seuchenrechtlicher vorschriften for a discussion of how biosecurity intertwines the field of public health with the security sector, see also fidler and gostin preemption, precaution, preparedness: anticipatory action and future geographies politics of catastrophe: genealogies of the unknown policing hearts of darkness: aspects of the international sanitary conferences a world of emergencies: fear, intervention, and the limits of cosmopolitan order some elements of a sociology of translation: domestication of the scallops and the fishermen of st brieuc bay pandemic influenza: preparedness, response, and recovery; guide for critical infrastructure and key resources european centre for disease prevention and control smallpox and its eradication from international sanitary conventions to global health security: the new international health regulations biosecurity in the global age: biological weapons, public health, and the rule of law leviathan or the matter, forme & power of a common-wealth ecclesiasticall and civil the scientific background of the international sanitary conferences - resilience and solidarity: our best response to crisis. address to the nd world health assembly mapping medical disasters: ebola makes old lessons preparing for the next emergency two regimes of global health governing insecurity: contingency planning, protection, resilience funktionen und folgen formaler organisation the global outbreak alert and response network a predictable unpredictability: the h n pandemic and the concept of 'strategic uncertainty' within global public health fear (the spectrum said) the tree of knowledge: the biological roots of human understanding assembling around sars: technology, body heat, and political fever in risk society regulating epidemic space: the nomos of global circulation defining epidemics in computer simulation models: how do definitions influence conclusions? a society of organizations responding to global infectious disease outbreaks: lessons from sars on the role of risk perception, communication and management emerging infectious diseases/emerging forms of biological sovereignty who checklist for influenza pandemic preparedness planning. geneva: who. ---. c. who global influenza preparedness plan: the role of who and recommendations for national measures before and during pandemics welt ohne krankheit: geschichte der internationalen gesundheitspolitik - key: cord- - acymenq authors: cameron, lydia c.; bonis, benjamin; phan, chi q.; kent, leslie a.; lee, alysha k.; hunter, ryan c. title: a putative enoyl-coa hydratase contributes to biofilm formation and the antibiotic tolerance of achromobacter xylosoxidans date: - - journal: npj biofilms microbiomes doi: . /s - - - sha: doc_id: cord_uid: acymenq achromobacter xylosoxidans has attracted increasing attention as an emerging pathogen in patients with cystic fibrosis. intrinsic resistance to several classes of antimicrobials and the ability to form robust biofilms in vivo contribute to the clinical manifestations of persistent a. xylosoxidans infection. still, much of a. xylosoxidans biofilm formation remains uncharacterized due to the scarcity of existing genetic tools. here we demonstrate a promising genetic system for use in a. xylosoxidans; generating a transposon mutant library which was then used to identify genes involved in biofilm development in vitro. we further described the effects of one of the genes found in the mutagenesis screen, encoding a putative enoyl-coa hydratase, on biofilm structure and tolerance to antimicrobials. through additional analysis, we find that a fatty acid signaling compound is essential to a. xylosoxidans biofilm ultrastructure and maintenance. this work describes methods for the genetic manipulation of a. xylosoxidans and demonstrated their use to improve our understanding of a. xylosoxidans pathophysiology. cystic fibrosis (cf) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (cftr) protein that resides at the apical surface of many epithelial cell types. cftr defects result in abnormal chloride and bicarbonate transport, increasing mucus viscosity in the pancreas, paranasal sinuses, digestive tract, and most notably, the lower airways. viscous mucus, due to its impaired clearance and abundant nutrient bioavailability, in turn becomes chronically colonized by pathogenic bacteria that are the leading cause of mortality among the cf population. for decades, pseudomonas aeruginosa and staphylococcus aureus have been extensively characterized and recognized as the primary airway pathogens. more recently, however, many other multidrug-resistant opportunistic bacterial species have received attention for their association with cf disease progression. among them, achromobacter xylosoxidans is notable given its association with poor pulmonary function scores and apparent patient-to-patient transmissibility. [ ] [ ] [ ] this aerobic, gram-negative opportunistic pathogen has been found to colonize anywhere from to percent of cf subjects, though its prevalence has risen in recent years, , - sparking a renewed interest in its pathophysiology. of particular concern is the intrinsic and acquired resistance of a. xlyosoxidans to multiple classes of antimicrobial agents, including aminoglycosides, beta-lactams, carbapenems, chloramphenicol and fluoroquinolones, which presents a significant burden for infection control. , , [ ] [ ] [ ] [ ] also complicating treatment of a. xylosoxidans is its ability to form robust biofilms. [ ] [ ] [ ] thought to be the predominant in vivo lifestyle among cf pathogens, biofilms confer a highly protective growth environment that shields pathogens against environmental stress, antimicrobials and the host immune response. in addition, nutrient and oxygen gradients throughout microcolonies (or aggregates) result in slowed metabolism among biofilm cells which can also potentiate drug tolerance. the recalcitrance of biofilm cells to antibiotic exposure likely enhances the persistence of a. xylosoxidans during chronic infection of the cf lung. relative to canonical cf pathogens, comparatively little is known about the molecular mechanisms of biofilm formation and maintenance in a. xylosoxidans. this is due, in part, to the paucity of genetic tools available for manipulation of its genome. therefore, the objectives of this study were two-fold: first, we sought to develop a tractable system to genetically manipulate a. xylosoxidans. using this system, our second objective was to take a transposon mutagenesis approach to study the molecular basis of a. xylosoxidans biofilm formation. in doing so, we identified several gene products essential for biofilm development. we chose to further characterize a gene identified most frequently in our screen (echa) encoding a putative enoyl-coa hydratase that, when disrupted, leads to a decrease in biofilm accumulation and increased susceptibility to multiple classes of antibiotics. to identify genetic determinants of biofilm formation in a. xylosoxidans, we generated a mutant library of strain mn using random transposon mutagenesis employing the minimariner transposable element. from this library, , mutants were screened for altered biofilm formation using an established crystal violet (cv)/microtiter dish assay. after an initial round of screening, putative biofilm-defective mutants were identified that were then re-tested (n = ) to verify mutant phenotypes. after secondary screening and eliminating mutants with general growth defects, transposon mutants were confirmed to be defective in biofilm accumulation (fig. , table ). to identify dna sequences (~ - bp) flanking transposon insertion junctions, mutants were screened by arbitrarily primed pcr. insertions were mapped to loci predicted to encode various classes of gene products, including membrane proteins, glycosyltransferases, flagella, transcriptional regulators, in addition to several proteins with no assigned function (table ). arbitrary pcr of three transposon insertion loci generated sequences with no homology to published gene sequences, and we were unable to obtain transposon-flanking sequences for five additional mutants. of note, a putative enoyl-coa hydratase, an exonuclease, and a propionate hydroxylase were each hit multiple times. we took interest in the gene encoding a putative enoyl-coa hydratase (axylo_ ) given the importance of homologous proteins in fatty acid signal biosynthesis and biofilm development among diverse bacterial species. [ ] [ ] [ ] [ ] [ ] [ ] [ ] we named this gene echa (for enoyl-coa hydratase a) and sought to characterize its effects on a. xylosoxidans biofilm physiology in greater detail. generation and complementation of an echa mutant genetic tractability has been challenging for a. xylosoxidans due its inherent resistance to multiple classes of antibiotics and inducible efflux system as a second line of defense. , , we therefore sought to develop a robust genetic system for the generation and complementation of marker-free deletion mutants that would facilitate further study of the genetic basis of biofilm formation. given that tetracycline resistance was an effective marker to generate the transposon mutant library, the tetracycline resistance cassette from pex tc was pcr amplified and ligated into the mobilizable suicide vector psmv generating pbmb (empty vector) and pbmb (echa deletion construct; supplementary fig. ). attempts to construct a complementation vector using broad host-range cloning plasmids were unsuccessful. we therefore introduced echa into pbmb (pbbr mcs- ::alca) forming pbmb ( supplementary fig. ), placing echa expression under the control of an alcohol-inducible promoter. using these vectors, we generated a markerless deletion mutant and complement of a. xylosoxidans mn lacking echa. a. xylosoxidans biofilm ultrastructure is mediated by echa to confirm that the biofilm-deficient phenotypes of transposon mutants -a , -f , and -e (table ) were a result of mutations in echa, we re-assessed biofilm formation by the markerless Δecha mutant using the cv assay described above. as before, Δecha exhibited a significant decrease in biofilm biomass accumulation relative to the wildtype (p = . ) after h (fig. ) . complementation with pbmb restored the wildtype phenotype (p = . ), confirming a role of echa and its gene product in a. xylosoxidans biofilm development. enoyl-coa hydratases are central to the biosynthesis of a class of fatty acid signaling molecules, or diffusible signal factors (dsfs), that have been described in diverse bacterial species for their role in mediating virulence, motility and biofilm development. [ ] [ ] [ ] [ ] [ ] [ ] , in p. aeruginosa, for example, the dsf cis- -decenoic acid (cis-da) is known to play a critical role in biofilm dispersion. , therefore, to test whether the observed biofilm impairment phenotype in a. xylosoxidans was also mediated by a dsf-like signaling mechanism, we exogenously added synthetic cis-da ( nm) to the Δecha mutant during biofilm development. despite having no effect on the wt strain, addition of cis-da to Δecha resulted in a significant restoration of biofilm biomass relative to the untreated control (p = . ) (fig. ) . these data suggest that biofilm formation in mn is directly mediated by a dsf-like metabolite generated by an enoyl-coa hydratase. since the cv staining approach used in the initial mutant screen relies on a dye that stains not only cells, but all biomass adhering to the microtiter plate, we elected to use additional biofilm assays to further characterize the biofilm phenotype of Δecha, and whether disruption of the putative enoyl-coa hydratase negatively impacts a specific stage of biofilm development (e.g. attachment, matrix production, maturation). we first used a modified adhesion assay to test for defects in attachment to the surface of a chamber slide. under static conditions, Δecha exhibited no difference in surface attachment relative to the wildtype, demonstrating that initial stages of biofilm formation are not affected (fig. a) . we then used a common colony morphology assay that relies on congo red staining as a macroscopic means of identifying the capacity for extracellular matrix production. , over the course of days of growth, biofilm colonies showed no apparent differences in morphology or color between strains, suggesting that the echa gene product has negligible effect on matrix production in a. xylosoxidans. finally, we used scanning electron microscopy to visualize microcolony ultrastructure in mature biofilms grown for hours under static conditions (fig. c) . relative to wt, the Δecha mutant exhibited notable phenotypic differences in biofilm architecture, corroborating observations made using the microtiter plate assay. first, the mutant strain demonstrated less robust biofilm growth and reduced surface coverage relative to wt. in addition, higher magnification images revealed a less-dense packing order to mutant biofilm cells, suggesting that cell-cell signaling mediated by enoyl-coa hydratase-derived metabolite is central to biofilm ultrastructural development in a. xylosoxidans. Δecha exhibits increased susceptibility to antibiotic treatment biofilm-associated bacteria can be orders of magnitude more tolerant to antibacterial compounds than their planktonic counterparts. given its defect in biofilm formation and more diffuse nature of mature microcolonies shown by sem, we hypothesized that the Δecha mutant would exhibit increased susceptibility to antimicrobial treatment. to test this, mature biofilms were grown statically in -chamber coverslip slides for h and treated with various concentrations of two commonly used therapeutics to which a. xylosoxidans is tolerant, levofloxacin and tobramycin (fig. ) . despite not showing any difference in minimum inhibitory concentrations for planktonic cells of the wt and mutant strains ( μg/ml for tobramycin; μg/ml for levofloxacin), live/dead staining of treated biofilms, visualization by microscopy (fig. a) , and quantification of dead biomass (fig. b ) revealed a statistically significant increase (p < . ) in susceptibility of the Δecha biofilm to both antibiotics relative to wt. these data demonstrate a central role of echa in biofilm antibiotic tolerance. a. xylosoxidans is recognized as an emerging nosocomial pathogen and is associated with a range of infections, including bacteremia, endocarditis, meningitis, and pneumonia in immunocompromised individuals. in addition, the prevalence of a. xylosoxidans is now estimated to be as high as % among individuals with cf, [ ] [ ] [ ] [ ] [ ] [ ] which is of increasing concern given its reported correlation to lung function decline, patient-to-patient transmissibility, and multi-drug resistance phenotypes. [ ] [ ] [ ] [ ] unfortunately, little is known about the physiology and molecular biology of this pathogen, and a greater understanding is critically needed to inform new treatment strategies. this study is an important step in that direction as it identifies several molecular determinants of biofilm growth, thought to play a critical role in the persistence and pathogenesis of a. xylosoxidans in the cf airways. the inherent and acquired drug resistance of achromobacter spp. not only limits therapeutic approaches but has also led to a paucity of tools (i.e. selection markers) available for molecular biology studies of a. xylosoxidans and manipulation of its genome. here, we systematically screened numerous vectors and determined that among antibiotics tested, only tetracycline and gentamicin resistance markers were compatible with both e. coli and a. xylosoxidans. tet r and gent r cassettes were then used to generate (to our knowledge) the first transposon mutant library and a null deletion strain in a. xylosoxidans, facilitating a detailed study at the molecular level. these tools pave the way for addressing critical questions about the physiology of this bacterium and its role in pathogenesis. screening of our transposon library revealed several genetic determinants of biofilm formation. statistically, genome coverage in our library was unsaturated (~ %), though hits in genes in diverse functional categories suggests that the regulation of biofilm development in a. xylosoxidans integrates a complex network of cellular processes and environmental stimuli. several homologs of genes identified in this screen have been linked to biofilm formation in other bacteria. for example, flgb, encoding a flagellar basal body rod protein, has been implicated in biofilm development in both bordetella bronchiseptica and p. aeruginosa. it is noteworthy that other flagellar-associated genes are downregulated in a. xylosoxidans biofilms relative to planktonically grown cells. in addition, lysr family regulators, such as axylo_ that was identified in our screen, have been implicated in biofilm morphology and mucosal colonization in other respiratory pathogens such as p. aeruginosa, burkholderia cenocepacia, and klebsiella pneumoniae. [ ] [ ] [ ] further characterization of these and other genes identified in our screen (see table ) will undoubtedly generate new insights into the many aspects of achromobacter biofilm physiology that remain uncharacterized. we took most interest in axylo_ (echa), encoding a putative enoyl-coa hydratase (one of eight in strain mn ) with homology to dspi in p. aeruginosa. dspi and other bacterial homologs (e.g. rpff in x. campestris) are key enzymes used in the synthesis of diffusible signaling factors (dsfs) that are monounsaturated fatty acids of medium chain length containing a cis- double bond thought to be central in their mechanism of action. these metabolites broadly regulate bacterial behaviors such as motility, iron uptake and virulence, , , and data presented here adds to the growing appreciation of their widespread role in biofilm development across diverse gramnegative bacteria. while synthetic cis-da was able to partially rescue the biofilm defect in Δecha, the specific identity of the fatty acid signal produced by a. xylosoxidans remains to be determined. it is also possible that a. xylosoxidans may respond to multiple dsf-like signals produced endogenously or exogenously, as interspecies signaling mediated by cis- fatty acids has also been reported. ongoing work is now aimed at characterizing the signal structure, its biosynthetic pathway, sensing mechanism(s) and downstream phenotypic effects in addition to biofilm development. respiratory pathogens that adopt a biofilm lifestyle in vivo exist in a protective environment against antimicrobials and host defense mechanisms. furthermore, biofilm cells generally reduce their metabolic activity relative to planktonic cells, further enhancing their intrinsic resistance to therapy. a. xylosoxidans has been shown to form robust biofilms both in vivo and in vitro, , which is corroborated by microscopy data presented here. however, our data demonstrating a heightened susceptibility of Δecha to antibiotic treatment suggest that interfering with fatty acid-mediated cell-cell signaling may represent a viable approach to managing a. xylosoxidans biofilms in the lower airways, particularly when used in combination with conventional cf antibiotics. here we elected to test two compounds to which a. xylosoxidans has high resistance, levofloxacin and tobramycin, though it will be interesting to test whether this effect holds true for other antibiotics with different mechanisms of action. in summary, we developed a robust genetic system for use in a. xylosoxidans that we leveraged to generate a transposon mutant library. further study of this library revealed a suite of genes essential for biofilm development in vitro and invoked an essential role for a putative enoyl-coa hydratase in biofilm ultrastructure and tolerance to antimicrobials, likely mediated by a fig. disruption of enoyl-coa hydratase activity leads to increased antibiotic susceptibility. a mature a. xylosoxidans biofilms were visualized by fluorescence microscopy after h exposure to levofloxacin ( μg/ml) and tobramycin ( μg/ml). deletion of echa resulted in increased susceptibility to both compounds. b integrative densities of green (live) and red (dead) cells were used to calculate % dead biomass for each biofilm treated with various concentrations of antibiotic (bars represent standard deviation, n = for each treatment) l.c. cameron et al. cis- fatty acid signaling compound. while a clear picture of the clinical impact of a. xylosoxidans in cf airway disease is only beginning to emerge, the continued study of the molecular basis of its biofilm formation and physiology will undoubtedly lead to improved treatment strategies for this important respiratory pathogen. and escherichia coli were grown in lysogeny broth (lb) at °c unless otherwise specified. when necessary, growth media were supplemented with gentamicin at μg/ml (e. coli) or μg/ml (a. xlyosoxidans), ampicillin at μg/ml (e. coli) or μg/ml (a. xylosoxidans), carbenicillin ( μg/ml), tetracycline ( μg/ml), or chloramphenicol ( μg/ml). e. coli strain β was supplemented with μm diaminopimelic acid (dap). the transposon delivery vector ptntet containing the hyperactive mariner transposon was introduced by transformation into the e. coli donor bacterial strain β . a. xylosoxidans mn and e. coli β (carrying ptntet) were grown overnight in lb and lb containing chloramphenicol ( mg/ml) and μm dap, respectively, at °c. cells were combined in a donor-to-recipient ratio of : , centrifuged at × g for min, resuspended in μl fresh lb-dap and spot-plated ( μl) onto lb agar. mating proceeded for h at °c, at which point cells were harvested and resuspended in ml of lb. cell suspensions were diluted : , and μl aliquots were plated on lb-tetracycline agar and allowed to grow for h at °c. colonies were randomly selected for downstream biofilm assays. to identify determinants of biofilm development, transposon mutants were screened using a modified crystal violet (cv) assay approach. briefly, individual mutants were transferred to single wells of a -well microtiter plate containing μl lb per well, and incubated while shaking at °c. following h of growth, μl was transferred to μl abt medium [ mm (nh ) so , mm na hpo , mm kh po , mm nacl, mm mgcl , . mm cacl and . mm fecl supplemented with . % casamino acids and . % glucose] in a new microtiter plate, and grown for an additional h at °c in a humidified chamber. plates were first measured spectrophotometrically (od ) to determine culture cell density. supernatants were discarded, and plates were washed x with ultrapure water. plates were dried in a biosafety hood for . h and stained with μl of . % cv for minutes. plates were then washed x to remove excess stain, air-dried for h, and μl % acetic acid was added to each well. following a min incubation, cv absorbance was measured spectrophotometrically (od ) and normalized to culture density. wells exhibiting less than % absorbance than the wildtype (mn ) were considered putative hits ( total) and were subjected to a secondary screen using the same protocol (n = for each mutant). transposon mutants showing a significant reduction in biofilm growth ( total, as determined by a mann-whitney u test) were stored for further characterization. biofilm growth of mn , its Δecha derivative and complemented strain (see below) were also tested using the same microtiter assay. in these experiments, growth medium was supplemented with % ethanol to drive expression of the alca promoter in pbmb (below). when indicated, cis- decenoic acid (f d, carbosynth) was also added at a concentration of nm. arbitrary pcr and mutant sequencing an arbitrary-pcr-based approach was used to identify sequences flanking transposon insertion sites. the pcr method involved two rounds of reactions, with the first using a primer unique for the mariner transposon and one degenerate primer (pair , table s ). the second round included nested primers (pair ) unique to the transposon and ' end of the arbitrary primer for amplification of pcr products obtained in the first round. pcr products were sequenced at the university of minnesota genomics center (umgc) and were mapped to the a. xylosoxidans mn genome (accession#prjna ). to generate a deletion vector compatible with a. xylosoxidans, psmv was first digested using apai. a tetracycline resistance cassette was then amplified from pex tc using primer pair (supplementary table ) and digested with apai. vector and insert ( : ratio) were then ligated using t ligase, transformed into e. coli uq and selected for on lb agar containing tetracycline ( μg/ml). colonies were screened using primers m f and tetr (pair ) to confirm insertion orientation. one plasmid, pbmb , was selected for further use. to generate an in-frame, unmarked deletion of axylo _ (echa), kb sequences flanking echa were pcr amplified using primer pairs and (supplementary table ). these flanking regions were combined and cloned into pbmb digested with spei and xhoi using gibson assembly, resulting in pbmb (supplementary fig. ). this plasmid was then chemically transformed into uq , and positive ligations were screened by pcr. pbmb was then transformed into e. coli strain wm and mobilized into a. xylosoxidans mn by conjugation. recombinants were selected for on lb-tetracycline agar and double recombinants were selected for on lb agar containing % sucrose. complementation was achieved via exogenous expression of echa (ax_ ) from pbbr mcs- . to do so, an alcohol-inducible promoter, alca was first amplified from pgga using primers alca_f and alca_r (supplementary table ) before digestion with restriction enzymes hindiii and bamhi. ligation into similarly digested pbbr mcs- yielded pbmb (pbbr mcs- ::alca). echa was then amplified from a. xylosoxidans mn genomic dna using primers echa_f and echa_r, and digested with bamhi and saci before ligation into pbmb using t ligase. the resulting vector, pbmb (pbbr mcs- ::alcaecha; supplementary fig. ), was transformed into e. coli uq . this vector was then introduced into a. xylosoxidans mn via conjugation with an e. coli donor strain wm , and transconjugants were selected on lb agar containing μg/ml gentamicin sulfate. all constructs and positive transformants were verified by sanger sequencing. a modified attachment assay was used to assess early attachment of a. xylosoxidans to a polystyrene substratum. briefly, mn and its Δecha derivative were grown for h at °c in abt followed by dilution : into fresh medium. cells were then grown to mid-log phase (od = . ) before dilution in abt to an od of . . μl of each culture was added to an -chamber coverslip slide (ibidi, # ) and incubated at °c for h. following incubation, slides were rinsed twice with μl of pbs to remove unattached biomass, and attached cells were stained using syto (invitrogen) in pbs according the manufacturer's protocol. substrata were imaged using an olympus ix inverted fluorescence microscope with a transmitted koehler illuminator and a x objective lens (olympus). four images per strain per biological replicate (n = ) were captured on a hamamatsu orca camera, and post-acquisition analysis was performed using fiji software by calculating the integrated density of syto . colony biofilm assay mn and Δecha were grown overnight in lb medium, diluted : , and μl was spotted on nutrient agar containing % tryptone, % agar, μg/ml coomassie brilliant blue, and μg/ml congo red. plates were incubated at °c for days and monitored daily for colony morphology. overnight cultures were diluted : in fresh lb medium and were added to -well microtiter plates containing autoclaved aclar fluoropolymer film (electron microscopy sciences, hatfield, pa). biofilms were grown for h at °c, shaking at rpm, and prepared for sem using cationic dye stabilization methods. , briefly, aclar membranes containing biofilm growth were washed three times in . m sodium cacodylate buffer, and submerged in primary fixative ( . m sodium cacodylate buffer, ph . , % paraformaldehyde, % glutaraldehyde, % sucrose, . % alcian blue gx) for h. samples were washed three more times prior to a minute treatment with secondary fixative ( % osmium tetroxide, . % potassium ferrocyanide, . m sodium cacodylate, ph . ). after three final washes, biofilms were chemically dehydrated in a graded ethanol series ( , , , , [ ×] and % [ ×]) before co -based critical point drying. aclar membranes were attached to sem specimen mounts using carbon conductive adhesive tape and sputter coated with~ nm iridium using l.c. cameron et al. the leica ace magnetron-based system. biofilms were imaged using a hitachi s- field emission sem with an operating voltage of kv. biofilm antimicrobial susceptibility testing was performed using a chamber slide assay. briefly, mn and Δecha were grown in lb overnight, diluted : and grown to an od of . before dilution to an od of . . in total μl of each culture was then added to each well of an ibidi -chamber coverslip slide and incubated at °c in a humidified chamber. after h, medium was replaced with fresh lb and incubated for an additional h. media was gently aspirated from each well, replaced with lb containing either tobramycin or levofloxacin ( , , , and μg/ml), and incubated at °c for h. cells were washed in sterile pbs to remove unattached biomass, stained for minutes using the baclight live/dead viability assay (life technologies, #l ) and visualized by fluorescent imaging as described above. integrated density for syto and propidium iodide (pi) for each image was determined using fiji, and percentage of dead biomass was determined by (average integrative density of pi)/(average integrated density of pi+ integrated density of syto ). data were generated using four biological replicates (n = ). this work was previously published as a preprint. reporting summary further information on research design is available in the nature research 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xanthobacter autotrophicus py using a new, highly efficient transposon mutagenesis system that is functional in a wide variety of bacteria the ars detoxification system is advantageous but not required for as(v) respiration by the genetically tractable shewanella species strain ana- fiji: an open-source platform for biological-image analysis high-resolution visualization of the microbial glycocalyx with low-voltage scanning electron microscopy: dependence on cationic dyes enterococcus faecalis produces abundant extracellular structures containing dna in the absence of cell lysis during early biofilm formation effect of high-dose antimicrobials on biofilm growth of achromobacter species isolated from cystic fibrosis patients a putative enoyl-coa hydratase contributes to biofilm formaton and the antibiotic tolerance of achromobacter xylosoxidans acknowledgements pgga (addgene plasmid # ) was a gift from dr. jan lohmann (heidelberg). we thank clayton summitt for assistance with data analysis and other members of the hunter lab for critical review of the manuscript. parts of this work were carried out in the characterization facility, university of minnesota, which receives partial support from nsf through the materials research science and engineering centers (mrsec) program. this study was supported by the university of minnesota, a nhlbi pathway to independence award to rch, american society for microbiology undergraduate research fellowships to akl and lak. supplementary information accompanies the paper on the npj biofilms and microbiomes website (https://doi.org/ . /s - - - ).competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/ . /. key: cord- -n axd bq authors: rusoke-dierich, olaf title: travel medicine date: - - journal: diving medicine doi: . / - - - - _ sha: doc_id: cord_uid: n axd bq before travelling to other countries, thorough travel advice should be provided. not only information about diseases of specific countries but also general advice for travelling should be given on this consultation. before travelling to other countries, thorough travel advice should be provided. not only information about diseases of specific countries but also general advice for travelling should be given on this consultation. the following topics should be included in the travel advice consultation: vaccinations (general and country specific) country-specific diseases malaria prophylaxis mosquito prophylaxis (wearing bright long-sleeved clothes, avoiding perfume, staying in air-conditioned rooms, using a mosquito net, using insect repellents, staying inside at dawn and dusk) food consumption and drinking overseas (no consumption of ice cubes, uncooked meals, salads and food, which is exposed to flies, limited alcohol consumption) uv protection (using sun cream, avoiding sun exposure between . and . o' clock, remaining in shaded areas, wearing a hat and covering skin) fitness assessment for travelling, flying and diving challenges of different climates and their effects on the personal health (dehydration, hyperthermia) medications thrombosis counselling counselling on symptoms on return, which require review (fever, skin changes, abnormal bleeding, lymphadenopathy, diarrhoea) sexual transmitted diseases contraception rabies the following items should be asked to enable to give the appropriate advice: risk assessment of the travel in a particular country (transport, area of stay/ rural or resort, reason for travelling, appropriate conduct overseas, pre-existing diseases and medications) vaccination status accomodation and stopovers duration of the stay the vast majority of up-to-date travel information and information about tropical disease are available on who (world health organization) or cdc (centres for disease control and prevention) websites. information on these websites are frequently updated. before giving appropriate advice based on these online resources, it should be checked, which medications are available in the particular countries. hence, recommendations need to be adjusted individually. usually, a medication record is required at the customs. however, it might be sufficient, if the original medication box has the patients and prescribing doctors details (. table . ). malaria is a tropical disease transmitted by the female anopheles mosquito. the distribution of malaria is primarily in the tropics and subtropics of africa, central and south america, asia, papua new guinea and the western pacific islands. as popular diving spots are located in these areas, malaria prophylaxis and advice should be given. the who (world health organization) estimates the worldwide number of people affected by malaria with about million and , , deaths ( ). the plasmodium parasites need temperatures above °c in order to complete the entire growth cycle. therefore, malaria occurs in some places only seasonal. additionally, there are differences in anopheles species regarding the affinity to the host and their local distribution. some genetic factors are protected against malaria. for example, sickle cell anaemia gives a certain protection against p. falciparum and duffy negative blood group against p. vivax. it appears that after recurrent malaria infections, the body adapts to the disease. this means that an infection is possible, but the symptoms of malaria seem to be reduced. children and pregnant women have an increased risk of being affected by malaria. additionally, children have a high mortality rate. during pregnancy the resistance against malaria is reduced. it also poses an increased risk for the unborn child (low birth weight). anopheles is active especially at sunrise and sunset. different kinds of mosquitoes are rather active during the day and can transmit other diseases such as dengue. especially p. falciparum and p. vivax have resistances against antimalaria drugs. there are different plasmodium pathogens: p. falciparum: worldwide tropical and subtropical distribution, mainly in africa; pathogen of severe malaria causes million deaths per year; rapid growth in the blood with haemolysis and emboli due to cytoadherence of affected erythrocytes; - days of incubation, irregular fever spikes. p. vivax: mainly in asia, latin america and some countries in africa; the disease can be activated after months or years. incubation period of - days; fever spikes every days. p. ovale: mainly west africa and the western pacific islands. similar to the p. vivax, it can also infect people with duffy-negative blood group; incubation period of - days; fever spikes every days. p. malariae: worldwide distribution; typical -day cycle, untreated can lead to lifelong chronic malaria; incubation period - days; fever spikes every days. p. knowlesi: southeast asia, mainly infected animals. after the anopheles mosquito aspirates with gametocytes infected blood, the gametocytes develop to gamete in the mosquito's intestines. in the blood of the mosquito, the microgametes (male) penetrate the macrogametes (female), forming zygotes. then cells are changed to an elongated, motile ookinete. this evolves into an oocyst. after the oocyst bursts, sporozoites are released and get in the saliva of the mosquito. the entire cycle inside the mosquito takes - days. if sporozoites enter the human bloodstream through the saliva of the . symptoms of malaria appear after the incubation period. the incubation period varies depending on the pathogen. it can be between a few weeks and also takes up to several months or even a year (p. vivax or occasionally p. ovale). malaria can be divided in three different forms: malaria tertiana: pathogen: p. vivax and p. ovale; fever every second day with one day without fever, spontaneous remission after max. years malaria quartana: pathogen: p. malaria; fever every third day with days without fever, no spontaneous remission malaria tropica: pathogen: p. falciparum, irregular fevers due to the lack of synchronisation of the parasite reproduction, severe form of malaria (malaria maligna) with high fatality, recurrence up to years the fever has a specific pattern. in the first hour, strong rigors and increasing fever typically develop. the fever can reach °c and more for duration of about h. it is often associated with flushing, vomiting and nausea. the fever stage is followed by an approximately -h stage of severe sweating with decreasing fever. severe forms of malaria can be fatal in within few days. causes of death are cerebral malaria, respiratory failure with adrs and kidney failure. the main reason of these complications is the cyto-adherence ("bonding") of the erythrocytes. it results in a failure of the microcirculation followed by ischaemia of vital organs. . the treatment depends on the severity and the pathogen. in complicated malaria, admission to the intensive care should be considered, if more than one of the following criteria exists: inability of the oral intake of medication parasite load of erythrocytes > % severe symptoms of malaria (see table above) the treatment options of complicated malaria are: artesunate (allowed only in some countries): . mg/kg/bw iv; first dose on admission, repeated after and h, minimum duration of therapy h and then once a day, till oral therapy is tolerated. or combination of quinine + doxycycline or clindamycin. quinine: Ȥ first dose: mg/kg/bw iv over h or mg/kg/bw iv over min with subsequent administration of mg/kg/bw iv over h. Ȥ maintenance therapy: mg/kg/bw iv over h three times a day, beginning h after the completion of the first dose. Ȥ exemption: if the patient received three or more doses of quinine in the last h or had an mefloquine prophylaxis in the last h or received a mefloquine treatment in the last days. + doxycycline: mg iv twice daily for days (iv or oral) or clindamycin: Ȥ initial dose: mg/kg/bw Ȥ maintenance dose: mg/bw every h for days (iv or oral) after clinical improvement medication can be changed to a complete cycle of the oral therapy of an uncomplicated malaria (riamet ® or quinine with doxycycline or clindamycin). uncomplicated malaria can be handled on the normal ward. outpatient therapy with close supervision can be considered under the following conditions: parasite load of erythrocytes < %. age > months. no co-morbidity. pregnancy is excluded. ability of oral medication intake. p. falciparum is excluded. clinically stable under medical therapy for the last h. a daily blood smear is necessary during treatment to follow the process of the disease. the patient can be discharged from the hospital and continue treatment at home; if oral therapy is tolerated, a clinical improvement is achieved and the parasite count decreases. a week and a month after discharge, blood smears should be repeated. primaquine as eradication therapy is approved in some countries. it is the only drug that can be used to eliminate hypnozoites, which are the dormant forms of the malaria parasites that occur with p. ovale and p. vivax. because primaquine causes haemolysis in g- -pd deficiency, g- -pd status prior therapy needs to be established. if an eradication with primaquine is required in patients with g- -pd deficiency, a dose up to mg weekly for weeks, with monitoring for haemolysis, could be considered. in children methaemoglobinaemia can be provoked by giving primaquine. a single dose of primaquine mg for p. falciparum, p malaria and p. knowlesi can be given to sterilise the gametocytes. if malaria caused by p. vivax or p. ovale or co-infection with these parasites is suspected, a -day treatment with mg of primaquine twice a day is recommended. before commencing holidays overseas, medical advice should be given in order to assess the malaria risk of the particular country. in nearly all tropical areas, there is a risk of getting infected with malaria. in some tourist areas, this risk might be small, but infection is still possible. in particular day trips to more remote areas pose a risk. some areas have malaria outbreaks and therefore should be avoided. in general, mosquito bites should be avoided to minimise the risk of any mosquitoborne infections. mosquitoes transmitting malaria are mainly active at night, sunrise and sunset. however, mosquito bites are also possible throughout the day. long-sleeved shirts, long pants and closed shoes cover the skin and provide protection against insect bites. insect repellent for the skin and clothes offer additional protection. higher concentrations offer better and longer protection. the protection period of a normal insect repellent lasts usually only - h. slow release products can prolong the effect. mosquitoes avoid air-conditioned rooms. so staying in air conditioned rooms itself provides certain protection. spraying insecticides in rooms and surroundings can be helpful to repel and minimise the quantities of mosquitoes. the bed should be covered with a mosquito net (. fig. . ). chemoprophylaxis is important, because the main cause of malaria deaths is still inadequate chemoprophylaxis. there are different drugs for chemoprophylaxis available. they are subject to the travel location and the parasite's resistances to certain drugs. in addition, they differ in side effects, dosage and cost. except malarone ® , all other drugs for the chemoprophylaxis against malaria have to be taken weeks after leaving the country as they aren't sufficiently effective against the primary liver stages of malaria. mefloquine (lariam ® ) is the only malaria prophylaxis without absolute contraindication in pregnancy. diving (decrease in vigilance); - weeks (at least week) before entering the malaria-endemic country and weeks after return; lariam ® is a category b medication and is the only medication against malaria without absolute contraindication in pregnancy. the use in the first trimester should only be considered, if the expected benefits justify the potential risk to the foetus. however, recent studies suggest that even in the first trimester this medication is safe to take. the dengue virus is an arbovirus. it has four different serotypes (denv - ). dengue has a worldwide distribution in the tropics and subtropics, especially in asia and south america. approximately - million cases and about , with serious complications per year occur. there is a % mortality, which can be reduced to % with timely diagnosis and appropriate treatment. it has an increased risk for children under years and persons with previous dengue infections. the dengue virus is transmitted by the aedes aegypti mosquitoes. these mosquitoes mainly bite at day and in twilight (. fig. . ). z symptoms the incubation period is - days. there is a wide range in severity of dengue symptoms. the majority of infections cause minor symptoms. but dengue infections can be also quite severe (. table . ). in particular recurrent infections with dengue are associated with complications and severity of the disease. it is important for the treating doctor to remember that after the initial fever, the critical phase follows. therefore, the patient must be monitored closely during this time. the disease goes through three stages: fever phase (day - ): sudden high fever °c occasional associated with bradycardia; myalgia mainly in the spine, arms and legs ("breakbone fever"), headache; retrobulbar pain; rigors; metallic/bitter taste; vomiting; and dehydration. . critical phase (day - ): normal temperature with possible mild fever later on, leucopenia, exanthema, petechiae and lymphadenopathy. severe dengue: abdominal pain, spontaneous bleeding, volume shift in to the peritoneal space ("plasma leak"), pleural effusion, hepatomegaly (≥ cm), rapid increase in haematocrit and decreasing thrombocytes, shock (dengue haemorrhagic shock = dhs or dengue shock syndrome = dss), increased bleeding (dengue haemorrhagic fever = dhf) and organ failure (particularly liver). remission (after days lasting sometimes for weeks): risk of hyperhydration is given when extravascular fluid is reabsorbed without reducing the intravenous fluid administration. in particular in long remissions, fatigue and depression may be present. normally there are no long-term damages after a dengue infection, and the vascular changes recover completely. z treatment there is no medication available to treat dengue directly. the diagnosis of dengue can be demonstrated by pcr in the initial phase and using igm and igg a few days later. due to severe complications, the haematocrit, coagulation parameters, leukocytes and platelets have to be tested daily. thrombocytes < , cells/mm can rise the suspicion of dhf. if pleural effusion is suspected, a cxr should be obtained. by tightening a blood pressure cuff petechiae can be provoked (medium pressure of the systolic and diastolic pressure for min). this can be used as a diagnostic tool. an increase of the haematocrit of > %, pleural effusion, ascites or hypoproteinaemia could be a sign for extravascular fluid loss. the extravascular fluid loss is typically found in the initial phase. hence, fluid replacement therapy is crucial in this phase. as the extravascular fluid loss can come to an end quite quickly, a complication of the fluid replacement therapy is hyperhydration. decrease of haematocrit of > % after fluid administration can represent a fluid excess and hyperhydration. hence, careful monitoring of the fluid balance and weight are necessary. the therapy is adjusted according to its severity. if necessary, dic, blood loss or shock require specific treatment. like dengue, chikungunya is a mosquitoborne disease. the species transmitting the chikungunya virus (chikv) are aedes aegypti in the tropics and subtropics and aedes albopictus in colder regions (. fig. . ). these mosquitoes bite day and night, but mainly in the early morning hours and late afternoon. the incubation period is between and days. the symptoms are similar to that of dengue. patients suffer from sudden fever with headache, skin rash, fatigue, strong limbs and muscle pain. affected joints often are swollen. the symptoms generally last for few days but can persist for weeks and years. the disease has no long-term effects. for diagnosis rt-pcr and virological methods can be used in the initial phase. later, it can be diagnosed by igm and igg. igm peaks after - weeks and can be detected up to months. the treatment requires analgesia only. . yellow fever is a disease transmitted mainly by the aedes aegypti mosquito but also by other mosquitoes or ticks. the pathogen is a rnacontaining flavivirus. it has approximately , infections with approximately , deaths annually. % of cases occur in africa and the remaining % in south america. the risk of getting infected with yellow fever is with : - in africa and higher than : in south america (. fig. . ). the transmission occurs in rainforest areas (jungle or sylvatic cycle), where mosquitoes transfer the virus from monkeys to humans, in endemic areas of the savannah (savannah or intermediate cycle) either transferred from monkeys or human to humans via mosquitoes or in urban areas from human to human via mosquitoes. the incubation period is - days. the disease has two phases. the acute phase comes with fever, headache, myalgia, headand backache, loss of appetite, nausea, vomiting and diarrhoea. the second phase occurs only in approx. % of infected humans within the next h. jaundice, abdominal pain and vomiting are rapidly developing, followed by diffuse bleeding (epistaxis and gi bleeding) and multi-organ failure (mainly kidneys). if symptoms of the more severe second phase develop, % of the patients die within the next - days. patients who survive usually recover without significant organ damage. the diagnosis can be made via a blood or tissue biopsy of the liver. there is no cure for yellow fever and only supportive measures can be taken. however, a very effective life-vaccination (stamaril ® ) is available. only authorised doctors are authorised to prescribe and give the vaccine. severe side effects of these vaccinations are severe allergic reaction ( : ), vaccine-associated neurotropic disease/post-vaccinal encephalopathy ( : ) and vaccine-associated viscerotropic disease/ multi-organ failure ( : ). for travelling into countries where yellow fever is endemic, vaccination is mandatory. the side effects seem to be age-related and occur increasingly with progressive age or in young children. the vaccination is contraindicated in children . · other mosquito-borne diseases below month and during pregnancy. analysis of yellow fever vaccines adverse events demonstrated an increased frequency of serious adverse events in persons age years and older. the risk of viscerotropic side effects in < years is : , in a population of - years of age : and in > years of age : . a failure to be vaccinated or being documented can lead to a refusal of entry into other countries or to a certain time in quarantine when leaving the area where yellow fever occurs. if there is a clinical indication against receiving yellow fever vaccine (e.g. children < month or poor immune status), a written medical exemption can be granted, to enable to travel to these countries without vaccination. absolute contraindications for a yellow fever vaccination are: allergy against the vaccine or egg protein age < months immunodeficiency neoplasia transplantations immunosuppressive therapies relative contraindications for a yellow fever vaccination are: age - months age > asymptomatic hiv infections and cd + t lymphocytes - /mm ( - % of the total in children < years of age) pregnancy lactation aedes aegypti spreads also the zika virus. however, it is also sexually, intrauterine and perinatal transmitted. currently the main distribution is countries in south and north america as well as the caribbean islands, singapore and some countries in south pacific islands. symptoms of zika infection may be fever, rash, arthralgia, myalgia, headache and conjunctivitis. but in most cases, an infection is asymptomatic (~ %). these symptoms are lasting for several days to a week. the incubation period is - days but is likely to be a few days to a week. the diagnosis can be made via pcr or serology. blood pcr can be detected only in the first week of the disease. urine pcr can detect the virus up to weeks. there is no specific treatment available. deaths are unlikely. there is a potential risk during pregnancy, as microcephaly or other birth defects (~ %) may develop. the zina virus cane be also transferred via semen and can affect unborn life. ross river virus (rrv) is transmitted by the bites of culex annulirostris, aedes vigilax, aedes normanensis and aedes notoscriptus in australia, papua new guinea, parts of indonesia and the western pacific islands. the main transmission time is in the humid summer month from december till march. the main symptoms are fever, rash, headache, myalgia, arthralgia and fatigue. the initial symptoms with fever last usually for - weeks. myalgia and arthralgia usually last longer. symptoms of fatigue and depression can be late complications. the incubation time is between days and weeks. the diagnosis is made with igm. there is only symptomatic treatment available. barmah forest virus (bfv) is transmitted by the same species as the rrv. it mainly can be found in australia. many people don't develop any symptoms. the incubation time is - days. if symptoms appear, they are similar to the one of rrv. the initial symptoms last for - weeks, and the arthralgia and myalgia may last for months. the diagnosis is made with igm. there is only symptomatic treatment available. sindbis virus (sinv) is related to the chikungunya virus. it is mainly transmitted via the culex and culiseta mosquitoes. it can be found in europe, africa, asia and oceania. the symptoms and the duration of the symptoms are quite similar to rrv and bfv. the diagnosis is made with igm. there is only symptomatic treatment available. the o'nyong-nyong virus (onnv) is related to the chikungunya virus but is restricted to africa. it has similar symptoms as the chikungunya virus but has additionally mainly cervical lymphadenopathy, and the affected joins rarely show signs of an effusion. most of the gastrointestinal tract infections are caused by poor hygienic conditions of the travel destination. occasionally ingested seawater can cause intestinal infections too. the main transmission routes are either food-borne or by contact. however, the most common cause for gastrointestinal infections is eating contaminated food. old, warmed up food, salads, unpeeled fruits, poorly cooked food, contaminated water (ice and already opened bottles with refilled water) and ice cream often have substantial quantities of pathogens and pose a risk. hence, the best protection against gi infections is avoiding contaminated food or drinks. usually gastrointestinal infections last for a few days and are self-limiting. if diarrhoea contains blood or mucus in combination of high fever for more than days, more thorough assessment is required. blood and mucus without fever are most likely related to a parasitic disease. if fever is present, it's most likely a bacterial or viral disease. but also climate change by itself or dehydration may be caused by autonomic dysregulation gastrointestinal symptoms such as nausea, weakness, vomiting and diarrhoea. with dehydration the dci risk increases. rehydration and supply of certain electrolytes such as sodium, chloride and potassium are the most important treatments for gastroenteritis. fatigue is a common associated symptom. tannins of black tea boiled for more than min might be beneficial for diarrhoea. the consumption of bananas is recommended because of the high content of potassium. but the best options are rehydration preparations in form of drinks, powders or icy poles. loperamide may slow down the peristaltic and give some relief from diarrhoea. probiotics may support recovery. a low fibre diet is rec-ommended in the active phase of diarrhoea. administration of antibiotics is rarely necessary and indicated. it only is used for serious illnesses or symptoms. reservoir: poultry or meals prepared with egg incubation: - h symptoms: fever, vomiting nausea, diarrhoea, occasionally blood and mucous in the stool duration: - days treatment: symptomatic; azithromycin g od for days or ciprofloxacin mg bd for days or ceftriaxone g od reservoir: water and food incubation: - weeks symptoms: headache, myalgia, bradycardia, roseola in the abdominal area, continuous fever - °c, porridge -like diarrhoea, intestinal bleeding and decrease of the fever after weeks treatment: symptomatic; azithromycin g od for days or ciprofloxacin mg bd for days or ceftriaxone g od; vaccination available reservoir: human, flies, food and faeces incubation: - days symptoms: fever, diarrhoea, sometimes with blood and mucus in the stool and severe abdominal pain treatment: symptomatic; ciprofloxacin bd for days, norfloxacin mg bd for days or bactrim / mg bd for days reservoir: food and water incubation: - days symptoms: mild to severe diarrhoea with fever and blood and mucous in the stool, most common cause for diarrhoea overseas treatment: symptomatic; norfloxacin mg od and ciprofloxacin mg od reservoir: food (particular strawberries) and water incubation: - weeks symptoms: diarrhoea like raspberry jelly, no fever! blood and mucous in the stool, risk for developing a liver abscess treatment: symptomatic, asymptomatic carrier, paromomycin mg tds for days; invasive, tinidazole g od for days or metronidazole mg tds for to days . . cholera (vibrio cholerae) reservoir: contaminated food and water incubation: - days symptoms: often mild gi symptoms, - % develop severe symptom with nausea vomiting, rice water-like diarrhoea and severe dehydration, mortality risk of - % treatment: rehydration, electrolyte substitution; vaccination available; azithromycin g single dose, ciprofloxacin g single dose reservoir: food (in particular sea food) and water incubation: - days symptoms: initial phase ( - days)flulike symptoms, gastrointestinal, hepatomegaly; hepatic manifestation ( - weeks), no jaundice (approx. %), jaundice ( %) with dark urine, jaundice, pruritus; hepatitis a has no chronic form, rarely fatal (fatality is age dependent) treatment: symptomatic, bed rest, avoidance of liver toxic substances (alcohol, medication); vaccination available japanese encephalitis is caused by a flavivirus, which is transmitted by mosquitoes (culex particularly c. tritaeniorhynchus). the hosts are usually pigs and water birds. in humans there are usually not sufficiently high concentrations of virus to serve as a host. the distribution is the asia, especially in rural areas. epidemics occur every - years (. fig. . ). the transmission can occur throughout the year but frequently peaks in the rainy season. there are about , cases per year. only about % of the patients are symptomatic. however, if symptoms develop, the mortality rate is - %. approx. - % of patients who survive have long-term neurological or psychiatric complications. mild courses of japanese encephalitis may be accompanied by mild fever and headache. severe cases show high fever, neck stiffness, photophobia, headache, disorientation, coma, convulsions, spastic paralysis or death. consequential damages may be behavioural disorders, convulsions, paralysis and speech disorders. the diagnosis can be established with blood tests and lumbar puncture. there is currently no treatment option. the vaccination is usually well tolerated and available for prophylaxis. there are various tropical diseases, which are present in poorer countries causing more or less severe symptoms. these diseases are termed "neglected tropical diseases" (ntd). the more common ntds are summarised in this chapter. there are three main conditions caused by these pathogens. the african trypanosomiasis (sleeping sickness) is transmitted by the tsetse fly. the distribution is only in some countries of the sub-saharan africa. seventy percent occur in the democratic republic of congo. tsetse flies are mainly found in rural areas. there are two forms causing sleeping sickness, t. brucei rhodesiense and t. brucei gambiense. t. brucei gambiense has an incubation period of months to years and t. brucei rhodesiense weeks to months. the initial phase is the haemolytic-lymphatic phase, in which pathogens replicate in tissues, blood and lymphatic tissues. symptoms are intermittent fever, headache, myalgia and pruritus. additionally, a painless, indurated chancre on the skin - days after the bite and lymphadenopathy (axillary and inguinal) can be associated. in the second phase, the cns affected causes continuous headache, behavioural disorders (mood swings and depression), delirium, sensitivity disorders, coordination problems and disruptions of the sleeping cycle (daytime somnolence). the diagnosis is mainly made clinically. only for the t. b. rhodesiense, a blood test (centrifuged or wet preparation) to data . detect the parasite is available. examination of buffy coat increases sensitivity. a biopsy of the lymph node to detect the pathogens can be diagnostic for t. brunei gambiense or be used for a culture and pcr. the card agglutination test for trypanosomiasis (catt) is a field test suitable for mass population screening in endemic areas for t. b. gambienses but has a low specificity and is hence only used for identifying suspected cases. all diagnosed patients need to have their cerebrospinal fluid examined for staging, which influences treatment options (. table . ). the treatment is dependent on the pathogen and the staging. if untreated, infections of both forms lead to coma and death. leishmaniasis has three forms: visceral, cutaneous and mucosal (kala-azar). there are about different pathogens, from which approx. are held responsible for these diseases. the disease is transmitted by mosquitoes or sandflies (phlebotomus and lutzomyia). the cutaneous form is the most common one, which causes skin ulcerations. typically this form appears weeks to months after the initial mosquito bite. initially papules are formed, which later ulcerate. they can be painful or painless. the visceral form affects organs, especially the liver, spleen and bone marrow. therefore, this form can be quite dangerous. the changes occur within months and years. hepatosplenomegaly and pancytopenia develop. the mucus form is rare. ulcerative changes of the mucous membranes (e.g. nose, mouth and throat) are typical for this. endemic areas for leishmaniasis are east africa, some arabic countries, india, bangladesh, brazil and some other south american countries. historically, the diagnosis was made by taking a biopsy (skin, bone marrow or other tissues) for culture. now pcr or serological testing with high sensitivity replaced biopsies for making diagnosis. as the visceral disease is fatal without treatment, it needs to be treated in any case. all other forms require normally no treatment. following medication is available: pentavalent antimonial (sb v ) compounds ( mg per day iv or im for days) liposomal amphotericin b ( mg od iv on day - , and ) miltefosine (in adults > kg mg times daily for days) azoles (fluconazole mg od for weeks, itraconazole mg bd for days, ketoconazole mg od for at least days) paromomycin (uncommonly used) pentamidine isethionate (uncommonly used) the chagas' disease is transmitted via an insect bite ("kissing bug") or by contaminated food. it occurs in central and south america. it has . an acute and chronic phase. in the acute phase within - weeks after the infection, localised swelling of the area of the insect bite (skin or mucous membranes), lymphadenopathy, bilateral orbital oedema, meningoencephalitis and myocarditis can occur. - % of all infections become chronic, causing arrhythmias with risk of "sudden death", cardiomyopathy and enlargement of the oesophagus (megaoesophagus) or of the colon (megacolon) even after years or decades. the cardiomyopathy consists of fibrosing myocarditis, causing arrhythmia (rbbb, left anterior fascicular block, st changes, premature ventricular beats and bradycardia) and ventricular failure. the diagnosis in the acute phase is made by a blood smear (thick and thin) to visualise the parasite. a serological test is also available. treatment is recommended in the acute phase and in patient up to the age of and no advanced cardiomyopathy with chronic chagas' disease (. table . ). in age groups above , benefits and risk need to be outweighed. worm infections are a major problem in underdeveloped countries. they occur mainly in rural areas. these conditions may cause insignificant symptoms but also lead to serious consequences or even cause death. because some dive sites are located far away from tourist centres, these infections should be discussed before travelling. this kind of roundworm is found in the tropical and subtropical regions of africa and southeast asia. the transfer follows on oral intake of eggs by contaminated food. the larvae are entering the bloodstream after hatching in the intestine. they reach the lungs via the blood and penetrate the lung tissue, and the larvae can be coughed up. if the sputum is swallowed again, the larvae reach the intestine, mature there within the next - months and lay eggs, which are then excreted via the faeces. the adult worms live about - years. infection is usually asymptomatic. however, abdominal pain, flulike symptoms, allergic skin manifestations, malnutrition, productive cough and a stridor can occur. the diagnosis can be made by examining the faeces (eggs, worms) or sputum (larvae). hookworms are found in tropical and subtropical regions of africa and latin america. the transmission is percutaneously or orally by ingestion of contaminated soil. in contaminated soil the larva is able to survive for about - weeks. larvae can penetrate the skin and enter the blood and reach the alveoli in the . lungs. from there they ascend in the airways, are swallowed again and finally get into the intestines. there larvae mature to adult worms. the worms attach themselves to the wall of the intestine and feed on blood. the eggs are excreted in the faeces and reach again the soil. the eggs can survive up to years. common symptoms are pruritus and rash at the entry site, abdominal pain, diarrhoea, weight loss, anaemia and extreme fatigue. the diagnosis can be made of the faeces. z filariasis filariasis has a worldwide distribution in tropical and subtropical regions. it is caused by wuchereria bancrofti and brugia malayi. it is transmitted by mosquitoes. the infective filariform grow inside mosquitoes and enter via its saliva during the bite. they migrate to the lymphatic vessels and lymph nodes where they develop into adults. they can live there for about years. the female worms produce microfila, which are circulating in the blood. absorbed by mosquitoes they develop within - weeks to the infective filariform. initially there are no symptoms. later lymph oedema in extremities or genitals is a common symptom. in men hydrocele can develop. the skin typically swells and hardens ("elephantiasis"). the diagnosis is made via the blood. detection in the blood smear has to be performed at night, as larvae only circulate in the blood at night. there is also a serological detection of anti-filaria igg available for diagnosis. the treatment with dec is the drug of choice. concurrent disease of loa loa or onchocerciasis is a contraindication for dec, because of the serious side effects (encephalopathy and deaths). ivermectin is used as a prophylaxis, but not as a therapy. z schistosomiasis (bilharziose) schistosomiasis can be found in tropical and subtropical regions worldwide. in addition to malaria, it is the most common parasitic disease. the parasite schistosoma is housed in freshwater snails. by being exposed to freshwater in these regions, infections can occur. the eggs are excreted in urine or faeces of the host. they hatch under optimal conditions and release miracidia. these miracidia infect freshwater snails and develop into sporocysts. these develop into cercariae and get released into the water, where they can penetrate the skin of the host. there, they shed their tail and become schistosomulae and migrate to the liver. in the liver they mature into adults. the paired adult worms migrate to the bowel and bladder, where they lay the eggs. a rash ("swimmers itch") may develop at the entry site on the skin. suprapubic pain and haematuria, abdominal pain, myalgia, fever, swelling of the lymph nodes, liver and spleen enlargement and eosinophilia can be additional symptoms. the risk of bladder cancer is increased with schistosomiasis. the diagnosis can be made in the stool and urine. the maximum excretion of eggs in the urine is between and pm. z trichuriasis (whipworm) whipworms have a worldwide distribution in the humid tropics. the eggs are orally absorbed via soil or unwashed vegetables or fruits. the whipworm grows in the large intestine. the eggs are excreted via the faeces. in the soil the eggs pass through various stages before getting absorbed again. the symptoms are abdominal pain, chronic diarrhoea, nausea, vomiting, inflammation of the intestine, anaemia and eosinophilia. the diagnosis is made with a stool sample. the treatment on the infection is dependent on the parasite (. table . ). leptospirae are long, motile spirochetes. they have a worldwide distribution, but infections occur more commonly in tropical and subtropical regions. they spread through infected urine, which enters water or soil. leptospires can survive for several weeks and months. infections can be caused by contact with either direct contact with the urine or other body fluids except saliva as well as with contaminated soil and water. the bacteria enter the body through the skin or mucous membranes. a broken skin increases the risk of infection. increased risk is after heavy rainfall or flooding. the incubation period is usually - days, but can range from - days. symptoms vary greatly. usually sudden onset of headaches, fever, chills, myalgia, nausea and vomiting, diarrhoea, rash and jaundice are common signs of the first phase for - days. if the patient doesn't recover the second phase (weil's disease) develops, with renal failure, ards, hepatomegaly, jaundice, haemorrhage and meningitis. this has a fatality rate of - %. untreated symptoms can persist for several months. treatment is either doxycyclin mg bd or benzylpenicillin . g qid or ceftriaxone g od for days. infections caused by rickettsia, orienta, ehrlichia, neorickettsia, neoehrlichia and anaplasma are summarised as rickettsial infections. rickettsias are divided into the typhus group and the spotted fever group. orienta make up the typhus group. the reservoir is found in mainly animals, like rodents, but some species are found in fish. the vector is commonly ticks. in scrub typhus the vectors are larval mites. others have fleas and lice as a vector. infection occurs either by bites of the vectors or by direct contact, inoculation or inhalation of contaminated fluids or faeces. the clinical presentation varies. mild symptoms are headache, myalgia, abdominal pain, cough and rash. some rickettsial infections, . q-fever is a zoonosis caused by the protozoa coxiella burnetii. the bacterium is quite resilient due to its sporelike life cycle and remains virulent for months even up to more than a year. the primary reservoir is cattle, goats, sheep and other wildlife like kangaroos, rats and cats. rarely is it transmitted by tick bites or by ingestion of unpasteurised milk or dairy products. the incubation time is usually - weeks but can range from days to weeks. the initial acute q-fever comes with sudden onset of high fever up to °c, headache (retrobulbar), myalgia, chills, non-productive cough and sweats. the symptoms settle within - days. % of all infections are however asymptomatic. often thrombocytopenia and abnormal lfts are found. complications are ards, endocarditis and meningoencephalitis. the diagnosis is based on detecting phase ii and phase i antibodies (igg) weeks apart. the initial test (phase ii) should be taken at the end of the first week of illness. igm and igg rise almost at the same time. a fourfold rise is diagnostic. an initial negative titre doesn't rule out q-fever. seroconversion occurs usually between days and but is almost always present by days. pcr testing can be used in the first weeks but before antibiotic administration. however, a negative pcr result doesn't rule out q-fever. chronic q-fever develops in . - %. it can result in endocarditis, aneurysms, osteomyelitis, hepatitis, neurogic (mononeuritis, optic neuritis), pulmonary (interstitial fibrosis, pseudotu-mor) and renal (glomerulonephritis) disease. chronic q-fever usually develops shortly after the infection. however, chronic endocarditis may not come apparent until - years or even longer. chronic fatigue syndrome is described in approx. %. typically in chronic q-fever, the initial igg titre is increasing (> : ). the treatment for acute q-fever is doxycyclin mg bd for days or for at least days after fever subsides and until clinical improvement. as serological confirmation takes time, treatment should not be delayed. early treatment is effective at preventing severe complications. for chronic q-fever, months of doxycyclin mg bd and hydroxychloroquine mg tds is recommended as standard treatment. rabies has an almost worldwide distribution. more than % of deaths occur in africa and asia. about % are children under years of age. dogs are the main vectors. in asia, there is also a risk of transmission through monkeys. in addition to other diseases, like the lyssavirus, bats or flying foxes can transfer rabies. it is transmitted by bites or scratch wounds but also by inoculation of saliva onto mucous membranes or eye of an infected animal. thorough cleaning of the wound and vaccination within hours can prevent the disease. the incubation period is usually - months but can be less than week and more than a year. initial symptoms include paraesthesia in the wound area. the disease can pass in two forms. the hyperactive form ( %) shows up with hyperactivity, manic behaviour, paranoia, hallucinations, delirium, hydrophobicity and occasionally aerophobia (triggered by the extremely painful spasms in the larynx area). the paralytic form ( %) is characterised by a slow but steady increasing paralysis. the paralysis begins in the area of the infection. the diagnostics can be established on the animal that has inflicted the wound. the tissue samples of the animal are taken from the brain (brainstem and cerebel-lum). the diagnosis in humans is difficult and unreliable. investigation of blood (antibodies), saliva (pcr), spinal fluid (antibodies) and skin biopsies (rabies antigen) are available. the vaccine and the immune globulin can be given during pregnancy. typical side effects of the vaccine are headache, myalgia, malaise, fatigue and nausea. treatment after potential infection (postexposure prophylaxis pep) includes: irrigation of the wound for a minimum of min and washing of the wound with water, soap, iodine or other disinfecting substances rabies vaccine rabies immunoglobulin into the wound area within days after the first vaccination following data should be recorded when a rabies vaccine is given overseas: address, email and telephone of the practice or hospital date of vaccinations batch number, name of the vaccine and manufacturer how many vaccinations are given application: subcutaneous or intramuscular injection who recommends the following approach with potential rabies after animal contact: vaccination against rabies is recommended for: travellers, who for more than month in areas, in which rabies is present professions that deal with bats or fruit bats professions, in which might get with rabies in contact (e.g., veterinary surgeon or nurse) laboratory workers who handle objects with rabies or lyssavirus after animal contact category + pre-exposure prophylaxis (prep) includes three vaccinations on day , and - . the dose is . ml intramuscularly or subcutaneously. the vaccination lasts for years. follow-up vaccinations (post-exposure prophylaxis = pep) include four vaccinations on day , , and . the dose is . ml intramuscularly. immunocompromised patients should receive five vaccinations with an additional vaccination on the th day. with previous vaccinations, two vaccinations are recommended on day and after exposure. it is not recommended to change the brand or the manufacturer during the course of vaccinations. however, it is possible, if that particular vaccine is not available. immunoglobulin should be administered with the first vaccination. the dose is iu/kgbw. the immunoglobulin preferably should be given in proximity of the wound. the immunoglobulin can be diluted, if the wounds is large, to enable to cover the entire wound area. the immunoglobulin is not recommended, if the first vaccination was given more than days ago, if prep or pep was completed or if an adequate serologic detection of vnab titres (≥ . iu/ml) is present. to avoid infection, no animals should be fed. bringing your own food or carrying items like handbags, water bottles, etc. should be avoided, if you stay in the range of monkeys. distance should be maintained to stray cats and dogs. the middle east respiratory syndrome (mers) is caused by a corona virus. corona viruses can cause mild flulike symptoms but also severe symptoms like the severe acute respiratory syndrome (sars). the mers-cov occurs . · mers mainly on the arabian peninsula (iran, jordan, kuwait, lebanon, oman, qatar, saudi arabia, united arab emirates and yemen). but through international travel, it can spread worldwide. recently it resulted in some cases in korea. mers has % mortality. the disease is transmitted through droplets or direct contact. the mers-cov also has a wide range of symptoms, from mild common cold symptoms and infections of the upper respiratory tract to a rapidly progressive pneumonitis, respiratory failure, septic shock and multi-organ failure. it seems the mers-cov has a low virulence, since the transmission occurs usually only through close contact by human to human, such as the care of a person suffering from mers. camels seem to be the original reservoir. mild forms with fever and mild respiratory symptoms, mers should be considered, if close contact with infected people existed prior to these symptoms. mers can be asymptomatic but also lead to respiratory failure and death. typical symptoms include fever, cough and shortness of breath. pneumonia or pneumonitis is often associated with mers. sometimes gastrointestinal symptoms such as diarrhoea and vomiting can occur. it has a high mortality of %. the treatment depends on the severity of the disease. caution in contact with camels in affected countries should be taken. eating insufficient heated camel meat and milk should be avoided. a suspicion of mers should be considered in individuals with the following risk profile: fever and pneumonia/pneumonitis and stay in endemic areas or contact with a symptomatic person from an endemic area within days before onset of symptoms fever and pneumonia/pneumonitis and hospitalisation in endemic areas or contact with camels and camel products in an endemic area within days before onset of symptoms fever and pneumonia/pneumonitis and contact with a mers diseased person within days before onset of symptoms cluster of patient (especially medical personnel) with severe respiratory symptoms with unclear aetiology tuberculosis is caused by an acid-resistant mycobacterium. m. tuberculosis is responsible for tuberculosis in more than %. it has global distribution but occurs more frequently in countries with low hygienic standards. tuberculosis spreads around the globe through international travel and immigration. it also shows a rising rate of resistances to conventional therapies. the time between the initial infection and tuberculin conversion takes approx. the diagnosis can be made with the tuberculin skin test (tst/mendel mantoux). days after the strictly intradermal injection of the substance, the induration at the injection site is measured. an induration of > mm may be suggestive of tuberculosis. it is considered a positive test if either the patient has a radiological proof, had close contact with someone with tuberculosis, and has symptoms of tuberculosis, is hiv positive or suffers from immunodeficiency. an induration > mm is considered as positive, when the patient who travelled to a country with high tb prevalence is an iv. drug user, homeless and a resident of nursing home or prison and has diabetes mellitus, silicosis, m. hodgkin's or end-stage renal failure. an induration > mm is considered as evidence of tuberculosis without any risk factors or symptoms. the tst can be negative in the first weeks after an infection as well as in patients suffering from miliary tuberculosis, m. hodgkin, sarcoidosis, viral infections, and lowered immunity, receiving an immunosuppressive therapy or at high age. a false-positive test can occur after multiple tsts, after vaccination against tuberculosis and infection of other mycobacteria. the interferon-γ test (quantiferon ® tb gold) offers an alternative testing method. this test has the same sensitivity as the tst but a higher specificity. moreover, this test is a confirmation test and isn't affected by previous bcg-immunisations. it consists of three parts, the control (to determine the baseline-interferon-γ), mitogen control (determining the ability of an immune response) and antigen detection (detection of prior infections). a cxr may demonstrate caverns or hilar lymph nodes, but is not a diagnostic tool to exclude tuberculosis. the treatment duration of uncomplicated tuberculosis is months, of complicated tuberculosis - months (. table . ). it's a combination treatment of different drugs. medications for the tuberculosis treatment are: isoniazid: mg/kgbw, max. mg /d; side effects: elevated serum transaminases, polyneuropathy, prophylaxis to avoid side effects of pyridoxine - mg/d a vaccination bcg vaccine is not recommended due to its side effects and the lack of efficacy. all vaccinations should be given days before travelling. minimum time for a sufficient protection is weeks (. divers alert network (dan) is a non-profit organisation for divers. they provide medical information and articles, diving insurance, life insurance and travel insurance. they also offer courses, support and research. dan has an international hotline for support and coordination of diving accidents but also for general medical advice overseas. european underwater and baromedical society (eubs) is a european organisation for diving and hyperbaric medicine. they provide guidelines for hyperbaric treatment and training of medical professionals for the hyperbaric medicine. the german organisation for diving and hyperbaric medicine is the "gesellschaft für tauch-und Überdruckmedizin" (getÜm). . . single dose certificate is valid for years, a new vaccination may be required after years to renew the certificate they provide guidelines for hyperbaric treatment and training of medical professionals for the hyperbaric medicine. brazil; office: tel: + - - - , emergency-hotline: + - - - . japan: japan marine recreation association, kowa-ota-machi bldg office: tel: + - - - , f ax southern africa: private bag x , halfway house, midrand eubs: webmaster@eubs.org gtuem: c/o bg-unfallklinik, professor-kuentscher-str. , d- murnau spums: st kilda road uhms: us highway , suite dan: www. diversalertnetwork. org dan europe: www. daneurope. org emedicine yellow fever chikungunya virus middle east respiratory syndrome (mers) parasites -african trypamosiasis (also known as sleeping sickness) parasites -american trypanosomiasis (also known as chagas disease) parasites -trichuriasis (also known as whipworm) air embolism of the brain in rabbits pretreated with mechlorethamine an examination of the critical released gas concept in decompression sickness accessed middle east respiratory syndrome (mers) middle east respiratory syndrome coronavirus (mers-cov) key: cord- -e phpqes authors: nan title: cis annual meeting: immune deficiency & dysregulation north american conference date: - - journal: j clin immunol doi: . /s - - -z sha: doc_id: cord_uid: e phpqes nan mutations in the genes encoding proteasome subunits (psmb , psmb , psma and psmb ) have been identified as the cause of candle syndrome. these mutations lead to malfunction of the proteasome, which results in buildup of cellular waste products. it is hypothesized that dysregulation in the interferon (ifn) signaling pathway in response to this waste is the driving mechanism of the inflammatory response, and may serve as a therapeutic target in these patients. objectives: to describe a case of suspected candle syndrome successfully treated with tofacitinib. methods: retrospective chart review was conducted with respect to diagnosis, treatment and response. results: a -month old caucasian male was admitted to the hospital for evaluation of profound anemia. his medical history was significant for extreme prematurity (born at weeks from premature labor), intraventricular hemorrhage grade iv that resulted in hydrocephalus needing ventriculoperitoneal shunting, and developmental delay. he was noted to have a hemoglobin of . g/dl during a neurosurgical evaluation for routine shunt revision. he developed hemodynamic decompensation and required hospital admission for packed red-blood cell transfusion. review of systems was remarkable for intermittent pruritic macular rash, daily temperature fluctuations (fever to hypothermia), joint pain/swelling/ stiffness of multiple sites, poor weight gain, irritability, irregular breathing, abdominal distention, and regression of gross motor milestones (no longer rolling over, sitting without support, or pulling up to stand). workup excluded infections and lymphoproliferative malignancies, and he met clinical criteria for systemic juvenile idiopathic arthritis. his initial laboratory studies showed systemic inflammation (wbc x ^ /ul, hgb . g/dl, platelets x ^ /uul, crp . mg/dl, sedimentation rate mm/h, ferritin ng/ml). imaging studies revealed serositis with right-sided pleural and pericardial effusions. he also had myositis supported by imaging and elevation of muscle enzymes (ast u/l, aldolase . u/l). the patient was started on pulse iv methylprednisolone mg/kg daily x days, followed by oral prednisolone mg/kg/day and anakinra mg/kg/day with partial improvement and he was discharged home. he was readmitted . weeks later due to concerns for macrophage activation syndrome (ferritin , ng/ml) in the setting of a gastrointestinal infection and anakinra was increased to . mg/kg/day. however, he continued to have persistently elevated inflammatory markers and so the dose was increased again to mg/kg/day. three months after initial presentation, he had an upper respiratory and ear infection and became ill with generalized rash, increased work of breathing, and poor perfusion. anakinra was considered a treatment failure at that time. he required several doses of pulse steroids and initiation of tocilizumab mg/kg iv every weeks with improvement on systemic symptoms. methotrexate mg/m² weekly was added soon after for persistent arthritis and inability to wean systemic steroids. he continued to have abnormal inflammatory indices, including ferritin ( , ng/ml) and il- levels ( , pg/ml, normal - ). proband only whole exome sequencing revealed a single heterozygous mutation in the psmb gene (c.- g>a), a published pathologic variant. based on this finding, the patient was started on tofacitinib . mg orally twice a day with a dramatic response. laboratory markers of inflammation normalized, and he was able to walk within the first month of treatment. further genetic testing to detect an additional proteasome subunit variant, as well as functional testing on a research basis to demonstrate an interferon signature are being pursued. conclusions: this case highlights the value of early genetic studies in patients with autoinflammation so that initiation of targeted therapy is not delayed in efforts to achieve control of symptoms and evade future complications. this case also illustrates the challenges in diagnosing monogenic autoinflammatory disorders in young patients that present with recurrent fevers, generalized rash, arthritis, and systemic inflammation that mimic systemic juvenile idiopathic arthritis. our experience contributes to the understanding of janus kinase inhibition in type i interferonopathies. of his recurrent infections and etiology of myasthenia gravis. results of the ct chest are notable for a thymoma. thymectomy with biopsy reveals benign pathology with a mixture of type a and b cells. he continues to have persistent fatigue, generalized weakness, diplopia, diarrhea and recurrent respiratory infections after thymectomy. immunoglobulin and lymphocyte subset panels reveal hypogammaglobulinemia with absent b cells. endoscopy reveals villous atrophy and blunting without evidence of celiac disease, inflammatory bowel disease or infection suggesting autoimmune enteropathy. the constellation of clinical and laboratory features are consistent with good syndrome with evans syndrome, seronegative myasthenia gravis and autoimmune enteropathy. the patient is started on immunoglobulin replacement therapy and pyridostigmine with resolution of recurrent infections and improvement of fatigue, generalized weakness and diplopia. three years later his fatigue and evans syndrome recur with new onset loss of appetite and a thirty pound weight loss. repeat immunologic labs were notable for elevated cd , borderline low cd and highly elevated cd cells with low absolute number and fraction naïve cd and cd cells suggesting worsening combined immunodeficiency with peripheral t cell expansion. a bone marrow biopsy reveals large granular lymphocytic (lgl) leukemia and he is started on methotrexate. serum antibodies targeting ifn, and il- are negative four years after removal of thymoma. conclusions: this case is consistent with a classic presentation of good syndrome represented by thymoma, t and b cell-mediated immunodeficiency, increased susceptibility to infections and autoimmune manifestations of evans syndrome, myasthenia gravis and autoimmune enteropathy. in this case the combination of evans syndrome, autoimmune enteropathy and lgl leukemia as malignancy further worsen prognosis and is typically not seen together in good syndrome. this case depicts well the crossroad of infection, autoimmunity and malignancy in late onset immunodeficiencies. introduction/background: dedicator of cytokinesis (dock ) deficiency is a known cause of autosomal recessive hyper-ige syndrome with a combined immunodeficiency. most of the mutations in dock are lossof-function homozygous or compound heterozygous point mutations or deletions. dock deficiency has been associated with low lymphocyte counts with impaired antibody responses, as well as eosinophilia, recurrent bacterial and cutaneous viral infections, malignancies, and severe atopy. we report the case of a year old man with history of hyper-ige syndrome, severe atopy, eosinophilia, and antibody deficiency, phenotypically atypical for dock , who was noted to have two variants of unknown significance in the dock gene. objectives: we report the case of a year old man with history of hyper-ige syndrome, severe atopy, eosinophilia, and antibody deficiency, phenotypically atypical for dock , who was noted to have two variants of unknown significance in the dock gene. methods: a year-old man presented to us for evaluation of known hyper-ige syndrome. he had a long history of elevated ige, peripheral eosinophilia, severe atopic dermatitis, food allergies, asthma, severe eczema since early childhood which failed to respond to methotrexate, mycophenolate, cyclosporine, and omalizmuab, but ultimately responded to intravenous immunoglobulin (ivig). his infectious history included mrsa skin infections and one episode of pneumonia, and he reported a history of fungal skin infections but the history was unclear. initial immune workup revealed eosinophilia of , ige level (as high as , ), igg level , igm level , and iga level . he had no random antibodies to streptococcus pneumonia serotypes, but he had protective antibodies to diphtheria and tetanus. lymphocyte subsets showed cd , cd , cd , cd . he had normal mitogen stimulation to pha but decreased mitogen stimulation to candida. dna testing for a stat mutation was negative. results: we found two missense variants of uncertain significance in the dock gene ( .p.v i, nm_ . :c. g>a and .p.l v,nm_ . :c. c>g ). the first variant had previously been reported in the clinvar database as a variant of uncertain significance, and the second variant had not been previously reported in the literature to our knowledge. our assay could not determine if the two dock variants were on the same allele or on different alleles. dock protein expression testing is currently pending. conclusions: our patient presented with history of elevated ige, eosinophilia, atopy, severe eczema, and cutaneous mrsa and fungal infections. he was noted to have variants of uncertain significance in the dock gene. homozygous or compound heterozygous pathogenic variants in dock are associated with an autosomal recessive hyper-ige syndrome and combined immunodeficiency with clinical features of recurrent bacterial infections, cutaneous viral infections, severe atopic disease, as well as susceptibility to malignancy. our patient does not have all the typical features of dock deficiency and he seems to have a less severe phenotype. notably, he does not have the cutaneous viral infections or malignancy often seen in dock mutation hyper-ige cases. our case demonstrates new missense mutations, which have not previously been described in the literature, possibly causing a milder phenotype of dock deficiency. a case of igm deficiency and adult-onset still's disease negative. previous biopsy of her cervical and thoracic lymphadenopathy was unremarkable for malignancy. during her hospitalization, serum immunoglobulins were performed, which demonstrated normal levels of igg and iga, with igm level of < mg/dl (reference range - mg/dl), consistent with selective igm deficiency. liver function tests revealed an elevated aspartate aminotransferase (ast) of u/l and an alanine aminotransferase (alt) of u/l with a total bilirubin of . mg/dl and an alkaline phosphatase of u/l. her ferritin was elevated at g/l. the patient fulfilled yamaguchi criteria for aosd with three major criteria of evanescent rash, intermittent fevers in a quotidian pattern, bilateral arthralgias in the hips, knees, and ankles. she also met two minor criteria of liver abnormalities and lymphadenopathy. conclusions: selective igm deficiency is an uncommon immunodeficiency disorder associated with increased risk for autoimmune disorders. the recognition of co-morbid autoimmune illnesses in an immunodeficient patient is often complicated by a paucity of examples in the literature and potential confounding of laboratory serology analysis. we report the first case of a patient with selective igm deficiency and aosd. introduction/background: anaphylaxis to protamine is an uncommon but life-threatening complication of cardiac surgery and insulin therapy. here we present a case of recurrent protamine hypersensitivity during vascular surgery. objectives . recognize clinical signs of protamine hypersensitivity . recognize recurrent hypersensitivity to protamine as a serious complication of anesthesia methods: a year old man with a history of diabetes, previously on nph insulin, hypertension, hyperlipidemia, chronic smoking, and peripheral artery disease with multiple vascular interventions was admitted to undergo a right lower extremity saphenous vein graft bypass. three years earlier during a similar intervention, the patient had developed intraoperative hypotension after protamine sulfate administration. protamine was subsequently held for additional surgeries, however the patient was able to tolerate protamine with slower infusion one year later. for the current vascular surgery, the patient was pretreated the day of surgery with diphenhydramine and dexamethasone, and a test dose of protamine was infused prior to full dosing. the patient initially appeared to tolerate the full protamine dose, but quickly developed facial erythema and angioedema. due to concern for laryngeal edema he remained intubated and was transferred to the surgical intensive care unit, where he received additional diphenhydramine and dexamethasone. his symptoms resolved and he was successfully extubated the next morning. results: anaphylaxis to protamine is an uncommon but lifethreatening complication of cardiac surgery and insulin therapy. protamine sulfate is a polypeptide used widely to neutralize heparin anticoagulation during cardiac and vascular surgeries, and in nph insulin. severe anaphylactic or anaphylactoid reactions caused by injection of protamine sulfate are well documented in literature, and the product contains a black box warning for such. the pathophysiologic mechanisms underlying these reactions are not clear, but ige-mediated hypersensitivity appears to play a role in many reactions, and prior sensitization or cross-sensitization (eg, to fish) have been suggested. type b adverse drug reactions are idiosyncratic drug reactions and are often unpredictable, as in our patient who previously tolerated protamine but subsequently developed an adverse reaction. hypersensitivity reactions during anesthesia should be thoroughly studied to identify the responsible drug and minimize exposure in recurrent surgeries. conclusions: this case illustrates the potential for severe reactions even with newer protamine formulations, and highlights the unpredictable nature of type b adverse drug reactions. it is important for clinicians to exhibit awareness of the potential adverse effects of protamine sulfate in such situations. introduction/background: x-linked lymphoproliferative syndrome type (xlp- ) is a rare primary immune deficiency caused by loss of function in the x-linked inhibitor of apoptosis protein (xiap). common reported manifestations include recurrent hemophagocytic lymphohistiocytosis, splenomegaly, crohns-like inflammatory bowel disease, and transient hypogammaglobulinemia without reductions in major t cell or b cell repertoires, with the exception of inkt cells and mait cells. however, with only~ known cases worldwide, we are likely only beginning to understand the phenotypic spectrum of this disease. objectives: to describe additional manifestations of xlp- that expand our current understanding of its phenotype. methods: a year-old male with adult-onset, treatment refractory ulcerative colitis was evaluated in the immunology clinic for a history of recurrent sinopulmonary infections, skin abscesses, and recurrent ebv and vzv infections. extensive laboratory testing was performed in the course of his evaluation, including lymphocyte immunophenotyping, lymphocyte proliferation and cytotoxicity studies, quantification of total immunoglobulin levels and specific antibody function, hiv testing, and genetic testing. results: laboratory testing was significant for persistent cd lymphocytopenia ranging from - cells/mcl (rr: cells/mcl). total b cell count was normal but b cell subsets showed an elevation in the percentage of naïve b cells (range: . . %), low non-switched memory b cells (range: . - . %, rr: . - . %), and low to low-normal switched memory b cells (range: . %- . %, rr: . - . %), a pattern that has been seen in some autoimmune diseases. genetic testing with a commercial immune deficiency panel (invitae corp) showed a pathogenic mutation in xiap [exon , c. c>t (p.arg *)]. this mutation has previously been reported to cause a premature stop codon and reduced xiap function. the patient was referred for hematopoietic stem cell transplant and is currently awaiting transplant with a matched unrelated donor. conclusions: xlp- is typically reported as having normal t cell, b cell, and nk cell counts, but the presence of persistent cd lymphocytopenia in this patient illustrates that this is not always the case. our patient also had abnormalities in his b cell repertoire that have not been previously reported in xlp- . additionally, xlp- has been associated with crohns disease and celiac-like bowel diseases, while our case indicates that the phenotype may also include ulcerative colitis. ( ) submission id# a case report: enteroviral encephalitis as a consequence of partial humoral immunodeficiency in a chronic lymphocytic leukaemia patient treated with rituximab hadeil morsi, st immunology st , oxford university hospitals introduction/background: enteroviral (ev) infections are prevalent and usually self limited or cause mild gastrointestinal manifestations. however , in the context of primary antibody deficiency , rare cases has been reported to develop meningoencephalitis and been linked to poor outcome with fatality or chronic course. ev meningoencephalitis is even far rare reported in the era of rising secondary humoral immunodeficiency as a consequence of b cell depleting therapy e.g. rituximab and lymphoproliferative malignancies. limited treatments for ev encephalitis are available to date, apart from intravenous immunoglobulin replacement which has variable efficiency. objectives: studying such rare cases of ev meningoencephalitis as a consequence of antibody deficiencies would help to develop guidelines for intravenous immunogobulin replacement for treating these infections to improve outcome as well as predicting patients at higher risk who should be considered for prophylactic immunoglobulin therapy. methods: herein, we report a rare case of proven enteroviral meningoencephalitis following rituximab based therapy for b-cell chronic lymphocytic leukaemia and an uneventful six months period of follow up. he was found to have persistent absent b cells six months after completing six cycles of fludarabine, cyclophosphamide and rituximab therapy. interestingly, he had partial pneumococcal igg serotypes deficiency, whilst his total igg, igm and iga were all within normal limits throughout the course of the disease. the patient was treated empirically with intravenous immunoglobulin when his subtle confusion progressed to overt behavioural changes. initially his level of consciousness continued to deteriorate and he was not communicating. results: fortunately enough, the patient did have a remarkable improvement of gcs within couple of days and a slower recovery of higher mental functions e.g. memory and calculations in the next couple of months. conclusions: early suspicion and detection of entervorial meningoencephalitis in patients at risk of secondary antibody deficiency is crucial for timely ivig replacement and better outcome. patients with haematological malignancies and those on b cell depleting immunotherapy should be screened for pneumococal igg serotypes as part of secondary immunodeficiency workup. further studies on enteroviral neurological meningo/encephalitis are required to optimise ivig therapy and prognostication. furthermore, such studies provide an important asset to reveal the underlying mechanisms for humoral/b-cell mediated protective response against ev compared to other t-cell mediated viral immunity, whilst highlighting the mechanisms of immunodeficiency in cll and immunotherapy. ( ) submission id# a comparison of immune reconstitution following human placenta-derived stem cells (hpdsc) with umbilical cord blood transplantation (ucbt) vs. ucbt alone in pediatric recipients with malignant and non-malignant diseases introduction/background: ucbt is a safe and effective treatment in children (geyer/cairo et. al bjh, ) . however, due to a limited concentration of hematopoietic progenitor cells (cd +) in ucb, ucbt has been associated with delayed hematopoietic reconstitution and a higher incidence of engraftment failure. hpdscs contain a rich population of hpcs, are low in hla class i/ii expression and t-cells, and have regenerative, anti-inflammatory, and immunosuppressive properties (cairo et al bmt, ) . objectives: to determine whether ucbt + hpdsc (vs. ucbt alone) is associated with enhanced hematopoietic and immune cell reconstitution in children with malignant and non-malignant diseases. methods: immune cell reconstitution at days + , , and was assessed in children who received ucbt with hpdscs at nymc (nct , ind# ). minimum tnc was x ^ /kg ( / hla match) or . x ^ /kg ( - / hla match). immune cell subset counts at these time points were compared to those from a historical population of pediatric recipients of ucbt alone (geyer/cairo et. al bjh, ) . results: twenty four patients years were enrolled. mean age was (range, . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] years. malignant diseases = , non-malignant diseases = . fourteen patients received myeloablative conditioning (mac) and ten patients received reduced toxicity conditioning (rtc). there were no severe adverse events associated with hpdsc infusion. two patients with non-malignant disease receiving rtc using alemtuzumab experienced primary graft failure. probability of neutrophil engraftment was . %, median day . of evaluable patients at day , the probability of platelet engraftment in neutrophil engrafted patients was %, median day . ( - ) . at days , , and , mean percent donor chimerism in whole blood was , , , and %, respectively. average percent of whole blood hpdsc chimerism was % at day and < % at beyond day . one patient with malignant disease relapsed. month overall survival was . %. there was no significant difference in cd , cd , cd , cd and cd immune cell reconstitution following ucbt + hpdsc vs. ucbt alone (image ). conclusions: these results suggest that ucbt ± hpdsc results in similar immune cell reconstitution. a larger cohort with extended follow-up would be required to confirm these preliminary findings. supported by a grant from celgene cellular therapeutics. a decade of disseminated abscesses due to mycoplasma faucium in a patient with activated pi k syndrome (apds ) introduction/background: pik r monoallelic mutations are known to be responsible for apds-like syndrome, a rare form of primary immunodeficiency presenting as combined immunodeficiency or hyper-igm like phenotype. this study reports a patient carrying heterozygous pik r mutation with early onset and long-term disseminated abscesses due to mycoplasma faucium in both peritoneal abscess and skin, with generalized involvement in neck and both upper extremities. objectives: we describe clinical management of retroperitoneal and skin abscesses before molecular diagnosis was available in a patient with a primary immunodeficiency. identification by rdna s in so-called sterile abscesses may confirm the clinical suspect of an oportunistic infection. furthermore, this study offers insight on the pik r suspicion even in the absence of higm-like phenotype. methods: a -year-old girl with -year history of recurrent peritoneal effusion, which had been drained repeatedly was admitted in our institution for a -year history of multiple supurative cutaneous and lymph node-abscesses (fig & a-c). she had prior diagnosis of agammaglobulinemia under standard subcutaneous immunoglobulin replacement therapy and subcutaneous interferon-gamma treatment. on physical examination at the age of years, she was stunted (weight and height below the rd percentile), with facial, arm skin abscesses and right fistulized axillary lymphadenopaties, - cm hepatomegaly and giant splenomegaly results: she had her first immunological work up at the age of years during one isolated episode of knee arthritis and first episode of skin abscesses. serum immunoglobulins revealed panhypogammaglobulinemia (igg< . mg/kg, iga < . mg/kg, igm < . mg/kg) with low b cell count. on her back, there was a x cm, elastic, neither painful nor tender mass. after proper assessment by ct scan and mri she had her retroperitoneal abscess drained percutaneously, and healed with sclerotherapy (percutaneous alcohol and polidocanol instilation) by the interventional radiologist ( fig b) . analysis of drained pus as well as pus of skin abscesses was made by s rdna pcr, having coincidence of . % with mycoplasma faucium . combination antibiotic therapy (doxycycline and ciprofloxacin) was started with favourable response. unfortunately skin abscesses then relapsed. t-cell phenotype only showed t-cell lymphopenia with senescent (tem & temra expansion) phenotype. whole exome sequencing revealed a heterozygous mutation, previously reported (c. + g>t) conclusions: in summary, this report emphasizes the suspicion of a combined immunodeficiency in the presence of multiple abscesses by mycoplasma, the usefulness of rdna s in order to achieve proper objectives: we describe a -year-old male patient with novel heterozygous mutation of ep gene; his first manifestations were initially characterized by infections, cytopenia and hypogammaglobulinemia suggesting a common variable immunodeficiency (cvid), but later on, persisting lymphopenia was suggestive of a combined immunodeficiency. methods: the patient was born to unrelated healthy italian parents at weeks gestation with adequate weight for gestational age. shortly after birth, he underwent several surgical procedures due to interventricular defect, aortic coarctation, double outlet right ventricle, open botallis duct, and gastroesophageal reflux. at the age of four, he came to our attention due to stomatitis. clinical examination revealed dysmorphisms (microcephaly, wide forehead, sparse eyebrows, high nasal root, low-hanging columella, thick lips, micrognathia), splenomegaly (spleen diameter . cm at abdominal ultrasound), and severe developmental delay. in the course of the infectious episode, blood tests showed leukopenia associated with neutropenia (white blood cells . /mm ; neutrophils / mm ) and thrombocytopenia (platelets . /mm ). analysis of bone marrow aspirate revealed normal differentiation of both myeloid and erythroid lineages. treatment with high doses immunoglobulin resulted in increase of platelet counts (up to /mm after month), while neutrophil counts spontaneously returned to normal when the infection resolved. however, thrombocytopenia relapsed ( /mm ) after months and intravenous high-doses of corticosteroids did not achieve normal platelets count. despite oral corticosteroid treatment started at the age of six, two episodes of autoimmune hemolytic anemia occurred. during the following six years of follow-up, the patient experienced recurrent infections (stomatitis, upper respiratory tract infections, and skin abscesses), but none of the episodes has required hospitalization. but, at the age of ten, he was admitted to the hospital because of severe cultures negative diarrhea. despite immunoglobulin replacement therapy was started at the age of fourteen, he was admitted twice due to bilateral pneumonia requiring continuous positive airway pressure and, a few months later, acute respiratory failure with evidence of mycoplasma pneumoniae and rhinovirus infections. immunological evaluation under chronic corticosteroid treatment at different time points showed persisting lymphopenia, with lymphocyte counts ranging from /mmc to /mm , thrombocytopenia (platelets ranging from /mm to /mm ), undetectable anti-diphteria and anti-tetanus toxoid antibodies, and splenomegaly. interestingly, analysis of isohemoagglutinins, revealed low titers of anti-a ( : ) at years of age, but normal immunoglobulins (igg mg/dl, iga mg/dl, and igm mg/dl). at the age of seven, reduced mitogen proliferation, hypogammaglobulinemia (igg mg/dl; iga mg/dl, igm mg/dl), increased cd +tcr+cd cd t-cell counts ( . / . %) and impaired fas mediated apoptosis as measured in two separate assays (table ) . at the age of fourteen, evaluation of b-cell subsets showed increase of cd locd lo cells and reduction of switched memory b-cells. analysis of t-cell compartment unveiled a decreased proportion of cd +ccr +cd ra+ recent thymic emigrants (rte) cells and ccr +cd ra+ naive cells, with prevalence of effector memory t-cells (ccr -cd ra-) ( table ) . interferon signature gene expression showed borderline levels of ifi (data not shown). because of the decrease of igg and the infectious episodes ivig treatment was started at age of fourteen. a molecular investigation performed by whole exome sequencing (wes) revealed a novel heterozygous missense mutation (nm_ . :c. t>c , p.met thr) in the exon of the gene ep encoding the histone acetyltransferase (hat) protein p . results: few immunological reports are available in rsts patients - . in keeping with previous data , , , our patient presented with progressive b-and t-cell lymphopenia, hypogammaglobulinemia with poor antibody response but also reduced naïve t cells, evans syndrome, splenomegaly, and defective lymphocyte apoptosis with increased dnt. at the age of seven, the patient presented the features of cvid. flow cytometry revealed expansion of cd hicd locd lo b cells, that is frequently associated with splenomegaly in cvid patients , and reduced switched memory b-cell, previously reported in a rsts patient with crebbp mutation (fig. s ). lougaris et al. reported expansion of cd locd lo b-cells in nf-kb haploinsufficiency , and this suggests in our opinion that alterations in the nf-kb pathway due to ep mutations may affect b-cell differentiation. compared to healthy controls and cvid patients with predominant infectious complications, upregulation of interferon responsive genes in cvid subgroup with noninfectious complications (i.e hematologic autoimmunity, lymphoproliferation) and lymphopenia with reduced total b cells and switched-memory b cells has been demonstrated . borderline levels of ifi expression under corticosteroid treatment represent a novel finding in rsts. interferon signature may identify and better characterize subgroup of rsts patients with autoimmune cytopenias and lymphopenia. at the age of fourteen, analysis of lymphocyte subsets revealed decreased total cd +, cd +, cd +, and of both naïve cd + and cd + cells. elevated and persisting igm levels were also observed (fig. s ). according to our data, increased igm levels may be related to high proportion of terminal differentiated igm+ cells. these data (infections requiring hospitalization, immune dysregulation, lymphopenia, reduced naïve t cells, and reduced proliferation to mitogen) together with clinical history (fisher evans syndrome and lymphoproliferation) and exclusion of known syndromic immunodeficiencies, suggested a diagnosis of combined immunodeficiency . conclusions: our case underlines the value of wes in patients with difficult phenotype-genotype correlation. no rsts typical traits were present and, prior to wes, several syndromes and immunodeficiencies were excluded. our report expands the phenotypic spectrum of ep mutations, thus in syndromic patients with clinical and immunological overlap between cvid and cid ruling out ep mutation should be advisable. furthermore, immunological work-up should be taken into consideration in rsts patients, in order to early identify immunological abnormalities that may lead to severe immune-hematological complications. introduction/background: interferon gamma receptor (ifngr )-related disorders are rare variants of mendelian susceptibility to mycobacterial diseases. although hematopoietic stem cell transplantation (hsct) is curative, it is complicated by high rates of delayed or failed engraftment thought to be due to high concentrations of interferon (ifn)-gamma. umbilical cord blood transplantation additionally increases risk of graft failure. objectives: describe a pediatric patient with non-functional ifngr who successfully underwent umbilical cord blood transplantation. methods: direct clinical care of described patient with additional electronic medical record chart review. results: the patient is a -month-old boy of yemeni descent who initially presented with significant hepatosplenomegaly and extensive lymphadenopathy, including a large mediastinal mass. he then developed salmonella enteritidis sepsis requiring numerous antimicrobials, vasopressor support, intubation and continuous renal replacement therapy. his evaluation showed a hyperinflammatory state with elevations in ferritin, ifn-gamma, scd , il- , il- , il- , il- and il- levels. maximal ferritin and ifn-gamma levels reached . ng/ml and pg/ml (normal < pg/ml), respectively. flow cytometry revealed normal expression of ifngr and il r but absent ifn-gammastimulated stat phosphorylation, suggesting defective ifngr signaling. genetic testing showed a previously unreported homozygous mutation in ifngr (c. + t>c) which affects a donor splice site in intron and is predicted to cause absent protein function. dexamethasone and a single dose of alemtuzumab ( . mg/kg) were given to decrease inflammation. he then underwent allogeneic hsct using a / human leukocyte antigen matched umbilical cord unit following a reduced-toxicity conditioning regimen of alemtuzumab ( . mg/kg), fludarabine ( mg/m ) and busulfan (auc mg/l*h). plasma ifn-gamma was undetectable prior to starting conditioning and on the day of transplant. neutrophil engraftment occurred on day + with day + posttransplant chimerism analysis of peripheral blood myeloid cells showing the presence of donor cells only. conclusions: these early results suggest that umbilical cord blood transplantation may be feasible in patients with ifngr -related disorders provided adequate control of inflammation is gained prior to transplant. introduction/background: introduction: btk is a cytoplasmic tyrosine kinase that activates phospholipase c (plc ) via phosphorylation, which ultimately leads to the activation of nfk, which is essential for b cell development and survival. mutations in btk lead to x-linked agammaglobulinemia (xla). in addition to the pleckstrin homology and tyrosine kinase domains, btk contains two src homology domains, sh and sh , which are essential for btk function. we describe a novel btk mutation (c. a>t) resulting in v a substitution in the sh domain that results in aberrant xla function with nearly normal btk protein expression. objectives: case report/results: the male proband presented with recurrent otitis media, persistent fevers and neutropenia beginning in the first year of life with an igg level of mg/dl and a lack of b cells ( cell/mm ), as demonstrated by flow cytometry. btk protein expression in monocytes, also determined by flow cytometry, was equivalent to controls. family history is significant for a maternal uncle with history of recurrent sinus infections and pneumonias with low iga and igm, low to normal ige, and an absent vaccine response. flow cytometry also showed an absence of b cells and essentially normal btk protein expression in his monocytes compared to controls. targeted high throughput sequencing of both proband and the uncle revealed a previously unreported missense mutation in exon , leading to the substitution of an aspartic acid residue for a valine (v d.) the mutation is in the highly conserved sh domain of btk (conservation phylop conservation score . .) the probands mother and his sister were shown to be carriers of the same mutation and had normal serum immunoglobulin levels and normal numbers of b cells. methods: we hypothesized that if the btk v d mutant protein was non-functional, the female carriers of the mutation would only express wild type (wt) btk in their b cells while their monocytes would express both wt and mutant btk. to test this hypothesis cd + b cells and cd + monocytes were purified from pbmc by fluorescence activated cell sorting (facs) from the sister, cdnas were generated from the respective populations of cells, and the btk cdna was sequenced using high-throughput sequencing. results: at a read-depth greater than , , cd + b cells demonstrated btk expression only from the wt allele (~ % wt btk) whereas both the wild type and mutant allele of btk were expressed at approximately equal levels in monocytes. conclusions: discussion: these results define a novel mutation in btk that nominally affects protein expression, but alters function. the v a substitution is found in the d structural element of the sh domain that is part of a hydrophobic phosphotyrosine binding pocket. a mutation in the adjacent residue, y s, has been shown to alter protein conformation and decrease binding affinity to plc by roughly -fold resulting in xla. our study, using a carrier harboring the c. a>t mutant btk, demonstrated that in contrast to mononuclear cells, b cells only expressed the wt allele. this is consistent with the loss of function of v a btk protein, thereby causing xla in both the proband and affected uncle. introduction/background: pyoderma gangrenosum (pg) is often associated with systemic autoimmune diseases but it has rarely been reported with common variable immune deficiency (cvid). while genetic analysis has been increasing in both disease domains, there has been little investigation into the genetic components associated with the cooccurrence of these entities. heterogeneous nfkb mutations have recently been identified in familial cases of cvid, though rarely have they been associated with pg. objectives: this case describes a novel nfkb mutation that may link both cvid and pg, and bolsters the recent identification of heterogeneous nfkb mutations in cvid. methods: a -year-old woman with a history of frequent skin infections in childhood presented with persistent, infected wounds following cholecystectomy. upon admission, she was started on broad spectrum antibiotics but continued to have fevers and leukocytosis. labs were also notable for elevated crp ( ; normal (n): . - ) and esr ( ; , with low c ( ; n: - ), c (< ; n: - ) , and ch (< ; n:> ). wound cultures grew multi-drug resistant coagulase negative staphylococcus. despite broad spectrum antibiotics, the wounds failed to heal. dermatology was consulted and punch biopsy revealed a dense neutrophilic infiltrate and no identifiable pathogens, which supports the diagnosis of pg. the patient was started on high dose steroids ( mg/kg/ day) and had a rapid response with decreased skin inflammation and lesion expansion. unfortunately, the patient developed posterior reversible encephalopathy syndrome (pres) on steroids and therefore pg treatment was changed to infliximab as recommended by dermatology. further laboratory testing found that the patient also had low igg ( ; n: - ) and iga ( ; , and a diagnosis of cvid was made given her clinical history of recurrent skin infections. genetic testing was pursued to evaluate additional pg therapy options and this revealed a heterozygous mutation in nfkb (c.a g; p.q q), located base pairs upstream of the splice donor site for exon twenty-one. while this mutation has not been previously identified as a pathogenic variant, similar mutations in this gene have been linked to autosomal dominant cvid. the patients father also carried a similar mutation but without any evident clinical phenotype. results: nfkb plays a crucial role in both immune and inflammatory responses. this case highlights a novel mutation in nfkb that has not been previously described as a disease-causing change. other mutations resulting in nfkb haploinsufficiency have been associated with cvid and rarely with concurrent pg, as in this case. based on the location of the mutation, it is expected that the variant causes obliteration of the normal splice site and therefore results in defective mrna that encodes p / p . interestingly, studies on p knockout mice show decreased levels of igg, iga, and ige but not igm and our patient similarly had low levels of igg and iga but normal igm. conclusions: further studies are needed to determine if there are other links to this novel nfkb mutation in patients with cvid and pg. ( ) submission id# a rapid flow cytometric analysis of dna repair proteins reveals a radiosensitive phenotype in bcl b deficiency associated with severe combined immunodeficiency (scid). introduction/background: we present the second report in the literature of a patient with immunodeficiency, dysmorphic features, growth retardation, and a homozygous variant in the dna ligase i (lig ) gene with associated absence of full-length lig protein. results: this is a now year old girl who was the fourth child of parents who are first cousins. she has one healthy older brother, a second older brother who died within hours of birth of meconium aspiration, and an older sister who died at six months of age of an upper respiratory illness / pneumonia after a history of congenital anemia, poor weight gain, cardiomegaly and hepatomegaly. she was born at weeks and spent the first five weeks of life in a neighboring hospital neonatal intensive care unit (nicu) for hepatomegaly, mild cardiomegaly (previously identified on fetal ultrasound) and congenital anemia (requiring transfusions). her exam was and remains notable for weight, height, and head circumference below rd percentile, prominent forehead, hypotelorism with epicanthal folds, downslanting palpebral fissures, and low set, posteriorly rotated and prominent ears. after discharge to home from the nicu, her course was subsequently complicated by poor weight gain, chronic diarrhea beginning after her first rotavirus vaccine, and multiple deep vein thromboses. she then became critically ill at months of age with respiratory failure, and was transferred to our institution for respiratory oscillator support. absolute lymphocyte count on admission to our institution was . k/μl (total wbc . k/μl, anc . k/μl) with agammaglobulinemia. she was diagnosed with and treated for pneumocystis jirovecii pneumonia, gradually weaned from oscillator to room air, and was discharged home five weeks later on . mg/kg every other week igg replacement. she has had no serious infections requiring hospitalization in the months since. alc has remained persistently below . k/μl with a corresponding uniform deficiency of t, b, and nk cells and no detectable trec positive t-cells. whole exome sequencing identified homozygosity for a c. g>a coding region variant in the lig gene not previously reported in the literature. a fibroblast cell line was successfully established and western blot shows an absence of full-length lig protein. further molecular characterization is in progress. conclusions: this second reported case provides further evidence for dna ligase i deficiency as a distinct clinical entity comprising immunodeficiency, dysmorphic features, and growth retardation. introduction/background: chronic mucocutaneous candidiasis (cmc) is associated with a heterogeneous group of primary immunodeficiencies. autosomal dominant stat gain-of-function (gof) mutations have been identified in up to % of patients with cmc. these mutations lead to impaired il a/f t cell immunity although the underlying mechanism is unclear. there seems to be no genotype-phenotype correlation. recently, jak inhibitor therapy has been reported to improve cmc and autoimmunity in patients with stat gof mutation. objectives: we describe an infant with cmc associated with a novel stat gof mutation. results: a -month-old girl was referred to our immunodeficiency clinic with chronic diaper rash since weeks of life, failure-to-thrive, and history of labial abscess complicated by rectolabial fistula. she was subsequently diagnosed with food protein-induced enterocolitis syndrome triggered by cows milk-based formula. laboratory evaluation revealed normal cbc with differential, lymphocyte subsets, mitogen response, immunoglobulin levels, antigen response to candida, and neutrophil oxidative burst assay. whole exome sequencing identified a de novo heterozygous variant in stat (c. t >c, p.cys arg) . further evaluation of this mutation revealed increased gas (gamma activation sequence) reporter activity in response to ifng stimulation suggesting that this is a gain-of-function mutation. the patient later developed significantly elevated liver enzymes while on fluconazole treatment, candida parapsilosis sepsis, granulomatous lesions in the liver, splenic lesions, intermittent thrombocytopenia and n o r m o c y t i c a n e m i a . s e p s i s a n d l i v e r l e s i o n s r e s o l v e d on amphotericin treatment but other findings, including tpn dependency persisted. we are planning to initiate a jak inhibitor therapy, ruxolitinib. conclusions: this case is a possible genotypic and phenotypic expansion of cmc due to stat gof. professor, the university of british columbia introduction/background: b cell cll/lymphoma b (bcl b) is a zinc finger protein transcription factor with a multitude of regulatory functions in the integumentary, central nervous, cardiac, and immune systems. it is critical for t cell lineage commitment, development, differentiation, survival, and function. in addition, it also specifies the identity and function of innate-like lymphocytes, including t cells, innate lymphoid cells (ilcs), and invariant natural killer t cells (inkt). however, little is known about its function in the human immune system, especially in the context of immune disorders. objectives: to understand the immunopathogenesis of a novel p.c y bcl b variant. methods: research study protocols were approved by our institutional research ethics board. two members of the family were enrolled (the index patient and her father). written informed consent for genetic testing and participation was provided by the parents for the child. genetic, bioinformatic, proteomic, and biochemical analyses were performed. results: we have identified the second described case of immune disease caused by a de novo heterozygous damaging variant of bcl b (p.c y). this young girl presented with intellectual disability, microcephaly, severe atopy, eczema, alopecia totalis, and brittle nails. extensive clinical immunophenotyping of patient blood showed initially unremarkable b and t cell populations. however, the patient possessed abnormal rare innate-like lymphocyte populations (inkt, dn t cells). using mass cytometry (cytof), a technique capable of concurrently analyzing parameters in a single cell, we were able to examine various innatelike lymphocyte populations, including t cells, ilc - , and nk cells. we found that the patient possessed severely compromised numbers of t cells, thus potentially implicating the p.c y variant in t cell development and function. conclusions: the identification of decreased t cells in a patient with a p.c y variant of bcl b suggests that bcl b is important for human t cell development and provides novel insights into the roles of both bcl b and t cells in regulating atopy and autoimmunity. introduction/background: adenosine deaminase deficiency caused by mutations in ada gene is a newly recognized disorder. it is associated with a spectrum of vascular and inflammatory phenotypes, ranging from early onset recurrent stroke to systemic vasculopathy or vasculitis. objectives: we describe a year old female patient with features of early onset immune thrombocytopenia (itp), autoimmune hemolytic anemia (aiha), chronic splenomegaly and variable abdominal lymphadenopathy. she was diagnosed with evans-syndrome and treated with rituximab at and month of age. from years of age she developed recurrent infections, hypogammaglobulinaemia with specific antibody deficiency, progressively decreasing class-switched memory b cells, and increased cd +cd -cd -//t cells ( %). differential diagnosis included common variable immunodeficiency (cvid) or autoimmune lymphoproliferative syndrome and therefore a broad search for causative genetic defect was initiated. the parents are first cousins of middle-eastern origin suggesting an autosomal recessive inheritance. patient was stable on long-term mycophenolate mofetil (mmf) and immunomodulatory dose ( g/kg/ month) ivig treatment. methods: genomic dna of the patient was sequenced with next generation sequencing technology. a panel of genes linked to primary immunodeficiency was analyzed. the identified variant was confirmed by sanger sequencing. results: genetic testing revealed a homozygous pathogenic mutation in the ada gene with one base pair duplication in exon (c. dup. p.arg alafs* ) that creates a frame shift starting at codon arg . the new reading frame ends at a stop codon positions downstream, likely resulting in a truncated protein. plasma ada activity of the patient was markedly reduced ( . mu/ml, normal . - . ) and confirmed the diagnosis of ada deficiency. the parents of the patient are heterozygous carriers of the same mutation. unlike most previously reported cases, this patient had an extended phenotype with no neurological evidence of vascular pathology, however brain mri revealed two silent lacunar infarct or vasculitis related changes. we speculate whether the long-term mmf or ivig therapy might be protective against vasculitis. conclusions: ada deficiency may present with a wide spectrum of clinical phenotypes beyond classical vasculopathy. the diagnosis should be considered in patients with hematological autoimmune disease, splenomegaly and/or cvid like presentation. better understanding of pathophysiology of ada deficiency may help diagnosis and targeted treatment. professor, university of california, los angeles ca introduction/background: adenosine deaminase (ada) deficiency as a cause of severe combined immunodeficiency (scid) is distinct from other forms of scid in several ways. historically, survival and clinical outcome of infants with ada scid have been inferior compared to infants with other scid genotypes. there are multiple treatment modalities available for ada-scid, including enzyme replacement therapy (ert), allogeneic hematopoietic cell transplant (hct) and experimental autologous transplant of gene corrected cells (in recent years preceded by low dose busulfan), designated as gene therapy (gt). in addition, there is a growing body of evidence for effects of ada deficiency on non-immunologic organ systems that may contribute to the historically poorer outcomes of these infants. therefore, it is important to evaluate the cohort of patients with ada scid separately from other scid cases. objectives: to capture incidence and treatment trends and to compare outcomes following available treatments for this rare inborn error of metabolism and other forms of scid, the primary immune deficiency treatment consortium (pidtc), a network of north american immunology and transplant centers, has collected standardized data for analysis. methods: ada scid patients, first treated between through , were enrolled from centers (range - subjects/site). ada accounted for % of the total pidtc scid patients treated during that time. patients were entered into either a retrospective protocol (pidtc , n= ) or a prospective protocol starting in (pidtc , n= ) . ada-scid patients who received an hct as first therapy entered either of two strata, as with other scid patients in pidtc studies, based on whether their initial presentation met definitions for typical (n= ) or leaky scid (n= ); in contrast, patients initially treated with either ert or gt were entered into a separate stratum (n= ). results: sixty-four patients ( % of all ada-scid enrollees) had ert as first therapy, but only in this cohort received ert as sole therapy; went on to have subsequent hct (n= ) or gt (n= ). there were various combinations of treatment cycles among these ada-scid patients. most received hct {+/-subsequent treatments} ( %), ert followed by hct {+/-subsequent treatments} ( %), or ert followed by gt {+/-subsequent treatment} ( %); several patients received multiple successive treatment modalities, representing either failure of initial treatment or planned progression from ert to cellular therapy. two-year survival has improved over time from % in ( - to % - (p= . ) (figure ). the survival for all other non-ada scid patients registered by pidtc over these two eras were: % ( - ) and % ( - ) . hct (either as sole therapy or after ert) accounted for > % of cellular therapies between and ; in contrast, since , gt was used as commonly as hct (n= vs. n= , respectively). there was a trend toward better two-year survival for patients receiving gt as first cellular therapy since ( %, n= , all after initial ert) compared to those receiving hct over the same time period ( %, n= , either as first therapy or after ert), although this did not achieve statistical significance (p= . ). conclusions: this study reveals the improved prognosis for patients with ada scid in recent years and the emergence of gt as a new treatment modality. further analyses are investigating the impacts of prior infection and treatment modality, including effects of conditioning, on outcomes (survival, event free survival, clinical outcomes and completeness of immune reconstitution) for ada-scid in successive eras. this study may identify optimal treatment approaches for future ada scid patients. sponsored by the pidtc, a member of the rare diseases clinical research network (rdcrn) and funded by u ai (niaid and ordr, ncats, nih). dbk has potential financial conflict of interest as a member of the scientific advisory board for orchard therapeutics and an inventor on intellectual property which ucla has licensed to orchard therapeutics related to gene therapy for ada scid. jp discloses that her spouse is employed at invitae, a dna sequencing company. intern, imam abdulrahman bin faisal university introduction/background: patients with diabetes mellitus are immunologically vulnerable population to develop different types of microbial infections. immunization has an important role in infection prophylaxis. in fact, vaccines containing thymus-dependent antigens protect patients with diabetes as they produce massive and complex immune response and feature immunologic memory. the recommended vaccinations for patients with diabetes mellitus are influenza vaccination yearly and pneumococcal vaccination. in observational studies, influenza vaccine has been shown to be similarly effective in adults < years of age with diabetes as in older patients with or without diabetes [ ] . among immunocompetent elderly, vaccine efficacy of the -valent pneumococcal conjugate-vaccine (pcv ) was modified by dm with higher vaccine efficacy among subjects with dm [ ] . the hepatitis b vaccination should be given to unvaccinated adults with diabetes mellitus who are ages to years. for older patients administration only after assessment of benefits and risks of acquiring hepatitis b virus (hbv). in fact, one review suggests that dm is associated with the progression of severe liver outcomes in adults with hbv [ ] . on the other hand, tetanus and diphtheria vaccinations should be updated. in addition to, vaccinations such tick-borne encephalitis, meningococcal infections and other infections that put in risk diabetic patients travelling abroad. accordingly, theres a variability of vaccines that can offer a preventive method to reduce morbidity, mortality, and medical expense. in our multicenter study among eastern province saudi arabia evaluated the degree of adherence of the physicians to the immunization recommendations for adult patients with diabetes mellitus type and type to increased awareness of the immunization importance in diabetic patients. objectives: to increased awareness of the immunization importance in diabetic patients. in fact, patients with diabetes mellitus are immunologically vulnerable population to develop different types of microbial infections. immunization has an important role in infection prophylaxis. methods: this is a cross sectional study involving adult patients with type and type diabetes mellitus using a questionnaire. patients will be recruited from outpatient clinics including the primary care clinics and inpatient words of king fahd hospital-al khobar and other centers in the eastern province saudi arabia. after an informed consent, baseline data will be collected. patients will be then asked if they received the recommended vaccines and who was the provider. their knowledge regarding the needed immunization will be also tested. they will then be asked about frequency of upper respiratory tract infections and pneumonia they had over the last years. results: we are expecting to find low adherence to the recommended immunizations by the physician. we may also find that patients who received the vaccines has low incidence of related infections. conclusions: increased awareness of the immunization importance in diabetic patients and adherence to immunization as part of standard care of adult patients with diabetes mellitus that offer a preventive method to reduce hospitalizations, mortality, and medical expense. - , - , and - bases. repeatability and reproducibility of the assays were . and . , respectively. . % of the target regions were covered with over x sequencing depth. we showed that the assays had . sensitivity to detect single-exon deldups and . sensitivity to detect copy number aberrations covering two or more exons. using acmg guidelines for variant classification a diagnosis was established in % patients that were sent for comprehensive panel analysis. conclusions: conclusions: our results demonstrate the analytic validity of the developed tests and show that the technology is well-suited for clinical diagnostics of inherited eye disorders. it also demonstrated a cost-effective diagnostic tool to simultaneously diagnose various types of mutations from snvs to copy number variations. introduction/background: loss of function (lof) and null mutations in orai and stim cause a rare autosomal recessive immunodeficiency by abolishing calcium release-activated calcium (crac) channel function and store-operated ca + entry. the clinical presentation is characterized by scid-like disease, dental enamel defects, muscular hypotonia and anhidrotic ectodermal dysplasia. objectives: here we present the outcome of calcium assessments performed on lymphocytes from an adult patient with unusual infections and a purported novel single pathogenic variant in orai . methods: the ca + response in lymphocytes following activation by varying concentrations of non-cross-linked anti-cd was assessed by flow cytometry. results: the patient was a year old female with a history of seasonal and medication allergies and recurrent sinus infections, who had recently developed an acute infection of her right first metatarsal joint. cultures from the joint space grew neisseria gonorrhea and atypical mycobacteria at two different time points. hiv screening was negative. follow-up testing with the mantoux test and quantiferon gold suggested that she also had latent tuberculosis infection, for which she was started on rifampicin therapy. immunologic evaluation revealed normal complete blood count and differential, normal t, b and nk cell counts, normal immunoglobulin g, a, and m levels, as well as normal responses to polysaccharide vaccines and normal t cell proliferative responses to mitogen and antigen stimulation. however, given the identification of atypical organisms from joint fluid cultures as well as latent tuberculosis (despite the lack of significant risk factors), genetic testing was recommended by her local physicians to rule out an underlying primary immunodeficiency. an invitae primary immunodeficiency panel identified a single novel pathogenic variant in orai . she was then referred to our institution for further evaluation. so far, individuals identified as heterozygous for lof or null mutations in either orai or stim have lacked any phenotype associated with crac channelopathy. however, there have been reports of abnormalities in calcium response in parents of a number of these patients, who are heterozygous for the disease-causing mutation. to examine the functional effect of the observed mutation in our patient, her freshly isolated pbmcs were loaded with indo- and the ca + response of her lymphocytes were assessed by flow cytometry. this showed a dose-dependent decrease in the patients t cell response to non-cross-linked anti-cd in comparison to the normal control, i.e. there was a clear decrease in the patients ca + response at microgram/ml in comparison to the normal control, but the decrease was rectified upon stimulation with microgram/ml or more of anti-cd . conclusions: these findings provide functional support for the identification of a new pathogenic mutation in orai . nevertheless, it is not yet clear if the mutation has any mechanistic role in the patients recent clinical presentation. introduction/background: patient : years old boy, born to nonconsanguineous parents, with hypothesis of autoimmune encephalitis (vasculitis), which was not characterized. csf has already been routed to barcelona times and to vienna time. refractory epilepsy remains (uses drugs yet). he had continuous fever during the entire hospitalization. he had adhd (took ritalin for years) and central auditory processing deficit. one year ago he began to have fever for days, improving later but evolving with bilateral otitis, predominantly on the left ear, accompanied by sinusitis. soon after that he began presenting epilepsy that worsened severely. he was interned and took acyclovir ev, associated with hydantoinate (had stevens-johnson by the drug, suspending and improving quickly). in march had uti + amo, several infections requiring meropenem + vanco, among others. received flebogamma mg/ day/ days, twice; he also received weekly rituximab for a few months. in use of carbamazepine, clobazan, phenobarbital, levotiracetam and vigabatrin times a day. personal antecedents: allergic rhinitis, bronchial asthma, recurrent otitis media, iga deficiency. patient : male, years old, born to non-consanguineous parents, with history of repeated infections in the upper respiratory tract from one year of age with prolonged dry cough and sore throat in all episodes, treated with dexamethasone without improvement, followed by antibiotics with resolution of the condition. at age five, in february , he had a new episode of sore throat and cough that lasted three months, improving spontaneously thereafter. in june , new episode of sore throat with elevated fever and whitish plaques in the tonsils, being prescribed benzetacil, without improvement in three days. he returned to the same emergency room, the antibiotic was replaced by zinnat (axetil-cefuroxime), but within the hospital he began to convulsionate, entering into an epileptic crisis, being hospitalized for days in this service and being transferred to another hospital specialized in pediatrics, intensely investigated and treated with partial improvement of the condition. he remained in coma, not walking and talking for some months, recovering slowly with physical therapy and speech therapy. of relevant exams have: reduced iga, but before it was normal (probably induced by anticonvulsants); full-body magnetic resonance imaging demonstrates generalized lymphadenomegaly and hepatosplenomegaly; pet-ct showing signs of hypoperfusion in temporal (right), occipital and cerebellum regions (suggestive of hypoperfusion -vasculitis?) my first impression was of possible mevalonate kinase (mvk) or autoimmune lymphoproliferative syndrome (alps) deficiency. with these tests described above, i believe that the first diagnostic suspicion is that the epilepsy was triggered by hemophagocytosis in the central nervous system and consequent extremely severe epilepsy, triggered by ebv infection. objectives: to compare the clinical and genetic similarities and differences of both patients. methods: both patients wer submitted to whole exome sequencing looking for the genetic alterations associated to the disease of these patients. results: wes of patient showed an allelic variant in the gene of rai (c. g.a; p.arg gln) possibly pathogenic and that could be related to the clinical features of the patient. wes of patient showed an allelic variant in the gene of cd (c. g>a; p.arg his) heterozygous and of uncertain significance. another allelic variant was found in the gene of btk (c. g>a; p.arg gln) classified as of uncertain significance and hemizygous (as btk is in chromosome x). the expression of both proteins evaluated by flow cytometry is normal, decreasing but not abolishing the possibility of pathogenicity. conclusions: the similarity of the clinical presentations is striking, but the genetic alterations are totally different, leading to the presentation of this abstract. (inf-) have been associated with adult onset immunodeficiency in patients of asian origin. pathogens that cause infections in these patients include mycobacterium avium-intracellularae (mai), non-typhoidal salmonella, cytomegalovirus, penicillium marneffei, and varicella zoster virus. methods: chart review of one patient results: we present a thirty-two-year-old filipino female, with sjogrens syndrome, penicillin and vancomycin allergy, and shellfish allergy suffering from recurrent mai spinal osteomyelitis. after three months of conservative management of back pain, mri showed an abscess at l /l and l / s vertebrae, which was diagnosed as acid fast bacilli on biopsy. she was treated for mycobacterium tuberculosis with rifampin, isoniazid, pyrizinamide, and ethambutal with subsequent change in antibiotic therapy after six weeks once cultures grew mai. mri showed spread of abscess to l -s vertebrae. three months into treatment, she was found to have a new abscess at a different spinal site, and antibiotics were again changed. two months later, she had recurrence of disease with multiple large iliopsoas abscesses, cutaneous fistulas, insufficiency fractures of the sacrum bilaterally, and osteonecrosis of the l vertebra, requiring extensive surgical debridement. medications were adjusted and she was referred to immunology eight months after initial presentation to infectious disease. laboratories were notable for elevated igg ( mg/dl) and iga ( mg/dl) and decreased cd ( cell/ul) and cd / ( cell/ul) cells. serum electrophoresis showed low albumin, elevated gamma fraction, and polyclonal gammopathy. specific antibody titers, lymphocyte proliferation assay, ch , and immunofixation were within normal limits. cytokine panel was significant for elevated il- receptor cd ( pg/ml), il- ( pg/ml), and il- ( pg/ml), and normal tnf, inf, il- , il- , il- , il- , il- , il- , il- , and il- . further serologic testing was positive for autoantibodies to inf-. patient continues treatment with iv antibiotics and is awaiting enrollment in a rituximab trial. conclusions: autoantibodies to inf-should be considered in patients of asian origin presenting with adult onset immunodeficiency, particularly those with severe or recurrent infection with mai. a high level of suspicion is required to make this diagnosis: failure to consider this disease entity leads to delay in diagnosis with potentially significant consequences for the patient. allergy/immunology, university of south florida at johns hopkins all childrens hospital introduction/background: autoimmune and inflammatory conditions are common in cvid. these have been associated with increased morbidity and mortality. objectives: we sought to further understand and evaluate the prevalence of autoimmune and rheumatologic manifestations in patients with common variable immunodeficiency (cvid). methods: we performed a retrospective analysis of cvid patients with rheumatologic/autoimmune complications in the partners healthcare cvid cohort. we evaluated baseline patient characteristics as well as autoimmune and rheumatologic complications in this cohort of patients. results: in the partners cvid cohort, / ( %) had autoimmune or rheumatologic disease. autoimmune cytopenias were reported in / ( %) patients, including coombs positive autoimmune hemolytic anemia (n= ), idiopathic thrombocytopenic purpura (n= ), and autoimmune neutropenia (n= ). autoimmune thyroid disease was reported in / ( %) patients, including hypothyroidism (n= ) and hashimotos thyroiditis (n= ). inflammatory arthritis was present in / ( %), most commonly seronegative rheumatoid arthritis (ra) (n= ), followed by inflammatory arthritis (n= ), seropositive ra (+rf or +ccp antibody) (n = ), psoriatic arthritis (n= ), and juvenile idiopathic arthritis (n= ). systemic autoantibody disease was diagnosed in patients ( %), with diagnoses including vasculitis (n= ), systemic lupus erythematosus (n= ), polymyalgia rheumatica (n= ), antiphospholipid syndrome (n= ), mixed connective tissue disease (n= ), crest/ scleroderma (n= ), myositis (n= ), sjogrens syndrome (n= ), and discoid lupus erythematosus (n= ). inflammatory neuropathy was diagnosed in patients, with small fiber polyneuropathy (n= ), uveitis (n= ), myasthenia gravis (n= ), bells palsy (n= ), and multiple sclerosis (n= ). autoimmune skin conditions were diagnosed in patients with diagnoses including psoriasis (n= ), alopecia (n= ), and vitiligo (n= ). while the mean igm was higher in the patients with autoimmune/rheumatologic manifestations than in other cvid patients ( vs mg/dl), this difference did not reach statistical significance (p= . ). conclusions: autoimmune and rheumatologic complications are present in over half of patients with cvid. increased vigilance for autoimmune and rheumatologic complications is important as survival outcome are worse in cvid patients with non-infectious complications as previously described. further evaluation of these patients to understand the mechanism of immune dysregulation is essential, as this may promote targeted therapies and improve clinical outcomes. introduction/background: immunoglobulin concentrates have been successfully used for decades to treat patients with primary or secondary immunodeficiency disorders. this treatment has substantially decreased the frequency of life-threatening infections in these patients. octanorm is a newly developed maltose-formulated subcutaneous immune globulin (human) . % liquid for the treatment of patients with primary immune deficiency (pid) and secondary immune deficiency (sid). objectives: biochemical and physico-chemical properties were investigated. methods: molecular size distribution of monomers, dimers, polymers and fragments were determined (according to european pharmacopeia (ep) monograph . ) by size exclusion chromatography (sec). igg and igg subclass concentrations were quantified by respective nephelometric methods. functionality of the igg was demonstrated by measurement of fc function, opsonophagocytosis and fc gamma receptor binding assays. dynamic light scattering measurement and size exclusion chromatography were used to characterize the integrity of the igg molecule. measurement of potential procoagulant activity was done by natem and tga (fxia-like activity). the capacity of the octanorm manufacturing process to robustly inactivate/remove pathogens was investigated in spiking experiments with prions and viruses. results: octanorm contains more than % of human igg and is characterized by an especially low content of polymers and aggregates, low viscosity, low isoagglutinin titres, low iga and igm contents with a broad spectrum of antibodies against infectious agents. it has a distribution of immunoglobulin g subclasses closely proportional to that in native human plasma. in the final product, potential procoagulant activity is not detectable. functionality and physico-chemical properties of the igg molecules were demonstrated by state-of-the-art methods. virus safety of octanorm is obtained via a combination of three validated orthogonal methods as part of the manufacturing process: cold-ethanol fractionation, solvent/ detergent (s/d) and ph treatment. a substantial depletion of prions during the manufacturing process was demonstrated. conclusions: octanorm is a state-of-the-art subcutaneous immunoglobulin. based on the excellent stability the intended shelf life of octanorm is months stored at + °c to + °c protected from light. within its total shelf life the product can be stored at room temperature up to + °c for up to six months. efficacy and very good tolerability of this new subcutaneous normal immune globulin . % were shown in a clinical phase iii study performed in centers in north america and europe. professor, sapienza university of rome introduction/background: primary antibody deficiencies (pad) are characterized by defective ig production resulting in high susceptibility to bacterial infections, especially caused by s. pneumoniae and h. influenzae. there is a limited evidence on the rate of microbial airway epithelial colonization and on the role of bacterial carriage on the development of recurrent respiratory tracts infections in such populations. objectives: the aim of this study was to investigate the prevalence of s. pneumoniae and haemophilus influenzae colonization in pad adults in italy and its clinical and immunological correlates. methods: nasopharyngeal and oropharyngeal swabs were obtained from cvid and patients with idiopathic primary hypogammaglobulinemia (iph) over years of age and under ig replacement treatment during the period october -april . presence of s. pneumoniae and h. influenzae was investigated using conventional cultural methods and rt pcr. s. pneumoniae isolates were serotyped by the quellung reaction; capsular type of h. influenzae isolates was determined by pcr. the pattern of associations between the two species and potential risk factors were investigated. respiratory infections rate was recorded over months of follow up. results: among cvid prevalence of carriage assessed by traditional culture was % and % for s. pneumoniae and h. influenzae, respectively. rt pcr allowed to identify a higher rate of carriage of s. pneumoniae and h. influenzae compared to standard culture. cvid and iph had not different rate of pneumococcal colonization, whereas cvid had higher rate of h. influenzae carriage identified by a culture methods and rt pcr. no synergistic association between s. pneumoniae and h. influenzae colonization was observed. among cvid, s pneumonia and h. influenzae carriage were associated to low iga and igm levels. cvid under antibiotic prophylaxis did not have an increased prevalence of carriage. no association was found between carrier status detected by culture and having chronic lung disease, bronchiectasis or the rate of infections during the follow up. rt pcr identified merely the association between igm levels and h. influenzae carriage. antibiotic resistance from isolated stains was also assessed. conclusions: this is the first study assessing the prevalence of s. pneumonia and h. influenzae carriage in cvid. objectives: to discuss clinical challenges in a subject with congenital hair hypoplasia detected on newborn screening with preserved t-cell mitogen responses but declining naive t-cell counts. parents are jehovah's witnesses, which adds complexity to clinical decisionmaking. methods: immune evaluation included complete blood count, lymphocyte subsets, flow cytometry to define t-cell subsets, immune globulins, repeat trec assay from peripheral blood, human immunodeficiency virus (hiv) and cytomegalovirus (cmv) dna pcr, screen for maternal engraftment, chromosome microarray analysis, lymphocyte proliferation to mitogens, t-cell proliferation to interleukins, t-cell receptor v-beta diversity analysis by spectratyping, and thymic ultrasound. results: repeat trec assay from peripheral blood on day confirmed undetectable trecs. cd + thymic emigrants were low at (reference - cells/ul). naïve cd + and cd + t cell compartments declined as did naïve cd + t cells from to cells/ul and naïve cd + t cells from to cells/ul. lymphocyte proliferation to mitogens was preserved except for the cd + response to pokeweed mitogen. interleukin proliferation of cd + lymphocytes was slightly decreased after stimulation with anti-cd ( %, normal > %). however, t-cell proliferation with other interleukins (il- , anti-cd +il- , anticd as %cd ) was preserved. the t cell receptor repertoire had intermediate diversity. antibody replacement therapy was prescribed for declining igg with no history of severe infections except for rhinovirus prior to discharge from the nicu at weeks, during which she required continuous positive airway pressure (cpap) therapy. the viral load for cmv and hiv dna were undetectable. breastfeeding was discontinued early because the mothers cmv serology revealed positive cmv igg. the child has had intermittent anemia, which is common in patients with chh. [ ] tests for autoimmune hemolytic anemia were not performed because the patient required ivig from an early age. the parents of the child are jehovahs witnesses, complicating requests for blood transfusions. conclusions: this case highlights challenges in clinical decision making for a newborn with chh identified on nbs. t-cell function is preserved, but declining naïve t cell counts and absent trecs lead us to consider hematopoietic stem cell transplant (hsct). indication and timing of elective hsct is unclear and may depend on the natural progression of disease, including infections and anemia division of immunology and allergy, the hospital for sick children introduction/background: cartilage-hair hypoplasia (chh), caused by mutations in the ribonuclease mitochondrial rna-processing (rmrp) gene, is associated with diverse immune abnormalities including combined immune deficiency (cid). most patients with chh are managed with supportive measurements, while few have received allogeneic hematopoietic stem cell transplantations (hsct). the progression of the immune abnormalities and the impact of hsct in patients with chh and cid have not been well characterized. objectives: to characterize the progression of the immune abnormalities and the impact of hsct in patients with chh and cid methods: the clinical and laboratory findings of siblings diagnosed in infancy with chh and cid due to the common a>g mutation in rmrp, including the effects of hsct performed in of them, were compared. results: both patients suffered from recurrent respiratory infections at early age with reduced t cells numbers and responses. patient # immune function continued to deteriorate leading to hsct from an hla-matched sibling at . years of age. the patient suffered acute and chronic graft versus host disease of the skin with residual mild joint contractures and scleroderma-like skin changes. seven years after hsct patient # has normal immune function. immune evaluations of patient # in the first years of life indicated mild improvement. the patient did not have a suitable related hsct donor and the family elected to continue with supportive care. at years of age, patient # is clinically well and thriving with persistent t cell abnormalities. conclusions: close monitoring of immune function in early life for patients with chh and cid as well as the availability of suitable donors assists in determining management, including hsct introduction/background: leukocyte adhesion deficiency (lad) represents a group of distinct inherited disorders, which inhibit the normal extravasation of neutrophils and their recruitment to sites of infection or inflammation. objectives presentation of case: the patient is a girl of years old with no history of primary inmunological disease (pid) in family members, including a healthy brother (currently years old). she received all the immunization schedule until year old (according to peruvian schedule). she had several admission to the hospital since newborn; the st hospital admission was at days of life with diagnosis of: sepsis, pneumony, onphalitis (leukocytes count: ), she had more hospital admission and the leukocytes count were always above . in summary, she presented other infections besides the ones admitted at hospital like: episodes of sepsis, episode of pneumony, episodes of cellulitis (left eye, left elbow, right thigh and vaginal ( ), episodes of otitis, episodes of tonsillitis, episodes of diarrhea, episodes of rhinoadenoiditis, episodes of sinusitis, episodes of gingivitis and episode of whooping cough. she received different antibiotics for treatment, even broad-spectrum as vancomycin, meropenem and cefepime. the diagnosis of leukocyte adhesion deficiency (lad) was made at year months by clinical features like delaying in separation of umbilical cord, recurrent infections and persistent leukocytosis> . flow citometry was taken at years old, resulting cd b/cd : , %. results: discussionf: or the severe phenotype, in which leukocytes express < % of normal levels of cd , death occurs at an early age because of severe infection unless patients receive bone marrow transplant. however, it does not happened with her so we suspected in a possible reversion in lad so we took a second flow citometry at years old and the results were: total leukocyte: ; linfocyte: cd b+/cd +: , %; granulocyte: cd b+/cd +: , % and the conclusion was c receptor (cd b/cd ) absent in linfocyte, monocyte and granulocyte. molecular study was taken at years old, resulting a homozygous substitution c. c>t identified in exon , causing a nonsense mutation: p.arg , confirming lad diagnosis. she is, currently, receiving profilactic antibiotic and antimicotic which has reduced considerably recurrent infections. it seems that our patient may have a mixed phenotype due to a clinical expression, which may not require hematopoietic cell transplantation (hct) despite features of the severe type. conclusions conclusion: we conclude that the clinical evolution of this patient is unsual because she has severe lad , she has not transplanted yet, profilaxis treatment has improved to decrease frecuency of infections, she exceeded life expectancy and last flow citometry confirmed that lad was not reverted. , ph.d. , luigi d. notarangelo, md , troy r. torgerson, m.d. ph.d. , ph.d. , hans d. ochs, m.d. , m.d., ph.d. introduction/background: patients with x-linked hyper-igm syndrome (x-higm) due to cd ligand (cd l) deficiency often present with low blood neutrophil counts. however, even when not neutropenic and despite immunoglobulin (ig) replacement therapy, cd l-deficient patients are susceptible to life-threatening infections by opportunistic pathogens, suggesting impaired function of phagocytes, and requiring novel therapeutic approaches. objectives: to analyze whether peripheral neutrophils from cd l-deficient patients display functional defects and to explore the in vitro effects of recombinant human interferon (rhifn)-on such cells. methods: we investigated the microbicidal activity, respiratory burst and transcriptome profile of neutrophils from cd l-deficient patients. in addition, we evaluated whether the lack of cd l in mice also affects neutrophil responses. results: neutrophils from cd l-deficient patients exhibited defective respiratory burst and microbicidal activity which were significantly improved in vitro by rhifn-. similar to humans with cd l deficiency, cd l-deficient mice were found to have defective neutrophil responses. moreover, neutrophils from cd l-deficient patients showed reduced cd protein expression and a dysregulated transcriptome profile suggestive of impaired differentiation. conclusions: our data suggest a non-redundant role of cd l-cd interaction in neutrophil development and function that could be improved in vitro by rhifn-, indicating a potential novel therapeutic application for this cytokine. methods: in this study, through the use of healthy livers, paired peritumoural tissues (pt) and intratumoural tissues (it) from hcc patients. results: increased expression of cd on nk cells was observed in intratumoral but not peritumoral regions, along with increased expression of its ligand cd and a poor prognosis. human cd + nk cells exhibited functional exhaustion, showing decreased ifn-and tnf-productions, impaired cytolysis in response to in vitro stimulation, and high gene expression of il- and tgf- with low expression of t-bet, il- , perforin and granzyme b by global transcriptomic analysis of sorted cd + and cd -nk cells. blocking tgf- specifically inhibited cd expression and reversed the dysfunction of nk cells. in addition, we compared other two receptors, cd and tigit, which share common ligand cd with cd , and found cd plays a more important role in nk exhaustion. conclusions: these findings indicate that human cd + nk cells have features of functional exhaustion, suggesting that cd -cd blockade has the potential to restore immunity against liver tumors by reversing nk cell exhaustion. introduction/background: mutations in ncf (encoding protein p phox of the nadph oxidase) result in an autosomal recessive form of chronic granulomatous disease (cgd), a rare genetic disease with impaired phagocyte production of reactive oxygen species and recurrent infections. diagnosis of p phox cgd is based on abnormal dihydrorhodamine assay and absence of p phox protein by immunoblotting; however, these assays fail to diagnose carriers of ncf mutations. instead, carrier status is inferred after the birth of a child with p phox cgd. furthermore, identification of the specific genetic defect in patients with p phox cgd is complicated by two highly conserved (> %) pseudogenes. the ncf gene has a gtgt at the start of exon , while the pseudogenes (ncf b and ncf c) delete one gt (gt). in p phox cgd, the most common mutation in ncf is gt causing c. _ delgt; p.tyr fsx . sequence homology between the wild type gene and pseudogenes precludes using standard sanger sequencing to identify specific mutations in ncf . objectives: to identify phenotypic and genotypic differences that facilitate the diagnosis of patients and carriers with p phox cgd. methods: expression of p phox in neutrophils is determined by fixing and permeabilizing whole blood with intraprep, and then incubating with either anti-p phox antibody or its corresponding isotype. alexafluor -conjugated secondary antibody is used to detect the target antigen. expression of p phox is based on the mean fluorescence intensity of cells within the neutrophil population gated using forward and side light scatter. differential expression of p phox by flow cytometry is validated using quantitative immunoblotting. to screen for the gt mutation, a droplet digital polymerase chain reaction (ddpcr) with two distinct probes recognizing either the wild-type gtgt sequence or the gt sequence was used to quantitate the ratio of gtgt vs. gt copies. a second ddpcr reaction established copy number by comparing one probe for an invariant region of ncf /ncf b/ncf c and a second probe for the single-copy telomerase reverse transcriptase gene, tert. the results of these two assays were combined to determine the total number of gtgtcontaining and gt-containing ncf copies. results: analysis of p phox expression in permeabilized neutrophils determined that neutrophils from p phox cgd patients had negligible p phox expression; neutrophils from p phox cgd carriers exhibited~ % of p phox expression compared to healthy volunteers independent of the mutation in ncf . of all p phox cgd patients tested by ddpcr, . % ( / ) exhibited copies of gtgt, . % ( / ) exhibited copy of gtgt (compound heterozygotes with non-gt mutation), and . % ( / ) exhibited copies of gtgt (two non-gt mutations). moreover, ddpcr can identify the carriers among kindreds within the gt p phox cgd families. unexpectedly, among normal subjects tested, only . % exhibited the expected copies of gtgt per total ncf /ncf b/ncf c copies, designated / ; a significant number exhibited more than two copies of gtgt ( . % with / , . % with / ); others exhibited ncf /ncf b/ncf c copy number variation ( . % with / and . % with / ). conclusions: flow cytometric analysis of neutrophil intracellular p phox staining provides a quick method to identify patients and carriers of p phox cgd. droplet digital pcr can be used to identify patients and carriers of p phox gt, the most common mutation in p phox cgd copy number variation is observed at the ncf locus among normal subjects tested. introduction/background: defects in the cd subunits of the tcr/cd complex account for a small percentage of the scid presentations. cd deficiencies are characterized by profound and t-cells lymphocytopenia, and normal numbers of b-and natural killer (nk) cells in the peripheral blood (t-b+nk+ scid). thymocyte development from double negative stage to double positive stage results arrested. affected individuals typically present with severe and opportunistic infections in the early infancy. objectives: to evaluate possible underlying immunodeficiency disorder in the setting of chronic ebv infection. methods: here we report our experience of a patient with an atypical presentation of cd deficiency. results: a -year-old girl previously healthy, first generation americanborn of gambia immigrants was referred to our clinic for further evaluation of chronic ebv infection. one year before presentation, she developed bilateral parotid enlargement, cervical, axillar, and hilar lymphadenopathy, bronchiectasis, and pulmonary nodules. relevant previous studies included: ebv viremia (pcr quant , copies), ebv igg and ea positive whereas igm and ebna were negative. peripheral blood phenotyping was not suggestive for malignant process and cervical lymph node biopsy was consistent with a reactive process without evidence of a clonal lymphoproliferative disorder and bal studies positive for ebv only. hiv and tb both negative. she was otherwise thriving with no history of recurrent infections and family history was negative for consanguinity or immunodeficiency. our evaluation revealed: normal blood counts, ebv pcr quant , copies, normal immunoglobulin levels and vaccine titers. she had a normal lymphocyte subsets, but skewed cd ra/ro consistent with low thymic output (very low cd naïve t cells ( . %), low cd + naïve t cells ( . %), and elevated temra ( . %)), poor mitogen, and antigen responses. all these findings were suggestive of a scid like phenotype; therefore, scid next generation sequencing panel was pursued, and showed a novel homozygous splice site mutation (c. + g>t) in the cd gene. conclusions: a homozygous mutation in the cd gene might not necessarily imply profound t-cell lymphopenia. though this patient did not present with classic clinical course, and immune findings; her inability to clear ebv with persistent significant viremia does support a t-cell immune deficiency. she remains at risk for ebv-associated lymphoproliferative disorder, infections, and autoimmunity. bmt will be a curative treatment option. however, it is difficult to predict evolution of clinical phenotype given the atypical presentation. this case illustrates the importance of contextual interpretation of clinical findings, laboratory data, and genetic analysis for treatment approach. introduction/background: pol is multi-subunit polymerase that includes both pole with catalytic activity and additional pole , and . this holoenzyme plays a key role in proofreading damaged dna and is required for proper dna replication in proliferating cells, such as lymphocytes. germline mutations are linked to rare cause of primary immunodeficiencies whereas somatic mutations are described in colon cancer. primary immunodeficiencies are reported pole- deficiency in members of a large consanguineous french kindred and a palestinian female with fils (facial dysmorphism, immunodeficiency, livedo, and short stature). all reported cases with pole deficiency have homozygous intronic splice site variant (c. + a>g) that result in a deletion of exon which lead to subsequent frame shift (from p.s v onwards) and a premature stop codon at position ; this transcript results in a degraded product. the proportion of the pole transcript in t lymphoblasts is significantly lower ( %) in patients then carriers or healthy individuals. objectives: hereby we describe the clinical progression and treatment challeneges of the palestinian female with pole- deficiency secondary to homozygous g.g + a>g substitution. results: initially patient presented with viral and recurrent ear infections and cmv viremia. with age, patient had less episodes of infections even with intermittent pause in immunoglobulin replacement therapy (igrt), however had multiple admissions for fever of unknown origin with negative cultures but increased ferritin level ( ng/ml) and low platelet count ( , count/ml). autoimmune and inflammatory complications were not reported among the french kindred. her skin also worsened with poor wound healing and scarring throughout that hinders igrt via subcutaneous route. furthermore, igrt with intravenous administration has resulted in symptoms of aseptic meningitis likely related to the underlying inflammatory state. in addition, patient shows decline in immune dysfunction. initially, she had normal immunoglobulin g (igg) however by years of age, she developed hypogammaglobulinemia with low igm unlike in the french family with patients. also, pneumococcal, diptheria, tetanus titers were non-protective off of immunoglobulin replacement therapy (igrt). beyond low switched memory b cells, patient also developed low b cell by yo age. t cell dysfunction continued to decline from decreased lymphocyte proliferation to antigens at yo age to fully absent lymphocyte proliferation to antigens and mitogens. naïve cd and cd compartments continue to be preserved. currently management challenges include treatment strategies for thrombocytopenia, inflammation and progressive skin disease that complicates the proper selection of route for optimal igrt. conclusions: beyond progression of immunological decline, our patient developed inflammatory phenotype with age. the progression of her immunological decline may be related to further decrease in the proportion of the wild type pole transcript and yet to be examined. overall, clinical follow up is essential in patients with pole- deficiency as phenotype can change with age and may pose new challenges. longitudinal follow up studies are needed to uncover the potential role of germline pole and pole pathogenic variants in cancer susceptibility. introduction/background: x-linked hyper-igm syndrome (xhigm) is one type of primary immunodeficiency diseases, resulting from defects in the cd ligand/cd signaling pathways. objectives: here, we retrospectively reviewed clinical, laboratory and genetic characteristic of xhigm in chinese population, thus further improving diagnosis and treatment for xhigm. methods: we collected and analyzed chinese patients, who were diagnosed and followed up in hospitals affiliated to shanghai jiao tong university school of medicine from to . targeted gene capture combined with next-generation sequencing technology and sanger sequencing were used to find out related gene mutation. results: the median onset age of these patients was months (range: days months). thirty-six percent of them had positive family histories, with a shorter diagnosis lag. the most common symptoms were recurrent sinopulmonary infections ( patients, %), neutropenia ( patients, %), protracted diarrhea ( patients, %), and oral ulcer ( patients, %). ten patients had bcgitis. six patients received hematopoietic stem cell transplantations and four of them had immune reconstructions and clinical remissions. twenty-seven unique mutations in cd l gene were identified in these patients, with novel mutations. conclusions: to our knowledge, this report provides the largest cohort of patients with xhigm in china. mutation analysis is an important tool for xhigm diagnosis. infants with scid are asymptomatic at birth and unless prompt diagnosis of the disease is made they may horrifically be vaccinated. simultaneous appearance of two live vaccine associated infections in one person is rarely reported. objectives: in this study we present two infants with scid, who received bcg and oral polio vaccines early in life before the diagnosis of immune deficiency was made. both patients developed localized and disseminated infections originating from the bcg vaccine (bcgitis and bcgiosis, respectively) and in addition were diagnosed with chronic fecal secretion of vaccine-derived polio virus (vdpv); alarmingly, in both cases, the vdpv underwent reverse mutation of attenuated sites to the neurovirulent genotype. the rarity of concomitant infection from two live vaccines in one recipient, together with the multiple complexities originating from these infections in immunodeficient infants, led us to report these cases and to inquire the pathogenesis that underlies this unique condition. methods: immunological evaluation:: cell surface markers of peripheral blood mononuclear cells (pbmcs) were measured by immunofluorescent staining and flow cytometry serum concentrations of immunoglobulins were measured using nephelometry. quantitative analysis of the tcr v repertoire was performed by means of flow cytometry. quantification of t cell receptor excision circles (trecs) was determined by real-time quantitative (rq)-pcr. genetic analysis: genetic diagnosis of scid was made for patient by direct sanger sequencing of candidate genes and retrieval of mutation in rag , and for patient by wes (whole exome sequencing), followed by validation of the dna cross-link repair c (dclre c) mutation using sanger sequencing. poliovirus detection and characterization: stool samples were collected monthly and transported to the national poliovirus laboratory located at israel central virology laboratory (icvl) for polio detection and characterization. results: in both patients, immunological workup revealed undetectable serum iga and igm levels with normal igg levels (table ) , lymphocyte immune-phenotyping using flow cytometry revealed complete absence of t and b cells with presence of nk cells (table ) . trecs, a dna marker of naive t cells and thymic output, were absent in both patients. the diagnosis of scid was made. we initiated prophylactic antibiotic (trimethoprim-sulfamethoxazole) and anti-fungal (fluconazole) treatment, as well as monthly intravenous immunoglobulin (ivig) infusions. genetic workup: the t-b-nk+ scid phenotype in patient led us to search for a mutation in the rag complex genes. indeed, a sanger sequencing of the rag gene, revealed a g t homozygous mutation which predicts an amino acid substitution from glycine to valine in position (fig. ). for patient , who had a similar t-b-nk+ scid phenotype, we identified a homozygous mutation in the dclre c gene (del. bp, c. -cttt) (fig. ) , using wes. the genetic evaluation confirmed the diagnosis of scid due to rag deficiency in patient and artemis deficiency in patient . clinical course: during hospitalization patient developed disseminated bcg related disease with skeletal lesions involving the phalanx, tibia and maxillary bones, as well as involvement of the spleen, liver and pancreas. anti-tubercular therapy with isoniazid, rifampicin, ethambutol and ciprofloxacin was initiated. due to lack of response, empirical trial of g-csf (granulocyte colony-stimulating factor), in order to enhance macrophage activity . the patient showed good response to this combination therapy. patient developed a palpable rigid mass on her left shoulder with surrounded redness at the site of the bcg vaccine at the age of months. the clinical diagnosis of bcgitis was established and triple antitubercular therapy with isoniazid, rifampicin and ethambutol was initiated with good response. throughout their hospitalization, both infants suffered from intermittent diarrhea. pcr for enterovirus was performed and detected the presence of type and type vaccine-derived poliovirus (vdpv) in patient and , respectively. during the follow-up, stool samples collection revealed accumulation of several polio virus mutations and some of the neuro-virulence attenuation sites were reverted to the neuro-virulent genotype .fortunately, both patients did not show any signs of flaccid paralysis. precautionary measures of isolation were taken to prevent spread of the vdpv. eventually, both patients underwent allogeneic bone marrow transplantation (bmt): patient had bmt without pre-conditioning, from a matched sibling donor. due to engraftment failure, a second bmt was repeated, this time successfully. on follow up examination her t cell repertoire showed a normal tcr v polyclonality and trec was detected, indicating the emergence of new t cells. due to ongoing low immunoglobulin levels this patient is still on regular ivig-infusions and prophylactic antibiotic treatment, as well as isoniazid. currently, she is well, her bcgitis is not active and her stool specimens are negative for polio. patient underwent an urgent haplo-identical bmt with alpha-beta t cell depletion without pre-conditioning, due to her unstable medical condition. flow cytometry analysis six months post bmt revealed lymphopenia of . % ( /mm³) with low mature t cells (cd %) and absent b cells. trecs were barely detected. microsatellite analysis as a marker for engraftment revealed a stable donor chimerism of %. the patient is still on immunosuppressive therapy doing well, her bcgitis is not active and her stool specimens are negative for polio. conclusions: these cases highlight the importance of early recognition of scid by neonatal screening or thorough family anamnesis, and the need to further defer the timing of administration of live vaccines. introduction/background: measurement of b cells, b cell subsets and specific antibodies produced in response to vaccination are key tests used to investigate immune system function. specific antibody production may indicate b cell functionality. objectives: in this study the correlation between the different b cell subsets and antibody responses to pneumovax® in an immunocompromised population was investigated. methods: b cell subsets were assessed by flow cytometry and pneumococcal responses measured using the vacczyme pneumococcal capsular polysaccharide (pcp) igg, iga and igm elisas (the binding site group, birmingham, uk) in primary immunodeficiency patients (pid) vaccinated with pneumovax®. lower limits of normal were defined as follows: b cells: . %; naive b cells: . %; non-switched memory b cells: . %; switched memory b cells: . %; pcp igg: mg/l; pcp iga: u/ml; and pcp igm: u/ml. results: the correlation coefficients between percentage of b cells/b cell subsets and igg, iga or igm pneumovax® responses ranged from - . to . . the percentage of the cohort achieving a normal b cell and normal igg, iga or igm response to pneumovax® was %, % and % respectively. b-cell responses were measureable in the remaining patients but they did not produce normal concentrations of pcp igg, iga or igm. further stratification of patients who achieved normal percentage of switched or un-switched b cells but who failed to achieve normal igg, iga or igm responses to pneumovax® were %, % and %, respectively, for un-switched and %, % and %, respectively, for switched b cells. conclusions: the combined measurement of b cells and response to vaccination are required to provide a detailed insight into these disorders. introduction/background: this is a -year-old boy of african american heritage with multiple congenital malformations who was diagnosed with very early-onset inflammatory bowel disease, which proved to be resistant to treatment. subsequent testing revealed a heterozygous mutation in exon of ctla . this variant has not been previously reported in the literature in individuals with ctla -related disease. heterozygous mutations in ctla cause a disease of immune dysregulation. clinical presentation is variable and may be characterized by enteropathy, hypogammaglobulinemia, granulomatous lymphocytic interstitial lung disease, lymphocytic organ infiltration in non-lymphoid organs, autoimmune cytopenias, and recurrent infections. early onset colitis has been reported with ctla -related disease and is unique to our patient's initial clinical presentation. objectives: this is a -year-old boy with cloacal exstrophy of the urinary bladder, omphalocele, imperforate anus, polydactyly, and sacral agenesis who was diagnosed with very early-onset inflammatory bowel disease at six months of age. he underwent cloacal exstrophy closure, omphalocele repair, and colostomy placement in the first week of life. at six months of age, he presented with dark tarry stools. upper endoscopy and colonoscopy revealed polyps, ileitis, and colitis. he was p-anca positive and started on sulfasalazine. unfortunately, he continued to have symptoms suggestive of active colitis, prompting a change to prednisone and azathioprine. despite therapy, his colitis persisted leading to chronic bloody diarrhea and growth failure. initial immune evaluation consisted of a normal complete blood count, serum immunoglobulin, lymphocyte subsets, and neutrophil oxidase burst assay. foxp analysis by flow cytometry showed a moderately elevated percentage of foxp +cd + cells in the cd + t cell population, but the regulatory t cell immunophenotype was normal. because suspicion was high for a monogenic immunologic disease to explain his symptoms, genetic sequencing was performed. a candidate gene panel was sequenced by next generation sequencing, and a heterozygous mutation in exon of ctla (c. g>a; p.arg gln) was found. this variant has not been previously reported but is predicted to be pathogenic in exac and polyphen databases. results: the patients diagnosis of ctla- haploinsufficiency associated with very early-onset inflammatory bowel disease has provided opportunity for targeted treatment of his specific molecular defect. given his poor response to treatment thus far, the patient will be started on abatacept. abatacept is fda approved for the treatment of rheumatoid arthritis but has been used successfully for the treatment of disease-related manifestations of ctla- haploinsufficiency. abatacept is a ctla- fusion protein formed by the igg fc region linked with the extracellular domain of ctla- ; it replaces the defective protein in ctla- haploinsufficiency. in addition, given other manifestations of ctla- haploinsufficiency including lymphoproliferative disease in non-lymphoid organs, particularly the brain and lung, we have initiated further evaluation of these organs to evaluate for disease-specific manifestations. conclusions: the protein cytotoxic t lymphocyte antigen- (ctla- ) is an essential negative regulator of t cells. heterozygous mutations in ctla cause a disease of immune dysregulation. clinical presentation is variable and may be characterized by enteropathy, hypogammaglobulinemia, granulomatous lymphocytic interstitial lung disease, lymphocytic organ infiltration in non-lymphoid organs, autoimmune cytopenias, and recurrent infections. inflammatory bowel disease may be associated with certain variants in ctla , but the literature remains limited, both by number of papers published as well as by ethnic subsets studied. clinicians who are presented with children who have early-onset colitis, and particularly inflammatory bowel disease that is difficult to treat, should consider possible genetic abnormalities, such as ctla- haploinsufficiency, as these can impact therapeutic decision-making and outcomes. introduction/background: dock deficiency is an autosomal recessive combined immunodeficiency syndrome associated with recurrent infections, eczema and other atopic diseases. the infections are usually viral, bacterial and fungal resulting in predominantly cutaneous and sinopulmonary manifestations. homozygous or compound heterozygous deletions or mutations in the dock gene ( p ) lead to abnormal cytoskeletal organization and impaired function of dendritic cells and lymphocytes. an aniline derivative belonging to the group of synthetic sulfones, dapsone has been employed in the treatment of chronic skin diseases characterized by an accumulation of neutrophils and eosinophils. methods: chart review of one patient results: we present a four-year-old male with severe eczema, persistent asthma, allergic rhinitis, as well as peanut and egg allergy suffering from recurrent skin abscesses and prurigo nodularis. abscesses began at age months and required prolonged courses of antibiotics, eight in total prior to presentation. other infectious history included otitis media and lymphadenitis. there was no history of pneumonia or other severe infections. skin abscesses responded to oral antibiotics, but recurred shortly after completing extended courses of treatment. laboratory results including quantitative immunoglobulins, specific antibody titers, myeloperoxidase staining, neutrophil oxidative burst and complement were within normal limits. laboratories were notable for elevated ige ( iu/ml) and eosinophilia ( eosinophils/microl). lymphocyte immunophenotype was significant for mild elevations in cd and cd . dock genetic sequencing by genedx revealed a heterozygous missense mutation in exon (c. c>a, amino acid change p.ala asp). abscess cultures grew methicillin sensitive staphylococcus aureus (mssa) and enterococcus faecalis. mssa was sensitive to ampicillin/sulbactam, cefazolin, gentamycin, moxifloxacin, oxacillin, rifampin, tetracycline, and vancomycin. isolate was resistant to ciprofloxacin, clindamycin, erythromycin, levofloxacin, penicillin, and trimethoprim/ sulfamethoxazole. the patient was initially treated with emollients, mupirocin washes, topical steroids, anti-histamines, bleach baths, and cephalexin three times a day. he improved clinically but was unable to tolerate cephalexin for more than ten days secondary to abdominal pain. cephalexin, with addition of probiotic, was attempted several months later and again had to be discontinued because of abdominal pain and vomiting. due to limited antibiotic options, dapsone was started after ruling out glucose- -phosphate dehydrogenase deficiency. dapsone was initiated at a dose of . mg/kg, and the patient was monitored weekly for hemolytic anemia. after two weeks of treatment, anemia was noted and the dose was decreased to mg/kg. he has continued on dapsone mg/kg once a day, with significant improvement in abscess number and severity. he has not required other systemic antimicrobials since starting dapsone. conclusions: dapsone may be considered as a treatment option for children with heterozygous dock mutation and recurrent abscesses, particularly those requiring prophylaxis, long term treatment and lacking antibiotic options. introduction/background: autosomal dominant hyper-ige syndrome (ad-hies) is a rare complicated primary immunodeficiency disease (pid). signal transducer and activator of transcription (stat ) gene mutation is found to cause ad-hies. tlr / signaling plays multiple roles in b cell proliferation, activation, class-switch recombination, and cytokine and antibody production. however, little is known about b cell response to tlr / agonist in patients with ad-hies. objectives: here, we aim to study the response of b cells from ad-hies patients to the tlr / agonist. methods: pbmcs were isolated from peripheral blood of ad-hies patients and age matched healthy controls. pbmcs were stimulated with tlr and tlr agonist (r and cpg odn , respectively), then b cells were analyzed for proliferation, the expression of certain surface markers (cd , cd and cd ), intracellular immunoglobulin levels (igm and igg) and intracellular cytokine levels (il- and il- ) by flow cytometry. results: in response to tlr / agonist, proliferative capabilities of b cells were reduced in ad-hies patients compared with those in age-matched healthy controls. besides, defective costimulatory molecule cd expression was observed in b cells from ad-hies patients. furthermore, significantly lower igm and igg levels, and il- production was detected in b cells from ad-hies patients. however, there was no significant difference in b cell apoptosis between ad-hies patients and healthy controls. conclusions: these data demonstrated that stat gene mutations in ad-hies patients contributed to impaired b cell tlr / signaling, and further affected b cell proliferation, activation, cytokine secretion and antibody production. introduction/background: antibody function is most commonly measured by a rise in antibody titers in response to antigen introduced by vaccination or natural infection. specific antibody deficiency is defined as normal serum levels of immunoglobulins with reduced or absent antibody response to antigens, often after administration of the pneumococcal vaccine polyvalent (pneumovax® ). a paucity of information exists about measurement of antigen-antibody binding, or avidity, as a measure of antibody function, including persons with recurrent sinopulmonary infections who have normal response to immunization with pneumococcal vaccine polyvalent. objectives: the aims of this study are to identify and evaluate children with recurrent sinopulmonary infections who had appropriate rise in pneumococcal antibody titers following immunization with pneumococcal vaccine polyvalent but low response by avidity, and to assess response with igg replacement therapy in these patients. methods: a retrospective chart review involved eight children with recurrent sinopulmonary infections with discordant pneumococcal antibody and avidity results following vaccination with pneumococcal vaccine polyvalent. these eight children subsequently received igg replacement therapy. results: the mean age of subjects was . (range - ) years. the mean number of serotypes with a normal antibody response (> . ug/ml) among children following immunization with pneumococcal vaccine polyvalent was . (range - ) of serotypes while the mean number of serotypes with a normal avidity response ( . ) was . (range - ) of serotypes. igg replacement was administered subcutaneously in children. the mean igg level was mg/dl. local reactions were all mild and observed in / ( %) children. no serious adverse events were reported. all children experienced a marked reduction in respiratory illnesses while on igg replacement therapy. conclusions: discordance between pneumococcal antibody titers and pneumococcal avidity titers was identified in eight children with recurrent respiratory illnesses. in children with recurrent sinopulmonary infections despite normal antibody response to pneumococcal vaccine polyvalent, measurement of pneumococcal avidity may identify patients with poor pneumococcal antibody function. igg replacement in these children was well tolerated and associated with a decrease number of respiratory infections. introduction/background: exome sequencing (es) is a powerful genomic tool that can be used to identify novel molecular causes of disorders with multiple etiologies. immunologic disorders are clinically and genetically heterogeneous, and therefore present unique diagnostic challenges both in the clinic and in the laboratory. objectives: the objective of this study was to assess the utility of es for determining the genetic etiology of immunological disorders and describe diagnostic yield and outcomes of exome sequencing (es) for patients with immunologic disorders and immunologic phenotypes. methods: a retrospective review was performed of individuals referred for clinical es for primary abnormalities of the immune system and individuals with additional phenotypes where multiple immunologic features were reported as part of the clinical picture, as determined during internal curation. analysis of clinical es data was performed by boardcertified clinical geneticists and all variants reported were confirmed by a secondary methodology. positive es outcomes required a pathogenic or likely pathogenic variant in a gene with autosomal dominant or x-linked inheritance, or compound heterozygous or homozygous pathogenic or likely pathogenic variants in a gene with recessive inheritance. results: the most common clinical indications for es in this cohort were hypogammaglobulinemia ( %), neutropenia ( %), immune dysregulation ( %), lymphopenia ( %), and combined immunodeficiency ( %). the gender distribution was % male (n= ) and % female (n= ); % of cases were pediatric (< years, n= ) and % were adult (>= years, n= ). positive results were reported in cases ( %), comparable to the overall diagnostic yield of es at our laboratory (retterer et al., ) . this included / ( %) of cases submitted as proband-only, / ( %) submitted as a trio, and / ( %) submitted as duo, quad or alternative family structure. diagnostic results spanned different genes and recurrently reported genes with identified pathogenic variants included flg (n= ), rag (n= ), sbds (n= ), lrba (n= ), stat (n= ), and pik cd (n= ). variants possibly associated with the phenotype, but not considered diagnostic, were reported in % of cases (n= ), while % of cases (n= ) had reportable findings in a candidate gene. conclusions: these results support that exome sequencing for individuals with immunologic-based phenotypes has similar diagnostic utility as the overall rate for clinical es. immunologic es cases with trio family structures have a higher diagnostic yield than proband-only cases, as inheritance information improves confidence in classification of variants as pathogenic or likely pathogenic. genetic heterogeneity, as demonstrated by the large number of distinct genes represented in this cohort of diagnostic es cases and rapid candidate gene discovery make es a valuable tool for genetic diagnosis in patients with immunological disorders. introduction/background: vaccination response to the -valent polysaccharide vaccine (ppsv ) is often used in the diagnosis of common variable immunodeficiency (cvid). unfortunately, ppsv titers are often difficult to interpret and many cvid patients are started on igg replacement therapy (igrt) before adequate evaluation. unlike ppsv titers, the enzyme-linked immunosorbent spot assay (elispot) is independent of igrt and can provide an ex vivo functional measurement of specific antibody production on the b cell level. objectives: develop and test an elispot assay to better determine vaccination response to ppsv compared to ppsv titers in cvid patients on igrt, healthy controls, and igrt patients without immunodeficiency. methods: an elispot assay was successfully optimized and used to evaluate the ppsv -specific b cell response in healthy adult controls. elispots were performed on day , and day (when plasmablasts are best evaluated). ppsv titers were measured on day , day , and day . for igrt patients, flow cytometry for b cell subpopulations will be performed to further validate the assay. results: normal controls demonstrated a significant increase in ppsv antibody spot forming units (sfu) between day and after ppsv vaccination. ppsv titers showed generally robust initial titers at day , and no significant change at day , with day results pending. conclusions: here we optimized an elispot assay that functionally measures the specific antibody response to ppsv in normal controls with ppsv titer results pending. we are actively recruiting patients on igrt (both with cvid and without immunodeficiency) for comparison. we hope to validate our assay as a useful alternative to ppsv titers that may be particularly useful when patients are on igrt. previous studies have demonstrated bal may be a sensitive diagnostic method for treatment failures of clinically diagnosed pneumonias, even if performed under treatment with empiric antibiotics, and can lead to a culture-directed change in antimicrobial therapy in the majority of cases. however, it has also been reported that at least in one piddchronic granulomatous disease (cgd)the diagnostic yield of bal was inferior to that of other diagnostic methods (marciano et al., ) . further information on the diagnostic yield of bal and other invasive procedures to obtain a specific organism diagnosis in pidd patients with suspected pulmonary infection is needed. objectives: to characterize the yield of diagnostic procedures used in pidd patients with pneumonia or other suspected pulmonary infections at the ucsf benioff childrens hospital. methods: we screened our database of pidd patients (encompassing patients seen from september , to september , cared for by the pediatric immunology service at the university of california, san francisco to identify patients with history of at least bal or other invasive diagnostic procedure (fna or open lung biopsy) for etiologic diagnosis of suspected pulmonary infection. if multiple bals were performed during a single episode of illness, only the first was used for this analysis. results: we identified pidd patients with history of at least one bal or other invasive diagnostic procedure, for a total of events. most procedures (n= ) were performed at our institution, with documented in outside hospital records. patient diagnoses included cgd ( ), nemo deficiency ( ), cvid ( ), stat -deficient ad-hies ( ), dock deficiency ( ) , and mhc class ii deficiency ( ) . of bals, / ( %) grew a predominant organism, but only / ( %) were positive for an organism believed to be causative by providers and/or for which the overall result of the bronchoscopy affected antimicrobial treatment. bal yield was highest in patients with a clinical and/or radiologic diagnosis of pneumonia ( / , %). yield was poor ( / , %) in minimally or chronically symptomatic patients referred for bal for interval changes on chest ct (i.e. suspected fungal infection). our nemo deficiency cohort had the highest rate of positive organism isolation by bal ( / , %) for diagnosis of pulmonary infection. in our cgd cohort, / ( . %) bals grew a causative organism. lung biopsy yielded positive organism isolation in / cases ( / in cgd, burkholderia cepacia and nocardia cyriacigeorgica; / in cvid) and fna in / cases of cgd (aspergillus fumigatus). fna or biopsy was done concurrently or after bal in cases; in / ( %), fna or biopsy, but not bal, was positive for a causative organism. conclusions: at our institution, bal overall had a % rate of causative organism isolation in pidd patients, but had up to a % rate of organism isolation in those with clinical and/or features of pneumonia. our rate of causative organism isolation was slightly higher than in previous reports. however, in specific instances, biopsy was still required to make a definitive diagnosis. bal may have limitations in certain populations of pidd patients, such as in cgd, but it may be a reasonable starting point in the diagnosis of pneumonia or worsening pulmonary disease in pidd. prospective research is needed to evaluate whether fna or lung biopsy, though more invasive, could result in overall shorter time to institution of appropriate directed therapy and shorter hospitalizations for specific pidd patients. introduction/background: prior to the introduction of newborn screening, cases of severe combined immunodeficiency often presented with severe or disseminated infections. herein, we report an infant from india who presented for evaluation and treatment of a periorbital mass, presumed to be malignant. however, he was found to have disseminated bacille calmette guerin (bcg), as well as multiple other infections, and was eventually diagnosed with x-linked scid. results: a -month old boy was born to unrelated parents in india. he initially presented with growing periorbital mass and fever for two weeks. pet scan showed hypermetabolic areas in the bone marrow, spleen, mesenteric lymph nodes, and left shoulder, along with the periorbital mass. biopsy of the mass revealed numerous b lymphocytes without malignant transformation. there was no evidence for malignancy on bone marrow biopsy, though numerous granulomas and a significant decrease in t lymphocytes were seen. peripheral blood flow cytometry showed a complete lack of t cells. the bone marrow biopsy was reexamined, and innumerous acid-fast bacilli were found. cultures grew mycobacterium bovis, and he was diagnosed with disseminated bcg disease. genotyping revealed a novel splice site variant in il rg, consistent with x-linked scid. further evaluation revealed multiple other infections. these included extended-spectrum beta lactamase-produce e. coli bacteremia, human metapneumovirus, cytomegalovirus, and pneumocystic jiroveci. he was also tested for vaccine-associated poliovirus because he had received the oral polio vaccine, but this was negative. four-drug therapy was started for the bcg, and the periorbital lesion completely resolved within several weeks. additionally, he was treated with intravenous ribavirin for the human metapneumovirus, and sulfamethoxazole-trimethoprim for the p. jiroveci. although the cmv was initially treated with ganciclovir, the virus eventually developed resistance and required treatment with foscarnet. due to his many infections, he underwent haploidentical stem cell transplant plus donor lymphocyte infusion from mom. this transplant failed to engraft, but a matched unrelated donor was eventually found, and our patient received a second bone marrow transplant. he currently is showing signs of engraftment, and is continuing to be treated for his multiple infections. conclusions: disseminated mycobacterial disease due to the bacille calmette-guérin (bcg) vaccination has been noted in cases of xlinked scid previously, often consisting of lymphatic, skin, and pulmonary manifestations. however, there is a paucity of published cases presenting with multiple other co-infections. nor are there reports of disseminated bcg presenting as a localized mass. this case highlights the unique considerations when evaluating a patient with immunodeficiency from another country, where vaccination practices and epidemiology differ. specifically, unusual presentations of infections or masses may warrant investigation for severe immunodeficiency. prototypic t-b+nk+ immune phenotype is caused by mutations in the iil ra gene. the il signaling has an important role during t-cell development in the thymus, contributing to cell proliferation and survival. in addition, in mouse models, the rearrangement of the t cell receptor genes, specifically the gamma locus (trg), has been shown to be regulated by il signaling. similar to other scid phenotypes, patients with il ra deficiency are predisposed to acquire opportunistic infections early in life and to display poor outcome and death, unless their immune system is restored by mean of hematopoietic stem cell transplantation (hsct). while most patients with il ra mutations have full il ra deficiency resulted in a severe t cell depletion, some have a partial deficiency with residual cells or leaky phenotype, or even present symptoms later in life. objectives: here we report two non-related infants detected by the israeli national newborn screening program for scid. despite having similar il ra missense mutation (f l) they displayed distinct clinical and immunological course, resulted in a completely different treatment approaches; observation in one patient with an unusual recovery, and hsct in the other patient methods: patient lymphocytes were examined for subset counts, thymic output (via excision circles), t cell receptor repertoire diversity (tcrb) and il ra expression and function. the pathogenic il ra mutation was found by whole exome sequencing (wes). high throughput immunesequencing was performed to characterize the trg repertoire. results: we established patients' diagnosis by validating the pathogenic il ra mutation, and showing profoundly impaired t cell immune work up and abnormal il ra expression and function, determined by stat phosphorylation assay. all these measurements improved over time for the patient with less severe clinical presentation, while remained low in the patient with the severe phenotype. characterization of their trg immune repertoire using high throughput immune-sequencing revealed restriction of t cell receptor repertoires of both patients upon their initial diagnosis, compared to healthy controls. however, skewed usage of variable (v) gene segments and abnormalities of the cdr length distribution were more prominent in the patient with the severe phenotype. conclusions: these studies illustrate the gap that exists in our understanding of other non-genetic parameters that may influence disease course and severity in patients harboring a similar genetic defect. furthermore, the results reinforce the role of il- signaling not only in cell proliferation but also in trg rearrangements introduction/background: dock immunodeficiency syndrome is primary immunodeficiency disease caused by loos of function mutations in the dock gene, which was known to play a critical role in the survival, proliferation and function of several types of immune system, especially lymphocyte. dock immunodeficiency syndrome is the most common cause of autosomal recessive hyper-immunoglobulin e syndromes (hies) and mainly expressed as recurrent infections and severe allergic disease affecting the skin. in addition, autoimmune features including systemic lupus erythematosus, hemolytic anemia or idiopathic thrombocytopenic purpura may be presented in dock immunodeficiency syndrome. objectives: we report a case of -month-old boy diagnosed with dock immunodeficiency syndrome, which was initially expressed as sle without recurrent skin infections. methods: a child with atopic dermatitis was admitted to another hospital because of fever lasting more than days accompanied by swelling of the hands and foot. he developed whole body edema, perioral purpura and oliguria. complete blood count was normal and blood urea nitrogen , creatinine and albumin levels were normal on his laboratory findings. however, c-reactive protein level was high at . mg/dl, coagulation parameters were abnormal (prothrombin time . sec; activated partial thromboplastin time . sec, d-dimer . ug/ml). so he was transferred to our hospital for further examination and treatment on the th day of fever. additionally, ulceration of the tonsil and maculopapular rashes on the abdomen and both legs were observed in physical examination. we suspected a meningococcal infection and administered antibiotics. however, no bacterial isolates were identified in the blood and csf culture test. fever persisted despite the administration of antibiotics. we checked immunoglobulin level, complement level and autoantibodies based on fever of unknown origin. immunoglobulin g, m and a were normal, complement fractions c , c , and ch were low at . mg/ dl, . mg/dl and . u/ml, respectively. antinuclear antibodies were positive at : with homogenous fluorescence. anti-ds dna antibody was positive at . . the tests for anti-ssa, anti-ssb, anti-ribonucleoprotein, anti-scleroderma , and anti jo antibodies were all negative. he developed leukopenia and thrombocytopenia over time. results: he was treated with steroid satisfied diagnostic criteria for systemic lupus erythematosus (sle) and fever subsided. he was confirmed lupus nephritis by renal biopsy later. because of onset of sle at the young age, we performed diagnostic whole exome sequencing and multiplex ligation-dependent probe amplification assays. conclusions: he was confirmed dock gene deletion (a deletion on one allele and point mutation on the other allele). he is preparing for hematopoietic stem cell transplantation due to autoimmunity and nonreversible parenchymal organ damage form infections although he has not yet experienced a life-threatening infection. introduction/background: during immunological investigation, it is important to distinguish those individuals who may hav e hypogammaglobulinemia (hypo) without fulfilling the criteria for the severe antibody deficiency common variable immunodeficiency (cvid) e.g. unspecified hypogammaglobulinemia from those with cvid. objectives: since low igg concentrations may support a diagnosis of cvid, we sought to investigate whether the measurement of additional igg subclass antibodies (iggsc) may provide further discrimination between patients with cvid and those with hypo. methods: iggsc concentrations were measured in serum samples from cvid patients (n= , : . m:f, median age . years, range - ) and hypo patients (n= - , : . m:f, median age . years, range - ). results: cvid patients had lower median iggsc concentrations for all iggsc: igg mg/dl (range - ) vs mg/dl (range - ); igg - mg/dl (range - ) vs mg/dl (range - ); igg - mg/dl (range - ) vs mg/dl (range - ); igg mg/dl (range . - ) vs mg/dl (range . - ). this was significantly lower for igg and igg (p= . and . respectively). a higher percentage of cvid patients had iggsc below the lower limit of the normal range compared to hypo patients: igg ( vs . %); igg ( vs . %); igg ( vs . %); igg ( . vs . %). % of cvid patients had low concentrations of or more iggsc vs only . % of hypo patients (p= . ). . % and . % of hypo patients had low levels of or iggsc, respectively. conclusions: igg subclass measurements may have some utility in distinguishing cvid patients from hypogammaglobulinemia patients. introduction/background: the thymus is often removed during cardiac surgery for repair of congenital heart disease, but the extent of tissue removed varies between procedures and surgeons. previous studies have shown decreased t cell counts after thymectomy but there is limited data on the effect of thymectomy on t cell receptor excision circle (trec) levels and infection risk. objectives: to determine the effect of partial and complete thymectomy during cardiac repair surgery on trec levels and infection risk. methods: a retrospective study of electronic medical records was performed on children who received cardiac surgery before age one at new york presbyterian/morgan stanley childrens hospital between / / and / / . patients with heart transplant or primary immunodeficiency were excluded. data was recorded on trec levels (abnormal trec < copies/μl on new york state newborn screen), number of positive cultures, viral pcr panels, and infiltrates on chest x-ray. patients were followed for a minimum of six months after cardiac surgery. study was irb approved. results: cardiac surgery was performed on patients and data was available for patients. of patients included, had a partial and had a complete thymectomy. trec levels after surgery were recorded for patients. only % of patients had an abnormal trec level on newborn screen. there was no difference between partial and complete thymectomy on risk of abnormal trec (p = . ) and mean total number of infections at months (p = . ). conclusions: thymectomy rarely causes low trec levels in children undergoing cardiac surgery. complete thymectomy does not significantly increase infection rates in these children compared to partial thymectomy. these findings are possibly due to presence of ectopic thymic tissue or thymic regeneration and are reassuring for children undergoing complex cardiac surgeries. however, long-term follow-up of these children will be necessary to determine residual function of the thymus and clinical response. introduction/background: new sequencing techniques have revolutionized the identification of the molecular basis of primary immunodeficiency disorders (pid), not only by establishing a gene-based diagnosis, but also by facilitating defect-specific treatment strategies, improving quality of life and survival, and allowing factual genetic counseling. because these techniques are generally not available for physicians and their patients residing in developing countries, collaboration with overseas laboratories has been explored as a possible, albeit cumbersome, strategy. objectives: we sought to determine whether blood collected by guthrie cards could be shipped across continents by regular airmail to a cliaapproved laboratory for confirmatory testing. methods: blood was collected and blotted onto the filter paper of guthrie cards by completely filling three circles. we enrolled male patients with presumptive x-linked agammaglobulinemia (xla) cared for at the vietnam national children's hospital, their mothers and several sisters for carrier analysis. dbs were stored at room temperature until ready to be shipped together, using an appropriately sized envelope, to a cliacertified laboratory in the us for sanger sequencing. the protocol for sanger sequencing was modified to account for the reduced quantity of gdna extracted from dbs. results: high-quality gdna could be extracted from every specimen. btk mutations were identified in of patients studied, confirming the diagnosis of xla in % of the study cohort. type and location of the mutations were similar to those reported in previous reviews. the mean age when xla was suspected clinically was . years, similar to that reported by western countries. two of mothers, each with an affected boy, had a normal btk sequence, suggesting gonadal mosaicism. conclusions: dbs collected on guthrie cards can be shipped inexpensively by airmail across continents, providing sufficient high-quality gdna for sanger sequencing overseas. using this method of collecting gdna we were able to confirm the diagnosis of xla in of vietnamese patients with the clinical diagnosis of agammaglobulinemia. introduction/background: znf is a positive regulator of stat expression. it has recently been described that nonsense mutations in znf account for the stat -like phenotype in four autosomalrecessive kindred. patients presented with reduced stat expression and diminished th cell numbers, in absence of stat mutations. objectives: here, we decribed a turkish case having nonsense mutation in znf developed dual malignancy in years. results: a -year-old female patient presented with severe eczema, recurrent cold skin abscesses, herpetic skin lesions, sinopulmonary infection, otitis media and hearing loss. the parents are cousins. two younger sisters of the index case had eczema and recurrent skin infections since their infancy period. physical examination of the patient revealed severe eczema, high palate, micrognathia, maxillary hypoplasia and hearing loss. laboratory findings showed reversed cd /cd ratio, high serum ige level ( . u / l) and low igg level ( mg / dl). the patient was diagnosed as hyper ige syndrome and ivig therapy ( mg / kg, every weeks) was initiated because of igg subgroup deficiency and recurrent sinopulmonary infections. at the age of , a polypoid mass filling the left nasal cavity was detected in her examination. paranasal sinus ct revealed a mass obliterating the left nasal cavity, left ethmoid sinus and frontal sinus. immunohistochemical stains showed a small round cell malignant tumor in the nasal cavity. she was treated with chemoradiotherapy successfully. a homozygous nonsense mutation has been detected at exon in the znf gene (c. c> t) (kindly provided by grimbacher's lab) very recently. she developed papillary thyroid carcinoma two years after completing the cancer therapy. conclusions: the relationship between znf defect and cancer development is unknown. the development of a second malignancy in this patient for a short time of completing the therapy might imply us a tendency for malignancy in znf patients. additionally, the likelihood of increased radiosensitivity in these patients should be taken into consideration. introduction/background: prometic % igiv contains purified igg, % as monomer; with a distribution of igg subclasses proportional to that in native human plasma. we report the interim results from a phase trial in the usa of prometic % igiv in adults and children with pidd. objectives: this was a phase , single-arm, open-label, multicenter trial to evaluate the safety, tolerability, and efficacy of prometic % igiv in adults and children with pidd. methods: adults and pediatric subjects with pidd on a stable dose of igg replacement therapy ( - mg/kg) for at least months with serum igg trough levels > mg/dl were included. subjects received prometic % igiv every to weeks for approximately year at the same dose and schedule as their previous igg replacement therapy. results: an interim analysis was conducted when data were available on adult subjects who received at least one dose of prometic % igiv (total of infusions), with subjects receiving at least months of treatment (exposure = . subject years). at this time, pediatric exposure was only . subject years. there were no serious bacterial infections (sbis) reported, and rate/yr of infections other than sbis was . which was comparable to rate while on commercial product ( . ) all subjects achieved an igg trough level > mg/dl. there were no deaths, and no subject had a study drug-related serious adverse event or an adverse event that resulted in permanent discontinuation of study drug. a total of adverse reactions (ar) ( . /infusion) occurred in subjects ( . %), with infusions ( . %) associated with an ar. most infusions ( . %) were completed without a rate reduction. most ars were mild or moderate in severity, with severe ars ( . /infusion) occurring in subjects ( . %). the most frequent ars were headache ( . % subjects or . /infusion) and fatigue ( . % subjects or . /infusion). conclusions: in adults treated with prometic % igiv, there were no sbis and infusions were well tolerated. director, sean n. parker center for allergy and asthma research at stanford naddisy foundation, professor of medicine and pediatrics, stanford university introduction/background: desensitization to food allergies is being studied in clinical trials using oral immunotherapy (oit). there are limited data regarding the immune changes associated with successful oit. epigenetics involves heritable changes in gene function without modification of the underlying dna sequence. this is mediated by methylation, histone modification, or changes in microrna. objectives: to study methylation changes in the loci of four key genes of immune cells involved in allergy, interleukin (il- ), interferon gamma (ifn-g), forkhead box protein (foxp ), and interleukin (il- ), comparing baseline to post-oit. methods: we completed a phase , randomized, placebo-controlled, multi-food oit trial using omalizumab, an anti-ige biologic, to facilitate desensitization for multi-food allergic individuals. double-blind, placebo-controlled food challenges (dbpcfcs) to multiple foods were conducted at entry and after weeks of treatment, the primary endpoint. omalizumab (n= ) or placebo (n= ) was administered for weeks, with oit for - foods starting weeks after the beginning of omalizumab or placebo. after weeks ( weeks of oit), participants underwent dbpcfcs to their offending foods. treatment failures (n= ) were offered open-label omalizumab. pyrosequencing of bisulfite treated genomic dna purified from pbmcs from each participant at baseline and post-oit was undertaken to investigate changes in methylation. results: forty-four participants achieved successful desensitization, defined as passing dbpcfcs to or more foods following oit. we found that the - cpg site in the il- promoter region is hypermethylated over time during successful multi-food oit (fdr-adjusted p < . by wilcoxon signed-rank test). the median % of methylation at baseline was . (interquartile range . %) and was . post oit (interquartile range . %). there were no statistically significant (with a significance level of fdr adjusted p value of . ) changes in the il- , foxp , or ifn-g loci in the cpg sites we studied. conclusions: these preliminary results suggest that one immune mechanism involved in successful desensitization may involve suppression of th function by hypermethylation of il- in immune cells in the peripheral blood. introduction/background: atopic dermatitis (ad) is a common chronic inflammatory skin disorder afflicting from infancy to adults with itching, scratching, and lichenification. objectives: we investigated the effects of esculetin from fraxinus rhynchophylla on atopic skin inflammation. methods: for induction of atopic skin inflammation, we exposed the ears of female balb/c mice with house dust mite (dermatophagoides farinae extract, dfe) and , -dinitrochlorobenzene (dncb) during weeks. results: oral administration of esculetin reduced dfe/dncbinduced atopic skin inflammation symptoms based on ears swelling and scratch numbers. the immunoglobulin (ig) e, igg a, and histamine levels in serum were decreased and inflammatory cell infiltration in skin tissue was reduced by the esculetin. it suppressed th , th and th responses by inhibiting the production of inflammatory cytokines such as tumor necrosis factor (tnf)-, interferon (ifn)-, interleukin (il)- , il- , il- and il- in the ear tissue. further, we investigated the effects of escueltin on activated keratinocytes, one of the most representative cells for studying pathogenesis of acute and chronic atopic skin inflammation. as results, esculetin suppressed gene expression of th , th and th cytokines and activation of nuclear factor-b and signal transducer and activator of transcription in tnf-/ifn-stimulated keratinocytes. conclusions: taken together, the results imply that esculetin attenuated atopic skin inflammation, suggesting that esculetin might be a potential therapeutic candidate for the treatment of ad. introduction/background: autoimmunity is often seen in common variable immune deficiency (cvid) with immune thrombocytopenic purpura (itp) being the most frequent manifestation at a prevalence of - % in cvid patients. in such patients, itp is often recognized and treated long before cvid, the implications of which are unknown. primary itp is a clinicopathologic diagnosis which includes an evaluation of other conditions that may mimic it including cvid. currently, it is unknown how frequently cvid is evaluated during the diagnostic workup of itp and what percentage of those patient actually have cvid. objectives: the two main objectives of this study were to determine the number of itp patients that had an igg level checked during their clinical course and if the globulin fraction can be used as a marker for hypogammaglobulinemia in itp patients at the time of diagnosis. methods: a retrospective chart review was undertaken at a large academic medical center of patients with a new diagnosis of itp between january and january . igg levels were collected and globulin fractions were calculated as the difference between serum total protein and albumin within days of the initial itp diagnosis. results: six hundred and twenty-three patients were found to have a new diagnosis of itp in the given timeframe. of these, only ( . %) had igg levels checked at any point during their clinical course. twelve of the ( . %) had hypogammaglobulinemia with only of the ( %) having a formal immunologic follow-up evaluation. two were diagnosed with a primary immunodeficiency ( cvid and ctla deficiency). globulin fractions were calculated on patients at the time of itp diagnosis. mean calculated globulin fraction in hypogammaglobulinemic patients was . (range . . ), versus . (range . . ) in patients without hypogammaglobulinemia (p= . ). conclusions: the diagnosis of cvid is often delayed from the onset of symptoms which can include autoimmune conditions such as itp. our data indicate that clinicians do not routinely check igg levels at the time of itp diagnosis which should be considered standard of care based on the current guidelines. our data suggest that although calculated globulin fractions were significantly lower in hypogammaglobulinemic patients, the variability was substantial and hypogammaglobulinemic patients would be missed using this as an indicator of low immunoglobulins. future directives include a prospective study using igg levels checked at the time of itp diagnosis, with formal evaluation for cvid in any hypogammaglobulinemic patients to evaluate the true prevalence of cvid among itp patients. introduction/background: autoimmune lymphoproliferative syndrome (alps) is a disorder characterized by immune dysregulation due to the rupture of lymphocyte homeostasis, which occurs as a result of mutations in the apoptotic pathway mediated by fas. this disease is sometimes misdiagnosed due to its variable phenotypic expression and the overlapping of symptoms with many other hematological and immunological disorders. ( ) a patient diagnosed with evans-fisher syndrome (sef) was referred to the laboratory for immunity studies. this is a patient who from infancy had multiple admissions for severe sepsis, with severe anemia and severe thrombocytopenia. in the evaluation of the history of the disease by the work team, a series of clinical data was observed that suggested the possibility of the patient presenting an alps, so it was decided to incorporate as part of the study, the quantification of the cells t cd + cd -cd -(double negative t cells or dnt) that express tcr +. the differentiation pathways of dnt tcr + and the role of fas in this process are not clear, some authors hypothesize that they may be represented by direct descendants of chronically activated positive simple t cells, with deregulated co-receptors, in a state of differentiation in which they were destined to perish by fas-mediated apoptosis. ( ) ( ) ( ) ( ) the population of tcr + dnt cells required for diagnosis must be derived from the particular study of each laboratory in their populations, but several working groups agree that the pathological limit values are , % in total lymphocytes or , % in cd + lymphocytes. ( ) evidence shows that dnt alps cells are not simply accumulated in senescent withdrawal; they and their precursors remain active and proliferate under the influence of activation signals. ( ) although numerous genetic deficiencies lead to lymphoproliferation of t cells, only that caused by a defective fas pathway is dominated by negative double t cells. ( ) in clinical immunology, the distinction between cd ra + and cd ro + cells is particularly useful for determining the state of the "naive" cell compartment in relation to its thymic origin. primary immunodeficiency disorders are characterized by a decrease / absence of thymus performance, and often involve a decrease in cd ra + t lymphocytes. scientific evidence suggests the important role of the "naive" subpopulation in the origin and maintenance of self-reactive effectors in the periphery. ( ) regulatory t cells are able to effectively control autoreactive t cells, especially when negative thymic selection is defective. its differentiation and function is controlled by foxp . the decrease or absence of regulatory t cells leads to autoimmune diseases, specifically those mediated by cd + t cells, and to lymphoproliferation characterized by multiorgan inflammation and other autoimmune disorders. ( , ) objectives general: establish the diagnosis of a pediatric patient with suspected primary immunodeficiency due to dysregulation, an autoimmune lymphoproliferative syndrome (alps). specific: evaluate the tcr + dnt population, relevant in the diagnosis of alps. quantify cell populations that exhibit markers of activation, differentiation and regulation of the relevant t cells in the study of this disease. methods: monoclonal antibodies (mab) cd fitc / cd pe / cd pc triple labeled (beckman coulter), tcrapc, cd pe, cd rapercp, cd rofitc, cd fitc, cd rope, cd apc, cd f, cd pe, cd percp, cd pe, cd pe, cd percp, cd fitc, cd fitc were used , hla dr and the cd fitc / cd pe / foxp apc regulatory t cell kit, all from miltenyi biotec. the trial included the use of a healthy control. both samples were processed in unison to ensure reproducibility. the samples were acquired in a beckman coulter gallios cytometer, with the use of the kaluza program, version . . the analysis strategy included the formation of overlapping "gate" windows for the quantification of dnt tcr + and regulatory t cells. results: the patient studied showed . % of tcr + negative double t cells, in relation to . % of the healthy control. the presence of this population of t cells in patients diagnosed with evans syndrome, even in the absence of lymphoproliferation, is consistent with alps. ( ) (graph ) the presence of increased tcr + dnt and lymphoproliferationexpressed as lymphadenopathies and splenomegaly of noninfectious or malignant cause, of at least months of evolution; plus the typical immunohistological findings found in the patient's lymph node biopsy (paracortical hyperplasia), the presence of autoimmune cytopenias (hemolytic anemia and thrombocytopenia) and hypergammaglubulinemia, led to the "probable diagnosis" of alps in the patient studied. the accumulation of dnt in the lymph nodes and other peripheral organs is accompanied by qualitative changes in the composition of the t cell repertoire, so that the immunophenotype performed included markers of activation, differentiation and regulation. the evaluation of the activation on t lymphocytes and nk cells showed that there was a slight decrease in the expression of the receptor for il- , cd . the cd + cd + population was expressed in greater percentalthough discretely-in the patient than in the healthy control, which was directly related to the presence of hypergammaglobulinemia, elevated iga and the presence of autoantibodies. it was also highlighted by the high expression of the cd + cd + autoreactive population in the patient studied ( . % vs . %). in order to know the impact on cell differentiation, the subpopulations of effector and memory cells for cd + and cd + t lymphocytes were evaluated by combining the cd ra and cd ro isoforms. cd ra is expressed in naive t lymphocytes. particularly, the cd + cd ra + population has an essential function as a suppression inducer, and it is diminished in the patient studied, in relation to the control. ( . % vs. . %, respectively) the same behavior was shown by the cd + cd ro + memory and effector cells, . % vs . %. in the cd + population itself, it was possible to confirm the presence of a clone that expressed both receptors (cd ra and cd ro), probably a temra population (terminally differentiated effector memory cells). when comparing the results, it was unexpectedly found that there was a slight decrease between patient and control ( . % vs . %). as the characteristic phenotype of this cell population could not be corroborated, conclusive assessments could not be made. the combination of cd isoforms was also used to study cd + t lymphocytes, but similar behavior between patient and healthy control was observed. (the lab results are shown in table ) the analysis of the regulation of the immune response showed a decrease in the cd + cd + population and the expression of the foxp transcription factor in the patient with respect to the control. ( . % vs. . %, . % vs. . %, respectively) (graph ) conclusions . the markedly high quantification of cd -cd -tcr + t lymphocytes allowed to define the "probable diagnosis" of autoimmune lymphoproliferative syndrome in the patient under study. . the increased activation of cd + cells and the presence of the cd + cd + self-reactive population, largely responsible for autoimmunity and lymphoproliferation in the alps, could be confirmed. . the decrease in the cd + cd ra + t-cell suppressor-inducing population evidenced corroborated its involvement in this disorder by primary immunodeficiency, in relation to the thymus dysfunction that originates and maintains self-reactive effectors in the periphery. . the decrease in the expression of the foxp transcription factor observed, points towards a low regulation of the response that leads to autoimmunity and to lymphoproliferation, specifically mediated by cd + t cells. allergy and immunology arnp, nicklaus children's hospital allergy and immunology division director, nicklaus children's hospital introduction/background: specific antibody deficiency syndrome is characterized by a weak antibody response to bacterial polysaccharide antigens when no other immune system abnormalities can be found. low titers to pneumococcal vaccine have become one of the most frequently recognized immune abnormalities in pediatric patients with recurrent sinopulmonary infections. nonetheless, insufficient data and lack of consistent testing of the response to pneumococcal polysaccharides continues to affect the optimal diagnosis and management of this specific antibody deficiency. objectives: to characterize the pre-and post-immunization igg antibody trend for each specific serotype included in the pneumococcal -valent conjugate vaccine (pcv ), as well as others that are routinely tested, in a cohort of pediatric patients with recurrent sinopulmonary infections. secondarily, to understand differences in the immune response to the vaccine booster between age groups. methods: this retrospective review identified patients with recurrent sinopulmonary infections. in this cohort, required an immune workup, and were found to have low pneumococcal titers needing a pcv vaccine booster. baseline pneumococcal serotype-specific antibody titers at initial visit and weeks after the vaccine booster were obtained. patients were categorized by age: years, - , - , and - . an adequate response to the pneumococcal conjugate vaccine was deemed to be a -fold increase over baseline and/or a post-immunization titer of . μg/ml or greater. results: overall, pcv booster provided a significant improvement in the number of protective titers, increasing from . ( % ci: . - . ) serotypes at baseline to . ( % ci: . - . ) serotypes at weeks (p < . ). this increase correlated with improved clinical outcomes % showed no signs of recurrent infection after the first booster and % after a second dose. all those who did not improve clinically suffered from co-morbidities (genetic abnormalities and rheumatologic diseases). post-immunization antibody concentrations were significantly higher than at baseline for all serotypes (p < . ) and only , n, and f did not exhibit a greater than -fold increase (p > . ) weeks following the booster. across age groups, only , f, and v showed pre-immunization differences in titers. there were no differences between ages in post-immunization titer levels for all serotypes. similarly, all age groups had a comparable number of baseline titers (p = . ) and at follow-up (p = . ). conclusions: in pediatric patients with recurrent sinopulmonary infections, an additional pneumococcal booster proved to be effective in the protection of these children from further infections. the pcv booster substantially increased titer levels and concentrations, and significantly improved clinical outcomes, independent of age. this investigation has provided us with a better understanding of the response after booster vaccination, and its role in the protection of patients from recurrent sinopulmonary infections. further studies are needed to elucidate whether a fifth dose of pcv should be optional as part of the vaccination schedule of patients with recurrent sinopulmonary infections. introduction/background: many primary immunodeficiencies (pids) share overlapping presentations, complicating clinical diagnosis. due to the ability to include many genes in one assay and the rapid turnaround time that these panels allow, expanded next-generation sequencing (ngs) panels are valuable in facilitating the diagnosis of patients with pids. objectives: we aimed to determine the clinical utility of an expanded ngs panel for the genetic diagnosis of patients with suspected pids. methods: we performed a retrospective analysis of the clinical utility of a -gene pid ngs panel used in a clinical diagnostic laboratory. from april to october , panels were ordered for patients with suspected or known pids. results: seventy-four pathogenic or likely pathogenic (p/lp) variants were identified in ( . %) patients. eight ( . %) of the p/lp variants were copy number variations ( deletions, duplications). fifty-two patients ( %) had p/lp variant, and patients ( . %) had p/lp variants. of positive patients, . % (n = ) were heterozygous carriers for autosomal recessive conditions where a second variant was not identified. twenty-two patients had p/lp heterozygous variants in genes with autosomal recessive and autosomal dominant inheritance patterns, in which the positive findings may or may not explain the patient's phenotype. for example, variants in this category were heterozygous variants in tnfrsf b (taci). genetic diagnoses were established or likely in % of patients with p/lp variants ( . % of all patients). five patients were heterozygous for a single p/lp variant and a variant of unknown significance in the same autosomal recessive gene. four patients in whom a genetic diagnosis was determined were also heterozygous carriers for a second, unrelated condition. one patient was found to have two distinct genetic diagnoses. variants of uncertain significance (vus) were identified in most ( . %) patients. the average turnaround time from test requisition to return of results was days. in total, % percent of genetic diagnoses were for conditions that are treatable with hematopoietic cell transplantation. conclusions: these results illustrate the utility of broad ngs panels for the diagnosis of patients with pids. introduction/background: severe combined immunodeficiency (scid) is a life-threatening immune deficiency manifest by extreme susceptibility to infection. early diagnosis and definitive treatment with either hematopoietic cell transplant (hct) or, in select cases, gene therapy (gt) has been shown to significantly improve survival. newborn screening for scid has allowed for the opportunity to promptly identify these patients before significant infections occur. at ucsf, newly diagnosed infants with scid are admitted to the hospital for management and remain in isolation for definitive treatment and until adequate immune reconstitution occurs. previous work by our group demonstrated up to % of these parents experience psychosocial trauma manifested by depression and post-traumatic stress disorder (ptsd). the psychosocial challenges that contribute to depression and ptsd in these parents have not been qualitatively described. objectives: to understand the range of experiences and feelings of parents/caregivers of infants with scid diagnosed by newborn screening throughout their prolonged hospitalization and isolation in order to better support patients, parents, and their families. methods: voluntary participation was elicited from parents of children with scid who were status post hct/gt for one year or longer. semi-structured, in-person interviews lasting approximately - minutes were conducted with parents; interviews were recorded and transcribed. parents were asked to discuss their experiences from first notification of an abnormal screening result through discharge after hct or gt. emerging themes were identified from the transcribed interviews. results: we interviewed mothers and fathers of infants with scid. six infants received hct, while one underwent gt for ada-scid. all children were alive and well at the times when interviews were conducted. overall, once admitted, parents reported feeling well supported by the medical team and support staff. however parents identified a number of stressful events. uniformly reported key stressors included: receiving the first phone call regarding their childs abnormal newborn screening results, preparing for hct, coping with prolonged isolation, and transitioning from hospital isolation to care with ongoing isolation at home. other challenges described reflected the additional stressors of caring for a newborn, including coping with postpartum depression. overall, we identified three major themes encompassing the challenges faced by parents of hospitalized scid patients: (i) loss of normalcy and control over multiple aspects of life; (ii) prolonged waiting periods (especially the wait between diagnosis and hct and between hct and evidence of t cell engraftment); and (iii) perceived lack of guidance on realistic expectations during the hospital stay . parents sought and relied on peer support from other scid parents to learn about coping with the nuances of daily life as a parent of an infant with scid. conclusions: we identified multiple psychosocial stressors and challenges uniquely faced by parents and caregivers of infants diagnosed with scid by nbs. parents described barriers to caring for their own physical and mental health, which can be especially harmful to parents experiencing postpartum depression. recognizing these challenges allowed for the identification of opportunities to improve both healthcare delivery to their children and institutional support for future families affected by scid (table i) . emphasis should be placed on providing parents with scid-specific resources as early as the time of diagnosis, connecting parents with scid support networks, and facilitating access to psychosocial and mental health services for caregivers introduction/background: chronic granulomatous disease (cgd) is caused by a genetic defect that impairs phagocyte function. this disease results in recurrent infections and granuloma formation. rarely do patients develop cutaneous symptoms, unless associated with autoimmune disorders such as systemic erythematous lupus. previously described cutaneous findings include granulomas, abscesses, photosensitivity, malar rash, discoid lupus, vasculitis, and rarely vesicular rashes. here we describe two term infants diagnosed with x-linked cgd who present, in addition to frequent infection, with a unique papulopustular skin rash initially diagnosed as non-classic appearing eczema refractory to usual eczema treatment and antibiotics. objectives: to characterize cutaneous findings in x-linked cgd and emphasize the importance of considering further work in patients who present with similar rashes in conjunction with concerning features for primary immunodeficiency. methods: each infant was diagnosed with cgd based on abnormal dhr testing and/or genetic evaluation. after obtaining consent from both families, we have documented photographs of the development of rash in two newly diagnosed infants with cgd. one infant underwent cutaneous biopsy with resulting pathologic evaluation. results: our first patient presented at months of age with episodic fever with chronic leukocytosis, iron deficiency anemia, thrombocytosis, elevated inflammatory markers, proctocolitis with elevated calprotectin, and rash. egd with flexible sigmoidoscopy showed no signs of inflammatory bowel disease, the left colon had macroscopically raised erythematous lesions but was microscopically normal with no signs of active colitis. he was initially diagnosed with fpies and eczema in the setting of diagnosis of various infections (otitis, upper respiratory illness). the skin rash was described as non-pruritic, generalized pink-purple papulopustular lesions with a pink base, most prominent on upper and lower extremities, mostly sparing the trunk. skin biopsy histopathology revealed an essentially unremarkable appearing epidermis and within the dermis, there was a superficial perivascular lymphohistiocytic infiltrate. eosinophils and plasma cells were not abundant. the histologic changes were thought to be non-specific, but could be seen in a drug reaction or urticaria. a viral exanthem could also demonstrate these changes. giemsa stain and a stain for mast cell tryptase revealed normal numbers of mast cells in the biopsy specimen. he was not on any oral medications at the time and did not respond to treatment with topical moisturizers, oral antihistamines, topical steroids, or any standard for eczema care. he had a maternal uncle who passed away in infancy from infection. heightened clinical suspicion for primary immunodeficiency led to obtaining a neutrophil respiratory burst assay which was consistent with cgd and genetic testing was positive for x-linked cgd. pathogenic mutation nucleic acid change was c. c>t; hemizygous; amino acid alteration was p. arg>stp within the cybb gene locus. he had no other features of auto-immune disease including negative ana obtained later in his clinical course and his colitis diagnosed as cgd-associated colitis. his rash did not respond to systemic treatments for his colitis, oral antibiotics, and during hsct. our second patient presented at months of age with persistent fevers, inguinal lymphadenopathy, leukocytosis, elevated inflammatory markers, non-bloody diarrhea and rash. there was a family history of autoimmune gi disease. he presented to the hospital with fever of unknown origin with concern for infection, atypical kawasakis, and drug rash. his rash was described as a blotchy, pink, papular rash most prominent on the upper arms but also present on lower arms, legs, and chest, faint on face. there is some induration and thickness to the rash in some areas (confluent on the arms with more erythema and induration) and more faint pink papules (scattered on legs) elsewhere. a biopsy of his inguinal lymph node showed granulomatous lymphadenitis with neutrophilic abscess formation, and culture was positive for serratia marcescens. neutrophil respiratory burst assay was consistent with cgd and genetic testing was positive for x-linked cgd, mutation with the cybb gene. pathogenic nucleic acid change was c. + g>a; hemizygous; amino acid alteration was p. deletion of exon . the rash remained unchanged with treatment for infection, initiation of antifungal and bacterial prophylaxis, along with topical steroid therapy. conclusions: in patients who present with frequent infections and who have unusual cutaneous findings that do not fit with common infant rashes, consideration of work up for cgd should be pursued. specifically, generalized papulopustular rash with a negative skin biopsy can be misdiagnosed as atopic dermatitis, and in the right clinical context cgd should be considered. introduction/background: chronic lung disease in common variable immunodeficiency (cvid) is heterogeneous, and it is a leading cause of morbidity and mortality in this population. currently, the diagnosis and monitoring of chronic lung disease in cvid rely on radiographic findings, biopsy and/or pulmonary function tests. fractional exhaled nitric oxide (feno) is a noninvasive biomarker of airway inflammation, and it has been widely utilized to aid the in-office diagnosis, characterization, and management of inflammatory airway disease, such as asthma. however, exhaled nitric oxide in cvid patients with chronic pulmonary complications has not been examined. objectives: we aimed to determine fractional exhaled nitric oxide levels in cvid patients. methods: we measured exhaled nitric oxide in cvid patients with or without chronic lung disease. results: feno measurements were obtained in cvid patients (mean age: , range - ). five patients had no known lung disease; patients had chronic lung disease (bronchiectasis, n= ; lung granulomas, n= ; hepatopulmonary syndrome, n= ). four patients were on inhaled steroid (bronchiectasis, n= ; lung granulomas, n= ), patients were on systemic steroid (lung granulomas, n= ; hepatopulmonary syndrome, n= ), and patient was on oral budesonide (no known lung disease). feno was elevated (> parts per billion [ppb]) in of patients, of whom had known chronic lung disease. patients with granulomatous lung disease had higher feno (mean . ppb, range - ppb) compared to patients without known lung disease (mean ppb, range - ppb, p= . ), patients with bronchiectasis (mean . ppb, range - , p= . ), and the patient with hepatopulmonary syndrome ( ppb). feno levels remained elevated in granulomatous lung disease in patients with inhaled or systemic steroid use. conclusions: this is the first report of feno measurements in cvid patients. feno is elevated in a subgroup of cvid patients, and it may differ according to the underlying lung pathology. further investigation is warranted to determine the utility of feno in the diagnosis and management of chronic lung disease in cvid. professor of pediatrics, university of british columbia introduction/background: whole exome sequencing (wes) has revolutionized the discovery, diagnosis, and treatment of primary immune deficiency diseases (pids), a group of disorders with high genetic and phenotypic heterogeneity. current bioinformatic analysis approaches for wes rely heavily on population databases to exclude variants present in the populations represented by these databases. this approach may confound the ability to detect true disease causing variants, and conversely, flag variants that are absent from population databases only by virtue of originating from minority populations. ultimately, the pathogenicity of novel variants can only be mechanistically established through rigorous biochemical and functional validation. objectives: to evaluate the pathogenicity of a novel homozygous variant in caspase recruitment domain family member (card ) (c. g>t, p.c y), in a patient with features of combined immunodeficiency (cid). methods: research study protocols were approved by our institutional review board. six members of one family (the affected child, healthy siblings and their parents) were enrolled. written informed consent for genetic testing and participation was provided by the parents for their children. genetic, bioinformatic, biochemical and immunological investigations were performed. results: targeted sanger sequencing confirmed the presence of the homozygous card variant c. g>t, p.c y in the index patient (and subsequently in one apparently healthy sister). each parent was found to be a heterozygous carrier of the variant. nfkb activation in vitro was found to be normal for both the index patient and sister, and was indistinguishable from unaffected family members. conclusions: although multiple in silico tools predicted the c. g>t, p.c y card variant to be pathogenic, the patients b and t cells did not have aberrant nfkb activation in vitro, as would be predicted given the central role of card in nfkb activation. this practical experience highlights how imperative it is to functionally characterize novel variants found by wes, even when variants are predicted to be damaging. chief, clinical immunology -faculdade de medicina do abc introduction/background: gata is a zinc finger transcription factor essential for embryonic and definitive hematopoiesis. heterozygous mutations leading to gata deficiency were first described in . the age of clinical presentation ranges from early childhood to late adulthood, with most of them in adolescence to early adulthood. patients present clinical findings as monocytopenia, nontuberculous mycobacterial infections, myelodisplasia, viral (mainly hpv and ebv), fungal and bacterial infections. patiens may arise with many phenopytes, showing how complex is the effect of this transcription factor. objectives: we report a patient with gata mutation. methods: report: a year old female patient was referred due to a mycobacterial non-tuberculosis pneumonia. she was a healthy child until puberty. at the age of she presented genital herpes and recalcitrant vulvo-vaginal warts (hpv). at , she complained of lower back pain with no diagnosis for months, medication was unnefective, and she developed ischemic stroke. hemiparesia was mild and temporary. endocarditis with no agent identified was also diagnosed. therapy with acetilsalicilic acid was mantained until the age of . five years later, vaginal hpv spread through vulve and anal region evolving to neoplasia. she was submitted to surgery, radio and chemotherapy until october . one year later, profound cytopenias led to hospitalization. during that time, she had cough and fatigue and pulmonary tuberculosis was diagnosed. despite therapy with rifampin, isoniazid and pyrazinamid, there was no improvement. bronchoalveolar lavage was positive for mycobacterium avium. main immunological evaluation showed: monocytes ( %), t cells /mm ; cd +: ( , %), cd +: ( %); b cells /mm ( , %); nk: /mm ( , %); normal immunoglobulin levels; incomplete response to pneumococcus serotypes; igm and igg positive for cmv; negative serologies for hiv, ebv and htlv / . molecular analysis identified an heterozygous mutation c. c>tp.(thr met) in gene gata . results: we report a patient with gata mutation. the same gata mutation was previously described in national institute of health nih-usa (n= ) and in australia (n= ). conclusions: it took almost years for diagnosis suspicion. gynecologists should be warned about this diagnosis in order to improve patients prognosis. early diagnosis is crucial with adequate prophylaxis, prompt treatment of infection, surveillance of malignancy, and, moreover, family screening and genetic counseling. this is the first report of this mutation in latin america. introduction/background: severe combined immunodeficiency (scid) due to adenosine deaminase (ada) deficiency was the first human monogenic disease to be approached with gene therapy, and ongoing research advances over years led to the approval by the european medicines agency of a stem cell gene therapy product for its treatment using a gammaretroviral vector (gv), strimvelis®. despite the high success rate using gvs for ada gene transfer without vector-related complications, the development of leukoproliferative complications from the use of gvs in gene therapy of other disorders led us to develop lentiviral vectors (lvs) to deliver the corrective ada sequence/cdna (corrigan-curay et al, mol ther ; modlich et al, mol ther .). lvs, typically derived from hiv- and devoid of all viral genes, can be produced in a self-inactivating (sin) configuration, in which the viral long-terminal repeat enhancers are absent, eliminating the major identified cause of insertional oncogenesis due to gvs. we developed a lv (efs-ada) that carries a normal human ada cdna (codon-optimized) and demonstrated in pre-clinical studies its efficacy to transfer and express the ada protein, with evidence of significantly decreased potential for insertional mutagenesis compared to gammaretroviral vectors using the immortalization (ivim) assay and in murine bone marrow transplant models (carbonaro et al, mol ther, ) . this new lv was evaluated for safety and efficacy in parallel clinical trials of gene therapy for ada scid performed at sites in the u.s (university of california, los angeles and the national institutes of health {nih} clinical center) and u.k. (university college london/great ormond street hospital) enrolling subjects between and . we report here results from the patients treated in the u.s. between - . objectives: this is a prospective, non-randomized phase i/ii clinical study to assess the safety and efficacy of efs-ada lentiviral vector stem cell gene therapy in ada-scid subjects older than month. methods: subjects with ada-scid were enrolled, screened to document eligibility and underwent bone marrow harvest ( - cc/kg). bone marrow was processed to isolate cd + cells, which were pre-stimulated by overnight culture in serum-free medium containing c-kit ligand, flt- ligand, and tpo followed by culture with the efs-ada lentiviral vector overnight. subjects received a single dose of busulfan ( mg/kg) iv for reduced intensity conditioning. cells were removed from culture, washed, formulated and administered by iv infusion at least hours after busulfan administration. enzyme replacement therapy (ert) with pegylated bovine ada (peg-ada) was continued for one month post-transplant and then stopped. subjects were followed over months to assess safety and efficacy end-points. results: with - months follow-up, overall survival is %. eventfree survival has also been %, as all subjects are alive, remain off peg-ada ert, and none have required a second transplant. successful engraftment of gene-corrected cells was observed in all subjects at months, and persisted over the months of observation, based on vector gene marking in granulocytes and peripheral blood mononuclear cells (pbmcs), changes from baseline in rbc ada enzyme activity, and levels of metabolic detoxification of deoxyadenosine nucleotides. immune reconstitution was observed in all subjects and was sustained over the two years of observation, based on improvement of peripheral blood absolute lymphocyte counts and lymphocyte subsets (t, b and nk cells, and naïve cd + t cells). eighteen of patients have been able to stop receiving immunoglobulin replacement therapy. subjects who had routine infections all recovered with standard of care treatment, and there were no severe or opportunistic infections. conclusions: conclusions: gene therapy using the efs-ada lv has a favorable safety profile and was efficacious in this trial. a current followon trial at ucla is using a cryopreserved formulation of the cell product and pharmacokinetic-adjusted busulfan dosing, sponsored by the california institute for regenerative medicine (clin - ) and orchard therapeutics. acknowledgements: this study was supported by research grants from the national institutes of health (u ai ; p hl - ; nhlbi gtrp rsas # and ) and the california institute for regenerative medicine (cl - ; fa - ); support from the ucla david geffen school of medicine human gene and cell therapy program and the ucla eli & edythe broad center of regenerative medicine and stem cell research; and funding from the nhgri intramural program. dbk has potential financial conflict of interest as a member of the scientific advisory board for orchard therapeutics and as an inventor on intellectual property which ucla has licensed to orchard therapeutics related to gene therapy for ada scid. introduction/background: x-linked severe combined immune deficiency (scid-xl) is a rare monogenic primary immunodeficiency disorder (pid) where male infants are born without an adaptive and innate immune system. it is a life-threatening disease due to patients inability to fight viral and bacterial infections. scid-xl is driven by any of the two hundred known pathogenic mutations in the interleukin gamma receptor (il rg) gene, which function is required for proper development of t, b and nk cells. the most effective treatment for scid-xl, if performed in the first few months of life, is allogeneic hematopoietic stem cell transplantation (allo-hsct). this treatment is limited by absence of match donors, incomplete immune reconstitution, graft versus host disease and the need for long-term immunosuppression. an alternative curative treatment for scid-xl would be genome editing-based gene therapy by ex vivo genome correction of the patients long-term hematopoietic stem cells (lt-hscs) prior to autologous stem cell transplantation (auto-sct). objectives: here we report a proof-of-concept genome editing-based approach for correcting scid-xl disease. methods: using a crispr/cas -raav platform, we deliver a fulllength codon optimized il rg complementary dna (cdna) at the endogenous start site in cd + hematopoietic stem and progenitor cells (hspcs). results: using an optimized genome editing protocol we achieved > % genome editing as early as h and a median of % genome targeting while retaining > % viability and promoting greater than % ex vivo expansion of cd + hspcs from healthy donors. we demonstrate that our approach retains proper il rg signaling function in t-cells derived from healthy male donors and rescues the lymphopoietic defect from a patients derived mobilized cd + hspcs both in vitro and in vivo. we further show robust in vivo primary human engraftment potential and multi-lineage hematopoietic reconstitution of il rg gene targeted hspcs: a median of % (bone marrow), % (spleen) and % (liver) of il rg genome targeted engrafted cells was achieved at four months following primary transplantation into nsg mice and a median of . % - % was detected at months from secondary transplants of il rg targeted hspcs, thus achieving clinical levels of editing of lt-hscs. lastly, our observation of ( ) an intact hematopoiesis derived from il rg targeted cd + hspcs combined with ( ) a normal karyotype analysis and ( ) our deep analysis of potential off-target activity that showed only off-target sites of no known functional significant with < . % frequency of off-target activity presents strong evidence for the safety of our genome editing approach. conclusions: in sum, this pre-clinical study provides specificity, toxicity and efficacy data supportive of continued development of genome editing to treat scid-xl. objectives: to determine the risk of gvhd in msd hsct for scid patients compared to matched related donor (mrd). methods: retrospective cohort study comparing msd with mrd and the outcome of gvhd in all scid patients who underwent hsct between and . all statistical analysis was done using ibm spss statistic software. results: scid patients underwent hsct, ( %) received gvhd prophylaxis. gvhd occurred in ( . %); / ( %) had gvhd prophylaxis compared to / ( %) that did not, p value = . . acute gvhd occurred at a higher rate in msd / ( . %) compared to mrd / ( . %) p value = . . we analyzed outcome also according to period of hsct. first periods was to ; hsct, msd: , mrd: ; all had gvhd prophylaxis and there was no difference in gvhd. the second period was to : hsct, had msd and had mrd, gvhd prophylaxis was used in . % in msd and % in mrd, p value = . . gvhd was significantly higher in the msd ( . %) compared to mrd ( . %) odds ratio of . ( ci . to . ) p value = . . conclusions: gvhd prophylaxis in msd transplant may have a role to be considered in scid patients. introduction/background: xiap deficiency (also known as x-linked lymphoproliferative disease type xlp ; mim: ) is an x-linked primary immunodeficiency associated with mutations in the gene encoding the x-linked inhibitor of apoptosis (xiap; mim: ). the pathophysiology is characterized by immune dysregulation, usually triggered by epstein-barr virus (ebv) infection. primary ebv infection is followed by hemophagocytic lymphohistiocytosis (hlh) with high grade persistent fever, splenomegaly, hematologic cytopenias and hepatitis. most patients die during this acute phase, and those who survive usually evolve with hypogammaglobulinemia, recurrent infections, cytopenias, inflammatory bowel disease (ibd) and low counts of inkt cells. dysbiosis of intestinal microbiota is believed to fuel ibd and possibly contribute to the initiation and/or perpetuation of the disease. experimental studies have provided solid evidence to support a role for the indigenous gut microbiota in the pathogenesis of autoimmune diseases, thereby raising the possibility that an altered gut microbiota is an environmental risk factor for xiap disease. objectives: in this study, we sought to investigate the identity and abundance of the bacteria in gut microbial communities in a years old male patient with xiap deficiency. case presentation: at years of age, the patient presented with positive serology of active ebv infection, hlh, severe hepatitis, encephalitis and myocarditis. after recovery, the patient evolved well with few manifestations for several years. approximately years ago, the patient showed slow progression of hypogammaglobulinemia predisposing him to infections of the upper and lower respiratory system that required intravenous immunoglobulin replacement. immunological evaluation revealed reduction (but not absence) of inkt cells. at that time, the patient presented with intermittent diarrhea and abdominal pain that became more frequent and severe. after evaluation as ibd, the patients had been treated but without much improvement of diarrhea or resolution of his pain. after approximately months, the patient presented noted pain and fistulas lesions at the scrotal and gluteal regions. the exact causes of ibd in xiap deficiency are not known, but the abnormal activation of the mucosal immune system due to exaggerated response to the commensal bacteria associated to the dysregulation of nod and nod signaling might play an important role in the development and maintenance of the inflammatory status. methods: the gut bacterial composition was assessed by targeted metagenomic from the patients stool sample, collected before initiation of ibd antibiotics therapy. the s rrna amplified and its sequences were analyzed using a bioinformatic pipeline based on mothur software. we determined the bacterial community composition using , filtered reads using illumina miseq platform. results: according to the ezbiocloud database, the obtained dataset included operational taxonomic units at % dissimilarity, distributed among the following groups: bacteroidetes ( . %) with . % of b. dorei, , % b. vulgates, and . % b. fragilis, firmicutes ( . %), proteobacteria ( . %), bacteria_uc ( . %), actinobacteria ( . %). conclusions: increased abundance of bacteroides species including b. dorei and b. vulgatus have been implicated in inflammation in several gut diseases such as ulcerative colitis, irritable bowel disease, and celiac disease. although this experience is limited to a single patient, the results of the present study suggest an association between altered gut microbiota and the pathogenesis of ibd in xiap disease and may be of relevance to the future development of novel therapeutic strategies for xiap deficiency. ( ) submission id# hematopoietic stem cell transplantation in patients with primary immune regulatory disorders: a primary immune deficiency treatment consortium (pidtc) and inborn errors working party (iewp) study introduction/background: primary immune regulatory disorder (pird) is a newly recognized group of immune-mediated diseases with prominent features of autoimmunity, autoinflammation, and non-malignant lymphoproliferation in addition to immunodeficiency. the clinical manifestations of pirds are frequently difficult to manage and hematopoietic cell transplantation (hct) can be considered as a treatment option, often in those with the most severe disease. we sought to aggregate data from patients who have undergone hct for genetically defined pird or features of immune dysregulation. objectives: we sought to aggregate data from patients with pird who have undergone hct in order to determine the quantity of patients, clinical manifestations, indication and hct, and overall outcome. methods: a questionnaire based survey was sent to all primary immunodeficiency treatment consortium sites and hct referral centers in europe to determine the quantity and characteristics of patients with pirds who have undergone hct. the survey captured clinical manifestations, timing and indication of hct, strategy of hct, and outcomes from - . results: patients from centers ( in north america and in europe) were included with either known genetic defects or considered to have immune dysregulation regardless of gene defect. known genetic defects were identified in subjects, while had symptoms of immune dysregulation but lacked a genetic diagnosis. the mean age of onset of disease was years (range - years). clinical manifestations included gastrointestinal disorders ( %), failure to thrive ( %), dermatitis ( %), hematologic cytopenias ( %), and lymphoproliferative disease ( %). recurrent infections ( %), immunodeficiency ( %), autoimmunity ( %), and autoinflammation ( %) were also common. organ specific autoinflammation occurred most commonly in the lung ( %) and brain ( %). the median age of hct was years ( - years). graft sources included matched unrelated donors ( %), matched related donors ( %), mismatched unrelated donors ( %) and haploidentical donors ( %). reduced or minimal intensity conditioning was used in % of transplants. five-year overall survival was % and the majority of survivors had resolution of symptoms that led to transplantation. among those patients that died, infection was the most common cause. conclusions: based on our survey data, pird patients commonly develop clinical features of autoimmunity, autoinflammation, and susceptibility to infection at a young age. hct can be successful and lead to disease resolution. however, further studies to define the appropriate patient, timing of hct, donor selection and pre-hct conditioning regimen are necessary to improve outcomes of patients with pird. introduction/background: pathogenic variants in tnfrsf b (taci) are relatively common (found in about % of the population) but have been seen in about - % of cvid patients, interestingly in both homozygous and heterozygous states. recent articles suggest that the heterozygous state increases the risk of developing autoimmunity due to an effect on autoreactive b cell selection and activation. objectives ) illustrate the importance of genetic testing in patients with difficulty to treat inflammatory disease ) present a case report of inflammatory bowel disease associated with a pathogenic mutation in the tnfrsf b gene results: yo wf was diagnosed with crohns disease due to the chronic abdominal pain, vomiting, and bloody stools since years of age. intestinal biopsy revealed inflammatory changes in the entire intestine with active, submucosal lymphoid hyperplasia, neutrophilic cryptitis with focal areas of crypathic damage, and submucosal epithelioid granulomas more predominantly seen in the colon and rectum. she was started on immunosuppressant medications but developed anaphylactic reaction to both infliximab and adalimumab. she was then treated with azathioprine, mesalamine, methotrexate, and oral budesonide. despite these medications, she continued to have frequent relapses, - episodes a year and required periodic systemic corticosteroid bursts. other biologics, vedolizumab and ustekinumab, were also tried without success, and she subsequently underwent a colectomy. her postoperative course was complicated by ards, poor abdominal wound healing, and sepsis. due to her complicated clinical course, immune work up was performed which revealed a normal cbc and lymphocyte subpopulations, but hypogammaglobulinemia with low isohemagglutinin titers and specific antibody levels. comprehensive genetic testing ruled out chronic granulomatous disease and other known primary immunodeficiencies but revealed a rare missense mutation in tnfrsf b (taci). this variant, c t (p.cys arg) (rs , exac . %) is likely pathogenic. this heterozygous variant has been seen in both cvid cases and unaffected relatives but significantly more common among cvid patients. moreover, the studies on b cells of these relatives showed impaired function. increased number of autoreactive b cells were also found in the bone marrow of heterozygous individuals and these cells could give a risk of developing autoimmunity. conclusions: in difficult-to-treat autoimmune diseases, identifying the underlying immune defect may aid in the treatment decision. in this case, b cell targeted treatment such as anti-cd monoclonal antibody could be beneficial. introduction/background: defects in immunoproteasome caused by biallelic or digenic loss-of-function mutations in proteasome catalytic subunits cause an autoinflammatory disease identified as chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (candle) associated to an increased interferon type i gene signature. the proteasome maturation protein (pomp) is a chaperone for both standard and immuno-proteasome assembly and is critical for the incorporation of catalytic subunits. here, we characterize and describe pomp-related autoinflammatory immunodeficiency disease (praid) in two unrelated patients and identify the underlying genetic mechanism of disease. objectives: determine the genetic cause and mechanism of disease in two patients with pomp variants . methods: whole-exome sequencing (wes) was performed to identify a genetic cause of our patients dysregulatory syndrome. proteasome assembly and catalytic function was assessed by sds-page and native gel respectively, using patient derived cell lines. expression of interferon type i-induced genes was measured by rt-qpcr. pomp protein was identified by western blot. results: we identified two unrelated individuals with a unique syndrome characterized by neonatal onset autoinflammation, neutrophilic dermatosis, autoimmunity, and combined immune deficiency with severe systemic viral and bacterial infections. immunologic evaluation for both individuals revealed elevated immunoglobulins, low cd + t cell numbers and extremely low b cell counts with persistently high titers of autoantibodies and increased expression of interferon type i-induced genes. in both individuals, truncating heterozygous de novo frameshift variants in pomp were identified by wes and confirmed by sanger sequencing. most mrna transcripts with premature termination codons should undergo nonsense-mediated decay (nmd), however in both of our patients, cdna sequencing revealed these transcripts escaped nmd. the expression wildtype and truncated versions of pomp protein was further confirmed by western blot. transfection of mutant constructs into an otherwise healthy cell line recapitulated an increased interferon signature suggesting a dominant negative mechanism. conclusions: we define praid in two unrelated individuals characterized by neonatal onset immune dysregulation and combined immunodeficiency caused by truncating variants in pomp in which transcripts that escape nmd result in a truncated protein that leads to a dominant negative (i.e antimorphic) allele. to our knowledge, praid is the first inherent defect of immunity mechanistically characterized by nmd escape. introduction/background: parvovirus viremia may occur in pediatric heart transplant patients who underwent thymectomy and developed secondary t cell lymphopenia. high dose intravenous immunoglobulin (hdivig) has been used to treat parvovirus infection in these cases. objectives: we aim to review different routes of immunoglobulin treatment in pediatric cardiac transplant patients with parvovirus viremia and compare the patients immunological phenotype. methods: data from three pediatric heart transplant patients with parvovirus viremia in a tertiary care center was reviewed including t cell counts, parvovirus viral load, route, dosage, and frequency of immunoglobulin treatment. results: all three patients received hdivig. patient and tolerated the treatment and viremia improved. patient developed recurrent aseptic meningitis from hdivig treatment and his viral load remained > million copies/ml. compared to the other two cases, patient had a much lower t cell count that likely contributed to the persistence of viremia. to improve his quality of life and reduce healthcare costs, a facilitated subcutaneous immunoglobulin (scig) treatment option was explored. scig treatment was well tolerated and led to a dramatic decrease in parvovirus viral loads in patient . conclusions: most pediatric cardiac transplant patients with persistent chronic parvovirus viremia respond well to hdivig, scig may serve as an alternative treatment option in refractory cases, especially in those with severe t cell lymphopenia. severe combined immunodeficiency (scid), by detecting tcell receptor excision circles (trecs) from dried blood spots (dbs) on routine newborn screening (nbs). this has lead to improved estimates of the incidence and prevalence of scid, decreased diagnostic delay, and improved patient outcomes. preliminary studies outside the us have demonstrated that nbs can be adapted to include screening for b-cell deficient infants before symptom onset by quantifying kappa-deleting recombination excision circles (krecs) on dbs. objectives: we report results of the initial characterization of a high throughput triplex trec/krec/rnasep assay run in , samples in new york state (nys). methods: dbs from anonymous, de-identified infants were included in the current study. dna from patients with confirmed primary immunodeficiencies (n= ), including, but not limited to, x-linked agammaglobulinemia (n= ) and scid(n= ) were obtained from the centers for disease control and prevention, and clinical immunologists working with the nbs programs in massachusetts, minnesota, wisconsin, and nys, were used as positive disease controls. all dbs were extracted and processed according to current nys nbs protocols. a trec/krec/rnasep triplex assay was designed and optimized to minimize reagent use, and maximize target amplification. cycle threshold (ct) was determined, and error detection cutoffs were identified to optimize sensitivity. results: pcr efficiency, assay quantification, intra-assay reproducibility, and error detection rates all met nys nbs standards. error detection rate for the triplex trec/krec/rnase p assay is %, comparable to the current error detection rate of % for the current duplex trec/rnase p assay. samples falling into the error detection range are repeated (analysis in process) to determine a receiver operating characteristic curve. conclusions: we show that the high-throughput trec/krec/rnase p triplex assay is feasible in a large, racially and ethnically diverse population in nys. compared to the current duplex assay, this assay has favorable performance characteristics and provides additional immunologic characterization. due to assay optimization, we were able to add the krec test at no additional cost. work is underway to further characterize other assay parameters such as sensitivity and specificity, in preparation for adoption of the triplex assay as part of routine nys nbs. highly accurate wiskott-aldrich syndrome diagnosis via rapid flowbased was protein staining samuel chiang , sue vergamini , ammar husami , marianne ifversen , kejian zhang , jack bleesing, md, phd , yenan bryceson , rebecca marsh, md introduction/background: wiskott aldrich syndrome (was) is a rare xlinked hemizygous disease commonly associated with symptoms of immune deficiency. diagnosis is based on clinical parameters including thrombocytopenia and reoccurring infections, but currently does not include any disease specific marker. as the only permanent treatment for was is hematopoietic stem cell transplantation, it is imperative that a swift and accurate diagnosis be made. absent or lowered was protein (wasp) levels have been reported in sporadic was cases. however, no systemic evaluation exists to date on the accuracy of wasp quantification for was diagnosis. objectives: to determine the accuracy of wasp staining in predicting was genetic abnormalities. methods: we retrospectively evaluated results from a rapid whole blood flow cytometry based assay on a cohort of suspected was patients and compared relative wasp staining levels to was genotype. roc curves as well as accuracy calculations were generated. results: a total of patients with normal and patients with a genetic abnormality in was were collected. missense mutations were most common but insertions, deletions, and gross mutations were also found ( fig a) . comparing was sequencing results to whole blood wasp expression levels provided an . % sensitivity and % specificity for a combined accuracy of . % when juxtaposed against genetic sequencing. when variants of unknown clinical significance (vucs) were removed, the sensitivity improved to . % (fig b) . conclusions: staining for wasp is a quick, simple, and accurate assay for the prediction of genetic was defects. introduction/background: cytotoxic t lymphocyte antigen (ctla ) is an inhibitory co-receptor essential for regulatory t cell (treg) function and a central regulator of t cell proliferation and expansion. ctla haploinsufficiency is a recently described autosomal dominant disease, in which heterozygous ctla mutations result in severe immune dysregulation with variable age of onset and a wide array of clinical manifestations. herein we describe atypical findings in a patient with a novel pathogenic variant in ctla . objectives ) to understand whether the novel ctla variant identified is pathogenic and ) to describe eosinophilic gastrointestinal inflammation and exocrine pancreatic insufficiency as possible manifestations of ctla haploinsufficiency. methods: next generation sequencing (blueprint genetics ©) was used to identify the ctla variant. polyphen and sift (blueprint genetics ©) were used for in silico analysis for the prediction of the effect of this genetic variant on protein structure/function. flow cytometry was used to evaluate ctla expression of regulatory t cells. results: a -year-old boy with type diabetes mellitus and autoimmune thyroiditis presented with abdominal pain, diarrhea, and weight loss. initial studies revealed markedly elevated peripheral blood eosinophils (> cells/μl) and exocrine pancreatic insufficiency (< μg elastase per gram of stool). prominent eosinophilic inflammation was appreciated in biopsies of the stomach, duodenum, jejunum, and terminal ileum. no parasitic infection or inciting drug/food trigger was identified. additional blood studies revealed normal total quantification of t cells but with increased memory t cells ( %, cd +cd +cd ro+) and decreased treg ( %, cd + cd +cd hifoxp hi). b cell quantification and serum immunoglobulin (ig) levels were unremarkable, save a modestly elevated ige level ( iu/ml). comprehensive next-generation sequencing of genes associated with primary immune deficiency revealed a novel heterozygous missense mutation (c. g>a, p.asp asn) affecting the last nucleotide in the ligand binding domain (exon ) of ctla . subsequent analyses revealed decreased ctla expression in the patients t cells compared to healthy controls as well as evolving hypogammaglobulinemia. treatment consisted of methylprednisolone and parenteral nutrition followed by sirolimus and abatacept to which the patient responded favorably. conclusions: we report a -year-old boy with a history of type diabetes mellitus and autoimmune thyroiditis presenting with hypereosinophilia, eosinophilic gastroenteritis, and exocrine pancreatic ins uff iciency as unique m anifestat ions o f ctla haploinsufficiency. although not previously reported in individuals with ctla haploinsufficiency, peripheral blood eosinophilia and eosinophilic inflammation of the gastrointestinal tract have been observed in patients receiving ipilimumab (ctla blocking antibody) suggesting a potential mechanism for the aforementioned findings. severe exocrine pancreatic insufficiency is a rare but observed manifestation in individuals with type diabetes mellitus. whether severe exocrine pancreatic insufficiency would be expected t o o c c u r m o r e f r e q u e n t l y i n i n d i v i d u a l s w i t h c t l a haploinsufficiency and type diabetes mellitus is unclear; however, our reported case and surveillance of others with ctla haploinsufficiency could elucidate incidence and prevalence of this manifestation. introduction/background: mild asymptomatic hypogammaglobulinemia during pregnancy is a well-described phenomenon due to hemodilution. in patients with known humoral primary immunodeficiency such as common variable immunodeficiency, women require an upwards titration of their immunoglobulin replacement dose. isolated symptomatic hypogammaglobulinemia during pregnancy in a patient is not well described in the literature. objectives . understand the physiology of igg during pregnancy. . define a rare entity with a likely genetic predisposition that manifests as hypogammaglobulinemia isolated in pregnancy. . management of this entity with defining goals of treatment. results: year old gravida para female presented with progressive hypogammaglobulinemia restricted to pregnancy starting with rd pregnancy, recurrent otitis media requiring sets of myringotomy tubes, recurrent sinusitis, and streptococcal pharyngitis. her son has common variable immunodeficiency (cvid) requiring immunoglobulin replacement (igrt). prior to conception, her igg was mg/dl. during th week of pregnancy, her igg level was mg/dl. during weeks of pregnancy, she complained of fatigue, and developed an episode of sinusitis that required different antibiotic treatments. at that time, her igg was mg/dl. she was started on subcutaneous igrt maintain igg troughs of > mg/dl. she remained infection-free during her pregnancy and igrt was stopped a few months after delivery with her serum igg level returning to pre-pregnancy levels. patient was initially evaluated during her rd pregnancy with recurrent streptococcal pharyngitis. at that time, her igg was mg/dl. diphtheria, haemophilus influenza, mumps, measles and rubella titers were protective, tetanus titer was non protective with / antipneumococcal titers protective. she was vaccinated with pneumovax and tdap with development of protective titers and remained infectionfree. igrt was not given and post-delivery, her igg levels improved to mg/dl. she was seen one and half years later, for prenatal counseling for her th pregnancy. her immune evaluation included an igg . she demonstrated protection to tetanus, diphtheria and streptococcal pneumoniae. at weeks of gestation, she developed recurrent upper respiratory infections requiring antibiotics. her igg was mg/dl. at weeks of gestation, her igg was mg/dl. igrt was recommended, but patient refused at that time. after delivery, igg improved to mg/dl. conclusions: decreased immunoglobulin levels during pregnancy are welldescribed phenomenon which can be attributed to hemodilution of pregnancy. igg transport to fetus generally begins in the second trimester and reaches its pinnacle in the third trimester. patients with known cvid require dose adjustments (higher) during pregnancy as they can be more symptomatic during this time. here we describe a patient who has an almost normal immune evaluation except for mildly low iga during absence of pregnancy, but during pregnancy, develops recurrent infections with significantly low igg level. her family history of a son with cvid, new daughter with low igg levels like to be transient hypogammaglobulinemia of infancy (thi), another daughter with thi suggests a genetic b-cell defect that manifests as cvid with mildly low iga and hypogammaglobinemia during metabolic stress such as pregnancy. there is only one similar report of a single pregnancy of transient symptomatic hypogammaglobulinemia during pregnancy. such patients should be adequately worked up and treated during pregnancy with igrt to decrease maternal and fetal mortality. introduction/background: card encodes a scaffold protein in lymphocytes that links antigen receptor engagement with downstream signaling to nf-b, jnk, and mtorc . germline mutations in card are known to give rise to distinct primary immune disorders in humans, including scid (null mutations), b cell expansion with nf-b and t cell anergy (benta; gain-of-function mutations), and severe atopic disease (loss-of-function, dominant interfering mutations). objectives: here we report our experience with an expanded cohort of patients harboring novel heterozygous card mutations that extend beyond atopy to include other immunologic phenotypes not previously associated with card mutations. methods: cell transfections and primary t cell assays were utilized to evaluate signaling and function of card variants. results: we demonstrate that in addition to severe atopy, heterozygous missense mutations in card associated with dominant negative activity can present with immunologic phenotypes similar to those observed in stat lof, dock deficiency, common variable immune deficiency (cvid), congenital neutropenia, and immune dysregulation, polyendocrinopathy, enteropathy, x-linked (ipex) syndrome. evaluation of rare or novel card variants found in affected patients showed that dominant negative activity was largely confined to the card or coiled-coil domains, but did not always manifest in atopic disease. conclusions: these results illuminate a broader phenotypic spectrum associated with card mutations in humans, and underscore the need for functional studies to demonstrate that rare gene variants encountered in expected and unexpected phenotypes must nonetheless be validated for pathogenic activity. introduction/background: increasing number of states have been screening for severe combined immune deficiency (scid) as part of the expanded newborn screening program for nearly a decade. in the era of newborn screening, patients with scid present often asymptomatically and are prepared for early hematopoietic stem cell transplantation (hsct). with advances in genetic testing, mutations in over genes have been associated with development of the scid or leaky-scid phenotype. objectives: to present unique cases of hypomorphic x-linked scid where no known pathogenic mutations were identified on initial genetic testing, the il r gamma-chain as protein was expressed, but subsequent testing months later revealed pathogenic il rg mutation affecting either translation or protein function. to expedite earlier identification of pathogenic il rg mutation, we propose screening with x-inactivation studies in maternal t lymphocytes and assessing gamma-chain function by evaluating il r signaling in select male infants with abnormal newborn screen for scid, specifically those with scid phenotype but no identified pathogenic gene mutation on initial genetic testing, and the presence of gamma-chain expression. male patient a presented during the first week of life after his newborn screen was found to be abnormal with undetectable t cell receptor excision circle (trec) count. a phenotype of t-b+nk-scid was established and the patient was sent for bone marrow transplant evaluation. genetic testing revealed a novel hemizygous missense mutation in il rg (p.glu gln), which was a variant of unknown significance. the patient had common gamma-chain expression by flow cytometry on b and nk cells. he underwent haploidentical hsct with his father as donor. unfortunately, his transplant was complicated by prolonged neutropenia, slow t cell reconstitution, and eventual graft failure. to review his next treatment options, it was necessary to prove that his il rg mutation was pathogenic. male patient b presented with concern for an underlying immune deficiency after being hospitalized for peumocystis jirovecii pneumonia. his newborn screening for scid was inadequate at birth and follow up was delayed. retrospective analysis of the newborn screening card at birth confirmed absence of trecs. his phenotype was also t-b+nk-, consistent with x-linked scid. he also proceeded to hsct with a haploidentical parent donor. despite hhv- viremia pre-hsct which persisted posttransplant, he has had appropriate t cell engraftment. comprehensive genetic testing on whole exome level did not reveal any known mutations contributing to his phenotype. patient did have expression of gamma-chain by flow cytometry on t, b and nk cells. however, further testing revealed an il rg utr deletion of aa that, based on similar findings from a prior study can possibly lead to mrna abnormalities. to expedite the association of scid phenotype with x-linked disease, implying gamma-chain pathology, we obtained x-inactivation studies in maternal t-cells that showed severe skewing in both cases. furthermore, il r signaling was impaired on b-cells in each case. the combination of these two assays proved that both patients carry a pathogenic il rg mutation. conclusions: in the era of newborn screening for scid, we are discovering that phenotypic variability of scid patients can be very broad and caused by hypomorphic mutations in the common chain gene. exonbased genetic testing cannot exclude all variants and novel variants of unknown significance have to be evaluated by additional assays, including functional studies for causal effect. it is important to expedite early proof of association with gamma-chain pathology, especially in the era of gene therapy. we propose that in male infants with abnormal scid newborn screening and no known or previously described pathogenic mutation on genetic screen, evaluation continues for hypomorphic il rg mutations. the probability of this process can be increased by a simple screening test for x-inactivation of maternal t lymphocytes. allergy / immunology, allergy / immunology associates inc. / case western reserve university introduction/background: igg -related disease (igg -rd) is an immunologic disorder with multiple clinical presentations previously thought unrelated. it is characterized by the frequent presence of tumor-like swelling of the affected organs and several histopathological findings including tissue lymphoplasmacytic infiltrates with predominantly igg -positive plasma cells and lymphocytes, storiform fibrosis and obliterative phlebitis. humoral immunodeficiency is a term that encompasses several disease entities associated with impaired antibody production. it is suspected in patients who present with recurrent, frequently severe, sinopulmonary infections with encapsulated bacteria, which leads to evaluation of quantitative immunoglobulin levels and vaccine responsiveness. despite a few reports of primary immunodeficiency in patients with serum igg elevation, no adult case has been reported of igg -rd in a patient with concomitant humoral immunodeficiency. objectives: to present a unique case with the presence of concomitant igg -related disease and humoral immunodeficiency. methods: comprehensive chart review of our patient and all performed exams. literature review for igg -related disease, igg elevation in humoral immunodeficiency and concomitance of igg -related disease with humoral immunodeficiency. results: our patient is an -year-old caucasian male with relevant past medical history of chronic bronchitis who was referred to our practice after several episodes of pneumonia in the previous years, with six courses of antibiotics just in the year prior for recurrent sinopulmonary infections. blood tests revealed hypergammaglobulinemia and low level of vaccine responsiveness. chest ct showed multiple bilateral pulmonary nodules and hilar and mediastinal lymphadenopathy. sinus ct showed left maxillary sinus opacification. pulmonary function testing was normal. he later presented with left eye edema, proptosis, diplopia, and painless submandibular salivary gland enlargement. laboratory investigation showed an igg of mg/dl, igg of mg/dl. the patient denied any history of pancreatitis or abdominal pain and abdominal ultrasound was normal. biopsy of a salivary gland was normal. mri of the left orbit was obtained, showing lacrimal gland enlargement. based on the patients recurrent infections, lack of response to tetanus immunization, and limited, non-sustained response to pneumococcal immunization, the patient was started on ivig therapy. the patient was also diagnosed with possible igg -rd based on his salivary gland enlargement and orbital disease in association with hypereosinophilia and increased plasmablast levels. oral prednisone mg daily was started for four weeks, later followed by slow steroid taper (reducing mg every two weeks) with considerable improvement in left eye swelling and proptosis. a few months after discontinuation of the steroids, the orbital disease returned to its previous severity. left lacrimal gland biopsy confirmed igg -related disease, with many areas showing greater than one hundred igg -positive plasma cells per high-power field. after another course of steroids, oral prednisone was weaned to a maintenance dose of mg daily and the patient became asymptomatic from his ophthalmologic complaints with normalization of his ophthalmologic exam. his last checked igg was mg/dl. igg was still elevated at . mg/dl , but given controlled symptoms the patient was spaced to monthly ivig infusions and continued on that daily steroid dosage. conclusions: our patient, initially diagnosed with a humoral immunodeficiency, was later also diagnosed with biopsy-proven igg -related disease, which is a novel association of this two diseases in an adult patient. previous rare reports of association between elevated serum levels of igg and patients with concomitant humoral immunodeficiency were in the presence of isolated igg elevation and not in the presence of igg related disease. this novel association creates a therapeutic dilemma since the patient in question is hypergammaglobulinemic, yet needs ivig, which can lead to side effects such as thrombosis due to a hyperviscous state. the description of additional concomitant cases of both diseases and further understanding of their pathophysiology will be crucial to create awareness and obtain earlier diagnosis, to refine therapeutic options and design adequate treatment protocols. igm and iga anti-pneumococcal capsular polysaccharides as prognostic tool for common variable immunodeficiency: a longitudinal study. professor, sapienza university of rome introduction/background: the clinical spectrum of cvid ranges from a poorly symptomatic form to severe phenotypes characterized by high susceptibility to infections, autoimmunity, granulomatous inflammation, lymphoproliferative disorders, and malignancies. due to high prognosis heterogeneity, prognostic factors are required. objectives: with the aim to identify additional prognostic factors, we evaluated the anti-polysaccharide iga and igm responses by elisa assay in cvid in a longitudinal study over a -year period. methods: patients were immunized at baseline with the -valent pneumococcal polysaccharide vaccine (pneumovax®). twenty healthy donors (hd) were also included. results: as expected, cvid patient had lower igm/iga response than hd. for cvid, four immunological phenotypes were identified by postvaccination igm and iga levels: igm and iga responders ( %), igmhigh responders ( %), igm-low responders ( %) and non-responders ( %). to simplify, we analysed igm-high group with igm and iga responders and igm-low with non-responders. during the follow up, concomitant cvid-related conditions, immunoglobulin serum levels, respiratory infections and outcome were recorded by medical files. cvid igm-low/non-responders developed more frequently respiratory, gastro enteric and autoimmune manifestation and malignancies in comparison to igm-high/igm and iga responders (respectively, pneumonia: % vs % ; chronic diarrhea: % vs %; autoimmunity % vs %). autoimmune cytopenias were not found in the igm-high/igm and iga responders group. eleven ( %) patients died during the study time. survival analysis according to the igm/iga responder status showed that the -years estimated survival for igm-high/igm and iga responders vs igm-low/non-responders group was respectively: % vs %, % vs %, % vs %, % vs %, % vs %, % vs %, % vs %. interesting, in our series only two deaths were due to infective complications: five were consequent to malignancies, one to autoimmune cytopenias and three to not-cvid related conditions. conclusions: in conclusion, even if patients could not raise the protective humoral level, in cvid the anti-polysaccharide iga and igm responses could represent a prognostic factor, individuating groups of patients with less immunological impairment, lower risk of comorbidities and better survival. introduction/background: gaucher disease (gd) is a rare autosomal recessive disorder characterized by a defective function of the catabolic enzyme -glucocerebrosidase (gba) leading to a progressive accumulation of its substrate-glucocerebroside (gc) -in various organs in particular in mononuclear phagocite system. hepatosplenomegaly and cytopenia represent the most common features of the disease. moreover, gd patients also show hyperinflammatory features -secondary to machrophages engorgement and actviation-hypergammaglobulinemia, and a immune-dysregulation involving b , t and nk cells. since clinical phenotpye can be subdolous, symtoms can overlap with alps, however, few data are available on specific immunity pattern in these patients. objectives: to evaluate immune-phenotype and other alps parameters in a cohort of patients with gd methods: we evaluated lymphocytes subsets, immunophenotypic and serological features of alps (dnts, tcr alfa/beta b , b-memory cells, tregs/hla-dr ratio, il- , il- ), and test of apoptosis in a cohort of patients with gd followed-up at igg. results: patients ( in treatment, not) were studied. dnts and tcr alfa/beta b + resulted to be > . % of t-lymphocytes and > % in / ( %) and in / ( %), respectively. b-memory cells and t-regs/hla-dr ratio were < % and < in / patients ( %). / evaluable ( %) had all these parameters concomitantly alterated. / ( %) evaluable patients were resistant to apoposis. il- was pathological in / ( %) patients. all patients had normal levels of il- and sfas. conclusions: this study shows that some patients with gd may present an immune-dysregulation pattern that can overlap with alps features. therefore, the differential diagnosis of gd should be taken into consideration by clinicians during diagnostic work-up of patients with an alpslike phenotype. introduction/background: allogeneic hematopoietic stem cell transplantation (hsct) using unrelated and haploidentical donors is complicated by increased rates of graft-versus-host disease (gvhd) and slow immune reconstitution. selective depletion of alpha/beta t lymphocytes and b cells is a recently developed method of graft manipulation that retains mature natural killer (nk) and gamma/delta t lymphocytes, both of which may exert a graft-versus-leukemia effect and protection against life-threatening infections. objectives: to describe the rate and quality of immune reconstitution, incidence of transplant-related complications, including viral reactivation and gvhd, and overall outcomes following tcr-alpha/beta-and cd depleted hsct for hematologic malignancy in pediatric patients. methods: forty patients of median age . years ( . - . ) underwent hsct for acute myeloid leukemia (n= ), acute lymphoblastic leukemia (n= ), and myelodysplastic syndrome (n= ). grafts were from unrelated (n= ) and haploidentical (n= ) donors. tcr-alpha/beta and cd depletion was performed with the miltenyi clinicmacs plus system. median cd + cell dose was . x /kg ( . - ), and median cd + cell dose was . x /kg ( . - . ). conditioning was with myeloablative busulfan or total body irradiation, cyclophosphamide, and thiotepa. twenty of unrelated donor hscts and / haploidentical hscts also included antithymocyte globulin x . no patient received post-transplantation gvhd prophylaxis. all but patients received rituximab on day + per protocol for recipient positive epstein barr virus (ebv) serology. results: all patients engrafted. median time to neutrophil engraftment was ( - ) days, and median time to platelet engraftment was ( - ) days. one patient experienced graft rejection on day + , and twelve patients relapsed at a median of ( - ) days. overall survival was / ( %) at a median of . ( . - ) months follow-up. two ( %) patients developed grade iii or higher acute gvhd, and ( %) patients developed extensive chronic gvhd. cumulative incidence of cytomegalovirus (cmv) and adenovirus reactivation were / ( . %) and / ( . %), respectively. nine ( . %) patients developed bk hemorrhagic cystitis +/-viremia. ebv reactivation was not observed. median total, myeloid, t cell, and b cell donor chimerism were all % (ranges - %, - %, - %, and - %) at year post-hsct. immune reconstitution of all cells lines was rapid ( table ) . eighteen of ( %) patients had detectable t cell receptor excision circles (trecs) by months with a median trec count of ( - ) per ^ cd t cells, and recovery of the naïve t-cell compartment was observed by months in / ( %) of patients. t cell function as measured by response to pha was normal by months in / ( %) patients and continued to increase steadily with time. despite rituximab on day + for / ( . %) patients, there was rapid b cell reconstitution. nineteen of ( . %) patients had present switched memory b cells at months, and / ( . %) surviving patients are off immunoglobulin replacement at a median of ( - ) months. conclusions: selective tcr-alpha/beta and cd depletion of haploidentical and unrelated grafts results in high engraftment and rapid immune reconstitution with low incidence of gvhd in children with hematologic malignancy. cd depletion and routine post-hsct rituximab on day + are effective at preventing ebv reactivation. introduction/background: hyper-igd syndrome (hids; ) is an autosomal recessive disorder characterized by recurrent episodes of fever associated with lymphadenopathy, arthralgia, gastrointestinal disturbance, and skin rash. the diagnostic hallmark of hids is a constitutively elevated level of serum immunoglobulin d (igd), although patients have been reported with normal igd levels. the disease is associated with mutations in the gene of mevalonate kinase. objectives: male patient, years old, born to non-consanguineous parents, with a history of severe diarrhea since childhood, followed by respiratory infections and pneumonias. moreover he presented several episodes of severe abdominal pain, with intestinal obstruction since seven months old, needing surgical intervention due to acute abdomen. this picture repeated several times until years of age, being submitted to new surgeries due to intestinal suboclusion. at and years he had gastroenteritis and then pancreatitis. years ago new severe acute gastroenterocolitis with suboclusion, submitted to laparotomy and resection of a little part of the gut. he has frequent diarrheas, triggered by coffee and tea. usually evacuates times a day. he was evaluated by several pediatric immunologists in childhood, who distrusted alpha heavy chain disease. he had an intense reaction to the bcg vaccine, and then did not make any more vaccines (only opv (sabin) in the campaigns). he was tonsillectomized at years of age. he had measles, chickenpox, and mumps (at years old). cellulitis at years, repeating several times since then. he presented improvement of pneumonias but still has sinusitis and otitis (approximately times a year introduction/background: pitthopkins syndrome is a rare neurological disorder caused by mutations the tcf gene on chromosome q . clinical features include severe intellectual disability, constipation, microcephaly, and seizures. features distinguishing it from other neurodevelopmental syndromes such as rett syndrome and angelman syndrome include breathing abnormalities (either apneic episodes or hyperventilation) and atypical facial features. typical facial dysmorphism includes bitemporal narrowing, deep-set eyes, an m shaped upper lip, and widely spaced teeth. although a very rare diagnosis (slightly over reported cases), it is not known to be associated with underlying humoral or cellular immunodeficiency. there is only one report of igm abnormalities described in a patient with pitt-hopkins syndrome. objectives: to present a case of pitthopkins syndrome with humoral immunodeficiency. methods: this is a case presentation of a patient with pitt-hopkins syndrome requiring immunoglobulin replacement therapy. results: year old female with genetically diagnosed pitt-hopkins syndrome who presented to our office for immunological evaluation in the setting of recurrent sinopulmonary infections. she was placed on chronic antibiotics by the department of otholaryngology for approximately two years prior to presentation. she was found to be hypogammaglobulinemic (igg: mg/dl, iga: mg/dl, igm: mg/dl), had non-protective titers to streptococcus igg antibody ( / titers protective greater than . mcg/ml) despite booster vaccination with pneumovax, and had borderline tetanus ( . iu/ml) and diphtheria titers ( . iu/ml). given this laboratory evaluation and her recurrent illness, immunoglobulin replacement therapy (igrt) was started. whole exome sequencing was completed to assess for any other genetic cause of her immunodeficiency. the only abnormality was her previous known pathologic variant p.q hfsx c. _ delga (gln his) in exon in the tcf gene. she continued to have infections despite therapeutic igrt. chronic antibiotic treatment was initially tapered, however needed to be reintroduced as ivig alone was not stopping her infections, despite a igg level over mg/dl. conclusions: humoral immunologic deficits are not known to be associated with pitt hopkins syndrome. there has been one case report of a patient with poliomyelitis-like syndrome following an asthma attack in a patient with pitt hopkins syndrome, which was treated with igrt and resulted in a nearly complete recovery. however, igrt was not used for reasons of underlying immunodeficiency. to our knowledge this is the first patient with pitt hopkins syndrome with persistent hypogammaglobulinemia and frequent infections requiring immunoglobulin replacement therapy. it remains unclear why the patient continued to have infections despite igg levels > mg/dl, yet with the combination of igrt and prophylactic antibiotics the patient remains healthy. introduction/background: gata deficiency is a rare disease that typically presents in late childhood or early adulthood with heterogeneous phenotypes including emberger syndrome. emberger syndrome is characterized by lymphedema and predisposition for myelodysplastic syndrome (mds) and acute myeloid leukemia (aml). over % of patients with gata mutations have immune deficiencies. definitive diagnosis of gata deficiency is made by gene sequencing, and treatment includes infection control and potentially hematopoietic stem cell transplant (hsct). objectives: the goal of this report is to contribute data to the small documented cohort of patients with gata deficiency to aid in diagnosis and management of this rare, heterogeneous disorder. methods: we present a case series of three siblings with identical gata mutations with variable phenotypes. a usidnet query resulted patients with gata mutations. results: three siblings ( year-old female (a), year-old male twin (b), and year-old female twin (c)) and their mother had congenital deafness. clinical symptoms include (a) h n influenza requiring mechanical ventilation, warts, and hypogammaglobulinemia, (b) streptococcus pyogenes neck abscess, warts, acne, and mds, and (c) lymphedema and acne. all three patients had absolute monocyte count (amc) of and lymphopenia without documented lymphoid cell dysfunction. a mutation on exon , c. delg, confirmed the diagnosis. all three patients were started on mycobacterial prophylaxis with azithromycin and recommended hpv vaccination. the usidnet query average age of symptom onset was years, and average age at diagnosis was . years. . % ( / ) of patients had a family history of gata deficiency, % ( / ) of patients had warts, . % ( / ) had lymphedema and only patient had sensorineural deafness. % ( / ) of patients had amc of . functional data was limited. conclusions: life threatening infections as well as hematologic malignancies have been reported in patients with gata deficiency, which can be successfully treated with hsct. to our knowledge, the gata mutation detected in this family has not been previously reported. the clinical presentation in these three patients was heterogeneous despite identical genotypes, and diagnosis occurred years after initial symptoms. variable phenotypes were found in the usidnet gata deficiency cohort as well. a high index of suspicion for the disorder and early recognition of clinical manifestations and laboratory abnormalities may aid in timely diagnosis of gata deficiency, with potential for improved outcomes. consulting medical advisor, immune deficiency foundation introduction/background: as individuals with antibody deficiencies age they are susceptible to developing shingles. patients with antibody deficiencies are advised not to receive live viral vaccines such as zostavax, the shingles vaccine. objectives: our survey aimed to determine the frequency of shingles and use of zostavax in common variable immunodeficiency (cvid) and hypogammaglobulinemia patients. methods: , email invitations delivered to members of the immune deficiency foundation database requesting participation in an online survey about zoster, influenza and varicella experiences. data from individuals age years old or older with cvid (n= ; mean age years old; % female; % white non-hispanic) or hypogammaglobulinemia (n= ; mean age years old; % female; % white non-hispanic) were analyzed. results: close to one fifth ( %, n= ) of adults age or older with cvid or hypogammaglobulinemia (n= ) had received shingles vaccination. the majority of those who were vaccinated reported receiving zostavax once ( %, n= ), while % (n= ) received a booster vaccination as well. mild side effects (e.g., skin rash and muscle pain) were only reported by % (n= ) of vaccine recipients after receiving their first vaccination. no side effects were reported after receiving the booster vaccination and no hospitalizations were reported as a result of receiving zostavax. when comparing shingles diagnosis and shingles vaccination, of the adults age years old and older, % (n= ) had been diagnosed with shingles, and of those diagnosed % (n= ) did not receive zostavax. of adults age years old or older, % (n= ) reported a shingles diagnosis. similarly, the cdc reports almost % people in the united states will develop shingles during their lifetime. more than half ( %, n= ) of those ever diagnosed with shingles reported experiencing shingles once, % (n= ) had shingles twice and % (n= ) had shingles three or more times. respondents with more than three shingles episodes were more likely to report their rash lasted more than two months and their blisters became infected. conclusions: almost % of adults with cvid or hypogammaglobulinemia reported receiving zostavax despite recommendations against vaccinations for immunodeficient individuals. however, side effects in those pi patients who received the shingles vaccine appears minimal. though it is possible these individuals were vaccinated prior to diagnosis of their pi; additional patient and physician education on live vaccines and immunodeficiency may be needed as well. the approval of the new non-live virus component varicella zoster vaccine may be of benefit to patients with pi. introduction/background: inclusion body myositis (ibm) is a rare disorder characterized as an inflammatory myopathy with endomysial inflammation and numerous red-rimmed vacuoles seen on biopsy. five cases of ibm have been described in the literature in patients with common variable immunodeficiency (cvid). objectives: to make immunologists aware of ibm as a complication of cvid, that may be incorrectly diagnosed as myositis or autoimmune neuropathy. methods: case description results: we report a -year-old man with common variable immunodeficiency on gammaglobulin replacement who presented complaining of progressively worsening lower extremity pain, weakness, and fatigue. he states that over the last couple of years, it has become difficult climbing and descending steps, arising from a seated position, and has begun experiencing frequent falls. his creatinine kinase level was found to be elevated at u/l and he continued to be lymphopenic with total lymphocyte counts ranging from to k/ul (cd + t-cells %, cd + t-cells %, cd + bcells %). his esr ranged from to mm/h. anti-glutamic acid decarboxy antibody (gad) was initially elevated at . u/ml and rose to . u/ml within months suggestive of a neuropathy. based on an electromyography (emg) and a muscle biopsy, he was diagnosed with polymyositis. he was treated with high dose steroids with no improvement. his intravenous gammaglobulin dose was then increased from mg every weeks to mg per day for straight days every weeks. months later, his creatinine kinase level dropped into the normal range (< u/l), however, he continued to complain of worsening weakness. physical exam showed decreased muscle bulk in forearms and quadriceps bilaterally, lack of a quadriceps tendon reflex, strength / in flexor digitorum profundus and / in hip flexors, and a broad-based gait. histology and electron microscopy of a repeat muscle biopsy identified rimmed muscle vacuoles as typically noted in inclusion body myositis. conclusions: inclusion body myositis is a potential rare complication of common variable immunodeficiency. it can mimic polymyositis and inflammatory demyelinating disorders. high dose steroids and ivig are of no clinical benefit in ibm, despite decreasing serum creatinine kinase levels, which may raise a false impression of a clinical benefit. objectives: in this abstract we present the case of the development of generalized cmv infection in a child with scid. girl n. at the age of months entered the children's infectious clinical hospital with complaints of cough, high febrile temperature for days, refusal to eat. from the anamnesis of life the girl from the st pregnancy, birth, was born full term in weeks gestation, birth weight g. for months of life, a bad increase in body weight was noted and at the time of admission, the weight in months was g. according to the parents, the child had atopic dermatitis. from the anamnesis of the disease on . . , the temperature rose to . °c, there was a cough and a mucous discharge from the nose. then the child refused to eat, the body temperature rose to . °c. at this time, the girl's mom was borne by the ari. january , patient was hospitalized in the hospital with a diagnosis: acute respiratory viral infection, acute rhinitis, pharyngitis, acute bronchitis, toxicosis of - degrees. acute pneumonia? atopic dermatitis, infant form. on january , , due to the worsening of the condition associated with the increase in oxygen (o ) -dependence, the child was transferred to the department of anesthesiology and resuscitation. methods: in the general analysis of blood upon admission, leukocytes are . x / l, hemoglobin is g / l, platelets are x / l, esr is mm / hour, stabs are % (abs. - . x / l), segmented - % (abs- . x / l), lymphocytes - % (abs . x / l), monocytes - % (abs - . x / l). in a biochemical study, the total protein is g / l, total bilirubin is . mol / l, urea is . mmol / l, creatinine is mol / l, lactate dehydrogenase is u / l, alt is u / l, asat - e / l, crp - . mg / l. radiography of the lung from / / -data in favor of interstitial pneumonia. the study of the acid-base ph state is . , pc is . mmhg, po is . mmhg, lactate is . mmol / l. a blood test was performed using the elisa and pcr method for markers of hsv, cmv, enterovirus and toxoplasmosis. ultrasound of the abdominal cavity revealed moderate hepatomegaly, signs of thickening of bile, splenomegaly. moderate diffuse changes in the renal parenchyma (toxic-inflammatory?). the minimum amount of free fluid in the abdominal cavity. ultrasound of the brain revealed signs of subependimal microcyst on the right. according to the immunogram, a sharp decrease in cd + % ( - %) was detected, activated t-lymphocytes (cd + hla-dr +) were . % ( - %), t helper / inducers (cd + cd - . % ( - %) and t suppressors / cytotoxic (cd + cd -) . % ( - %), a high ratio of tx / tc (cd + cd +) was detected . % ( . - . ), cytotoxic non-t cells (cd -cd +) - , , an increase in the number of b-lymphocytes (cd +) - . % ( - %), natural killers (cd + cd +) - . % ( - %), natural t-killers (cd + cd + cd +) - . ( - %), leukocyte gates (cd + cd -) - % ( - %). the absolute content of t-lymphocytes was . x / l, b -lymphocytes - . x / l. the number of thymic migrants (cd + cd ra + cd +) was not detected ( %). according to the results of the immunogram the diagnosis is made: severe combined immunodeficiency (t-b + nk +). / / ct scan of the chest was diagnosed ct signs of a polysergic two-sided inflammatory process in the lungs (figure ). when blood was sown for sterility on january , , staphylococcus epidermidis was isolated in an amount of , sensitive to linezolid, gentamicin resistant to amoxicillin, amoxicillin / clavulonic acid, and ciprofloxacin. on january , , cmv dna was detected in an amount of . × copies / ml. results: since the arrival clarithromycin was administered at a dose of mg / kg per day in divided doses from / / to / / . from / / to / / . change of antibacterial therapy for azithromycin intravenous at a dose of mg / kg per day once a day. . . - / / the state of the child is very severe with negative clinical and laboratory dynamics despite the ongoing therapy. antibacterial therapy was changed to meropenem in a dose of mg / kg intravenously every hours, linezolid at a dose of mg / kg per day and oseltamivir at a dose of mg / kg per day from / / to / / . . . - . . substitution therapy with an octagam in a dose of . g / kg was intravenously dripped. the patient's condition without significant dynamics. based on the results of pcr on cmv, ganciclovir was administered at a dose of mg / kg intravenously drip times a day. / / due to a decrease in platelet count, the platelet mass is transfused and there was a rash all over the body at night, which is associated with the development of the "graft versus host" reaction (gvhr). despite the ongoing therapy, a fatal outcome occurred. the main diagnosis: primary immunodeficiency (severe combined immunodeficiency, t b + nk +). complications: sepsis. septic shock. spon: ards, renal failure, dis, thrombocytopenia, anemia . two-sided lower-lobe pneumonia. generalized cmv infection. gvhd, acute dermal form. concomitant: atopic dermatitis, infant form. conclusions: the peculiarity of the described clinical case was that the patient's first symptoms of scid developed in the first months of life and were manifested by a bad weight gain, atopic dermatitis and the development of a life-threatening generalized cytomegalovirus infection with the development of bilateral low-grade pneumonia, respiratory insufficiency and acute cutaneous gvhd form, after transfusion of unirradiated platelet mass. an expanded immunological study confirmed the diagnosis of scid. methods: this study included patients ( boys and girls) aged months to years. the reasons for entering the hospital were manifestations of severe hepatitis (in children), acute respiratory infection ( children) and patient with symptoms of infectious mononucleosis. all patients were examined according to clinical protocols and given the severity and atypicality of the course of any infectious diseases, patients underwent immunological examination of the blood and they were consulted by an immunologist. all children were diagnosed with congenital immunodeficiency. results: in cases, the trigger for the realization of the immunodeficiency state was infection (e. meningoseptica + kl. pneumonia + b. pertussis; cmv; veb); in patients, giant cell hepatitis occurred. in patients, despite the ongoing therapy, the disease had an unfavorable (lethal) outcome ( patient with hepatitis and patient with generalized cmv infection). conclusions: thus, it should be noted that timely diagnosis of a congenital defect of the immune system and thus timely therapy will avoid adverse outcomes. interferon gamma (actimmune®) effects on severe burkholderia cepacia pneumonia in variant x-linked chronic granulomatous disease professor of pediatrics, university of utah associate professor of clinical pediatrics, keck school of medicine at the university of southern california professor of medicine, university of utah introduction/background: interferon gamma (ifnγ; actimmune®) has been proven to significantly decrease the overall number of infections in patients with chronic granulomatous disease (cgd) when given prophylactically (nejm : , ) . therapy with ifnγ has also been employed to treat severe overwhelming infections in some instances, such as severe aspergillosis with success (jid : , ) . we report here, two patients with very severe burkholderia cepacia (b. cepacia) infection, one of whom was placed on a respirator for approximately two weeks and another who was on extracorporeal membrane oxygenation for an extended period of time. both were treated with ifnγ (actimmune®) in addition to appropriate antimicrobial therapy in an attempt to affect these lifethreatening infections. objectives: the objective of this presentation is to describe two very severe variant x-linked cgd patients with b. cepacia pneumonia who were treated with ifnγ. in addition, we measured both super oxide production as well as nitric oxide production in the stimulated or unstimulated phagocytes from these patients in the presence or absence of interferon gamma. methods: case histories of both patients were reviewed in respect to the severity of their infection, the time spent on a respiratory or extracorporeal membrane oxygenation, the antimicrobial therapy administered, and the clinical results following the administration of interferon gamma as adjunctive immunomodulatory treatment. a standardized neutrophil oxidative burst assay was employed using cytochrome c reduction to measure super oxide production. in addition, nitric oxide was measured in the phagoctyes of the patients after stimulation with phorbol myristate acetate (pma) in the presence or absence of ifnγ using daf- fluorescence dye to detect the production of intracellular nitric oxide. results: a -year-old male developed a left lobar pneumonia and was admitted to primary children's medical center's intensive care unit and treated with iv cefotaxime, clindamycin, later, vancomycin and azithromycin were added. ct scan revealed a left-sided pneumonia and moderate parapneumonic effusion. subsequently, the patient decompensated, was intubated, and placed on respirator therapy. broncheoalveolar lavage and blood grew b. cepacia. neutrophil dihydrorhodamine fluorescence (dhr) demonstrated an intermediate broad peak of fluorescence with a small peak of unactivated cells, while the mother's dhr showed a broad intermediate peak suggesting the carrier state of variant x-linked cgd. both the patient, a . month old younger brother, and the carrier mother were found to have a g to a splice site mutation in exon of the gp -phox gene at position c. confirming the diagnosis of x-linked variant cgd. after approximately weeks on the respirator, ifnγ (actimmune®) therapy was instituted with significant improvement of the patient's lung function. he was taken off the respirator approximately days after the ifnγ therapy was instituted. following addition of ifnγ to his pma-stimulated neutrophil, there was a % increase in superoxide production and a fold increase in nitric oxide in his monocytes. the second patient was a -year-old male who presented with fever and cough and was diagnosed with right-sided middle and lower lobe pneumonia with cavitations. bronchoalveolar lavage grew b. cepacia and nocardia. following increasing ventilatory and circulatory collapse he was placed on extracorporeal membrane oxygenation and treated with - antimicrobial agents. after days of such therapy, ifnγ therapy was initiated and he was weaned from ecmo after days and has remained essentially healthy since then when he is on ifnγ prophylaxis. dhr revealed a broad intermediate peak in the patient and normal and intermediate peaks in the mother suggesting variant x-linked cgd in the patient and the carrier state of x-linked variant cgd in the mother. targeted sequencing revealed a g to a splice site mutation in exom of the cybb gene at position c. confirming the diagnosis of x-linked variant cgd. following addition of ifnγ to this patient's pma stimulated phagocytes, there was a % increase in superoxide production and a % increase in monocyte nitric oxide production. conclusions: two male variant x-linked cgd patients with splice site mutations in the cybb gene and severe life-threatening b. cepacia pneumonia, one on a respirator and one on ecmo were administered ifnγ (actimmune®) and each responded dramatically within - days recovering their respiratory capacity and coming off of assisted ventilation and ecmo, and have continued to do well when on ifnγ (actimmune®) therapy. introduction/background: hyqvia is a recombinant human hyaluronidase (rhuph )-facilitated subcutaneous immunoglobulin (ighy) % replacement therapy for patients with primary immunodeficiency diseases (pidd). objectives: to acquire long-term safety data on ighy, and assess prescribed treatment regimens and administration in routine clinical practice, a global postauthorization safety study (pass) is being conducted. methods: this is an ongoing prospective, non-interventional, open-label, uncontrolled, multicenter study initiated in the united states in november to assess local and systemic effects of ighy within a routine clinical setting. patients aged years with pidd who have been prescribed and/ or have started ighy are eligible for enrollment. patients are followed according to standard clinical practice and their treatment regimen is at the discretion of the treating physician. the presence of anti-rhuph antibody titers is evaluated on a voluntary basis. results: as of august , patients had been enrolled at us study sites. there were no serious aes which were deemed treatment related. sixteen patients experienced a causally related non-serious local ae ( . %; . events/patient-year, . events per infusion) and patients experienced a causally related non-serious systemic ae ( . %, . events/patient year, . events per infusion). of the patients with immunogenicity data, had positive binding antibody test to rhuph (titers : ); no neutralizing rhuph antibodies were detected. conclusions: this interim analysis of prospectively-collected data of ighy use in routine clinical practice indicates that ighy is well tolerated with no treatment-related saes and has not been associated with neutralizing anti-rhuph antibodies in patients with pidd. introduction/background: idiopathic thrombocytopenic purpura (itp) and/or hemolytic anemia accompanied by splenomegaly occurs in up to % of patients with common variable immunodeficiency (cvid). treatments include steroids, other immune suppressants and rituximab. however, in some that do not respond, splenectomy may be performed. while splenectomy is known to be associated with an increased risk of infections or thromboembolic events, studies in other conditions (hemolytic disorders, hereditary spherocytosis, etc), also suggest an increased risk of pulmonary arterial hypertension (pah) after this procedure. objectives: while splenectomy is known to be associated with an increased risk of infections or thromboembolic events, studies in other conditions (hemolytic disorders, hereditary spherocytosis, etc), also suggest an increased risk of pulmonary arterial hypertension (pah) after this procedure. methods: we report three cases of pah following splenectomy for cytopenias in patients with cvid. results: the first is a yo female, with long standing cvid complicated by interstitial lung disease, nodular regenerative liver disease and itp post splenectomy. the second is a yo man with severe bronchiectasis, cirrhosis/nodular regenerative liver disease and itp post splenectomy. the third is a yo woman with cvid (taci compound) complicated by cirrhosis/nodular regenerative liver disease, lung nodules and evans syndrome. all developed severe pah requiring chronic medications. pah in these patients is best classified as multifactorial, group v. conclusions: whether due to thrombus formation, continued cytopenias, and/or vascular changes, we suggest that pah may be a long-term complication of splenectomy in complex cvid. connective tissue, skeletal and vascular abnormalities. two isoforms exist, stat , a amino acid protein, and stat , a amino acid protein produced by alternative splicing of exon resulting in a frame shift and truncated protein at the c-terminus. objectives: we follow patients from families with stat mutations leading to altered c-terminal proteins. the patients have high ige but milder features of ad-hies. methods: clinical data were collected. stat sequencing, stat functional assays as well as lymphocyte phenotyping were performed. results: patient is a year old man diagnosed with hies as a child due to eczema, recurrent boils and high ige ( s iu/ml). he has tortuous and dilated coronary arteries, however denies lung infections, cmc, retained teeth, scoliosis, minimal trauma fractures, or hyperextensible joints. as an adult he developed avascular necrosis of both hips. whole exome sequencing revealed a novel splice mutation, c. + g>t at the end of exon causing skipping of the nucleotide exon as well as utilization of the stat alternative splice acceptor in exon , resulting in a nucleotide deletion. the mutant stat protein product has a amino acid, in-frame deletion encompassing both y and s phosphorylation sites. no stat is made from the mutant allele. lymphocyte phenotyping was unremarkable, however total stat protein levels were decreased in ebv transformed b cell lines and there was decreased y phosphorylation after stimulation. patient (family ) was healthy until diagnosed with severe, refractory coccidiodies pneumonia complicated by pneumothorax with prolonged bronchopleural fistulae at age years. this led to an immune evaluation in which he was found to have elevated ige ( iu/ml, ref . - . iu/ ml). he had one perianal abscess, primary teeth requiring extraction and mild scoliosis, but denies cmc, bacterial pneumonias, eczema, or minimal trauma fractures. stat sequencing revealed a single base insertion in the transactivation domain, c. _ insc causing a frameshift in the stat isoform, p.r pfsx , occurring immediately after the s phoshporylation site. the deletion occurs within the alternatively spliced region of exon , providing an intact, wild-type stat . stimulation with il- or il- showed reduced pstat at y , and elevated pstat which is often seen in other ad-hies patients. lymphocyte phenotyping was unremarkable and th cell analysis showed low-normal levels of th cells while ebv transformed b cells showed reduced total stat levels. his mother and infant sister also share this mutation -the month old infant had normal ige, and intermittent rashes; his mother has high ige ( iu/ml), recurrent sinopulmonary infections (complicated by tobacco use) but without bronchiectasis or pneumatocele, and denies cmc with the exception of pregnancy related vaginal candidiasis. conclusions: loss of function and gain of function mutations in stat lead to distinct syndromes, but it appears that stat mutations affecting the isoform expression, such as those reported here, can also lead to immune dysregulation with incomplete features of ad-hies. these stat mutations will allow us to better understand the relative roles of the isoforms stat and stat in somatic and immune cell signaling. physicians and is associated with immunological defects aswell. mutationsin the kmt d and kdm a genes are the most common genetic changes that lead to kabuki syndrome but for many cases the genetic basis remains unknown. objectives: recognize the varied presentation for a unique immunodeficiency syndrome methods: this is a case series. results: this is a case series describing three patients with ks and their clinical presentations which predominantly involve immunodeficiency and autoimmunity. our first patient is a -year-old male with autoimmune hemolytic anemia (aiha) at a young age, recurrent respiratory infections and hypogammaglobinemia which led to a diagnosis of cvid at the age of six. in addition, he has dysmorphic facial features, intellectual disabilities, and short stature but it was not until his late twenties where he was found to have a missense mutation in kmt d (p.arg cys) which has been described in patients with ks. the second patient is a -year-old female with hypogammaglobinemia, evan's syndrome, short stature, and severe complications which include granulomatous-lymphocytic interstitial lung disease, pulmonary hypertension, and chronic kidney disease. she was also found to have a missense mutation in kmt d (p.arg cys) in her early thirties and passed away from complications of her disease. the third patient is a -year-old female with a history of low iga/igg and poor vaccine titers, aiha, neutropenia, pulmonary nodules, and developmental delay who was diagnosed with cvid in her mid-twenties. for her immunodeficiency and autoimmunity, she was treated with immunoglobulin replacement, rituximab and cyclosporine and was found to have a missense mutation in kmt d (p.cys trp). this mutation was a de novo mutation in this patient and has also been reported in another patient with ks. conclusions: these cases highlight that the presentation of ks is varied and frequently includes immunodeficiency and autoimmunity in addition to the characteristic short stature and developmental delay. a diagnosis of ks remains challenging due the diversity of symptoms and disease severity, the need for genetic testing, and due to overlapping clinical presentations with other developmental conditions. thus, often times, like in these cases, the diagnosis of ks is delayed. chief medical officer, rocket pharma introduction/background: lad-i is a rare disorder of leukocyte adhesion, resulting from itgb gene mutations encoding for the beta- integrin component cd . cd deficiencies prevent integrin dimerization and endothelial leukocyte adhesion, essential for extravasation and antimicrobial activity. severe lad-i (< % of normal neutrophil [pmn] cd levels) is characterized by recurrent serious infections and early mortality unless treated by allogeneic hematopoietic stem cell transplant (hsct). mortality for severe lad-i was reported as % by age in an initial multicenter retrospective study. moderate lad-i ( - % of pmn cd levels) is more indolent; although most patients (pts) survive childhood with recurrent skin and mucosal surface infections; mortality by age can exceed %. lad-i is characterized by umbilical cord complications (delayed separation and omphalitis), poor wound healing and leukocytosis. objectives: reports regarding lad-i have been published in recent decades but no recent comprehensive prognostic assessments are available. we sought an updated understanding of severe lad-i with emphasis on prognosis in the absence of hsct, hsct outcomes and association of cd expression with clinical features. methods: we created a database of all published lad-i cases via pubmed searches and review of available references. results: three hundred twenty-three lad-i cases were reported between - in publications ( case-reports; largest series n= ). the nations reporting the most cases were iran (n= ), usa (n= ), and india (n= ); the highest number of publications were from us centers ( ). pts were considered to have severe lad-i, moderate and were not classified. pmn cd expression levels was reported for cases and was < % in patients ( %) and >= % in pts. four pts with cd > % were considered to have severe lad-i (cd % range . . ). gender was noted for pts; ( %) were male. age at presentation was reported for cases. for pts with cd < %, median presentation was age m (range . - m); for pts with cd >= %, median presentation was age m (range . - m). infection details and cd % were available for ( %) cases. the most frequent infections in pts with cd < % were respiratory tract ( %), sepsis ( %) and otitis media ( %) and for pts with cd >= % they were periodontal ( %), otitis media ( %) and sepsis ( %). perianal skin infections and necrotic skin ulcers were noted in > %. umbilical complications were more frequent in severe lad-i ( of pts with cd < % [ %] and of with cd >= % [ %; p = . ]). for severe lad-i pts with years of follow-up (or death prior to y), there was correlation between absence of umbilical complications and survival to m (p < . ). wbcs were reported in cases (median x /l; range x /l). there were limited correlations between cd expression and wbc (r < . ) and between cd and cd expression (r < . ). mutation analyses were reported in cases with > gene locations noted and mutations on exons , and accounting for % of specified cases. in cases, cd expression was > %; in of cases where cd expression was noted, at least one cd moiety was reported as < %. we sought to understand whether prognosis for severe lad-i in the absence of hsct is similar to the initially-reported % survival to age . there were severe lad-i cases (per investigator assessment or cd < %) for whom survival to years was reported, of whom died prior to age ( % mortality). mortality was similar for the subset of cases reported since ( %, deaths). early mortality was substantially lower in patients with cd >= % and the majority of pts with cd > % survived to adulthood. outcomes for pts who received hsct were consistent with recent series; phenotypic correction was reported in % of pts with hla-matched sibling donors. mortality was % overall ( % for hlamatched sibling recipients). for pts receiving haploidentical hsct there was % mortality and % received subsequent hsct. conclusions: severe lad-i remains a life-threatening condition with limited -year survival in the absence of allogeneic hsct. umbilical complications and granulocytosis are frequent early manifestations; respiratory tract, ear, sepsis, oral and skin infections are common. hsct is potentially curative; transplant-mortality and other complications are frequent, especially in haploidentical recipients. diverse itgb mutations result in lad-i, and genetic evaluation may be valuable for diagnosis and prognosis. rapid identification of pts with potential lad-i (unusual or severe infections in infancy, granulocytosis and umbilical complications) is essential to enable referral to centers with disease expertise. objectives: to determine the phosophorylation of stat in ad-hies patients with known stat mutations. methods: peripheral blood mononuclear cells (pbmcs) were collected from ad-hies patients under irb-approved institutional research protocols. pbmcs were then stimulated with il- or il- for , and minutes. cells were surface stained for cd , cd , cd , cd and cd . they were then fixed, permeabilized and stained with anti-y -stat to evaluate for phosphorylation of stat at position y . cells were then washed and data acquired using flow cytometry. results: pbmcs from one ad-hies patient with a stat sh domain mutation (p.y c) demonstrated normal y phosphorylation after il- stimulation. pbmcs from another ad-hies patient with a dbd mutation (p.h y) exhibited highly reduced stat phosphorylation after il- stimulation and absent phosphorylation after il- stimulation. conclusions: these findings highlight that, in the context of ad-hies, the domain location of the stat mutation does not predict stat phosphorylation potential following stimulation and challenges the current paradigm. ( ) submission id# cheng sun associate professor, institute of immunology introduction/background: as the predominant lymphocyte subset in the liver, natural killer (nk) cells have been shown to be highly correlated with the outcomes of patients with hepatocellular carcinoma (hcc). previously, we reported that nk cells were decreased and functional deficiency in hcc. however, the mechanism underline remains unknown. objectives: in this study, through the use of healthy livers, paired peritumoural tissues (pt) and intratumoural tissues (it) from hcc patients, methods: we have evaluated the expression of cd and its co-ligand receptor btla on hepatic cd + t cells and nk cells. results: decreased expression of cd on nk cells was observed in intratumoural but not pt regions, along with nk cell dysfunction, poor prognosis and tumour metastasis. human cd + nk cells exhibited functional activated, high capacity of ifn-secretion and nk mediated immunity by global transcriptomic analysis of sorted cd + and cd -hepatic nk cells. blocking tgf- specifically reversed the ifn-production of cd + nk cells. in addition, this decreased cd expression is predominantly on cd bright nk cells. conclusions: these findings indicate that cd expression reduction contributes to nk cell exhaustion and tumour immune escape, suggesting that cd has the therapeutic potential for fighting liver cancer. introduction/background: the low number of circulating lymphocytes in the blood is a marker for cellular immunodeficiency in young children. ethnicity also affects the lymphocyte count and ethnicity-specific lymphocyte norms have been used in many countries. this study analyzed the lymphocyte counts in a large cohort of infants and young children from the arabian peninsula. objectives -to define the normal lymphocyte counts in arab children -to define the possible cutoff lymphocyte count that define lymphopenia. methods: this is a cross-sectional analysis of the lymphocyte counts in , arab children. the age groups were: day, - months, - months, - years, - years, - years, - years, and - years, % females. we analyzed the first blood count performed during their visit to the abu dhabi seha ambulatory healthcare services between april and october . the median, th percentile and th percentile counts were calculated. the th percentile lymphocyte count was used to define lymphopenia. the kolmogorov-smirnov test, a non-parametric test, was used to compare lymphocyte counts between groups. statistical significance was defined by a two-tailed p< . . results: the median counts were higher during infancy. the variability (disparity) of the counts (reference intervals) progressively decreased from birth to years of life. the th percentile lymphocyte counts were relatively constant from birth to years ( . - . x /l) and from to years ( . - . x /l), table . the lymphocyte counts were similar in boys and girls. the lymphocyte counts were compared to those from five other studies. conclusions: arab children have lower lymphocyte counts ( th percentile) than children in the united states, brazil and south africa, but their counts are similar to children in china and uganda. our study results support the development and use of ethnicity-specific lymphocyte count standards. the implication of our results is that using these lower cutoff values for lymphopenia will prevent a large number of arab children from having unnecessarily investigated for immunodeficiency. introduction/background: chronic granulomatous disease (cgd) is a rare phagocytic defect caused by mutations in the nadph oxidase system leading to reduced or absent reactive oxygen species production. in addition to specific infectious susceptibility, patients with cgd are predisposed to hyperinflammation in response to infectious agents, autoimmunity, colitis, and other forms of autoinflammation. cgd patients with mutations in p phox are at increased risk of diabetes and cardiovascular disease. hyperinflammatory and auto-inflammation responses are often difficult to predict and manage. intracellular adhesion molecule- (icam- ) and e-selectin are endothelial adhesion markers that facilitate the adhesion and transendothelial migration of leukocytes and elevations in these markers have been associated with cardiovascular disease, glomerular injury, and thrombotic events. expression of adhesion molecules is induced by pro-inflammatory cytokines and are associated with a hyperinflammatory state. objectives: to determine if e-selectin and icam- are elevated in patients with cgd and to determine of endothelial adhesion markers could serve as a biomarker of inflammatory disease in cgd patients. methods: thirty-eight pediatric and adult subjects with cgd ( xlinked (xl-cgd), p phox deficient and p phox deficient, and x-linked cgd carriers were enrolled. e-selectin (pg/ml) and icam- (ng/ml) were measured from the plasma of patients via sandwich elisa. results: all cgd patients had histories of severe infection, active infection, chronic colitis, or other autoimmune disease at time of evaluation. nine p phox deficient cgd patients had history of diabetes and/or early onset cardiovascular disease. one subject with x-linked cgd had undergone hematopoietic stem cell transplantation (hsct). plasma levels of e-selectin were significantly elevated above healthy controls (median , pg/ml) in subjects with xl-cgd (median , pg/ ml, p< . ), p phox deficient cgd (median , pg/ml, p= . ) and p phox deficient cgd ( , pg/ml). plasma levels of icam- were also elevated above healthy controls in subjects with xl-cgd (median . ng/ml), p phox deficient cgd (median . ng/ml), and p phox deficient cgd ( . ng/ml), although none were statistically significant. plasma quantities of e-selectin and icam- increased further in those p phox deficient patients with diabetes and/or cardiovascular disease (median e-selectin , pg/ml, icam- . ng/ml) but neither reached statistical significance. e-selectin and icam- quantities in female carriers of xl-cgd and in xl-cgd cured by hsct were similar to values found in healthy controls. conclusions: immune dysregulatory features and hyperinflammation in cgd can be difficult to predict and manage. the endothelial adhesion markers e-selectin and icam- are elevated in patients with xl-cgd and p phox deficient cgd that worsens with presence of early onset cardiovascular diseases and resolves post-hsct. elevations in e-selectin and icam- in the serum of cgd patients may serve as surrogate markers of inflammation and suggest a chronic endotheliopathy in cgd patients. introduction/background: the phenotypic presentation of ctla haploinsufficiency was only recently described. the management of these patients in the medical literature is limited to anecdotal case reports. we aimed to detail our experience with short and long term immunomodulatory therapy in treating autoimmune cytopenias in the background of ctla impairment. objectives: we aimed to assess the efficacy of mtor inhibitors in treating autoimmune cytopenias in patients with ctla haploinsufficiency. methods: we retrospectively identified patients with proven ctla mutations and documented refractory autoimmune cytopenias while receiving care at nih clinical center (from july to august ). the complete (cr) and partial (pr) clinical response was assessed after six weeks of treatment and defined as hgb > g/dl, platelets > k/ul and hgb > g/ dl, platelets > k/ul, respectively without transfusion requirement. results: all analyzed patients failed or exhibited disease recurrence on at least one prior medical therapy, including: corticosteroids, rituximab, romiplostim and eltrombopag. the initial response rate to evrolimus and/or sirolimus was % ( cr and pr). one of the partial responders had recurrence of idiopathic thrombocytopenic purpura at four months while on rapalogs. overall, we used mtor inhibitors in patients for a total of patient years to treatm multiple modalities. the top three most common recorded adverse events were: clostridium difficile colitis (n= , in patients), lipid abnormalities (n = , patients required treatment) and bacterial pneumonia (n= , in patients). conclusions: our limited retrospective data suggests that mtor inhibitors might be efficacious in the treatment of autoimmune cytopenias in ctla haploinsufficent patients. further prospective studies are required to assess safety and efficacy of mtor inhibitors in this patient population. association with frequent upper respiratory tract infections and pharyngitis. from years of age she continued to have recurrent febrile episodes in the absence of infection, with fever of - degrees celsius on a monthly basis, normally lasting - days. she also developed episodes of urticaria, temporally unrelated to her febrile episodes. the rash was noted to be induced by exposure to the cold, and would generally last - days with some clinical response to antihistamines and naproxen. her clinical picture then progressed and she developed joint pain and swelling, particularly affecting her hands and feet. over the following years the patient continued to have recurrent episodes of urticaria as well as progressive joint involvement and limitation. there was some initial improvement with naproxen and prednisone. her symptoms were refractory to subsequent treatment with methotrexate, leflunomide, infliximab and tocilizumab and she remained corticodependent. immunological work up including lymphocyte phenotype, immunoglobulins, vaccine responses to both protein and live vaccines and ch were normal. there was no evidence of raised inflammatory markers with esr - mm/h, crp < . mg/l and ferritin ug/l. autoimmune workup including ana, ena, anti-dna, anca and c and c was normal. the initial differential diagnoses included systemic juvenile arthritis, periodic fever syndrome or cryopyrin associated periodic syndromes (caps). the patient therefore had a periodic fever gene panel that identified a heterozygous mutation in exon of nlrp , c. c>t (p.arg trp). whilst mutations in nlrp are known to be associated with familial cold autoinflammatory syndrome, this was reported as a variant of unknown significance. we therefore proceeded with functional in vitro testing to demonstrate pathogenicity. the patients monocytes showed increased secretion of il b upon stimulation with lps as compared to healthy donors. when tested in a luciferase reporter assay, the mutated nlrp partially lost the capacity to inhibit the nfkb pathway. overall these in vitro studies show that this nlrp mutation results in defective regulation of the inflammatory response. the patient was commenced on anti-il therapy with canakinumab, with no clinical improvement so was therefore discontinued. we report a case of familial cold autoinflammatory syndrome due to a mutation in exon of nlrp , that to this point has remained refractory to multiple treatment modalities. functional testing was able to demonstrate mutation causality and defective regulation of the inflammatory response. objectives: authors aim to evaluate the spectrum of clinical phenotypes associated with nemo hypomorphic mutations within the usidnet registry. methods: investigators obtained demographic, laboratory, and clinical data on patients with a defect in nemo within the usidnet registry. results: there were male patients within the usidnet registry with a diagnosis of eda-id attributed to nemo hypomorphic mutation. of these, were associated with a known variant in nemo (e x, f l, m v), and were associated with previously unreported variants (d v, e del, c. - g>c). for patients, a mutation was not specified. most reported having an affected family member (n= , %). median age of symptom onset and diagnosis were year (iqr . - y) and years (iqr - y), respectively. median age at most recent visit was years (iqr - y). infections, additional clinical features, treatments, and outcomes are summarized in table . skin manifestations (n= , %) and pulmonary complaints (n= , %) were common, with eczema (n= , %) and asthma being most prevalent (n= , %). gastrointestinal conditions (n= , %) were also frequently reported, and included non-specific diarrheal illness, enteropathy, colitis, enteritis, and inflammatory bowel disease. neurologic features (n= , %), including seizures, hearing defect, peripheral neuropathy, and encephalopathy, were unexpectedly common, suggesting a previously unrecognized disease association. conclusions: we observed that allergic diseases, including asthma and eczema, were common in patients with nemo mutation. notably, varied neurologic features were more prevalent than previously reported. this study highlights the potential of cross-institutional registry analysis to deepen our understanding of extremely rare genetic diseases. coordinated effort across institutions is required to better characterize the spectrum of clinical phenotypes associated with hypomorphic mutations in nemo. mycobacterial lysate (ml) and purified protein (ppd) in the diagnosis of patients with mendelian susceptibility to mycobacterial disease (msmd) elimination of this infection depends mainly on the success of the interaction between macrophages and infected t lymphocytes. patients with mendelian susceptibility to mycobacterial disease (msmd) present severe and recurrent infections due to impaired signaling of the ifn/il- axis. objectives: our aim was to evaluated the ifn/il- axis of patients with clinical history suggestive of msmd using mycobacterial lysate (ml) and purified protein (ppd) by elisa assay. methods: samples of patients (n= ) with a clinical history suggestive of msmd arrived in our laboratory. for the diagnosis, ml of blood diluted ( : ) in rpmi culture medium supplemented were used. the samples were distributed in two different plates, one used in the dosage of il ( h) and the other in the ifn dosage ( h). thus, they were stimulated with ml ( ug/ml for the h plate and ug/ml for the h plate) and with ppd ( ug/ml for the h plate and ug/ml for plate of h). at the same time, in half of the wells stimulated with lm and ppd, the cytokine ifn ( iu/ml) or il p ( ng/ml) were added in the plate. next, the plates were incubated at °c and % co² and the supernatant were collected and quantified by the elisa assay. the results were evaluated by the statistical mann whitney u test. results: six of the patients presented alterations in the evaluation of the ifn/il- axis. the age of the diagnosis of male patients ranged from to years. the only two female patients diagnosed were and years old. the clinical history was heterogeneous: had lymph node hyperplasia, pneumonia, colitis, herpes zozter, another had a urinary tract infection and bcgitis. the pathogens isolated were the m. tuberculosis, m. abscessus, m. gordonae, m. genavense and m. konsossi species found in different patients. statistical analysis of the ifn-/il- axis evaluation by elisa was performed.the results of the dosage of ifn were significant in samples with lm and lm plus il- (***). similar results were observed in samples treated with ppd and ppd plus il- (**) when compared with healthy controls. in the il- dosage, statistical difference was observed in the samples with lm (***) and with lm plus ifn (*). in samples stimulated with ppd, the results did not show statistical differences (ns.) but ppd + ifn (*) were significantly different. conclusions: six patients were diagnosed by the evaluation the il- / ifn axis. the use of micobacterial lysate (ml) showed reliable results to the diagnosis of patients with il /ifn pathway defects. the genetics diagnosis will be performed. introduction/background: invasive infections due to mycobacterial species are a feared complication in patients with t-cell deficiency or phagocyte disorders, and treatment is frequently complicated by antimicrobial resistance. cd + th t-cell immunity is known to be critical to antimycobacterial defense; accordingly, adoptive t-cell immunotherapy with mycobacteria-specific t-cells (mst) may be a beneficial therapy for combating these infections. objectives: to determine if ex vivo expansion of t-cells targeting common mycobacterial antigens is feasible from healthy donors, and whether the same antigens are recognized by patients with primary immunodeficiency (pid) and invasive mycobacterial infections. methods: peripheral blood mononuclear cells (pbmc) from healthy donors were pulsed with overlapping -mer peptide libraries encompassing five mycobacterial antigens (ag b, ppe , esat- , cpf , adk) and expanded for days with cytokines il- and il- . expanded msts were tested for specificity against the targeted antigens via ifn-g elispot, multiplex cytokine analysis, and flow cytometry. pbmcs from pid patients with invasive mycobacterial infections were similarly tested for presence of t-cells recognizing the tested mycobacterial antigens. a minimum of spots per x cells above negative control was considered specific on elispot. results: ten healthy donors and eight patients with pid were tested. specificity against - mycobacterial antigens (median ) was confirmed in all ten healthy donors, with a mean . -fold cellular expansion during the -day culture. msts were predominantly cd + t-cells (mean/sd: +/- %), with both central memory (mean/sd . +/ - . %) and effector memory (mean/sd . +/- . %) populations. there was no clear difference in antigen specificity between bcg immunized (n= ) and bcg naïve (n= ) healthy donors. six of pid patients had no detectable immunity to tested mycobacterial antigens. one patient with combined immunodeficiency has a low detectable specificity to ag b (mean spot forming colonies[sfc]), and a patient with nfkb haploinsufficiency mounted a response against ag b (mean sfc) and ppe (mean sfc). conclusions: mycobacteria-specific t-cells can be rapidly expanded from healthy donors utilizing a protocol that could easily be translated to a good manufacturing practices facility. the majority of tested pid patients lacked immunity to the targeted antigens. adoptive immunotherapy with msts derived from third-party healthy donors may be a beneficial adjunctive therapy for pid patients with invasive mycobacterial infections. is an innate immune deficiency, primarily affecting the phagocytic compartment, and presenting with a diverse phenotypic spectrum ranging from severe childhood infections to monogenic inflammatory bowel disease. dihydrorhodamine (dhr) flow cytometry is the standard diagnostic test for cgd, and correlates with nadph oxidase activity. while there may be partial genotype correlation with the dhr flow pattern, in several patients, there is no correlation. objectives: in such patients, assessment by flow cytometric evaluation of nadph oxidase-specific (nox) proteins provides a convenient and rapid means of genetic triage (table) . methods: we performed dhr flow cytometry and nox flow cytometry on granulocytes and monocytes of cgd patients. results: phenotypic and laboratory patient data shown in table. *p had decreased p phox (% and mfi) monocytes, but not in granulocytes. all other siblings (p , p , and p ), and mother (p ) had relatively higher p phox (%) monocytes, but still lower than the healthy control. p had normal %p phox monocytes, comparable to control. however, p -p , and p had normal p phox (mfi) in monocytes and granulocytes. p - , and p have normal %gp phox+ granulocytes, while p and p have modestly decreased %gp phox in monocytes. p - , and p have all moderately decreased gp phox protein (mfi) in granulocytes and monocytes compared to healthy control, with a single population for protein expression. p , p and p have bimodal populations for gp phox in monocytes but not in granulocytes, with a larger positive population, and a much smaller negative population. the data from p - suggest that the amount of gp phox does not necessarily correlate with neutrophil oxidative burst, as measured by dhr. also, not all cybb variants affect p phox protein expression, though both proteins are membrane-bound. the cyba vus in p does not appear to have affected p phox protein expression in either monocytes or granulocytes. conclusions: the atypical clinical presentation of some cgd patients can make genotype-phenotype correlation with dhr flow data challenging. genetic testing, while necessary, can take several weeks. however, nadph-oxidase specific-protein flow assessment offers a rapid alternative to identification of the underlying genetic defect, and can be utilized as a reflex test to an abnormal dhr flow. further, it can provide insight into correlation between oxidative burst relative to protein expression in granulocytes and monocytes. head, division of immunology and allergy, the hospital for sick children introduction/background: adenosine deaminase (ada) is a ubiquitous enzyme important for purine metabolism. few studies have indicated that ada deficiency, in addition to causing profound lymphopenia and susceptibility to infections, is associated with neutrophils abnormalities. objectives: determine whether ada deficiency directly affects neutrophils and what are the mechanisms involved. methods: peripheral blood (pb) and bone marrow (bm) from . weeks old ada-deficient (ada-/-) mice that closely recapitulate the phenotype observed in ada-deficient patients, as well as ada+/-littermates were used to study neutrophils development and function. some experiments were supplemented with -week old ada-/-mice, maintained until weeks of age with ada enzyme replacement, and littermates. results: the number of neutrophils in pb of ada-/-mice at . and weeks of age was similar to ada+/-mice. the function of pb neutrophils from ada-/-mice, determined by oxidative burst, was also normal. the percentage of lin-/c-kit+/sca + hematopoietic progenitor cells in bm demonstrated significant reduction in ada-/-mice compared to littermates ( . ± . % and . ± . %, respectively, p= . ). moreover, expansion of bm isolated from ada-/-mice in methylcellulose resulted in significantly less cd b+/ly- g+ neutrophils compared to healthy controls ( . ± . % compared to . ± . %, respectively, p= . ). proliferation of bm cells, determined by brdu incorporation into cells dna, was higher in ada-/-mice than in littermates, possibly contributing to the normal neutrophil numbers in pb of ada-/-mice. conclusions: ada deficiency directly affects neutrophil development. further studies will help understand the significance of these effects and potential therapies for ada-deficient patients. objectives: we present here the clinico-pathologic features of a -yearold female with a heterozygous fancd gene deletion/mutation with evidence of cellular and humoral immune dysregulation. methods: we evaluated the patient using standard immunology anatomic, cellular, and biochemical functional assays. results: the patient has multiple dysmorphias including total anomalous venous return (repaired), mesomelia, absent ear canal, radial ray dysplasia, and short stature. her medical history is significant for an episode of pneumococcal sepsis despite adequate vaccination. whole exome sequencing demonstrated deletion of exons - and a pathologic mutation (c. g>a, p.arg gln). repeated blood samples and immunophenotyping demonstrated severe lymphopenia. there were markedly low cd + t-cell counts with a low cd :cd ratio ( . ). changes in the composition of the b-cell population included: significantly diminished absolute total b-cells, elevated immature cells, low levels of transitional cells, and undetectable advanced b-cell populations. there was no immunogenic response to pcv- or varicella/tetanus/ diphtheria vaccination. the nk-cell count was unaffected and demonstrated normal spontaneous and stimulated cytotoxic response. bone marrow analysis demonstrated hypocellularity without dysplasia. conclusions: we report here a pediatric patient with a novel fancd deletion/mutation presenting with severe lymphopenia in two cell compartments (b and t cells) and susceptibility to invasive bacterial infection. the findings are suggestive of combined immune deficiency. the cellular immune profile suggests that fancd may be involved in the transition of immature b and t cells to mature cells, a process that requires substantial dna recombination. additional genetic and biochemical evaluation is needed to further characterize this rare clinical finding. introduction/background: familial hemophagocytic lymphohistiocytosis type (hlh) is a rare fatal condition due to a mutation in the pfr gene on chromosome q - inherited in an autosomal recessive pattern which results in overactivation of the immune system. symptoms usually manifest before year of age. a -year-old previously healthy male presented with acute onset of bilateral lower extremity pain and weakness, with subsequent inability to ambulate over a -day period. neurological exam demonstrated decreased lower extremity power, sensation, absent patellar and achilles reflexes, and a wide-based gait. objectives: not applicable methods: not applicable results: laboratory data was significant for neutropenia and thrombocytopenia, elevated levels of ferritin and serum cd , and a positive ebv pcr. patient was initially treated with ivig for possible ebv-driven guillain-barre syndrome with some improvement in neurologic symptoms as well as thrombocytopenia. a mass of retroperitoneal lymph nodes were noted on spinal mri. testing was negative for alps and bone marrow biopsy was negative for leukemia/lymphoma. further work-up revealed absent perforin expression in cytotoxic cells and normal sap protein expression on staining, with poor nk function. genetic testing revealed a pathogenic mutation in the pfr gene with additional variant of unknown significance. the patient was treated with rituximab for persistent ebvand returned with worsening lower extremity weakness. on mri, focal enhancements were found in the brain as well as worsening mass compression of the lumbosacral nerve roots. nerve root biopsy showed histiocytes with hemophagocytosis and a dense lymphohistiocytic infiltrate which stained positive for cd , cd , and granzyme b, with some loss of cd and no perforin. bone marrow biopsy was negative for hemophagocytes. conclusions: the patient was diagnosed with worsening familial hlh with cns involvement. hlh-directed chemotherapy (dexamethasone, cyclosporine, etoposide, intra-ommaya methotrexate/hydrocortisone) was started and hsct was performed. familial hlh is a rare and often lethal disorder that generally presents at a very young age. the acute onset and severity of presentation as occurred in this previously healthy adolescent is uncommon. familial hlh should be considered even in older patients with unexplained overactivation of the immune system. is the most profound form of primary immune deficiency, and is usually fatal in the first year of life without treatment. newborn screening for scid using quantitative analysis of t-cell receptor excision circles (trecs), has become the accepted method to facilitate early diagnosis and treatment in most of the united states, as scid babies typically do not make trecs. ikbkb deficiency is a rare form of autosomal recessive scid found in the northern cree first nations people of canada, where t cells develop normally but are non-functional. trec analysis is expected to be normal in ikbkb scid, and does not identify these cases. objectives: the objective of our study was to determine the feasibility of targeted genetic newborn testing for ikbkb deficiency. methods: we implemented a pilot project of prospective targeted genetic testing for the previously described homozygous ikbkb mutation (c. dupg) in newborns from small northern manitoba communities. between and , dna was extracted from dried blood spots of newborns, and targeted sanger sequencing of the mutationharbouring ikbkb exon was performed. results: all infants born in the selected communities underwent testing. fifty-five infants ( . %, or / . ) were found to be heterozygous carriers. one affected infant was identified, and underwent hematopoietic stem cell transplant before onset of infections. our findings are consistent with the predicted homozygosity for this mutation ( / . x / . x / = / births). conclusions: we demonstrated that targeted newborn testing for ikbkb deficiency was feasible, and provided the first prospective estimate of the ikbkb mutation carrier frequency in select manitoba northern cree first nations populations. we suggest that if we are to capture all babies with scid in manitoba, future newborn screening should be universal and include both trecs and direct mutation testing for population-specific mutations, including the first nations ikbkb mutation. high throughput analysis for trecs and targeted mutations will be introduced for universal newborn screening in manitoba. introduction/background: immunoglobulin class-switch recombination (csr) and somatic hypermutations (shm) are prerequisites of antibody and immunoglobulin receptor maturation and diversity within the adaptive immune system. the mismatch repair (mmr) machinery, consisting of homologues of mutsa, mutla, and mutsb (msh /msh , mlh /pms , and msh /msh , respectively) and other enzymes, is involved in csr, e.g. as backup of nonhomologous end-joining repair of activation-induced cytidine deaminaseinduced dna mismatches, and furthermore, in addition to errorprone polymerases, in the repair of shm-induced dna breaks. in line, a varying degree of antibody deficiency, from iga or selective igg subclass deficiency, to common variable immunodeficiency and hyper-igm syndrome have been shown in small numbers of patients with constitutional mmr deficiency (cmmrd) in addition to the known severe cancer predisposition due to genomic instability of patients with biallelic loss-of function mutations in one the mmr components. objectives: to elucidate the clinical relevance of primary immunodeficiency (pid) in cmmrd, we collected history and laboratory data of a novel cohort of consecutive patients from families with homozygous mutations in pms (n= ), msh (n= ), and mlh (n= ) reported to the consortium care for cmmrd (c cmmrd) between and , most of whom manifested with typical malignancies during childhood. methods: retrospective chart review according to a specific questionnaire and extended routine immunological analyses were performed with irb approval from the medical university of graz, austria. results: none of the presented patients fulfilled any classical or extended clinical warning signs of pid (infections, immune dysregulation, inflammation). furthermore, analyzing multiple specific laboratory parameters of the humoral and cellular immune system, we could not detect a uniform pattern of abnormalities. importantly, our data do not confirm previous suggestive evidence of iga or igg subclass deficiency, a specific antibody formation, or a b memory cell maturation defect. results of next generation sequencingbased detection of impaired class switch recombination and somatic hypermutations are pending. the t cell subsets and receptor repertoires were unaffected. together, neither clinical nor laboratory parameters were suggestive of pid in the present series of novel cmmrd patients. conclusions: we conclude that patients with cmmrd do not generally show a clinically relevant pid that could facilitate early diagnosis. on the contrary, these data support the prospect of potentially successful immune therapy of malignancies in the context of cmmrd. introduction/background: ada- deficiency is immune dysregulation diseases caused by an autosomal recessive mutation on cecr gene characterized by polyarteritis nodosa, childhood-onset, early-onset recurrent ischemic stroke and fever. objectives: report a new mutation on the cecr gene resulting in ada- deficient children. methods: female, -year-old, presented history of multiple ischemic strokes at one-year-old associated with recurrent fever and livedo racemosa. she has no siblings and parents are not consanguineous. results: the laboratory evaluation shows red cell= . x /ml, hemoglobin= . g/dl, leucocytes= . x /ml, neutrophils= . x / ml, lymphocytes= . x /ml and plateletes= x /ml. ige< ui/ml, igg= mg/dl, igm= . mg/dl and iga= . mg/dl. subsets lymphocytes shows cd += . %, cd /cd = . , cd + = % and cd / = . %. due the clinical history, we performed cecr gene sequence homozygous substitution located at position - of acceptor splice site intron , c. - g>a. this is a high conservative region with no alteration along phylogenetic studies and predicted to be pathogenic. to confirm the functional alteration, the ada- activity was tested in dried plasma spot showing . mu/g protein confirm the gene loss of function and the mutation pathogenicity. conclusions: the authors presented a novel mutation of cecr gene, the first one described in splice site causing gene loss of function and confirmed by extremely reduced ada activity. introduction/background: severe combined immunodeficiency (scid) is the most severe form of primary immunodeficiency characterized by severe, life threatening infections during early infancy. scid is a medical emergency associated with significant mortality if hematopoietic stem cell transplantations is not instituted early in the course of the disease. scid is a genetically heterogeneous disease caused by mutations in more than different genes. different genes are implicated in different ethnic populations and geographical locales depending on the rates of consanguinity and endogamy in these populations. however, following the institution of newborn screening of scid in almost all state of the us and the widespread use of next generation sequencing in primary immunodeficiency diseases ar-scid due to mutations in rag and rag genes are found to be more prevalent than reported earlier. objectives: we performed a retrospective analysis of scid cases diagnosed at our centre and referred to us from other centres to determine the clinical, immunological and genetic basis of the disease in these cases. genetic variants both recurrent and novel were analysed in detail. methods: fifty six ( ) of the suspected patients met the esid diagnostic criteria. the clinical features, immunological defects and the gene sequencing results of these patients were analysed. gene sequencing was performed at the our centre and other collaborative centres at dept of pediatrics and adolescent medicine, queen mary hospital, hong kong, national defense medical college, saitama japan, kazusa dna research centre, chiba, japan and duke medical university centre, usa. mutations were detected in of the patients. mutations were classified as recurrent or novel after checking different databases such as exome aggregation consortium (exac), human gene mutation database (hgmd) and other relevant scid databases. the effect of novel, previously unreported mutations was determined using in-silico prediction tools such as sift and polyphen . functional studies were also performed in few cases to determine the effect of novel mutations. results: mutations were detected in patients. mutations were more common in genes causing autosomal recessive form of scid than the x-linked variant. mutations were detected in il rg gene in patients followed by mutation in the rag gene in patients, dclre c in patients and rag in patients. mutations were also detected in ada gene ( patients, mutations), il r ( patients), stim , pnp and nhej ( patient each). nine novel mutations were detected. three in il rg gene, in rag , in ada and one each in the nhej and il r gene. conclusions: autosomal recessive form of scid was more common in our cohort compared to x-linked form of the disease. mutations in rag and rag genes were the commonest ( patients) followed by mutation in il rg gene ( patients). nine novel mutations in different pid genes were detected in our cohort of scid patients introduction/background: rasgrp is a guanine-nucleotide exchange factor which phosphorylates ras-gdp to the activated form ras-gtp in response to t-cell receptor stimulation, resulting in ras activation. mutations in the gene coding for rasgrp have been recently described in four patients with profound t-cell deficiency, resulting in recurrent bacterial and viral infections, autoimmunity and malignancy. here we describe a two-year-old male presenting with recurrent sino-pulmonary infections, found to have two variants in rasgrp , one not previously described. objectives . to describe a case of combined immunodeficiency with two pathogenic compound heterozygous rasgrp mutations. . to compare the clinical phenotype of rasgrp deficiency of our patient with that of previously described cases. . to argue for early hematopoietic stem cell transplantation in view of the increased susceptibility to epstein barr virus (ebv) induced lymphoma in patients with rasgrp deficiency. methods: a two-year-old male was referred to seattle children's immunology clinic for recurrent otitis media and two episodes of pneumonia. the diagnosis of combined immunodeficiency was considered based on a profound t-cell deficiency during immune evaluation and he was started on azithromycin and tmp/smx prophylaxis. at age ½ years he was hospitalized with a bladder outlet obstruction and found to have two abdominal masses. biopsies were obtained and he was diagnosed with an ebv driven b cell lymphoproliferative disorder. in addition, his csf and bone marrow were considered positive based on pcr and staining, respectively. treatment with cyclophosphamide, prednisone, rituximab, and intrathecal methotrexate was initiated. due to poor csf ebv clearance, intrathecal therapy was escalated to rituximab. results: initial laboratory evaluation showed elevated igg ( mg/dl), normal number of cd b-lymphocytes, and adequate response to tetanus, prevnar- and varicella vaccine. b-cell phenotyping showed elevated immature/transitional b-cells. a profound t-cell defect was identified with cd t-cell lymphopenia ( /mm ), elevated cd t-cells ( /mm ), inverted cd /cd ratio ( . ) and absent proliferation in response to mitogens (pha, anti-cd ) and antigen (tetanus). forty-three percent of peripheral blood t-cells were / positive. t-cell phenotyping revealed decreased cd and cd naïve t-cells with elevated proportion of cd + t-effector memory t-cells. cervical lymph node and retroperitoneal mass biopsies showed atypical lymphoproliferation without malignant transformation. exome sequencing revealed two variants in rasgrp . the first variant (c. + g>a) is located at a splice site predicting an unstable transcript targeted for degradation. the second variant (c. c>t) is a novel mutation resulting in a stop codon. conclusions: recurrent sino-pulmonary infections are often a presentation of antibody deficiency. in this case, further investigation showed a profound t-cell defect, resembling that reported in patients with homozygous nonsense mutations in the catalytic domain of rasgrp and patients with homozygous insertion mutations leading to a premature stop codon at the bzip domain. our patient had biallelic mutations in rasgrp downstream of the catalytic domain, both leading to unstable transcripts. he developed an ebv induced atypical lymphoproliferative disorder, a complication reported in one rasgrp deficient patient whose disease progressed to b cell lymphoma and unsuccessful hsct. another patient developed ebv induced lymphoproliferative disorder after hsct for ebv-positive hodgkin lymphoma. two additional patients presented with recurrent infections, developed b-cell lymphoma and one was successfully transplanted. we are preparing the patient for hsct after chemotherapy for his lymphoproliferative disease to correct the underlying immune defect given that these patients are at high risk of developing lymphoma following ebv infection. introduction/background: immunodeficiency-centromeric instabilityfacial anomaly is a group of rare genetic disorders typically involving agammaglobulinemia. type four is caused by variants in the hells gene. the five patients previously reported with icf have fit the phenotype of agammaglobulinemia. here we report a patient with novel phenotype including neutropenia and neuroblastoma. objectives: describe a unique presentation of immunodeficiencycentromeric-instability-facial anomaly syndrome to further expand our understanding of this disease. methods: retrospective chart review results: six-month old male was transferred to our tertiary care facility for ongoing chronic respiratory infection, chronic diarrhea, and failure to thrive. his past medical history was significant for -week prematurity due to rupture of membranes requiring a two month nicu stay for bronchopulmonary disease. upon discharge, he was bottle feeding and on room air. he had recurrent congestion for three months with two courses of antibiotics and one and a half weeks of diarrhea leading up to admission for difficulty breathing. he was found to have multiple infections including rhinovirus and parainfluenza virus on nasal wash, pjp pneumonia, norovirus, and pseudomonal cellulitis of his nose causing significant destruction. although previous laboratory studies revealed a normal absolute neutrophil count (anc), his anc quickly dropped to cells/ul. his igg, iga, and igm were undetectable. while his total b cell count ( cells/ul) was normal, he lacked any switched memory b cells. he had near normal total t cell count (cd cells/ul) and cd count ( cells/ul) and a markedly decreased cd count ( cells/ul) and poor proliferative response to low concentrations of phytohaemagglutinin and pokeweed mitogen. a . cm x . cm x . cm paraspinal mass was found on chest ct, which was subsequently characterized as mibg-avid with a curie score neuroblastoma. metastatic evaluation including bone marrow aspirate and biopsy was negative for malignancy. however, marked granulocytic hypoplasia and maturation arrest were present suggesting severe congenital neutropenia or, less likely, immune-mediated. whole exome sequencing detected homozygous variant of unknown significance in the hells gene (p.m t). he was treated with intravenous immunoglobulin and g-csf with clinical and laboratory improvement. his neuroblastoma was initially observed, then subsequently removed due to a > % increase in size. pathology confirmed mycn non-amplified favorable histology. he remains in remission months after resection. he is currently awaiting bone marrow transplant for his immunodeficiency. conclusions: the significance of this case report is the novel presentation of icf . neutropenia and malignancies have been reported in immunodeficiency-centromeric-instability-facial anomaly syndrome (icf ) but not icf . this case report thus expands upon the clinical picture of icf patients to include neutropenia and malignancies, and further describes the immunodeficiency. associate professor, university of south florida -johns hopkins all childrens hospital introduction/background: identification of newborns with severe combined immunodeficiency using state wide newborn screening (nbs) began in florida in . abnormal results require extensive confirmatory diagnostic testing and prophylactic antimicrobial medications are needed to effectively evaluate and treat the infant. several barriers have been identified within government-sponsored health insurance programs that impede delivery of these evaluations and medications, often resulting in delays and/or inpatient hospitalization in order to provide timely and appropriate care. objectives: determine the cost differential between the initial evaluation and treatment of a scid patient detected by newborn screening in the inpatient versus outpatient setting. methods: the cost utilization of inpatient versus outpatient management of newly identified scid patients from nbs were analysed to include the cost of confirmatory testing and initiation of prophylaxis within the inpatient versus outpatient setting. laboratory tests included assessment of t cell immunity with quantitative and functional assessment, immunoglobulin measurement, genetic testing for scid variants, evaluation for maternal engraftment, and hla typing. medications included ig supplementation, pentamidine, fluconazole, and acyclovir. we compared the actual cost of inpatient stay and inpatient evaluation versus the approximate cost that would have been accrued if the patient were not admitted to the hospital. results: from - , infants with government-sponsored health insurance had abnormal nbs and were confirmed to have scid after evaluation at our institution. all infants were admitted into the hospital for initial evaluation and initiation of appropriate medications for an average of days. total average cost of medication administration for days was $ , , total cost of laboratory testing was $ , , and average inpatient stay averaged $ , per patient. conversely, the cost that would have been accrued in the outpatient setting for medication would have been $ , for days. laboratory testing costs would be no different as an outpatient. in total, the cost for inpatient evaluation was $ , versus $ , as an outpatient. conclusions: standard laboratory assessments and medications are necessary for infants identified with scid by population based nbs. despite government sponsoring of the florida nbs program, unnecessary barriers exist by government sponsored insurers that lead to a delay appropriate care. inpatient admission alleviates these barriers, but significantly increases cost. we advocate that standard ambulatory scid outpatient evaluation and initial treatment be authorized in children identified with scid through nbs without delay. patients with cd g mutations reveal a role for human cd g in treg diversity and suppressive function. introduction/background: integrity of the tcr/cd complex is crucial for positive and negative selection of t cells in the thymus, and for effector and regulatory functions of peripheral t lymphocytes. genetic defects that reduce, but do not abrogate tcr signaling, are associated with a variable degree of immune deficiency and immune dysregulation. in particular, while cd d, cd e, and cd z gene defects in humans present mainly with severe immune deficiency, cd g mutations lead to milder phenotypes, mainly characterized by autoimmunity. however, the role of cd , encoded by cd g, in establishing and maintaining immune tolerance has not been elucidated. objectives: we aimed to investigate abnormalities of treg cell repertoire and function in patients with genetic defects in cd g with evidence of clinical autoimmunity. methods: high throughput sequencing (hts) was used to study composition and diversity of the t cell receptor (trb) repertoire in treg, conventional cd + (tconv), and cd + cells from patients with cd g mutations and in healthy controls. treg function was assessed by studying their ability to suppress proliferation of tconv cells. results: treg cells of patients with cd g defects had reduced diversity, increased clonality, and reduced suppressive function. the trb repertoire of tconv cells from patients with cd g deficiency was enriched for hydrophobic amino acids at position and of the cdr , a biomarker of self-reactivity. overlap between treg and tconv cell repertoires was observed in cd g mutated patients. conclusions: the treg and tconv cell repertoire of patients with cd g mutations is characterized by a molecular signature that may contribute to the increased rate of autoimmunity associated with this condition. introduction/background: a common concern with b cell-depleting therapies is their potential effect on humoral immunity. although there have been reports of prolonged hypogammaglobulinemia in adult patients receiving rituximab, little is know about this phenomenon in children. objectives: we sought to assess humoral immunity in children receiving rituximab and determine risk factors leading to low immunoglobulin levels and infections. methods: we conducted a retrospective study on all pediatric patients ( years) who received rituximab for the first time between january to december in a single tertiary pediatric hospital. charts were reviewed and data was collected prior to rituximab treatment and at , , and > months after treatment. patients who received rituximab after hematopoietic cell transplantation (hct) or for a malignancy and those with an underlying primary immune deficiency (pid) at the time of treatment were excluded. results: in total, patients received rituximab during the study period. of those, were excluded (hct: n= , lymphoma: n= , pid: n= ). sixtyeight patients were eligible. indications for rituximab treatment were renal disease (n= ), neurologic disease (n= ), hematologic disease (n= ), rheumatologic disease (n= ), ebv control (n= ), other (n= ). one patient who died from autoimmune encephalitis days after rituximab was excluded from the follow-up study. at any time after rituximab treatment, low igg was present in / ( . %), low iga in / ( . %), and low igm in / ( . %) of patients. over a year after their last rituximab dose, / ( . %) of patients still had low b cell counts for age, and / ( %) had low memory b cell counts (cd + among cd + cells: mean value = . % +/- . % sd). hospitalisation for infection was required in / ( . %) patients in the year following rituximab treatment, which was associated with having either low igg ( . % vs . %, p= . ) or low iga ( . % vs . %, p= . ), but not with low igm levels ( . % vs . %, p= . ). also, receiving a treatment with more than one rituximab cycle was a risk factor for low igg ( . % vs . %, p= . ). conclusions: hypogammaglobulinemia following rituximab treatment was frequent, and the presence of low igg and iga were associated with a higher risk of serious infection in this context. introduction/background: subcutaneous immunoglobulin (scig) replacement therapy for patients with primary immunodeficiency (pid) is usually administered once a week. however, a variety of dosing regimens can be used to provide flexibility for patients. objectives: we used pharmacokinetic (pk) analysis to evaluate the pk characteristics of weekly and biweekly (once every weeks) scig administration in patients with pid. methods: this pk substudy was part of a prospective, open-label, phase study (nct ) in patients with pid treated with igpro (hizentra®, csl behring, bern, switzerland). a noncompartmental analysis of serum igg concentrations was used to calculate pk parameters and compare pk outcomes on weekly and biweekly dosing. results: of the patients included in the pk substudy, provided samples for both weekly and biweekly regimens. the dose-adjusted area under the concentration-time curve was comparable for both treatment regimens: . and . (h*g/l)/mg for the weekly and biweekly regimens, respectively. the igg clearance was also similar, being . for the weekly and . ml/h for the biweekly regimen. median peak igg concentrations occurred later with the biweekly regimen ( . days) compared to . days for the weekly regimen. igg trough levels were close for both treatment regimens, with arithmetic means slightly lower for biweekly than for weekly regimens, at . vs. . g/l respectively. the minimum igg concentrations within a dosing interval were also comparable, with arithmetic means of . and . g/l for the weekly and biweekly treatment regimens, respectively. conclusions: biweekly and weekly hizentra® administration at the same total monthly igg doses resulted in similar igg exposures. pharmacokinetics, efficacy, tolerability and safety of a new subcutaneous human immunoglobulin . % in primary immune deficiency introduction/background: patients with primary immune deficiencies (pid) require life-long replacement therapy with immunoglobulins (ig) to prevent severe infections and irreversible complications. in addition to safety and efficacy, tolerability and convenience of administration of ig products are essential factors in patient acceptance. a new . % ig preparation (octapharma, lachen) was developed for subcutaneous administration (scig) derived from the established manufacturing process of octapharmas intravenous ig (ivig) brand octagam®. objectives: primary outcome was to assess efficacy of a new . % subcutaneous human immunoglobulin preparation in preventing serious bacterial infections. secondary endpoints included evaluating tolerability and safety, determining the pk profile, the number and rate of other infections and changes in quality of life measurements. methods: a prospective, open-label, single-arm phase study involving patients was conducted at centers in north america and europe. pid patients who were stable on ivig treatment for at least months and with igg trough levels . g/l underwent a -week wash-in/wash-out period consisting of weekly scig doses . times the previous ivig dose (based on published conversion rates for marketed scig products), followed by a -week efficacy period ( scig infusions in total). of patients enrolled had complete pharmacokinetic assessments at different time points: before the switch from ivig to scig (pkiv), after the wash-in/wash-out phase (pksc ) and at week of the efficacy period (pksc ). results: patients (age: - years; mean age . years; . % female) receiving a total of , scig infusions ( . g/kg/week in young children ( years and < years of age) and . g/kg/week in adults; average overall: . g/kg) were included in the full analysis sets. no serious bacterial infections were recorded. among the other infections observed during the efficacy period only one infection was graded as severe (bronchiolitis due to rsv virus), which led to hospitalization ( days). all other infections were mild ( . %) or moderate ( . %) in intensity. infection rate per person-year was . . of the reported adverse events, only were assessed as being related to the study drug; all of these events were non-serious. five non-study drug related serious adverse events were reported in patients ( . %). serum igg trough levels were nearly constant during the study with a minimum trough level of . g/l and mean trough plasma concentrations of . ± . g/l and . ± . g/l for pksc and pksc . median igg trough levels after scig treatment were . to . g/l higher compared to ivig treatment prior to enrollment. a dosing conversion factor (dcf) of . was determined by auc (area under the curve) measurements, allowing dose adjustment to achieve bioequivalence between ivig and scig dosing. improved quality of life measurements, utilizing sf- v , were observed in both physical and mental health parameters when compared from the first to last scig infusion. conclusions: this study demonstrated that the new subcutaneous human normal immunoglobulin . % is well tolerated, safe and effective in patients with pid. introduction/background: atopic dermatitis (ad) is a chronic, relapsing, inflammatory skin disorder with associated pruritus that affects percent of children in the united states. severe atopic dermatitis refractory to conventional therapy can be concerning for an underlying immunodeficiency, especially in infants. increased incidence of hypogammaglobulinemia has been associated severe ad. a handful of cases describe a correlation with transient hypogammaglobulinemia of infancy (thi), but a thorough immunological evaluation is often missing to further understand this relationship between ad and characterization of thi. objectives: define various phenotypes of thi with their clinical presentation and laboratory findings. compare thi vs. thi associated with severe a.d. methods: a case series of six patients was conducted at a single academic center from / / to / / for patients with severe atopic dermatitis with low igg levels. all available immunological laboratory data were retrospectively collected during this time period. descriptive statistical analysis was utilized for data comparison. results: of the six patients, four had no infectious history. of the remaining two, one had recurrent skin abscesses associated with his poorly controlled atopic dermatitis requiring oral antibiotics and one patient had two episodes of staphylococcus aureus superinfection of the eczema. at the time of presentation, the mean age was months with mean igg of mg/dl and mean ige of , ku/l. iga and igm were within normal age cut offs. all patients had normal protein and polysaccharide specific antibody titers after completion of vaccination series. mean cd + count was , /ul with normal cd +, cd +, cd + and cd + cell counts. three patients were tested for lymphocyte mitogen proliferation and complement function which were normal. two patients, who were tested, had normal phagocyte work up. mean age of igg improvement to igg> mg/dl, was months. patients had total protein and albumin levels with mean . ku/l and . ku/l, respectively which eventually normalized. four patients improved with skin care and dietary modification to hypoallergenic formula. one patient improved with extensively hydrolyzed formula and three patients improved with amino acid formula. one patient improved with aggressive skin care. one patient, who was noncompliant with dietary recommendations and aggressive skin care, did not improve. conclusions: one prospective study described an increased incidence of hypogammaglobulinemia in patients with atopic dermatitis compared to controls regardless of the severity. no further prospective studies have characterized hypogammaglobulinemia in this population. according to the primary immunodeficiency practice parameters, children with thi often present with frequent viral and bacterial respiratory illnesses, low igg levels and normal vaccine responses. in one study, the period of hypogammaglobulinemia spontaneously corrects to normal by mean age of months with all patients reaching normal levels by months. these patients may have low igm or iga and decreased t or b cells, which eventually normalize. management is often with antibiotic prophylaxis and if refractory to prophylaxis or unable to tolerate, igg administration (igrt) based on severity of symptoms is recommended. we describe a less severe variant of thi associated with severe atopic dermatitis and characterized by very transiently decreased igg levels with earlier resolution than typical thi, normal igm and iga levels, normal specific antibody levels and normal t, b and nk cells. these patients typically do not manifest recurrent viral or bacterial respiratory illnesses. they also do not require antibiotic prophylaxis or igrt. severe atopic dermatitis maybe a positive prognostic indicator for patients with thi and is associated with an earlier self-resolution of hypogammaglobulinemia, lack of typical infections, normal iga, igm and normal t and b cell numbers. a possible mechanism for thi associated with severe ad may be transdermal loss of protein which was supported with mildly low total protein and albumin levels rather than immaturity of the immune system in patients with true thi. introduction/background: in recent years it was found that heterozygous mutation in pik cd gene produces an autosomal dominant primary immunodeficiency characterized by onset of recurrent sinopulmonary and other infections in early childhood with defects in both b-and t-cell populations and a special susceptibility to uncontrolled viral infections. many patients develop chronic lymphoproliferation and there is also an increased susceptibility to b-cell lymphomas. here we present a patient assumed as a common variable immunodeficiency with heterozygous mutation in pik cd gene. objectives: to describe a case of a patient assumed as a common variable immunodeficiency with heterozygous mutation in pik cd gene. results: year old woman with personal history of severe and recurrent upper and lower respiratory infections, chronic pulmonary disease with bilateral bronchiectasis, chronic diarrhea without diagnosis, mild osteopenia, focal lesion in right hepatic lobe, atopic dermatitis and anemia. she was following up in other center and in she was diagnosis with common variable immunodeficiency (cvid) and started treatment with intravenous immunoglobulin (ivig), but she referred low adherence to it. she did not referred history of lymphoproliferation nor significant viral infections. she has a daughter with spherocytosis who required esplenectomy and also had bronchiectasis and cvid, she deceased at years old because pulmonary infection. other daughter and sons referred healthy. in our first immunologic studies we found severe hypogammaglobulinemia (igg mg%, no dosable iga and igm) with absent of b cells in peripheral blood. we started with high doses of ivig ( mg/k/month) and antibiotic prophilaxis with improvement of the functional respiratory test and without new infections. we are planning colonoscopy to study her chronic diarrhea. thinking that her clinical picture could be other than cvid we order a genetic study. a nextera exome capture and next generation sequence with illumina hiseq was made and an heterozygous mutation in pik cd gene (chr : . . , p.pro ala) was found. family and functional studies are still pending. conclusions: due that clinical presentations of primary immunodeficiencies are becoming more complex, its diagnosis is a challenge for immunologist now a days. studies with next generation sequence is a very useful tool in indefinite cases, especially when more than one member in the family are involved. chief, laboratory of clinical immunology and microbiology, national institute of allergy and infectious diseases, national institutes of health introduction/background: heterozygous gain-of-function mutations in pik cd as well as heterozygous pik r mutations that affect interaction of p with p lead to constitutive hyperactivation of the pi k pathway and cause activated pi k delta syndrome type or type (apds , apds ), respectively. we describe a female with apds with short stature, diffuse lymphadenopathy, recurrent upper respiratory tract infections, elevated igm, persistent ebvand cmv viremia and disseminated toxoplasmosis who gave birth to a genetically affected daughter with severe congenital toxoplasmosis. objectives: to characterize the molecular and cellular defects underlying severe toxoplasmosis in this family methods: investigation of the molecular basis of the disease was performed through whole exome sequencing, and results were validated by sanger sequencing. functionality of the pi k pathway was assessed by analyzing akt and s phosphorylation in freshly isolate b cells with and without stimulation with anti-igm. an excisional lymph node biopsy from the affected mother was stained with anti-pd antibody to detect t follicular helper cells, and with igm and igg specific antibodies to analyze the proportion of isotype-specific b and plasma cells results: whole exome sequencing of maternal dna with targeted analysis of pid genes identified a heterozygous mutation at an essential donor splice site of pik r (nm_ . :c. + g> a). sanger sequencing confirmed the presence of this mutation in both the mother and her child. functional studies on b cells freshly isolated from both patients confirmed an increase in baseline akt and s phosphorylation, suggesting constitutive activation of the pi k-mtor signaling pathway. a lymph node biopsied from the mother contained numerous pd- + tfh cells and igm+ plasma cells conclusions: toxoplasma gondii is an obligate intracellular parasite that is usually only symptomatic in immunocompromised hosts. severe toxoplasmosis has been reported in the following primary immunodeficiencies: cd ligand deficiency, tap deficiency, cvid, nfkb deficiency, and immunodeficiency due to anti-ifn-autoantibodies. importantly, toxoplasma infection was previously reported in a month-old infant with apds , and ocular involvement has been described in a -year-old patient with apds . this, however, is the first report of systemic and severe congenital toxoplamosis in a mother and child with apds. to evade innate host defenses, t. gondii induces the activation of the pi k/akt signaling pathway, reducing intracellular reactive oxygen species and creating an intracellular environment that is hospitable to parasite survival and proliferation. therefore, we postulate that the pik r mutation may lead to a hospitable cellular environment for toxoplasmosis replication. this work was partially supported by the division of intramural research, niaid, nih (protocol # -i- ). additional authors of this work are: ottavia delmonte and kerry dobbs, from the laboratory of clinical immunology and microbiology, niaid, nih. introduction/background: aplaid (autoinflammation and plc-gamma- -associated antibody deficiency and immune dysregulation) is a term that was proposed for the newly discovered autoinflammatory condition resulting from a pathogenic missense variant, ser tyr, in the cterminal sh (csh ) domain of plc-gamma- in order to distinguish it from plaid, a distinct clinical entity that results from intragenic deletions of portions of the csh domain of the same protein. plc-gamma- is a phosphodiesterase that is predominantly expressed in hematopoietic cell lines and acts on pip to produce ip and dag in the pkc and ras/raf/ erk pathways. the only formally published case of aplaid described a father-daughter pair with an autoinflammatory clinical syndrome affecting the skin, mucosa, eyes, pulmonary and gastrointestinal systems. objectives: to describe the aplaid phenotype resulting from a novel genetic variant of the phosphodiesterase plc-gamma- in two unrelated families methods: clinical review and case presentation results: we report a -year-old female with a long-standing history of recurrent pneumonia, cellulitis and cystitis with obstructive lung disease characterized as bronchiolitis and dynamic airway collapse, with negative alpha- -antitrypsin testing. she had a history of childhood onset granuloma annulare and pressure-induced urticaria, as well as episcleritis. immune testing revealed low igm ( mg/dl), elevated baff levels and a low percentage of cd + memory b-cells, prompting sequencing of plcg , which identified a c. t>a, p.met lys variant in the calcium binding c domain. her -month-old daughter, who has a history of bullous skin lesions, failure to thrive, febrile episodes and recurrent respiratory infections was likewise found to have this plcg variant. like her mother, the daughter also has low igm and elevated baff levels. similar symptoms of recurrent sinopulmonary infections and hypogammaglobulinemia, have been described in an unrelated family that shares this same plcg variant in a doctoral thesis by rozmus. this work also describes functional analysis, in which this particular variant of plc-gamma- was demonstrated to have dysregulated plcgamma- activity leading to aberrant intracellular calcium signaling and increased apoptosis of immature b-cell subsets. conclusions: we describe our experience in evaluation and treatment of this family with a previously undiagnosed disorder. together, these new cases add to the expanding body of knowledge regarding plc-gamma- and its importance for the development of autoinflammatory and primary immunodeficiency conditions. ( ) submission id# present a case.non-celiac gluten sensitivity is an emerging entity with symptoms similar to celiac disease, but without positivity in specific diagnostic tests. it is considered more common than celiac disease patients with sigad have a greater risk of concomitant autoimmune disorders than health individuals. sigad was previously to be associated with celiac disease, but not usually in non -celiac gluten sensitive. objectives: describir a case ataxia non celiac gluten sensitivity, methods: clinic case description. results: he patient has negative serology test for gluten, but clinically respond as celiac disease. conclusions: all patient has negative serology test for gluten, but clinically respond as celiac disease, could has non-celiac disease, this case described here ,it suggest than this entity should to thought before than the celiac disease, since not-celiac disease is it more common. associate professor, federal university of rio de janeiro introduction/background: primary cutaneous actinomycosis is a rare condition caused by gram-positive filamentous bacteria and generally occur after traumatic inoculation. objectives: to report an unusual etiology of skin lesions in a patient under anti-tnf-alpha therapy. methods: we report the case of a -years-old female receiving conventional doses of adalimumab for ankylosing spondylitis who presented, two weeks before referral for dermatological assessment, with an erythematous nodule with purulent discharge on right pretibial region and pruritic erythematous plaques with scaling and peripheral pustules in the trunk and nose tip. results: a fungal etiology was suspected and scraping specimens from several cutaneous lesions were submitted to direct microscopic examination and culture, which were negative for fungi. cutaneous biopsy of the pretibial lesion was sent for histopathology and microbial cultures. adalimumab was interrupted and empirical therapy with oral terbinafine ( mg/day) was started with close clinical follow-up. a folliculitis reaction pattern without granulomas was observed during histopathological examination and gram-positive cocci were isolated from biopsy sample and further identified as saccharopolyspora sp. by molecular typing. sulfamethoxazole+trimethoprim was added and discontinued after one week due to a cutaneous rash. terbinafine ( mg/day, p.o) was used for months with complete clearing of all skin lesions and very good tolerability. spondyloarthritis signs and symptoms were unremarkable and no anti-inflammatory or immunomodulating treatment was necessary. conclusions: increased risk of skin actinomycotic infections in patients under anti-tnf therapy is not consistently reported in the literature. nevertheless, they should be included as a differential diagnosis of atypical skin lesions in individuals under anti-tnf therapy. where we aim to determine the prevalence, incidence, characteristics, treatment and outcomes of primary immunodeficiency disease (pid) patients in qatar. pids are rare heterogeneous disorders of the immune system that result in an increased susceptibility to infection, immune dysregulation and occasionally, to cancer. objectives: determine the range of pids with important epidemiological data in qatar after analyzing the database and creating a registry. methods: this is a retrospective study of pid patients followed at hamad medical corporation from to using medical records. all patients who were diagnosed with a pid irrespective of age were included. patients were classified according to the international union of immunological societies expert committee on pid. the data is captured under sectionsa) patient demographics including age, gender, ethnicity b) clinical presentation, c) immunodeficiency profile including age at diagnosis, type of immunodeficiency, family history d) treatment modality and e) lab/genetic data. results: we registered patients ( females and males) over a span of four years. mean age at onset, diagnosis and diagnostic delay were . , . and years respectively. majority of the patients were arabs % followed by people from the asian subcontinent %. antibody deficiency was seen in %, immune dysregulation %, well defined immunodeficiency (at, hige, digeorge,wiskott aldrich) % and t/b cell cid %. rare diagnoses (ipex, msmd) were recorded whereas no cases of toll like receptor and complement deficiency were seen. consanguinity rate was (n = , % = ) and first degree cousin marriage (n = , % = ). family history was positive in % (n= ) of the patients. maximum diagnostic delay was seen in scid ( % > months) and agammaglobulinemia ( % > months).during the patients life, infection was the most common presenting complaint ( %) followed by sinopulmonary disease ( %) and gi-tract manifestations ( %). the most common infections were pneumonia ( %), otitis media and conjunctivitis ( % each) followed by failure to thrive ( %) and sepsis ( %).microbial isolates particularly seen as causative agents of infections were p.aeruginosa and salmonella ( %) each, mrsa and e.coli ( %) each. a genetic defect was confirmed in % of ataxic telangiectasia and % of scid patients. active infections were treated and prophylactic antibiotics were prescribed in cases ( %). prophylactic antibiotics were prescribed to % of patients with immune dysregulation and to % of well-defined syndromes. out of patients who had hsct, % had successful transplants. ivig was given to % of the total pid patients with humoral immunodeficiency patients receiving the most ( %). one patient had gene therapy and two required interferon gamma treatment for msmd. mortality rate at st year of life was % whereas total mortality rate was %, excluding cases that passed away before pid was diagnosed. conclusions: the estimated prevalence of pid in qatar is found to be per . over the years, physicians have become increasingly aware of pid and survival rate has improved. initiation of newborn screening for scid and agammaglobulinemia will lead to earlier diagnosis and initiation of therapy with better outcomes. introduction/background: agammaglobulinemia is typically associated with a near absence of b cells secondary to a developmental block in bone marrow, and, usually but not always, manifests in early life. most of such patients are males with x-linked agammaglobulinemia (btk deficiency). females with agammaglobulinemia, of either autosomal recessive or dominant inheritance, are rare. objectives: we present and discuss the differential diagnosis for the conflicting clinical and immunological phenotypes of two adult females who present with infections, cytopenias, and agammaglobulinemia with low to normal b cell count. methods: retrospective chart review of clinical and laboratory data results case : a -year-old female who, at age years, had evans syndrome (autoimmune thrombocytopenia and hemolytic anemia) that resolved after steroid and high-dose gammaglobulin treatment. at age years, immunologic workups revealed a complete absence of serum immunoglobulins (igg, iga, igm) and low b cells ( %) (normal range - ). however, three years prior, patient had detectable igm ( mg/ dl). b cell subset analysis showed an expansion of cd hi lo b cells ( %, normal . - . %), with a marked decrease in switched memory b cells ( . %, normal - %). additional immunophenotyping revealed a reduced frequency of naïve cd + ( . %, normal > %), cd +( %, normal > %) t cells, and normal lymphocyte proliferative responses to mitogens and anti-cd , anti-cd /anti-cd , and anti-cd /il- . case : a -year-old female with recurrent upper respiratory tract infection, undetectable igg, iga, igm and ige, and no response to vaccinations (tetanus and pneumococcal). thrombocytopenia ( x count/microliter) was noted once during her thirdtrimester pregnancy without evidence of pre-eclampsia. postpartum cd + t cell count was low ( cell/microliter, normal - cell//microliter). immunophenotyping revealed normal b cell count with reduced frequency of naïve cd + ( . %, normal > %) and cd + ( %, normal > %) t cells. conclusions: we report two adult females who present in early adulthood with recurrent infections and cytopenias. both had agammaglobulinemia and decreased naïve t cells suggestive of late-onset combined immunodeficiency (locid). the presence of peripheral b cells makes autosomal recessive defects in b cell receptor signaling (lamda , iga, igb, igm, blnk) less likely. differential diagnosis of locid with cytopenias includes ctla- haploinsufficiency, gain-of-function pik cd mutations, ikaros defects and autosomal recessive rag deficiency. both patients remain at risk for developing autoimmune complications. a molecular diagnosis will facilitate targeted therapy for the underlying defect. professor, director of the laboratory of childhood immunology, ku leuven introduction/background: complement factor properdin (cfp) is a soluble glycoprotein which has a unique known role as a positive regulator of the alternative complement pathway by binding and stabilizing the inherently labile c /c convertase enzymes. mutations in the cfp gene lead to aberrant protein expression or to expression of a dysfunctional protein which results in high susceptibility to pyogenic infections especially neisseria meningitidis. properdin-deficient individuals are at greater risk of fulminant meningococcal disease, with mortality rates as high as %. objectives: case report: a year old belgium boy from nonconsanguineous parents presented with achronic purulent cough and recurrent infections of upper and lower airways. he suffered from a severe pneumonia at the age of . ct thorax showed bronchiectasis of the right middle lobe and left lower lobe. in addition, he had recurrent acute otitis media since the age of year old. immunological work-up showed an absent ap activity, with normal ch , c and c . results: genetic and functional analysis: sanger sequencing of cfp gene identified a hemizygous c. t>g, p.y g (cadd score . , msc_cadd . ) mutation in the patient and his mother. properdin elisa (hycult biotech) showed absent and % of the normal healthy control value of serum properdin concentrations in patient and the healthy mother, respectively. conclusions: conclusion: we diagnosed properdin deficiency in a y old boy presenting recurrent lower and upper respiratory tract infections. ap testing, added to ch , should therefore be part of initial workup for patients with recurrent severe respiratory tract infections. indeed early diagnosis allows for appropriate prolonged antibiotic prophylaxis and immunization to reduce the risk of fatal meningococcal disease, to reduce or prevent organ damage and to allow for genetic counselling in the family. - , - , - and - ) and child depression inventory (cdi) were implemented to both children and parents in addition to sociodemographic data form; beck depression inventory (bdi), beck anxiety inventory and zarit caregiver burden scale were implemented only to parents. results: the depression inventory parent and child form values of the patient group with primary immunodeficiency were significantly higher than the control group (p= , and p= , ). according to beck depression and anxiety inventories, it was seen that the depression and anxiety inventory scores of the parents of the patient group were higher than the control group (p= , and p= , respectively). it was determined that the cdi parent form scores of the patients with hospitalization history were statistically higher than the patients without hospitalization history ( , ± , and , ± , ; p= , ). bdi scores were significantly higher in the group which received ivig (p= , ). while the quality of life of the patients compared to their parents was perceived as worse than the healthy children in all dimensions (p= , p< , and p< , ), the quality of life of the children was worse only in psychosocial and total quality of life fields (p< , p= , and p= , ). it was seen that quality of life of children physical health scores of only the patients with hospitalization history were statistically significantly lower ( , ± , and , ± , ; p= , ) . while no statistically significant difference was found in terms of quality of life of the child scores between the groups which received and did not receive ivig replacement treatment, psychosocial and total qualify of life scores of parents were statistically significantly lower in the group which received ivig replacement treatment (p= , p= , ). zarit care giver burden scale scores were similar in patient and control groups. although both groups were on the limits of mild to moderate caregiving burden, it was seen that the scores of the group which received ivig were significantly higher than the group which did not receive ivig (p= , ). conclusions: as a conclusion, we think that it will be appropriate to inform and monitor the entire family in relation to psychosocial difficulties and care giving burden they may experience in time as well as the medical aspects of the disease in order to develop a holistic approach to children with primary immunodeficiency. introduction/background: chronic lung disease is the most common complications of cvid, affecting - % of patients. it includes bronchiectasis affecting % of patients and granulomatous lymphocytic interstitial lung disease (glild) in to %. both are associated with an increased morbidity and mortality. pulmonary functional studies and ct scans have been proposed as screening procedures for lung involvement in cvid. the definitive diagnosis of glild is established histologically, but it is too invasive in patients with radiological abnormalities and no symptoms. objectives: we aim to describe the pulmonary complications of our cohort of patients with cvid comparing them with subjects affected with other types of hypogammaglobulinemia. methods: we reviewed all clinical records of the patients with a diagnosis of hypogammaglobulinemia of any cause until december . we looked at all pulmonary function tests (pft), -minute walk tests and ct scans performed. we classified patients according to the ct scan pulmonary disease pattern. we compared the demographic data and pulmonary characteristics of each group and intended to describe similarities and differences between them. results: we collected patients and ct scans from patients. patients were grouped for further analysis as follows: no ct performed: ; normal ct: ; bronchiectasis: ; glild: and bronchiectasis + glild: . eight patients ( %) had no ct including six with cvid, one with x linked lymphoprolipherative disease (xlp) and one had a syndromic deletion in chromosome . the median age of symptom onset was . yo, with a median delay to diagnosis of year. nine subjects ( %) had ct scans with no chronic disease, six had cvid, rituximab induced hypogammaglobulinemia (rih) and mgus with hypogammaglobulinemia (mguswh). the median age of symptom onset was years old with a median delay to diagnosis of years. nine patients ( %) presented persistent bronchiectasis, with cvid, with an igg deficiency (sigg d) and with xlp. median age at symptoms onset was yo and median delay to diagnosis years. seven patients ( %) had glild; all afected with cvid. median symptoms onset was years old, and median delay to diagnosis was years. one patient ( %) was included in the bronchiectasis and glild group. she was referred with a diagnosis of rih, in the context of a pulmonary malt lymphoma. however, her ct did not show a typical ct lung lymphoma pattern, lymphocyte monoclonality was never shown and she had a reduced pre-treatment low gamma globulin concentration and a history of respiratory infections since adolescence, we believed her diagnosis is cvid. pft with an obstructive pattern identified patients with bronchiectasis (p< . ) and patients with glild had a significantly lower basal and post minute walk o saturation (p< . , n= ) conclusions: using a systematic approach, we identified that roughly % of patients with hypogammaglobulinemia have chronic pulmonary ct abnormalities. half of them had bronchiectasis, that were associated to hypogammaglobulinemia of any cause, a longer disease course and a reduction in fvc and fev with a shorter distance reached in the minute walk test. the other half had glild, spirometries in this group were useless, but o saturation was significantly lower, basal and after the minute walk test. we found a high frequency of pulmonary disease in our cohort and a disease progression study is now in place. hies) is a primary immunodeficiency characterized by eczema, sinopulmonary infections, and musculoskeletal and vascular abnormalities. care of patients with this disease is largely supportive with the use of prophylactic antibiotics and topical eczema therapies. the use of replacement immunoglobulin is increasing. hematopoietic stem cell transplant (hsct) is being considered more frequently, but many questions remain regarding which patients should undergo transplant. as pulmonary complications are a leading cause of morbidity and mortality in this disease, and potentially improved with replacement immunoglobulin and hsct, we sought to examine more closely the patients with more frequent pulmonary hospitalizations and structural lung disease. objectives: to determine the rates of pulmonary complications in our large cohort of ad-hies patients, and examine the relationship between immunologic markers and pulmonary disease. methods: we retrospectively reviewed the records of ad-hies patients seen more than one time at nih between and . there were pediatric patients under the age of years. we reviewed the number of and cause for hospitalizations, and reviewed radiology reports for structural lung disease including bronchiectasis and pneumatoceles. we correlated these findings with specific antibody responses and lymphocyte phenotyping, such as the number of memory b lymphocytes. results: the patients range in age from years to years, with a median age of years. patients had chest cts, with percent having bronchiectasis, and percent having cavitary lesions. percent of patients had both. these abnormalities were more prevalent in patients with low memory b cells. percent of patients receive replacement immunoglobulin. in patients not receiving replacement ig, percent had appropriate response to pneumococcal vaccine. during this time period, there were hospitalizations, the majority of which were associated with pulmonary infections. the rate of overall hospitalization was higher in the group with low memory b cells (p= . ), but there was no difference associated with age. conclusions: immunologic abnormalities may assist in determining the long-term prognosis of patients with ad-hies, and can be considered in management plans such as the use of immune globulin and consideration for hsct. introduction/background: purine nucleoside phosphorylase (pnp) deficiency is an autosomal recessive disorder affecting the purine salvage pathway causing a (severe) combined immunodeficiency disorder, autoimmunity, and neurological symptoms. patients typically present in the first few years of life with frequent infections and a failure to thrive. decreased plasma levels of uric acid are suggestive, while neutropenia is a rare complication. prognosis is generally poor with few patients making it to adulthood without treatment. treatment options are limited to general supportive care and hematopoietic cell transplantation. methods: we present the cases of three patients, one sister and two brothers, from a consanguineous french canadian family. they presented in early adulthood with nearly identical histories of repeated pneumonias and chronic rhinosinusitis. one patient had necessitated filgrastim to treat a parainfectious neutropenia. they did not report any neurological abnormalities or symptoms of autoimmunity. results: a primary immune deficiency was suspected. initial evaluation showed normal immunoglobulin levels, a lack of response to vaccination, positive ebv ebna iggs, and auto-antibodies. the patients were severely lymphopenic with reduced numbers of t, b and nk cells ( l: , cd + %, cd + %, cd + %, cd + %, cd -cd + %). in particular, they had a severe depletion of naive t-helper recent thymic emigrant cells (cd +cd ra+cd + %). additional studies revealed increased urinary inosine, guanosine, deoxyinosine, and deoxyguanosine, severely reduced erythrocyte pnp activity ( % residual activity), and a normal uric acid level. a homozygous, missense mutation, c. c>g (p.his asp) was found and described as pathogenic in silico. the diagnosis of pnp deficiency was made; tmp-smx prophylaxis and ivigs were started. conclusions: theses cases are atypical for a number of reasons. the first is the relatively benign immunological course, the lack of neurological symptoms, and the advanced age at the time of the diagnosis, possibly due to residual enzymatic activity. in addition, pnp deficiency has been classically described as causing a marked reduction of t cells with a relative sparing of b cells. however, b cell lymphopenia have been reported, particularly with latter presentations. while this specific mutation had been identified once previously in the literature, as a compound heterozygote, these are the first confirmed cases of homozygotes. the pathogenicity of the mutation was not only suggested in silico, it was confirmed biochemically. in addition, in vitro functional studies have demonstrated the importance of his for the binding of pnp to its substrate, particularly in the formation of the early transition state. indeed, pnp (p.his asp) has been shown to have a greatly reduced affinity for its substrates and catalytic activity. further study is needed to identify the optimal management for these patients. introduction/background: whole exome sequencing has become an integral part of diagnosis and treatment of rare immunodeficiency diseases. one of these diseases is tetratricopeptide repeat domain a(ttc a) mutations; a rare disorder associated with multiple intestinal atresias and severe combined immunodeficiency. histologic assessment of organ biopsies of patients with ttc a deficiency suggest it may play a role in multiple organs as it is expressed in the cytoplasma of intestinal, thymus and pancreatic cells. many patients with ttc a deficiency have moderate to severe combined immunodeficiency. nine patients described in the literature with ttc a mutations have been treated with hematopoietic stem cell transplant, mostly at a young age. objectives: this case report describes a rare presentation of an adolescent with ttc a deficiency, her clinical presentation, immune evaluation and treatment with eventual referral to bone marrow transplant at the age of . methods: retrospective chart review under irb approval was performed. whole exome sequencing performed at baylor texas childrens hospital and mutations were confirmed by sanger sequencing. extensive immune and nk cell phenotyping were performed by flow cytometry and nk cell function was tested using standard cr release cytotoxicity assays. results: a year old female was referred to immunology for severe refractory warts, history of severe diarrhea with epithelial dysplasia but no atresia, failure to thrive requiring parenteral nutrition until years old and multiple infections with staphylococcus aureus and candida. at years of age, she developed rapidly worsening pulmonary function that improved with monthly pulse methylprednisone. her immune phenotype demonstrated a combined immunodeficiency including severely low cd , cd , b and nk cells. she had no igd-cd + memory b cells and undetectable igg, iga and igm, isohemagglutinins and vaccine titers to diphtheria and tetanus. her mitogen proliferation response was low and she had abnormal tcr v-beta repertoire. she was referred to the texas childrens hospital center for human immunobiology for the nk cell evaluation and reasearch clinic (near) the patient was found to have impaired nk cell lytic function and terminal maturation. this was demonstrated by the decreased frequency of cells that matured to the cd dim subset and was accompanied by decreased expression of the lytic effector molecule perforin and the fc receptor cd . this phenotype was conserved when we isolated cd + hematopoietic precursors and performed nk cell differentiation in vitro. whole exome sequencing demonstrated a compound heterozygous mutation in the ttc a gene, including a c. + _ + aagt mutation, which has been previously described as a pathologic founder mutation in the french canadian population. the second mutation is a previously unreported c. g>a (p.e k), a variant of unknown significance. mutations were confirmed by sanger sequencing, and parents are carriers of the mutations. she was referred for hematopoietic stem cell transplantation with a / unrelated donor option. conclusions: ttc a deficiency is a genetic mutation associated with high morbidity and mortality leading to gut atresia and dysfunction in combination with severe immunodysfunction. we have described a unique case of ttc a deficiency with a novel mutation which is associated with intestinal epithelial dysplasia with no atresias and a late presentation and evolution of combined immune deficiency. nk cell assessments show significantly impaired terminal maturation suggesting a critical role for ttc a in human nk cell development. introduction/background: ras-associated autoimmune leukoproliferative disorder (rald) is a rare condition with significant overlap of clinical and laboratory findings with malignant disorders, notably juvenile myelomonocytic leukemia (jmml) and chronic myelomonocytic leukemia (cmml), but with much better prognosis without specific therapy. we report a case of rald in a month-old male infant originally suspected to have jmml. results a -month-old male infant, born to an unrelated ethiopian father and turkish mother, presented with splenomegaly and leukocytosis ( , /l), monocytosis ( , /l), and thrombocytopenia ( , /l); hemoglobin was gm/dl and fetal hemoglobin concentration . %. family history indicated early death of three paternal uncles, two at age - months and one at age years. jmml was suspected and bone m a r r o w a sp i r a t i o n w a s p e r fo r m e d ; h ow e v e r, r e s u l t s o f immunophenotypic testing with flow cytometry analysis and cytogenetic testing with fish did not support this diagnosis. patient had a normal b level. serum igg ( . mg/dl [nl . - . mg/dl]) and igm ( . mg/dl [nl . - . mg/dl] levels were elevated and iga was normal. lymphocyte subset analysis showed % cd [nl - %], % cd [nl - %], % cd [nl - %] positive cells with normal percentages of cd and cd /cd cells; there were no cd -/ cd -t cells detected. given the findings of leukocytosis, monocytosis, splenomegaly, hypergammaglobulinemia, and a dearth of cd -/cd -t cells, the patient appeared to meet criteria for rald. mutation testing with next generation sequencing showed an nras missense mutation [c. g>t; pgly cys] in % of t cells and % of myeloid cells. the presence of the nras mutation, in the absence of other typical jmml findings or rasopathy syndrome features, supported the diagnosis of rald. there was no therapeutic intervention and, at one year of age, the patient was reported to remain clinically well; he relocated to ethiopia with his parents. conclusions rald is a nonmalignant clinical syndrome originally classified as a subtype of autoimmune lymphoproliferative syndrome (alps) but subsequently distinguished to be a separate entity due to lack of double negative t-cells, a mutation in fas/fasl/caspase- , or consistently elevated serum b levels. though also present in % of jmml patients, a somatic mutation in ras signaling protein (kras or nras), which controls b-cell tolerance and production of autoantibodies, is present in all reported cases of rald. while all previously reported cases of rald had somatic kras or nras mutations, our patient's nras mutation was present in more than % of t cells and myeloid cells, suggesting a heterozygous germline mutation; this has not previously been reported in rald and needs to be further confirmed ( ) the primary objective of this study was to evaluate hct outcomes in patients with was who underwent hct since in north america. we hypothesized that survival after hct from alternative donors such as cord blood has improved compared to published results, but that overall survival would be superior in patients who undergoing hct at a young age. methods: patients were enrolled on pidtc protocol , a multicenter retrospective natural history study of patients treated for was in north america since . clinical features, disease status, hct type and post-hct outcomes were analyzed in patients who underwent hct at pidtc centers between - . descriptive statistics such as median and range for continuous variables and counts and percentages for categorical variables were used to summarize characteristics of the study population. in addition, kaplan-meier curves were used for estimating survival probabilities results: diagnosis of was was confirmed by an expert review panel for eligibility. mutation in the was gene was available for patients, including nonsense (n= , %), frameshift (n= , %), missense (n= , %), splicing (n= , %), gross deletion (n= , %), in-frame deletion (n= , %), pr complex (indel) (n= , %). donor types included matched sibling (n= , %), unrelated adult volunteer bone marrow or peripheral blood stem cells (n= , %), umbilical cord blood (n= , %) and other related donors ( each, phenotypically matched related, haploidentical) . median age at time of hct was . months (range, . . months). the vast majority of patients received busulfan containing conditioning regimens ( %), with some receiving other myeloablative ( %) or reduced intensity regimens ( %). with a median follow-up of . years, overall survival was excellent with -year and -year survival probabilities of % ( % ci, - %) and % ( % ci, - %), respectively. survival was similar at year for recipients of hct from matched sibling ( %, % ci, - %), matched unrelated donor ( %, % ci, - %) or umbilical cord blood ( %, % ci, - % see figure) . importantly we confirmed that survival at year was better in patients who were < years old (n= ) compared to those who were years old (n= ) at the time of hct ( % versus %, respectively p= . ). this difference persisted when only unrelated donor recipients were analyzed ( % vs %, p= . ). overall the percentage of patients in our study who underwent hct at a young age was high ( %) compared to the literature ( % in moratto et al, , blood) . the rate of second hct was only % (n= ). cumulative incidence of acute grade - at days, acute grade - at days and chronic graft-versushost disease at year were % ( % ci - %), % ( % ci - %) and % ( % ci - ), respectively. conclusions: outcome of hct for was since shows excellent overall survival for all donor types, including umbilical cord blood with very low rates of second hct. importantly, hct at a younger age (< years old) continued to be associated with superior survival supporting the provision of hct earlier in the course of the disease. further analysis of the complete cohort is planned to determine whether age at hct has decreased in the modern era compared to pre- and to analyze factors associated with platelet and immune reconstitution, donor chimerism and autoimmunity. introduction/background: comel-netherton syndrome is a rare disease hallmarked by congenital ichthyosis, atopy, trichorrhexis invaginata (bamboo hair) and, within the past years, has been defined as a primary immunodeficiency. specific, rare genetic polymorphisms (c. t>a, pl h) in the fc receptor iiia (fcgr a) on natural killer (nk) cells have been shown to decrease nk cell function. patients with these defects present with susceptibility to severe and recurrent viral infections, however, not all fcgr a mutations result in this clinical phenotype. here we report on a child with a mixed genotype, presenting with congenital ichthyosis, atopy and recurrent bacterial and viral infections. methods: immunophenotyping of lymphocyte subpopulations were evaluated by flow cytometry. genomic dna was sequenced using next generation sequencing. all detected variants were then sequenced using sanger sequencing. cd dual epitope assay and evaluation of nk cell maturation was also performed. intravenous immunoglobulin replacement therapy was initiated. results: our patient presented at months of age for evaluation of food allergy. she has congenital ichthyotic erythroderma with pruritic atopic dermatitis-like skin eruptions and a history of hypernatremic dehydration immediately following birth, thin and easily broken hair and a history of failure to gain weight with appropriate catch-up growth following formula fortification. she has had multiple episodes of acute otitis media and recurrent upper respiratory tract infections associated with wheezing. t-, b-and nk cell quantitation by flow cytometry was normal, including naïve and memory b cells. immunoglobulins and vaccine antibody levels to haemophilus influenza type b, tetanus and streptococcus pneumoniae were normal. lymphocyte proliferation to mitogens was normal. natural nk cell cytotoxicity was initially decreased, however, subsequent cytotoxicity testing performed at months of age was normal. whole exome sequencing revealed a homozygous mutation in spink (c. - a>g) and a homozygous missense mutation in the first immunoglobulin domain of fcgr a (c. t>a), both variants of unknown significance and under additional investigation. this homozygous mutation in spink was not detected in the patients healthy, unaffected sibling. conclusions: this is a case of an infant with a clinical phenotype consistent with comel-netherton syndrome, however genotyping has revealed homozygous mutations in spink and fcgr a, suggestive of a possible mixed genotype. a recent large study investigating the use of whole exome sequencing to identify variants implicated in primary immunodeficiency found that in % of families, more than gene contributed to the immunodeficiency phenotype. it should therefore be kept in mind that variability in an individuals clinical phenotype may be attributable to the presence of a mixed genotype. introduction/background: cartilage-hair hypoplasia (chh) is a rare autosomal recessive disease caused by mutations in the rmrp gene and can manifest with scid. allogeneic hct is a curative therapeutic option for scid associated with chh. bordon et al. reported an overall survival of % ( / patients) following hct with a predominantly myeloablative conditioning regimen with busulfan and cyclophosphamide. data on outcomes of hct using a ric regimen are limited. objectives: we herein report our experience with allogeneic ric hct for scid associated with chh. methods: we reviewed records of all patients who underwent allogeneic hct for scid associated with chh at our institution, with a ric regimen containing alemtuzumab, fludarabine and melphalan. results: five patients ( male, female) underwent allogeneic ric hct for chh at median age of months (range, months years). all patients had biallelic mutations in the rmrp gene, and met pidtc criteria for scid, prior to hct. two patients were diagnosed by newborn screening for scid. one patient received serotherapy only (rituximab mg/m daily x days, anti-thymocyte globulin . mg/kg daily x days) conditioning for initial hct but developed graft failure and subsequently received a ric regimen for second hct. all patients received a ric regimen consisting of alemtuzumab mg/kg over days(n= ) or mg/kg over days (n= ) and fludarabine mg/kg (weight < kg, n= ) or mg/m over days (n= ), and single dose of melphalan . mg/ kg(weight < kg, n= ) or mg/ (n= , dose reduced by % for preexisting sclerosing cholangitis with grade liver fibrosis). patients received matched (n= ) or - allele mismatched (n= ) bone marrow grafts and all but grafts were from unrelated donors. all patients received cyclosporine and steroids for gvh prophylaxis. all patients engrafted with full donor chimerism. three patients developed mixed chimerism, but continue to maintain donor t cell chimerism > %. two patients developed vod of the liver (one patient developed mild vod whereas the patient with pre-existing sclerosing cholangitis developed severe vod). one patient developed grade skin gvhd and one patient developed limited chronic skin gvhd. none of the patients developed liver or gi-gvhd. all patients remain alive at a median follow up of years (range months- years) with good t-cell and b-cell immune reconstitution. conclusions: our experience suggests that allogeneic ric hct with alemtuzumab, fludarabine and melphalan for scid associated with chh is curative, offers durable t-cell engraftment, low gvhd along with excellent survival and might be preferable over a myeloablative conditioning regimen, to further limit toxicity in young infants, especially in the era of newborn screening. refractory thrombocytopenia in a patient with wiskott-aldrich syndrome despite hematopoietic stem cell transplantation: eltrombopag as a therapeutic option. mauricio chaparro-alzogaray , marcela estupiñan-peñaloza , gisela barros-garcía , oscar correa-jimenez pediatric hematologist/oncologist, hsct unit, fundación homi hospital de la misericordia pediatric hematologist/oncologist, fundación homi hospital de la misericordia pediatrics resident, universidad nacional de colombia introduction/background: wiskott-aldrich syndrome (was) is an xlinked disorder characterized by: immunodeficiency, eczema and hemorrhage due to thrombocytopenia [ ] . hematopoietic stem cell transplantation is the treatment of choice, with an overall survival of approximately % regardless of the source of the stem cells [ ] . eltrombopag has been used as a pre-transplant stabilization therapy [ ] . in our knowledge, there are no reports of its use for the management of post-transplant autoimmune cytopenias in was. objectives: to present a clinical case in which the complexity of diagnosis and management of wiskott-aldrich syndrome is highlighted, as well as the usefulness of eltrombopag in post-transplant persistent thrombocytopenia. methods: clinical case presentation and review of literature. results: clinical case: -month-old infant with a history of thrombocytopenia identified at the third day of life (positive serology and viral load for cmv), bacteremia due to serratia marcescens at months, intermittent diarrhea from months, multiple platelet transfusions, ivig cycles, and ambulatory management with corticosteroids; who consulted the er for a -day of bloody diarrheic stools, generalized petechiae and fever. he was irritable with generalized petechiae in the lower extremities, diaper area dermatitis with signs of superinfection. admission cbc: wbc: /mm , neutrophils /mm , lymphocytes /mm , monocytes /mm , hb . g/dl, ht . % mcv . fl, mch . g/dl, mcmh . %, platelets /mm mpv fl. bone marrow aspiration was performed that ruled out proliferative syndrome. immunological profile with normal immunoglobulins and flow cytometry showed t lymphocytosis with cd / cd inversed ratio, direct coombs was positive. he progressed to refractory thrombocytopenia, with intracranial bleeding and transfusion platelet requirement every h. molecular diagnosis of wiskott-aldrich syndrome was made and it was decided to carry out an allogeneic transplant of unrelated umbilical cord. he showed adequate response, % chimerism; however, he persisted with important cytopenias, without response to management with ivig and corticosteroids, so that on day + he restarted eltrombopag that he received prior to transplantation. from day + he received rituximab for weeks. he continued with eltrombopag months post-transplant. currently without cytopenia and without complications, he completed the post-transplant year without additional complications. conclusions: was represents a great diagnostic challenge in pediatric clinical practice. despite the therapeutic option of transplantation of hematopoietic stem cells, patients may persist with different complications, within these; autoimmune cytopenias will require additional therapies. eltrombopag, in addition to being used as a pretransplant transient measure, is useful for the management of post-transplant persistent thrombocytopenia in these cases. references: . immunol allergy clin north am. ; ( ) methods: retrospective study of medical records in two centers of immunology results: in our center we have p with pid, with made retrospective study of medical records, up today we have registered p ( %). according to this record, these diseases are distributed in the following way: predominantly antibody disorders: p ( %), predominantly t cell deficiencies: p ( , %), phagocytic disorders: p ( . %), complement deficiencies: p( . ), other well pid: p( . ), autoimmune and immune dysregulation syndromes: p( . %), unclassified immunodeficien cies: p( . %). predominantly antibody deficiencies are the most common pid, which comprise more than half of our all p. these group is represented by: specific iga deficiency (sad): p, specific igg deficiency: p, transient hipogammaglobulinemia: p, common variable immunodeficiency (cvid): p, agammaglobulinemia linked x: p, agammaglobulinemia unknown causes: p, secondary hipogamma globulinemia: p, selective igm deficiency: p, subclasses deficiency: p, cd l deficiency: p, hyper igm unknown causes: p, other hyogammaglobulinemia: p. among them, selective iga is the most common pid.in our cohort of unclassified immunodeficiency, we have p with auto inflammatory syndrome, such as family mediterranean fever, hyper igd syndrome and candle like-syndrome and p with nk deficiency. in all our pid p, are under replacement gammaglobulin (gg)treatment, p use intravenous gg and p use subcutaneous gg conclusions: the lasid registry model represents a powerful tool to improve health policies, showing that are under diagnosed and should receive more attention. more data are needed to define the exact prevalence of pid to avoid underestimation of these diseases due to under reporting. as different reports in different countries, in our centers predominantly antibody deficiencies are the most prevalent. although the number of patient diagnosed with pid, is growing. many physicians still know little about these disorders. introduction/background: a number of anticonvulsant medications have been shown to cause hypogammaglobulinemia. lamotrigine is a phenyltriazine anticonvulsant medication that is approved to treat seizure disorders and bipolar disorder. objectives: we describe a patient who developed hypogammaglobulinemia secondary to lamotrigine use. methods: we performed a chart review and case-based literature review. results: a- -year old female presented to immunology clinic for evaluation of panhypogammglobulinemia. she was initially evaluated by general internal medicine for lightheadedness and fatigue and was found to have serum igg of mg/dl ( - mg/dl), igm mg/dl ( - mg/dl) and iga mg/dl ( - mg/dl). the patient reported a history of recurrent sinus infections occurring around twice per year. she reported resolution with oral antibiotics. she denied any history of pneumonia and was never hospitalized for treatment of infection. she did report increased number of recurrent upper respiratory infections; around - per year for the last several months. she denied any family history of primary immune deficiency. she was never prescribed corticosteroids or immuno-modulators. her past medical history was significant for bipolar disorder type for which she was prescribed lamotrigine mg oral twice daily seven months prior to her initial visit. the medication was prescribed prior to the onset of recurrent sinus infections. she had protective post-vaccination titers against tetanus, diphtheria, and acellular pertussis and non-protective pre-vaccination pneumococcal titers. post vaccination pneumococcal titers were not obtained due to the cost of the pneumonia vaccine. the patient was started on prophylactic azithromycin three times weekly. lamotrigine was discontinued and the patient switched to lurasidone due to concern for anticonvulsant-induced panhypogammaglobulinemia. her serum immunoglobulin levels increased after . months off of lamotrigine (igg mg/dl, igm mg/dl, iga mg/dl), and she is currently asymptomatic without further infections. conclusions: lamotrigine is likely responsible for the reversible hypogammaglobulinemia in this patient. serial immunoglobulin levels should be checked in all patients who experience recurrent sinopulmonary infections while on lamotrigine. in two separate reports, lamotrigine induced hypogammaglobulinemia began within months and months of starting therapy respectively. in another study, hypogammoglubinemia was reported in % of patients taking lamotrigine. further studies are needed to accurately describe onset and frequency of hypogammgeobulinemia in these patients. currently, it is unclear whether post vaccine titers are protective in patients with lamotrigine induced hypogammaglobulinemia. introduction/background: the association of vaccine strain rubella virus with cutaneous and sometimes visceral granulomatous disease has been reported previously in patients with various primary immunodeficiency disorders (pids). the majority ( / ) of these pid patients with rubella positive granulomas had dna repair disorders, namely ataxia telangiectasia (at) (n= ) or nijmegen breakage syndrome (nbs) (n= ) or rag (n= ) and rag (n= ) deficiency. objectives: to support this line of inquiry, we provide additional descriptive data on the previously reported nbs patients as well as additional previously unreported patients with rubella virus induced cutaneous granulomas and dna repair disorders as well as additional previously unreported pid patients with rubella virus induced cutaneous granulomas. methods: we provide in-depth descriptive data on the previously reported nbs patients as well as additional previously unreported patients with rubella virus induced cutaneous granulomas and dna repair disorders including at (n= ) and dna ligase deficiency (n= ). we also provide in-depth descriptive data on additional previously unreported pid patients with rubella virus induced cutaneous granulomas, including cartilage-hair hypoplasia (n= ), mhc class ii deficiency (n= ), whim syndrome (n= ), and coronin- a deficiency (n= ). results: the median age of the patients is . years (range - ). the majority are females ( %). cutaneous granulomas have been documented in all cases while visceral granulomas (spleen and liver) were observed in cases. t cell and b cell lymphopenia as well as hypogammaglobulinemia or impaired antibody formation were present in most patients. all patients had received rubella virus vaccine. the median age at presentation of cutaneous granulomas was months (range - ). the median duration of time elapsed from vaccination to the development of cutaneous granulomas was months (range - ). the diagnosis of rubella was made by pcr in % of patients and by immunohistochemistry in the remainder. one patient was confirmed to have vaccine strain rubella virus. hematopoietic cell transplantation was reported in three patients. rubella associated complications did not contribute to death among those patients who died ( %). conclusions: of the now cases, ( %) share the diagnosis of a dna repair disorder and confirm that chronic rubella virus infection is associated with cutaneous granuloma formation. analysis of patients with dna repair disorders and other pids with this complication will help clarify determinants of rubella pathogenesis, identify specific immune defects resulting in chronic infection and may lead to defect-specific therapies. introduction/background: introduction/background: hizentra® is a % liquid igg product approved for subcutaneous administration in adults and children greater than two years of age who have primary immunodeficiency disease (pidd). limited information on use of hizentra® is available for children who have received hematopoietic stem cell transplantation (hsct). objectives: objectives: the aims of this study are to determine the safety and efficacy of hizentra® in pediatric patients post hsct, and to characterize reasons for switch from intravenous igg (ivig) to subcutaneous (scig) delivery following hsct. methods: methods: a retrospective chart review involved pidd infants and children (mean age . [range . to . ] months) status post hsct who received hizentra®. ten patients received hizentra® by pump administration, and patients by manual push. results: results: hizentra® administered weekly to children included an average of infusions (range - ). the mean dose was mg/kg/ weeks. the mean igg level was mg/dl while on hizentra® in patients compared to a mean trough igg level of mg/dl in patients during immunoglobulin administration prior to hizentra® of most children. four patients naïve to igg therapy were started on hizentra®. average infusion time was . (range - ) minutes for manual entry and (range - ) minutes for pump entry, and the average number of infusion sites was . (range to ). local reactions were mild and observed in / ( . %) children. ten patients had no local reactions. no serious adverse events were reported. the rate of serious bacterial infections (sbi) was . per patient-year while receiving hizentra®, similar to reported efficacy studies. the reasons for switch from ivig to scig in patients (some patients had multiple reasons) were improved igg serum levels and physician desire for steady state serum igg levels (n= ), loss/lack of venous access (n= ), patient/caregiver preference (n= ), home site of care preference (n= ), and physician preference for scig (n= ). conclusions: conclusions: hizentra® is a safe and effective option in children who have received hsct. reasons for switch from ivig to scig included improved serum igg levels, desire for steady state serum igg levels, and patient/caregiver preference. consulting medical advisor, immune deficiency foundation introduction/background: it is currently unknown how many persons with primary immunodeficiency (pi) receive the seasonal influenza vaccination, and what proportion becomes ill. additionally, the effect of immunoglobulin (ig) replacement therapy on the frequency of influenza diagnosis and severity of symptoms is not known. objectives: the current study sought to measure the prevalence of seasonal influenza vaccination and diagnosis among persons with pi, specifically those with antibody deficiencies: x-linked agammaglobulinemia (xla), common variable immunodeficiency (cvid) and hypogammaglobulinemia. methods: , email invitations were delivered to members of the immune deficiency foundation database requesting participation in an online survey regarding their zoster, influenza and varicella vaccination experiences. data from persons with xla (total n= ; children age< ; n= ; adults age> , n= ; % male; % white non-hispanic), cvid (total n= ; children age< , n= ; adults age> , n= ; % female; % white non-hispanic), and hypogammaglobulinemia (total n= ; children age< , n= ; adults age> , n= ; % female; % white non-hispanic) were analyzed for the - influenza season. results: overall, % (n= ) of the sample received a seasonal influenza vaccination during the - influenza season. persons with xla were less likely to receive an influenza vaccination ( %, n= ), than persons with cvid ( %, n= ) and hypogammaglobulinemia ( %, n= ). a fifth of respondents ( % overall, % of children and % of adults) who attend school and/or work stayed home at some time to avoid seasonal influenza. though the majority ( %, n= ) were not diagnosed with influenza, when stratified by age, children were twice as likely to be diagnosed than adults. of the children sampled, % (n= ) were diagnosed with seasonal flu compared to % (n= ) of the adults that were sampled. the role of replacement ig therapy in protection against flu can best be examined in the patients with xla since these patients cannot make specific antibodies to vaccine and only % (n= ) received the vaccine. although % (n= ) of the individuals with xla were exposed to flu, only % (n= ) were diagnosed with influenza; two of whom were not receiving ig therapy at time of diagnosis. concerning influenza severity, individuals receiving ig therapy at the time of flu diagnosis tended to have modestly milder symptoms than those not receiving ig treatment (e.g., less likely to report sore throat ( % versus %, p<. )), but the sample size is too low to draw firm conclusions. conclusions: a high proportion of antibody deficient persons received a seasonal influenza vaccination for the - influenza season. in addition to vaccination, many individuals attempted to avoid influenza infection by remaining home from school and/or work. there was a suggestion that ig replacement therapy may partially protect xla patients from symptomatic flu, although the role of cell mediated immunity in protection against flu is not clear. a prospective study could determine if ig replacement therapy partially protects against clinical flu symptoms in xla and cvid patients using two groups those that receive the current flu vaccine vs. those that do not receive the flu vaccine. introduction/background: fanconi anemia (fa) is an autosomal recesive or x-linked genetic disorder, characterized by cytopenia or bone marrow failure, this will lead to a severe anemia, neutropenia and thrombocytopenia, requiring frequent interventions with diversified therapies, including hematopoietic stem cell transplant (hsct). a complete immune reconstitution is requiere for a success transplant. one of the main upcoming events asociated after the hsct, is the secundary immunodeficiency, which is asociated with a significant morbility and mortality in the patients. to rich a successful alogenenic-hsct, is impending a complete reconstitution of the t cells immunity, for this it is crucial the presence of factors like; thymic activity of the hsct recipient, biological features of the allograft (eg, degree of histocompatibility, number and type of infused donor t cells) and preparative regimens. objectives: describe the kinetics of the immune reconstitution in fa patients after alogenic-hsct, as well as the infections associated during this process and other comorbilities. methods: we decribed the lympocyte population (t, b and nk) in pb measured by flow cytometry in fa patients on days + , + , + , + , + and + post alogenic hsct from a match related donor. the conditioning was base on fludarabine mg/m , cyclophosphamide mg/kg and antithymocyte globulin rabbit mg/kg. infectious-disease survillance for virus was determined by the quantification by dna pcr-rt for cmv, ebv, adenovirus, as well as the presence of galactomannan and candida sp antibodies, or isolation of fungus or bacterial culturing in the cases of infectious disease of known or uknown aetiology. results: the lymphocyte population mesurement was performed in a total of five fa patients who undergo to an allogenic hsct, all of them received stem cells from a match related donor and the source was bone marrow. successful engraftment was observed in all patients, there were no deaths reported. after the hsct was performed, the kinetics of recovering for the distinct lymphocytes subsets was the the following: nk cells (cd +cd +) were the first to recover, followed by cd+ t cells, b lymphocyte and finally cd+ t cells (figure ) all of them rich normal values and remain stable. three out of five patients presented infectious disease: two of them were cmv positive, one patient has a concurrent detection of adenovirus, and in the other was detected aspergillus, in both of them it was presented at day + . the third one developed acute gvhd which progressed to a chronic gvhd, at day + was diagnosed on him listeria monocytogenes meningitis, at that momento he has just cd+ +cd and cd + reconstituded. there is no new infectous disease detected in any of the patients after they reconstituted cd + t cells. conclusions: complete immune reconstitution is the decisive for the presence of several morbilities and mortalities, mainly because of oportunistic infecctions and gvhd. we show that the kinetics of recovery of the different populations of lymphocytes follows those patterns also described for patients with other hematological malignancies: early recovery of nk cells, followed by effector cytotoxic t cells and b cells, and finally, cd + t-helper cells. the utility of post-transplant monitoring of pb-lymphocyte subsets for improved follow-up of patients undergoing bmt and prevent opportunistic infections. introduction/background: early detection of primary immunodeficiency diseases (pids) before serious infection is of utmost importance and a question of patient survival. one of the main organs involved in pids is skin with mucocutaneous manifestations being one of the common complaints in pids. the presenting skin symptoms may serve as an essential element in early diagnosis of pids. objectives: this study aims to determine characteristics, frequency, nature and incidence of skin manifestation in pid patients seen in qatar. methods: this retrospective study was conducted at hamad medical corporation the only tertiary hospital in qatar from january to july .the subjects included were pid patients < years old, who had dermatological complaints. information collected included dermatological diagnosis, gender, age at onset of signs and symptoms, age at definite diagnosis, family history of related disease, any lab results such as pathology or viral studies obtained from the clinical medical record. patients were diagnosed and classified according to clinical and laboratory criteria by the international union of immunological societies primary immunodeficiency committee. results: a total of patients were studied and skin/mucocutaneous manifestations were found in patients ( %) with male to female ratio of . : . age at onset of skin manifestations ranged from to years. skin manifestations were divided in two categories according to presentation before pid diagnosis (primary manifestations n= , %) and during the disease period (secondary manifestations n= , %). the type of pids predominantly having primary manifestation were scid (n= , %) cvid and cgd (n= , %, each), xla, at and griscelli syndrome (n= , %, each).various manifestations which were atypical in their presentation and persistently found in patients who did not respond to any effective treatment (n= ) led to the basis of clinical immunological study and diagnosis including rare diagnosis such as ipex. whereas secondary manifestations were primarily reported in scid (n= , %) cgd, at and digeorge syndrome (n= , %, each). the nature of skin manifestations varied in both groups, primary manifestations notably had % cutaneous infections comprising of thrush ( %) stomatitis ( %) viral rashes ( %) bacterial rashes ( %) impetigo ( %) and fungal rash ( %). eczema/atopic dermatitis were % and other minor miscellaneous skin alteration cases found were apthous mouth ulcer ( ) erythroderma ( ) gvhd like rash ( ) alopecia ( ) psoriasis ( ) and scleroderma ( ) . secondary manifestations were identified as % cutaneous infections % eczema and infantile seborrheic dermatitis ( ) pruritus ( ) gvhd and alopecia ( ) each. the causative microorganisms were confirmed in % of the cases via lab cultures whereas common infections such as chicken pox and herpes were validated through clinical symptoms. none of the cases were biopsied. overall, highest skin infection was seen in ataxia telangiectasia ( ). other pids with prominent cutaneous manifestations included cgd ( ), digeorge syndrome ( ), hyper ige syndrome ( ) and scid (rag or , cases). more than two types of skin manifestations were found in % patients over the due course of their illness. conclusions: an awareness of various cutaneous and skin disorders associated with pid which are persistent and unresponsive to treatments among dermatologist and family physicians is crucial to raise suspicion for early detection, timely management and prevention of complications. introduction/background: ataxic telangiectasia (at) is a rare neurodegenerative autosomal recessive disease associated with immunodeficiency, poor coordination and disability. ataxia telangiectasia has a diverse clinical heterogeneity which may often lead to an incorrect diagnosis or late detection of the disorder resulting in exposing the patient to unnecessary radiation from various sources. objectives: we present a female child from the asian subcontinent that was seen for the first time with ulcerative skin lesions, failure to thrive and marked lymphopenia. methods: a search of the pubmed database was carried out, using different combination of the terms "ataxia", "telangiectasia", typical , atypical and "presentation" results: four-year-old pakistani girl, product of first degree nuptial presented with generalized vesicular rashes mainly on abdomen and scalp which turned into ulcers with residual hypo pigmented lesions and diarrhea. she had short stature, failure to thrive (weight and height < th percentile) and no previous infection or family history of primary immunodeficiency diseases (pid). mild delay in milestones was observed especially in speech. immunizations were up-to-date including bcg. on examination she had mild ocular telangiectasia but no cutaneous involvement. investigations revealed lymphopenia ( . ) and eosinophilia. workup for diarrhea including duodenoscopy revealing subtotal villous atrophy however the biomarkers for celiac disease were negative. immune system workup showed high igg levels, normal iga, ige and igm level. igg subclasses: low igg , and , antibody titers to h.influenza and pneumococcus vaccine were relatively low. lymphocyte subsets showed low cd ,cd and high nk cells ( %), low naïve t cells ( %) and inverted cd /cd ratio.t cell function with post pha was . % but had normal response to cd . alfa feto protein was requested due to continued low cd count and the level was found to be high: . iu/ml. next-generation sequencing (ngs) showed homozygous mutation for trp fs in chromosome of atm gene confirmed by whole exome sequencing. immediate treatment with ivig was started leading to mark improvement of skin lesions, diarrhea and weight gain. conclusions: the index of suspicion of at should be highlighted when deciphering lymphocyte subset. currently there is no neonatal screening for pid diseases and next generation sequences in qatar. once implemented it may prove beneficial in discovering cases from early infancy and reaching upon a definite diagnosis. introduction/background: chronic granulomatous disease (cgd) is a genetic disorder of the nadph oxidase complex in phagocytes which results in impaired production of microbicidal reactive oxygen species that can lead to recurrent life-threatening bacterial and fungal infections. the majority of affected patients in the united states have a x-linked defect in the cybb that encodes gp phox protein followed by an autosomal recessive defect in the ncf gene that encodes p phox . x-linked female carriers show populations of neutrophils and following lyonization, and severe skewing of x-chromosome inactivation can get cgd type infections. a recent study has shown that the lower dihydrorhodamine (dhr) percent in female carriers predicts a higher infection risk but carrier state on its own predicts autoimmunity results: a -year-old female presented for an evaluation of recurrent fevers. she had a history of several lobar pneumonias in childhood. at age , she developed erythematous bumps on her abdomen that were initially non-bothersome but later became painful. she then developed recurrent fevers, chills, and rapid weight gain. biopsy of the skin lesions showed subcutaneous panniculitis-like t cell lymphoma. she was treated with chemotherapy and found to be in complete remission. she had no previous family history of recurrent infections. at the completion of her chemotherapy, her -year-old son became septic in the hospital and was then diagnosed with cgd from mutated cybb gene. post chemotherapy, the patient had a prolonged course with an atypical lung infection that required bronchoscopy and video thorascopic surgery, however no bacteria was ever identified. she did improve after a long course of ivantifungals. evaluation for immunodeficiency was done after she had a recurrence of low grade fevers, cough, and fatigue. dhr flow cytometry with phorbol myristate acetate (pma) was done which showed . % neutrophil oxidative burst activity after stimulation, consistent with a highly skewed lyonization pattern in x-linked cgd. conclusions: this case demonstrates an interesting lesson in a woman with recurrent infections since childhood whose clinical picture was confounded by her history of malignancy. the abnormal lung infections prior to malignancy were alarming and gave cause to do additional immunology testing. however, it begs the questions, if she did not have a son, would she have been diagnosed with severe x-lined carrier disease. introduction/background: measurement of the specific antibody response following vaccine challenge provides clinicians with a better understanding of the adaptive immune response in individuals undergoing immunological evaluation. objectives: we hypothesised that after classification of primary immunodeficiency (pid) patients based on vaccine response (vr), further division using igg subclass (iggsc) - measurements may identify additional patients with abnormal b cell function and patients with different frequencies of infection at presentation. methods: vrs and serum iggsc concentrations were quantified using the vacczyme anti-pneumococcal polysaccharide (pcp) igg elisa and human iggsc liquid reagent kits (the binding site group limited, uk) in pid patients ( : . m:f, median age . years, range - ). all patients were immunised with pneumovax® (sanofi pasteur msd). the lower limits of published normal iggsc ranges were used as cut-off values (igg . g/l, igg . g/l and igg . g/l). pcp concentrations equal to or less than mg/l post-vaccination was considered an abnormal response. results: agreement between vr and iggsc measurements was % (p= . ). the frequency of respiratory tract infections at presentation among pid patients with a normal pcp igg vr was % and % among patients with an abnormal vr. subsequently, four separate groups could be identified by including iggsc measurements. the frequencies of infections at presentation were for the vr+/iggsc+ group (n= ) % vs. % for the vr+/iggsc-group (n= ); and % for the vr-/iggsc+ group (n= ) vs. % for vr-/iggsc-individuals (n= ). conclusions: these results confirm that vr and iggsc measurements are independent serum biomarkers of humoral immunity. taken together the vr and iggsc results provide more detailed information about the immune status that may influence diagnosis, treatment and monitoring decisions. introduction/background: hizentra® is a % liquid igg licensed for subcutaneous administration in adults and children greater than two years of age who have primary immunodeficiency disease (pidd). subcutaneous immunoglobulin (scig) use in autoimmune conditions is reported. stiff person syndrome (sps), a rare neurologic disorder characterized by fluctuating muscle spasms and rigidity, is mediated by autoantibodies to glutamic acid decarboxylase (gad). symptoms of sps have been shown to improve after administration of intravenous immunoglobulin (ivig) however, there is a paucity of information regarding use of scig in sps. objectives: the aim of this study is to describe the use of hizentra® in patients with sps, including indications for scig, clinical characteristics of patients, and clinical and laboratory response to scig. methods: a multicenter retrospective chart review examined patients with stiff person syndrome treated with hizentra®. results: sps was diagnosed in patients at and years. both patients were started on ivig, steroids, and rituximab prior to initiation of weekly hizentra® (ages and years). the average dose of hizentra was mg/ kg weekly. the average igg level while receiving ivig was , . mg/dl, similar to the average igg level while on hizentra was , mg/dl. the number of administration sites ranged from - , and duration of infusions ranged from - minutes. serum anti-gad antibody levels prior to hizentra® were u/ml and u/ml respectively. anti-gad antibody level during treatment with hizentra (available for one patient) was > , u/ml, and , u/ml following discontinuation of hizentra®. one of the reasons for switching to hizentra® was lack of response while on ivig. the average number of hizentra infusions were . patients reported improvement in spasticity related to sps while on hizentra®, and one patient had improvement in seizures. one patient discontinued hizentra® in favor of intravenous immunoglobulin (ivig) due to physician preference. the most common side effects were local reactions including pain, pruritus, and redness. no serious adverse events were reported. conclusions: hizentra® was associated with improved symptoms in sps in both patients including decreased spasticity, and improved seizure frequency in one patient. serum anti-gad levels did not decrease following administration of hizentra. hizentra® was well tolerated in patients with sps, with most side effects reported as mild. hizentra® may be considered as an alternative to ivig treatment in patients with sps. introduction/background: combined immunodeficiency (cid) has been associated with a spectrum of secondary gastrointestinal manifestations, including infectious and non-infectious causes. abatacept, a soluble ctla -igg fusion protein targeting t cell activation, has demonstrated benefit in the treatment of autoinflammatory manifestations in patients with cid due to underlying heterozygous germline mutations in ctla and lrba. objectives: herein, we report two patients with cid characterized by low immunoglobulin levels, low class-switched memory b cell counts, and low peripheral naïve cd + t cell counts. both patients suffered from severe and persistent diarrhea complicated by protein-wasting and malnutrition, ultimately diagnosed as biopsy-consistent autoimmune enteropathy. the clinical history of patient was also notable for granulomatous lung disease and autoimmune cytopenias. methods: a t-regulatory cell disorder was considered in both cases, however, ctla and lrba were found to be unaffected by whole exome sequencing (patient ) and/or flow cytometry (patient ). due to progressive worsening of the autoimmune enteropathy despite management with chronic steroids (both patients), combination rituximab/mycophenolate mofetil (patient ), and total parenteral nutrition (tpn) complicated by recurrent infections (patient ), abatacept was trialed at mg sc weekly. results: in both patients, the start of abatacept therapy produced a dramatic improvement as measured by decreased stool frequency, improved weight gain, and decreased protein-wasting. patient has been maintained on this monotherapy for over one year, with improvement in the gastrointestinal as well as pulmonary autoinflammatory complications. the only documented adverse event has been hepatitis b reactivation, managed with tenofovir and abatacept continuation. patient has required azathioprine/abatacept combination therapy for clinical stabilization and is without a significant adverse event to date. conclusions: we conclude that abatacept may be a relatively safe and effective therapeutic in the management of severe autoimmune enteropathy in the background of cid, even when used outside of the classical clinical context of ctla or lrba haploinsufficiency. objectives: as increasing the outbreak of allergic diseases, study for treatments comes to the force. in this research, we aimed to assess the effects of sg-sp , a derivative of gallic acid, on mast cell-mediated allergic inflammation using various animal and in vitro models. methods: ovalbumin-induced systemic anaphylaxis and immunoglobulin e (ige)-induced passive cutaneous anaphylaxis are standard animal models for immediate-type hypersensitivity. oral administration of sg-sp hindered the allergic symptoms in both animal models. these inhibitions were deeply related to the reductions of histamine and interleukin- . results: sg-sp reduced degranulation of mast cells and expression of inflammatory cytokines in a dose dependent manner. sg-sp showed better anti-allergic effects compared to gallic acid and dexamethasone. down regulations of intracellular calcium level and nuclear factor-b activation by sg-sp were causative of the reduction of allergic mediators. to anticipate the exact target of sg-sp , phosphorylation of proteins involved in mast cell signalling was assessed. sg-sp suppressed the activations from lyn and was aggregated with high affinity ige receptor (fcri). conclusions: from these results, we assured that sg-sp directly interact with fcri. all together, we propose that sg-sp might be a therapeutic candidate for allergic disorders. ( ) submission id# systematic assessment of pain in patients with primary immune deficiency using validated pain questionnaires: a prospective study. introduction/background: the number of primary immunodeficiency (pid) patients is rising dramatically because of good medical care as well as increased awareness. around in live births are affected. more than disorders have been discovered so far and this number is expected to rise in the coming years. as with any other chronic illness, pid patients are also prone to acute and chronic pains. chronic pain is a big challenge and lack of understanding of the etiology and underlying mechanisms limit our ability to diagnose or treat it effectively. objectives: to systematically assess chronic pain in patients with pid using validated questionnaires and to try to understand the underlying mechanisms of neuropathic versus non neuropathic pain. methods: short-form mcgill pain questionnaire (sf-mpq) is a recognized way to ascertain different pain characteristics as well as severity. a validated arabic version of the sf-mpq was used to prospectively assess chronic pain in patients with pid. furthermore, a validated arabic version of the neuropathic pain questionnaire-short form (npq-sf) was also used to assess neuropathic pain and to differentiate it from nonneuropathic pain. a total of patients with pid were included. results: males: females were . %: . % respectively. mean age was . years. commonest diagnosis was combined immune deficiency in % of the patients followed by common variable immune deficiency which was . %. chronic pain was found in % of the patients that participated in the study. % of the patients who complained of pain found it to be tiring and exhausting. % each had aching or heavy or sharp pain. % had cramping pain, % had tender pain and % characterized their pain as throbbing or burning. % felt shooting pain, % had splitting pain and % had gnawing pain. % found it to be frightening and % described it as being sickening in itself. the commonest pain complaint was abdominal pain in % of the patients followed by headache % and chest pain %. pain attributed to neuropathy was present in about . % of the study population. most patients described that they had been experiencing pain for at least - years. conclusions: this is the first international study to understand the prevalence, duration and severity of chronic pain among pid patients. a significant number of patients reported ongoing pain. this is the first time any kind of pain has been studied systematically in the patients with primary immune deficiency. treating pain should have a major impact on improving the patients quality of life. introduction/background: primary immunodeficiency (pidd) patients, particularly those with severe t cell defects, are at increased risk of infections. bone marrow transplant also contributes to significant t cell lymphocytopenia, which can be associated with similar risks. several prophylactic measures, which vary per institution, are placed on these patients in order to prevent infections. we report on a likely outbreak of rapid growing nontuberculous mycobacteria (ntm) at our institution, with a suspected association to tap water objectives: to recognize nontuberculous mycobacteria as a threat to patients with immunodeficiency disorders methods: case series of consecutive patients admitted to one institution with similar infections results: in a period of months, / to / , patients admitted to our institution were found to have rapid growing ntm species on central line culture or bronchoalveolar fluid. these cultures were obtained due to fever and symptoms of systemic infection. per institutional practices no peripheral blood samples were obtained at the time of initial culture. all had significant t cell dysfunction: wiskott-aldrich syndrome ( ), an undefined combined immunodeficiency ( ) , and three patients post bone marrow transplantation (bmt), one months ( days) post an unrelated cord blood transplant for farber syndrome, one days out of a matched related transplant for epstein barr virus (ebv)-associated lymphoproliferative disorder, and one patient with high risk neuroblastoma days out of his second tandem autologous transplant. both allogeneic transplant recipients had received serotherapy. all species were rapid growing, identified as mycobacterium mucogenicum (n= ), immunogenum (n= ), or abscessus-chelonae complex (n= ). the patients had not shared rooms, caregivers, invasive procedures, or medications from the same batch. three ( %) cases met cdc criteria for hospital acquired infections (hai). all patients were in general ward rooms, and were on standard precautions given diagnosis had not been established (n= ) or they were considered outside the typical window of strict bmt precautions (n= ). ntm species were subsequently isolated from hospital tap water. this has resulted in a significant increase in infection precautions, including the use of sterile water only for cares, as well as bottled water for drinking (with no use of ice machines), for all patients being evaluated for pidd or with significant lymphocytopenia. conclusions: ntm are a threat to patients with pidd. tap water is a potential source of mycobacterial infections in pidd patients. minimizing exposure risk to water sources containing ntm is very important in this population. patients with concern for pidd or significant t cell lymphocytopenia should take steps to avoid ntm exposures, including the use of sterile water for cares, and bottled water for drinking. introduction/background: purine nucleoside phosphorylase (pnp) deficiency is a rare autosomal recessive condition leading to severe combined immunodeficiency and neurologic impairment, typically presenting in early childhood. the condition is progressive and typically fatal in the first or second decade of life without hematopoietic stem cell transplantation (hsct). objectives: to illustrate the clinical and laboratory presentation of pnp deficiency with a novel mutation in the pnp gene via a case study. results: case: a four year old female of hispanic descent, with a past medical history of spastic diplegia, initially presented with chronic nasal congestion, recurrent sinusitis, and cough. laboratory studies were significant for an alc cells/mcl ( - cells/mcl). she was lost to follow-up for two years, before returning to medical attention for pneumonia. evaluation of lymphocyte subsets at age years revealed cd + cells/mcl ( - cells/mcl), cd + cells/mcl ( - cells/ mcl), cd + cells/mcl ( - cells/mcl), and cd + cells/mcl ( - cells/mcl). t cell mitogen stimulation to pha measured by flow cytometry was % of control. pnp activity was nearly absent and urine guanosine and inosine were significantly elevated. gene sequencing revealed a homozygous c. a>g mutation in the pnp gene. prophylactic antimicrobials were started. despite strong recommendation for hsct, the patient was again lost to follow up until age years, at which time she was found to have progressive lymphopenia, bronchiectasis, and developmental delay. t cell mitogen stimulation to pha measured by thymidine uptake assay was % of control and pnp functional activity was undetectable. the patient underwent a / matched unrelated hsct six years after her initial presentation. one year post-transplant, the patient continues on immunoglobulin replacement therapy. her course was complicated by cutaneous gvhd, treated successfully with topical corticosteroids. conclusions: this case study illustrates the progressive nature of pnp deficiency in the first decade of life. our patient is notable in that she survived without significant medical intervention to the age of years. her presentation at age years was not unlike those previously reported in the literature, with muscle spasticity, ataxia, and recurring bacterial infections. to the authors knowledge, this case reports a novel mutation in the pnp gene. introduction/background: severe congenital neutropenia (scn) is a primary immunodeficiency disease characterized by early onset recurrent infections, persistent severe neutropenia and congenital genetic defect. severe idiopathic neutropenia (sin) is a rare disease defined by persistent severe neutropenia, in the absence of an identifiable etiology. objectives: here, we aim to find out clinical, laboratory, genetic characteristic and remission status in children with scn and sin in chinese population. methods: in this study, we enrolled chinese children who experienced severe neutropenia longer than months without any virus infection or auto-immune antibodies from june to july in hospitals affiliated to shanghai jiao tong university school of medicine. their clinical, laboratory and molecular characteristics were analyzed and the patients were followed up to observe their remission status. targeted gene capture combined with next-generation sequencing technology was used to find out related gene mutation. results: patients in this study had a mean age of . ± . months. molecular analysis revealed that patients had associated mutations of scn, including elane and g pc . among patients with continuous follow-up, one died for unknown reason. ten patients have recovered from sin (r-sin) with mean neutropenia duration of . ± . months. scn patients had more frequent infection ( . ± . times per year) than sin ( . ± . times per year, p= . ) and r-sin patients (p= . , . ± . times per year). scn patients had significantly higher count of anc and monocytes than sin (p= . ) and r-sin patients (p= . ). however, there was no difference in anc and monocytes counts between sin and r-sin patients. bone marrow examinations demonstrated a myeloid maturation arrest at the myelocyte-metamyelocyte stage in scn patients, while most of sin and r-sin patients were normal. conclusions: our study indicated that, patients with mild infection, lower anc, monocytes count and normal bone marrow are likely to be sin. whereas others with relatively more severe infection, higher anc, monocytes count and maturation arrest in bone marrow are inclined to be scn. introduction/background: patients with qualitative and/or quantitative defects of humoral immunity often require immunoglobulin (igg) replacement therapy (igrt). usual starting doses range between - mg/kg and the dose is adjusted as needed depending on the patient. there is a paucity of information about whether and how extreme bmi (obese or underweight) and route of administration may affect a patients rate of infections. objectives: the objective of the study is to determine whether rate of infection is associated with bmi, dose or route of administration in patients receiving igrt. methods: this is a retrospective chart review from december -october . we included patients between the age of month and years old who were evaluated initially and had at least one follow up evaluation. data reviewed included the route of administration, dose, infection history, igg serum levels, height and weight to calculate bmi, gender, age and diagnosis requiring immunoglobulin replacement therapy. participants were excluded if the type of immunodeficiency is unknown or if the participant had incomplete data for the requested data fields. the number of infections between visits was modeled using poisson regression as a function of dose, route of administration and the bmi with the log of the follow up interval as an exposure offset. results: eighty-five patients were eligible, and preliminary results for patients are presented here. the mean infection rate per weeks of follow up was . in obese patients, . in overweight patients and . in underweight/normal weight patients adjusted for route of administration; however these rates do not differ significantly from one another. the mean infection rate per weeks of follow up differed by administration route; . infections for ivig versus . infections for scig when adjusting for bmi (p< . ). the mean infection rate per weeks of follow up was not associated with dosage. conclusions: overweight patients may experience more infections than obese or underweight patients, regardless of administration route. ivig patients may have a lower rate of infections compared to scig patients regardless of bmi. work is ongoing to complete analyses for the remainder of the eligible patient population. ( ) submission id# introduction/background: primary immunodeficiencies (pids) are a rare group of genetic disorders associated with a tendency to infectious diseases and an increased incidence of cancer. there is no data regarding the prevalence of malignancies in patients with pid in turkey. objectives: in this study, we aimed to evaluate patients who diagnosed as pids and developed malignancies, and calculate the estimated frequency of malignancies associated with pids in turkey. methods: forty five patients who were diagnosed with malignancy in the follow-up period of pid at the four tertiary immunology clinics between and years were included in the study. the data were obtained retrospectively from the hospital records and the database of esid online patient registry. results: the prevalence of malignancies in patients with pid was found as . % ( / ). the male to female ratio of the patients was / , the median age was . years (minimum: . , maximum: ) and the median age at which patients get diagnosed with malignancy was years (minimum: . , maximum: ) . there was no cancer history in their family members. the most common type of pid which associated with malignancy was ataxia telangiectasia (n= , . %). non-hodgkin lymphoma was the most common malignancy (n = , . %) in our group (table ) . ebv quantitative pcr was positive in lymphoma cases ( . %). the median number of x-rays and ct scans in patients with at and bloom syndrome before malignancy developed were (minimum: , maximum: ) and (minimum: , maximum: ), respectively. two cases had dual malignancies (papillary thyroid cancer and anal adenocancer). twenty cases were treated with chemotherapy, cases with hematopoietic stem cell transplantation, cases with radiotherapy, and cases with surgical treatment, treatment information of patients was not reached. remission was detected in cases, while resistance to therapy in cases and recurrences in two patients were observed. four patients are still on chemotherapy. twenty cases died. conclusions: the tendency of malignancy in patients with pids is due to the deficiency in the immune response that lead to failed surveillance against oncogenic viruses, premalignant/malignant cells, or both. lymphoid malignancies are the most common malignancies associated with pids. pids-associated malignancy incidence has increased in recent years because of that improved survival of the patients. this study is the largest cohort investigating the association of malignancy in patients with pid in turkey. additionally, we first reported tendency to malignancy in a patient with znf deficiency. introduction/background: next-generation sequencing (ngs) has become an integral tool in the evaluation of primary immunodeficiency disorders (pid). we describe a patient with a previously described pathogenic foxp variant who met clinical and laboratory criteria for cvid. objectives: describe a patient with a previously described pathogenic foxp variant who met clinical and laboratory criteria for cvid. results: case description: an -year-old male born premature at weeks presented with a history of recurrent infections. family history was negative for immunodeficiency. the patient developed recurrent acute otitis media beginning at year of age, three episodes of pneumonia beginning at years of age, and recurrent sinus infections requiring treatment on average four times a year beginning at years of age. initial immunologic evaluation at age was notable for: igg mg/dl (reference - mg/dl), igm mg/dl (reference - mg/dl), iga mg/dl (reference - mg/dl), ige iu/ml (reference - iu/ml). lymphocyte subpopulations were normal. specific responses to vaccines showed: protective antibody titers to diphtheria, but not to tetanus or pneumococcal antigens. he did not respond to booster vaccination and was started on ivig with significantly reduced frequency of infections. at age , while on ivig, he developed oral ulcers (biopsy consistent with ulcerative eosinophilic granuloma), abdominal pain, and recurrent arthralgias involving ankles, elbows, hips and the sacroiliac joint. magnetic resonance imaging (mri) was consistent with sacroilitis. subsequent imaging was consistent with chronic relapsing osteomyelitis (crmo). gastrointestinal biopsies showed severe active chronic pangastritis with antralized oxyntic gastric mucosa with enterochromaffin cell hyperplasia; suggestive of autoimmune gastritis. plasma cells were present throughout the gastrointestinal tract. ngs (bcm-ngs, baylor miraca genetics laboratory, houston tx) identified a hemizygous pathogenic missense variant, c. g>a (p.r q) in the x-linked foxp gene, that has been reported previously in patients with immunodysregulation, polyendocrinopathy, enteropathy, x-linked (ipex) syndrome. flow cytometry studies showed a decreased percentage of treg cells of total cd expressing cells, . % (reference . - . %), but normal foxp expression within these cells, % of treg cells with foxp expression (reference - %). interestingly, treg cell subset phenotyping obtained at the same time showed a normal percentage of natural tregs, . % cd t cells (reference . - . %), as well as normal percentage of naive tregs, . % cd t cells (reference . - . %). up to this point, he had not had any signs of diabetes, thyroid disease or frank enteropathy involving the small or large intestine. conclusions: we report a pathogenic foxp variant, occurring in a patient with a cvid-like phenotype, autoimmune gastritis, and an association with crmo. this case demonstrates the increasing utility of ngs, which can profoundly impact prognostic and therapeutic considerations. ( ) submission id# the natural history of patients with profound combined immunodeficiency (pcid): interims analysis of an international prospective multicenter study. immunodeficiency in this patient group. so far, patients were transplanted after enrolment, overall patients died ( in the hsct group, in the non-hsct group). analysis of the hsct decisions revealed the divergent decisions in patients with similar disease burden, favoring an ongoing prospective matched pair analysis of patients with similar disease severity with or without transplantation. so far, neither the genetic diagnosis nor simple measurements of t-cell immunity emerged as good predictors of disease evolution. conclusions: the p-cid study for the first time defines and characterizes a group of patients with non-scid t-cell deficiencies from a therapeutic perspective. since genetic and simple t-cell parameters provide limited guidance, prospective data from this study will be an important resource for guiding the difficult hsct decisions in p-cid patients. ( ) submission id# the plasma contact system and its role in common variable immunodeficiency: an explorative study. introduction/background: a growing body of evidence suggests that the contact system is involved in the activation of various vascular and immunological pathways and acts as an interface to help regulate allergic reactions, coagulation, complement, innate immunity and inflammation. as demonstrated in mice experiments, contact activation and high molecular weight kininogen (hk)-derived peptides increased homing of t and b lymphocytes into lymph nodes, which suggests an important area of research for understanding the contact systems role, specifically fxii, in immune-mediated inflammation and immune dysregulation. this novel mechanism prompted further inquiry into its role of various human disease states characterized by inflammation. plasma hk cleavage has been proposed as a useful and minimally invasive biomarker in various inflammatory disease states. this pathway has not been explored in cvid, in which inflammatory complications are found in one-third of patients with an unidentified genetic cause. characterizing the contact system biomarkers in cvid patients could elucidate a role in pathogenesis. objectives: assess the presence of contact activation at baseline in sera from cvid patients with and without inflammatory complications compared to healthy controls. methods: cvid patients were recruited in the outpatient setting and the measurement of cleaved plasma hk (chk) levels was determined by western blot analysis, under reducing conditions, with quantitation of total and chk bands using an odyssey imaging system (licor). a one-way anova test for differences among the studied groups will be applied. c inhibitor levels, c and c levels and high-sensitivity crp were also measured as comparable biomarkers for inflammation. results: to date, cvid patients were studied, with and without inflammatory complications. repeated determinations of cleaved hk% (chk%) revealed an average of . % (range: . - . %) in cvid patients with inflammatory complications and those without complications averaged . % (range: . - . %). healthy controls had an average chk of . % (n= , range: . - . %). conclusions: cleaved kininogen detected in the sera of cvid patients was found at similar levels compared to healthy controls (chk< %). findings suggest that systemic activation of the contact system might be absent in cvid, however, future considerations include developing detection methods for local tissue activation. ( ) submission id# the underlying primary immunodeficiencies and lung diseases, and low cd and cd counts are associated with recurrent pneumonia in hiv negative lymphopenia patients. clinical fellow, yale university school of medicine introduction/background: lymphopenia can be considered as primary or acquired immunodeficiency. lymphopenia is associated with a considerable increase in susceptibility to infections and the treatment focus for lymphopenia is mainly consists of prophylaxis and treatment of opportunistic infections. aids is the most well known cause of acquired lymphopenia and the cd count serves as an effective surrogate marker for disease progression and guideline for prophylaxis for hiv positive lymphopenia patients (hplp). hiv negative lymphopenia patients (hnlp) have been following the same prophylaxis guideline for hplp patients. however, it is unclear whether same prophylaxis guideline will be appropriate for both groups since the underlying immune mechanisms are different between these two groups. objectives: we aimed to define the optimal treatment and prophylaxis guideline for hnlp. in this study, we compared the clinical phenotypes and absolute counts of lymphocyte subsets between hnlp with recurrent pneumonia (pna), recurrent upper respiratory infection (uri) and no pulmonary infection. methods: electronic medical records of hnlp (n= ) seen at an academic immunology clinic between the year of and were reviewed retrospectively. lymphopenia was defined as absolute cd count less than /μl. the age, absolute counts of cd , cd , cd , cd , nkt and nk cell counts, history of antibiotic or antiviral prophylaxis use, autoimmune disease, lung disease, immunosuppressive therapy use, hypogammaglobinemia and ivig therapy use were compared between patients with recurrent pna (n= ), recurrent uri (n= ) and no pulmonary infection (n= ). results: this study showed that patients with recurrent pna had significantly lower absolute cd , cd , nk cell counts and age compared to the patients with recurrent uri ( table ) . none of the clinical phenotype was significantly different between these two groups. when we compared the patients with recurrent pna vs no infection, all lymphocyte subset counts and age were similar between these two groups except the frequency of underlying lung disease which was significantly higher in the recurrent pna group (table ) . lastly, we grouped the recurrent pna and uri groups together as the pulmonary infection group. when we compared the pulmonary infection group with the no infection group, lymphocyte subset counts were not significantly different, however, infection group showed significantly higher incidence of pid and the trend of lower rate of is therapy use than the no infection group (table ) . conclusions: the initial study has several limitations. this was a retrospective study from a single clinic and patient population was limited to patients. despite these limitations, we believe that this study provides valuable messages regarding prophylaxis guideline for pulmonary infection in nhlp; the underlying pids including icl and cvid, lung diseases particularly bronchiectasis and the absolute cd and cd counts less than /μl and / μl, respectively, are associated with recurrent pna and the patients with these risk factors likely will benefit from antibiotic prophylaxis. in addition, patients with acquired lymphopenia due to chronic use of is therapies without underlying pid or lung disease less likely develop pulmonary infection despite of low cd and cd counts. further investigations are crucial to elucidate the clinical significance of our initial observation by increasing the patient population and analysis of detailed immunologic and genetic profile of these patients which will likely reveal immunological markers and genes that are involved in the pathogenesis of both primary and hiv negative acquired lymphopenia. director, neuro-oncology program, division of hematology/oncology, ucsf benioff children's hospital oakland, ca introduction/background: gata is a hematopoietic transcription factor, required for the development and maintenance of a healthy stem cell pool. heterozygous mutation of gata has been associated with different but overlapping syndromes affecting both myeloid and lymphoid cell lines including aplastic anemia, myelodysplastic syndrome, acute myeloid leukemia, pulmonary alveolar proteinosis and lymphedema. it is also associated with immunodeficiency and susceptibility to mycobacterial, fungal, and viral infections. hematopoietic stem cell transplantation (hsct) is the only available cure which should ideally occur before patients develop neoplasia, severe infections or lung disease. objectives -to describe the clinical presentation of a genetically confirmed case of gata mutation -to describe the characteristic hematological and immunological profile of patients with gata mutation -to emphasize the importance of high index of suspicion and early diagnosis in improving outcome with hsct methods: case: a -year-old female presented with prolonged fever, fatigue, nonspecific rash, and unremarkable clinical exam. laboratory evaluation was significant for mild pancytopenia with profound monocytopenia. bone marrow analysis was remarkable for hypocellularity without evidence of dysplasia or malignancy. peripheral blood flow cytometry showed decreased t-and b-cells and absent nk cells. results: the constellation of pancytopenia, marked monocytopenia and absent nk cells, were suggestive of gata deficiency. sanger gene sequencing of gata revealed a heterozygous nonsense mutation (p.arg *). conclusions: mutation of gata is the underlying defect in overlapping clinical syndromes and is associated with immunodeficiency and malignant predisposition. incidence of organ dysfunction, infections and neoplasia increases with age. confirming the diagnosis during the phase of marrow hypocellularity/monocytopenia and pursuing hsct prior to malignant transformation may improve patient outcome. introduction/background: reduction in child mortality has kept pace with improved immunization and nutritional status. however, there are children with severe infections who have no identifiable reason. the search for children with pid was initiated in a tertiary care referral hospital from a region with no documentation of the prevalence of this disorder, but with a high rate of consanguinity. financial constraints, poor availability of laboratory facilities or therapeutic options locally, limited awareness among pediatricians and vast distances between premier centres were major obstacles. objectives to identify children with primary immune deficiency and study the spectrum of pid in north kerala to prevent infectious and other complications in these children to provide curative therapy whenever feasible methods . acquisition of knowledge and skills to diagnose and manage pids . establishment of an immune deficiency clinic . liaison with centres across the country offering diagnostic tests and stem cell transplant . formation of project team and submission of a project to the central government . improvement of diagnostic facilities at home institution results: children with recurrent, severe or persistent infections or with two or more esid warning signs were screened for pid. severe combined immune deficiency was diagnosed in children, wiskott -aldrich syndrome in children, chronic granulomatous disease in children and x linked agammaglobulinemia in children and leucocyte adhesion deficiency in children. prophylaxis with ivig was initiated in children and stem cell transplant was done for children. conclusions: in a country with resource constraints, limited awareness among health care providers and vast distances, it is possible to make a difference to the lives of families of children with pid by networking across centers with expertise in immunological and molecular genetic diagnostic methods and life -saving therapeutic modalities like stem cell transplantation. introduction/background: complete thymic aplasia is a rare cause of scid, and requires thymic transplantation for curative treatment. because thymic transplantation is not widely available, there can be significant delay between diagnosis and curative therapy placing the infant at risk of invasive life threatening infections. objectives: describe modalities of therapy for adenoviremia in a patient with scid due to thymic aplasia. methods: chart review was performed. treatment including hlapartially matched third party cytotoxic t cell therapy and matched related hematopoietic cell transplant were assessed for treatment of life threatening adenoviremia in a scid patient with thymic aplasia. we identified an infant with a mutation in tbox transcription factor (tbx ) (c. _ dup ) by way of state newborn screening for scid. she had severe t cell lymphopenia and abnormal t cell function. at months age, she developed adenoviremia with associated fulminant hepatitis, and an initial viral load of million copies/ml. despite prolonged therapy with cidofovir, viral load increased to as high as million copies/ml. treatment with two infusions of partially hlamatched third-party cytotoxic t cells specific to adenovirus ( / and / hla matched, with hla class ii-mediated antiviral restrictions) led to partial clinical improvement without viral clearance. due to continued severe adenovirus-related hepatic dysfunction, unmanipulated bone marrow from an hla-identical sibling was infused without conditioning ( million cd + cells/kg and . x cd + cells/kg), at months of age. after an initial surge in adenoviral loads attributed to massive viral lysis, the degree of viremia progressively declined and was < , copies/ml within weeks of marrow infusion. antiviral tcell activity against adenovirus was detected at low level in peripheral blood via ifn elispot at weeks post-marrow infusion. she subsequently developed acute cutaneous and hepatic gvhd responsive to tacrolimus and steroids without recrudescence of viral illness. conclusions: delay in curative therapy in scid substantially increases risk of invasive life threatening infections. one strategy of allogeneic hct can be to eradicate severe infection in scid by providing the necessary t cell directed therapy against infectious agents. antigen-specific partial immune reconstitution can be achieved with hct in patients with thymic aplasia but concern regarding the development of full immunologic t cell diversity in athymic patients remains. senior technician, sanquin bloodsupply amsterdam introduction/background: chronic granulomatous disease (cgd), an inherited disorder of granulocyte function caused by a failure of intracellular superoxide production, normally presents as severe recurrent bacterial and fungal infections in the first years of life. the majority of affected individuals are diagnosed before the age of years, although patients may remain undiagnosed until adulthood despite the early onset of the symptoms objectives: investigation of fungal infection in adult cgd patients methods: nbt and dhr for detection of cgd and molecular analysis for detection of type of mutation in cgd and fungal characterization. results: we report here the detection of causative fungal infections in adult patients with cgd. in the first patients we found paecilomyces formosus infection in an adult patient ( years old female) with undiagnosed cgd who was referred to the shahid beheshti university hospital (tehran, iran) complaining of cough, dyspnea and fever for weeks prior to admission. microscopic analysis revealed branching solitary phialide with ellipsoidal conidia with long chain arrangement and many chlamydospores which mostly resemble botryotrichum species but during subcultures on potato dextrose agar (pda) and sabouraud dextrose agar (sda), phialides typical of paecilomyces species appeared. typically, paecilomyces spp. rarely cause infections in humans and if these fungi are detected in blood urine or cerebrospinal fluid cultures they are considered as contaminants. the second patient was a -year-old man was referred to the hospital with weight loss, fever, hepatosplenomegaly and coughing. he had previously been diagnosed with lymphoadenopathy in the neck at age and prescribed antituberculosis treatment. bal and serum galactomannan tests were negative. low, subnormal levels of ros were produced following stimulation of purified neutrophils with the phorbol ester pma. genomic dna was extracted and gene scan was used to determine the ratio between the number of exon sequences of neutrophil cytosolic factor (ncf ) gene, which encodes p phox, and the number of -ncf exon sequences. in addition, the fungal culture was disrupted with glass beads and dna was extracted. the dna sequence results were blasted using the ncbi genebank database, which showed % similarity to an aspergillus terreus isolate in the gene bank fungal library with accession no . conclusions: thus, despite its current relative rarity in older patients, the presence of fungal infection is changing our understanding of the diagnosis, management and outcome of cgd. greater appreciation of the potential of fungal infection in older cgd patients is important in regions, such as iran, where tuberculosis is endemic and that sarcoidosis and cgd are considered as differential diagnosis. the demonstration of the successful patient-orientated treatment after using sequencing to confirm cgd and to identify the presence of the specific infectious agent emphasises the importance of adopting this approach across the region. introduction/background: primary immunodeficiencies (pi) represent a heterogeneous group of over genetically distinct disorders which interrupt normal host-defense mechanisms and predispose to significant morbidity and mortality. presently, we are only able to screen for severe t-cell deficiencies at birth; however, the most common forms of pi often go undetected for years leading to adverse patient outcomes and excessive healthcare spending. given the relatively high incidence of pi in the general population, informatic measures could be useful for determining individual risk for pi and facilitating earlier, correct diagnosis and appropriate treatment across the spectrum of pi. objectives: the purpose of this study was to test the jeffrey modell foundations spirit (software for primary immunodeficiency recognition intervention and tracking) analyzer on the texas childrens health plan. major aims were to identify individuals with medium-high risk for pi, assess the clinical characteristics of at risk patients and determine if risk identification led to diagnosis of pi over a month period. methods: after removing all known pi diagnosed patients from the database, , individual texas childrens health plan enrollees were screened for risk of pi with the spirit analyzer using relevant, weighted icd and icd codes. patient characteristics are shown in table . following identification of medium-high risk (mhr) individuals, letters were sent to their primary care physicians to alert them of patient risk. a second analysis of the mhr individuals was performed months later. detailed chart reviews were conducted on mhr individuals to further assess clinical features of this group. the study was approved by the baylor college of medicine institutional review board (study h- ). results: of the original cohort, ( . %) were identified as mhr for pi. from that group, ( . %) were accessible for analysis and ( . %) had electronic health records for review. in the months following the first analysis, ( . %) were diagnosed with a pi. (figure ) another patients had concerning diagnoses coded warranting further investigation. (figure ) concerning diagnoses included: cellulitis( ), abscess ( ), recurrent otitis media( ), recurrent sinusitis ( ), osteomyelitis ( ), and mastoiditis ( ) among others. in total patients had a pi diagnosis or a history concerning for pi ( . % of main cohort; . % of mhr cohort). conclusions: the spirit analyzer is effective at identifying persons at risk for pi and facilitates diagnosis. potential mhr yield by the analyzer is over %. these patients are treatable and will benefit from targeted intervention once identified. the analyzer can also highlight concerning conditions worthy of additional assessment. future work should focus on longitudinal healthcare outcomes for patients diagnosed with pi via spirt and physician perspectives on the utility of the tool. introduction/background: genetic variants in card contribute to several diseases caused by dysregulation of the adaptive immune system. dominant-negative, loss-of-function and gain-of-function variants in card variants lead to primary immunodeficiency disease. however, primary immunodeficiency disease caused by card gain-of-function variants often progresses to b-cell malignancy. the clinical course and treatment options depend on the type of card mutation. unfortunately lymphocyte immunophenotyping and traditional proliferation assays can't distinguish the variant effect or predict the likelihood of malignancy. objectives: to use multiplexed functional assays for determining variant effect to distinguish between dominant-negative, loss-and gain-offunction effects in card . our ultimate goal is to generate a variant function map that can be used to guide diagnoses and treatment of immune dysregulation caused by mutations in card . methods: we co-delivered crispr/cas ribonucleoprotein complexes with libraries of single-stranded oligonucleotide repair templates thus generating lymphoma cell populations containing all possible singlenucleotide variants (~ different protein coding changes) in the nterminal amino acids of card . following culture with and without bcr pathway inhibitors, we used next generation sequencing to quantify variant abundance before and after culture, both with and without bcr pathway inhibitors. results: due a requirement for card in these lymphoma cells, those with dominant-negative and loss-of-function card variants grow more slowly, whereas those with gain-of-function variants grow faster in the presence b cell receptor pathway inhibitors. by tracking the relative abundance of each variant in the population by next generation sequencing over multiple conditions, we determined the functional effect of each. we assessed the functional effects of thousands of card variants in parallel. this enabled us to confirm several previously reported gain-offunction and dominant-negative variants in card , as well as identify several additional novel variants. finally, we evaluated previously undescribed dominant-negative, loss-of-function and gain-of-function variants during differentiation of primary human b cells and during nf-b signaling. conclusions: the results of our experiments demonstrate the utility of multiplexed functional assays for determining variant effect in proteins where distinguishing between dominant-negative, loss-and gain-offunction effects are required to guide diagnoses and treatment. introduction/background: primary immune-deficiencies (pids) are a heterogeneous group of nearly monogenic inborn errors of immunity. in recent years, whole exome sequencing (wes) became a valuable diagnostic approach for the identification of molecular defects in patients with clinical manifestations suggestive of pid. this approach provides a definitive diagnosis and may help in genetic counseling, prenatal diagnosis and pre-symptomatic identification of patients with a potentially lethal disease. the diagnostic yield using wes was found to be~ % in rare mendelian disorders and~ % in pids. we present here a markedly higher yield, of %, in pids with a high percentage of consanguinity ( % in this study). objectives: using the whole exome sequencing (wes) approach in israeli pid patients with high percentage of consanguinity to identify the genetic causes underlying their diseases for better diagnosis and clinical management. methods: wes was performed on genomic dna obtained from wbc of immune deficient patients with potential genetic causes. the sequencing data was analyzed bioinformatically. each of the discovered mutations was validated and the familial segregation was confirmed, using sanger sequencing. results: the probands ( males and females) ranged in age from weeks to years with a mean of . years. of them, patients are jewish ( %), palestinians ( %) and ( %) is from a greek ethnicity (cyprus). based on their clinical and immunologic phenotype at the time of initial evaluation, patients were assigned to one of seven pid groups: (i) humoral immunodeficiency patients ( %); (ii) combined immunodeficiency (cid) ( %); (iii) cid with syndromic features ( %); (iv) scid ( %); (v) congenital defects of phagocytes ( %); (vi) immune dysregulation ( %) and (vii) phenocopy of pid ( %). we identified mutated alleles, all in coding regions, that are highly likely to be causative in of the patients, achieving a % molecular diagnostic yield. among the patients, the mode of inheritance in patients ( %) is autosomal recessive and in is compound heterozygote ( %). four patients ( %) harbor a non-inherited mutation on one allele, either de novo or somatic. the inheritance of the mutation in one patient ( %) is x-linked. the high rate of bi-allelic inheritance ( % of the alleles) is mainly due to the high frequency ( %) of consanguinity among the studied cohort. twenty eight mutated genes were identified in this study. of them, were found to be novel in causing an inherited disease in man. interestingly, some genetic defects in known genes were found in patients with atypical phenotypes. in patients ( % of the total number of patients and % of the wes diagnosed) the discovery of the genetic cause led to a change of therapy, towards a more targeted and personalized one. the revised treatments included bone marrow transplantation, conditioning protocols, reduced intensity of immune suppression, and prevention of unnecessary treatments due to their possible deleterious outcome. conclusions: except of being a useful tool for diagnosing and deciphering novel or atypical forms of pids, our wes study demonstrates an immediate and powerful impact on patient therapy in pids. early intervention with bone marrow transplant or gene therapy is critical and best when the infant in uninfected. newborn screening for scid and t-cell lymphopenia has been implemented in states. the screening test is performed from dried blood spots collected at birth and involves pcr quantification of circular dna byproducts of t-cell receptor gene rearrangement, t-cell receptor excision circles (trec). trecs are generated during t-cell maturation in the thymus and are indicative of naïve t-cell output. assays and protocols to measure trecs vary by state and there is no standardized guideline at this time. washington state is unique in that it is one of few states where all newborns undergo two or more independent newborn screens, the first by which igrt treatment helps in these patients is unreported. here we present the first case of an mbl deficient patient with other complicating conditions (low igg and multiple cf polymorphisms, ciliary dyskinesia) who did not show improvement on igrt, thus refuting current literature. results: a -year-old caucasian male patient presented due to recalcitrant warts of the hands and feet. he had childhood otitis media status post tympanoplasty, mild molluscum contagiosum, and eczema. also, history of migraines with a negative brain mri. a trial of weekly pegylated interferon alpha mcg for his warts was discontinued due to significant neutropenia and depression. physical exam was notable for bulky skin colored warts on the lateral and dorsal fingers and dorsal hands including periungual skin. clusters of verrucae were noted on the feet. initial laboratory testing was notable for mild hypogammaglobulinemia with protective specific antibodies. lymphocyte subset analysis revealed a predominantly t cell lymphopenia with decreased naïve and memory cd and cd subsets, normal absolute numbers of b cells but with low memory subsets, and normal numbers and function of nk cells. whole exome sequencing ultimately revealed homozygous r q mutations in the cecr , a mutation previously described as causing ada deficiency. ada activity was about %. conclusions: ada deficiency is a relatively newly defined genetic defect, with a clinical phenotype that continues to evolve with newly diagnosed cases. our patient had not had evidence of vasculitis or stroke, but had recalcitrant warts with lymphopenia as his primary presentation. approach to therapy for those without vasculitis or significant cytopenias remains unknown. introduction/background: whole exome sequencing has added greatly to our library of primary immune deficiencies. however, interpretation of findings is not always straightforward. clinicians need to understand the limitations of this diagnostic tool and be familiar with the next steps in order to achieve a diagnosis. objectives: a year old male presents for evaluation of bronchiectasis of bilateral lower lobes and poor growth. past medical history was significant for gerd with poor weight gain during infancy and childhood, oral thrush, recurrent respiratory infections and bilateral partial hearing loss. the bal showed abundant neutrophils and was positive for strep pneumoniae and haemophilus influenza. methods: serum immunoglobulins were normal with an elevated iga ( ) and mildly elevated ige ( ). tetanus igg was protective ( . iu/ml) but post-vaccine responses to the pneumococcal polysaccharide vaccine were poor with protective titers achieved to / serotypes. lymphocyte phenotyping was remarkable for a complete absence of b and nk cells. cd :cd ratio was inverted ( . ) and the majority of the cd + t cells were memory phenotype. mitogen proliferation was essentially normal. due to the absence of b cells and despite normal total serum immunoglobulins, the patient was started on sc ig weekly and antibiotic prophylaxis with trimethoprim-sulfamethoxazole and fluconazole. clinically he demonstrated good weight gain and a reduction in cough and respiratory symptoms. results: whole exome sequencing was performed. a single base mutation in ada (c. t>c) was identified in one allele. this mutation (p.l p) is known to be deleterious and when homozygous is associated with typical scid. however, this mutation was not present on the other allele. secondary analysis looking for large deletions and duplications failed to identify any abnormality in sequence in the normal allele. but the expression of this allele was markedly diminished causing us to suspect that its function might be impaired. functional testing was performed and demonstrated that there was no ada activity in the patients red blood cells, thus confirming a diagnosis of combined immune deficiency due to ada deficiency. conclusions: null mutations in ada result in an absence of ada function with profound cd cell lymphopenia and dysfunction. clinically affected infants have typical scid. hypomorphic mutations may lead to partial function of ada with more variable immune defects. interestingly, most patients with ada deficiency have neurologic complications, frequently hearing loss. we believe that most likely this patient has had some degree of spontaneous reversion of the mutation in the normal allele leading to a less severe phenotype. as reported previously in a case involving a mutation in il rg chain, normal function was not restored and the patient remains with a combined immune deficiency. this case highlights an important limitation of whole exome sequencing and the need for confirming the impact of a genetic defect with a functional assay. introduction/background: the autosomal dominant hyper-ige-syndrome (ad-hies) is a rare primary immunodeficiency and multisystem disorder resulting from heterozygous loss-of-function mutations in the stat gene. ad-hies is characterized by skeletal dysplasia, recurrent pulmonary and skin infections (e.g. staphylococcal abscesses, eczematoid dermatitis) due to an increased susceptibility to bacteria and fungi. since many patients do rather well on anti-infective prophylaxis and supportive care, and early case reports suggested no benefit of allogeneic hematopoetic stem cell transplantation (hsct), ad-hies patients are rarely referred for hsct. the literature still contains only a handful of patients who underwent hsct. all these patients had experienced severe disease related complications before hsct and consequently the benefit of hsct was reported to be variable. objectives: currently, the general consensus is to only consider patients with severe pulmonary disease for hsct. it could however be postulated that transplanting patients earlier in their disease course and correcting their immunodeficiency before permanent organ damage due to infectious complications has occurred may extend life-expectancy and improve the quality of life of ad-hies patients. methods: we report on a year old female ad-hies-patient who presented with bronchiectases following recurrent pneumonias, one pneumatocele, and normal pulmonary function tests. her past medical history also included recurrent skin infections, serious haemoptysis, pathological fractures and atopic eczema. considering that lung infections are the major life-limiting complication in ad-hies and are potentially positively influenced by hsct, our patient had enquired about a transplant. after extensive discussion of the pros and cons and obtaining full informed consent from her and her family, she underwent an elective hsct. she received bone marrow from an hla-matched sibling donor after a reduced-intensity conditioning consisting of alemtuzumab ( x , mg/kg), treosulfan ( x g/ m ), fludarabine ( x mg/m ) and thiotepa ( x mg/kg), and graftversus-host disease (gvhd) prophylaxis with cyclosporine a (csa) and mycophenolate mofetil (mmf). results: the peri-transplant course was complicated by acute lower gastrointestinal tract bleeding and renal failure of unknown origin. continued kidney function impairment led to early tapering and discontinuation of csa on day + after hsct in the absence of any acute gvhd. neutrophils and platelets engrafted on days + and + respectively. she is currently on day + , free of gvhd or infection, exhibiting full donor chimerism and recovered kidney function. in view of the preexisting pulmonary damage she currently remains on antibiotic prophylaxis and inhalation therapy. conclusions: this ad-hies patient who underwent hsct with few pre-hsct disease complications and relatively little permanent organ damage may add to our understanding of whether early hsct will lead to improvement of quality of life and possibly increased life-expectancy in ad-hies patients. it remains to be elucidated whether her rather uncommon peri-hsct complications are connected to her underlying disease. future research should be directed at identifying ad-hies patients at high risk of severe pulmonary complications early, so these could be referred for timely hsct. objectives: this is a case study of a patient who had refractory ibd symptoms and recurrent infections who was found to have xiap deficiency and mefv variant mutations. methods: a year old male presented at age with recalcitrant ibd unresponsive to multiple medications including steroids, mesalamine, azathioprine, infliximab, adalimumab, methotrexate, and vedolibumab. in addition, he developed severe infections from a combination of immune dysregulation and immunosuppressant medications. currently he is on ustekinumab but still has severe abdominal symptoms. patient does not have a history of hemophagocytic lymphohistiocytosis (hlh). family history was significant for ibd in both his mother and sister. he has had persistent lymphopenia which ranged between to cells/ mm . t/b/nk panel showed decreased cd t cells ( cells/mm ) with normal cd , cd , and cd /cd ratios. b and nk cells were normal in quantity. t cell antigen/mitogen assays showed normal response to all mitogens (pha, pwm, con a) and most antigens (tetanus, candida, hsv, vzv, adv) but low response to cmv antigen. igg was elevated at , , but iga, igm, and ige were normal. his ebv dna pcr is negative. results: exome sequencing revealed a novel xiap hemizygous variant at position c. g>a (p.=?) of unknown significance and a mefv heterozygous variant. functional testing performed at medical college of wisconsin showed no expression of xiap on lymphocytes and a defect in nod pathway. given the xiap deficiency, bone marrow transplant was discussed as an option for refractory ibd and prevention of hlh. rituximab was also offered to decrease the possibility of hlh. currently the patient is in the decision making process of both treatment options. conclusions: genetic evaluation with clinical exome in early onset refractory ibd, family history of ibd, and recurrent infections demonstrated a novel mutation in the xiap gene with possible contribution of mefv variant as well. the absence of xiap protein expression and abnormal functional assay of the nod pathway confirm the pathogenicity of the mutation. identification of this genetic variant will help guide future therapeutic options and prognosis for this patient. introduction/background: x-linked agammaglobulinemia (xla) is a primary immunodeficiency disease caused by mutations of bruton tyrosine kinase (btk), which is essential in b cell maturation. xla is typically associated with bacterial infections of upper and lower respiratory systems, enteritis and increased risk for malignancies. objectives: to present the evolution and treatment of a complicated flexispira (helicobacter bilis) infection with delayed diagnosis in an xla patient. methods: clinical and laboratory features of a patient with xla evaluated at the national institutes of health. results: a -year-old male patient with xla presented for initial evaluation of indurated lower extremity and torso lesions. he was diagnosed with xla at age years secondary to bacterial sepsis pneumonia and empyema. after starting ivig at age , he was well without significant infections except for recurrent otitis media. at years of age, he developed right leg edema below the knee, which progressed to patchy skin thickening and discoloration. a tissue biopsy at years of age revealed marked fibrous thickening of the subcutaneous septum with diffuse infiltrate of eosinophils with negative cultures for bacterial, fungal, and mycobacterial infections and thought to be consistent with eosinophilic fasciitis. mri demonstrated infiltration in the superficial and deep muscle compartments with fibrosis. symptoms persisted despite empirical treatment with iv antibiotics and steroids. at years of age his left leg became involved with similar findings, while under treatment including multiple immunosuppressants (dapsone, methotrexate, tacrolimus, hydroxychloroquine, iv cyclophosphamide, remicade, cytoxan, and enbrel) targeting eosinophilic fasciitis. at age , he developed ulcerations over the left shin and ankle and was diagnosed with chronic multifocal osteomyelitis based on mri findings. physical exam was notable for bilateral leg swelling below the knees with woody appearance and induration, hyperpigmentation, and tenderness. indurated and nodular lesions without discoloration were noted above the waist line, on right forearm and above right nipple. skin biopsies of right lower extremity and right forearm were positive for numerous spirochetal-like organisms with warthin-starry stain. treatment was initiated with meropenem and gentamicin. gentamicin was discontinued due to vestibular ototoxicity six weeks later and doxycycline was added. initial response was followed by worsening of symptoms and intolerance to treatment, which led to the addition of tigecycline and azithromycin. with continued progression, seven months into initial evaluation, chloramphenicol and nitazoxanide were added to the regimen. due to the persistence of flexispira organisms on skin biopsy warthin-starry stains, fresh frozen plasma (ffp) was introduced four months later ( months after initial evaluation) as an adjunctive treatment. cultures performed at the cdc were negative; however, pcr and sequencing resulted in identification of helicobacter bilis. units of ffp was infused weekly for over three years, then reduced to every two weeks, with goal igm levels > mg/dl. subsequently, labs including cytopenias, inflammatory markers, and immunoglobulins improved as well as a negative warthin-starry stain. symptoms have improved with almost complete resolution of findings with only residual small areas of discoloration over both lower extremities. conclusions: xla immune deficiency is associated with flexispira (helicobacter bilis) infections, with typical appearance of discoloration and induration, which may evolve to osteomyelitis due to delayed treatment. although typically observed over the lower extremities, immunosuppressive treatment may lead to further expansion above the waist line. approach to therapy with weekly to bimonthly ffp infusions in addition to antibacterial treatment has proven to be beneficial in controlling the infection. higher igm levels resulting from the ffp may also provide antibacterial effects. introduction/background: x-linked severe combined immunodeficiency (scid) is a well described primary immunodeficiency associated with mutations in the common gamma chain. patients with x-linked scid classically present with profoundly low or absent t cells and nk cells with a variable number of b cells. the lymphocytes that are present typically have a proliferation index < % control when stimulated with mitogens and antigens. patients must undergo corrective therapy with bone marrow transplant (bmt) or gene therapy to avoid the life-threatening infections that are associated with the nearly absent adaptive immune system. objectives: a -week-old boy presented to childrens national immunology clinic for initial evaluation of critical result on newborn screen. methods: targeted partial exome sequencing was performed on a -dayold patient who was picked up via trec assay on the maryland newborn screen. flow cytometry was completed at childrens national and proliferation studies completed at cincinnati childrens hospital diagnostic immunology lab. results: flow cytometry revealed markedly decreased lymphocytes with nearly absent cd + t cells and low cd + t cells with a r e l a t i v e i n c r e a s e i n c d + c d r o t c e l l s ( r a t i o cd ra:cd ro: %: %). the b and nk cells were within the reference range for age. mitogen proliferation studies showed a mild decrease to pha and normal responses to pokeweed and cona ( cpm, cpm, and cpm, respectively). partial exome sequencing revealed a hemizygous nonsense substitution in il rg (c. c>t, p.r ) . maternal engraftment accounted for % of the t cells. the patient was started on prophylaxis with ivig, bactrim, and fluconazole with the plan to proceed with bone marrow transplant. as patient approached bmt maternal engraftment became absent in whole blood and repeat proliferation studies revealed normalization of the response to pha (stim index) with continued normal responses to cona and pokeweed. the patients flow cytometry values and ratios remained unchanged. patient completed a reduced intensity preparative regimen of busulfan, fludarabine and alemtuzumab prior to receiving his / matched unrelated donor bone marrow transplant. conclusions: it remains to be determined why initial proliferation studies showed > % function with improvement over time in a patient with a well-described genetic mutation causing scid ( ) director, division of intramural research, nih/niaid introduction/background: the advent of next-generation sequencing (ngs) has led to a proliferation of newly discovered genetic diseases and expanded phenotypes of known immunodeficiencies. availability of specific gene panels or whole exome sequencing (wes) with targeted analysis based on broad phenotypes, coupled with clinicians increasing awareness has led to higher utilization of ngs. published reports from high throughput sequencing labs indicate exome analysis identifies causative mutations in only - % of probands. results: we have seen several patients who underwent high quality ngs in whom causative mutations were not identified. two separate families with multigenerational histories of leukemia, aplastic anemia, myelodysplastic syndrome, and cytopenias suggestive of gata deficiency had myeloid gene panel screens at commercial labs without causative mutations identified. targeted gata sequencing in the first family identified a novel change in gata , c. g>t, p.l l. cdna analysis demonstrated this synonymous variant resulted in aberrant splicing leading to a frameshift and premature termination. the wes bioinformatics pipeline failed to recognize the splice mutation. in the second family, pcr amplification spanning the terminal exons revealed a shortened pcr product with a base deletion fully encompassing the penultimate exon and leading to a amino acid in-frame deletion. the deletion spanned all capture probes for the exon resulting in only the wild-type allele being captured and sequenced. additionally, capture kits targeting only coding regions of genes fail to capture deep intronic mutations such as those seen in gata (hsu, ) or in the untranslated region of ikbkg, encoding nemo, (mooster, ; hsu, submitted) . lastly, even with good capture and sequencing, the presence of pseudogenes may confound downstream sequence alignment as seen in ncf , encoding p phox preventing recognition of disease causing mutations. conclusions: with ngs becoming more widely available as a clinical diagnostic tool, it is important to remember that wes results, unlike many laboratory tests, are not binary. inadequate bioinformatics pipelines, deletions, intronic or untranslated mutations, and pseudogenes can all mask the presence of causative mutations. targeted panel captures or analysis will miss novel genes. astute clinicians need to recognize the limitations of the current technology and pursue alternate assays when suspicions warrant. a cell based assay for the detection of autoantibodies to il- in human serum. matt phillips, phd , vijaya knight, md, phd senior scientist, national jewish health director of immunology and complement adx labs, national jewish health introduction/background: patients with chronic candida infections are typically deficient in some aspect of il- signaling. one mechanism, which has recently come to light, is through the production of il- autoantibodies, particularly in patients who already suffer from specific autoimmune diseases. objectives: our objectives are to develop a diagnostic assay to accurately and easily detect il- autoantibodies in patient serum. methods: we developed a cell-based reporter assay using hek blue il- cells (invivogen) to detect the ability of patient serum to block il- receptor signaling. once stimulated with il- , the cells secrete alkaline phosphatase (ap) into the surrounding media which is detected by invivogen hek blue media and an absorbance reader. addition of serum containing blocking antibodies inhibits secretion of ap. results: we were able to demonstrate, using a single patients serum with known il- autoantibodies, the inhibition of il- signaling in hek blue il- reporter cells. we further characterized the sensitivity of our assay with a commercially available anti-il- monoclonal and found it to be sensitive to between . x - m and . x - m. conclusions: loss of il- signaling can lead to problematic immune deficiencies including difficulty in clearing extracellular pathogens such as candida. some people who have an immune deficiency in the il- pathway may have developed autoantibodies to il- and thus have difficulty generating an appropriate immune response. we have developed a relatively low maintenance, cost effective, and simple test for detecting il- autoantibodies in human serum. alternations in repertoire of t and b cell subsets in patients with partial recombination activating gene (rag) deficiency with autoimmunity and history of viral infections introduction/background: patients with partial deficiency of recombination-activating genes or (rag / ) can present with a wide spectrum of primary immunodeficiencies including combined immunodeficiency with granuloma and/or autoimmunity (cid-g/ai). prior case reports have highlighted alterations in b and t cell compartments; however comprehensive characterization of t and b cell receptor repertoires of lymphocyte subsets regarding diversity and autoreactivity has not been reported. objectives: defects in v(d)j recombination due to rag deficiency results in a skewed t and b cell repertoire that may be further modified by viral infections and promote inflammatory or autoimmune phenotype. methods: peripheral t and b cell compartments were sorted from two patients with combined immunodeficiency secondary to hypomorphic rag and rag mutations. b cells were stimulated with cd l, cpg and il- to transition to antibody secreting cells (ascs), mimicking viral infection. repertoire analysis and single cell cloning of bcr heavy and light chain variable regions from sorted b cell populations has been performed. repertoire of t cell subsets (treg and follicular helper) were also examined results: we noted skewing towards proximal j usage in all b cell compartments (mature naïve, marginal zone, cd -/low and memory) of two rag deficient patients compared to healthy controls. b cell clones with v - v genes with low rate of somatic hypermutation expanded during b cell development. after in vitro stimulation mature naïve b cells from rag deficient patient were capable of transitioning to antibody secreting cells and enriched for polyreactivity to dsdna, insulin, lps and ifn cytokine ( to %) compared to healthy control ( to %). in connection to altered b cell compartments, restricted repertoire of regulatory t cells and an expanded and skewed follicular helper t subset were detected. conclusions: our data indicate that patients with partial rag deficiency have skewed t and b cell subsets that can further be altered towards antibody secretion and polyreactivity after stimulation such as viral infections. chief of the division of allergy and immunology, division of allergy immunology, the childrens hospital of philadelphia, philadelphia, pa usa introduction/background: the clinical features of q . deletion syndrome include virtually every organ of the body. t cell lymphopenia, as a consequence of thymic hypoplasia, is the most commonly described immunologic feature and is most prominent in childhood. later in life, t cell exhaustion may be seen and secondary deficiencies of antibody function have been described in patients with q . deletion syndrome. objectives: the role of deletion breakpoints in determining q . deletion syndrome immunophenotype is unknown. in this study, we examined the effect of q . deletions with and without tbx on lymphocyte counts. methods: lymphocyte counts were compared between total q . patients with tbx -containing deletion (a-b, a-c, a-d deletions), and a total of patients with tbx -noncontaining deletion (b-d, c-d, d-e, d-f deletions). lymphocyte counts of patients with q . deletions were compared to a set of patients with a q . duplication including the tbx locus. lymphocyte subset counts for each group were analyzed by t-test. results: cd counts were significantly lower in the tbx -deleted cohort compared to the other two cohorts (mean cells/mm in tbx deleted cohort, cd cells/mm in the non-tbx deleted cohort, and in the duplication cohort, p< . for all). similarly, cd counts were lower in the tbx -deleted patients compared to the other two cohorts. there were no significant differences in cd , cd , and nk cell counts between the three cohorts. conclusions: these represent the first data to examine t cell counts in q . deletion syndrome patients with different breakpoints. our data highlights an important role for tbx or other genes in the a-b region in regulating t cell production. paracoccidioidomycosis associated with a heterozygous stat mutation and impaired ifn-immunity introduction/background: mutations in genes affecting ifn-immunity have contributed to understand the essential role of this cytokine in the protection against intracellular bacteria and fungi. however, inborn errors in stat , which controls il- responses, have not yet been reported. objectives: to determine the underlying genetic defect in a family with a history of paracoccidioidomycosis (pcm) disease. methods: genetic analysis was performed by whole-exome sequencing and sanger sequencing. stat phosphorylation and translocation from the cytosol to the nucleus, as well as ifnrelease by patient lymphocytes were assessed. the effect on stat function was evaluated by site-directed mutagenesis using a lymphoblastoid b cell line (b-lcl) and u a cells. microbicidal activity of patient monocytes/macrophages was also analyzed. results: a heterozygous missense mutation, c. a>t (p.e v) in stat was identified in the index patient and her father. patients and fathers lymphocytes showed reduced stat phosphorylation and nuclear translocation as well as impaired ifn-production. in accordance, b-lcl and u a cells carrying the stat mutant displayed reduced stat phosphorylation. patient's and father's pbmcs and macrophages (alone or in the presence of t cells) displayed impaired fungicidal activity compared with those from healthy controls that improved in the presence of recombinant human (rh) ifn-, but not rhil- . conclusions: our data suggest autosomal dominant stat deficiency as a novel inborn error of il- -dependent ifn-immunity associated with susceptibility to pcm disease. profound b cell lymphopenia in gof-stat that improves post ruxolitinib associate professor, university of south florida -johns hopkins all childrens hospital introduction/background: subjects with gain of function signal transducer and activator of transcription (gof-stat ) mutations have a variable clinical phenotype including combined immunodeficiency (cid). ruxolitinib, a janus kinase / inhibitor has been successful at treating immune dysregulation in subjects with gof-stat . two subjects with profound b cell and/or t cell lymphopenia as a major manifestation are described, one of which was successfully treated with ruxolitinib. objectives: to discuss gof-stat mutations, their effect on the immune system, and the potential benefit of ruxolitinib in these subjects. methods: retrospective chart review was performed. results: subject (c. a>g) is a year old male with a history of recurrent shingles, chronic mucocutaneous candidiasis (cmc), pneumocysitis jiroveccii pneumonia, varicella zoster meningitis, severe enteropathy, cerebral aneurysm and lymphoproliferation, and autoimmune hypothyroidism. he has profound lymphopenia predominantly affecting t and b cells (cd + cells/ul, cd + cells/ul, cd + cells/ul, cd + cells/ul, cd + cells/ul). subject (c. g>t) is a year old male with a history of severe cmc, recurrent pneumonia, enteropathy, and autoimmune thyroiditis. he had severe b cell lymphopenia (cd + cells/ul, cd + cells/ul, cd + cells/ul, cd + cells/ul, cd + cells/ul). treatment with ruxolitinib . mg bid led to clinical improvement of enteropathy and increased b cell counts in subject (cd + cells/ul). ruxolitinib has not yet been initiated in subject . both subjects were treated with anti-microbial prophylaxis and immunoglobulin supplementation. conclusions: combined immunodeficiency with variable degrees of b and t cell lymphopenia and hypogammaglobulinemia can be profound in subjects with gof-stat mutation. despite proper anti-microbial prophylaxis, this immunodeficiency can lead to severe infections. in addition to treating the autoimmune and immune dysregulatory features, treatment with ruxolitinib can improve the cid present and potentially reduce infectious susceptibility. igg -related disease (igg -rd), its common mimickers and response to anti-il -(reslizumab) treatment rachel eisenberg, md , arye rubinstein, md-phd fellow in allergy and immunology, montefiore medical center chief division of allergy and immunology, albert einstein college of medicine and montefiore hospital, bronx, ny, usa introduction/background: we describe a complicated case of igg related disease (igg -rd) both in its presentation and novel treatment objectives: to review the common mimickers of igg -related diseases which often lead to delayed diagnosis and treatment. to discuss novel therapeutic treatments for igg -related disease results: a -year-old woman with a history of thyroid disease, sicca symptoms, lipodystrophy, relapsing parotid enlargement, asthma and erdheim chester syndrome initially presented with recurrent bacterial and fungal sinusitis despite multiple sinus surgeries. immunologic workup was notable for lymphopenia of /ml, cd count of ( - cells/ul normal range) and elevated igg of ( . - . normal range). imaging was notable for nasal septal perforations and hypoplastic maxillary sinuses. there was high suspicion for igg disease however the patient was lost to follow up during which time she developed cachexia, eosinophilic pleural effusions ( % eosinophils), lung mass and a parotid mass with predominant t cell infiltrate misdiagnosed as follicular lymphoma. features consistent with igg -rd included > % ratio of igg /igg and a predominant t cell infiltrate a biopsied lung mass showed igg plasma cell > /hpf also consistent with igg -rd. bone marrow biopsy was within normal limits. the patient was treated with rituximab, an effective treatment for igg -rd. on treatment her igg levels normalized, however she developed recurrent large eosinophilic lung effusions requiring repeat drainage. fractional exhaled no (feno) was elevated to ppb. she was started on reslizumab at a dose of mg/ kg resulting in marked improvement in her respiratory status along with normalization of peripheral eosinophilia and reduction of feno to the normal level of ppb. conclusions: igg -rd is a fibro-inflammatory condition which can affect any organ system and is diagnosed via tissue histology showing igg positive plasma cells and a typical morphologic pattern. this case outlines the common mimickers of igg -rd often leading to a delayed diagnosis. before the final diagnosis of ig -rd was made by us, the patient carried multiple diagnoses including: thyroid disease, recurrent parotid enlargement and seronegative sjogrens. these diagnoses in hindsight may have been mikuliczs syndrome, kuttners tumor and/or riedels thyroiditis which are common manifestations of igg -rd. chronic sinusitis, atopic diseases, peripheral eosinophilia and destructive osseous lesions as noted in our patient are seen in up to % of patients with igg -rd. destructive bony lesions and eosinophilia can mimic granulomatous polyangiitis, which was ruled out in our patient. her cachectic appearance and diagnosis of lipodystrophy can be explained by destruction of osseous tissue in the craniofacial skeleton which was later confirmed on imaging. lymphoid inflammatory infiltrates are commonly seen in igg -rd and are can be misdiagnosed as a follicular lymphoma as in our case. a novelty in this case is the successful treatment with reslizumab targeted at the eosinophilic component of the disease. on reslizumab our patients asthma was for the first time controlled, pleuritis improved, fractional exhaled no (feno) normalized and her cachexia is improving. treatment with reslizumab should be considered in patients with igg -rd who manifest with eosinophilic respiratory disease. introduction/background: ikbkb deficiency (c. dupg in exon ) is a rare autosomal recessive form of severe combined immune deficiency (scid) originally described in canadian infants of northern cree descent. ikbkb scid is characterized by normal lymphocyte development, but impaired t-cell activation, along with innate immune defects. objectives: to report the clinical presentation, immunologic phenotype, and outcomes for patients with confirmed or suspected ikbkb scid due to this founder mutation. methods: we retrospectively reviewed hospital records dating back to of patients with confirmed homozygous ikbkb mutations, as well as patients suspected to be affected due to their clinical presentation and family relations to molecularly confirmed cases. results: fifteen patients were included. they presented early in life (average age months) with invasive and disseminated viral, bacterial, mycobacterial, and fungal infections. patients had concurrent and multiple infectious organisms, with a notable predilection for candida, gram negative organisms, and mycobacteria. four patients in our cohort received bcg vaccination at birth, resulting in fatal disseminated m. bovis infection in all, and additional patients succumbed to atypical mycobacterial infection following hematopoietic stem cell transplant (hsct). one newborn was identified in through new initiatives for targeted newborn screening for the mutation. immunologic features at presentation included normal to elevated lymphocyte counts with normal to elevated cd , cd , and cd t cells. when tested, response to pha varied from absent ( / ), to low ( / ), to normal ( / ). most patients had hypogammaglobulinemia, most often of the igg isotype ( / ). six had assessment of trec levels, and all had values above thresholds for screening programs. eight patients died before they could undergo hsct, and received transplants in the setting of ongoing severe, lifethreatening infections. only patient underwent hsct prior to the onset of infection. in our cohort, there are only long-term survivors. conclusions: ikbkb deficiency is a severe form of scid with early onset of invasive and disseminated multi-organism infection. the immunologic phenotype is characterized by normal to elevated lymphocyte numbers which do not meet pidtc criteria for scid, variable (and sometimes normal) mitogen response, normal trec levels, and low igg levels. the disease is universally fatal without hsct, however, conclusions regarding efficacy and long-term outcomes of hsct are uncertain given the small sample size. immune -dysregulation mimicking systemic lupus erythematosus in a patient with lysinuric protein intolerance introduction/background: lysinuric protein intolerance (lpi) is an inherited aminoaciduria caused by defective amino acid transport in epithelial cells of the intestine and kidney due to bi-allelic, pathogenic variants in slc a . the clinical phenotype of lpi includes failure to thrive and multi-system disease including hematologic, neurologic, pulmonary and renal manifestations. individual presentations are extremely variable, often leading to misdiagnosis or delayed diagnosis. here we describe a patient that presented with suspected immunodeficiency in the setting of early-onset systemic lupus erythematosus (sle), including renal involvement, who was subsequently diagnosed with lpi post-mortem. objectives: describe a clinical a patient with lysinuric protein intolerance that presented as early-onset systemic lupus erythematosus (sle), including renal involvement and primary immunodeficiency. methods: after informed consent was obtained, dna samples were obtained from the proband and his parents. trio whole exome sequencing was performed to identify a cause of early onset autoinflammation resembling systemic lupus erythematosus. results: the male proband had a history of failure to thrive starting at months of age, recurrent bacterial otitis media, and one episode of severe bacterial pneumonia requiring hospitalization. he presented at months of age with multifocal pneumonia, anemia (hgb . mg/dl) and mild thrombocytopenia. initial laboratory studies revealed low albumin ( . mg/dl), elevated ldh ( ), and mild hepatomegaly. renal and liver function testing was initially normal. immunologic evaluation for suspected primary immune deficiency showed normal immunoglobulin titers, low c ( ) and low c ( . ). lymphocyte phenotyping revealed low b cell counts ( . % of total lymphocytes) with t cells and nk cells within the normal range. despite antibiotic therapy, the patient worsened, developing fevers, a generalized erythematous rash, edema and nephrotic syndrome with oliguria. renal biopsy uncovered glomeruli with accentuated, global thickening and diffuse, peripheral capillary loops, as well as focal spiculated defects, there was endothelial swelling and other signs of acute damage including epithelial flattening, adluminal irregularity and extensive intraluminal proteinaceous detritus. endocapillary proliferative lesions, extracapillary crecents or tubular atrophy was not observed. immunofluorescence studies were positive for c , igg and c q granular deposits, mainly at the mesangium, interpreted as lupus nephropathy. endothelial swelling and massive, subepithelial electron-dense deposits with spike formation from the basement membrane were noted on electron microscopy, mimicking a stage ii membranous pattern of injury. autoantibodies included ana ( : ), anti-dsdna, smith, ssa and rnp were positive in agreement with the diagnosis of sle. immunosuppressive therapy with high dose iv corticosteroids and cyclophosphamide was initiated. despite this, the patient developed pancytopenia, elevated ferritin levels, increased triglycerides, and low fibrinogen. bone marrow biopsy displayed erythrocyte phagocytosis by macrophages, confirming a diagnosis of hemophagocytic lymphohistiocytosis (hlh). the patient subsequently died despite aggressive immunosuppression with high dose methylprednisolone and high dose iv immunoglobulin and dialysis. samples were collected from the deceased patient and his parents for research whole exome sequencing. trio analysis identified compound heterozygous missense variants in slc a . ammonia levels were not evaluated during the patients hospitalization. conclusions: lysinuric protein intolerance is a severe metabolic disorder that can present with protean systemic features including primary i m m u n o d e f i c i e n c y. i m p a i r e d l y m p h o c y t e f u n c t i o n , hypocomplementemia, immune-mediated glomerulonephritis, autoantibodies, and hlh are known complications of lpi. exactly how ineffective amino acid transport triggers these systemic inflammatory features is not yet understood. lpi should be considered in the differential diagnosis of early-onset sle, particularly in the absence of response to immunosuppressive therapy. director, national institute of immunohaematology introduction/background: chronic granulomatous disease (cgd) is a primary immunodeciency disorder with recurrent pyogenic infections and granulomatous inflammation resulting from loss of phagocyte superoxide production. mutations in any one of the five structural genes of the nicotinamide adenine dinucleotide phosphate (nadph) oxidase complex viz. cybb and cyba encoding for membrane bound gp phox and p phox; and ncf , ncf , and ncf encoding for cytosolic components p phox, p phox, and p phox respectively, have been found to cause cgd. the relative incidence of these gene defects varies significantly depending on the ethnic background of the population. identification of molecular defect is important for patient management as well as for prenatal diagnosis in the affected families. the present study was aimed at studying the pattern of underlying genetic defects in a cohort of indian patients affected with cgd. objectives .to identify the underlying genetic defect in patients with chronic granulomatous disease in india . clinical,immunological and molecular characterisation . to utilise this information for genetic counselling and prenatal diagnosis of the affected families methods: eighty-seven (n= ) patients with abnormal nbt and dhr were included in this study. in case of male patients, mothers were first screened for carrier status to rule out x-linked cgd (xl-cgd). those patients where mother is not showing mosaic pattern were suspected for autosomal recessive cgd (ar-cgd) and were screened for the ratio of ncf gene to pseudo ncf gene by genescan analysis. additionally, evaluation of nadph oxidase components expression by flow cytometry also helped us to determine the underlined genetic defect and it is validated by dna sequencing of respective genes. results: eighty-seven patients were molecularly characterized to identify disease causing mutation which includes: novel mutations in cybb, in cyba, in ncf gene in our cohort. . % (n= ) of the patients belonged to xl-cgd. . % (n= ) of the patients are suspected to have ncf gene defect among which; homozygous delgt mutation was identified in patients. . % and . % patients showed abnormal p phox and p phox expression suggesting defect in cyba gene and ncf gene respectively. spectrum of mutations involve: % of delgt mutations, % nonsense, % missense, % deletion, % insertion, % other than homozygous delgt mutations. male to female ratio is . : . consanguinity is noted in % of the patients. conclusions: despite the male predominance ar-cgd is more common ( %) as compare to xl-cgd ( %) in this cohort of indian patients, which is distinct from the western data. % are the novel mutations suggesting, a wide heterogeneity in the nature of mutations in indian cgd patients. flow cytometric evaluation of nadph oxidase component is used as a secondary screening test to identify cgd sub-group. molecular characterisation of cgd genes was not only used in the confirmation of diagnosis but also in genetic counselling and pre-natal diagnosis in affected families. novel nlrc gain-of-function mutation presenting with neonatal enterocolitis and autoinflammation, with positive clinical response to rapamycin and anakinra. senior investigator, viral immunology section, national institute of neurological disorders and stroke senior investigator, translational neuroradiology section, national institute of neurological disorders and stroke staff clinician, neuroimmunology clinic, national institute of neurological disorders and stroke staff clinician, laboratory of clinical immunology and microbiolology, niaid, nih introduction/background: cytotoxic t-lymphocyte antigen- (ctla- ) is an essential negative regulator of the immune response and its function is critical for immune homeostasis. uni-allelic mutation in the ctla gene leading to reduced function or expression of ctla- , termed ctla- haploinsufficiency, can lead to systemic immune dysregulation with wide spread clinical disease, but with variable clinical penetrance. the neurological manifestations of ctla- haploinsufficiency are not known. objectives: to perform detailed phenotyping of the neurological manifestations of ctla- haploinsufficiency. methods: a retrospective review and prospective collection of clinical, imaging, cerebral spinal fluid, and pathological specimens was performed in a cohort of genetically confirmed patients (n= ) with ctla mutations who are followed at the national institutes of health. neurological symptoms and exams were collected on patient visits and from historical records. the data collected included brain mris and spinal cord mris that were visually inspected for evidence of inflammation. cerebral spinal fluid values were obtained from patients including flow cytometry in patients. pathological tissue from brain biopsies of inflammatory lesions was examined from patients. results: central nervous system (cns) inflammation was found in / ( %) of the cohort. common clinical manifestations from the patients with cns inflammation were headaches / and seizure / . focal deficits were rare. mri findings included contrast enhancing neuroinflammatory lesions in the brain / , brainstem/cerebellum, and spinal cord / . figure a -c show representative inflammatory lesions. lesions were multifocal in / patients and / had recurrent inflammatory lesions on longitudinal follow up. lesions were, at times, extremely large, / with a lesion > cm^ . leptomeningeal enhancement (lme) was seen in / patients and clearly preceded intraparenchymal lesion development in patients. figure d shows a site of lme (green chevron) that develops into an intraparenchymal lesion (yellow chevron), mris are separated by days. spinal fluid analysis showed a lymphocytic pleocytosis (mean cells/mm^ ) with the presence of oligoclonal bands in patients. pathological features included a mixed cellular infiltrate, predominantly lymphocytes or plasma cells, with little evidence of demyelination or necrosis (figure e). conclusions: the neurological manifestations of ctla- haploinsufficiency include recurrent and, at times, severe neuroinflammation. however, even large lesions and lesions in eloquent anatomical locations had little to no focal clinical defects resulting in a striking clinical-radiological dissociation. future studies into the mechanisms of cns-related disease may reveal important information related to peripheral and central immune system functioning. conditioning with anti-cd immunotoxin in a mouse model of hypomorphic rag deficiency allows complete reconstitution of the immune system with lack of toxicity enrica calzoni, md , cristina corsino, technician , marita bosticardo, phd , yasuhiro yamazaki, md phd , hsin-hui yu, md phd , lisa ott de bruin, md , john manis, md , rahul palchaudhuri, phd , david scadden, md , luigi d. notarangelo, md treated mice and % in cd -sap/ treated mice. at sacrifice, in the bm we observed strong selective advantage for donor b cells at all developmental stages, but in particular in the most mature subsets, in both cd -sap and cd -sap/ treated mice, rescuing the block in development at pre-b cell stage found in untreated f l mice. donor engraftment in bm hsc reached levels around % and % in cd -sap and cd -sap/ treated mice, respectively. donor chimerism in t and b cells in the spleen was also higher than % in both cd -sap and cd -sap/ groups. in the thymus, full donor chimerism was achieved in both cd -sap and cd -sap/ treated mice, starting at the dn stage and persisting at dp, sp and sp . importantly, in both treatment groups, t cell development was corrected both in terms of subset distribution and absolute numbers to levels comparable to those of wt mice. finally, the thymic epithelial cell compartment was also fully reconstituted, with a normal number, distribution and maturation of both cortical and medullary thymic epithelial cells. conclusions: in conclusion, we show here that conditioning with cd -sap immunotoxin, alone or in combination with rads tbi, is safe and leads to full reconstitution of the immune system in rag hypomorphic mice, suggesting that this conditioning regimen should be considered for testing in clinical setting. introduction/background: foxp is a key transcription factor for the maintenance of immune tolerance. foxp mutations result in dysfunction of foxp + regulatory t cells (tregs) causing immune dysregulation, polyendocrinopathy, enteropathy, x-linked (ipex) syndrome, a severe early onset autoimmune disease, which can be fatal if not promptly diagnosed and treated. our recent international study analyzing the longterm outcome in i.e. patients of the two currently available treatments, pharmacological immune suppression and allogeneic hematopoietic stem cell (hsc) transplantation, showed poor long-term disease-free survival or overall survival limitations, respectively (barzaghi f. et al, jaci, ) . ipex syndrome is a good candidate for gene therapy as it has been demonstrated that reconstitution of wild-type treg cells can control the disease. however, foxp expression is highly regulated, and its safe and physiological expression in treg and teffector (teff) cells is challenging. lentiviral-mediated (lv) foxp gene transfer successfully converts ipex patients-derived cd + t cells into treg-like cells (cd lv-foxp t cells) with stable suppressive capacity (passerini l. et al, sci transl med, ) . these ex vivo converted tregs are ideal as a short term cell-based therapy for ipex patients, but this approach does not reestablish regulated foxp expression in teff cells, that also likely contribute to the ipex pathology. thus, we are further characterizing cd lv-foxp t cells and, at the same time, developing gene editing strategies for ipex, whereby autologous t cells or hscs are genetically modified or corrected, respectively, and reinfused into the patients. objectives: to provide more effective treatments for ipex patients, we are i) optimizing lv-foxp gene transfer in t cells to be suitable for clinical use, and ii) establishing a novel foxp gene editing in hscs and testing both approaches in preclinical models. methods: lv-foxp gene transfer can be obtained in cd + t cells activated polyclonally or in an antigen-specific manner. the vector construct is bidirectional, foxp expression is under the ef a-promoter and the truncated form of ngfr, used as marker gene, is under cmv promoter. foxp gene editing is performed using a combination of crispr/cas , a chemically modified sgrna targeting foxp , and an aav packaged homologous donor dna template and the efficacy and safety of the resulting construct is tested in different cell types in vitro and in humanized mice. results: we demonstrate that cd lv-foxp t cells can successfully be generated specific to different antigens. this result opens to new potential clinical benefit of cd lv-foxp t cells with more safe and specific regulatory effect than polyclonal cd lv-foxp t cells. we are currently adapting the protocol to optimal in vitro production for clinical use and assessing dose, survival and efficacy of the cd lv-foxp t cells using different in vivo models. due to the wide distribution of identified mutations throughout the foxp gene, we have designed a gene editing strategy that uses homology directed repair to insert the coding sequence of the foxp gene at the start codon of the endogenous mutated foxp gene. this strategy permits regulated expression of the inserted wild-type, functional foxp protein in patient cells independent of the location of the downstream mutation. using this site-specific gene knock-in, we find that the system effectively targets expression of foxp in different cell types, namely tregs, teff cells and primary human cord blood-or bone marrow-derived hscs. gene editing of normal donor and ipex tregs and teff cells allowed us to test for regulated gene expression and for establishment of normal treg suppressor function and t cell proliferation upon activation. additionally, preliminary results demonstrate that gene edited hscs can be transplanted into nsg mice for long-term reconstitution. conclusions: our results show the feasibility of different gene therapy approaches for ipex syndrome. in addition, they suggest that cd lv-foxp t cells, either polyclonal or antigen-specific, could be applied not only in ipex but also in immune mediated diseases of different origins. the results from the foxp gene editing support the use of crispr/ cas to treat ipex syndrome patients with autologous edited hscs. this gene editing approach may also be applied to treat other pediatric monogenic blood and immune disorders. human pi kgamma deficiency with humoral defects and lymphocytic infiltration of barrier tissues andrew takeda, bs , william comrie, phd , yu zhang, phd , paul tyler, bs , koneti rao, md , carrie l. lucas, phd graduate student, yale university clinician, niaid, nih assistant professor of immunobiology, yale university introduction/background: the phosphatidylinositol -kinase (pi k) signaling pathways play a key role in transducing signals from a diverse array of stimuli by producing the pip second messenger. class ib pi k is primarily activated by g protein-coupled receptors (gpcrs), and this class is comprised of the p gamma catalytic subunit in complex with the p or p regulatory subunit. in contrast to the class ia pi k subunits, inherited mutations in the genes encoding the class ib subunits have not been described. objectives: given the leukocyte-restricted expression pattern of p gamma, we hypothesized that mutations affecting this kinase may be found in cohorts of patients with rare immunodeficiency disorders. our objective was to identify such mutations and determine molecular, biochemical, and cellular derangements in patients with mutated p gamma. methods: we used whole-exome sequencing of families to identify inherited gene mutations and determined the mechanistic basis of disease using biochemical assays to assess effects on protein function and cellbased assays to define functional defects with disease relevance. results: we identified a patient (here called a. ) harboring compound heterozygous mutations in pik cg, the gene encoding p gamma, who presented in early life with autoimmune cytopenias and eczema and, at the age of years, developed cryptogenic organizing pneumonia and prominent t cell infiltration of the lungs. she also has a history of skin infections, lymphadenopathy/splenomegaly, eosinophilia, defective antibody production, and more recently, lymphocytic colitis. she inherited a frameshift pik cg mutation from her mother and a missense mutation resulting in an r p amino acid substitution from her father. expression of p gamma protein was lost, and stability of its p binding partner was reduced. despite defective t cell signaling responses to chemokines (i.e., gpcr stimulation), chemotaxis of patient t cell blasts in vitro was normal. intriguingly, the frequency of peripheral blood treg cells was low in patient a. , and her cd t cells more frequently expressed the tissue-homing cxcr chemokine receptor. consistently, serum levels of cxcr ligands were elevated in patient a. . moreover, we found augmented inflammatory cytokine production from m -polarized macrophages differentiated from patient a. monocytes or from thp cells treated with p gamma inhibitor or stably expressing pik cg shrna. conclusions: we report the first human with loss of pi kgamma activity and present her clinical presentation with notable t cell infiltration of barrier tissues. based on our analyses, we propose that loss of p gamma activity in humans causes t cell-intrinsic effects of reduced tregs and increased tissue-homing propensity and the t cell-extrinsic effect of augmented inflammatory responses in macrophages. together, these consequences of p gamma deficiency drive aberrant accumulation of t cells in lung and gut. introduction/background: pulmonary disease is a frequent complication across many primary immunodeficiencies (pidds), however its impact on the quality of life (qol) in pidds is not well characterized. objectives: to ascertain the types of infectious and non-infectious pulmonary complications occurring in pidds and to determine how these complications affect qol. methods: we analyzed the pulmonary complications, disability descriptions, and clinical status of subjects with pidds in the usidnet registry using descriptive statistics. karnofsky or lansky performance indices (n= ) and promis qol data (n= ) were also analyzed. the t-test/mann-whitney test and chi square test were utilized to compare continuous and categorical variables, respectively. results: infectious pulmonary disease was reported in a majority of subjects ( . %), most commonly pneumonia ( . %) and bronchitis ( . %). non-infectious pulmonary disease was reported in . % of all subjects, most commonly asthma/reactive airway disease ( . %), bronchiectasis ( . %) and interstitial lung disease ( . %). pulmonary insufficiency was listed as a cause of disability in . % of all subjects with pidds, with highest rates of this disability in subjects with immune dysregulation ( . %). lower karnofsky/lansky performance scores were observed in subjects with pneumonia, lung abscess, bronchiectasis, interstitial lung disease, and emphysema/copd as compared to without these disorders (p< . ). promis qol metrics were largely similar among subjects with and without pulmonary disease, although physical function scores were significantly worse in those with copd/emphysema (mean= . +/- . ) as compared to without (mean= . +/- . , p = . ). promis physical function scores were also worse in subjects with non-infectious pulmonary disease (mean = . +/- . ) compared to those with infectious pulmonary disease only (mean = . +/- . , p= . ). a significantly greater percentage of patients with a history of copd/emphysema ( . % vs. . %) or interstitial lung disease ( . % vs. . %) were deceased as compared to those without a history of these disorders (p< . ). conclusions: both infectious and non-infectious pulmonary disorders cause significant morbidity in pidds and are associated with higher mortality in this population. infectious and non-infectious pulmonary complications were often associated with worse karnofsky/lansky scores while there was limited impact on promis qol measures. latin-american consensus on the management of patients with severe combined immunodeficiency, part : supportive measures during the time from diagnosis to definitive treatment. juan carlos bustamante ogando , armando partida-gaytán , francisco espinosa rosales , lasid "consensus on scid" study group pediatric allergy and clinical immunology specialist, clinical immunologist and researcher at primary immunodeficiency research unit, national institute of pediatrics pediatric allergy and clinical immunology specialist, researcher at primary immunodeficiency research unit, national institute of pediatrics pediatric allergy and clinical immunology specialist, president, fundación mexicana para niñas y niños con inmunodeficiencias primarias (fumeni) result in a majority of patients with late diagnosis, more comorbidities and reduced access to curative treatments. the interventions during such period are vital to keeping optimum health status to improve the probability of success of curative therapies. many interventions are not supported by clinical trials, are based mainly on clinical experience, and there are no clinical guidelines to standardize such treatments. objectives: to generate a consensus on the supportive care of patients with scid, from the diagnosis until a curative treatment is given, under a latin-american perspective taking into account particular challenges for our region. methods: in a first step, we gathered available information about scid diagnostic and therapeutic guidelines from two sources: a) literature search and b) personal communications with pid experts from europe and usa. next, we developed an expert consensus through a modified delphi technique (electronic and anonymous). we used google® forms® to gather the information and microsoft office excel® for the analysis of agreement through kappa coefficient and rounds concordance through repeated measures analysis of variance (anova). results: we gathered an expert panel of subjects from latin-american countries (argentina, brazil, chile, costa rica, mexico, and peru) including the primary centers caring for scid patients. we generated a document with agreed diagnostic and therapeutic interventions grouped in topic-domains (i.e. protective and isolation methods to decrease the risk of infections, antimicrobial prophylaxis, immunoglobulin treatment, immunizations, nutritional aspects, antimicrobial treatment, blood derivatives use, routine laboratory workup, imaging and other studies, conventional multidisciplinary approach). we also included nonagreed interventions, but where relevant arguments are shared, to allow for particular clinical scenario decisions. conclusions: this is the first document of its type, and it intends to standardize clinical care of latin-american patients with scid, reduce disease burden and ultimately improve health outcomes. we see this effort as a starting point for the continuous improvement of our professional care to such patients and is intended to help as a tool not only for immunologists but for primary care physicians and other specialists involved in scid patient's care. this work will hopefully be published during as a lasid collaborative work, and it will help as a guide for clinicians caring for scid patients not only in latin america but in other world regions. also in the future, this consensus may be improved by collaboration from immunologists worldwide. no significant difference in hospitalizations one year before vs. year after treatment for prophylactic antibiotics (p= . ) or igrt (p= . ). baseline igg was higher in prophylactic antibiotics vs. igrt ( . vs. . mg/dl, p= . ) . sex, severity of sad, igg subclasses deficiency, and lymphocyte counts were not significantly different between treatment groups. conclusions: prophylactic antibiotics are not inferior to igrt in preventing infections in some sad patients. while, clearly some patients with sad will need igrt, our date indicate that larger prospective studies are needed to identify patients who will benefit most from igrt vs prophylactic antibiotics alone. richard and barbara schiffrin presidents distinguished professor of microbiology and director, institute for immunology, university of pennsylvania introduction/background: t cell thymic development is dependent on signals received via the pre-tcr complex and we here report the first case of pre-tcr alpha (ptcra) autosomal recessive t cell immunodeficiency in an infant with a positive scid newborn screen (nbs). objectives: we sought to uncover the mechanistic links between ptcra mutations and immune dysfunction. methods: the patient was tracked clinically, with serial clinical immunophenotyping and t cell function testing. in addition, we performed deep immunophenotyping with mass cytometry and single cell rna sequencing to delineate the molecular circuitry underlying her immune phenotype. results: the patient presented with t cell lymphopenia and impaired response to mitogen stimulation. hsct was considered, but she did not meet clinical criteria and remained healthy, so she was watched closely on prophylaxis while awaiting genetic testing. response to serial mitogen stimulation remained between~ - %, response to serial cd /cd activation was normal and tcrv spectratyping was normal. whole exome sequencing revealed two mutations in ptcra. no prior human cases of ptcra deficiency have been published, but a mouse model bears a striking resemblance to this case (fehling et al, nature, ) , with elevated t cells and decreased t cells. her mitogen stimulation responses became persistently normal around years of age with stable t cell lymphopenia, elevated t cells and normal switched memory b cells. anti-fungal prophylaxis was halted, and she remained on atovaquone alone with persistent t lymphopenia. she was able to mount an antibody response to rabies vaccine at . years and was weaned off scig replacement and is planned to initiate vaccination. deep immunoprofiling with mass cytometry (cytof) demonstrated a unique immunophenotype. single cell rna sequencing confirmed normal cd and cd tcr clonotypic diversity but increased clonotype diversity in t cells and increased and transcript levels. in addition, cd naïve, cd memory and cd naive t cells demonstrated both increased numbers of expressed genes and transcriptomic diversity, with altered cytoskeletal and tcr proximal signaling pathways across t cell subsets versus control. this may reflect a peripheral role for ptcra, a durable imprint of thymic signaling events mediated by ptcra, evidence of homeostatic proliferation or a combination of the above. conclusions: scid nbs led to identification of homozygous variants in ptcra causing a novel t cell immunodeficiency characterized by t cell lymphopenia, altered proximal tcr and cytoskeletal signaling and increased number of altered t cells. we will continue to pursue the mechanism of these mutations by developing ipsc and studying their t cell differentiation capacity in vitro, as well as further defining her immunometabolic phenotype. rag hypomorphic mouse mutants show partial preservation of thymocyte development but peculiar abnormalities of thymic epithelial cell phenotype introduction/background: the recombination-activating gene (rag) and rag proteins are essential for v(d)j recombination. in the absence of these proteins, the development of b and t cells is blocked at early progenitor stages, resulting in severe combined immunodeficiency (scid). hypomorphic mutations in rag , allowing residual activity, result in delayed-onset combined immunodeficiency with residual development of t and b lymphocytes, associated with autoimmunity and/or granulomas (cid-g/ai). objectives: to study in details the effect of rag hypomorphic mutations at the early stages of t cell development, we have generated mouse models carrying mutations described in patients with cid-g/ai (r q, r w, f l) (niaid animal protocol: lcim e). methods: we performed an extensive evaluation of the thymic phenotype in the mouse models. results: the number of total thymocytes was found to be drastically reduced in all three models. however, two of these mouse models (r q and f l) retained a significant level of rag activity, and resulted in the development of mature t cells in the thymus, while the mouse model carrying the r w mutation had minimal rag activity and presented a phenotype more similar to that of complete rag knockout mice. in r w mice, almost all thymocytes were blocked at the double negative (dn) stage and there were virtually no mature t cells, as found in rag ko mice. on the other hand, r q and f l mice presented double positive (dp) and single positive (sp) and sp cells. the cross talk between t cells and thymic epithelial cells (tec) in the thymus is fundamental for the development and maturation of both types of cells. in rag -/-mice, and consequently in the absence of mature t cells, tecs cannot complete their maturation, and the mtec subset is virtually absent. these results were also observed in the r w mouse model. instead, in r q and f l mice, the residual rag gene activity allows development of a reduced number of mature t cells. although the number of tecs was markedly reduced in r q and f l mice, ctecs and mtecs were both present, but with an excess of ctecs. furthermore, mtecs were predominantly mhc-iihigh (mtechi), and only a minority of mtecs were mhc-iilow (mteclo) cells, the opposite of what found in adult wt mice. finally, mtechi cells from rag mutant mice were found to express aire to levels and frequencies comparable to those of wild-type (wt) mice. conclusions: our results show that tec in mouse models carrying rag hypomorphic mutations are affected both in terms of absolute numbers and in terms of subset distribution and maturation state. to further investigate the functional consequences of impaired cross-talk between thymocytes and tecs in rag mutant mice, we have performed rnaseq in sp and tecs sorted from r q, f l and wt mice. analysis of the gene expression profile in tec may thus provide novel insights in the mechanisms that govern normal and pathologic thymic t cell development. vedolizumab for autoimmune enteropathy in primary immunodeficiency: a case series of outcomes introduction/background: gastrointestinal complications are common in patients with primary immunodeficiency. infections are the leading cause, but autoimmune enteropathies including inflammatory bowel disease (ibd)-like colitis, sprue-like enteropathy, and nodular lymphoid hyperplasia (nlh) have been recognized in a subset of these patients. to date, there is no established treatment for these noninfectious disorders. vedolizumab is a humanized monoclonal antibody that binds to the alpha- beta- integrin, inhibiting the migration of memory tlymphocytes across the endothelium into inflamed gastrointestinal parenchymal tissue. it is fda approved as first-line therapy for inflammatory bowel disease. the safety and efficacy of treating autoimmune enteropathy with vedolizumab in patients with concurrent primary immunodeficiency (pid) has not previously been reviewed. objectives: to review the outcomes of a series of patients with hypogammaglobulinemia and autoimmune enteropathy following vedolizumab therapy methods: patients ( male, female) at mount sinai with enteric biopsies demonstrating inflammatory enteropathy with t cell infiltrates have been treated with vedolizumab. results: five of the seven patients completed induction therapy. one patient was recently started on therapy. therapy was aborted in one patient who developed acute hepatitis during induction. another developed severe cytomegalovirus enteropathy, prompting discontinuation. two patients discontinued therapy due to response failure. at present, two patients remain on therapy at months with symptomatic improvement. conclusions: vedolizumab was effective in cases, but had no benefit or deleterious side effects in subjects. its effectiveness in another patient is presently under investigation. introduction/background: ataxia telangiectasia (at) is an immunodeficiency most often associated with t cell abnormalities and abnormalities in serum immunoglobulin levels, primarily iga. there is a subset of patients with a hyper-igm phenotype, some with cutaneous granulomas, which may reflect a distinct clinical phenotype. a yearold female presented for evaluation of concern for immunodeficiency because of frequent illnesses, presumed to be viral. she was found to have an ataxic gait, some speech delay, mild ocular and ear pinna telangiectasia, and an ulcerative rash on the left upper and right lower extremity. initial blood work showed elevated -fetoprotein levels ( ng/ml), elevated serum igm ( mg/dl), low igg (< mg/dl), and iga ( . mg/dl). objectives: to determine if the atm mutations in this patient are associated with perturbations in the frequencies, distributions and functions of b and t cell subsets which account for the observed phenotype. methods: next generation sequencing was used to identify the mutations in the atm gene. b and t cells were purified from the patients peripheral blood by positive selection. intracellular staining for foxp and t-bet was performed. b cells were activated in the presence of polyclonal f(ab) rabbit anti-human igm, multimeric, soluble, recombinant-human cd l, gardiquimod (tlr agonist), or cpg (tlr agonist). the treg suppression assay was carried out by co-culturing cd +cd hicd lo tregs and cd +cd cd + responder t cells at a : ratio in the presence of beads loaded with anti-cd , anti-cd , and anti-cd for . days. results: next generation sequencing revealed two pathogenic mutations in the atm gene, a novel mutation creating a premature stop codon [c. dela,(p.lys asnfs )], and a nonsense mutation [c. c>g, (p.tyr ter)]. proliferative responses of pbmc to mitogens (pha, cona, pwm) were reduced to roughly half of the control responses; the response to tetanus was normal whereas the response to c. albicans was absent. serum cytokine analyses demonstrated elevations in levels of tnf ( . pg/ml) and il- ( pg/ml); levels of ifn, il- , il- , il- , and il- were below the limits of detection. b cell abnormalities included markedly increased percentages of cd locd lo cells ( %) expressing t-bet and fas. activation of these cd /low b cells through the b cell receptor, tlr and tlr , and cd was decreased in response to all of the stimuli as evidenced by a lower percentage of b cells expressing the activation markers cd and cd relative to healthy control samples. the frequency of unswitched cd +igd+ memory b cells was also increased ( %). among the naive b cells, the proportion of cd + cd cd cd +igmhi transitional b cells that newly emigrated from the bone marrow (bm) was found to be diminished to . % of the naive b cell compartment. in the t cell compartment, there was a decreased frequency of total cd + cells but normal absolute numbers of cd +cd + t cells. there was also a decreased proportion of naive cd +cd +cd rocd l+ t cells and a striking increase in the cd +cd +cd ro+ memory t cells ( %). this appeared to be largely attributed to the increased proportion of cd +cd +cd ro+cd l effector memory t cells ( %). the circulating t follicular receptor (ctfh) cell frequency in the patient was -fold higher ( %) than the average for healthy donors but icos expression levels were normal. treg frequency was decreased but suppressive capacity was not impaired. conclusions: the mutations in atm described here add to the growing understanding of the heterogeneity in degree and complex nature of the immunodeficiency seen in patients with at. these mutations resulted in perturbations in frequencies and distributions of normal and atypical b and t cell subsets, which can explain some immunologic aspects of the clinical phenotype in this patient. the immunophenotype seen here may also differentiate at patients with granulomas from those without cutaneous lesions. supported in part by grifols, the joanne siegel memorial fund, the dreizessen fund (to ewg), grants from niams t ar - (km) and niaid r ai (em). introduction/background: a -month-old male presented with pancytopenia, b cell deficiency and developmental delay. he was born at weeks with weight of . kg. he was severely anemic with a hb of . , and transfused on day of life. he received hep b and bcg vaccines without complications. a month later he had a hb of . with a febrile illness. a bone marrow aspiration performed at days, showed dyserythropoiesis without hemophagocytosis, and normal numbers of precursors. t and b cells were decreased. further evaluation with repeat bone marrow showed decrease in all cell lineages. exome sequencing of the family showed homozygous variant in mysm (c. _ delp. (lys arg/s* ) omim: * ) in the patient. both parents and hla-matched sister, were heterozygous for the same variant in mysm . treatment consistent of replacement immunoglobulin, packed rbcs, and g-csf. there was no history of recurrent viral or severe bacterial infections except for - episodes of urinary tract infections, which were treated with antibiotics. physical exam revealed low set ears, sunken and wide set eyes, depressed nasal bridge, mild micrognathia, frontal bossing, and cm x cm cafeau-lait spot noted behind left knee. he was pancytopenic with a wbc count ranging /mcl to /mcl, and anc ranging from /mcl to /mcl (on intermittent g-csf). hb = . gm/dl requiring transfusions every - weeks, and platelet count was , /mcl. b cell deficiency was confirmed with total b cell count of cells/mcl. b-cell maturation was essentially normal. t cell counts were normal with ageappropriate distribution of naïve and memory t cells, and t cell function. there was normal t cell receptor repertoire diversity. objectives: to assess defect in dna repair using a flow cytometry-based assay in a patient with mysm deficiency. methods: patients with mysm deficiency are reported to have increased genomic instability. deb testing, and telomere length analysis revealed normal results. defects in the dna repair pathway were assessed using a flow cytometry-based assay measuring phosphorylation of atm (patm), smc (psmc ) and h ax (gh ax) without irradiation, or h or h after low-dose ( gy) radiation using a cs source. the analysis was performed in t, b and nk cells. results: the patient had higher patm and psmc in t cells compared to the experimental controls (hc) at h post-irradiation. also, the amount of gh ax in nk cells was significantly higher than hc at h post-irradiation. interestingly, the patients b cells showed approximately % of b cells with constitutive gh ax even without irradiation, and this subset increased slightly to % at h after irradiation. the mfi (amount) of gh ax also increased at this time-point. at h post-irradiation, there was normal dephosphorylation in healthy control lymphocyte subsets. however, the patients t cells did not de-phosphorylate completely and showed higher residual patm, and psmc in both t and b cells. also, both t and b cells, at h, demonstrated a small subset of t cells ( %) with constitutive gh ax without irradiation, which increased to % after irradiation. there was also an increase in gh ax mfi in the irradiated sample. in b cells, % showed constitutive gh ax without irradiation at h, and this increased to % after irradiation, with a corresponding increase in mfi. conclusions: in summary, this rapid flow analysis revealed defects in the dna repair pathway, including higher patm, psmc and h ax phosphorylation in t, b and nk cells at h post-irradiation. at h, only t cells showed a residual subset with patm expression. but, psmc , a downstream target of atm, revealed higher levels in t, b and nk cells at h post-irradiation. this assay, which allows lineage-specific analysis, permitted dissection of dna repair defects, in individual lymphocyte subsets revealing heterogeneity within the cell subset to radiation susceptibility. the practical benefit of this rapid multi-parameter flow assay is selection of appropriate conditioning regimen for hematopoietic transplantation, as was the case with this patient. this has significant practical implications for treatment of patients with radiosensitive immunodeficiencies. ctla- haploinsufficiency-associated inflammation can occur independently of t-cell hyperproliferation introduction/background: cd and ctla provide opposing proliferative signals to t cells. we identified an -year-old female subject (s ) with heterozygous deletions of cd and ctla and multi-organ inflammatory disease characterized by a lack of t cell infiltrates in affected organs. inflammatory disease was remarkably responsive to s ctla -ig therapy. objectives: our goal was to characterize the immunologic consequences of combined deletion of cd and ctla , specifically assessing t cell proliferation and treg function in comparison to patients with alps associated ctla haploinsufficiency. we further sought to explain how this s s inflammatory diseases could occur without a pathologic t cell infiltrate and why they were amenable to ctla -ig therapy. methods: we performed phenotypic analyses of subject t cells and innate lymphoid cells (ilcs). we functionally characterized subject t cells. we created serum cytokine profiles. we stained and analyzed tissue biopsies. results: cd and ctla expression on s t cells were half that of control t cells. s t cells were hypoproliferative. s tregs were scarce and lacked suppressive function similarly to alps tregs. s tregs could suppress autologous t responder cells, likely due to their poor proliferative capacity. s colonic biopsies featured significantly fewer infiltrating intraepithelial lymphoid cells than biopsies from an alps patients. unlike alps patients whose colonic gland infiltrates were overwhelmingly t cells, s intraepithelial lymphoid cells were neither t cells nor b cells, suggesting the presence of ilcs. indeed, a greatly expanded population of type innate lymphoid cells (ilc ) and prototypical ilc cytokines were identified in s peripheral blood. ilc frequency and cytokine levels decreased in response to treatment with ctla -ig, corresponding with marked improvement in enterocolitis, hepatitis and pericarditis. conclusions: we report a novel genetic syndrome of combined cd /ctla deletion and describe the immunolopathologic correlates of this disease. dual ctla -and cd -haploinsufficiency results in a phenotype of multi-organ inflammatory disease characterized by ilc expansion in the setting of t-cell hypoproliferation and quantitative and qualitative treg defects. our patients clinical response to ctla -ig parallels published mouse studies and suggests the existence of additional stimulatory b receptor(s) preferentially expressed on ilc s over conventional t-cell populations. diagnosis of radiosensitivity and dna repair defect in dna ligase iv deficiency with a rapid flow cytometry assay. introduction/background: dna ligase deficiency (lig -scid) is one of several monogenic defects affecting dna repair, and causing lymphopenia (t-b-nk+) and a radiosensitive scid (rs-scid) phenotype. the assignment of a timely diagnosis is vital in the management of patients with rs-scid. laboratory assessment of radiosensitivity is laborious, and utilizes fibroblasts (non-hematopoietic) or lymphoblastoid cell lines, and can take several weeks to months for results. objectives: we demonstrate for the first time, the application of a flow cytometric-based kinetic analysis of phosphorylated h ax (h ax) in lymphocyte subsets, especially nk cells, for the diagnostic assessment of lig -scid. methods: simultaneous measurement of multiple dna repair markers phosphorylated (p) atm, smc and h ax (h ax) was performed by flow cytometry to assess dna repair defects in a -year-old korean female. the patient was evaluated for recurrent fevers, chronic respiratory tract infections, chronic diarrhea, and rash. genetic testing revealed compound heterozygous variants (nm_ , c. g>t, p.trp cys and nm_ , c. a>t, p.asp val) in lig . functional assessment (phosphorylation) was measured in t and nk cells (b cells were absent), before irradiation (background control), or after low-dose ( gy) irradiation ( and hours). results: we observed maximal h ax generation at hour post-irradiation, with progressive dephosphorylation at hours post-irradiation in healthy controls. the patient showed normal frequencies (%) of t cells and nk cells positive for h ax ( . % and . % respectively); (controls (n= ) t cells = . % and %; nk cells = . % and . %), but increased intracellular levels (mean fluorescence intensity, mfi) of h ax (t cells = . and nk cells = . ) compared to controls (t cells = . and . ; nk cells = . and . ) at hour post-irradiation. however, more importantly, at hours post irradiation there was a lack of dephosphorylation in a substantial proportion of lymphocytes ( % of t cells and % of nk cells) compared to healthy controls (t cells= . % and . %; nk cells = . % and . %). further, while there was dephosphorylation of h ax at h in patient lymphocytes as compared to h, the amount, as measured by mfi, remained elevated at h (t cells = . , nk cells = . ) compared to controls (t cells = . and . ; nk cells = . and . ). the data from patm and psmc were uninformative for the evaluation of lig -scid. conclusions: flow-based kinetic analysis of h ax is a useful marker for the diagnosis of lig -scid, and can be performed with a small amount ( cc) of blood, and provides a result in - days, facilitating rapid assessment of radiosensitivity in this condition. human plcg haploinsufficiency results in nk cell immunodeficiency and herpesvirus susceptibility objectives: we aimed to investigate the cause of disease in three patients from two kindreds with recurrent or severe herpesvirus infections and nk cell dysfunction. methods: we used exome sequencing and mass cytometry (cytof), as well as traditional immunologic techniques, to investigate the genetic causes, immune cell subpopulations/signaling, and nk cell function of these patients. we additionally used mouse models, crispr cell lines and in vitro assays to assess the role of plcg haploinsufficiency in disease. results: kindred a consisted of two patients presenting with hsv susceptibility and autoimmunity. kindred b consisted of one patient with severe cmv myocarditis and adenoviral hepatitis. both kindreds were evaluated for nk cell function and showed reductions in target killing in spite of normal cytotoxic granule degranulation against the same target. microscopy analysis suggested that granule mobility was reduced in at least one kindred. cytof revealed reductions in plcg phosphorylation after receptor crosslinking in the nk cells of both kindreds. kindred a also presented with a reduction in naïve b cells without perturbations in immunoglobulin output, b cell memory formation or class switching. trio whole exome sequencing was performed and revealed rare heterozygous plcg mutations in both kindreds. functional analysis, as well as mouse and crispr models, support a functional haploinsufficiency as a cause for nkd in these patients. conclusions: heterozygous loss-of-function point mutations in plcg have not been previously investigated as a cause of nk cell deficiency or recurrent herpesvirus infection. thus, these patients represent a novel immunodeficiency involving plcg haploinsufficiency, nk cell dysfunction, and herpesvirus susceptibility. hypomorphic rag mutations alter the pre-immune repertoire at early stages of lymphoid development introduction/background: human rag deficiency is associated with a spectrum of clinical phenotypes. while the most severe forms of rag deficiency manifest with severe combined immune deficiency or omenn syndrome since the first weeks of life, more recently patients have been identified who present to medical attention at a much older age predominantly with symptoms of autoimmunity and/or inflammation. many of the mutations associated with this atypical syndrome are found in the c-terminal domain (ctd) of the rag gene and allow residual development of t and b cells. these patients have an abnormal peripheral t and b cell repertoire, but how this is affected by abnormalities in the composition of the pre-immune repertoire vs. antigen-mediated selection and homeostatic proliferation in the periphery is unknown. objectives: in order to investigate whether mouse models with hypomorphic mutations in the rag ctd recapitulate the phenotype observed in patients with cid-g/ai, and to study how these mutations affect repertoire composition, cell selection and survival during t and b cell development, we generated three mouse models carrying homozygous rag mutations (f l, r q, and r w), corresponding to human mutations (f l, r q, r w) previously reported in patients with late-onset combined immune deficiency with granuloma and/ or autoimmunity (cid-g/ai). methods: mice were generated using crispr/cas mediated gene editing. t and b cell development, including apoptosis was studied by flow cytometry. immunoglobulins in naïve mice, baff levels and specific antibody responses were measured by elisa. serum igm autoantibodies were measured using a microarray (utsw). analysis of t cell receptor (trb) repertoire in several sorted t cell populations and immunoglobulin heavy chain (igh) repertoire in pre-b cells was performed by adaptive biotechnologies. in order to be able to detect both dj and vdj rearrangements, pro-b cells and spleen b cells were sequenced using high-throughput genome-wide translocation sequencing-adapted repertoire sequencing (htgts-rep-seq). analysis of vk-jk rearrangements in pre-b cells was performed by pcr amplification. results: immunological characterization showed partial development of t and b lymphocytes, with persistence of naïve cells, preserved serum immunoglobulin, but impaired antibody responses and presence of autoantibodies, thereby recapitulating the phenotype seen in patients with cid-g/ai. by using high throughput sequencing, we identified marked skewing of igh v and trb v gene usage in early progenitors, with a bias for productive rearrangements after selection occurred, and increased apoptosis of b cell progenitors. this suggested that more alleles remained in germline configuration. moreover, in the rearranged igh loci, the distal v gene segments were preferentially rearranged already at the earliest stages of b cell development, a finding that has not been previously reported. in addition, rearrangement at the igh locus was impaired, and polyreactive igm antibodies were detected. conclusions: in conclusion, this study demonstrates that hypomorphic rag mutations reported in cid-g/ai cause abnormalities of the primary b and t cell repertoire. these changes may affect survival and selection of t and b cells, and thereby contribute to the immune dysregulation often seen in patients with cid-g/ai. senior clinician, nih/niaid/lcim introduction/background: autosomal dominant hyper ige syndrome (ad-hies) is a primary immunodeficiency due to loss of function stat mutations. disease manifestations include recurrent skin and pulmonary infections, eczema, mucocutaneous candidiasis, as well as tion and in vitro studies demonstrated that the enhanced signaling could be controlled by ruxolitinib, an approved jak / inhibitor. informed by these experimental data, the patients were treated with ruxolitinib with remarkable improvement in a variety of clinical end-points, including hematological profiles and growth parameters. conclusions: this characterization of a human jak gain-of-function mutation expands our current understanding of the role of jak in eosinophil biology, hematopoiesis and immune function. chronic granulomatous disease, ornithine transcarbamylase deficiency and x-inactivation is a primary immune deficiency characterized by defects in the nadph oxidase enzyme complex resulting in a susceptibility to a narrow spectrum of bacteria and fungi. mutations in cybb encoding gp phox and located at xp . are associated with the most common form of cgd. deletions and rearrangements in this region are associated with other genetic diseases such as mcleod syndrome (xk), retinitis pigmentosa (rpgr), duchenes muscular dystrophy (dmd), ornithine transcarbamylase deficiency (otc), and x-linked mental retardation (tspan ). patient phenotype depends on the extent and position of the deletion, creating a "contiguous x-chromosome gene deletion syndrome. objectives: we report a four-year-old female, who presented with symptoms of x-linked cgd and otc deficiency with a large, contiguous multi-gene deletion on the x-chromosome. methods: we report a four-year-old female, who presented with symptoms of x-linked cgd and otc deficiency with a large, contiguous multi-gene deletion on the x-chromosome. the patient is the second born of a set of non-identical triplets from a clomiphene assisted pregnancy at weeks gestation. (weight at birth: lbs oz). after a day stay in the nicu, and cpap for one day she was discharged home. by the second month, she began having problems gaining weight and had persistent vomiting. at months, she was admitted with a pneumonia diagnosed as methicillin resistant staphylococcus aureus by lung biopsy. during that hospitalization, the mother noted an enlarging lesion on the infants left hand which was biopsy proven serratia marscecens osteomyelitis and was treated with intravenous cefepime for weeks. at this point a dihydrorhodamine assay (dhr) showed only . % positive cells (nl - %) and she was diagnosed as a carrier of x-linked cgd. bactrim was initiated for antibacterial prophylaxis but it was discontinued due to recurrent diarrhea; she was unable to tolerate antifungal prophylaxis as well due to liver function abnormalities. she continued having frequent vomiting episodes, irritability, failure to thrive and development delay. ulcerations in esophagus were confirmed by egd and colonoscopy, probably related to persistent emesis. diarrhea was unresolved. at months of life she was admitted with acute encephalitis, a serum ammonia level of and elevated urine orotic acid. results: given the demonstrated carrier status for cybb and apparent otc deficiency, comparative genomic hybridization was performed, revealing a deletion from xp . to xp . this . mb loss includes genes that are known to cause disease. since the diagnosis, the patient has been on reduced protein diet, resolving her diarrhea, she has subsequently grown and is on th percentile for weight and height. her dhr is now % positive. at years she was diagnosed with cone rod dystrophy. currently she is on bactrim for cgd prophylaxis and l-citrulline for the otc deficiency. she continues to gain weight, and has shown great improvement in her development delay and no new cgd-related infections have recurred. conclusions: this case reminds us that x-linked carriers with large deletions may be symptomatic and genetic analysis to determine other affected genes can be important for medical management. introduction/background: wiskott-aldrich syndrome (was) is a rare and severe x-linked disorder with variable clinical phenotypes correlating with the type of mutations in the was gene. the long-term prognosis of this syndrome is generally poor, with hematopoietic stem cell transplantation (hsct) remaining the only curative choice. the syndrome is poorly characterized in china. objectives: we retrospectively reviewed patients with was referred to our hospital from to , and summarize their clinical manifestations and genetic features. methods: sixty-four children suspected to be was from unrelated families were enrolled in this study. the clinical data of children were reviewed in the present study. distribution of lymphocyte subsets from peripheral blood and was protein (wasp) expression in peripheral blood mononuclear cells was examined by ow cytometry (fcm). wasp mutations were identified by direct sequencing of pcramplified genomic dna results: among patients with primary immunodeficiency diseases (pid), ( . %) were finally diagnosed as was with gene identified. the mean time of diagnosis was . months (range, . - . ). the common onset clinical manifestation was diarrhea, and most patients had recurrent upper respiratory tract infection, otitis media, pneumonia, and skin abscess. one patient had nephrotic syndrome and no patient with malignancy. all patients had classical was phenotype with was clinical scores - . total mutations in wasp were identified, including novel mutations. six patients received hsct, five survived, with one died because of gvhd. compared with the other patients without wasp mutations, was patients had lower numbers of cd + t cells and b cells, and higher eos and ige level. there was a negative association between the number of b cells and the was clinical scores. conclusions: in china, diagnosis of was has improved over the last decade, although a much higher number of cases had been expected. establishing more diagnostic centers dedicated to the care of pid will facilitate early, correct diagnosis and better care of was in china. regulatory cells (tregs) are precisely quantified by measuring demethylation of the treg-specific-region of foxp . more recently, we have identified highly cell type-specific dna regions of demethylation for further cell populations. objectives: this novel technology allows the implementation of differential immune phenotyping into the newborn screening procedure. here, we aimed at epigenetically quantify multiple immune cell types in different biological samples including dried blood spots and samples from patients with pid with immundysregulation, where currently no approach is available. methods: using cell type-specific demethylation sites, we developed epigenetic qpcrs for quantification of t-, treg, b-, nk-, monocyte and granulocyte cell population. epigenetic qpcr is applicable for relative and absolute quantification in whole blood and dried blood spots using isolated, bisulfite converted dna. results: we demonstrated > % concordance with flow cytometric analyses of the same fresh blood samples from healthy subjects. we have validated the method in children with symptoms of immundysregulation resulting from monogenic defects, including for example foxp , cd , stat , ctla , and leading to treg/teffector cell imbalance where treg deficiency has been difficult to assess by flow cytometry. furthermore, we tested dried blood spot (guthrie card) samples from healthy newborns and patients with diverse pids and correctly identified / pid patients, indicating that this method holds promise for newborn screening. conclusions: the method we established for immune cell quantification based on epigenetic cell type-specific markers, is feasible and reliable in biological samples either fresh, frozen or archived, including dried blood spot. the analysis of further immune cell types introduces an innovative opportunity to diagnose a variety of pids and immunodysregulatory disorders as early as newborn screening. pancytopenia and immunodeficiency with mds in an infant due to samd l mutation we present our data on behalf of the pcid study consortium of the inborn errors working party worth profound combined immunodeficiencies (p-cid) are inherited diseases with impaired t-cell function leading to infections, immune dysregulation or malignancies. genetic, immunologic and clinical heterogeneity make patient specific decisions on indication and timing of hematopoietic stem cell transplantation (hsct) difficult. objectives: since the pcid study recruits non-transplanted p-cid patients aged - years to prospectively compare natural histories of age and severity-matched patients with or without subsequent transplantation and to determine whether immunological and/or clinical parameters may be predictive for outcome methods: our prospective/retrospective international observational multicenter study recruits pediatric p-cid patients to identify biomarkers and clinical parameters that are predictive of outcome. results: so far > growth hormone deficiency comprised the majority of neuroendocrine cases ( %, / ). other notable neurologic diagnoses included headache ( %, / ), seizure ( %, / ), cerebrovascular accident ( %, / ), and neurologic tumors ( . %, / ). in addition to somatic neurologic conditions, many cvid patients ( . %, / ) had reported diagnoses of depression, anxiety, and post-traumatic stress disorder. conclusions: our findings suggest that neurologic diagnoses are more common in cvid patients than previously recognized. patients with neurologic autoimmune disease appear to have a more severe phenotype with earlier age at symptom onset. many cvid patients had depression, anxiety clinical outcomes of human herpesvirus reactivation after hematopoietic stem cell transplantation prognostic factors and outcome of epsteinbarr virus dnaemia in high-risk recipients of allogeneic stem cell transplantation treated with preemptive rituximab cytomegalovirus in hematopoietic stem cell transplant recipients daratumumab controls life-threatening post-hsct autoimmune haemolytic anaemia objectives: we present a month old pancytopenic male who was diagnosed with myelodysplastic syndrome (mds) with monosomy due to samd l mutation. methods: whole exome sequencing was performed on a male, who presented at months of age with findings concerning for a bone marrow failure (bmf) syndrome despite a normal bmf genetic panel. results: the patient presented at months of age, with severe pancytopenia, fevers, e. coli bacteremia, pancolitis, and echtyma gangrenosum. bone marrow showed severe aplasia with occasional macrophages.he was treated with antibiotics, as well as steroids, etoposide and cyclosporine for presumed hemophagocytic lymphohistiocytosis (hlh). a bone marrow failure and hlh genetic panels were normal conclusions: this is one of the first reported cases of samd l mutations causing mds since its initial discovery earlier this year. samd l mutation should be considered in patients who present with pancytopenia and monosomy mds. as new defects continue to be identified, further evaluation outside of typical bmf panels may be relevant primary immune deficiency disease in patients over age : an analysis from a proprietary immunology patient registry roger ucla school of medicine director objectives: to characterize the prevalence of pidd among older individuals using a patient database maintained by the consortium of independent immunology clinics (ciic), comprised of specialty immunology outpatient practices in the us. methods: patients with pidd were identified in the ciic database using icd- codes d conclusions: our data suggest that pidd in patients over age may be more prevalent than previously reported introduction/background: the patient is a -month-old boy, born at + weeks gestation to non-consanguineous parents of italian origin. he was admitted to the intensive care unit at days of age with profuse bloody diarrhoea, weight loss, severe metabolic acidosis and acute renal failure. he had a rapid respiratory deterioration necessitating intubation, ventilation and inotropic support. the patient developed features of macrophage activation syndrome with: (i) prolonged fever > . °c, (ii) hepatosplenomegaly, (iii) bicytopenia (anaemia and thrombocytopaenia), (iv) hypertriglyceridemia, (v) high ferritin ( , ) and (vi) haemophagocytosis on bone marrow smear. he also presented with three interesting features (i) a macular erythematous rash that slowly resolved and was replaced by reticulo-livedoid rash, (ii) a marked hypereosinophilia and (iii) no significant elevation of hladr/cd + t cells on lymphocyte immunophenotyping ( - %). the patient underwent rectal biopsy which confirmed the presence of eosinophils, but without significant inflammation or architectural changes. stool microscopy showed presence of partially necrotic intestinal epithelial cells. immune work up demonstrated global t lymphopaenia without balanced subpopulation and eliminated a familial hemophagocytic lymphohistiocytosis (normal perforin and cd a expression on cd + t-cell and nk cells). circulating foxp + cd +cd lowcd + t cells were within normal range. a dihydrorhodamine reduction assay was normal. chief, laboratory of clinical immunology and microbiology, national institute of allergy and infectious diseases, national institutes of health introduction/background: hematopoietic stem cell transplantation (hsct) has been used for the treatment of hematologic malignancies and primary immunodeficiencies (pid), for several decades with increasing efficacy. however, toxicity related to conditioning regimens based on the use of chemotherapy and/or irradiation to ensure engraftment of donor cells, remains a significant problem. recently, an alternative, potentially low-toxicity, approach has been proposed, which makes use of an immunotoxin targeting cd -expressing cells, which include hsc and more mature leukocytes. this approach is particularly attractive for leaky forms of severe combined immune deficiency (scid), with residual production of dysfunctional t and/or b cells, such as atypical forms of rag deficiency. objectives: we have developed a mouse model carrying a hypomorphic mutation in the rag gene (p.f l) resulting in a combined immunodeficiency with signs of autoimmunity, recapitulating the phenotype seen in patients. methods: using this model, we have tested the efficacy of conditioning with an anti-cd immunotoxin (cd -sap) alone or in combination with low irradiation ( rads; cd -sap/ ), and compared these regimens to a myeloablative dose of irradiation ( rads) [niaid protocol lcim e]. following conditioning, the mice were transplanted with wild-type (wt) bone marrow (bm) lineage-negative cells and followed over time to evaluate immune reconstitution. results: conditioning with cd -sap alone or with cd -sap/ led to a consistent engraftment of donor t and b cells in the peripheral blood (pb) of f l that increased overtime, reaching % donor chimerism at weeks. myeloid (cd b+) and nk cells in pb of f l mice also showed high level of donor engraftment that remained stable at around % in cd -sap around hours of life and the second after one week. there is no data on the utility of one versus two screens for scid screening. here we present our data evaluating whether patients with scid or t-cell lymphopenia were identified on the first or second trec screen. objectives . determine the benefit of a second trec screen in identifying scid and t-cell lymphopenia at birth. . examine outcomes of trec screening in washington state since implementation. methods: results of scid newborn screening performed in washington state between january and june were reviewed retrospectively with the staff of the washington department of health laboratory where the trec assay is performed. trec thresholds (copies/μl) were defined as follows: absent ( ), low ( - ), borderline ( - ) and normal (> ). all trec assays were run with a beta-actin control to assure sample adequacy. a screen is considered abnormal if there is one low/ absent trec or two borderline trec. newborns with abnormal trec screening have follow-up diagnostic testing consisting of lymphocyte flow cytometry to evaluate numbers of naïve and mature t cells, b cells, and nk cells, performed at seattle childrens hospital. results: a total of positive trec screens were found in washington state between january and june . five patients who did not have diagnostic flow cytometry testing were excluded from the analysis (one protocol deviation, one lost to follow-up and three who died before testing could be performed). the first screen was abnormal in forty three patients, while the second screen was abnormal in patients. five patients had an abnormal third or fourth trec drawn for other newborn screen follow-up. three patients with scid were identified, all with abnormal values on first screen. fortyfive patients with t-cell lymphopenia were identified; from the first screen and from the second screen. there was one patient with mhc ii deficiency was missed by both first and second screens because she did not have t-cell lymphopenia. the false positive rate with the first screen was % versus % with subsequent screens. the false positive rate dropped to % with two abnormal trec. the positive predictive value of scid or t-cell lymphopenia with the first abnormal trec was % versus % with two abnormal trec. average age of collection among infants with a positive screen was . hours for the st nbs and . days for the nd nbs. live viral vaccines were postponed in three patients who had an abnormal secondary screen (one with idiopathic t-cell lymphopenia, one with ectrodactyly-ectodermal dysplasia-clefting (eec) syndrome and one with q . deletion). one of these was started on pjp prophylaxis. conclusions: the practice of obtaining a second nbs from all newborns in washington state has led to increased identification of patients with tcell lymphopenia but did not result in identification of additional patients with scid. the false positive rate of the first and subsequent newborn screens was similar and decreased in patients with two abnormal trec. interventions including delaying live viral vaccines and pjp prophylaxis were instituted in patients who had a normal initial trec but abnormal secondary screen and documented t cell lymphopenia. two trec screens in all newborns can result in identification of additional patients with t-cell lymphopenia who may require intervention and additional follow-up. it is not yet clear whether the cost of a second mandatory scid newborn screen is balanced by the additional sensitivity gained by this approach. introduction/background: mannose-binding lectin (mbl) is a multimeric lectin that recognizes a wide array of pathogens independently of specific antibody, initiates the lectin pathway of the complement system, and acts as a proinflammatory mediator. mbl deficiency is reported to increase the frequency of infections in patients with impaired immune systems or cystic fibrosis (cf) patients. mbl replacement is experimental and unavailable. immunoglobulin replacement therapy (igrt) is controversial in the treatment of mbl deficiency; however, there are no reports of its efficacy or role in this condition. we describe a cf carrier patient with mbl deficiency, ciliary dyskinesia and mildly low igg who did not respond to igrt. objectives . understand when mbl deficiency can be symptomatic. . define the relationship between mbl deficiency and cf. . describe the treatment options of mbl deficiency. . define the efficacy of igrt and mbl deficiency. methods: a single case report. results: year old girl with mbl deficiency, cf carrier state with polymorphisms ( - a>g cf variant with t/ t and m v polymorphism) and ciliary dyskinesia (diagnosed by ciliary biopsy) who initially presented at eight years of age with recurrent sinusitis, recurrent otitis media status post tympanostomy tube placement, reactive airway disease and tracheomalacia. she had nine episodes of recurrent sinusitis with six negative sinus cultures and one positive for pseudomonas aeruginosa. she required a total of nine courses of antibiotics. labs showed several mbl levels < ng/ml on three occasions, normal ch , ah , igg - mg/dl (low normal for age), normal iga, igm, t cells, b cells, nk cells, and robust specific antibody titers. despite adequate pulmonary hygiene including nebulized levalbuterol, budesonide, dornase alfa, ipratropium, hypertonic saline and compression vest twice daily, she continued to have recurrent bronchitis and cough. in addition, even with sinus rinses and intranasal corticosteroid, she continued to have - episodes of sinusitis yearly. for this reason, she underwent bilateral total ethmoidectomies and maxillary antrostomies with modest reduction in frequency of sinus infections and symptoms. however after two years, her bacterial sinusitis recurred. four episodes were positive for methicillin staphylococcus aureus or pseudomonas aeruginosa. this did not improve significantly with a trial of intranasal mupirocin. due to increased frequency of bacterial sinusitis refractory to traditional therapy, she was started on subcutaneous igrt dosed approximately mg/kg/month dosing maintaining igg troughs around mg/dl. after a six month trial, she did not have improvement in the frequency of sinusitis, bronchitis and otitis media. she required - courses of antibiotics for bacterial upper respiratory tract infections and igrt was stopped. prophylactic antibiotics and repeat sinus surgery were instituted. conclusions: majority of the patients with low/deficient mbl levels do not manifest significant symptomology due to the redundancy of the innate immunity. the increased susceptibility to infections is thought to be due to additional factors that compromise other components of the immune system. specifically in cf patients, mbl deficiency is associated with earlier colonization with pseudomonas, more rapid decline in lung function and earlier death secondary to end-stage lung disease. there are no reports of combined mbl and cf carrier symptomatic patients similar to this patient. there are no validated age-corrected values for mbl levels in pediatric patients. the clinical relevance of these levels to infection frequency, severity, or treatment of mbl deficiency remains to be proven. it has been proposed that < ng/ml is considered deficient in children. mbl therapy is still experimental and not commercially available. management when provided for severe or frequent infections includes prompt treatment with antibiotics, prophylactic antibiotics, appropriate vaccinations, and a trial of igrt. there are no reported cases describing the efficacy of igrt in mbl deficiency, and the mechanism subsequent genetic analysis identified the presence of a de-novo heterozygous mutation in the nucleotide binding domain of nlrc (c. g>c, p.val leu). a mutation involving this amino-acid position has already been described but with a different substitution pattern in a boy and his father who presented with mas (p.val ala) ( ) . in order to prove the causality of this mutation, our team generated thp- cell lines expressing the two different mutations through gene-editing with the crispr system. in this system the mutation p.val leu, as well as the p.val ala mutation, were responsible for spontaneous activation of caspase , as evidenced by flica assay. the patient was treated with iv methylprednisone mg/kg/day. he continued to have progression of his inflammatory state, and was therefore commenced on anakinra. following confirmation of mutation, he was started on rapamycin, reasoning that (i) through autophagy induction, rapamycin could potentiate the action of anakinra ( , ) and (ii) through mtor inhibition counteract the effect of il- on t-cells ( ). with a combinatory therapy of anakinra up to mg/kg/day and ramapycin (with trough levels of - ng/l), the patient showed a marked clinical improvement, allowing weaning of steroids and establishment of enteral feeds. ferritin levels reduced to - ng/ml. we observed a significant decrease in il- plasmatic level following treatment initiation (pre-vs post-treatment levels of pg/ml and pg/ml, respectively). we report a novel nlrc gain-of-function mutation, presenting with neonatal enterocolitis and autoinflammation with improvement under combinatory therapy of anakinra and rapamycin. to our knowledge this is the first case to report the use of rapamycin in this disease, with what appears to be encouraging results. further studies are required to elucidate the potential role of rapamycin in the management other inflammasome disorders. introduction/background: allogeneic hematopoietic stem cell transplantation (hct) is currently standard treatment for patients with severe combined immunodeficiency (scid), with -year overall survival > % for typical scid (heimall blood ). previous studies revealed that poor clinical outcomes correlated with poor long-term t cell reconstitution, including low cd t cell counts and low naïve cd + t cell counts (pai nejm ) . we hypothesized that t cells developing in a poorly reconstituted immunologic environment would show features of chronically activated t cells with increased expression of inhibitory co-receptors. we further hypothesized that the intensity of the conditioning regimen would correlate with the expression of inhibitory co-receptors. objectives: to characterize the t cell phenotype of scid patients at > years post-hct and to investigate the impact of conditioning regimen on the quality of t cell reconstitution and the expression of inhibitory coreceptors methods: we analyzed scid patients - yrs (median yrs) after hct. we excluded from the analysis patients with chronic graft-versushost disease (gvhd), chronic dna viral infections or patients who had received donor lymphocyte infusion or boost in the months prior to study. scid genotypes (n) included il rg/jak ( ), rag /rag / dclre c ( ), ada ( ), il r ( ) and other/unidentified ( ). nine patients had received a reduced intensity (ric) or myeloablative (mac) conditioning regimen, while had received either no conditioning or immunosuppression only (none/is). poor t cell reconstitution was defined as cd t cell counts below cells/mm ( patients). t cell phenotype, including expression of inhibitory co-receptors, was assessed by flow cytometry. results: compared to patients with cd counts above cells/mm , patients with low cd counts had low naïve cd ra+ccr + t cells (p= . ) and high cd ra-ccr -t effector memory (tem) cells (p= . ), low numbers of naïve thymic cd ra+ cd + t cells, low numbers of trecs and a less diverse t cell repertoire (p< . ). additionally, they had an increased frequency of cd t cells expressing pd ( % vs %), ctla ( % vs . %), cd ( % vs %, p= . ), and b ( % vs %, p= . ) inhibitory co-receptors. increased inhibitory receptor expression was associated with a differentiation profile skewed toward a tem phenotype, reduced t cell diversity, increased markers of t cell activation, and the development of a highly exhausted cd high pd- high t cell population. more importantly, a fraction of ccr + cd ra+ cd t cells expressed pd ( % vs %, p= . ) and b ( % vs %, p= . ) in patients with low cd counts, suggesting that some naïve t cells of poorly reconstituted patients were chronically activated. inhibitory receptor expression did not increase with hla disparities between the donor and recipient, a history of gvhd after transplant or the infection status of the patient prior to transplant. however, inhibitory co-receptor expression was correlated with conditioning regimen, with increased frequency of b + cd t cells in unconditioned patients ( % vs % in none/is and ric/mac patients respectively, p= . ). conversely, ric/mac conditioning was associated with higher naïve cd t cell numbers (p= . ), higher naïve thymic cd ra+ cd + t cell numbers (p= . ), a more diverse t cell repertoire (p= . ) and low expression of inhibitory co-receptors. ric/mac conditioning was also associated with improved naïve t cell generation and limited expression of inhibitory receptors in il rg/jak patients ( % vs % for b , % vs % for cd in ric/mac versus none/is il rg/ jak patients respectively), a genotype permissive to t cell engraftment. conclusions: collectively, our results suggest that the expression of inhibitory co-receptors may be a biomarker of poor t cell reconstitution in transplanted scid patients. further, we propose that lack of conditioning limits t cell reconstitution, which correlates with increased expression of inhibitory receptors on circulating cd t cells. antibodies targeting inhibitory receptors are now available in clinical trials to treat cancer and viral infections. it will be necessary to evaluate the relationship between inhibitory receptor expression, t cell function and clinical outcome, to see if a selected group of scid patients could benefit from these immunotherapies. wallace chair, chief of allergy immunology, children's hospital of philadelphia introduction/background: prophylactic antibiotics (abx) and immunoglobulin replacement (igrt) are commonly used to treat specific antibody deficiency (sad), but the optimal therapy is not established. objectives: to compared outcomes (number of infections and hospitalizations) in sad treated with igrt vs. prophylactic antibiotics. methods: two-center, retrospective chart review of sad patients from jan -may . we excluded patients with hypogammaglobinemia and/or other immunodeficiency diagnosis. characteristics and treatment were reported, rates of infections/hospitalizations among treatment groups were compared using linear regression model. results: sad patients included. mean age was years, % were females. ( . %) received prophylactic antibiotics, ( . %) received igrt, ( . %) did not receive any specific treatment. number of infections decreased from . (year before treatment) to . (year after treatment) in prophylactic antibiotics group (p= . ), and from . to . in igrt group (p< . ). various musculoskeletal and vascular abnormalities. little is known of gynecologic-obstetric complications, but with improved therapies women are living longer making reproductive health more pertinent. objectives: to learn more about obstetric and gynecological health in women with stat loss of function. methods: we prospectively interviewed and retrospectively reviewed medical records of adult women with ad-hies evaluated at the nih between - . results: of patients aged - years (mean years), women were interviewed, and chart reviews were performed on . age of menarche in our cohort was consistent with the national average ( . vs. . years). five of patients ( . %) reported having worsening lung symptoms with menstruation, and of participants ( %) reported worsening eczema during menstruation. with regard to routine health maintenance, of women reported having regular cervical cytology testing; ( %) reported an abnormal result. of these , had hpv that responded to treatment or was hpv only not requiring treatment; had reactive changes due to yeast and ascus that was subsequently normal. mastitis and breast abscesses occurred in women. eleven women reported vulvar cysts/abscesses requiring drainage. nine women had progestin-releasing iuds placed, in some to suppress menses-associated vulvar eczema/abscess flares; no infectious complications were reported from progestin iud use. over % of women chose not to conceive given underlying disease. of women with pregnancies, women had live births, of ( %) had miscarriages, and of ( %) experienced recurrent pregnancy loss. three women whose pulmonary symptoms worsened during pregnancy were diagnosed with progression of parenchymal lung disease post-partum. one woman experienced worsening of skin manifestations. other reported postpartum complications included one wound infection (after caesarean) and one hemorrhage leading to hysterectomy. conclusions: as women with ad-hies are living longer, significant infectious and disease-related exacerbations related to both menstruation and pregnancy were observed in this patient population. it is important to focus on maintenance of their gynecologic and obstetric health and carefully monitor for these morbidities. finally, while these women may choose to attempt pregnancy, the risk of recurrent pregnancy loss and worsening disease warrants discussion. rna sequencing identifies aichi virus as the cause of chronic infection with lymphoproliferation in a patient with x-linked agammaglobulinemia objectives: we here present an xla patient with a complicated course in whom we detected aichi virus (aiv ). methods: case report: the patient was diagnosed with xla at age years, based on agammaglobulinemia with absent b cells and a known pathogenic mutation in btk (c c>t, p.r c). he had been suffering from recurrent respiratory and gastrointestinal infections since the age of months. he was started on immunoglobulin (ig) substitution, which resulted in complete control of infections with igg trough levels at g/l. however, at years of age he developed unexplained fever, refractory temporal epilepsy, hepatitis, progressive nephromegaly with chronic renal failure, splenomegaly, episodic diarrhea and growth failure. ultrasound identified multiple focal lesions in the liver, spleen and kidney. a liver biopsy showed severe chronic hepatitis with initial perisinusoidal fibrosis. serial kidney biopsies showed variable oligoclonal cytotoxic t cell infiltrates, suggestive of chronic viral infection. standard diagnostics failed to reveal a pathogen in blood or stool samples and on biopsies. results: we finally resorted to rna sequencing on a kidney biopsy sample. this technique identified aiv , a kobuvirus of the family picornaviridae, with a high read number. subsequently pcr confirmed the presence of aiv in both liver and spleen. results for cerebrospinal fluid, blood and feces are pending. conclusions: aiv is a picornavirus responsible for self-limiting gastroenteritis in humans. confirmatory pcrs on blood, csf and stool samples are ongoing. however, given the unequivocal result of the rna sequencing and confirmatory pcrs in other affected organs, we believe that the complications in this xla patient can be explained by aiv chronic infection. these findings confirm the potential of next generation sequencing techniques to identify infectious agents in patients with primary immunodeficiency. characterization and successful treatment of a novel autosomal dominant immune dysregulatory syndrome caused by a jak gain-offunction mutation. introduction/background: janus kinase (jak ) plays an essential, nonredundant role in the jak/stat signaling cascade, a key pathway in the control of hematopoiesis and immune function. significant progress has been made in elucidating the role of jak , but gaps in our knowledge still persist. to date, somatic gain-of-function mutations in jak have been linked to t-cell acute lymphoblastic leukemia. objectives: to understand and treat human jak gain-of-function mutations. methods: research study protocols were approved by our institutional review board. four members of the family (the affected children and their parents) were enrolled. written informed consent for genetic testing and participation was provided by the parents for their children. genetic, bioinformatic, biochemical and immunological investigations were performed. results: we describe the first known patients carrying a germ-line gainof-function mutation in jak . the clinical phenotype includes severe atopic dermatitis, markedly elevated peripheral blood eosinophil counts with eosinophilic infiltration of the liver and gastrointestinal tract, hepatosplenomegaly, autoimmunity, and failure to thrive. functional analysis established the gain of function phenotype caused by the muta- introduction/background: herpesviridae infection after hsct for hematologic malignancies, specifically cytomegalovirus (cmv), epstein-barr virus (ebv) and human herpes virus- (hhv- ), have been associated with various outcomes including post transplant lymphoproliferative disorder (ptld), graft-versus-host disease (gvhd) and mortality ( ) ( ) ( ) . patients with primary immunodeficiency may have different incidence and outcomes with respect to herpesviridae given their differences in age at transplant, conditioning choices and underlying disease susceptibility to this viral family. objectives: the objective of this study was to describe the incidence and outcomes of cmv, ebv and hhv- post hsct for the primary immunodeficiency population. methods: a single center retrospective chart review of primary immunodeficiency registry patients (research ethics board protocol no. ) who received hsct from january december was undertaken. patients who received gene therapy were excluded. antiviral prophylaxis was given according to institutional protocol. demographic and clinical data were collated and analyzed with microsoft excel . the primary outcome was incidence and time to dnaemia for cmv, ebv and hhv- . results: sixty one patients who underwent hsct for primary immunodeficiency from january -december were reviewed. diagnoses are noted in table . the average age of transplant was . months (range . - . ). transplant recipients received busulfan and cyclophosphamide conditioning ( with anti-thymocyte globulin (atg), with alemtuzumab added), transplants received busulfan, fludarabine and either atg or alemtuzumab, and received atg alone. six transplants were unconditioned. . % ( / ) of patients developed gvhd requiring systemic immune suppression. the overall incidence of cmv, ebv and hhv- post hsct for primary immunodeficiency was . % ( / ), . % ( / ) and . % ( / ) respectively. in those with severe or profound combined immune deficiency the incidence of cmv was . % ( / ), ebv . % ( / ), and hhv- . % ( / ). in the patients with chronic granulomatous disease, cmv incidence was % ( / ), and ebv and hhv- were both % ( / ). in recipients with pre-transplant negative pcr for ebv, cmv and hhv- (r-), time to dnaemia post transplant is seen in figure . ptld was seen in patients with rag and il r, both of whom were d+r-for ebv status with ebv dnaemia, and one of whom died attributed to ptld. two year mortality for all patients was % ( / ), with mortality . % ( / ) for those with either cmv, ebv or hhv- dnaemia vs . % ( / ) in those without (p= . ). disseminated cmv prior to transplant was attributed to cause of death for one case. conclusions: cmv incidence was rare, likely from screening for cmv negative hsct donors. cmv, ebv and hhv- dnaemia was not associated with differences in mortality in this cohort. ebv incidence was common, and ptld incidence was similar to previously published outcomes for hsct for other disease ( ) . the advent of cytotoxic t cell therapy for ebv may help abrogate this risk. professor, senior physician, ulm university medical center, pediatrics, germany introduction/background: new-onset aiha occurs in - % of pediatric patients post-hsct. incomplete immune recovery may predispose to immune dysregulation following hsct including autoimmune cytopenias. although prednisolone or other immunosuppressive drugs control most episodes, some patients respond incompletely to first or second line therapies including rituximab. objectives: we describe an innovative therapy for post-bmt aiha refractory to proteasome inhibition. three patients responded to anti-cd antibody (daratumumab) therapy after failing treatment with bortezomib. methods: we retrospectively evaluated data from three patients treated with daratumumab for post-transplant aiha. patients and were treated according to the positive response reported for patient . results: aiha occurred between - months following hsct. daratumumab was curative in patients, the third one had only transient response and relapsed months after this treatment had been initiated. following daratumumab patients no longer required any prbc transfusions. conclusions: in potentially life-threatening aiha in the context of hsct daratumumab may be an effective rescue therapy in combination with rituximab. early post-natal thymus development is strictly dependent on the level of foxn expression in tec these infants present severe t cell lymphopenia early in life, but in most cases their immune system gradually normalizes. however, the role of foxn haploinsufficiency in causing this phenotype is unclear. objectives: to analyze t cell development and tec phenotype in nu/+ mice of various age, in order to investigate whether foxn haploinsufficiency in mice results in impaired thymic development early in life, followed by progressive normalization, thereby recapitulating the human phenotype. methods: we analyzed groups of nu/+ and +/+ littermates, divided by age: day, - days, weeks. the number of etp and the distribution of cortical and medullary tecs (ctecs, mtecs) were analyzed by flow cytometry. maturation of mtecs was further assessed by staining for mhc-ii and aire. real-time pcr was used to analyze the expression of ccl , cxcl , dll , and scf, four key foxn target genes. the study was performed in accordance to niaid animal protocol lcim e. results: at day and - days of life, nu/+ mice showed a dramatic reduction of etps, both in terms of frequency and absolute numbers, as compared to +/+ mice. however, by weeks of age, the frequency and count of etps were comparable in nu/+ and +/+ mice. a slight but significant reduction in the frequency and absolute count of mtecs was observed in all three groups of nu/+ mice. additionally, the ratio between mtec expressing high levels of mhcii (mtechi) and those expressing low levels of mhcii (mteclo) was always higher in +/+ mice as compared to nu/+ mice. moreover, mtechi cells in nu/+ mice expressed lower levels of aire, the gene crucial for thymic negative selection of autoreactive t cells. finally, as compared to wild-type littermates, nu/+ mice showed reduced thymic expression of ccl , cxcl , dll , and scf at day . reduced expression of ccl persisted at day , but normalized at weeks. conclusions: these data indicate that foxn haploinsufficiency in mice leads to impaired thymic colonization by etps and abnormalities of tec differentiation and maturation early in life, followed by progressive normalization, thereby recapitulating what observed in newborns with heterozygous foxn mutations. these observations have important implications for the management of these infants, who should be monitored closely without rushing to definitive treatment for scid. introduction/background: foxn is the master regulator gene for the development and maturation of the thymic epithelial cells (tecs). by inducing the expression of chemokine receptors such as ccl and cxcl , foxn also allows the migration of early thymic progenitor (etp) cells from the bone marrow. lack of foxn leads to the nude (nu)/severe combined immunodeficiency (scid) phenotype in humans and mice. recently, a number of newborns have been identified with low t cell receptor excision circles (trecs) at birth, associated with heterozygous foxn mutations. these infants present severe t cell lymphopenia early in life, but in most cases their immune system gradually normalizes. however, the role of foxn haploinsufficiency in causing this phenotype is unclear. objectives: to analyze t cell development and tec phenotype in nu/+ mice of various age, in order to investigate whether foxn haploinsufficiency in mice results in impaired thymic development early in life, followed by progressive normalization, thereby recapitulating the human phenotype. methods: we analyzed groups of nu/+ and +/+ littermates, divided by age: day, - days, weeks. the number of etp and the distribution of cortical and medullary tecs (ctecs, mtecs) were analyzed by flow cytometry. maturation of mtecs was further assessed by staining for mhc-ii and aire. real-time pcr was used to analyze the expression of ccl , cxcl , dll , and scf, four key foxn target genes. the study was performed in accordance to niaid animal protocol lcim e. results: at day and - days of life, nu/+ mice showed a dramatic reduction of etps, both in terms of frequency and absolute numbers, as compared to +/+ mice. however, by weeks of age, the frequency and count of etps were comparable in nu/+ and +/+ mice. a slight but significant reduction in the frequency and absolute count of mtecs was observed in all three groups of nu/+ mice. additionally, the ratio between mtec expressing high levels of mhcii (mtechi) and those expressing low levels of mhcii (mteclo) was always higher in +/+ mice as compared to nu/+ mice. moreover, mtechi cells in nu/+ mice expressed lower levels of aire, the gene crucial for thymic negative selection of autoreactive t cells. finally, as compared to wild-type littermates, nu/+ mice showed reduced thymic expression of ccl , cxcl , dll , and scf at day . reduced expression of ccl persisted at day , but normalized at weeks. conclusions: these data indicate that foxn haploinsufficiency in mice leads to impaired thymic colonization by etps and abnormalities of tec differentiation and maturation early in life, followed by progressive normalization, thereby recapitulating what observed in newborns with heterozygous foxn mutations. these observations have important implications for the management of these infants, who should be monitored closely without rushing to definitive treatment for scid. introduction/background: immunoglobulin g rd is an immunemediated disease most commonly seen in middle-aged and older men. clinical features include autoimmune pancreatitis, salivary gland disease, orbital disease and retroperitoneal fibrosis. pathologic features include a lymphoplasmacytic infiltrate enriched in igg -positive plasma cells and fibrosis in a storiform pattern. laboratory evaluation usually reveals an elevated serum igg concentration, and glucocorticoids are often used as treatment in early stages of disease. however, disease may recur off of steroids, and prolonged illness or steroid non-responsive disease is usually treated with rituximab. objectives: the objective of this case presentation is to discuss a presentation of a igg rd is a young adult male. results: year old male adopted from thailand, initially presented to hospital with five days of intermittent abdominal pain, night sweats and worsening fatigue. ct abdomen and pelvis revealed bronchiectasis in lung bases, hepatic masses and soft tissue infiltration in the porta hepatis extending into the liver, pulmonary nodules (read as possible metastases), bilateral renal masses, retroperitoneal nodes and ileocolic intussusception secondary to possible lymphoma. one year prior to presentation he developed new onset bilateral cervical lymphadenopathy. biopsy revealed a heterogenous lymphoid population and lymphoma was ruled out. he had a repeat lymph node biopsies during the admission, which was all negative for malignant cells, and was compatible with reactive lymphoid tissue with plasmacytosis. immunophenotype showed % lymphocytes with normal cd /cd ratio, and % cd -,cd -, tcr ++ t cells. immunohistochemical stain of a lymph node fine needle biopsy showed a mixture of cd + b and cd + t cells, abundant cd +, igg+ plasma cells(pcs) , with some igg+ pcs(ct of the neck and chest was completed for possible staging and revealed cervical lymphadenopathy, nasal polyposis, a soft tissue mass vs. enlarged lateral rectus muscle (left orbit), and mediastinal lymphadenopathy. a diagnosis of autoimmune lymphoproliferative syndrome (alps) was presumed by the hematologic/oncologic team given his elevated igg ( , mg/dl) and elevated vitamin b (> ) with multiorgan involvement. alps panel revealed a mutation in faslg (allele , c.- c>t) which is a variant of uncertain clinical significance. alps criteria/scorewas + for / criteria (cd +cd +/hla dr ratio < . ). he had been referred to the nih for further management of alps before initial presentation to our office. immunologic work up revealed immunoglobulins of igg: , mg/dl, iga: mg/dl, igm: mg/dl; / protective streptococcal titers (> . mcg/ml); hib: . mg/l; negative quantiferon gold, and hiv-serology. at that point igg rd vs. castleman disease was suspected instead of alps. further work up showed a normal il- level, and human herpes virus was also negative. igg subsets showed an elevated igg ( mg/dl), igg ( mg/dl), igg ( mg/dl) and normal igg ( . mg/dl). excisional lymph node biopsy was recommended to rule out castleman syndrome or igg rd. a left salivary gland was removed and showed mainly igg positive pcs with no multicentric lymphocytic infiltrates. dilution of the patients serum to determine if there was a prozone affect that minimized the igg level showed an elevated igg level of . mg/dl. this is in the st percentile of all cases of igg rd in terms of serum igg concentration. he was diagnosed with igg rd, a form previously called mikulicz disease, which is comprised of lacrimal and parotid gland enlargement. rituximab was given initially because of the severity of his disease. after two cycles he showed decreased lymphadenopathy, notable weight gain and marked decrease in fatigue. conclusions: igg rd is a rare and complex immunologically based disease process rarely seen in children or adolescents. patients with igg rd often undiagnosed at initial evaluation. normal serum igg levels are seen in % of patients with igg rd and thus a normal igg level should not be used as a biomarker to make the diagnosis of igg rd or in treating this disease. furthermore, the possibility of having a prozone affect in measuring igg needs to be considered and evaluated. meticulous correlation of clinical, pathologic, and imaging findings is required to make the diagnosis. modelling human immune deficiency from novel missense mutations with orthologous heterozygous mutations engineered in mice by crispr/cas introduction/background: next generation sequencing has resulted in substantial progress in identification of mendelian immune deficiency syndromes. in some cases, however, putative causal mutations occur in single kindreds, or even individual patients. under these circumstances, functional analysis of patient derived cells combined with in vitro analysis of genetically manipulated cell lines can provide additional evidence in support of genetic causation, but this might not be conclusive. objectives: understanding how genetic defects result in complex syndromes of immune deficiency and immune dysregulation can be impossible to achieve in vitro. one method for overcoming these obstacles is to generate accurate mouse models of human immune deficiency methods: mouse models of human immune deficiency are a valuable tool in which the murine genome is engineered to introduce a mutation orthologous to that discovered in the patient. we have applied this strategy to elucidate causation and mechanism of immunological defect in several mutations affecting the nf-kb pathway. results: so far, defects in both canonical and non-canonical pathways of nf-kb activation have been shown to cause immune deficiency, often associated with immune dysregulation. we describe a known defects and novel putative defect identified in the canonical nf-kb pathway conclusions: crispr-cas mouse models can be used to elucidate mechanism of disease and provide compelling evidence that mutations are causative. introduction/background: primary immune deficiencies (pid) with or without immundysregulation are rare diseases resulting from monogenetic aberrations leading to either infections or autoimmune manifestations or both. early diagnosis and treatment are crucial for reducing morbidity and mortality. beside genetic diagnosis not commonly yet performed, current standard methods for early diagnosis are dependent on either fresh samples or limited to certain cell types. to overcome those limitations, especially in newborn screening, a novel technology of methylation-based qpcr can be applied. among the known epigenetic modifications, dna demethylation is the most stable and genomic loci with highly cell type specific demethylation sites can be identified. differential methylation can be measured from blood samples of limited availability, or with suboptimal storage. we previously showed that thymic-derived t population, and determine whether there are unique diagnostic and treatment considerations within this demographic. whitney goulstone , elizabeth tough executive director, canadian immunodeficiencies patient organization lpn, alberta health services introduction/background: primary immunodeficiency diseases (pids) represent a significant collection of immune system disorders that increase susceptibility to infection, which in some cases are serious or life-threatening. patients with pid often require immunoglobulin g (igg, commonly referred to as ig) replacement therapy to prevent infections and associated comorbidities. pid treatment, in addition to symptoms and associated social and emotional impacts, has a significant impact on patients quality of life (qol). information available on the real-world diagnosis, management, and outcomes of the canadian pid patient population is limited. objectives: to better understand diagnosis and treatment of canadian patients with pid, we surveyed canadian patients with pid associated with the canadian immunodeficiencies patient organization (cipo) the primary goal of the survey was to gain insight into the canadian pid patient population with regards to demographics, diagnosis, treatment, including regimes, qol, and communication and support . methods: the authors conducted a cross-sectional survey to measure health-related qol in a cohort of patients with pid. eligible participants were identified through the canadian immunodeficiencies patient organization (cipo). the questionnaire consisted of questions that covered patient-reported outcomes including diagnosis, qol, treatment regimes, and communication. results: surveys were returned by patients with pid. participants conveyed significant impact on qol including personal, occupational, financial, emotional, and social impacts as a result of pid symptoms, risks, and treatment logistics, limitations, and side effects. the most common diagnoses were related to b-lymphocyte disorders ( . %). common treatments include intravenous immunoglobulin (ivig; in hospital) and subcutaneous immunoglobulin (scig; at home), in addition to antibiotics and antifungals as required. respondents reported feeling average before treatment on a scale of - (mean . ± . ) with increased health after treatment (mean . ± . ). respondents felt current treatment was convenient (mean . ± . ) and were comfortable with self-infusions (mean . ± . ). conclusions: patients with pid are not uncommon in the canadian community, and in these patients pid is associated with a significant impairment in qol. experiences range with regards to a particular treatments advantages and disadvantages, cost, travel, and convenience. respondents hope to achieve improved qol through the following solutions: better treatment, improved infusions, gene modification, more research and clinical trials, a cure, and education and outreach. improved financial, medical, and social supports were also requested. introduction/background: severe combined immunodeficiency (scid), the most severe form of t cell immunodeficiency, is detectable through quantification of t cell receptor excision circles (trecs) in dried blood spots (dbs) obtained at birth. for many professional, humanitarian and financial reasons, newborn screening (nbs) for scid is warranted. implementation of this screening test is highly important where high frequency of consanguinity is known to exist. objectives: since october , israel has conducted national scid nbs. this important, life-saving screening test is available at no cost for every newborn in israel. methods: herein, we describe two years results of the israeli scid newborn screening (nbs) program. validation includes cbc, lymphocyte subsets, trec (in a different method), t cell receptor (tcr) repertoire and response to mitogenic stimulation. whole exome sequence (wes) for genetic detection, and next generation sequencing (ngs) to demonstrate tcr clonality are used as well, in some unsolved cases. results: of , births screened, ( . %) had abnormal trec in their first screen ( terms, pre-terms), ( . %) had a repeated abnormal trec also in their second screen and were referred for a validation process. fourteen scid patients were diagnosed, so far, through the nbs program in its first years, revealing an incidence of : , births in the israeli population. consanguine marriages and muslim ethnic origin were found to be a risk factor in affected newborns, and a founder effect was detected for both il r and dclre c deficiency scid. other diagnoses were made as follows: cases were found to have t cell lymphopenia; cases were diagnosed with syndromes; cases were found to have secondary t cell lymphopenia; cases were pre-term infants and cases were considered as false positive with normal evaluation. lymphocyte subset analysis and trec quantification in the peripheral blood appear to be sufficient for confirmation of typical and leaky scid and ruling out false positive results. detection of secondary targets (infants with non-scid lymphopenia) did not significantly affect the management or outcomes of these infants in our cohort. we could also report several perspectives regarding t cell development in non immunodeficient newborns that emerged from the accumulated data. conclusions: already in a short term, trec nbs in israel has achieved early diagnosis of scid and other conditions with t-cell lymphopenia, facilitating management and optimizing outcomes. this program has also enabled gaining insights on t cell development in health babies. (hct) . surprisingly, % of these infections were acquired during the interval between confirmation of the scid diagnosis and hct (heimall, blood ). to investigate further, in late we surveyed pre-hct management practices for scid patients at pidtc centers. physicians representing north american centers responded, including immunologists, transplant specialists and who identified as both. % of centers lacked a standard procedure for the management of infants with a positive nbs result. to confirm the scid diagnosis, basic t, b and nk cell flow cytometry was performed by most. testing for naïve t cells was generally included, but testing of lymphocyte mitogen proliferation was inconsistent. specialists were notified of a patients positive nbs test at median age . days ( - days) , and management of patients as scid was started at median age days ( - days). when unconditioned hct was anticipated, % of respondents began planning as soon as possible after diagnosis, while % awaited genetic testing results before hct. respondants consistently implemented pre-hct prophylaxis with trimethoprim/sulfamethoxazole ( %), fluconazole ( %) and immunoglobulin infusions ( %), although timing of initiation varied. palivizumab was used by %. there was little consensus regarding viral monitoring. although most physicians screened for cmv by blood pcr ( %), only about half routinely screened for ebvor adenovirus. while % of physicians started prophylaxis against double-stranded dna viruses in all patients, % did so only in selected situations, such as active genital hsv at the time of delivery, or when the mother was cmv-seropositive. although all centers used only acyclovir as antiviral prophylaxis, doses and timing varied widely: from - mg/kg/day, divided into or doses, continued in most centers until immune reconstitution. finally, % of physicians recommended that cmvseropositive mothers stop breast-feeding. there was no consensus on where patients should reside prior to hct. hospital or home were favored equally, although need for a reliable family was indicated by % as a criterion for home-based management. for hospitalized patients, % of centers required patients to be in a reverseisolation/positive-pressure room and over half required staff to wear gown, gloves, and mask. with regard to visitors, % required parents/ relatives to perform hand hygiene. approximately % did not require a gown, gloves, or mask for visitors. finally, there was no consensus on allowing siblings or friends to visit, but the majority permitted grandparents or other adult relatives. this survey revealed wide variability in the diagnostic pathway, viral surveillance, and isolation practices for scid patients, although pretransplant prophylaxis with immunoglobulin, fluconazole, and trimethoprim/sulfamethoxazole were utilized consistently. we conclude there is considerable opportunity to develop diagnostic and pre-hct management pathways for scid. prospective tracking of management practices could reveal which are important for avoiding pre-hct infections. evidence-based practice guidance is needed to maximize the potential to bring each scid patient identified via nbs to hct infection-free. ikaros/ikzf is an essential transcription factor expressed throughout hematopoiesis. in humans, somatic mutations in ikzf are linked to b-cell acute lymphoblastic leukemia (all), and germline heterozygous haploinsufficient mutations cause common variable immunodeficiency-like disorder with incomplete penetrance. herein, we report seven unrelated patients with an earlyonset novel combined immunodeficiency associated with de-novo, fully-penetrant, germline heterozygous dominant negative mutations affecting amino acid n in ikzf dna binding domain. patients presented with different infections, but pneumocystis jirovecii pneumonia was common to all. one patient developed a t-cell all. additional findings included decreased b-cells, neutrophils, eosinophils and myeloid dendritic cells as well as t-cell and monocyte dysfunction. t-cells exhibited a profound naïve/recent thymic emigrant/ t-helper phenotype and were unable to evolve into effector memory cells; monocytes failed to respond to different stimuli or facilitate t-cell activation. this new defect expands the spectrum of human ikzf -associated immunodeficiency diseases from haploinsufficient to dominant negative. the beta subunit of the il- receptor (il rb; cd ) is essential for il- and il- mediated signal transduction in a variety of hematopoietic cell types including t and nk cells. here we report the clinical and immunologic phenotypes of two siblings born to consanguineous parents harboring a variant in il rb, resulting in autoimmunity, with lymphoproliferation of cd + t and cd hi nk cells, and decreased regulatory t cell frequency. the proband presented with inflammatory enteropathy, failure to thrive, and disseminated cmv infection at months of age, and later developed atopy, lymphocytic interstitial pneumonitis, and red blood cell autoantibodies. his younger sister presented with severe autoimmune hemolytic anemia and cmv viremia at months of age, and later developed lymphocytic interstitial pneumonitis. whole exome sequencing and chromosomal microarray studies revealed a homozygous deletion within the highly conserved wsxws motif of the extracellular domain of il rb (c. _ delcctggagcc, p.pro _ser del). the deletion results in reduced il rb expression (cell surface and intracellular) in t and nk cells. the functional consequences of the defect include complete impairment of stat phosphorylation through the il- receptor but only partial impairment through the il- receptor, as well as a compensatory increase in serum il- and il- levels (> pg/ml). cd + t cell proliferation responses to in vitro t cell receptor (tcr) stimulation were reduced compared to an age-matched, healthy control, but partially rescued by supraphysiologic levels of il- and il- . despite reduced cd + t cell proliferation responses, the proband displayed a t cell population skewed toward cd + t cells, with an oligoclonal expansion of effector memory cells. arguing against effects of pervasive cmv infection alone, the sister displayed similar phenotypic and functional abnormalities at birth, prior to her cmv infection. our data suggest that the identified hypomorphic mutation in il rb results in a survival advantage for those cd + t and cd hi nk cells most sensitive to the exuberant serum il- and il- levels produced in response to the defect. these surviving cd + t and cd hi nk cells have great lymphoproliferative potential, leading to multisystem autoimmunity and inflammatory complications. therefore, we describe il rb deficiency as a novel primary immunodeficiency disease with prominent immune dysregulation and selective cd + t and cd hi nk cell lymphoproliferation. key: cord- -o qg qta authors: mocchegiani, eugenio; malavolta, marco title: role of zinc and selenium in oxidative stress and immunosenescence: implications for healthy aging and longevity date: - - journal: handbook of immunosenescence doi: . / - - - - _ sha: doc_id: cord_uid: o qg qta aging is a complex process that includes gradual and spontaneous biochemical and physiological changes which contributes to a decline in performance and increased susceptibility to diseases. zn and se are essential trace elements that play a pivotal role in immune functions and antioxidant defense and, consequently, are claimed to play also a role in successful aging trajectories. consistently with their nature of essential trace elements, a plethora of data obtained “in vitro” and “in vivo” (in humans and animal models) support the relevance of zn and se for both the innate and adoptive immune response. moreover, zn and se are strictly involved in the synthesis and regulation of activity of proteins and enzymes, e.g., metallothioneins (mt) and glutathione peroxidase (gpx), that are necessary for our endogenous antioxidant response. this is clearly important to protect our cells from oxidative damage and to slow the decline of our immune system with aging. age-related changes affecting tissue levels of zn and se may indicate that the risk of zn and se deficiency increases with aging. however, it is still unclear which of these changes can be the consequence of a “real deficiency” and which can be part of our physiological compensatory response to the accumulating damage occurring in aging. furthermore, the upregulation of antioxidant proteins (zn and se dependent) may be a manifestation of self-induced oxidative stress. by the way, zn and se dependent proteins are modulated not only by nutritional status, but also by well-known hallmarks of aging that play antagonistic functions, such as the deregulated nutrient sensing pathways and cellular senescence. thus, it is not an easy task to conduct zn or se supplementation in elderly and it is emerging consistent that these kind of supplementation requires an individualized approach. anyway, there is consistent support that supplementation with zn using doses around mg/day is generally safe in elderly and may even improve part of immune performances in those subjects with a baseline deficiency. regarding se supplementation, it may induce both beneficial and detrimental effects on cellular immunity depending on the form of se, supplemental dose, and delivery matrix. the nutritional association of supplements based on “zn plus se” is hypothesized to provide additional benefits, but this will likely need a more complex individualized approach. the improvement of our knowledge around screening and detection of zn and se deficiency in aging could lead to substantial benefits in terms of efficacy of nutritional supplements aimed at ameliorate performance and health in aging. biological aging is a process driven by molecular damage, molecular heterogeneity, and metabolic imbalance which determinates increased susceptibility to diseases and impaired adaptation to changes in environmental conditions (rattan ) . alterations in the immune functions play a fundamental role in aging. the term "immunosenescence" has been coined to describe the changes that occur with age in the immune system and that lead to a higher incidence of infection, cancer, and autoimmune disease (fulop et al. ; pawelec et al. ) . immunosenescence involves a shift in function of both adaptive and innate immune cells, which can be resumed in a reduced capacity to recognize new antigens and in the occurrence of systemic low-grade chronic inflammation. this phenomenon, termed "inflamaging" (franceschi et al. ) is likely the result of immunosurveillance of chronic infections (chronic antigen stress) (pawelec et al. ) combined with accumulating senescent cells which, in turn, are a rich source of pro-inflammatory factors collectively termed the "senescence-associated secretory phenotype" (sasp) (campisi ) . in analogy with aging, immunosenescence in humans is very heterogeneous and can be described as a complex mosaic resulting from the interaction of a variety of environmental, stochastic, and genetic-epigenetic variables. it is likely that, through a favorable combination of these factors, centenarians avoid or delay the major inflammation-driven age-related diseases, such as cardiovascular disease (cvd), diabetes mellitus (dm), alzheimer disease (ad), and cancer (bürkle et al. ) despite showing some markers of inflammation. diet and nutritional factors are known to play a role in the preservation of our immune system in aging. deficiencies of both macro-and micronutrients in aging is strictly related to global impairments of the immune functions and appearance of age-related diseases (lesourd ) . several causes contribute to micronutrient deficiencies in elderly. first of all, the poor socioecon omic condition present in a large part of old people may lead to a consumption of inexpensive foods deficient in micronutrients (kant ) . the gap is worsened by loss of appetite, lack of teeth, intestinal malabsorption, and subclinical diseases that lead to the final result of frailty, disability, and mortality (semba et al. ) . by contrast, changes in diet and exercise patterns appears to be effective in the prevention of late-onset nutritionrelated conditions, especially when these are instituted early in life (chernoff ) . available data do indicate that essential vitamins and trace elements are necessary for normal immune function (maggini et al. ), but conclusive studies demonstrating that vitamin or mineral supplements can boost immune function are currently lacking. hence, there is evidence that further supplementation of micronutrients beyond the recommended daily intake can improve immune function (dharmarajan ) . however, clear roles exist for vitamin and trace element supplementation in states of deficiency and in subgroups of older adults at high risk for deficiency. by the other way, the prevalence of certain nutritional deficiencies in the geriatric population (e.g., zinc and selenium) is sufficiently high that certain supplements may be indicated (ames ) . we herein review the role of zinc and selenium for our immune system (buttriss ) and describe the mechanisms through which they affect immunosenescence. epidemiological and clinical evidence have shown that in most developing countries deficiencies of these micronutrients are partly responsible for the severity of infectious disease, morbidity, and mortality in malnourished children (bhaskaram ; bailey et al. ) as well as in elderly (meydani ) and critically ill patients (mertens et al. ) . these two trace elements display a common pivotal role in establishing the cellular antioxidant response as well as in mounting a proper immune response, which in turn may be useful to prevent excessive accumulation of senescent cells in aging and to reduce the senescence-associated increase of chronic inflammatory mediators. biological role of zinc zinc is one of the most important trace elements in the body, although its presence in nature does not exceed . % (mills ) . the major characteristics of zinc include a highly concentrated charge, a small radius ( . a), no variable valence (low risk of free radical production), ready passage from one symmetry in its surroundings to another without exchange, rapid exchange of ligands (on and off reactions), and binding mostly to s-and n-donors in biological systems. these properties enable zinc to play a major biological role as a catalyst. zinc is essential for the activity of more than enzymes influencing the activity of zinc dependent antioxidant enzymes, such as superoxide dismutase (sod) and various organ functions having a secondary effect on the immune system (rink and gabriel ) . zinc is present in "zinc finger domains" of transcription factors and in a multitude of proteins, peptides, enzymes, hormones, and cytokines, which act in maintaining body homeostasis (coleman ; berg and shi ) . zinc also regulates mrna stability (taylor and blackshear ) and extracellular matrix (vallee and falchuk ) . zinc binds enzymes, proteins, and peptides with different binding affinity (kd) ranging from À to À mol/l (mocchegiani et al. ) . some of these enzymes may be activated by zinc, as for instance the thymic hormone named thymulin, which loses its activity in absence of zinc (fabris et al. ) , while others may be inactivated. zinc also regulates cell cycle and apoptosis (fraker ) with an optimal range of concentration that has been well defined by studies on genomic stability "in vitro." these studies, performed on various cellular models, suggest that genomic instability is minimized when zn concentration in culture medium is comprised between and μm (ho and ames ; ho et al. ; sharif et al. ) . although, it is currently unknown whether these results can be translated "in vivo," where additional factors including blood composition, genetic background, aging, and diseases can affect the response, these studies "in vitro" confirm that an excess of zn is dangerous for cellular health in equal measure to zn deficiency. a very recent study altered the zinc balance within caenorhabditis elegans to examine how changes in zinc burden affect longevity and healthspan in an invertebrate animal model. the authors found that increasing dietary zinc levels decreased the mean and maximum life span of the worms, whereas treatment with a zinc-selective chelator increased their mean and maximum life span. the life span modulating effects were mediated by daf- , hsf- , and skn- proteins, which suggest the involvement of the insulin/igf- pathway. these results are somewhat supporting the idea that zinc itself has a role as an antagonistic pleiotropy player (kumar et al. ) . by the way, it has been just recently hypothesized that reduced zinc levels in serum during human aging may reflect the homeostatic shade from a general systemic "growth and reproduction" status typical of juvenile age to a "repair and maintenance" status that evolved to preserve health status during old age . the cellular basis for the impact of low and high zn status has been studied for many years but it is only recently that the complexity of zn homeostasis has been appreciated. the advance in the knowledge around the major intracellular zn binding proteins, namely the metallothioneins (mt) and the discovery, cloning, and characterization of zn transporter proteins, has highlighted the role of free zn ion and the fluxes of zn ions between extracellular, cellular, and intracellular compartments. indeed, these zn signals can initiate pathways which have tight analogies with those discovered for calcium signaling, albeit in the sub-and nanomolar range (haase and rink ) . metallothioneins (mt) are a group of low-molecular-weight metal-binding proteins who have high affinity for zinc (kd = . Â À m) (kagi and schaffer ) . mt exist in different isoforms characterized by the length of amino acid chain: isoform i, ii, iii, e iv mapped on chromosome in man and on chromosome in mice with complex polymorphisms (west et al. ). the more common isoforms are i and ii; the isoform iii, also called growth inhibitory factor (gif), is mostly expressed in brain tissues while the isoform iv is restricted in squamous epithelia. mt contain cysteines which bind seven zinc atoms through mercaptide bonds that have the spectroscopy characteristics of metal thiolate clusters (maret and vallee ) . the zinc/cysteine clusters are of two different types. in the beta-domain cluster, three bridging and six terminal cysteine thiolates provide a coordination environment that is identical for each of the three zinc atoms. in the alpha-domain clusters, there are two different zinc sites; two of them have one terminal ligand and three bridging ligands, respectively, while the other two have two terminal and two bridging ligands (maret and vallee ) . mt can act as an antioxidant since zinc-sulfur cluster is sensitive to changes of cellular redox state and oxidizing sites in mt (reduced thiol groups) induce the transfer of zinc from its mt-binding sites to those of lower affinity in other proteins (kagi and schaffer ) . in response to oxidative or nitrosative stress, zn-mt release free zinc ions. the released free zinc ions are used to confer the biological activity to some zincdependent antioxidant enzymes and to other proteins involved in dna repair, as well as to upregulate the gene expression of various factors involved in the antioxidant response (mocchegiani et al. ). hence, mt are able to transduce stress signals into free zinc ion signals which are immediately compensated by the activity of zn transporters and subsequently downregulated by a feedback mechanism that involves the gene expression of mt themselves. this mechanism is also involved in regulation of nitric oxide (no) pathways (bogdan et al. ) as no induces the release of zinc from mt, via s-nitrosylation (zangger et al. ) . the release of zinc by mt, via s-nitrosylation, contributing to raise the intracellular free zinc ions concentration, plays a crucial role in modulating the production of pro-inflammatory cytokines and in the activation of immune cells (rink and haase ) . free zinc ion levels are also regulated by special proteins named zinc transporters, which in turn appear to be also specifically involved through regulation of cellular zinc homeostasis via influx, efflux, or vesicular sequestration (cousins and mcmahon ; eide ) . two families of zinc transporters have been identified. the znt family decreases cytoplasmic zinc concentrations by secretion, sequestration, or efflux, whereas the zip family increases cytoplasmic zinc influx or release of stored zinc (eide ) . the huge network based on zn transporters and mt suggest how much critical is a tight regulation of zinc ion availability. moreover, the timely release, efflux, and influx of zn ions is known to virtually affect all aspects of innate and adaptive immunity. several molecular targets, including phosphatases, phosphodiesterases, caspases, and kinases, suggest that zinc ions are like a second messenger that regulate signal transduction in various kinds of immune cells (mocchegiani et al. ; haase and rink ) . this phenomenon clearly explains why in vivo zinc deficiency alters the number and function of neutrophil granulocytes, monocytes, natural killer (nk), t, and b cells. the redox properties of mt contribute to make this protein a crucial defense against ionizing and uv radiations (cai et al. ) , heavy metals (mercury, cadmium), lipid peroxidation, reactive oxygen species, oxidative stress caused by anticancer drugs, and conditions of hyperoxia (sato and kondoh ) . this protective role of mt has been studied especially in young-adult mt knockout mice (null mice) for short periods of exposure to toxic metals, such as cadmium for weeks (habeebu et al. ) or mercury (single injection and the effect of mercury analyzed days after the injection) (satoh et al. ) , or to anticancer agents for - h (kondo et al. ) , or in presence of an excess of zinc or zinc deficiency for weeks (kelly et al. ) . expression of mt is mediated by zinc (directly via mre responsive elements in the promoter), glucocorticoids (via glucocorticoid responsive element in the promoter), and other stressor agents as well as from inflammatory cytokines, such as il- , il- , ifn-a, tnf-a (davis and cousins ) . these findings clearly suggest the existence of a strong interplay between mt and the immune system. an important link between cytokines and mt seems to be stat (signal transducers and activators of transcription) responsive element observed in the promoter of mt as well as the nitric oxide pathway. it is remarkable that cytokines reported to increase inducible no synthase (inos) expression (il- and tnf-a) have also induced the expression of mt (zangger et al. ). this induction is likely the indirect consequence of the release of free zinc from preexisting mt via s-nitrosylation caused by no. this process is particularly important for the production of thymic hormone (thymulin) from thymic epithelial cells. indeed, il- induces thymic uptake of zinc (coto et al. ) , which is subsequently made available for the activity of thymulin by a process that involve the redox status of mt (savino et al. ) . mt have been also found to be released to the extracellular environment in a number of different compartments, including cell culture media, serum, urine, bronchoalveolar spaces, liver sinusoids, and inflammatory lesions. albeit a very small amount of mt can be found in the extracellular environment (e.g., around μm in serum), these proteins may support the beneficial movement of leukocytes to the site of inflammation representing a "danger signal" for the immune cells and modifying the character of the immune response when cells sense cellular stress (yin et al. ) . hence, mt are involved in several mechanisms that support immune function. what is still uncertain is the role of these proteins in aging and immunosenescence. in particular, there have been concerns around the possibility that mt may retain their protective role during chronic stress and inflammatory conditions associated with the major disease of aging (mocchegiani et al. a ). in the case of cancer, for example, mt have been proven as an important barrier against carcinogenesis (dziegiel et al. ; malavolta et al. ) but, once malignant cells are formed these proteins may help cancer cells to survive and proliferate. several studies have disclosed mt expression as a prognostic factor for tumor progression and drug resistance in a variety of cancers, including breast, prostatic, ovarian, head and neck, nonsmall cell lung cancer, melanoma, and soft tissue sarcoma (eckschlager et al. ; dziegiel et al. ). this action is consistent with the original biological role of mt, as these proteins are part of the defensive intracellular stress response. additionally, elevated levels of extracellular mt, as detected at sites of inflammation and in certain types of neoplastic lesions, have been shown to display an immunosuppressive function (youn and lynes ) . by contrast, there are cases where mt have been found to provide additional benefit in chronic inflammation, especially in cancer therapy. indeed, while cancer cells overexpressing mt appear in general less sensitive to chemotherapeutic drugs, cancers where mt are progressively silenced are more sensitive to drugs following expression of specific mt isoforms in presence of zinc (pedersen et al. ; arriaga et al. ) . these studies suggest that there is not a common role and that different mt isoforms might be differently associated with either chemoresistance or sensitivity to cancer drugs. in the case of other chronic age-related diseases, it was hypothesized that the expression of mt, continuously stimulated by the persistence of inflammatory mediators (e.g., il- ), could lead to a detrimental sequestration of zinc ions with potential functional consequences on the immunity of frail aged individuals (mocchegiani et al. a, b) . observational studies performed (a) in pbmcs from centenarians, which display lower mt expression compared to elderly aged - years, (mocchegiani et al. a ); (b) in pbmc from down's syndrome (syndrome which presents some features of accelerated aging and immunosenescence) subjects, which display levels of mt comparable to elderly subjects (mocchegiani et al. a) , and (c) studies in mice models of athropic thymus, which display overexpression of mt (mocchegiani et al. b) , have contributed to argue the hypothesis against a preserved functional role of mt in aging. however, it has been more recently documented that mt expression declines with "in vitro" aging of pbmcs, thus suggesting that the low levels of mt observed in pbmcs of centenarians could simply be the consequence of the increasing accumulation of senescent cells in blood or other phenomena related to epigenetic changes. conversely, the increased levels of mt observed in pbmcs from elderly subjects could be the physiological consequence of a stress response induced by inflammatory agents. a general pro-longevity role of mt emerges also from recent studies in mice. a longevity phenotype has been shown in mt transgenic mice that overexpresses the mt- isoform (mt -tg) in the c bl/ j background , as well as in cardiac-specific mt transgenic mice that overexpress the human mt isoform in the fvb background (yang et al. ) . more recently, the findings that mt ko mice display a shortened life span can be considered a definitive proof that constitutive mt expression from birth to death may play a beneficial role in longevity, at least in mice, independently by the strain studied (kadota et al. ) . however, it is still uncertain if this situation may be comparable in humans and, most importantly, there are no examples of mt-inducible mice models, which might unravel the consequence of reactivation of mt expression in late life. anyway, association studies of snps of mt genes point out a role of these proteins in human longevity or in the susceptibility to agerelated diseases but the functional role is far to be clarified (giacconi et al. ; cipriano et al. ) . taking inspiration from the role of mt in cancer, it should deserve appropriate consideration for future investigation the role of mt in cellular senescence. currently, there is common agreement on the negative role of excessive accumulation of apoptosis-resistant senescent cells in aging and in several age-related diseases. this evidence is supported by the rejuvenating effects observed after inducible removal of senescent cells in transgenic animals (baker et al. (baker et al. , as well as after treatment of mice with senolytic drugs (chang et al. ; roos et al. ) . while late passage senescent lymphocytes clones may display reduced expression of mt protein , some models of oncogene-induced senescence (collado et al. ) and senescent endothelial cells (malavolta et al. unpublished observation) appear to overexpress mt genes. in analogy with the role shown by mt in resistance to apoptosis of cancer cells, it might be possible to build the hypothesis that these proteins can play a role in the resistance to apoptosis of senescent cells. investigation around this hypothesis might be important to optimize the development of senolytic drugs before human studies. increasing evidence suggest that zinc transporters play a major role in intracellular zinc homeostasis, which is critically involved in the signaling and activation of immune cells. while few gerontological studies have specifically addressed the role of zinc transporters, there is evidence that they may play a role in the process of progressive dysregulation of immune responses associated with aging. a recent flow cytometry assay showed that uptake of extracellular zinc is reduced in pbmcs taken from elderly subjects compared to the younger counterpart (giacconi et al. ). this observation argues for a possible downregulation of zinc importers' or upregulation of zinc exporters' function in aged immune cells. in agreement with this observation, a study performed in mice showed reduced zip expression and dysregulation in splenocytes from aged mice (wong et al. ). most importantly, the age-specific zip dysregulation correlated with an increase in zip promoter methylation while reduced zip expression was shown to enhance pro-inflammatory response. similarly, a correlation between the level of methylation at slc a (znt ) promoter region and age has been reported in humans (coneyworth et al. ). moreover, methylation of the znt promoter region resulted in reduction of znt expression and was hypothesized to contribute to the decline in zn status observed with aging. in addition to epigenetic changes occurring with aging, another potential link between immunosenescence and zinc transporters regards the regulation of zip in the liver. zip expression is upregulated through il- , and this mechanism was shown to be responsible of the hypozincemia that accompanies the acute-phase response to inflammation and infection (liuzzi et al. ) . psychological stress, a putative recipe for accelerated aging, has been shown to induce zinc accumulation and upregulation of zip and metallothionein in rat liver (tian et al. ). since chronic inflammation, particularly driven by increased il- , is a usual event in old age (mocchegiani et al. ) , it cannot be excluded that this process may contribute to hypozincemia, depressed immune response, and risk of infection in the elderly. however, it should be noted that a gene expression study on two major zn transporters (znt and zip ) in leukocytes obtained from two different age groups of korean women indicated major changes following supplementation then baseline changes between the two groups (andree et al. ) . hence, it is still uncertain which zn transporters are more likely involved in age-related changes and, most importantly, which is the real impact of these changes on immune function and health of elderly subjects. "in vitro" studies since the discovery that the crude zinc balance is negative in old mice (mocchegiani et al. ) and in old human (turnlund et al. ), a number of studies involving zinc supplementation have been performed. most of these studies have been focused on the effect of zinc on immune function, as "in vitro" experiments have provided considerable support on the immune-modulatory effects of zinc. when pbmcs are stimulated with zinc, il- , il- , and tnf-α, soluble (s)il- receptor and ifn-γ are released (ibs and rink ) . the secretion of il- , il- , and tnf-α is induced directly by zinc in monocytes and is independent by the presence of lymphocytes (driessen et al. ) . however, the effect of zinc on monocytes may depend upon external stimulation. in fact, zinc inhibits lps-induced tnf-α and il- β release from primary human monocytes and monocytic cell lines through the inhibition of cyclic nucleotide phosphodiesterase activity (von bulow et al. ) , suggesting that zinc may display also some anti-inflammatory properties. the dose of zinc used is also a critical variable. in serum-free culture medium, pharmacological concentrations (> μm of zinc) stimulate monocytes but prevent t cells from activating, perhaps due to the lower intracellular content in t cells than in monocytes (ibs and rink ) . treatment with zinc "in vitro" generally displays also beneficial effects on cell survival but the effect largely depends upon the cell type and the dose of zinc used. the optimal concentration of zinc "in vitro" from genomic stability studies performed in different cellular models point out at concentrations in the range - μm (sharif et al. ) . it seems that both apoptosis prevention and induction are mediated by pathways involving zinc and/or zinc-dependent enzymes (clegg et al. ; wiseman et al. ) . therefore, the modulation of the zinc homeostasis plays a key role not only in preventing apoptosis, when oxidative stress is low, but also in inducing apoptosis, when oxidative stress and cellular damage is high. this mechanism could play a role, for example, in the elimination of virally infected or malignant cells (fraker and lill-elghanian ) . the pro-apoptotic function of zinc has been documented under condition of persistent oxidative damage in pbmc from young-adult humans as well as in very old age (ostan et al. ) . the key player that mediates the activity of zinc in this case appears to be the tumor suppressor p , which needs zinc for site-specific dna binding and proper transcriptional activation (hainaut and mann ; loh ) . the response to zinc during stress condition appears be cell-type dependent. experiments in thymocytes have shown that zinc from up to μm prevents old thymocyte apoptosis induced by dexamethasone or serum deprivation (provinciali et al. ) , whereas the direct introduction of free zinc as zincpyrithione inside thymocytes induces apoptosis (mann and fraker ) . it is likely that the continuous presence of intracellular free zinc ions induced by zn-pyrithione can be interpreted by the cell as a presence of irreversible damage with subsequent activation of pro-apoptotic pathways. old literature documented that zinc supplementation performed throughout the whole life span of rodents is able to delay some age-related cell-mediated immune modifications, such as the decreased circulating thymic hormone levels (iwata et al. ). however, an immunomodulatory effect of zn supplementation has been shown with a relatively short time of treatment in old mice. for example, μg/ml zn ++ in the drinking water induced thymus regrowth and functionality (dardenne et al. ; mocchegiani et al. ) as well restoration of nk cell cytotoxicity (mocchegiani et al. ) in old mice in just month. that the benefit of zinc supplementation upon the immune functions in old mice is not to consider an epiphenomenon comes by the analysis of the rate of survival in old zinc-treated mice. old mice (inbreed balb/c mice) treated with daily zinc at the dose reported above in drinking water from the pre-senescent age ( - months of age) display a significant increment of the median and maximal life span (up to months vs. - of controls) (mocchegiani et al. b ). the increased longevity was largely due to significant decrements of deaths due to cancer and infection in the middle age. part of the effects shown in aged mice can be the consequence of an improved thymopoiesis (wong et al. ). however, increased longevity after zinc supplementation was also reported in the short lived thymectomized and nude mice, which display a negative crude balance of zinc (mocchegiani et al. ). taking into account that the liver extrathymic t cell pathway is prominent in nude, thymectomized and old mice (abo et al. ) , it is likely that these effects of zinc may be in part the effects of zinc on this extrathymic pathway (mocchegiani et al. ) . conversely, in aged c bl/ mice, zinc supplementation ( mg/kg for days) was shown to increase thymopoiesis, as assessed by increased total thymocyte numbers (wong et al. ). the improved thymic output was mediated in part by reducing the age-related accumulation of immature cd (À)cd (À)cd (+)cd (À) thymocytes, as well as by decreasing the expression of the thymosuppressive cytokine named stem cell factor. a slightly increased survival has been also recently documented in old (age ! months) c bl/ j mice supplemented for the whole life span with mg/l of zinc in drinking water. however, survival curves of supplemented mice displayed an increased variability and were markedly lower compared to those of transgenic mice overexpressing mt independently of zinc supplementation . the results of mice overexpressing mt are similar to those obtained in worms with zinc chelation (kumar et al. ) . these data may reinforce the idea that zinc has a potential antagonistic pleiotropic. in particular, zinc may act on nutrient-sensing pathways (e.g., insulin/igf- -like receptor pathway and pi k/akt/mtor pathway) with the final result of promoting growth and proliferation. inhibition of this pathway has been proposed as a pro-longevity treatment while its enhancement may help elderly, which in turn display excessive downregulation of nutrient-sensing pathways as a consequence of compensatory phenomena normally occurring in aging. this point of view could explain the beneficial effects observed by supplementation in elderly and old mice. with regard to elderly, there is consistent support that zinc supplementation may slightly impact immune system, but real benefits appears to be evident only in the case of well-documented nutritional zinc deficiency. an exhaustive picture of the zinc supplementation in elderly by different studies has been reported by haase and rink ( ) . many attempts have been performed in the past using different doses of zn for various treatment duration (duchateau et al. ; sandstead et al. ; bogden et al. ; prasad et al. ; boukaïba et al. ; cakman et al. ; fortes et al. ) . immunostimulative effects of zinc have been documented using pharmacological doses largely exceeding the rda. for example, duchateau et al. ( ) and sandstaed et al. ( ) reported an improvement in response to skin-test antigens and taste acuity with doses of zinc around mg/day for month. however, in the majority of the studies, zinc supplements were used in the range proposed by the rda while attempting to not exceed the tolerable upper intake level (usually a range from to mg/day). prasad et al. ( ) and boukaniba et al. ( ) have found an increment of thymulin activity and improvements in response to skin-test antigens and taste acuity (zinc dose = mg/day for months); bodgen et al. ( ) have reported no benefit exclusively for increased lymphocyte mitogen proliferative response (zinc dose = mg/day for year); cakman et al. ( ) have found enhanced ifn-γ production by leukocytes (zinc dose = mg/day for days); fortes et al. ( ) report an increased number of cytotoxic t lymphocytes (zinc dose = mg/day for days). a long-term ( months) supplementation trial with two doses of zinc ( and mg/day) in elderly was recently conducted by the zenith study (hodkinson et al. ). zn supplementation of mg/day significantly lowered b-lymphocyte at month but not later. zn supplementation of mg/day significantly increased the ratio of cd to cd t lymphocytes at month . overall, these findings suggest zn supplementation has minimal longterm effects on immune status of healthy elderly persons. in the framework of a recent european study focused in zinc and immunosenescence, elderly subjects to be supplemented with zinc were chosen according to the presence of a pro-inflammatory genotype for il- and low plasma zinc (mocchegiani et al. ) . supplementation (with mg/day of zn-aspartate for +/À days) was carried out in old subjects (age > years) who presented stable low plasma zinc levels ( . μm at baseline and at year follow-up) and in c-carriers for il- - g/c with unstable plasma zinc ( . μm at baseline and > . μm at year follow-up). most evident effects of zinc supplementation on immune system comprised both corrective, but also noncorrective effects on known immunosenescence markers, including: ( ) an improved susceptibility of t cells to activation-induced cell death (aicd) (varin et al. ); ( ) an increased circulating levels of il- , mcp- associated with increased nk activity that were partly dependent on il- - g/c and + mt a snps (mariani et al. ) ; ( ) an increase of th and th cells in thawed samples and a slight reduction in th /th ratio in fresh whole blood lymphocytes ); ( ) a marked increase in both basal as well as stress-induced hsp levels in lymphocytes from healthy elderly donors with a higher impact on cd + cells (putics et al. ) ; and ( ) reduced spontaneous cytokine release in pbmcs and defects in termination of inflammatory activity ). in the same population, giacconi et al. ( ) studied the influence of zip gln/arg/leu (rs ) polymorphism on zinc homeostasis and inflammatory response following zinc supplementation. leu-(arg arg genotype) elderly showed higher inflammatory markers at baseline than carriers of other alleles that were reduced after zinc supplementation. later, the influence of + a/g mt a snp on plasma ages and ros production by pbmcs at baseline and after zinc supplementation was also studied ). + g+ carriers showed increased plasma ages and ros production in pbmcs at baseline with a significant interaction of genotype on zinc supplementation only in limited markers of intracellular zinc status. more recently, a randomized, double-blind, placebo-controlled study (with mg zn/day) has been performed on n = nursing home elderly (aged ! year;) with low serum zinc concentration (serum zinc < μg/dl) versus n = placebo ( mg zn/day) group. in this case, the treatment group showed an increase in serum zinc concentration that was associated with an increase in the number of t cells (barnett et al. ) . it is also noteworthy to mention the effects of a zinc fortified milk (designed to integrate mg zinc/day for months) on the cytokine production of pbmcs obtained from very old subjects (age > years). in unstimulated pbmcs from these subjects a significant percent change ( p < . or p < . ) of cytokine release is evident and is related to a good functioning of the cell-mediated immunity ( . % il- p and . % ifn-γ) coupled with increase of anti-inflammatory cytokines ( . % il- ) and decrease of pro-inflammatory cytokines ( . % il- α) ). thus, it seems evident from these studies that physiological dose of zinc for a long period or high doses of zinc for short periods might induce limited effects on immune response. although the body of proof of an impact of zinc on immunosenescence is consistent, there is limited knowledge on the long-term effects and outcome (e.g., protection from infection) of zinc supplementation in elderly. current evidence suggests that benefits on immune response and health outcome are likely to be obtained with doses around the rda in elderly with documented zinc deficiency. unfortunately, we do not have in this moment a reliable assessment of zinc status that can undoubtedly identify subjects with zinc deficiency. taking into account this lack of knowledge, it is recommended to educate elderly to consume an appropriate diet with foods that contain the necessary zinc requirements. the effect of zinc supplementation may be related to the levels of other cations such as cadmium, lead, calcium, iron, manganese, and copper. the beneficial effects of zinc on ameliorating toxicity of cadmium and lead, the accentuation of zinc deficiency by administration of calcium and phytate, and production of hypocupremia by excessive zinc intake in humans and animals are some examples of competition phenomena between these cations (hill ) . such a competition occurs because these ions have similar valence shell electronic structure and therefore could be antagonist to each other. for instance, the competition between zinc and iron (fe++) occurs at the level of cysteine-histidine ligands for the formation of iron or zinc "fingers" proteins (prasad ). if iron is in excess, a preferential binding of iron than zinc to the metal free-protein occurs. excess of zinc or zinc deficiency impairs dna-protein interactions of zinc fingers domains with their cognate dna target sites. in these conditions the production of some transcriptional factors like sp or tfiiia is altered (thiesen and bach ) . similar transcriptional alterations occur in excess or deficiency of copper (prasad ) . this reinforces the notion of the relevance of interactions between zinc and copper as well as with other metals in the immune efficiency (sandstead ) . in order to avoid interference with copper homeostasis, it has been proposed to not exceed - times the rda with zinc supplementation and to perform alternate cycles of supplementation not longer than - months (licastro et al. ; feillet-coudray et al. ). combination of zinc in complex micronutrient supplements appears to produce minimal and contrasting effects. health status was recently investigated in older french adults years after a period of daily nutritional-dose supplementation with antioxidant nutrients (assmann et al. ) . during - , participants received a daily combination of vitamin c ( mg), β-carotene ( mg), vitamin e ( mg), selenium ( μg), and zinc ( mg) or placebo. healthy aging was assessed in - with multiple criteria (e.g., absence of major chronic disease and good physical and cognitive functioning). supplementation was associated with a slightly greater healthy aging probability among men, but not among women or all. similarly, another study assessing a micronutrient mix ( mg vitamin e, mg vitamin c, mg beta-carotene, and mg zinc/day for weeks) on immune function parameters of a population of subject aged - years showed only an increase of delayed-type hypersensitivity (dth) responses (in particularly in the oldest subjects) without any change in responses to systemic tetanus and oral typhoid vaccination, phagocytosis, oxidative burst, lymphocyte proliferation, and lymphocyte subset distribution (wolvers et al. ) . importantly, vitamin and mineral supplements (including zinc) in older women were found to be associated with increased total mortality risk compared with nonusers in other studies (bjelakovic and gluud ; mursu et al. ) . these results reinforce the idea that nutritional guidelines instead of supplementation might be the preferred choice, while keeping interventions with micronutrients only for short periods in case of documented deficiencies. selenium is considered since long time ago an essential dietary element for the prevention of some diseases, including cancer and infections (schwarz ). in food, selenium derives from vegetables and animal products and in particular from the consumption of seafood, liver, and cereals. however, in vegetables and cereals the amount of selenium varies in soil in different countries and geographical regions (wasowicz et al. ) . indeed, selenium deficiency and related diseases have been well documented in geographic regions where the soil content is low, such as the chinese province of keshan (li et al. ) . from this region of china, in fact, was taken the name of the keshan disease, a pathology characterized by selenium deficiency and the presence of mutated strains of coxsackievirus . mammals can use both inorganic and organic selenium as a nutrient. most of the biological functions of selenium are attributed to selenoproteins, which contain selenocysteine residues responsible for their specific activity. selenoproteins are present in every cell type. the human selenoproteome consists of selenoproteins, mostly involved in antioxidant defense systems (kryukov et al. ) . glutathione peroxidases (gpxs), a family of the selenoproteins, protect cells against oxidative damage by catalyzing the reduction of hydrogen peroxide and other hydroperoxides (hall et al. ; brigelius-flohé ) . five seleniumdependent gpx isoforms exist in humans and four isoforms in mice. gpx is found in the cytosol of almost all cells and catalyzes the reduction of free hydroperoxides. gpx is expressed in the gastrointestinal tract and has a substrate specificity similar to gpx ; gpx is an extracellular enzyme found in plasma and reduces membrane-bound phospholipid hydroperoxides (brigelius-flohé ) . gpx is expressed in various tissues, and reduces phospholipid hydroperoxide and hydrogen peroxide using also thiols, such as -mercaptoethanol, cysteine, and homocysteine, other than gsh as reductant agents (roveri et al. ). the isoform gpx seems to be specifically expressed in embryonic tissues and olfactory epithelium (kryukov et al. ) . it also exists as a selenium-independent isoform, gpx , which is an epididymis isoenzyme present in mice and humans (hall et al. ), but its mrna was found to be not translated into functional protein in human epididymis (ghyselinck et al. ) . selenium is also involved in the thioredoxin system, a major enzymatic system that plays an important role in maintaining the redox state of the cell (holmgren ) . this system is highly complementary to the gsh system in protecting against oxidative stress (watson et al. ) . it comprises basically of thioredoxin (trx) and the selenoprotein thioredoxin reductase (tr) and uses the reducing power of nadph to act as a potent antioxidant system as well as a general disulfide redox system (rundlof and arner ) . mammalian tr maintains trx in a reduced state (holmgren ) and reduces a variety of other substrates including nondisulfides. the thioredoxin system protects the cell against oxidative stress through a variety of mechanisms. trx can directly quench single oxygen and scavenge hydroxyl radicals (das and das ) , or reduced trx can indirectly serve as an electron donor for trx peroxidase. in addition, human tr is directly capable to efficiently reduce lipid hydroperoxides, hydrogen peroxide, and organic hydroperoxides using nadph, especially in the presence of catalytic amount of selenocysteine, thus serving as an important alternative to the gpx pathway for the elimination of harmful hydroperoxides (björnstedt et al. ) . trx system is also critical for signal transduction (arner and holmgren ) and in the restoration of the reduced form of several antioxidant compounds, including ascorbic acid, lipoic acid, and ubiquinone (nordberg and arner ) . in this context, selenomethionine, a potent catalytic antioxidant in biological system and an aminoacid occurring in proteins in place of methionine (walter and roy ) , reacts more efficiently than methionine (padmaja et al. ) with oxidants forming methionine selenoxide which, in turn, is effectively and rapidly reduced to seleniomethionine by glutathione (assmann et al. ) . in contrast, methionine sulfoxide, which is produced by the oxidation of methionine in presence of oxidants, is not simply reduced by gsh, but it requires a specific enzymatic reaction catalyzed by methionine sulfoxide reductase (levine et al. ) . since selenomethionine can occur in proteins such as hemoglobin (beilstein and whanger ) , these residues may play a defensive role against peroxinitite. another selenoprotein, which reduces phospholipid hydroperoxides in the presence of thiols, is the selenoprotein p (sep) (burk et al. ) . sep is expressed in many tissues and represents the major plasma selenoprotein, which contains % of the total plasma selenium in the form of selenocysteine. sep protects endothelial cells against damage from peroxynitrite and transports selenium from the liver to peripheral tissues. last, but not the least in order of importance, is a class of selenoproteins (iodothyronine deiodinase enzymes), which catalyze the peripheral deiodination of thyroxin (t ) to , -triiodothyronine (t ). these enzymes play crucial roles in determining the circulating and intracellular levels of t and, consequently, the control of growth, development, differentiation, metabolism, and finally also the immune response (kohrle ; beckett and arthur ) . immunologically, the ability of selenoproteins to protect the host from oxidative stress is vitally important, as many host defense systems rely on the microbiocidal effects of macrophage-or neutrophil-generated free-radical species. oxidative species are generated through general metabolism, during the metabolism of xenobiotics and during exposure to ultraviolet radiation (uv) in sunlight. inflammation as a process to clear infection and damaged tissue also generates great oxidative stress. if antioxidant systems are not functioning correctly, host cells will be damaged (mckenzie et al. ) . taking into account that chronic inflammation and oxidative stress are features of aging (franceschi et al. ) , it is likely that se mediates protective effects through upregulating the antioxidant defenses and host immune responses. the influence of selenium on the immune function can be, in part, attributed to the same selenoproteins involved in the protection against oxidative damage and, in part, to still undefined biochemical pathways. the antioxidant gpxs have probably a role in protecting neutrophils from ros that are produced during inflammation arthur ) . selenium supplementation, in mice, increases the expression of subunits alpha (p ) and/or beta (p / ) of il- receptor (il- r) from activated lymphocytes and nk cells, thereby enhancing proliferation and clone expansion of cytotoxic precursor cells. in vitro, selenium enhances the release of tumor necrosis factor (tnf), il- , and il- from lps-stimulated macrophages (beckett et al. ). however, one of the most widely investigated associations between selenium and the immune system is the effect of the micronutrient on neutrophil function. neutrophils produce superoxide-derived radicals to take part in killing of microbes. this type of process is a balance between the production of sufficient radicals to kill invading organisms and the systems that protect the neutrophils themselves from the radicals. thus, although selenium deficiency does not seem to affect neutrophil number, certain aspects of their function are defective (turner and finch ) . neutrophils from selenium-deficient mice, rats, and cattle are able to ingest pathogens in vitro but are less able to kill them than are neutrophils from selenium-sufficient animals. this defective function has been associated with decreased cytosolic gpx (gpx ) activity in the neutrophils, which allows the free radicals that are produced in the respiratory burst to kill the neutrophils themselves . this is the reason why selenium deficiency may involve oxidative damage of biomolecules, such as lipids, lipoproteins, and dna, that is well known to play a role in many age-related diseases. anyway, selenium deficiency is a condition mainly attributed to geographical factors that include a low selenium content in the soil or to long-term parenteral nutrition rather than aging itself. selenium levels in blood have been studied in different age-related diseases such as cancer, cardiovascular disease, and immune dysfunctions. additionally, as for many other micronutrients, se inadequacy likely due to malnutrition or intestinal malabsorption may occur in older people (seiler ) . however, few data reported a marked selenium deficiency in old subjects. in the baseline results of the su.vi.m.ax study (involving women aged - and men aged - ), it was reported that fewer than % of the volunteers had a serum se status under . μm, which has been proposed as the threshold for se sub-deficiency and that age was associated with increased serum se concentrations in women (arnaud et al. ) . however, in the same study, large part of the population displayed a value of blood selenium below the range of levels ( . - . μm) proposed as optimal for gpx and selenoprotein p (combs ; thomson ). in the eva (etude du vieillissement artériel) study, a longitudinal study that explored the relationships between plasma selenium and mortality in an elderly population for over years, mortality rates were significantly higher in individuals with low selenium [increments = . μm; relative risk (rr) = . ; % ci = . - . ] (akbaraly et al. ) . when the underlying causes of death were considered, an association with low selenium and cancer-related mortality was found. the same authors suggest that plasma selenium could be an indicator of longevity in the pre-aging period of life. survival curves illustrate that the relationship between plasma selenium and mortality remained pertinent during the entire -year period (akbaraly et al. ) . however, the mechanism of this potential relationship is still unclear. other authors observed selenium deficiency in elderly people in relation to hypothyroidism (olivieri et al. ) . interestingly, most of human healthy centenarians seem to display selenium values equal or greater than the lowest values reported in normal elderly (savarino et al. ) . the relevance of selenium in the etiology of cardiovascular diseases has been also extensively studied. selenium metabolism is potentially involved in several protective biochemical pathways related to cardiovascular disease, such as reduction of ldl levels and lipoprotein oxidation, inhibition of foam-cell formation, and shift in prostaglandin production from prostacyclin to tromboxane (alissa et al. ) . however, wei et al. ( ) found no association between death for cardiovascular diseases and baseline selenium status in a cohort of adult individuals (mean age, years) with a mean serum concentration of . μm. finally, an intriguing point is the association between selenium deficiency, immune response, and increased incidence of infections in adults and elderly. in a prospective study performed in patients with systemic inflammatory response syndrome, a % decrease in plasma selenium concentrations was observed compared to reference range (forceville et al. ). moreover, low levels of selenium in these patients were associated to morbidity and mortality while survivors at follow-up displayed slightly increased selenium levels. the interrelationships between selenium deficiency, impaired immune response, and infections have been clearly shown in experimental animals. an inoculated avirulent virus in selenium-deficient animals turns into a virulent one due to genomic changes within the virus, provoking an impaired humoral immune defense (beck ). an enhanced oxidative stress, caused by selenium deficiency, is the likely reason of possible viral genetic changes (beck et al. ) and increased progression of viral infections with subsequent impaired immune defense (daniels ) . regarding the role played by selenium deficiency in viral infections it is also noteworthy to mention the: (i) the emergence of newly recognized human disease agent (coronavirus) that causes sars from guangdong province of china (lashley ) as well as from northern vietnam (reynolds et al. ) , where significant areas of overt selenium deficiency exist (xia et al. ) and (ii) the increased risk of enhanced virulence of influenza virus in elderly (ellis et al. ) associated with a possible selenium deficiency (seiler ) . therefore, selenium deficiency may be considered a risk factor for age-related diseases (cancer, cardiovascular diseases, and infections). such a risk is of relevance in elderly because accumulating data suggest that persistent infection with varicella-zoster virus (vzv) (arvin ) , epstein-barr virus (ebv) (stowe et al. ) , and particularly cmv (mcvoy and adler ) impacts upon the immune system in aging and may contribute to the immune risk phenotype (irp), which predicts remaining longevity in the very elderly (pawelec et al. ) . specific study on these aspects should be encouraged taking into account the possible relevant implications for public health. in vitro studies cellular studies with selenium provide evidence that, in analogy with zinc, it may have anticarcinogenic and antioxidant effects at low concentrations, whereas at concentrations higher than those necessary for nutrition, it can be genotoxic and carcinogenic (valdiglesias et al. ) . se toxicity "in vitro" depends primarily on se compound and dose but also on exposure time and cellular model. conversely to the reputation of an antioxidant and protective trace element, various "in vitro" studies found that se compounds can induce dna damage (biswas et al. ; machado et al. ) and produce oxidative stress (wycherly et al. ). an investigation on chromosome breaking activity of various se compounds demonstrated that all may induce this kind of damage with an efficacy that follows this order: selenious acid > sodium selenite > se dioxide > selenic acid > sodium selenite (nakamuro et al. ) . as a consequence, it is not surprising that selenocompounds (mainly sodium selenite but also semet, se dioxide, and methylseleninic acid) induce cell death in various mammalian cell lines (valdiglesias et al. ). an intensive investigation on apoptosis-inducing effects of organic selenium derivatives showed that breast carcinoma cells were highly sensitive to the organic selenium compounds, manifesting apoptosis at . μm of selenium while nontumorigenic mammary epithelial cells required concentration above μm. cell lines derived from hepatoma and neuroblastoma showed intermediate sensitivity and colon carcinoma cells were more resistant to selenium-induced apoptosis (jariwalla et al. ) . a role for se in dna repair has been also documented, likely as a consequence of its modulatory activity on nrf pathway both in tumor and normal tissues (zhang et al. ) . however, the concentrations of selenium compounds, in combination with the medium components and the biological physiology of the cell, affect the redox potential of the compounds that may switch from pro-oxidant to antioxidant (cemeli et al. ) . a recent paper investigated the ability of selenium supplementation in vitro (in the form of na seo nm or nm) to modify monocyte cell viability and ros production under condition of oxidative stress (induced by paraquat mm and s-nitroso-n-acetyl-dl-penicillamine mm) (leighton et al. ) . the experiments indicated that selenium supplementation, especially with the lower dose tested, was effective in improving cell viability and ros quenching ability of monocytes. a previous study addressing the influence of selenium supplementation "in vitro" on macrophages reported enhanced phagocytosis, degranulation, and production of superoxide anion after phorbol myristate (safir et al. ) . in general, it is not easy to give an interpretation of the results "in vitro," especially when addressed at generation of ros and immune function. indeed, ros are not only used to kill pathogens, but they have been recently recognized as important mediators of cell signaling and cell to cell communication in phagocytic and nonphagocytic immune cells . so, even if we know that se compounds in vitro may affect ros production and phagocyte function (huang et al. ) , the overall rationale for supplementation strategies can only derive from "in vivo" supplementation studies. furthermore, there is evidence that as in the case of zinc the expression of several selenoproteins involved in antioxidant defense is specifically affected in response to cellular senescence (yona et al. ; malavolta et al. ; provinciali et al. ) . taking into account that senescent cells accumulate with aging and that cellular senescence is a phenomenon that displays antagonistic pleiotropic effects (campisi ) on physiological and pathological processes, it is likely that many changes observed with aging and attributed to changes in selenium status involve the process of cellular senescence and that their impact on overall health are rather difficult to be correctly predicted. multiple experimental studies in animal models have revealed that se-deficiency impairs immune response to infection, cancer, and other stimuli. for example, se deficiency was reported to reduce cd + t cell response in mice challenged with a peptide/adjuvant (hoffmann et al. ) , to increase tumor growth and burden in a breast cancer mouse model (chen et al. ) , to increase type i allergic response in a mouse model of cutaneous anaphylaxis (arakawa et al. ) , and to increase immunotoxicity of arsenic (rodríguez-sosa et al. ) . the il- and interferon-γ pathways appear to be among the pathway more responsive to dietary selenium intake in mice (tsuji et al. ) . although excessive releases of il- and interferon-γ have been associated with inflamaging, moderate increases and adequate response of these cytokines are known play a pivotal role in host defense through the ability to activate macrophage cell functions thus contributing to keep under control infectious diseases. decreased dietary selenium can change a normally avirulent b coxsackievirus (cbv / ) into a virulent virus (cbv / ) by inducing changes in viral genoma, especially in viral rna polymerase (duarte et al. ). the mutated virus can infect heart muscle and cause myocarditis with the likely development of dilated cardiomyopathy and death (beck and levander ; jun et al. ) . similar to the data reported for coxsackie virus, selenium intake and selenium status affected the host immune response, the mutation rate of the viral genome, and the pathology in mice infected with the human influenza a/bangkok/ / (h n ) virus (nelson et al. ) . however, the impact of selenium status on the outcome of the immune response appears to depend on the virulence of the applied influenza a virus strain. indeed, opposite results with an increased mortality of the selenium adequate group were observed using a the influenza a/puerto rico/ / virus, which is highly pathogenic in mice (li and beck ) . with regard to cancer, many studies have investigated the effects of selenium in carcinogen-exposed animals showing a reduction in tumor incidence and/or pre-neoplastic endpoints (reid et al. ) . various animal experiments have been conducted by combining experimental selenium deficiency with treatment with carcinogens, such as , -dimethylhydrazine (dmh) or dimethylbenz(a)anthracene (dmba), and comparing the results with animals fed with higher content of selenium in the diet. in general, se deficiency appears to affect dmh toxicity with, however, no inhibition of tumor development by nutritional se ( . ppm se) (pence and buddingh ) . three relevant papers report a greater development of carcinoma by dhm or dmba in various organs (colon and mammary gland) of rats under selenium deficiency in comparison with rats treated with ppm of se (jacobs ; liu and milner ; mcgarrity and peiffer ) . these findings further suggest the ability of dietary selenium to inhibit the in vivo metabolism of carcinogens dmba or dmh and, consequently, to inhibit the development of the tumor. selenium supplementation in old mice was also shown to counteract immunosenescence (roy et al. ) . in c bl/ jnia mice aged months, supplementation with selenium ( . ppm as sodium selenite for weeks) corrected age-related defect in lymphocyte proliferation likely as a consequence of an increased number of high-affinity il- receptors. unfortunately, few trials have been carried out up in elderly with a clear focus on immunosenescence. although selenium supplementation improved lymphocyte mitogen responsiveness of institutionalized elderly individuals (peretz et al. ) , it is difficult to conclude on a specific beneficial effect of selenium in immunosenescence. a finnish study adding selenium to fertilizer has shown only an increased selenium status in the general population (young, adult, old) (aro et al. ) without any clear beneficial effect on general health. a recent randomized, doubleblinded, placebo-controlled clinical trial was undertaken to test the effects of se supplementation on a variety of parameters of anti-flu immunity in healthy subjects aged - years (ivory et al. ) . the authors used six doses of se (in different form from to μg/day) for weeks in six groups of individuals with plasma se levels < ng/ml who received flu vaccine (at week ). in this trial, se supplementation resulted in both beneficial and detrimental effects on cellular immunity to flu that was affected by the form of se, supplemental dose and delivery matrix. in general, the dose-dependent increase in t cell proliferation, il- and il- secretion after in vivo flu challenge were contrasted by lower granzyme b content of cd cells or by inhibition of tnf-α synthesis. another recent study was conducted to evaluate the effects of selenium supplementation ( μg selenium supplements for weeks) on metabolic profiles, biomarkers of inflammation, and oxidative stress of patients with type diabetes and coronary artery disease aged - years (farrokhian et al. ) . supplemented patients displayed a significant decrease in insulin, homeostasis model of assessment-insulin resistance, homeostatic model assessment-beta cell function, serum hs-crp, and a significant increase in quantitative insulin sensitivity check index score and plasma total antioxidant capacity concentrations. the positive impact on these biomarkers is, unfortunately, not complemented by a long-term follow-up. however, reduced cardiovascular mortality at -years follow-up was observed in another supplementation trial performed on elderly with selenium and coenzyme q (alehagen et al. ) . in agreement with the protective role of selenium a sweden study performed in elderly subjects showed that persons with serum se in the lowest quartile had % and % increased risk for all-cause and cardiovascular mortality, respectively (alehagen et al. ). this confirms the reduced incidence of cardiovascular diseases observed in previous studies after supplementation with selenium combined in multivitamins supplements (czernichow et al. ; shenoy et al. ) . there is also evidence that a number of hiv-positive patients suffer from deficiencies in vitamins including selenium (coodley ) . enhanced gpx and gsh activity (delmas-beauvieux et al. ) were observed in hiv patients receiving oral selenium supplementation. this finding suggests that gpx and gsh activity may represent natural inhibitors of (aids) viruses and that selenium supplementation may play a role in prevention antiviral strategies (rayman ) . of interest, supplementation with multivitamins and trace elements, including se, during treatment of pulmonary tuberculosis reduced mortality in subjects co-infected with hiv (range et al. ) . large part of the other studies involving selenium supplementation in humans have been conducted in adult subjects with the aim to prevent or counteract various type of cancer. past studies showed that supplementation with μg/day of organic selenium in randomized subjects showed preventive effects in the incidence and the mortality from various types of cancer (prostate, colorectal, and lung cancer) (clark et al. ) . another large supplementation trial with lower amount of selenium ( μg) performed in lixian (north china) showed a small but significant reduction in total and cancer mortality was observed in subjects receiving selenium supplement (blot et al. ) . the impact of se supplementation was better in women than men and, interestingly, the effects were more pronounced in persons under the age of years compared to individuals older than years. moreover, supplementation with mg/day of sodium selenite during therapy of patients with squamous cell carcinoma of the head and neck resulted in a significantly enhanced cell-mediated immune responsiveness (better response of lymphocytes to stimulation with mitogen, generation of cytotoxic lymphocytes, and destruction of tumor cells) (kiremidjian-schumacher et al. ) . considering these results, it might be assumed that younger persons might be more amenable to a protective effect of selenium supplementation. however, a recent large selenium and vitamin e cancer prevention trial (select) carried out in north america has reported unexpected and very concerning findings, especially the increased the risk of high-grade prostate cancer among men with high selenium status (kristal et al. ) . perhaps, the most conclusive data regarding the story of selenium and cancer can be retrieved from an excellent systematic review that included prospective observational studies as well as randomized clinical trials (vinceti et al. ) . the authors of this systematic review concluded that the inverse association between selenium exposure and the risk of some types of cancer cannot be taken as evidence of a causal relation, and that all these studies have many limitations, including issues with the form and the assessment of selenium, heterogeneity, confounding and other biases. moreover, randomized clinical trials reported inverse, null, and direct associations have been reported for some cancer types which overall yielded inconsistent results with even harmful effects of selenium exposure. hence, to date, no convincing evidence suggests that selenium supplements can prevent cancer in humans. dietary zinc and selenium are important nutritional factors for the immune response in protecting against the appearance of age-related diseases. there is a number of biochemical processes in which selenium and zinc interact. selenium compounds that have the capacity to form selenol(ate)s catalytically couple with the glutathione/ glutathione disulfide and metallothionein/thionein redox pairs to either release or bind zinc. (maret ) . when mt are oxidized by glutathion disulfide (gssg) or other disulfides free zinc is released in the cytosol. however, the efficiency of this chemical reaction seems very low even at high concentrations of gssg in the absence of selenium. in contrast, the release of zinc from mt may occur very rapidly in presence of selenium compounds have the capacity to form selenol(ate)s (maret ) . the mechanism of the reaction was suggested to proceed through an activated selenenyl sulfide r-se-s-g intermediate which, in turn, oxidizes the zinc-thiolate cluster of mt to form r-se-s-mt with the concomitant release of zinc during the oxidation (chen and maret ) . the selenol group is subsequently released by the attack of a nearby thiol group of mt that convert r-se-s-mt into thionein generating a catalytic cycle of oxidative zinc release from mt. other oxidized selenium compounds, such as selenoxide and selenic acid, may be directly reduced by mt through the formation of a r-se-s-mt intermediate and the concomitant release of zinc, followed by the formation of an inter-or intramolecular disulfide bond (jacob et al. ; chen and maret ; klotz et al. ) . selenium compounds also catalyze the release of zinc from mt in peroxidation and thiol/disulfide-interchange reactions. in presence of t-butylhydroperoxide, gpx catalyzes the oxidation of mt with subsequent zinc release, suggesting that mt may serve as reducing agents for gpx (or at least some gpx isoforms) in alternative to gsh (jacob et al. ) . these results suggest that zinc release is a significant aspect of the therapeutic antioxidant actions of selenium compounds in anti-inflammatory and anticarcinogenic agents. hence, the assessment of intracellular labile zinc and selenoproteome during a challenge with oxidative compounds might be useful to understand the physiology of successful aging. while there are no studies specifically addressing these aspects in humans, it is of interest to consider the results of a recent study that examined elements in the brain, heart, kidney, and liver of mammalian species, and studied their correlation with body mass and longevity (ma et al. ) . zn levels in liver and kidney showed strong, positive correlations with species maximum lifespan and maximum lifespan residual whereas liver se was the only element correlating negatively with all measures of longevity, although the correlations were relatively weak. it is interesting to relate this study with the finding that the naked mole rat (a rodent model of delayed aging with a life span > years) is characterized by a reduced utilization of selenium due to a specific defect in gpx expression (kasaikina et al. ) . a possible interpretation of these results is that there is a delicate balance between the beneficial aspects of se, its toxicity, and other systems that support maintenance functions. supplementation studies with zinc and selenium in humans are also poor, especially with a focus on immunosenescence. girodon et al. ( ) determined the effects of a long-term (for years) daily supplementation with zinc ( mg) plus selenium ( μg) on immunity and the incidence of infections in a large number (n. ) of institutionalized elderly people (> years). the main results of the study were: ( ) selenium-deficient patients decreased from about % to - % in the selenium supplemented group after months of supplementation with respect to placebo group; ( ) antibody titres after influenza vaccine were higher in groups that receive trace elements; and ( ) trace element-supplemented patients were those who remained most free of respiratory tract infections than placebo group. these findings suggest that low dose supplementation of zinc and selenium provides significant improvement in elderly patients by increasing the humoral response after vaccination and decreased influenza compliances (respiratory tract infections) with thus a possible impact on the maintenance of health conditions during aging. however, conversely to the conventional belief and claims that supplementation with zn and se may improve health conditions there are important studies that observed lack of clear effects. the large su.vi.max trial conducted with a supplement of antioxidant including zinc and selenium reported no effects on health-related quality of life after months of supplementation in french adults (briançon et al. ). in addition, supplementation with low or medium doses of zinc and selenium provided no benefits, neither were able to correct low zinc or selenium status in hemodialysis patients (tonelli et al. ) . in conclusion, conversely to the past belief that zinc and selenium supplements could be used as elixir of long life, recent epidemiologic and laboratory studies are changing our perception of the biological effects of these essential trace elements. while several studies suggest that correcting zinc and selenium deficiency in aging provide a beneficial impact on immunosenescence, it is evident that controversial finding exists on the "real" necessity of micronutrient supplementation (dangour et al. ) . the critical step of supplementation strategies with zinc and selenium still remains the diagnosis of zinc and selenium deficiency, as there are no validated methods to assess their status in aging. however, in the case of documented deficiency, it seems to make sense to provide selenium supplements associated with zinc and even with additional supplements. indeed, micronutrient deficiencies are usually not isolated and some biochemical mechanisms involved in the action of selenium are under the control of zinc ion bioavailability. in other words, the attempt to correct selenium deficiency alone may result in lack of beneficial effects due to the presence of zinc or other micro-and macronutrient deficiency. many points still require further investigations, first of all, the possible involvement of mt, zinc, and selenium in antagonistic pleiotropic mechanisms that regulate mammalian life span. one of these mechanisms is cellular senescence, which has gained considerable attention in these last years as a useful anti-aging target. useful tools are available, such as no donors and zinc fluorescent probes (zinpyr- and fluozin- ) to help researcher in the quantitative assessment of labile zinc and on the functional role of mt haase et al. ) in senescent cells. these tools applied in supplementation trials performed in animal models that allow to visualize, sort, and selectively eliminate senescent cells (demaria et al. ) could help to clarify these mechanisms. additional insight could be provided by the investigation of serum trace elements associated with mt induction in pbmc obtained from centenarian's offspring in the framework of markage project (bürkle et al. ) . the results may help to understand the physiological mechanisms involved in healthy aging and may help to understand new strategies for nutritional or pharmacological intervention aimed at increased 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signaling acknowledgments the study has been partially funded through the revenues of the " per mille" donations received by inrca through the italian ministry of health. the authors declare that there are no conflicts of interest. key: cord- -wsjob p authors: wang, yan-hang; lv, hai-ning; cui, qing-hua; tu, peng-fei; jiang, yong; zeng, ke-wu title: isosibiricin inhibits microglial activation by targeting the dopamine d /d receptor-dependent nlrp /caspase- inflammasome pathway date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: wsjob p microglia-mediated neuroinflammation is a crucial risk factor for neurological disorders. recently, dopamine receptors have been found to be involved in multiple immunopathological processes and considered as valuable therapeutic targets for inflammation-associated neurologic diseases. in this study we investigated the anti-neuroinflammation effect of isosibiricin, a natural coumarin compound isolated from medicinal plant murraya exotica. we showed that isosibiricin ( − μm) dose-dependently inhibited lipopolysaccharide (lps)-induced bv- microglia activation, evidenced by the decreased expression of inflammatory mediators, including nitrite oxide (no), tumour necrosis factor-α (tnf-α), interleukin- (il- ), interleukin- β (il- β) and interleukin- (il- ). by using transcriptomics coupled with bioinformatics analysis, we revealed that isosibiricin treatment mainly affect dopamine receptor signalling pathway. we further demonstrated that isosibiricin upregulated the expression of dopamine d / receptors in lps-treated bv- cells, resulting in inhibitory effect on nucleotide binding domain-like receptor protein (nlrp )/caspase- inflammasome pathway. treatment with dopamine d / receptor antagonists sch ( μm) or sultopride ( μm) could reverse the inhibitory effects of isosibiricin on nlrp expression as well as the cleavages of caspase- and il- β. collectively, this study demonstrates a promising therapeutic strategy for neuroinflammation by targeting dopamine d / receptors. dopamine, as a crucial neurotransmitter, can transmit neuronal signals to regulate neurological function by acting on specific dopamine receptors [ , ] . currently, the dopamine receptor family is considered to play a key role in various advanced neural functions, including emotion, reward and autonomic movement [ ] . in addition, increasing attention has been paid to the role of the dopamine signal system in immunomodulation and inflammation control [ , ] . previous studies have shown that the genetic and pharmacologic regulation of dopamine receptors can have potential anti-inflammatory effects. for instance, dopamine d receptor (drd ) activation is widely involved in the alleviation of inflammatory bowel diseases [ , ] , pancreatic inflammation [ ] and arthritis [ ] . specifically, drd /d have been found to be widely expressed not only in neurons and t cells [ ] but also in microglia [ ] , indicating that drd /d might also be potential therapeutic targets for neuroinflammation in the central nervous system (cns). recently, the pharmacologic activation of drd was shown to exert an obvious inhibitory effect on neuroinflammation induced by intracerebral haemorrhage, by regulating the nuclear factor-κb (nf-κb) signalling pathway [ ] . moreover, new therapies based on drd /d agonists have been developed for experimental autoimmune encephalomyelitis [ ] . therefore, treating the microglia-derived inflammatory response by targeting dopamine receptors appears to be a promising therapeutic strategy for neuroinflammation. in particular, dopamine receptors have been found to be highly associated with the inflammasome signalling pathway. some previous research has suggested that spinal cord injury induces inflammatory cytokine production by activating the nucleotide-binding domain-like receptor protein inflammasome pathway, which is significantly suppressed by drd agonists [ ] . moreover, drd can also block nlrp inflammasome activation in a β-arrestin-dependent manner [ ] . collectively, these findings provide a potential therapeutic strategy for treating the neuroinflammatory response by targeting the drd-mediated inflammasome pathway. murraya exotica l. (rutaceae) has traditionally been used in china for the treatment of infectious diseases and pains [ ] . modern pharmacological studies have suggested that murraya koenigii and m. exotica possess a wide range of biological activities, including anti-inflammatory, antinociceptive, antioxidative stress, anti-mutagenic stress, and chondroprotective and anticancer metastasis effects [ , ] . isosibiricin is a natural bioactive coumarin compound isolated from m. exotica. several studies have indicated that the coumarin derivatives isofraxidin, osthole and urolithin can exert obvious anti-inflammatory activities both in vitro and in vivo [ ] [ ] [ ] . therefore, in this study, we explored the mechanism of the anti-neuroinflammatory effects of isosibiricin in a bv- microglial model and highlighted that isosibiricin can significantly inhibit the production of multiple inflammatory mediators induced by bacterial lipopolysaccharide stimulation via targeting the drd /d -dependent inflammasome pathway, providing a potential therapeutic strategy for inflammation-related neurological disorders. mouse strains balb/c mice ( - weeks old) were purchased from the vital river laboratories (beijing, china). all mice were raised in a specific pathogen-free environment. the bv- cell line was purchased from the cell bank of peking union medical college in china. the cells were cultured in dulbecco's modified eagle's medium containing % fetal bovine serum, u/ml penicillin and mg/ml streptomycin. the cell culture conditions were maintained at °c in a % co incubator under % absolute humidity. nitrite oxide analysis bv- cells were treated with isosibiricin ( , , and μm) with or without lps for h. then, the supernatants were used to analyse the production of nitrite oxide (no) with a nitric oxide analysis kit (jiancheng bioengineering institute, nanjing, china) enzyme-linked immunosorbent assay bv- cells were treated with isosibiricin ( , , and μm) with or without lps. after treatment for h, μl of the cell supernatants was centrifuged at × g for min to evaluate the accumulation of tumour necrosis factor-α (tnf-α). in addition, μl of the cell supernatants was also centrifuged at × g for min to evaluate il- levels h after treatment. the two experiments were carried out with enzyme-linked immunosorbent assay (elisa) kits (excell bio company, shanghai, china) following the manufacturer's protocol. the cells were collected and rna was extracted by the rnaprep pure cell/bacteria kit (tiangen biotech, beijing, china). target analyses were carried out by the yuanquan yike company (beijing, china). amino allyl-labelled antisense rna was prepared and purified prior to hybridization. purified, coupled antisense rna was quantified using nanodrop nd- to explore the targets affected by isosibiricin. transcriptome analysis pathway analysis [ ] on isosibiricin was also performed by the yuanquan yike company. after the targets of isosibiricin were determined by amino allyl-labelled antisense rna hybridization, kyoto encyclopedia of genes and genomes (kegg) pathway enrichment was used to perform the pathway analysis. signal pathways significantly influenced by isosibiricin compared with lps were selected. western blotting after treatment, the cells were lysed in ice-cold ripa buffer containing % protease inhibitors (macgene, beijing, china). whole-cell proteins were collected by centrifugation at × g for min. the concentration of the total proteins was detected by a protein assay kit (transgen, beijing, china). protein solutions of equal concentration were separated by %- % sds-polyacrylamide gel electrophoresis and transferred to pvdf membranes. next, the pvdf membranes were blocked with % skim milk at °c for min. subsequently, the membranes were incubated with primary antibodies at °c for h. after incubation with a second antibody for h, the membranes were developed with supersignal west femto maximum sensitivity substrate. the protein bands were imaged by a tanon imaging analysis system (tanon, shanghai, china). relative densitometry analysis was carried out by imagej software. reverse-transcription pcr the cells were collected and total rna was extracted using the rnaprep pure cell/bacteria kit (tiangen biotech, beijing, china). the mrna was reverse transcribed using a cdna synthesis kit (transgen, beijing, china). reverse-transcription pcr (rt-pcr) was performed using a rt-pcr superkit (transgen, beijing, china) on an agilent technologies stratagene mx p system. a single cycle was carried out at °c for s, °c for s and °c for s, and this cycle was repeated times. the relative transcriptional levels of drd and drd normalized to those of gapdh was calculated by the comparative −ΔΔct method [ ] . dopamine receptor agonist analysis hek cells stably expressing drd /gα were cultured in -well plates for h. after the supernatant was removed, fluo- / am, a calcium fluorescence probe, was added at °c for min in an incubator under % absolute humidity. the fluo- /am was removed and a calcium buffer solution was added. flexstation ii was used to measure the levels of gα protein activation induced by isosibiricin ( , . nm, . nm, nm, nm, nm, μm and μm) at nm. dopamine was used as a positive control. the ec was analysed by graphpad prism. all experiments were carried out by the national center for drug screening (shanghai, china). nine balb/c mice were randomly divided into the control, lps and isosibiricin groups. the isosibiricin group was treated with isosibiricin ( mg/kg body weight). after h, both the lps group and the isosibiricin group were injected intraperitoneally with lps ( mg/kg body weight). then, the brain tissues were sectioned and stained with specific antibodies. cycle adenosine monophosphate analysis bv- cells were treated with μm isosibiricin with or without lps for h. then, the cell lysates were used to analyse the level of cycle adenosine monophosphate (camp) with a camp direct immunoassay kit (bioway, beijing, china) all experiments were carried out at least three times in triplicate. statistical analyses were performed by using one-way analysis of variance with graphpad prism . software. multiple comparisons were performed by comparing the mean of each column with the mean of every other column. student's t-test was used without correcting for multiple comparisons. all data were expressed as the means ± s.d. a p-value < . was considered to be significant. in this study, the anti-inflammatory effect of isosibiricin (fig. a) was measured by using an no release assay. as shown in fig. b , lps obviously stimulated no release, which was significantly inhibited by isosibiricin ( , and μm) in a concentrationdependent manner. to evaluate the effect of isosibiricin on other inflammatory factors, the levels of tnf-α and il- were further analysed by elisa. as shown in fig. c , d, tnf-α and il- expression was significantly increased upon lps treatment; however, isosibiricin ( , and μm) inhibited tnf-α and il- production in a concentration-dependent manner. moreover, cox- and inos expression was markedly upregulated in lpstreated bv- cells, and isosibiricin reduced cox- and inos expression in a concentration-dependent manner (fig. e) . taken together, these findings indicated that isosibiricin can attenuate neuroinflammation by inhibiting the expression of multiple inflammatory mediators in lps-treated bv- cells. isosibiricin upregulates drd /d expression in lps-treated bv- cells and balb/c mice to investigate the potential mechanism of the anti-inflammatory effect of isosibiricin, transcriptomics coupled with bioinformatics analysis was performed to explore the anti-inflammatory signalling pathway of isosibiricin in lps-treated bv- cells. the results suggested that genes, including upregulated genes and downregulated genes ( genes related to inflammation and genes related to the synthesis, storage, release, termination of dopamine and the expression of drds) were significantly changed by isosibiricin compared with lps. notably, dopamine receptors were most strongly related to isosibiricin treatment (fig. a) . moreover, kegg pathway analysis was carried out to explore the pathway affected by isosibiricin. as shown in fig. b , dopamine activation of the neurological reward system was strongly influenced by isosibiricin. therefore, we speculated that isosibiricin might exert an anti-inflammatory effect by targeting the dopamine receptor signalling pathway. nevertheless, isosibiricin did not directly target or regulate the functions of dopamine receptors (supplementary table ). thus, we hypothesized that isosibiricin can regulate the expression of dopamine receptors. as shown in fig. c , d, lps downregulated the mrna expression of drd and drd , and this was significantly reversed by isosibiricin. in addition, isosibiricin also upregulated drd /d expression in the ca region and cortex of lps-treated balb/c mice (supplementary fig. s a) . collectively, these observations revealed that isosibiricin can effectively upregulate drd and drd expression in vitro and in vivo, which might represent a crucial antiinflammatory mechanism. isosibiricin inhibits the nlrp /caspase- inflammasome pathway in lps-or nigericin-treated bv- cells and lps-treated balb/c mice it has been reported that the expression of the pro-inflammatory mediator il- β significantly increases in drd -null mice compared with wild-type mice [ ] . in addition, drd agonists are able to inactivate the nlrp inflammasome pathway [ ] . thus, we detected the effect of isosibiricin on the nlrp inflammasome pathway. as shown in fig. a , isosibiricin significantly reduced nlrp , caspase- , il- β and il- expression in a concentrationdependent manner, indicating that isosibiricin exerts an inhibitory effect on the nlrp /caspase- inflammasome pathway. moreover, the nlrp inflammasome inducer nigericin was used to activate bv- cells and we found that nigericin significantly increased nlrp , caspase- , il- β and il- expression, which was the predicted pathways (a) and targets (b) of isosibiricin inferred by the differential expressed gene-based method. c, d isosibiricin upregulated dopamine d receptors (c) and dopamine d receptor (d) expressions in lps-induced bv- cells. ## p < . vs. control group. **p < . vs. lps group effectively inhibited by isosibiricin (fig. b) [ ] . animal experiments were also carried out to evaluate the anti-inflammatory effect of isosibiricin. as shown in fig. c , isosibiricin significantly inhibited inflammasome-related il- β and il- induced by lps in the ca region and cortex of balb/c mice. these results suggested that isosibiricin exerts an anti-inflammatory effect via the inhibition of the nlrp inflammasome pathway. drd /d inhibitors reverse the isosibiricin-mediated inactivation of nlrp /caspase- inflammasome pathway considering the effect of isosibiricin on dopamine receptors (fig. c, d) and the inflammasome pathway (fig. a, b) , we hypothesized that isosibiricin might inhibit the inflammasome pathway through dopamine receptors. therefore, drd and drd inhibitors, sch and sultopride were used to investigate whether drd /d are involved in isosibiricinmediated anti-inflammatory effects. we found that the anti-inflammatory effect of isosibiricin was significantly reversed upon treatment with sch and sultopride. nlrp , caspase- , il- β and il- expression was markedly increased by sch and sultopride (fig. a) . furthermore, sch and sultopride were also used in nigericin-treated bv- cells. similar to what was observed in the lps-induced experiment, nlrp , caspase- , il- β and il- expression was reversed in the sch -and sultopride-treated groups compared with the isosibiricin-treated group (fig. d) . taken together, the results suggested that drd and drd inhibitors are able to reverse the isosibiricin-mediated inhibition of the nlrp /caspase- inflammasome pathway. dopamine agonists (das), such as dopamine, act on dopamine receptors to regulate a great diversity of biological functions [ ] . fig. isosibiricin inhibits nlrp /caspase- inflammasome pathway in vitro and in vivo. a isosibiricin inhibited inflammasome-related nlrp , pro-caspase- , caspase- , pro-il- β, il- β, pro-il- and il- expressions in lps-induced bv- cells. quantification for caspase- , il- β and il- expressions were relative to gapdh in lps-induced bv- cells. b isosibiricin inhibited inflammasome-related nlrp , pro-caspase- , caspase- , pro-il- β, il- β, pro-il- and il- expressions in nigericin-induced bv- cells. quantification for caspase- , il- β and il- expressions were relative to gapdh in nigericin-induced bv- cells. c isosibiricin inhibited inflammasome-related il- β and il- in the ca region and cortex stained with the specific antibodies il- β and il- . arrows indicate the activated inflammatory factors. ## p < . vs. control group. **p < . vs. lps group or nigericin group in the past decade, das have been mainly used clinically for the treatment of parkinson's disease and, recently, the development and clinical application of das for various other neurological diseases have begun to draw attention from researchers [ , ] . neuroinflammation is an inflammatory response in the cns characterized by increased microglial over-activation [ , ] . thus, the development of anti-neuroinflammatory agents is of great importance for the treatment of inflammation-related neurological disorders, such as motor paralysis, amyotrophic lateral sclerosis and dementia [ ] [ ] [ ] . in the current study, we reported that isosibiricin exerts a significant anti-neuroinflammatory effect on lps-treated bv- cells. lps is a common inflammation inducer that is used to investigate different types of inflammatory pathology, such as tissue inflammation [ ] , vascular inflammation [ ] and myocarditis [ ] . dopaminergic neurons play an important role in the pathological process of parkinson's disease [ ] , and lps is widely used to induce neuroinflammation to establish models of parkinson's disease. in addition, some references have indicated that dopamine receptors are also involved in the regulation of the classical lps signalling pathway [ ] [ ] [ ] . therefore, lps was used to induce neuroinflammation and damage dopamine receptors in the substantia nigra [ ] . the potential mechanism of isosibiricin may involve the upregulation of drd /d expression and the further inactivation of the nlrp /caspase- inflammasome pathway, which results in the blockage of the production of multiple inflammatory mediators. in addition, we used pipeline pilot to measure the solubility, intestinal absorption and blood-brain barrier (bbb) level of isosibiricin. isosibiricin exhibited good solubility in water at °c and was easily absorbed by the intestine. the bbb level was , which means that isosibiricin can pass through the bbb. thus, our study provides an attractive therapeutic methodology for inflammation-associated neurological disorders, as well as an anti-neuroinflammatory lead compound that may become a clinical candidate. dopamine receptors were recently discovered as key therapeutic targets involved in immunoregulation and the inflammatory response [ ] . previously reported active compounds were shown to mainly activate or inhibit dopamine receptors [ , ] ; however, we found that isosibiricin did not directly activate drd /d but significantly upregulated the expression of drd /d mrna, further promoting drd /d -dependent inflammasome signalling pathway inactivation and blocking inflammatory mediator production. in addition, the level of camp was regulated by drd /d , but we detected that isosibiricin did not affect the level of camp ( supplementary fig. s b ). these mechanisms are quite different from those of current small molecular regulators, which directly activate drd /d [ ] . however, the molecular mechanism by which drd mrna expression is regulated still needs to be explored. we speculate that some transcription factors, such as nf-κb, nrf and creb, might be involved in this process. dopamine receptors, including five subtypes from drd to drd , are seven-transmembrane-spanning g-protein-coupled receptors [ , ] . based on their structures and pharmacological characteristics, dopamine receptors are divided into two major categories: dopamine d -like receptors (drd and drd ) and d like receptors (drd , drd , and drd ) [ ] . notably, the specific drd inhibitors sch (for drd ) and sultopride (for drd ) significantly reversed the inhibitory effect of isosibiricin on the nlrp signalling pathway, further supporting the observation that isosibiricin suppresses neuroinflammation by blocking the nlrp / caspase- inflammasome pathway. it is worth mentioning that the regulatory effect of drd inhibitors on the isosibiricin-dependent inhibition of il- β production was more significant in lps-induced bv- cells than in nigericin-induced bv- cells. a possible explanation may be that the mechanism by which lps induces inflammasome signal activation differs from that of nigericin. fig. dopamine d / receptor inhibitors reverse isosibiricin-mediated inhibition of nlrp /caspase- inflammasome pathway. a sch and sultopride reversed isosibiricin-mediated inhibition of nlrp , pro-caspase- , caspase- , pro-il- β, il- β, pro-il- and il- expressions in lps-induced bv- cells. quantification for caspase- , il- β and il- expressions was relative to gapdh in lps-induced bv- cells. b sch and sultopride reversed isosibiricin-mediated inhibition of nlrp , pro-caspase- , caspase- , pro-il- β, il- β, pro-il- and il- expressions in nigericin-induced bv- cells. quantification for caspase- , il- β and il- expressions was relative to gapdh in nigericin-induced bv- cells. ## p < . vs. control group. $$ p < . vs. lps group or nigericin group. **p < . vs. the isosibiricin group isosibiricin regulates drd / to inhibit neuroinflammation yh wang et al. meanwhile, isosibiricin-mediated anti-neuroinflammation was highly associated with the canonical lps-induced neuroinflammation pathway. of course, further studies are needed to elucidate this speculation. in conclusion, our observations demonstrated that isosibiricin exerts a marked anti-neuroinflammatory effect via targeting the drd /d -dependent nlrp /caspase- inflammasome pathway. this finding indicates that isosibiricin may a promising candidate molecule for that treatment of neuroinflammation, and that drd /d may be a key target for inflammatory programmes in neurological diseases. dopamine promotes ascorbate release from retinal neurons: role of d receptors and the exchange protein directly activated by camp type (epac ) modulatory effect of dopamine receptor on the neurosecretory dahlgren cells of the olive flounder, paralichthys olivaceus a concise review of the conflicting roles of dopamine- versus dopamine- receptors in wound healing exercise decreases oxidative stress and inflammation and restores renal dopamine d receptor function in old rats dopamine d -like receptor antagonist attenuates th -mediated immune response and ovalbumin antigen-induced neutrophilic airway inflammation role of dopamine and d dopamine receptor in the pathogenesis of inflammatory bowel disease dopamine d receptor polymorphisms in inflammatory bowel disease and the refractory response to treatment dopamine d receptor signalling controls inflammation in acute pancreatitis via a pp a-dependent akt/ nf-κb signalling pathway dopamine d receptor is involved in alleviation of type ii collagen-induced arthritis in mice dopamine, t cells and multiple sclerosis (ms) microglial dopamine receptor elimination defines sex-specific nucleus accumbens development and social behavior in adolescent rats activation of dopamine d receptor suppresses neuroinflammation through αb-crystalline by inhibition of nf-κb nuclear translocation in experimental ich mice model dopamine d receptor agonist a- inhibits nlrp inflammasome activation, controls inflammation, and alleviates histopathology in a rat model of spinal cord injury α-synuclein disrupts the antiinflammatory role of drd via interfering β-arrestin -tab interaction in astrocytes selective activation of nociceptor trpv channel and reversal of inflammatory pain in mice by a novel coumarin derivative muralatin l from murraya alata anti-inflammatory, analgesic and antiulcerogenic effect of total alkaloidal extract from murraya koenigii leaves in animal models evaluation of anti-inflammatory and antinociceptive activities of murraya exotica isofraxidin inhibits interleukin- β induced inflammatory response in human osteoarthritis chondrocytes osthole protects against acute lung injury by suppressing nf-κb-dependent inflammation urolithin a attenuates proinflammatory mediator production by suppressing pi -k/akt/nf-κb and jnk/ ap- signaling pathways in lipopolysaccharide-stimulated raw macrophages: possible involvement of nadph oxidase-derived reactive oxygen species transcriptome analysis of dairy goat mammary gland tissues from different lactation stages an integrated proteomics and bioinformatics approach reveals the anti-inflammatory mechanism of carnosic acid suppression of neuroinflammation by astrocytic dopamine d receptors via αb-crystallin the inflammasome drives gsdmd-independent secondary pyroptosis and il- release in the absence of caspase- protease activity behavioral and cellular dopamine d and d receptor-mediated synergy: implications for l-dopa-induced dyskinesia a randomised, open-label, crossover study of the dopamine agonist, pramipexole, in patients with sleep bruxism efficacy and safety of low-and high-dose cariprazine in acute and mixed mania associated with bipolar i disorder: a double-blind, placebo-controlled study formononetin inhibits neuroinflammation and increases estrogen receptor β (erβ) protein expression in bv microglia neurotropin inhibits neuroinflammation via suppressing nf-κb and mapks signaling pathways in lipopolysaccharide-stimulated bv cells (-)-β-caryophyllene, a cb receptor-selective phytocannabinoid, suppresses motor paralysis and neuroinflammation in a murine model of multiple sclerosis disease origin and progression in amyotrophic lateral sclerosis: an immunology perspective nstearoylethanolamine protects the brain and improves memory of mice treated with lipopolysaccharide or immunized with the extracellular domain of α nicotinic acetylcholine receptor suppressive effects of pelargonidin on lipopolysaccharide-induced inflammatory responses emodin attenuates lipopolysaccharide-induced injury via down-regulation of mir- in h c cells tubeimoside i protects dopaminergic neurons against inflammation-mediated damage in lipopolysaccharide (lps)-evoked model of parkinson's disease in rats spinal dopaminergic projections control the transition to pathological pain plasticity via a d /d -mediated mechanism dopamine induces il- -dependent il- production via d -like receptor on cd naive t cells and d -like receptor antagonist sch- inhibits cartilage destruction in a human rheumatoid arthritis/scid mouse chimera model effects of antiglaucoma drugs glc , a novel dopamine d agonist and d antagonist, and timolol on endotoxininduced tnf-alpha release in serum of rats modulation of m /m polarization by capsaicin contributes to the survival of dopaminergic neurons in the lipopolysaccharide-lesioned substantia nigra in vivo stimulation of dopamine d receptors in the nucleus accumbens inhibits inflammatory pain long-term treatment with dopamine d receptor agonists induces a behavioral switch that can be rescued by blocking the dopamine d receptor antagonism of the d dopamine receptor enhances tremor but reduces voluntary muscle activation in humans ck oppositely modulates l-dopa-induced dyskinesia via striatal projection neurons expressing d or d receptors dopamine receptor subtypes differentially regulate autophagy predicting subtype selectivity of dopamine receptor ligands with three-dimensional biologically relevant spectrum characterization of an invertebrate-type dopamine receptor of the american cockroach, periplaneta americana this work was financially supported by the national natural science foundation of china (numbers , and ) and the drug innovation major project (number zx - - ). kwz, pft, and yj designed the research. yhw and hnl conducted majority of the experiments. qhc coordinated the experiments. kwz and yhw wrote the manuscript. kwz provided suggestions for the experimental design and manuscript. the online version of this article (https://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users.competing interests: the authors declare no competing interests. key: cord- -r tg s authors: john, maya; shaiba, hadil title: shiny framework based visualization and analytics tool for middle east respiratory syndrome date: - - journal: advances in data science, cyber security and it applications doi: . / - - - - _ sha: doc_id: cord_uid: r tg s people in the middle east have been affected by the middle east respiratory syndrome coronavirus (mers co-v) since . new cases are continuously reported especially in the kingdom of saudi arabia, and the risk of exposure remains an issue. data visualization plays a vital role in effective analysis of the data. in this paper, we introduce an interactive visualization application for mers data collected from the control and command centre, ministry of health website of saudi arabia. the data corresponding to the period from january , to february , was used in the present work. the attributes considered include gender, age, date of reporting, city, region, camel contact, description and status of the patient. the visualization tool has been developed using shiny framework of r programming language. the application presents information in the form of interactive plots, maps and tables. the salient feature of the tool is that users can view and download data corresponding to the period of their choice. this tool can help decision makers in the detailed analysis of data and hence devise measures to prevent the spread of the disease. mers is a viral disease that was first discovered in when a patient in saudi arabia was diagnosed with critical respiratory distress and kidney trouble [ ] . the infection can either be asymptomatic or show symptoms such as cough, fever etc. along with difficulty in breathing [ ] . the disease has been brought under control in all middle eastern countries except saudi arabia where each month, new cases are still reported. studies have confirmed that this disease is zoonotic and camels are its significant reservoir [ , ] . people in close contact with infected camels and health care professionals who care for infected patients have a high risk of acquiring the infection. many cases of community and household acquired infections have been reported from saudi arabia [ ] . the ministry of health, saudi arabia has issued guidelines for infection prevention, control and management. to the best of our knowledge, there is a lack of interactive visualization tools for mers data visualization in saudi arabia. this work deals with developing an application where users can interactively view information about the infection in the form of plots, tables and maps. the data used in this study was obtained from the saudi ministry of health website and it includes the cases reported in saudi arabia from january , to february , . by viewing the data visualizations, users can analyze mers cases better, find trends, monitor the disease and help authorities set detection and prevention guidelines. the mers data analyzed consists of cases reported during the first two months of the year . the information is present in the form of a pdf file corresponding to the cases reported each week. the cases reported contains details which include the date of reporting and patient information such as personal information, demographic information, camel contact details, description of infection and status of the disease. the latitude and longitude of the regions and cities are stored in separate .csv files. the details of the attributes present in the database are shown in table . shiny is an r package used to create interactive web applications [ ] . the shiny framework is a reactive programming model where browser refreshing is not required to instantiate the output changes when the user modifies the input. it can be used to build web applications without using javascript coding. shiny combines the computational power of r programming language with the highly interactive nature of the web. leaflet package is used to create interactive maps in r based on the javascript library leaflet [ ] . these interactive maps can be rendered in r markdown, shiny apps, rstudioide and r console as it was developed in association with htmlwidgets. the leaflet( ) function is used to create the map widget. the different types of layers which can be included in the map widget are map tile, markers, lines, polygon, popups, raster images, color legends, layer groups, layer control etc. the maps created can be zoomed or downsized interactively. the googleviz package in r facilitates the use of google chart apis [ ] . interactive charts can be incorporated into web pages using google charts. the data stored in r data frames can be visualized in the form of google charts without uploading the information to google. an internet connection is required to view the output rendered by this package. the mers data visualization tool was developed using r programming language. the tool consists of three sections (tabs) namely "different cases analysis", "miscellaneous analysis" and "summary" section. the users can choose the period for which they would like to visualize the mers data. in the case of different cases analysis, the user can view the information as pie charts and maps, or tables. the users can view details about either all the cases together or recovered cases, death cases or hospitalized cases separately. the details regarding the cases can be viewed with respect to all cities, all regions or cities within a region. in the case of the option "cities within a region", the user has to choose a region from the drop down list provided. unlike conventional pie charts, the pie charts created using googleviz package are interactive in nature. by pointing the cursor over a portion in the pie chart, one can understand the actual number of cases in that particular part of the chart. in the map, places are marked based on longitude and latitude of that place. on clicking the marker displayed in the map, the name and number of cases in that place will be displayed as a pop-up. based on the number of cases, markers are assigned colors such as red, orange or yellow. the colors red, orange and yellow represent "large number of cases", "moderate number of cases" and "few number of cases" respectively. the information in figs. and is presented in the form of pie chart and map. the screen shot of the page corresponding to different cases analysis is shown in fig. . the figure depicts the analysis of all mers cases reported for the first two months of the year with regard to cities. it can be inferred from fig. that during that period . % of the patients recovered from the disease and the maximum number of cases ( %) were reported from the city of wadi aldawasir. the analysis based on all cases reported in "all cities within riyadh region" during january to february is shown in fig. . it can be observed from the figure that majority of cases in riyadh region were reported from the city of wadi aldawasir. nearly % of the infected people in riyadh region recovered from the disease. the application page with table output is shown in fig. . it can be observed from the table that people died due to mers in riyadh region during the first two months of the year . the table which displays the information is interactive in nature. when the user clicks a column name in the table, the records in the table are sorted based on values in the clicked column and displayed accordingly. the table also has provision for searching values and selecting the number application page corresponding to "different cases analysis" tab for cities within a region of records to be displayed in a page. the users can also download the details contained in the table. the different cases analysis will help decision makers in gathering information regarding the cases reported in different places. this type of analysis is essential to identify disease prone areas and hence take measures to curtail the spread of infection. the miscellaneous analysis consists of analysis based on age, camel contact and months. the users can analyze the data for all cases, death cases or recovered cases. depending on the user's choice, the analysis corresponding to all cities, all regions or cities within a region are displayed. analysis based on age. the infected people are divided into three age categories namely below years, - years and greater than years. in the case of age based analysis, the information is displayed both in the form of pie chart and stacked bar chart. the pie chart represents the number of cases corresponding to each age group. the stacked bar chart depicts the number of cases in each age group corresponding to the location type selected. the screen shot corresponding to age category wise analysis is shown in fig. . the figure corresponds to the analysis corresponding to all cases reported with respect to cities for the first two months of the year . it is evident from the pie chart fig. that the percentage of cases reported is . %, . % and . % for the age group below years, - years and above years respectively. the age category wise helps in understanding the number of cases reported among people of different age groups. this type of analysis will help in identifying which group has more mortality rate and health authorities may conduct extensive analysis of causes leading to death. analysis based on camel contact. camels have been identified as a carrier of the disease, and hence it is essential to perform analysis of patient cases involving camel contact. depending on the choice of the user, the information is displayed for "all cases", "death cases" or "recovery cases". the analysis is carried out for "all cities", "all regions" or "cities within a region". the details are displayed as pie chart, map and table. the pie chart represents the numbers of cases corresponding to the location type selected by the user. the places where camel contact cases are reported are marked in the map. this can help in analyzing whether the cases pertaining to people with camel contact are clustered in a region or not. the table displays the details of infected people who had contact with camels. the data visualization based on camel contact is shown in fig. . the figure depicts the analysis corresponding to all cases with regard to cities in saudi arabia. it can be observed from fig. that % of the camel contact based cases were reported from the city of wadi aldawasir. camel contact based analysis of mers patients is highly essential to identify places where people in contact with camels have been infected with the disease. this will help health authorities in taking more measures to spread awareness about the precautionary methods to be taken when handling camels. the infected camels in such regions may be identified and isolated to prevent the spread of the disease. month-wise comparison of the data can be carried out for "all cases", "death cases" and "recovery cases". the stacked bar chart for cases reported in different months is plotted based on the location type specified by the user. when the cursor is moved to a region in the bar chart, the number of cases in the particular month will be displayed corresponding to the location. the screenshot corresponding to month-wise comparison is shown in fig. . the figure portrays the analysis corresponding to death cases reported in various regions. the maximum number of death cases were reported in riyadh region during february. monthwise analysis is useful in identifying whether the infection is related to climate. summary based visualization gives a graphical summary of the count of the "status of the patients" for different attributes. the information is depicted in the form of stacked bar charts where the charts are plotted based on the frequency of status of patients corresponding to different values of the attributes. this visualization will help the users in getting an overall idea regarding the distribution of data with regard to the status of the patients. figures and depict the screenshots of the visual summary of data for the first two months of the year . figure confirms the earlier findings that riyadh region and specifically wadi aldawasir city has the highest number of mers cases and death cases. figure shows that most mers patients are male and that people below years are less likely to get infected by mers. elderly patients are more prone to die of mers. the majority of patients were not in contact with camels and death rate was low in the case of patients in contact with camels. many patients acquired the infection from healthcare facilities and high death rate is reported among this group. the users can view information corresponding to their period of interest. the interactive feature of the pie chart prevents the chart from being cluttered with description. the use of maps to represent the information will give an idea regarding the spatial distribution of mers cases. these maps will help the health authorities to identify the areas with large number of cases and hence alert the hospitals and the general public regarding that. the interactive nature of the table helps the users in analyzing the data as per their requirement. a salient feature of the application is that the users can easily download details corresponding to the period of their choice. analyzing the data based on camel contact will aid health authorities to identify the areas where many such cases are present and hence intensify the awareness programs to reduce the rate of infection. in this paper, we have created an interactive visualization tool for mers co-v infection cases based on details of cases reported in saudi arabia. the attributes used include the date of reporting, city, region, age, gender, description of the disease, camel contact and status of the patient. the user can view details regarding all cases, recovered cases, death cases or hospitalized cases for all the cities, all regions or cities within a region. our tool provides the flexibility to view information in the form of charts and maps, or downloadable tables. the analysis is also carried out based on age group, camel contact and month-wise cases. by viewing the maps, users can easily differentiate between places based on the number of cases. moreover, the users can view a visual summary of the number of cases for different values of attributes based on the status of the patients. understanding and analyzing the disease related information can help decision makers in setting guidelines for preventing and controlling the spread of disease. location-based analysis of the infection is highly essential in formulating region specific awareness programs to reduce the rate of infection. isolation of a novel coronavirus from a man with pneumonia in saudi arabia main factors influencing recovery in mers co-v patients using machine learning mers-cov infection of alpaca in a region where mers-cov is endemic middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission key: cord- -ebcu ywq authors: xu, jianguo; yuan, zhiming title: inaugural editorial: towards evidence-based biosafety and biosecurity date: - - journal: nan doi: . /j.jobb. . . sha: doc_id: cord_uid: ebcu ywq nan inaugural editorial: towards evidence-based biosafety and biosecurity we are pleased to introduce the journal of biosafety and biosecurity, the first dedicated periodical addressing these subjects, published by keai press. china has experienced significant biosecurity and biosafety challenges and is the only nation that has been subjected to a bioweapon assault. although the significant economic growth experienced by china has facilitated remarkable improvements in hygiene as well as food and water quality, the outbreak of severe acute respiratory syndrome (sars) led to an infectious disease epidemic. in march of that year, as sars spread beyond chinese borders, the world health organization (who) issued a global alert. the sars epidemic served as a timeous practical reminder to both china and the world that emerging infectious diseases could significantly threaten national and global safety and security. however, this was not the end of the story for the sars virus. in , a case of laboratory-acquired sars infection was reported, and a number of containment failures occurred in high-security laboratories in singapore, taiwan, and mainland china. [ ] [ ] [ ] [ ] [ ] the sars outbreak in north china resulted from a series of flaws in the biosafety protocol at a national institute in beijing, resulting in infection of four laboratory workers. this has been the most serious biosafety event to date. , in recent years, reports of outbreaks of african swine fever along the chinese-russian border fueled growing concerns that this livestock infection could spread to china. in , these fears were realized, and within a very short time african swine fever had spread to many chinese provinces. given the large scale of china's pork sector, the economic impact of such a disease has the potential to be economically devastating. recently, international political authorities and scientific organizations have both recognized the significance of and need for improved biosafety and biosecurity. the united nations secretary-general's mechanism for investigation of alleged use of chemical and biological weapons has been proposed as the foundation on which to build a protocol to gauge the potential for biothreats and bioweapons, and a network of designated laboratories focused on supporting such investigations is under development. national legislation regarding biosecurity has been discussed and implemented in several countries. [ ] [ ] [ ] [ ] in , the chinese government incorporated biosecurity as a specific national security domain. similarly, in , the united states (u.s.) issued the new national biodefense strategy, and the united kingdom (u.k.) issued the biological security strategy. despite political recognition of the importance of biosecurity and biosafety, their implementation lacks a thorough empirical evidence base. we propose that the scope of biosecurity and biosafety should include all relevant areas with the potential to cause death, social disruption and panic, economic breakdown, and/or national crisis (e.g. emerging infectious diseases, bioweapons, bioterror, laboratory biosafety, antibiotic-resistant bacterial super-strains, harmful invasive plant or animal species, misuse of synthetic biological technology, misuse of human genetic information, etc.). the u.s. national biodefense strategy definition of biological threats includes catastrophic disease outbreaks, regardless of whether they are naturally occurring, accidental, or deliberate in origin (https://www.whitehouse.gov/wp-content/uploads/ / /national-biodefense-strategy.pdf). the u.k. biological security strategy definition of biosecurity includes significant outbreaks of disease and events precipitated by an accidental release from biological facilities, deliberate biological attack, and animal or plant disease outbreaks with the potential to significantly impact the economy, environment, and society (https:// www.gov.uk/government/publications). in order to prevent and adequately prepare for potentially significant future biosecurity or biosafety threats, a dedicated scientific subject area is required. optimal research strategies and technologies should be developed and employed to secure our nation and our world against such threats. these are the aims underlying the launch of this journal. the scientific community is invited to work with us in bringing together research and technology that will inform an improved understanding and implementation of biosafety and biosecurity. victims of japan's notorious unit sue who issues rallying cry to keep fight against sars on track early influenza in europe and sars escaping from high security laboratories! recent singapore sars case a laboratory accident china dumps cdc head, probes lab infectious diseases. mounting lab accidents raise sars fears infectious diseases. second lab accident fuels fears about sars emergence of african swine fever in china arrival of deadly pig disease could spell disaster for china united nations secretary-general's mechanism. rev sci tech analysis of australia's new biosecurity legislation eu is a key source of biosecurity risk to the uk biosafety and biosecurity in european containment level laboratories: focus on french recent progress and essential requirements biobanking and biosecurity initiatives in africa the authors declared that they have no conflicts of interest to this work. key: cord- - b eazd authors: merzendorfer, hans; cohen, ephraim title: chitin/chitosan: versatile ecological, industrial, and biomedical applications date: - - journal: extracellular sugar-based biopolymers matrices doi: . / - - - - _ sha: doc_id: cord_uid: b eazd chitin is a linear polysaccharide of n-acetylglucosamine, which is highly abundant in nature and mainly produced by marine crustaceans. chitosan is obtained by hydrolytic deacetylation. both polysaccharides are renewable resources, simply and cost-effectively extracted from waste material of fish industry, mainly crab and shrimp shells. research over the past five decades has revealed that chitosan, in particular, possesses unique and useful characteristics such as chemical versatility, polyelectrolyte properties, gel- and film-forming ability, high adsorption capacity, antimicrobial and antioxidative properties, low toxicity, and biocompatibility and biodegradability features. a plethora of chemical chitosan derivatives have been synthesized yielding improved materials with suggested or effective applications in water treatment, biosensor engineering, agriculture, food processing and storage, textile additives, cosmetics fabrication, and in veterinary and human medicine. the number of studies in this research field has exploded particularly during the last two decades. here, we review recent advances in utilizing chitosan and chitosan derivatives in different technical, agricultural, and biomedical fields. chitosan, a polymer of β( - )-linked glucosamine ( -amino- -deoxy-o-glucose) units, is a biopolymer with unique characteristics due to the presence of free amino groups on its backbone. it is obtained by partial deacetylation of chitin, which is found in the cell walls of unicellular and filamenous fungi and in extracellular matrices and skeletal deposits of many protozoan and metazoan organisms including algae, choanoflagellates, sponges, corals, cephalopods, and arthropods. commercially, chitin is extracted from the waste shells of marine crustaceans such as shrimp and crab. a significant proportion is used to produce chitosan, which, in contrast to chitin, is soluble in water at a slightly acidic ph and is easy to modify chemically to increase solubility at neutral ph and to add new functionalities. chitosan and its derivatives have many desirable properties such as antioxidative and antimicrobial effects, mucoadhesiveness, biodegradability, and biocompatibility and can be manufactured in various formulations including hydrogels, films, membranes, porous sponges, nanoparticles, and nanofibers. moreover, chitosan is considered a harmless compound, as it has received the generally recognized as safe (gras) status by the us food and drug administration (fda), and it has been approved as a food additive in several asian countries ). in the european union, chitosan is registered as a basic substance, and the use of chitosan hydrochloride is considered by the european food safety authority (efsa) as having neither harmful effects on human or animal health nor any negative effects on the environment (european commission ). therefore, chitosan-based materials have been adopted worldwide in numerous applications in water treatment; food, cosmetic, and textile industry; biosensor engineering; plant protection; pharmaceutical industry; and regenerative medicine. they are used as flocculants, ion exchangers, chelating agents, coating materials, drug carriers, and scaffolds for tissue engineering. during the past years, many companies have started to develop chitosan-based products, and some have already successfully launched them for commercial purposes. this review is intended to summarize recent developments in the use of chitosan-based materials for potential and effective applications in different technical, environmental, agricultural, and biomedical fields. pollutants in water, industrial wastewater, and reclaimed wastewater for crop irrigation have presented severe environmental and medical problems all over the world. such contaminants include various heavy metal ions (copper, cobalt, manganese, chromium, mercury, lead, arsenic, cadmium, and nickel), dyes (mainly azo dyes like malachite green, methyl violet, or methylene blue), oil spills, and a variety of pharmaceuticals and endocrine-disrupting compounds. among the various methods used as remedial measures to treat polluted water and wastewater, the potential of chitosan-based composites as efficient adsorbent, flocculating and chelating agents has been widely investigated. the presence of free hydroxyls and amino groups in many structural forms of chitosan-derived composites facilitates adsorption of pollutants such as dyes, metals, and organic compounds. chitosan derivatives like carboxymethyl chitosan and graft polymerization are a prevalent strategy to add a variety of functional groups to the composite. magnetic particles are embedded usually as nanoparticles in the complex core to facilitate regeneration and reuse of adsorbent composites by applying external magnetic field. was capable to adsorb heavy metals like cu(ii) but also cd(ii) and pb(ii) from aqueous solutions (peng et al. ) . chitosan-algal biomass composite microbeads (sargın et al. b ), a binary chitosan/silk fibroin composite (ramya and sudha ) , and cotton fibers functionalized by triethylenetetramine (teta) and carboxymethyl chitosan form composites and hybrids for adsorption of cu (ii) from water (niu et al. ) . microcapsules composed of phytopathogenic (ustilago sp.) fungal spores immobilized in cross-linked chitosan matrix (sargın et al. a ) and a binary complex of chitosan and emu egg shells (anantha and kota ) were shown to remove copper ions from aqueous solutions. chitosan complexed with clays, ceramic minerals, and carbon-based materials was used to enhance absorbance of heavy metals from aqueous solutions. a nanocomposite that consisted of chitosan-montmorillonite (pereira et al. ) and silica gel/chitin and chitosan with nano-hydroxyapatite was used as adsorbents for cu(ii) (rajiv gandhi et al. ) . nanocomposites containing chitosan-poly (vinyl alcohol)-attapulgite were also used for removal of cu(ii) from aqueous solutions . furthermore, a recyclable magnetic microsphere composed of cross-linked chitosan-rectorite (a clay mineral) and fe o was studied for adsorption of cu(ii) and cd(ii) (xie et al. ) , and chitosan-zeolite composite hydrogel beads were examined for cu(ii) sorption (djelad et al. ) . a particular interesting recyclable composite with chelating capacity consists of core magnetic (fe o )-silica particles combined with cross-linked chitosan. its porous and highly specific surface area contributed by activated carbon carrier showed an excellent adsorption capability for cu + ions . a recyclable nanocomposite with a core xanthated fe o chitosan grafted on graphene oxide introduced sulfur groups to the composite using carbon disulfide (liu et al. a) . other sorbent composites that were prepared and studied are a recyclable composites containing chitosan grafted on a core of fe o -hexadecyl trimethoxysilane (liu et al. b ), a flocculant composed of poly(acrylic acid) grafted on chitosan (saleh et al. ) or beads containing chitosan-poly(vinyl alcohol) and zno (xu et al. a) . a sophisticated composite was prepared by using magnetic nanoparticles on the surface of polystyrene as core, coated with chitosan crosslinked by glutaraldehyde followed by grafting polyethylenimine on the complex surface . this submicron composite is recyclable and exhibits good adsorption capacity for cu(ii) ions. highly selective adsorption of copper ions from aqueous solutions was achieved by the ion-imprinting polymer method (kong et al. ) . microspheres of magnetic cores of fe o with a shell of cross-linked chitosan and graphene oxide were used to imprint cu + ions. zarghami et al. ( ) prepared cu(ii) ion-imprinted membranes composed of cross-linked chitosan/poly(vinyl alcohol) for adsorption of the metal from aqueous solutions. a similar ion-imprinted technique was reported for selective adsorption of pb(ii) from a recycling wastewater unit (hande et al. ). textile, leather, paper, and food industries discharge a plethora of environmental pollutants such as synthetic dyes. a variety of chitosan-based composites was examined as promising adsorbents of hard to remove industrial dyes. chitosan per se contains functional groups for interaction with pollutants including dyes. adding more functional groups by modifying chitosan (cross-linking of chitosan layers, direct chemical modification, or graft polymerizationsee chapter ) improves adsorption capability. molecular imprinting technique was devised as selective adsorbent of pollutants. composites' core of iron oxide magnetic nanoparticles like maghemite (γ-fe o ) and magnetite (fe o ) offers a way to recover the adsorbent scaffolds for reuse. again, since the published articles are enormous in number, only essential parameters and basic blocks of adsorbing chitosan-based composites are included. methyl orange as a model anionic azo dye was adsorbed by films of cross-linked chitosan/nanonized maghemite from aqueous solution (jiang et al. ) . improved adsorption of the same anionic dye was achieved by preparing a magnetic chitosan grafted with multi-walled carbon nanotubes (zhu et al. ) , and magnetic chitosan grafted with graphite oxide nanocomposite was able to adsorb the toxic azo dye, reactive black (travlou et al. ) . chitosan modified by ethylenediamine (zhou et al. ) or polyaniline (abbasian et al. ) grafting was able to adsorb other anionic azo dyes like orange , acid orange acid and red and direct red , respectively. a magnetic complex of chitosan and zirconium oxide was a potent adsorbent of food anionic azo dyes like amaranth and tetrazine (jiang et al. a ). moreover, a complex composite adsorbent was prepared by grafting chitosan with poly[poly(ethylene glycol) methyl ether methacrylate] (tsai et al. ) . the functionalized groups added to chitosan contributed to improved removal of the azo dye reactive orange from water. recyclable composite microspheres composed of cross-linked chitosan grafted with glutamic acid and having a core of fe o nanoparticles coated with silica adsorb cationic dyes like methylene blue, crystal violet, and light yellow gl (yan et al. ) . similarly, an amphiphilic n-benzyl-o-carboxymethyl chitosan composite with a core of iron oxide nanoparticles was prepared for adsorption of methylene blue, crystal violet, and malachite green (debrassi et al. ) . the cyclic oligosaccharide β-cyclodextrin (β-cd) was added to chitosan-based composites as it provides a hydrophobic inner cavity and a hydrophilic exterior. magnetic chitosan-β-cd with grafted graphene oxide to enlarge surface area exhibited an improved adsorption of methylene blue as a model dye from water (fan et al. ) . molecular imprinting technique is of interest to selectively remove dyes from aqueous solutions. the molecule or ion used as templates will be subsequently removed, and a recognition site is generated. alizarin red served as template molecule, and imprinted magnetic chitosan nanoparticles showed improved adsorption of the dye (fan et al. ). pharmaceutical, endocrine-disrupting compounds and personal care products have become a new class of hazardous environmental pollutants (grassi et al. ) and have emerged as an extensive global concern. they are discharged as municipal and hospital effluents, from manufacturing industries, and found in water, reclaimed wastewater, and even in crops irrigated by reclaimed water (paltiel et al. ) . pharmaceuticals, endocrine disruptors, and personal care products and their chemical transformation derivatives are characterized as stable, persistent compounds that are biologically active at very low concentrations. the challenging goal has been to completely remove the above micropollutants from wastewater following conventional cleaning methods. laboratory research including adsorption by chitosan-based composites has been high on the agenda (amouzgar and salamatinia ) . zhang et al. ( ) used a rather simple crosslinked magnetic chitosan-fe o composite to examine the sorption of three pharmaceutical compounds from contaminated water. the absorbance analysis showed effective sorption of diclofenac (a nonsteroidal anti-inflammatory drug) and clofibric acid (an antilipemic agent) but not of carbamazepine (an antiepileptic medication). pharmaceuticals in water can be present as cationic, anionic, and neutral forms at different ph values. thus, zhang et al. ( ) in a more recent study devised an innovative, more complex three-dimensional chitosan-based scaffold. a magnetic core of chitosan-fe o was grafted with polymeric arms of either the polycation [poly( -methyl acryoxyethyl trimethyl ammonium chloride], the polyanion poly (acrylic acid), or the neutral polymer poly(methylmethacrylate). the polycationic extension was cost-effective in removal of diclofenac from water due to charge attraction . further, magnetic composite pellets with grafted clay (bentonite) and activated carbon were prepared to examine possible cost-effective removal of cationic and anionic pharmaceuticals (arya and philip ). the composite was effective as a sorbent for the beta-blocker (atenolol), the antibiotic (ciprofloxacin), and the lipid regulator (gemfibrozil). a variety of chitosan composites have been tested for the removal of other drugs. cross-linked chitosan grafted with sulfonate or n-( -carboxymethyl) groups was used as a sorbent to remove the dopamine agonist pramipexole dihydrochloride from polluted water . chitosan-poly(acrylic acid)-graphite oxide nanocomposite showed adsorption of dorsolamide, a carbonic anhydrase inhibitor for eye treatment (kyzas et al. ) . adsorption of nonsteroidal anti-inflammatory drugs ibuprofen and ketoprofen was studied using porous composite beads prepared of chitosan-mil (cr) (zhuo et al. ) . using the antiepilectic carbamazepine as template, the magnetic molecular imprinted technique, based on chitosan-fe o nanoparticles, was applied for selective sorption of the drug (zhang et al. c) . chlorophenols are endocrine-disrupting chemicals, used inter alia in manufacturing pharmaceuticals that are found in wastewaters (sin et al. ) . excellent adsorbing capability was demonstrated using a cross-linked chitosan-salicylic acid-β-cd composite. composites of chitosan-γ-cd were capable of adsorbing the endocrine disruptors, polychlorophenols, and bisphenol a (duri and tran ) . composite films prepared by blending microporous carbon fibers with cross-linked chitosan/polyvinyl alcohol were examined as sorbents of bisphenol a from water (bilgin simsek et al. ) . finally, soares et al. ( b) proposed an interesting and unusual concept of using low-cost magnetic chitosan-based scaffold for absorbing and removing oil spills following initial skimming from water. in addition, the composite, which had a core of magnetic nanoparticles with a shell of chitosan-silica hybrid, effectively adsorbs nonpolar organic solvents. biosensors are essentially analytical devices that convert biological reactions or interactions into measurable signals. basically, the biosensors' constructs consist of a biological sensing element associated and intimately interfaced with a transducer that converts a signal in one form of energy to a signal of another form. such signals should be proportional to the amount of analyte within a certain concentration range. electrochemical biosensor devices, for example, possess advantages as being simple and relatively cheap while offering rapid detection and high sensitivity and further being amenable to miniaturization. biosensors have been developed not only as analytical tools for medical purposes of clinical detection but also for applications in food industry and environmental monitoring. chitosan, and to a much lesser extent chitin, has several advantageous qualities in the design of biosensors. the polysaccharides are biocompatible, have functional groups pliable to chemical modification, and can be easily deposited on the surface of the transducer as adhesive thin films for the immobilization of recognition elements (enzymes, antibodies, dna, whole cells, and cell organelles). addition of carbon tubes, graphite, and graphene oxide to the composite increases electron transfer to the transducer and enhanced mechanical strength as well as water permeability and retention. since there is a vast array of biosensors based on chitosan in their constructs, the following provide only representative devices. glucose detection and monitoring is of paramount importance in the medical field. a variety of biosensors, constructed with chitosan and using immobilized glucose oxidase for the detection of glucose levels, were reported. a glucose electrochemical sensor was prepared with glucose oxidase immobilized on the composite of chitosan-carbon nanotubes ). an amperometric glucose biosensor composed of multilayered chitosan biofilms-gold nanoparticles-glucose oxidase on platinum (pt) electrode was devised ). the biocompatible gold nanoparticles helped in directing the transfer of electrons to the transducer. yang et al. ( ) devised a different glucose biosensor composed of pt electrodeglucose oxidase-fe o -chitosan-nafion. zhang et al. ( c) prepared an electrochemical biosensor for glucose with chitosan-graphite composite and the addition of magnetic fe o nanoparticles on pt-coated indium tin oxide (ito) glass electrode. shrestha et al. ( ) devised a glucose biosensor with a glassy carbon electrode on which a nanocomposite film of glucose oxidase immobilized on chitosan and on which a graft of polypyrrole-nafion and multi-walled carbon nanotubes was deposited. electrochemical biosensors using other oxidases and various constructs were fabricated to monitor food and medically important compounds. for instance, a lactate biosensor was generated using lactate oxidase and a nanocomposite structure of chitosan-polyvinylimidazole-os-carbon nanotubes (cui et al. ) . glutamate and xanthine oxidases as recognition elements immobilized on chitosan/graphene oxide-polymerized riboflavin were constructed as glutamate and hypoxanthine biosensors (celiesiute et al. ). in addition, a xanthine biosensor based on immobilization of xanthine oxidase on chitosan-polypyrrole-gold nanoparticles was fabricated by dervisevic et al. ( ) . tkac et al. ( ) developed a selective galactose biosensor with a rather simple configuration of chitosan-single-walled carbon nanotubes and immobilized galactose oxidase. a sensitive amperometric nanocomposite biosensor for cholesterol detection was constructed using a matrix of pt nanoparticles deposited on multi-walled chitosan-carbon nanotubes complexes with immobilized cholesterol oxidase (tsai et al. ) . a similar construct was proposed by medyantseva et al. ( ) for the detection of antidepressant monoamine drugs using immobilized monoamine oxidase. dai et al. ( ) developed an electro-chemiluminiscent biosensor to detect choline by immobilizing choline oxidase on a chitosan/titanate nanotubes composite film. finally, a biosensor for measuring ethanol was prepared using alcohol oxidase immobilized on chitosaneggshell film (wen et al. ). the biosensor monitored the decrease in oxygen level vs ethanol concentration. a number of electrochemical biosensors were similarly constructed to immobilize various dehydrogenase enzymes (zhang et al. ). the nanocomposite scaffold film, attached predominantly to glassy carbon electrodes, consists of chitosan, multi-walled carbon nanotubes, and nad + as cofactor. the signal current is based essentially on electrooxidation of the formed nadh. among the large list of enzymes suffice it to mention nad-dependent alcohol (lee and tsai ; zhang and gorski ) , lactate (tsai et al. ) and glutamate (hughes et al. ) dehydrogenases, and fad-dependent glucose dehydrogenase (monosik et al. ) . in contrast to the above enzyme-based biosensors, a nonenzymatic electrochemical device for monitoring glucose was formulated (al-mokaram et al. ). the construct, which was based on a nanocomposite film composed of polypyrrole-chitosantitanium dioxide nanoparticles on ito glass electrodes, involved redox reactions and exhibited improved glucose oxidation and high electron transfer kinetics. other biosensors detecting and measuring diverse compounds were formulated, for example, nitrite biosensor based on cu-containing nitrite reductase immobilized on viologen-chitosan that catalyzes the reduction of nitrite (quan and shin ) . horseradish peroxidase immobilized on alumina nanoparticles-chitosan composite was devised to detect phenolic compounds . wang et al. ( ) developed a biosensor to detect and measure glucose, galactose, and glutamate in human blood by using their corresponding oxidases immobilized on chitosan-prussian blue composite film. the biosensor used prussian blue as a good catalyst to form hydrogen peroxide by electroreduction. biosensors to detect catechol as well as other phenolic compounds were based on immobilized tyrosinase on a film of chitosan-nickel nanoparticles . a biosensor for detection of chlorophenol that includes immobilized laccase on zno-chitosan nanocomposite was prepared by mendes et al. ( ) . nanocomposite of functionalized graphene oxide (enriched with carboxylic moieties)-polypyrrole-chitosan film was constructed to detect hydrogen peroxide using screen-printed carbon electrodes (akhtar et al. ). such a device was able to electro-catalyze the reduction of hydrogen peroxide. teepoo et al. ( ) constructed an electrochemical biosensor to detect and monitor hydrogen peroxide by using horseradish peroxidase immobilized on a chitin-gelatin nanofiber composite. another biosensor for hydrogen peroxide that used immobilized catalase on chitosan-β-cyclodextrin (with ferrocene in its cavity) was fabricated by dong et al. ( ) . it was based on chitosan-functionalized graphene oxide (enriched with carboxylic moieties)-polypyrrole nanocomposite able to electrocatalytically reduce hydrogen peroxide. detection and quantification of trace amounts of carcinogenic and toxic metallic ions are of great challenge and importance. a cross-linked chitosan-carbon nanotube sensor was developed for the determination of cd(ii) and hg(ii) (janegitz et al. ) . sugunan et al. ( ) prepared a biosensor made of chitosan-gold nanoparticles to detect cu(ii) and zn(ii), and ahmed and fekry ( ) used a construct of chitosan-α-fe o nanoparticles sensor to detect ni(ii), as(ii), and pb (ii). biosensors were developed to detect and determine organophosphorus (op) pesticides as well. for instance, stoytcheva et al. ( ) prepared a device based on op hydrolase immobilization on a chitosan-carbon-nanoparticles-hydroxyapatite nanocomposite. a nanocomposite immunosensor to monitor the op compound, chlorpyriphos, is based on immobilized anti-chloropyriphos monoclonal antibody on multi-walled carbon nanotubes-chitosan-thionine (as electronic mediator) (sun et al. b ). an intricate electrochemical immunosensor for the detection and monitoring of the fungal hepatocarcinogen, aflatoxin b , as model antigen was developed by masoomi et al. ( ) . the construct scaffold involved chitosan-gold nanoparticles, immobilized polyclonal anti-aflatoxin b , and a magnetite core that can enable regeneration of the immunosensor. biosensors based on chitosan/multi-walled carbon nanotubes hybrid films were developed largely by babaei and colleagues to determine and quantitate drugs and neurotransmitters: acetaminophen and mefenamic acid (babaei et al. ) , dopamine and morphine (babaei et al. a) , paracetamol (babaei et al. b ), l-dopa (babaei and babazadeh ) , and -hydroxytryptamine and dopamine . the polycationic nature of chitosan films in immunobiosensors is also exploited to immobilize polyanionic polymers such as nucleic acid sequences and proteins. singh et al. ( ) devised an electrochemical dna biosensor to detect typhoid which was constructed by surface immobilizing salmonella typhi single-stranded (ss) dna on graphene oxide/chitosan/ito nanocomposite as a bioelectrode. the biosensor was capable of distinguishing between complementary, noncomplementary, and one base mismatch sequences. a similar electrochemical dna biosensor was developed for the detection of escherichia coli :h . it was prepared with immobilized e. coli ss-dna using a graphene oxide/chitosan hybrid nanocomposite. an electrochemical immunobiosensor to detect botulism neurotoxin a was reported by afkhami et al. ( ) . the sensor consisted of a gold nanoparticles/chitosan/graphene nanocomposite with immobilized antibodies to quantify the bound neurotoxin. to detect α-fetoprotein in human serum, an immunosensor was fabricated in which the α-fetoprotein antigen was immobilized on a film of a gold nanoparticles/carbon nanotubes/chitosan nanocomplex to quantify protein levels using a competitive immunoassay format (lin et al. ). giannetto et al. ( ) fabricated a competitive electrochemical immunosensor to detect hiv -related capsid protein p in human serum. the p antigen was immobilized on gold-free single-walled carbon nanotubechitosan complex for the interaction with a mouse monoclonal anti-p , which was used for competitive immunodetection. liu et al. ( ) developed an immunosensor to detect carcinoembryonic antigen, which is based on corresponding antibodies immobilized on chitosan-gold nanoparticles. finally, qiu et al. ( ) reported an immunosensor to detect hepatitis b surface antigen, which was constructed on the basis of a gold nanoparticles/chitosan/ferrocene biofilm with immobilized hepatitis b antibodies. . beneficial properties of chitosan for possible use in agriculture, food, and textile industry the wide-ranging antimicrobial, antiviral, and antioxidant activities, induction of defense systems in plants, and stimulation of plant growth by chitosan, chitosan oligomers, chemically modified chitosan and their composites have indicated their potential use in agricultural practices (el hadrami et al. ; malerba and cerana ) . pre-and postharvest treatment of coating seeds, fruits, and vegetables by edible chitosan-based films effectively improve germination and plant vigor and prolonged shelf life and storage quality of food products ). preservation by chitosan-based coating also expanded to include meat, eggs, dairy products, and seafood (friedman and juneja ). other promising practices such as delivery and slow and sustained release of chitosan-based encapsulated agrochemicals (fertilizers, micronutrients, pest control agents, and genetic materials) have been widely investigated (malerba and cerana ) . there are several comprehensive reviews that summarize the potential use of chitosan, its derivatives, and chitooligosaccharides in agriculture as related to their broad-spectrum antimicrobial and antioxidant activities (aider ; cota-arriola et al. ; li et al. a; xing et al. ; liaqat and eltem ) . such beneficial activities were demonstrated in a variety of agricultural products like preservation of vegetables, fruits, cereals, dairy products, eggs, meat, and seafood friedman and juneja ) . chitosan per se has antimicrobial activity that depends on higher degree of deacetylation, low molecular weight (its oligosaccharides), increased protonation at low ph, and the type of microorganisms (katiyar et al. ) . the antimicrobial efficiency is enhanced by adding essential oils (extracted from lemon, lemon grass, cinnamon, or rosemary) (duan and zhang ; xing et al. ; yuan et al. ) or by adding metal ions like silver or copper brunel et al. ; kumar-krishnan et al. ; choudhary et al. a; sharma ) particularly to chitosan-based nanoparticles (friedman and juneja ; cota-arriola et al. ) . the mode of action is mainly attributed to electrochemical interactions between the positively charged chitosan and the negative surface charge of bacterial cells leading to membranes disruption (xing et al. ) . in addition, penetration and binding of nanochitosan with microbial dna that impact mrna and protein synthesis were proposed (rabea et al. , malerba and cerana ) . scavenging of free radical and reactive oxygen species by chitosan and its derivatives is responsible for its antioxidative effects (guo et al. ; ngo and kim ) . scavenging of superoxide and hydroxyl radicals by chitosan and its derivatives was demonstrated by several studies (xie et al. ; guo et al. ; yen et al. ; wan et al. ) . furthermore, chitosan acts as a biogenic elicitor of various enzymes that detoxify reactive oxygen species (malerba and cerana ) and induces the formation of antioxidant and fungicidal phytoalexins (yamada et al. ; hadwiger ; xing et al. ) . chitosan and its derivatives were shown to activate plant immunity enzymes (catalase, peroxidase, superoxide dismutase, phenyl oxidase, phenylalanine ammonia lyase) that are capable of detoxifying reactive oxygen species (hadwiger ; xing et al. ; malerba and cerana ) . such activation engages different signal transduction pathways that involve a variety of second messengers. other defense responses include pathogenesis-related proteins, phytoalexins, proteinase inhibitors, lignin synthesis, or callose formation (el hadrami et al. ; hadwiger ) . induction of programmed cell death and hypersensitivity-associated responses by chitosan and chitooligosaccharides was documented (zuppini et al. ; vasilêv et al. ; zhang et al. ) , as well as activation of plant defense genes via the octadecanoid pathway leading to jasmonate synthesis (doares et al. ; rakwal et al. ) . chitosan induces hydrolase enzymes such as chitinase and β- , glucanase able to destroy chitin/glucan-containing fungal cell walls (ma et al. b; xing et al. ) . controlled and sustained release of chitosan-encapsulated agrochemical such as fertilizers, micronutrient, pesticides, and genetic materials was demonstrated by a plethora of investigations (kashyap et al. ) . food products coating by films of edible chitosan derivatives (plus a variety of additives) prolong their shelf life with concomitant improvements in storage quality yuan et al. ) . a number of examples linked to chitosan-coated pesticides given below indicate the potential of the eco-friendly techniques in plant protection against phytopathogens, insects, and weeds: controlled release of insecticides like the botanicals azadirachtin being encapsulated in the complex carboxymethyl chitosan-ricinoleic acid (feng and peng ) and rotenone wrapped in oleoyl carboxymethyl chitosan (kamari and aljafree ); nanoparticulate chitosan-β-cyclodextrin, which encapsulated carvacrol and exhibited high acaricidal and repellency activities (campos et al. ) ; and controlled release of avermectin conjugated to n,o-carboxymethyl chitosan or avermectin coated by silica cross-linked chitosan composite (he et al. ) . encapsulation of the neonicotinoids imidacloprid (li et al. a; lim and ahmad ) and acetamiprid (yan et al. ) , malathion, and spinosad (el badawy et al. ) by chitosan-alginate capsules exhibited prolonged release of the insecticides. slow release of the fungicide carbendazim against the phytopathogens sclerotinia sclerotiorum using chitosan/β-cd-epichlorohydrin (wang et al. a ) and hexaconazole encapsulated by chitosan nanoparticles against rhizoctonia solani (chauhan et al. ) was demonstrated. ilk et al. ( ) reported the antifungal and antioxidant activities of kaempferol encapsulated in lecithin-chitosan nanoparticles against fusarium oxysporum. in addition to their slow release property, chitosan composites also protect pesticides from photodegradation. nanoparticles of chitosan-beeswax protected deltamethrin from photodegradation (nguyen et al. ) , and a similar protective effect of avermectin was demonstrated for a silica/chitosan copolymer (he et al. ) . likewise, composites of chitosan with a variety of clays (montmorillonite, attapulgite, bentonite, and kaolinite), safe anionic dyes (fast green and naphthol yellow s), and photo-stabilized fungal conidia of the insect biocontrol agent aschersonia spp. were reported (cohen et al. ) . chitosan composites were found to be useful carriers of herbicides facilitating soil sorption as in the case of paraquat associated with chitosanalginate nanoparticles (silva mdos et al. ) or slow release of paraquat encapsulated in tripolyphosphate-generated chitosan nanoparticles (grillo et al. ). moreover, encapsulation of metolachlor in blended gel beads of cross-linked carboxymethyl cellulose and carboxymethyl chitosan was effective in slow release of the herbicide as a model compound (dong et al. ) . finally, slow release of atrazine encapsulated in carboxymethyl chitosan/bentonite gel was demonstrated (li et al. a ). the modulated release of encapsulated fertilizers is important for enhanced growth of plants while reducing environmental problems of their excessive use. experiments were accompanied by swelling rates of composites, fertilizer loads, and kinetics of release. examples are chitosan-xanthan tablets (melaj and daraio ) or chitosan-starch beads (perez and francois ) as carriers of potassium nitrate that serve as model fertilizer; slow release of npk fertilizers aggregated on chitosan nanoparticles (corradini et al. ) and application on leaf surfaces enables translocation via stomata into the phloem (abdel-aziz et al. ); efficient controlled slow release of water soluble npk fertilizers coated by chitosan with an additional outer coating by poly (acrylic acid-co-acrylamide) (wu and liu ) . this composite also exhibited improved water absorption and retention. noppakundilograt et al. ( ) examined the controlled release of npk fertilizer granules embedded in a hydrogel composed of poly(vinyl alcohol) and then chitosan and a third layer of acrylamide and acrylic acid following cross-linking of chitosan by glutaraldehyde. controlled release of urea by a variety of chitosan-based composites was established. urea dispersed with humic substances in chitosan (araújo et al. ) , urea encapsulated in chitosan-acryamide (siafu ) , urea release from adduct of silk fibroingelatin-chitosan hydrogels (rattanamanee et al. ) , urea smectite clay chitosan composite (puspita et al. ) , and urea-kaolinite mixed with chitosan (roshanravan et al. ) were tested for controlled release of the fertilizer. beneficial effects of preharvest chitosan-based seed coating and foliar treatment were reported by el hadrami et al. ( ) . chitosan-coated artichoke seeds, for example, induced better germination, stimulated root system growth, and were effective against a number of pathogenic fungi (ziani et al. ). bhaskara reddy et al. ( ) demonstrated induced resistance to seed-borne fusarium graminearum followed by improved germination and vigor in wheat seeds coated with chitosan. soybean seeds coated by chitosan had anti-feeding effects and protected against several insect pests (zeng et al. ) , and coating rice seeds increased antifungal effect, stimulated seeding growth, improved root system, and increased crop yield (zeng and shi ) . tomato seeds coated with chitosan resulted in resistance to infection by inducing plant defense mechanisms (benhamou et al. ) . chickpea seeds treated with chitosan-silver nanoparticles promoted germination and increased biomass, chlorophyll, carotenoids, and protein contents as well as amylase activity and defense enzyme activities (anusuya and banu ). similar effects were demonstrated in maize seeds coated with cu/chitosan nanoparticles (saharan et al. ; choudhary et al. b ). the antimicrobial activity of chitosan was targeted for use to improve preservation of a large variety of vegetable and fruit crops as well as of eggs, meat, and dairy products (devlieghere et al. ; friedman and juneja ; yuan et al. ) . chitosan with added compounds such as plant materials and animal proteins (formulations of chitosan with additions of tapioca starch, hydroxypropyl cellulose, pectin, and fish gelatin) was used to develop edible films. such films in addition to their antimicrobial and antioxidant activities also keep food products from loss of moisture and oxygen penetration (aider ; duan and zhang ) . postharvest coating of vegetables and fruits with chitosan and additional essential oils (extracts from lemon, rosemary, lemon grass, bergamont, cinnamon, oregano, and thymine), which by themselves exert antimicrobial and antioxidant activities, improved storage quality and prolonged the shelf life of products . controlling postharvest decay during storage was reported also for additives such as olive oil, glacial acetic acid, green tea extract, and lactic acid yuan et al. ) . microbial contaminations are a serious problem in food industry, because foodborne bacteria and fungi are associated with food spoilage and food poisoning leading to economic losses and human health risks. using appropriate food packaging materials with antimicrobial properties may prevent or at least slow down bacterial and fungal growth. for this reason, a variety of biopolymers has been tested to identify alternative materials to the classical nondegradable plastic packaging materials, which have caused serious environmental issues due to their inappropriate disposal. optimal alternative materials should be environmentally safe due to biodegradability and biocompatibility. as chitosan-based material combine antimicrobial properties with biodegradability and biocompatibility, they are the focus of research in food packaging. moreover, chitosan-based materials have food-preserving antioxidant activity and film-forming ability, which allows the production of transparent foils and bags. different methods have been established during the past decades to fabricate chitosan films including casting, coating, extrusion, and layer-by-layer synthesis, and the resulting materials have been evaluated for their antimicrobial and antioxidant activity and for their optical, mechanical, barrier, and thermal characteristics. chitosan has also been combined with other functional materials resulting in composite films with tremendous preservative properties that can be utilized for the packaging of different foods such as vegetables, fruit, and meat. for a comprehensive overview on this topic, the reader is referred to an excellent review article published recently by wang et al. ( ) . pure chitosan films are frequently based on dispersions of chitosan nanoparticles (ali et al. ) , to which plasticizers, such as glycol (leceta et al. ) , and/or surfactants, such as tween (martins et al. ) , are added to modify the mechanical properties and to emulsify auxiliary compounds. in addition, chitosan nanofibers have been fabricated as a packaging material and tested for their antimicrobial activity. for instance, arkoun et al. ( ) examined the antimicrobial activity of chitosan/polyethylene oxide nanofibers produced by an electrospinning process. they showed that the chitosan nanofibers were efficient in inhibiting growth of e. coli, staphylococcus aureus, lysteria innocua, and s. typhimurium, however at ph . , which was below the pka of chitosan, limiting the applicability to slightly acidic food. importantly, the authors demonstrated that the antibacterial effects were irreversible, suggesting a bactericidal rather than bacteriostatic mechanism. combinations of chitosan and other natural polysaccharides have been frequently used to fabricate functional films with applications in food packaging. these biopolymers comprise of cellulose and various cellulose derivatives, alginate, cyclodextrin, glucan, mannan, pectin, starch, and xylan. chitosan/cellulose films revealed improved mechanical properties while maintaining excellent antimicrobial properties . also chitosan/hydroxypropyl methylcellulose (hmpc) films exhibit significant antimicrobial activity. for instance, möller et al. ( ) examined the antimicrobial effects of chitosan/hpmc films against listeria monocytogenes and found that bacterial growth was completely inhibited on the film. similarly, chitosan/carboxymethyl cellulose films showed superb food preservation properties when tested on packaged cheese (youssef et al. ) . antimicrobial chitosanalginate films have a great potential for food packaging as well, particularly because they show improved gas exchange and water vapor permeability properties when prepared by a layer-by-layer electrostatic deposition approach (poverenov et al. a ). martiñon et al. ( ) studied the effectiveness of antimicrobial multilayered coatings consisting of chitosan, pectin, and trans-cinnamaldehyde at different concentrations to extend the shelf life of fresh-cut cantaloupe and found that certain compositions were effective in preventing bacterial growth and spoilage. lorevice et al. ( ) produced chitosan nanoparticles and combined them with different methyl pectin matrices to generate nanocomposite films and tested the mechanical, thermal, and barrier properties. the results showed that the nanocomposite film improved mechanical characteristics when compared with conventionally produced pectin films, making these novel materials promising for food packaging production. similarly, chitosan/cyclodextrin films with inclusions of essential oil have been reported to possess desirable mechanical properties for food packaging . moreover, this material showed significant antimicrobial activities against a variety of pathogenic bacteria. chitosan films have been also combined with a variety of proteins including casein (khwaldia et al. ) , gelatin (poverenov et al. b; noorbakhsh-soltani et al. ) , collagen , kidney bean protein ), lactoferrin (brown et al. ) , and lysozyme (yuceer and caner ) , as well as with antibacterial peptides such as nisin . in addition, chitosan was blended with antimicrobial and antioxidant extracts from bee wax (velickova et al. ) and plants, such as citrus (iturriaga et al. ) , thyme (talon et al. ) , and maqui berry (genskowsky et al. ) , as well as with essential oils including clove bud oil, cinnamon oil, and star anise oil . other approaches in fabricating chitosan-based films employed grafts, blends, or casts using synthetic polymers such as poly(vinyl alcohol) , poly (lactic acid) (pal and katiyar ) , poly(ethylene) (reesha et al. ) , poly(ethylene oxide) (kohsari et al. ) , poly(styrene) (lopez-carballo et al. ) , poly(propylene) (cavallo et al. ) , poly(caprolactone) (alix et al. ) , and poly(acrylonitrileco-acrylamide) (kumar et al. ) that led to improved mechanical and thermal properties. however, these synthetic polymers are not readily degraded in nature; hence concerns regarding the environmental safety have been raised. guo et al. ( ) developed new edible antimicrobial films using microemulsions in combination with high-pressure homogenization processing. the films were made of chitosan, allyl isothiocyanide, and barley straw arabinoxylan, which were used as film-forming, antimicrobial, and emulsifying agents, respectively. the material was tested to be efficient in preventing growth of l. innocua. to improve antibacterial activity, chitosan-based films were synthesized as composites with metals, minerals, and other inorganic compounds. youssef et al. ( ) produced chitosan-silver and chitosan-gold (cs-au) nanocomposites films, which showed enhanced antimicrobial activity against gram-positive (s. aureus) and gram-negative bacteria (pseudomonas aerugenosa), fungi (aspergillus niger), and yeast (candida albicans). in another study published by al-naamani et al. ( ), poly(ethylene) films were coated with zinc oxide/chitosan nanocomposite, which completely inactivated and prevented the growth of food pathogens. in an approach based on a solution cast method, sanuja et al. ( ) fabricated a chitosan-based nanocomposite film using nano zinc oxide and neem essential oil, which improved mechanical, physical, barrier, and optical properties. moreover, zhang et al. ( ) prepared chitosan/titanium dioxide composite films, which were found to possess significant antimicrobial activity against e. coli, s. aureus, c. albicans, and a. niger. xu et al. ( c) employed a different strategy by synthesizing chitosan/graphene oxide nanocomposites with titanium dioxide and analyzed their antimicrobial and food-preserving efficacies. they showed that the material effectively prevented bacillus subtilis and a. niger biofilm formation presumably by disrupting cellular membranes. in addition, they demonstrated that the nano-coating could be applied as a cling film, which delays loss of moisture in fruits and vegetables and inhibits polyphenol oxidase activity and thus enzymatic browning but increases superoxide dismutase activity, which protects against reactive oxygen species. next to these materials, chitosan-montmorillonite composites, chitosan/nanosilica films, and manifold combinations of chitin, metals, and minerals have been tested. in addition, numerous chemical chitosan derivatives have been explored for their properties to screen for new films suitable in food packaging. these derivatives include carboxymethyl chitosan and quaternized chitosan such as ( -n-hydroxypropyl- trimethylammonium chloride) chitosan ). due to their versatile and unique physicochemical and biological properties, chitosan, its multiple derivatives, and their adjunct complexes (addition of functional groups) have attracted considerable attention for possible use of eco-friendly materials in the textile industry. they are relatively inexpensive, biocompatible, biodegradable, and nontoxic and readily adhere to textile fabrics and usually demonstrate antibacterial activity. certain formulations retain moisture as well as impart thermal stability and uv protection. chitosan per se or blends of chitosan-based composites deposited onto textiles fabrics were mostly tested for durable antibacterial activity (nearly all antibacterial studies include e. coli and s. aureus that represent correspondingly gram-negative and gram-positive bacteria). coated thai silk fabric with chitosan using radio frequencies plasma treatment exhibited antibacterial effects (wongsawaeng et al. ) , and polyester/cotton fabric treated with chitosan can be used as an alternative to the antibacterial triclosan (ranganath and sarkar ) . chitosan grafted on cotton (ferrero et al. ) or on wool (periolatto and ferrero ) fabrics using uv irradiation bestowed antibacterial activity after many washing cycles. chitosan reduced to nanoparticles and applied onto wool fabric imparted durable antibacterial and bestowed shrink proofing (yang et al. ) . nanonized chitosan applied onto cotton exhibited, in addition to antibacterial activity, also thermal stability, uv protection, as well as improved dye-binding ability (hebeish et al. ). periolatto et al. ( ) demonstrated antibacterial effects and laundry durability of cotton and silk fabrics by uv curing with -hydroxy- -methylpropylpropane- -one as photoinitiator of the photochemical reaction. chitosan possesses abundant potential, in particular, for use in medical textiles and sportswear. for example, a blend of chitosan (short fibers) with cotton (long fibers) yarn by spinning technology is desirable for medical applications (lam et al. ) . gauze bandages for wound dressing were prepared by electrospinning of chitosan nanofibers and cotton fabric (nawalakhe et al. ) . plasma treatment was applied to improve adhesion by increased cross-linking between the two fiber systems imparting subsequent durability (nawalakhe et al. ) . pure chitosan microfibers produced by wet spinning process was aimed for possible stable -d scaffold woven or nonwoven textile fabrics to be used in regenerative medicine such as bone and cartilage engineering (toskas et al. ) . lam et al. ( ) examined a blend of chitin fibrils with cotton jersey fabric and showed reduced rigidity that may provide comfort to patients with epidermolysis bullosa skin disease. likewise, chitosan-coated textile fabrics improved atopic dermatitis disease by restraining skin microbiome (lopes et al. ) . sonochemical deposition was used by petkova et al. ( ) to coat cotton fabrics with a hybrid of chitosan and zno nanoparticles. this complex showed improved antibacterial activity, slow release of the metal and washing stability, and postulated as effective treatment for hospital textiles to prevent transfer of pathogens. similarly, the hybrid of chitosan and silver nanoparticles deposited onto cotton fabric demonstrated antibacterial effects and laundry durability befitting their possible use for medical textiles and sportswear . ali et al. ( ) proposed to use chitosan nanoparticles that are able to pick and retain silver ions in medical textile applications. a polyester fabric coated with this complex hybrid imparted enhanced antibacterial activity. nanonized chitosan applied onto cotton exhibited in addition to antibacterial activity, also thermal stability, uv protection as well as improved dye-ability (hebeish et al. ) . a large number of publications signified and reported beneficial properties of chitosan and chitosan-based formulations with possibly great potential to treat textile fabrics. such valuable features include protecting a variety of fabrics with emphasis on medical textiles, production of aromatic and flame-retarding fabrics, as well as dye removal and treatment of textile wastewater. table . summarizes inter alia nanochitosan, chitosan nanometal complexes, or chitosan derivative composites with metals and other substances, which were treated onto textile fabrics (notably cotton), and depicts their conceivable potential for the textile industry. chitin and, in particular, chitosan and its derivatives provide advantageous properties in the cosmetic area. they are biocompatible and adhere to surface components of the skin and hair, forming elastic films with moisturizing and water retention capabilities. they can serve as vehicles for encapsulated cosmetic ingredients and their controlled delivery and release and formation of gels in mixtures with water and alcohol and have some antimicrobial, antioxidant, and anti-inflammatory activities, with the additional important benefit of low cytotoxicity jimtaisong and saewan ; aranaz et al. ) . chitosan and its derivatives are included in cosmetic formulations and products for mainly care and protection of the skin and hair but inter alia in tooth enamel and tooth lacquer, nail lacquer, lipsticks, cleansing and bath materials, toothpaste, mouthwash, chewing gum, deodorants, and breath refresheners (dutta et al. ). aging of the skin, viewed as wrinkling, dryness, loss of elasticity, dehydration, and hyperpigmentation, is the result of long-term exposure to sunlight uv, which mainly forms reactive oxygen species. protection from photoaging is a major drive in the cosmetic industry. for example, chitooligosaccharides per se were able to protect uv-irradiated hairless mouse skin from photoaging damage (kong et al. ) . gel formulation of chitosan microparticles served as a delivery system for the sustained release of the hydrophilic sunscreen, phenylbenzimidazole sulphonic acid (gomaa et al. ) . the cosmetic gel formulation of blended chitosan, collagen, and aloe vera, with antibacterial and antioxidant effects, proved useful in the regeneration and rejuvenation of the skin using cultured mouse fibroblast (rajashree and rose ). microspheres composed of carboxymethyl chitosan/collagen peptides-calcium chloride protected mice skin and thymus lymphocytes from uv-b radiation damage thermally stable blend grande et al. (continued) (liu et al. b) . a cosmetic cream formulation composed of quaternized carboxymethyl chitosan-montmorillonite nanocomposite bestowed good uv protection and additional moisturizing and water retention effects . there is a possible cosmetic use of neutralized chitosan in citrate buffer film for skin exfoliation (libio et al. ). chitosan and various chitosan derivatives, as active ingredients, were examined in cosmetic hair care products like shampoos, permanent wave agents, hair conditioner, styling lotions, rinses, hair colorant, hair sprays, and hair tonics (dutta et al. ; aranaz et al. ) . they can adhere to the negatively charged hair keratin forming a transparent elastic film that covers hair fibers endowing smoothness, softness, and also mechanical strength (dutta et al. ) . a blend of chitosan and two other biopolymers like collagen and hyaluronic acid that forms a thin film over hair surface provides enhanced mechanical strength and improved conditioning of the treated hair (sionkowska et al. ). chitosan as a targeting vehicle to hair follicles in the skin was demonstrated with entrapped minoxidil, a medication to treat hair loss (gelfuso et al. ; matos et al. ) . microparticles and nanoparticles of chitosan-encapsulating minoxidil enabled its controlled release. a large number of possible applications of cosmetic formulations containing chitosan and its derivatives have been patented. it is noteworthy that formulations containing chitosan are already in the busy cosmetic market. removal and degradation of anionic azo dyes Škorić et al. ( ) ab antibacterial effects, cs chitosan, lbl layer by layer deposition, β-cd β-cyclodextrin a photochemical reaction by uv generating cross-linked polymers because chitosan and many of its derivatives are nontoxic, biocompatible, biodegradable, and highly versatile polymers, a large assortment of possible biomedical applications have been explored, of which some were implemented into therapeutic strategies by the pharmaceutical industry. to obtain optimal materials for the delivery of drugs, several factors have to be considered including the stability of the bioactive agents, absorption properties and mucoadhesiveness, gelling properties, particle sizes, permeation and transfection-enhancing properties, efflux pump inhibition, tissue targeting, residual toxicity of the final products, as well as release kinetic profiles. chitosan derivatives have been developed into different kinds of pharmaceutical excipients used for the production of tablets and capsules (illum ; werle and bernkop-schnurch ) , suppositories (caramella et al. ) , sprays (osman et al. ) , ointments (kang et al. ) , eye drops (basaran and yazan ) , and wound dressings (bano et al. ) . the drugs are usually encapsulated by ionotropic gelation, spray drying, emulsion solvent evaporation, and coacervation (panos et al. ). chitosan-based excipients have been found useful in tablet disintegration and drug dissolution (illum ) and in enhancing penetration and absorption properties (thanou et al. ; van der merwe et al. ; sahni et al. ) . most importantly, certain dosage forms allow the controlled release of drugs (jennings et al. ; fonseca-santos and chorilli ). these include chitosan-based hydrogels (knapczyk ; kristl et al. ; berger et al. ; ishihara et al. ; elviri et al. ) and micro-/nanoparticles for drug delivery (hamman ). here, we will focus on the applications of chitosan-based matrices in drug delivery for cancer, immune, and gene therapy, and we will summarize some recent advances in tissue engineering ( fig. . ). chitosan-based materials can be used in various forms as drug delivery system ( fig. . ). tablets are probably the most favorable and accurate dosage form, which are moreover easy to fabricate and handle. a simple method for their production is homogenization of the drug and chitosan and compressing the resulting mixture to tablets. however, it has to be considered that due to the alkaline conditions in the distal intestine, drug absorption is restricted to the more proximal regions of the gastrointestinal tract when pure chitosan is used which precipitates at an alkaline ph (sakkinen et al. ; dhaliwal et al. ) . therefore, more ph-insensitive formulations using higher-charged chitosan derivatives such as trimethylated chitosans or thiolated chitosan conjugates have improved absorption properties along the gastrointestinal tract. although there is still a lack of robust data in human volunteers, some studies indicate that tablet formulations using highercharged chitosan derivatives increase bioavailability due to improved mucoadhesiveness and better protection of the drug from degrading enzymes (van der merwe et al. ). chitosan-based tablets have been also examined for their use in vaginal drug delivery, mainly as carriers for antiviral and antifungal therapeutics (el-kamel et al. ; senyigit et al. ; frank et al. ). however, the antimicrobial properties of chitosan may negatively affect the vaginal microflora, and hence long-term treatment should be critically evaluated (raafat and sahl ) . as chitosan-based hydrogels facilitate equal distribution and increase mucoadhesiveness, permeation, and bioavailability, they are effective formulations for eye drops to administer therapeutic drugs in ophthalmology (krishnaswami et al. ) . chitosan-based formulations used in eye care include hydrogels, nanoparticles, and liposomal and colloidal systems (de campos et al. ; de fig. . chitosan-based drug delivery systems campos et al. ; diebold et al. ; gupta et al. ) . for similar reasons, they are also in use for nasal drug delivery, which is impaired by high turnover and secretion rates (illum ) . notably, chitosan-coated lipid micro-and nanoparticles have been developed for nose-to-brain delivery of a variety of therapeutic drugs (casettari and illum ; sarvaiya and agrawal ) . chitosan-based nanoparticles are also a promising carrier for buccal drug delivery, which has the advantage of avoiding the hepatic first-pass metabolism and degradation in the gastrointestinal system (sandri et al. ) . polymeric carriers generally have the potential advantage of prolonged release times of low-molecular-weight drugs. because chitosan is additionally susceptible to hydrolysis by lysozyme in the blood serum, which facilitates drug release, and exhibits no toxic or hemolytic effects when applied parenteral (nordtveit et al. ; richardson et al. ) , chitosan-based formulations are also suitable carriers for controlled drug release when administered by intravenous injection (thanoo et al. ). conventional chemotherapeutics are frequently not very effective in reaching the tumor cells, as solid tumors are not well supplied with blood, and lack lymphatic vessels, which results in and decreased convective flow in the interstitial fluid. to overcome these problems, novel drug delivery systems have been designed. these carriers are capable of encapsulating high concentrations of the cytotoxic compound within a macromolecular matrix that specifically targets the cargo to the tumor cells where the drugs are finally released in a controlled manner. this concept profits from the epr (enhanced permeability and retention) effect, the phenomenon that macromolecules preferentially accumulate in solid tumors, probably because they have a defective vasculature and lack effective lymphatic drainage (matsumura and maeda ) . chitosan-based nanoparticles have many properties that make them suitable carriers for anticancer drugs. next to their great chemical flexibility, allowing the design of selective carriers, chitosan-based materials evidently exhibit also the epr effect depending on the tumor microenvironment (yhee et al. ) . moreover, they are degraded inside the body into fragments which can be cleared by the kidney (kean and thanou ) , and several studies suggested that chitosan itself has antitumor effects (qi and xu ; yao et al. a) , making this polymer a highly suitable supplementary antitumor drug and drug carrier. indeed, chitosan-based nanocomposites can be used to deliver hydrophilic and hydrophobic drugs such as doxorubicin hydrochloride and paclitaxel, respectively (kim et al. ; yousefpour et al. at anti-tumor effects, cs chitosan, tu tumor conjugate and examined the antitumor effects in vivo in macrophage tumor cells implanted into balb/c mice. the authors observed an improved therapeutic efficacy of dextran-doxorubicin loaded chitosan nanoparticles, which is probably due to the prolonged circulation time and/or drug accumulation at the tumor sites. in another study published by yousefpour et al. ( ) , doxorubicin was conjugated to chitosan using succinic anhydride as a cross-linker. in a second step, the resulting self-assembled chitosan-doxorubicin conjugate nanoparticles were conjugated with trastuzumab, a monoclonal antibody to the human epidermal growth factor receptor + (her +), via lysine thiolation and subsequent linking of the derived thiols to chitosan. the trastuzumab conjugated chitosan-doxorubicin nanoparticles selectively targeted her + cancer cells resulting in enhanced uptake when compared to chitosan-doxorubicin particles and the free drugs. in another study, pluronic f , a block copolymer of hydrophobic polyoxypropylene flanked by two chains of hydrophilic polyoxyethylene, was grafted onto chitosan to generate a copolymer micelle that can encapsulate doxorubicin (naruphontjirakul and viravaidya-pasuwat ) . the resulting chitosan-pluronic micelles carrying doxorubicin showed a high drug loading capacity and revealed a higher cytotoxic activity to mcf breast cancer cell lines in vitro than the free drug. another approach to deliver doxorubicin specifically to cancer cells was reported by varshosaz et al. ( ) . the research team fabricated dual targeted nanoparticles loaded with doxorubicin and magnetic nanoparticles to treat breast cancer. for this purpose, the nanoparticles were produced via a layer-bylayer technique and functionalized with a bioconjugate of chitosan/poly(methyl vinyl ether maleic acid) and luteinizing hormone-releasing hormone (lhrh) to target corresponding receptors on the surface of mcf breast cancer cells, which presumably take up the particles by endocytosis. the targeted nanoparticles increased the cytotoxicity of doxorubicin about twofold in lhrh-positive cancer cells. in a similar approach, folate-grafted chitosan-coated magnetic nanoparticles were loaded with doxorubicin to target human glioblastoma u cells in athymic balb/c nude mice in a subcutaneous tumor model system . guiding the injected nanoparticles to the tumor by a magnetic field significantly decreased tumor growth by controlled delivery of doxorubicin to the cancer cells and demonstrated the feasibility of magnetic nanoparticles to direct the localization of drug release. mesoporous aluminosilicate zeolite (zsm- ) chitosan core-shell nanodisks loaded with doxorubicin were used as ph-responsive drug delivery systems against osteosarcoma that release the drug after upon endosomal acidification . recently, radmansouri et al. ( ) showed that doxorubicin-loaded electrospun chitosan/cobalt ferrite/titanium oxide nanofibers could be used for localized melanoma cancer therapy. the fastest release of doxorubicin from prepared magnetic nanofibers was observed at acidic ph when an alternating magnetic field was applied. as mentioned above, chitosan-based nanoparticles can also be modified to carry hydrophobic drugs such as paclitaxel. for this purpose, hydrophobic side chains are grafted onto chitosan. for instance, kim et al. ( ) used glycol chitosan nanoparticles that were hydrophobically modified with β-cholanic acid and incorporated paclitaxel. the resulting nanoparticles showed sustained drug release, and following injection into the tail vein of tumor-bearing mice, tumor growth was impaired. in a subsequent study, the same research team used this system as carrier for cisplatin, which is also poorly soluble in water. the hydrophobically modified glycol chitosan nanoparticles loaded with cisplatin exhibited the epr effect, as they accumulated in solid tumors, and was proven to have a high antitumor efficacy in a tumor-bearing mice model (kim et al. ) . paclitaxel was also encapsulated in chitosan-containing glyceryl monooleate core-shell nanoparticles, which were generated by the emulsification/evaporation technique (trickler et al. ) . using this drug delivery system, the authors observed a -fold increase in cytotoxicity, when determining the ic values in a human breast cancer cell line. another common hydrophobic anticancer drug is -fluorouracil, which has been widely used to treat different kinds of solid tumors. in a study by zheng et al. ( ) , polyelectrolyte nanoparticles based on chitosan and polyaspartic acid sodium salt were used to encapsulate -fluorouracil testing various conditions for nanoparticle preparation such as temperature, ionic strength, ph and cross-linker concentration, and different loading methods. the optimized nanoparticles showed sustained drug release in vitro and in vivo. rajan et al. ( ) prepared hyaluronidase- -fluoruracil-loaded chitosan-polyethylenglycol-gelatin copolymers as a targeted drug delivery system and examined particle size, distribution, morphology, and drug loading capacity. the nanoparticles showed less cytotoxicity than free -fluorouracil when applied to colon cancer cells for a few hours. another approach for controlled drug delivery used molecular surface imprinted graft copolymer of chitosan with methyl methacrylate, which was prepared by free-radical polymerization with -fluorouracil as template molecule (zheng et al. ). the ph dependency and the kinetics of drug release suggested that this chitosan-based carrier is optimal for orally applied colon-specific drug delivery. a similar strategy to achieve colon specificity was used recently by bashir et al. ( ) . they synthesized ph-responsive semi-interpenetrating network hydrogels of n-succinyl-chitosan via schiff base mechanism using glutaraldehyde as a cross-linking agent and embedded poly(acrylamide-co-acrylic acid). the hydrogel exhibited a porous structure and ph-dependent swelling properties. the hydrogel was effectively loaded with -fluorouracil, and the determined drug release was ph-dependent as well, with high release rates at ph . and low rates at ph . . in many cases, chitosan nanoparticles have been conjugated with tumor-specific ligands to mediate active targeting of cancer cells, which is expected to increase therapeutic efficacy, accelerate drug release to selected sites, prevent unwanted drug release before arrival at the target sites, and diminish adverse side effects of chemotherapeutic drugs. active targeting can be accomplished by functionalizing chitosan-based nanoparticles and hydrogels using tumor-targeting ligands, which bind to specific receptors that are specifically present on the surface of cancer cells. proper ligands of such kind include cytokines, peptides, folic acid, hyaluronic acid, biotin or avidin, and antibodies (prabaharan ) . here, we will discuss only a one example for each of these ligands to illustrate active targeting. deng et al. ( b) synthesized monodispersed and ph-sensitive chitosan-silica hollow nanospheres, loaded them with antitumorigenic tumor necrosis factor α (tnf-α), and conjugated them with an antibody to epidermal growth factor receptor , which is overexpressed in about % of all women suffering from breast cancer (owens et al. ) . subsequent drug release studies demonstrated that the nanospheres delivered cytotoxic tnf-α to mcf- breast cancer cells and suppressed tumor with high therapeutic efficacy. due to the acidic microenvironment inside solid tumors, tnf-α is gradually released from the nanospheres, binds to the tnf-α receptor, and activates a signaling cascade which induces programed cell death. in a study published by shen et al. ( a) , doxorubicin and verapamil were combined in chitosan nanoparticles to achieve an integrated treatment for cancer and doxorubicin-induced cardiomyopathy in the process of cancer therapy. for this purpose, chitosan shells coated on magnetic nanoparticles were loaded with both drugs and entrapped into poly(lactic acid-co-glycolic acid) nanoparticles conjugated with a cyclo(arg-gly-asp-d-phe-lys) (crgd) peptide targeting α v β integrin, which is highly expressed on activated endothelial cells of newborn vessels during tumor angiogenesis as well as in some tumor cells . near-infrared laser irradiation was sufficient to trigger drug release within an acidic microenvironment. cytotoxicity assays performed in vitro suggested that crgd-conjugated nanoparticles exhibited a greater growth inhibitory potential in cancer cell lines than the free drug or control nanoparticles likely due to crgd-mediated targeting of tumor cells. in vivo imaging and biodistribution studies further showed that the nanoparticles preferentially accumulated in the tumor tissue under magnetic guidance. finally, in vivo data for tumor regression along with electrocardiogram recordings and histopathology observations indicated that the crgd-conjugated polymer-coated magnetic nanoparticles could have a high therapeutic potential as a dual-drug delivery system for the treatment of both cancer and doxorubicinmediated cardiotoxicity. recently, a novel disulfide-linked chitosan-g-methoxy poly(ethylene glycol) copolymer was successfully synthesized, which was suggested to have excellent properties for redox-responsive drug delivery (antoniraj et al. ). redoxresponsive -fluorouracil-loaded nanoparticles were synthesized by ionic gelation method, and folic acid was used to functionalize the nanoparticles for receptortargeted drug delivery, as cancer cells commonly express high-affinity folate receptors on their surface. the -fluorouracil-free nanoparticles showed improved hemocompatibility, and the -fluorouracil-loaded nanoparticles conjugated with folic acid had a high cytotoxicity to mcf breast cancer cells, presumably due to intracellular internalization because of folic acid conjugation, which is expected to enhance the cellular uptake of the nanoparticles. many types of cancer cells overexpress different isoforms of hyaluronic acid receptors, which leads to enhanced binding and internalization of hyaluronic acid, as reported for instance in breast tumor cells (bourguignon et al. ) . to exploit this fact for targeting tumor cells, jain et al. ( ) prepared hyaluronic acid-conjugated chitosan nanoparticles loaded with oxaliplatin and encapsulated in eudragit s coated pellets for effective delivery to colorectal tumors. in immunodeficient c bl mice model with ht- cancer cells injected into the ascending colon, relatively high local drug concentrations were found in the colon tumors after oral administration of the oxaliplatin nanoparticles, and the concentrations increased with prolonged exposure time. coupling of hyaluronic acid onto the surface of chitosan nanoparticles was found to make them more specific for delivery of the anticancer drug to the tumor of the colon. several studies revealed that also biotin and avidin possess tumor-targeting properties. biotin receptors are overexpressed in many tumor types characterized by rapid division rates and aggressive growth (russell-jones et al. ) , and avidin (a highly glycosylated protein) is recognized by lectins expressed on the surface of tumor cells (yao et al. ) . for this reason, bu et al. ( ) prepared chitosan nanoparticles conjugated with either biotin or both biotin and avidin as tumortargeted carrier system for the delivery of trans-resveratrol. pharmacokinetic experiments revealed that avidin-biotin-loaded nanoparticles rapidly accumulated in the liver after injection, while the delivery nanoparticles conjugated only with biotin was attenuated. cytotoxicity assays using hepg cells further uncovered that compared to trans-resveratrol solution and unconjugated chitosan nanoparticles, both biotin and avidin-biotin loaded nanoparticles significantly improved anticancer activity, but the latter combination exhibited a higher cytotoxicity. thus, it was proposed that the synthesized nanoparticles conjugated with avidin and biotin may be a potent drug delivery system particularly to targeting hepatic carcinoma. finally, xiao and yu ( ) developed a glycol/chitosan nanobioconjugate loaded with gemcitabine and conjugated with anti-egfr and anti-chitosan antibodies to target pancreatic cancer cells and cause aggregation. administration of the chitosan conjugates efficiently blocked tumor growth and metastatic spread in human pancreatic cancer cells. gene therapy requires the transmission of nucleic acids (dna or rna) into the target cell to mediate expression of therapeutic genes or to silence gene expression by rna interference. however, negatively charged phosphates of nucleic acids impair permeation through the plasma membrane, which is negatively charged as well. in addition, unprotected nucleic acids are highly susceptible to degradation by nucleases. hence, delivery of nucleic acids into cells relies on non-viral or viral vectors, which drastically improves transfection and protects from enzymatic degradation (wivel and wilson ) . as viral vectors have the risk of causing adverse side effects such as immune reactions and malignant transformation, many efforts have been made to develop non-viral vectors for gene delivery, among them are ample examples of different chitosan-based nanoparticles. actually, unmodified chitosan is not an effective carrier for the transfer of nucleic acids due to its low solubility in water and instability of dna/rna chitosan complexes at physiological ph. thus, chitosan requires chemical modification or grafting to convey appropriate physicochemical properties to the resulting complex. chitosan modifications that have been used to design chitosan-based carriers for gene or sirna delivery include quaternization by alkylation of tertiary amines, reaction with -chloroethylamine hydrochloride and n,n-dimethyl- chloroethylamine hydrochloride, conjugation with polyethylene glycol, poly (amidoamine) or rgd dendrimer grafting, modification with phosphatidylcholine, or combinations of these modifications. among the quaternized chitosan derivatives, n-trimethyl chitosan and its derivatives have been extensively studied for their suitability in gene delivery, because they are reasonably soluble in water, have comparably little tendency to form aggregates, and exhibit a high loading capacity for nucleic acid under physiological conditions. in a systematic study published by germershaus et al. ( ) , the physicochemical properties of chitosan, n-trimethyl chitosan, and poly (ethylenglycol)-n-trimethyl chitosan were analyzed and compared. using cell lines derived from mouse embryonic fibroblasts as a transfection system for plasmid dna, the authors observed a significant increase in transfection efficiency when n-trimethyl chitosan nanoparticles were used to deliver plasmid dna instead of chitosan nanoparticles. in addition, grafting poly(ethylenglycol) onto n-trimethyl chitosan further improved transfection efficiency, stabilized the particles, decreased particle size, and reduced cytotoxicity when compared to unmodified n-trimethyl chitosan nanoparticles. zheng et al. ( ) prepared folate-conjugated n-trimethyl chitosan nanoparticles and compared cellular uptake and transfection of plasmid dna (pdna) in vitro with non-conjugated n-trimethyl chitosan nanoparticles using folate overexpressing kb and skov cells and folate receptor-deficient a and nih/ t cells. the folate-n-trimethyl chitosan/pdna complex showed a decrease in cytotoxicity in comparison to pdna complexes made of polyethylenimine. moreover, folate conjugation increased transfection efficiency and folate receptormediated endocytosis by kb cells and skov cells when compared to non-conjugated n-trimethyl chitosan nanoparticles. exploring further possible improvements of n-trimethyl chitosan-based gene delivery systems, zheng et al. ( ) synthesized arginine, cysteine, and histidinemodified trimethyl chitosan nanoparticles to form complexes with pdna. using hek cells, they evaluated stability, cellular uptake, endosomal escape, release behavior, nuclear localization, and in vitro and in vivo transfection efficiencies. the cysteine-modified n-trimethyl chitosan nanoparticles turned out to be the most promising candidates for gene delivery due to sufficient stability, high cellular uptake, and glutathione-responsive release-favoring mechanism in combination with preferable nuclear distribution. addition of sodium tripolyphosphate to the cysteine-modified nanoparticles was further effective to compromise certain disadvantageous attributes for pdna delivery. n-trimethyl chitosan nanoparticles were also employed in drug-sirna co-delivery using a metastatic breast cancer cell line (eivazy et al. ) . in this study, the authors tested simultaneous delivery of sirna to silence the gene encoding the high mobility antigen (hmga- ) and the anticancer drug doxorubicin to boost therapeutic anticancer effects. they found that dual delivery of hmga- sirna and doxorubicin by trimethyl chitosan nanoparticles significantly inhibited breast cancer cells growth. a very recent study developed novel strategies in fighting lung cancer by rna interference mediated gene silencing of the gene encoding the anti-apoptotic protein, survivin. for this purpose, ni et al. ( ) designed nanoparticles consisting of baclofen functionalized n-trimethyl chitosan as polymeric carriers, tpp as ionic cross-linker, and sirna to survivin. baclofen was used to target the nanoparticles to non-small lung cancer cells that overexpress the gaba b receptor, which specifically binds baclofen (zhang et al. b ). the sirna-loaded nanoparticles increased the uptake of survivin-sirna through the interaction with gaba b receptor and efficiently induced apoptosis and gene silencing. the authors further encapsulated the sirna-loaded nanoparticles into mannitol microparticles for dispersion in the hfa- a aerosol to allow administration by pressurized metereddose inhalers. pulmonary delivery of sirna is expected to avoid serum-induced degradation, reduce systemic side effects, and improve therapeutic efficacy. zhong et al. ( ) hypothesized that the addition of amino residues to chitosan could improve stable complex formation with negatively charged sirna enhancing transfection and gene silencing efficiency. for this purpose, they prepared a novel chitosan derivative (mixnch) combining -chloroethylamine hydrochloride and n, n-dimethyl- -chloroethylamine hydrochloride with chitosan and examined the physicochemical properties of the resulting nanoparticles. using a hepatocellular carcinoma cell line (hepg ), gene transfection efficiency of mixnch/midkine-sirna nanoparticles and inhibition of hepg cell proliferation were analyzed. they found that midkine-sirna delivered by mixnch nanoparticles was able to significantly reduce both mrna and protein levels of the midkine growth factor, resulting in a significant decrease of cell proliferation in hepg cells. guzman-villanueva et al. ( ) evaluated the capability of different-sized chitosan derivative-based polyplexes to carry, internalize, and release sirna in human adenocarcinomic epithelial cells. for this purpose, they first prepared nphthaloyl-chitosan or n-phthaloyl-oligochitosan, reacted them with polyethylene glycol and hydroxybenzotriazole in dmf, and then cross-linked the polymers using ecd. finally, the n-phthalimido groups were removed by the reaction with hydrazine monohydrate, and the products were purified by dialysis against water and ethanol. both the chitosan-and oligochitosan-based polyplexes exhibited biodegradability, low cytotoxicity, and resistance to enzymatic degradation up to h. when loaded with sirna, the oligochitosan-based polyplexes drastically increased cellular internalization of the sirna and gene silencing compared to naked sirna. to improve the transfection efficiency of chitosan-based gene delivery systems, deng et al. ( a) fabricated a dendronized chitosan derivative using a coppercatalyzed azide alkyne cyclization reaction of propargyl focal point poly (amidoamine) dendron with -azido- -deoxy-chitosan. the resulting dendronized chitosan nanoparticles exhibited higher water solubility and buffering capacity than native chitosan and showed lower cytotoxicity and enhanced transfection efficiency in transformed human embryonic kidney and nasophyryngeal carcinoma cell lines than commonly used polyethyeneimine. as already mentioned above, the rgd motif can be used for targeting chitosanbased nanoparticles to tumor sites via the interaction with integrin α v β . utilizing this fact, kim et al. ( ) produced a dendrimeric rgd peptide/polyethyleneimine grafted chitosan copolymer, which was soluble in water. the copolymer was nontoxic to mammalian cells and erythrocytes in the absence and presence of plasmid dna. moreover, it was found to transfect cells involving microtubuledependent macropinocytosis and clathrin-mediated endocytosis. finally, injecting copolymers complexed with psirna-hbcl to silence the gene for the human antiapoptotic bcl protein into balb/c-nu mice carrying a pc prostate tumor xenografts, markedly inhibited tumor growth. thus, the copolymer was suggested to be a good candidate to develop a specific targeted gene delivery system. to confer membrane-like properties to chitosan-based gene delivery systems, li et al. ( ) grafted phosphorylcholine and macrocyclic polyamine onto chitosan to obtain water-soluble nanoparticles. chitosan grafted with both compounds were more efficient in binding and protecting plasmid dna than chitosan grafted only with phosphorylcholine or macrocyclic polyamine. the authors also demonstrated that phosphorylcholine and macrocyclic, polyamine-grafted chitosan had a positive net charge and can, therefore, wrap dna to yield nanoparticles of about nm in diameter. finally, the dna-loaded nanoparticles significantly increased cellular uptake and transfection rates in transformed human embryonic kidney cells when compared to chitosan/dna complexes. a similar transfection strategy was published by picola et al. ( ) , who inserted phosphorylcholine and increasing numbers of diethylaminoethyl (deae) groups into the polymer. the resulting chitosan nanoparticles were water soluble at physiological ph and less cytotoxic than lipofectamine, a commonly used transfection reagent. they further could form complexes with plasmid dna, and the transfection efficiencies of the nanoparticles with high deae substitution rates tested in hela cells were in the same range as determined for lipofectamine. when the nanoparticles were loaded with sirna, they were able to induce gene silencing, with efficiencies highly dependent on the n/p ratio. chitosan-based materials have been recognized to be potent adjuvants for immunotherapy, because they non-specifically stimulate immune responses in the host organism and therefore have antiviral, antimicrobial, and antitumor properties ). the adjuvant potency of chitosan is comparable to incomplete freund's adjuvant, and it has stronger immune-stimulatory effect than aluminum hydroxide, which is frequently used in vaccines though it shows adverse side effects such as neurotoxicity (zaharoff et al. ). the mechanism of how chitosan triggers immune responses involves phagocytosis-dependent activation of the nod-like receptor family, pyrin domain containing (nlrp ) inflammasome, which finally induces a robust interleukin- β response (bueter et al. (bueter et al. , . carroll et al. ( ) described another mechanism by which chitosan stimulates the activation of dendritic cells inducing cellular immunity. they found that chitosan promotes the intracellular release of dna, which involves the cgas-sting pathway. as a result, type i interferon is secreted activating the expression of interferoncontrolled genes. due to the release of the cytokines that stimulate dendritic cells, the cellular immune system is elicited. due to its mucoadhesiveness, chitosan and its derivatives are considered effective for mucosal administration, which includes oral, nasal, as well as ocular antigendelivery routes. however, other routes are also anticipated to be effective in provoking immune response including subcutaneous (borges et al. ; scherliess et al. ) , intraperitoneal (chang et al. ) , intravenous (shi et al. ) , and intratumoral injections (zaharoff et al. ) . evidently, innate immune responses are stimulated by chitin, chitosan, or derivatives (peluso et al. ; tokura et al. ; lee et al. ; lee ). this includes the activation of alveolar macrophages with the release of cytokines such as interleukin (il)- , tumor necrosis factor-α, or il- , leading to inf-γ production predominantly released by natural killer cells (shibata et al. a, b) . however, also humoral and cellular adaptive immune responses are triggered by some antigens when co-administered or encapsulated in chitosan-containing micro-and nanoparticles (tokura et al. ; van der lubben et al. ; arca et al. ; mori et al. ) . in particular, wen et al. ( ) found that the stimulatory effect on the humoral and cellular immune system by chitosan results in a balanced th /th response. however, care has to be taken in assessing the properties of chitosan-based adjuvants, as many studies do not provide sufficient data on the chemical and physical characteristics, preparation and formulation procedures, as well as potential impurities (vasiliev ) . this is particularly critical, as immune responses appear to depend on these parameters as uncovered by scherliess et al. ( ) , who reported that the degree of immune response varied when chitosans of different qualities were used. the immune-stimulatory effect of chitosan is affected by the combination of molecular weight, solubility, particle size, and viscosity as well as deacetylation degree. preclinical studies performed predominantly in mice models suggested that chitosan-containing antigen-delivery systems are promising adjuvant platforms for mucosal vaccination against human pathogenic viruses such as influenza (read et al. ; svindland et al. ; sawaengsak et al. ; liu et al. a) ; hepatitis a, b, and e (jiang et al. ; tao et al. a; tao et al. b; soares et al. ); human papilloma virus ; and poliovirus (ghendon et al. ). (choi et al. ) . using chitosan nanoparticles targeted to dendritic cells via antibodies to the dec- surface receptor, raghuwanshi et al. ( ) successfully delivered plasmid dna carrying the cdna for the n protein to trigger immunization against the severe acute respiratory syndrome coronavirus (sars-cov). simultaneous comparison of targeted formulations using intramuscular and intranasal routes revealed that intramuscular administration induced a more potent systemic igg response compared to intranasal administration. solid evidence substantiating the advantages of chitosan as an efficient adjuvant for nasal vaccination originates from clinical examinations on a norovirus vaccine, which demonstrated the ability of a chitosan/monophosphoryl lipid-based antigen delivery system (chisys®) to induce immunity against the gastroenteric norovirus infections after immunization (smith et al. ) . chitosan-derived adjuvants were also used in combination with antigens derived from bacterial toxins, such as diphtheria toxoids (mcneela et al. ; schipper et al. ) , tetanus toxoids (ahire et al. ; pirouzmand et al. ) , and dermonecrotoxin (jiang et al. ). in addition, the potential of various vaccine formulations against anthrax were evaluated in female balb/c mice (malik et al. ) . encapsulating protective antigens (pa) in trimethyl-chitosan nanoparticles and administering them by subcutaneous, intramuscular, and intraperitoneal injections resulted in a strong igg antibody response (th -biased) when combined with immune-stimulatory cpg oligodeoxynucleotides or polyinosinic-polycytidylic acids. interestingly, without the immune-stimulatory nucleic acids, the pa-loaded trimethyl-chitosan nanoparticles led to a th -biased immune response. many studies have explored the adjuvant properties of chitosan in vaccines against cancer. in a study published by wen et al. ( ) , the effects of chitosan nanoparticles on the immune response triggered by an ovalbumin antigen in mice were analyzed. as the administration of the chitosan nanoparticles did not only increase cytokine levels of th (il- and ifn-γ) and th (il- ) cells, but also stimulated natural killer cells, the authors suggested that chitosan is a promising adjuvant for cancer immunotherapy by promoting both humoral and cellular immune responses. this hypothesis was confirmed by highton et al. ( ) , who demonstrated that immunization with an ovalbumin/chitosan hydrogel had antitumor effects in an intracaecal mice cancer model. after subcutaneous injection of the ovalbumin/chitosan vaccine, the authors detected cd + t memory cells specific for ovalbumin and observed decreased tumor growth in contrast to unvaccinated control mice or mice that were vaccinated with dendritic cells and ovalbumin. zaharoff et al. ( ) analyzed antitumor effects in mice using a bioluminescent orthotopic bladder cancer model, after repeatedly administering chitosan/il- into the bladder and comparing the antitumor efficacy of this treatment with that of an established adjuvant therapy applied to treat bladder cancer based on attenuated mycobacteria (bacillus calmette-guerin therapy). determination of the urinary cytokine spectrum and immunohistochemical analysis resulted in the identification of cytotoxic t cells and natural killer cells as effector cells responsible for tumor regression. in contrast to the bacillus calmette-guerin therapy, chitosan/il- treatment utterly prevented recurrence of the disease. more recently, yao et al. ( b) prepared mannosylated chitosan nanoparticles and loaded it with a plasmid to produce a vaccine against gastrin-releasing peptide (grp), whose receptor is overexpressed in various cancer cells. the nanoparticles were intranasally administered in a subcutaneous mice prostate carcinoma model to evaluate the efficacy on inhibition of the growth of tumor cells. cell binding and cellular uptake assays revealed that the mannosylated chitosan nanoparticles facilitate targeting to antigen-presenting cells, promoting receptor-mediated endocytosis via the mannose receptor. due to antigen representation, enhanced tumor regression was observed as a result of the production of high titers of anti-grp antibodies. a similar strategy was finally used by lai et al. ( ) to test an immune therapy against hepatocellular carcinoma in a h tumor-bearing mice model. they synthesized folate-conjugated chitosan nanoparticles and loaded them with a plasmidencoding mouse interferon-γ-inducible protein- (ip- ). they found that ip- plasmid exhibited efficient antitumor activity, prolonging the survival time in h tumor-bearing mice. the antitumor effects were likely due to different effects. next to the secretion of ifn-γ and ip- , inhibition of regulatory t cells, suppression of angiogenesis, inhibition of cancer cell proliferation, and activation of apoptosis contributed to tumor growth inhibition. tissue engineering is an increasingly important interdisciplinary field in regenerative medicine, which aims to create replacements for damaged tissues based on the combined knowledge provided by physicians, biologists, and engineers. most approaches employ scaffolds made from biocompatible polymers, which are colonized by cells of the respective tissue. ideally, the scaffold increases adherence, proliferation, and differentiation of colonizing cells. chitosan and its derivatives offer ample benefits to generate cell and tissue supporting matrices, which include chemical versatility, antimicrobial activity, biocompatibility, biodegradability, and neglible toxicity (ahsan et al. ) . chitosan can be produced to form sponge-like scaffolds using rather simple phase separation techniques including freeze-drying (aranaz et al. ) , gas foaming (kaynak bayrak et al. ) , and electrospinning procedures (qasim et al. ) . the presence of a system of interconnected pores with appropriate diameters facilitates vascularization and tissue integration. moreover, chitosan-based scaffolds can be synthesized in combinations with ample natural and synthetic polymers resulting in matrices exhibiting special characteristics. due to their positive surface charges they open the possibility to fabricate polyelectrolyte complexes with anionic polymers such as glutamic acid (fang et al. ) , hyaluronic acid (lalevee et al. ) , dextrane sulfate (kulkarni et al. ) , heparin (almodovar and kipper ) , dermatan sulfate (rasente et al. ) , and chondroitin sulfate (tsai et al. ) . particularly the presence of glycosaminoglycans, which are naturally found in extracellular matrices, is known to modulate the activity of cytokines and growth factors by binding to the polymers (zaman et al. ) . otherwise, chitosan-based scaffolds can be loaded with cytokines and growth factors to attract cells and stimulate tissue regeneration (sun et al. a; bader et al. ; choi et al. ) . finally, they further open the possibility of controlled degradation and resorption in physiological environments, designing mechanical properties that match the conditions found in the respective tissue, and determination of desired sizes and shape by easy fabrication procedures. therefore, chitosan-based scaffolds have numerous applications in tissue engineering, and we will review recent progress in using these materials for tissue regeneration and wound healing. . . . bone, cartilage, and tooth repair bone defects can either be congenital or result from trauma, infection, cancer, or failed orthopedic surgical procedures (venkatesan and kim ) . the grafts used to bridge bone defects can be autografts (bone material from other body regions of the same patient) or allografts (bone material from decedents). however, both materials have disadvantages: autografts require bone harvesting from healthy tissues and may cause complications of wound healing and pain, and allografts may result in immunogenic rejection and have the risk of transmitting viral diseases from the donor to the recipient. due to these concerns, scientists around the world are searching for alternative materials as bone graft substitutes. as described before, chitosan-based materials have valuable properties for orthopedic applications. chitosan itself has the capacity to increase bone regeneration rates (muzzarelli et al. b ); however, it cannot fully substitute natural bone material. therefore, different composite scaffolds have been developed to assure porosity for vascularization and nutrition, facilitate the formation of new bone material (osteoconductivity), guarantee structural integrity during ingrowth at the site of implantation, and orchestrate biodegradation with bone regeneration (venkatesan and kim ) . in addition, chitosan-based composites can be loaded with cells and growth factors that promote osteoconductivity and hence facilitate bone regeneration. one of the most important chitosan grafting that has been used in bone tissue engineering is hydroxyapatite, which by itself stimulates bone regeneration, provided that the scaffold has a microporous structure (woodard et al. ). hydroxyapatite grafting of chitosan can be easily achieved by coprecipitation from homogeneous mixtures of precursor (deepthi et al. , and references therein). one of the first researchers who tested combinations of chitosan and hydroxyapatite was michio ito, who examined the use of chitosan-bonded hydroxyapatite paste for treatment of periodontal defects (ito ) . in , ge et al. ( published a remarkable study, in which they tested different combinations of air-and freezedried chitosan/hydroxyapatite materials that were colonized by osteoblasts and implanted into rats. the material was found to be nontoxic and biodegradable and to stimulate mineralization. the explanted material that was colonized by osteoblasts before implantation showed newly formed bone material containing proliferating osteoblasts that recruited surrounding tissue to grow in. in another study published by oliveira et al. ( ) , three-dimensional macroporous hydroxyapatite/chitosan bilayered scaffolds of inorganic and organic deposits were produced in a stepwise procedure and examined with regard to their mechanical properties and cytotoxicity to mouse fibroblast-like cells. moreover, in vitro cell culture studies using goat marrow stromal cells revealed that the macroporous hydroxyapatite/chitosan composite is a suitable material that promotes attachment, proliferation, and differentiation into osteoblasts and chondrocytes. additionally, three-layered porous materials of collagen, hydroxyapatite, and chitosan were produced and characterized as an artificial bone matrix (wang et al. b) . when testing murine pre-osteoblast cell line (mc t -e ) grown on this matrix, the cells proliferated significantly more rapidly than cells grown on a pure chitosan matrix (teng et al. ). in addition, higher levels of alkaline phosphatase (secreted by osteoblasts) were determined, which is indicative for bone regeneration. similar results were obtained when osteoblasts were cultured on hydroxyapatite/chitosan nanocomposites and osteocalcin as a marker for late osteoblastic differentiation, and mineralized bone matrix formation was determined (chesnutt et al. ). further studies characterized hydroxyapatite/chitosan hybrids with additional blend materials such as montmorillonite (katti et al. ) , polylactic acid ), cellulose and carboxymethyl cellulose (liuyun et al. ; jiang et al. b) , gelatin (sellgren and ma ; maji et al. ; lee et al. ) , nylon ), polygalacturonic acid (khanna et al. , marine sponge collagen (pallela et al. ) , collagen ), alginate (jin et al. ; kim et al. ; liao et al. ) , chondroitin sulfate (venkatesan et al. a; ), hyaluronic acid (hu et al. , fibroin (lima et al. ; ran et al. ; ye et al. ), poly- -hydroxybutyrate-co- -hydroxyvalerate (zhang et al. b) , fucoidan (lowe et al. ) , β-tricalcium phosphate (shavandi et al. ; oryan et al. ) , graphene oxide , β-cyclodextrin (shakir et al. ) , β- , -glucan , whitlockite (zhou et al. a) , zoledronic acid (lu et al. ) , and zirconium dioxide (balagangadharan et al. ) . also, three-dimensional hydroxyapatite/chitosan-carbon nanotube scaffolds were shown to be promising materials for bone regeneration (im et al. ) . naturally, many of these combinations have been tested also in the absence of hydroxyapatite (park et al. a; jiang et al. ; arpornmaeklong et al. ; venkatesan et al. b; deng et al. ; azevedo et al. ; dinescu et al. ; listoni et al. ; georgopoulou et al. ; koç demir et al. ) . several studies showed that various growth factors such as transforming growth factors (tgfs), vascular endothelial growth factors (vgefs), bone morphogenic proteins (bmps), insulin-like growth factors (igfs), and platelet-derived growth factors (pdgfs) have major impacts on vascularization and osteoblast activities and thus have been employed to stimulate bone regeneration (yun et al. ) . however, there are limitations in maintaining therapeutic concentrations due to the short halflife of the growth factors in vivo. this can be effectively prevented by the controlled release of the growth factors from porous chitosan composite matrices, which have been demonstrated to stimulate bone formation. for instance, pdgf-bb is an important osteogenic growth factor in the process of bone regeneration, as it stimulates mesenchymal cell proliferation and differentiation and mediates chemotaxis of osteoblast. park et al. ( a, b ) produced a porous chitosan or chondroitin- -sulfate/chitosan sponges releasing pdgf-bb to stimulate bone regeneration. the release rate of pdgf-bb increased proportionally with increasing concentrations loaded onto the sponge, and pdgf-bb retained its chemotactic activity regardless of being loaded onto the sponge or added freely to test solution (park et al. a ). finally, osteoblast proliferation was found to be stimulated in pdgf-bb-loaded chondroitin- -sulfate/chitosan sponge compared with that of the unloaded sponge. pgfs have also been used in combination with other growth factors. as vegf is known to prolong cell survival, osteoblast proliferation, differentiation, and migration next to its effects on angiogenesis, it has been suggested that the combined action of vegf and pdgf can accelerate the bone healing process even more efficiently. de la riva et al. ( ) established a system based on brushite-chitosan capable of controlling release kinetics for these two growth factors. after implanting the chitosan scaffolds loaded with the growth factors into rabbits with femur defects, release kinetics and tissue distribution of radiolabeled vegf and pdgf were determined. analyzing bone repair histologically revealed that the combined use of vegf and pdgf promoted bone regeneration most effectively. also, the controlled release of igf- and bmp- by the enzymatic degradation of the porous chitosan scaffold stimulates bone healing and regeneration in rabbits considerably (nandi et al. ) . when the chitosan particles were loaded only with one of the two growth factors, the effect was found to be more pronounced for igf- than for bmp- infiltrated matrices. in a very recent study, chitosan/biphasic calcium phosphate scaffolds functionalized with bmp- -encapsulated nanoparticles and the rgd tripeptide were produced using a desolvation technique (gan et al. ). in vitro cell culture and in vivo implantation tests demonstrated that rgd and bmp- synergistically increased cell adhesion and spreading via integrin binding triggering differentiation of osteoblasts. increased bone healing was also observed, when porous chitosan scaffolds were loaded with resolvin d , a potent lipid immune modulator derived from both eicosapentaenoic acid, and implanted into rats with a femur defect (vasconcelos et al. ) . obviously, resolving d administration in the acute phase of the innate immune response to the bio-implant had beneficial effects during bone tissue repair. impairment of the articular cartilage is frequently due to sport-related injury, disease, trauma, and tumor. if not treated successfully, it may result in osteoarthritis, which increasingly affects also younger individuals (muzzarelli et al. ) . in contrast to bone regeneration, cartilage healing is limited by the lack of vascularization and poor proliferation rates of chondrocytes. injection of hyaluronan into the joints of arthritic patients is known to improve their function, as it restores viscoelasticity and flow of the synovial fluid, helps to normalize hyaluronan production, and finally reduces pain and inflammation. chitosan easily forms polyelectrolyte complexes with hyaluronan and chondroitin sulfate, which are important building blocks particularly of the hyaline cartilage found on the surface of joints. therefore, combinations of chitosan and hyaluronan and/or chondroitin sulfate may be useful in cartilage healing. in a first attempt to realize this idea, kuo et al. ( ) synthesized a highly elastic, macroporous, and chitosan-containing gelatin/chondoitin- -sulfate/ hyaluronan (gch) cryogel scaffold, which mimics the extracellular matrix composition of the cartilage. furthermore, in vitro cell culture studies suggest that chondrocytes proliferate and redifferentiate within the porous matrix of the cryogels. although chitosan reduces cell proliferation, it stimulates the secretion of sulfated glycosaminoglycans and type ii collagen. in addition, they performed in vivo studies culturing chondrocytes on the gch chitosan cryogel and implanting the material into rabbits with an articular cartilage defect (kuo et al. ) . after months, the defect in the chondrocytes/cryogel group was completely covered with semitransparent tissue, which had similar characteristics as the native cartilage. a large variety of other chitosan-based materials lacking glycosaminoglycans have been suggested as suitable scaffolds for cartilage tissue engineering and some of them have been tested as mesenchymal stem cell carriers. such materials include scaffolds containing n,n-dicarboxymethyl chitosan (mattioli-belmonte et al. ) , chitosan/gelatin and/or alginate complex (xia et al. ; bhat et al. ) , poly(l-lactide)/chitosan microspheres (lao et al. ; haaparanta et al. ) , polyethylene oxide/chitin/chitosan scaffolds (kuo and ku ) , genipin-cross-linked chitosan/silk fibroin sponges vishwanath et al. ) , chitosan/polyester-based scaffolds (alves da silva et al. ) , chitosan/ collagen type i scaffolds (gong et al. ) , chitosan/poly(epsilon-caprolactone) blend scaffolds (neves et al. ; filova et al. ) , chitosan/poly(l-glutamic acid) scaffolds , polyvinyl alcohol/chitosan composite hydrogels (dashtdar et al. ) , poly(n-isopropylacrylamide)/chitosan hydrogels (mellati et al. ) , viscoelastic silk/chitosan microcomposite scaffolds (chameettachal et al. ) , and chitosan/graphene oxide polymer nanofibers (cao et al. ) . as in the case of bone repair, strategies using various chitosan-based scaffolds loaded with growth factors such as tgfs (kim et al. ; choi et al. ) , igfs (zhao et al. ), bmps (mattioli-belmonte et al. , and basic fibroblast growth factor (bfgf) (tan et al. ) have been tested for cartilage repair. in an interesting pilot study, qi et al. ( ) produced an injectable chitosan/polyvinyl alcohol gel and examined its structure and physicochemical properties. the resulting material exhibited low cytotoxicity and good biocompatibility. next, the gel was mixed with rabbit bone marrow stromal cells (bmscs) that were transfected with an adenovirus to produce tgf-b , and rabbits with cartilage defects were injected with this mixture. after weeks, the defects appeared to be fully repaired. the regenerated tissue was almost indistinguishable from the native cartilage. in another study, a demineralized bone matrix was conjugated with mesenchymal stem cell (msc) e affinity peptide (eplqlkm) and combined with a chitosan hydrogel for cartilage engineering. cell culture and implantation experiments demonstrated that the developed material has a high chondrogenic capacity facilitating tissue repair of cartilage defects (meng et al. ) . in a more recently published study, the proliferation and differentiation of multipotent dental pulp stem cells into chondrocytes were investigated to generate cartilage-like material. in this case, a porous chitosan-xanthan gum matrix was employed as a scaffold and loaded with kartogenin to promote chondrogenic differentiation (westin et al. ). the manufactured scaffold exhibited favorable characteristics for cartilage tissue engineering, such as high porosity, low cytotoxicity, and mechanical properties compatible with those characteristic of cartilage. very recently, agrawal et al. ( ) reported the in vitro generation of cartilage-like material by seeding human mesenchymal stem cells on freeze-dried porous silk fibroin/ chitosan scaffolds and culturing them in a spinner flask bioreactor under dynamic conditions. the team was successful in preparing a cartilage construct of mm thickness, which roughly corresponds to the thickness of a native articular cartilage. due to its unique properties, chitosan has also emerged as a scaffold for potential applications in dental medicine. chitosan-based hydrogels and nanocomposites have been used as anti-erosive and enamel-repairing additives in dentifrices and chewing gums (shibasaki et al. ; arnaud et al. ; ganss et al. ; ruan et al. ) , for reduction of dental bacterial biofilm formation (jahanizadeh et al. ) , for guided tissue regeneration to treat periodontal diseases such as periodontitis lotfi et al. ) , as dentin-bonding agent (fawzy et al. ) , as modification of dental restorative materials and implants (petri et al. ; ali et al. ; ibrahim et al. a) , and as scaffold for stem cell-based tissue regeneration (yang et al. b; asghari sana et al. ; soares et al. a) . periodontitis is a chronic inflammation of the gum, which ultimately may lead to the loss of periodontal tissues and teeth (pihlstrom et al. ) . current therapeutic strategies mainly rely on good oral hygiene (brushing and flossing), plaque removal, and in more severe cases local application of antibiotics and surgical intervention including open flap debridement, osseous surgery, as well as guided tissue and bone regeneration. chitosan-based materials turned out to be very useful for periodontal tissue regeneration. such materials comprise methylpyrrolidinone chitosan (muzzarelli et al. a) , chitosan scaffolds coated with a bioactive hydroxyapatite (ang et coimbra et al. ; fraga et al. ; miranda et al. ) , injectable thermosensitive chitosan/β-glycerophosphate/hydroxyapatite hydrogels , chitosanbased risedronate/zinc-hydroxyapatite intrapocket dental films (khajuria et al. ) , asymmetric chitosan/tripolyphosphate cross-linked membranes , porous chitosan/collagen scaffolds (yang et al. b ), mucoadhesive electrospun chitosan and thiolated chitosan nanofibers (samprasit et al. ) , chitosan-coated titanium surfaces (campos et al. ) , chitosan modified glass ionomer restoratives (petri et al. ), chitosan-intercalated montmorillonite/poly(vinyl alcohol) nanofibers (ghasemi hamidabadi et al. ) , and polyhydroxybutyrate/chitosan/ nano-bioglass nanofiber scaffolds (hashemi-beni et al. ) . in one of the first studies that used growth factors to promote dental pulp stem cell differentiation, porous chitosan/collagen scaffolds prepared by freeze-drying were used and loaded with a plasmid vector encoding the human bmp- gene (yang et al. b ). the stem cells grown in this scaffold were successfully transfected by the plasmid vector, which led to the formation of bmp- triggering odontoblastic differentiation as indicated by the activation of specific marker genes encoding steocalcin, bone sialoprotein, dentin sialophosphoprotein, and dentin matrix protein . the chitosan/collagen scaffolds with stem cells were subcutaneously implanted into the back of balb/c mice. after weeks, the material was explanted and evaluated by immunohistochemistry. in the gene-activated scaffold group, there were still transfected cells detectable showing the upregulated gene expression when compared to pure scaffold groups. cutaneous wound healing is a complex process in which the skin repairs itself. the process is divided into different stages, which include blood clotting (hemostasis), inflammation, cell migration and proliferation, and tissue remodeling (maturation). the wound healing process may be delayed or completely fail leading to non-healing chronic wounds, which are frequently found in patients with diabetes, venous or arterial diseases, infections, and age-related metabolic deficiencies. bedsore and burns behave differently, as the healing process is complicated by coagulation, necrosis, and infections. small, non-severe wounds may be treated with chitosancontaining ointments (kweon et al. ) , topical gels (alsarra ) , and/or wound dressings (jayakumar et al. ) . for instance, kang et al. ( ) reported the synthesis of silver chloride nanoparticles stabilized with chitosan oligomer for an ointment that was tested on burn wound healing in a rat model. burn wound healing of rats treated with this ointment was superior to rats treated with pure vaseline or chitosan ointments. more severe wounds may require removal of necrotic tissue and surgical wound closure using suturing techniques. if the defects are too large to be covered in this way, autologous avascular mesh grafts, microvascular flap grafts, mikroskin grafts, and/or cultured epithelial grafts are transplanted to the wound site (chua et al. ) . however, surgical intervention is only possible up to a critical size. to cover large-sized skin defects, artificial grafts produced by tissue engineering techniques are required. this type of wound dressings must protect from infections, absorb excess exudates, and facilitate oxygen and nutrient exchange. in addition, the material must be nontoxic, non-allergenic, non-adherent, and biocompatible. chitosan-based materials are a good choice for wound dressings, as they fullfil most of the criteria mentioned above, and they are known to promote wound healing by activating platelets when getting into contact with blood (periayah et al. ) . moreover, chitosan-based scaffolds can be loaded with growth factors to facilitate skin repair by promoting cell adhesion and proliferation (lu et al. ) . several in vitro, preclinical and clinical studies actually demonstrated that chitosan-based hydrogels films, powders, and dressings, as well as artificial skins, accelerate wound healing and reepithelialization (patrulea et al. ) . however, the precise mechanism of action in promoting wound healing is still under debate. next to chitosan-mediated immunomodulation, the type of functionalization contributes to wound healing. chitosan-based scaffolds used to promote wound healing comprise hydrogels, films, micro-and nanoparticles, nanocomposite materials, and micelles, and many biocompatible chitosan derivatives have been tested including n-carboxybutyl chitosan (dias et al. ), hydroxybutyl chitosan (hu et al. , fluorinated methacrylamide chitosan (wijekoon et al. ; patil et al. ; akula et al. ) , and chitosan/polyvinyl alcohol materials (charernsriwilaiwat et al. ; wang et al. b ). in addition, numerous chitosan hybrid materials have been synthesized for wound healing, which comprise chitosan or carboxymethyl chitosan/gelatin hydrogels patel et al. ) , chitosan/heparin/poly(γ-glutamic acid) composite hydrogels , chitosan-hyaluronan composite sponge scaffolds (sanad and abdel-bar ; tamer et al. ), heparin-chitosan complexes (kratz et al. kweon et al. ) , chitosan-fibrin nanocomposites (vedakumari et al. ) , polyvinyl alcohol/chitosan/fibroin-blended sponges (yeo et al. ) , polyvinyl alcohol/starch/chitosan hydrogels with nano zinc oxide (baghaie et al. ) , poly(caprolactone)/chitosan/poly(vinyl alcohol) nanofibrous sponges (gholipour-kanani et al. ) , chitosan/poly(ethylene glycol)-tyramine hydrogels (lih et al. ) , chitosan-alginate polyelectrolyte complexes (wang et al. ; hong et al. ; caetano et al. ; kong et al. ) , porous keratin/chitosan scaffolds without and with zinc oxide (tan et al. ; zhai et al. ) , nanotitanium oxide/chitosan complexes (peng et al. ) , chitosan/collagen hydrogels and sponges (wang et al. a; cui et al. ; ti et al. ) , chitosan green tea polyphenol complexes (qin et al. (qin et al. , , dextran hydrogels loaded with chitosan microparticles (ribeiro et al. ) , chitosan/polycaprolactone scaffolds zhou et al. b) , chitosan oleate ionic micelles (dellera et al. ) , castor oil polymeric films reinforced with chitosan/zinc oxide nanoparticles (diez-pascual and diez-vicente ), sponge-like nano-silver/zinc oxide-loaded chitosan composites , gellan gum-chitosan hydrogels (shukla et al. ) , chitosan-silica hybrid dressing materials (park et al. ), chitosan/bentonite or tourmaline nanocomposites (devi and dutta ; zou et al. ) , chitosan/ gelatin/chondroitin- -sulfate films with and without zinc oxide (cahu et al. ) , chitosan/polyvinylpyrrolidone/cellulose nanowhiskers nanocomposites (hasan et al. ) , α-tocopherol-loaded chitosan oleate nanoemulsions (bonferoni et al. ) , chitosan-based liposome formulations (mengoni et al. ) , and electrospun chitosan/polyethylene oxide/fibrinogen biocomposites (yuan et al. ) . topical application of anti-inflammatory and antioxidant curcumin, which is a component of many curry powders, has been shown to promote wound healing, significantly preventing oxidative damage in tissues (gopinath et al. ). chitosan-alginate sponges have been used to deliver curcumin for dermal wound healing in rat. loading curcumin onto the chitosan sponge enhanced the therapeutic healing effect when compared to other carriers like cotton gauze. similarly, injectable nanocomposite hydrogels composed of curcumin, n,o-carboxymethyl chitosan, and oxidized alginate possess many characteristics that promote wound healing including exudate absorption and immobilization and activation of growth factors. nano-curcumin, which is released slowly from the hydrogel in a sustained manner, evidently stimulates fibroblast proliferation, angiogenesis, and collagen production, supporting the healing process when tested in a mice model (li et al. b) . wound dressings made of chitosan/poly-γ-glutamic acid/pluronic/curcumin nanoparticles also promoted collagen formation and tissue regeneration (lin et al. ) , and collagen-alginate scaffolds impregnated with curcumin-loaded chitosan nanoparticles proved promising in the treatment of various pathological manifestations of diabetic wounds (karri et al. ) . most recently, zhao et al. ( ) prepared a thermosensitive chitosan/β-glycerophosphate hydrogel loaded with β-cyclodextrin-curcumin and demonstrated improved healing of infected cutaneous wounds in rats, which may be due to the combination of antioxidative, antimicrobial, and anti-nf-κb signaling effects. as silver has been demonstrated to have potent antimicrobial activities with no reports on bacterial resistances, several laboratories prepared chitosan wound dressings impregnated with silver to prevent wound infections and promote wound healing (graham ) . indeed, a wound dressing composed of nano-silver and chitosan improved wound healing in rats better than a silver sulfadiazine dressing, which led to unwanted higher silver levels in the blood than the chitosan-silver dressing (lu et al. ) . in another study, abdelgawad et al. ( ) combined silver nanoparticles that were embedded in chitosan with polyvinyl alcohol to produce antimicrobial nanofibrous material for wound dressing. the material with the highest chitosan-silver nanoparticle content was tested against e. coli and showed significant antibacterial activity. a combined antibacterial/tissue regeneration response triggered by functional chitosan-silver nanocomposites was also reported for thermal burns (luna-hernandez et al. ) . to increase antibacterial activity wound dressings, several groups combined chitosan-silver-based materials with sulfadiazine, a sulfonamide antibiotic. topical administration of chitosan-based hydrogels containing silver sulfadiazine improved burn and wound healing capacities in different studies (nascimento et al. ; chakavala et al. ; aguzzi et al. ; lee et al. ; el-feky et al. a) . besides inhibiting gram-negative bacteria such as e. coli, chitosan-based dressings carrying silver sulfadiazine also inhibited the growth of gram-positive bacteria as well as fungi such as c. albicans on an infected wound (el-feky et al. b) . several studies reported that wound healing is accelerated when chitosan hydrogels are combined with adipose-derived or mesenchymal stem cells. altman et al. ( ) grew human adipose-derived stem cells onto a chitosan/silk fibroin scaffolds and used it in a cutaneous wound healing model. they found that this regimen significantly enhanced wound healing, increasing micro-vascularization and differentiation into epidermal epithelial cells. in another study, an artificial dermis was fabricated by culturing human adipose-derived stem cells on a poly (l-glutamic acid)/chitosan scaffold (shen et al. b) . notably, the seeded stem cells maintained their capability to proliferate, produce extracellular matrix, and secrete cytokines including transforming growth factor β and vascular endothelial growth factor. the artificial dermis was used to cover wounds that have been generated before in streptozotocin-induced diabetic mice. the artificial dermis significantly accelerated wound closure and healing in diabetic mice. tong et al. ( ) used a different stem cell-based strategy to generate a skin substitute promoting wound healing. they manufactured a collagen-chitosan sponge scaffold to culture bone marrow-derived stem cells, which were pre-treated by hypoxia to induce the expression of pro-angiogenic cytokines. when the skin substitute was used to treat wounds generated in diabetic rats with hindlimb ischemia, wound healing was enhanced in comparison to scaffold-only controls or skin substitutes that were generated with normoxic stem cells. a novel strategy for wound healing involving exosomes was reported recently. exosomes are small secretory membrane vesicles that are involved in cell-to-cell communication. stem cell-derived exosomes can improve wound healing and promote skin regeneration by stimulating cell proliferation and migration, angiogenesis, and reepithelization and modulating immune responses (phinney and pittenger ) . based on these observations, shi et al. ( b) isolated exosomes derived from gingival mesenchymal stem cells and encapsulated them in chitosan/silk hydrogel sponge. the combination of the exosomes and hydrogel was effective in promoting skin wound healing in a diabetic rat model by inducing reepithelialization, vascularization, and neuronalization paralleled by the remodeling of the extracellular matrix. corneal damage can be the result of different diseases and injuries and may lead to a reduction or even loss of vision. currently, the only therapy to cure vision loss after irreversible corneal damage is a surgical procedure where the cornea is replaced by donated corneal tissue. frequently, the entire cornea is replaced in a surgical intervention called penetrating keratoplasty. as there is a shortage of corneal donors and there is a certain risk associated with the surgery and graft rejection, new types of corneal replacements are examined including materials containing chitosan. actually, topical application of chitosan or chitosan/n-acetylcysteine to the eye is known to enhance corneal epithelial proliferation and migration during the wound healing in rabbits (fischak et al. ) . this process appears to involve the activation of the extracellular signal-regulated kinases (erk) pathway (cui et al. ) . another study evaluated the effects on corneal epithelium regeneration by combination of exogenous recombinant human serum-derived factor- α (rhsdf- α) with a thermosensitive chitosan/gelatin hydrogel and analyzed the underlying mechanism (tang et al. ) . conducting in vitro experiments, the team showed that rhsdf- α enhanced stem cell proliferation, chemotaxis, and migration, as well as the expression of related genes in limbal epithelial and mesenchymal stem cells (lescs and mscs). in vivo experiment using an alkali burn-injury rat model further revealed enhanced corneal epithelium regeneration and increased local expression of growth factors known to be essential for corneal epithelium repair. the underlying mechanism by which rhsdf- α released from the chitosan/gelatin hydrogel stimulates corneal regeneration may involve activation of c-x-c chemokine receptor type (cxcr ) expressing cells (lescs and mscs) and chemotactic attraction of these cells to the sites of lesion via the binding of rhsdf- α to the cxcr receptor. chen et al. ( ) had considered a tissue-engineering scaffold made of collagen, chitosan, and hyaluronic acid as a potential replacement for corneal tissue. to study cytocompatibility in vitro, they cultured rabbit limbal corneal epithelial cells, corneal endothelial cells, and keratocytes on the polymer complexes and demonstrated that the corneal cells were able to attach, migrate, and proliferate. to evaluate biocompatibility in vivo, they implanted the polymer complex into the corneal stroma of rabbit eyes and inspected ocular reactions. overall, the polymer complexes exhibited transparency and good biocompatibility, as they were degraded and absorbed within the corneal tissue while maintaining transparency. in another study, poly(ethylene glycol)-stabilized carbodiimide-cross-linked collagen-chitosan hydrogels were tested for biocompatibility and host-graft integration. for this purpose, rafat et al. ( ) performed in vitro and in vivo studies demonstrating excellent biocompatibility when analyzing human corneal cells, dorsal root ganglia from chick embryos, or subcutaneous implants. the hydrogel scaffold was also studied as corneal substitute by implanting it into the cornea of pig eyes and monitoring them for months. the substitute was seamlessly integrated into the cornea with regeneration of host corneal epithelium, stroma, and nerves. liang et al. ( ) prepared a blend membrane composed of hydroxyethyl-chitosan, gelatin, and chondroitin sulfate. the membrane exhibited good transparency, ion and glucose permeability, and cytocompatibility for corneal endothelial cells, which formed a monolayer on the membrane in cell culture. in vivo animal experiments revealed that the membranes were characterized by biodegradability and a good histocompatibility suggesting that the membranes may be employed as carriers for corneal endothelial cell transplantation. similar results were obtained for chitosan/ polycaprolactone, chitosan/poly(ethylene glycol), silk fibroin/chitosan, carboxymethyl chitosan/gelatin/hyaluronic acid, as well as hydroxyethyl-chitosan blend membranes, which were tested as potential scaffolds and carriers for bovine, ovine, and rabbit corneal endothelial cells, respectively guan et al. ; ozcelik et al. ; liang et al. ; xu et al. ) . using an allogeneic rabbit model of stromal destruction caused by bacterial keratitis, chou et al. ( ) tested the hypothesis that intra-stromal injection of keratocyte spheroids manufactured on chitosan coatings has higher therapeutic efficacies than eye drop instillations or isolated cell injections. the results of clinical observations and histological studies performed weeks after the surgical intervention showed that, in comparison to a treatment relying only on antibiotics, intrastromal grafting of keratocytes provides additional benefits due to improved preservation of cellular phenotypes, secretion of collagen matrix, and retention of the graft. in a stem cell therapeutic approach published by chien et al. ( ) , human corneal fibroblasts (keratocytes) were reprogrammed into human-induced pluripotent stem cells (ipsc) using a feeder cell-free culturing system. to increase ipsc delivery and engraftment, the researchers generated an injectable thermogelling carboxymethyl-hexanoyl chitosan nanogel with seeded ipscs and showed that viability and pluripotent properties of the reprogrammed ipscs were maintained in the hydrogel system. they further demonstrated that the reprogrammed ipscs grown on the hydrogel could be used to enhance corneal wound healing efficiently. this strategy opens the possibility for a personalized therapy for human corneal damage when ipscs are reprogrammed from cells derived from corneal surgical residues. the plethora of favorable characteristics of chitosan outlined in this chapter prompted many researchers around the world to employ chitosan-based materials also in the reconstruction of peripheral nerves to improve healing of nerve damage caused by accidents or diseases. although therapeutic interventions to peripheral nerve repair have yielded some progress during the past years, a full recovery of nerve function is usually not achieved. current therapies mostly rely on microsurgical techniques, which either try to directly establish a tension-free connection between the ends of severed nerves (epineural, fascicular, and grouped fascicular repair) or bridge larger nerve defects by autologous grafts (cable grafts, trunk grafts, and vascularized nerve grafts) (matsuyama et al. ; houschyar et al. ). the various neurosurgical techniques used to connect nerve ends are challenging, and the therapeutic results are frequently not satisfactory. many studies have provided evidence that various types of conduits, such as veins, pseudo-sheaths, and bioabsorbable tubes, are helpful in bridging shorter gaps by promoting nerve regeneration. after bridging nerve gaps with hollow conduits, the lumen between the nerve ends becomes filled with fibrin, and macrophages and other cells are attracted, which create a favorable microenvironment for vascularization and neuronalization. chitosan-based conduits have been extensively analyzed for this purpose. an early electrophysiological and histological study on nerve regeneration using rat sciatic nerve defects demonstrated that pure chitosan/collagen conduits were superior in bridging cm nerve defects to that of control groups (wei et al. ) . the chitosan/collagen film was found to be degraded about months after the surgery. in a methodologically similar study, wang et al. ( ) generated an artificial nerve graft composed of a chitosan conduit and tested them to bridge a cm dog sciatic nerve defect. in contrast to the previous study, the conduit was filled with longitudinally arranged filaments of polyglcyolic acid. the team found that the sciatic nerve trunk was successfully reconstructed in dogs treated with the chitosan/polyglycolic acid graft with reinnervation of the target skeletal muscle. in a case report on a -year-old man with a cm median nerve defect in the distal forearm, implantation of chitosan/polyglycolic acid graft promoted nerve regeneration and functional reconstruction, so that the patient was able after months to fully use the injured hand during daily activities . other chitosan-based conduits have been successfully used to guide and promote nerve generation in various in vitro and in vivo models. these materials include chitosan/gelatin and chitosan/poly(l-lysine) polyelectrolyte-based scaffolds (martin-lopez et al. ) , chitosan-gold nanocomposites (lin et al. ) , chitosan/polylactic acid films (xie et al. ) , chitosan/poly( -hydroxybutyrate-co- -hydroxyvalerate) nanofibers (biazar and heidari keshel ) , porous chitosan-poly(p-dioxanone)/silk fibroin copolymers (wu et al. ) , poly(d,l-lactide-co-glycolide) sleeves with multifilament chitosan yarn or a microcrystalline chitosan sponge core (wlaszczuk et al. ) , chitosan/ hyaluronic acid hybrid materials (li et al. a) , porous chitosan-γ-glycidoxypropyltrimethoxysilane hybrid membranes (shirosaki et al. ) , hydroxyapatite-coated tendon chitosan tubes with adsorbed laminin peptides (itoh et al. ) , and hyaluronic acid doped-poly( , -ethylenedioxythiophene) nanoparticles in a chitosan/gelatin matrix ). an evident upgrade of chitosan-based conduits is lumenal loading with neurotrophic factors such as nerve growth factor (ngf), ciliary neurotrophic factor (cnf), or brain-derived neurotrophic factor (bdnf), which are all secreted by schwann cells that support growth of neuronal cells (houschyar et al. ). in addition, fibroblast growth factor (fgf), glial growth factor (ggf), and vascular endothelial growth factor (vegf) were reported to have positive effects on nerve regeneration. yang et al. ( ) immobilized ngf on genipin-cross-linked chitosan and tested the material for cytotoxicity using primary cultured schwann cells and for neuronal differentiation of pc cells in response to ngf release. subsequently, wang et al. ( a) demonstrated that genipin-cross-linked chitosan conduits loaded with ngf can be successfully used to bridge -cm-long sciatic nerve defects in rats as revealed by electrophysiological assessment, behavioral analysis, and histological examination weeks after the surgery. similar results were obtained, when ngf-containing microspheres were implanted into chitosan conduits to repair a cm defect of the facial nerve in rabbits . in another ngf-based approach, chao et al. ( ) combined an autologous vein conduit with a chitosan-β-glycerophosphate-ngf hydrogel. the researchers surgically reconstructed a -mm-long defect of a rat facial nerve with an autologous vein and then injected the chitosan-β-glycerophosphate-ngf hydrogel into the lumen of the conduit. facial nerve regeneration was as efficient as in control groups, which were transplanted with an autologous nerve, but significantly better than in control groups where the vein conduit was injected with ngf only. shen et al. ( ) used a polylactic/polyglycolic acid chitosan nerve conduit loaded with cnf to repair larger canine tibial nerve defects in crossbred dogs and evaluated nerve regeneration by general inspection, electrophysiological, immunological, and histological analyses months after the surgery. nerve regeneration was significantly improved in animals that were treated with cnf-loaded polylactic/ polyglycolic acid chitosan conduits when compared to groups treated with the polylactic/polyglycolic acid chitosan conduits. the results were similar to controls groups that were treated with autologous nerve grafts, suggesting that the artificial nerve conduit is a promising alternative for bridging nerve defects. furthermore, zhao et al. ( ) hypothesized that tacrolismus-loaded chitosan enhances peripheral nerve regeneration through modulation of the expression profiles of neurotrophic factors. to test this hypothesis, they loaded tacrolismus onto chitosan conduits and examined nerve regeneration of sciatic nerve injury in a rat model. they found significant regeneration of sciatic nerves with normal morphology but higher density of myelinated nerve fibers in rats treated with tacrolismusloaded chitosan. the underlying mechanism seems to involve bdnf signaling, because nerve regeneration was paralleled by an increased expression of bdnf and its corresponding receptor (trkb) in the motor neurons in the spinal cord. the membrane-bound cell adhesion molecule l is known to promote neurite growth and prevent neuronal apoptosis, a function which can be mimicked by a recombinant chimeric version of this molecule called l -fc (roonprapunt et al. ) . loading l -fc to an artificial chitosan/polyglycolic acid conduit, xu et al. ( ) studied guided regeneration of rat optic nerves. they found that the implanted chitosan/polyglycolic acid conduit was degraded and absorbed. when l -fc loaded conduits were implanted to bridge a defect caused by surgical intervention, axonal regeneration and remyelination were significantly improved when compared to control groups that were treated with conduits lacking l -fc. nerve regeneration can be additionally promoted using chitosan-based scaffolds as conduits seeded with stem cells that express neurotrophic factors and can differentiate into nerve cells. zheng and cui ( ) tested chitosan conduits of such kind combined with rat bone marrow mesenchymal stem cells to evaluate their potential for the reconstruction of -mm-long rat sciatic nerve defects. they demonstrated that the combination of chitosan and mesenchymal stem cells alone was sufficient to improve nerve regeneration and functional recovery. moreover, some of the mesenchymal stem cells were found to have differentiated into neural stem cells. similar results were obtained when injured rat sciatic nerves were treated in this way, and the nerve repair was monitored electrophysiologically and histomorphologically (moattari et al. ) or by noninvasive magnetic resonance neurographic imaging (liao et al. ). in addition, chitosan-coated poly- -hydroxybutyrate conduits combined with human bone marrow mesenchymal stem cells were recently shown to be efficient in promoting nerve regeneration in this rat model of sciatic nerve injury (ozer et al. ) . improved nerve regeneration was also reported for chitosan/poly(lactic-co-glycolic acid) scaffolds seeded with autologous bone marrow mesenchymal stem cells to treat injuries of dog sciatic nerves and rhesus monkey median nerves (xue et al. ; . in another approach, zhu et al. ( ) used chitosan conduits filled with bone marrow mesenchymal stem cells and evaluated nerve regeneration and neuronal survival when injured lumbosacral nerves were bridged with this material. they found that this treatment enhanced sacral nerve regeneration and motor function and weeks after the surgery. moreover, the mesenchymal stem cells prevented cell death of motor neurons in the anterior horn of the spinal cord, thereby improving the motor function in rats treated with the mesenchymal stem cell-seeded chitosan conduit. finally, a clinical study performed with patients suggests that defects in chronic spinal cord injury can be successfully bridged with peripheral nerve grafts combined with a chitosan-laminin scaffold and co-transplanted bone marrow-derived mesenchymal stem cells, which enhanced recovery (amr et al. ) . using chitosan/silk fibroin scaffolds grafts seeded with adipose-derived stem cells, wei et al. ( ) examined regeneration of surgically injured rat sciatic nerves. implantation of this conduit significantly improved axonal regeneration and functional recovery in comparison to control groups. the positive effect was partially attributed to the differentiation of adipose-derived stem cells into schwann cells, which additionally secrete neurotrophic factors and prevent apoptosis. nie et al. ( ) investigated axonal regeneration and remyelination using a chitosan/gelatin-based conduit combined with tgf-β and schwann cells. for this purpose, they bridged a -mm defect of a rat sciatic nerve and examined nerve regeneration based on functional recovery, electrophysiological measurements, retrograde labeling, and immunohistochemical analysis. the obtained data indicate satisfactory functional recovery of the injured sciatic nerve. meyer et al. ( ) filled chitosan ( % degree of acetylation) conduits with a gel containing hyaluronic acid and laminin (nvr-gel) and added genetically modified neonatal rat schwann cells as cellular delivery system for neurotrophic factors. testing the chitosan conduits in the rat sciatic nerve model revealed that the chitosan conduit, which only is filled with the nvr-gel, was insufficient to promote nerve regeneration in contrast to autologous nerve grafts. notably, delivery of fgf by seeded schwann cells genetically modified to overexpress this factor improved nerve regeneration significantly. unexpectedly, schwann cells expressing gdnf did not show positive effects in this experimental setup. recently, zhu et al. ( ) used skin-derived precursor schwann cells to seed chitosan/silk scaffolds for bridging a -mm-long rat sciatic nerve gap. the artificial graft exhibited significant promoting effects on peripheral nerve repair and hence constitutes an alternative to other stem cell-based approach promoting nerve regeneration. numerous studies reported favorable effects of chitosan-based materials for a wide range of applications. doubtless, the controlled and targeted delivery of drugs to specific tissues has a great potential in biomedicine, and first clinical trials with chitosan-based drug carrier systems revealed promising results for the therapy of a variety of diseases including diabetes and cancer and also mainly because adverse side effects are reduced. the antimicrobial activity of chitosan and its derivatives is particularly important when the polymer is used for textile fabrication, food packaging, wound dressings, and tissue engineering. however, the underlying mechanism of antimicrobial activity is not fully understood. one prominent explanation is the assumed interaction of chitosan's positively charged amine groups with the negatively charged surface of bacteria and fungi, which might impair the movement of ions across membranes and hence disrupt cellular integrity. although the proposed mechanisms seem plausible, there is a clear lack of experimental data that would provide evidence at a molecular level reminding us to continue basic research on the mode of actions. the studies conducted so far indicate that the antimicrobial activity of pure chitosan is not sufficient to prevent microbial infections completely in vivo. however, chitosan and its derivatives can be combined with other antimicrobial compounds including essential oils (krausz et al. ) , polyphenols (madureira et al. ) , tretinoin (ridolfi et al. ) , metal ions (sanpui et al. ; tran et al. ) , lysozyme , or antibodies (jamil et al. ) , to prevent bacterial of fungal infections. thus, chitosan appears to be an ideal adjuvant polymer for design and production of new materials exhibiting intrinsic antimicrobial properties for a large variety of potential applications in the chemical, pharmaceutical, food, and textile industry. similarly, the observed immunestimulatory effects of chitosan need further investigation, as there may be a certain risk to develop allergic or even anaphylactic reactions after oral ingestion (kato et al. ) . however, it has to be noted that overall the beneficial characteristics exceed possible side effects due to some allergic potential. finally, the antitumor activity of chitosan also needs to be analyzed in more detail. recently, li et al. ( b) provided some evidence indicating that chitosan activates dendritic cells, which subsequently secrete pro-inflammatory cytokines and thereby enhance immune surveillance by natural killer cells. accordingly, the antitumor effects of chitosan can be enhanced by specifically targeting dendritic cells by attaching mannose to the surface of chitosan nanoparticles (shi et al. a) . different pattern recognition receptors that are expressed on the surface of dendritic cells are potential receptors for chitosan. this includes toll-like receptors, c-type lectin receptors, and other molecules, which are known to recognize specific molecular patterns, particularly those associated with pathogens. however, currently it is not known how dendritic cells recognize chitosan. in summary, it has to be noted that chitosan is a highly promising material for a variety of applications in industry and medicine. while chitosan-based materials have been commercially launched as packaging and coating material in food industry, as an ingredient in cosmetics, and as ion exchanger in water treatment and are approved for human dietary use and wound dressing, their commercial applications in medicine as drug delivery systems or scaffold for tissue engineering are pending. nevertheless, there are clinical phase / trials, and depending on their outcome, some products may reach first approval by the health authorities in near future. grafting of aniline derivatives onto chitosan and their applications for removal of reactive dyes from industrial effluents nano chitosan-npk fertilizer enhances the growth and productivity of wheat plants grown in sandy soil antimicrobial wound dressing nanofiber mats from multicomponent (chitosan/silver-nps/polyvinyl alcohol) systems impedimetic immunosensor for the label-free and direct detection of botulinum neurotoxin serotype a using au nanoparticles/graphene-chitosan composite in vitro cartilage construct generation from silk fibroin-chitosan porous scaffold and umbilical cord blood derived human mesenchymal stem cells in dynamic culture condition solid state characterisation of silver sulfadiazine loaded on montmorillonite/chitosan nanocomposite for wound healing chitosan microparticles as oral delivery system for tetanus toxoid characterisation of composite films fabricated from collagen/chitosan and collagen/soy protein isolate for food packaging applications preparation and characterization of a nanoparticles modified chitosan sensor and its application for the determination of heavy metals from different aqueous media chitosan as biomaterial in drug delivery and tissue engineering chitosan application for active bio-based films production and potential in the food industry: review functionalized graphene oxidepolypyrrole-chitosan (fgo-ppy-cs) modified screen-printed electrodes for non-enzymatic hydrogen peroxide detection fluorinated methacrylamide chitosan hydrogels enhance cellular wound healing processes synthesis and characterization of chitosan and silver loaded chitosan nanoparticles for bioactive polyester double layer coatings: a new technique for maintaining physico-chemical characteristics and antioxidant properties of dragon fruit during storage exploring antibacterial activity and hydrolytic stability of resin dental composite restorative materials containing chitosan active pseudo-multilayered films from polycaprolactone and starch based matrix for food-packaging applications coating electrospun chitosan nanofibers with polyelectrolyte multilayers using the polysaccharides heparin and n,n,n-trimethyl chitosan the development of non-enzymatic glucose biosensors based on electrochemically prepared polypyrrole-chitosantitanium dioxide nanocomposite films chitosan-zinc oxide nanoparticle composite coating for active food packaging applications chitosan topical gel formulation in the management of burn wounds ifats collection: human adipose-derived stem cells seeded on a silk fibroin-chitosan scaffold enhance wound repair in a murine soft tissue injury model chitosan/polyester-based scaffolds for cartilage tissue engineering: assessment of extracellular matrix formation a short review on presence of pharmaceuticals in water bodies and the potential of chitosan and chitosan derivatives for elimination of pharmaceuticals a sensor for simultaneous determination of dopamine and morphine in biological samples using a multi-walled carbon nanotube/chitosan composite modified glassy carbon electrode selective simultaneous determination of paracetamol and uric acid using a glassy carbon electrode modified with multiwalled carbon nanotube/ chitosan composite preparation and characterization of sdf- alpha-chitosan-dextran sulfate nanoparticles wound healing properties of pva/starch/chitosan hydrogel membranes with nano zinc oxide as antibacterial wound dressing material the effect of active ingredient-containing chitosan/ polycaprolactone nonwoven mat on wound healing: in vitro and in vivo studies chitosan/nano-hydroxyapatite/nano-zirconium dioxide scaffolds with mir- - p for bone regeneration chitosan: a potential biopolymer for wound management ocular application of chitosan smart and fragrant garment via surface modification of cotton fabric with cinnamon oil/stimuli responsive pnipaam/chitosan nano hydrogels ph responsive n-succinyl chitosan/poly (acrylamide-co-acrylic acid) hydrogels and in vitro release of -fluorouracil induction of systemic resistance to fusarium crown and root-rot in tomato plants by seed treatment with chitosan structure and interactions in covalently and ionically crosslinked chitosan hydrogels for biomedical applications chitosan treatment of wheat seeds induces resistance to fusarium graminearum and improves seed quality supermacroprous chitosan-agarose-gelatin cryogels: in vitro characterization and in vivo assessment for cartilage tissue engineering development of chitosan-crosslinked nanofibrous phbv guide for repair of nerve defects chitosan beads immobilized manganese peroxidase catalytic potential for detoxification and decolorization of textile effluent carbon fiber embedded chitosan/ pva composites for decontamination of endocrine disruptor bisphenol-a from water removal of hexavalent chromium from wastewater using a new composite chitosan biosorbent alpha tocopherol loaded chitosan oleate nanoemulsions for wound healing. evaluation on cell lines and ex vivo human biopsies, and stabilization in spray dried trojan microparticles alginate coated chitosan nanoparticles are an effective subcutaneous adjuvant for hepatitis b surface antigen cd interaction with tiam promotes rac signaling and hyaluronic acid-mediated breast tumor cell migration antimicrobial activity of lactoferrin against foodborne pathogenic bacteria incorporated into edible chitosan film complexation of copper(ii) with chitosan nanogels: toward control of microbial growth trans-resveratrol loaded chitosan nanoparticles modified with biotin and avidin to target hepatic carcinoma chitosan but not chitin activates the inflammasome by a mechanism dependent upon phagocytosis spectrum and mechanisms of inflammasome activation by chitosan chitosan-alginate membranes accelerate wound healing evaluation of chitosan-based films containing gelatin, chondroitin -sulfate and zno for wound healing preparation and characterization of homogeneous chitosan-polylactic acid/hydroxyapatite nanocomposite for bone tissue engineering and evaluation of its mechanical properties acidic ph resistance of grafted chitosan on dental implant chitosan nanoparticles functionalized with β-cyclodextrin: a promising carrier for botanical pesticides fabrication of chitosan/graphene oxide polymer nanofiber and its biocompatibility for cartilage tissue engineering mucoadhesive and thermogelling systems for vaginal drug delivery flame retardant multilayered coatings on acrylic fabrics prepared by one-step deposition of chitosan/montmorillonite complexes the vaccine adjuvant chitosan promotes cellular immunity via dna sensor cgas-sting-dependent induction of type i interferons chitosan in nasal delivery systems for therapeutic drugs preparation of a milk spoilage indicator adsorbed to a modified polypropylene film as an attempt to build a smart packaging a strategy to employ polymerised riboflavin in the development of electrochemical biosensors antimicrobial controlled release systems for the knitted cotton fabrics based on natural substances development and in vivo evaluation of silver sulfadiazine loaded hydrogel consisting polyvinyl alcohol and chitosan for severe burns effect of visco-elastic silk-chitosan microcomposite scaffolds on matrix deposition and biomechanical functionality for cartilage tissue engineering comparison of adjuvant efficacy of chitosan and aluminum hydroxide for intraperitoneally administered inactivated influenza h n vaccine facilitation of facial nerve regeneration using chitosan-beta-glycerophosphate-nerve growth factor hydrogel electrospun chitosan/ polyvinyl alcohol nanofibre mats for wound healing development of chitosan nanocapsules for the controlled release of hexaconazole study on biocompatibility of complexes of collagen-chitosan-sodium hyaluronate and cornea in vitro proliferation and osteogenic differentiation of human dental pulp stem cells in injectable thermo-sensitive chitosan/betaglycerophosphate/hydroxyapatite hydrogel quaternized carboxymethyl chitosan/organic montmorillonite nanocomposite as a novel cosmetic ingredient against skin aging preparation and characterization of antimicrobial cotton fabrics via n-halamine chitosan derivative/poly( -acrylamide- -methylpropane sulfonic acid sodium salt) self-assembled composite films composite chitosan/ nano-hydroxyapatite scaffolds induce osteocalcin production by osteoblasts in vitro and support bone formation in vivo corneal repair by human corneal keratocyte-reprogrammed ipscs and amphiphatic carboxymethyl-hexanoyl chitosan hydrogel covalently conjugated transforming growth factor-beta in modular chitosan hydrogels for the effective treatment of articular cartilage defects chitosan as an immunomodulating adjuvant on t-cells and antigen-presenting cells in herpes simplex virus type infection bioengineered keratocyte spheroids fabricated on chitosan coatings enhance tissue repair in a rabbit corneal stromal defect model cu-chitosan nanoparticle boost defense responses and plant growth in maize assessment of cu-chitosan nanoparticles for its antibacterial activity against pseudomonas syringae pv. glycinea skin tissue engineering advances in severe burns: review and therapeutic applications photostabilization of an entomopathogenic fungus using composite clay matrices sodium hyaluronate/ chitosan polyelectrolyte complex scaffolds for dental pulp regeneration: synthesis and characterization a preliminary study of the incorporation of npk fertilizer into chitosan nanoparticles controlled release matrices and micro/nanoparticles of chitosan with antimicrobial potential: development of new strategies for microbial control in agriculture highly sensitive lactate biosensor by engineering chitosan/ pvi-os/cnt/lod network nanocomposite development of chitosancollagen hydrogel incorporated with lysostaphin (cchl) burn dressing with anti-methicillinresistant staphylococcus aureus and promotion wound healing properties chitosan promoted the corneal epithelial wound healing via activation of erk pathway biocompatible electrochemiluminescent biosensor for choline based on enzyme/titanate nanotubes/chitosan composite modified electrode pva-chitosan composite hydrogel versus alginate beads as a potential mesenchymal stem cell carrier for the treatment of focal cartilage defects chitosan nanoparticles: a new vehicle for the improvement of the delivery of drugs to the ocular surface. application to cyclosporine a the effect of a peg versus a chitosan coating on the interaction of drug colloidal carriers with the ocular mucosa local controlled release of vegf and pdgf from a combined brushitechitosan system enhances bone regeneration removal of cationic dyes from aqueous solutions using nbenzyl-o-carboxymethylchitosan magnetic nanoparticles an overview of chitin or chitosan/nano ceramic composite scaffolds for bone tissue engineering development of chitosan oleate ionic micelles loaded with silver sulfadiazine to be associated with platelet lysate for application in wound healing dendronized chitosan derivative as a biocompatible gene delivery carrier hollow chitosan-silica nanospheres as ph-sensitive targeted delivery carriers in breast cancer therapy a silk fibroin/chitosan scaffold in combination with bone marrow-derived mesenchymal stem cells to repair cartilage defects in the rabbit knee novel electrochemical xanthine biosensor based on chitosan-polypyrrole-gold nanoparticles hybrid bio-nanocomposite platform preparation and characterization of chitosan-bentonite nanocomposite films for wound healing application chitosan: antimicrobial activity, interactions with food components and applicability as a coating on fruit and vegetables mucoadhesive microspheres for gastroretentive delivery of acyclovir: in vitro and in vivo evaluation antibacterial activity of hybrid chitosan-cupric oxide nanoparticles on cotton fabric supercritical solvent impregnation of natural bioactive compounds in n-carboxybutyl chitosan membranes for the development of topical wound healing applications ocular drug delivery by liposome-chitosan nanoparticle complexes (lcs-np) wound healing bionanocomposites based on castor oil polymeric films reinforced with chitosan-modified zno nanoparticles in vitro cytocompatibility evaluation of chitosan/graphene oxide d scaffold composites designed for bone tissue engineering sorption of cu (ii) ions on chitosan-zeolite x composites: impact of gelling and drying conditions oligogalacturonides and chitosan activate plant defensive genes through the octadecanoid pathway characterizations of blend gels of carboxymethylated polysaccharides and their use for the controlled release of herbicide construction and characterization of a chitosan-immobilized-enzyme and beta-cyclodextrin-included-ferrocene-based electrochemical biosensor for h o detection application of chitosan based coating in fruit and vegetable preservation: a review supramolecular composite materials from cellulose, chitosan and cyclodextrin: facile preparation and their selective inclusion complex formation with endocrine disruptors chitin and chitosan: chemistry, properties and applications the impact of the codelivery of drug-sirna by trimethyl chitosan nanoparticles on the efficacy of chemotherapy for metastatic breast cancer cell line (mda-mb- ) evaluation of released malathion and spinosad from chitosan/alginate/gelatin capsules against culex pipiens larvae chitosan in plant protection alginate coated chitosan nanogel for the controlled topical delivery of silver sulfadiazine using chitosan nanoparticles as drug carriers for the development of a silver sulfadiazine wound dressing chitosan and sodium alginate-based bioadhesive vaginal tablets eco-friendly finishing agent for cotton fabrics to improve flame retardant and antibacterial properties chitosan microparticles for sustaining the topical delivery of minoxidil sulphate kinetics of hexavalent chromium removal from water by chitosan-fe nanoparticles assessment of antibacterial and antioxidant properties of chitosan edible films incorporated with maqui berry (aristotelia chilensis) chitosan/gelatin scaffolds support bone regeneration gene delivery using chitosan, trimethyl chitosan or polyethylenglycol-graft-trimethyl chitosan block copolymers: establishment of structure-activity relationships in vitro chitosan-intercalated montmorillonite/poly(vinyl alcohol) nanofibers as a platform to guide neuronlike differentiation of human dental pulp stem cells chitosan as an adjuvant for poliovaccine tissue engineered poly(caprolactone)-chitosan-poly (vinyl alcohol) nanofibrous scaffolds for burn and cutting wound healing innovative gold-free carbon nanotube/ chitosan-based competitive immunosensor for determination of hiv-related p capsid protein in serum removal of the heavy metal ion chromiuim (vi) using chitosan and alginate nanocomposites chitosan microparticles incorporating a hydrophilic sunscreen agent use of synovium-derived stromal cells and chitosan/collagen type i scaffolds for cartilage tissue engineering dermal wound healing processes with curcumin incorporated collagen films the role of silver in wound healing thermoplastic blends of chitosan: a method for the preparation of high thermally stable blends with polyesters endocrine disruptors compounds, pharmaceuticals and personal care products in urban wastewater: implications for agricultural reuse and their removal by adsorption process chitosan/ tripolyphosphate nanoparticles loaded with paraquat herbicide: an environmentally safer alternative for weed control surgical repair of a mm long human median nerve defect in the distal forearm by implantation of a chitosan-pga nerve guidance conduit use of a silk fibroinchitosan scaffold to construct a tissue-engineered corneal stroma the synthesis and antioxidant activity of the schiff bases of chitosan and carboxymethyl chitosan antibacterial chitosan coating on nano-hydroxyapatite/polyamide porous bone scaffold for drug delivery influence of radiation crosslinked carboxymethyl-chitosan/gelatin hydrogel on cutaneous wound healing development of a novel reagentless, screenprinted amperometric biosensor based on glutamate dehydrogenase and nad(+), integrated with multi-walled carbon nanotubes for the determination of glutamate in food and clinical applications characterization of chitosan/tio cano-powder modified glass-ionomer cement for restorative dental applications loading of chitosannano metal oxide hybrids onto cotton/polyester fabrics to impart permanent and effective multifunctions kaempferol loaded lecithin/chitosan nanoparticles: preparation, characterization, and their potential applications as a sustainable antifungal agent chitosan and its use as a pharmaceutical excipient nasal drug delivery-possibilities, problems and solutions biomimetic three-dimensional nanocrystalline hydroxyapatite and magnetically synthesized single-walled carbon nanotube chitosan nanocomposite for bone regeneration chitosan hydrogel as a drug delivery carrier to control angiogenesis in vitro properties of a chitosan-bonded hydroxyapatite bone-filling paste hydroxyapatite-coated tendon chitosan tubes with adsorbed laminin peptides facilitate nerve regeneration in vivo active naringin-chitosan films: impact of uv irradiation curcumin-loaded chitosan/ carboxymethyl starch/montmorillonite bio-nanocomposite for reduction of dental bacterial biofilm formation beg am ( ) design and development of ligand-appended polysaccharidic nanoparticles for the delivery of oxaliplatin in colorectal cancer cefazolin loaded chitosan nanoparticles to cure multi drug resistant gram-negative pathogens development of a carbon nanotubes paste electrode modified with crosslinked chitosan for cadmium (ii) and mercury(ii) determination chitosan microencapsulation of various essential oils to enhance the functional properties of cotton fabric biomaterials based on chitin and chitosan in wound dressing applications chitosan coatings to control release and target tissues for therapeutic delivery controlled release of bordetella bronchiseptica dermonecrotoxin (bbd) vaccine from bbd-loaded chitosan microspheres in vitro in vitro evaluation of chitosan/poly(lactic acidglycolic acid) sintered microsphere scaffolds for bone tissue engineering novel chitosan derivative nanoparticles enhance the immunogenicity of a dna vaccine encoding hepatitis b virus core antigen in mice removal of methyl orange from aqueous solutions by magnetic maghemite/chitosan nanocomposite films: adsorption kinetics and equilibrium synthesis of novel biocompatible composite fe o /zro /chitosan and its application for dye removal biomimetic spiral-cylindrical scaffold based on hybrid chitosan/cellulose/nano-hydroxyapatite membrane for bone regeneration microintumescent mechanism of flame-retardant water-based chitosan-ammonium polyphosphate multilayer nanocoating on cotton fabric utilization of carboxymethyl chitosan in cosmetics in vivo evaluation of porous hydroxyapatite/chitosan-alginate composite scaffolds for bone tissue engineering glutaraldehyde cross-linked magnetic chitosan nanocomposites: reduction precipitation synthesis, characterization, and application for removal of hazardous textile dyes amphiphilic chitosan derivatives as carrier agents for rotenone antimicrobial silver chloride nanoparticles stabilized with chitosan oligomer for the healing of burns curcumin loaded chitosan nanoparticles impregnated into collagen-alginate scaffolds for diabetic wound healing chitosan nanoparticle based delivery systems for sustainable agriculture a future perspective in crop protection: chitosan and its oligosaccharides a case of anaphylaxis caused by the health food chitosan synthesis and characterization of a novel chitosan/montmorillonite/hydroxyapatite nanocomposite for bone tissue engineering microwave-induced biomimetic approach for hydroxyapatite coatings of chitosan scaffolds biodegradation, biodistribution and toxicity of chitosan effect of locally administered novel biodegradable chitosan based risedronate/zinc-hydroxyapatite intra-pocket dental film on alveolar bone density in rat model of periodontitis a green approach to constructing multilayered nanocoating for flame retardant treatment of polyamide fabric from chitosan and sodium alginate cartilage regeneration by novel polyethylene oxide/chitin/chitosan scaffolds incorporation of chitosan in biomimetic gelatin/ chondroitin- -sulfate/hyaluronan cryogel for cartilage tissue engineering preparation of water-soluble chitosan/heparin complex and its application as wound healing accelerator recent modifications of chitosan for adsorption applications: a critical and systematic review environmental friendly technology for the removal of pharmaceutical contaminants from wastewaters using modified chitosan adsorbents removal of dorzolamide from biomedical wastewaters with adsorption onto graphite oxide/poly(acrylic acid) grafted chitosan nanocomposite anti-tumor immune response of folate-conjugated chitosan nanoparticles containing the ip- gene in mice with hepatocellular carcinoma polyelectrolyte complexes via desalting mixtures of hyaluronic acid and chitosan-physicochemical study and structural analysis effect of fiber length and blending method on the tensile properties of ring spun chitosan-cotton blend yarns a pilot intervention with chitosan/cotton knitted jersey fabric to provide comfort for epidermolysis bullosa patients chitosan modified poly(l-lactide) microspheres as cell microcarriers for cartilage tissue engineering characterization and antimicrobial analysis of chitosan-based films chitin, chitinases and chitinase-like proteins in allergic inflammation and tissue remodeling preparation of multiwalled carbon nanotube-chitosan-alcohol dehydrogenase nanobiocomposite for amperometric detection of ethanol chitin regulation of immune responses: an old molecule with new roles chitosonic((r)) acid as a novel cosmetic ingredient: evaluation of its antimicrobial, antioxidant and hydration activities chitosan/polyurethane blended fiber sheets containing silver sulfadiazine for use as an antimicrobial wound dressing oxygen plasma treatment on d-printed chitosan/ gelatin/hydroxyapatite scaffolds for bone tissue engineering water-based chitosan/melamine polyphosphate multilayer nanocoating that extinguishes fire on polyester-cotton fabric chitosan-alginate as scaffolding material for cartilage tissue engineering chitosan-alginate hybrid scaffolds for bone tissue engineering controlled release and retarded leaching of pesticides by encapsulating in carboxymethyl chitosan/bentonite composite gel in situ injectable nano-composite hydrogel composed of curcumin, n,o-carboxymethyl chitosan and oxidized alginate for wound healing application antibacterial mechanism of chitosan and its applications in protection of plant from bacterial disease chitin, chitosan, and glycated chitosan regulate immune responses: the novel adjuvants for cancer vaccine chitosan grafted with phosphorylcholine and macrocyclic polyamine as an effective gene delivery vector: preparation, characterization and in vitro transfection preparation, characterization, and insecticidal activity of avermectin-grafted-carboxymethyl chitosan preparation and adsorption properties of magnetic chitosan composite adsorbent for cu + removal chitosan conduit combined with hyaluronic acid prevent sciatic nerve scar in a rat model of peripheral nerve crush injury the natural product chitosan enhances the anti-tumor activity of natural killer cells by activating dendritic cells fabrication and characters of a corneal endothelial cells scaffold based on chitosan tissue-engineered membrane based on chitosan for repair of mechanically damaged corneal epithelium peripheral nerve repair: monitoring by using gadofluorine m-enhanced mr imaging with chitosan nerve conduits with cultured mesenchymal stem cells in rat model of neurotmesis preparation, bioactivity and mechanism of nano-hydroxyapatite/sodium alginate/chitosan bone repair material chitooligosaccharides and their biological activities: a comprehensive review films based on neutralized chitosan citrate as innovative composition for cosmetic application rapidly curable chitosan-peg hydrogels as tissue adhesives for hemostasis and wound healing development of ca-alginate-chitosan microcapsules for encapsulation and controlled release of imidacloprid to control dengue outbreaks preparation, characterization and biological test of d-scaffolds based on chitosan, fibroin and hydroxyapatite for bone tissue engineering sciatic nerve repair by microgrooved nerve conduits made of chitosan-gold nanocomposites sensitive amperometric immunosensor for alphafetoprotein based on carbon nanotube/gold nanoparticle doped chitosan film enhancing catalytic performance of laccase via immobilization on chitosan/ceo microspheres development of chitosan/poly-gamma-glutamic acid/pluronic/ curcumin nanoparticles in chitosan dressings for wound regeneration differentiation potential of mesenchymal stem cells from equine bone marrow cultured on hyaluronic acid-chitosan polyelectrolyte multilayer biofilm recent advances in chitosan and its derivatives as adsorbents for removal of pollutants from water and wastewater the direct electron transfer of glucose oxidase and glucose biosensor based on carbon nanotubes/chitosan matrix integrin α(v)β( )-targeted cancer therapy ultrasensitive amperometric immunosensor for the determination of carcinoembryonic antigen based on a porous chitosan and gold nanoparticles functionalized interface amperometric biosensors based on alumina nanoparticles-chitosan-horseradish peroxidase nanobiocomposites for the determination of phenolic compounds chitosan conduits combined with nerve growth factor microspheres repair facial nerve defects conjugating influenza a (h n ) antigen to n-trimethylaminoethylmethacrylate chitosan nanoparticles improves the immunogenicity of the antigen after nasal administration effect of collagen peptidescarboxymethyl chitosan microspheres on ultraviolet induced damages a novel reusable nanocomposite adsorbent, xanthated fe o -chitosan grafted onto graphene oxide, for removing cu(ii) from aqueous solutions the efficient removal of cu(ii) from aqueous solutions by fe o @hexadecyl trimethoxysilane@chitosan composites preparation and biological properties of a novel composite scaffold of nano-hydroxyapatite/chitosan/carboxymethyl cellulose for bone tissue engineering chitosan coated textiles may improve atopic dermatitis severity by modulating skin staphylococcal profile: a randomized controlled trial silver ions release from antibacterial chitosan films containing in situ generated silver nanoparticles chitosan nanoparticles on the improvement of thermal, barrier, and mechanical properties of high-and low-methyl pectin films biological evaluation (in vitro and in vivo) of bilayered collagenous coated (nano electrospun and solid wall) chitosan membrane for periodontal guided bone regeneration preparation and characterization of chitosan-natural nano hydroxyapatite-fucoidan nanocomposites for bone tissue engineering construction, application and biosafety of silver nanocrystalline chitosan wound dressing preparation of quaternary ammonium salt of chitosan nanoparticles and their textile properties on antheraea pernyi silk modification healing of skin wounds with a chitosan-gelatin sponge loaded with tannins and platelet-rich plasma enhanced antibacterial and wound healing activities of microporous chitosan-ag/zno composite dressing high-activity chitosan/nano hydroxyapatite/zoledronic acid scaffolds for simultaneous tumor inhibition, bone repair and infection eradication combined antibacterial/tissue regeneration response in thermal burns promoted by functional chitosan/silver nanocomposites fabrication and characterization of kidney bean (phaseolus vulgaris l.) protein isolate-chitosan composite films at acidic ph chitosan and oligochitosan enhance the resistance of peach fruit to brown rot guided bone regeneration with tripolyphosphate cross-linked asymmetric chitosan membrane interleukin/chitosan (jy) adjuvant enhances the mucosal immunity of human papillomavirus l virus-like particles in mice current state on the development of nanoparticles for use against bacterial gastrointestinal pathogens. focus on chitosan nanoparticles loaded with phenolic compounds development of gelatin-chitosan-hydroxyapatite based bioactive bone scaffold with controlled pore size and mechanical strength chitosan effects on plant systems trimethyl chitosan nanoparticles encapsulated protective antigen protects the mice against anthrax chitosan, gelatin and poly (l-lysine) polyelectrolyte-based scaffolds and films for neural tissue engineering development of a multilayered antimicrobial edible coating for shelf-life extension of fresh-cut cantaloupe (cucumis melo l.) stored at c influence of α-tocopherol on physicochemical properties of chitosan-based films a non-enzymatic nanomagnetic electro-immunosensor for determination of aflatoxin b- as a model antigen chitosan nanoparticles for targeting and sustaining minoxidil sulphate delivery to hair follicles a new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs peripheral nerve repair and grafting techniques: a review n,n-dicarboxymethyl chitosan as delivery agent for bone morphogenetic protein in the repair of articular cartilage a mucosal vaccine against diphtheria: formulation of cross reacting material (crm( )) of diphtheria toxin with chitosan enhances local and systemic antibody and th responses following nasal delivery fattakhova an ( ) determination of antidepressants using monoamine oxidase amperometric biosensors based on screen-printed graphite electrodes modified with multi-walled carbon nanotubes preparation and characterization of potassium nitrate controlledrelease fertilizers based on chitosan and xanthan layered tablets poly(nisopropylacrylamide) hydrogel/chitosan scaffold hybrid for three-dimensional stem cell culture and cartilage tissue engineering determination of chlorophenol in environmental samples using a voltammetric biosensor based on hybrid nanocomposite a composite scaffold of msc affinity peptide-modified demineralized bone matrix particles and chitosan hydrogel for cartilage regeneration a chitosanbased liposome formulation enhances the in vitro wound healing efficacy of substance p. neuropeptide peripheral nerve regeneration through hydrogel-enriched chitosan conduits containing engineered schwann cells for drug delivery a chitosanhyaluronic acid hydrogel scaffold for periodontal tissue engineering tumour targeted delivery of encapsulated dextrandoxorubicin conjugate using chitosan nanoparticles as carrier chitosan-film associated with mesenchymal stem cells enhanced regeneration of peripheral nerves: a rat sciatic nerve model antimicrobial and physicochemical properties of chitosanÀhpmc-based films amperometric glucose biosensor utilizing fad-dependent glucose dehydrogenase immobilized on nanocomposite electrode the vaccine adjuvant alum inhibits il- by promoting pi kinase signaling while chitosan does not inhibit il- and enhances th and th responses osteoconduction exerted by methylpyrrolidinone chitosan used in dental surgery osteoconductive properties of methylpyrrolidinone chitosan in an animal model chitosan, hyaluronan and chondroitin sulfate in tissue engineering for cartilage regeneration: a review pyrazole-based compounds in chitosan liposomal emulsion for antimicrobial cotton fabrics protein growth factors loaded highly porous chitosan scaffold: a comparison of bone healing properties evaluation of chitosan gel with % silver sulfadiazine as an alternative for burn wound treatment in rats plasma-assisted preparation of high-performance chitosan nanofibers/gauze composite bandages chitosan/poly(epsilon-caprolactone) blend scaffolds for cartilage repair antioxidant effects of chitin, chitosan, and their derivatives photoprotection for deltamethrin using chitosancoated beeswax solid lipid nanoparticles preparation of magnetic composite based on zinc oxide nanoparticles and chitosan as a photocatalyst for removal of reactive blue a high photo-catalytic activity of magnetic composite based on chitosan and manganese-doped zinc oxide nanoparticles for removal of dyeing wastewater gabab receptor ligand-directed trimethyl chitosan/tripolyphosphate nanoparticles and their pmdi formulation for survivin sirna pulmonary delivery axonal regeneration and remyelination evaluation of chitosan/gelatin-based nerve guide combined with transforming growth factor-beta and schwann cells novel recyclable adsorbent for the removal of copper (ii) and lead(ii) from aqueous solution applications of chitosan for improvement of quality and shelf life of foods: a review a comparative study of gelatin and starch-based nano-composite films modified by nano-cellulose and chitosan for food packaging applications multilayer-coated npk compound fertilizer hydrogel with controlled nutrient release and water absorbency degradation of fully water-soluble, partially n-acetylated chitosans with lysozyme novel hydroxyapatite/chitosan bilayered scaffold for osteochondral tissueengineering applications: scaffold design and its performance when seeded with goat bone marrow stromal cells chitosan/gelatin/platelet gel enriched by a combination of hydroxyapatite and beta-tricalcium phosphate in healing of a radial bone defect model in rat spray dried inhalable ciprofloxacin powder with improved aerosolisation and antimicrobial activity her amplification ratios by fluorescence in situ hybridization and correlation with immunohistochemistry in a cohort of breast cancer tissues ultrathin chitosanpoly(ethylene glycol) hydrogel films for corneal tissue engineering regenerative potential of chitosan-coated poly- -hydroxybutyrate conduits seeded with mesenchymal stem cells in a rat sciatic nerve injury model nanoamphiphilic chitosan dispersed poly(lactic acid) bionanocomposite films with improved thermal, mechanical, and gas barrier properties biophysicochemical evaluation of chitosanhydroxyapatite-marine sponge collagen composite for bone tissue engineering human exposure to wastewater-derived pharmaceuticals in fresh produce: a randomized controlled trial focusing on carbamazepine new drug delivery systems based on chitosan controlled release of plateletderived growth factor-bb from chondroitin sulfate-chitosan sponge for guided bone regeneration platelet derived growth factor releasing chitosan sponge for periodontal bone regeneration the accelerating effect of chitosan-silica hybrid dressing materials on the early phase of wound healing preparation and optimization of chitosan-gelatin films for sustained delivery of lupeol for wound healing fluorinated methacrylamide chitosan hydrogels enhance collagen synthesis in wound healing through increased oxygen availability chitosan as a starting material for wound healing applications chitosan-mediated stimulation of macrophage function composite nano-titanium oxide-chitosan artificial skin exhibits strong wound-healing effect-an approach with anti-inflammatory and bactericidal kinetics nanoporous magnetic cellulose-chitosan composite microspheres: preparation, characterization, and application for cu(ii) adsorption chitosan-montmorillonite biocomposite as an adsorbent for copper (ii) cations from aqueous solutions chitosan-starch beads prepared by ionotropic gelation as potential matrices for controlled release of fertilizers in vitro capacity of different grades of chitosan derivatives to induce platelet adhesion and aggregation cotton filter fabrics functionalization by chitosan uv-grafting for removal of dyes antimicrobial chitosan finish of cotton and silk fabrics by uv-curing with -hydroxy- -methylphenylpropane- -one sonochemical coating of textiles with hybrid zno/chitosan antimicrobial nanoparticles preliminary study on chitosan modified glass ionomer restoratives concise review: msc-derived exosomes for cell-free therapy chitosan derivatives for gene transfer: effect of phosphorylcholine and diethylaminoethyl grafts on the in vitro transfection efficiency immunoadjuvant potential of cross-linked dextran microspheres mixed with chitosan nanospheres encapsulated with tetanus toxoid layer-by-layer electrostatic deposition of edible coating on fresh cut melon model: anticipated and unexpected effects of alginate-chitosan combination effects of a composite chitosan-gelatin edible coating on postharvest quality and storability of red bell peppers chitosan-based nanoparticles for tumor-targeted drug delivery chitosan/beta- , -glucan/hydroxyapatite bone scaffold enhances osteogenic differentiation through tnf-alpha-mediated mechanism evaluation of the potential of chitosan/beta- , -glucan/hydroxyapatite material as a scaffold for living bone graft production in vitro by comparison of adsc and bmdsc behaviour on its surface chitosan-modified smectite clay and study on adsorptiondesorption of urea electrospinning of chitosan-based solutions for tissue engineering and regenerative medicine in vivo antitumor activity of chitosan nanoparticles chitosan/poly(vinyl alcohol) hydrogel combined with ad-htgf-beta transfected mesenchymal stem cells to repair rabbit articular cartilage defects chitosan green tea polyphenol complex as a released control compound for wound healing the antioxidant property of chitosan green tea polyphenols complex induces transglutaminase activation in wound healing a label-free amperometric immunosensor based on biocompatible conductive redox chitosan-ferrocene/gold nanoparticles matrix a nitrite biosensor based on co-immobilization of nitrite reductase and viologen-modified chitosan on a glassy carbon electrode chitosan and its antimicrobial potential-a critical literature survey chitosan as antimicrobial agent: applications and mode of action doxorubicin hydrochloride -loaded electrospun chitosan/cobalt ferrite/titanium oxide nanofibers for hyperthermic tumor cell treatment and controlled drug release peg-stabilized carbodiimide crosslinked collagen-chitosan hydrogels for corneal tissue engineering dendritic cell targeted chitosan nanoparticles for nasal dna immunization against sars cov nucleocapsid protein hyaluronidase enzyme core- -fluorouracilloaded chitosan-peg-gelatin polymer nanocomposites as targeted and controlled drug delivery vehicles studies on an anti-aging formulation prepared using aloe vera blended collagen and chitosan removal of copper(ii) using chitin/chitosan nano-hydroxyapatite composite octadecanoid signaling component "burst" in rice (oryza sativa l.) seedling leaves upon wounding by cut and treatment with fungal elicitor chitosan adsorption of cadmium (ii) and copper (ii) ions from aqueous solution using chitosan composite a novel chitosan-tussah silk fibroin/ nano-hydroxyapatite composite bone scaffold platform with tunable mechanical strength in a wide range evaluation of durability to laundering of triclosan and chitosan on a textile substrate dermatan sulfate/chitosan polyelectrolyte complex with potential application in the treatment and diagnosis of vascular disease role of chitosan on some physical properties and the urea controlled release of the silk fibroin/gelatin hydrogel fabrication of zno incorporated chitosan nanocomposites for enhanced functional properties of cellulosic fabric effective nasal influenza vaccine delivery using chitosan application of magnetic chitosan composites for the removal of toxic metal and dyes from aqueous solutions development and characterization of an ldpe/chitosan composite antimicrobial film for chilled fish storage nanocomposites based on chitosan/ silver/clay for durable multi-functional properties of cotton fabrics synthesis of chitosan incorporated neem seed extract (azadirachta indica) for medical textiles dextran-based hydrogel containing chitosan microparticles loaded with growth factors to be used in wound healing potential of low molecular mass chitosan as a dna delivery system: biocompatibility, body distribution and ability to complex and protect dna chitosan-solid lipid nanoparticles as carriers for topical delivery of tretinoin soluble cell adhesion molecule l -fc promotes locomotor recovery in rats after spinal cord injury enhancement of nitrogen release properties of urea-kaolinite fertilizer with chitosan binder amelogenin-chitosan matrix for human enamel regrowth: effects of viscosity and supersaturation degree vitamin-mediated targeting as a potential mechanism to increase drug uptake by tumours application of a biopolymer chitosan-poly (propylene)imine dendrimer hybrid as an antimicrobial agent on the wool fabrics cu-chitosan nanoparticle mediated sustainable approach to enhance seedling growth in maize by mobilizing reserved food potential prospects of chitosan derivative trimethyl chitosan chloride (tmc) as a polymeric absorption enhancer: synthesis, characterization and applications scintigraphic verification of adherence of a chitosan formulation to the human oesophagus preparation of poly (chitosan-acrylamide) flocculant using gamma radiation for adsorption of cu(ii) and ni(ii) ions a review on chitosan-based adsorptive membranes mucoadhesive electrospun chitosan-based nanofibre mats for dental caries prevention chitosan-hyaluronic acid composite sponge scaffold enriched with andrographolide-loaded lipid nanoparticles for enhanced wound healing buccal penetration enhancement properties of n-trimethyl chitosan: influence of quaternization degree on absorption of a high molecular weight molecule the antibacterial properties of a novel chitosan-ag-nanoparticle composite synthesis and characterization of zinc oxide-neem oilchitosan bionanocomposite for food packaging application efficiency of chitosan-algal biomass composite microbeads at heavy metal removal microfungal spores (ustilago maydis and u. digitariae) immobilised chitosan microcapsules for heavy metal removal chitosan as a suitable nanocarrier material for anti-alzheimer drug delivery intranasal chitosan-dna vaccines that protect across influenza virus subtypes preparation and characterization of cotton fabrics with antimicrobial properties through the application of chitosan/silver-zeolite film in vivo evaluation of chitosan as an adjuvant in subcutaneous vaccine formulations diphtheria toxoid and n-trimethyl chitosan layer-by-layer coated ph-sensitive microneedles induce potent immune responses upon dermal vaccination in mice perfusion conditioning of hydroxyapatite-chitosan-gelatin scaffolds for bone tissue regeneration from human mesenchymal stem cells evaluation of chitosan based vaginal bioadhesive gel formulations for antifungal drugs nano-hydroxyapatite/beta-cd/chitosan nanocomposite for potential applications in bone tissue engineering enhanced antibacterial efficacy of silver nanoparticles immobilized in a chitosan nanocarrier a novel squid pen chitosan/hydroxyapatite/beta-tricalcium phosphate composite for bone tissue engineering ciliary neurotrophic factor-coated polylactic-polyglycolic acid chitosan nerve conduit promotes peripheral nerve regeneration in canine tibial nerve defect repair crgd-functionalized polymeric magnetic nanoparticles as a dual-drug delivery system for safe targeted cancer therapy accelerated healing of diabetic wound using artificial dermis constructed with adipose stem cells and poly (l-glutamic acid)/chitosan scaffold hydrodynamic delivery of chitosan-folate-dna nanoparticles in rats with adjuvant-induced arthritis enhanced antitumor immunity by targeting dendritic cells with tumor cell lysate-loaded chitosan nanoparticles vaccine gmsc-derived exosomes combined with a chitosan/silk hydrogel sponge accelerates wound healing in a diabetic rat skin defect model ph response of human dental plaque to chewing gum supplemented with low molecular chitosan alveolar macrophage priming by intravenous administration of chitin particles, polymers of n-acetyl-d-glucosamine, in mice chitin particle-induced cell-mediated immunity is inhibited by soluble mannan: mannose receptor-mediated phagocytosis initiates il- production challenges for nerve repair using chitosan-siloxane hybrid porous scaffolds high-performance glucose biosensor based on chitosan-glucose oxidase immobilized polypyrrole/nafion/functionalized multi-walled carbon nanotubes bio-nanohybrid film fabrication of apigenin loaded gellan gum-chitosan hydrogels (ggch-hgs) for effective diabetic wound healing silicone doped chitosan-acrylamide coencapsulated urea fertilizer: an approach to controlled release fertilizers paraquat-loaded alginate/chitosan nanoparticles: preparation, characterization and soil sorption studies novel genipin-cross-linked chitosan/silk fibroin sponges for cartilage engineering strategies degrading endocrine disrupting chemicals from wastewater by tio photocatalysis: a review graphene oxide-chitosan nanocomposite based electrochemical dna biosensor for detection of typhoid sustainable fragrance cum antimicrobial finishing on cotton: indigenous essential oil preparation and characterization of collagen/chitosan/hyaluronic acid thin films for application in hair care chitosan-based microparticles for immobilization of tio nanoparticles and their application for photodegradation of textile dyes chitosan: a promising safe and immune-enhancing adjuvant for intranasal vaccines odontogenic differentiation potential of human dental pulp cells cultured on a calciumaluminate enriched chitosan-collagen scaffold chitosan-silica hybrid nanosorbents for oil removal from water oral hepatitis b vaccine: chitosan or glucan based delivery systems for efficient hbsag immunization following subcutaneous priming a nanotechnological approach to biosensors sensitivity improvement: application to organophosphorus pesticides determination embedment of silver into temperatureand ph-responsive microgel for the development of smart textiles with simultaneous moisture management and controlled antimicrobial activities heavy-metal ion sensors using chitosan-capped gold nanoparticles promotion of angiogenesis by sustained release of rhgm-csf from heparinized collagen/chitosan scaffolds an amperometric immunosensor based on multi-walled carbon nanotubes-thionine-chitosan nanocomposite film for chlorpyrifos detection antimicrobial and mechanical properties of beta-cyclodextrin inclusion with essential oils in chitosan films the mucosal and systemic immune responses elicited by a chitosan-adjuvanted intranasal influenza h n vaccine antioxidant edible films based on chitosan and starch containing polyphenols from thyme extracts mitoq loaded chitosan-hyaluronan composite membranes for wound healing gelatin/chitosan/hyaluronan ternary complex scaffold containing basic fibroblast growth factor for cartilage tissue engineering fabrication and evaluation of porous keratin/chitosan (kcs) scaffolds for effectively accelerating wound healing a facile fabrication of multifunctional knit polyester fabric based on chitosan and polyaniline polymer nanocomposite thermosensitive chitosan-based hydrogels releasing stromal cell derived factor- alpha recruit msc for corneal epithelium regeneration evaluation of immunostimulatory effects of n-( -hydroxy) propyl- -trimethylammonium chitosan chloride for improving live attenuated hepatitis a virus vaccine efficacy n-( -hydroxy) propyl- -trimethylammonium chitosan chloride: an immune-enhancing adjuvant for hepatitis e virus recombinant polypeptide vaccine in mice electrospun chitosan-gelatin biopolymer composite nanofibers for horseradish peroxidase immobilization in a hydrogen peroxide biosensor three-layered membranes of collagen/ hydroxyapatite and chitosan for guided bone regeneration cross-linked chitosan microspheres: preparation and evaluation as a matrix for the controlled release of pharmaceuticals oral drug absorption enhancement by chitosan and its derivatives controlled release of thymosin beta using a collagen-chitosan sponge scaffold augments cutaneous wound healing and increases angiogenesis in diabetic rats with hindlimb ischemia ultraviolet protection cotton fabric achieved via layer-by-layer self-assembly of graphene oxide and chitosan the use of single walled carbon nanotubes dispersed in a chitosan matrix for preparation of a galactose biosensor immunological aspects of chitin and chitin derivatives administered to animals hypoxia pretreatment of bone marrow-derived mesenchymal stem cells seeded in a collagen-chitosan sponge scaffold promotes skin wound healing in diabetic rats with hindlimb ischemia pure chitosan microfibres for biomedical applications bactericidal effect of iron oxide nanoparticles on staphylococcus aureus graphite oxide/chitosan composite for reactive dye removal a novel nanoparticle formulation for sustained paclitaxel delivery immobilization of lactate dehydrogenase within multiwalled carbon nanotube-chitos anhanocompo site for application to lactate biosensors amperometric cholesterol biosensors based on carbon nanotube-chitosan-platinum-cholesterol oxidase nanobiocomposite antitumor efficacy of doxorubicin released from crosslinked nanoparticulate chondroitin sulfate/chitosan polyelectrolyte complexes poly(poly(ethylene glycol) methyl ether methacrylate) grafted chitosan for dye removal from water trimethylated chitosan as polymeric absorption enhancer for improved peroral delivery of peptide drugs targeted delivery of doxorubicin to breast cancer cells by magnetic lhrh chitosan bioconjugated nanoparticles chitosan porous d scaffolds embedded with resolvin d to improve in vivo bone healing chitosan-induced programmed cell death in plants chitosan-based vaccine adjuvants: incomplete characterization complicates preclinical and clinical evaluation chitosan-fibrin nanocomposites as drug delivering and wound healing materials impact of chitosan-beeswax edible coatings on the quality of fresh strawberries (fragaria ananassa cv camarosa) under commercial storage conditions chitosan composites for bone tissue engineering-an overview chitosan-amylopectin/hydroxyapatite and chitosan-chondroitin sulphate/hydroxyapatite composite scaffolds for bone tissue engineering preparation and characterization of chitosancarbon nanotube scaffolds for bone tissue engineering optimization and evaluation of silk fibroin-chitosan freeze-dried porous scaffolds for cartilage tissue engineering application adsorption of dyes and heavy metal ions by chitosan composites: a review antioxidant activity of high molecular weight chitosan and n,oquaternized chitosans chitosan-based biosorbents: modification and application for biosorption of heavy metals and radionuclides chitosan-poly(vinyl alcohol)/attapulgite nanocomposites for copper (ii) ions removal: ph dependence and adsorption mechanisms chitosan-alginate pec membrane as a wound dressing: assessment of incisional wound healing chitosan/prussian blue-based biosensors dog sciatic nerve regeneration across a -mm defect bridged by a chitosan/pga artificial nerve graft acceleration of diabetic wound healing with chitosan-crosslinked collagen sponge containing recombinant human acidic fibroblast growth factor in healing-impaired stz diabetic rats synthesis and characterization of collagen-chitosan-hydroxyapatite artificial bone matrix synthesis and evaluation of collagenchitosan-hydroxyapatite nanocomposites for bone grafting synergistic antimicrobial activities of natural essential oils with chitosan films repairing rat sciatic nerve injury by a nerve-growth-factor-loaded, chitosan-based nerve conduit novel chitosan-polycaprolactone blends as potential scaffold and carrier for corneal endothelial transplantation kinetics and functional effectiveness of nisin loaded antimicrobial packaging film based on chitosan/poly(vinyl alcohol) thermosensitive behavior and antibacterial activity of cotton fabric modified with a chitosan-poly(n-isopropylacrylamide) interpenetrating polymer network hydrogel development of chitosan/poly(vinyl alcohol) electrospun nanofibers for infection related wound healing chitosan and β-cyclodextrinepichlorohydrin polymer composite film as a plant healthcare material for carbendazimcontrolled release to protect rape against sclerotinia sclerotiorum (lib.) de bary hyaluronic acid doped-poly( , -ethylenedioxythiophene)/chitosan/gelatin (pedot-ha/cs/gel) porous conductive scaffold for nerve regeneration emerging chitosan-based films for food packaging applications bridging peripheral nerve defect with chitosan-collagen film chitosan/silk fibroin-based tissue-engineered graft seeded with adipose-derived stem cells enhances nerve regeneration in a rat model application of a biosensor for monitoring of ethanol chitosan nanoparticles act as an adjuvant to promote both th and th immune responses induced by ovalbumin in mice thiolated chitosans: useful excipients for oral drug delivery differentiation of dental pulp stem cells into chondrocytes upon culture on porous chitosan-xanthan scaffolds in the presence of kartogenin fluorinated methacrylamide chitosan hydrogel systems as adaptable oxygen carriers for wound healing methods of gene delivery poly(d, l-lactide-co-glycolide) tubes with multifilament chitosan yarn or chitosan sponge core in nerve regeneration environmentally-friendly rf plasma treatment of thai silk fabrics with chitosan for durable antibacterial property the mechanical properties and osteoconductivity of hydroxyapatite bone scaffolds with multi-scale porosity preparation and properties of chitosan-coated npk compound fertilizer with controlled-release and water-retention amperometric glucose biosensor based on layer-by-layer assembly of multilayer films composed of chitosan, gold nanoparticles and glucose oxidase modified pt electrode mechanical properties and permeability of porous chitosan-poly(p-dioxanone)/silk fibroin conduits used for peripheral nerve repair removal of cu(ii) ions from aqueous water by l-arginine modifying magnetic chitosan integration of lysozyme into chitosan nanoparticles for improving antibacterial activity tissue engineering of cartilage with the use of chitosan-gelatin complex scaffolds gemcitabine conjugated chitosan and double antibodies (abc-gc-gemcitabine nanoparticles) enhanced cytoplasmic uptake of gemcitabine and inhibit proliferation and metastasis in human sw pancreatic cancer cells antibacterial hybrid materials fabricated by nanocoating of microfibril bundles of cellulose substance with titania/chitosan/silvernanoparticle composite films sub-micron-sized polyethylenimine-modified polystyrene/fe o /chitosan magnetic composites for the efficient and recyclable adsorption of cu(ii) ions antioxidant activity of water-soluble chitosan derivatives in vitro and in vivo evaluation of a biodegradable chitosan-pla composite peripheral nerve guide conduit material synthesis and adsorption behavior of magnetic microspheres based on chitosan/organic rectorite for low-concentration heavy metal removal chitosan antimicrobial and eliciting properties for pest control in agriculture: a review chitosan-based coating with antimicrobial agents: preparation, property, mechanism, and application effectiveness on fruits and vegetables international optic nerve regeneration in polyglycolic acid-chitosan conduits coated with recombinant l -fc selective recognition of -hydroxytryptamine and dopamine on a multi-walled carbon nanotube-chitosan hybrid filmmodified microelectrode array durable antibacterial cotton modified by silver nanoparticles and chitosan derivative binder antibacterial property and biocompatibility of chitosan/poly(vinyl alcohol)/zno (cs/pva/zno) beads as an efficient adsorbent for cu (ii) removal from aqueous solution electrochemical dna biosensor based on graphene oxide-chitosan hybrid nanocomposites for detection of escherichia coli o :h the graphene oxide and chitosan biopolymer loads tio for antibacterial and preservative research carboxymethyl chitosan/ gelatin/hyaluronic acid blended-membranes as epithelia transplanting scaffold for corneal wound healing joint use of a chitosan/plga scaffold and mscs to bridge an extra large gap in dog sciatic nerve induction of phytoalexin formation in suspension-cultured rice cells by n-acetyl-chitooligosaccharides removal of various cationic dyes from aqueous solutions using a kind of fully biodegradable magnetic composite microsphere mechanochemical modification of kaolin surfaces for immobilization and delivery of pesticides in alginate-chitosan composite beads a practical glucose biosensor based on fe o nanoparticles and chitosan/nafion composite film preparation and application of nanochitosan to finishing treatment with anti-microbial and anti-shrinking properties nerve conduits based on immobilization of nerve growth factor onto modified chitosan by using genipin as a crosslinking agent a novel tyrosinase biosensor based on chitosancarbon-coated nickel nanocomposite film chitosan/collagen scaffold containing bone morphogenetic protein- dna supports dental pulp stem cell differentiation in vitro and in vivo preparation and controllable release of chitosan/vanillin microcapsules and their application to cotton fabric release of doxorubicin by a folate-grafted, chitosan-coated magnetic nanoparticle ph-responsive mesoporous zsm- zeolites/ chitosan core-shell nanodisks loaded with doxorubicin against osteosarcoma avidin targeting of intraperitoneal tumor xenografts preparation, characterization, and cytotoxicity of various chitosan nanoparticles preventative vaccine-loaded mannosylated chitosan nanoparticles intended for nasal mucosal delivery enhance immune responses and potent tumor immunity application of silk fibroin/chitosan/nanohydroxyapatite composite scaffold in the repair of rabbit radial bone defect antioxidant properties of chitosan from crab shells the effects of pva/chitosan/fibroin (pcf)-blended spongy sheets on wound healing in rats effects of tumor microenvironments on targeted delivery of glycol chitosan nanoparticles enhanced wettability and moisture retention of cotton fabrics coated with selfsuspended chitosan derivative targeted delivery of doxorubicin-utilizing chitosan nanoparticles surface-functionalized with anti-her trastuzumab chitosan nanocomposite films based on ag-np and au-np biosynthesis by bacillus subtilis as packaging materials enhancement of egyptian soft white cheese shelf life using a novel chitosan/carboxymethyl cellulose/zinc oxide bionanocomposite film self-assembled high-strength hydroxyapatite/ graphene oxide/chitosan composite hydrogel for bone tissue engineering chitosan films and coatings containing essential oils: the antioxidant and antimicrobial activity, and application in food systems development of electrospun chitosan-polyethylene oxide/fibrinogen biocomposite for potential wound healing applications antimicrobial lysozyme-chitosan coatings affect functional properties and shelf life of chicken eggs during storage administration of growth factors for bone regeneration chitosan solution enhances both humoral and cell-mediated immune responses to subcutaneous vaccination intravesical immunotherapy of superficial bladder cancer with chitosan/ interleukin- intratumoral immunotherapy of established solid tumors with chitosan/il- incorporation of heparin-binding proteins into preformed dextran sulfate-chitosan nanoparticles modification of wool fabric using prepared chitosan-cyanuric chloride hybrid cu(ii) removal enhancement from aqueous solutions using ion-imprinted membrane technique preparation and application of a novel environmentally friendly organic seed coating for rice application of bioactive coatings based on chitosan for soybean seed protection international keratin-chitosan/n-zno nanocomposite hydrogel for antimicrobial treatment of burn wound healing: characterization and biomedical application amperometric ethanol biosensors based on chitosan-nad(+)-alcohol dehydrogenase films carbon nanotube-chitosan system for electrochemical sensing based on dehydrogenase enzymes oligochitosan induces programmed cell death in tobacco suspension cells repair of an articular cartilage defect using adipose-derived stem cells loaded on a polyelectrolyte complex scaffold based on poly(l-glutamic acid) and chitosan expression of gamma-aminobutyric acid receptors on neoplastic growth and prediction of prognosis in non-small cell lung cancer sorption of carbamazepine from water by magnetic molecularly imprinted polymers based on chitosan-fe o removal of selected pharmaceuticals from aqueous solution using magnetic chitosan: sorption behavior and mechanism in-situ birth of mscs multicellular spheroids in poly (l-glutamic acid)/chitosan scaffold for hyaline-like cartilage regeneration biocomposite scaffolds for bone regeneration: role of chitosan and hydroxyapatite within poly- -hydroxybutyrate-co- -hydroxyvalerate on mechanical properties and in vitro evaluation multifunctional glucose biosensors from fe o nanoparticles modified chitosan/graphene nanocomposites adsorption of pharmaceuticals on chitosan-based magnetic composite particles with core-brush topology preparation of chitosan-tio composite film with efficient antimicrobial activities under visible light for food packaging applications a composite hydrogel of chitosan/ heparin/poly (gamma-glutamic acid) loaded with superoxide dismutase for wound healing experimental study on chitosan mediated insulinlike growth factor gene transfection repairing injured articular cartilage in rabbits fk -loaded chitosan conduit promotes the regeneration of injured sciatic nerves in the rat through the upregulation of brain-derived neurotrophic factor and trkb efficacy of thermosensitive chitosan/betaglycerophosphate hydrogel loaded with beta-cyclodextrin-curcumin for the treatment of cutaneous wound infection in rats use of chitosan conduit combined with bone marrow mesenchymal stem cells for promoting peripheral nerve regeneration nanoparticles based on the complex of chitosan and polyaspartic acid sodium salt: preparation, characterization and the use for -fluorouracil delivery receptor mediated gene delivery by folate conjugated n-trimethyl chitosan in vitro exploring advantages/disadvantages and improvements in overcoming gene delivery barriers of amino acid modified trimethylated chitosan surface molecularly imprinted polymer of chitosan grafted poly(methyl methacrylate) for -fluorouracil and controlled release development of hybrid-type modified chitosan derivative nanoparticles for the intracellular delivery of midkine-sirna in hepatocellular carcinoma cells adsorption of acid dyes from aqueous solutions by the ethylenediamine-modified magnetic chitosan nanoparticles comparative study of porous hydroxyapatite/chitosan and whitlockite/chitosan scaffolds for bone regeneration in calvarial defects functional poly (epsilon-caprolactone)/chitosan dressings with nitric oxide-releasing property improve wound healing preparation, characterization, adsorption kinetics and thermodynamics of novel magnetic chitosan enwrapping nanosized γ-fe o and multi-walled carbon nanotubes with enhanced adsorption properties for methyl orange repair and regeneration of lumbosacral nerve defects in rats with chitosan conduits containing bone marrow mesenchymal stem cells skin derived precursor schwann cell-generated acellular matrix modified chitosan/silk scaffolds for bridging rat sciatic nerve gap adsorption of three selected pharmaceuticals and personal care products (ppcps) onto mil- (cr)/natural polymer composite beads application of bioactive coatings based on chitosan for artichoke seed protection in vitro and in vivo evaluation of the chitosan/tur composite film for wound healing applications chitosan induces ca + -mediated programmed cell death in soybean cells acknowledgment the authors are grateful to subbaratnam muthukrishnan for critically reading the manuscript. guo m, jin tz, yadav mp, yang r ( ) antimicrobial property and microstructure of microemulsion edible composite films against listeria. int j food microbiol : - gupta h, velpandian t, jain s ( ) ion-and ph-activated novel in-situ gel system for sustained ocular drug delivery. j drug target : - guzman-villanueva d, el-sherbiny im, vlassov av, herrera-ruiz d, smyth hd ( ) key: cord- -tpm i authors: goodin, douglas g.; jonsson, colleen b.; allen, linda j. s.; owen, robert d. title: integrating landscape hierarchies in the discovery and modeling of ecological drivers of zoonotically transmitted disease from wildlife date: - - journal: the connections between ecology and infectious disease doi: . / - - - - _ sha: doc_id: cord_uid: tpm i changes in landscape and land use can drive the emergence of zoonoses, and hence, there has been great interest in understanding how land cover change and the cascade of ecological effect associated with it are associated with emerging infectious diseases. in this chapter, we review how a spatially hierarchical approach can be used to guide research into the links between landscape properties and zoonotic diseases. methodological advances have played a role in the revival of landscape epidemiology and we introduce the role of methodologies such as geospatial analysis and mathematical modeling. importantly, we discuss cross-scale analysis and how this would provide a richer perspective of the ecology of zoonotic diseases. finally, we will provide an overview of how hierarchical research strategies and modeling might be generally used in analyses of infectious zoonoses originating in wildlife. zoonotic diseases, or zoonoses, are infectious diseases transmitted to humans from animals and may be bacterial, viral, or parasitic in origin. approximately % of the pathogens associated with infectious diseases in humans have originated through spillover from wildlife-e.g., ebolaviruses, hantaviruses, coronaviruses, henipaviruses (jones et al. ; lloyd-smith et al. ; smith et al. ; woolhouse and gowtage-sequeria ) . since , zoonotic pathogens represent the bulk of the outbreaks in human populations in both number ( % versus % by vector-borne pathogens) and diversity (smith et al. ) . the recently reported increase in zoonoses has been attributed to a variety of reasons, although a major driver is changes in land use resulting from the increased demands of human populations on the natural environment through agriculture intensification and deforestation (jones et al. ; woolhouse and gowtage-sequeria ) . land alteration for food production, broadly defined to include both agriculture and pastoral activities, has produced profound changes in the type and structure of the earth's vegetation cover. it has also altered the way humans interact with their environment, for example, in some geographical areas suppressing wildlife-human interaction (e.g., contact with large predators common among hunter-gathers) and instead favoring contact between humans and peridomestic species such as rodents, which are common reservoirs for many zoonotic pathogens. numerous examples of emergent zoonoses have often accompanied land clearance, and hence, there has been great interest in understanding how land cover change and the cascade of ecological effects associated with it are ecologically associated with emerging infectious diseases (mcfarlane et al. ; woolhouse and gowtage-sequeria ) . a persistent question in modeling of pathogen emergence is what is the spatial scale at which these processes occur? how do we measure and integrate the impact of processes that occur at varying and nested spatial scales that result in the observed disease distribution (watts et al. ) ? for example, climate (which can vary in scale from local to continental) and landscape are each associated with disease patterns and have been cited as factors in zoonotic disease outbreaks. land cover change and land-use intensification often occurs at finer scales than climate and can thus be thought of as nested within the climate system. at still finer scales, population dynamics and habitat interactions of the pathogen and its reservoir communities (and, indeed, with the human populations vulnerable to disease transmissions from these communities) occur within the climate and landscape scales and are influenced, but not necessarily determined, by them. this hierarchical nesting ( fig. . ) of these processes complicates the overall study and modeling of the spillover and transmission dynamics of wildlife pathogens to human populations. however, there are existing bodies of theory which can help shed light on these processes. hence, we review some of these conceptual ideas, focusing especially on landscape epidemiology, a body of theory first developed in the s and more recently updated to include modern tools and techniques for studying both disease and environmental process (ostfeld et al. ) , and hierarchy theory, a framework which incorporates the idea of multiple, nested spatial scales (allen and starr ) . in the following, we will review how a spatially hierarchical approach can be used to guide research into the links between landscape properties and zoonotic diseases. this will show how landscape concepts have been used to analyze the occurrence and spatial patterning of zoonotic diseases and how many of these studies are being conducted at particular spatial scales. this review will be followed by an illustration of how an integrated, cross-scale analysis might be employed to gain a deeper understanding of the ecology of zoonotic diseases, using an example from our own research. as earlier noted, methodological advances have played a role in the revival of landscape epidemiology. we will therefore discuss the role of methodologies such as geospatial analysis and mathematical modeling-again using examples drawn from our current research. we will show how cross-scale analysis might be employed to gain a deeper understanding of the ecology of zoonotic diseases. finally, we will provide an overview of how hierarchical research strategies and modeling might be generally used in analyses of infectious zoonoses originating in wildlife. landscape epidemiology is the study of spatial patterns of disease and disease risk arising from underlying environmental causes. the fundamental concepts of landscape epidemiology, first proposed by evegeniĭ pavlovskiĭ, stem from the idea that the spatial occurrence of disease could be understood by studying landscape and environmental factors associated with the disease (pavlovskiĭ ). pavlovksiĭ's ideas have undergone a revival, stimulated in part by widespread availability of geospatial data, analysis tools, and models. in particular, satellite remote sensing data analyzed within a geographic information systems (gis) framework has equipped landscape epidemiologists with a powerful suite of tools for analyzing environmental patterns associated with disease occurrence. landscape epidemiology has also benefited from the theoretical perspective of landscape ecology, another relatively new discipline that attempts to understand the relationship between spatial pattern and ecological process (meentemeyer et al. ) . for example, landscape fig. . illustration of hierarchical nesting of possible key components in pathogen emergence in wildlife. each process outlined with a "box" represents a distinct spatial scale to consider in developing models epidemiologists have made use of ecological concepts such as fragmentation to analyze disease vectors (brownstein et al. ; reisen ). one aspect of landscape ecology that remains relatively unexplored in infectious disease applications is the concept of spatial hierarchy. hierarchy in ecology is a multifaceted theory incorporating elements of nonlinear dynamics and complexity; for a comprehensive treatment of hierarchy theory, see allen and starr ( ) . the fundamental concept of hierarchy theory is that processes occurring at finer scales (i.e., "lower" in the spatial hierarchy) are constrained by processes at higher levels. hierarchical levels can also be distinguished by the rates at which ecological processes occur-faster at finer scales, slower at coarser ones. hierarchy theory in ecology arose as a response to the need for a rigorous method of handling middle-number systems, that is, systems whose components are too few to treat statistically but too many to address with classical newtonian mathematics. hierarchy provides a framework by which these middle number systems can be decomposed into a series of manageable units, whose environmental drivers can be characterized by the scale (and thus the rate) at which they occur. such a framework is amenable to the study of landscape epidemiology, since the linkages between environmental factors and disease are often multivariate, nonlinear, and not confined to a specific spatial scale. spatial scale is a crucial aspect of hierarchy theory. any discussion of the relationship between ecological process and scale therefore must first define how the term is used and to what characteristic dimensions these terms apply. these definitions are complicated by the number of ways that the term "scale" is used. scale is frequently referred to by descriptive adjectives such as "large" or "small," which meentemeyer ( ) noted can have opposing meaning depending on whether one is referring to cartographic scale (where small scale refers to less spatial detail) or ecological scale, where smaller scale equates to smaller spatial area and greater detail. ecologists generally distinguish between the grain and extent of a process, where extent refers to the area over which a process occurs, and grain denotes the smallest resolvable component of the process. typically, grain can be described as either "fine," indicating small resolvable elements, or "coarse," indicating larger elements. thus, we might characterize the scale process like tropical deforestation as occurring at relatively large extent, because a large area is affected, but at relatively fine grain, because the individual deforested units can be quite small. csillag et al. ( ) argue that for ecological uses of scale, it is preferable to adopt specific terminology to distinguish between extent and grain. in hierarchy theory, spatial extent is probably the more commonly used sense of scale (jenerette and wu ) . in describing spatial hierarchies, it is often useful to apply descriptive names to realms of scale. terms such as "global," "continental," "regional," and "local" are often used, although the precise areas referred to often vary. for our review of hierarchy in infectious disease analysis, we will operationally define continental scale as areas exceeding km , regional scales as ranging from km - km , local scale from - km , and microscale < km . the lower end of the microscale represents the general size range of a small rodent's world. landscape analyses across spatial scale frequently use remote sensing data (kitron et al. ; wu ) . currently, imagery is available from an array of orbital sensors with widely varying spatial, temporal, and spectral resolutions (table . ). grain size (or resolution, as it is more commonly termed in remote sensing) is an engineered property of these sensors, dependent on the optical characteristics of the sensor and orbital characteristics of the platform. theoretically, there are no limitations to the extent of any of these systems, although practical limitations (particularly cost of data acquisition) dictate a rough correspondence between extent and resolution. thus, the extents listed in table . are based on these practical limitations. the variety of remote sensor data available has certainly facilitated hierarchical landscape analysis, but in some sense, it has also imposed limitations. each remote sensor represents a "window" through which ecological process at some combination of grain size and extent can be observed. these limitations also extend to the range of spectral wavelengths each sensor can detect and the number and width of bands in which these wavelengths are detected. combining these discrete views into an integrated picture is a central challenge for hierarchical analysis of infectious disease processes. there is a large body of work relating infectious disease to environmental factors. most of these studies have concentrated on a single class of causative factor operating over a characteristic spatial scale. in the following, we will briefly review several examples which represent different classes of landscape factors that have been shown to impact zoonotic viral emergence. since our intent is to show how landscape spatial hierarchies influence disease processes, we will concentrate on examples that show the influence of landscape and land cover processes at various spatial scales, using the concept and terminology of scale developed in the previous section. for convenience, we will group the literature reviewed here into two categories. first, we will review the relationship between land cover disturbance (including anthropogenic and natural disturbance) and zoonotic disease emergence. included in this category are causes due to landscape structure, including natural landscape barriers, land cover change and disturbance, and fragmentation. this category also includes disturbance due to agricultural practices. the second class of landscape impacts that we will review are climate-driven landscape changes, which include climate-influenced changes in vegetation phenology patterns as well as persistent or transient superficial changes such as flooding. processes contained within these two categories are not exclusive; however we will review them based on their predominant process. landscape structure refers to the pattern and arrangement of habitats on the earth's surface. like all landscape variables, structure is complex. it varies with scale and per organism. the same landscape might be structurally very different for birds, small mammals such as rodents, and larger animals, and since zoonotic disease reservoirs vary in their body size and habitat, structural effects on disease also vary. structure can arise naturally due to topographic, edaphic meaning related to the soil, or climatic conditions, as well as through human landscape alteration. both types of structural changes can be relevant to disease processes. landscape structure has been related to zoonotic disease at a variety of spatial scales. at regional scale, russell et al. ( ) show how natural landscape barriers such as rivers and preferred habitat can limit rabies spread by raccoons and be used to manage wildlife through vaccination efforts. smith et al. ( ) used landscape heterogeneity as a predictor of the spread of rabies. langlois et al. ( ) showed that the distribution of hantavirusbearing rodents in north america was influenced by landscape fragmentation. their analysis was unusual in that the extent of the study was continental, but the grain size was local ( km). estrada-peña and oteo ( ) showed that landscape structure, particularly landscape connectivity, showed a strong influence on the abundance of lyme disease vectors in spain. land cover changes are known to affect zoonotic diseases through controls on the population dynamics of reservoir species (especially wild mammals) as well as disease vectors (patz et al. ) . giraudoux et al. ( ) used regional-scale land cover changes in france and china to show how host mammal communities affect transmission dynamics of the endoparasite echinococcus multilocularis. using the rompa (ratio of optimal to marginal patch area) hypothesis of lidicker and others, it has been shown that regional-scale landscape dynamics of intermediate host species can in turn affect parasite egg survival and transmission (lidicker ) . the giraudoux et al. ( ) study also considered the role of landscape change on establishing minimum thresholds, which they termed "filters" or "screens" of suitability for disease transfer. agriculture practices led to the nipah virus outbreak in malaysia (epstein et al. ; pulliam et al. ) . using a combination of field, laboratory, and modeling approaches, these efforts have supported the hypothesis that emergences of viruses such as nipah are due to ecological and not evolutionary drivers. these findings underscore the importance of having multidisciplinary teams work together to build predictive models for discovery of the relationships between anthropogenic environmental change and the transmission or spillover of infectious agents. climate-driven landscape change in this context refers to the effect of atmospheric processes (most notably precipitation) on the habitat of zoonotic host organisms. variations in rainfall magnitude and frequency have notable effects on vegetation phenology, causing variations in surface greenness that can be tracked using remotesensing instruments (de beurs and henebry ; reed et al. ). the emergence of several zoonotic diseases including hantavirus pulmonary syndrome (hps) , argentine hemorrhagic fever (simone et al. ) , and bolivian hemorrhagic fever (kilgore et al. ) can be clearly linked to landscape. several studies have linked climate-driven changes to patterns of disease occurrence at different spatial scales. two groups have evaluated the relationship between temporal patterns of normalized difference vegetation index (ndvi) and occurrence of ebola virus in west africa (pinzon et al. ; tucker et al. ) . they found that ndvi trajectories showed distinctive "trigger events" prior to occurrences of the disease in humans and apes, which they hypothesized might be used to forecast conditions conducive to outbreaks of ebola hemorrhagic fever. the remotely sensed data for this analysis came from the noaa-avhrr sensor, with continental spatial extent and observation grain size (pixel resolution) of km . the observed ndvi trajectories were related to precipitation patterns, reinforcing the link between climate and disease occurrence. estrada-peña and oteo ( ) and estrada-peña et al. ( ) used coarse resolution vegetation index data to model and predict the continental-scale relationship between climate-driven landscape change and lyme disease. again, the resolution and extent of this study were consistent with the idea that climate constrains disease processes at higher levels in the spatial hierarchy. at finer spatial scales, glass et al. ( ) used patterns of reflectance in landsat thematic mapper (tm) data to statistically model the presence of hps in the southwestern united states. at this spatial scale, the spectral response of the surface incorporates climatic factors (especially antecedent precipitation) but also integrates structural and compositional factors of the vegetation canopy itself. models based on these techniques have shown some utility for predicting hantavirus cases (glass et al. ) . one of the important advantages of a hierarchical approach is that it allows a multifactorial explanation for the occurrence of the infection and disease. the environmental, landscape, and climatic processes each contribute to processes that may alter species interactions within their habitat. these extrinsic factors can alter the reservoir population dynamics, drive extinction, and affect maintenance (persistence) of the microorganism. essentially, these factors can create constraints within their spatial scale and across scales. in this section, we explore an example of hierarchical observation that can be used in conjunction with modeling to test hypotheses regarding the effects of environment, landscape, and climate upon zoonotic pathogen distribution. for this we draw upon our work and others in the study of hantavirus with a focus on south america palma et al. ). hierarchy theory suggests that processes at coarser grain also occur over longer time frames and can often be assumed to be static with respect to finer-scale processes. the phylogeographical patterns of south american hantaviruses within the southern cone ("el cono sur," a subcontinental region roughly defined as consisting of the country argentina, often including chile, plus sometimes considered to include uruguay and paraguay) mapped with environment at the coarsest spatial scales . in other words, the phylogenetic clades of hantaviruses from the southern cone of south america appear tied to coherent spatial patterns consistent with subcontinental-scale biogeographic features such as the major biomes ). in fig. . , the locations of strains from three major subclades of south american hantaviruses are shown in the context of the major biomes based on the world wildlife fund terrestrial ecoregions data (olson et al. ) . for example, we find members from one subclade (i.e., laguna negra, rio mamoré, and alto paraná viruses) carried by rodent reservoirs that span the tropical grass and shrubland and dry broadleaf forest areas along the western, central regions of south america johnson et al. ; richter et al. ; yahnke et al. ) . in contrast, a second subclade of rodents that harbor jabora, maporal, and necocli hantaviruses inhabit mainly the moist broadleaf forest biome stretching from venezuela and colombia into paraguay de oliveira et al. ; fulhorst et al. ; londoño et al. ) . knowing the extent of prevalence of these closely related viruses over this vast region would certainly be fascinating from a phylogeographical point of view. it is interesting that these viruses are not yet associated with cases of hps. there are numerous strains of hantaviruses that have been identified in the atlantic forest (extends along the eastern coast of brazil and into eastern paraguay) and in the temperate grass and shrubland of argentina. the third clade of rodents, those that harbor the juquitiba, oran, and lechiguanas viruses, resides in the more humid lower chaco (a region that encompasses the flooded savannas of southern paraguay and forms a transitional environment between the arid gran chaco) and temperate coniferous forests in argentina, brazil, and uruguay (de araujo et al. ; delfraro et al. ) . andes, maciel, and pergamino viruses reside in rodents in the temperate grass and shrubland biome in argentina (bohlman et al. ; gonzález-ittig et al. ) . andes virus is also found in chile in mediterranean woodland and shrub biome (torres-perez et al. ) . araraquara viruses are associated with rodents that largely reside within agriculturally transformed areas of brazil (i.e., sugarcane production fields) that were formerly areas of moist broadleaf forest biome (de araujo et al. ; de sousa et al. ; suzuki et al. ). coarse-scale analysis has shown a relationship between hantavirus genetics and broad ecological pattern. narrowing this view to a more regional scale begins to reveal how land cover (and land cover disturbance) affects the spatial variability of hantaviruses. like all zoonotic disease, the ecology of each species of hantavirus is closely related to that of its host organism; thus, generalization of virus-landscape relationships cannot be made without considering the habitat characteristics of the reservoir host. we do not have abundant data on the microhabitat characteristics of many host species (lozada and guthmann ) . however, general habitat types defined at grains sizes of~ km might be useful to study preferences among various (olson et al. ) . selected strains of closely related hantaviruses are presented from three distinct subclades as indicated by the color of the yellow or pink dot at their location. the viruses represented by the pink dot and pink with yellow represent two distinct lineages from one subclade host species, and be relevant to the landscape epidemiology of hantaviruses, regardless of whether certain land cover types are more closely associated with the presence of the virus. a regional-scale analysis of rodent reservoirs of hantaviruses in paraguay showed that the host species do indeed show patterns of land cover preference, even when land cover is mapped into very general categories. the most common hantavirus host rodents in paraguay, akodon montensis and oligoryzomys spp., both showed disproportionately high probabilities of occurring in areas subjected to large-scale agricultural disturbance . even more significant, however, was the fact that rodents found to be antibody positive for hantavirus (indicating exposure to the virus at some point) were more likely to be associated with the human-disturbed land cover types. this relationship held even when the underlying differences in habitat preference were controlled. this finding suggests that some aspect of land cover disturbance or change increases the likelihood that a member of a host species will be infected with the virus. while analysis at the regional scale shows that human land cover alteration is associated with hantavirus presence, discovering the specific nature and causes for this observation cannot be addressed at this scale. questions of causation must be addressed at finer spatial scales, in large part because that is the scale to which most host populations and individuals are interacting with their environment (lozada and guthmann ; owen et al. ). simply presumed, because rodents species have specific requirements with respect to habitat, and because each hantavirus species seemingly persists only in certain rodents species, one might assume that a predictive map can be made for of the prevalence of a particular hantavirus based on knowledge of the associated rodent species. unfortunately, for most rodents, we know very little about their general life cycles and other important biological information that is critical for modeling (e.g., age at sexual maturity, birth rates, litter sizes). further, we have only a minimal amount of information on their habitat preferences, although some recent studies have indicated that fine-grained evaluations are necessary to understand rodent species distributions and community composition lozada and guthmann ; poindexter et al. ; schnell et al. ). at the microscale level, pathogen survival and reproduction depend on the dynamics within the reservoir host, which in turn depend on the habitat or the reservoir. a rodent may live within an approximate km range for most of its life barring fire, flooding, and other natural disasters. in the case of a viral pathogen, the virus must overcome physical barriers of the host and be compatible with cell receptors to gain entry into a target cell (allen et al. ). viral replication depends on host and viral genetics and other host factors such as prior pathogen or other immunogenic exposure, nutritional status, coinfection, age, sex, reproductive status, and the host immune response (allen et al. ). these and other factors determine outcome when a pathogen enters a host, with the possibilities including severity of any associated disease and whether the pathogen either is to be cleared by the host immune system, or the pathogen will persists in the host. for example, the survival of hantaviruses in nature depends on maintenance of persistent infections within their specific rodent reservoir. hantaviruses infect and persist only in the rodent reservoir with which the virus has coevolved, and the infection is believed to last the life of the animal (meyer and schmaljohn ) . notably, persistent infection of rodent reservoirs by hantaviruses shows continuous virus replication, without complete clearance by the immune system, and no pathological changes vaheri et al. ) . humans are not a natural reservoir for these viruses, and as such humans typically become infected only upon contact with aerosolized excreta from the rodent reservoir. in humans, hantavirus infection can result in severe disease although outcomes vary with different hantaviral species. the molecular basis for different disease outcomes in humans has been attributed to difference in receptor preferences of nonpathogenic and pathogenic hantaviruses. models that connect data on the outcomes associated with immune response in reservoir versus human hosts are just beginning to be developed. mathematical models are valuable tools for synthesizing information and testing hypotheses to provide insight into how and why disease outbreaks or spatial patterns of infections might arise in wildlife populations. several books and review articles summarize some of the modeling efforts on zoonotic infectious diseases (alexander et al. ; allen et al. ; grenfell and dobson ; heesterbeek et al. ; hudson et al. ; lloyd-smith et al. ) . a variety of modeling formats, deterministic and stochastic, have been applied to the study of zoonotic diseases. these models include compartmental, agent-based, individual-based, metapopulation, network, and ecological niche, but these classifications overlap. agent-based, individual-based, and metapopulation models may be classified under network models, where the nodes are infectious agents, individuals or populations, connected via an underlying network (riley et al. ) . simple compartment models (seir-susceptible, exposed, infectious, and recovered) are connected via a dispersal network in what is often called a patch model or metapopulation model (allen et al. ; arino et al. ; mccormack and allen b) . ecological niche models contain less detail about individual dynamics, and instead they are closely related to landscape, presence/absence data, and gis-based climatic and environmental data (alexander et al. ; peterson ) . mathematical tractability and the complexity of interactions among pathogens, reservoirs, and human hosts and the environment have often restricted the model formulation to the reservoir host and to a single spatial scale-landscape, population, or within-host. coupling temporal scales (hours to years or longer), biological complexity levels (genes to cells to ecosystems), and spatial scales (local to global) have been a continuous challenge to modelers . recent theoretical investigations on coupling within-host and between-host models are advancing (feng et al. ; gilchrist and coombs ; mideo et al. ) . hantaviruses and rabies are two examples where spatial patterns of infection involving multiple species have been observed haydon et al. ; rhodes et al. ; smith et al. ) . spatially explicit computer simulations that incorporate landscape heterogeneity and spatial genetic structure are being applied to study control of rabies and other zoonotic diseases (alexander et al. ; parratt et al. ; real and biek ; rees et al. ) . models with multi-host species and multi-pathogens are being investigated. for example, mathematical models for hantavirus infection in rodents have been studied in the context of multiple host species, spatial spread, and environmental variability (abramson and kenkre ; abramson et al. ; allen et al. a allen et al. , b, mccormack and allen a, b) . these models have shown that those random or seasonal variations which impact an ecosystems carrying capacity for a particular rodent species can trigger outbreaks when rodent densities and contacts rates are high (allen et al. b ). in addition, theoretical analyses have shown that when multiple species, as opposed to a single species, are involved in the transmission process, there may be a dilution or amplification effect that impacts disease persistence (dobson ; mccormack and allen a) . models for spatial spread among discrete patches have shown the importance of there being at least one patch where the disease persists (arino et al. ; allen et al. ; mccormack and allen b) . wu and loucks ( ) have suggested that the most significant contribution of hierarchy theory is as a framework for explicitly incorporating heterogeneity and scale into ecological analysis. in this chapter, we have tried to show some ways in which hierarchical consideration of spatial (and to some extent, temporal) scale can be incorporated into ecological analyses of zoonotic diseases. our review of the literature also suggests some of the opportunities in infectious disease research resulting from the hierarchical consideration of scale but also some of the challenges. spatial patterns of zoonotic hosts, the pathogens they harbor, and the host-zoonosis relationships may appear quite different depending on the scale at which we view them. although patterns derived from different size scales are often referred to as "emergent" patterns, it is unclear whether the newly recognized large-scale or the small-scale patterns might not be emergent from scale levels either above or below them. although we could view these scales to be nested subsets of one another, it may be more useful both conceptually and in practice, to view the patterns derived from different scales as complementary information requiring integration, rather than as contradictory results requiring amelioration. one area of opportunity for the application of hierarchical concepts in zoonotic disease ecology corresponds to scale-related questions facing the global change research community in general; how does global environmental change, especially global warming, manifest itself spatially? it has long been recognized that the spatial distribution of both individuals and communities of species is linked to climate and that these are two-way linkages; however much uncertainty remains about where, when, and how species will respond to climate change (potter et al. ) . fundamentally, the issue reduces to one of scale; the potential impacts of climate change are most often conceptualized at the macroscale (i.e., regional or global) but operate across a range of scales including those more proximate to the host or reservoir organisms (ashcroft et al. ; diffenbaugh et al. ; suggitt et al. ) . adoption of the complementary, hierarchical view of scale provides a framework to address these and similar questions in disease ecology. model selection among the wide array of potential formats depends on the virushost ecological system being investigated, data availability, and the questions to be addressed. many challenges remain in model formulation, analysis, and simulation of zoonotic disease dynamics that relate to landscape and climate and the wide range of temporal and spatial scales (allen et al. ; buhnerkempe et al. ; heesterbeek et al. ; lloyd-smith et al. ; pellis et al. ) . addressing the challenges of scale can be met through mathematical and computational approaches and methods being developed in a variety of fields including computer science, ecology, geography, immunology, genetics, mathematics, statistics, and virology. this will require the continued close collaboration across numerous disciplines to converge toward models that reflect the immense biological diversity of pathogen-host ecology. spatiotemporal patterns in the hantavirus infection traveling waves of infection in the hantavirus epidemics modeling of wildlifeassociated zoonoses: applications and caveats hierarchy: perspectives for ecological complexity mathematical models for hantavirus infection in rodents the impact of environmental variation on hantavirus infection in rodents a habitat-based model for the spread of hantavirus between reservoir and spillover species mathematical modeling of viral zoonoses in wildlife a multi-species epidemic model with spatial 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the emergence of nipah virus: a lethal bat-borne zoonosis spatial dynamics and genetics of infectious diseases on heterogeneous landscapes remote sensing phenology. in: noormets a (ed) phenology of ecosystem processes: applications in global change research modelling the effect of landscape heterogeneity on the efficacy of vaccination for wildlife infectious disease control landscape epidemiology of vector-borne diseases rabies in zimbabwe: reservoir dogs and the implications for disease control geographical range of rio mamoré virus (family bunyaviridae, genus hantavirus) in association with the small-eared pygmy rice rat (oligoryzomys microtis) five challenges for spatial epidemic models spatial control of rabies on heterogeneous landscapes habitat use and demographic characteristics of the west mexican cotton rat (sigmodon mascotensis) environmental determinants of the small mammal assemblage in an agroecosystem of central argentina: the role of calomys musculinus predicting the spatial dynamics of rabies epidemics on heterogeneous landscapes global rise in human infectious disease outbreaks habitat microclimates drive fine-scale variation in extreme temperatures identifying rodent hantavirus reservoirs peridomestic small mammals associated with confirmed cases of human hantavirus disease in southcentral chile climatic and ecological context of the - ebola outbreaks uncovering the mysteries of hantavirus infections (disease/disorder overview) multiscale, resurgent epidemics in a hierarchical metapopulation model host range and emerging and reemerging pathogens hierarchy and scaling: extrapolating information along a scaling ladder from balance of nature to hierarchical patch dynamics: a paradigm shift in ecology patterns of infection with laguna negra virus in wild populations of calomys laucha in the central paraguayan chaco the ecology and evolutionary history of an emergent disease: hantavirus pulmonary syndrome acknowledgments cbj and ljsa acknowledge the support of the national science foundation grants (nsf) dms- and dms- . cbj and rdo acknowledge the support of the national institutes of health grant (nih) i . cbj, ljsa, rdo, and dg acknowledge support from nih r tw - through the nih-nsf ecology of infectious disease program. rdo was partially supported by the programa nacional de incentivo a los investigadores (conacyt, paraguay). funding this study was funded by national science foundation grants (dms- and dms- ) and national institutes of health grant (r ai ). this work was supported by a grant from the fogarty international center (r tw - ) under the nih-nsf ecology of infectious diseases initiative.conflict of interest douglas g. goodin declares that he has no conflict of interest. colleen b. jonsson declares that she has no conflict of interest. linda j. s. allen declares that she has no conflict of interest. robert d. owen declares that he has no conflict of interest.ethical approval this article does not contain any studies with human participants or animals performed by any of the authors. key: cord- -r en s authors: watanabe, chiho title: health impact of urban physicochemical environment considering the mobility of the people date: - - journal: health in ecological perspectives in the anthropocene doi: . / - - - - _ sha: doc_id: cord_uid: r en s most of the current environmental health researches assumes that exposure to the environmental agents occurs either in the residence or workplace, neglecting the mobility of the people due to commuting and daily activities. mobility of the people varies in terms of spatial and temporal range, that is, from momentary short ones to generation-scale long ones. focusing on the daily movement of the people, various methods for grasping the mobility, which also range from simple observational methods like time allocation to methods with advanced technology like global navigation satellite systems, will be reviewed. referring environmental health studies examining the health effects of either air pollution or heat, importance of the mobility of the people is discussed. assessing the mobility will open a new research avenue for the study of infectious diseases as well as noncommunicable diseases. depending on the media (most of the case, relevant media should be food, water, air, soil, etc.) as well as the agent of concern, a variety of the methods exists to evaluate the exposure, and exposure evaluation has been recognized as an independent research field. the procedure for exposure evaluation can be largely classified into two categories, that is, measuring environment and measuring organism; these are the procedures called as environmental monitoring and biological monitoring. depending on the media and the agent, one or both of these methods are utilized. for example, both (organic) mercury from fish and cadmium from rice are heavy metals exposed through food consumption. therefore, by monitoring the concentration of these metals in respective major food source, combined with food consumption data, exposure (ingested amount of mercury/cadmium) could be estimated. biological monitoring for these metals is also possible by measuring the concentration of mercury in the hair (which reflects the concentration in the blood, and approximates the concentration in the brain) or cadmium in urine (which reflects the concentration in renal cortex). detailed discussions about these two categories of monitoring are beyond the scope of this book, and interested readers should refer to existing textbooks. unlike the case for arsenic in water or mercury in the fish, many of the major "classical" air pollutants like nox, sox, co, pms (either . or ) do not have appropriate biological exposure markers, and we need to rely on the environmental monitoring. this is also true for the exposure to temperature or to noise, both of which are associated with atmospheric exposure. in conventional environmental health study or environmental epidemiology, environmental monitoring of the outdoor atmosphere at the residential area of the participants has been used as surrogates for individual/population exposure to the agents of concern. implicit assumption here is that people would stay in their residence, and variation of the exposure due to their daily mobility is, if any, negligible. although this assumption is not realistic at all, this approach has been successful in a sense evidenced by the existence of numerous epidemiological findings in this area in the past. in fact, exposure to pollutants or physical factors associated with air/atmosphere is heavily influenced by the location of the individual. assume we would like to estimate an individual's exposure to nitric oxides, most of the case, individuals would not stay in the home whole day, particularly in urban settings, commuting into city center or business districts for workplace or for schooling, thereby exposing themselves to environments that are different from their own residential neighborhoods. in this sense, kwan [ ] has suggested that research involving geographical components should reconsider conventional methods to estimate the exposure, referring to environmental health/epidemiology (together with the research on segregation or on the issue of accessibility). richardson et al. [ ] pointed out that due to the accumulation of highly sophisticated spatial and spatiotemporal technology like gis, gps, remote sensing, and computer cartography, collectively termed as geographic information science, it becomes possible to model the disease process involving multiple spatiotemporal data obtained in different disciplines. likewise, the exposure to environmental factors could be evaluated using such spatiotemporal data. spatial resolution of various types of environmental data becomes so high that heterogeneity within the area of commuting distance could be documented. in addition, recent technical progress in downscaling of the climate models (see [ ] ) enables the researchers to predict differential impact of climate change within relatively small areas. health? apart from the environmental data, to consider the mobility of the people, we need to collect the information on the mobility of people with enough spatiotemporal resolution. mobility of people can mean wide variety of phenomenon in terms of time, space, and context as listed in fig. . . time scale of the mobility can range from moment to moment as exemplified by the ecological momentary assessment (later in this chapter) to years, even including hundreds of centuries (like out of africa, the expansion of our ancestors)! duration of the sojourn time should be considered as an independent factor and can also range from few seconds to generations; for example, workplace exposure to hazardous substances (including radioactive materials) would be evaluated in terms of minutes, while the effects of regional migration fig. . schematic classification of various type of human mobility by "relevant" spatial and temporal scales. for example, "migration' takes place with relatively long time, ranging from months to multiple generations and traveling relatively long distance, while "outdoor/indoor" discrimination is needed even a couple of meters apart. 'ema' stands for ecological momentary assessment (see text) may emerge after several generations. accordingly, spatial scale could range from a few meters to thousand kilometers. a few meter matters when micro-scale environments such as indoor (including air-conditioning) vs. outdoor, inside and outside of vehicle are considered, for example. at larger scale, proximity to major roads or any combustion facility could exert significant influences on exposure to noise or air pollutants. far larger scale can change the environment as a whole; an important aspect in today's environmental health is that people can make a global scale travel within h. mobility of people also entails the change in the context; by changing the location, the socioeconomic and cultural aspects of the neighborhood would change, which may affect the meaning of material environment and, in turn, health status of the moving individuals. at the same time, change in location means the change in activities of the individuals; for example, individuals working at outside road construction site might have much higher physical activity levels (and increased ventilation rate) and higher exposure to heat, dust, and noise than staying at home. while it is fairly difficult to consider all of these potential factors, in case of atmospheric exposure, geographical location of the individual should be the factor to consider in the first place, since it does provide the basis of the air which she or he inhales in every moment. in this chapter, main focus will be on daily mobility of people for commuting and for other daily life activities. geographical location is crucial for some physicochemical environmental factors other than air pollutants. one such example is heat environment, which consists of temperature, humidity, air flow, and radiation, and varies even with a very small scale, reflecting the variation of land use or land cover, local topography (layout of surrounding buildings, for example), or elevation. in addition, numerous heat sources are found in human-built environment, including exhaust gases, waste heat, etc. as a result of combined effects of these factors, most of the urban centers have warmer environment, compared to the surrounding areas, termed as heat island. mobility of individuals has been dealt with in many research areas. in urban planning, grasping mobility of individuals is crucial to create an appropriate layout for transportation, public space/facilities, and private houses. in public health, mobility of individuals sometimes play key role in the spread of diseases (described later). also, mobility has been one of a classical topic in the area of human ecology since it is associated with the question of how a population utilizes the environment spatially as well as temporally (time allocation studies). mobility is also associated with energetics (utilization of somatic energy) as a part of physical activities in general. as such, many methods to grasp the mobility of individuals have been developed (table . ), which will be discussed below. the simplest method is field observation. time allocation studies observe the individuals in the targeted field and record the location and type of activity for a given period, which is useful to answer some of the basic questions in human unlikely large ecology or other related fields as noted above. while this method is in a sense "perfect" since the observer can obtain whatever details she or he wants, obvious disadvantages include time-labor intensiveness, biased behavior due to the presence of observing researcher. a simplified variation of the time allocation is the spot-check method, in which researcher will observe the people's activity in certain fixed location(s) (e.g., see [ ] ). despite its simplicity and easiness, spot-check method could provide valuable quantitative information. activity diary is another classical method, which relies on the self-recorded diary. this would solve the issue of laborintensiveness for researcher and could cover much larger number of individuals, but like other self-reporting methods, reliability and accuracy of the record are the main problems to be considered. activity diary is useful when researchers are interested in qualitative aspects of the moving behavior. person-trip is another method, which has long been utilized especially urban planning as well as urban studies (e.g., see ( [ , ] )). person-trip uses a predetermined, formatted questionnaire to be either self-recorded or recorded by interviewers, through which location of the individual, purpose of travel (translocation), and method of travel (either walk, bicycle, private car, public transportations) for a given period will be obtained. while this method has problem of bias and/or inaccuracy due to recall, brevity and simplicity made this method popular, particularly when a large population needs to be covered. many governmental (both national and local levels) surveys utilize this method to quantify the volume of mobility, for example, traffic volume of vehicles. gps (global positioning system) has been used as if it was a generic term, but actually gps is a name of a system developed in usa. generic name for the systems is global navigation satellite systems (gnss), which is referring to any system that locates specific targets by use of the combination of signal-detecting device and a group of satellites. in this book, the "targets" are basically human individuals, but they could be animals (so-called activity logger) or traffic vehicles. spatial resolution of contemporary gps is fine enough to locate individuals for many study fields, and it solves the issues of time-labor intensiveness and false or biased report. thus, this has been used in various research areas including anthropology, human ecology, and presumably sociology, and urban studies (see [ , ] ). another advantage is that gps uses electrical data processing, which also alleviates the risk/errors associated with data transfer. on the other hand, in some area like inside building or underground malls the signal for gps are generally weak and difficult to detect. although the gps could provide very rich and useful information regarding the mobility, individuals rarely own the device, and researchers need to provide and distribute it to the participants. this has been overcome by the recent propagation of gpsequipped mobile phones and alike. mobile phone per se can be also utilized as a tool in capturing the location of individuals without using gps device since every call made by mobile phone is registered by nearby relay station, hence generating a record regarding approximate location of the mobile phone users at the time of the call. by collecting such records of local calls, researcher can trace or reconstruct the translocation of individuals. major advantage of this method is the fact nowadays a large proportion of populations own the mobile phone even in developing countries and in very remote areas. on the hand, researchers need to negotiate with mobile phone company to obtain such record, and the major barriers for such negotiation are as following: ( ) conflict with the protection of privacy information (even when the information is provided in anonymous manner) may arise, ( ) often there are two or more mobile phone companies operating in a given area, and ( ) since the information is provided in anonymous manner, demographic parameters of the mobile owner are not known to the researcher. effort has been made to overcome the last point, in which an algorism has been developed to estimate the demographic attributes of a mobile phone owner through the pattern of mobility, although the feasibility as well as the accuracy of estimated attributes needs further investigation (arai, shibasaki, in preparation). wesolowski et al. [ ] compared the mobility data obtained by a person-trip type survey with the one obtained by mobile phone; both conducted in kenya over the same period. reflecting the nature of the methods, numbers of the participants were and , in the survey and mobile phone analyses, respectively. while the person-trip type survey was cross-sectional in nature, the mobile phone records followed the movement of the people for months. while the resultant two data sets agreed in some aspects of the travels such as ( ) most visited areas (in terms of districts) or ( ) overall relative frequency of individuals with different number of travels, they disagree other aspects such as the number of mobile phone subscribers in the area as much as ten times. in this section, a couple of examples will be presented that incorporates the mobility of the individuals/groups in estimating exposure to physicochemical factors in the air. as the physicochemical factors, air pollutant and heat will be discussed. in the final part, a rather classical, different approach to trace individual exposure will be also introduced. a relatively large spatial scale study has been conducted covering approximately × km area in belgium [ ] , which compared regional exposure estimates for two representative air pollutants, nox and ozone, under two alternative assumptions. first, exposure assumed to occur in the residential place of the participants (static exposure), that is, the mobility of the participants is neglected. second, mobility of the people was taken into account in estimating exposure (dynamic exposure). information on mobility was obtained through activity diaries collected from residents, which is then extrapolated to a synthetic (but reflecting the demographic structure of actual) population of approximately millions. the target geographical region consisted of zones ( municipalities) whose average area was km , and the location of each individual has been predicted for week ( days) by -hr interval. pollutant concentrations are estimated for no and ozone using air pollution models (i.e., an emission model combined with a dispersion model) for a year by h interval and × km resolution with finer resolution along major roads. based on the location data and pollution data, time-weighted exposure estimate was calculated under two conditions: with and without taking peoples' mobility into account. to estimate the municipality-wise health impacts of the exposure to these air pollutants, the time-weighted exposure estimate was converted into the respiratory mortality using the information of existing epidemiological data, which is then converted into years of life lost (yll) following the burden of diseases framework by who. the analyses revealed the pollutant-specific regional difference in the pollutant concentration; that is, for no , urban zones had higher concentrations than rural zones regardless the age and gender, and for ozone, it was vice versa. as expected, urban and industrial zones have much larger population in daytime, which was in contrast with the surrounding zones. as the results, dynamic exposure for no for the whole population was slightly higher than static exposure, while for ozone it was vice versa. while the difference between static and dynamic exposure were statistically significant, the difference was small and reached only up to %. in terms of health impact, again the difference between the two methods was statistically significant but small ( . % increase for no and . % decrease for ozone). at the municipality level, the maximum difference between the two approaches reached as large as %, where dynamic was higher than static, and larger differences were usually observed in rural areas. for ozone, maximum difference was only % (dynamic was lower than static). while the extent of impacts shown in this study might not be so remarkable, the results demonstrated that mobility of people could have significant impact on the estimate of health impact by air pollution and that such an impact could be more remarkable at smaller scale. as pointed out by the authors, considering the mobility of people in air pollution issue inevitably connects the issue of transportation (and urban planning) with health issues, which is also commended from the viewpoint of eco-health. similar dynamic-vs-static comparison was conducted with much smaller sample size in western new york, and as naturally expected the difference between the two approaches depended on the pattern of spatiotemporal pattern of air pollutants, pm . , as well as on that of behaviors [ ] . the human early-life exposome (helix) is a multi-country (eight countries), multi-cohort project in europe to characterize early life exposure to various chemical and physical environmental factors and to associate them with health consequences in early life ( [ ] project url: http://www.projecthelix.eu/en). involving , mother-child pairs, the project would try to grasp the whole picture about the exposure as much as possible, and the use of time-space activity information is planned, which will be utilized to estimate the participants' exposure to not only air pollutants but also noise, uv radiation, temperature, and built environment/green space etc. basically, the environmental data is collected from (ground) monitoring stations and/or remote sensing. in addition, smartphone-based "personal exposure monitoring kit" has been developed that enables to capture not only the location of mothers and children but also their physical activities and air pollution by built-in accelerometer and sensors for uv and pm . . recent progress of this project can be found at the following url: https://www.isglobal.org/en/web/guest/healthisglobal/-/custom-blog-portlet/prova/ / . helix is an ambitious attempt, which needs large amount of budget ( . m euro, according to the web page), time, and manpower. considering the nature of current "environmental exposure," that is, long term, multiple species, and mild to moderate (rather than severe) level, probably such an extensive effort is required to elucidate the relationship between environmental agents and health consequences. heat is another environmental factor, which might have some relevance to the mobility issue, since urban heat, or heat island, is a ubiquitous phenomena common to most of the big urban areas, which would pose additional heat burden to urban dwellers as well urban commuters under the influence of climate change (global warming). usually, effects of heat are considered to be immediate or short, which is different from those of air pollutants, whose effects can be both short term and long term. although not involving mobility assessment, laaidi et al.'s study on the heatmortality relationship [ ] is worth to be discussed here. this study analyzes the relationship between all-cause mortality among the elderlies living in paris, france, or nearby area and land-surface temperature captured by satellites for a period of a heat wave occurred in august . based on a case-control study of pairs of mortal-alive elderly people (age > ), they found elevated odds ratio of mortality with increased land surface temperature (lst) of the residence area. of noteworthy, the elevated odds ratio was associated with minimum (night time) lst averaged over either days (whole observation period) or days preceding the reported deaths but not with any lsts averaged over days preceding deaths or the day of death. this result suggested that the effect of heat might not be limited to immediate effects but might be "cumulative" to some extent. also, approximately . °c of increase in temperature was associated with a significantly elevated odds ratio more than two, showing relatively potent effect of temperature on mortality. in this study, spatial resolution of the lst was km , and the case and control are matched, in addition to age and sex, for residential area, which contains - pixels, allowing the temperature comparison between the case and control. targeting the elderlies, mobility would be less important than in younger generations. we have conducted a study in which mobility of the people is considered in heat exposure issue in a subtropical urban area. this particular issue will be discussed in the next chapter. in the area of industrial health, exposures of the factory workers to air-borne chemicals peculiar to the factory are monitored with device, which is "worn" by each worker. many types of such devices have been developed for various solvents or gaseous pollutants, among which the γ-radiation monitor is the best known. cumulative exposure of each individual to the target chemical/radiation is quantified by analyzing the amount of chemical collected/absorbed by the device. such personal device has been used for appropriate control of worker's exposure to hazardous chemicals, but which could be extended into surveys in general population. while this method provides the estimate of individual exposure, if used in general population, distribution and retrieval of the device could be labor taking, and the quantified results would not give any hint of the potential sources of the exposure, since it only provides the cumulative exposure rather than temporally tracing the individual exposure. potential use of mobility information would not be confined to the issues that have been discussed in this chapter so far. these examples will be discussed in this section. many infectious diseases are transmitted through direct or indirect human-to-human contact. mobility information has been utilized in the development of the models for propagation of some of the infectious diseases like influenza (direct) or malaria (indirect). in developing models, however, most of the attempts have based on simulations under plausible assumptions about the parameters, and not so much have been done using actual mobility data. malaria is one of the diseases that will be propagated by mosquitoes (indirect human contact). propagation of the malaria agent (plasmodium) occurs when a mosquito (anopheles) sucked blood from an infected human individual and then bites an intact individual. since the range of area that traveled by a mosquito is relatively limited (approximately - km/day for anopheles) [ ] , mobility of infected human individuals should play some roles in the propagation of the agent, particularly for long distance propagation. wesolowski [ ] tried to elucidate the role of human mobility in the propagation of malaria in kenya. based on mobile phone records (either call or text) of about million people for year, they reconstructed the mobility patterns of the people and combined this information with spatial prevalence data of malaria cases. location of the people (mobile users) is followed based on approximately , cell towers located in settlements in kenya. travels (change of the location) beyond the border of each participant's "primary settlement" (presumably where the residence is located) was counted and used for data analyses. a malaria prevalence map with km resolution for has been used to classify the settlements according to their prevalence, then, combining the two types of information, that is, travels and prevalence observed in various settlements, they have estimated the proportion of the infected travels, which actually transported the malaria from one settlement to another. in this way, they could identify the source areas and the sink areas; the former supply the malaria, while the latter accept it. with such an analyses, they could show there were several distinct sources and sinks for malaria in kenya. nairobi, the capital, and the area around the victoria lake were serving as the most remarkable sink and the source, respectively. also, they observed that travel of people reflected the regional population density and regular travel, which is different from the travel of the parasites, where the lake regions serve as the primary source of the parasites, which flew into its surrounding areas and the capital area. based on these observations, the authors suggested that the elimination program must take the imported case into account for the program to be successful. in addition, they demonstrated that this method could identify the "hot spot" (settlement), which shows distinct export and import of malaria incidence compared to adjacent settlements and that it can also provide useful information for elucidating the mechanisms of smaller scale transmission. such an analysis provides a good example demonstrating the huge potential of using spatial analyses in the area of disease propagation. they applied the same approach to model and predict the dengue epidemics in pakistan [ ] . mobility of approximately million subscribers was followed for months in across tehsils (small politically defined areas in pakistan), and analyzed with more than , reported cases observed in tehsils over months in . focus of the analyses was the spread of the disease from southern region, where the warm climate supports the existence of vector mosquito throughout the year, to the northern regions with greater seasonality that limits the transmission. the authors calculated the "dengue suitability" of each region mainly based on daily temperature, which was combined with the probability of importing the infection by travelers from epidemic area to estimate the regional (spatiotemporal) risk of dengue epidemic. the results considering the mobile phone data are compared with those obtained from conventional "gravity" model, in which the travel volume of the people depends on the population sizes of and distance between the two regions (beginning and end of the travel). the results of the two models sometimes differ widely; among the two regions that experienced real dengue epidemics in , import of infection could not be predicted by the gravity model, while the model with mobile phone information could. also, the risk map generated for entire pakistan showed substantially different pictures between the two models. part of the reason of such differences is related with the observation that contrary to conventional mobility model, the mobile phone data showed no decay of travel volume with increasing distance of the regions. overall, this research demonstrated the importance of grasping mobility based on real observational data to understand the spread of infectious disease at the level of a country. for the diseases that are propagated through direct human-to-human contact, mobility of individuals among the population at stake should be much more crucial than the case of malaria as described above. for example, in the outbreaks of sars and mers, identifying the "index case" would be important. many quantitative models have been proposed to explain the spread of disease, but many of them have not taken the spatial information into account. as explained in this section, modeling with peoples' mobility for infectious diseases has not been explored so much, while it is a promising field for the future. at a larger spatial scale, spread of infectious diseases is associated with international travels. using the data for international airline travelers, potentially "hot" areas for the spread of zika virus have been identified. potential threat of importation of the virus from americas to africa and asian countries was demonstrated [ ] . relative importance of noncommunicable diseases (ncds) has been increasing both in developed and developing countries; to name a few, ischemic heart diseases, stroke, diabetes mellitus, and various types of cancer are the big ones in this category. it has been quite well established that obesity and hypertension are associated with higher risk of these diseases, which in turn are associated with imbalance in the energetics. numerous reports have been published regarding metabolic aspects of individuals at high risks for the ncds. in these reports, activities are mainly evaluated with activity diary, pedometer, or accelerometer wore by the subjects. while these lines of information would provide valuable data to demonstrate the association between inactivity and risk factors of ncds, a major defect is that it would not easily identify where in the daily life of the individual potential problem lies (i.e., leverage point). gps information or mobile phone call record might be useful in reconstructing daily activity of individuals, since they can provide the information regarding the speed of the translocation, by which researcher can make a reasonable guess if the individual moved actively (i.e., walking or bicycling) or passively (i.e., driving a car, using public transportations). this is a relatively unexplored area, which might bear potential public health importance both in developed and developing countries. ecological momentary assessment (ema) refers to the methods of collecting data from individuals, who are in their daily lives (thus, ecological), providing real-time data (thus, momentary) repeatedly [ ] . this is often enabled by using ict devices that can prompt a series of questions to participating individuals to report their physical and/or mental conditions to the researchers. the devices can be also connected with sensors for physiological or clinical information like heart rate, blood pressure, or body temperature, blood glucose, or blood oxygenation [ ] , thereby health "events" like arrhythmia, asthma attack, and panic episodes can be recorded. in addition, ema device has been connected with physical sensors to air pollution. with this kind of device, chronological data, which can be associated with special health events, can be collected, accumulated, and later be related with physiological and environmental conditions (like air pollution) where the individual was in, revealing hidden association between the health event and certain patterns of preceding environmental and/or behavioral conditions. this methodology has been successful in clinical settings, particularly highlighted in clinical psychology, and its application to environmental health may generate a unique opportunity to grasp individual's "dose" and "response" simultaneously. by combining with environmental monitoring information provided by satellites as well as ground monitoring stations, potential of using ema will be greatly enhanced in the area of environmental health. as noted in the beginning of this chapter, environmental health concerns the relationship between environmental condition and health consequences, spatial information is an indispensable component of this field. conventional environmental health studies have dealt with the spatial aspects as represented by administrative units, which is basically a qualitative variable and black box so to speak, in nature. more quantitative aspect of the spatial distribution is worth to be focused in environmental health, and recent progress in information and communication technology including data processing enables us to develop a new type of research. in this way, spatial information becomes much more manipulative in the analyses with its implication being much clearer. refining spatial information is only a method to improve the accuracy of exposure estimate, hence, associated with other progresses in this field. for example, conventional environmental health study only focused on a very limited number of environmental factors, most often only a single factor, and the dose-response relationship was evolved around this single factor. this is most likely due to the fact that problems in the past were mostly associated with a single environmental agent like one chemical species. contemporary issues, however, involve multiple factors that are converged on a single endpoint. in such a case, approach adopted in the helix study might be useful, although we yet to know what can be obtained with this approach. refining exposure estimate should be considered in such a context to characterize comprehensive exposure. while potential of this field is enormous, especially to be combined with other relevant techniques like ema, there are a couple of issues that needs constant attention. first, as the technology (both hard and soft) advances, more attention should be paid for the importance of the issue of privacy. this is not only saying that full attention should be paid to protection of privacy, but also ( ) considering the benefit for the people obtained through such information and ( ) letting people know both aspects (goods and bads) of mobility information, thereby enabling them to choose appropriate reaction towards such investigation. finally, it should be emphasized that mobility information obtained in the ways described in this chapter might evoke a new discussion about what "true" exposure is and to what extent we need to know about the exposure. as partially discussed before, if you would like to quantify the exposure of an individual as much as possible, you need to actually chase this individual to see how the individual and the environment at a given moment is faced with each other. for example, merely wearing a fine-pore mask would substantially change the exposure to certain air pollutants, which could not be picked up by the approaches discussed in this chapter. after all, required fineness of the quantitative evaluation totally depends on the objectives of the specific research. potential for zika virus introduction and transmission in resource-limited countries in africa and the asia-pacific region: a modelling study downscaling climate models health impact assessment of air pollution using a dynamic exposure profile: implications for exposure and health impact estimates issues of commuter transport in developing countres flight performance of the malaria vectors anopheles gambiae and anopheles atroparvus beyond space (as we knew it): toward temporally integrated geographies of segregation, health, and accessibility the impact of heat islands on mortality in paris during the august heat wave urban form and the ecological footprint of commuting. the case of barcelona gender difference in daily time and space use among bangladeshi villagers under arsenic hazard: application of the compact spot-check method health and the mobile phone spatial turn in health research ecological momentary assessment the human early-life exposome (helix): project rationale and design quantifying the impact of human mobility on malaria quantifying travel behavior for infectious disease research: a comparison of data from surveys and mobile phones impact of human mobility on the emergence of dengue epidemics in pakistan geospatial estimation of individual exposure ot air pollutants: moving form stati monitoing to activity-based dynamic exposure assessment key: cord- -os faovj authors: peghin, maddalena; danziger-isakov, lara title: prevention and treatment of respiratory virus infection date: - - journal: infectious diseases in solid-organ transplant recipients doi: . / - - - - _ sha: doc_id: cord_uid: os faovj there is increasing recognition of infections caused by respiratory viruses (rvs) as a major cause of morbidity and mortality in solid organ transplant (sot) recipients, especially within the thoracic and pediatric population. in addition to their direct, cytopathic, and tissue-invasive effects, rvs can create an inflammatory environment, autoimmune responses, resulting in acute and chronic rejection, although this relationship remains controversial. a laboratory diagnosis in sot with respiratory syndrome should be performed with nucleic acid amplification tests on respiratory specimens, mainly nasopharyngeal swabs (nps) and bronchoalveolar lavage (bal). treatment options remain limited and consist of supportive care, reduction of immunosuppression, and, if available, antiviral therapy. the use of immunomodulatory agents remains a clinical dilemma. since treatment options for rvs are limited, maximizing prevention measures against viral infections in sot is mandatory. the main preventive strategy against influenza remains the administration of yearly inactivated influenza vaccine in all sot. the aim of this review is to summarize the evidence-based recommendations on the diagnostic, preventive, and therapeutic strategies to decrease the burden of rv infections in sot recipients. respiratory viruses (rvs) are increasingly recognized as a major cause of morbidity and mortality in solid organ transplant recipients, especially within the lung transplant population. respiratory viral infections are typically caused by rhinovirus (rhvs), coronavirus (cov), respiratory syncytial virus (rsv), influenza (flu), parainfluenza (piv), human metapneumovirus (hmpv), and adenovirus (adv) ( table . ). respiratory infections can also be caused by viruses less commonly associated with the respiratory tract such as cytomegalovirus (cmv), human herpesviruses (hsv , hsv ), and varicella zoster virus (vzv) that will be discussed in another chapter (chap. ) . a detailed discussion of other newer respiratory viruses (table . ) is beyond the scope of this chapter, since they have not been widely studied in immunocompromised patients and their clinical impact is not fully understood. however, these viruses should be considered in the differential diagnosis of patients presenting with severe lower tract disease, especially if clinical history indicates potential exposure. the newer rvs are more challenging to diagnose since they are not included in the routinely available diagnostic tests and optimal management has not been defined. the definition of rv disease includes ( ) a new onset of symptoms and ( ) at least one respiratory symptom and ( ) the clinician's judgment that the illness is due to an infection [ ] . an upper respiratory tract infection (urti) is defined with the onset of sore throat, rhinorrhea, or hoarseness. a lower respiratory tract infection (lrti) is defined as new onset of shortness of breath, cough, sputum, rales, hypoxemia, and/or wheezing. when symptoms of lrti are associated with a new pulmonary infiltrate (on chest radiograph or chest computed tomography), pneumonia is distinguished from tracheobronchitis. many common respiratory viral infections in sot patients are mild, self-limiting upper respiratory tract infection (urti) and do not require hospitalization. however, compared to immunocompetent hosts and due to alterations in cellular and humoral immunity, infections can cause protracted symptoms with greater risk of progression to lrti, prolonged periods of viral shedding, and increased mortality. in sot, lrtis have been associated with increased risk of adverse complications and subsequent development of fungal, viral, and bacterial superinfections [ ] . although these complications may appear in the context of any type of transplantation, pediatric, lung, and heart-lung transplantation recipients appear to have the greatest risk of respiratory viral infections with more severe courses and complications [ ] [ ] [ ] . in addition to their direct, cytopathic, and tissue-invasive effects, rvs can create an inflammatory environment that leads to local and systemic microbially determined immune modulation (mdim) [ ] . mdim may increase the alloimmune and autoimmune responses that increase susceptibility to other opportunistic infections and are associated with the development of acute and chronic rejection. the greatest risk appears from data in lung transplant recipients, although data on this topic in the literature are conflicting [ , , ] . in transplantation overall, rhv and cov are the most common etiological agents, causing mostly mild urti, with lrti less frequently described. in contrast, flu and other paramyxovirus (rsv, piv, and hmpv) have a greater association with lrti and particularly acute and chronic rejection in adult lung transplant recipients [ , ] (tables . and . ). outcomes of infection are associated strongly with site of involvement, net state of immune suppression, and availability and use of antiviral agents. the clinical diagnosis of rvs can be difficult, since sot recipients often present with mild or atypical symptoms and signs, which are often overlapping and not always specific for any one viral agent. fever can be absent in sot with pneumonia or can be the sole presenting sign. in addition bacterial and fungal coinfections may occur. the distribution of rv infections throughout the year suggests that seasonal patterns of rv circulation in sot are similar to those circulating in the general [ , ] . consequently, vigilance regarding circulating community rv infections is required while caring for sot recipients. rapid and reliable laboratory diagnosis is required in sot with respiratory syndrome to significantly impact on patient care and management. the ideal method of sampling has also come into question, as the yield of viral specimen may differ depending on the specimen source. all sots with suspected rv infection should have a nasopharyngeal sample tested by pcr, including nasopharyngeal swab (nps), wash, or aspirate. between common respiratory specimens collected from the upper respiratory tract, nps are preferred, since they are practical for widespread use and comparable in sensitivity to nasopharyngeal aspirates or bronchoalveolar lavage (bal) for the detection of all major rvs [ , , ] . nps should be collected diligently by trained staff, using the standardized procedures of the centers for disease control and prevention (cdc) (https://www.cdc.gov/urdo/downloads/ speccollectionguidelines.pdf; https://www.youtube.com/watch?v=dvjnwefmhje) [ ] . if upper tract samples fail to document the rv cause of the respiratory illness and clinical or radiologic evidence of lower tract involvement exists, bal should be performed for rv testing [ ] . the array of diagnostic tools for rvs in immunocompromised patients has greatly increased over the last few years, and diagnosis can be performed using realtime pcr (rt-pcr) techniques, antigen detection, and serology (table . ) [ ] . the sensitivities of contemporary molecular diagnostic techniques have been substantially improved, allowing for the rapid simultaneous detection of a wide variety of conventional and emerging rvs in respiratory samples. at present, realtime multiplex nucleic acid amplification testing (multiplex nat) based on the rt-pcr technology is the preferred diagnostic tool for studying rvs in immunocompromised patients and is incorporated into many of the current guidelines [ , ] . both laboratory-developed and commercial rt-pcr assays are currently available, differing in specificity and sensitivity (ranges from % to %, with best sensitivity seen for flu and lower sensitivities for adv and piv). with the aim of overcoming technical complexity of pcr-based testing, fully automated rt-pcr instrument for rapid detection of rv has been tested in immunocompromised patients with promising results with a turnaround time of approximately - h [ ] . therefore, clinicians should be aware of the performance characteristics of the assay performed (https://www.cdc.gov/urdo/downloads/speccollectionguidelines. pdf). of note that regarding adv, negative testing from the upper or lower airway may not exclude infections particularly for sot with disseminated disease if there is limited to no involvement of the respiratory tract. rt-pcr should be applied on respiratory specimen, blood (quantitative viral load testing), and other compartments depending on clinical presentation (urine, cerebrospinal fluid). it is important to remember, however, that despite the excellent sensitivity, poorly collected samples may yield false-negative results, and results may greatly vary depending on the quality of the swab. the high sensitivity of these methods also has drawbacks, such as frequent detection of viruses in asymptomatic individuals and prolonged detection of viruses in patients who have already clinically recovered [ , ] . the major challenge is to determine association between the presence of microbial nucleic acids and a clinical syndrome in individual patients. quantification of the virus may be a helpful result interpretation, since high viral loads are associated with the presence of symptoms and may be related to the severity of the clinical symptoms [ ] . antigen detection techniques, which include immunofluorescence (if) and immunoassay (ia), are fast and have high specificity but are only available for specific viruses and their sensitivity less than molecular methods. this technique is not available for viral respiratory infections caused by rhv or cov and is moderately complex, and interpretation of results is subjective [ ] . a number of commercial ia are available for rsv and flu (a and b) and require little technical expertise. however, false-negative and false-positive results can be generated. a low prevalence of circulating virus within the community decreases the positive predictive value of the test. for flu, rapid ia has shown high specificity but low sensitivity ( - %) as compared to other assays, making them suboptimal for sot recipient, particularly in clinical decision-making for antiviral therapy [ ] . antiviral susceptibility testing for rvs is primarily focused on influenza, and both phenotypic and genotypic assays can be tested, although such testing is not widely available in local or commercial labs. antiviral resistance is of considerable concern among immunocompromised patients infected with influenza virus, and testing should be strongly considered in sot undergoing treatment who fails to have an appropriate clinical response within - days of initiating antiviral therapy or who has a relapsing course despite ongoing therapy. in the absence of available treatment options and of strong evidence of effectiveness for any particular therapy, treatment strategies differ widely among centers [ ] . limited understanding of ( ) risk factors for progression to severe lrti and poor outcomes and ( ) indirect inflammatory effects of viral infection impact opinions on appropriate interventions for respiratory viral infections. rv infections, particularly cause by influenza virus, are a risk factor for subsequent bacterial and fungal superinfections. in cases of lrti, secondary infections must be ruled out and appropriately treated, and initiation of oral or nebulized antifungal prophylaxis to prevent invasive fungal infections should be evaluated in high-risk patients [ , , ] . management in transplant patients is generally focused on reduction of immunosuppression feasible to speed resolution of viral infection. treatment options for rvs are limited (tables . and . ). resistance patterns may change and affect recommended antiviral strategies. consequently, clinicians should consult national health authority regularly for updated recommendations, especially for influenza. in our opinion treatment efforts should be always performed in any sot with lrti or in lung transplant and heart-lung transplantation recipients both with urti and with lrti, due to increased morbidity and mortality [ ] . reconstitution of the immune system appears to be important in overcoming rv infections. clearly, the currently available treatment option is a clinical dilemma [ , , ] . there are numerous reports in the literature citing the use of intravenous immunoglobulin (ivig) as part of therapy for viral infections in immunocompromised patients. hypogammaglobulinemia has been associated with an increased risk of opportunistic infections in sot, but not to community-acquired rvi. however, some experts recommend considering the addition of ivig for severe rv infection in sot [ ] . the use of monoclonal antibodies is limited to the treatment of rsv. immunotherapy including transfer of rv-specific t lymphocytes from healthy donors is under investigation and has been reported to be safe and effective when performed early in the course of the infection for hmpv, adenovirus, rsv, and piv. at the same time, virus-associated immune modulation may sometimes be deleterious in rvs due to local inflammatory responses. adjunctive therapy with corticosteroids has been purposed for sot with influenza and rsv and for lung transplant recipients with any rvs with lrti because of the risk of both acute and chronic rejection [ ] . treatment options for rvs are limited, and maximizing prevention measures against viral infections in sot is mandatory. rvs are potential community and nosocomial pathogens that can be spread by staff or visitors with mild upper respiratory illness. overall awareness among sot, healthcare personnel, family members, and caregivers about the potential deleterious outcomes of rv infections in sot and the importance of early detection of infection may have a significant impact on the incidence of rv infections and risk of transmission [ ] . strict adherence to hand hygiene, contact precautions, and respiratory droplet isolation are required to reduce rv nosocomial spread and outbreaks during hospitalization (table . ) (https://www.cdc.gov/infectioncontrol/guidelines/isolation/). the appropriate length of isolation for patients with laboratory proven rvs is debated, as prolonged shedding is a common finding in sot patients, but viral load thresholds for infectivity are unknown. infection control measures should be maintained until the patient is discharged home or until pcr is negative. stringent hygiene precautions should be also applied in community settings, where sot recipients should avoid close contact with individuals with respiratory tract infections [ ] . the influenza virus is currently the only carv that can be prevented with vaccination [ ] . prevention and treatment of specific rvs three main viral strains have been recently associated with human infection, namely, influenza a/h n , influenza a/h n , and influenza b. influenza infection in sot causes significant morbidity and mortality compared to general population [ ] . in studies performed in h n pandemic, the proportion of patients who required hospitalization varied between % and %, and one of every five patients suffered severe complications with - % mortality [ ] . the mainstay of treatment for influenza a and b are the neuraminidase inhibitors (nai), mainly oseltamivir (tables . and . ) [ ] . doubling the treatment dose of oseltamivir in hospitalized patients with influenza does not seem to increase virologic efficacy, except perhaps for influenza b infections or in case of oral absorption concerns, with no evidence of emergence of oseltamivir resistance [ , ] . zanamivir is used less frequently than oral oseltamivir, likely due to the inhaled delivery route, although it has shown better activity against influenza b and few cross-resistance with oseltamivir. regarding intravenous formulations, if available, intravenous zanamivir or peramivir can be considered in sot recipients who are severely ill despite oral oseltamivir, in case of concerns with oral absorption, although experience with these drugs in sot recipients is lacking [ ] . parenteral zanamivir is currently available in europe, and a single dose intravenous peramivir has been approved in the united states for treatment of uncomplicated influenza infections. however, peramivir use in sot likely would require repeated dosing or switching to oral oseltamivir to complete therapy. nai resistance is currently uncommon ( . - . % of isolates), especially for influenza a/h n and influenza b viruses, but remains an area of growing concern. in case of high-level oseltamivir resistance (such as h n viruses strains with h y substitution), peramivir usually preserves reduced susceptibility, but zanamivir is usually active. another common resistance mutation (h y in h n ) confers resistance to both oseltamivir and peramivir, but not zanamivir. therefore, peramivir should not be used in patients with oseltamivir resistance unless the isolate is proven to be susceptible [ ] (tables . and . ). das , an inhaled sialidase potentially inhibiting influenza and parainfluenza infection, has shown promising in vitro results of activity against oseltamivir-resistant influenza strains but failed to show superiority compared to placebo in previous studies in healthy subjects with influenza infection [ ] . treatment should be initiated as soon as possible since antiviral therapy is most likely to provide benefit when initiated within the first h of illness in sot, with a reduced rate of influenza-associated complications (admission to icu, use of invasive ventilation, and death) [ ] . however, benefit has been demonstrated even with delayed treatment, and most experts endorse influenza-specific antiviral treatment at any point in the illness. further, treatment should not be delayed while awaiting diagnostic testing results or if a rapid antigen ia test is negative when clinical symptoms are suggestive of infection due to the poor sensitivity of rapid antigen tests (table . ) [ ] . in general, duration of antiviral therapy should be at least days for sot patients although some data suggest that longer duration (≥ days) may be required, particularly in critically ill patients, those with pneumonia and persistent viral shedding. aside from advances in supportive care, no specific adjunctive therapies are routinely recommended. corticosteroids have been shown to decrease the need for mechanical ventilation and progression to lrti but at the cost of prolonged viral shedding and risk for invasive fungal coinfection. corticosteroids are not routinely recommended but should be used if indicated for another reason such as concurrent acute rejection [ ] . the main preventive strategy against influenza in sot recipients remains the administration of yearly inactivated influenza vaccine. all transplant recipients and candidates, as well as family members, close contacts, and healthcare workers, should receive the influenza vaccine to provide herd immunity [ , ] (table . ). influenza vaccines are available in inactivated (intramuscular or intradermal administration) and live-attenuated (intranasal) formulations. the liveattenuated vaccine is not recommended for immunocompromised recipients and close contacts, due to a potential risk of dissemination of the vaccine [ , ] . current guidelines recommend the standard injected inactivated influenza for sot starting - month posttransplantation with option for administration as early as month posttransplantation in an outbreak setting. if influenza vaccine was administered earlier than months posttransplantation, when it is likely to be less effective, consideration may be given to administering a second dose of vaccine later in the influenza season [ , ] . an association between vaccination and the development of the de novo antibodies and graft rejection is unproven. a higher-dose vaccine in pediatric sot and a booster strategy weeks after standard influenza vaccination in adult sot have shown to induce an increased antibody response compared with standard single dose. whether or not protection is increased by use of higher-dose vaccine, adjuvants, booster doses, or quadrivalent versus trivalent vaccines constitutes an area of active research [ ] . clinical failure of influenza vaccination in sot recipients has not been extensively studied, but most of the studies clearly suggest a reduced immune response in sot, with a seroconversion rate that varies between % and %, although this is also dependent on the match between the vaccine and the circulating strains [ ] . vaccination has shown to attenuate adverse outcomes among sot recipients with a lower incidence of pneumonia and shorter length of hospital stay [ , ] . beyond influenza vaccination, pre-exposure or postexposure chemoprophylaxis with either oseltamivir or zanamivir is approved (tables . and . ) and may be considered [ ] . caution should be used with prescribing oseltamivir for prophylaxis in patients exposed to an index case because prophylaxis has been associated with emergence of resistant mutants; therefore, monitoring and empiric therapy are generally recommended in these cases [ ] . respiratory syncytial virus has long been recognized as a concerning pathogen in immunocompromised hosts. in sot, rsv infection typically manifests as an urti with progression to lrti in - %. risk factors for more severe disease after organ transplantation include infection in children under a year of age or lung transplantation [ , ] . the use of ribavirin (rbv) for the treatment of rsv infection is controversial. in immunocompromised patients (mainly hematopoietic stem cell transplant recipients), rbv has been shown to decrease progression to lrti when given to patients with urti. among sot, the greatest experience with rbv is with lung transplant recipients. based on published reports as well as self-reported treatment strategies in surveys from sot centers, lung and heart-lung recipients often receive rbv for both rsv-related urti and lrti [ ] . due to lack of clear evidence of efficacy, wide variation in the management of rsv exists including variability often dependent on availability of the inhaled, intravenous, and oral rbv formulations [ ] . intravenous and inhaled rbv are not available in most european countries. oral ribavirin appears to be an effective, well-tolerated alternative to intravenous or inhaled ribavirin, providing potential cost savings and reducing length of hospital stay [ ] (tables . and . ). aln-rsv , a small interfering rna that targets the rsv nucleocapsid messenger rna, has shown some early promise in potentially preventing chronic rejection in lung transplant recipients with rsv; this agent is no longer being developed clinically. in addition, there are a number of other small molecule therapies in various stages of development including early clinical trials [ ] . immunomodulators have also been investigated. experts recommend considering the addition of an antibody preparation (palivizumab) and ivig with or without corticosteroids for severe rsv infection in sot, although data are limited to support this recommendation [ , ] . a systematic review reported that any form of rbv, alone or in combination with an immunomodulatory agent, was effective in preventing progression from urti to lrti, with a trend toward better outcomes with inhaled rbv plus an immunomodulatory with monoclonal (palivizumab) or polyclonal antibody preparations (ivig) ( table . ). prevention in addition to the general preventive measures, the only fda-approved agent for the prevention of severe rsv infection in high-risk patients under the age of years is palivizumab [ , ] . survey data suggest that antibody-based prophy-laxis is used among pediatric transplant centers in young candidates and recipients. however, guidelines regarding the use of this agent in older children and adults do not exist, and the high combined with a lack of clear evidence of efficacy in sot recipients precludes its wide-scale use (table . ). in sot patients piv, most commonly piv , is able to cause more serious and even fatal infections, which mostly occur in patients after lung transplantation [ ] . an outbreak of piv infections in a kidney transplant unit demonstrated that all infections were mild and symptoms resolved spontaneously without associated mortality [ ] . treatment there are no currently approved antiviral treatments for parainfluenza disease. treatment is supportive and includes reduction in immunosuppression. oral, aerosolized, and intravenous rbv and/or ivig and corticosteroids have been used off-label in piv with variable results and no impact on mortality [ ] . das has been used to treat piv infections in immunocompromised patients and has shown encouraging results including reduction in piv quantitative viral load and overall outcomes [ ] . clinical trial results are pending. prevention outbreaks caused by piv have been reported previously [ ] , and patients with known or suspected piv should be isolated with standard contact precautions. there are no approved vaccines or prophylactic antiviral agents. human metapneumovirus has a clinical pattern similar to rsv and is a significant cause of disease in transplant recipients [ ] . hmpv has been associated with lrti (pneumonia) and high hospitalization rates [ ] . treatment there is no approved drug for the treatment for hmpv respiratory infection. supportive therapy is the main treatment although rbv alone or with ivig could be considered for the management of lrti and severe cases of hmpv in sot [ ] . prevention there are no approved vaccines or prophylactic antiviral agents. rhinovirus has more than serotypes in different species: a, b, and the more recently characterized c. rhinoviruses are the leading cause of community-acquired rv infections, and that finding is in agreement with the knowledge that this rv is the primary cause of acute viral respiratory illnesses [ , ] . infections with rhinovirus are usually mild and self-limiting urti, although significant lrti has been described in lung transplant recipients [ , ] . prolonged shedding for over months with minimal symptoms has been reported in lung transplant recipients. treatment no specific treatment is approved for rhinovirus infection. prevention there are no approved vaccines or prophylactic antiviral agents. coronavirus generally results in self-limited disease but may progress to lrti. the most common types of hcov are oc , e, hku , and nl . severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) are novel coronavirus that have been responsible for recent acute respiratory syndrome epidemics. treatment there are no antivirals licensed for the treatment of hcov infections, and therapy consists of supportive care. rbv has been used for the treatment of lrti caused by coronavirus during the outbreak of sars, and the use of rbv in combination with interferon-α- a on mers-cov has been reported. however, this combination has not been reported in sot, and there are no specific data to recommend rbv for the treatment of cov infection in sot recipients [ ] . prevention there are no approved vaccines or prophylactic antiviral agents. adenovirus is a double-stranded dna virus of the family adenoviridae, with subgroups (a-g) and serotypes. in contrast to many of the other community-acquired rvs, adenoviral infection can occur from primary acquisition or through reactivation. the transplanted organ is typically the site of infection, and pneumonia is most frequent in lung transplant recipients [ ] . of note, commercial rt-pcr assays differ in sensitivity and specificity for adenovirus (adv), and quantitative adv pcr from blood may also be obtained to aid in diagnosis (tables . and . ). treatment treatment is supportive and includes reduction in immunosuppression. the optimal timing for therapeutic intervention during the course of illness is unclear. existing data suggests that cidofovir and brincidofovir, an orally bioavailable lipid conjugate of cidofovir, may provide the highest likelihood of antiviral efficacy. brincidofovir appears to have increased in vitro and in vivo efficacy against adv for treatment of serious infections with less renal and bone marrow toxicity than cidofovir (table . ). rbv does not appear to have significant anti-adv activity in humans and is generally not recommended to treat serious adv infections. the use of ivig remains controversial because it does not appear to have a clear benefit at this time. adoptive t-cell transfer has generally been limited to a few centers (predominantly in hematopoietic stem cell transplantation) and has been reported to be safe and effective when performed early in the course of the infection [ ] . prevention there are no approved vaccines or prophylactic antiviral agents. longitudinal prospective surveillance using molecular diagnostics is needed to understand the true epidemiology and clinical spectrum of respiratory viral diseases in sot, particularly in non-lung population. optimal timing, duration, and treatment indication for rvs are a dilemma that needs to be clarified in clinical practice. the efficacy of adjuvant immunogenic therapies remains controversial. maximizing prevention and infection control measures against rvs in sot is essential (table . ). table . key points for rv infections in sot epidemiology and clinical presentation • there is increasing recognition of infections caused by rvs as a major cause of morbidity and mortality in sot • in addition to their direct, cytopathic, and tissue-invasive effects, rvs can create a microbially determined immune modulation the impact of rvs in acute and chronic rejection remains controversial, with the greatest risk in lung transplant recipients • pediatric solid organ, lung transplant, and heart-lung transplantation recipients appear to have the greatest risk of both rvs infections and more severe complications • rhinovirus and coronaviruses are the most common etiological agents • influenza and other paramyxovirus (rsv, piv, and hmpv) have a greater propensity to produce lrtid diagnosis • diligent collection of respiratory specimens and knowledge of the limitations of the assay used by your laboratory are essential for interpreting the results • nasopharyngeal swabs (nps) are preferred for the detection of all major rvs • bronchoalveolar lavage is the preferred specimen for diagnostic testing in lrtid with negative nps • laboratory diagnostic methods include virus culture, rapid antigen detection tests, the reverse-transcriptase polymerase chain reaction (rt-pcr) and other nucleic acid amplification assays, and serology • nucleic acid amplification tests, mainly rt-pcr, are the best diagnostic tools for studying rvs in sot treatment • treatment options remain limited and consist of supportive care, reduction of immunosuppression and, if available, antiviral therapy • the use of immunomodulatory agents (intravenous immunoglobulin, monoclonal antibodies, rv-specific t lymphocytes or augmented corticosteroid therapy) is a clinical dilemma • sot should receive antiviral therapy with a neuraminidase inhibitor (nai, either oseltamivir or zanamivir) when influenza is suspected, before laboratory confirmation • treatment of influenza with m inhibitors (amantadine and rimantadine) is not recommended for high resistance rates • treatment for influenza with nai should be initiated as soon as possible (< h) for optimal benefit, however and all symptomatic patients should receive antiviral therapy, irrespective of symptom onset • duration of therapy should be minimum of days, but longer duration of therapy (≥ days) may be required in critically ill patients • double dosing of oseltamivir is not recommended but may be considered in severe cases or in case of insufficient response to therapy • the use of intravenous zanamivir or peramivir can be considered in patients not responding to oseltamivir therapy or for whom oral absorption is a concern • in case of high-level oseltamivir resistance, zanamivir is usually active • the efficacy of ribavirin (aerosolized, intravenous, or oral) for the treatment of rsv infection in sot recipients has not been determined • in severe cases of lrtis with rsv, piv, and hmpv infections in sot recipients, therapy with ribavirin (aerosolized, intravenous, or oral) may be used, alone or in combination with an immunomodulatory agents prevention • maximizing prevention infection control measures against rvs in sot is mandatory • sot recipients should avoid close contact with individuals with respiratory tract infections • the main preventive strategy against influenza remains the administration of yearly trivalent inactivated influenza vaccine in all sot recipients and their relatives • influenza vaccination is safe in sot recipients, even in the early posttransplant period • oseltamivir may be used as pre-exposure and postexposure prophylaxis in selected patients • the use of palivizumab for prevention of rsv infection is not recommended in adult sot recipients flu influenza, hmpv human metapneumovirus, lrtid lower respiratory tract infectious disease, nps nasopharyngeal swab, piv parainfluenza, rbv ribavirin, rsv respiratory syncytial virus, rvs respiratory viruses, sot solid organ transplant table . (continued) influenza and other respiratory virus infections in solid organ transplant recipients epidemiology and immediate indirect effects of respiratory viruses in lung transplant recipients: a -year prospective study incidence and outcomes of respiratory viral infections in lung transplant recipients: a prospective study respiratory viral infections in pediatric solid organ and hematopoietic stem cell transplantation respiratory viruses in lung transplant recipients: a critical review and pooled analysis of clinical studies from the classic concepts to modern practice rna respiratory viruses in solid organ transplantation nasopharyngeal viral pcr in immunosuppressed patients and its association with virus detection in bronchoalveolar lavage by pcr respiratory virus detection in immunocompromised patients with filmarray respiratory panel compared to conventional methods antiviral agents for the treatment and chemoprophylaxis of influenza -recommendations of the advisory committee on immunization practices (acip) current practices for treatment of respiratory syncytial virus and other non-influenza respiratory viruses in high-risk patient populations: a survey of institutions in the midwestern respiratory virus collaborative update in the treatment of non-influenza respiratory virus infection in solid organ transplant recipients vaccination in solid organ transplantation outcomes from pandemic influenza a h n infection in recipients of solid-organ transplants: a multicentre cohort study global update on the susceptibility of human influenza viruses to neuraminidase inhibitors antiviral treatments a prospective intervention study on higher-dose oseltamivir treatment in adults hospitalized with influenza a and b infections a five-year prospective multi-center evaluation of influenza infection in transplant recipients influenza vaccination in solid-organ transplant recipients a comprehensive review of immunization practices in solid organ transplant and hematopoietic stem cell transplant recipients reduced incidence of pneumonia in influenza-vaccinated solid organ transplant recipients with influenza disease rsv prevention and treatment in pediatric lung transplant patients: a survey of current practices among the international pediatric lung transplant collaborative oral ribavirin for respiratory syncytial virus infection after lung transplantation: efficacy and cost-efficiency updated guidance for palivizumab prophylaxis among infants and young children at increased risk of hospitalization for respiratory syncytial virus infection parainfluenza virus infection in adult lung transplant recipients: an emergent clinical syndrome with implications on allograft function parainfluenza infections early after kidney or simultaneous pancreas-kidney transplantation parainfluenza virus in the hospitalized adult successful outcome of human metapneumovirus (hmpv) pneumonia in a lung transplant recipient treated with intravenous ribavirin key: cord- -sy cpff authors: mitrovic, stéphane; feist, eugen; fautrel, bruno title: adult-onset still’s disease date: - - journal: periodic and non-periodic fevers doi: . / - - - - _ sha: doc_id: cord_uid: sy cpff adult-onset still’s disease (aosd) is a rare but clinically well-known, polygenic, systemic autoinflammatory disease. it is typically characterized by four main (cardinal) symptoms: spiking fever ≥ °c, arthralgia or arthritis, skin rash, and hyperleukocytosis (≥ , cells/mm( )). however, many other clinical features are possible, and it can appear in all age groups with potentially severe inflammatory onset accompanied by a broad spectrum of disease manifestation and complications. hence, it remains a diagnostic challenge, and the clinician should first rule out infectious, tumoral, or inflammatory differential diagnoses. determination of the total and glycosylated ferritin levels, although not pathognomonic, can help in diagnosis. new biomarkers have recently been described, but they need to be validated. the disease evolution of aosd can be monocyclic, polycyclic, or chronic. in chronic disease, a joint involvement is often predominant, and erosions are noted in one-third of patients. many progresses have been made in the understanding of the pathogenesis over the last decades. this chapter provides a comprehensive insight into the complex and heterogeneous nature of aosd describing the identified cytokine signaling pathways and biomarkers. it also discusses the current evidence for the usage of biologics in aosd to provide guidance for treatment decisions, taking into account both the efficacy and the safety of the different therapeutic options. adult-onset still's disease (aosd was first described in the early s, by bywaters, as an inflammatory condition occurring in young adults. the disease was very similar to childhood-onset still's disease, today called systemic-onset juvenile idiopathic arthritis (sojia), described a century ago by sir still [ ] . although the exact pathogenic mechanisms of the disease remain unknown, substantial advances have been made first to confirm the homology between aosd and sojia, and then the disease became the archetype of nonfamilial, or sporadic, systemic autoinflammatory disorders (said) [ ] [ ] [ ] . by definition, aosd affects people older than , either de novo or those with a history of systemic jia [ , , ] . in the latter, a disease-free interval between the childhood episode and the adulthood recurrence differentiates aosd from persistent sojia. in most patients, aosd is characterized by four cardinal symptoms: spiking fever, an evanescent salmon-pink maculopapular rash, arthritis, and a white blood cell count (wbc) ≥ , /mm , mainly neutrophilic polymorphonuclear cells (pmns). other features are sore throat or pharyngitis, myalgia, lymph node or spleen enlargement, pleuritis or pericarditis, multivisceral involvement, elevated levels of liver enzymes, and other hematologic abnormalities [ , , ] . none of these symptoms is disease specific, so diagnosis of aosd is difficult, and clinicians must first rule out neoplastic, infectious, or inflammatory conditions. to date, epidemiologic data about aosd have been scarce and imprecise, because of the heterogeneous clinical presentation of the disease and the complex assessment of diagnosis. the disease occurs worldwide, and no specific familial aggregation has been reported. aosd satisfies the definition of an orphan disease because historically its prevalence was estimated between one case per million in europe [ ] and ten cases per million in japan [ ] . in the japanese study, the incidence was estimated at about one case per million in the mid- s. however, because of the substantial advances in aosd diagnosis as well as differential diagnoses during the last two decades, these prevalence and incidence estimates lack robustness, and new studies are clearly needed to update these figures. initially, the disease was characterized as affecting exclusively young adults (i.e., - years of age [ ] ); however, more recent series identified cases in adults older than and even elderly people [ , [ ] [ ] [ ] [ ] . the sex ratio is almost balanced, with only a slight female predominance [ , , , [ ] [ ] [ ] ]. clinical expression aosd is traditionally characterized by four "cardinal" symptoms (three are clinical, one is biological), with many other manifestations possibly associated [ , , ]. fever is constant when the disease is active, except in patients with established disease, for whom residual inflammation could remain without it. typically, fever starts suddenly, and temperature rapidly reaches °c or more, associated with shivers. it evolves with daily evening spikes for more than a week ( fig. . ). patients usually experience rapid deterioration of general health as well as significant weight loss. of importance, fever may be the only clinical symptom of aosd as a potential diagnosis in patients with fever of unknown origin [ ] . joint pain or arthritis is the second most common symptom, with synovitis, usually with concomitant fever spikes, occurring in more than two-thirds of patients. all joints can be involved, including sacroiliac and distal interphalangeal joints. in some patients, the presentation is that of a bilateral symmetrical rheumatoid arthritis (ra)-like polyarthritis [ , ] . synovial fluid analysis displays high cellularity, > cells/mm , which confirms joint inflammation. when performed, synovium biopsy reveals only nonspecific synovitis. during the evolution, arthritis becomes erosive in one-third of patients; in these patients, isolated bilateral carpal ankylosis (i.e., without structural damage of metacarpophalangeal or proximal interphalangeal joints, in contrast to ra) is very suggestive of aosd ( fig. . ) [ , , ]. skin rash is typically salmon pink-colored macular or maculopapular (figs. . and . ). the rash is transient, mainly visible during fever spikes on the proximal limbs or trunk, and rarely involves the face, palms, or soles of the feet [ , , ] . no specific histological feature has been described. misdiagnosis with drug allergy is frequent and usually attributed to nonsteroidal anti-inflammatory drugs (nsaids) prescribed for joint symptoms or fever. complete regression without scars is the rule. atypical patterns also reported are urticarial or pruritic with dermographism [ , , ] . presence of purpuric lesions should lead to urgent coagulation workup because these lesions are suggestive of the aosd hematologic complications of hemophagocytic syndrome or reactive hemophagocytic lymphoproliferation (rhl), disseminated intravascular coagulation (dic), or thrombotic microangiopathy (tma), also called thrombocytic thrombocytopenic purpura or moschcowitz syndrome [ , ] . sore throat (odynophagia) and sometimes pharyngitis are classical symptoms, concomitant with fever (and not just a few weeks previous, e.g., in post-streptococcal arthritis) [ ] . all microbiological test results are negative. the presence of this symptom in such a systemic context seems to exclude lymphoma or another neoplastic disease [ ] . diffuse and symmetrical lymphadenopathy is possible, eventually associated with splenomegaly or even hepatomegaly. however, large and nonsymmetrical lymphadenopathy should lead to the exclusion of malignant lymphoma by biopsy: reactive polyclonal lymphoid hyperplasia is the usual pattern in aosd [ , ] . kikuchi necrotizing lymphadenitis has been described in a few cases [ ] . myalgia is frequent, but myositis or polymyositis is exceptional [ , , , ] . finally, as in many other inflammatory disorders, nonspecific manifestations have been reported: abdominal pain related to deep lymphadenitis, aseptic peritonitis or rarely acute pancreatitis, pleural effusion or pericarditis, or interstitial lung infiltrates [ , ] . high serum ferritin level, an indicator of macrophage activation, has been frequently reported [ , , [ ] [ ] [ ] [ ] . although hyperferritinemia was first considered suggestive of aosd, several studies showed that hyperferritinemia, whatever the threshold used, has poor positive predictive value in isolation without a suggestive context [ , ] . besides total ferritin level, the diagnostic performance of glycosylated ferritin (gf) level has been investigated [ ] . the gf level normally represents more than half of the total ferritin level. in inflammatory conditions, the concentration of gf decreases and usually ranges from % to %; this decrease has been related to the saturation of glycosylation mechanisms due to hyperferritinemia. however, in aosd, gf level is typically markedly decreased (below %), which suggests a more specific phenomenon. more extensive data revealed a sensitivity and specificity of gf ≤ % for aosd diagnosis of % and %, respectively [ ] . the combination of both hyperferritinemia and gf level ≤ % yielded a sensitivity and specificity of % and %, respectively. of note, although serum ferritin level fluctuates according to systemic inflammation, gf level remains low several weeks to several months after disease remission [ ] . however, as mentioned previously, low gf level is not completely specific to aosd and is observed in other inflammatory processes, such as severe systemic infections [ ] . moreover, the gf level is usually low, ≤ %, in hemophagocytic syndromes, regardless of the cause, such as infection, neoplasm, or inflammation [ ] [ ] [ ] . results of immunological workups performed to exclude other connective tissue diseases or inflammatory joint diseases must be negative. approximately ten years ago, mcgonagle and mcdermott formulated the hypothesis of two main pathogenic mechanisms underlying immune-mediated inflammation against the self and proposed a new classification for immunological diseases contrasting autoimmunity and autoinflammation [ , ] . autoimmunity referred to adaptive immunity and was defined as aberrant dendritic, b-and t-cell responses in primary and secondary lymphoid organs leading to tolerance break and development of immune reactivity toward native antigens (with autoantibodies in most cases), whereas autoinflammation referred to innate immunity and was defined as dysregulated activation of macrophages and neutrophils in response to a danger signal leading to tissue damage. these categories represent a continuum, which allowed for a new classification of immune-mediated inflammatory disorders that was refined in the following years [ , ] . although autoimmune diseases were rather easily diagnosed based on autoantibody or autoantigen-specific t and b cells, no specific biomarker exists for said. the definition mainly relies on similarities with monogenic, hereditary periodic fever syndromes, which were at the origin of the concept. they include several key features: intense inflammation with periodic fever, tissue inflammation depending on the disease, increased leukocyte and neutrophil count, elevated erythrocyte sedimentation rate (esr) and c-reactive protein (crp) level, and, more recently, a pathogenic role of inflammasome and response to interleukin- (il- ) blockade [ ] . besides monogenic familial syndromes, crohn's disease was the first nonfamilial polygenic aid, mainly tissue specific, with predominant gut involvement [ , ] . a few years later, still's disease was described as another nonfamilial aid, becoming one of the most characteristic polygenic systemic said [ ] . the mechanisms underlying aosd pathogenesis are largely hypothetical, but, from the evident pathogenic pathway seen in sojia patients and the growing understanding of said, a pro-inflammatory cascade can be proposed (fig. . ) [ , , , , ] . the starting point is likely specific danger signals, such as pathogen-or damageassociated molecular patterns (i.e., pamps or damps). danger signals are transmitted to macrophages and neutrophils via specific toll-like receptors that activate specific inflammasomes, likely nod-like receptor family pyrin domain containing (nlrp ), leading to caspase activation and overproduction of active il- [ ] [ ] [ ] [ ] . this step seems to be central to the aosd pathogenesis and leads to intense innate immune cell activation and overproduction of several pro-inflammatory cytokines including il- , il- , tumor necrosis factor (tnf), as well as il- and il- . several factors actively contribute to an amplified inflammatory response, often referred to as the cytokine "burst" or "storm" [ , ] . in addition to il- itself, conferring retrograde activation of macrophages and neutrophils; different alarmins, such as the s protein-s a protein seeming to be more specific to still's disease in children-and advanced glycosylated end (age) products are involved in these processes [ , [ ] [ ] [ ] [ ] [ ] . besides amplification mechanisms, said pathogenesis has been suggested to involve deficiency or failure in regulatory or antiinflammatory mechanisms: deficiency in t regulator or natural killer cells, insufficient il- production, and deficiency in resolution lipid mediators, soluble receptors of ages (rages), or other resolution-associated molecular patterns [ ] [ ] [ ] [ ] . [ , , ] . in addition, patients often experience odynophagia or pharyngitis just before the start of the disease or the relapse, which could correspond to the infectious danger signal that will trigger toll-like receptors and start the intense inflammatory process. in contrast to monogenic, hereditary, periodic fever syndromes, the underlying genetic background of aosd is largely unknown. the disease is present in different geographic regions and different ethnic groups [ , , , , ] . association studies have suggested a potential predisposing genetic background. hla-bw was the first identified as a susceptibility antigen and associated with a mild self-limiting pattern of the disease [ ] . hla-dr was found more prevalent in aosd cases versus healthy controls, and hla-drw was associated with the occurrence of proximal arthralgia [ ] . however, no functional analysis has confirmed these hypotheses [ , , ] . recently, two major findings have been reported in sojia patients. firstly, a homoallelic missense mutation in the enzyme laccase (multicopper oxidoreductase) domain-containing -lacc -has been identified in sojia patients from five saudi consanguineous families [ ] . although familial sojia is not the rule, this study raises the potential role of this laccase in the pathogenesis of sojia. secondly, a large association study performed in sojia children and healthy controls identified a strong association between sojia and different hla-drb * haplotypes which all contain glutamate [ ] . this finding is more challenging since it would involve adaptive immunity in the aosd pathogenesis which was not expected [ ] . finally, substantial advances have been made in exploring the human genome, which will facilitate the identification of potential mutations in genes involved in autoinflammatory pathways, including de novo germinal mutations or somatic mutations occurring during fetus development or after birth [ , ] . several severe manifestations have been described in aosd, which explains the potentially pejorative prognosis of the disease [ , ] . the disease can be lifethreatening because of the predominant involvement of one organ or of systemic complications with multiple organ failure from the outset, while the diagnosis isn't clearly confirmed. the articular signs may often fall by the wayside because of the gravity of the complications of such clinical forms, which can contribute to misdirect the clinicians who are not familiar with aosd. these complications are given in table . , and the most frequent and relevant ones are discussed in more detail. this is a common complication of aosd at the time of diagnosis, right after treatment introduction or during the course of the disease. in systemic-onset jia, it is also called macrophage activation syndrome. this serious complication should be suspected in a patient with persisting fever (in contrast to evening spiking fever) and decrease in initially elevated leukocyte and neutrophil counts [ , , ] . several other manifestations can be associated. the key issue with rhl is to determine whether its occurrence is related to aosd intense inflammation or to concomitant infection, potentially favored by immunomodulators introduced because of aosd. the list of possible infections is quite long, with viral reactivation at first place. aosd can be complicated by two serious coagulation disorders, mainly at the acute phase of the disease. the first disorder, disseminated intravascular coagulation (dic), is not rare and may occur at a frequency of - % [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this diagnosis should be suspected with the combination of thrombotic events and cutaneous or mucosal bleeding and sometimes specific organ involvements, such as fulminant hepatitis, cardiac or respiratory failure, or stroke. hemostasis workups reveal platelet and coagulation factor consumption, increased thromboplastin time, and high d-dimer levels. the other rare but quite severe coagulation disorder is thrombotic microangiopathy (tma). it must be suspected with unexplained multiorgan failure or stroke, related to multiple small thrombi leading to tissue ischemia and mechanical hemolytic anemia [ , , ] . acute vision impairment, such as blurred vision related to purtscher-like retinopathy, is frequently the first symptom [ ] . key diagnostic tests display thrombocytopenia by platelet consumption, anemia, and schistocytes (fragmented red blood cells), which are specific to this diagnosis. organ imaging may reveal multiple infarctions. in addition, adamts enzymatic activity needs to be tested because acquired deficiency has been found to predispose to tma [ , [ ] [ ] [ ] . tma has been mainly described during aosd flare, related to intense inflammation or to concomitant infection with shiga toxin-producing microorganisms [ ] . although pleural effusion or pericarditis is frequent, other serious cardiac or pulmonary manifestations have been described. specific attention should be dedicated to a rare and very severe aosd manifestation, pulmonary arterial hypertension (pah), with several cases recently reported [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . pah, either idiopathic or occurring with aosd or other connective tissue diseases, is thought to be related at least in part to immune-related alteration of the endothelium with overproduction of il- , il- , il- , and tnf [ , , , , ] . pah may occur at aosd onset or later and seems to predominate in women. the diagnosis must be suspected with unexplained and often rapidly progressing dyspnea [ , , , , ] . electrocardiography is rarely contributive, eventually disclosing signs of right atrium hypertrophy. echocardiography is more useful for detection by measuring systolic pulmonary artery pressure, which is > mmhg, and the exclusion of left ventricle dysfunction. the formal pah diagnosis remains the right heart catheterization [ , ]. while liver abnormalities are most often limited to mild to moderate increases in aminotransferase activity which is frequent (up to % of cases), fulminant and fatal hepatitis have been reported [ , ] . the potential role of aspirin, nonsteroidal anti-inflammatory drugs (nsaids), or methotrexate has been mentioned by some authors [ ] . thus, clinicians should closely monitor liver function from the onset of the disease and particularly after prescription of potentially hepatotoxic drugs. self-medication should be avoided. liver biopsy, if performed, reveals nonspecific portal infiltrates of lymphocytes, plasma cells, and polymorphonuclears [ , , ] . hepatocytic lesions or massive necrosis has been described in fulminant hepatitis with rapidly progressive hepatic insufficiency. liver transplantation was necessary in a few exceptional cases [ , ] . amyloid a amyloidosis is becoming extremely rare owing to the better ability of aosd treatments to control inflammation [ ] [ ] [ ] [ ] [ ] . it could be observed in refractory patients or in patients who did not have access to adequate treatments for long periods of time. multiple organs can be involved. many other symptoms attributed to aosd have appeared in the literature. as these are almost exclusively in case reports, drawing conclusions is difficult: -ophthalmologic involvement related to sicca syndrome, conjunctivitis, uveitis, or episcleritis [ , , ] . -neurological manifestations, such as ischemic stroke, aseptic meningitis, or encephalitis [ ] . such symptoms might reveal hepatic failure or coagulation dysfunction, as in dic, rhl, or tma. -renal involvement limited to isolated proteinuria or related to glomerular or interstitial nephritis [ , ] . acute renal failure is possible in the context of severe myositis, hepatic insufficiency, or hematological complications [ , , [ ] [ ] [ ] . -secondary amyloid a amyloidosis (aa amyloidosis) is nowadays exceptional and is related to sustained uncontrolled inflammation [ ] [ ] [ ] [ ] [ ] . aosd is an exclusion diagnosis. because of presentation heterogeneity and of the lack of specificity of the clinical and biological manifestations, many other diagnoses might be evoked and afterward ruled out, before reasonably considering aosd (table . ) [ , , , ] . before finally diagnosing aosd, it is thus essential to first rule out neoplastic, infectious, or inflammatory conditions that can mimic the disease. infectious diseases represent the most complex issue in differential diagnoses because aosd therapies can be highly deleterious. the most misleading diagnoses are probably deep occult infections (e.g., biliary tract abscess or bone marrow infestation) or subacute endocarditis [ ] . whole blood culture, urine testing, and synovial fluid culture seem the minimal workup, while other tests might be considered depending on the context. among malignancies, malignant lymphomas-either hodgkin's disease or non-hodgkin's lymphomas-are probably the most misleading and the first to exclude if nonsymmetrical, fixed, and indurated lymph node enlargement is present [ , ] . less frequently, systemic inflammatory conditions have been described in the course of lymphoid leukemia or in paraneoplastic syndromes associated with solid cancers of either the lung or the breast [ , ] . autoimmune diseases that can mimic aosd include vasculitis, especially polyarteritis nodosa, and polymyositis [ ] . for patients with mainly chronic arthritis, the distinction between seronegative ra and aosd can be difficult. besides autoimmune diseases, hereditary or nonfamilial autoinflammatory syndrome (ais) can present with symptoms close to aosd because of the combination of fever, skin lesions, and arthritis in addition to other symptoms that differ among the ais [ ] [ ] [ ] . although the hereditary ais often begins during childhood, the diagnosis tends to be made when the individual is an adult; examples are hyperimmunoglobulin d (igd) syndrome or tnf receptor-associated periodic syndrome (traps). different mutations have been associated with different hereditary ais, but, to date, none has been associated with aosd. however, the similarities between these diseases and aosd might suggest common pathogenic pathways and place aosd as a nonfamilial, sporadic form of ais. finally, in rare cases, the severe and systemic drug hypersensitivity reaction can mimic aosd. the presence of urticarial skin lesions and increased eosinophilic pmn count are suggestive features, as is exposure to a potential agent within the previous days or weeks. after comprehensive investigations, no clinical sign or biological abnormality can be considered aosd specific enough to ascertain the diagnosis. thus, classification criteria may be helpful, although they have been developed more for clinical research than diagnosis. two sets of criteria have been validated (table . ). the yamaguchi criteria set, published in , is the most widely used [ ] ; however, it contains exclusion criteria that are problematic in clinical practice. the fautrel criteria set has the advantage of including ferritin and gf levels as diagnostic biomarkers and does not require exclusion criteria [ ] . in a recent validation study, both sets showed high sensitivity and specificity [ ] . the most relevant serum biomarkers identified to date are listed in table . . ferritins are the major biomarkers for aosd. serum ferritin level is a key biomarker to assess disease activity, but probably less relevant for diagnosis since its specificity is limited and no clear threshold has been identified so far [ , ] . the diagnostic value of glycosylated ferritin level, with the threshold of gf≤ %, is much more interesting [ , ] . (continued) identifying the disease subset might orientate the therapeutic strategy c serum ferritin levels are significantly higher in the systemic subtype [ ] , but high ferritin levels after adequate treatment may predict chronic articular course [ ] d calprotectin levels help rule out rheumatoid arthritis, but further studies are needed to validate it as a diagnostic biomarker because of no statistical difference between aosd and septic patients, although the populations were small [ ] e elevated plasma levels of il- β, il- , and tnfα have been found during aosd, but the cytokine profile is not specific and cannot differentiate aosd patients from those with sepsis f s a was found an efficient diagnostic and monitoring biomarker in systemic juvenile arthritis, but further studies are needed for validation in aosd procalcitonin, a marker of severe systemic infection, was also found elevated in patients with active aosd and does not appear relevant to distinguish acute infection from aosd flare [ , ] . serum level of calprotectin (i.e., s a /s a protein) could be an additional disease activity biomarker because alarmins seem to play a key role in aosd pathogenesis. however, it is not specific to aosd and may be elevated in many other inflammatory conditions [ , , , ] . finally, serum amyloid a protein (saa) is an inflammatory biomarker found predictive of amyloidosis [ ] . several research works assessed serum cytokine levels. high serum levels of il- , il- , and il- have been found in systemic forms of aosd and can be considered activity biomarkers. however, their additional value on top of crp level and other inflammatory biomarkers is unclear, and these cytokines are clearly not specific to aosd. thus, they are not recommended in routine investigation. of importance, il- seems to play a key role in rhl. age products and rages, involved in aosd pathogenesis, may be elevated in other inflammatory disorders. high serum levels have been associated with polycyclic or chronic/progressive evolution patterns [ ] . serum level of soluble cd , a macrophage activation biomarker, is elevated in aosd but is not specific to the disease. finally, several chemokines (c-x-c motif chemokine ligands and , macrophage inhibitory factor) have been found to be diagnostic biomarkers for aosd, which needs to be confirmed in larger patient samples [ ] . aosd evolution can be quite diverse and currently is impossible to predict. several patterns of evolution have been described, mainly based on case series and not robust epidemiological studies [ , , , , ]: it combines systemic manifestations and joint involvement: the initial flare evolves over a few weeks; remission is achieved with steroids or other immunomodulatory agents after a few days or weeks; and treatments can be progressively tapered and then stopped without relapse after a few months. the pattern may represent - % of cases. it is characterized by aosd relapses after a few months or years, under immunomodulatory treatment or after its discontinuation. in this pattern, one classical presentation is a first flare during childhood-diagnosed sojia, followed by sustained drug-free remission for several years, and then a relapse in adulthood. in most of these cases, recurrences combine systemic and joint involvement. the pattern represents - % of cases. in this form, a continuous inflammation is responsible for chronic and frequently erosive joint involvement with regular systemic flares. this pattern was the most frequent, estimated at - % of cases, in old series [ , , ] . although no new estimates are available, this pattern should be less likely with the most recent targeted therapies. the prognosis of aosd is dominated by potential life-threatening events as well as progressive functional impairment. the life prognosis is dominated by the severity of the following visceral involvements, in isolation or combination: hematological complications, such as dic, tma, or severe rhl; fulminant hepatitis with rapid liver failure; and acute respiratory distress syndrome, extensive myocarditis, or multiorgan failure [ , ] . importantly, exacerbation of these serious symptoms has been reported within the first days of treatment initiation, especially of biological therapies. this necessitates a close and very careful monitoring of such patients at the start of treatment. in addition to organ involvements, the development of secondary amyloidosis of the aa type remains possible in long-standing refractory chronic articular aosd [ ] . functional prognosis depends mainly on erosion and joint destruction in chronic articular aosd, which affects approximately one-third of patients [ , , ] . no clear predictor of erosive joint disease has been identified, but, as joint involvement is often limited, functional prognosis in aosd seems to be less severe than in ra or other inflammatory arthritides [ ] . corticosteroid side effects are also common because steroids are usually prescribed in high doses and the long term, which might contribute to the overall prognosis in aosd. aosd is rare, and no randomized controlled trial has been performed. thus, the only information on therapy is from observational studies and retrospective case series. recently, the management of aosd benefited from the proofs of the efficacy of targeted biotherapies. although nsaids, including aspirin, have been effective in systemic jia, they are rarely effective in aosd; only % of patients have achieved control with this therapy [ , , ] . of the nsaids, indometacin - mg/day seems to be the most effective [ , ] . liver enzymes must be monitored at the initial stages of the disease because severe hepatitis has been suggested to be related to treatment with nsaids [ , , ] . acetaminophen may help to reduce fever, although it is often not enough to suspend it. other analgesics might be necessary to relieve intense myalgia and joint paint. once the diagnosis is established, corticosteroids are usually required to induce symptom remission. optimal dosing relies on medium to high doses (i.e., . - mg/ kg/day of prednisone equivalent) [ , , , , ] . patients with serious visceral involvement might achieve a quick response with intravenous infusion of high-dose methylprednisolone [ , , ] . response to corticosteroids is often dramaticwithin a couple of hours or a few days [ , ] . the consensus is lacking on a therapeutic tapering scheme once clinical remission is achieved. however, owing to the potentially serious side effects of cumulative corticosteroid therapy, many others currently recommend to achieve the dose of . mg/kg/day after weeks of therapy and to stop completely after three months. if this is not possible, the response should be considered inadequate, and a diseasemodifying treatment, especially a targeted therapy, should be started (fig. . ) [ ]. methotrexate is used as an immunomodulatory agent in rheumatoid arthritis. it is efficient in controlling aosd disease activity and allowing for steroid dose sparing [ , [ ] [ ] [ ] . however, whether methotrexate can prevent or limit joint structural damage in the erosive form of aosd, as has been shown for ra, is unclear. the presence of liver enzyme abnormalities does not contraindicate methotrexate prescription, but close biological monitoring is necessary [ , , , ] . of importance, methotrexate can be associated with anti-il- -or anti-il- targeted therapies. different targeted therapies have been used to treat refractory aosd (table . ). the medical need is high, keeping in mind the unsatisfactory rates provided by classical therapies with nsaids or conventional disease-modifying antirheumatic drugs (dmards). the use of glucocorticoids is also limited by the known longterm safety issues, especially with higher doses. in fact, for at least - % of patients, the disease cannot be controlled by glucocorticoids even when combined with conventional dmards, such as methotrexate. the use of il- inhibitors for aosd contributed to the revival of considering this mode of action in rheumatology and now represents the primary choice for treating autoinflammatory diseases in general. however, it took surprisingly long until clinical development moved forward from cohort studies to randomized, placebocontrolled trials. currently, two il- blocking compounds are the focus of phase / phase studies with different designs. anakinra, a recombinant il- receptor antagonist, was the first biologic showing beneficial effects in treating the systemic and articular features of aosd in many case series, uncontrolled trials, and different national surveys. although the evidence for its efficacy has not been proven by controlled trials, the overall number of more than published cases provides convincing results. in fact, a clear and sustained improvement was described for systemic and also arthritic symptoms in most treated cases [ , [ ] [ ] [ ] [ ] [ ] . systemic features seem to show a more rapid response, and longer exposure seems usually required for improvement of arthritis. of major importance for long-term safety were consistent reports of a reduction or even discontinuation of glucocorticoids use as well as nsaids. in this context, meta-analyses from eight case series and three national surveys meeting predefined quality standards and including more than anakinra-treated aosd patients revealed a remission rate of approximately % and a reduced use of steroids in approximately % of patients [ ] . recently, a large observational retrospective multicenter study from italy added evidence for a strong impact on disease activity with the pouchot score as well as clinical and serological features regardless of sex, age, disease pattern, or co-medication [ ] . since september , a randomized, double-blind, placebo-controlled, multicenter, phase study in the united states has recruited patients for investigating two different doses of anakinra ( mg/kg/day [max mg/day] or mg/kg/day [max mg/day]) to evaluate its efficacy and safety in patients newly diagnosed with sojia and aosd. the primary end point, american college of rheumatology (acr ) response, has already been evaluated at week ; however, the overall treatment period is short, with only weeks of exposure followed by a -week safety follow-up [ ] . of note, anakinra is the only il- blocker for which we have substantial long-term results in terms of efficacy and safety in aosd [ ] , and it has now been approved in this indication. in contrast to monogenic autoinflammatory diseases, in aosd, remission can continue in some cases even after treatment is stopped. however, a relatively high withdrawal rate of % has been reported owing to loss of response over time and also to frequent injection site reactions to the required daily administrations. the other approved biologic for aosd is canakinumab, a fully human antibody against il- β [ ] . furthermore, canakinumab is also approved for treating other autoinflammatory diseases including cryopyrin-associated periodic syndromes, familial mediterranean fever, tnf receptor-associated periodic syndrome, and hyper-igd syndrome as well as sojia. although the overall experience with canakinumab in aosd is still limited, the reported response of systemic features and arthritis was rapid and sustained over many months to years in most patients, frequently allowing for tapering of steroids [ ] [ ] [ ] . of note, canakinumab was found highly effective for patients with aosd who were difficult to treat, including those showing failure of nsaids, glucocorticoids, and other il- inhibitors. with these promising results, a placebo-controlled, randomized, multicenter, phase study of canakinumab in aosd is ongoing [ ] . the dosage of canakinumab with monthly injections of up to mg/kg body weight (maximum dos of mg) is based on the pharmacokinetic profile in adolescent patients with sojia. the primary aim is to investigate the efficacy of canakinumab in patients with aosd and active joint involvement in terms of the proportion of patients with a clinically significant reduction in disease activity (disease activity score in joints [das ] > . ) following a treatment period of weeks. the overall placebocontrolled period is weeks, but nonresponders can be unblinded and show treatment rescue at week . the core study is followed by an optional long-term extension over years to provide additional safety results in the adult population. in summary, the published data for anti-il- agents in refractory aosd clearly show a consistently high rate of full or partial remission with the additional achievement of lowering or stopping glucocorticoid medication [ ] . inhibition of il- signaling is possible by two different approaches, the direct neutralization of the cytokine or the blockade of the respective receptor. none of these mechanisms has been investigated in aosd in a controlled setting of clinical trials in detail. for the two different il- receptor antagonists currently available in daily practice for treating rheumatic diseases, only the tocilizumab aosd case series has been published and reported at conferences [ ] [ ] [ ] [ ] . to summarize, the observed anti-inflammatory effects were strong, rapid, and sustained for most of the cases. usually, the systemic features disappeared entirely, but also the arthritic manifestations improved, and the serologic activity decreased [ , ] . however, because of limited data, the likelihood of response cannot be estimated as clearly as for il- inhibitors and seems to be between % to % in terms of full remission. a recently published meta-analysis of ten original studies ( individuals) on the efficacy of tocilizumab and aosd revealed overall high partial and complete remission rates of - %, respectively. tocilizumab prevented new flares, was well tolerated, and could significantly reduce the need for corticosteroids [ , ] . whether other il- inhibitors have similar effects remains unknown. as shown for ra treatment, with aosd, differences could exist with respect to safety, especially in comparison to the direct cytokine inhibitors. also, the advantages or disadvantages that distinguish il- and il- inhibition are unclear. although clearly il- as well as il- inhibitors have high response rates and represent alternative approaches in aosd, we have no way to predict the individual effects and define the ideal treatment algorithm. a novel compound is tadekinig alfa, a recombinant human il- binding protein [ ] . this drug was tested in healthy volunteers and patients with psoriasis and ra in phase i studies and demonstrated a good safety and tolerability profile with only mild adverse events at the injection site. because of the postulated role of il- in the pathogenesis of aosd, it was a logical step to investigate the effects of tadekinig alfa in this condition. a first open-label, dose-finding phase study involving multiple centers in europe was designed to capture safety information as the primary outcome. two doses ( and mg) were administered during weeks, and patients were followed up for more weeks. as a secondary outcome measurement, the efficacious dose at weeks was defined as normalization of body temperature and decrease of crp level by % or more of baseline values. although limited by the low patient number and short observation period in the study, the safety profile of tadekinig alfa was overall acceptable, with injection site reactions and infections being the most frequent adverse events. in terms of efficacy, reduced level of crp as well as other inflammatory markers (including il- , ferritin) was detected in some patients. furthermore, an improvement in skin rash and a slight but significant reduction in the prednisolone doses were reported. in summary, inhibition of il- by tadekinig alfa could be a promising new approach in asod that needs to be investigated in a controlled setting. many other treatments have been tried in aosd, with varying degrees of success. stimulated by encouraging results from other chronic inflammatory joint diseases, especially ra, tnf inhibitors have been the first biologics used for aosd [ ] [ ] [ ] [ ] [ ] [ ] . however, driven by the deceptive impression that aosd, with a predominant arthritic phenotype, is a subgroup of seronegative ra, the results from uncontrolled trials involving usually small cohorts of patients were inconsistent. since favorable outcomes have been seen in only a few patients without any specific characteristics, tnf inhibitors can only be considered third-line drugs, preferentially for patients with chronic arthritis. cyclosporine a has been proposed before the era of biotherapies, in patients with systemic features of aosd or with rhl, with an interesting efficacy. however, the tolerance of this drug limits its use, but it certainly can be of interest in complex or refractory situations. there is henceforth no place for intravenous immunoglobulins in aosd treatment, owing to the efficacy of the other therapies available. the strategy we propose for aosd patients is summarized in fig. . . management should include, firstly, symptomatic care in a medicine department or intensive care unit (icu); secondly, active and rapid search for an infection; and, thirdly, introduction of high-dose steroids. since rhl has described under il- therapy, it appears prudent to introduce this treatment only after rhl control by a few days of high-dose steroids. dic is a medical emergency whose care combines: . supportive measures, which could include platelet transfusion, coagulation factors (or fresh frozen plasma), and fibrinogen (in cryoprecipitate) infusion to control severe bleeding or sometimes heparin if no active bleeding is present [ ] . . specific anti-inflammatory agents often decided in multidisciplinary rounds, with mainly high-dose corticosteroids, either intravenous (methylprednisolone mg/kg/day) or oral (prednisone mg/kg/day) [ ] . to limit steroid exposure, the addition of other immunomodulatory agents, such as il- or il- blockers or cyclosporine, is recommended [ , , [ ] [ ] [ ] . tma treatment is complex and mandatorily includes [ ]: . supportive care in an icu, with plasma exchange being central [ ] . high-dose steroids intravenously and then orally other immunomodulatory agents proposed for refractory cases include anakinra [ ] , tocilizumab [ ] , intravenous immunoglobulins, cyclophosphamide, azathioprine, cyclosporine a, and rituximab [ , ] . despite this treatment, mortality associated with tma remains high, up to % [ ]. their treatment combines supportive care, high-dose steroids, and frequently il- or il- inhibitors. regarding pah, early diagnosis is central for the prognosis of this complication, with mortality remaining as high as % [ ] . the diagnosis includes [ , , , , , , ]: . hospitalization in an icu and rapid referral to a specific reference center for multidisciplinary round discussions. . vasodilating therapies frequently combining endothelin receptor antagonists (bosentan, ambrisentan), phosphodiesterase- inhibitors and other guanylate cyclase stimulators (sildenafil or others), and prostacyclin analogues. . high-dose steroids, initially intravenously and then orally. . rapid introduction of biologic immunomodulatory agents targeting il- or il- or other immunosuppressive agents, such as methotrexate, azathioprine, cyclophosphamide, cyclosporine, or rituximab. however, these treatments must be carefully monitored because a few cases of pah worsening have been described after their introduction. pah deserves to be well known by physicians caring for aosd patients because it was not described in most of the initial aosd case series for several reasons: now better diagnostic tools for pah; differential efficacy of large-spectrum immunomodulatory agents, such as steroids, as compared with more targeted therapies, such as il- or il- blockers; and exposure to 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part ii. management, outcome, and prognostic factors carpal arthritis with ankylosis in late onset still's disease a controlled study of the long-term prognosis of adult still's disease methotrexate treatment in patients with adult onset still's disease-retrospective study of japanese cases low dose methotrexate treatment in adult still's disease corticosteroid sparing effect of low dose methotrexate treatment in adult still's disease rapid responses to anakinra in patients with refractory adult-onset still's disease il -receptor antagonist anakinra provides long-lasting efficacy in the treatment of refractory adult-onset still's disease anakinra treatment in patients with adult-onset still's disease is fast, effective, safe and steroid sparing: experience from an uncontrolled trial interleukin- receptor antagonist (anakinra) treatment in patients with systemic-onset juvenile idiopathic arthritis or adult onset still disease: preliminary experience in france anakinra in adult-onset 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infliximab: first experiences infliximab in the treatment of adult still's disease refractory to conventional therapy club rhumatismes et inflammation. tumour necrosis factor alpha blocking agents in refractory adult still's disease: an observational study of cases tocilizumab in refractory adult still's disease: refractory asd and tocilizumab tocilizumab in adultonset still's disease: the israeli experience tocilizumab for the treatment of adult-onset still's disease: results from a case series treatment of refractory adult-onset still's disease with tocilizumab: report of two cases and review of the literature a pilot study on tocilizumab for treating refractory adult-onset still's disease. sci rep efficacy of tocilizumab therapy in korean patients with adult-onset still's disease: a multicentre retrospective study of cases efficacy and safety of tocilizumab with inhibition of interleukin- in adult-onset still's disease: a meta-analysis the effect of tocilizumab on preventing relapses in adult-onset still's disease: a retrospective, single-center study open-label, multicentre, doseescalating phase ii clinical trial on the safety and efficacy of tadekinig alfa (il- bp) in adult-onset still's disease successful treatment of refractory adult onset still's disease with rituximab refractory adult-onset still disease successfully treated with abatacept efficacy of abatacept in a refractory case of adult-onset still's disease adult onset still's disease-the evidence that anti-interleukin- treatment is effective and well-tolerated (a comprehensive literature review) s.m. has received consultant fees from bms and pfizer. e.f. has received consultant fees from abbvie, biogen, bms, celgene, janssen, lilly, medac, msd, nordic pharma, novartis, pfizer, roche, sanofi-aventis, swedish orphan biovitrum, and ucb, as well as a research grant from bms, lilly, novartis, pfizer, and roche.b.f. has received consultant fees from abbvie, biogen, bms, celgene, janssen, lilly, medac, msd, nordic pharma, novartis, pfizer, roche, sanofi-aventis, swedish orphan biovitrum, and ucb, as well as research grants from abbvie, lilly, msd, and pfizer. key: cord- -of vx og authors: saghazadeh, amene; rezaei, nima title: the physical burden of immunoperception date: - - journal: biophysics and neurophysiology of the sixth sense doi: . / - - - - _ sha: doc_id: cord_uid: of vx og the previous chapter introduced the immunoemotional regulatory system (immers). also, there was a brief discussion about psychological states/psychiatric disorders that so far have been linked to the immers. the present chapter considers another aspect of the immers in which physiological states/physical diseases can be fit to the immers. such as pemphigus [ , ] . further, human studies provided evidence pointing to the increased development of emotional problems and edr-related disorders in patients with various types of aids, such as sle and multiple sclerosis (ms), in a disease state/severity-dependent manner [ ] [ ] [ ] [ ] [ ] [ ] . for example, among patients with childhood-onset sle, % manifest neuropsychiatric sle (nsle). mood and anxiety disorders were the most common psychiatric conditions with the prevalence rate of % and % [ ] . even about % of patients with sle without cns manifestations suffer from psychological distress compared with % in controls. it is, thus, not surprising that both emotional coping and depressive symptoms were correlated with non-nsle [ , ] . interestingly, there was an increased activation of the brain regions related to emotion regulation/processing (e.g., the amygdala and superior temporal) in sle patients. however further analyses led to identifying this increased activity of emotional circuit as a consequence of cns involvement by sle [ ] . among patients with ms, emotional troubles were more than twofold more likely to occur in patients who had an exacerbation or progressive nonremitting ms compared to stable patients. this was reflected by an increased rate of using emotion-focused coping styles in patients with relapsingremitting multiple sclerosis (rrms) compared to stable patients [ , ] . mood disturbance was correlated negatively with sil- r levels and positively with joint pain in patients with ra [ ] . consistent with data from human studies, animal experiments have also supported the link between emotionality-related behaviors and aids. clearly, aids result from immdr. interestingly, the aids-related immdr has been observed in the specific brain regions associated with emotional behaviors, particularly anxiety-and depressive-like behaviors [ ] . in this manner, the link between aids and immers is strengthened. a high rate of increased emotionality and emotional-like behaviors in aids led to propose the term autoimmune-associated behavioral syndrome (aabs). studies emphasize the pivotal role of cytokines and neuroendocrine factors in the pathogenesis of aabs [ ] . b-cell-activating factor (baff) transgenic mice model, which is used as an experimental model of systemic lupus erythematosus (sle), rheumatoid arthritis (ra), and sjögren syndrome, exhibited an anxious phenotype along with the following changes in immune brain signaling, such as increased igg titers in the hippocampus, hypothalamus, and cortex and increased cd (as a maker of activated microglia/macrophages) and gfap (as a maker of activated astrocytes) immunoreactivity in the hippocampus in mice at . - months of age, but not in young ( months of age) mice [ ] . eae models of ms showed an increase in levels of il- β and tnf-α in the hypothalamus. this indicates an inflammatory central basis behind anxiety-and depressive-like behaviors [ ] . these emotional deficits were shown to display before the onset of ms [ , ] . consistently, behavioral problems usually manifest before symptoms of impaired cognitive and motor performance in dementia. moreover, this model showed an early (at day ) and a meaningful increase in circulating cytokine levels and cd + t cell counts. of note, these inflammatory markers began to decrease in the periphery (at day ) almost when their infiltration in the cns (at day ) started [ ] . in the mrl-lpr model of aid, which is a well-documented model of emotional deficits [ , ] , a reduced preference to glucose, and as an index of emotionality, was detected in -to -week-old mice [ ] . this deficit could be diminished by immunosuppressive treatment with cyclophosphamide and was pronounced by means of chronic administration of il- [ ] . along with psychosocial stressors, either chronic and acute, and social networkrelated factors (i.e., social ties, social conflict, and social support), the experience of unpleasant emotions, including anger, depression, sadness, and stress, or, in general, extremely exciting emotions, often promptly, pulls susceptible individuals into a steep road leading to cardiovascular events, particularly acs (for review see references [ ] [ ] [ ] ). for example, emotional stress is ranked as the second most common neuropsychological cause of acute myocardial infarction (ami) owing to its record in approximately - % of these patients [ , ] . at the molecular levels, these patients have shown increased levels of the proinflammatory cytokines (e.g., tnf-α, il- , il- , il- , and il- ) and decreased levels of the anti-inflammatory cytokine (il- ). thus, it is not surprising that the inflammatory response and respective cytokines are supposed as one of the possible mechanisms linking the experience of negative emotions or er-related disorders and the progression of cardiovascular diseases, of course along with the neuroendocrine system and apoptosis signaling pathways [ , , [ ] [ ] [ ] [ ] . it is to be noted that when patients with cardiovascular diseases are stratified according to their emotional background, some cytokines are more highlighted than others, for example, tnf-α and il- , but not il- , considering depressive symptoms in chf patients [ ] . cognitive reappraisal is found to correlate positively with the engagement of the lateral and prefrontal regions and inversely with the engagement of the amygdala and medial orbitofrontal cortex [ ] . the role of the inflammatory cytokine il- in the progression of cardiovascular diseases is widely appreciated [ ] . studies show that il- is significantly involved in efforts to arbitrate between the sides of the relationship between the reappraisal-related activation of the dorsal anterior cingulate cortex and preclinical atherosclerosis (evaluated by carotid artery intima-media thickness and inter-adventitial diameter) in healthy individuals [ ] . the normative aging study carried out a -year follow-up study in older men (mean . ± . years). there was a dose-response relationship between negative emotions, evaluated by the minnesota multiphasic personality inventory (mmpi), and incidence of coronary heart disease (chd) within the duration of the study (p = . ) [ ] . meanwhile, the circulating levels of il- were positively associated with the reappraisal-related activation of the dorsal anterior cingulate cortex in healthy subjects [ ] . however, higher reappraisals and suppressions were positively and inversely associated with the serum levels of crp, respectively [ ] . on the other hand, the reflection of watching the ice hockey match, as a real-life emotional excitement, on serum levels of endothelin- (et- ) and il- was more pronounced in spectators with coronary artery disease compared with healthy spectators [ ] . patients with type d personality display concurrently two absolute opposite, positive and negative, tendencies towards the experience and the expression of negative emotions by themselves and in front of other people, correspondingly. meta-analysis studies and comprehensive literature reviews have revealed that this type of personality is positively associated with contracting cardiovascular conditions and their consequent mortality and morbidity, as well as with a constellation of non-cardiovascular complaints (for details, see [ ] [ ] [ ] ). also, individual studies, either cross-sectional or follow-up, have providence evidence of increased levels of proinflammatory cytokine tnf-α and its receptors, stnfr and stnfr [ ] [ ] [ ] [ ] , an enhanced il- /il- ratio, and decreased levels of anti-inflammatory cytokine, il- , in chf patients with type d personality compared to those without type d personality [ ] . interestingly, in chf patients, the inflammatory effect of type d personality appears to resemble closely the effect of aging. there was a similar increased pattern of stnfr and stnfr in younger chf patients with type d personality and older patients without this personality trait [ ] . plasminogen activator inhibitor- (pai- ) is a factor contributing to thrombosisrelated cardiovascular diseases in elderly people. both cytokines and hormones take part in the regulation of the gene expression of pai- (for review, see reference [ ] ). in a model of premature immunosenescence, mice were assigned to either fast or slow group if the amount of time taken to explore the first arm of the maze was ≤ s or > s, correspondingly [ ] . when compared to fast mice, slow mice expressed high emotional response to stress and had lower life span [ ] . at the immunological levels, slow mice showed a reduction in proliferative response to concanavalin a (con a) and related release of il- and il- β and nk cell activity, while increasing the production of tnf-α [ ] . an investigation on women who had to undergo breast biopsy indicated that this procedure should be considered as an emotional stressor if the final diagnosis is determined benign. in parallel with this emotional stress, the immune system prepares itself before the procedure and seeks for ways to prolong this preparation even months after the procedure. this is a reflection of the joint regulation of our body by both the immune system and the emotional brain [ ] . the immune system responds to this challenge by decreasing nk cell activity, decreasing production of ifn-γ, and increasing production of il- , il- , and il- [ ] . further, there was a significantly positive relationship between mothers with breast cancer and their adult daughters on distress levels. this persuaded scientists to investigate the immune profile and its association with distress in daughters' group. daughters' distress levels were inversely associated with il- , il- , and ifn-γ production and also with il- -induced natural cytotoxic activity (nca) [ , ] . further, nca activity and the production of th cytokines were both negatively related to the emotional distress degree [ ] . antoni and his colleagues accomplished a genome-wide transcriptional analysis on leukocyte samples taken from women subject to the treatment of stage -iii breast cancer and demonstrated that the negative affect, evaluated by the affects balance scale (abs), was significantly associated with greater than % increased expression of leukocyte transcripts such as proinflammatory marker-related genes [ ] . another multiplex analysis on circulating concentration of cytokines identified the il- profile as the predictor of physical and cognitive functioning and also the vascular endothelial growth factor (vegf) profile as the predictor of emotional functioning [ ] . further, the experience of childhood emotional neglect/abuse was associated with lower levels of nca at the first evaluation after breast cancer surgery [ ] . emotional processing and expression (evaluated by emotional approach coping scale), respectively, tended to be inversely and positively correlated with plasma levels of il- , soluble tnf-receptor type (stnf-rii), and crp in male patients with prostate cancer [ ] . however, among those correlations, two of them, the correlations of emotional expression with il- and crp, were not found significant (p < . ) [ ] . in vivo model of ultraviolet-b light-induced squamous cell carcinoma concluded that the high stress and anxiety levels can leave mice prone to the more considerably progression of the tumor through increasing the expression of immunosuppressive (ccl and t regulatory cells) and angiogenic (vegf: vascular endothelial growth factor) markers and decreasing the expression of antitumor immune markers (ctack/ccl , il- , and ifn-γ) [ ] . on the other side, a peripheral tumor, by itself, could lead to a reduction in the hippocampal function, as reflected in increased depressive-like behaviors and memory impairment. this was, at least in part, underpinned by triggering an inflammatory process both in the hippocampus (↑il- β, ↑il- , ↑il- , and ↑tnf-α) and in the circulation (↑il- , ↑il- β, and ↑il- ) [ , ] . this process was found to be significantly strengthened in infection models compared to peripheral tumor models, explaining the presence and absence of the sickness state in these models, respectively [ ] . using hospital anxiety and depression scale (hads) test, it was estimated that nearly half of patients with hemodialysis (hd) were in a depressive mood, which was significantly higher than what was reported for control group [ ] . also, there was a higher production of il- in hd patients with anxiety (hads≥ ) than those without anxiety (hads≤ ) [ ] . at least one major psychiatric illness, particularly depression, affects greater than % of hiv patients [ ] . by virtue of the fact that specific substances of abuse aggregate further the situation of hiv patients at the neuropathological level [ ] , the inverse correlation between affect regulation and regular substance motivates us to utilize er as a therapeutic intervention in this population [ ] . hiv patients encounter commonly with situations where the social self is threatened. this threat causes shame feelings, which have been associated with increased proinflammatory cytokines [ ] . both emotional and environmental factors affect the gut [ ] . similar to that mentioned for asthma, this affect is mediated by crf and similar neuropeptides and also by mucosal mast cells [ ] . immunoregulatory factors along with genetic and environmental factors contribute to the pathogenesis of inflammatory bowel disease (ibd). patients with ibd confront various er-related problems in their social life in a disease severity-dependent manner, such as higher sensitivity to negative emotions, fewer dropping into bar/disco and delayed falling in love, and experiencing more depressive and anxious symptoms, not only compared to controls, but also compared to patients with other chronic conditions [ ] [ ] [ ] [ ] [ ] . neuroimaging studies have indicated a reduction in both the volume and the activation of brain regions related to emotional processing [ , ] . the gray matter (gm) volume of both the frontal cortex and the anterior midcingulate cortex was reduced in patients with crohn's disease (a type of ibd) compared to controls. more interestingly, disease duration was found to correlate with the gm volumes of some brain regions, importantly, limbic areas [ ] . also, patients with ulcerative colitis (another type of ibd) showed reduced activity, evaluated by bold signal, within the amygdala, thalamic regions, and cerebellar areas during the emotional visual task, compared to the control group [ ] . for the first time in , cohen and his colleagues demonstrated that higher psychological stress is associated with lower resistance to respiratory viruses (rhinovirus type , , or , respiratory syncytial virus, or coronavirus type e) in a dose-response manner [ ] , while positive emotional style (pes), but not negative emotional style (nes), was found to correlate inversely with susceptibility to common cold and upper respiratory infections following exposure to rhinoviruses and influenza a virus in a dose-response manner [ , ] . various regression analyses showed that this correlation is independent of prechallenge virus-specific antibody, virus type, age, sex, education, race, body mass, season, and nes, optimism, extraversion, mastery, self-esteem, purpose, and selfreported health [ , ] . by contrast, childhood socioeconomic status, as assessed by "the number of childhood years during which their parents owned their home," was found to correspond negatively with both the risk of illness and infection and, in a word, with vulnerability to common colds [ ] . this finding along with approximately the similar increased risk of common colds in "those whose parents did not own their home during their early life but did during adolescence" in "those whose parents never owned their home" [ ] indicate that (a) the childhood period takes more impression of socioeconomic status of their family than other lifetime periods (e.g., adolescence) such that (b) it would influence the mind-body background of future life. meanwhile, pes and nes were negatively and positively related to the subjective report of unfounded symptoms of common cold, respectively [ , ] . however, the basal protein levels of all the investigated proinflammatory cytokines, e.g., il- β, il- , and il- , were associated with illness symptoms/ signs after exposure to rhinoviruses. however, il- was the best cytokine which could predict nasal symptoms/signs [ ] . further, daily evaluation of emotional style and cytokine production in infected individuals on each one of days after exposure to rhinoviruses and influenza virus showed that the production of inflammatory cytokines including il- , il- β, and tnf-α was negatively related to positive affect (pa) on that day or on the next day [ ] . neurological diseases including parkinson's disease (pd) and alzheimer's disease (ad) are accompanied by serious shortfalls in emotional processing in a severity-dependent manner. for example, patients with frontotemporal dementia (ftd) represent a poor recognition of several basic emotions, e.g., anger, sadness, disgust, fear, and contempt. also, patients with the probable ad are more likely to fail to recognize fear and contempt compared with controls [ ] . in patients with mild ad, the recognition of more basic emotions are missed, and they are less able to differentiate between some emotions, e.g., happiness and sadness [ ] . apathy in patients with ad was found to correlate positively with dysfunction in the prefrontal and anterior temporal regions [ ] . regarding memory recall, individuals with ad presented no preference to recall better emotional memories other than nonemotional ones, standing in stark contrast to healthy subjects, either young or older [ ] . experimental models provided evidence that there are deficits in the emotional memory performance in ad, which can be diminished by treatment with cytotoxic necrotizing factor (cnf ) [ ] . this pleasant effect of cnf was accompanied by a reduced il- β expression in the hippocampus, along with other encouraging events, especially enhancing the energy amount evaluated by the atp (adenosine triphosphate) levels [ ] . there is a spectrum of behavioral problems in patients with ad [ ] . agitation is the second most common behavior in ad, after apathy. it has been associated with cognitive impairment [ ] . inflammatory changes appear to pave the way for agitation. there were higher il- β levels and decreased nk cell activity in both the morning and evening periods corresponding with preagitation and agitation phases of ad [ ] . even, esterling and his colleagues demonstrated changes in the immune profile of ad patients' spousal caregivers, either former or current. there was a reduced response of enriched nk cells to either ril- or rifn-γ cytokines in patients' caregivers than controls [ ] . interestingly, this response was related positively to the emotional and tangible social support levels [ ] . women with severe or morbid obesity had significantly increased levels of proinflammatory markers il- and hscrp in a bmi-dependent manner, which were closely related to anxiety and depression subscales of neuroticism, even after the bmi adjustment [ ] . since these patients had to undergo gastric surgery, these markers were measured again after surgery. interestingly, decreased levels of il- and hscrp were correlated with lower anxiety and depressive behaviors postoperation [ ] . the long-term maternal exposure ( weeks before mating and during pregnancy and lactation) to a high-fat diet (hfd) led to a decrease in the basal serum levels of cort in offspring [ ] . in addition, the steps toward normalizing the stress-induced cort levels were made with the lower speed in long-term hfd-exposed offspring than standard chow diet (sd)-exposed offspring at the end of stress challenge [ ] . regarding inflammation-related markers, the increased expression of il- and il- ra in long-term hfd-exposed offspring than chow-exposed ones in the amygdala was found in both females and males [ ] , while changes in the expression of nf-kb and i-kappa-b-alpha (ikba) were observed only in female, but not in male, offspring [ ] . mice subjected to short-term ( - weeks) hfd also exhibited anxiety-like behaviors in addition to learning and memory impairments and had significantly higher levels of homovanillic acid-a metabolite of dopamine-in their hippocampus and cortex but without any alteration in the gene expression of inflammatory markers [ ] . further, chronic western diet (wd) intake led to the increased responsiveness to lps, which was represented in higher and more prolonged protein/mrna measures of il- in both plasma and hypothalamus, while there was no significant difference in the plasma levels of other proinflammatory cytokines such as tnf-α, il- β, and ifn-γ between wd and sd groups [ ] . in parallel with the increased expression of il- , there was significantly increased mrna expression of socs- , which belonged to the suppressors-of-cytokinesignaling (socs) family of proteins, in the hypothalamus in wd than sd mice [ ] . however, the lps-induced mrna expressions of tnf-α and ifn-γ in the hippocampus were significantly higher in wd than sd mice. also, lps augmented the levels of adipokines, e.g., cst, leptin, and resistin, more significantly in wd mice when compared with mice exposed to sd [ ] . altogether, both short-term and long-term obesity in either young adult or maternally can lead to display disturbed anxiety-like behaviors and impaired learning/memory, and brain inflammation might be one of the reasons behind these hfd-related events [ ] [ ] [ ] [ ] . chronologically, at first, learning/memory and then anxiety-like behaviors are impaired, and disturbance in depressive-like behaviors is subject to exposure to an immune challenge, such as lps [ ] . a high age-adjusted prevalence rate of ~ % is estimated for metabolic syndrome (visceral obesity, dyslipidemia, hyperglycemia, and hypertension) in the united states [ ] . both er-and edr-related subscales have been associated with the metabolic syndrome factor [ ] . even, a disease pathway involving edr can be proposed, which is triggered by low socioeconomic status (ses), followed with low reserve capacity for high negative emotions and eventuated in the metabolic syndrome factor [ ] . inflammation plays a major role in metabolic syndrome [ ] . it has also been indicated that this role is not performed in the periphery merely, but a mice model of metabolic syndrome proved the presence of central inflammation (↑tnf-α, ↑il- β, and ↑il- ) in the hippocampus, explaining the anxiety-like behavior in this model [ ] . a possible pathogenic pathway for metabolic syndrome is initiated by emotional stress and ensuing enhancement in the levels of proinflammatory cytokines, e.g., il- , il- , and tnf-α. then, these cytokines lead to increased levels of ngf, which in turn stimulates a series of cascades toward insulin resistance and finally resulting in diabetes mellitus (for review, see [ ] ). skin diseases are frequently associated with troubled er, reflecting in problematic emotional expression. for instance, patients with psoriasis, who are more likely to be alexithymic, employ more control over negative emotions and more avoidance of emotional closeness and intimacy compared with controls [ ] . it may explain the negative relationship between psoriasis symptoms and affective expression in a severity-dependent manner [ ] . when patients with atopic dermatitis were compared to healthy controls, the effect of psychological stress on the various immune parameters, such as ↑ eosinophil count, ↑ cd + /cd + and cla + t cells, and ↑ cytokines (il- and ifn-γ), was significantly strengthened [ ] . the route from edr to immdr in acnegenesis has been explained elsewhere [ ] . in summary, emotional distress would make sebocytes prone to the increased expression of receptors for crh, melanocortins, b-endorphin, vasoactive intestinal polypeptide, neuropeptide y, and calcitonin gene-related peptide, and then the production of proinflammatory cytokines are stimulated by means of these receptors and binding to their ligands. this notion that sleep disturbances are, irrespective of their cause, seen as chronic stressors is supported by evidence at different levels, e.g., immunological, neuropathological, and neuroimaging studies (for review, see [ ] ). to assess the effects of sleep and its efficiency on susceptibility to respiratory infections, cohen and his colleagues conducted an investigation on healthy individuals and recorded at first their sleep duration and its efficiency within consecutive days and then made them exposed to rhinoviruses, and after days postchallenge, calculated the rate of clinical cold development [ ] . this investigation indicated that those who had average sleep duration (asd) ≤ hours were three times more susceptible to clinical cold than those with asd≥ hours. further, there was a . -fold increased risk of clinical cold in individuals with sleep efficiency < % compared to those with an efficiency of ≥ %. therefore, it is well expected that circadian arrhythmia has been associated with edr-related parameters, e.g., decreased social motivation/functioning, decreased exploratory anxiety, and decreased emotional functioning [ , ] . in a cancer population, the presence of circadian arrhythmia was associated with decreased levels of all the investigated cytokines, e.g., tnf-α, tgf-α, and il- [ ] . alexithymic patients were more likely to suffer from severe forms of stroke when compared with non-alexithymic patients. this alexithymia trait appears to contain an inflammatory component either as a cause or an effect [ ] . study of patients with a first-ever symptomatic ischemic stroke revealed that (a) the circulating levels of il- were correlated positively with the severity of alexithymia, (b) stratification of patients made this correlation more statistically significant in those who had right hemisphere lesions, and (c) these increased il- levels were pronounced in alexithymic (tas- score ) than non-alexithymic patients [ ] . in patients hospitalized for orthopedic injuries, the use of emotion-focused coping was found to correlate positively with the levels of proinflammatory cytokines, il- and il- , whereas it was negatively correlated with tgf-β levels [ ] . compared with controls who received placebo, circulating levels of cytokines, in particular, il- , were increased at hours after the first-ever typhoid vaccination. it coincided with significant mood impairment [ , ] . it has been elucidated that when subjects perform psychological tasks (i.e., stress condition) after injection, the il- response is inversely related to optimism in either the typhim vi typhoid vaccine or saline placebo group [ ] . in response to the implicit emotional face perception task, there was an increased activity in the subgenual anterior cingulate cortex (sacc) along with reduced functional connectivity between sacc and reduced activity within the 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serum cytokines correlated with altered behavior, serum cortisol rhythm, and dampened -hour rest-activity patterns in patients with metastatic colorectal cancer disease outcome, alexithymia and depression are differently associated with serum il- levels in acute stroke cytokine levels as potential biomarkers for predicting the development of posttraumatic stress symptoms in casualties of accidents inflammation causes mood changes through alterations in subgenual cingulate activity and mesolimbic connectivity neural origins of human sickness in interoceptive responses to inflammation dispositional optimism and stressinduced changes in immunity and negative mood key: cord- -g earfl authors: song, xiuchao; liu, miaohua; song, hao; ren, jinshen title: dynamical behavior of an svir epidemiological model with two stage characteristics of vaccine effectiveness and numerical simulation date: - - journal: advances in intelligent systems and interactive applications doi: . / - - - - _ sha: doc_id: cord_uid: g earfl an svir epidemiological model with two stage characteristics of vaccine effectiveness is formulated. by constructing the appropriate lyapunov functionals, it is proved that the disease free equilibrium of the system is globally stable when the basic reproduction number is less than or equal to one, and that the unique endemic equilibrium of the system is globally stable when the basic reproduction number is greater than one. in human history, infectious diseases have repeatedly brought great disaster to human survival. in recent years, the outbreak of some new infectious diseases (sars, influenza a (h n ), influenza a (h n ), etc.) has caused a great impact on people's lives. vaccines are biological agents made from bacteria, viruses, tumor cells and so on, which enable antibodies to produce specific immunity. vaccination can provide immunity to those who are vaccinated, can eliminate the spread of some diseases (such as smallpox) [ ] . in recent years, more and more authors study the epidemiological models with vaccination [ ] [ ] [ ] [ ] . some authors assume that vaccine recipients will not be infected [ , ] ; some other authors assume that vaccine recipients may still be infected [ , ] , but the probability of being infected is smaller than before vaccination. in fact, for some infectious diseases, the vaccinated individuals would not be infected for some time after vaccination. however, bacteria or viruses mutate as time goes by, and the efficacy of the vaccine is correspondingly affected, which makes it is possible for the vaccinated individuals to be infected. for example, the new h n influenza virus mutates more quickly, and the effectiveness of the vaccine depends largely on the extent of the virus mutation [ ] . based on the above facts, we assume that vaccine effectiveness has two stage characteristics: in the first stage, the vaccinated individuals will not be infected; in the second stage, the vaccinated individuals will be infected, but the probability of infection will be smaller than before vaccination. therefore, this paper studies the epidemiological model with two stage characteristics of vaccine effectiveness, on the basis of getting the basic reproductive number, by using appropriate functionals, the stability of the model is proved by the algebraic approach provided by the reference [ ] . in this work, we study the following epidemiological model: the model ( ) has the same dynamic behavior with the following system existence of equilibria obviously, system ( ) has a disease free equilibrium p ðs ; v ; v ; Þ, where it can be found the unique endemic equilibrium p à ðs à ; v à ; v à ; i Ã Þ from the following equations, and i à satisfies the following equation theorem. when r the p ðs ; v ; v ; Þ is global stable. and p à is global stable when r [ . proof. the global stability of p is firstly proved. consider the following lyapunov functional where for simplicity, denote using the algebraic approach provided by the reference [ ] , we will prove the function hðx ; x ; x Þ . firstly, we can get five groups x x g and the product of all functions within each group is one, then we have as the nonnegativity of b i ði ¼ ; . . . Þ; b must satisfy the following condition maxf ; pes c À kv g b minflð À qÞa; l v ; pes c g; it is easy to prove the existence of the positive number b . so hðx ; x ; x Þ and hðx ; in summary, when r \ we have l ; and when r ¼ ; we get l ; and l ¼ if and only if s ¼ s v ¼ v v ¼ v . the largest invariant set for ( ) on the set fðs; v ; v ; iÞ x : s ¼ s ; v ¼ v ; v ¼ v g is fp g. using the literature [ ] , we can prove the theorem. the numerical simulations on system ( ) were carried out. we can see that if r , then p ðs ; v ; v ; Þ is global stable (fig. ) and p à is globally stable when r [ (fig. ) . world health organization: immunization against diseases of public health importance two different vaccination strategies in an sir epidemic model with saturated infectious force stability analysis for sis epidemic models with vaccination and constant population size global stability of an epidemic model with latent stage and vaccination global analysis for a general epidemiological model with vaccination and varying population characterization of h n influenza a viruses isolated from humans long-term immunogenicity of hepatitis b vaccination in a cohort of italian healthy adolescents an algebraic approach to proving the global stability of a class of epidemic models reproduction numbers and subthreshold endemic equilibria for compartmental models of disease transmission the stability of dynamical systems acknowledgements. this paper is supported by the research fund of department of basic sciences at air force engineering university ( ). key: cord- -qwe ne q authors: poritz, mark a.; lingenfelter, beth title: multiplex pcr for detection and identification of microbial pathogens date: - - journal: advanced techniques in diagnostic microbiology doi: . / - - - - _ sha: doc_id: cord_uid: qwe ne q multiplexed nucleic acid-based tests for infectious disease have become a standard part of clinical laboratory practice. these tests provide a comprehensive syndrome-based approach to determine the etiological agent of disease. the technology underlying these different systems is reviewed here with a special focus on the biofire filmarray® platform. the literature on the clinical utility and cost-effectiveness of these platforms for respiratory, blood culture, and gastrointestinal infections is discussed. although there are reports showing a clear benefit to the patient or to the healthcare system from adopting a syndromic molecular approach, it is also apparent that clinical laboratories and healthcare providers are still learning how to take full advantage of the new systems. finally, some improvements to this technology that should appear in the next few years are discussed. these include automated pathogen-specific surveillance based on aggregating the data from these systems, a move toward point-of-care syndromic testing, and further decreases in time to result of the tests. over the last decade, a number of manufacturers have developed multiplexed in vitro diagnostic (ivd) platforms that can detect the nucleic acid signatures of many of the organisms responsible for infectious disease. some of these testing platforms offer limited test menus (i.e., influenza a, influenza b, and rsv), while others are designed to detect a more comprehensive set of potential pathogens that can cause a particular infectious disease syndrome (e.g., respiratory, gastrointestinal, sepsis, meningitis) [ ] [ ] [ ] . this chapter will describe fda-cleared and/or ce-marked multiplex assays that are designed to detect a comprehensive set of pathogens associated with a particular infectious disease syndrome (≥ assays/ test). these include the biofire (salt lake city, ut) filmarray ® system [ ] , the genmark (carlsbad, ca) esensor xt- ® [ ] and eplex® [ ] , and the luminex (austin, tx) xtag® [ ] , nxtag ® [ ] , and verigene® systems [ ] . in addition to being comprehensive with respect to pathogens responsible for a particular syndrome, these multiplex panels offer the advantage of superior test sensitivity and specificity. many of these panels have been designed to be easy-to-use, allowing molecular testing to be performed in moderate or low complexity settings and eliminating barriers that prevented many laboratories from being able to perform molecular assays on-site. another important benefit of multiplex panel is the fast time to result. molecular multiplex tests require hours to perform instead of the days required for culture-based methods. these systems differ in particular details (the exact turnaround time, sample throughput, cost per sample, and number of target pathogens detected). these differences are due to the underlying technologies used for the detection of the pathogen nucleic acid. however, all these systems share the common attribute that the incremental cost of each additional assay in the test cartridge (in materials, labor, and quality control (qc) testing) is small compared to the total manufacturing costs for the disposable. this has enabled ivd manufacturers to develop broad test panels that include organisms which have not been a part of standard testing protocols because of the technical limitations of existing methods. nonetheless the availability of syndromic infectious disease panels poses hard questions for clinicians and the healthcare system overall: does the wealth of information in a comprehensive test improve the treatment of an individual patient, and how can the economic value of this improved treatment be measured? commercially available fda-cleared multiplex nucleic acid-based tests for infectious agents include systems from luminex, genmark, and biofire (now a subsidiary of biomérieux) ( table ) . at present, all such systems combine the sequential steps of: . nucleic acid purification from the appropriate human sample matrix (e.g., nasal swab, blood or blood culture, stool) . cdna synthesis (reverse transcription) to convert viral rna to dna, if necessary . multiplex pcr to amplify molecules of the pathogen nucleic acid . specific detection of the expected amplicons to confirm that the correct target nucleic acids have been identified the different systems vary mainly in whether the nucleic acid purification steps are integrated into the same cartridge that is used for amplification (verigene, eplex, and filmarray) and in how the specific detection of amplicon is achieved. the luminex xtag and nxtag systems use a fluorescent signal generated after hybridization of the amplicon to fluorescently encoded bead arrays to detect a specific amplicon [ , ] . the verigene system uses hybrid capture of the amplicons on a microarray with detection by gold nanoparticle probes [ ] . the genmark esensor xt- and the eplex systems use electrochemical detection of the target amplicon hybridized to a specific gold microelectrode [ ] . the filmarray system is described in more detail below. the filmarray pouch and chemistry the filmarray system performs sample-to-answer multiplex nucleic acid testing for infectious disease. to accomplish this, the filmarray pouch ( fig. ) integrates all of the steps of nucleic acid purification, nested multiplex pcr amplification, and automated data analysis into a closed system [ ] . the pouch is created by welding two sheets of plastic film together with heat in such a fashion that fluid can move between the working areas of the pouch via channels left in the plastic. a hard plastic fitment attached to the film (indicated in fig. b) provides the enzymes and buffers needed to perform the biochemical reactions in the pouch. other reagents (# , # , and # in fig. a ) are inserted between the film layers during manufacture of the pouch. the filmarray reagents are lyophilized in the pouch, and the pouch is stored under vacuum before use. this benefits the end user in two ways. first the freezedrying process stabilizes the pcr reagents so that the pouch has a shelf life, at ambient temperature, in excess of year -which simplifies the logistics of acquiring and storing the pouches. second vacuum storage ensures that the wells of the fitment are also under vacuum. thus the user does not need to control the volume of hydration fluid or sample that is injected into the pouch -which simplifies the steps needed to load a pouch. lysis and homogenization of bacteria, spores, and viruses occur in the filmarray pouch by means of vigorous agitation in the presence of zirconium beads (# in fig. a ) and a denaturing buffer. dna and rna in the sample are purified by binding to silica magnetic beads (# in fig. a) , the beads are washed to remove proteins and other pcr inhibitors, and the nucleic acids are eluted into a buffer compatible with reverse transcription and pcr [ ] . the eluted material hydrates a pill (# in fig. a ) that contains all of the primers needed for reverse transcription (for rna targets) and first stage pcr. the primers in this pill are specific to each pathogen target (assay) contained in the pouch. the filmarray system performs nested, multiplex pcr in two stages (fig. ) . reverse transcription and first stage pcr (pcr ) are performed in the same reaction volume and in a multiplex format, combining primer sets for all assays for a filmarray panel in one mix. following pcr amplification, the reaction is diluted approximately -fold to reduce the concentrations of the outer primers, nonspecific products, and first-stage pcr chemistry. the diluted reaction is then combined with a fresh pcr master mix and flooded over a -well array, where a second stage pcr occurs (pcr ). each well of the array contains assay-specific primers that anneal within the first-stage outer amplicon. at the end of pcr , a dna melt curve analysis of the amplicons is performed using a nonspecific dna-binding dye, lcgreen® plus [ ] , that fluoresces in the presence of double-stranded dna. the presence of a specific melt curve with tm (melting temperature) in the range predicted for that specific amplicon confirms the presence and identity of the products made in each well. the mechanics and chemistry of the filmarray pouch have been described in greater detail elsewhere [ ] . here we highlight some of the features of the system that have contributed to the robustness of the test. the combination of a denaturing lysis buffer and "bead beating" with zirconium beads has been shown to lyse organisms and release intact total nucleic acid (dna and rna) from spores [ ] as well as from the cerebral spinal fluid (csf), stool, blood culture media, and whole blood. the nested pcr of the multiplex chemistry makes the filmarray test remarkably resistant to pcr inhibitors that may remain after nucleic acid purification. even modest levels of amplification in first-stage pcr can still be detected as an amplicon in the inner, nested product of pcr . the dilution step following the firststage amplification reduces the complexity of the input material to the second-stage reaction enough that, in most cases, the dna-binding dye detects the presence of a single amplicon in the reaction (the melt curve analysis serves as an additional filter for the correct product). the filmarray pouch and instrument contribute to the sensitivity of the overall system in several important ways. first the nucleic acid sample purification starts with a relatively large volume of the sample ( μl for the rp and gi panels, μl for the bcid), and, nominally, all of the nucleic acid purified from this volume is delivered to the combined reverse transcription -first-stage pcr reaction. unlike some benchtop protocols, there is no dilution from the purified nucleic acid into an rt step and a second dilution into a pcr. secondly the pouch is controlled by pneumatic actuators (described below) that can move liquid between blisters of the pouch in seconds. this minimizes the time during which nucleic acid could be degraded or pcr primers could bind to an incorrect dna or rna target and thus generate specific "nonspecific" amplicons. to the same end, the filmarray pouch achieves a true mechanical hot start. in both the first stage and second stage pcrs, the primers do not come in contact with the dna polymerase/mg, dntp mixture until both components are at or above the temperature at which polymerization will take place. this minimizes the formation of primer dimers, and higher-order multiplex primer structures in the first-stage pcr, which compete with the correct amplicons for pcr reagents. this enhances the specificity of the amplification reactions and thus increases the sensitivity of the individual pcr assays. all filmarray pouches have at least two internal controls that demonstrate the proper functioning of the pouch. one is a small amount of synthetic dna spotted onto the second stage pcr array along with primers to amplify this target. this "pcr " control only monitors the function of the second stage pcr. a more important control for pouch function is generated by freeze-drying a small number of cells of the yeast schizosaccharomyces pombe into the well of the pouch fitment that receives the sample. nucleic acid purified from these yeast cells must pass through all the steps of the pouch. outer primers in the first-stage pcr and inner primers spotted onto the second-stage pcr array are designed to amplify a spliced messenger rna from the s. pombe cells (in the filmarray bcid pouch which does not contain reverse transcriptase, the s. pombe target is a genomic dna sequence). biofire has shown during the development of several different pouches that artificially induced failure modes that prevent detection of a pathogen organism in a sample also prevent detection of the yeast control. the filmarray instrument controls the movement of liquid through the pouch using pneumatically-actuated bladders and seals that force liquid from a pouch blister or prevent liquid from leaving a blister, respectively. the hydrated reagents in the pouch fitment are introduced into the pouch blisters via additional pneumatically actuated pistons. the amplification reactions are thermocycled using inch square peltier devices situated adjacent to the first-and second-stage pcrs (# and # of fig. a , respectively). to detect the melting of the second-stage pcr amplicons, a blue led illuminates the array, and a camera with a filter to detect green light observes the signal generated by the dna-binding dye lcgreen® plus. multiplex panels are particularly attractive to clinicians because they provide a comprehensive and accurate test result in a short period of time, and the test panels have been designed to match the clinical syndrome (i.e., respiratory infection, infectious gastroenteritis). however, given the increased cost of these tests, it is important to demonstrate their impact to patient management and their cost-effectiveness. it is intuitive to believe that a rapid, accurate, and comprehensive diagnostic test should improve patient management by shortening the time to the most effective therapy, by preventing inappropriate therapy (especially empiric antimicrobials), by reducing additional diagnostic testing (e.g., imaging studies), by improving the use of infection control measures, and by reducing patient length of stay. of particular importance, these tests can reduce the unnecessary use of empiric antimicrobials by reducing the time to pathogen-directed therapy. use of broad-spectrum antibiotics for patients with serious illnesses and the inappropriate use of antibiotics in the outpatient setting "just in case" are important drivers of antibiotic resistance which is one of the major healthcare threats of our time. in this section, we will review what is known about the clinical utility and cost-effectiveness of these multiplex panels. because the clinical implication for each syndromic panel is different, the discussion is presented by syndrome. currently there are three vendors with multiplex respiratory panels that are both fda-cleared and ce marked (biofire diagnostics, salt lake city, utah; luminex, austin, texas; and genmark, san diego, california) and several more that are ce-marked (seegene, seoul south korea; curetis, holzgerlingen, germany, fast-track diagnostics, sliema, malta). these panels include assays for several viral pathogens (e.g., influenza, respiratory syncytial virus (rsv), human rhinovirus, parainfluenza viruses, adenovirus, coronaviruses, etc.) and some also include selected bacterial targets (e.g., mycoplasma pneumoniae, bordetella spp, legionella pneumophila). all of the fda-cleared test are limited to testing nasopharyngeal swab (nps) samples, while several of the ce-marked tests include a larger range of respiratory sample types (e.g., bronchoalveolar lavage, sputum, etc.). prior to the availability of multiplex respiratory panels, testing for viral respiratory pathogens relied on viral culture, direct fluorescent antigen (dfa) testing, enzyme immunoassays (eias), and traditional pcr assays. while viral culture was the gold standard, it has several limitations, including that it is a complex test requiring highly skilled laboratory workers to both set up the test and interpret the results, it is slow (taking days to complete), and only a limited number of human pathogens can be grown in viral cultures. dfa tests can be performed directly on the patient sample and can have a fast turnaround time; however, these tests are also technically complex, and the range of pathogens is limited. eia assays can be performed in a variety of ways and are used for rapid antigen tests. rapid antigen tests are fast and simple to use but are known to have poor test sensitivity and a limited test menu. traditional pcr assays are highly sensitive and specific; however, they are complex and require highly trained laboratory staff and specialized laboratory facilities. in addition, each test is ordered independently placing a large burden on the ordering clinician to select the correct test or to order multiple individual tests. due to their complexity, these tests are commonly sent to specialized reference laboratories which can increase cost and slows the time to result. the introduction of multiplex respiratory panels has allowed for comprehensive testing (ability to test viral pathogens that do not grow in cell culture and the ability to simultaneously test for bacterial and viral pathogens) in a shorter time frame. the easy-to-use systems allow the testing to be performed by laboratory workers without specialized molecular skills and in laboratories without specialized equipment and facilities. these systems allow testing to be performed closer to the patient and therefore further reduce the time to test result by reducing the need to transport samples and eliminating the delays associated with batch testing. multiplex respiratory panels have the potential to improve patient management and lower overall healthcare costs by improving use of influenza antivirals, reducing inappropriate use of antibiotics and antivirals, reducing use of healthcare resource (e.g., additional laboratory or imaging procedures), informing appropriate infection control practices, and reducing length of hospital, emergency department, and intensive care unit (icu) stay. several studies have evaluated the effect on patient and healthcare outcomes linked to the use of a multiplex respiratory panel. xu et al. [ ] showed that replacing dfa testing with on-demand use of the filmarray rp for children presenting to the emergency department (ed) resulted in dramatic reductions in test turnaround time ( vs . h), timely (defined as within h of discharge from the emergency department) administration of oseltamivir for % of patients testing positive for influenza, effective use of cohorting for admitted patients, and a potential saving of h of ed boarding time. similarly, in a pre-/post-intervention study of pediatric patients admitted through the ed, rodgers et al. [ ] compared several outcome measures when the filmarray rp replaced the use of three clinicianordered traditional pcr tests (prodesse assays for, flua/b/rsv, piv , , , and hmpv). the study demonstrated that use of the filmarray rp resulted in a significant increase in the number of patients with positive test results ( . % vs . %, p < . ) and a % reduction in time to result ( . vs . h, p < . ) when compared to use of traditional pcr tests. these improvements lead to a mean reduction in length of hospital stay of . days for patients with positive test results, reduced duration of antibiotics for patients with positive test results ( . vs . days, p < . ) or when results were reported in < h ( . vs . days, p < . ), and an overall reduction in healthcare costs of $ /patient. another study of pediatric patients reported significant reductions in the duration of antibiotic use ( vs days, p < . ), use of chest radiographs ( % vs %, p < . ), and an increase in appropriate use of isolation measures [ ] . similar findings have been observed in studies of adult patients. brendish et al. [ ] performed a prospective randomized study of adult patients presenting to the emergency department (ed) with respiratory symptoms over two respiratory seasons. in the control arm, patients were tested for respiratory viruses at the clinician's discretion using nine traditional pcr assays performed at a reference laboratory. in the intervention arm, all patients were tested with the filmarray rp, and testing was performed in the ed. the study demonstrated the expected increase in pathogen detection ( % vs %, p < . ) and reduction in time to result ( . vs . h, p < . ) but failed to show the expected reduction in the proportion of patients that received antibiotics ( % vs %, p = . ). however, there was an increase in the proportion of patients that received a short course of antibiotics (< h, % vs %, p = . ) especially for patients with positive filmarray rp test results. patients in the intervention group also had a mean reduction in hospital length of stay of . days ( . vs . d, p = . ) with the shortest length of stay observed in patients with positive filmarray rp results. the use of influenza antivirals was the same in both groups ( % vs %, p = . ); however, the intervention group had a significant increase in the number of influenza-positive patients that received influenza antivirals ( % vs %, p = . ) and a reduction in the use of antivirals for patients that were influenza-negative ( % vs %). in another study evaluating adult patients with a positive influenza result on a multiplex respiratory panel, rappo [ ] reported a significantly lower odds ratio for hospital admission (p = . ), a reduced length of stay (p = . ), reductions in antimicrobial duration (p = . ), and a reduction in the number of chest radiographs (p = . ). there is currently only one study evaluating the use of multiplex panels in an outpatient setting. greene et al. evaluated the difference in use of antibiotics and antivirals for adult outpatients tested with the filmarray rp that were [ ] positive for influenza, [ ] positive for non-influenza pathogen, or [ ] negative for all pathogens [ ] . they observed significant increases in the use of influenza antivirals ( . % vs . - . %, p < . ) and reduced use of the antibiotics ( . % vs . - . %, p = . ) for individuals with a positive result for influenza. however, detection of non-influenza viral pathogens did not lead to a reduction in the use of antibiotics when compared to those with no pathogen detected ( . % vs . %). the authors suggest that either influenza testing alone is more cost-effective for this patient population or that additional education and/or antibiotic stewardship is needed to drive appropriate use of antibiotics in the outpatient setting. blood culture panels are designed to test positive blood cultures with the aim to provide a faster time to organism identification. in addition, these panels include assays for selected antibiotic resistance genes providing important information to guide antibiotic therapy. there are currently three vendors with multiplex blood culture panels that are both fda-cleared and ce-marked (biofire diagnostics; luminex and accelerate diagnostics, tucson, arizona), while genmark (san diego, california, usa) and curetis (holzgerlingen, germany) have panels that are ce-marked. these panels include assays for gram-positive bacteria, gram-negative bacteria, yeast, and antibiotic resistance markers or, in one case (accelerate pheno™ system), antibiotic susceptibility test results. the curetis unyvero bcu blood culture application cartridge® panel is the most comprehensive (with identification of ~ bacteria and resistance markers) followed by the biofire filmarray blood culture identification panel (identification of ~ bacteria and resistance markers). the genmark eplex and luminex verigene systems provide separate panels that are specific to gram-positive bacteria, gram-negative bacteria, or yeast with selection of the appropriate panel determined by blood culture gram stain. all of these tests can be used with a variety of different blood culture media and blood culture systems. prior to the availability of multiplex blood culture panels, pathogen identification was performed using classic standard culture-based systems with phenotypic (or maldi-tof) identification followed by traditional growth-based antimicrobial susceptibility testing. these tests are the gold standard methods and are very reliable; however, they suffer from a slow time to result ( - days after the positive blood culture) and technical complexity. molecular assays for specific pathogen identification, such as staphylococcus aureus and enterococci, have been in use for some time and have shown improvements in patient outcomes [ ] [ ] [ ] [ ] . another method that reduces time to organism identification is using maldi-tof to identify bacteria directly from minimally processed blood cultures without the need to subculture to agar plates. the maldi-tof identification is very comprehensive, and the reduced time to bacterial identification has also been shown to improve patient outcomes [ , ] . while individual molecular assays and maldi-tof identification have both been shown to improve patient outcomes, they are both technically complex and require specialized skills. as a result, these test methods are typically performed during standard laboratory working hours and are typically not used on night shifts when staffing is limited. molecular multiplex panels offer the advantage of a fast time to organism identification along with ease of use. all of the fda-cleared and ce-marked panels use unprocessed blood culture media and provide identification results within - h of test initiation. due to the ease of use, these panels can be used by laboratory staff without specialized molecular biology skills; however, because these panels do not identify all possible pathogens, the results must be interpreted in conjunction with the blood culture gram stain, and traditional culture and sensitives must still be performed. furthermore, appropriate adjustments to antimicrobials rely on proper test interpretation and a good understanding for the local patterns of antimicrobial resistance (e.g., antibiogram). treatment adjustments should be based on local guidelines developed by an antimicrobial stewardship program (asp) team with an in-depth understanding of the capabilities of the test being used, the local antibiogram, and the local patient populations. as with the individual molecular assays and the maldi-tof identification, numerous studies have shown that use of multiplex molecular blood culture panels dramatically reduces the time to organism identification [ ] [ ] [ ] [ ] which drives more appropriate pathogen-directed therapy. pathogen-directed therapy includes antibiotic escalation (the addition or change of dose when the current therapy is ineffective against the identified organism) and antibiotic de-escalation (discontinuation of unnecessary empiric antibiotics). a prospective randomized study conducted at the mayo clinic showed that antibiotic escalation occurred more quickly with or without real-time asp oversight ( h with bcid and asp, h with bcid only, and h without bcid or asp, p = . ); however, optimal antibiotic de-escalation requires oversight by an asp ( h with bcid and asp, h with bcid only, and h without bcid or asp, p < . ) [ ] . the finding that multiplex molecular blood culture panels paired with an asp results in a faster time to optimal antibiotic therapy (most narrow effective therapy) has been confirmed in several additional studies [ , ] . the use of molecular multiplex blood culture panels has also been shown to reduce unnecessary treatment due to contaminated blood cultures [ , ] . reducing the use or duration of unnecessary antibiotics is important to reducing the incidence of antibiotic resistance. the cost of molecular multiplex blood culture panel and the fact that they do not replace existing testing have been raised as reasons to not use them; however, studies of the overall healthcare cost prove the panels to be cost neutral [ ] [ ] [ ] or to reduce overall healthcare cost [ , , [ ] [ ] [ ] [ ] . while the evidence is strong that multiplex panels dramatically reduce time to organism identification and time to optimal antibiotic therapy, the evidence is inconsistent with regard to reductions in patient mortality and length of hospital stay, mostly likely because the management of patients with sepsis is complex and multifactorial. however, a recent meta-analysis [ ] of studies using rapid molecular methods to test positive blood cultures found a small but statistically significant reduction in patient mortality when the results were used as part of an asp (or . , % ci . - . ) but not when used outside of an asp (or . , % ci . - . ). the improvements were seen for patients with gram-positive (or . , % ci . - . ) or gram-negative bacteremia (or . , % ci . - . ), but not for patients with candidemia (or . % ci . - . ). the study also found that time to effective therapy decreased by a weighted mean difference of − . h ( % ci − . to − . ) and that length of hospital stay decreased by − . days ( % ci − . to − . ). currently there are three vendors with multiplex gastrointestinal panels that are both fda-cleared and ce-marked (biofire diagnostics; luminex, and bd, sparks, maryland) and several more that are ce-marked (seegene, soul, south korea; mobidiag, finland; serosep, limerick, ireland). some of these panels include assays for bacteria, viruses, and parasites in one test (filmarray gi panel, biofire diagnostics; luminex ggp, luminex; verigene enteric pathogens test, luminex), while others provide separate panels for bacterial, viral, or parasitic pathogens (allplex™, seegene; bd max®, bd; amplidiag®, mobidiag; entericbio® realtime dx, serosep). most tests are performed with raw stool samples or stool in transport media. the use of fecal swabs with transport media is also common, and direct testing of rectal swabs is desirable. current testing for infectious gastroenteritis includes many tests (stool culture, ova and parasite examination (o&p), enzyme immunoassays) that are technically complex and suffer from low diagnostic yield. the gold standard for stool pathogen testing is stool culture; however, stool culture has low diagnostic yield, is technical complexity, and has a long time to result. the yield for stool cultures is reported to be between . % and . % with a cost per positive result of $ to $ [ ] . another commonly ordered test is o&p, which requires the collection and testing of three different stool samples. this method is also known to be technically difficult, to have low sensitivity, to be improperly used, and to have a low diagnostic yield of . % [ ] . as a result of the low diagnostic yield, clinicians often order multiple tests for the same stool sample, or perform testing sequentially until a causative pathogen is identified. as an example, a study conducted at a children's hospital found that a median of three tests (range - ) were ordered per stool sample [ ] . to make matters worse, several studies have shown that clinician test ordering practices for gastroenteritis are problematic, in part due to the complexity of which pathogens are covered by what test [ , [ ] [ ] [ ] . the use of culture-independent molecular multiplex panels has increased the diagnostic yield for stool testing due both to increased test sensitivity and an expanded test menu. studies using the filmarray gi panel identified a pathogen in - % of stool samples [ , , ] . some of the assay requires specialized molecular laboratories and personnel (bd max, luminex ggp, allplex); however, some are designed to be simple to use (filmarray gi panel, verigene enteric panel) and to provide a fast time to result (as little as h from test initiation). while these tests have the benefits of offering a comprehensive and accurate result in a relatively fast time period, the impact to patient care is largely unknown; however, one recent pre-/post-implementation study highlighted several important improvements when the filmarray gi panel was used to test pediatric and adult inpatients [ ] . these included an increase in diagnostic yield from . % to . % and an improved time to result from a mean of . h to . h when compared to traditional clinician ordered tests. when compared to a matched historical control group, implementation of the filmarray gi panel led to a reduction in the number of additional stool tests ( . vs . , p = . ), a trend toward shorter duration of antibiotics ( . days vs . day, p = . ), significantly fewer imaging studies ( . vs . , p = . ), and a reduction in the length of hospital stay after sample collection ( . days vs . days, p = . ). reduced length of stay was more pronounced for the adult population ( . days vs . days, p = . ). recent studies have also shown that these tests have important advantages for infection control. in a recent retrospective study, the filmarray gi panel was used to test frozen stool samples that had previously been tested for rotavirus and c. difficile for infection control purposes [ ] . the study showed that % of the samples contained pathogens that should have required infection control measures, including norovirus, rotavirus, and c. difficile. of these patients, % were under no or inadequate contact precautions, for a total of patient days. conversely . % of the patients with negative results by the filmarray gi panel were unnecessarily placed under contact precautions for a total of patient days. this study illustrates that without a rapid comprehensive test result, contract precautions are not rationally applied, leading to both an increased risk of nosocomial infections and unnecessary costs associated with inappropriately applied contact precautions. these are important factors when considering the care of patients in hospitals and in long-term care facilities. use of the luminex xtag gastrointestinal pathogen panel (gpp) has been compared with conventional laboratory testing for hospitalized patients and was found to be cost-effective because the increased cost of the laboratory testing was more than offset by the cost saving for elimination of unneeded contact precautions. importantly, the cost savings was directly related to the time to test result [ ] . the national institute for health care excellence (nice) in the uk recently published a very comprehensive health economics assessment of molecular multiplex panels [ ] . they reported that there was considerable uncertainty in their models; however, the luminex ggp panel was determined to be cost-effective for use in community-acquired and traveler diarrhea and both the filmarray gi panel and the luminex ggp were found to be cost-effective for use in inpatients with diarrhea. these models will be updated as new information (such as the recent study by beal [ ] ) becomes available. further clinical utility studies will highlight the importance of the different pathogens detected by the multiplex panels. however, independent of this work, continuing improvements to these ivd systems are likely to increase their value in the clinical infectious disease setting. in contrast to previous generations of infectious disease ivd platforms which used cell culture, microscopy, or immunoassay technology, the current generation of multiplex systems are all highly automated and computerized [ , [ ] [ ] [ ] ] . this opens the possibility of exporting the results of a patient test directly to an internet (or "cloud") database. a pilot version of such a system has been achieved with development of filmarray trend [ ] . trend aggregates result from geographically dispersed clinical laboratories and displays the results on a website (www.syndromictrends.com) in close to real time, resulting in a form of infectious disease "weather map." the pilot project summarized the respiratory pathogen results for the filmarray rp from > , patient samples acquired over years ending in july from clinical laboratories in the united states (fig. ) . similar to social media-based disease reporting systems, the data is syndrome-based [ ] . however, a unique feature of the trend dataset is that analysis is pathogen-specific. the filmarray rp patient test results demonstrate that a number of viruses (rsv, hmpv, piv, and cov) show seasonal occurrence that is similar to influenza and thus can be confused with influenza when a symptomatic diagnosis of influenza-like-illness is made. this has important implications for treatment of influenza and for determining the efficacy of influenza vaccination and thus emphasizes the value of multiplex testing for respiratory symptoms. the filmarray trend data also show that % of the filmarray rp tests detect the presence of two or three pathogens in a single sample. the importance of this result for patient treatment is not currently clear. clinical studies that focus on patients presenting with more than one pathogen are needed. data from the filmarray gi panel are also available at the www.syndromictrends.com website. over time, additional filmarray ivd panels will be added to trend, thus enabling the tracking of the pathogens that those panels detect. in the us fda cleared the filmarray rp-ez panel [ ] . this panel has clinical laboratory improvement act (clia)-waived status and thus can be used in settings close to the patient including low-complexity outpatient settings. in other work, the time to result for the rp panel has been decreased from min for rp v . to min for the rp panel [ ] . speed, ease of use, and a comprehensive test menu are all critical features if the possibilities of point-of-care syndromic testing are to be fully realized [ ] . in the longer term, additional simplification of the test setup procedure as well as reductions in the time to result should further expand the number of outpatient settings able to use multiplex testing. the data from farrar and wittwer [ ] on "extreme" pcr conditions (cycle times below s) suggest that reductions in the time to result are limited by the instrument and not by the chemistry and that substantial improvements are still possible. as noted earlier, the cost of multiplex diagnostic systems is not driven by that of the primers needed for each assay, so the number of assays in a test is limited by assay format and the ability to separate the signal for each analyte. the depth of multiplex achieved in pcr multiplexes for infectious disease has steadily increased from the earliest, manually performed multiplexes, compared to the those being developed for the automated systems of today. the point will soon be reached in which the feasible number of assays in a test begins to exceed the number of distinct pathogens that need to be tested for in any particular syndrome. however, there are situations (e.g., hiv drug resistance testing or hpv serotyping) where the ability to detect a limited number of point mutations would add great value to the test result. there is also great interest in the direct detection of sepsis pathogens from blood because the hours saved by not having to culture the bacteria reduces the risk of incorrect antibiotic treatment. with a combination of direct enrichment of the bacteria from the blood and careful attention to removing endogenous bacteria from the test manufacturing process, it should be possible to achieve the necessary sensitivity to detect bacterial pathogens directly from blood, without the time and labor of culturing the sample. an infectious disease society of america policy paper from [ ] made a number of recommendations for the key characteristics of future infectious disease ivd tests. the current generation of multiplex tests already meets many of these goals (direct testing from easily accessible sample types, able to rule out infection with high certainty, based on clinical syndromes). the technical improvements described above suggest that meeting many of the remaining goals (rapid testing, point-of-care syndromic testing, improved outbreak detection) will be accomplished in the next decade. emerging technologies for the clinical microbiology laboratory multiplex polymerase chain reaction tests for detection of pathogens associated with gastroenteritis evaluation of four commercial multiplex molecular tests for the diagnosis of acute respiratory infections an automated nested multiplex pcr system for multi-pathogen detection: development and application to respiratory tract infection comparison of the genmark diagnostics esensor respiratory viral panel to real-time pcr for detection of respiratory viruses in children the eplex® respiratory pathogen panel (rp)* comparison of the luminex respiratory virus panel fast assay with in-house real-time pcr for respiratory viral infection diagnosis comparison of luminex nxtag respiratory pathogen panel and xtag respiratory viral panel fast version for the detection of respiratory viruses comparison of three commercial rt-pcr systems for the detection of respiratory viruses clinical evaluation of the luminex nxtag respiratory pathogen panel bead-based suspension arrays for the detection and identification of respiratory viruses selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles electronic detection of nucleic acids: a versatile platform for molecular diagnostics rapid and simple method for purification of nucleic acids high-resolution genotyping by amplicon melting analysis using lcgreen evaluation of the filmarray(r) system for detection of bacillus anthracis, francisella tularensis and yersinia pestis implementation of filmarray respiratory viral panel in a core laboratory improves testing turnaround time and patient care impact of a rapid respiratory panel test on patient outcomes 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hospital-acquired enterococcal bacteremia: delivering earlier effective antimicrobial therapy the clinical impact of rapid, direct maldi-tof identification of bacteria from positive blood cultures integrating rapid pathogen identification and antimicrobial stewardship significantly decreases hospital costs randomized trial of rapid multiplex polymerase chain reaction-based blood culture identification and susceptibility testing impact of a rapid multiplex polymerase chain reaction blood culture identification technology on outcomes in patients with vancomycinresistant enterococcal bacteremia benefits of adding a rapid pcr-based blood culture identification panel to an established antimicrobial stewardship program clinical impact and provider acceptability of real-time antimicrobial stewardship decision support for rapid diagnostics in children with positive blood culture results potential clinical impact of the film array meningitis encephalitis panel in children with suspected central nervous system infections clinical and economic impact of antimicrobial stewardship interventions with the filmarray blood culture identification panel rapid testing using the verigene gram-negative blood culture nucleic acid test in combination with antimicrobial stewardship intervention against gram-negative bacteremia stewardship approach for optimizing antimicrobial therapy through use of a rapid microarray assay on blood cultures positive for enterococcus species prospective intervention study with a microarray-based, multiplexed, automated molecular diagnosis instrument (verigene system) for the rapid diagnosis of bloodstream infections, and its impact on the clinical outcomes the effect of molecular rapid diagnostic testing on clinical outcomes in bloodstream infections: a systematic review and meta-analysis practice guidelines for the management of infectious diarrhea physician use of parasite tests in the united states from to and in a utah cryptosporidium outbreak in how well does physician selection of microbiologic tests identify clostridium difficile and other pathogens in paediatric diarrhoea? insights using multiplex pcr-based detection survey of physician diagnostic practices for patients with acute diarrhea: clinical and public health implications management of suspected infectious diarrhoea by english gps: are they right? general practitioner practices in requesting laboratory tests for patients with gastroenteritis in the netherlands multicenter evaluation of the biofire filmarray gastrointestinal panel for etiologic diagnosis of infectious gastroenteritis spectrum of enteropathogens detected by the filmarray gi panel in a multicentre study of communityacquired gastroenteritis a gastrointestinal pcr panel improves clinical management and lowers health care costs multiplex gastrointestinal pathogen panels: implications for infection control a cost benefit analysis of the luminex xtag gastrointestinal pathogen panel for detection of infectious gastroenteritis in hospitalised patients integrated multiplex pcr tests for identifying gastrointestinal pathogens in people with suspected gastroenteritis (xtag gastrointestinal pathogen panel, filmarray gi panel and faecal pathogens b assay). diagnostics guidance detection of influenza a and b with the alere i influenza a & b: a novel isothermal nucleic acid amplification assay. influenza other respir viruses automated real-time collection of pathogen-specific diagnostic data: syndromic infectious disease epidemiology infectious disease surveillance in the big data era: towards faster and locally relevant systems fda clearance for filmarray respiratory panel ez (rp ez) fda clearance for filmarray respiratory panel (rp the point-of-care laboratory in clinical microbiology extreme pcr: efficient and specific dna amplification in - seconds better tests, better care: improved diagnostics for infectious diseases key: cord- -u fnzy authors: nevarez, javier g. title: differential diagnoses by clinical signs—crocodilians date: - - journal: mader's reptile and amphibian medicine and surgery doi: . /b - - - - . - sha: doc_id: cord_uid: u fnzy nan the first signs of illness in captive crocodilians are usually nonspecific in nature. these include anorexia, lethargy, and death. in commercial operations, the workers may notice excess food remaining from the day before, which is sometimes followed by a perceived change in the behavior of the animals. one should not underestimate these observations because most workers are well tuned to the daily routine and behavior of the animals. a visit to the ranch or farm should be performed during feeding time to avoid additional stress to the animals. this also allows observation of the feeding and water change practices. at this time, a thorough history and a collection of animals for diagnostics and necropsy can be obtained. in addition to routine samples, tissues should be frozen for possible bacterial, fungal, or viral cultures. the list of differentials for nonspecific clinical signs must be narrowed after diagnostic results are obtained. however, one must remember that husbandry and disease go hand in hand. most systemic diseases of captive crocodilians are thought to be secondary in nature. bacterial and fungal diseases are commonly encountered. the majority of data available on bacterial disease in crocodilians are from studies involving the american alligator (alligator mississippiensis). table . presents a large number of isolates from diseased and nondiseased animals, including a survey of fecal coliforms. huchzermeyer et al. also have extensive data on enteric bacterial and fungal isolates from african dwarf crocodiles (osteolaemus tetraspis). important bacterial infections reported in crocodilians include mycoplasma alligatoris, mycoplasma crocodyli, and chlamydia spp. fungal organisms are often opportunistic invaders of the integument and respiratory system, but primary fungal infections are also reported. viral diseases are likely underdiagnosed in reptile medicine because of the challenges in diagnostic techniques and scarce information. poxvirus, west nile virus (wnv), and herpesvirus have been reported as pathogens in crocodilians. an adenovirus-like infection in captive nile crocodiles (crocodylus niloticus) has also been reported. coronavirus, influenza c virus, and paramyxovirus have been identified with transmission electron microscope in the feces of crocodilians, but their significance remains to be determined. finally, evidence exists of seroconversion to paramyxovirus and eastern equine encephalitis virus in crocodilians. toxicities are not common in captive crocodilians. lead toxicity is reported from alligators fed lead-shot nutria. clinical signs include weakness, lethargy, anorexia, and death. feeding of nutria is no longer common practice. for privately owned and zoo animals, there are additional differentials to be considered. a faulty or broken heater can lead to sudden death from electrocution. this can also present a risk for humans, therefore, it is important to isolate, protect, and inspect all equipment within the enclosure. a thermostat malfunction can lead to altered water temperatures that can affect the animal's behavior. in colder climates, animals may become lethargic if the heater is not working properly. conversely, if a thermostat fails, allowing the heater to remain on, the animals' behavior may change because of increased temperatures. at first they may appear agitated but eventually anorexia and lethargy will occur if overheated. overheating may also occur as a result of inappropriately sized or placed heat lamps. foreign bodies should be a differential for anorexic crocodilians, especially those in outdoor exhibits where trash, coins, and other objects may be tossed in by the public. it is also important to pick up and secure all tools such as buckets and hoses, which may also be ingested by animals. physiological anorexia can be observed as part of the normal reproductive cycle, in particular during the later stages of egg development. however, dystocia (egg binding) can lead to a more prolonged anorexia that may be the first indication that the animal is ill. unless the actual mating date is known or the reproductive cycle is monitored by ultrasonography, it can be very difficult to ascertain what constitutes true dystocia. in animals housed outdoors in their natural climate, dystocia should be considered if oviposition has not occurred for days past the expected date. for those housed indoors or outside their natural climate, behavior together with plasma biochemistry, hematology, and diagnostic imaging can be used to aid in the diagnosis of dystocia. secondary nutritional hyperparathyroidism can also cause animals to be lethargic and anorexic. this is more common in juvenile animals. older animals may succumb to secondary renal hyperparathyroidism. another phenomenon seen in captive crocodilian operations is runting, the lack of growth and failure to thrive of some animals within a group. in some operations, animals are separated by size and some buildings contain only the runts. a clear size difference is observed in same-age animals between the runts and the otherwise healthy ones. these animals crocodile (crocodylus porosus), and freshwater crocodile (crocodylus johnstoni). in caimans, poxvirus is characterized by -to -mmdiameter, gray to white, coalescing to macular skin lesions. these can be found on the head, palpebrae, maxilla, mandible, limbs, palate, tongue, and gingiva. [ ] [ ] [ ] other signs observed include palpebral and generalized edema. resolution of clinical signs was observed weeks after improvement of husbandry in one case and after months in another case, with no changes in husbandry. lesions in crocodiles are described as -to -mm-diameter, yellow to brown, wartlike, sometimes firm, and unraised to raised nodules with occasional shallow ulcers. these lesions can be found on the head, palpebrae, nostrils, sides of the mouth, oral cavity, limbs, ventral neck, and coelom and at the root of the tail. , resolution of lesions was reported to occur as early as to weeks. light microscopy reveals epithelial hyperplasia, acanthosis, hyperkeratosis, and necrosis, and at times borrel and bollinger's bodies are also visible. [ ] [ ] [ ] [ ] [ ] secondary bacterial and fungal infections may also be present. horner reported the use of an autogenous vaccine to treat poxvirus in nile crocodiles. no specific treatment recommendations exist. maintaining appropriate husbandry is essential in the prevention and resolution of poxvirus in crocodilian farms. west nile virus (wnv) has been reported to cause lymphohistiocytic proliferative syndrome of alligators (lpsa). , lpsa skin lesions are a chronic manifestation of wnv infection and occur in animals that have survived a wnv outbreak. this production problem affects the quality of the hide and consequently decreases profit for alligator ranchers. gross lesions can be seen as multifocal, mm to mm, gray to red foci on the ventral mandibular, abdominal, and sometimes tail scales ( fig. . ) . they can be found on any section of a scale and do not appear concave or convex with respect to the scale's surface. routine microscopic examination reveals dermal nodular lymphoid proliferation with perivascular cuffing. similar lesions can be found in other tissues besides the skin. identification of the lesions is complicated by the fact they are sometimes not seen until the hides are removed from the animal. therefore antemortem identification alone is not very accurate. are not as hardy in the captive environment and are potentially more susceptible to disease. dominance by other animals, environment, and even incubation factors contribute to the presence of runts. integumentary disease is often secondary to poor water quality, poor enclosure design, stress, and immunosuppression or a combination of these factors. lacerations, abscesses, and draining tracts can all be observed in captive and wild crocodilians. consequently, these open wounds can serve as a nidus for bacteria and fungi. an additional factor in commercial operations or small enclosures is the accumulation of a fatty slime layer on the surface of the water when meat supplements, in particular chicken, are fed in addition to a dry commercial diet. this slime attaches to the skin of the animals and the enclosure, creating an environment for bacterial and fungal growth. the same is true for accumulation of biofilm on the surface of the enclosure. a soap or detergent can be used as a surfactant to help reduce the fatty layer on the water surface. any product used must be nontoxic, or the animals must be removed from the enclosure while in use. fungal dermatitis is also common because many fungi thrive in the water column and environment of captive alligators. in most instances, both bacteria and fungi are present in skin lesions. culture and sensitivity testing can be frustrating in these cases because of the mixed flora found in the lesions. the first step in addressing integumentary abnormalities is to analyze water quality and make improvements as needed. in addition to improvements in water quality, antimicrobial or antifungal therapy may be necessary. medicating a large group of crocodilians is challenging because it must usually be done orally by mixing the medication with feed. in zoos or private collections, individual crocodilians can be medicated by injecting the prey item or via injection. a pole syringe can be used in large or aggressive animals. there is a lack of pharmacokinetic data for oral antimicrobials in crocodilians, and it has been shown that oral tetracycline is not effectively absorbed in american alligators. these factors pose a challenge for effective treatment and the potential for promoting antimicrobial resistance. therefore a combination of good hygiene, water treatments with disinfectants, and improved husbandry is recommended. a number of parasites are known to affect the skin of crocodilians. paratrichosoma spp. are capillaroid parasites that cause a zigzag lesion on the skin. these lesions appear to be purely cosmetic. this parasite is known to affect various crocodile species in the wild and is believed to have a stage of its lifecycle dependent on soil; therefore, it is not observed in captive scenarios where animals are kept on concrete. regardless of the etiology, good hygiene is essential in the prevention and treatment of integumentary disease in crocodilians. the etiology itself is at times not as important as what conditions predisposed them to the disease. a herpesvirus was identified via tem from saltwater crocodiles (crocodylus porosus) with concurrent pox virus and bacterial infection of the skin. dermatophilosis causes brown-red to ulcerative lesions most commonly located between scales on the ventral skin ( fig. . ) . most of the cultures appear to resemble dermatophilus congolensis. , these filamentous bacteria do not respond well to antibiotic therapy; therefore, intensive hygiene practices are necessary to prevent and control outbreaks. parapoxvirus or poxlike viruses have been identified in five different crocodilian species: spectacled caiman (caiman crocodilus fuscus), , brazilian caiman (caiman crocodilus yacare), nile crocodile, , saltwater this is one of the most common presentations of captive crocodilians, second only to integumentary disease. respiratory signs can often be confused with neurological signs, and a clear distinction must be established. however, respiratory disease may accompany neurological disease as a consequence of weakness leading to aspiration. clinical signs associated with respiratory disease in crocodilians may include dyspnea, tachypnea, nasal discharge, excessive basking, abnormal swimming (either in circles or on one side of the body), and anorexia, among others. as the animals become weak, the basihyoid valve does not function properly and they can aspirate food or water. the pharyngeal anatomy of crocodilians makes aspiration unlikely in healthy animals. therefore, if aspiration is suspected, evaluation for an underlying illness should be pursued. a number of rhinitis and pharyngitis syndromes have also been described in some crocodilian species. most respiratory infections are either bacterial or fungal in origin. an early report showed a fungal pneumonia associated with beauveria bassiana in two alligators, and fusarium moniliforme was found in another case. one well-documented respiratory pathogen is mycoplasma alligatoris. clinical signs are nonspecific and include lethargy, weakness, anorexia, white ocular discharge, paresis, and edema (facial, periocular, cervical, limbs). pneumonia, pericarditis, and polyarthritis are often diagnosed on necropsy. pathogenicity of m. alligatoris has been documented for a. mississippiensis and for the broad-nosed caiman (caiman latirostris). , other crocodilian species closely related to alligators are also potentially susceptible. helmick et al. reviewed antimicrobial susceptibility for m. alligatoris. a second mycoplasma species, m. crocodyli, is known to affect nile crocodiles. lesions are similar to those observed with m. alligatoris. some studies have examined the use of an autogenous vaccine for m. crocodyli, but work is still needed to determine its true efficacy. , currently antemortem diagnosis is difficult and requires a combination of serology and tissue biopsy for pcr. therefore most cases are confirmed via histopathology and pcr postmortem. neurological deficits are not commonly seen in crocodilians and deserve special attention if encountered. clinical signs may include swimming in circles or on one side of the body, abnormal flotation, lethargy, ataxia, head tilt, and muscle tremors. anorexia often accompanies the neurological signs. thiamine deficiencies should be considered in animals fed a frozen fish diet. signs of thiamine deficiency typically include severe lethargy that leads to coma. hypoglycemia has also been reported as a cause of neurological signs in alligators. these animals have muscle tremors, loss of the righting reflex, and mydriasis. stress seems to be the main contributing factor. the author has also observed neurological signs associated with low oxygen levels inside alligator enclosures. neurological signs may also be observed after ingestion of metal foreign bodies, including coins, containing lead or zinc. west nile virus causes neurological signs in captive-reared american alligators raised indoors. , presence of antibodies, but no clinical signs, is reported from free-ranging and captive crocodilians housed outdoors. , wnv in crocodilians is transmitted by mosquitoes and horizontally via fecal shedding. this represents an opportunity for zoonosis in captive operations. strict building quarantine and hygiene strategies should be implemented to prevent spread to other animals in the facility. wnv is a reportable disease, and one should contact the state veterinarian when diagnosis is confirmed. wnv can affect an animal of any age, but hatchlings often experience severe per-acute mortalities of up to % to %. wnv has two disease stages in alligators, acute and chronic. clinical signs during the acute stage include swimming in circles, head tilt (fig. . ) , muscle tremors, weakness, lethargy, anorexia, and death. animals that survive the acute infection go on to develop lpsa skin lesions, the chronic stage of wnv. there is no treatment. definitive diagnosis is accomplished with reverse transcriptase polymerase chain reaction (rt-pcr) of liver and brain. however, it must be noted that rt-pcr can detect viral rna from a commercially available wnv vaccine for alligators (bivi, st. joseph, mo). therefore vaccine status must be known before diagnosis. maternal transfer of antibodies has also been observed in hatchlings. veterinarians should take additional precautions during necropsies of suspect animals. aggressive mosquito control and vaccination have limb fractures and partial amputations can be seen after altercations. it is possible for animals that have lost limbs after a fight to survive to the point of complete healing (fig. . ). these lesions however may also lead to nerve or muscle damage and consequent paresis or paralysis. as mentioned in the respiratory signs section, m. alligatoris and m. crocodyli cause polyarthritis, which can be evident antemortem. nutritional secondary hyperparathyroidism can occur in young crocodilians not being fed whole prey or pelleted diets. affected animals may also be runts of the group and have concurrent disease. the innate requirement of ultraviolet b light for the production of vitamin d in crocodilians is not known. they appear to be able to obtain appropriate levels from their diet, but further research in this area is needed. gout also occurs in crocodilians (figs. . a and b) . clinical signs can be nonspecific in nature, but limb paresis/paralysis and joint enlargement may be observed. a case of gout with concurrent suspected hypovitaminosis a in crocodile hatchlings has been reported. deficiencies of vitamins a, b, c, or e can also lead to a variety of musculoskeletal disorders. these are not commonly seen when commercial diets are fed in addition to meat products. anorexia is likely the most common clinical sign associated with gastrointestinal disease in captive crocodilians. foreign body ingestion, gastric ulcers, enteritis ( fig. . ) , and trauma to the oral cavity can be observed in crocodilians. ingestion of foreign bodies is more common in wild crocodilians. however, it must also be a differential in captive specimens. malfunction of water pumps, water filters, or construction can lead to the presence of foreign bodies in the enclosures. in outdoor exhibits, the public is often responsible for the presence of foreign objects that are thrown into their enclosures. these can then be ingested by the animals and cause severe problems in the case of nails and other sharp objects (fig. . ). infectious enteritis may be difficult to assess grossly because crocodilians have very thick intestinal walls normally. however, crocodilians appear to have a characteristic response to insult of the gastrointestinal tract. accumulations of fibrous material or necrotizing lesions are commonly seen on necropsy (fig. . a and b ). this reaction is aggressive and can even lead to obstructions caused by the fibrous material. obstruction can also occur with fecal impactions and torsions. gastric ulcerations and abnormalities of the gastric mucosa are also routinely observed on necropsy and may be associated with stress and diet. finally, the intestinal tract contains a large amount of peyer's although not commonly reported from crocodilians, the author has worked on a number of alligator cases with acid-fast organisms consistent with mycobacterium sp. in the lungs. these animals had evidence of pneumonia on necropsy as shown by multiple white foci, to mm in diameter, on the lung parenchyma. other cases of pulmonary and enteric mycobacterial infections have been suggested. difficulties of growing mycobacterium sp. make its definitive diagnosis a challenge. musculoskeletal disease can be the result of alterations in the incubation temperature or environment (fig. . ) notice the red mucosa. patches and likely represents a site of aggressive inflammatory response to infectious agents. a novel herpesvirus was reported from an american alligator with lymphoid follicular inflammation of the cloaca. chlamydiosis has been reported from c. niloticus (south africa and zimbabwe), c. porosus (papua new guinea), and c. siamensis (thailand). [ ] [ ] [ ] the author has also diagnosed chlamydiosis in a. mississippiensis (united states). although it has been proposed as a chlamydophila psittaci-like strain in c. niloticus, the chlamydia species affecting crocodilians is likely a novel specie(s). infection usually leads to hepatitis and/or conjunctivitis (fig. . ), but lung and spleen can also be infected. morbidity and mortality can exceed %, resulting in significant economic losses for farmed animals. treatment with oxytetracycline is anecdotally reported to be effective in crocodiles. this creates significant concerns for possible antimicrobial resistance, zoonosis, and the safety of the meat from these animals. a novel herpesvirus was reported from an american alligator with lymphoid follicular inflammation of the cloaca. a herpesvirus was identified via tem from saltwater crocodiles (crocodylus porosus) with concurrent pox virus and bacterial infection of the skin. in captive c. porosus and freshwater crocodiles (c. johnstoni) from australia, three novel herpesviruses have been identfied. systemic lymphoid proliferation. chlamydiaceae was also identified from conjunctival and pharyngeal tissues of some of the c. porosus cases. of interest is that these syndromes occur concurrently with other infectious agents and have been associated with significant morbidity and mortality of infected animals. see www.expertconsult.com for a complete list of references association of west nile virus with lymphohistiocytic proliferative cutaneous lesions in american alligators (alligator mississippiensis) detected by rt-pcr hypoglycemic shock in captive alligators west nile virus in farmed alligators west nile virus in alligator ranches from louisiana west nile virus infection in crocodiles prevalence of neutralizing antibodies against west nile virus (wnv) in monkeys (ateles geoffroyi and alouatta pigra) and crocodiles (crocodylus acutus and c. acutus-c. moreletti hybrids) in mexico wnv vaccination of alligators, 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single-dose oral administration in the american alligator (alligator mississippiensis) isolation of dermatophilus sp. from skin lesions in farmed saltwater crocodiles (crocodylus porosus) pathology of skin diseases in crocodiles pox like skin lesions in captive caimans pox virus infection in captive juvenile caimans (caiman crocodilus fuscus) in south africa poxvirus dermatitis outbreak in farmed brazilian caimans (caiman crocodilus yacare) poxvirus in farmed nile crocodiles poxvirus infection in nile crocodiles (crocodylus niloticus) poxvirus infection in two crocodiles establishing an association between west nile virus exposure and the development of lymphohistiocytic proliferative syndrome of american alligators, alligator mississippiensis isolation of aeromonas hydrophila from the american alligator, alligator mississippiensis aeromonas-induced deaths among fish and reptiles in an eutrophic inland lake diseases of the eye and ocular adnexae in reptiles immobilization, blood sampling, necropsy techniques and diseases of crocodilians: a review initial antibiotic therapy for alligator bites: characterization of the oral flora of alligator mississippiensis isolation of edwardsiella tarda from a sea lion and two alligators gram-negative septicemia in american alligators (alligator mississippiensis) infectious diseases and pathology of reptiles mycoplasma alligatoris sp. nov., from american alligators key: cord- -bn x authors: cox-georgian, destinney; ramadoss, niveditha; dona, chathu; basu, chhandak title: therapeutic and medicinal uses of terpenes date: - - journal: medicinal plants doi: . / - - - - _ sha: doc_id: cord_uid: bn x terpenes, also known as terpenoids are the largest and most diverse group of naturally occurring compounds. based on the number of isoprene units they have, they are classified as mono, di, tri, tetra, and sesquiterpenes. they are mostly found in plants and form the major constituent of essential oils from plants. among the natural products that provide medical benefits for an organism, terpenes play a major and variety of roles. the common plant sources of terpenes are tea, thyme, cannabis, spanish sage, and citrus fruits (e.g., lemon, orange, mandarin). terpenes have a wide range of medicinal uses among which antiplasmodial activity is notable as its mechanism of action is similar to the popular antimalarial drug in use—chloroquine. monoterpenes specifically are widely studied for their antiviral property. with growing incidents of cancer and diabetes in modern world, terpenes also have the potential to serve as anticancer and antidiabetic reagents. along with these properties, terpenes also allow for flexibility in route of administration and suppression of side effects. certain terpenes were widely used in natural folk medicine. one such terpene is curcumin which holds anti-inflammatory, antioxidant, anticancer, antiseptic, antiplasmodial, astringent, digestive, diuretic, and many other properties. curcumin has also become a recent trend in healthy foods and open doors for several medical researches. this chapter summarizes the various terpenes, their sources, medicinal properties, mechanism of action, and the recent studies that are underway for designing terpenes as a lead molecule in the modern medicine. terpenes, also known as isoprenoids are the largest and most diverse group of naturally occurring compounds that are mostly found in plants but larger classes of terpenes such as sterols and squalene can be found in animals. they are responsible for the fragrance, taste, and pigment of plants. terpenes are classified on the basis of organization and number of isoprene units it contains (see footnote ). an isoprene unit is a building block of terpenes that is a gaseous hydrocarbon that contains the molecular formula c h (see footnote ). terpenes and terpenoids are terms that are often used interchangeably but the two terms have slight differences; terpenes are an arrangement of isoprene units that are naturally occurring, volatile, unsaturated -carbon cyclic compounds that give off a scent or a taste to defend itself from organisms that feed off of certain types of plants (see footnote ). terpenes have many functions in plants such as a thermoprotectant, signaling functions, and not limited to, pigments, flavoring, and solvents but also have various medicinal uses (yang et al. ) . table . shows the different types of terpenes discussed in this chapter along with an example of that terpene. terpene is a natural compound with various medical properties and found in both plants and animals (gershenzon ) . among natural products that mediate antagonistic and beneficial interactions within the organism, terpene play a variety of roles (gershenzon ) . terpene protects many living organisms like microorganisms, animals and plants from abiotic and biotic stresses (gershenzon ) . terpene can ward off pathogens, predators, and competitors. living organisms use terpene for multiple reasons like medicinal purposes and communications about food, mates, or enemies (gershenzon ) . it is impressive how different organisms use terpene for common purposes even though terpene contain many forms and varieties (gershenzon ) . so far only a small percentage of terpene is investigated (franklin et al. ). cannabis is one of the most common sources for the medicinal terpene (franklin et al. ). this plant contains many medicinal properties like anticancer, antimicrobial, antifungal, antiviral, antihyperglycemic, analgesic, anti-inflammatory, and antiparasitic (franklin et al. ) . terpene is also used to enhance skin penetration, prevent inflammatory diseases (franklin et al. ) . nowadays modern medication use large scales of terpene for various treatment drugs (franklin et al. ) . there are commonly used plants like tea (melaleuca alternifolia), thyme, cannabis, salvia lavandulifolia (spanish sage), citrus fruits (lemon, orange, mandarin) etc. that provide wide range of medicinal values (perry et al. ) . tea tree oil has increased in popularity in recent years when it comes to alternative medicine (perry et al. ) . tea tree oil is a volatile essential oil and is famous for its antimicrobial properties, and acts as the active ingredient that is used to treat cutaneous infections (carson et al. ) apart from the flavor that gives to food, essential oil contain antimicrobial properties (bound et al. ) . thyme is one of plants that synthesize terpene alcohols and phenols which contain powerful antibacterial and antifungal properties (bound et al. ) . terpene synthesized from cannabis also long served as medicines (perry et al. ) . they also contain psychoactive properties and used against many infectious diseases (perry et al. ) . salvia lavandulifolia is famous for anti-dementia (current memory-enhancing) drugs by enhancing james and dubery ( ) cholinergic activity via inhibition of cholinesterase (perry et al. ) . in vitro examination method was used to study the effects of constituent terpenes on human erythrocyte acetylcholinesterase (perry et al. ) . some of the medicinal properties of terpenes are listed in table . . important properties associated with terpene are difficult to overstress (franklin et al. ). there are many important uses with terpene and these include antiinsect properties, antimicrobial properties and anti-herbivore properties (franklin et al. ) . terpene can be extracted through plants and thorough some insects (franklin et al. ) . without using harsh chemicals that could potentially contain side effects, terpene is a healthy alternative to ward off insects (franklin et al. ). there have been many pesticides made for killing domestic pests like lice, or mites (franklin et al. ) . in these cases, it is very important to make sure that these pesticides do not affect humans in harmful ways (franklin et al. ). there are many options like shampoo, sprays, lotions that were manufactured against pests that include one or more terpenes that are employed in the instant invention (franklin et al. ). these naturally occurring terpenes are generally not modified they were used in their raw form and the environment protection agency in the usa classified as "gras" which mean generally regards as safe (franklin et al. ). certain terpene is highly effective against both lice and lice eggs and there is a less than significant chance of resistance developing against this terpene based pesticides; reason for this is their observed modes of action (franklin et al. ). silva et al. ( ) unlike other types of pediculosis medication this terpene based instant inventions are not neurotoxins (franklin et al. ) terpenes are also used combined with terpene aldehyde called citral. citral derives from an essential oil that is extracted from lemongrass (cymbopogon citratus) (franklin et al. ) . citral possesses antibacterial and antifungal properties, while lemongrass possesses anti-insect properties (franklin et al. ) . a series of anti-insect formulation contain many terpenes (franklin et al. ) most of these pesticides are a mix of terpene and citral (franklin et al. ). table . consists of what these terpenes include. antimicrobial properties or the ability to kill or stop growth of a microorganism in terpenes are commonly used in traditional and modern medicine (himejima et al. ). there are many terpenes with antimicrobial activities (himejima et al. ). the following plants produce terpenes which have antimicrobial properties: pinus ponderosa (pinaceae), spices (sage, rosemary, caraway, cumin, clove, and thyme), cretan propolis, helichrysum italicum, rosmarinus officinalis, and so on (himejima et al. ) . these antimicrobial terpenes can also be used against food borne pathogen like escherichia coli, staphylococcus aureus, and bacillus cereus (himejima et al. ) . pinus ponderosa cell extract contain wide-ranging antimicrobial activities (himejima et al. ) . after steaming and distillation from pinus ponderosa cell extract, a distillate and a residue are obtained (himejima et al. ) . the distillate consists of monoterpenes and some sesquiterpenes while the residue consists of four diterpene acids (himejima et al. ) . it was also reported that when a physical damage is caused to the pine tree or any other terpene containing tree from insect attacks, resin which contains terpene secret to protect the tree from further damage (himejima et al. ) . five different kinds of terpene can be isolated from cretan propolis, they are, the diterpenes, , -dinor- -oxo- ( )-labden- -oic acid and a mixture of labda- ( ), e-dien- -carboxy- -yl oleate, palmitate and triterpene (popova et al. ). spectroscopic analysis and chemical evidence has been used to establish the structures of the different compounds (popova et al. ). these compounds that were isolated from terpene was tested for its antimicrobial activity against bacteria like gram positive and gram negative (popova et al. ). it was all tested for human pathogenic fungi which has broad-spectrum antimicrobial activity (popova et al. ). helichrysum italicum essential oil was analyzed using gas chromatography and mass spectrometry to fraction into terpene and terpenoid. fifty two compounds, including hydrocarbons of the oil; α-pinene ( . %), α-cedrene ( . %) aromadendrene ( . %), β-caryophyllene ( . %), and limonene ( . %), neryl acetate ( . %), -methylcyclohexyl pentanoate ( . %), -methylcyclohexyl octanoate ( . %), and geranyl acetate ( . %) were identified (mastelic et al. ). the smallest of terpenes are monoterpenes. they contain the compound c h , come from different flowers, fruits and leaves and are known as the main component of essential oils, fragrances and many structural isomers (see footnote ). monoterpenes are also the most fragrant of all the classes of terpenes (see footnote ). examples for the types of monoterpenes found in natural scents are α-pinene, which imparts scent to pine trees, and limonene from citrus plants (see footnote ). what is thought to be one of the main purposes of monoterpenes is to attract pollinators or to serve the purpose of repelling other organisms from feeding off of plants. they also may be related to the flowering process of the plants (loreto et al. ) . they are isolated from their plant sources by distillation with steam and have a boiling points in the range of °c to °c (see footnote ). monoterpenes are purified using fractional distillation at pressures that are reduced or use another process in order to form a crystalline derivative (see footnote ). many studies test the hypothesis of high emissions of monoterpenes under high temperatures using the leaves of quercus ilex, also known as evergreen oak (table . ). the evergreen tree is native to the mediterranean area where it has to survive under hot and dry conditions and synthesis of these monoterpenes may have been an adaptive mechanism for the plants to survive under heat stress. this tree does not emit isoprenes but it emits monoterpenes and is able to handle different environmental stresses such as drought, salt, and heat (see footnote ). a particular study done by loreto et al. ( ) were conducted to visualize monoterpene production in response to high temperatures and to see if thermotolerance is increased with monoterpenes (loreto et al. ) . in this study, the leaves were exposed in °c intervals ranging from the temperatures °c to °c and leaves were kept under conditions in which inhibited or allowed monoterpenes to synthesize (loreto et al. ) . the results that were found in this experience was a discovery of seven most abundant monoterpenes which was emitted at the maximum temperature of °c and decreased its abundance over time as the temperatures increased and α-pinene had the greatest abundance of emittance at °c as well as other terpenes but greatly reduced over higher temperatures (loreto et al. ) . at °c the monoterpenes, myrcene and limonene had higher emission rates compared to temperatures around °c (loreto et al. ) . photosynthesis was also decreased when the leaves were exposed to any temperature that was higher than °c and at °c showed a loss of co and recovery occurred around °c (loreto et al. ) . overall, the monoterpenes showed that their optimal temperature for emission was around - °c (loreto et al. ) . researchers prove that the emission of monoterpenes is under enzymatic control due to their optimal temperatures (loreto et al. ) . sesquiterpenes, containing the chemical formula c h , are much larger compounds than monoterpenes and are much more stable in comparison. they are isolated by distillation with steam or by extraction and purified by methods such as vacuum fractional distillation or gas chromatography (see footnote ). oxidation or rearrangement of isoprene units that are made to sesquiterpenes produce the corresponding sesquiterpenoids (see footnote ). sesquiterpenes are naturally occurring and found in plants, fungi, and insects and act as a defensive mechanism or attract mates with pheromones in insects (see footnote ). acyclic compounds of sesquiterpenes such as farnesans can be used as a natural pesticide for insects and also as pheromones for some insects and mammals such as elephants, to attract mates or to mark their territory (see footnote ). sesquiterpenes have a vital role in plant growth hormones and signaling properties in response to its environment (giraudat ) . abscisic acid has a role in plants such as development, germination, cell division, and synthesis of protein storage and signalling (giraudat ) . it also plays a role in plants in response to various environmental stresses. it regulates the closure of the stoma by regulating ion channels and exchange of water across the plasma membrane (giraudat ) . cyclic adp-ribose signals abscisic acid in response to drought-stressing conditions from the environment (giraudat ) . abscisic acid is not unique to plants, it has shown to be present in the central nervous system of other organisms such as pigs and may play a role in humans as a pro-inflammatory cytokine and stimulator of insulin release in the human pancreas (chadwick et al. ) . gossypol is a sesquiterpene that is found in cotton plants. it has anticancer properties and can potentially inhibit fertility in male humans which is why it must be removed from essential oils and various other products before human use or consumption. avarol, a sesquiterpenoid that has shown to have antimicrobial and antifungal uses, is effective against the aids virus in humans (see footnote ). the medicinal properties of sesquiterpenes typically come from flowering plants that are included in the asteraceae family, which include, but not limited to sunflowers, marigolds, and daisies. this family of flowers is a significant resource for potent sesquiterpene lactones, which are usually found in the leaves and the flower portion of plants and are constantly being produced at high levels (chadwick et al. ) . the role of sesquiterpenes in these flowering plants are not solely made for human use but for the purpose of protecting the plant from predators and are produced de novo in response to microbial attack and ultraviolet ray protection (chadwick et al. ) . their bitter taste is a defense mechanism against herbivores from feeding on them but some have sweet tastes or tastes that are pleasant to certain organism for the purpose of spreading their seeds and being fertilized in different areas (chadwick et al. ) . sesquiterpenes have many uses in traditional, western medicine because they contain so many anticancer, antiplasmodial, and anti-inflammatory activities (chadwick et al. ) . sesquiterpenes lactones are able to reduce stomach ulcers in some people and are also present in powerful antimalarial drugs (chadwick et al. ). artemisinin, a metabolite produced from artemisia annua, which contains sesquiterpene lactone produced in the roots and shoots of the plants, is used in drugs to treat malaria (chadwick et al. ) . other uses of this family of flowers is for treatment of bacterial infections, migraines, and to improve skin (chadwick et al. ) . lettuce opium has been used for many years as a painkiller (chadwick et al. ). diterpenes are naturally occurring chemical compounds that contain the molecular formula, c h . diterpenes have physiologically active groups such as vitamin a activity well as plant growth hormones that regulate germination, flowering and switch reproductive cycles (from asexual to sexual reproduction) of plants (lee et al. ) . they can also be classified as a phytol, which is an oxygenated acyclic diterpene. over diterpenoids have been isolated from euphorbia plants, which is a very diverse genus of flowering plants (popova et al. ). diterpenes have many therapeutic benefits such as antitumor, cytotoxic, and anti-inflammatory (vasas and hohmann ) . they are present in anticancer drugs such as taxol, and the tumor promoter, phorbol (vasas and hohmann ) . tanshinones are a class of diterpenes that are isolated from dried roots or rhizomes of an herb in traditional chinese medicine called salvia miltiorrhiza also known as danshen or tanshen (zhang et al. ) . tanshinones were first isolated in the s, and since then, more than chemicals have been identified and split up into two groups: lipophilic and hydrophilic compounds (zhang et al. ) . tanshinones have recently been extensively researched for their anticancer properties in vitro and in vivo (zhang et al. ). their potential use as an anticancer drug comes from their broad range of activities such as anti-proliferation and inhibiting adhesion, migration, and invasion (zhang et al. ) . analogues of tanshinone have been synthesized in many clinical trials because they have many anticancer attributes (lee et al. ) . this herb has been used in many asian countries for preventative and therapeutic solutions to many diseases such as heart disease, vascular diseases, and arthritis (zhang et al. ) . tanshinones may also reduce inflammation and increase immune responses (zhang et al. ) . cafestol and kahweol are diterpene alcohols that are found in the oil derived from coffee beans. these chemical structures are very similar but only differ by an extra double bond that is present in kahweol's chemical structure. researchers have reported that coffee lowers the risk of depression in women, prostate cancer in men, stroke, diabetes, and some cancers (see footnote ). it is thought that the antiinflammatory and antioxidant properties of these particular diterpenes are responsible for such events (see footnote ). coffee benefits the liver as well by lowering liver enzymes that are in response to inflammation and damage and may offer some protection against liver cancer as well (see footnote ). the adverse result of these diterpenes is that they raise cholesterol level, but it seems to be limited to coffee that has been unfiltered and has oily droplets of cafestol and kahweol (see footnote ). filtered coffee may not have much impact on cholesterol levels (see footnote ). triterpenes are composed of three or six isoprene units and have the chemical formula c h which includes steroids and sterols with squalene being the biological precursor of all triterpenes (see footnote ). triterpenes are produced by animals, plants, and fungi. they play a role as precursors to steroids in animal and plant organisms, and are derived from mevalonic acid (see footnote ). saponins come from the skins of many plants and have emulsion like properties that make them excellent detergents in the human digestive system (see footnote ). chemical structures of steroid saponins are similar to hormones that are produced in the human body (see footnote ). the medicinal uses of triterpenes are not quite as recognized as other different types of terpenes but their uses are being continuously investigated by researchers. their properties have been studied for anticancer, antioxidant, antiviral, and anti-atherosclerotic activities (nazaruk and borzym-kluczyk ) . some studies have shown that there is promising potential for the use of triterpenes for people with diabetes by aiming to reduce glucose levels and also by reducing sweetness inhibitors in sweet and high calorie foods (nazaruk and borzym-kluczyk ) . saponins have detoxification properties and act as a diuretic for the kidneys and wound healing properties (nazaruk and borzym-kluczyk ) . tetraterpenes are also known as carotenoids that have the molecular formula c h and can be in the category of terpenes because they are made from isoprene units. most carotenoids are highly unsaturated and for this reason, they are extremely difficult to isolate and purify (see footnote ). they are found in all different types of fungi, bacteria, and plants and mainly responsible for red, yellow, or orange fatsoluble plant and animal pigments (see footnote ). one of the most crucial and common tetraterpene is beta-carotene that contributes to the yellow pigment in carrots. it is important to mammals especially because it is a precursor in producing vitamin a and other important terpenoids for vision (see footnote ). higher order terpenes have been shown to increase thermotolerance (singsaas ) . the permeability of the thylakoid membranes increase at higher temperatures and this happens by an increase in cyclic photophosphorylation around photosystem ii (singsaas ) . when the temperature of the atmosphere continues to rise, the photophosphorylation system is not able to keep up with protons leaking, which causes the transmembrane gradient to drop and a reduction in atp synthesis occurs (singsaas ) . all these events can potentially cause lowering in the rubisco activation state due to an inhibition of rubp regeneration (singsaas ) . the mep pathway, also known as the non-mevalonate pathway or methylerythritol phosphate pathway, is a metabolic pathway for isoprenoid biosynthesis that creates the products isopentenyl pyrophosphate (ipp) and dimethylallyl pyrophosphate (dmapp). this pathway occurs in the chloroplasts and produce monoterpenes, specific sesquiterpenes, diterpenes, and carotenoids (zhang et al. ) . the vital application of this pathway is to develop antimicrobial agents to target diseases such as malaria and sexually transmitted diseases (hunter ) . since this pathway does not occur in humans, it is a valuable resource to develop antibacterial and antiparasitic drugs (seemann et al. ). the first steps of this pathway involve pyruvate and d-glyceraldehyde -phosphate to produce doxp which is catalyzed by -deoxy-d-xylulose- -phosphate (dxs) (hunter ) . -deoxy-d-xylulose- -phosphate reductoisomerase, otherwise known as ispc, coverts doxp to mep. from mep, it reacts with ctp to create -diphosphocytidyl- c-methyl-d-erythritol (hunter ) . a phosphate is released in this reaction and then reacts with atp-dependent ispe to make -dipho sphocytidyl- c-methyl-d-erythritol -phosphate and adp and then reacts with the enzyme ispf to create c-methyl-d-erythritol , -cyclodophosphate (hunter ) . the enzyme requires metal cations. then finally, in the least understood step of the reaction, the two enzymes, ispg and isph make the two products, ipp and dmapp by using a two-electron reduction (hunter ) . the pathway is regulated by control of repression or activation of gene expression via feedback loops within the pathway or by effector molecules which target an enzyme or downstream activities (hunter ) . the mva pathway or mevalonic acid pathway occurs in the cytosol. it is responsible for the synthesis of sterols, specific sesquiterpenes, and also may play a role in the synthesis of transhinones (zhang et al. ) . in gram-positive bacteria, the genes in the metabolic pathways such as mva are organized into operons and are thought to be regulated by transcription (hunter ) . the use of cannabis is increasing for medicinal uses that commonly treat pain, the side effects of chemotherapy in cancer patients such as nausea, anxiety and depression, and its uses and benefits are continuously being researched by scientists (cathcart et al. ) . there are at least compounds that come from the cannabis plant that are regarded as cannabinoids that cause psychotropic effects in the human brain due to cb receptors (klein et al. ) . the main active ingredient, delta- tetrahydrocannabinol, otherwise known as thc, is a psychoactive agent and is a focus for controversy in society because it binds to the human endocannabinoid receptors in areas of the brain such as the hippocampus and the frontal cortex, which are responsible for memory, cognition and attention. how thc works is by taking the place of endocannabinoids, naturally occurring chemicals in the human body (see footnote ). one of the most common and well known molecules that thc replaces in the human body is called anadamide (see footnote ). to this day, scientists are researching to discover the exact role of this molecule in the human body. cannabidiol, or cbd is also a common ingredient in cannabis but compared to thc, it is a non-psychoactive and it can potentially reduce the effects of thc (klein et al. ). cbd does not bind to the same receptors as thc does in the human body and it works by inhibiting faah or the enzyme fatty acid amide hydroxyls (see footnote ). this enzyme is responsible for degrading anadamide in the body and by inhibiting faah, cbd increases natural endocannabinoids already in the human system (klein et al. ) . cbd is thus an agent that works for depression, anxiety and neuroprotective effects (klein et al. ) . what are major components in cannabis are the monoterpenes that are responsible for many different medicinal properties. one of the main uses for thc is the potential for cancer treatment and can play a role in reducing size of tumors (see footnote ). thc can also reduce inflammation caused by certain diseases in patients. other conditions that thc can help but are not limited to are adhd, arthritis, migraines, and glaucoma (see footnote ). it can also improve the symptoms in individuals that suffer from hiv by helping their appetite and thus causing weight again, improving their depression symptoms and their quality of life (lutge et al. ). terpenes have been shown to have a favorable antiplasmodial activity. with the rising malarial infections and drug resistance, terpenes have gained more attention towards it through antiplasmodial activity (nogueira and lopes ) . the interesting mechanism behind the terpene activity is that it binds to the hemin part of infected erythrocytes and kills the parasite just like the famous antimalarial drug chloroquine (orjih et al. ; kayembe et al. ) . hemin is made of iron which is necessary for the plasmodium development in the erythrocytes. though hemin breaking enzymes are not yet found in plasmodium, it could be one reason why hemin binding accounts for parasite lysis (ginsburg and demel ) . another study suggests that drug-hemin complex binds to phospholipid layers thereby disrupting the respective membrane structure and causing cell lysis (ginsburg and demel ) . moreover, it is also known that hemin can affect the carbohydrate metabolism of the parasites, which could lead to lysis of parasites (rodriguez and jungery ) . thus, terpenes can be designed to be promising drugs for malaria. different kinds of terpenes show different effects on the parasites. for instance, beta-myrcene the most common terpenes, is proven to have in vitro antiplasmodial activity (kpoviessi et al. ). beta-myrcene from cannabis sativa, the plant which is high in terpenes, does not show an anti plasmodial effect but extracts from stem, leaves, and seeds of clove basil showed a good antiplasmodial activity (small ; kpoviessi et al. ). additionally, it was also reported to have antitrypanosomal activity when tested against trypanosoma brucei brucei (habila et al. ) . this data leads to the fact that terpenes are effective against pathogenic protista. limonene regarded as the second most commonly found terpene, also possesses antiplasmodial activity against plasmodium falciparum. limonene achieves its goal by targeting the intermediates of the active isoprenoid pathway of the parasite. isoprenoid pathway plays a major role in parasite survival by mediating cell signaling, protein translation and several other biological processes (jordão et al. ) . specifically, the isoprenic products that are inhibited from being synthesized are dolichol and ubiquinone (goulart et al. ). the isoprenoid pathway of parasites is distinct from that found in mammals, which makes limonene a reliable constituent of antimalarial drug (goulart et al. ). thus, the host cell pathway will not be affected by the administration of the drug. pinene, commonly found monoterpene in pine trees is composed of two classesalpha-pinene and beta-pinene. both the classes of pinene were reported to be effective against the w strain of plasmodium falciparum, which is resistant to chloroquine (boyom et al. ) . of particular interest is the increase in antiplasmodial activity of pinene in cumin seed oil with increase in the distillation time. the study concluded that the optimal distillation time for increased antimalarial activity is - and - . min (zheljazkov et al. ) . further investigation is needed to ascertain if distillation time is just increasing the yield of pinenes in the oil or improving the bioactivity of pinenes. the next most abundant terpene, caryophyllene has the ability to both prevent and cure malaria. caryophyllene is an active component of insect repellents especially for mosquitoes and other blood-feeding diptera (maia and sarah ) . recent studies ensured that silver nanoparticles synthesized from caryophyllene are highly effective against plasmodium falciparum (kamaraj et al. ). thus, terpenes could be a safer and a cost effective alternative for malarial treatment. the emerging viral diseases have necessitated the research for new effective antiviral agents such as terpenes. as a result, scientists evaluated various terpenes for their properties, among which monoterpenes showed a good result. monoterpenes are terpene classes that possess two isoprene units. they form a major constituent of essential oils in plants which indicates monoterpenes play a major role in defense for plants (grabmann ) . a study evaluated the in vitro antiviral activity of several essential oils extracted from south american plants (duschatzky et al. ) . the oil extracts were tested against three major human viruses-herpes simplex virus- (hsv ), dengue virus type , and junin virus. the oils that were proved to be virucidal were mainly composed of monoterpenes, namely, carvone, carveol limonene, alphaand beta-pinene, caryophylene, camphor, beta-ocimene, and one sesquiterpene which is germacrene (duschatzky et al. ) . a similar study in analyzed the essential oils of seven plants from lebanon for in vitro antiviral activity (loizzo et al. ). the viruses under investigation were hsv and severe acute respiratory syndrome corona virus (sars cov). the results were positive for antiviral effects, and the major constituents were alpha-and beta-pinene, beta-ocimene, and , -cineole (loizzo et al. ) . following this, a study on salvia cedronella also had similar results which suggested , -cineole, α-pinene, caryophyllene oxide, and sabinene to be the major components of virucidal oils (alim et al. ). functional data from these studies reveal that a few monoterpenes are shared by various plants for antiviral properties (alim et al. ). these shared monoterpenes could be of importance as they are present universally. of particular interest is the single main monoterpene that is contributing to the virucidal activity. this was studied by astani et al. ( ) using eucalyptus, tea tree, and thyme essential oil extracts (astani et al. ). they suggested that monoterpene hydrocarbons have a slightly higher virulent activity compared to the monoterpene alcohols against hsv- . the monoterpenes with the highest virucidal activity were identified to be alpha-pinene and alpha-terpineol (astani et al. ). the mechanism behind the virucidal activity was suggested to be direct inactivation of free viral particles. however, the study concluded that more than isolated single monoterpenes, a mixture of monoterpenes are more effective and possessed lesser toxicity to host cells (astani et al. ). this was further bolstered by another study which evidenced the virucidal property of a combination of monoterpenes obtained from melaleuca alternifolia (zamora et al. ). the activity was tested against a human flavivirus west nile virus. the results were positive both in vivo and in vitro. the underlying mechanism was predicted to be induced cell cycle arrest at g or g phase. this indicates that a mixture of monoterpenes could act as a better antiviral agent rather than a single monoterpene (zamora et al. ) . recent studies have shown that triketone-terpene adducts also exert antiviral, antimicrobial and antitumor activity (chen et al. ). these adducts are obtained from myrtaceae as secondary metabolites in the form of sesquiterpenes called myrtucomvalones a, b, and c. the terpene adducts successfully inhibited the respiratory syncytial virus (rsv) (chen et al. ) . the bioactive terpenes present in various plants have shown various results for antiviral property. it would therefore be important to look for various plant source rather than various monoterpenes for therapeutic purposes. researchers are also focusing on synthesizing terpene hybrid from fungal sources as they are presumed to have antiviral and uv protective properties (yuan et al. ) . terpene synthesis from fungi can lead to cost effective and limited labor methods (yuan et al. ). the medicinal benefits of terpenes are not limited to pathogenic diseases. terpenes are widely acclaimed for their anticancer activity too. an early study concluded that a combination of monoterpenes, diterpenes and sesquiterpenes can be effectively used to treat cancers that occur in colon, brain, prostate gland, and bones. it also claimed that administration of terpenes in humans inhibited the growth of prostate cancer cells and sensitized the tumor in such a way it becomes susceptible to radiotherapy (see footnote ). the major advantage of this treatment was that, the drug can be administered through several routes among which oral and topical were most preferred (see footnote ). among the different kinds of terpenes, limonene is well recognized as an anticancer agent. limonene is a bioactive food component found in citrus peels, orange peels, and several other citrus fruits (jirtle et al. ) . studies have reported limonene to exhibit strong cancer inhibition activity both in vitro and in vivo. the mechanism behind limonene activity is still under investigation. a study by jirtle et al. ( ) reported that limonene acts through induction of transforming growth factor b- and mannose- -phosphate/insulin-like growth factor ii receptors (jirtle et al. ). in contrast a study by bishayee and rabi ) structural studies on limonene reported that they are lipophilic and have the tendency to be deposited in fatty tissues when administered orally. this indicates that limonene can act as an excellent chemopreventive drug for cancer as it can be deposited in the body (miller et al. ) . another study in concluded that limonene acts by suppressing the expression of breast tumor cyclin d (miller et al. ) . this lead to cell cycle arrest and mitigated proliferation of cancer cells in women with early stages of breast cancer (miller et al. ) . recent study showed that limonene from pinecones can kill lung cancer cells in vitro by apoptotic mechanism that is activated through caspase- pathway (lee et al. ) . these findings indicate a novel application of limonene towards fighting and preventing cancer. not just limonene, but also its metabolite perillyl alcohol is also said to exhibit antitumor activity in pancreatic cell lines through apoptotic mechanisms (sobral et al. ; dalessio et al. ) . apart from limonene, the terpene thymoquinone has all been widely studied for its chemoprotective and chemotherapeutic activity. thymoquinone is found to be an active constituent of the volatile oils of an annual herbaceous plant called nigella sativa (black cumin) (majdalawieh et al. ). the pathways affected by thymoquinone to exert its antitumor properties are p , pparγ, mapk, nf-κb, pi k/akt, and stat signaling pathways (majdalawieh et al. ) . thymoquinone has been proved to be anticancerous against several cancers such as breast cancer, skin cancer, non-small cell lung cancer, bile duct cancer, and brain cancer. the basic mechanisms underlying the cancer inhibition is apoptosis and cell cycle arrest (sobral et al. ; khader and eckl ) . most of the cancer related studies were performed using thermoquinone obtained from the n. sativa extracts. a study showed that thermoquinone can be obtained in larger amounts from the mint family, namely, monarda didyma and monarda media (taborsky et al. ) . thus, thermoquinone from alternative sources has to be tested for its precious potential in cancer therapy. other terpenes that have reported cytotoxic effects on cancer cells include alloocimene, camphor, beta-myrcene, pinene, alpha-and gamma-thujaplicin, terpinene, thymohydroquinone, carvone, camphene, and cymene (sobral et al. ) . terpenes being natural compounds are unlikely to affect the healthy cells or create a side effect, which attracts many researchers to exploit its capability in cancer treatment. diabetes is one of the widely prevalent diseases in the world. it is affecting both children and adults in both developing and developed nations (you and henneberg ; narayan et al. ) . the social and economic burden of diabetes continues to grow and it is expected to rise rapidly in developing countries (sarwar et al. ) . in usa, diabetes is one of the leading causes for visual impairment, limb amputation, renal diseases, heart diseases and death (saddinne et al. ) . diabetes can be of two types-type (where the immune system of the body acts against the insulin-producing organs) and type (where the insulin produced cannot be used by the body or insulin is produced in low amounts). although there are several medications available, their use is limited due to their adverse effects. some of the commonly found side-effects include low blood sugar, vomiting, nausea, diarrhea, bloating, and weight gain. this led to the research for natural products to be used as effective antidiabetic medication. phytochemicals from the medicinal plants have been recommended for treating type diabetes, of which terpene forms a major constituent (jung et al. ) . medicinal plants of oriental morocco were studied for their antidiabetic property in rats. the report showed that terpenes, terpene diols, and terpene diol glucosides form major components of the extracts of plants under study (bnouham et al. ) . a similar study on medicinal plant and their natural products that were reported from - was conducted by jung et al. . this study was focused on non-insulin-dependent diabetes mellitus (type ), and it proved that terpenes along with few other secondary metabolites such as alkaloids and flavonoids exhibit antidiabetic potential (jung et al. ) . the most promising terpene compound for treating diabetes is called andrographolide which is a diterpenoid lactone (brahmachari ) . this compound forms the major component of the leaves of the small herbaceous plant andrographis paniculata. a. paniculata is an asian plant that has already been reported to be used in traditional medicines for its therapeutic nature (brahmachari ) . the terpenoid acts by reducing the plasma glucose and increasing the utilization of glucose by the body in diabetes mellitus rats (gupta et al. ). the actual mechanism by how it does this is it activates the alpha-adrenoreceptors to increase the release of an opioid peptide beta-endomorphin (brahmachari ) which is reported to be secreted in low amounts in diabetic rats (forman et al. ) . this increased secretion in turn activates the opioid μ-receptors. these receptors can effectively curb the hepatic gluconeogenesis (glucose synthesis from non-carbohydrate precursors) and elevate the utilization of glucose by muscles. finally, this results in a reduced plasma glucose concentration (brahmachari ) . andrographolide is also observed to prevent the secondary complications of diabetes such as diabetic retinopathy, a condition that will lead to blindness (brahmachari ) . it significantly weakens the retinal angiogenesis and inflammation during the development of the disease (brahmachari ) . moreover, it can also fix the impaired or extended estrous cycle in diabetic rats (reyes et al. ) . andrographolide was orally administered in all the above studies. this indicates its efficiency for being used as a lead molecule in the future drugs designed for treating diabetes mellitus. another widely known terpene is curcumin obtained from curcuma longa which commonly called turmeric (nabavi et al. ) . it exhibits high antidiabetic property and acts by quashing the oxidative stress and inflammation. by regulating the polyol pathway, it can also reduce the plasma glucose and levels of glycosylated hemoglobin (nabavi et al. ) . moreover, curcumin is also reported to activate the enzymes present in the liver that are essential for glycolysis, gluconeogenesis, and lipid metabolism (zhang et al. ) . alike andrographolide, curcumin is also reported to reduce the complications of diabetes (nabavi et al. ) , for example, liver disorder which is a common manifestation of diabetes type (zhang et al. ) . curcumin treats these disorders by reducing the liver weight and lipid peroxidation products. further, it is also reported to normalize the levels of fetuin-a in serum that contributes to insulin resistance and fatty liver in diabetic rats (zhang et al. ) . other complications that can be attenuated by curcumin include diabetes associated-retinopathy, microangiopathy, neuropathy, and nephropathy (zhang et al. ) . these findings confirm that curcumin is likely to be used in the future for diabetes treatment. depression has become a serious health concern by contributing to the emerging mental and emotional disorders throughout the world. it is hitting both the developed and developing countries. depression can pave way to various health issues from alcoholism to heart diseases (holden ) . it is also said to increase the rate of mortality significantly in breast cancer patients (hjerl et al. ) . moreover, depression immobilizes its victims thereby leading to economic loss (holden ) . by analyzing the social and economic burdens caused by depression, researchers have stepped out towards finding novel stress-relieving drugs. synthetic drugs have serious side-effects and unintended interactions with the body that negatively affects the treatment outcome (jawaid et al. ) . hence this necessitated the need for natural drugs. terpenes serves as one of the most relevant bioactive compound for treating depression and therefore can open doors for designing natural or synthetic antidepressant drugs (bahramsoltani et al. ) . twenty-five percentage of antidepressant drugs prescribed by doctors are obtained from herbs through various extracts (saki et al. ) . to estimate the important compounds contributing to the antidepressant effect, saki et al. ( ) performed an electronic database based study. the results revealed that terpenes formed a major part of the extracts of medicinal plants that exerted antidepressant effects (saki et al. ) . thus, scientists focused on identifying the active principles of plant extracts contributing to the antistress effects. different plant had different acting compounds. among the several terpenes, linalool and beta-pinene are commonly found to be active principles (both guzmán- gutiérrez et al. ; guzmán-gutiérrez et al. ) . they were discovered from the extracts of medicinal plants litsea glaucescens and tagetes lucida and flowers of lavender (appleton ; guzmán-gutiérrez et al. ; guadarrama-cruz et al. ). these monoterpenes act by interacting with the ht a receptors of the serotonergic pathway. serotonins are important in the fact that their release and re-uptake levels can be altered to overcome stress (chaouloff ; guzmán-gutiérrez et al. ) . they also interact with adrenergic receptors of the body that play a major role in stress-induced behavioral changes (pandey et al. ; guzmán-gutiérrez et al. ) . another interesting finding is the interaction of beta-pinene with dopaminergic receptors namely d receptors. this is the mechanism followed by most of the antidepressant drugs available in the market (guzmán- gutiérrez et al. ) . a more interesting study would be to examine the beta-pinene and linalool efficiency through inhalation tests. this is because these monoterpenes are aromatic compounds that generally have an enhanced activity when inhaled as they can directly hit the central nervous system (guzmán gutiérrez et al. ) . apart from monoterpenes, sesquiterpenes also exhibit antidepressant effects. one striking example is beta-caryophyllene which was proved to ameliorate the depressive symptoms in mice (bahi et al. ) . the underlying mechanism of this compound is binding to a receptor called cb and activating it. cb is found in the brain and immune cells and plays a major role in regulating depressive-related disorders (bahi et al. ) . thus beta-caryophyllene curbs depression by acting as a cb receptor agonist (bahi et al. ) . other terpenes that have effective antidepressant properties include hyperforin which is present in the extracts of hypericum perforatum (subhan et al. ) . it has been shown that the extracts of h. perforatum differ in their antidepressant potential with the difference in concentration of hyperforin present in the extract (laakmann et al. ). similar to many other antidepressants hyperforin acts by inhibiting the neuronal uptake of mood regulators such as serotonin, dopamine and norepinephrine. in addition, it also has its own unique mechanism of controlling depression by inhibiting the neurotransmitters gaba and l-glutamate uptake (müller et al. ) . another fascinating antidepressant plant is valeriana wallichii, which is a short perennial herb. this plant not only reduces the stress and anxiety levels but also improves the symptoms of depression in humans (bhattacharyya et al. ). the major components of valeriana extracts are terpenoids called maaliol, patchouli alcohol, and -acetoxypatchouli alcohol (subhan et al. ) . the terpenoid-less extract of valeriana was found to be devoid of antidepressant activity which indicates that terpenes are the active components involved in reducing the depression (subhan et al. ). folk medicine has always been an eye-opener for designing novel drugs for diseases. to be more specific, almost three-fourths of the plant-based drugs were created based on the knowledge of folk medicine (table . ) (efferth et al. ) . realizing this fact, western worlds are now turning back into old medicines and bioactive plant components to treat modern diseases (efferth et al. (efferth et al. , . this has boosted the export rates of chinese medicinal products (based on traditional chinese medicine) from china to other developed nations. plants used in traditional chinese medicine (tcm) are being extensively studied for their secondary metabolites and their therapeutic properties (efferth et al. ). one of the active principles of tcm products is terpenes (liu and jiang ) . due to their large availability and diversity, terpenes contribute the most to industrial and medicinal applications among all the secondary metabolites of plants (zwenger and basu ) . paclitaxel is one of the most successful terpenes available in the market today (efferth et al. ) . it is made out of yew trees which is a medicinal tree used in tcm. raw material from yew contains taxol (brand name of paclitaxel) which is used in the treatment of cancers in breast, lung, ovary, pancreas, cervix, and blood (see footnote ). , two variations of this drug are used now in chemotherapyconventional paclitaxel and albumin-bound paclitaxel (see footnote ). the advantage of the latter is that concentration increases in tumor cells at a rate higher than that of the former (see footnote ). the mechanism of anticancer activity is described as disruption of microtubules in the mitotic spindle, which will lead to incomplete chromosome separation thereby causing cell death (see footnote ). in tcm and ayurveda (herbal medicinal science mainly developed in india), healers used the twigs and barks of the tree to make a special kind of tea that can be given to patients suffering from cancer. however due to the slow growing nature of yew tree, paclitaxel nowadays is produced by coalescing the products of endophytic fungus that grows under the tree and the bark of the tree , (heinig et al. one more common terpene present in the drugs used in tcm is pinene (wu et al. ) . pinene exhibits therapeutic properties such as anti-inflammatory, antiseptic, anticancer, and antibiotic properties. , the source for pinene is eucalyptus and other related coniferous trees (see footnote , sartorelli et al. ) in olden days, the juice from the bark of eucalyptus was collected and mixed in water, milk or wine to be used as a drug (see footnote ). currently, they are extracted in the form of oil and sold in the form of syrups and lozenges. as eucalyptus oil contains several monoterpenes, a study analyzed the different constituents of eucalyptus oil for its effectiveness against bacteria. here it was concluded that alpha-pinene is the best monoterpene with the highest inhibitory activity (sartorelli et al. ) . recently scientists are studying another primary terpene in eucalyptus called cineole. cineole is reported to improve the memory power, cognitive performance and attenuate the symptoms of alzheimer's disease in humans (see footnote ; moss and oliver ) . in addition, studies also showed that cineole is capable of improving the health of bronchitis patients by reducing their cough (fischer and dethlefsen ) . this is in agreement with the fact that eucalyptus oil was used as an expectorant in ayurvedic medicine. it is also known that local brazilians used the eucalyptus leaves to treat several human diseases such as cancer (mathias et al. ) . further reports also suggest that eucalyptus oil has been involved in ancient indian ayurvedic and greco-european medicine systems (see footnote ). ayurveda is a popular medicine system which originated about years ago in india. the ayurvedic medicines are based on medicinal herbs, minerals, and metals (see footnote ) along with diet regimes such as vegetarianism (caldecott ) . this system of medicine has proven to cure chronic disorders that could not be treated by western medicine (sharma et al. ) . interestingly a lot of medicinal plants used by ayurvedic practitioners owe their therapeutic property to their terpene contents. one good example is turmeric, a family of ginger which is regarded as "golden goddess" by medical practitioners (see footnote ). it has numerous therapeutic properties that includes anti-inflammatory, antioxidant, anticancer, antiseptic, antiplasmodial, astringent, digestive, diuretic, and many more (see footnote ). recently, scientists discovered that most of the turmeric's properties are laid out by the yellow-colored terpene-curcumin (kocaadam and Şanlier ) . studies are now trying to create curcumin analogues to improve the effects and activity of natural curcumin (kocaadam and Şanlier ) . another popular example is clove which was used by both ayurveda and tcm as a painkiller in dental cases. it was applied topically on cavities to relieve toothache and abdomen to treat digestive problems (alqareer et al. ) . the essential oil of clove is mostly composed of eugenol, a bioactive terpene that is responsible for clove's aroma (alqareer et al. ) . eugenol by itself is said to enhance the blood circulation in the body and improve metabolism (see footnote ). thus, based on the above data we can conclude that various terpenes have been in use even before their discoveries by modern science, due to their amazing medicinal properties. a schematic summary of different terpenes and their medicinal uses, that we discussed, is provided below in fig. . . 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and anti-cancer activities curcumin and diabetes: a systematic review distillation time as tool for improved antimalarial activity and differential oil composition of cumin seed oil plant terpenoids: applications and potentials holy basil/ tulsi eugenol, β-elemene, β-caryophyllene and germacrenerestores functions of nervous system increases fertiltity used to treat asthma and cold key: cord- - pmts ui authors: nema, vijay title: microbial forensics: beyond a fascination date: - - journal: dna fingerprinting: advancements and future endeavors doi: . / - - - - _ sha: doc_id: cord_uid: pmts ui microbiology has seen a great transition from culture-based identification of microbes using various biochemical and microscopic observations to identify and functionally characterize the microbes by just collecting the dna and sequencing it. this advancement has not only moved in and around microbiology but has found its applications in fields which were earlier considered to be the remote ones. forensics is one such field, where tracing the leftover evidence on a crime scene can lead to the identification and prosecution of the culprit. when leftover microbes in the biological material or objects used by the culprit or the person in question are used to correlate the identity of the individual, it takes us to the new field of science—“microbial forensics.” technological advances in the field of forensics, molecular biology, and microbiology have all helped to refine the techniques of collecting and processing of the samples for microbiological identification using dna-based methods followed by its inference in the form of evidence. studies have supported the assumption that skin or surface microflora of an individual is somewhat related with the microflora found on the objects used by that individual and efforts are ongoing to see if this is found consistently in various surroundings and with different individuals. once established, this technique would facilitate accurate identification and differentiation of an individual or suspect to guide investigations along with conventional evidence. legal investigations are not only the field where microbial forensic could help. agriculture, defense, public health, tourism, etc. are the fields wherein microbial forensics with different names based on the fields are helping out and have potential to further support other fields. tracing the leftover evidences on a crime scene remained the only way of getting to the culprit. the history and evolution of forensic sciences has been very fascinating and interesting for even those who do not understand the science behind it. the journey started with william james herschel sometimes in when he recognized that the fingerprints remain unique to the individuals. it was used on legal and administrative documents then and was published in with all evidences and their analysis [ ] . it was gradually picked up for all possible uses including crime scene investigations. fingerprints remained the landmark evidence in all forensic investigations and are still playing important roles. additional techniques came in to support forensics in the form of dna fingerprinting, wherein dna matching became useful in various cases. dna fingerprinting is used to establish a link between biological evidence and a suspect in a criminal investigation [ ] [ ] [ ] [ ] [ ] . dnaor genetic-fingerprinting relies heavily on the principle that no two individuals share the same genetic code. recently, there has been a new discipline co-emerged with culture-independent techniques of identifying microbes. this new discipline looks for microbes and tries to co-relate them with individuals as like fingerprints or dna. there is a constant interaction of individuals with microbes in their surrounding, and they leave microbes into their surroundings. whenever there is a physical contact, bacteria hop across from the skin to the material used. the microbial communities attached with an individual's skin or other sites are being explored, and preliminary evidences suggest that they are unique and can identify individuals or the material used by them in a few cases [ ] [ ] [ ] . this became the base for coining of a new term "microbial forensics." however the field has other dimensions too and would be discussed in detail in this chapter. the microbial forensics rely on the inputs from various fields of basic and applied sciences. these include microbiology, genetics, bioinformatics, forensic science, immunology, population genetics, biochemistry, molecular biology, epidemiology, etc. along with the law enforcement, public health, policy, and intelligence communities. bacterial density on the human skin may be as high as cells/cm [ ] and can be freely transferred to surface which comes in contact. evaluating the traces of skin microbiome left on questioned objects may be useful for forensic identification. calculating the distances from samples and their donators, it seems possible to estimate whether items or palm prints belong to one specific person or not. this has become possible because of the culture-independent method of tracing all microbial species present in a given environment. this technique used here is called nextgeneration sequencing, and the population of microbes no matter cultivable or nonculturable is called microbiome. in this process, total dna of the given environment or object is taken out, and the specific gene for prokaryotes, i.e., srrna gene, is amplified. this gene has a property like a clock with fast-moving arm and slowmoving arm. this concept has been explained by the work of karl woose who named this gene as a biological clock [ ] [ ] [ ] [ ] . the gene contains highly conserved regions to identify the microbes with consistency and at the same time some variable regions to differentiate between closely related microbes. the technique has advantages over dna from individuals as the microbiome dna is abundant in touch dna, and these organisms are much more stable because of complicated cell wall structures. hence, it might be easier in certain instances to extract bacterial dna than human dna from surfaces target samples. another good reason for this is the higher abundance of bacterial cells on the skin and shed epidermal cells as compared to human cells. there are some surfaces like fabrics, smudged surfaces, or highly textured surfaces from where obtaining clear fingerprints is very difficult. bacterial dna can still be found there and can help in solving the purpose [ ] . microbiology and forensic science were always considered to be different fields, and establishing a link between the two for better investigative power was not thought of earlier. microbiology is simply defined as study of microorganisms wherein microorganisms are the organisms that exist as single cells or cell clusters and must be viewed individually with the aid of a microscope. microbiology classifies microbes into various groups, and based on their characteristics and physiology, they could be assigned different genera and species. most of the microbes, especially the saprophytic ones, depend on other substrates and interact specifically with their environment and habitat to gain important nutrients for their survival. this requirement remains very specific and unique with respect to one community of microbes and here comes a link with forensic sciences. forensic science explores about those unique things (belongings or body remains) which could be co-related with the career (criminal) of those things, for instance, fingerprints or dna. because microbial communities on a particular individual's body are expected to be unique, identifying that individual with leftover microbial traces on the material used by the individual is a possibility and is being confirmed using various experiments. however, culturing of all those microbes and identifying them is a near impossible task. the reason is that all microbes do not grow on known culture media, and some which can grow are sometimes overtaken by the competition with other microbes in the same community. hence collecting the total dna and amplifying for the microbial signature gene is a good solution ( fig. . ). this technique is called metagenomics. further, a sequencing technique that sequences the metagenome is called next-generation sequencing (ngs). ngs also known as high-throughput sequencing, represents different modern sequencing technologies for sequencing dna and rna much faster than sanger sequencing and with lower economical and technical inputs. identifying individuals is important in forensic science, and various developments have supported it from time to time. however, while making use of dna, its quantity becomes crucial for accurate detection with desired quality for prosecution of crime. a smart offender can be cautious enough to decrease or degrade the traces of biological leftovers like blood, semen, etc. along with the fingerprints from the crime scene, which can complicate offender detection. hence although fingerprints and dna fingerprints remain very precise techniques, the availability of raw material becomes limiting factor in their use. microbes can help in such scenario as their dna is not as easily destroyed as a human dna and remains available on surfaces at crime scene or on objects used. the pattern of bacterial dna is dictated by the surrounding environment and the individual's microbiome [ , ] . it is possible that the different bacterial patterns or the type of bacteria with typical physiology could discriminate individuals with different lifestyles. hence, bacterial dna analysis may serve as a complimentary technique in cases where standard dna identification is partially informative [ ] . along with this, technologies like pcr, real-time pcr, mlst (multilocus sequence typing), mlva (multilocus vntr analysis), fish (fluorescence in situ hybridization), and microarrays are being employed based on the need of the case or the availability of the infrastructure. additional new methodologies like matrix-assisted laser desorption/ionization-time of flight (malditof), gas chromatography-mass spectroscopy (gc-ms), and liquid chromatography-mass spectroscopy (lc-ms) are also well established in resolving minor difference in bacterial strains. investigation of a crime scene is not just potential application wherein microbial forensics can play an important role. bioterrorism, biosecurity, biometry, medical forensics, etc. are the upcoming fields wherein definitive detection of microbes and their correlations may help a lot. research on the transmission of microbes between human and surrounding environments has proved to be a property with potential for microbiome to be used in forensic investigations. in some cases, typical human microbial associations have been used to relate individuals to objects they have used [ ] . pattern of associated microbes with the surfaces at home and the family living and working with them have shown a predictive correlation to an extent that family's home and that individuals within a home can be differentiated [ ] . similar is the case with smartphones which rather remain in contact with specific individual and for longer durations [ ] . interestingly the microbial communities were different on the top and bottom of the phone differentiating the individuals and surface flora. an interesting property of the deposited microbiome is that it changes with a constant rate on a particular surface and can be utilized for forensic calculations. work by metcalf and co-workers have revealed that postmortem, the microbiome of animal hosts changes radically, but the pattern is much more predictable [ ] . this can help tracing the time and direction of the events. lax and co-workers worked to determine if the surface type and individuals have an effect on the microbial communities and have found that microbial community structure was determined both by surface type and participant. another aspect of microbial forensics is its role in bioterrorism. herein the microbial forensics could be defined as "the discipline of applying scientific methods to the analysis of evidence related to bioterrorism, biocrimes, hoaxes, or the accidental release of a biological agent or toxin for attribution purposes" [ ] . microbial forensics, while dealing with bioterrorism, concentrate on identification of the agent or toxin and/or the mode of its production and dissemination. in addition, traditional forensic methods are used in conjunction to reach the goal of identifying the perpetrators of the crime. around , microbial species or strains are listed as dangerous for humans [ ] . building individual diagnostic methods for these many numbers of agents is an impossible task, and hence ngs is the only tool to do massive parallel sequencing and to identify unknown pathogens, microorganisms modified to create panic, and pathogens in complex communities or samples with low abundance. with the global expansion of trade and communication, frequent movement of individuals from one country to other is unavoidable. in such scenario carriage of an endemic pathogen or drug resistance to a new geographical region is a looming threat. recent outbreaks and transmission incidents of sars and ebola have raised an alert. microbial forensic has a crucial role in such cases wherein the status is not declared by individuals, is not detected by routine quarantine, or is a new pathogen altogether. detection of microbial drug resistance or the emerging resistance is becoming increasingly important for human health. similar things are true in the case of plant pathogens and food borne diseases [ , ] . the foodborne diseases have always been a substantial global challenge to public health. a huge population worldwide become sick of foodborne illnesses every year with a substantial burden on public health as well as on economy. addressing this problem has many steps, out of which one age-old problem is the rapid identification of the food source of the contamination. in a classic laboratory study, we could trace back the source of the foodborne outbreak, but the finding could not be utilized in helping the troubled ones immediately [ ] . this is due to the infrastructural limitations and technical challenges in identifying the pathogens. a technology which was considered to be reliable and was used till recent past was pulsed-field gel electrophoresis (pfge). however its resolution in pinpointing the source of the outbreak has not been satisfactory. recent employment of whole-genome sequencing (wgs) in such investigations has shown promise. a retrospective study by us food and drug administration's center for food safety and applied nutrition (fda-cfsan) in could provide a far better resolution of the causal factors. all the isolates were sequenced on the illumina miseq. wgs using illumina could distinguish all of the isolates which looked exactly the same. ultimately they concluded that the isolates from the outbreak were most closely related to a -year-old historical isolate that was linked to a processing facility only km away from the source of the outbreak [ ] . this could not only allow newer findings but also traced back the source of contamination to further allow the rectification. such cases are sometimes accidental but are often criminal too, and tracing back the source would prevent such cases. in bio crimes, serious disease outbreak by natural occurrence or intentional may result in harm or death, causing disruption, creating fear, and affecting economic well-being. microbial forensics thus plays an important role in consumer protection, food security, and even in litigation. agriculture and agricultural goods are also the susceptible area for microbial interventions and hence are important in terms of microbial forensics. there could be deliberate misuse of microbes or their products affecting flora and fauna which is important for agriculture. this could be given a name such as "agroterrorism" [ ] . a field of investigation emerged against this threat for investigating into the violations and used scientific knowledge and technology to do so. this was given the name of bioforensics [ ] . another aspect of microbial forensic application to the foodborne pathogens is to trace pathogens in cash crops especially spices and other costly ones. van doren and co-workers have studied reported illness outbreaks from canada, denmark, england and wales, france, germany, new zealand, norway, serbia, and the united states which occurred due to consumption of pathogen-contaminated spice during - . the outbreaks were reported from a few developed countries only. the reason for not including other countries was that those countries did not have updated technology to investigate and report similar findings. it was reported that these outbreaks resulted in human illnesses, hospitalizations, and deaths. infants/children were the primary population segments impacted by % ( / ) of spice-attributed outbreaks [ ] . the economic aspect associated here is the detection of pathogens after shipment and then the recall of the material. this involved a huge cost. recent development in microbial forensics in agricultural sciences also aids in pest control as well as deliberate introduction of pests along with food imports or use of pathogens as anticrop bioweapons. different companies are coming up with molecular detection tools for rapid detection of specific pathogens in such products. for instance, hu and co-workers have compared and evaluated the effectiveness of the molecular methods ( m molecular detection system (mds) and ansr pathogen detection system (pds)) for the detection of salmonella in egg products and compared the same with culture methods to find that the molecular methods are the superior and faster ones [ ] . budowle and co-workers have established a criterion comprising a foundation for investigators to establish, validate, and implement high-throughput sequencing (hts) as a tool in microbial forensics [ ] . likewise, design principles for an effective microbial forensics program for law enforcement and national security purposes have been provided [ ] . microbial pathogens or toxins can be used to commit acts of terror; they can be used as weapons for execution of a crime. in biological warfare, transmissible lethal agents are used to attack the targeted populations. the impact of the bioterrorism was seriously considered after the anthrax attack in the united states in . this incidence in the united states helped the world understand that bioterrorism can have drastic and global impacts. microbial forensics has a role in such cases by applying scientific methods for the analysis of evidence from such a bioterrorism attack. microbial forensic in conjunction with epidemiology can try to decipher if an outbreak is natural, accidental, or intentional [ , ] . for instance, study by price and co-workers found that the bacillus anthracis injectional anthrax cases were originated from heroin users in scotland [ ] . ou and co-workers used molecular tracking of hiv to report for the first time about the passage of hiv infection from dentist to patient after invasive healthcare procedure [ ] . a spanish anesthetist infected patients with hepatitis c virus which could be found using phylogenetic and molecular clock analysis [ ] . in the ebola outbreak, the origin and transmission could be traced using bioforensic methods [ ] , etc. other important and relatively new aspect of microbial forensics in medicine and medicolegal field is "thanatomicrobiome" (thanatos-death) that studies the microorganisms found in internal organs and cavities upon death. the thanatomicrobiome tries to investigate the total microbial communities including bacterial and fungi from all the body locations of decomposing corpses. these studies are important in providing evidence in medicolegal death investigations [ ] . by doing this, the concept of human postmortem microbiome project (hpmp) has also been introduced which would create a consortium of research projects to identify and characterize the thanatomicrobiome and epinecrotic communities (e.g., epithelial tissues, body cavities, and the alimentary canal), relating to human decomposition with a potential of finding a state-of-the-art, more dependable, and molecular way of determining the time of death [ ] . it has been shown that typical characteristics and pattern of the human microbiome might identify individuals and remain constant in the individual but differ from the others [ , , ] . this means that individuals might be specifically and consistently identified using their microbiome. however microbiome-based identifiability is still a long way to go. franzosa and co-workers have suggested a few means to achieve the target of identifiability using microbes. the identification of a "metagenomic code" that remains unique for an individual for a longer duration of time and stands true for a sizable population is the key [ ] . hence microbiome establishment, structure, personalization, and temporal stability are the standard terms and areas to work upon. there are some untraveled avenues which start from microbial forensics. emergence of antimicrobial resistance, prediction and prevention of future outbreaks, etc. are some of the fields wherein a surveillance system making use of the principles of microbial forensics can help. antimicrobial resistance in microorganisms emerges naturally. however, antimicrobial exposure due to human practices in healthcare, agriculture, sanitation, industrial processes, travel, and other fields contributes significantly. timely detection of pathogens harboring resistance can mitigate the onward transmission among individuals and among different geographical locations. timely detection of pathogens like hiv, severe acute respiratory syndrome (sars) virus, and pandemic influenza could have avoided the big health emergencies which we have witnessed in recent past. similar is the case with the pandemic spread of sars coronavirus in and h n influenza in resulting in substantial economic loss. microbial forensics can play a crucial role in such cases by checking the emergence in real time and suggesting measures to prevent transmission. however, a lot of investment is required to place such services at all vulnerable points or check points. technological advances especially in the field of metagenomics have paved way for identification of potential human pathogens among other species and have attained good predictive power regarding transmissibility and virulence of the novel microbes. the science of microbial forensics is in its infancy and needs much more that what has already been done. a few things which needs to be taken care as preparation before we take this as a routine science are protocols and procedures for collecting specimens at the attack site, recognizing that an attack is occurring and diagnosing the disease, analysis of specimens in contained facilities, quality assurance and control. although microbial forensics involve techniques or methodologies from basic laboratory sciences, the problems in question, processes engaged in and expected outcomes need more than that. there are efforts and continuous need for validating all the tools and techniques involved which should be acceptable to peers and stakeholder from scientific, legal, and policy making side. the optimization of methods to answer key questions pertaining to investigative and legal needs is a must to satisfy the criterion of acceptability. meeting these challenges will allow the establishment of a complementary and reliable method to compensate for the lacuna of dna fingerprinting and fingerprinting as discussed earlier. meeting the challenges needs the consorted efforts from global communities of workers from basic sciences, epidemiologists, forensic experts, medical experts, legal experts, and a big team of technology developers. the most reliable technique till date for microbial forensics is metagenomics-a culture-independent approach for identifying and enumerating microbes. metagenomic have been an outstanding technique for sequencing the genomes of unculturable microbes, which represent the vast majority of microorganisms, particularly from environmental samples. however, technological advancement identifies the rare taxon is awaited. as microbial metagenomics is undergoing a formative phase as a diagnostic technique, optimization of methods and their validation remain a challenge. other aspects of this are the leadership role and generic availability of tools and techniques. countries with major resources would be able to take lead in basic research, while the resource-limited setting may not be able to adapt microbial forensics owing to its monitory needs. availability of equipment and techniques for rapid and precise molecular diagnosis is important for controlling and responding to the needs of microbial forensics. till date, next-generation sequencing is the only technology that seems promising for microbial forensics. but the instrument and the reagents remain very costly as compared to biochemical tests and a few basic molecular assays being used in forensic laboratories. moreover, expert workers and bioinformatics analysis of the huge data generated after massive parallel sequencing or next-generation sequencing require a devoted facility and expertise. microbial forensics may, in most of the cases, be associated with the detection of causal pathogen in cases of biological terrorism, but microbiome associated with individuals and objects used or touched by them at crime scenes may also be used as a tool in providing forensically relevant information. this science has got its diverse utility in the field of medicine, agriculture, trade especially in food articles, etc. as discussed in this chapter. the evidence and the literature available till date indicate a definitive linkage between an individual and microbial communities inhabiting that individual skin or other body parts. the science is progressing toward identifying these communities, next steps of which would be to see if these communities have something in common and that common character has something to do with the individual. later work in this field may identify chemical entities and their microbial connections to land up to the final conclusions about signature communities of microbes and their forensic potential. however, we may have to continue our search to see those days soon. the origin of fingerprinting dna 'fingerprinting' of captive family groups of common marmosets (callithrix jacchus) forensic application of dna 'fingerprints' application of dna "fingerprints" to paternity determinations the man behind the dna fingerprints: an interview with professor sir alec jeffreys demographic study of a wild house sparrow population by dna fingerprinting forensic identification using skin bacterial communities comparison of bacterial dna profiles of footwear insoles and soles of feet for the forensic discrimination of footwear owners forensic analysis of the microbiome of phones and shoes microbial ecology of human skin in health and disease microbiology's scarred revolutionary - ) phylogenetic structure of the prokaryotic domain: the primary kingdoms towards a natural system of organisms: proposal for the domains archaea, bacteria, and eucarya microbial forensic analysis of bacterial fingerprint by sequence comparison of s rrna gene cohabiting family members share microbiota with one another and with their dogs is human dna enough?-potential for bacterial dna longitudinal analysis of microbial interaction between humans and the indoor environment mobile phones carry the personal microbiome of their owners a microbial clock provides an accurate estimate of the postmortem interval in a mouse model system expansion of microbial forensics risk factors for human disease emergence practical value of food pathogen traceability through building a whole-genome sequencing network and database singleton sequence type , an emerging clonal group of listeria monocytogenes associated with three multistate outbreaks linked to contaminated stone fruit, caramel apples, and leafy green salad isolation and characterization of heat resistant enterotoxigenic staphylococcus aureus from a food poisoning outbreak in indian subcontinent tracing origins of the salmonella bareilly strain causing a food-borne outbreak in the united states a methodology for assessing the risk posed by the deliberate and harmful use of plant pathogens in characterization of the threat resulting from plant pathogen use as anticrop bioweapons: an eu perspective on agroterrorism foodborne illness outbreaks from microbial contaminants in spices evaluation of m molecular detection system and ansr pathogen detection system for rapid detection of salmonella from egg products validation of high throughput sequencing and microbial forensics applications designing an effective microbial forensics program for law enforcement and national security purposes microbial forensic investigation of the anthrax-letter attacks review of the scientific approaches used during the fbi's investigation of the anthrax letters molecular epidemiologic investigation of an anthrax outbreak among heroin users economou an ( ) molecular epidemiology of hiv transmission in a dental practice genomic variation landscape of the human gut microbiome the thanatomicrobiome: a missing piece of the microbial puzzle of death human thanatomicrobiome succession and time since death the long-term stability of the human gut microbiota molecular evolution in court: analysis of a large hepatitis c virus outbreak from an evolving source identifying personal microbiomes using metagenomic codes key: cord- -u gsa lg authors: divatia, j. v.; pulinilkunnathil, jacob george; myatra, sheila nainan title: nosocomial infections and ventilator-associated pneumonia in cancer patients date: - - journal: oncologic critical care doi: . / - - - - _ sha: doc_id: cord_uid: u gsa lg nosocomial infections or healthcare-acquired infections are a common cause of increased morbidity and mortality among hospitalized patients. cancer patients are at an increased risk for these infections due to their immunosuppressed states. considering these adverse effects on and the socioeconomic burden, efforts should be made to minimize the transmission of these infections and make the hospitals a safer environment. these infection rates can be significantly reduced by the implementing and improving compliance with the “care bundles.” this chapter will address the common nosocomial infections such as ventilator-associated pneumonia (vap), catheter-associated urinary tract infections (cauti), and surgical site infections (ssi), including preventive strategies and care bundles for the same. the term "healthcare-associated infections" (hcais) is commonly used to refer to the whole spectrum of infections that a patient acquires from the healthcare environment including hospitals, intensive care units, hospice, nursing homes, etc. nosocomial infections or hospital-acquired infections (hais) are defined by the centers for disease control and prevention (cdc) as "those infections that were not present in carrier state or incubating state at the time of admission and manifest h after hospital admission" [ ] . these infections are often unrelated to the primary cause of hospital admission and can present even after the hospital discharge of the patients [ ] . as per the cdc criteria for surveillance, nosocomial infection sites can be of types affecting over infection sites that can be differentiated on the basis of microbiological and clinical criteria [ ] . patients in intensive care units (icus) are more vulnerable to nosocomial infections. the extended prevalence of infection in intensive care (epic ii) study showed a prevalence of infections within the icu as high as % [ ] . nosocomial infections are associated with worse outcomes including increased length of hospital stay, long-term disability, and increased mortality rate, and are associated with increased antibiotic use and antibiotic resistance [ ] . due to the multiple risk factors like immunosuppression, disrupted skin and mucosal barriers, recurrent hospital visits, exposure to multiple antibiotics, and the presence of invasive lines and other devices, cancer patients, irrespective of whether they have solid or hematologic malignancies, are at high risk for nosocomial infections [ ] . as cancer patients are increasingly being admitted to icus for management of diseaseand treatment-related complications, the incidence of nosocomial infections is also increasing in icus caring for cancer patients [ ] . the common nosocomial infections are catheter-related bloodstream infection (crbsi), catheter-associated urinary tract infections (cauti), surgical site infections (ssi), and ventilator-associated pneumonia (vap). this chapter will focus on vap, cauti, and ssi, and central line-related bloodstream infection will be dealt with separately. hospital-acquired infection is common across all parts of the world, with an estimated incidence of - % in developed countries and up to % in developing countries [ ] . data from the international nosocomial infection control consortium (inicc) suggests that among developing countries, the crbsi rates were . per , central venous catheters (cvc)-days, ventilator-associated pneumonia (vap) rates were . per , ventilator-days, and the catheter-associated urinary tract infection (cauti) rates were . per catheter-days [ ] . with increased awareness and constant vigilance, there has been a steady and gradual decrease in the incidence rates of hospital-acquired infections with a % reduction in central line-associated bloodstream infections (clabsi) rates and a % reduction in surgical site infections (ssi) [ , ] . table shows the national health safety network (nhsn) and inicc benchmarks for various hospital-acquired infections [ ] . although nosocomial infections can be caused by a variety of organisms including bacteria, virus, fungi, and parasites, bacterial infections are the commonest. these agents may be commensals in the patient or may originate from an exogenous source and spread via cross infection. hospital-acquired pathogens are often resistant to most antibiotics (multidrug resistant) or at times extremely drug resistant or pan drug resistant, thereby increasing the treatment costs, antibiotic use, and antibiotic resistance. this is evident from microbiology data demonstrating an increasing incidence of nosocomial infections that are caused by multidrug-resistant bacteria over the years [ , ] . the common pathogens are gram-negative bacteria including pseudomonas, klebsiella, and acinetobacter and gram-positive bacteria like methicillin-resistant staphylococcus aureus (mrsa), coagulase-negative staphylococci, and enterococci. invasive candidal infections also occur in those with indwelling catheters, lines or contaminated abdominal surgeries [ ] . the other common nosocomial organisms are clostridium difficile, vancomycin-resistant enterococci, anaerobes, and enterobacter. nosocomial infections result in an increased mortality and morbidity to the patients with a significant effect on the treatment cost due to the need for higher antibiotics and prolonged icu and hospital length of stay. these infections are responsible for - % of all death causes in neonates in developing countries and - % in the united states [ ] . as per the world health organization (who) report, nosocomial infections result in direct financial losses of approximately € billion in europe and $ . billion in the united states. the risk factors for developing nosocomial infections are: (a) patient factors such as extremes of age, immunosuppression due to malignancy, acquired immunodeficiency syndrome (aids), patients requiring emergency admission to the intensive care unit (icu), duration of stay more than days, chronic illness like renal failure, diabetes mellitus, chronic liver disease, presence of indwelling catheters, ventilation, total parenteral nutrition, trauma, abdominal surgeries, and impaired functional status [ , ] (b) organizational factors such as the poor environmental hygiene inside the hospital or icu, lack of efficient infection control measures, inadequate manpower such as an inadequate nurse to patient ratio or inadequate waste management staff, and inadequate equipment for patient use (c) iatrogenic factors such as ignorance regarding infection control practices, lack of training in infection control, etc. [ ] treatment and prevention of nosocomial infections strategies for the prevention of nosocomial infections as majority of the patient risk factors for developing nosocomial infections cannot be modified, care should be given for focused education and training to the hospital staff, by distribution of education materials regarding healthcareassociated infections and basic infection control policies including identifying the need of isolation, types of isolation, barrier nursing, hand hygiene, etc. [ ] an infection control committee should be formed headed by an infection control nurse and hospitalinfection control policies should be laid down. the infection control committee should be entrusted with the responsibility of formulating and implementing "care bundles" for common nosocomial infections that can be adopted from health organizations like cdc, who, etc. and modified as per hospital policy. across the world, implementation of such "bundles of care" and adherence to these bundles have been proven to significantly reduce nosocomial infections, especially in the developing countries [ , ] . the infection control team should conduct audits and give necessary feedback regarding compliance with hand-washing and other infection control policies. [ ] . an antibiotic stewardship program should be initiated with a multidisciplinary team, with members such as an infectious disease specialist, a clinical pharmacist with training in infectious disease, a clinical microbiologist, an information system specialist, and an epidemiologist, with a policy for regulating higher antibiotic prescription. review of practice of antibiotic prescription, ensuring environmental decontamination with surface cleaning, air filtration and decontamination of water source, increasing strength of healthcare personnel (improving nurse to patient ratio and increasing waste management staff), and regular training and feedback to hospital staff are some important measures that can be adopted at an institutional level for reducing the nosocomial infections inside the hospital. the treatment of common nosocomial infections and bundle of cares will be discussed under respective sections in the chapter. previously nosocomial pneumonia was considered as a spectrum of high-risk diseases comprising of ventilator-associated pneumonia, non-ventilator-associated hospital-acquired pneumonia, and healthcare-associated pneumonia. the terminology "healthcare-associated pneumonia (hcap)" was introduced by the infectious diseases society of america (idsa) in for patients in the community to be considered at high risk for mdr pathogens similar to those associated with hap. these patients, in spite of not being hospitalized, were still considered as high risk in virtue of their interaction with the healthcare system. over years, increasing evidence suggested that this could be a false assumption that also led to inappropriate use of antibiotics [ , ] . probably this group of patients needs to be considered as a high-risk group when they present to the emergency department with communityacquired pneumonia. hence the current guidelines do not consider hcap as a part of hap [ , ] . the current idsa guidelines recommend the use of two mutually exclusive termsventilator-associated pneumonia and hospitalacquired pneumoniathereby avoiding the terminology of "non-ventilator-associated hospital-acquired pneumonia." the terminology has been explained in fig. . nosocomial pneumonia (hap and vap) is the most common hospital-acquired infection in the developed world, with a prevalence of % [ ] . vap contributes almost % of all cases of nosocomial pneumonia and is a major cause of increased morbidity and mortality. the attributable mortality rates of vap range from % to % across both developing and developed countries [ ] . although hospital-acquired pneumonia (hap) is generally considered to be less severe than vap, patients who develop complications of hap have mortality rates similar to those of vap. in the icu, data suggests that treatment of vap is the main reason for antibiotic usage, with more than % of antibiotic use in icu being for vap [ ] . vap also significantly prolongs ventilation days, hospital length of stay, and treatment costs as compared to patients who do not develop vap. ventilator-associated pneumonia (vap) is defined as pneumonia occurring after - of intubation and ventilation, associated with a new or progressive infiltrate on chest x-ray along with fever, altered leucocyte count, and changes in sputum characteristics for which a definitive causative agent can be found [ ] . early vap occurs in the initial days of ventilation (within - h) and is more likely to be caused by antibiotic-sensitive bacteria. late vap (occurring after days) is likely caused by bacteria which are likely to be multidrug resistant. however, this distinction might not hold true always as patients who are hospitalized for more than days prior to intubation will probably harbor multidrug-resistant bugs [ ] . vap is the most common nosocomial infection in patients who are mechanically ventilated with rates of % being reported among patients admitted in multiple hospitals across the united states [ , ] . the international nosocomial infection control consortium (inicc) data from the developing world suggests that the overall vap rate was . per , ventilator days with a pooled crude excess mortality of . % [ ] . the incidence increases with duration of ventilation. the risk of vap is highest during the initial days of ventilation ( % per day), which gradually decreases over time ( % per day from fifth to tenth day and % afterward). older data suggested that most of the vap episodes occurred in the initial part of icu stay itself (early vap) probably because of the increased practices of short-term ventilation in majority of patients [ ] . recent studies however suggest the converse with an increase in the late vap rates (as much as % of total vap) [ ] . the data on vap is difficult to assimilate for surveillance reporting due to the technical issues in diagnosing vap from radiologic criteria alone and in differentiating vap from other conditions such as pulmonary edema or acute respiratory distress syndrome. hence the cdc has laid down a set of epidemiological definitions called ventilator-associated events (vaes). [ ] . this is a surveillance system to prevent underreporting of the complications (including vap) occurring in mechanically ventilated patients, irrespective of their origin or mechanism, and should not be used in the clinical management of patients. ventilator-associated events are defined for a period of weeks and require patients to be ventilated for a minimum of days, with at least days of clinical stability, to be assessed for vae. vaes are further classified into ventilator- ventilator-associated condition (vac) is defined as days of worsening oxygenation, assessed by an increase in peep requirement more than cm of h o or an increase infio requirement more than . , after an initial clinical stability (of h) or improvement. any vac associated with either a change in temperature or leucocyte count (fever > c or hypothermia < c, or leukocytosis > , / mm or leukopenia < , /mm ) and that requires addition of a new antibiotic for at least days is an infection-related ventilator-associated complication (ivac). an ivac, with a positive microbiological test from respiratory tract specimens, is called possible vap (pvap). a positive microbiological test is defined as a positive microbiological culture in specimens, meeting the threshold of quantitative or semiquantitative culture, without purulent respiratory secretions; or a representative lower respiratory tract sample that is visibly purulent, but the positive culture does not meet the thresholds as per quantitative or semiquantitative criteria; or positive pleural fluid culture or lung tissue culture or a positive test result for legionella or viruses implicated in respiratory diseases. organisms such as candida species, coagulase-negative staphylococcus (cons), and enterococcus species can be considered as positive microbiological test only if isolated from pleural fluid or lung tissue and not from sputum, endotracheal aspirates, bronchoalveolar lavage, or protected specimen brush specimens. positive microbiological test of normal/respiratory flora should be ignored [ ] . vap is defined as a pneumonia occurring in a patient on ventilator for at least calendar days before the onset of a vae, with same duration of ventilation, and the patient was on ventilator on the day of the event, or a day prior [ ] . a complex interplay between host factors, microbiology of the oropharyngeal flora, and the presence of endotracheal tube is responsible for the development of vap. this is summarized in fig. . after hospitalization and antibiotic administration, the normal flora of the upper respiratory tract is replaced by exogenous aerobic gramnegative flora due to alterations in host defense properties. these organisms colonize in the oropharynx from multiple sources (see table ). the normal cough reflex is hampered by the endotracheal tube, and these pathogens gain access to the lower respiratory tract through micro-aspiration along the endotracheal cuff, aided by the ventilator gas flow. the stomach is an important source of bacterial colonization. change in the acidic ph of the stomach due to drugs favors colonization with these virulent bacteria, and with regurgitation of gastric contents, they pool in the oropharynx and reach the lower respiratory tract by micro-aspirations. the risks are further increased in the absence of adequate cuff seal or with multiple attempts of intubation. these virulent bacteria are usually difficult to treat owing to the thick biofilm that they produce alongside the endotracheal tube. the presence of this biofilm hampers antibiotic penetration and increases antibiotic use and antibiotic resistance [ , ] . although increased pharyngeal colonization with virulent bacteria, micro-aspiration, and biofilm formation all contribute to the risk of developing vap, it is the host's immune response to these pathogens that determines whether vap will develop or not. immunosuppression is common in critically ill patients due to dysfunction of monocytes and t cells [ ] . apart from that, critically ill patients have an over expression of c a that leads to neutrophil dysfunction and reduced phagocytic activity, again predisposing them to severe infections [ ] . other contributory factors to the development of vap include advanced age, emergency intubation for surgery or trauma, severity of illness and organ dysfunction, immunosuppressant drugs, previous antibiotic exposure, presence of nasogastric tubes (resulting in sinusitis), and preexisting illness like diabetes mellitus, chronic lung disease, and chronic renal failure. the etiology for vap varies between icus and hospitals which highlights the importance of knowing local infection and susceptibility patterns. the duration of hospital stays before intubation, length of icu stays, and duration of ventilation are also significant as they determine the nature of flora causing vap (see table ). vap before days (early-onset vap) is often caused by microbes similar to the organisms causing community-acquired pneumonia like streptococcus pneumoniae, haemophilus influenzae, methicillin-sensitive s. aureus (mssa), and susceptible enterobacteriaceae [ ] . late-onset vap is usually caused by microbes from the hospital environment. they are usually the aerobic gramnegative bacilli like klebsiella, pseudomonas, acinetobacter, enterobacter, and e. coli, while mrsa is rarely implicated [ ] . the odds that they are multidrug resistant is high, and hence these infections are more difficult to treat. this difference between early and late vap may not always be clinically relevant, and there are increasing reports of lack of difference in microbiology and mortality across both groups [ , ] . patients who are previously exposed to healthcare environment such as those who received antibiotics within the preceding months, those who are currently hospitalized for more than days, those who are on immunosuppressants, or those who have immunosuppressive states such as chronic renal failure, diabetes mellitus, aids, etc. are prone to multidrugresistant infections irrespective of the onset of vap. vap is diagnosed in patients who are being ventilated or was on a ventilator recently and develops signs of infection such as fever or hypothermia, leukocytosis or leukopenia, and worsening in gas exchange along with the appearance of new infiltrates on radiologic imaging and changing nature (increase in purulence) of the tracheobronchial secretions [ ] . these signs are highly nonspecific and may be also associated with various noninfectious causes. moreover, the sensitivity and specificity of routine icu x-rays is much lower than the conventional x-rays. interobserver variability in interpreting x-ray findings also affects their accuracy as a diagnostic tool. hence the diagnosis of vap in icu lacks sensitivity and specificity and may result in both over diagnosis or underdiagnosis. however, if the clinical suspicion of pneumonia is high, empiric antibiotics should be administered immediately as delay in antimicrobial treatment leads to increased mortality [ , ] . to aid in the diagnosis and to rationalize the use of empirical antibiotic therapy for vap, a clinico-radiologic criterion was proposedthe clinical pulmonary infection score (cpis). the cpis (table ) consists of six clinical and laboratory parameters with scores range from to . a score has a sensitivity of % and a specificity of %, for the presence of vap [ , ] . although seemingly simple and straightforward, calculation of cpis score also varies substantially from observer to observer, hereby limiting its routine use in clinical trials [ ] . the current idsa guidelines suggest the use of clinical criteria rather than cpis score for initiating and stopping of antibiotics [ ] . a microbiologic diagnosis can be made by gram staining and culture of the tracheal aspirate or lower respiratory secretions obtained by direct/ non-direct bronchoscopic alveolar lavage (bal). bronchoscopic techniques like bal, mini-bal, and protected specimen brush (psb) specimens provide reliable lower respiratory tract samples, and quantitative cultures of these samples may help to differentiate colonization from true infections. technically it has the advantage of identifying the pathogens correctly, leading to less antibiotic exposure and thereby minimizing antibiotic resistance. however, bronchoscopy and sample collection require expertise and still result in false negative reports. blind sampling requires less expertise and infrastructure and is easy to perform. however, blind sampling is likely to produce false positive results with colonizing organisms, thereby increasing inappropriate antibiotic use and promoting antibiotic resistance. the available data remains conflicting with no benefits of one method over the other with respect to mortality, length of icu stay, and mechanical ventilation days [ , ] . the idsa recommends blind methods of sample collection, while the european guidelines recommend bronchoscopicdirected methods [ , ] . the threshold values for cultured specimens recommended by cdc for the diagnosis of pneumonia are mentioned in table . numerous biomarkers for infection/inflammation have been studied in vap including erythrocyte sedimentation rate (esr), c-reactive protein (crp), procalcitonin, pro-adrenomedullin, lps-binding protein, soluble-triggering receptor expressed on myeloid cells (strem-) , presepsin, etc. other than esr, crp, and procalcitonin, the use of other biomarkers is not widely practiced out of research field [ ] . procalcitonin, a precursor hormone of calcitonin, is actively produced by neuroendocrine cells in the lung and intestine on exposure to bacterial endotoxin and inflammatory cytokines. the level peaks at h and may aid in early identification of infections as compared to blood culture. procalcitonin is not useful in cases of viral or fungal infections and in cases of localized bacterial infections. it is also elevated in noninfectious conditions such as burns, major surgery, end-stage renal failure, etc. [ ] . procalcitonin testing is expensive and serial measurements make it even more expensive. based on the current data, procalcitonin levels should not be used to rule out an infection or influence the decision of antibiotic initiation. the main role of procalcitonin is in its role as a guide for early stoppage of antibiotics, thereby preventing unwanted exposure to antibiotics [ , ] . the current guidelines do not recommend the use of these biomarkers over clinical criteria for a diagnosis of vap [ , ] . idsa recommends coverage for methicillinsensitive staphylococcus aureus and gramnegative bacilli including pseudomonas for patients with suspected vap, pending culture and sensitivity reports [ ] . mrsa coverage is not usually required, unless there is an increased risk for mdr organisms such as recent antibiotic exposure, septic shock, acute respiratory distress cpis clinical pulmonary infection score, ards acute respiratory distress syndrome, pao partial pressure of alveolar oxygen, fio fraction of inspired oxygen, cxr chest x-ray table threshold values for cultured specimens used in the diagnosis of pneumonia bronchoalveolar lavage: more than colony-forming unit/ml protected specimen brush: more than colony-forming unit/ml nondirected bronchoalveolar lavage obtained from (blind) specimens: more than colony-forming unit/ml endotracheal aspirate: more than cfu/ml open lung biopsy/transthoracic or transbronchial biopsy: more than colony-forming unit/g tissue syndrome (ards) prior to the current episode of vap, acute kidney injury requiring renal replacement therapy, or in case of high infection rates of mrsa in the hospital, i.e., > - %. in patients without risk factors for gram-negative mdr infection, such as those without any structural lung disease or those who have not received antibiotics in recent past, a single antipseudomonal agent that also covers mssa will be appropriate. only in patients with underlying structural lung disease like bronchiectasis or cystic fibrosis or those having higher risk for mdr infection, dual antipseudomonal coverage should be given [ ] . recommended empirical antibiotics are those with antipseudomonal and mssa activity such as ceftazidime, cefepime, piperacillintazobactam, fluoroquinolones such as levofloxacin, carbapenems such as meropenem or imipenem, etc. aminoglycosides are not recommended as monotherapy for vap. in case mrsa is suspected, linezolid or vancomycin may be used. with the increased incidence of infections due to mdr gram-negative pathogens, there has been a resurgence of polymyxins in the treatment of vap. they may be particularly useful in places with increased baseline mdr acinetobacter rates [ ] and for empiric therapy in patients with septic shock or high risk such as neutropenic patients or those who have hypersensitivity to beta-lactams. current evidence suggests that different doses or dosage schedules might be required for various bacteria, depending on the pharmacokinetic/pharmacodynamic parameters [ ] . the idsa guidelines recommend the addition of nebulized colistin along with intravenous route for managing vap [ ] . regarding newer antibiotics, daptomycin is inactivated in the lungs and hence is not recommended for vap. tigecycline monotherapy in the doses as per the label is not recommended for hap or vap [ , ] . doxycycline and fosfomycin have not been studied for hospital-acquired mrsa as standalone treatments [ , ] . antibiotics should be changed according to culture and sensitivity reports and may be administered for a total duration of days, or fewer, perhaps guided by procalcitonin levels. the european guidelines for vap recommend a total treatment duration of - days for all immunocompetent hosts in the absence of complications such as empyema, lung abscess, or necrotizing pneumonia, if initial empiric therapy was adequate and their response to treatment has been good irrespective of the microbiological etiology. patients infected with pseudomonas, carbapenem-resistant enterobacteriaceae, and acinetobacter, those on antibiotics such as tigecycline and colistin, and immunocompromised hosts will require a prolonged duration of treatment [ ] . a simplified algorithm for antibiotic selection is shown in fig. guidelines for managing vap and hap were published by the ats and idsa in , while the european respiratory society/european society of intensive care medicine/european society of clinical microbiology and infectious diseases guidelines were published in . while addressing nosocomial pneumonia including hap and vap, both guidelines concur with each other except for a few points. the ats guidelines do not make any new recommendations regarding vap prevention and encourage the use of clinical criteria to decide on initiation and discontinuation of antibiotics rather than use of clinical pulmonary infection score (cpis) score. the ats guidelines recommend the usage of noninvasive or minimally invasive techniques for microbiological investigations of vap. risk factors for mdr pathogens are described by ats as prior hospitalization and organ failure such as septic shock ards and requirement of dialysis prior to vap onset. they recommend combination therapy for target therapy and set a duration of treatment for hap and vap as days. the european guidelines mention prevention strategies for vap but do not make any recommendation on the use of chlorhexidine for selective oropharyngeal decontamination. the european guidelines introduce the concept of "low probability of hap" and suggest the usage of cpis score to identify the same. they endorse invasive sampling over noninvasive sampling, as it might help to avoid overdiagnosis and unnecessary antibiotic exposure. the risk of mdr pathogens is described based upon local prevalence rates of mdr organisms and presence of septic shock. although they recommend days of treatment, they suggested prolonged treatment for selected patients. both guidelines agree on the need of appropriate empiric antibiotic treatment, the need for de-escalation, and the need to minimize antibiotic exposure [ , , , ] . the antimicrobial drug concentration in the lung is the most important factor that determines the efficacy of the antibiotic treatment. drugs delivered reach the lung parenchyma by bulk flow, permeation, active transport, and passive diffusion [ ] . patient factors such as parenchymal inflammation, volume of distribution, renal function, and drug factors such as water solubility, tissue penetration, molecular weight, inactivation of drug in local site, etc. are important factors in deciding the further efficacy of these drugs. hydrophilic drugs like beta-lactams, aminoglycosides, and colistin attain less concentrations in the lung even after administering of therapeutic dose, while linezolid, fluoroquinolones, and macrolides concentrate well inside the lung. hence the pharmacokinetic (pk) and pharmacodynamic (pd) parameters of drugs should be kept in mind while determining loading dose, dosing frequency, and dosing route. alternate routes such as nebulization may be tried as an additional measure to improve the lung deposition of the antibiotics [ ] . the exact time frame of when to expect resolution of symptoms of vap after initiation of treatment is unclear and varies upon the symptoms or signs that are being monitored for resolution. the lack of improvement in clinical condition and/or clinical parameters such as fever, tachypnea, oxygenation, etc. after initiation of treatment can be either due to poor response to treatment/persistence of infection or due to a secondary infection. typically, non-resolving pneumonia is common in elderly patients or in those with comorbidities, underlying immunosuppression, chronic lung disease, or infection with virulent/drug resistant pathogen. treatment factors such as inappropriate initial therapy (either drug or its dose, route, frequency, and duration) are other important factors responsible for vap. workup for non-resolving pneumonia should be undertaken, including microbiological workup for mdr pathogens and imaging for complications such as lung abscess or empyema, while ruling out noninfectious causes of fever and radiologic infiltrates. once an infective etiology is confirmed, optimizing antibiotics as per the pk/pd principles with a hike in antimicrobial coverage will be needed to manage non-resolving pneumonia [ ] . infection control programs form the most crucial step in the prevention of vap [ ] . vap rates can be reduced by proper decontamination of ventilatory equipment and practice of infection control measures during care of the mechanically ventilated patient. a brief outline of the steps to reduce vap is given below. . adherence to the five moments of hand hygiene as recommended by the world health organization (who) [ ] . avoiding intubation and re-intubation by the judicious use of noninvasive ventilation and high-flow nasal oxygen helps reduce the risk of development of vap. . preferring the use oral route than nasal route for intubation, thereby reducing the chances of nosocomial sinusitis and vap. . avoid routine stress ulcer prophylaxis as alteration in gastric ph is associated with increased microbial colonization in the stomach. . enteral feeding reduces the gut translocation of endogenous bacteria and reduces bacteremia. caution should be taken to avoid overdistension of the stomach and monitor gastric residual volumes if there are signs of feed intolerance. . daily oral hygiene with . - % chlorhexidine gel reduces the pathological colonization of oral flora. its role is supported by evidence of multiple meta-analyses of randomized controlled trials which are open-labelled trials [ ] . role of selective decontamination of the gut is controversial in areas with high antibiotic resistance. elevation of the head end of the bed by - has shown to reduce vap rates significantly and is recommended by many professional societies [ ] . continuous low-pressure suction of the subglottic secretions above the endotracheal cuff is useful [ ] . silver-coated endotracheal tubes may prevent bacterial colonization and biofilm formation though the current evidence is weak. [ ] iii. vap bundle the term "bundles" in critical care refers to collective group of practice statements, each with high level of evidence in itself; when practiced together, they result in better patient outcome by the consistent delivery of these practices and avoidance of individual preferences or practices [ , ] . infection surveillance, hand hygiene, semi-recumbent positioning, early extubation, ensuring adequate cuff pressure, and continuous subglottic suctioning have been proven to be simple and efficient methods that help to reduce vap rates significantly [ ] . the initial vap bundle suggested by ihi comprised of five components: head end elevation of bed, daily interruption of sedation combined with assessment of the likelihood of weaning, prophylaxis for stress ulcer and deep vein thrombosis, and daily oral care with chlorhexidine [ ] . table represents the suggested practice from scottish intensive care society [ ] . they differ from the classical institute for healthcare improvement (ihi) vap bundle by not suggesting peptic ulcer prophylaxis or dvt prophylaxis as they have no direct relation with vap rates. similarly, the implementation of a bundle for vap as proposed by the international nosocomial infection control consortium (inicc) also led to substantial reduction in vap rates in multiple countries including india, kuwait, saudi arabia, etc. [ , , ] . the bundle proposed by the inic consortium included the following elements: . adherence to guidelines for hand hygiene . patient nursing in a semi-recumbent position, with head of the bed elevated at - . use of weaning protocols and daily assessment of readiness to wean . regular oral care with chlorhexidine . minimization of the duration of mechanical ventilation and use of noninvasive ventilation if feasible . preferable the orotracheal route instead of nasotracheal route for intubation . endotracheal cuff pressure monitoring and attempts to keep it at least cm h o . care of ventilator circuits and removal of condensates from circuits while keeping the ventilator circuit closed . avoiding scheduled changes of ventilator circuits and changing them only if they are visibly soiled or malfunctioning . prevention against gastric overdistension . avoidance of stress ulcer prophylaxis similar to the inicc study, a spanish group reported successful reduction of their vap rates by more than half with the implementation of a vap bundle among icus across the country [ ] . the bundle they used is similar to other vap bundles and notably avoided the dvt and peptic ulcer prophylaxis of the ihi recommendation [ ] . they had seven mandatory recommendations including staff training in airway management, hand hygiene in airway management, monitoring cuff pressure, chlorhexidine oral care, positioning in bed, striving to reduce ventilator days, and discouraging scheduled changes of ventilator circuits. they also added "highly recommended measures" such as selective decontamination of the digestive tract, subglottic suctioning, and short-course antibiotics for patients intubated with altered sensorium. the data published seemed robust and the reduction in vap rates was sustained and significant. these data suggest that vap bundles are pragmatic, are easy to implement and adhere to, and are effective in reducing the vap rates substantially across the world including developed and developing countries. adapting evidence-based vap bundles that are tailor-made to suit the prevailing practices and hospital policies does not affect the effectiveness of the program [ , , ] . with an increased awareness against vap and with active infection control measures, the incidence of vap is on a declining trend. there has been an increased incidence in hap due to an increased use of noninvasive devices for respiratory support such as noninvasive ventilation and high-flow nasal oxygen [ ] . currently hap is one of the leading causes of nosocomial infections that in turn leads to prolonged hospital stay and increased treatment costs. a recent study found that the incidence of hap is . per patient days, occurring in both wards and the intensive care units [ ] . hap is classified into icu acquired and non-icu acquired hap, with icu acquired hap having an increased incidence of mdr pathogens increased incidence of septic shock, and worse outcomes as compared to non-icu hap [ ] . the term "non-icu acquired pneumonia (niap)" has been recently proposed and refers to a specific subset of hap patients who developed pneumonia outside icu and has an estimated incidence of . - . cases per admissions [ ] . hap differs from vap with respect to the microbiology, diagnostic investigations, and mortality. the microbiological diagnosis is by culture of a pathogen identified from a representative sputum sample. there are no data to suggest invasive sampling techniques like bronchoscopy over simple sputum collection; rather some data suggest that they may be harmful and do not improve outcomes [ ] . the use of rapid diagnostic methods such as polymerized chain reactionbased technologies in hap has yielded promising results especially in the choice of antibiotics and detection of antibiotic resistance. however further studies are needed to confirm the benefits of such systems over the possible disadvantages such as false diagnosis of colonization [ ] . the microbiology of hap differs from vap, and there seems to be an increased incidence of s. pneumoniae and respiratory viruses and a lower incidence of mdr gram-negative pathogens [ ] . the ats/idsa guidelines recommend antipseudomonal therapy for most patients with hap. this may lead to unnecessary use of broad spectrum antibiotic therapy [ ] . the ers guidelines suggest that antipseudomonal treatment is not necessary for initial empiric treatment of most patients with hap, in the absence of risk factors or septic shock [ ] . thus, hap patients need to be treated based on factors such as local epidemiology, surveillance cultures, presence or absence of mdr risk factors, and septic shock [ , ] . infections involving any part of the urinary system, from the urethra to kidney, are labelled as urinary tract infections. they comprise more than one third of all nosocomial infection. in the intensive care units, uti comprises of - % of all nosocomial infections and is the third most common nosocomial infection occurring in the icu [ ] . icu patients require a catheter for reasons such as immobility, strict intake output charting, etc. and retain it for prolonged duration. each catheter day is associated with a - % increase in the risk of acquiring a catheter-associated urinary tract infection (cauti). the cdc guidelines classify cauti as symptomatic urinary tract infection or asymptomatic urinary tract infection as follows: cauti is an infection occurring in a patient with an indwelling catheter of more than h duration before the event, with the catheter remaining in situ or removed h prior at the time of the event. signs and symptoms of infection such as fever, suprapubic tenderness, loin pain, andin those patients without the catheterincreased urinary frequency, urgency, and dysuria and a significant bacteriuria should be present [ ] . significant bacteriuria is defined as a urine culture with no more than two species of organisms identified, of which at least one of which is a bacterium which has a colony-forming unit count more than cfu/ml [ ] . the most common pathogens associated with cauti are enterobacteriaceae. in the setting of icus, candida, enterococcus, pseudomonas, klebsiella, and e. coli become increasingly prevalent and are often drug resistant [ ] . cauti as well as other nosocomial infections prolongs hospital stay, increases treatment cost, and increases mortality. as with an endotracheal tube for vap, the indwelling catheter is the main risk factor for uti. other risk factors include female sex, severity of current illness, age greater than years, presence of diabetes mellitus, altered rft with serum creatinine level more than mg/dl, location of catheter insertion, nonadherence to aseptic precautions of catheter care, etc. [ ] . laboratory examination will demonstrate pyuria irrespective of symptoms and urine wbc > cells/microl. quantitative urine wbc > cells/microl has low sensitivity but retains high specificity for the likelihood of getting a positive microbiological culture. a proper sample of urine should be sent for culture when cauti is for obtaining culture results. samples should be collected from the "needleless site" after applying aseptic precautions or with a needle from the aspiration port. the idea of changing the catheter before sample collection has been suggested in some studies but currently cannot be recommended [ ] . the empiric antimicrobial therapy for cauti depends on the presentation, i.e., whether they are symptomatic/asymptomatic and also upon the complications if any. antibiotics for asymptomatic bacteriuria do not prevent the progression to symptomatic cauti nor its complications. as the risk of antibiotic resistance is high, patients with asymptomatic bacteriuria are usually not treated with antibiotics except in pregnancy and in patients undergoing surgical procedures of the lower urinary tract [ ] . for symptomatic bacteriuria, the choice of antibiotic will depend on patient's risk factors for mdr infection and ongoing antibiotics. a -to day duration of intravenous antibiotic therapy is generally advocated and can be switched to oral route as per the sensitivity reports if the patient can tolerate oral medications [ ] . candiduria is a common occurrence among hospitalized patients, with candida being isolated from almost one third of the total samples among hospitalized patients. candiduria with symptoms and signs of infection including fever, leukocytosis or leukopenia, shock, etc. should be evaluated for disseminated candidiasis. treatment for otherwise asymptomatic candiduria is not required except in the high-risk population such as neutropenia or urinary tract instrumentation. imaging should be obtained in patients with diabetes mellitus or urinary tract abnormalities in case of persistent candiduria as they have a high risk for developing fungal balls. treatment should be guided by the culture and sensitivity reports. fluconazole - mg daily is recommended for susceptible strains for a total duration of - days. fluconazole-resistant strains should be treated with amphotericin b ( . - . mg/kg per day) for days. lipid formulations of amphotericin b do not penetrate the kidney and cannot be used for the treatment of fungal cauti. the data on efficacy of echinocandins is still evolving, and the preliminary data shows clearance of candiduria with micafungin. further data is required for making any recommendation [ ] . strategies for reducing cauti include strict aseptic techniques including hand hygiene for insertion and maintenance of catheters and maintaining a closed drainage system, monitoring the insertion of urinary catheters for appropriate indications, development of cauti bundles [ ] (table ) for placement, daily check list, early removal, encouraging other alternatives such as intermittent catheterization, and condom catheter. a recently conducted study in the united states demonstrated that a cauti bundle could reduce the catheter use and cauti rates in non-icu acute care settings [ ] . other methods proposed to reduce the incidence of cauti were to use urinary catheters coated with antibiotics or to use urinary catheters with silver impregnated in it. silver compound was found to reduce biofilm formation on the catheter significantly. however, the use of both types of catheter was not associated with a reduction in uti rates or any other meaningful benefit [ ] . surgical site infections (ssis) are those occurring at the incision site and/or extending to deeper tissue spaces or adjacent organs within days of a surgery or within days if the procedure involved prosthetic material implants. they are further classified into superficial ssi, deep ssi, and organ/space ssi (table ) [ ] . these are the most common healthcareassociated infections in patients undergoing surgery, with an incidence of about % [ ] . studies have estimated that more than half of these infections are preventable, if appropriate measures were taken [ ] . the most common organisms responsible for ssi are staphylococcus aureus (mrsa and mssa), e. coli, coagulase-negative staphylococci (cons), pseudomonas, etc. [ ] . the inicc data from developing countries shows a significantly higher incidence of ssi, as compared to the data from developed countries. the incidence rates reported by the inicc group are . % after hip prosthesis, . % after cardiac surgeries, . % in abdominal hysterectomy, . % in other abdominal surgery, and . % after ventricular shunt [ ] . patients at risk include those who are elderly patients; those with history of skin or soft tissue infection, recent radiotherapy, diabetes, obesity, alcoholism, and preoperative hypoalbuminemia; those who are current smoker; or those having immunosuppression. other risk factors include emergency procedure, wounds of increasing complexity, prolonged surgeries, contaminated environmental surfaces, lack of strict asepsis in the operating room, inappropriate antibiotic with respect to choice/timing/weight-based dosing, impaired glycemic control, etc. [ ] . maintaining strict asepsis during wound handling and timely administration of the correct antibiotics in appropriate doses are the most important factors to prevent ssi. other measures such as hand hygiene, skin antisepsis, avoiding shaving of hair (if necessary, to clip), and use of double gloves and other barrier devices are also recommended by various societies for reducing ssi. surgical antimicrobial prophylaxis (amp) is the administration of a short-course antibiotic to reduce the microbial burden at the time of skin incision. an ideal amp program has to select the antibiotics that are active against the likely pathogens at the surgical site and administer the optimum dose at correct time, so that adequate serum and tissue concentrations are achieved. it is recommended that the full dosa should be administered within min of the surgical incision and re-dosed as per the half-life of the drug or in case of blood loss more than one third of circulating blood volume. cefazolin as a single agent is the recommended drug of choice for cardiothoracic and upper gastrointestinal surgeries, surgery of non-obstructed small bowel, cesarean section, orthopedic surgery, spinal surgery, and neurosurgery. in patients with allergy to cefazolin, aminoglycosides such as gentamycin may be used. infective endocarditis prophylaxis is restricted to specific procedures in patients with few high-risk cardiac conditions. the cdc recommendation for prevention of ssi is outlined in table [ ] . table strong recommendations from cdc regarding prevention of ssi bath with water and soap on the night prior to surgery administer amp only if indicated, and amp to be administered in the correct time and correct dose such that optimal bactericidal concentration of the agents is established in the serum and tissues when the incision is made administer amp for all caesarean section procedures use an alcohol-based antiseptic agent for surgical site preparation unless otherwise specified avoid application of all antimicrobial agents including ointments, solutions, or powders to the surgical incision use of antibiotic (triclosan)-coated sutures strict glycemic control in the perioperative period with target blood glucose levels less than mg/dl in all patients irrespective of diabetic status maintain perioperative normothermia optimize tissue oxygenation by maintaining normothermia and euvolumia provide an increased fio during surgery and in the immediate postoperative period after extubation transfuse blood and blood products as per transfusion thresholds avoid additional amp after closure of the surgical incision in the operating room cdc centre for disease control and prevention, amp antimicrobial prophylaxis nosocomial infections increase patient's morbidity, hospital and icu length of stay, treatment costs, and mortality. they are also responsible for increased antibiotic use, leading to antibiotic resistance and outbreaks of multidrug-resistant infections. implementing and enforcing infection control measures is the pivotal step toward curbing the nosocomial infection. increased awareness, health education, and adhering to care bundles have been proved to be efficacious in reducing nosocomial infections. impact of the international nosocomial infection control consortium (inicc)'s multidimensional approach on rates of ventilatorassociated pneumonia in intensive care units in hospitals of cities of the kingdom of saudi arabia impact of the international nosocomial infection control consortium (inicc) 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society of america ventilator associated pneumonia respective impact of implementation of prevention strategies, colonization with multiresistant bacteria and antimicrobial use on the risk of early-and late-onset vap: an analysis of the outcomerea network institute for healthcare improvement: how-to guide: prevent ventilator-associated pneumonia institute for healthcare improvement: evidence-based care bundles n.d institute for healthcare improvement, i. institute for healthcare improvement: how-to guide: prevent ventilator-associated pneumonia defining antibiotic dosing in lung infections trends in antibiotic use and nosocomial pathogens in hospitalized veterans with pneumonia at medical centers empirical antibiotic therapy for ventilator-associated pneumonia ventilator-associated pneumonia in the icu management of adults with hospitalacquired and ventilator-associated pneumonia: clinical practice guidelines by the infectious diseases society of america and the american thoracic society nosocomial infections and their control strategies nosocomial infections: epidemiology, prevention, control and surveillance severe sepsis bundles prevalence of and risk factors for hospital-acquired infections in slovenia-results of the first national survey the prevalence of and risk factors for healthcare-associated infections in slovenia: results of the second national survey. zdravstveno varstvo estimating health care-associated infections and deaths in u.s. hospitals reappraisal of routine oral care with chlorhexidine gluconate for patients receiving mechanical ventilation benefits and harms of treatment of asymptomatic bacteriuria: a systematic review and meta-analysis by the european association of urology urological infection guidelines panel impact of inappropriate antibiotic therapy on mortality in patients with ventilatorassociated pneumonia and blood stream infection: a meta-analysis early onset pneumonia: a multicenter study in intensive care units treatment failure in ventilator associated pneumonia subglottic secretion suction for preventing ventilator-associated pneumonia: an updated meta-analysis and trial sequential analysis silver-coated endotracheal tubes and incidence of ventilator-associated pneumonia the nascent randomized trial new guidelines for hospital-acquired pneumonia/ventilator-associated pneumonia: usa vs guidelines for prevention of hospital acquired infections effectiveness of a multidimensional approach for prevention of ventilator-associated pneumonia in adult intensive-care units from cities in india: findings of the international nosocomial infection control consortium (inicc) attributable mortality of ventilatorassociated pneumonia: a meta-analysis of individual patient data from randomised prevention studies comprehensive evidence-based clinical practice guidelines for ventilator-associated pneumonia: prevention antibiotic treatment of hospitalacquired pneumonia bronchoscopic or blind sampling techniques for the 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control consortium's multidimensional approach on rates of ventilator-associated pneumonia in intensive care units in hospitals of cities within argentina a program to prevent catheter-associated urinary tract infection in acute care controlling antimicrobial use and decreasing microbiological laboratory tests for urinary tract infections in spinal-cord-injury patients with chronic indwelling catheters impact and effect of nosocomial infections: a review inhibition of daptomycin by pulmonary surfactant: in vitro modeling and clinical impact biomarkers in sepsis impact of quantitative invasive diagnostic techniques in the management and outcome of mechanically ventilated patients with suspected pneumonia immunoinflammatory response in critically ill patients: severe sepsis and/or trauma a systemic review on surgical site infections: classification, risk factors, treatment complexities, economical and clinical scenarios international ers/esicm/escmid/ alat guidelines for the management of hospitalacquired pneumonia and ventilator-associated pneumonia: guidelines for the management of hospitalacquired pneumonia (hap)/ventilator-associated pneumonia (vap) of the summary of the international clinical guidelines for the management of hospital-acquired and ventilator-acquired pneumonia estimating the proportion of healthcare-associated infections that are reasonably preventable and the related mortality and costs pharmacokinetics of antibiotics in the lungs international study of the prevalence and outcomes of infection in intensive care units the prevalence of nosocomial infection in intensive care units in europe procalcitonin as a diagnostic marker for sepsis: a systematic review and meta-analysis. lancet infect dis national trends in patient safety for four common conditions nosocomial infections in the icu: the growing importance of antibioticresistant pathogens surgical site infections: control., causative pathogens and associated outcomes who|my moments for hand hygiene. who. world health organization ventilator-associated pneumonia: the clinical pulmonary infection score as a surrogate for diagnostics and outcome key: cord- - ofi mg authors: wei, yuwa title: human rights issues date: - - journal: issues decisive for china&#x ;s rise or fall doi: . / - - - - _ sha: doc_id: cord_uid: ofi mg contemporary china is plagued by a wide range of human rights related issues and problems. in addition to those arising in the areas of religious toleration, judicial practice, treatment of labor and forced abortion, which were extensively reported by the media in the past, some newly emerged problems concerning human rights violation are much more alarming, due to the size of population affected and the degree of challenge caused to the public’s psychological endurance and confidence in the social ethnics and administration of the nation. most of all, these problems concern nearly every chinese citizen’s well-being and impact on their personal prosperity, as well as the prosperity of the nation as a whole. these problems are mainly associated with failures in environmental protection, food safety, and medical security. first white paper on human rights being released in , the white papers of the following years were arranged as a series sequentially informing the progress made and the actions taken in human rights promotion in each year. nevertheless, it is an undeniable fact that contemporary china is plagued by a wide range of human rights related issues and problems. in addition to those arising in the areas of religious toleration, judicial practice, treatment of labor and forced abortion, which were extensively reported by the media in the past, some newly emerged problems concerning human rights violation are much more alarming, due to the size of population affected and the degree of challenge caused to the public's psychological endurance and confidence in the social ethnics and administration of the nation. most of all, these problems concern nearly every chinese citizen's wellbeing and impact on their personal prosperity, as well as the prosperity of the nation as a whole. these problems are mainly associated with failures in environmental protection, food safety, and medical security. achieving modernization has been the core economic objective and a solemn promise of the chinese government. urbanization has been a major means of reaching this goal. this course has been accelerated since the s with an average urban population growth rate of more than . %. nowadays, the number of urban population has reached . million, account for . % of the total population of the nation. in the meantime, china has scored an extraordinary gdp growth rate ranging from the lowest . % to the highest % between and . if these are to be seen as modernization or development, the modernization or development droughts and storms, deforestation, desertification, etc., affected more than two billion people and the economic toll multiplied compared with the previous decades. this trend has an even acuter presentation in china. in the past three decades, china experienced extraordinary economic growth, accompanied by rapid industrialization and urbanization. the emergence of many city clusters has resulted in centralized and condensed population in urban areas. the economic development, together with urbanization, has generated substantial environmental problems. nowadays, china is facing nearly all of the world's ecological challenges including climate change, desertification, deforestation, declining water resources, acid rain, soil erosion, air and water pollution and biodiversity loss. apart from increasingly frequent natural disasters caused by climate change, air and water pollution has become a paramount concern to the general public, which is acutely perceived in their daily life. as mentioned in the beginning of this section, the cost of environmental degradation in china is massive and profound, from people's lifespan to economic toll. the link between environment and human rights can be established on basis that physical environment is a precondition for living a life of dignity and worth. good environment is a pre-requisite for the enjoyment of fundamental human rights. in china, for a long time, human rights violations and environmental degradation have not been treated by central and local governments as related issues and addressed in a coherent and integrated way. it has become evident that environmental protection and human rights are prima facie interrelated, interconnected, and mutually responsive, since they both serve the purpose of improving and sustaining the well-being of humanity. human well-being has a basic requirement for safe food, clean air and drinking water, safe shelter and protection from diseases. human activities such as rapid industrialization and urbanization result in environmental degradation including rising of sea levels, air and water pollution, drought, flooding, which in turn, affects people at a large number. at the international law level, the vital role that environment plays in guaranteeing individuals' fundamental human rights has been recognized at a much earlier time. the stockholm declaration of , nairobi declaration of , world charter for nature of , earth summit of , johannesburg conference on sustainable development of , un conference on sustainable development of have all acknowledged the interdependence between environment and human rights by conveying the message that environment may lead to long term impact on humanity and violation of human rights. the human rights council in its resolution / of march and resolution / of march focused specifically on human rights and climate change, pointing out that environment change impairs and undermines directly and indirectly the effective enjoyment of human rights. traditional international environmental laws commonly address environmental issues through creating and determining rights and obligations between states. human rights laws, on the other hand, place individuals' rights at the center of the focus. the establishment of the linkage between human rights and environment enables individuals victimized by environmental degradation to seek remedy on human rights violation ground, which traditional environmental laws gives little focus. it is unfortunate that in china's priority agenda, both human rights protection and environment protection have given way to economic development for quite a while. the link between environmental damage and human rights violation has not been articulately addressed either. indeed, it is a high time for china to adopt a comprehensive, human rights based approach to formulate environmental policies for clarifying human rights obligation in environment protection and for identifying and promoting good practice in environment management. there are signs that current central government shows the interest in considering human rights law enforcement as a means of achieving the purpose of environment protection. "environment protection" have become hot words in china's political life since the th national congress of the communist party of china (nov. - , ) . in his keynote report in the opening ceremony, hu jin tao, the president of china by then, emphasized the term "ecological civilization" for fifteen times. his successor, the current president xi jin ping, in his speeches at the national people's congress and at the chinese people's political consultative conference , called for environment protection and pointed out that protecting environment is as important as protecting productive forces. with the initiative from top leaders, the governments of all levels have become more and more mobilized to change their focus and give particular importance to environment protection. although the areas like beijing, hebei and shanxi are still frequently blanketed by hazardous smog and problems caused by environmental degradation and climate change are still acute, people are more willingly to adopt an optimistic attitude when speaking likely environmental improvement in the coming decades. nowadays, with the growth of economic prosperity, common chinese people enjoy an ever-abundant supply of food products and are keen to broaden their dining experiences. in today's china, eating occupies nearly all occasions and businesses. visitors to china are likely to find that eating is arranged as a highlight for them by the hosts. some research suggests that the current food consumption pattern in china even poses as an exception to engel's law, that is: as the income of an average chinese family rises, the proportion of income spent on food increases, rather than decreases. some other research finds that in fact, the share of chinese households' income spent on food fell from over half in the s to about one-third nowadays. nevertheless, one-third of household income being spent on food is still spectacularly excessive, compared with . % in the u.s., . % in the u.k., and . % in singapore. while all levels of the population take any possible opportunities to indulge with feasts, disturbing facts concerning food safety emerge. this article concerns itself with some of the most severe problems associated with food safety in china, which mainly refer to the cases involving deliberate contamination of food for profits. these include problems such as manufacturing and trading imitation food (e.g., selling pork as beef), processing and selling contaminated food, adding chemical additives into food products for improving visual appeal, taste, or simply for falsely increasing nutritional value (e.g., adding melamine into baby formula to inflate the protein levels), and producing and trading tainted food (e.g., profiting from recycled oil mainly dredged up from restaurant gutters). worst of all, the above problems are not sporadic but are widespread and rampant, and in some cases, systematic. it needs to be borne in mind that the above issues are the most severe ones and only account for a small part of the avalanche of food scandals. here, incidents relating to sanitation or foodborne illness outbreaks caused by negligence in manufacture and service processes are not taken into account, though they are also serious problems. the right to safe food is a basic human right for all individuals. in addition, safe food should be regarded as an issue of national security for the reason that toxic food poses a serious threat to public health, which in turn leads to destructive social and economic consequences, and eventually has an adverse impact on the national interests of a country. it is high time for the chinese to review their policies and strategies in dealing with social issues, including social justice, equal distribution, anticorruption and clean governments, social ethics and public morality, and public psychology. regulation is surely a necessary instrument to control illegal conduct. however, given the fact that policies and laws are always watered down or compromised in implementation, it is thus particularly important for policy makers to take all the above issues into consideration when shaping the nation's regulatory infrastructure for food safety. when serious food problems have a devastating impact on a large proportion of the population, it is necessary to assess the situation from a human rights perspective while searching for solutions. this section examines china's food safety issues in connection with human rights awareness in the country. it investigates the social, political and cultural causations of the food scandals, and explores possible solutions. before the s, under the planned economy, food problems that arose from manufacturing were extremely rare and the regulatory focus was on sanitation occurring in the service and consumption processes, where the food safety administration was part of the duties of the sanitation and anti-epidemic departments. although it appeared that a legal framework was largely put into place, the food industry was, in fact, less effectively regulated, due to the loose context of the laws and the gaps between the laws and their practice. the situation has deteriorated since the beginning of the new millennium with the introduction of the practice of quality inspection exemption or the regime of self-inspection by enterprises themselves, an initiative launched to reduce the operational costs of food enterprises. the self-inspection regime was a result of the administrative authorities exercising their supervisory powers in an abusive manner while in the discharge of their quality control duties concluding with the imposition of unnecessary and excessive burdens on food producers. however, instead of disciplining administrative conduct, the policy makers opted for a compromise by abolishing food quality inspection. research has shown that the managerial costs in chinese firms are chiefly caused by defects inherent in the governance of these firms. without fundamental improvements in the corporate governance system, xu, supra note . see, for detailed discussions on managerial costs and corporate governance in chinese firms, yuwa wei, "directors' duties under chinese law -a comparative review", ( ) university of the costs are likely to continue to inflate. later instances of food safety crises have proven that those who maintain the argument that relaxation in quality control can reduce food companies' operational costs are simply barking up the wrong tree, where the most likely result is noncompliance. it took the government years to realize that giving enterprises free rein in regards to food quality control was an unwise decision. it was at least partly responsible for the current culpable culture in the food industry. in the aftermath of a series of food scandals, policy and regulatory mechanisms have been introduced as the main crisis control efforts. on the current law imposes full responsibility on food producers and manufacturers to ensure that their products meet safety standards throughout each process, including purchase of raw materials, production, storage, package, transport and delivery. in addition, they need to faithfully record the safety management throughout the whole course. however, china's experiences reveal that without fierce scrutiny, the industry is unlikely to comply with such requirements. under the current regime, the supervision and administration are given to local governments at or above the county level, who formulate annual plans for food safety and undertake food sampling inspection. specific branches of government, including the departments of agriculture administration, quality supervision, industrial and commerce administration, and the food and drug administration carry out the routine work of food sampling inspection. ( ) china, § ( ) , an english version is available at <www.fdi.gov.cn/ _ _ _ _ .html>. the section states: "the food producers and traders shall, in accordance with laws, regulations and food safety standards, carry out productive and operational activities, establish and improve the food safety management system and adopt efficient management measures to ensure food safety. the food producers and traders shall be responsible for the safety of produced and trade food themselves as well as society and the public, and also bear social responsibility." ibid, at § . ibid, at § . persons with direct responsibility in the above mentioned governments are subject to disciplinary punishment comprising of the recording of serious demerits, demotion, dismissal or expulsion, in the event of a failure to perform their supervisory and administrative duties, particularly where the failure causes a serious social effect. the same penalties are imposed directly on persons responsible for the above mentioned government branches, in the case where negligence is committed, daily supervision and inspection of food samples are omitted, abuse of power occurs, or favouritism is shown in the supervisory and administrative processes. from the above overview of the new regulatory regime, a few inadequacies can be identified. first, the legal (regulatory) framework is far from comprehensive. the supervisory model of food safety mapped out in the new laws can be summarized as: relying mainly on the segment-based approach, supplemented by a categorybased approach. in other words, the administration of food quality is undertaken by multiple inspection and administrative agencies, exercising their authorities in different stages of food production and services and targeting different types of foods. such an arrangement poses a potential risk in regards to conflict of powers, though the new laws allude to the benefits of efficient coordination among these agencies by identifying their authorities and duties in different stages of production and services. given the gaps in the existing laws, it is very likely that china will encounter further challenges in food supervision caused by its inefficient administrative system. speaking from a broader perspective, china is not the only country that has applied a multi-agency approach to food supervision and administration. the united states and japan have also adopted a scheme of assigning duties of food administration to multiple governmental agencies and departments. however, their comprehensive and detailed stipulations on division of responsibilities, quality standards, transparency and public scrutiny have ensured effectiveness of the system and smooth coordination among the agencies. moreover, it is noteworthy that even in countries such as the united states, there are increasing calls for combining their multiple agencies into a single agency to further the operational efficiency. it is therefore necessary for china to improve its food safety administration system to further enhance its regulatory regime on the one hand, and to rationalize the administrative chains and procedures in food supervision on the other. second, the current legal regime does not provide sufficient remedies for victims of food safety, nor does it effectively encourage consumer and community participation in food safety control. the laws have introduced monetary remedies for consumers consisting of damages and a fine of ten times that of the commodity's value. however, the accused are liable for the fine only where evidence shows that they have produced defective products and are at fault. what is more outrageous is that the onus of proof is on the consumers in such cases. the amount of ten times the value of the goods has already been criticized as being too insignificant to be a deterrent. the requirement that consumers need to prove the culpability of producers in making defective foods renders it nearly impossible for the consumers to benefit from the fine, not to mention the conceivable negligible impact that the provision has on food producers. third, the regulatory regime has loopholes and demonstrates too much leniency towards liable producers and traders, as well as those administrative personnel who fail to discharge their duties or show favoritism. among government staff of relevant departments, only those directly responsible for administrative negligence or abuse of powers that result in serious social impact may face disciplinary action. criminal penalties imposed on food producers for breaches of the law are also weak. moreover, no single criminal penalty is available to penalize delinquent public servants. the criminal law of china only punishes the conduct of producing for instance, in the u.s., the powers of food administration are divided amongst the u.s. department of agriculture, the food and drug administration and the environmental protection agency. see chamus burnside-savazzini, "training in an integrated food safety system: focus on food protection officials", food safety magazine (april/may ), <www.foodsafetymagazine.com/ article.asp?id= &sub=sub >. the people's congress of the people's republic of china, supra note , § . the food safety law does not address the issue of burden of proof. hence, the rules of general law apply, rendering that the burden of proof lies upon he who brings the claim. see tonghai liu, "on the shortcomings of the foot safety law and some suggestions on improvement", blog of tonghai liu on legal study net (october , ), <http://wq.zfwlxt.com/ newlawyersite/blogshow.aspx?itemtypeid=fdf ea- b- - ea- bfb &itemid= de f - bd- f - bdc- f e &user= >. the people's congress of the people's republic of china, supra note , § . and selling foods causing serious food poisoning and diseases, and the conduct of producing and selling foods mixed with toxic or harmful non-food substances. it can thus be observed that the scope of application of the criminal law provisions is limited to offences occurring in food production and sale. this leaves the conduct of processing, packaging, storing and transporting defective or harmful foods unpunished. furthermore, the amounts of the penal fines provided in the criminal law are even less than those stipulated in the food safety law, and are subject to a higher threshold in application. finally, the most essential issue to be addressed by the chinese is the enforcement of the laws. according to a survey carried out by the national survey research center at renmin university of china in , % of people interviewed attributed the cause of food scandals to administrative inefficiency and supervisory failure. in the meantime, judicial failure to rigorously implement food laws has cast a further shadow over effective law enforcement in the future. it is reported that courts are reluctant to support consumers' claims for damages up to or times the amount of the commodity price provided for in the law of the people's republic of china on the protection of consumer rights and interests and the food safety law of the people's republic of china . many manufacturers also promise the criminal code of china states: "whoever produces or sells food that does not confirm to hygiene standards in a manner that is sufficient to cause a serious food-poisoning accident or any serious disease caused by foodborne bacteria shall be sentenced to fixed-term imprisonment of not more than years or criminal detention, and concurrently or independently be sentenced to a fine of not less than half of the sum obtained through sale and not more than twice of that. if the offence causes serious harm to human health, the offender shall be sentenced to fixed-term imprisonment of not less than years and not more than years, and concurrently be sentenced to a fine of not less half of the sum obtained through sale and not more than twice of that. if the consequences are especially serious, the offender shall be sentenced to fixed-term imprisonment of not less years or life imprisonment, and concurrently be sentenced to a fine of not less than half of the sum obtained through sale and not more than twice of that or confiscation of property." a person who knowingly mixes the food to be produced or sold with toxic and harmful non-food stuffs is subject to the same penalties. : "when any manufacturer produces any food not conforming to the food safety standards or sells any food knowing its nonconformity with the food safety standards, the customer can demand the manufacturer or the seller to pay a penalty times of the paid amount, in addition to the compensation for the loss thereof." the amount of the compensation provided for in article of the law of the people's republic of china on the protection of consumer rights and interests is up to two times upon occurrence of fraud. junhai liu, the deputy president of china consumer association pointed out that due to judges' misunderstanding of legal reasoning, consumers' that an amount of compensation of up to times the amount of the commodity price will be paid to consumers upon violation of food safety. however, such claims are likely to be rejected by courts. some judges insist that such an amount appears to be punitive and thus does not have legal basis in contractual relations. the stance of courts in such cases renewed the public's suspicion of judicial corruption. the worst scenario concerns events involving arrest and conviction of consumer advocates. the zhao lianhai case is a typical example. zhao lianhai was a man who became an activist after his son experienced kidney problems linked to contaminated baby formula. he was convicted of inciting public disorder by setting up a web site to help other parents with sick children share information, seek compensation, and organize protests. it is clear that despite legislative efforts, tackling the food safety problem is an overwhelming challenge. compared to assessing legislative improvements, evaluating law enforcement in china presents a far more demanding task and is entangled with a number of social, political, economic and cultural issues. more discussions on enforcing food safety law in china will be carried out in section iii of this article. in summary, much work needs to be done to develop an efficient and effective food safety regulatory regime. this is not a mere legal task, but a social project involving reform of the administrative culture, a change in the business environment and transformation of the perception of the right to safe food for the public. human rights are rights inherent to all human beings, and they are inalienable and fundamental. the international community has reached a common understanding on the universality of human rights. for promoting and protecting human rights and fundamental freedoms of individuals, governments are obliged to act in certain ways or to refrain from specific acts. the most common universal human rights claims for compensation provided in the above provisions are rarely supported by courts in the past. see social and cultural rights , the right to food is recognized as part of the right to an adequate standard of living. the rome declaration on world food security and world food summit plan of action reaffirms everyone's right to "have access to safe and nutritious food, consistent with the right to adequate food and the fundamental right of everyone to be free from hunger" and describes food security as: "[w]hen all people, at all times, have physical and economic access to sufficient, safe and nutritious food to meet their dietary needs and food preferences for an active and healthy life." it is therefore clear that as a part of human rights, the right to food is inalienable and indivisible from other fundamental rights including the right to life, the right to health, the right to know, ( - nov. , rome) . here, "right to know" is a term used in environmental law, referring to the legal principle that the individual has the right to know the chemicals to which they may be exposed in their daily living. it comprises two aspects -community right to know (environmental hazard information) and workplace right to know (workplace hazard information). nowadays, the term is used in the field of human rights in reference to the right of access to information concerning citizens' human rights, or their rights under freedom of information laws. for instance see sandra coliver, the justifiable to say that the right to food incorporates at least the following dimensions: availability, accessibility and adequacy. in other words, food must not only be available as a resource and affordable in order for people to be able to satisfy their dietary needs, but must also be safe for human consumption and free from contaminants. everyone must have access to food that is not only nutritionally adequate, but also free from harmful substances, so that it does not endanger people's lives or health. the states parties to the international covenant on economic, social and cultural rights have agreed to assume the obligation to progressively realize the right to adequate food. the responsibility is interpreted by the committee on economic, social and cultural rights as comprising three types of obligations: respect, protect and fulfil/facilitate. a state government must not interfere with individuals' efforts to access food. it must also protect its people from infringement of their right to adequate food by others. the government should help those who do not already enjoy the right to food by creating opportunities for them to provide for themselves. in the case where people are unable to enjoy the right to food due to events out of the individuals' control (natural disaster for example), the government ought to provide food to them directly. china is a signatory of the universal declaration of human rights and a contracting party of the international covenant on economic, social and cultural rights , the convention on the elimination of all forms of discrimination against women and the united nations convention on the rights of the child . china also signed the international covenant on civil and political rights but has yet to ratify it. the government of china is under international obligation to facilitate a pre-warning system regarding threats to the right to adequate food, and to improve coordination between different government and food safety agencies, as well as improve their accountability. violation of the right to food refers right to know: human rights and access to reproductive health information (article -university of pennsylvania press, london-philadelphia, ) ; and jacqueline klosek, the right to know (praeger, westport, ) . article of the universal declaration of human rights states: "everyone has the right to freedom of opinion and expression; this right includes freedom to hold opinions without interference and to seek, receive and impart information and ideas through any media and regardless of frontiers." jean ziegler, "what is the right to food?", right to food (august , ) <www.webcitation. org/ cmsst c>. not only to shortage of food, lack of infrastructure, mal-distribution and inadequate access to food, but also to protection from contaminated and harmful food. most of the recent food scandals in china involve rampant violations of the right to food in the dimension of adequacy -provision of food for safe consumption. while immoral and illegal conduct of individuals and enterprises is ultimately responsible for the outbreak of food safety crises, the government is accountable for inefficiencies in monitoring the enterprises and disciplining personnel in charge of administrating the food industry. as the most populated country on the earth, the chinese government has always paid great attention to food issues and regarded provision of enough food to the people as the first priority. after decades of effort, china has mostly solved the problem of feeding its people. however, new problems associated with agriculture and food have arisen since the commencement of market oriented reforms. it is reported that due to accelerated industrialization and urbanization, arable land has been shrinking and farmland capacity has declined. this constitutes a threat to china's future food security. the government has introduced the national land consolidation plan ( ) ( ) ( ) ( ) ( ) that aims to increase the effective area of arable land by . million hectares before , though the enforcement of the plan faces challenges such as lack of mechanisms for protection of cultivated land and inefficiencies in the management of land tenure rights. it appears that china has made a significant effort to achieve food sufficiency in the country. food availability and accessibility have been a top agenda item for the government in the past and will continue to remain a central issue in the future. the adequacy aspect received far less attention. the recent outbreak of food scandals is chiefly a result of a lack of oversight within the food industry. this can be partly attributed to the understaffing of supervisory authorities, partly to the misfeasance, malfeasance, nonfeasance and unprofessionalism of administrative officers. it is noteworthy that administrative personnel's unsupportive attitude and behavior towards businesses has been the cause of changes in inspective practice, resulting in a switch of models from official inspection to enterprise self-inspection. under the enterprise self-inspection regime, the working manner of administration is even more unprofessional. for instance, in the recycled buns case, when doing their sample inspection, the governmental inspectors, once arriving at a food factory or workshop, would only come into the administrative office and wait for the factory managerial staff to provide the samples to be examined. it is no surprise that they did not get any clues over the years. in one of the clenbuterol contaminated pork s z gu and y j zhang, "food security in china", in sun honglie and shidong zhao (eds.), area studies -china: regional sustainable development review (vol. , ) incidents, the problematic pigs were sold from henan pig farms to jiangsu province and had to go through several checkpoints conducted by the trade bureau, health inspection station, veterinary station, etc. however, some inspectors acted as middlemen between pig sellers and slaughterhouses for a commission of rmb yuan per pig, which caused wide dissemination of the contaminated pork and pork products. the recycled buns case showed workers in a filthy workshop "recycling" buns by throwing the stale buns into a vat, adding water and flour, and repackaging them to be sold anew. the clenbuterol contaminated pork case involves pig farmers feeding pigs with pig feed mixed with clenbuterol used to make the pigs have leaner bodies, which sell for a higher price. other food scandals involve melamine-contaminated baby milk; pork sold as beef after it was soaked in borax; rice contaminated with cadmium, arsenic-laced soy sauce; popcorn and mushrooms treated with fluorescent bleach; bean sprouts tainted with an animal antibiotic; and wine diluted with sugared water and chemicals. the alarming fact is that most of the food related offences were first discovered and disclosed by journalists, and not the personnel in charge of the surveillance authorities, or even the police or national security authority. it is inexcusable for administrative agencies to claim that ill-training, poor quality equipment and understaffing are the main causes for such an appalling incidence of food scandals in the country, since journalists are by no means in a position to be better trained and better equipped than food inspection agencies or police. as a matter of fact, detection and disclosure of food safety problems is not the expertise of journalists, nor their major business. respect for human rights is becoming a universal principle of good government. this is because many human rights require activity on behalf of government. these activities relate to the duties to respect, protect and fulfil human rights in order to provide the conditions necessary for prosperity and well being of individuals. a good government is committed to protection of the rule of law. hence, it is the state government's responsibility to make good laws and to establish able, professional and corruption-free administrative institutions, with a public service team to guarantee the implementation of the laws. although china's government is quick in making legislative efforts to curtail misconduct in the food industry, it needs to demonstrate its determination and capability of ensuring that the laws are fully abided by and it is accountable for building a law enforcement agency comprised of highly motivated, dedicated, well-trained law enforcers. a typical national food control system comprises the following components: food law and regulation, food control management, inspection services, laboratory services and information, education, communication and training. the capacity of food safety control in a nation is demonstrated by effectiveness, efficiency and sustainability. in china's case, apart from legislative efforts, much needs to be done in the following aspects: law enforcement involves certain members of society acting in an organized manner to promote adherence to the law by investigating and punishing persons who violate the rules and norms governing that society. the enforcement of the law consists of a system comprised of the courts and procuratorates, the judicial and public security agencies, and the administrative agencies, with their descending hierarchy of departments, bureaus, sub-bureaus, and stations. with regard to enforcement of food laws and regulations, administrative mechanisms play a more substantial role, compared with other enforcement instruments. the key is to enhance food control management. this involves development of food control strategies, implementation of risk analysis, operation of a food control program, securing funds and allocating resources, and development of emergency response procedures. development and implementation of food control strategy is concerned with defining food control objectives, identifying priorities of competing interests relating to public investment, state economic interest, food import and export, farmers and producers. an integral, effective food control strategy is particularly important to china, since it will improve the coherence of multiple existing administrative agencies in the country and reduce confusion, duplications of effort, inefficiencies in performance and waste of resources. for the time being, a crucial task china food and agriculture organization of the united nations and world health organization, assuring food safety and quality: guidelines for strengthening national food control systems (joint fao/who publication) - (rome ), <ftp://ftp.fao.org/docrep/fao/ /y e/ y e .pdf>. see food and agriculture organization of the united nations and world health organization, supra note , at . ibid. faces is to consolidate its food control policy at the macro-level by standardizing food quality certification, management, and rating, and in the meantime, to improve the market price system. a market entry certification system should also be put in place. another important task to deal with is to establish a uniform food safety risk analysis scheme to ensure compliance with food regulations and standards. unlike developed countries, the food safety problems in china occur in every stage from food production to service, typically consisting of contamination of raw food materials, cross contamination in manufacture, improper storage and preservation, and unhygienic practices in services. this requires a wide-ranging risk analysis system capable of identifying risk factors at every stage and of providing solutions. in doing so, there is the need to coordinate the risk assessment activities currently exercised by different administrative agencies and to develop a comprehensive scheme for food safety risk assessment, in which a professional team comprising food researchers, toxicological analyzers, pathological analyzers and inspectors play an essential role. attention should also be paid to food safety risk communication improvement. insufficient food risk communication in the past was chiefly responsible for a crisis in customer confidence with china's food industry. until this point, no substantial improvement has been made in this regard. in summary, the flaws of this situation can be attributed to a lack of government guidance, silence on the part of scientific academia, and unreliability of entrepreneurs. addressing this issue, the minister of health recently announced that a plan for food safety risk communication was in development and the press was expected to continue to play a leading role in this aspect. however, without access to channels for obtaining scientific information, the press's capacity for publishing fair and accurate reports is considerably undermined. it is therefore necessary to base risk communication on a system of interactive exchange of information and opinions among all stakeholders, including risk assessors, risk managers, consumers, industries, the academic community and other interested parties. this requires the government to allocate sufficient funds to finance and staff risk communication institutions of various specialities. china has more than million farmers and more than , food production companies. hence, to some, the food production system seems too vast to see world health organization/food and agriculture organization of the united nations, "about risk analysis in food", <www.who.int/foodsafety/micro/riskanalysis/en/>. allow for meaningful inspection at all stages of the food production process. however, the establishment of a national database for food supervision and inspection and a platform for food safety risk assessment enabling all interested parties to follow the development of food safety will absolutely make a difference to china's food industry. by the end of , the ministry of health formed the expert committee for national food safety risk assessment, which was a significant step toward improvement of china's food safety control. it is also reported that the state council of china has revealed its intention to establish a database of food companies' safety records. according to the plan, companies with poor safety records will be blacklisted and publicized, and will be subject to punishment. better regulation mechanisms, legal and standard systems, as well as technical support systems will also be introduced to improve the overall food safety management level. now, the world is waiting to see how well the plan will be realized. improving inspection services for food safety control is another key element for enhancing the food safety control system in china. food inspection service is a means of ensuring that the food supply is safe for consumers and meets legislative requirements. it is also an instrument for decreasing hazardous intoxicants in foods, including pathogenic bacteria, viruses and parasites, chemical contaminants and mycotoxins. the primary duty of the food safety inspection service is to inspect food manufacturing, processing and handling facilities, importing/exporting of foods and the monitoring of a company's facilities for compliance with the national legal and regulatory requirements. in some countries, the food inspectorate also conducts the functions of investigating food poisoning and injury cases and fraudulent marketing practices, as well as handling consumer complaints. of individual inspectors and the quality of their work. it is thus important to make sure that inspectors have detailed guidance and rules to follow when discharging their responsibilities. their powers and the procedures to be followed should be well defined. inspectors should receive up-to-date training in the new technologies used in processing and manufacturing, including what is required for the control of these technologies in order for them to function at maximum effectiveness and to assure proper performance. they must also be able to evaluate the performance of equipment and instruments used in production to guarantee that they are appropriately controlled and monitored. the impotence of china's food safety inspectorate has been widely reported and acutely recognized by consumers. it is officially perceived that the food safety inspection administration suffers from insufficient resources, a lack of qualified personnel, and deficient coordination among layers of governments and among various institutions, particularly among those administrative agencies at the provincial level and below. apart from the above-mentioned vices, three more factors must be taken into account: corruption, shirking and laziness. china's public services have long suffered from a number of bureaucratic problems such as authoritarianism, routinism, elitism, corruption, shirking of responsibility, deceit, laziness, formalism, red tape, nepotism, seeking special privilege, overstaffing, duplication, ineffectiveness, and over-centralization. upon close study, one can discern that corruption, shirking and laziness are the most prominent problems that induce or exacerbate the other problems. these deep-rooted problems penetrate the food safety inspection system and eventually take their toll on public confidence in the food industry. the government of china has pledged to enhance the ability of the inspectorate. in its food safety decision june , , the state council announced that it planned to take years to improve the nation's food safety and food control management. according to the state council's statement, food safety will become a measure of local governments' performance in their annual assessments, in the hope that incorporating food safety as one of the evaluation criteria will make local governments and officials aware of their responsibilities. however, given the institutional decline, erosion of authority of the central government, and loss of control over local agencies and agents in the era of the market economy, effectively rooting out corruption in the food safety administration will certainly demand much greater efforts based on resolute determination. to a certain extent, it represents a social problem and needs to be tackled on a broader scale. to curtail food safety problems on a broader scale, it is necessary to promote general respect for universal human rights in the society. from this point of view, improvement of food safety can be seen as a social project. in the past three decades, in making the transition from a planned economy to a market economy, china placed much emphasis on economic gains and profitability. along with gdp growth, extreme utilitarianism, fickleness and mammonism have become more and more evident and have turned into a driving force in chinese people's daily life. the food safety outbreaks are merely typical consequences of the erosion of social morality and business ethics. food safety outrages spark a new round of calls to promote public morality and human rights. many chinese have come to realize that without a morally healthy social environment, mere economic prosperity does not guarantee a successful society and true advancement. in contrast, it may only carry with it the seeds of corruption and decay. to bring changes to the broader system, the general public needs to step out from their family-centred tradition and self-interested sphere, and endeavor to shape a society where individual equality is upheld, respect for others' human rights is generally aware of, and public interests are greatly valued. the construction of such a society requires the combined efforts of the government, private sectors and the community. while the government is accountable for formulating policies and initiatives, facilitating state agencies and mechanisms, and allocating resources, the business sector and the community are expected to participate in shaping a good society by promoting sustainable business practices, interacting with the government and influencing the policies of governance. it is worth noting that the constitution of china only implicitly addresses citizens' rights to safe food. article provides that "the state . . . promotes health and sanitation activities of a mass character, all for the protection of the people's health"; and article states: "citizens of the people's republic of china have the right to material assistance from the state and society when they are old, ill or disabled." compared with some nations that incorporate the right to healthy food into their constitutions, china is much behind in providing constitutional safeguards for food security. given the challenge of tackling food problems, it may be necessary for china to introduce provisions to protect food safety in the constitution. in the case of combating food safety problems, the food industry shares responsibility with governmental agencies in achieving the objectives of a national food control strategy. involving the industry in national food control activities might be instrumental in overcoming potential problems. the food industry is responsible for the implementation of rules regulating agriculture, food manufacturing and processing practices, as well as a food quality and safety system. the industry should also be obliged to educate and train all employees in food handling and make sure that they understand general food quality and safety system. it can also provide information to consumers through food labeling and advertising. in china's case, the food industry may need to go through a long journey to correct its practices which may also involve a change in the business culture. as members of the community, consumers have a right to quality and safe food and also have responsibilities to prevent food related health hazards. consumers need to understand there is no such thing as an absolutely safe food supply. the right to food for consumers also requires a responsibility to play a role in food control. consumers can actively contribute to food safety control by providing valuable information to the authorities, usually by complaining about product deception and poor quality and by reporting injury and illness caused by food. the authority then needs to provide them with a channel to let their dissatisfaction be known. consumer organizations are expected to play an important role in representing the consumer in the development of a national food control strategy and also in bringing the concerns of consumers to the attention of policy makers and the industry. the chinese government needs to take proactive action in order to ensure that consumers are heard and taken into account. in summary, combating food safety problems depends on a combination of mechanisms including training, business ethics education, administration and supervision, public scrutiny, civil litigation and penal punishment. in other words, combating the food safety problem require a collective effort from the entire nation. the government is certainly accountable for all mandatory activities necessary to ensure the quality and safety of food, including enacting food regulations, operating food inspectorates, organizing analytical services, and enforcing rules. the participation of other stakeholders and interested parties including the industry, consumers, communities and even the society as a whole, is equally crucial. the battle will happen across a gamut of fields involving governmental conduct, social ethics, public relations, business practice, and consumer culture. the medical insurance system of a state directly affects its people's key human rights, namely the right to life and the right to health. the notion of modern medical insurance can be traced back to the introduction of the sickness insurance program in in germany. the antecedents of similar kinds existed in the form of charity or welfare in some civilizations, such as friendly societies organized by european trade guilds, fraternal organizations, the english poor law , the prussian common law , and the civil war pension program . the sickness insurance program in germany came as a result of the medical reform movement and a series of political debates and compromises afterwards. the sickness insurance act was enacted in to facilitate the program. the law allowed the largest segment of workers to benefit from access to public health care through insurance funds. in the event of accidental injury, illness or old age, workers were entitled for minimum indemnity for medical treatment and sick pay for up to weeks. this initial system was financed by workers and employers, with the employers contributed one third, while the workers contributed two thirds to insurance funds. in germany passed the accident insurance act covering serious and fatal accidents occurring on the job in certain industries. the act went a step further by placing the entire cost of the liability on the employers. the significance of the german health insurance program introduced in the s lay in that it was for the first time in history that a national health insurance system was institutionalized. following the german practice, industrialized countries commonly introduced social security programs in the following decades, i.g., the uk the medical insurance program as an indispensable part of the modern social security system has carried human societies into a new era where the government of a country provides economic assistance to persons faced with unemployment, disability, or agedness for the purpose of advancing the benefits of the members of the community as a whole. the key feature of the scheme is: it is imposed and controlled by the government. before the introduction of social security schemes, the dominant mode of support for people unable to provide for themselves was charitable relief proffered by benevolent societies, sometimes with financial help from the authorities. social security comprises two arms: social assistance and social insurance. the rationale behind modern social security programs is the necessity to maintain social and economic justice. with industrialization, social security has gone through a change from charity to a state responsibility. guaranteeing a certain amount of social well-being and economic security to every individual member in the community has become a duty of a modern state. nowadays, social security has emerged as a human right enshrined in the universal declaration of human rights, which states: after the establishment of the socialist state in , as part of the social welfare or security package china introduced a state funded, central planned health care system, which provided a universal coverage of health care to the chinese population. an innovative feature of the system was the operation of the so call barefoot doctors at health clinics at the village and township level, which served the mass rural citizens accounting for three quarters of the total population. this system was held up as a model for providing universal health care in the developing world in the s by the world health organization (who) and inspired who to launch the "health for all by a.d." program. under this scheme, public health in china improved dramatically, with the citizens' life expectancy increasing from to years, infant mortality decreasing from to per live births. the system operated in china for nearly years until the market oriented medical reform took place in the s. as a result, china's health care program has experienced a change from a scheme performing universal social welfare function to a model that is a mixture of free market and medical insurance. however, the reform turns to be a failure which makes china's health care scheme one of the least equitable systems in the world. care reforms ciency and abuse. its sustainability posed a challenge to the leadership in the post-mao era, which had an economic reform scheme at the top of the agenda. by the s, the administrative and financial support for the health care was removed from the national agenda through decentralization before a nationwide health insurance system was provided. with local governments having little incentive to promote local healthcare, individuals of rural areas and urban non-salaried residents primarily relied on out-of-pocket spending for medical and health services. in the meantime, prices for provision of medicines, diagnostic tests, surgical implants, and specialized care were deregulated. as a direct impact, many chinese were in short of medical services and supplies since they simply resolved to not seeking medical help. it was reported in , about % of the chinese people refused to see a doctor when ill and % of those refused to go to hospital, mainly out of financial concerns. there was still worse to come. after the collapse of the previous model, no strategy was adopted to mitigate the lack of integration and coordination in the later medical system. due to a lack of reliable local clinics, people had to wait in long queues for basic health care, with no scheduled appointments and little privacy. after all the inconvenience, what they could receive was usual a very brief face to face consultation of a few sentences or a couple of minutes. during this period, health care was seen "as a consumption activity rather than a fundamental right of the people or a responsibility of the state". indeed, this unsustainable, reactionary approach had to be subject to review and amendment. as a result, in , the new cooperative medical scheme (ncms) for rural population, mainly funded by the government, was launched. since , a health insurance scheme for urban non-salaried residents, especially for children and old people has been implemented. although china's health care system has appeared to get back on the track of government subsidy since the mid- , it is far from offering accessible, affordable, equitable and quality health care services with a universal coverage. some believe that the current health care reform is unlikely to solve the fundamental problems of accessibility, affordability, equity and quality. as long as the buck-passing polity does not fully change and the health policy process is still influenced by interested groups, the problem of dis- see jason shafrin, "an olympic post: the history of the chinese healthcare system", healthcare economist (august, ), available at <http://healthcare-economist.com/ / / page/ />. parity inherent in china's health care system will continue to affect the country's social environment. the worst part of china's health care system is the rampant medical corruption stemming from the medical reform over the past decades, particularly in the period between the mid- s and the mid- s. the usual scenario is that doctors and other staff expect to be paid extra fees (usually in the form of "red envelops" ) to perform operations and take kickbacks from pharmaceutical firms and medicalequipment suppliers. the practice is so prevalent that an unsullied doctor nearly does not exist. almost all practitioners and medical staff tell the same story as the defence of their unclean hands -low salary or underpayment. whistle blowers also tell that doctors have economic income targets allocated by hospitals and hospital administrators for them to meet and exceed. only upon completion of their quotes, they are paid benefit bonuses which can be much higher than their basic salaries. in other words, they are under pressure to charge patients extra fees and to recommend patients to go through unnecessary but expensive tests and procedures as well as purchase expensive medicines. nonetheless, such excuses are not sound enough to defeat professionalism and patients' human rights. besides, on many occasions, they are free to make a choice. for instance, a surgeon or a nurse can always hold up professional dignity by declining a red envelop because it is commonly given privately. moreover, nothing can justify their procurement of briberies and kickbacks from pharmaceutical companies or health care companies. facing a choice between campaigning for improving income or work environment and becoming corrupt, chinese doctors have had less difficulty in choosing to compromise their professional morality in the first place. moreover, nothing can defend the corruptive and negligent conduct of the personnel in governments and the health care system, which have caused further spread of epidemics such as aids. a nation has every reason to become alarmed when seeing medical profession to be infested by the epidemic of corruption. the governments and citizens all have a responsibility to trace the deep root of the phenomenon in their society, culture, policy and ethics in order to find the cures. access to quality health care services is a basic right in a civilized society. it is essential to one of the core human rights -human right to health. the human right to health care means that hospitals, clinics, medicines, and doctors' services must be accessible, available, acceptable, and of good quality for everyone, on an equitable basis, where and when needed. hence, universal access, availability, acceptability, dignity, non-discrimination, accountability, and transparency should be the fundamental principles upon which a health care system is built. many countries have upheld health care as a basic human right in their laws. unfortunately, the failure of the health care scheme has deprived many chinese citizens this right for more than two decades after the medical reform. this renders china's medical reform a truly disgraceful story. when people are deprived their access to hospital and health care services, their human rights, including their rights to health, life and information are potentially compromised and threatened. china is a contracting party to the international covenant on economic, social and cultural rights (icescr) and the united nations convention against corruption (uncac), a signatory of the international covenant on civil and political rights (iccpr). it is thus under treaty obligation to protect individuals against corrupt acts of third parties. though corruption and abuse of the health care scheme committed by practitioners and professionals in hospitals, insurance companies and suppliers of medical and health care goods and services impact on patients' human rights, the guilty individuals are not in direct breach of the uncac. instead, the uncac is directed primarily at member states rather than non-state actors. hence, it is the obligation of the chinese government to prevent corruption involving governments, industries, companies and individuals in fight against corruption and to raise public awareness of the matter. due to the fact that corruption has invaded into every corner of the chinese society, citizens have faced an unfavorable physical and social environment. fraud in project approvals and failure to apply emission control measures are responsible for the human right to health means that everyone has the right to the highest attainable standard of physical and mental health, which includes access to all medical services, sanitation, adequate food, decent housing, healthy working conditions, and a clean environment. xiangming zhou, a study of the health care right (phd thesis, jilin university, ) . see part ii -articles and uncac. rising pollution in the country. some professions including teachers, doctors, lawyers and those safeguarding consumptive goods are not only important to people's lifestyle and well-being and society's progress, but also play a social role in which trustworthiness is an expectation in every tradition. in china, these professions, because of the rampage of the unethical performance conducted by a majority of the people in the trades, have definitely failed to honor their part of social compact with society, which in turn exacerbates human rights related problems. it is a high time for china to review the ethical conduct of these key professions. the chinese government has an obligation to protect citizens' human rights to health, access to healthy food, safe drinking water, clean air, and a healthy environment. in discharging this obligation, effective mechanisms combating corruption in various forms need to be established in order to sustain the country's social stability and economic prosperity. china -pig farmers told to drug 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environment programme ( ) nairobi declaration on the state of worldwide environment united nations environment programme universal declaration of human rights international covenant on economic, social and cultural rights united nations general assembly ( b) international covenant on civil and political rights convention on the elimination of all forms of discrimination against women draft world charter for nature convention on the rights of the child what are human rights? www.ohchr.org/en/issues/pages/whatarehumanrights.aspx usda -economic research service ( ) percent of consumer expenditures spent on food, alcoholic beverages and tobacco that were consumer at home, by selected countries three prescriptions for treating the symptoms of the medical reform directors' duties under chinese law -a comparative review world meteorological organization, timely access and response to accurate early warnings are crucial for minimizing impacts of natural disasters johannesburg declaration on sustainable development and plan of implementation of the world summit on sustainable development: the final text of agreements negotiated by governments at the world summit on sustainable development a study of china's legal system for food safety a study of the health care right chinese vs. western perspectives: understanding contemporary china food safety becomes national priority, china daily what is the right to food? right to food key: cord- - xfkse authors: bindenagel Šehović, annamarie title: human rights and state responsibilities date: - - journal: reimagining state and human security beyond borders doi: . / - - - - _ sha: doc_id: cord_uid: xfkse this chapter lays out an argument that citizens’ human rights are the responsibility of the corresponding state, meaning that citizens of a territorial state claim particular rights that state is obliged to deliver. in return, in an aspect which is often neglected in analyses of human security, citizens also owe allegiance to the state. citizens’ rights have been expanded to encompass not only physical protection within a territory but also a host of economic and welfare provisions. despite the increasingly international discourse on human security rights, their legal home remains with the national state vis-à-vis its citizens. the chapter argues that the rules of the state-based order are shifting, with no clear loci of responsibility and accountability for human security. the introduction sketched the origins and elements of human security. this chapter lays out one argument to make the case that citizens' human rights are the responsibility of the corresponding state, meaning that citizens of a territorial state claim particular rights that state is obliged to deliver. as outlined in the introduction, these rights have been expanded to encompass not only physical protection within a territory but also a host of economic and welfare provisions (hösle ; slaughter ; cescr ; icpcr ) . however, especially with regard to the latter, not all of these rights are equally or legally encoded into national law. thus despite the increasingly international discourse on human security rights, their legal home remains with the national state vis-à-vis its citizens. in return, in an aspect which is often neglected in analyses of human security, citizens also owe allegiance to the state. this includes submitting to civic codes such as police ordinances and taxation, as well as to the military draft when instituted : without such a reciprocal relationship between states and citizens, it might not be possible to guarantee territorial or other human security protections. of course, the necessary existence of such a relationship does not preclude its potential for abuse by either party (see also howell ) . this reciprocal relationship is based on a state-citizenship centric order, and that not only at the national level, but also internationally. in other words, citizenship here is dependent upon its conferral by a territorial state, which derives its contours from its citizenry. this chapter thus assumes that the current national/international governmentality order continues to be based upon this state-citizens relationship, with a twist. that is, while the national/international legal order rests upon the pillars of state-citizen reciprocity with regard to rights and obligations, this exchange does not reflect the more complicated reality. that is, the rules of the state-based order are shifting, with no clear loci of responsibility and accountability for human security. the hypothesis presented here argues that a bifurcated evolution wherein rights have ascended up the international agenda but not necessarily at the national level, and state or sovereign obligation has been diffused between state and nsas without clarifying where the locus of the final guarantee of protection lies, describes the current status. this has led to a diffusion of the guarantor status of the national state, with elements of power in governance-agency, scope, mechanisms, and normative context-diverging. this leads to two questions: first, if state a acts as a guarantor to the human security of citizens of a, the same holds for state b and citizens of b; but what happens to citizens of state a residing in state b, or vice versa? second, what are the consequences of human security provision to citizens of a or b by nsas, notably when nsas go bankrupt or depart? both of these questions point again to the need to clarify the relationship between citizens and states in order to conceive of suitable answers. as outlined in the introduction, the idea of human security as the remit of the state is inextricable from the notion of state sovereignty. sovereignty as a concept has been debated since its inception, and each idea of it has made various assumptions as to what it entails and what it excludes. the majority of scholars (krasner ) of westphalia-influenced definitions of sovereignty include requisites such as the state enjoying a monopoly of power capable of defending its territorial borders against external aggression; even these have rarely been absolute in practice (krasner , - , ) . in describing this 'compound' myth, anne-marie slaughter defines westphalian sovereignty as "the right to be left alone, to exclude, to be free from any external meddling or interference" (slaughter , ). yet the same sovereignty that offers the option to opt out is also the ticket to inclusion in the inter-national community of (equal) states. the westphalian definition also invokes "the right to be recognized as an autonomous agent in the international system, capable of interaction with other states and entering into international agreements" (slaughter , ) , the responsibility for whose implementation resides squarely with those signatory states. this reveals the schism between what robert keohane ( ) called formal and 'operational' sovereignty, and what i referred to as the divergent 'final guarantee' and 'functional' sovereignty with regard to the governance accountability problem (gap) (Šehović ). it acknowledges that westphalian sovereignty is not absolute, and rather that "it is now a platitude that the ability of governments to attain their objectives through individual action has been undermined by international political and economic interdependence" (keohane, quoted in slaughter , ) . the eu exemplifies political and economic interdependence, a model partially replicated to differing degrees by the african union (au), asean (association of southeast asian nations), and mercosur (the common market of select south american states) in a quest to confront threats and maximize opportunities. both in theory and in practice then, this means that, on the one hand, states increasingly cannot-and often do not want to-fully guard against external interference. on the other hand, states (should also) acknowledge that the sources of such interference include not only other states but also activities of nsas, from crime syndicates and cyber surveillance and mercenaries to human rights' campaigners, as well as cross-border challenges such as (infectious) disease spread and migration. these interdependencies and their potential both for cooperation and for conflict directly influence a state's ability not only to control its own territory (see krasner, interdependence sovereignty, pages - ) but also to the "security, economic stability and a measure of prosperity, clean air and water, and even minimum health standards" (slaughter , ) that are the hallmarks of hösle's expanded definition of sovereignty (hösle ) and the integral components of human security. human security's main argument places the emphasis of security on the human as opposed to the state. the central assumption underscoring human security is that "when a human faces a threat, so does international security" (burgess and gräns , ; kerr , ; undp ). yet the two are necessarily in dialogue with each other: first of all, states in the inter-national remain the arbiters of human security (hösle ; un declaration ; undp ) , regardless of whether the point of departure is human-or state-centric; and second, as members of the international community of (equal) states, these are themselves increasingly subjected to trial by their peers. "states can no longer assume that if they refrain from interfering in the affairs of other states they will remain free from interference themselves" (slaughter , ) . furthermore, governments increasingly understand that they often cannot afford to look the other way; that fundamental threats to their own security, whether from refugees, terrorists, the potential destabilization of an entire region, or a miasma of disease and crime, may well have their origins in conditions once thought to be within a state's exclusive domestic jurisdiction. (slaughter , ) as the post-cold war era has shown, both intra-and inter-state conflict have coincided with the spread of disease. this has been evident in the former yugoslavia, in rwanda and somalia, in iraq and syria (intrastate conflict by the numbers ; human security centre). these conflicts have seen the increase in cross-border spread of disease such as evd, h n , hiv, measles (notably in continental europe, and the us), mers-cov, and sars, to name a few examples. this incidence salience of the insight that: "states can only govern effectively by actively cooperating with other states and by collectively reserving the power to intervene in other states' affairs" (slaughter , ) . it has been backed up by the normative evolutions first from rights to responsibility, to the r p, to, arguably at this moment, the responsibility to respond. this captures the essence of a continual conversation between human security and sovereignty. therein, "internally, a government has a responsibility to respect the dignity and basic rights of its citizens," and "externally, it has a responsibility to respect the sovereignty of other states" (slaughter , ) , except when a state heeds the (r)evolution rewriting sovereignty as control to sovereignty as responsibility. daniel philpott describes this shift as part of an ongoing process. he attributes this revolution in sovereignty to "prior revolutions in ideas about justice and political authority" (philpott , ) . the post-cold war reordering of the world proffers a multitude of examples of this progress: from emergent multipolarity (flockhart ) to the rise of nongovernmental organizations (ngos) and nsas, from the human rights debates to gain access to hiv treatment to those to usher in the r p (iciss ), reconceptualizations of internal and external state responsibility have been pitted against each other. though the state remains legally dominant, theoretical and philosophical evidence underscored by empirics points to two key unresolved tensions: the locus of the responsibility for human security and the scope of human security, particularly in reaching non-citizens. on the theoretical side, foucault presciently identified emergent 'governmentality' (faubion and rabinow ), anticipating the collaborative governance that would emerge as states and nsas sparred and cooperated in response to ever more global challenges to human security. the s, amid the (western) euphoria of the 'end of history' (fukuyama ) , witnessed an initial acknowledgment that states alone could not meet the rising number of international and increasingly global challenges-from the multiplication of intra-state conflict and the proliferation of weapons to water management. rosenau introduced the idea of 'governance without government' (rosenau and czempiel ), maintaining that governance 'regimes' composed both of states and nsas would form to tackle specific issues in the international realm. nsas have long been engaged in shoring up or tearing down state sovereignty, with (hösle ) or without the consent of the state. while on the one hand a tension exists between theory and practice of state sovereign obligation with regard to human security, it also means that though threats to human security abound on the part of both state and nsas, precedents likewise exist for mitigating these to the benefit of human security. to a large extent, rosenau has been proven correct: if nsas are included, then a plethora of organizations exist dedicated to treating hiv/aids, providing water and sanitation, and even administering public transportation in municipalities around the world. however, these are not regimes in the sense that they have a central organizational structure, that their interventions are legally binding, or that any mechanisms are in place to ensure the continuation of their work if and when they opt out. this is not a central theme of risse's work, which focuses on 'areas of limited statehood' (risse ) . here nsas might perform functions theoretically if not in practice associated with state responsibility for human security. yet they are not bound to such actions, for instance, of service delivery and health care. critically, instead of shoring up states' lack of capacity, nsas have contributed to the fragmentation of their power-including their ability to guarantee traditional and human security: ngos' [nongovernmental organizations'] role and influence have exploded in the last half-decade. their financial resources and-often more importanttheir expertise, approximate and sometimes exceed those of smaller governments and of international organizations. "we have less money and fewer resources than amnesty international, and we are the arm of the u.n. for human rights," noted ibrahima fall, head of the u.n. centre for human rights, in . "this is clearly ridiculous." today ngos deliver more official development assistance than the entire u.n. system (excluding the world bank and the international monetary fund). in many countries they are delivering the services-in urban and rural community development, education, and health care-that faltering governments can no longer manage. (matthews ) nonetheless, risse assumes that nsas will continue their activities. that these nsas might be accountable not to the human beings they serve, but otherwise, or that they might be dependent upon funding sources whose priorities are prone to shift, remains under-analyzed. it leaves unanswered the questions of what happens to the state-citizen relationship when they do not. krasner attempts to corral some of these disparate responses to the sovereign redrafting by delineating four elements of sovereignty: westphalian, juridical, domestic, and interdependence (krasner ; czempiel and rosenau ) . none directly deal with the engagement between sovereignty and human security explicitly, yet they are critical in highlighting their exchange. whether the four 'sovereignties' can be meaningfully divorced from one another and applied in an empirical sense to state or human security remains unproven: while theory must conform to practice, so, too, must practice inform theory (see box . ). while keohane's divide between formal and functional sovereignty alludes to some of the problems with distilling sovereignties listed above, they are not thereby resolved (keohane ) . similarly, slaughter's network theory, taking nsas into account, revives some of the same solutions put forward by foucault and rosenau. likewise my gap thesis, while identifying the lack of accountability between state and nsas with regard to the guarantee of human security to citizens, it did not deal with the same responsibility to non-citizens. this points to a new stage in philpott's (r)evolutions in ideas: while each of the conversations between sovereignty and human security introduced above acknowledges the limits of westphalian absolutism, each fails to account for their (re)imagining beyond borders. on the philosophical side, scholars have wrangled with this conceptually in various terms. the human rights agenda, which both precedes and parallels that of human security, is itself an outgrowth of a historical trajectory of political theology. referring to "to the connections between religion (in the broadest sense, including philosophy as well) and legally structured power," political theology is of "special importance in the western world and influenced the development of juristic concepts, especially those concerned with public law" (hösle , ; schmitt ) . public law, inextricable from the relationship between states and subjects, then states and citizens, is vested with antecedents of values-with morals and their changing interactions with politics (hösle , ; carlson and owens ) . • what is the value of westphalian sovereignty where a state cannot control its territory? • what role does juridical sovereignty play when a state is only partially recognized by its peers? (see kosovo) • what does domestic sovereignty mean if (a) a portion of the citizenry is excluded from, for instance, health care? (b) non-citizens have no recourse to rights (to education, health, justice)? • what is interdependence sovereignty if borders are porous or surveillance systems are technologically or politically incompatible? christianity in the west, particularly after the treaties of westphalia largely ended internecine wars on the european continent, contributed immensely to the conversation and construction of sovereignty, as related to human rights and human security. hösle argues that christianity estranged citizens from their state and universalized their rights' claims. through the idea of all human beings as god's children, a broader as well as existentially deeper diffusion of the universalistic and individualistic ideas of hellenism-and thereby eliminated a possible identification with any state that does not include all human beings and is not constituted in accord with the principles of christianity. (hösle , ) christianity can arguably be made responsible for two things (hösle , ) : first, a politics free of religious and ritual considerations, taken further through the enlightenment; and second, an intensive moralization of the religious, demanding "an influence on politics that went far beyond what was conceivable for ancients" (hösle , ) . the latter finds its echo in the articulation and demand for individual human rights delivered by the state. thus although the notion of a christian theocracy likewise receded with the secularization of westphalia, the ideas of universal human rights and of a universal claim to human security have wound their way through various (r)evolutions in sovereignty right up to this present reimagining. returning to the core of the conversation between sovereignty and human security, theory and philosophy back up the urgent need to practically respond to the three main tenets of state and human security: ( ) ensuring the territorial and physical security of citizens; ( ) protecting lives and livelihoods through basic economic stability, health, and welfare; and ( ) bearing accountability internally and to the international community (hösle ; risse ) . assuming that states remain the final arbiter of such securities, articulating, delegating, and assuming respective state and human rights and responsibilities are key to reimagining and implementing human security beyond borders. states, sovereignty, human security-all are predicated upon a relationship of rights and responsibilities between citizens and states. the tension in this reciprocal relationship is not new. it can be divided into three broad shifts dating from westphalia through to the last major global reordering in the s, which ended the second wave of democratization (strand et al. ) and inaugurated the third wave of liberal, democratic capitalism based on state sovereignty. the first shift, demarcated but by no means consolidated with the treaties of westphalia in , ordered responsibility, for territorial and physical protection in the name of state sovereignty, at the level of the state. the second shift, from circa the s, occurred at the height of the second wave of democratization, and in the name of 'self-determination' (unpo ) . this meant on the one hand that especially newly minted states could cling in particular to the westphalian notions of 'nonintervention,' a stance reemphasized by both blocs at the height of the cold war. on the other hand, however, the existence of the post-world war ii un and its emerging norms and values spread the notion that state responsibility includes human rights discourse if not its translation into practice. this pre-exposure arguably paved the way for the s shift, which, in the words of the independent commission on human security, refers to the 'vital freedom,' explicitly tied state responsibility to "protecting people from severe and pervasive threats, both national and societal, and empowering individuals and community to develop the capabilities for making informed choices and acting on their own behalf" (ogata and cels , ) . what remained formally the same throughout these transitions, and became all the more pronounced as state subjects/constituents became citizens, is the onus placed on the state to assume responsibility for the security of those citizens. while a constituent refers to a voter within a particular area, a citizen is a (political) member of a state. this has two implications: first, a constituent must not be a citizen. indeed, a constituent might receive physical security within a territory in return for heeding the obligation to serve that same territorial state's security in the event of war. this leads to the second point: a citizen might have more privileges, such as the right to vote. yet the obligation to serve the state-by taxation and/or by (required) military service-remains. so, too, does the threat of the revocation of citizenship if an individual serves in the armed forces or swears allegiance to another state. as is quoted on the inside of every us passport: . loss of u.s. citizenship: under certain circumstances, you may lose your u.s. citizenship by performing, voluntarily and with the intention to relinquish u.s. citizenship, any of the following acts: ( ) being naturalized in a foreign state; ( ) taking an oath or making a declaration to a foreign state; ( ) serving in the armed forces of a foreign state; ( ) accepting employment with a foreign government; or ( ) formally renouncing u.s. citizenship before a u.s. consular officer overseas. (authors' passport) (generous) provisions do exist that allow dual citizenship. some states, the us among them, allow citizens to renounce their citizenship. others, such as iran, do not. while citizenship obligation has long been linked to a measure of state responsibility for protection, such as consular services overseas, it has not been synonymous with citizenship rights. by its very exclusivity, citizenship does not and cannot confer universal, inalienable rights. the concept and enactment of human security attempt to rebalance those obligations into an equation wherein state sovereign responsibilities meet individual human rights (bergman ; kerr ; nef ; undp ) . the revolution of human security and rights-based development lies in their universalism. states become the bastions not only of ultimate responsibility for the extent of the provision of rights for what is possible within their capacities but also, arguably, for the highest standard internationally. president franklin d. roosevelt's now-famous "four freedoms speech" of preceded the call for human security in the undp and again in the publication of the report "human security now" by the commission on human security (roosevelt ; ogata and cels ) . from the very beginning of the post-world war ii period, article of the un charter and article of the un universal declaration of human rights (udhr) encoded the principles of human security, including an emphasis on the right to health, which is central to the case studies presented in chaps. and : everyone has the right to a standard of living adequate for the health and well-being of himself and of his family, including food, clothing, housing and medical care … and the right to security in the event of … sickness, [and] disability … motherhood and childhood are entitled to special care and assistance. (udhr ) the centrality of health among global policy priorities is reiterated in the constitution of the world health organization (who) in ; the international covenant on economic, social and cultural rights (icescr); the undp; and the adoption of the ihr in and most recently updated in . the icescr-as well as the convention on the elimination of all forms of discrimination against women (cedaw), the convention on the rights of the child, and the world trade organization's doha declaration on "trade-related aspects of intellectual property rights," which allows for the production of generic versions of essential medicines under certain conditions before patent protection runs outappears to provide an implicit obligation on the part of states to improve health and to establish and secure health as a human (security) right. however-and crucially-none of them prescribes an explicit obligation. similarly, the ihr emphasize the universal and expanding right of each individual citizen (of the world) to the highest standard of health. in fact, the ihr, having gone into effect in , require their signatory state parties to "develop public health capacities to detect and respond to public health emergencies of international concern (pheic), with states required to cooperate in building these capacities" (who ) . "however, the regulations do not provide incentives, sanction states for failing to cooperate, or allocate responsibility" (gostin and friedman , ) . no specific or enforceable obligation to ensure that individuals attain physical and mental health and no guidelines for how the state's obligations are to be discharged exist (davies ) . this situation obviously creates problems for the implementation of the right to health within the remit of a state's responsibility to provide (human) security. nonetheless, these agreements have transformed normative ideas into principles of action (icescr , article ). yet real implementation lags, lost in the opaque realm between theoretical and practical responsibility. the consequences are particularly obvious with regard to states' responses to threats to human security of, but not only of, health. in the narrow sense, human security is limited to physical protection and the creation of conditions conducive to human welfare, but stops short of full protection and provision. whether or not defending those values parallels interests that reach to the hindu kush (löfflmann and vaughan-williams ; maull ) , the assertion of which resulted in the then german defense minister struck ( struck ( - tendering his resignation, remains a point of contentious debate, not just in germany. kaldor et al. ( ) attempt to work this into an especially value-based foreign policy strategy for the eu that nonetheless takes state interests into account. this morphs into the broader conceptualization of human security, wherein an equal level of priority is given to any type of threat (thakur , ) . critics argue that such prioritization of all is equal to prioritization of none. liotta and owens present one attempt to differentiate between threats, risks, and vulnerabilities as part of this debate (liotta and owens ) . they arguably all converge and infringe upon human security which demands a response. a gap emerges between theory of protecting human security and its practice. it begs the questions: for whom? how far? by whom? the (inter)national system based on sovereign states continues to operate under the assumption that "governments have a responsibility for the health of their peoples which can be fulfilled only by the provision of adequate health and social measures" (who ) . critically, "while only states are parties to the covenant, and thus ultimately accountable for compliance with it, all members of society-individuals, including health professionals, families, local communities, intergovernmental and non-governmental organisations as well as the private business sectorhave responsibilities regarding the realisation of the right to health" (who, ihr ) . as lake notes with regard to judicial processes in the congo, "the de facto assumption of power by these diverse sets of actors has created opportunities through which non-state actors can enter and influence juridical processes by engaging in tasks normally reserved for representatives of the sovereign government. these activities would not be possible in contexts where the state had greater reach" (lake , ) . this exacerbates the problem of responsibility because merely counting the number of convictions of a prioritized crime or the number of people inquiring about health treatments and antiretroviral medications for hiv, for example, "tells us little about the dynamics of power" that determine the necessary response to the problem (including the problem definition) at hand (lake , ) . lake notes that "on a broader scale, it could also be argued that the involvement of international actors in micro-level governance activities in dr [democratic republic of] congo has served not to build capacity but in fact to further relieve the congolese state of its responsibilities to provide basic goods and services to its citizens." indeed, because a litany of "international and domestic organizations ready to engage in this work, there may be little incentive for the central government to re-invest its own time and resources into developing a functional state apparatus" (see also keohane ; lake , ) . such developments actively undermine state's sovereignty and capacity to exercise responsibility, leading to absurdities such as indonesia's claim to 'viral sovereignty'-the idea that viruses belong to the state in which they originate. it was invoked to prevent and delay sharing data and sam-ples of h n influenza also due to the anticipated costs of being branded a state of contagion amid exclusion from research and treatment benefits. indonesia's was an ill-fated attempt by the state to seize control over information pertaining to the outbreak, its domestic response, and its interdependence sovereignty-notably its ability to regulate any potential medical interventions and possible patents created externally and sold (back) to indonesia. these examples all iterate the theory and practical reality in the still state-centric international system that there are roles that only the state-at least among today's polities-can perform. states are the only nonvoluntary political unit, the one that can impose order and is invested with the power to tax…. moreover, it may be that only the nation-state can meet crucial social needs that markets do not value. providing a modicum of job security, avoiding higher unemployment, preserving a livable environment and a stable climate, and protecting consumer health and safety are but a few of the tasks that could be left dangling in a world of expanding markets and retreating states. (matthews ) assuming then the necessary vitality of a responsible sovereign state to the guarantee of access to rights, any reworking of state and human security must take states into account even while rising to the challenge of responding to and guaranteeing human security beyond states. placing the responsibility for human security beyond states requires flexible relocation of that responsibility itself. although state sovereignty continues to be the building block of local, national, and international relations and global governance, its real power to enact responsibilities and assume accountability for the provision of the rights of its citizens has arguably waned-not uniformly but almost regardless of whether the state in question is considered consolidated, fragile, or failing/failed. consequently, the ostensibly sovereign state is ultimately responsible for the traditional, territorial security and physical security of the populace within its borders. in addition, it is accountable for both of these securitizations both internally and externally (i.e., within the international community of states). however, the same state is increasingly confronted with nsas that both demand its action and assume some of its functional responsibility-but not state(-citizen) accountability. as such, the statecentric international governance system faces the challenge of responding to both internal and external rights' demands and responsibility duties. the next chapter will further explore these conceptual challenges and analyze possible levels of such a reordering of human security responsibilities beyond borders. see also the cambridge dictionary entry for "citizenship: the status, rights and duties of a citizen, especially of a particular country on concepts and paradigms in mixed methods research human security the sacred and the sovereign global politics of health the coming multi-order world the end of history and the last man. the national interest (spring) morals and politics international commission on intervention and state sovereignty (iciss). . the responsibility to protect the international relations and security network intrastate conflict by the numbers human security: a new strategic narrative for europe hobbes's dilemma and institutional change in world politics: sovereignty in international society human security sovereignty: organized hypocrisy organizing hypocrisy: providing legal accountability for human rights violations in areas of limited statehood ebola: a crisis in global health leadership why human security? the whitehead journal of diplomacy and international relations winter/spring narrating identity, border security, and migration: critical focus groups and the everyday as problematic power shift germany's uncertain power: foreign policy of the berlin republic human security and mutual vulnerability: the global political economic of development and underdevelopment revolutions in sovereignty: how ideas shaped modern international relations paradoxien der souveränität: die konstituive norm, auf der die heutige staatenwelt gründet-dass nämlich staaten souverän sind-gilt uneingeschränkt nicht mehr. was heisst das? internationale politik state of the union address (the four freedoms) governance without government: order and change in world politics human security-protecting and empowering the people the concept of the political hiv/aids and the south african state: the responsibility to respond sovereignty and power in a networked world order democratic waves? global patterns of democratization international covenant on civil and political rights unrepresented nations and peoples organization (unpo) constitution of the world health organization world health organization key: cord- -s zk l a authors: mihaylova-garnizova, raynichka; garnizov, vasil title: refugee crisis as a potential threat to public health date: - - journal: defence against bioterrorism doi: . / - - - - _ sha: doc_id: cord_uid: s zk l a the refugee crisis in europe continues to persist despite recent data, showing a drop in the number of refugees seeking asylum. the eu has called this as “an unprecedented displacement crisis” and has aimed at devising a comprehensive approach to tackle it, which has been widely criticized. concerns about public healthcare aspects of the crisis have permanently entered the media and policy discourse even though no systematic association between migration and the importation of infectious diseases has been recorded. in this context, the literature has not filled the existing gap between discourse and evidence, and almost no publications with reliable empirical data exist, both thematic (epidemiology) and geographical (eastern europe and bulgaria). among the existing publications, the focus has been on tb and hiv (odone et al., euro j public health ( ): – , ). in light of this, the aim of this research is to contribute to the debate by providing an overview of the refugee situation in bulgaria, as a primary entry-point for refugees entering the eu. in order to achieve this, the article analyses the case of the refugee camp in city of harmanly, close to the bulgarian-turkish border, and assesses the public health risks related to this specific situation. based on a study of patients with different symptoms we aim to draw wider implications about the linkages between public health and migration. the in-depth review of this specific case shows that both the probability and impact of migration on public health increases when the hosting country is relatively poor, the domestic public healthcare system is not efficient, and there is lack of trust in the government and public services. the study contributes to understanding better these risks in order to identify potential mitigation strategies in the region and the eu as a whole. the european migrant/refugee crisis in - deeply challenged the political, economic and healthcare systems in the european union (eu). looking at these developments, we are faced with the key problem of lack of data on the health profiles of migrants, in particular -of refugees. immediately after the crisis began there were many publications that insist, without extensive evidence, that migrants do not pose a health threat to the host population. almost no publications could be found with reliable empirical data, especially epidemiological, including for bulgaria. the absence of reliable and robust data is even more problematic at times of crisis since public health systems cannot prepare adequately. the lack of objective information also opens up opportunities for the emergence of public myths and psychoses, including disinformation that can lead to political destabilization, which could in turn affect national security. the purpose of our article is to collect, summarize and present epidemiological data related to migrants in bulgaria and, on the basis of this information, to analyze the potential risks to public health (including risks to migrants) and to assess the capacity of bulgaria's healthcare system to cope with the refugee crisis. migration is a phenomenon known since antiquity. thanks to it, new countries have emerged and new communities have formed. but what is happening today is extraordinary because millions of people constantly change their place of residence for various economic and non-economic reasons, going beyond the scope of our research. the bottom line is that at the end of , almost million individuals were forcibly displaced worldwide. twenty million of them are refugees. for the first time, turkey has become the country with the largest number of refugees ( fig. . ) . as far as europe is concerned, with the exception of certain high-income member states, most countries did not have serious difficulties with migrants. this was fundamentally altered after the series of events in north africa and the middle east, known as the arab spring, as well as the civil war in libya, and especially since the syrian conflict began in . based on regular easo annual reports, since one can see the changing structure of migration trends altogether. until then migration was mainly from lower income countries to countries with a higher income and the greatest percentage of migrants represented the share of those arriving from western balkan countries. after , migration started affecting not only highincome countries but also all european countries with the highest percentage of asylum seekers being refugees from syria ( fig. . ) . as a result, europe has faced a unique influx of refugees, asylum seekers and other migrants since the establishment of the eu: . million people arrived in the eu only in . since the beginning of , , , applications have been recorded in the eu+ (eu plus norway and switzerland). even though this is % less than in the same period of , when , , applications were lodged, this highlights the extent of the challenge [ ] . this is a not an easy situation to be rationally understood since it is sensitive to strong public opinion and intense debate. even more, from the point of view of public health 'this refugee situation is unparalleled since the end of ww ' [ ] . in this context, often the public healthcare aspects of migrant crises enter the broader public discourse, regardless of expert opinion. the diverse discourses have been addressed by multiple international organisations. in dr. zsuzsanna jakab, who regional director for europe, clearly persons applied for international protection in the eu highlighted that there is no systematic association between migration and infectious diseases stating that: 'communicable diseases are primarily associated with poverty. refugees and migrants are exposed mainly to the infectious diseases that are common in europe, independently of migration. the risk that exotic infectious agents, such as ebola virus or middle east respiratory coronavirus (mers-cov), will be imported into europe is extremely low. experience has shown that, when it occurs, it affects regular travellers, tourists or health care workers rather than refugees or migrants' [ ] . in the european commission also addressed the healthcare aspects of migrant crises, declaring that refugees are actually the ones at risk, rather than a burden on health systems. dg sante made it explicit that measures to protect refugees' health are being taken 'not out of unfounded fears that they might spread infectious diseases' or 'place a burden on health systems. their [refugees'] health is at risk, not the health of eu citizens' ([ ] , emphasis added). who and ec's stance on public health challenges of large-scale migration and preparedness of countries in the european region could be seen as too optimistic. who insisted that 'the health systems in the countries receiving migrants are well equipped and experienced to diagnose and treat common infectious diseases; they should also be prepared to provide such health care to refugees and migrants. should a rare exotic infectious agent be imported, europe is well prepared to respond, as shown over the past years in responses to imported cases of lassa fever, ebola virus disease, marburg virus disease and mers' [ ] . at the same time, events in germany on the weekend of - september demonstrated the opposite: large-scale migration provoked serious malfunctioning of both political and administrative systems including police, social services, housing, public healthcare [ ] . after these events, reported in depth by robin alexander, the responses across europe quickly escalated. once the eu-turkey deal was agreed, balkan border closures organised by austria followed by hungary, serbia and macedonia left some - , migrants blocked in the informal camp in idomeni on the greek-macedonian border [ ] . from the beginning of march to the end of may asylum seekers there survived in poor sanitary conditions with very limited running water, no lavatories, and insufficient food, despite efforts by ngos such as 'hot food idomeni' and medical care from 'médecins sans frontière'. the unhealthy and overcrowded conditions at the camp have given rise to infections and in march some cases of hepatitis a have been reported [ ] . posters with the message 'greece will offer you accommodation, food and healthcare' (written in arabic, farsi and pashtun) were posted in march but the final decision was taken only on th of may. in reality, initial reactions and follow up to the idomeni situation were limited since there was no confirmed information on the epidemiological situation in idomeni, no risk assessment and no further public health measures were taken. in contrast to who and ec statements, bulgarian authorities never declared their readiness to manage a large-scale migrant influx. until bulgaria had not been a country of interest to migrants and, as a result, there was scarce experience of responding to a migrant crisis. rather, the country was a source of migrants towards western europe. bulgarian membership of the eu on january , led to an increase in the flow of migrants to bulgaria and a slow but sustainable decrease of bulgarian emigrants. this situation has changed radically since the start of the civil war in syria in . the geographical position of the country, which shares a km border with turkey, makes it a natural primary point of entry for refugees entering the eu ( fig. . ). moreover, turkey is the largest host country to syrian refugees. the dramatic increase of refugees in bulgaria led to a peak in . compared to , when the number of people legally seeking asylum in bulgaria was , saw an increase to , a % increase from , with the largest share of the migrants, arriving from syria - %. indeed, migrants account for only . % of migrants in the eu , but the ratio is approximately migrants per million people ( fig. . ) . at the same time, the majority of migrants did not intend to stay long in bulgaria, which they saw only as a stage of their journey to western europe. many of the migrants entering bulgaria in moved to france, germany and sweden, probably due to the lower incomes, but also the lack of previous settlers and migrant communities [ ] . the total number of those remaining in bulgaria is relatively low, compared to greece, italy, and germany ( fig. . ). it should be noted that for the period from to , a major share of refugees originated from syria, while in , the ratio changed, and most of those arriving, were fleeing afghanistan and iraq (fig. . ) . in terms of the demographic profile of those arriving, migrants in bulgaria are relatively less educated and the share of men is approximately equal to that of women and children figs. . and . . following the - crisis, in february a who assessment mission to bulgaria took place to access the country's capacity to address the public health implications of sudden large-scale influxes of migrants [ ] . the mission concluded that the level of assistance provided to the migrants by the bulgarian government has clearly improved but the medical response system is very fragile and not fully prepared to respond to a possible new and larger influx of migrants. the main weakness of the response system is the weak provision of primary health care through clinics in migrant centres or the assignment of general practitioners (gp) to migrants due to the fact that clinics are understaffed and interpretation services are not always available. as a consequence, the first recommendation of who is the revision of the national plan for crisis management with a focus on reorganizing primary health care services and rationalizing the use of available resources [ , p. ]. to put this into context, in the following section we provide an overview of the institutions and personnel involved in refugee and migrant management. upon arrival in bulgaria, the asylum seekers first face the authorities of the ministry of interior. by law, migrants can be held for up to hours at a location, where basic health screening is conducted, and from there they are directed to the registration and reception centre (rrc), the transit centre (tc) or detention centres where primary care is carried out. primary healthcare in detention centres is the responsibility of the medical institute of the ministry of interior, situated in sofia, while medical staff in rrc and tc should be provided by the state agency for refugees ( fig. . ) . according to bulgarian legislation, access to public health services requires payment of health insurance contributions. asylum seekers and persons applying for refugee status are funded by the state agency for refugees. in case they have acquired refugee status or the right to asylum, they are obliged to pay health insurance themselves to access the basic package of health care to which bulgarian citizens are entitled under the insurance system. however, the vast majority of refugees cannot cover their health insurance. for this reason, in march the bulgarian government passed a normative act creating an obligation for municipalities to cover health insurance expenses of migrants with official refugee status already granted. after strong public outcry the government withdrew the proposed act and this issue remains open. moreover, even when migrants fulfil their financial contribution, the number of refugees with an assigned gp is limited due to language barriers and the reluctance of many gps to take on patients whose residence is likely to be temporary [ , p. ] . the other important recommendation of the who assessment mission to bulgaria is the revision of the disease surveillance early warning and response system, introducing syndromic surveillance to increase early detection of outbreaks and effectively monitoring selected disease trends [ , p. ] . such a syndromic surveillance system has not been created yet and it is unknown whether the government plans to implement one. at the moment bulgaria only conducts surveillance and control of communicable diseases (cd) under the authority of the ministry of health (fig. . ). however, this information system does not include relevant information on the spread of infectious diseases among migrants/refugees. in the current paper we manage to fill the gap with operational reports provided by the chief state health inspector, angel kunchev from regional health inspections (rhi). these reports include epidemiological data from rhi-haskovo and rhi-sofia for . actually, these two administrations cover the two territories of the country in which almost all migrants are situated and thus the data can be seen as representative for the country. during based on these tests on the territory of rhi-sofia the following communicable diseases have been diagnosed (table . ). despite their limited capacity, authorities conduct basic health screening at the border, along with health services in the migrant reception and detention centres. screening at rhi-haskovo and rhi-sofia showed prevalence of intestinal parasites (giardiasis, ascaridiasis, blastocystosis) and our data indicates that the majority of refugees posed very limited infectious risk. therefore, the findings confirm who at the same time, contrary to analysis by experts, public opinion reacted to the migrants situation as high-risk, demonstrated by the conflict in harmanli migration rrs. residents of the town of harmanli protested on th november, demanding the camp's closure after local media reported on the suspected existence of communicable diseases on site: 'an artificially created tension led to this, following misleading reports that the centre is a hearth of infection,' petya parvanova, head of the bulgarian refugee agency, was quoted by reuters as saying. the government declared that no medical reason for quarantine existed but the authorities took the decision to temporarily close the camp and restricted the free movement of migrants with the aim to calm down town citizens. in response, on th of november some - migrants (from the officially announced registered in the camp) clashed with the police and the gendarmerie. as a result, most of asylum seekers have been moved to other centres. meanwhile, on - th november , immediately after the first articles in the local media and first signs of discontent, a special medical mission from the military medical academy was sent to verify the health status of the migrants concerned. the main findings of the military medical team study were as follows [ ] : • very high share of patients with skin and infectious diseases: patients with different symptoms; as a result of the inspection, the military medical team has diagnosed the following diseases, ordered according to incidence (table . ). the conflict in harmanly town illustrates some of the weaknesses reported in who's assessment of bulgaria's capacity to manage a large migrant influx, notably risk communications and work with local media. even though a special highly qualified medical team was sent to check the medical status of refugees and eventually to refute speculations of an epidemic, the conflict escalated instead of calming down. one explanation for this is the low levels of trust in the authorities and institutions in bulgaria, which also includes lack of trust in healthcare professionals. in such circumstances, both clear risk communication and medical expertise are not sufficient to resolve a growing crisis. in discussing the risks to public health, five topics are commonly considered: infectious diseases, vaccination, antimicrobial resistance, noncommunicable disease and bioterrorism (fig. . ) , as well as the linkages between them. with few exceptions, migrants are not at increased risk of transmitting communicable diseases [ ] . however, infectious diseases can spread when new migrants live together in communal, close-quarter settings [ ] . according to a recent survey in eu/eea countries, screening for infectious diseases among migrants is currently directed towards predominantly human immunodeficiency virus (hiv), tuberculosis (tb), hepatitis b, hepatitis c, gonorrhoea, syphilis, measles and rubella, malaria and chagas disease. these diseases were selected because the european surveillance system (tessy) collects data disaggregated by migrant status or because evidence suggests that they may disproportionately affect migrants in the eu/eea [ ] . other diseases that could be considered include vaccine-preventable diseases, cholera, malaria, helminths and intestinal protozoa (semenza et al. ) [ ] . epidemiological data from bulgaria do not confirm the severity of tb, hiv and sexually transmitted diseases, but confirm the importance of the screening of parasitological diseases. a potential risk to public health is the possibility of outbreaks of vaccine-preventable diseases (vpd) in a population coming from countries where immunization coverage is low [ ] . vaccinations to consider among migrants include: measles, poliomyelitis, meningococcal disease, and diphtheria/tetanus/pertussis. the risk of the spreading of vaccine-preventable diseases among the local population should not be underestimated too. the varicella vaccination, which is not included in bulgarian routine vaccination programs because the majority of individuals in temperate climates develop natural immunity from previous infection before adolescence, is a good example, especially when taking into account cases registered in the harmanli refugee camp. other possible threats could be recognized in the cholera epidemic in iraq [ ] and poliomyelitis in the syrian/lebanon border refugee camp [ ] . population displacement can also threaten global vpd eradication and elimination efforts [ ] . antimicrobial resistance (amr) is not a disease in itself but a complication of the treatment of a disease. in situations such as the crowded settings with poor hygienic conditions found in refugee camps, infections can easily occur and spread; whether they are caused by resistant pathogens depends on their origin, which can be the environment, animals, food or humans [ ] . amr is becoming a global concern with amr strains associated with new resistance mechanisms emerging and spreading worldwide [ ] . the journey the refugees undertook, crowded conditions in refugee camps or settlements, and the lack of regular medical care, are prime drivers of the spread of amr among this vulnerable group especially in multidrug resistance tb cases among refugees [ , ] . the range of potential risks that can be associated with refugees is inevitably wideranging. it includes communicable diseases, trauma, dermatological disease injuries associated with the journey, environment, changes in climate, especially during the winter, and last but not least, mental health and psychological problems [ ] . who assessment in bulgaria reported on physical and psychological trauma, the consequences of post-traumatic stress disorder; dehydration, nutrition disorders and hypothermia; absence or interruption of treatment for chronic diseases. particularly high health risks are faced by vulnerable groups of migrants, including the elderly, people with disabilities, pregnant women and young children [ ] . last but not least, migrant's ncd treatment requires substantial resources that create additional economic pressure on the health systems of affected countries. there are multiple causal relations between (forced/irregular) migration and terrorism -but these are generally complex [ ] . while the link between terrorism and migration is widely discussed, where in the context of the migration inflow hypothesis immigrants are an important vehicle for the diffusion of terrorism from one country to another [ ] , the link between bioterrorism and migration is hardly found in official and scientific publications. however, bioterrorism as a risk and policy measures to address it is discussed in official documents of the bulgarian government, particularly in the annual report of the ministry of health, which names as one of its aims to 'protect the country from importation and distribution of infections with high epidemic risk by creating and maintaining mechanisms for timely and adequate response to health threats of a biological nature, including bioterrorism'. the eu also includes bioterrorism in the list of new threats but not specifically in the framework of migration but in the general context of cross-border threats: 'under eu law on cross-border health threats, existing mechanisms coordinate preparedness for serious cross-border threats to health, linking member states, eu agencies and scientific committees through the early warning and response system. the health security committee, which coordinates member states' responses to threats, may act as a focal point on vulnerabilities in public health, to enshrine hybrid threats, in particular bioterrorism [ ] . therefore, there is a wider recognition that migration and bioterrorism have to be dealt with in a broader institutional and policy context. despite these general considerations of bulgarian authorities and the eu health security committee, none of our past experience, including the data from our research, convinces us that there is a clear link between bioterrorism and migration processes. therefore, in the risk assessment, the likelihood of migrants being instrumental in a bioterrorist attack was assessed as very low (see table . ). our findings confirm who and dg sante expectations that migrants do not pose a significant risk to the local population. at the same time some specific, health risks should be taken into consideration and categorised by varying degrees of probability and impact. the risk assessment should be made by a wide range of specialists based on detailed logitudinal data. our assessment, based on the public data at our disposal, published and analysed in our article, is summarised in table . . the assessment of these risks may seem overwhelming or unrealistic to the external observer, but it seems realistic when considering the geographical location of the country, the institutional weakness and the fragile public health capacity. die getriebenen: merkel und die flüchtlingspolitik: report aus dem innern der macht cholera in iraq strains the fragile state does immigration induce terrorism? migration, refugees, and health risks refugees and antimicrobial resistance: a systematic review annual report on the situation of asylum in the european union annual report on the situation of asylum in the european union european asylum support office. annual report on the situation of asylum in the european union annual report on the situation of asylum in the european union latest asylum trends european centre for disease prevention and control. assessing the burden of key infectious diseases among migrant populations in the eu/eea. stockholm: ecdc joint framework on countering hybrid threats a european union response population movement is a challenge for refugees and migrants as well as for the receiving population vaccine-preventable diseases in humanitarian emergencies among refugee and internally-displaced populations health case study -bulgaria military medical academy the refugee crisis in the middle east and public health europe's migration challenges: mounting an effective health system response state agency for refugees with the council of ministers links between terrorism and migration: an exploration, icct research paper public health needs of migrants, refugees and asylum seekers in europe, : infectious disease aspects european parliament, briefing. martin seychell, deputy director general of the european commission directorate for health and food safety, the public health dimension of the european migrant crisis war and infectious diseases: challenges of the syrian civil war unhcr ( a) un agency for refugees. winter operations cell -daily report asylum trends : levels and trends in industrialized countries. office of the united nations high commissioner for refugees joint report on a mission of the ministry of health of bulgaria and the who regional office for europe migration and health: key issues antimicrobial resistance key: cord- -jckfzaf authors: walsh, patrick f. title: intelligence and stakeholders date: - - journal: intelligence, biosecurity and bioterrorism doi: . / - - - - _ sha: doc_id: cord_uid: jckfzaf this chapter underscores the need for more explicit and strategic engagement of stakeholders (scientists, clinicians, first responders, amongst others) by the intelligence community. the chapter argues that the intelligence community will increasingly rely on their expertise to build more valid and reliable assessments of emerging bio-threats and risks. however, the discussion also identifies some of the limitations and challenges stakeholders themselves have to understanding complex threats and risks. agricultural scientists and veterinarians) can all be critical stakeholders for intelligence communities. without them it would be almost impossible to see how the ic alone can fulfil its mission to identify, prevent, disrupt and treat potential and emerging bio-threats and risks. indeed as seen in chapter 'the scientific community' brings a lot of expertise to the intelligence community about how to assess bio-threats and risks in a number of different ways and contexts. these include understanding potential risks through gof experiments, the development of biosensors and knowledge about weaponisation, pathogenicity and transmissibility of various bio-agents. chapter also surveyed briefly the role of scientists working in epidemiology and forensics as providing central roles in the prevention, disruption and treatment of bio-threats and risks. additionally, chapter , highlighted the critical role the scientific community plays in helping the intelligence community better frame their understanding of potential threats and risks emerging from the fast paced changing biotechnology and synthetic biology sectors. this chapter provides a thematic analysis of how important stakeholders can contribute to reducing current and emerging bio-threats and risks. in contrast to chapter , which focused on what internally the intelligence community can do to better equip itself to manage bio-threats and risks, this chapter surveys what important external stakeholders can bring to the table to improve intelligence capability and to reduce bio-threats and risks themselves. paraphrasing research impact scholar mark reed's definition, i define a stakeholder of the intelligence community as any person, organisation or group that is affected by or can affect a decision, action or issue relevant to preventing, disrupting or treating bio-threats and risks (reed : ) . specifically, i am referring to stakeholders in the scientific, research, clinical, policy, first responder and private sectors that can provide capability, expertise to the intelligence community and/ or contribute to biosecurity through their own actions. in particular, the thematic analysis of the role of stakeholders in this chapter is organised around three sub-headings: prevention, disruption and treatment. traversing the literature and interviews with a select number of stakeholders shows there that there is a large and diverse number of individuals and organisations that could potentially play a role in either preventing, disrupting or treating future bio-threats and in the biological context, surveillance is the ongoing collection, analysis, and interpretation of data to help monitor for pathogens in plants, animals, and humans; food; and the environment. the general aim of surveillance is to help develop policy, guide mission priorities, and provide assurance of the prevention and control of disease. in recent years, as concerns about consequences of a catastrophic biological attack or emerging infectious diseases grew, the term bio surveillance became more common in relation to an array of threats to our national security. bio surveillance is concerned with two things: ( ) reducing, as much as possible, the time it takes to recognize and characterize biological events with potentially catastrophic consequences and ( ) providing situational awareness-that is, information that signals an event might be occurring, information about what those signals mean, and information about how events will likely unfold in the near future (gao : ). this definition highlights how the functions and roles of biosurveillance has changed from a more narrow concern of mapping disease in the public health sector to represent a diverse array of knowledge and capabilities that are vital in understanding bio-threats in the national security context. the definition also underscores the ongoing multiple challenges in improving bio-surveillance capabilities and their utility in the national security context. three key challenges in particular remain for improving national bio-surveillance capabilities and they are: methodological, information sharing and integration issues. the information sharing and integration issues have already been discussed in chapter so this section will focus on the bio-surveillance methodology issues. by methodological issues, i am referring to both the technical methods (biosensors) and the broader different disciplinary approaches to biosurveillance that now inform debates amongst stakeholders on how to improve bio-surveillance capabilities. from a technical perspective, there has been a range of bio-sensor research from inside and outside the ic to detect the release of dangerous pathogens into the environment. perhaps the most well-known of these initiatives-biowatch was developed by dhs in with the aim to detect aerolised bio attacks for high risk bioagents in major us cities. the program however, has had mixed success relating to the reliability of results and the delay in the publication of these once samples were collected from the field (gao (gao , . the dhs tried to speed up the detection times from the first generation manual systems to gen acquisitions, which promised speedier autonomous systems though testing difficulties remained. further analysis, however, of alternatives by the dhs as showing any advantages of an autonomous system over the current manual system were insufficient to justify the cost of a fully technology switch (gao : ) . in the us, research continues to improve the robustness, sensitivity, specificity, timeliness and cost of biosensor equipment. while conventional pcr based methods and immunoassay are still being used other biochemical, microbiological and genetic solutions are being trialled such as the incorporation of antibodies and peptide molecules, which may greatly reduce detection times to minutes instead of several hours (kim et al. ) . leaving aside efforts to improve aerolised biosensors, the expected rapid growth of synthetic biology and biotechnology and the potential (however unknown) that bioengineered material may be used maliciously in a way that threatens public safety or national security may shift the focus into other scientific research that can detect signals of bio-engineering including types of changes, location and possibly in the future where changes were made. in july , iarpa commissioned a new program-finding engineering linked indicators (felix) to meet such objectives. iarpa is seeking interest from a range of scientists (synthetic biologists, micro biologist, immunologist, statisticians and computer scientists) to carry out - research projects addressing the two main focus points of felix (eaves ) . if this research can produce reliable results, it will provide another useful collection and analysis point for the ic by allowing the detection of previously undetectable signatures of bio-engineered material in bio-criminal and terrorism cases. in addition to the various technical innovations in biosensors, a range of other bio-surveillance methods have been deployed. in the late s, the us cdc pioneered syndromic surveillance systems, which were initially aimed at improving the early warning of infectious diseases and bio-terrorism and have now evolved to include situational awareness (buehler et al. ) . similar syndromic surveillance systems have developed in other 'five eyes' countries such as the uk's real-time syndromic surveillance team (resst), which collects four national syndromic surveillance systems from several sources. additionally and more recently, the robert koch institute is creating an early warning system based on machine learning and natural language processing that will include 'appealing' interactive web applications and be linked to the german electronic reporting and information system demis (robert koch institute ). syndromic surveillance systems are a critical adjunct to traditional public health lab surveillance as they strive to provide real time or near real time collection, analysis and dissemination of health data to enable early identification and management of public health threats as they are not based on lab confirmed diagnoses-and assess a wider set of health related data including: clinical signs, absenteeism, pharmacy sales or animal health production collapse (buehler ) . a clear benefit of syndromic surveillance is it can be cheaper, faster and potentially more transparent then a state's public health lab surveillance system. however, as with the use of big volumes of data more broadly in the ic, data quantity, quality and structural variation all impact on the utility, accuracy and timeliness of some rapid epidemic intelligence from internet based surveillance methods (yan et al. ) . increasingly these syndromic surveillance systems rely on the use of big data, machine learning and analytics. additionally, web based epidemic detection systems like biocaster portal developed by the national institute of informatics in tokyo (collier ) and canada's global public health intelligence network (gphin) an event based surveillance system which looks at news feeds globally have also contributed to syndromic surveillance systems (mawudeku et al. ) . several event based internet surveillance systems have grown in number in the last decade. using pubmed, scopus and google scholar data bases, o'shea's study found based internet systems all using different technology and data sources to gather data, process and disseminate it to detect infectious disease outbreaks (o'shea ). in line with the broader ic development of exploiting social media analytics discussed in chapter , in dhs piloted another approach to bio-surveillance. the pilot involved dhs trialling various social media analytics from self-reported information on facebook and twitter to determine pandemics and acts of terrorism given social media feeds can provide close to real time reporting of symptoms, sickness access to hospital or pharmaceuticals (insinna ) . additionally, other private companies have entered the biosurveillance space-providing novel methods for capturing bio-surveillance data. wilson's discussion of how a private company (veratect corporation) assessed signal recognition in global media reports to provide warning on the emergence of the h n influenza pandemic shows how the ic warning culture methodology can be employed usefully along with what he described as the 'risk adverse forensically oriented response culture favoured by traditional public health practitioners' (wilson : ) . the veratect case shows that the private sector has a role in developing better bio-surveillance capability as well. as can be seen from the brief discussion above about different methodological approaches to bio-surveillance. there are also different views amongst bio-surveillance scholars and practitioners about the merits of each, particularly in their abilities to predict the 'next pandemic'. can for example, a national bio-surveillance system informed by one or more methods discussed above predict the emergence of the next pandemic or outbreak, particularly novel new viruses? some scientists argue that the prediction of a micro-evolutionary process of some biological agents such as a virus (i.e. a short term emergence or cross species transition) is incredibly difficult given evolutionary and epidemiological timescales are fundamentally different. geoghegan and holmes argue that instead it would be better to build surveillance capability that 'assesses the fault line of disease emergence at the human-animal interface, particularly those shaped by ecological disturbances' ( : ). others have argued differently. scientists working on the usaid funded predict and the global virome project examine disease hotspots globally in order to sequence (rather ambitiously) almost all the viruses in birds and mammals that could potentially spill over into humans. in particular, researchers working on the global virome project believe that prediction of which viruses might spill over from animal to human health is possible. geoghegan and holmes in response argue focusing on disease hotspots relies on very small amounts of data that can be unreliable given they are rare events. they give the example of saudi arabia which has not classically been a hotspot, yet mers recently jumped into humans from camels there. sequencing these viruses may provide useful evolutionary information, but geoghegan and holmes argue it won't necessarily provide early warning of what is going to affect us (geoghegan and holmes ) . other scientists are trying to change the ecology of disease, which presumably in some cases would make the early warning of some pandemics easier. in recent years, the scientific community has increasingly exploited crispr gene editing techniques to change the genetic makeup of malaria mosquitoes. additionally, advances in gene drives have recently been shown to change the ecological parameters of disease. gene drives are artificial 'selfish' genes that can force itself into % of an organism's offspring instead of the usual %. currently there is a global research effort funded by the gates foundation to cause female mosquitoes to become sterile within generations or year. the objective would be to release the genetically altered mosquitoes into malarial areas by (regalado ) . there are concerns by the fbi however that gene drives could be misused to create a 'designer plague' (ibid.). in addition to the 'predictability' challenges presented by various bio-surveillance methods, there are also differences in opinion amongst members of the bio-surveillance community about what an effective bio-surveillance system looks like. on what metrics can an 'effective bio-surveillance' system be evaluated given the multiple methodological approaches and systems that have developed for bio-surveillance? clinician and public health security specialist jim wilson has argued that the development of an effective global surveillance and response system is probably at least a decade or more away (wilson : ) . in the interim, we are left with multiple approaches of varying validity and reliability. so based on the current fragmented bio-surveillance efforts how do we learn the lessons that need to be learnt that will enable the implementation of the long awaited national bio-surveillance capabilities? how do we know if progress is being made to that goal? importantly, beyond national efforts, how do we assess the current capability of state, local agencies to contribute to a national bio-surveillance capabilities? where are the gaps and vulnerabilities in the current sub-national bio-surveillance and detection systems? (gao ) . compounding the current challenge of evaluating bio-surveillance capabilities in order to construct a viable national approach is that different bio-surveillance systems have been created for different end users (e.g. animal and human). the blue ribbon project report into animal health detailed information sharing challenges in animal health bio-surveillance and its integration with other bio-surveillance data including in human health (blue ribbon report : ) . this lack of integration makes it difficult to assess how information collected for animal or agricultural bio-surveillance could improve national approaches to bio-surveillance, particularly in scenarios where the emergence of disease could be an intentional or a malevolent act. different approaches to bio-surveillance have been informed by multi-disciplinary perspectives, which can be both a strength and weakness to developing a national perspective. current efforts across the 'five eyes' to develop fully national and integrated bio-surveillance capabilities remain works in progress and the political will to steward them into being seems insufficient. for example, in the us a program designed to provide a national bio-surveillance and integration system was eliminated in the president's budget request for fy (blue ribbon report : ). any evaluation of the effectiveness of various methods and approaches for building a national bio-surveillance capability also needs to consider how national efforts can both enhance and lever off global bio-surveillance capabilities. gaps and impediments in global biosurveillance have become increasingly evident to the world in the wake of the largest ebola epidemic ever-in which these challenges impacted the ability to prevent, detect, and respond. under the looming threat of mers-cov, leishmaniasis, influenza, multidrug-resistant tuberculosis, and plague, the global public health community now realizes the urgent need to address shortcomings in global bio-surveillance and the broader public health security system. properly preparing for the next major outbreak hinges on our willingness to transform global health surveillance systems and those of countries with fragile health infrastructures (shaikh et al. : - ) . in some respects, similar challenges in developing national bio-surveillance capabilities exist in those at the global level including: siloed systems, inadequate training and technical expertise, different information and communication technology (ict) standards, concerns over data sharing and confidentiality, poor interoperability, and inadequate analytical approaches and tools. there is likely not one bio-surveillance method, technique or tool that is going to detect in real time disease outbreaks, particularly unusual ones which might imply malicious intent. a fully integrated approach to bio-surveillance may rely on more than one method or capability which together can provide reliable and valid bio-surveillance data and early warning at the national and global level. it may mean investigating ways that older legacy systems can be integrated or at least made interoperable with newer more mobile platforms such as mobile or wireless health technologies particularly in the developing world (shaikh et al. ) . it should be clear by now that improving bio-surveillance capabilities is essential to improving the prevention of natural and suspicious outbreaks of disease. it is important for the 'five eyes' intelligence and law enforcement communities to understand broadly the theoretical and practical developments in bio-surveillance so that they are able to more effectively lever relevant knowledge on bio-threats and risks. a second cluster of stakeholders that are useful in the prevention of bio-threats and risks (both natural and malicious) are those working in national, regional and global health. the ebola epidemic ( ) ( ) was a recent reminder of the consequences of weak public health capability and infrastructure in failing to prevent, identify and respond quickly to infectious disease. the ebola epidemic also had a catalytic effect on many public health authorities, practitioners and researcher's views about the capability of the traditional un response to global health crisis mainly coordinated through the who. many public health watchers are now arguing the need for a broader more effective focus-not just on prevention and response to infectious disease, but one that also included reframing the focus as a human security issue. adherents to this view make a compelling point when seen through the ebola case that continues to have significant impact on the economic and social stability of countries impacted (sparrow ; marston et al. ; who ; mmwr ) . beyond west africa, similar vulnerabilities in capabilities such as diseases surveillance, detection, contract tracing, clinical care, community engagement and communications exist globally as was also seen with the proliferation of zika in latin american/caribbean and mers in the middle east. in , the commission on a global health risk framework for the future that met after the ebola crisis estimated . billion per year investment would be needed for better detection and response tools. the same commission report also estimated that the economic cost for global pandemics per year was $ billion (schnirring ; dzau and sands ) . effective national bio-surveillance relies on not only what 'five eyes' countries can do to improve the scientific and technical capability of bio-surveillance, but also how they can improve bio-surveillance globally particularly in at risk areas. beyond effective bio-surveillance, effective prevention of pandemics whether natural, accidental or malicious relies on good global (multilateral), regional and national public health responses. there are several multilateral instruments, institutions and initiatives that are relevant, but i will focus here on what have become the key ones rather than attempting to traverse in detail all major international health initiatives struck since / . they include who international health regulations (ihr), un security resolution , the global health security agenda (ghsa), the biological weapons convention (bwc) and the australia group. the who international health regulations ( ) entered into force in june to prevent, protect against, control and provide a public health response to the international spread of diseases (detect, assess, notify events has a biosafety and biosecurity function) and includes all members of the un. the ihr has improved accountability of countries about progress towards building national core public health capability targets in several areas including, but not limited to: surveillance systems, creating rapid response teams, border management. however, the ihr annual reporting process has been by self-assessment of core capacities to the world health assembly (wha) by all state parties, which has resulted in incomplete or not credible reporting for some member states. the commission on global health risk framework for the future also expressed concerns over the self-assessment monitoring tool of the ihr, because questions are binary (yes/no) answers and recommended that who devise a regular independent mechanism to evaluate country performance against benchmarks (ghrf commission : ). for example, a country can 'tick yes' for having a national public health legislation, but other dependent legislation (biosecurity, food safety, environmental health) may not be in place-thereby reducing overall the country's ability to manage health crisis or for the global community to understand and respond to capability and information gaps in that country (ibid.). some countries continue to be slow or uneven in their reporting of ihr ( ) attributes. in , one study showed that the african region was well below global averages across all attributes measures with no african state reporting full implementation (kasolo et al. : - ) . the second multilateral instrument relevant to our discussion here is the un security council resolution ( , which calls on all states to prohibit non-state actors from developing, acquiring, manufacturing, possessing, transporting, transferring or using nuclear, chemical or biological weapons and their delivery systems. more importantly and specific to bio-threats only, the bwc has historically played the most significant role in preventing the weaponisation of biology. the bwc was established in and seeks to prohibit the development, production, acquisition, transfer, stockpiling and use of biological and toxin weapons (gerstein ; chevrier and spelling : - ) . in , there was an attempt by some member states to introduce a verification process, but this was vetoed by the us following inspection of soviet sites under the tripartite agreement between the soviet union, usa and the uk. the us arguing it could be difficult to certify that a state's biological program was merely defensive rather than offensive. the us also had concerns that inspection to labs could be disruptive or provide opportunity for industrial espionage against legitimately operating biotechnology companies (gerstein : ) . historically there has been a mixed record by some 'five eyes' intelligence countries in assessing verification and therefore noncompliance of the bwc. koblentz surveyed the role of intelligence (particularly humint) in assessing the former soviet union's offensive bio-weapons program between and which resulted in an incomplete picture of moscow's program (koblentz : ) . additionally, as discussed in chapter , in several 'five eyes' intelligence communities (us, uk and australia) incorrectly assessed that iraq had a mobile offensive bio-weapon capability. intelligence collection on its own can either over or under-estimate such capabilities. between yearly review conferences, several initiatives and activities have been introduced (confidence building measure, meetings of experts, information exchanges) to improve the effectiveness and the implementation of the convention. however, state parties are only encouraged to implement relevant national legislation and other measures to prohibit prevent the development, production, stockpiling or transfer or use of bio weapons. how they precisely undertake measures is at the discretion of individual state parties. the bwc has been criticised for several reasons over the years. some of this is warranted, while other criticisms seem to not take into account that the bwc is different from its chemical and nuclear counter proliferation counterparts. as gerstein argues, 'material is the centre of gravity for nuclear discussions and intent being the center of gravity for biological issues' (gerstein : ) . developing nuclear weapons leaves a large recognizable footprint, whereas the development of an offensive biological weapon requires virtually no specialised equipment (ibid.). the first major criticism of the bwc is that it has no verification mechanism or any other mandatory provisions for monitoring compliance. a second complaint is that for many years (until ) , it lacked an implementation capability to help states fulfil their obligations. since , the convention has had a small three team implementation support unit (isu) based in the united nations office for disarmament affairs in geneva which aims to 'assist, coordinate, and magnify the implementation efforts of the states parties to help states parties help themselves' (lennane : ) . in reality though, the isu does not have 'capacity for analysis and coordination other than for the collection of the annually submitted confidence building measures, posting them to the website and organising and attending conferences' (gerstein : ) . historically there has also been a low number of party states submitting their annual confidence building measures. although the bwc isu was able to report that a record number ( ) annual confidence building measures were submitted in , this only represented . % of all state parties submitting that year. though the trend line seems to be going up from a low in of (bwc newsletter : ). a third criticism of the bwc is that it has moved slowly since inception and further questions remain about its relevance strategically and operationally in preventing bio-threats and risks into the future. such questions are likely fundamental to its long term viability. however despite shortcomings, the bwc has nonetheless created a normative institution for reducing the risk of biological or toxin weapons being used or developed by state and non-state actors (lennane : ) . more importantly, as developments in biotechnology continue at a pace, the bwc does provide a venue, where the security implications of dual-use technology can be assessed which will be critical in 'mitigating these emerging threats' (gerstein : ) . the bwc still does have an important role in reducing weaponisation of biology in the future, though its poor funding particularly of the isu means that other multi-lateral measures are needed to amplify the work of the convention. in addition to the above historic/traditional proliferation arrangements of the bwc, other international regimes have been implemented such as the australia group (established in ) and the proliferation security initiative (established in ). both have a broader counter proliferation objectives beyond biological weapons to chemical and nuclear. the australia group member countries have collaborated on the development of lists of technologies and materials that could be used in the development of chemical and biological weapons. member countries then commit to monitor the export or transfer of these materials. the australia group maintains common control lists for dual use bio-equipment, technology, software, bio agents and plant and animal pathogens as the basis for promoting common standards and regulations (australia group common control list handbook ). the australia group works in concert with the bwc. the psi was a bush administration initiative that sought to supplement existing non-proliferation regimes, but seeks to enforce these by interdicting and seizing illegal weapons or missile technology in planes or ships carrying cargo. the psi also includes intelligence sharing and joint operational activity (national institute for public policy ). turning the focus slightly away from multi-lateral counter proliferation measures, other multilateral initiatives have focused on improving global health security. in some respects the ghsa provides a bridge between traditional, narrow security approaches to biological weapons and a wider securitisation of global health. the ghsa was established in by the obama administration and is a multi-sectoral approach to global health security seeking to include governments, international organisations and non-government organisations. ghsa was set up in part to 'advance further the ihr implementation through focused activities to strengthen core capacities and to ensure a world safe and secure from global health threats posed by infectious disease; where we can prevent or mitigate the impact of naturally occurring outbreak and intentional or accidental releases of dangerous pathogens' (heymann et al. (heymann et al. : . ghsa is a refreshing approach not only because it seeks to establish a global framework and capacity to assess, measure and sustain advances in global preparedness for epidemic threats, but it also addresses biosecurity as a public health priority-thereby linking public health and health security, development, defense and agricultural sector (cameron ) . the underlining logic of ghsa suggests that the same attributes needed to prevent, detect and respond to deliberate use of a bio agent are those required to manage a natural or accidental outbreak of a biological agent. ghsa also includes technical targets aligned to three areas: prevention, detection and response (heymann et al. (heymann et al. : . like earlier initiatives, such as the us sponsored global health initiative (ghi), which was discontinued by the obama administration in due a lack of financial and technical authority to leverage and coordinate multiple us agencies-the ghsa will need to secure ongoing funding beyond from major donors including the us. at a november ghsa ministerial meeting in uganda, assembled governments signed onto an extension of the ghsa for another five years. us secretary tillerson had issued public support for continuing it, but at the time of writing no commitment by the us for future financial support (beyond fy ) has been made. ghsa holds promise, but in addition to ongoing funding challenges, those member states signed up to it will need to ensure effective governance is in place to align funding to global health priorities articulated by the who, world bank, imf and other donors in order to avoid duplication and promote an effective approach to international health security capabilities (paranjape and franz ; . in summary, this discussion of multilateral security and global health initiatives demonstrates that there is a diverse number of stakeholders working in these sectors, which can play a role in preventing biothreats and risks-whether they are natural pandemics or a malicious attack from a biological weapon. it's clear that the 'five eyes' intelligence communities have worked extensively with other member states in counter-proliferation institutions such as the bwc and the australia group for several decades, but what remains still under developed is how global health security stakeholders and intelligence communities can work more collaboratively for the mutual goal of global health security regardless of whether the risks are natural pandemics or result from a bio-terror attack or theft of a dangerous select agent from a lab. more trusting and formalised contact between both global health security stakeholders and those working in the security and intelligence communities can only be mutually beneficial to preventing major bio-threats and risks. the final cluster of stakeholders that can help prevent bio-threats and risks are of course those that specialise in biosafety and its promotion in their research institutes, biotechnology companies, universities and medical facilities. promoting biosafety in environments that work with select agents and other facilities that work with less dangerous material which can still cause harm relies on consistently high risk management practices. in all 'five eyes' countries there has historically been in place biosafety risk management procedures and practices to prevent accidental infection, accidental release, or intentional misuse of biological substances. however, as noted in chapter in the last two decades the expansion in synthetic biology, biotechnology and biological science research has meant there are now more people working in more locations on dangerous pathogens-not just in well-regulated liberal democracies such as those in the 'five eyes' countries, but also in developing countries; where biosafety and biosecurity capabilities and practice may be less established such as parts of africa, the middle east, pakistan and former soviet states (gronvall et al. ; shinwari et al. ) . just in terms of the scale of this expansion of facilities working with dangerous pathogens-in the us alone, there is thought to be thousands of bsl labs and in china the number of such labs is increasing too (nature editorial : ). the us and other 'five eyes' countries such as canada have invested in cooperative engagement programs since / in several former soviet union states. the us defense threat reduction agency (dtra) has lead efforts in georgia to reduce bio-risk by securing/consolidating pathogens, training scientists in biosafety and biosecurity technology, regulation and detection. likewise, the cdc has been involved in building public health capacity there as well as in armenia and azerbaijan (bakanidze et al. : ) . as important as building biosafety capacity is in developing countries, it is clear that much more still needs to be done to build biosafety capacity in 'five eyes' countries-including finding better ways to understand and manage comprehensively threats and risks in the biosciences environment. biosafety experts such as salerno and gaudioso argue for more comprehensive risk management systems across the global bioscience community 'to avoid an accident that jeopardizes the entire bioscience enterprise' (salerno and gaudioso : xv) . their argument is that such a system would supplement existing national and international biosafety regulations by risk managing fully at an organisational and unit level every single potential incident rather than by generic risk hazard assessments that are currently done by most facilities today (ibid.: ). others have also called for more systematic tools and approaches for managing biosafety incidents in labs dealing with particular dangerous pathogens such as marburg virus (dickmann et al. ) . still others have argued that while 'security awareness is high among employees who work with biological select agents and toxins, it is not pervasive across the entire life research community' (grphyon scientific : . such a statement does not seem to be hyperbole if one looks at some of the cases of biosafety and security lapses since / (gao (gao , . there have been several lapses at cdc between and . in june , dozens of workers in cdc could have been potentially exposed to live anthrax that hadn't been killed before being shipped from cdc's bioterrorism rapid response and advanced technology (brrat) bsl to a bsl lab in its bacterial special pathogens branch. cdc investigations determined that at least cdc staff members may have been exposed to viable anthrax cells or spores though no illness or deaths occurred (cdc ). the same report found several breaches of biosafety process and procedure including failures of policy, training, supervision, judgement and even scientific knowledge (ibid.). similarly, biosafety lapses cases involving cdc labs occurred in january when an unintentional cross contamination strain of low pathogenic avian influenza a (h n ) with a strain of highly pathogenic avian influenza a (h n ) was shipped from cdc to the usda (schnirring ) . further biosafety breaches were detected in july -this time at the national institute of health campus in bethesda maryland; where viable smallpox vials were discovered improperly stored (dennis and sun a ). an additional five improperly stored vials were also found at the nih-three were select agents (burkholderia pseudmomallei, francisella tularensis and yersinia pestis ) (dennis and sun b). in the nih cases despite their age, they were still viable organisms which could have caused illness. their theft could have also posed a bio-threat and risk to the community. then after a hiatus where biological material was suspended being sent between bsl and bsl labs live transfers commenced again. after a further internal cdc review (cdc a, b) some additional safety measures were put into place, however there was a subsequent lapse when a specimen of chikungunya virus was shipped from a high secure lab in fort collins to a lower level one which had not been killed (young ) . similarly, in the pentagon shipped live anthrax spores from the dugway proving ground in utah to states and one international location that were also meant to have been killed (burns ) . it was later found that dugway and the us dod had been shipping nationally and internationally live anthrax for more than years-often without adequate safeguards. other reports suggested that some samples were sent by federal express (sisk ) . similarly in november , the us hhs discovered that a private lab had 'inadvertently sent a toxic form of ricin to one of its training centres multiple times since putting training staff at risk' (gao : ). similar biosafety lapses have occurred in the uk resulting in investigations since of government, university and hospital labs (sample ) . as noted in chapter , one possible bio-threat and risk pathway could be the theft of biological substances or information from a biosciences institution. lapses in biosafety arrangements demonstrate, at least in some cases, biosecurity vulnerabilities that could make the theft or even infiltration of a threat actor into high containment lab easier. thefts from labs have occurred in the past by an insider, and a motivated insider can compromise biosafety for a range of reasons. bunn and sagan's edited book insider threats provides a useful taxonomy for thinking about 'insider threats' (bunn and sagan ). they can be: self-motivated insiders, who at some point decide to become a spy or thief. insiders can also be recruited insiders, who are already inside an organisation, but become convinced to become part of a plot. finally, an infiltrated insider might be associated with some adversary of the organisation and join it with the purpose of carrying out a malicious act against it. bunn and sagan also refer to inadvertent or non-malicious actors, who pose a threat by making mistakes without really intending to do so-such as leaving a password lying around. finally, the authors refer to a 'coerced insider', who remains loyal in intent, but knowingly assists in theft or sabotage to prevent hostile acts against themselves or their loved ones (ibid.: ). the insider threat that was posed by bruce ivins' activities in a high containment lab (that resulted in amerithrax in ) demonstrates the potentially high threat and risks associated with an insider. the ivins case provides a useful case study in how an organisation's security procedures and other organisational and cognitive biases can miss for several years risks posed by an insider threat actor (stern and schouten : - ) . since the amerithrax incident, significant investment has been made to close the biosafety vulnerabilities revealed by it. increasingly since / and amerithrax, a number of policies, procedures and normative behaviour have developed in the scientific community to promote biosafety and biosecurity. these have ranged from safety regulation codes such as the us biosafety in microbiological and biomedical laboratories (bmbl ) to more formal legislative and oversight regulations. the latter will be addressed in chapter . there are also technical and policy improvements that can be made in securing both physical and remote access to labs including computer systems that house data, which are at risk of theft or being hacked (gryphon scientific : berger : - ; slayton et al. : - ) . leaving aside discussion of some of the formal legislative and regulatory instruments for promoting biosafety, the development and maintenance of effective risk management across the biosciences also relies on an organisational culture that treats biosafety and biosafety as an equal priority to other deliverables. a culture of accountability at all levels must also exist if effective risk management can prevent, identify and treat bio-threats and risks promptly. a rogue insider threat, who may have been assessed as appropriate to work with select agents and seems initially to follow all the relevant biosafety regulations and procedures could still pose a risk if they have not embraced the organisation's normative cultural biosafety values. it is critical then in order to stop opportunities for insider threats, that the organisation promote relevant biosafety cultural values as much as and perhaps more than adherence to formal biosafety regulations. risk management measures must of course be measured against the ability of scientists to carry out its functions. effective engagement with local law enforcement and relevant domestic security intelligence organisations in each 'five eyes' country to help scientists build viable biosafety cultures will likely remain important in addition to internal organisation biosafety initiatives. stern and schouten provide a number of useful suggestions for improving policies and procedures that may help improve biosafety cultures across the biosciences enterprise ( : - ). two that i think would be helpful are, one: developing standard operating procedures for proactively identifying vulnerabilities including using 'red team' exercises to explore how systems could become exploited. in other words, what motivators (financial, psychological, religious, and political) might drive an insider threat and are there ways to assess the signs of such an evolving threat? the other is to 'ensure personnel reliability programs incorporate ongoing assessments of counterintelligence vulnerabilities, including vulnerabilities to self-ascribed whistle-blowers or attention seekers' (ibid.: ). effective biosafety and biosecurity training is also crucial as the number of labs working with select agents or other dual use bio-agents proliferate globally, particularly in locations with fragile states. more consistent approaches to training will also be important so nations can be confident that as many scientists as possible regardless of the country or the context in which they work understand what bio-risks and threats may emerge and how to prevent or mitigate against them (sture et al. ). as discussed above there are multiple stakeholders in the scientific community, global health security and biosafety fields that can play a critical role themselves in preventing bio-threats and risks as well as supporting the operational efforts of the intelligence community to prevent these. while prevention of bio-threats and risks is one critical dimension that stakeholders can play central roles another is disruption. although the intelligence community can use a range of knowledge, technologies and methodologies from stakeholders in the scientific community, to prevent bio-threats and risks, we have to accept that it will not be possible to detect every criminal or terrorist act. nonetheless, some of the techniques, practices, technologies and knowledge available from stakeholders in the scientific community will still be useful to disrupting bio-threats and risks. in other words prevention may not always be possible yet measures can be put into placewhich can detect threats early enough to reduce their impact. similar to preventing bio-threats and risks, disrupting them will also rely on seeking advice from stakeholders involved in bio-surveillance, public health and biosafety research, amongst others on disrupting them as well. for example, as discussed earlier iarpa's commissioning of research into detecting signals of bioengineering changes (felix) may result in better capability for the intelligence community in not only preventing bioengineering changes that make it easier for terrorists to carry out attacks on populations, critical infrastructure or biotechnology companies, it could also help detect and disrupt the planning stages for such attacks. additionally as noted earlier, if a high containment lab has a strong biosafety culture it is more likely that disruption of a biothreat may be possible just by colleagues speaking up about suspicious activities in their working environment rather than any elaborate disruption knowledge and techniques, procedures the intelligence community might have in place to disrupt such threats. but knowledge, technologies, techniques and practice for disruption of bio-threats and risks cannot just come from scientific stakeholders in the biosciences, it should also come from other fields and practitioners working in other areas where successful disruption operations has taken place. these areas include criminology, policing, engineering, legislation, cyber, counter-intelligence amongst others. in this section, we examine briefly what other stakeholders and discipline perspectives might the intelligence community learn from that can provide better capabilities for the disruption of bio-threats and risks. are there lessons to be learnt from other stakeholders, disciplines or even other threat contexts that might be relevant to disrupting biothreats that might not have been initially detected? since / , there are three stakeholder and discipline groups, which are investigating and applying disruption strategies to threats and risks and their knowledge might be relevant in disrupting threats and risks in the bio context. these are criminology, counter-terrorism and cyber. we will explore each briefly to see how stakeholders (researchers and practitioners) have developed disruption strategies in each and how they might be employed against bio-threats and risks. insights from criminology and the practical application of disruption for crime prevention has provided a supplementary approach to traditional law enforcement approaches of prosecution against certain crimes through the courts. disruption is not a new concept in criminology and law enforcement practice, though it can be difficult to define in all law enforcement contexts (ratcliffe : ) . its meaning at least in the criminology/policing/law enforcement contexts can partly be traced back to broader desires-initially by uk law enforcement followed later by other 'five eyes' countries in the late s and early s to move law enforcement away from its traditional reactive mode to offending to one driven by intelligence. this concept of law enforcement or policing being intelligence driven or led gained significant traction in the criminology and policing literature (walsh ; ratcliffe ; innes and sheptycki ) . it was driven initially in the uk by the desire for governments to maximise efficiencies and reducing costs by increasing the use of intelligence to drive strategic and operational decision-making. the implementation of intelligence led policing models into operational policing across 'five eyes' countries has had mixed results partly due to cultural, financial and leadership issues in agencies that have attempted to put intelligence at the centre of strategic and operational decision making in policing (walsh ; ratcliffe ) . nonetheless, despite historical challenges in adopting intelligence led approaches, increasing fiscal constraints and the ever increasing demands on law enforcement in managing both high volume crimes and complex operating environments in counter-terrorism, cyber and organised crime meant, at least in many national law enforcement agencies; a greater demand for an intelligence driven approach (walsh ) . this intelligence driven approach, which promulgated proactive disruption of crime strategies was in part an admission that not all crime could be prevented or the offenders prosecuted. additionally, in many law enforcement agencies such as the australian federal police (afp), the growing volumes of information collected have given intelligence a more central role in triaging the significance of information, value adding to it and guiding investigators to targets and operations that are high priority; or have the greater likelihood of successful prosecution outcomes. in complex organised crime cases such as transnational drug trafficking, people smuggling and even terrorism and cyber threats, which we discuss shortly-intelligence driven disruption strategies have become increasingly popular for many 'five eyes' law enforcement agencies. this has particularly been the case where it can be difficult to dismantle completely the organised crime group-or to even know the full extent of the group's network. disruption operations that attempt to take down threat actors with key roles (e.g. facilitator, financier, and logistics) may nonetheless reduce the threat posed by the organised crime network even if the network continues to exist. additionally, with some organised crime networks, it may be difficult to secure sufficient evidence for prosecution against a more serious offence such as drug importation, but there may be sufficient intelligence that can be used to make the criminal environment more hostile for the group's illicit enterprise by arresting key group members for lesser offenses such as unexplained wealth or migration irregularities. while disruption of crime does seem like a useful tool in preventing or reducing the impact of offenders, the criminology literature demonstrates it has been difficult to evaluate the effectiveness of intelligence driven disruption strategies. ratcliffe cited an rcmp disruption attributes tool, which attempts to examine where the disruption activity is aimed at (core business, financial, personnel) and whether the kind of disruption for one or more of these attributes is high, medium or low in impact (ratcliffe : ) . however, such tools are largely subjective and qualitative-making it difficult to accurately measure the impact of intelligence driven disruption measures. the other concern about disruption strategies is that they may just cause displacement, where other criminal enterprises take the place of those removed by law enforcement or as innes suggest, 'disrupting a network may just provide a vacuum for more dangerous offenders to step in' (innes and sheptycki : ) . finally, the literature suggest that employing effective disruption strategies rely on proactive collection and valid analysis that can led to both timely strategic and operational outcomes that in turn result in threat mitigation and harm minimisation. so are there benefits for the intelligence community working on bio-threats and risks to investigating research and practice for disrupting threats in the organised crime context? the answer is a qualified 'yes'. much of course depends on the nature of the threat and risk posed. clearly as with any crime, it is hard to disrupt a bio-threat, when it's still in the head of the offender. however, we do know that criminal and terrorist acts don't just happen spontaneously. there usually involve predicate steps taken by the offender. some of these might happen in very compressed periods while in other offences planning may take years. either way, and regardless of whether these can be detected by the intelligence community, there is likely to be some signs in the predicate planning stages of an impending threat/risk that can provide the intelligence community opportunities for disruption. it is difficult to say in which bio-threat cases disruption strategies will be most successful. much will depend on how quickly the intelligence community can collect and analyse information that may be indicative of an evolving bio-threat and risk. as discussed previously, good collection and analysis is contingent on having robust core intelligence processes in place and more importantly effective intelligence governance. both are needed to ensure intelligence efforts are coordinated across multiple internal intelligence community stakeholders, with relevant knowledge-as well as ensuring information and expertise from external stakeholders (the scientific community) is available to provide earlier warning signs of an emerging bio-threat. while it is important not to over-play the potential for success of the kind of disruption strategies used against traditional organised crime groups, there are likely bio-threat scenarios where disruption strategies may make a difference. arguably, disruption of bio-threats could be on a continuum with the individual threat actor on one end and a sophisticated organised group on the other. at the individual level one could have the scenario of a lone terrorist actor or a mad/bad scientist. while it may seem difficult to get early warning of the malicious act of mad/bad scientist, we saw in the earlier discussion on 'insider threats' that it may be possible to disrupt their activity before you reach an amerithrax style attack. twenty/twenty is hindsight with the bruce ivins amerithrax case, but the lessons learnt from this incident do provide guidance on the sources of collection and analysis required from within the intelligence and scientific communities to aid the disruption of this kind of bio-threat. it does not mean that all similar cases of 'insider threats' will be detected, prevented or disrupted, but a more careful collection and analysis of 'odd' behaviour or unusual security lapses by a scientist working in a high containment lab could reveal areas of vulnerabilities. detection both of abnormal changes to an individual's psychological profile and/or in their working environment can provide opportunities for those vulnerabilities to be disrupted. at the other end of the bio-threat scale, a more organised bio-criminal or terrorist planned event may resemble in some respects other illicit criminal markets and networks (drugs, identity fraud, money laundering) and thereby present opportunities for disruption. again this is not to suggest that disruption of organised bio-threat scenarios will be always be possible. as discussed in earlier chapters, since / , even with state based wmd programs the intelligence community has had a mixed record in detecting them and uncovering the intention and capability of non-state actors to exploit dual use technology for malicious end remains difficult. however, disruption could be useful in some bio-crimes where there is a bigger network of actors involved in the illicit business. for example, in crime scenarios where food suppliers are not registered legally to import food into a 'five eyes' country because it poses a biosecurity risk, there may be opportunities for parts of the intelligence community (particularly national law enforcement agencies) to work with agriculture, animal health, food regulatory agencies and relevant scientific stakeholders to disrupt illicit food suppliers from a country of concern. equally there may be opportunities for disruption of activity from non-compliant biotechnology providers in a 'five eyes' country, who provide dual use equipment to a company overseas with a questionable profile that resides in a country vulnerable for terrorist infiltration. in addition to useful knowledge that can be gained from criminology and law enforcement practice there are also perspectives on disruption from contemporary counter terrorism studies that may have utility in the bio-threat and risk context. as noted above, since / law enforcement agencies across the 'five eyes' countries have been increasingly deploying disruption strategies in countering terrorism given the preservation of life demands an earlier interception of attacks preferably at the planning stage. as innes suggest in the case of counter terrorism operations, one aim is to overtly disrupt planned attacks, which has many effects including sending a message to other terrorist groups that they may be next, reassuring the community and if possible deploying countering violent extremism (cve) strategies in communities where future attacks may arise (innes et al. : ) . in the uk in particular, a key plank in its counter terrorism strategy has been disruption both at the strategic and tactical level. at the strategic level, disruption has involved a number of initiatives from arresting persons of interest, legislative action and enhanced surveillance (innes et al. : ) . in addition to global influence of groups such as al qaeda and islamic state, the growth in lone actor attacks-some across the us and european countries from s to late s (danzell and montanez : ) has also been a significant catalyst for enacting further stringent legislative measures such as detention without trial and control orders (walsh ). all 'five eyes' countries have also adopted further legislative changes that allow disruption of terrorist attacks by reducing thresholds law enforcement and intelligence agencies need for reasonable suspicion in order to access both electronic and human intelligence (humint). governments desire to do something to reduce the threat and risks posed by terrorists by creating increasingly proactive, flexible and permissive legislative environments has also raised concerns about the role of intelligence, secrecy and privacy. these issues will be discussed as they relate to the bio-threat and risk context in chapter . but legislation is only one plank in effective counter terrorism and the scale and pace of actual and potential terrorist attacks suggest other disruption strategies are required at the tactical level. innes et al. suggest such strategies might include: 'prosecution against an individual or a network for offences other than those they were principally being investigated for and/or interfering with the operations of the criminal enterprise in cases where there is insufficient evidence to secure prosecution ' ( : ) . they add that, at the tactical level, disruption strategies can 'interfere with the ability of suspected adversaries to operate effectively and efficiently' (ibid.). innes et al. suggests that tactical disruption functions at 'near event interdiction', which can mitigate or minimise harms associated with the actual or planned terrorism attack (ibid.). other counter-terrorism disruption strategies in 'five eyes' countries have included the creation of cve policies and interventions as well as the disruption or take down of social media venues advocating politically motivated violence or recruitment to jihadist groups. regardless of the complexity of post / terrorist attacks-such as the multi-site attacks in paris orchestrated by a group; or the knife attack against two police officers in australia in by one individual-disruption strategies employed by law enforcement and national security intelligence agencies are also likely to be usefully employed in the bio-threat and risk context. just how useful strategic and tactical disruption strategies used in conventional counter-terrorism will be in the bio-threat context depends on the nature of the intent and capability of individual threat actor(s) and the risks posed by their actions. the effectiveness of disruption strategies in the bio-threat context like conventional terrorist attacks are contingent on a range of variables that are unique to that event. in the bio-threat context, leaving aside large levels of uncertainty about the future threat trajectory for bio-terrorism, effective disruption will rely on law enforcement and intelligence agencies understanding how the intention, capability and opportunities of threat actors operating in a particular environment-make an attack possible. intention, capability and opportunities will differ along the threat continuum from individual to group and from state to non-state actor. for example, in the research facility, hospital or high containment laboratory environment, intention, capabilities and opportunities may be shaped by actors that are internal, external or an indirectly involved in the facility (perman et al. : ) . threats can also be as perman suggest overt or clandestine (ibid.). in some cases, if a scientist is motivated politically (for religious, environmental or political reasons) to commit an act of violence by using a biological agent it may be easier to disrupt their activities if they are public about their agenda. however, in the case of a clandestine plan it could be very difficult to disrupt an attack launched externally or internally in a contained lab. nonetheless, as we saw with historical cases of lone actor threats such as the bruce ivins amerithrax incident there are likely predicate steps in the process to carrying out an attack which are revealable. similarly, in the lesser known case of dr. larry ford, who was suspected of murdering his business partner in a biotech company-the police subsequently found a cache of weapons, white supremacist writings and allegations that he attempted to infect six mistresses with biological agents (perman et al. : ) . again even in cases of lone actors such as this whose attack planning is more clandestine; there may well be an abundance of 'warning intelligence' that if collected and assessed in time might be useful in disrupting a lone actor planned attack. while it can be difficult to disrupt a lone actor plot, more elaborate ones by a group of conspirators could in some circumstances provide greater opportunities for interception and disruption by law enforcement and intelligence agencies. this is because in plots involving multiple actors there are more stages before the attack can be carried out. some stages such as communications, procuring supplies and transport also provide points of vulnerability, where threat actors can be exposed to authorities and disrupted. so an external threat such as a terrorist attack against a high containment laboratory might involve communications amongst group members, financing of the plan, purchasing of explosives and surveillance of the facility's perimeters. each stage presents opportunities for disruption providing intelligence and information is available to law enforcement and intelligence agencies. similarly a theft of intellectual property or biological material from a private sector biotechnology company might result from either an external criminal group; or state actor pressuring or paying an employee to steal information on their behalf. again, intelligence may exist already about the criminal group or the compromised employee that provides opportunities for disruption. in an ideal world of course, it would be desirable if all potential biothreat and risk scenarios could be prevented early in the intent stage, where they are mainly an idea in a perpetrator's head. pre-employment screening, including criminal checks and select agent risk assessments will show up some individuals, who are not suitable to access and work with dangerous biological agents. this will have an early disruptive effect but it is not fool proof. people can lie about their circumstances in security suitability checks allowing them the ability to access and plan malevolent acts in a secure biological facility rather than just thinking about them. once operating inside a facility-depending on the nature of the planned attack it can be very difficult for law enforcement and the intelligence community to respond quickly enough to disrupt the attack before its fully implemented. in all threat scenarios (simple to complex) in addition to the mandatory background checks for workers, each scientific institution needs to develop a full suite of threat assessments that can be updated regularly on different threat actors, including but not limited to: visitors, criminals, lone actor attacks (internal and external), terrorist and issued motived groups, international terrorists groups and foreign powers (perman et al. : ) . these threat assessments should be developed by an institution's internal security department in collaboration with local law enforcement. the relatively low number of threat scenarios that have taken place involving bio-agents since / will likely mean that there will be many intelligence gaps in assessing the intent, ability and opportunity of different threat types. however, providing baseline threat assessments will begin to build pictures of threats scenarios that should help promote better biosafety measures as well as opportunities to disrupt threats earlier should they begin to emerge. in summary, law enforcement and intelligence agencies working on bio-threats and risks of the future can learn a lot from their counter terrorism colleagues. since / , countering terrorism continues to produce lessons for the law enforcement and intelligence communities on how more effectively to disrupt emerging terror plots before they are implemented. the knowledge gained from investigating conventional terrorism attacks that don't involve biology can help those working on future bio-threats and risks by seeing how to optimise the legislative, intelligence, investigative and community response to terrorism while also learning lessons from contemporary counter terrorism efforts. in particular, the increase in lone actor terrorist attacks in the westoften with short notice underscores that either an insufficient amount of intelligence or types of intelligence that cannot be revealed in court often exists. in these cases, other tactical disruption strategies are gaining traction amongst 'five eyes' countries to mitigate the threat and harm posed by terrorists. similarly, given the complexity of threat scenarios that could arise from the exploitation of dual use biotechnology, it may be difficult in some cases to collect sufficient solid 'evidence' or use bio-forensics to attribute confidently for a conviction on bioterrorism or bio-criminal activity. nonetheless, the various counter terrorism strategies discussed above point to ways threat actors may be disrupted on lesser offences while also providing a greater intelligence dividend on other individuals involved. the final knowledge area and stakeholder group that intelligence agencies and investigators working with bio-threat and risks may learn more from is cyber security. as koblentz and mazanec ( ) suggest there are a lot of common characteristics between biological and cyber weapons including but not limited to: difficulty of attribution and how multiple technologies can be used for offensive, defensive and civilian applications ( - ). both authors argue because of these similarities there is likely a lot cyber can learn from how bio-threats have been managed historically. this is undoubtedly true, though in this section the focus will be the opposite-i.e. what can intelligence and investigative agencies working on bio-threats learn from the cyber threat and capability landscape? even a cursory review of the literature suggest that there are a number of areas where current cyber research and practice could inform the 'five eyes' intelligence communities understanding of current and emerging bio-threats and risks. space does not allow an exhaustive discussion on all of them, but there are three cyber areas in particular; where i believe those working with bio-threats and risks could benefit greatly from knowing more about in order to learn the lessons from the cyber context as well as identifying good intelligence and investigative practice. these areas are: the dark web, cyber terrorism and cyber espionage. i will discuss each briefly in turn. turning to the dark web environment first here we are referring to the content on the internet that is 'not indexed by standard search engines' (weimann : ) . much of the dark web is hidden or blocked and can only be accessed by specialised browsers. given the relative anonymity it provides, the dark web has seen the proliferation of child pornography, credit card fraud, identify theft, drugs and arms trafficking amongst other illicit offences. the dark web only emerged in recent years though law enforcement and intelligence agencies have made some in roads into its penetration and disruption. the fbi's shut down of the dark web site silk road, which operated between february and october was to that point the largest and most sophisticated anonymous online market place for illicit drugs (zajácz ) . new technological solutions are also being developed to better identify, collect and analyse illicit activity on the dark web, including darpa's memex software, which helps catalogue dark web sites (weimann : ) . nonetheless, all 'five eyes' intelligence communities will need to continue to develop their collection, analytical and investigative capabilities in the dark web content to profile more accurately various illicit market places in order to orchestrate impactful disruption activity across multiple markets. although it is unknown, at least in an unclassified sense the extent to which illicit markets exist that could benefit bio-threat actors (criminals or terrorists), undoubtedly law enforcement and intelligence agencies, who are given a watching brief on emerging bio-threats and risk should be exploiting the dark web more for opportunities for disruption. a first step might be first to map the bio-terrorism literature and identify researchers, who have access to bioterrorism agents/disease research, domain, institutions, countries and emerging topics and trends in bioterrorism agents/disease research. chen shows how by using informatics research it might be possible to use knowledge mapping techniques, to analyse productivity status, collaboration status and emerging topics in the bio-terrorism domain (chen : - ) . additionally, other intelligence and investigative teams that are working on non-bio threats such as conventional terrorist attacks, terrorism financing, drug trafficking or even child sexual exploitation may come across offenders, who have links to others interested in exploiting dual use biological agents for malevolent objectives. so the work currently going on by intelligence agencies working on broader cyber security issues such as cybercrime or cyber terrorism is directly relevant to improving collection and analysis against emerging bio-threats and risks. developments in the second area cyber-terrorism provides another opportunity for bio-threat intelligence and investigative teams to learn off their colleagues working on cyber threats. in the past we often think about the classical 'bio-terrorism' attack involving the aerolising and dispersal of a dangerous pathogen like anthrax into a crowded place. this mode of attack may still be chosen in the future by a terrorist group (leaving aside for a minute the technical difficulties of such an attack). though committed acts through cyber opens up other choices for a bio-attack. cyber security specialist's knowledge of cyber terrorism is still developing. we have seen for example groups like the taliban and is increasingly use computers for recruitment, propaganda and communications, but it remains difficult to know empirically how many of the current virtual attacks such as ransomware can be attributed to terrorist or led to deaths or impacted critical infrastructure in significant ways. such attacks could just as easily be attributed to cyber hackers (criminals) or state sponsored espionage both issues we will return to shortly (riglietti ; bernard ; heickerö ) . nonetheless, it is clear that terrorism groups are increasing their use of computers including the dark web given they know that intelligence communities are monitoring the surface internet and social media. in august , al-aan tv reported a laptop belonging to a tunisian member of is captured in syria contained thousands of documents from the dark web including pages about making biological weapons in a way to impact the biggest number of people (weimann : ) . there have also been cases where is has carried out a series of cyber-attacks, 'exclusively computer based, which in one instance even led to the disclosure of private information regarding us government officials, from private conversations to work and email addresses' (riglietti : ) . the final area of cyber security that is useful for bio-threat intelligence and investigative teams to reflect on relates to cyber hacks and espionage. putting hacks and espionage together is not meant to suggest that both are always linked-though we have seen in the russian interference in the us presidential election they can be. china too is playing an increasingly sophisticated and aggressive cyber espionage strategy aimed at political interference and stealing intellectual property (inkster ) . there seems little doubt that the extent of hacking (unauthorised access to a computer or network) being perpetrated by state and non-state actors is on the rise and network vulnerabilities across the civil and military space remain. in a recent article, fbi assistant special agent in charge (chicago), todd carroll said the average time between an unauthorised user getting inside a network and the user being detected is days-'a lifetime in cyber means'. todd went on to say that % of business owners don't have a dedicated employee or vendor monitoring for cyber-attacks (stone ) . we have also seen in recent years the growth in malware and ransomware attacks across the globe. for example, in the wannacry ransomware attack caused , infections across countries (locking down banking, energy and manufacturing systems) (schilling ) . the dark web also provides terrorist and criminal groups opportunities to operate botnet campaigns in anonymity that can remotely operate networks of computers to commit attacks on other systems including critical infrastructure. again there is insufficient space to provide a full survey of all the cyber hacking and espionage threats, and indeed what to do about them is beyond the scope of this chapter (clarke and knake : - ) . nonetheless the hacking attacks-whether they are state sponsored (espionage) or non-state actors (terrorists or criminals) provide another rich source of knowledge to be collected and assessed that can be used by those working on emerging bio-threats and risks. for example, it would seem unwise for bio-threat intelligence and investigative teams to not learn from the fast changing angles of cyber-attack from hackers given how the physical security of biological institutions, their intellectual property and the kinds of biological products produced in such facilities is reliant on secure cyber systems. we have seen in recent years the take down of government websites involving ransomware attacks on both government and private sector networks. increasingly more information is being shared and stored via the cloud. what would be the impact of a major ransomware attack that locks down the entire bio-surveillance capability of a public health authority such as cdc do to maintaining national health security? could a cybercriminal group infiltrate the network of a major biodefense company steal ip and sell it to a terrorist group on the dark web? could research stored via the cloud on non-secure networks relating to the genetic sequences of pathogens be stolen by a terrorist group or state actor to engineer bio-weapons? (blue ribbon project : - ) . in all the three areas discussed above, a fuller development of links between those working in the cyber intelligence collection and analysis streams, and those who might examine emerging bio-threats and risks is a necessary first step in bringing relevant knowledge and practice from cyber security to bio-threat stakeholders. in this final section the attention is turned to what kind of stakeholders play a role in treating bio-threats and risk? second, in performing these roles, how can they help the 'five eyes' intelligence communities build better capability (knowledge, practice and technology) about treating actual or emerging bio-threats and risks? as we have seen so far the management of bio-threats and risks is potentially a crowded enterprise with many stakeholders (beyond the intelligence communities) playing critical roles. in this section, i have grouped them into three 'types of stakeholder': first responders, science and technology stakeholders and security stakeholders. these are not three distinct clusters of unique stakeholders that do not interact with each other. depending on the nature of the bio-incident that has occurred, one would expect to see a close interaction amongst the various knowledge brokers and practitioners from each group. for example, a release of a synthetically manufactured select agent in an airport should result in the combined strategic and tactical contributions from first responders, engineers and security personnel rather each being delivered in isolation. an uncoordinated delivery of knowledge, practice and expertise to treat an unfolding bio-threat/risk from multiple stakeholders will not result in the best outcome for mitigating the risk or disrupting future potential of similar threats occurring. again as with previous sections, the focus here is not a deep exploration of the specific knowledge, practice or technology of all stakeholders involved potentially in the treatment of bio-risks. this would be an impossible task. instead this section will explain briefly what each of the three broad stakeholder categories (first responders, science and technology and security) can do broadly to treat bio-risks (current or potential), what intelligence communities can learn from this in ways that extend their capabilities to manage bio-threats and risks. the label 'first responders' is a descriptor for a much broader range of stakeholders including: fire/hazmat, paramedics, emergency responders, health and hospital service providers. each would play a different role in both responding to and treating a bio-incident depending on the type of biological hazard, their jurisdictional and legislative responsibilities and fiscal capacity. in all 'five eyes' countries with perhaps the exception of new zealand (with a smaller population and only one national government) the complexity of response will be particularly governed by the overlapping roles that various local, state and federal first responders might play. obviously in the us with multiple federal, state and local agencies, the coordination of first responder efforts to a bio-incident presents more challenges than other 'five eyes' countries such as australia and the uk with less agencies and jurisdictions. there is not an abundance of academic literature on the role of first responders in treating bio-threats and risks. this lack of evidence makes it difficult to assess accurately what first responders can do to treat bio-threats and risks, what the challenges are and what the intelligence community can learn from these important stakeholders. there is however, some research available that can increase the intelligence communities' understanding of first responder capabilities to treat bio-threats and risks as well as illuminate some of the challenges in doing so. this research should provide at least a start to what the intelligence community can learn from first responders as they deploy their knowledge and practice to disrupt and treat bio-threats and risks. / and the amerithrax incident provided a catalyst for law enforcement and public health agencies to work closer together to respond to an unfolding threat. since amerithrax, across the 'five eyes' countries further work has been done to better coordinate the work of law enforcement and public health agencies on treating bio-threats and risks. but such efforts have not involved routinely the broader spectrum of national security intelligence agencies, who have tended to play a more strategic and adhoc role compared to their law enforcement counterparts. overall, policy, coordination and legislative efforts to bring first responders and members of the intelligence and law enforcement community together have had only mixed success for a number of reasons. in , a study of how law enforcement and public health agencies in the us, canada, uk and ireland work together on bio-threat incidents identified several common barriers to improving multi-agency responses (strom and eyerman ) . these included cultural, legal, structural, communication and leadership barriers (ibid.: ). ten years on from strom and eyerman's research, other researchers have made similar observations about the ability of first responders to manage effectively a bio-threat incident and to work with law enforcement and intelligence community on such tasks. but it's not just the capability issues raised above, other research points to other technical challenges to treating the impact of bio-threats and risks in the physical environment. for example, research by chemists and environmental engineers show that given the varying nature and strains of the bacteria-the science for assessing risk of exposure may not be able to provide a fully accurate risk assessment of a building's vulnerability or resilience to a bio-attack nor-in some cases whether first responders have effectively 'cleaned the environment up after exposure' (canter ; taylor et al. ) . a lack of effectiveness in responding to a biothreat incident in a local area obviously can have broader public sector implications in both treatment and preparedness of bio-risks. for example, gerstein ( : ) citing a study by advocacy group trust for america's health reported that states and dc scored / or lower on a scale for preparedness. additionally, since / major disease outbreaks such as sars and ebola have also demonstrated fragility in parts of the world, including some 'five eyes' country's public health response capability, which remains a concern if there was a major bio-terrorist event. the blue ribbon study project report raised similar concerns about the capability of certain responders including those local, state and federal agencies that might be involved in decontaminating sites following a bio-incident. in the us, the report raised similar coordination issues between federal, state and local agencies in which first responder agency would take the lead in decontaminating and remediating environments and how other agencies would get involved to ensure the attack site was deemed safe for people to return (blue ribbon study project : ). one underlying theme arising from the studies mentioned on first responder's roles in treating bio-threats and risks is that the intelligence community must share more information with emergency services on the nature of the threat they are meant to respond to. this is not to suggest that in all the 'five eyes' countries that no sharing is going on. my selected interviews with law enforcement and intelligence officials in each country did not give the impression that no sharing was going on with first responders. however it is clear if the local fire officers or emergency staff in a hospital are meant to better respond to a bio-incident they will need regular, consistent, reliable, real-time information and intelligence. this is vital to them safely securing the scene, or rapidly diagnosing and treating infected patients while also keeping themselves safe. importantly too, the more intelligence they receive will likely be helpful in first responders preserving any relevant evidence from the scene that might be needed by the either the law enforcement and intelligence communities. gerstein makes a valuable point when referring to improving bio-preparedness and response activities, when he suggests that first responders need to be seen as part of a complex system rather than each representing a series of programs (gerstein : ) . in addition to the range of knowledge and practice the intelligence community can learn from first responders, arguably the biggest lesson they can learn is to seek to better understand the 'linkages among disparate disciplines (biodefense, public health, emergency management), government, industry, the scientific community and themselves to better support first responders' (ibid.). if the 'five eyes' intelligence communities were able to create the necessary national health security coordination arrangements suggested in chapter such as the health security coordination council and the national health security strategy, then through these institutions further intelligence sharing mechanisms could be established to improve information flow between the intelligence communities and first responders at federal, state and local levels. however, first further research is required to investigate how law enforcement and intelligence communities work currently with first responders to identify and as much as possible ameliorate the cultural, legal, communication and leadership barriers that persist. a second cluster of knowledge and stakeholders for treating bio-threats and risks could be loosely described as 'science and technology' stakeholders. in earlier sections, under the relevant headings (prevention and disruption), significant space was devoted to how our intelligence communities can learn from a range of stakeholders working across a diverse array of disciplines (including bio-surveillance, public health, biosafety, criminology, counter terrorism and cyber). in each of these disciplines, discussion included exploration of relevant science, technology and knowledge useful for the intelligence community in preventing and disrupting bio-threats and risks. some of that discussion, for example bio-surveillance, biosafety and strengthening global health is also relevant to our focus here in treating bio-incidents. however, in this section the focus is not what the intelligence community can learn from stakeholders working in the above disciplines, but rather what they can learn from disciplines more removed from the biological sciences or relevant social sciences (e.g. engineering or security studies). what can the intelligence community learn from physical, mechanical or environmental engineering? there are multiple roles engineering specialties could play and are playing in preventing, disrupting and treating bio-threats and risks. for one and historically, the us dod has relied on engineers, microbiologists to provide advice on weaponisation of biological agents under a range of scenarios and conditions (state actor and terrorists threats). for example, even pre / , between and dtra funded project bacchus to see if a team of scientists and engineers, who allegedly did not have extensive experience in bio-weapons could make bio-weapon facility using just commercially available items. the objective was to see if the team could make anthrax successfully without the detection of the intelligence community, though it was later revealed that this team did have substantive technical knowledge and support throughout this project (vogel : - ) . engineers have also long been engaged in studying aerolisation dynamics, which has become increasingly a multi-disciplinary collaboration of environmental engineers, biomedical engineers, microbiologists, chemists and epidemiologists (xu et al. ) . related to aerolisation studies has been the work of hardware and software engineers-many of whom came from the aerospace and automotive industries that have brought their skills into modelling bio-terrorism attacks to help first responders predict how airborne particles might move through sections of a city under certain weather and windflow conditions (thilmany ) . other engineering studies, sometimes referred to bio-protection studies have been important in the design of the heating ventilation and air conditioning (hvac) systems used to resist biological contaminants. much of this research became activated after the amerithrax incident, and is designed at reducing the health consequences from airborne contaminants by augmenting heating and air conditioning systems (ginsberg and bui ) . another focus of engineering led research relates to improving the portability, speed and reliability of bio aerosol monitors for pathogens. one recent study has been working on a device that would be fully portable and automated-capable of detection of selected air-borne microorganisms on the spot-within to minutes depending on the genome and particular strain of the organism (agranovski et al. ). in this last sub-section in our exploration of what other stakeholders may be useful in treating bio-threats and risks we turn our attention to the role of security officers. i am conscious in the discussion above regarding prevention and biosafety much was said about the role of security officers and managers in promoting biosecurity and biosafety across all sectors of the bio-sciences enterprise (e.g. research centres, hospitals, biotechnology companies, public and private labs). in this section, we focus instead on the role of security officers and managers across the broader economy-beyond biosciences. as argued in previous chapters, in addition to taking a one health perspective to bio-threats and risk, 'five eyes' intelligence communities and their law enforcement colleagues need to also understand the potential development of biothreats and risks beyond the technical world of biotechnology and labs to include also in their wider social, economic and community contexts. hence in this section, we are referring to the role of security officers and companies that work across the international, national, state and local economies in each 'five eyes' country. given the trajectory of most (if not all) future bio-threats is unknown, our intelligence communities need to be forging more formalised (less adhoc) relationships with security officers in a range of non-biotechnology industries (banking, mining, food supply, agriculture, critical infrastructure). as nalla and wakefield ( ) argue several factors have increased the role of private security since the second world war. increased economic wealth, enhanced security technology (alarms, access control and cctv), in addition to an increase in the control by a number of private sector companies of publicly accessible places have, amongst other factors all contributed to the growth in private sector security (ibid.: ). while it is difficult to generalise 'as the functions of security officers/agencies are as varied as the organisations that employ them' (ibid.: ), their functions and roles cut across many facets of each 'five eyes' nation to include office buildings, warehouses, shopping malls, education establishments, residential complexes and critical infrastructure. one often thinks of the classic scenario of a security guard standing in front of a physical gate, which is one role of many others which might also, depending on their functions include traffic control, surveillance, responding to emergencies, security vetting. in the security role of complex large companies, airports and electricity plants, it is likely that the security officers will have a deep understanding of their physical and virtual security environments and this kind of expert knowledge would be integral for them and the intelligence community gaining threat awareness, prevention, surveillance, disruption, treatment and recovery to bio threats and risks which may manifest in their operating environment. historically however, the relationship between intelligence communities (including law enforcement) and private sector security has not been optimal partially because a lack of trust between both (ibid.: ). however, several studies on private and public sector security do show several areas of improvement across each 'five eyes' country. some of these improvements have been initiated by governments such as in the uk making significant cuts to policing in the late s and mid- s and seeking the private sector security sector to pick up more cheaply what were considered less core policing such as offender management and transfers of prisoners. in other cases, governments were interested in engaging with the private sector to extend their own security and intelligence collection capabilities with terrorism. connors et al. ( ) , wakefield ( ) , and rigakos ( ) provide more detailed analysis of a range of factors that have been involved in building partnerships with private sector security companies in the us, uk and canada respectively. / and of course subsequent terrorist attacks in many western countries has seen a more focused attempt by 'five eyes' countries to reach out to the private sector-including private sector security given many attacks occur in public places owned or managed by the private sector. threats as well to public and privately owned critical infrastructure (aviation, power, water, and telecommunication) have also influenced 'five eyes' government's closer liaison with the private sector. for example in the us, dhs has established a private sector office to provide government advice on relevant security issues to the private sector as well as promoting public-private partnerships. in australia, since / parts of the australian intelligence community, particularly asio has developed closer links with the private sector. in australia's attorney general's department created the business-government advisory group on national security to provide a vehicle for the government to discuss a range of national security issues and initiative with ceos and senior business leaders (dpm &c : ). the group later ( ) evolved into the australian governments industry consultation on national security (ibid.). more recently ( ) the australian government released its strategy for protecting crowded places from terrorism. this significant policy document was developed in close partnerships with federal, state and local governments, the intelligence community and the private sector. the key objective being to assist owners and operators to increase the safety, protection and resilience of crowded places across australia (anzctc ). an interesting aspect of this strategy is that it places the primary responsibility for protecting sites and people on private sector businesses. similar policy articulations have been declared in the uk's counter-terrorism strategy (hmg ) and canada's approach to counter-terrorism (canadian government ). in summary, it's clear that various agencies of the 'five eyes' intelligence communities and their broader law enforcement counterparts have increased their liaison and implemented various initiatives with private sector industry. what is less clear is the nature and extent of these as they relate to the prevention, disruption and treatment of potential bio-threats and risks. much is unknown, for example, about whether intelligence and law enforcement communities are actively working in partnership with the private sector beyond the classical threat typologies of basic terrorist's tactics, improvised explosive devices or vehicle born attacks. given the low probability high impact nature of the evolving bio-threat environment, it is likely that many private sector companies (banking, shopping malls, mining, hotels) see little need to include bio-threats in their security risk management plans or indeed consult with intelligence and law enforcement communities on them. while it is important not to be alarmist on low probability threats that are more likely on balance to effect the biosciences community rather than the broader private sector economy, it seems unwise for the latter not to consider the impact of such bio-threats on their operations and to at least have formalised dialogues on these with the intelligence community. but such a dialogue will in the future rely on several factors identified already by researchers coming together to develop more effective public-private crime prevention strategies. prenzler and sarre list several factors including: a common interest in reducing a specific crime, leadership, mutual respect, information sharing based on high levels of trust in confidentiality and formalised mechanisms for consultation and communications (prenzler and sarre : ) . this chapter surveyed the role of external stakeholders external (to the 'five eyes' intelligence communities) in preventing, disrupting and treating bio-threats and risks. depending on the particular bio-threat a diverse array of stakeholders could provide knowledge, skills and capabilities to the intelligence community. the large number of disciplines and stakeholders with relevant technical knowledge suggest that they will continue to play a critical role in the prevention, disruption and treatment of bio-threats and risks. in many cases, such as in biosurveillance, forensics and even engineering the scientific and technical stakeholders discussed here may play a greater role than the traditional intelligence and investigative response to managing bio-threats and risks. the chapter also highlighted that although each 'five eye's intelligence community has a wealth of knowledge to tap into from stakeholders, however in most cases all stakeholder groups are faced with their own theoretical and practical limitations. analysts and investigators working on bio-threats and risks need to understand these limitations while also seeking to build deeper and more formalised partnerships with scientific, technical and cross disciplinary stakeholders. in the final chapter , we shift the focus away from the practice and processes involved in interpreting bio-threats and risks to oversight and accountability issues. given the legislative, ethical and normative challenges modern intelligence practice faces, particularly in understanding the potential threat trajectory of synthetic biology, what role can oversight and accountability play in achieving the objectives of the intelligence communities in liberal democracies? miniature pcr based portable bioaerosol monitor development australia's strategy for protecting crowded places from terrorism. anzctc, australian government biological weapons-related common control lists biosafety and biosecurity as essential pillars of international health security 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months of the epidemic and forward projections signal recognition during the emergence of pandemic influenza type a/h n : a commercial disease intelligence unit's perspective. intelligence and national security utility and potential of rapid epidemic intelligence from internet-based sources labs cited for 'serious' security failures in research with bioterror germs silk road: the market beyond the reach of the state key: cord- - caevjvh authors: falanga, annarita; galdiero, massimiliano; morelli, giancarlo; galdiero, stefania title: membranotropic peptides mediating viral entry date: - - journal: pept sci (hoboken) doi: . /pep . sha: doc_id: cord_uid: caevjvh the means used by enveloped viruses to bypass cellular membranes are well characterized; however, the mechanisms used by non‐enveloped viruses to deliver their genome inside the cell remain unresolved and poorly defined. the discovery of short, membrane interacting, amphipathic or hydrophobic sequences (known as membranotropic peptides) in both enveloped and non‐enveloped viruses suggests that these small peptides are strongly involved in breaching the host membrane and in the delivery of the viral genome into the host cell. thus, in spite of noticeable differences in entry, this short stretches of membranotropic peptides are probably associated with similar entry‐related events. this review will uncover the intrinsic features of viral membranotropic peptides involved in viral entry of both naked viruses and the ones encircled with a biological membrane with the objective to better elucidate their different functional properties and possible applications in the biomedical field. significant improvements have been achieved in recent years in the understanding of the multiple alternative ways of virus entry into susceptible cells. [ , ] the strategies employed by viruses to enter cells are different according to the presence or absence of a lipid bilayer surrounding the virus. enveloped viruses present a membrane bilayer while non-enveloped viruses lack this membrane and present on their surface only capsid proteins. the mechanism of cell invasion by the two groups of viruses is rather diverse and the foremost difference is the direct consequence of their distinctive physicochemical state at the interface that occurs at the time of the encounter between the virus and the cell membrane; in particular, two lipid membranes confronting each other in the case of enveloped viruses as opposed to a layer consisting only of proteins that face a lipid layer in the entry of naked viruses. enveloped viruses entry exploits direct fusion with a cellular membrane through the involvement of specialized viral fusion proteins, present on the viral membrane and the consequent transfer of the nucleocapsid into the cytoplasm. [ ] on the other hand, the entry of non-enveloped viruses, which lacking the outer viral membrane are unable to take advantage of the cellular mechanism of membrane fusion, involves the activation of viral lytic factors that induce cell membrane rupture. [ ] the major features in membrane interaction by enveloped and non-enveloped viruses are reported in figure . notwithstanding the different mechanisms of penetration, the key step is the entry process and the modification of the cell membrane which allows the viral genome to penetrate into the host cell and start the replication cycle in the appropriate cellular compartment. [ , ] the most critical barriers for viral penetration and replication are the plasma membrane, the cytoplasm, and any other membrane that needs to be crossed in order to have access to the sites where viral replication or assembly take place. the overall picture of the mechanism of viral entry is becoming increasingly complete; in fact, depending on their dimension and structure, they have acquired different strategies to penetrate and take control of cell functions. [ , ] the molecular details of the interactions at the interface of virus and cell surfaces are quite complex and highly variable, but there is a common idea that only a limited number of pathways allowing viruses to reach the sites of penetration exist, with enveloped and non-enveloped viruses presenting different and unrelated processes, but with general principles driving all fusion events. the main difference between fusion peptides of enveloped viruses and lytic factors of non-enveloped viruses is the fact that fusion peptides promote membrane fusion while lytic factors promote membrane disruption. in this review, we summarize current knowledge on the mechanism of membrane fusion of both enveloped and non-enveloped viruses with a focus on common principles. fusion peptide derived from enveloped viruses and lytic peptides from non-enveloped viruses are described with an effort to find undisclosed differences and similarities. we conclude reporting ground breaking applications of membranotropic peptides (both fusion and lytic peptides) which open a new exiting field of research. enveloped viruses depend on membrane fusion for cell penetration. [ ] the two main routes used by enveloped viruses to enter the cell are the endocytic and non-endocytic pathways. [ ] most enveloped viruses undergo endocytosis while only few are able to fuse directly to the plasma membrane. [ ] viruses that use the endocytic route for cellular internalization are able to escape into the cytosol avoiding lysosomal degradation. therefore, penetration invariably involves membrane fusion mediated by specific viral glycoproteins (catalysts) with the main difference being that viruses using endocytic pathways fuse their envelope with the endosomal membrane from the luminal side. [ ] thus, membrane fusion constitutes the essential and ubiquitous mechanism of entry of enveloped viruses, irrespective of their route of entry. [ , , ] viral fusion proceeds through a hemifusion stalk with merging of proximal leaflets, which culminates in the opening and expansion of a pore connecting the two sides of the membrane. [ ] the overall process is supported by a catalyst responsible of the lowering of the transition barriers and fusion proteins constitute the catalytic agents fulfilling this function. [ ] the entry involves several other critical steps which include cellular receptor or co-receptor binding, internalization, uncoating, and release of viral nucleic acids at the proper site of replication. [ , , ] various factors can mediate an efficient interaction between cells and viruses; for example, cholesterol rich domains are platforms for the entry of many enveloped viruses such as the influenza virus [ , ] and many viral membranes contain much more cholesterol than the mammalian plasma membranes from which they are derived. [ , ] viral fusion proteins undergo significant rearrangements from the pre-fusion to the post-fusion conformations which are triggered by either receptor binding, proteolytic cleavage or low endosomal ph, and eventually determine the exposure of previously sequestered hydrophobic peptides, loops, or patches, able to interact with and destabilize one or both the opposing membranes. [ , ] crystallographic data on pre-and post-fusion structures of viral fusion proteins has resulted in the identification of at least three distinct classes based on their three-dimensional organization and mechanism of fusion ( figure ). [ , ] class i fusion proteins includes many of the most studied human pathogens such as influenza virus, human immunodeficiency virus (hiv), and ebola virus; the key features is the requirement of a proteolytic cleavage to initiate fusion, [ ] activating these proteins which form an extended intermediate on the surface of the virion with the fusion peptide at the n-terminus of the protein engaged in the interaction with the target membrane and forming a link between the two fusing bilayers. the pre-hairpin structure is a unique state in which the fusion protein is simultaneously connecting two distinct membranes, the target membrane through the fusion peptide or patches and its own viral membrane through its transmembrane domain (tm). further conformational changes produce trimers of hairpins with a central a-helical coiled-coil structure. [ , ] the -helix formation is related to the opening of the fusion pore and provides the major driving force for the process. thus, peptides able to prevent -helix formation, interfering with refolding of the fusion glycoproteins act as potent viral entry inhibitors. [ , ] class ii fusion proteins, which include flaviviruses, alphaviruses, and bunyaviruses, are consistently different from those of class i and are mainly composed of b structures. [ ] these fusion proteins are often heterodimers or homodimers lying almost flat on the virion surface. [ ] they are organized in three globular domains: domain i is char- class iii fusion proteins have a mixed secondary structure with the central a-helical trimeric core similar to class i and two fusion loops located at the tip of an elongated b-sheet similar to class ii fusion proteins; the main representatives of class iii fusion proteins are protein g of vesicular stomatitis virus (vsv), [ , ] gb protein of herpes simplex virus (hsv), [ ] and of epstein-barr virus (ebv), [ ] and gp from insect-cell baculovirus. [ ] the post fusion structure is characterized by the presence of an internal fusion peptide in domain i organized in two hydrophobic fusion loops flanked by a b-sheet domain with a pleckstrin-like fold (domain ii). domain ii is nested with the largely a-helical domain iii, which is composed of trimers that give rise to an elongated, rod-like shape molecule. the a-helical domain is inserted in domain iv which is made of b-sheets and a very long c-terminal extension (domain v). interestingly, the multicomponent herpesvirus fusion machinery requires the presence of gb, gh/gl, and gd and multiple cellular receptors are engaged in the entry pathway in a cascade of molecular interactions. [ ] gh/gl and gb are part of the core fusion machinery and need to cooperate in order to trigger the initial lipid destabilization which culminates in the fusion of the two bilayers. [ ] [ ] [ ] despite the fact that it is still debated whether gh is merely a fusion regulator or it plays a more direct role in the fusion process, studies leave little doubt that also the gh/gl complex undergoes dynamic rearrangements during the fusion process. [ ] the crystallographic post-fusion structure of gb shows that it is a canonical class iii fusion protein; [ ] and several synthetic peptides derived from gb induce the fusion of large unilamellar vesicles and inhibit herpes virus infection. [ , , ] irrespective of their structural differences, the three classes of fusion proteins seem to induce membrane fusion by essentially the same generic mechanism and a common refolding pathway is highly suggestive for the conservation of several phases of the process. the non-enveloped viruses exploit a different mechanism for entry because they lack a membrane which surrounds the protein capsid; as a consequence they require capsid-dependent mechanisms for penetrating the cell membrane or for exiting from the endosome. [ , ] the entry process is much less known and involves a series of triggers producing conformational and structural rearrangements which end in the exposure and/or release of lytic factors. low ph, receptor interactions, protease cleavage, chaperone-assisted morphological changes, divalent cation chelation, or any combination of these factors which take place at the appropriate site of membrane penetration are essential triggers to produce the activated viral intermediate. [ ] the mechanism of membrane disruption involves proteolytic cleavage of the coat protein and programmed exposure/release of small membrane-lytic peptides. in this scenario, viral penetration is mediated by short, membrane interacting, amphipathic and/or hydrophobic sequences present in proteins undergoing a conformational modification which allows the exposure of these domains and their interactions with membranes. [ ] similarly to enveloped viruses, capsid proteins seem to be trapped in a metastable state waiting for a trigger to expose their membrane active peptides and the membrane penetrating ability of these sequences is fundamental for entry but at the same time their premature exposure has to be avoided before host cells provide the triggers. flock house virus (fhv) is one of the simplest and most studied non-enveloped viruses. the infection starts with the binding to one or more host cell receptors leading to a receptor-mediated endocytosis mechanism. [ ] the receptor binding induces a conformational change that initiates uncoating or viral disassembly once the particle is exposed to low ph within the endocytic pathway blocking this event often inhibits infection. the immature provirion fhv is initially assembled from copies of the coat protein alpha and, subsequently, it undergoes autoproteolytic cleavage to generate the mature infectious virion which contains a large n-terminal fragment, b ( amino acids), and a small c-terminal fragment, g ( amino acids). [ ] the capsid shell is constituted by the central region of b forms, while the g peptides are the amphipathic helices non-covalently associated with the capsid interior. the g peptides are responsible of the interaction with the membrane and its local disruption which ends in viral entry. these peptides are composed of an n-terminal amphipathic helix separated by a proline-glycine-proline turn from a hydrophobic c-terminal region. the amphipathic g helices are located in the interior of the capsid and when fhv enters cells through receptor mediated endocytosis, the g peptides are exposed and determine the disruption of the membrane with consequent release of the viral genome in the cytosol. [ ] this is a dynamic process with g peptides continuously, transiently, and reversibly exposed to the exterior of the capsid; g peptides may not only be exposed but also released from the virus particle which may represent a common paradigm of non-enveloped virus entry. in this dynamic process g peptides may continuously "sample" the environment until they encounter the appropriate cellular trigger; at this point the virus undergoes an irreversible conformational change in which the g amphipathic helices insert into the target membrane, allowing the viral rna to enter the cytoplasm. [ ] | m em b ra n otrop i c p ep ti d es many studies are devoted to the comprehension of the mechanism of insertion of fusion peptides, loops, or patches into monolayers inducing nipple formation and curvature in the target cellular membrane through many synergic interactions. the insertion in one leaflet of a closed bilayer will cause the increase of the surface area of the leaflet and the formation of a spontaneous curvature, which is one of the driving forces to reduce the energetic barrier needed for the achievement of fusion. [ ] fusion and/or membranotropic domains in viral fusion proteins contain aromatic residues which together with alanines and glycines [ ] contribute to the interaction with just one bilayer leaflet. many biophysical and structural techniques using synthetic analogues and model membranes have been used to determine physiologically relevant states during membrane partitioning. [ , ] the two structural motif widely found in fusion proteins and able to produce membrane curvature are the amphipathic a-helix and tilted peptides. [ , ] the segregation of hydrophobic and polar residues on the two opposite sides of the amphipathic a-helix is responsible of the superficial insertion into the upper monolayer which modifies the pack- similarly, also tilted peptides are characterized by an asymmetric distribution of hydrophobic residues, which again produces a modification of the organization of the membrane into which they insert. [ ] the membrane bound conformation of the influenza virus fusion peptide is characterized by the presence of a hairpin of two tightly packed, antiparallel a-helices; [ , ] the asymmetric insertion is determined by the fact that hydrophobic residues insert into the proximal membrane leaflet and polar residues project outward. the precise angle of the kink between the two arms depends on the sequence, the ph, and the lipid environment and determines the functional features necessary for activity. as a matter of fact, the compact hairpin structure drives favorable insertion, while its expanded structures promote subsequent membrane destabilization. [ ] canonical class ii and class iii fusion peptides correspond to loops which do not undergo conformational changes upon insertion into the target membrane; the interaction clearly involves few hydrophobic residues, and the insertion into the outer leaflet of the membrane is superficial and probably inadequate to destabilize membranes. thus, a cooperative effect attained upon fusion activation is the only explanation for activity. [ ] the idea of a single fusion peptide being exclusively responsible for the membrane perturbing activity has been overwhelmed by evidences supporting the concerted action of different membranotropic peptides, [ ] which together with the canonical fusion peptide are involved in the modification of membrane curvature. [ ] [ ] [ ] [ ] [ ] [ ] many viral fusion proteins present an additional hydrophobic membrane proximal region at the intersection between the ectodomain and the transmembrane anchor (tm), the so-called mper (or pre-tm); [ ] the unusual clustering of aromatic amino acid in these regions prompted the idea of their strong involvement in the fusion process. indeed, peptides corresponding to the pre-tm region partition into membrane interfaces and likely cooperate with fusion peptides and tm domains during apposition of membranes, enhancing the overall hydrophobicity of the environment and contributing to the distortion of the lipid membranes required for fusion. [ ] the pre-tm of hsv- gh interacts strongly with membranes; [ ] the pre-tm of gp of foamy virus induces fusion of model membranes; [ ] the aromatic domain of the glycoprotein s of severe acute respiratory syndrome virus (sars) partitions into lipid membranes and perturbs their integrity; [ ] the pre-tm region of the gp protein from ebola promotes perturbations of membranes when in a helical structure. [ ] hsv fusion involves both gb and gh and several membranotropic sequences are present in both glycoproteins, [ , ] although the precise role played by each of these regions remains to be elucidated. the two fusion loops at the tip of the domain ii of gb constitute a structural subdomain with the hydrophobic amino acids forming a crest lined on both sides by charged residues; [ ] the concerted use of the two peptides produced a significant distortion of the target membrane bilayer, while when they were used separately they presented a lower membrane penetration. [ ] the two charged residues represent a novel feature of fusion peptides with the presence of hydrophilic residues on either side favoring insertion. [ ] fusion of inner and outer monolayers is clearly involved but not formation of pores, indicating that bilayer perturbation not complemented by leakage is a typical feature of viral fusion peptides which violate the host membrane without compromising its integrity. [ ] several gh peptides are able to interact with membranes and play a role in the process. among these, gh , represents a key achievement in grasping the role of hydrophobic viral peptides. [ ] [ ] [ ] [ ] the peptide contains residues critical for interaction such as aromatic residues (tryptophan and tyrosines) which are known for their preferred location at the membrane interface and for their ability to facilitate oligomerization [ ] together with numerous hydrophobic residues (glycines, leucines, alanines) which are critical for membrane insertion; [ ] at the c-terminus there is an arginine residue which is key for establishing peptide-lipid interactions. gh penetrates into membranes from its n-terminal side, and assumes an amphipathic helical conformation. [ ] the n-terminal histidine acts as a switch for triggering viral fusion and strongly enhances the fusion activity; [ ] the role of the histidine has been reported also for paramyxoviruses [ ] and togaviruses. [ ] yao et al. [ ] in order to investigate how the fusion peptide (fp) and lipids also play a key role in this process, generating membrane curvature thanks to their physico-chemical properties. cholesterol is key as it selectively intercalates into the leaflet of the bilayer favoring its distortion without producing unfavorable hydrophobic/hydrophilic interactions. [ ] the secondary structure of the fusion peptide of hiv changes according to the cholesterol content in the membrane, being a-helical in the absence of cholesterol, but shifting to a b conformation with the increase of cholesterol. [ ] both the fully a-helical and the fully b-structured peptides are able to insert deeply inside the membrane, while the mixed secondary structures, present at intermediate cholesterol concentrations, are more superficially inserted into lipid bilayers and less effective in inducing membrane fusion. [ ] it is likely that different secondary structures and domains with different content of cholesterol might be involved in different stages of fusion. [ ] probably, lipid phase discontinuities between liquid ordered and disordered domains containing cholesterol produce membrane defects with exposed hydrophobic surfaces favoring a deeper insertion of fusion peptides, and promoting membrane fusion. [ ] in conclusion, the clear view is that membrane fusion is a very complex process involving several domains of the fusion proteins which interact directly or indirectly with biological membranes, and contribute to the merging of the viral envelope and cell membrane. some peptides (as the lytic peptides of nodaviruses, picornaviruses, and reoviruses) can be generated by an autocatalytic cleavage step of a precursor, whereas others can be generated from the proteolytic activity of cellular enzymes. [ , ] essentially we can classify lytic peptides in amphipathic a-helices and myristoyl groups; or we can classify them according to their mechanism of membrane action in those causing transient modification of the cellular membrane, pore formation, and total disruption of the limiting membrane. [ ] although being clearly different among themselves and with fusion peptides of enveloped viruses, they present notable similarities. incubation of hela cells with various peptides corresponding to the c terminus of the l protein of papillomavirus, determines the entrance of propidium iodide into cells. [ ] the incubation of the adenovirus internal protein vi with liposomes loaded with a fluorophore, caused the release of the entrapped fluorophore. [ ] similarly, incubation of vp * of rotavirus with liposomes entrapping a fluorophore caused its release, suggesting that vp * is sufficient to perforate the lipid vesicles. [ , ] the membrane disrupting g peptide of fhv also triggers the release of the fluorophore. [ , ] as for enveloped virus fusion peptides, the amphipathic a-helix seems to be a key structural motif also for non-enveloped viruses. the n-terminal amino acids of the fhv g peptide form an amphipathic a-helix which is commonly referred to as g , [ ] while the g peptide comprises also a c-terminal region which is commonly considered to play a supporting role for the correct positioning of g . the g peptide assumes a random coil conformation in solution, while it adopts a kinked helical conformation in model membranes; similarly to other membranotropic peptides also viral lytic factors seem to be able to adopt a membrane-active conformation when interacting with the lipid bilayer. [ , ] the g peptide is able to spontaneously partition into lipid bilayers and increases the membrane permeability of liposomes; [ , [ ] [ ] [ ] ] in particular, it mediates liposome lysis through the insertion only into the outer leaflet of the lipid bilayer, and locating parallel to the membrane surface, with the hydrophobic face of the helix packed against the membrane surface. [ , ] a concentration dependent membrane leakage process [ ] similar to other non-enveloped viruses such as poliovirus vp [ ] and reovirus l n [ ] is observed. similarly to the influenza [ ] and hiv gp [ ] fusion peptides from enveloped viruses, the amphipathic region of g peptide presents a kinked helical structure in solution. the rigid, boomerang like structure assumed by the influenza fusion peptide in lipid environment is required to promote membrane fusion; [ ] in fact, abolishing the kink in the structure or making it flexible eliminates membrane fusion [ ] probably for the failure of the fusion peptide to insert deep into the lipid bilayer and pack against the hydrocarbon moieties. at neutral ph the g peptide is able to cause membrane disruption; while at low ph it is only able to alter its location relative to the capsid, but does not increase its membrane interacting ability. [ ] surprisingly, thanks to a kinked structure and a tight alignment of the hydrophobic residues on one side of the peptide at low ph similar to influenza virus, fhv g peptide shows localized perturbation of lipid arrangements with no proof of pore formation. [ , ] while membrane destabilization requires the simple insertion of the fusion peptide into the outer leaflet of the lipid bilayer, leakage needs both a deeper insertion and an interaction between peptides inside the membrane. the amphipathic region of g peptide oligomerizes in bilayers [ ] with a low content of cholesterol, which being more fluid would promote association between peptides. [ ] the presence of the g c-terminus is absolutely necessary for virus entry; [ ] as a matter of fact, truncations, or point mutations in the c-terminal region of g determine a disordering of the pentameric bundle formed by the n-terminal amphipathic helices of g in fhv particles and hamper in vitro and in vivo membrane lysis. the pentameric bundles constitute the viral "membrane attack module," and an essential function of the g c-terminal region is to maintain this module in its correct conformation [ ] (figure ). the g and g peptides cause a similar localized disorder of the target bilayer and the presence of the cterminal hydrophobic helical region in full-length g make the peptide effective at concentrations achievable in the context of viral infections. the kinked helical structure seems to be a common trait of enveloped and non-enveloped viruses and differences in length and angularity of the helices as well as differences in the amino acid content may cause variations in the mechanism of interaction with the bilayer; the presence of this motif in viral proteins may signal a membrane associated role for this component during a certain step of the viral life cycle which adds to eventual other roles. adenovirus requires acidic ph for exposure of the amphipathic helix contained in its protein vi and disrupts endosomal membranes to release its nucleocapsid. [ ] mutations in the amphipathic helix reduce infection and endosome escape, supporting the view that both the hydrophobic character and a-helical structure are key to allow maximal membrane disruption. the n-terminal amphipathic helix of protein vi, as fhv g peptides, lies parallel to model membranes with the hydrophilic face interacting with the phospholipid head groups, and probably causes disruption of the bilayer by introducing positive curvature in figure schematic representation of fhv capsid (a). an expanded view of the crystallographic structure (pdb: ftb) of one subunit (a protein) showing the location of the amphipathic region of g peptide in yellow (b). schematic representation of a protein, which undergoes auto cleavage during maturation producing b and g (c) with relative sequence of g peptide membranes. [ ] mutations in the amphipathic region of protein vi, which prevent insertion into the membranes, severely affects membrane penetration and cellular entry. [ ] proteins vp and vp play a fundamental role in membrane penetration by poliovirus [ ] with the exposure of amphipathic a-helical nterminal approximately amino acid region of vp being necessary for liposome binding; [ ] while the n-terminus of poliovirus vp contains a hydrophobic myristoyl (c acyl) group for insertion into membranes. [ ] following receptor binding, the amphipathic a-helix within the n-terminal portion of vp , is exposed and probably forms pores to transfer the genome to the cytoplasm. interestingly, vp also performs a role in membrane penetration and the hypothesis is that vp primarily function is to secure the particle to the limiting membrane, while vp participates directly in pore formation. mammalian orthoreovirus protein l contains a small hydrophobic peptide (l n) with a myristoyl group at its n-terminus. [ ] disruption of cellular membranes, requires the complete dissociation of l n peptides from the particle; auto-cleavage of l during reovirus entry generates l n peptides that are linked to an n-terminal myristoyl group. after lipid association, the l n peptide changes conformation from an extended to a b-strand rich secondary structure. [ ] l n is able to generate size-selective pores in erythrocyte or liposomal membranes. it is likely that the b-hairpins in l n associate with the membranes forming a b-barrel pore. [ ] probably, membrane disruption caused by l n is similar to that of b-barrel toxins. [ ] it is highly probable that a future more detailed knowledge of the mechanism used by lytic peptides of non-enveloped viruses will lead to the discovery of more common features between enveloped and nonenveloped membranotropic peptides. membranotropic peptides thanks to their adaptness to interact with the membrane, are opening the way for numerous applications. [ , ] their ability to bind lipid membranes is correlated to their simultaneous hydrophobic and amphipathic nature, while their insertion into the bilayer is due to their capability to change conformation according to the environment; moreover, they are able to penetrate deep into the hydrophobic core but do not span the bilayer in a pore-like manner; on the contrary, they tend to self-associate at the interface between the membrane and the aqueous compartments. below are reported main applications. one of the main applications of fusion peptides of enveloped viruses is as inhibitors of viral penetration. their ability to directly interact with the hydrophobic surfaces present on cell membranes and/or fusion proteins allows them to interfere with virus entry. [ , ] the inhibition mechanism is still unclear but it is likely that inhibition of infectivity is correlated to inactive aggregates formed between the fusogenic stretches present in the viral protein and in the peptides. in particular, the formation of aggregates is related to their ability to oligomerize or to mimic the mode of binding of their original domains in their partner protein; thus, stabilizing a pre-fusion intermediate and preventing merging of the bilayers. [ , [ ] [ ] [ ] self-oligomerization of fusion peptides has been proposed to be responsible of inhibition by several groups. [ , ] hiv fusion peptides form structurally defined oligomeric complexes which have been considered responsible of inhibition; [ , ] moreover, mutants of the native sequence with a lower helical content and tendency to self-associate into b-sheets are able to inhibit membrane fusion with different magnitude and at various stages. [ ] virip is a peptide designed to target gp fusion peptide and thus block hiv- infection; it has undergone clinical studies and was demonstrated to be as active as peptides targeting the coiled-coil. [ , ] its clinical evaluation represents the proof of concept that membranotropic sequences could inhibit viral replication in infected individuals and may have potential clinical effectiveness. moreover, the knowledge that several domains are implicated in the fusion mechanism and may interfere with the intramolecular interactions between the several domains, clearly demonstrates that they all represent potential targets for the design of entry inhibitors. [ ] membranotropic peptides are emerging as delivery vectors. [ ] [ ] [ ] until now, the most widely used delivery vectors are cationic cell penetrating peptides (cpps), which enter essentially by endocytosis causing the entrapment of the cargo into endosomes with only minor quantities of the cargo able to reach the target where to exert the biological function. on the contrary, membranotropic peptides are internalized by direct penetration of the membrane and thus determine immediate bioavailability of the delivered molecule. fusion membranotropic peptides are particularly noteworthy because they can physically interfere with the membrane hydrophobic interior forming bulges that protrude from the membrane and ease contacts between fusing bilayers; in particular, they are able to translocate molecules through the plasma membrane directly into the cell, promoting lipid-membrane reorganizing processes, and causing local and temporary membrane destabilization with subsequent reorganization, circumventing the endosomal entrapment by favoring the escape from the endosome. [ , ] the internalization mechanism is also related to the toxicity of the internalized drug and the development of resistance. the gh is able to directly translocate across the membrane bilayer and to transport several cargos such as quantum dots, [ ] liposomes, [ ] dendrimers, [ , ] nanoparticles; [ ] it is also able to cross the bbb in vivo. [ , ] mpg is an amphipathic peptide composed of the fusion peptide of hiv- associated to an hydrophilic domain with positively charged residues derived from the nuclear localization sequence (nls) of simian virus (sv ) large t antigen (pkkkrkv), through a spacer (wsq). [ , ] in vitro mpg is able to deliver both sirna and dna after just h. [ ] the principal internalization mechanism was shown to be independent of the endosomal pathway and to involve the study of the internalization of g peptide derived from fhv [ ] revealed that this is mediated by relatively high cell surface adsorption leading to enhanced macropinocytic uptake and cytosolic distribution and also revealed a higher efficiency of internalization compared with tat. [ ] influenza virus fusion peptide has been used for increasing transfection efficiency, its ph-dependent fusogenic and endosomolytic activities are able to enhance lysosomal degradation before the contents of the endosomes are delivered to lysosomes. [ ] the development of industrial applications in drug delivery is probably one of the most exciting and fastest growing fields, with the possibility of these peptides to pass through the bbb and become an important player in the fight against all pathologies correlated to neurosciences. scientists envisage also a possible use of viral membranotropic peptides as an alternative to classical antibiotics in order to combat the antibiotic resistance problem. [ ] antimicrobial peptides (amps) are widely exploited and represent attractive candidates for the development of anti-infective agents; [ ] [ ] [ ] recently, attention has been devoted also to the exploitation of membranotropic peptides derived from viral fusion proteins as antibacterial drugs. in fact, some amps with helical structure seem to share high sequence (preference for alanines and glycines) and structure (amphipathic a-helix) similarity with fusion peptides and suggest a convergent evolution correlated to their ability to disturb lipid bilayers. [ ] the fusion domain of influenza virus was evaluated for its antibacterial activity; analysis showed that the amidation of the c-terminus is a key factor to render the fusion peptide an antibacterial peptide and optimization of the amphiphilic balance can improve efficacy. [ , ] the antibacterial activity of viral membranotropic peptides is not yet widely evaluated and much work is still open in this field; in particular, their mechanism of perturbation of membrane bilayers may allow the design of novel sequences with the ability to denature the membrane bilayer of bacteria which will add to their many roles. development of effective vaccines against viruses is another worldwide concern. a potent vaccine needs to be able to induce both [ ] in vivo prime-boost immunization enhanced humoral and cellular immune responses, suggesting the promising application of membranotropic peptides as vaccine candidates in future (table ) . the membrane entry of enveloped and non-enveloped viruses employs fundamentally different mechanisms, although common themes have emerged in the entry process. this similarity is essentially represented by the presence/exposure of small membranotropic peptides which cause membrane disruption and/or promote membrane fusion. entry involves membrane fusion versus perforation, but cellular triggering factors and structural intermediates appear to share some similarities. interestingly there is also some similarity with the mechanism used by bacterial toxins to cross biological membranes in order to reach the cytosol; in fact, many toxins, undergo conformational changes which allow them to initiate the translocation process. [ , ] how exactly both enveloped and non-enveloped viruses overcome host cell membrane barriers to deliver their genomes remains an intriguing problem. comprehensive structural and biochemical studies on enveloped viruses have brought to the conclusion that a unifying mechanism for host cell entry exists; where a membranotropic fusion loop, peptide, or patches catalyze fusion of the two membranes. in contrast, interaction of non-enveloped viruses with host cells during entry is less defined; while membrane active peptides have been discovered as necessary elements for entry in several well-studied non-enveloped virus capsids. in conclusion, it is now evident that the success of membranotropic peptides further stimulates challenging research on the unraveling of the many roles and applications that could be developed for both enveloped virus fusion peptides and small lytic peptides in nonenveloped viruses; membranotropic peptides are attracting increasing attention from the scientific community and their future will be dictated by the progresses in their industrial applications. http://orcid.org/ - - - entry of enveloped viruses into host cells: membrane fusion biophysical modulation of peptide-membrane interactions author biographies annarita falanga received the degree in food science and technology at the university of naples he earned his phd in virology from the university of cambridge (uk) in . he was appointed lecturer in microbiology in at the department of animal health of the faculty of veterinary medicine of the university of naples 'federico ii'. he moved to the faculty of medicine of the second university of naples where he was appointed associate professor and then full professor of microbiology giancarlo morelli is a full professor of chemistry at the university of naples 'federico ii she carried out research activities at the columbia university of new york in - . in she earned the position of assistant professor of inorganic chemistry at the university of naples and since she is associate professor. since , she is adjunct professor at loyola university chicago. research activities focus on antimicrobial peptides and drug delivery mechanisms. how to cite this article membranotropic peptides mediating viral entry key: cord- -vgjz ts authors: nan title: th international congress of the european association for endoscopic surgery (eaes) sevilla, spain, – june date: - - journal: surg endosc doi: . /s - - -x sha: doc_id: cord_uid: vgjz ts nan laparoscopic cholecystectomy is one of the most commonly performed operations worldwide. bile duct injury (bdi) is a rare but very serious complication of the procedure, with a significant impact on quality of life and overall survival. the high frequency of bdi with laparoscopic cholecystectomy was first considered to be a consequence of the initial learning curve of the surgeon, but it later became clear that the primary cause of bdi is misinterpretation of biliary anatomy. intraoperative cholangiography (ioc) has been advised by many authors as the technique reduces the risk of bdi. however, the procedure has inherent limitations and is therefore reserved for select cases. fluorescent cholangiography using indocyanine green(icg) is a novel approach, which offers real-time intraoperative imaging of the biliary anatomy. a comparative study was contacted by administering icg intravenously or intrabiliary during the operation. forty patients scheduled to undergo an elective lap. cholecystectomy were randomly divided in two groups: in group a icg was administered in a dose . mg in ml solution intravenously hour before surgery. in group b icg was injected intrabiliary in a . mg/ml solution mixed with the patient's bile. also, we observed and analysed the following parameters, liver function, b.m.i, asa score and possible complications, before and after operation. results: group a. intravenous icg was administered in patients. there was no any reaction and the extrahepatic biliary anatomy was identified well. there was no bdi or any complication related to the procedure. group b. icg was injected intrabiliary in patients during the laparoscopic procedure. in all but one patient the extrahepatic biliary tree was delineated very well. in one patient part of icg solution was injected into the gallbladder wall and this resulted in a partially confusing image. there was no bdi and no postoperative complication conclusions: fluorescence cholangiography can be used during laparoscopic cholecystectomy to obtain fluorescence images of the bile ducts following intrabiliary injection during the operation orintravenous injection h before the procedure. the later technique is more easy to perform and does not require catheterization of the biliary tree. endoscopia digestiva chirurgica, policlinico universitario ''a. gemelli'', rome, italy; ihu, strasbourg, france; camma group, icube, university of strasbourg, cnrs, ihu strasbourg, strasbourg, france; ircad, strasbourg, france; digestive and endocrine surgery, nouvel hopital civil, university of strasbourg, strasbourg, france; digestive and endocrine surgery, ihu-strasbourg, strasbourg, france aim: surgical societies are united in promoting the critical view of safety(cvs) during laparoscopic cholecystectomy(lc). nonetheless, reports have shown a discrepancy between the operative reports and the correct application of cvs, which may explain the stability of bile duct injury rates. therefore, surgeons and computer scientists at our institution are developing a machine-learning algorithm to automatize cvs assessment. however, the lack of a consistent cvs video assessment framework limits the ability to generate data to train the artificial intelligence. here we describe and test a method for cvs evaluation in videos. method: between march and july , consecutive videos of lc performed at nouvel hospital civil(strasbourg, france) were recorded. two independent reviewers assessed the achievement of cvs in the s video sequences preceding clipping of cystic duct and artery. in addition to the 'doublet view' method, a 'binary' video evaluation method was tested: each of the criteria composing the cvs( structures entering the gallbladder, clearance of the hepatocystic triangle and lower part of the cystic plate) was classified as achieved or not. if the criteria were met, then the cvs was considered achieved. inter-rater agreement for cvs and for each of the criteria was evaluated. results: twenty-two videos( fundus first and partial lc, and broken videos) were excluded from the cvs analysis. cvs elements were assessable in all but one s videos sequences( . %). after mediation, cvs was achieved in / ( . %) of lc. the cystic plate was identified in only . % of videos. inter-rater agreement using the doublet view vs. the binary method was as follows: . %(? = . ) vs. . %(? = . ) for cvs achievement, . %(? = . ) vs. . %(? = . ) for the structures, . %(? = . ) vs. . %(? = . ) for the hepatocystic triangle and . %(? = . ) vs. . %(? = . ) for the cystic plate ( fig. ) . conclusions: reliable cvs assessment is crucial to generate consistent data for machine-learning algorithms aiming at decreasing bile duct injury after cholecystectomy. our binary cvs video assessment method showed higher inter-rater reliability than the doublet view, originally described for assessment of photos. further studies are on going to validate the cvs assessment in videos and support our initial results. surg endosc ( ) the vital role of surgeries in healthcare requires a constant attention for improvement. surgical process modeling is an innovative and rather recently introduced approach for tackling the issues in nowadays complex surgeries, involving complex logistics, much technology, and large teams. surgical process modeling allows for evaluating the introduction of new technologies and tools prior to the actual development and is beneficial in optimization of the treatment planning and treatment performance in operating room. in this study, we first discuss the concepts associated with surgical process modeling, aiming to clarify them and to promote their use in future studies. next, we apply these concepts to analyze the procedure of challenging interventions, minimally invasive liver treatment (milt) methods, with the ultimate goal of improving and optimizing the treatment procedure. the procedure model of current treatment activities and planning of various milt methods and the associated techniques, are analyzed and combined into a generic procedure model of milt, which provides a firm foundation for qualitative and quantitative analysis of different milt procedures. the generic procedure model is validated by data from erasmus medical center (rotterdam, the netherlands) and oslo university hospital (oslo, norway) . the proposed procedure model is designed to be a basis for improvement of the procedure and to determine how and where the new technologies can be best, effectively and efficiently, employed in the clinical practices prior to and/or during actual development of the new technologies for milt. as a conclusion, the current work illuminates the importance of surgical process modeling for improving different aspects of treatment procedures and provides an overview of various modeling strategies that can be used to establish surgical process models. the generic procedure model of various milt methods, including laparoscopic liver resection, laparoscopic liver ablation and percutaneous ablation, is introduced and validated which is a basis for introduction of the optimized procedure model of milt objective: to determine the most appropriate time to start total laparoscopic living donor right hepatectomy (tldrh) based on the experience with laparoscopic liver resection (llr). summary background data accumulation of experience in llr is essential before starting tldrh to ensure donor safety. methods: we retrospectively reviewed data of and consecutive patients who underwent llr and donor hepatectomy, respectively, between and . operative outcomes of laparoscopic major hepatectomy (lmh) were compared between two periods based on tldrh introduction (phase i - vs phase ii . learning curve of llr was evaluated using the cumulative sum (cusum) method to determine the optimal time of tldrh introduction. conclusion: accumulating an experience of at least lmh cases is needed in low-volume lt centers before starting tldrh to ensure donor safety. introduction: the number of surgical adverse events is still too high. an important number of these adverse events occur within the operating room (or) and are in fact preventable. in order to reduce adverse events in the or, we simply need to know what went well and what can be done better. the aim of this study was to analyze and debrief a predefined selection of surgical procedures, with the use of an operating room 'black box', to identify commonly observed safety threats and resilience support events. methods: in the period - , predefined gastro-intestinal laparoscopic cases were recorded by the or black box'. the postoperative surgical team assessment record (star) questionnaire was used. the recordings were analyzed by specifically trained raters, using the systems engineering initiative for patient safety (seips) model of work system and patient safety to identify relevant safety threat and resilience support events. qualitative data analysis was used to identify the most commonly discussed events during the team debriefings. results: in only . % (n = ) of times or team members, when asked direct following surgery, indicated that they had noticed aberrations (n = ) during the case. a mean number of . (sd . ) relevant positive and negative events (e.i. aberrations) per surgical procedure were identified using the black box performance report. on average, . (sd . ) of events identified by the black box were rated as safety threats. most events discussed during the team debriefings were related to communication. conclusion: these results once again highlighting the importance of clear and closed-loop communication in the operating room. theatre staff underestimated the number of aberrations occurring in the or, when asked to retrieve from memory. postoperative structured team debriefing may be important for resolving incorrect assumptions between operating team members to avoid future unnecessary miscommunication. background: the eaes has recently published an intraoperative adverse event classification to assist the direct measurement and routine reporting of minimal access surgery interventions. we aimed to explore the clinically validity and reliability of the classification. methods: a prospective evaluation utilising case videos and clinical data from a completed multi-centre laparoscopic total mesorectal excision surgery randomised controlled trial was performed (isrctn ). enacted adverse events identified with the observational clinical human reliability analysis technique were graded with the eaes classification by two blinded, independent assessors. test-retest reliability was explored using grades previously applied during the development of the classification with intraclass correlation co-efficients calculated. clinical validity was assessed using -day morbidity events, the clavien-dindo classification and the highest eaes grade per case. results: laparoscopic cases ( h of surgery) contained error events which were all successfully categorised. excellent inter-rater and test-retest reliability was seen (icc . , % ci . - . , p \ . and icc . , % ci . - . , p \ . respectively. % of patients experienced post-operative morbidity (median event, range [ ] [ ] [ ] [ ] [ ] [ ] . labelling analysed cases by their highest eaes classification grade gave % grade , % grade and % grade procedures. % of grade cases developed a morbidity event, but this significantly increased in grade and operations ( % and %, p = . ). the number of complications and highest recorded clavien-dindo grade increased with each additional grade ( . ± . vs. . ± . vs. . ± . , p = . and median vs. vs. , p = . respectively). anastomotic leak and re-operation were correctly captured by the allocated eaes grade ( . % vs. . % vs. %, p \ . and % vs. % vs. %, p \ . respectively). there was a significant rise in length of stay observed with increasing eaes grade (median vs. vs. days, p \ . ). conclusion: in the context of major laparoscopic surgery, the eaes intraoperative adverse classification is seen to be a clinically valid and reliable assessment method. psychological medicine, nuhs, singapore, singapore aims: neurobiological feedback in surgical training could translate to better educational outcomes such as measures of learning curve. the variation in brain activation of medical students when performing laparoscopic tasks before and after a training workshop is not properly studied before and we planned to do this using functional near infrared spectroscopy (fnirs) which is a non-invasive optical brain imaging tool that measures cortical oxygenation change which is used as a marker of pre-frontal cortex activity (pfca). methods: this randomised controlled trial examined the pfc activity differences in two groups of novice medical students during the acquisition of basic laparoscopic tasks. 'trained-group' had standerdised oneto-one training on the tasks, while the 'untrained-group' had no prior trainining and was just shown a video of the tasks. the pfca was measured pre and post intervention using a portable fnirs device. primary outcome was the difference in the pfca pre and post intervention. secondary outcomes were the differences in pfca between the tasks and between the sexes. results: trained and untrained medical students with an equal sex distribution and a comparable age distribution were invovlved in the study. all students were right handed. trained group had a significantly attenuated pfca in the 'precision-cutting' (p = . ) and 'suture-insertion' (p = . ) tasks compared to the untrained group. subgroup analysis based on sex revealed significant attenuation in pfca in trained females compared to untrained females across of the laparoscopic tasks: 'pegstransfer' (p = . ), 'precision-cutting' (p = . ), 'suture-insertion' (p = . ). no significant pfca attenuation was found in male students who underwent training compared to untrained males. conclusion: a standardised laparoscopic training workshop promoted greater pfca attenuation in female medical students compared to males. this suggests that female and male students respond differently to the same instructional approach. these results may have implications for surgical training and education such as a greater focus on one to one surgical training for female students and use of pfca attenuation as a form of neurobiological feedback as a measure of learning curve in surgical training. robot assisted versus laparoscopic advanced suturing learning curve e. leijte , i. de blaauw , c. rosman , s.m.b.i. botden surgery, radboudumc, nijmegen, the netherlands; pediatric surgery, radboudumc, nijmegen, the netherlands aims: compared to conventional laparoscopy, robot assisted surgery is expected to have most potential in difficult areas and demanding technical skills as minimally invasive suturing. this study was performed to identify the differences in the learning curves of laparoscopic versus robot assisted advanced suturing method: novice participants, with the knowledge of basic surgical procedures, were recruited and performed three suturing tasks on the eosim laparoscopic augmented reality simulator or the robotix robot assisted virtual reality simulator. each participant performed an intracorporeal suturing, tilted plane needle transfer and anastomosis needle transfer task. to complete the learning curve, all tasks were repeated for maximal twenty repetitions or until a plateau was reached three consecutive times. clinical relevant and comparable parameters regarding time (seconds), movements and safety were recorded. intracorporeal suturing was used to visualize and compare the learning curves between the groups. results: forty-six participants completed the learning curve, of which laparoscopically and robot assisted. when comparing the suture time, the plateau was reached much faster in the robot assisted group ( - repetitions) than the laparoscopic group ( ) ( ) ( ) repetitions) as shown in figure . there was a significant difference in 'time per suture', during the whole learning curve with median values of versus (first knot), versus (fifth) and and (eighteenth), all with a p \ . . however, the parameter 'adequate surgical knot' was reached earlier in the laparoscopic group than in the robot assisted group. first: % versus %, fifth: % versus %, and eighteenth: % versus %. when assessing the 'needle out of view' parameter, the robot assisted group scored a median of . and . s during the first, respectively eighteenth knot, and the laparoscopic participants had their instruments out of view for and s during the first respectively eighteenth knot. conclusion: the learning curve of minimally invasive suturing can be reduced with the use of robot assisted surgery, with a specific reduction in operation time. the rate of adequate knots seemed to remain lower in robot assisted surgery, although this could be due to the virtual reality aspect of the simulator. introduction: endoscopic sleeve gastroplasty (esg) is a novel promising bariatric endoscopy treatment. gastric volume reduction and delayed gastric emptying are the mechanisms driving weight loss. however, little is known about the factors influencing the effectiveness of weight loss overtime. the present study aims at evaluating the correlation between endoscopic suture appearance and excess weight loss (ewl%) at and months follow up. patients and methods: all patients who underwent follow-up endoscopy at and months after esg were included. esgs were classified in groups according to endoscopic appearance of the gastric sutures: optimal (group ) when all stitches were in place and tights; suboptimal (group ) when one or more stiches were displaced; loose (group ) when all the sutures were completely disrupted. bmi at enrollment and ewl% at and months were recorded and compared to the endoscopic appearance. results: a total of patients were included in the analysis. at months, ( . %) patients had an optimal esg, ( . %) had a suboptimal sleeve and ( . %) had complete sutures failure. bmi at enrollment and ewl% were respectively . ± . and . ± . % for group , . ± . and . ± . % for group and . ± . and . % ± . % for group . twenty five patients had months egds: ( %) presented an intact esg and were classified in group , ( %) in group and ( %) in group . twelve months ewl% was respectively . ± . %, . ± . and ± . %. initial bmi significantly correlated with suture status at both (rho - . ; p \ . ) and months (rho - . ; p = . ) follow-up. furthermore, the sutures' appearance itself correlated with ewl% at both time points (rho ? . ; p = . and rho . ; p = . respectively). conclusion: our preliminary results show that the aspect of the endoscopic suture has a significantly impact on ewl% at and months after esg. furthermore, bmi at enrollment seems to predict endoscopic suture duration overtime. larger studies and longer follow-up are needed to further validate our preliminary findings. background and aim: endoscopic sleeve gastroplasty(esg) is a relatively novel endoscopic procedure that reduces the gastric lumen with proven less complications and less months weight loss compared to laparoscopic sleeve gastroplasty (lsg) . at present there are no studies investigating the role of multidisciplinary approach in esg. the aims of the present study were to evaluate the role of multidisciplinary assessment(ma) prior esg, weight loss outcomes, quality of live improvements and adverse events. material and methods: from may to may all patients that underwent esg were retrospectively evaluated from a prospective database. until september before esg only psychiatric evaluation was requested, while after this date we adopted the guidelines of the italian society for obesity surgery and all patients were evaluated on a multidisciplinary fashion prior esg. the multidisciplinary team was composed by:gastroenterologist, surgeon, psychiatrist, endocrinologist and dietitian. patients were divided in two groups:group were patients with esg before ma and group were patients with esg after ma. we compared this two groups in terms of weight loss outcomes, quality of live improvements and adverse events. quality of live was measured with the bariatric analysis and reporting outcome system(baros).all procedures were done with the apollo overstitch suturing system(apollo endosurgery) and a double channel gastroscope olympus tgif- (olympus japan).all procedures were done in general anesthesia and with insufflation of co . all patients had ambulatory visit t , and months after esg and weight loss outcomes were measured in terms of excess weight loss (%ewl),the total body weight loss (%tbwl) and baros scale were assessed. statistical analysis was done with chi-square test and \ . value was considered significant. results: patients were identified ( female; mean age . , range - ) . mean bmi at inclusion was . (range . - . ). mean %ewl and %tbwl at months was . and . respectively (table ) .non procedure related complications were observed. comparing the two groups there was significant(p \ . ) difference in terms of %ewl and %tbwl (table ) ,with better results in group . there was also a significant improvement in the baros scale in the patients in group . conclusions: ma before esg has a fundamental role in terms of better procedure outcomes for both weight loss and quality of live in obese patients. gastroenterology, hadassah medical center, jerusalem, israel, israel aims: the over-the-scope clip (ovesco) is a novel endoscopic tool that enables non-surgical management of gastrointestinal defects. the aim of this study was to report our experience with ovesco for patients with staple line leaks following laparoscopic sleeve gastrectomy (lsg). methods: a prospectively maintained irb-approved institutional database was queried for all patients treated with ovesco for staple line leaks following lsg from to . primary outcome was complete resolution of leak following ovesco as defined by return to complete oral nutrition and no evidence of leak on imaging. secondary outcome was the number of additional endoscopic or surgical procedures needed following ovesco. results: twenty-five patients ( males, females) were treated with ovesco for staple line leaks following lsg. the median age was years (range - ), and mean body mass index was kg/m . nine patients ( %) were referred from an outside hospital. the median time from index operation to leak diagnosis and from leak diagnosis to ovesco was days (range - ), and days (range - ), respectively. all patients had upper staple-line leaks near the gastroesophageal junction. initial treatment included antibiotics- patients; computed tomography guided drainage and antibiotics- patients; and laparoscopic drainage- patients. ovesco led to final resolution of leak in patients ( %) within days of clip deployment (range - ). leaks which persisted following ovesco were eventually resolved with a combination of ovesco and stent- patients ( %), total gastrectomy and esophago-jejunostomy- patients ( %), and endoscopic suturing- patient ( %). one mortality was noted in a patient who suffered multiorgan failure. the number of additional endoscopic sessions ranged from to (median ). no procedure related complications were noted. all patients were treated with total parenteral nutrition and the total length of stay was days (range - ). conclusions: despite its low success rate, ovesco should be part of the bariatric surgeon's non-surgical armamentarium in treating staple line leaks following lsg. r. bademci , r. vilallonga , p. alberti , r. renato , c. yuhamy , s.s. cordero , l. posadas general surgery, istanbul medipol üniversitesi, istanbul, turkey; bariatric surgery, vall d'hebron, barcelona, spain background: in cases of morbid obesity, treatment is generally applied as either a surgical or endoscopic approach. the number of primary obesity surgery endolumenal (pose) procedures is increasing but the reliability and effectiveness is unclear as yet. the aim of this study was to present a series of cases that required revision surgery due to pose failure and to reveal possible alternative surgeries. materials and methods: a retrospective comparison was made of the data of obese patients with pose failure and conversion to surgical procedures between and in respect of operation, medical illness and bmi results. results: the patients comprised % females, % males with a mean age of . ± . years and mean follow-up period of . ± . months. on average, patients lost . ± . kg, with an average excess weight loss of . %. conclusion: no firm conclusions can be drawn from such a small group. although sg seems to be a safe procedure and should be considered as the first technique to be applied following pose failure, it is possible to perform gastric bypass on patients with this endoscopic precursor. introduction: the population of post bariatric surgery patients is rapidly increasing worldwide. due to the altered anatomy post roux-en-y gastric bypass (rygb), conventional endoscopic management for choledocholithiasis is challenging. these patients are now commonly managed by means of a laparoscopic assisted ercp. although effective, this requires significant resource utilization and potential morbidity related to the need for surgical intervention. we present our preliminary experience with a purely percutaneous management of choledocholithiasis in bariatric patients post-rygb. methods: a retrospective single center review identified five patients with choledocholithiasis after bariatric rygb who underwent percutaneous cbd access and treatment by interventional radiology. four patients underwent percutaneous transhepatic cbd access while one patient underwent percutaneous trans-cholecystic cbd access. in three of the five patients conscious sedation alone was sufficient to perform the procedure. results: all patients had radiologically confirmed choledocholithiasis and were clinically symptomatic prior to intervention. the biliary tree was successfully accessed percutaneously and cleared in all five patients. in the four patients where a percutaneous transhepatic access was utilized, three patients required only fluoroscopic balloon sphincterplasty and sweep of the cbd to clear the ductal stones, while the fourth required percutaneous cholangioscopy assisted lithotripsy for clearance. in the fifth patient with non-dilated intrahepatic bile ducts a trans-cholecystistic approach into the cbd was utilized with percutaneous cholangioscopic assistance to clear the ductal stones. all procedures were completed successfully with no post procedure complications. conclusion: percutaneous clearance of cbd stones in bariatric patients presents a minimally invasive alternative to current surgical practice. the use of conscious sedation and the purely percutaneous approach may potentially reduce morbidity and resource utilization for this increasingly common clinical scenario. laparoscopic narbona-arnau procedure to control the gerd after lsg- years results of a prospective study i.c. hutopila, c. copaescu background: after the laparoscopic sleeve gastrectomy (lsg) alone or associated with calibration of the esophageal hiatus, for some patients the reflux symptoms worsen postoperatively due to development of a hiatal hernia (hh) or due to the recurrence of the hh previously repaired. for these situations, when the conservative treatment fails, are proposed some surgical solutions, one of them cardiopexy with teres ligament-narbona arnau. objective: is to establish a standardized laparoscopic technique for cardiopexy using the teres ligament (narbona arnau technique) and to analyze the procedure's outcomes. methods: the study was performed in a bariatric and metabolic center of excellence-ponderas academic hospital. all the patients undergoing narbona arnau procedure to control gerd after lsg since were included and prospectively analyzed. the selection criteria included lsg patients, presenting hh and symptomatic gerd. preoperative investigations were upper gastrointestinal endoscopy, radiological contrast study, ph-metry, computed tomography with oral contrast. results: patients were included into the study. gerd and hh were preoperatively documented in all the cases. one patient was excluded after years of follow up after being converted to a laparoscopic roux-en-y gastric bypass, for intense relux symptoms. no incidents during surgery. for cases laparoscopic narbona arnau technique was performed concurrent with re-sleeve gastrectomy and gastric curvature plication. without postoperative complications. postoperative follow-up at months, , and years, the percentage of patients without gerd symptoms and free of treatment with ppis was , %, , %, , %, respectively . %. at years postoperatively the upper gi endoscopy showed remission/ improvement of the degree of esophagitis for patients. for the same period of follow-up, the ph-metry highlighted a normal value of demeester score for . % o patients (all the patients had preoperatively high de meester scores). no objective signs of hiatal hernia recurrence at imagistic investigations and upper gastrointestinal endoscopy were encountered. conclusions: complete preoperative evaluation is mandatory for choosing the optimal intervention. laparoscopic narbona arnau technique after lsg is proved to be a good option for the treatment of symptomic gerd, but further studies with high-volume patients are necessary. introduction: the aim of this study was to investigate the influence of baseline glycated hemoglobin level (hba c) level in bariatric patients on postoperative outcomes. we found scarce of clinical data regarding influence of baseline hba c on bariatric surgeries postoperative morbidity and readmission what was inspiration to conduct this multicenter retrospective study. methods and procedures: retrospective cohort study analyzed patients who underwent laparoscopic: sleeve gastrectomy (sg), roux-en-y gastric bypass (rygb) or mini-gastric bypass (mgb) for morbid obesity in seven referral bariatric centers. patients were divided into groups depending on preoperative hba c : hba c \ . %; . - . % and c . %. primary endpoints: influence of hba c level on perioperative ( -days) and postoperative ( -months) morbidity rates, operation time, length of hospital stay (los) and readmission rate. results: study group included , % females and % males. median age was ( - ) years. median hba c was . ( . - . ). hba c \ . % was present in % patients, hba c . - . % in %, and hba c c . % in %. percentage of male patients increased in groups from % in hba c \ . % to % in hba c c . % significantly. same tendency through groups we observed in case of bmi and age. uncontrolled diabetes (hba c c . %) was present in . % patients, while . % patients were not on antidiabetic medications despite having hba c c . %. median operative time in patients was significantly longer than in hba c \ . % and hba c . - . %. -days morbidity rate was . % and did not differ groups significantly, as -months morbidity rate (excl. -days) of . % . los did not differ groups significantly. patients having hba c in range of . - . % and with hba c c . % did not have significantly increased odds for perioperative morbidity, -months postoperative morbidity as compared with those with hba c \ . %. patients with hba c c . % had increased or for prolonged los as compared to those with hba c \ . % (or . ; % ci . - . ). hba c did not influence or for readmissions. patients with baseline hba c c % had significantly increased chances for hospital readmission (or . , % ci . - . ). conclusion: baseline level of hba c did not influence chance for perioperative morbidity, -months postoperative morbidity and prolonged los. patients with hba c c % have increased chance for hospital readmissions. surg endosc ( ) :s -s introduction: surgical resection is crucial for curative treatment of rectal cancer. through improvements in treatment and minimally invasive techniques, -year survival improved to over % of patients. the most recently introduced surgical technique is robotic-assisted surgery (ras). ras and conventional laparoscopy (cl) seem equally effective in terms oncological control. however, ras possibly provides further advantages e.g. d vision or the endowrist function, which have the potential to maximize the precision of surgery and thus has benefits for functional outcomes such as sexual function as well continence. therefore, the aim of this systematic review and meta-analysis was to compare functional outcomes of cl and ras for rectal cancer. materials and methods: this review was done according to the prisma and amstar guidelines andregistered with prosper-o(crd ). the search was planned with the pico criteria and conducted on medline (via pubmed), web of science and central. two independent reviewers first screened titles and abstracts and then eligible full-texts. inclusion criteria were original studies, comparative studies for cl vs. ras for rectal cancer as well as reporting of functional outcomes. quality assessment was done with the newcastle-ottawa-scale for non-randomized studies and the cochrane tool to assess risk of bias for randomized trials. results: the search retrieved hits, of which studies with patients met inclusion criteria. preliminary results yielded a lower rate of urinary retention for ras (odds ratio (or)[ %-confidence interval (ci)] . [ . , . ] ) while there were no differences for ileus (or[ci]: . [ . , . ] ). erectile function (iief) showed no differences after (mean difference (md)[ci] . [- . , . ] , (md[ci] . [- . , . ] ) and months (md[ci] . [- . , . ] ). in terms of urinary problems (ipss) there were no differences postoperative (md[ci] - . [- . , . ]) and month postoperative md[ci] - . [- . , . ] ), but advantages for the cl group after months md[ci] - . [- . , - . ] ). discussion: ras and cl seem to provide similar functional outcomes after rectal cancer surgery. however, the results need to be interpreted carefully as none of the studies had any functional outcome defined as primary endpoint. future studies should evaluate both surgical approaches in terms of functional outcomes and should be appropriately powered. methods: from april to november , laparoscopic right colectomy with intracorporeal anastomosis were performed in our surgical department. all patients in both groups were perioperatively managed using an eras pathway. seventy-two patients had the enterotomy closed with a single layer running suture of filbloc tm (assut europe). these patients were matched with patients who underwent intracorporeal right colectomy with enterotomy closed with a 'hybrid' double layer technique (first layer interrupted stitches in maxon tm - (covidien), second layer using a running suture in pds tm .intraoperative variables, anastomotic leak rate, morbidity and mortality rates were analyzed. results: the two groups were homogeneous with respect to demographics, body mass index (bmi), american surgical association score (asa) as well as for tumor stage. in the barbed group, median operating time was . min vs . min in the hybrid group (p = . ). anastomotic leak occurred in ( . %) patients in the hybrid vs ( . %) patients in the barbed group (p = . ) all patients required a reoperation. intraoperative findings at shows in ( . %) cases in the hybrid group a leak at the enterotomy closure, while an intact staler access was observed in both patients in the barbed group. no difference was observed with respect to non-infectious complications between the two groups (p = . ). patients in the hybrid group experienced a longer hospital stay when compared to the barbed group (p = . ). a re-admission occurred in the hybrid due an intraabdominal collection, while no re-admission was observed in the barbed group. no patient died in the postoperative period. conclusion: our results shows that the use of knotless barbed suture for enterotomy closure after laparoscopic intracorporeal right colectomy is safe, reproducible and associated with shorter operative time. aims: the accurate measurement and staging of rectal cancer, in particular the distal margin of low rectal tumours, is of paramount importance to optimise oncological surgical resection whilst preserving function. it is well recognised that the lower the tumour, the greater the technical challenges, operative time and the possibility of a temporary or permanent stoma. accurate localisation of the tumour is also essential to assist the multi-disciplinary team when considering neo-adjuvant chemoradiotherapy (crtx). the objective was to compare tumour height as reported on magnetic resonance imaging (mri) with endoscopic measurement. methods: a retrospective analysis of rectal tumour heights on pre-operative endoscopy and mri in patients undergoing radical colorectal surgery with curative intent over years from january . rectal tumours were identified as within cm of the anal verge (av). all mri measurements were reported by one of two specialist gastrointestinal radiologists. measurements were taken from the lowermost point of the tumour to the av. endoscopic measurements were as recorded by endoscopists including rectal surgeons, general surgeons, gastroenterologists and a clinical nurse specialist endoscopist. results: records of eighty one patients with histologically confirmed rectal adenocarcinoma were reviewed. median age was years ( to ). twenty three patients had or more endoscopies. on mri the median tumour height from the av was . cm ( . - cm) . on endoscopy the median tumour height was cm ( - cm) . on comparing endoscopy with mri, the median difference was cm ( - cm) . for over a third of patients ( %) tumours were lower on mri than endoscopy, median difference . cm ( . - cm) . only rectal surgeons documented tumour height in relation to the rectal folds. the majority of the repeat endoscopies were performed by surgeons to locate tumours more accurately pre-surgery. on no occasion was it documented whether the tumour had been measured during insertion or withdrawal of the endoscope. conclusions: precise localisation of rectal tumours is imperative to plan complex surgery and give informed counsel to patients. this study demonstrates the urgent need for a standardised protocol for all endoscopists to use while recording the distal extent of rectal tumours. objectives: the aim of the present rct was to compare the incidence of genitourinary (gu) dysfunction after elective laparoscopic low anterior rectal resection and total mesorectal excision (lar ? tme) with high or low ligation (ll) of the inferior mesenteric artery (ima). secondary aims included the incidence of anastomotic leakage and oncological outcomes. background: the criterion standard surgical approach for rectal cancer is lar ? tme. the level of artery ligation remains an issue related to functional outcome, anastomotic leak rate, and oncological adequacy. retrospective studies failed to provide strong evidence in favor of one particular vascular approach and the specific impact on gu function is poorly understood. methods: between june and december , patients who underwent elective laparoscopic lar ? tme in italian nonacademic hospitals were randomized to high ligation (hl) or ll of ima after meeting the inclusion criteria. gu function was evaluated using a standardized survey and uroflowmetric examination. the trial was registered under the clinicaltrials.gov identifier nct . results: a total of patients were randomized to hl (n ) or ll (n ). gu function was impaired in both groups after surgery. ll group reported better continence and less obstructive urinary symptoms and improved quality of life at months postoperative. sexual function was better in the ll group compared to hl group at months. urinated volume, maximum urinary flow, and flow time were significantly (p \ . ) in favour of the ll group at and months from surgery. ultrasound measured post void residual volume and average urinary flow were significantly (p \ . ) better in the ll group at months postoperatively. time of flow worsened in both groups at months compared to baseline. there was no difference in anastomotic leak rate ( . % hl vs . % ll). there were no differences in terms of blood loss, surgica l times, postoperative complications, and initial oncological outcomes between groups. conclusions: ll of the ima in lar ? tme results in better gu function preservation without affecting initial oncological outcomes. hl does not seem to increase the anastomotic leak rate. introduction: robotic single-site cholecystectomy (rssc) has been known to have some advantages such as reducing stress of the surgeon compared to single incision laparoscopic cholecystectomy (silc). however, there are few studies comparing the perioperative outcomes of these two operative methods. patient and methods: between march and february , rssc and silc were performed for benign gallbladder disease in our center. propensity score matching was performed to control variables including sex, age, body mass indes (bmi), diagnosis, american society of anesthesiologist (asa) score and cohorts were selected among the silc group through : matching. the perioperative data of these patients were analyzed retrospectively. the diagnosis was classified into acute cholecystitis, chronic cholecystitis, and gallbladder polyp. results: patient demographics between the two groups were evenly matched. total operation time including docking time was slightly longer in rssc group ( . min vs. . min, p \ . ), but real working time except the docking or set-up was shorter in rssc group ( . min vs. . min, p \ . ). conversion to additional robotic arm or additional port was frequent in silc group ( vs. cases, p = . ). intraoperative bile spillage rate ( . % vs. . %, p = . ) and postoperative hospital stay ( . days vs. . days, p = . ) were comparable in both group. conclusion: both surgical procedures performed safely. but the rssc demonstrated the better performance of the operation with shorter working time and the advantage of overcoming unexpected difficulties during the surgery with low conversion rate compared to silc. even though laparoscopic cholecystectomy(lc) is the gold standard procedure for cholelithiasis, patients are still suffering from various causes of pain. one of main causes is high pressure by pneumoperitoneum which makes peritoneal stretching and diaphragmatic irritation. however, there are few well-designed studies for evaluating pneumoperitoneum. therefore, we conducted a study to compare the postoperative pain after lc at serial different pressure methods. a prospective randomised double blind study was done in patients with benign gallbladder disease. they were divided into groups. each patients underwent lc with different pneumoperitoneum method; group a: far-low ( - mmhg), goup b: low ( - mmhg) and group c: standard pressure ( - mmhg). three groups were compared for pain intensity, duration, analgesic requirement and complications. post-operative pain score was significantly least in far-low pressure group as compared to low or standard pressure group during late periods ( , h). but, there were no pain score difference between far-low and low groups during early period ( , , , h) even though scores of standard group were significant higher than those of low group. number of patients requiring rescue analgesic doses and intraoperative complications were not significantly different among groups. this study demonstrates reducing the pressure of pneumoperitoneum results in reduction in intensity of post-operative pain. this study also shows that low pressure technique is safe with comparable rate of intraoperative complications. however, in immediate postoperative period, there is limitaton of pain relief after low pressure surgery. therefore, there may need new alternatives for pain. background and aim: anatomical hepatectomy with the glissonian approach is widely accepted as an important technique to ensure surgical safety and curability of the carcinoma. however, the histomorphological structure of the hepatic connective tissue is not sufficiently understood by surgeons. this study aimed to clarify the hepatic connective tissue structure using modern tissue imaging and analytical techniques. materials and methods: in total stained thin slices were loaded onto the computer and were reconstructed as dimages and analyzed. results: when the liver capsule enters the liver at the hepatic hilum, it becomes a sheath which envelops the portal pedicle. the hepatocytes in a row that constitute the periportal limiting plate at the edge of the hepatic lobule are firmly supported by the framework of the reticular fiber. the hepatic lobule and the portal area are in contact via the periportal space of mall. the framework of the limiting plate plays a role of a capsule of hepatic lobule (proper hepatic capsule) on the side in contact with the portal area. the binding site between the hepatic capsule and proper hepatic capsule (ppbs) is loose binding and is a layer that is easy to apply to surgical procedures. in order to enter between the liver capsule which became the sheath of the portal pedicle and the proper hepatic capsule at the hepatic hilum, the liver capsule must be dissected to reach the surface of the proper hepatic capsule. then, on the one hand, the portal pedicle is firmly gripped and pulled, on the other hand, the hepatic parenchyma covered by the proper hepatic capsule is pushed to expand between the portal pedicle and the liver parenchyma. at this time, the portal area (glisson's sheath) branched from the sheath of the portal pedicle into the gap of the hepatic lobule breaks like a string. with this dissecting plane, dissecting layer can reach to the next branch of the portal pedicle without entering into the portal pedicle or liver parenchyma. conclusion: understanding the connective tissue constituting the liver and conducting surgery turns the laparoscopic systematic hepatectomy into a standardized procedure. background: postoperative pancreatic fistula (popf) is the primary contributor to morbidity after distal pancreatectomy (dp). to date, no techniques used for the transection and closure of the pancreatic stump showed a clear superiority over the others. this study aimed to compare the rate of popf after pancreatic transection conducted with the reinforced stapler (rs) and ultrasonic dissector (ud) following dp. method: consecutive patients underwent dp from to were retrospectively reviewed. we included dps where pancreatic transection was performed by rs or ud and excluded dps extended to the pancreas head. to overcome the absence of randomization, we conducted a propensity matching analysis according to risk factors for popf. results: overall, patients met the inclusion criteria. the rs was employed in patients and ud in cases. after the one-to-one propensity matching, patients were selected from each group. the matched rs and ud cohort have no differences in baselines characteristics except for the mini-invasive approach, that was more common in the ud group ( % vs. %, p = . ). overall, patients ( %) developed a popf, a grade b ( %) and ( %) a grade c. in the rs group the rate of popf was % (n = ) and the ud group % (n = ) with a p \ . . conclusion: the results of this study suggest that the use of rs for pancreatic transection, reduces the risk of postoperative pancreatic fistula. a randomized trial is needed to confirm these preliminary data. aim: this study compares clinical and cost outcomes of robot-assisted single port and open longitudinal pancreaticojejunostomy (rlpj and olpj) for chronic pancreatitis. single incision mis needs more manual skills than conventional multiport operation. the advantage of better operation course is d vision and dedicate instrument. this paper aims to evaluate the feasibility and safety of the robot-assisted single incision with single port platform for chronic pancreatitis. materials and methods: clinical and cost data were retrospectively compared between open and ralpj. we collected patients since july, to september, . the patient was supinely placed in reverse trendelenburg position. the assistant surgeon was located between patient's legs. under general anesthesia a trans-umbilical . cm skin incision was made. a single incision advanced access platform with lagis port, glove portÒ (nelis, s. korea) and gelpoint combined with the da vinci si and xi surgical system (intuitive surgical, sunnyvale, ca, usa) pure or plus one was performed. the three arms, no. , no. , and da vinci scope, were in dwelled through the glove portÒ. pneumoperitoneum of mmhg was established through the port. a rigid -degree up scope was used during operation. results: twenty-one patients underwent lpj: open and ralpj. no robot-assisted cases converted to open were noted. patients undergoing ralpj had less intraoperative blood loss, a shorter surgical length of stay, less postoperative pain and lower medication costs. operation supply cost was higher in the ralpj group. no obvious difference in hospitalization cost was found. conclusions: versus the open approach, ralpj performed for chronic pancreatitis shortens hospitalization, less postoperative pain and reduces medication costs; hospitalization costs are equivalent. a higher operative cost for ralpj is mitigated by a shorter hospitalization and less pain control. robotassisted puestow procedure using single port platform is feasible and safe method. the total procedures by da vinci robotic system are safe and easily performed in highly selected patients. general surgery, hospital universitario infanta sofia, madrid, spain; general surgery, hospìtal quirón la luz, madrid, spain aims: the concomitant presence of abdominal wall midline hernias and diastasis recti is frequent. diastasis recti might be a risk factor not only for having but for recurrence of midline hernias. most open surgical procedures not consider the treatment of both pathologies, nor laparoscopic most spread out approaches. the author presents a novel endoscopic, extraperitoneal and retromuscular hernioplasty technique and its preliminary results. methods: a serie of patients is presented. a ct abdominal wall study is performed preoperatively. they all presented abdominal wall midline hernias in presence of a [ . cm concomitant diastasis recti. there were females and males. a totally endoscopic, extraperitoneal and retromuscular repair was performed, that included a midline anatomic restoration, tension-free hernia gap closure, omphaloplasty and skin treatment, if needed in every case. the tension-free massive-meshed hernioplasty included a bilateral totally endoscopic posterior components separation when needed. no drainages were used. all procedures included a bladder catheterization. results: all patient were dispatched within a period under h. no reoperations were needed in postoperative period. postoperative pain was measured by an eva scale. % of the patients have no pain medication after - h dispatching from hospital. % of the patients have a skin suffusion or hematoma. a male patient presented a temporary abdominal asymmetry due to a unilateral posterior component added to his technique. the mean following-up is to months ( - months) . no recurrence was observed. conclusions: preliminary results demonstrate this new approach to be a safe, feasible and a reproductible procedure. the 'terra' novel technique could provide of a new minimally invasive approach to abdominal wall midline hernias repair in the presence of a diastasis recti. only time and new results can predict the spreading out of this 'third way'. results: this study comprised males and females. mean age was years (range - years) and mean body mass index was . gh and mh were found intraoperatively in and cases respectively. mean operative time for all hernias (gh/mh) was min (range - min); min for gh (range - min); and min for mh (range - min). in . % of cases, hernia operative measurement was larger than preoperative size, especially in cases of incisional hernias ( . %). in . % of cases, laparoscopy found additional abdominal wall defects previously undetected by physical examination and by us-and/or ct-scan. a composite mesh and a non-composite mesh (up to cm in size) were used in . % and . % of cases respectively. the ethicon securestrap?? absorbable fixation device straps for sm fixation were employed in . % of cases. mean length of hospital stay was . days. mean follow-up time was months (range - months). in our study, there was one early (\ days) postoperative seroma ( . %), plus one late, small ( cm) symptomless recurrence, but neither needed reoperation. conclusion: the sutureless sm technique facilitates intra-abdominal introduction, as well as the handling and fixation of large/very large meshes. this new approach is safe and fast, even in cases of gh/mh repair. aims: any ventral hernia (vh) combined with rectus muscle separation (rms) must be repaired along with repairing the rms, otherwise there is a high risk for hernia recurrence. open rms repair is vast and traumatic surgery and laparoscopy is not effective. at a new era of repairing abdominal wall hernia by assisted endoscopy started with wolfgang reinhold's milos procedure. these procedures are somewhat complexed and real reconstruction of the linea alba (la) was limited, which done better by ferdinand koeckerling's elar technique. we perfected the elar technique to be fully endoscopic with wide mesh fusing to the muscles immediately by fibrin glue: extended endoscopic hernia & linea alba reconstruction glue (eehlarglue), achieving a low traumatic mis for vh and rms with excellent surgical and cosmetic results. methods: our eehlarglue is a totally endoscopic based technique used since . penetrating with optiview trocar and co pressure to the anterior rectus sheet (ars) level is followed by an extensive endoscopic dissection of the sub-cutaneous fat tissue from the ars. three trocars are inserted at the supra-pubic line enabling the dissection up to the xiphoid and costal margins laterally. any hernia sac is dissected, and the content reduced back to the abdominal cavity. relaxing incisions of the ars are performed longitudinally in the lateral aspect. the la is reconstructed by running two layers of non-absorbable sutures from xiphoid to pubis. a light mesh x cm is applied over the repair and the mesh is fused immediately to the muscles by fibrin glue. results: patients underwent the eehlarglue with follow up of months. all had significant rms of - - cm combined with primary or recurrent vh. recovery was smooth with - days of simple analgesics and return to regular activity within - days. no one had recurrent vh, but two males had limited rms and two early cases seroma formation. conclusions: our eehlarglue enables endoscopic vh repair and la reconstruction with extrastrength received by immediate mesh fusion to muscles with fibrin glue. thus, achieving low traumatic mis, easy recovering and very effective results-a perfect solution for patients with vh combined with rms. results: twelve blinded prospective rcts were used. when compared to tep repair, tapp repair has comparable seroma formation rates (chi = . ; (p = . ); ci - . , . ; i = %) and post-op pain at h (chi = . ; (p = . ); ci - . , . ; i = %). however, tep repair is associated with a significantly shorter operative time (chi = . ; (p = . );ci . , . ; i = %), post-op pain at hour (chi = . ; (p = . ); . , . ; i = %) and shorter hospital stay (chi = . ; (p = . ); ci . , . ; i = %). conclusion: tep is significantly better than tapp repair with regards to operative time, post-op pain at h and hospital stay. there is no significant difference with regards to post-op pain at h and seroma formation. background: primary hyperhidrosis (ph) is a neurological condition characterized by excessive sweating most often of the face, palms or axillae . palmar hyperhidrosis is treated through sympathetic chain clipping or transection .we aiming to compare the efficacy and results obtained with both techniques. patients and methods: sixty four patients underwent of sympathetic procedures from march to february . the patients were categorized into two groups: right sided transection sympathectomy and left sided clipping . patients were evaluated to compare the rates of success, satisfaction, compensatory sweating and recurrence either with transection or clipping of the t andt ganglion .mean follow up was ? _ months. results: sixty four patients males and females undergoing electro-coagulation sympathectomy on the right side and clipping on the left side. with mean age was years (range to years). all patients had balanced demographic data . no statistical difference between the two groups according rate of success. compensatory sweating was observed in patients ( . %) overall with cases of severe unsatisfied compensatory sweating. recurrence was reported in one case with transection and cases in clipping. satisfaction was occurred in cases in transection group and cases in clipping group .pnumothorax was occurred in cases in transection group compared to one case in clipping. no gustatory sweating and over dryness were reported in both groups. conclusion: both thoracoscopic sympathetic transection and clipping of t t ganglion are safe and effective procedure in palmar hyperhidosis treatment. with no differences regarding recurrence rate,satisfaction and incidence compensatory sweating. keywords: thoracoscopic sympathectomy,palmar hyperhidrosis, clipping, compensatory hyperhidrosis. introduction: primary ventral hernias and ventral incisional hernias pose a challenge for surgeons throughout the ages. even though minimally invasive surgery and hernia repair have evolved rapidly, there is no standardized method that adequately decreases postoperative complications. hybrid hernia repair is a surgical repair, which has not been adopted widely. it combines both a laparoscopic and open component allowing sac excision, primary defect repair as well as laparoscopic mesh insertion. aims: to evaluate the short-term and long-term outcomes of hernia recurrence for patients undergoing hybrid ventral repair (hvr) for the treatment of primary and incisional ventral hernias. methods: between october- and june- , hybrid vhr was performed in -patients at st mary's hospital, imperial college london. the medical records of these patients were reviewed retrospectively for demographics, comorbidities, prior surgeries, body mass index (bmi), hernial defects, hybrid technique used; mesh selection, operative time, complications and recurrences over a -year follow-up. results: twenty-four patients who underwent hybrid vhr were included with surgery performed by two surgeons. the mean age is -years with a mean bmi of . kg/m . % had incisional hernias and % had primary hernias. the number of hernia defects ranged from to , with the average mesh size used was x cm. extensive adhesionolysis was performed in % of patients. -day postoperative complications; patients developed post-operative seroma, paralytic ileus in , pain control in and urinary retention in patient. there were no conversions to open procedures. the mean length of hospital stay was -days. none of the patients developed chronic pain and only one recurrence over the -year follow-up period. conclusions: the hybrid technique for vhr is safe and feasible, and has important benefits over an open or purely laparoscopic approach, including a low rate of seroma formation, chronic pain and fiveyear hernia recurrence. future investigation may include randomized controlled trials, to fully evaluate the benefits of hybrid vhr, with careful assessment of patient-centred end-points including quality of life and postoperative pain. surgery, medical faculty-university of tetove, tetove, macedonia; general medicine, medical faculty-university of tetove, tetove, macedonia; anestesiology, medical faculty-university of tetove, tetove, macedonia; surgery, clinical hospital-tetove, tetove, macedonia laparoscopic cholecystectomy is widely used operative technique and it's characterized with less postoperative hospitalization and side effects. duration of the hospitalization after laparoscopic surgery depends on several factors of which pain and physical weakness are the most important. dexamethasone is well known; not only for its anti inflammatory effects but at the same time for analgesic and antiemetic effects, although the mechanism of this effects are not clarified yet. objectives: the aim of our study is the evaluation of analgesic effect of dexamethasone on reducing postoperative pain after laparoscopic surgery. patients and methods: in this study, patients aged - years old undergoing laparoscopic surgery, were classified into two groups, patients in each group. the first group were treated with a intravenous injection of mg dexamethasone preoperatively and another dose the next day after operation. the second group received a intravenous injection of normal saline. we evaluated the dose of consumed analgesics and antiemetic's drug during the first h in both groups. results: according to our experience results the total dose of tramadol in a postoperative period in dexamethasone receiving group was smaller than in normal saline group. measure of postoperative pain was assessed using the paper-based vas scale. our result shows that the intensity of post operative pain in a period during first h, after surgery in a group of patients treated with dexamethasone was lower compared with the group of patients treated with normal saline. nausea and vomiting during the first h was significantly lower in the dexamethasone group than in the normal saline group. surgery, hospital quiron sagrado corazon, sevilla, spain; surgery, hospital virgen macarena, sevilla, spain; surgery, hospital virgen del rocio sevilla, sevilla, spain aims: closing the defect (cd) during laparoscopic ventral hernia repair (lvhr) could be related to a reduction of seroma formation or bulging (hernia mesh) compared to conventional lvhr. but tension of the midline may contribute for some authors to a higher incidence of pain, recurrence in medium size defects and suggest to perform a component separation (cs) for restoring the midline in medium-large defects.we have developed a new technique for restoring the midline in medium ventral hernias (lira technique) and weanalyzed our results in terms of pain and recurrence compared to our conventional cd series (ccd). methods: we conducted a prospective controlled study of lvhr with ccd from january to december and a prospective controlled study performing lira technique from january to january . we analyzed and compared both techniques in medium size defects ( - cms) in terms of postoperative pain ( , days, , months and year) using a visual analogue scale (vas), bulging (return to prior distance among rectus muscles with the mesh in the sac in ct that didn't need surgical treatment)and recurrence (by physical examination and tomography). results: ccd was performed in patients (mean age was . ± . years old and mean bmi was . ± . kg/m ) and lira technique in patients (mean age was . ± . years old and mean bmi was . ± . kg/m ). the mean average follow-up in both series was year. mean average vas in ccd was . ± . ( day), . ± . ( days) . ± . ( month) . ± . ( months ) and at year. in lira series vas was . ± ( h) . ± . ( days), . ± . ( month), ( months) and ( year) . there are cases of bulging in ccd series and recurrence. bulging and recurrence were absent in lira series. conclusions: lira technique might be a safe procedure in medium size defects for restoring the midline in lvhr, and could be related to a lower pain rate compared to ccd with no recurrence or bulging. surg endosc ( ) :s -s background: the desire of pediatric surgeon to reduce incision related morbidity and pain while achieving good cosmetic results has recently led to the introduction of single incision pediatric endo-surgery [sipes] and needlescopic surgery. intracorporeal suturing and knot tying during sipes remains challenging. the aim of this study is to introduce a novel and simple technique for intracorporeal suturing of the pediatric inguinal hernia after needlescopic disconnection of hernia sac using just needles rather than laparoscopic instruments. it is an imitation of the principles of sewing machine. methods: the first author discussed the idea of the technique with the co-authors and a demonstration was done on a silicon pad before application of the technique on children with congenital inguinal hernia [cih] for peritoneum closure after needlescopic disconnection of the hernia sac. the main outcome measurements were; feasibility of the technique, knot quality, suture placement accuracy, performance and suturing time and recurrence rate. results: the sutures were snugly applied to the ridges of silicon pad with good approximation and the knot was firmly tightened in all experiments. after applying and mastering the technique on a silicon pad, we shifted to use it on children with hernia defect. all operations were completed by the needlescopic technique without the need for insertion of any laparoscopic instruments. the time required for suturing of the peritoneum around internal inguinal ring [iir] and knot tying, decreased considerably from min s in the first operation to less than min after the fifth operation and stabilized at approximately minute s. no major intraoperative complication and no recurrence. the primary end-point was to compare clinical outcome as well as cost effectiveness study between both groups. results: a total of patients were enrolled ( of them underwent tapp and olr). drop out occurred in cases ( of tapp and of olr group). patient characteristics were statistically similar between the groups. tapp procedure had less early post-operative pain (p = . ), a shorter length of stay (p = . ) and less postoperative complications (p = . ) when compared with the olr approach. a slightly higher recurrence rate in the tapp group was found. additionally, there is a trend towards a higher postoperative quality of life and less chronic pain in the tapp group. conclusions: tapp procedure for bilateral inguinal hernia effectively reduces early postoperative pain, hospital stay and postoperative complications. cannizzaro hospital, catania, italy aim: the purpose of this study was to evaluate the long-term results in terms of safety and efficacy of a new technique to repair incisional ventral hernias with a self-gripping mesh, after a mean follow-up period of months. methods: a retrospective, single-centre study was performed from june to june . all patients undergoing elective incisional ventral hernia repair were included. hernias were diagnosed based on clinical examination at the outpatient clinic. in case of doubtful diagnosis, ct-scan was used to confirm the diagnosis. the component separation technique and, when needed, tar were performed. the self-gripping mesh was placed in sublay position (overlap cm) with the self-gripping surface face down. in all cases drainage tubes were placed in retromuscular and supraaponeurotic position. the following characteristics were collected: age, sex, body mass index (bmi), smoking, comorbidities, number of previous surgical operations, defect size (ehs classification), mesh size, postoperative complications, duration of follow-up. all patients were interviewed by telephone every six months. when patients complained recurrence or other symptoms, visits were organized and when there was the doubt of recurrence a ct-scan was performed. results: a total of patients were included in this study, males, mean age was years. % of patients had bmi [ , smokers and diabetics were respectively % and %. the mean defect size was cm . component separation technique was associated with tar in patients. in cases the size of mesh was cm, while in patients the size of mesh was cm and in cases this was cm. in the other patients the mesh sizes were tailored to defect dimensions. subcutaneous seromas occurred in patients, they were treated conservatively in cases and with percutaneous punction in cases. long-term follow-up demonstrated recurrences in one case, while in another one ct-scan revealed a bulging. no cases of mesh infection, pain or sensation of mesh. conclusions: this study with a mean follow-up period of months demonstrated that the use of self-gripping mesh in sublay position is safe and effective to treat incisional ventral hernias. aim: morgagni hernias present technical challenges. the laparoscopic approach was described at first in , however, as they are uncommon in adult life and, little data exist on the optimal method of surgical management. this study purpose was to analyse a method for laparoscopic repair of morgagni giant hernias using laparoscopic primary closure. methods: this case series describes a method of laparoscopic morgagni hernia repair using primary closure. in all patients a laparoscopic transabdominal approach was used. the content of the hernia was reduced into the abdomen and the diaphragmatic defect was closed with a running laparoscopic suture using a self-fixating suture. clips were placed at the edges of the suture to secure the pledged sutures to both the anterior and posterior fascia. demographic data as age, gender and bmi were collected. operative data (operative time, rate of conversion, blood loss) and post-operative data (short and long term complications, length of hospital stay, need of readmission and reoperation) were recorded. results: retrospectively collected data about patients were analysed. there were ( . %) male and ( . %) females. the median bmi was . ± . kg/m . median operative time was ± min. there were no intraoperative complications nor conversion to open surgery. patients began a fluid diet on the first post-operative day and were discharged after a median hospital stay of ± . days. in a median follow up of months we did not observe any recurrences. conclusions: transabdominal laparoscopic approach with primary closure of the diaphragmatic defect is a viable approach for repair of morgagni hernia. in our experience, the use of laparoscopic transabdominal suture fixed to the fascia allowed the closure of the defect laparoscopically with minimal tension on the repairs. can we predict the success of the laparoscopic approach in the adhesive small bowel obstruction? c. tellez marques, e. sebastian valverde, e. membrilla fernandez, l. grande posa, i. poves prim general surgery, parc de salut mar-hospital del mar, barcelona, spain aims: the laparoscopic approach in the acute adhesive small bowel obstruction and internal hernias (asbo) has shown superior to laparotomy in terms of morbidity and hospital stay. especially, in patients who present simple adhesions or internal hernias. according to this, the aim of the study is to determine those preoperative factors associated with simple adhesions and internal hernias, and consequently, improve the success of the laparoscopic approach in asbo methods: a retrospective study of patients who underwent urgent surgery for asbo was conducted from january to may . we compare preoperative variables between single adhesions and internal hernias vs complex adhesions. a p value \ . was considered statistically significant. results: we analysed patients who underwent surgery for asbo, ( %) by laparoscopy and ( %) by laparotomy. conversion rate in laparoscopy was . %. . % of patients presented a single adhesion or internal hernia; and . % were considered complex adhesions. sex and age did not correlate with the type of adhesions. previous surgery (p \ . ), number of previous surgeries (p \ . ), asa (p \ . ) and previous abdominal wall mesh (p = . ) were significantly associated with complex adhesions. laparoscopy as the only surgical history was significantly associated with simple adhesions (p = . ). only appendectomy (p = . ) or supramesocolic (p = . ) previous surgeries tended to present single adhesions but it did not reach statistical significance. the need for intestinal resection was not related to the type of adhesions (p = . ). there was a significant correlation between the findings in the ct (computed tomography) and the type of adhesion found (p = . ). signs of ischaemia on ct were related to the need for intestinal resection (p \ . ). in the multivariate analysis, the number of previous surgeries, asa and ct scan findings were identified as independent factors related to the type of adhesion. conclusions: according to our study, a lower number of previous surgeries, asa i-ii and internal hernia in the ct scan are associated with single adhesions and internal hernias. patient selection is a key factor for the success of laparoscopic approach in asbo. aims: there aims of this study were: (i) to compare england with the united states in the utilisation of minimal access surgery (mas) and in-hospital mortality from four common abdominal surgical emergencies (appendicitis, incarcerated or strangulated abdominal hernia, small or large bowel perforation and peptic ulcer perforation). (ii) within england to evaluate the influence of mas upon in-hospital and long-term mortality. methods: between and , the rate of mas and in-hospital mortality for four abdominal surgical emergencies were compared between the united states and england. univariate and multivariate analyses were performed to adjust for underlying differences in baseline patient demographics. results: , admissions in england for four abdominal surgical emergencies were compared to an estimated , , admissions in the united states. after adjustment for patient demographics, mas was used less commonly england for three conditions; appendicitis (odds ratio (or) . , % ci . - . ), abdominal hernia (or . , ) and small or large bowel perforation (or . , ). in-hospital mortality in multivariate analysis, was increased in england compared to the united states for three conditions; abdominal hernia (or . , % ci . - . ), small or large bowel perforation (or . , ) and peptic ulcer perforation (or . , % ci . - . ). in england, after adjustment for patient demographics, open surgery was associated with increased in-hospital mortality for three conditions; abdominal hernia (or . , % ci . - . ), small or large bowel perforation (or . , % ci . - . ) and peptic ulcer perforation (or . , . similarly open surgery was associated with increased long-term mortality for three conditions; abdominal hernia (hr . , % ci . - . ) , small or large bowel perforation (hr . , % ci . - . ) and peptic ulcer perforation (hr . , % ci . - . ). conclusions: minimal access surgery was used less commonly and inhospital mortality was increased in england compared to the united states for common abdominal surgical conditions. given the benefits of mas shown in this large study, strategies to enhance adoption of mas in emergency conditions in england need to be optimised and include appropriate patient selection and improved surgeon mas training and experience. surg endosc ( ) :s -s background: in the treatment of inguinal hernias, there is little hard evidence concerning the economic reimbursement in the diagnosis-related-group (drg) era. factors that affect whether a hospital may earn or lose financially depending on open or laparoscopic approach is still underexplored. the aim of this study is to provide a reliable analysis of in-hospital costs and reimbursements in inguinal hernia surgery. methods: this retrospective study analysed the -year experience in inguinal hernia repair in patients undergoing open lichtenstein (ol), laparoscopic totally extraperitoneal unilateral (utep) or bilateral (btep) hernia repair. demographics, results, costs and drg-based reimbursements were recorded and analysed. results: during the study period, patients underwent ol, patients utep and patients btep. the average total cost amounted to eur in ol, eur in utep and eur in btep groups (p \ . *). the hospital reimbursement amounted to eur, eur and eur in the ol, utep and btep groups respectively (p \ . *). finally, the mean hospital earnings were eur, eur and - eur for each patient in ol, utep and btep respectively (p \ . *). conclusions: in-hospital costs were higher in utep and btep as compared to ol. the drg-based reimbursement provided adequate compensation for patients with unilateral inguinal hernia, whereas hospital earnings were profitable in ol group only, and led an overall financial loss in the btep group. surgeons should be conscious that clinical advantages of the laparoscopic approach are not adequately compensated for, from an economic point of view. aims: umbilical hernias are common anatomical defects in swine which become a suitable model for surgical training and research in the field of surgical meshes. the aim of this study was to develop a surgical protocol for a successful laparoscopic implantation of stem cell-coated surgical meshes. methods: large white pigs, weighing - kg and with congenital abdominal hernia were anesthetized for the surgical procedures. non absorbable polypropylene surgical meshes were coated with fibrin glue (fg) (control group) or with fg admixed with porcine bone marrowderived mesenchymal stem cells (fg/bm-mscs). approximation of hernia's borders was performed by intracorporeal suture. the meshes were carefully rolled inside the trocar for laparoscopic implantation. the surgical implantation was performed by laparoscopy using helicoidal staples. laparoscopic inspections and biopsies of the tissue surrounding the mesh were performed at , and days post-implantation. at day , the animals were euthanized and macroscopically evaluated. ultrasonography was used at day , , and to evaluate the size of the hernia. the biopsies were then processed for the histological analysis. results: ultrasonography demonstrated that the mean size of umbilical hernias before mesh implantation was . ± . cm. a decrease in hernia mean size was observed at day and . the laparoscopic procedures allowed a successful mesh implantation in all animals. in most of cases, the implantation site did not show excessive inflammation or tissue adhesions. but one animal showed hernia maintenance. one animal had peritoneal and implant-site infection. foreign body reaction was noted in the histological analysis, although no significant difference was found between the control, and bm-msc group. conclusions: the anatomical similarities between humans and pigs in umbilical hernias make this animal model useful to: i) improve minimally invasive surgical procedures for hernia treatment; ii) evaluate new surgical meshes, and iii) introducing stem cell therapy to hernia surgical repair. the laparoscopic approach is efficient and safe for the implantation of stem cellcoated meshes. gene and protein expression analysis are required to evaluate the molecular changes between the conventional and the stem cell surgical approach. aims: fluorescence angiography with indocyanine green (icg) is used as a marker in the assessment of tissue perfusion, being more frequently used in colorectal procedures. this technology has shown to be a good technique to reduce complications related to vascular supply to the anastomosis. in esophagogastric procedures blood supply to the gastric pouch, jejunum and esophagus could be evaluated by icg fluorescence imaging. it could be also used in bariatric surgery to evaluated the anastomoses, during gastric bypass, and blood supply to the gastroesophageal junction and the angle of his during sleeve gastrectomy. methods: we have collected data during gastric resection due to adenocarcinoma and bariatric procedures that were performed by the same surgeon, using icg fluorescence to evaluate blood supply. the icg was infused before performing the anastomosis in order to evaluate the need to change the transaction line (tl). we analyzed those cases in which the tl was changed and the number of leaks in those cases that we changed this line. results: all the cases were performed by laparoscopic approach: subtotal gastrectomy (sg), total gastrectomy (tg), gastric sleeve (gs) and gastric bypass. there were no changes regarding the tl before performing the anastomosis in any of the four types of procedures (sg, tg, gs, gb). in the analyzed data there is anastomotic leak in one sg procedure ( . %). conclusions: icg fluorescence angiography could be helpful in assessing blood supply during gastrointestinal anastomosis, although we have not find an influence in the results during bariatric and gastric procedures. however, we do not have the sufficient evidence to determine the value of this technology in this entities, being needed more volume and data to improve the significance of the results. aims: hyperspectral imaging (hsi) combines a spectrometer with a camera to analyze the tissues' optical properties in a broad wavelength range, without the need for a contrast agent. it provides extensive real-time information about tissue physiology, including oxygen saturation (sto ). fluorescence-based enhanced reality (fler) is a software solution providing a dynamic, quantitative analysis of the signal evolution of a systemically administered fluorophore, during fluorescence angiography (fa) . the aim of this study was to compare the performance of hsi and fler to assess bowel perfusion, in a porcine, non-survival model of bowel ischemia. methods: in pigs, an ischemic small bowel segment was created and imaged after hour of ischemia. the imaging modalities were applied sequentially to the same area.hsi was performed first, to acquire the sto spectra, by means of the tivita tm system (diaspective vision, pepelow, germany), which provides a spectral range of - nm and a nm resolution. subsequently, fa was performed using a nir-capable laparoscopic camera (d-light p, karl storz, germany), after intravenous injection of . mg/kg of indocyanine green (icg; infracyanine, serb, paris, france). the fluorescence flow was recorded during s, then the slope of the fluorescence flow was analyzed using a proprietary software to obtain a virtual perfusion cartography. the virtual cartography was overlaid onto real-time images to obtain the enhanced reality effect. ten adjacent regions of interest (rois) were selected from hsi datasets and were superimposed to fler-generated cartographies using a custom plug-in software function, allowing for a quantitative comparison of both imaging modalities. hsi was repeated after icg injection. results: the r correlation coefficient between hsi-sto and the fler slope was . . at control hsi after icg injection, the correlation coefficient dropped significantly (r . ). the interference of icg on hsi imaging was clearly identified in the spectral curves. conclusion: sto given by hsi provided results comparable to those obtained with fler in our bowel ischemia model, without the need to inject a contrast agent. icg interferes with hsi datasets, disrupting sto values. surgical treatment is one of the most effective options for treatment of giant hiatal hernia. laparoscopic approach became is a 'gold standard' over the time demonstrating all advantages of minimally invasive techniques over the open procedures. however the utility of robotic operations still remains controversial. aim of the study: evaluate the initial experience of robotic fundoplication in compare to laparoscopic procedures. materials and methods: since the january till the december of thirty operations were operated on. mean age was . ( - ), among them ( %) were female and ( %) were males. mean bmi was . ( . - . ) . laparoscopic procedures were performed in patients ( st group), robotic procedures with davinci system were performed in patients of the second group. nissen fundoplication modified was performed in patients, toupet fundoplication was used for patients. results: the median operative time in laparoscopic group was min, in robotic group- , min. there were no statistical differences between two groups (p = . ). blood loss was minimal in both groups. mean postoperative hospital stay was . days ( - days) in the st group and , days ( - days) in the second. there were no statistical differences between two groups (p = . ). postoperative course was uneventful in all patients of both groups. surgical stress response is associated with systemic inflammatory syndrome, sepsis, multiorgan dysfunction syndrome. robotic assisted surgery has been introduced to overcome the limitations of conventional laparoscopy. this technique has potential advantages over laparoscopy, such as increased dexterity, three-dimensional view, and a magnified view of the operative field. these advantages could result in limited intra-abdominal trauma and hence in attenuated surgical stress response over conventional laparoscopy. aims: this study aimed to synthesize data on the effect of robot assisted surgery on surgical stress response. methods: electronic databases were searched with the search terms 'surgical stress', 'stress response', 'oxidative stress', 'robotic assisted surgery', 'c-reactive protein', 'interleukin ', 'interleukin ','cortisol',;'oxidative stress markers', 'antioxidants', 'antioxidant status', 'mda', 'glutathione', 'cortisol', 'acute phase response' up to and including march . results: one hundred forty studies were identified and their title and abstract were reviewed. one randomized controlled trial, six non randomized comparative studies, one experimental study and one case report met inclusion criteria. data were discordant. one prospective trial concluded that cortisol and il- were lower in laparoscopic assisted distal gastrectomy compared with robot assisted distal gastrectomy in another study comparing robotic assisted laparoscopic radical prostatectomy with open radical prostatectomy based on plasma measurements of il- , il- a and c-reactive protein, it was demonstrated that robotic assisted laparoscopic radical prostatectomy induces lower tissue trauma than open radical prostatectomy. in another study, it was reported reduced expression of genes associated with surgical stress response in patients treated with robotically assisted radical prostatectomy compared with patients treated with open prostatectomy. the case report concerned a case of polymyalgia rheumatic after robotic assisted laparoscopic prostatectomy. the experimental trial demonstrated that cortisol and substance p were significantly higher with open thoracic approach versus robot assisted thoracoscopic oesophageal surgery. conclusion: further research is needed to elucidate the effect of robotic surgery on surgical stress, based on a well standardized protocol for the measurement of surgical stress response. purpose: tissue compression is essential to prepare the tissue for proper staple formation. this study evaluates the risk factors of compression injury on the circular stapling line in vitro. methods: to reproduce the artificial bowel wall, a collagen plate was prepared by mixing collagen extracted from porcine with glycerin. artificial collagen plates with mm and mm in the thickness were made for dry and healthy condition and immersed plates in the tap water for min to make wet and edematous condition. circular stapler (cdh a, ethicon, usa) was applied in the collagen plates (dry and wet condition) and optimal compressions. compression line was evaluated for compression injury score. risk factors for excessive compressions and unacceptable injury were analyzed. results: in the dry condition, optimal compression didn't cause unacceptable injury. in the wet condition, excessive compressions were occurred in . % with optimal approximation. unacceptable injury was significantly different in proper and excessive compression cases as . % and . %, respectively. on the univariate analysis, thickness ( mm), wet condition, proximal side, maximal compression, and excessive compression were associated with unacceptable injury. on the multivariate analysis using logistic regression model, excessive compression was significant independent factor to cause tissue injury (p \ . ) and this significance was also proved in the optimal compression group (p = . ). background: minimal invasive appendectomy gained much popularity due to its better cosmoses, early recovery and less wound site infections. single incision laparoscopic appendectomy (sila) has many disadvantages such as, long operative time, bad ergonomics, surgical site infections, high conversion rate and port site hernia. needlescopic appendectomy (na) using mediflexÒ facial closure needle expected to be more superior over sila. here in we compare our results of needlescopic appendectomy with single-incision one. material and methods: one hundred and twenty patients with acute non complicated appendicitis were randomly assigned to na and sila children for each group during the period between january to october . the main outcome measurements included, demographics, operative time, intraoperative complication, conversion rate, post-operative hospital stay, surgical site infection, port site hernia and cosmetic results. results: a total of children underwent appendectomy. there were children who underwent na and children who underwent sila. there were no difference in age ( . vs . years, p = . ), weight ( . vs . kg, p = . ) and hospital stay ( . vs . days, p = . ) between the two groups. there were no intraoperative complication during the two surgical approaches. operative time for na group is significantly shorter than single incision group ( . vs . min, p = . ). no single case of conversion in na group and cases needed conversion in sila group. seven cases of sila showed surgical site infection. cases of sila group presented with port site hernia. the na group was superior as regard ergonomics. the two groups showed equal excellent cosmetic results. conclusion: needles scopic appendectomy and sila are comparable as regard cosmetic results and hospital stay. na proved to be safe, applicable, repetitive and superior over sila as regard better ergonomics, less operative time, absence of surgical site infection and port site hernia. aims: to objectively analyze the surgical performance and surgeon's ergonomics in the use of a novel flexible laparoscopic instrument during intracorporeal suture, and compare it with the use of a conventional laparoscopic needle holder. methods: three experienced laparoscopic surgeons performed five laparoscopic sutures on an organic tissue using the novel flexible instrument (flexdexÒ) and five sutures using a conventional needle holder with axial handle. the new device is based on a mechanical design with no electrical components, which transfers the surgeon's hand, wrist, and arm movements to the instrument tip in an intuitive manner. the use of the instruments was organized in a random fashion. prior to the study, participants conducted a -minute training session with the new flexible instrument. execution time and quality of the suture were assessed for each repetition. besides, flexion and radioulnar deviation of the wrist were recorded using an electrogoniometer (biopac systems, inc.) attached to the surgeon's hand and forearm. the intensity of the forearm's muscle activation was also analyzed by means of a myo armband (thalmic labs). results: surgeons required more time to perform the intracorporeal suture using the novel laparoscopic instrument ( . ± . s vs. . ± . s; p \ . ), but the quality of the suture was similar with both instruments. the wrist flexion ( . ± . °vs . ± . °; p \ . ) and wrist ulnar deviation ( . ± . °vs . ± . °; p \ . ) were significantly lower when using the flexible instrument. during the suturing tasks, the use of flexdexÒ instrument led to a higher muscular activation of the flexor ( . ± . vs . ± . rms; p \ . ) and extensor ( . ± . vs . ± . rms; p \ . ) muscle groups of the forearm. conclusions: the presented novel instrument allows surgeons to perform robotic-like laparoscopic suturing. we believe that with a longer training period surgeons could potentially reduce surgical times with this device. the preliminary results of this study suggest that the use of this new instrument provides a quality of the suture similar to that obtained with a conventional laparoscopic needle holder and an ergonomically more adequate wrist posture. aims: the intraoperative real-time evaluation of tissue perfusion is one key element for successful visceral surgery. traditionally, tissue evaluation is performed visually by surgeons. newer devices for objective quantification have in majority been based on the application of the fluorescent dye indocyanin green (icg). a novel method derived from geographic research is hyperspectral imaging (hsi). the aim of this study was the evaluation of hsi as a promising method for the evaluation of tissue perfusion and its implementation in the evaluation of the gastric conduit during esophagectomy in a porcine model. methods: the hsi camera records a dimensional data cube from a dimensional surgical situs obtaining wavelengths between and nm. the absorption at different wavelengths is tissue-specific and influenced by the amount of oxygenated haemoglobin and other pigments. a software calculates different indices in real-time including oxygen saturation. a porcine model (n = ) is used for esophagectomy with gastric conduit formation. ischemia is induced artificially by magnets simulating staplers. different shapes of the gastric conduit and anastomosis formation are evaluated for perfusion metrics in order to obtain recommendations for the optimal formation of esophagogastrostomy. conclusion: hsi is a promising method for intraoperative evaluation of tissue perfusion that does not require application or injection of any agents. the preliminary results in this study showed that the gastric conduit receives its main blood supply from the gastroepiploic arteries and not via the mucosa. further results from the current evaluations enable formation of an optimized gastric tube and esophagogastrostomy in esophagectomy. surg endosc ( ) pediatric surgery, al azhar university, giza, egypt; pediatric surgery, beni suef university, beni suef, egypt background: varicocele is one of the most common causes of infertility. many surgical interventions are used for varicocele ligation including open and conventional laparoscopic multiport or single incision techniques. the aim of the study is to present a new needlescopic lymphatic sparing varicocele ligation using mediflexÒ facial closure needle and gauge vascular access cannula. material and methods: twenty-two male children with bilateral varicocele of grade ii-iii. all children were counseled by clinical examination, doppler ultrasonography, abdominal ultrasonography, and routine laboratory investigations. testicular lymphatics were delineated by subcutaneous injection of / cm methylene blue in anterior wall of the scrotum min prior to surgery. the testicular vessels (both vein and artery) were ligated one cm above the deep inguinal ring using two mediflex needles with preservation of lympatics. the main outcome measurements included; operative time, hospitalization, testicular atrophy, hydrocele formation, recurrence of varicocele and intra or postoperative complication. results: a total of twenty-two male children with grade ii-iii varicocele subjected to needlescopic lymphatic sparing technique. twenty one were bilateral. background and aims: even if the clinical outcomes of robotic rectal resections are under investigation, the related robotic costs have not yet been well addressed, and the differences between the robotic rectal resection costs and the laparoscopic approach are still not well known. we have therefore performed a prospective comparative study of robotic rectal resections (rrr) and laparoscopic rectal resections (lrr) performed at our centre with the aim to evaluate the cost-effective outcomes of robotic versus laparoscopic surgery. study design: this is an observational, comparative prospective non-randomized study which includes patients that underwent laparoscopic and robotic rectal resection reaching a minimum of months of follow up from february to march , at the sanchinarro university hospital, madrid. an independent company performed the financial analysis and fixed costs were excluded. outcome parameters included surgical and post-operative costs, quality adjusted life years (qaly), and incremental cost per qaly gained or the incremental cost effectiveness ratio (icer). the primary end-point was to compare clinical outcome as well as cost effectiveness study between both groups. results: a total of rrr and lrr were included. the mean operative time was significantly lower in the lrr approach ( versus min; p = . ). the main pre-operative data, overall morbidity, hospital stay and oncological outcomes were similar in both groups, except for the readmission rate (rrr: . %, lrr: . %;p = . ).the mean operative costs were higher for rrr ( . versus . €; p = . ); however, the mean overall costs were similar ( . € for rrr and . € for the llr; p = . ). mean qalys at year for rrr group ( . ) was higher than that associated with lrr ( . ) (p = . ). at a willingness-to-pay threshold of , € and , €, there was a . % and . % probability that rrr group was cost-effective relative to lrr approach. conclusion: this study provides data of cost-effectiveness differences between rrr and lrr approach showing a benefit for the rrr aim: the efforts were aimed to the introduction of novel surgical technologies to overcome the intrinsic anatomical and technical constraints of rectal surgery. this was the case of the introduction into the clinical practice of laparoscopy and later on of robotic surgery for rectal surgery. however, whether robotic surgery is actually superior to laparoscopy is still debated. the aim of this study was to compare d laparoscopy and robotic surgery for rectal cancer on technical and oncological outcomes. methods: this was a single-center, prospective, randomized controlled trial. all patients more than years of age undergoing elective surgery for rectal cancer situated from to cm from the anal verge were included. patients undergoing abdominal perineal amputation and/or with t and/or m tumours were excluded. patients were randomized before surgery into two arms: arms a ( d laparoscopy) and arm b (robotic), and gave their consensus to the study. demographic data, data regarding the tumour, operative and post-operative data were collected. patients with a follow up shorter than months were excluded as well. results: twenty patients were enrolled in arm a and in arm b in the period time of one year. patients' population of the arms was homogeneous as concerns demographic characteristics and stage of the disease. robot-assisted rectal resection results in comparable operative time ( . vs min; p = . ). the conversion rate was significantly lower for arm b ( vs p = . ). postoperative morbidity was comparable between groups. hospital stay was comparable but time required to resolve post-operative ileus was shorter in arm b ( . vs . days, p = . ). overall survival and disease-free survival were comparable between arms ( . % vs . %, p = . , and . % vs . %, p = . , respectively) conclusions: d laparoscopy and robotic surgery are two viable options for rectal surgery. robotic surgery can add some in terms of post-operative outcomes and ergonomics. aim: currently, robotic surgery for rectal cancer is a surgical operation that is being performed worldwide. we also introduced robotic surgery in . however, after robotic surgery, we observed a rise in creatinine kinase (ck), which is unlikely to happen in other surgeries. we studied the postoperative complications of rectal cancer patients who underwent either robotic surgery or laparoscopy during the same period of time. methods: from january to november , patients underwent surgery using robotassisted rectal resection (da vinci si cases and xi cases) and patients underwent laparoscopic rectal resection. in this study, abdominoperineal resection, intersphincteric resection, and lateral lymph node dissection were excluded. result: the operation time for the robotic surgery group was significantly longer than that for the laparoscopic group ( min vs. min; p \ . ). the ck value of the robotic surgery group on pod was significantly higher than that of the laparoscopic group ( iu/l vs. iu/l; p \ . ). in addition, one case of compartment syndrome was observed in the laparoscopic group. there were no significant differences in age, body mass index, intraoperative bleeding, tumor invasion depth, urination disorder, or postoperative hospital stay. in robotic surgery, it is considered that the increase in ck value is caused by the extended operation time, contact of the patient's cart with the left thigh of the patient, and the extra force applied to the abdominal wall caused by the displacement of the remote center. conclusion: in robotic surgery, it is suggested that the measurement of postoperative ck value is important. therefore, an attempt to shorten the operation time and paying attention to the surgical field are necessary to improve the outcomes. aims: anastomotic leak remains as one of the most important and life threatening post-operative complications in colorectal surgery. this complication has important consequences, both acute and long term, longer hospital stay, re-intervention, and increased morbidity and mortality. among all different circumstances that have been related to this entity, blood supply is an important factor that might have influence. fluorescence with indocyanine-green (icg) is used as a marker in the assessment of tissue perfusion in colorectal surgery which might reduce the numbers of leaks. methods: a multicenter analysis of the experience of centers in spain is collected in order to assess the value of icg in colorectal anastomosis. colorectal procedures were performed using icg to evaluate vascular supply in the anastomosis. icg was infused before performing the anastomosis analyzing the number of cases in which the transection line (tl) was changed. we also analyzed the number of leaks in those cases that we changed this line. results: out of the cases performed, cases were performed by open surgery, by laparoscopy, by single-port and with transanal total mesorectal excision(tatme). the following procedures were performed: right colonic resection(rc), splenic flexure partial resection(sf), left colonic resection(lc), subtotal colectomy(sc), total colectomy(tc), hartman reversal surgery(hr), low anterior resection with partial mesorectal-escision(lar) and ultra low anterior resection with total mesorectal-escision(ular). leak rate(lr) was . % ( . %rc, . %lc, . %sc, . %lar, . %ular). overall lr was . % in colonic surgery and . % in rectal surgery. the tl was changed due to icg in . % of the cases ( . %rc, . %sf, . %lc, % tc, . % lar, . % ular), being . % in colonic resection and . % in rectal resection. the relation between leaks and the cases in which the tl was changed, were % ( . %rc, %lc, . %ular). conclusion: icg fluorescence may play a role in anastomotic tissue perfusion assessment. the lr after colorectal surgery might decrease using icg to detect the proper tl before to perform the anastomosis. however, we do not have the sufficient evidence to determine that the changing transaction line can lead to avoid leaks. surg endosc ( ) aims: to analyse the value of postoperative day crp as an early predictor of safe discharge in robotic rectal cancer surgery. methods: a retrospective analysis was performed, including patients who had undergone robotic total mesorectal excision (tme) in a single centre over a -year period (may -september . patients who had a permanent stoma (abdominperineal resections or hartmann's procedure) were exluded from the study, leaving patients for further analysis. as the los is currently used as a performance tool in assessing outcomes in colorectal surgery (with a cut-off established at days), we compared the crp values in these groups. results: fourty one percent of patients were discharged home within days. they had an earlier peak of crp on postoperative day (pod) (median . , ) . the group of patients that were discharge home after days ( %) had a crp peak on pod (median , ). on pod , the group of patients that went home within days had a lower crp ( - -vs. - -) compared to the group of patients that were discharge after days, p = . ). conclusions: a crp peak on pod in robotic tme can predict an early and safe discharge (los within days). background: purposelateral pelvic lymph node dissection (lpnd) is suggested to treat suspected lymph node metastasis in pelvic side-wall in patients with rectal cancer who underwent preoperative chemoradiotherapy (crt). however, technical difficulties make it possible that lateral pelvic lymph nodes (lpns) are not dissected completely and, thus, remained in the narrow pelvis. near-infrared fluorescence imaging (fi)-guided surgery is expected to help visualization and complete excision of nonvisible lymph nodes during cancer surgery. this study aimed to evaluate the efficacy of fi using indocyamine green (icg) to identify lpns during robotic lpnd. methods: rectal cancer patients who were suspected lpn metastasis and had received preoperative crt were prospectively enrolled. icg in a dose of . mg was injected around tumor preoperatively. all procedures were performed with a totally robotic approach. after completing lpnd, fi was checked again for identifying remained lpns and resecting them completely. results: the lpns were successfully detected in ( . %) of the patients. however, after accounting for eight cases, having finished adjusting icg injection, the lpns were successfully detected in ( . %) of patients. the fi-guided lpnd group (n = ) showed similar mean operative time for unilateral pelvic dissection and complication rate, compared to patients who underwent conventional robotic lpnd (n = ). however, the mean number of unilateral harvested lpns was . in the fi-guided lpnd group, which was greater than the mean of . in the conventional group. lpn metastasis was identified in % of the fi-guided lpnd group, which was higher than that of the conventional group, . %. conclusion: fi-guided lpnd identifies lymph nodes in pelvic side-wall with great reliability. this contributes to increased number of lpns yield compared to conventional robotic lpnd. this technique should be considered to dissect them completely by preventing subsequent missing of nonvisible lpns. aims: to compare the medium-term oncological outcomes of laparoscopic total mesorectal excision (l-tme) vs. robotic total mesorectal excision (r-tme) for rectal cancer. methods: a retrospective analysis was performed including patients who underwent l-tme or r-tme resection between - . patients with disease stage iv at diagnosis or r resection were excluded. patients were initially included, and cases of r-tme were matched based on age, gender, stage and time of follow-up with an equal number of patientswho underwent l-tme. we compared -year disease free survival (dfs) and overall survival (os). in adittion, a multivariate analysis was performed in order to idenfity independent prognostic factors for -year dfs and os. results: pathological outcomes were similar between groups. however, major complications were lower in the robotic group ( . % vs. . %, p = . ), highlighting the anastomotic leakage rate, which was . % in the r-tme vs. . % in the l-tme group (p = . ). overall, the -year dfs rate was % in the laparoscopic group and % in the robotic group (p = . ). the -year os rate was % in the l-tme groups and % in the r-tme group (p = . ). for disease stage iii, -year dfs was significantly higher in the r-tme group. os was also significantly superior in the robotic group for every stage, reaching % in the stage iii. in the multivariate analysis, r-tme was a significant positive prognostic factor for distant metastasis (or . %ci . , . , p = . ) and os (or . %ci . , . , p = . ). conclusions: r-tme for rectal cancer can achieve better oncological outcomes compared to l-tme, especially in stage iii rectal cancers. the robotic approach has demonstrated to be a significant positive prognostic factor for local recurrence and overall survival, due to the better postoperative outcomes. however, a longer follow-up period is needed to confirm the oncologic findings. university hospital for visceral surgery, university of oldenburg, oldenburg, germany; bremen spatial cognition center, university of bremen, bremen, germany aims: in clinical settings, realistic assessment of one's own abilities can enhance performance and promote patient safety, especially in surgical residents, who inevitably have to acquire skills during real surgery. this study thus implemented the global assessment of laparoscopic skills (goals) questionnaire with the aim to explore divergences between resident self-evaluation and specialist's evaluation on laparoscopic performance, as a first step to implement the goals questionnaire as a tool for constructive and objective feedback. methods: between july and october , seven residents from the university hospital for visceral surgery at the pius-hospital oldenburg participated in this study. at the end of every laparoscopic operation where the resident acted as the primary surgeon, the resident and the supervising surgeon independently evaluated the resident's operative performance using the goals questionnaire. the five dimensions evaluated were depth perception, bimanual dexterity, efficiency, tissue handling and autonomy. a cumulative goals-score (with being the highest possible score) was calculated for n = laparoscopic operations. resident's year of training, the level of case difficulty and the type of laparoscopic procedure performed was also analysed. results: residents overestimated their laparoscopic abilities in . % of the operations (goals-scores: residents: median = , mean = . ; specialists: median = , mean = . ; p \ . ). residents in the first three years of surgical training were more likely to overestimate their performance (residents: median = . , mean = . ; specialists: median = , mean = . ; p \ . ) than those with more than three years of surgical experience (residents: median = , mean = . ; specialists: median = , mean = . ; p = . ). goals score differences did not depend on case difficulty and laparoscopic procedure. conclusions: surgical residents tend to overestimate their intraoperative laparoscopic performance when compared to specialist evaluation. overestimation was found to depend on one's own laparoscopic experience and seem to disappear with gained expertise. these results signify the importance of individually adapted training and the greater need for objective feedback for surgical residents. this approach could in return increase the skill acquisition rate of the resident and in return contribute towards enhancing patient safety. introduction: the delivery of safe surgical care is dependent of various, complex and interrelated factors. substantial data exist regarding the impact of training in human factor skills on surgical outcomes. however, except for the standardized time-out process, the best way to go about improving these skills remains unclear. the aim of this study was to gain more insights in the theatre staff's perception of human factors and their importance on surgical outcome in the operating theatre. methods: the surgical team assessment record (star) questionnaire was used to study the role of human factors, such as communication, situational awareness and organization, contributing to surgical team performance. the self-assessment questionnaire was filled out by the theatre staff, directly after the surgical procedure. conditional logistic regression was used to identify the impact of the role in the operating theatre on the yes versus no answers. results: some questionnaires were completed. the theatre staff rated their team's performance with a median of (iqr . , -point likert scale). the surgical fellows (n = ) rated their personal factors significantly lower compared to the rest of the operating team (median versus , p-value \ . ). the staff surgeon (n = ) indicated significantly more often that there were many distractions ( . %, yes n = ) and noticed aberrations ( . %, yes n = ) during the surgical procedure (pvalue \ . ) when compared to the rest of the operating team. most aberrations reported by the surgeons were related to technical performance. conclusions: human factors play an important role in the surgical environment. situational awareness may be less developed in members of operating teams, compared to the surgeon of that team. further work is needed to elucidate the impact of human factor skills on team performance. a team-based approach to safety interventions is recommended. future studies should determine what type of aberrations and distractions are most relevant and valuable to embark on with team training. dept. of digestive surgery, school of medicine, tokushima university, tokushima, japan; dept. of digestive surgery, tokushima university, tokushima, japan background: the qualitative evaluation for laparoscopic training of medical students was performed using rubric evaluation, and weak points in conjunction with the lack of anatomical knowledge were derived. to conquer these weak points, virtual reality (vr) ? augmented reality (ar) training for understanding of regional anatomy was investigated. materials and methods: one hundred and six students in th grade of tokushima univ. participated basic laparoscopic task training (gummy band ligation, beads transfer, delivery of beads, gauze excision) with training box and sham laparoscopic cholecystectomy with virtual simulator. rubric evaluation, as qualitative evaluation, which includes the evaluation standards for each maneuver were performed before and after basic task training and sham operation. the group which self-evaluation was higher in a rubric evaluation was investigated. the d image of vessels and bile duct obtained from mdct of real patient was projected in reality space with microsoft hololens. training of ar image using hololens was performed for understanding of regional anatomy. after training of regional anatomy with hololens, sham laparoscopic cholecystectomy was performed again, and quality of procedure was evaluated by rubric. anatomical questions were. results: rubric evaluation in basic task training showed no difference between self-evaluation and evaluation by tutor before and after practice. in sham laparoscopic cholecystectomy, several students showed higher score than tutor, especially in part of extension of operation field by elevation of the gall bladder, exposure of triangle of calot, and exposure of cystic duct. after ar training, all students showed high score in questions related regional anatomy during operation. especially, rubric evaluation of students who showed high self-evaluation in sham operation showed same score with tutor. conclusions: as rubric evaluation showed weak points of detailed parts of maneuver, and vr ? ar was useful for understanding details of regional anatomy for laparoscopic training. background: the eaes has recently published an intraoperative adverse event classification to aid reporting of minimally access surgery events. this includes capture of non-consequential errors. we aimed to investigate the clinical impact of these apparent 'near miss' events. methods: case videos and clinical data from a completed multi-centre laparoscopic total mesorectal excision randomised controlled trial was utilised (isrctn ). the eaes classification was applied by two blinded assessors to all enacted adverse events identified on video analysis using the observational clinical human reliability analysis technique. the total number of grade (non-consequential) errors were compared with the number and nature of day morbidity events (graded with the clavien-dindo system) and length of stay. results: cases ( h of surgery) contained error events of which ( . %) were classified as eaes grade (median per case, interquartile range - , range - ). there were significantly more inconsequential errors recorded in patients that developed any early morbidity event than those who had an uneventful post-operative recovery (median . (iqr - ) vs. ( - ), p = . ). a stepwise increase in the sum of eaes grade errors is seen for each additional day morbidity event reported ( . vs. vs. vs. , p = . ) and the highest clavien-dindo grade experienced ( vs. vs. vs. . p = . ). positive correlation is observed between the sum of eaes grade a errors and length of post-operative stay (r s = . , p = . ). conclusion: in the context of major laparoscopic surgery, near misses are commonplace and correlate with surgical outcomes. this may represent a novel surrogate assessment method for intraoperative performance. aims: diagnostic laparoscopy (dl) is an under-utilised procedure that can replace non-therapeutic exploratory laparotomies in many contexts. to date, no validated education programme for dl exists. this study seeks to evaluate the feasibility, acceptability and face, content, construct validity of the laplat curriculum (laparoscopic learning for abdominal trauma; a simulationbased curriculum for trauma dl). this is in addition to the development of a novel d-printed bench-top model for abdominal inspection. methods: this prospective and observational pilot study involved novice medical students and junior doctors. surgeons from the uk and international (n = ) were involved in a two stage delphi-process to determine the components of the training course which were used to formulate a final curriculum. in the absence of an adequate model for abdominal inspection, a novel dprinted abdominal inspection model was designed and produced. after an introductory familiarisation session as well as pre-course cognitive lectures, the novices performed tasks on a virtual reality and bench-top simulator with repetitions of each in a half-day session. outcome measures for construct validity were total time to complete task, accuracy, percentage of horizon maintained and economy of movement. face and content validity as well as acceptability was evaluated by a qualitative and quantitative survey. results: face, content and construct validity as well as acceptability was established. face validity was demonstrated in all components of the course (including pre-course cognitive content and technical tasks) in addition to content validity. all also met an acceptability threshold of / on a -point likert scale. one-way anova tests demonstrated construct validity in all tasks (p \ . ) with learning curves in reducing time observed. using a performance improvement metric, one-way anova tests showed similar rates of improvement per participant between most tasks (p [ . ). the course was rated overall mean . / (± . ). conclusion: this pilot study has demonstrated the feasibility, acceptability and face, content and construct validity of the laplat curriculum as well of the novel d-printed abdominal inspection model. randomised controlled trials are needed to establish higher-quality evidence, as part of a wider curriculum with transfer needed to the clinical environment. surgery, regional institute of gastroenterology and hepatology, cluj-napoca, romania; anesthesiology-surgical propedeutics, university of agricultural sciences and veterinary medicine, cluj-napoca, romania; radiology, regional institute of gastroenterology and hepatology, cluj-napoca, romania; urology, training and research center, prof. dr. sergiu duca, cluj-napoca, romania; general surgery, training and research center, prof. dr. sergiu duca, cluj-napoca, romania aims: to evaluate the benefits of systematical use of ex vivoliver model and ct imaging in the planning process for swine laparoscopic liver resections done by residents during training programs. methods: twenty four general surgery residents were equally divided into two groups: first one which performed laparoscopic liver resections without planning stage and the second one which systematically used anatomical data from a swine liver model and interactive ct scans d reconstructions. the planning stage included an interactive tutorial for establishing the strategy for the next resection followed by performing open liver dissection and the same resection on an ex vivoswine model. a total of twelve models were used during this step. afterwards, laparoscopic procedures were performed on sixteen anesthetized domestic pigs, two swine for every team, composed of three residents. both groups were part of a dedicated and continuous training program and used the same 'step by step' protocol for resections. results: the average time for imagistic planning was . min and for open dissection and resection was . min. all teams successfully completed the interventions and followed the standardized protocol without trainers' interventions and with no conversions. the second group obtained better results regarding the time needed for completion and blood loss. also, when the planning stage was applied the resection was more accurate and less functional parenchyma was removed. the 'warming up' by adding the imagistic and anatomical data to the core protocol offer more clarity before laparoscopic liver resections. this also makes an upgrade for our 'step by step' protocol and provides sufficient data to admit this planning stage as mandatory for laparoscopic liver resection on swine during a training program. introduction: submucosal tunnel endoscopic resections (ster) had been increasingly performed for treatment of gastric subepithelial tumors. one of the limitations for ster is the risk of incomplete tumor resection due to close dissection and bridging of tumor capsule. endoscopic full thickness resection (eftr) allowed complete resection of the tumor with margins to prevent recurrence. this study aimed to review the techniques and outcomes of eftr for treatment of gastric subepithelial tumors. method: patients who received endoscopic resection for gastric subepithelial tumors were recruited. the gastric subepithelial tumors were considered eligible for endoscopic resection with size \ mm. all patients received preoperative assessments including eus and ct scan to define the extend of tumors and the proportion of extra and intralumenal components. all the procedures were performed under general anesthesia with co insufflation. eftr started after injection with mucosal incision up to % of tumor circumference, followed by submucosal dissection to identify tumor margin. further dissection was performed using esd devices. after adequate exposure of lateral margins, incision into muscularis propria was performed to achieve full thickness resection. luminal defects were closed by either clips, clip-loop crown method or overstitch suturing. results: from to , patients received eftr for gastric subepithelial tumors. the mean age was . years, and were male. the gist were located at greater curvature ( ), cardia ( ) , lesser curve ( ) and antrum ( ) . the mean size was . mm ( - mm) . most of the eftr were performed in operation theatre while two were done at endoscopy. the mean hospital stay was . days, and mean operative time was min ( - mins). there was no conversion to laparoscopy. closure of luminal defect were performed mostly with clips ( ), followed by overstitch ( ) and clip and loop crown closure ( ) . most patients resumed full diet on day , and all the pathologies confirmed gist tumors with clear resection margins. conclusion: endoscopic full thickness resection is technically feasible and safe procedure for treatment of gastric gist. future research should focus on refining the techniques of eftr and closure of the defect. next generation endoscopic intervention (project engine), osaka university, suita, japan; gastroenterological surgery, osaka university, suita, japan; research & development, -d matrix, ltd., chiyoda-ku, tokyo, japan; research & development, fuso pharmaceutical industries, ltd., cyuou-ku, osaka, japan background: hemostatic peptides have received increased attention. self-assembling peptides (tdms) comprise synthetic amphipathic peptides that immediately react to changes in ph and/or inorganic salts to transform into a gelatinous state. since tdms do not carry a risk of infection, their clinical application as new hemostatic agent is expected to increase. the first generation of these peptides (tdm- ) is currently used as a hemostatic agent in europe. however, tdm- exhibits slow gel-formation and low retention capabilities on tissue surfaces. the second generation (tdm- ) was therefore developed to encourage faster gel-formation and better tissuesealing capabilities, and we subsequently verified its usefulness and increased performance relative to tdm- in preclinical open surgery. aim: the aim of this study was to verify the efficacy of tdm- in terms of its hemostatic effect in endoscopic surgery. materials and methods: evaluation of the hemostatic effect in endoscopic surgery (animal study) was performed using eight female ( kg) pigs in spine position. following systemic heparinization, we established a bleeding model by utilizing flexible endoscopic grasping forceps on the anterior wall of the stomach and duodenum. in the hemostasis method, an endoscope with a distal hood was brought into contact with the bleeding point, and ml tdm- was applied to the wound. after tdm- gelation, the endoscope was removed, and the acute hemostatic effect (after min) was confirmed. histologic evaluation was subsequently performed on resected specimens. results: in the endoscopic bleeding model, of the cases ( . %) showed complete hemostatic effects on the anterior wall of the stomach, whereas on the anterior wall of the duodenum, of cases ( %) showed complete hemostatic effects. moreover, none of the gels were displaced from the anterior walls of the stomach and duodenum, and histologic evaluation confirmed no infiltration of inflammatory cells. the new self-assembling peptide (tdm- ) displayed improved hemostatic effects relative to the previous generation (tdm- ) in endoscopic surgery. tdm- had potential usefulness for upper gastrointestinal bleeding. our future work will assess its usefulness for laparoscopic surgery. objective: indocyanine green (icg) is a dye used in medicine since the mid- s for different applications in ophthalmology, cardiology and hepatobiliary surgery; thanks to its selective hepatic uptake and biliary excretion, it can be used to evaluate hepatic function in patients scheduled for hepatic resection surgery. the aim of this study is to evaluate the efficacy and the feasibility of icg guided surgery in the intra-operative localization of liver tumors, comparing the pre-operative radiological aspect, the intra-operative visualization and the post-operative histopathological features of the tumors. materials and methods: icg was intravenously injected for a routine liver function test (limonÒ) in patients who underwent hepatic resection surgery for primitive and secondary liver tumors in the period between november and september . for each patient was performed an intraoperative visualization of the stain both in vivo and ex vivo, using a nearinfrared imaging system. all the images were recorded. results: a correct differentiation between liver parenchyma and tumor area was obtained in . % of cases. five patients were not evaluable due to widespread uptake or complete absence of uptake; it was probably the first cases enrolled in the study for which we were not able to set doses and timing of administration of icg. in patients in which the method had been feasible, we observed a prevalence of nodular pattern in patients with hepatocellular carcinoma ( %) and a predominance of rim pattern in both cholangiocarcinoma ( %) and metastasis ( %). furthermore, in patients with hccs well-intermediate differentiated (g -g ) was found predominantly a nodular pattern ( . %), whereas in poorly differentiated ones was prevalent a rim appearance ( %). regarding radiological correlations, the only one patient who presented an atypical radiological feature in pre-operative evaluation, showed a lesion with no icg captation in intra-operative visualization. conclusions: icg fluorescence imaging is a safe, minimally invasive and quite inexpensive method, that can be easily administered for routine evaluation of pre-operative liver function. it can be a useful support tool in the intra-operative detection of liver tumors, especially in laparoscopic surgery where it is not possible to directly touch the tissue. surgery, bundang cha medical center, seongnam-si, korea; surgery, severance hospital, seoul, korea; surgery, nhimc ilsan hospital, ilsan, korea; surgery, seoul national university bundang hospital, seongnam, korea; surgery, asan medical center, seoul, korea backgrounds & aims: robotic surgical system had been widely accepted in various surgical field with the expectations of overcoming the limitation of laparoscopic surgery. however, robotic liver resection had not generalized, so far. thus, this study aimed to evaluate the feasibility and safety of robotic major liver resection by prospective multicenter study. methods: from july to december , five surgeons who were novice in robotic liver resection but experienced a lot in open and laparoscopic liver resection in five tertiary hospitals performed cases of robotic major anatomical liver resection. perioperative patient's clinical data and surgical data were prospectively collected. results: cases of left hemihepatectomy, case of extended left hemihepatectomy, cases of right hemihepatectomy, cases of right anterior sectionectomy, cases of right posterior sectionectomy, and one cases of central bisectionectomy were performed. the most common indications were hepatocellular carcinoma for cases following intrahepatic cholangiocellular carcinomas for cases, liver metastases for cases, sarcoma for case, intraductal papillary neoplasms for cases, mucinous cystic neoplasm for case, hemangioma for case, and intrahepatic duct stones for cases. surgical resection margins for all tumor cases were negative. total average operation time was . ± . min and estimated intraoperative blood loss was . ± . ml (minimal to ml). in terms of severe surgical complication, there were cases of postoperative fluid collection treated with drainage and one case of bile leakage treated with percutaneous trans-hepatic biliary drainage. only one case out of cases was converted to the conventional open left hemihepatectomy because of bleeding. conclusions: in this study, robotic anatomic major liver resection might be safely performed even by robotic beginners but advanced open and laparoscopic liver surgeons. surgical technique: with the patient at °on right lateral decubitus, access is gained through the path of the percutaneous drainage catheter after opening of the aponeuroses of the oblique and transverse muscles of the abdomen. a mm laparoscopic trocar is inserted and a cavity is created with pneumoretroperitoneum at mmhg. it is accessed with an optic of °and mm, and the work space is extended with aspiration and hydrodissection. with mm grippers, the necrotic material is removed, washed and drained. a two light silicone probe is left, one light for drainage and another one for washing. results: the mean age was . background: minimally invasive surgery has achieved worldwide acceptance in various fields, however, pancreatic surgery remains one of the most challenging abdominal procedures. in fact, the indication for robotic surgery in pancreatic disease has been controversial. the present study aimed to assess the safety and feasibility of robotic pancreatic resection. methods: we retrospectively reviewed our experience of robotic pancreatic resection done in sanchinarro university hospital. clinicopathologic characteristics, and perioperative and postoperative outcomes were recorded and analyzed. aim: this work aims to study the contact pressure between the moving capsule and a synthetic small intestine in order to provide design guidance for prototyping the self-propelled capsule robot for small-bowel endoscopy. method: since small-bowel peristalsis consists of peristaltic contraction and wave distension, the contacts between the capsule and the small intestine are multimodal. we consider three contact cases for the capsule robot. case : the capsule moves on a flat small intestinal surface; case : the capsule moves in a collapsed intestine with a flat surface support; and case : the capsule moves in a surrounded small intestine. by considering these three contact cases, experimental testing and finite element analysis (fea) were conducted by measuring the contact pressure between the small intestine and the capsule. introduction: traditional laparoscopic instruments have limited degrees of freedom and are not ergonomic. this results in severe limitations in performing complex, and even simple tasks in surgery, limiting many surgeons from performing a variety of minimally invasive procedures. handx tm is a hand-held, electromechanical smart instrument with robot-like features. the instrument is composed of a sophisticated user interface that enables unrestricted hand movement, and a novel, motor driven articulating tool that is controlled by the interface. the instrument is . mm in diameter, lightweight, and can be easily moved between laparoscopic trocars and perform complex motions in the surgical field. after the regulatory process was completed we have tested the device clinically through a structured, approved, clinical trial. materials and methods: after irb approval patients were recruited to the trial. we have included a variety of procedures that require suturing and complex tissue manipulation. two experiences surgeons performed all procedures. after completing each procedure the surgeons completed a detailed standard usability (sus) questionnaire. results: procedures were completed successfully without complications or device malfunction. there were female and male patients with an average bmi of . procedures performed were right hemicolectomis with intra-corporeal anastomosis, paraesophageal hernia repairs and fundoplication, diagnostic laparoscopies, tapp procedures, ventral hernias with fascial suturing, and laparoscopic cholecystectomies. the average performance score was . / . the results suggest that the handx device is safe and easy to use and may offer a simple solution for enhancing minimal invasive surgery capabilities and possibly reduce conversion rates while maintaining current standard surgery flow.the handx could potentially extend the surgeon's abilities to access hard to reach anatomy and perform complex maneuvers and present a cost-effective alternative to large console-based robotic systems. objective: endoscopic submucosal dissection (esd) has become widely accepted treatment for rectum neuroendocrine neoplasm. the aim of this study is to evaluate the safety and efficacy of esd with dental floss-assisted suspension traction for rectal neuroendocrine neoplasm. methods: we retrospectively reviewed the medical records of the patients, who underwent esd for rectum neuroendocrine neoplasm at endoscopy center of zhongshan hospital, fudan university. the data of operation time, r resection and adverse events were collected analyzed.in dfs-esd group: after the mucosa was partly incised along the marker dots, the next step was to construct traction device, similar to others in esd, with dental floss and hemoclip. the dental floss was tied to any arm of the metallic clip. the hemoclip was attached onto the incised mucosa, another hemoclip was attached onto normal mucosa opposite to the lesion in the same way. the submucosa was clearly exposed with the traction of dental floss and the resection could proceed. results: patients were enrolled in the study. there were patients treated by esd with dental floss-assisted suspension traction and patients treated by conventional esd. the average tumor size was ( . ± . )cm in both group. the operation time was . ± . min in conventional esd group and ( . ± . ) min in dfs-esd group (t = . , p = . ). according to pathological grading about rectal neuroendocrine neoplasm, there were grade (g ) and grade (g ) in conventional esd group while grade (g ) and grade (g ) in dfs-esd group (? = . , p = . ). among cases in this study, all the basal resection margins were negative, the en blot resection rate was % and the curative resection rate was %. however, pathological results showed tumor tissue close to the burning margin in cases of conventional esd group and in cases of dfs-esd group (? = . , p = . ). conclusions: esd with dental floss-assisted suspension traction for rectum neuroendocrine neoplasm can assist exposing tumor borders, provide good vision during the procedure and offer clearer anatomic structure, so as to simplify operation, reduce operation time and ensure the negative basal margin. it is especially suitable to be promoted in primary hospitals. surg endosc ( ) aims: force feedback and assessment provides detailed insight into tissue manipulation skills. the aim of this study is to evaluate learning curves for basic laparoscopic skills based of force and motion learning curve patterns. morevover, we aimed to detect the favourable time span for this curriculum for each individual trainee. methods: in this prospective cohort study, first year surgical residents participated in a three week at home training course. a mobile box training was equipped with forcesense system for objective force, motion and time based assessment. the system provides seventeen unique metrics. the training goal was set by the mean score of proficient laparoscopic surgeons. each repetition was captured and made available for analyses. continuous force feedback was provided during training. curve fitting was used to estimate the learning curve plateau and the number of repetitions needed to approach the plateau phase and to reach proficiency level. finally, a comparisson between novices and experts was executed. results: a total of attempts, executed by residents were captured and analyzed. significant improvement of motion analysis parameters (e.g. path length and time) was observed for all training tasks, except for the fifth tasks. tissue manipulation skills (i.e. maximum and mean applied force) significantly improved by training tasks , and . learning curve analysis revealed various shapes and lengths of the individual learning curves. a large range in learning curve plateaus was found between trainees and between tasks. each trainee managed to accomplish the preset goals within three weeks. conclusion: force-and motion based assessment provides insight into both tissue manipulation and instrument handling skills. when combined in learning curve analysis, these parameters effectively show progression towards proficiency for each individual trainee over time. we emphasize the variation in learning curves between trainees. therefore, we recommend individually tailored courses provided with objective force-and motion-based learning curve tracking. aims: the posterior retroperitoneoscopic adrenal access represents a challenge in orientation and working space creation.the aim of this experimental acute study was to evaluate the impact of computer-assisted quantitative fluorescence imaging on adrenal gland identification and perfusion assessment in the posterior retroperitoneoscopic approach. methods: six pigs underwent synchronous (n = ) or sequential (n = ) bilateral posterior retroperitoneoscopic adrenalectomy (pra, n = ). fluorescence imaging was obtained via intravenous administration of ml of indocyanine green (icg) using two near-infrared camera systems. fluorescence-based visualization of adrenal glands before vascular division (n = ), after main vascular pedicle ligation (negative control, n = ) or after adrenal division (n = ) was followed by completion adrenalectomy. one of the animals had undergone icg injection h previously, during another study. the dynamic evolution of fluorescence signal intensity over time was recorded and analyzed using a proprietary software. the computed color-coded perfusion cartography was superimposed onto real-time images obtained by corresponding left (l) and right (r) camera systems. the slope of fluorescence signal intensity evolution over time in the regions of interest (roi) served to assess adrenal perfusion by means of quantitative fluorescence signal analysis. results: in the retroperitoneum, the adrenal glands were promptly highlighted after primary intravenous icg administration or showed an increase in fluorescence signal intensity upon reinjection (both glands in a recovery pig and one gland in the sequential approach). after left adrenal main vascular pedicle ligation, the gland displayed low perfusion (blue; rois a -a in figure ), while a weak fluorescence signal after completion adrenalectomy suggests perfusion via collateral vessels. with intact vascular supply, the caudal segment of the right adrenal (a ) gland showed a significantly higher perfusion rate (red) than the ischemic cranial segment (a ). quantitative analysis of logarithmic fluorescence intensity showed a statistically significant difference between perfused and ischemic zones (p = . ) allowing to assess gland vascularity. kidneys (k) and adrenal glands showed distinct perfusion curves ( figure ). conclusions: prior to dissection, fluorescence imaging allows to easily discriminate the adrenal gland from surrounding retroperitoneal structures. during adrenal gland surgery, icg injection complemented by a computer-assisted quantitative analysis helps to distinguish between wellperfused and low-perfused segments. giant adrenal tumors:technical considerations and surgical outcome a. giordano, g. alemanno, c. bergamini, p. prosperi, v. iacopini, a. dibella, a. valeri sod chirurgia d'urgenza, aou careggi, firenze, italy objectives: giant adrenal tumors are tumors with size more than cm. these are rare cancer associated with malignancy in % of cases. the size of these tumors is an important topic in literature because of their higher probability of malignancy and possible technical limitations of laparoscopic approach. we report our center's experience on laparoscopic adrenalectomy. materials and methods: in the last ten years we performed about adrenalectomies for benign and malignant adrenal tumors. of these were giant tumors. the medium size was . cm ( - cm). tumors were on the left adrenal gland and on the right. there were women and men, the average age was ( - years). of these cancers were laparoscopically removed and with open approach. cases of open conversion. results: betweenn the tumors laparoscopically removed we recorded cases of carcinoma, endothelial cysts, adenomas ( with aldosterone and with cortisol hypersecretion), myelolipomas, pheochromocytomas and metastases from lung carcinoma. the surgical outcomes in these patients were optimal in terms of good pain control and hospital stay (median days). the average time of the intervention was min with very low blood less ( ml). no postoperative complications were recorded. the removal of the adrenal gland necessitated or trocars. in the dissection and resection phases we always used radiofrequency scalpel. the follow up after and months didn't show local recurrences. conclusions: laparoscopic adrenalectomy offers significant advantages over the open approach. the size of these tumors is still at the center of debate for the choice of the technique. the tumor size is only a predictive parameter of possible malignancy. the laparoscopic approach is a safe and feasible method in terms of surgical and oncological outcomes also for the giant adrenal tumors, only if performed by expert surgeons and in high-volume centers. vascular or adjacent organs infiltration is a contraindication to the laparoscopic approach. aims: adrenal gland size greater than cm is considered a contraindication to laparoscopic adrenalectomy (la). aim of the present case-control study is to compare the surgical outcomes in patients undergoing la for adrenal gland measuring = cm versus = . cm in diameter. methods: from january to august , las were performed in the two authors' centers which follow an identical treatment protocol. eighty-one patients with an adrenal gland size = cm (intervention group) were included in the study. based on body mass index (bmi) class [ kg/m ) , lesion side (right or left), surgical technique (anterior transperitoneal for right and left-sided lesions, anterior transperitoneal submesocolic for left-sided lesions) and lesion type (conn-cushing, pheocromocytoma, primary adrenal cancer or metastases, other type of lesion), patients with an adrenal gland lesion measuring = . cm in diameter were included (control group) and paired to the intervention group. results: comparing the intervention and control groups, statistically significant differences were observed in mean lesion size ( conclusions: the only significant difference between the two groups was the operative time which was longer in the intervention group. conversion and complication rates were also higher in the intervention group but the difference was not statistically significant. based on the present data, adrenal gland size measuring more than cm in diameter is not a contraindication to a laparoscopic approach. ; and orthopaedics and urologists for the remaining . %.the costs from these claims, differed from to % of the total damage burden per year. the review of medical charts of claims related to laparoscopic gynaecologic surgery showed that % of claims were filed for visceral and/or vascular injuries ( % bowel injuries, % ureter). % of the injuries were entry-related. a delay in diagnosing injuries was the primary reason for financial compensation. conclusion: evaluating and learning from complications and claims will improve medical health care. in contrast to overall trends and developments considering medical claims, claims concerning laparoscopic surgery decreased, possible due to a rising learning curve. considering laparoscopic surgery, extra caution is required at moment of entry and the early recognising complications and at pre-operative counselling from patients. the aim of the study was to determine indications and contraindications for laparoscopic splenectomy in abdominal trauma patients and to analyze results of the operations. patients and methods: the study involved patients with spleen injury grade iii who were admitted in our institute in the years of - . the patients were divided on two groups. laparoscopic splenectomy was performed in patients (group i) and 'traditional' splenectomy was carried out in patients (group ii). there was no difference in the demographic data and trauma severity between the two groups.non-invasive investigations, such as laboratory investigations, serial abdominal ultrasound examinations (us), x-ray in multiple views and computed tomography (ct) had been performed before the decision about necessity of an operation was made. results: patients after laparoscopic operations had better recovering conditions compare to patients with the same injury after 'traditional' splenectomy. neither surgery related complications no mortalities were registered in both groups. laparoscopic splenectomy was more timeconsuming operation than 'traditional' splenectomy. we suggest that as experience of laparoscopic splenectomy is gained the operation time will be reduced. conclusion: laparoscopic splenectomy is a safe feasible operation in patients with spleen injury. the operation is indicated in patients with spleen laceration more than cm of parenchymal depth with moderate continuing bleeding or expanding hematoma and contraindicated in patients with hemodynamic instability and high bleeding rate (more than ml/h on serial us examinations). the isolated hydatid disease of the spleen is a quite rare condition, liver and lungs being the most common locations. the treatment requires usually splenectomy, open or laparoscopic. there are few reports in the literature describing a spleen-preserving type of surgery. we present a case of a female patient, y.o., with a large cystic lesion of the spleen, cm in diameter. lab tests and ct scan confirmed that is a hydatid cyst. after albendazole treatment and vaccination the patient was referred to us for surgical treatment. the procedure was performed under general anesthesia and laparoscopic approach was performed with the intention to preserve the spleen. after the cyst was identified and adhesiolysis was done, the area was isolated from the rest of the abdominal cavity with sponges with a betadine solution in order to prevent contamination. a needle aspiration of the cyst allowed the evacuation of ml of purulent content, an indicator of a dead cyst. betadine solution was injected into the lesion. laparoscopic excision of the cyst was performed using advanced electrocoagulation devices and the spleen removal was not deemed necessary. two drainage tubes were placed in the remnant cavity. an abdominal ultrasound was performed in the third postoperative day and no collections were identified. the postoperative outcome was uneventful; the patient was discharged in the th postoperative day. the conclusion is that in selected cases, with the cyst located in the anterior part of the spleen, with proper equipment and experienced laparoscopic teams, the cyst can be successfully treated without splenectomy. deep neuromuscular block was induced with rocuronium . mg/kg. in group , forty patients were enrolled for reversal of profound neuromuscular block during thyroid surgery (sugammadex mg/kg, after identification of vagus nerve). in group , thirty-five patients were enrolled profound neuromuscular block during thyroid surgery(without reversal of nmbd). tof-watch acceleromyograph was recorded in response to adductor pollicis muscle for ulnar nerve stimulation in patients with both groups; recovery was defined as a train-of-four (tof) ratio = . .to prevent laryngeal nerve injury during the surgical procedures, all patients were neurophysiologically detected using ionm. results: the total duration of surgery was higher in group than group ( . ± . , . ± . ;p \ . ). the mean time to recovery of the tof ratio to . was higher in group than group ( . ± . , . ± . ; p \ . ). the mean duration of vagus reverse (v : , milisecond) was higher in group than in group ( . ± . , . ± . ; p \ . ). no significant difference was found between left and right v -v and r -r values in group following nerve monitoring, whereas in group , a significant difference was found between left v -v , left r -r and right v -v values ( introduction: oeosphagogastric oncology trials have often lacked robust methods of monitoring and surgical quality assurance (sqa), leading to difficulty in interpretation of trial results. this study aims to assess expert opinion regarding challenges to sqa in oncology trials and potential mitigating strategies. method: a purposive international cohort of expert stakeholders with experience in oncology trials were recruited including: surgeons; oncologists; trial methodologists, and; trial managers. semi-structured interviews were thematically analysed using grounded theory. spss was utilised to assess differences between trial stakeholders' opinions. results: emergent themes were identified and consensus themes emerged on qualitative analysis of stakeholder responses. key consensus challenges to implementation of sqa in oncology trials included: insufficient resources; limitations of surgical volume in centre selection; differing oncological beliefs and resistance to change adoption; overly prescriptive protocols and standardisation contributing to difficulty in surgeon recruitment; and cultural factors leading to difficulties in providing and receiving feedback. seminal consensus mitigating strategies to overcome challenges to sqa in oncology trials included: trial centre selection according to case volume (n = , %); requirement for specific centre attributes for inclusion in trials including specialist centre designation and participation in national audit (n = , %); consideration for surgeons learning curve in surgeon selection (n = , %); flexible standardisation of trial operating (n = , %); operation manual utilisation to aid standardisation of surgical interventions (n = , %); case monitoring using video (n = , %) or photographs (n = , %); direct intraoperative observation by an expert (n = , %), and; histopathological assessment of resected specimens (n = , %). other methods of monitoring surgical quality advocated included: recording post-operative outcomes; lymph node yield; case report forms; and real time data monitoring (n = , %). oncologists were significantly more likely to state the importance of standardisation of surgery in oncology trials (p \ . ), and trial methodologists significantly more likely to advocate consideration of surgeons' learning curve in surgeon selection (p \ . ). conclusion: surveying international expert stakeholder opinion revealed a wide variety of perceived challenges across all domains of surgical quality assurance. proposed mitigating solutions require consensus opinion to formulate a framework to aid design of sqa measures within future oncology trials. research group did not register a single case of ega leakage while patients in control group (? \ , ). had the leakage which was stopped by means of 'endovac' system. there were cases of esophagus postoperative strictures which developed months after the surgery in the research group which was less than in the control group which saw cases of strictures of ega (? \ , ). months after surgery, the number of post-operative strictures increased in both groups, but was lower in the research group and amounted to cases in the research group and cases in the control group (? \ , ). there were cases of esophagus postoperative strictures which developed months after the surgery in the research group which was less than in the control group which saw cases of strictures of ega (? \ , ). neither of the groups had any cases of post-operative mortality. purpose: to investigate the prognostic effects and risk factors of the omission and delay of postoperative chemotherapy of ii/iii gastric cancer (gc), with the goal of providing a reference for interventions of related departments. methods: the clinicopathological data of patients undergoing radical gastrectomy for ii/iii gc were collected and retrospectively analyzed. we defined the chemotherapy delayed until more than days after radical gastrectomy and the complete omission of chemotherapy as unacceptable chemotherapy initiation (uac group), while the chemotherapy conducted within days of radical gastrectomy was defined as acceptable chemotherapy initiation (ac group). the survival between the two groups was compared, and the trends and risk factors of uac were analyzed. results: the total number of patients who underwent totally laparoscopic distal gastrectomy with uncut roux-en-y and delta shaped billroth-i anastomosis was and , respectively. the mean reconstruction time was longer in uncut roux-en-y than in delta shaped billroth-i, ( . ± . vs. . ± . min, p \ . ). the uncut roux-en-y was used more cartridge than delta shaped billroth-i anastomosis ( . ± . vs. . ± . , p \ . ). however there was no significant differences in operation time, estimate blood loss, number of retrieved lymph node and postoperative course between reconstruction methods. postoperative complications more than clavien-dindo grade iiia occurred in cases ( . %) of postoperative early complications and cases ( . %) of late complications. the endoscopic findings showed excellent short and long-term outcomes in terms of very low incidence of bile reflux and reflux-induced remnant gastritis in uncut roux-en-y compared with delta shaped billroth-i anastomosis. conclusions: uncut roux-en-y gastrojejunostomy was a useful reconstruction method with totally laparoscopic distal gastrectomy for cancer, especially for diverting enteral contents from the remnant stomach and preventing remnant gastritis. therefore, it is recommended for young patients with early stage disease who have a long time to live after distal gastrectomy for cancer. operative technique: the seromuscular layer above the tumor is dissected, while the mucosa is kept unbroken. when seromuscular layer is dissected all around the tumor, the full layer is lifted, and the mucosa is stretched. the mucosa is then transected with a stapling device to execute fullthickness resection of the specimen. finally, the seromuscular defect is repaired by hand-sewn suture. results: since december , clean-net has been performed in patients with gastric smts. all tumors were resected en-blocwithout rupture. the average operation time ranged from to min with an average of . min. the postoperative course was uneventful. microscopically the surgical margin was tumor-negative (r resection) in all cases. the margin width was small with an average of . mm ± . . conclusions: clean-net is a useful option in the laparoscopic surgical treatment of gastric smt, when excessive sacrifice of the healthy gastric wall surrounding the endophytic tumor should be avoided. background: the type of fundoplication-complete or partial is still controversial for the surgical treatment of gerd. laparoscopic toupet ( wrap) fundoplication has less post op dysphagia and gas bloating compared to nissen fundoplication ( wrap) and is advised to be the procedure of choice when esophageal manometry findings are abnormal, however it is considered by some less effective and more difficult to perform. the aim of this research was to determine in the functionality and efficacy of the different types of fundoplication. methods: explanted pigs stomachs weighing - kg were studied. two different studies of the les were performed: distensibility and failure point (occurrence of reflux according to volume added to the stomach). for both studies we first disrupted the lower esophageal sphincter using a rigiflex tm dilating balloon. we then performed three different fundoplications-nissen, toupet, dor and measured the distensibility of the egj after each fundoplication. the failure point was determined following each fundoplication type. results: we used pig stomachs for the distensibility study and pig stomachs for the failure point study. there was no statistically significant difference between the nissen and toupet fundoplications when distensibility was measured, however the egj was more distensible following dor fundoplication (p = . for nissen, . for toupet). when the failure point was measured, nissen fundoplication was significantly more effective than toupet, and toupet was significantly more effective than dor (p = . ,p = . respectively) conclusions: we studied the differences between the mechanical effects on the egj following three different fundoplications, encompassing , , and of the esophagus. we demonstrated that there is a significant difference between dor fundoplication and nissen/toupet when distensibility was measured. there was no difference in the distensibility of the egj following a or wrap. there was, however a significant difference of effectiveness between all three fundoplications. these findings suggest that the and fundoplications have similar functionality while the wrap mechanically prevents possible reflux and support proponents of toupet fundoplication rather than nissen due to the similar functional results while decreasing the post op dysphagia and gas bloating complications. surg endosc ( ) aim: to describe patients undergoing surgical treatment of incident gastro-oesophageal reflux disease and the use of anti-reflux treatment in a danish population-based cohort. methods: all adult danes - undergoing upper endoscopy and receiving a diagnosis of gerd within days were identified. patients with previously diagnosed gerd, peptic ulcer-disease, barrett's oesophagus or cancer of the gastrointestinal tract were excluded. in this study, only patients undergoing anti-reflux surgery within two years of gerd-diagnosis were subsequently included. age, sex, charlson comorbidity index (cci), anti-reflux surgery (primary and re-operative) and endoscopic dilatation were identified using the danish national patient registry. mortality was identified using the national civil registry. pharmacological treatment of gerd (proton pump inhibitors, h \ su \/su-blockers and other prescription anti-reflux drugs) as well as use of nonsteroid anti-inflammatory drugs (nsaid) and anti-thrombotic treatment were identified using thethe danish national prescription registry. all data was linked on an individual level using the unique identification number that all danish citizen are assigned to at birth or first immigration. results: a total of first-time fundoplications were performed, hereof . % performed laparoscopically (n = ) and . % performed using open technique (n = ). at one-year followup, . % (n = ) had undergone endoscopic dilatation and . % (n = ) had undergone reoperation. the -day mortality was \ . %. patients had a median age of years ( - years) and were predominately male ( . %-n = ). a total of . % had cci (n = ). diagnoses were gerd with esophagitis ( . %-n = ), gerd without esophagitis ( . %, n = ) and gerd without specification ( . %, n = ). before initial endoscopy, , % (n = ) used at least one type of anti-reflux drug, dropping to . % (n = ) in the year after anti-reflux surgery. however, even when censoring patients with barrett's esophagus or peptic ulcer disease after initial endoscopy and patients undergoing concomitant treatment with nsaids or antithrombotic drugs, . % still used at least one type of anti-reflux drug after surgery. conclusion: in this population-based study, anti-reflux surgery was safe and lowered the use of pharmacological treatment. however, even when adjusting for competing reasons for use of antireflux drugs, . % used at least one type of anti-reflux drug one year after surgery. the new approach to perform nissen fundiplication m. paranyak, v. grubnyk surgery, odessa national medical university, odessa, ukraine nearly % of patients who undergo laparoscopic anti-reflux surgery at long-term follow-up need for surgical reintervention mostly because of hiatal hernia (hh) recurrence, wrap migration or disruption. purpose: the aim of our prospective study was to evaluate and compare several technics of wrap fixation and determine whether modified nissen fundoplication(mnf) reduce failure rate in the long term follow up. materials and methods: this was a prospective, randomized, controlled trial. from november to october one hundred and thirty-eight gerd patients who underwent anti-reflux surgery were divided into two groups. excluded criteria for our study ware diagnosed hiatal hernia (hh) type iii. in the i group which include patients we performed the following manipulations: nf was supplemented with suturing wrap to the diaphragmatic crura ( patients) on each side using two non-absorbable stitches. such technique permit us to create more symmetrical wrap. in case of weak conditions of crura or short esophagus ( patients) fundoplication wrap was sutured to the body of stomach using two non-absorbable stitches on each side. control group ( patients) underwent classic nissen fundoplication (nf) without wrap fixation. all patients were assessed before and after surgery using validated symptoms and quality of life (gerd-hrql) questionnaires, -h impedance-ph monitoring and barium-swallow. results: baseline characteristics were similar between groups. there were no conversion to open procedure or mortality. mean hospitalization was . days ± . days. at , months (range -- ) of followup, the overall rate of complications after mnf was , % ( hh reccurence) and nf , % ( hh reccurence, slipped wrap). patient in mnf group show significant improvement in gerd-hrql score, from . ± . (preoperatively) to . ± . (postoperatively) (p? \ ? . ). complete ppi independence was achieved in %. in the ii group of patients mean gerd-hrql score decline from . ± . (preoperatively) to . ± . (postoperatively), postoperative ppi treatment was necessary in %. conclusions: according to our study mnf minimized risk of slipped wrap and intrathoracic migration of the wrap and can make positive impact on reducing the failure rate of laparoscopic anti-reflux surgery. aims: comparative evidence across laparoscopic antireflux procedures does not exist. aim of this project was to identify direct comparative evidence between laparoscopic antireflux procedures and synthesize evidence using network meta-analytical methods. methods: the databases of medline, amed, central, opengrey were interrogated. pairwise meta-analyses for each pair of interventions using a random-effects model and network metaanalysis in stata was performed using the mvmetacommand and self-programmed stata routines. differences between direct and indirect evidence were explored by comparing direct and indirect estimates though computing the inconsistency factor within each closed loop of evidence. the ranking probabilities for all treatments of being at each possible rank for each intervention were computed using the mvmetacommand in stata. a hierarchy of the competing interventions was obtained using rankograms. quality of evidence was assessed using grade-nma and the cinema application. results: forty-three publications reporting on randomized trials and some patients were identified. the network of treatments formed a closed loop between °, °and anterior °; and star network between °and other treatments; and between anterior °and other treatments. laparoscopic °, °, anterior °and anterior °were equally effective in the control of heartburn and this was supported by low quality of evidence according to grade-nma. the odds for dysphagia were lower for anterior °(high quality evidence), anterior °( moderate quality evidence), °(moderate quality evidence) and proton-pump inhibitors (moderate quality evidence) compared to °. the odds for gas-bloat were lower for °and anterior °compared to °(low quality evidence). the odds for regurgitation, morbidity and reoperation were similar across treatments, albeit these were associated with very low quality evidence. anterior °had a % probability of being the best treatment in terms of dysphagia. conclusion: under consideration of treatment effect estimates, evidence quality as assessed with grade-nma and other parameters, anterior °, anterior °and °should be preferred over °. further research needs to focus on the comparison between °and °/ °. aims: we have recently demonstrated that the tension of crural closure can be reliably measured intraoperatively (alsgbi conference december ). the aims of this study were to further characterise tension at the diaphragmatic hiatus from our prospective pilot study of patients. methods: a prospective analysis was performed of patients undergoing laparoscopic hiatal hernia repair between april and december . patients underwent crural tension measurement intra-operatively. patients had a pre-operative ct scan of the abdomen within one-year of surgery. hiatal surface area (hsa) was measured intraoperatively and a sauter-fh universal digital force gauge was used to measure the tension of crural closure during cruroplasty. outcome measures included the mean tension of the crural closure and the presence of muscle splitting during the cruroplasty. results: for all patients, the mean crural tension measurement was . n and the mean hsa was mm . pre-operative ct was positively correlated with post-dissection intra-operative hsa (r = . , p = . ), however, strength of association was weak (r = . ) and ct consistently overestimated the size of hiatal defect intra-operatively (mean of differences mm , p = . ). crural tension was positively correlated with age (r = . , p = . ), hiatal height (r = . , p \ . ), hiatal width (r = . , p \ . ) and hsa (r = . , p \ . ). crural tension was correlated to the hiatal width to height ratio to assess the shape of defect and there was positive correlation (r = . , p = . ). tension was calculated for the posterior and anterior halves of the suture cruroplasty. anterior tension was significantly higher when compared to posterior tension ( . n vs . n, p \ . ) . patients had evidence of muscle splitting during the cruroplasty. the group with muscle splitting were significantly older ( vs , p = . ), had larger hsa ( mm vs mm , p \ . ) and higher crural tension ( . n vs . n, p \ . ). the lowest observed mean crural closure tension causing muscle splitting was . n. conclusion: there is now a possibility to optimise this operation with objective measures years after it was first described. initial findings suggest that crural closure up to * n could be the permissible tension threshold for suture cruroplasty and higher tension may benefit from the use of mesh reinforcement. background: endoscopic submucosal dissection (esd) and endoscopic full thickness resection (eftr) are advanced endoscopic techniques which can be time consuming using traditional endoscopic instruments. a new endosurgery platform, designed by fortimedix surgical, was developed featuring flexible articulating instruments to use in combination with a standard flexible endoscope. the platform is intended to perform endoscopic cutting, dissecting, and hemostasis. aim: evaluate feasibility of the platform in the upper gi-tract. project description: the platform was tested in a dry esophageal model as well as a second series with a porcine esophagus and stomach. the system has an external docking station affixed to the operative table to stabilize both flexible instruments for the right and left hand of the surgeon. at the tip of the endoscope, a cap containing instrument lumens is attached to allow advancing and removing the flexible instruments. the endoscope with the cap and instrument lumens attached is advanced via an overtube with outer diameter . mm. in the first series, flexibility and range of motion of the endeffectors was assessed. additionally, the ability to advance the instruments to the intraluminal target area from the docking station and along the scope was evaluated. in the second series, the functional capabilities of the system and instruments were evaluated in a porcine model. preliminary results: : in the dry model, the platform was adequately deployed to the target then range of motion was tested as well as cutting and grasping gastric wall with instrumetn triangulation achieved. the grasping forceps provided enough force to pull the mucosal wall and expose the dissection plane. in the pig model, the distal esophagus and stomach could successfully be accessed and platform deployed. esd was performed using newly designed flexible articulating scissors, dissection-hook, and graspers with good triangulation and sufficient grasping force with traction/counter-traction. the new fortimedix surgical endo-surgery platform applied to a standard flexible endoscope is feasible to perform esd. future studies are planned to determine learning curve and compare it to traditional endoscopic instruments. background: in laparoscopic surgery, we usually observe the organs in the same direction to avoid a mirror-image situation. therefore, we are unable to recognize how far the dissection has proceeded on the other side of the target organs or lesions, especially when the plane of dissection is under the mesentery or organs. this becomes a problem not to understand how far the dissection has progressed and how much more dissection is needed. aim: to solve this problem, we developed a laparoscopic device with tip illumination. project description: the device is configured by the long and narrow part made of polycarbonate resin and a battery-powered light-emitting diode to illuminate the tip by shining light through the polycarbonate resin. during the surgery, the tip of the device is inserted into the deepest part of the dissection area, and the transmitted light indicates how far the dissection has progressed. the tip of the device has a prism structure and light is emitted in a direction perpendicular to its axis. tip position can thus be more clearly identified even with insertion in the same direction as the laparoscopic view. to verify the utility of this instrument, laparoscopic surgeries were performed in a porcine model and cadavers. preliminary results: we performed some laparoscopic surgery such as the medial-to-lateral approach to the white line of the left side of the descending colon for sigmoidectomy, dissection of the posterior surface of the pancreas to the upper edge of the pancreatic body or splenic artery for distal pancreatectomy, and the separation of the anterior surface of the inferior vena cava from the liver to the area between the right and middle hepatic vein for right hepatectomy. we quickly and easily identified the deepest part of the dissection area even if identification had been difficult using other techniques such as placing gauze in the deepest position, inserting forceps into the dissection area or simply depending on the experience of the operator. background: recent advancements within surgery have seen artificial intelligence transform traditional approaches. robotic assistive devices have demonstrated particular success, as safe and cost effective, and are widely supported via industry and local government as a step closer to the future standard of practice. an example of seamless and touchless robotic assistive technology is based on touchless and interactive eye tracker glasses worn by the surgical team thereby enabling the team to perform wider surgical tasks, more efficiently and reduced human error. we introduce a perceptually-enabled, smart operating room (smart-or) based on a novel real-time framework for theatre-wide d gaze localisation in a mobile fashion. this framework enables dynamic gaze based user interaction with a robotic scrub nurse to facilitate meaningful practical integration of human and technology intra-opertively. aims: we tested participant acceptability of a novel robotic scrub nurse during simulated surgery. project description: surgeons performed segmental resection of pig colon and handsewn end-to-end anastomosis while wearing eye-tracking glasses to select surgical instruments on a screen. the robotic scrub nurse(rn) picked up and transferred the instrument to the surgeon. the study compared human nurse(hn) vs rn. gaze-screen interaction was based on a d gaze framework we developed with synergy of conventional wearable eye-tracking, motion capture system and fixed in space rgb-d cameras for real-time d reconstruction of the environment. nasa-tlx and van der laan's technology acceptance questionnaires were collected and analysed using anova. preliminary results: overall, teams of surgeons(st) and scrub nurses(sn) participated. nasa tlx feedback for st and sn revealed no significant difference between in mental, physical or temporal demand. importantly, st and sn reported no significant difference in task overall performance. st reported more significant frustration with rn vs hn. van der laan's scores showed positive usefulness and satisfaction scores in using the rn platform. overall, all outcomes were more positive by sn vs rn. conclusions: this is the first platform of its kind. overall, quantitative and qualitative feedback was positive. the source of frustration has been understood and we believe it can be improved by appropriately modifying robot behaviour. importantly,there was no difference on perception of performance. background: endoscopic tumor resections in the gi tract may be facilitated by more advanced instruments for dissecting and suturing. we have focused on developing an endoscopic suturing technique using a standard flexible pediatric endoscope with new, flexible instruments allowing for complex end-effector movements. aim: perform flexible endoscopic suturing using a standard flexible scope in the gi tract project description: a standard flexible pediatric endoscope and a standard gastroscope were used for testing the new technique. via an overtube, the endoscope and newly designed fortimedix surgical flexible instruments (needle holder; grasper) with a diameter of mm were inserted into the esophagus. suture training was performed in an experimental setting in a box in the dry lab and porcine model . the flexible needle holder was advanced into the esophagus next to the scope, and a suture of the esophageal wall was performed, followed by extracorporeal knot-tying with knots. the test series consisted of training with both resident trainees and surgeons to evaluate the learning curve. each participant performed sutures on the box model and in the pig-esophagus. feasibility, duration of the different steps, and handling problems were documented. preliminary results: test series (box training on esophago-gastric explant) with prototype showed good feasibility. suturing was possible in out of attempts. median duration for single bite: min ( - ); knot-tying: min ( ) ( ) ( ) ( ) ( ) ( ) ( ) . test series (training in pig-model) with prototype showed improved feasibility with better flexibility of instrument shaft: median duration of double bite: min ( - ); knottying: min ( ) ( ) ( ) ( ) ( ) , overall duration intraluminal esophageal double bite suture and closing with knots: median duration: min ( - ). the new flexible endosuture instruments seem feasible to use and perform dependable intraluminal sutures. the training period and learning curve is short and the objective is to apply this system clinically for closure of perforations and fistulas. school of mechanical and aerospace engineering, nanyang technological university, singapore, singapore; general surgery, national university hospital, singapore, singapore; gastroenterology, national university hospital, singapore, singapore; surgery, chinese university of hong kong, hong kong, hong kong background: ideally, endoscopic suturing should mimic surgical closure as the latter is stronger than most endoscopic closure devices. however, endoscopic suturing is challenging due to the confined endoluminal space and lack of dexterity of current endoscopic instruments. we have developed a novel robotic suturing device to overcome these problems. aim: this animal study aims to demonstrate the feasibility of this device in closing perforations. method: the trial was conducted on an anaesthetized live pig. a double-channel colonoscope was first inserted into the rectum. following saline lift, a mm submucosal incision was created in the rectum to simulate a perforation. the robotic suturing device and grasper were inserted into the two colonoscope channels, allowing the endoscope to remain in position for tool exchanges or needle reloading. both the effectors were intuitively tele-operated by the user via a robotic master console. this robotic suturing device manipulated a curved, double-point needle (with a cm - vicryl suture) to penetrate tissues at desired orientations. the needle could be switched between both jaws of the device through a locking mechanism. this facilitated passing the needle through tissues to form stitches or through suture loops to form surgical knots. the articulated joints and five degrees of freedom allowed dexterous steering to reach targets and triangulation with other tools in a confined space. the robotic grasper facilitated handling of tissue and suture. result: a total of four running stitches were performed and secured with a surgical knot by passing the needle through suture loops. the suture was cut and the needle was removed by the robotic grasper through the channel. min and min were required to stitch and tie the knot respectively. there was no complication. conclusion: our novel endoscopic robotic device can suture perforations resulting from complex endoscopic procedures. as our suturing method is similar to laparoscopic and robotic suturing, closure using our device is expected to be as strong as a surgical through-and-through closure. when developed further, this device can be used to close full-thickness resection sites and orifices in transluminal endoscopic surgery. modelling a collaborative robot with the ieee sdc standard for combined focused ultrasound and radiation therapy j. berger, m. unger, l. landgraf, a. melzer medical faculty, university hospital leipzig, innovation center computer assisted surgery, leipzig, germany background: surgical robotics require a smooth integration into the operating room (or) . for this propose the ieee sdc(service-oriented device connectivity) standard has been developed in the or.net project. in preparation for a combined focused ultrasound and radiation therapy (fus-rt) we have shown concepts and evaluations to position ultrasound and interventional devices with collaborative kuka arms. however, the safe and intraoperative cooperation with multiple different or-devices (e.g. an irradiation unit) requires a more sophisticated exchange of the robot's information and functionality. aim: to realize a safe clinical integration, the aim of this work is to implement and evaluate a dynamic connection between the kuka robots and other devices using the vendor-independent sdc communication standard. project description: a kuka lbr iiwa r robot (kuka ag, germany) was modeled inside the sdc standard for medical device communication. the interconnection with other devices was implemented and evaluated on a mobile platform to position a clarius l wireless ultrasound transducer (clarius mobile health corp, canada). all necessary information of the robot was represented in the medical device description of the sdc standard to be shared via network. for each joint of the robot arm the position, torque, stiffness, damping, velocity and functional-states were represented, resulting in a total of parameters. the software was implemented in c ?? on a standard pc accessing the kuka controller cabinet with ros (robot operating system) via ethernet. the accessibility of each parameter, as well as activation commands for planning and movement were tested with an sdc-consumer application. preliminary results: the sdc-provider functionality of the robot was successfully implemented, allowing for dynamic changes of the robot state during interventions. all appliances (sdc standard compatible) in the robots network can react to state changes and send movement and planning commands to the robot via activations. after testing, % of the defined parameters are safely accessible. implementing the medical device communication for the kuka robot enables its integration into any networked operation room that supports the sdc standard. it is, therefore, ready to be set up and evaluated for the application of fus-rt in a clinical environment. background: assessment of perfusion of the left colon with fluorescence during anterior resections for cancer changes surgical decisions in up to % of cases. use of fluorescence has been shown to be associated with lower leak rates, and improved short-and long-term outcomes with reduced costs. given the high incidence of colorectal cancer, fluorescence-guided perfusion assessment could be of great importance in contemporary surgical practice. however, there is currently no standardisation of this technique which represents a significant limitation to widespread adoption. aim: to standardise fluorescence-guided perfusion assessment in rectal anterior resection through a computer vision algorithm. project description: videos were collected by a single surgeon in a referral centre for colorectal cancer treatment. perfusion assessment was used before proximal colon division to identify the best location for transection. a bolus of indocyanine green was injected intravenously and a near-infrared camera used to assess perfusion through fluorescence. photographs of fluorescent imaging of the colon were analysed using a non-supervised learning algorithm called 'k-means clustering'. the first step was to digitally subtract all background pixels, leaving only the area of interest of the colon. this area was then subsegmented into 'clusters' corresponding to perfused and nonperfused areas. a mathematical model was applied based on the sub-clusters centres to select the area for transection with optimal perfusion of the proximal colon. preliminary results: representative images of proximal colon under perfusion assessment were presented to expert surgeons. the optimal point for transection was selected based on their clinical judgement on previously delimited areas indicated by random letters. this was compared with the results from the automated segmentation using the algorithm ( fig. ). the area identified for section by the algorithm included the area selected by the expert surgeons in . - % of test cases. these results need to be further validated due to high risk of overfitting. next steps include the collection of multicentre data with a standardised fluorescence perfusion assessment. after robust training, the algorithm will be validated on real-time clinical data to ensure improved outcomes for patients, which is our ultimate goal. background: endoscopic submucosal dissection (esd) is a flexible endoscopic technique that allows for an en bloc removal of lesions of the gastrointestinal (gi) tract. these procedures are typically time consuming due to the difficult control of the tools, and they often require around min for removing lesions, that can reach - cm in diameter. the probability of intestinal perforation exceeds % and the hemorrhage risk ranges from . % to . %. a flexible robotic endoscope may offer a solution to overcome these limitations, by improving the degrees of freedom (dof) and operational efficiency. aim: within this clinical panorama, the aim of this project is presenting the development of a novel miniaturized robotic device to be coupled to the tip of a traditional endoscope for the surgical dissection of gi neoplasms. project description: the robotic platform consists of the miniaturized robot, the actuator housing (hereafter called external platform), the control unit and the master console (i.e.,two geomagic touch phantom) to allow the user driving and control (figure a ). during the operation, one surgeon stands close to the patient to maneuver the endoscope for exploring the gi tract and reaching the target area. another surgeon operates the miniaturized robot through the master console, carrying out the surgical procedure. the robot has been designed to be coupled to the tip of traditional flexible endoscopes of . mm in diameter. it exploits the flexibility of the endoscope for navigation through the intestine and integrates two-active robotic arms (i.e.,cautery and gripper) extending the dofs, and thus enhancing the efficiency during complex tasks such as manipulation and surgical tissue dissection. furthermore, the endoscope provides the optical system for visual feedback and one working channels for conventional instruments. preliminary results: firstly, a mock-up that faithfully reproduces the miniaturized robot has been realized using a d printer machine (projet mjp , d system, inc.) to verify the feasibility of the design solution. after verifying the potentiality of the d printed prototype, a final device, with the same features (i.e.,dof and geometry) of the d printed prototype, has been designed, fabricated and assembled ( figure b ). background: virtual and augmented reality has been widely used in many fields mainly for entertainment purposes. we think that it could be beneficial to use augmented reality in medical practice. aim: the aim of this study was to evaluate usefulness of d holographic images of patients anatomy displayed using augmented reality goggles during endovascular aortic repair (evar). project description: one of the major challenges during endovascular procedures is working on two dimentional x-ray images of three dimentional vascular anatomy. using d holograms of patients anatomy could be beneficial during the evar procedure and could make the orientation in vascular anatomy easier for surgeon. we performed two endovascular aortic repairs with the assists of microsoft hololens -smart glasses using augmented reality. we used carna life application created by polish company medapp. it was one of the first use of holograms during vascular procedures in the world (second and third stent-graft implantation using holographic imaging in the world). results: two patients with abdominal aortic aneurysms, -years old male and -years old female, were operated on. holograms of patient's anatomy made from preoperative angio ct scans by polish company medapp were displayed during the procedures using microsoft hololens. holograms could be displayed in any place and configuration using augmented reality, which means that the images did not interfere with the surgeon's field of vision. microsoft hololens use voice commends which permits the surgeon staying sterile. stent-graft implantations were successful. both patients were discharged three days after the procedure and the hospitalization was uneventful. seeing precise patient's vascular anatomy reconstructions in three dimention certainly helped us to navigate in a vascular tree. we believe that in the future this technology would enable to reduce the operation time and need for radiation. background: interaction with electronically controlled operating room (or) systems embedded in modern surgical environments is everyday practice for surgeons performing minimally invasive surgery (mis). while there is a non-sterile operating nurse available in the or, capable of interacting with these systems upon request by the surgeon, this indirect control is mostly slow, prone for error and disrupting surgical workflow. facing an unanticipated and unwanted outcome may cause distress emotions. distress emotions are undesirable when performing surgery, since they may impact available cognitive workload. furthermore, they may result in negative communication, hampering or-team empowerment and effective leadership. both factors are known to negatively influence quality and safety in the or. aim: the aim of the tedtrial is to investigate what setup best enables surgeons to interact with the endoscopic operating room setup during surgical procedures. as a result, disruptions of workflow, delays and errors may be reduced. outcome parameters will be objectified using medical data recorder (mdr) derived output and biometric analysis using hexoskinÓ. subjective evaluation of outcome parameters is done using questionnaires. project description: the tedcubeÓ system is a plug-and-play device enabling wearable sensors to act as a wireless alternative for a regular computer mouse, therefore enabling direct hands-free and sterile control of the or. the study is an observational trial with three different arms: intervention group ) direct interaction by surgeon with or environment using tedcubeÓ and myo tm armband, intervention group ) direct interaction of surgeon with or environment using tedcubeÓ and plantronicsÓ wireless microphone headset. the third arm is the control group using indirect interaction of surgeon with or environment using third-person computer interaction. main endpoint of study is the number of workflow disruptions due to the operation of laparoscopic or equipment. secondary endpoints are error rate, delay, team communication, subjectively reported frustration and satisfaction with the system and objectively measured stress as symptom of frustration and anger as distress emotions. preliminary results: primary and secondary endpoints of study are compared among groups. it is anticipated that reduction of miscommunication, error and delay may result in a reduction of distress emotions. trial start is expected q . anticipating the automated intraoperative tissue recognition: intraoperative tissue classification using hyperspectral imaging and machine background: iatrogenic injuries may occur despite a sound expertise in surgical anatomy. hyperspectral imaging (hsi) is an emerging optical method, combining the use of a camera system with a spectrometer. hsi analyzes optical properties of tissues and acquires d data sets with two spatial dimensions (x, y) and one spectral dimension (?). the data sets contain information about tissue physiology, composition, and perfusion. those spectral features coupled with machine learning algorithms might allow for automatic tissue recognition. aim: assessing the ability of an hsi-based machine learning to discriminate the hyperspectral features of different tissues during neck and abdominal surgical procedures. methods and procedures: fourteen pigs underwent laparotomy (n = ) or neck dissection (n = ). twenty data sets were acquired in vivo from abdominal organs and from neck structures by means of a customized hyperspectral camera (diaspective vision, germany). different anatomical structures were manually outlined by a surgeon using an image manipulation software (gimp). each pixel contained a hyperspectral curve and each curve was composed of bands (from to nm with a nm resolution). the curves were normalized using the standard normal variate method. a logistic regression machine learning (ml) algorithm was used to train the model to discriminate tissues, based on the hsi spectral features. the efficacy of the prediction model was tested using the k-fold (k = ) cross-validation. results: a large number of tissue-related hyperspectral curves could be extracted ( thyroid, vagal nerve, fatty tissue, cartilage, carotid artery, muscle, carotid vein, portal vein, biliary tract, gallbladder, hepatic artery, pancreas, duodenum, abdominal adipose tissue). the algorithm used min to 'learn' all data sets, and prediction was provided as an immediate output. overall, prediction accuracy was and % for neck and abdominal structures respectively. in particular, biliary ducts could be identified with a % accuracy and the vagal nerve with an % accuracy (see figure for details). background: a gaze-controlled robotic endoscope is innovative technology with myriad potential applications in the rapidly advancing field of flexible endoscopy. improvements to the current flexible device to allow examination of the gastrointestinal tract whilst minimising procedural discomfort and complications are desirable. aim: to use a gaze contingent framework to manipulate a flexible endoscope through a simulated upper gastrointestinal tract (ugit) model. description: a flexible gastroscope (karl storz pks) was attached to a ur axis robotic arm (universal robots), mounted onto a rail and placed on top of a surgical table. two cogwheel shaped dials were d printed and placed onto the up/down and left/right wheels on the head of the gastroscope ( figure ). robotization of these controls was achieved by using two motors (dynamixel rx- f) to steer the distal tip. this system allows users to operate a robotised flexible endoscope using gaze control. gaze interaction with the screen was based on a d gaze framework we developed with the synergy of conventional wearable eye-tracking, motion capture system and fixed in space rgb-d cameras for d reconstruction of the environment. users are able to control endoscope movements without handling the device. the distal tip of the gastroscope was controlled using eye gaze technology. the ur robot was used to enable shaft rotation (initiated by fixed head movements) and linear movements were triggered using a joystick handle (up for forward movement, down for endoscope withdrawal). pause and retroflexion of the endoscope are achieved by moving the joystick left and right respectively. users were asked to navigate an endoscope through an ugit model (chamberlain group) simulating a diagnostic gastroscopy using gaze control and targeting ten points scattered through the stomach. results: four expert endoscopists and one novice used gaze control to successfully navigate a gastroscope through a simulated ugit. all were able to intubate the oesophagus and accurately locate ten targets placed in the fundus, body, antrum and pylorus of the stomach. conclusion: gaze control endoscopy is a feasible concept. it allows ergonomic, user-friendly and intuitive control whilst maintaining the benefits of a flexible endoscope. background: image-guided needle biopsies and histopathological evaluation are the gold standard for the diagnosis of liver neoplasms. most often, however, these are reserved for suspicious, but not diagnostic, situations. radiomic may help to characterize tumor biology by correlating imaging features with relevant tumor-biology information. features derived from radiomic analysis may provide complementary information to support clinical decisions, especially in situations where tissue analysis cannot be performed or is inconclusive. aim: the goal of our technology is to exploit computational capabilities for image analysis in order to identify radiomic features useful for characterizing liver lesions and to identify relevant information related to patient prognosis. project description: patients derived from an internal database and patients randomly extracted from the cancer archive liver dataset were included in this study. lesions were extracted from those volumes using expert annotations ( secondary vs primary; well differentiated vs non-well differentiated). lesions were then split into training and testing sets. first order statistical features were computed and a lasso regression step was performed to reduce the number of features. both logistic regression and random forest models were built using cross-validation to predict the target classes on the test set. preliminary results: only features namely the energy and the volume of the lesion were sufficient, when combined in either model, to predict the differentiation grade on the test set with an f -score of . (± . ). we are currently working on the addition of higher order statistical features to the analysis in order to differentiate primary from metastatic tumors and identify complementary features that may assist clinical decisions in patients with inconclusive hepatic lesions. objective of the technology or deviceideally, the use of medical simulators could provide trainees with initial background information about indications for procedures, endoscopic technique, and early hands-on training experience that could shorten the initial critical learning curve. rationale for using ex vivo models is that in the beginning of the learning curve, the most important issue is having an initial exposure to the basic movements and maneuvers. our objective of is to create a stomach model from renewable polymer, which would closely simulate normal human stomach with gastric pathology for endoscopic diagnostic or interventional skill acquisition/evaluation. description of the technology and method of its use or application stomach model is based in several steps; the first one is in the in-silicodesign of the overall shape, after that we d print the positive two halves of it. the interior detail is obtained shaping the d printer parts with ceramic putty. once concluded, this elaborated part will serve as a template in order to build injection bleeding moulds. in the injection bleeding moulding a mesh is placed between layers in order to provide structural attachment points as stiches or several pathological models that will be incorporated after the casting process. we have developed for these instance polyp moulds, fistulae structures in order to attach endoscopic clamps. the two halves are closed once the pathological models are placed inside via a thermic-fusing and stitching creating a leak proof stomach model. preliminary results if available: our models were evaluated by international experts in ircar/ihu france in interventional endoscopy course and were favorable accepting for next trails in these prestigious institutions. conclusions: future directionsa new endoscopic training model of stomach was made and will be evaluated and validated for feasibility in mastering diagnostic and interventional endoscopic skills. clinical trials will be necessary to compare the ability of the simulator to perform training compared with traditional methods of training in endoscopic procedures. background: endoscopes are the eye of surgeons in minimally invasive surgery (mis). conventional endoscopes are mostly chopstick-like and are steered by the assistant. this limits the field of view and results in issues such as endoscope-instrument fencing, surgeon-assistant coordination. existing robotic endoscope holder enables solo-surgery, however endoscope remains blocking the instrument movement and impairs the operational safety. flexible endoscope such as the endoeye provides angulation at the tip and could enlarge the field of view. however, its steering the view is much more complex compared to the rigid endoscope. aim: to provide an intuitive robotic flexible endoscope with enhanced safety. project description: in this work, we present a robotic flexible endoscope for mis with enhanced safety. in the proof-of-concept system, it contains a flexible endoscope module and a robot manipulator. the endoscope contains a proximal rigid shaft and a distal flexible bending section. it is installed onto the patient side manipulator (psm) of the da vinci research kit (dvrk). visual servoing is adopted to achieve autonomous instruments tracking. during the tracking process, movements of the manipulator as well as the endoscope are minimized to save space for the operation and avoid instrument-endoscope fencing. the endoscope could also be controlled by the surgeon. a foot pedal is used to switch between the tracking-mode and control-mode. preliminary results: a prototype was developed and tested experimentally. in tracking a volume of * * mm , the spaces required by the flexible endoscope are . % (inside the trocar) and . % (outside the trocar) of that occupied by the rigid endoscope. evaluation with the fls tasks involved subjects. all of the participants completed the tasks under the tracking-mode without failure. in the ex-vivo test with porcine stomach, the endoscope successfully guided the detection, dissection and knotting autonomously. background: fluorescence imaging allows to visualize deep-seated anatomical structures, using a deeper tissue penetration of near-infrared (nir) compared to visible light. the most commonly used fluorescent substance, indocyanine green (icg), is not naturally excreted by the urinary system and requires retrograde stent placement and injection. lighted catheters have been proposed to help visualise the ureter. fluorescent dye-coated ureteral catheters could well represent a more effective and less expensive solution. icg is unsuitable for coating materials. aim: to develop a stable fluorescent coating for catheters to be used intraoperatively, working in the same nir window as icg, to facilitate its use with clinically available systems. project description: the coating was developed based on poly(methyl methacrylate) (pmma), a biocompatible polymer, and on specifically designed fluorescent dyes exhibiting icg-like optical properties. three nir dyes (substances a, b, and c) were tested in order to find the optimal one, in terms of fluorescence signal intensity, and were compared to icg in a polymer form and to an icg-based reference card (green balance tm ). the fluorescent coating was applied onto common ureteral stent materials: hydrophilic-coated ultrathaneÒ, silicone-coated latex, and pvc. the coating process involved cycles of immersion into the respective dyes blended in pmma polymer (icg, substances a, b, and c), followed by a drying phase. the various tubes were partly inserted into a porcine ureter, next to the icg-based reference card. images were taken in white light and nir modes using the d-light p camera system (karl storz), at a fixed camera-to-target distance. the fluorescence signal intensity was measured for the different regions of interest (each material/coating combination inside and outside of the ureter, reference card) using proprietary software and normalised against the reference card. preliminary results: the signal intensity was significantly higher for all new substances as compared to icg. substance a showed the strongest fluorescence signal intensity among the tested coatings in all tested conditions and materials and was identified as the ideal candidate to undergo further evaluation and in vivo testing. background: endoscopic resection(er) of early gastric cancers provides tremendous patient advantages. however, post-resection findings of deeper sub-mucosal(sm) and/or lympho-vascular invasion can necessitate a second, surgical intervention. we propose that pre-resection evaluation of the submucosal architecture under the tumour can provide critical information for staging and operative planning. we evaluate three techniques to assess the submucosal architecture underlying the gastric mucosa in a pig model. aim: to evaluate three needle-based methods of evaluating the sm before er. project description: acute pigs were used. a simulation of sub-mucosal tumours (endoscopically and eus visible bleb) by injecting the sm with cc of undyed nac. a linear eus was use for all procedures. the tumours were marked and labelled according to geography. methodology: after creating the tumours, anterior lesions were evaluated using the following g needle-based modalities: confocal microscopy(cm) using the through-the-needle cellvizio (mauna-kea) system; mini-biopsy(mb) using the micro-biopsy forceps moray (us endoscopy) and fine-needle biopsy(fnb). results: cm examinations were video recorded in all a positions. submucosal vascular visualisation was possible in all cases, excellent in / . mb was performed in lesions with a total of biopsies obtained from each lesion (total = ). fnb was performed once in the anterior lesions and twice in the posterior lesions with different needle brands. therefore, there was a total of biopsies collected. passes were performed in each biopsy (total = ). each pass constituted - insertion/withdrawal movements combined with fanning, slow pull technique, no suction and suction ( - cc air negative pressure) to collect the material. all material were sent to an animal anatomo-pathologist blinded to the acquisition method. mean time of confocal examination was min sec ( ' ' '- ' '') . mbtook a mean time of min and fnb was a mean of min for each biopsy. cm identified different patterns of vessels in relation to the probe position (superficial/reticular, middle cross-roads or deep/longitudinal). conclusion: eus-fnb, cm and mb are three potential methods to assess the sub-mucosal space underlying the gastric mucosa. cm offered the most architectural information but required more time to perform. these method's may have a role in better staging patients for appropriate er. background: the overall and disease-free survival of patients with rectal cancer is dependant on its staging, and adequate selection of the treatment strategy. mri has a proven efficacy in rectal cancer local staging and recognition of the adverse prognostic features. however, it can be difficult to utilise it as a navigation tool for surgeons, as it represents a complex three-dimensional pelvic space with a series of individual two-dimensional images. d image reconstruction has been successfully adopted in other surgical fields to overcome these limitations. aim: our primary aim is to develop a bespoke automated generation of patient-specific d pelvic models, which will improve surgical planning and navigation, patient interaction and surgical education. true-size, rotatable d models will offer a more realistic three-dimensional representation of the surgical space and its complex relationships, allowing for a more confident surgical rehearsal and potentially better utilisation of minimally invasive techniques in rectal cancer management. our secondary aim is to develop a large multipurpose database of the d models of male and female pelvis in health and in the disease. project description: our multidisciplinary team consists of colorectal surgeons, radiologists specialising in pelvic mri imaging and computer scientists. virtual d pelvic models are generated based on standard d dicom mri images routinely used for rectal cancer staging, which guarantees the high fidelity of cancer delineation. segmentation of the pelvic anatomy is performed with the use of itk-snap, an open-access, multi-platform software. machine learning technology is then employed to automate the d model generation, making it time-efficient, allowing for its clinical application. preliminary results: in the initial stage, using the manual segmentation, we have created ten models of normal male and female pelvic anatomy. a good inter-rater agreement level was found, which proves reproducibility of the approach applied. various machine learning algorithms are being explored to fully automate the process of d model generation, which will allow for their use in clinical practice and in development of the d colorectal database. the technology will be further implemented in creation of dynamic models of functional pelvic floor disorders. surgery, toho university omori medical center, tokyo, japan; surgery, neuchâtel hospital, neuch tel, switzerland background: laparoscopic gastrojejunostomies are time-consuming and require a specific training. alternatively, sutureless anastomosis can be achieved by means of endoscopically delivered magnetic rings. objective of the study: assessing the feasibility and reproducibility of an endo-laparoscopic gastrojejunostomy technique, using magnets coated with a fluorescent biocompatible polymer. methods and procedures: four pigs ( acute, survival models) and one cadaver were included in this study. the anastomotic device was composed of two magnetic rings ( x x mm; attraction force newton), each one attached to a cm long thread. the distal ring was inserted endoscopically into the first duodenum, and the extremity of the thread was clipped to the gastric mucosa. twenty-four hours later, a two-port laparoscopy ( mm, mm) was performed, using a near-infrared (nir) laparoscope (d-light-p; karl storz). the magnet's position in the jejunum was detected thanks to the transluminal fluorescence of the dye. magnetic interaction with the metallic tip of the laparoscopic grasper allowed to catch the ring and bring the bowel loop to the future anastomotic site on the gastric wall. simultaneously, the proximal magnet was delivered to the gastroesophageal junction using a flexible endoscope. the magnet was carefully advanced into the stomach allowing precise connection with the distal ring. in one cadaver the procedure was repeated. the sole variation was that, in order to reach the second jejunal loop, the distal magnet was placed using a gastroscope inserted through a transgastric port. in two acute animals, the distal magnetic ring was introduced into the jejunum via an enterotomy. the anastomotic procedure (from the distal magnet detection via fluorescence to the magnetic connection using a hybrid approach) was reiterated times. survival animals were followedup for days and underwent control endoscopies and ct-scans. results: the procedure was easy to standardize and reproducible, with a mean anastomotic procedure time of . ± . min. there were no technical problems and magnetic connection could be precisely directed in all cases, at both the anterior and posterior gastric wall. no complications occurred during the survival period and the anastomoses were patent by day . transluminal fluorescence allowed for a rapid detection of the magnet. colorectal cancer is the fourth most common cancer in high-income countries counting [ . deaths worldwide. survival rate reaches % in case of early diagnosis, falling down to % in case of advanced stage. conventional colonoscopy screening is limited by invasiveness, pain and often need of sedation. wireless capsule endoscopy enables inspection without discomfort, but passive locomotion often leads to incomplete and/or false negative results. the european endoo project (grant agreement ) aims to develop a novel system that overcomes most of the drawbacks of conventional colonoscopy, maintaining accurate and reliable diagnosis and therapy. the system is composed of an active robotic platform that magnetically drives a soft-tethered capsule; magnetic guidance is achieved through the magnetic localization of the capsule in combination with a closed-loop control that maintains an optimal and safe link between the capsule and the magnetic end-effector. a stereoscopic camera is integrated in the capsule for enhanced diagnosis though d reconstruction and automated detection of lesions/pathologies. the different modules of the endoo medical platform are illustrated in the figures. the robotic guidance systemconsists of an anthropomorphic manipulator that controls the capsule through an external permanent magnet. the robot, positioned on a dedicated trolley, is equipped with sensors for performing safe human-robot collaboration. the medical workstationincorporates: screens, buttons and pedals for visualization and command initiation, a joystick for system teleoperation and a back-end for fluidic control and data communication. the soft-tethered capsuleembeds an internal permanent magnet, magnetic sensors, an accelerometer, white and infrared illumination and an hd stereoscopic vision system with two wide-angle customized optics. a controller serves as the main control unitfor performing real-time communication and closed-loop control of the robot, localization system, capsule and physician commands. the synergistic cooperation of academic, industrial and clinical partners within the project allowed to develop and validate the system in in-vitro \/i [ , exvivoand preliminary cadaver sessions, performing comparisons with state-ofthe-art commercial colonoscopes. in conclusion, the endoo medical platform provides: reduced procedural pressures, user-friendly procedures, similar functionalities and performances of commercial devices, comparable procedural times and considerably lower costs with a new painless approach. background: this study is aimed at the comparison of the process of manual and robotic-assisted positioning of the electrode performing radiofrequency ablation with the usage of multifunctional robot-assisted surgical platform. under the control of the surgical navigation system. the main hypothesis of this experiment was that the use of a collaborative manipulator will allow to position the active part of the electrode relative to the center of the tumor more accurately and from the first attempt. we also check the stability of the electrode's velocity during insertion and consider some advantages in ergonomics using the robotic manipulator. methods: sphere-shaped tumor phantoms measuring mm in diameter were filled with contrast and inserted in cow livers. livers were used for the robotic experiment and an equal quantity for manual. the livers were encased in silicone phantoms. analysis of ct data gave the opportunity to find the entry and the target point for each tumor phantom. this data was loaded into the surgical navigation system that was used to track and record the position of the rf-electrode during the operation for further analysis. results: standard deviation of points from the programmed linear trajectory totaled in the average . mm for the robotic experiment and . mm for the manual operation with a maximum deviation of . mm and . mm respectively. standard deviation from the target point was . mm for the collaborative method and . mm for manual method. the average velocity was . mm/s for the manipulator and . mm/s for the manual method, but the standard deviation of the velocity relative to the value of the average velocity was . mm/s and . mm/s respectively.thus, in two criteria out of three, the manipulator is superior to the surgeon, and equality is established in one. surgeons also noticed advantages in ergonomics performing the procedure using the manipulator. conclusions: this experiment was produced as part of the work on the developing of the robotic multifunctional surgical complex. we can confirm the potential advantages of using robotic manipulators for minimally invasive surgery in case of collaborative practice for cancer treatment. surg endosc ( ) background and aims: laparoscopy has reduced tactile feedback compared to open surgery. in neuropsychological literature there is increasing evidence that visual and haptic information converge to form a mental representation of an object. through the combination of these inputs, this representation is believed to be more refined and robust. we investigated whether tactile exploration of a lifelike anatomical object before executing a laparoscopic action on this object in a laparoscopic box trainer improves performance of this action. description: a randomized prospective cohort study with two groups (a ? b) of ten laparoscopically naïve medical students was conducted. we compared the groups for baseline characteristics and performance, using a basic laparoscopic task (post and sleeve). to investigate the effect of haptic exploration, students performed ten repetitions of a laparoscopic needle action on a lifelike silicone caecum model (applied medical, rancho santa margerita, usa). group a did a pre-test visual exploration of the model. in group b manual exploration of the anatomical model was added to the visual exploration before executing the task. the box trainer was equipped with the forcesense tm (medishield, delft, the netherlands) system for skill assessment using objective force, motion and time parameters. results: baseline characteristics and-laparoscopic performance were comparable (p [ , ) . performances of trials on the anatomical model were captured and parameter outcomes were compared between groups. significantly less force (maximal force, maximal impulse, mean force and force volume) was exerted by the 'touch' group (p \ . ) (fig. ). this group also completed the task with less distance travelled by the instruments (p \ , ). there was no significant difference in time needed to complete the task (p = , ). conclusion: this study showed that, when performing a laparoscopic task on an anatomical model, pre-task haptic exploration of the model results in the use of significantly less force and less movement. adding haptic exploration to a laparoscopic training curriculum could therefore result in more efficient and more refined learning of laparoscopic actions. this, in turn, could lead to better, quicker and safer performance of laparoscopic operations. . esophagogastroscopy was performed before gabe and -week post-procedure assessing gastric abnormalities. weight and fasting plasma ghrelin were obtained at baseline, -, -, -and -months post-index procedure. after months, the sham group was unblinded and received gabe. both gabe and sham crossover to gabe groups were followed for months and received lifestyle therapy (behavioral-diet education). preliminary results: gabe was successful in all patients with no serious complications. significant, progressive weight loss was observed at and maintained at months. ghrelin in gabe group decreased by % ( . pg/ml) compared to baseline and months levels. weight-loss was approximately . % greater in the gabe group versus sham at months ( table ) . itt = intent-to-treat, pp = per-protocol analysis preformed using independent-sample t-test and à paired-sample t-test conclusions: gabe using eles is safe, accompanied by significant and so far maintainable weight loss. gabe using the eles demonstrated a reduction in ghrelin levels. aims: transanal total mesorectal excision (tatme) is the latest colorectal approach that continues to be in the spotlight. this study aims to describe the technique in depth by identifying and understanding technical advantages, errors and adverse events. methods: detailed video analysis using observational clinical human reliability analysis (ochra) was completed on clinical tatme cases performed by international surgeons. error frequency and error pathways leading to adverse events were described. tatme expert surgeons were interviewed and engaged in a workshop to elicit error-reducing mechanisms. results: overall technical errors and adverse events per procedure on average occurred ± . (range -- ) and ± . (range - ) times respectively. inadequate insufflation and poor camera optics were the most frequent set-up problems. instrument handling errors consisted most commonly of excessive grasper movement during the pursestring phase ( times total), inappropriate force applied ( times) with the energy device during the rectotomy, inappropriate force with the grasper ( times) and excessive movement with the energy device ( times) during tme dissection. incorrect dissection planes were created during tme dissection mostly due to insufficient retraction ( times) which didn't allow adequate exposure of the tissue planes. the most frequently occurring consequence was bleeding (mean: times per procedure). rectal perforation ( cases), vaginal wall injury ( cases), and prostatic injury ( cases) were also recorded. adverse events regularly occurred as a result of poor set-up/exposure, inappropriate retraction and/or instrument movement and incorrect plane surgery. error-reducing mechanisms and 'technical tips' describe specific steps and actions, both set-up/equipmentrelated and technique-related, that aim to prevent errors from occurring and avoid adverse consequences. ochra and individual feedback with error-reducing mechanisms developed by this study have been implemented into the national training programme for tatme. conclusion: tatme is an advanced complex procedure during which technical errors and their consequences are not infrequent. tatme requires knowledge of anatomy 'bottom-up', familiarity with its specialised equipment and technical skill working in a narrow space. appropriate structured training and mentorship are therefore recommended. surg endosc ( ) objective: insufficient vascular supply is one of the main causes of anastomotic leak in colorectal surgery. icg has been shown to provide information on tissue perfusion, identifying a well-perfused location for colonic and rectal transections and thus possibly reducing the leak rate. objective of this study is to evaluate the usefulness of intraoperative assessment of anastomotic perfusion using intraoperative indocyanine-green dye (icg) angiography in patients undergoing left-sided colon or rectal resection with colorectal anastomosis. methods: this randomized trial involved patients undergoing laparoscopic left-sided colon and rectal resection randomized : to intraoperative icg or to subjective visual evaluation of the bowel perfusion without icg (clinicaltrials.gov nct ). the primary aim was to assess whether icg angiography could lead to a reduction in anastomotic leak rate. secondary outcomes were possible changes in the surgical strategy and postoperative morbidity. results: after randomization, patients were excluded. accordingly, patients were included in the analysis; in the study group, and in the control group. icg angiography showed insufficient perfusion of the colic stump, which led to extended bowel resection, in cases ( %). an anastomotic leak developed in patients ( %) in the control group and in patients ( %) in the study group (p = n.s.). conclusion: intraoperative icg fluorescent angiography can effectively assess vascularization of the colic stump and anastomosis in patients undergoing colorectal resection. this method led to further proximal bowel resection in cases, however its role in reducing anastomotic leak rate should be studied in further research. endoscopic sleeve gastroplasty (esg) is a promising endoscopic bariatric procedure carried out with the application of transmural sutures resulting in a gastric reduction and gastric shortening. sutures are placed in u shape fashion, from the incisura to the fundus, which is preserved, using an over the endoscope suturing platform (overstitch, apollo endosurgery, austin, texas, usa). the choice of right lankmarks for suturing the gastric wall is extremely important for the efficacy and safety of the procedure. flexible endoscopy suffers from little anatomical reference points. correct spatial relation to precisely target the insertion of the helix device used for retraction and correct orientation of the full thickhness tissue bite require a good undrestanding of the anatomy of the stomach and sourrounding organs including vascular structures that could be inadvertently injured (left lobe of the liver, gallbladder, spleen, short gastric vessels, pancreas, transverse colon). surgeons by training can 'see' the anatomy beyond the gastric wall and undrestand whether they work in a safe layer or whether an underlying structure should be spared. this video illustrates all the potential risks realted with a wrong chioce of endoscopic landmarks when performing esg with respect to gastric and abdominal anatomy. introduction: central bisectionectomy, anterior sectionectomy, and posterior sectionectomy are technically demanding procedures in minimally invasive approach because of difficult expoure and extensive parenchymal transection planes. with limited robotic instruments including absence of cusa, these procedures have been rarely perfomed by robotic approach. method: consecutive robotic central bisectionectomy, anterior sectionectomy, and posterior sectionectomy were performed. patients were all males and were , , and -years-old, respectively. pathologic diagnoses were all hepatocellular carcinomas of each . , . , and . cm diameter. operative settings were identical for the three kinds of procedure. the patients were placed in supine with a reverse trendelenburg and right side elevation. umbilical -mm camera port, three -mm ports and additional -mm assistant port were used. glissonian approach and icg fluorescence image clearly demarcated the resection planes. parenchymal transection was performed using the maryland bipolar dissector and harmonic scalpel. the rubber band self-retraction method and third arm of robot system helped for stable and excellent exposure of surgical planes result: there were no conversions to laparoscopic or open surgery. the operative time was , , and min and estimated intraoperative blood loss was , , and ml. the pathologic surgical margin was . , . , and . cm. the length of stay after surgery was , , and days and there were no postoperative complications. conclusion: robotic central bisectionectomy, anterior sectionectomy, and posterior sectionectomy are still demanding procedures with long operative time. however, these procedures could be performed safely in regard to short-term perioperative outcomes. robot surgical system provided several benefits for anatomical hepatectomies including a stable and excellent operative field and clear surgical planes. suprapubic hernias (less than cm above the pubic arch in the midline) require important anatomical knowledge because of complexity of their repair and low incidence, by approximately % of all hernias. the problem to repair this type of hernias is that inferior margin of the defect is very close to pubic symphysis, consequently, mesh overlap is often inadequate. treatment of suprapubic hernias is controversial because of limited evidence in the literatura. this video shows the case of a -year-old female patient with suprapubic hernia with a defect of x cm. we performed a laparoscopic repair with a bilateral peritoneal flap of the groin region (as it is perfromed during tapp) for proper view of the pubic symphysis, cooper's ligaments, epigastric and major vessels, nerves and meticulous dissection the space of retzius. the defect was repaired by reconstructing the middle line with a running sutures. subsequently, titanium helical tacks were used to fix the mesh to the pubis and cooper and following the double-crown technique having special attention when fixing the mesh near to inguinal chanal, due to the possibility of causing chronic pain. the peritoneal flap was fixed over the mesh with abdsorbable fixation devices and seal with fibrin glue. laparoscopic repair of suprapubic hernias can be considered as the first option in treatment, because it endeavors to join the advantages of a minimally invasive approach and it is associated to low recurrence. the main advantages are that allows a proper visualization the anatomy and a proper fixation of the mesh. background and aim: thoracoscopic esophagectomy has been performed for two decades and becomes widely spread. we evaluate our cases who undergone the thoracoscopic esophagectomy and consider the future prospective of this operation.transient recurrent laryngeal nerve palsy after lymphadenectomy in this surgery is not rare and induces not only hoarseness but also aspiration or pneumoniae. new method to avoid this complication is desired. patients and methods: patients who received thoracoscopic esophagectomy in our institute from march to october were enrolled and studied retrospectively. operative indication is an all of the clinically resectable cases including with a neoadjuvant treatment or definitive chemoradiotherapy before surgery. overall survival rate of the patients with thoracoscopic approach and with thoracotomy until was analyzed. long term outcome of the patients with thoracoscopic esophagectomy was compared to the result from comprehensive registry of esophageal cancer in japan. short term results of the perioperative parameters were analyzed between left lateral decubitus position and prone position.we had introduced intraoperative nerve monitoring system for prone esophagectomy from . results: there was no significant differences of the survival rate between thoracoscopic group and thoracotomy group based on pathological stage. year survival without neoadjuvant treatment was . % (pstagei), . %(pstageiia), . %(pstageiib), . %(pstageiii), respectively. year survival rate of cstageii and iii with neoadjuvant chemotherapy was . % and year survival rate of the salvage esophagectomy after failure of definitive chemoradiotherapy was . %. every outcomes are as good as any reported results in esophagectomy. in the comparison of the lateral position with the prone position, total blood loss was significantly lower in prone position. inflammatory response after surgery was improved more rapidly in prone group, therefore, prone position is recommended as a minimally invasive procedure for thoracoscopic esophagectomy. transient recurrent laryngeal nerve palsy was observed % of patients. conclusion: thoracoscopic esophagectomy will develop further as a standard operation for esophageal cancer. nerve monitoring is useful for detecting recurrent nerve and avoiding nerve injury. background: laparoscopic total mesorectal excision (tme), in a wide female pelvis is usually technically easier than in a narrow male pelvis. however, this is not always the case, as the uterus and adnexae may obscure the views and hinder safe dissection, especially in obese patients. techniques such as graspers through additional ports or suspension with sutures through the broad ligament may potentially cause injury or need additional ports/assistants. aim: we present a novel technique using a self-retaining gynaecological uterine manipulator to improve access during deep pelvic laparoscopic surgery in female patients. technical tip: the operation is commenced in the standard manner for a laparoscopic rectal excision. once pelvic dissection is commenced, whenever it is felt that uterine retraction would be advantageous (depending on the level of the rectal tumour, size of the uterus and ovaries, obesity etc.) a self-retaining uterine manipulator (as shown in the video) is used. the tip of this disposable device is introduced into the uterus after dilatation of the uterine cervix. once the balloon at the tip has been inflated, the instrument is secure and hence there is no need for active manipulation by an assistant. the shaft can be rotated to allow anteversion/retroversion of the uterus to varying degrees as required to aid dissection. as the video depicts clearly, it acts as a self-retaining retractor for the uterus and is removed at the end of the operation. though the procedure is being demonstrated by a gynaecologist in the video, the instrument is quite easy to insert and some of our colorectal team have been trained as well. conclusion: the self-retaining uterine manipulator is an efficient tool for uterine retraction in laparoscopic rectal surgery and we have been using it routinely in tme in females for the past years, with no complications. this was previously published as a technical tip in the journal of minimal access surgerybut has never been submitted for peer review as a video. the authors present a video of two clinical cases treated by trans-axillary endoscopic approach. methods: a years-old male and a -year-old male presented with intermittent dysphagia and frequent reflux (class ii of lahey). one had a history of recurrent respiratory infections. the disease was characterized by oesophagogastroscopy (egd) and oesophagogram. trans-axillary approach with areolar port. step-by-step as follows: (i) dissection anteriorly to the pectoralis major muscle (ii) isolation of the anterior border of sternocleidomastoid muscle (iii) omohyoid muscle's isolation (iv) identification of the thyroid's upper pole (v) zd isolation (vi) myotomy of the cricopharyngeal muscle (vii) zd's resection with stapler and its withdrawn with sac. results: both cases progressed without complications. complete local recovery was verified in both cases one month after the procedure. conclusion: this technique seems feasible and reproducible, allowing zd diverticulectomy with a better cosmetic result and perhaps lower surgical site infections (ssi). in the authors' knowledge, this approach to dz has never been published. background: gastric leak occurs in - % of patients who undergo roux-en y gastric bypass (rygb) for morbid obesity. the pathophysiology may be related to gastric ischemia, fistula, or ulcer.gastric leak is a severe complication of gastric bypass (gbp) that is associated with significant morbidity and mortality. fistula may have several clinical impacts, depending on patientrelated factors, fistula characteristics, onset time, and therapy proposal. abdominal drainage, gastrostomy, and revisional surgery constitute the traditional approaches to dehiscence and fistula closure, with variable results. methods: we present a video of a clinical case of -year-old lady with body mass index of kg/m who underwent roux-en-y gastric bypass and h later presentedtaquicardia and right cuadrantum pain. the ctscan inform a apical leak at the gastric pouch level. the video shows the relevant aspects of a revisional surgery and the key points to drain the fistula and close de defect laparoscopically. results: after monts, the patient achieved succesful results, defined as a stabel clinical situation with image evidence of gastric fistula remision. conclusions: gastric bypass (gbp) is one of the most efficient bariatric interventions in morbidly obese patients. the most severe risk of this procedure seems to be the staple line leak, and the management of this complication can be very arduous. without any guidelines it is very difficult to determine the right procedure addressing the staple line leak after gbp. laparoscopic sleeve gastrectomy (lsg) has become the most commonly performed operation worldwide as a primary bariatric/metabolic procedure. however, conversion to other surgical procedures such as roux-en-y gastric bypass (rygb) or one anastomosis gastric bypass (oagb) have been described as treatment options for inadequate weight loss after lsg and unresolved co-morbidities or complications such as leak, stricture, and severe gastroesophageal reflux disease (gerd). we present two clinical cases of weight regain and severe gerd and dysphagia, which account for the main indications to reversal of lsg to either oagb or rygb. aims: we show in the video the surgical technique that we perform by laparoscopic aproach, in order to construct a roux-en-y polipropilene banded gastric bypass lrygb-b. methods: we are performing this procedures within a prospective randomized trial that is design to compare the long term results of lrygb-b versus the standard laparoscopic roux-en-y gastric bypass.the video shows our technique in a case of a years old female with a bmi of kg/m . first we create a vertical gastric pouch of about - ml, and a polypropylene mesh ( x mm) is placed - mm proximal to the anastomosis around the gastric pouch, with the help of a laparoscopic band retractor. after that a cm roux-en-y limb is constructed in an antegastric antecolic fashion, been the lenght of the biliary limb cm. a mm gastroyeyunal anastomosis is performed with a linear stapler, and the enterotomy and gastrostomy are closed with a / barbed running sutures. jejunojejunostomy anastomosis is constructed in similar fashion, but with a lenght of - mm. the petersen space and the mesenteric defect are closed with polipropilene / sutures. results: patients has been operated following this technique, and there has been no complications related to the polipropilene band. (the ramdomized prospective trial is still ongoing). conclusions:the video shows a reproductible easy way to perform a lrygb-b using a polipropilene mesh. introduction: a -year old female patient presented at our clinic two years after initial rouxen-y gastric bypass. she had had a preoperative bmi of , and had a significant weight loss which resulted in a bmi of , at two years postoperatively. she currently suffered from severe dumping with glycaemia levels dropping to mg/dl. pharmacological treatment with metformine, sandostatine and acarbose did not yield any results. on top of these problems she felt less restriction, could eat large portions and had gained kg in the last three months. objective: the usual approach for severe dumping-related hypoglycemia would be to undo the gastric bypass. this patient however was extremely anxious to regain weight, so we sought other options. we assumed that by adding more restriction and slowing down the emptying of the gastric pouch we could alleviate some-if not all-of the dumping related symptoms and prevent further weight regain. methods: in this video we present the banding of a gastric pouch for severe dumping after rouxen-y gastric bypass. results: although unconventional, the banding of the pouch yielded excellent results. the slower pouch emptying and reduced portions resulted in a near complete remission of all symptoms. as an additional benefit we found a slight weight loss of four kilograms six weeks postoperatively. conclusion: the usual treatment of severe dumping-related hypoglycemia would be an undo of the gastric bypass. in this case however the patient was extremely anxious to regain weight, being very pleased with the results her gastric bypass had yielded. in agreement with both the patient and treating endocrinologist we attempted a different approach. the slower pouch emptying and increased restriction offered another way to alleviate the dumping and deep hypoglycemia while concomitantly resulting in weight maintenance. aim: the aim of this video is to present a novel surgical technique to avoid stent migration after endoscopic placement in patients with leakage subsequent to laparoscopic sleeve gastrectomy (lsg) . methods: this video shows the case of a patient (bmi , kg/m ) who developed an upper gastric leakage days after lsg. a ct scan showed a small leakage at the eg junction complicated by intra-abdominal abscess. a ct guided percutaneous drainage of the abscess was performed. a stent placement was attempted endoscopically three times and failed for migration. we decided to place laparoscopically a non adjustable gastric ring (nagr) around the stomach, in order to avoid stent migration.first of all the stent is replaced endoscopically in order to cover the fistula tract. the patient is placed in a half sitting position and the pneumoperitoneum was obtained using a veress needle in left subcostal space. a port technique is used as in standard laparoscopic sleeve gastrectomy.the procedure starts with the mobilization of adhesions, the fistula is identified in the upper part of the tubule.the gastric tubule is isolated and the lesser omentum is opened. the blunt needle at the tip of the ring is passed retrogastrically, a tourniquet can be useful is the positioning turn out to be difficult. the nagr is then closed over the gastric tubule containing the stent. a drain is finally placed. results: the stent was removed after weeks. a gastrointestinal ct scan with oral contrast showed a complete resolution of leakage. after months the patient was in a good condition with bmi , kg/m . the stent was endoscopically removed after weeks. a gastrointestinal ct scan with oral contrast showed a complete resolution of the leakage. after months the patient was in a good condition with bmi , kg/m . conclusions: this new technique is feasible and effective, as shown in this video; however the nagr can lead to complications, so a strict follow up is needed and if any complication appears, should be considered to remove laparoscopically the ring. introduction: in this case, we will discuss the case of a year old male patient who underwent a laparoscopic cruraplasty and gastric plication resulting in a weight loss of kg. other medical history reported insulin-dependent diabetes, reflux esophagitis and sleeping apnea with cpap. two years after gastric plication the patient presented with passage problems, gastro-esophageal reflux and epigastric pain. to this end a swallow test was performed revealing a large fundus with a restricted passage of contrast. due to the persistent complaints and the abnormal findings on barium swallow a surgical re-intervention was needed. objectives: despite the current bmi of and the age of the patient, conversion from a gastric plication to a roux-en-y gastric bypass was performed. several other surgical options were considered, including an undo of the gastric plication or a dilatation with a resizing of the fundus. methods: in the video we describe the laparoscopic approach for a conversion of a gastric plication to a roux-en-y gastric bypass. results: at months follow-up the patient showed a weight loss of kg and the resolution of his earlier symptoms. the patient had a normal oral intake without any gastro-esophageal reflux or epigastric pain. conclusion: after a gastric plication, partial loosening of the sutures and stenosis are both wellknown complications. as presented in the video, it is apparent that a laparoscopic undoing of gastric plication is not as straightforward as it seems. firm adhesions between folds can compromise the procedure and inhibit a complete separation of the tissues. we believe that in these cases the best surgical approach is to convert to a roux-en-y gastric bypass. laparoscopic sleeve gastrectomy (lsg) is a relatively new surgical approach in the weight loss surgeon's armamentarium. in literature there is a consensus about the importance of mobilizing completely the gastric fundus before transection. the resg (revised sleeve gastrectomyresleeve) may be a valid option for failure of primary lsg. we focused the attention on the consequences that can have an incomplete resection of gastric fundus during an operation of sleeve gastrectomy and how they can be solved by the repetition of this procedure. a sleeve gastrectomy was performed in an obese -year-old woman (bmi = ). three days after the operation, an upper gi x-ray with gastrografin did not show any abnormalities. three months after the surgical procedure, the woman referred frequent episodes of vomiting and a significant weight loss ( kilos). an upper gi x-ray with gastrografin demonstrated the presence of multiple communicating cavities of the gastric fundus. the esophagogastroduodenoscopy (egd) showed that the gastric tube close to the esophagogastric junction was separated from a recess ( - cm in diameter) by an incomplete septum. a severe hypokalemia and consequent ecg abnormalities were treated with intravenous infusion of potassium. then, we performed a laparoscopic operation. the gastric tube was completely released along the suture line of the previous operation and, especially, the posterior surface of the upper part until the left crus of diaphragm became evident. under the guide of the bougie, the recess was removed. results: the clinical course was regular, and the patient was discharged on third post-operative day after an upper gi x-ray with gastrografin which demonstrated the absence of leakage and a normal gastric tube. after year, the patient was very satisfied with the operation. conclusions: the complete mobilization of the gastric fundus allows to see clearly which part should be resected to obtain an adequate gastric tube and facilitate a correct placement of the stapler. in our experience, in patients with a residual fundus, an upper gi x-ray with gastrografin and an egd are needed to exclude the presence of stenosis. then, a resleeve gastrectomy is an efficient and safe procedure to treat this post-lsg complication. weight regain is one of the main problems in bariatric surgery. we have many surgical option but when we evaluate patients with long follow up and bmi of superobese patient before the first surgery, the weight recidivism can arrive up to - % at years.in most cases the first surgery is a restrictive procedure, and in many cases sleeve gastrectomy.here we present a case of weight regain after laparotomic super-magenstrasse (that we consider like a sleeve gastrectomy except for remnant removal) with a big incisional hernia. after a complete multidisciplinary re-evaluation we decided to perform an oagb (one anastomosis gastric bypass) but in this case we decided to create a functional exclusion to the duodenal transit by positioning a minimizer ring. this solution is effective in food diversion and guarantee gastric and duodenal endoscopic exploration in case of need. we think that this technique can represent an option to take in account for selected cases. at the end of bariatric procedure we perform a laparoscopic repair of incisional hernia with mesh in the hope to avoid future surgery and post operative small intestine herniation. patient rejected additional bariatric procedures and in fact she has gained kg two years later (bmi . ). conclusions: lagb gastric erosion is uncommon ( . - %) . intraoperative (such as perigastric approach) and patient related factors (smoking, alcohol…) have been described as risk factors. the most frequent clinical presentation is weight loss failure; band and port issues (such as infection) are also frequent. erosion is infrequent to present as an acute event (\ %: peritonitis, abscess…) or asymptomatically (\ %). diagnosis is mostly performed under upper endoscopy. the most common therapeutic technique is removal of the band (by endoscopy or surgery), repair of the stomach, if needed, and band replacement (at least three months later). some authors have performed immediate replacement but the incidence of recurrent erosion seems to be higher. other options are lagb removal alone or conversion to different bariatric procedure. for endoscopic removal, it has been advised to wait until the band buckle is in the stomach and is sometimes very difficult. replacement of the band is not associated with weight regain. she reports years of evolution presenting moderate intensity heartburn that was exacerbated during the night as well as submit occasional rejurgutation. the intensity of the symptoms is attenuated by maintaining a diet without irritants and improving feeding times. denies hematochezia, unintentional reduction of weight, dysphagia or early satiety. the patient has suffered from obesity since childhood, after pregnancy she had progressive weight gain and difficulty in controlling blood sugar, so she is scheduled a gastric bypass roux-en-y . preoperative endoscopy was performed, evidencing submucosal tumor in the gastroesophageal junction at of the dentary arch, approximately cm in diameter. an endoscopic ultrasound was performed, demonstrating subepithelial lesion of the gastroesophageal junction, hypoechoic, with well-defined borders, pseudobilobulated, . cm x . cm, and dependent on the external muscular layer. a fine needle aspiration is performed in which spindle cells are identified, leiomyoma is likely diagnosed. it is programmed for laparoscopic resection of submucosal gastric tumor, gastric bypass and laparoscopic cholecystectomy. a tumor at the level of the gastro esophageal junction of approximately . cm is identified in the surgery, which can be resected by laparoscopy without complications. the patient is discharged after days of postoperative stay. the final histopathological result: leiomyoma of . cm with free edges. cd (-) gog (-) caldesmon (?)s (?). background: fifty percent of patients who have undergone gastric bypass, posterior reversal and sleeve gastrectomy and finally complete hiatoplasty presents symptomatic gastroesophageal reflux disease. surgical reinforcement of the lower esophageal sphincter is necessary to prevent acid reflux. here, we describe ligamentum teres cardiopexy, a surgical technique that reinforces the lower esophageal sphincter and restores its competence with a new valve, in patients with previous conversion of sleeve gastrectomy to gastric bypass and hiatal hernia repair. methods: we present the surgical techhnique performed to a patient with initial gastric bypass who underwent sleeve gasterctomy for hipoglycemias and hiatoplastia for severe gerd. persistent gerd requested to undergo ligamentum teres cardiopexy. in this procedure, the ligamentum teres is released from its umbilical connection and the hernia reduced by manual traction, freeing the last - cm of esophagus in the abdomen. the distal ligamentum teres is fixed with one stitch to the apex of the angle of his, one at the gastroesophageal junction, and one joining the gastric fundus to the esophagus. the remainder of the ligamentum teres is fixed over itself with four to six stitches, forming a necktie cardiopexy. the procedure concludes with diaphragmatic crus closure. results: after months, the patient achieved successful results, defined as resolution of gerd, no protonpump inhibitor (ppi) use, and manometry measurement over mmhg after surgery. conclusions: ligamentum teres cardiopexy combined with closure of the gastric crus is a late alternative treatment for gastroesophageal reflux disease in patients with previous sleeve gastrectomy and hiatal hernia. general surgery, ponderas academic hospital, bucharest, romania introduction: as metabolic surgery techniques evolve during the years, we have to face more and more patients with complications ands uboptimal results after the older/initial procedures. vertical banded gastroplasty(vbg) is one of those procedures that gain momentum during the initial experience in bariatric surgery, but has proven to have dissapointing results and a lot of complications, nowadays surgeons having to deal with difficult revisional operations. aim in this video: we want to present from our experience the difficulties encountered during the revisional surgery, rouxen y gastric bypass (rygbp)aftervbg, and the tips and tricks that will make this a safer and easier procedure. objective: after thorough preoperative assessment and a review of the literature multiple treatment options were considered. the procedure of choice ended up being a laparoscopic adjustable gastric banding, with the objective to achieve optimal weight loss with the lowest risk for complications. methods: in this video we present the placement of an adjustable gastric banding in a patient with a cirrhotic liver and portal hypertension and the possible pitfalls. results: postoperatively there were no complications and patient had a satisfying weight loss both months and year postoperatively. in a short review of the literature we've found that bariatric surgery is feasible in patients with portal hypertension as long as the patient is not decompensated or has bleeding varices. conclusion: cirrhosis and portal hypertension are no absolute contraindication for banding, sleeve or rny gastric bypass as long as the patient is not decompensated or has bleeding varices. the type of surgery is dependent on patient and surgeon-related factors. the aim should be to achieve optimal weight loss with the lowest possible surgical risk in this type of patients. surg endosc ( ) :s -s introduction: in this case, we will discuss on a year old female patient who had undergone a laparoscopic nissen fundoplication years ago due to gerd grade b. because of morbid obesity a n-sleeve gastrectomy was performed year ago resulting in a weight loss of kg. at presentation she had regained all the lost weight, resulting in a bmi of , . the patient history also reported insulin-dependent diabetes and obstructive sleep apnea with cpap. gastroscopy was performed showing a large residual fundus but no esophagitis. on the subsequent upper gi series a relatively wide sleeve with an intact nissen-collar was detected. objectives: a laparoscopic conversion to a roux-en-y gastric bypass was performed. other potential surgical treatment options are a sadi procedure or a sleeve gastrectomy with transit bipartition (santoro procedure). methods: in the video we describe the laparoscopic approach for a conversion of a n-sleeve to a roux-en-y gastric bypass. results: at month follow-up the patient presented with a weight loss of kg. the patient had good restriction on oral intake and did not have any reflux-related symptoms or complaints. conclusion: conversion from a n-sleeve to a roux-en-y gastric bypass is a challenging procedure. the largest pitfall during the creation of the gastric pouch is to staple a double fold of the nissen fundoplication. we believe that in these rare cases of weight regain after n-sleeve, the best surgical approach is to convert to a roux-en-y gastric bypass. four years later, in , a laparoscopic conversion to roux-en-y gastric bypass was performed because of weight regain. she now presents with satisfactory and stable weight loss over the last few years. she was recently diagnosed with a brca- mutation for which she underwent a bilateral ovarectomy and mastectomy. the patient's brother was also diagnosed with this mutation and died of pancreatic cancer at the age of . genetic counseling advised a twoyearly follow-up because of an increased risk up to % of developing pancreatic cancer. control gastroscopy showed a normal esophagus and gastric pouch. control ct scan revealed hypertrophic stomach creases in the excluded stomach. these results prompted a laparoscopy-assisted gastroscopy of the excluded stomach which uncovered hypertrophic stomach glands and intestinal metaplasia on biopsy. methods: in this video we demonstrate the laparoscopic approach for complex revisional bariatric surgery. conversion from rny gastric bypass to a sleeve gastrectomy in a patient who already underwent a vbg. the focus of the video is on a manual gastro-gastrostomy with partial gastrectomy of the fundus and part of the stomach where the old vbg-band was placed. results: after , months follow-up the patient had no complaints and a stable weight. upper gi series shows a normal passage of contrast through the sleeve gastrectomy. conclusion: endoscopic surveillance of the remnant stomach and echo-endoscopy of the pancreas is no longer possible after rny gastric bypass. in cases where the need for such a surveillance arises after a rny bypass a patient-tailored approach is necessary. in our patient a laparoscopic conversion from a rny gastric bypass to a sleeve gastrectomy was performed. this approach keeps the patient's wish for weight loss intact while enabling further surveillance through natural-orifice endoscopy. a -year-old morbidly obese japanese woman with a body mass index of kg/m suddenly complained of swallowing difficulty months after laparoscopic roux en y gastric bypass surgery with retro-colic roux limb route. an internal hernia of the defect of the transverse mesocolon was suspected by computed tomography, and emergency intervention was performed. the surgery revealed no internal hernia. however, strong inflammation and adhesion were observed between the transverse mesocolon and the retrocolicroux limb. in addition, the roux limb on the oral side of the adhesion site was dilated and bent.the adhesion between the transverse mesocolon and the flexed roux limb was dissected, linearized and re-fixedby suturing to the transverse mesocolon. however, since the difficulty of oral intake persisted re-do surgery was performed again. after resecting the roux limb involved in the severe inflammation, a 'new' roux limb was lifted to the cephalad via the ante-colic route. finally, the gastric pouch and roux limb were re-anastomosed with - absorbable sutures in an interrupted full thickness single layer manner. in the present case, we experienced difficulty with both adhesiolysis and determining the accurate target line to resect at the 'old' gastrojejunostomy. however, blocking the blood flow of the 'old' roux limb facilitated the accurate recognition of the target line. esofagogástrica, cirugía general y ap digestivo, hospital regional universitario de málaga, malaga, spain; hepatobiliopancreática, hospital regional universitario de málaga, malaga, spain introduction: marginal ulcer is one a serious complications after a bariatric gastric bypass. tobacco, non-steroidal anti-inflammatory drugs (nsaids) and helicobacter pylori (hp) infection are known risk factors. methods: we present a -year-old women operated years before of bariatric surgery with a gastrojejunal (gy) bypass technique due to intraoperative dehiscence of the staple line after attempting a vertical gastrectomy (sleeve). she has persistent vomiting and epigastralgia from months after the intervention, affecting his quality of life. upper gastrointestinal endoscopy (uge) was performed, describing an ulcer in the gy anastomosis. she started hp eradication treatment, treatment with proton pump inhibitors (ppis), tobacco and nsaids were discontinued, but she had slight improvement. after months the uge was made again, which show peptic esophagitis and marginal ulcers. the plasma gastrin level was normal. due to the persistence of symptoms despite conservative treatment, we decided reoperation by laparoscopy. we found herniated bowel in petersen space, which were reduced and the space was closed. we proceeded to truncal vagotomy. the gy anastomosis was resected ( fig. ) and performed again. finally, we perform antrectomy. the pathological anatomy showed ulceration. she was diacharged home on the th postoperative day without any complications. results: a marginal ulcer after bariatric surgery appears in the jejunal mucosa of the g-y anastomosis. the symptoms are epigastric pain, nausea and vomiting. acid, tobacco, nsaids and hp infection has an important role in their development \ sup [ . \/sup [ the first treatment is medical, discarding out the risk factors, but if it is not effective, it will be surgical, resecting the previous anastomosis. the usefulness of vagotomy is debatable, but the percentage of success increases. in our case, we perform antrectomy to avoid retained antrum syndrome. the hernia through petersen space is a cause of intestinal obstruction and abdominal pain as the case presents. although we believe that the symptoms were mainly caused by the marginal ulcer, the internal hernia was probably a symptomatic cause. conclusion: the treatment of a marginal ulcer is medical, eliminating the risk factors, but if it is not effective, the surgery is indicated. results: bowel's measurements and confection of the gastric pouch are identical in both cases. in the first case, intestinal anastomosis is performed in the inframesocolic compartment once small bowel has been divided. in the second case, such union is made next to the gastrojejunal anastomosis with the bowel uncut, making the section once no leakage has been found conclusions: laparoscopic roux-en-y gastric bypass is currently considered one of the technique of choice in the surgical treatment of morbid obesity. there are variations and alternatives for its realization. to know them can allow to individualize the technique to each type of patient. we present a clinical case of a year old female. she had a vertical banded gastroplasty procedure (in another clinic) years ago with an initial weight loss of kg in a period of months. she was seen at our clinic because she was suffering from dysphagia to solids and general diffuse abdominal pain for the last month. at physical exam we found a bmi of and nothing else called our attention. we did an upper gi endoscopy and egd transit; we concluded that a gastric bypass would offer her the best results. therefore, we converted her vertical banded gastroplasty into a gastric bypass laparoscopically. she had an uneventful postoperative period and was discharged home without complications. aims: sadis emerged as a modification of biliopancreatic diversion with duodeno-ileal switch (bpdds) in which after sleeve gastrectomy (sg), the duodenum is anastomosed to an ileal loop in a billroth-ii fashion. sadis has promising outcomes for weight loss and comorbidity resolution in morbidly obese patients avoiding the high morbidity of biliopancreatic diversion with duodenal switch. clinical case: -year-old patient, subjected to bariatric surgery two years ago, including a sleeve gastrectomy (sg). despite this operation and dietary and hygienic modifications, the patient gained weight in recent months, reaching a bmi of kg/m and an overweight of kg. an endoscopy was carried out on her, which provided evidence of a gastric remnant of moderate size with flexible tissue, normal peristalsis, and fast disposal speed. the case was discussed in a joint session, leading to the decision to apply revision surgery. the decision was taken to apply sadis, a novel technique that had never been used before in andalucia. the rate of weight re-gain after the use of classical techniques such as sleeve gastrectomy (sg) or the roux-en-y gastric bypass (rybg) is considerable high. revision surgery due to weight re-gain is necessary in many of these cases. sadis emerged as a simplified alternative to the use of bpdds as revision surgery following a gv due to weight re-gain with good short-term results, in terms of both weight control and comorbidity control. since only one anastomosis needs to be applied, chirurgical time diminishes, as well as the rate of surgery-related complications. moreover, it could be used, through laparoscopy, for patients who have undergone previous, complex abdominal surgery. conclusion: sadis showed a promising short-term weight loss outcome and comorbidity resolution rate but long-term data are missing and there is currently a high level of technical variability. on the other hand, further studies are required to measure its cost-effectiveness compared to the currently popular bariatric procedures, sg and rygb. aims: the lps sleeve gastrectomy is the most common bariatric surgery technique because it has a low surgical complexity and acceptable weight loss results. however, - % of patients present with an insufficient weight loss, weight regnances, reflux or dysphagia. in these cases, it is recommended to perform a second bariatric surgery to combine a component of malabsorption such as gastric bypass or duodenal switch. the video describes the technique of a laparoscopic biliopancreatic diversion with duodenal switch with a previous laparoscopic sleeve. the objective is to describe the safety of the technique and the subsequent success of it. methods: a -year-old female patient presented morbid obesity with a bmi of after performing a laparoscopic sleeve gastrectomy in . initially, she presented a percentage of excess weight loss of %, reaching a bmi of after two years of follow-up. after this, she suffered a reganancia of all the weight lost despite diet and exercise, presenting a bmi . a study was made with tegd where no complications of the previous surgery or symptoms of gastroesophagical reflux or dysphagia were observed. the lps duodenal switch is proposed in the obesity unit committee in , without immediate postsurgical complications. the patient presented a favorable postoperative period and was discharged three days postoperatively. results: at the present time, the patient has achieved a % excess weight loss and has a bmi of . . presents good oral tolerance with stools a day without urgency. it doesn't present protein deficioncies. vitamin deficiencies are orally supplemented. the lps duodenal switch is a technique that can be performed after a sleeve gastrectomy safely in cases of insufficient weight loss or weight reganancia. the patients presented a greater weight loss after the duodenal switch than after the gastric bypass, observing a lost of excess weight of % compared to %. the differences being statistically significant. weight regain after gastric bypass is a challenging problem. a number of revisional surgical options have been reported. this is a case of a year-old woman years after lrygb. her initial bmi was , lowest after surgery- , at presentation- . the video shows a robotassisted laparoscopic conversion of rygb to loop duodenal switch. the roux limb is transected and dissected to the gastrojejunostomy. the gastrojejunostomy is resected and the gastric pouch is recreated over a bougie. the gastric blood suply is confirmed with icg. a gastro-gastrostomy is created to restore gastric continuity and a sleeve gastrectomy is performed. the duodenum is devided and a duodeno-ileostomy is created cm from the ileocecal valve. the remaining roux limb is resected. the patient recovered uneventfully. conversion of rygb to loop duodenal switch requires creation of as little as two anastomoses, in comparison to standard ds, which requires four. it is a safe option for patients with weight regain after lrygb. methods: irb approval and informed consent have been obtained. a dissection is conducted to separate the descending mesocolon of the gerota's plan from the medial aspect to the peritoneal lining to the left parietal gutter. the peritoneal layer is incised parallel to the vessel and close to the colonic wall. the dissection is continued anteriorly up to reach the resected parietal gutter. a passage into the mesentery of the upper rectum is created for the allocation of the stapler and the dissection of the rectum. these maneuvers permit to straighten the mesentery simplifying the identification and cutting of the sigmoid arteries. a caudal-to-cranial dissection of the mesentery is performed from the sectioned rectum to the proximal descending colon by a sealed envelope device. it can be very useful to mobilize the colon in any direction: laterally, medially, or upward. the dissection is performed along the course of the vessel up to the proximal colon, with progressive sectioning of the sigmoid arterial branches. the specimen is extracted by a pfannenstiel incision. the anastomosis is performed transanally with a circular stapler according to knight-griffin technique. results: we performed a laparoscopic segmental colectomy using this approach for patients with benign sigmoid lesions: diverticulitis, flat polypoid lesions (no lift-up sign), and bowel endometriosis. the mean operative time and blood loss were . ± . min and ± ml, respectively. there were not a single conversion to open surgery and no any leakage or stricture. only cases of intraluminal bleeding and case of wound infection (treated conservatively) were observed. conclusion: we consider this approach to be safe and useful for segmental colectomy to be performed sectioning the sigmoid artery close to the colonic wall. aims: to show a clinical case with a video of a patient was operated for colon cancer in hepatic angle by a single suprapubic incision (ssilrh). methods: a -year-old male assessed for abdominal pain and weight loss. on physical examination: a painful mass was detected in the upper right quadrant. the colonoscopy revealed an ulcerated lesion in the hepatic angle and the biopsy revealed a moderately differentiated adenocarcinoma. in the abdominal ct a mass of x cm was observed (figure). the patient was operated with ssilrh technique, as shown in the attached video. results: the patient was placed in the supine position and with the legs separated. the surgeon is placed between the patient's legs. a transverse incision of the skin was made in the middle line of . cm to cm above the pubis. the underlying fascia was divided transversely, the rectus abdominal muscle was exposed, a purse-string suture placed in the fascia. an mm reusable trocar was inserted for the chamber, a mm reusable flexible trocar was placed at the o'clock position and another trocar was placed at the o'clock position. the ileocecal valve was released from the peritoneal parietal foil, as well as the mesocolon right by a lateral to medial approach to the second portion of the duodenum. the hepatic angle was also dissected from lateral to medial. for the anastomosis, the mm trocar was replaced with a mm trocar and a stapler was placed. a mm °chamber was inserted through the mm flexible trocar. the small intestine was divided as well as the proximal transverse colon with endogia. an intracorporeal ileocolic anastomosis was performed. the piece was removed through the suprapubic incision. he was discharged after days without complications. the histological studies confirmed a differentiated adenocarcinoma of x x cm. the surgical margins were free, without infiltrated lymph nodes ( / ) with stage pt n . the ssilrh technique allows a complete resection of the mesocolon and complies with the oncological principles. they can present with abdominal pain, nausea, acute abdomen, symptoms of intestinal obstruction or asymptomatic with incidental diagnosis. their diagnosis can be difficult. the objective is to demonstrate the safety and efficacy of the laparoscopic approach in this infrequent pathology. material and methods: we present a video of the surgical intervention of a -year-old patient, with functional dyspepsia, with a casual diagnosis of a pseudocystic mass of the right colon after performing a ct scan: giant diverticulum of the hepatic colon angle with fecaloid content inside it under tension the patient goes to the emergency room for acute abdominal pain, pending colonoscopy, antibiotic treatment is established, and a laparoscopic approach is decided upon after the patient's evolution. results: intervention: complete laparoscopic approach, trocars. large size tumor in the right colon, diverticular in appearance, with stony content inside, with locoregional adenopathies, oncological radical right hemicolectomy, manual intracorporeal anastomosis, correct postoperative, hospital discharge. on the th day. definitive pathological anatomy: giant diverticula on areas of intense mucosal ulceration, free edges. conclusion: the laparoscopic approach of the symptomatic diverticula of the right colon is safe and effective. introduction: minimally invasive transanal surgery (tamis) is a surgical technique whose established indications are the complete exeresis of rectal polyps that are not resectable endoscopically or early rectal neoplasms with good prognosis criteria. transanal devices with gel platform facilitate dissection in this field. however, one of the drawbacks of this approach is the oscillation of the right nerve, which hinders dissection and prolongs the surgical time. material and methods: we present the case of a patient with a central depression neoformation, located cm from the anal margin in the posterior aspect of the rectum in a male patient. the lesion occupies % of the circumference and was considered unresectable endoscopically. the endoscopic biopsies showed a tubulovillous adenoma with moderate dysplasia. results: an exeresis of full thickness of the rectal wall is performed, with subsequent suture of the defect. we show in the video the use of a glove interposed in the pneumoperitoneum gum to maintain the stability of the neumorectum and the technique of dissection and suture, as well as the stability of the neumorectum with this technique throughout the procedure. the use of a glove as a reservoir to stabilize the nemorectum is an economical and easy-to-use method that can safely replace extra devices. aims: endometriosis is a gynecologic disorder defined by the presence of endometrial glands and stroma outside the uterine cavity. deep infiltrating endometriosis (die) invades mm to the retroperitoneum of the pelvic sidewalls, the rectovaginal septum, or the muscularis of the bowel, bladder or ureters. the rectum is being the most common bowel site of involvement. for symptomatic die, medical therapy should always be the first-line treatment. therefore, a minimally invasive approach using laparoscopy is considered the gold standard option and challenging aiming at complete disease excision. also, there are several advantages of natural orifice specimen extraction when compared with abdominal incision that may directly impact the postoperative results of these young patients. methods: we report a case of a -year-old female with a -month history of chronic pelvic pain, dyschezia and rectal bleeding. these symptoms were refractory to hormonal, antispasmodic and opioid therapy. magnetic resonance imaging reported a nodule x cm invading the rectal wall cm to the dentate lane. we performed a laparoscopy and we found the nodule at the uterine posterior wall invading the rectal anterior wall. the nodule was invading into the rectum in a large area so we proceeded with segmental resection and added hysterectomy and salpinguectomy because it was the preference of the patient. the anastomosis was created intracorporeally and the specimen was removed through the vagina performing in this way a totally laparoscopic procedure with natural orifice specimen extraction. results: the total operative time was h, the postoperative stay was uneventful and the patient was discharged on day four. the pathological report showed an endometrioma cm length predominantly involving colonic muscularis propria. conclusion: laparoscopic surgery is a safe and feasible approach for the surgical management of deep infiltrating endometriosis of the rectum and the gold standard for female young patients that often need multiple surgeries. in addition natural orifice specimen extraction avoids potential complications of abdominal incisions. week-day surgery, university, sapienza, ospedale sant'andrea, rome, italy; urology, clinica mater dei, rome, italy aims: we describe a case of a patient affected by a mass in the left kidney and a diverticular stenosis of the sigma. methods: a years old woman complained abdominal pain in the left flank of the abdomen and in the left iliac fossa radiated to the hypogastrium, with fever and no passing flatus. contrast enhanced computer tomography scan (ct-scan) showed a cm mass of the superior pole in the left kidney and a colonic diverticulitis with thickness of the wall and a microperforation of the sigma. she underwent to medical therapy with resolution of the diverticulitis. after weeks a laparoscopic nefrectomy and sigmoidectomy was planned. patient was positioned on the right flank. this position was kept for both the procedures. we performed four trocar accesses along the left subcostal region and a periombelical incision for the specimen extraction. results: post-operative course was uneventful. patient was discharged in post-operative day. istopathological exam showed a renal cell carcinoma confined to kidney with no positive lymph nodes and a diverticular stenosis of the sigma. laparoscopy allowed to perform two fine procedures in a critical situation using few trocar incisions and obtaining good results. background: hartmann procedure consists in a sigmoidectomy followed by a terminal colostomy. stoma is associated with complications and suboptimal quality of life, so the restoration of colonic continuity should be at least considered in any case. open restoration has been associated with significant morbidity and mortality. many authors have described the advantages of laparoscopic hartmann reversal. we want to go a step further showing our experience using a combined laparoscopic and transanal approach in an attempt to improve the surgical technique in a patient with previous abdominal surgeries and a rectovaginal fistulae. methods: the transanal and laparoscopic team work simultaneously. by the abdominal approach a pericolostomic incision is made, the distal affected colon is resected and a purse string suture is performed around the anvil of the eea mm single-use stapler with . mm staples (autosuture, covidien). a mm umbilical trocar is located for a °camera and a gelport laparoscopic system (applied medical) with two mm trocars is introduced through the colostomy wound. hard pelvic adhesiolysis was performed and splenic flexure was also mobilized.the gelpoint path transanal access platform (applied medical) is introduced through the anal canal with three trocars in a triangle position. the proximal rectum and mesorectum are dissected until the peritoneal reflexion. the previous stapler line with the resected tissue is then exteriorized throught the anus. the distal rectum is prepared with a circumferential purse string suture. the vaginal defect was sutured transanally. the proximal colon and the anvil are extracted through the rectal stump and connected to the circular stapler, performing an end-to-end anastomosis. results: the total operative time was h. the postoperative stay was uneventful and the patient was discharged on day . conclusions: as in patients with rectal cancer, dissection of the stump in hartmann reversal procedure may be better and associated with shorter operative time. as with any new surgical procedure, it is probably too early to draw conclusions but nowadays transanal combined with laparoscopic approach seems to be a safe and feasible technique to perform a hartmann reversal, especially in challenging cases. intravenous and endoluminal contrast enhanced ct revealed the presence of a large retroperitoneal fluid and gas collection, due to diverticular perforation, extended from pelvis to iliac bifurcation, involving the left urether. no hydrosoluble contrast media leakage or massive pnuemoperitoneum were present. after an initial conservative treatment without significant improvement an emergency laparoscopic left colectomy with primary anastomosis and laparoscopic retroperitoneal collection drainage was performed. the laparoscopic approach was very challenging due to the obesity of the patient and the presence of the abscess. the patient was discharged on pod after requiring re-intervention for dehiscence of the left iliac mini-laparotomy on pod . conclusion: diverticular perforation in obese patients adds a further challenge to its laparoscopic treatment and deserves an aggressive surgical approach since its outbreak. although intracorporeal anastomosis has been demonstrated to be safe and effective after right colectomy, limited data are available about its efficacy after left colectomy for colon cancer located in splenic flexure. there are few studies comparing patients who underwent laparoscopic left colectomy with intracorporeal anastomosis or with extracorporeal anastomosis. anyway literature shows that there is no significant difference between intracorporeal anastomosis and extracorporeal anastomosis about oncological result. as for right hemicolectomy, intracorporeal anastomosis seems to show a trend towards a faster recovery after surgery due to the shorter time to flatus and lower post-operative pain expressed in the mean vas scale. laparoscopic left colectomy with intracorporeal anastomosis is associated with a lower rate of post-operative complications as for right colectomy. literature results could suggest that a complete laparoscopic approach could be considered a safe method to perform laparoscopic left colectomy with the advantage of a guaranteed faster recovery after surgery. as usual further randomized clinical trials are needed to obtain a more definitive conclusion. we show a video of a years old patient with a pure splenic flexure colon cancer who underwent to a laparoscopic left hemicolectomy with intracorporeal anastomosis. case presentation: here we describe a case of a year old asthmatic and hypertensive lady with an asa score of iii who presented to emergency after a right knee replacement with a four day history of lower abdominal pain. she was septic upon arrival to the resuscitation roomimmediately prompting the hospital's local septic management protocol. a ct scan of her abdomen showed a rectosigmoid perforation with free intra-abdominal air and fluid. the patient underwent laparoscopic hartmann's procedure within h of admission. after an uneventful postoperative recovery the patient was discharged home after a total of days of hospitalisation. she was followed up at surgical outpatients with no adverse events over the course of the subsequent months. conclusion: this case exhibits the feasibility of laparoscopic hartmann's procedure as a surgical modality for hinchey stage iv diverticulitis. the positive outcome supports the claim that for experienced surgeons laparoscopic hartmann's procedure remains a safe and viable option for elderly comorbid patients in the emergency setting. introduction: mesenteric cysts are a very infrequent pathology, they usually present an anodyne clinic, and their diagnosis is reached casually. objectives: to demonstrate the safety and efficacy of the laparoscopic approach, in cases with intra-abdominal cysts of benign etiology, using material with mini-instruments, reducing surgical aggression, maintaining its safety and efficacy.material and method: clinical case: a -year-old man with no personal history of interest. in the last two months he presented episodes of pain in the right hypochondrium, exploration without findings, us-ct scan: a cystic tumor of cm. in hepatic colon angle compatible with uncomplicated benign mesenteric cyst, tumor markers and normal colonoscopy. evidence of interest is exposed. given the evolution it is decided tto. elective surgical. result: intervention: laparoscopic approach, trocars, two of . mm, optics of mm °, benign cystic tumor, with colloid content of more than cm. of diameter in antimesenteric border of colon, which is not possible to separate, mobilization and resection is carried out by endo-gia, including a portion of the colonic wall, appendectomy, extraction in a pocket. good postoperative course, alt to nd day. definitive ap: mesenteric cyst, absence of malignancy. the laparoscopic approach is a valid and effective alternative in cases of benign intra-abdominal cystic pathology, the use of mini instruments reduces surgical aggression, favoring the recovery of the patient. male, yr, wuth doblue post-operative coloanal stenosi and women, with ultralow rectal neoplatis stenosis ( cm from anla verge). both patients were discharge ater days from prosthesis positionins without pain and complications. the first patient, with protection ileostomy, showed fecal incontinence before the operation and was performed prosthesis positioning because rectal losses of infected material and fever. fecal incontinence was showed also after procedure but he had not fever. second patient, yr, with ultralow rectal tumor, after prosthesis positioning was submitted to radiotherapy and she decided for not to be operated and she survives after months in ful well-being. conclusion: endoscopic prosthesis positioning is a consolidated procedure for treatment of bowel obstruction. this study demonstrated that this procedure is safe and this kind of prosthesis is suitable for correct positioning. results: we present a case of a years old man with faecal occult blood test positivity that was diagnosed by colonoscopy of a villous lesion at cm of anal verge. biopsies were taken showing a tubulovillous adenoma with high grade dysplasia. a rectal mri was done showing the lesion fixed to the postero-lateral left side of the lumen at cm of anal verge. no pathological lymph nodes were reported. extension study was negative. the case was presented in multidisciplinary committee agreeing in local excision. in october the procedure was done without incidents. the patient was placed in lithotomic position finding a lesion occupying of the lumen. resection was done without incidents and posterior suture with continuous barbed sutures. he presented an uneventful recovery being the patient discharged in rd postoperative day. definitive pathological findings showed a ptis with negative margins. after three months of followup the patient remains with good functional results and waiting for the first endoscopic revision. conclusions: tamis is a safe and feasible technique with low morbidity that gives us an alternative for early rectal cancer or big rectal lesions much less invasive than techniques used until now. complete mesocolon excision and d lymphadenectomy are two fundamental points in the oncological surgery of right colon cancer. most of the adenopathic recurrences of colon neoplasia in tumors located in the hepatic angle and the ascending colon are located near the head of the pancreas and the vascular axis of the superior mesenteric vein due to an alleged incomplete dissection. we present a case of right colon neoplasia where we performed a laparoscopic right hemicolectomy associated with a d lymphadenectomy. we use medial to lateral dissection of the mesocolon focused on the dissection of the superior mesenteric vein with the identification of ileocolic vascularization, right colic vessels and henle's trunk. this approach is safe and facilitates a correct resection of the mesocolon, which is approached following the embryological plans and a vascular ligature near the bifurcation. the performance of an extended lymphadenectomy allows a wider resection of the mesocolon and the excision of a greater number of lymph nodes, all of which can contribute to a greater survival. the efficacy of pc treatment is related with a properly preoperative imaging diagnosis of the disease, but the poor sensitivity for identifying small peritoneal metastasis are the major obstacle to achieve a complete resection and that leads to peritoneal recurrence. imageguided surgery using icg, could represent an advance in the detection of small peritoneal nodules. there are only a few clinical studies that have analyzed the role of icg for the staging of pc, specially from ccr, and nearly in all of them the selected approach were exploratory laparotomy. this study presents a laparoscopy case, as a non-invasive way of cs in selected patients with limited pc. a new category, tis, was created for low-grade appendiceal mucinous neoplasms (lamns) that invade or push into the muscularis propia by ajcc cancer staging th ed. management of these tumors depends on stage and histology. traditionally, laparotomy was the most recommended approach, however, if laparoscopy is safe, it could be used. the laparoscopic appendectomy should be done with 'not touch' technique and a radical approach has been recently proposed for its treatment. the laparoscopic radical appendectomy should start by exploring complete abdominal cavity. grasping of appendix should not be done. complete resection of mesoappendix is obligated. cequectomy with stapled endogia is necessary. the specimen must be extracted in an endobag. methods: we report a case of a year-old female patient with a personal history of three caesarean sections. this patient was studied due to chronic abdominal pain. a computerized axial tomography was performed, showing an appendix increased in size and a thick wall. the colonoscopy evidence a lesion that protrudes from appendiceal base which is biopsied. results: a laparoscopic way was used and large and width appendiceal was viewed ( x cm). furthermore, a rounded right anexial tumor was also found. a radical not touch laparoscopic appendectomy with stapled cequectomy was done. the intraoperative study was mucinous appendiceal tumor without serose affection. the final result was ptisnx (lamn) without resection margins affected. after h of admission, the patient is discharged without incidents. conclusion(s): minimally invasive surgery in lamns is possible if it is performed with enough experience, following specific rules and tips to manage this tumors. a correct follow-up should be carried out using tumor markers and computer tomography (ct). introduction: resection of both benign and malignant colovesical fistulae can be particularly challenging and carry with it specific surgical considerations. often there is a large inflammatory mass sat within a narrow pelvis, limiting specimen mobility and consequently access to dissection plains. additionally, with the underlying inflammatory process, the ureters may be displaced anatomically and be at risk of injury. aim: to demonstrate a streamlined and reproducible approach to the laparoscopic management of both benign and malignant colovesical fistula, with specific emphasis on the different modalities for bladder repair. method: the following method portrays an overall technique which is adapted dependant on the clinical scenario and specific intra-operative findings: approach to abdominal cavity in standard fashion.identification of right ureter.poster-medial mobilisation of the mass to facilitate delivery out of the pelvis followed by visualisation of the left ureter on the medial and lateral sides before division of the fistula.division of the fistula in benign disease or resection of the bladder dome in malignant disease.transverse laparoscopically sympathetic suprapubic skin incision.vertical incision through linea alba to deliver bulky specimen.intra/extracorporeal repair of bladder dome. results: all of the considered cases were successfully completed with a laparoscopic approach, irrespective of the malignant status of the disease in question. conclusion: both benign and malignant colovesical fistula disease can make the laparoscopic approach to resection challenging, especially when encountering a bulky mass in a narrow male pelvis. the stepwise and streamlined approach considered here can help facilitate successful and safe laparoscopic completion without the necessity to convert to open. background: primary neoplasms of the retrorectal space are very rare. they are located in anatomically difficult area to be addressed, hence a complete evaluation of the lesion is required to determine the extent of resection and the appropriate surgical approach, which include posterior, abdominal and combined abdominoperineal, depending on the characteristics of the lesion. objective: to show a combined laparoscopic abdominoperineal approach of retrorectal tumor. method: we present a video of a combined laparoscopic abdominoperineal resection of a lowlying retrorectal tumor in a -year-old female without prior abdominal surgery. conclusion: retrorectal tumors are infrequent. their anatomical location can make difficult the surgical approach. preoperative imaging can provide useful information for surgical planning. in the recent years, minimally invasive surgical approach has been proposed. laparoscopic approach is feasible and safe, but it is important to select adequately the patients. background: adult intussusception is a rare clinical event representing only - % of all bowel obstruction cases and % of all intussusceptions and the occurrence of adult intussusception due to colonic cancer is even more rare. aim: we present this case of malignant colo-colic intussusception and literature review to increase the awareness of the incidence of colocolic intussusception due to colonic cancer. case report and literature review: our patient is a years old female was admitted to our hospital due to central abdominal pain, cea level of , she was further investigated with ct scan of the abdomen and pelvis which raised the suspicion of mid transverse colon intussusception due to large polypoid lesion. she was further assessed with urgent colonoscopy which confirmed mid transverse colon tumour with biopsies confirmed adenocarcinoma. laparoscopic extended right hemicolectomy with lymph node dissection was performed. upon laparoscopic exploration it was found that the colocolic intussusception was evident as described on the ct scan and as clearly shown on the video. histologically, the transverse colon carcinoma was a moderately differentiated adenocarcinoma, with no lymph node involvement ' out lymph nodes', tnm staging of pt pn pm and r resection. intussusceptions of the colon in adult are frequently found in the ileocecal portion or sigmoidal colon but rarely in the transverse colon. only two cases of adult intussusception of the transverse colon caused by colonic cancer have been reported. overall cases on literature review reported showing colo-colic intussusception due to colonic malignancy. conclusion: colo-colic intussusception due to colorectal cancer is a rare clinical event, however it should be included in the differential diagnosis of colonic obstruction. laparoscopic surgery is safe in malignant colocolic intussusception. aims: single-incision laparoscopic colectomy (silc) aims to achieve better cosmetic outcomes, less pain, and faster recovery compared to multi-port laparoscopic colectomy, but it also has several limitations, especially the technical difficulties. we report our experience with singleincision robotic right hemicolectomy via video presentation. methods: we arranged robotic-assisted single-incision right hemicolectomy for a -year-old female patient with ascending colon tumor. the operation was performed with gloveport singleport device and a three-arm da vinci robotic surgical system through a small midline umbilical incision. colectomy was proceeded by a medial-to-lateral approach along with one or two accessory instruments for maintaining sufficient bowel traction or surgical field exposure. after vessel ligation, complete colon mobilization and right side omentum division, the robotic arms were undocked to perform anastomosis extracorporeally. results: the operation was performed successfully without drainage tube placement. the total operative time was min. the bowel movement returned on post-operative day ,and the patient tolerated normal soft diet on post-operative day . she was hospitalized for days after operation. the pathology report revealed colon adenocarcinoma (t n m , tumor size . cm), and lymph nodes were harvested. conclusions: single-incision robotic colectomy (sirc) approach seems feasible and safe in treatment of ascending colon cancer. this surgical option provides less pain and wound scar for the patient. moreover, it also achieves further benefits for the surgical procedures compared to silc. reasons being, first, it has better instruments flexibility and precision with endo-wrist, as well as less instruments clashing. second, the improved camera stability achieved through the use of the robotic arm is unattainable through manual hand-controlled methods. third, roboticassisted approach gives us an ergonomic environment, which enables the operator to control the arms while sitting by the console, and also to reassign them whenever they cross each other or block the surgical view. in spite of the advantages above, we still need to sincerely consider each patient's situation for proper management. recently, indocyanine green (icg) fluorescence has been introduced in laparoscopic colorectal surgery to provide detailed anatomical information.the aim of our study is the application of icg imaging during laparoscopic colorectal resections: to identify sentinel lymph node, for studying its prognostic value on nodal status, to facilitate vascular dissection when vascular anatomy of the tumor site is unclear and to assess anastomotic perfusion to reduce the risk of anastomotic leak. after tumor identification ml of icg solution ( . mg/kg) is subserosal peritumoral injected. a full hd image s camera, switching to nir mode, in about min displays fluorescence: the sln is identified and the sln biopsy (slnb) is performed.when tumor is in difficult site, as hepatic or splenic flexure, ml of icg solution ( , mg/kg) is intravenous injected. in about - s a real-time angiography of tumor area is obtained; on this guide, vascular dissection and pedicle ligation is performed.after anastomosis, another ml of icg solution is injected to confirm anastomotic perfusion. if there is an ischemic area, a new anastomosis is performed. from november , patients were enrolled: left colectomy, right colectomy, transverse resections, and resections of splenic flexure. in ten cases, intraoperative angiography led to the identification of vascular anatomy. in two cases the anastomotic perfusion wasn't good and the surgical strategy was changed. four postoperative complications occurred, of which one anastomotic leak, due to a mechanical problem. from november , patients were enrolled to perform the slnb: right colectomy, left colectomy, transverse resection and splenic flexure resections. the sln was identified in cases. cases were found to be n to the conventional examination and were subjected to ultrastaging. icg-enhanced fluorescence imaging is a safe, cheap and effective tool to increase visualization during surgery. it's recommended to reduce the incidence of anastomotic leak, to facilitate the assessment of vascularization in order to perform oncological resections, and to perform the slnb to study its clinical role on nodal status and for the sln ultrastaging in order to identify the micrometastases. background: surgical emptying of lateral pelvic lymph nodes (llnd) is a strategy used differently when compared the approaches to rectal cancer in the west and eastern countries. there is evidence that = mm lymph nodes in lateral compartment should be removed, even in the setting of neoadjuvant chemoradiation. minimally invasive surgery with nerve-sparing technique and sharp dissection with minimal bleeding may help overcome the significant complexity of the procedure that may have been a technical obstacle to implementation in the past. the standardization of the technique may help implementation with shorter learning curves and excellent surgical outcomes. methods: a -year-old male with distal rectal cancer underwent neoadjuvant crt for a mrt cn m mremvi ? mrcrm ? disease. there was one left obturator node of mm prior to crt. following weeks of crt completion, the patient underwent tatme for the primary disease followed by left lateral node dissection by laparoscopy. results: the present video illustrates the most relevant surgical steps to perform lateral node dissection. the procedure has been didactically divided into steps. the left ureter is identified and retracted using a vessel loop (step ). identification of the common iliac vein and dissection with subsequent identification of psoas and internal obturator muscles (step ). identification and dissection of accessory vessels. ( step ) identification of obturator nerve and obturator vessels (step ). blunt dissection of obturator nerve (step ). identification and ligation of obturatory vessels. (step ) umbilical artery is skeletonized to allow identification and clearance of fatty tissue along superior vesical arteries, internal iiliac artery/vein, inferior vesical artery and internal pudendal artery (step ). postoperative course was uneventful. conclusion: standardization of lateral-node dissection for rectal cancer has paramount importance. laparoscopic lateral-node dissection for rectal cancer provides optimal anatomical view and allows safe dissection of the nodes of interest. aims: the aim of this video is to describe our technique using fluorescence to assess the lymph flow to ensure a complete mesocolic excision and central vascular ligation in order to provide expertise to contribute to the standardization of this new tool. methods: laparoscopic right colectomy with total excision of the mesocolon was proposed in all cases. for the detection of lymph flow, we injected indocyanine green dye ( milliliter of milligrams dye dilution in milliliter of distilled water) into the subserosal to submucosal layer around the tumor at point with a -gauge injection laparoscopically after trocar insertion, and observed the lymph flow using a near-infrared system (visera elite ii, olympus) after injection. we also performed a total mesocolic excision with central vascular ligation in the region where the lymph flow was fluorescently observed. results: ( %) patients were included. no intraoperative or postoperative complications presented. no adverse effects were reported due to the infusion of indocyanine green. the lymph flow was visualized intraoperatively in a satisfactory way helping the surgeon in decision making to determine an appropriate separation line of the mesentery. the section line of the mesocolon was modified in ( %) case based on the findings obtained by fluorescence. the mean operative time was ( ) min. the morphometric laboratory data of the specimens to audit the correct complete mesocolic excision were satisfactory according to the oncological standards. conclusion: fluorescence lymphography during colorectal surgery was feasible and reproducible with a minimum of added complexity. fluorescence-guided surgery may be a helpful technique for determining an appropriate total mesocolic excision in colon neoplasms. aims: this video shows our technique for complete mesocolic excision (cme) during right colectomy for cancer. methods: in this video, a years old patient underwent a laparoscopic right colectomy with cme for a cancer of the ascending colon diagnosed with a colonoscopy performed after positivity to fecal occult blood test (fobt). after ct scan staging we obtained d printed models to clarify patient's vascular anatomy. patient was placed in supine position, trocars were inserted in left quadrants as for standard right colectomy. cme is performed by sharp dissection between the visceral fascia that covers the posterior lay of the mesocolon and the parietal fascia that covers the retroperitoneum (toldt's fascia). the ileo-colic vessels are used as landmark to identify the right anterior surface of the superior mesenteric vessels. with a caudo-cranial approach, the mesocolon is sharply dissected and the root of tributaries venous is ligated, up to the inferior margin of the pancreas. the gastro-colic trunk is dissected out with ligation of the right colic vein, while the gastroepiploic vein is preserved (harvesting the sixth group lymph node). the pancreas-duodenum fascial plane is entered and all the lymphoid tissue around the vessel surface is harvested. procedure is completed with ileo-transverse intracorporeal stapled anastomosis. results: in our experience, between april and december , laparoscopic right hemicolectomies with cme were performed. we had no major intraoperative vascular lesions. no patients needed intraoperative blood transfusion. compared to our series of standard right colectomies we did not notice any significant difference in post-operative complications. the follow-up is too short to demonstrate if the cme approach has a better oncological outcome compared to standard right colectomy. conclusions: laparoscopic cme is feasible, although it requires a higher expertise level of surgical know-how. the quality of evidence is limited and does not consistently support the superiority of cme as compared to standard right colectomy. better data are needed before cme can be recommended as the standard of care for colon cancer resections. h. bando gastroenterological surgery, ishikawa prefectural central hospital, kanazawa, japan aim: in case of right-sided transverse colon cancers, it is necessary to dissect the lymph nodes around the root o f the middle colic vessels. but in this area there are dangerous organs, for example : pancreatic head, duodenum, and gastrocolic trunk. it is the point of our technique that we resect the accessory right colic vein and middle colic vein, and then dissect pancreas head and duodenum at early step of the operation. method: we perform the operation by five trocars. the first step is to transect the great omentum, and confirm the lower edge of pancreas.there are much adhesion between mesocolon of transverse colon and stomach, great omentum. it is very important to dissect the adhesion accurately. secondly, the mesocolon is incised at lower edge of pancreas. it is possible to detect the lower edge of pancreas in obese people. the anterior surface of superior mesenteric vein is exposed. the accessory right colic and middle colic vein are resected. and then front face of surgical trunk, pancreas, and duodenum is dissected caudally as possible. the superior mesentery artery is resected below the mesocolon after flip up of transverse colon. this approach is safe and feasible, because the dangerous organs are handled by direct vision. by that, extraction of intestine is easy from small incision. afer flip up of transverse colon, the mesenteric of ileum is incised. the root of ileocecal vessels is exposed and these are resected. the peritoneum of the front of superior mesenteric artery is incised, and the lymph nodes around the surgical trunk are dissected. this dissected area is easily connected with the one done beforehand. uniquely we resect the mesocolon and major omentum from the root of dissected vessels to resected side of transverse colon. and then right-side colon is dissected medial approach. conclusion: we dissect the dangerous organs in advance. that prevent major injury of them. background: good visualisation of the operative field is a fundamental requirement for safe laparoscopic colorectal surgery. over the past years of the senior author's experience, camera systems have evolved from single to three chip, high definition (hd) and most recently, the k system. in parallel, the rest of the infrastructure such as cables, processors, monitors etc. have also undergone improvements, resulting in improved image quality. aim/methods: we present a video of a case of laparoscopic total mesorectal excision (tme), performed with strict adherence to our previously published 'stepwise approach to laparoscopic colorectal surgery' which places particular emphasis on safety aspects. tme was performed in a year old male patient with history of previous abdominal as well as robotic prostatic surgery. the procedure was filmed with all components including the camera head, cables, processing unit, screens as well as the recording/mixing decks being k. multiple external k cameras were also used. live transmission to a remote audience as part of our masterclass was achieved using appropriate bandwidth and projection on to k screens. results: feedback from the operating team as well as from the live audience was that the image quality was far superior to hd systems. the k system accorded a degree of clarity well beyond usual expectations. the depth of field also appeared to be different initially, but within a few minutes of starting the procedure and acclimatisation, the effects were appreciable. the clarity of the image which showed the fine details of the dissection planes and anatomical landmarks as well as the vibrancy of the vasculature gave a distinct three-dimensional effect to the picture. this excellent visualisation added one more layer of safety and complemented our stepwise approach for a successful procedure. conclusion: the laparoscopic k system, in our practice, proved to bea beneficial visualisation tool to enhance the accuracy of dissection. vital structures appeared to be more vivid and clearer with dissection planes being more easily apparent. in our opinion the laparoscopic k system when combined with a systematic approach enhances safety, especially in complex laparoscopic colorectal surgery. accumulating evidence suggests that laparoscopic surgery for colon cancer has feasibility and efficacy equal to or over conventional laparotomy. for cases with pasthistory of laparotomy, especially history of colon resection, however, there is almost no evidence for laparoscopic recolectomy for metachronous colon cancer. since , we have been used submucosal local injection of indocyanine green (icg) around primary colorectal cancer by using intraoperative endoscopy, and complete mescolic excision (cme) have been convincingly carried out, which was clarified by completely resected icg positive area. although evidence on the oncological efficacy of icg guided surgery has not yet been clarified, since it can be easily judged whether cme is performed clearly, it is considered that icg guided surgery for primary colon cancer is useful for education. recently, we are applying this to ensure convincing cme for patients with colorectal cancer who had a history of colic resection. the representative case is as follows. a -year-old female was diagnosed as advanced sigmoid colon cancer, and laparoscopic sigmoidectomy with high tie of the inferior mesenteric artery was performed years ago. then she was diagnosed as the metachronous descending colon cancer. the feeding artery of the new tumor should be the left colic artery, however, the left colic artery was already resected and genuine feeding artery was not identified by preoperative examination. by injecting icg into submucosa endoscopically during operation, it was clearly observed that the lymphatic flow from the tumor was directed to the inlet portion of the inferior mesenteric vein (imv). re-cme was performed by ligating the inlet of imv. intraoperative icg was also useful for clarifying the borderline for adhesion detachment of pastoperation between the mesentery and retroperitoneum (figure) . interestingly, icg flow in the mesentery direct to of the anus side was disrupted clearly at the past anastomotic site. we believe that laparoscopic surgery under icg guidance is potential useful tool that can confirm evidence to date more intuitively in real time. further studies, ideally randomized controlled trials, are required for define the oncological usefulness of icg guided surgery for re-do colectomy. the operation movie will be presented at the meeting. background: laparoscopic lateral pelvic node dissection (llpnd) is a minimally invasive alternative to open surgical therapy for advanced low rectal cancer patients. in this video, we demonstrate the technique of llpnd for rectal cancer patients with suspicion of lln metastases after neoadjuvant chemo-radiation. methods: the principle of this approach is en bloc resection with bilateral peritoneum. the peritoneum is incised lateral to the ureter following the line between external and internal iliac vessels. in the next step, llpnd dissection of the regional lymph node and high ligation of inferior mesenteric vessels were performed. a contralateral llpnd was performed in the same manner as a mirrored technique. after extracting the specimen, an end-to-end double-stapled circular anastomosis was performed. results: the procedure was done safely without any complications.the surgical duration was mins, and the blood loss was ml. the number of harvested lateral pelvic lymph nodes was . the tnm stage was ypt an m . conclusion: this approach enables extended resection during lymph node dissection, allowing autonomic nerve preservation. it is maybe a helpful approach in the treatment of locally advanced rectal cancer with a lateral lymph node metastasis. aims: the aim is to present an inspection method where the anastomosis vascularity is testing simultaneously using the indocyanine green fluorescent angiography intraluminal and intraperitoneal. methods: sixty-five year old female patient underwent standard laparoscopic-assisted low anterior rectal resection for rectal carcinoma. the proximal end of the bowel and the stump of the distal rectum were checked using near-infrared fluorescence imaging with d-light camera. after making sure of adequate perfusion of the bowel, the end-to-end stapled anastomosis was performed under the laparoscopic visualisation. the d-port proctoscope was inserted into the anus. the second icg injection was administered. the perfusion of the anastomosis in transabdominal way and viability of the mucosa in transanal way was evaluated with two d-light cameras simultaneously. the anastomosis was determined cm from the anal verge. an air-water leak and tension of the bowel tests were performed. after evaluation of anastomosis viability with fluorescence imaging, after negative air-water leak and tensions testing, the decision was made by surgeon not to perform preventive ileostomy. results: the patient had no complains for the first three days postoperatively. nevertheless, crp level was growing and was . mg/l on the second postoperative day, and . mg/l on the th postoperative day. the patient complained of the pain in the right iliac area and below symphysis on the th postoperative day. the abdominal and pelvis computed tomography scan with oral contrast was performed which denied our thoughts about the anastomotic leakage. intravenous cefuroxime and metronidazole antibiotics were prescribed. the crp level was . mg/l on the th postoperative day. the patient was discharged on the th postoperative day without preventive ileostomy. conclusion: using the original, standardized colorectal anastomosis inspection method we can determine which patient doesn't need the preventive ileostomy after low colorectal anastomosis. the important causes of anastomotic leak are local ischemia and staple line defect. the purpose of this study was to investigate the combination of methods aimed to reduce the risk of anastomotic leak after anterior resections for rectal cancer. methods: we retrospectively analyzed perioperative outcomes of the first patients, who underwent modified laparoscopic anterior resection with partial mesorectal excision for rectal cancer without preventive stomy. operative technique was modified and included routine preservation of the left colic artery (fig )(aimed to improve anastomotic blood supply), manual suture invagination of the 'dog ears' (fig ) (aimed to reduce the risk of staple line defects), transperineal pelvic drainage and pelvic peritoneum reconstruction (aimed to reduce the risk of reoperation in case of leakage). anastomotic leak rate, reoperation rate, left colic artery preservation rate, additional operative time (time required for left colic artery preservation, 'dog ears' invagination and pelvic peritoneum reconstruction), blood loss, morbidity and mortality were analyzed. results: ( . %) patient developed an asymptomatic leakage, which was managed conservatively. there was no postoperative mortality and no reoperations. median additional operative time was min for the first procedures and min for the last procedures. left colic artery preservation was successful in ( . %) patients. median blood loss was ml. conclusions: additional techniques used in our modification of laparoscopic anterior resection are safe and may lead to improved perioperative outcomes. however, they are associated with increased operative time, which may be reduced with a better learning curve. introduction: parastomal hernias are a significant cause of post abdominal ostomy morbidity with an overall life-time incidence exceeding %. the complications can range from a bulge resulting in stoma bag leakage, to life threatening bowel obstruction. the prevent-trial sought to determine if prophylactic utilisation of polypropylene mesh would decrease the incidence of parastomal hernias, with initial results demonstrating that it was safe to use in permanent end stomas. aim: to demonstrate a reproducible and streamlined technique for laparoscopic parastomal hernia repair with intraperitoneal funnel mesh, and assess the outcomes with the clavien-dindo (cd) classification tool. method: parastomal hernia repairs ( colostomy, ileostomy) were considered, with the following approach adopted for each: swab sutured in stoma orifice to prevent wound contamination.sharp dissection of the stoma using parachute technique.stoma end refreshed followed by change of gloves and instruments.lateral stay sutures placed to tighten sheath later on.pneumoperitoneum temporarily created to assess/divide adhesions.funnel mesh placed in-situ, orientated in the optimal intraabdominal position, and sutured to the peri-colic fat to prevent slip.medial suture placed to narrow the sheath further.pneumoperitoneum re-created and mesh fixed in place with double crown laparoscopic tacks.redundant portion of end stoma excised and stoma formed. results: at median follow up of months: no recurrence.no reported symptoms of pain or decreased stoma functionality.one superficial wound infection treated with drainage at bedside (cd = grade ) conclusion: laparoscopic parastomal hernia repair with intraperitoneal funnel mesh for permanent end stomas yielded good outcomes in our patient cohort. a streamlined and reproducible approach ensures that the technique can be adopted for both prophylactic, primary and recurrent repair. parastomal hernias are common and can be associated with significant morbidity. when taking this into account, in conjunction with the recommendations of the initial results of the prevent-trial, one may consider prophylactic utilisation of a mesh in patients receiving a permanent end stoma. general surgery, rambam medical center, haifa, israel year old, female patient referred to our institution with common bile duct stricture, caused by iatrogenic injury during laparoscopic cholecystectomy. during last year, patient suffered from recurrent episodes of ascending cholangitis. recently, she underwent ercp and severe stricture of middle cbd was diagnosed. plastic stent was inserted through the cbd. mrcp also showed severe stricture of cbd with dilatation of biliary tree, proximal to the stricture. due to severe and resistant (did not resolved by recurrent dilatation) structure of middle cbd, she was referred to operation. patient underwent da vinci robot-assisted excision of the cbd stricture, hepaticojejunostomy and extracorporeal jejunojejunostomy of roux-an-y limb. total operating time was min. day three after operation patient started regular diet and was discharged home on day four. final pathology has shoved part of cbd with severe inflammation. aims: extrahepatic biliary duct resection for the treatment of bismuth i and ii stage klatskin tumor is the standard surgical technique [ ] . methods: a years old patient present at emergency room (er) with right upper abdominal pain with an elevation of the inflammatory markers at the blood exams and fever. the patient was submitted to a computer tomography (ct) that shows a tumor involving the lower tract of the principal bile duct. an endoscopic retrograde cholangio pancreatography (ercp) with biopsy (intraductal papillary neoplasm of the bile duct,ipnb with high-grade dysplasia) and stent placement was performed. considering the good general conditions of the patient and an absence of vascular and nodal invasion at the preoperative imaging, a minimally invasive surgical resection of the biliary tract with cholecystectomy was performed. results: a four port laparoscopic biliary tract resection with cholecystectomy was performed with lymphadenectomy of the hepatic hilum. no vascular or liver infiltration was found. the hepatic hilum was completely skeletonized. the resection of the biliary duct was performed with adequate free margin. a biliary reconstruction with roux-en-y technique was performed and a fully laparoscopic hepatico-jejunal anastomosis was done. and abdominal retro anastomotic drain was placed. the operative time was min. the postoperative course was complicated by a low rate biliary leakage that was treated conservatively. the patient was discharged at post operative day in good general conditions. the histological examination revealed a moderately differentiated in situ cholangiocarcinoma of the principal bile duct with the involving of the cystic duct with free resection margin (pt bn r ). conclusions: laparoscopic resection of the biliary tract is a challenging procedure that allows, in expert hands, to achieve in selected cases negative pathological margin, complete linfonode retrieval and entero-biliary bypass. injury to the extrahepatic bile duct during bile duct or hepatic surgery can be reduced by better real-time visualization. recently, indocyanine green (icg) fluorescence imaging has been used in laparoscopic hepatobiliary surgery. we applied icg fluorescence imaging in patient with huge hepatic cyst which severely deviated extrahepatic bile duct. the patient had received laparoscopic cholecystectomy and huge hepatic cyst stuck firmly with peri-hepatic structures including bile duct. icg fluorescence imaging correctly identified the common hepatic duct and remnant cystic duct and allowed for more meticulous and easier dissection. therefore, icg fluorescence imaging may guide a safe and accurate dissection and excision in hepatobiliary surgery. results: total patients who underwent ercp were , and . percent ( cases) had a first failed ercp and of then were unsuccesfull in the second intent of ercp. intrahospitalary stay was more than days in the percent, in the . percent was to days, with and average of days. conclusions: before, during or after lcbde, ercp remains the gold standard for manegement of choledocolitiasis confirmed by clinics, laboratory and imagenology. lcbde is a very good option that requires experience and specific skills, and especialized equipment. in years the rate of sucess in our hospital was . % and there were no posoperatory complications such as: biliar peritonitis, pancreatitis or liver abscess. aims: easier intraoperative recognition of the biliary anatomy may be accomplished by using near-infrared (nir) fluorescence imaging after an injection of indocyanine green (icg). neither radiological support nor additional intervention such as opening the cystic or common bile duct is required, making it an easy and real-time technique to use during surgery. the aim of this video is to describe our experience in fluorescence-guided cholangiography in different clinical situations. methods: intravenous injection of icg is used to illuminate extrahepatic biliary anatomy. however, the simultaneous enhacement of liver parenchyma can disturb the visualization of clinical details. the key is in the used dose of icg, the route of administration and the time since its infusion. in the first case, a scheduled cholecystectomy is shown in which a dose ( ml of mg dye dilution in ml of distilled water) administered intravenously h before the intervention was used. the second case shows an urgent cholecystectomy in which the dose ( ml of mg dye dilution in ml of distilled water) was administered intragallbladder during surgery. all patients underwent laparoscopic cholecystectomy with traditional four-port technique. all procedures were performed using a -degree mm laparoscope with nir imaging capability (visera elite ii, olympus). results: there were no intraoperative or postoperative complications. there was no increase in operative time due to the use of icg. in the first case, a clear identification of the cystic duct and the main bile duct was obtained thanks to the biliary excretion of the icg and the intravenous clearance. in the second case, the identification of the cystic duct, the main bile duct and the cystic artery occurred due to the intravesicular absorption of icg. conclusion: fluorescence-guided cholecystectomy clarifies the dissection plane. it can be considered to increase the safety of laparoscopic cholecystectomy. being aware of the doses, times and possible routes of administration is basic to universalize the technique and give it utility in different scenarios. introduction: mirizzi syndrome type is an uncommon cause of obstructive jaundice caused by an inflammatory response to an impacted gallstone in hartmann's pouch or the cystic duct with a resultant cholecystocholedochal fistula. the obstructive biochemical changes can be caused by direct extrinsic compression from the impacted gall stone or from the fibrosis caused by advanced chronic cholecystitis, or for the established fistula. objective: we present a case of a mirizzi type syndrome with choledocholithiasis which was solved by laparoscopy approach. material and methods: a -year-old female patient with no past medical history. the history of present illness begans with the presence of icteric dye since the last days; she received symptomatic treatment with poor improvement. a liver and biliary tract ultrasound was performed with report of a mm coledochus, mm wall gallbladder. then an endoscopic retrograde clolangiopancreatography was performed with successful endoscopic sphincterotomy and removal of gallstones. but the patient jaundice persisted after the procedure. the patient underwent cholecystectomy and laparoscopic common bile duct exploration, where the findings were a mirizzi type according to the csendez classification, chronic cholecistitis and choledocholithiasis. results: in this laparoscopic approach we performed a partial cholecystectomy, bile duct exploration with removal of residual gallstones. the closure of the choledocotomy was performed with simple knots using vycril . . a subhepatic drainage was left. the patient showed adequate clinical evolution. after days the patient was discharged. conclusions: it is important to properly identify the anatomy at the time of surgery to avoid injury of the common bile duct. operative treatment of mirizzi syndrome type includes either laparoscopic or open subtotal cholecystectomy or placement of a t-tube or choledocoplasty. near-infrared fluorescent cholangiography (nirf-c) is an innovative intra-operative imaging technique that allows a real-time enhanced visualization of the extrahepatic biliary tree by fluorescence. thanks to the development of laparoscopes/endoscopes with light sources emitting infrared frequencies, it is possible to visualize anatomical structures (vessels, ureters, bile ducts, etc.) through the luminous intensity of substances (fluorescein, blue of methylene, indocyanine green) which are injected into the patient. this technology may be considered as an important teaching tool for laparoscopic surgery, especially for young surgeons in their surgical learning curve and it could lead to reduce the risk of iatrogenic bile duct injuries during laparoscopic cholecystectomy. the following video is characterized by a series of intra-operative images of biliary anatomy by fluorescence, having an important educational interest, while also detecting anatomical variations of the cystic duct. a. umezawa, minimally invasive surgery center, yotsuya medical cube, tokyo, japan aims: laparoscopic cholecystectomy(lap-c) for cholecystolithiasis has become standard. however, serious bile duct injury has been reported as a complication. repeated colic and chronic inflammation in cholecystolithiasis lead to the so-called difficult gallbladder conditions, such as dense fibrosis and scarring of the tissue. dissection of calot's triangle includes the risk of bile duct injury. critical view of safety (cvs) is the most well-known land mark for safe cholecystectomy. in the revised tokyo guidelines (tg ), important land marks and bailout procedures had been proposed. those are for the difficult gallbladder which are not able to achieve cvs. methods land marks: baseline of segment of the liver and sulcus rouvier should be confirmed. the gallbladder wall itself is also useful landmark. bailout procedure: when the dissection of calot's triangle is considered impossible, bailout procedures should be considered. subtotal cholecystectomy which leave the neck is one of option. the fundus first technique is another approach. however, because fundus first technique has a possibility of leading to serious bile duct injury, it should stop by the neck. in this video, first case shows the importance of landmarks from near miss cases of misidentified injuries. second case shows bailout procedure, subtotal cholecystectomy with fundus first technique. result: in the atrophic gallbladder (case , near miss), it is liable to misidentify the junction of common bile duct as the gallbladder neck. the neck and common hepatic duct were lifted together easily. with confirming the landmark, misidentification was corrected and bile duct injury was avoided.in the case , since the calot's triangle was obscured due to repeated cholecystitis, dissection of gallbladder was performed from the bottom to the neck, and was excised with the cervical portion remained. the remaining neck was reconstituted.in each case, intraoperative cholangiography was performed, and it was confirmed that there was no bile duct injury. without postoperative complications, those patients were discharged pod as usual lap-c. conclusion: during lap-c for difficult gallbladder, the most annoying part is bile duct injury. confirming landmarks and switching bailout procedures can be contributory to avoid bile duct injury and to achieve safe lap-c. aims: choledocholithiasis is an important cause of morbidity and is present in about % of patients submitted a cholecystectomy. his treatment should be done in the same operative time, avoiding the morbidity and hospitalization time and costs of multiple procedures.the transcystic approach is preferable to prevent morbidity associated to choledochotomy.large stones can preclude this procedure. the use of laser lithotripsy to stone fragmentation is an option to provide transcystic extraction. methods: we present a video of laparoscopic transcystic common bile duct (cbd) exploration for choledocholithiasis. results: female patient, years old with a previous hospitalization for acute cholangitis with choledocholitiasis.submitted to laparoscopic cholecystectomy with intraoperative cholangiography that showed the presence of stone in distal cbd with cm size. the use of holmium laser lithotripsy made the stone fragmentation and provided his extraction by transcystic route using a basket.the patient was discharged at th postoperative day, with no complications. conclusion: the use of laser lithotripsy for large cbd stones is safe and effective, making possible the transcystic approach and preventing the choledochotomy morbidity. surg endosc ( ) :s -s gallbladder adenocarcinoma is rare and extremely aggressive. its' incidence is higher in elder females and its progression is rapid and silent with a dismal prognosis if diagnosed at advanced stages. we present the case of a years-old female with dyspeptic complaints. the abdominal ultrasound revealed a cm solid lesion of the gallbladder suspect for malignancy. the ct confirmed the presence of a vegetant mass on the free border of the gallbladder fundus with x mm. we performed a radical cholecystectomy with lymphadenectomy and liver bed excision. the post-operative period was complicated with a urinary tract infection, with full recovery after antimicrobial treatment. the histological sample revealed an adenocarcinoma of the gallbladder (t bn m ) and the patient remains asymptomatic and tumour free months after the surgery. gallbladder cancer treatment depends of the stage and clinical presentation of the disease. complete surgical excision is the only curative treatment and should include a limited hepatectomy and portal pedicle lymphadenectomy. laparoscopic surgery might be an option in early stages, although it is challenging and requires both expertise in hepato-biliary and laparoscopic surgery. seen at the emergency room for a two month history of abdominal pain associated with jaundice. she is evaluated by the surgical team and diagnosed with acute cholecystitis and moderate risk for choledocholithiasis. the initial surgical plan was cholecystectomy with intraoperative cholangiogram. during surgery, firm adhesions are found from the gallbladder to omentum. friable tissue with edema and easy bleeding. difficulty is encountered during the dissection of calots'triangle. an intraoperative cholangiogram is done through hartmans'pouch without identifying correctly the biliary tract. therefore, an endoscopic retrograde cholangiopancreatography (ercp) is done to visualize the correct anatomy. during the ercp, a stenotic common hepatic duct is found and no stones are visualized. a biliary endoprosthesis is placed. she is discharged asymptomatic. a month later, the patient is back in the emergency room with abdominal pain. after an abdominal ct scan, we found that the endoprosthesis had migrated to the th portion of the duodenum. a second ercp is done and this time we found a big stone ( . - cm) aims: when training in the residency you watch your teacher perform laparoscopic cholecystectomy with ease, and even yourself perform several steps. but as a young surgeon, when confronted with a patient with acute cholecystitis, you're filled with emotions, and you do not know where to start the gallbladder dissection. the aim of this presentation is to show to young surgeons that you can, and must achieve, critical view of safety when performing laparoscopic cholecystectomy for acute cholecystitis. methods: we present the case of a years old female patient, bmi of . , who presented with a grade ii (moderate) acute cholecystitis. following tokyo guidelines, we initiated antibiotics and general supportive care, but without clinical improvement. the patient was proposed for laparoscopic cholecystectomy. results: at initial exploration we identified a cm long gallbladder, with a thick wall, difficult to manipulate. we opted for an anterograde cholecystectomy, in our opinion the best option in acute cholecystitis. the dissection was started with hook electrocautery and then continued with a combination of blunt dissection with the aspirator and with the hook. when reaching the pedicle, blunt dissection was used in order to appreciate the anatomy of the cystic duct and cystic artery. after correct identification of these structures they ware clipped and cut. a drainage tub was then placed, and the abdomen deflated. conclusion(s): as a young surgeon, when dealing with acute you must maintain your calm, and try to achieve critical view of safety before transecting the cystic duct and cystic artery. this can be achieved with a combination of blunt and sharp dissection, keeping your camera clean and with a good collaboration with the assisting surgeon. conclusions: here, an easy and reproducible method is described for future macroscopic analysis by the surgeon following a cholecystectomy. in addition, we depict several frequent macroscopic abnormalities in order to provide surgical colleagues with some cases of abnormal macroscopic gallbladders. the left hepatectomy is a demanding and difficult procedure, still limited to reference centers. the caudal approach and exposure of the middle hepatic vein is a reliable way to achieve a safely and reproductible left hepatectomy. with this technique, exposing the middle hepatic vein, we believe that we can perform a safe and feasible laparoscopic left hepatectomy increasing the quality of this hepatectomy. we present a -year-old woman with an intrahepatic and common bile ductlithiasiswhich was previously submitted to an ercp. with an unsolved intrahepatic lithiasis the patient was proposed to alaparoscopic left hepatectomy. the minimally invasive approach for alpps in a patient with a large hepatocellular carcinoma in a liver with severe steatosis is shown. during the first stage a partial alpps is performed. pve is performed in postoperative day one. after days from the first stage both liver volume and function (by hida scan) are re-assessed. right hepatectomy (second stage of alpps) is then conducted by laparoscopic aproach. hepato-bilio-pancreatic, centro hospitalar são joão, porto, portugal a year old woman with a previous history of anxiety and catheter ablation to treat heart arrhythmias, was studied for for multiple pancreatic cysts incidentally discovered on a routine ultrasound. an mri was performed showing multiple cystic tumors throughout the pancreas, the largest of which was mm. this led to a suspicion of multi-focal, side-branch intraductal papillary mucinous neoplasm (ipmn), with minimal dilatation of the main pancreatic duct. an echo endoscopy was subsequently performed indicating probable multifocal ipmn. a fna was carried out during this procedure, with aspiration of cystic content which was sent for cea analysis and cytology. cytology was compatible with mucinous neoplasm with mild atypia and cea u/ml. a splenic preserving total laparoscopic pancreatoduodenectomy was proposed. the procedure was uneventful and the patient was discharged on the th post-operative day. pathology revealeded a mm ipmn, with severe dysplasia and foci of microinvasive ductal adenocarcinoma of mm-pt n r . indocyanine green immunofluorescence guided laparoscopic partial hepatectomy y. tai obtaining negative tumor margin during laparoscopic hepatectomy has always been a very challenging topic for surgeons in that the surgeons are not able to palpate the tumor during laparoscopic surgery. although intraabdominal echo is available, but it demands great experiences and skills. with the guidance of icg immunofluorescence, surgeons can avoid failure of not obtaining enough negative margins nor resect too much healthy liver. icg is often used to estimate the liver function prior to hepatectomy traditionally. it binds to plasma protein and has a peak absorbance at nm and emits fluorescence with a wavelength of approximately nm. icg is preferentially retained in or around biliary malignancies due to impaired biliary excretion of hepatocytes in the affected area. we performed icg immunofluorescence guided laparoscopic partial hepatectomy on a years old male who suffers from hcc located at segment and . icg was injected days prior to the operation day. while evaluation of liver is performed, it also allowed us to use a high-end laparoscopic camera system equipped with integrated filters for detection of near-infrared fluorescence. during the surgery, we were able to clearly locate the borders of malignancies through the use of integrated filters combine with icg injection. the pathology study also confirmed that the adequate tumor free margin ([ . cm) were obtained in both tumors and the patient's condition was stable as well. icg immunofluorescence guidance enables surgeons to obtain optimum result in tumor resection through laparoscopic surgery. it also has the ability to detect bile leakage. with the use of icg immuofluorescence, surgeons will have higher chances to achieve adequate negative margins. background: parenchymal sparing hepatic resection has the advantage of preserving valuable tissue in chemotherapy-treated livers, assuring an adequate future remnant volume without compromising long-term survival. moreover, the laparoscopic approach offers the decreased postoperative morbidity of minimally invasive surgery. whenever technically feasible, this kind of procedure should be considered a suitable alternative to the classic major hepatectomy for the treatment of multiple colorectal liver metastases. methods: -year old male with a previous history of laparoscopic sigmoidectomy in november for a pt n m sigmoid adenocarcinoma. a control scanner three years later showed liver metastases in segments v, viii, ii and caudate lobe. after chemotherapy (xelox), control mri and pet scans showed a good response. he was proposed for a laparoscopic parenchymal-sparing liver resection. results: total operative time was h and min with no intraoperative complications. patient presented a right atelectasis as the only postoperative complication and was resolved with respiratory therapy. he was discharged in days. pathology report showed that lesions on segment v and viii had no viable tumor ( % fibrosis) and lesions on segment ii and caudate lobe had moderately differentiated adenocarcinoma. margins were free in all the lesions. after a month follow up, the patient has no recurrence and normal liver function tests. conclusion: minimally invasive liver resection is possible in patients with multiple bilobar liver metastases and allows to perform parenchymalsparing surgery safely. difficult localization of lesions such as the caudate lobe are not a contraindication for this type of surgery. laparoscopic approach for perihilar cholangiocarcinoma is still poorly reported in the literature due to technical challenges secondary to the combination of major hepatectomy, lymphadenectomy and biliary confluence resection. despite this, in selected cases it can be a good option to provide a short term benefit to patients. the video reports the case of a perihilar cholangiocarcinoma with involvement of left bile duct and therefore requiring left hepatectomy. komagome hospital, bunkyo-ku,tokyo, japan aims: segmentectomy is an anatomic liver resection, in which the tertiary branches of the glissonean pedicles are selectively transected. however, the branching pattern of the tertiary branches varies depending on the case, particularly in segment (s ) and segment (s ). the extrahepatic approach to the glissonean pedicle from the hepatic hilum is very difficult depending on the branching pattern. furthermore, the distance of exposing the secondary branches that are to be preserved becomes longer, and there is an increased risk of biliary leakage and delayed biliary stricture due to excessive traction in laparoscopic surgery. therefore, laparoscopic s and s segmentectomy are considered technically difficult. we standardized the intrahepatic glissonean pedicle approach for laparoscopic s and s segmentectomy. methods: we standardized the intrahepatic glissonean pedicle approach for laparoscopic s and s segmentectomy. we identify the targeted glissonean pedicle intrahepatically after the parenchymal transection along the major hepatic vein or its branch running on the intersegmental plane, referring to the preoperative simulation by d imaging. (a)s segmentectomy; after the mobilization of the right lobe, the glissonean pedicles of s (g ) can be approached from the dorsal side by transecting the parenchyma between the ivc and the right hepatic vein. after the division of the g , the parenchyma is transected along the demarcation line and the rhv from the root side to the peripheral side. (b)s segmentectomy; first, the parenchyma is transected along the middle hepatic vein (mhv) from the root side to the peripheral. g is typically detected on the right dorsal side of the mhv. after the division of the g , the liver parenchyma is transected along the demarcation line and the rhv from the root side to the peripheral side. results: we have experienced cases of laparoscopic s segmentectomy and cases of laparoscopic s segmentectomy. conclusion: our approach to the g and the g is safe and very useful. laparoscopic anatomical segmentectomy of right anterior section is technically demanding because it is difficult to dissect the deep tertiary branches of right anterior portal pedicle (rapp). we present three cases of laparoscopic anatomical segmentectomy using the extrafascial and transfissural approach: ) anatomical resection of segment , ) anatomical resection of the ventral area ) anatomical resection of segment dorsal area. the extrafascial and transfissural approach means that the liver parenchyma along the fissure lines is opened, then the surgeon can confirm the glissonean pedicles and territory directly. the extrafascial and transfissural approach in laparoscopic anatomical segmentectomy of right anterior section is feasible and effective because this technique can easily be approached to the deep tertiary branches of rapp. repeated liver resection has significant role in patients with recurrent hepatocellular carcinoma (hcc) in several situations. laparoscopic redo surgery is becoming safer along with advance in surgical technique. we have performed laparoscopic re-resection for limited intrahepatic hcc recurrence. the aim of the present study was to investigate its significance comparing with first laparoscopic liver resections. subjects: patients with limited intrahepatic hcc recurrence after open hepatectomy underwent laparoscopic liver re-resection (n = ). methods: adhesion between abdominal wall and visceral organs was carefully divided, after the first laparoscopic port was safely inserted. adhesion between diaphragm and liver surface or between previous liver cut surface and colon or duodenum was also minimally dissected. approach to the glisson's pedicles at the hepatic hilum was often difficult due to previous surgical procedure, thus pringle's maneuver was generally applied. dissection of hepatic parenchyma approaching to the target glisson's branch was often preceded under the ultrasound-guidance. liver resection was performed using lcs, biclamp, and cusa using intermittent block of the hepatic inflow. operation time, intraoperative bleeding, morbidity, mortality, and postoperative hospital stay were compared with those in patients who underwent first laparoscopic liver resection during the same period (n = ). results: operation time was significantly longer in the re-resection group, possibly due to the adhesiolysis. meanwhile, no significant difference was detected in intraoperative bleeding, morbidity, mortality and postoperative hospital stay between the first and the redo surgeries. methods: the donor was a -year-old gentleman who decided to donate part of his liver to his wife suffering from viral liver cirrhosis and hepatocellular carcinoma. his bmi was . kg/m and the preoperatively estimated donor's right liver volume was ml, representing . % of his entire liver. with the recipient's weight of kg, the graft to recipient weight ratio (grwr) was . %. the liver had classic hilar anatomy except that the right posterior intrahepatic duct seperately joined to the left main hepatic duct. after isolation and clamping of right hepatic artery and portal vein, indocyanine green of . mg was injected intravenously. results: the total operation time was min and the estimated blood loss was ml without transfusion. indocyanine green fluorescence image clearly demonstrated the anatomical demarcation between the lobes and visualized the running of the biliary tree. his postoperative course was uneventful and discharged postoperative day . conclusion: real-time indocyanine green fluorescence image may be particularly helpful to delineate anatomical surgical plane and to determine the appropriate division point of hepatic duct during laparoscopic living donor hepatectomy. surg endosc ( ) the correct management of intraoperative volemic status is essential in laparoscopic liver resection in order to control bleeding and to perform even complex procedures with a good profile of safety. central venous pressure is not really reliable in laparoscopy, due to presence of the pneumoperitoneum and patient position. monitoring of haemodynamic parameters via vigileo system is a minimally invasive method to control stroke volume variation, cardiac output, cardiac index and oxygen delivery in order to optimize the anaesthesiological management by controlling venous bleeding and avoiding tissutal ischemia. introduction: non-hydatid liver cysts represent a heterogeneous group of disorders that differ in their etiology, prevalence and clinical manifestations.within them, the simple hepatic cyst is the most frequent.the majority of simple cysts are an incidental finding during the performance of an imaging test for another unrelated cause and few of them are symptomatic or are associated with complications, and surgery is not necessary in most of them. described various therapeutic approaches so far there is no consensus about the optimal treatment of simple symptomatic, complicated or growth-showing liver cysts during its follow-up. currently the laparoscopic approach is widely used for the management of cysts hepatic, with results similar to open surgery but with the advantages of laparoscopy. objectives: to demonstrate the safety and efficacy of the laparoscopic approach in the approximation of complicated simple hepatic cysts.material and method: clinical case: a -year-old female patient with a history of: giant hiatus hernia intervention with laparoscopic nissen, fibromyalgia, previous ischemic colitis. hospital admission due to pneumonia and right pleural effusion with us: simple cyst x x mm in segment v hepatic, with dilatation of biliary radicals adjacent to the cyst, distended gallbladder with irregular walls in the hepatic side. ct: cystic lesion in segment iv-v of the liver, which has increased in size, with small microabcesses adjacencies to the lesion, thickening of the gallbladder wall, to assess cholecystitis. antibiotic treatment is established with good evolution, deciding surgery. results: intervention: complete laparoscopic approach, trocars, edematous cholecystitis, large retroyuxta vesicular cyst,with thickened walls with serous content. cholecystectomy maintaining the cyst wall, puncturing and taking samples for cytology and biochemistry of the contents, resection of the cyst wall, partial flare of its internal surface, negative intraoperative biopsy, epipoplasty, with drainage placement.correct postoperative course.pathological anatomy: simple biliary cyst with negative cytology, ck ?, ck -, calretina-. conclusion: the treatment of choice of complicated simple hepatic cysts is laparoscopic.we recommend performing an intraoperative biopsy of all resected liver cysts to confirm its nature,we propose cyst enucleation as the best surgical treatment. objective: the objective of the following case is to present a patient with symptomatic polycystic liver disease, which was solved by laparoscopy approach and the management of its complications. material and methods: the case reported is about a years old female patient with abdominal pain in upper right quadrant associated to asthenia, adynamia and hyporexia. ct scan reported heterogeneous liver with multiple ovoid images with regular edges defined which the biggest one measure x x mm with volume of cc on segment and , which comprises stomach, and the other one in segment with a volume of cc and others small sized located in segment , and b. results: in this laparoscopic approach, we performed a cyst unroofing of the two biggest cysts as well as cholecystectomy because of firm and lax adhesions. the patient evolved with fever in the th day postsurgical day and biliary leaking in a volume of cc in hrs. an ercp (endoscopic retrograde cholangiopancreatography) was asked for that was carried out by finding leak at the intrahepatic biliary duct therefore; esphinterotomy with placement of plastic endoprotesis was performed. the patient evolved without complication and was discharged at the th day. conclusions: only symptomatic polycystic liver disease needs to be treated. the choice of treatment is not yet standardized, for voluminous cysts the unroofing ideally by laparoscopy is the gold standard and the ercp is the elected treatment when the biliary leak appears as a complication. introduction: laparoscopic liver resection (llr) for tumors located in the posterosuperior segments of the liver (segments (s) or ) is a challenging procedure. especially, llr for s is difficult because the access of instruments is limited, bleeding control is not feasible, major llr is sometimes required, and obtaining sufficient resection margin is not easy. to overcome this obstacles, we performed llr in s with a lateral approach using intercostal trocars. to obtain competent resection margin, llr through right hepatic vein (rhv) first approach was performed for . cm mass located near the rhv in a year old female. case: after full mobilization of right liver including all short hepatic veins and caudate lobe, rotate the whole liver completely to the left side to approach to the root of rhv. one intercostal trocar was inserted to access the lesion. parenchymal transection started from the confluence of hepatic vein and then, followed along rhv with ligating several small branches from rhv. resection margin was demarcated after localization using laparoscopic ultrasonography. after completion of parenchymal dissection using cusa and ultrasonic shears, hemostatic agents were applied and drain was inserted. operation time and estimated blood loss were mins and ml. the patient was discharged without any complication on postoperative day . final pathological assessment confirmed clear resection margin (safety margin : . cm). conclusion: laparoscopic s segmentectomy with hepatic vein first approach technique is safe and recommended to obtain better resection margin. aims: simple liver cysts are the most common cystic lesions of the liver. most are diagnosed casually in image tests such as ultrasound or computerized tomography, most of which are asymptomatic and do not require treatment. in symptomatic patients (abdominal distension with palpable mass, abdominal pain, dyspnea, jaundice, etc.) the clinical manifestations are usually due to the growth of the cysts or the compression of neighboring structures. liver function tests are usually not altered. intracystic complications occur in less than % of cases and malignancy is exceptional. in this video, we present the case of a symptomatic patient with polycystic liver disease including a large size hepatic cyst. material and methods: -year-old woman with a personal history of arterial hypertension, saos, partial hysterectomy due to endometrial cancer, who was referred to our department complaining of supraumbilical pain and abdominal distension with palpable mases. abdominal ultrasound showed cholelithiasis and multiple simple hepatic cysts. in ct scan, multiple hepatic cysts were found, the largest one of about cm of larger diameter. echinococcus granulosus serology test was negative. there was also no evidence of cancer disease in pet scan. results: a laparoscopic approach was performed with four trocars, three of mm and a hasson trocar inserted thought a umbilical small incisional hernia. aspiration and wide unroofing of the large size cyst and smaller accessible ones was done. the patient also underwent cholecystectomy with intraoperative cholangiography and umbilical eventroplasty. the patient recovered uneventfully and is asymptomatic one year after surgery. conclusion: simple liver cysts rarely require treatment. in some cases, especially in large, complicated and symptomatic simple liver cysts, surgery is indicated. laparoscopic fenestration treatment is the best choice. aims: liver resection is the preferable initial treatment option for solitary or limited multifocal hepatocellular carcinomas. surgical indications for laparoscopic liver resection (llr) are the most important consideration, like liver function, tumor size (diameter less than cm) and location (easy technical access like in the left lateral section or on the surface of the inferior region). partial liver resection or left lateral sectionectomy are the typical procedures for such tumors and are considered the best way to begin llr. with accumulating experience and technical advancement, llr has been performed for tumors larger than cm and for others locations. some requirements to perform llr are to have experience in liver surgery and laparoscopic also, adequate technology and intraoperative ultrasound. methods: a -year-old male smoker, ex-parenteral drug users with chronic hcv liver disease child-a stage. he is diagnosed with a single lesion of cm in segment iii of the liver, biopsied twice without conclusive diagnosis and with a three-phase ct suggestive of hepatocarcinoma li-rads with data of portal hypertension (pht) and mild ascites. after the study is commented on tumor committee deciding surgical intervention. results: a laparoscopic resection of segment iii was performed with trocars. liver is explored by intraoperative laparoscopic ultrasound. vascular control was performed using the pringle technique. liver transection was done with sonostar until identification of intraparenchymal segment iii vascularization, which is sectioned with endogia ( mm) with seamguard. after the resection, we perform hemostasis control with electrocoagulation and hemostatic material. intraoperative bleeding of ml. favorable postoperative evolution, high on the th postoperative day. ap: cm trabecular hepatocarcinoma moderately differentiated pt b, r resection. conclusions: llr allows major liver resections with low morbidity and mortality and the advantages of laparoscopic surgery. an efficient learning curve can be achieved by a parallel evolution of procedures and indications (according to modified bclc staging system and treatment strategy). studies suggest that llr results in less blood loss, shorter postoperative hospital stays, lower abdominal wall trauma and lower incidences of ascites accumulation and postoperative liver failure. with respect to oncological considerations, tumor margins are adequately maintained during llr. v. drakopoulos, s. voulgaris, i. iliadis, k. botsakis, p. trakosari, v. vougas st department of surgery and transplantation unit, district general hospital of athens « evangelismos » , athens, greece introduction: laparoscopic surgery is gaining acceptance in the treatment of liver metastasis. laparoscopic treatment of liver metastasis often presents technical difficulties and requires an extensive learning curve. material-method: we present the case of a year old woman presented with a liver metastasis in section of the liver. the patient had been submitted to a laparoscopic low posterior resection in february . patient underwent laparoscopic left lateral hepatectomy, with the use of three trocars (umbilical mm, and two in the midclavicular line bilaterally.) left lateral hepatectomy was conducted with the use of a linear stapler. the postoperative period was uncomplicated and the patient remains in good condition three months after surgery. conclusion: laparoscopic approach seems to be safe for treatment of liver metastasis, offering better surgical field view and less postoperative complications. year survival rate after laparoscopic hepatectomy is compared to the open approach. general surgery, chang gung memorial hospital kaohsiung division, kaohsiung, taiwan purpose: laparoscopic hepatectomy is a quickly growing method for liver tumor because of modern technology. but for the ihd thrombosis, it is still technique dependent. the video was tried to share our experience for special case. material and method: one y/o female patient suffered from fever episode and image show s cm hcc with right anterior ihd obstruction r/o tumor thrombosis, hilum ln enlargement, double right portal vein, hilum adhesion with duodenum, no ascites . lab data : no-b, no-c child a, afp , icg clearance rate . %, plt . heart, lung function exam normal. the laparoscopic right total hepatectomy and hilum ln dissection was conducted. results: laparoscopic approached was performed. the hilum ln dissection was done with vessel and bile duct isolation. hilum ln frozen show negative malignancy. hemi-vessel control was done with resecting the vessel. right hepatectomy was done with preserving middle hepatic vein. the right anterior and posterior ihd was opened and tumor thrombosis was removed from right anterior ihd carefully. the stump of ihd was closed by suture separately. the total op time was min with cc blood loss. post op minimsl bile leakage was found in the drain at day . the patient discharged at day with drain. conclusions: laparoscopic hepatectomy may be a feasible method for hcc even with ihd tumor thrombosis. surg endosc ( ) introduction: the progressive laparoscopic learning in gastric surgery and the great development of instruments and laparoscopic material that facilitates the realization of advanced procedures, has led to an increase in the use of laparoscopy in the treatment of gastric cancer. material and methods: we present the case of a -year-old man without amc with a history of ischemic heart disease who enters our surgery department for cholangitis secondary to choledocholithiasis. ercp is requested during his admission that describes a gastric lesion from which a biopsy is taken, making it impossible to access vater papilla to perform sphincterotomy and lithiasis extraction due to the existence of duodenal diverticula. the result of pathological anatomy of the gastric lesion was compatible with adenocarcinoma. negative extension study. the clinical case is presented in a committee of multidisciplinary tumors and it is decided to perform surgical intervention of both pathologies. a subtotal gastrectomy was performed with a roux-en-y reconstruction. surgical time of min. choledochotomy was performed with lithiasis extraction, as well as intraoperative exploration of the bile duct and main conduits by means of a choledochoscope. results: income of days, with a clavien ii. the definitive pathological anatomy was an ai stage with a total of isolated nodes without evidence of neoplasia in any of them, therefore it does not require adjuvant treatment. the patient is asymptomatic, with nutritional supplementation with follow-up in ccee of surgery. conclusions: in our case, there were no serious postoperative complications when performing gastric resection and bile duct exploration with drainage of the same. from the oncological point of view, the number of lymph nodes extracted and the surgical margins are similar to those obtained in patients in whom we perform open surgery; therefore, although it is a single clinical case, laparoscopy in expert surgeons is a safe and effective technique. the puestow procedure was initially proposed to alleviate the pain in patients with chronic pancreatitis and dilated wirsung duct. its objective is to provide an efficient drainage of the pancreatic fluids and, in the meantime, to preserve the pancreatic tissue and minimize the risk of endocrine and exocrine pancreatic insufficiency. aims: to describe the particular technical aspects and the efficacy of totally laparoscopic puestow procedure in patients with cystic duodenal dystrophy. methods: a years old patient presenting diffuse epigastric pain, vomiting and weight loss was diagnosed at endoscopic ultrasound and biopsy with cystic duodenal dystrophy. a conservative treatment was decided with octreotide and opioids. however, due to the persistence of symptoms surgery was performed. results: due to the association of a dilated wirsung duct, the patient was submitted to a puestow procedure. the surgical procedure was completed in a minimally invasive manner; after dissecting the anterior surface of the pancreas an intraoperative ultrasound was performed in order to identify the wirsung duct. therefore, the pancreatic parenchyma was transected along the wirsung duct, a totally laparoscopic pancreato-jejunostomy on roux en y limb being performed. the early postoperative outcome was uneventful, the patient being discharged in the sixth postoperative day. at one month and six months follow up the need for opioid treatment significantly diminished. a kinking of the enteral anastomosis required a laparoscopic intervention one year after with a very good evolution after. conclusions: totally laparoscopic puestow procedure seems to be a safe and efficient method in order to treat symptomatic patients with cystic duodenal dystrophy in whom a dilated wirsung duct is present. aims: the approach to the intraductal papillary mucinous neoplasm (ipmn) is various, from a radiological follow-up with magnetic resonance (rm) to the surgical treatment with a pancreatic resection [ ] . the surgical approach is various and depends on the localization of the lesion and on the surgical skills [ ] . methods: a years old patient was admitted at the chi possy-saint germain-en-laye with an acute pancreatitis. at the ecoendoscopy was found a pancreatic cystic at the junction of the pancreatic body and tail with a wirusng diameter of mm. a second episode of acute pancreatitis occurred a few months later. after that episode the patient was submitted to a computer tomography (ct) that found a cystic lesion of cm with an increasing dilatation of the wirsung duct. the serum ca - was ui/ml. a laparoscopic sils distal pancreatectomy with spleen conservation was performed. results: a trans-umbilical incision was performed with the positioning of the gelpoint sils platform with the placement of trocars. a distal pancreatectomy with a spleen preservation and without a standard linfadenectomy was performed. the pancreatic stump was closed with an endo-gia mm with seamguard device. any drain was placed. the post-operative course was uneventfull. a ct scan was performed in …. post-operative day which didn't show collections. the patient was discharged in -…… post'operative day. the histological examination shows an ipmn with low grade dysplasia. no invasive carcinomatoses cells were found. the distal pancreatic sils resection with spleen conservation is a feasible and safe technique that combine all the advantages of the minimally invasive laparoscopic approach with the esthetic advantages of the sils approach. pancreato-duodenectomy is a complex surgery, requiring several anastomoses to reconstruct the digestive tract. due to its technical complexity, the laparoscopic approach is not yet the goldstandard and there remains some controversy about its oncological safety. worldwide experience is limited, and its safety and effectiveness are yet under evaluation.we present the clinical case of a years-old woman with a prior history of epilepsy. she was studied due to painless obstructive jaundice and a cm pancreatic head tumour was diagnosed on imaging, causing cbd and wirsung channels' dilatation. the tumour was considered locally resectable and she was proposed for a radical pancreato-duodenectomy.we present the main steps of the surgery including the oncological resection with lymphatic basin clearance and totally laparoscopic reconstruction.the post-operative was uneventful, and the histologic sample revealed a ductal adenocarcinoma (t ) with an r resection and / lymph nodes invaded. although technically demanding, laparoscopic pancreato-duodenectomy is safe and effective requiring teams with experience both in pancreato-biliary and laparoscopic surgery. chronic pancreatitis is characterized by a progressive pancreatic fibrosis with loss of endocrine and exocrine function. one of its main symptoms is debilitating pain. surgical drainage of a dilated pancreatic duct is an option to consider in cases of refractory pain. longitudinal pancreato-jejunostomy allows an effective decompression of the pancreatic channel and a significant improvement in the quality of life. we present the clinical case of a years-old lady with a prior history of gallstones. she was treated for an acute pancreatitis in may , followed by recurrent relapses of pain and enzymatic elevation. she required opioid use for partial pain control and a significant kg decrease on body weight due to 'fear of eating'. the endo-ultrasonography and the mri revealed a chronic pancreatitis with an mm wirsung duct with ductal stones and an atrophic body and tail. we proposed a laparoscopic longitudinal pancreato-jejunostomy. the surgery was performed with trocars, with the surgeon on the right side of the patient. we performed a trans-mesocolic cm pancreato-jejunostomy. the post-operative was uneventful, and the patient was discharged on the th post-operative day, asymptomatic. laparoscopic longitudinal pancreato-jejunostomy, although effective is a technically demanding surgery but brings the benefits of a minimally invasive approach. background: preservation of spleen in distal pancreatectomy is also useful from the maintenance of platelets and the prevention of overwhelming post splenectomy infection. we have performed laparoscopic spleen preservation distal pancreatectomy: lspdp to benign and low-grade tumors of the pancreatic body tail. the aim of this study was to report our surgical experience with the method of svp: splenic vessel preservation and wt: warshaw technique of lspdp, describe our techniques with videos. method: there are three points of our surgical technique. , precede pancreatic dissection, improve the mobility of the pancreas. , confirming the courses of splenic artery and classified them into two major types. , preserving the left gastro-epiploic vessels and short gastric vessels.the postoperative cases of lspdp which performed from april to september was retrospectively studied. result: of consecutive patients were performed lspdp at our institute, were svp and were wt. ages, gender and bmi were similar for two groups. there were no significant differences in operative time, blood loss and length of stay after surgery. comparing pathological finings, wt was associated with a slightly large tumor lesion (median mm vs. . , p = . ). among the median observation period of months, splenic infarction was observed in case in svp and cases in wt. however, they were focal splenic infarctions, they did not need surgery or drainage. there were no cases in which late onset of splenic artery occlusion or esophageal / gastric varices. conculusion: after performing lspd, the function of the spleen was good in all cases. both svp and wt were safe and feasible procedures. this is the case of a -years-old lady presenting with recurrent abdominal intractable pain she has been suffering from for the last years. msct revealed pancreatic calcifications from mm to - mm and dilatation of the main pancreatic duct in the body of the pancreas up to mm. the patient underwent laparoscopic local resection of the head of the pancreas combined with longitudinal roux-en-y pancreaticojejunostomy-a technique known as frey's procedure. it is recognized as an effective therapeutic option for the surgical treatment of patients with persistent pain caused by chronic pancreatitis.after performing the posterior wall of the pancreaticojejunal anastomosis we've faced an intraoperative complication such as volvulus of the roux limb causing serious ischemia of the limb. we were forced to remove all previous sutures in order to untwist the roux limb, thereafter the pancreaticojejunostomy was started anew.the purpose of this video is to demonstrate that frey's procedure can be performed in a minimally invasive fashion, which provides all the well-known advantages of this approach. we demonstrate that even such serious intraoperative complication as volvulus of the roux limb can be managed without conversion. our center has an experience of over laparoscopic frey's procedures, however this is the first case where we encountered with such complication and we believe this is an experience worth sharing.yet we would like to underline that this approach should be used by highly skilled minimally invasive surgeons experienced in intracorporeal suturing which is the most challenging stage in frey's procedure. v. tomulescu, i. hutopila, c. copaescu spleen preserving distal pancreatectomy (spdp) is commonly applied in patients with benign or low-grade malignant tumors in the body and tail of the pancreas. two surgical techniques for spdp have been described. the first technique was described by kimura (spleen preserving distal pancreatectomy with splenic vessel preservation-spdp-svp) and preserves the main splenic artery and vein and excises the tail of the pancreas and those small, short vascular connections to the body;the second technique was described by warshaw and involves resection of the splenic vein and artery before distal pancreatectomy, and conservation of theshort spleno-colic and gastric vessels to keep normal blood flow for the spleen (spleen-preserving distal pancreatectomy with splenic vessel resection-spdp-svr). we present the case of a years old female with / mm tumor of the pancreatic tail on ultrasonography. ct scan confirmed the tumor and endoscopic ultrasonography with fna have shown a solid pseudopapillary tumor. due to the low grade malignancy we have decided to perform a laparoscopic spleen preserving distal pancreatectomy with splenic vessels preservation (lspdp-svp). for lspdp-svp the difficulty is related with the splenic vessels dissection and manipulation. primary dissection and control of main trunk of splenic artery and vein will help to quickly control bleeding during vascular rupture in small vessels dissection. optimal stapling of any tissue requires an adequate tissue compression time to allow elongation of the tissue being compressed, smooth firing of the instrument, consistent staple line formation balanced against the risk of increased tissue tearing and excessive tensile strength. this is why, for pancreatic division, we prefer choosing a cartridge loaded with higher staplers. the pancreatic stump transection line is evaluated for bleeding and when it is needed, hemostatic clips are applied. histology report confirmed a solid pseudopapillary tumor t nomxl v r at this moment with month good follow up. in conclusion lspdp-svp is safe, reproductible and demonstrated very good outcomes when certain indications are respected. surg endosc ( ) aim: advances in minimally invasive surgery has permitted to perform complex techniques by this approach, being the laparoscopic duodenopancreatectomy (lpd) one of these. the aim of this communication is to present a surgical technique video for a complete laparoscopic pd, showing the most important steps of the resective and reconstructive phase, with the anastomosis realized completely by laparoscopy. methods: a surgical technique video is presented showing the main steps for the lpd and a complete laparoscopic reconstruction with an hepaticojejunostomy, duct-to-mucosa pancreatic-jejunostomy and a gastrojejunostomy. results: an years old woman with past medial history of arteria hypertension, dyslipidemia, type ii diabetes mellitus and a breast cancer treated in with lumpectomy and axillary lymphadenectomy plus radiotherapy, recently diagnosed of and adenocarcinoma of the head of the pancreas. the ct scan showed a neoplasia localized in the head of the pancreas without extension to other organs. a laparoscopic pd was indicated after a multidisciplinary committee evaluation. a supraumibical hasson trocar was used for the pneumoperitoneum, three mm trocars and two mm trocars were used. lpd was performed. the resective phase was done following the conventional steps of the open whipple procedure and for the reconstructive phase, a child limb was used for a termino-lateral hepatico-jejunostomy with an absorbable / monofilament; a duct-to-mucosa pancreatic-jejunostomy with an absorbable / monofilament and finally a latero-lateral mechanical gastro-jejunostomy was performed. surgical time was min. postoperative course without complications and the patient was discharged on the th postoperative day. definitive anatomopathological exam: intraductal tubulopapilar neoplasia, x x mm, with wide high grade epithelial dysplasia. free margins. ptisn ( / ). conclusion: laparoscopic pd is a feasible procedure with a high technical requirement which should be performed in specialized centres with high experience in hepatobiliary surgery and in advanced laparoscopic procedures, because of its high morbidity and mortality. conclusions: robotic assistance in whipple may overcome limitations of laparoscopy and offer a minimaly invasive approach to this procedure potentially resulting in lower blood loss and less morbidity. we need further prospective randomized trials in order to determine the exact role of robotics in pancreatic surgery. aims: distal pancreatectomy is the standard curative treatment for symptomatic benign, premalignant, and malignant disease of the pancreatic body and tail. the most obvious benefits of a laparoscopic approach to distal pancreatectomy include earlier recovery and shorter hospital stay. spleen-preserving distal pancreatectomy should be attempted in case of benign disease. laparoscopic spleen-preserving distal pancreatectomy (lspdp) is expected to be less invasive than laparoscopic distal pancreatectomy with splenectomy. however, there are few reports regarding the details of the procedure for lspdp, and its safety remains unclear. this study aimed to evaluate the feasibility and safety of lspdp. methods: retrospective analysis of surgery treatment of patients was made. lspdp was conducted in the period from to in the department of laparoscopy surgery of state institution o.shalimov national institute of surgery and transplantology. the average age was : . years, the body mass index (bmi) was . ± . results: laparoscopic distal pancreatectomys was performed in % of cases, were attempted in female and male patients. postoperative pathological examinations revealed cases of serous cystadenoma in the body and tail of the pancreas, case of serous oligocystic adenoma, case of mucinous cystadenoma, case of neuroendocrine tumor (insulinoma), and case of solidpseudopapillary neoplasm. complications related to the surgery were like acute pancreatitis with -fold increase normal plasma amylase confirmed by ct- cases, fluid collection- cases, pancreatic fistula (grade a)- cases. the operation time was . min, (range - min) blood loss of . g (range - g), mean hospital stay was . days (range - days). conversion to laparotomy was in case. mortality was . conclusion: laparoscopic spleen-preserving distal pancreatectomy is minimally invasive, safe, and feasible for the management of benign pancreatic tail tumors, with the advantages of earlier recovery and less morbidity from complications. aims: a pancreatic pseudocyst is an encapsulated, mature fluid collection occurring withing the pancreas that have a well-defined wall minimal or no necrosis secondary to pancreatic injury and mediated by the enzimatic and inflammatory disruption of pancreatic tissue. it is a common complication of acute and chronic pancreatitis. we present the case of a pancreatic pseudocyst located within the body of the pancreas due to recurrent necrotic pancreatitis. the objective of this video is to show the minimally invasive surgical approach of this entity. methods: a -year-old man without medical history was admitted to hospital in the digestive service on times for acute necrotizing pancreatitis. after study in which is evidenced cholelithiasis and pseudocyst in pancreatic body of cm maximum diameter and formation of two peripancreatic collections without signs of superinfection, cholecystectomy is indicated. magnetic control cholangiography was performed after surgery and it showed an increase in the size of the pancreatic pseudocyst, suspecting wirsung's duct disruption. therefore, endoscopic retrograde cholangiopancreatography (ercp) was performed by placing a plastic pancreatic prosthesis and performing a sphincterotomy. after hospital discharge, the patient is re-admitted due to recurrent abdominal pain without analytical alteration. tc abdominal observed an increase in the pseudocyst from to cm. this case was discussed in a multidisciplinary committee and surgical intervention was decided. results: laparoscopic approach is decided and four trocars were placed. initially, a gastrostomy was performed with liquid outlet. an aspiration of the liquid and quistogastrostomy with mm endogia was made. the patient progresses favorably, being high on the tenth postoperative day, without complications. conclusions: almost every pancreatic pseudocyst improves spontaneously and needs no specific treatment. draining is indicated when secondary symptoms to compression, complications or rapidly enlarging are found. depending on the complexity of the pseudocyst, its communication with wirsung's duct and the existence of ductal injury, it may perform a percutaneous, endoscopic or surgical drainage. the goal of pancreatic debridement is to excise all dead and devitalized pancreatic and peripancreatic tissue while preserving viable functioning pancreas, controlling resultant pancreatic fistulas, and limiting extraneous organ damage. only the surgical procedure is definitive. case: a y old male presents with intermittent low retrosternal pain and progressive dyspnea with exercise since a couple of months. cardiac investigation was negative and gastroscopy showed a grade b esophagitis. he was treated medically but with only partial response. on a thoraco-abdominal cat-scan the diagnosis of a left sided bochdaleks' hernia was made. the hernia includes the left kidney (with blood vessels and ureter), transverse colon and small intestine which are positioned in the left lower thoracic cavity with the left lung considerably compressed. method: given the clear correlation between the patients' complaints and these anatomical findings, he was referred to our service of abdominal surgery. we performed a laparoscopy with the patient in lithotomy position and the surgeon between the legs. the patient was tilted to his right side. mobilization of the spleen was necessary to gain maximal access to the hernia. we were able to reduce all the herniated content, freed the margins of the defect, reduced the hernia sac and repositioned the kidney intra-abdominally. the defect was manually closed with non-resolvable stitches and covered with a mesh which was secured with tackers. result: postoperatively the patient recovered well with adequate pain relief and pulmonary support. he could leave the hospital after days. control cat-scan on day postoperatively shows an intact lining of the diaphragm with normal positioning of the intra-abdominal organs. on follow-up weeks after surgery the patient had regained normal activities and was symptom free. conclusion: a symptomatic left sided bochdaleks' hernia in adults with an ectopic intrathoracic kidney is extremely rare. we hereby state that, during a laparoscopic repair, the kidney can also be safely reduced, which has almost never been described in literature yet, enhancing pulmonary recovery, improving access for mesh placement and thus diminishing recurrence rate. aims: large incisional hernias repair involves an actual problem for surgeons to face. anterior component separation has been an important method allowing to close the fascia defects without tension while also having underlay mesh reinforcement.therefore, we present a case of incisional hernia reparation performing endoscopic anterior component separation with advantages compared with open approach. method: we present the case of a -year-old woman, bmi kg/m , with previous laparoscopic gastric sleeve and posterior reintervention using open approach. the patient presented a cm size incisional hernia m w . a ct scan was performed, confirming a midline incisional hernia containing colon, with an herniary defect of cm. full minimal invasive abdominal wall repair was proposed. a cm size incision was made in left iliac region to reach the aponeurosis of external oblique muscle. we placed a balloon trocar and subcutaneous pneumo-dissection with mmhg pressure was performed; then, we placed a mm trocar in left lumbar space. the aponeurosis of external oblique muscle was incised and anterior component separation from inguinal to subcostal area was achieved. an extensive intermuscular dissection was performed to achieve complete midline closure. we performed the same procedure on the right side. then, with laparoscopic approach using v-loc n° suture, we completely closed the midline. eventually, we placed a x cm ptfe-c mesh fixed with a double crown of tackers and fibrin glue. results: postoperatory course was uneventful and the patient was discharged h after surgery without any remarkable event during his postoperative stay. the patient has been followed up for months without any complication or recurrence in ct scan, confirming the correct minimally invasive reconstruction of the abdominal wall. conclusions: trends in abdominal wall reconstruction and complex-hernia repairs have advanced rapidly in recent years. the goal is to perform a complete abdominal wall repair with no tension in midline incisional hernias. endoscopic anterior component separation and laparoscopic eventroplasty with closure of the defect, leads to a complete wall reconstruction without tension and avoids drawbacks due to primary close defect in those patients with herniary defects wider than cm. aims: endoscopic technique is a valid and safe approach for the treatment of abdominal wall defects. to combine the advantages of complete endoscopic extraperitoneal surgery with those of sublay mesh repair we propose totally endoscopic sublay anterior repair (tesar), a safe and feasible approach for the treatment of ventral and incisional midline hernias. methods: from may to september patients were referred to our unit for clinical and radiological diagnosis of midline ventral or incisional hernia and selected for tesar. exclusion criteria were: complicated ventral or incisional hernia (i.e. incarcerated hernia), maximum defect width [ cm, contraindications to general anesthesia. the procedure consisted of suprapubic access with trocars, complete endoscopic pre-aponeurotic dissection, isolation and reduction of the hernial sac, bilateral incision of the medial rims of recti aponeurosis and dissection of retromuscular plane to create the retromuscular space, sublay non-absorbable mesh positioning and anterior aponeurosis reconstruction. one drain was always placed in the retromuscular space and one drain in the subcutaneous space. results: all procedures were completed with endoscopic approach, with no conversion to laparoscopy or open surgery. no intraoperative complications were registered. total mean operative time was ± . min. no post-operative major complications were registered. only one subcutaneous seroma was registered ( . %), and treated conservatively. the mean postoperative stay was . ± . days. at post-discharge clinical checkups drains were checked and removed when indicated. no wound complications nor recurrence were registered to date. cosmetic and functional results were successful in all patients. conclusions: tesar is a safe and feasible technique for the extra-peritoneal sublay repair of ventral hernias with totally endoscopic approach. it provides accurate hernia repair with good outcomes in terms of resolution of symptoms and post-operative complications. r. mizuno, m. kondo backgrounds: abdominal incisional hernia is found in more than % after abdominal surgery, and risk factors such as wound infection, obesity, elderly, high abdominal pressure are pointed out. laparoscopic hernia repair using intraperitoneal onlay mesh (standard ipom) is becoming widespread in japan since the insurance release in , and our hospital is actively working on it. recently, ipom plus procedure which also carries out fascia suture in addition to laparoscopic mesh placement has been introduced. aims: we report the clinical results of laparoscopic abdominal incisional hernia repair in our hospital. methods: we performed hernia repairs using a mesh for cases from january to september . of these, cases were standard ipom and cases were ipom plus. there was no significant difference in the patient background such as gender, age, bmi, etc, and in the intraoperative findings such as hernia orifice diameters and adhesions. surgical time, postoperative hospital stay, and the rate of complications such as seroma, mesh bulging, postoperative pain, hernia recurrence were compared and examined between the two groups. results: as a result, in ipom plus group, the operation time was longer and the incidence rate of postoperative pain was higher, but the incidence of mesh bulging was significantly lower. also, in some cases since , the ' u reverse stitch method ' is used as an ingenuity of fascia suture in ipom plus. conclusions: laparoscopic abdominal incisional hernia repair has the advantage of being able to reliably confirm the hernia orifice from the intraperitoneal side?it is excellent in the identification of the fragile part of the abdominal wall and in the visibility of the restoration range. with regard to the ipom plus procedure which has been introduced in the last few years, although the operation time is extended, it has usefulness such as reduction of mesh bulging. from the viewpoint of cosmetic surgery, usage of ipom plus will increase in the future. introduction: incisional hernia is one of the most common complications after abdominal surgery. several methods have been introduced, and yet, there is no consensus on the best method of repair. we present a novel method for hernia repair which uses the retromuscular sublay mesh repair through a single incision at the pubic area to improve cosmesis. methods: medical records of patients who underwent single-port retrorectal incisional hernia repair from may to december were reviewed. patients were placed in supine position and a cm incision was made in the pubic area below the panty line. a flap is made upwards until the defect is found and bilateral rectus sheathes are dissected. a mesh is then placed between the posterior rectus sheath and the muscle. results: a total of patients with midline incisional hernia underwent single-port retro-rectal incisional hernia repair. mean age was . ± . years with an average bmi of . ± . . all the patients had midline hernia defect with an average of . ± . cm. mean operation time was . ± . min and estimate blood loss was . ± . ml. there was no postoperative complication, and ( %) patients were discharged on the day of surgery. conclusion: the single-port retrorectal incisional hernia repair is safe and effective while providing good cosmesis to selected patients with incisional hernia. aims: closing hernia defect during laparoscopic hernia repair is a vast extended technique nowadays. however, this technique is associated with mesh placemnt intraabdominally in contact by the abdominal content. nowadays there is a trend to recontruct the midline and to avoid a mesh intraabdominally in those cases suitable for it, as a new step forward of minimally invasive abdominal wall reconstruction. laparoscopic sublay approach with retromuscular placement of a mesh without mechanical fixation after reconstruction the linea alba migth be considered an option in primary hernias of the midline. methods: we present a case of a year old male with an umbilcal hernia of centimeter in diameter associated with rectus diastasis. a laparoscopic approach was performed, using one and two millimeter trocars placed on the left flank. the first step was to open the lateral side of the posterior fascia of the left rectus muscles, dissecting the retromuscular plane until we reach the linea alba getting into the preperitoneal space where the sac was diseected preserving the integrity of the peritoneum. the contralateral posterior fascia was also dissected all the way to the semilunaris line. the midline was closed, including th hernia defect, using a running double loop suture (maxon-loopÒ). a self gripping mesh (progripÒ) is placed in the retromuscular space in a sublay position ( cm long, cm wide). last, we close the fascia of the left rectus muscle using a barbed suture (v-locÒ). results: surgical time was min, being discharged of the hospital on postoperative day . pain was controlled with conventional analgesia and no postoperative complications, nor seroma was detected. conclusions: sublay approach for ventral hernia can provide a midline reconstruction, reestablishing abdominal function and avoiding the use of intraabdominal meshes and traumatic fixation, decreasing postoperative complications and pain. aims: lumbar hernia is one of the rare cases that most surgeons are not exposed to. hence the diagnosis can be easily missed. this is often related to previous surgery as lumbotomies or primary in the superior lumbar triangle. this leads to delay in the treatment causing increased morbidity. we report a case of adquired lumbar hernia in a middle-aged woman repaired by laparoscopic approach. methods: a years old woman with surgical history of a myelomeningocele surgery by posterior approach over years ago, a laparoscopic left nephrectomy years ago with a left colostomy due to a left colon injury during this procedure. a hartmann reversal by laparoscopic approach months later. patient showed a large lumbar mass over cms in the left lumbar region and a large scar near to spinal cord. it was soft in consistency, reducible and expansible on coughing and straining with defined borders. computerized tomography showed a large defect in the superior lumbar fascia over cms in the grynfeltt-lesshaft triangle with the left colon inside. results: patient was placed in a full lateral decubitus position. in order to optimize exposure, a lumbar roll was placed under the lumbar region. a capnoperitoneum ( - mmhg) was built up. one mm and two mm trocars were used and positioned in the left mid axillary line. a optic was used. adhesions were removed and toldt fascia was opened in order to expose the hernia defect bounded by quadrates lumborum, erector spinae muscles, rib and serratus. hernia content was carefully extracted from the sac using a ligasure maryland (covidien medtronic-usa). hernia defect was measured and an intraperitoneal mesh (dinamesh-ipom feg textiltechnik mbh, aachen, germany) was positioned and sutured by tackers to the margins included the bone. patient was discharged in h with a low pain rate and without complications. there is not recurrence in months follow-up. conclusion: laparoscopy might be a safe and feasible approach for repairing lumbar hernias, either primary or adquired, with a low rate of pain and complications s surg endosc ( ) after pneumoperitoneum is done, three mm trocars are placed on the left flank. the defect is delimited by drawing it over the skin of the patient with aid of an intramuscular needle and intraabdominal vision. posterior fascia is opened longitudinally at its medial edge and the retromuscular space is dissected. the arcuate line of douglas and the epigastric vessels are identified. from this point, transversus abdominis fascia is sectioned cranially cm medial to the semilunar line, preserving the neuro-vascular pedicles that reach the rectus abdominis laterally. at supraumbilical level, transversus abdominis fibers advance behind rectus abdominis, so they need to be sectioned to access to the space below the ribs. lateral dissection of this space enables a tensionfree closure at midline. once the procedure is repeated on the contralateral side using two mm and one mm trocars on the right flank, a continuous suture of the posterior fascia is performed with a barbed suture. the anterior fascia is closed with a slowly-absorbable monofilament loop-type suture. finally, a double-layer polypropylene mesh is placed at retromuscular level without any suture and fibrin glue is applied. results: the patient was discharged hous after surgery. no recurrence has been presented to the moment. conclusions: the section of the aponeurotic plane from the arcuate line of douglas enables a more accurate dissection of the retrotransversus plane without sectioning its fibers except for its cranial end, preserving the innervation and vascularization of the abdominal wall. this technical modification aims to simplify a complex laparoscopic procedure allowing its estandarization. aims: the authors present a video with their standardized laparoscopic ventral hernia intraperitoneal mesh (ipom) hernioplasty procedure but introducing a novel laparoscopic technique for tension releasing while hernia gap closure and midline anatomical restoration. methods: a years old male patient with a bmi presents a symptomatic ventral hernia recurrence after a sigma colic cancer open surgery. a ct scan study showed a cm transverse diameter midline ventral hernia. a laparoscopic ipom hernia repair procedure is performed using mm instruments and a mm camera. when checking tension while midline restoration suturing, we decide to add a tension-releasing maneuver: a totally laparoscopic transverse abdomini muscle release (taltar). this maneuver allow right rectus posterior sheath to advance some distance to the midline, in order to provide a tension-free midline closure. a double-faced ready-to visceral contact mesh is now placed and fixed. case and technical details are shown in the video. results: the patient was discharged from hospital within a period of h with a rate in a eva acute pain visual scale. in a year follow-up, there has no been an anatomical or clinical recurrence. no chronic pain, anatomical recurrence, lateral asymmetry, umbilical or abdominal wall complications have been reported with this technique. conclusions: depending on the patient characteristics, anatomical hernia factors and surgeon mini invasive experience, a taltar maneuver could be a safe and feasible option for releasing tension when midline anatomical laparoscopic closure. more studies are needed in order to standardized this approach. aims: when primary ventral hernia and simultaneous diastasis recti are diagnosed, there is no consensus among the international surgical community on the surgical treatment regarding indications or surgical technique. however, if diastasis recti is symptomatic of or is associated with midline hernias, the corrective surgery of both pathologies at the same time could be the most recommended option. when we only correct the herniary defect, we risk performing a reparation on an anatomically weak tissue, so the rate of hernia recurrence may increase. we propose a minimally invasive access using totally endoscopic retromuscular hernioplasty. by developing this technique, several advantages are provided, such as no peritoneal opening without intraabdominal access, no mesh fixation needed and simultaneous solving of both pathologies. method: we present the case of a -year-old man, with bmi kg/m and no previous medical history complaining of ventral hernia with associated recti diastasis. a cm size umbilical hernia was diagnosed with a cm size supraumbilical diastasis recti associated. full endoscopy retromuscular hernioplasty was proposed. a cm size incision was made in left hypocondrium, openned the anterior rectus sheath and retracted the rectus muscle. we placed a balloon trocar and open the homolateral retromuscular space after placing two mm trocars in left lumbar space and epigastric position. we crossed-over the linea alba and achieve contralateral retromuscular space. after this step, the hernia sac was reduced and we extended the dissection cm caudal to the hernia ring. both medial posterior rectus sheaths were sutured with running barbed suture n° and a x cm size light-weight, big pore, polipropilene mesh was placed in retromuscular space and unrolled properly with enough overlap. a drain was placed and the anterior rectus sheath incision was closed. results: the patient was discharged h after surgery without remarkable events during his postoperative stay. he has been followed up for months remaining asymptomatic. conclusions: totally endoscopic retromuscular ventral hernia repair in men with umbilical hernia and diastasis recti associated, is feasible and reproducible procedure with several advantages compared to traditional laparoscopic ipom in terms of pain and mesh position. aims: parastomal hernia (ph) is one of the most frequent long-term complications of stoma formation, occurring in %- % of patients. surgical treatment for parastomal hernia is the only cure but a fairly difficult field with a recurrence rate ranging from % to % of cases. due to its advantages, the number of laparoscopic mesh repairs for parastomal hernia has gradually increased over the past decade. according to this common complication, we report a case of laparoscopic reparation of ph using the sugarbaker technique. method: we present the case of a -year-old patient with surgical antecedent of laparoscopic low anterior resection due to rectal cancer, presenting in postoperative period an anastomosis leakage with severe peritonitis was identified and a laparotomy with end colostomy was performed. the postoperative course was uneventful. during the follow-up the patient showed a centimetres size paraestomal hernia, being a m w incisional hernia confirmed with ct scan.the patient underwent full laparoscopic hernia repair, performing a sugarbaker technique, exposing parastomal hernia completely to measure the hernia ring size ( centimetres) and the midline associated defect ( centimetres). a x cm size ptfe-c was selected to allow a -cm overlap over two defects. results: using this approach, the bowel loop was pushed into the abdominal wall and appropriate place between the mesh edge and the abdominal wall is left to allow the bowel loop to pass through. postoperatory course was uneventful and the patient was discharged h after surgery without any remarkable event during his postoperative stay. he has been followed up for months without realizing any clinical signs or alterations in ct scan. compared with traditional open surgical repairs, laparoscopic repair has certain advantages including its safe operation, postoperative rapid recovery, fewer complications, and lower recurrence rate. however, it still faces challenges regarding parastomal hernia treatment, and there is a need to improve existing surgical techniques. aims: nowadays, the principal disadvantages of laparoscopic approach in hernia repair are the use of intraabdominal meshes and traumatic fixation. first, intraabdominal meshes involve the contact of the prosthesis with the intestinal loops with the consequent risk of adhesion and fistula. also, using helicoidal sutures in prosthetic fixation produces adhesions to the tackers and a non-negligible incidence of chronic pain. when it comes to lead to better results, placing the mesh in retromuscular space avoids the drawback of contact with the loops, and using self-fixation meshes may decrease the rate of acute and chronic pain. accordind to this facts, we present a case of laparoscopic ventral hernia repair with transabdominal retromuscular mesh placement without traumatic fixation. methods: we present a -year-old patient with a cm diameter hernia showed in preoperative ct scan, m w , with diastasis recti associated. the patient underwent laparoscopic surgery using transabdominal retromuscular route. one mm and two mm trocar were placed in left flank. the posterior rectus sheath on the left side is opened starting cms far from the left egde of the defect. once the retromuscular space is dissected, the hernia ring is dissected and the hernia sac reduced, we continue with the dissection in retromuscular space on the side. craniocaudal dissection is achieved cm distal to the defect margins. the hernia defect with the anterior rectus sheath and the diastasis recti were closed using v-loc running suture. self-adhesive mesh was subsequently placed. the mesh should be overlap cm from the margins of the defect, covering the defect widely, with grips facing upwards. finally, we closed the posterior rectus sheath with peritoneum on the left side with v-loc running suture. results: the postoperative course was uneventful and the patient was discharged h after the surgery. after months of follow-up no clinical or radiological recurrence was showed. conclusions: the combination of laparoscopic approach, retromuscular mesh placement and the use of self-fixation meshes, seems to be an actual useful solution, combining the advantages of each item and avoiding the use of intraabdominal meshes and helicoidal sutures. aims: laparoscopic ventral hernia repair has clear advantages over open repair, including less post-operative pain and earlier return to normal activity. however, a prolonged surgeon learning curve is necessary to perform this technique effectively. robot assistance may improve outcomes of minimally invasive ventral hernia repair with improved three-dimensional visualization and enhanced dexterity with articulating instrumentation. we report a case of robotic rives-stoppa epigastric hernia repair in order to demonstrate the feasibility of the robotic approach. methods & results: a -year-old man came to our attention for the presence of a palpable mass in the epigastric region. the abdominal ct scan showed the presence of an epigastric hernia with herniation of omental content, and the presence of diastasis recti. the patient was then submitted to a rives-stoppa robotic hernia repair under general anesthesia. the da vinci-si surgical system (intuitive surgical inc., sunnyvale, ca, usa) was brought into position over the head of the patient and docked after placement of the ports. three trocars were placed in the hypogastric region along the transtubercular line. a fourth trocar was placed in the left iliac fossa and used by the assistant. the operation started with an extended adhesiolysis and hernia reduction. then, the retromuscolar dissection began by incising the posterior sheath starting from cm above the pubic symphysis. an extended dissection of the rives space was performed to create a correct housing for the mesh. the hernia defect and the diastasis recti were closed using a - absorbable barbed suture. a phasix st tm mesh (bard inc./davol inc., warwick, ri) was positioned in the retromuscular plane, and was anchored with absorbable sutures and glue. the midline incision was closed using a - absorbable barbed suture. the operative time was minute. the postoperative period was uneventful, and the patient was discharged home on the second post-operative day. conclusions: robotic rives-stoppa ventral hernia repair is feasible, safe, and effective when a standardized approach is performed. whether robotics may improve the outcomes of minimally invasive ventral hernia repairs, including lower recurrence rates, decreased post-operative pain, or shorter surgeons' learning curve, will require careful prospective investigation. aims: the authors present a video with a left chronic bochdaleck hernia classical hernioplasty repair but performing a mini invasive thoracoscopic approach and mm instruments. methods: a years old female patient come to hospital due to chronic left dorsolumbar pain. a ct scan study showed a chronic left diaphragmatic bochdaleck hernia. a lateral right decubitus thoracoscopic repair is performed using mm instruments and a mm camera. case and technical details are shown in the video. results: the patient was discharged from hospital within a period of h with no pain and a clean chest x-ray. in a year time follow-up, not an anatomical or clinical recurrence has been reported. neither chronic pain or respiratory complications happened, with in this period of time. conclusions: depending on the patient characteristics, anatomical factors and surgeon mini invasive experience, left bochdaleck hernia mini invasive thoracoscopic hernioplasty repair using mm instruments could be a safe and feasible option. more studies are needed in order to standardized this approach. surg endosc ( ) abdominal wall surgery has expanded exponentially in the last decade. many techniques have been developed, mainly in minimally invasive surgery. laparoscopic ventral and incisional hernia repair (lvihr) has become a common procedure because of its feasibility and safety but unfortunately, it is not free of complications. chronic postoperative pain and bleeding are frequent complications, prolonging hospital stay and altering quality of life of the patients. absorbable or non-absorbable tacks are the usual method of mesh fixation and sometimes combined with transfascial sutures to secure the mesh. these mechanical fixations pierce the abdominal wall causing nerve or vessel injuries. some studies showed no differences between absorbable tacks, non-absorbable tacks or transfascial sutures concerning postoperative remarkably high pain. some authors consider that a non-penetrating fixation of the mesh getting an effective mesh-abdominal wall interface will reduce significantly the postoperative pain after a laparoscopic ventral hernia repair. tissue glues are used in different medical treatments and also have been used successfully for extra peritoneal mesh fixation in laparoscopic inguinal hernia repair, open ventral hernia repair but not so in laparoscopic ventral hernia repair in spite of good results published in the literature. cyanoacrylate and its derivatives are 'synthetic glues' and classified as medical devices with stronger adherent properties than fibrin glues. experimental studies have reported good results compared with suture fixationand also tissue toxicity doesn't lead to an increased foreign body reaction. some authors have studied the use of cyanoacrylate in laparoscopic inguinal hernia repair but unfortunately, clinical trial reports in ventral and incisional hernia repair were not found in the literature because the lacking of experimental studies that guarantee the safety of intra-abdominal mesh fixation and the interaction of the glue with the intra-abdominal tissue. our group developed an experimental study demonstrating the feasibility, safety and effectiveness of the cyanoacrylate using for intraperitoneal mesh fixation and after this conclusion, started a clinical study. this video shows the methodology for laparoscopic mesh fixation with only glue in our first cases. aims: small epigastric hernias, associated or not with the rectus abdominis diastasis, and small umbilical hernias are common in middle-aged women, particularly with past history of pregnancy. the aim of this video is to illustrate a new extraperitoneal approach to these clinical situations. methods: patients between the ages of and years old, with epigastric hernia orifice up to cm, with or without associated umbilical hernia (up to cm), were chosen for this procedure. the surgery begins with a vertical umbilical incision for the umbilical hernia's correction, and dissection of the pre-aponeurotic plane. two mm trocars (mini-laparoscopy instruments) are introduced at both flanks to enlarge the pre-aponeurotic plane towards the xiphoid appendix. in this way epigastric hernial defects are isolated. the surgery proceeds with defect suturing with braded suture, midline invagination and mesh placement if necessary. results: all patients had an eventful post-operative period and were discharged home at postoperative day . the aesthetic and functional results are optimal conclusion: for selected cases with high aesthetic motivation this technique seems to be feasible and with optimal cosmetic results. this technique allows the mesh placement both in-lay and onlay, protecting it from surgical site infections often present at the classical approac bochdalek hernia is a rare entity in adults. fewer than have been reported in medical literature, the majority of which were incidentally diagnosed. as such, the optimal repair of a symptomatic hernia is unknown. we present a case of adult bochdalek hernia repair. methods: a -year-old obese male patient with a years of chronic dry cough and left lung opacity in chest x-ray. a large posterior and lateral bochdalek hernia with herniation of intestinal loops and fat to the left hemithorax was seen in chest and upper abdominal ct scan. the hernia extended to mid-thorax, caused significant atelectasis of left lung. eighteen months later, due to appearance of chest and abdominal pain following a recent motor vehicle accident, a repeat chest ct was done and a slight enlargement of the hernia was shown. results: the patient was operated laparoscopically, positioned in a semi-right lateral decubitus with double lung intubation. a large left posterior and lateral diaphragmatic hernia which contained transverse and descending colon with omental fat was seen. they were pulled in to the intraperitoneal space carefully. the defect was measured to be * cm. it was reduced to * cm by suturing with a non-absorbable v-loc suture . advancing the camera to the thoracic cavity showed the left lung to be severely atelectatic. after selective recruitment lung was well expanded. a symbotex composite cm mesh was fixed to the defect area by suturs and laparoscopic tacker. the operation and post-operative course were uneventful. chest x-ray demonstrated the bowel below the diaphragm. the patient was discharged on pod . at -month follow-up, chest x-ray was normal. objective: to demonstrate the safety and efficacy of the standardized laparoscopic approach in the treatment of large parastomal hernia. currently, this approach is recognized as the one of choice in parastomal hernia pathology, being controversial which is the best technique of choice: keyhole vs sugarbaker. material and method: clinical case: a -year-old woman with a history of laparoscopic abdominoperineal amputation due to rectal neoplasia (pt n ), a year ago, with symptomatic parastomal hernia with incarceration episodes and inflamation changes in the stomal orifice.tac: large hernia parastomal with intestinal content inside. surgical treatment is decided. result intervention: complete laparoscopic approach, right lateral partial decubitus, trocars, dissection of the hernia defect and reduction of the content, partial mobilization of the pre-stomal colon, with bleeding at the level of the vascular origin, requiring careful hemostasis to avoid ischemia of the colostomy, herniorrhaphy with stitches with extracorporeal knotting, placement of polypropylene/pvdf mesh,fixed with irreabsorbable tackers with administration of biological glue at the edges of the mesh. correct postoperative, discharge at the rd day. asymptomatic and without hernia recurrence at one year of follow-up. conclusions: the technique of sugarbaker using a laparoscopic approach is a safe and effective alternative in the treatment of parstomal hernias. objetives: laparoscopic ventral hernia repair provides advantages in term of low infection rates and postoperatory stay when is compared with open repair. trends in laparoscopic abdominal wall surgery is to complete defect closure without tension in midline. closing the defect in ventral hernias wider than - cms creates high tension in midline and postoperatory pain. it's proposed different techniques to solve this drawback. laparoscopic posterior component separation makes the defects closure easier with no tension and placing the mesh extraperitoneally. methods: years old woman with previous total hysterectomy, a m m w midline incisional hernia was clinically diagnosed and confirmed with ct scan. full laparoscopic abdominal wall repair with defect closure was proposed. trocars in left side were placed and posterior rectus sheath right side in the defect margin is freed. once the lateral edge of the rectus sheath is reached, the posterior rectus sheath is incised, dividing the posterior aponeurotic sheath of the internal oblique muscle. this allows access to the plane between the internal oblique and the transversus abdominis muscles. it's is made the same steps in the left side with trocar on the right flank. the posterior rectus sheath both side is reapproximated in the midline and cms polipropilene mesh is placed and unfolded properly. it's fixed using cyanocrilate glue. one drain is left in retromuscular position and mm trocar wounds are sutured. results: postoperatory course was uneventful. hospital stay h. the drain was removed in day after surgery. after months follow-up no complication or recurrence were identified. methods: this video will show the evidence of gangrenous jejunal segment due to superior mesenteric vein thrombosis in a patient with history of breast ca on hormonal treatment.in this video, the gangrenous segment was resected and primary anastomosis was done using endogia mm. results: a second look after h revealed to be negative for any further ischemic bowel. conclusion: therefore, laparoscopy in acute abdomen is diagnostic and for treatment. introduction: gastric pseudo-volvulation is a rare entity of paraesophageal hernia that is characterized by migration of the stomach into the posterior mediastinum. this clinical-radiological picture has severe complications so in certain cases should be operated urgently. another small group of patients are asymptomatic, although the current literature recommends their regulated surgical intervention. we present a gastric pseudo-volvulation in the mediastinum, with a laparoscopic approach, showing that by systematizing the surgery, it is possible to perform this type of intervention with relative ease and safety material and methods: we present a video of an urgent laparoscopic approach in a female patient of years with a personal history of hypertension, smoking and dyslipidemia. with a hiatus hernia diagnosed more than ten years ago. he went to the emergency department due to significant symptoms of heartburn and reflux, as well as incoerctable vomiting and difficulty feeding one week of evolution. a simple abdomen and postero-anterior chest radiograph was performed, showing a paraesophageal hiatus hernia with almost the entire stomach included in the mediastinum. a thoraco-abdominal axial tomography corroborated giant hiatus hernia with pseudovolvulation and incarceration data. urgent intervention was decided by laparoscopic approach in which hiatus hernia reduction and esophageal abdominalization were performed. closure of pillars and reinforcement with bioabsorbable mesh. gastric and gastropexy toupet of anterior face to anterior peritoneum of abdominal wall. results: the patient had a post-operative h without incident, discharged with a crushed diet. the follow-up and evolution has been acceptable without notable complications. conclusion: the laparoscopic approach, in extreme cases of paraesophageal hiatus hernia with incarceration of the stomach and pseudovolvulation of it, is a correct, safe and effective alternative in experienced groups. surg endosc ( ) case report of incarcerated hiatal hernia. years old female was admited to the hospital due to severe chest pain and vomiting for about six h. physical examination and lab test showed no abberations. chest xray revaled incarcerated stomach above the diaphragm. she was rushed to the or. laporoscopic approach was used, the stomach was removed from the chest and nissen fundoplication was performed. day after surgery patient was asymptomatic, got full oral diet. she was discharged on postoperative day two, without a need of any analgetics. gastroduodenoscopy was performed weeks after surgery and showed proper image of oesophagus, stomach and duodenum, neither signs of hiatal hernia nor inflamation were present. laparoscopic approach is good way to treat incarcerated hiatal hernias and is related with shorter lenght of stay, lesser postoperative pain and better patient comfort. and it should be procedure of choice in this kind of cases. she was operated open technique using a cm long incision in right iliac fossa and the appendix was phlegmonous. the patient began feeling bad from the second day postoperative having temperature over °c, pain and increasing crp. the general condition worsened the next day when the temperature went up till . °c, extreme generalized pain and crp: . the ct abdomen control indicates signs for generalized peritonitis and rises the suspicion for a forgotten large gauze. the patient is operated using laparoscopy technique: identifying and taking out the foreign body, doing adhesiolises, extensive lavage and in the end inserting one drain in douglas. the video is presenting what king of special graspers can be used but also tips and tricks when speaking about identifying the anatomy but also dissection in acute and inflamed environment. postoperatively the patient began to feel better and in the th day was released home. conclusion: this case illustrates that even after open surgery, laparoscopy is a viable solution with the condition that there is available experience in minimally invasive surgery. introduction: foreign bodies can enter inside the human body by different mechanisms such as ingestion, aspiration, trauma or in some cases due to medical procedures. they are potentially life-threatening events, the diagnosis could be challenging and its management depends on their location. case report: a -year-old male was referred to our hospital due to chronic abdominal pain. he had cholelithiasis, medical history of acute pericarditis and past surgical history of left adrenalectomy, left nephrectomy, distal pancreatectomy and colon resection due to an adrenal adenocarcinoma (stage t n m ).abdominal radiograph showed a foreign body in the left lower quadrant of the abdomen, as an incidental finding. this was not detected in ct scans during ten years of oncology follow-up. ct scan revealed an extraintestinal metallic curved object in the right lower quadrant. this finding was not related to any surgical intervention or trauma. diagnostic laparoscopy was performed: the foreign body seemed to be a guidewire, it was included into the omentum and almost stuck to the abdominal wall. the guidewire was reached and carefully extracted through a mm trocar without any evidence of intra-abdominal organ injury. then an elective cholecystectomy was also performed due to his medical history of symptomatic cholelithiasis.the procedure lasted min. the hospital discharge was on the third postoperative day and no complication was registered. conclusion: is extremely rare to discover a guidewire that had migrated into the peritoneal space without abdominal injuries.this case report demonstrates the technical feasibility, safety and minimal postoperative morbidity associated with minimal invasive laparoscopic removal. aims: the authors present a video with their standardized laparoscopic groin hernia transabdominal preperitoneal hernioplasty (tap) procedure but using mm instruments and mm camera approach. methods: a years old male patient with a bmi presents a symptomatic bilateral groin hernia for months. us study showed an indirect bilateral inguinal hernia. a laparoscopic tap hernia repair procedure is performed using mm instruments and a mm camera. a selfgripping mesh preperitoneal hernioplasty and peritoneal flap barbed-sutured hermetic closure was performed. case and technical details are shown in the video. results: the patient was discharged from hospital within a period of h with a rate in a eva acute pain visual scale. in a year follow-up, there has no been an anatomical or clinical recurrence. no chronic pain, anatomical recurrence, umbilical or abdominal wall complications have been reported with in this period of time. conclusions: depending on the patient characteristics, anatomical factors and surgeon mini invasive experience, a laparoscopic bilateral hernia repair using mm instruments, could be a safe and feasible option. more studies are needed in order to standardized this approach. results: during tapp approach a direct hernia relapse was identified, the previous mesh was included on preperitoneal space and some non-absorbable sutures to inguinal ligament were identified. stitches and nearly total mesh removal (only the part surrounding cord elements was left in place) were performed. x heavyweight polypropylene mesh was employed fixed with gubran Ò and the flap was closed with running sutures. patient was discharged uneventfully the same day. seven months later he did not need analgesics and had no physical impairment. conclusions: post inguinal hernia repair chronic pain can be severe and disabling, and is becoming more prevalent. the origin is complex and meshes and sutures could play a role. the management is multimodal and demanding. for refractory patients, surgery may be an option. laparoscopic, open and mixed approaches have been employed. they usually combine mesh removal and substitution (often in different planes) and groin nerve therapies. nowadays, triple neurectomy seems to be the most effective treatment (more than % pain relief). generally, removal of mesh alone does not lead to lasting pain relief or has worse outcomes compared with associated neurectomy. introduction: mesh repair of inguinal hernia is sometimes followed by adverse effects such as mesh migration, chronic groin pain or recurrence. removal of the mesh is necessary in selected cases. we affront this cases by tapp intervention. methods: we present a video with two intreventions of inguinal recurrent hernia by laparoscopy (tapp). we remar the points to decide explant the mesh or not to explant. the conditions to decide the explant were the proximity to the main vessels in inguinal area (espigastric and femoral vessels) and the plication of the mesh. results: and conclusion as we show in the video, the explant of the mesh is only conditioned by the plicature of the mesh for its migration and recurrence, accompanied usually with pain. we don't remove any time the mesh or the plug if it is in the triangle of doom with firm adhesions to the main vessels. we cover the previous mesh with a new ligthweigth d mesh and closing at the end the preitoneum over the new reparation. introduction: tep technique isn't a controversial area in surgical practice for inguinal hernias anymore, but a fully accepted method. the use of general anesthesia has been the mainstay of laparoscopic hernia repair, but epidural anesthesia is not a contradiction to properly selected patients. material-method: the approach of the extraperitoneal area achieved without use of a dilation balloon, but via the indroduction of the camera and the dissection of the regional structures. trocars ports were used: a mm trocar through the umbilicus for the camera, exactly as in sils (single incision laparoscopic surgery), another one mm is placed in the midline between the umbilicus and pubis, the last mm trocar is placed in the midclavicular line ipsilateral with the hernia. the key for every operation was the tension free technique with placement and fixation of a mesh x cm. in / cases the mesh was placed with tacks on the inside of the inferior epigastric artery-vein complex. all patients were dismissed from the hospital in h, no drain was placed and no major postoperative complications took place. conclusion: tep is a demanding technique with serious learning curve. the use of a dilation balloon for insertion in the extraperitoneal area is not prerequisite. tep is an appropriate method both for first appearing and recurrent inguinal hernias. epiduralanesthesia instead of general anesthesia is no a contradiction for properly selected patients. aims: the aim of this study was to investigate the effects of preperitoneal carbon-dioxide (co ) insufflation during tapp (transabdominal preperitoneal) repair. materials and methods: male patients with inguinal hernia were include in our study. we obtain laparoscopic access at the umbilicus and introduce mm port. two mm working ports are placed lateral. diagnostic laparoscopy of the entire abdomen is necessary to rule out other pathology or contraindications for surgery. using aspiration needle we insuflate carbon-dioxide ( mmhg) preperitoneal at the level of anterior superior iliac spine while decrease abdominal gas pressure to mmhg. same procedure is made lateral to the umbilical artery. results: we found that preperitoneal carbon-dioxide (co ) insufflation during tapp facilitate the future parietalisation and even can reduce operating time in future improvements of the technique. there were no intraoperative complications related to this procedure. we did not found any potential risk of the technique when is use by trained surgeons. aims: laparoscopic inguinal hernia repairs (lihr) are performed more and more frequently because they offer some advantages; however, we cannot forget their specific complications. lihr are associated sometimes with peritoneal tears that can lead to bowel obstruction. we present two cases of bowel obstruction related to peritoneal defects post tapp procedure and review peritoneal closure, bowel obstruction and options to repair defects. a year-old male was scheduled for tapp due to bilateral relapse. two x tio mesh tm fixed with securestrapÒ, employed also for peritoneal flap closure, were employed. three days later he was readmitted with bowel obstruction with ct suggesting 'adhesions'. a year-old male had bilateral tapp in another centre. seven days later he presented with bowel obstruction. ct showed metallic tackers and suggested 'adhesions' results: first case: after four days of conservative treatment failure, a revisional laparoscopy showed ileum herniation through a peritoneal defect and firm adhesions to the mesh. bowel was labouriously separated and the peritoneal defect closed with two running sutures. he was discharged on the \ sup [ th \/sup [ postoperative day and three years later he is asymptomatic. second: after two days of conservative treatment failure, on laparoscopy, ileum was filmy adhered to polipropilene mesh through a big defect on flap closure. defect was closed with interrupted sutures. as tears persisted, an omental flap was created to cover the area. patient was discharged on the th day and continues asymptomatic three years later. conclusions: lihr bowel obstructions can be divided in adhesive disease and herniation. herniation can be early (through peritoneal defects) or late (trocar site). international guidelines recommends a thorough closure of peritoneal incision or bigger tears (grade b). the closure can be achieved with staples, tacks, running suture, or glue. these last two methods are more time-consuming but less painful. running suture seems to be the best, due to its low costs, tightness and low pain but sometimes can be technically difficult. low intra-abdominal pressures (= mmhg) facilitate suturing. when a herniation appears, careful bowel management is needed and running sutures are recommended. if tears persist, an omental flap can be useful. aims: application of a single port robotic platform to perform an entirely transanal tatme/ tata. methods: the following video demonstrates how a totally transanal proctosigmoidectomy is performed using a novel, single port (sp) robotic platform was used to carry out a totally transanal proctosigmoidectomy, single port robotic tatme/tata. a -year-old female patient with a clinical t n b rectal cancer at the cm level, status post neoadjuvant chemoradiotherapy ( cgy, xeloda) is presented. shown here is the open transanal dissection followed by docking of the sp robot, implementation of the single port instruments (fenestrated bipolar forceps, cadier, scissors, camera, clip applier) through a gelpoint path to complete a totally transanal proctosigmoidectomy including transanal tatme, ima/imv transection, splenic flexure release, and left colonic mobilization, loop ileostomy, and handsewn coloanal anastomosis. results: blood loss was cc. pathology demonstrated a moderately differentiated, rectal adenocarcinoma. the total mesorectal excision was complete (grade ), margins were negative, and all lymph nodes were negative for metastatic carcinoma. the patient was discharged on postoperative day after an uncomplicated hospital course. there was no postoperative morbidity or mortality. conclusions: application of the single port robot to transanal tatme/tata (sprtatme) is presented here. while much work remains to be done to validate the sp robot's safety, this first demonstration of a totally transanal tatme/tata establishes its feasibility and utility. this single port platform stands to greatly expand the application of natural orifice transluminal endoscopic surgery (notes). as shown, the sp robot offers more than sufficient visualization, technical control, and adequate reach to perform such an operation. we present an exciting new avenue by which to complete operations in an entirely transanal fashion, which are classically performed via a combined transanal and transabdominal approach. methods: this video shows the utilization of a new robotic platform to perform transanal endoluminal microsurgery, rtem. presented here is a year old woman with a recurrent rectal adenoma at the cm level, status post a previous tem resection in october . demonstrated is the utilization of the sp robot through a gelpoint path in order to perform a partial fullthickness and full-thickness resection. the robot is introduced through a mm in diameter cannula via a four-channel face-plate. the instruments' two-jointed mobility at the elbows and wrists as well as the novel navigation system are well demonstrated. the docking of the sp robot, utilization of the dissecting devices, and closure of the defect is shown. results: sprtem was performed with a blood loss of cc, and the patient was discharged on postoperative day . there was no postoperative morbidity, mortality, or moderate/severe pain. pathology showed tubular adenoma with low-grade dysplasia in a non-fragmented specimen with negative margins circumferentially. conclusion: initial experience using the sp robot for rtem is demonstrated here. the robot provides wonderful visualization and operative control to the surgeon. articulation of the robot's wrists and arms have the potential to facilitate technical aspects of the procedure. rtem stands as an exciting development in the field of transanal endoluminal surgery. introduction: the application of robotic approach in the esophageal surgical field is in its first phase. the microsuturing and microdissection capabilites of the robotic system can potentially overcome the traditional limitation of the laparoscopic surgery thus enhancing the indications of minimally invasive surgery. methods: we have performed a retrospective analysis of our prospectively maintained database that included patients who underwent robotic-assisted esophagectomy for malignant disease between and . results: ten out of sixteen patients had squamous cell carcinoma meanwhile six had adenocarcinoma. ten mckeown's and six ivor lewis were performed. the mean operative time was min ( - ) and the median blood loss was ml ( - ). no patients required conversion nor intraoperative transfusion. the morbidity rate was / ( . %) : a transitory laryngeal nerve paresis, a pneumotorax and pneumonia. the mean hospital stay was (range - ) days. an r resection rate of . % was achieved with a mean lymph node yield of ( - ). the -year disease free survival was . %, wheres the the -year overall survival was . %. conclusions: robotic assisted minimally invasive esophagectomy (ramie) is safe and feasible, it offers promising results while preserving a good oncology adequacy. this video shows our technique for the treatment of an esophageal diverticolum using a robotic left sided transthoracic approach, followed by a heller myotomy and dor fundoplication using a transabdominal approach. our case is a year old male, who suffered from severe dysphagia, halitosis and gastric reflux who on endoscopic and radiological investigations was found to have low grade and a cm wide esophageal diverticulum, cm from the lower esophageal sphincter. initially conservative management was attempted, however following poor compliance and the persistance of symptoms after year of therapy, surgical intervention was indicated. the operation was performed using the minimally invasive robotic system of the davinci siÒ, starting with the thorax time. the patient is positioned in left side decubitus. the camera-trocar is insert in the thorax via the fifth intercostal space the, two mm and one mm robotic trocars are added. the lung is liberated from pleural adhesions and the esophagus is then prepared exposing the diverticulum which is successfully removed with an endo-giaÒ. the esophageal muscle fibers, near the suture line is reinforced with separated vicryl stitches and the resected piece is extracted via endo-bag. a fr thoracic drainage tube is then placed and the trocar accesses repaired. the patient is the put in supine position with a °anti-trendelemburg angle. three robotic trocars (two mm and one mm) are placed and the robot docking is made from the patient left shoulder. the lesser omentum is divided to visualize and prepare the gastric-esophageal junction (gej) sparing the vagus nerve. the heller myotomy is then performed for cm over the gej and cm under it. the mucosal integrity is assured via laparoscopic and contemporary gastroscopic view. the gastric fundus is attached to the distal esophagus completing the dor fundoplication. post-operative care comprehends the removal of the thoracic drainage during the first post-operative day, the pain management and the progressive realimentation. the hospitalized period lasts day and the patient was dismissed without complications occurred. the uniportal video assisted lung lobectomies gained popularity all over the world during the last years. the technique is safely applied for peripheral pulmonary lesions, under cm, but more and more complex cases are being approached while the indications continue to evolve. our aim is to present the particular aspects of this technique in an -year-old female patient with a giant bullous lesion located in the lower lobe of the right lung. the preoperative work-up for this case is presented and commented. a multidisciplinary surgical team consisting of thoracic and pediatric surgeons was involved. a single . cm length incision in the fourth intercostal space was used for the access. due to the fact that the lesion involved almost the entire lobe and the margins were very close to the hilum, we have decided and performed a right lower lobectomy. dissection and stapling were quite difficult. all the anatomical structures had small dimensions, forcing us to perform an 'artery first approach' in a very narrow space. no complications during or after surgery were encountered. the patient was discharged after four days and she went to school on the sixth day. histopathological examination showed that the lesion was a type ccam (congenital cystic adenomatoid malformation). conclussion: the uniportal video assisted lung lobectomy was safety applied for a giant bullous lesion of the right lung. aim: dunbar syndrome, celiac trunk (ct) compression syndrome, caused by median arcuateligament is a rarely diagnosed disease because of its nonspecific symptoms, which cause adelay in the correct diagnosis. the aim of the study was to demonstrate the usefulness andadvantages of laparoscopic approach in the treatment of dunbar syndrome. methods: we performed laparoscopic release of ct in the department of general, minimallyinvasive and elderly surgery in olsztyn in . all of three patients suffered from severepain of abdominal cavity before the surgery. results: in two cases, there were a complete remission of the symptoms. in one case, there was animprovement. all patients reported relief of symptoms in the first days after the operation.there were no postoperative complications. conclusions: the laparoscopic treatment of dunbar seems to be safe and feasible procedure. thelaparoscopic surgery alone can often eliminate discomfort, while angioplasty and stentimplantation are no longer necessary. introduction: the advances in robotic surgery have permitted the application of such technology to various surgical fields, one of the last of these being hernia surgery. we present a case video of the treatment of a dual-hernia using a robotic retromuscular ventral hernia repair(rrvhr) using the davinci siÒ robotic system. the case report demonstrates the evolution of the trans-abdominal robotic umbilical prosthetic (tarup) in that it utilises a 'double docking' technique to allow the positioning of a large retromuscular mesh. methodology: our patient is a -year-old male who presented with chronic epigastric pain. the abdominal ct confirmed two abdominal wall hernias; an epigastric and supra-umbilical hernia with visceral contents and wall defect diameter of cm and . cm, respectively. using the minimally invasive robotic system of the davinci siÒ we adapted the well known retromuscular mesh technique. the operation was initially intraperitoneal with access to the retromuscular preperitoneale space using a right sided longitudinal incision.(as per standard tarup technique). we proceed with the dissection of the retro-muscular space until the left lateral edges of the rectus sheath, creating a preperitoneal space for the placement of a specifically modified ultrapro polypro-leneÒ x cm mesh. following this we repositioned the davinci siÒ in a symmetrical manner, with ports placed in the retromuscular space. the mesh is positioned and the peritneum subsequently closed with a v-lock sutureÒ. finally we opted for a negative pressure jackson-pratt drain, inserted preperitoneally. results: the patient was discharged on the nd post-operative day without complication follow up continued until months post operatively during which the patient remained asymptomatic, without signs for hernia recurrance . conclusion: the technique highlighted in our video demonstrates the utility of the robotic system in hernia repair. specifically the approach proved a success as it facilites the placement of the mesh totally extra-peritoneally with closure of the posteriore sheath without tension. the added advantages are that the port-sites are distant from the mesh thus reducing infective risk. additionally this technique allows the treatment of large peritoneal defects. surg endosc ( ) aim: to analyse the performance of a robotic fellow during a robotic total mesorectal excision (tme) at the end of the fellowship, and subsequently compare it with their mentor. methods: the fellow is exposed to robotic colorectal lists per week. during the fellowship, assessment of performance is recorded in a structured proforma covering aspects of autonomy, tissue handling and dissection. at the end of the fellowship, areview of cases performed by the fellow and the mentor was carried out in a blindly manner (video footage). results: robotic tme training was divided into modules in order of complexity and the trainee had to achieve sequential proficiency in each module, before progression. docking of davinci robotic system. inferior mesenteric artery exposure and ligation, development of medial to lateral plane and inferior mesenteric vein division. left colonic and splenic flexure mobilization. pancreas identification. rectal dissection (tme). qualitative assessments were recorded by the mentor; the fellow was 'able to perform with verbal help' most of the steps from early on. by the end of the fellowship, all steps were performed in a similar manner in terms of quality and oncological integrity when compared with the mentor. conclusions: at completion ofan advanced robotic colorectal fellowship, high quality trainees can perform every step of the tme dissection in a similar manner with the trainer, when assessed blindly, without compromising oncological integrity. aims: to find safe and simple method in robotic rectal low anterior resection with low tie arterial ligation and lymph node dissection around the root of inferior mesenteric artery. methods: we performed robotic rectal low anterior resection (rlar) by davinci si system in eight patients with rectal cancer. we applied low tie arterial ligation, just caudally to the origin of the left colic artery in all cases. during the procedure, we used tilepro function of davinci si system which enabled to display two other visual informations through external inputs under the normal -dimensional surgeon console view. preoperative d-ct vessel branching simulation video and intra-operative real time ultra sound navigation view were displayed simultaneously under normal operative camera view in the surgeon console. results: left colic artery preservation was completely done in all cases. the mean time to find and expose the left colic artery from the first incision in sigmoid mesentery was min, which was drastically shorter than conventional method. this method needed lesser mobilization of inferior mesenteric artery (ima), and may be less invasive to autonomic nerve around the root of ima which is very important for ejaculation function. conclusion: robotic rectal low anterior resection with low tie arterial ligation was performed safely and in short time, using tilepro intra-operative navigation method. preoperative d-ct vessel branching simulation video and intra-operative real time ultra sound navigation view were very useful in the procedure. we present the method in video. nerve sparing tme and pelvic neuroanatomy for colorectal surgeons p. tejedor, f. sagias, j.s. khan aim: to describe the critical points in which the pelvic nerves can be damaged during a total mesorectal excision (tme) for rectal cancer and the benefits of robotic surgery for identifying these points. methods: there are critical points regarding pelvic neuroanatomy: superior hypogastric plexus (shp): located in front of l -s . the ganglionic sympathetic fibres form the right and left sympathetic trunk, travel along the anterior surface of the aorta and coalesce in the shp at the level of the inferior mesenteric artery (ima). superior hypogastric nerves: they take an anterolateral course into the pelvis. there is an avascular 'holy plane' around the rectum between these two nerves. inferior hypogastric plexus (ihp): lies over the posterolateral pelvis, almost parallel to the internal iliac arteries. this can be identified at the lower end of the rectum. neurovascular bundles(of walsh): in front of the denonvillier's fascia, at and o'clock position. they are responsible for erectile function. results: lack of knowledge or identification of key structures at these points can lead to increased risk of nerve damage and translate into poor functional outcomes. the ima is dissected up to the origin from aorta and here the shp can be seen. care is taken to avoid any damage to these structures. the tme plane is found at the back of ima as the inner most dissectible layer between mesorectum pelvic fascia. right and left superior hypogastric nerves are identified. dissection is carried out posteriorly, laterally and anteriorly. ihp is identified at the lower third of the rectum, when the dissection is about to reach the pelvic floor. care should be taken in not to go too far lateral and damage this plexus. in the anterior dissection, plane is carried in front of the denonvilliers' fascia. the neurovascular bundles can be seen at and o'clock position and the surgeon has to be careful to stay inside that plane in order to avoid damage. conclusions: the precise dissection in robotic surgery results in minimal tissue damage and better visualization and preservation of the pelvic nerves. aims: to describe and evaluate new contributions and eventual advantages of icg fluorescence to perform an icg guided bilateral pelvic lymph node dissection in a patient who underwent low-anterior-resection for rectal carcinoma. we also present the basic steps to avoid ileostomy during rectal surgery in which icg and ghost ileostomy play an important role. methods: a -year-old male patient was referred to our hospital due to abdominal pain and significant changes in usual bowel habits.colonoscopy showed a no obstructing cm middle rectal mass, which was reported as an adenocarcinoma.ct scan and mri revealed a mm polyp in the anterior rectal wall which was located cm from the anal verge. it was involving mucosa and sub-mucosa with muscularis propia invasion. no pathological lymphadenopathies or hepatic metastatic disease were found (stage t n ).a laparoscopic ultra-low-anterior resection plus icg lateral lymphadenectomy with total mesorectal excision was performed. a complete splenic flexure mobilization was performed to achieve a safe tension-free anastomosis. transection line of the proximal rectum was checked after icg intravenous injection. icg was injected around the tumor by inserting an anoscope, just before the surgery. after the dissection of the rectum, lateral lymphadenectomy was performed assisted by icg. an end-to-side anastomosis was made. and a vascular loop was passed around the terminal ileum to create a ghost ileostomy.the procedure lasted min. reactive protein c was monitored to identify an initial leak. the patient was discharge in postoperative day and no complication was detected. results: pathological exam reported a rectal adenocarcinoma. pelvic lymphadenectomy results were: negative nodes, negative nodes and negative nodes from right lymph node dissection, left lymph node dissection and rectosigmoid resection specimen respectively. no metastatic disease was found (stage t n m ). conclusions: in our experience, icg fluorescence imaging system offers important contributions to rectal surgery furthermore than evaluating vascular supply to the anastomosis. lymphatic mapping of the lateral lymph nodes and avoiding ileostomy could be a potential important use in the future. larger studies and more specific evaluations are needed to confirm its role in colorectal surgery and to find its limitations. background: robotic surgery for colorectal cancer is an emerging technique. potential benefits as compared to conventional laparoscopic surgery have been demonstrated. innovative robotic technologies have helped surgeons overcome many technical difficulties of conventional laparoscopic surgery such as hand-eye coordination, a two-dimensional view, and a restricted range of motion. robotic-assisted surgery was established as a new approach to minimally invasive surgery, overcoming these limitations. the following video shows a total robotic sigmoidectomy step by step on the basis of ourexperience. intervention: a -year-old male patient with no previous medical historyand a colon adenocarcinoma, cm from the anal verge, no distant metastases. it was decided to perform a robotic sigmoidectomy. target anatomywas located andwe proceededto the exposure of the mesenteric vessels from medial to lateral. a cautery wasused to open the peritoneum,up to the origin of the inferior mesenteric artery, and caudally past the sacral promontory.the vessels weretransected by ligasuretm. we performedthe complete release of the colon taking care to avoid injury to retroperitoneal structures. we usedligasuretm to section the mesocolon in order to prepare the transection of the proximal colon. indocyanine green was used to check the correct vascularization. an endogia tristapletm was used to divide the colon. subsequently, we sectioned the rectumand extracted the specimen through itwith no need to make any auxiliary incisions. we introduced the anvil of the suture device to perform the anastomosis. we sectionedand close the rectum with an endogia tristapletm. finally we opened the proximal colon to introduce the anvil,making a pursestring to fix it and create a side to end anastomosis. outcome: the surgery took min. the patient started oral intake h after surgery and left the hospital on the rd postoperative day. pathological examination ruled out a colon adenocarcinoma pt n . conclusion: total robotic sigmoidectomy is safe and feasible and can be a procedure of choice to achieve a good surgical qualityand avoid assistance incisions in patients with colon cancer. surg endosc ( ) with more and more data now advocating wait and watch policy for these patients which require close radiological and endoscopic follow-up but unfortunately around % of them have regrowth of tumour which will require surgical intervention. the use of robot for cancer resections is becoming more frequent especially in narrow spaces like in an obese male pelvis. the reason being better -dimensional views, more angulation of the instruments and exclusion of tremors, which in turn leads to better dissection and preservation of hypogastric nerves. in this video, we present a robotic low anterior resection for rectal re-growth in an obese -years old male patient. he was offered neoadjuvant chemoradiotherapy after discussion in mdt. he had an complete response with chemoradiotherapy and was decided to offer him watch and wait regime. unfortunately, he developed rectal re-growth in the first year of his follow up. imaging showed t lesion with no distant metastasis and was later confirmed on histology as well. after mdt discussion he was offered robotic low anterior resection. the video starts by showing the clinicopathological features of patient including his radiological and endoscopic images. robotic port sites are shown. the edited video starts with rectal dissection after ligation of inferior mesenteric artery and vein with emphasis on narrow pelvis and preservation of hypogastric nerves, seminal vesicles and intact presacral fascia. postoperative histology was ypt no and patient was discharged home after days with no postoperative complications. background: minimally invasive surgery for colon resection has improved patient outcome, however a minilaparotomy still is necessary to extract the specimen. this report describes a new approach that combine laparoscopic parellel overlap stapling left colectomy with natural orifice specimen extraction surgery, with the aim to minimize abdominal wall trauma. method: laparoscopic left colectomy for malignant diesease was performed using a standard five-port technique. after releasing the left colon via laparoscopy, divide the proximal and distal of specimen with -echelon, and put distal sigmoid colon and proximal transverse colon together. open sigmoid colon cm apart from distal margin, and incise transverse colon at proximal margin. take transverse colon and sigmoid colon side-to-side anastomosis via -echelon. incise posterior vaginal fornix to get into the abdominal cavity and extract specimen through vaginal. outcome parameters such as complications, conversions, operative time, postoperative recovery, and postoperative pain were prospectively recorded in a database. results: surgery was performed for patients with left-colonic carcinoma. no perioperative complications or conversions occurred. the median operating time was min. the median visual analogue scale score of postoperative pain was , and of patients needed analgesia on postoperative day . the median postoperative hospital stay was days. for malignancies, tissue margins were oncologically adequate, the averge number of harvested lymph nodes were . . the -week follow-up period was uneventful. conclusion: the described technique, a combination of laparoscopic parellel overlap stapling and natural orifice surgery, has the potential to avoid incision-related morbidity of the minilaparotomy in laparoscopic left colon resections. background: open surgical skills training has been well established over centuries, however, there are some significant differences in laparoscopic surgical skills training. it is an obvious advantage that the trainee and the trainer have the same view; however, some of the hurdles include the differences in tactile feedback, hand eye co-ordination, spatial awareness, depth perception and maximizing assistance. aim: we present a video highlighting some of the key challenges faced in laparoscopic colorectal surgical training, show-casing our systematic, structured approach. our approach: we have developed a structured approach starting with junior surgical trainees and progressing through to consultant level as per the levels below: level : attend courses/ workshops level : master camera work level : contra-lateral assisting level : intermediate level trainee-start operating with trainer scrubbed. the trainer is an additional member of the scrub team and stands on the same side as the trainee (does not replace any assistant) level : advanced level trainee-gradual progression from level . trainer un-scrubbed but standing next to the monitor throughout the procedure. level : trainer in theatre but out of sight of the trainee, with little interference level : progression to trainer-once proficiency is achieved at level / , the trainee is trained to become a trainer, for the junior and intermediate level trainees. within each level the complexity of the procedure increases as the trainee progresses through the level. junior trainees (years - of surgical training) are taken through levels - , intermediate (middle years of training) level or and advanced (last - years) up to levels . this way of training allows multiple members of the team to be trained simultaneously in every case. each operating list is preceded by team briefings where the role of every member of the team is clearly identified and followed by individual and collective feedback. conclusion: this training ladder proved very successful through the years. the feedback from trainees at all stages has been consistently positive. several trainees who have progressed to independent consultant practice, in the uk and abroad, are adopting this approach in their practice. introduction: despite the potential microsuturing capabilities of the robotic surgery, most of the esofago-jejunostomy after robotic total gastrectomy are still performed extracorporeal or through mechanical staplers. this can increase the cost of the procedure, the risk related to a improper functioning of the stapler. methods: we reviewed our prospectively maintained database analyzing patients from april to september , who underwent robotic total gastrectomy with hand-sewn esophagojejunostomy for gastric cancer. results: a total of patients were included in the study. the mean estimated blood loss was ml ( - ). the overall operative time was min ( - ). length of hospital stay was days ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . no conversion was necessary nor anastomotic leakage occurred. the morbidity rate was / ( . %) and included a subhepatic abscess and wound infection trough pfannenstiel incision. a r resection rate was achieved in all cases. the mean of lymph node yield was ( - ). the -year disease free survival was %, the -year overall survival . %. the robotic-assisted hand-sewn esophago-jejunostomy is a safe and no time-consuming technique. it avoids the complication related to the stapler firing and it offers cosmetic benefit to the patient in terms of extraction site. introduction: colorectal endoscopic submucosal dissection (esd) is increasingly practiced for treatment of early colorectal neoplasia. however, colorectal esd is difficult to perform due to lack of retraction as well as instability especially over hepatic flexure. dilumen eip is an external flexible sheath introduced during colonoscopy to stabilize environment for esd. this video demonstrated the use of dilumen eip for performance of colonic esd at the ascending colon method and results: this is a years old lady who received screening colonoscopy and found a mm lateral spreading tumor (lst) type iia lesion at ascending colon distal to ileocecal valve. under general anesthesia, patient received colonic esd using dilumen eip. due to significant looping, the dilumen device was introduced with the techniques of double balloon enteroscopy. after identification of the lst, the balloon in the front would be deployed to the proximal to the lesion while both balloons would be insufflated and created a stable environment. the esd procedure started after submucosal injection with normal saline in mix with indigocarmine, epinephrine and hyaluronate. mucosal incision was performed over the anal side of the lesion, and after adequate submucosal dissection, clips were applied to attach the mucosal flap to the sleeve of proximal balloon and achieved retraction. the submucosa was adequately exposed for dissection using dual knife jet. this enhanced submucosal dissection especially at one area with significant fibrosis. after the procedure, complete closure of the mucosal defect was performed by clips and assisted by the front balloon. the pathology confirmed intramucosal adenocarcinoma with clear resection margins. discussion: the dilumen eip device stabilized the environment within the colon with the double balloon and provide adequate retraction for performance of colorectal esd. surgery, kobe city medical center general hospital, kobe, japan background: robotic surgery has been widely spread all over the world, but robotic gastrectomy is not common and difficult because of complex anatomy and wide-ranging operation fields. in addition, it had been performed only under a few high-volume centers for reasons of the limitation of national health insurance in japan, which means medical expenses not covered by insurance. the situation was changed from this april, so we started robotic gastrectomy to reduce complications more rather than laparoscopic gastrectomy. we report results and aim to present the methods in detail using da vinci si surgical system. methods: we place five trocars, one is umbilical endoscopy port, and other four ports are placed at the reverse trapezoid, almost fan-shaped. using the arm number , the organ can be lifted up so that sharp lymphadenectomy is able to be done by almost a scissor as the arm number while applying the countertraction by the arm number . in order to achieve a clear and bloodless lymphnode dissection while maintaining the oncological safety, we think not only the ultrasonic coagulating scissor but also the electrocautery of the scissor is very essential in robotic surgery. less postoperative complication such as pancreatic fistula or pancreatitis might be derived from robotic surgery because we can avoid pressing the pancreas during the suprapancreatic dissection of lymph nodes. the billroth i reconstruction can be performed using da vinci endowrist stapler under stable and inflexible surgical fields without needing help of surgical assistant. results: from october to december , patients with gastric cancer were operated robotic gastrectomy, included total gastrectomy. there was no conversion to open surgery and no conversion to other procedures derived from intraoperative complications, and the overall operation time is gradually decreasing from the th case. we are now on the way of learning curve shortening operation time, but robotic gastrectomy is no less safer and adequate than laparoscopic surgery. we will show our robotic procedures including lymphadenectomy around subpyloric and suprapancreatic area, and reconstruction with several important points in our video purpose: this report describes the benefits and drawbacks in the use of a novel articulating device (artisential), which has a multi-degree wrist freedom like the davinci endowrist, in performing complete single-port d lymph node dissection (lnd) in single-incision distal gastrectomy (sidg). methods: the artisential was used in performing sidg with d lnd for patients with advanced gastric cancer. all operations were performed by a single surgeon using a threedimensional camera and a passive scope holder in place of a scopist. the artisential was used mainly in the sb and suprapancreatic lnd, an area that is relatively far from the single port. in certain cases when the pancreas needed to be pushed down, such as obese male patients, the intraabdominal organ retractor was used to lift the tissue and the artisential to push the pancreas. operative results and short-term outcome were analyzed. results: twelve patients underwent the procedure without any intraoperative events, conversion to conventional laparoscopy, or surgery-related complications including postoperative pancreatic fistula. all patients underwent single port d lnd by complete exposure of the portal and splenic vein. mean operation time was . ± . mins. and mean number of retrieved lymph nodes was . ± . . the artisential was found to be useful in grasping the tissues behind the pancreas and the major arteries throughout most of the lnd. the articulating motion also allowed the narrow single-port field of view to be clearly seen without the instrument body obstructing the camera. conclusion: the use of artisential in sidg appears feasible and reproducible, and is mandatory in performing a complete d lnd in sidg. the video shows a case of laterally spreading tumour of the rectum with preoperative benign histology, paris classification -is g (granular type), ut n eus stage, kudo type iv, nice type . the neoplasm measured x cm, and extended from to cm from the anal verge, mainly located on the posterior wall. according to our local policy the indication was a transanal full-thickness excision. this was performed with the medrobotics flexÒ robotic system, used here for the first time outside the united states.the system technology utilizes an articulated multi-linked scope that can be steered along non-linear, circuitous paths in a way that is not possible with traditional, straight scopes. the maneuverability of the scope is derived from its numerous mechanical linkages with concentric mechanisms. this enables surgeons to perform minimally-invasive procedures in places that were previously difficult, or impossible, to reach. with the flexÒ robotic system, surgeons can operate through a single access site and direct the scope to the surgical target. once positioned, the scope can become rigid, forming a stable surgical platform from which the surgeon can pass flexible surgical instruments. the system includes on-board d hd visualization. the flexÒ robotic system contains two working channels to accept a number of different surgical and interventional instruments including monopolar and bipolar electrodes, scissors and graspers for tissue manipulation.the video shows the introduction of the dedicated rectoscope, the connection of the flexible robot, and the way to operate the device performing a full-thickness excision, including suturing of the rectal defect by means of two running sutures by a v-lock / thread. while illustrating the technique the authors will comment pros and cons of the use of the device. background: hepatobiliary procedures using a minimally invasive approachare demanding, especially in major hepatectomies. the use of da vinci surgical system allows to overcome some of the kinematics limitations of the direct manual laparoscopy maintaining the potential advantages of a minimally invasive approach . we herein present a case of left hepatectomy and local lymphadenectomy for hepatocellular carcinoma, carried out with the use of the da vinci xi. methodology: a -years old man with a long-lasting hbv chronic infection and ct scan and mri finding of a -cm solid neoplasia of the left hepatic lobe and gallbladder stones, was operated with the da vinci xi platform. the patient was placed in a supine position, with °anti-trendelenburg inclination. the trocars were positioned according with the intuitive indication for the upper quadrants surgery. results: the procedure was successfully completed in min.at first, an intraoperative us scan with the use of tile-pro technology was done to determinate the tumor extension. the hepatic parenchyma transaction and the local lymphadenectomy were performed with monopolar scissors and bipolar grasps. the left hepatic vein section was performed with an endoscopic vascular stapler. there were no surgical complications or need for conversion to laparoscopy or laparotomy. the post-operative course was uneventful and the patient was discharged days after surgery. conclusion: the da vinci xi can facilitate some technically demanding procedures and ultimately widen the range of application of minimally invasive surgery such as hepatic surgery. besides the well-known advantages provided by robotic surgery on d imaging, increased range of motion and augmented surgical dexterity, one of the most interesting and innovative features of robotic technology is the digitalization of the operative view; furthermore the tile-pro multiinput display allows the surgeon a d view of the operative field along with the ultrasound exam for a precise understanding of anatomy and vascularity and of tumor location. during the last few years, robotic surgery as well, as the latest innovation of minimally invasive procedures, takes its position in this particular field with the benefits of overcoming the limitations of conventional laparoscopy. our aim is to demonstrate the advantages of robotic surgery in procedures of hepatectomies, on occasion of a robotic hepatectomy performed by our team. methods: we present video fragments of a robotic left lateral hepatectomy procedure in an elderly female patient with a symptomatic gigantic haemangioma of the left hepatic lobe. we emphasize on the technical aspects and the advantages that the surgeon gains applying the robotic techniques in such procedures. results: the procedure was completed with minimal blood loss and the patient presented an uncomplicated post-operative course, with discharge on the third postoperative day, minimal need of analgesics and full recovery. conclusions: the excellent three-dimensional and high quality visualization that the robotic system offers, combined with the flexibility and the accuracy of the robotic instruments (especially on suturing), provide to the surgeon an important aid, in order to avoid serious complications, such as intraoperative bleeding and post-operative bile leaks. the restriction of the limitations of conventional laparoscopy is far more beneficial and promising for the evolution and the future of minimal invasive liver surgery. aims: the new da vinci xi surgical cart allows multi-quadrant and complex surgical interventions in a minimally invasive fashion. we present a case of robotic appleby left pancreatectomy using this platform and its specific operating bed. methods: a -years old woman with ct scan finding of a -mm hypo-vascular neoplasm of the pancreas body underwent surgery with the use of the new da vinci xi with four arms upper quadrants trocar' disposition. results: the procedure was successfully completed in min. the pancreatic body was mobilized in order to expose the portal-mesenteric axis. the gland was transected using a robotic endo-stapler as well as the splenic vein. after evaluating the patency of collateral circles with intra-operative ultrasound, the common hepatic artery and the celiac artery were transected. then we increased the right tilted position and the neoplasia was detached from the gastric body by a tangential gastric resection using the robotic endo-stapler. finally, the operation was accomplished with the transection of the posterior attachment of the spleen and the pancreatic tail. no conversion or intra-operative complications were recorded. the post-operative course was uneventful and the patient was discharged days after surgery. the da vinci xi with its specific tools helps in performing challenging procedures such as appleby operation for locally advanced pancreatic cancer. in our experience, the robotic endo-stapler permits the operating surgeon to directly control the transaction phase whereas the specific operating bed allows to perform minimally invasive multi-quadrant surgery and to obtain a better exposition of the operating field. results: the whipple procedure was successfully completed in min. thanks to the dvtm the patient's position changed during the intervention to improve the exposure, with the instruments left inside the abdomen and without undocking the robot. the dissection of the pancreatic head from the portal vein and the section of the retroportal lamina were performed with the use of the endowrist vessel sealer device. a personal modified end-to-side pancreatojejunostomy was carried out, with / prolene and gore-tex double layer suture. no intra-operative complications occurred and no conversions to laparoscopy or laparotomy were required. the postoperative course was uneventful. conclusions: the use of the new fully wristed vessel sealer extend makes easier difficult maneuvers such as the fine dissection of the pancreatic head from the portal vein and the section of the retroportal lamina, enabling an optimized approach for vessels sealing and cutting and tissue bundles. moreover, the dvtm allows patient's movements without undocking the system or removing instruments from the abdomen, enhancing the surgical workflow. background: necrotizing pancreatitis is a devastating illness which can develop in up to % of patients who suffer from pancreatitis. it carries great morbidity with an associated mortality rate between to %. many of these patients require drainage of fluid collections to treat sequela related to pain, per-os tolerance, and source control of sepsis if infected. the step-up approach to treatment of this disease has trended towards minimally invasive techniques, considering the morbidity of open debridement. as such, many centers have implemented the use of transgastric debridement via endoscopic cystogastrostomy. this technique, while effective in draining fluid and particulate necrotic tissue, has difficulty in resection of large necrotic tissue, due to instrument and anatomic limitations. current endoscopic accessories designed for polypectomy or foreign body extraction, for example, are not optimal for performing necrosectomy. to overcome this obstacle, additional access sites can be utilized to assist debridement. we describe the first laparoscopic assisted transgastric endoscopic necrosectomy through a percutaneous gastrostomy in a year old male with infected pancreatic necrosis secondary to biliary pancreatitis. aim: to investigate the feasibility of utilizing gastrostomy access to assist in debridement during endoscopic necrosectomy. methods: the patient previously underwent an open necrosectomy and gastrostomy tube placement for acute emphysematous pancreatitis. post-operatively, there was a persistent and enlarging cm infected walled-off necrosis (won). therefore, endoscopic cystogastrostomy was performed using a lumen-apposing metal stent. results: frank pus was evacuated. initial endoscopic necrosectomy was technically challenging due to the large volume of solid necrotic tissue. repeat endoscopic debridement utilized a surgical laparoscopic grasper via the gastrostomy site to aide solid debris extraction (video). this allowed for complete necrosectomy and resolution of the won. the patient did well and was discharged subsequently. conclusion: this is another emerging minimally invasive technique in the step-up approach for debridement and drainage of won. the use of the gastrostomy as a utility port for accessory instruments not only enhanced the technical aspects of the procedure but increased its efficacy as well. further experience is needed to validate the utility and reproducibility of this technique. objective: the presentation of the minimally invasive surgical approach for pancreatic necrosectomy guided by videoretroperitoneoscopy or var (video assisted retroperitoneoscopic), established in our center, as one of the option of the step-up approach treatment for acute necrotizing pancreatitis (anp) methods: the placement of the patient on the operating table should be in decubitus, with right lateral inclination, at - °on the horizontal surface. the pancreatic cell is approached using the drainage catheter previously placed by radiological control (ultrasound or ct) as a guide, which will allow access to the cavity with safety. an incision of - cm is made around the previously placed catheter, crossing the subcutaneous cellular tissue and muscular fascias, dissolving the musculature. it continues in a blunt dissection, until a loss of resistance is appreciated which generally coincides with the outflow of necrotic or purulent material. once the retroperitoneal cell is accessed, a mm trocar is placed and a pneumoretroperitoneum is performed. the -mm trocar allows the joint use of a mm and °optic and the surgical material that allows debridement and cleaning. the aspiration and hydrodissection of the necrotic material, and the extraction of the solid component of the necrosis are proceeded. once the collection is drained and the necrotic material removed, a wash and drain system is placed, like a -way foley type probe. conclusions: in conclusion, the var is an alternative surgical technique, valid and reproducible in the treatment of anp, which offers comparable results and even superior, in some series, to those of open surgery, with satisfactory results in terms of morbidity and postoperative mortality. aim: lung subsegmentectomy is suitable for small and deep, non-palpable lung nodules. since it is difficult to intraoperatively detect the arteries, veins and bronchi of the subsegment, as well as the intersubsegmental borders, complete video-assisted thoracic surgery (vats) for lung subsegmentectomy is challenging. we use preoperative three dimensional ct to detect the arteries, veins and bronchi of the subsegment before conducting complete vats subsegmentectomy, and perform intraoperative bronchoscopy to detect the bronchi and intersubsegmental borders. i would like to describe our experience of complete vats combined subsegmentectomy for a non-palpable lung nodule. methods and results: the patient was a -year-old woman. during health screening, a small groundglass opacity was observed in her right lung on chest ct. the nodule was mm in diameter and was located in s b (horizontal subsegment of the posterior segment) near s (the anterior segment). we preoperatively diagnosed the lesion as well-differentiated adenocarcinoma, and planned combined subsegmentectomy for s b and s a (lateral subsegment of the anterior segment) of the right upper pulmonary lobe. before the operation, the locations of vessels were confirmed by three-dimensional ct angiography. video-assisted thoracoscopic surgery was performed using four ports: two cm ports in the th intercostal space in the post-axillary line and in the angulus inferior scapulae line for the operator, a cm port in the th intercostal space in the mid-axillary line for the assistant, and a cm port for the camera in the th intercostal space in the mid-axillary line. the cm port was also used for removal of the resected specimen. intraoperative bronchoscopy was used for detecting the subsegmental bronchi. she was diagnosed with primary lung cancer (adenocarcinoma in situ, nonmucinous) postoperatively. the tumor was pathologically graded as tisn m . no tumor recurrence has been noted in follow-up of twenty two months. conclusions: the combination of preoperative three-dimensional ct angiography, intraoperative bronchoscopy and complete video-assisted thoracoscopic surgery can be used for performing lung combined subsegmentectomy. aims: minimally invasive surgery is increasingly widespread for the diagnosis and treatment of abdominal pathology. laparoscopy is a diagnostic resource for those cases in which mass biopsy is not approachable through image-guided puncture, and is often therapeutic in the same act. it avoids the morbidity and mortality associated with laparotomy, favoring the early treatment of malignant processes. methods: we present a case of a year old male who was incidentally diagnosed with an oval-shaped pelvic mass in the right lateral wall of the pelvis, adjacent to the vascular bundle of the right external iliac at its origin( centimeter), without sign of infiltration of surrounding structures. no other pathological findings on the abdominal computerized tomography and magnetic resonance imaging were found. due to its localization, it was not accessible to percutaneous biopsy. the first diagnostic impression was a benign tumor of the nerve sheath (schwannoma), without being able to rule out other diagnostic possibilities. to provide a definitive diagnosis the patient was subjected to an elective laparoscopic resection of the tumor. surgical procedure was performed using a millimiter and two millimeter, umbilicus for the optical system and operative on hypogastrium and left iliac fossa respectively. acleavage plan between the tumorand rightiliac vesselswas found. the exeresis of the masswas achieved, and it was extracted using an endo-bagÒ through the umbilical port site. a drain was put in the surgical bed. results: the patient had a short, uneventful post-operative course, being discharged on postoperative day . pathological examination revealed a lymphatic node with metastasis of poorly differentiated carcinoma, with suspected urothelial lineage. cystoscopy was performed with the finding of a centimeter lesion on the right ureteral orifice with calcifications on the surface. biopsies were taken, confirming the bladder origin of the tumor. conclusions: both diagnostic and therapeutic laparoscopy is useful on pelvic masses because of the direct vision into this narrow anatomical space, especially in obese patients, providinga detailed view that makes easier to isolate and spear the anatomical structures surrounding the tumor, minimizing the risk of tumor rupture and bleeding. surg endosc ( ) aim: indocyanine green (icg)-enhanced fluorescence has been introduced initially in laparoscopic surgery to provide detailed anatomical information during laparoscopic cholecystectomy and to evaluate vascular supply to garantee correct anastomotic perfussion in order to reduce the risk of anastomotic leak. the uses of icg are increasing, specially in hepatic and oncological surgery in order to identify centinel lymph node and lymphatic mapping.we propose the use of icg imaging during complex laparoscopic colorectal resection in cases presenting ureter obstruction, to prevent iatrogenic ureteral injury. methods: we present a case of a year old female previously diagnosed of pelvic endometriosis with severe pain and symptoms related with episodes of pseudo-occlusion .a colonoscopy was performed finding sigmoid cancer in an area of endometriosis in a narrow colon with difficulties to perform a complete colonoscopy that could be related to the process of pseudo-occlusion. the biopsy was informed as an adenocarcinoma.the ct-scan showed a dilatated left ureter in an area next to the sigmoid colon.we propose a preoperative strategy with a bilateral double j stent insertion, finding a ureter obstruction caused by the endometriosis.icg was injected through the ureteral catheter, guiding us during the surgery to avoid a iatrogenic ureteral injury. results: a laparoscopic left colectomy was performed. the icg allows us to follow the ureter during the surgery, disecting the colon properly from the area attached to the ureter. the prestenotic area of the ureter was marked dilatated up to two centimeters allowing the icg to identify it from the anatomic structures of the areas and guarateeing that there was not spill of icg out of the ureter avoiding a postoperative leak of urine. conclussions: when tumors, or another entities like endometriosis, produce a ureteral occlusion, icg could be injected through a j stent, allowing us to identify and to avoid an injury.icg fluorescence imaging is a safe, cheap, and effective tool to increase visualization during surgery, offering additional information of the anatomy in colorectal surgery. in this video we are going to show three cases of the robotic treatment of splenic artery aneurism and the evolution of the technology that we relied on for the preoperative planning and intraoperative navigation. our preoperative evaluation evolved from a tridimensional virtual reconstruction with augmented reality to patient specific anatomical d printed models, initially made of rigid materials and afterwards made of malleable materials, in order to reproduce hollow anatomical structures such as vessels, feasible to simulate the planned surgical plan. the choice of a robotic approach, in selected cases, allowed to restore the continuity of the splenic artery after the exclusion or excision of the aneurism, in order to preserve the spleen. aims: tumor-induced osteomalacia (tio) is a rare paraneoplastic syndrome in which patients presents bone pain, fractures and muscle weakness caused by the fibroblast growth factor (fgf- ), a phosphate and vitamin d-regulating hormone. in tio, fgf- is secreted by mesenchymal tumors that are usually benign but small and difficult to locate. when medical treatment is unsuccessful, surgical treatment is indicated and conclusive.this video shows our technique for tio surgical treatment guided by indocyanine green (icg) fluorescent angiography. methods: the patient is an -years-old woman with tio confirmed by blood sampling of fgf- , gallium- pet/ct scan and abdominal ct scan which identified a highly vascularized mm nodule in the mesentery along the ileo-colic vascular axis.the patient was scheduled for a diagnostic laparoscopy with tio removal.the patient was placed in supine position. three trocars were introduced in the left quadrants. identified the last ileal loop, following ileo-colic vessels, a small mesenteric bulge was found. the icg fluorescent angiography confirmed the localization of the ipervascularised nodule and helped to define its edges. the nodule was removed through monopolar energy and the hemostasis was optimized through bipolar energy. the specimen was extracted through an endobag using one of the trocar access. results: the postoperative course was uneventful and the patient was discharged on postoperative day . histopathological examination showed an extrasurrenalic paraganglioma. conclusion: tio are often difficult to locate for surgical removal. icg fluorescent angiography allows to facilitate tio localization and removal. the minimally invasive technique decreases perioperative morbidity and mortality. laparoscopic removal guided by icg angiography should be considered when tio needs to be removed and is difficult to locate. aim: this video shows our technique to perform laparoscopic resection of a voluminous left paraaortic paraganglioma. methods: the patient is a -years-old man with a recent medical history offever, lumbar pain and haematuria.abdomen ct scan, performed during admission at emergency department, revealed a x cm left paraaortic retroperitoneal mass with pseudo-aneurysm. after procedure of angiographic embolization (with spermatic artery sparing), the patient was scheduled for a laparoscopic resection of paraaortic tumor. the patient was placed in the right flank position. three trocars ( mm) in the abdominal midline and one trocar in left hypocondrium were placed. at initial examination of the abdominal cavity, voluminous left paraaortic mass arising in the contest of left mesocolon was found, dislocating posteriorly kidney vessels. the parietal peritoneum was divided and the paraaortic lesion was dissected on the aortic plane from medial to lateral and from down to up, preserving the inferior mesenteric vessels; the mobilization was carried on to splenic vein. the vessels, supplying the mass and arising directly from aorta, was isolated and taped with vascular clips. on the inferior margin of the lesion a large vessel, probably connected with previously embolized pseudoaneurysm, was dissected with vascular linear stapler. the mobilization was completed through difficult dissection from aortic plane and mesocolic posterior surface. the colonic perfusion was verified with fluorescence angiography. specimen was extracted through an endobag.a drain was left in pelvis. postoperative day .histopathological examination showed a morphological and immunoistochemical pattern for benign paraganglioma. conclusion: laparoscopic resection of paraaortic paragangliomas is feasible by skilled surgeon. the minimally invasive technique decreases perioperative morbidity and mortality. careful preoperative planning and surgeon's experience with vascular dissection and visceral mobilization are mandatory for a good outcome. aims: posterior retroperitoneal endoscopic approach has been considered for many years as a very complex and unsafe surgical technique. often attributed to a difficult location and visualization of retroperitoneal structures. in addition, surgeons were forced to work in a small and easily altered space due to discontinuous flow with constant changes of the retroperitoneal vision. lately this approach is emerging thanks to technological advances, mainly better visualization laparoscopic cameras and high definition screens, as well as continuous flow insufflators of co , maintaining stable and smoke-free cavity uninterruptedly. methods: it shows a management of a potentially serious complication and the reproducibility of the technique through the retroperitoneal approach. results: to operate with high pressure of neumoretroperitoneum allows to contain the hemorrhage and to value with relative serenity and security, the best surgical option to repair said injury being laborious due to the reduced workspace. conclusions: the posterior or retroperitoneal approach is feasible, safe and fast. although the possibility of injuring the vena cava in right adrenalectomy remains one of the most serious and feared complications. as shown in the video, posterior retroperitoneal endoscopic approach allows repair of vascular injury correctly and safely. methods: four patients undergoing adrenalectomy, two of them with right adrenal pathology and two left. minimally invasive access, endoscopic approach, is exposed in all of them. results: in the first two surgeries, right gland is shown. initially, transabdominal approach, which requires mobilization and separation of the liver to access the retroperitoneal space and subsequent proceed to adrenal extirpation. later, right retroperitoneal approach is observed, with a meticulous sealing of the adrenal vein prior to complete the dissection of the gland, despite the small cavity created by co . in the second part, both left adrenal approaches are exposed. transabdominal pathway is necessary to mobilize left colon and spleen to access a narrow space above the upper edge of the pancreas to locate adrenal gland. this is very different in posterior adrenal approach. conclusions: posterior or retroperitoneal approach is feasible and safe, allowing access to adrenal glands, located in retroperitoneal space, without across peritoneal cavity and its disadvantages. colon and small intestine mobilization is not necessary, with a lower rate of intestinal lesions and postoperative ileus. in the same way, liver or spleen mobilization is avoided. aims: when performing a laparoscopic adrenalectomy, especially in the setting of pheochromocytoma, one of the most important steps is to gain control of the adrenal vein early on in the procedure before great manipulation of the adrenal gland. we present the case of a year old female with episodic headaches and tachycardia and severe uncontrolled hypertension, found to have elevated plasma and urine metanephrines with ct scan localizing a . cm right sided adrenal nodule. the patient was prepared preoperatively with phenoxybenzamine until mildly orthostatic with dry mucous membranes and was taken for laparoscopic right adrenalectomy. methods: after positioning our patient in left lateral decubitus, ports were placed inferior to the costal margin. the right lobe of the liver was mobilized and retracted cephalad and the ivc was exposed. careful and meticulous dissection was carried up the ivc, however no main adrenal vein was encountered. the adrenal gland was then dissected circumferentially and was removed in an endoscopic retrieval bag. there was no difficulty in hemostasis and the patient was deemed to be hemostatic prior to withdrawal of the ports and extubation. results: our patient had no issues with hemodynamic stability and her blood pressure was within normal ranges during and following the case. her hemoglobin was stable postoperatively with . immediately post op and . on discharge. her pre-op hemoglobin was . . conclusions: our video demonstrates a right adrenal gland that was congenitally missing a main adrenal vein. it is very possible that small venous branches were taken with dissection however we believe this report to be important to note in the literature for surgeons performing adrenalectomy. surg endosc ( ) aims: adrenal cysts are the most frequently identified adrenal cysts, although they are a rare entity. typically they are presented by abdominal pain or palpable mass, but nowadays, cystic lesions of the adrenal gland are more often discovered incidentally by radiologic studies. adrenal cysts have an extensive differential diagnoses, which makes a difficult definitive diagnosis and a difficulty in later management. the management of an adrenal cyst can be summarized in three fundamental pillars: discard the functional status of the cyst, evaluation of eventual malignancy by images, and avoid possible complications (hemorrhage, infection), especially in large cysts . methods: clinical case: a -year-old male patient, with no history, studied for nonspecific pain in the right hypochondrium, without other accompanying symptoms. an abdominal ultrasound was performed, a cystic lesion in hcd without being able to identify the origin was seen. complementary explorations of interest are shown (ct), the biochemical study discards functionality of the lesion, negative serology for hydatidosis. the minimally invasive approach is the gold standard in the surgical treatment of adrenal pathology, so a laparoscopic approach is proposed for this patient. aims: endometriosis is a high incidence disease (approximately % of women) with a large impact on women's quality of life and fertility. endometriosis nodules surgical treatment is necessary every time there is evidence of active disease. the aim of this video is to present a minimally invasive technique for the resection of an endometriosis nodule from the abdominal wall. methods: a -years-old woman, with past history of endometriosis and a c-section, presents at the office with a palpable nodule at the rectus abdominis left lateral border, close to the umbilical scar. she had complaints of exuberant catamenial pain and magnetic resonance imaging (mri) showed a mm nodule compatible with endometriosis depot. this technique uses trocars ( ? mm) placed at the pfannenstiel scar. stepby-step as follows: (i) dissection of the pre-aponeurotic plane and isolation of the lesion (ii) lesion excision and its removal with sac (iii) closure of the aponeurotic defect braded suture. results: the post-operative period was uneventful and the patient was discharged home at post-operative day one. the aesthetic result was excellent and the patient was asymptomatic one month after the procedure. conclusion: endometriosis of the abdominal wall is related to previous c-section, is a rare event (incidence . - . %) and usually located in the subcutaneous fat underlying the scar. the presence of nodules in the depth of the muscle is much uncommon and particularly in this clinical case, the nodule was located cm cephalad from the previous pfannenstiel scar. this technique seems easy and reproducible in the authors' opinio. aims: general surgeons often face gynecological pathological findings, either along with other abdominal pathology, or as primitive cases that need laparoscopic expertise. with this particular presentation, our goal is to demonstrate the essential laparoscopic skills and the basic operative strategy that a general surgeon should be familiar with, in order to manage such cases. the presentation is made on occasion of a woman with multiple uterine fibromatosis of the pelvis, who was treated by our team. methods: we present video fragments of the laparoscopic excisional procedure for multiple uterine fibromyomatosis of the pelvis, highlighting the proper strategy in order to conclude the operation effectively and uneventfully, in a minimally invasive fashion. results: patients with multiple, large or other complex forms of uterine or pelvic fibromas can effectively be treated with a minimally invasive approach, with minimal blood loss, very fast recovery and minimal postoperative pain and complications. % of pregnancies require emergency surgery for a non obstetric indication, including acute appendicitis, cholecystitis, adnexal torsion, choledoco-lytiasis, hernias, intestinal obstructions, oncologic pathology or other less frequent indications. laparoscopic approach is the preffered surgical option for the patologies presented above. aims: to present the technical particularities and to analyze the outcomes of the emergency operations in pregnant women operated in hospital. method: a retrospective study including all the pregnant women operated in our hospital between - was performed. the preoperative workup and the surgical indication was discussed by a multidisciplinary medical team. the anesthesic and the obstetrical risk and their management was evaluated and specifically planned for each patient. the intraoperative and post-operative outcomes were recorded. results: patients with gestational age between weeks and weeks who underwent emergency laparoscopic procedures were included in the study. out of the cases we have performed appedectomies, cholecystectomies, adnexal torsions. with a min mean operating time, we had no major intraoperative complications; the technical challenges are presented and discussed. the hospital stay was , days ( - days). no major complications were associated with the laparoscopic approach in these cohort. one pre-term labour in a weeks gestational patient was post-operatively encountered. conclusion: laparoscopic surgery can be the first option for pregnant woman with non obstetrical surgical emergencies; challenges in diagnostic, management and surgical techniques of the multidisciplinary team are expected. the objective of this presentation is to demonstrate step by step the technique to the oncologic surgeon and gynaecologist in training, including some tips and pitfalls. this is a laparoscopic transperitoneal approach in a woman with advanced cervical cancer (figo ib ) that will be treated with exclusive radio-chemotherapy. the purpose of the laparoscopic lumbo-aortic lymph node staging is to define the irradiation field. in this indication false negative in pet ct ranges from to % (depending of the existence of pelvic fixation or not). the limits of this lymphadenectomy are: both ureters as the lateral limit of the dissection, iliac bifurcation as the caudal limit and renal veins as the cranial one. since the tumour is cervical and not ovarian, both ovarian veins are not resected. in the pathologic report, lymph nodes were examined free of cancer spread. the patient have had a radio-chemotherapy with restriction of the irradiation field on the pelvis. lymphocele is a frequent complication that only sometimes needs treatment ranging from dietary changing to percutaneous drainage. if conversion to laparotomy for bleeding this technique loose its benefice but this is a rare complication. this technique is feasible and safe but requires advanced laparoscopic skills. objectives: although extremely rare, isolated splenic metastases are being increasingly diagnosed due to the improvement of imaging, survival times, and surveillance of oncologic patients. this video alerts to the growing diagnostic dilemma with primary lesions of the spleen, particularly in patients with history of cancer, and reviews the laparoscopic splenectomy 'step-by-step'. case-report: -year-old male patient diagnosed with rectal cancer (g adenocarcinoma at cm of the anal verge) after a colonoscopy for rectal bleeding. thoracic and abdominal ctscan and pelvicmri, showed a ct n lesion, without distant metastases, except for a mm suspicious splenic lesion. cea- . ng/ml. after neoadjuvant therapy, a complete response was verified at the th week post-crt with a stable splenic lesion, and a 'watch-and-wait' program was initiated with no evidence of disease at the rd month. pet-ctscan did not show active metabolic features, despite an increase in the splenic lesion. in mdt, elective laparoscopic splenectomy was proposed and afterwards performed uneventfully. with the patient in semi-right lateral tilt, we approached the spleen inferiorly by dividing the splenocolic ligament. then we continued upwards, dividing the gastrosplenic ligament and exposing the splenic hilum, which was then carefully dissected, clipped and divided. finally the splenorenal ligament was divided and the spleen was extracted within an endobag, through a small pfannenstiel incision. pathologic report revealed a splenic lymphangioma. the patient is currently under a 'watch and wait' protocol surveillance with no signs of regrowth or relapse disease after year and months of follow-up. conclusion: one out of five colorectal carcinomas are metastatic at their presentation. isolated metastases to sites other than liver, lung or axial skeleton, are extremely rare, but can be found in the spleen. although the rare splenic secondary involvement is usually associated with breast, lung, melanoma, and gynecologic malignancies, if we consider solitary splenic metastases, colorectal and ovarian carcinomas are important sources. also, imaging including percutaneous biopsy, is frequently insufficient to clarify the nature of splenic lesions. for all these reasons, the decision-making process about this issue can be a true challenge, and will probably end up with laparoscopic splenectomy. therefore, surgeons must be familiarized with a standardized technique. sarcoidosis is a multisystem disease of unknown etiology characterized by the formation of noncaseating granulomas. sarcoidosis should be considered in the differential diagnosis of lymphoid disease. indications for diagnostic splenectomy includes a suspicion of a neoplasic process. the less invasive laparoscopic approach is the gold standard. case report: a -year-old female was referred to a general surgery department to complete a study to rule out lymphoid neoplasia. followed by hematology for cytopenias. biopsy of bone marrow and adenopathies were negative for lymphoid process. patient presented ct with multiple solid ( - mm) lesions in spleen, in thorax showed no pathological changes. laparoscopic splenectomy was performed. access with optical trocar, in mammary line. triangle -mm trocars after pneumo under vision. section with ligasure of gastroesplenic ligament with short vessels and phrenic-splenic ligament. identification and preservation of pancreatic tail. section of splenic vessels at hilar level (branches) with ligasure. lower pole release. release of posterior part with gerota and diaphragm. incision by aid helps in bag without fragmenting. review of hemostasis, extraction of trocars under direct vision. intraoperative findings: spleen with normal external appearance, not megalic. postoperative evolution: satisfactory. first hours without incidents and with analytical control without anemization. tolerance and mobilization starts without incidents. the histopathology report shows granulomas formed by epithelioid histiocytes with the presence of multinucleated giant cells of the foreign body type, in some perisinusoidal granulomas the giant cells with the presence of asteroid bodies in their interior. the material has been revised with the extension of special studies. conclusion epithelioid granulomatosis, non-necrotizing, which suggests sarcoidosis. the procedure lasted min. the hospital discharge was on the next postoperative day and no complication was registered. conclusion: splenectomy can be performed in a classic way, but at present the less invasive laparoscopic approach is the gold standard. indications for splenectomy include splenic tumours of unknown origin, suspicion of a neoplastic process, and splenomegaly. sarcoidosis should be considered in the differential diagnosis for lymphoid disease. postoperative pathological examination confirms the diagnosis. week-day surgery, university, sapienza, ospedale sant'andrea, rome, italy aims: we describe an interesting case of a female patient affected by a suspected echinococcus granulosus large cyst of the spleen. methods: a years old woman complained abdominal pain and a sense of gravity in the upper left abdominal quadrant. computed tomography scan(ct-scan) showed a centimetre (cm) cyst of the spleen with thickness of the wall and contrast enhancement uptake referred to an echinococcus granulosus cyst. the sierological blood test assessment, antigens and antybody markes, for echinococcus granulosus infection was negative. a laparoscopic procedure was planned. the patient was positioned on the right flank, four trocars were inserted along the left subcostal region of the abdomen: one millimetre (mm) trocar for camera, one mm for the assistant, and two of mm for instruments. a periombelical minilaparotomy was performed for the specimen extraction. results: post-operative course was uneventful. patient was discharged in third post-operative day. istopathological exam showed a simple epithelial cyst of the spleen. conclusions: laparoscopy is safe and feaseable in case of large cyst of spleen in condition of unclear nature of the cyst. laparoscopy permits to explore the abdominal cavity and to assess the cyst characteristics in a lack overlap between the radiological exam and blood test examination. surg endosc ( ) we describe laparoscopic splenectomy for recurrent splenic cyst after laparoscopic marsupialization and partial resection of splenic cyst. the patient was a -year-old woman with abdominal discomfort and with a -cm palpable mass in the left upper and inferior quadrant. she undergone years ago in another country a laparoscopic operation for splenic cyst. abdominal computer tomography revealed a cystic lesion of the spleen with concomitant huge splenomegaly. serology and oncological marker were negative. we performed laparoscopic splenectomy for the recurrent splenic cyst. the operation took min. histologic examination of the resected spleen revealed a chronic hematoma. the patient had no abdominal symptoms during months of follow-up. postoperative long term follow-up and examination by ultrasound or computed tomography is required after surgical treatment for splenic cyst to exclude the possibility of recurrence after spleen-preserving surgery. hand-assisted surgery is a recognized technique that combines the advantages of laparoscopic approach with the tactile feedback of the laparotomic one. it proved beneficial especially for the treatment of megaspleens due to lymphoma localization, thanks to safer handling of splenic vessels, major bleeding control and more effective detachment of superior splenic pole from the diaphragmatic dome. here we show an hand-assisted splenectomy for megaspleen reaching the omolateral anterosuperior iliac spine due to lymphoproliferative disease, in which the hand, inserted through a right subcostal minilaparotomy, was very useful during the dissecting manoeuvres, the splenic artery recognition and ligation and the isolation of the superior pole of the spleen from the gastric fundus and diaphragm. in any case of huge spleens, the specimen bagging is very difficult to perform in a pure laparoscopic way, not to mention the inexistence of capable endobag; besides, a minilaparotomy would be necessary for the spleen extraction. hand-assisted approach allow to overcome this not underestimable technical difficulty, reducing operative time with similar aesthetic and functional results to that of laparoscopic approach. aim: the evolution of technology and its application to the minimally invasive surgery of the thyroid gland offers new surgical techniques, like the transaxillary approach. this new procedure is still being implemented in our environment and has recently begun to be incorporated into our surgical practice. the objective of this case is to explain step by step how to carry out a right transaxillary endoscopic thyroidectomy and emphasize in the most relevant tips to take into account. also, current indications and limitations of this technique will be addressed. methods: a -year-old woman is referred for evaluation of a right thyroid nodule without any associated symptomatology. the blood test shows normal thyroid profile. cervical ultrasound is performed identifying a . cm single right nodule with well-defined edges and presence of peripheral vascularization. no other nodules are identified. fine needle aspiracion (fna) of the nodule describes a bethesda iii. after evaluation, a right transaxillary endoscopic thyroidectomy was performed. results: dissection begins in the subcutaneous plane above the pectoralis major muscle until identification of the sternocleidomastoid muscle. dissection continues towards the prethyroid muscles in order to perform a lateral approach of the thyroid gland. section of the upper pole allows better exposure of the recurrent laryngeal nerve (rln) which is being monitored intermittenly. identification and preservation of the parathyroid glands is the next step. surgery is completed with the section of the inferior pole of the thyroid along with the istmus. the postoperative period was uneventful and patient was discharged at h after surgery. final pathology revealed a cm nodule without malignancy. conclusion: surgical treatment of the thyroid gland by transaxillary approach may be indicated in previously selected patients with benign pathology, offering the advantages from minimally invasive techniques (shorter recovery time, shorter incision length, etc.). further research is required to make a better assessment of the minimally invasive approaches in thyroid surgery. we present the video of a thoracoscopic esophageal leiomyomaenucleation. it has been widely demonstratedthe advantages of theminimally invasive approach in surgery. esophageal thoracoscopic surgery has been suggested as an alternative to open procedures, presenting less surgical trauma, lower risk of bleeding, less postoperative pain, lower wound infection and lower pulmonary morbidity, showing similar oncologic outcomes. although leiomyomas are the most commonof benign tumors of the esophagus, they are relatively rare, presenting an incidence of - per . autopsy series. in our case, the patient was diagnosed of leiomyoma located at the medium third of the esophagus. he referred a history of months of dysphagia for solid and liquids and retrosternal pain. the complementary studies were esophagoscopy, esophagography, ct and endoscopic ultrasonography. the patient was operated by a thoracoscopy approach using ports. it was completed the enucleation of the tumor following the closure of the muscular layer. methylene blue test confirmed no leaks. the patient was discharged on third day postoperative developing no incidences. pathology report: leiomyoma cm size, actin and desmin positive; s- , cd and cd negative. we want to demonstrate the advantages of a minimally invasive approach in this kind of pathology. aims: this video shows our technique to perform thoracoscopic enucleation of large esophageal leiomyoma. methods: the patient is a -years-old woman with a six months history of progressively worsening dysphagia. chest ct scan revealed a cm lesion of middle esophagus with extrinsic compression of mucosa and no increased fdg uptake on fdg-pet scan. barium swallow study showed a lateral deviation of thoracic esophagus due to extrinsic compression. endoscopic ultrasound confirmed the suspicion of esophageal leiomyoma. patient was scheduled for a thoracoscopic enucleation of esophageal tumor. she was placed in prone position and one-lung ventilation was employed. three trocars were placed in intercostals spaces on right hemithorax. azygos vein was identified and transected between vascular clips. esophagus was circumpherentially isolated from mediastinal structures. after myotomy, the lesion was dissected from submucosal-mucosal layer. since air leak test excluded injury of internal layer, muscular layer was closed with a continuous suture. the specimen was extracted through an endobag. a drain was left in place. results: the postoperative course was uneventful and the patient was discharged on postoperative day . final pathological examination confirmed esophageal leiomyoma. conclusion: thoracoscopic surgery in prone position allows removal of large esophageal tumor with several advantages. the minimally invasive technique decreases perioperative morbidity and mortality. introduction: spontaneous esophageal perforation is life threatening disease and requires emergent surgical treatment. recently, the efficacy of minimally-invasive surgery such as laparoscopic and thoracoscopic surgery for esophageal perforation has been reported. we report a novel technique of minimallyinvasive abdominal and left thoracic approach (malta) for spontaneous esophageal perforation. case presentation: -year-old male, who had been under hemodialysis due to iga nephropathy, complained of chest pain after vomiting several times. since the ct scan showed left hydropneumothorax and pneumomediastinum, and the gastrografin study demonstrated extravasation from left side of esophagus, we diagnosed him with the spontaneous esophageal perforation and planed emergent surgery. the patient was placed in the reverse trendelenburg position, and the legs were split, with the left side of the upper body lifted in order to perform thoracoscopy and laparoscopy simultaneously. first, we explored the thoracic cavity through a mm port in the left th intercostal space and added other ports. we identified the rupture site mm in size on the left wall of the lower esophagus and sutured the mucosa and the muscle layer with a running suture respectively. we covered the perforation section with pericardium fat and irrigated the cavity with physiological saline. then transferred to the abdominal cavity, no contamination was found in the abdominal cavity. a feeding tube was inserted into stomach through the round ligament of the liver and the operation was completed. the total operative time was min and the amount of intraoperative bleeding was ml including pleural effusion. postoperatively, the patient experienced left empyema pleurae but no other severe complications and was discharged on postoperative day . conclusion: we experienced a rare case of spontaneous esophageal perforation of a patient under hemodialysis. malta is an effective procedure for emergent esophageal operation because of great visual field of the chest and abdominal cavity without expanding contamination. introduction: digestive caustic injury is associated with high morbidity and mortality with stenosis in the long term. surgical treatment involves resection of the esophagus and reconstruction with the stomach, colon or jejunum. coloplasty provides several advantages but its vascularization is complex and involves anastomosis. classically, vascular assessment was achieved by palpation through laparotomy and color evaluation. indocyanine green (icg) allows a minimally invasive intraoperative angiography in real time. methods: a -year-old female with medical history of caustic ingestion and subsequent esophagogastric stenosis, carrier of feeding jejunostomy. . thoracoscopy (prone position): dissection of the esophagus from the hiatus to the upper thoracic inlet. . laparoscopy: patient in the supine position, placement of five trocars. total non-oncological gastrectomy, post-pyloric section of the duodenum and omentectomy were completed. mobilization of the righ, transverse and descending colon. measurement of the transverse colon with a tape (distance from the neck and the esophageal hiatus). individualization of the righ, middle (with its branches) and left colic arteries and placement of clamps at the right colic, right branch of the middle colic and left colic arteries. cc of icg were injected allowing for an assessment of the colon vascularization. section of the right branch of the middle colic artery. proximal section of the ascending and distal colon near the splenic angle, preserving the marginal arch. silk point to join the staple line of the descending colon and the pylorys. side-to-side mechanical antiperistaltic anastomosis between the distal endo of the coloplasty and the jejunum. finally an anastomosis between the ascending and descending side-to-side mechanical anastomosis using an assistance incision in the left flank was performed. . cervical dissection: extraction of the surgical especimen under laparoscopic control. vascular assessment with icg is performed before and after the side-to-side anastomosis is performed. results: there were no intraoperative complications. the patient was discharged on postoperative day . discussion: we describe the first case of total minimally invasive colonic interposition with icg assessment of the vascularization. this technique, although technically demanding, avoids the drawbacks of the open surgery and allows for a precise assessment of the vascularization of the graft. surg endosc ( ) introduction: large pedunculated fibrovascular polyps are uncommon, mostly benign, intraluminal massess, usually located in the upper esophageal tract. most frequent reported clinical manifestation is dysphagia, followed by regurgitation, chest pain and intestinal bleeding. ct scan, and mri are the key in the diagnostic work-up revealing a sausage-shaped intraluminal mass. endoscopy with ultrasonography and biopsy add important information for the diagnosis and pedicle location. surgical excision is deemed due to potentially life-threating complication related to airway obstruction. the most frequent polyp resection is performed through cervical esophagotomy or by direct esophagectomy. however, this approach is related to a high morbidity and mortality rate. in the last years, few excisions have been reported by a endoscopical approach with a lower post operative complication. material and methods: this video shows the surgical steps of a trans-oral endoscopic surgical resection of a giant ( cm) pedunculated polyp in a year old man. the procedure was performed under general anesthesia. a flexible endoscope probe was used and the distal end of the polyp was extracted through the oral cavity with a loop. the endo-gia stapler was used to cut the base of polyp and finally removed. the anatomo-pathological study confirmed the diagnostic of a fibrovascular polyp with no evidence of malignancy. results and conclusions: the patient had an uneventful recovery with no recurrency at years of follow up. this minimally invasive approach is a safe and feasible procedure to treat large esophageal fibrovascular polyps avoiding the complications related to more aggresive procedure. introduction: leiomyomas are the most common mesenchymal tumors affecting the esophagus and they usually grow in the mid to distal third of it. they tend to be asymptomatic, but sometimes they can grow to enormous size and produce dysphagia. case report: -year-old male asymptomatic patient was referred to our hospital due to an incidental finding. ct scan revealed a x mm rounded submucosal tumor on the dorsal side of the lower third of the esophagus. upper gastrointestinal endoscopy revealed a cystic lesion in the lower esophagus cm from the incisor teeth, with normal overlying mucosa. an endoscopic-ultrasound-guided fine-needle-aspiration of the mass was performed, which was reported as a likely leiomyoma.conservative treatment was performed, no growth was detected during eleven years of follow-up. but it became symptomatic, the patient complained of progressive dysphagia caused by compression so surgical resection was decided.laparoscopic enucleation of esophageal leiomyoma was performed. the tumor was reached by transhiatal dissection. a careful dissection of the mass was performed, preserving the vagal branches. an intraoperative endoscopy was performed to verify the integrity of esophageal mucosa and that the tumor was completely resected. the muscular layer was sutured after enucleation using absorbable suture material and the hiatus was closed with non-absorbable suture material. a dor fundoplication was also performed. a swallow test with a water-soluble contrast was obtained on postoperative day one. no pathological findings were found so the patient was asked to drink.histopathological exam revealed a tumor measuring mm mm mm consistent with leiomyoma.the procedure lasted min. the hospital discharge was on the third postoperative day and no complication was registered. conclusion: surgical excision is the mainstay of treatment and is recommended for symptomatic leiomyomas and those greater than cm. this case report demonstrates the technical feasibility, safety and minimal postoperative morbidity associated with minimal invasive esophageal surgery. introduction: total esophagectomy by means of minimally invasive surgery has proven to be a valid and effective alternative for performing this procedure. however, this procedure is not implemented in most centers. objective: demonstrate the technique of a total esophagectomy by endoscopic surgery for a benign esophageal stenosis. material and methods: clinical case: a -year-old female patient diagnosed with double esophageal peptic stenosis, treated on several occasions with endoscopic dilation by digestive, showing in the last endoscopy: severe esophagitis with stenosis impassable to cms. additional tests of interest are exposed. resolved: intervention: right thoracoscopy in prone position, dissection and complete mobilization of the thoracic esophagus, section of the azygos vein, pleural drainage. laparoscopic time, trocars, gastrolysis respecting the right gastroepiploic vessels, broad kocher until the cava is identified, vascular section of the vessels left gastric, full mobilization of the stomach, subxiphoid minilaparotomy, beginning of the cervical time with dissection of the cervical esophagus, section and fixation of this to a tube, externalization of the piece by abdominal route, creation of the gastric tubular with successive loads of gia, ascending posterior mediastinal plasty with manual esophago-tubular anastomosis, with placement of drainages and feeding jejunostomy. right operative with radiological control with gastrografin on the th day, discharge from hospital on the th day. asymptomatic one year after surgery, with radiological control without alterations. conclusions: the approach of esophageal peptic stenosis with minimally invasive surgery is safe and effective, adding the advantages inherent to this type of technique. (figs. , ) , showing a cystic lesion in the gastric submucosa with a well defined, medial and superior to the lesser curvature of the stomach with exophytic growth. it causes extrinsic compression of the cardia in the gastric body. within the differential diagnosis are gastric duplication cysts or gastrointestinal stromal tumor. a biopsy was taken, discarding the presence of neoplastic cells. finally, a study of digestive transit showed extrinsic compression at the cardial level, which causes difficulty in passing contrast. a laparoscopic approach was performed beginning with the dissection of the abdominal esophagus. the presence of a cystic lesion on the anterior face of the abdominal esophagus was identified (figs. , ) . we proceeded to the complete resection of cyst. the surgery was completed with °f undoplication anterior dor. the patient went home on the rd day without incident. results: duplication cysts are congenital malformations of the gastrointestinal tract contiguous with the esophagus, which can communicate with the esophageal lumen. most are diagnosed in childhood, but when it is diagnosed in adults, they used to be symptomatics. it is more frequent in men. although the pathogenic mechanism is unknown, it is caused by an anomaly during embryonic development. they are located in the thoracic esophagus, at the level of the lower and posterior mediastinum and, less frequently, in the abdominal esophagus, as in our case. they can give digestive symptoms(epigastralgia, vomiting) or respiratory symptoms. the diagnosis is made with eda, ct, and ecoendoscopy of choice, although it may be incidental. the treatment of choice in symptomatic patients is complete resection or cystic enucleation. in asymptomatic surgical is not defined, because it can cause complications, and, malignization due to degeneration is very infrequent. conclusion: the most of duplication cysts are diagnosed in childhood, although it's more frequent in adults to be symptomatic. surgical treatment can cure this disease. however, the choice between these becomes difficult in young patients, where the low incidence does not allow get series of long patients and decisions must be based on results achieved in adults. objetivos: to demonstrate the safety and efficacy of the laparoscopic approach in this infrequent pathology, pointing out the importance of having standardized the procedure to achieve better results. material and methods: case report: a -year-old man with progressive dysphagia until almost complete afagia, with clinical, endoscopic, radiological and manometric diagnosis,compatible with typical primary achalasia. chagas negative serology,we show the complementary studies of interest.dilatation is not performed, preoperative symptomatic treatment with calcium channel inhibitors. intervention: laparoscopic approach, trocars, aberrant left hepatic artery with signs of severe esophagitis,opening of the gastroesplenic-hepatic ligament, no retroesophageal window, dissection of the hiatus and inferior mediastinal, preservation and mobilization of the left hepatic artery and the anterior vagus,meticulous disection of the cardia,standardized myotomy: first proximal cms. with adequate simultaneous traction of both edges of the myotomy, then distal myotomy including - cm, including selectively the distal oblique fibers of les, tutorization with fouché and methylene blue to confirm good step and absence of leakage, dor-type funduplication, pts on each side, fixed to both pillars, hiatalmediastinal drainage. egd on the st day of normal po, dischargeat rd day, asymptomatic and with normal radiological control at year of age. conclusions: laparoscopic mh should be the first therapeutic option, in patients with primary achalasia, even in young patients. the length of myotomy, especially distal to ueg is one of the most important aspects of surgery, most authors (pellegrini) recommend that the myotomy extend - cm in the stomach, even up to cm below the ueg to achieve an effective disruption of the eei.the standardization of the procedure is fundamental to increase safety and effectiveness in these more complex cases. aims: the surgical treatment of giant hiatal hernias is a complex and demanding procedure, not only in terms of performing the operation in a minimally invasive abdominal fashion by avoiding thoracic approaches, but also concerning the management of large hiatal defects which contribute to high recurrence rates. our aim is to present our surgical technique for the reconstruction of such hiatal hernias, exploiting the benefits of the robotic approach and also to highlight the technical aspects of non-absorbable mesh placement in order to bridge effectively the hiatal defects. methods: we present video fragments from a procedures selected from a series of cases of robotic reconstruction of giant hiatal hernias performed by our team, in which a non-absorbable meshes were utilized to restore the hiatal gap. we emphasize on the clear benefits of robotic surgery in these cases and on the strategy of how to avoid high recurrence rates. results: all of our patients, who underwent reconstruction of giant hiatal hernias with this particular technique, experienced very good early post-operative results, very short hospital stay and no recurrence in a -month follow-up. conclusions: the robotic approach for the treatment of large hiatal hernias offers great advantages to both surgeons and patients, by eliminating the restrictions of conventional laparoscopic surgery, minimizing intra-operative incidents and post-operative complications. large hiatal defects are very effectively closed with the use of advanced suturing techniques and non-absorbable meshes in a tension-free bridging fashion. aims: mckeown esophagectomy is commonly used for invasive esophageal carcinoma. as the morbidity and mortality rates for esophagectomy are persistently high, minimally invasive esophagectomy in prone position is expected to reduce respiratory postoperative complications. there is still limited experience for the use of minimally invasive approaches in patients undergoing surgery after neoadjuvant chemoradiation and many concerns about the feasibility, safety, and oncological outcomes of these procedures are still present. methods: we present the case of a -year-old female with a middle third esophageal squamous cell carcinoma, who received neoadjuvant chemoradiation. she underwent laparoscopic and thoracoscopic (prone position) mckeown esophagectomy with hand-sewn esophagogastric anastomosis through a left lateral cervical incision. results: the operation was completed successfully, with no conversion to open surgery. the operative time was h with minimal blood loss and the patient was fed on day and discharged on day post-op. r resection was achieved and the number of total harvested lymph nodes was ( positive nodes, n ). conclusions: minimally invasive mckeown esophagectomy in patients with esophageal cancer and prior chemoradiation is feasible and safe procedure with acceptable oncological outcomes. results: preliminary results demonstrated that minimally invasive ivor-lewis esophagectomy procedure, provided of a better postoperative pain control and less respiratory complications. in order to standarise our procedure, the video shows how three different types of esophagogastric anastomosis are performed, depending on the patient characteristics, anatomical factors and safety and comfort for the surgeon: manual termino-terminal, mechanical termino-terminal and mechanical latero-lateral. conclusions: in our way to standardization, we are still looking for the best type of anastomosis, even though, we find out that, manually performed anastomosis are easier to performed, when the section in esophagus is lower, involving medium and inferior third. in the other hand, mechanical termino-terminal anastomosis seemed to be an ideal option for upper sections. more studies are needed in order to standardized one anastomosis, for all cases. because esophagectomy with radical lymphadenectomy is highly invasive, thoracoscopic esophagectomy (te) is attracting attention as a less invasive procedure. we first performed te with the left decubitus position in . in we developed a hybrid of the prone and left lateral decubitus positions for te, and a total of patients underwent te with a hybrid position . we introduced te with a hybrid position for the following three reasons: ( ) mobilization and lymphadenectomy around the middle and lower esophagus are easier in the prone position. thanks to artificial pneumothorax and the gravity, the middle and lower mediastinum are opened, and which give us good surgical field. ( ) lymphadenectomy along the left recurrent laryngeal nerve (rln) is more reliable and precise when performed in the left lateral decubitus position. we can dissect lymph node around the rln higher position in the upper mediastinum. ( ) unexpected events requiring conversion to thoracotomy (e.g., massive bleeding, injury of other organs, dense intrathoracic adhesion, resection of adjacent organs) are easier to deal with in the left lateral decubitus position. the patient is fixed on the operating table with the semi-prone position and we can easily change patient positions from the left lateral decubitus position to the prone position and vice versa using rotation system of the operation table. the upper mediastinal procedure including lymphadenectomy along the right and left rln is performed with the patient in the left lateral decubitus position, while the middle and lower mediastinal procedures are performed with the patient in the prone position with artificial pneumothorax ( mmhg). theabdominal procedures wereperformed by hand-assisted laparoscopic surgery (hals) and gastric tube reconstruction through aposterior mediastinal route was performed as a standard surgical procedure in our institution. the magnifying effect of thoracoscope enables us to perform more precise surgery and preserve nerve and vessels, and a hybrid position is thought to be feasible and effective methods. ivor lewis esophago-gastrectomy is a standard procedure for the treatment of distal esophageal cancer. among the years, the surgical community standardized the mininvasive abdominal phase. the thoracic phase is much more complex because usually all surgeons get in trouble in the phase of esophago-gastric anastomosis. in fact, is still very difficult and tricky to perform a mechanical circular anastomosis due to problems with the correct handling of the circular stapler through the minitoracotomy and is also difficult to place the anvyl in the proximal esophagus. the linear anastomosis (side-to-side) is a little bit easier but not so effective as the circular anastomosis in terms of leak rate. we think that robotic approach with its endowrist can allow us to overcome these limits and that a tailored double layer hand sewn esophago-gastric anastomosis could be the right choice. we treated patients with this approach and they were all uneventful in the post operative period except for a case of chylothorax we treated successfully with lipiodol injection in inguinal lymphnodes. we need more cases to analyze the technique in terms of leak rate and major complications but we think this is a promising and cost-effective procedure for robotic approach. aim: anastomotic leakage is one of the most dreaded complication after esophagectomy. indocyanine-green near-infrared angiography (nir-icga) intraoperative use has been recently introduced for visceral perfusion evaluation. in this video we present our technique for gastric conduit fashioning according to the nir-icga blood supply evaluation in a total minimallyinvasive ivor-lewis esophagectomy. methods: a years-old man affected by a siewert adenocarcinoma (ct , n , m ) underwent a preoperative neoadjuvant treatment according to cross protocol. at restaging ct-scan no more pathologic nodes were evident (ct , n , m ). the patient was submitted to a total minimally-invasive ivor-lewis esophagectomy. surgical procedure: after pneumoperitoneum induction and trocars insertion, the lesser omentum was opened and a lymphadenectomy at stations a, , , and performed. the esophagus was dissected at diaphragmatic hiatus with lymphadenectomy at station , and . the larger omentum was opened along the right gastroepiploic arcade, that was preserved, the short gastric vessels divided and gastric fundus mobilized. by evaluating the presence of an intense fluorescence at nir-icga, a tailored partial tubulization of the stomach was performed with multiple linear stapler firing. a right thoracoscopy was performed through trocars. the azygos vein ligated and divided. the mediastinal pleura was opened and the esophagus was dissected entirely with an en-bloc excision of nodes at stations , and . nodes at stations , , and were removed separately. the esophagus was sectioned above the azygos vein level and a purse-string fashioned. the cardia and the gastric tube were pulled up and a minithoracotomy performed. a new nir-icga was repeated to verify the good blood supply and tailor the site of anastomosis on a well perfused area. the stomach was opened and a circular stapler inserted. after the end-to-side esophago-gastric anastomosis fashioning, the tubulization was completed by linear stapler firing and the specimen removed. results: the post-operative course was uneventful and the pathologic examination revealed a cardial adenocarcinoma (ypt ,n , r ). conclusions: nir-icga is an interesting and easy-to use tool for surgeons. nevertheless in literature is still not clear which is the best parameter to measure the blood supply. large studied are needed. aim: uses and application of indocyanine green (icg) fluorescence in the field of surgery are growing exponentially. the safety and feasibility of its usage has been proven in several areas and various pathologies of surgery and surgeons are starting to incorporate it into their common practice. however, there are still several aspects to define regarding this technology. we present different uses of icg in the specific area of esophageal cancer. methods: we used icg fluorescence at different moments of a two-field minimally invasive esophagectomy. first of all, peritumoral injection of icg may offer a lymphatic mapping, both in the abdominal phase of the surgery and the thoracic one, improving lymph node dissection by allowing a more targeted and less morbid approach that includes all relevant nodal stations. at the moment of the gastric section, intravenous injection provides assessment of gastric conduit perfusion, therefore optimizing the construction of the graft to avoid the inclusion of poorly perfused areas that may increase the risk of leak of the anastomosis. besides that, the esophagogastric anastomosis can be tested in the thoracic phase of the operation in order to check an adequate perfusion and prevent further complications. results: we consider that icg fluorescence is a promising technology that could be easily introduced in the surgical routine of the esophageal surgeon as an instrument to assess the anastomosis perfusion. icg is also feasible in detecting lymph node drainage from the esophagus, although its technique of application needs to be defined. conclusions: icg fluorescence has opened a new world of possibilities in all the different surgical specialties. its use in the esophagectomy is safe, simple and feasible. in a near future, its application to esophageal cancer surgery could improve survival by predicting and preventing anastomotic leak and guiding in a tailored lymphadenectomy. further research is needed to demonstrate these promising applications. introduction: the oesophagectomy is currently still mandatory in the curative treatment of the malignant oesphagic pathology. this procedure is defined by important morbidity and mortality. the minimally invasive approach aims to reduce the complications without repercussion on the oncological outcomes, however it's not exempt from them being a demanding surgical technique like it is. aim: we present the video of three complications after a three-stage oesophagectomy (mckeown-like) with the thoracic stage via thoracoscopy and the minimally invasive surgical solution for both of them. methods: all three cases represent a three-stage oesophagectomy for malignant esophageal pathology. the first one was a -year-old male who suffered an intraoperative left main bronchus injury. the second case was a -year-old male with no intraoperative complications whatsoever. nonetheless, on the second postoperative day, milky drainage started to appear through the thoracic tube. the third and final case represents an intraoperative hemorrhage, which is the most common complication of this kind of surgery. results: the first case was diagnosed and treated intraoperatively with the use of an adhesive matrix. during the postoperative period the patient showed no further complications. the second case was a chylothorax, diagnosed on the second postoperative day. it was treated initially with conservative measures. due to bad evolution, he underwent surgery on the tenth postoperative day. we can see how we ligated the stump of the thoracic duct in the original surgery and then how we repaired the unexpected leak. after the second surgery, the patient was discharged on the sixth day. the last patient was also diagnosed and treated intraoperatively successfully, with no repercussion whatsoever in the postoperative time. conclusions: the minimally invasive surgery has many advantages in the upper gastrointestinal field. it is a demanding technique, so it is important to be able to treat the complications that may arise with this approach. surg endosc ( ) aims: the use of icg fluorescence is incrising in surgery, mainly as a test of vascular supply in colonic anastomoses. during the last years, other potential uses have been described, such as the identification of the sentinel lymph node and lymphatic mapping in oncological surgery. these advances could allow a better staging in order to decide the most appropriate treatment to each patient. gastric cancer is one of the fields where this could play a key role in the near future. we present a case of a patient who underwent a laparoscopic total gastrectomy with icg-guided d lymphadenectomy, where a personalized lymphatic mapping was performed. methods: a -year-old male patient underwent gastroscopy for gastric discomfort, and a gastric carcinoma was detected at the greater curvature of gastric body. endoscopic biopsy was informed as diffuse type gastric adenocarcinoma. the preoperative staging was completed with echoendoscopy and ct-scan (t bn m ). we decided to perform a laparoscopic total gastrectomy with icg-guided lymphadenectomy. the preoperative day, a gastroscopy was performed to inject . mg of icg in four submucosal areas around the tumor. results: intraoperatively, the lymphatic mapping marked by icg was checked, allowing the identification of the territory of drainage of the tumor to lymph nodes at the lesser curvature, the greater curvature and the splenic artery. a d lymphadenectomy and a total laparoscopic gastrectomy with roux-en-y reconstruction was performed. during the lymphadenectomy, we were able to observe marked lymph nodes in territory , and also observed that the paraaortic lymph node behind the celiac trunk did not become green and the lymphadenectomy at this area was not continued. the patient presented no postoperative complications, and was discharged on the seventh postoperative day. the histological results showed a diffuse type gastric adenocarcinoma pt n and isolated lymph nodes, being one of them possitive (corresponding to the adenopathy marked at the greater curvature). conclusions: lymphatic icg-mapping in gastric cancer is a potential revolutionary advance that could ensure a correct lymphadenectomy, avoiding lymph node understaging. it is necessary to continue carrying out studies that will allow developing protocols to define appropriate lymphadenectomy based on icg-mapping. introduction: petersen's hernia is one of severe postoperative complication after gastrectomy, which may result in massive resection of small intestine. it is considered an essential proscedure to close the petersen's defect for all cases after such reconstruction after gastrectomy as roux-en y method. we report a case of petersen's hernia after radical gastrectomy, which was repaired laparoscopically. patient: the patient was -year-old male, who underwent laparoscopic distal gastrectomy for gastric cancer (d lymph node dissection followed by roux-en-y reconstruction) two years ago. the closure of petersen's defect was not performed in the initial operation. he was aware of abdominal pain and visited emergency unit in our hospital. abdominal ct scan showed internal hernia of petersen's hernia. surgical procedure: in laparoscopic examination, dilation of small intestinal and mastoid ascites was observed. massive small intestine including y-limb entered into petersen's defect from left to right side. we carefully pulled through the small intestine and confirmed absence of ischemic change in the whole small intestine. then the petersen's defect was closed by continuous suturing with - non-absorbable barbed suture. results: the operation time was min and the estimated blood loss was ml. oral intake was started from the next day of the operation. there was no postoperative complication. the patient was discharged on the th postoperative day. conclusion: we could safely perform laparoscopic repair for petersen's hernia. regarding technical points in the procedure, it is important to judge the direction of the small intestine into the petersen's defect, to manage the dilated small intestine gently, and to close the petersen's defect by laparoscopic suturing. introduction: in this case we present a -year old male with a history of morbid obesity, sleep apnea and psychiatric affliction including alcohol and nicotine abuse. in he underwent a laparoscopic roux-en-y gastric bypass. the results were satisfactory, with no complications post-surgery, and a steady weight loss over time (pre: kg, post: kg). in november , he presented with complaints of dysphagia and weight loss ( kg in months). laryngoscopic examination by the otorhinolaryngologist was negative. he was referred to gastro-enterology for gastroscopy. biopsies showed a mildly differentiated adenocarcinoma of the gastro-esophageal junction with submucosal invasion. objective: after negative staging assessment, multiple treatment options were considered. the route of choice ended up being a laparoscopic radical gastrectomy with esophago-jejunostomy, with the objective to achieve optimal oncological results. methods: the procedure is demonstrated in this video. the gastric pouch as well as the remnant stomach, greater and lesser omentum were resected laparoscopically. due to the invasion of the carcinoma into the distal esophagus, a segment of the esophagus was resected as well. following anatomopathological examination on frozen section, the resection margins were reported malignancy free. results: postoperatively, there were no complications. ct scan with contrast showed no signs of leakage. anatomopathological examination confirmed the tumor to be a mildly invasive and poorly differentiated adenocarcinoma with local signet-ring cell differentiation (pt bn ). there was no need for adjuvant therapy. oral intake was sound. conclusion: adenocarcinomas of the gastric pouch are rarely seen following gastric bypass. this patient presented with complaints of dysphagia, and an adenocarcinoma was diagnosed. consequently, the patient had a total gastrectomy at our hospital. the surgery was performed laparoscopically, and was executed with success. to conclude, it is feasible to treat adenocarcinomas after gastric bypass laparoscopically via total gastrectomy and omentectomy. year old, female patient presented with upper abdominal discomfort and microcitic anemia. an ulcerative lesion was found on gastroscopy examination in body of the stomach (near the grate curvature). biopsy was done and pathology result showed poorly differentiated adenocarcinoma. chest computed tomography (ct) was without any significant findings. abdominal ct showed the lesion in stomach without enlargement of regional lymph nodes. her blood laboratory examinations were within normal limits, including serum cea. patient underwent laparoscopic total gastrectomy with modified d lymphadenectomy and roux-en-y esophagojejunostomy. total operating time (ort) was min. three days after operation, patient has developed none st elevated mi and respiratory failure. she was intubated. on day after operation she was extubated, on day patient started regular diet and was discharged home on day . final pathology result confirmed poorly differentiated adenocarcinoma of the stomach. this video shows our favourite technique for laparoscopic d subtotal gastrectomy. we usually perform the procedure with or trocars; after the coloepiploic detachment we perform the gastric transection first! this manoeuvre provides a perfect view for the lymphadenectomy. at the end of our dissection we transect the duodenum with seamguard reinforcement. before going on with the reconstructive phase, we prepare the roux en 'y' with double loop technique, usually without dividing the mesentery. then we remove the specimen through a periumbilical - cm minilaparotomy; we think it's important anyway to check margins and distance from the tumor before going on with the reconstructive phase. from the same minilaparotomy we retrieve the prepared limb for roux en y and we perform a side to side mechanical linear anastomosis outside.then we proceed with performing the anastomosis between the gastric pouch and the alimentary limb by laparoscopy. we like very much this technique for the increased exposition of tissues during lymphadenectomy. laparoscopic roux en 'y' d surg endosc ( ) a body-tc was performed in which axillary, mediastinal adenopathies and images suggestive of hepatic metastases were identified. the biopsy confirms a gastrointestinal stromal tumor. the case discussed in a multidisciplinary committee and the pet-ct study was completed, subcardial gist t n m was diagnosed. neoadyuvance was decided with imatinib for one month and surgery was performed using a laparoscopic approach. the approach was performed with trocars ( mm supraumbilical, two mm subcostal left and right, mm subxifoid and mm left flank). gastrectomy was performed with d lymphadenectomy following the oncological principles of subcardial tumors. the piece was removed in a bag by extending the mm port to mini-laparotomy. esophagogastric anastomosis was performed by hand assisted circular mechanical suture. methylene blue test was carried out. no nasogastric tube left, but drainage tutoring the esophagogastric anastomosis was left. results: the postoperative evolution was favorable. oral tolerance without incidents at fourth postoperative day. the patient was discharged without incidences on the seventh postoperative day. the pathological study of the piece was reported as subcardial gastrointestinal stromal tumor cm with respected surgical margins and lymph nodes free of malignancy, postoperative diagnosis of t n m . one month after surgery, the patient has adequate oral tolerance. she does not report gastroesophageal reflux and at months remains asymptomatic and with good evolution. conclusions: laparoscopic proximal gastrectomy is a technique that is not currently used but can be performed through a laparoscopic approach. it is a safe technique with good clinical and oncological results, especially in the early gastric cancer and gastrointestinal stromal tumors. however, long-term studies are necessary. laparoscopic gastrectomy is a perfectly safe option nowadays for the treatment of gastric cancer. every year the percentage of the laparoscopic approach is rising not only in the east but also in the west. we present a case of a year old female patient with a gastric tumor of the antrum-g adenocarcinoma with a ct n m staging. we perform a subtotal laparoscopic gastrectomy with a d lymphadenectomy and roux-en-y anastomosis. the patient begin clear liquids on the first post operative day and was discharged on the th. the final anatomopathological result of the specimen was a adenocarcinoma (g )-pt n . there were nodes resected all negative. the case was discussed in multidisciplinary team and was decided for clinical follow up with no further treatments. the patient was evaluated one month after surgery with no complaints and will continue the follow up. upper gi surgery, university of verona, verona, italy laparoscopic endoscopic cooperative surgery is an option in medium size submucosal cancers invading the muscular layer, mainly in border area were wedge resections are nor feasible.in this video we report a case of prepiloric gist treated with news technique (nonexposed endoscopic wall-inversion surgery).we think that this technique is feasible and safe and should be considered a valid option with a view to preserving the organ. aim: laparoscopic wedge resection or partial resection is a safe and feasible stomach preserving approach to gastric submucosal tumors (smt) such as gastrointestinal tumors (gist), and it has been widely performed recently. however, it should not be applied to the tumors at cardia in order to avoid stenosis or disruption of anti-reflux mechanism. we have introduced percutaneous endoscopic intragastric surgery (peigs) for smt at cardia since to preserve function of cardia. we will report the tips, techniques, and clinical result of our peigs. methods: from september to august , seven patients with smt at cardia underwent peigs in our hospital. we insert the mm port at umbilicus and investigate the abdominal cavity at first. then the incision is extended to . cm and lap-protector tm is equipped with the site to perform mini-laparotomy. using peroralendoscopy, a stomach was insufflatedand incision is made at an anterior wall of gastric body under direct vision. additional lap-protector tm is placed into the stomach so that the stomach is fixed on the abdominal wall. it enables us to keep direct access to gastric lumen and stable operative field. ez access tm is attatched on lap-protector tm , and intragastric operation is started. subsequently, two trocars are inserted using funada's gastropexy instrument. tumor is dissected by using energy devices, avoiding injury of capsule of the tumor. the defect of the gastric wall is closed by intragastric suturing. we should take particular care not to damage egj during the suturing, inserting peroral endoscopy in and out. results: the mean operation time was ( - ) min and the amount of intraoperative bleeding was . ( . - . ) ml. the maximum diameter of tumors was ( - ) mm. one case was low risk gist and otherwise were leiomyoma. the postoperative course was uneventful in all cases, without leakage or stenosis. total hospital stay was ( . - ) days. no patient had symptoms of esophagitis. conclusions: peigs for smt at gastric cardia is a feasible and safe approach, preserving function of cardia. our procedure achieves great stability and excellent visualization during the operation, which may have led to the fine surgical results. laparoscopic lymphadenectomyin gastric cancer is considered a feasible and safe procedure. data on the compliance of lymphadenectomy in the various lymph node stations is not yet well understood; moreover it is not clear if there are particular conditions relate to the patients impairing the oncological results. this video reports the use of the icg for the lymph node dissection of station number in a case of obese patient and a case of a cirrhotic patient. fist patient, m.a., was a year old man with distal cancer ct n and a bmi. second patient, t.d., was a year old man with distal cancer ct n and a alcoholic cirrhosis child b . in both cases, intraoperative endoscopy was performed to min before the onset of lymph node dissection. . mg of icg was injected into the submucosal layer in quadrants of the primary tumor. a laparoscopic subtotal gastrectomy was conduced with d lymphadenectomy. lymph note navigation were analyzed by novadaqÒ detector. using navigation system we removed the n basin. in both cases dissection were effective and pancreatic surface were easily detectable. number of lymph nodes retrieved was in the case of obese patient and in the case of cirrhotic patient. pathological tnm were pt n ( / n ?) in the first case and pt n ( / n ?) in the second. no n ? metastases were detected in n station for both cases. no pancreatic fistula was recorded. icg lymph node navigation system should be considered a valid support for the surgeon for completion of a correct lymphadenectomy in surgical challenging cases. aims: morgagni hernia is the rarest of congenital diaphragmatic hernias ( - %). its presentation is rare in adults and its finding is usually incidental. it was first described by giovanni b. morgagni in . it is located in the anterior region of the diaphragm. it is caused by a congenital defect in the fusion of the transverse septum of the diaphragm and the costal arches. the need for surgery depends on the presentation, it is recommended early repair before the development of complications. classically, the surgical approach was thoracotomy or laparotomy. currently, the tendency is to use a minimally invasive approach, which has shown good results, lower morbidity and faster recovery. the need to repair the defect with a mesh is controversial, recommended when it is not possible to close the defect without tension. the objective of this video is to demonstrate the safety and efficacy of the laparoscopic approach for the repair of this type of hernia, as well as the different types of mesh that can be used. aims: the treatment of the non-metastatic gastro intestinal stromal tumour (gist) is the surgical resection [ ] . the duodenal gastro-intestinal stromal tumour (gist) is a relatively rare clinical entity. from all the resected gist, only % are duodenal [ ] . the clinical presentation could vary from the most common acute gastro intestinal (gi) bleeding, chronic anaemia, but also acute abdomen caused by tumour rupture and intestinal obstruction. methods: a years old patient present at the emergency department of the chi poissy-st germain-en-laye (paris, france) with acute gastrointestinal bleeding. at the laboratory exams the haemoglobin was . g/ dl. the patient perform a computer tomography (ct) which shows two hyper vascularised lesion at the th duodenum; this lesions has an intra and extraluminare growing. the ct scan didn't show any other abdominal lesions. the patient were submitted to a minimally invasive surgical operation with the multiport laparoscopic technique. results: the resection of the th and th duodenum and of the first centimetre of jejunum was performed with a four trocar laparoscopic technique. a latero-lateral duodeno-jejunal mechanical anastomosis was performed. the operative time was min. the patient start with oral alimentation on the third post-operative day after a ct scan with oral contrast that was negative for anastomotic dehiscence and collections. the post-operative course was globally uneventful and the patient was discharged at fifth post-operative day without complications. the histological examination shows a low risk gist, cd positive and with a ki- inferior of % (classification tnm th edition pt aims: gastric volvulus are a rare condition that sometimes represent a diagnostic challenge for surgeons. here we present the video of a recent case of a gastric volvulus in our area that was treated with a minimal invasive approach. methods: we report the case of a -year-old woman who was admitted in the emergency room (er) with epigastric transfixing pain and impossibility to vomit that had started h prior to the admission. the physical exam showed good vital signs, and her abdomen was soft, with a tendency to tenderness with palpation in the epigastrium without guarding or rigidity. her lab tests were normal and the conventional radiology showed a double gastric bubble. we run an urgent computed tomography scan (ct scan) and a upper gastrointestinal (gi) endoscopy that showed a big type ii hiatal hernia that was complicated with a gastric volvulus. results: first, a nasogastric (ng) tube was placed for decompression of the stomach at the time when the endoscopy was made. the patient experienced a great improvement of the pain with that initial measure and remained stable. after almost a day of appropriate resuscitation, she underwent urgent surgery: we performed a hernia reduction, resection of the hernia sac, hiatal closure and a gastropexy and nissen fundoplication. the patient suffered no complications in the immediate postoperative time and was discharged after six days. conclusion: gastric volvulus are an uncommon emergency that we need to keep in mind. a simple abdomen x-ray can be very helpful, given that the double gastric bubble sign is a typical sign of this condition. it's always mandatory to perform an upper gi endoscopy in order to get to the diagnose and place a ng tube promptly. the surgery can be safely delayed in stable patients with no signs of ischemia, and a laparoscopic approach is associated with a shorter hospital stay and good long-term outcomes in this kind of patients. aims: during laparoscopic treatment of hiatal hernias the dissection can be complicated, but even more so the closure of the pillars, especially in giant hiatus hernias with a large defect. the use of prosthesis is controversial due to the lack of long-term studies and the possibility of secondary complications. the aim of this video is to demonstrate the safety of mesh hiatoplasty in hiatus hernia surgery. methods: we present the case of a -year-old woman with hypertension, hypothyroidism and right hemicolectomy for neoplasia years ago. the patient presented with malnutrition, with a weight loss of kg in the last months. a gastroscopy was performed in which a large hiatus hernia, that caused gastric volvulation, was shown. the upper gastrointestinal oral contrast study revealed esophageal tertiary waves and good passage to the gastric chamber, with an organoaxial volvulation of the stomach that was completely included in the thoracic cavity. results: through a five trocar laparoscopic approach, a large paraesophageal type iv hiatal hernia ( cm hiatal orifice) with complete herniation of the stomach and greater omentum to the mediastinum was observed. after reduction of the hernia content, complete dissection with partial resection of the sac was performed. an extended mediastinal dissection of the esophagus, with descent of the esophagogastric junction until achieving an abdominal esophagus of - cm, was carried out. after posterior and anterior phrenorrhaphy with nonabsorbable sutures, dislacement of the right pillar without diagragmatic opening was evidenced. it was decided to reinforce the hiatorraphy using a c shape composite mesh fixed with nonabsorbable sutures. the procedure was completed with a nissen-type fundoplication. postoperative course was uneventful and the patient remains without hernia recurrence months after the intervention. conclusion: prosthetic reinforcement in hiatal hernia repair can be a very useful resource in large hiatal defects in which a stress-free hiatus closure cannot be achieved. however, its use must be individualized according to the characteristics of the patient, the quality of the tissues involved and the result of simple hiatorraphy. aims: heller myotomy is an advanced laparoscopic surgical technique for the treatment of achalasia, a rare disease in which long time is needed to achieve the learning curve. surgical simulation, using animal models with an anatomy similar to humans, could help improving surgeon performance in a shorter time. the aim of our video is to show an ex-vivo and in-vivo animal model for heller myotomy training. methods: a cadaveric porcine model was developed using a tissue block including the esophagus and the stomach, without the diaphragm, mounted in a physical laparoscopic simulator. training procedures were also performed in an in vivo porcine model. experiments were carried out in the ' jesús usón' minimally invasive surgery centre in cáceres. results: the surgical technique is described step by step, first in the esophagus-stomach ex-vivo model and later in the live animal model. conclusion: surgical simulation using cadaveric an live animals offers a realistic representation, allows training in a safe environment, and can be very useful for advanced laparoscopic training in low incidence pathologies. introduction: esophagic perforation is one of the least frequent complications after laparoscopic nissen fundoplication, but it remains one of the most dreaded because of its morbidity and mortality rates and its technically difficult reparation. aims: to present how the authors dealt with an iatrogenic esophagus perforation post laparoscopic nissen fundoplication. methods: the authors report the clinical case of a -year-old woman who underwent a laparoscopic nissen fundoplication because of a symptomatic large hiatus hernia in a different hospital. the second postoperative day and after resuming oral intake, she presented respiratory and hemodynamic instability. she needed a chest tube that drained purulent content. the patient was referred to our hospital for clinical management. an urgent ct scan with oral contrast was performed without showing any leakage. results: in spite of the results, as the patient was unstable, she underwent an emergent diagnostic laparoscopy. it was found a small anterior esophagus perforation with right mediastinic collection. a running suture of the perforation was carried out and the nissen fundoplication was converted to a dör fundoplication. the operative time was min. she went to the intensive care unit for ten days. five days after the surgery, she was given methylene blue with no exteriorization through the drainages. as postoperative morbidity, she suffered from a right pneumonia and a thoracic collection that was treated by thoracic surgeons. the patient was finally discharged on the th postoperative day. she did well at home. she attended follow-ups without clinical reflux or any other particular condition. conclusions: esophagic perforation is a life-threatening complication that can be managed laparoscopically if it is detected soon after surgery and an expertise is available. surgical treatment of achalasia fails in - % of patients. the most frequent responsible cause is a previous incomplete myotomy, followed by fibrosis and aspects related with antireflux procedure. revisional surgery can represent a greater difficulty and a challenge. in this video we show revisional surgeries after heller's myotomy with dor fundoplication for achalasia. all cases presented a recurrence of the symptomatology and a revisional surgery was proposed. surgeries were characterized by the presence of a herniation of the previous fundoplication, fibrosis around the prior myotomy and/or the formation of a pseudodiverticulum. we show the steps followed and the aspects to consider during the dissection. these cases demonstrate that laparoscopic reoperation for achalasia is feasible, even after open surgery. aims: upside-down stomach (uds) is a rare type of large paraoesophageal hernia, characterized by migration of the entire or large parts of the stomach into the posterior mediastinum. uds is associated with severe complications like strangulation or volvulus development, possibly leading to acute gastric outlet obstruction and incarceration. surgical repair is the only curative treatment option and therefore recommended for these patients. standard procedure includes a hiatoplasty followed by an anti-reflux procedure. in a variety of studies, the use of mesh proved to be superior with respect to reduction of anatomical recurrences. methods: a -year old woman presented to us with reflux symptoms, dysphagia, dyspnea and tachyarrhythmias. she reported a weight loss of kg in the last months. ct scan and esophagogastroscopy showed a large paraoesophageal hernia, measuring approximately cm, with intrathoracic uds. results: we performed a laparoscopic hernia repair with dissection of the hernia sac from the posterior mediastinum, tension-free intrabdominal reposition of stomach and distal esophagus and hiatoplasty with biologic mesh (tutomesh tm ) augmentation. finally, a toupet fundoplication was performed to recreate the antireflux valve. in consequence of pronounced adhesions, a lesion of the muscularis of the distal esophagus occurred during surgery. the esophageal mucosa was unaffected. we treated the lesion laparoscopically with a simple interrupted suture (vicryl tm - ). an intraoperative patent blue v leak test did not identify any leaks. the recovery was uneventful. the patient was discharged on day after surgery and was free of symptoms in her follow-ups. conclusions: patients with large hiatal hernias and uds can be treated successfully and effectively with laparoscopic mesh repair. intraoperative complications can be handled laparoscopically in a safe manner. iatoplasty followed by nissen fundoplication represent the gold standard treatment of hiatal hernia; however it has been reported high hernia recurrence rate, especially in case of giant hiatal defects. in order to reduce recurrence rate, various techniques of hiatoplasty reinforcement have been implemented, such as prosthetic materials apposition. however, in literature have been reported various mesh complications such as esophageal and proximal stomach erosion and late esophageal perforation after ischemia, especially in case of synthetic non absorbable materials.in this video we are going to show the repair of a huge hiatal hernia by hiatoplasty and positioning of an absorbable biosynthetic 'bio a' mesh which is replaced by connective soft tissue over six months, therefore decresing complications and recurrence rate.as usual, we start with the mobilization of gastric fundus and isolation of diaphragmatic pillars by sectioning the aderences between them and herniated viscera. we proceed then with intrabdominal esophagus mobilization and higher mediastinal dissection in order to obtain an adequate esophageal lenght. after the exposition of the hiatus, we approximate the pillars with some non absorbable stitches and we reinforce the hiatus positioning a 'u' shaped bio a mesh over the esophagus, simply fixed to crura with single stitches. then we go on performing nissen fundoplication.the use of this prosthetic material appears to be cost-effective; first series in literature show very low complication rate and less recurrences of hiatal hernia than hiatoplasty without reinforcement. this video demonstrates the technique of laparoscopic identification and complete dissection of the sac of a totally intrathoracic stomach. identification of the sac is performed centrally by scoring the peritoneum overlying the arch of the diaphragmatic hiatus. the inferior edge is retracted and series of blunt dissection is undertaken. once the areolar tissue of the avascular plan is visualized; a raytec sponge is placed and advanced cephalad to expose the extra-saccular space. this raytec is kept in place to allow carbon dioxide to infiltrate and further delineate the anatomy. we then proceed with dissection at the left crus and right crus. complete reduction of the stomach can be achieved without grasping it. this can be performed by applying caudal retraction on the sac. this maneuver exposes the plane outside the sac. this space is divided into two compartments (right and left) separated by a septum which indicates the proximal extent of the sac. this plane is avascular and blunt dissection can easily free the hernia sac from the mediastinal structures and the pleura. this was followed by excision of the sac and hiatal repair which is reinforced with bioabsorbable mesh. the proximal short gastric vessels were then divided and a standard toupet fundoplication was performed. v -upper gi-reflux-achalasia introduction-objectives: the mixed giant hiatus hernias with paraesophageal component are hernias of difficult surgical correction, the laparoscopic approach of these implies a greater experience of the surgical team, given the complexity involved in its management, being the recurrence a complication that neither the use of meshes in this surgery has been able to avoid. in very high-risk patients, the gastric anterior pexy (boerema) may be an alternative to treat or alleviate the symptoms of these large hiatal hernias, although the frequency of recurrence with this technique is very high. material and methods. objective: the objective of the following case is to present a patient with type- hiatal hernia, barrett's esophagus without dysplasia and situs inversus. method: a laparoscopic partial fundoplication was performed on a -year-old male patient with a history of situs inversus totalis, who was seen in our general surgery service presenting a clinical history of retrosternal pain, heartburn and regurgitation. an endoscopy was performed, which reported hiatal hernia type and incompetence of lower esophageal sphincter, with squamocolumnar junction biopsies with report of barrett's esophagus without dysplasia. results: surgical time was programmed, for toupet type fundoplication; there were lax adhesions from omentum to wall, the lax hiatus and already known situs inversus. partial funcuplication was performed, with the usual technique adapted for structural anatomical abnormality, postoperative course without complications, oral initiation at the next day, drainage penrose type was set draining just a little serohematic liquid, diet was progressed and patient was discharged on the third postoperative day without complications. conclusion: situs inversus is the mirror image of situs solitus, which presents subdivision in situs inversus totalis, which is the most usual form, characterized by the mirror location of the intraabdominal and thoracic organs including the heart; in the case presented, the patient was referred with situs inversus and barrett's esophagus, performing laparoscopic fundoplication. the gold standard of surgical management is laparoscopic in benign esophageal pathology. gastroesophageal reflux disease (gerd) is a condition that reduces the quality of life and can causedisorders associated with acid reflux, such as bronchial asthma, barrett's esophagus and esophagealadenocarcinoma. gerd is often caused by existing of hiatal hernia. in rare instances gerd is associatedwith type iv hiatal hernias in which the part of stomach and other organs migrate into mediastinum.nowadays this condition can cause problems for some surgeons.patient was a -year-old man. he was diagnosed with hiatal hernia in . the condition had beenhaving asymptomatic course until . patient takes omeprazole mg for years. he startedexperiencing chest pains when inhaled and dyspnoea in june . co-morbidities were: arterialhypertension, chronic obstructive pulmonary disease (copd) and obesity (body mass index was . kg/m ). the posterior mediastinum contained the part of stomach, large bowel and small bowelaccording to chest roentgenography. sizes of esophageal hiatus were mm. in our clinical centerwas performed laparoscopic removal of hernia, cruroraphy, mesh repair of the esophageal hiatus andnissen fundoplication in july. during the surgery stomach, the part of small intestine, greatomentum and transverse colon were relocated into abdominal cavity. after cruroraphy, repair of theesophageal hiatus with prolene mesh was performed. the patient was in intensive care during h.total enteral feeding was initiated on second day. patients had been discharged within days aftersurgery. patient was re-examined by a surgeon in september , there were no signs of a reccurence.this case report shows an efficiency and feasibility of the laparoscopic approach to the treatment gerdassotiated with a large defect in the phrenoesophageal membrane, allowing other organs, such as colon,great omentum and small intestine to enter the hernia sac. aims: the authors present a video with their standardized laparoscopic hiatal hernia repair and anti-reflux nissen procedure but using mm instruments and mm camera approach. methods: a years old female patient with a bmi presents a symptomatic gastro esophageal reflux disease (gerd) for years. manometry and ph-metry showed a lack of esophageal motor disorders and a severe acid pattern with a . demeester score. panendoscopy study showed a cm hiatal hernia and a los angeles-grade esophagitis. she decided not to go with ppi treatment anymore. a laparoscopic hiatal hernia repair and standardized nissen procedure is performed using mm instruments and a mm camera. case and technical details are shown in the video. results: the patient was discharged from hospital within a period of h with a rate in a eva acute pain visual scale. in a year follow-up, there has no been an anatomical or clinical recurrence. no chronic dysphagia, anatomical recurrence or abdominal wall complications have been reported with in this period of time. conclusions: depending on the patient characteristics, anatomical factors and surgeon mini invasive experience, hiatal hernia and anti-reflux mini invasive standardized repair using mm instruments, could be a safe and feasible option. more studies are needed in order to standardized this approach. background and aims: parastomal herniation a very frequent complication in stoma patients. in isolated parastomal hernias (type i or iii)* a laparoscopic sugarbaker repair with intraperitoneal mesh is our preferred technique. if a concomitant incisional hernia is present (type ii or iii)* we currently opt for a retromuscular mesh repair as described by pauli. we adopted a minimal invasive approach using the robotic platform. methods: we performed a robot-assisted laparoscopic pauli repair for a wide incisional henria needing component separation and a small parastomal hernia (type ii)*. a non-slit retromuscular mesh was placed after a bilateral tar (transversus abdominus release) and lateralization of the colon. results: the operation was performed robot-assisted laparoscopically with x trocars without the need to convert to an open procedure. the procedure lasted min. the patient was discharged from the hospital on post-operative day three. one month after the repair our patient presented with a parastomal skin infection for which she received surgical cleaning and wound dressing. ct scan three months postoperative shows good positioning of the mesh with a reinforced abdominal wall. conclusions: the modified sugarbaker repair in parastomal herniation is feasible following a pauli approach (retromuscular mesh positioning) completely laparoscopic, albeit robotically assisted. short-term follow up is promising. long-term postoperative follow-up in these patients is needed. methods: case presentation results: a -year-old caucasian female patient was admitted on emergency due to a progressive alteration of her physical condition and weigh loss, caused by intermitent and intense epigastric pain and vomisments. symptomes occurred several years prior to admittance, but used to be mild and rare. during the last months, the described episodes became more intense, lasted longer and occurred more frequently. percutaneous ultrasound raised the suspicion, while thoraco-abdominal ct revealed an enormous intrathoracic morgagni hernia and gastric volvulus inside the hernial sack. after a careful preoperative preparation (restoring of the nutritional and hydric inballances, amelioration of respiratory parameters), laproscopy confirmed the diagnosis; we gently reintegrated the herniated organs from the thoracic hernia into the abdominal cavity (small bowel, large omentum, transverse-doloco-colon, and thisted stomach); a laparoscopic exploration of the hernial cavity was followed by a thorough hemostasis. do to patient's frailty, we decided to leave the hernial sack in situ. surgical direct repair of the defect technique was done by using extracorporeally tied separate sutures through separate skin incisions. the postoperative outcome was completelly uneventful; patient was discharged on postoperative day . barium contrast at mounths followup showed a slight esophageal diskynesia, but normal gastroduodenal passage; due to aerocolia, the normal position of the transverse colon could be confirmed aswell; no signs of reccurrence where detected. conclusions: although very rare, a morgagni hernia should be suspected and included in the differential diagnosis of patinets with dispeptic syndrome and epigastric/thoracic symptomes. thoraco-abdominal ctscanning is the imagistic technique of choice. laparoscopic approach became the gold standard, being far mora addvantageous as compared to laparotomy or thoracoscopy. direct suture with extracorporeally separate sutures through separate skin incisions was chosen for being less time consuming; for the same resons, the hernial sack was left in situ, with no consequences so far. aims: the giant fibrovascular polyp is one of the rarest benign lesions of the oesophagus. the most common locations of origin are the upper oesophagus or crico-pharyngeal region. the lesion is more common in elderly population, particularly men. symptoms include dysphagia, odynophagia. management options include surgical resection or endoscopic removal with endoloop. we aim to demonstrate the optimal management of these rare lesions using an endoscopic approach. method: we demonstrated the management of -year old patient with a giant oesophageal polyp, excised by minimally invasive endoscopic resection. the patient was placed in supine position and tracheal intubation was performed under general anaesthetic before an endoscopic approach was taken. the oesophago-duodenoscopy visualised a cresenteric shaped lumen due to an intraluminal mass occupying two thirds of the oesophageal diameter. the procedure was a multidisciplinary approach with the upper gi surgical and gastroenterology consultants. the polyp stalk was located in the oesophagus at the level of t and an endoloop was manipulated to surround the polyp. the polyp was then separated from the stalk by cauterisation and resected from the patient. the stalk was then injected with adrenaline to prevent haemorrhage. results: the excised specimen was a cm polyp with stalk originating from the t -t level. histology confirmed diagnostic suspicions of a benign pedunculated fibrovascular polyp. the polyp was covered by non-keratinising squamous epithelium with a discoloured tip demonstrating ulceration. there was no evidence of dysplasia or neoplastic process. the video shows a case of laterally spreading tumour of the rectum with preoperative benign histology, paris classification -is g (granular type), ut n eus stage, kudo type iv, nice type . the neoplasm measured x cm, and extended from to cm from the anal verge, mainly located on the posterior wall. according to our local policy the indication was a transanal full-thickness excision. this was performed with the medrobotics flexÒ robotic system, used here for the first time outside the united states. the system technology utilizes an articulated multi-linked scope that can be steered along nonlinear, circuitous paths in a way that is not possible with traditional, straight scopes. the maneuverability of the scope is derived from its numerous mechanical linkages with concentric mechanisms. this enables surgeons to perform minimally-invasive procedures in places that were previously difficult, or impossible, to reach. with the flexÒ robotic system, surgeons can operate through a single access site and direct the scope to the surgical target. once positioned, the scope can become rigid, forming a stable surgical platform from which the surgeon can pass flexible surgical instruments. the system includes on-board d hd visualization. the flexÒ robotic system contains two working channels to accept a number of different surgical and interventional instruments including monopolar and bipolar electrodes, scissors and graspers for tissue manipulation. the video shows the introduction of the dedicated rectoscope, the connection of the flexible robot, and the way to operate the device performing a full-thickness excision, including suturing of the rectal defect by means of two running sutures by a v-lock / thread. while illustrating the technique the authors will comment pros and cons of the use of the device. surg endosc ( ) the video shows the use of a barbed suture of novel concept. this first prototype thread developed together with assut europe (roma, italy) is characterised by a bidirectional / barbed suture, of cm length overall, with two needles mm in diameter, and circumference. in order to fix the thread to the tissue, on the exact midline of the thread a small ( mm) ball of the same material of the thread is fused with heat. this button as the entire thread is made of polydioxanone, a monofilament material for long-term absorption, self-retaining. this small advancement offera a consistent advancement in endorectal surgery helping in making transverse wound closures as easy as never before. the first stitch is placed on the transverse midline of the rectal wall defect, this way approximating proximal and distal edges. the button keeps the thread under tension allowing the completion of a running suture towards one of the two lateral ends of the would. when the first end is reached the needle is cut and the other needle is grabbed in order to perform the other half of the running suture keeping the right tension on the thread, with no risk of lumen stenosis. the two lateral ends of the suture are self-blocked passing them through the last stitch. no need for clips, knotting or buttonholes to pass through. while illustrating the technique the authors will comment pros and cons of the use of the device. background: the usefulness of robotic surgery has been largely reported; however, there are not enough reports on gist's treatment. the aim of this study is to report a single center experience on gastric gist's robotic resection. gastrointestinal stromal tumor (gists) are the most common mesenchymal tumors (overall incidence - % of all gastrointestinal malignant tumor). they are most frequently located into the stomach. complete surgical resection still remains crucial for patients with gists. in cases of difficult localization of tumor (e.g. posterior wall, his angle) and bigger tumor (more than cm in diameter), there still exist apparent difficulty and limitation to conduct laparoscopic resection. robotic assistance provides wide movements of its arms and was recognized particularly useful in case of difficult tumor localization, especially for those positioned at the posterior side of the stomach wall or around the lesser curvature. methods: six consecutive patients were analyzed focusing on safety (conversion/complications rate; hospital stay), oncological (margin resection, recurrence rate) and feasibility (operative time, technical tip and tricks) profile of robotic approach. results: the mean operative time was ± min, the mean hospital stay ± days. conversion rate was nihil. no intra and post-operative (mean follow-up months) complications were registered. in all cases, the resections were classified as r . conclusions: our experience supports the usefulness of robotic system in case of gist located at anatomically difficult gastric portion, such as lesser curvature or fundus close to gej, confirming the excellent oncological outcomes ( % r ) and the encouraging safety profile ( %). regarding the operative time our data are similar or better as compared to those reported by the previous literatures and didn't differ by the most recent published data for laparoscopic gastric resection. the anatomical hand-sewn reconstruction performed by precise hand-sewn suture instead of the usage of mechanical staplers represents a real great advantages of robotic resection. in our series, no patients suffered from stenosis or leakage after operation. background: we describe the use of a different suture from those historically used in the elaboration of a widely spread surgical technique such as the nissen fundoplication, for the treatment of pathological gastroesophageal reflux or symptomatic hiatal hernias. in our unit we have implemented the use of / irreabsorbable barbed suture to close the diaphragmatic pillars and the °fundoplication with the same continues suture. aim: the objetive of the use of the irreabsorbable barbed suture in the nissen fundoplication is to shorten the surgical times, which would achieve benefits for the patient and the institution, increasing the number of ambulant patients and the number of patients to be operated the same surgical session. objective: the goal of the present study was to demonstrate that intraoperative icg fluorescent imaging is a safe technique that can be used in laparoscopy establishing the exact location of the lymphocele and reducing intraoperative risks. method: fifty milligrams of icg dissolved in ml of saline solution was injected via percutaneous drainage placed into the lymphocele to decompress transplanted kidneys weeks before a laparoscopic lymphocele marsupialization procedure. results: during the first exploratory laparoscopy, in the flank and right iliac fossa, near the renal grafts, fluorescence was identified in raised areas that were the internal side of the lymphocele lobes. the lymphocele wall was dissected and ml of serous fluid was aspirated after puncturing. a cm breach was then made in the cyst wall using the ultracision harmonic scalpel (ethicon us). afterwards, a pedicle of the omentum in the lymphocele core was interfered with and fixed by stitches. conclusions: laparoscopic surgery seems to be the preferred surgical option for the treatment of primary symptomatic lymphocele after kidney transplantation. intraoperative icg fluorescent imaging is a safe technique to establish the exact location of the lymphocele and reduces the risk of damaging urinary structures during surgery. conclusions: this method proved safe and risk-free, easily reproducible and without the need for a different toolkit than the one of a modern operating theatre. the preliminary analysis shows a strong correlation between the perfusional data so far obtained and the early outcome of the graft. thus opening the way to further analysis aimed to a future better management of post-operative immunosuppressant and support therapy. these results are quite encouraging, even if our study is at an initial stage. conclusions: results show a persistent dread across specialities regarding ai. rather than be seen as threat, ai should be embraced by clinicians as it will ease the ever-increasing daily workload faced. this will enable clinicians to focus their skills on patient centred activities, interventional procedures and development. despite current regulatory hurdles, ai implementation in medicine is unavoidable. this coming revolution presents a unique opportunity for endoscopist and radiologist to refocus their expertise in novel areas. project description: from february to july a three-armed proof of concept study was conducted at three hospitals, in three groups of patients; recurrent ventral hernia, aortic aneurysm, and healthy controls. patients were measured once at the outpatient clinic using an electronic nose based on three metal oxide sensors. measurement data were compressed to low-dimensional vectors using a tucker like algorithm, and used to train an artificial neural network (ann) to provide a classification between patients (? ) and healthy controls (- ). preliminary resultsa total of patients (hernia n = , aneurysm n = ) and controls were included in the study. based on receiver operating curve (roc) analysis, the ann could differentiate between recurrent hernia patients and controls with the following details: area under the curve (auc) . , sensitivity . , specificity . . aortic aneurysm patients and healthy controls could be differentiated with an auc of . , sensitivity of . , and specificity of . . the aeonoseÒ enose can reliably distinguish patients with weak collagen (recurrent hernia and aortic aneurysm patients) from healthy controls. validation of these results in a prospective cohort study is required before clinical application of the device. background: laparoscopic and endoscopic cooperative surgery (lecs) has been performed gastric submucosal tumor (smt) or duodenal tumor. although minimally invasive surgery using thoracoscopy has been the usual approach for esophageal smt, the treatment method combined thoracoscopy and endoscopy has not been established. in addition, submucosal endoscopic tumor resection (set) for esophageal smt was reported using the technique of submucosal tunnel. aim and project description: we planned to resect large esophageal smt located in the upper or middle thoracic esophagus by a combined endoscopic and thoracoscopic approach. because set is only recommended for tumors up to mm in size owing to the limited submucosal space available and the left thoracic approach is restricted by the aortic arch and the trachea. preliminary results (case presentation): a -year-old woman was diagnosed with a benign schwannoma of length mm originated from either the submucosal or the muscular layer of the middle thoracic esophagus by endoscopic ultrasonography, enhanced computed tomography, and ultrasound-guided fine-needle aspiration biopsy. since the tumor had increased in the years and she had a symptom of dysphagia, we planned to resect it by a combined endoscopic and thoracoscopic approach. on endoscopy in the supine position, a submucosal tunnel was created mm proximal to the cranial edge of the tumor, and its only oral end was dissected from the mucosal and muscular layers. this was followed by the resection of the entire tumor by left-sided thoracoscopic procedure in the prone position. endoscopic mucosal closure was achieved by using clips. no postoperative complications were observed. large benign esophageal tumors can be safely excised with minimally invasive surgery by using a combination of endoscopy and thoracoscopy. background: haemorrhage remains a major cause of morbidity and death in all surgical specialties. in laparoscopic surgery. relatively small amounts of blood can obscure the view of the operative field and increase the risk of injury to surrounding structures. excessive bleeding often leads to longer hospital stays, increased healthcare service utilisation, and higher healthcare costs, among other negative consequences. aim: the aim of this study was to analyse the feasibility of purastatÒ, a new synthetic haemostatic device, made of self-assembling peptides in laparoscopic colorectal surgery. project description: this was a prospective observational non-randomised study. consecutive patients undergoing laparoscopic colorectal surgery were enrolled. informed consent was obtained from all patients inclusion criteria was the need employ a secondary method of haemostasis when traditional methods such as conventional pressure or utilization of energy devices to control the bleeding were either insufficient or not recommended/ appropriate due to proximity to ureter, pelvic/sacral veins and other important structure. preliminary results: twenty patients were enrolled ( males ( %) and ( %) females). mean age was years (± , years). all patients were undergoing elective laparoscopic colorectal cancer surgery ( right hemicolectomy, sigmoid colectomy, anterior resection). we utilised mls of purastatÒ in all patients. the mean area of application was , cm (± . cm ) with the amount of purastatÒ needed per centimetre being . mls. the mean time to apply the product was secs (± s), whereas the mean time to achieve haemostasis was , secs (± . s). there were no post operative complications in this cohort of patients. mean operative time overall was min (± . min). none of the patients experienced delayed post-operative bleeding and the mean hospital stay was days (± . ). in conclusion, according to the purpose of this preliminary study, we have demonstrated that purastatÒ can be easily used in laparoscopic surgery and it is a safe, effective haemostatic agent. this is a feasibility study and additional controlled studies would be useful in the future. during last three years the laparoscopic method of surgery in the presence of common bile duct stones was carried out. after performance of intraoperative cholangiography and visualization of stones in the common bile duct laparoscopic, choledochotomy and bile duct stones extraction was undertaken in patients, using flexible choledochoscopy control. in all patients with gallbladder stones was then performed laparoscopic cholecystectomy. results: laparoendoscopic intervention on common bile duct was successfully performed in patients ( . %) and the operation was completed by common bile duct drainage by kehr. in patients due to technical difficulties conversion to open surgery was carried out. postoperative morbidity in the form of bile leakage were diagnosed in patients ( . %). in three cases they stopped spontaneously in - days after the operation. patients were operated on repeatedly and additional suturing on choledocholithiasis was carried out. postoperative mortality was . %. the death of the patient of years was caused by acute cardiovascular failure. institute for image guided surgery, ihu-strasbourg, strasbourg, france; hepato-digestif, nouvel hôpital civil, strasbourg, france; image guided surgery, nouvel hôpital civil, strasbourg, france; gastroenterology, chu-besancon, besancon, france; gastroenterology, chu-lyon, lyon, france; gastroenterology, clinic de trocadero, paris, france background: eus is difficult to learn and has a steep learning curve. therapeutic eus (teus) is even more so. simulators, ex-vivo models and phantoms are the most common current teaching modalities but are felt by many to be unsatisfactory for high-level training. aim: we designed a training curriculum for teus that uses high-fidelity animal models and present a validation study performed by teus experts. project description: different simulated pathologies were created in each of acute pigs. teus experts performed therapeutic procedures in two or more animals over two days. each intervention was evaluated simultaneously using a structured survey by an non-expert observer. data included demographics and procedure details as well as likert-scale evaluation of the quality, realism and education utility of the simulations. global evaluation of the experience was captured from the experts as written comments. all data was consurrently registered and subsequently analysed by two blinded surgical educators. methodology: three types of models were created using surgical access: -tumors (injection of types of hydrogel), -retro-gastric collections ( - cm long intestinal loops filled with oatmeal, oil-water and gel), -obstructions (bile duct and ureteral ligations days prior to experience). gastric, pancreatic and liver tumor models were used for fna and fnb practice. retrogastric fluid collections and choledochal/ureteral obstructions were used for cyst gastrostomy, hepaticogastrostomy, gallbladder drainage and kidney drainage. results: experts age: - , median intervention time min , total of interventions evaluated, overall quality of experience: ( %) ranked - (excellent), ( %) from - (good), ( %) from - (poor), / procedures were successfully completed. models were rated good to excellent quality ( - ) in ( %), poor quality in ( %). for % ( ) of the interventions the model was considered not good enough to be repeated (solid retrogastric tumor and peripheral hepatic lesion). conclusion: high-fidelity live animal models with simulated pathologies are considered to be excellent training tools by experts and may provide a better learning experience for teus. surg endosc ( ) in our short videos, we present scenarios in which this technology helped: to distinguish a significantly dilated cystic duct from the cbd, to identify an anterior cystic artery in the contest of acute inflammation, to identify a luksha duct, to exclude a leak after endoloops positioning on cystic duct. intra-operative augmented visualisation of biliary anatomy with indocyanine green cholangiography is an essential technology tool with the potential to extend the hour window of safety for emergency cholecystectomies, with significant logistics benefits. introduction: endoscopic resection of subcardial polyps has its limitations; especially when it is necessary to dry out the entire gastric wall. uniportal intragastric surgery is a good alternative for the exeresis of subcardial premalignant lesions with gastric preservation. patient and method: we present a video with two cases. technique: we perform a laparoscopy to explore the entire abdominal cavity, then we open a . cm hole in the great curvature; a cm incision is made in left hypochondrium and the uniportal device is placed inside de stomach. we inject serum in the submucosa with a endoscopic needle. when submucosa is completely separated from muscular lay; a submucosal exeresis can be made; but when there is not a complete separation after injection, then we perform a entire wall resection with a cm circular margin. if a complete wall resection is made, then we close the defect with a barbed suture. results: case : years old male with a , cm subcardial polyp, preoperative biopsy was informed as severe dysplasia. in the laparoscopy we saw an unknown lesion in the great curvature that looked like a gist. we placed the uniportal device intragastric and proceed to the submucosal serum injection. as the submucosal lay was completely separated from muscular, then we performed a submucosal exeresis. we close the gastrotomy and made the resection of the great curvature lesion with endostapler. the pathological analysis confirmed the severe dysplasia in subcardial lesion and gist in the great curvature lesion. patient was discharged with no complications after days. case : years old male with a cm subcardial polyp. preoperative ecoendoscopy suggested a muscular layer infiltration but only severe dysplasia was found in the previous biospsy. laparoscopy did not found more lesions, and uniportal intragastric device was placed. a complete wall resection was made, and the defect was closed with manual barbed suture. pathologyst confirmed severe dysplasia and unaffected margin. patient was discharged with no complications after days. conclusion: uniportal intragastric surgery is feasible and safe and may be useful for subcardial premalignant lesions when endoscopic resection is very difficult or not feasible. introduction: the role of icg in bariatric surgery is still unclear. knowing the lack of perfusion in the gastric pouch could be of great interest in revisional surgeries; but the question remains: is the current icg technology reliable enough to make intraoperative decisions in bariatric surgery? methods: we have carried out a check of tissue perfusion with icg fluorescence in several cases of primary and revisional bariatric surgery. a solution of , mg/kg was injected intravenously and icg fluorescence was performed. we looked for the correct staining of the entire gastric pouch and the intestinal loop trying to rule out areas of tissue ischemia. results: the cases in which the test was performed showed a minimal delay of - s between the intestinal loop stain and the pouch. a correct staining was observed in all but one case shown in the video. is the case of a years-old male patient who was operated years earlier in another center; at that time he underwent a sleeve gastrectomy. we evaluated the patient for persistent gastroesofhageal reflux of years of evolution with esophagitis. we offered revisional surgery to perform gastric bypass and hiatal closure. fundus was dilated so a funduplasty was performed instead of using endostappler in the vertical side of the pouch. manual anastomosis gastric bypass was performed. when the icg test was performed, a corner of the pouch does not stain green (an area of , cm) . so the decision was to resect that part and redo the anastomosis or wait and see. it was decided not to resect and the patient was discharged two days later with no complications and good outcome with a months follow up. conclusion: icg fluorescence may be useful in bariatric surgery in the future but more evidence is needed of its usefulness in making intraoperative decisions. background: lymph node status is one of the key prognostic factors in patients with colorectal cancer, and remains the most important selection criteria for adjuvant chemotherapy. it is believed that at least % of node negative patients will suffer disease recurrence within the first years after surgery. this may be due to understaging of lymph node status. sentinel lymph node mapping is widely used for staging of breast cancer and melanoma, with injection of colloid tc and isosulfan blue (ib). however, indocyanine green (icg) fluorescence guidance is a new technical approach to this issue, with promising results for detection of aberrant lymphatic drainage outside of the planned resection. the icg lymphography has the advantage of offering a good visualization of the lymphatic channels but there are problems in order to identify the lymphatic nodes. aim: the objective of the experimental study is to investigate the possibility to detect the sentinel lymph nodes after the injection of different solutions with indocyanine green in the subserosal colonic layer in the pig. project description: twelve female large white pigs were operated with laparosocpic approach and spies optic filter (karl storz, germany). indocyanine green was injected in the subserosa of the colonic wall ( ml at points). lymphatic flow was observed at - - - - and min, searching for the migration of the icg by the lymphatic channels and its introduction in the sentinel nodes. preliminary results. the identification of the sentinel nodes is very difficult with the solution of icg-sterile water. with this technique we can see the lympjatic channels but not the lymphatic nodes. the adition of % human albumin as a transporter of the icg is very helpful for the correct identifiaction of the lymphatic channels at - - min and the correct visualization of the lymphatic nodes at min after the bowel injection. addition of other transporters like dextran solutions may be helpful too but the time to the correct visualization is longer. there was significant difference among the three groups as regards postoperative les and postoperative ph metery.the incidence of persistent dysphagia was significantly higher in the group i. postoperative gerd symptoms were significantly higher in group iii ( . %. p \ . ). recurrent achalasia was significantly higher in group i ( patients . %, patients in group ii ( . %) and nil in group iii (p \ . ). in conclusion: longer myotomy on the gastric side ([ . cm) ensures complete division of the les with better outcomes in term of resolution of dysphagia but may be associated with higher postoperative gerd. therefore, a myotomy length of . to . cm on the gastric side provides a balance between relieve of dysphagia and development of postoperative gerd. c/t scan of his abdomen revealed a large groin hernia with signs of small bowel obstruction and collapsed distal bowel. emergency theatre was organised for this patient with anaesthetic assessment prior to his surgery. initial plan of local exploration with possibility of small resection was changed once he was under full anaesthetic with muscle relaxation. his abdominal girth provided an opportunity to utilise laparoscopic intervention as an initial approach. laparoscopy with degree lens revealed moderately distended loops of small bowel and a large omental mass along with a loop of small bowel incarcerated in right direct inguinal hernia site. background: robotically assisted surgery is a rapidly developing modality of minimally invasive surgery with proven advantages in the management of cancer. despite its increasing prevalence, there is still an ongoing debate regarding its future role in colorectal surgery. while the prospective randomised multi-centre studies provide research evidence for its potential efficacy, an assessment of its effectiveness and realistic outcomes in everyday clinical practice can add an important perspective to this discussion. the international robotic colorectal registry will allow to compile and pool the international robotic colorectal experience. aims: the aim of the international robotic colorectal registry is to monitor the safety and outcomes, as well as the quality of specimen of robotically assisted colorectal surgery for malignant and benign diseases of the colon and rectum. the primary endpoint is a composite oncological failure. the secondary endpoints include anastomotic leak, resection margin involvement, conversion rate, operative time, post-operative -day morbidity and mortality, long term oncological outcomes, quality of life, functional outcomes and cost-effectiveness. project description: the international robotic colorectal registry is a multicentre web-based, online secure database. the registry has been awarded an ethical approval by a relevant national committee. all surgeons performing robotic or robotically-assisted surgery are invited to participate. the data collected includes patient demographics, cancer characteristics, operative details, histology of the specimen, wound healing, post-operative therapy, readmission, quality of life and functional (bowel, urinary and sexual) outcomes. all the sensitive patient information is encrypted before its introduction into the database. preliminary results: so far, twenty robotic colorectal centres have joined the international robotic colorectal registry. the preliminary results will be published once patients have been enrolled. univariate and multivariate analyses will be performed to identify possible risk factors for poor outcome.the possibility to record open, laparoscopic or other minimally invasive colorectal procedures will facilitate comparison of the outcomes of the robotically assisted surgery and other modalities. the registry will also allow each surgeon enrolled to monitor their skill progression and outcomes over the time. results will be published in surgical literature and presented internationally. background: gastric outlet obstruction (goo) due to benign strictures is an uncommon surgical entity today. this paucity relates to the decrease in its aetiological factors in the modern era as well as to advances in both prevention and medical as well as endoscopic treatments of such condition. the most common of causes relating to peptic ulcer disease, has been subdued for decades with quality acid control medications. on the other hand advances in gastroscopic dilatations skimmed even more the frequency of these cases from arriving to surgical intervention. aim: this presentation gives an update on the standing of this pathology and its surgical management today. it will also shed a light on our early experience in this condition at the royal hospital of muscat in oman. project description: a case series of all patients with goo, who were surgically managed between and results: there were a total of patients, males and females. the cause of obstruction was peptic ulcer disease in , corrosive injury in , iatrogenic perforation in and idiopathic hypertrophic stenosis in . emergency presentation was seen in . management included jaboulay pylorpolasty in , resection in (distal gastrectomy in , total gastrectomy in ) or a bypass (gastrojejunostomy) in . in of the above, the procedure was done by laparoscopy. post operatively, temporary gastric paresis delayed recovery in , however all symptoms resolved in . there were no recurrences at minimum of years of follow up. in spite of advances in medications and gastroscopy interventions, we still seem to identify this condition within our population. although infrequent, they demand awareness from surgeons since they could be managed successfully, especially laparoscopically, with minimal morbidities and early recovery. the introduction of advanced laparoscopy to the unit's setup in recent years, made such option feasible with satisfactory and durable outcomes. background: gists of the upper gi are found mainly in the stomach ( - % of cases) and small intestine ( %). duodenal gists however, comprise a smaller subset with a frequency of to %. the optimal surgical procedure for duodenal gist is still evolving. since wide margins and extensive lymphadenectomy are not required, restrain from more radical resections in this area would be a valid option. aim: this is a video case report of a patient with a gist involving the third part of the duodenum treated by laparoscopic lateral duodenectomy and end-side roux-en-y duodenojejunostomy case report: years lady presented with recurrent mid abdominal postprandial pain with anorexia, nausea and occasional vomiting an ultrasound showed well defined hypoechoic mass of . . cm at the right para-aortic region . ct scan defined the mass as retroperitoneal, intimately related to the pancreas uncinate process and the third part of duodenum with no clear cleavage line between them. an mri endorsed the diagnosis of gist of the duodenum. she was operated upon through a laparoscopic lateral duodenectomy including the gist at the third part of the duodenum. a frozen section confirmed the clear margins . reconstruction was done by a roux-en-y duodenojejunostomy with the alimentary limb taken cm from the dj flexure. she had an uneventful post operative recovery and was discharged well. the histology confirmed a low grade gist tumour hence no further treatment was needed. at follow up six months later, she was doing well and gaining weight. conclusion: complex anatomy of the pancreatico-duodenal area makes conserving the duodenum for tumours rather than a major resection a challenging option. in our case however, with the disease in the third part being of a moderate size, a lateral duodenal wall resection including the mass was possible rather than a segmental resection. this procedure could be an ideal choice for benign, moderate sized tumours in the third and fourth part of the duodenum. background: during laparoscopic colectomy, laparoscopic lymph node dissection and extracorporeal intestinal anastomosis is commonly performed. an umbilical incision of - cm and wide-range mobilization of the intestinal tract is required for extracorporeal anastomosis. previously, we introduced intracorporeal overlap anastomosis in june as a minimally invasive treatment. here, we report its short-term outcomes. aim: we retrospectively compared the surgical outcomes of cases of extracorporeal anastomosis and cases of intracorporeal anastomosis, all of which were performed between june to may . procedures: after lymph node dissection and sufficient mobilization of the intestinal tract, the proximal and distal intestines were resected perpendicularly to the intestinal tract with a -mm linear stapler. the anastomosis was performed after the specimen was extracted from an umbilical incision. the opposite sides of the mesenteric margin cm from the staple line of the one intestinal tract, and cm from the staple line of the other intestinal tract, were marked, and then the respective intestinal tract was positioned to join the opposite mesenteric sides together. an insertion hole was made in the intestinal tract at the marked site. side-to-side anastomosis with a linear stapler was performed, and then the insertion hole was closed with a linear stapler after several temporary sutures. preliminary results: in the extracorporeal anastomosis group, the mean operation time, blood loss, and post-operative days were min, ml, and . days, respectively. furthermore, there were three intraoperative cases of bleeding ( . %), and two postoperative cases of lymphorrhea ( . %) that occurred. however, in the intracorporeal overlap anastomosis group, the mean operation time, blood loss, and post-operative days were min, ml, and . days, respectively. additionally, there were no cases of intraoperative complications, and only one postoperative case of lymphorrhea ( . %). conclusion: intracorporeal overlap anastomosis in laparoscopic colectomy is safe and feasible, and can be used as a minimally invasive treatment. nowadays d-printing it's not a new technology any more but with an exponential developing. there are beliefs that in % of everything that will be produced will be d-printed. in medical field this technology knows the same exponential developing. first used in orthopedics and maxilo-facial surgery now d-printind is used in many other fields for different reasons, like preoperative training models, surgical special instruments, in medical education, etc.. liver surgery is in continuous developing and this is the reason why we need experimental liver model for training and testing. a best liver experimental model should heave liver consistency, to be flexible, to heave the same ultrasound feedback, to be cheap and easy to be reproduced. this is why we developed a liver experimental model made of gelatin by a simple recipe, using a dprinted mold, created after a human liver ct-scan. first was made the segmentation of the liver. after segmentation we create the d virtual liver model and the negative image of the liver, which was used for creation of the pieces of the liver mold, with connections between them and a hole on the top to pour the gelatin solution. ( ) ethical concerns on learning and training with real patients and substitutives such as animals, and ( ) reconciling time devoted to learning with clinical practice, considering the european work time directives. simulation in medical education is and has been the preferred route to address both pedagogical needs. virtual simulation has proven to be a valid tool for training; however, current systems restrict usage to tasks and modules offered, without possibility of personalization. we present the minimally invasive surgery simulator scenario editor (mis-sim) an environment where users can create, edit and run virtual reality tasks designed for medical training. the environment features an editor allowing users to develop learning tasks, defining its learning objectives and task goals in an easy way. a first proof of concept has been implemented for surgical training and training activities (demostrators and short courses) have been carried out in three european sites: spain, the netherlands and hungary. during training activities, different exercises have been created and uploaded to the contents' database. trained technical skills include handeye and bimanual coordination, instrument handling and pulling. preliminary results with users have shown mis-sim training potential, although some functionalities should be made easier. personalization has been highlighted as the key added value of mis-sim with respect to the current competitions in the market: the ability for target users to use virtual reality based learning tools while remaining in complete control of the learning process. mis-sim aspires to break the barrier between vr and medical education by empowering users to create their own tasks. with mis-sim teachers/course creators and learners (healthcare professionals & future healthcare professionals) will benefit from an innovative tool to ( ) create personalised medical learning contents tailored to preferred learning styles, allowing the creation of individualize learning paths; ( ) improve the efficiency of training by focusing on the training needs of the learners and ( ) share and sell vr-based didactic contents. c. tiu , p. sánchez-gonzález , m. chmarra , d. gutiérrez , c. guzmán-garcía , l. sánchez-peralta , g. wéber , f. sánchez-margallo , b. pagador , j. dankelman aims: currently surgical training is largely based on the improvement of technology enhanced learning solutions. the progress of engineering and the diversification of training facilities outside the operating theater results in an even greater contribution of technology in the future. the main reasons to encourage these changes are increased efficiency of simulators and directly increased patient's safety. the goal assumed by the easier project is to develop multi-skill, online platforms for minimally invasive surgical (mis) procedures-based on common pedagogical principles with reference value in a multinational space. the platform will allow the connection of external assets (such as simulators) to centralize all training data from residents. this work presents the milestones of the project during its first year of life. methods: the consortium's activity started with a knowledge elicitation process organizing brainstorms and workshops including experts in mis and interventional techniques, from spain, romania and hungary. this experience led to the formulation of a questionnaire that was implemented online and sent via email to surgeons and residents from the participating countries. results: accumulated experience was used to define the pedagogical needs of the platform. the pedagogical needs form the starting point for defining the technical requirements and specifications. based on them, the design of the platform has been achieved, including its architecture and communication protocol between external assets design, facilitated by the use of state of art educational standards. discussions and conclusions: next steps include the implementation of the easier platform, as well as the definition of cases studies selected by the clinical partners in spain, romania and hungary to solve applications of the platform dedicated to cholecystectomy, lumbar puncture and arthroscopy. a pedagogical model, built by the experience of the consortium, is being used to guide instructional design of the course. finally, results will be validated in a multi-center validation study.the easier project will create a training platform with reference value for european surgery. the structure of the consortium, based on the confluence between collectives with clinical, technological and pedagogical experience, will generate a complex learning tool in surgery embodying technology-based training systems with clinical experience. background: the treatment of groin hernia is an important part of our daily surgical activity. aim: we proposed to evaluate outcomes of the laparoscopic trans abdominal pre peritoneal treatment (tapp) of the groin hernia. project description : one hundred and fifty patients who underwent a tapp for a groin hernia were included in a retrospective study between january and november . results: the gender ratio was . the average age was , years. twenty percent of patients had a history of abdominal surgery. the operative indication was a unilateral hernia in % of cases, associated with an umbilical hernia in % of cases, a recurrent groin hernia in % of cases and a bilateral inguinal hernia in % of cases. the conversion rate was . %. the hernias were classified according to the ehs classification in l type in % of cases, l in % of cases, l in % of cases, m %, l r in % of cases and f r in % of cases. a contralateral inguinal hernia was discovered in % of patients. a polypropylene mesh x cm was fixed by a stapling in % of cases and by a suture in % of the cases. the average operation time was min. the hospital stay average was , day. an antalgic treatment was prescribed in % of patients. the average time to return to normal physical activity was days. a postoperative seroma was noted in % of patients. no cases of mesh suppuration were noted. chronic pain was noted in two patients. no recurrence was noted with an average follow-up of months. conclusion: laparoscopic treatment of the groin hernia by tapp had good results concerning the postoperative pain, early recovery of physical activity and aesthetic damage. a larger setback is needed in our study to evaluate the recidivism rate. background: surgeon's training in ultrasound is viewed and understood differently in different parts of the world. if in united states the need for surgeons' training was accepted and taken over by the american college of surgeons, in europe the practice is completely different from one country to another, from one city to another, from one department to another within the same premises hospital. in some european countries, surgeons currently use ultrasound for diagnosis-especially in urgency, for follow-up, intraoperative, or as guidance for many surgical gestures. during this time, access to ultrasound of other surgical specialties-gynecology, urology, ophthalmology-is considered natural. material and method: once the decision to initiate an ultrasound course for surgeons was taken, a team of experts with technical or clinical expertise in ultrasound was organized. at the initiative of the technology commission, the courses were to be organized at the eaes congresses or others communication events endorsed by eaes. starting from the importance of each ultrasound application in surgery, it was decided to develop different modules to solve different training needs. at this time, the course offered at seville covers the capitols like abdominal ultrasound, guided punctures and trauma. a module dedicated to intraoperative ultrasound is under construction and will be available in november . the course has a skill abilities dominant character, two thirds of it being thought of as a hands-on application on stationary, classical ultrasounds with large screens and also on small size wireless actual devices. results: after his debut in frankfurt in , the course was resumed in london and in bucharest, twice. in this process, new modules and better teamwork skills have been developed. the participants' satisfaction quizzes, coming from all continents, were really encouraging. for the intraoperative ultrasound module the team approach is unique. students will have the opportunity to practice on live animals both laparoscopic and open abdominal procedures conclusions: the ultrasound for surgeons course initiated by eaeas was received with interest. the team will seek to inspect the real needs of training surgeons in this field and will complement and diversify the current platform surg endosc ( ) preliminary results: a total of procedures ( - , ± . ) were carried out in patients. the indications included acute ( leaks following esophageal resection, rupture of the strictured anastomosis following pneumatic dilatation) and chronic conditions (esophagopleurobronchial and gastropleurobronchial fistulas following the resection of esophageal diverticulum and sleeve gastrectomy). the initiation of the therapy was in , and day in case of acute conditions, and after years of the duration of the unsuccessful therapy in chronic cases. the successful closure was observed in patients, patient passed from mods and ards. in case, the initiation of evac was provided as a combined surgical and endoscopic intervention (ct proven distant intraabdominal abscesses). in chronic cases, was discontinued due to the haemophagocytic syndrome of unknown etiology, in the second one, success in reduction of the lesion and symptomatology with long term duration was observed following just applications of evac, despite minimal remanent leakage. the success is to our experience linked to early initiation of the therapy and presumes complex intensive care. the future investigation should specify the timing including preemptive use of evac and the combination of evac with other endoscopic, interventional and surgical therapeutic modalities. the aim of this feasible study is to investigate complete robotic esophagectomy with total mediastinal lymph node dissection (retm) only by the robotic arms. methods: the patient is placed hemi-prone position with one lung ventilation by blocking balloon tube under general anesthesia. the robotic trocar for st arm of da vinci xi surgical system is placed in the th intercostal space (ics) on the scapular line, the trocar of nd arm is placed in the th ics on the posterior axillary line, rd arm trocar is placed in the th ics on the middle axillary line, th robotic arm trocar is placed rd ics on the middle axillary line, and assistant trocar only for taking in and out of gauze is placed in the th ics on the middle axillary line. on the upper mediastinal lymph node dissection, robotic camera exchanges from nd to rd robotic arm to close and identify anatomical structures. esophagectomy with lymph node dissection starts from middle and lower mediastinum to upper mediastinum including along bilateral recurrent laryngeal nerves. all procedure perform under close-up view along the robotic enhanced anatomy to preserve organ functions. background: complete stenosis of the duodenal lumen secondary to a surgical suture in the treatment of a duodenal ulcer is a rare complication. the usual surgical resolution corresponds to a gastrojejunostomy associated or not, to an antrectomy with vagotomy, as a treatment for the peptic disease. the endoscopic resolution of this complication requires the use of complex maneuvers and specific therapeutic instruments. aim: to describe the endoscopic resolution of iatrogenic occlusion after raffia of perforated duodenal ulcer. description: a -year-old man was admitted to the emergency service for four days of pain and abdominal distension associated with abundant retention vomiting. performed ten days ago of a perforated duodenal ulcer, in which manual raffia was performed in two planes and drainage. abdomen and pelvis ct showed great distension and diffuse thickening of the gastric wall. the endoscopy showed abundant gastric retention content, pylorus, and bulb edema, and complete closure of the duodenal lumen, secondary to suture material; it was possible to count three suture threads. with a tipped papillotome and electrocautery, all the suture threads were sectioned, identifying a filiform opening through which a hydrophilic guide is inserted under fluoroscopy until it is sure to overcome the stenosis; we dilated the trajectory with a dilator of mm in diameter and cm in length. with a contrast medium, we observed an adequate trajectory and installed a partially covered duodenal metal stent (hanaro stent) of cm in length by mm in diameter in order to sustain the dilation. he was sent to home with inhibitors of the proton pump. after two weeks, the stent was removed, without complications. he was controlled two weeks after withdrawal, and, with pharmacotherapy, the patient was asymptomatic, making a normal life. conclusion: in this case, the result was positive. satisfactory results can prevent major surgery, which reduces the risk and possible complications. a new range of non-invasive medical tools with a remarkable improvement on the existing market. a manual laparoscopy, with the important novelty of having a bending head with a high degree of movement. this head can get multiple spatial positions to work in surgery and is very easy to use, with the same scissors thimble who controls it, so its performance and learning is very simple and intuitive. the tools can be easily reusable and they can be cleaned and sterilized by ordinary methods, very ergonomic and lightweight . the generic type models we initially have developed are focused in general surgery, but gradually we will develop new applications and different heads for specific medical conditions such as arthroscopy, laryngoscopy, otolaryngology, ophthalmology, orthopedics . its operation is simple and functional, simply moving the scissors thimbles, where they have a dual role, combining the head tilt and the action of opening and closing of this is achieved.the design of this tool allows us to work with some degrees of unparalleled freedom from the existing tools. our instruments replicate the movements of the robot with a simple handheld mechanical instrument, our philosophy is to position our instruments in between a long empty field between the surgeon and the robot. the tip of our instruments are providing the surgeons with angulations impossible to reach with the traditional instruments unless applying huge movements from the hands of the surgeon. we consider that this devices will have a very fast acceptance from the market as this robotic type movements can be managed by the surgeon through the traditional mm trocars, without the need to change to a new surgical technique, just with the traditional method and a very brief training. background: diverticula of the middle thoracic esophagus are infrequent, its etiology may be secondary to traction or pulsion mechanisms. when the etiology is a mechanism of pulsion, they are associated with esophageal motor disorders and its prevalence is estimated between . % and . % of the population . they are rarely symptomatic and the diagnostic is usually incidental. the most common symptoms are episodes of food impaction, chest pain or bronchoaspiration.diverticulectomy and esophageal myomectomy by minimally invasive approach is the treatment of choice in those with large size or associated symptoms. aim: to describe a clinical case of esophageal diverticulum solved by minimally invasive surgery approach. clinical case description: a -year-old patient with a history of epilepsy and hypothyroidism consulted for atypical chest pain and dysphagia to liquids and solids. a study with esophagogastroduodenoscopy was performed: cm from the dental arch, a large wide-mouth diverticulum was identified. we complete the study with an esophagram with barium: voluminous diverticulum in the right lateral face of the middle esophageal and a thoracic ct scan showing esophageal diverticulum located in the carina, from . x . cm to cm from the esophagogastric junction. due to the suspicion of associated motor disorder, high resolution manometry was performed showing a significant motor disorder with alteration of peristalsis and exit obstruction with incomplete relaxation of the inferior sphincter and superior hypertonic sphincter. preliminary results: the patient underwent surgery: diverticulectomy and complete esophageal myotomy by thoracoscopy minimall invasive approach. the patient evolved favorably and was discharged after days with a previus control esophagram without pathological findings. currently, it remains asymptomatic months after surgery. , rectus muscles are re-approximated from xiphoid to pubis using laparoscopic running self-locking, pds sutures to restore anatomy and physiologic function of the abdominal wall. unlike the standard access to the abdominal cavity executed with lateral access, the lap-t technique is performed through sopra-pubic aesthetic approaches, using one mm and one mm bariatric ( cm) instruments laterally, and one mm camera in the middle.the entire procedure is performed in gas-less laparoscopy, with laryngeal mask and intra-peritoneal liquid anesthesia. the repair is consolidated placing an intra-peritoneal semi-absorbable mesh. preliminary results: in all cases abdominal functioning was successfully restored; no higher pain related to the continuous laparoscopic suturing has been reported compared to bridge ipom laparoscopic repair, while allowing for a more physiologic outcome and a stronger repair. the use of miniaturized instruments allowed for minimal tissue trauma and accurate surgical gestures; the tiny trocar sites did not require skin suturing and might reduce the risk of trocar hernias. no intra operative bleeding, no seroma formation, chronic pain, nor mesh infection have been recorded. % follow up at months, % at months with no recurrences observed. the lap-t technique allowed for a sound and anatomic reconstruction, reduced trauma, faster recovery and more satisfactory aesthetic results. surg endosc ( ) background: anastomotic leakage is a serious complication, associated with significant morbidity and mortality. one possible cause of anastomotic leakage is insufficient vascular supply. markers of sufficient perfusion include pink color of the bowel wall, visible peristalsis, palpable pulsations and bleeding from the marginal arteries. these signs are subjective and may be misinterpreted even by experienced surgeons. aim: the assessment of bowel perfusion with the use of indocyanine-green fluorescence angiography might be helpful in decreasing the number of anastomotic leaks. project description: we report a case report of a middle-aged patient without significant medical history who was treated by transanal total mesorectal excision (tatme) for rectal carcinoma. the patient underwent neoadjuvant treatment with radiochemotherapy. during the surgical procedure, indocyanine-green fluorescence angiography showed adequate perfusion of the bowel. the postoperative phase was uneventful and the patient was discharged home on the th postoperative day. preliminary results: indocyanine-green fluorescence angiography is a safe, cost-effective and feasible tool for assessment of tissue perfusion during colorectal resections. background: to properly learn how to perform a laparoscopic suture, along with safe tissue handling, applying an appropriate magnitude of the force on the tissue is essential. for this reason, it is fundamental to investigate and validate if training with real-time visual force feedback improves the suturing performance of laparoscopic novice surgeons. capturing all of the forces applied in laparoscopic in surgery in an unobtrusive way has been difficult in the past. sensor has supplied a novel force-sensing film (forcefilm) that can detect all of the forces applied with laparoscopic instruments without changing the surgical workflow or operation of the instruments. aim: to evaluate the effect of visual force feedback on surgical performance, applied force and surgeon's ergonomics during training of laparoscopic suturing using the sensor technology (sensor medical laboratories ltd.). methods: twenty novice laparoscopic surgeons participate in this study. they perform a laparoscopic suture on an ex vivo stomach tissue from a pig. participants are assigned, in a random fashion, to either group that receives visual force feedback (a) or the control group (b) without visual force feedback. five training trials (t -t ) are carried out in order to assess the learning curve. in addition, an evaluation pretest (t ) and posttest (t ), without visual force feedback but recording the force applied, will be performed after the training trials. the applied force on the tissue and visual force feedback of each instrument are provided by means of the sensor technology. it accurately measures the forces exerted on the tissue from the instrument tip and wirelessly communicates the force information to the surgeon via visual force-feedback. during each trial, several parameters are evaluated such as execution time, applied force, surgical performance, and mental and physical workload. preliminary results: laparoscopic training using visual force feedback leads to an improvement of suturing skills with a reduction of the applied force and therefore providing a potentially positive effect on patient outcomes and surgeon's ergonomics. background: hiatal hernia is a frequent disorder, characterized by a protrusion of any abdominal structure other than the esophagus into the thoracic cavity through a widening of the diaphragmatic hiatus. current anatomic classification is mainly based on the location of the gastroesophageal junction and the presence of a true hernial sac, differentiating sliding from paraesophageal hernias. there is no solid evidence to support an association between gastric carcinogenesis and peh. however, chronic reflux is considered as one of the strongest risk factors of developing adenocarcinoma of the esophagus and proximal stomach. aim: herein, we report a case of an -year-old caucasian female with dysphagia, regurgitation and heartburn accompanied by an iron deficiency anemia, a remarkable total body weight loss and recurrent lower respiratory tract infections due to microaspirations. subsequent work-up with ct, upper endoscopy and barium esophagram confirmed the presence of synchronous distal gastric adenocarcinoma and a giant paraesophageal hernia with complete intrathoracic stomach. after mdt discussion and keeping in mind the patient's age and comorbidity, a d laparoscopic distal gastrectomy with a synchronous hernia reduction with posterior cruropexy was scheduled. project description: the patient was placed in a supine position, five thoracoscopic ports were introduced, and a diagnostic laparoscopy of the abdominal cavity was performed. the stomach was identified through the dilated hiatus into the left thorax. the hernia sac was dissected away from mediastinal structures, then excised to untwist the stomach. after reduction of the stomach to abdominal cavity, a total d ? gastrectomy with a roux-en-y reconstruction was performed. maintenance of optimal vision during minimally invasive surgery is crucial to maintaining operative awareness, efficiency and safety. hampered vision is commonly caused by laparoscopic lens fogging (llf) and lens condensation which has prompted the development of various antifogging fluids and warming devices. numerous tricks have been proposed to overcome this issue, such as heating the scope into a sterile thermos flask filled with hot water, or using one of the commercially available antifogging solutions. however, whether one method is superior to another remains elusive. as most surgeons know, none of these tips are totally efficient, as they don't treat the cause: the temperature difference. taking into account this need, we have developed ehs (endoscopy heater system), a thermoadjustable system by microcontroller, which is implemented in the manufacturing process of the rigid endoscope focused on laparoscopy. the technology enables the self-modulation of the temperature of the endoscope within the different conditions during the surgery, avoiding the % of laparoscopic fogging. with the adoption of ehs surgeons get a clear field of vision avoiding continues repetitions of extraction and insertion of the endoscope in the body during the intervention. in this way the risks of the patients are reduced with a more efficient and shorter duration procedure. ehs also represents an alternative that meets sustainability criteria by reducing energy costs and eliminating much of the waste currently generated by this procedure. therefore, this innovation will disrupt the laparoscopic device market by enhancing safety and effectiveness without introducing new components that could complicate surgical procedures. case report: we presented the case of a year old women with chronic coloenteric fistula. conservative treatment was unsuccessful. the orifice was then closed with two subsequent clips, and the patient recovered well. to our knowledge, this is the first successful case of coloenteric fistula treatment with ovesco discussion: ovesco system is a technique that enables the closure of gastrointestinal defects (perforation sites, leaks, fistulas) . after the system application, the patient can be treated at home as was the case with our patient. a successful closure of the leak or fistula is possible when no extraluminal abscess is present. in our case, we had a cavity (previous sinus or abscess) that drained into the small bowel, thereby forming the coloenteric fistula. this allowed us to succeed with a fistula closure, as the cavity could drain into the small bowel conclusions: looking through the reports, one notes that the success rate of the otsc system procedure for insufficiency of anastomosis or colorectal fistula was - %, but only nine successful reports of chronic colorectal fistula were found). a % success rate is reported if the clip is placed within a week of occurrence of the leak . on considering the financial side, clips could reduce costs and time of hospitalization and avoid patients having to undergo a surgical repair . the major advantage of ovesco clips seems to be their ability to grasp more tissue compared to the standard clips and their strong grip on the wound margins because of their sharpened teeth. the drawback of the clips in fistula sealing is their incomplete grasp when the tissue is fibrotic. most authors agree that ovesco is not very appropriate for fistulas larger than - mm. inguinal hernia repair is one of the most performed interventions in minimally invasive surgery. in this opportunity we report a new technique through the use of innovative devices such as the robotic clamp and magnetic deviceswith this technique and thanks to the magnetized devices and the robotic clamp we have demonstrated to reduce the surgical time between - min as well as to optimize the ergonomics of the surgeon.we explain the technique with a demostrative video and exposition of the devices that are necessary for make it.with this new technique we get a greater capacity of mabiobra for dissection of the peritoneum and later a greater facility for the suture of the same in the repair of the inguinal hernia. a motorized and computerized laparoscopic tool that can be customized to the specific surgeon and procedure a. szold aim: surgeons have different levels of skill and use instruments for different tasks, but laparoscopic instrument are commonly simple mechanical instruments that allow limited degrees of motion, and the same instruments are used regardless of the surgeon or the task. robotized articulating instruments so far have added degrees of freedom, but perform in a standard way for all users and procedures. technology: human xtensions has developed a \ u[hand-held \/u [ motorized smart laparoscopic instrument, that was recently introduced in human procedures. the device has several features that enable to customize it to the user and procedure. the degrees of freedom can be reduced from to , the scale of rotation motion has options that can control both speed and range of rotation, a feature especially useful for the variable types of suturing tasks. results: the variable features were tested in different procedures requiring suturing and grasping. the combination of all optional settings made the instrument customizable to the different skill levels of the surgeons. as such, it enabled to control the complexity of the device and take the surgeon through the learning curve until full control of all features was achieved. in addition, the combination of different controls was used for performing specific tasks requiring different levels of maneuverability. in september , the results of a bomss survey regarding the routine use of pre-operative bariatric surgery were published. they found that % of units surveyed considered routine preoperative ogd completely unnecessary. as part of newly launching bariatric services in a single isolated centre we protocoled that all bariatric patients had to undergo pre-operative ogd, including a clo test, and reviewed if the ogd findings had influenced our surgical choice of operation and any necessary treatment before surgery. all patients embarking on the bariatric programme since its launch in jan to sept were included and had an ogd. the results of these ogds and all the clo tests were reviewed. these ogds were all performed by a single consultant to minimise any potential subjective differences. of the patients, ( %) tested clo positive of which had normal findings on ogd. patients had a hiatus hernia, gastritis, oesophagitis, gastritis and oesophagitis, had other findings e.g. ulcers, polyps' or nodules. the positive clo patients underwent eradication of h pylori. studies have shown that this is a treatable and preventable cause of gastritis/ gastric cancers and potential surgical complications causing prolonged hospital stay in % of patients. knowing about the presence of a hiatus hernia prior to surgery also contributed to the surgical planning, including allowing time for the concurrent correction of the hiatus hernia in the operation. all patients with demonstrable oesophagitis ( %) had their operative choice changed to roux en y gastric bypass thus aiming to prevent post-operative reflux which would have been exacerbated had they undergone a sleeve gastrectomy instead. carrying out a pre-operative ogd had a significant impact in operative choice and additional treatment before surgery and therefore should be advised in all patients. general surgery department, ahievran university, kirsehir, turkey the majority of fatalities worldwide in people under the age of years are caused by trauma . blunt mechanisms account for . to . %of injuries [ ] [ ] [ ] [ ] ,with the abdomen being affected in . to . % of all traumatic injuries. this case contribute to the literature: a patient with sleeve gastrectomy has distorted anatomy at duodenogastric junction, if has bat,her/his small bowel perforation(sbp) will occur on more distal segment. this a unique case before unpublished. years old female who had sleeve gastrectomy years ago presented to emergency department sustained blunt abdominal trauma (bat). when she arrived pyschical exam (pe) revealed an abdominal guarding, tenderness, normal vital signs but those increased h later. wbc values also increased h later. hb was normal.fast showed in cm thickness fluid early in douglas pauch (dp), mm in supravesical, mm diameter in dp h later. abdominal ct: mm diameter fluid in interloop, dp free air at th h from the accident. a diagnostic laparoscopy(dl) was done with diagnossed acute abdomen.there were a sbr-located cm from treitz, intraperioneal fibrin deposits and fluid-repaired with a primary suture.the patient discharged on days without any event. repeat ct scans are recommended for patients with initial suspected bowel injury. we could not do this; cause ct exam could taken in rush hours only but we did repeatly pe that peritoneal iritation signs increased,resulted a dl,surgical therapy. according to the literature; dl may be a good treatment option in these patients, to reduce morbidity or mortality, time to surgery has been emphasized. long interval between presentation and surgery was found to be associated with complications. very few reports of isolated jejunal transection following blunt abdominal trauma have been published in literature. the literature mentioned; the patients with sbp are hemodinamically stable on arrival to the hospital like our case are, a rupture of the jejunum was seen just distal to the duodenal-jejunal flexure but there were a perforation cm below treitz ligament and caused me to think the patient had sleeve gastrectomy and some brid around gastric and duodenal proximal jejunal part of intestines and also caused a new descended treitz ligament. normally external forces across to spine produce a blast effect on small bowel between treitz and ileocolic ligament. introduction: sasi bypass is a novel metabolic/bariatric surgery operation based on minigastric bypass and santoro's operation.it can be offered for patients with weight regain after sleeve gastrectomy. sleeve gastrectomy (sg)is a commonly performed bariatric procedure.weight regain following sg is a significant issue.yet,the understanding of this phenomenon is still unclear.rates of regain ranged from . % at years to . % at years.sasi bypass was an option for some candidates having sg done years back and failed to achieve the required weight loss or having weight regain.in sasi bypass, resleeve gastrectomy of the dilated gastric pouch is done followed by a side to side gastro-ileal anastomosis. the aim of this study is to report the clinical results and the outcomes of sasi bypass as a therapeutic option for patients with weight regain after sg methods: we conducted a retrospective study for morbidly obese patients having history of sg done more than years back and failed to achieve and/or to maintain the required bmi. exclusion criteria:patients with recent history of laparotomy(less than months). procedure was done at sidra hospital in kuwait from november to november . using ports, resleeve gastrectomy was performed over fr bougie tube starting cm above the pylorus then gastro-ileal anastomosis (side to side)was performed cm above the pyloric ring to an ileal loop counted cm from the ileocaecal valve. data was collected from the patients including:weight loss progress,laboratory full results. discussion and results: during the study period: morbidly obese patients with a mean bmi of ± kg/m were evaluated. -%ewl(excess weight loss)reached % at one year. -diabetes was cured in the known diabetic patients (type )within months,and the one known type diabetic patient had better control and less insulin daily doses(results were guided by glycated haemoglobin results every months). follow up laboratory results were normal in % of patients (all were kept on regular vitamins and proteins supplementation)-one patient had postoperative leak(day )from the anastomotic line that was treated conservatively. conclusion: sasi bypass is a promising operation that offers a good weight loss for morbidly obese patients having weight regain after sg conclusions: our study demonstrates a good agreement between the degree of liver steatosis and monocytes fat accumulation as well as between plin levels in liver and circulating monocytes. this suggests that ectopic fat deposition is a generalized feature of insulin resistance in obesity. sg reverses monocyte fat accumulation and restores insulin signalling, which correlates well with insulin sensitivity. moreover, circulating mmp levels significantly dropped after sg suggesting that the state of generalized inflammation characterizing obesity normalizes. her stomach stapled,a foreign tube like body was seen on cut surface of the stomach.the foreign body seen in dissected stomach wall is the tube is in placed a gastric banding insuflation tube.a laparatomy was made and the tube is extracted her stomach sutured primarily,nasogastric decompression,peritoneal drainage was made.her peritoneum was drained.she has a septic condition, leave in icu for a long period.her general status being well and discharged from hospital in days.we learn after the operation not before;she had a gastric adjustable banding and extraction the gastric band, but the tube of gastric band is not removed. alkhaffaf et al present a case of fistulation of the lagb tubing into the jejunum a review of the published data to identify the salient learning points with this and similar rare complications fistulation from lagb tubing is a rare complication that tends to follow removal of an infected port. the clinical presentation is nonspecific, rendering the preoperative diagnosis difficult. the tube and band can be removed laparoscopically, with closure of the small bowel fistula site. securing the tubing to the abdominal wall fascia after intentional detachment from the port might reduce the incidence of this complication.katherine j et al report a late and rare complication of a small bowel obstruction in a -year-old woman from an lagb placed for years. although not a common complication, one that could easily see the safety record of lagb patients tarnished if this small subgroup of patients is not acted upon promptly by emergency departments' unfamiliar lagb surgery. in our case we already made esophagogastroduodenoscopy before operation, ofcourse take past medical history from the patient.the patient hide past operation (gastric banding and removing band and port but leaving insuflation tube). ). there was no difference in the two groups regarding follow-up rate. basic demographics were the same, and other long-term results were similar between the groups. regression models for both post-op complications and failure as defined by baros score did not show that gender is a risk factor. discussion and conclusions: in our study, revisional sleeve surgery were similar. we did not see any significant difference in post-op complications, success of the operation as defined by baros, or subjective feeling of the patients. we do believe that gender-specific outcomes should be taken into consideration in optimizing patient selection and preoperative patient counseling, and that in the case of a sleeve post a band gender is not a risk factor for complication or failure of the procedure. objective: the internal hernia is a rare but a potentially fatal complication of laparoscopic roux-en-y gastric bypass (lrygb). the aims of this study are: ( ) to determine the impact of mesenteric defects closure on the incidence of internal hernia after lrygb; ( ) to determine the symptoms, characteristics and management of internal hernias after lrygb. the median interval between lrygb and reoperation was months in group a and months in group b. the median percentage of excess weight loss (%ewl) was % vs %, respectively (p = . ). the median percentage of total weight loss (%twl) was % vs %, respectively (p = ns). patients, % ( in group a), were admitted to the emergency room with acute abdomen pain. a ct scan was performed in patients, %, and showed signs of occlusion in all cases. the most common symptoms were abdominal pain and vomiting. the surgery was performed using laparoscopy in patients, %, and using laparotomy or conversion in patients, %. conclusions: the closure of mesenteric defects during lrygb is recommended because it is associated with a significant reduction in the incidence of internal hernia. our study intends to analyze the long term results of sleeve gastrectomies performed by d laparoscopic approach. materials and methods: a prospective cohort study was conducted to perform gastric sleeve for morbid abesity. all surgeries were performed by the same surgeon over a period of two years. the operating surgeon is a senior most laparoscopic surgeon with vast experience in laparoscopic surgery. during two years period, cases were operated using d laparoscopy system. scientific calculation was done using spss release . windows software. results: patients, female ( %) and male ( %), with median age of . ± . ( - ) the excess weight lost (ewl) was % in the first year, % in the second, % in the third, % in the fourth, % for the fith year, % for the sixth and % for the seventh. postoperative complications were stenosis of the sleeve always located in the incisura and treated with endoscopic dilatation except one that required conversion to oagb. three leakages, all of them reoperated with drainage and introducing prosthesis by endoscopy in the same act. we have never had a postoperative bleeding of the sleeve. conclusions: d gastric sleeve laparoscopy is a safe and feasible technique for morbid obesity and related pathologies. the ewl is correct in long time. complications are rare but is necessary to have a good level of suspicious in order to a rapid solution. the worst complication is the leak of the sleeve. the oversewn of the gastric section is a good technique to avoid this complication. surg endosc ( ) aims: leak is one of the common complications of laparoscopic sleeve gastrectomy that result prolongation of hospital stay, morbidity and even mortality. methods: i report new approach for the treatment of leaks presented to me post laparoscopic sleeve gastrectomy with laparoscopic roux en y bypass to the leak site at the level of gastroesophageal area. this new approach is possible and feasible, and avoids stenting due to high failure rate, prolonged hospitalization and saves life of patients. results: all leaks healed days from surgery due to well vascularized small intestinal patch, except for leaks that healed after weeks of conservative treatment. aims: analyse the effect of one anastomosis (mini) gastric bypass (mgb/oagb) in the treatment of gastro-esophageal reflux in patients previously submitted to laparoscopic sleeve gastrectomy (sg). methods: a retrospective analysis was performed on the data of patients who underwent mgb/ oagb after a previous sg at policlinico san marco, italy, from january to june . a total of patients, female and males ( % f/ % m) underwent mgb/oagb after sg, due to the development of significant gastro-esophageal reflux disease (gerd), refractory to proton pump inhibitors (ppi), detected with the gerd questionnaire (gerd-q) and esophagogastroduodenoscopy (egds). in three patients ( %) a weight regain was also observed (mean bmi . kg/m , range . kg/m - . kg/m ). mean patients age was . ( - years old). before sg none of the patients had declared symptoms of gerd or was subjected to a therapy with ppi, preoperative egds did not show signs of esophagitis. mean bmi of the patients who developed gerd without weight regain was . kg/m ( kg/m - . kg/m ) at the time of surgery, with a medium ewl% of % ( . - . %). patients were treated unsuccessfully with ppi for at least six months before programming revisional surgery. mean gerd-q score was . results: after mgb/oagb, with a mean follow up of months ( - months), mean bmi was . kg/m and gerd-q score was . however, five patients out of ( %) developed an anastomotic ulcer or a grade c esophagitis. we did not observe any post-operative immediate complication nor any death. conclusion: mgb/oagb is a simple, effective and safe surgical procedure for patients who underwent a previous sg and who developed gerd, with satisfactory results in the short and medium post-operative time, even if there is still concern regarding the complications linked to biliary reflux. v.s. kyosev aims: laparoscopic adjustable gastric band (lagb) was one of the common techniques in bariatric surgery worldwide. the advantages included the possibility of regulation, ease of placement, acceptable weight loss and low rate of perioperative complications. a late complication of lagb is penetration of the gatric band through the gastric wall and migration into the lumen of the stomach. hereby, we present three cases of gastric band migration following lagb. methods: from to we observed cases of gastric band migration in between and years after lagb placement. the patients were hospitalized in surgical department complaining of sudden sharp epigastric pain, nausea and vomiting, with symptoms onset in the last few days. all patients underwent abdominal ultrasound examination, x-ray investigation of the abdomen with oral contrast administration, fibrogastroscopy. in cases the imaging studies revealed gastric band migration into the stomach's lumen and in case-obstruction of the jejunum by the gastric band. all patients underwent laparoscopic surgery. results: two of the patients underwent gastrotomy, extraction of the gastric band and roux-and y-gatric bypass. the patient with jejunal obstruction underwent laparoscopic enterotomy, extraction of the gastric band and cholecystectomy due to concomitant cholecystitis. two of the patients had no additional perioperative complications and were discharged at the th postoperative day. one patient developed fever, left pleural effusion and partial insufficiency of the gastrointestinal anastomosis in the early postoperative period without the need of surgical treatment. the patient was discharged on th postoperative day. all patients were prescribed a diet and monthly blood test of ion balance. conclusions: lagb was one of the most common treatment methods due to the epidemic spread of morbid obesity in western countries. detailed knowledge on possible lagb complications is essential for the treatment of these patients. the diagnosis of lagb complications is often delayed due to its relative rarity and nonspecific clinical manifestations, but in most of the cases it requires emergency surgery for management of life-threatening conditions. results: there was conversion in patients (short mesentery of the small intestine). such postoperative complication like anastomotic leak in patient ( . %) and staple line bleeding in patient ( . %), which was managed laparoscopically. compensation for type diabetes was achieved in ( %) patients, improvement was recorded in ( . %), dyslipidemia in ( . %) and ( . %) patients, arterial hypertension in ( . %) and ( %) patients respectively, what led to metabolic syndrome resolution in ( %) patients. the liquid is allowed to take after day. average postoperative hospitalization- . ± . days. %ewl for months %. conclusions: laparoscopic mini gastric bypass is an effective method of surgical correction of body weight and metabolic disorders in patients with morbid obesity and allows to receive an adequate and stable correction of arterial hypertension, lipid and carbohydrate metabolism, which are components of a metabolic syndrome. introduction: over time laparoscopic sleeve gastrectomy lsg has become the most popular bariatric operation worldwide. a critical step during lsg is ensuring sleeve-size consistency. gastrisail device (gastric positioning system) is a three in one surgical device replacing the standard bougie used in lsg for the application of suction, decompression and to serve as a sizing guide for gastric sleeve creation. the aim of this study is to evaluate the possible merits of gastrisail device in lsg over the standard laparoscopic sleeve gastrectomy. methods: a prospective study of patients randomly divided into two groups: group a composed of twenty patients who undergo lsg with the use of gastrisail and group b composed of twenty patients who undergo lsg with the standard bougie without the use of gastrisail comparing both according to operative time, consistent sleeve formation, delineation and visualization, intraoperative and post-operative complication rates, the lenght of hospital stay,gastric pouch design and percentage of excess weight loss (%ewl). results: regarding intraoperative time, the mean time was . ± . and . ± . for group a and b respectively,while no patients in group b had consistent sleeve formation, patients ( %) had consistent sleeve formation. delineation and visualization were accomplished in % of group a patients, was not accomplished at all in group b patients. the alignment of the stomach was reached in patients in group a but no patients at all in group b, the mean of hospital stay was . ± . and . ± . for group a and b respectively, the smaller tube design illustrated by gastrograffin x-ray at rd post-operative day was accomplished in patients ( %) and patients ( %) in group a and b respectively. there was no significant difference in %ewl in both groups. conclusion: the use of gastrisail device is superior to the standard lsg in consistent sleeve formation, visualization, delineation and good alignment and accomplishment of a small tube design while no significant difference in %ewl. bariatric surgery has spread all over the world. since japan has few patients with morbid obesity compared with western countries, it has been implemented only in limited facilities. however, bariatric surgery in japan is rapidly spreading recently, and many facilities are about to install bariatric surgery. effects of bariatric surgery are known to last for a long time, but some cases require reoperation which is called revision surgery due to late complications or rebound. because of thick subcutaneous and visceral fat, open surgeries are not even always a good solution to make surgery easier in morbid obese patients and all procedures must be completed laparoscopically. therefore, especially in revision surgery, the incidence of complications tends to be increased. as the number of bariatric cases to be increased in japan, cases requiring revision surgery is likely to increase. in revision surgery, it is necessary to select the procedure according to patient condition, and it is necessary to familiar well with those procedures. we will present cases that underwent revision surgery in our department and show the clinical outcome. we have done four revision surgeries after sleeve gastrectomy so far. operative indications are mid-gastric stenosis and rebounding disease. for stenosis cases, we performed roux-en y gastric bypass with distal stomach resection, and for rebounding cases, we performed re-sleeve gastrectomy with duodenal-jejunal bypass. average interval from initial operation to revision surgery is months in rebounding cases, and months in stenosis cases. duration of operation was min in average, and mean estimated blood loss was ml. no postoperative mortality was observed. in rebounding cases, excess bmi loss at year after surgery was . % in average, and both cases achieve diabetes remission at year. one cases of mid-gastric stenosis required a nutritional support with formula diet temporally. in particular after sleeve gastrectomy, revising to roux-en y gastric bypass, re-sleeve gastrectomy, and adding the duodenal-jejunal bypass will be the main techniques. along with an increase of bariatric surgery in japan, it is necessary to acquire sufficient knowledge and skills to carry out revision surgery. methods: we present the case of a year old woman who underwent lsg after lagb removal and lgcp. the patient underwent preoperative endoscopy and barium swallow, with no sign of stomach perforation or erosion. we emphasize that the patient, had undergone three operations of gastric band placement, gastric band removal and gastric plication before sleeve gastrectomy. however, a successful lsg was achieved. results: no severe postoperative complications were mentioned. conclusion: weight loss in the first year was % of the excess weight.sleeve gastrectomy after gastric band removal and gastric plication, for morbid obesity seems to be safe and efficient, especially in casesof absence of gastric erosion. surg endosc ( ) department of surgery, patients were observed with serious septic complications many years after gastric banding operation. we detected a female dominance ( female, male) in patients with a mean age of . years. the leading symptoms were: dysphagia, upper abdominal tenderness and pain, spontaneous fistula formation, fever, masked septic signs, bowel and urinary obstruction. patients underwent video-endoscopy, chest and abdominal ct (computed tomography), fistulography and cystoscopy. results: in still morbid obese patients, laparoscopic procedures were performed with a conversion rate of %: atypic gastric and cardia resection in cases, gastric suture in cases, small bowel resection and suture in - cases. in one case, fistulectomy, abscess evacuation and combined urinary bladder suture and drainage were carried out. the duration of the surgeries were over h with minimal blood loss (\ ml). the foreign bodies were completely removed in every case. intraoperative complication was not occurred. early physiotherapy were promoted, oral feeding were gradually built up from the th postoperativ day depending on the type of the operation. early postoperative complications included recurrent fistula formation (n = ) and wound infection (n = ). all the fistulas were closed after conservative treatment. average hospital stay was days, regular check-ups were held on the rd, th and th months of follow up. conclusion: gastric banding is the most common, routine and safe technique for the treatment of morbid obesity. the development of late, severe septic complications draws attention to the crucial importance of follow up. the surgical management of these patients is recommended in specialized centers in regard to difficult operative conditions and atypic treatment options. aims: single anastomosis duodeno-ileal bypass with sleeve gastrectomy (sadi-s) has been proposed as an alternative to biliopancreatic diversion with duodenal switch (bpd-ds) in order to maintain the outcome of the original procedure simplifying the technical complexity and to avoid potential complications. moreover, it potentially represents the more natural second step bariatric procedure after sleeve gastrectomy (sg). we aimed to report the initial experience with sadi-s of our high volume bariatric center. methods: retrospective analysis of patients who underwent bariatric procedure between july and november was conducted. the primary aim was the evaluation of the safety of sadi-s, defined as the rate of postoperative complications. the secondary endpoint was the bariatric efficacy of the procedure, defined as percentage excess weight loss (%ewl). results: among patients who underwent bariatric procedures at our institution ( . %) patients were scheduled for sadi-s. all patients had multiple comorbidities. initial indication for sadi-s was failed sg in patients (median pre-sg bmi . kg/m \ sup [ \/sup [ ; median months after initial operation respectively) and primary procedure in patients (median pre-operative bmi . kg/m ). the surgical procedure was accomplished with robotic-assisted approach in cases (median operative time min) and with laparoscopic trocars standard approach in the remaining cases (median operative time min). the duodeno-ileal anastomosis was fashioned using a double layer hand-sewn running sutures. no patients showed early post-operative complications, the median postoperative stay was days. at a mean follow up of months the median %ewl was . . to date no patients experienced surgical. one patient develop wernicke encephalopathy months after surgery, but he was non-compliant to multivitamin supplementation. conclusions: at least in a high volume bariatric center sadi-s, both as second step after sg and as primary surgical option, seems to be a safe and effective bariatric metabolic procedure based on solid physiopathologic principles. on the other hand, longer follow-up is necessary to support the use of this procedure as a better alternative to bpd-ds. m.r. elkeleny , a. abo khozima git and bariatric surgery, faculty of medicine, alexandria university, alexandria, egypt; git surgery department, faculty of medicine,alexandria university, alexandria, egypt four bariatric cases . female patient with intragastric balloon, minor leak from the balloon leading to ballon migration to the jejunum;hance, small bowel obstruction occured. emergency diagnostic laparoscopy was done, enterotomy and extraction of the balloon, direct repair of enterotomy and balloon extraction through mm port site. . male patient presented after days of lsg with small bowel obstruction due to entrapment of small bowel loop through one of the port sites;therefor, emergency laparoscopy was done with reduction of the herniated segment and closure of the port site. . female patient presented with stricture of the ogj after re-sleeve gastrectomy managed by balloon dilatation which recur after weeks .she was managed by expandable metallic stent for weeks with good response and the stent was removed. . -year-old male patient presented with sever peripheral neuropathy following months after sleeve gastrectomy, and the patient was getting worse; thus, he used wheel chair. he has been making good progress on vitamin b complex injections. the aim of our study was to compare histopathological findings of gastric specimens to preoperative clinical symptoms and to conclude about the need for ugi endoscopy as a routine prior to surgery. methods: the last two years, morbid obese patients were selected to undergo laparoscopic sleeve gastrectomy (lsg) in our institution. for the needs of our study, all of them had ugi endoscopy and were reviewed for upper gi symptoms. histopathological reports obtained according to our protocol, after surgery. results: gastric histology from specimens revealed: no findings in / patients ( . %), gastritis in / patients ( . %) and focuses of incomplete intestinal metaplasia without dysplasia in / patients( . %). finally, two minor leiomyomas with low cellular proliferation rate were fully excised in a patient's specimen. there was no inconsistency between preoperative symptoms and gastric histology, while leiomyomas found were no reported to ugi endoscopy due to size. conclusions: some of the patients with clinical features of food intolerance, gastroesophageal reflux disease, and peptic ulcer disease had finally findings in histopathology of their stomachs. history of helicobacter pylori infection implements a raised incidence of mucosa pathology as well. because only one case revealed carrying significant pathology (leiomyomas), we consider that is safe to proceed with surgery in an otherwise asymptomatic patient based on his previous medical records and blood tests. aims: splenic abscess following laparoscopic sleeve gastrectomy (lsg) is a rarely seen complication. the aim of our study was to present a case of splenic abscess in a morbid obese patient who underwent lsg. as the main concern in these cases is leakage from the staple line, we present our diagnostic and treatment approach. methods: a -year-old, female morbid obese patient (bmi . kg/m ), without any predisposing risk factors, underwent elective lsg in our department. following an uneventful course, she was discharged at the nd postoperative day. however, at the th postoperative day, she readmitted to our unit with high temperature of . o c, left upper quadrant tenderness and leukocytosis. contrast computed tomography (ct) revealed an abscess at the upper pole of the spleen , cm in maximum diameter, without leakage from the staple line. results: the patient was treated with broad-spectrum antibiotics and radiological percutaneous drainage of the abscess. although there was a partial clinical improvement, a week later, a new ct scan revealed the continuous presence of the abscess. despite the stable general condition of the patient a laparoscopic splenectomy was performed and a gradual recovery was followed. the presence of splenic abscess without splenic trauma or leakage from the gastric staple line, is an extremely rare complication and only a few cases have been previously reported. the cause has not yet clarified, but the proposed mechanism involves infarction of the spleen, due to vascular compromise and subsequent infection. most of the reported splenic abscesses were diagnosed during the late postoperative period. in our report we present a case of early onset, hence highlighting the need of clinical awareness for early diagnosis and treatment. introduction: obese surgical patients with obstructive sleep apnea (osa) have a higher risk of peri-and postoperative desaturations and subsequent morbidity and mortality. currently, the best perioperative management of patients without known osa remains unclear. although routine osa screening has been advocated, sleep studies are costly and time consuming. we hypothesized that bariatric patients can be safely monitored on a surgical ward by continuous postoperative pulse oximetry without preoperative screening for osa. objectives: to evaluate outcomes of continuous postoperative pulse oximetry without preoperative osa-screening, and to compare the results to outcomes of patients with osa and continuous positive airway pressure (cpap) treatment. methods: all patients who underwent bariatric surgery between and were included in this single-center retrospective cohort study. all patients were postoperatively monitored with continuous pulse oximetry on the surgical ward. patients with less than two documented saturation measurements were excluded. patient files were reviewed for osa diagnosis, cpap usage and perioperative details. primary outcomes were -day complication rates, intensive care unit admissions due to cardiopulmonary causes and postoperative desaturations of spo \ %. secondary outcomes were icu admissions following all causes, length of stay and rates of reoperation and readmission. results: in total, patients were included. patients ( %) were preoperatively diagnosed with osa, ( . %) were cpap users. complications occurred in . % of patients without osa and in . % with osa(p = . ). desaturations were documented in . % and % (p \ . ), respectively. in both groups, patient was admitted postoperatively to the icu for cardiopulmonary causes that could be related to osa (p = . ). both recovered without further complications. icu admissions, regardless of cause, occurred in . % of patients without osa and in . % with osa(p \ . ). no significant difference between groups was observed in complications based on clavien dindo classification, length of stay, reoperation-and readmissions-rates. conclusions: these findings suggest that continuous postoperative pulse oximetry without preoperative osa-screening is a safe perioperative management strategy for bariatric surgical patients. future studies are needed to assess cost-effectiveness of pulse oximetry vs. routine preoperative osa-screening in a prospective clinical setting. background: the pathology of colon is one of the most pressing and socially significant problems of modern health care, because it leads to reduction of the working population employed in manufacturing, in some cases to disability and reduced quality of life. mini invasive surgery of the colon has a great advantage: speed recovery, shorter hospital stay and better cosmetic results, the quickest return of patients to work. as a result, mini invasive endovideosurgery is firmly established in clinical practice of coloproctology. objective: the choice of optimal surgical method for treatment of colostasis, achievement of favorable outcomes of treatment. introduction: laparoscopic roux-en-y gastric bypass (rygb) is one of the most important bariatric surgical procedures performed worldwide and it can produce an important loss of weight with reversal of metabolic disorders like diabetes and dyslipidemia. even though it has good results, some complications occur after gastric bypass. a rare but serious complication of rygb is the so-called postprandial hyperinsulinemic hypoglycemia. its prevalence has been estimated less than % of cases and its pathophysiology remains unclear. methods: the aim is to present a case series of reversal surgery in patients with severe hiperinsulinemic hypoglycemia after rygbp in the hospital general universitari de la vall d'hebron. unit of endocrine-metabolic and bariatric surgery (eac-bs center of excellence for bariatric and metabolic surgery by ifso). it is a retrospective analysis of a prospective database same surgical team. we present in this study, the main features of those patients. results: between and , patients underwent a laparoscopic reversal procedure to normal anatomy and age mean was year ( years to years). mean preoperative body mass index (bmi) was . kg/m (range - . kg/m ) and were women. all patients presented hypoglycemia symptoms years after and the longest was years after the procedure. the first step of the standard approach was a laparoscopic reversal to normal anatomy with resection of the alimentary rygb limb in cases. a concomitant sleeve-like gastrectomy (sg) was added. four patients presented postoperative complications: gastrogastric anastomosis leak ( ) introduction: laparoscopic sleeve gastrectomy is the most performed bariatric procedure, but complications might interfere with patient's long-term evolution based on its compliance and tolerance, surgical attitude and unpredictable evolution. materials: we present the case of a female obese patient, with type ii diabetes mellitus and blood hypertension, with multiple, sequential bariatric minimally-invasive interventions: sleeve gastrectomy in complicated by postoperative acute gastric dilation and mediogastric stenosis, reoperated for viscerolysis and cholecystectomy, with endoscopic gastric dilations, initially converted to functional one anastomosis gastric bypass ( cm limb), with a non-adjustable gastric ring positioned instead of stapled division. the last operation was complicated months after by persistent biliary gastro-esophageal reflux, chronic abdominal pain, and gas bloat syndrome. in the patient underwent conversion to laparoscopic r-en-y gastric bypass, with gastro-enteral anastomosis resection, band removal and viscerolysis. results: conversion to r-en-y was complicated by biliary leakage post-viscerolysis, treated with laparoscopic approach in the \ sup [ th \/sup [ po day. after multiple surgical and endoscopic interventions, the patient presents short-term favorable outcomes, with no reflux or abdominal pain, with further weight loss and diabetes improvement. conclusion: bariatric surgery has unpredictable evolution in same cases, and conversion to r-en-y seems to be the best solution. lgcp is widely used in developing countries due to its lower cost and good results. material and methods: we performed in our department lgcp for morbid obesity. excess weight loss (%ewl) was % at month after surgery and % at one year. in cases revision surgery was needed for different complications and in cases for inadequate weight loss or weight regain after month follow up. in cases we performed sleeve gastrectomy (in cases after taking down the plication) and in cases we performed a re-plication in one row. results: the rate of revision surgery was % overall and % for inadequate weight loss (excess weight loss \ %) or weight regain. major complications occurred only in one patient (leak with abscess) but it was solved by laparoscopy. minor complications as vomiting and nausea appeared in patients ( %) and were solved with medication. after one year follow up %ewl in these cases was %. conclusions: revision surgery after lgcp is possible. a new plication or sg was the option in our series with good results. further studies are needed to evaluate the use of lgcp in the armamentarium of bariatric surgery. background: roux-en-y gastric bypass (rygb) is one of the most commonly performed bariatric procedures around the world.however, rygb it sometimes carries the risk of rarebut serious long-term complications such as malnutrition and liver failure. we report a case of laparoscopic reversal of rygb. methods: in march , a laparoscopic rygb was performed for a -year-old female without comorbidities and with a bmi of kg/m . all laboratory test results at the preoperative evaluation were within the normal range. abdominal ultrasound revealed moderate hepatic steatosis and oral endoscopy a hiatal hernia with grade b esophagitis. one year later, patient experienced an important weight loss of kg (from to kg) with a bmi of kg/m . however, patient presented general weakness, abdominal pain, ascitis lower extremitiy edema, anemia, progressive caloric and protein malnutrition, vitamin (a, d), mineral (copper) and folic acid deficiencies, nonalcoholic steatohepatitis (nash) and liver function was progressive worsening. results: a laparoscopic reversal of gastric bypass was performed. the operation was successfully performed via laparoscopy. operating time was min. postoperative was uneventful and patient discharge home at day . hepatic biopsy revealed nash with steatohepatitis of % (fibrosis f - / ). eight months after reversal of gastric bypass, patient has improved her clinical situation (no asthenia), maintains of weight ( kg) and has improved her nutritional status and liver function parameters. conclusion: laparoscopic reversal of rygb is technically feasible and might be performed safely after thorough preoperative evaluation in carefully selected patients with malnutrition and liver failure. conclusion: laparoscopic sleeve gastrectomy it's a safety obesity procedure before major abdominal hernia repair. it's a minimally invasively technique with an absence of anastomoses. these factors prevent fewer complications, without using the small bowel, and skin problems and allow resolution of obesity-associated co-morbidities. body weight loss after surgery may be an opportunity to repair the severe loss of domain incisional hernia. bibliography borbély, y., zerkowski, j., altmeier, j., eschenburg, a., kröll, d. and nett, p. general surgery, benhazi medical center, benghazi, libia, general surgery, royal bahrain hospital, manama, bahrain obesity is a worldwide epidemic with an increasing incidence trends and as a consequence obesity related health problems become priority to healthcare authorities in all the countries. laparoscopic gastric plication is an emergent restrictive procedure which claimed to be low cost because they do not need staplers and carries less complications as compared to laparoscopic sleeve gastrectomy. we present here a years female who was operated for morbid obesity four months back where she underwent laparoscopic gastric plication with no immediate post operative complication and her wight loss was adequate. two days before presentation to our emergency department she started to complains of sever attacks or upper abdominal pain and vomiting.clinical examination was unremarkable apart of abdominal tenderness in left upper abdomen. all blood routine were normal and all inflammatory markers were within normal range.ct abdomen showed large cystic lesion around the greater gastric curvature containing fluid and raised possibility of collection. patient was admitted to hospital, in despite of medical treatment her pain persists and necessitate immediate laparoscopic exploration. gastro-gastric hernia at the greater curvature through loosen ethibond suture that was used to plicate the stomach in the previous surgery. we released the suture to liberate the strangulated stomach which is not gangrenous. re-plication was not possible because of the extensive gastric wall edema and as preoperative discussion with the patient she refused conversion to sleeve gastrectomy no intervention was done. post surgery patient was free of symptoms and tolerating oral diet and discharged home on third post operative day with no complications. gastro-gastric herniation could progress to gastric wall gangrene which will result in high morbidity and even mortality. high index of suspicion is required to diagnose the condition . preoperative patient counseling is important to explore the surgical options if deemed necessary to convert to another bariatric procedure. k. chouillard, a. d'alessandro, l. chahine background: bariatric surgery is the best available, long-term treatment for morbid obesity. currently, laparoscopic sleeve gastrectomy (sg) is the most commonly performed bariatric procedure in france. despite its safety and efficacy, long-term complications of sg are not rare including gastro-esophageal reflux disease (gerfd), twisting, stenosis, insufficient weight loss, and weight regain. the goal of this study was to analyze the pattern and short-term results of surgical revision in patients with sg. methods: revisional bariatric surgery, regardless of its motivation, was always a multidisciplinary decision after clinical, biological, endoscopic, and radiological assessment. patients who had revisional surgery after sg were retrospectively identified and subsequently divided in subgroups according to preoperative body mass index ( we aim to present the management and the particular aspects of the surgical technique in a gastrobronchial fistula after gastric sleeve . the mean time between intervention and diagnosis is . - . months. methods: between and , laparoscopic gastric sleeve resections were performed in our bariatric center. we had one case of gastrobronchial fistula associated with an inferior lobe abscess of the left lung, diagnosed months after the gastric sleeve. the patient was subject for medical treatment for h, than a laparoscopic intervention was performed in order to drain the lung abscess and the gastric fistula and to place a feeding jejunostomy. . months after this intervention ( . months after gastric sleeve) a laparoscopic roux-en-y fistulojejunostomy was performed. the evolution was monitorized with blood tests, upper gi contrast series and ct scans. results: the surgical drainage of the lung abscess, along with the antibiotherapy, controlled the infection and allowed the lung cavity to reduce in size, and thus the drainage tubes introduced in the thorax through the diaphragmatic orifice were retracted progressively. also, the feeding jejunostomy allowed a proper nutrition for the patient with a good recovery. however, . months after the drainage intervention, the gastric fistula was not healed, and a decision to interrupt the communication with the lung cavity was made, by creating a laparoscopic fistulojejunostomy. after this, the evolution was favorable, with the healing of the lung cavity, oral feeding was permitted and the jejunostomy was suppressed. conclusions: the treatment of the gastrobronchial fistula is complex (medical, endoscopic or surgical), phased and long lasting until healing. surgery was our initial choice for treatment due to the existence of the lung abscess, which needed to be drained. key words: gastrobronchial fistula, lung abscess, laparoscopy, fistulojejunostomy s.i. filip, i. hutopila, c. copaescu introduction: leakage remains one of the most dreadful complications in metabolic surgery. the main cause of leakage is poor tissue oxygenation due to inadequate vascular perfusion. the study of intraoperative tissue perfusion in real time due to icg enhanced fluorescence could provide valuable information for the surgeon in order to prevent postoperative fistula. aim: to present our experience in using icg enhanced fluorescence in laparoscopic bariatric surgery material and method: in cases of gastric sleeve, cases of gastric bypass and in cases of revisional surgery or redo cases we used intraoperative icg mediated fluorescence to assure the optimal vascularization of the involved tissues. in our video we present intraoperative aspects before and after using icg in different cases. results: in all cases of primary gastric sleeve and gastric bypass with intraoperative use of icg we did not encounter inadequate perfusion. in one case of redo gastric bypass after failed vertical banded gastroplasty for morbid obesity despite intraoperative laparoscopic normal aspect of the gastro-jejunal anastomosis, icg mediated fluorescence allowed to identify an unexpected ischemic anastomosis and we could prevent consecutive postoperative leakage. discussion: presented cases are discussed and result with referral to literature is made. conclusion: intraoperative use of icg is a valuable tool in assessing the perfusion of the tissues and provide essential information for the surgeon in order to avoid postoperative leakage. , including patients, hemostasis with clips has been performed in all cases. however, among these cases nine patients required reoperation for early postoperative bleeding. in five cases a bleeding source from the stapled line was identified while in cases no identifiable source was found. during the second period ( to present) patients were submitted to bariatric surgery and hemostasis was performed by over sewing with a running suture. among these cases reoperation for postoperative bleeding was needed in cases ( . %), but no bleeding from the staple line being encountered ( %). the difference has statistical significance. no significant complications related to the use of this type of reinforcement were encountered. conclusions: over sewing the gastric stapled line in bariatric surgery is superior to hemostatic clip application in preventing the postoperative bleeding from the stapled line postoperative bleeding. a protocol of active search of the bleeders during the bariatric procedure should be implemented and respected in all the cases. gastroenterological surgery, saitama medical university international medical center, hidaka-shi, saitama, japan intestinal endometriosis is a rare disease which is associated with about to % of patients with endometriosis, and it is favorable to the rectum and sigmoid colon. here we report cases (shown in the table) underwent laparoscopic resection for intestinal endometriosis. there were no postoperative complications in all cases, and all patient was discharged on - \ sup [ th \/sup [ postoperative day. before the operation, of patients were diagnosing intestinal endometriosis, and it was difficult to preoperatively diagnose. among them, the symptoms at the time of menstruation were clear was one case. in case of submucosal tumor, preoperative diagnosis seems difficult. additional image examination at menstruation may be useful for diagnosis. d dissection was performed for case , , because malignant disease could not be denied as a preoperative diagnosis. of them were strongly doubted endometriosis in surgical findings. in intestinal endometriosis surgery, pelvic adhesions and fibrosis are often advanced. in the sigmoidectomy, the average operation time was min and the blood loss was ml. in the rectal resection, the average operation time was min and the blood loss was ml. in case and , pelvic adhesion was severe, residual rectum could not be straightened, and side to side anastomosis was performed. in intestinal endometriosis surgery, intestinal anastomosis method should be considered flexibly. conclusion: laparoscopic surgery for intestinal endometriosis was safe, but technically difficult because of fibrosis and adhesion. it is important to accurately diagnose from clinical symptoms and image also intraoperative findings. anastomotic method should be decided according to the case. aim: the aim of the study was to identify and highlight some of the complications one can encounter in bariatric surgery-specific-sleeve gastrectomy and discuss the therapeutic options one has at his disposal. methods: the study was retrospective. we identified a number of patients which had a sleeve gastrectomy done in our clinic for a year period. of these had important surgical complications encountered during the surgery or in postoperative care. results: the group included patients, with an average bmi of [ kg / m . average hospital stay was days, with an average of . days which increased to days when fistulas were encountered. the most frequent surgical complications were bleeding from the gastric suture ( ) and gastric fistula ( cases). other complications encountered were wound hematoma. surgery was required in of the cases of bleeding and of the fistula cases required reintervention. one case was resolved with endoscopic stenting. conclusions: laparoscopic gastrectomy is considered a safe procedure with good results for the patient. although complications are rare they pose a series of technical difficulties for the surgeon due to the weight of the patient and frequent comorbidities which come with obesity. a thorough understanding of the symptoms and good follow-up ensures the best results. aims: to achieve additional weight loss or to resolve band-related problems, a laparoscopic adjustable gastric banding (lagb) can be converted to a laparoscopic roux-en-y gastric bypass (rygb). there is limited data on the feasibility and safety of routinely performing a single-step conversion. we assessed the efficacy of this revisional approach in a large cohort of patients operated in a high-volume bariatric institution. to the best of our knowledge this series represents the largest single-center study on conversion from lagb to rygb methods: between october and december , a total of patients who underwent lagb removal with rygb were identified from a prospectively collected database. in all cases, a single-stage conversion procedure was planned. the feasibility of this approach and peri-operative outcomes of these patients were evaluated and analyzed. results: a single-step approach was successfully achieved in ( . %) of the patients. during the study period, there was a significant increase in performing the conversion from lagb to rygb single-staged. no mortality or anastomotic leakage was observed in both groups. only patients ( . %) had a -d complication: most commonly hemorrhage (n? = ? / ), with no significant difference between the groups. conclusion: converting a lagb to rygb can be performed with a very low morbidity and zero-mortality in a high-volume revisional bariatric center. with increasing experience and full standardization of the conversion, the vast majority of operations can be performed as a single-stage procedure. only a migrated band remains a formal contraindication for a one-step approach. surg endosc ( ) . six months after surgery the mean hrql score, was ( - ) in patients underwent to lsg and . ( - ) in patients underwent lgb. twelve months after surgery the mean postoperative questionnaire score was ( - ) in patients who underwent lsg. at ph-manometry the mean percentage time of acid reflux in orthostatism was . (range - . ) and in clinostatism . (range . - . ). the mean demeester score at the distal electrode was . ( . - . ). conclusions: in asymptomatic patients, complete gerd evaluation before bariatric surgery allows better selection of surgical procedure, to reduce the postoperative occurrence of severe or de novo gerd. postoperative gerd evaluation provides useful data regarding the impact of lsg on gastroesophageal reflux. a larger patient sample size is required. aims: vertical calibrated gastrectomy (usually know as gastric sleeve) as unique technique gives better results than the roux y bypass in terms of improvement of anthropometric measures, reduces comorbidities and has a lower rate of postsurgical complications, with an improvement of quality of life. material and methods: an observational, longitudinal, retrospective and comparative study with patients, aged - years,during a period of years. everyone must comply with the protocol of the unit. demographics of the population and the anthropometric data will be measured in the presurgical consultation, the month and the year after the surgery: weight, height, bmi, weight loss percentage,bmi percentage and percentage of excess weight lost. we took data on the cardiovascular risk by the framingham score. the quality of life is measured by baros scale. mayor comorbidities are hypertension, diabetes, dyslipidemia. complications will be measured in absolute frequencies. for de statistical study, we apply type t student or chi square being statistically significant p equal to or less than . . results: there was not statistically significant difference between the techniques of surgery month (p = , ), but they were evident to the year of the same (p . ). not gender or age differences were apparent. mayor complications did not appear in gastrectomy (no leaks), highlighting the number of bleeds with this surgical technique. the bypass there were two leaks. there was no statistically significant difference in cardiovascular risk (p = , ) between the two techniques. there was a more significant decrease in number of comorbidities in gastrectomy against the bypass, with a total disappearance of patients with dyslipidemia. there were no statistically significant differences in baros score, although it was higher in gastrectomy. conclusions:-the vertical gastrectomy as unique technique can be considered superior in the short term, as well as safe, according to the aec quality parameter. we think it will be necessary to continue their studies into the medium-long term. aims: analyze the impact of different bariatric surgeries technics in carbohydrate metabolism and pancreatic beta cell population of none obese adult wistar rats. methods: we used twenty healthy not obese adult wistar rats divided in five groups randomly assigned. each with n = . the control groups were divided into fasting control (f) and sham (surgical control). the surgical groups were separated into vertical gastrectomy (gs), % resection of the middle small bowel (ri ) and gastric bypass (gb). in each group was assessment: beta cell mass modifications, pancreatic islets histomorphometry, proliferation, apoptosis and neogenesis in beta-cell pancreatic population; intraperitoneal glucose test tolerance, body weight and food intake. statistical analysis as evaluated using mann whitney test. results: the malabsorptive and restrictive group have a significantly smaller increase weight than the control groups. the intraperitoneal tolerance glucose test reports incremental glucose area under curve (auc) was significantly higher in the malabsorptive group and lower in the restrictive group compare to the control groups during the second (p \ . ) and third (p \ . ) month of the study. the beta-cell mass was significantly higher in the ri group compared with control groups respectively. there was a significantly increased number of beta-cell per pancreatic insulin positive area in gs and gb. proliferation was significantly increased in ri and gb group, and significantly decreased in sg compared. there was no significantly difference during apoptosis assessment among surgical and control groups. in neogenesis differences between groups were assessed qualitatively by the presence pdx - expression, being higher in rygb. the endocrine pancreas in our model is altered by the anatomical and functional conditions arising from surgical techniques. carbohydrate metabolism conditions are affected by temporary adaptive processes due to surgical alternatives. there is a hyperplasia and hypertrophy of the beta cells in surgeries with a malabsorptive component, as well as greater neogenesis. these results could explain part of the existing relationship between the enteropancreatic axis and the existing incretins. m. buza, c. copaescu introduction: nowadays, we have high volumes of obese patients for whom surgery is the answer, but unfortunately the psychological evaluation has no standard recomandation in preoperative evaluation of bariatric patients. it is argued that surgery success, in addition to the operation itself, relies on behavioral changes and that one of the goals of the preoperative assessment is to prepare the patient for the postoperative period, aiming to optimize surgical results. aim: although no formal standard exists in the literature, there is growing recognition of the important elements to be addressed and the appropriate means for collecting the necessary data to determine psychological readiness for these procedures. methods: information regarding the components of the clinical interview and the specific measures used for psychological testing are discussed. given the limited data on predicting success after surgery, determining psychological contraindications for surgery is addressed. additionally, the multiple functions served by the psychologist during this assessment procedure are highlighted along with the value of this procedure in the patients' preparation for surgery as well as the postoperative follow-up. in our center of excelence for bariatric and metabolic surgery (coe) we introduced since a mandatory pre-and postoperative psychological evaluation for all patients addressing the metabolic program. results: psychological evaluation of patients before bariatric surgery is a critical step, not only to identify contraindications for surgery, but also-and more so-to better understand their motivation, readiness, behavioral challenges, and emotional factors that may impact their coping and adjustment through surgery and the associated lifestyle changes. postoperative follow-up is necessary. the psychological evaluation of the patient undergoing bariatric surgery is an invaluable piece of the larger pre-and post-surgical assessment, aiming better results in the short and long term after bariatric surgery. introduction: a mesenteric cyst is defined as a benign abdominal tumors that is located in the mesentery of the gastrointestinal tract, identified in * of , hospital admissions. mesenteric chylous cysts are rare pathologic entities that often present with unspecific symptoms. the preoperative diagnosis requires all the common abdominal imaging techniques. usually the correct diagnosis may be made only at the operation stage or during the histological examination. all mesenteric cyst should be resected in order to avoid their complications, complete surgical resection is recommended and curative in the majority of cases with a low risk of local recurrence. the laparoscopic approach is the gold standard in the treatment of intraabdominal mesenteric chylous cyst. laparoscopic resection provides less pain, shorter hospital stay, and early recovery for the patient. case report: we report a case of -year-old saudi woman who presented to our clinic complaining of upper abdominal pain and mass in the epigastrium for one week, no history of nausea, vomiting, or recent changes in bowel habits. her medical and family histories were clear and she had never had any abdominal interventions. abdominal palpation revealed a smooth-surfaced mass palpable in the left upper quadrant, ultrasonography and with computed tomography of the abdomen revealed an approximately mm unilocular cyst closely related to the mesentery in the left side of upper abdomen not related to the pancreas .the cyst was excised by laparoscopy complete surgical excision to avoid recurrence within healthy borders, it is contained milky white fluid. the histopathological findings were chronic inflamed mesenteric cyst. a review of the literature considering this rare entity was also performed to evaluate our treatment strategy. conclusion: mesenteric chylous cysts represent a diagnostic challenge and they should be considered when a physician encounters an intraabdominal mass. usually the correct diagnosis may be made only at the operation stage or during the histological examination. the treatment of choice is the complete surgical excision that can be safely performed by laparoscopy. surg endosc ( ) background: diverticulum of appendix is relatively rare, and appendiceal diverticulitis was reported to have a higher risk of perforation than appendicitis. in the us and europe, because of the high risk of perforation, preventive appendectomy is recommended to appendiceal diverticulosis, even if the patient has no abdominal pain. methods: we retrospectively reviewed the records of post-operative patients, who were diagnosed appendicitis or appendiceal diverticulitis on the pathological findings in our institution from january to october . all patients were performed computed tomography (ct) before operation. patients underwent laparoscopic surgery, including appendectomy, cecal resection, ileocecal resection and right hemicolectomy, while patients underwent open surgery. total of cases of appendiceal diverticulitis were analyzed in our study. result: patients had abdominal pain before surgery. patients were diagnosed appendiceal diverticulitis by preoperative ct. all patients underwent laparoscopic surgery ( appendectomy, cecal resection, and ileocecal resection). on the pathological findings, perforation of appendix was found in patients and the pseudo type of diverticula with no muscle layer was found in all patients. patients with appendicitis were treated surgically during the same period. among them, a perforation of appendix was found in cases. the perforation rate was . %. on the other hand, the perforation rate of appendiceal diverticulitis was . % in our study. conclusion: the perforation rate of appendiceal diverticulitis was higher than of appendicitis in our study. for the examination of the treatment strategy, including preventive appendectomy, the accumulation of more cases will be expected. case presentation: a -year-old man was referred to our hospital with right lower quadrant abdominal pain for days. his fever was . °c. his white blood cell count was , / ll, and c-reactive protein level was . mg/dl. ct revealed multiple diverticula of cecum and appendix. micro-abscess and free air were found around appendix. we diagnosed this case as appendiceal diverticulitis and laparoscopic appendectomy was performed. a perforation was found in resected appendix. microscopic study revealed a pseudo-diverticulum. the inflammation of appendix was stronger in serous membrane side than in mucosa side. this finding accorded with appendiceal diverticulitis. introduction: in order to reduce the abdominal trauma and the length of scar incisions (also during laparoscopic surgery) many approaches during the last decade has been proposed, such as single access laparoscopic surgery (sals). the aim of our paper was to update the data of our previous paper with a greater cohort of patients and a longer follow-up, also showing the single access laparoscopic left colectomy (salc) technique in particular with inferior mesenteric artery preservation imap (valdoni's technique). materials and methods: we made a retrospective analysis from october and october of all patients who underwent a sals approach for colorectal disease in the department of general and mininvasive surgery of san camillo hospital of trento. statistical analysis was performed using ibm spss statistics . continuous data were expressed as mean ± standard deviation (sd). categorical data were expressed as absolute number and percentage. the results are presented as -tailed values with statistical significance if p values \ . results: from october until october , salc for colorectal surgery were performed in our unit. of this , were for left colectomy. in cases we performed an imap. the salc with imap were performed only in case of benign disease. the mean operative time was . ± . . only one intraoperative complication were recorded, that was a splenic capsule tear, resolved with apposition of fibrillar haemostats. according to clavien dindo classification there were in particular grade ii complications, a bleeding solved with blood transfusion and one pancreatitis solved with medical therapy; grade iiia complications that was anastomotic bleeding solved endoscopically (the two complications raised in patients with imap) and iiib complications due to anastomotic leakage which needed reoperation. the mean length of incision was . ± . cm. logistic regression did not show any correlation between imap and any complications. conclusion: in conclusion, salc is a safe but very challenging technique which need a longer learning curve than the conventional laparoscopic one. in laparoscopic colectomy, also, imap seems to be safe and effective without correlation with post-operative complications also if performed in single access laparoscopic approach. aims: to describe an infrequent anatomical variation that can give rise to diagnostic and therapeutic difficulties. methods: patient with ivermark syndrome (situs ambiguus and polysplenia) with acute appendicitis and bibliographic review results: a -year-old male who consulted for flank and right hypochondrium pain of h of evolution, associated with nausea without vomiting, no fever noir other symptoms. to the physical examination good general condition. painful to palpation selectively on the flank and right hypochondrium, with involuntary defense and positive decompression at this level. the signs of rovsing and psoas were negatives. in the analytical performed leukocytes of , with neutrophils in % and rpc (reactive protein c) in mg/l. abdominal ct (computed tomograph): cecum and the ilio-cecal valve were visualized at the subhepatic level with tubular structure on the side and seemed to correspond to the cecal appendix which is increased in size ( mm), with findings suggestive of acute appendicitis. sigma and descending colon located in right hemiabdomen. second per duodenal portion located anterior to the superior mesenteric artery. superior mesenteric vein located to the left of the superior mesenteric artery, rotating around it, (radiological signs compatible with intestinal malrotation). no free fluid collections nor pneumoperitoneum. laparoscopic appendectomy on phlegmonous acute appendicitis without incidents. correct post-operative course, being discharged at h; the pathological anatomy was reported as acute appendicitis in phlegmonous. conclusions: ivermark syndrome is a genetic alteration with a multifactorial inheritance pattern, characterized by an alteration in the situation of the mesenteric vessels, which leads to abnormal rotation of the intestine during the embryonic period and alteration of the situation of different intra-abdominal organs, without a specific pattern that is pathognomonic, is associated with congenital heart anomalies between and %. reaching adulthood only between and % of them. a case of acute appendicitis is presented in a patient with this anomaly, which can lead to diagnostic and therapeutic difficulties due to the anatomical variations involved. abdominal tomography is the image method that provides the best performance for the diagnosis of acute pathologies in this type of patients. background: the clinical manifestations which occur in relation to decompression during scuba diving are variable. mild symptoms have often been reported in gastrointestinal tract. this is one of the severe cases with gastrointestinal barotrauma. ischemic colitis caused by air embolism very rare, therefore it is to be reported and discussed. case presentation: a -year-old man visited our emergency room with diffuse abdominal pain and bloody diarrhea days ago. the patient was a skilled diver who took seafood through diving for years. two days before presenting, the patient had severe abdominal pain just after diving for h at a depth of meters. he was immediately transferred to a local hospital for hyperbaric oxygen therapy, but there was no improvement with the symptom. abdomen ct angiography showed terminal ileal, ascending, sigmoid colonic and rectal decreased enhancement with wall thickening. sigmoidoscopy showed diffuse huge ulcerative lesions and ischemic changes on mid rectum and sigmoid colon. emergent subtotal colectomy and temporary loop ileostomy were done, and pathologic findings revealed diffuse mural infarct with serosal abscess formation in whole colon and transmural infarct in terminal ileum. conclusion: surgical approach could be one of the treatment options, though it depends on severity of the symptoms and the patients' conditions. colonic lipomas are extremely uncommon benign tumours, with an incidence ranging between . % and . %. although they are most frequently asymptomatic, when colonic lipomas are [ ?cm, they may present symptoms such as constipation, abdominal pain or rectal bleeding. most colonic lipomas typically occur in middle aged women and are located in the ascending colon and the caecum, while occurrence in other parts of the colon and rectum is rare. in this case report, we describe a lipoma that caused descendent bowel intussusception. a -year-old male presented with longstanding history of constipation. personal history of interest included active smoker, hypertension, hypercholesterolemia, psoriasis with joint affectation and reiter syndrome. he had had no previous surgery. he attended the emergency services on th july with a two-day bowel obstruction, without fever or nausea, being attended by our surgical emergency unit. he had been assessed during the previous months by gastroenterology, with a colonoscopy that showed a cm submucosal lesion that partially occluded descendent bowel, with inconclusive biopsy. an abdominal contrast-enhanced computed tomography (ct) was performed, confirming a welldefined mass located in splenic flexure of descendent bowel, conditioning a large bowel intussusception, nevertheless with no obstructive acute signs. the surgery was scheduled a few weeks later, performing a laparoscopic segmental resection with primary anastomosis including oncologic margins. the patient evolved satisfactorily in the postoperative period and was discharged six days after the surgery without any complications. likewise, he was monitored on a regular basis at our outpatient department and was free of symptoms at the -month follow-up visit. the histological analysis revealed a cm ulcerated lipoma affecting % of bowel circumference. the molecular study, using fluorescent in situ hybridation (fish) showed no mdm gene amplification. laparoscopic segmental resection of the large bowel is a safe and feasible technique for the treatment of large bowel intussusception caused by a colonic lipoma. the complete removal of the lipoma will condition the prognosis. furthermore, in the future, endoscopic surgery using colonoscopy could be employed when having a certain preoperative diagnosis of lipoma. surg endosc ( ) introduction: acute appendicitis is one of the most common abdominal surgical emergency, the diagnosis of which mostly relies on conventional methods such as physical examination and blood tests. the use of ultrasonography and ct abdomen aids in more precise diagnosis especially in patients with atypical presentation or in elderly. aim: this study aims to evaluate the ability of the neutrophil/lymphocyte ratio (nlr), platelet/lymphocyte ratio (plr) and mean platelet volume (mpv) in predicting the diagnosis of acute appendicitis. methods: retrospective analysis of prospectivly maintained data of all patients ( ) admitted with acute appendicitis to the emergency department at a tertiary hospital in the middle east between january till september . medical records and database of patients,who had appendicectomy for clinically and radiologically proven appendicitis, were reviewed. the retrieved data included patient's demographic and laboratory values of white blood cells (wbc), neutrophil (n), lymphocyte (l), and platelet (p) along with their ratios for comparison. results: spss version was used for tabulating the data. the recommended cutoff value of the nlr, plr and mpv in predicting the diagnosis of acute appendicitis was decided by using receiver operating characteristic (roc) curve analyses. at least for nlr, the confidence interval (ci) was . which is percentage of the positive values, since the confidence limit was between to %. our results showed that the laboratory parameters were fairly significant since the confidence interval was . in predicting the diagnosis in our population. conclusion: although appendicitis is a clinical diagnosis but laboratory parameters specially nlr, plr and mpv can be used as an adjunct in the diagnosis of acute appendicitis. literature is scarce concerning the validity of such parameters in our part of the world and prospective randomized controlled trials are needed to prove the efficacy of such rationale. objective: tumors of the cecal appendix represent a subset of colonic neoplasms whose early diagnosis is a real clinical challenge. correspond to . % of all gastrointestinal tumors and their prognosis depends on the type of injury, being the most frequent variety the carcinoid type. appendix involvement in endometriosis is rare, accounting for % of all endometriosis cases, and sometimes mimicking cecal tumors. methods: a -year-old woman with a history of hypothyroidism due to autoimmune thyroiditis and atrophic gastritis with gastric neuroendocrine tumors resected by endoscopy that in the digestive unit reviews, tac with double contrast was requested, showing a lobulated lesion in the cecum adjacent to the ileocecal valve, with contrast enhancement of approximately mm, suggestive of tumor. the colonoscopy evidenced a protruding appendicular osteum with inflammatory aspect that was biopsied. the pathological anatomy of the biopsy reports chronic congestive colitis with edema of the own blade and minimal acute activity, with moderate local eosinophilia.the case was presented in the multidisciplinary oncology committee and it is decided, due to the patient's background, to perform surgery on the lesion. laparoscopic right hemicolectomy was performed, with extracorporeal latero-lateral mechanical anastomosis with endogia signiaÒ mm. results: the patient evolves favorably, with good oral tolerance and depositional habit. she is sent home at the sixth postoperative day. the pathological anatomy reports tumor injury in the appendicular ostium compatible with endometriosis at the base of the cecal appendix implantation, ruling out malignant tumor pathology. conclusions: gastrointestinal tract endometriosis represents - % of cases, being most frequently located in the rectal-sigmoid region. appendix involvement in endometriosis is rare, accounting - % of all endometriosis cases and presents a preoperative diagnostic challenge, because sometimes mimicking a carcinoid cecal tumor. in our case, due to the patient's history, we assumed that the cecal lesion was a carcinoid tumor, so we performed a laparoscopic right colectomy, but if we had known that it was an endometriosis, we could have performed an appendectomy, although in both cases the laparoscopic approach gives us some benefits compared to the open approach aims: the natural history and predictive factors associated with chronic anastomotic complications have not been clearly studied. the aim of this study was to evaluated the predictive factors related to chronic anastomotic complications methods: from january to december , a total of patients who underwent anastomotic leakage were enrolled in this study. all patients underwent anterior resection with or without defunctioning stoma due to colorectal cancer. the patients received follow-up by clinical examination and abdominopelvic computed tomography (ct). they underwent a follow-up ct every months for the first year and then every months for the next years after that. complicated group (cg) underwent chronic anastomotic complications. normal group (ng) didn't underwent chronic anastomotic complications like stricture, fistula, chronic sinus, etc. results: there were no significant differences in gender, age, preoperative chemoradiotherapy and operation type between two groups. low rectum lesion and defunctioning stoma at the time of primary surgery were more frequent in cg (p = . , . ). there were no significant differences in type of anastomotic leakage, international leakage grade and ct findings at the time of diagnosis of anastomotic leakage. however, abnormal ct findings at the time of month were more frequent in cg group (p \ . ). in multivariate analysis, abnormal ct finding at the th months was only significant factor related to chronic anastomotic complications. conclusions: abnormal ct findings at the th month associated with prediction of chronic anastomotic complications. aims: acute appendicitis is the most common cause of acute abdomen requiring surgical intervention in the world. nowadays, standard treatment of acute appendicitis involves a surgical approach, eitherlaparoscopic or open.the purpose of the present study is to evaluate the safety of a discharge within less than h after performing appendectomy as a result of an uncomplicated acute appendicitis. conclusions: patients who undergo appendectomy (open or laparoscopic) for acute uncomplicated appendicitis, without surgical incidents and an adequate social/family network, can be discharged in less than h without a higher risk of post-operative complications or readmissions than patients with longer postoperative stays. it will be necessary to conduct more prospective studies with higher level of evidence that could corroborate our results. aims: median arcuate ligament syndrome (mals), also known as the celiac axis compression syndrome, is a rare condition caused by to the compression of the celiac trunk and the nerves located in this area (celiac plexus) by the median arcuate ligament. it is believed that mals is caused by the median arcuate ligament compression of the celiac plexus nerves over the celiac trunk, but another probably cause may be the lack of blood flow to the organs supplied by the celiac artery, however, this theory is controversial. the first clinical sign of mals is the apparition of postprandial abdominal pain in the upper abdomen. this typical pain forces patients to avoid eating, which can lead to loss weight (often more than pounds). other associated symptoms may include nausea, diarrhea, vomiting and delayed gastric emptying (a delay in food moving from the stomach into the small intestine). in relation to this uncommon condition, we present a clinical case of laparoscopic management of mals. methods: we present a -year-old patient with complaints of recurrent epigastric pain, postprandial vomiting and loss weight. blood tests and gastroscopy were performed to help ruling out more common causes of his symptoms, such as gastroesophageal reflux disease (gerd), gastritis or gastroparesis. as a part of the differential diagnosis, mals was suspected and a mesenteric doppler ultrasound was ordered to check blood flow through the celiac trunk and evaluate a possible compression of the celiac plexus. also, an angio-ct scan was also performed to confirm the diagnosis. once the mals was diagnosed, we decided to perform a laparoscopic approach as definitive surgical procedure. results: the patient was discharged h after surgery with no remarkable events during his postoperative stay. he has been followed up during months, remaining asymptomatic. conclusions: laparoscopic approach in mals offers a superior visualization during the surgery and involves lower morbidity in compare to open approach, which makes it an optimal treatment for this condition. aim: pilonidal sinus is a common disease with annoying and often painful symptoms. traditional surgical techniques for its treatment are characterized by either intense postoperative pain and prolonged wound-healing periods (wide resection, marsupialization) or unsatisfying aesthetic results (advancement or rhomboid flaps). 'endoscopic pilonidal sinus treatment' (epsit) is a new minimally invasive technique which utilises the meinero scope, primarily designed for the endoscopic treatment of complex perianal fistulas in a technique known as vaaft. we present our experience and outcomes in three treatment centers in northern greece. methods: between july and november we treated patients with pilonidal sinus using the epsit technique. the mean age of patients was , and % of them were male. patients were treated in the acute phase with the presence of pilonidal abscess. all operations were performed by two laparoendoscopic surgeons specifically trained in the technique. most patients were treated on a day-case basis. postoperative wound care included daily tract irrigation with ml of saline for a total of days. results: there were no immediate postoperative complications. medium postoperative pain was . on a vas scale. % of patients were discharged on the same day, patients remained in hospital for one day mainly due to social reasons. return to daily activities was immediate. in a maximum follow-up of months we observed recurrences. conclusions: epsit is a promising minimally invasive technique for the treatment of pilonidal sinus. what makes it mostly attractive is the minimal amount of postoperative pain, the excellent cosmetic result and the fast recovery with return to daily activities. introduction: isolated acute chylous peritonitis is a rare event. when presented as an acute abdomen warranting surgical intervention, it is often difficult to determine the cause pre-operatively. here, we report a case of acute chylous peritonitis due to meckel's diverticulitis presented with the clinical features suggestive of acute appendicitis. presentation of the case: a -year-old female presented with abdominal pain and clinical features consistent with acute appendicitis underwent diagnostic laparoscopy. she was found to have four-quadrant chylous peritonitis and ileus caused by an inflamed meckel's diverticulum adhered underneath a loop of small bowel and mesentery leaking chyle. after uneventful postoperative recovery, she was discharged at post-operative day two with oral antibiotics and was advised to take a low-fat diet. aims: perforated diverticulitis with purulent peritonitis (hinchey iii) has traditionally been treated with surgery including colon resection and stoma (hartmann procedure) with considerable postoperative morbidity and mortality. laparoscopic lavage has been suggested as a less invasive surgical treatment. methods: a -year-old woman with a -day history of abdominal discomfort exacerbed during the last h. ct scan showed neumoperitoneum accompanied by free fluid and a cm collection adjacent to descending colon showing diverticula suggestive of covert perforation. after h of non-response to medical treatment, associated with the impossibility of percutaneous drainage through interposition of intestinal loops, colon and lumbar vessels, urgent surgical intervention is decided. results: laparoscopic lavage of all quadrants was performed with saline, l or more, of body temperature, until clear fluid was returned. two non-suction j-pratt drains were placed. intravenous antibiotics were continued for a minimum of h, then oral antibiotics were continued for week. oral fluids were commenced on the first postoperative day and solids were subsequently introduced, depending on clinical progress. conclusion: laparoscopic management is reasonable alternative to the traditional open resection for hinchey grade ii-iii perforated diverticulitis with generalized peritonitis. this approach has a low mortality rate despite patient co-morbidity and disease severity. benefits include stoma avoidance and minimal wound infection. subsequent elective resection is probably unnecessary and readmission in the medium term is uncommon. background: constipation and fecal incontinence are common annoying complications after pull through procedures for hirschsprung disease (hsd). many causes could be the etiology of these problems. perineal descent syndrome could be the major hidden cause of these complications. the aim of this study is to evaluate the role of perineal descent syndrome in the development of post pull through constipation and fecal incontinence in addition to evaluate the role of laparoscopic rectopexy for treatment of these problems. \ b[patient and methods: \/b [ patients treated with pull through for hsd over the period of five years. out of the patients presented with constipation and fecal incontinence. patients with constipation and patients with fecal incontinence. rectal exam, anorectal manomety, defecography, contrast enema, rectal biopsy, emg, proctoscopy and endorectal ultrasound were performed to all patients. patients with stricture, missed aganglionic segment, injured internal anal sphincter, and loss of the sensory mucosa above the dentate line were excluded from the study. anterior wall rectopexy was performed for anterior wall rectocele. posterior wall rectocele was treated by retro rectal mesh rectopexy. emg is repeated weeks and months after surgery. outcome measurements included constipation, fecal incontinence and pudendal nerve latency. results: cases of post pull through constipation and fecal incontinence. patients with constipation and patients with fecal incontinence. patients with stricture, patients with missed aganglionic segment, patients with loss of anal sensory sensation and patients with injured anal sphincter were excluded from the study. defecography showed patients with anterior rectocele ( males and females) and patients with posterior rectocele ( males and females). the patients mean age . ± . years . emg showed prolonged pudendal nerve conduction in all cases. anterior wall and retro rectal rectopexy were performed laparoscopically without complications. constipation was resolved in all patients after surgery. all patients showed fully control in defecation. pudendal nerve latency decreased in all patients. conclusion: perineal descent syndrome proved to be a major hidden cause of post pull-through constipation and fecal incontinence. laparoscopic rectopexy showed a good solution of these complications. cystic lymphangioma is a rare entity. the surgical indication is determined by the size and symptomatology, and consists of the complete exeresis of the tumor. the laparoscopic approach is feasible in these cases, allowing a broad visualization of the anatomy, accessibility to the retroperitoneum in the context of a minimally invasive approach and a better recovery of the patient, without providing an increase in morbidity compared to the conventional. in this way we defend as a technique of choice laparoscopic surgery against these rare tumors for the general surgeon in the abdominal cavity, betting on a minimally invasive surgery. aims: laparoscopicposterior sutured rectopexy is one of the accepted treatment options for fullthickness rectal prolapse. recently, reduced port surgery(rps) has beenan emerging concept that, compared with conventional multiple port surgery (mps), yields reduced postoperative pain and improved cosmesis. the aim of the study is to evaluate the feasibility and safety of rps for fullthickness rectal prolapse. methods: rps was performed by single-incision plus one puncture, using internal organ retractor(ior) to secure operative field. straining one ior by - strings in - directions makes it possible to retract the internal organs three-dimensionally. this multi-directional flexible retraction could secure good operative field. from to , patients (rps: cases, mrs: cases) underwent laparoscopicposterior suture rectopexyfor total rectal prolapse. shortterm outcomes were compared between the two procedures. results: there was no significant difference between rps and mps in median operative time ( vs . min, respectively, p [ . ). the median blood loss volume was not significantly different between rps and mps groups ( . vs . ml, p \ . ). the duration of median hospital stay after surgery was not significantly different between two groups ( . vs days, respectively, p [ . ). the frequency of complications after surgery were not different between them. conclusions: reduced port lap-rectopexy can be a good therapeutic option for total rectal prolapse. a prospective, randomized, controlled trial should be conducted to confirm the superiority of this procedure over mps. the piccolo project proposes a new compact, hybrid and multimodal photonics endoscope based on optical coherence tomography (oct) and multi-photon tomography (mpt) combined with novel red-flag fluorescence technology for in vivo diagnosis and clinical decision support. for its development it includes different phases of validation. within this framework, the present study has as main objective: to characterize a model of rat colonic hyperplasia, which will be used for the development and validation of the previously mentioned endoscopic technology. secondary objectives: procure the reproducibility of the model chosen and determine the optimal time, after induction of the model. material and methods: animals (rattus norvegicus), wistar, males and females \ -yearold, randomly distributed. group (n = ): by laparotomy, a non-resorbable suture (silk / ), not stenosing, is placed through the wall of the colon. group (n = ): by endoscopy, a . mm long segment of a polymeric catheter is inserted, which is fixed to the wall of the colon by means of a suture. group (n = ): by means of endoscopy, a self-expanding and uncoated metallic stent are placed in the colon. group (n = ): a superficial laser resection of the colonic mucosa is performed by endoscopy. group (n = ): as an extension of the most optimal model. weekly, the animals were anesthetized again to perform a colonoscopy, which determined the degree of mucosal growth in descending colon and colonic biopsies were extracted weekly ( weeks). results: group . growth around the sutures after the second follow-up, diagnosed as hyperplastic polyps after a histopathological analysis. aim: the role of laparoscopy in the management of generalized appendicular peritonitis is controversial. this is due mainly to the lack of scientific data. through this study and a laborious bibliography research, we proposed to report our experience in terms of postoperative results, in the laparoscopic treatment of generalized appendicular peritonitis and to try to identify the risk factors associated with the occurrence of global morbidity and conclude on the feasibility of this technique in its treatment. methods: we conducted a retrospective study including all cases of generalized appendicular peritonitis managed laparoscopically, in the general surgery department of charles nicolle hospital between january and december . results: we identified patients. the mean age was . years. one fifth of the cases required a midline conversion ( . %). the mean operative time was . ± , min. the overall morbidity rate was % including surgical complications. there were no deaths. in uni-variate analysis, comorbidity, crp [ mg /l, operative time exceeding min and midline conversion were significantly associated with postoperative morbidity. co-morbidity, diabetes, asa score [ , delay of consultation [ days, intra-abdominal abscess and operative time exceeding min were significantly associated with medical complications. the univariate analysis also revealed that crp [ mg /l and midline conversion were predictive of surgical complications.the multivariate analysis identified the midline conversion as the only independent factor significantly associated with post operative morbidity (odds ratio = . , % confidence interval [ . - . ] ). conclusion: based on our results, it appears reasonable to continue the laparoscopic management of diffuse appendicular peritonitis. however, enhance this technique is basic in order to reduce midline conversion rate and to shorten operative time, which can lead to post operative complications. aims: currently, acute appendicitis is the most common surgical emergency. laparoscopic appendectomy is the usual procedure to treat acute appendicitis. the aim of this study is to evaluate the safety of electrocoagulation in the treatment of mesoappendix in laparoscopic appendectomy. methods: we have retrospectively studied a prospective database of operated patients of appendecectomy in emergency surgery unit. we have reviewed laparoscopic appendectomies from june st, to december st, . the mesoappendix was electrocoagulated in every laparoscopic appendectomy. the statistical analyses has been done with spss . version. results: our group consists of patients of which . % were male and . % were female. the average age was . years with a standard deviation of . % and p was . years. the most common total stay was day ( patients). the usual post-operative stay was one day ( ). we classified the diagnosis in complicated apendicitis ( patients) and no complicated apendicitis ( patients). the conversion rate was . % ( ). the main surgical complications were: surgical wound infection ( . %); intraabdominal abscess ( . %); and bleeding ( %). only one of the patients that suffered bleeding had complicated appendicitis. the medical complications were catheter sepsis ( . %); respiratory infection ( . %); cardiologicals ( , %); and paralytic ileus ( . %). the treatment of mesoappendix with electrocoagulation is safe and effective since the complications rate is very low. even so, it would be necessary to conduct more prospectives randomized studies in order to get enough evidence about the treatment of mesoappendix with monopolar electrocoagulation. introduction: the difficulty of resection of the rectum is determined by its anatomical relationships, intimately in contact with the bladder, seminal vesicles, prostate and urethra in the case of the male, vagina in the woman and nerve structures that will give defecatory, genital and urinary functionality. this structure creates a big impediment due to problems of visualization and difficult dissection, in such a way that conventional surgical techniques instigates a series of complications derived from this difficulty. we propose a new approach in rectal surgery in patients with inflammatory bowel disease. material and methods: a -year-old man with a history of ulcerative colitis developed a severe acute outbreak refractory to treatment. a total laparoscopic colectomy with a terminal ileostomy was performed in . in he was notified for reconstruction. we evidenced a rectal stump of about cm with signs of inflammatory disease at the mucosal level. a transanal proctectomy was performed with confection of 'j-pouch' and ileoanal anastomosis about cm from the anal margin by laparoscopy. the postoperative courses favorably, being discharged on the sixth day. currently in follow-up in digestive and general surgery, he is asymptomatic and he has an optimum level of quality of life valued by the sf- weeks after the intervention. conclusions: our service introduces the transanal approach to the performance of proctectomy in cases of inflammatory disease, a technique that provides clear advantages by improving visualization and the identification of anatomical structures. in this way, a safe dissection of the pelvis is achieved, adjusted to the serosa of the rectum, with preservation of the mesorectum and the hypogastric plexus, and with the consequent improvement of the genital and urinary function. the result is an equally safe surgery, which implies little increase in operative time and with better and shorter postoperative recovery.the conservation of the pelvic innervation avoids disorders of ejaculation, vaginal lubrication and bladder and rectal motility. the transanal approach for the performance of proctectomy provides benefits in terms of the preservation of the hypogastric plexus, minimizing the anatomical difficulties involved in rectal surgery and maintaining urinary and sexual function. aims: to evaluate the feasibility and outcomes of laparoscopic appendicectomies in both simple and complicated appendicitis, given the increasing trend towards a laparoscopic approach in the last four decades for the treatment of acute appendicitis. we present data from a district general hospital over a -year period. methods: we retrospectively analysed a single consultant's continually updated database of laparoscopic appendicectomies between / / and / / ( months). patient demographics, investigations, intraoperative findings and postoperative outcomes were recorded and analysed. complicated appendicitis was defined as the formation of appendiceal mass or abscess with or without perforation and peritonitis. results: cases of laparoscopic appendicectomies were identified during the specified period. the median patient age was (range - years). true positive rates for uss and ct were % and %, respectively. the rate of negative appendicectomies was %. transanal minimally invasive surgery (tamis) has been used for the treatment of rectal neoplasms such us benign polyps and early rectal cancer. when the tumour is located in the upper rectum or close to the rectosigmoid junction, this approach may be technically dificcult.we present a video of a tamis resection of a large polyp located cm from the anal verge. after properative examination and ct and mri were performed, the patient was prepared for surgery, and a trasnanal minimally invasive surgery was proposed.resection of the polyp was performed with the aim of an endogia and conventional laparoscopic materials. total resection of the polyp with free margin was possible. the postoperative pathology report confirmed a high grade displasia villo-tubular adenoma with a lesion free margin. tamis resection of tumours located above the rectosigmoid junction may be a safe and feasible technique in selected patients. aims: pelvic organ prolapse (pop) is a very relevant problem for women's quality of life and has a prevalence of about % defined by symptoms and up to % when established by physical examination. nowadays, sacrorectopexy for posterior pop and sacrocolpopexy for apical pop are considered the gold standard techniques. recently, we have seen that laparoscopic lateral suspension is a feasible procedure for apical pop, obtaining a success rate higher than % at one year. these results are similar to what we can achieve with sacrocolpopexy. methods: we herein present the case of a -year-old woman with apical and posterior pop, this was provoking an important impact on her quality of life, with obstructive defecation (needing digitations) and urinary incontinence. we proposed sacrorectopexy for her posterior pop and laparoscopic lateral suspension for her apical pop. in the video we can see how we perform a ventral mesh sacrorectopexy, following d'hoore technique; and a laparoscopic lateral suspension with preperitoneal dissection, following the technique described by the team headed by dubuisson and veit-rubin. we used laparoscopic ports ( , , . and . mm). results: patient was discharged home on the second postoperative day and has not had any sign of recurrence or extrusion after more than two years of follow-up. in addition, she has not suffered lower urinary tract symptoms, constipation or pain. conclusions: we present a case in which we have carried out a laparoscopic lateral suspension instead of a sacrocolpopexy for an apical pop, obtaining good short-term and long-term results. we consider it is very soon to assess this technique's efficacy and it has to be validated in studies with larger source of patients. nevertheless, we think this procedure might become an excellent alternative to sacrocolpopexy for apical pop. aims: laparoscopy is a minimally invasive approach with low morbidity. the aim is to show the usefulness of the laparoscopic approach for massive intra-abdominal abscesses, which it is controversial. we report three patients who underwent emergency laparoscopy for peritonitis or massive intra-abdominal abscesses not amenable to percutaneous approach that were suspected to be caused by acute diverticulitis. methods: all patients had diagnosis of acute diverticulitis (hinchey ii-iii grade) with pelvic abscesses situated between sigma and bladder or diffuse peritonitis. the patients with hinchey ii grade had failed conservative management with antibiotics. they underwent emergency laparoscopy under general anaesthesia, with three abdominal ports. intra-abdominal abscess cavities were exposed and the purulent exudate was sampled and aspirated. copious irrigation was performed under direct vision and thorough examination without other findings. the procedure was completed laparoscopically in all cases. results: all patients had favourable evolution. one of them had a properly drained faecal fistula which changed to a purulent fistula on the twentieth postoperative day. this patient underwent laparoscopic left colectomy three months later because he had have a new episode of acute diverticulitis. other two cases showed very good clinical evolution, without evidence of fistula in postoperative period and they were complete asymptomatic one month later. conclusion: in our experience laparoscopic drainage is a feasible, safe, and effective for the treatment of pelvic abscesses and diffuse peritonitis secondary to acute diverticulitis. n. pinheiro, a. ziegler introduction: solitary rectal ulcer syndrome (susr) is characterized as a rare disease whose pathophysiology remains uncertain. it was first described in by cruveilhier and his clinicopathological feature was reported in by mandigan and morson, where he is associated with defective disorders, internal rectal prolapse, and psychological changes. according to works about % of the patients are asymptomatic. when symptomatic the diagnosis can be made through physical examination, clinical history and, often, confirmed by endoscopy with biopsies. treatment depends on the severity of the symptoms and the existence of associated rectal prolapse. according to the literature, conventional surgical options include local excision, rectal mucosectomy, retopexy, and segmental colonic resection. rolato: a -year-old male complaining of anal bleeding at bowel movements years ago. he performed, several times, conservative treatment, but without improvement. he sought proctological care and underwent colonoscopy, in which he showed an ulcerated lesion on the anterior wall of the distal rectum. new investigation with videodefecogram revealed colorectal intussusception with associated mucosal prolapse, being considered the factor causing the ulcer. elected by the sacropromontofixação. evoluiu with improvement of anal bleeding, mucorrhea and anal discomfort. after a proctological examination, which was normal, a control colonoscopy performed after months of surgery revealed rectal mucosa, with residual scarring and disappearance of the submucosal nodule present in the initial examination. reassessed after months, the patient is asymptomatic. conclusion: rectal solitary ulcer whose causal factor was a colorectal prolapse (intussusception) with mucosal exteriorization through the anal canal, which was individually treated with sacropromontofixation. j.p. mali, p.j. mentula, a.k. leppäniemi, v.j. sallinen approximately - % of patients diagnosed with colonic diverticulitis have an intra-abdominal abscess as a complication. abscess diameter of - cm is generally accepted as a cut-off determining the choice of treatment between antibiotics alone and percutaneous drainage. the aim of this study was to analyze the treatment choices and outcomes of patients with diverticular abscesses. this was a retrospective cohort study which was conducted in helsinki university hospital, an academic teaching hospital functioning as secondary and tertiary referral center. patients with computer tomography-verified acute left-side colonic diverticulitis with intra-abdominal abscess were collected from a database containing all patients treated for colonic diverticulitis in our institution during - . altogether, suitable patients were included in analyses. those treated primarily with percutaneous drainage or antibiotics alone ( and patients, respectively) were further compared in regards to treatment results. the main measured outcomes were need of emergency surgery and -day mortality. abscesses under mm were mostly treated with antibiotics alone with high success rate ( out of , %). in abscesses over mm, the use of emergency surgery increased and use of antibiotics alone decreased with increasing abscess size, but the proportion of successful drainage remained at - % regardless of abscess size (figure ). there were no differences in failure rate, -day mortality, need of emergency surgery, permanent stoma, recurrence, or length of stay in patients treated with percutaneous drainage versus antibiotics alone, even when groups were adjusted for potential confounders. white blood cell count = . * /l, abscess diameter = mm, and corticosteroid medication were independent risk factors for failure of treatment with antibiotics alone. patients without these risk factor had % and patients with one risk factor had % success with antibiotics alone. percutaneous drainage as treatment for large abscess does not seem to be superior to treatment with only antibiotics. majority of patients with abscesses over mm in diameter undergo surgery as primary intervention. introduction: even today, 'chronic appendicitis' is a clinical term that is not widely accepted nor well documented amongst the medical community. its etiology is the presence of a mass (e.g. fecal mass, hyperplasia of lymphatic tissue, etc.) that continuously and partially obstructs appendix lumen. it is presented as a low intensity, intermittent, with exacerbations and remissions, abdominal pain that is located at the right iliac region. the pain lasts up to several months and it is usually underestimated by the patient. its diagnosis is based on imaging examination. appendectomy is the treatment of choice for chronic appendicitis. the operation is challenging for the surgeon who has to cope with an intensively inflamed area around the appendix without the ease of access to that area. purpose: to present our laparoscopic approach to a chronic appendicitis case and to review the literature. case report: a -year-old woman is hospitalized due to chronic appendicitis. the patient was treated conservatively with the use of intravenous antibiotics in two separate hospital admissions dated and months back respectively. eight weeks after the last exacerbation, she underwent a laparoscopic appendectomy. results: even though the procedure was planned six months after the first episode, the laparoscopy revealed a severe inflammation of the appendix, which was extended to the caecum and the surrounding preperitoneal tissues. although the difficultness of the operation it was completed successfully laparoscopically. the histological examination confirmed without any doubt the existence of 'chronic appendicitis'. the patient was discharged uneventfully the third postoperative day. conclusions: chronic appendicitis is an existing clinical entity that the surgeon may come through during his career. in the hands of experienced laparoscopic surgeon, the laparoscopic approach is feasible and safe. introduction: ventriculo-peritoneal shunting (vps) used in the treatment for hydrocephalus is associated with several complications.the exact cause of such extrusion is not known. visceral perforation is an unusual but serious complication with consequeces such as peritonitis, meningitis or encephalitis. management involves prompt removal of shunt, intravenous antibiotics, an adequate recovery gap so that cerebrospinal fluid culture is sterile and then followed by shunt replacement on opposite side. aim: multidisciplinary approach of extrusion of vps through anus by laparoscopic and external ventricular drainage. case exposure: a -year-old woman had a vps inserted year ago after excision of gangliocytoma due to lhermitte-duclos disease. she was admitted in the emergency department without symptos after trans-anal protrusion of vps catheter. the neurological and abdominal evaluation was normal. laboratory tests did not reveal disorders and abdominal ct-scan suggested perforation, itshowed the insertion of the end of the catheter in sigma, without pneumoperitoneum or intraabdominal free fluid. cranial ct-scan did no describe sings of hydrocephalus. the patient underwent an emergencysurgical intervention. first of all, antibiotic therapy was initiated and neurosurgery's team was performed an external ventricular drain and they disconnected the proximal catheter side. after that, an exploratory laparoscopy was performed. it revealed a microperforation and collection beside to an appendix' base due to the proximity with the catheter. additionally, the catheter was freed from adhesions at the point of entry into the colon and after careful dissectionwe release the vps from colon with a . cm transmural trajectory at the sigmoid level. no free fluid was seen and rest of the bowel appeared normal. the distal end was removed through the anus and the proximal end through a laparoscopic port. we performed a laparoscopic segmental cecum resection and an extracorporeal colo-colonic anastomosis was performed for a mini-pfannestiel laparotomy of assistance. there were no complications in the postoperative period, being discharge on the th day. conclusion: the multidisciplinary approach and the laparoscopic support in the diagnosis and treatment of patients with colon perforation caused bay vps catheter is a feasible and safe option in third level centers. background: acute appendicitis continues to be the most common source of complicated intraabdominal infection worldwide. the high incidence of postoperative complications and dissatisfaction with the results of treatment in cases of complicated appendicitis and peritonitis gave the reason for conducting this study. aim: to evaluate the effect of different laparoscopic trocars position in case of laparoscopic appendectomy for diffuse appendicular peritonitis for the incidence of postoperative complications methods: the results of laparoscopic treatment of patients with acute appendicitis complicated by diffuse peritonitis were analyzed. the first group consisted of ( %) patients operated by triangulation access (type trocar placement according sages guidelines for laparoscopic appendectomy (sages qla). the second group consisted of ( %) patients operated by sectorisation access (type sages qla). postoperative complications were classified by clavien-dindo classification. results: the duration of the operation for the analyzed groups was . ± . vs . ± . min. there were no deaths among this group of patients. the incidence of postoperative complications for both group was . %. postoperative complications in the triangulation and sectorisation group were % and . % respectively (p . ). clavien-dindo iiib complications were noted in . % (n- ) patients and presented with intra-abdominal abscesses (iaa). all patients with iaa were operated in sectorisation group. conclusion: sectorisation trocar placement increases the incidence of intra-abdominal complications for laparoscopic appendectomy for diffuse appendicular peritonitis. introduction: the diverticular disease of the colon is a chronic entity with a variety of abdominal symptoms that can present with recurrent episodes of acute diverticulitis (ad). the prevalence of diverticulosis is not influenced by gender and increases with age, which, according to the increase in life expectancy, explains the accumulation of cases in western countries. the classic diagnostic-therapeutic algorithm of the disease is it has been based on the hinchey classification, the use of antibiotics and the intervention of hartmann (ih) at the acute time and elective colectomy in the multirecurrent cases. the use of laparoscopy with washing and drainage is actualymore extended in cases with peritonitis. objectives: to demonstrate the safety and efficacy of the laparoscopic approach, in cases with diverticular disease complicated by severe inflammatory plastron with 'covered' perforation, with several recurrent episodes. material and method: case report: a -year-old man with ap-diverticulitis years ago with complete resolution and normal control colonoscopy. he presents in the last two months three compatible episodes of acute diverticulitis, exploration with plastron-mass in hypogastrium without defense, tac-marked thickening of a segment of cms. of medium sigma, collection not drainable in mesosigma, of cm, which loses the plane of cleavage with loops of thin neighbors with a linear tract that suggests fistulization. evidence of interest is exposed. given the evolution, it is decided surgical elective treatment. result intervention: preoperative ureteral double catheterization, laparoscopic approach, is exposed by video, rectosigmoid resection by diverticular plastron, with negative io biopsy, mechanical colorectal anastomosis. good postoperative course, discaharge at th day. defini-tive ap: perforated diverticulitis, absence of malignancy. the laparoscopic approach is a valid and effective alternative in cases of complex and severe diverticular disease. aim: tamis resection has been described for the treatment of rectal neoplasms, wether benign or early malignant tumours. since tamis appearance, many different indications have been reported.we aim to show an special indication as seen in this video of a tamis resolution of a rectal stenosis non treatable by endoscopy. method: we present a video of a female patient, previously treated for a large rectal adenoma treated by trasnanal apporach, with a postoperative sepsis which required lateral colostomy and trasanal drainage. after surgery, the patient suffered from a rectal stenosis which couldn' t be solved by endoscopy, so the patient was sent back for a surgical treatment.we decided to performed a trasnanal apporach by tamis and a long and circunferiential stenosis around cm from the anal verge was seen.we performed a rectotomy by electrocautery in the posterior rectal wall until the perirectal fat was seen and the stenosis was passed. a dilatation with a foley catether was also performed. results: postoperative course was uneventful and after months she was prepared for colostomy closure with no complications and remains asymptomatic nowadays. conclusion: tamis approach of rectal stenosis may be a safe and feasible technique in selected cases if conservative treatments fail. iatrogenic endoscopic colon perforation it is a severe, but rare complication of colonoscopy. the incidence of this complication is estimated to be . - . % for diagnostic colonoscopies and . - % for therapeutic colonoscopies. the management of these complications depends on the size of the lesion,the time elapsed between the lesions were produced and diagnostic of the lesions and associated pathology. the treatment can be consevative,endoscopic or surgical(clasic/ laparoscopic) in our sevice in last years we treated cases with iatrogenic colon perforation after diagnostic colonoscopies. all lesions were at sigma level. one case was admission in our service at days after a diagnostic colonoscopy.the pacient was operated clasic,in emergency,we found a fecaloid peritonitis,a perforation at sigma level.we made a colostomy, lavage, drainage but the pacient died after days. in cases we made the operation at maximum h after the lesion was diagnosticated by the endoscopist(directly visualisation).we didn't made radiologic investigation.the pacients were operated laparoscopic,we made suture,lavage,drainage. evolutions of the pacients were good. conclusion: iatrogenic colonic perfortion are rare,but severe complication. laparoscopic surgery can be a choice in treatment of this complication introduction: complicated diverticulitis with fistula is responsible for about % of surgical procedures in diverticular disease and is commonly found in patients with diverticulitis of the sigmoid colon. colovesical fistulas are the most frequent ( %), with highest incidence in males. only a third of these patients have a history of diverticulitis. in most cases, treatment is surgical, and colectomy is performed, whether or not in association with vesical recession. case report: year old male with pneumaturia and fecaluria for the preceding months. the colonoscopy identified a diverticulitis of the sigmoid colon and the subsequent pelvic mri suggested a colovesical fistula. the cystoscopy was not able to identify any fistulous opening, but a double j catheter was placed in the left ureter, as surgical treatment had been proposed. a subsequently abdominal pain motivated a preoperative ctscan which revealed a pneumoretroperitoneum and a fluid collection near the left ureteral tract. the multidisciplinary team on the case decided to perform a percutaneous nephrostomy, followed by an exploratory laparoscopy. the fistula tract was identified and a laparoscopic sigmoidectomy with partial cystectomy was performed, as well as a ureterorenoscopy (with double j replacement). there were no intra or postoperative complications, and the patologic repport had no signs of malignancy. video of the surgical procedure is presented. conclusion: a laparoscopic approach to complicated diverticulitis with colovesical fistula is safe and effective when performed by experienced colorectal surgeons. introduction: diverticular disease is characterized by its high prevalence, being one of the most frequent causes for hospital admission when it comes to gastrointestinal pathology. even though it is more frequent in older patients, there has been an increase in incidence amongst lower age groups. the approach to the disease has also suffered changes in the last few years, showcasing a tendency for less invasive options, deferring elective surgery to later in the course of the disease. this study examines the therapeutic approach to diverticular disease in our hospital. methods: retrospective analysis of demographic data, therapeutic options and surgical outcomes in patients admitted for diverticular disease between january and june . results: patients(n = )were included in the study: ( %)were male and ( %) were female, with an average age of years. patients( %) underwent medical treatment, with surgery reserved for the remaining patients, of which were emergencies, with the other being elective( %).about % of patients were only admitted once, %were admitted twice and % had or more episodes. for the single-admission group, the most common treatment was medical( %of cases), as was the case for the group with or more episodes(in which %of cases were subjected to medical treatment). in the patient group with episodes, %were submitted for surgery, most of which elective.as far as the surgically operated group is concerned, no statistically significant differences were found with regards to patient sex. age, however, was significantly greater for the group that underwent emergency surgery vs that submitted to elective ( . years vs . years,p \ . ).the most common procedure overall was colic recession, with hartmann's operation standing out as the most frequent for the emergency surgery group. length of hospital stay was again higher for the emergency group (vs elective; vs ,p \ . ),as well as the morbidity rate. no statistically significant differences were found with regards to mortality rate. conclusion: knowledge of the natural history of diverticular disease led to changes in the approach to treatment, with a tendency to adopt a less aggressive therapeutic. despite controversy around aspects such as selection of patients for elective surgery, among others, it is key to the approach to diverticular disease that existing recommendations are taken into account, treatment is individualized and outcomes are closely monitored. surg endosc ( ) aims: the objective of this analysis is to establish if there is differences after the procedure of laparoscopy appendicectomy comparing the use of endoloop (el) vs. endostapler (es) in complicated and non-complicated acute appendicitis. methods: we performed a retrospective analysis of a prospective database of patients from february to june . we divided the patients in two groups: depending on that the procedure of the appendicectomy was with endoloop or endostapler. the groups were created selecting patients in order to be homogeneous as to perforation appendix rate, thus a propensity score was performed for sex, age and perforation rate. an univariant analysis was carried out in regard to the differences in the use of el vs es in the apparition of abdominal complication, as well as hemorrhage, ileo, surgical wound infection, collection, reintervention or hospital readmission.qualitative variables were expressed in terms of absolute frequencies and percentages and mean values and standard deviation were used to express quantitative variables. introduction: intramural haematomas can develop anywhere within any the gastrointestinal tract . these are most frequently associated with blunt trauma above the level of the sigmoid colon and very rarely occur in the rectum . spontaneous, non-traumatic haematomas are a rare clinical condition usually secondary to haematological blood disorders or anticoagulant therapy . case summary: a -year old gentleman presented to the emergency department with a day history of worsening lower abdominal pain and bloody stool. he presented twice within the previous week with worsening, generalised abdominal pain. the patient had been taking regular aspirin and clopidogrel following insertion of coronary artery stents. on clinical examination, he was guarding with a distended, generally tender lower abdomen but all observations were stable, afebrile. an initial computer tomography of the abdomen reported pneumoperitoneum with haemorrhagic ascites; a differential diagnosis being perforated sigmoid colon with a large localised haematoma. the patient underwent an emergency laparotomy and hartmann's procedure (appendicectomy, sigmoid colostomy and rectal stump). he recovered well with no significant post-operative complications. histology reported the rectal perforation macroscopically associated with an opened haematoma and no evidence of malignancy. the appendix shows reactive appendicitis with serousal inflammation background: ulcerative colitis (uc) is one of the risk factor of developing sporadic colorectal cancer. approximately % of uc patients develop an acute attack of severe colitis, and % of these patients require colectomy. one third of the patients will not respond to steroid therapy. thus, a long-term follow-up has been recommended. case report: we reported a single case of completed years follow of colorectal cancer related ulcerative colitis on years old female patient undergoing emergency operation ( staged total colectomy and j-pouch ileo-rectal anastomosis) after year no responsed of medical treatment before, presenting with bloody diarrhea and anemia. there was no post operative complication reported. pathologic finding was early adeno carcinoma, closed follow up was done each year and for another five years later, no progression of the disease was found in this period and the patients has good quality of life after this procedures. is becoming a standard and feasible surgical method worldwide. % of patients with crohn's disease (cd) and % patients with ulcerative colitis (uc) will require an operation during their life. over the last decade, there have been many studies documenting the safety and feasibility of the laparoscopic approach for ibd in well-selected patients. methods: patients with a cd with the tight stenosis in the distal ileum and/or ileo-colon or various colon and rectum stenosis, patients with uc with ineffective medical therapy, steroid dependence or dysplasia underwent the lcs. from to , ileocolic resections, hemicolectomies, subtotal colectomies and restorative proctocolectomies with ileopouchanal anastomosis were performed either totally laparoscopically or laparoscopically assisted (n = ).the average time of the procedure was min ( - min), average blood loss ml ( - ml) and the conversion to laparotomy was in . %. average return time of the bowel function was . days ( - days) and the average hospital stay was . days ( - days). complications occurred in patients ( . %). cases of the early ileus due to adhesions, cases of the anastomotic bleeding threated conservatively, case of the instrumental perforation of the small bowel, cases of the incisional hernia in minilaparotomy and wound infections occurred. conclusion: in well-selected patients with ibd, thanks to superior short-and long-term outcomes, the laparoscopic approach should be considered a safe and effective method when performed by experienced surgeons. supported by mo . aim: over % of patients with crohn's disease (cd) will require a surgical resection within years of their diagnosis and one quarter will have another resection for disease recurrence. laparoscopy should by preferred approach in surgery in cd due to reduced morbidity, faster recovery time, shorter hospital stay and reduction in adhesions and hernial formation. methods: patients with cd with the tight stenosis in the distal ileum and/or ileo-colon or various colon stenosis were indicated for the laparoscopy. from january to november we performed ileocolic resections, hemicolectomies and subtotal colectomies either totally laparoscopically or laparoscopically assisted. the average time of the procedure was min ( - min). the average return time of the bowel function was . days ( - days) and the hospital stay was from to days. complications occurred in patients ( . %) . in cases early ileus developed due to adhesions, in case was anastomotic bleeding threated conservatively, the incisional hernia in minilaparotomy occurred in cases and wound infections occurred. conclusion: minimally invasive surgery is becoming a gold standard in cd. it is safe and feasible in well-selected patients thanks to short-and long-term outcomes. laparoscopic approach for recurrent disease is still in debate. supported by mo . aim: the aim of the study was to observe when laparoscopy is avoided when treating surgical complications of crohn disease. methods: we did a retrospective study which included all of the patients diagnosed and operated in our clinic for complications of crohn disease during a period of years. results: we identified a number of patients operated for complications of crohn disease. of these were operated by minimally invasive procedures. we observed that laparoscopy was avoided in the case of intestinal fistulas (p = , ). also when sepsis associated the surgical complication-laparoscopy was avoided (p = , ). age under years represented another factor to avoid laparoscopy (p = , ). conclusions: although laparoscopy offers numerous advantages careful selection of the patients is of utmost importance so the safety of the procedure can be ensured. retroperitoneal sarcoma represents approximately - % of all sarcomas and less than . % of all neoplasia. radiotherapy and chemotherapy still do not represent valid therapeutic alternatives; therefore radical surgery remains the only valid option. complete surgical resection is the only potential curative treatment modality for retroperitoneal sarcomas. the ability of complete resection of a retroperitoneal sarcoma with tumor grading remains the most important predictor of local recurrence and disease-specific survival. hypoglycemia is a rare but potentially lifethreatening presentation of soft tissue tumors the etiology of hypoglycemia may be difficult to diagnose, assays for insulin-like activity (ila) were found to be high in the extract of tumor tissue, while insulin was not detected in significant concentration neither in the same extract nor in his serum. the most likely mechanism of hypoglycemia appears to be production of insulin like substance and increased utilization of glucose by the tumor. laparoscopic surgery represents an alternative technique for radical resection of such tumors rather than traditional surgery. only few cases of retroperitoneal tumors resected laparoscopically were reported in the literature. we report a rare case of years old male presented to ed unconscious due to hypoglycemia.he was resuscitated and admitted for further investigations. hypoglycemic attack recurred again during the same evening of admission. initial investigations were within normal except for serum glucose mg/dl ( . mmol/l). his tsh, glucagon & cortisol levels were within normal, insulin and c-peptide levels were undetectable. only hypokalemia ( . meq/l). he tested negative for the anti-insulin antibodies. his abdominal ultrasound as well as his ct scans showed the presence of a large retroperitoneal tumor ( cm cm cm) with a heterogeneous contrast effect. a glucose supplement was required to maintain the plasma glucose level within normal limits during which complete resection of the tumor which was performed laparoscopically. diagnosis of such hypoglycemia inducing retroperitoneal fibrosarcoma represents great challenge especially when patients presents only with hypoglycemia and no other abdominal symptoms, management using minimal invasive technique to resect and remove such tumors from the retroperitoneal region shows superiority in recovery and limitation of complications when done by experienced surgeons. solitary fibrous tumor (sft) is a rare fibroblastic mesenchymal neoplasm, tipically arising from the pleura, less frequently from other anatomic sites. sft is an indolent neoplasm, but it have been described cases of greater aggressiveness in terms of local recurrences and more rarelly of distant metastases. among the various extrapleural sites, intrabdominal, retroperitoneal localization is the most common site, followed by the pelvis soft tissues and parenchymatous organs. the most common clinical finding of intraabdominal localization is a palpable mass, and the pain is the most frequently associated symptom. the diagnosis is performed by imaging, but the histological as well as immunohistochemistry characterization of the lesion is the latest goal. furthermore, histological features are used to attempt to identify the patient with a hight risk of malignant evolution of the tumor. the gold standard treatment is surgical approach, meanwhile there are no evidences about the efficacy of any adjuvant treatment. we present the case of a -year-old man affected by symptomatic tfs arising from mesosigma treated by surgical radical excision. finally, we propose a review of the literature of last decade. background: laparoscopic right hemicolectomy involves making an additional incision to remove the specimen and perform the anastomosis. recently, natural orifice specimen extraction surgery (noses) has been reported as an alternative approach without any additional incisions or extensions, may lead to better outcomes compared to conventional laparoscopic right hemicolectomy. in this video, we aimed to evaluate the safety and feasibility of noses for laparoscopic right hemicolectomy. methods: we describe the technique with transvaginal specimen extraction and d lymph node dissection in laparoscopic right hemicolectomy by this video. we performed intracorporeal anastomosis combined with a transvaginal route of specimen extraction after medial-to-lateral mobilization. transverse transvaginal posterior colpotomy was performed under aid with visualization. the specimen was pulled into the sterilized plastic bag, passed transvaginally. the vaginal incision was then closed with a running suture. results: the operation time was min and the hospital stay was days. an excellent postoperative recovery was demonstrated and has shown future potential for less incision. the pathologic tnm stage is t n m . conclusions: this video has shown that laparoscopic right hemicolectomy with the noses technique is feasible and safe for selected cases. the long-term benefits of this procedure need to be more evaluated. recently, indocyanine green (icg) fluorescence has been introduced in laparoscopic colorectal surgery to provide detailed anatomical information.the aim of our study is the application of icg imaging during laparoscopic colorectal resections: to identify the sentinel lymph node (sln) to search micrometastases that can be missed with the conventional pathological exam, and to assess anastomotic perfusion to reduce the risk of anastomotic leak. after tumor identification ml of icg solution ( . mg/kg) is subserosal peritumoral injected. a full hd image s camera, switching to nir mode, in about min displays fluorescence: the sln is identified and the sln biopsy (slnb) is performed.after the transection ml of icg solution is injected to confirm the stumps perfusion. if there is an ischemic area, a new resection is performed.after the anastomosis is performed, another bolus of icg is intravenous injected to confirm the anastomotic perfusion.when the sentinel node is negative for cancer metastases by conventional histological examination, ultrastaging is performed by serial sections. when no micrometastases are identified on these sections, immunohistochemical techniques are applied. from november , patients were enrolled: left colectomy, right colectomy, transverse resections, and splenic flexure resections. in two cases, one left colectomy and one right colectomy, the anastomotic perfusion wasn't good and the surgical strategy was changed. four postoperative complications occurred, of which one anastomotic leak, due to a mechanical problem. from november , patients were enrolled to perform the slnb: right colectomy, left colectomy, transverse resection and splenic flexure resections. the sln was identified in cases. cases were found to be n to the conventional examination and were subjected to ultrastaging. the serial sections showed micrometastases in two cases. in the other cases the immunohistochemistry was performed but the exam is still in progress. icg-enhanced fluorescence imaging is a safe, cheap and effective tool to increase visualization during surgery. it's recommended to assess the anastomotic perfusion in order to reduce the incidence of anastomotic leak, and to perform the slnb for the sln ultrastaging in order to identify micrometastases. methods: for the last years, tem was performed on patients' with early rectal cancer. there were women and nine men, age to . localization of tumors was - cm from anus. mean size of tumors was . cm. full thickness excision was performed in all patient with suturing of mucosa. during follow-up in three patients' metastasis in lymph nodes of mesorectum were detected. all of these patients were re-operated: laparoscopic colectomy with total mesorectal excision (tme) was done. for the last year in patients with early stage rectal cancer we used indocyanine green (icg) with fluorescent imaging for mapping sentinel lymph node. icg was injected in four quadrants to submucosa around the tumor. during the laparoscopy, sln was detected and removed with morphological examination. results: among nine patients in patients, sln was negative. tem was performed in these patients with good results. after - months no recurrence or metastasis were detected in these patients. in two patients with positive sn laparoscopic tme was performed with low colorectal anastomosis. anastomotic complication was occurred in one patient. conclusion: tem procedure is highly effective in selected group of patients with early rectal cancer. mapping and examination of sln can clarify indication for the tem in the patients with early rectal cancer. purpose: laparoscopic surgery for colorectal cancer provides better short-term benefits and similar long-term outcomes compared with conventional open surgery. unlike minimally invasive surgery, natural orifice specimen extraction (nose) can provide additional advantages by reducing morbidity and postoperative pain related to the surgical extraction site. this study aimed to evaluate the efficacy and safety of a nose procedure using needlescopic instruments for colon cancer surgery. methods: between november and february , patients underwent laparoscopic nose using needlescopic instruments. the first port for the camera was placed at the umbilicus. a -mm or -mm port was inserted in the right lower quadrant. a -mm or -mm port was inserted in the right upper quadrant. individual needlescopic forceps for the assistant were inserted into left upper and lower quadrant ports. thus, a total of ports were placed. the superior rectal artery and inferior mesenteric vein were ligated with clips, and colonic mobilization was performed using a medial to lateral approach. after rectal stump irrigation, the distal rectum was transected using an endoscopic linear stapler. the proximal colon and associated mesentery were transected. after the rectal stump was opened, a wound retractor was pulled through the anus and inserted in the rectal lumen. the resected specimen was transanally extracted through this route. an anvil was intracorporeally attached to the proximal colon, and the open rectal stump was reclosed using an endoscopic linear stapler; colorectal anastomosis was then performed using a double-stapling technique. results: of the patients, were male and were female, with a median age of years ( - years). median body mass index was . . the tumor site was in the sigmoid colon in patients and rectosigmoid colon in patients. median operative time was min and blood loss was ml. there was no conversion to open surgery. no postoperative complication was observed. median postoperative hospital stay was days ( - days). conclusions: nose surgery using needlescopic forceps is an easily performed type of reducedport surgery with a conventional port arrangement. this procedure is feasible for the selected patients. introduction: splenic flexure colon cancer accompanying obstruction is usually managed stent insertion as a bridge to surgery and left hemicolectomy, or subtotal colectomy. however, stent insertion can fail more often than in sigmoid colon because it requires longer colonoscopic approach in the circumstance of impossible bowel preperation. although subtotal colectomy has advantage in the aspect that it is -stage treatment, it needs open surgery in most cases, right colon has to be sacrificed without oncologic neccesity, and preoperative staging and evaluation can be insufficient. despite colostomy is reluctant procedure when considering quality of life, in splenic flexure colon cancer obstruction, we can obtain prompt stabilization of patient state, suffient time to preoperative staging and evaluation, and also we can achieve minimally invasive surgery by using colostomy site as mini-laparotomy and close colostomy before discharge. colostomy site, tumor location, and minilaparotomy site for next radical surgery have to be considered comprehensively before making colostomy incision. colostomy site has to be appropriate as mini-laparitomy site for feasibility of laparoscopic left hemicolectomy and the colostomy has to be included in the specimen with caution to prevent unneccessary lengthening of the specimen. we experienced cases which were treated succefully in this strategy and report them. result: temperary loop transverse colostomy and laparoscopic left hemicolectomy via colostomy site in splenic flexure colon cancer obstruction has advantage of quick stabilization of patient's status, suffient preoperative staging and evaluation, achieving minimally invasive surgery, and also rapid colostomy closure before discharge. our tatme procedure for locally advanced low rectal cancer following chemoradiotherapy y. nakamoto , r. okamoto , f. kimura , h. yanagi , t. nakajima , h. yoshie , n. yamanaka surgery, meiwa hospital, nishinomiya, japan, surgery, yoshie clinic, itami, japan background: short-course chemoradiotherapy using hyper-fractionation method (scrt; gy/ fraction/ days ? s- or xeloda) is performed to secure circumferential resection margin (crm) due to tumor shrinkage, reduction of cancer cells with viability, reduction of radiation hazard for resectable locally advanced lower rectal cancer (more t or n ). the patient underwent radical surgery after one month of scrt. for more locally advanced lower rectal cancer (t or n ), induction chemotherapy is performed before scrt. for patients with poor efficacy of chemotherapy, we also do normal fraction gy radiotherapy. methods: we introduced tatme from last august, and cases were performed so far. in all cases temporary stoma has been constructed, and intersphincter resection (isr) is based on partial isr avoiding total isr considering postoperative anal function. if possible, colonic j-pouch is added, and pelvic floor repair may be added for esr cases and older people. at first one team preceded with the anal operation and shifted to the abdominal procedure, now it is done with two teams with the advantage of getting good visual field from both sides when there is difficulty identifying the right dissecting layer. tatme is very useful in cases such as large tumor, obesity, and narrow pelvis. furthermore, when it is difficult to identify the dissecting layer by scarring after crt, it is more possible to control the crm/drm of cancer. results: cases of isr, case of apr, case of tpe were performed, and in of these cases lateral lymph node dissection was also performed (one side , both sides ). postoperative complications were anastomotic leakage, pelvic floor infection, perineum infection, and bowel obstruction. conclusions: tatme for locally advanced lower rectal cancer is useful even after chemotherapy and scrt. background: although many studies have demonstrated similar perioperative outcomes for single-incision laparoscopic surgery (sils) and conventional laparoscopic surgery (cls) for colon cancer, few have directly compared the costs of them. we aimed to compare costs between sils and cls for colon cancer. methods: we analyzed the clinical outcomes and overall hospital costs of patients who underwent laparoscopic surgery for colon cancer from july to september at severance hospital; were used for analysis after propensity score matching. the total hospital charge, including fees for the operation, anesthesia, preoperative diagnosis, and postoperative management was analyzed. results: the total hospital charges were similar in both groups ($ . vs. $ . , p = . ). however, the patients' total hospital bill was higher in the sils group than in the cls group ($ . vs. $ . , p \ . ) mainly due to the difference of the cost of access devices. there was no difference in the additional costs associated with readmission due to late complications between the two groups ($ . vs. $ . , p = . ). conclusions: sils for colon cancer yielded similar costs as well as perioperative and long-term outcomes compared with cls. therefore, sils can be considered a reasonable treatment option for colon cancer for selective patients. aims: technology improvements in medicine allow the development of new minimally invasive approaches. despite every single advantage of these new devices they also can cause technical problems and difficulties for the surgical team. well known from last few years-laparoscopic assisted transanal total mesorectal excision for distal rectal cancer is perfect example for a quite new procedure, based on the combination of forgotten old surgical principles and technology advances. the aim of the study is to analyze the rate of technical problems during the procedure and to measure the impact of them on the operative time. methods: we conducted prospective observational study related to technical problems during the procedure. for the period between september and november in the department of endoscopic endocrine surgery and coloproctolgy at military medical academy-sofia have been performed laparoscopic assisted transanal total mesorectal excisions. we used standard local preoperative work up and postoperative care protocols. we defined technical problem as intraoperative event different from complication leading to delay in operative time. every technical problem during the procedure was recorded and time for resolving the problem was measured in seconds. results: overall technical problems occurred in of the cases. most of them were related to the insufficient smoke evacuation during the cases. the second most common technical problem were the excessive rectal stump spasms during the procedure-this complication occurred in of the patients. mean delay of the procedure related to technical problems is min. in our series we experienced only one intraoperative complication which was specimen perforation during the dissection. three complications occurred in postoperative period-two urinary retentions and one perianastomotic abscess, without need of reoperation. conclusion: technical problems during the procedure can be source of delay in operative time. correct use of devices in operating room is the key to reduce technical issues. technical problems can increase the rate of intraoperative near miss events and complications during the transanal total mesorectal excision. surg endosc ( ) aims: anastomotic leak after rectal cancer surgery constitutes a severe complication associated with poorer oncologic outcome and quality of life. preoperative assessment of the risk for anastomotic leak is a key component of surgical planning, including the opportunity of creating a defunctioning stoma. methods: studies on rectal cancer surgery published between and were systematically reviewed according to the preferred reporting items for systematic reviews and meta-analyses of individual participant data (prisma-ipd) guidelines. with the aim to generate a score for anastomotic leak, all available per-operative covariates were used as independent factors in a logistic regression model with anastomotic leak as dependent variable. a receiver operating characteristic curve (roc) analysis was generated. we selected as threshold the value that allowed a missing rate of anastomotic leak \ %. the predictive power of the previously selected cut-off was validated in an independent set of patients. results: twenty-six centers provided individual data on patients. with a threshold value of the roc corresponding to . in the training set, the area under the roc curve (auc) was . (p \ . ). sensitivity and specificity of the model's probability [ . to identify anastomotic leak were . % and . %, respectively. accuracy of the threshold value was confirmed in the validation set with . % of sensitivity and . % specificity. conclusions: we trust that, with further refinement using prospective data, this nomogram based on preoperative risk factors may assist surgeons in decision making. the score is now available online (http://www.real-score.org). in ( . %) cases laparoscopic interventions were performed in patients with diverticular colon disease. in the group of patients with colorectal cancer localization of the tumor in the right parts was observed in ( %) patients, in the left-in ( %), in the rectum- ( %). results: in the adenocarcinoma of the sigmoid colon, performed a left laparoscopic hemicolectomy ( cases) and resection of the sigmoid colon ( ). was executed high clipping and intersection of the lower mesenteric vessels, aorto-iliac lymphatic dissection. in the standard scope, lymph node dissection was performed with removal and testing of not less than epi-, para-and mesocolical lymph nodes (max ). the average length of the laparoscopic stage is ± min. laparoscopic right hemicolectomy ( cases) was performed in accordance with the principles of cvl (central vascular ligation) and cme (complete mesocolic excision). intracorporal ileotransversoanastomosis was formed by a semimanual method with endogia universal and v-lock suture material. the average length of the laparoscopic stage was ± min, the open phase was ± . in the tumor of the lower and middle ampullary parts of the rectum ( cases) after neoadjuvant chemoradiotherapy, was executed a laparoscopic total mesorectumectomy. conclusions: the use of minimally invasive technologies in colorectal surgery provides a complete revising of the abdominal organs, adequate scope of resection and lymph nodes dissection in surgical interventions. background: it is thought that complete mesocolic excision (cme) improves the oncologic outcomes for colon cancer. but, precise mesenteric mobilization from retroperitoneum and safe ligations at the origins of central vessels are considered to be technically difficult in single port surgery(sps). to resolve this problem, we utilize retro-mesenteric medial approach for right side colon cancer. herein, we introduce this technique and assess its outcomes. operative procedure: the multi-trocar platform is placed in the umbilical site. d laparosopy is inserted from one of this channels. the surgeon manipulates instruments via the other channels. st step: right colonic mesentery is mobilized medial to lateral from the head of the pancreas and retroperitoneum along the embryonic plane. nd step: the origins of ileocolic and right colic vessels are divided and central lymph node dissection is achieved. rd step: hepatic flexure is taken down from cranial. and right lateral attachment is dissected away and cme is achieved. th step: specimen is extracted and anastomosis is performed using a functional end to end anastomosis extracorporealy. results: from april to december , consecutive patients underwent sps-cme with right side colon cancer. there were in stage i, in stage ii, in stage iii and in stage iv. the mean operative time was min. the mean estimated blood loss was ml. there was no conversion to open surgery. additional port was placed in patients ( . %). intraoperative bleeding was occurred in patient. anastomotic leakage was observed in patient ( . %), intestinal obstruction ( . %) and wound infection in ( . %). conclusion: these results suggest that retro-mesenteric medial approach in single port surgery with right side colon cancer is useful and safe technique. aims: this multicenter, randomized controlled trial (simple trial) aimed to investigate the quality of life (qol) and patient satisfaction of single port laparoscopic surgery (spls) for colon cancer, compared with multiport laparoscopic surgery (mpls). methods: patients with histologically diagnosed adenocarcinoma in cecum, ascending and sigmoid colon were eligible for this trial. eligible patients were randomly assigned to the spls or mpls group at a ratio of : . qol was measured with the eortc qlq-c third edition (korean version) preoperatively and postoperatively at month , , and . in addition, patient satisfaction was surveyed with a five-point questionnaire at postoperative month. to exclude the impact of adjuvant chemotherapy on qol, subgroup analysis for patients with or without adjuvant chemotherapy were carried out. (clincaltrials.gov identifier: nct ) results: total patients were randomly allocated into the spls group (n = ) and mpls group (n = ). in total patients, global health status and five functional scale steadily increased and nine symptom scales also gradually improved over time. but, nausea/ vomiting and appetite loss temporally deteriorated at postoperative month. pain score was significantly worse in the mpls group ( . in the spls group vs. . in the mpls, p = . ) at postoperative month and appetite loss score was significantly worse in the spls group ( . vs . , p = . ) at postoperative month. except for that domains, all the other items of qol between groups were not different until postoperative months. patient satisfaction was significantly higher regarding the operation (p = . ) and the abdominal wound (p = . ) in the spls group. in patients without adjuvant chemotherapy, some items of qol (global health status, physical functioning, role functioning, emotional functioning, fatigue and pain) were significantly better in the spls group at postoperative month. since postoperative month, all of qol domains (except pain score) were similar between groups. conclusion: although postoperative pain was temporarily better in the spls, most of qol domain were similar between the spls and the mpls group until postoperative month. in patients without adjuvant chemotherapy, spls showed better outcomes in some of functional scales and symptom scores at postoperative month. coloproctological surgery, juntendo university, tokyo, japan; gastroenterological surgery, juntendo university, tokyo, japan introduction: laparoscopic surgery causes less postoperative pain compared with pain after laparotomic surgery, and its low invasiveness should be considered for pain control. we have previously controlled postoperative pain by epidural anesthesia. in this study we compared postoperative multimodal analgesia centering on acetaminophen in patients who underwent laparoscopic colorectal cancer surgery with the conventional method. subjects: the subjects were patients who underwent laparoscopic colorectal cancer surgery between january and june . surgery was performed under epidural anesthesia in patients and multimodal analgesia in : periodic acetaminophen administration ? transverse abdominis plane (tap) block in , periodic acetaminophen administration ? local anesthesia of the wound in , and periodic acetaminophen administration ? intravenous patient-controlled analgesia (ivpca) in . the operating roomoccupying time, postoperative pain (nrs), frequency of taking analgesics as needed, and postoperative nausea were investigated for days after surgery and the duration of urethral catheter placement and postoperative intestinal movement were investigated in the epidural anesthesia and multimodal analgesia groups. results: while the time from entering the operating room to initiation of surgery was significantly shorter, the time from completion of surgery to leaving the room was significantly longer in the multimodal analgesia group. there was no difference in the operating room-occupying time. the frequency of postoperative pain was significantly lower in the multimodal analgesia group on postoperative day (pod) . the frequency of taking analgesics as needed was significantly lower in the multimodal analgesia group on pod , , and . no significant difference was noted in the duration (number of days) of urethral catheter placement or postoperative nausea between the groups. regarding postoperative intestinal movement, discharge of gas occurred significantly earlier in the epidural anesthesia group. the total number of incidents of complications in the epidural anesthesia group was . discussion: in laparoscopic colorectal cancer surgery, the effect of multimodal analgesia centering on periodic administration of acetaminophen without epidural anesthesia for postoperative analgesia was sufficient compared with the effectiveness of epidural anesthesia. this approach to analgesia may be useful because none of the potential complications of epidural anesthesia occur. surg endosc ( ) in the last years the application of new technologies like d vision or virtual reality have provided to surgeons the possibility of establish a preoperative surgical plan of each surgery and of each patient. these advances are specially useful in minimally invasive colorectal surgery due to the variability in location, anatomical relationship with other organs and vascular variants of these type of surgeries. the aim of our work is to built a digital -dimensional virtual model of the colorectal ct scan imagen of patients with colorectal cancer. the virtual models are obtained from the preoperative ct scan. the ct scans that we use to this work are general electric healthcare revolution gsiÒ and siemens somatom perspective Ò and the size of each image is mm. a medical software let us build a reconstruction of colorectal digital images where a radiologist has marked the exact image of the tumor so we obtain a d reconstruction which can provide an enhanced understanding of crucial anatomical details like the exact location of the tumor and the relationship with other organs and structures of the patient which can be selectively displayed or hidden. this information has an important applicability into clinical practice since it lets surgeons estimate the colorectal anatomy, tumor size and relationships, providing key landmarks to choose the most appropiate surgery, the best trocar location and a safer dissection specially in some cases whose location can change the kind of surgery radically. we present some cases where virtual models were crucial for the preoperative and intraoperative surgical plan, showing the potential interest of these d reconstructions in colorectal surgery. in conclusion the ct scan colorrectal image reconstruction can provide an enhanced understanding of crucial anatomical details of the colon and tumor location and relations which could contribute to choose the best surgical option and to improve safety in colorectal surgery. background: anastomotic leakage (al) after colorectal procedures are a common surgical experience and represents a significant burden both for patients and surgeons. the incidence of al has been reported to vary between . % to up to %, with rates for the colon and rectum of - % and - %, respectively. they, not only add to potential postoperative patient morbidities and to overall costs of postoperative patient care, but also are considered a quality indicator in colorectal surgery. aim: we aimed to evaluate the clinical burden associated with anastomotic leaks following colorectal surgery. methods: we conducted a retrospective analysis of colorectal patients who underwent conventional or laparoscopic colorectal surgery for colorectal cancer (crc), from january st, to december st, in a single colorectal centre (centro hospitalar de leiria). patient demographics, intraoperative and postoperative aspects were collected and analysed. all statistical analysis will be conducted using stata software (statacorp lp). results: in our cohort of pts, developed a clinical al ( . %), mostly males ( %), with an average age of ± . . male gender and conversion were independent risk factors. the group with al had a higher lohs ( . days vs, . -p \ . ). out of al have been detected after the discharge. the mean diagnostic day was the eighth, and mode estimated at day . when compared with a control group, wcc, eosinophils and crp were statistically significant different in al group, at day and . conclusion: in the present study, no statistically significant risk factors for al in crc surgery were detected, except for male gender and conversion. clinical methods and biomarkers were useful for early diagnosis. technology combined with experience and common sense may be the embodiment of the clinical method. conclusions: our regional screening program has significantly improved early diagnosis and quickened surgical treatment of crc. thanks to this, we obtained an earlier stage at diagnosis, a less invasive surgical approach, and a lower rate of complications and emergency surgery need were obtained also with an improvement in both os and dfs. introduction: surgeons are increasingly being faced with the problem of treating elderly colon cancer patients. we evaluated the outcome of silc in patients of over years with colon cancer with a propensity score matched comparison to assess its perioperative and long-term oncological outcomes. methods: this retrospective cohort study analyzed our experience with silc for colon cancer over years. eighty-seven patients of over years with colon cancer who electively underwent silc were included in this study (elderly group). eighty-seven patients were then chosen out of a collective of patients less than years old in a propensity score matched design (younger group). short-term clinical outcomes in both groups were compared and verified its long-term oncological outcome. results: american society of anesthesiologists score and post-operative complication rate were significantly higher in elderly group. however, the other short-term clinical outcomes including post-operative hospital stay were equivalent in two groups. the rates of -year cancer specific survival were . % in elderly group and . % in younger group, respectively, and the -year overall survival rates were . % and . %, respectively. no significant differences were seen between two groups. conclusions: our initial experiences suggested the oncological and clinical safety of silc in patients of over years with colon cancer. however, further studies are needed to demonstrate the advantages of this procedure compared to conventional laparoscopic colectomy. aim: some clinical trials have reported the safety and efficacy of laparoscopic colectomy for colon cancer. on the other hand, transverse colon cancer was excluded in these trials because of the difficulty of laparoscopic colectomy for transverse colon cancer. in this presentation, we report the tips for laparoscopic colectomy for transverse colon cancer. tips: in our department, transverse colon cancers has been resected by laparoscopically so far. to complete cvl and cme, lymph nodes around middle colic artery should be resected, however many important structure, duodenum, pancreas, superior mesenteric vein (smv) and so on, may be obstacles. this is most difficult point for this surgery. our surgery is as follows. mobilization of ileum and ascending mesocolon from caudal sideconfirm duodenum and pancreasexpose smv and ligation root of ileocecal artery and veindissect lymph nodes around smv and ligation of middle colic artery and accessary right colic veinconfirm pancreas from caudate side of transverse mesocolon and incise the peritoneum along the caudal side of the pancreasdissect lymph nodes sufficiently by dissection from both side of transverse mesocolonmost important point. to dissect lymph nodes safely, confirmation from both side of transverse mesocolon is necessary and dissection should be performed along important structure, smv, pancreas and so on. introduction: we have developed and previously reported single-incision plus one port laparoscopic anterior resection of the rectum (sils ? -ar) as a reduced port surgery in which we can utilize the incision for drainage as an additional access route for laparoscopic procedures including the transection the lower rectum. a consecutive experience from its introduction of sils ? -ar for rectal cancer is reviewed, and its -year oncological outcomes are evaluated retrospectively. methods: one hundred and forty-one patients ( female) with a mean age of . years adopted the sils ? procedure for rectal cancer. a lap protector (lp) was inserted through a . cm transumbilical incision; an ez-access was mounted to the lp and three -mm ports were placed. a -mm port was inserted in the right lower quadrant. results: one hundred and thirty-six patients ( . %) completed with sila ? -ar. the tumor locations in the rectosigmoid, rectum above the peritoneal reflection (ra), and rectum below (rb) were , and , respectively. the median follow-up interval was months. aims: colovesical fistulae came from inflammatory disease or cancer and do have a significant morbidity. the most common location is the sigmoid colon and the most common aetiology is diverticulitis. the treatment of choice is a surgical procedure. the aim was studying compare laparoscopic approach in patients diagnosed by benign (diverticulitis) and malignant (colon adenocarcinoma) colovesical fistulae. methods: from january to march all characteristics of surgical patients with diverticular and colon adenocarcinoma colovesical fistulae were reviewed. patient details (sex, age, symptoms, diagnosis, medical history and anaesthetic risk), surgical approach, hospital stay and complications were recorded. both groups were compared with significance level set at p \ . . results: nine laparoscopic ( %) and open approaches ( %) in diverticular colovesical fistulae were performed, with a conversion rate of %. the procedure done was sigmoidectomy. there were also performed laparoscopic ( %) and open approaches ( %) in colon adenocarcinoma colovesical fistulae. the procedures done were sigmoidectomy, pelvic exenteration, left colectomy, low anterior resection and loop colostomy. comparison between the two groups didn't show significant differences in characteristics but did show significant differences regarding the approach, with more cases performed by open approach in colon adenocarcinoma colovesical fistulae (p = . ). conversion rate didn't show significant differences. patients diagnosed for malignant colovesical fistulae had more complications, cases ( %), ( %) i-ii and ( %) iii-iv-v according to clavien dindo classification, manifesting significant differences (p = . ). laparoscopic approach didn't show significant differences regarding complications. conclusions: generally, surgical approach with colonic resection and partial or total cystectomy is the treatment of choice in colovesical fistulae, although vesical resection can be avoided if it is suspected benign aetiology. whenever laparoscopic approach is performed by experienced surgeons, is feasible in colovesical fistulae and the morbidity and mortality numbers are acceptable. laparoscopic approach allows the advantages of a minimally invasive treatment but implies clinical trials to stablish stronger evidence. aims: laparoscopic right hemicolectomy became the standard of care for treating cecum, ascending and proximal transverse colon cancer in many center. most centers use multiport laparoscopic colectomy with extracorporeal resection and anastomosis (mce). single-incision laparoscopic colectomy with intracorporeal resection and extracorporeal (sci) remains controversial. the aim of the present study is to compare these two techniques using propensity matching analysis. methods: this study analyzed patients who underwent laparoscopic right hemicolectomy including mce surgeries and sci surgeries from december to december . short-term outcomes were recorded. postoperative pain was evaluated using a visual analogue scale (vas) and postoperative analgesic use as outcome measure. results: the length of skin incision in the sci group was significantly shorter than in the mce group: median (range) ( - ) cm verses ( - ) cm (p \ . ). the vas score after surgery was significantly less in srhi than in mrhe. significantly fewer patients required analgesia after srhi after surgery. there were no significant differences in operative time, intraoperative blood loss, the number of lymph nodes removed and postoperative courses between the groups. the cost effectiveness was significantly cheaper in srhi than in mrhe. conclusions: sci for right colon cancer is safe and technically feasible. sci reduces the length of skin incision and postoperative pain compared with conventional mce. aim: this study was designed to clarify the utility of laparoscopic surgery for advanced lower rectal cancer after neoadjuvant chemoradiotherapy (ncrt). patients and methods: we investigated -year disease-free survival rate, operative outcomes and recurrence risk factor in patients with lower rectal cancer (ct - , n - ) who underwent laparoscopic surgery after ncrt from to december in kitasato university hospital. results: of patients, patients underwent low anterior resection (lar), patients underwent intersphincteric resection (isr) and abdominoperineal resection (apr). there were anastomotic leakage, and urinary disorder and sexual dysfunction. ypcr rate was . %, but patients ( . %) had recurrence ( liver, lung and lymph node and local recurrence; there is some overlapping). ypt and lymph node metastasis were detected as a recurrent risk factor. the -year relapse-free survival rate (rfs) was . % and the -year overall survival rate (os) was %. conclusion: in this examination, ypt and lymph node metastasis were risk factor for recurrence. the operative outcomes, -year rfs and the -year os are relatively good results. we will conduct further follow-up, and it is necessary to investigate a long term prognosis. laparoscopic surgery is warranted for rectal cancer after ncrt. surg endosc ( ) introduction: synchronous colorectal neoplasia presents an incidence ranges from % and %. classically its surgical treatment consisted in the realisation of a subtotal colectomy (stc), however, several authors have proposed that in certain occasions the realisation of two segmental resections with two anastomoses was not accompanied by an increased risk of anastomotic failure. the objective of this study was to compare the feasibility and safety of the laparoscopic approach of synchronous colorectal neoplasia using two different techniques: stc versus two segmental resections with two anastomoses. methods: we retrospectively reviewed the clinical data of patients over years of age who underwent colorectal surgery between and at a single center. we included patients with a synchronous colorectal neoplasia who underwent laparoscopic surgery, either stc or double resection (dr). results: a total of patients met the inclusion criteria. mainly males ( %) with an average age of years, with a scale of the american association of anesthesiologists superior to ii in % and with an average body mass index of kg / m . the mean operative time was min in the dr and min in the stc, the stc resulted in a higher conversion rate ( % vs %) and intraoperative bleeding ( % vs %), in addition to a postoperative period with more complications, only % of the patients undergoing stc didn't present any complication while % of the patients with a dr didn't present any complication. % of the stc presented anastomotic failure and only % of the dr. the mean hospital stay was days in the dr and . in the stc. in the dr, an average of cm of colon was resected with an average of . lymph nodes, while in the stc, cm of colon was resected with an average of . resected nodes. conclusions: the double resection with two anastomosis is a less aggressive surgery, with fewer complications and a shorter hospital stay, providing similar oncological results. there were no differences in morbidity, re-operations or hospital stay. regarding tumor stage there were no differences between the three groups. as for the resected nodes, we found a mean of in stc, in lc and sr with no statistical difference. there were no differences in the affected nodes among groups. in our patients we didn't find differences in the recurrences rate or in the distant metastases rate.the average follow-up was months (range: - ), with no differences in overall survival. conclusion: segmental resection of splenic flexure neoplasias is safe and feasible, with no differences in morbidity or in the oncological outcomes compared with more aggressive surgeries. introduction: the evaluation of perfusion in colorectal anastomosis is still a field of study and progress for the development of new modalities that allow reducing the ratio of dehiscence or anastomotic leakage (al) in said surgery. our objective with this work is to highlight the utility of indocyanine green (icg) in the said evaluation after colo-rectal surgery. methods: we present a series of cases of colorectal surgery (benign and malignant disease) intervened in the period between and . the population sample has been homogenized according to age criteria, risk factors and comorbidity. a retrospective database has been developed with the spss v. software for the evaluation of the results obtained. the primary outcome measure was al rate with at least month of follow-up. results: a significant reduction in the incidence of al was observed in patients who underwent colo-rectal surgery (p = . ). low al rates were shown in rectal cancer surgery (p = . ). there was no significant decrease in the al rate when colorectal procedures for benign and malignant disease were combined. conclusions: the use of the image by fluorescence with indocyanine green is a safe, reproducible and relatively simple method with which to evaluate the perfusion of the colorectal anastomosis as well as reduce the rate of anastomotic leak in the postoperative period. large well-designed randomized control trials are needed to provide evidence for its routine use in colorectal surgery. introduction: currently colonoscopy is the gold standard investigation for colonic evaluation. although caecal intubation is one of its quality indicators, it is not attained in up to % of cases. this remains a significant concern. limited data are available on the follow-up of patients with incomplete colonoscopy. aims: to assess colonoscopy completion rate, the reasons for incomplete colonoscopy, and the methods used to complete colonic evaluations after incomplete colonoscopy. methods: we performed a retrospective study of incomplete colonoscopies in our unit over a one year period ( ) these results compare favorably with published data. few statistically significant differences between groups suggest varying modalities of treatment broadly result in similar qol. this data highlights a need for well-delivered support programmes for specific issues, for example stoma care and sexual dysfunction. future studies will need to include a baseline questionnaire to truly measure the impact of surgery and measure quality in an increasingly elderly and comorbid population. splenic flexure cancer (sfc), comprising the tumours raised in the distal transverse colon and proximal descendingcolon, accountfor to %of all surgically treated colorectal cancers.in cme forsfc, dissection of both the transverse and descending mesocolon must be considered. however, the use of laparoscopic surgery as a curative treatment for sfc, has never been investigated in adequate controlled trials, because of difficulty in deciding on the appropriate operative procedure, as well as technical difficulties with laparoscopic lymph node dissection. the aim of this multicenter study is to evaluate the oncologic effectiveness of laparoscopic segmental resection with cme with for cancer located at the splenic flexure. we performed a retrospective analysis of all cases of sfc treated with a laparoscopic segmental resection with cme in five different institution. intra and post operative were evaluated. patientes were evaluated, the mean operative time was . ± . min. a total of ( . %) conversions occurred, due to splenic artery lesion, one for difficult adesyolisis and three due to locally advanced tumour. recurrence was observed in ( . %) patients. there was a significant association between disease stage and recurrence (p \ . ) with a higher proportion of stage iv patients in the recurrence group ( . % vs . %). at days follow-up no mortalitywere recorded.during a median follow-up of months (range - ), deaths occurred (all of them for disease progression). keplan mayer curves showed a compareble suvival with other colo-rectal cancer. in conclusion, laparoscopic segmental resection with cme and cvl seems to be an oncologically safe and effective procedure for treatment of sfc. it may be regarded as the standard surgical method for elective management of this disease. in the future, more tailored patient-and tumor-specific segmental resection might be achieved with the use of routine lymph node road mapping. it is very important to establish a minimum number of lymph nodes to analyse for a correct staging. it has been established as . the treatment of colorectal cancer is essentially surgical. the review of the medical literature indicates that laparoscopic colorectal surgery is a safe procedure that has not found significant differences in the survival rate from open surgery. aim: the aim of our study is to compare the outcomes of laparoscopic and open resection for colorectal cancer surgery evaluating lymph node assessment. methods: the patients were collected in our hospital during the period from / / to / / and the number of lymph nodes obtained in lymphadenectomy has been studied comparing the laparoscopic and laparotomy approaches. results: interventions were performed. were laparotomic, were laparoscopic and converted laparoscopic (fig ) . the average number of nodes found in these interventions was , . nowadays, the recommendations to obtain a proper lymphadenectomy is to find more than lymph nodes. analysing our procedures, surgeries had obtained a good lymphadenectomy. according to the approach, , % of the interventions ( ) are laparotomy, , % ( ) are laparoscopic procedures and , % ( ) are by reconverted laparotomy (figure ). the average number of lymph nodes isolated was similar. laparotomy approach found , nodes while , nodes were found in laparoscopy. converted laparoscopy found , ( figure ). conclusion: the treatment of colorectal cancer is essentially surgical. today, there are a lot of studies that support that laparoscopic surgery has a survival rate similar to laparotomy surgery. according to our study, the data collected indicates that the number of isolated lymph nodes in both approaches is very similar. to sump up, laparoscopic colorectal surgery is safe and has demonstrated oncological adequacy comparable to open approach and better short-term outcomes due to a less invasive approach. background: laparoscopic low anterior resection highlights the advantages of laparoscopic surgery (better surgical field, less bloodloss, less postoperativepain, better cosmeticresult). defunctioning ileostomy prevents anastomotic leakage in low rectal cancers, butincreases morbidity, degrades thequality of life and requires a second surgery for its closure. method: in the last months we performed laparoscopic low anterior resections for rectal cancer, whithout performing any protectiveileostomy, afterchecking the anastomosis intraoperatively( men, women. average age: years). the typical placement of trocars included one supraumbilical mm trocar, two right sided mm trocars in the midclavicular line, one mm in the left midclavicular line and one mm trocar in the suprapubic midline which is also used for specimen removal, after a cm transverse extension of the incision. we present themain stages of the procedure (dissection andmesorectal excision, division of the rectum with linear stapler using the 'chinese hat-parnex' technique, creation of an end-to-end intracorporeal anastomosisusing circular stapler under direct laparoscopic vision). results: no major postoperative complication was observed. the mean operative time was min ( - ) and free surgical margins were achieved. in one case a conversion to open surgery occured. the average length of hospital stay was days ( - ). conclusions: the laparoscopic approach facilitates access to the middle and lower rectum, total mesorectal excision and avoidance of ileostomy if possible. it is a demanding operation with extended learning curve, and requires adequate experience in laparoscopic surgery and colorectal surgical oncology. background: in colorectal cancer, local excision is an attractive treatment option, but additional resection is considered when lymph node metastasis(lnm) is expected at high rate. in lower rectal cancer, advanced surgery techniques are required, so it is often difficult to make judgments. the aim of the current study is to assess the reliability of laparoscopic surgery for submucosally invasive rectal adenocarcinoma (pt ) analyzing short-term outcomes and long-term survival. method: this cohort study analyzed patients who underwent laparoscopic rectal resection for submucosally invasive rectal adenocarcinoma (pt ). conversion rate and functional and oncologic outcomes were analyzed. data on long-term results and survival were evaluated. result: surgical procedure was low anterior resection / intersphincteric resection / abdominoperineal resection: / / , and conversion to open surgery was needed for ( . %) patients. sphincter-preserving procedures were performed in ( . %) patients. there were no perioperative mortalities and positive resection margin. the mean length of hospital stay was . days. complications beyond clavien-dindo grade iii occurred in ( . %) patients,the anastomotic leakage rate was . % ( / ). the positive lymph node metastasis rate was . % ( / ). high tumor budding (p = . ), lymphatic invasion (p \ . ), and mucinous /poor histological differentiation (p = . ) were significantly associated with lymph node metastasis on univariate analysis. on multivariate analysis, only lymphatic invasion was associated with lymph node metastasis (p \ . ).the median follow-up time was months (range, - months), recurrence free survival rates was . % ( / ). conculusion: the outcomes of this study suggest that laparoscopic surgery can be used for safe and radical resection of submucosally invasive rectal adenocarcinoma (pt )?and the absence of lymphatic invasion, budding, and mucinous /poor histological differentiation are each associated with low risk of lnm. risk stratification models integrating these factors need to be investigated further. conclusions: this study highlights the complex nature of sarcopenia, as well as its common incidence. minimally invasive surgery had a higher incidence of sarcopenia than that of open surgery when both were performed within an enhanced recovery setting. despite colorectal patients being a typically well-nourished cohort at low risk of complications, there may well be benefit from interventional strategies such as perioperative immunonutrition or pre-habilitation to reduce the incidence of this poor prognostic indicator. backgrounds: urinary dysfunction is frequently observed after rectal resection and justifies urinary drainage. the concept of enhanced recovery after surgery (eras) has been widely spread from the early s. however, the optimal duration of postoperative urinary drainage is unknown. aims: the aim of this study was to comprehend short-term outcome of early removal of urinary catheter after robotic rectal surgery (rrs). patients and methods: (patients) the data of consecutive patients who underwent rrs at two hospitals between april and november were retrospectively reviewed. the main indication of rrs was the patients who need rectal mobilization with autonomic nerve preservation regardless of benign or malignant disease. perioperative management: none of the patients received epidural anesthesia for postoperative analgesia. our basic principle was to remove urinary catheter on postoperative day (pod) . after removal of urinary catheter, trans-urethral catheterization (tuc) was performed in the following situations: ) no autonomous urination over h after removal ) the decrease in urine volume (\ ml/ hr) ) the appearance of subjective symptoms like abdominal distension. when tuc was required even once, residual urine volume was measured with ultrasonic examination device since then. results: twenty seven male and female were included. the median age of patients and bmi were years old and . kg/m , respectively. the surgical procedures included anterior resection (n = ), intersphincteric resection (n = ), abdominoperineal resection (n = ), hartmann's procedure (n = ), and total coloproctectomy (n = ). only one patient received lateral pelvic lymph node dissection. urinary catheter was removed on pod in cases ( . %), on pod in cases ( . %). although tuc was needed in three cases ( . %) immediately after removal, tuc was no longer needed within three days in all three patients. late dysuria was observed in two cases ( . %), and bladder overdistension was suspected in these two cases. conclusions: our study showed that urinary catheter could be safely removed on pod after rrs. however, careful follow-up observation to avoid bladder overdistension is essential after removal. introduction: intersphincterian low rectal resection is a valid alternative to lower rectal cancers located at about - cm from the anus. methods: we present cases from our personal experience for tumors localized - cm from the anus. of them required preoperative radiochemotherapy. in cases, abdominal surgery was performed laparoscopic, having the surgical specimen extracted transanal. lone star device was used for the perineal procedure in all cases. cases required a manually, separate wires anastomosis; the others cases benefited from mechanical anastomosis performed endoanal with - mm circular stapler. we performed complete mesorectum excision in all cases, ligation at the origin of inferior mesenteric artery, complete mobilization of left splenic flexure and lateral protective ileostomy. all pacients underwent inspection rectoscopy before transit reintegration, and cases were reintegrated over a period of - weeks, except for cases which developed a colo-anal fistula, that closed under conservative treatment over a period of - months. results: there were no postoperative anal incontinence. in one case, a relative anal stenosis occured, which required endoscopic dilation. there was case of tumor recurrence and required abdominoperineal resection. conclusion: literature data sustain a - / ratio for very low rectal resection versus rectum amputation. the limit resection under the tumor is accepted as . cm. very good functional results by considering oncological principles, is a sustainable argument for choosing this kind of procedure as an alternative of rectum amputation. in the few studies conducted on crcs, the reported rate of sln micro-metastases is up to - %. the aim of this ongoing prospective study is to assess the predictability of the ex-vivo nirf sln mapping and of the research of micrometastases in nnd crc patients to propose adjuvant chemotherapy. materials and methods: fifty-eight patients undergoing standard oncological crc laparoscopic resection have been prospectively enrolled in two centre. as previously described by the authors, the intact surgical specimen was extracted and opened longitudinally and ml of indocyanine green (icg; mg/ml) was injected submucosally at four corners around the tumor in order to identify the lymphatic pathway and the slns. each sln presenting as negative at conventional histological analysis, was further investigated with ultrastaging techniques including serial sectioning and additional immunohistochemistry, in order to detect the presence of micrometastases. results: thirty patients were n ? , and were nnd. overall, a total of lymph nodes were retrieved. a total of sln were identified (mean . per case) and of those were nnd. after ultrastaging investigations, micrometastatic cases were found in nnd patients. the patients were so upstaged to n . sln located deeper in the mesenteric and mesorectal fat could easily be identified by nirf (even after nchrt). conclusions: in our preliminary series, the ex-vivo nirf sln mapping rightly predicts the status of loco-regional nodes, as confirmed by the histological investigations. the micrometastases' identification let selected patients to undergo the adjuvant treatment with the aim to reduce the risk of recurrence. ( / ) in lateral node positive group and . % ( / ) in lateral node negative group. four of local recurrence were lateral lymph node recurrence. two patients recurred the other lateral side of previous lpl, then they were laparoscopically resected and no recurrence ( , months). two patients recurred the same side after lpl were not curable because of liver metastasis and extensive invasion to the common iliac vessels. conclusion: selective lpl for rectal cancer was safe and good local control for lateral lymph node positive patients. also curable local recurrence resection was possible for non-treated lateral lymph node recurrence. intestinal malrotation is an embriologic anomaly generally discovered in the first months of life due to bowel obstruction. adult presentation is rare and its association with colon cancer is far more rare. we report a case of a years old man affected by asymptomatic intestinal malrotation incidentally found during an abdominal computed tomography (ct) performed for retroperitoneal colonic perforation in a patient with an endoscopically diagnosed aenocarcinoma of the caecum and a large polyp of the descending colon. preoperative vascular anatomic study allowed us to plan a laparoscopic approach safely also with adequate lymphoadenectomy. the abdominal cavity was entered throught a right flank mm optical trocar on the transverse umbilical line. three additional mm trocars were placed in right iliac fossa, right and left hypocondrium respectively. exploratory laparoscopy confirmed midgut malrotation and a fresh flogistic area at the descending colon perforation site. caecum and ascending colon were on midline and attached due to adhesions to sacral promontory. ileocolic artery (ica), middle colic artery (mca) and ima were selectively ligated but not at their origins due to aberrant anatomy. laparoscopic subtotal colectomy with intracorporeal stapled ileosigmoid anastomosis were carried out (endogia mm, double layer / polyglicolic acid suturing of the breech). the anisoperistaltic nature of the anastomosis is due to the disposition of the mesenterium which did not allow an isoperistalting orientation of the two resected stumps. the specimen was extracted throught a pfannestiel incision. the postoperative course was complicated by intestinal obstruction conservatively treated with slow bowel function's restoration. the patient was discharged from the hospital in th postoperative day. unexpectedly specimen histology revealed two villous adenomas with high grade dysplasia. lymphnodes were retrieved from the specimen (ptisn ). to date our case is the only fully laparoscopic colonic resection reported in literature in malrotation as well as the first intracorporeal stapled ileo-sigmoid anastomosis for such disease. the median hospital stay was days. in-hospital mortality was nil. the overall morbidity was %. the median length of follow-up was months. conclusions: our preliminary results suggest that robotic-assisted surgery for colorectal cancer can be carried out safely and according to oncological principles. robotic surgery is advantageous for both surgeons (in that it facilitates dissection in a narrow pelvis) and patients (in that it affords a very good quality of life via the preservation of sexual and urinary function in the vast majority of patients and it has low morbidity and good midterm oncological outcomes). in rectal cancer surgery, the robotic approach is a promising alternative and is expected to overcome the low penetration rate of laparoscopy in this field. aims: postoperative inflammation have been reported as one of the independent prognostic factors in several types of malignancies.the aim of this study is to clarify the impact of laparoscopic approach on postoperative inflammatory status after surgery for colorectal cancer, and to analyze the association between postoperative inflammation and prognosis in patients with colorectal cancer. methods: a total of patients with stage l-lll colorectal cancer (crc) who underwent curative surgery were retrospectively analyzed. the maximum crp value measured between the times of surgical resection and discharge was defined as 'max crp'. the optimal cut-off value of max crp that best predicts rfs was determined to be mg/dl by the minimum p-value approach. methods: trainees working in this firm were responsible for data collection. patients who underwent emergency surgery during the calendar year of had the following details collected-the presence or absence of a complication in the -day post-operative period, the type of complication and description of complication along with the grade of the complication (see fig. .) . patients who underwent intermediate to major surgery were followed up at outpatients and were specifically asked for the occurrence of complications from the point of discharge up until the outpatient appointment. with one centralised national hospital-the people who were discharged and subsequently experienced considerable or major complications invariably represented back to hospital via the a&e department. results: a total of emergency surgeries were performed by this surgical firm in , % of these being done laparoscopically. of these cases- patients experienced post-operative complications within the first days after their procedure. this equated to a complication rate of . %. the most common complications were abdominal pain, nausea & vomiting, and wound infection. there were complications for each of these categories. post-operative bleeding occurred in cases with fistulas or leak of an anastomosis occurring in cases. death of a patient occurred in instances once as a result of post-operative bleeding from the site of anastomosis after a whipple's procedure, the nd occurred subsequent to post-operative bleeding from a peptic ulcer and in the rd case occurred in an instance of faecal peritonitis as a result of anastomotic failure after a roux-en-y bypass for a patient with pancreatic malignancy. conclusion: the davien-clindo classification proved to be simple, efficient and useful in analysing post-operative outcomes. the results indicate that despite the emergency setting & elderly cohort of patients-minimally invasive surgery proved to be a safe and viable option. conclusions: in this prospective study, we observed greater rates of detection of adenomas among endoscopists. screening colonoscopy on symptomatic and/or high risk group for crc is valuable in early detection and the prevention of crc. large sample size and long period of screening colonoscopy was needed. limitation of our study was the small sample size and no use of high detention endoscopy. results: the mean intraoperative blood loss volume was significantly less in the lap group than in the open group ( vs. ml, respectively, p \ . ). the mean operative time was not significantly different between the lap group and the open group ( vs min, respectively, p = . ). the incidence of severe postoperative complication (grade or higher in the clavien-dindo classification) was lower in the lap group ( / ( %) vs / ( %), respectively). the mean postoperative hospital stay was significantly shorter in the lap group than that in the open group ( vs. days, respectively, p = . ). conclusions: lap-tpe can be a safe and feasible procedure. background: amyloid light chain (al) amyloidosis is a rare protein deposition disorder with an incidence ranging between - cases per million people. it can present insidiously with localized or multisystem symptoms and usually occurs later in life. prognosis is poor as al typically presents at an advanced stage. intestinal pseudo-obstruction is a rarely reported complication of al amyloidosis. here we report a case of al amyloidosis which was identified during surgery for intestinal pseudo-obstruction. case presentation: a year old male presented to the emergency department with a month history of abdominal pain and distension, as well as marked swelling of his lower limbs. this had worsened in the previous weeks and he had developed intermittent diarrhoea. ct showed ileitis with marked dilation of the proximal small bowel. laparatomy revealed small bowel that was grossly distended that rapidly developed multiple petechiae and subsequent haematomas upon handling. two days later a repeat laparotomy was performed and . m of ishaemic small bowel was resected. histology showed amyloid deposition with positive congo red staining. subsequent cardiac events led to an echo being performed that showed concentric left ventricular hypertrophy attributed to amyloid deposition within the myocardium. free serum light chain ratio was sent and confirmed the diagnosis of al amyloidosis. he has recently been started on a treatment regimen consisting of cyclophosphamide and dexamethasone. discussion: systemic al amyloidosis frequently involves the gastrointestinal tract, typically presenting with chronic diarrhoea and associated malabsorption. only case presenting with pseudo-obstruction has been reported in the literature. al amyloidosis presents insidiously with non-specific symptoms depending on which organs are affected. treatment aims to prevent further deposition of protein within the organs. prognosis is determined by the organs that are affected and the extent of protein deposition within them. cardiac involvement holds the worst prognosis ultimately causing sudden cardiac death. the mainstays of management are early identification and treatment implementation to prevent protein build up and subsequent organ failure. conclusion: a diagnosis of amyloidosis should be considered in patients with intestinal pseudo-obstruction to expedite the diagnosis of al amyloidosis and improve survival. aim: in the management of locally advanced rectal cancer (larc), the achievement of a complete total mesorectal excision (tme) with clear resection margins was demonstrated to be the main predictor of overall and disease-free survival. predicting surgical difficulty in larc patients may be of particular importance to choose the best surgical approach. this study proposes a mri-based score to identify preoperatively larc patients with a high risk of having a difficult surgery. methods: this is a retrospective study based on the european mri and rectal cancer surgery (eumarcs) database, including patients with mid-low larc who were treated with neoadjuvant chemoradiation therapy and laparoscopic tme with primary anastomosis. data on pre-treatment and restaging through magnetic resonance imaging were available for all patients. surgical difficulty was defined as high or low grade taking in to account operative (e.g. duration of surgery), and postoperative factors (e.g. hospital stay). score accuracy was evaluated by estimating sensitivity, specificity and area under the receiver operating characteristics curve (aroc). results: seventeen ( . %) of larc patients were graded as high surgical difficulty. the eumarcs score was developed using the following significant predictors of surgical difficulty: bmi [ , interspinous distance \ . mm, ymrtstage = t b, and male sex. the score ranged from to . the cut-off score to best differentiate patients with a high probability of difficult surgery was = points. this cut-off value showed the best balance in sensitivity and specificity. the eumarcs score demonstrated high accuracy (aroc: . ) conclusions: the eumarcs score was found to be sensitive and specific in predicting surgical difficulty in larc patients who were candidate for laparoscopic tme. the score has the advantage of considering patient and cancer related characteristics that can be all assessed preoperatively and it can be useful in the decision making process. this score has not yet been externally validated. background: recently published two non-inferiority randomised control trials has raised questions on laparoscopic surgery for rectal cancer, showing lower quality pathological specimens to those achieved using an open technique. locally advanced rectal cancers add to the level of difficulty for laparoscopy approach. our study was aimed to assess feasibility of laparoscopic rectal surgery, comparing short term outcomes, quality of surgical specimen, morbidity and mortality, between propensity score match groups of locally advanced and early rectal cancers. methods: prospectively acquired data from consecutive patients undergoing laparoscopic surgery for rectal cancer at the minimally invasive colorectal unit in united kingdom between and . locally advanced rectal tumours were identified as t b or t with pre-operative mri scans. all the patients were operated by the same team and the procedures were performed laparoscopically. : propensity score matching was performed to create a perfect match in terms of tumour height. results: total of laparoscopic rectal resections were performed during the study period, out of which patients had locally advanced (la) disease and were propensity-score matched for tumour height with non-locally advanced (nla) patients. median operative time was higher for the la surgery group ( min vs min p = . ). however, conversion to open surgery (p = . ), readmission (p = . ), re-operation (p = . ), clinical anastomotic leak (p = . ) and -day mortality rates (p = . ) were all equivalent between the two groups. r resection was achieved in % of la group as compare to % of nla group (p = . ). conclusion: this study demonstrate that standardised approach to laparoscopy is safe and feasible in locally advanced rectal cancers. comparable post-operative short-term clinical and pathological outcomes were seen between la and nla groups. aims: the application of colorectal cancer screening programs, has showed a decrease in recurrence and mortality. for this reason, these programs are being implemented at a national level in the different spanish regions, as has happened in our community.to present the initial short-term results on the morbidity of the immediate postoperative period to days of colon cancer, mortality and hospital stay after the implementation of a screening program in our center. methods: a retrospective study was performed. patients aged between and years were included in the study, diagnosed with colon cancer. they underwent minimally invasive surgery, in most cases, with any type of colonic resection, from january to december . all patients were diagnosed, conventionally or through a screening program, the latter according to the plan implemented in our community. the sample was divided into two groups of patients according to the way of being diagnosed (group si screening = patients, group no screening = patients) and they were compared according different variables: dependent factors of the patient, factor of type colon cancer, factors of colon cancer resection and follow-up. results: both groups were comparable in all study variables. regarding the variables included in the follow-up, no statistically significant differences were found in terms of postoperative mortality-clavien-dindo v. however we found differences statistically significant in postoperative morbidity (p = . ) and in its classification according to clavien dindo i-iv (p = . ). the complications analyzed independently, such as anastomotic dehiscence (p = . ) or postoperative ileus (p = . ), have also presented significant differences, unlike surgical wound infection (p = . ). conclusion: at our center, the application of the screening program has not influenced in the initial stage of colon cancer or its surgical approach. however, we have found a lower overall morbidity rate and minor complications, justified by a lower incidence of anastomotic dehiscence and postoperative ileus. background: colorectal carcinoma is one of the most common malignancies. surgery is the only definitive method to achieve cure for this illness and can be performed via an open or a laparoscopic approach. the pros and cons of each approach have been discussed extensively, with the oncologic efficiency of the laparoscopic approach being one of the leading topics. objective: the aim of this study was to establish oncological non-inferiority of the laparoscopic approach to colorectal cancer. primary outcome measure was defined as number of harvested lymph nodes. secondary outcome measures were medium-term disease free and overall survival as well as length of hospital stay, time to oral feeding and short-and long-term complication rate. methods: this was a single center retrospective chart review. all consecutive patients who underwent colon or rectal resection due to colorectal carcinoma at hadassah medical center between the years - were included. patients who were operated on for recurrent disease or who had metastatic disease at the time of surgery were excluded. patients were divided into three groups according to the surgical approach: laparoscopic, open or converted. medium-term oncological outcomes were the same for all groups. time to oral feeding, length of hospital stay, short-and long-term complication rate were all significantly improved in the laparoscopic group. conclusions: we were unable to prove non-inferiority of the laparoscopic approach regarding the number of harvested lymph nodes. however, all surgical approaches yielded a high number of harvested lymph nodes which is most probably oncologically sufficient, as reflected by the non-existent difference in medium-term oncological follow up. this study supports previous studies showing the superiority of the laparoscopic approach regarding short term recovery and overall complications rates. aims: two non-inferiority randomised control trials have questioned the utility of laparoscopic surgery for rectal cancer by failing to prove that pathological markers of high quality surgery are equivalent to those achieved by open technique. we intend to present short and long-term postoperative outcomes from the largest single surgeon series of consecutive patients undergoing laparoscopic tme for rectal cancer. we describe the standardised laparoscopic technique developed by the principal surgeon, and the short-term outcomes from three surgeons who were trained in and subsequently adopted the same approach. methods: prospectively acquired data from consecutive patients undergoing surgery for rectal cancer by the principal surgeon (ap) at the minimally invasive colorectal unit in portsmouth between and were analysed along with data acquired between and from surgeons (tq,nf,ah) at three further international centres. end-points were overall and diseasefree survival at years, and early post-operative clinical and pathological outcomes. results: consecutive patients underwent laparoscopic tme surgery by the principal surgeon (ap). at years overall survival was . % (dukes' a = . %; b = . %; c = . %); disease-free survival was . % (dukes' a = . %; b = . %; c = . %). post-operative length of stay, lymph node harvest, mean operating time, rate of conversion, incomplete resection, major morbidity and day mortality were not significantly different between the principal surgeon and those he had trained when subsequently in independent practices. conclusion: laparoscopic tme produces excellent long-term survival outcomes for patients with rectal cancer. a standardised approach has the potential to improve outcomes by setting bench-marks for surgical quality, and providing a step-by-step method for surgical training. results: analysis of association of tumor location (sigmoid, right or left colon), operation time, blood loss, extraction site, type of surgical sutures used for wound closure with postoperative complications or specimen quality either did not show significant correlation or could not be conducted due to data nature. unexpectedly, a significant difference was demonstrated between two surgical teams in terms of hernias. majority of cases- ( . %) were performed by surgeon (s ), surgeon (s ) operated on ( . %) patients, nevertheless minilaparotomy closure was usually performed by junior members of the team. conversion rate was . % for s and . % for s (p = . ). operation time and blood loss were smaller in s group compared to s ( . ± . min vs . ± . min, p = . and . ± . ml vs . ± . ml, p = . respectively). specimen quality and early postoperative complications did not differ. postoperative hernia rate was . % for s and . % for s (p = , ). both surgeons used the same specimen extraction sites and materials for wound closure. hernias were more frequent after vertical minilaparotomy- % ( of patients), and in converted patients , % ( of patients), compared to , % ( of ) in transverse minilaparotomy group. there was no association of hernias and wound infections. conclusions: our study demonstrates, that besides consultant dependent surgical surrogates, steps which are often performed by other members of surgical team (such as wound closure) may contribute to complication rate as well. more thorough supervision of wound closure may be needed. aims: laparoscopic complete mesocolic excision (cme) right hemicolectomy is considered a demanding procedure and it is actually adopted in few centers from the west. the aim of the present study is to analyze the safety of laparoscopic cme right hemicolectomy and to compare its short-term results with standard right hemicolectomy in a single western center. methods: prospectively collected data from patients who underwent laparoscopic cme right hemicolectomy between june and november were retrospectively analyzed (cme group) and compared with data from patients submitted to standard laparoscopic right hemicolectomy between april and november (s group). results: no differences were observed between the cme and the standard right hemicolectomy groups in terms of clinical characteristics. in the cme group, . % of patients were = years old, . % of patients were asa class , . % of patients had = comorbidities, . % of patients had bmi [ and . % of patients had = previous abdominal surgeries. no differences were observed in terms of duration of surgery ( ± min vs. ± min; p = . ) and intraoperative complications ( . % vs. . %; p = . ) between cme and s groups; mean blood loss was lower in the cme group ( . ± . ml vs . ± . ml, p = . ). the percentage of overall ( . % vs. . %; p = . ) and severe (clavien-dindo = ) complications ( . % vs. . %; p = . ), redo surgery ( . % vs. . %; p = . ) and readmission ( . % vs. . %; p = . ) was comparable between cme group and s group. a significant difference was observed in the length of specimen ( ± mm vs. ± mm; p \ . ) as well as in the length of proximal ( ± mm vs. ± mm; p = . ) and distal margins ( ± mm vs. ± mm; p = . ) in favor of the cme group. the number of lymph nodes harvested was slightly higher in the cme group ( . ± . vs. . ± . ; p = . ) as it was for the percentage of cases with less than retrieved lymph nodes ( . % vs. . %; p = . ), although these differences did not reach statistical significance. conclusions: this study represents one of the few western experiences demonstrating the safety of laparoscopic cme right hemicolectomy. cme technique showed good short-term results and better quality specimens when compared with the standard procedure. aim and background: peritoneal dissemination of colorectal cancer (pc) makes the complete resection of cancer lesions impossible. in such cases, multidisciplinary therapy is essential with mainly chemotherapy. preoperative diagnosis of pc is usually uncertain by ct or mri image. for diagnosis of pc needs surgical materials with laparotomy. but the laparotomy and resection of pc with general anesthesia tends to make impossible for immediate chemotherapy. less invasive diagnosis of pc is necessary and expected.endocytoscopy (ec) makes the histological diagnosis with precise images gained by high magnification (x ). as a preliminary examination, ec diagnosis for resected specimens of pc were evaluated. methods: two cases of pc diagnosed in operation were evaluated. under general anesthesia, laparotomy was conducted. peritoneal dissemination lesions obviously diagnosed as pc were resected. immediately the lesions were stained by methylene blue solution for to s. ec observation was done according ec classification ) and ecv classification ). results: in two cases, ec observation was successfully done. images of dilated surface microvessels of a nonhomogeneous caliber or arrangement were observed in nbi ec corresponding to ec-v . histopathological diagnosis of resected specimens was metastatic colorectal carcinoma in peritoneum in both cases. conclusions: histological diagnosis for pc is gained by ec with resected specimen. as the result of this investigation, ec examination via camera port in laparoscopic operation might be possible for diagnosis for pc of colorectal cancer in vivo. aims: the aim of this presentation is to demonstrate and analyze surgical complications, arising during laparoscopic colorectal resections for cancer and to analyze the reasons of adverse events. methods: we demonstrate videos from our surgeries, where different types of complications occurred and share our classification of types of mistakes, that may lead to intraoperative complications and ways to prevent them. results: we divide mistakes in laparoscopic colorectal resections into two large groups-'false strategy' and 'dangerous techniques'. the first includes poor diagnosis, too extensive or insufficient extent of surgery and improper enthusiasm in using platforms. prevention of first type mistakes is in thorough training and peer-review of each consultant practice. second type of mistakes includes two subtypes : 'faulty habits'-use of unsafe techniques (blind port insertion, poor vascular exposure prior to clipping, not obtaining 'critical views', unsafe use of energy and stapling devices etc.) and 'failure in a certain case'-when despite correct general approach a complication occurred (misinterpretation of fascial layers or vessels). prevention of 'faulty habits' lies in supervised training in high volume colorectal departments including dedicated surgical devices training. to avoid 'failure in a certain case' standardization of surgical procedure is essential, as the most efficient way to prevent this type of mistake is 'pattern recognition'ability of a surgeon to compare the picture he sees during a procedure with a 'standard' view, he used to have during previously performed standard surgeries-this is apparentely impossible when every procedure is done differently. regular reviews of own surgeries recording and other surgeons' procedures may also fascilitate pattern formation. in case a complication occurres we use the four step course of action: preservation of the view, temporary control, decision on conversion, permanent control. conclusion: as popularity of laparoscopic colorectal resections is growing rapidly the number of intraoperative compliactions is increasing as well. we demonstrate videos of complications and our approach to classification of possible mistakes. systematic aproach to reasons, underlying certain mistakes helps to produce a strategy to reduce intraoperative complication rate. introduction: the drains placement inside the abdominal cavity has traditionally been carried out to evacuate hematic remains or postoperative collections. there is no scientific evidence of the prophylactic use of drainage in elective colorectal cancer (ccr) surgery to avoid anastomotic complications or other complications. however, it is traditionally used. when the anastomotic leak is produced, it is generally agreed that drainage system should be used for therapeutic purposes. aims: the aim of this study is to evaluate the effectiveness of the use of prophylactic drainage in elective surgery of ccr. we would check if they avoided the appearance of complications, and if they are useful when the anastomotic leak appears. methods and results: we analyzed the data collected in our hospital from / / to / / . we studied the number and type of interventions in which prophylactic drainages were placed, the appearance of anastomotic complications and if these drains were effective. interventions were performed during this period of time. % of these procedures had used prophylactic drainage ( interventions). this percentage was up to % in patients who have performed a left colon surgery as a sigmoidectomy or rectal procedure. during this period, there were cases of anastomotic leakage. in all of them had been placed drainage but only of them were effective. conclusions: we have seen that prophylactic drainage is a common practice independently of the location of the anastomosis. the last multimodal rehabilitation guidelines recommended the nonuse of drains systematically above the peritoneal reflection with a high level of scientific evidence. they cause discomfort to the patient and delay early mobilization. however, it may be useful to use drains in the first h of a pelvic floor procedure. there is not enough evidence to show sistematic drainage after colorectal anastomosis prevents complications of the anastomosis or other complications. aims: colonic cancers of the splenic flexure is uncommon and associated with poor prognosis. several studies were published aimed to identify the optimal surgical option for the best oncological outcomes. however, whether an extended colectomy or a segmental resection is required is still controversial. the aim of this study is to analyse the outcome of the two different approaches through the experience of a single centre. materials and methods: retrospective data of consecutive patients with diagnosis of colonic cancer situated at the splenic flexure of our department between and were analysed. based on type of surgical procedure, patients were enrolled in arm a (segmental resection) and arm b (extended resection). arm a patients were treated with segmental resections with a wide mobilisation of the transverse and descending colon and ligation of the left colic artery, sparing the middle colic artery and the inferior mesenteric artery. functional lateral to lateral anastomosis was performed extracorporeally. arm b patients were treated with more extended colectomies, both associated with central vascular ligation. results: out of patients included, were allocated in arm a and in arm b. patients' population of the arms was homogeneous as concerns demographic characteristics and stage of the disease. operative time was comparable ( , min vs min, p = , ). the length of the specimen was significantly shorter in arms a ( , vs , , p = , ) . the number of harvested lymphnodes did not differ between the two groups ( , vs p = , )postoperative short term complications was comparable in both arms ( vs , p = , ). no postoperative mortality was observed. overall -year survival and disease free survival rates were similar in arm a and b ( . % vs . %, p = , and , % vs , %, p = , ). hospital stay was similar in the two groups (p = , ). conclusions: despite a shorter length of surgical specimen after limited resections, postoperative complications, lymph node harvest, and survival were comparable in both.in our opinion the extracorporeal anastomosis is functional to both the achievement of a cleaner operative field and a better control of the resection margins. incidence of neuroendocrine tumours in the rectal area has increased in recent years.before the onset of minimally invasive colorectal surgery, these lesion had to be treated by a more radical technique when not suitable for endoscopic resection.selection of the cases is mandatory in order to achieve good results not only surgical, but also oncological. we present our series of neuroendocrine tumoirs treated by tamis approach, including technical aspects, deffect closure techniques and data regarding pathological findings.all cases were low grade carcinoid tumours. resection with free margin was obtained in all cases. defect closure was performed in all cases. the tumours were settled , and cm form the anal verge. postoperative course was uneventful, ann no adyuvant therapy was needed.tamis apporach for rectal neuroendocrine tumours is a safe and feasible technique. proper selection of the cases is mandatory in order to achieve good results. surg endosc ( ) aim: to assess the safety and efficacy of single layer of barbed vs double layer 'hybrid' (interrupted and running) suture for the closure of anastomotic stapler access enterotomy after laparoscopic right colectomy with intracorporeal anastomosis. methods: from april to november , laparoscopic right colectomy with intracorporeal anastomosis were performed in our surgical department. all patients in both groups were perioperatively managed using an eras pathway. seventy-two patients had the enterotomy closed with a single layer running suture of filbloc tm (assut europe). these patients were matched with patients who underwent intracorporeal right colectomy with enterotomy closed with a 'hybrid' double layer technique (first layer interrupted stitches in maxon tm - (covidien), second layer using a running suture in pds tm - (ethicon). intraoperative variables, anastomotic leak rate, morbidity and mortality rates were analyzed. results: the two groups were homogeneous with respect to demographics, body mass index (bmi), american surgical association score (asa) as well as for tumor stage. in the barbed group, median operating time was . min vs . min in the hybrid group (p = . ). anastomotic leak occurred in ( . %) patients in the hybrid vs ( . %) patients in the barbed group (p = . ). all patients required a reoperation. intraoperative findings show in ( . %) cases in the hybrid group a leak at the enterotomy closure, while an intact staler access was observed in both patients in the barbed group. no difference was observed with respect to noninfectious complications between the two groups (p = . ). patients in the hybrid group experienced a longer hospital stay when compared to the barbed group (p = . ). a re-admission occurred in the hybrid due an intraabdominal collection, while no re-admission was observed in the barbed group. no patient died in the postoperative period. aims: lymph node status is one of the key prognostic factors in patients with colorectal cancer, and remains the most important selection criteria for adjuvant chemotherapy. it is believed that at least % of node negative patients will suffer disease recurrence within the first years after surgery. this may be due to understaging lymph node status. sentinel lymph node mapping is widely used for staging of breast cancer and melanoma, with injection of colloid tc and isosulfan blue (ib). however, indocyanine green (icg) fluorescence guidance is a new technical approach to this issue, with promising results as it is not influenced by body mass index or lymphatic invasion. intraoperative fluorescence icg navigation also aims for detection of aberrant lymphatic drainage outside the planned resection. the icg lymphography has the advantage of offering a good visualization of the lymphatic channels but there are problems to identify the lymphatic nodes. our objective with this study is to rate the use of the intraoperative lymphogram in cases of elective colorectal surgery to evaluate if there were changes in the surgical attitude regarding the performance of lymphadenectomy. methods: indocyanine green was injected into the submucosal layer around the tumor at points with?a -gauge localized injection before lymph node dissection and the lymph flow was observed at , and min after injection, using a near-infrared camera system. in addition, a complete mesocolic excision with central vascular ligation guided the region where the lymph flow was observed to be fluorescent. the following table summarizes the procedures carried out as well as the lymphadenectomy performed before and after the use of icg. in brief, after the application of intraoperative icg it was observed that in % of patients additional lymph nodes were obtained after the expansion of the surgical plan, moreover % affected lymph nodes were spotted after the expansion of the surgical plan. conclusions: intraoperative real-time visualization of the lymph flow using indocyanine green fluorescence imaging during laparoscopic colon cancer surgery is feasible and a helpful technique for lymph node mapping which may lead to intraoperative changes in lymphadenectomy. tamis resection of rectal tumours has proven to be a sefe and feasible technique, specially for lesion located in the mid and low rectum.when the tumour is located in the upper rectum, and specially near the colorectal junction, tamis resection may be more difficult, not only due to technical aspects, but also due to the risk of a free perforation, specially when a full thickness resection is performed.we present our results of tamis resections of lesions located around the colorectal junction. four resections where performed with the aim of an endostpaler in order to achieve full resection without the risk of a free colonic perforation.in cases, an abdominal combined laparoscopic exploration was made, in order to help and assure proper resection of the lesion as well as avoiding intraoperative complications.distance from the anal verge ranged from to cm.postoperative course was uneventful in all cases, and a complete specimen resection was obtained in all cases.tamis resection of tumours located in the rectosigmoid junction may be a safe and feasible technique in selected patients. methods: between january to april , patients with diagnosis of right colon adenocarcinomas underwent right hemicolectomies. the data was analysed for patients demographic, histology, type of surgical approach, intraoperative details (length of surgical procedure, blood loss, blood transfusion, conversion rate) and short-term post-operative outcomes including complications. introduction: postoperative ischemic colitis is a life-threatening vascular gastrointestinal condition, that mainly occurs after cardiovascular surgery. we present a surprising case following a laparoscopic rectum resection. case report: a -year-old diabetic patient with upper rectal adenocarcinoma undergoing laparoscopic anterior rectal resection (partial mesorectum excision) and mechanical anastomosis following chemotherapy / radiotherapy. after h postsurgery he presented abdominal pain, distension and fever. on adominal computed tomography (ct) scan (contrast enema) no anastomotic leakage (al) finfings were revealed. neither digital palpation nor proctosigmoidoscopy ( th day) showed al signs. the patient clinical situation improve with conservative treatment (antibiotics, digestive rest …), c-reactive protein levels decreased and the blood cultures were negative. on the th day he was discharged presenting semiliquid stools. eight days later he needed hospital readmission: air and feculent/purulent discharge from the previos abdominal drainage orifice. ct scan: no evidence of dehiscense found although rectum and sigmoid colon distention and an image of a 'large fecaloma' were observable. on the th day of hospitalization he expulsed a large malodorous segment of tissue with necrotic asppearance (image) through the anus with surprising histologic features: 'complete-thickness necrotic colonic wall'. further rectosigmoidoscopy: complete anastomosis, signs of ischemic colitis proximate to the anastomosis and a fistulous orifice. surprisingly, the patient progressed favorably, being discharged the th day for ambulatory control with a low debit enterocutaneous fistula. histopathological diagnosis: ypt n m . follow up: the fistula discharge quantity increased maintaining diarrheal stools through anus along with persistent anemia and malnutrition. a exploratory laparotomy was schedule. fistulous tract towards a small stenotic segment of colon inmediately proximal to the colorectal anastomosis was identify and resected. finally a terminal colostomy was performed. subsequent postoperative without incidents. currently the patient is asymptomatic. comments: it seems indisputable that a colon segment, proximal to the anastomosis, was necrosed and expelled through a colorectal anastomosis. the mechanism seems inexplicable to us. it is even more disconcerting that there was no disruption of the anastomosis. objectives: fluorescence-guided surgery has emerged as a new imaging modality to improve the detection of liver and lymph node metastasis in colorectal cancer. in right-sided colon cancer, the standard lymphadenectomy should reach the ileocolic vessels and the right branch of the middle colic vessels. the purpose of this study is to perform an objective estimation of lymphatic drainage and metastatic lymphonodes in right-sided colon carcinoma through indocyanine green (icg) lymphography. methods: patients with right-sided colon adenocarcinoma were included, excluding those in stage iv, t and those who underwent urgent surgery. cc of icg peritumoral were injected using a peripheral intravenous catheter at the beginning of the intervention. the lymphatic drainage mapping of the tumor was identified. lymphadenectomy of the ileocolic vessels and right branch of the middle colic vessels was performed extending it to the left branch and origin of middle colic vessels if it was shown in the mapping. results: patients were included. the average age was . in patients the tumor was located in the ascending colon and in patients in the hepatic angle. in patients, the mapping showed lymphatic drainage to ileocolic vessels and right branch of the middle colic vessels. in patients ( %) it showed drainage to the left branch and origin of the middle colic artery, therefore extended lymphadenectomy was performed at that level. in patients, the postoperative period was uneventful. patient presented infection of the surgical wound and another patient developed a cm perianastomotic collection treated with percutaneous drainage. the anatomopathological report showed nodal metastasis in of the patients ( %) in whom lymphatic drainage was observed in the territory of the middle colic vessels with icg. these patients presented the tumor in the hepatic angle. therefore, of the patients with right-sided colon carcinoma ( %) presented nodal metastasis in the territory of the middle colic vessels. conclusions: fluorescent lymphography may improve the results of lymphadenectomy in colon cancer. in patients with tumors of the hepatic angle, lymphadenectomy extended to the left branch and origin of middle colic vessels, could be an adequate alternative. introduction: over the last decade, the common principles of surgical treatment in colon surgery are central vascular ligation (cvl) and complete mesocolic excision (cme). however, the superior mesenteric vessels anatomy, while performing the right colectomy is characterized by wide variability, which can lead to complications, especially during minimally invasive surgical intervention. objective. the purpose of this study is describing vascular variations around the superior mesenteric artery and vein-middle colic, right colic and ileocolic vessels, henle trunk in the laparoscopic right colectomy. materials and methods: the study was held in the 'dobrobut' clinic and o. o. bohomolets national medical university, department of general surgery (kyiv, ukraine) during the - period. patients were included to the study, females ( . %), males ( . %) in the average age of , ± , years. all the patients underwent the laparoscopic right colectomy (cme ? cvl) with d lymph node dissection. recorded video materials from each laparoscopic right colectomy were analyzed during the study. results: ileocolic vessels were the most stable. there were typical anatomical position in all cases. . % of cases, ileocolic vein was identified anteriorly to the ileocolic artery, while . % being posteriorly. right colic vein was absent in . % of cases. right colic vein drainage was to henle trunk and inferior mesenteric vein in . % and . % respectively. the right colic artery was present in % of patients, it's origin was superior mesenteric artery in . % and . % the middle colic artery. the middle colic vein was present and drained to superior mesenteric vein in % of cases. same as the middle colic artery with the superior mesenteric artery origin. henle trunk was present in . %, gastro-pancreato-colic trunk in . % of cases gastro-pancreatic trunk in . %, gastro-colic in . %. conclusions: knowing the options of surgical vessels anatomy, while performing the right colectomy, altogether with surgeons preparation, using the ct-scan data can reduce the risk of iatrogenic damage and complications risks. introduction: the enhanced recovery after surgery (eras) protocol was designed to accelerate convalescence, reduce morbidity and shorten the length of hospital stay (los). one of its major interventions is balanced perioperative fluid therapy. the impact of this single intervention on short-term outcomes is widely discuss. aim: the aim of this study was to assess the impact of perioperative fluid therapy on short-term outcomes. material and methods: the analysis included consecutive prospectively registered patients operated laparoscopically for colorectal cancer between november and january . patients were divided into two groups: balanced (= ml) or unbalanced ([ ml) perioperative fluid therapy. all patients were treated according to eras protocol. study outcomes were: recovery parameters, morbidity rate, los, -day readmission rate. results: group consisted of and group of patients. there were no statistically significant differences between the groups in terms of demographic and operative parameters. morbidity was lower in group ( . % vs . %, p = . ). patients in group were discharged home earlier than in group ( vs days, p \ . ). moreover, we observed differences in recovery parameters between the groups: tolerance of an oral diet on the st postoperative day ( % vs. %, p = . ) and patient mobilization on the day of surgery ( % vs. %, p = . ). -day readmission rate was lower in group ( . % vs. %, p = . ). conclusion: a balanced perioperative fluid therapy on the day of surgery may be associated with faster convalescence, lower morbidity rate, shorter los and lower -day readmission rate. methods: a retrospective analysis was performed including patients who underwent lcs or ocs for cancer treated as emergency in a single centre between and . patients who underwent palliative surgery were excluded. lcs were : propensity score-matched based on pposum and stage of disease with ocs. short-term outcomes included oncological quality, length of hospital stay (los) and postoperative mortality. for long-term outcomes, -year overall and disease free survival (os and dfs) rates were analyzed. results: during the study period, a total of emergency colorectal resections were performed. of them, % (n = ) were coloniccancers. lcs were matched to an equal number of ocs.median age was ( ) years and % were females. median follow-up was ( ) months. the majority of resections were right hemicolectomies ( %), followed by sigmoid resections ( %) and subtotal colectomies ( %). operative time ( ( ) background: total mesorectal excision (tme) offers the best reported rates for local recurrence and survival in patients with rectal cancer. our series from a single high-volume center, assessed the feasibility, safety and long-term oncologic adequacy of laparoscopic total mesorectal excision methods: we reviewed the prospective database of consecutive unselected patients undergoing laparoscopic tme for rectal cancer between and at the department of general surgery, onze-lieve-vrouwziekenhuis hospital (olv), campus aalst, belgium. the objective of the present study was to evaluate the effectiveness of laparoscopic tme, with an emphasis on perioperative variables and long-term oncological outcomes. results: pts with mid and distal rectal cancer up to cm from the anal verge had laparoscopic tme resection. patients ( . %) underwent a sphincter-preserving surgery and the remaining patients ( . %) had an abdominoperineal resection. end-to-end anastomoses: pts ( %), j-colonic pouch: pts ( % introduction: the rica clinical pathway (intensified recovery in abdominal surgery), also called surgical multimodal rehabilitation, is the application of a series of perioperative measures and strategies in those patients who are going to undergo a surgical procedure with the objective of reducing secondary stress to the surgical intervention. in this way, we achieve a better recovery of the patient and significantly reduce complications and morbidity. objective: s to analyze, through our database of patients undergoing crc, the percentage of postoperative ileuses and the following quality indicators: the postsurgical hospital stay, the anastomotic leak, and the infection of the surgical site. to check if the implantation of the rica pathway has meant an improvement in our postoperative hospital stay and with that, a lower sanitary cost. methods and results: we analyzed the data collected from those patients who underwent ccr in our hospital between / / and / / , during which time we implemented the rica clinical pathway. the average hospital stay was days. of the patients, . % presented anastomotic leak, . % infection of the surgical wound and . % paralytic ileus. we have verified how the average hospital stay increases with the appearance of anastomotic leak ( . days), infection of the surgical wound ( . days) and paralytic ileus ( . days). when we divided this -month period into two halves to see the impact of the implantation of the clinical pathway, we obtained the following results: the post-surgical hospital stay in the period from / / to / / was . . the stay from / / to / / was . . the implantation of the rica clinical pathway is providing us with important advantages in our clinical practice, with greater postoperative comfort and an improvement in our quality indicators, such as the decrease in the average hospital stay of our patients. on the other hand, after starting its implementation we have encountered the resistance to change clinical habits and the one that requires a multidisciplinary participation, so adherence to this is being progressive, and requires periodic audits to reinforce and consolidate our achievements, and identify our points of improvement. however, tem has not yet achieved widespread use. recently, transanal minimally invasive surgery (tamis) using single-port surgery devices has been reported. initially facilitated by existing single-port surgery devices, two platforms for transanal access, the gelpoint Ò path (applied medical, rancho santa margarita, ca, usa) and the sils tm port. the gelpoint Ò path is the only platform to be specifically designed for tamis y tatme. objetive: in the present study, usesa gelpoint Ò path was performed in patients with lower rectal neplasms. results: complete full-thickness excision was performed in all cases of tamis and free margins over rectal cancer. on two cases no neoplasm was visualizad. the patient characteristics, operative techniques and operative outcomes were evaluated. the mean age of the patients was . years (range - ). the mean operating time were min (range - ). patients was selsted for tatme, for tamis and two patients for evaluation and biopsy if was necesary. additional transabdominal rectal resection was not performed, and adjuvant chemoradiotherapy was performed in all cases. tamis using a gelpoint Ò path was revealed to be easy and safe to perform. although only a small number of cases were treated, and the operation was demonstrated to be sufficiently feasible. conclusion: gelpoint path is a good tool for colorectal surgery in tatme, tamis and evalluation of anastomoses or de novo lesions introduction: several improvements in rectal cancer treatment, in the last decades, resulted in a markedly increased survival. nevertheless, surgery remains the prevalent treatment and to % of operated patients experience some kind of functional abnormalities. as nowadays we acknowledge the importance to focus not only on survival rates but also on quality of life, we craved for a precise, reproducible, simple, clear and user-friendly tool for evaluating bowel function in rectal cancer patients after sphincter saving operation. therefore, we performed a thorough translation with cultural adaptation of the patient reported outcome tool, low anterior resection syndrome (lars) score, to the portuguese language (lars-pt) and population. methods: according to the current international recommendations, we designed this study encompassing three main phases: (i) cultural and linguistic validation to european portuguese; (ii) feasibility and reliability tests of the version obtained in the previous phase; and (iii) validity tests to produce a final version. the questionnaire was completed by patients from six portuguese colorectal cancer units, and completed it twice. results: the portuguese version of lars score showed high construct validity. regarding the test-retest, the global intraclass correlation showed very strong test-retest reliability. looking at all five items, only items and presented a moderate correlation. lars score was able to discriminate symptoms showing worse quality of life in patients submitted to preoperative radio and chemotherapy. conclusion: lars questionnaire has been properly translated into european portuguese, demonstrating high construct validity and reliability. this is a precise, reproducible, simple, clear and user-friendly tool for evaluating bowel function in rectal cancer patients after sphincter saving operation. therefore, his sistematic use should be implemented. oesophagectomy is the mainstay of curative treatment for oesophageal cancer and post-oesophagectomy diaphragmatic hernia (podh) represents a potentially life-threatening surgical complication characterized by an underestimated occurrence rate and unknown related risk factors. this study analyses the experience of two tertiary designated centers in order to evaluate key elements concerning development and treatment of podh. a cohort of consecutive patients affected by a clinically resectable oesophageal cancer (any t, any n and m ) underwent ivor-lewis oesophagectomy between march and april according to three different approaches: totally open incision procedure (oilo), hybrid (hilo) and totally mininvasive to esophagectomy (milo). all population was retrospectively observed in the context of a postoperative calendarised follow-up in order to record the incidence and postrepair results of podh. patients underwent ivor-lewis oesophagectomy for cancer and ( . %) developed podh within a median follow-up period of months ( - ). surgical repair was generally applied by the mean of laparoscopic cruroplasty ( %) with a conversion rate of %. postoperative morbidity did not include early recurrences but exclusively cardio-pulmonary complications ( patients) with one case of respiratory failure leading to death. the discharge was reached after a median hospital stay of days ( - ) while recurrences ( %) occurred over a median followup period of . months. a wide univariate analysis identified statistically significant associations between podh occurrence and the administration of preoperative chemoradiotherapy, the complete pathological response (cpr) and a lymph node harvest (lnh) larger than stations (p-value of . , . and . respectively). the strong influence of an extended lnh was confirmed by the multivariable analyses ( . ) along with cpr which should however be considered as longer survival-related bias. the minimally invasive surgery and the neoadjuvant chemoradiotherapy represent a considerable part of multimodal treatment for oesophageal cancer presenting a not statistically significant association with podh development while a lnh including more than nodes resulted to be an independent risk factor mirroring the extent of surgical demolition in oesophagectomy. l. barbulescu aim: to asses the safety and effectiveness of robotic total meso-rectal excision vs laparoscopic total meso-rectal excision and to analyse the primary outcomes. methods: the operative, post-operative and oncological outcomes were evaluated to assess the effectiveness of both techniques of tme. in our center were performed robotic rectal resections and laparoscopic resections from january to present. results: the rtme was associated with longer operation time, early bowel movements, lower risk of conversion and shorter hospitalization. the statistical equivalence was seen between rtme and ltme for non-oncological variables like blood loss, morbidity and reintervention risk. the oncological variables such as number of harvested nodes and positive circumferential resection margin risk were also comparable in both groups. the length of distal resection margins was similar in both groups. conclusion: rtme in patients with rectal cancer was associated with a lower rate of conversion and less incidence of urinary retention. the operative time in rtme was significantly longer than in ltme. the initial oncological and function outcomes of rtme seem to be equivalent with ltme. c. athanasiou aims: two randomized controlled trials failed to show non-inferiority of the laparoscopic total mesorectal excision (ltme) compared to open. ltme becomes particularly challenging in low rectal cancers and in narrow pelves. many surgeons report that robotic tme (rtme) may be beneficial in that setting. our aim was to systematically review the literature and compare the pathologic outcomes of open, laparoscopic and robotic tme for rectal cancer methods: medline, embase, scopus, cochrane library and web of knowledge databases were searched for randomized controlled trials (rct) reporting patholologic outcomes of open, laparoscopic or robotic tme with no language restriction. our primary outcome was quality of tme on macroscopic assessment of the specimen. secondary outcomes included positive circumferential resection margin, distance to radial margin, number of lymph nodes and positive radial margin. the included studies were quality assessed and the jadad score was reported. the grade approach was used to rate the certainty of each network estimate. results: fourteen rcts were included in our study. seven rcts compared the otme to the ltme, six compared the ltme and rtme and one study the otme to the rtme. no statistical significant difference was found in quality of tme when the the ltme was compared to the otme or = . ( . , , ) or the rtme or = . ( . , . ) . no difference was found in pcrm for the laparoscopic or = . ( . , . ) or the robotic approach or = . ( . , . ) when compared to open. distance to radial margin and number or lymph nodes didn't differ between the groups. conclusions: no significant advantage on pathologic specimen quality has been found with the robotic approach. the ltme doesn't seem to compromise the quality of the specimen. h. samura , j. arakaki , k. sugata , y. hori , y. nagamine , f. kohagura , h. motonari , s. kameyama , t. ishimine division of digestive and general surgery, urasoe general hospital, okinawa, japan; department of surgery, urasoe general hospital, okinawa, japan colorectal cancer often invade adjacent organs and it is known that prognosis improves with resection of the involved organ. we report our experience of invaded adjacent organ resection, which include seminal vesicle, uterine and bilateral appendages, posterior wall of the vagina and bladder wall. method: although the range of resection is predicted by image study preoperatively, at the time of operation, it was decided by palpation with a forceps. each operation is evaluated by operation time, blood loss, blood transfusion volume, postoperative complication, postoperative hospital stay, and short term prognosis. result: resection cases of seminal vesicle, posterior vaginal wall, uterine and bilateral appendages and bladder wall were , , and , respectively. the results are shown in the order of seminal vesicle / vaginal posterior wall / uterine / bladder. median age was , , and years old. the median operation time was , , , min, the median blood loss was , , , ml, and only one case of uterine and bilateral appendages resection required the blood transfusion. the average postoperative hospital stay was , , , days. nine cases have postoperative complication, that include delayed wound healing, anastomotic leakage and rectovesical fistula, postoperative ileus, chyle ascites and neurogenic blodder. all of those were improved with conservative treatment. the mean hospital stay in complication cases was days ( - ) and ( - ) days without complications. the median observation period was days ( - ), and there was no local recurrence. all of the case of stage iv were dead. there was no local recurrence and all patient without stage iv are alive, it seems that the resection range was sufficient. conclusion: even with adjacent organ invasion colorectal cancer, it was possible to determine the resection line by palpation with laparoscopic forceps manipulation, and possible to resect margin free of cancer. laparoscopic low rectal resection with/without diverting ileostomy p. ihnát, m. tesar, p. ostruszka, p. gunková, p. vávra background: the construction of diverting ileostomy (di) is recommended to avoid septic complications of anastomotic leakage. the aim of our study was to assess the benefits and risks of di constructed during laparoscopic low anterior resection (lar). methods: retrospective clinical cohort study was conducted in university hospital ostrava, czech republic. all patients undergoing laparoscopic lar with tme because of rectal cancer within a -year study period were assessed for study eligibility. results: a total of patients ( patients without di, patients with di) after laparoscopic lar were enrolled into the study and underwent analysis. both study subgroups were comparable in terms of demographic and clinical features. postoperative -day morbidity was significantly lower in patients without di ( . % vs. . %, p = . ). anastomotic leakage frequency was higher in patients without di ( . % vs. . %, p = . ); surgical intervention was necessary in . % of patients without di. stoma-related complications were noted in . % of patients with di; some patients had more than one complication. surgical intervention because of stoma-related complications was needed in patients ( . %). distinctive complications of di laparoscopic construction (small bowel obstruction due to di semi-rotation around its longitudinal axis) was noted in patients ( . %). mean stoma period (interval between lar and di reversal) was more than months in our study; only . % of patients were reversed without delay (= months). postoperative morbidity after di reversal was . %; re-laparotomy was needed in . % of patients. conclusions: despite benefits of di in protecting low rectal anastomosis, ileostomy construction remains fraught with many stoma-related complications and long stoma periods associated with significantly decreased quality of life. aims: single port laparoscopic is a minimally invasive surgical technique that joint the cosmetic advantages with the well recognized benefits of the standard laparoscopic approach [ ] . we describe a laparoscopic single port hartmann reversal in a patient by the use of the umbilical colostomy site for surgical access [ ] . methods: a years old patient was submitted to a laparoscopic single port hartmann procedure with an trans-umbilical colostomy for a recurrent sigmoid volvulus that was treated at the beginning by endoscopic de-rotation. after three months the patient was reevaluated for a hartmann reversal with a laparoscopic single port technique. after routine skin preparation and laparoscopic setup, the colostomy is mobilized from its mucocutaneous border, and the anvil of a circular stapler is secured to the distal lumen. by the use of a gelpoint system with trocars, the intra-abdominal adhesiolisis in performed. the splenic flexure is mobilized to achieve a sufficient mobilization of the left colon that allows the fashion of a tension free anastomosis. the rectal stump is mobilized to the mid rectum, starting from the posterior mesorectal fascia around to the anterior rectal wall. a tension-free colorectal anastomosis is secured with a standard circular mm stapling device inserted transanally. the colostomy wound is closed. the operative time was min. results: the postoperative course was uneventful, the patient was discharged at forth postoperative day, oral intake started on postoperative day three. conclusions: single port laparoscopic hartmann reversal thought the umbilical stoma site is a minimally invasive surgical option that is safe in selected patients and offer the best cosmetic results. [ the progressive evolution of surgical techniques and oncologic protocols on rectal cancer disease facilitates surgeons to challenge the skills for anus preservation in low rectal cancer surgery. the laparoscopic surgery is already one of the best ways to reach the pelvic floor and to try procedures, which were previously difficult to apply through open surgery. the anastomotic leakage has particularly high occurrence if the anastomosis is performed in the anal or distal rectum area. it is evident that although the fecal diversion does not decrease post operatory mortality, it significantly reduce the risk of anastomotic leak and the risk of a second major surgery when the leak occur. diverting stomas are low-risk procedures from a technical point of view, but they potentially expose the patients to postoperative morbidity, impacting the patients' quality of life. it is not easy to decide whether the fecal diversion is needed or not. this decision must be made on a case to case basis, trying to apply the stomas only when they are really needed. we report our initial experience by living a transmesenterial cotton loop around the pre terminal ileum which extremities are turned out usually through the lateral trocar wound in laparoscopy or by applying a dedicated mini incision in open surgery. the purpose is to perform (in case of suspected fistula), a mini invasive diverting procedure, by widening the loop wound and by pulling up the ileum in a lateral loop ileostomy. we applied this procedure to consecutive patients with low colorectal anastomosis and in two of them we performed a lateral loop ileostomy with good results. we believe this can be an alternative that needs to be standardized. purpose: sarcoidosis is a chronic, multisystem inflammatory disorder with unknown aetiology characterised by noncaseating granulomas within involved organs. gallbladder involvement in sarcoidosis is extremely rare and literature review revealed only reported cases to date. in this paper, we present a case of gallbladder associated sarcoidosis. method: a -year-old lady was known to the clinic for regular surveillance of liver steatosis and incidental gallbladder polyps. the largest polyp was mm at presentation in and has grown to mm in . in view of worsening symptoms of biliary colic and growing polyps, a laparoscopic cholecystectomy was performed. results: laparoscopic cholecystectomy was unremarkable and specimens of the gallbladder and lymph nodes were sent for histology. histological examination revealed chronic cholecystitis with polypoid cholesterolosis of the gallbladder and noncaseating granulomata within a lymph node, which strongly suggest sarcoidosis. conclusion: in conclusion, we report a case of incidental finding of gallbladder sarcoidosis over the course of treatment of biliary colic and symptomatic gallbladder polyps. therefore, the definitive treatment for patients with symptomatic gallbladder sarcoidosis is a cholecystectomy. the surgical management of cholelithiasis can be associated with significant morbidities. despite the relatively low incidence of bile duct injuries during laparoscopic cholecystectomy, the total number is large due to the high frequency of the operation. the subtotal cholecystectomy with its variants is a well known bailout strategy to the surgical community. however, there is no agreement on when and how to perform these procedures. indeed, the majority of surgeons will adopt these solutions when there is a struggle to identify the critical view of safety. this struggle results increases the risk of injuries. we hypothesize that a primary intent gall bladder lithotomy and disconnection (glad) when the dissection of the gb pedicle is anticipated difficult dissection is a safe and feasible strategic option. methods: out patients elevtively admitted to aberdeen univesity hospital with gall stone disease between march and november , consecutive patients were operated with glad procedure based on intraoperative criteria. the primary outcome was the operative time. secondary outcomes were length of hospital stay, the criteria to do this procedure will be explained, the outcomes will be listed. indocyanine green is a molecule that becomes fluorescent when excited struck with light of a specific wavelength in the infrared spectrum (nir-infrared), allowing the visualization of anatomical structures in which it has accumulated. the aim of the study is the application of icg enhanced fluorescence in laparoscopic cholecystectomy in order to identify the anatomy of the biliary tract, to reduce the risk of iatrogenic lesions and the conversion rate. the study involves laparoscopic cholecystectomy for cholecystitis and gallstones of main biliary tract. the evening before the surgery, a vial of icg ( mg) diluted in ml of saline solution was intravenous injected. during the procedure, after opening the calot triangle, switching to the nir mode on the camera, the anatomy of the biliary tract and in particular of the main biliary tract is visualized. the cystic duct and cystic artery are isolated, their section between clips is cut and the cholecystectomy is performed. from january patients were enrolled: cases of acute cholecystitis, cases of gallstones of main biliary tract, undergoing preoperative ercp. in cases of cholecystitis, the angiography allowed the visualization of the main biliary tract. in one case, an abnormal course of the cystic duct was identified. in two cases of gallstones of common bile duct, it favoured the visualization of the biliary tract anatomy. all cases were completed with laparoscopic technique. there were no intra-and post-operative complications. icg-enhanced fluorescence is a safe, effective, cheap and rapid tool that can also be applied in small hospitals with no need for training. its use does not extend the time of surgery and allows the visualization of the anatomy of the biliary tract, especially in situations where it can be altered by reducing the conversion rate and potentially the risk of iatrogenic lesions of the main biliary tract. case presentation: patient is a year old female with no significant past medical or surgical history presented to the emergency department with a day history of worsening sharp right upper quadrant pain with associated nausea, vomiting, and po intolerance. the pain started a few months prior, however it was self-limited with diet modifications. an ultrasound demonstrated a contracted gallbladder with a mm gallbladder wall. white blood cell count was within normal limits and total bilirubin was slightly elevated to . mg/dl. no palpable mass was noted on physical exam. an mr cholangiopancreatography was performed which demonstrated a dilated gallbladder measuring . x . cm, a severely thickened gallbladder with a small intramural collection and multiple gallstones. the patient proceeded with a laparoscopic cholecystectomy. intraoperatively, the omentum was densely adhered to the gallbladder and needle decompression of the gallbladder was unsuccessful due to the wall thickness. the gallbladder was subsequently removed without any complications. patient's remaining hospital course was uncomplicated. surgical pathology returned demonstrating acute on chronic cholecystitis. discussion: cholecystomegaly or 'giant gallbladder' disease is a rare pathology encountered in the surgical world. there have been few reported cases, most of which occurred in the elderly ([ years). kuznetsov et al. defined an enlarged gallbladder to have a volume of - cc and a giant gallbladder as exceeding cc (the average weight of the liver). the etiology remains unknown, however certain factors exist to allow the gallbladder to reach this size without life-threatening sequela. preoperative imaging, such as mr cholangiopancreatography, is important to differentiate biliary pathology and delineate anatomy. removal of the gallbladder is recommended to prevent the development of complications like cholangitis or bowel obstruction. the cause of cholecystomegaly still remains uncertain and warrants further research. the management and treatment remains similar to acute cholecystitis. aims: mini-laparoscopic cholecysectomy (mlc) is considered to be the best variant of minimizing surgical trauma and improving cosmesis in laparoscopic cholecystectomy. the most challenging techniqual step of mlc is clipping the cystic duct. it may be impossible or unsafe when diameter of cystic duct exceeds mm, which is common in severe chronic colecystitis or acure cholecystits. there is very limited data in the literature about the use of mlc in acute cholecystits. the aim of study was to access the first results of new technique of mlc. methods: five women with the mean age of years ( - ) underwent mlc. the st -mm troacar was inserted in the umbilicus and used for the camera and removal of the gallbladder. the nd -mm troacar was inserted in subxyphoidal area and used for the main working instruments, including medium-large polymer clip-applier (hem-o-lok type). the rd and th -mm troacars were placed in right subcostal area and used for mini-graspers (karl storz). in initial procedures we used conventional -mm clip-applier with adopted medium-large titanium clips. to improve safety, we aplied -mm hem-o-lok type clip-applier for the last patient with acute cholecystitis. in this case the diameter of cystic duct was , mm. the clipping was performed successfully. the -mm drain was placed via subcostal troacar incision. also, in this case we applied original technique of removal of the bladder using wound retraction instrument (karl storz). results: in all the cases there were no intra-or postoperative complications. the mean duration of procedures was min ( - min). the postoperative stay was days in every patient. the patients estimated their pain on postop day as 'almost absent' and cosmethic results mo postop as 'exellent'. conclusions: . new technique of mlc alowed to perform the clipping of cystic duct safely, which is essential in acute calculous cholecystitis. was conducted in department of surgery lumhs jamshoro. all the patients having age = year of age, either gender presented with history of abdominal pain, nausea and vomiting and were diagnosed as cholelithiasis included in the study and were planned either for mini-laparoscopic cholecystectomy and conventional laparoscopic cholecystectomy were explored for outcome while the patients with empyema gallbladder, gangrene, mucocele gallbladder and adhesions were excluded from the study. results: during one year study period, total five hundred patients were diagnosed as cholelithiasis with means age . ± . (sd). of five hundred, ( . %) were underwent for mini-laparoscopic cholecystectomy with ( . %) were males and ( . %) were females. the outcome were measured as postoperative pain (vas) . ± . , size of wound (umbilical mm, epigastrium mm and subcostal mm), excellent cosmetic results, mean ± sd for hospital stay (hrs) and operative time (minutes) was . ± . and . ± . , early return to work ( . %), minor oozing ( . %), port size hernia ( . %). remaining ( . %) were underwent for conventional laparoscopic cholecystectomy with ( . %) were males and ( . %) were females. the outcome were measured as postoperative pain (vas) . ± . , size of wound (umbilical mm, epigastrium mm and subcostal mm), mean ± sd for hospital stay (hrs) and operative time (minutes) was . ± . and . ± . , early return to work ( . %), port size hernia ( . %) along with zero ( %) mortality. conclusion: it has been concluded that mini-laparoscopic cholecystectomy is superior and feasible than conventional laparoscopic cholecystectomy and has decreased early postoperative incisional pain, avoided late incisional discomfort and safe procedure with nearly scarless wounds with superior cosmetic effect especially for young female patients. objective: to determine the outcome of immediate versus late laparoscopic cholecystectomy in acute cholecystitis at tertiary care hospital hyderabad / jamshoro sindh pakistan patients and methods: the descriptive case series study of one year ( - ) was conducted in department of surgery lumhs jamshoro. all the patients having age = year of age, either gender presented with history of abdominal pain, nausea and vomiting and were diagnosed as acute cholecystitis (cholillthiasis) included in the study and were planned for laparoscopic cholecystectomy and were explored for outcome as immediate (within h) and late components ([ weeks). the frequency and percentage was calculated for categorical variables and mean ± sd was calculated for numerical variables. as this was descriptive case series so there was no any statistical test of significance was applied. results: during one year study period, total one hundred patients were diagnosed as acute cholecystitis with means age . ± . (sd). of one hundred, % were females and % were males. the immediate outcome reported as tissue fragile %, pancreatitis %, slipage of ligature of cystic duct %, empyema gallbladder %, mucocele % and gangrenous gallbladder % while the late outcome reported as adhesions %, cholecystoduodenal fistula and mirizzi syndrome % and %, gallstone ileus %, perforated gallbladder % and cholidochiolithiasis % while the mean ± sd for hospital stay (days) in immediate as . ± . while in late outcome (days) during acute cholecystitis . ± . and after surgery ( weeks later) as . ± . respectively. conclusion: it has been concluded that early lc for acute cholecystitis with cholelithiasis is safe, low cost and feasible intervention and offering the additional benefit of shorter hospital stay and reduce the economical burden. surg endosc ( ) :s -s general surgery, chang gung memorial hospital kaohsiung division, kaohsiung, taiwan background: the treatment of common bile duct (cbd) stones is challenging while unclear hepatic hilum anatomy especial experience of previous laparotomy. a minimally-invasive approach choledocholithotomy is feasible, but can be difficult and converted for the unclear anatomy of the biliary tree. near-infrared (nir) cholangiography by systemic administration of indocyanine green (icg) can enhance the visualization of the biliary tree anatomy but is limited by the high intensity of background fluorescence signal coming from the liver. nir fluorescence cholecysto-cholangiography by direct biliary tree administration of the icg can enhance the biliary tree without background noise signal. we created the nir cholangiography via different route according to patient situation : systemic circulation or biliary tree injection to see the feasibility of those application. material and method: ten patients who suffered from obstructive jaundice due to cbd stone and patients received percutaneous biliary tree drainage as first treatment and patients received endoscopic biliary tree drainage. those patients received laparoscopic choledocholithotomy as definite treatment after acute infection phase. patients received biliary tree icg injection via drain tube and patients by systemic injection. visualization and fluorescence patterns around cbd was recorded. results: in our series, one patient received previous gastrectomy and patients had previous biliary tree surgery. background: laparoscopic cholecystectomy (lc) has become the gold standard for the treatment of gallstone disease. multiple studies have confirmed its safety, lc at index admission is still not widely practiced in ireland. we present our experience in performing index cholecystectomy at cuh after the start of acute care surgery program in may . aim: the aim of this study is to determine the safety of laparoscopic cholecystectomy at index admission, complications,re-admissions, and los. methods: electronic records, theatre records and imaging reports were searched to enroll all patients who underwent lc for gallstone disease at index admission from may to october . patient demographics, indication for surgery, postoperative complications, readmission and conversion rate were recorded.in addition timings of mrcp and ercp, imaging findings, and los were also noted. results: a total of patients underwent lc during the study period. median age was years ( - ). male to female ratio was : . . ( %) patients had acute cholecystitis, ( %)had acute biliary pancreatitis, ( . %)biliary colic and ( . %) had cholecystitis with signs of cbd obstruction. ( . %)patients had obstructive jaundice and one with adenomyomatosis. patients ( %) had preop mrcp while ( %) underwent pre-op ercp. all except patients undergoing ercp had preprocedure mrcp. patients had pre-op cholangiograms. in terms of complications, ( . %) patients had bile leak and one( . %) had re-operation. one patient had the post-op hematoma which was drained percutaneously, one patient had procedure abandoned because of bradycardia upon induction of anesthesia. there was no common bile duct injury, no conversion to open and no days mortality was reported. the average length of hospital stay has been days. ( to days). conclusions: laparoscopic cholecystectomy at index admission for cholecystitis, choledocholithiasis, and biliary pancreatitis, has been a safe and feasible treatment option in our hospital. a safe practice can be ensured by adherence to a care pathway and a multidisciplinary, consultant-led service. index cholecystectomy service can be provided safely across the country to prevent diseaserelated morbidity and multiple re-admissions in patients awaiting interval surgery. when to use the two-stage surgery to treat choledocholithiasis: the size aims: the treatment of choledocholithiasis has been provided by various of studies worldwide. the most common accepted minimal invasive treatment was two-stage treatment using endoscopic retrograde cholangiopancreatography before or after laparoscopic cholecystectomy(ercp ? lc), and one-stage treatment with laparoscopic exploration of the common bile duct(lcbde). in fact, despite several large studies have been published in recent years, the debate for the ideal treatment of choledocholithiasis is way from being concluded. we aim to find the proper treatment option for the patients with variable sizes of choledocholithiasis. methods: we retrospectively analyzed patients who underwent treatments for cholidocholithiasis in our institute between january , and july , . the patients who received either ercp and lc in the same admission, and the patient who received lcbde, irrespective of trans-cystic(ltcbde), or choledochotomy(lcd), were included. the data was analyzed with chi-square test and mann-whitney u test. results: the stone size of the ercp ? lc group is significantly smaller than the lcbde group. we further analyzed the ercp failure case, and the group of stone size [= . mm has a significantly higher rate of procedure failure. the failure rate is increasing with the stone size. conclusions: both the treatment of lcbde and ercp ? lc have similar safety and success rate, and the rate of residual stone was also similar in both group. however, the failure rate for ercp is significantly increased when stone size is larger than . mm in this study. aims: the xanthogranulomatous cholecystitis (xc) is a rare entity that can cause doubt in the choice of surgical treatment, because of differential diagnosis with gallbladder carcinoma (gc). methods: a -year-old patient presented acute abdominal pain in the right upper quadrant, nausea and low-grade fever with signs of peritonitis. he had elevated pcr, leukocytosis with neutrophilia. abdominal ultrasound showed an acute xanthogranulomatous cholecystitis. a laparoscopic cholecystectomy was decided but it was converted to open surgery due to the difficulty in the dissection, with fundus embedded in the hepatic bed and intraoperative finding of hilar adenopathic conglomerate .the postoperative period was torpid, with abdominal pain, jaundice, elevated bilirubin and enzymes of cholestasis. postoperative abdominal tomography showed injury in the iv segment of the liver suggestive of neoplasia. metastatic adenopathic conglomerate at the hepatic hilum caused extrinsic biliary obstruction with hepatic failure later so an internal-external drain was placed in the bile duct. the patient was died a week later. the pathological anatomy reported a stage four of gc. results: xc is a rare, non-neoplastic, inflammatory and destructive entity of the gallbladder wall, considered a variant of chronic lithiasic cholecystitis. it may be due to extravasation of bile or ulceration of the mucosa, causing an inflammatory reaction and fibrosis, with xanthomatous cells. the prevalence is to % in the resected gallbladders. it is more frequent in - years oldfemales. its clinical presentation does not have specific characteristics that differ from cholelithiasis, except for the weight loss. radiologically it is characterized by nodular thickening and increased attenuation of the vesicular wall with signs of cholecystitis, indistinguishable from a vc. the xanthogranulomatous inflammatory foci infiltrate the hepatic parenchyma, having an invasive behavior; hence, it mimics a neoplastic disease. the confusion in diagnostic and the risk of gc (up to %) makes treatment contentious. conclusions: the xc can simulate an advanced gc that sometimes makes us wonder if we should perform a radical surgical treatment; when presented in an emergency situation, our therapeutic decision can focus on solving the acute problem and be conditioned by the patient's general condition. single port transumbilical laparoscopic surgery (sptls) is a techinque that has been around for about years. although the enthusiasm for this type of surgery seems to have diminished in recent years it is expected to rise considering the recent development of sophisticated devices for its execution . we report retrospectively our year experience with procedures performed by sptls technique. in a private practice setting in mexico city. procedures include cholecystectomy ( ), appendectomy ( ), inguinal hernia tapp and tep ( ), hiatus and esophageal ( ) , sleeve gastrectomy ( ), colon ( ), gyn ( ) . different access platforms were employed. we explain our selection criteria for the application of the technique and describe the evolution of the instruments employed during the past years, from laparoscopic conventional to curved and bendable; regular scopes to extra long telescopes with different angles. or time, top bleeding, conversion rate, the need to employ an extra trocar, complications, pathology reports, scheduled or urgent kind of surgery and length of hospital stay were recorded from the beggining; patients variables such as bmi, asa status, tep risk, satisfaction with the procedure and other were recorded . we describe the evolution of our technique, and our learning curve with cholecystectomies. we compare our group of sptls transvaginal assisted laparoscopic hysterectomy (tvalh) patients vs tvalh multiport patients. we explain the feasibilty, and efficiency of the procedure in our hands compared to other series. background: in japan, the severity of acute cholecystitis(ac) is assessed by the severity classification of the tokyo guidelines (tg ). the value of c-reactive protein (crp) is not included in the severity classification criteria. the first line treatment, according to tg , for mild (grade i) to moderate (grade ) ac is laparoscopic cholecystectomy, but laparoscopic surgery may not be feasible in some cases due to adhesion or local inflammation of the gall bladder. aim: the aim of this study is to assess the effect of crp on the open conversion rate in laparoscopic cholecystectomy for acute cholecystitis.method: we conducted a retrospective study. patients who were diagnosed with ac and treated with emergent laparoscopic cholecystectomy between june and may in our institution are included. we set the cutoff value for crp at mg/dl and compared the open conversion rate. secondary endpoints are amount of bleeding, operation time, post-operative course (peak in body temperature and inflammatory markers) and the frequency of complications according to the clavien-dindo classification. results: out of patients had a crp value greater than or equal to mg/dl. the median crp values for the crp \ group and crp = group were . and . , respectively. the open conversion rate of the crp = group was significantly higher than that of the crp \ group ( / , / , p = . ). the most common reason for these conversions was local adhesion ( / ) . there were no differences in the amount of bleeding, operation time, post-operative course, and frequency of complications with clavien-dindo grade ii or higher. background: reports about clinical value of fluorescent cholangiography using indocyanine green (icg) during single-incision laparoscopic cholecystectomy (silc) were increasing. we report clinical value and pitfalls of fluorescent cholangiography during silc for the patients with the infraportal type of the right posterior bile duct. methods: our silc procedure utilized the sils-port with an additional -mm forceps through the umbilical incision. before silc, ml of icg ( . mg) was administrated by intravenous injection. for fluorescent cholangiography, icg fluorescent laparoscope system was used. results: we performed fluorescent cholangiography during silc in patients with the infraportal type of the right posterior bile duct. all procedures were completed successfully. the interval from the injection of icg to the first obtained fluorescent cholangiography before the dissection of calot's triangle ranged from to min. detectability of infraportal type of the right posterior bile duct before dissection in claot's triangle was . % (n = ) and that during dissection in calot's triangle was . % (n = ). the infraportal type of the right posterior bile duct could be identified under fluorescent cholangiography only when it joined into the common hepatic duct. conclusions: utilization of fluorescent cholangiography can lead silc to safe even for the patients with the infraportal type of the right posterior bile duct. its benefit is emphasized when the infraportal type of the right posterior bile duct joins into the common hepatic duct. aims: due to the development of laparoscopic surgery and the progress made in surgical treatment ofhydrocephalus, surgeons may come across patients with ventriculoperitoneal (vp) shunt, as candidates for laparoscopic procedures. according to this fact, we report a case of an unusual complication of laparoscopy surgery that can appear in this kind of patients. methods: we present a case of a -year-old man with medical history of normotensive hydrocephalus with vp shunt, that came to the emergency room complaining of abdominal pain and fever since two days. blood test showed an elevation of infection parameters and inflammatory markers, and the ultrasound study revealed an emphysematous cholecystitis. therefore, we decide to carry out an emergency laparoscopic cholecistectomy. the patient did not present any adverse event during the surgery or the immediate postoperative period, being discharged the third postoperative day and evaluated ambulatory one month after the surgery with no complications. two months after surgery, the patient returned to the emergency room presenting alteration in consciousness and fever. results: during the study of the pacient, an abdominal ct was performed, showing a complete section of the vp shunt in the subcutaneus space of the upper abdominal wall and intraperitoneal migration of the remaining catheter. the patient was transferred to neurosurgery to carry out an emergent replacement of the ventriculoperitoneal shunt. after surgery and intravenous antibiotic treatment, the patient evolved favourably and was discharged a few days later. conclusions: the rate of serious complications associated with a laparoscopic approach is overall low and up to % of them occur during the abdominal access for camera or port placement and may not be recognized until postoperative period. vp shunts should not be a contraindication for laparoscopic surgery. however, laparoscopy approach must be carry out with good anesthetic and monitoring facilities and taking several previous considerations, such as verifying the proper functioning of the vp shunt, identifying the path of the catheter within the abdominal wall to avoid inadvertent damage to the catheter during trocar placement and ensuring that the intraperitoneal portion of the catheter is not twisted or obstructed prior to decompression of the abdomen. surg endosc ( ) introduction: since advantages of robotic surgery is being more emphasized, robotic cholecystectomy (rc) cases are increasing. ajou group had introduced a method called which technique places the trocars transversally on the bikini line and it makes cosmesis and pain beneficial. however, rc with low incision port has several limitations. therefore, we changed port placement which may be a one of safe tehniques for rc. method: this study retrospectively reviewed data for patients who received rc with port changing method (rcpc, n = ) and rc with low incision port (rcli, n = ) from february -february and surgical variables were analyzed. results: patients in both groups had similar demographic features and indications for surgery. the rcpc group required no conversions to conventional robotic surgery and no additional operation, whereas the rcli group had one incisional hernia ( . %) and two bowel perforation ( . %) cases. length of stay ( . ± . vs. . ± . days, respectively; p = . ) did not significantly differ between the rcpc and scli groups. however, the rcpc group had shorter operative time ( . ± . vs. . ± . min; p = . ) than the rcli group, although the parameters mentioned above were not statistically significant. conclusion: robotic cholecystectomy with bikini line incision has some limitations even though it has cosmetic benefits. whereas robotic surgery with changing port method is one of safe and feasible procedures for performing robotic cholecystectomy. also nothing more to say that it gains cosmesis effect and escapes complications. mini surgery, odessa medical university, odessa, ukraine the aim of the study was to optimize the diagnostic and therapeutic tactics for yatrogenic injuries of the extrahepatic bile ducts. methods: patients were examined. typical manifestations were jaundice, cholangitis, biliary peritonitis, external biliary fistula, subhepatic abscess.cholecystectomy main cause of damage.a visual, manual and x-ray examination of the hepato-choledochus and cholangioscopy were performed. ultrasound, endoscopic retrograde cholangiopancreatography, fistuloholangiography or percutaneous transhepatic cholangiography play a leading role in diagnosing. the results: high damage to the bile duct was detected in . % of patients, low-in . %.percutaneous transhepatic drainage under ultrasound control was performed in . % of patients.emergency laparotomy, sanation of the abdominal cavity and external drainage of the bile ducts were performed with bile peritonitis. recovery operations produced . % of patients. reconstructive interventions were performed in . % of patients after - weeks after the first stage. the covery operations were successful in . % of patients. . % of the sick had complications in the form of biloma. a scar stricture formed in . % of patients after - months. patient underwent recanalization of the stricture zone with a dilatation balloon through interchangeable transhepatic drainage. balloon dilatation was performed retrogradely through the large duodenal papilla in patients. deaths in the postoperative period was not observed. conclusions: the surgical team should be strengthened by an experienced surgeon when intraoperative diagnosis of yatrogenic damage to the bile ducts.the operation should be completed by external drainage of the bile duct and the abdominal cavity in the absence of an experienced specialist.recovery operations are shown only with lateral injury of the ducts.the patient must be sent to a specialized institution for radical surgical treatment after stabilization of his general condition. aim of the study sub-hepatic bile collections, biloma and hematoma are rare complications and we present our experience in treatment this complications. material and methods: from laparoscopic cholecystectomy performed in our clinic, three patients (two women and one men) to whom it was performed laparoscopic cholecystectomy, came back two weeks later after they were released from the hospital because of epigastric discomfort, fever and nausea. results: clinical examination after rehospitalization showed tenderness in the epigastrium and right subcostal region. in all patients were measured high levels of leukocytosis and crp . an ultrasound examination of the abdomen revealed a large hypoechoic collection in the sub hepatic space, after the abdominal ct scan was performed, the density of the collection did not indicate the presence of blood in two patients. percutaneous drainage of the collection in both patients was realized under us guidance and - fr catheter was inserted in the sub hepatic region. in the first patient cc of bile-stained liquid, and in the second patient cc of biliary liquid was drained. in a third patient h after surgery signs of significant hypotension and limited tenderness at the right subcostal region occurred. a complete blood count (cbc) showed a decrease in the level of haemoglobin to . g%. ultrasound examination revealed a fluid collection in the sub hepatic space, which is also confirmed by computed tomography. laparotomy was performed and the large sub hepatic hematoma was evacuated. after that the fr abdominal drain was inserted into the sub hepatic space. the postoperative course of all three patients was not complicated. conclusion: sub hepatic biloma and hematoma are rare complications of laparoscopic cholecystectomy, while early diagnosis followed by percutaneous drainage or open laparotomy is the only way to resolve these complications. ( ), hemoperitoneum . % ( ) . the average number of days of hospitalization was . days. there was no mortality at days. conclusion: in the emergency setting the rendezvous technique has an adequate success rate of cannulation and clearence of the bile duct, an acceptable surgical time, few complications, these being more frequent in those patients with inflammation of the gallbladder and without associated mortality at days. there is a need for controlled randomized studies with a greater number of patients recruited and follow-up to determine the usefulness of this technique. intraoperative cholangiography could serve as a fundamental solution to avoid the bile duct injury during laparoscopic cholecystectomy. however, it is difficult to identify the cystic duct to which the contrast catheter should be inserted in cases with high degrees of adhesion around the calot's triangle. in these cases, it is not possible to conduct cholangiography from the cystic duct. for these types of cases, intraoperative cholecystography may serve as an option. however, since the bladder is a bag-like organ that expands when liquids are entered, directly inserting a contrast dye into the bladder would make the bladder itself expand, which makes it impossible for to maintain enough pressure in the contrast dye to flow into the cystic duct, extrahepatic bile duct, and intrahepatic bile duct. also, since it is difficult to control leakage of the contrast dye from the catheter insertion site, it is not possible to obtain enough images to sufficiently understand the anatomical characteristics of the bile duct in many cases. therefore, cholecystography is not generally recognized as a method to be used during surgery. in our facility, we insert the contrast catheter through the bladder after stretching the gallbladder neck as much as possible, hold the gallbladder neck with a removable intestinal clamp, and then apply the contrast dye to the bile duct. through this method, it is possible to insert enough contrast dye into the cystic duct, extrahepatic bile duct, and intrahepatic bile duct to understand the anatomical characteristics of the bile duct, allowing us to obtain appropriate images of the biliary tract. because this method uses equipment that is highly versatile, we believe that it is inexpensive and convenient. during this presentation, we will also conduct a case presentation of the methods of bladder contrasting that we utilize in our facility during laparoscopic cholecystectomy. introduction: retrieval of a thick walled gallbladder during a difficult laparoscopic cholecystectomy (lc) for an acute or chronic calculous cholecystitis can be exasperating. it increases operative time and often necessitates enlargement of mm port to deliver the specimen. the 'in-situ cholecystotomy', which we wish to call the 'delhi maneuver' is very helpful in improving the ergonomics of specimen retrieval, saves time and conserves cosmesis. patients & methods: one hundred and ten patients of acute or chronic calculous cholecystitis were placed randomly in groups. a disposable transparent plastic bag was used in all cases to retrieve the gallbladder specimen through the - mm port using a rampley's sponge holding forceps. retrieval was done using conventional technique in patients (group b). the delhi maneuver was used in the remaining patients (group a). it involved cutting the gall bladder inside the plastic bag in a certain fashion, delivering the gallstones in the bag, and removal of gallbladder preceding the stones. the retrieval time, number of insertions of sponge holder, any rupture of plastic bag as well as the number of cases needing port enlargement were noted. results: the average time taken by delhi maneuver (group a) was min as compared to was min by conventional method (group b). the number of insertions of sponge holder ranged from - in group a (mean ) and - in group b (mean ). four patients needed port enlargement in group a ( %) while patients needed enlargement in group b ( . %). there were incidences of bag rupture in group a ( %) and in group b ( %). the delhi maneuver improved the ease and speed of specimen extraction at laparoscopic cholecystectomy for thick walled gallbladders. it also decreased the need for port enlargement for specimen retrieval. the bile duct injuries are a very complex desease to confront, the inciian managment is to clasificate the injury and to identifie the mechamism of the injury. it's important for the optimal heal of the patient to have a multidisciplinary approach including internal medicine, surgery, endoscopy and interventional radiology specialists. the laparoscopic cholecystectomy responsible for %- % of them.this is a retrospective study on the incidence, classification and management of bile duct injuries in a private sector hospital in monterrey nl. mexico. in this study, bile duct injuries were identified in years of experience in a single center. were categorized using the strasberg classification. variables were evaluated such as type of injury, mechanism of injury, hospital stay, if the surgery was scheduled or of emergency, the moment in which the surgeon evidenced the injury, the way in which the surgeon became aware of the injury performed. the type of management that was given to this lesion was also studied and the days of intrahospital stay and the number of reinterventions or procedures performed were compared.the average age of the patients was years, patients belonged to the female sex, although there were lesions of all kinds in this work, there was a greater incidence in strasberg type a lesions, which represented % of the lesions. the most common diagnosis presented was cholecystolithiasis. in surgeries the evidence and repair of the bile duct was in the same intervention aims: bile leak is a rare but recognised complication after laparoscopic cholecystectomy. this usually occurs after a difficult procedure complicated by adhesions, unusual anatomy or if the surgeon is inexperienced or unfamiliar with the anatomy. this video aims to demonstrate the laparoscopic diagnosis and treatment of this complication particularly for surgical trainees. methods: we report a case of significant bile leak occurring soon after a straightforward laparoscopic cholecystectomy due to very short cystic duct (cd). the procedure was carried out uneventfully but the cd was clipped flush with the bile duct. the patient was discharged on the day of surgery feeling well but readmitted with abdominal pain h later. results: after readmission the patient underwent a ct demonstrating only a small amount of fluid suggestive of a small collection. she was treated conservatively but suddenly deteriorated and a repeat ct confirmed significant intraperitoneal fluid. a diagnostic laparoscopy was carried out urgently confirming a cd stump bile leak where the clips had sloughed off causing the leak. two litres of bile was aspirated with copious irrigation and a latex t-tube inserted into the cbd. patient made a full and rapid recovery. conclusions: this is a rare complication and learning opportunities for trainees are therefore infrequent. this video demonstrates a successful laparoscopic approach to management of postoperative bile leak showing t-tube insertion technique and highlighting the need for careful cd closure techniques during laparoscopic cholecystectomy when the duct is very short. about - % of bile duct stones could not be extracted using conventional endoscopic techniques (baloon, sphincterotomy). there is lower success rate in elderly patients; among the biggest challenges are intrahepatic stones, size of stone is large, etc. aims: to present the case of a recurrent intrahepatic lithiasis and its management using spyglass choledochoscopy.to expose, other cases and the main outcome and complications of other difficult cases of bile duct stones that solvedusing this choledochoscope vs. the traditional one and the beneffits. we present a case of years old male who presented with cholangitis caused by an intrahepatic stone that required multiple sessions of endoscopic retrograde cholangiopancreatography with spyglass for clearance. one year later, he presented again with cholangitis, that required another session of spyglass lithotripsy and cholecistectomy. conclusions: besides ercp, there are different approaches to treat difficult bile duct stones, as transhepatic percutaneous drainage, surgical techniques, or other endoscopic techniques (doubleballoon, enteroscopy). ercp and sphincterotomy are the first step of endoscopic treatment with more than % of success rate, and a low mortality and morbility rate; other steps include some lithotripsy techniques, or the use of biliary stent as a bridge before definite treatment. spyglass is a visualization & intervention system used when common ercp has been unsuccessful, and it is first line for better and direct image of biliary ducts, with °range of motion, with multiple advantages like the concomitant use of lithotripsy devices. aims: the number of elderly people has increased, because of the strong association between age and gallstone disease, both prevalence and incidence of this disease are increasing. this presentation aims to review our current management options of octogenerian patients with acute cholecystitis. methods: we retrospectively analyzed octogenerian patients who were admitted to the our hospital with the diagnosis of acute cholecystitis between january and october . the patients were initially allocated to four different treatment groups as follows: immediate surgery, delayed surgery, medical treatment and cholecystostomy. differences in the outcomes between the treatment groups were evaluated. results: there were males ( . %) and females ( . %) with a mean age of . years (range - years). the patients had different co-morbid diseases, especially hypertension ( , . %) cardiovascular disease ( , . %) and diabetes mellitus ( methods: a retrospective observational study where were analyzed patients older than years who underwent urgent surgery for ac who fulfilled an indication for surgery according to tokyo guidelines . the type of cholecystitis, stay and postoperative complications, the type of intervention, the conversion rate, the need for reoperation and re-admissions in patients older than years were analyzed and compared with those of patients operated on for cholecystitis younger than years. outcomes: a total of patients were registered, older than years ( %) and younger ( %). in cases, cholecystitis were complicated ( . %), cases older than years ( . %) and in cases younger than years ( . %). the approach was laparoscopic in % of the cases older than years, with a conversion rate of . %, not finding statistically significant differences with younger than years ( % laparoscopies with . % of conversions). % of patients older than years had some type of postoperative complication, not finding statistically significant differences in patient younger than years ( %); being the most frequent complication the intrabdomintal abscess ( . % of patients [ years, and . % of those \ years = '' span = '' [ being not statistically significant with % ci. any patient older than years required re-entry after discharge, compared to patients younger than years who were re-entered, not being statistically significant; and any patient older than years required reintervention, while it was necessary to reoperate patients younger than years ( %), being not statistically significant. mortality was very low, finding case in older than years ( . %) and case in younger ( . %), not obtaining statistically significant differences. the postoperative stay in patients younger than years of age has a median of days and in older than years a median of days, not finding statistically significant differences with % ci conclusions: laparoscopic cholecystectomy is safe and effective in the treatment of elderly patients with (ac), there being no differences with younger patients. introduction: significant bile leak is an uncommon but serious complication of laparoscopic cholecystectomy. our study aims to evaluate the efficacy of relaparoscopy in treating symptomatic bile leak and biloma formation. material and methods: patients presenting with postoperative bile leak after different operations on extrahepatic biliary tree from january to december were reviewed retrospectively (in total, , laparoscopic surgical interventions were performed for the period under study). the sites of bile leaks were the cystic duct stump in thirty seven patients, the bile ducts of luschka in fifty two, liver beds in cases after hepatectomy, in had small injury of cbd, and seven patients with tubular stenosis of the common bile duct. results: three main approaches of mini-invasive treatment of bile leakage was used: ( ) percutaneous puncture with or without drain under ct-scan or ultrasound guidance in patients; ( ) endoscopic management in patients (in patients ( . %) were managed with ercp alone and fifteen ( . %) were treated with a percutaneous intervention followed by ercp. endobiliary stent placement was performed after es in patients and without es in twenty seven patients ( ) relaparoscopy has been performed in patients, in cases of biliary peritonitis. conclusions: relaparoscopy was the ultimate method of treating postoperative complications of laparoscopic surgery in . % of patients. in general, this method, as well as laparoscopic intervention, is highly effective in the diagnosis and correction of postoperative complications, with minimal surgical trauma for the patient, with great therapeutic effect and subsequent rapid social rehabilitation of patients. introduction: laparoscopic operations have already become routine, even for pancreatoduodenectomy for periampular cancer. for unresectable cases, endoscopic bibliary stenting or hepaticojejunostomy are usually used. these methods are quite expensive and may be accompanied by complications. materials and methods: laparoscopic cholecystogastroanastomosis was performed in patients with unresectable periampullary cancer. there were females and men and average age was , . the indications for surgery in all patients was unresectable periampullary cancer and biliary hypertension with preserved patency of the cystic duct. the level of bilirubinemia ranged from to lmol/l (the average level was , lmol/l). we used -port technique. optical trocar was placed in the right iliac region, one mm above the navel and one mm in the right hypochondrium after punction gallblaber and aspiration of bile, we cut the apex of the gallbladder and gastric antrum up to . cm and performed cholecystogastroanastomosis with barbed-suture v-loc. results: we had not conversion to open surgery. the average operation time was min. postoperative stay was average days and on median follow-up of month. post-operatively, there were no major morbidity and nil mortality. we had cases of leakage of bile through drainage for up to - days, which spontaneously stopped. all patients showed a decrease in the level of bilirubinemia. patients were later radical operated (pancreatoduodenectomy), while they did not have such phenomena as cholangitis, pancreatitis, inflammation of the hepatoduodenal ligament elements, which we often observe after endoscopic biliary stenting. conclusions: laparoscopic cholecystogastroanastomosis is safe, effective and feasible for patients with periampular cancer and obstructive jaundice. aims: surgeons with the expertise and resources to perform laparoscopic common bile duct exploration often prefer the 'one stage approach' over endoscopic retrograde cholangio-pancreatography (ercp) for the management of common bile duct (cbd) stones. this case series aims to evaluate the effectiveness of lcbde in a single benign upper gastrointestinal (gi) unit. methods: all patients with suspected and confirmed pre-operatively cbd stones who underwent a lcbde between january and october were included. lcbde was performed on the basis of pre-operative suspicion of cbd stone confirmed by intra-operative imaging. results: patients with confirmed choledocolithiasis had lcbde during this time period. the indications for lcbde were deranged liver function tests, dilated cbd or confirmed stones on preoperative imaging. median age was (range - ), % of whom were female. % of patients had confirmed cbd stones pre-op. % of cases were performed as emergencies and conversion rate to open was . %. choledocotomy was performed in % of cases. in % of these t-tube was left in situ. transcystic approach was used in the remaining %. despite positive intraoperative imaging no stones were found on cbd exploration in cases ( %). in patients stones were unable to be cleared with lcbde. the overall morbidity was %. % of patients had gallstone related complications. overall mortality was % (due to bile leak). / patients required re-intervention with re-look laparoscopy (n = ) or ercp (n = ). patients re-presented within months with cbd stones. overall median length of stay was days. conclusions: our case series demonstrates that lcbde is an effective and safe treatment for choledocolithiasis in both the elective and emergency settings. complication rates are comparable with therapeutic ercp ( % specific complications) followed by laparascopic cholecystectomy ( % day morbidity). the variability in anatomic location of subvesical bile ducts puts them in danger during hepato-biliary operations. its prevalence varies between % and %. the origin and drainage of these ducts were limited mainly to the right lobe of the liver, but great variation could be seen. some authors think of them as small bile ducts that drain directly into the body of the gallbladder; others consider them to be networks of miniscule bile ducts between the liver capsule and the gallbladder. recent studies suggest that clinically relevant bile leaks complicate approximately . - . % of cholecystectomies. injury to a subvesical duct is one of the most common causes of cholecystectomy associated bile leak and occurs as often as major bile duct injuries and leaks from the cystic duct stump. indeed, recent studies suggest that about % of clinically relevant bile leaks are caused by inadvertent injury to a subvesical bile duct. there are four types of subvesical bile ducts, including ( ) superficial variations of segmental and sectorial bile ducts, ( ) superficial or intercommunicating accessory bile ducts, ( ) hepaticocholecystic ducts, and ( ) aberrant bile ducts.we present a case of year old patient who developed a coleperitoneum after a routine daycase colecystectomy due to the inadvertent injury of a hepatocholecystic duct. a superior comprehension of ductal anatomy is essential in preventing and managing operative injury to the subvesical ducts, although some times is unavoidable. nowadays, the diagnosis of liver cancer is primarily radiological, as recommended by the principal international societies. in doubtful cases or due to the clinician needs, diagnostic evaluations can eventually be completed with a liver biopsy. the goal is to perform the examination, or the examinations, that guarantee the most elevated sensibility and specificity levels being as little invasive as possible. nevertheless, even using the best radiological tools, the diagnosis is not certain, due both to device limitations and radiology experience. recently, various diagnostic algorithms have been proposed, relating with contrast enhancement characteristics, different radiological techniques, blood examinations and cross evaluations from different radiologists. one of the most recent algorithm purposed is liver imaging reporting and data system (li-rads), that evaluates ct and mri imaging to classify hepatic lesions in different diagnostic categories, in order to perform a better and more precise diagnosis of hcc or other liver benign or malignant lesion. through a retrospective study, we evaluated and compared preoperative imaging and post-operative histological reports. results reveal that li-rads routine use increases hcc diagnosis up to %. background: we previously developed a modified difficulty scoring system (dss-ihd) of laparoscopic liver resection (llr) for patients with intrahepatic duct (ihd) stone. we validated dss-ihd in patients who underwent llr for hepatolithiasis. methods: dss-ihd was based on the extent of liver resection ( to ), stone location ( to ),atrophy of liver parenchyma ( to ), ductal stricture \ cm from the bifurcation ( to ), and combined choledochoscopic examination for remnant ihd ( to ). results: the dss-ihd ranged from to and divided to -level groups of low group (score * ; n = ), intermediate group ( objective: improving the surgical treatment of patients with cholangiogenic abscesses of the liver through the application of minimally invasive technologies. material and method: in the presented study presented results of treatment of patients with biliary liver abscesses. surgical interventions for hepatic abscesses were performed simultaneously with the elimination of the primary pathological process of the biliary system, which caused the occurrence of cholangitis, or in the near future (up to days) after biliary drainage drainage. among patients with biliary liver abscesses, treated with minimally invasive methods, revealed abscesses of the right hepatic lobe, -abscesses of the left hepatic lobe, -abscesses and right and left hepatic lobes. single abscesses were detected in patients, and in -two or more abscesses. in terms of liver abscesses, more than cm were detected in patients, more than cm in patients. drainage of the biliary tract was carried out endoscopically transpapillary and (if the endoscopic approach was unsuccessful) with transcutaneous transhepatic approach. results: drainage under ultrasound guidance was performed on patients with solitary and patients with two or more cholangiogenic abscesses of the liver. laparoscopic interventions were performed on patients. among the patients operated on using minimally invasive technologies, occurred complications ( . %). patient died due to the development of biliary sepsis ( . %). conclusion: percutaneous drainage of liver abscesses under ultrasound control is appropriate not only for single abscesses, but also for their larger number, which has many advantages over other interventions. it was proved possibility of simultaneous drainage of liver abscess and bile duct. percutaneous drainage of the liver abscess, drainage of the biliary tract and laparoscopic surgical intervention are complementary aspects in the treatment of liver abscesses of biliary origin. after laparoscopy residual calculus can be removed endoscopically in more favorable conditions after stabilization of the patient's condition is achieved and the infection-associated disorders are eliminated. in case of localization of abscesses in the marginal segments of the liver, laparoscopic atypical resection of the liver with an abscess is most desirable. general surgery, rambam medical center, haifa, israel background: recently robotic surgery has emerged as one of the most promising surgical advances. despite its worldwide acceptance in many different surgical specialties, the use of robotic assistance in the field of hepatobiliary (hbp) surgery remains relatively unexplored. our study presents single institution's initial experience of robotic assisted surgery for treatment of benign hepatobiliary pathologies. methods: a retrospective analysis of a prospectively maintained database on clinical outcomes was performed for consecutive patients that underwent robotic assisted surgery for benign hbp disease at rambam medical center during - . results: there were robotic assisted surgical procedures performed for benign hbp pathologies during the study period. there were anatomical robotic liver resections for symptomatic hemangiomas, cases of giant liver cyst, robotic assisted surgery for type i choledochal cyst, case of benign (iatrogenic) common bile duct (cbd) stricture, cases of robotic (cbd) exploration due to large intra choledochal stones and cases of cholecystectomy for cholelithiasis. the median postoperative hospital stays for all procedures were . days (range - days). general morbidity (minor) was %. there was no mortality in our series. conclusion: robotic surgery is feasible and can be safely performed in patients with different benign hbp pathologies. further evaluation with clinical trials is required to validate it's real benefits. most liver cysts are asymptomatic and tend to have a benign clinical course. however, symptomatic or complicated liver cysts sometimes require surgical intervention. needle aspiration is safe and can be the lease invasive procedure, this procedure is however associated with a high failure rate and rapid recurrence. surgical approach is the crucial and provides definitive treatment for such cysts. thirteen cases were nominated from shonan kamakura general hospital between january and december . mean age and body mass index (bmi) were . and . , respectively. all patients have had any complaint such as upper abdominal pain, dyspnea, and fever. two cases were clinically diagnosed as the infectious cyst and serum crp was elevated before surgery. additional cholecystectomy was planned for one case of chronic cholecystitis with gallbladder stones. all cases were prompted the reduced port surgery (rps) and cases were performed rps with trans-vaginal approach (hybrid notes) and case was chosen in single port surgery. cyst unroofing was performed for all cases. mean operation time and blood loss of all cases were . min. and . ml, respectively. no surgical complication has been occurred in all cases, an infectious cyst case was however required additional drainage for infectious control after surgery. although statistic difference was not shown, fewer blood loss and shorter hospital stay was seen in non-infectious cases, compared to laparotomy cases. mean hospital stay after surgery of whole cases, non-infectious cases, infectious cases was . , . , . days, respectively . no recurrence of any symptom was shown in any cases in observation period ( - days) . laparoscopic unroofing is the definitive treatment for the complicated or symptomatic liver cyst. however, for the infectious cyst, infection control such as intensive drainage and/or administration of antibiotic before surgery may be needed to avoid additional treatment, leading to longer hospital stay. laparoscopic unroofing of liver cyst can be the first choice for symptomatic or complicated liver cyst. also, reduced port surgery can be nominated to achieve less invasiveness. introducction: laparoscopic liver resection (llr) has been increasing since it was first reported in . three international expert consensus conferences on llr surgery were held in louisville, ky, usa, in , morioka, japan in and southampton, uk, in . while most initial minimally invasive liver resections were typically done for benign lesions in anterior o left segments, llr is currently being applied for major anatomic resections, malignancy, cirrhosis and liver donor hepatectomy. clinical case report: this is a -year-old male patient with a history of hta and liver cirrhosis due to hepatitis b virus. hepatocarcinoma is diagnosed in liver segment vi with a size of cm . in the digestive study the patient presents a child a stage, meld \ , without signs of portal hypertension. complete analytical with normal afp and cea . markers. after presentation of the patient in a multidisciplinary committee and being a stadium according to the early bclc classification, laparoscopic surgery with segment vi resection was decided. discussion: laparoscopic liver resection is becoming widely accepted for the treatment of hepatocellular carcinoma. liver resection is a first-line option in very early and early-stage disease. many meta-analysis have shown that llr is better than open liver resection in terms of short-term outcomes for patients with child-pugh a cirrhosis, solitary tumors, and minor resections. in the long-term setting, the results demonstrate that a minimally invasive approach is comparable to an open approach in terms of overall. in conclusion, the current evidence conclude than llrs for hcc are safe and may be considered a standard practice in specific settings. results: there were women ( %) and men ( %). the age of patients ranged from to years. the patients underwent complex examination including abdominal ultrasound, esophagogastroduodenoscopy, and some of them underwent ct (computed tomography). all patients in the first stage were performed antegrade external drainage of biliary tracts with x-rays of the biliary tracts, and specifying the level and extent of the block.total miniinvasive interventions were hold. two patients in connection with the uncoupling of equity ducts were performed antegrade bilobar stenting with preliminary split external bile release.there were complications after carried out interventions in cases, which were associated with dislocation of holangiostomic drainage in patients ( . %); with acute cholecystitis in patient ( . %); with hydrothorax in patients ( . %); perihepatic biloma in case ( . %). patient ( . %) had a recurrence of obstructive jaundice due to germination of endobiliary stent in the late period after stenting. lethal outcome appeared in patient. conclusions: ultrasound examination allows us to determine the level of obstruction of the biliary tract, to substantiate the tactical position in the application of mini-invasive technologies. antegrade miniinvasive technologies in the treatment of tumor lesions of the proximal bile ducts allow timely and effectively stop biliary hypertension and to determine further treatment strategy. acknowledgements this study was supported by the russian science foundation under project ? - - . background: repeat hepatectomy is an effective treatment, with long-term surgical outcomes for recurrent hcc and colorectal liver metastasis(crlm). however, the efficacy of a minimally invasive surgical approach for recurrent liver tumor is not yet confirmed. the purpose of this study is to examine the efficacy of laparoscopic repeat hepatectomy(lrh) compared with open repeat hepatectomy(orh) for recurrent liver tumor. we retrospectively analyzed the clinicopathological features and short-term surgical outcomes between lrh and orh. methods: from to , patients with liver cancer underwent repeat hepatectomy. of those patients, patients underwent partial hepatectomy, patients were undergone laparoscopically, and patients underwent open hepatectomy. we compared the clinicopathological and surgical parameters in the lrh group with those in the orh group. results: there were no significant differences in patients' gender, age, viral infection status, child-pugh classification, tumor size, tumor number, and tumor location in the two groups. the operative times were similar, but blood loss was significantly lower in lrh group ( vs. ml, p \ . ). the postoperative hospital stay was significantly shorter in the lrh group ( . vs. . days, p = . ). postoperative complications(cd = a) were observed only in the orh group, with a complication rate of . %. conclusions: we demonstrate that lrh reduces blood loss and postoperative complications compared with orh. lrh might be a feasible and effective procedure for the selected patients. background: the liver is the most common site of metastatic disease with up - % of all cancers having the potentiality for sending liver metastasis during the disease. consequently, increasing value for surgical resection of hepatic deposits of different types of cancers, the need for accurate evaluation of the extent of hepatic metastasis was established for choosing the most suitable patients for surgery and in planning the extent of hepatic resection. the aim of this work is to evaluate the role of intra-operative ultrasound in the detection of hepatic deposits in intra-abdominal malignancies with special emphasis on its accuracy, sensitivity, specificity. patients and method: this study was carried out on thirty patients who were admitted to the gastrointestinal surgery unit, main alexandria university hospital with intra-abdominal malignancies for whom elective open surgical intervention was recommended in the period from st of september till the th of march . results: in the present study consisted of males ( . %) and females ( . %). their mean age at admission was . ± . years. six of the included patients ( %) were found to have hepatic lesions by using ious including the four cases ( . %) already detected by preoperative imaging. two cases ( . %) were newly discovered in the operative room by using ious. conclusion: the current study has proved that ious demonstrates superior lesion detection over the various non-invasive preoperative imaging modalities causing significant impact on change of the planned surgical strategy laparoscopic approach to the liver has become an integral part of surgery. two consecutive international consensus meeting recommends major hepatectomy has been on the expert hands. tumors located in the right posterior section are considered to be difficult for laparoscopic resection. patients and methods: since , until , cnuhh has been performing laparoscopic hepatectomies including major hepatectomies. among major ones, there are rh, lh, rps, ch, and as. we analyze data on patient demographics, tumor characteristics, operative date, and posterior outcome retrospectively. results: during - , laparoscopic rps were performed. the diagnosis were hcc in and crlm in patients. median operative time was min, and median blood loss was ml. no blood transfusion was occurred. median tumor size was mm, and median resection margin was . mm. six of the patients ( %) were cirrhotic on pathology. there was no conversion and was no postoperative mortality. median hospital stay was . days. conclusion: laparoscopic rps is known challenging procecedure. strict preoperative planning and operative procedure is mandatory. even though it should be performed by the experienced hands both on hepatic surgery and laparoscopic skill, it can be an good option for treatment of the tumor locating over right posterior section. purpose: previously we developed a new sponge (named endoractor) as an organ retraction device in laparoscopic surgery in and have reported that it is useful in various surgical procedures including rectal surgery we confirmed that it is also useful in laparoscopic radiofrequency ablation of the liver in terms of pulling and protecting organ, so we report it materials and methods: a case is an -year-old female with liver cirrhosis. she had primary hepatocellular carcinoma in s lesion with a diameter of . cm very close to the inferior vena cava and middle hepatic vein root and in s lesion with a diameter of . cm we thought she could not put up with hepatic resection because of her poor hepatic reserve capacity. and we could not expect treatment effect by embolization therapy since contrast effect was poor. so we decided to select ablation therapy in the puncture and ablation of the s tumor, since there was concern about the thermal damage of the middle hepatic vein and the cooling effect by the inferior vena cava, we would dissect the right coronary mesentery sufficiently and pull the liver apart from the inferior vena cava and the middle hepatic vein as much as possible using our endoractor also, in the puncture and ablation of the s tumor, it was feared that the stomach would be thermally damaged, so we would place endoclactor between the liver and the stomach to protect the stomach results: when ablating the s tumor, we could pull the liver securely without slipping, so we did not cause thermal damage to the middle hepatic vein. and there was no cooling effect by the inferior vena cava, so we could obtain sufficient cautery margin. in ablation of s tumor, we were able to puncture by stabilizing the lateral segment of the liver on our endoractor, and avoid thermal damage of the stomach conclusion: it seems possible to perform safe and reliable puncture and ablation by using our endoractor as well in laparoscopic radiofrequency ablation surg endosc ( ) surgical reinterventions in patients with complicated hepatic hydatid cysts usually occur as a result of diagnostic or technical failures during the initial procedure. according to recent studies, the most common complication after liver hydatid cyst surgery is local sepsis at the residual cavity and long-term biliary leak. we report the case of a -year-old male with a history of liver hydatid disease four years before the current episode, admitted in our surgical department for intense upper right quadrant pain. abdominal ultrasonography, ct and mri scans revealed three cysts in the gastrosplenic ligament, in liver segments vii-viii, and ii-iii respectively, sized between and cm. the intraoperative aspect during laparoscopy was strongly suggestive for liver hydatid disease. laparoscopic fenestration with tunneling for the hepatic cyst in segment viii, partial cystectomy in the left liver lobe and ideal cystectomy in the gastrosplenic ligament were performed. postoperatively, the patient displayed a constant biliary drainage output of - ml from the cavity remnant in the segment viii. conservative therapy for external biliary fistula and concomitant treatment with albendazole for months were initiated. evolution was slowly favorable with decreased biliary drainage to ml two months after surgery and complete symptom resolution five months after hospital discharge. aims: this study aimed to evaluate the effectiveness of fluorescence imaging with indocyanine green (icg) during laparoscopic deroofing of hepatic cysts. methods: this was a single-center, case-control study. we included patients who underwent laparoscopic deroofing between november and october . imaging with and without icg fluorescence was performed in (icg group) and (non-icg group) patients, respectively. icg was intravenously administered between min and . h before surgery. we performed a standard laparoscopic procedure. we detected a thin bile duct on the hepatic cyst on using intraoperative icg fluorescence imaging. we adjusted the resection line of the cyst wall and ligated the bile duct at the point at which it crossed the resection line. data on age, sex, cyst size, resected cyst size, operative time, estimated blood loss, post-operative hospital stay, complications, and recurrence were compared between the groups. results: the mean cyst size was ± . and ± . mm, the mean resected cyst size was ± . and ± . mm, and the mean operative time was . ± . and ± . min in the icg and non-icg groups, respectively. using icg fluorescence imaging, the bile duct was detected on the cyst wall in patients ( %). all surgeries were completed laparoscopically, and no post-operative complications occurred in either group. recurrence of the hepatic cyst occurred in one patient ( %) of the non-icg group. conclusions: fluorescence imaging with icg is used widely in hepatobiliary surgery for intraoperative identification of biliary and vascular anatomies. this method does not require complicated techniques or instruments. icg fluorescence imaging may facilitate the prevention of intra-or post-operative complications, such as biliary leakage, in laparoscopic surgery. in this study, icg fluorescence imaging was found to be effective in detecting the bile duct on the cyst wall intraoperatively, allowing for wider resection of the cyst and avoiding inadvertent injury. our study suggests that wider resection of the cyst wall might prevent recurrence of hepatic and that icg fluorescence imaging could ensure procedural safety. abdominal ct showed: large hepatic cyst ( x , x cm size), with no malignity signs, that occupies practically the whole right liver, causing subsegmentary atelectasis of the middle lobe, superior and inferior cava vein compression, and displacement of right kidney, pancreas and right atrial. due to breath involvement, a percutaneous drainage is performed achieving clinical improvement and reduction of the size of the injury. the patient was released but a cyst superinfection occurred; once this problem was solved, the drainage was removed. results: in light of the complication, surgical treatment was decided, which confirmed the large cyst located in right posterior hepatic segments with tight diaphragmatic adhesions. we carried out the cyst evacuation and a wide laparoscopic resection of the cyst walls, until the posterior area of the cava vein, combining supra and infrahepatic access. the patient was released on the sixth postoperative day and continues asymptomatic. conclusions: simple cysts can be approached in a no surgical way (punction-aspiration with/ without sclerosing products injections) or in a surgical way (cyst wall fenestrations, cystectomy or liver resections). a conservative treatment will obtain symptomatic relief but with a high risk of recurring. recurrence is the main drawback of unroofing. cystectomy is the better option but may be too complicated depending on the cyst's location. to our patient, we carried out a wide laparoscopic unroofing (even though its posterior localization) to minimize recurrence possibilities. in conclusion, laparoscopic resection of the cyst wall is a simple and effective approach in symptomatic or complicated cases. background: single-incision laparoscopic surgery or laparoendoscopic single-site surgery is emerging as an alternative to conventional multiple-incision laparoscopic surgery. it has a potential benefit of less postoperative pain and faster recovery compared with conventional multiple-incision laparoscopic surgery. single-incision laparoscopic hepatectomy (silh) has been reported in only a few small series and the majority were minor resections. case report: a y/o male patient is a case of chronic viral hepatitis b and early cirrhosis of liver. two atypical hepatocellular carcinomas (up to . cm in diameter) located at the junctions of segments & and segments & were impressed by liver magnetic resonance imaging (mri). we performed single-incision laparoscopic anatomical hepatic resection of the right posterior section via a -cm transverse incision on the right middle abdominal wall. inflow control was carried out with an extra-glissonian approach before parenchymal transection. the glissonean pedicles of segments and were divided by linear staplers respectively as well as a major branch of the right hepatic vein in segment . the operative time was min and the estimated blood loss was ml. the pathologic examination revealed two foci of hepatocyte dysplasia with a safe margin of cm. the patient was discharged eight days after the surgery uneventfully. conclusion: single-incision laparoscopic anatomical right posterior sectionectomy is feasible and safe by experienced laparoscopic surgeons. it provides a fast recovery but needs a long operative time. the mortality in the patient with liver cirrhosis is very high. the aim of this work was to decrease mortality and morbidity by using endoscopic local heamostasis and laparoscopic operations, in the patients with bleeding from cirrhosis by variceal bleeding. methods and material: we observed patients with cirrhosis complicated by variceal bleeding during years. there were patients with child phue a, ones with child phue b, ones with child phue c. all the patients were performed prolonged endoscopic heamostasis with conservative therapy. the main methods that we used were the ligation in cases, sealing in cases, sclerotherapy in cases. in cases we couldn't stop the bleeding with band ligation method and introduce the danis stents into esophagus and stopped the bleeding successfully. to prevent the re-bleeding we performed the laparoscopic dissection the abdominal part of esophagus with suturing the venous vessels, coagulations and dissection of short gastric vessels between stomach and spleen, clipping the left gastric artery and vein in the patients. in patients we performed laparoscopical suturing the variceal veins by introducing the laparoscopic trocars into the stomach. in cases with varices vien of stomach, with non-effective local endoscopic heamostasis we performed laparoscopic resection the fundal part of stomach. results: endoscopic local heamostasis were successful (in %) in cases. the relapse of bleeding were in patients. patients died. there was no mortality after laparoscopic operations. there were cases for trocar wounds infection, cases of subphrenic abscess. goals: the advance of laparoscopic surgery also includes the more complex procedures of abdominal surgery such as those affecting the liver and pancreas. there are multiple indications that laparoscopy has in hepatobiliopancreatic surgery, both in benign and malignant pathologies. material and methods: we present the video of a -year-old male patient with a history of right hemicolectomy due to disease-free intestinal lymphoma who, in the control analysis by his attending physician, detects the elevation of tumor markers. an extension study was started showing a hepatic lesion in the caudate lobe with a pathological anatomy suggestive of hepatocarcinoma and an adenopathy suspicious for malignancy adjacent to the right renal vein. the clinical case is presented in a multidisciplinary tumor committee and it is decided to perform surgery. a laparoscopic caudate lobe resection was performed, previously performing intraoperative ultrasound and a lymphadenectomy of the portal territory, vena cava and exeresis of adenopathy of the right renal vein. introduction: major vascular complications during laparoscopic surgery occur approximately in one in cases, but mortality rate can reach - %. most major vascular injuries lead to conversion to laparotomy but successful laparoscopic repair is also possible. simulation training improves laparoscopic performance and possibly reduces surgeons mental strain. materials & methods: during two editions of advanced laparoscopic training course participants had a task to control a major vessel damage (damage). before the task an educational video explaining the methods of obtaining haemostasis was shown. the algorithm of the 'damage' task was as follows: without previous preparation a cm injury of a major vessel was done with l-hook electrocautery. after the injury participants were free to control the damage the way they wanted. heart rate of the participants was measured with an ear electrode. measurements were carried out times-before the injury, immediately after, and afterwards obtaining vessel control. after participants were interviewed for their feelings after the 'damage' task. results: there were vessel injuries in animals. one animal died during the 'damage' task min after desuflation due to relapse of bleeding. there was no conversion to open procedure. temporary vessel control was obtained with different methods. all participants used vicryl . or pds ii . suture for final hemostatic purposes. heart rate of the participants before injury were - ± . bpm, immediately after the injury it rose to - ± . bpm, and after obtaining vessel control were in the range - ± . bpm. a statistically significant difference was found between the ratio of the first and second hr measurement (p = . , t = - . ), and second compared to the third (p = . , t = . ) measurement. participants judged their experience on a -point scale ( - was not helpful at all; -was extremely educative). the educational value of the task received points in cases and points in one case. conclusion: participants feel stress during major vessel bleeding even in animal model, and this stress can result in a serious intraoperative mental strain and significantly increase heart rate. participants found the 'damage' task very useful for their daily practice. the aim of study was to improve the results of treatment of patients with hepatic echinococcal cysts by using of argon plasma coagulation. methods: the analysis of treatment results of patients was put into the basis of this study. it was ( . %) men and ( . %) women in total. an average age of them was . ± . years. the main difference between groups was a way of liver parenchyma coagulation in order to make reliable hemostasis. in main group the final stage of surgical intervention on liver was argon plasma coagulation. it was performed to ( . %) patients. alternatively, monopolar coagulation was performed to ( . %) patients (comparison group). results: in main group in the . % cases pericystectomy was conducted. the resecting surgeries was performed to . % cases. in comparison group was conducted in . % cases. in early postoperative period in main group the complications were observed in . % of cases. the same parameter was . % in comparison group. it led to relaparomies. the forming of external biliary fistulas was observed in ( . %) patients in main group and in ( . %) patients in comparison group. however, all the fistulas have closed spontaneously on th- th day in both groups. hernias of abdominal wall and peritoneal adhesions that manifested by intestinal obstruction of different degree were considered as complications of late postoperative period. these values were % and . % in main group versus % and . % in comparison group, respectively. the resection of hepatic echinococcal cysts with further application of argon plasma coagulation on the cyst bed was accompanied by complications quantity decrease in patients that underwent surgery in early as well as in late postoperative period. in this case more positive dynamics of functional liver values improvements was observed. aims: indocyanine green (icg) fluorescence imaging has been reported as a reliable and safe navigation tool in laparoscopic hepatectomy. however, the factors affecting the sensitivity of tumor detection with icg fluorescence imaging is relatively unclear. the aim of the present study is to analyze the factors of successful icg fluorescence in laparoscopic hepatectomy. methods: this is a retrospective single-center study. this study population consisted of laparoscopic hepatectomies from january to november undertaken at kurashiki central hospital. we excluded patients whose tumors were located more than mm from the liver surface, those who did not receive icg fluorescence imaging, and those who were not injected with icg dye ( . mg/kg) intravenously within days of surgery. the pinpoint endoscopic fluorescence imaging system was used to detect the tumor location. we evaluated the relationship between successful fluorescence and the timing of injecting icg before operation, tumor size, icg r , liver damage and bmi. results: following exclusion, patients were eligible for analysis. among the tumors resected, icg fluorescence imaging detected tumors ( . %), including hepatocellular carcinomas and liver metastases. icg fluorescence imaging detected all tumors in the patients injected with icg to days before hepatectomies . icg fluorescence imaging detected all tumors which were more than mm in diameter. there was no relationship between indocyanine green fluorescence with icg r , liver damage and bmi. conclusions: the injection of icg to days before operation and a tumor size of more than mm can be factors in successful fluorescence in laparoscopic hepatectomy. introduction: cysts in the liver have a wide variety of aetiologies. it is important to characterize the cystic lesion before treating it. the simple cyst has a low prevalence and is more frequent in women. fenestration is a useful option for the treatment of simple cysts in selected patients. case presentation: a -year-old woman was referred to our hospital with a one-year history of intermittent, right upper quadrant pain, with no other associated symptoms. computed tomography and magnetic resonance imaging showed a large cyst ( , x , cm) in the right of the liver. the cyst presented lobulated morphology, smooth edges and well delimited. there were other smaller cysts in the left lobe. hepatic function in blood analysis was normal. biomarkers, tumor markers and hepatitis virus markers were negative. outpatient follow-up and symptomatic treatment of pain was decided. after six months of follow-up, the pain persisted, so surgical treatment was proposed. a laparoscopic fenestration was performed, widely resecting the free wall of the cyst. there was no evidence of a connection to the bile duct. there were no complications. on days she was discharged. discussion: some giant hepatics cysts become symptomatic due to mass effect. persistence of pain is an indication of surgical treatment. laparoscopic fenestration is an alternative for the management of simple hepatic cysts. aim: laparoscopic liver resection for malignant pathology such as colorectal cancer metastases has been a matter of discussion for several groups in the last years. it has been proposed as a safe and feasible treatment but subjects like short and long term outcomes and oncologic results have not been adequately assessed. methods: we performed an observacional retrospective study of patients undergoing laparoscopic liver resection for colorectal metastases in our center. from november to november a total of patients underwent laparoscopic liver resection. data for resection margin, hepatic and extrahepatic recurrence and both disease free survival and overal survival were collected. patients were discussed in a multidisciplinary group with oncologist, radioterapic oncologist and surgeons. the surgical procedures were perfomed by the same team in all the cases to minimize bias. results: a total of patients ( . %) were non resectable at the time of surgery.the mean overall survival was months with a maximum of months. we got a mean of disease free survival in our patients of . months. the hepatic recurrence was %, most of them in high risk patients, and from this group . % underwent a new liver resection. major complications took place in patients ( . %) two biliar leaks, one bowel perforation, two hepatic failure, one evisceration and three respiratory insufficiency needing urgent surgery in three of the cases. mean hospital stay was . days. a mean of days of this stay were in an intensive care unit. conclusions: laparoscopic liver resection for colorectal liver metastases could be a feasible technique when perfomed by trained surgeons. it improves the postoperatory recovery with a reduction of hospital stay and less postoperatory pain without increasing the development of major complications or mortality in the first days after surgery. we got good oncological results that have been improving with the experience acquisition of the surgical team. aged to underwent surgery for cirrhosis with massive refractory ascites child c ( - ), without obvious signs of hepatic encephalopathy. major etiological factors were: viral hepatitis c ( patients ( . %)), b ( patients ( . %)), b ? d ( patients ( . %)), toxicity ( patients ( , %) ). to prevent possible bleeding at the first stage, endoscopic filling of esophageal varices with fibrin glue was performed in patients ( . %). after testing the effectiveness of varices filling, in the following - days decompression surgery of thoracic lymphatic duct was performed under local anesthesia to improve lymphatic drainage from liver and abdominal organs. simultaneously, laparoscopic sanitation of abdominal cavity was performed, with complete evacuation of ascites fluid, rinsing and drainage. fractional post-surgery rinsing was repeated daily for - days towards removing peritoneum edema and improving its absorptive properties. results evaluation was performed , and months after surgery, based on criteria of liver reserves and ascites volume. results: post-surgery mortality from liver failure was . % ( patients) . other patients died of the same cause the following - months. annual survival rate was . %. complete ascites regression over - months after surgery was noted in patients ( . %), significant regression and stabilization in ( . %), moderate regression with need for periodic decompressive laparocentesis in cases. in all patients, functional liver reserves and life quality significantly improved. conclusions: the use of the given technique of refractory ascites correction, in patients with depleted liver cirrhosis, by laparoscopic sanitation with post-surgery fractional rinsing of abdominal cavity, with simultaneous decompression of thoracic lymphatic duct showed very high efficiency and deserves establishment as a clinical practice. t. urade, hepato-biliary-pancreatic surgery, kobe university, kobe, japan aim: anatomical liver resections guided by a demarcation line after portal staining or inflow clamping of the target territory were established as essential methods for the curative treatment of hepatocellular carcinoma (hcc) and then subsequently applied to other malignancies. however, laparoscopic anatomical liver resection (lalr) is much more difficult to reproduce these procedures and to confirm demarcation of the hepatic segment visually on the monitor. recently, laparoscopic fluorescence imaging system has been used as a tool for real-time intraoperative navigation in llr. the aim of this study is to demonstrate how to perform lalr using indocyanine green (icg) fluorescence imaging. methods: three patients underwent pure lalr using icg fluorescence imaging. the following operative procedures were performed: partial liver resection for hcc, segmentectomy for liver metastasis and right anterior sectionectomy for hcc. in all patients, preoperative d simulation images from dynamic ct were reconstructed using a d workstation to decide on cutting points of the glissonean branches. after mobilization of the liver, intraoperative ultrasonography was performed to identify the location of the tumor and glissonean pedicles corresponding to the tumor-bearing hepatic region. we dissected or transected the hepatic parenchyma to encircle the glissonean pedicles. after clamping or closure of them, . mg of icg was injected intravenously to identify the boundaries of the hepatic segments under near-infrared light. parenchymal transection was started according to the demarcation on the liver surface. the lateral aspect of the parenchymal transection was carried out based on the demarcation between non-fluorescing and fluorescing liver parenchyma as far as possible. results: in all the cases, demarcation lines on the liver surface could be visualized clearly after injection of icg. in addition, boundaries of cone units, segments and sections could be recognized to some extent because the tumor-bearing hepatic region became non-fluorescing parenchyma during parenchymal transection. these procedures were completed successfully, and the postoperative courses were almost uneventful. aim: sintrahepatic cholangiocarcinoma is the second most common primary liver cancer after hepatocellular carcinoma (hcc). although the laparoscopic approach of these tumours is not frequent due to its complexity, it is performed increasingly by hepatic surgeons.traditionally, the abdominal surgery in cirrhotic patients has been reserved to selected cases secondary to the high rate of complications. the advance on the treatment of the hcc on liver cirrhosis and the higher safety when performed by laparoscopic approach has encourage some surgeons to extend surgery to child b-c or portal hypertension patients. methods: we present a male of years old, diagnosed in of liver cirrhosis accompanied with portal hypertension. on mri in was found a solid lesion of mm located on segment ii hepatic. biopsy confirmed the diagnostic of intrahepatic cholangiocarcinoma. after a liver function evaluation (child c, meld ), an hepatic chemoembolization was performed. sequentially ct scans indicated a complete radiologic response. after years of follow up, mri showed a recurrence of mm between segment ii and iii of the liver.on multidisciplinary committee liver resection was decided due to suitable liver function and low aggressiveness of the tumour. a laparoscopic left lobe liver resection was performed. sonastarÒ and ligasure tm were used to perform the liver transection and endo gia tm for portal and hepatic veins sections. the surgery develop was complicated due to trend to bleeding that finally was achieve through cauterization. results: early after the surgery, the patient presented a haematic debt through the drain of cc accompanied of hypotension, therefore an emergent surgery was indicated. an exploratory laparoscopy was performed finding hemoperitoneum and diffuse bleeding of the liver surface that was controlled. the patient had a proper recovery and was discharged on the th day post-surgery. the analysis of the specimen showed a . cm cholangiocarcinoma with a . cm margin of resection. conclusion: there is an augmented risk of complications on liver resection of cirrhotic patients with portal hypertension. the laparoscopic approach allows to reduce potential complications, despite bleeding continuous to jeopardize this surgery, this option could be proposed on selected patients. introduction: accessory spleen itself is found in approximately % to % of the population. most ( %) are located near the splenic hilum but intrapancreatic accessory spleens (ipas) are the second most frequent location ( . %) of accessory spleens. in adults, ipas are clinically silent. they may become clinically important because of their radiographic similar appearence of cancer. intrapancreatic accessory spleen is a rare cause of pancreatic pseudotumors and is located in the pancreatic tail in approximately % to %. ipas can be difficult to differentiate radiologically from hypervascular pancreatic tumors such as pancreatic endocrine neoplasms because theycan share a similar enhancement pattern. as a result, most of the reported cases of ipas have been diagnosedonly after distal pancreatectomy was completed. material and methods: we present the case of a -year-old male patient with a history of large vessel vasculitis followed-up for rheumatology, which showed a pancreatic nodule in a control ct so he was referred to digestive for study. an echoendoscopy was performed. it showed, at the level of the tail, in the third distal, a lesion of x mm, hypoechoic, with rounded morphology and well-defined edges that can not be biopsied given the absence of adequate window for the realization of fine needle aspiration biopsy (fnab). based on these radiographic findings, the differential diagnosis included a pancreatic endocrine tumor. due to the high suspicion of malignancy and the absence of biopsy, he was referred to general surgery for scheduled surgery. a laparoscopic corporocaudal pancreatectomy was performed without incidents and the definitive histology showed an intrapancreatic accessory spleen in the pancreatic tail that excluded the presence of cancer. conclusion: intrapanceratic accesory spleen is a challenging diagnosis to make and it should be included in the differential diagnosis of pancreatic neoplasm. its early identification precludes surgical resection. however, the preoperative diagnosis of ipasmay be difficult, and distal pancreatectomy is a safe and relatively simple operation, most of the reported cases of ipas being diagnosed correctly only after surgery there are various options for treating pps. this paper describes our tailored and methodological approach to laparoscopic drainage of pancreatic pseudocysts based on an anatomical classification. methods: we adopted the laparoscopic approach in patients who had pps requiring surgical drainage. the laparoscopic method had been decided according to preoperative computed tomography (ct) and intraoperative findings. the results shown represent median (range). the aim of this work was to decrease mortality and morbidity in patients with combined trauma. methods and material: for years patients were brought to our clinic with combined trauma. everybody was performed ct and ultrasound examination. patients were performed open laparatomic operation due to massive liver rupture, spleen rupture and massive trauma of bowels, pancreas and kidney with massive bleeding. in circumstances we didn't found the trauma of the abdominal organs and the massive abdominal bleeding after ct observation. those patients were cured conservatively. in circumstances with combined trauma after ct examination we performed laparoscopic operation. in circumstances from the patients, who we started laparoscopic operation in, we conversed to laparotomy, due to massive liver rupture, and trauma spleen and hollow organs. in those circumstances we performed urgent laparotomies with suture ligation of bleeding points, suturing of liver and hollow organs and drainage of abdomen cavity. results: we performed laparoscopic operation in patients. in circumstances with trauma of liver we performed laparoscopic electro coagulation and argon-plasma coagulation. in circumstances with trauma of liver we performed electro coagulation with packing the omenture to its surface. in circumstances with trauma of spleen we performed argon plasma coagulation and used fibrin glue. after laparotomic operations mortality were in circumstances, morbidity were in patients. after laparoscopic operation mortality were in circumstances of severe combined trauma with multiple abdominal trauma and morbidity in patients. conclusion: laparoscopic operations in patients with combined trauma decrease mortality and morbility. aims: in laparoscopic distal pancreatectomy, getting away liver and stomach from the surface of the pancreas is sometimes difficult. when we separate the pancreatic body from the retroperitoneum, we must not injure the pancreas to prevent breaking a tumor. when we cut the dorsal side of the spleen from the retroperitoneum, we rarely cut into the spleen accidentally. based on our experiences, we gradually explored a set of procedural operation steps to resolve these problems. our three-step maneuver simplifies the procedure and improves the efficiency and safety of laparoscopic distal pancreatectomy. methods: as the first step, to get away the liver we sutured the round ligament of liver and crus of the diaphragm using - pds and the both ends were tugged form the outside of the body through both side of the xiphoid process. and the stomach was hung from the outside using two nylon thread like a bridge, so we could see the surface of the pancreas body with a good view. the second step was a rolling up maneuver of the pancreas. when we separate the pancreatic body and tail from the retroperitoneum, we rolled the pancreas with gauze for use in laparoscopic surgery and lifted the gauze up in only one assistant's forceps. then we could find the correct line for dissection clearly. the last step was a hanging maneuver of the spleen. when we cut the dorsal side of the spleen from the retroperitoneum, we hanged the hilum of spleen with cotton tape. with this technique we could find easily the correct line to dissect. results: the operation time was h and min and the estimated blood loss was a little. we did not injure the tumor or spleen in this operation. the patient recovered uneventfully after short hospitalization. conclusion: our three-step maneuver can be effective to perform laparoscopic distal pancreatectomy. about - % of patients with pancreatic collections will develop walled off necrosis, with an associated - % mortality. there are multiple options for intervention and drainage, usually the outcomes after endoscopic drainage are related with the nature of the collections. aims: to evaluate and present the rol of endoscopy in pseudocyst and walled off necrosis treatment, and favorable outcomes. methods and results: we present a case of a years old male, who presented biliary pancreatitis treated with cholecystectomy and transoperative cholangiogram weeks ago. he continued with persistent abdominal pain; his ct scan showed a big walled off necrosis; he was taken to surgery for an endoscopy-assisted laparoscopic cystogastrostomy with necrosectomy, he was discharged days po. conclusions: the step-up management of walled off necrosis has proven to be a better option than conventional surgical or endoscopical techniques alone; by reducing complications and mortality vs conventional necrosectomy. the use of endoscopic treatments reduce the pro-inflamatory response. drainage of walled off necrosis can be done by a transpapilar or transmural endoscopic apporach each one with its own advantages. some authors avoid the use of endoscopy in walled off necrosis because of a higher rate of complications, re-interventions and a greater lenght hospital stay. in our experience, we have achieved excellent results with this combined technique. nearest and long- patients underwent chemotherapy after electroporation procedure. day mortality was . % (n = ) in electroporation group. it was found that erreversible electroporation improved local recurrence-free survival ( and months, respectively, p = . ) and distant recurrence free survival ( and months, respectively, p = . ) . overall survival was and months, respectively (p = . ). conclusion: irreversible electroporation of locally advanced pancreatic cancer is safe. four month chemotherapy followed by surgical procedure is associated with good local response and better overall survival compared with chemotherapy alone. these data will be validated in further multicenter study. introduction: pancreatic pseudocysts are the most frequent complication of acute or chronic pancreatitis. usually asymptomatic, they can be managed conservative or, in case of complications, by several methods, endoscopic, percutaneous or by surgery. material and method: we present the case of a years old patient known with an episode of acute pancreatitis five years ago, who was hospitalised now for an upper gastrointestinal bleeding with hematemesis. the upper endoscopy showed a subcardial bulking with an erosion of the posterior gastric wall, with signs of recent bleeding, managed by clipping. patient work-up showed a cm pancreatic pseudocyst at endoscopic ultrasound. taking into consideration the history of the patient, the size and the complication of the cyst, the patient was proposed for a drainage intervention. results: a minimally invasive approach was decided. using ultrasonography guidance, a posterior gastrotomy was performed with the cystotome, establishing the comunication with the pancreatic pseudocyst. dilatation of the path with mm cre baloon, with partial evacuation of turbid liquid. the drainage consisted in pigtail fr plastic stents. the patient was discharged the following day in a good health condition.the endoscopic ultrasound control at weeks showed complete resolution of the pancreatic cyst and was followed by stent removal. the endoscopic drainage of the pancreatic pseudocyst represents the first treatment option as an alternative to the surgical intervention, being minimally invasive, with low risk and fast recovery. clinical case report: a -year-old man was admitted to the hospital with a diagnosis of severe acute pancreatitis and multi-organ failure. during the first month patient has in uci and non invasive procedures were attempted: enteral feeding by a nasoduodenal tube was started and antibiotics were administered to control sepsis. on day , percutaneous drainage was performed for large retroperitoneal abscess. on days, endoscopic transgastric necrosectomy was performed and the left collection was resolved. due to the multi-organ failure persistence and the evidence of size increase of the right retroperitoneal collection, a vard was decided.the right collection was accessed following the previously pigtail catheter. a mm trocar was placed to create retro-pneumoperitoneum with a pressure between - mmhg. a trocar of mmhg was placed, purulent content was aspirated and a debridement was performed. irrigation and aspirate was performed with normal saline and povidone-iodine solution. drainage was used to perform washes with physiological saline and urokinase.on days, the ct confirmed collection resolution. on days he was discharged. after months, the patient is in good clinical condition. discussion: drainage of the retroperitoneal abscesses via laparotomy is highly invasive and risky. vard enables radical necrosectomy and drainage less invasively. in this patient, the complete resolution of the right collection is obtained with retroperitoneal debridement without complications. we conclude that careful retroperitoneal necrosectomy is a valid alternative for the management of right collections. aims: in this study we analyze laparoscopic approach for hepatocellular carcinoma in order to clarify iwe can take advantage in some outcomes as complications, postoperative recovery or long-term survival outcomes. methods: a retrospective case consecutive study has been taken analyzing: age, sex, body max index, comorbidity, surgical extension and tumor size. the outcomes analyzed were: operation time, intraoperative blood loss, blood transfusion, postoperative morbidity and mortality, intensive care stay, hospital stay, tumor size, r resection, conversion rate, early reintervention, disease-free survival rate, overall survival rate results: in this study patients were analyzed males and females with ages between and years (mean age ) and diverse comorbidities: arterial high pressure ( / ; %), diabetes ( / ; , %) ; dislipemy ( / ; , %) , hepatophaty measured as liver cirrhosis ( / ; , %). all of them underwent laparoscopic liver surgery, in cases non-anatomical resection was performed while in the other a segmentectomy was performed. in cases the laparoscopic was strict, in and assistance incision was needed. operative time was - min (mean: min). blood loss mean was , g/dl and only intraoperative transfusion were needed. massive blood loss was reported in case. postoperative medical complications were observed: hepatic failure and renal insufficiency and in case we observed a postoperative hemorrhage that needed an urgent reintervention. the mean of intensive care stay was day and hospital stay was . days. about oncological outcomes r resection was achieve in / ( %), r in / ( %). at years / cases were free disease, dead by progression of disease and dead by other causes. aim: the purpose of this study is to analyze our initial experience with laparoscopic duodenopancreatic resection. introduction: laparoscopic procedures have advanced to represent the new gold standard in many surgical fields. laparoscopic pancreatoduodenectomy and laparoscopic distal pancreatectomy(ldp) are advocated to improved perioperative outcomes, including decreased blood loss, shorter length of stay, reduced postoperative pain and expedited time to functional recovery. however, the indication to minimally invasive approach for pancreatic surgery is often benign or low grade malignances. material and method. the steps of ldp procedures are similar to the open procedure. we perform destructive part of procedure totally laparoscopically and we prefer to do reconstructive part of procedure using hand-assisted techniques. for the period - , we have been perform pd, ( %) we have done with laparoscopic approach. ( %) of patients were operated totally laparoscopic and ( %) of patients were operated by handassisted techniques. results: a significantly higher conversion rate was encountered when lc was done - weeks after es, as compared to week after ercp. it is estimated that pancreatitis after ercp affects roughly three to percent of patients and many endoscopists quote a post-ercp pancreatitis rate of - %. however, - % is probably a more realistic answer for the majority of ercp endoscopists. wise endoscopists inform their patients that there is a spectrum of post ercp pancreatitis severity, from mild ([ % of cases) to severe ( - % of cases). in mild forms, pancreatitis after ercp may resolve itself. conclusion: endoscopic retrograde cholangiopancreatography is a procedure used to diagnose and treat disorders involving the pancreatic and bile ducts. acute pancreatitis is the most common and feared complication of endoscopic retrograde cholangiopancreatography. the assumption is that the duration of the laparoscopic method is longer, but on the other hand the patient have better wound healing and fewer possibility of developing postoperative hernia . the postoperative period is much more simple due to the significantly shorter hospitalization and the faster recovery, and according to patients the level of pain is much smaller as well. however the oncology results are the same. introduction: spiegel hernias are a rare, representing only between . % and % of all abdominal wall hernias. due to its location, below the spiegel line, its diagnosis requires a high index of suspicion. the physical examination only detects % of the spiegel hernias and, in many occasions, imaging tests are necessary for the diagnosis. goals: our objective is to describe the case of an urgent laparoscopic repair of a case of high grade bowel obstruction secondary to a spiegel hernia. material and methods: we present the case of a -year-old male patient with no medical history that comes to the emergency department of our center due to an eight hour evolution of abdominal discomfort associated with nausea without vomiting or other symptoms. the patient was afebrile and hemodynamically stable at all time. on physical examination, the abdomen is soft and depressible, painful on the left flank where a tumor compatible with spiegel's hernia is palpable. in the blood count there is no leukocytosis nor alteration of inflammatory parameters. an abdominal computed tomography (ct) scan was requested from the emergency department which demonstrated a high-grade small bowel obstruction caused by an entrapped loop of distal jejunum conditioned by a left-sided spiegel hernia. given the situation, an informed consent was obtained, and the patient was taken to the operating room for emergency laparoscopic repair. we performed a laparoscopic hernioplasty with ventralpatch mesh between oblique major and transverse and primary closure of defect in continuous suture. after this, the evolution of the patient is favorable, with good oral tolerance and re-establishment of intestinal transit, being able to be discharged h after surgery. the spiegel hernia is a rare entity that requires a high index of suspicion for its diagnosis. despite the limited evidence published in the literature on the laparoscopic repair of incarcerated spiegel hernias, the studies published so far suggest that the laparoscopic repair is a valid alternative to the classic approach when it is performed by a well-trained laparoscopic surgeon. introduction: repair of lateral abdominal wall hernias (both primary and incisional) can be challenging due to the complexity of anatomy, issues with fixation and the low incidence of such cases. a good understanding of abdominal wall and retroperitoneal anatomy, coupled with proficient laparoscopic technique is essential for successful repair via the minimally invasive approach. methods: a retrospective review of a prospectively maintained database was performed to identify patients with lateral abdominal wall hernias who underwent laparoscopic repair from january to july . results: patients with hernias were identified ( primary, incisional). mean patient age was (range - ) and mean bmi was . kg/m (range . - . ). according to ehs classification, the incisional hernia defects were located at subcostal (l , n = ), flank (l , n = ), iliac (l , n = ) and lumbar (l , n = ) regions. background: it is commonly admitted that laparoscopic surgery has the advantage of abdominal wall preservation. however, the increased use of laparoscopy has resulted in certain complications specifically associated with the laparoscopic approach, such as trocar-site incisional hernia. until today, it is not finally clarified 'patient-dependent' factors contributing to the occurrence of postoperative hernia after laparoscopic abdominal surgery. methods: between and , patients were operated due to trocar-site incisional hernia in one surgical centre. 'the patient-depending' factors which caused postoperative trocar site incisional hernia data was collected and retrospectivily analysed. results: port site incisional hernia occurred in % ( patients) after the use of trocars with mm or larger diameter. the presence of metabolic syndrome was the decisive factor in the development of postoperative incisional hernia in % ( patients). in % ( patients) the postoperative hernia occurred on the background of a long cough symptoms caused by chronic obstructive pulmonary diseases. the cause of postoperative hernia in % ( patients) of patients was the condition of lifting a one-time severity or heavy physical work. in % ( patients) of postoperative patients hernia developed due to prolonged constipation of chronic inflammatory colon diseases. conclusions: thus, when the aponeurosis of the trocars is adequately closed, the reason of the occurrence of postoperative hernias was caused by patient-dependent factors which increase intra-abdominal pressure. for this method, small midline incision cm in length - cm away from hernia orifice was carried out initially. dissection of intraperitoneal adhesion was carried out by sils with sils device. subsequently after closure of initial laparotomy unilateral anterior rectus sheath was incised from the same incision and dissection of retro-rectus space up to preperitoneal space was done under laparoscopic vision. dissecting the other side was carried out by same fashion. initial dissection of linea alba could be done by open surgery from initial incision. further dissection of linea alba, retro-rectus space, and hernia orifice was carried out by sils. defect closure of anterior and posterior rectus sheath using barbed suture was also done by sils and self-grip mesh was inserted. additional trocar to assist retro-rectus dissection, defect closure, and decompression of intraperitoneal cavity was inserted as required. aims: the laparo-endocsopic approach of inguinal hernia contiue to bring many clarifications concerning inter-parieto-peritoneal space of this region through in vivo exploration, obtained by magnification by means of specific optic intrumentation. our study aimed to revalue the in vivo fascias, to establish their embryological correspondences and to reunite the variable nomenclature existing in the classical anatomy of this region. these observations find their applicability in tapp and tep hernia procedures, as the old anatomical descriptions are no longer operative. methods: we have tried to identify the structures that delimit the anatomical regions of retzius and bogros in recording of tapp procedures performed on men, on the right side, for small indirect hernias on patintes with clear view of the structures. additional, a review of literature on this subject has been performed through a search in the detabases according to the following keywords: bogros space, retzius space, preperitoneal approach, urogenital fascia. results: retzius and bogros are the medial and lateral compartments of the inter-parietalperitoneal space, located between the transversal fascia and the parietal peritoneum. these narrow, virtual spaces are best highlighted today with the help of insufflation techniques during laparo-endoscopic procedures. a competent and careful dissection confirms a 'deep and superficial' stratification, highlighting embryonic relics derived from the uro-genital fascia: urinaryprevesical fascia and spermatic fascia. in addition, the real retzius space is located previously and the real bogros space is located behind this strcuture. the confluence area of the two spaces is a critical point of laparo-endoscopic dissection, its non-recognition may 'wander' the dissection. conclusions: literature data in this topic reflects a certain terminological confusion using general terms such as 'preperitoneal tissue' or 'arreolar tissue' to denote what we consider to be the urogenital fascia or its prologations. the data obtained were synthesized in several drawings and diagrams very useful in training surgeons to use tapp / tep techniques. aim: spigelian hernia containing epiploic appendage is really rare entity. in this paper, we present a very rare case of spigelian hernia involving epiploic appendage performed laparoscopic hernia repair. case report: a -year-old woman presented to the emergency department with sudden onset abdominal pain in the left lower quadrant. on physical examination, she had a small, palpable tender mass in the left lower abdominal quadrant. temperature and white blood cell count were normal. an inflamed epiploic appendage with an oval shape, a fatty core, and a central thin hyperdense line in the hernia sac was detected on abdominal computed tomography. its intraabdominal relationship with the normal wall of the sigmoid colon was well appreciated (figure a, b) . diagnostic laparoscopy was performed. (figure ) adhesions between the sac and epiploic appendage are released using sharp dissection. a peritoneal flap is then created (figure ). laparoscopic tapp repair was used without closing the defect (figure ) . the patient was discharged on th days uneventfully. aims: morgagni's hernia is an in infrequent, congenital, anterior or retrosternal diaphragmatic defect. the right side is the most frequently affected, up to % of cases. it represents between and % of congenital diaphragmatic hernias. in childhood, they usually attend asymptomatically or with respiratory symptoms. up to % are diagnosed in adulthood, incidentally or after gastrointestinal obstruction debut. the treatment is surgery, which can be by laparoscopic or open approach.we present a case of laparoscopic approach with intra-abdominal mesh placement of giant morgagni's hernia diagnosed in senile age. methods: -year-old woman with a history of advanced alzheimer's dementia, partially dependent in daily life activities and institutionalized who consulted for intermittent episodes of oral diet intolerance associated with vomits of one month of evolution. abdominal examination was anodine. chest radiograph revealed a right lower lung field mass with fluid collected. thoracoabdominal scan showed small bilateral pleural effusion and large, right anterolateral morgagni's hernia, which contains dilated segment of transverse colon and greater omentum . results: laparoscopic approach was performed. hernia was reduced and hernia sac was removed. the defect was repaired with a dual-component (absorbable and non absorbable) mesh anchored with intracorporeal suture. patient recovered and was discharged days after surgery. conclusion: laparoscopic approach for morgagni's hernia reapir is secure and offers the advantages of less post-opertive pain, faster recovery and short postopatory stay. introduction: recently, laparoscopic operations for ileus are increasing. we have undergone laparoscopic operation to adhesive ileus with umbilicar incision at the beginning. the umbilicar incision at the beginning makes it possible to secure the laparoscopic field by peeling the adhesion under direct view, and makes it easy to repair damage to the intestinal tract. surgical procedure: at first, the umbilicus - cm incision was made and peeled the adhesion as much as possible under direct vision. secondly, ez access was set and inserted one mm port, therefore laparoscopic operation was performed with or pieces of mm ports. when the repair or resection of small intestinal due to damage is necessary, it is pulled out through the ez access. objective: to investigate the possibility of problems of laparoscopic ileus operation to adhesion ileus by umbilicar incision at the beginning. introduction: small bowel obstruction (sbo) during pregnancy is a rare condition with an incidence of . - . % and in around % of cases it is most caused by adhesions from previous abdominal surgery. other diagnosis, such as, hernias, malignancy, volvulus or intussusception are extremely rare. when sbo occurs in pregnancy, it carries a significant risk to mother and fetus. its diagnosis of can be difficult to make as symptoms are often attributed mistakenly to the pregnancy. goals: a case report of congenital bowel obstruction during the second trimester of pregnancy handled by laparoscopy. material and methods: we report the case of a year old woman with a history of chronic lung disease, pregnant because in vitro fertilization ( ? weeks) who attended the emergency department with abdominal pain and bloating accompanied by nausea and vomiting for two days. on physical examination she showed a distended, soft, depressible and painful abdomen without peritonism. laboratory tests were normal. a nasogastric tube was placed with generous output fecaloid intestinal contents. abdominal ultrasound by expert radiologists in abdomen showed a moderate amount of free abdominal fluid with normal uterus moderate and sbo to the ileum because of intestinal adhesion. this results were confirmed with an magnetic resonance imaging (mri). results: the patient was operated by laparoscopic approach with three trocars. the main problem was discovered. we founded a congenital adhesion which conditionated the obstructive syndrome. postoperative recovery was uneventful and the patient was discharged h after surgery. conclusion: the non-obstetrical acute abdomen in pregnant patient is a reality that occurs in one of every pregnancies. its diagnosis in more difficult than in nonpregnant patients requiring or high index of suspicion. the laparoscopic approach of acute abdomen during pregnancy is a valid and safe option, even in the early hours after diagnosis of bowel obstruction when it is performed by a well-trained laparoscopic surgeon. aim: intestinal malrotation (im) without midgut volvulus in adults is a rare clinical entity, which is the result of an incomplete rotation of the small bowel during embryogenesis, due to the nonlysis of the ladd bands. these ligaments spread between the duodenum and caecum and do not allow the gastrointestinal tract to take its normal position into the peritoneal cavity. im appears in to - newborns and is usually asymptomatic. diagnosis is usually made in the first month, and presents with findings of an acute abdomen, small bowel ileus and volvulus. im in adults is a rare entity. most of the times it is asymptomatic, but it can cause chronic abdominal discomfort and constipation. we present the laparoscopic management of an adult patient with intestinal malrotation. methods: our patient, a year old female, presented to the emergency room with a -month history of abdominal pain and nausea. all blood tests were normal. an abdominal mri showed intestinal malrotation without volvulus. due to persisting symptoms, she underwent a diagnostic laparoscopy with complete lysis of the ladd bands. the only unusual finding was a slight oedema of the duodenum. results: her symptoms settled postoperatively and she was discharged on the nd postoperative day. since her discharge, she has not developed any similar abdominal pains or complaints. conclusions: symptomatic intestinal malrotation in adults is an unusual clinical entity, but it is definitely one of the differential diagnoses we need to consider in case of chronic abdominal symptoms. the management consists of the division of the ladd bands, and this procedure can be performed safely with laparoscopy. many small intestinal obstructions are due to adhesions after laparotomy, but small bowel obstructions without history of open surgery is relatively few. in diagnostic imaging such as preoperative ct examination, the cause is diagnosed to some extent, but details are sometimes unknown unless operative observation is actually made. in many institutions, laparoscopic surgery is also actively introduced into the operation to relieve bowel obstruction, and its effectiveness is beginning to be recognized. we examined the usefulness of laparoscopic surgery for patients with small bowel obstruction without history of laparotomy from experience in our hospital. aim: from december to october , we searched cases of laparoscopic surgery for a small bowel obstruction without previous laparotomy at our hospital, and clinical findings, surgical results, and postoperative course were examined. results: there were ten cases. eight men and two women. the median age was years ( - yrs.) . reasons for intestinal obstruction were adhesions cases, internal hernia cases, persimmon stones case, small intestine tumor case. four cases of adhesions were emergency surgery. there were cases of emergency surgery and waiting surgery. five laparoscopic operations were completed and five cases during laparotomy transition. the median surgical operation time was min ( - min), and the median bleeding amount was g ( - g). there was no fatal case after operation, only one complications of ileus. the median length of hospital stay was days ( - days) . conclusion: laparoscopic surgery for intestinal obstruction with no history of laparotomy was thought to be a safe and effective procedure. although the transition to laparotomy would be higher in case of emergency, but there was no case of large incisional laparotomy. conclusions: laparoscopic surgery for sbo reduces postoperative complications and contributes to shortening the postoperative hospital stay and to decreasing the rate of recurrences, although it is a retrospective study, which is a safe and a useful approach. furthermore, first episode of sbo without previous operation seems to be an appropriate indication for laparoscopic surgery. background: postoperative adhesion after abdominal surgery may cause intestinal obstruction, chronic pain, or female infertility, which constitutes the major problems after surgery. adhesion formation are reported to be reduced by laparoscopic surgery and the use of anti-adhesion barriers. seprafilm composed of sodium hyaluronate carboxymethylcellulose bioresorbable membrane has been widely used to date, especially in open surgery. the characteristics of seprafilm, which is easily stick when wet, conversely brittle when dry cause it difficult to deliver into the abdominal cavity via the small incision in laparoscopic surgery. therefore, seprafilm is not much used in laparoscopic surgery. although various methods of insertion of seprafilm have been reported, some need special devices, or some acquire skill. methods: we adopted the pre-moistening technique for the replacement of seprafilm in consecutive cases of laparoscopic gastrointestinal surgery. a sheet of seprafilm was cut into equal pieces. to soften the sheets, one of the pieces was placed on a folded wet gauze until it became naturally curled then it was reversed, and the same procedure was repeated. softened sheet is easily to deliver into the abdominal cavity via a small incision by pushing with digital finger. moistened sheet expands naturally in the abdominal cavity. one or two pieces were needed to cover the incision. this process took only a few minutes. results: in all cases, the sheets were successfully introduced into the abdomen and spread widely enough to cover the incision. there have been no adverse effects, no postoperative complications, or gastrointestinal obstruction due to adhesion in the observation period of median two years. conclusions: short term outcomes were good after applying this technique. however, to record the incidents of intestinal obstruction and chronic pain, over years observation is indispensable. long term follow-up studies are required to clarify the usefulness of the anti-adhesive barrier in gastrointestinal surgery. b. east, rd department of surgery, motol faculty hospital, prague, czech republic aim: since when the ipom acronym was used for the first time our views at intraperitoneal mesh positioning has changed several times. despite growing evidence on its possible long term consequences it is still preferred method at some centres for large number of patients. the aim of this study is to point out the pitfalls of this method but also show that ipom is a good technique but only for highly selected cohort of patients. methods: this is a review of the literature focusing on the indications and complications of ipom pointing out controversies among the published articles over last two decades. some mesh material characteristics are being discussed as they are basic for understanding this complex and highly sensitive issue. results: a wide range on indications of ipom from little umbilical to large incisional hernias is advocated by many. however, some opinion leaders promoting this technique as universal and ideal for everyone just few years ago are advising to avoid it if possible lately. a necessary overlap has also been questioned recently. despite improving anti-adhesion barriers and methods of fixation in may a surgical mesh has become classified as risk class iii by the eu parliament and council on medical devices hoping to prevent physiomesh like incidents in the future. the need for post market registries and long term follow up is obvious. conclusion: us as surgeons implant a mesh in our patients and therefore we should be aware of its possible long term effects. no mesh on the market has a long term safety evidence especially in the intraperitoneal space. ipom is a good technique but possess a significant risk of long life complications and therefore should be spared only for those unfit for other methods of repair, patients with too high mesh infection risk, obese or older patients. introduction: acute appendicitis in elderly patients is relatively uncommon and could represent an underlying neoplasm. hence patients over the age of are often referred for a follow-up colonoscopy after management of acute appendicitis. the current routine use of computed tomography (ct) scans in the evaluation of suspected acute appendicitis in elderly patients prior to surgery coupled with intra-operative findings at laparoscopy question the role of follow-up colonoscopy for these patients. aims: to determine the role and optimal timing of colonoscopy in early detection of colorectal neoplasia after treatment of acute appendicitis in elderly patients. methods: all patients aged years and above with confirmed appendicitis admitted to our hospital during the period / / to / / were included. follow-up colonoscopy, diagnosis of colorectal neoplasia and its location in this patient cohort was evaluated. results: number of people aged and above in olol who had appendectomies from the dates / / to / / = . out of them / ( %) had full colonoscopy within years of the appendectomy.of them of the colonoscopies done were maleand were females. / ( %) of these colonoscopies were completely normal. colonoscopy identified colorectal carcinoma in ascending colon ( . %). other pathologies identified included: benign polyp ( %), polyp with low grade dysplasia ( %) and others ( . %) (lymphocytic colitis, ulcerative colitis, medication related ulceration, diverticulosis, melanosis coli, haemorrhoids). conclusions: in elderly patients above years of age: there may be an increased risk of colorectal cancer after acute appendicitis. only % of this patient cohort underwent colonoscopy after appendectomy. the current recommendations suggest the need for follow-up colonoscopy in elderly patients post acute appendicitis. further studies are needed to decide whether routine colonoscopy is indicated after acute appendicitis patients over years. introduction: it is generally accepted that the main aetiology of appendicitis is obstruction due to appendicoliths in adults and lymphoid hyperplasia in children. in contrast, incidental appendicoliths have been reported to occur in up to % of the asymptomatic population. controversy still exists regarding the association of appendicolith and appendicitis. is the appendicolith a causative factor or merely an incidental finding? aims: to determine the association between the presence of appendicolith and acute appendicitis (perforated or non-perforated) vs healthy appendix. methods: we collected the data retrospectively from the electronic records of all appendicectomies performed between january and december in our institution. data collected included: age, sex, appendix histology and the presence of appendicolith. interval or incidental appendicectomies were excluded from this study. we analysed the data using spss software version . results: during the study period appendectomies were performed (males: , females: , age range: - years). cases were histologically confirmed cases of acute appendicitis and of these, were perforated. a normal appendix was identified in cases. the remaining cases were due to chronic appendicitis, sub-acute appendicitis, lymphoid hyperplasia, parasitic infestation, and neoplasm. appendicolith was found in cases, of which were found in a normal appendix and were found in an inflamed appendix. out of the cases of appendicolith with normal appendix: cases were aged between and years old, cases were aged between and years old and case was aged between and years old. out of cases of appendicolith with acute appendicitis, cases were aged between and years old, cases were aged between and years old and case was aged over . conclusions: appendicolith may merely be an incidental finding and is not the primary cause of appendicitis. no significant correlation between gangrenous/perforated appendicitis and the presence of appendicolith. contrary to popular belief appendicoliths are more common in paediatric appendicitis than in adult cases. further research is recommended. over the last years, patient satisfaction surveys have gained increased popularity. nowadays, respect for patients' needs is central to our health care system. hospitals use patient satisfaction surveys to assess quality of care. many hospitals routinely survey patient satisfaction but relatively little data has been published. our acute surgical assessment unit operates from am to pm monday to friday and in its first year saw surgical patients, of whom were discharged and were admitted to the hospital for further management. aims: to assess the levels of satisfaction of patients attending asau at our lady of lourdes hospital. methods: a random sample of patients seen in the asau was surveyed to determine their level of satisfaction and the experience they had whilst attending asau. a novel self-reported patient satisfaction questionnaire was developed and used to assess patients' opinion regarding the treatment they received, the doctor's explanation of their condition, the waiting time and the service in asau. also the questionnaire encouraged patients to suggest improvements to the service. aim: sintestinal obstruction is a very common cause of presentation to an emergency department. the most common cause in patients with prior abdominal surgery are adhesions, but the list of differential diagnosis is large. internal hernia is a very rare cause of obstruction, with a reported incidence of between . and . %. the herniation related with broad ligament defects is even more uncommon. methods: we report the case of a -years-old woman with antecedents of liver transplant, tubal ligation and appendectomy. the patient was admitted refering abdominal pain in the epigastrium of h duration, accompanied by nausea and vomiting. on physical examination, abdomen was depressible, tender in the right low quadrant, without evidence of peritoneal irritation. laboratory studies were normal except for an elevated leukocyte count with a left shift. computed tomography (ct) revealed dilated small bowel loops with a transition point in right lower quadrant. radiological diagnosis was intestinal obstruction, with fibrous adhesion as the most probably aetiology. management was conservative at the beginning, with intravenous hydration, nasogastric tube and administration of gastrografin (diatrizoate) without a good response. results: at h, an exploratory laparoscopy was perform, finding dilatation of small bowel loops and a cm defect in the right broad ligament in which a segment of ileum was herniated. ileal segment was liberated without evidence of ischemia. the hernial defect was closed by laparoscopy with simple silk stitches. the postoperative course was excellent, tolerating oral feeding next morning. the patient was discharged h after surgery. conclusions: internal hernias of the broad ligament are an extremely rare cause of intestinal obstruction, but must be added to the differential diagnosis for female patients due to the risk of intestinal strangulation and perforation. even if clinical and radiological diagnose is difficult, ct is the best tool to delineate the cause and location of the obstruction. laparoscopy allows reduction of the hernia and closure of the defect with minimal invasiveness. because of that, the laparoscopic approach of bowel obstruction should be considered as the first choice if there is the suspicion of an internal hernia, without signs of necrosis or perforation. the laparoscopic approach is a safe and effective tool in the management of postoperative complications. it is well tolerated in critically ill patients and avoids respiratory and wound related morbidity associated with laparotomy. it also reduces diagnostic delay and a considerable number of unnecessary laparotomies, with a high resolution rate and minimal morbidity. it thus represents a valid and necessary alternative in surgeon's armamentarium. in the management algorithm of our institution we always choose the laparoscopic technique as the fisrt tool in case a reoperation is necessary. , small bowel obstruction ( . % vs . %), and colorectal cancer obstruction ( . % vs . %) was found higher for acs unit group, and also progressively higher during the last years. conclusion: according to our study, laparoscopic approach in abdominal emergencies shows an upward trend, and surgeons from acs units seem to have higher rates of laparoscopy than general surgeons in emergency procedures. background: incarcerated and strangulated hernias present a major problem in emergency medicine. there is scarce data about the role of laparoscopy in the management of these patients. laparoscopic repair offers the benefits of the ability to survey the incarcerated organ and to evaluate its viability, apart from the obvious advantages of laparoscopic surgery. the use of mesh repair in these emergent operations is also a major concern, due to the un-sterile conditions in which they are performed. objective: to evaluate the safety and short-term efficacy of laparoscopic emergent repair of incarcerated hernias. methods: retrospective review of prospectively collected data of all the patients who underwent emergent laparoscopy due to an incarcerated hernia between november and october . results: during the study period, patients underwent emergent laparoscopy due to incarcerated hernias ( females, males). had incarcerated inguinal hernias, and had incarcerated umbilical hernias. mean age was . . all inguinal hernias were repaired in the tapp approach, and using an absorbable mesh. all umbilical hernias were repaired using the ipom approach. patients had bowel obstruction, had incarcerated omentum, and one patient had incarcerated urinary bladder. patients underwent resection of an ischemic organ ( bowel, urinary bladder, omentum). mean hospital los was . days. during the follow up period there were no mortalities, and no recurrences. one patient had a wound infection that resolved with antibiotics. conclusion: laparoscopic emergent repair of incarcerated hernias is a safe and feasible approach. further studies with longer follow up time need to be conducted, in order to evaluate the added benefit of the laparoscopic approach. gibraltar is a small overseas british territory with a residential population of approximately , inhabitants, that increases up to , daily due to incoming tourists and cross-frontier workers. as a geographically isolated center we have to provide a varied service including emergency surgery, and elective operating such as colectomies, gastrectomy's etc. one of the challenges faced is the limited stock of red blood cell (rbc) units within gibraltar and reliance on platelets (plt) from across the border from spain. given the immanent brexit we need to prepare for the challenges we will face in these times of political and distribution uncertainty. a prospective audit of all blood use within gibraltar was carried out over months. the number and type of units requested, the number of units given, the speciality, location and indication for requests was recorded. introduction: the use of laparoscopic surgery in abdominal emergencies, such as in trauma, has had a slow acceptance. the advantages with this approach include less postoperative pain, faster recovery, quicker return to everyday activities, and fewer complications. we have collected the cases and indications of laparoscopy in abdominal trauma in the main hospitals in the andalusian capitals and compared with the national registry material and methods: a total of patients who underwent laparoscopic surgery in the main hospitals of seville, cordoba, malaga, cadiz, huelva, jaen, granada and almeria were analyzed. they have been compared with the traumas archived nationally by the spanish association of surgeons taking into account age, sex, score of the american society of anesthesiologists, hemodynamic stability and mechanism of injury. the intra and postoperative variables were compared between groups. results: at the national level, the main cause of abdominal trauma were traffic accidents, therefore, it was the patients who had a greater number of laparoscopies ( . %), followed by stab wounds ( , %) and run over ( . %). in our series, the average age of the patients is years and % are male. only eco-fast was performed in % of the patients, being positive in . % of the cases. as they were stable patients, in % of the cases a tac was possible. in our data, % of the laparoscopies were performed for therapeutic purposes as well as being diagnostic, thus avoiding a posterior laparotomy. conclusion: slaparoscopic surgery for abdominal trauma, either blunt or penetrating, is safe and technically feasible in hemodynamically stable patients. we found that laparoscopic surgery was associated with shorter operative time, lower estimated blood loss and faster return to normal diet. based on our findings we establish the indications of laparoscopy in these patients aims: submucosal aneurysm of small intestine is extremely rare, but its rapture can be lifethreatening. due to the unstable hemodynamics and unknown site of bleeding, emergency laparotomy has been widely performed for the rupture. we will present case reports and show the strategy for minimally invasive treatment for ruptured aneurysm. methods: we experienced two cases of ruptured submucosal aneurysm resected by laparoscopic surgery. case is a -year-old male who was taken to our er with massive hematochezia. ct showed arterial bleeding in the small intestine and angiography revealed bleeding from the ilial artery. selective embolization using gelatin sponge and micro coil was performed and hemostasis was obtained. video capsule endoscopy found the hemispheric elevated lesion with protrusion at the top in the ileum. using balloon assisted enteroscopy, the site of aneurysm was marked with injecting india ink, which allows surgeons to accurately and easily identify the part of small intestine with aneurysm. subsequently, a single incisional laparoscopic assisted partial ileectomy was performed for the purpose of definitive diagnosis and preventing re-bleeding. the ileum with aneurysm was easily identified in laparoscopic exploration owing to the marking, and it was taken out from the incision to perform resection. case is a -year-old female who was transferred to our emergency department with sudden onset of massive melena. ct and angiography were perfomed, and bleeding from the rd jejunal artery were confirmed. subsequently, therapeutic embolization was performed in the same way as case . enteroscopy revealed submucosal elevation similar to case in the jejunum. we carried out endoscopic tattooing, followed by single incisional laparoscopic assisted partial jejunectomy. results: the operative time in case and case were min and min, respectively, and the amount of blood loss was both ml. the postoperative course was uneventful in both cases. case was discharged on the postoperative day , and case was on postoperative day . conclusions: our experience indicates that ruptured submucosal aneurysm of the small intestine can be effectively managed by a laparoscopic surgery with combination of therapeutic embolization and enteroscopic evaluation, which is safe and minimally invasive. background: laparoscopic bilateral inguinal hernia repair may be completed with one large selffixating mesh crossing the midline in front of the bladder. no studies have investigated in detail whether preperitoneal mesh placement induces temporary or more lasting urinary symptoms. methods: urinary and hernia related symptoms were evaluated preoperatively and postoperatively at , and months in patients using the iciq-mluts questionnaire and eurahs-qol score. results: voiding symptoms and bother scores were unchanged at or months, but there was significant improvement at months compared with preoperative findings (symptoms p \ . ; bother score p \ . ). incontinence symptoms improved at month (p \ . ) but not at or months, with a bother score significantly improved at month (p \ . ) and months (p \ . ). diurnal and nocturnal frequency did not change significantly postoperatively, but months nocturnal bother score was decreased (p \ . ). eurahs-qol scores showed significant improvement in all domains for all measurements compared to previous measurements. postoperative symptoms were improved at months, compared with preoperative pain scores (- . ), restriction of activity (- . ) and cosmetic scores (- . ) these findings were statistically significantly (p \ . ). at months, there were no patients with severe discomfort (score = ) for any of domains. no recurrences were diagnosed with % clinical follow-up at months. conclusion: placing a large preperitoneal self-fixating mesh for bilateral groin hernia repair did not cause new urinary symptoms and demonstrated significant improvement in voiding symptoms at months. incontinence and nocturnal bother score were significantly improved. introduction: tep/tapp hernia repair is an increasingly widely used surgical methods for minimally invasive treatment of inguinal hernia. tep advantages to tapp are noincision of the parietal peritoneal sheet, therefore no need for its recovery-sewing or sticking at the end of the procedure, and no need for attachment of the prosthetic mesh to the structures of the anterior abdominal wall, which results in a reduction in the financial cost of operation.various types of meshes with different characteristics are used, depending on the surgeon's preferences.the aim of this study is to highlight mesh-related postoperative complications, which can be serious and life-threatening. material and methods: a retrospective cohort study of cases of unilateral or bilateral tep and tapp hernia repair performed at the university hospital for the period - with a study of early and late postoperative complications potentially causally related to the implanted prosthetic mesh and methods of their treatment. results: for a -year period tapp ( bilateral) and tep ( bilateral) have been performed. three complications (clavien-dindo iva, ivb and v) were found, of which were early postoperative (up to pod)-one in tapp- pod small bowel adhesive ileus due to suture dehyscense of the peritoneal sheet and adhesion of a bowel loop to the surface of polypropylene mesh.one in tep- pod-a large preperitoneal hematoma with haemorrhagic shock at years old female in anticoagulant therapy-an open revision of the preperitoneal space and definitive haemostasis; followed in pod established bladder lesion from erosion from the edge of self-locking polypropylene mesh. suture and drainage performed, but the patient died of decompensation of concomitant diseases. a late complication- months after bilateral tep-erosion of soft polypropylene mesh of sigma (probable undetectable lesion of the peritoneum) with faecal peritonitis-hartmann procedure with laparostoma followed by restitution but persistent chroniosepsis with established abscess in retzii. months after-revision with abscess incision and extraction of infected meshes. discussion: use of biologic meshes is quite expensive, however synthetic non-resorbable meshes implanted in preperitoneal layout is a prerequisite for specific severe postoperative complications. inguinal hernia repair is one of the most performed procedure all over the world, with more than million procedures performed each year, it represents one of the top three most performed procedures. the lichtenstein procedure is one of the first procedures that a young trainee in general surgery learn, not only for its reproducibility and for the great numbers of procedures that could be done in each department, but also because during inguinal hernia repair the trainee learn a lot of skills which are the basis of major surgical interventions. the surgeon's performance for any procedure could be evaluated by way of established learning curves that can predict the minimum number of procedures required to reach the same intra and post-operative outcomes as an experienced surgeon performing the same technique. the aim of our multicentre study was to analyse how many cases are required to stabilize operating time (ot) and intra and post-operative complication rates over the course of the learning curve period for a lichtenstein procedure. from january to december all lichtenstein procedures from four different institutions were recorded in a prospective maintained computer database. the results of the first consecutive procedures performed by three different trainees (group a; group b; group c) were compared with the same numbers of procedures by two senior surgeons of the same institutions (group e, group f). cusum analysis was performed to evaluate the achieving of learning curve. no differences in terms of biometric and hernia type were recorded between the five groups. cusum analysis showed that the trainees achieve the learning curve between the - procedures. no intra or post-opertive complications were recorded during the training period.in conclusion after our analysis we found that at least procedures are needed for the trainees to achieve the learning curve for lichtenstein procedures. background: since its first description in the s, the total extraperitoneal (tep) technique has established itself as a popular endoscopic method for the repair of inguinal hernias. the tep repair is generally viewed as a technically-demanding procedure requiring adequate experience to minimize and handle complications. in this case report, we describe an uncommon complication of urethral injury, which was successfully repaired laparoscopically. case report: mr r is a year old gentleman with no significant past medical history who presents to the department of general surgery, tan tock seng hospital, with a two-month history of a reducible right inguinal hernia, associated with some tenderness. an ultrasonography confirmed the diagnosis of a fat-containing indirect right inguinal hernia. in view of persistent pain, mr r was counseled for a laparoscopic repair of his right inguinal hernia. as mr r was able to empty his bladder just prior to surgery, no urinary indwelling catheter (idc) was inserted. an infra-umbilical incision was made to access the posterior rectus sheath and a balloon was used to bluntly dissect the pre-peritoneal plane. on inspection of the operating field, persistent pooling of blood was noted in the retropubic space. careful inspection revealed a defect in a tubular structure just inferior to the bladder neck. an idc was inserted, which confirmed a . cm defect in the pre-prostatic urethra. decision was made for primary repair using absorbable sutures in two layers. the bladder was subsequently filled via the idc, which did not reveal any leak. we then completed the right inguinal hernia repair using a mesh. mr r made an uneventful recovery and was discharged on post-operative day with instructions to keep the idc in-situ for two weeks. the idc was removed after two weeks and a micturating cystourethrogram was performed, which showed no filling defects along the urethra and no contrast leaks. discussion: though uncommon, urethral injuries can be a complication of laparoscopic tep repair. the key to managing these complications is in the early identification of such injuries intra-operatively. with early recognition and careful assessment, such complications can be managed laparoscopically with minimal post-operative morbidity. aim: the purpose of this study is to report surgical technique and outcome of hybrid tapp procedure (a combination of tapp and ipom) for inguinal hernia patients complicated with preperitoneal space adhesion. methods: hybrid tapp procedure is applied if peritoneal dissection or closure of the peritoneum is difficult due to severe adhesion. the peritoneum should be dissected as much as possible. for the site where adequate dissection was achieved, the collagen mesh is placed outside the peritoneum. in the part where dissection was difficult it is placed inside the peritoneal cavity. in order to prevent mesh migration, the mesh should be directly fixed to the cooper's ligament with a tacker. for this purpose, the peritoneum around the cooper's ligament must be well-dissected, even if it is strongly adhered, so that the ligament can be exposed. the crucial points in the hybrid tapp procedure are fixation of the mesh and prevention of the bowel herniation into the preperitoneal space. at the site where peritoneal dissection is possible, the mesh is directly fixed on the fascia using a tacker. if it is difficult, the mesh is placed in the peritoneal cavity and fixed over the peritoneum. if there is a risk of migration along with peritoneum, transcutaneous full-thickness fixation can be performed using non-absorbable sutures. the preperitoneal space should be closed tightly as soon as possible in order to prevent the bowel herniation into the preperitoneal space. at closure of the preperitoneal space, the peritoneum is fixed on the collagen mesh using non-absorbable sutures. objective: show a tapp approach using a self-fixating mesh( x cm. progrip tm laparoscopic self-fixating mesh, medtronic) with bipolar peritoneal defect sealing, avoiding the use of tackers and performing an easy and sutureless peritoneal closure. material and methods: years old male, asa ii, medical history of beta-latacm allergy, high blood pressure, dyslipidemia and bilateral knee surgery. diagnosed of bilateral inguinal hernia at consultation due to inguinal disconfort. surgical site infection prophylaxis with iv vancomycin. balanced general anesthesia. supine decubitus position with shoulder supporting to allow a forced trendelemburg. degree optical device with trocars disposition: one mm umbilical trocar and mm trocar in both flanks, same distance and height to umbilical trocar. peritoneal opening and flap creation with monopolar energy, blunt maneuvers and pneumoperitoneum dissection. anatomical landmarks identification(cooper's ligament, epigastric and iliac vessels, hernia defect and spermatic cord elements). reduction of hernia sac content(pseudosac in this case, direct hernia) and complete peritoneal dissection to achive a correct mesh placing. mesh is folded in parts(one inferior part, two superior parts) in vertical axis outside the abdomen to facilitate the posterior intraabdominal maneuvers. introduction: into abdominal cavity with grasping forceps and correct unfolding mesh assesment: medially(pubic bone), caudal(cooper's ligament) cranial(more than cm of hernia defect/ deep inguinal ring) and lateral(anterior superior iliac spine). finally, we use a bipolar forceps to close de peritoneal defect. in order to facilitate this step, its necessary to decrease pneumoperitoneum pressure and to use the grasping forceps to bring together both peritoneal flap edges prior to bipolar energy sealing. results: min. surgical procedure. h hospital discharge, no complications. routine outpatient follow up(week, month, months and month later) with an epididymitis episode months after surgery(treated with oral ciprofloxacin). conclusions:-this procedure is an easy implementation technique once the intraabdominal mesh unfolding procedure control is reached.-the use of a self-fixating mesh avoid the use of tackers and its potential disadvantages(e.g. increasing postoperative pain).-bipolar peritoneal sealing offers a quick, easy, cheap and safe peritoneal closure, avoiding the contact of the mesh with the viscera in the same manner. results: we performed procedures within patients. the average age was years. twenty six percent of hernias were bilateral, , % were inguinoscrotal and % in the right side. the median asa score was . the conversion rate was , %. the average duration of the procedure was , min min. overall morbidity was %. there were seromas ( , %) . on -year follow-up, one recurrence ( , %) was found and chronic postoperative pain in one case . we had no mortality. in the univariate analysis, male sex, inguinoscrotal hernias, hernias classified as nyhus a were significantly associated with overall postoperative morbidity. a chronic obstructive pulmonary disease was the only variable significantly associated with the occurrence of medical complications. conclusion: given these results, the tapp technique is a good alternative in the treatment of groin hernias. however, enhancing this approach is essential to reduce the operating time and the postoperative outcomes. introduction: studies have emphasized the impact of a strong safety culture on patient outcomes. consequently, many interventions focus on improving the safety culture, of which teamwork and safety climate are important ingredients. it is known that differences in culture and safety attitudes may also impact teamwork. implementations of safety interventions, such as a ' black box', are dependent upon these differences. the aim of this study was to assess the safety culture at the operating theatre complex, along with the theatre staff's attitude towards a specific quality improvement intervention, a black box in the operating room as a tool for structured team debriefing. methods: the validated dutch version of the hospital survey on patient safety culture was administered to all healthcare professionals working in the operating room complex at one academic medical centre. this survey was supplemented with questions regarding the use of a 'black box', a medical data recorder in the operating room, to measure the staff's attitude towards this quality improvement tool and its potential contribution to patient safety. aims: the aim of the study was to compare two methods of treatment of dunbar syndrome: thelaparoscopic release of median arcuate ligament alone and the hybrid method consisting ofsurgery and percutaneous stent implantation to celiac trunk. methods: we performed laparoscopic release of ct in the department of general, mini-mallyinvasive and elderly surgery in olsztyn in - . all of patients suffered from severepain of abdominal cavity before the surgery. three patients underwent doppler percutaneousangioplasty of the ct with stent implantation one month after the laparoscopy. results: all patients reported relief of symptoms in the first days after the operation. in two cases fromboth groups, there were a complete remission of the symptoms. in one case respectively,there was an improvement. there were no postoperative complications. the results of both methods do not show the differences therefore the surgery alone seems tobe a safe and feasible procedure. it increases the comfort of the patient and brings theopportunity for normal functioning. the method of wedge resection of lungs in patients with limited forms of chemo-resistant pulmonary tuberculosis is developed. in order to evaluate the efficacy, patients underwent surgery (the main group). for comparison, the data on similar operations in patients, made according to the traditional method (with the help of a cardboard weaving machine yo- ) were selected. compared the duration of the stage of resection itself, the frequency of need for additional hemostasis of the parenchyma sutures, the degree of deformation of the pulmonary tissue in the seam area, the frequency of postoperative complications and reoperations, the duration of postoperative inpatient treatment. the developed method, in comparison with the traditional one, has the following advantages: simultaneously leak proofness and hemostasis with minimal electrothermal damage to tissues are provided and there is no need for additional hemostasis, there are no negative effects of manual stitching of parenchyma of lung with abandonment of foreign material, a significant reduction in the duration of wedge resection of the lung from . to . min, a decrease in the number of postoperative pulmonary-pleural complications is achieved by . % and caused by them reoperations-by . %, shortening the duration of postoperative inpatient period of treatment from . to . days. introduction/aims: laparoscopy is a diagnostic and therapeutic resource that is largely used in elective gastrointestinal surgery due to its well-known advantages over the classic open approach. nevertheless, there is still some discussion about its application in emergency surgery. our aim is to analize the use of the laparoscopic approach by the members of the surgical emergency unit from our medical center. methods: a descriptive research based on the data of patients who required emergency surgery, that was performed by the members of the surgical emergency unit of a spanish hospital between november and may , was conducted. these data were analyzed according the pathology that motivated the surgical procedure and the chosen form of surgical approach (open versus laparoscopic). results: out of the patients in whom emergency surgery was performed, suffered from a pathology that actually allowed the laparoscopic treatment. laparoscopy was used in . % of these patients. according to pathology, the most common were acute appendicitis and cholecystitis, in which the laparoscopic approach was used, respectively, in % and % of the cases. regarding other less frequent pathologies, such as gastroduodenal perforation, bowel obstruction, diverticulitis and pancreatitis, laparoscopy had a less significant role. according to the year, a general tendency to increase the use of the laparoscopic approach was found, most notably in the cases of acute appendicitis and cholecystitis (with rates above % in ). conclusions: despite our positive results in the terms of the implementation of the laparoscopic approach in emergency surgery, there is still room for improvement, especially in regards of the less common pathologies. furtheremore, additional studies are needed in order to identify the factors that have had an effect, in favour or detriment, in the development of emergency laparoscopy in our center. aims: laparoscopic surgery, which produces small scars, has become widespread. when performing surgery through small laparoscopic incisions, a surgeon manipulates tools inserted into the abdomen through ports. for minimally invasive accurate procedure, the port as the pivot point should be stabilized on the abdominal wall. however, these laparoscopic incisions are loaded while manipulation because it is difficult for the port to be fixed on. thus, it is necessary for the patient friendly manipulation to be fixed the port mechanically. we developed a new pivot restraint device (prd) attached to a trocar for guiding the tool. the purpose of this study is to evaluate both of reducing the operating time and the load of the port with the prd experimentally. methods: the prd uses gimbal mechanism for two rotating axes and a linear guide mechanism for the insertion axis though into the forceps. in the experiment, the left hand forceps with or without the prd and the right hand forceps without the prd were set on the training box. the box had a measuring system created with a pressure sensitive sensor for the continuous force (resolution . n, fps) applied to abdominal wall fulcrum. the experiment task was performed as following three steps. ( ) the surgeon lifted the g weight for s at the initial position using the right hand forceps. ( ) the weight was transferred from the right hand forceps to the left hand forceps, and held for s. ( ) the weight was moved to the predetermined position, held for s, and returned to the initial position. the surgeons were five endoscopic specialists and five non-specialists. the operating time and the time ratio exceeded n for the left hand forceps were measured. two grouped datasets with or without the prd were compared using two-sided t-test. results: the prd was associated with both of reducing the operating time ( . s vs. . s; p \ . ), and the load of the port ( . % vs. . %; p \ . ) at the statistical analysis. conclusion: the prd could be used for reducing the operating time and the load of the port in minimally invasive accurate procedure. background: pathophysiological changes during laparoscopic surgery and positive pressure pneumoperitoneum (pp) may include (beside cardiovascular changes) elevated intra-thoracic as well as intracranial pressures. however, the possibility of physiological and functional cerebral impairment under pp is still debated. aim: to study the effects of pp on brain activity during different modes of anesthesia and ventilation during laparoscopic cholecystectomy (lc). patients and methods: thirty patients undergoing elective lc were divided to those who were ventilated by intermittent positive pressure ventilation (ippv, pt.) and by high frequency jet ventilation (hfjv, pt.). in those under hfjv we used total intravenous anesthesia (tiva). in those under ippv we either used inhalational anesthesia or tiva. intra-ocular pressures were detected in both eyes, trans-cranial doppler was used to measure the changes in flow of the middle cerebral artery, and cerebral oxygenation (o saturation) was measured too. each parameter was detected during anesthesia before surgery, several times during surgery under pp and after co evacuation. a novel computerized signal analysis by a continuous recording through a single electrode was done to explore cerebral cognitive activity during surgery. results: all surgeries went uneventful and without complications, pp was set to mmhg, and each patient was positioned in a degree anti-trendelenburg posture. cerebral perfusion and oxygenation were not changed significantly during pp. intra-ocular pressures decreased during anesthesia and increased during pp, but to a lesser extent under tiva. however, pressures during pp did not exceed pre-surgical values. we did not observe changes in cognitive brain activity during pp, although enhanced cerebral activity was seen under hfjv. conclusions: increased intra-abdominal pressure during laparoscopic surgery was not accompanied by decreased cerebral functions, maybe due to cerebral circulatory auto-regulation. changes in cerebral cognitive functions under hfjv might be explained either by the different cerebral effects of tiva in comparison to inhalational anesthesia, or due to dissimilar hemodynamic changes during hfjv. aims: gallstone ileus (gi) is a rare complication of cholelithiasis and accounts for . - % of small bowel obstructions. intermittent and non-specific presentation often results in late diagnosis. the triad of rigler is pathognomonic (pneumobilia, small bowel obstruction and ectopic gallstones), so an image test is usually mandatory in order to assure the diagnose. our aim is to expose our experience regarding this topic to show that a minimally invasive approach is feasible in selected cases. methods: since january we treated cases of gi, of whom ( %) underwent laparoscopic surgery. in all cases a ct was made to reach diagnosis. enterolithotomy alone is our preferred procedure for the resolution of this pathology. here we present a descriptive analysis of our data in those cases where a laparoscopic treatment was attempted. epidemiological variables, surgical technique, postoperative complications, days until hospital discharge, recurrence, etc. has been collected. results: % of patients were female( ) and % male ( ). mean age was . size of gallstones varied from to mm and ct located them all in the ileum. two conversions to open surgery were made ( %), in one case because the gallstone could not be found and in the other case due to the need of an intestinal resection. in two cases ( %) la aparoscopic-assisted surgery was performed using a pfannestiel incision for the gallstone extraction and enterorrhaphy. only one case was total laparoscopic approach ( %). two cases needed an intestinal resection and anastomosis, one of them was complicated with a leak that needed reintervention. there were two cases of recurrence during the follow-up time. hospital stay varied from to days, mean of days. conclusion: the widespread use of ct facilitates early diagnosis with high sensitivity detecting rigler's triad. a totally laparoscopic procedure might be ideal for patients specially with solitary stones even though a laparoscopic-assisted approach is an easier technique for surgeons with less experience in laparoscopic surgery. although experience in minimally invasive surgical treatment of gi is still developing, it may be recommended in selected cases and experienced hands. introduction: most of surgical interventions in hospitals in the world, where laparoscope is used, it is common that the vision inside the human body is constantly interrupted by fogging in laparoscope tip. the laparoscope fogging is caused by the difference of temperatures between the optic tip and the abdominal cavity. material and method: we replaces the traditional laparoscope for the ehs (endoscope heater system) with resistance between the internal and external tube that maintains the temperature of laparoscope at ( - °celsius) without modifying the external architecture of traditional laparoscope. results: ehs does not generates any waste like other anti-fog systems, like liquids, plastics covers or electric heater. reduces intervention time, can keep same instruments or accessories for the intervention. all of the above means a saving of resources with have a positive environmental impact. conclusions: the discomfort transmitted by surgeons about the fogging in laparoscopy tip make success of the product and it will replace the current laparoscope which is fogged. aim: synchronous locally-advanced low rectal cancer and prostate adenocarcinoma represent a rare condition and a challenging situation for colorectal surgeons and urologists. the simultaneous resection of both adenocarcinomas after long-course chemoradiation therapy combines two major surgical procedures associated with a potentially increased postoperative morbidity. in the other hand, simultaneous resections minimize the risk of difficult dissections, which are expected if the two procedures are scheduled sequentially. in the past decade, robotic-assisted minimally-invasive surgical techniques have been increasingly used to treat both rectal and prostatic malignancies. especially in case of prostatic malignancy, the robotic approach is considered the treatment of choice because it is associated with significantly lower blood loss and transfusion rate, and much greater functional outcomes compared to laparoscopy. methods: we present the case of a -year-old male patient (bmi: . ) diagnosed with a histologically proven locally-advanced rectal adenocarcinoma (ct an ) located at cm from the anal verge and concurrent histologically proven prostatic adenocarcinoma [gleason score of ( ? )] located in the postero-basal right lobe. the preoperative total-body computed tomography (ct) scan showed no evidence of metastatic disease. after discussion in a multidisciplinary meeting, the patient received a long-course neoadjuvant chemoradiation therapy (ncrt). at the restaging positron emission tomography / magnetic resonance imaging (pet-mri), the rectal lesion was classified as ymrt n . preoperatively, the surgical difficulty was assessed as high, based on the calculation of the eumarcs score (equal to / ). moreover, due to the high-risk status of the prostate cancer (gleason ), it was decided not to preserve the neuro-vescular bundles during the radical prostatectomy. results: the patient was operated on after weeks from completion of ncrt by using the da vinci robot system si with a single docking approach, as previously described, in order to address both cancers. conclusions: this video shows the main surgical steps of the simultaneous robotic resection of the low rectal adenocarcinoma first, of the prostatic carcinoma then, and the mechanical colo-anal anastomosis followed by drain positioning and ileostomy. this video demonstrates the perioperative safety and feasibility of the minimally invasive robotic approach in case of extended and challenging oncologic resections. general surgery, rambam medical center, haifa, israel year old, male patient presented with melena, without abdominal pain, nausea or vomiting. patient underwent colonoscopy and tumor was found in ascending colon (near the hepatic flexure). biopsy from the tumor has showed moderately differentiated adenocarcinoma. his blood laboratory examinations were within normal limits except of hgb level- . . cea and cea - were normal. abdominal computed tomography was normal . patient underwent da vinci robot-assisted right hemicolectomy with extracorporeal anastomosis. total operating time was min. three days after operation patient started regular diet and was discharged home on day four. final pathology result confirmed diagnosis of moderately differentiated adenocarcinoma. introduction: one of the goals of colorectal surgery is to decrease the number of leaks once an anastomosis has been performed. this life-threating entity after elective surgery has been related to the clinical history of the patients, the location of the tumor and to technical reasons, specially due to tension in the anastomosis or to lack of vascularization. tension could be identified during surgery, while vascular supply is evaluated by the surgeons based on a subjective analysis of the color of the colon/ileum. fluorescence tries to make these subjective parameter more objective in order to avoid an anastomosis with lack of vascularization, decreasing the numbers of leaks related to this factor. patients and method: the study presents a quasi-experimental analysis made from january to october in two hundred and eighty-five patients who underwent elective colerectal surgery, performing either a colo-rectal, ileo-rectal or intracorporeal ileo-colic anastomosis. vascular supply was eveluated using indocianyne green (icg) in one hundred and forty-five patients, while one hundred and forty subjects were operated in a previous period without using this technology, being considered the control group. the number of time that the attitude changed and the number of leaks were collected. results: out of the cases performed, were right colectomies (rc), left colectomies (lc) and rectal excision (re). in % the transection line was changed ( , % in rc, , % in lc and , % in rr) . in comparison with the control group, the icg group had a significantly less indicence of anastomotic leak compared to the control group ( , % vs. , %, p = , ), lower rate of terminal stoma after reoperation ( , % vs. , %, p = , ), a shorter length of hospital stay ( days vs. days, p = , respectively), and a low morbidity and mortality. conclusions: the rate of leaks after colorectal surgery decrease using icg to detect the proper transsection line before to perform the anastomosis in comparison with control group. these findings might influence in the final results although it is necessary in the future to find a system that provides greater objectivity by quantifying icg. aims: anastomotic leaks continue being one of the most important complications when a colorectal surgery is performed. this complication is usually related to the level and type of resection, the patient clinical history and surgical technique, where tension and vascular supply are the most important. indocyanine green (icg) fluorescence angiography seems to be helpful in order to evaluate the vascularization at the resection margins. methods: we have collected data on colorectal procedures that were performed by the same surgeon using icg fluorescence angiography to evaluate vascular supply to the anastomosis. in order to asses in which of the different type of colorectal procedure has more value to be used, we analyzed the type of surgical procedure, the percentage change in the resection margin and the number of anastomotic leaks (al). results: all of the cases were performed by laparoscopic approach: left colonic resection (lc), right colonic resection (rc), splenic flexure partial resection (sf), low anterior resection with partial mesorectal escision (lar), ultra low anterior resection with total mesorectal escision (ular) and total colectomy (tc). there was a change of transection line (ctl) in lc ( , %), rc ( %), sf ( , %) and ( , %) in rectal anastomosis (lar, ular and tc). as far as al we found: lc ( , %), rc ( %) and , % in rectal procedures. lc, sf and rectal procedure showed more ctl and less al, while rc showed less ctl and more al. conclusion: icg fluorescence angiography as an additional tool to try to reduce the anastomtic leak rate seems to have more value in the procedures that involve the left colon and the rectum, since that is where we have observed the greatest number of ctl, this could be explained by the riolan's arcade and the variability of the vascular anatomy. however, it seems that this is a line of research should continue developing with longer and larger studies, so in that way we can have more significant results. retrorectal tumors ara rare and often found incidentally. the majority of retrorectal tumours are benign, but they have potential for malignant transformation and therefore should be resected when found. a case of a -year-old female patient with a retrorectal tumor is showed. the tumor was found incidentally on ct scan of the abdomen for evaluation of non specific right side abdominal pain. a mri was also performed and imaging was informed as a probably congenital retrorectal tumor (tailgut cyst) there was no evidence of involvement or invasion of other structures the tumor was palpable at rectal examination. a transanal minimally invasive surgery (tamis) approach was proposed. preoperative preparation was done with a full mechanical and oral antibiotic bowel preparation. preoperative parenteral antibiotics werw administred. under general anesthesia, lithotomy position. the contour of the tumor is not visible due to the small size. palpation of tumor and placement of clips to lolocate was done. placement of gel point path and rectal insufflation. a longitudinal incision was made to the posterior left side of rectal wall. the insufflation of the perirectal extraperitoneal space allowe for excellent exposure of the tumor. the tumor was disected with ligasure. then the tumor was extracted transanally.the proctotomy was closed in a single layer with reabsorbible monofilament continuous suture (pds). no complications after the procedure. the patient was discharged at days. discusion: traditionally, the retrorectal tumors have been resected using a posterior parasacrococcygeal approach, an abdominal approach or a combined abdominal and posterior approach. with the advent of minimally invasive surgery, laparoscopyc approach has been described too. however, tamis approach is feasible, with low pain, morbidity, fester recovery and excellent cosmetic (no scare) results. it can be accomplished using standard laparoscopic equipment, with transanal access. we think that perhaps it could be the gold standar approach for this tumors. aimes: robotic-assisted laparoscopic surgery (rals) is a promising advanced technology that can overcome the inherent limitations of conventional laparoscopic surgery (cls). its advantage includes free-moving multijoint forceps, a motion scaling function, high-quality three-dimensional imaging, and stable camera work by an operator. this study aimed to clarify the short-term outcomes of rals for rectal tumors. methods: this study group comprised patients who underwent rals for rectal tumors (cancer in patients and gastrointestinal stromal tumor in patient), excluding ones with distant metastasis from november through december . the clinicopathological findings and short-term outcomes in rectal tumors were analyzed. results: the median operative time was min ( - ). the median console time was min with a median blood loss was ml ( - ). conversion rate was . % ( / ). the median postoperative hospital stay was days ( - ). patients ( . %) had postoperative complications. patients ( . %) had lymph nodes metastases. the mean harvested lymph node was . . the r resection rate was % ( / ). conclusions: these results suggest that rals for rectal tumors is safe and feasible, and the perioperative outcomes are acceptable. introduction: anastomotic healing defects are a feared complication which might have a fatal impact on the patient. fundamental conditions for proper anastomotic healing include sufficient blood supply. fluorescent angiography using indocyanine green in the spectrum of near infrared light facilitates the monitoring of tissue perfusion during a surgery. aim: a presentation of the results of our non-randomized study in which we assessed prospectively obtained data from a perioperative assessment of anastomosis perfusion by fluorescent angiography using indocyanine green during robotic rectal cancer surgery. method: thirty patients with rectal cancer, who underwent a robotic resection with primary anastomosis, were consecutively included in the study between april , and june , . the study included patients facing a least invasive surgery with a guaranteed payment by a health insurance company. during the surgery, we monitored and assessed the quality of the perfusion of the resection line of the sigmoid colon and subsequent anastomosis by means of fluorescent angiography using indocyanine green in the spectrum of near infrared light. the data were obtained prospectively and subsequently analyzed. results: between april , and june , , we consecutively included rectal cancer patients in the project: men and women. monitoring of the perfusion of the resection line and anastomosis was successful in all cases and perfusion quality was satisfactory across the sample. perfusion insufficiency requiring a change in the resection line level or anastomosis adjustments was not detected with any patient. in two cases ( . %) of tme, we gave up the planned protective ileostomy owing to quality perfusion of the anastomosis. one patient ( . %) suffered from defective anastomosis healing without clinical symptomatology (type a). we found no technical complications related to fluorescent angiography or undesirable effects due to the application of indocyanine green. conclusion: even though we did not register insufficient perfusion in our sample and hence we did not have to change the resection line level or adjust the anastomosis, we may state that fluorescent angiography performed by an experienced colorectal surgeon may potentially reduce the frequency of complications linked to defective anastomosis healing.supported by mo aims: the aim of our study is to demonstrate whether robotic surgery has any influence on the reduction of complications in the aged population undergoing rectal cancer. methods: we performed a retrospective analysis of a prospective database of patients who underwent robotic surgery for rectal cancer. we divided our population in groups: under year old, between and year old and above year old. we recorded complications in each group intra and post procedure. qualitative variables were expressed in terms of absolute frequencies and percentages and mean values and standard deviation were used to express quantitative variables. the analysis of data was applying fisher's exact test or chi-squared test for qualitative variables and variance analysis or student'-t test for quantitative variables. statistically significant values of p \ . underwent multivariate logistic regression analysis. results: the present study included patients ( males).seventy seven patients were under year old, patients were between and year old and patients were above year old. the analysis showed conversion rates of . %, . %, . %, and complication rate of . %, . %, and . % in each group. univariate analysis showed no differences between the three groups. nevertheless, there were statistical differences from bmi, asa and neoadjuvant therapy. in multivariant analysis only neoadjuvant therapy was significant. conclusions: robotic approach do not decrease complications in elderly population. introduction: it has been described the advantages of total transanal mesorectal excision (tatme), with better visualization and access to the lower rectum. we use this access whith the gel point path device, to repair a rectovaginal fistula with stenosis of low rectal anastomosis in two patients, that would be difficult by conventional abdominal approach method: we show our surgical technique for repair a rectovaginal fistula with stenosis of low rectal anastomosis in two female patients operated due to rectal neoplasia. one of the patients underwent prior chemo-radiotheratpy. rectoscopy and image test was performed at the patients prior the intervention. no recurrence signs are recorded at mri.we describe the operation technique: a new anterior rectal resection was performed with a combined transanal (gel point path) and abdominal minimally invasive approach. redo anastomosis whith eea stappler was performed, vaginal repair and epiploplasty. the intervention was especially laborious due to the fibrous tissue. pathology: fistulous path without tumor infiltration in the two patients. at two months, a opaque enema show permeability and absence of leaks in the two patients. the ileostomy was closed at three months. discusion: we believe that transanal access through the gel point path can be a good option for rectovaginal fistula and stenosis of low rectal anastomosis, allowing a better visualization and acces, and making more easy a very difficult intervention. introduction: tamis or transanal minimally invasive surgery for polyp resection has increased fame for several situations in which adenomas with or without dysplasia cannot be removed with conventional colonoscopy. in this video we show the step by step technique performed with the da vinci xi system. material and methods: in this video we show the setting and the location of the patient-side cart and the arms to perform the resection of polyps in different patients and how to develope the procedure. results: after placing the patient-side cart the arms are connected to ports and the camera, double fenestrated grasper and scissors are connected to the arms through a transanal gel-port device. a line is described around the polyp with monopolar energy to determine the place of the dissection. the scissor is exchanged by a robotic harmonic wrist instrument and the complete dissection is performed. the wound is closed using a robotic needle holder and a suture. results: transanal robotic surgery could be safely performed after a standardized technique is stablished. aims: robotic rectal cancer surgery has demonstrated to obtain at least the same results than laparoscopic surgery. however, robotic surgery is associated with high rates of costs, specially when conversion to opened surgery occurs. the goal of this study is to create a predictor nomogram of conversions for robotic rectal cancer surgery. methods: we performed a retrospective analysis of a prospective database of patients who underwent robotic surgery for rectal cancer from october to november . we performed a bivariant analysis and detected the variables which were related with the conversion: body mass index (bmi) and the t. we divided the patients of the population in two groups depends on obesity (bmi of kg/m ) and on t (t - /t - ). we registered conversions in each group calculating the pretest risk. we performed likelihood index (lr ?/-) for under and above kg/m of bmi, adding in a second step the lr of t; obtaining the prediction index for four groups by using a standardize nomogram. results: the present study included patients ( males). were under bmi of kg/m and above. regarding t, were with a tumor of t - and with t - . the analysis showed a conversion rate of the statistical sample of %. univariant analysis showed significative differences in the bmi (p = . ) and t (p = . ). a nomogram was performed; as regards the bmi, the positive likelihood index in the group of bmi [ a prediction index of conversion of % (lr ? , ) and in bmi \ the prediction index of conversion is % (lr- , ). adding the t group data, for bmi [ and t - the conversion prediction rate is . % (lr- , ); for bmi [ and t - the conversion prediction is % (lr ? , ). bmi \ and t - the conversion prediction is % (lr- , ); imc \ and t - , the conversion prediction is %. conclusion: a standardize nomogram with the variable bmi and t facilitates the selection of patients for robotic surgery in rectal cancer avoiding conversion to open surgery. background: d-laparoscopy is proven to improve performance in dry laboratory settings, especially for novice surgeons due to better depth perception. however, the benefits for experienced laparoscopic surgeons are still discussed. aim: the aim of this study is to compare the results of right hemicolectomy (rc) using a conventional ( d hd) laparoscopic system with rc performed using a d laparoscopic system in terms of duration, complications and results. material and methods: from all laparoscopic right hemicolectomies performed in our clinic we selected all procedures performed by the same team of consultant surgeons using the same technique and divided them in groups. the study group comprised of all patients operated using our d einstein vision . system; all other patients which were operated using our standard wolf hd laparoscopy system comprised the control group. all patients were retrospectively analyzed in terms of patients characteristic, or time, duration of operation, intra-and postoperative complications, length of hospitalization, pain score, necessity of analgesics and number of lymph nodes retrived. risk factors for complications (bmi, smoker, diabetes, copd, bph) were also registered. results: there were patients included in the study group, while the control group comprised of patients. mean operation time in the study group was . min in the study group, while mean or time was . min. mean operation time in the control group was . min, while mean or time . min. one reintervention was noted in the control group and two in the the study group; no conversion to open surgery was noted. there were no significant differences regarding patient characteristics, pain score, wound complications, hernia rate, length of hospitalization or number of lymph nodes removed. conclusions: there were no significant differences regarding the outcome of rc using d laparoscopy; total or time was significantly higher in the study group due to the time needed to set up the d-laparoscopy unit. this is biased by the fact that the d system needs to be set up manually while the conventional hd system is integrated in the or. also, there was no significant difference in complication rate. background/purpose: robotic approach can be a treatment option for patients with pelvic recurrence after primary resection for rectal cancer. however, data regarding patient selection, complication rates, and oncologic outcomes are rarely reported. we aimed to present initial experience and to evaluate feasibility, safety, and oncologic outcomes of robotic salvage surgery for recurrent rectal cancer. methods: ten patients who underwent robotic salvage surgery for local recurrence at the anastomotic site, lateral pelvic side-wall, or lateral pelvic lymph nodes (lpns) were retrospectively evaluated from a prospectively maintained database. results: two patients underwent pelvic mass excision with en bloc resection of anastomosis and redo-anastomosis, and eight patients underwent lateral pelvic lymph node dissection (lpnd) for lpn metastasis; one of these eight patient underwent additional en bloc resection of anastomosis. all patients achieved r resection. the median operation time was min and the median estimated blood loss was ml. there were no conversions. as for intraoperative complications, one patient experienced ureter injury during lpnd because the metastatic lpn was closely abutting to the ureter. the median hospital stay was days. in six patients who underwent lpnd, the median number of harvested lymph nodes was (range - ) and the median number of metastatic lymph nodes was (range - ). with median follow-up months, one patient developed lung and pelvic recurrence at months after salvage operation and seven patients remained in disease-free state at the last follow-up. conclusion: initial experience of robotic salvage surgery for pelvic recurrence in rectal cancer indicated that it is safe and feasible. therefore, the robotic approach can be considered as a treatment option for the treatment of local recurrence in selected patients. introduction: there is uncertainty regarding the effects of simulated patient death. several reports showed increased cognitive load and poorer learning outcomes, and others increased performance without causing stress to learners. we have not found any report studying the impact of animal death in the simulation lab. methods: this was an observational cohort study to assess the emotional and cognitive load of surgeons who experienced animal death in the simulation lab. seventy-four faculty and residents from different surgical specialties training minimally invasive surgery participated in the study. one cohort consisted of surgeons whose animal died during surgery, and the other by those whose animal survived. emotions were assessed using the scale for mood assessment and cognitive load with nasa task load index. results: twenty percent of participants experienced mortality while training anti-reflux surgery ( cases) and other procedures ( cases). causes of death included intraoperative pneumothorax (n = ), hemorrhage (n = ), and cardiac dysrhythmias (n = ). participants exposed to animal death had higher levels of sadness and anxiety, and lower levels of happiness (p [ . ). cognitive load was slightly higher in the exposed cohort (p [ . ). conclusions: these findings suggest that mortality in the animal lab do not have a significant effect on cognitive workload and emotions of surgeons training complex laparoscopic procedures. introduction: the visuospatial profiles of expert laparoscopic surgeons remain unaccounted in the current literature for as the influence of visuospatial ability on laparoscopic learning has mainly been investigated in medical students or novice surgeons and using simulators as means of performance measurement. such knowledge is critical, as without understanding how clinical experience may impact visuospatial processes in surgeons, we hinder our efforts to utilize the available knowledge to support surgical education for the future. this study is aiming to explore the development and influence of visuospatial processes on intraoperative laparoscopic learning. method: the study reports the interim baseline results from the ongoing longitudinal study throughout a -year period of training on laparoscopic surgery. data from surgeons including residents undergoing training were captured and compared to specialists who are working in departments of general and visceral surgery at two large hospitals. the mean experience of the surgical residents was years. the mean laparoscopic experience among the senior surgeons is years, with each surgeon performing an average of laparoscopic procedures per week. visuospatial ability was tested using mental rotation test (mrt), guay visualization of views tests (gvvt), spatial perspective taking and spatial orientation test (ptost) and pictorial surface orientation (picsor). spearman correlation coefficient was used in this study with a p-value of significance at \ . . results: senior surgeons have an overall good visuospatial profile, in the sense that they performed close to optimum on all measurement scales. the spearman rho revealed a significant correlation between scores on gvvt and picsor (r = . , p = . ) and between ptost and picsor (r = -. , p = . ). a significant correlation between years of laparoscopic experience and ptost score was also observed (r = . , p = . ). when comparing residents and senior surgeons, no significant difference on the mrt was observed (m = . , sd = . ), nor between baseline scores of senior surgeons and resident surgeons on all tests. conclusion: the results of this study carry important clinical and theoretical implications, as the results hint towards the idea that intraoperative laparoscopic experience lends little to no influence over the development of visuospatial ability. learning models and laparoscopic technical skills, how to adapt each case to improve objectives: according to da. kolb learning is the result of how people perceive and then process what they have perceived. the aim of this study is to identify the personal characteristics of learning in of the participants in a course of laparoscopic technical skills according to the styles described by kolb. methods: between june and november , participants performed a h course distributed over five consecutive days performing laparoscopic manual intestinal anastomosis in endotrainer. they all filled in kolb's learning style test adapted to spanish. the anastomoses were performed in 'ex-vivo' swine intestines. in each anastomosis we evaluated the quality at the end and execution time. the test and quality variables were analyzed through statistical studies. results: in our study, % of the participants were women and % wew men. %were staff surgeons and % were resident. the median age among residents was years and among the staff years. the most frequent learning model in the sample studied was converging ( %). the predominant model among women was assimilating ( %), which, however, represented only % in men. in men, converging model was predominant ( %). among the staff, the most frequent model was diverging ( %). adaptation style prevailed among residents ( %), being rare among the staff ( %). the mean time of the anastomosis was min for both the adapter model and the assimilator, min, for the convergent and divergent models. the quality of the anastomosis performed by each participant was % for the adapter model, % for the assimilator model, % for the convergent model and % for the divergent model. the predominant style in our study was convergent. among women, the most frequent model was assimilator wheras in men it was the least frequent. in the residents, the most frequent model was adapter however, it was very rare in adjuncts. among residents we do not find divergent styles. the highest quality of the anastomosis was achieved by those who worked with an assimilating style. knowing previously the training style we can individualize the teaching methodology in order to improve competences. aims: assess whether laparoscopic appendicectomies (la) are a superior option to open appendicectomies (oa). specifically, comparing the time taken, complication rates and whether it is more appropriate to perform an la overnight, as opposed to oa. finally, to find out how a range of outcomes differs between different grades of surgeon. methods: an information request was sent to the clinical coding department to derive patient identification numbers for all appendicectomies over a ten-month period ( total surgeries). these numbers were then inputted into the hospital information system where the electronic operation note is present, and specific outcomes were derived and analysed. results: % of operations were oa and % were la. mean la times for consultants, sas and spr were . , and min respectively and oa , and min respectively. their respective conversion rates were %, % and %. oa had a complication rate of . %, la was . %. conclusion: oa are performed more than la. spr doctors had the slowest completion times for la but the lowest conversion rates. sas doctors had the fastest completion times for la and oa but higher conversion. la takes longer than oa but has lower complication rates; key factors when performing at night. key statement: laparoscopic appendicectomies require more surgeon-hours and have the potential to be converted to open, however the rates of complications and serious complications are significantly lower. background: paper based resources have been the standard sources for information for centuries. however, more and more people (patients and staff alike) are looking online for information. while the internet often provides excellent resources, there is often conflicting and confusing material of doubtful veracity. trainee staff and patients/carers should be able to access reliable resources whenever and wherever they are. the aim of this project was to create a high-quality resource fulfilling these needs. aim: we present a video demonstrating our integrated colorectal education website ( http://www.colorectaleducation.com/). our approach: high quality health care provision requires highly trained staff as well as wellinformed patients. information resources for these two groups are usually accessible from different repositories. our integrated website provides a common platform for all those involved in colorectal surgery, to use, learn and reflect on. users are directed to separate sections for patients and colorectal professionals. multiple disclaimers prevent patients accidentally stumbling across clinical/ operative information, whilst providing access to those who wish to do so. trainees struggle with balancing their educational needs with their service commitments. this website gives them the opportunity to view detailed operative training videos on the go. many of videos are chapter based allowing them to stop and re-start with ease. modules are also available for nurses providing them access to relevant educational material. the modular design of the website allows us to build upon it with more topics planned to be added over the next eighteen months. the resource also has detailed chapterised videos for patients due to undergo various colorectal procedures. all have been approved by a multi-professional panel including patients and are designed to provide information, offer support and to allay any anxiety. videos with the care pathway and previous patients' experiences are accessible on demand. conclusion: on demand information has now become the norm with the use of smart phones/ tablets. this website provides patients, surgical trainees and other healthcare professionals access to information and education in a clear and reliable format anywhere in the world. colorectal education, on demand and just a click away! objective: in the last decade the growing interest in robotic surgery is evident as shown by several published articles. the aim of the present study is to evaluate the main outcome of a single center experience and to describe the organizational system we have progressively established in our center in order to improve the development of robotic program in all surgical area. materials and methods: we report a case series of patients who underwent robot-assisted surgery at sanchinarro university hospital since the beginning of the program (october ) until november main patient demographic characteristics, type of surgery, peri and postoperative data and follow-up were evaluated. results: a total of robotic procedures were performed for a total of patients. the prevalence of malignant disease was %. a total of pancreatic surgery were performes; liver resections (mean operating time: min); gastrectomy (mean operating time min); esophagectomy (mean operating time: min); colorectal resections ( rectal resections, sigmoidectomy hemicolectomies right, left colectomy) (mean operating time: min); nissen procedures (mean operating time: min), esofagheous myomectomy for achalasia (operating time: min); adrenalectomy (mean operating time: min); three biliary surgery for benign desease, splenectomy. eight partial resection of the duodenum, one yeyunal resection, one mesenteric cyst resection and retroperitoneal tumor have been performed. conversion rate was %, total morbidity have been %. there has been no peri and postoperative mortality up to days after surgery. the average hospital stay and intensive care were respectively days (range - days) and . days (range - days). conclusions: the organizational model defined in our center is facilitating the constant and progressive development of the robotic program. a broad and flexible availability of the robotic system, a progressive increase of young surgeons joining this technology as well as the institutional and departmental economical effort are the points with which the robotic system may increase its development in a surgical department. aims: endoscopic surgery has been widespread in the field of general surgery. however, in japan, there is no standard program for endoscopic surgery training, and its competency has not been considered for the acquisition of board certified surgeon. the purpose of this survey was to investigate the current situation of endoscopic surgery training and autonomy of young surgeons for endoscopic surgery in japan. methods: the survey was planned to target general surgery members of the japan society for endoscopic surgery (jses) who was post graduate year or less. after approval by the ethics committee of jses, the request for the participating in survey was mailed to object members. questionnaire responses were available in print or online media. the contents of the questionnaire consisted of items, about the conditions of endoscopic surgical training, experienced case number, and the self-assessment of autonomy from to point by zwisch scale in specific procedures of endoscopic surgery. results: the total response rate was . % ( / ). sixty five answers were excluded due to inadequate response and answers were analyzed. of the questionnaire respondents, % were male and % were female. the ratio of board certified surgeon was %. although % of the teaching hospitals had simulators for basic training of endoscopic surgery and % of the respondents practiced basic skill of endoscopic surgery, only % teaching hospitals had specific training programs for endoscopic surgery. the surgeons who operated cases of laparoscopic appendectomy and inguinal hernia repair and cases of laparoscopic cholecystectomy, right hemicolectomy and sigmoidectomy, felt confident to perform each procedure independently. regarding with laparoscopic rectal resection and gastrectomy, even though the surgeons who had cases of experience, they didn't had confidence to perform those procedures independently. conclusions: this study is the first national survey to investigate the status of endoscopic surgery training in japan and the autonomy of young surgeons for endoscopic surgery. in order to develop a training system for not only basic skills but also advanced procedures of endoscopic surgery, cooperation of each teaching hospital, academic surgical society, medical specialty board is necessary. currently there is a debate about what is the most optimal work schedule for residents of general surgery, it is important to respect the free time of residents to avoid burnout, however it is also important have enough exposition to clinical cases that allow a satisfactory development in the clinical practice. this becomes even more important when we talk about the learning of surgical skills. this is where the laparoscopic simulation industry opens a large area of opportunity, for a reasonable price it is possible to practice basic laparoscopic skills without compromising patient safety. this is a pilot study that was carried out during the period from january to june , in a public hospital in monterrey, nl, mexico, the composition between the execution of the standardized exercises of the fls (fundamental laparoscopic surgery) in an endoscopic simulator was performed to residents of general surgery (from first to fifth year) hrs before being on call vs these same residents post call. a series of questions was asked to each resident in each measurement, so in this way they answered the same questions twice, then a comparison of the results of both questionnaires was made. the results of the exercises were assessed and rated by the same person using the criteria established in the fls for the scores of each exercise and for the final grade. an average age of years was obtained, measurements were taken of residents of which are male and female. on average, the residents before be on call performed the exercises with h of having slept while the post call performed the exercises with . h of having slept, the residents before be on call had on average . h without sleep while the post call had h without sleep. the average number of hours worked per week is h, measured by the time in and out of the hospital. in this study, conclusive results were obtained regarding the null relationship of sleep deprivation with the performance of laparoscopic skills in surgical residents. aim: 'precision cutting' is one of skills tasks of the fundamentals of laparoscopic surgery (fls) program, which is cutting a circle on a piece of gauze under laparoscope and assessed by completing time (maximum time limit: s). there is no definition of quality of the final product. the aim of this study is to develop an assessment tool of laparoscopic precision cutting and test its reliability. method: an assessment tool of laparoscopic precision cutting was developed with four items based on completion, degree of deformation, degree of being pulled, and overall appearance of the final product of laparoscopic precision cutting by experts' meetings. the scale of each item was points likert scale. a descriptive sheet with a legend and a text description for each scale (fig) was attached for assessors' reference. for our high school entry medical students, they gained hands-on experiences of laparoscopic skills first time by attending a -hour course at minimally invasive surgery training center, national taiwan university hospital (ntuh). we invited students to participate this study after this training. we collected participants' final products of ' precision cutting' station and assessed them by using this assessment tool. this study was proved by institutional review board, ntuh (irb no: rinb). results: students were enrolled between february to june . two non-medical assessors and a senior surgeon were invited to assess the products. the mean score and cronbach' s alpha value of each item were as followed: completion . ± . , . ; degree of deformation . ± . , . ; degree of being pulled . ± . , . ; and overall appearance . ± . , . . conclusions: in summary, we successfully developed an assessment tool for laparoscopic 'precision cutting' and showed its reliability. the tool could provide qualitative descriptions for objective feedbacks. validating this tool in a large scale is undergoing. purpose: to evaluate whether the participants who experienced this scenario could recall an interventional scenario for testing trainees' situational awareness and intra-operative decision making when they participated this training again. methods: we designed an iodm training course for junior surgical trainees and nurses by using live pigs since sep . in the first simulation, we created an interventional scenario and then provided an educational session. a researcher disconnected the ekg monitor on purpose for creating a scenario that the pig would lose vital signs when the team nearly finished a diagnostic laparoscopy. if the team did not aware the situation after . min, a researcher would remind the team (fig). we used a new developed assessment tool of iodm and an assessment tool for nontechnical skills for surgeons (notss) for self-evaluations and objective assessments. we also discussed with them about their reactions while encountering this interventional scenario. results: between sep to june , teams participated this training and experienced this interventional scenario. fourteen nd year surgical trainees have experienced it before. only one participant ( %) recalled it and made a quick decision while encountering this interventional scenario again. the results of iodm assessment and notss did not show statistical difference comparing their self-assessments in the first and second year. based on the analysis of the discussions, most of them remembered this this interventional scenario and reminded themselves to react it properly before the simulation. however, when they were the primary surgeon of diagnostic laparoscopy, they focused on performing this procedure and tutoring their junior trainee. they had no capacity in their brain to notice the change of vital signs. in addition, although they increased their situation awareness in clinical settings after the st time iodm training, they did not show this ability in the simulation. conclusions: recalling of an interventional scenario for testing situational awareness of surgical trainees was very poor ( / , %) among the nd year surgical trainees. qualitative analysis of discussions showed their brain capacities were occupied by performing new procedures and tutoring others. how to enhance trainees' situational awareness should be addressed. aims: a well-designed learning curve is essential to measure the progress of surgical abilities. learning curves are very important to test the skills of trainees. however, there are still no welldefined criteria for developing good learning curves. as a result, many authors use subjective evaluation criteria. the purpose of this review is to analyse this field of surgical education and to identify the key criteria for good learning curves. methods: learning curves were investigated in the field of laparoscopic and robotic minimally invasive surgery. surgery of appendectomy, cholecystectomy, cholectomy, inguinal hernia repair and gastrectomy were considered. the type of surgery, the year of publication, the design of the study, the surgeon's experience (resident, young or senior), the surgical technique, the number of patients involved in the study and the suggested learning curve by the different studies were taken into account. in the selection of articles, more importance was given to those based on the activity of young surgeons or residents. results: the literature analysis showed conflicting results. the different learning curves for the same surgery may be due to the different evaluation criteria considered. only a few studies investigate the learning curves of young surgeons and residents. conclusions: the data available in the literature on learning curves are contradictory. several factors need to be evaluated in order to create more accurate learning curves. we suggest the introduction of checklists with a score for each parameter to be examined, in order to develop more objective and standardized learning curves. aim: the uk training programme for transanal total mesorectal excision (tatme) has completed its first round of training. the study aim was to design a reporting platform that provided trainees with video-assisted feedback in a clear, concise and useful manner to support their training. methods: an established method of video analysis called observational clinical human reliability analysis (ochra) was used to assess the surgical performance of the trainees during their clinical tatme cases. a reporting form for the ochra results was designed identifying areas of difficulties in each procedure and providing error reduction mechanisms. this was piloted during the national training programme for tatme in the uk. results: the ochra reporting form underwent three modifications before the content and format was agreed upon. the final version is divided into three sections: a. case details, b. ochra findings, and c. suggested error-reducing mechanisms. for part b the tatme procedure was divided into four phases of the operation: . pursestring, . rectotomy, . tme dissection, and . connected phase when the abdominal and transanal teams work together synchronously. for each phase, ochra findings described the most frequently occurring technical inaccuracies/errors, number of consequential errors/adverse events and the most frequent and serious consequences encountered. suggested error-reducing mechanisms in part c were developed and established by an expert workshop and individual interviews with international surgeons experienced in tatme.trainee and mentor feedback stated that the reporting form had a clear format, easy to follow and understand. the error-reducing mechanisms were particularly useful and allowed the trainee to focus on improving specific technical aspects in their subsequent cases. conclusion: video analysis using ochra can provide a wealth of information on surgical performance, especially for trainees at the start of their learning curve. as an exploratory study, validation of the reporting platform is required; however, its potential to offer detailed, individualised feedback to enhance training is promising. laparoscopic pelvic surgery training program-using a new concept d-printed versatile pelvi-trainer r.c. elisei , f. graur , c. popa , e. mois , l. furcea , n. al hajjar general surgery, bistrita emergency county hospital, bistrita, romania; general surgery, regional institute of gastroenterology and hepathology ,,prof. o. fodor,,, cluj-napoca, romania pelvic laparoscopic surgery (rectal, urological, or gynecological laparoscopic surgery) is an advanced surgery which require advanced skills, not easy to acquire. there are a lot of training programs for advanced laparoscopic skills but many of them are not affordable for most of surgery residents in eastern europe, where the training programs are far behind from those in western europe. because of that those training programs need to be improved and optimized. in the european union we want equal and high skilled surgeons. this is why we designed a new concept of pelvi-trainer, a versatile one in order to offer the residents the possibility to achieve advanced laparoscopic skills like perfect coordination, precise movements, ability to cut and suture after a well defined route, all of them in the pelvis tight space. we d-printed this pelvitrainer which has multiple characteristics: cheap and easy to produce, easy to be used, versatile because offer the possibility to achieve the skills named above, and many others, but also to train on real ex vivo animal rectum (suine model). we also believe that with a proper training a medical student and a young surgery resident are able to achieve the same skills like experienced surgery residents or specialists. in order to demonstrate that we need a study to compare the time to perform or more exercises in this new concept pelvi-trainer by the medical students, young and experienced residents and surgery specialists. what we want to achieve with this training program project is to have more and more skilled surgeons in advanced laparoscopy and an equal laparoscopic surgery training all over the country, close to the level of training in the western europe. also we want this training program to make a standardization of the pelvic laparoscopic surgery training first in our country and then in other countries if possible. aims: the objective of this systematic review is to provide an evidence-based overview of the different components of laparoscopic training curricula, emphasizing the value of objective forcebased assessment and how this in implemented in modern laparoscopic training. methods: bibliographic databases of pubmed and embase were searched till april to identify studies reporting on evidence-based laparoscopic skills training. abstracts of retrieved studies were reviewed by two authors independently and those meeting the inclusion criteria were selected for full-text review. results: the search yielded a total of individual records. a total of articles were included. the articles were divided into nine different categories, which include 'metrics', 'benchmark criteria', 'measurement systems', 'timetable', 'training modalities', 'camera settings', 'training tasks', 'serious gaming', and 'competition'. a descriptive analysis of the data is provided. motion analysis parameters, such and path length and time are frequently validated and used for assessment. the results of validation studies on tissue manipulation parameters, such as maximum force and mean force show proved their discriminating power between different levels of proficiency. however, implementation of these metrics remain restrained. conclusions: numerous studies on laparoscopic skills training have been conducted over the years. nevertheless, no consensus is reached towards the use of objective assessment tools. although the value of validated metrics is described well, implementation of objective metrics is limited. we recommend to consider objective force-and motion metrics for feedback and assessment during laparoscopic skills training. surgery, regional institute of gastroenterology and hepatology, cluj-napoca, romania; anesthesiology, university of agricultural sciences and veterinary medicine, cluj-napoca, romania; radiology, regional institute of gastroenterology and hepatology, cluj-napoca, romania aims: the aim of the study was to create a new easy learning method of swine liver anatomy for residents in training. based on human liver surgical anatomy we put 'face to face' the similar structures and also the differences using ex vivo porcine models and ct reconstructions from live pigs. methods: having in mind the human liver anatomy, in the first stage we used data obtained from dissection of twelve porcine liver models to create an anatomical pattern, which summarized the most important surgical information. in the second stage, anatomical data obtained from ct scans of twelve living anesthetized pigs were analyzed. the ct reconstructions and volumetry data were added to the gross anatomy pattern to create a more complex learning module. results: the residents established the most frequent description of swine liver anatomy by putting together the information from ex vivo model dissection. the liver parenchyma is divided into four main anatomic lobes: left lateral, left medial, right medial and right lateral. all those lobes are connected only in the posterior part, which allows a very good separation between them by deep fissures. just as in humans, we found eight distinct segments with independent vascularization and biliary drainage. portal vein has a specific 's' shape; in most cases hepatic artery was found like a trifurcation and extrahepatic biliary tree has a very thin wall. in the right hemi-liver, the inferior vena cava passes through the liver parenchyma. most frequent, we found five hepatic veins which are running completely intraparenchymal. the imagistic data offered a very useful d reconstruction with anatomical positions of the vascular-biliary tree and liver segmentation and gave us the possibility to create practical scenarios for resections. perhaps the most important information was to discover and see the section plan and to calculate the volume of the remaining liver after resection. conclusions: the anatomical-imagistic pattern based on \ i[ex vivo \/i [ model disections combined with imagistic data offers a unique mindset before intervention. the concept 'human \ i[vs \/i [ swine' to create an easy method of learning for residents in training can be applied to swine liver anatomy. the learning of surgery is traditionally based on the behaviourist model . goals are set, standards of care fixed, with regular assessments of the level achieved. the teacher exercises control over the student, imposing rules and models, supported by 'reinforcing' actions (reward or punishment). the theory of skinner's program of education, from , is reflected in surgical learning. it foresees a gradual progression by level of difficulty, following a transmission-imitation model . these theories seem currently outdated to face the new challenges of medicine and surgery and to keep up with technological developments. bruner, one of the theorists of the constructivist model, proposed in a method of collaborative learning between those who teach and those who learn. the goal of the method was to improve strategic problem solving. the comparison between various perspectives (between teacher and student), allowed the learner to better absorb knowledge and improve critical thinking. in kapur published on the theory of 'productive failure'. this model makes the error of a single person useful for all his colleagues, privileges the practice of theoretical knowledge, contextualised learning as opposed to abstract learning, and 'guided' practice compared to a 'guided' theory. bruner and kapur's systems favour creativity, critical analysis of a problems origin, and the practical use of knowledge. they represent a hypothesis of learning, based on constructive discussion and a continuos 'give-take' feedback system. in order to put these new models into practice in the clinical context, one may hypothesise and propose the adoption of a formal discussion of clinical cases that are complicated or difficult. thereby making the theoretical lessons more collaborative, intuitive and inclusive. in the surgical field, one could adapt such a concept to surgery simulation, virtual reality and anatomical models. aim: large hiatal hernias have a surgical indication when the patients suffering disabling symptoms such as anaemia, dyspnea, chest pain, gastric reflux. several studies showed that in the case of large hernias the placement of a prosthesis was safe and could protect against recurrence. mini-invasive surgery is the preferred approach for hiatal hernia repair and anti-reflux procedure and the toupet fundoplication has been shown to be the best surgical technique for the hiatal hernias repair.the laparoscopic approach is currently the surgical gold standard but is burdened by technical difficulties especially in the case of large hiatal hernias. the robotic system is designed to overcome some technical difficulties of laparoscopy and the studies available in literature report the safety and effectiveness of the robotic approach in complex hiatal hernias repair. methods: we present the case of a grade iv hiatal hernia treated with a robotic approach in a years old woman (bmi: kg/m ). the medical history consisted of a road accident with a probable mechanism of deceleration, three years before. the patient had been suffering from dyspnea for three years. due to the recent discovery of an anaemia, the patient was subjected to an endoscopic examination with the identification of a voluminous grade iv hiatal hernia. a subsequent computed tomography (ct) scan showed also the partial herniation of the transverse colon. results: the patient underwent to surgery by using the da vinci robot system siÒ (intuitive surgical, sunnyvale, usa) with a single docking approach. the surgery consisted in the liberation of the hernial sac, the placement of a goretex prosthesis and the packaging of a toupet fundoplicatio. the surgery was performed without complication. conclusions: the robotic approach in the hiatal hernia surgery seems to be a valid alternative to laparoscopy, especially in complex cases. the surgical ability in robotic surgery is of paramount importance. general thoracic surgery, kawasaki municipal hospital, tokyo, japan aim: video-assisted thoracoscopic surgery (vats) with carbon dioxide (co ) for mediastinal surgery is known to improve the visualization of medaistinal space. we report our experiences with two cases that underwent vats thymectomy using co insufflation under the one-lung ventilation general anesthesia by double lumen tube. methods: the instruments that were used for vats thymectomy were only the -mm -degree rigid thoracoscope, maryland jaw energy device, cotton made-dissectors, and straight endoscopic grasping forceps. they were used through sealed ports designed for laparoscopic surgery. lowpressure co insufflation set at mmhg were used for compression of surround tissue of mediastinal tumor during the releasing procedure. results: the patients were an -year-old male and a -year-old female. thoracoscope with the mmhg co insufflation provides excellent visualization of the medaistinal space and operation could be done smoothly without any hemodynamic compromise. their pathological diagnoses were thymic cancer and thymoma, type b . the operative times were min and min. the postoperative courses were uneventful and the patients were discharged on day th and rd . conclusion: we have just begun to routinely use co insufflation for mediastinal tomorectomy and present our early experiences of successful vats thymectomy by utilizing co \ su \/su insufflation. aims: this retrospective study aims to evaluate the feasibility of single-incision thoracoscopic surgery (sits) for primary spontaneous pneumothorax (psp), using a novel multichannel port (x gateÒ). methods: between october and november , ten patients who underwent sits using x gateÒ. nine patients were male and was female, with mean age of . ± . years old. a . cm incision is placed in the middle axillary line on the th or th intercostal space, depending on the lesions. postoperative outcomes of these patients were compared with those of patients with psp who underwent conventional three-port video-assisted thoracic surgery (vats). results: there were no conversions from sits to vats. mean operative time of sits group was significantly shorter than that of three-port vats group ( . ± . min vs . ± . min, p = . ). mean number of staplers used in surgery was . ( ) ( ) ( ) ( ) in sits group and ( ) ( ) ( ) ( ) ( ) in vats group (p = . ). mean duration of postoperative drainage was also shorter in sits group ( . ± days vs . ± . days, p = . ). no recurrence and wound infection were observed in sits group. conclusion: sits using x gateÒ is feasible when performed for selected patients with psp. x gateÒ provides good visualization of intrapleural space and esthetic outcomes, as well as a superb maneuverability by decreasing mutual interference of surgical instruments. although conventional three-port vats for psp is well established, sits using x gateÒ can be a permissible alternative. further examinations are required to evaluate efficacy of sits using x gateÒ. aims: haemorrhage remains a leading cause of potentially preventable death in trauma. in particular non-compressible torso haemorrhage is approximated to cause - % of mortality in civilian trauma patients with otherwise survivable injuries and % in war setting. we performed a literature review to assess the potential for using endovascular stenting in traumatic venous injuries and explore the evidence of their efficacy and safety with different venous injury patterns. methods: systematic online search of pubmed performed using key words'endovascular stent', 'venous injury', trauma, penetrating, blunt, abdominal and pelvic. inclusion criteria included all studies that explored the use of endovascular stents following traumatic abdominopelvic venous injuries. english language studies were used. results were presented according to prisma guidelines. results: of the studies generated by the search,there were only four case reports in the literature documenting the use of endovascular stents in traumatic venous injuries dating back to and most recently . the four cases included three retrohepatic ivc injuries, two secondary to blunt trauma and one penetrating; whilst the final case a blunt injury at the ilio-caval bifurcation. all four cases reported successful deployment of stents via the femoral or internal jugular veins, with subsequent resolution of haemorrhage. length of time taken for stent insertion ranged from to min. three of four patients made full recoveries and discharged from hospital, with one patient subsequently dying of a brain injury independent of the successful venous stent insertion. no complications were reported at up to months follow up in remaining cases including stent leak, stenosis or migration. conclusion: endovascular venous stents have been used successfully in managing complex abdominopelvic traumatic venous injuries. in particular retrohepatic venous injuries refractory to hepatic packing and vessel embolization, which are not amenable to direct surgical repair due to anatomical location. however before endovascular stenting can be added to the arsenal of interventional radiologists for abdomino-pelvic trauma, further development of stents custom made for venous injuries as well as prospective studies examining their long term safety and outcomes is needed. tracheal papilloma is a rare neoplasm growing from the tracheal or bronchial epithelium and has no specific clinical presentations. this is a -year-old female who complained of progressive dyspnea for about months. physical examination was unremarkable and the there was no abnormal finding by the chest plain film. chest computed tomography was arranged and revealed a mass lesion located at the tracheal lumen with more than % luminal obstruction. we used fiberoptic bronchoscopy to evaluate the airway and found a mass lesion with pedicle originated from the posterior tracheal wall. cryotherapy was considered for the tumor mass removing to establish a patent airway. the pathologic report revealed tracheal papillomatosis without any malignant component. dyspnea was immediately improved and the patient chose closely observation after the bronchoscopic cryotherapy. aims: recent advances in laparoscopic surgery, both in techniques and instrumentation material, have led to the emergence of innovative technological fields, among which robotic surgery stands out.one of the handicaps of this surgery is its high cost as well as the long learning curve. in this stage a new tool arises, the flexdex semi robotic arm, which combines the precision and the range of movements of robotic surgery with the greater availability, simplicity of use and learning of conventional laparoscopic surgery.the objective of this study is to evaluate the efficacy and safety of the flexdex device in different laparoscopic procedures. methods: flexdex's is a three-axis gimbal technological device integrated in a conventional laparoscopic instrument that translates the surgeon's hand, wrist, and arm movements from outside the patient into corresponding movements of an end-effector inside the patient's body.the greater accessibility provided by the flexdex allows the surgeon to perform sutures in areas of difficult access where mobility with conventional laparoscopic instruments is not optimal. the comfort of the surgeon remains fundamental in any type of surgery, even more when we are in anatomical locations with complex access, especially for the realization of sutures. here is where surgical innovation instruments such as flexdex provides ergonomic comfort for the surgeon and improves the patient's safety, especially in high-risk situations, such as when performing anastomosis. results: we present a prospective series of laparoscopic procedures carried out by the same surgical team being the initial experience in our environment in the use of the flexdex semi robotic arm for the realization of complex anatomical sutures.this is a case series of patients to whom different surgical techniques requiring manual suture have been performed. these being tapp procedures, nissen-type fundoplicature and reinforcements of colorectal anastomosis. it is important to note that in none of the cases complications were recorded conclusions: flexdex can provide an excellent alternative to the robotic systems in complex surgical procedures, offering surgeons the precision and control they desire while maintaining the balance of cost, outcome and patient benefit. background: a new single-port device (fsis-flexible-single-incision-surgery) is presented. this new platform has three working channels, two for rigid instruments and one for the flexible endoscope. the channel for flexible instruments offers a pneumatic sealing to avoid the air's leak of the cavity (abdomen, rectum, vagina) . in this study the preclinical data are shown testing the feasibility and safety for laparo-endoscopic instruments. methods: experimental evaluation of feasibility and safety in two stages. in the first stage a working channel with pneumatic sealing was tested in simulators to use a flexible endoscope. in the second stage (animal model) the single incision device that makes possible to use laparoscopic instruments and flexible endoscopes was tested. the measured variables were: time of the procedure, co employed, adverse intraoperative events, grip's losing, losing of pneumatic sealing, feasibility and safety of the procedure for the surgeon. results: the hysterectomy and double adnexectomy was done with a median time of . min. the median of the co consumption was . litres. only in one case ( . %) the surgeon had problems with the abdominal navigation of the endoscope that was easily solved. the grip's lose wasn't a major problem. the median size of the skin incision was . cm. the median surgeon' score for the feasibility was and for the safety was . . conclusions: the surgeons considered that the use of the device was very feasible and safe. the fsis-device is a universal platform for single-incision-surgery for surgeons and gastroenterologists and for abdominal, rectal and vaginal access. aim: despite the near-infrared fluorescence (nirf) via the intravenous administration of indocyanine green (icg) improves the visualisation of the cystic duct (cd) and the extrahepatic biliary tract (ebt), the back fluorescence of the liver reduces the signal-to-noise ratio.we have modified the technique of nirf cholecystocholangiography with intragallbladder icg injection by using the arrow-karlan tm balloon cholangiography catheter instead of the purse string at the gallbladder's fundus. this procedure allows a high rate of visualisation of the ebt, with few cases of icg leakage.aim: of this study is to confirm the feasibility of this different technique and to analyse the icg spillage from the gallbladder and to identify the ebt. methods: we enrolled nine patients undergoing laparoscopic cholecystectomy for cholelithiasis. the gallbladder was perforated with the cholangiogram catheter, the balloon inflated with . ml of saline and tightened. the bile was drained and the icg bolus injected. a titanium clip was the placed on the catheter strict closely to the gallbladder in order to prevent the catheter dislocation. results: the cd and the ebt were visible before dissection in / and / patients respectively. after dissection the cd was visible in all the patients and the ebt again in / patients. there was only one icg spillage due to a tardive positioning of the clip. in a case of inflamed gallbladder this technique helped in the identification of the dissection plane. conclusions: our preliminary results of this ongoing study confirm the feasibility of this different approach as a possible alternative to the purse string and a good visualisation of ebt. introduction: robotic-assisted surgery is a promising technique for overcoming the limitations of laparoscopic surgery, especially with regards to complex and advanced surgical procedures. here, we describe the establishment and implementation of our robotic upper gastrointestinal (gi) and hepato-pancreato-biliary (hpb) surgery program within our center of excellence for minimally invasive surgery as well as the first-year results. method: robotic-assisted surgery was performed using the davinci xi surgical system tm and performed by two surgeons specialized in minimally invasive surgery (db and tk). our robotic surgery program of upper gi and hpb surgery was established in three steps: ( ) first, surgical procedures with easier degree of difficulty were performed robotically, including cholecystectomy, minor gastric resections and fundoplications. ( ) then, pancreatic distal resections, enucleations, adrenalectomies and atypical liver resections were robotically performed, as procedures with moderate degree of difficulty. ( ) finally, advanced and highly complex procedures were performed, including right hemihepatectomy, complex pancreatic head resections (including portal vein resections), total gastrectomy and esophagectomy. data collected from july till july were retrospectively analyzed with regard to conversion rate, morbidity (clavien dindo grade £ ) and mortality. results: within the first year, a total of robotic assisted upper gi and hpb resections were performed. the first step of establishing our robotic surgical program included eight procedures. here, conversion rate, morbidity and mortality were %. within the second step of establishment procedures were performed. conversion rate, morbidity and mortality were %, % and %. the last step included of advanced and highly complex procedures. these procedures resulted in a conversion rate of %, % morbidity and % mortality. conclusion: our stepwise approach enables a safe implementation of a robotic surgical program for upper gi and hpb surgery with low morbidity and no mortality even for highly complex procedures. however, highly complex procedures required a high conversion rate, which might be caused by the early stage of experience. the standard surgical procedure of choledochal cyst is a complete excision of the cyst with rouxen-y hepaticojejunostomy and laparoscopic surgery had been increasingly used. this is still a challenging way to perform anastomosis due to the small diameter of bile duct and the possibility of bile leak or stricture. robotic system can overcome the shortcomings of laparoscopy with providing three-dimensional view, magnification, and articulated instruments. from jan to dec , patients underwent robotic cyst excision and hepaticojejunostomy by single surgeon. we reviewed the clinical data and compared with laparoscopic outcomes of early (from to ) and late (from to ) group, retrospectively. patients of robotic series were all female with mean age . years and bmi . . the mean size of cyst was . . cm, and todani type ia , ic and iva , respectively. total trocars were used with robotic working arm and assist and camera. the mean operative time . ± . min, and it was similar with late laparoscopic group ( ± . min) and significantly shorter than early group ( ± . min).there were no open conversion in robotic and late laparoscopic group, however, the early laparoscopic group involved % of conversion rates. the hospital length was ± . days in robotic group, and it was similar with late group ( ± . ) and more shorter than early group ( . ± . ). in robotic series, postoperative complications occurred patients. one case included cholangitis which was resolved after conservative treatment. bile leakage was developed in patient, and treated with drain that inserted intraoperatively. last cases showed incisional hernia at postoperative months, and was corrected by laparoscopic herniorrahphy. complications (n = ) in late laparoscopic group included hepaticojejunostomy stricture and stone, bleeding of jejunal branch, portal vein thromobosis, acute pancreatitis, and adhesive ileus. there were no mortaility case in any groups.robotic surgery of choledochal cyst is a safe and feasible option with short-term results that are comparable to laparoscopic approach. general surgery, sanchinarro university hospital, madrid, spain background: the incidental detection of benign to low-grade malignant small pancreatic neoplasms increased in the last decades. the surgical management of these patients is still under debate. the aim of this paper is to evaluate the safety and feasibility of robotic enucleations. methods: we retrospectively reviewed our prospectively databases from november . demographics, pathological characteristics, perioperative outcome, and medium-term follow-up of patients who underwent robotic pancreatic enucleations were collected. results: patients were included. the mean age of the patients was years ( - ). the median body mass index was ( - ). ten lesions were located in the pancreatic head, in the pancreatic body, in the pancreatic tail. operative time was min (range - ), no intraoperative transfusion were needed and in one patient conversion to open approach was needed. in three patients grade b pancreatic fistula occurred. the mean postoperative stay was , days. conclusions: robotic enucleation is a feasible and safe approach, with low incidence of morbidity. the results of surgical treatment of patients with pulmonary tuberculosis were evaluated depending on the prevalence of the tuberculosis process and the type of surgical intervention used. according to the results of the questionnaire, people operated on pulmonary tuberculosis in the period from to years ago, the frequency of cases of tuberculosis reactivation, the complicated course of the remote postoperative period, as well as the mortality and causes of lethal outcomes were assessed. it was found that after sublobular resection and lobectomy, treatment failure was noted at . %, relapse of tuberculosis- . %, pleural empyema- . %, bronchial fistula- . %, cardiovascular insufficiency-in . % operated. the mortality rate was . % with a total clinical efficacy of . %. after combined resection and bylobectomy, treatment failure was noted at . %, relapse of tuberculosis- . %, pleural empyema- . %, bronchial fistulae- . %, cardiovascular failure- . % operated. the mortality rate was . % with a total clinical efficacy of . %. after pneumonectomy, treatment failure was noted at . %, relapse of tuberculosis- . %, pleural empyema- . %, bronchial fistulae- . %, cardiovascular failure- . % operated. the mortality rate was . % with a total clinical efficacy of . %. robotic reduced-port splenectomy using single-site platform j.h. lee background: in the era of minimal invasive surgery, single incision laparoscopic splenectomy can offer some advantages compared to conventional laparoscopic splenectomy. but it requires expertise in minimally invasive techniques due to technical difficulties. the da vinci robotic reduced-port splenectomy using single-site platform permits greater freedom of movement and higher levels of accuracy than previous laparoscopic surgery through two small incisions. methods: we performed a retrospective review of all patients who underwent robotic reduced-port splenectomy using single-site platform at our institution between january, and november, . one cm periumbilical incision was made for glove port insertion and the other incision was made at left side of abdomen for additional mm port insertion.the surgical technique is much same as open procedure. short gastric artery was ligated, firstly. splenic artery and vein were ligated individually. during the surgery, any stapling device was not used. vessel sealer was used for hemostasis and mobilization of spleen. a specimen was removed through umbilical port site within lap-bag. result: eight patients ( female and male) with median age of . years underwent robotic reducedport splenectomy using single-site platform (one case with combined robotic cholecystectomy for gall bladder stones without additional trocar). the indications were; hematological disease (n = ), splenic mass (benign n = , malignant n = ). preoperatively measured spleen size was ranged . cm to cm (mean cm). there were no intraoperative complications and open conversion. mean operative time was min. (range - min) including docking (mean min) and console time (mean min) mean blood loss was under ml. mean hospital stay was . days after surgery. one patient underwent oral anticoagulation therapy only for portal vein thromobisis without any symptoms, and thromobisis was resolved at month follow-up ct scan. there were no clavien-dindo class iii or above postoperative complication. conclusions: robotic reduced-port splenectomy using single-site platform seems to be feasible and effective. it seems to overcome certain limits of previous robotic or conventional single-site laparoscopic splenectomy and single-site only robotic splenectomy. we think mm additional port allows to use endo-wrist da vinci instruments such as vessel sealer which enhances dissection efficiency andsafety of procedures. aims: inguinal lymph node dissection carries an important risk of post-operative complications, mainly related with wound complications and long term lymphedema. the minimally invasive approach aims to reduce the morbidity of this procedure, avoiding the traditional groin incision but still allowing a full access to the lymph node basin. the authors aimed to describe their videoassisted inguinal lymph node dissection (vilnd) cases, comparing the surgical outcomes with a sample of open inguinal lymph node dissection (oilnd) cases. methods: we performed a retrospective descriptive study that compared the data from patients submitted to vilnd since (the year in which this technique was first performed in our institution) with the patients submitted to oilnd in and . gynaecologic and urologic malignancies were excluded. the statistical analysis was performed using spssv Ó, with a p value \ . indicating statistical significance. results: a total of cases of inguinal lymph node dissection were analysed, . % of which vilnd (none of them requiring conversion to the open approach). melanoma was the primary tumour in % of patients. the vilnd and oilnd groups had no statistically significant difference between them regarding age, body mass index, smoking status or the reason for lymph node dissection-clinically detected lymph node vs. positive sentinel node biopsy. the mean of isolated lymph nodes in the vilnd ( . ) and oilnd ( . ) groups was also not statistically different (p = . ). there was no difference in the rate of post-operative seroma, wound dehiscence or lymphedema. the rate of surgical site infections was higher in the oilnd group- % vs. . % during post-operative hospital admission (p = . ); . % vs. . % after discharge (p = . ). conclusions: in our population of patients we conclude that the main advantage of the videoassisted approach regarding surgical morbidity lies in the reduction of the infection rate, as the published literature also confirms. the equivalent number of lymph nodes retrieved in both groups points toward the oncological safety of the minimally invasive procedure, that we hope to study further in the future after a longer follow up period. objectives: to evaluate the clinical feasibility of tumor localization technique with radio-frequency identification (rfid) clip marker methods: we developed the proto-type rfid integrated endoscopic clip (rfid-clip) and probe to detect it on serosa surface during the laparoscopic surgery. a pig weighing kg was used as the specimen for the in-vivo test. endoscopist performed the application of the rfid-clip on porcine gastric mucosa. after then, the surgeon tried to find the location of rfid-clip using the detection probe and marked with the electrocautery. after the gastrectomy with cm margin (each to proximal and distal), we confirmed the prediction of rfid-clip location and accuracy of resection. results: rfid-clip location was detected and recorded on the exact site of clip application. detection range was very short and we confirmed there are almost no differences between actual clip location and our prediction. this result might arise from using the low-frequency rfid tag to increase the accuracy through reduction of the range. however, some rfid-clip were not detected because of the issue of clipping trouble, not rfid tag. conclusions: this is a basic study to evaluate the clinical usefulness and feasibility of the new localizing technique. we confirmed the possibilities of this system and it could be the helpful option to provide the information of exact location for the minimally invasive surgery or early gastrointestinal tumors. background: the advantages of laparoscopic posterior retroperitoneal adrenalectomy (lpra) have been described in the literature. the aim of this study was to compare the clinical outcomes of lpra and robotic posterior retroperitoneal adrenalectomy (rpra) and determine the differences that could affect the outcomes. methods: we retrospectively analyzed adrenalectomy cases at asan medical center from to . there were lpra and rpra cases, and their clinicopathological features and surgical outcomes were compared. results: in lpra, there was a positive relationship between operation time and male gender, early period of experience, adrenal tumor size, and pheochromocytoma. in rpra, adrenal tumor size and pheochromocytoma were the only factors affecting the operation time. when the adrenal tumor size was = . cm, the operation time of lpra was shorter than that of rpra (p = . ). when the tumor size was [ . cm, there was no significant difference in the operation time of lpra and rpra (p = . ). conclusions: rpra is a feasible and technically safe approach for benign adrenal diseases. the use of rpra could benefit patients with large tumors and provide comfort by overcoming the factors contributing to a longer operation time in the laparoscopic technique. methods: twenty years experience at the american university of beirut medical center for laparoscopic adrenalectomy. a total of cases were done laparoscopically with no conversion and minimal complication. the average operative time is mins.the video will show the various steps used for lap redo (lt) adrenalectomy for a cm pheochromocytoma using the lateral position and through trocars. attempt to remove the pheochromocytoma in iraque was complicated by cardiac arrest treated successfully and patient referred to the american university of beirut medical center. results: patient had smooth postoperative course following laparoscopic adrenalectomy and patient discharged days later with no complications. conclusions: even large adrenal masses can be completed laparoscopically in advanced experienced centers in laparoscopy. surg endosc ( ) aims: the adrenocortical of uncertain malignancy neoplasm is a spectrum of classification for adrenal tumors whose histopathological diagnosis is uncertain. clinical case: we present a year old patient with constitutional syndrome and severe hypercortisolism and hypokalemia reason why she was admitted to icu for episodes of ventricular fibrillation. no other medical history of interest except refractory hypertension to treatment. the tc showed a left adrenal mass of . . cm with microcalcifications, areas of necrosis and hemorrhage, no infiltrating, without disease to distance. the surgery was a laparoscopic left adrenalectomy with no evidence of infiltration and no lymph nodes. the histopathology lesion presented a dense proliferation cellular of cortical type, with incomplete fibrous, without vascular or capsular invasion, with a % ki ; positivity vimentin and cd . all epithelial markers, were negative. all this leads to the diagnosis of a neoplasm of uncertain malignancy potential adrenocortical. during the postoperative period, the patient presents a crisis of adrenal insufficiency that was treated with intravenous replenishment corticoidea and later orally with good clinical response. discussion: the adrenal carcinoma has a low incidence ( . %), incidence peak around the years, the most frequent is the mixed secretory. they are - % of the adrenal incidentalomas. it is usually presented to the diagnosis as a locally advanced tumor with metastases (to liver, lung, retroperitoneal ganglia and bone). may present clinically due to hormonal hyperproduction; or be non-functioning tumors. the adrenal carcinoma poses a great difficulty at the time of the diagnosis pathological, and includes as differential diagnosis to other abdominal tumors. the distinction between corticoadrenal adenoma and adrenal carcinoma is sometimes difficult, so it has been defined a spectrum of intermediate category called adrenocortical neoplasm of intermediate or uncertain malignancy. it is obtained with the weiss criteria, being necessary at least of them for confirm the diagnosis of adrenal carcinoma. this category has a low risk of local recurrence or metastasis, but it needs a narrow follow-up. conclusion: adrenal carcinoma of uncertain malignancy implies a new category in those tumors of difficult classification. aims: multiple endocrine neoplasia type (men ) is an autosomal dominant disorder with an estimated prevalence of per , in the general population. among patients suspected to have a pheochromocytoma, the diagnosis is rarely confirmed and only % is presented bilaterally. we present bilateral laparoscopic adrenalectomy in patients with men . method: a -year-old woman with a family history of medullary thyroid cancer and breast cancer. personal history: hypertension, medullary thyroid cancer, breast cancer, laparoscopic cholecystectomy. appendectomy. after a study by endocrinology and suspicion of bilateral pheochromocytoma, discussing the case in a multidisciplinary committee, bilateral adrenalectomy was decided by laparoscopic approach. selective alpha- -adrenergic blocking agent (doxazosin) were utilized before surgery. under general anesthesia left adrenalectomy was performed first in right lateral decubitus position. mmhg pneumoperitoneum was started with the verres needle and trocars ( mm umbilical, mm subxifoid and mm left subcostal).once dissection was completed the gland was placed in a plastic bag and extracted through one of the trocars incisions, then the position of the patient was changed to left lateral decubitus for the right adrenal approach. another right subcostal mm trocar was used. adhesiolysis of previous cholecystectomy was performed to right adrenal approach. adrenal veins were divided between metallic clips.no drainage was employed. results: the procedures were successfully performed without conversion. surgical time was min and hospital stay was days. had a clinical reversion with control of blood pressure monitored by endocrinology conclusions: currently, the laparoscopic approach is the technique of choice for the management of adrenal pathology.lateral decubitus transperitoneal approach is the procedure of choice in most cases. bilateral laparoscopic synchronous adrenalectomy is feasible and safe with good results as in our patient. traditionally the treatment of hyperparathyroidism for patients with familial hyperparathyroidism was subtotal parathyroidectomy or total parathyroidectomy and auto transplantation. in the era of minimally invasive parathyroidectomy, the removal of only abnormal glands guided by preoperative localizing studies has been suggested. aims: this systematic review aimed to investigate the role of focused minimally invasive parathyroidectomy in the treatment of patients with familial hyperparathyroidism. methods: electronic databases were searched with the search terms 'men i', 'familial hyperparathyroidism', 'men a','hyperparathyroidism-jaw tumor syndrome', 'parathyroidectomy', 'minimally invasive ', for the time period up to and including december . full publications, including clinical trials randomized or not, retrospective studies, case series, case reports that provided relevant data met inclusion criteria. results: thirty five possibly relevant studies were identified. abstracts were reviewed and fifteen articles were excluded. twenty studies, that met inclusion criteria were retrieved in full text and included in the systematic review, including three retrospective cohort studies i.e. two presenting data on meni associated hyperparathyroidism and the third study on familial hyperparathyroidism and seventeen small case series or case reports. the two retrospective studies on meni hyperparathyroidism included patients treated either with focused minimally invasive parathyroidectomy or with the conventional approach. these studies presented conflicting data with one supporting and the other negating the focused minimally invasive parathyroidectomy due to the failure of localization studies to identify enlarged parathyroid glands in a great number of patients. conclusion: undoubtedly, the idea of minimally invasive parathyroidectomy in patients with hereditary and familial hyperparathyroidism is interesting. this idea is especially challenging in the case of meni. existing data suggest that focused mimimally invasive parathyroidectomy is feasible under the condition of exact preoperative localization studies. the main advantage of this approach is the minimization of the risk of postoperative hypoparathyroidism. however, data are limited and further research is needed before valid conclusions can be drawn on the suitability of this approach. objective: resection of pheochromocytomas is a challenging procedure due to hemodynamic lability, tumor vascularity and malignant potential.given the technical challenges for resection of large pheochromocytomas, there were hesitations about using the laparoscopic approach for these tumors during the first decade of laparoscopic surgery. however, improvement in imaging modalities,better pharmacological preparation,advances in anaesthesia and laparoscopic surgery rendered laparoscopic surgery for pheochromocytomas safe and efficient. our aim was to evaluate surgical outcomes in patients with pheochromocytoma and to validate the role of laparoscopic surgery in the treatment of these tumors. design: a total of procedures for pheochromocytoma were performed between january -september . the preoperative diagnosis, operative details, complications, length of hospital stay, morbidity and follow up were retrieved from the hospital records of patients who underwent adrenalectomies for benign and malignant adrenal tumors in the same period. preoperative localization was established in all patients with computerized tomography (ct) or magnetic resonance imaging (mri), while iodine - -metaiodobenzyguanidine(mibg) scan was reserved for ambiguous cases where paraganglioma or metastatic disease was suspected. endocrinological evaluation and complete adrenal dynamic testing were performed to determine whether the tumor was functional or not. results: eighty-seven tumors were removed from patients. one patient with meniia underwent bilateral resection of pheochromocytomas in two stages. tumor size in laparoscopic procedures ranged from . cm to . cm (mean . cm). forty-three patients had benign disease, potentially malignant (based on pass), malignant with metastasis. eight were in the context of a familial syndrome. sixty -eight patients underwent laparoscopic adrenalectomy, patients had open approach from the start for recurrent pheochromocytoma or large benign tumor, patient had open approach due to inoperable malignant pheochromocytoma and patients had conversions from laparoscopic to open procedure. nine patients received sodium nitroprusside intraoperatively to treat hypertension. one patient developed pulmonary embolism, and succumbed month later. there were no recurrences for the benign tumors during the follow-up period. conclusions: laparoscopic resection of pheochromocytomas despite its increased level of difficulty compared to that of other adrenal tumors, is a safe and effective procedure. aim: the concept 'large' in transperitoneal lateral laparosopic adrenalectomy (tlla) has been evolving along time, ranging from to - cm depending on different authors. on the other hand, some authors discourage laparoscopic surgery in larger tumors due to the increased risk of malignancy in those larger than - cm, referring to malignancy in out of or cases. paragangliomas are rare tumors originated in extra-adrenal chromaffin cells, with an incidence of - cases per million inhabitants. they can appear in any location between neck and pelvis. sympathetic paragangliomas are usually functional and catecholamines producers. we present a movie of surgical intervention of a -year-old patient who, in study for refractory hypertension, presented paraganglioma producing norepinephrine, whose approach was performed laparoscopically. -year-old woman studied by nephrology for refractory hypertension. on physical examination, only obesity standed out. in blood exams, levels of normetanephrine were observed in plasma of pg/ml and aldosterone pg/ml. abdominal scintigraphy was performed in which there was no evidence of increased activity at adrenal level. abdominal ct shows retroperitoneal extra-adrenal tumor of inter-aortocava location immediately below renal vessels with dimensions of . . cm. after preparation, she was operated. laparoscopic access was performed under exhaustive monitoring. an heterogeneous, polylobulated tumor of cm, located interaortocava, intimately adhered to left renal vascular pedicle, was observed. a cattell-braash and kocher maneuver was performed, with exposure of inferior cava and aorta to iliac bifurcation. complete tumor excision was performed after clipping arterial and venous tributary branches. after the operation, the pacient presented favorable evolution being discharge on the second postoperative day with good control of blood pressure levels. laparoscopic approach of retroperitoneal paragangliomas is a safe technique, which allows minimally invasive access, with consequent improvement in postoperative results. the exact location of lesions and their relationships with surrounding structures, as well as their functional behavior, are very important when considering the best therapeutic strategy for these patients. we present the case of a -year-old obese male patient referred for adrenalectomy after being diagnosed with left adrenal incidentaloma. abdominal mri showed a . / . / . left adrenal mass with normal hormonal levels. after preoperative workup, the patient underwent standard laparoscopic adrenalectomy. the lateral to medial dissection and mobilization of the spleen and pancreatic tail was difficult due to the abundance of peritoneal and pararenal fat. the anatomy was peculiar: the bulky pancreatic tail was located well inferior to the splenic hilum and was visible throughout the intervention and the spleen was quite elongated-long axis = cm. the exposure of the adrenal gland was therefore cumbersome. the operating time was min and blood loss ml. the abdominal drainage was maintained for h. before discharge the patient underwent a control abdominal us examination that only showed a thin line of left pleural fluid. the patient was readmitted days after discharge for chest pain, fever ( . °c) and malaise with no abdominal signs. the emergency ct scan diagnosed left basal pneumonia with minimal pleural effusion and a / cm fluid collection between the spleen and diaphragm while the blood test showed leukocytosis. the patient was treated for pneumonia with an apparent clinical benefit for three days and lowered white cell count but his condition worsened during the forth day. repeat abdominal us demonstrated that the abdominal collection increased in size therefore the patient underwent emergency surgery. during laparoscopic exploration, the collection was unveiled as being pancreatic juice (more than times the normal serum levels of lipase and amylase). after thorough lavage, two drainage tubes were positioned in the left subphrenic space. the postoperative course was uneventful under antibiotic treatment for pneumonia and pancreatic antisecretory medication. the patient was discharged after days with minimal pancreatic drainage and the drainage tube was extracted after more days. the aim of the study was to develop the algorithm and the choice of the method of endoscopic treatment of a combined pathology of uterine leiomyoma and adenomyosis depending on the reproductive plans. methods: the study involved patients with a combined pathology of uterine leiomyoma and adenomyosis. indications for conservative myomectomy were: the size of the uterus is more than weeks. pregnancy; multiple leuomatous nodes and adenomyotic foci up to cm in size; hemorrhagic and pain syndromes, anemia, compression of the adjacent organs; suspected node malfunction; submucous leiomyoma deforming the uterine cavity with foci of adenomyosis; subserous, cervical isthus nodes and foci of adenomyosis; the presence of endometrial hyperplasia, tumors of uterine appendages; growth rate of uterine leiomyoma more than weeks pregnancy for the year; the growth of uterine leiomyoma on the background of drug treatment; infertility associated with leiomyoma and uterine adenomyosis.the laparoscopic myomectomy of the subserous node on the 'leg' with a size of more than cm and nodes of more than cm of intramural location is shown with an interest in preserving the organ.the hysterectomy is indicated for women after years of age who insist on hysterectomy, with a combination of uterine leiomyoma with atypical endometrial hyperplasia. results: the conservative myomectomy and removal of adenomyotic foci were performed in ( . %) patients: from hysteroscopic access- , vaginal access- , laparoscopic access- , abdominal access- in the presence of reproductive plans.the hysteroscopic myomectomy was performed in ( . %) patients, hysterectomy in ( . %) patients: from laparoscopic access- , from vaginal access- , from abdominal access- in the absence of reproductive plans. conclusions: the choice of surgical treatment of uterine leiomyoma and adenomyosis depends on the reproductive plans of the woman and the severity of the lesion.the laparoscopic method of treating a combined pathology of uterine leiomyoma and adenomyosis in the presence and absence of reproductive plans is a priority for women. surgery, policlinico ,,paolo giaccone,,, palermo, italy background: breast cancer in females represents the most frequent neoplasm in all age groups. the risk of getting breast cancer (mc) increases with age. the brca and the brca genes (tumor-suppressor genes, autosomal dominant transmission at high penetrance) alone justify from % to % of cases of hereditary breast cancer. methods: from january to june we have analyzed patients with brca mutation. all patients had in common a genetic mutation of brca or brca tumor suppressor genes. results: the frequency of germline mutation on brca ( patients: %) was identical to brca gene ( patients: %). of the analyzed patients were women ( . % of patients) brca and brca , and men ( . %) all with brca mutation. conclusions: prophylactic surgery must be seen as a way to put the patient in the condition to implement the most appropriate treatment. further studies will be necessary to support the validity of prophylactic surgery in patients with mutations in brca and brca genes. introduction: laparoscopic hysterectomy is a safe surgical technique for removing the uterus with or without including the ovaries and fallopian tubes. laparoscopic surgery of endometrial cancer is a safe method, with the mean time of recovery being two days only. material-method: the case of a yr old woman with metrorrhagia and anaemia (ht , %) due to adenocarcinoma of the endometrius is presented. the patient underwent a laparoscopic hysterectomy and oophorectomy. trocar ports were used during the procedure (a mm transumbilical port, similar to the port used in single incision laparoscopic operations, two mm ports at the level of the anterior superior iliac spines, and a mm port in the middle of the imaginary line between the pubic symphisis and the umbilicus). the uterine vessels and the uterine ligaments were ligated and dissected by using a thermal energy source. the patient's postoperavite course was uneventful. the patient continues to be in good condition, months post-surgery. conclusion: laparoscopic hysterectomy seems to be a safe method for addressing endometrial cancer, as it offers the surgeon a better surgical field, is tissue friendly and causes fewer postoperative complications. it is considered to be a less traumatic operative method, as due to zooming in the picture there is greater accuracy in handling the tissue, and blood loss is minimal. m. shahin background: hysterectomy is one of the most frequently performed surgical procedure. though there are three approaches in hysterectomy (open, vaginal and laparoscopic), still there are controversies regarding the optimal route for performing it. methods: this prospective comparative study included obese patients subjected for panhysterectomy as a treatment. the forty-two patients were allocated into two groups: group (a) subjected to laparoscopic pan-hysterectomy, group (b) subjected to open pan-hysterectomy. results: there was significant difference between the two groups regarding mean operative time, blood loss, analgesic requirements and hospital stay, while no significant difference regarding intra-operative complications. conclusions: laparoscopic hysterectomy in obese patients has emerged as a viable, safe and better alternative to open hysterectomy amongst appropriately trained surgeons. general: endometriosis in the inguinal region is rare. the usual presentation is that of a woman in the reproductive age group. it accounts for . - . % of patients affected by endometriosis. the groin swelling is usually slow growing, painful with exacerbations during menses. the incidence of inguinal endometriosis on the right side is - % as compared to the left. aim: to present our laparoscopic approach for the treatment of the diagnostic dilemma. case presentation: a -year-old woman presented with a palpable mass in the right groin. the swelling was associated with a dull aching pain. the patient was suffering from increasing pain over the swelling during menstruation. she had undergone cesarean section some years ago and the scar had healed by primary intention. mri scan revealed a nodular hypoechoic lesion at the level of the internal inguinal ring with the absence of vascular flow around the lesion. results: since inguinal endometriosis was in the differential diagnosis and it may be associated with pelvic or intraperitoneal endometriosis, a laparoscopic approach was decided. the procedure was successfully completed laparoscopically following the transabdominal preperitoneal approach. the endometriosis was found, after dissecting the internal inguinal ring, firmly adhered to the round ligament. it was excised en bloc with the round ligament. a preperitoneal polypropylene mesh was inserted to protect for future inguinal hernias due to extensive dissection at the level of the internal inguinal ring. no intraperitoneal endometriosis was appreciated. histopathology revealed endometriosis of the round ligament. the patient was uneventfully discharged the next day. on follow up the patient was asymptomatic. conclusions: round ligament endometriosis is a rare entity. it is a disease of specific interest to the physician. it can be confused with an inguinal hernia and thereby pose a diagnostic dilemma. we recommend considering endometriosis in the differential diagnosis of groin swellings in women. the transabdominal preperitoneal approach is feasible and safe in the hands of an advanced laparoscopic surgeon. introduction: sentinel node biopsy is the newest accepted method for surgical staging of early stage endometrial and cervical cancer. aim: to evaluate the role of the technique of indocyanine green (icg) identification of the sentinel lymph nodes in cases of early endometrial cancer. material and method: five patients with early endometrial and cervical cancer were introduced in a prospective study. icg was locally injected during the laparoscopic exploration. novadac pinpoint near to red technology was used. guided biopsies were performed into the marked sentinel nodes and histological results were evaluated. results: sentinel lymph nodes were easily identified by using icg and near-infrared technology. technical details are described. no associated complication was encountered. conclusion: sln mapping using icg in uterine cancers is demonstrated as an effective and safe procedure. laparascopic extraction of an intraperitoneal gossypiboma following c/s and a retroperitoneal gossypiboma following pyeloplasty n. ozlem general surgery department, ahievran university, kirsehir, turkey gossypibomas are forgatten foreign bodies,iatrogenic.their symptoms are different where they are. they extracted with laparotomy in the past but now we can some article mentioned their extraction was made with laparoscopy. case : y o female has abdominal pain after c/s for . years. a gossypiboma was extracted with laparoscopy above umblicus.a superficial surgical site infection existed,drained,subsided. case : yo m had a pyleoplasty operation years ago.a gossypiboma was extracted with retroperitonescopy,no postoperative event. basibuyuk et al reported retroperionescopic extraction of a gossypiboma from single port in first time.althoug every effort taken the incidence of foreign body detected in the body is about . - . %.they are most frequently localized in the intraabdominal cavity followed by tracheobronchial area,pleural cavity,pararenal area,vagina,spinal chord, neck, femur,breast,bladder,pancreas,and they may cause local irritation,and infection.tactile sense is absent in laparoscopy. all radiologic examinations(usg ct pet mri etc) be used to detect.we used usg ct.in the end laparoscopy make the diagnosis and remove gossypibomas in our cases with less postoperative pain and cosmosis. justo et al the computerized tomography (ct) scan is the most useful method for diagnosis; however, sometimes the preoperative diagnosis remains uncertain even after the imaging exam. in that case, laparoscopy arises as a valuable diagnostic tool, as well as a prompt treatment option. concerning gossypiboma, prevention is preferred rather than treatment. notwithstanding, there is no highly reliable prevention system. counting sponges is a method based on staff communication during the surgery with only % sensibility. routine surgical postoperative x-ray (spox) constitutes an early detection system, but the need to incorporate a radiopaque marker and to expose the whole surgical field to maximize its efficacy limits its use. more recently, electronic dispositives based on barcode detection and other technological adjuncts for counting sponges are being developed. none of these prevention systems are reliable when used alone. our education and research clinic was a state hospital before. no surgeon followed above instruction.but now we use all. multiple procedures and surgical teams, long operations and non-elective operations are the evidenced risk factors.c/s operation was learned full opened of ostium of cervix of the patient. urology, japan, nagoya, japan aims: some scoring systems have been suggested to standardize the renal tumor characteristics. among them, renal score is widely used in partial nephrectomy. whereas diameter-axis-polar (dap) score was developed to be more significantly related with postoperative renal function. our study compared dap score with renal score in robotic partial nephrectomy (rpn) outcomes. methods: records of patients who underwent rpn at nagoya daini red cross hospital between april to october were analyzed retrospectively. those include three oncocytomas. accordingly, we calculated the estimated glomerular filtration rate (egfr) just before rpn and month postoperatively in patients. we compared two nephrometry scores with warm ischemic time and change in egfr. results: in our institution, four surgeons performed rpn. according to dap score, patients were high, were middle and were low. according to renal score, were high, were middle and were low. the median warm ischemic time was min ( - ). the median egfr decreased from . ( . - . ) to . ( . - . ) ml/min/ . m . there were no significant differences in warm ischemic time and percentage change in egfr between renal score groups (p = . and . ) but significant differences between dap score groups (p \ . and p \ . ). univariate and multivariate analyses were used to identify factors influencing postoperative renal function. that confirmed that dap score was independent poor predictors of change in egfr after rpn. conclusions: dap score is simpler estimate system than renal score. our study suggested that dap score is a useful scoring system for preoperative evaluation of renal tumor for rpn. further investigation is needed to better understand preoperative dap score. aims: retroperitoneal primary tumors comprise a great variety of neoplasm with different histological typologies, with insidious clinical symptoms and little specificity in most cases. its diagnosis is established through imaging tests and anatomopathological study is needed so complete surgical resection is the treatment of choice. the aim of the video is to demonstrate the safety and efficacy of the minimally invasive approach in patients with retroperitoneal lesions. methods: a -year-old female patient who, in the course of an abdominal pain at the right iliac fossa suspected of possible acute appendicitis, is diagnosed with a right retroperitoneal tumor, compatible with primary neurogenic tumor on a ct. radiographic imaging is a key component of the evaluation of a patient with a retroperitoneal mass, a ct scan is necessary to evaluate the primary site as well as to rule out metastatic disease. after complete biochemical study, nonfunctioning tumor is determined. the study is completed with mri where the lesion is located below the right kidney, in front of the right psoas muscle and lateral to the inferior vena cava, and without contact with these structures. ??it is in intimate contact with the ovarian vein. the complementary tests and iconography of interest of the case are exposed. surgical intervention is proposed with a laparoscopic approach. results: full minimally invasive approach in left lateral decubitus position: trocars-lateral laparoscopic transabdominal approach. laparoscopic liberation of the right colon, kocher maneuver until the inferior vena cava is visualized, identification of a tumor of approximately cm in the right infrarenal region, lateral to the right ureter, which includes the gonadal vessels. resection of the tumor in block with margins previous dissection and clipping of the proximal and distal gonadal vessels with ligasureÒ. the patient presented a successful postoperative recovery, being discharged h after the intervention. definitive result of the specimen: leiomyosarcoma, grade of the fnclcc with negative margin. the laparoscopic approach is a safe and effective technique in the approximation of retroperitoneal tumors, a radical oncological criterion is always needed with correct margins of resection especially in those of uncertain etiology. we started endoscopic thyroidectomy using the lifting method in and have developed single incision endoscopic thyroidectomy (siet) via chest (c-) or axillary incision (a-) by our original retractor since . we created a new approach in . recently, we have applied this method to parathyroid surgery. in this study, we present our method and results in parathyroid surgery with regard to surgical outcome and patients' complaints. method: endoscopic parathyroidectomy of c-siet was performed in patients with hyperparathyroidism (primary , secondary ) in new approach (mean age , male female ). single parathyroid adenoma was diagnosed using ultrasonic device, preoperatively. the patient is placed in a supine position with the neck extended. mm vertical incision is made in anterior chest. flexible endoscope (olympus co. japan) is used through mm trocar detached the retractor. in new approach, the parathyroid and thyroid are exposed through the avascular space between sternal head and clavicular head of sternocleidomastoid muscle. both of the skin and sternal head are lifted up by our original retractor (takasago medical co. japan). parathyroid adenoma behind the thyroid is resected using an ultrasonic scalpel. i would like to present our c-siet procedure. results: no scars in the neck were left in all cases. benign and hemi lateral parathyroid adenoma sized from mm to mm (mean: . mm) were operated. mean operation time is min. in new approach. there was no complication. parathyroid hormone levels decreased in all patients immediately after operation. conclusion: it is a little possible to make recurrent nerve palsy in this approach. new approach is useful to operate and make the working space wider without stress to find out of parathyroid adenoma. our original retractor can be introduced easily in most hospital, because it is not so expensive. most of women satisfied cosmetic results because of hidden scars. objectives: radiofrequency ablation (rfa) is a novel and developing technique for the treatment of parathyroid hyperplasia/adenoma in the context of secondary hyperparathyroidism (hpt) to chronic kidney disease (ckd) and there is little literature on the subject. the purpose of this study is to determine its usefulness by contributing a case carried out in our hospital. methods: we selected a case of secondary htp in a patient of years old with ckd who presented a parathyroid adenoma detected clearly by ultrasound scanning. the patient was dismissed for surgery due to high surgical risk due to his comorbidities. rfa of a right inferior parathiroid adenoma was performed. intact parathyroid hormone (ipth) was measured before arf and min after de procedure, calcium and phosphorus were measured the day after. the treatment was considered effective if ipth levels decreased at least % min after rfa and calcium levels decreased the day after. results: ipth level before rfa was pg/ml. ipth level after min of rfa was pg/ ml, this meant a % reduction (normal values - pg/ml). calcium levels were from . at the baseline to . the day after (normal values . - . mg/dl) and phosphorus from . to . mg/dl (normal values . - . mg/dl). the patient presented dysphonia as a complication that improved with corticosteroid therapy. we are currently waiting for the next analytical controls at , and months after the proceidure. conclusions: rfa of parathiroid adenomas for treating secondary hpt in patients with ckd is feasible in selected patients. this treatment may reduce the morbidity that surgery supposes, it is developed in an outpatient regime avoiding hospital admission and this contributes to a reduction of health costs. however, a longer follow-up is necessary to verify the good results in our case. splenectomy is one of the treatment strategy for advanced portal hypertension due to liver cirrhosis. after splenectomy, thrombocytopenia is dramatically ameliorated, and liver function parameters have also been improved in several clinical settings. however, the mechanism underlying such a phenomenon remains unclear. the aims of the present study was to analyze histological changes of the liver after splenectomy in human, and to speculate the underlying mechanism. subjects and methods: cirrhotic patients with hepatocellular carcinoma (hcc) who had undergone laparoscopic splenectomy prior ( weeks- months) to hepatic resection were analyzed (n = ). non-tumorous liver specimens obtained at hepatectomy were histologically investigated. liver tissues from cirrhotic hcc patients who underwent only hepatectomy were used as controls (n = ). results: after splenectomy, significant leukocytosis, especially increase in monocytes, was observed in addition to thrombocytosis. in the non-cancerous liver tissues, many round-shaped cd -positive macrophages accumulated after splenectomy, while this phenomenon was merely observed in patients without splenectomy. the macrophages were cd ? (m marker) and cd -cd ? , suggesting their anti-fibrotic population. the accumulated macrophages existed around fibrous scar as well as ck ? epcam ? cells spreading out from the ductular reactions (dr). as a result, the number of ki -positive hepatocytes significantly increased after splenectomy. the amount of platelets detected in the liver did not change even after splenectomy. finally, remarked attenuation of the established liver fibrosis was detected after relatively long duration. the accumulated macrophages expressed metalloproteinase (mmp)- and fibroblast growth factor (fgf)- , suggesting these molecules may possibly participate in resolution of established fibrosis and hepatocyte proliferation. conclusion: splenectomy in cirrhotic patients with portal hypertension ameliorate liver fibrosis, and stimulate liver regeneration. the mechanism possibly include hepatic accumulation of anti-fibrotic cd -positive macrophages and stimulation of dr-derived ck ? epcam ? progenitor-like cells. in patients with advanced splenic fibrosis, splenectomy could be a feasible therapeutic modality. the paper tries to establish the role and the opportunity of using laparoscopy in regard with abdominal contusions, as well as its indications or contraindications, combined in a therapeutic algorithm. we analyzed two groups of patients with abdominal contusions divided over two -year periods, - ( patients) and - ( patients) respectively. we have separated the two periods because starting from we have established a strategy for dealing with cases of abdominal contusions where we included diagnostic and / or therapeutic laparoscopy and nonoperative management. the investigation was done by fast echography, ct scan, simple abdominal radiography, peritoneal lavage puncture, and sometimes arteriography. in the second period we determined the diagnostic and therapeutic laparoscopy indications: suspicion of hollow or parenchymal organ injury, or mesentery injury, the presence of hemoperitoneum or fluid in the peritoneal cavity in a stable patient without major hemorrhage, apparent with unique injuries, without immediate vital risk and without other associated severe trauma. we have associated in this last period the nonoperative management for patients with grade and lesions of parenchymal organs that do not have fluid in the peritoneum, or only a very discreet quantity. in the first period, all patients were treated by classic surgery, resulting in unnecessary laparotomies where no visceral lesions were revealed. in the second period, we applied non-operative management to patients out of , patients with grade and splenic injuries, and patients with grade and hepatic lesions. diagnostic laparoscopy was performed in cases, in of them without evidence of lesions, and in other cases of grade lesions no therapeutic action was required. therapeutic laparoscopy was required for one case of splenectomy and one of hepatorrhaphy. diagnostic laparoscopy is useful in abdominal contusions, if certain indications are followed and in selected patients. in our study, with the introduction of modern therapeutic strategies, unnecessary laparotomies were completely avoided, some lesions being even treated by laparoscopy. the new algorithm introduced allowed % of patients to avoid laparotomy. aims: about cases of splenic hamartoma have been described in the literature since it was first described by rokitansky in , it is a rare benign tumor. it is usually a casual finding in laparotomies or autopsies. they are usually asymptomatic, but there are few symptomatic splenic hamartomas and they can be associated with haematological alterations, being in some cases associated with spontaneous splenic rupture and acute abdomen, two thirds of them have multiple tumors. there are no specific data that allow the preoperative diagnosis of this entity, which is performed after the anatomopathological study of the surgical specimen, which must be extracted entirely, this together with the size of the spleen makes the laparoscopic approach difficult. the aim of this video is to demonstrate the surgical technique of a complete laparoscopic approach for this type of lesions, without the need for assistance laparotomies (handport). methods: clinical case: a -year-old man admitted to internal medicine due to fever and left lumbar pain. additional explorations of interest are discussed, including: thrombopenia of probable peripheral origin secondary hypersplenism (fna of bone marrow), ct: splenomegaly with splenic masses, which deform the splenic contour, compatible with atypical hemangiomas, without being able to discard other vascular splenic tumors. results: complete semi-laparoscopic approach, trocars, multilobulated splenomegaly ( x cm.), mechanical vascular section, complete bag extraction after minilaparotomy on the left flank. the patient presented a successful postoperative recovery, being discharged on the th po day. abdominal ultrasound at st week with portal vein thrombosis, which resolves after treatment with heparin. definitive result of the specimen: multiple splenic hamartoma. asymptomatic one year after surgery. the laparoscopic approach is a valid and effective alternative to splenic benign tumor lesions. the size does not contraindicate this type of approach, although the complete extraction of the spleen is recommended for its pathologic study. we recommend eco-doppler control per week, given the risk of portal thrombosis with an existing laparoscopic post-splenectomy. objectives: splenic cysts are a rare entity, currently described between - cases in literature. a female patient's case is hereby presented, giant splenic cyst treated by conservative laparoscopic surgery obtaining good results. method: years old female, without any relevant medical history, examined after abdominal pain on the left hypochondriac region, nausea, postprandial swelling and mass sensation. after exploration the presence of such mass was ratified, the rest of exploration found no relevant findings, no record of previous traumatism nor any other relevant incidence. diagnosis was made through ultrasound and computerized tomography, the existence of a big splenic cyst is confirmed, cm by cm, on the superior section of the spleen, negative results after parasitism test, normal haemogram, coagulation and biochemistry levels. patient was intervened using laparoscopic surgery, performing the deroofing technique on the cyst (two liters of orangey amber serous liquid that was sent for analysis) as well as extirpation of superior wall of the cyst, which was sent to pathological anatomy, a saline solution was used to cleanse the cavity, omentum and drainage were then set in place. results: patient evolved satisfactorily, hospital discharge and drainage withdrawal after h. regular check-ups, after and months, patient presents no symptoms nor recurrence. pathological anatomy confirmed primary splenic cyst and the extracted liquid as cystic. conclusion: splenic cysts are primary ( %) or secondary ( %). diagnosis is performed through imagery tests, cat scan the being standard test used. regarding her treatment there is no clear consensus, due to the fact that up to a few years ago, complete splenectomy was the recommended treatment, techniques with preservation of the spleen are currently being widely recommended through laparoscopy in literature. among the conservative techniques percutaneous aspiration, with or without the injection of a sclerosing agent, partial splenectomy, marsupialisation, cystectomy, decapsulation, unroofing or fenestration can be found. the main issue is recurrence rates. few cases of primary giant splenic cysts treated by laparoscopic decapsulation can be found in literature, this treatment being simple and quick to perform, resenting a recurrence rate lower than other techniques such as aspiration and marsupialization. introduction: technology's progress and its application in the minimally invasive surgery of the thyroid gland offers us new surgical approache's like the transaxillary approach. this new technic still unusual in our environment and has recently begun to be incorporated into our surgical practice. the objective of this case is to explain step by step how to carry out a right transaxillary thyroidectomy and emphasize in the most relevant tips to take into account. also we going to review the main limitations we observed so far. statement of the case: we present the case of a -year-old woman referred for evaluation of a left thyroid nodule without associated symptomatology. the blood test shows normal thyroid profile. cervical ultrasound is performed identifying a . cm single right nodule with welldefined edges and presence of peripheral vascularization . no other nodules are identified. fna of the nodule describes a bethesda iii. after evaluation we decide to perform a left transaxillary thyroidectomy. discussion: surgical treatment of the thyroid gland by transaxillary approach may be indicated in previously selected patients, offering the advantages from minimally invasive techniques (shorter recovery time, shorter incision length, etc.). surely, more evidence and experiencie is required to make a better assessment of minimally invasive approaches in thyroid surgery. surgery, taipi city hospital, yan-ming branch., taipei, taiwan; surgery, taipei city hospital, taipei, taiwan the first endoscopic thyroidectomy was performed in using a cervical approach. since then, various remote-access method, have been developed for thyroid surgery to avoid scarring of the neck. trans axillary approach(taa),bilateral axillo-breast approach(baba),and retroauricular approach(raa) are common in use. the main benefit of these procedure is that there are no visible scar that is one of the drawbacks of conventional kocher's incision. however,these methods require more dissection and longer operation time than conventional thyroidectomy transoral thyroidectomy(tovet) is a new approach and has become popular in recent years, however,most surgeons peformed a single procedure because of the limited patients and the learning periods sine ,more than cases were performed,patients received endoscopic thyroidectomy(et) procedure at our hospital. we compare the surgical procedure of bilateral axillo-breast approach(baba) with transoral vestiblar approach(tovet) in our hospital both performed by one single surgeon .the surgen has expended eaqual amounts of time with these two procedures. the patient seletion process,operation time, operation procedure and approach,learnig experience, consmetic effect,onaologic consideration and surgical outcome were discussed yhroughly. presenting a case of a thyroid metastasis from an ovarian carcinoma, we conducted a review of the literature without finding similar reported cases. case: a -year-old woman consults for progressive asthenia, weight loss and ascites. abdominal ct finds a conglomerate in the pelvis involving the ovaries and peritoneal implants, the largest up to cm. an omental epigastric lesion biopsy and paracentesis is performed resulting in adenocarcinoma and omental metastasis from ovarian neoplasm, associated with ca of . patient starts neoadjuvant therapy with carboplatin-paclitaxel. in image controls there is a favorable response. three months later, intervention was carried out; laparotomy hysterectomy ? double anexectomy ? omentectomy ? appendectomy ? pelvic and paraaortic lymphadenectomy.the anatomopathological study shows a low-differentiated endometrioid carcinoma, omentum infiltration and absence of metastatic lymphatic involvement. while getting the maintenance treatment with bevacizumab the patient presented symptoms of arthritis and hypercalcemia was detected ( . ) with pth . a gammagraphy was performed and an increased uptake area was detected in the lower pole of rtl, suggestive of a parathyroid adenoma. we initially proposed the possibility of performing radiofrequency ablation but in a previous thyroid ultrasound we visualize nodular lesions in rtl compatible with adenoma and a mass in the superior mediastinum that seems to correspond the area of greatest uptake in the gammagraphy so finally the procedure is dismissed and surgery is proposed. during the intervention we found a hard consistency nodule in the inferior pole rtl and lymphadenopathies of hard consistency in right vi area that are sent for intraoperative anatomopathological study with the result of adenocarcinoma metastasis without identifying origin. a total thyroidectomy, parathyroidectomy and central ganglion drainage is performed with the result of a parathyroid adenoma, lymphatic invasion of ovarian-grade latent carcinoma and extensive vascular permeation by carcinoma of the thyroid. the patient maintains oncological treatment with carboplatin-caelix. in the last follow-up, the pth and calcemia remains normal. conclussion: although some cases of neoplasic thyroid involvement associated with struma ovarii have been published, no cases similar to the one described are found, neither in our experience, which is why it is an exceptional case. the aim of the study was to evaluate the effectiveness of the use of embolization of the splenic artery in order to prevent portal bleeding. methods: the study included patients, who had esophageal varices bleeding, which developed as a result of decompensated cirrhosis of the liver of various etiologies of classes b and c according to child-pugh. patients were divided into groups. the main group included ( . %) patients who underwent endoscopic ligating of bleeding varix and in order to prevent recurrence of bleedingembolization of the splenic artery with gianturco coils. the comparison group consisted of ( . %) patients who received only drug therapy. to assess the effectiveness of the treatment, the patient's condition was monitored for months. results: the average age of patients in the comparison group was . ± . years. using only drug therapy, we stopped bleeding in ( . %) patients. in all cases, at the end of treatment, we received an improvement in clinical and laboratory parameters. ( . %) patients died. the duration of treatment was . ± . days. the average age of patients in main group was . ± . years. performing endoscopic ligation of bleeding varices, we stopped bleeding in ( . %) patients. in all cases, at the end of treatment, we received an improvement in clinical and laboratory parameters. ( . %) patients died. the duration of treatment was . ± . days. a statistical analysis of mortality and duration of treatment revealed a significant difference (p \ . ) between the groups in both indicators. after splenic artery embolization in all cases managed to achieve a reduction in blood flow of - %. after months among patients in the comparison group, bleeding relapse occurred in ( . %) cases. in the main group, this indicator was . % ( patients). the indicator in the main group was significantly (p \ . ) different from the same indicator in the comparison group. conclusion: performing embolization of the splenic artery in patients after endoscopic hemostasis of variceal bleeding allows to reduce the pressure in the portal system, which in turn leads to a decrease in the frequency of bleeding recurrences. thoracoscopic esophagectomy for aortoesophageal fistula y. ebihara, t. shichinohe, y. kurashima, s. murakami, surgery ii, hokkaido university, sapporo, japan background: aortoesophageal fistula (aef) is an uncommon but one of highly fatal conditions. there are surgical, endoscopic and interventional radiological treatment options, however, definitive treatment is the surgical intervention. video-assisted thoracoscopic surgery (vats) has been gradually accepted as a substitution for thoracotomy to reduce the invasiveness of the surgery as radical surgery for esophageal cancer. we aimed to evaluate a feasibility of vatsesophagectomy (vats-e) for aef in this study. introduction: achalasia is the most common motility disorder of the esophagus. heller's cardiomyotomy associated with a antireflux technique is the treatment of choice in patients with this disease; however, a small group of patients could present a recurrence of the symptoms being necesary a new surgery, what is an important challenge for most of the surgeons. we report the case of recurrence after a laparoscopic miotomy and dor fundoplication as a paradigm for the appropiate management in this kind of patients. methods: a years old female, who underwent a previous miotomy and a dor fundoplication in due to an achalasia.six years after surgery, the patient showed epigastric pain and dysphagia. the study of the patient was performed with: barium swalow, phmetry, manometry, ct-scan and mri showing a recurrence of her disease.the patient was transfered to our center where she underwent a new surgery.the key points of the new surgery includes the next steps: dissection of the previous adhesions, dissection of the dor's partial fundoplication, avoid dissection of the anterior esophageal wall at the leve lof the hiatus (the area of previous myotomy) in order to avoid perforation of the esophagus, lateral and posterior dissection of the distal esophagus, lateral myotomy at the rigth wall of the esophagus and a toupet's funduplicatury. all of thisis procedures are done under intraoperative endoscopy in order to confirm a good passage to the estmach and to identify a perforationic supervision. results: following theseis steps several patients have been operated in our center with excellent results. in all of these cases, including the patiente presented previously, the symptoms have dissapeared. conclusions: achalasia is a rare motility disorder of the esophagus, being recurrences an important challenge for surgeons. a great proper therapeutic strategy using the different diagnostic exams and the supervison by a group of experts in this kind of entity are the basis in order to obtain good results in these situations. aims: re-do fundoplication is usually performed for recurrent reflux symptoms due to wrap failure or recurrent hiatus hernia. conversely, persistent dysphagia may occur early due to tight wrap/crural repair which should be avoided by good surgical technique. a small group of patients however may suffer progressive dysphagia due to weakening motility (especially in older patients), fibrosis of the wrap or a combination of the two. this video demonstrates the successful treatment of this problem with a laparoscopic conversion from nissen to posterior toupet fundoplication. a year old man underwent an uncomplicated laparoscopic nissen fundoplication in with complete resolution of reflux symptoms. he re-presented years later, still free of reflux but suffering progressive dysphagia and troublesome regurgitation. investigations demonstrated intact wrap and no mechanical obstruction, but confirmed low-amplitude peristalsis. a trial endoscopic dilatation improved symptoms for days before recurrence, suggesting likely wrap fibrosis (which would reduce elasticity and impede passage of food bolus), justifying consideration for a conversion from nissen to toupet. results: this video demonstrates the expected adhesions between fundoplication and inferior surface of left lobe liver, mobilisation and division of the nissen fundoplication, and reconstitution of a posterior toupet fundoplication. the patient made a good recovery and was discharged the following day. three-and six-month follow-up confirmed complete resolution of symptoms with no recurrence of reflux. conclusion: laparoscopic re-do surgery for late-onset progressive dysphagia is a safe and viable option. patients must be thoroughly investigated and carefully selected for an appropriately tailored procedure. they should also be advised of the increased risks associated with re-do surgery. the anatomy can be unpredictably distorted by variable adhesions and this operation should therefore only be performed by laparoscopic surgeons experienced in both primary and re-do fundoplication. methods: i report unusual iatrogenic injury of cervical esophagus that resulted with complete resection post total thyroidectomy for papillary ca of thyroid patient presented days post surgery to our center. the video will show the steps used to treat this unusual complication by neck exploration, laparoscopic trans hiatal esophagectomy with creation of gastric tube with preservation of the right gastroepiploic artery and the neck anastomosis between the cervical esophagus and stomach. were open and minimally invasive esophagectomies. of the patients, were for squamous cell carcinoma, were adenocarcinoma and were of other histological diagnosis such as gastrointestinal stromal tumor and schwannoma. the median length of stay for patients who underwent minimally invasive esophagectomies was days ( to days) while the median length of stay for patients who underwent open esophagectomies was days ( to days). the minimally invasive group had a shorter icu stay of day. for day morbidity, the minimally invasive esophagectomy group had patients who encountered anastomotic leaks, with post operative pneumonia while the open esophagectomy group had patient with anastomotic leak, patient with post operative stricture and patient with delayed gastric emptying. there were mortalities in the minimally invasive group while there were no mortalities in the open group. conclusion: our data show that patients who underwent minimally invasive esophagectomies had a shorter duration of hospitalization with similar perioperative morbidity rates. minimally invasive esophagectomy is a viable surgical option for a select group of patients. aims: there has been an increasing tendency towards minimally invasive surgery for esophageal cancer. our aim was to evaluate the results of the thoracoscopic approach (ta) and compare them with the ones of open approach (oa) at our institution. methods: retrospective review of all patients who underwent esophagectomy due to esophageal cancer (adenocarcinoma or squamous cell) between and were included. patients with siewert iii tumors and those who didn't need a thoracic approach were excluded. results: during the study period were performed esophagectomies, through ta. in . % of these, the abdominal stage was done by laparoscopy. when comparing ta versus oa, there were no statistically significant differences in the baseline characteristics of the two groups (mean age, median body mass index, ecog performance status, asa score, smoking status, diabetes mellitus, pulmonary disease, histologic type, clinical staging and neoadjuvant chemo and radiotherapy). regarding outcomes, there were no significant differences in need of intraoperative transfusion, median intraoperative blood loss, operative time and length of stay. although not significant, in ta group there was a tendency for higher overall morbidity ( . % versus . %, p = . ); major morbidity-ctcae - ( . % versus . %, p = . ); anastomotic leak ( . % versus . %, p = . ) and re-intervention rate ( . % versus %, p = . ). on the other hand, in ta group there was a tendency (although not significant) towards lower rate of respiratory complications ( . % versus . %, p = . ), lower rate of r margins ( . % versus . %, p = . ) and higher median of lymph nodes removed ( versus , p = . ). conclusions: in our series, outcomes of ta were similar to oa, with a tendency towards lower respiratory complications, lower rate of r margins and higher number of lymph nodes removed in ta group. the impact of these findings in survival remains to be seen. the tendency towards higher morbidity may be related to the learning curve, since this were the first cases performed at our center. background: esophagectomy is a surgical procedureburdened by a high morbidity rate. the effect of minimally invasive (mi) approach on elderly patients is still not clear. aim: of this study was to analyze the impact of mi approach on post-operative course according to the patient age. methods: a consecutive series of patients underwent to elective oncological esophagectomy between and . all data were entered into a prospective database. patients submitted to -flield or trans-hiatal esophagectomywere excluded andonly ivor-lewisopen, hybrid or totally minimally invasive esophagectomywere. patients were stratified according to age in groups:group a(= years) patients, group b ([ and \ years) and group c (were = years) .clinical and pathological factors influencing surgical outcome were evaluated. complications were classified according to clavien-dindo (cd). results: as expected outcomes worsened with patients age(cd = b: . % group a, % group b and % group c. p = . ), mortality ( % group a, % group b and . % group c. p = . ) and length of stay ( days group a, days group b and days group c. p = . ).a statistically significant higher incidence of anastomosticleaks was observed among patients submitted to totally mi esophagectomy in group c vs a and b that were respectively , %, % and %. major respiratory complications were not statistically different among these three sub-group. conclusions: old age has a significant impact on outcomes afteresophagectomy. in this subset of patients a mi approachcould also increasepostoperative morbidity. elderly patients should be carefully selected before to be submitted to mi esophagectomy. introduction: esophagectomy is a major surgical procedure with morbidity and mortality related to the patient's condition, stage of the disease, complementary treatments, and surgical experience. minimally invasive esophagectomy (mie) may lead to a reduction in perioperative morbidity and mortality with very good quality of life. material and method: we present the experience of the center of excellence in esophageal surgery regarding totally mie through thoracolaparoscopic modified mckeown three-stage approach followed by esophageal reconstruction by gastric intrathoracic pull-up and cervical esophagogastric anastomosis used for the treatment of thoracic esophageal cancer. results: in the last years, mie was performed initial, in our clinic with extracorporeal preparation of the gastric conduit with reduced lung complications and hospital stay. we introduced the totally minimally invasive esophagectomy with laparoscopic-assisted feeding jejunostomy using a d high definition camera. operative times were: thoracic- min, abdominal- min and cervical- min with a total of min. the augmented d high definition image provided an excellent visual field, that allowed an accurate identification of dissection plans and extensive periesophageal and perigastric lymphadenectomy. the short-term outcomes of the totally minimally invasive esophagectomy procedure were very encouraging with early feeding on jejunostomy and the control of cervical anastomosis was usually performed in the th day postoperative and the patients were discharged in the th day postoperative without any symptomatology. at the first and third-month follow-up was not reported any major complications. the long-term oncological results are being evaluated. conclusions: the totally minimally invasive approach using advanced technology of endoscopic surgery allowed for these patients a simple postoperative evolution, no major complications, and a good recovery after an extensive surgery. the solid experience in open esophageal surgery of the upper gastrointestinal surgeons provides a fast learning curve of complex minimally invasive surgical procedures with reduced perioperative morbidity. long-term follow-up should confirm the results from the literature regarding the survival, which is expected to be for these patients at least equivalent with outcomes after open esophagectomy. introduction: esophageal fistulas, benign or malignant, represent a real challenge for the surgeons and gastroenterologists, regarding the treatment and the outcome. in these cases, endoscopic treatment is the first line approach, being less invasive and sometimes avoiding the need for surgery. this includes clips, stents, glue and even suture. material and method: we have analyzed esophageal fistulas in patients with benign or malignant pathology, diagnosed and treated in the first months of . the management of this complication included a self-expandable esophageal metallic stent. we have evaluated the diagnosis, the surgical intervention, the timing until the development of the leak, the localization and management of the fistula. results: were postoperative leaks and spontaneous esophageal fistulas. the localization was cervical in one case, thoracic in cases and abdominal in cases. for the postoperative fistulas, in patients the treatment included at least one surgical reintervention with lavage and drainage, beside the insertion of an esophageal metallic stent. in the other cases, endoscopic treatment and antibiotic therapy was enough. in cases, the stent migrated needing repositioning. days mortality was %, both patients from postoperative group. conclusions: esophageal fistulas represent a severe complication, usually in patients already immunocompromised. endoscopic management, including self expandable esophageal metallic stent, can be the main approach, by stopping the contamination and by permitting the early per oral feeding. disadvantages include the possibility of migration and the need of removal after - weeks. methods: five hundreds and one patients with esophageal cancer who underwent mie from to at our department were eligible. we considered the risk factors of complications of pneumonia, anastomotic leakage, and hoarseness after surgery, and the risk factors of difficulty of surgery. results: the risk factors of postoperative complications in univariate analysis were more than years old (odds ratio: . , p = . ), more than ii in asa-ps (odds: . , p \ . ), more than g of bleeding (odds: . , p = . ), more than min. of operation time (odds: . , p \ . ), and colon reconstruction (odds: . , p = . ). the one in multivatiate analysis was more than ii in asa-ps (odds: . , p = . ). the risk factors of much bleeding were colon reconstruction (odds: . , p \ . ), and more than of lymph node dissection (odds: . , p = . ). the risk factors of long operation time without cervical lymph node dissection were neo-adjuvant therapy (odds: . , p \ . ), more than of lymph node dissection (odds: . , p = . ), and colon reconstruction (odds: . , p \ . ). the ones with cervical lymph node dissection were more than pstage iii (odds: . , p \ . ) and more than of lymph node dissection (odds: . , p = . ). conclusions: considering those risk factors, we should perform perioperative management more carefully. method: sa -year-old man with a tobacco and alcoholic habit was suspended for years, under treatment for arterial hypertension, who consults for a logical dysphagia of months of evolution. he is diagnosed of stenosing esophageal distal third epidermoid carcinoma txn m . it is decided to place a prosthesis that is effective and subsequent neoadjuvant qt-rt, after weeks of its completion the surgery is performed. results: the surgery is performed in times, initially by laparoscopy. the esophageal hiatus and the greater curvature are dissected preserving the right gastroepiploic, and lymphadenectomy of the celiac trunk with pedicle section of the left gastric. gastric plasty is performed with a section of lesser curvature towards fundus. it is continued by thoracoscopy. a section of the azygos vein is performed, dissection of the esophageal middle and lower third and lymphadenectomy. gastric plasty is promoted, proximal esophagus section and latero-lateral intrathoracic gastro-oesophageal anastomosis. the anatomopathological study reports ypt and pn with / adenopathies, and disease-free surgical margins. he was discharged without complications on the th day and did not require re-entry. conclusions: ivor-lewis endoscopic surgery is safe and meets oncological criteria in selected patients with distal esophageal neoplasia and performed by an experienced esophagogastric unit. background: the rates of thoracoscopic esophagectomy performed in the prone and left lateral decubitus positions are similar in japan. we retrospectively reviewed short term outcomes of thoracoscopic esophagectomy for esophageal cancer performed in the left lateral decubitus position under artificial pneumothorax by co insufflation in a single institution. this study aimed to evaluate the feasibility of applying this procedure. methods: between july and december , patients with esophageal cancer underwent thoracoscopic esophagectomy in the left lateral decubitus position under artificial pneumothorax by co insufflation. the thoracic procedure is performed as follows:the lymph nodes around the right recurrent laryngeal nerve are dissected. on the cranial side, the lymph node dissection is advanced to the level of the inferior thyroid artery. then, the assistant rotates the trachea toward the ventral side, and the lymph nodes around the left recurrent laryngeal nerve are dissected. the middle and inferior mediastinal lymph nodes are dissected including supradiaphragmatic lymph nodes and the dorsal lymph nodes around the thoracic descending aorta. then, the esophagus is transected using an automatic suture device. finally, the tracheal bifurcation area lymph nodes are dissected. we retrospectively analyzed these patients. results: the completion rate of thoracoscopic esophagectomy was . %, and the procedure was converted to thoracotomy in five patients, due to hemorrhage,severe adhesion. the mean intrathoracic operative time, intrathoracic blood loss, and number of dissected mediastinal lymph nodes were . min, . ml, and . , respectively. postoperative complications included pneumonia ( . %), anastomotic leakage ( . %), and recurrent nerve paralysis ( . %). postoperative ( d) mortality was / ( . %) due to ards and nomi, respectively. conclusions: standardization of the procedure for thoracoscopic esophagectomy in the left lateral decubitus position under artificial pneumothorax by co insufflation, with a standardized clinical pathway for perioperative care led to favorable surgical outcomes. introduction: recently thoracoscopic surgery has become widespread even in chest procedure in thoracic esophageal cancer surgery. as an advantage of minimally invasive esophagectomy, it is possible to perform sophisticated procedures due to its magnified visual effects. on the other hand, short-term perioperative safety and oncological safety are still unclear. in cases where abnormal anatomy or comorbidity in the thoracic cavity is observed, it is thought that it is necessary to carry out thoracic surgery which ensures safety while keeping in mind the transition to transthoracic surgery. here, we report on esophageal resection of the thoracic esophageal cancer accompanied by a mm saccular aneurysm inside the aortic arch. patient: a -year-old man visited a nearby doctor with a chief complaint of discomfort during swallowing. upper gastrointestinal endoscopy examined middle cervical esophageal cancer and received referral to our hospital. ct revealed a mm saccular aneurysm inside the descending aorta in contact with the thoracic esophagus. preoperative diagnosis was middle thoracic esophageal cancer; -iic ct bn m stageia (uicc th). we performed thoracoscopic esophagectomy and lymph node dissection as curative surgery. the anterior surface of the aorta was exposed from the lower mediastinum and descended ascendingly, reaching the lower end of the saccular anus at the head level of the lower pulmonary vein. peeling off the esophagus dorsal side along the margin of the saccular sac and performing esophageal resection. conclusion: we reported thoracoscopic esophageal resection for thoracic esophageal cancer with chest descending aortic saccular aneurysm. thoracoscopic surgery, which can fully exploit close magnification effect, seemed to be useful for anatomically disqualified cases. introduction: anastomotic leakage from oesophagojejunal (oj) anastomosis after total gastrectomy is associated with a high morbidity and mortality rate. leakage rates reported vary between ?% and ?% but lack of consensus in management. in the past, it often required surgical intervention or radiologically abscess drainage that will keep patients fasted with external drain for a long duration. recently, variable endoscopic options-oesophageal stents, clips, fibrin glue and endoluminal vacuum therapy had been introduced with variable outcomes. here, we presented a case of oj anastomotic leak management with combination innovative endoluminal and radiologically technique to insert double pig-tailed catheter. aim: to introduce the feasibility of double pig-tailed catheter for drainage and management of oj anastomosis leak. a year old man presented with two months history of dysphagia. upper endoscopy (ogd) showed suspicious cardio-oesophageal lesion. histology biopsy confirmed with adenocarcinoma. ct-scan of thorax, abdomen and pelvic showed irregular thickening at cardiooesophageal junction with regional lymphadenopathy. no distant metastases. he underwent uneventful d total gastrectomy. on th post-operative day, patient had spike fever and newly developed atrial fibrillation. urgent ct-thorax, abdomen and pelvis with oral omnipaque. it showed lower mediastinal gas-containing fluid adjacent to oj anastomosis within the left retrocrural space suspicious for leak. ogd evaluation showed pin-hole oj leak. guidewire inserted via endoscopy into left retrocural space under radiologically guidance. double pig-tail fr cm subsequently inserted via seldinger approach over guide wire. the proximal end of pig-tail pushed into left retrocural space and distal end positioned into efferent jejunal limb with crocodile jaw through endoscope. diluted contrast injected and passed down to efferent limb with minimal leak. outcome: after double pig-tail insertion, patient started on clear feed on st day post-insertion. one week later, he was started on full feed. repeat upper endoscopy and stent removal done two weeks later. contrast injection showed small blind ended sinus tract from anastomosis toward left pleural space without obvious leak. conclusion: radio-endoscopic is a novel minimally invasive technique that allows insertion of double pig-tailed internal drainage to control oj anastomosis leak. it allows early enteral nutritional feeding and avoid external drainage. background: the number of gastric cancer (gc) survivors, especially long-term survivors, is increasing. how best to evaluate the diseasespecific survival (dss) of gc survivors over time is unclear. we aimed to assess changes in the conditional survival of patients with gc after curative intend gastrectomy and the evolution of the impact of well-known risk factors. methods: clinicopathological data from , patients who underwent curative intend resection for gc at four specialized centres (three in china and one in italy) and from the surveillance, epidemiology, and end results (seer) database were retrospectively analysed. changes in the patients' -year conditional disease-specific survival (cs ) were analysed. we used time-dependent cox regression to analyse which variables had long-term effects on dss and devised an accurate, dynamic dss predictive model based on the length of survival. results: the median follow-up time was months, and disease-specific death occurred in , cases ( . %). the dss of the patients after surgery was dynamic, and most of the disease-specific deaths occurred within the first years after surgery. based on -, -, -, -and -year survivorships, the cs of the population increased gradually from % to . %, . %, . %, . %, and . %, respectively. subgroup analysis showed that the cs of patients who had poor prognostic factors initially demonstrated the greatest increase in postoperative survival time (e.g., n b: . %- . %, ? . % vs. n : . %- . %, ? . %). time-dependent cox regression analysis showed the following predictor variables constantly affecting dss: age, the number of examined lymph nodes, t stage, n stage and site (p all \ . , years after gastrectomy). the influence of prognostic factors on dss and cs changed dramatically over time. based on data from several large global centres, we developed an effective model for predicting the dss of gc patients based on the length of survival time. this model can provide personalized long-term follow-up strategies for patients. methods: we retrospectively analyzed clinicopathological data for rgc patients who underwent radical gastrectomy from centers. the prognosis prediction performances of the ajcc th and ajcc th tnm staging systems and the trm staging system for rgc patients were evaluated. web-based prediction models based on independent prognostic factors were developed to predict the survival of the rgc patients. external validation was performed using a cohort of chinese patients. result: the mean number of retrieved lymph nodes was . , and in . % of patients, the number was = . the predictive abilities of the ajcc th and trm staging systems were no better than those of the ajcc th staging system (c-index: ajcc th vs. ajcc th vs. trm, . vs. . vs. . ; p [ . ). within each staging system, the survival of the two adjacent stages was not well discriminated (p [ . ). multivariate analysis showed that age, tumor size, t stage and n stage were independent prognostic factors for overall survival (os), disease-specific survival (dss) and disease-specific survival (dfs). based on the above variables, we developed web-based prediction models, the huang os model, the huang dss model and the huang dfs model, which were superior to the ajcc th staging system in their discriminatory ability (cindex), predictive homogeneity (likelihood ratio chi-square), predictive accuracy (aic, bic), and model stability (time-dependent roc curves). the stratified analysis showed that regardless of whether more or fewer than lymph nodes were retrieved, the predictive performances of the web-based prediction models were still better than those of the other three staging systems. a decision curve analysis showed that the huang model provided better net benefits than the other three staging systems. external validation showed predictable accuracies of . , . and . , respectively, in predicting os, dss and dfs. conclusion: the ajcc tnm staging system and the trm staging system did not enable good distinction among the rgc patients. we have developed and validated visual web-based prediction models that are superior to these staging systems. objective: to perform competing risk analysis and evaluate cancer-and noncancer-specific mortality in patients with gastric cancer after radical surgery. methods: a total of patients from our department (as training set) and a total of patients from the surveillance, epidemiology, and end results (seer) database (as validation set) were enrolled in the study. the cumulative incidence of cancer and noncancer-specific mortality was determined by univariate and multivariate competing risk analysis. results: the five-year cancer-and noncancer-specific cumulative incidence of death (cid) in the training set were . % and . %, respectively, which were significantly lower than that in the validation set ( . % and . %, respectively). multivariable analysis showed that age, tumor site, tumor size and ptnm stage were independent predictors of gastric cancer-specific mortality and overall survival, whereas age was an independent predictor of gastric noncancer-specific mortality. noncancer-specific cid surpassed cancer-specific cid for ptnm stage i patients after approximately years of surgery, but never for stage ii and iii patients. moreover, for stage i patients, the time point when noncancer-specific cid surpassed cancer-specific cid become earlier as age increasing, with only . years after surgery for patients more than years of age. conclusions: age is an independent predictor of gastric cancer-and noncancer specific mortality and overall survival for patients after radical surgery. for patients with stage i gastric cancer, noncancer-specific mortality is a significant competing event, with an increasing impact as age increases. aim: of the study was to analyse the possibility of function preserving gastrectomy based on the sentinel lymph node (sln) concept. methods: during last years in two clinics odessa national medical university we used mapping procedures in the patients with early gastric cancer. there were men and women, age to years, mean age . ± . years. blue dye was injected into quadrants of the submucosal layer surrounding the primary lesion using an endoscopic puncture needle in patients. blue lymphatic vessels and blue-stained lymph nodes can be identified by laparoscopy within min. of the blue dye injection. we used . % indocyanine green in patients, which we injected by intraoperative endoscopy. new technology indocyanine green (icg) fluorescent imaging was used for sln mapping in this patients. results: amany patients, in which we used blue dye for mapping sln, positive sln was in patients, negative-in patients. in all patients distal gastrectomy (dg) was performed with d lymphdissection. from patients with negative sln in patients metastasis in other lymph nodes were detected.among patients in whom we used icg fluorescent mapping positive sln were detected in patients. laparoscopic-assisted distal gastrectomy with d lymph node dissection was performed in these patients. in patients with negative sln partial wedge resection was performed in patients, segmental pylorus preserving gastrectomy was performed in patients. during follow-up period from to months no recurrences or metastasis were detected in these group of patients. qol in this group of patients was much better, than in patients with conventional distal gastrectomy. conclusions: icg fluorescent method is highly effective for detection of sln. in the patients with early gastric cancer function preserving gastrectomy based on sln navigation may be promising strategy to achieve better results. laparoscopic procedure taking advantage of robotic gastrectomy for gastric cancer to prevent pancreatic fistula gastrointestinal surgery and surgical oncology, ehime university, toon-city, japan backgrounds and aims: analysis of japanese national clinical database (ncd) showed that laparoscopic gastrectomy(lg) had rather increased pancreatic fistula (pf) compared with open gastrectomy. on the other hand, last year, multicenter collaborative research result of robotic gastric cancer surgery(rg)was shown that the complications including pf were significantly decreased as compared with lg. in this study, we have employed a new easy to use device in lg to minimize pf during suprapancreatic lymph nodes dissection requiring pancreatic retraction and compared with conventional lg and rg. materials and methods: internal organ retractor (aesculapÒ) to grasp the gastropancreatic fold and the suprapancreatic peritoneum to imitate davinci's forceps was guided with a thread outside the body. patients(jan. * nov. ) were divided into three groups as follows, group lg- (n = ), lg using the standard devices, group lg- (n = ), lg using organ retractor, group rg (n = ). amylase value in drain(d-amylase) and the volume in drainage, intraoperative bleeding, postoperative hospital stay, incidence of cd (] grade iii) were compared among three groups. results: data are indicated as lg- /lg- /rg(mean ± sd), respectively. on the day and third day after surgery, d-amylase were ± / ± / ± and ± / ± , ± (iu/l). d-amylase was significantly lower in lg- and rg group than in lg- the day after surgery. the operation time was significantly longer in rg, ± / ± / ± (min). bleeding volume and hospital stay did not differ among groups. pancreatic fistula (cd ] grade iii)was observed only in lg- group at (%) . discussion: pf(grade]cdiii), which may lead to mortality, occurred in lg- group. a significant elevation of d-amylase on the st postoperative day was prevented in lg- just like rg, which seemed to lead to prevent pf afterwards. the multijoint forceps is known to be an advantage of rg but it cannot be reproduced by lg using a linear forceps. however, another advantage such as vertical grasping and lifting of the gastropancreatic fold at rest could be mimicked by lg using this device, which seemed to enable a safe lymph node dissection and lead to reduce the pancreatic damage. conclusion: this inexpensive and easy to use method taking the advantage of rg seems to reduce surgeon's fatigue and tissue damage(pf). the study presents comparison of perioperative outcome between different surgical approaches for gastric adenocarcinoma (ac). methods: retrospective cohort of patients that underwent gastrectomy for (ac) at rambam hospital during - . patients data was collected based on demographic characteristics, bmi, operating room time (ort), number of lymph nodes (ln), length of hospitalization (loh), and perioperative complications. results: study population included patients after total gastrectomies, of them robotic and partial gastrectomies, of them robotic. age, gender and bmi were similar between patients who underwent any type of procedures. median length of hospitalization (loh) for robotic total gastrectomy was . days and it was significantly shorter than both laparoscopic total gastrectomy (ltg) . days (p = . ) and open total gastrectomy (otg) . days (p \ . ). similar significant differences in (loh) between the groups were observed among patients who underwent partial gastrectomy, but the comparison between robotic and laparoscopic procedures was limited due to small numbers of (lpg). median(ort) was significantly longer among robotic gastrectomies compared to open, the difference was min in total gastrectomy group and min in partial gastrectomy group (p \ . for both differences), but the difference in(ort) between laparoscopic and robotic procedures were smaller and non-significant. the number of dissected (ln) was similar between the procedures in total gasrectomies. in partial gastrectomies, the number of dissected (ln) was even higher among both laparoscopic and robotic gastrectomies compared to open (p \ . ).) conclusions: robotic total and partial gastrectomies for gastric (ac) are associated with oncologically adequate lymphadenectomy and faster patient recovery, but longer ort. objectives: during esophagojejunostomy using a circular stapler after latg, placement of the anvil head via the transabdominal approach proved difficult. the authors report on a method modified for laparoscopy-assisted, esophagojejunostomy performed by placing the pretilted anvil head(orvil) via the transoral approach. methods: between january and november , esophagojejunostomy was performed using orvil in patients after latg. the anesthesiologist introduced the anvil while observing its passage through the pharynx. during the anastomosis, we kept the jejunum fixed in position with a silicone band lig-a-loops, thereby preventing the intestine from slipping off the shaft of the stapler. results: esophagojejunostomy using the orvil was achieved successfully in all patients. no other complications, such as hypopharyngeal perforation and/or esophageal mucosal injury, occurred during passage. the postoperative complications of anastomosis were leakage in two patients and stenosis in patients, in whom mild relief was achieved using a bougie. conclusions: esophagojejunostomy using the orvil is a simple and safe technique. gastrointestinal tract surgery, fukushima medical university, fukushima-shi, japan; surgery, ohara general hospital, fukushima-shi, japan background: juvenile polyposis of the stomach is a very rare disease, and its malignant potential has been reported previously and total gastrectomy has been recommended as a standard treatment. recently, the usefulness of laparoscopic surgery for this case has been reported, however this type of surgery is thought that maintaining the surgical space is difficult because of distended and thickening stomach. case presentation: eight years ago, a -year-old woman who had no family history of gastrointestinal polyposis had been diagnosed with gastric polyposis and polyp-related anemia and received twice endoscopic submucosal dissection to early gastric cancer in another hospital. she had received an annual upper gastrointestinal endoscopy and she had taken iron supplements for anemia caused from the occasional bleeding from the polyps. however, the number of the polyps had increased over time. because she had a loss of appetite, she admitted to our hospital. enhanced computed tomography showed gastric wall thickening and multiple gastric polyps without lymphadenopathy or distant metastasis. colonoscopy showed no specific findings. she was diagnosed as the juvenile polyposis of the stomach, and she received laparoscopic total gastrectomy with roux-en y esophagojejunostomy. in operative findings, although there were the excessive distention and congestion of the stomach, standard laparoscopic surgery could be performed. the resected specimen revealed multiple variously sized polyps throughout the stomach except for lesser curvature and fundus and the histopathological examination revealed that all polyps were hyperplastic polyps without containing cancer. she was discharged on postoperative day . we successfully performed laparoscopic surgery to treat a rare case of juvenile gastric polyposis. introduction: we report a novel technique for combined use of laparo and thoracoscopy for faradvanced adenocarcinoma of esophagogastric junction (aeg). case presentation: a 's years old man presented with far-advanced aeg. an esophagogastroduodenoscopy revealed a type lesion with the entire circumference around esophagogastric junction (egj). contrast radiography revealed a severe stenosis in the egj and wall irregularity from egj to cardia. computed tomography revealed a stenosis of egj, suspected invasion into the left side diaphragm and some lymph nodes metastases at the abdomen. we diagnosed siewert type ii aeg (ct an m , cstage iiia : japanese classification of gastric carcinoma ver. ). surgical technique :the patient was placed in the reverse-trendelenburg position with the left upper body lifted and legs spread, under general anesthesia. the tumor was huge, exposed from the serous membrane and invaded the left crus. first we performed from laparoscopic proximal gastrectomy using five ports. then, three ports were added in the th, th, and th intercostal spaces with the patient in the same body position, and performed thoracoscopic lower esophagectomy under artificial pneumothorax with intrathoracic pressure of - mmhg, which allows the ventilation of both lungs. the lower esophagus was resected under the thoracoscopic view to ensure an adequate margin. following this resection, intrathoracic esophagojejunostomy was performed by using the laparo-and thoracoscopic techniques. the operative time was min, and the blood loss was g. he was discharged on the th day after the operation without any postoperative morbidity. the histopathological diagnosis was pt bn am , p , pstage iv. after adjuvant chemotherapy with capecitabine and oxaliplatin, ramcilumab monotherapy is undertaken now. ct revealed solitary lung metastasis in months after the operation. conclusion: malta for locally advanced aeg invading the surroundings could be performed safely. introduction: despite being the pioneer in laparoscopic surgery, europe did not have similar surgical experience compared to east asia due to decreased exposure to gastric cancer. several studies on minimally invasive gastrectomy for gastric cancer have been conducted in europe. however, some of them did not analyse total gastrectomy as a distinct entity combining both distal and total gastrectomies; moreover, most of them do not provide data on full five-year follow up for each patient. baltic countries stand in between east and west in terms of gastric cancer incidence: incidence rate per , is . in united kingdom, . in lithuania and . in japan. this exposure to gastric cancer provides unique opportunity to investigate the role of laparoscopic gastrectomy. therefore, a case-control study was designed to evaluate laparoscopic (ltg) versus open total gastrectomy (otg), comparing short-term surgical and long-term oncologic outcomes. surgery, jeju national university, school of medicine, jeju, korea; surgery, chosun university, school of medicine, gwangju, korea objective: although mcv (mean corpuscular volume) levels are known to be associated with the prognosis of various diseases, few study investigated mcv as prognostic factor after gastric cancer surgery. the aim of this study is to address the prognostic value of mcv in gastric cancer who underwent curative gastric cancer surgery. methods: patients (june -december ) with stage i, ii, and iii cancer were consecutively included in this study. all patients underwent curative gastric cancer surgery including subtotal gastrectomy or total gastrectomy. overall survival (os), disease-free survival (dfs) and postoperative complications rate were compared between mcv [ group and = group. results: of all patients, the mean mcv was fl (normal range, to fl). the dfs was significantly higher in the high-mcv ([ ) than low-mcv group(= ) (p \ . ) group. there was no significant difference in postoperative complications when compared with clavien-dindo scale. the survival rate of the high mcv group was higher but there was no significant difference. conclusions: mcv may be a predictive factor after gastric cancer surgery. unlike previous studies, patients with low mcv group showed lower dfs. more research is needed on the significance of mcv in variety of disease. methods: and materials. for years we observed cases with gist of stomach and duodenum. seven patients were brought to clinic with the bleeding and two patients were brought to clinic with vomiting and compensate stenosis. in all circumstances we done the ct, mrt and endoscopic examinations of stomach and duodenum with biopsy . in two circumstances we performed endoscopic operation. in one circumstance we successfully take off the gist from the duodenum endoscopically. during the operation we use the endoscopic instruments. in another circumstances,after endoscopic excision the tumor appear the bleeding which was stopped by endoscopic local heamostasis, by putting clipps on the vessels. in circumstances the tumors were in stomach. in circumstances we performed laparoscopic wedge resection the tumors by staplers. in circumstances when the tumor was very big and situated in the fundus of stomach, we performed laparoscopic resection of the fundal part of stomach by using laparoscopic staplers and 'liga sure' sealing. in circumstance we took off the tumor by putting laparoscopic trocars inside the stomach for instruments and for visualization tumor. after excision the tumor and took it of the stomach we sutured the holes in the stomach. we have no mortality after laparoscopic operation. there were no malignisation in all circumstances. we have cases morbidity. in circumstance the bleeding from the stomach that was stopped endoscopically. in circumstance there was wound infection. the aim of the study to decrease the morbidity in the patients with perforated ulcers of the stomach and duodenum. we observed patients with perforated ulcers of stomach and duodenum. women were , men were . average age about years. patients had perforation ulcer of stomach and duodenum. patients had perforations with bleeding. all patients were divided in two groups. the first groups patient operated laporocopically, in the second group patients operated traditionally. results: there were no mortality in the group that operated laparoscopically. in the group that were operated traditionally one patient died after rebleeding. the average stay in hospital in the group that were operated laporoscopically about days. in the groups with traditional operations, were about days. the morbidity in the first group were in cases. pneumonia in cases, suppuration of the troacar points were in cases. in the second group pneumonia were in cases, suppuration of the operation wound were in cases, subdiaphragmatic abscess was in cases. conclusion: laporoscopic operation in during treatment decrease the mortality, morbidity and hospital staying in the patients the perforated ulcer of stomach and duodenum . of the patients of the third group ( . %) were operated about ulcer rebleeding in the hospital, and ( . %)-about the profuse bleeding ulcer. noonr patient had recurrent bleeding. the average treatment time for patients in group was . ± . days. conclusions: the development of hemorrhagic shock in patients with peptic ulcer bleeding significantly increases the risk of rebleeding and mortality. the application of endoscopic hemostasis allows to reduce the risk of rebleeding and mortality compared with conservative antiulcer therapy. surgical treatment can achieve reliable hemostasis, but accompanied by higher mortality and longer duration of hospital treatment. tan tock seng hospital is second largest hospital in singapore. it is affiliated to two medical schools in singapore and it is a training hospital for both undergraduates and postgraduates. minimally invasive surgery for both benign and malignant diseases of upper gastrointestinal tract becomes more and more popular nowadays. in our department, all the residents have to view the step by step instructional videos of mininally invasive surgeries before they can assist in the cases or perform on their own under the supervision of consultant surgeons. the viewing of the instructional videos help them with better understanding of the procedures. the viewing of videos help them with the importance of steps, standardization of steps. with the help of instructional video, they can not only assist better in the surgery but also reduce the learning curve when they start doing the procedure themselves after the graduation from the residency programme. this is the step by step instructional video of laparoscopic repair of perforated duodena ulcer for surgeons-in-training rotated to our department. in general duplication cysts are rare developmental congenital disorders of the gi tract. three morphological criteria should be met in order to confirm the pathological diagnosis: . they should be attached to the stomach's wall and should be the continuation of it, . at least one of the muscle layers of the stomach's wall should be included and .it should have normal gastric mucosa. the treatment is either enucleation or partial gastrectomy. aim: present our minimally invasive approach to a rare prepyloric submucosal cystic lesion causing gastric outlet obstruction. case report: a -year-old female with vomiting, weight loss and in bad general condition was diagnosed after a full work-up (blood tests, endoscopies, eus, ct and mri) with a submucosal cystic tumor. this cyst first was thought to be a duplication cyst. since the patient was young, our intention was to offer the least invasive surgical technique in order to spare gastrectomy and billroth anastomosis. results: the procedure was completed laparoscopically with enucleation of the cyst through a gastrotomy on the anterior wall of the stomach. after the enucleation of the cyst the gastric mucosa was sutured back and then the gastrotomy was closed with continuous sutures. the result of the pathological report confirmed a rare case of a heterotopic pancreatic cystic lesion. the postoperative course of the patient was uneventful and was discharged with instruction for her diet the th postoperative day. the patient months post-operative has no symptoms. conclusion: in such benign conditions and especially in young patients, gastrectomies could be avoided if possible and give their place to less invasive approaches in order to reduce lifelong risks and morbidity. trangastric enucleation of the cyst although a demanding approach is safe and could be considered as a 'gentler' technique with reduced morbidity. background: pancreatoduodenectomy is considered to be very invasive for early superficial duodenal tumors (sdts), which have a lower risk of lymph node metastasis. partial duodenal resection with endoscopic submucosal dissection for sdts is an attractive technique but it is associated with a high risk of complications. the full-thickness resection of the duodenum wall including laparoscopic and endoscopic cooperative surgery has risk of spreading tumor cells and digestive juices into the abdominal cavity. we have developed novel technique for sdts to decrease the risk of exposure to abdominal cavity of tumor cells and digestive juices, called nonexposed duodenum laparoscopic and endoscopic cooperative surgery (neo-dlecs). aim: the aim of this study is to evaluate the feasibility and safety of neo-dlecs for sdts. surgical procedure: the attachment of the transverse mesocolon was freed from the head of the pancreas and retroperitoneal tissues under laparoscopy. the duodenum and the head of the pancreas were mobilized from the retroperitoneum using the kocher maneuver. a standard esd was performed for the sdt using endoscope. the serosa of the esd ulcer bed was reinforced using the laparoscopic hand-sewn suturing technique in the seromuscular layer around the resected area. after completing the procedure, the endoscope was inserted and passed over the resected area to confirm that there was no stenosis or leakage. methods: ten consecutive patients with sdt underwent neo-dlecs in our institute between march and march . the clinicopathological features of the patients and surgical outcomes were prospectively collected and retrospectively analyzed. results: pathological diagnosis was adenocarcinoma for six patients, adenoma for three patients, and neuroendocrine tumor grade for one patient. the median tumor size was ( - ) mm. the median operative time was . ( - ) min. the median blood loss was ( - ) g. there were no conversions to open surgery in this series. intraoperative perforation was found in two cases during the esd procedure. however, all perforations were closed and reinforced using hand-sewn sutures. no postoperative complications were above grade in the clavien-dindo classification system. conclusions: neo-dlecs is safe and feasible and can be an option for surgical sdt resection. aims: wilkie's syndrome is caused by the entrapment of the rd part of the duodenum between the aorta and the superior mesenteric artery (sma). surgery is indicated for chronic cases and failure of conservative management, being reported a laparoscopic duodenojejunostomy as a minimally invasive option. methods: all cases treated by laparoscopic duodenojejunostomy in our centre because of chronic wilkie's syndrome were recorded. results: females and male underwent a laparoscopic duodenojejunostomy, with a mean age of years (range - ). all patients presented abdominal pain, and weight loss was identified in most of them. a reduced aortomesenteric angle measured by ct scan was the key for the diagnosis (mean angle . degrees, range - ). conventional laparoscopic approach was performed in two patients, the other two patients underwent a sils port approach. mean time of surgery was . min (range - ) and length of stay was days (range - ). after a mean follow-up of . months (range - ), patients improved their symptoms. conclusions: surgery is the mainstay in complicated or refractory cases of sma. laparoscopic duodenojejunostomy has the advantages of the laparoscopic approach (including rapid recovery time, reduced post-operative pain and shorter hospital stay) and it is feasible, safe and effective. in mexico in , gastric cancer represented the rd cause of death; it may manifest in a variety of histologic, anatomic, and genetic patterns, which influences the surgical approach. until now gastrectomy with curative intent is the only treatment that offers potential cure in gastric cancer. in recent years, laparoscopy has emerged as an important modality in the surgical management. in multiple trials no significant difference in recurrence, long-term survival and disease-free survival was observed when compared to the standard open gastrectomy. we present the case of a year old man. with a smoking history of pack years, suspended years earlier. he presented unspecific upper gastrointestinal symptoms; an upper endoscopy was made observing a suspicious depressed lesion of cm located in the greater curvature between the body and the antrum, the biopsy resulted in a poorly diferentiated signet-ring cell carcinoma of the stomach. an endoscopic ultrasound and a thoracoabdominal ct scan showed no evidence of enlarged adenopaties or metastatic disease. initially a diagnostic laparoscopy was made, there was no evidence of carcinomatosis, nor free intraperitoneal fluid; so the greater omentum was dissected towards the splenic and hepatic flexure; a d lymph node dissection was performed, and a subtotal gastrectomy with reconstruction of roux en y was done; intraoperative endoscopy was done to identify the lesion, so adequate margins could be obtained. the patient had a good post operative evolution and was discharged home at th day tolerating oral intake. minimally invasive techniques have proved equivalency of oncologic results when compared to the conventional approach; these techniques are becoming the preferred approach in the treatment of well-selected patients with gastric cancer and have a role in definitive staging, curative resection, and lymphadenectomy. appropriate selection of patients and optimal technical approach are paramount for good outcomes. most data of laparoscopic gastrectomy come from eastern countries, where the prevalence is higher; however western experience is growing along with evolution and development in surgical instruments and new technology. wilkie syndrome is a rare cause of high intestinal obstruction, resulting from the compression of the duodenum between the abdominal aorta and the superior mesenteric artery. the main symptoms are nausea and vomiting, weight loss, early satiety, abdominal distension and epigastric pain. historically, the barium study and arteriography were the diagnostic tests used; more recently the angiotac has shown greater sensitivity. the diagnostic criteria are: dilated duodenum, duodenal compression by the superior mesenteric artery and aortomesenteric angle less than degrees. patients with an acute condition usually respond to conservative treatment (decompression, correction of hydroelectrolyte alterations, nutritional support…). however, those with chronic symptoms usually require surgery preferably with laparoscopic approaches of duodenojejunostomy or the strong's procedure. the strong procedure mobilizes the duodenum by dividing the ligament of treitz. once the duodenal-jejunal junction is mobilized, the duodenum is positioned to the right of the superior mesenteric artery and it is preferred because it provides less morbidity due of the maintaining of the integrity of the gastrointestinal tract, but it has a failure rate of %. gastrojejunostomy allows gastric decompression, but does not relieve duodenal compression, so digestive symptoms may persist, leading to the appearance of a blind loop syndrome or recurrent peptic ulcers. on the other hand, the duodenojejunostomy, which according to some series may be the procedure of choice, may obtain a success rate higher than %. we advocate to initiate the surgical approach with the strong procedure and if it fails to perform to a duodenojejunostomy. during this procedure, gastro-esophageal reflux was evaluated and assigned to severe, moderate and slight category. if the reflux was observed slightly up to cervical esophagus, the case was assigned to moderate category. if the reflux was observed intensely up to cervical esophagus, the position was returned to head high position for the safety and the case was assigned to severe category. the anti-reflux surgery was considered in the moderate and severe categories. results: we have performed laparoscopic nissen procedure in cases. the outcome was assessed by reflux test performed on - postoperative day, and the results showed the reflux was disappeared in every cases. median follow-up period of this study was months ( - months) . in cases ( . %) ppi was restarted before months after the anti-reflux surgery. in cases ( . %) ppi was restarted after the anti-reflux surgery during the whole follow-up period of this study. the bmi of the patients had no relationship to the needed restart of ppi. to evaluate the degree of esophagitis objectively before and after the anti-reflux surgery we designed 'the esophagitis score'. in this scoring method, a number from - was assigned according to the degree of esophagitis along with the la classification. the results of the study have shown that the reflux esophagitis was improved obviously after the anti-reflux surgery even in the ppi restarted group (p \ . ). discussion: to extract the gerd patients who really need anti-reflux surgery is important. reflux test is feasible because of its convenience and visual effects for the patients. the results of the laparoscopic nissen fundoplication were good. background: laparoscopic paraesophageal hernia repair with fundoplication has become more and more popular nowadays due to less morbdity and mortality with shorter length of hospital stay. discussion: tan tock seng hospital is the second largest hospital in singapore. it is affiliated to two medical schools in singapore and it is a training hospital for both undergraduates and postgraduates. in our department, all the residents have to view the step by step instructional videos of mininally invasive surgeries before they can assist in the cases or perform on their own under the supervision of consultant surgeons. the viewing of the instructional videos help them understand the procedures better. the videos can also help them recognize the important steps and standardized safe approach. with the help of instructional video, they can not only assist better in the surgery but also reduce the learning curve when they start performing the procedure themselves during their training period. this is the step by step instructional video of laparoscopic paraesophageal hernia repair with fundoplication for surgeons-in-training who are posted to our department. conclusion: the step by step instructional video on laparoscopic paraesophageal hernia repair with fundoplication can help the surgeons in training reduce their learning curve and improve their surgical skills so that they can perform the procedure safely. the human immunodeficiency virus (hiv) is a neurotropic virus. there have been reports of patients with hiv who have esophageal motility problems, sometimes associated with opportunistic infections. the absence of contractility is defined as a major motility disorder according to the chicago v . classification, which is characterized by normal esophagogastric union relaxation and % peristalsis failure. we present the case of a -year-old male patient with a history of acquired immunodeficiency on treatment with efavirenz, emtricitabine and tenofovir. he presented progressive dysphagia, gastroesophageal reflux and pyrosis of months of evolution. physical examination showed no alterations. upper endoscopy is done reporting a normal esophagus and diffuse chronic gastritis. the esophagogram reported inadequate esophageal motility with contrast stasis and a delayed emptying. the esophageal manometry reported an upper esophageal sphincter with high resting pressure. the middle and distal esophagus showed absence of peristalsis with a pan-esophageal pressurization pattern. the lower esophageal sphincter presented normal resting pressure and borderline relaxation ( %). the integrated relaxation pressure was less than mmhg. the diagnostic impression was absence of contractility (chicago classification v . ).medical management was initiated with inhibitors of the proton pump, isosorbide dinitrate and injections of botulinum toxin without success. it was decided to program the patient for a heller myotomy with toupet fundoplication. a trans-surgical endoscopy revealed a complete myotomy with no leakage or obstruction. the patient went home on the second postoperative day tolerating a solid diet.heller myotomy by laparoscopy with partial fundoplication is safe in the treatment of patients with hiv and esophageal motility disorders, reporting a mortality of . %. the effect of endoscopic treatments prior to surgery is controversy aims: epiphrenic diverticulum represents an infrequent entity and it is usually associated with esophageal motility disorders, such as achalasia, distal esophageal spasm, nutcracker esophagus or hypertensive lower esophageal sphincter. nowadays, epiphrenic diverticulectomy, esophageal myotomy and partial fundoplication is the gold standard technique; although it supposes a challenging procedure and it may provoke lots of complications. approach for diverticulectomy usually depends on the distance from the upper border of the diverticulum's neck to gastroesophageal junction, considering that thoracoscopy should be carried out when this distance is more than cm. methods: we presentthecase of a -year-old male patient, with a bodymass index of anda medical history of diabetes, smoking and alcoholism. his symptoms were mainly regurgitation and dysphagia. upper endoscopy showed esophageal dilatation and the presence of a diverticulum with its neck cm over the gastroesophageal junction. ct scan confirmed these findings and manometry showed achalasia. in the video we can see how we perform a laparoscopic diverticulectomy with esophageal myotomy and dor fundoplication. results: patient was discharged home on the second postoperative day with no complication. after more than two years of follow-up, he has not suffered regurgitation, heartburn, dysphagia or chest pain. conclusions: we present a case with an epiphrenic diverticulum secondary to achalasia in which we performed a laparoscopic diverticulectomy, esophageal myotomy and dor fundoplication. some authors suggest that the correction of the underlying motility disorder is the key in the management of these patients and they do not recommend concomitant diverticulectomy for all cases. however, we consider that the complete procedure, adding diverticulectomy, supposes the gold standard and it is feasible to perform for teams which are skilled in esophageal and gastric laparoscopic surgery, despite its high morbidity rates. purpose: a laparoscopic wedge resection for a gastric submucosal tumor closed to gastroesophageal junction or involved to gastroesophageal junction is technically challenging and more aggressive compared with tumors in other sites of the stomach. a gastroesophageal reflux disease would be more prevalent after laparoscopic wedge resection of a gastric submucosal tumor in gastroesophageal junction because of the destruction to low esophageal sphincter. we hypothesized that a prophylactic anti-reflux surgery after this surgery would be less prevalent the gastroesophageal reflux disease (gerd) and more improve the quality of life of the patients. the aim of this study is to analyze our experience with prophylactic anti-reflux surgery after laparoscopic wedge resection for a gastric submucosal tumor of gastroesophageal junction materials and methods: we retrospectively collected data from patients who diagnosed with submucosal tumor of near the gastroesophageal junction underwent laparoscopic wedge resection between january and december . the patients were divided into groups according to operation with prophylactic anti-reflux surgery (group a) and without one (group b). results: there were no difference in the frequency of the preoperative gerd symptoms between the groups, whereas postoperative gerd symptoms and postoperative use of acid suppressive medications were more frequent in the group b (p = . , p = . ). however, there were no differences in the follow-up endoscopic findings in terms of reflux esophagitis and hill's grade between the groups. in group a, postoperative mean low esophageal sphincter (les) pressure was . ± . . the les pressure was dropped until mmhg in the only one patient. however, there was no reflux symptom in this patient. conclusions: the prophylactic anti-reflux surgery after laparoscopic gastric wedge resection of gastroesophageal junction is an effective method of prevent gastroesophageal reflux symptoms. background: the most critical obstacle is a pancreatic leakage(pl). the most cause of pl might be an activation of pancreatic juice by the mixing of pancreatic juice and intestinal fluid because of the anastomosis technique, the difference of anastomosis between pancreatic duct and caliber of jejunum, and the topple of jejunal mucosa. aim: in this study, we devised the new anastomotic method of pancreato-jejunostomy, so called ' pancreatic stent sliding guide' (pssg) method using a pancreatic duct stent. we would like to demonstrate its method and results. (operative procedure) the cases of hybrid laparoscopic pancreatico-duodenectomies (pd) were done by shuriken-shaped umbilicoplasty with pssg. the pancreatic duct stent, which is fit for a diameter of pancreatic duct, is used for the direct puncture without any incineration. the aims of direct puncture are both the avoidance of the enlargement of anastomotic opening and disturbance of blood flow. the contralateral of anastomotic opening is also punctured and the stent is pulled out of the jejunum. the - pds with the needles at both ends is used for anastomotic thread. firstly, the eversion anastomosis of posterior wall is done by sliding the needle on the stent. and then the anastomosis of anterior wall is done by the same way. the stent of contralateral side is cut and the hole is closed. materials and methods: the cased of pancreato-jejunostomy by pssg method were done by february . the average of patient's age was y.o. the disease of patients were pancreatic cancer (n = ), bile duct cancer (n = ), and papilla vater cancer(n = ). the pancreatic leakage by the isgpf were grade : ,a: ,b: ,c: respectively. in the same periods, we underwent the more ten cases of open pd by pssg method. the pl were only one case of grade a and there were none of clinical pl. conclusion: our new device of pancreato-jejunostomy by pssg might be very effective for the decrease of pl from the view point of machanisms of pl even for laparoscopic pd. year old, male patient presented with upper abdominal discomfort and pain, without nausea, vomiting or weight loss. an sub mucosal lesion was found on endoscopy examination in first part of the duodenum. endoscopic ultrasound has showed . cm sub mucosal lesion in first part of duodenum (anterior wall and close to pylorus). cytology examination from the lesion has showed neuroendocrine tumor. computed tomography of abdomen and chest were normal. his blood laboratory examinations were within normal limits. patient underwent da vinci robotic partial gastrectomy with intra corporeal billroth ii gastrojejunostomy. total operating time (ort) was min. three day after operation patient started regular diet and was discharged home on day fife. final pathology report confirmed diagnosis of carcinoid tumor with ki less than %. surg endosc ( ) :s -s p -robotics & new techniques-education integrated education for colorectal disease-a digital solution for a digital age united kingdom aims: surgical plume has problem in poor visibility of the operative field, inclusion of harmful chemical substances, and biological risk. it is desirable that plume should be removed appropriately to minimize these risks. we assessed whether these problems can be solved by using commercialized evacuator semi-quantification of residual chemicals in the abdominal cavity: was performed using industrial smoke tester by aspirating the intra-abdominal plume onto filter papers and digitizing the stains. ( ) detection of dna in the exhausted gas from the evacuator: the hepa filter, which was interposed at the inlet or outlet of the evacuator, was analyzed using pcr method to detect any dna derived from porcine tissues. results: ( ) laparoscopic visualization: judgement score were . vs. . for ec and . vs. . for us (evacuator: on vs. off, both p \ . ), indicating the visualization was significantly better in the use of the evacuator on both devices general surgery, royo villanova hospital general surgery minimally invasive surgery centre, jesús usón minimally invasive surgery centre methods: i report my experience at the american university of beirut medical center for laparoscopic adrenalectomy cases, left adrenalectomy and cases for right adrenalectomy. three out of the series are large adrenal of cm, and all of these were completed laparoscopically.the video will show the steps of this procedure.a large rt. adrenal mass measuring cm, wt. gm was removed laparoscopically using trocar techniques. the lateral position facilitated the exposure and ease of dissection. the mass was removed by extending one of the trocar site with muscle splitting using endocatch mm. results: patient was discharged home days after surgery. the operative time was hour. pathology revealed carcinoma with no involvement of the capsule or vascular invasion patients (male: n = ; female: n = ) underwent minimally invasive adrenalectomy (tp: n = ; rp: n = ) at our institute. mean patient age was . years ( - years). besides comparing operative (intraoperative blood loss, previous abdominal surgeries, conversion rate, operative time, tumor size) and perioperative factors (time of hospitalization, time to oral intake, histology, postoperative complications) in each group, perioperative outcomes of a learning curve (lc)-the first procedures in both groups-was also analyzed in terms of tumor size, significantly larger lesions were removed with tp (tp: . ± . mm vs rp: . ± . mm; p = . ). the number of asa (american society of anesthesiologists) ii patients were significantly higher in the tp group while there were significantly more asa iii patients in the rp group conversions ) showed no significant difference. the analysis of lc showed a significant difference in previous abdominal surgeries min vs rp: . ± . min; p = . ] all favoring the tp approach. conclusion: both methods proved to be feasible and safe in terms of minimally invasive adrenalectomy. based on our own experience the tp approach resulted in improved operative time and conversion rates to demonstrate the safety and efficacy of the laparoscopic approach in the treatment of large splenomegaly. currently, this approach is recognized as the one of choice in benign splenic pathology, being controversial in the face of a massive splenomegaly or neoplastic pathology. material and method: clinical case: a -year-old man followed in the dept. of internal medicine for a hepatosplenomegaly of probable lymphoproliferative origin. additional explorations of interest are provided. result: intervention: complete laparoscopic approach, right lateral partial decubitus, massive splenomegaly, ? cm, splenuncle of - cm that is resected, section of short vessels, dissection of the splenic hilum, vascular section with endogias, splenectomy with full extraction in a pocket through reduced laparotomy in the left flank for anatomopathological study the aim of this video is to demonstrate the safety and efficacy of the laparoscopic approach in the treatment of large splenomegaly. currently, this approach is recognized as the one of choice in benign splenic pathology, being controversial in the case of a massive splenomegaly or neoplastic pathology it can transform into adenocarcinoma. patients and methods: between and we performed laparoscopic nissen fundoplication (lars) in cases of gerd. in cases of gerd patients be was proved by endoscopy and histological examination. the demeester score was higher ( . versus . , p \ . ), and bile re?ux was measured more frequently among the be patients on the other hand during the . years long endoscopic follow up early barrett carcinoma developed in patients, . months after the lars. both patients underwent a limited surgical resection of the distal esophagus and esophagogastric junction, regional lymphadenectomy, and reconstruction by interposition of an isoperistaltic jejunal segment. there were no complication. histological examination was shown pt n stage disease in both cases. oncological follow up was months long ( . y) and both patients are still disease free. conclusions: although lars can affect regression in a part of be patients, progression to adenenocarcinoma can also occur. endoscopic surveillance is important in the case of be to recognize early cancer, to perform limited surgical resection with low morbidity and long overall-and disease free survival gastric cancer development a nomogram for predicting the conditional probability of survival after d lymphadenectomy for gastric cancer this study aimed to devise a nomogram to predict the conditional probability of cancer-specific survival (cpcs) in gastric cancer (gc) patients after gastrectomy with d lymphadenectomy. methods: clinicopathological data for , gc patients who underwent d lymphadenectomy in a large-volume eastern institution (the training cohort) were analysed. cancer-specific survival (css) was predicted using cox regression models. a conditional survival nomogram was constructed to predict cpcs at and years post-gastrectomy. two external validations were performed using a cohort of , chinese patients and a cohort of italian patients. results: in the training cohort, the -year cpcs was . % immediately post-gastrectomy and increased to . %, . %, . % and . % at , , and years post-gastrectomy, respectively. multivariate cox regression analyses showed that age; tumour site, size and invasion depth; numbers of examined and metastatic lymph nodes; and surgical margins were independent prognostic factors of cancer-specific survival (all p \ . ) and formed the nomogram predictor variables. internal validation showed that the conditional nomogram exhibited good discrimination ability at and years post-gastrectomy (concordance index, . and . , respectively) gastric cancer does non-compliance in lymph node dissection affect oncological efficacy in gastric cancer patients undergoing radical gastrectomy? univariate and multivariate analyses revealed that non-compliance was an independent risk factor for os. logistic regression analysis demonstrated that the extent of gastrectomy, primary tumour site, history of intraperitoneal surgery, bmi and open gastrectomy were independent preoperative predictive factors for non-compliance. cox analysis demonstrated that age, pt, pn, and the extent of gastrectomy independently affected os in patients with noncomplaint lymphadenectomy. however, os was significantly better in the compliant group than in the non-compliant group regardless of the recommendation for chemotherapy. stratified analysis demonstrated that os was significantly better in chemotherapy patients than in patients without chemotherapy and stage ii patients (pt n /n m and pt n m ) in whom chemotherapy was not recommended. conclusion: non-compliance is an independent risk factor after radical gastrectomy for gc we prospectively collected and retrospectively analysed the medical records of patients with proximal gc who underwent lspsd. the data were split / , with one group used for model development and the other for validation testing. results: of the patients enrolled in this study, ( . %) required laparoscopic haemostasis treatment. a multivariate analysis determined the following preoperative adverse risk factors for the model group: gender, preoperative n stage, and terminal branches of the splenic artery (spa), and we developed a scoring system based on these findings. each of these factors contributed point to the risk score. the intraoperative laparoscopy hemostasis rates were . , . , . , and . % for the low-, intermediate-, high-, and extremely high-risk categories, respectively. there were statistically significant differences among groups (p \ . ). with the increase in risk, both blood loss volume (blv) and operative time (min) of lspsd increased significantly (p \ . ).the area under the receiver operating characteristic curve for the score of intraoperative laparoscopic haemostasis was . . the observed and predicted incidence rates for intraoperative laparoscopic haemostasis were parallel in the validation set. conclusions: this simple we compared the survival of src patients with that of tubular adenocarcinoma patients according to bmi. results: the -year survival of src was significantly worse than that of wmd (p \ . ) but superior to that of pd (p \ . ). bmi-stratified analysis showed that in the high-bmi group, the prognosis of src was similar to that of wmd (p [ . ) and better than that of pd (p \ . ). in normal-bmi patients, src had a worse prognosis than wmd (p \ . ) but a more favorable prognosis than pd (p \ . ). src among low-bmi patients displayed much poorer survival than did both wmd (p \ . ) and pd (p = . ). multivariate analysis indicated that the risk of death was lowest for src patients with a high bmi and highest for src patients with a low bmi baseline characteristics were compared in a -patient rspshl cohort and a -patient lspshl cohort. one-to-four propensity score matching was performed to determine between-group differences. result: in total, patients were matched, including patients who underwent rspshl and who underwent lspshl. no significant differences in baseline characteristics were observed between these groups after matching. significant differences in total operative time, estimated blood loss (ebl), splenic hilar blood loss (shbl), splenic hilar dissection time (shdt), and splenic trunk dissection time were detected between these groups (all p \ . ). furthermore, no significant differences were evident between rspshl and lspshl in the overall noncompliance rate of lymph node (ln) dissection ( the highest body temperature within week after operation was used to establish diagnostic thresholds for high body temperature and low body temperature, which was obtained by x-tile software. the study used cox regression to analyze the influence of high body temperature on -year dfs. results: a total of patients were included in the analysis. the diagnostic threshold for high body temperature was defined as °c; patients with a high postoperative body temperature were allocated to the high temperature group (htg), while another patients were allocated to the low temperature group (ltg) cao department of gastric surgery, fujian medical university union hospital, fuzhou, china background: laparoscopic surgery for remnant gastric cancer third step: baring of the right side of the esophagus. fourth step: exposure of left gastroepiploic vessels and lns dissection in the splenic hilar area. fifth step: baring of the left side of the esophagus. the above procedure was performed for rgc patients with stage ct - an /? disease. results: there was no conversion to open surgery. mean operation time was . ± . min, mean blood loss was . ± . ml, and mean times to first flatus p -upper gi-gastric cancer a novel prognosis prediction model after gastrectomy for remnant gastric cancer: development and validation using international multicenter databases fuzhou, china; department of gastrointestinal surgery the model calibration was accurate in predicting -year survival. dca showed that the model has a greater benefit. the results were also confirmed by bootstrap internal validation. in external validation, c-statistics and dca showed good prognostic performance in patient datasets from participating institutions. moreover, we verified reliability of the model in an analysis of patients with different eln counts p -upper gi-gastric cancer a novel abdominal negative pressure lavage-drainage system for anastomotic leakage after r resection for gastric cancer while risk of gastric cancer for ppi users was higher than non-ppi users when duration between - year, = year, = year and = year. the risk of gastric cancer when duration = year(rr = . )and duration = year(rr = . )are higher than risk of gastric cancer when duration between - year (rr = . ). according to location subgroups meta-analysis,risk of non-cardiac gastric cancer for ppi users higher than non-ppi users conclusion: based on a systematic review with meta-analysis, we found the correlation between long-term use of ppi and the risk of gastric cancer and long-term use of ppi may increase the risk of non-cardiac gastric cancer when duration = year p -upper gi-gastric cancer age-adjusted charlson comorbidity index (acci) is a significant factor for predicting survival results: there were patients included in the analysis. the high-acci and low-acci groups had significant differences in preoperative abdominal surgery history, asa grade, tumor size, tumor stage, histologic type, age and comorbidity (all p \ . ). the incidence of postoperative complications was . % in the high-acci group and was significantly higher than that in the low-acci group (p = . ). the overall survival rate (os) and cancer-specific survival (css) rate in the low-acci group were both higher than those in the high-acci group (p \ . ). univariate and multivariate analyses showed that the acci was an independent risk factor for os and css (p \ . ). furthermore, a combination of the tnm staging system and acci showed a trend toward higher prognostic value and higher auc for os and css than the tnm staging system alone (p \ . ). conclusions: the acci was an we aimed to investigate the clinicopathological features and prognosis of patients with mgc and the impact of postoperative adjuvant chemotherapy on long-term survival. methods: the clinical and pathological data of patients diagnosed with gastric adenocarcinoma and undergoing radical gastrectomy from stratified analysis showed that, in advanced gastric cancer (agc), the -year os rates of mgc without adjuvant chemotherapy and sgc without adjuvant chemotherapy were . % and . %, respectively, with a statistically significant difference (p = . ). the -year os rates of advanced mgc after adjuvant chemotherapy and of advanced sgc after adjuvant chemotherapy were . % and . %, respectively, and the difference was not statistically significant (p = . ). the -year os rate of advanced mgc after adjuvant chemotherapy was significantly higher than that of patients without adjuvant chemotherapy ( . % vs. . %, p = . ). conclusions: mgc is a poor prognostic factor after radical gastrectomy for gastric cancer background: whether the tumor-node-metastasis (tnm) staging system is suitable for patients with node-negative gc is still controversial. the modified staging system established by rpa showed good prognostic performance in a variety of cancers. the application of rpa has not been reported in the prognostic prediction of gc. methods: node-negative gc patients who underwent radical resection at fujian medical university union hospital (n = ) and sun yat-sen university cancer center (n = ) with an at least -year follow-up information were selected as the training set. rpa was used to develop a modified staging system. patients from the surveillance, epidemiology, and end results databases (n = ) were selected as the external validation set. results: the -year overall survival (os) rates of patients with th ajcc-tnm stage ia-iiia in the training set were ia %, ib %, iia %, iib % and iiia %. multivariate analysis (mva) showed that larger tumor size, older age, and deeper depth of invasion were independent risk factors for os in patients with node-negative gc (all p \ . ). patients were reclassified into rpa i, rpa ii, rpa iii, and rpa iv stage based on rpa, the -year os rates were %, %, %, and %, respectively, with significantly difference (p \ . ). two-step mva showed that the rpa staging system was an independent predictor for os (p \ . ) were retrospectively collected. patients were classified into two groups according to bmi of \ kg/m ( patients; high bmi group) and = kg/ m ( patients; low bmi group). for these patients, clinicopathological variables were analyzed using propensity score matching to mitigate the selection bias: sex, age, asa physical states, clinical stage, laparoscopy-assisted total gastrectomy (latg) or totally laparoscopic total gastrectomy (tltg), d lymph node dissection, combined resection of other organs, method of anastomosis, jejunal pouch reconstruction. the surgical results and postoperative outcomes were compared and examined between the two groups. results: a total of patients were matched for the analysis. contrary to our expectations, there were no differences in the surgical results about operative time and estimated blood loss (low bmi . ± . min, high bmi . ± . min; p = . , low bmi . ± . g, high bmi . ± . g; p = . , respectively). furthermore, there was no significant difference in postoperative outcome of complication (clavian-dindo [ iiia) and the length of postoperative hospital stays (low bmi cases, high bmi cases baiocchi general surgery, university of brescia-spedali civili, brescia, italy background and aim: recently indocyanine green (icg) was introduced in clinical practice as a fluorescent tracer. the use of icg for sentinel lymph node (ln) mapping was investigated in lots of fields such as breast methods: we conduced a single center prospective trial. we included patients with gastric cancer candidate to surgery. icg was injected intraoperative or the day before surgery, via submucosal or subserosal. total or subtotal gastrectomy was performed open, laparoscopic or video-assisted access. during gastric cancer standard lymphadenectomy we studied lymphatic flow and ln bright in vivo and ex vivo japan introduction: in japan, the number of elderly patients with gastric cancer has been increasing in correlation with the increase in average age of the population. the aim of this study is to assess the safety and efficacy of laparoscopic gastrectomy for cancer in elderly patients compared with the short-term outcome in the nonelderly. method: we reviewed patients who underwent laparoscopic gastrectomy (dital gastrectomy,proximal gastrectomy,total gastrectomy)between ).the incidence of advanced cancer(stageiior more)was higher in elderly patients there were no significant differences in the operating time,blood loss and postoperative hospital stay. there were no significant differences in the incidence of postoperative morbidity. conclusion: in elderly patients, there was a tendency of reduction surgery being selected according to individual condition, but there was no significant difference in the short-term outcome.hence,we conclude that laparoscopic gastrectomy is indicated even in elderly patients. p -upper gi-gastric cancer improved technique of vacuum therapy and carried out ltg . a patient factor (the gender, the age and bmi), an operation factor (operation time, the bleeding amount, lymph node dissection and conjurer), a coincidence related complication (clavien dindo classification, sutural insufficiency of grade more than , anastomotic stricture, anastomotic region bleeding and reflux esophagitis) and the post-operatively length of stay were considered . result: cs crowd met cases ( . %) and ls cluster ( . %) cases. years old of age medians ( - ), men and women were examples ( . %), examples ( . %) and bmi median . ( . - . ) by a patient factor, and a significant difference didn't admit by two groups for days, the post-operatively average length of stay was days by ls group by cs group. conclusion: operation time was short for a coincidence by linear stapler more than a coincidence by circular stapler in comparison of an esophagoenterostomy way in ltg on the day before the operation, we endoscopically clipped several points located cm proximal to the tumor edge to cover about half of the tumor. after lymph node dissection, we incised the stomach with an endoscopic linear stapling device,including the previously placed clips. reconstruction was performed in all patients who underwent billroth i or roux-en-y procedures. result: no complications were observed during pre-operative endoscopic clipping or intraoperatively p -upper gi-gastric cancer small intestinal tumors after laparoscopic surgery in our hospital small intestinal tumors are rarely observed, accounting for about - % (malignant cases: - %) of all gastrointestinal tumors. therefore, occasionally, their diagnoses can be difficult. however, recently, capsule and balloon endoscopes have been widely employed were examined regarding patient backgrounds, diagnostic methods, pathological findings, postoperative courses, and prognoses. results: the subjects consisted of males and females, with a mean age of years. their chief complaints were black stools the median distance from the treitz ligament or bauhin valve was cm ( - ) postoperative complications were abdominal abscess ( cases; . %) and surgical site infection (ssi), hemorrhage, and paralytic ileus ( case each; . %). pathological diagnoses were lymphoma metastatic small intestinal tumor ( cases; . %), and granuloma, lipoma, peutz-jeghers polyp, clear cell sarcoma, malignant mesothelioma, and ectopic pancreas most patients were diagnosed in bleeding, complicated by anemia and black stools. however, as most tumors were relatively close to the treitz ligament and bauhin valve, almost a half could be diagnosed with a small intestine endoscope before surgery patients were classified as popf and no-popf according to their grade b or c popf status. popf was diagnosed according to international study group of pancreatic fistula (isgpf) criteria or clinical findings. patient characteristics, intraoperative parameters, electrosurgical device type, pathological findings, and early postoperative outcomes were compared. electrosurgical devices were classified asthunderbeat (tb) or laparosonic coagulating shears (lcs) based on energy sources. results: eighteen patients developed grade b or c popf. among them, ( . %) and ( . %) were diagnosed with popf according to isgpf criteria and clinical findings ), operation time (p = . ) and electrosurgical device type (p = . ) were significant risk factors for popf following lag ) and tb device (or, . ; % ci ) were independent risk factors for popf following lag. conclusions: operation time and tb use significantly affect the risk of popf and should be considered in future clinical studies. p -upper gi-gastric cancer feasibility and nutritional benefits of double flap with no-knife stapler reconstruction after laparoscopic proximal gastrectomy for gastric cancer were analyzed. receiver operating characteristic curves were generated, and by calculating the areas under the curve(auc) and the c-index, the discriminative ability of crps during different periods were compared, including pre-crp, postoperative days , , and postoperative maximum crp (post-crp max ). a decision curve analysis was performed to evaluate the clinical utility. result: ultimately, patients were included this study and the median follow-up time was ( - ) months. for postoperative recurrence, the auc and c-index of pre-crp were . and . , respectively, significantly higher than the other crps, all p \ . . among = ''''''the = '''' post-crps = '''' post-crp = '''' sub = similar findings were observed for overall survival. conclusion: both pre-crp and post-crp max , cheap and easily obtained, are independent predictors of recurrence for gc. act significantly prolonged the rfs for stage ii/iii gc patients with high-prep p -upper gi-gastric cancer robot-assisted gastroduodenal surgery: a single center experience robot-assisted gastroduodenal surgery (ras) was introduced to overcome the technical limitations of conventional laparoscopy. it provides a d-amplified view to the surgeons and an increased ability to control the operative field by manipulating optics, as well as enhanced mobility and precision of instruments. the aim of the present study is to evaluate the main outcome of a single center experience in gastroduodenal robotic surgery. materials and methods: we report a case series of patients who underwent robot-assisted gastroduodenal surgery at sanchinarro university hospital between conclusions: robot-assisted gastroduodenal surgery is a safe and feasible technique in experienced centers with advanced robotic skills. in the literature, there are only few reports of robotic assisted gastroduodenal resection. further studies are necessary to better confirme our results. p -upper gi-gastric cancer atypical methods: retrospective review of ogd reports before and after the introduction of the new guidelines. inclusion criteria: all elective ogds. exclusion criteria: emergency ogds and elective therapeutic ogds. data recorded: patient demographics, endoscopist, indication, number of photos, anatomical site photographed, pathology identified and whether pathology photographed or not. results: ogds reviewed, before the guidelines (group ) and afterwards (group ). the most common indication was reflux ( %) in group and anaemia ( %) in group clinical utility of systematic pre treatment staging laparoscopic exploration methods: all locally advanced gastric adenocarcinoma managed in surgical oncological unit between st january and th november were prospectively enrolled in the study. in the absence of emergency surgery or preoperative contraindications, all patients with curative intent underwent either preoperative chemotherapy followed by surgical exploration in the intent of curative gastrectomy (g)or systematic pretreatment laparoscopic exploration (l) benkabbou surgical department the patient background (age, gender, bmi) and c-stage of the preoperative factor were matched using propensity score matching method, and the surgical results were compared and examined. results: thirty rg groups matched rag cases. the operation time (rag / lg) was significantly longer in the rag group as . ± . min / . ± . min (p \ . ). amount of blood loss was not significantly different each other; ml / ml (p = . ). pathologically t a case was involved in cases in rag and cases in lg. the extent of lymph node dissection (d ? / d ) was / cases in both groups conclusions: rag in our clinical experiences can be safely introduced and short-term results are comparable to those of lg. verification of superiority of robotic surgery including long-term results seems to influence the future of robotic surgery conclusions: totally laparoscopic gastrectomy is feasible method in terms of surgical outcomes. furthermore, totally laparoscopic total gastrectomy is not technically difficult in advanced gastric cancer such as early gastric cancer and safety method. key words: gastrectomy, reconstruction, laparoscopic surgery, stomach neoplasm aims: meckel's diverticulum (md) is one of the most common congenital anomalies of the small intestine caused by an obliteration defect of omphalomesenteric duct. the objective of this study was to review surgical treatment and clinical outcomes of md, and evaluate the safety and feasibility of minimal invasive surgery (mis) in md. methods: we performed a retrospective analysis of medical record for patients who underwent meckel's diverticulectomy at six hallym-university-affiliated hospitals between d), as well as the average of drainage stay. patients who underwent laparoscopic repair required significantly less parenteral analgesics than the open group.the mean postoperative stay was significantly shorter for laparoscopic group (mean, . d) than the open one.morbidity of medical and surgical complication was higher in open groups ( vs ). the most common complication in both groups was medical complication.more case of pneumonia was occurred in open groups compared to laparoscopic groups methods: a retrospective study using our prospective database was designed to analyse all the resected md in our centre. epidemiological data, clinical setting, diagnostic test and histological results were reported. results: md was resected in patients, males and females, with a mean age of . years ( . - . ). in cases, a laparoscopic approach was chosen. eighty-seven percent of the patients had a presurgical imaging test (ultrasounds, ct-scan or meckel's scan) background: perforated peptic ulcer (ppu) is a substantial health problem with significant postoperative morbidity up to % and mortality up to % worldwide. aims: this study aimed to estimate the sensitivity scoring systems for prognosis morbidity of patients operated for ppu with diffuse peritonitis. methods: a total of patients were underwent emergency repair for ppu with diffuse peritonitis in pirogov russian national research medical university's surgical clinics during - years. different scoring systems used to predict outcome in ppu patients were identified: boey score, peptic ulcer perforation (pulp) score, asa, mannheim peritonitis index (mpi), world society of emergency surgery sepsis severity score (wses score). to quantify the strength of the concatenation of prognostic score and morbidity we use odds ratio (or) with % ci ), respectively. pulp score and asa score have good prognostic value in relation to morbidity, but less than boey, mpi and wses sss. patients with pulp [ had or with % ci of p -upper gi-gastroduodenal diseases gastrostomy tube placed by laparoscopy as a new therapeutic option for continuous intestinal infusion treatment with levodopa/ carbidopa we present year outcomes of our initial consecutive patient cohort. methods: patients were identified in a prospectively maintained irb-approved database ( - ). post-operative eckardt scores and a -point validated system questionnaires were obtained via telephone interviews one patient required reoperation for failed myotomy. the mean eckardt score at years was . (± . ), with all fourteen patients having an eckardt score \ . all patients reported significant improvement in their quality of life. classic gerd symptoms (heartburn and regurgitation) were present in ( . %) patients. proton-pump inhibitors are being used by % of with patients with excellent symptom control. seven patients returned for a repeat egd (median . yrs) with patients having normal anatomy and having la grade a esophagitis ( patient on ppi). barrett's esophagus was not detected. conclusion: long-term results from our early experience with lhm are excellent and durable with only one patient requiring re-intervention in years until recently the esophagectomy was the only choice in treatment of patients with end-stage achalasia. developing of minimally invasive techniques such as a laparoscopic heller miotomy and peroral endoscopic myotomy (poem) allowed to use them as a treatment options. aim: to present an experience of treatment of patents with end-stage cardiac achalasia. materials and methods: since . till the laparoscopic heller myotomy was performed in , and esophagectomies were performed in patients with failed previously myotomy made in other clinics. gastric tube was used to replace the esophagus in patients underwent esophagectomy after skeletization of crura posteriorly to esophagus, two separated rectangular patches of parietene progrip mesh (covidien) measuring x . - cm were attached to the posterior surfaces of the crura. the patches were fixated themselves due to special hooks. than continious twodirections suture was placed through both crura along with the patches using self-gripping v-loc - suture (covidien). the same suture was used for construction of nissen fundoplication wrap . cm long. aditional anchoring stich through the wrap and esophageal wall was placed using ti-cron - suture (covidien). d laparoscopy was used while suturing using richard wolf epic system. results: all the procedures were performed successfully. there were no cases of bleeding from the suturing points either from the crura and the fundus wall. there were no crural dehiscence while suturing, even if the distance between crura was more than cm. the mean duration of suturing facilitated by d laparoscopy was min (range, - min) for crural repair, and min (range, - min) for fundoplication. there were no excessive postoperative pain in all the patients. there were no disphagia month postop in every patient. conclusions: . the new technique of posterior buttress of crural repair using small patches of parietene progrip mesh and v-loc suture showed feasibility and safety. . the use of d in such case most commonly manifested symptoms are cough, sore throat, hoarseness, dysphonia, globus and only % patients with lpr have typical gerd symptoms. also ppi therapy are less effective in patients with lpr in comparison with patients which have typical features of gerd. purpose: to compare the outcomes between surgical treatment and conservative therapy in patients with laryngopharyngeal reflux. materials and methods: for the period chesarev faculty surgery # , federal state autonomous educational institution of higher education i.m. sechen, moscow, russia p -upper gi-reflux-achalasia a case of a primary parahiatal hernia associated with a type i hiatal hernia emergency county hospital parahiatal hernia is a rare disease that occurs when an abdominal organ protrudes through an opening adjacent to an anatomically intact esophageal hiatus. the herniated organ is usually the stomach, although cases of omental and colonic herniation exist we report the case of a -year-old woman which accused epigastric pain, starting years prior, pseudo-angina, heartburn and bloating. based on imagistic findings the patient was diagnosed with a parahiatal hernia and an associated type i hiatal hernia. patient underwent surgery and a cm diameter defect in the diaphragm lateral to the left crus was discovered, through which - % of the stomach had herniated. the hiatal orifice was slightly enlarged but anatomically intact, with an associated small sliding hiatal hernia. we performed closure of the defect, hiatoplasty and a floppy-nissen fundoplication pneumatic dilation with mm balloon was performed under general anesthesia. radiological contrast control and endoscopy reevaluation revealed a perforation just above the squamo-columnar junction. a minimally invasive approach was decided. an fully covered esophageal stent was inserted. radiological control after days reveals left pleurisy and migration of the stent. the same day was performed an endoscopic repositioning of the stent with clip fixation. left pleural puncture was performed and clear fluid was extracted. the condition of the patient got worse and she was transferred on icu ( . ). we performed left pleurostomy and initial exploratory laparoscopy-no intraperitoneal lesions. due to difficult transhiatal access to the inferior mediastinum the surgery was converted to open-perisophageal mediastinal abscess was found, evacuated and drainage and jejunostomy were performed. after a week, the patient presented progressive altered condition, febrile syndrome. thoraco-abdominal ct-scan showed left pleural effusions. left pleurostomy was performed, with extraction of fetid fluid. continuous lavage was instituted. on st of august, the pleurostomy tube drained gastric content, and the clinical examination revealed signs of generalized peritonitis. laparotomy was performed with lavage, drainage and posterior decompression gastrostomy. results: postoperative evolution was favorable, with the suppression of pleural drainage in . and discharge in . with alimentation exclusive on jejunostomy. one month later, she had normal clinical and radiological examination evaluation of efficacy was performed with reflux symptom index (rsi) specific for extraesophageal symptoms, subjective satisfaction and occurrence of dysphagia and gas-bloat syndrome. a rsi score [ was considered as pathological. results: rsi significantly decreased after surgery ( and msa as compared with total fundoplication; , % of patients were satisfied with surgery: a comparison between techniques showed superiority of msa objective: to demonstrate the efficacy of hiatorraphy without the use of meshes in thegiant paraesophageal hiatus hernia, as well as the standardization of our technique, with thetechnical steps that we make successively. material and method: clinical cases: -year-old man,with symptomatic hiatal hernia with progressive intolerance and dysnea. egd: the stomach rotated in a giant hiatal hernia.gastroscopy not completed due to endoscope loop formation within giant hiatal hernia with gastric volvulation. ct: large hiatal hernia,combined volvulation (axial axial mesenteric organ), the stomach in a right subpulmonary situation. results: intervention: laparoscopic approach.hh of large paraesophageal size,double organoaxial-and-mesenteric volvular component,gastric walls very thickened and adhered to the mediastinum.reduction of all content and the sac,is adhered to the pleura, extended med-iastinal esophageal disection, up to vein pulmonary and get enough abdominal esophagus and rule out the presence of an short esophagus,posterior-anterior and left tutorized modified hiatorraphy with stitches in ''u'' with non-absorbable suture on teflon reinforcement patches.nissen fixed to both pillars, intramediastinal drainage.egd at the st day with esophageal stenosis due to inflamation of the nissen, resolved with medical treatment. dischage at th day.asymptomatic and without radiological recurrence after months of follow-up. conclusions: in giant and paraesophageal hiatus hernias, modified primary hiatorraphy together with mediastinal esophageal dissection extended can be an effective and safe alternative, and can be advised as a technical gesture prior to a collis nissen and-or placement of a hiatal-hiatoplasty mesh united states of america aim/background: prescribed opioids for pain control have been implicated as major contributors to addiction through their illicit use. efforts to reduce opioid prescriptions and measure their impact on outcomes are novel. we analyzed how patient outcomes are affected with reduced opioid prescriptions following laparoscopic foregut surgery narcr: %), length of hospital stay (narcs: . days vs. narcr: . days), -day readmission rates (narcs: % vs. narcr: %) and perioperative complication rates. additionally, no significant qol outcome differences between the groups were reported at one month postoperatively. conclusion: our study supports reducing opioid prescriptions as a strategy to counter illicit drug use and addiction patients who underwent paraesophageal hernia repair at a tertiary referral center were analyzed retrospectively. demographic data, asa classification, characteristics of peh, onset of symptom, dysphagia severity score, characteristics of fundoplication (partial vs. total; laparotomy vs. laparoscopy; emergency vs. elective) and surgical outcome (length of stay, complication and -day mortality) were recorded and reviewed. results: patients were included; % were female (mean age of . years old and mean body mass index of . ). mean onset of symptom was . weeks after peh repair, dysphagia severity scores were changed . from . . conclusion: in our series, the dysphagia severity scores reduced after surgery upper gi surgery, the catholic university of korea mary's hospital, incheon city, korea p -upper gi-gastric cancer general surgery biopsy from the mass has showed poorly differentiated signet ring cell adenocarcinoma. chest computed tomography revealed mm thoracic aortic aneurism. abdominal computed tomography showed mm infra renal aortic aneurism and no evidence of metastatic disease general surgery year old, female patient presented with upper abdominal pain, weight loss ( kg during last three month), without nausea or vomiting biopsy was done and pathology result showed intestinal type, her -negative adenocarcinoma of the stomach. chest and abdominal computed tomography (ct) were normal. endoscopic ultrasound (eus) revealed cm lesion with invasion to the muscularis propria (mp) she was treated by neo adjuvant chemotherapy ( cycles carboplatine ? fu). patient underwent laparoscopic partial gastrectomy with modified d lymphadenectomy and billroth ii gastrojejunostomy. total operating time (ort) was min. three day after operation patient started regular diet and was discharged home on day fife. final pathology result confirmed intestinal type, modified differentiated adenocarcinoma of the stomach economou st surgical department general, visceral and transplant surgery, section minimally invasive surgery, heidelberg university hospital, heidelberg, germany; department of surgery, iuliu hatieganu university, cluj-napoca, romania; general and visceral surgery, klinikum mittelbaden, baden-baden, germany surgery, toyonaka municipal hospital, toyonaka city, osaka, japan; gastroenterological surgery, osaka university, osaka, japan; next generation endoscopic intervention, osaka university, osaka, japan aims: uncomplicated healing of anastomoses in colorectal surgery is the basis for early adjuvant oncology therapy. the basis for proper healing is good blood flow. we use by robotic surgery foor control the firefly by intuitive. since january , icg has used for blood flow in laparoscopic bowel surgery for the d-light system of storz. method: use of icg wants to accurately determine the resection line for free colon operations based on good blood circulation. we use icg pulse in two batches and color detection using d light from storz to verify blood flow. the first dose is given after the skeletalisation of the intestines intraabdominally and the second after the colic anastomosis to verify its vitality. results: in the period under review we performed laparoscopic operations on the free colon, % of the operations were elective. we had . % leakage across the set, however, in the subset of elective operations, we had only a leak of . %. conclusion: in an unselected set of colorectal operations, leakage was . %, but only . for elective operations. in our group there was a clear effect of using icg in elective laparoscopic resections with an intracorporal anastomosis, the effect was not shown in others, probably due to leakage were factors other than blood flow. objective: right hemicoloctomy (rhce) is the first choice in treating the right colon cancer. complete mesocolic excision with extended lymph node dissection at the roots of superior mesenteric artery (sma) branches enables removal of all lymphatic tissue and prevents local recurrence. previously variability of sma branches was demonstrated. the aim of presented study was to compare the distribution of sma branches in two ethnically different cohorts methods: preoperativect scans with vascular d reconstruction were assessed in patients ( - years) from russia and patients ( - years) from turkey with right colon cancer operated in - . the distribution of ileocolic artery (ica), right colic artery (rca) and middle colic artery (mca) was investigated. results: ica and mca could be found on ct scans in all patients, whereas rca had significantly different distribution between patient cohorts: it was visible in ( %) of turkish patients and only in ( %) of russian patients (p = . ). conclusion: these results suggest that there might be ethnical differences in sma branches distribution. in turkish patients all named sma branches ate visible on ct scans in %, whereas in russian patients only in %. the majority of patients from russia don't have rca. ica and mca could be found in all patients regardless ethnicity. knowing the variant of sma branching before the operation can help plan extended lymph node dissection. the national training programme in laparoscopic colorectal surgery (s-micras-lapserb) in serbia was set up to introduce standardized and structured training in laparoscopic colorectal surgery. method: an assessment based structured training programme (lapserb) started in . series of hands on supervised workshops were conducted for four different hospitals using the structured training by single trainer. this study aims at retrospective analysis of prospectively collected data for patients undergoing colorectal resections. we look at short-term clinical and pathological outcome of patients within laparoscopic colorectal resections performed in national training program. results: during the period november until november , laparoscopic colorectal resection was performed in ( male and female) patients. mean age of patients was . ( - ). the most common indication was colorectal cancer ( patients, . %), ( . %) patients were operated due to the colorectal polyps not suitable for endoscopic resection and ( . %) was operated due to ibd. there were ( . %) right colonic, ( . %) left colonic /tme and ( . %) other resections. average number of lymph node harvested in patients with colorectal carcinoma was . . there were / ( . %) r resections mean duration of hospital stay was . days ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . postoperative complications were encountered in / patients ( . %). overall, mortality rate was . % ( / ). conclusions: this study demonstrates successful and safe adoption of laparoscopic technique for colorectal resections. short term clinical and pathological outcomes are compared to published data and shows wider adoption at the national level. standardization of operative technique and structured training remains the key in success. introduction: femal adnexal tumor of probable wolffian origin (fatwo) was first described in . it is a tumor of mesonephiris wolffian duct origin. fatwo is rare tumor which is usualle benign. in the literature has been reported cases and only cases of recurrent disease. next rare tumor in pelvic localisation is sex cord-gonadal stromal tumor of sertoli cells. methods: we present case report of women who presented metastatic fatwo with duplicity of sertoli tumor. results: -year old women underwent years ago exstirpation of tumor in the left broad ligament. histologically there was rare benign fatwo. this year was indicated adnexectomy at gynecology department. during the operation was done bilateral adnexectomy and discovered tumor of anterior wall on upper rectum. microscopic examination showed sertoli tumor on the left ovary. afterward we completed next examinations. colonoscopy without any abnormality. on ct scan was tumor cm without contact to rectum wall, without distant metastasis. the same was described on rectal ultrasonography-normal wall of rectum, tumor probably from uterus. at diagnostic laparoscopy was tumor mass cm, with necrosis arising from anterior wall of the rectum. next small metastasis on pelvic peritoneum. we performed debulking of this big tumor and metastasectomy, there was no infiltration to muscularis propria of the rectum. patient did not have any postoperativ complications. microscopic examination of the rectal tumor and small peritoneal metastasis showed metastatic fatwo. after weeks she underwent laparoscopic second look operation. there were small metastasis on pelvis peritoneum. we removed two biggest metastasis and the rest was destroyed wit j plazma. microscopic examination showed in this metastasis sertoli tumor. conclusion: our patient has metastatic fatwo and sertoli tumor. fatwo is so rare, that in the literaure is not enough information for observation or adjuvant therapy. in one case was described imatinib mesylae (gleevec) therapy with good results. surgeons must be ready to meet new diagnosis. bochdalek hernia is a type of congenital diaphragmatic hernia. in most cases, it is diagnosed during the neonatal period. we present a case of laparoscopically treated congenital bochdalek hernia that led to jejunal strangulation in an adult . case: an fifty eight year old obese(bmi = . ) female admitted for gradually worsening right flank pain, vomiting and respiratory distress for one day. there was no trauma history to chest or abdomen. the past medical history were old cva, well controlled hypertension and dm. she was hemodynamically stable. her right flank was very tender. there was no abdominal distension. initial cbc tests showed leukocytosis ( , /ul) , but electrolytes were normal. chest pa revealed right side diaphragmatic hernia. there was poorly enhanced, herniated small bowel in the right hemithorax on chest ct scan. the patient was taken for emergency operation. on laparoscopy, the normal liver was displaced leftward because of herniated bowel. there was incarcerated jejunum and omentum which could not be reduced. so we widened a cm sized posterolateral diaphragmatic defect first, and then we could reduce the strangulated jejunum( cm in length) and omentum. there was no hernial sac. the defect was closed with -o prolene. finally, the strangulated jejunum was resected and anastomosed extracorporeally. the hospital progress of the patient was not eventful. on post-operative day , the patient was allowed soft diet. the patient was discharged on post-operative day without any complication. conclusion: congenital diaphragmatic hernia is an uncommon condition in adults, but you should keep in mind the diaphragmatic hernia as a cause of intestinal obstruction and respiratory distress in an adult. prompt surgical intervention is required to a favorable outcome. laparoscopic repair of bochdalek hernia is a good management option. aims: intestinal obstruction is one of the most frequent abdominal conditions in the emergency department (ed). up to % of patients having undergone a laparotomy will have an episode throughout their lives, of which % up to % will respond to conservative management. the laparoscopic approach is widely accepted and supported by the studies published up todate. it is recommended in patients with suspected single band, who have less than one previous laparotomy and less than h of clinical evolution. our objective is to validate in our experience that these premises are the appropriate ones in the election of candidates for a minimally invasive approach. methods: we present a series of cases admitted with symptoms compatible with adhesive intestinal obstruction in the ed of a third level hospital during months. all patients underwent abdominal ct to rule out the possible causes of obstruction. emergency surgery was indicated because of failure of a conservative medical treatment or for the findings of the complementary tests. results: a initial laparoscopic approach was performed in the patients, with a conversion rate of % of the cases (resection and anastomosis was required in patients, due to loop suffering or intestinal tumor not seen in the ct). among the patients who required laparotomy, % had more han h of clinical evolution before de surgery and % had free fluid in the tc. as surgical complications: intestinal perforations were produced secondary to the manipulation. there was recurrences of obstruction in the following months. conclusion: the laparoscopic approach is feasible in selected cases and experienced hands. acording to our results it is recommended to perform it in only in patients with less than h evolution and with a single band image in the ct without free fluid. the intestine should be explored avoiding the manipulation of the most extensive loops to prevent complications and keep in mind the possible conversion to laparotomy in case of complications. aim: the aim of the study was to evaluate whether physiologic and operative severity score for the enumeration of mortality and morbidity (possum) is useful to predict the risk of complications in patients older than years. methods: we performed a retrospective study of patients older than years old diagnosed with acute abdomen who were admitted to the department of general, minimally invasive and elderly surgery in olsztyn between may and october . results: the most common disagnosis was ileus. the mortality rate in surgery department was %.after relocation to the intensive care unit, the overall mortality rate was . %. the patients who died a short time after surgery had mortality rates greater than % and morbidity rates greater than % according to possum. conclusions: this study shows that possum seems to be a valuable scale to predict the risk of death after surgery in older patients. patients with higher mortality and morbidity scores should be very carefully selected for surgery. aims: diaphragmatic hernia in adulthood is rare. the most common causes are blunt and penetrating trauma. we present an intraoperative video of the laparoscopic repair of an adult onset, non-traumatic, diaphragmatic hernia in a patient with splenomegaly. method: a year old woman was referred to upper gastrointestinal surgery with epigastric burning and pain in the left side of her chest, radiating to the left shoulder, for one year. there was no recent or distant history of trauma. she has a past medical history of treated hepatitis c, cirrhotic liver disease, splenomegaly, thrombocytopenia and iron deficiency anaemia. gastroscopy was interpreted as a fundal diverticulum. ct abdomen/pelvis with intravenous (iv) contrast showed this to be a left diaphragmatic defect with herniated stomach causing a volvulus, which lay immediately above an enlarged spleen. a ct three years prior to this showed no diaphragmatic hernia. the patient had some symptomatic relief with a proton pump inhibitor and oral antacids, however due to her persistent symptoms surgery was undertaken. the patient had laparoscopic repair of the diaphragmatic hernia. ports were as follows: mm umbilical, mm left upper quadrant, mm right upper quadrant, mm left iliac fossa. a left posterior diaphragmatic defect was found, just above the enlarged spleen, containing the incarcerated fundus of the stomach. the hernia was reduced by gradual dissection of the sac. a cm nonabsorbable polypropylene mesh (proceed) was used to cover the defect a cm margin. this was tacked in place with protack. a single french robinson drain was left in situ. results: the procedure was uncomplicated. oral diet was introduced on post-operative day and the drain was removed and patient discharged on day . there were no post-operative complications. conclusions: the video shows an effective dissection of a left sided diaphragmatic hernia and mesh repair, overcoming multiple technical challenges secondary to splenomegaly and portal hypertension. aims: creating laparoscopic anastomosis is a challenging surgical skill with high clinical relevance. to assure efficient training and enhanced learning curves, constructive and objective feedback is essential. currently there is no appropriate instrument to assess the surgical performance while creating laparoscopic anastomosis. the aim of this study is to develop and validate the anastomosis-objective structured assessment of technical skill (a-osats) score. methods: to obtain an international expert consensus for a procedure specific checklist (psc) for laparoscopic anastomosis, a modified delphi survey with an integrated analytic hierarchy process is currently being performed. each a-osats sub step is assigned a specific weight to determine its importance to the final outcome of the anastomosis. to validate the a-osats score, a laparoscopic side-to-side small bowl anastomosis with a linear stapler and hand-sewn closure of the enterotomy was chosen and is performed by surgeons with varying degrees of laparoscopic experience on a live porcine model. all performances are recorded and rated twice using the a-osats by two blinded experts. results: the final a-osats score includes a weighted psc developed by the modified delphi survey and the already validated global rating scale of previously published osats scores. four key steps (bowel placement, creation of enterotomies, stapling, closure of enterotomy) and sub steps, as well as their definitions, were established during the delphi survey. to validate the a-osats, surgeons ( experts, intermediates, novices) have participated in the study so far. preliminary results showed significant differences between all three levels of laparoscopic experience (novices: . ± . ; intermediates: . ± . , experts: . ± . ; p \ . ) for the overall a-osats score as well as the psc itself (novices: . ± . , intermediates: . ± . , experts: . ± . ; p = . ). conclusions: the a-osats is a weighted score that objectively assesses surgical skill during the creation of laparoscopic anastomosis. preliminary results confirm construct validity of the proposed score. furthermore, by offering the possibility to differentiate single aspects during the procedure, the a-osats allows focused feedback to enhance one's performance. minor changes in weights are expected after the last round of the delphi survey. interand intrarater reliability will be assessed after final inclusion of all participants.aims: to apply augmented reality technology from three-dimensional colon models as preoperative planning method in colorectal surgery. method: from three-dimensional anatomical models of the colon we have developed holograms of augmented reality. the models were obtained from ct images (siemens somatom perspective Ò) with abdominal image cuts with mm thick. the recovery of the images was in dicom format and the processing to achieve the three-dimensional reconstruction was performed with the programs osirixÒ and horosÒ, which made a complete segmentation of the colon surface, and a modification of the image density. in this way models d were obtained of the isolated colon, and in relationship with the bone structure. the application colon d ar was designed (increased hyper experience-visualizer with slam technology) creating a hologram of augmented reality to scale : from each three dimensional model to make a projection of it on the abdomen of the patient by modifying the position in height of the reconstruction, using the bone pelvis as anatomic reference point to calibrate the placement of the hologram. results: in the preliminary phase (from october to december ) holograms of augmented reality were developed in patients with colorectal cancer (right colon, left colon, transverse colon and rectum) to complement the radiological reconstruction with the virtual model. in the application phase (from january ) the holograms developed are going to be applied as a method to improve preoperative study. conclusions: three dimensional reconstruction of the tumor in the preoperative plan of colorrectal surgery combined with hyperreality technology allows to develop models of augmented reality in order to improve colon anatomy knowledge and to plan the surgical technique.aims: laparoscopic adrenalectomy has become the standard of care for most adrenal masses. we report a case of laparoscopic adrenalectomy for left adrenal adenoma. methods: we present the case of a -year-old caucasian female patient with an asymptomatic, left-sided adenoma, that was incidentally detected during abdominal ultrasound. no headaches, palpitations, tachycardia, tremor, dizziness or vomiting were reported. pre-operative blood tests confirmed that the tumor was a non-secreting one and a ct-scan revealed a . . cm left adrenal mass. laparoscopic surgical excision was proposed. the patient was placed in semilateral right-sided decubitus position. four trocars ( epigastric- mm & subcostals- mm & mm) were used, without the use of a liver retractor. the adrenal vessels were clipped not only with the standard laparoscopic clips, but also with the hem-o-lok ligation system. results: the operation lasted for h with minimal blood loss. the patient's post-operative course was uneventful and she was finally discharged four days post-operatively. histology report ensured that it was adenoma of the adrenal cortex. aims: since the first laparoscopic adrenalectomy in (gagner), the laparoscopic lateral transabdominal approach has proved to be the one of choice. it provides an easy anatomical orientation, overall the technique is similar to other traditional laparoscopic procedures. on the other hand, the posterior retroperitoneoscopic adrenalectomy (pra), described in (waltz), has proven to be a safe technique and effective for the surgical management of several adrenal pathologies. the advantages include direct access to the adrenal gland, without the need for visceral mobilization or lysis of adhesions from previous abdominal operations and the ability to perform a bilateral adrenalectomy without repositioning the patient. currently there is controversy about which is the approach of choice, having to take into account the learning curve necessary for the retroperitoneal approach and the reduced number of patients with adrenal pathology subsidiary of surgical management. the objetive is to demonstrate the safety and efficacy of the standardized laparoscopic approach of the left adrenal gland with trocars for selected cases. methods: clinical case: -year-old man, resistant hypertension despite concurrent use of three antihypertensive agents, with biochemical and radiological diagnosis of left adrenal adenoma with primary hyperaldosteronism. demonstrative video of the technical steps in a standardized way that we propose for laparoscopic left adrenalectomy only using trocars. results: full laparoscopic surgical approach in right lateral decubitus position: trocars-lateral transabdominal approach. steps: . laparoscopic liberation of the splenic flexure of the colon for the colo-spleen-pancreato-gastric en block mobilization until identification of the left pillar, . dissection of the medial border of the gland, identification of left renal and diaphragmatic vein, as well as the adrenal vein which is dissected and clipped, . dissection of the lateral edge of the adrenal gland, . lower pole dissection of the gland completing the resection with ligasureÒ. the patient presented a successful postoperative recovery, being discharged h after the intervention. asymptomatic, the patient does not need antihypertensive drugs at year follow-up. conclusion(s): the standardization of the procedure allows reducing the number of trocars, maintaining the safety and effectiveness of the minimally invasive approach. aims: cortical-sparing adrenalectomy is a suitable treatment for hereditary and sporadic bilateral pheochromocytoma, in cases of low risk of malignancy, to reduce the possibility of adrenal insufficiency assuming the chance of local recurrence. the aim of the study is to analyze the functional results of partial adrenalectomy by retroperitoneal endoscopic approach in singleadrenal patients or patients requiring bilateral adrenalectomy. methods: prospective study between january and october including pheochromocytoma patients diagnosed with low risk of malignant mutations. all patients agreed to be included in the study. experienced endocrine surgeons who have been trained in minimally invasive endocrine surgery performed the procedure using the same surgical technique. demographic variables and clinical characteristics were collected, subsequently carrying out the descriptive analysis of the data. results: a total of eight patients were registered, five associated with men type syndrome and three in the context of vhl syndrome. retroperitoneoscopic resection was performed without laparoscopic or open conversion and no postoperative complications; the average hospital stay was . days. preservation of the functional cortex without corticosteroids was achieved in ( . %) of out cases with a follow-up of . ± months. today, these seven patients have a preserved adrenal function without hormone replacement. conclusions: cortical-sparing adrenalectomy by the retroperitoneal endoscopic approach, in expert hands, is safe and feasible for the treatment of hereditary and sporadic pheochromocytoma in a context of low malignancy, making it possible to avoid the need for corticoid replacement in most cases. biomedical sciences, university of west attica, athens, greece partial adrenalectomy has been suggested for patients benign adrenal tumors especially in the case of hereditary syndromes, like multiple endocrine neoplasia type , von hippel-lindau disease and neurofibromatosis type i. aims: this systematic review aimed to investigate the role of partial adrenalectomy in the treatment of hereditary pheochromocytoma. methods: electronic databases were searched with the search terms 'men ii', 'von hippel lindau', 'neurofibromatosis', 'laparoscopic partial adrenalectomy', 'robotic assisted partial adrenalectomy' for the time period up to and including december . full publications, including clinical trials randomized or not, retrospective studies, case series, case reports that provided relevant data met inclusion criteria results: thirty five possibly relevant studies were identified. abstracts were reviewed and fourteen articles were excluded as they were review articles or articles presenting data on open partial adrenalectomy. twenty one studies, that met inclusion criteria were retrieved in full text and included in the systematic review. eight studies presented data on partial adrenalectomy in patients with von hippel lindau including two case series with median follow up ranging from to . years and six case reports. thirteen studies presented data on partial adrenalectomy in patients with men ii, including two case series and eleven case reports. recurrence rate was estimated at about % for pheochromocytoma. overall steroid dependence rate was estimated at %. conclusion: minimally invasive partial adrenalectomy is a therapeutic option especially in patients with heritable pheochromocytoma, given that tumors are often bilateral, tumors are commonly benign and severe morbidity and mortality may be associated with life-long steroid replacement therapy such as the possibly lethal addisonian crisis . however, data are limited, follow up is not standardized and not appropriately reported and rcts are difficult to be done due to the rarity of the disease. a multinational registry on the short term and long term outcomes of partial adrenalectomy in hereditary pheochromocytoma would be a significant source of knowledge. results: patients were operated on after an average of months with complaints. in both groups, the leading symptoms were severe dysphagia and severe regurgitation. no intraoperative complication was detected. in the transoral group, one patient had to be reoperated on for bleeding, another patient developed pneumonia in the transcervical group. the average duration of the surgeries ( . vs. min, p \ , ), the time to oral feeding ( . vs. . days, p \ , ) and the mean hospital stay ( . vs. . days, p \ , ) were significantly shorter in the transoral group than the transcervical group. patients were completely symptomless postoperatively. after transcervical treatment, complaints were developed in cases (moderate dysphagia and hoarseness). after transoral surgery, recurrent symptoms were observed in patients, had to be reoperated transcervically due to severe regurgitation. conclusion: transoral stapler diverticulostomy is a fast procedure and offers short hospital stay especially in comorbid, aged patients and intermedium diverticulum size. in the long term, some of the patients may require reintervention due to persistent regurgitation. the transcervical approach has higher perioperative morbidity, which can be performed in patients with less than cm or large diverticulum size. aims: complex hiatal hernias, either implicating large hiatal defects or concerning cases of recurrence, often need apart from the primary closure of the hiatal gap, the re-enforcement of the crura with the use of meshes. our aim is to demonstrate the surgical technique for the on-lay placement of the absorbable mesh (phasix tm st mesh /bard) in challenging cases, presenting both the laparoscopic and the robotic approach. methods: we present video fragments from procedures of laparoscopic and robotic reconstruction of complex hiatal hernias, performed by our team, in which an absorbable mesh was utilized in an on-lay fashion. results: patients having undergone a minimally invasive surgical approach (laparoscopic or robotic) for the treatment of complex hiatal hernias with the use of an absorbable mesh, had an uneventful post-operative course and very short hospital stay and recovery time. the -month follow up revealed no recurrences or late complications. conclusions: treating complex cases of hiatal hernias with a minimally invasive approach can be proven quite challenging, with high recurrences and possible complications rate. a proper surgical technique, either laparoscopic or better (based in our primary experience) robotic, by experienced surgical teams and the use of meshes with the right strategy, minimizes the complications, offers all the benefits of minimally invasive surgery and reduces the recurrence rates. aims: several flexible endoscopic techniques for symptomatic zenker's diverticulum have been developed during the last decade. thulium laser has limited tissue penetration and may decrease the risk of perforation. this study reports the first use of thulium laser through flexible endoscopy for cricopharyngeal (cp) myotomy. aims were safety and efficacy of flexible endoscopic thulium laser myotomy and quality of life (qol) changes after treatment. methods: a retrospective review of a prospectively collected database of patients who underwent thulium laser septum division for symptomatic zenker's diverticulum was done. demographic data, presenting symptoms, diverticulum characteristics, and intraoperative data were analyzed. functional outcome swallowing scale (foss) and m.d. anderson dysphagia inventory (mdadi) questionnaires were administered to determine severity of dysphagia and its effect on qol, both preoperatively and during follow-up visits. all the operations were carried out under general anesthesia. a continuous laser configuration and an emissionpower of w was used in non-contact mode. once the mucosa was opened, the fibers of the cricopharyngeal muscle were divided until the buccopharyngeal fascia was visibile. results: between march and september , patients ( males) underwent flexible endoscopic cp myotomy with thulium laser. mean age was ± . , mostly males ( . %). seven patients ( . %) presented with recurrent diverticulum after previous transoral or open treatment. mean diverticulum size was . ± . cm. preoperative main symptoms were dysphagia ( . %), regurgitation ( . %), and cough ( . %). foss score was = in patients ( . %). mean mdadi global and composite score were . ± . and . ± . . complete division of the septum was achieved in all patients. mean hospital stay was . ± . days. there was only one perforation treated conservatively. no -days mortality was observed. at median follow-up of months, foss was = in ( . %) patient and mdadi global and composite score were . ± . and . ± . . all main symptoms were significantly reduced and qol significantly increased. conclusions: flexible endoscopic approach with thulium laser is a safe and effective treatment option for zenker's diverticulum either as a primary treatment or as a rescue therapy. objective: this study sought to explore prognostic factors for patients with borrmann type iv gastric cancer and to establish a predictive model for survival benefit of postoperative adjuvant chemotherapy in such patients. method: this study reviewed the clinical data of patients who underwent curative surgery at fujian medical university union hospital from to for borrmann type iv gastric cancer using a prospective database. cox regression analyses were performed to identify prognostic factors that formed the basis for a nomogram and risk groups. establishment of risk groups to identify patients with borrmann type iv gastric cancer who would benefit from adjuvant chemotherapy. results: patients who underwent r resection were included in this study.multivariate analysis showed that bmi, tumour differentiation, pt stage, pn stage, and asa score were independent prognostic factors. patients in the act-group had longer os than patients in the sagroup, although the p-value for this difference was marginally above the threshold for statistical significance ( . % vs. . %, p = . ). stratified analysis showed that there was no significant difference in os between the act-group and the sa-group for each ajcc stage (stage ii: . % vs. . %, p = . ; stage iii: . % vs. . %, p = . ).a nomogram was established based on these independent risk factors, and nomogram scores were used to divide all patients into a high-risk group (score [ ), an intermediate-risk group ( \ score = ) and a low-risk group (score = ).further stratified analysis based on ajcc stage showed that the -year survival rate was higher in the adjuvant chemotherapy group than in the surgery alone group for low-and intermediate-risk patients in each ajcc stage, while high-risk patients in stage iii did not significantly differ. objective: this study sought to explore the prognostic factors for smoking patients with gastric cancer and to establish a predictive model for the survival benefit of postoperative adjuvant chemotherapy in such patients. methods: we studied patients who were diagnosed from september to september at union hospital of fujian medical university. cox regression analyses were performed to identify prognostic factors. the kaplan-meier method was used to assess the effect of smoking history on the benefit of adjuvant chemotherapy after gastric cancer surgery. a decision tree algorithm was used to identify smoking patients who benefited from postoperative adjuvant chemotherapy. results: the median follow-up time for the whole group was . months, and the average age of all the included patients was . years.multivariate analysis showed that age (p \ . ), bmi (p \ . ), degree of tumor cell differentiation (p \ . ), and ajcc stage (p \ . ) were independent risk factors for the prognosis of smoking patients. based on these independent risk factors, a decision tree model for the benefit of adjuvant chemotherapy for smokers with gastric cancer was established, and the smoking patients were divided into the low-risk patients . %), medium-risk patients ( - year os, . %) and high-risk patients ( - year os, . %) (p \ . ). conclusion: cigarette smoking may reduce the efficacy of adjuvant chemotherapy after gastric cancer surgery. our decision tree model is simple and effective for identifying smokers who would benefit from adjuvant chemotherapy. objective: our study investigated the effect of lymph node (ln) noncompliance on the longterm prognosis of patients after laparoscopic total gastrectomy (ltg) and explored the risk factors of ln noncompliance. methods: the clinicopathological data of gastric cancer (gc) patients who underwent ltg with d lymphadenectomy from june to december were prospectively collected and retrospectively analyzed. the effects of ln noncompliance on the long-term prognosis of patients with gc after ltg were explored. results: the overall ln noncompliance rate was . %. ln noncompliance was significantly correlated with age, bmi, asa score, tumor size, macroscopic tumor type and tnm staging (p values \ . ). the survival rate of patients after ltg with ln compliance was significantly superior to that of patients with ln noncompliance (p = . ). the stratified analysis of tnm stage indicated that there was no difference between the os of stage i patients with ln compliance and those with ln noncompliance; os of stage ii/iii patients with ln compliance was significantly better than that of those with ln noncompliance. cox regression analyses showed that ln noncompliance was an independent risk factor for os. logistic regression analysis showed that high bmi ([ kg/m ) was an independent risk factor for preoperative prediction of ln noncompliance in cstage ii/iii patients. compared with patients with a low bmi (bmi \ kg/m ), those with a high bmi were more likely to show ln noncompliance during surgery, especially during the dissections of # , # a and # a ln stations. conclusion: ln noncompliance was an independent risk factor for poor prognosis in patients with advanced gastric cancer (agc) after ltg. patients with high bmi were more likely to have ln noncompliance, especially during the dissections of # , # a and # a ln stations. ln tracing was recommended for these patients to reduce the rate of ln noncompliance. aim: to study the differences in pathology, survival, and recurrence between special remnant gastric cancer (srgc) and nonspecial rgc (nrgc). method: a total of rgc patients were analyzed in hospitals in china from january to july .we compared the -year overall survival (os) disease-free survival (dfs) rates and used two-step regression explore the influence of the rgc categories on patient outcomes. results: all of the patients divided into srgc group (group s) (n = ) and nrgc group (group n) (n = ). the r resection rate and lymph node (ln) dissection number of group s were significantly higher than group n (p \ . ). the difference in -year os was not significant (p = . ), but the -year dfs of group s was worse than group n (p = . ). twostep multivariate analyses showed nrgc was an independent risk factor for poor dfs. of the patients who had undergone r resection, patients ( . %),suffered recurrence, and the recurrence rate of group s was significantly higher than group n (p = . ), moreover, the ln recurrence rate of group s was significantly higher than group n (p = . ). cox regression analysis showed that age, ca level, n stage and category of rgc were independent risk factors for rgc recurrence. conclusion: srgc has a higher r resection rate and ln dissection number than nrgc, but among patients who had undergone radical gastrectomy, srgc patients had worse dfs and a higher tendency for ln recurrence; thus, they should be treated differently in the clinic. objective: the aim of this study was to report our institution's experience with a novel abdominal negative pressure lavage-drainage system (anplds) for anastomotic leakage (al) after radical gastrectomy (rg) for gastric cancer (gc). background: al is a severe complication associated with high morbidity and mortality after rg for gc. the optimal creation of drainage in al patients after rg remains controversial. methods: the study enrolled patients who underwent r resection for gc at our institution between and . anplds was routinely used for patients with al after january . al rates and postoperative outcome were compared before and after the anplds therapy. we used multivariate analyses to evaluate clinicopathological and perioperative factors for associations with al and failure-to-rescue (ftr) after al. results: al occurred in patients ( / , %), leading to deaths. the al rate was similar before ( - , period ) and after ( - , period ) the implementation of anplds ( . % vs . %, p = . ). age and malnourished were independently associated with al. the ftr rate and abdominal bleeding rate after al occurred were respectively . % and . % for the entire period, but compared with period , it significantly decreased at period ( . % vs . %, p = . ; . % vs . %, p = . , respectively). what's more, only anplds therapy was an independent protective factor for ftr after al. conclusion: our experience demonstrates that anplds is feasible and cost-effective for the management of al after rg for gc. objective: to apply the principles of the 'metro-ticket' paradigm to develop a novel tnm staging system (ntnm) for gastric cancer (gc). background: the 'metro-ticket' prognostic tool for hepatocellular carcinoma has been proven to predict outcome, but a similar concept has not been investigated for gc. methods: the ntnm considered the distance from the origin on a cartesian plane incorporating the pn (x-axis) and pt (y-axis) stages. gc patients undergoing radical resection at fujian medical university union hospital (fmuuh) (n = ) were included. the ntnm was validated using external cohorts from the sun yat-sen university cancer center (sysucc) (n = ) and surveillance, epidemiology, and end results (seer) (n = ) databases. results: ntnm classes with the same distance from the origin have same stage; the stage increases with this distance. among all patients, . % (n = ) were restaged in the ntnm compared with the th edition of the ajcc-tnm classification; . % (n = ) were downstaged in the ntnm compared with the th edition. the ntnm provides significant survival differences between stages (all p \ . ). the survival difference between stages ib and iia was especially large for the ntnm (p \ . ) compared to the th and th editions (p = . ). the concordance index and hazard ratio increased successively with the ntnm stage. similar findings were observed in both external cohorts. conclusion: compared with the ajcc-tnm classification, the 'metro-ticket' ntnm for gc is easier to remember and provides some improvements; therefore, the ntnm may be considered for adoption in future editions of the ajcc-tnm classification. objective: to investigate the prognostic value of complete blood count (cbc)-based biomarkers for patients with resectable gastric cancer (gc). methods: patients with gc who underwent curative resection between december to december were included. estimated area under the curve (auc) and multivariate cox regression models were used to identify the best cbc-based biomarker. time-dependent receiver operating characteristics (t-roc) analysis was used to compare the prognostic impact. results: based on multivariate analysis, the lymphocyte-monocyte ratio (lmr) and hemoglobin (hb) level were the independent prognostic factors (both p \ . ). based on the lmr and hb level, we established the cbc-based inflammatory score (cbcs). higher cbcs was associated with older age, female sex, higher american society of anesthesiologists (asa) score, proximal tumor location, larger tumor size, later stage and vascular involvement (all p \ . ). univariate analyses showed that higher cbcs was also associated with poorer overall survival (os), which was consistent in each stage (all p \ . ). multivariate analysis revealed that the cbcs was a significant independent biomarker (p \ . ). furthermore, t-roc curve of the cbcs was superior to that of the prognostic nutritional index (pni), systemic immune-inflammation index (sii), modified glasgow prognostic score (mgps) and c-reactive protein/albumin ratio (crp/ alb) throughout the observation period. conclusion: preoperative lmr and hb were optimal cbc-based biomarkers for predicting os in gc patients after curative resection. based on the lmr and hb, we developed a novel and easily obtainable prognostic score called the cbcs, which may improve the prediction of clinical outcomes. purpose: the aim of this study was to evaluate the prognostic value of the eighth ajcc tnm staging classification for patients with gastric cancer who had already survived for years. patients and methods: patients who underwent radical gastrectomy at a large eastern center were considered. the prognostic value of staging systems were assessed and compared. additional external validation was performed using a dataset from the surveillance, epidemiology, and end result (seer) database. results: the -year overall survival (os) rate for patients in the training set was . %. with the prolongation of the survival time after surgery, the -year os improved significantly (p \ . ). however, there were no significant differences in survival curves among patients who have survived years after surgery. the auc and c of the eighth ajcc classification for predicting of -year os decreased gradually after surgery and appeared stable after years. for patients who survived years after surgery, we constructed a new tnm staging system (ntnm) according to the survival curves of t stage and n stage. a -step multivariate analysis showed that ntnm, age and sex were independent prognostic factors. the ntnm demonstrated superior prognostic stratification, with higher c-statistic and likelihood ratio chi-square scores and lower aic values than those of the ajcc classification. similar results were observed in the external validation set. conclusion: the ntnm predicted an additional survival more accurately than did the ajcc classification for patients who have survived years after surgery; this may guide decisions regarding surveillance. objective: to investigate the relationship between preoperative sarcopenia and systemic inflammation and evaluate the prognostic impact of these factors on patients with resectable gastric cancer (gc). methods: patients with gc who underwent radical gastrectomy between december and december were included. a multivariate cox regression analysis was performed to identify the prognostic factors. a novel prognostic score (slmr) was developed based on preoperative sarcopenia and the lymphocyte-monocyte ratio (lmr), and its prognostic value was evaluated. results: in total, patients with resectable gc were included in the study. on multivariate analysis, preoperative sarcopenia and the lmr were shown to be independent prognostic factors (both p \ . ). a low lmr was an independent predictor from sarcopenia (p \ . ). based on preoperative sarcopenia and the lmr, we established the slmr. an elevated slmr was associated with older age, higher asa scores, larger tumor size, advanced stages and vascular invasion (all p \ . ). multivariate analysis revealed that the slmr was a significant independent predictor (p \ . ). we incorporated the slmr into a prognostic model that included tumor size and tnm stage and generated a nomogram, which accurately predicted -and -year survival for gc patients. objective: to explore whether adjuvant chemotherapy is still needed in patients aged less than years with pt n - and pt / n gastric cancer. methods: multi-center cohort data of patients with gastric cancer who underwent radical gastrectomy were analyzed. kaplan-meier curves and cox regression were used to analyze the relationships between chemotherapy and prognosis. additionally, nomograms to predict the benefit of chemotherapy were established. results: in total, , patients with pt n - and pt / n gastric cancer were included. patients ( . %) were aged \ years. the -year overall survival (os) was not significantly different between the \ years of age group and = years of age group ( . % vs. . %, respectively; p = . ). lymph node (ln) metastases (hr . ; p = . ) and ln dissection number \ (hr . ; p \ . ) were independent risk factors for the os of patients aged \ years. adjuvant chemotherapy did not improve the -year os for patients aged \ years with pt n - and pt / n gastric cancer (p = . ). however, chemotherapy showed a significant benefit (p = . ) when there were ln metastases and/or ln dissection number was \ . two nomograms were constructed, and the calculated difference was the potential benefit of adjuvant chemotherapy for the patients aged \ years. conclusions: ln metastases and ln dissection number \ were independent prognostic risk factors of patients aged \ years with pt n - and pt / n gastric cancer. patients with these risk factors may benefit from the addition of adjuvant chemotherapy. objective: the choice of reconstruction after distal gastrectomy remains controversial. we have performed roux-en-y (r-y) method after laparoscopic distal gastrectomy(ldg) as a standard since , but we have performed billroth ii (b-ii) method in an increasing number of cases, depending on the patient. we retrospectively investigated the outcomes of patients with b-ii method after laparoscopic distal gastrectomy in our hospital. methods: patients who underwent b-ii and r-y reconstruction after ldg from january to december were included. the patient characteristics, surgical outcomes, and postoperative outcomes between the procedures were retrospectively analyzed. we also compared extend of gastritis on endoscopy and loss of body weight after surgery at year. results: b-ii / r-y : / . b-ii was selected in the elderly patients with poor asa-ps (p \ . ). in surgical outcomes, operative time was shorter for b-ii than r-y (p \ . ), and blood loss was also smaller (p = . ). in postoperative outcomes, there were significant differences in complications (?grade ) (b-ii vs. r-y: . vs. . %, p = . ) and length of stay (b-ii vs. r-y: median . vs. -day, p \ . ). there was significant difference in presence of gastritis between b-ii ( . %) and r-y ( . %) (p \ . ), but no significant difference in loss of body weight (p = . ). conclusion: b-ii reconstruction may be an adequate procedure for high-risk cases because of its shorter operative time and the absence of severe complications. background: numerous studies have shown that the short-term efficacy of three-dimensional ( d) laparoscopic radical gastrectomy (lg) is comparable to that of two-dimensional ( d)-lg. whether d-lg affects the recurrence pattern after surgery has not been investigated. using data from a prospective clinical trial, the present study compares the recurrence patterns between d-lg and d-lg. methods: from january to april , a total of patients were recruited for the clinical trial (nct ). the recurrence types, the first recurrence time and recurrence-free survival (rfs) were compared between the two groups. multivariate analyses of factors associated with rfs were performed to identify whether d-lg affects the recurrence patterns. results: ultimately, patients were analyzed ( in the d-lg group and in the d-lg group), and there were no differences in the clinicopathological data between the two groups. distant metastasis was the most common type of recurrence. there were no significant differences between the two groups in the recurrence types, the first recurrence time or rfs (all p [ . ). according to the th american joint committee on cancer tumor-node-metastasis (tnm) staging system, both groups were stratified into pathological (p) i, ii, and iii stages. the stratified analysis showed that there were no statistically significant differences in rfs between the d group and the d group among patients in each subgroup (all p [ . ). the multivariate analysis of rfs showed that pathological tnm (ptnm) stage and lymphovascular invasion were independent risk factors (all p \ . ). the multivariate analysis of post-recurrence survival (prs) showed that adjuvant chemotherapy was an independent protective factor (p = . ). conclusions: distant metastasis was the most common type of recurrence after lg. the postoperative recurrence patterns, rfs and prs after d-lg were similar to those after d-lg. purpose: the aim of this study is to evaluate the efficacy of delta-shaped anastomosis compared to circular stapler anastomosis in laparoscopic distal gastrectomy with billroth i reconstruction (ladg-bi). method: this is a single-center randomized controlled study. eligibility criteria included histologically proven gastric adenocarcinoma in the lower third of the stomach, clinical stage i tumor. patients were preoperatively randomized to circular stapler anastomosis or delta-shaped anastomosis. the primary endpoint is the number of analgesics use during days after surgery. we compared the surgical outcomes of the two groups. postoperative qol was evaluated using the postgastrectomy syndrome assessment scale- . this trial was registered at the umin clinical trials registry as umin . results: between december and september , patients (delta-shaped anastomosis , circular stapler anastomosis ) were enrolled. there was no difference in the number of analgesics use during day after surgery (median : delta-shaped anastomosis vs. : circular stapler anastomosis, p = . ). there was no difference in the overall proportion with in-hospital grade ii-iiib surgical complications ( %: delta-shaped anastomosis, %: circular stapler anastomosis). there was no operation-related death in either arm. regarding postoperative qol evaluated month after surgery, diarrhea subscale was significantly worse in delta-shaped anastomosis than in circular stapler anastomosis. conclusion: we did not demonstrate the advantage of delta-shaped anastomosis in terms of postoperative pain. since delta-shaped anastomosis tended to cause postoperative abdominal symptoms related to diarrhea, we should carefully apply the delta-shaped anastomosis to ladg. introduction: the use of a three-dimensional( d) camera for laparoscopic surgery has been reported in literature. however, there are only few comparative studies demonstrating its benefits, and no reports on the application of d vision to single-incision laparoscopic surgery. this study aims to compare d vision to the previous two-dimensional( d) system in solo single-incision laparoscopic distal gastrectomy(sidg). methods: medical charts of gastric cancer patients who underwent solo sidg from february to december were retrospectively reviewed. patients were grouped into either d group or d group depending on the type of camera used. all the operations were performed by a single surgeon using a flexible camera(olympus, japan), fixed onto a passive scope holder without the use of a scopist or an assistant. operative data, postoperative outcome, and early complication were analyzed. results: ninety had their operations under d vision and used the d scope. in both groups, there was no difference in age, body mass index, staging, and other demographic or histopathologic criteria. operative time was significantly faster in the d group( . ± . vs. . ± . mins., p = . ) and ebl was also less( . ± . vs. . ± . ml, p = . ). patients in the d group started small fluid diet faster( . ± . vs. . ± . postoperative days, p = . ), and were discharged faster( . ± . vs. . ± . postoperative days, p = . ). early complication was also less in the d group( . % vs. . %) but there was no statistical significance(p = . ). conclusion: the use of the d camera improves operative outcome and hospital stay in patients undergoing solo sidg. the frequency of anastomotic leakage after gastrectomyreaches - %. at the same time, mortality in this group of patients reaches %, and the use of aggressive methods of surgical treatment for the treatment of anastomotic leakage increases the mortality rate from to %. since , vacuum-assisted closure has been used to treat anastomotic leakage of various localizations. the essence of this method is based on the creating a local negative pressure, which is transmitted to the drip cavity through a special porous spongy system. the negative pressure created in the closed cavity, allows you to remove exudate, helps to reduce tissue swelling, improvesmicrocirculation, which in turn contributes to the development of granulations and wound healing with separation of the fistulous course. failures in using the method of vacuum therapy in anastomotic leakage are associated with the great difficulty of delivering a polyurethane sponge with a drainage tube to the leakage zone. in this regard, we have developed an improved method of endoscopic local vacuum therapy, in which the delivery of a polyurethane sponge was carried out with the help of a thread through a pharyngeal ring, a leakage zone and brought out through a drainage tube. this technique has been successfully used in the treatment of four patients with anastomotic leakage after operations on the upper part of the digestive tract. for complete healing of the cavity of the leakage and defect of the organ wall, it took , , and sessions of replacing the vac system, respectively (average . ± . ). there were no complications during the endoscopic local vacuum therapy. when the control endoscopic studies after months after the completion of the treatment at the site of defects of the seams of the anastomoses formed tender scar tissue without signs of narrowing of the organ. aims: enhanced recovery after surgery pathways are safe and effective for patients undergoing gastrectomy. this study aimed to identify perioperative factors influencing the adherence to the protocol, the postoperative course, and the consequent length of stay. methods: between and , patients were referred to our institution for gastric cancer. among these, patients underwent atypical gastric resection and were excluded from this analysis. were assigned to either total or distal gastrectomy and represent the study population. all patients were managed with a standardised perioperative pathway according to eras principles. according to data from the literature and based on our clinical experience, patients with optimal adherence to eras protocol may fit the criteria for discharge within ninth postoperative day, that was considered our ideal threshold for hospital discharge. data were retrospectively collected and analysed from a prospectively maintained database. statistical analyses were performed using spss version for macintosh. the v test, with a significance level of . , was used to investigate the association between the outcome and perioperative categorical variables. when parametric assumptions were met, student's two-tailed t-test was used to compare the means of continuous variables; otherwise, the mann-whitney test was performed. a significance level of . was chosen. logistic binary regression with a backward selection procedure and selection criteria of p-value \ . were exploited to determine significant predictors. results: preoperative, intraoperative and early postoperative variables were considered. among all, multivariate regression analysis revealed that incomplete preoperative immunonutrition, failure to extubate the patient at the end of surgery, intraoperative crystalloids infusions [ ml and blood transfusion [ ml, surgery duration [ min, and failure to mobilise patients within h from surgery were associated with delayed discharge. the logistic regression model was statistically significant (p \ . ) and correctly classified . % of cases. sensitivity and specificity were . % and . %, respectively. conclusions: results seem to be clinically rational and focus the attention on the importance of some perioperative clinical issues for the management of postoperative course. these variables could be considered as clinical goals to be reached in order to get an early discharge. objectives: the purpose of this study is to confirm the safety of laparoscopic gastrectomy with intraperitoneal cisplatin administration as a treatment for advanced gastric cancer with potential for peritoneal seeding. methods: from july to august , patients with advanced gastric cancer who underwent ip chemotherapy after diagnostic laparoscopy were retrospectively studied. all patients underwent laparoscopic gastrectomy with ip chemotherapy or ip chemotherapy alone after a diagnostic laparoscopy. gastrectomy was performed for palliative purposes even with seeding. results: the average age of the patients was years. eight patients ( . %) had preop chemotherapy. curative resection (r ) was performed in patients ( . %). in diagnostic laparoscopy, cytology was performed in patients ( . %) and cy was ( . %). peritoneal metastasis was detected in patients ( . %). of the total cohort, the year os rate was . % and the median survival time was months. in the case of stage iiib and below, the -year os rate was %, but it was % in stage iiic-iv group. when the r resection group and the r - resection group were compared, the -year os rates were . % and . %, respectively. hematological toxicity such as neutropenia was not seen in all patients. the mean hospital stay was . days and adjuvant chemotherapy was performed in patients ( . %). background: radical proximal gastrectomy (pg) and lymph nodes dissection are indicated for selected gastric cancers at the upper third of the stomach. with the advent of laparoscopic surgeries, more and more pg were performed by laparoscopic apporaches. in the past years, our team has accomplished and reported the oncological outcome of laparoscopic distal gastrectomies in cases of clinical stage i gastric cancer in taiwan. through the evolution of surgical trechniques and team work, we have cruised the learning curve of laparoscopic gastrectomy and reconstruction. materials and methods: in this report,we would like to present our surgical experience of laparoscopic proximal gastrectomy for gastric cancer patients. from to , pateints with gastric cancer underwent laparoscopic gastrectomies by the same surgical team at the national taiwan university. among them,six consecutive pateints (male:female = : ) with gastric adenocarcinoma of the upper stomach underwent laparoscopic pg in . the demographics, dissection, reconstruction methods and peri-operative outcome are presented. all six patients tolerated the procedure well, onepatient had mild anastomotic stenosis and improved with one session of endoscopic dilatation. one patient needed temporary proton pump inhibitor for controlloing acid reflux. four of the patients were pathological stage i, and the rest two pateint were stage iia and iiia disease. there was no tumor recurrence until now. summary: laparoscopic proximal gastrectomy is technically safe for treating upper third gastric cancers. the long term oncological outcome deserve further observation. introduction: open gastrectomy (og) has long been the preferred surgical approach worldwide for treatment of gastric cancer (gc). nowadays, several randomized, prospective trials have confirmed improvements in postoperative outcomes for laparoscopic gastrectomy (lg) compared to open procedures, with similar oncologic outcomes. however, most part of these studies comes from the eastern countries. material and methods: a prospective non randomized study was conducted with all patients operated of gc at ramón y cajal university hospital from january to december . over patients enrolled, patients underwent lg and og. textbook outcome was defined as the percentage of patients who underwent a complete tumour resection with at least lymph nodes in the resected specimen and an uneventful postoperative course, without hospital readmission. results: a textbook outcome was achieved in . % of patients operated of gc. the outcome parameter 'no severe postoperative complication' had the greatest negative impact on the textbook outcome. a statistically higher number of patients with early cancer ( % vs. . %) and subtotal gastrectomy ( . % vs. . %) were found in the laparoscopic group. no statistically differences were found between open and laparoscopic approach regarding operating time, rate of microscopic margin positivity, hospital stay, number of retrieved lymph nodes, complications, reinterventions, mortality and readmissions. no statistical differences in textbook outcome were found between both groups ( . % vs. %; p = . ). conclusions: laparoscopic gastrectomy for treatment of gastric cancer seems to be safe and feasible with similar textbook outcomes compared to open gastrectomy. introduction: laparoscopic surgery has been increasing for treatment of gastric cancer. however, standardization of this minimally invasive approach has not been reached yet because of its technical difficulties and the concern about oncological safety. the aim of the study was to analyze the outcomes of our learning curve in this complex surgical technique. material and methods: the first consecutive cases of laparoscopic gastrectomy (lg) performed at our hospital from november to february were enrolled. patients were divided into two groups based on the period they were operated. training phase (tp) was considered between and ( cases) and more-developed phase (mdp) between and ( cases). conversion, lymphadenectomy and retrieved lymph nodes (ln), hospital length of stay, mean operative time, complications, reintervention and mortality rates were compared between the two phases of learning curve. results: the number of retrieved ln was higher in the mdp ( ± , vs. , ± , ; p = , ). furthermore, we have also found less complications ( , % vs. , %; p = , ), a decreased reintervention rate ( , % vs. , %; p = , ) and overall mortality ( , % vs. %; p = , ) in the mdp. there were no significant differences in conversion rate, mean operative time, and hospital length of stay between phases. conclusion: although we consider that our learning curve is not completed yet because the average of monitored parameters have not reached a steady state, the improvement on surgical parameters and postoperative course in the last two years have showed our results are near to the best results published in the literature. aims: lymph node (ln) dissection proves to be essential for oncological gastrectomy, given that the presence of ln metastases is very high, even for early gastric cancer ( . % for t a and . % for t b). this way, d dissection for advanced gastric cancer and d ? for early gastric cancer are the gold standard procedures. some teams are using indocyanine green (icg) lymphography to improve their ln dissections, claiming that this technique facilitates the harvesting of small fluorescent ln that, otherwise, would be difficult to identify by conventional laparoscopic methods. methods: we herein present the case of a -year-old man with a t b distal gastric cancer. endoscopic ultrasound discarded the presence of metastatic ln and ct scan showed no distant metastases. icg was administrated endoscopically the day before the surgery, an amount of mg was injected along the submucosal layer around the tumour. in the video we can see how we perform a laparoscopic distal gastrectomy with d ? ln dissection and roux-en-y reconstruction. icg lymphography helped us to complete our expected ln harvesting, especially for groups (infrapyloric) and (left gastric artery). thanks to this technique, we could resect ln that we might have obviate during a usual laparoscopic procedure. results: patient was discharged home on the sixth postoperative day without complications and with adequate oral tolerance. conclusions: we present a case in which we have performed a laparoscopic distal gastrectomy with d ? dissection and roux-en-y reconstruction. we used icg lymphography to help us to improve our ln harvesting. although it is soon to assess if this technique may increase the number of retrieved ln and in which stations might be more useful, we consider this is a harmless method that may help gastric teams to complete their expected ln dissections. introduction: gastrointestinal stromal tumor (gist) represents around . % to % of gastrointestinal neoplasms, with the mesenchymal tumor being more frequent than the digestive one.the gist can be produced from the esophagus to the anus, at any point, being the stomach of ( to %) and the small intestine ( to %) more frequent sites.it is characterized by the expression of the tyrosine kinase growth factor receptor,cd ,differentiating it from other mesenchymal tumors,which do not express it.it is accepted that its origin corresponds to the interstitial cells of ramón y cajal,which act as a pacemaker for intestinal motility.they are very heterogeneous tumors, which vary in size,morphology and biological behavior,being neoplasms with uncertain malignant potential.the incidence is between the fourth and sixth decades,being the distribution by gender similar. clinical case: female patient of years,who goes to the general surgery service,as interconsultation,after a veda,by dyspepsia.it is reported stomach:ceiling mucosa without alterations,at the level of the greater curvature is seen a tumor of cm,hard to the touch with the biopsy forceps,slightly irregular covered with mucosa of normal appearance. computed tomography: stomach body:rounded image of nodular aspect which does not present heterogeneous enhancement after administration intravenous iodine contrast extending to peritoneal region, measures x x mm liver:hypodense image without heterogeneous enhancement adjacent to this,a mm rounded image that is suggested to be studied with nmr. gadolinium nmr liver hypodense image with well-defined limits without heterogeneous enhancement of cystic aspect. gastric roof,heterogeneous formation,which enhances with gadolinium mmx m-mx mm,having to discard a gist. surgical technique laparoscopic partial gastrectomy. pathological anatomy and immunohistochemistry . cm injury with net edges.uncertain malignant fusocellular nodule, cd ??? actin-dog ??? s -no mitosis or invasion of the mucosa is observed. conclusion: a case of stomach gist is presented,which,the main symptom was dyspepsia,being the clinical presentation very variable,in relation to the place in which it is located. there is fletcher criteria for the risk of malignancy,this being less than . cm,very low risk,less than cm, the patient evolved favorably,without surgical complications.aims: to present the surgical procedure of resection of the lesser gastric curvature and its pedicle with laparoscopic surgery, fulfilling oncological criteria, carried out in the general surgery service of the hospital of torrecárdenas. methods: an -year-old man with prostate cancer treated with complete hormonal block and epoc, who consults for rectal bleeding of week of evolution. it is diagnosed of gist in gastric lesser curvature, x x cm, very vascularized and infiltrates the wall producing marked imprint on the fundus. it is tributary of left gastric artery. precise blood transfusion and presents hemodynamic stability, is decided surgical resection scheduled. results: the surgery is performed by laparoscopy, with a tumor of approximately cm, which is dependent on the lesser curvature. the esophageal hiatus and the lesser curvature are dissected with section of the left gastric pedicle. atypical gastrectomy of the lesser curvature including gist, making a gastric sleeve dependent on the greater curvature. the anatomopathological study reports pt pn with lymph nodes without adenopathies, and disease-free surgical margins. he was discharged without complications on the th day and did not require re-entry. conclusions: the laparoscopy surgery for atypical gastrectomy of lesser curvature is safe and meets oncological criteria in selected patients and performed by an experienced esophagogastric unit. aims: to present the surgical procedure of total gastrectomy with d lymphadenectomy with laparoscopic surgery, fulfilling oncological criteria, carried out in the general surgery service of the hospital of torrecárdenas. methods: a -year-old male with a tobacco habit who consults due to epigastric pain and constitutional syndrome of months of evolution. it is diagnosed of gastric adenocarcinoma t n m . neoadjuvant qt is decided, after weeks of its completion, scheduled surgery is performed. results: the surgery is performed by laparoscopy, showing a stenosing tumor in at gastric antrum of approximately cm. dissection of the greater curvature with section of the right gastroepiploic at its birth and duodenal section is performed. dissection of the lesser curvature with d lymphadenectomy, section of the pedicle of the left gastric and the distal esophagus. the transit is restored with latero-lateral esophageal-jejunal anastomosis and jejunojejunostomy. the anatomopathological study reports ypt a and pn with / adenopathies, and disease-free surgical margins. he was discharged without complications on the th day and did not require reentry. conclusions: the laparoscopy surgery for total gastrectomy with complete d lymphadenectomy is safe and meets oncological criteria in selected patients and performed by an experienced esophagogastric unit. background: in gastric cancer surgery, to secure surgical margin, it is necessary to accurately judge the position of the tumor. however, with conventional marking clips, it is difficult to identify the exact location of the tumor during laparoscopic surgery. purpose: we investigate whether icg (indocyanine green) fluorescence navigation method is effective and safe for determination of cutting line in laparoscopic gastrectomy. patients and methods: subjects underwent laparoscopic gastrectomy (including robot-assisted surgery) based on the icg method for gastric cancer in the period from april to december . the day before surgery, icg diluted times ( . ml of reagent ? . ml of distilled water) was injected at cm from the tumor edge and . ml at the four submucosal layers around. then clip to the same part. gastrectomy based on standard surgery is performed, and the position of the tumor and spread of icg are confirmed by icg fluorescence navigation during operation, and a cutting line is determined. the extent of icg from the tumor is again measured with the excised specimen and compared with the pathological margin. results: among the patients who underwent intraoperative pathological examination, they were negative in all cases except one. the spread of icg was . cm on average, and considering the marking position ( cm) from the tumor edge, securing of . cm or more was possible. the operation time was . ± . min and the estimated bleeding loss was . ± . ml. conclusion: laparoscopic gastrectomy with icg method can evaluate tumor position and spread easily and in real time during operation and it was effective for determining the cutting line in laparoscopic gastrectomy. epstein-barr virus (ebv) has been known as one of causal virus of gastric cancer. ebv-related gastric cancer considered to be about % of the entire gastric cancer, and it is rare that ebvrelated gastric cancer has multiple lesions. the patient was years old female. she was diagnosed with upper gastrointestinal endoscopy with lesion in the lower major stomach body, lower anterior wall of the stomach body, rear wall in the middle part of the stomach, rear wall in the middle part of the stomach, and lesser curvature of the stomach angle, as a result of biopsy, adenocarcinoma was observed from the former four. the patient underwent a robot-assisted total gastrectomy. adding a newly found lesion, the histopathological diagnosis was pt b in the lower major stomach body, pt b in the lower anterior wall of the stomach body, pt b on rear wall in the middle part of the stomach, pt b on rear wall in the middle part of the stomach, and pt a in lesser stomach body, pn , pstageia. pathological examination results showed that the four lesions were positive for tumor cells in eber in situ hybridization and were considered to be ebv-related gastric cancers. she was discharged on the th day after the operation without any postoperative morbidities.there has been no sign of recurrence without postoperative therapy for months. results: a -year-old female with no medical history of interest or allergies to medications, who consulted for palpable mass at mesogastric level to the left of the midline associated with abdominal pain of - months of evolution, without concomitants or relationship with the intake, valsalva or physical efforts, without change in the depositional habit or toxic syndrome. the abdominal ct (computed tomography) revealed a cystic mass in jejunum mesentery, defined edges, about cm in diameter and that does not capture contrast; likewise, there is no ascites, retroperitoneal adenopathies or other intra-abdominal or pelvic masses, radiology recommends completing the study with abdominal mri (magnetic resonance imaging) that informs of possible lymphangioma at the level of the jejune mesentery. surgical exeresis was decided, which was carried out by laparoscopic approach, with emptying of the lesion and enucleation of the lesion without incidents, the postoperative evolution was favorable being discharged at h. the pathological anatomy reported fibro-adipose tissue with presence of lymphatic dilatations associated with a cystic lesion without epithelial lining, with serous fluid and abundant macrophagic reaction compatible with mesenteric lymphangioma. conclusions: the mesenteric cyst is a rare pathology with an incidence ranging from / , to / , , predominating in the fourth decade of life. it is defined as any cystic lesion in the mesentery, and is subdivided according to its origin into lymphatic, mesothelial, urogenital, dermoid, and enteric and pseudocysts. most of the time they are asymptomatic although they can (as in our case) present with abdominal pain and even produce complications such as intestinal obstruction, volvulus, intracystic hemorrhage, infection, rupture, and even malignant transformation. for the diagnosis, the palpation can be of great help, showing mass of well-defined limits and partially mobile. the imaging test of choice is abdominal ultrasound / abdominal ct, supplemented by magnetic resonance imaging. the recommended treatment is surgical exeresis, considering laparoscopy as the first option; if it is complete, it can be considered as a curative treatment. purpose: gastrostomy(og) is an alternative method for nutrition support, especial for the patients with oral-esophagus route obstruction or dysfunction. the most operation were conducted by young surgeon or residents. laparoscopic gastrostomy(lg) was a new coming procedure and the skillful suture techniques were needed. the most the residents can't be qualified for this operation. we designed the method for laparoscopic gastrostomy to provide the traning opportunity of suture skill training and guarantee the patient's safety. material and method: laparoscopic gastrostomy procedure was done with two mm trocar. the lower body of stomach was chose. four point around gastrostomy wound were chose for subcutaneous fixation. the straight needle with - prolene was inserted into peritoneal cavity from upper point, then punctured the sero-muscular layer of stomach. the needle was retrieved out from the same point by guidance of gauge needle. the same way was used for other three points. one purse string around gastrostomy was created by one hand suture method and fastened by köckerling knot tier after insertion of fr foley tube. finally, the peritonization was finished by hand tie externally and knot were keep in subcutaneous layer material-method: we present the case of a year old woman who presented with melena, hematemesis, anemia (ht . %) and being haemodynamically unstable. after the stabilization of the patient, a gastroscopic examination followed, where it revealed a tumor of the fundus (adenocarcinoma). the patient was submitted to laparoscopic total gastrectomy and oesophagojejunal anastomosis, omega type (o), and intestinal anastomosis braun, with the usage of trocars (umbilical mm as inserted in laparoscopic surgery of a single incision, and two mm in the midclavicular line bilaterally). the oesophagojejunal anastomosis was conducted with the use of a linear stapler for the posterior wall and the convergence of the anterior wall with laparoscopic sutures in two layers. patient remains in well condition, months after the operation. conclusion: laparoscopic approach seems to be safe for treatment of gastric cancer of the fundus and of the gastroesophangeal junction, as it offers better surgical field view and less postoperative complications. the restoration of the continuity of the gastrointestinal tube with anastomosis of w type is considered safe alternative to the classic roux-en-y anastomosis. git and bariatric surgery, faculty of medicine, alexandria university, alexandria, egypt; git surgery, faculty of medicine, alexandria, egypt background: superior mesenteric artery syndrome is best described as compression of the third part of duodenum by the superior mesenteric artery, resulting in obstruction. the study of this rare medical condition was carried out since decades yet remain obscure. this study aimed to analyze different clinical presentations, diagnostic modalities, treatment approaches and outcomes, as well as to emphasize the importance of long term follow up. methods: thirty-five superior mesenteric artery syndrome cases were collected retrospectively from a facebook group called 'superior mesenteric artery syndrome awareness & support'. a questionnaire was designed using google form to obtain the demographics, presenting symptoms, risk factors and co-morbidities, investigations, means of treatment and the outcomes. data was entered into microsoft office excel for statistical analysis. results: the median age at diagnosis was years. the median body mass index was . kg/ m ;. the median time interval from symptom onset to initial diagnosis was months. the major presenting symptoms were abdominal pain ( . %), nausea ( . %), and vomiting ( . %). abdominal computed tomography scan with contrast ( . %) was commonly used for confirmation of diagnosis. thirteen cases ( . %) were congenital. thirty patients ( . %) had received treatment. the overall management success was only . %. surgical management ( . %) was the most used regimen. conclusion: diagnosis of superior mesenteric artery syndrome is established after a thorough assessment of the clinical presentations and confirmation with suitable imaging modalities. the choice of treatment should be dependent on the causes and severity as different patients respond differently to therapy. recurrence is possible in all patients thus a long-term follow up is required. aims: in the last hundred years much has been written on peptic ulcer disease and the treatment options for one of its most common complications: perforation. laparoscopic repair of perforated peptic ulcer has been gaining popularity in recent years. treatment for perforated ulcer can be performed laparoscopically in % of cases, making it possible to avoid a median laparotomy which can lead to wound infection and late eventration. methods: a -year-old male presented to emergency room with a three-hour history of progressively worsening epigastric pain and nausea. physical examination revealed rebound tenderness compatible with an acute abdomen. a ct scan showed: important pneumoperitoneum unable to define the drilling point; distended stomach with plenty of fluid inside and dense content fundus / body suggestive of active arterial bleeding . results: the patient was emergently taken to the operating room for diagnostic laparoscopy . perforation shown in greater gastric curvature associated blood remnants. gastrotomy for clot removal is done without observing active bleeding. the gastrotomy was repaired using standard stitches. all exudate was aspirated and the peritoneal cavity was irrigated with warm saline solution the patient had an uncomplicated post-operative course. jp drain was removed and he was discharged one week after surgery. conclusion: the role of laparoscopic surgery in emergencies is well documented. laparoscopic approach is indicated in any case of suspected gastroduodenal perforation and seems to offer the same advantages as for the vast majority of laparoscopic procedures. laparoscopic surgery may therefore have a real place in the treatment of perforated peptic ulcer. the aim: of our study was to evaluate of effectiveness of local injection of platelet-rich plasma for treatment of peptic ulcer bleeding with hemorrhagic shock in experiment. methods: the study was performed on wistar rats according to local and international rules for working with experimental animals. the average weight of animals was ± grams. in all animals our modification of type acetic acid ulcer (susumu okabe, ) was modeled. we randomly divide all animals in groups. rats with only modeled ulcer were included in group . rats with modeled ulcer and hemorrhagic shock after - . ml blood sampling were included in group . in group we included rats with modeled ulcer and hemorrhagic shock and performed local injection of platelet-rich plasma (local periulcelar injection of . ml of autologous platelet-rich plasma). on st, th and th day measurement of the ulcers square and morphological study were performed. results: the data we have received demonstrate a tendency of decrease of ulcers' square in all groups with time flow. we also compared sizes of ulcerative defects in all groups at every point of the study. on the st day of investigation there were no differences (p [ . ) between ulcers' square in all groups. on the th day we found out more rapid decrease of size in group (p [ . ). however, this tendency had no statistical significance. on the th day difference was larger and it was statistically significant this time (p \ . ). also the better ability to stimulate the activity of fibroblasts and revascularization in the young connective tissue with improving oxygenation in the ulcers and enhancing of cell proliferation, differentiation and accelerating of maturation of connective tissue and healing of ulcers was demonstrated in group . conclusion: platelet rich plasma reduces inflammatory response and stimulates proliferation of gastric epithelial cells on th day with the restoration of secretory activity and epithelialization of ulcers in . % of experimental animals on th day, the activation of the fibroblastic reaction during the all experiment and decreasing of ulcers' square. h. fujii, depat. of surgery, japanese red cross fukui hospital, fukui, japan introduction: in conjunction with charmant, a local eyeglass frame manufacturer, we developed novel devices called the fj (free jaw) clip to grasp organs in the abdominal cavity and the f (free) loop plus to pull thread extracorporeally from within the abdominal cavity. product summary: the fj clip is used to grasp organs in the abdominal cavity, a stainless steel, removable forceps for use in laparoscopic surgery. it provides a strong grip but rarely crushes organ tissue. the clip comes in two sizes, one for use in a -mm port and the other for use in a -mm port, and in two lengths, . mm and . mm, respectively. to pull out thread tied to the fj clip, we developed the f loop plus, which is a g by -mm-long special stainless needle with f . -mm niti alloy thread which is used pull suture threads from inside the abdominal cavity to outside the body. case: we performed cases of reduced port laparoscopic and endoscopic cooperative surgery (lecs). we performed reduced port surgery (rps) by making a . -cm incision at the umbilicus, inserting trocars ( mm and mm), and inserting another trocar ( mm) at the left side of the abdomen. we expanded the left hepatic lobe with a -mm fj clip for penrose drain placement, grasped the front wall of the gastric body with a -mm fj clip, applying traction toward the legs to pull up the tissues around the tumor, and resected all layers of the tumor via oral endoscopic submucosal dissection technique. the resected area was closed with a suturing device or interrupted sutures in the abdominal cavity. a year-old female was admitted to the emergency department with complaints of abdominal cramping pain, back pain and diarrhea for one day. she also had fever, ever up to °. in these two weeks, she felt occasionally epigastric pain. her past medical history included hypertension. on physical examination, she was conscious and alert. abdominal examination revealed diffuse tenderness and knocking pain over right flank. laboratory tests indicated an degraded white cell count of /cumm with % band forms, c-reactive protein of mg/dl and abdominal liver function tests (alanine aminotransferase: u/l, alkaline phosphatase: u/l, gammaglutamyl transferase: u/l) without hyperbilirubinemia. abdominal x-ray showed paralytic ileus. our presumptive diagnosis was acute peritonitis, based on the patient's symptoms. empirical antibiotics were administered immediately, and a computed tomography (ct) imaging study was performed. the ct scan showed a stick like foreign body noted between ventral side of pylorus and smv lumen, about . cm in length and associated with perifocal infiltration and segmental smv thrombus formation. (fig. ) however, there is no obvious pneumoperitoneum and no evident ascites is associated. an emergency exploratory laparotomy was performed, revealing stomach perforation at posterior wall with a cm fish bone thourgh pancreas into smv. localized inflammation and fibrosis were identified without obvious fluid accumulation( fig. - ) . removal of fish bone and simple closure of stomach perforation were performed. blood cultures revealed bacteroides thetaiotaomicron. three weeks later, she received a follow-up ct scan which showed smv obliteration with chronic pylephlebitis. aim: here we present a case report about the endoscopic treatment for iatrogenic gastric perforation secondary to a chest tube insertion. methods: a case report of a -year-old male with history of a road traffic accident. described injuries were severe brain injury with gcs \ at pre-hospital care arrival, thoracic injury with several rib fractures on the left hemithorax and hypoventilation on the left side. prior to hospital transfer a chest drain was inserted on the left side, and the patient was intubated. results: at hospital admission, the patient was hemodynamically stable and connected to a mechanical ventilator. thoracic exam showed persistent hypoventilation on the left chest. no other abdominal or pelvic injuries were found in the physical exam. a frontal chest x-ray revealed pneumothorax and the chest tube was not viewable. a further ct scan showed the chest drain placed in the abdominal cavity, into the stomach, besides a subdural hematoma, comminuted pelvic fracture of the pubic rami and a left sacroiliac fracture. during the first h in the icu, neurological worsening was observed, and a new cranial ct revealed enlargement of the subdural hematoma, for what the patient underwent decompressive craniectomy, with improvement thereafter. following a five-day period of stabilization after surgery, the patient was evolving satisfactorily, and the removal of the intragastric chest drain was considered. endoscopy was performed to confirm the placement of the drain, and it was removed under direct vision. approximately twenty five centimeters of the catheter were visualized in the gastric lumen, and then successfully removed. the patient recovered well and was discharged from icu to medical hospital ward after fourteen days, and a week later he was discharged home. conclusion: endoscopic management for gastric perforation after a chest drain insertion may result effective and can prevent open surgery morbidity. aims: intestinal infusion treatment with levodopa/carbidopa (duodopa) is a therapeutic option concerning the advanced parkinson disease cases with no response to the conventional treatment. the drug requires carrying out a gastrostomy either by percutaneous endoscopy way, or by laparoscopy -if the first one is not possible-. later, a duodenum-yeyunum tube is placed in order to infuse the duodopa gel continuously by a portable bomb. in this report, we explain the laparoscopic gastrostomy technique. method: sin this report, we include two patients with advanced parkinson disease: the first one is a year-old female patient suffering from an important gait disorder; and the second one is a year-old male patient with uncontrolled motor fluctuations. in both cases, a percutaneous endoscopic gastrostomy was proposed, but neither was feasible because of the non-traslumination between the gastric and the abdominal wall. under general anesthesia, neumoperitoneum by veress needle was performed. three main trocars and one accessory were placed. at the level of the gastric antrum, a cm incision was conducted to insert a gastrostomy tube, to be the guide for the drug infusion catheter. next, the gastrostomy is fixed to the abdominal wall by the stamm technique, externalizing the catheter through the accessory trocar in the medial line. results: on the first post-operative day, a duodenum-yeyunum tube is placed by endoscopic control through the gastric device. both patients got well satisfactorily, and no complications were described; and they develop a total normal life within the limitations of their underlying disease. conclusions: the duodopa intestinal infusion shows a significative improvement for the advanced parkinson disease symptoms, compared with oral medication; appreciating positive results referring to life quality. when the catheter placement by endoscopy way does not seem posible, gastrostomy by laparoscopy constitutes a valuable surgical option for the treatment of this kind of patients. peptic perforated ulcus (ppu) is a common surgical emergency and laparoscopic repair has been introduced as an alternative to open repair. it has shown good results and allows closure and peritoneal lavage, just like the open repair does but with the advantages of a minimally invasive surgery. the objective is to report the outcome of laparoscopic ppu in our hospital. methods: from january to october , patients with a clinical diagnosis of ppu were assigned to undergo laparoscopic repair. this retrospective study included all husm patients who underwent laparoscopic ppu repair by emergency surgeons. minimum follow-up of months is carried out. results: of the patients in this series, % were men and % were women, between and years of age at the time of surgery, average of years. the time between the manifestation of symptoms and surgery was [ to h in % of patients. in patients there was a history of previous ulcer or non-steroidal anti-inflammatories intake and up to % were smokers. a ct scan was performed in all cases to reach the diagnosis primary closure with simple suture plus omental patch was the elected technic ( %). the approach was performed with trocars in %, trocars in % and in case. cases ( %) were gastric ulcer, duodenal cases ( %) and in one case no perforation was found. the conversion rate was %, in two cases due to technical difficulty and in the other case because the level of the perforation was not found. the median postoperative stay was days although there were cases with intrabdominal complications. there was an exitus due to a metastasic pulmonary neoplasia diagnosed in the immediate postoperative period. there were no cases of recurrence in the follow-up time. conclusion: in most centers, including ours, the rate of laparoscopic management has gradually increased along with the improvement of technical skills. improvements in the outcome of laparoscopic ppu repair are to be expected with more experience surgeon and a good selection of the cases. general surgery, jzu hospital ,,sveti vracevi,, bijeljina, bosnia-herzegovina introduction: diverticulum is an outpouching of a hollow organ. gastric diverticulum is rare form od this disease. incidence of detection varies depending on investigation method. it has been reported in . % cases of autopsies, . % cases of gastroduodenal roentgenographies with contrast, and . - . % cases of upper endoscopies.small diverticula are usually asimptomatic, but bigger diverticula can cause variable symptoms such as abdominal pain, feeling of epigastric fullness right after meal, feeling of discomfort in upper parts of abdomen, and severe 'foetor ex ore' .diagnosis is usually established in procedures such as gastroduodenal roentgenographies with contrast, upper endoscopies and abdominal ct scan. case report: a -year-old woman came to our hospital because of feeling of discomfort and mild pain in upper abdomen that lasted for last year. diagnosis is established after ct scan of abdomen and upper endoscopy procedure. initially she has been prescribed conservative therapy (proton pump inhibitors). since the symptoms persisted, laparoscopic resection of the gastric divertuculum was performed using endogia stapler. considering the feeling of discomfort and abdominal pain dissapeared, the patient was discharged from hospital on the fourth postoperative day. conclusion: asymptomatic gastric diverticula doesn't require treatment. since gastric diverticulum can have complications such as bleeding, perforation and neoplasia, patient without symptoms should be monitored. initial therapy for symptomatic diverticula is conservative therapy (proton pump inhibitors). if conserative therapy doesn't procude expected results, laparoscopic resection of the diverticulum should be considered. introduction: the acute perforation of a gastric ulcer is a serious entity that requires urgent surgical treatment in most of the occasions, it is increasingly accepted that the approach of choice is laparoscopic, depending, above all, on the time of evolution of the process. objectives: to demonstrate the safety and efficacy of the laparoscopic approach in the perforation of a pyloric peptic ulcer, even in cases of severe peritonitis, by means of a standardized procedure, insisting on the sequential thorough washing of the cavity.material and methods: we present a video of the surgical intervention of a patient with acute abdomen, with a history of nsaid ingestion, exploration and ct-analysis compatible with perforation of hollow viscus, probably of gastric origin. results: intervention: complete laparoscopic approach, trocars. severe biliopurunitic peritonitis, by pyloric perforation 'acute', liquid culture, suture of the perforation, epipoplasty, sequential thorough washing of the cavity with physiological saline and placement of drainages.correct postoperative period, discharge from the hospital on the th day after completing antibiotic treatment. endoscopy and helycobacter test are performed on an outpatient basis with normal results. conclusion: the laparoscopic approach is safe and effective in acute and complicated gastric ulcer disease, even in cases of severe peritonitis. surgical procedure: the clean-net procedure involves the selective dissection of both the serosa and muscle layer using a laparoscopic monopolar endoscopic scissor. the preserved mucosal layer provides a mechanical barrier between the gastric lumen and peritoneal cavity that aids in the prevention of peritoneal cavity contamination with gastric contents. tumors are observed with an upper gastrointestinal endoscope with the injection of indocyanine green (icg) into peri-tumoral submucosal layers at points. selective seromuscular dissection is performed using a laparoscopic electrocautery monopolar scissor. the mucosa surrounding the gist is then resected using a endoscopic mechanical stapler to prevent exposure of the gastric lumen to the peritoneal cavity and peritoneal tumor cell seeding. results: there were males and female, and the average age was years. the operation time was min, the average bleeding volume was . ml, the postoperative hospital stay was . days. the mean tumor diameter was . mm, the final histopathological diagnosis was gist, schwannoma. there were no postoperative complications of clavien-dindo classification or more. conclusion: clean-net was found to be safe and useful for the treatment of gastric smt with ulceration. year outcomes: laparoscopic heller myotomy stands the test of time aims: laparoscopic cardiomyotomy leads to excellent relief of dysphagia in % of patients and avoids thoracotomy or laparotomy. methods: we present a video illustration of the procedure that was modified at the american university of beirut medical center. so far, patients underwent laparoscopic cardiomyotomy, age range of to years, with males and females. most of them have had previous balloon dilatation. results: all cases were successfully completed laparoscopically without complications. followup of months to years revealed excellent results with complete resolution of symptoms and no need for further medications. this will result in minimal post-operative pain and very short recovery period and is associated with low complication rate. conclusion: cardiomyotomy for achalasia is ideal for laparoscopic approach. magnification allows for precise division of muscle fibers. the new technique of hydro dissection and enseal for division of esophageal muscle allows for completion of the procedure without injury of the mucosa. therefore, adequate release of the obstructing segment followed by anti reflux procedure toupet will lead to excellent results with minimal morbidity and no mortality. aims: laparoscopic repair of huge hiatus hernia methods: twenty two cases of huge hiatus hernia presented to the american university of beirut medical center. patients underwent through trocars in the upper abdomen reduction of the hernial sac from the chest. special care was taken in the dissection of the mediastinum to keep the thoracic fascia and pleura intact. the defect was sutured primarily by -ethibond sutures reinforced by onlay prolene mesh u-shaped was fixed at the rt. and lt. crus and a floppy nissen fundoplication performed . results: the video presentation includes the technical aspects and the method of reducing and repair of huge hiatus hernia.aim: nowadays, there is little experience in the world of applying robotic surgical system (rss) in treatment of patients with hiatal hernia (hh) and reflux-esophagitis (re). the aim of study was to determine the possibility and feasibility of using rss in treatment of patients with hh. materials and methods: a total of patients underwent robot-assisted hh repair without mesh, followed by fundoplication with our original method ( °full symmetric wrap). the clinical and technical analysis did not reveal any advantages over similar laparoscopic procedures, so we abandoned the use of rss for hh type i, and these patients were excluded. there were ( %) patients with hh type iii and ( %) type iv. the surgeries were performed by experienced robotic upper gastrointestinal surgeon and conducted with the davinci si surgical system (intuitive surgical, sunnyvale, ca). results: average operation time was ± ( - ) min. the respondents' mean age was . ± . years (range - ) and bmi was . ± . (range . - . ) cm/kg . average blood loss was ± ( - ) ml. average hospital stay was ± . ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) days. the average follow-up time was ± . ( - ) months. postoperative x-ray imaging and upper gi endoscopy was conducted in all ( %) patients. there was no hh recurrence diagnosed. we did not observe a relapse of hh or clinical manifestations of re in the early (less than days) and long-term (more than months) postoperative periods. conclusion: we can conclude that robot-assisted surgery is safe, appropriate and justified in patients with hh type iii and iv. all procedures performed to the patients with giant hh revealed clear technical advantages of rss over similar laparoscopic operations: an enlarged d hd image, bendable instruments with endowrist technology allowed for precise dissection of tissues (hernial sac, cicatricial adhesions) in a narrow anatomical space-posterior mediastinum without damage to pleura, pericardium and vagus nerves. we believe that use of rss in treatment of patients with reflux esophagitis and/or hh type i is unjustified, due to the lack of proven advantages over laparoscopy. introduction: the presence of major anatomical obstacles -such as massive caudate lobe-in the confined operative field of laparoscopic hiatal hernia repair (lhhr) poses significant challenge to the foregut surgeon. aim: to provide a safe alternative for lhhr using a laparoendoscopic approach. method: this patient is a year old female, with bmi of . . her past medical history includes diabetes, hypertension and hyperlipidemia. she had gerd for years. her egd showed x cm hiatal hernia and class b esophagitis. manometry showed ineffective esophageal motility. we used the classic five ports approach for lhhr. we found a massive caudate lobe which was comparable to the size of an already enlarged left lobe of the liver. the operative strategies: terminating the procedure proceeding with the standard approach and taking the risk of bleeding from the caudate lobe itself or the inferior vena cava (ivc) with possible catastrophic outcome. using the laparoendoscopic approach. the following three steps facilitated the performance of safe and effective surgery. additional liver retractor this improved exposure and minimized manipulation of the caudate lobe. extracorporeal sliding arthroscopic knots (esak) esak are similar to the knots used in endoloop. they are tied extracorporeally and require a single insertion of the knot pusher as they do not unravel. transoral incisionless fundoplication (tif) we performed tif to avoid a limited operative field and to prevent excessive tissue manipulations associated with laparoscopic fundoplication. tif also preserves the angle of his and produces partial fundoplication which has less side effects of dysphagia and gas bloat syndrome. results: the operative time was min (lhhr min and tif min). there were no complications. patient discontinued omeprazole which she used daily for years. at months follow up, her gerd related quality of life (hrql) and gerd symptom (gerss) scores were ( vs ) and ( vs ). conclusion: the laparoendoscopic repair of hiatal hernia in the presence of anatomical obstacles is safe and effective. longer follow up is needed to assess the durability of this repair. gastroesophageal reflux disease (gerd) is a condition that reduces the quality of life and can causedisorders associated with acid reflux, such as bronchial asthma, barrett's esophagus and esophagealadenocarcinoma. gerd is often caused by existing of hiatal hernia. nowadays, some surgeons haddifficulties with the laparoscopic approach to treatment of recurrent hiatal hernias.patient was a -year-old man. he requested medical assistance with dysphagia, nausea after eatingand heartburn getting worse in a horizontal position. conservative treatment was not effective.transthoracic nissen fundoplication was performed in . the main complaints of the patientpersisted during the postoperative period. the upper half of stomach and s-like curved esophagus werelocated in the mediastinum according to multislice computed tomography of the thorax in august.in our clinical center was performed laparoscopic cruroraphy, cardiomyotomy and nissen fundoplicationin november. during the surgery the normal anatomical position of stomach has been restored, s-like curve of esophagus has been removed; a gastric cuff (collis-nissen) has been created and anteriorand posterior cruroraphy has been performed. the patient was in intensive care during h. anasogastric tube feeding was continued during the first h. passage of the contrast through theesophagogastric junction was free within h after surgery. patients had been discharged within days after surgery.this case report shows that at the current stage of surgery laparoscopic approach can be useful not onlyfor treatment of primary hiatal hernias-but also for treatment of recurrent ones. aims: laparoscopic heller myotomy procedure, completed with an anti-reflux procedure is technically demanding. we report a case of laparoscopic heller myotomy followed by a dor anterior fundoplication. methods: this is the case of a -year-old caucasian woman with gradual dysphagia for solids and liquids, accompanied by severe regurgitation and chest pain. an initial diagnosis of achalasia was made in , with the use of manometry and barium swallow. endoscopic dilatations were attempted pre-operatively with no clinical improvement. decision was made to perform a laparoscopic heller myotomy, combined with a standard dor anterior fundoplication. a -ports operation took place (one intra-umbilical mm trocar-single incision laparoscopic surgery (sils) technique, two -mm subcostal trocars, and one another mm subcostal trocar for the use of liver retractor). the operation lasted h and min. results: no post-operative complications were noted. the post-operative swallow test showed improvement of the esophageal patency. the patient started a liquid diet three days later and was discharged six days post-operatively. two months later the patient presented no complications. conclusions: heller's myotomy has demonstrated good long-term results in the treatment of esophageal achalasia and the laparoscopic approach has been well established in the last two decades. it is a very demanding operation to perform and the disease is relatively rare, making the learning curve difficult to achieve. aims: achalasia is a type of motor disorder of the esophagus due to degeneration of ganglion cells in the myenteric plexus, leading to failure of relaxation of the lower esophageal sphincter, accompanied by a loss of peristalsis in the distal esophagus. the association of a long-term achalasia and a large size hiatus hernia is an infrequent entity. among the therapeutic options is medical treatment, endoscopic treatment and surgical treatment associated with an antireflux procedure. the laparoscopic approach being the more indicated due to its better results in terms of morbidity, mortality and recurrences. the aim of the video is to show the effectiveness and safety of the laparoscopic approach in this infrequent pathology, pointing out the importance of performing a standardized procedure. methods: -year-old male patient, with personal history of chronic ischemic heart disease and obesity, diagnosed with long-term achalasia with moderate dilatation of the esophagus associated with giant hiatus hernia. the complementary explorations and iconography of interest are exposed. results: intervention: complete endoscopic approach, trocars. reduction of hernial content into the abdominal cavity, dissection of the hernial sac and esophageal lipoma. extended mediastinal esophageal dissection. complete resection of both the sac and lipoma, respecting the posterior vagus. heller's myotomy of cm, including cm distal to the ueg, perforation of mm of the mucosa at the ueg level, suture and blue methylene verification of the sealing. hiatorraphy and dor-type anterior fundoplication as antireflux technique. correct postoperative, with egd control on the rd po day and discharge on the th. asymptomatic at months after surgery. conclusion(s): for achalasia laparoscopic heller myotomy with a partial fundoplication should be the treatment of choice in patients who are at low surgical risk. the length of the myotomy, especially distal to ueg is one of the most important aspects of the surgery, to achieve an effective disruption of the les. the presence of a giant hiatus hernia makes the procedure difficult, increasing the risk of complications, such as perforation. standardization is essential to increase safety and efficacy in these complex cases. purpose: there is evidence that the application of mesh-reinforced hiatal repair has resulted in a significant reduction in recurrence rates in comparison with primary suture repair, at least in short-term follow-up. however, and instead of this, the standard of care for repairing large paraesophageal hiatal hernias (lphh) remains controversial because no clear guidelines are given regarding indications, mesh type, shape and position. the aim of this study is to evaluate our short-term outcomes in management of lphh with a biosynthetic monofilament polypropylene mesh surrounded by a high-purity and adherent titanium dioxide surface coating to enhance the biocompatibility (tio mesh tm ). methods: a retrospective study was conducted on our institution between december and october . data were collected on patients with lphh greater than cm in which a laparoscopic repair was carried on by primary suture and additional reinforcement with a tio mesh tm . clinical and radiological recurrences, dysphagia and mesh-related complications were investigated. results: there were females and males with a mean age of years (range, - years). all operations were completed laparoscopically. median postoperative stay was days. after a mean follow-up of months, patients developed clinical recurrence of reflux symptoms ( . %) and radiological recurrences ( . %). there were no mesh-related complications. conclusions: the use of tio mesh tm for laparoscopic repair of lphh is suited and with a reasonably low recurrence rate in this short-term study. additional long-term studies with enormous numbers carried out for years will be necessary to affirm whether this mesh is convenient in the prevention of recurrences and mesh related complications. background: surgery for refractory gastroesophageal reflux disease (gerd) has a satisfactory outcome, however sometimes fundoplication fails and redo surgery is required. several publications have investigated the feasibility of performing reoperative fundoplications using laparoscopic techniques. the aim of this study was to describe our experience in laparoscopic redo fundoplications in the last years. material and methods: we retrospectively reviewed consecutive patients who required laparoscopic redo fundoplication from january to august .the indications were recurrent symptoms of gastroesophageal reflux disease (gerd) ( . %), recurrent symptomatic paraesophageal hernia ( . %), dysphagia ( . %) and acute volvulus ( . %). results: all redo fundoplications (basically toupet . % and nissen . %) were completed laparoscopically. the mean operative time was min (range, - . min). a mesh was placed in % of cases. intraoperative and postoperative complication rates were . % and . % respectively. the mean hospital stay was days (range, - days). one patient ( . %) from the laparoscopic group required a third operation-one for acute recurrent paraesophageal herniation of the redo wrap one month after surgery, which was repair laparoscopically again. symptomatic outcome was successful in . % without any kind of proton bomb inhibitors therapy. conclusion: laparoscopic redo fundoplication is technically feasible and clinically effective with a reasonable low rate of postoperative complications p -upper gi-reflux-achalasia objectives: in recent years, balloon dilatation (bd) for diseases requiring correction of the impaired patency of the sphincter zones of the esophagogastroduodenal region has become widespread. purpose: to assess the effectiveness of the use of the balloon dilatation in patients with impaired sphincter zones of the esophagogastroduodenal region. materials and methods: in the institute department of surgery for the period from to , bd was performed in patients. of them diagnosed with achalasia of cardia (ac): - stage, - stage, - stage, - stage. patients diagnosed with pylorospasm, patients had compensated stenosis and patients had subcompensated ulcerative pyloroduodenal stenosis. there were males, females, average age ( . ± . ). bd was performed under endoscopic and / or x-ray control by 'boston scientific' balloons with a diameter of - mm, mm and mm, a course of - sessions with an interval of - days and a cylinder exposure of - min. evaluation of bd was performed using esophagogastroscopy, balloon manometry and x-ray passage of barium. results: in the course of the study, the existing indications were refined and new indications were developed for performing an endoscopic bd in pyloroduodenal stenosis and in ac. in patients with stage - ac, a positive result was noted in . % of cases already after the first session of bd. recurrences of ac after bd for up to years were established in ( . %) patients: at stage -in . %, at stage -in . %, at stage -in . % and at stage -in . %. repeated bd courses in case of ac recurrence in ( . %) cases turned out to be ineffective. recurrence of pyloroduodenal subcompensated stenosis was diagnosed in . % of cases in the period of months after performing bd. conclusions: bd is an effective method for correcting the permeability of the sphincter zones caused by the pathology of the esophagogastroduodenal region. keywords: balloon dilatation, achalasia of cardia, pylorospasm, ulcerative pyloroduodenal stenosis, recurrences. introduction: the reoperation in antireflux surgery significantly increases morbidity and mortality up to - %, reaching rates of % in patients undergoing or more surgeries. the advantages of laparoscopic surgery used in this surgical technique have amplified its acceptance and use, resembling its results in terms of feasibility, safety and efficacy of laparoscopic surgery to open surgery.objective: :evaluate the currently literature about antireflux surgery reintervention, focusing on the main indications of re-intervention, type of approach and morbidity and mortality of laparoscopic antireflux surgery. material and methods: a literature search was conducted in two electronic databases, med-line and embase. the search was limited to the period to . terms were used in relation to the procedure or intervention and the underlying disease. we chose observational studies (cohort, cases and controls and series of cases), where the main indication for antireflux surgery would have been gastroesophageal reflux disease. results: a total of studies were selected, most of them were case series ( . %), cohort studies ( %) and case-control studies ( . %). a total of patients. the main indications were anatomical faults, of these failures, recurrent hiatus hernia and sliding occupy the highest percentage, while physiological failures, failure in esophageal and gastric motility occur more frequently. the main type of approach was laparoscopic in %, the conversion rate was . % and the open approach was reserved for complex cases with more than one re-intervention . %, for abdomen . % and chest . %, this last for cases with high esophageal lesions that can not be repaired via trans-abdominal.the main complications were injuries to hollow viscera, such as: esophagus and stomach among others. these complications are related to the complexity of the procedure. mortality has remained low up to . %, however, the cause of death was due to medical complications and not related to the procedure. conclusions: this systematic review on reoperation in reflux surgery has confirmed that morbidity after reoperation surgery is higher than after primary surgery and reoperation indications increase with the use of new technologies (manometry) and the laparoscopic approach continues on the rise, with great adaptation to its use and improvement in results. aims: eras protocol is not commonly used in acute emergency procedures. elective lc is commonly performed as one day surgery, while in an emergency setting of acute cholecystitis, the in hospital stay averages , days. the aim of this trial is the application of eras protocol in patients with acute cholecystitis, undergoing laparoscopic cholecystectomy. methods: a randomized prospective trial was conducted in first surgical department of sismanogleion g.h.a. the study included patients, who were admitted with acute cholecystitis and underwent lc into h from their admission. preoperatively, they all received crystalloid isotonic solutions and antibiotics. . % were submitted to ercp, preoperatively, due to choledocholithiasis. the postoperative care included early mobilization into h after surgery, early fluid intake (into h) and early liquid food intake (into h). they all received systematically antibiotics, analgesics and antiemetic on demand. asa score was not an exclusion criterion. results: conversion to open procedure was necessary in . % of patients, whom were excluded from the study. all the rest were discharged into h from the surgery with the guidance to receive oral antibiotics for more days. readmission was necessary for patients, one week after the operation. the first one presented with bile leak and submitted to ercp with stent placement and percutaneous drainage of the intrabdominal collection. the second one presented with choledocholithiasis and underwent ercp with balloon catheterization. conclusion: it is commonly accepted that eras protocol in elective procedure enhances the postoperative recovery while reduces the in hospital stay and cost. in emergency condition eras cannot be applicated preoperatively. however, a modified post surgery application seems to have advantages equal to those observed in elective procedures. aim: laparoscopic cholecystectomy is the gold standard for the treatment of symptomatic cholelithiasis. administration of one single dose of chemoprophylaxis before an elective laparoscopic cholecystectomy is a broadly accepted practice. however, its value is currently questioned, especially in low risk patients. method: this study was conducted in a high volume surgical department. one hundred and twelve patients submitted to elective laparoscopic cholecystectomy were included in this research. a written consent was acquired after thorough patient briefing. half the patients that underwent surgical operation received one dose of antibiotics min prior to the incision and the other half did not receive any chemoprophylaxis. results: the age ranges from to years old. commonest concomitant diseases were arterial hypertension, type ll diabetes, hypothyroidism and respiratory deficiency. approximately % of patients were smokers and % were obese (bmi [ ). the duration of the operations was between and min. intra-operative gallbladder rupture was observed in patients (rate %). all the patients were discharged the first post-operative day and their monitoring continued for more days. in the chemoprophylaxis group, no surgical site infection or other major complication was observed. from the group that did not receive any antibiotics, one patient developed surgical site infection and specifically infection of the surgical port in the epigastrium, which was treated with drainage of the abscess and oral antibiotics administration. no other complications were recorded. conclusion: our study concluded no statistically significant difference between the two patient groups, which depicts that chemoprophylaxis may not be necessary in elective cholecystectomy operations. on the contrary, antibiotics increase the cost of hospital stay and are often accompanied by multiple mild or severe side effects. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -pj pal authors: ouyang, bo; dong, ying; chou, james j. title: structural and functional properties of viral membrane proteins date: - - journal: advances in membrane proteins doi: . / - - - - _ sha: doc_id: cord_uid: pj pal viruses have developed a large variety of transmembrane proteins to carry out their infectious cycles. some of these proteins are simply anchored to membrane via transmembrane helices. others, however, adopt more interesting structures to perform tasks such as mediating membrane fusion and forming ion-permeating channels. due to the dynamic or plastic nature shown by many of the viral membrane proteins, structural and mechanistic understanding of these proteins has lagged behind their counterparts in prokaryotes and eukaryotes. this chapter provides an overview of the use of nmr spectroscopy to unveil the transmembrane and membrane-proximal regions of viral membrane proteins, as well as their interactions with potential therapeutics. we will focus our discussion on two classes of transmembrane (tm) proteins encoded by viruses, viroporins (or viral channels) and membrane fusion proteins, as these proteins have been sought after as antiviral targets and they often exhibit peculiar structural features not seen in other membrane proteins. as implied by their name, the viroporin proteins can form channel-like structures in lipid bilayer that permeate ions or solutes. now over a dozen viroporins from various sources have been characterized, covering a wide range of ion substrates including h + , k + , na + , ca + , and cl − , as well as larger substrates such as rna. the exact function of ion channel activity in many viruses is not yet known, though their roles have been implicated in entry, virus assembly and virus release. the function of membrane fusion proteins is much better defined, that is, to enable viral entry by mediating virus-host membrane fusion. the viral fusion proteins have large extramembrane domains for receptor recognition and for grabbing onto the host cell membrane, but the function of their tm and membrane-proximal regions have been elusive. functional mutagenesis studies have suggested that, at least in the cases of hiv- and influenza a viruses, the tm domains (tmds) of fusion proteins are not merely membrane anchors, but play important roles in membrane fusion and viral infectivity. apart from the channels and fusion proteins, some viruses have developed enzymatic domains anchored to the membrane, e.g., the polymerases of the hepatitis c virus and the neurominidase of the influenza viruses. in these cases, the tmds are believed to only serve as membrane anchors and thus will not be discussed in this chapter. viroporins serve highly diverse functions in viral infection cycles (illustrated in fig. . ). early studies of viroporins have focused on only a few systems including b of poliovirus, k of togavirus, and m from influenza a virus (am ). the b is a well-known viroporin that can modify membrane permeability and allow the passage of ions and small molecules during later stages of viral infection [ ] [ ] [ ] . the k is a small, hydrophobic acylated protein that has been shown to form cation-selective channels when inserted into lipid bilayers [ ] . the k is proposed to be involved in virus budding, but the exact role of k in the viral life cycle is poorly characterized [ ] [ ] [ ] . the am proton channel is the most studied viroporins because ( ) it is the drug target of the first anti-flu drug (amantadine or rimantadine) and ( ) it is one of the smallest proton channels with proton selectivity motif not found in any of the proton channels from other organisms [ ] [ ] [ ] . since the definition of viroporin has been put forward, more and more viroporins have been classified (table . ). the influenza b virus encodes the bm proton channel that is a functional homolog of the am but cannot be blocked by the adamantane family of drugs [ ] . the human immunodeficiency virus type (hiv- ) vpu and the hepatitis c virus (hcv) p have been shown to form oligomeric channels to conduct cations [ ] [ ] [ ] [ ] [ ] . the b protein from the enterovirus (ev ), however, displayed altered channel activity, as it conducts anions (e.g., cl − ) instead of cations [ ] . paramecium bursaria chlorella virus (pbcv- ) has a viral k + channel named kcv that contains the canonical potassium selectivity filter [ ] and was proposed to adopt similar structure as kcsa [ ] . in addition to the ion channels, the vp from rhinovirus can form pores that ( ) . virion attaches directly host cell receptors on the membrane ( ) . and subsequently enters the cell by receptor-mediated endocytosis ( ) . acidification of the endosomal vesicles triggers conformational changes in the virion, resulting in fusion between the viral and the endosomal membranes, allowing the release of the nucleocapsid into the cytoplasm. the viral rna is then released into the cytoplasm and presented to the endoplasmic reticulum (er) ( ) . at the er, viral rna is translated into protein that is processed by viral and host proteases ( ) . after the viral replication complex is synthesized, rna synthesis begins by the transcription of an antisense viral rna followed by the amplification of viral rna ( ) . the newly synthesized rna is subsequently packaged by capsid protein, forming a nucleocapsid ( ) . virus assembly occurs on the surface of the er when the nucleocapsid buds into the er lumen, then transported through the golgi, where acidification induces virus maturation ( ) . mature viral particles are released in the neutral ph of the extracellular millieu (a) viral entry, which acidifies the virion interior immediately after endocytosis and facilitates rna release (b) dissipation of the proton gradient in the golgi and the trans-golgi network. the viroporins influenza a virus (iav) matrix protein (m ) and hepatitis c virus (hcv) p equilibrate the proton concentration with the cytosol to reduce the acidification of vesicular acidic compartments (c) viroporins play an essential role in assembly, budding and release of the viral particles (d) alteration of cellular ca + homeostasis. calcium uptake by the mitochondria can lead to dissipation of the inner-mitochondrial-membrane potential (ΔΨm), permeabilization of the outer mitochondrial membrane and, finally, the release of cytochrome c. in the cytosol, cytochrome c promotes the subsequent formation of apoptosome that is involved in apoptosis allow single-stranded viral rna to cross the membrane [ , ] , further expanding the list of substrates that viroporins transport. probably the best-defined roles of viroporins are that of the m proton channels of influenza a and b viruses, both of which involve equilibrating ph between compartments. one important role of the m is to equilibrate ph across the viral membrane during viral entry, which acidifies the virion interior immediately after endocytosis and facilitates rna release [ , ] (fig. . a ). another role of m is to equilibrate ph across the trans-golgi membrane of the infected cells during viral maturation, which preserves the pre-fusion form of the hemagglutinin fusion protein through de-acidification of the golgi lumen [ ] (fig. . b) . the roles of other cation-selective viroporins are less clear. one of the general roles proposed for viroporin is to depolarize either the er or plasma membrane to facilitate membrane curvature formation during virus budding [ , ] (fig. . c ). in the case of hcv, for example, the p -mediated channel activity has been reported to facilitate virus release [ , ] . another general role for viroporin is simply to cause cation leakage, which would induce cellular stress and programmed cell death [ ] (fig. . d ). in addition to ion permeation, several viroporins are known to participate in viral assembly through interaction with other proteins. for example, the influenza am and bm proteins both have significant cytoplasmic domains that are believed to recruit matrix proteins m during virus assembly [ ] [ ] [ ] . the hcv p protein has also been reported to interact with other non-structural proteins such as ns , and this interaction appears to be crucial for the production of infectious hcv particles [ , ] . pore formation indicated but a defined oligomeric state either does not exist or is not characterized enveloped viruses infect host cells by the fusion of viral and cellular membrane. depending on the type of the virus, fusion can occur either with the host plasma membrane or with the endosomal membrane. the membrane fusion must occur without consuming energy; it is catalyzed by major conformational changes of specialized envelope proteins generally known as viral fusion proteins. despite their diversity, all known viral fusion proteins convert from a prefusion state (dimers or trimers, depending on the class), through a key intermediate state (extended conformation), to a postfusion state (a trimer of hairpins) that brings the fusion peptide, attached to the target membrane, and the tm domain, attached to the viral membrane, into close proximity thereby facilitating the fusion of viral and target membranes ( fig. . a) [ ] . three distinct classes of viral fusion proteins (exemplified in fig. . b) have been defined based on structural criteria [ ] . the class i includes fusion proteins from some of the best characterized human pathogens, such as influenza and hiv- . these proteins are trimers of a single chain precursor that requires a proteolytic cleavage to generate two fragments. for example, the influenza a hemagglutinin (ha) contains the n-terminal fragment ha and c-terminal fragment ha [ ] , and the hiv- envelope protein is cleaved into the gp and gp fragments [ ] . the fusogenic fragment gp bears a hydrophobic fusion peptide capped by the outer receptor binding fragment gp [ ] . receptor binding accompanies cap protein release, allowing major conformational changes in the gp that ultimately leads to fusion [ ] . in the case of influenza, the cap ha is released by low ph of the endosome after endocytosis [ ] . the class ii fusion proteins are found on flaviviruses, alphaviruses, and bunyaviruses (e in flaviviruses, e in alphaviruses, gn and gc in bunyaviruses) [ , ] . although these fusion proteins are architecturally different from the class i fusion proteins, i.e., class ii fusion proteins are mostly made of β-sheets as opposed to the helical structures of the class i fusion proteins, they operate on the same physical principle. their fusion peptides are formed as loops at the tips of β-strands that serve as an anchor to insert into the host membrane after endocytosis. unlike class i proteins, which remain trimeric, their conformational change involves a change in oligomeric state from pre-fusion dimers to post-fusion trimers [ , ] . the reduced ph of the endosome induces a cascade of subsequent refolding and trimerization events that achieve membrane fusion [ ] . members of the class iii include g protein of vsv, gb of herpes simplex virus and epstein-barr virus (ebv), and gp of the insect-cell baculovirus [ ] . the class iii fusion proteins combine some of the features of class i and ii fusion proteins [ ] . each protomer is composed of five domains that contain a central trimeric coiled-coil, a hallmark of class i proteins in their postfusion states, three of the domains are predominantly made of β-sheets and their fusion peptide more closely resemble those of class ii proteins in that their fusion loops are found at the tips of extended β-strands [ ] . due to the essential roles of viral fusion proteins during infection, they have long been pursued as targets for antiviral intervention. for example, the approved small molecule drug arbidol is used as a broad-spectrum inhibitor of influenza a and b virus, as well as hepatitis c virus [ , ] . unlike many other broad-spectrum antivirals, arbidol has an established mechanism of action against the has in influenza a and b viruses that involves the inhibition of virus-mediated membrane fusion and thus viral entry [ ] . in addition, enfuvirtide (t , fuzeon) is an approved peptide drug that blocks the hiv- entry [ ] ; it is derived from the c-terminal heptad repeat region of the hiv- gp envelope glycoprotein, designated the c-peptide, that disrupts membrane fusion by competing with an intra-molecular interaction that is important for the refolding of gp during membrane fusion [ ] . apart from being therapeutic targets, another important medical use of the fusion proteins is vaccine development. viral fusion proteins are usually presented on the virion surface with high copy numbers, and thus are popular antigens for eliciting broadspectrum neutralizing antibodies. for example, the native conformations of the the n terminus of ha and the c-terminus of the ha ectodomain are labeled. blue arrow: position of fusion peptides inserted near threefold axis in prefusion form. only ha is shown on the bottom. the n-terminus (blue arrow; note that the fusion peptide is not part of the structure shown) and c-terminus of the blue-colored subunit are indicated class ii: dengue virus type e protein ( oan and ok ). the radial ("top") view shows just the "stem-less" ectodomain ( oan). colors: domain i: magenta; domain ii: cyan; domain iii: yellow; stem: cyan; colors for domains i, ii and iii are the same in the postfusion representation. a dashed cyan arrow on the postfusion trimer shows where the stem emerges from domain iii. red asterisks: fusion loops class iii: vsv g ectodomain ( j j and cmz). the three chains are in gold, sea green, and magenta. dashed lines show the location of a disordered, c-terminal segment that connects the folded protein to the transmembrane anchor. only the magenta-subunit c-terminal segment is shown on the bottom. the curved gold arrow indicates that in the transition from the conformation on the top to the conformation on the bottom, the domain bearing the fusion loops flips over by about ° to engage the host-cell membrane. red asterisks: fusion loops hiv- envelope glycoprotein have being pursued intensely as immunogens for b-cell based vaccine development [ , ] . for most single-pass tm proteins, the role of their tmds beyond membrane anchoring is unresolved, partly because of the difficulty in structural studies: they are notorious for resisting crystallization and due to their small sizes, unfeasible for visualization by cryo-electron microscopy (em). there have been only a few examples of crystal structures of the small tmds, including the tmd of the am [ , ] and the more recent crystal structure of the tm helix dimer of the glycophorin a protein [ ] . as cryo-em is rapidly approaching atomic resolution, it is becoming increasingly used to examine the structures of the much larger viral fusion proteins containing the tmd. most notably, a recent cryo-em study of the hiv- envelope glycoprotein (env) including the tmd achieved a high resolution structure of the prefusion state of the hiv- env [ ] . this study, however, showed that the tmd and the membrane-proximal regions of the env are disordered, possibly due to incompatibility between the membrane-associated regions of the env and the detergent used to solubilize the env. in this chapter we mainly focus on the use of nmr to study the tm and membrane-proximal regions of viral membrane proteins, including methods for structure determination and for investigating drug binding. the first challenge of nmr studies is preparing sufficient quantities of isotope labeled proteins and this is not trivial for small and hydrophobic proteins. a robust approach is to force highlevel expression of the hydrophobic peptides, which are toxic to cells, into inclusion bodies. in our experience, the most effective implementation of this approach is to express the peptide as a c-terminal fusion to the trple protein (which drives inclusion body formation) in the pmm-lr vector [ ] [ ] [ ] (fig. . a). the trple has an n-terminal ×his tag to permit nickel affinity purification. a methionine between the trple and the peptide is added for cleavage by cyanogen bromide so that the peptide can be released from the trple. purification of the peptide involves ( ) purifying the trple-peptide fusion protein from the inclusion bodies by nickel affinity, ( ) cleavage by cyanogen bromide of the fusion protein in formic acid, and ( ) separation of the peptide from trple or fusion protein by reverse-phase hplc. while the above protocol is quite general for hydrophobic peptides, it requires that the peptides have no methionine in the middle of the peptide sequence. if the native methionines cannot be mutated, an alternative method is to use soluble fusion domains (e.g., the maltose binding protein) with specific protease cleavage site for releasing the peptide. the purified hydrophobic peptides can be reconstituted in any membrane mimetic media, including detergent micelles, bicelles, and lipid nanodiscs. while nanodiscs are the closest mimic of native membrane, we find that the lipid/detergent bicelle system is a good compromise between having the capacity to provide a near lipid bilayer environment and generating good nmr spectra. previous studies on the bicelle system with , -dimyristoyl-sn-glycero- -phosphocholine (dmpc) as lipid and ( , -dihexanoyl-sn-glycero- -phosphocholine (dhpc) as detergent have shown that when the molar ratio of lipid to detergent (q) > . , the assembly reaches the ideal bicelle condition in which the lipid and detergents are well segregated [ , ] . at q = . , for example, the estimated diameter of the lipid bilayer region of the bicelle disc is ~ Å according to the equation describing bicelle assembly [ ] [ ] [ ] , and this size is sufficiently large to accommodate small tmds. remarkably, even bicelles of such size could generate nmr spectra of sufficiently high quality to enable full-scale structure determination, as was demonstrated for the structure fig. . a schematic of hydrophobic peptide sample preparation using hcv p as an example . trple plasmid with targeted peptide is transformed into and bl (de ) competent cells for expression . the cells were grown in m media . cells were collected by centrifugation and lysed by sonication . after lysis, the inclusion bodies and membranes were collected by centrifugation and dissolved in m guanadine buffer. insoluble aggregates were removed by centrifugation. the supernatant was then subjected to ni-nta purification. . the protein was eluted using mm imidazole . cyanogen bromide cleavage followed by dialysis in water . the protein was further separated by reverse-phase hplc or ni-nta . the lyophilized peptide was then dissolved in detergent and refolded by dialysis against the nmr buffer . nmr experiments were performed at spectrometers determination of the trimeric tmds of the fas death receptor [ ] and hiv- env [ ] . here we provide an example of bicelle reconstitution from a previous study on the tmd of hiv- env [ ] . the hydrophobic peptides (lyophilized) are first completely dissolved in strong organic solvent (e.g., hexafluoro-isopropanal) with calculated amount of dmpc. the solution is slowly dried to a thin film under nitrogen stream. the dried film is then dissolved in m urea containing calculated amount of dhpc. the denaturant is removed by dialysis, during which the dmpc: dhpc ratio is monitored by d nmr, and the loss of dhpc during dialysis is compensated by further addition of dhpc. owing to the small size of most of the tmds of viral membrane proteins, structure determination by nmr is normally straightforward. probably the biggest challenge is measuring inter-protomer restraints in cases of oligomer complexes. in particular, structure determination of symmetric oligomers faces the challenge of measuring noe-based h- h distances between structurally equivalent protomers having the same nmr peaks, which are needed as inter-protomer restraints. a proven way to solve this problem is using a mixed sample in which half of the protomers are ( n, h)-labeled and the other half c-labelled, for measuring, exclusively, noes between the n-attached h of one protomer and c-attached h of the neighboring protomer [ , ] (example from the tmd of the fas receptor shown in fig. . b). once the trimer solution is established, conventional n-and c-edited noe experiments can then be used to collect self-consistent backbone-sidechain and sidechain-sidechain noe restraints. in addition to structure determination, an important aspect of small tmds is interaction with membrane. the bicelle system allows accurate determination of the position of tmd in the bilayer region of the bicelles using a solvent paramagnetic relaxation enhancement (pre) analysis [ ] . in this method, titration of the water-soluble and membrane-inaccessible paramagnetic agent, gd-dota (gadolinium (iii) , , , -tetraazacyclododecane- , , , -tetraacetate), is used to generate the solvent pre. this approach is based on the notion that if the bicelle is sufficiently wide, the lateral solvent pre becomes negligible, thus allowing the use of measurable solvent pre to probe residue-specific depth immersion of the protein along the bicelle normal. at each titration point, a d h- n correlation spectrum is recorded to measure residue-specific pre. for each of the residues, the pre titration curve is fitted to exponential decay to derive the residue-specific pre amplitude (pre amp ). the pre amp vs. (residue position) along the trimer symmetry axis (parallel to the bilayer normal) is then analyzed using the sigmoidal fitting method [ ] to determine the tmd region that resides at the center of the bilayer as well as the thickness of the bilayer around the protein. for studying small molecule drug binding to viral membrane proteins, chemical shift perturbation (csp) induced by drug titration is a convenient probe for identifying the approximate binding site. but, cautions need to be taken due to complications associated with membrane-mimetic media. first, many small molecule inhibitors are hydrophobic and preferentially partition into detergent micelles or lipid/detergent bicelles, and thus the csp can be induced simply by alteration of the micelle/bicelle environment. second, in addition to csp caused by close ligand contact, csp could arise from changes in conformation and/or dynamics. hence, the more direct noe data is preferred wherever feasible. the simplest and the most sensitive approach for collecting protein-drug noe is using proteins that are n-labeled and completely deuterated so that noe between the protein backbone amide protons and drug protons could be measured unambiguously (fig. . c ). once the binding site has been unambiguously spotted, further contacts between the drug and protein sidechains can be measured using conventional c-edited methyl or aromatic noesy. details of these experiments have been described in studies that identified the drug binding sites in the influenza am and hcv p channels [ , , ] . structural studies of viroporins have been challenging because these small membrane proteins are typically dynamic and very hydrophobic. in the past years, multiple biophysical techniques including solution nmr, solid-state nmr, x-ray crystallography, and em have been used to gradually fill the structural gaps. taking the influenza am for example, there is now structural information in crystal and solution states, in lipid bilayer, under different phs, and bound to different small molecules. these complementary structural data allow elucidation of the functional mechanism from different view angles. the am of influenza a and the bm of influenza b are -and -residues single-pass membrane proteins, respectively, that form homotetramers in membrane [ ] [ ] [ ] [ ] [ ] . the sequences consist of three domains: an extracellular n-terminal domain, a transmembrane domain (tmd) and an intracellular c-terminal domain. these domains arrange into different structures. the only homologous sequence between the two proteins is the hxxxw sequence motif of the tmds that is essential for channel activity. the tm region of the am contains residues - . the unstructured extracellular segment of am is relatively conserved and has been sought after as a vaccine epitope [ ] [ ] [ ] [ ] [ ] . bm has a similar sized tmd (residues - ), but a much larger c-terminal cytoplasmic domain [ , ] . the bm cytoplasmic region also assembles into oligomers which are important for recruiting the matrix proteins to the cell surface during the viral assembly [ , , , ] . the first high-resolution structures of the am channel were reported concurrently by solution nmr spectroscopy and x-ray crystallography in [ , ] . subsequent x-ray and solution nmr studies also determined structures under different conditions [ ] and with different drug resistance mutations [ , ] . moreover, structural characterization by solid-state nmr studies generated the channel structures in the lipid bilayer environment [ ] [ ] [ ] . the above am structures solved under different conditions show significantly different conformations ( fig. . a) , suggesting that the tetrameric assembly of the am is quite dynamic and is sensitive to the reconstitution environment. the structural plasticity observed in am is likely present in many other viral membrane proteins, which could explain why structural study of this type of proteins has been so difficult. despite the differences, the experimental structures converged to a common mode of channel assembly: a left-handed four-helix bundle forms the channel pore, and that tetramerization of the four tm helices is further stabilized by intermolecular contacts between c-terminal amphipathic helices flanking the tmd [ , ] . this mode of assembly places the "h" and "w" of the hxxxw sequence motif inside the channel (fig. . a) . four imidazole rings of his , which are ph-gating features and are essential in transporting protons, are packed closely inside the pore (fig. . b) . moreover, packing of the trp indoles creates a channel gate, which occludes the c-terminal end of the pore. the structure of the bm channel was solved using solution nmr methods [ ] . although the overall assembly of the bm tmd is similar to that of the am tmd, i.e., both show left-handed helical packing, the two differ substantially in details ( fig. . c) . unlike the am , the bm channel shows strong coiled-coil characteristics with regular heptad repeats [ ] . in the bm structure, the two heptad repeats show that positions a and d are occupied mostly by hydrophilic residues such as serines and his ; positions g and e are occupied by hydrophobic leucines and phenylalanines, respectively, to allow for peripheral hydrophobic interactions between the leucines and phenylalanines (fig. . d ). this arrangement for coiled-coil assembly in membrane allows the tm segment to form a stable tetramer by itself and is the opposite to that of water-soluble coiled-coil tetramer, in which positions a and d are typically hydrophobic residues and positions g and e are polar residues [ ] . the histidine (his ) and tryptophan (trp ) of the hxxxw motif are also pore-lining in the inverse coiled-coil assembly in membrane. the cytoplasmic domain of the bm also oligomerizes into a left-handed coiled-coil tetramer, but it is water-soluble and shows strong bipolar distribution in the surface charges. this charged domain supports the interaction with the m matrix protein [ ] . the structural arrangement of the histidine and tryptophan inside the pore of the am and bm suggests that the two residues play an essential role in the channel selectivity and channel gating (fig. . e ). functional mutagenesis [ ] and nmr measurements [ ] showed that proton transport across the membrane through the am channel involves cycles of histidine protonation and deprotonation, and that the histidines serve as proton shuttling devices. not all histidines can be protonated at the same time in the narrow channel, as protonation of one histidine would increase the energy barrier for the protonation of another histidine. indeed, multiple pka values ( . , . and one below . ) have been detected in the am [ , ] . therefore, our current understanding of the proton conduction mechanism is that the minimally required unit for ph-dependent proton conduction in the am and bm is the his-trp complex. it was proposed in ref. [ ] that in the non-conductive state two pairs of histidines in the tetramer each share one proton, which explains structures of the influenza proton channels and mechanism of proton conduction. the many structures of the influenza m channel. the pdb codes rlf and kwx represent the solution nmr structures of the wildtype and the v a mutant determined using residues - . the c j and lbw are crystal structures of the tm domain (residues - ) determined at ph . and ph . , respectively. the structures l j and kqt were obtained using solid-state nmr using protein constructs that encompass residues - and residues - , respectively the high pk a ~ . . lowering ph results in the protonation of the third histidine from the n-terminal side, which, in turn, disrupts the two histidine dimers and leads to the proton conductive state. it was also proposed that protonation of histidines leads to cation-π interactions between the histidine and tryptophan [ , ] . the remaining question to be addressed is how does the third protonation affect the tryptophan conformation, allowing proton to be relayed to the c-terminal side of the tryptophan gate [ ] . the viroporin p encoded by the hcv genome is a -residue protein that oligomerizes in membrane to form cation-selective channels [ , ] , with higher selectivity for ca + than k + /na + [ , ] . the channel activity of p is important for the assembly and release of infectious viruses, although the molecular mechanism of this function remains unknown [ , ] . as in the case of am , structural characterization of p was confronted with challenges of coping with the hydrophobic and dynamic nature of the protein. earlier nmr studies of p under conditions that support the monomeric state of the protein showed that p has three tm helical segments: two in the n-terminal half of the sequence and one near the c-terminus [ , fig. . b isolated view of the pore-lining histidine (magenta) and tryptophan (cyan) sidechains in m and bm channels. images are from the high resolution crystal structure ( lbw) and nmr structure ( rlf) of m and nmr structure of bm ( kix) ] . although the monomeric state should not conduct ions, it could be involved in interacting with the ns protein during virus assembly [ , ] . the oligomeric form of p was first examined using single-particle em, which showed that p from hcv genotype a (jfh- strain) assembles into hexamers in , -diheptanoylsnglycero- -phosphocholine (dh pc) micelles and the complex adopts a flowerlike shape that does not resemble any of the known ion channel structures in the database [ ] . later, a more detailed structure of the p hexamer was determined by solution nmr using p from genotype a (euh strain) reconstituted in dodecylphosphocholine (dpc) micelles [ ] . consistent with the flower-shaped em images, the nmr structure shows a funnel-like architecture with six minimalist chains, each containing three helical segments: h , h , and h . the h and h form the narrow and wide regions of the funnel-shaped cavity, respectively, and the h helices wrap the channel peripheral by interacting with h and h (fig. . a) . the assembly strategy adopted by p differs significantly from those known channels from bacteria and eukaryotes. ion channels typically have two essential features: ( ) pore elements that support selective ion dehydration; ( ) a gate or constriction that prevents non-specific per- meation, but can open in response to regulating factor such as ph, voltage, ligand, or the ion of selection itself [ , ] . the channel interior of p has a number of strongly conserved residues that are likely candidates to serve the above functions. one suspect is asn , which forms a ring of carboxamide near the narrow end of the channel (fig. . b) . residue is asparagine in all strains except being substituted with histidine in genotype viruses. formation of a ring of carboxylates or carboxamides has been a recurring theme in prokaryotic and eukaryotic channels that have selectivity for divalent cations. for examples, the cora mg + channel has a pentameric ring of asparagines [ ] , and the calcium release-activated calcium (crac) channel orai has a hexameric ring of aspartic acids [ ] . these channels all have strong selectivity for divalent cations, although they can also conduct monovalent cations such as na + and k + . in addition to the asn ring, residues near the hinge between h and h (ser , asn , trp ) are in an arrangement that may also bind in addition to what appears to be the cation selectivity ring near the narrow, n-terminal exit of the channel, the wider, c-terminal entrance of the channel is decorated with a conserved ring of arginines or lysines (fig. . b) . placement of a positively charged ring at the entrance was anticipated because it may repel cations. but an earlier study reported that a designed tm barrel with internal argininehistidine dyads forms efficient cation selective channels [ ] because the immobile arginines can recruit mobile anions, which in turn facilitate cations to diffuse through the pore. it is interesting to note that a highly basic region containing arg , lys and lys in the pore was also found in the crac orai structure [ ] . one possible role of these basic residues in cation selective channels is binding and obstructing anions while allowing cations to diffuse into the pore. this mechanism would be consistent with the observation that replacing arg with negatively charged aspartic acid largely abrogated conductance [ ] . there are still many unanswered questions. does the nmr structure, solved in the absence of ca + and inhibitors, represent the open or closed state of the channel (if the two states exist)? how strong is the ca + selectivity of p ? or was p developed as a general, unspecific cation channel for the purpose of dissipating membrane potential? for example, another recently study reported p activity in dissipating proton gradients within cell membrane compartments [ ] . it is unclear what ion flux mediated by p plays a dominant role in the hcv life cycle. from a structural perspective, the funnel-like architecture is formed with multiple helical segments connected by hinges and short loops and we believe this flexibility can afford the dynamic opening and closing of the tip of the channel. probably the most intriguing aspect of small molecule interaction with viroporin is the finding that the adamantane derivatives amantadine and rimantadine have inhibitory effect on multiple viroporins including the influenza am and hcv p . amantadine (symadine) or rimantadine (flumadine) was the first licensed drug for treating influenza infections [ ] . in fact, the compound also played critical roles in the early days of functional characterization of the am channel [ ] [ ] [ ] . the bm channel is a functional and structural homolog of am but is not sensitive to the adamantane family of drugs [ ] . remarkably, the hcv p channel structure is completely different but showed detectable, though not strong, sensitivity to rimantadine [ , ] . the mechanism of how amantadine/rimantadine inhibit the am channel has been elusive for quite some time. previous confusion came mainly from the multiple binding sites that have been observed experimentally. in a crystallographic study of the am tmd (residues in the presence of amantadine, the drug density was found inside the channel near residue ser , but at structural resolution of . Å, it was difficult to confirm the position of amantadine binding [ ] . at the same time, however, a solution nmr study of a longer version of am (residues showed that rimantadine binds to an external, lipid-facing pocket around residue asp between adjacent tm helices [ ] . the two different binding sites obviously suggest very different mechanisms of inhibition. one is the drug directly blocking the channel passage, and the other is the drug binding to the external site allosterically favors the closed state of the channel. subsequent solid-state nmr measurements of the am in lipid bilayer showed that both sites exist, with higher affinity for the internal site [ ] . the strongest evidence that the internal site is the primary site of drug action came from functional studies using an am -bm chimera protein [ , ] . in this study, the authors constructed a chimera of m variants from influenza a and b viruses that contains only internal site showed that the chimera channel is still amantadine sensitive, indicating that the internal site is the primary site of drug inhibition. later, the complex structure of the tmd of the chimera with rimantadine was determined by solution nmr, providing a detailed view of the drug binding inside the channel pore [ ] . the drug binding site consists of eight methyl groups of the m tetramer (two from each subunit: val c γ h and ala c β h ) that form a deep internal hydrophobic pocket surrounding the adamantane cage of the drug (fig. . a ). the structure also shows that the nitrogen of the rimantadine amino group may form a hydrogen bond with the backbone carbonyl oxygen of ala of one of the four subunits. the terminal methyl group of the rimantadine is in the middle of the pore, facing the open space in the channel around the gly position. the structure also shows that rimantadine binding is slightly tilted: its vertical axis is on average ~ ° from the c symmetry axis of the channel. the tilt angle is consistent with the amantadine tilt in the m channel observed with solid-state nmr spectroscopy [ ] . in addition to blocking the am channel, the amantadine and its derivatives have also been shown to pose some inhibitory effects on the p channel conductance [ , fig. . the amantadine and rimantadine binding sites in the m and p channels (a) the precise nmr structure of the am -bm chimeric channel with rimantadine determined in dhpc micelles and at ph . . left panel: detailed illustration of the methyl groups (in green) that interact with the adamantane cage. right panel: surface representation for showing the hydrophobic pocket that fits the drug snuggly. one of the four subunits is omitted for drug visibility (b) the amantadine or rimantadine binding site of the p channel determined by nmr in dpc micelles and at ph . . left panel: the drug binds to six equivalent hydrophobic pockets of the p channel. right panel: a close view of amantadine docked into the binding pocket as determined using nmr noe restraints (c) comparison between adamantane binding sites of influenza m and hcv p channels top: the internal pocket that wraps around rimantadine in the am -bm chimeric channel. the am -bm chimeric channel is a well-behaved model system wth its n-terminal half is from influenza a m protein (sensitive to amantadine or rimantadine inhibition) and its c-terminal half is from influenza bm protein (insensitive to amantadine or rimantadine). on the right panel, one subunit of the tetrameric complex is removed to unveil the channel interior bottom: amantadine binds to the peripheral pockets between the h and h helices; and a representative pocket among six equivalent pockets in the p hexamer ]. the physical binding sites of amantadine and rimantadine have been identified in p of genotype a using intermolecular noe experiments [ ] . the noe data revealed that amantadine or rimantadine binds to six equivalent hydrophobic pockets (due to the six-fold symmetry of the p channel) between the pore-forming and peripheral helices (fig. . b) . in each site, leu , leu , and leu from h and val , val , and phe from h appear to form a hydrophobic pocket that wraps around the adamantane cage of the drug. the amino group of amantadine or rimantadine is facing the largely hydrophilic channel lumen. an important property of the drug binding site is that it consists of elements from different helical segments and from different monomers. as rationalized above, permeation of cations through the p channel may depend on opening the narrow end of the funnel, which in turn depends on the reorientation of the helical segments. the binding of adamantane derivatives to the pocket may inhibit channel activity allosterically by causing the channel to close. indeed, an nmr relaxation dispersion study showed that residues at the h -h hinge (phe ) and the narrow end of the cavity (val , leu ) experienced substantial chemical exchanges (k ex~ ± s − and ~ % excited state). this data is consistent with movements of the h helices that cause the tip of the funnel to open and close. more importantly, addition of rimantadine slowed down motion at the tip of the channel, as relaxation dispersion curve for val , which has significant chemical exchange in the apo state, is completely flat in the drug-bound state [ ] . the dispersion curve of phe is also significantly flatter, and individual curve fit yielded k ex value of ± s − . clearly, rimantadine binding makes the channel less dynamic. therefore, the rimantadine may thus act as a "molecular wedge" that prevents the dynamic "breathing" of the channel required for ion conduction. comparing the amantadine or rimantadine binding mode of hcv p to that of influenza am shows two fundamentally different mechanisms of drug inhibition. in the case of am , one drug binds to one channel. drug binding inhibits proton transport by directly blocking the channel passage; it also prevents channel from opening. in the case of p , amantadine and rimantadine are clearly too small to block the channel. they instead bind to six equivalent sites outside of the channel cavity, which can afford up to six drugs per channel. if rimantadine binding to this site is relevant to inhibition, as crudely suggested by previous functional mutagenesis, drug binding to these sites inhibits cation conduction with an allosteric mechanism, possibly by stabilizing the closed state of the channel. although the mechanisms of drug inhibition may be completely different, the structural bases that govern drug-binding affinity for am and p are actually similar, and they involve hydrophobicity and size of the pocket, and position of the drug amino group (fig. . c ). in the case of the am tetramer, the drug adamantane cage fits snuggly in a hydrophobic pocket formed by eight methyl groups from val and ala (two from each subunit), while the drug amino group forms polar contact with the backbone oxygen of ala and points to the polar region of the channel cavity. for the p channel, the adamantane cage is in contact with ten methyl groups and an aromatic group from the protein. these hydrophobic groups form a deep hydrophobic pocket that also matches closely the size of the adamantane cage. the amino group of amantadine or rimantadine points to the channel lumen; it is in position to form polar contacts with the electronegative groups such as backbone carbonyl of residues - . hence, having a greasy pocket for the adamantane cage and a nearby electronegative group to interact with the amino group may be a general requirement for amantadine or rimantadine binding. previous functional mutagenesis studies have suggested that the tmds of viral fusion proteins are not only limited to the function of membrane anchoring, but also involved in other functions such as membrane fusion or assembly of the fusion protein on the viral membrane. for example, sequence analysis of the ha from mutant viruses and site-specific mutagenesis of the fusion peptide identified a group of mutations in the n-terminal half of the influenza ha tmd severely affected membrane fusion (the hemifusion to pore formation) [ ] [ ] [ ] . in the case of hiv- , multiple lines of evidences suggest that the tmd of gp is not merely a membrane anchor, but plays critical roles in membrane fusion and viral infectivity [ ] [ ] [ ] [ ] [ ] . the amino acid sequence of gp tmd is also highly interesting. there is a gly rich motif in the tmd, which suggests some sort of oligomerization. even more peculiar is the presence of a conserved arginine in the middle of the predicted tm region. unlike the structural biology of viroporins, there is essentially no structural information of tmds of viral fusion proteins except for that of the tmd of hiv- env. hence, we focus on the discussion on the trimeric membrane anchor of the tmd of the hiv- envelope spike. hiv- envelope spike [env; trimeric (gp ) , cleaved to (gp /gp ) ] is a type i membrane protein that fuses viral and host cell membranes to initiate viral infection [ ] . the gp and gp are the receptor recognition and membrane fusion proteins, respectively. conformational changes in gp when triggered by binding to receptor (cd ) and co-receptor (e.g., ccr or cxcr ) lead to a cascade of refolding events in gp (similar to those illustrated in fig. . a) , and ultimately to membrane fusion [ , [ ] [ ] [ ] . the mature and functional env spikes, (gp /gp ) , are the sole antigens on the virion surface and thus important candidates for vaccine development [ , ] . the native prefusion conformation of hiv- env is recognized by most broadly neutralizing antibodies (bnabs) [ ] [ ] [ ] and it is generally believed to have the potential to induce such antibody responses. thus, the native conformation of env spikes on the surface of virions is extremely important to immunogen design in b-cell based vaccine development. a vast amount of structures of the ectodomain (ecd) of the gp /gp complex have been determined [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , but relatively little is known about the tm and membrane proximal regions of gp due to challenges of preserving the native-like folding of these regions in membrane mimetic media. it has been shown that truncations in the cytoplasmic tail (ct) of gp could alter the antigenic surface of the env ecd on the opposite side of the membrane [ ] , suggesting that the ecd, the tmd, and the membrane proximal regions are conformationally coupled, and thus the conformational stability of the tmd is an important consideration for immunogen design in b-cell based hiv- vaccine development. recently, the nmr structure of the gp tmd has been solved in bicelles (made of dmpc lipid and dhpc detergent; q = . ) [ ] using a gp fragment (residues - ) from a clade d hiv- isolate ug . , designated gp hiv d ( - ). the tmd forms a well-structured trimer, almost helical all the way from the n-to the c-terminal end. it shows two peculiar features not seen with other known oligomeric tm helices (tmhs) (fig. . a) . one unusual feature is that the tmd trimer appears to be stabilized by two separate packing modes (fig. . c) . the n-terminal half encompassing the gxxxg motif forms a coiled-coil trimer, whereas the c-terminal half is held together by a network of polar contacts, which we named the hydrophilic core. for the n-terminal coiledcoil, the helical wheel representation of the trimer clearly indicates packing of hydrophobic residues such as ile and val at the "a" and "d" positions of the heptad motif, forming a hydrophobic core. the gxxxg is a well-known motif that drives tmh dimerization [ , , ] . in the classic example of the glycophorin a tmd dimer structure, the two glycines of one tmh allow close packing with the gxxxg face of another tmh, resulting in a very strong tmh dimeric complex [ , ] . there has been no previous report, however, of the gxxxg involvement in tmh trimerization. in the coiled-coil region of the hiv- env tmd, only g is involved in the trimer assembly, i.e., its small sidechain allows a close vdw contact with v of the adjacent tmh. the other glycine, g , is on the periphery of the trimer facing outwards and its mutation to alanine or valine has essentially no effect on tmd trimerization, as well as env functions [ ] . therefore, the key difference in the structural role of the gxxxg motif between the glycophorin a tmd dimer and hiv- env tmd trimer is that both the glycines are required for forming intermonomer contacts in the dimer, while only one glycine is important for the trimeric assembly. tm segments of many viral fusion proteins contain a gxxxg motif or "smallxxxsmall" motifs ("small" refers to residues with a small side chain, such as, glycine, alanine, serine or cysteine) [ ] [ ] [ ] , suggesting that oligomerization of their tmds may be a common property. for example, recent biochemical evidence has shown that the tmds of hepatitis c virus envelope glycoproteins e and e form stable dimers or trimers that are also resistant to sds [ ] . no highresolution structure of any tmd oligomer from other viral fusion proteins has been reported, due to technical challenges for structural studies of such constructs in the context of lipid bilayer. the nmr structure of the hiv- env tmd may provide some clues for how other viral fusion proteins oligomerize in the membrane. another peculiar feature is the presence of three copies of arginine (r ) near the middle of the tmhs (fig. . b) , suggesting three unbalanced charges in the hydrophobic core of the membrane if the arg remains protonated. in the nmr structure, the tips of the long sidechains of these arginines are facing lipids and each of them is surrounded by three hydrophobic residues (l , l , and i ). it is also interesting to note that r occupies a "d" position in the coiled-coil, with its cβ facing inwards to the trimer interface. precise physical basis of how r is accommodated in the highly hydrophobic environment remains unclear, but the underlying mechanism must tolerate a lys residue, which is present in some viral isolates at this position. it is interesting to mention that the noe experiment for the r epsilon protons showed clear water noe even with a short noe mixing time of ms, suggesting that the arg is somehow hydrated in the membrane. a positively-charged arg or lys in the tm segment of viral fusion protein is present in some related enveloped viruses, including simian immunodeficiency virus, caprine arthritis and encephalitis virus, equine infectious anemia virus, visna virus, and foamy virus, as well as in hepatitis c virus [ ] [ ] [ ] [ ] [ ] [ ] , but absent in many others. it is therefore not a prerequisite for viral membrane fusion in general [ ] . functional mutagenesis indicated that the r a mutant of hiv- env showed some defect in cell-cell fusion, but it could be fully compensated by high env expression [ ] . moreover, this mutant has wildtype viral infectivity. in vitro infectivity and cell-cell fusion do not, however, mimic all the conditions under which the virus moves from one cell to another in an infected individual, nor are those assays particularly sensitive to physiologically relevant kinetic parameters. the structures of viroporins discussed above are all substantially different from any of the channel structures found in prokaryotes and eukaryotes, and are thus clean structural targets for developing antiviral compounds. the available structures also suggest that viroporins generally adopt minimalist architecture and can possess structural features compatible with selective ion transport. the viral channels, however, may not be as functionally robust or specifically regulated as some of their counterparts in prokayrotes and eukaryotes, because viruses often do not need intricate regulation of channel activities during their infection cycles. having minimalist structure also makes viroporins fragile and sensitive to the membrane environment. this is reflected by the conformational variations observed for the am channel in different reconstitution media. therefore, it remains important to examine these viroporins in more native environments, e.g., lipid bilayer and full-length proteins. as solid-state nmr continues to improve spectral resolution [ , ] , obtaining detailed structures of viroporins in liposomes should in principle be feasible in the near future. alternatively, the ideal bicelle system can be explored with solution nmr to revisit some of the viroporin structures determined previously in detergent micelles. future establishment of the nmr systems for viroporins under more native conditions would certainly provide versatile and effective platforms for investigating inhibitor binding. apart from the progress in structure determination, major challenges lie ahead in the functional aspects of viroporin research. the precise functional roles of many viroporins are still unclear. moreover, due to the lack of robust single-channel recording setups suitable for viroporins, the ion conductance properties of most viroporins have not been fully characterized. therefore, better definition of the functional roles and channel properties of viroporins will certainly draw greater enthusiasm for developing therapeutics that target this interesting family of membrane channels. the example from hiv- env suggests that tmds of viral fusion proteins can adopt very interesting structures, and the distinct structural features certainly allude to their roles in viral fusion protein assembly and incorporation into the envelope, as well as in the process of membrane fusion. the nmr structural study of the hiv- gp tmd in bicelles with q = . [ ] demonstrates that the structures of most of the viral fusion protein tmds can in principle be determined in essentially lipid bilayer environment using modern nmr techniques. moreover, the combined use of ideal bicelles (q ≥ . ) and solvent paramagnetic relaxation enhancement measurements can be used to determine the membrane partition of the tmds in the bilayer region of the bicelles [ ] . we believe the next major challenge lies in understanding the roles of the fusion protein tmds in the fusion mechanism. in the cases of the flu ha and hiv gp , for example, new experiments need to be designed to address questions such as, "does the tmd interact with the fusion domain in the hemifusion stage in which the two domains are presumably in close proximity?" and "does the tmd play a role in facilitating the conversion from the hemifusion to the pore formation?" understanding the structural properties and conformational stability of the tmd may also have far reaching implication to vaccine development and this has been recognized at least for hiv- . as mentioned above, truncations in the ct of the env could drastically alter the sensitivity of the env ecd to the known trimer-specific bnabs [ ] . the continuous structure from the n-to c-terminal ends of the env tmd, as observed by nmr, suggests that the env ecd can be structurally coupled via the tmd. indeed, it has also been shown that the env tmd can also modulate the antigenic structure of the ecd in a cell-cell fusion assay and a pseudovirusbased neutralization assay [ ] . the results show direct correlation that more disrupted tmd trimer led to less inhibition or neutralization by the trimer-specific bnabs that target only the ecd. the results suggest that the stability of the tmd trimer is an important consideration when designing immunogens for hiv vaccines. the hiv env tmd is, however, not directly linked to the env ecd. another important segment known as the membrane-proximal external region (mper) is the direct link that connects the tmd to the ecd, and thus it could in principle also affect the antigenic properties of the env ecd. the mper sequence is extremely conserved with five absolutely conserved tryptophans, suggesting that the mper probably also has interesting structural features. the conformation of the mper attached to the tmd trimer in a lipid bilayer environment is still unknown (fig. . d) . previous nmr studies of an isolated mper peptide in detergent micelle suggested that the mper is monomeric and folds into a kinked helix with many hydrophobic residues embedded in the micelles [ , ] . this of course would have a rather negative implication for the mper as a vaccine epitope because antibodies that bind to the mper must dig it out of the lipids, which could be accompanied with polyreactivities. the env tmd appears to be well structured and assembled strongly to trimers. hence, the structure of the mper when connected to the trimeric tmd and in the context of a lipid 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controls fusion activity contribution of the charged residues of hepatitis c virus glycoprotein e transmembrane domain to the functions of the e e heterodimer hiv- broadly neutralizing antibody extracts its epitope from a kinked gp ectodomain region on the viral membrane antibody mechanics on a membrane-bound hiv segment essential for gp -targeted viral neutralization the active oligomeric state of the minimalistic influenza virus m ion channel is a tetramer the oligomeric state of the active bm ion channel protein of influenza b virus evidence for the formation of a heptameric ion channel complex by the hepatitis c virus p protein in vitro oligomerization state and supramolecular structure of the hiv- vpu protein transmembrane segment in phospholipid bilayers the human immunodeficiency virus type vpu protein enhances membrane permeability severe acute respiratory syndrome-associated coronavirus a protein forms an ion channel and modulates virus release analysis of sars-cov e protein ion channel activity by tuning the protein and lipid charge viral weapons of membrane destruction: variable modes of membrane penetration by non-enveloped viruses viroporin-mediated membrane permeabilization. pore formation by nonstructural poliovirus b protein the hpv- e protein represses expression of stress pathway genes xbp- and cox- in genital keratinocytes hpv- e oncoprotein upregulates lipid raft components caveolin- and ganglioside gm at the plasma membrane of cervical cells the human papillomavirus type (hpv- ) e protein sensitizes human keratinocytes to apoptosis induced by osmotic stress viral encoded potassium ion channel is a structural protein in the chlorovirus paramecium bursaria chlorella virus- (pbcv- ) virion key: cord- -quns b authors: cui, shunji title: china in the fight against the ebola crisis: human security perspectives date: - - journal: human security and cross-border cooperation in east asia doi: . / - - - - _ sha: doc_id: cord_uid: quns b the outbreak of the ebola virus disease (evd) in west africa became one of the worst disease-driven humanitarian crises in modern history. the crisis turned the global securitization of health issues into unprecedented levels, at the same time, aligned closely with human security frameworks and thus has significant impacts on national foreign and aid policies. china has played a significant role in the global fight against ebola, indicating important changes in its foreign policy orientations. based on the lessons drawn from china’s operation in africa, it is argued that states must transcend their narrow national interest and seriously consider the dignity and well-being of vulnerable people. in september , when the crisis was at its peak, the number of weekly cases reached almost , . by january , , when the world health organization (who) officially declared the epidemic to be over, the crisis had lasted nearly two years, during which time more than , people were infected with the virus and more than , lives were lost, mostly in guinea, liberia and sierra leone (who b; cbs ) . faced with such a devastating humanitarian crisis, the entire international community has shown great courage in fighting the disease. after the august/september announcement by the who that ebola was a 'public health emergency of international concern' and the united nations security council (unsc) declaration that ebola was a 'threat to international peace and security,' many countries as well as international organizations, non-governmental organizations, companies and individuals participated in the fight against this unprecedented challenge to humanity. china played a significant role in the international efforts to halt the spread of the ebola disease. through four rounds of emergency aid supplied in april, august, september and october , a total of million yuan (about usd million) was contributed to west african countries by china. in addition to financial and material assistance, china also sent more than , medical personnel to the region to help with local epidemic prevention and control work (nhfpc a; undp a) . this was unprecedented in the history of chinese foreign assistance. in fact, china has often been considered as lacking a philanthropic culture, and its international aid and financing models are frequently criticized as resource-backed and tied to aid in a way that simply serves the business interests of the country. thus, the chinese case raises intriguing questions, especially in terms of human security: what are the main motives and driving forces behind these efforts, and how effective are they? this chapter aims to answer these questions through the lens of human security rather than from a general foreign policy perspective. for this purpose, it begins by laying out the criteria and framework for the analysis. this is followed by an examination of china's efforts to fight the crisis. the chapter then goes on to evaluate these policies with a focus on effectiveness, empowerment and motives. based on china's experiences in africa, the concluding section draws some lessons for future human security-oriented foreign policies. when the un security council adopted resolution on september , , declaring the outbreak of ebola in africa to be 'a threat to international peace and security,' the ebola crisis was no longer a mere health issue but an international security crisis (unsc ) . of course, it was not the first time that the security council had acknowledged the link between health and security. in , the council had recognized the hiv/aids pandemic in resolution (unsc , ) , and declared it to be 'a risk to security and stability,' although its main concern was on the 'regional effects' in africa (deloffre a) . resolution is important in terms of human security because the council clearly focused not only on issues such as preventing wars and control of the proliferation of weapons of mass destruction but also dealt with matters concerning human security such as disease (poku , ) . therefore, resolution constitutes a clear securitization of public health issues within the un system. following the ebola crisis, resolution and the creation of the united nations mission for ebola emergency response (unmeer) in pushed the scale and depth of securitization to an unprecedented level, while at the same time brought the securitization processes into close alignment with human security frameworks (snyder ) . the question to be asked in this context is, therefore: what is meant by human security, and what counts as appropriate foreign and aid policies toward human security threats? since its introduction in (undp ) , the concept of human security has increasingly been reflected in global governance and in the foreign and aid policies of many countries. yet the kind of definition one adopts, narrow or broad, will have very different operational and policy implications. in keeping with the approach to human security taken by jica and many east asian countries (see chapter in this book ; tanaka ; jica ) , this chapter takes a broader concept as its working definition and uses this to identify the three features of human security. first, the causes and effects of human security can be far-reaching and multifaceted, and if we underestimate the complexity of such threats, we can never have adequate policies toward human security practices. secondly, human security and human development are so closely related that, even though we can conceptually separate them, we must be aware of the interconnectedness between the two at the operational level (cui ) . amartya sen made the point comprehensively. he argued that on the one hand, human security demands both 'protection' of people from a variety of dangers, and 'empowerment' of people so that they can cope with, and when possible overcome, these hazards. on the other hand, human development is concerned with 'removing the various hindrances that restrain and restrict human lives and prevent its blossoming,' and hence goes beyond 'overarching concentration on the growth of inanimate objects of convenience' (chs , - ) . such a comprehensive understanding is at the heart of his conceptualization of 'development as freedom,' in which poverty, one of the central concerns of human security, is no longer premised solely on income, but seen as a non-fulfillment of basic human rights (sen ) . thirdly, when human security is threatened by conflict situations, natural disasters or pandemics, it is often the case that the most vulnerable people in society are the ones who are most threatened. given these features of human security, what kinds of policy tools are to be considered as most appropriate and effective? tanaka ( , - ) distinguishes between two types of human security instruments: 'fundamental measures' that affect the underlying causes of human security, and 'defensive measures' that affect consequences. this is very similar to johan galtung's ( ) distinction between 'positive peace' and 'negative peace,' because fundamental measures may bring positive peace, while defensive measures are more likely to bring negative peace. for galtung, peace can be defined in a negative way, meaning the absence of violence (both direct and structural). yet more importantly, peace can also be defined positively, that is, the construction of an appropriate environment for lasting peace. in other words, the conditions for positive peace may be built through a process analogous to the 'building of a healthy body capable of resisting diseases, relying on its own health forces or health sources' (galtung , - ) . drawing on these ideas, this chapter is framed around three measures by which human security policies can be assessed. the first measure is effectiveness. once human security threats have occurred, how should we respond to the problem more effectively? the issue here is how swiftly and decisively can a country reduce the negative impacts of disease and human suffering. this is in line with tanaka's defensive measure or galtung's negative peace. effectiveness means more than speed, scale and comprehensiveness; it also refers to the ability to cooperate with a variety of actors to tackle human security threats. speed and scale are extremely important; however, if they are pursued singlehandedly, their impact remains limited. cooperation is imperative when trying to effectively handle deadlier challenges. the ebola crisis is a good demonstration of how a problem goes beyond the capacity of a single state. unmeer was created specially to coordinate a variety of actors to fight the crisis more effectively. thus, both comprehensiveness and cooperation are required if human security policies are to be effective. the second measure is empowerment. if human security practices are only prepared to tackle problems once they emerge, only negative peace can be achieved. taking individual health as an example, although a person may be cured of a disease, if they do not build up their bodily conditions, illness will reoccur. empowerment is more closely related to dealing with structural violence as the underlying cause, building capacities, and creating a more secure environment, so that the occurrence of human security threats can be prevented or the likelihood of them occurring be reduced (cui ) . in this way, even if threats occur, people have the ability, or at least an increase in the ability, to address those threats on their own. thus, if the first evaluate of effectiveness is used to assess more short-term defensive approaches, empowerment is used to evaluate longer-term fundamental approaches to human security. thirdly, in addition to the above measures, motives or moral imperatives are important in assessing human security policies. traditionally, theorists, particularly realists, emphasized national interests when measuring national foreign policies. hans morgenthau ( morgenthau ( , argued explicitly that a 'foreign policy derived from the national interest is in fact morally superior to a foreign policy inspired by universal moral principles.' of course, morgenthau did not deny political morality and prudence, or the need for a logic of consequences to save policy makers from both moral excess and political folly. however, because most human security-related governance activities do not directly relate to national interests in the narrow realist sense, the notion of raison d'état provides poor guidance and does not fully explain the current global efforts to achieve human security. thus, the practice of human security requires a certain degree of consideration of those people who are socially vulnerable, and concern should be given to the possibility of their dignity being exposed to existential threats. the ebola virus disease (evd) in west africa broke out in december and the who put out its first alert in march . what followed was the largest, longest, and most severe and complex outbreak of the virus since it was discovered in (who a). although at the initial stage the impact of the outbreak was underestimated, following the declaration of emergency and threat by the who and the unsc in august and september , the international community has made great efforts and shown solidarity in the fight against the deadly disease. among the countries fighting evd, traditional donors, such as the usa and the uk, played a leading role. by october , the us government was ranked as the largest donor having contributed around usd million in aid, with the uk coming in second with about usd million (who a). yet, the crisis also saw intense media attention given to some non-traditional donors, like china. by october , china had contributed close to usd million in financial aid. although this was much lower than that of the usa, it was still above the contribution of traditional donor countries such as france, japan and canada (undp a; see also fig. . ). accordingly, china's role and its impact on human security merit more detailed examination. china's participation in fighting the ebola epidemic is regarded as being historic, marking the first time it offered such aid to help combat a foreign health crisis (tiezzi ) . the chinese government also admits that it was the largest medical aid program to be implemented by china at the time (nhfpc b). china's role in fighting ebola was particularly important in the early stages and was in stark contrast to the delayed response of the rest of the international community. for instance, even though the who was first alerted to the outbreak on march , , it was not until april that médecins sans frontières (msf) first warned that ebola was getting out of control. by june, the spread and scale of the epidemic was obvious to many experts, yet it was not until august that the who declared that it was a public health emergency. as a result, the international response generated criticism as being both too small and too slow (dearden ; grépin ) . in comparison, china's response was swift. there are two reasons for this swift response: first, given china's long-term medical cooperation with african countries, many doctors and medical staff were already present in african countries when the outbreak began. for example, when the epidemic first emerged in guinea, a chinese medical team of nineteen people from beijing's anzhen hospital was working at the china-guinea friendship hospital in conakry, the capital of guinea. during this time, one infected patient was treated in the hospital without anyone realizing it was ebola and was thus faced with the risk of death. secondly, the chinese experience with sars in and its struggle to stop its spread, coupled with an awareness of the weaknesses in the medical system in west africa, made chinese officials and medical experts particularly alert to pandemics in west africa. in fact, the weak healthcare systems in all three countries were emphasized by many medical experts after ebola broke out. as marie-paule kieny ( ) notes, ebola became epidemic in guinea, liberia and sierra leone in large part due to their weak healthcare systems. indeed, all these countries lack adequate numbers of qualified health workers, particularly in rural areas. other limitations included weak or absent rapid response systems, and a lack of electricity and running water in some health facilities (kieny ; who c) . in march , when the outbreak of ebola in africa began to be reported, the news placed policy makers and medical experts in china on high alert. immediately, high-level meetings were held with the ministry of health calling for discussions on the ebola virus and how to help africa deal with it. among those involved in the meetings was the deputy director of the center for disease control and prevention (cdc) in beijing, he xiong, who was a frontline veteran of china's battle with sars. in april , the chinese government announced its first emergency assistance plan, under which it would send disease prevention and control materials worth million yuan (about usd , ) to guinea, liberia, sierra leone and guinea-bissau; by may , the assistance had arrived (china daily, october , ; nhfpc a) . by june , , the ebola situation had become even more urgent as it had reached the liberian capital, monrovia. six days later, the death toll had risen to and it had officially become the worst ebola outbreak on record (nhfpc ). in august, the epidemic accelerated as the total number of cases reached almost , , with more than , deaths in guinea, liberia and sierra leone alone. cases of infections among american, british and spanish citizens were also reported. on august , , the who declared that the epidemic had gone from being an african problem to an 'emergency of international concern' (who b). this situation alerted the top chinese leaders. on august , , beijing announced its second round of assistance, whereby it would provide emergency anti-epidemic supplies worth million yuan (about usd . million) to the three most affected countries. the supplies were mainly medical protective clothing, sterilization equipment, drugs and other much-needed medical equipment and supplies. due to the urgency of the situation, china even used chartered planes to deliver medical supplies, which arrived on august , just one week after the announcement. the initial aid was followed by another three chinese medical teams dispatched across west africa to help with prevention and treatment. by this stage, more than medical workers had been dispatched (china daily, september , ) . the situation further developed and reached a devastating level. by september , the total number of infected cases had reached , including deaths (medical express, september , ) . two days later, the unsc declared the outbreak of ebola to be a 'threat to international peace and security' (unsc ). on september , the unmeer, the first-ever un emergency health mission, was formed. this mission was led by a full range of un actors, who utilized their expertise under the leadership of a special representative of the secretary general. it was in such crisis atmosphere that many countries pledged more aid and manpower to help. on september , us president barack obama announced 'major increases' in the us response to fighting ebola in africa including up to troops, material to build field hospitals, additional healthcare workers, community care kits and badly needed medical supplies (new york times, september , ) . in a speech to the un high-level meeting on the response to the evd outbreak on september , , japanese prime minister shinzo abe also promised that japan would provide , sets of protective gear for medical personnel working to combat ebola in africa. in october, japan used civilian aircraft to deliver , sets of protective gear to liberia and sierra leone (asahi shimbun, february , ) . in september, as its third phase of assistance, china increased its contribution significantly by opening a biosafety lab and providing protective treatment supplies and food assistance. additionally, to help sierra leone improve lab testing, china sent a laboratory team of (thirty doctors and twenty-nine laboratory technicians) to work at the sierra leone-china friendship hospital (china daily, september , ). on october , china announced its fourth round of emergency aid worth million yuan (usd million), which would mainly be used to finance the construction of a -bed treatment center in liberia, where the epidemic was most serious. as the chinese foreign ministry explained, the treatment center, which was completed on november , would be managed and operated by a medical team from the people's liberation army (pla) (xinhua, november , ). the treatment center was able to accept patients for observation and testing from december , (nhfpc b). the aid package also included sending medical equipment and materials, such as ambulances, motorcycles, , healthcare kits, , pieces of personal protection equipment, and other materials (larson ) . china continued its commitment to fighting ebola after these four phases of contributions, as lin songtian, director of the foreign ministry's african affairs department, stated, 'china's assistance will not stop as long as the ebola epidemic continues in west africa' (xinhua, october , ) . thus, in early november, the nhfpc announced that china planned to send medical workers and experts to west africa over the months that followed (xinhua, november , ) . in february, china handed over a p -level biolab to sierra leone as part of its continued contribution to fighting ebola; it also delivered a consignment of metric tons of food assistance for distribution to ebola patients at various treatment units across the country. evaluating china's role: an emerging human security-oriented foreign policy? from the above discussion, it is not difficult to see how china actively participated in the global efforts to address the ebola outbreak. how then should china's role be assessed through the specific lens of human security? what lessons can be drawn for future human security-oriented foreign policies? the following section will analyze china's role in terms of effectiveness, empowerment and motives. as previously argued, effectiveness comprises both comprehensiveness and the ability to enhance multiple-level cooperation. first, china's assistance is considered as comprehensive and wide-reaching. its contribution of personnel is particularly highlighted compared with many other countries, especially asian countries. at a news conference in seoul on november , , the world bank group president, jim yong kim, lamented the fact that although they may have the capacity, many asian countries were not doing enough to help. he called upon asian leaders to send trained health professionals to west african countries (aljazeera, november , ) . the lack of assistance by asian countries is true to some extent. japan, for example, while making significant financial and material contributions lagged behind many other countries in terms of the provision of personnel. by the end of , japan had sent a total of twenty japanese experts to participate in who missions to liberia and sierra leone, two self-defense force (sdf) personnel to the headquarters of the us africa command (africom) in germany to support liaison activities, and one to unmeer as a senior advisor (government of japan ). there were suggestions from japan's defense ministry that a ground sdf unit would be dispatched to join the fight in sierra leone. the plan was submitted to the prime minister's office on february , , and called for gsdf personnel to begin operations in april, with a possible maritime sdf contingent to serve as the base of operations. however, for many reasons japan did not go ahead with the plan. this decision generated some criticism because although prime minister abe promotes a vision of 'proactive pacifism,' he chose to put japanese lives and his government's own political interests ahead of global well-being (pollmann ; the japan times, february , ) . south korea made a large step in its contribution to international personnel at this time. in addition to the usd . million of assistance it had already provided, between december and april , seoul sent three emergency relief teams (a total number of thirty people), comprising mostly skilled military and civilian healthcare workers, to west african countries to carry out medical activities (the korea times, october , ; china news, april , ) . this represented the first time that the south korean government had sent an emergency relief team to fight the outbreak of an epidemic overseas. in comparison, china's participation was much swifter and of greater weight. the un secretary general ban ki-moon acknowledged 'the speed and breadth' of china's response and emphasized the commitment and dedication made by chinese medical staff to fighting ebola (china daily, february , ) . but, there were also other countries who made significant contributions, including personnel, to this global effort to fight ebola. since mid-september , the usa had shown renewed engagement and significantly enhanced the global scale of the fight against ebola. over the course of the epidemic, the usa deployed more than personnel to the affected region ; as a superpower and as a longstanding traditional donor country, the usa did play a leading role in this humanitarian effort. nevertheless, as a rising great power and as a non-traditional donor country, china's growing role in international aid and global governance is commendable for its willingness and comprehensiveness. secondly, in terms of cooperation, china's role is, however, less straight-forward. the complexity and devastation of the ebola crisis again demonstrated the value and necessity of cooperation among a variety of actors. of course, in the process of engaging in the global effort to fight ebola, china did cooperate with many countries and international and regional organizations by providing financial support to the un, the who and the au, and assisting them in playing leading and coordinating roles. china also made many bilateral and trilateral agreements to combat the unprecedented spread of ebola, including with the usa, france and the uk (focac ). the health ministers of china, japan and south korea also agreed to boost information-sharing on the ebola epidemic and countermeasures against other types of diseases, such as pandemic influenza (the japan times, november , ). however, in comparison with many traditional donor countries, china had less experience of coordinating with non-governmental actors, and the ebola crisis, in a sense, highlighted the shortcomings of china's private sector participation and its philanthropic shortfalls (rajagopalan ) . even though at the government level china contributed over usd million to fight ebola, at the private sector level it donated little to the cause. many firms and business people in china still assume that the chinese government should take the lead on international assistance. in a deeper sense, this philanthropic shortfall is the result of china's international aid tradition, which has been predominantly bilateral and government-to-government. this is clearly revealed in china-africa relations. since china began its assistance to africa in the s, it has been the government that has initiated the sending of medical practitioners and the building of roads and railways (chan , - ) . even at present, this tendency has not changed very much; hence, the aid commitment under the multilateral mechanism of the forum on china-africa cooperation (focac) is also realized mainly through a bilateral mechanism (xu ) . with global multilateral cooperation frameworks growing in sophistication, china faces the challenges of how to better integrate itself into the multilateral development framework. to what extent has china's approach to the ebola crisis contributed to empowerment rather than just protection? as argued earlier in this chapter, empowerment refers to a longer-term positive/fundamental approach to human security that looks at the underlying causes of human security threats and should ultimately lead to an improved capacity to overcome threats. strictly speaking, capacity building and empowerment may not be entirely identical, as the former is more related to organizational capabilities, while the latter is more concerned with people. yet, the two are closely related to each other, and building organizational capabilities such as proper health systems can directly and indirectly enhance individual health resilience in the long term. thus, this chapter examines the following two aspects in detail: china's effort in offering help to build public healthcare systems and its active engagement in african economic and social reconstruction. first, in fighting the ebola crisis, china has not only devoted itself to tackling the deadly disease but has also offered valuable help to african countries to improve their capacity to respond to public health emergencies. if china's four major rounds of assistance between april and october were dedicated mainly to stopping the spread of the ebola epidemic, since then china has focused more on long-term capacity building (embassy of the prc ). in november , china sent its public health training team to sierra leone to study the ways they could carry out training for public health professionals in west africa. the team was to smooth the way for large-scale training programs in the future. by august , chinese public health training teams had trained more than , residents, including medical staff, community healthcare workers, government officials and volunteers. using china's fight against sars as a way of sharing their experiences, the training teams were able to deliver useful prevention and control knowledge and skills to the participants (nhfpc a; embassy of prc ). indeed, empowerment through improving regional health systems has become an important part of the china-africa health cooperation. at the second ministerial forum of china-africa health development in early october , ministers emphasized the importance of african people being able to access quality essential health commodities, medicines, vaccines and medical services (nhfpc c). to that end, china pledged to send medical workers to africa in the next three years, and it encouraged ten of its large pharmaceutical and medical equipment enterprises to cooperate with various african counterparts, through measures such as technology transfers in the production, maintenance and distribution of quality pharmaceutical products (china daily, october , ) . secondly, along with establishing and improving public health systems, poverty reduction and economic and social reconstruction have become china's key goals in its efforts to address the ebola epidemic. the chinese foreign minister wang yi emphasized this point while on a visit to the three countries worst hit by ebola, by saying that 'poverty was the root cause' of the ebola outbreak (xinhua, august , ) . from china's perspective, the fundamental solution to preventing the reoccurrence of ebola and other epidemics of this kind is to find effective paths to eliminate poverty and achieve development as soon as possible. for this purpose, china's cooperation would prioritize areas such as infrastructure building, resumption of trade and export, food security and other areas to enhance their resilience to crises (xinhua, august , ; august , ) . of course, rebuilding the fragile health system and enhancing socio-economic reconstruction in these countries will not be easy and will require a much longer time-period and persistent efforts. in this sense, whether wang yi's visit to the african continent will lead to more substantial engagement from china, or whether it will bear positive fruits, remains to be seen. however, china's commitment to african development and to the recovery of the three countries was demonstrated at the forum on china-africa cooperation (focac). indeed, in the past three focac meetings, china has consistently doubled its financing commitment to africa-from usd billion in to usd billion in and usd billion in (sun ) . additionally, at the forum, china pledged usd billion worth of assistance and loans for african development. it also specified ten areas of cooperation and assistance, including agriculture modernization, public health and poverty reduction that it would engage in. in the declaration, china promised it would transfer agricultural technology to africa; cancel outstanding debt for some of the poorest african countries; help build an african center for disease control; and back cooperation between twenty chinese and african hospitals. china also hoped to explore the possibility of linking china's belt and road initiative and africa's economic integration. if these efforts can be materialized, they would certainly have a positive impact on africa. finally, why did china participate so actively in the global efforts to contain ebola? as discussed, realist notions of raison d'état cannot provide sufficient answers about the international efforts for human security purposes. the question is this: to what extent can china's role transcend this notion of national interests? of course, one cannot deny that china's national interests are growing in africa, including in the three most affected countries. when evd emerged, there were approximately , chinese nationals living in the three afflicted countries (beijing youth daily, august , ) . moreover, when chinese foreign minister wang yi visited the three countries in august to prepare for the post-ebola reconstruction, he promised more funding and joint projects including infrastructure building, and resumption of trade and export (xinhua, august , ) ; this was an indication of china's growing economic interests in these countries. however, national economic interests alone cannot explain china's proactive engagement in the global fight against ebola, given that these countries are the least developed countries in africa. if china was purely seeking economic interests in the area, it should have invested in countries that had a greater chance of return. therefore, we should examine the ways in which human security as a fundamental value has an increasing impact on national foreign policy and strategic choices. china's active participation in and significant contributions to the global fight against ebola indicates its 'growing position within the international community as a global actor in humanitarian aid' (undp a, ) . it also reflects some important changes in its foreign policy orientations, particularly in foreign aid strategy. this is clear in china's second white paper on foreign aid (wp ii) (state council of china ). in , china published its first-ever foreign aid white paper (wp i), which was already indicative of its effort and strategy to become a responsible great power in international society. by moving its focus from its own development to the provision of assistance to other developing countries, china is 'fulfilling its due international obligations' (state council of china , ), and is enhancing its image as a responsible great power (liu and huang ) . in wp i, the underpinning principles for china's foreign aid were clearly put forward: the 'five principles of peaceful coexistence' and the 'eight principles' for economic aid and technical assistance to other countries. china often defended its role as being 'an alternative to western donors who impose more conditions on recipients' (state council of china , - ) . by comparison, there are some noticeable modifications contained in the wp ii, which sets out the following two areas-'helping improve people's livelihood' (改善民生) and 'promoting economic and social development'-as its major foreign aid objectives (state council of china , - ) . of course, this does not mean that china has abandoned the principles of non-conditionality, non-interference, and respect for sovereignty, which continue to underpin the basic principles of china's foreign and aid policies. however, the growing emphasis on poverty reduction (减少贫困) and improvement of people's livelihood (改善民生) means that these are increasingly attuned to those values of human security which have been endorsed and promoted by the un and the international community (state council of china ; undp b). the shift also reflects some important changes in the way china assesses global security threats and identifies its national interests; this is increasingly in line with the broader definition of human security. as china's foreign aid specialist wang xiaolin argues, the trend of china's foreign assistance has changed significantly from being driven by ideology and only aiding socialist countries to being based on its assessment of global security challenges. china sees the global security agenda, such as poverty reduction and tackling climate change, as being part of its foreign aid agenda, and hence its foreign assistance is more consistent with the millennium development goals (now the sustainable development goals) (wang ) . importantly, as the global security agenda expands, human security norms such as poverty reduction and environmental responsibility have emerged and been institutionalized as important norms and institutions in international society (kozyrev ; kopra ) . the global adoption of mdgs ( ) and sdgs ( ) has hugely contributed to the institutionalization of a human security norm in international society. building on the success of the mdgs, the sdgs with goals and targets are particularly determined to eradicate poverty and hunger in all their forms, which are the core elements of human security. moreover, the sdgs are also truly global in nature and universally applicable, and all countries have a shared responsibility to achieve them (unga ) . in this way, human security norms have become an important and legitimate basis for moral claims within international society and even have an impact on 'the criteria for rightful membership' of international society (falkner and buzan , ) . given that china is so eager to build its global image as a responsible 'great power,' it cannot ignore these changes. thus, the argument can be made that china may not entirely abandon its national interests and would not promote its foreign aid purely out of altruistic aspirations; however, it does indicate the ways in which china assumes and identifies its national interests in the changing international environment of the twenty-first century. in other words, china is increasingly seeing its national interests and security in terms of the interests and security of international society as a whole; in so doing, china is showing a growing sense of raison de système in global international society, in which human security considerations are becoming an important part of its foreign policy projection. china's participation in the global effort to address the ebola outbreak provides us with several important lessons as to how human security-oriented foreign and aid policies should be conducted, especially in an environment of emerging and complex human security challenges. the destructive nature of the ebola crisis and the devastation it brought with it again demonstrated the changing nature of global security threats. as ginsburg vividly illustrates: 'it is shocking to realize that a tiny virus with just a handful of genes can fracture families, shred communities, destroy national economies and destabilize whole regions in just a matter of months. but this is what we are witnessing with ebola.' (guardian, october , ). diseases like ebola can become as serious and deadly as the threats caused by conflicts and even wars. moreover, as viruses know no borders, once a breakout occurs, it can easily affect people across countries, regions and worldwide. as is often the case, it is always the most vulnerable individuals and groups who are the most affected. given the complexity and potential destructiveness of infectious diseases, future health security governance should be prepared with greater care. first, it is imperative that early warning systems for future health crises should be developed at the national, regional and global levels, especially in low-and middle-income countries. one of the important lessons that was drawn from the ebola crisis was the weak (or even lack of) healthcare systems in the three most affected countries. according to anthony fauci, a health expert based in bethesda, usa: 'if there was a system to have recognized and stopped the outbreak that began with the child in guinea in december , we might have avoided the explosive outbreaks in sierra leone and liberia' (kupferschmidt ) . it is in this sense that the un secretary general ban ki-moon stressed 'the need to strengthen early identification systems and early action' (snyder ) . the east asian region has also been faced with many health and security challenges, for instance, the outbreaks of sars in and h n bird flu in - , both of which had the potential to turn into pandemics (fidler ) . the sars outbreak did indeed spark regional health security initiatives (caballero-anthony and amul , ). yet, to prepare for complex challenges in the future, more enhanced and sophisticated regional public health systems are required. secondly, the ebola crisis strongly demonstrated the value of cooperation between actors at various levels, including both state and nonstate actors (nsas). particularly, cooperation between external militaries and ngos is a notable development. both china and the usa deployed many troops to help control the epidemic. importantly, some ngos, such as msf, which had previously refused to work with national militaries, are now calling for military intervention as part of outbreak responses. in fact, it has been proven that with their adaptability, discipline, ability to operate in challenging environments and logistical capabilities, the military can be a particularly valuable resource during large-scale public health crises (edelstein et al. ) . however, it should be emphasized that in this situation the operationalization of the role of states and their militaries cannot be properly understood in the traditional sense of state centrism. importantly, as the most powerful state in the world, the usa acknowledges that global health security is a 'shared responsibility' that cannot be achieved by a single actor or sector of government (white house, september , ) . thus, in any future health security governance, ngos and other nsas should become important partners, and through state-ngo or public-private partnerships (ppp), they should achieve their common objectives through collaboration. in addition to public-private partnerships, future health security governance should also pay sufficient attention to the idea of 'local ownership,' which values cooperation between international actors and local actors. exploring the concept of 'local ownership' in the field of conflict resolution and peacebuilding, shinoda ( , ) argues that unless it is solidly rooted in local society, conflict resolution and peacebuilding would end up becoming 'superficial' and 'short-sighted.' this is true with international development cooperation, because the ultimate aim of such cooperation should empower stakeholders of a local society to enable them to take responsibility for dealing with the situation. finally, the close link between health security and poverty is another case in point. indeed, the fact that 'poverty and infectious diseases interact in subtle and complex ways' has long been recognized by scholars through various case studies (alsan et al. ). the ebola case further supported the argument that human security and human development cannot be treated in isolation. poverty can and has often been the root cause of many human security threats. in this sense, future human security governance must combine proper defensive measures with more fundamental measures so that human security can be achieved and sustained not only through protection, but more importantly, through empowerment. in all these cases, should future human security-oriented foreign policies be truly realized, states must transcend their narrow national interest and seriously consider the dignity and well-being of the most vulnerable people in society. china's foreign policy. the 'eight principles' were announced by premier zhou enlai in during a visit to africa. the principles 'established the notion of non-conditionality of chinese foreign aid the militarization of aid in a variety of global contexts has long been a concern for humanitarian actors, who worry that such actions violate important principles of ethical humanitarian aid, namely: 'neutrality' (not taking sides in a conflict); 'impartiality' (not discriminating in aid provision); and 'independence accordingly, several ngos have renounced working with military forces in the provision of humanitarian relief poverty, global health and infectious disease: lessons from haiti and rwanda asia 'not doing enough' to fight ebola japan weighs sdf mission to sierra leone to help in fight against ebola jin wan zhongguo gongmin zai xifei aibola yiqu shenghuo [approximately , chinese nationals living in the west africa ebola epidemic areas health and human security: pathways to advancing a human-centered approach to health security in east asia.' in routledge handbook of global health security who declares official end to ebola outbreak in west africa china engages global health 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international peace-keeping operations, sc/ with spread of ebola outpacing response, security council adopts resolution ( ) urging immediate action, end to isolation of affected states, sc/ remarks by the president at global health security agenda summit who (world health organization). a. ebola situation summary. geneva: world health organization b. statement on the st meeting of the ihr emergency committee on the ebola outbreak in west africa c. high level meeting on building resilient systems for health in ebola-affected countries, meeting report. december - ebola virus disease latest ebola outbreak over in liberia; west africa is at zero, but new flare-ups are likely to occur china pledges continuous aid to africa in anti-ebola efforts china uses anti-sars experience to fight ebola in w china plans , more staff to fight ebola in africa china's anti-ebola efforts further enhance china-africa community of common destiny wang yi: xifei sanguo zhansheng aibola zui genben de shi yao fazhan qilai wang yi tan xifei sanguo xing forum on china-africa cooperation: creating a more mature and efficient platform key: cord- -azrqz hf authors: ganasegeran, kurubaran; abdulrahman, surajudeen abiola title: artificial intelligence applications in tracking health behaviors during disease epidemics date: - - journal: human behaviour analysis using intelligent systems doi: . / - - - - _ sha: doc_id: cord_uid: azrqz hf the threat of emerging and re-emerging infectious diseases to global population health remains significantly enormous, and the pandemic preparedness capabilities necessary to confront such threats must be of greater potency. artificial intelligence (ai) offers new hope in not only effectively pre-empting, preventing and combating the threats of infectious disease epidemics, but also facilitating the understanding of health-seeking behaviors and public emotions during epidemics. from a systems-thinking perspective, and in today’s world of seamless boundaries and global interconnectivity, ai offers enormous potential for public health practitioners and policy makers to revolutionize healthcare and population health through focussed, context-specific interventions that promote cost-savings on therapeutic care, expand access to health information and services, and enhance individual responsibility for their health and well-being. this chapter systematically appraises the dawn of ai technology towards empowering population health to combat the rise of infectious disease epidemics. infectious diseases disrespect national and international borders. they pose substantial threats and serious repercussions to global public health security. while the asia-pacific region was generally regarded as the main epicenter of emerging infectious diseases, with outbreaks of avian flu, asian flu and severe acute respiratory syndrome (sars) [ ] , the recent and unexpected emergence of zika pandemic spurred global concerns about pandemic preparedness capabilities particularly as it relates to training and deployment of healthcare workforce at a massive level, worldwide. despite coordinated global efforts, containing the "red alert" pandemic of zika remained a challenge, as both healthcare workers and public health advocates were uncertain about such disastrous contagion causing serious complications including congenital microcephaly in newborns and neurological deficits in adults [ , ] . control measures were obtunded as public health advocates were initially speculative about the potential transmission route of zika, while clinicians in hospitals were irresolute, instituting multiple levels of care and management to tackle the complications of zika. this debacle gave rise to an urgent need to debate the circumstances under which the zika epidemic has challenged human intelligence behavior and capacity to battle the threat effectively and efficiently. as population explosions and uncontrolled human mobility across nations catalyzes rapid disease propagation, our next question is, what else above human intelligence could help resolve such unprecedented epidemic crisis? scientists believe that the time has come to institute analytic technologies-such as artificial intelligence (ai)-in healthcare to help prevent and resolve such large disease epidemics [ , ] . adaptive ai applications could mould human behavior to practice preventive behaviors and disease control strategies [ ] , thereby improving global health. this chapter will systematically discuss the dawn of ai technology in healthcare that could potentially empower the human population to tackle unprecedented infectious disease epidemics. the human population has witnessed four major revolutions till date (fig. ) ; the foremost being the first industrial revolution that introduced steam engine to the world [ ] . this was followed by the second industrial revolution that introduced electrical-energy based productions. the first information revolution was conceptualized during the third industrial revolution in the late th century. it was during this time that computers and internet-based knowledge began and has since then shaped human interactions. in early st century, the fourth industrial revolution accelerated the second information revolution. the entire phase of human daily functions transformed with the debut of ai, bringing together massive information flow from different specialties. these culminated in the rise of big data with systems integration across the internet of things (iot) and cloud computing systems. current revolutionary era is based on extreme automation for global connectivity, in which ai would definitely play an imperative role as a resource to utilize. at the peak of emergent multi-function contexts of ai and the rise of big data analytics, the united nations (un) in unified global experts to galvanize a dynamic consensus on the adoption and expansion of ai use in delivering good public care services [ ] . succinctly, various stakeholders were assembled together in another un meeting to assess the role of ai towards achieving sustainable developmental goals (sdgs) [ ] . from the healthcare perspective, massive data have been obtained from public health surveillance efforts with the advancement of ai. one major public health field that gained momentum to develop various ai applications for disease prevention was the infectious disease domain [ ] . the human population is currently able to access potentially useful massive data sources of infectious disease spread through sentinel reporting systems, national surveillance systems (usually operated by national or regional disease centers such as the center for disease control (cdc)), genome databases, internet search queries (also called infodemiology and infoveillance studies) [ ] [ ] [ ] , twitter data analysis [ , ] , outbreak investigation reports, transportation dynamics [ ] , vaccine reports [ ] and human dynamics information [ ] . with the influx of massive data volume, effective data integration, management and knowledge extraction systems are required [ ] . epidemic modeling and disease-spread simulations form new horizons to understand the effects of citizen behaviors or government health policy measures [ ] . a simple integrated effect of disease knowledge discovery is exhibited in fig. . as humans, we are able to perform simple essential tasks such as object detection, visual interpretation and speech recognition. our interpretation is instantaneous when we look at an object or image, or when we hear voices or noises surrounding us. our next question is-could ai perform these essential intellectual tasks as well? the answer is absolutely yes, but in a different mode of function. while human interpretation is solely dependent on cognitive functions, ai requires mathematical algorithms to automate machines for execution of such functions [ ] . machines here refer to programmable computers! an example is to visualize the cause of an outbreak; dengue, chikungunya or zika, of which these diseases are commonly caused by the vector mosquito. in massive epidemics, elimination of the vector is important, and human cognitive functions can never detect all mosquitos in an outbreak investigation area! however, this can be easily detected through deployment of ai in areas which have loads of mosquito vectors to facilitate control measures. figure exhibits how human and ai technology interpret the vector differently. while human interpretation is instantaneous, ai evaluates the same image as humans do, but translated into codes [ ] , facilitating massive detection. while ai aims to mimic human cognitive functions, it lacks intuitive behaviors. scientists postulate that such synthetic intelligence which could be on par with human intelligence can be called "computational intelligence." however, the primary goal [ ] of ai was to create a system programming that is capable to think and act rationally like humans, although such machines may lack intuitive or emotional capabilities. as such, ai has been appropriately defined in simple and straightforward terms, as "a branch of computer science that deals with simulation of intelligent behaviors as humans using computers [ ] ". in principle, there are three types of ai. if a machine is able to think as humans do and perform a task similar to human intellectual capabilities, then that machine functionality is referred to as artificial general intelligence [ ] . if a machine performs a single task extremely well, this is known as artificial narrow intelligence [ ] . if the same machine out-smart the best humans in all fields from scientific creativity to general wisdom or social skills, this is referred to as artificial super intelligence [ ] . at present, virtually all contemporary ai application systems utilize artificial narrow intelligence. there are numerous concepts to function underlying ai applications in healthcare. based on the required functions, these concepts are clumped together to automate a single application-such as tracking infectious disease health seeking behavior. the following sub-sections summarize key concepts of different ai subsets adopted in emerging literature of infectious diseases. machine learning (ml). ml is a subfield of ai that implies learning from previous experiences (fig. ) . the system finds solution to a problem by extracting fig. interpreting the vector from the human and ai perspective. source da silva motta et al. [ ] previous relevant data, learn from this data and predicts new outcomes [ ] . ml applications are sub-divided into three categories: i. supervised learning: uses patterns of identified data (e.g. training data) ii. unsupervised learning: finds and learns from patterns of data (e.g. data-mining that involves identification of patterns in large datasets) iii. reinforcement learning: an extension of supervised learning that "rewards" and "punishes" when an application interacts with the environment. table illustrates some common examples of supervised and unsupervised ml methods that are currently adopted and utilized to track health seeking behaviors during infectious disease epidemics [ ] . deep learning (dl). dl is a specific subset of ml that uses neural networks (fig. ) . in short, it is basically a synthetic replica of the human brain structure and functionality [ ] . dl can execute multiple functions like image recognition and natural language processing (nlp). the system is capable of handling large datasets of information flow. image recognition. ai has the capability to process large amount of data about characteristics of a particular phenomenon in the form of images or signals [ ] . motion images and sounds are examples of signals that could be analyzed using artificial neural networks (anns) [ ] . recently, researchers from the usa proposed a system that could rapidly identify potential arbovirus outbreaks (mosquito, ticks or other arthropod borne viruses) [ ] . the system identifies images of mosquito larvae captured and delivered by a group of citizen scientists. not only did the developed prototype facilitate collection of images, it also facilitated training of image classifiers for the recognition of a particular specimen. this sets a base for execution of expert validation process and data analytics. it was found that recognition of specimen in images provided by citizen scientists was useful to generate visualizations of susceptible geographical regions of arboviruses threat (fig. ) . the system was capable of identifying mosquito larvae with great accuracy. the rapid identification of potential outbreak to a susceptible community could alert preventive behaviors and policy drafting in the quest to control potential epidemics. natural language processing (nlp). nlp bridges the gap between languages that humans and machines use to operate. algorithms are built to allow machines to identify keywords and phrases in an unstructured written text. ai applications then interpret the meaning of these texts for actionable knowledge [ ] . expert systems (es). es incorporates expert-level competence to resolve a particular problem [ ] . the system is constituted of two main components, namely knowledge base and a reasoning engine. it solves complex problems through reasoning a set of incomplete or uncertain information through a series of complex rules. in recent years, fuzzy logic, a set of mathematical principles for knowledge representation was crafted to accelerate the evolution of es. such strategy was utilized by a team of researchers from south africa to improve predictions of cholera outbreaks [ ] . public reaction and behavior towards disease outbreaks could be difficult to predict. with the rise of big data analytics and a pool of ai applications in place, public health researchers were able to correlate population's behavior during an outbreak [ ] . the following examples illustrate real life applications of ai during disease epidemics: twitter, a free social-networking micro-blogging service has enabled loads of users to send and read each other's "tweets (short, -character messages)." as important information and geo-political events are embedded within the twitter stream, researchers now postulate that twitter users' reactions may be useful for tracking and forecasting behavior during disease epidemics. the zika pandemic. most of the world's populations are living in endemic areas for common mosquito-borne diseases. the zika pandemic between the years of and marked the largest known outbreak, reaching a "red-alert" warning of multiple complications requiring global public health interventions. in such exigencies, population health behaviors are important for potential control measures. daughton and paul postulated that internet data has been effective to track human health seeking behaviors during disease outbreaks [ ] . they used twitter data between and respectively to identify and describe self-disclosures of an individual's behavior change during disease spread. they combined keyword filtering and ml classifications to identify first-person reactions to zika. a total of , english tweets were analyzed. keywords include "travel," "travelling intentions," and "cancellations." individual demographic characteristics, users' networking and linguistic patterns were compared with controls. the study found variations between individual characteristics, users' social network attributes and language styles in twitter users. these users changed or considered to change their travel behaviors in response to zika. significant differences were observed between geographic areas in the usa, with higher discussion among women than men and some differences in levels of exposure to zika-related information. this finding concludes that applying ai concepts could contribute to better understanding on how public perceives and reacts towards the risks of infectious disease outbreak. the influenza a h n pandemic. signorini and colleagues in analyzed twitter embedded data for tracking rapid evolvement of public concerns with respect to h n or swine flu, while concurrently measuring actual disease activity [ ] . the researchers explored public concerns by collecting tweets using pre-specified search terms related to h n activity with additional keywords related to disease transmission, disease countermeasures and food consumption within the united states. they utilized influenza-like illness surveillance data and predicted an estimation model using supervised learning method in machine learning. the results showed that twitter was useful to measure public interest or concern about health-related events associated with h n . these include an observed periodical spikes related to user twitter activity that were linked to preventive measures (hand-hygiene practices and usage of masks), travel and food consumption behaviors, drug related tweets about specific anti-viral and vaccine uptake. they concluded that twitter accurately estimated influenza outbreak through ai applications [ ] . the integration of internet data into public health informatics has been regarded as a powerful tool to explore real-time human health-seeking behaviors during disease epidemics. one such popular tool widely utilized is google trends, an open tool that provides traffic information regarding trends, patterns and variations of online interests using user-specified keywords and topics over time [ ] . such adaptations formed two conceptualizations: the first was "infodemiology," defined as "the science of distribution and determinants of information in an electronic medium, specifically the internet, or in a population, with the ultimate aim to inform public health and public policy [ ] ;" the second was "infoveillance," defined as "the longitudinal tracking of infodemiology metrics for surveillance and trend analysis [ ] ." examining health-behavior patterns during dengue outbreaks. dengue is highly endemic across the south-east asian countries. recently, a group of researchers from the philippines conducted an infodemiology and infoveillance study by using spatio-temporal concepts to explore relationships of weekly google dengue trends (gdt) data from the internet and dengue incidence data from manila city between and [ ] . they subsequently examined health-seeking behaviors using dengue-related search queries from the population. their findings suggested that weekly temporal gdt patterns were nearly similar to weekly dengue incidence reports. themes retrieved from dengue-related search queries include: "dengue," "symptoms and signs of dengue," "treatment and prevention of dengue," "mosquito," and "other diseases." most search queries were directed towards manifestations of dengue. the researchers concluded that gdt is a useful component to complement conventional disease surveillance methods. this concept could assists towards identifying dengue hotspots to facilitate appropriate and timely public health decisions and preventive strategies [ ] . health-seeking behavior of ebola outbreak. an unprecedented ebola contagion that plagued most west african countries in marked the rise of global public health interest in pandemic preparedness interventions. millions of ebola-related internet hit searches were retrieved. with such high fluxes of health-seeking behavior using computers, a group of italian researchers' evaluated google trends search queries for terms related to "ebola" outbreak at the global level and across countries where primary cases of ebola were reported [ ] . the researchers subsequently explored correlations between overall and weekly web hit searches of terms in relation to the total number and weekly new cases of ebola incidence. the highest search volumes that generated ebola related queries were captured across the west african countries, mainly affected by the ebola epidemic. web searches were concentrated across state capitals. however, in western countries, the distribution of web searches remained fixed across national territories. correlations between the total number of new weekly cases of ebola and the weekly google trends index varied from weak to moderate among the african countries afflicted by ebola. correlations between the total number of ebola cases registered in all countries and the google trends index was relatively high. the researchers concluded that google trends data strongly correlated with global epidemiological data. global agencies could utilize such information to correctly identify outbreaks, and craft appropriate actionable interventions for disease prevention urgently [ ] . public reactions toward chikungunya outbreaks. the italian outbreak of chikungunya posed substantial public health concerns, catalyzing public interests in terms of internet searches and social media interactions. a group of researchers were determined to investigate chikungunya-related digital health-seeking behaviors, and subsequently explored probable associations between epidemiological data and internet traffic sources [ ] . public reactions from italy toward chikungunya outbreaks were mined from google trends, google news, twitter traffics, wikipedia visits and edits, and pubmed articles to yield a structural equation model. the relationships between overall chikungunya cases, as well as autochthonous cases and tweet productions were mediated by chikungunya-related web searches. but in the allochthonous case model, tweet productions were not significantly mediated by epidemiological figures, instead, web searches posed significant mediating tweets. inconsistent associations were detected in mediation models involving wikipedia usage. the effects between news consumption and tweets production were suppressed in this regard. subsequently, inconsistent mediation effects were found between wikipedia usage and tweets production, with web searches as a mediator. after adjustment of internet penetration index, similar findings were retrieved with the adjusted model showing relationship between google news and twitter to be partially mediated by wikipedia usage. the link between wikipedia usage and pubmed/medline was fully mediated by google news, and differed from the unadjusted model. the researchers found significant public reactions to the chikungunya outbreak. they concluded that health authorities could be made aware immediately of such phenomenon with the aid of new technologies for collecting public concerns, disseminating awareness and avoiding misleading information [ ] . expert systems are built upon the basis to act as a diagnostic tool to accelerate detection of infectious disease epidemics, determining the intensity or concentration of vector-agents within the triads of infectious disease dynamics. the malaria control strategy using expert systems. malaria constitutes a "red-alert" health threat to the african communities. a group of researchers from nigeria built an expert system for malaria environmental diagnosis with the aim of providing a decisional support tool for researchers and health policy-makers [ ] . as prevailing malaria control measures were deemed insufficient, this group of researchers developed a prototype that constituted components of "knowledge," "applications," "system database," "user graphics interface," and "user components." the user component utilized java, while the application component used java expert system shell (jess) and the java ide of netbeans. the database component used sql server. the system was able to act as a diagnostic tool to determine the intensity of malarial parasites in designated geographical areas across africa. the proposed prototype proved useful and cost-effective in curbing malaria spread [ ] . whereas ai is gaining increasing popularity and acceptance as a quick fix to the myriad of challenges faced with pandemic preparedness using traditional population-based approaches, it is not without its own limitations. even in resource-rich settings, there are challenges associated with building and updating the knowledge base of expert systems [ ] , providing high-quality datasets upon which machine learning algorithms can be premised, and ethical issues associated with data ownership and management [ ] . additionally, resource-limited settings are further plagued with constraints of poorly organized and integrated health systems, poor it and communication infrastructure, and socio-economic and cultural contexts [ , ] that significantly impact successful implementation of ai systems. beyond these, the dynamics of human behavior and other environmental covariates (such as mass/social media, public emotions, public policy etc.) may not only influence the accuracy of epidemic disease modeling frameworks but also impact health seeking behavior during epidemics [ ] . more than ever before, public health experts, it developers and other stakeholders must work together to address concerns related to scalability of ai for healthcare, data integration and interoperability, security, privacy and ethics of aggregated digital data. finally, the transparency of predictive ai algorithms have been called to question, particularly given their 'black box' nature which makes them prone to biases in settings of significant inequalities [ ] . perhaps, it may be premature to describe ai as the future of healthcare given it is still in its infancy, however, it has become increasingly difficult to not acknowledge the substantial contributions of ai systems to the field of public health medicine. notwithstanding current challenges with the widespread adoption of ai particularly in resource-limited settings, the use of ai in providing in-depth knowledge on individuals' health, predicting population health risks and improving pandemic preparedness capabilities is likely to increase substantially in the near future [ ] . further, the rapidly expanding mobile phone penetrance, developments in cloud computing, substantial investments in health informatics, electronic medical records (emrs) and mobile health (mhealth) applications, even in resource-constrained settings, holds significant promise for increasing use and scalability of ai applications in improving public health outcomes [ ] . public health policy, practice and research will continue to benefit from the expanding framework of infodemiology and infoveillance in analyzing health information search, communication and publication behavior on the internet [ , ] . advances in cryptographic technologies-including block chain is likely to allay fears and concerns with security, privacy and confidentiality of public digital data/information [ ] . there is no doubt that ai is and will continue to revolutionize healthcare and population health. from prevention and health promotion to diagnosis and treatment, ai is increasingly being deployed to improve clinical decision-making, enhance personalized care and public health outcomes. in particular, ai offers enormous potential for cost-savings on therapeutic care given its predictive accuracy of potential outbreaks and epidemics and ability to enhance positive health seeking behaviors (at individual and population levels) during epidemics predicated upon robust infodemiology and infoveillance frameworks supported by expert systems, machine learning algorithms and mobile applications. amazing as the future of ai in healthcare seems, there are significant legal and ethical concerns that need to be addressed in order to pave way for robust implementation and scalability across a variety of socio-cultural, epidemiological, health system and political contexts. tracking infectious disease spread for global pandemic containment an update on zika virus infection neurologic complications associated with the zika virus in brazilian adults will artificial intelligence solve the human crisis in healthcare? bmc artificial intelligence for infectious disease big data analytics the human behavior-change project: harnessing the power of artificial intelligence and machine learning for evidence synthesis and interpretation recommendations for implementing the strategic initiative united nations: looking to future un to consider how artificial intelligence could help achieve economic growth and reduce inequalities detecting influenza epidemics using search engine query data google trends in 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and global health: how can ai contribute to health in resource-poor settings? image recognition of disease-carrying insects: a system for combating infectious diseases using image classification techniques and citizen science fuzzy expert systems and gis for cholera health risk prediction in southern infodemiology and infoveillance: framework for an emerging set of public health informatics methods to analyze search, communication and publication behavior on the internet infodemiology and infoveillance: tracking online health information and cyber-behavior for public health using google trends to examine the spatio-temporal incidence and behavioral patterns of dengue disease: a case study in metropolitan manila assessing ebola-related web search behavior: insights and implications from an analytical study of google trends-based query volumes public reaction to chikungunya outbreaks in italy-insights from an extensive novel data streams-based structural equation modeling analysis building a computer-based expert system for malaria environmental diagnosis: an alternative malaria control strategy developing and using expert systems and neural networks in medicine: a review on benefits and challenges semantics derived automatically from language corpora contain human-like biases machine bias accounting for healthcare-seeking behaviors and testing practices in real-time influenza forecasts algorithmic transparency via quantitative input influence: theory and experiments with learning systems. security and privacy (sp) health intelligence: how artificial intelligence transforms population and personalized health tracking health seeking behavior during an ebola outbreak via mobile phones and sms adopting m-health in clinical practice: a boon or a bane? acknowledgements we thank the ministry of health malaysia for the support to publish this chapter. key: cord- -vok dni authors: chin, taylor; buckee, caroline o.; mahmud, ayesha s. title: quantifying the success of measles vaccination campaigns in the rohingya refugee camps date: - - journal: nan doi: . / sha: doc_id: cord_uid: vok dni in the wake of the rohingya population's mass migration from myanmar, one of the world's largest refugee settlements was constructed in cox's bazar, bangladesh to accommodate nearly , new refugees. refugee populations are particularly vulnerable to infectious disease outbreaks due to many population and environmental factors. a large measles outbreak, with over , cases, occurred among the rohingya population between september and december . here, we estimate key epidemiological parameters and use a dynamic mathematical model of measles transmission to evaluate the effectiveness of the reactive vaccination campaigns in the refugee camps. we also estimate the potential for subsequent outbreaks under different vaccination coverage scenarios. our modeling results highlight the success of the vaccination campaigns in rapidly curbing transmission and emphasize the public health importance of maintaining high levels of vaccination in this population, where high birth rates and historically low vaccination coverage rates create suitable conditions for future measles outbreaks. . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / doi: medrxiv preprint that occurs in other settings [ ] . figure : schematic representation of disease states in a model of a measles outbreak in cox's bazar. individuals are either susceptible to measles (s), infectious with measles (i), or recovered from and immune to measles infection (r), either due natural immunity from infection or prior vaccination. β represents the transmission coefficient, γ is the recovery rate, and µ is the birth and death rate. the model starts with one infected (and infectious) measles case (i.e., i( ) = ). the number of recovered individuals at time = was calculated as r( ) = n − s( ) − i( ). the model tracks the number of individuals that move between the compartments each day and is run for one year using the ordinary differential equations - . the transmission coefficient, β, was estimated according to the relationship β = r /n d, where n is the total population (i.e., s + i + r) and d is the average duration of infectiousness ( the model uses one infected case as the initial value for the number of individuals in the infected compartment, i( ). the initial value for the recovered compartment was calculated as (table ) . the model runs for days to reflect one year following the start of the outbreak. lhs was used to take into account uncertainty in parameter estimates; , values for each parameter were sampled from the assumed parameter probability distributions (table ) where vc is ≥ dose coverage of the measles, mumps, and rubella (mmr) vaccine, ve is the median effectiveness . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / doi: medrxiv preprint now. table reports all parameters and calculations used as inputs in the mathematical model. lhs was again used to take into account uncertainty in parameter estimates. for this analysis, the sir model was run for two years to estimate the impact of vaccination on the next outbreak. the cumulative number of cases over two years was calculated for vaccination coverage rates of %, %, and % of the at-risk population. cumulative cases were estimated as the cumulative proportion of the population that moves into the infectious compartment in the sir framework. distributions of our estimates for the cumulative number of cases at two years under the different vaccination rate scenarios are presented. to contrast these results with the scenario of the next measles outbreak in the absence of vaccination, the same model used to estimate the size of the outbreak in the absence of vaccination was used with two changes: ) the total population size was increased to , [ ] , and ) the proportion of the population that was assumed to be susceptible at the start of the outbreak was estimated as su instead of s (table ) figure ). vaccination therefore has the potential to avert between approximately , and , cases over two years depending on the coverage rate. figure b shows estimates in terms of boxplots to reflect the uncertainty in our model's results due to parameter uncertainty. . we find that nearly , cases were averted over the course of one year due to vaccination in the outbreak. the daily effective reproductive number declined from its maximum value of . at the end of week to below by the middle of week . when we apply our model to estimate the impact of vaccination on subsequent outbreaks, . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / doi: medrxiv preprint cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / doi: medrxiv preprint isolation of infectious cases were implemented during the outbreak, which would have the effect of shortening the it may be reasonable to assume that these interventions were not widely used given their infeasibility in this setting. secondly, the contraction of the serial interval described by kenah, lipsitch, and robins [ ] in high transmission settings is not considered in this analysis. instead, a constant serial interval distribution is assumed. for these reasons, second, reporting rates and how these rates changed over time are also not considered in this analysis. one possible scenario is that identification of measles was worse at the start of the epidemic relative to later in the epidemic, and that fewer measles cases were therefore reported in the early phase of the epidemic. if this were the case, the initial case counts may underestimate the true extent of measles transmission at the start of the epidemic, cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / doi: medrxiv preprint figure : histogram of estimated r values using lhs with , parameter combinations, where the maximum has been restricted to . summary statistics: . (mean), . (median), . ( . %) and . ( . %) . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / doi: medrxiv preprint cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / doi: medrxiv preprint situation report: rohingya refugee crisis, cox's bazar | url more- -rohingya-babies-born-bangladesh-refugee-camps-every-day-unicef the theory of measles elimination: implications for the design of elimination strategies. the different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures serial intervals of respiratory infectious diseases: a systematic review and analysis refugee settlements situation report: rohingya refugee crisis rohingya babies set to be born in overcrowded refugee camps in republic of korea pledges us$ , to scale up unfpa's response to the rohingya refugee crisis in s-response-rohingya-refugee-crisis-bangladesh prevention of measles, rubella large sample properties of simulations using latin hypercube sampling an age-structured model of pre-and post-vaccination measles transmission mortality and morbidity weekly bulletin (mmwb) waning antibody levels and avidity: implications for mmr vaccine-induced protection impact of public health responses during a measles outbreak in an amish community in ohio: modeling the dynamics of transmission health sector bulletin # rohingya crisis in cox's bazar the basic reproduction number (r ) of measles: a systematic review generation interval contraction and epidemic data analysis estimation of the reproductive number and the serial interval in early phase of the influenza a/h n pandemic in the usa weekly situation report # , rohingya refugees weekly situation report # , rohingya refugees situation report # , rohingya refugees epidemiological highlights key: cord- - oh tlr authors: markl, jürgen; sadava, david; hillis, david m.; heller, h. craig; hacker, sally d. title: evolution von genen und genomen date: - - journal: purves biologie doi: . / - - - - _ sha: doc_id: cord_uid: oh tlr der erste weltkrieg endete im november . die zahl der todesfälle in den vier kriegsjahren wurde jedoch schon bald übertroffen von den opfern einer massiven grippeepidemie, an der weltweit über mio. menschen starben – und damit mehr als doppelt so viele wie in den schlachten des ersten weltkriegs. die pandemie von / war insofern bemerkenswert, als die sterberate unter jungen erwachsenen, die einer grippe gewöhnlich mit viel geringerer wahrscheinlichkeit zum opfer fallen als kinder und greise, um das -fache höher lag als bei den vorherigen und später folgenden grippeepidemien. warum erwies sich dieses grippevirus speziell unter den normalerweise widerstandsfähigsten menschen als so tödlich? der virusstamm von löste im menschlichen immunsystem eine besonders starke reaktion aus. infolge dieser Überreaktion waren menschen mit einem leistungsfähigen immunsystem tendenziell stärker betroffen. der erste weltkrieg endete im november . die zahl der todesfälle in den vier kriegsjahren wurde jedoch schon bald übertroffen von den opfern einer massiven grippeepidemie, an der weltweit über mio. menschen starben -und damit mehr als doppelt so viele wie in den schlachten des ersten weltkriegs. die pandemie von / war insofern bemerkenswert, als die sterberate unter jungen erwachsenen, die einer grippe gewöhnlich mit viel geringerer wahrscheinlichkeit zum opfer fallen als kinder und greise, um das -fache höher lag als bei den vorherigen und später folgenden grippeepidemien. warum erwies sich dieses grippevirus speziell unter den normalerweise widerstandsfähigsten menschen als so tödlich? der virusstamm von löste im menschlichen immunsystem eine besonders starke reaktion aus. infolge dieser Überreaktion waren menschen mit einem leistungsfähigen immunsystem tendenziell stärker betroffen. in der regel können wir uns im kampf gegen viren schon auf unser immunsystem verlassen. die immunreaktion bildet auch die grundlage der impfung. seit haben spezielle impfprogramme gegen grippeviren dazu beigetragen, die anzahl und schwere der grippefälle in grenzen zu halten. allerdings wirkt der impfstoff eines bestimmten jahres vermutlich nicht gegen die viren des folgenden jahres. der grund: es entwickeln sich ständig neue stämme von grippeviren und sorgen für genetische variabilität in der population. würden diese nicht evolvieren, könnten wir eine dauerhafte resistenz gegen sie aufbauen. das würde die jährliche grippeimpfung überflüssig machen. da die viren aber evolvieren, müssen pharmaunternehmen jedes jahr einen neuen, anderen grippeimpfstoff entwickeln und in ausreichender menge bereitstellen. die immunantwort von wirbeltieren wird ausgelöst, wenn das immunsystem proteine auf der virenoberfläche erkennt. demnach kann das virus durch veränderungen seiner oberflächenproteine der immunabwehr entkommen. je größer die anzahl der veränderungen der oberflächenproteine ist, desto eher werden die virenstämme vom immunsystem nicht erkannt, können ihre wirte infizieren und haben damit einen vorteil gegenüber anderen stämmen. biologen verfolgen, wie sich die oberflächenproteine der grippeviren von jahr zu jahr ändern. dadurch beobachten sie die evolution in aktion. mit diesen erkenntnissen können sie dann wirkungsvollere impfstoffe entwickeln. durch die erforschung von rasch evolvierenden organismen hat man sehr viel über die molekularen grundlagen der evolution gelernt. die ergebnisse molekularer evolutionsstudien finden wiederum anwendung in der praxis, etwa bei der entwicklung besserer strategien zur bekämpfung tödlicher krankheiten. in "experiment: warum war die grippepandemie von / so schlimm?" in abschn. . und in "faszination forschung" am ende dieses kapitels finden sie antworten auf diese frage. das genom eines organismus oder virus setzt sich zusammen aus der gesamtheit aller seiner gene sowie sämtlichen nichtcodierenden abschnitten der erbsubstanz. bei eukaryoten finden sich die meisten gene auf den chromosomen im zellkern, es gibt aber auch gene in den mitochondrien und chloroplasten. bei organismen mit sexueller fortpflanzung vererben sowohl die männlichen als auch die weiblichen individuen gene der nucleären dna, hingegen werden mitochondrien-und chloroplastengene in der regel nur über das cytoplasma der eizellen weitergegeben. durch sequenzalignments können biologen bei einzelnen individuen oder arten auftretende nucleotid-oder aminosäuresubstitutionen nachweisen. wird alleine die anzahl der nucleotidsubstitutionen oder aminosäureaustausche zwischen sequenzen ermittelt, führt dies häufig dazu, dass die tatsächliche zahl der zugrunde liegenden veränderungen unterschätzt wird. damit genome von eltern an die nachkommen weitergegeben werden können, müssen sie zunächst repliziert werden. die replikation der dna verläuft jedoch, wie sie bereits wissen, nicht ohne fehler. fehler bei der dna-replikation -mutationenliefern einen großteil des ausgangsmaterials für evolutionäre veränderungen. mutationen sind eine grundvoraussetzung für das langfristige Überleben von organismenpopulationen, denn sie bilden die eigentliche quelle für die genetische variabilität, die es populationen ermöglicht, als reaktion auf veränderungen ihrer umwelt zu evolvieren. ein bestimmtes allel eines gens kann erst dann an nachfolgende generationen weitergegeben werden, wenn ein individuum, das dieses allel besitzt, überlebt und sich fortpflanzt. das betreffende allel muss in kombination mit zahlreichen anderen genen des genoms funktionieren, sonst wird es von der selektion rasch ausgelesen. darüber hinaus werden das ausmaß und der zeitpunkt der expression eines gens streng reguliert. aus diesem grund kann man die gene eines einzelnen organismus als interagierende mitglieder einer gruppe betrachten, unter denen arbeitsteilung herrscht, aber auch starke wechselseitige abhängigkeiten bestehen. ein genom ist also nicht einfach eine willkürliche ansammlung von genen, die auf den chromosomen in zufälliger reihenfolge angeordnet sind. vielmehr handelt es sich dabei um eine komplexe zusammenstellung miteinander interagierender gene, regulationssequenzen und strukturelemente. dazwischen liegen abschnitte aus nichtcodierender dna, die vermutlich kaum eine direkte funktion erfüllen. die positionen der gene unterliegen genau wie ihre abfolge einem evolutionären wandel; das gleiche gilt für den umfang und die lage der nichtcodierenden dna-abschnitte. all diese veränderungen können sich auf den phänotyp eines organismus auswirken. mittlerweile haben biologen die genome einer großen zahl von organismen einschließlich des menschen vollständig sequenziert. die in diesen sequenzen enthaltenen informationen tragen dazu bei, dass wir heute besser verstehen, wie und warum sich organismen unterscheiden, wie sie funktionieren und wie sie sich im verlauf der evolution entwickelt haben. das fachgebiet der molekularen evolutionsforschung beschäftigt sich mit der erforschung der mechanismen und konsequenzen der evolution von makromolekülen, insbesondere von nucleinsäuren (dna und rna) und proteinen. i i i i i i i i i i i i i i i t v i i i l l l l i i i i i zahl der aminosäuren an dieser position i i t t t t t t i t t t t i t v t t t t t l e t e i i i i i i i i i i i i i i i i i i i i i i i i i p p p p p p p p p p p p p p p p p p p p p p p p p p p p p i i i i i i i i i i i i i i i i i i i i i i i i i i i i i p p p p p p p p p p p p p p p p p p p p p p p p p p p p p t t t t t t t t t t t t t t t t t t t t t t t t t t t t t m m m m i i i i i i i i i i i i i v i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i v i i i i i i v i i i i i i hoch konserviert die genome aller organismen evolvieren im laufe der zeit. evolutionäre veränderungen lassen sich durch einen vergleich der nucleotid-und aminosäuresequenzen verschiedener arten nachweisen. durch analyse der molekularen evolution im laborexperiment unter kontrollierten bedingungen können biologen viele evolutionsprozesse direkt erforschen. sie sollten . . . ein sequenzalignment durchführen und dazu eine matrix erstellen können, in der die Ähnlichkeiten und unterschiede der sequenzen miteinander vergleichend gegenübergestellt sind. zeigen können, warum anhand der ermittelten zahl der nucleotid-oder aminosäureunterschiede zwischen zwei sequenzen häufig unterschätzt wird, wie viele veränderungen tatsächlich zwischen den sequenzen stattgefunden haben. ? . führen sie ein sequenzalignment der folgenden sequenzen durch und erstellen dann eine distanzmatrix, in der die zahl der identischen nucleotide und der unterschiede (einschließlich insertions-und deletionsereignissen) verglichen werden. . erklären sie, warum durch einfaches zählen der abweichenden nucleotide zweier sequenzen die tatsächliche zahl der nucleotidsubstitutionen seit der auseinanderentwicklung der sequenzen häufig zu gering eingeschätzt wird. verwenden sie dazu als beispiel einen vergleich ihres sequenzalignments der sequenzen a und b aus aufgabe . wie sie gesehen haben, befasst sich die molekulare evolutionsforschung mit der evolution von genen und proteinen, vergleicht nucleotid-und aminosäuresequenzen verschiedener organismen miteinander und rekonstruiert, welche veränderungen während der stammesgeschichte stattgefunden haben. als nächstes werden sie erfahren, wie sich genome verändern, und einige der folgen dieser veränderungen näher betrachten. wie sie in abschn. . erfahren haben, versteht man unter einer mutation jegliche veränderung des genetischen materials. eine mutationsform, die sich in einer population etablieren kann, ist die punktmutation, der austausch eines einzelnen nucleotids. viele solcher nucleotidsubstitutionen in der dna haben keine auswirkung auf ein protein -selbst dann nicht, wenn die veränderung an einem proteincodierenden gen erfolgt, denn für die meisten aminosäuren gibt es mehr als ein codon ( abb. . ). eine substitution, die nicht zu einer anderen aminosäure führt, bezeichnet man als synonyme, neutrale oder stille substitution ( abb. . a). synonyme substitutionen wirken sich nicht auf die struktur und funktion eines proteins aus (können allerdings andere auswirkungen haben, wie in abschn. . beschrieben) und unterliegen daher wahrscheinlich weniger dem einfluss der natürlichen selektion als andere formen der substitution. eine neutrale evolution unterscheidet sich von einer negativen (reinigenden) und positiven selektion dadurch, dass sie sich nicht auf die Überlebensfähigkeit und fortpflanzung des betreffenden organismus auswirkt. die fixierungsrate neutraler nucleotidsubstitutionen innerhalb von populationen ist unabhängig von der populationsgröße. durch vergleiche der raten synonymer und nichtsynonymer substitutionen kann man eine positive und negative selektion in proteingenen erkennen. die genome von organismen zeichnen sich durch eine sehr unterschiedliche größe aus, dagegen ist die zahl der proteincodierenden gene deutlich weniger variabel. der genetische code legt fest, welches codon welche aminosäure codiert ( abb. . ). eine nucleotidsubstitution, die zu einer veränderung der von einem gen codierten aminosäuresequenz führt, bezeichnet man als nichtsynonyme substitution ( teil vi . die unterschiede in den aminosäuresequenzen wurden jeweils paarweise in eine tabelle eingetragen. anschließend wurden die aminosäureänderungen in den evolutionären stammbaum eingetragen und die zahl der konvergenten Ähnlichkeiten zwischen jedem der artenpaare ermittelt. die ergebnisse können dann als distanzmatrix dargestellt werden. die matrix zeigt oberhalb der diagonalen für jedes artenpaar die anzahl der aminosäureunterschiede und unterhalb die anzahl der konvergenten Ähnlichkeiten. an den lysozymsequenzen der beiden arten mit einer gärkammer zur verdauung lässt sich die mehrzahl der konvergenten Ähnlichkeiten zwischen den einzelnen artenpaaren nachweisen; dabei zeigt sich einhergehend mit der unabhängigen evolution einer gärkammer zur verdauung eine molekulare konvergenz. abb. . in der anzahl der gene pro genom gibt es enorme unterschiede. diese abbildung zeigt die zahl der gene einer auswahl von organismen, deren genome schon vollständig sequenziert wurden, angeordnet nach ihren bekannten evolutionären verwandtschaftsbeziehungen. bakterien und archaeen weisen im normalfall weniger gene auf als die meisten eukaryoten. unter den eukaryoten besitzen vielzellige organismen mit organisierten geweben (pflanzen und tiere; die dunkelgrünen und blauen zweige) mehr gene als einzellige organismen (türkisfarbene zweige) oder vielzellige lebewesen ohne eine auffällige organisation der gewebe (gelbe zweige) entstanden. obwohl also die hoatzins und die säugetiere mit gärkammer in den letzten paar hundert millionen jahren keinen gemeinsamen vorfahren hatten, haben sie ähnliche anpassungen ihres lysozyms entwickelt, die es ihnen ermöglichen, ihren der gärung dienenden bakterien nährstoffe zu entziehen. k l k e y d n q n v pavian i r l r q y n d q n v mensch v r m r r y n d q n v ratte t r m y q y n d k n v rind v k l k e w d n r d l pferd v a m g g w n e k d l ursprünglicher zustand v r m r q w n d k n v durch in der größe der genome gibt es bei verschiedenen organismen bekanntlich erhebliche unterschiede. betrachtet man die großen taxonomischen kategorien, so ist eine gewisse korrelation zwischen genomgröße und komplexität der organismen zu erkennen. das genom des winzigen bakteriums mycoplasma genitalium umfasst nur gene. aus genen besteht das genom des bakteriums rickettsia prowazekii, dem erreger des fleckfiebers. hingegen besitzt homo sapiens ungefähr . proteincodierende gene. abb. . zeigt die zahl der gene einer auswahl von organismen, deren genome bereits vollständig sequenziert wurden, angeordnet nach ihren bekann-ten evolutionären verwandtschaftsbeziehungen. wie ihnen die abbildung zeigt, bedeutet ein größeres genom nicht eine höhere komplexität des phänotyps. (vergleichen sie beispielsweise den reis mit den anderen pflanzen.) es überrascht nicht, dass für den bau und die aufrechterhaltung der funktionen eines großen, vielzelligen organismus mehr und komplexere genetische informationen erforderlich sind als bei einem kleinen, einzelligen bakterium. Überraschend ist jedoch, dass einige organismen, etwa lungenfische, manche schwanzlurche und lilien, rund -mal mehr dna im zellkern aufweisen als beispielsweise der mensch. natürlich ist ein lungenfisch oder eine lilie nicht -mal komplexer aufgebaut als ein mensch. warum variiert die genomgröße dann so stark? die unterschiede in der genomgröße sind nicht so groß, wenn man nur jenen anteil der dna betrachtet, der tatsächlich proteine codiert oder sequenzen anderer rnas als mrnas festlegt. die organismen mit der größten menge an kern-dna (einige farne und blütenpflanzen) weisen zwar . -mal so viel dna auf wie die bakterien mit den kleinsten genomen, aber keine der arten besitzt mehr als -mal so viele proteincodierende gene wie ein bakterium. daher beruhen die meisten wie im vorherigen abschnitt erwähnt, besitzen die meisten vielzelligen organismen sehr viel mehr gene als der größte teil der einzelligen arten. aber die vielzelligen lebewesen sind aus einzelligen vorfahren hervorgegangen. wie kam es zu der zunahme der anzahl der gene innerhalb des genoms von vielzelligen organismen im laufe der evolution? eine solche zunahme kann vor allem durch zwei mechanismen zustande kommen: es können gene von anderen arten übertragen oder innerhalb einer art dupliziert werden. bisweilen können gene zwischen entfernt miteinander verwandten zweigen am stammbaum des lebens ausgetauscht werden. die duplikation eines gens bietet gelegenheiten für die evolution neuer funktionen. manche gene liegen im genom in multiplen kopien vor; diese evolvieren im laufe der zeit häufig gemeinsam. in viele genduplikationen betreffen jeweils nur ein gen oder wenige gene, bei polyploiden organismen (darunter zahlreiche pflanzen) wurden jedoch komplette genome dupliziert. bei einer duplikation sämtlicher gene ergeben sich unzählige möglichkeiten, neue funktionen zu evolvieren. genau das ist offenbar bei der evolution der wirbeltiere passiert. das genom der gnathostomata (kiefermünder -das sind alle wirbeltiere mit einem beweglichem unterkiefer) scheint von vielen wichtigen genen vier diploide sätze aufzuweisen. aufgrund dieser erkenntnis gelangten biologen zu dem schluss, dass sich bei dem vorfahren dieser gruppe zwei duplikationen des gesamten genoms ereigneten. diese duplikationen ermöglichten eine beträchtliche spezialisierung einzelner wirbeltiergene, deren expression heute in hohem maße gewebespezifisch erfolgt. zwar haben sich die vertreter der globingenfamilie in ihrer form und funktion diversifiziert, die mitglieder vieler anderer die ziffern geben die geschätzte anzahl an Änderungen der dna-sequenz entlang des betreffenden astes im stammbaum an. durch ein duplikationsereignis entstanden die und -globingen-cluster. abb. . ein genstammbaum der globingenfamilie. eine analyse mittels der methode der molekularen uhr legt nahe, dass sich die 'und "-globingen-cluster (blau bzw. grün) vor ungefähr mio. jahren auseinanderentwickelt haben -also relativ bald nach der entstehung der wirbeltiere genfamilien evolvieren hingegen nicht unabhängig voneinander. so besitzen beispielsweise fast alle organismen zahlreiche kopien (bis zu mehrere tausend) der gene für ribosomale rna. ribosomale rna (rrna) bildet das hauptstrukturelement der ribosomen und erfüllt als solches eine wichtige rolle bei der proteinsynthese. sämtliche lebewesen müssen -oft in großen mengen -proteine synthetisieren (vor allem während ihrer frühen entwicklung). durch den besitz zahlreicher kopien der rrna-gene wird gewährleistet, dass die organismen rasch viele ribosomen produzieren und dadurch eine hohe proteinsyntheserate aufrechterhalten können. die gene für ribosomale rna evolvieren wie alle anderen teile des genoms, und so sammeln sich in den rrna-genen verschiedener arten mit der zeit unterschiede an. innerhalb einer art sind sich die zahlreichen kopien der rrna-gene hingegen sowohl strukturell als auch funktionell sehr ähnlich. diese Ähnlichkeit ist auch sinnvoll, denn im idealfall sollte jedes ribosom einer art auf die gleiche weise proteine synthetisieren. mit anderen worten, innerhalb einer spezies evolvieren die vielen kopien dieser rrna-gene gemeinsam. dieses phänomen bezeichnet man als konzertierte evolution. www.life e.com/a . wie kommt es zu einer solchen konzertierten evolution? offensichtlich liegen ihr zwei verschiedene mechanismen zugrunde. einer davon ist ungleiches crossing-over. während der replikation der dna eines diploiden organismus im zuge der meiose lagern sich die homologen chromosomenpaare aneinan-der, und es kommt zu einer rekombination durch crossing-over ( abschn. . ). im falle hochrepetitiver gene wie der rrna-gene passiert es jedoch leicht, dass diese bei der paarung der chromosomen gegeneinander versetzt werden, weil so viele kopien der gleichen gene auf den chromosomen vorhanden sind ( abb. . a). infolgedessen erhält das eine der beiden homologen chromosomen beim crossing-over zusätzliche kopien des rrna-gens, während das andere chromosom entsprechend weniger kopien abbekommt. erfolgt in einer der kopien eine punktmutation in form einer basensubstitution, kann diese durch das ungleiche crossing-over schrittweise vermehrt und damit allmählich fixiert werden. umgekehrt kann sie durch das ungleiche crossing-over auch schrittweise verringert und schließlich eliminiert werden. so oder so bleiben die vielen kopien des gens einander sehr ähnlich. den zweiten mechanismus, der zu einer konzertierten evolution führt, bezeichnet man als gerichtete genkonversion. dieser mechanismus erfolgt mit viel höherer geschwindigkeit als ein ungleiches crossing-over und hat sich als primärer mechanismus für die konzertierte evolution von rrna-genen erwiesen. in dna-strängen kommt es häufig zu brüchen, welche dann wieder repariert werden ( abschn. . ). die gene für die ribosomale rna liegen während des zellzyklus die meiste zeit eng beieinander. kommt es zu einer beschädigung eines der gene, kann eine kopie des rrna-gens des homologen chromosoms als matrize zur reparatur der beschädigten kopie dienen; dabei ersetzt die als matrize fungierende sequenz die originalsequenz ( abb. . b). in vielen fällen erfolgt diese reparatur offenbar insofern recht einseitig, als häufig bestimmte sequenzen als matrizen verwendet werden. dadurch kann sich teil vi eine solche bevorzugt verwendete sequenz rasch auf alle kopien des gens ausbreiten. auf diese weise kann es passieren, dass sich eine veränderung, die in einer einzelnen kopie auftritt, rasch auf alle anderen kopien ausbreitet. aber ganz gleich, welcher dieser mechanismen der konzertierten evolution im konkreten fall zugrunde liegt: durch sie evolvieren die kopien eines hochrepetitiven gens nicht unabhängig voneinander. zwar treten nach wie vor mutationen auf, aber wenn diese in einer einzelnen kopie erfolgen, dann breiten sie sich entweder rasch auf sämtliche kopien aus oder gehen vollständig aus dem genom verloren. dieser prozess ermöglicht es, dass jede kopie über die zeit bezüglich sequenz und funktion ähnlich bleibt. durch horizontalen gentransfer können bestimmte funktionen von genen auch zwischen entfernt verwandten arten übertragen werden. genduplikation kann zur evolution neuer funktionen führen. manche hochrepetitiven gene durchlaufen eine konzertierte evolution, wodurch ihre gleichförmige funktionalität erhalten bleibt. sie sollten . . . beschreiben können, wie es durch horizontalen gentransfer zur Übertragung von genen zwischen verschiedenen linien kommen kann, insbesondere zwischen bakterien. anhand eines genstammbaums einer genfamilie ableiten können, wann in der stammesgeschichte einer gruppe von arten genduplikationen stattgefunden haben. erläutern können, inwiefern ein dupliziertes gen gelegenheiten für die evolution neuer funktionen liefert. die beiden prozesse, die einer konzertierten evolution zugrunde liegen, grafisch darstellen und ihre unterschiede aufzeigen können. ? gentransfers für den organismus, der durch diesen mechanismus neue gene erhält. für die ergebnisse von untersuchungen der molekularen evolution gibt es in der gesamten biologie praktische anwendungsmöglichkeiten, etwa, um grundlegende aspekte biologischer funktionen besser zu verstehen, oder für forschungen zur menschlichen gesundheit. die evolutionsgeschichte von genen liefert informationen zur funktion von proteinen. zebrafisch en b zebrafisch en a huhn en maus en mensch en zebrafisch en b zebrafisch en a huhn en maus en mensch en en en seeigel en lanzettfischchen en bei wirbeltieren entstanden durch eine genduplikation die beiden paralogen engrailed-gene en und en . in der zu den zebrafischen führenden linie traten weitere genduplikationen auf. anstatt mit genstammbäumen arbeiten viele biologen je nach fragestellung auch mit proteinstammbäumen, zum beispiel um die verwandtschaftsbeziehungen oder den funktionswandel homologer proteine zu analysieren. entsprechend zu den genen unterscheidet man orthologe proteine (gleiche funktion in unterschiedlichen organismen) und paraloge proteine (unterschiedliche funktion im gleichen organismus). so sind die 'und die "-untereinheit des menschlichen hämoglobins paralog ( abb. . ), die "-untereinheiten von mensch und maus dagegen ortholog. weiter vorne in diesem kapitel haben sie erfahren, wie biologen genabschnitte identifizieren können, die einer positiven bei den verschiedenen duplizierten genen für natriumkanäle der vielen kugelfischarten sind mehrere unterschiedliche substitutionen erfolgt, die zu einer resistenz gegen ttx geführt haben. in diesen genen sind aber auch zahlreiche andere veränderungen aufgetreten, die nichts mit der evolution der ttx-resistenz zu tun haben. biologen, die sich mit der funktion von natriumkanälen befassen, können eine menge über die funktionsweise dieser kanäle lernen (und auch über neurologische krankheiten, die durch mutationen in den genen für natriumkanäle verursacht werden), indem sie in erfahrung bringen, welche veränderungen für eine ttx-resistenz selektiert wurden. dazu vergleichen sie die raten synonymer und nichtsynonymer substitutionen bei den genen der verschiedenen linien, in denen sich eine resistenz gegen ttx entwickelt hat. in ähnlicher weise versucht man mithilfe der prinzipien der molekularen evolution die funktion sowie die diversifizierung der funktion zahlreicher anderer proteine zu verstehen. bei ihren untersuchungen der zusammenhänge zwischen selektion, evolution und funktion von makromolekülen kamen biologen schon bald auf die idee, sich die molekulare evolution in einer kontrollierten laborumgebung zunutze zu machen, um neuartige makromoleküle mit nützlichen eigenschaften herzustellen. das war die geburtsstunde der anwendungen der in vitro-evolution ("evolution im reagenzglas"). anschließend können sie feststellen, welche der gegenwärtig kursierenden influenzastämme die größte zahl von veränderungen in diesen positiv selektierten codons aufweisen. diese influenzastämme werden nämlich am ehesten überleben, sich vermehren und zukünftig grippeepidemien verursachen -und bilden somit die logischen ziele für neue impfstoffe. diese praktische anwendung der prinzipien der evolutionstheorie führt dazu, dass wirkungsvollere impfstoffe gegen grippeviren entwickelt werden -und damit alljährlich weniger menschen an grippe erkranken oder gar daran sterben. synapsenfutter: wenden sie an, was sie gelernt haben als genom eines organismus bezeichnet man die gesamtheit seiner gene, regulatorischen sequenzen und strukturelemente einschließlich der nichtcodierenden dna. das gebiet der molekularen evolution befasst sich damit, welche zusammenhänge zwischen der struktur von genen und proteinen und der funktionsweise von organismen bestehen. mittels sequenzalignment von nucleinsäuren oder proteinen verschiedener organismen lassen sich diese vergleichen und homologe positionen identifizieren. siehe abb. . ; activity . key: cord- -dpftawj authors: boin, arjen title: the transboundary crisis: why we are unprepared and the road ahead date: - - journal: nan doi: . / - . sha: doc_id: cord_uid: dpftawj modern societies rely on complex technological systems that are deeply intertwined with other complex systems that stretch across geographical, judicial and administrative borders. when threats emanate from this transboundary space, national governments are often surprised and discover that existing crisis management arrangements do not suffice. this article describes the political and administrative challenges that accompany transboundary crises. it argues that arrangements and processes that work reasonably well for “bounded” crises are unlikely to work in the case of transboundary crises. it formulates an agenda for political debate and academic research. the bottom line is that we need to rethink traditional crisis management arrangements in order to prepare for these increasingly common type of threats. much has been done in recent decades to prepare modern society for all sorts of crises and disasters. one might say that many societies have never been as prepared for crises and disasters as they are today. that is a good thing. but here is the bad news: modern societies are woefully underprepared for dealing with a type of threat that is on the rise. this is what i refer to as the transboundary crisis. the transboundary crisis effortlessly exceeds geographical, policy, cultural, public-private and legal boundaries that normally enable public managers to classify, contain and manage a crisis. it escalates rapidly and mutates constantly, creating confusion about causes and possible consequences. it ends up on many administrative tables, but it is not obvious which of those tables is or should be "in the lead." these features make a fast and adequate response difficult, to say the least. the transboundary crisis comes in many guises: • in , a mysterious virus spread from china, via hong kong, to countries causing hundreds of deaths. the sars virus caused a major health crisis in toronto. in all these examples, national governments were confronted with a crisis that had origins in faraway domains. however well prepared they may have been for traditional crises, they soon discovered that their response repertoire was insufficient in the face of the transboundary challenges. when the state has no answer to a crisis, consequences follow. in times of crisis, the public expects representatives of the state to take charge. it is, after all, a core task of the state to protect its citizens against the consequences of threat and calamity. if the state fails in this core task, the legitimacy of public institutions and the especially when the legitimacy of public institutions is already under question. the transboundary crisis is the ultimate nightmare for crisis managers. it marks the moment they discover their traditional crisis arrangements do not suffice in the light of the political-administrative challenges that this crisis brings. it sheds light on a structural governance deficit, which presents politicians with a pressing design, let alone management, challenge. this study offers a roadmap for a discussion about possible solutions. this roadmap hinges on a strategic choice between two options that emerge from our discussion of theory: move backward by decoupling from modern systems or move forward by strengthening transboundary crisis management capacities. transboundary crises may come in different guises, but they share common characteristics that make them difficult to manage: the transboundary crisis reaches across multiple countries and/or multiple policy areas. there is no defined geographical location (a "ground zero") or policy sector around which to organize. that creates diversity in perspective: what in one domain is experienced as a problem of scarcity may become a matter of public safety in another; what in one country is considered a local matter is chefsache in another country. the transboundary crisis brings a critical challenge to any administrative system that is based on boundaries and demarcation. by crossing borders, the transboundary crisis challenges borders. in a democratic state that is based on the principle of political accountability and makes use of the bureaucratic organization form, most organizations are organized around demarcated areas of expertise and authority. the bureaucratic organization is based on boundaries among task fields, responsibilities, divisions, departments and policy sectors. two mechanisms have been traditionally used to address blurring of borders: coordination (negotiating boundaries) and centralization (transcending boundaries). these mechanisms can be problematic in the best of times; they are especially problematic in a transboundary crisis. coordination mechanisms may work fine for complex problems and the traditional crisis. the mechanisms, however, do not work in the world of the transboundary crisis for two reasons. first, in a transboundary crisis, it is not clear who the critical actors are or should be, and what their authority in the matter is. second, it is hard to establish or negotiate ownership in a short time frame (time is always scarce in a crisis). we may thus say that the transboundary crisis robs bureaucracy of its most effective tool. centralizing emergency powers in the hands of a leader or a central body is the traditional catch-all solution. in the roman empire, unlimited powers were placed in the hands of a dictator. in modern democracy, crisis centralization is still a valued mechanism. but it comes with constraints: it is not easy to centralize power, and it does not happen often. moreover, the "high command" is not always defined clearly enough, and the mechanisms that should regulate such a concentration of power are cumbersome. a quick look at the legal terms that condition the authorization of exceptional violence (the deployment of special police units) makes clear that a tension exists between legal considerations and the required speed of action. this tension can often only be circumvented in an environment in which political actors know and trust each other. in a transboundary crisis, the nation state may not be the only actor. but how to centralize power in an international context? governments are reluctant to shift decision-making authority to international institutions. think, for example, of eu agencies, which in principle could play a decisive role during a transboundary crisis. but these organizations were never endowed with decision-making powers. we should, then, not be surprised that an eu agency such boin | as frontex accomplished so little during the immigration crisis that peaked in . in sum, transboundary crises pose a wide and deep challenge to the standing governance arrangements of democratic states. that is problematic. the state is left rudderless in a time when citizens look to their elected leaders and trusted institutions to navigate them through the storm. a transboundary crisis can thus rapidly become a crisis of legitimacy. a vicious cycle threatens. the effectiveness of the crisis response relies to a large extent on legitimacy. but the legitimacy of public institutions is already under attack. if institutions do not function effectively during a crisis, they lose even more legitimacy. the transboundary crisis makes vulnerable institutions even more vulnerable. what can be done to protect our country, our prosperity and our well-being in a world of new, unprecedented crises that effortlessly bypass existing lines of defence? to answer this question, we must understand the underlying drivers of the transboundary crisis. two books, both classics, provide a great starting point: barry turner's ( ) man-made disasters and charles perrow's ( ) normal accidents. both authors focus on the relentless modernization of socio-technical systems. we build increasingly complex systems that we connect to other complex systems with one goal: to enhance the efficiency of critical processes (such as food supply, transport, production chains, internet and energy), that is, to increase the speed of service delivery at ever-lower costs. the thesis that emerges from both books can be summarized as follows: these "highways of efficiency" become the "highways of failure" that allow, if not actually enable, routine disruptions to travel very quickly from one system to another. perrow's argument is simple and convincing. if a system becomes evermore complex, it also becomes evermore difficult to understand. when something goes wrong, it is not immediately clear for system operators what is happening. if a complex system is tightly connected with other systems, we know that even a small disturbance can affect the functioning of these other systems. by the time the problem is recognized in one system, others may have been infected. complexity and tight coupling create fertile ground for small incidents to jump from one domain to another and thus escalate into larger-scale crises. new threats exploit these "highways of failure." the rise of revolutionary technologies-artificial intelligence, dna editing, drones, d-printing, self-propelled cars, the "internet of things"-brings happiness and economic prosperity, but they also speed up failure and create unknown shortcuts for unprecedented threats. from this theoretical perspective, two options emerge. a country can move backward by decoupling from the modern complexity ecology or move forward by a strategy of protecting complexity. perrow opted for the way back: simplify and unbundle critical systems. if complexity and tight coupling are the problem, perrow's solution-decoupling-is the obvious solution: return to simpler systems that are isolated from other systems. by decoupling from modernity, many crises simply cannot happen. the choice for decoupling does not come free, however. withdrawal entails a decoupling from the benefits that complex systems generate. it is a costly affair, as economists are fond to explain. but this rational argumentation collides with the growing unease about the negative effects of modern systems. in times of uncertainty, the call for entrenchment behind hard borders may not be evidence based, but it is intuitively convincing and politically attractive. we should therefore expect that "decoupling" remains an option as long as modern systems generate uncontrollable hazards. an alternative (or complimentary) strategy is prepared in the face of complexity. if we accept that disturbances can and will emerge, perhaps we are better off investing in early detection and timely intervention. aaron wildavsky ( ) argued that enhanced resilience through a trial-and-error strategy, relying on the human capacity for innovation and learning, is a much more fruitful strategy than a short-sighted focus on prevention through entrenchment. resilience became a dominant feature of many policy efforts in the domain of safety and security. governments that are unable or unwilling to protect their citizens against systemic risks appeal to the ability of citizens (also organizations and communities) to "absorb shocks" and "bounce back" after a disruptive event. the underlying idea is that citizens know what is good for them. in this line of thinking, it is not necessary that government arranges full protection for all citizens from all those complex systems surrounding them. begins with understanding the many ways in which incidents can develop into cross-border threats. how are systems connected? how can a system be infected by a disruption in another system? how can one system be protected against another without undermining the fruits of the existing connection? we need to understand the vulnerabilities of complex and tightly coupled systems. vulnerability thinking is still in its infancy. intelligence services seek to recognize potential threats in a timely manner. hedge funds bet on "big data" to foresee political upheavals. the eu has more than detection and early warning mechanisms in place (but nobody knows if they are effective). research funded by the eu (horizon ) focuses on understanding escalation mechanisms in critical infrastructures. complexity researchers study "tipping points." other researchers focus on the ability of people in control rooms to detect early aberrations in critical processes. the need for detection mechanisms is clearly and widely acknowledged, but we await major breakthroughs. the transboundary crisis will continue to surprise us for the time being. the transboundary crisis is difficult to comprehend. the causes are hidden in system complexity and pile up when the dominoes start falling. to understand how a threat unfolds, where exactly and how quickly, it is necessary to bring together as much relevant information as possible, that is, to authorize, analyse and share it with the right parties-quickly and effectively. where critical information about the broken levees in new orleans disappeared. it took the federal authorities more than hours to understand that the city was under water. the solution does not lie in the development of new technologies. what is needed is an approach that helps information managers to quickly collect information from a variety of organizational domains. they must learn to locate sources of critical information; they must also learn to make sense of that information, which is likely to be difficult as the information emanates from very different sources. it is a truism that critical decisions must often be made quickly on the basis of very little information when in a crisis. from a legitimacy perspective (see below), it is important that those critical decisions are made by the appropriate officials or institutions. the transboundary crisis makes this quite tricky, as it challenges the underlying logic of bureaucracy (where responsibility is tied to a person, position or institution). when a crisis involves multiple actors, each with their own responsibilities, interests and working methods, it must be clear who is authorized to decide what. this rarely has been determined beforehand, as a transboundary crisis tends to involve a unique constellation of actors. as we have seen, the typical solution to this type of problem involves a combination of centralization and coordination. centralization is hard enough for the more routine crises; it is even less likely to work in the international arena. national governments do not lightly cede authority to international organizations (certainly not during a crisis where so much is at stake). the international arena has produced some innovative solutions: think of nato's article five boin | (centralization in case of defined threats) and the european central bank (centralization by stealth). the most important problem is that crisis centralization tends to create serious legitimacy issues. if the ownership on critical issues is allocated to an official or institution, and thus not allocated somewhere else or taken away from someone, it is crucial that the "crisis owner" can rely on political and public support. if this is not the case, the legitimacy of public institutions may come under pressure. without a solid legitimacy basis, effective crisis management becomes difficult. centralization that has not been subjected to processes of democratic deliberation and control is risky in this regard. the european central bank became increasingly powerful during the financial crisis and is hardly subject to democratic control. the impotence of the european parliament is telling. the european central bank has made decisions that large groups of citizens perceive as unjust. the lack of democratic control opens the door for politicization of the crisis response. coordination, on the other hand, may work. but that requires a degree of "instant trust" that is often lacking between actors that have never worked together before. promising initiatives can be found at the interface of public and private. for instance, the netherlands has the ict response board in which private parties work with government to prepare for cyber disasters. the louisiana business emergency operations center is another innovative example. the question is whether such practices can be scaled to the international level. theoretical progress is being made. by formulating transboundary crisis management as a collective action problem, we can apply theoretical insights from this body of research. this type of research may provide a view of the conditions under which crisis cooperation is possible, and the strategies that may be helpful. one such strategy, for example, is based on that idea of "instant trust"-unilaterally taking the first step, without being sure of reciprocity (majchrzak, jarvenpaa, & hollingshead, ) . but, overall, we can state that mechanisms for the delineation of authority are lacking for transboundary crises. in the absence of serious political discussion, real progress will likely only happen after a disastrous encounter with a transboundary crisis. the transboundary crisis is, of course, not a new phenomenon. crises have always traversed boundaries. think of the plague sweeping across europe, the flu epidemic, food shortages in the roman empire or the two world wars. the optimist may claim that the western world has managed the really big threats in the postwar era. the pessimist may argue that the connections among international systems have spectacularly increased, problems have become more complex, and the number of actors has increased dramatically. even if the pessimist is only half right, we may justifiably ask whether we are sufficiently prepared. the good news is that crisis management has professionalized in the past few decades. since the beginning of this century (after the millennium computer bug and / ), western countries have heavily invested in crisis management capacity, both in the public and private sectors. municipalities, schools, hospitals, businesses-crisis management is now firmly on the radar just about everywhere. more, even though public bureaucracies are not designed to deal with exceptional situations, "work arounds" have been created to deal with routine crises. i have argued, however, that these existing crisis management structures are no match for the transboundary crisis. when the system under threat becomes spread too far, and workarounds do not we leave it to the national crisis institutions. we can instruct these institutions to pay more attention to the transboundary dimensions of crises. it will not be easy to introduce a new way of working in institutions that are built around traditional practices that work for traditional incidents and crises. these organizations do not have to unlearn standing practices; they must learn to deal with the transboundary crisis. we must, in other words, make those institutions "ambidextrous." this will require a lot of work, because national crisis institutions have little affinity with transnational and cross-border crisis management. perhaps it is better to invest in organizations that are already active in cross-border domains. nato and the european union have in recent years developed crisis management capabilities which, in principle, are intended for cross-border crises. the eu, in particular, has developed in embryonic form abilities needed for a transboundary crisis. but these capabilities and assets are scattered across the many agencies and commission parts of the eu. and the crisis performance of the eu is not always considered effective or legitimate. in particular, the response to the financial crisis and the immigration crisis has become sources of controversy. the eu will need time to become an effective and legitimate actor in the crisis domain. . build transboundary crisis management institutions. think in terms of a new organization, with new people, a respected leadership, real powers and a realistic budget. an institute that invents and tests new forms of crisis management practices. new processes and forms of organization that can effectively address the transboundary crisis. the advantage is that a brand new institution can devise novel methods or ways of thinking from scratch. that is, at the same time, the downside: it will take a long time and the outcome is by no means guaranteed. we conclude by setting out a research challenge. it is important to study the different guises in which the transboundary crisis comes, which allow for classification. it may well turn out that some types of transboundary crises are more amenable to certain interventions (such as centralization and coordination, decoupling or resilience) than others. it may well be that certain types are more politically or analytically challenging. the research challenge is to find out how characteristics relate to preparatory and management efforts. we may discover that certain types are intractable, whereas others may lend themselves to early detection and intervention. by studying cases of actual crises and near misses, we should be able to enhance our understanding of the transboundary crisis. acknowledgment i wish to thank emery roe for his extensive and constructive comments, which greatly improved this article. this study has benefitted from funding provided by the european union's horizon research and innovation programme under grant agreement no (transcrisis). the politics of crisis management managing hurricane katrina: lessons from a megadisaster coordinating expertise among emergent groups responding to disaster man-made disasters searching for safety how to cite this article: boin a. the transboundary crisis: why we are unprepared and the road ahead key: cord- - rs e authors: dionne, audrey; le, cathie-kim; poupart, steffany; autmizguine, julie; meloche-dumas, léamarie; turgeon, jean; fournier, anne; dahdah, nagib title: profile of resistance to ivig treatment in patients with kawasaki disease and concomitant infection date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: rs e introduction: kawasaki disease (kd) can be associated with concomitant viral or bacterial infections. children with persistent or recurrent fever hours after the end of intravenous immunoglobulin (ivig) are considered to be resistant to treatment and are at increased risk for coronary complications. although concomitant infection does not affect coronary outcome, it is unknown how it influences the response to ivig treatment. methodology: retrospective cohort study between and in a tertiary pediatric university hospital, including children, of which ( %) had concomitant infection. results: children with concomitant infection were more likely to have fever hours after initial ivig treatment ( % vs %, p = . ) and to be treated with a second dose ( % vs %, p = . ). children with infection had higher c-reactive protein at the time of diagnosis ( vs mg/l, p = . ), and hours after ivig administration ( vs mg/l, p = . ). nevertheless, there was no statistically significant difference in the prevalence of coronary complications (z-score > . ) between children with and without concomitant infection ( % vs %, p = . ). conclusion: children with kd and concomitant infection are more likely to have persistent fever and elevated inflammatory markers after treatment. this association increases the likelihood of receiving a second dose of ivig but not the risk of coronary complication. accordingly, prospective studies to distinguish true ivig resistance from infection induced persistent fever is warranted. a a a a a kawasaki disease (kd) is an acute systemic vasculitis mostly affecting children younger than years old. it is the most important cause of acquired heart disease in children in developed countries [ ] . concomitant respiratory viral infections have been described in - % of patients, and bacterial infections were found in % of patients [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the clinical presentation of patients with and without concomitant infection is similar [ , ] . in one study, bacterial coinfection alluded to a trend towards a higher rate of resistance to intravenous immunoglobulin (ivig), without reaching statistical significance ( % vs %, odds ratio . ( % confidence interval . - . )) [ ] . the accepted definition of ivig resistance is based on the persistence or recrudescence of fever hours after the end of ivig infusion [ ] . however, it is unclear how concomitant infection influences the resolution of fever and response to ivig treatment. the aim of this study was to determine the impact of concurrent infection on the prevalence of ivig resistance and coronary outcome. this retrospective study included children with a diagnosis of kd between and followed at the sainte-justine's university hospital center (montreal, canada). inclusion criteria were a diagnosis of kd maintained at discharge based on current clinical practice and recommendations [ ] ; and echocardiography measurements of the coronary artery (ca) at onset, - weeks and > months after diagnosis. the main outcome was resistance to ivig treatment in children with, versus those without concurrent infection. secondary outcomes included duration of fever, progress of inflammatory markers and coronary artery complications. this study was approved by the institutional research ethics committee of the chu sainte-justine. the institutional research ethics committee waived the requirement for informed consent. medical charts were reviewed for demographic characteristics, clinical course, laboratory values and infectious workup. clinical kd criteria were reviewed and children classified as complete or incomplete kd, in the presence of fever and � clinical criteria for the latter. delay in ivig administration was defined as > days between onset of fever and ivig administration; and ivig resistance as persistent or recrudescent fever (> . ˚c) hours after the end of ivig infusion. concurrent infection was defined as a clinical diagnosis and proven concurrent infection as patients with a positive microbiologic testing or imaging study in the presence of clinical symptoms. testing for concomitant infection was based on clinical symptoms. urinary tract infection was diagnosed according to current american academy of pediatrics guidelines [ ] . gastroenteritis was defined as gastrointestinal symptoms with positive stool cultures for virus or pathogenic bacteria. children with respiratory symptoms and a positive respiratory virus multiplex reverse transcription polymerase chain reaction (influenza, parainfluenza, coronavirus, enterovirus, rhinovirus) were diagnosed with upper respiratory tract infection (urti). otitis media was diagnosed according to current american academy of pediatrics guidelines [ ] . children with clinical signs of exudative pharyngitis in the presence of a positive group a streptococcus culture were diagnosed with concurrent infection. bacterial adenitis was diagnosed in the presence of clinical symptoms with signs of abscess formation and/or necrosis on ultrasound. children with positive serology for acute infection (igm) with epstein-barr virus (ebv), cytomegalovirus (cmv), measles, parvovirus and mycoplasma before ivig administration were considered to have concurrent infection. pneumonia was diagnosed when chest x-ray, interpreted by a pediatric radiologist, confirmed the presence of a consolidation, in the presence of respiratory symptoms. echocardiographic ca size was reviewed for all children at onset of the disease, with follow-up studies for a minimum of months and up to one year after diagnosis of kd. ca zscores were calculated for the right coronary artery, left main coronary artery, left anterior descending artery and circumflex artery. ca aneurysm was defined as a localized dilatation of a portion with adjacent normal measurements, or an obvious saccular deformation of the ca. ca dilatation of non-aneurysmal segments was defined as a ca z-score > . [ ] , calculated according to dallaire and dahdah [ ] . early ca dilatation was defined as ca dilatation at onset up to one week following kd diagnosis, and late ca dilatation when persisting at months' follow-up. quantitative variables were summarized as mean ± sd and categorical variables as frequencies and percentages. the shapiro-wilk test was used to test for normal distribution. comparison of clinical and laboratory data between patients with and without concurrent infection was performed using the student's t-test for continuous variables with normal distribution or the mann-whitney u test for continuous variables with non-normal distribution. anova for repeated measures was used to describe the variation of temperature and laboratory values over time. the fisher or the χ tests were used for comparison of categorical variables. logistic regression was used to examine the association between concomitant infection and ca complications, controlling for confounder variables (ivig resistance). all analyses were performed with spss statistics version (ibm, chicago, illinois). a two-tailed p value of < . was deemed significant. the study included children ( male; %) aged . ± . years. the median number of diagnostic criteria was (range - ), with ( %) children having incomplete kd criteria. ivig were administered to ( %) patients, . ± . days after onset of fever. delay in ivig administration occurred in ( %) patients, and ivig resistance in ( %). concurrent infections were diagnosed in ( %) patients, of which ( %) were viral and ( %) bacterial. urti was diagnosed in ( %) children, caused by respiratory syncytial virus (n = ), parainfluenza (n = ), rhinovirus (n = ), influenza (n = ), enterovirus (n = ) and adenovirus (n = ). three children had multiple viruses detected. two ( %) children were diagnosed with viral gastroenteritis. three ( %) children had a clinical and biological profile suggestive of acute cmv infection (positive igm and negative igg), and ( %) of acute mycoplasma infection. otitis media was diagnosed in ( %) of patients, and pneumonia in ( %) children. group a streptococcus pharyngitis was diagnosed in ( %). four ( %) patients had bacterial adenitis, complicated by retropharyngeal pharyngitis in one child. one child was diagnosed with perforated appendicitis and underwent surgery, with pathology confirming the diagnosis. one child had escherichia coli pyelonephritis. concomitant infection was proven by microbiologic testing and/or imaging in ( %) of patients. characteristics of patients with viral versus bacterial infections are described in table . during hospitalization, antibiotics were empirically initiated in ( %) children, and completed in ( %). antibiotics were initiated on average ± days prior to ivig treatment, with antibiotics initiated prior to ivig treatment in the majority of cases ( , %). only a minority ( , %) were started on antibiotics after non-response to initial ivig treatment. age was similar between children with and without infection ( . ± . versus . ± . years, p = . ), with a similar proportion of male ( ( %) versus ( %) patients, p = . ). there was a similar proportion of children with incomplete clinical criteria among children with and without infection ( ( %) versus ( %), p = . ). there were ( %) children with infection who received antibiotics compared to ( %) without infection who received antibiotics (p< . ). of the former, ( %) completed antibiotic treatment versus ( %) of the latter (p< . ). most children with and without concurrent infection received ivig ( ( %) and ( %) patients; p = . ). delay in diagnosis and ivig administration occurred in ( %) children with concurrent infection versus ( %) without concurrent infection, p = . (table ) . finally, there was no difference in delay of ivig administration whether patients presented with complete versus incomplete clinical criteria ( % and %; in general, basic laboratory values were similar between children with and without concurrent infections. c-reactive protein was however higher in cases with concurrent infections compared to cases without concurrent infection ( ± versus ± mg/l, p = . ) ( table ) . similar results were found between patients with proven infection on microbiologic testing and/or imaging versus those without and results are presented in table . overall, ivig resistance occurred in ( %) patients. children with concurrent infection had higher rates of ivig resistance ( ( %) versus ( %) patients, p = . ), and higher temperature at hours (fig ) . they were also more likely to have fever > . ˚c at hours, than those without concurrent infection ( ( %) versus ( %) patients, p = . ). this was accompanied with higher crp at time of diagnosis, remaining similarly higher in the first hours after treatment (fig ) . ivig resistance was higher in patients with proven infection on microbiologic testing and/ or imaging than those without ( ( %) versus ( %) patients, p = . ). there was no difference in response to treatment between patients who had a proven infection on microbiologic testing and/or imaging study versus those with clinical diagnosis of infection ( ( %) versus ( %) patients, p = . ). resistance to initial ivig treatment in patients with bacterial infection was nearly double that in patients with viral infection, although not reaching statistical significance ( ( %) versus ( %) patients, p = . ). there was no difference in response to treatment between patients with complete and incomplete clinical criteria ( ( %) versus ( %) patients, p = . ). patients who received antibiotics during their hospital course, independent of infection status, were at higher risk of ivig resistance than those who did not ( ( %) versus ( %) patients, p = . ). however, neither receiving antibiotics prior to ivig therapy ( ( %) versus ( %) patients, p = . ) nor completing antibiotic course ( ( %) versus ( %) patients, p = . ) were associated with response to treatment. there was no significant difference in coronary artery complications (fig ) between patients with and without infection ( ( %) versus ( %), p = . ). this remained true even after adjusting for ivig resistance and number of ivig treatment (p = . ). resistance to ivig treatment was associated with an increased risk of ca complication both as a univariate ( % versus %, p = . ), and when adjusting for the presence of infection (p = . ). coronary artery dilation at time of diagnosis was found in ( %) patients, similarly distributed according to the presence or absence of concurrent infection ( ( %) versus ( %) patients, respectively; p = . ), and persisted in ( %) patients (in ( %) versus ( %) patients, respectively; p = . ). coronary aneurysms were diagnosed in ( %) patients, without significant difference between patients with and without infection neither ( ( %) versus ( %) patients, p = . ). while the risk of coronary aneurysm was similar between patients with viral versus bacterial infection ( ( %) versus ( %) patients, p = . ), patients with bacterial infection were more likely to have coronary artery dilation ( ( %) versus ( %) patients, p = . ). in this retrospective series, the presence of a concomitant infection was associated with a higher rate of resistance to ivig treatment. patients with concomitant infection were more likely to have persistent fever and slower normalization of inflammatory markers after ivig treatment. the administration of antibiotics did not decrease this risk of resistance to ivig therapy. however, concomitant infection was not associated with an increased risk of coronary artery complications. more precisely, the higher likelihood of repeated ivig treatment when concurrent infection was present is defined as recrudescence of fever hours after the end of ivig infusion based on expert opinion [ ] . our results challenge this definition since fever may be maintained by an infection, either viral or bacterial, that is not likely to respond to ivig. inflammatory markers are often used to help guide the decision about the need for additional treatment in kd. in our series, patients with concurrent infection presented higher crp levels at baseline, as well as hours after initial treatment. this is consistent with a retrospective study on the variability in response to ivig, which showed a higher rate of concomitant infection and higher crp levels in complete non-responders to ivig treatment compared to partial nonresponders and responders [ ] . thus, both the persistence of fever after ivig treatment and elevated inflammatory markers contribute to the increased likelihood of "ivig resistance" in patients with concomitant infections. interestingly, non-response to ivig therapy was associated with the use of antibiotics in a prior single-center retrospective study [ ] . one would have expected patients with concomitant infection, at least bacterial, to have lower rate of ivig resistance if the infection was controlled by antibiotics. both in prior reports and this series [ ] , the use of antibiotic was found to be associated with non-response to ivig treatment, independent of whether or not an infection was confirmed. however, it is not clear if it is just reflecting the underlying infection, or kd that is resistant to initial treatment. another hypothesis would be that the presence of infection triggers an additional inflammatory response at the molecular levels that impact response to treatment and outcomes. il- α and il- β have been shown to be essential in the development of kd [ ] . the il- pathways play a critical part of the host defense against microbial pathogens through activation of toll like receptors [ , ] . moreover, inositol-triphosphate -kinase c (itpkc) has a critical role in mediating nlrp expression and intracellular calcium, which is then responsible for il- β production [ ] . genetic polymorphism in itpkc is associated with higher il- β cytokines and treatment failure [ ] . this raises the question if concomitant infection increases resistance to ivig treatment by increasing levels of il- β cytokines. this could be very important as new treatment strategies are targeting il- blockade in recalcitrant kd. persistence of fever after ivig treatment is a strong risk factor for development of coronary aneurysms [ , ] . in this series, patients with resistance to ivig treatment had a higher risk of developing ca complication. notwithstanding, the rate of ca complication was statistically independent of the presence of infection in our series and in previous series [ ] . thus, the lack of increased risk of ca complication in the setting of persistent fever in patients with kd and concomitant infection argues for infection as the cause of fever as opposed to ivig resistance. it questions whether this "excessive" diagnosis of ivig resistance is well justified when there is a concomitant infection, and if the current definition of treatment resistance should be modified. retreatment with ivig or other immunosuppressive therapies may also have had a protective effect on the development of ca complication. however, the rate of ca complication was similar between patients with and without infection, even after accounting for the number of ivig treatment received. thus, other criteria need to be used to help with decision for retreatment of patients with persistent fever and concomitant infection. coronary artery dimensions may be a more useful marker for the need for additional treatment in this at risk population. in general, patients who are resistant to initial treatment receive a second dose of ivig ( g/ kg) [ ] . notwithstanding, at least % of children remain febrile despite multiple dose of ivig [ ] . these particular patients are at even greater risk of ca complications, and additional therapies are usually administered [ ] , including corticosteroids [ ] [ ] [ ] , anti-tnf alpha agents [ , ] , cyclosporine [ ] , cyclophosphamide [ ] and more recently anakinra (anti-interleukine ) [ , ] . the use of these additional therapies is based on effectiveness in other vasculitis, with no prospective clinical trials to show effectiveness on coronary artery outcome. in patients with concerns for concurrent infection, the benefit of those additional therapies should be carefully balanced against the increased risk of infection. it is important to make this distinction, in order to ) avoid using aggressive immunosuppressive therapies in patients with persistent fever due to uncontrolled infection and ) avoid not treating aggressively a patient with kd that is resistant to initial treatment and at increased risk of serious coronary artery complications. however, this distinction can be clinically very difficult to answer, and caution should be exercise to prevent both cardiac complications and adverse side effects of therapies. there are limitations to this study essentially related to the retrospective methods, and the diagnosis of concomitant infection. on one hand, in the absence of systematic testing for infections there is a potential underestimation of the actual concurrent infection rate, as some infections could not be identified. on the other hand, inclusion of only children with infectious workup at time of initial kd diagnosis could have falsely increased the rate of concurrent infection. however, the rate of concomitant infection in this series was similar to those previously published [ ] . moreover, positive testing for virus and/or throat culture in children could reflect a carrier status rather than actual concurrent infection. however, because infectious workup was performed based on children's symptoms, the positive results most likely represent an actual infection. serologies for different viral infections (cmv, ebv, mycoplasma) can be difficult to interpret in an acute inflammatory setting, with igm cross-reactivity. these infections could not be confirmed by pcr due to the retrospective nature. however, this could only have affected children, and statistical analysis excluding these children did not affect the results. moreover, whereas the aha definition of resistance to ivig treatment is based on persistent fever hours after the end of ivig infusion [ ] , other factors are considered in the decision whether or not to retreat patients, including inflammatory markers and ca dimensions. thus, some patients were classified as resistant to treatment based on the definition, but were not retreated and had favorable evolution. small sample size of sub-groups analysis and secondary aims limit the statistical power, and results should be interpreted in light of these limitations. in this study, patients with concomitant infection had a higher rate of resistance to ivig treatment. patients with concomitant infection had longer duration of fever and slower normalization of inflammatory markers after initial treatment. however, the presence of infection was not associated with an increased risk of ca complication. accordingly, the persistence of fever in kd and the definition of resistance to ivig should be regarded speculatively when concurrent infection is present. decision to intensify treatment in patients with concurrent infection should not only be based on persistent fever. coronary artery dimensions may be a more useful indication for treatment in this patient population. prospective studies are needed to better refine which children truly require additional therapies. conceptualization: audrey dionne, nagib dahdah. diagnosis, treatment, and long-term management of kawasaki disease. a scientific statement for health professionals from the american heart association concurrent respiratory viruses and kawasaki disease influenza 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tlr and il- signaling network at a glance inositol-triphosphate -kinase c mediates inflammasome activation and treatment response in kawasaki disease predictive risk factors for coronary artery abnormalities in kawasaki disease intravenous gamma-globulin treatment and retreatment in kawasaki disease. us/canadian kawasaki syndrome study group treatment options for resistant kawasaki disease coronary artery complication in kawasaki disease and the importance of early intervention: a systematic review and meta-analysis effects of steroid pulse therapy on immunoglobulin-resistant kawasaki disease steroid pulse therapy for children with intravenous immunoglobulin therapy-resistant kawasaki disease: a prospective study infliximab treatment of intravenous immunoglobulin-resistant kawasaki disease infliximab for intravenous immunoglobulin resistance in kawasaki disease: a retrospective study cyclosporin a treatment for kawasaki disease refractory to initial and additional intravenous immunoglobulin initial intravenous gammaglobulin treatment failure in kawasaki disease rational and study design for a phage i/iia trial of anakinra in children with kawasaki disease and early coronary artery abnormalities (the anakd trial) usefulness and safety of anakinra in refractory kawasaki disease complicated by coronary artery aneurysm key: cord- -zpf xjqi authors: walter, james m. title: thrombocytopenia in the intensive care unit date: - - journal: evidence-based critical care doi: . / - - - - _ sha: doc_id: cord_uid: zpf xjqi the evaluation and management of thrombocytopenia is a daily challenge for clinicians in the intensive care unit (icu). thrombocytopenia is incredibly common, present in upwards of % of icu patients. additionally, thrombocytopenia in the critically ill is rarely caused by a single etiology. several causes of thrombocytopenia in the icu including heparin-induced thrombocytopenia (hit) and thrombotic thrombocytopenic purpura demand urgent recognition and intervention. this chapter provides a general overview of thrombocytopenia in the icu and highlights important diagnostic and management considerations for some of the most common etiologies. point during their icu stay [ , , , ] . in general, icu patients who develop thrombocytopenia are sicker than patients with normal platelet counts, with higher illness severity scores, more need for vasoactive infusions, and more organ dysfunction [ , ] . the presence of thrombocytopenia in the critically ill has consistently been associated with poor outcomes. in a multicenter review of over critically ill patients, patients with severe thrombocytopenia (defined as a platelet count < × cells/l) had an adjusted hazard ratio for hospital mortality of . ( % ci, . - . ) compared to patients with normal platelet counts [ ] . the association between thrombocytopenia and mortality has been identified in multiple studies [ , , , ] . regardless of the absolute value, a fall in platelet count by > % from a patient's admission level identifies patients who may be up to times more likely to die during their hospital stay [ , ] . patients whose platelet count fails to recover during their icu course represent a particularly high risk group [ , ] . the presence and severity of thrombocytopenia is included in several validated severity scores including the multiple organ dysfunction score and the sepsis-related organ failure assessment [ , ] . the evaluation of thrombocytopenia in the icu is challenging as thrombocytopenia is both a common problem and rarely due to a single etiology. in a study of over icu patients with either absolute (platelet count < × cells/l) or relative (decrease in platelet count > %) thrombocytopenia who underwent extensive evaluation including bone marrow aspiration, % had or more identifiable etiologies for their thrombocytopenia [ ] . as such, a structured approach to the evaluation and management of thrombocytopenia is essential. a comprehensive review of the myriad causes of thrombocytopenia is beyond the scope of this review. what follows is a simplified approach to the critically ill patient with newonset thrombocytopenia (fig. . ). step : confirm true thrombocytopenia pseudothrombocytopenia occasionally, a low reported platelet count does not represent true thrombocytopenia. exposure to ethylenediaminetetraacetic acid (edta) in blood collection tubes induces a conformational change in the platelet surface protein glycoprotein iib/iiia [ ] . patients may develop igm autoantibodies to these newly exposed giib/iiia epitopes which causes in vitro platelet clumping. large platelet aggregates are not recognized by automated counters, leading to a falsely low reported platelet count. the identification of platelet clumps on a peripheral blood smear and re-drawing blood using heparin or citrate containing collection tubes can help confirm the diagnosis. step : is the patient bleeding? there is a consensus that patients with clinically significant bleeding should be transfused to a platelet count of > × • if platelet clumps seen on smear, repeat blood draw in non-edta collection tube • platelet goal > x cells/l in non-cns bleeding [ , , ] the transfusion threshold should be increased to × cells/l in patients with intracranial bleeding [ ] . thrombotic microangiopathy (tma) is a pathologic term used to describe microvascular thrombosis in the arterioles and capillaries [ ] . the key clinical manifestations of tmas are microangiopathic hemolytic anemia (maha) and thrombocytopenia. tmas are a diverse group of disorders that can be classified broadly as primary (thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, drug-mediated, etc.) or secondary to a systemic disorder (disseminated intravascular coagulation, severe hypertension, hemolysis with elevated liver enzymes and low platelets during pregnancy, etc.) [ ] . while diseases like thrombotic thrombocytopenic purpura (ttp) are uncommon, their prompt recognition is critical as delayed or missed diagnosis can lead to significant patient harm. ttp and disseminated intravascular coagulation (dic) will be reviewed here as examples of primary and secondary tmas respectively. ttp is characterized by a functional deficiency in a vwf cleaving protein termed, "an acronym for a disintegrin and metalloprotease with thrombospondin- -like-domains" (adamts ) [ ] . with a functional deficiency of adamts , large vwf multimers accumulate, triggering platelet adhesion, activation, and the formation of platelet rich microthrombi [ ] . ttp is a rare disease with an incidence of cases per million in the united states [ ] . roughly % of cases of ttp are acquired, caused by the production of igg autoantibodies against adamts [ ] . antibody production can be idiopathic (≅ % of cases) or driven by a variety of conditions including malignancy, human immunodeficiency virus infection, pregnancy, autoimmune disease, medications, and following organ transplantation [ ] . ttp has historically been associated with a clinical pentad of maha, thrombocytopenia, neurologic symptoms, renal impairment, and fever. in the modern era, this constellation of symptoms is rarely seen [ ] . while maha and thrombocytopenia are universally present, upwards of % of patients will be afebrile and % will have either normal mental status or renal function [ ] . presenting symptoms are often non-specific and include nausea and abdominal pain [ ] . initial laboratory testing in patients with suspected ttp should confirm the presence of hemolysis (e.g., an elevated lactate dehydrogenase level, low haptoglobin, elevated indirect bilirubin, and elevated reticulocyte index). in contrast to dic, coagulation parameters are typically normal. a peripheral blood smear should be reviewed to identify fragmented red blood cells called schistocytes -one of the histologic hallmarks of tma ( fig. . ) . renal and cardiac biomarkers should be obtained to screen for organ dysfunction. the role of adamts assays is debated [ , ] . a severely low level (< %) confirms the diagnosis of ttp. however, it is imperative that the decision to initiate therapy is made urgently on the basis of an initial clinical and laboratory evaluation without waiting for adamts activity levels to result [ ] . ttp was previously viewed as an almost universally fatal diagnosis. however, with the rapid initiation of plasma exchange, survival rates now approach % [ , ] . plasma exchange should be continued until platelet counts are > × cells/l for days [ ] . steroids and rituximab may have a role in the treatment of refractory and recurrent disease [ ] . dic is defined by the international society on thrombosis and haemostasis (isth) as "an acquired syndrome characterized by the intravascular activation of coagulation with a loss of localization arising from different causes. it can originate from and cause damage to the microvasculature, which if sufficiently severe, can produce organ dysfunction." [ ] the pathobiology of dic is complex and is driven by dysregulated coagulation and fibrinolysis pathways. a central component of dic is excessive tissue factor (tf) expression and thrombin generation. depending on the clinical scenario, this can be caused by the release of inflammatory cytokines including il- and il- , increased tf expression on mononuclear cells, injury to vascular endothelial cells, and exposure to pro-coagulant molecules (e.g. amniotic fluid) [ ] . activated platelets contribute to excessive thrombin generation and the formation of microvascular clots. concurrently, fragmented red blood cells (schistocytes) are identified by * the major anticoagulant systems (antithrombin, the protein c system, and tf pathway inhibitor) are dampened due to impaired synthesis and increased degradation of the relevant factors [ ] . finally, intrinsic fibrinolysis is impaired in part due to elevated levels of plasminogen activator inhibitor . the propagation of microvascular thrombi leads to organ ischemia and dysfunction-one of the clinical hallmarks of dic. [ ] by far, the most common underlying cause of dic is sepsis. depending on the patient population and definition used, - % of patients with sepsis develop dic [ , , ] . in cases series, sepsis is identified as a risk factor for dic in over % of patients [ ] . other important causes of dic include trauma, obstetric emergencies, malignancies, and liver failure among many others [ ] . dic is consistently identified as a risk factor for increased mortality both in patients with sepsis and in critically ill patients more broadly [ , , ] . the diagnosis of dic should be suspected in any critically ill patient with thrombocytopenia, abnormal coagulation parameters (e.g., a prolonged prothrombin and partial thromboplastin times), maha, and laboratory evidence of fibrinolysis (e.g., an elevated d-dimer and reduced fibrinogen) [ ] . while catastrophic hemorrhage is uncommon, most patients have evidence of bleeding, often at sites of intravenous access [ ] . diagnostic criteria developed by the isth are available to aid diagnosis (table . ) [ ] . in a prospective validation study, a score > had a sensitivity of % and specificity of % for the diagnosis of dic [ ] . a score should be calculated daily both to accurately confirm the diagnosis and to aid prognostication [ ] . the foundation of dic management is treatment of the underlying disorder. there is limited data to guide the administration of blood products in dic. in general, guidelines agree that platelets, fresh-frozen plasma, and a source of fibrinogen should be given to patients who are actively bleeding or those undergoing invasive procedures [ ] . a platelet count > × cells/l, prothrombin (pt) and partial thromboplastin time (aptt) < . times normal, and a fibrinogen level > . g/l are typical targets [ ] . recommendations for the use of heparin vary across guidelines [ ] . in general, heparin is reserved for patients with clinical evidence of thrombosis. a host of targeted interventions aimed at augmenting the major anticoagulant pathways have failed to show benefit in large randomized trials including recombinant tf pathway inhibitors and anti-thrombin iii [ , ] . early studies of activated protein c (apc) in patients with sepsis showed promise, especially in the subset of patients with dic [ , ] . however, the recent prowess-shock trial, which included over patients with septic shock, did not identify any benefit to the use of apc [ ] . a careful review of a patient's medication list is an essential step in the evaluation of thrombocytopenia in the icu. indeed, medications may contribute to over % of new onset thrombocytopenia in the critically ill [ ] . well over drugs have been linked to the development of thrombocytopenia [ ] . a list of notable drugs known to cause thrombocytopenia is provided in table . . drug-induced thrombocytopenia can be grouped into two major categories: drug-induced non-immune adapted from toh [ ] . a score of > is compatible with dic, scoring should be repeated daily. a score < is suggestive of non-overt dic, scoring should be repeated in the next - days data adapted from mitta [ ] , reese [ ] , and the university of oklahoma web resource https://ouhsc.edu/platelets/ditp.html thrombocytopenia and drug-induced immune thrombocytopenia [ ] . drug-induced non-immune thrombocytopenia is far more common and is characterized by dose-dependent suppression of bone marrow platelet production. representative medications include linezolid, chemotherapeutics, and immunosuppressive agents like azathioprine [ ] . drug-induced immune thrombocytopenia occurs through a variety of mechanisms. rarely, medications may induce autoantibodies that destroy host platelets in the absence of the drug. examples include gold salts and procainamide [ ] . more commonly, a drug will induce the production of antibodies that bind to an epitope on a platelet glycoprotein in the presence of the medication. many antibiotics including aztreonam, piperacillin, sulfonamides, and vancomycin likely act through this mechanism [ ] . heparin-induced thrombocytopenia (hit) is a particularly important example and is reviewed in detail below. finally, antiplatelet agents such as eptifibatide used in the treatment of acute coronary syndrome facilitate antibody-mediated destruction of platelets through their binding of glycoprotein iib/iiia [ ] . drug-induced immune-mediated thrombocytopenia typically occurs - days after exposure to the causative medication. thrombocytopenia is often severe with platelet counts falling to < x cells/l. mucocutaneous bleeding and systemic symptoms may be present [ ] . the diagnosis requires a high index of suspicion given the lag between when a drug is started and the subsequent fall in platelet count. identifying drug-dependent platelet reactive antibodies helps confirm the diagnosis; however, testing is time consuming and available at a limited number of centers. treatment is focused on the identification and removal of the causative medication. when the offending drug is removed, platelets typically begin to improve in - days. the role of steroids and intravenous immunoglobulin in the treatment of refractory drug-induced immune thrombocytopenia is controversial [ ] . a helpful website, https://ouhsc.edu/platelets/ditp.html, includes a curated list of all drugs associated with drugdependent platelet-reactive antibodies identified by the bloodcenter of wisconsin dating back to . hit is caused by the production of host igg antibodies against platelet factor (pf )-heparin complexes. the fc domain of these immune complexes binds to the platelet fcγ riia receptor causing platelet aggregation, platelet activation, and eventual thrombin formation [ ] . thrombocytopenia is caused by intravascular platelet consumption. while a diagnosis of hit is frequently considered for thrombocytopenic patients in the icu, it is relatively uncommon. for patients in the medical icu, the incidence may be as low as . % [ ] . the biggest risk factors for hit include the use of unfractionated heparin and cardiac surgery. in these settings, the incidence increases up to % [ ] . hit is unique among the common causes of thrombocytopenia in the critically ill in that it is characterized by thrombosis rather than bleeding. over % of patients with hit develop thrombosis, most commonly in the deep veins of the extremities and pulmonary arteries [ , ] . hit can also cause arterial thrombosis, thrombosis in unusual venous structures (e.g. mesenteric vessels), and myocardial infarction [ ] . hit should be suspected when platelet counts fall by at least % - days after the initiation of heparin therapy [ ] . an important caveat to this pattern is patients who have been previously exposed to heparin. host igg against pf heparin complexes can remain active for up to days. during this window, heparin re-exposure can produce a rapid drop in platelet count within hours [ ] . up to % of cases of hit may present in this manner [ ] . thrombocytopenia in hit is generally less severe than other causes of thrombocytopenia in the icu with levels rarely falling below × cells/l. [ ] up to % of patients exposed to heparin produce igg antibodies against pf -heparin complexes while only a small minority develop hit [ ] . given the costs associated with laboratory testing for hit and the potential risk of transitioning to a non-heparin anticoagulant agent, the diagnosis of hit should only be pursued in patients with an intermediate to high pre-test probability of having the disease [ ] . the most widely used pre-test probability assessment tool for hit is the ts score (table . ) [ ] . a score < is associated with a negative predictive value for hit of > % and obviates the need for further testing [ ] . patients with a score ≥ should undergo step-wise serologic testing. the initial serologic test for patients with an intermediate to high pre-test probability of hit is an enzyme-linked immunoassay (elisa) to detect hit antibodies. these assays are widely available and result in a matter of hours. igg-specific elisas have a sensitivity of % and specificity of % for the diagnosis of hit [ ] . results are typically reported quantitatively as an optical density (od). the higher the od threshold used to identify a positive test, the more likely a positive elisa will predict a positive functional assay. the commonly used od cutoff of . has a sensitivity of > % for hit [ ] . patients with a positive elisa should undergo confirmatory testing with a functional assay -typically a serotonin release assay (sra). a sra evaluates for in vitro activation of platelets in the presence of patient serum and heparin. a positive sra has a specificity of nearly % for the diagnosis of hit [ ] . the cornerstone of management for patients with either an intermediate to high pre-test probability of hit or a confirmed diagnosis is transition to a non-heparin anticoagulant. options include direct thrombin inhibitors (e.g., lepirudin, argatroban, and bivalrudin) and factor xa inhibitors (e.g., danaparoid and fondaparinux) [ ] . there is currently insufficient evidence to support the use of direct oral anticoagulant agents in this setting [ ] . warfarin is contraindicated in patients with hit until the platelet counts rises above x cells/l as warfarin reduces protein c levels and may exacerbate thrombus formation [ ] . platelet transfusions should be avoided if possible and are only recommended for patients who are actively bleeding or those undergoing an invasive procedure associated with a high risk of bleeding [ ] . sepsis is one of the most common causes of thrombocytopenia in the icu and may contribute to a low platelet count in up to % of casas [ ] . the incidence of thrombocytopenia in patients with sepsis varies by illness severity. in a multicenter prospective evaluation of patients with severe sepsis, thrombocytopenia was present in . % of patients [ ] . in patients with septic shock, the incidence of thrombocytopenia approaches % [ , ] . thrombocytopenia has consistently been associated with increased mortality in septic patients [ , , ] . multiple mechanisms cause thrombocytopenia during sepsis. decreased bone marrow production, hemophagocytosis, platelet consumption in microvascular beds, sequestration, and hemodilution may all contribute to varying degrees [ ] . septic patients are at high risk for dic which can further lower platelet counts. additionally, many medications routinely administered to septic patients including antibiotics are associated with thrombocytopenia. based on very low-quality evidence, the surviving sepsis campaign recommends prophylactic platelet transfusions in septic patients with a platelet count < × cells/l and × cells/l for patients at high risk of bleeding. a platelet count > × cells/l is recommended for patients who are actively bleeding or undergoing invasive procedures [ ] . dilutional thrombocytopenia is a well-recognized complication of massive transfusion. the incidence of severe thrombocytopenia (defined as a platelet count < × cells/l) may be as high as % when patients require more than red blood cell containing products [ ] . prompt damage control and transfusion of blood products in a balanced ratio ( : : of red blood cells:plasma:platelets) are important preventative strategies [ ] . step : evaluate support devices support devices used in critically ill patients may lower platelet counts through mechanical shearing. veno-venous extracorporeal membrane oxygenation (vv-ecmo) is increasingly utilized in the management of severe acute respiratory distress syndrome (ards). in a retrospective study of patients placed on vv-ecmo for respiratory failure, % developed thrombocytopenia [ ] . severity of illness and the platelet count at the time of cannulation were the strongest predictors of developing thrombocytopenia. in a large randomized trial of ecmo for severe ards, % of patients randomized to ecmo developed adapted from lo [ ] . a score ≤ suggests a low pre-test probability for hit, a score - an intermediate probability, and a score > a high probability severe thrombocytopenia (defined as a platelet count < × cells/l) vs % in the control arm [ ] . for patients placed on an intra-aortic balloon pump, roughly % will develop thrombocytopenia [ , ] . despite the high incidence of thrombocytopenia in critically ill patients, there is a paucity of data to guide when prophylactic platelet transfusion is indicated. indeed, a recent systematic review did not identify a single high-quality study that investigated the impact of prophylactic platelet transfusions on bleeding rates in critically ill patients [ ] . recommendations on prophylactic platelet transfusions in critically ill patients are largely extrapolated from studies in patients with hematologic malignancies. in a landmark trial of platelet transfusion thresholds in patients with acute myeloid leukemia undergoing induction chemotherapy, a transfusion threshold of × cells/l did not increase the risk of major bleeding and reduced the need for platelet transfusions by . % compared to a threshold of × cells/l. [ ] a recent cochrane review supports the conclusion that a restrictive platelet transfusion threshold is safe in patients with hematologic malignancies [ ] . guidelines by the american society of clinical oncology recommend a prophylactic platelet transfusion threshold of × cells/l in patients with malignancy [ ] . it is unclear, however, if data from patients with malignancies can be reliably generalized to the heterogeneous group of patients cared for in the icu [ ] . some have advocated for an approach which reserves platelet transfusions for critically ill patients with clinical evidence of bleeding (regardless of the actual platelet count) [ ] . however, there is insufficient data to support the safety or efficacy of this practice. there is equally limited evidence to guide the platelet count needed to limit bleeding complications during bedside procedures commonly performed in the icu. the american association of blood banks recommends a platelet threshold of × cells/l for patients undergoing central line insertion and × cells/l for patients undergoing lumbar puncture although both are weak recommendations supported by low-quality evidence [ ] . a recent cochrane review of platelet thresholds for patients undergoing central line insertion was unable to draw any conclusions given the complete lack of data on the subject [ ] . there is mounting evidence that thoracentesis can be safely performed by an experienced operator in thrombocytopenic patients without prophylactic platelet transfusions [ ] . platelet biology and functions: new concepts and clinical perspectives beyond thrombosis: the versatile platelet in critical illness platelets and the immune continuum coagulopathy in critically ill patients: part : platelet disorders thrombocytopenic disorders in critically ill patients time course of platelet counts in critically ill patients thrombocytopenia in medical-surgical critically ill patients: prevalence, incidence, and risk factors thrombocytopenia in critically ill patients receiving thromboprophylaxis: frequency, risk factors, and outcomes the frequency and clinical significance of thrombocytopenia complicating critical illness: a systematic review thrombocytopenia in the intensive care unit thrombocytopenia in patients in the medical intensive care unit: bleeding prevalence, transfusion requirements, and outcome thrombocytopenia and prognosis in intensive care platelet count decline: an early prognostic marker in critically ill patients with prolonged icu stays blunted rise in platelet count in critically ill patients is associated with worse outcome the sofa (sepsis-related organ failure assessment) score to describe organ dysfunction/failure. on behalf of the working group on sepsis-related problems of the european society of intensive care medicine multiple organ dysfunction score: a reliable descriptor of a complex clinical outcome epidemiology and outcome of thrombocytopenic patients in the intensive care unit: results of a prospective multicenter study pseudothrombocytopenia due to platelet clumping: a case report and brief review of the literature bleeding and coagulopathies in critical care guidelines for the use of platelet transfusions evidence-based platelet transfusion guidelines consensus on the standardization of terminology in thrombotic thrombocytopenic purpura and related thrombotic microangiopathies syndromes of thrombotic microangiopathy thrombotic thrombocytopenic purpura thrombotic thrombocytopenic purpura the incidence of thrombotic thrombocytopenic purpurahemolytic uremic syndrome: all patients, idiopathic patients, and patients with severe adamts- deficiency epidemiology and pathophysiology of adulthood-onset thrombotic microangiopathy with severe adamts deficiency (thrombotic thrombocytopenic purpura): a cross-sectional analysis of the french national registry for thrombotic microangiopathy thrombotic thrombocytopenic purpura: diagnostic criteria, clinical features, and long-term outcomes from through guidelines on the diagnosis and management of thrombotic thrombocytopenic purpura and other thrombotic microangiopathies comparison of plasma exchange with plasma infusion in the treatment of thrombotic thrombocytopenic purpura. canadian apheresis study group towards definition, clinical and laboratory criteria, and a scoring system for disseminated intravascular coagulation disseminated intravascular coagulation disseminated intravascular coagulation epidemiology of disseminated intravascular coagulation in sepsis and validation of scoring systems disseminated intravascular coagulation in sepsis trends in the incidence and outcomes of disseminated intravascular coagulation in critically ill patients ( - ): a population-based study the scoring system of the scientific and standardisation committee on disseminated intravascular coagulation of the international society on thrombosis and haemostasis: a -year overview thrombocytopenia in the icu: disseminated intravascular coagulation and thrombotic microangiopathies-what intensivists need to know prospective validation of the international society of thrombosis and haemostasis scoring system for disseminated intravascular coagulation what's new in the diagnostic criteria of disseminated intravascular coagulation? diagnosis and treatment of disseminated intravascular coagulation (dic) according to four dic guidelines efficacy and safety of tifacogin (recombinant tissue factor pathway inhibitor) in severe sepsis: a randomized controlled trial caring for the critically ill patient. high-dose antithrombin iii in severe sepsis: a randomized controlled trial efficacy and safety of recombinant human activated protein c for severe sepsis treatment effects of drotrecogin alfa (activated) in patients with severe sepsis with or without overt disseminated intravascular coagulation drotrecogin alfa (activated) in adults with septic shock identifying drugs that cause acute thrombocytopenia: an analysis using distinct methods how i evaluate and treat thrombocytopenia in the intensive care unit patient drug-induced thrombocytopenia in critically ill patients drug-induced immune thrombocytopenia drug-induced thrombocytopenia: update of clinical and laboratory data heparin-induced thrombocytopenia heparininduced thrombocytopenia in the critically ill patient treatment and prevention of heparin-induced thrombocytopenia: antithrombotic therapy and prevention of thrombosis, th ed: american college of chest physicians evidence-based clinical practice guidelines temporal aspects of heparin-induced thrombocytopenia impact of the patient population on the risk for heparininduced thrombocytopenia argatroban in the management of heparin-induced thrombocytopenia: a multicenter clinical trial evaluation of pretest clinical score ( t's) for the diagnosis of heparin-induced thrombocytopenia in two clinical settings predictive value of the ts scoring system for heparin-induced thrombocytopenia: a systematic review and meta-analysis evaluating thrombocytopenia during heparin therapy a prospective, observational registry of patients with severe sepsis: the canadian sepsis treatment and response registry thrombocytopenia is associated with a dysregulated host response in critically ill sepsis patients is thrombocytopenia an early prognostic marker in septic shock? sepsisassociated thrombocytopenia surviving sepsis campaign: international guidelines for management of sepsis and septic shock: complications of massive transfusion damage control resuscitation thrombocytopenia and extracorporeal membrane oxygenation in adults with acute respiratory failure: a cohort study extracorporeal membrane oxygenation for severe acute respiratory distress syndrome thrombocytopenia in patients treated with heparin, combination antiplatelet therapy, and intra-aortic balloon pump counterpulsation clinical implications of thrombocytopenia among patients undergoing intra-aortic balloon pump counterpulsation in the coronary care unit platelet transfusions for critically ill patients with thrombocytopenia the threshold for prophylactic platelet transfusions in adults with acute myeloid leukemia gruppo italiano malattie ematologiche maligne dell'adulto comparison of different platelet count thresholds to guide administration of prophylactic platelet transfusion for preventing bleeding in people with haematological disorders after myelosuppressive chemotherapy or stem cell transplantation platelet transfusion for patients with cancer: american society of clinical oncology clinical practice guideline update fresh frozen plasma and platelet transfusion for nonbleeding patients in the intensive care unit: benefit or harm? platelet transfusion: a clinical practice guideline from the aabb comparison of different platelet transfusion thresholds prior to insertion of central lines in patients with thrombocytopenia complications of thoracentesis: incidence, risk factors, and strategies for prevention key: cord- -yddcme z authors: khalil, m. title: herz und gefäße date: journal: p&#x e ;diatrie doi: . / - - - - _ sha: doc_id: cord_uid: yddcme z herzfehler gehören zu den häufigsten angeborenen fehlbildungen. heute erleben fast % dieser kinder das erwachsenenalter. dennoch sind angeborene herzfehler weiterhin mit einer hohen morbidität und mortalität vergesellschaftet, insbesondere wenn ein kritischer herzfehler vorliegt. dem pädiater obliegt nicht nur die rechtzeitige erkennung eines herzfehlers, sondern auch im zunehmenden maße die mitbetreuung dieser patienten im prä-und postoperativen stadium. das verständnis für die zugrundeliegende hämodynamik der jeweiligen herzfehler sowie die physiologischen veränderungen der kreislaufverhältnisse im laufe des lebens sind wichtig für die diagnosestellung und die klinische verlaufsbeurteilung. daher wird im folgenden kapitel besonderen wert auf die hämodynamischen zusammenhänge am beispiel ausgewählter herzfehler gelegt. neben primär azyanotischen und zyanotischen herzfehlern gehören entzündliche herzerkrankungen sowie herzrhythmusstörungen zum spektrum der kinderkardiologie. diese felder werden in grundzügen beschrieben und sollen dem kliniker eine hilfestellung für das diagnostische und therapeutische vorgehen geben. . angeborene herzfehler . . epidemiologie und Ätiologie j epidemiologie die prävalenz angeborener herzfehlern beträgt etwa , % aller lebendgeborenen und gehört damit zu den häufigsten angeborenen fehlbildungen (. tab. . ). die prognose der betroffenen kinder hat sich in den letzten jahren kontinuierlich verbessert. so erle ben heute fast % der kinder das erwachsenenalter. viele herzfeh ler können heute pränatal diagnostiziert werden. dies ermöglicht neben einer optimalen medizinischen versorgung des neugebore nen auch den betroffenen eltern sich auf die situation einzustellen. nichtsdestotrotz gehören angeborene herzfehler weiterhin zu den fehlbildungen, die mit einer hohen morbidität und mortalität vergesellschaftet sind. in deutschland ist die pränatale detektions rate niedrig und sehr heterogen verteilt, da sie im hohen maße von der erfahrung des untersuchers abhängig ist. umso wichtiger ist die sorgfältige durchführung der vorsorgeuntersuchungen durch den pädiater, um mögliche angeborene herzfehler frühzeitig zu detektieren. j Ätiologie die Ätiologie angeborener herzfehler ist komplex und noch nicht vollständig aufgeklärt. der größte anteil tritt als isolierte fehl bildung auf. in den meisten fällen geht man von einem multi faktoriellen geschehen aus. insbesondere die untersuchung von familienmitgliedern betroffener patienten lässt den schluss auf eine polygenetische oder multifaktorielle genese zu. das wiederho lungsrisiko für ein erneutes auftreten angeborener herzfehler be trägt für verwandte ersten grads - %. dies korrespondiert mit theoretischen mathematischen wahrscheinlichkeitsmodellen für multifaktorielle mechanismen. multifaktoriell bedeutet hier, dass kardiale fehlbildungen durch die wechselwirkung einer oder meh rerer gene mit umweltfaktoren verursacht werden. ungefähr - % der angeborenen herzfehler sind mit extra kardialen anomalien wie chromosomenanomalien, genetischen punktmutationen oder komplexen fehlbildungssyndromen assozi iert (. tab. . ). zu den chromosomenanomalien, die durch eine karyotypisierung detektiert werden, gehören z. b. trisomie (downsyndrom), trisomie (edwardssyndrom) und monosomie x (ullrich turnersyndrom). jede dieser chromosomenanomalien ist mit einer häufung eines spezifischen herzfehlers assoziiert, so z. b. tri somie mit avsd oder ullrichturnersyndrom mit der aortenis thmusstenose. die häufigeren chromosomalen syndrome sind auf mikrodeletionen zurückzuführen und können mittels insitu hybridisierung (fish) oder arraybasierter "comparative genom hybridisierung" (cgharray) dargestellt werden. diese sind v. a. die mikrodeletion q . (di georgesyndrome, syn. shprintzen syndrom, velokardiofaziales syndrom) mit conotrunkalen herzfehl bildungen (fallottetralogie, "double outlet right ventricle", truncus arteriosus, aortenbogenanomalien) und das williamsbeuren syndrom (v. a. supravalvuläre aortenstenosen und periphere pul monalstenosen). monogene syndrome, die mit herzfehlern assoziiert sind, sind u. a. rasopathien (z. b. noonansyndrom,) und jagmutationen (z. b. alagillesyndrom). zu den komplexen fehlbildungssyndromen gehören die vacterlassoziation, chargeassoziation und das goldenharsyndrom (. tab. . ). ein kleiner teil der angeborenen herzfehler kann auf teratogene noxen wie infektionen (z. b. röteln), stoffwechselerkrankungen symptome angeborener herzfehlern können unterschiedlich ausge prägt sein. leitsymptome sind neben herzgeräuschen, die typi schen merkmale der herzinsuffizienz und/oder zyanose. die erstuntersuchung eines kindes mit verdacht auf das vorliegen eines angeborenen herzfehlers hat zum ziel diesen verdacht auszuschlie ßen oder eine möglichst genaue verdachtsdiagnose zu stellen. wie bei jeder klinischen evaluation sollte man auch hier so systematisch wie möglich vorgehen (. tab. . ). herzfehler können in primär azyanotische und zyanotische herzfehler eingeteilt werden. eine zyanose kann bei der körperli chen untersuchung festgestellt werden und sollte durch die durch führung einer pulsoxymetrie bestätigt werden, insbesondere da eine gering ausgeprägte zyanose bei einer inspektion nicht erkennbar sein muss. sowohl azyanotische als auch zyanotische vitien können weiter unterteilt werden in vitien mit verminderter bzw. normaler oder vermehrter lungenperfusion. eine röntgenaufnahme des thorax zeigt dabei entsprechend entweder eine verminderte, nor male oder vermehrte lungengefäßzeichnung (. tab. . ). die auskultation mit der charakterisierung der herztöne bzw. herzgeräu sche sowie deren lokalisation grenzt die differenzialdiagnosen weiter ein. ein ekg kann eine rechts, links oder biventrikuläre hypertrophie aufzeigen oder durch einen pathologischen lagetyp hinweise auf spezifische herzfehler geben. schließlich wird die de finitive diagnose durch eine bildgebung, meistens reicht eine echo kardiographie aus, gestellt. azyanotische herzfehler mit druckbelastung des herzens sind am häufigsten durch eine aortenklappenstenose, pulmonalklappen stenose oder aortenisthmusstenose bedingt. seltener sind mitral, trikuspidalklappenstenose oder eine hypertrophe obstruktive kar diomyopathie. solange es sich nicht um kritische stenosen handelt sind die patienten relativ symptomarm. bei kritischen stenosen ist die obstruktion so stark ausgeprägt, dass eine ductusabhängigkeit be steht. die kritischen stenosen manifestieren sich somit bereits im neugeborenalter und werden im abschn. . . weiter beschrieben. ein herzgeräusch kann das einzige symptom sein. das herz reagiert auf eine stenose mit einer konzentrischen hypertrophie und später mit einer dilatation, um die vermehrte druckbelastung zu bewältigen und das herzzeitvolumen (hzv) aufrecht zu erhalten. das hzv kann dann jedoch nur noch begrenzt gesteigert werden. besteht eine hochgradige obstruktion kann das erste symptom eine synkope oder plötzlicher herztod während körperlicher belastung sein. > jede synkope unter körperlicher belastung muss schnellstmöglich auf ihre ursache hin abgeklärt werden. j zyanotische herzfehler zyanotische herzfehler können mit verminderter lungenperfusion oder vermehrter lungenperfusion einhergehen. dies ist insofern von bedeutung, da eine zyanose mit vermehrter lungenperfusion weniger ausgeprägt ist und daher klinisch übersehen werden kann. bei zyanotischen herzfehlern mit verminderter lungenperfusion liegt eine obstruktion des pulmonalen blutflusses auf pulmo naler, ventrikulärer oder atrialer ebene vor. zusätzlich besteht eine shuntverbindung (foramen ovale/asd, vsd, pda), die o armes blut an der lunge vorbei in den systemkreislauf leitet. typische zyanotische herzfehler sind die fallottetralogie, trikuspidalatresie, vitien mit singulärem ventrikel und die pulmonalstenose. säuglinge mit diesen vitien haben keine herzinsuffizienzzeichen und daher in der regel keine trinkschwierigkeiten oder gedeihstörung. der grad der zyanose ist dabei abhängig von der ausprägung der ob struktion. ist die obstruktion so ausgeprägt, dass die pulmonale perfusion nur durch einen offenen ductus arteriosus gewährleistet wird, han delt es sich um einen kritischen herzfehler ( abschn. . . ). bei zyanotischen herzfehler mit vermehrter lungenperfusion liegt keine obstruktion des pulmonalen blutflusses vor. die zyanose entsteht durch eine vollständige durchmischung von venösen und arteriellen blut oder fehlstellungen der großen gefäße (aorta, pul monalarterie). am häufigsten ist dies durch eine transposition der großen gefäße bedingt. eine vollständige mischung von arteriellen und venösen blut findet sich ebenfalls bei komplexen herzfehlern mit univentrikulären herzen, bei der totalen lungenvenenfehlmün dung oder dem truncus arteriosus. liegt keine pulmonale obstruk tion vor, zeichnen sich diese herzfehler durch ein klinisches misch bild aus zyanose und herzinsuffizienz aus. aufgrund der vermehr ten lungenperfusion kann die zyanose gering ausgeprägt sein und klinisch wenig imponieren. herzinsuffizienz ist ein klinisches symptom und kein eigenständiges krankheitsbild. eine herzinsuffizienz liegt vor, wenn das herz nicht in der lage ist ein ausreichendes herzzeitvolumen (hzv) zu er bringen, um den metabolischen bedürfnissen des körpers gerecht zu werden. das hzv ist das produkt aus schlagvolumen und herzfrequenz (hzv = sv×hf). diese parameter sind wiederum abhängig von: der vorlast, der myokardialen kontraktilität, der nachlast, einem synchronen rhythmus, der interventrikulären interaktion, der atrioventrikulären kopplung. eine herzinsuffizienz kann folge einer angeborenen oder erworbe nen herzerkrankung sein, die zu einer volumen oder druckbelas tung des herzens führt. ein kritischer herzfehler ist postnatal der häufigste grund für eine akute kardiale dekompensation. etwa % aller herzfehler sind kri tische herzfehler, die bereits für das neugeborene zu einem lebens bedrohlichen zustand führen können. um zu überleben benötigen kinder mit diesen herzfehlern einen herzchirurgischen oder kathe terinterventionellen eingriff. die sterblichkeitsrate liegt auch heute noch bei bis zu %. kritische herzfehler werden trotz verbesserter pränataldiagnos tik und postnataler klinischer untersuchungen immer noch unzu reichend detektiert. insbesondere die sensitivität der klinischen untersuchung in den ersten lebenstagen beträgt aufgrund eines möglichen symptomfreien intervalls ("diagnostische lücke") durch die verzögerte umstellung der fetalen auf die neonatale kreislauf physiologie nur ca. %. die meisten neugeborenen zeigen eine unterschiedlich ausgeprägte hypoxämie, die jedoch zu diesem zeit punkt häufig nicht mit einer klinisch erkennbaren zyanose einher geht. mittels pulsoxymetriescreening kann die postnatale "diagnos tische lücke" verkleinert werden, insbesondere wenn ein rechts j prognose und verlauf die spontanverschlussrate ist bei kleinen muskulären und perimem branösen defekten hoch. kleine muskuläre defekte verschließen sich häufig in den ersten lebensjahren. perimembranöse defekte ver schließen sich zu %. wichtig ist zu wissen, dass vsd im verlauf nicht größer werden. es besteht also eine relative verkleinerungstendenz. infundibuläre inlet und malalignmentvsd verschließen sich nicht spontan. bei nicht korrigierten, hämodynamisch relevanten isolierten vsd entwickelt sich eine fixierte pulmonale hypertonie und somit eine eisenmengerreaktion. um festzustellen, ob diese pulmonale hypertension irreversibel ist, kann die pulmonale widerstandserhö hung im herzkatheter mittels sauerstoffnobeatmung und prosta glandininfusion untersucht werden. besteht hier eine reversibilität kann der vsd auch in höherem alter noch verschlossen werden, ansonsten ist eine korrektur nicht mehr möglich. der vorhofseptumdefekt (asd) ist einer der häufigsten ange borenen herzfehler und hat als isolierter defekt einen anteil von ca. - % aller angeborenen herzfehler. mädchen sind häufiger be troffen als jungen. ist ein katheterinterventioneller verschluss nicht möglich, kann der asd mittels direktnaht oder durch einen patch operativ ver schlossen werden. liegen keine anderen zwingenden gründe vor, werden asd in der regel im vorschulalter verschlossen. partielle lungenvenenfehlmündungen treten selten isoliert auf, son dern häufig in kombination mit anderen kardialen anomalien. bei der partiellen lungenvenenfehlmündung drainiert eine oder ein teil der lungenvenen (aber nicht alle) anstatt in den linken vor hof in den rechten vorhof. die hämodynamik der partiellen lun genvenenfehlmündung entspricht der eines vorhofseptumdefekts ( abschn. . . . ). o reiches blut gelangt in den rechten vorhof. es ergibt sich eine volumenbelastung und dilatation der rechten herzhöhlen. sinusvenosusdefekte gehen anatomisch fast immer mit einer partiellen lungenvenenfehlmündung einher. der avsd hat einen anteil von ca. - % an allen angeborenen herzfehlern, und kommt gehäuft bei patienten mit trisomie vor ( - %). j prognose bestehen keine assoziierten fehlbildungen und besteht keine lun genvenenobstruktion, ist die körperliche belastbarkeit und die pro gnose sehr gut. der postoperative verlauf kann durch neu oder wie der auftretende pulmonalvenöse obstruktionen kompliziert wer den, und damit auf lange sicht die prognose verschlechtern. der truncus arteriosus communis (tac) hat nur einen anteil von etwa % aller angeborenen herzfehler und ist häufig ( %) mit einer mikrodeletion q assoziiert. j morphologie und hämodynamik beim tac ist die trennung zwischen aorta und pulmonalarterie unvollständig geblieben, d. h. es entspringt nur ein großes arterielles gefäß mit einer taschenklappe (truncusklappe) aus beiden ventri keln. zusätzlich liegt praktisch immer ein großer malalignment vsd vor, der truncus überreitet sozusagen den ventrikelseptumde fekt, und versorgt sowohl den systemischen als auch den pulmonalen kreislauf (. abb. . ). die truncusklappe ist häufig dysmorph verdickt, weist eine variable anzahl von taschen auf und ist häufig insuffizient. ein rechter aortenbogen ist häufig. es gibt unterschiedliche anatomische variationen des tac. am häufigsten entspringen aorta und pulmonalarterie aus einem ge meinsamen gefäß oder stamm. die pulmonalarterie zweigt sich dann kurz nach ihrem ursprung in ihre Äste auf. aufgrund des großen ventrikelseptumdefekts besteht ein druckangleich zwischen beiden ventrikeln. es kommt zu einer mischung von system und pulmonalvenösem blut. dieses misch man unterscheidet zwischen supraventrikulären und ventrikulären extrasystolen. supraventrikuläre extrasystolen nehmen ihren aus gang oberhalb des hisbündels, während ventrikuläre extrasystolen im oder unterhalb des hisbündels entspringen. supraventrikuläre extrasystolen sind definiert durch eine vor zeitige pwelle und einem schmalen kammerkomplex (vorwiegend) sowie eine postextrasystolische pause, die nicht vollständig kompen siert ist. einzelne isoliert auftretende supraventrikuläre extrasysto len erfordern in der regel keine therapie. sie sind u. a. gehäuft im neugeborenenalter zu beobachten. der befund kann nach - wo chen mittels ekg kontrolliert werden, evtl. sollten die elektrolyte bestimmt werden. finden sich gehäuft auftretende supraventriku läre extrasystolen in form von couplets und triplets, sollte eine weitere kinderkardiologische abklärung erfolgen. sog. blockierte supraventrikuläre extrasystolen treten fast aus schließlich im neugeborenenalter auf. die extrasystole trifft auf eine noch refraktäre kammer und wird daher nicht übergeleitet. dies führt zu einer deutlichen reduzierung der kammerfrequenz. eine therapie ist in der regel nicht erforderlich, die extrasystolen ver schwinden meistens im verlauf. ventrikuläre extrasystolen sind definiert durch den vorzeitigen einfall einer kammererregung. die kammerkomplexe sind meist verbreitert und deformiert. eine pwelle ist in der regel nicht erkennbar. bei einer erstmalig auftretenden ventrikulären extrasys tolie, auch im neugeborenenalter, sollten eine strukturelle herz erkrankung und eine myokarditis ausgeschlossen werden, elektro lyte sollten kontrolliert werden und die einnahme von arrhythmo genen medikamenten ausgeschlossen werden. kommt es unter körperlicher belastung zum verschwinden der isoliert auftretenden ves, handelt sich meist um eine benigne form der extrasystolie, die keiner weiteren therapie bedarf. liegen polymorphe ves, gehäuft auftretende ves in form von couplets oder salven oder kommt zu einem vermehrten auftreten der ves unter belastung oder in der erholungsphase nach belastung auf, muss eine umgehende weitere abklärung erfolgen. in diesen fällen besteht das risiko, dass die ves eine ventrikuläre tachykardie herzfehler mit links-rechts-shunt - azyanotische herzfehler ohne shunt - zyanotische herzfehler - zyanotische herzfehler mit verminderter lungenperfusion - zyanotische herzfehler mit vermehrter lungendurchblutung - entzündliche herzerkrankungen - dabei wird die akzessorische leitungsbahn elektrophysiolo gisch charakterisiert. besteht eine potenziell lebensbedrohliche gefährdung durch die eigenschaften dieser bahn av-knoten-reentry-tachykardie (avnrt) die avknotenreen trytachykardie ist die zweithäufigste tachykardie im kindesalter. es liegen zwei separate leitungsbahnen innerhalb des avknotens vor. eine von diesen bahnen hat eine schnelle Überleitungs geschwindigkeit, während die andere deutlich langsamer leitet. die avnrt ist im ersten oder zweiten lebensjahr selten und tritt ebenso wie die avrt paroxysmal auf. im ekg lässt sich während der typischen avnrt kein retrogrades p abgrenzen. aufgrund der leitungseigenschaften besteht kein erhöhtes risiko für einen plötzlichen herztod. eine indikation zur behandlung ergibt sich aus der rezidivhäufigkeit, den symptomen während der tachy key: cord- -mtl x gz authors: leung, t. i.; pendharkar, s. s.; chen, c.-y. a.; snyder, r. title: physician suicide: a scoping review to highlight opportunities for prevention date: - - journal: nan doi: . / sha: doc_id: cord_uid: mtl x gz objective: the aim of this scoping review is to map the current landscape of published research and perspectives on physician suicide. findings could serve as a roadmap for further investigations and potentially inform efforts to prevent physician suicide. methods: ovid medline, psycinfo, and scopus were searched for english-language publications from august , through april , . inclusion criteria were a primary outcome or thesis focused on suicide (including suicide completion, attempts, and thoughts or ideation) among medical students, postgraduate trainees, or attending physicians. opinion articles were included. studies that were non-english, or those that only mentioned physician burnout, mental health or substance use disorders were excluded. data extraction was performed by two authors. results: the search yielded , articles, of which articles passed to the full-text review round. the oldest article was an editorial from ; ( . %) articles were published from to present. authors originated from countries and ( . %) were opinion articles. most discussed were suicide risk factors and culture of practice issues, while least discussed themes included public health and postvention. conclusions: consistency and reliability of data and information about physician suicides could be improved. data limitations partly contribute to these issues. also, various suicide risk factors for physicians have been explored, and several remain poorly understood. based on this scoping review, a public health approach, including surveillance and early warning systems, investigations of sentinel cases, and postvention may be impactful next steps in preventing physician deaths by suicide. physician suicide is a significant problem for the medical community and general public and is poorly understood, suggesting that important knowledge and implementation gaps towards prevention remain. to address this gap, this literature review aims to describe the state of current knowledge and research on physician suicidal behaviors among medical students, postgraduate trainees, including residents and fellows, and physicians. suicide is estimated to occur at a higher rate among physicians than the general population and perhaps even other professions ( , ), however, estimates by profession vary ( ). several medical organizations have begun launching various initiatives to address physician well-being, yet efforts to address physician suicide remain organization-or institution-specific. the aim of this scoping literature review is to map the current landscape of published research and perspectives on physician suicide. findings could serve as a roadmap for informing further study, evidence-based policy, and interventions to prevent physician suicide. differs from other reviews such as a systematic review which gathers and assesses the quality of quantitative evidence to report on the effectiveness of a particular intervention in achieving a certain outcome ( ). the research question was broadly designed to gather and analyze articles that mention physician suicide. no date range for the search was specified. the initial searches were performed on august , in ovid medline, and october , in ovid psycinfo. authors contributed seed articles that they had previously identified as relevant to physician suicide ( , - ), which were analyzed by the medical librarian co-author. search terms and databases were then selected and tested based on this analysis. the search strategy also underwent a peer review process with two additional medical librarians. an updated search was run again on april , in ovid medline, psycinfo, and scopus. detailed search terms are available in appendix . inclusion criteria were english-language papers with a primary outcome, measure, or thesis focused on death by suicide or suicidal behaviors among physicians, including suicide attempts, and suicidal thoughts and ideation. physicians included medical students, postgraduate trainees (residents and fellows), and physicians at any career stage. opinion articles were also included, if they otherwise met inclusion criteria; these included perspectives, letters to the editor or their replies, essays, and viewpoints with a focus specifically on physician suicide. exclusion criteria were non-english publications and those only pertaining to physician burnout, mental health, substance use disorders, or other media, such as newspaper and magazine articles. the aim was to inductively identify key themes across the published literature included in this scoping review. during each subsequent round of review, tags could be added to articles. after all articles were reviewed and tagged by two authors (tl,sp,cyac), one of the authors (tl) re- reviewed all articles to add tags until a point of saturation was reached and no further topic tags could be added. tags were then condensed into a core set of themes. these themes were assembled into a framework based on their frequency of occurrence, resulting in a map of the most published themes about physician suicide. findings are summarized in narrative form. results the articles that met inclusion criteria ( figure ) covered a broad range of publication types over time and from countries worldwide. the earliest publications were editorials ( primarily sought to estimate epidemiology of suicidal behaviors among physician populations. in other words, death by suicide was most often studied among attending physicians because of availability of data on occupation and death, particularly in vital statistics or death certificates. however, the tools used to perform such estimates varied. deaths by suicide were most frequently estimated based on th or th revisions of the international classification of diseases (icd- or icd- ) codes on death certificates. other commonly used sources of data included membership masterfiles from physician organizations or associations ( ), published obituaries, and charts from medical records or forensic reports ( records, and any other information available, such as suicide notes ( ). such an approach can elucidate potential contributors to an individual's death, providing detailed contextual information, all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / doi: medrxiv preprint their life circumstances, and how they managed such circumstances up until their death. however, psychological autopsies are not systematically applied in the setting of deaths by suicide. case reports also may describe a psychological profile, in addition to medical and clinical aspects of the case ( , , ), but this was also infrequently published. in , gold et al examined data from the u.s. national violent death reporting system (nvdrs), which is a combination of information and data from death certificates, coroner and medical examiner reports, toxicology reports, and law enforcement reports ( ), the only study to triangulate multiple data sources to estimate suicide incidence. the wide variety of data sources and methods of estimation suggest that there is no standard. suicide clusters are suicides that occur near each other, typically with respect to time and geography. suicide contagion refers to spread of information about a suicide via media and other channels, which can increase suicide risk in a community. only three studies acknowledged these topics. two opinion papers described suicide clusters that occurred in a short time span and in a focused geographic region, one in winnipeg, canada ( ) and another cited suicides in australia that had been reported in news media ( ). both articles focused on intense or distressing working conditions as important factors in the suicide deaths but did not elaborate further on suicide clustering. in fact, the canadian article, along with one other case series of physicians who died by suicide while on probation in oregon state ( ), used the term "epidemic" rather than "clusters." nonetheless, all three papers referred to the concepts of suicide clusters and suggest contagion, even though they do not use these terms explicitly. these were articles were tagged with the public health theme. suicide clusters also appeared to follow physician subpopulations, although numbers were small in such case series and opinion articles. jewish dermatologists during the holocaust: some emigrated, some died during nazi rule, and others survived ( ). three of these dermatologists died by suicide, which was "very common among the doctors [under the nazi ordeal], especially those physicians of the older generation" ( ). another unexpected analysis described the confusing ethical circumstances of physicians as terrorists ( ) . overall, suicidal ideation was the second most studied thesis among articles included in this literature review, especially suicidal ideation among medical students. to assess suicidal ideation and attempts, surveys were most commonly used; overall, cross-sectional survey studies were performed (table ). validated surveys are available to assess suicidal ideation ( suicide attempts in general were poorly studied in any physician subpopulation, but suicidal behaviors among postgraduate trainees were the least studied of all, with resident physicians' behaviors studied more than fellows. among articles reviewed, only two validated tools, paykel's instrument and the beck hopelessness scale, were identified that were specifically designed to assess all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / doi: medrxiv preprint suicide attempts; otherwise, investigators developed survey items to assess respondents' self-reported attempts. physicians' risk factors for suicidal behaviors, especially mental health disorders, are among the most common topics of study. gold et al examined nvdrs data and identified certain risks unique to physicians compared to non-physicians: physicians were more likely to have a job problem preceding death by suicide, and a greater likelihood of known mental health disorders, yet no accompanying increased likelihood of antidepressant therapy ( ). additional risk factors potentially include professional setbacks prior to suicide death, such as facing complaints ( , ), feeling overloaded, working long hours, or being unable to cope with job responsibilities ( ), or experiencing disability as a result of medical illness ( , , ). related to these conditions, physicians are also at high risk of burnout, which has been found to be associated with suicidal ideation in u.s. medical students ( ) and dutch residents ( ), although no direct relationship between burnout and death by suicide has been established. to assess these risk factors, a variety of validated questionnaires were used to inquire about general health, burnout and stress, mental health (e.g. depression, anxiety or insomnia), substance use including alcohol use, and other measures of career, work, personality, and other life experiences (table ) . additionally, investigator-developed survey questions were also used, for example, to assess attitudes towards suicidal behaviors of a peer. because the earliest publications about physician suicide suggested an increased incidence compared to the general population, certain theories have been applied to attempt to explain the increased suicide risk among physicians. or provocative stimuli, including events triggering second victimization, such as patients' poor outcomes, death, and suffering. this can then lead to desensitization when exposed to death in general. role strain describes physician risks as a result of their direct work environment or professional norms; a mismatch between social and institutional norms and the physician's roles can manifest as an unrealistic expectation of perfect function at a maximum level of competence ( ). professional self-image and identity, along with self-stigma, are also considered relevant mediating themes ( , , ) . early studies of suicide rates by specialty suggested that psychiatry and anesthesiology had the highest suicide rates ( ). indeed, these two specialties occurred most frequently in this scoping literature review, followed by surgery or general surgery. however, the findings remain mixed due to data limitations in ascertaining suicide rates as noted previously ( - ). furthermore, no explanations for the association have been offered other than a proffered but unstudied hypothesis that some medical specialties may possess more acquired capability than others ( , ). the access and knowledge hypothesis is a commonly discussed risk factor for suicide death among physicians. physicians acquire specialized medical knowledge of the human body and have access to the means (e.g. prescription drugs) that can cause lethal self-harm. observational studies of means of all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / doi: medrxiv preprint suicide death suggest that firearm, prescription drug overdose, and hanging are among the most common methods used by physicians ( , , ), although this varies by country. psychological autopsies are consistent with the access and knowledge hypothesis. one set of psychological autopsies in the united kingdom examined physician suicides, finding that of the physicians died by self-poisoning and by self-injury (other than poisoning), as one physician used both methods ( ). in a finnish psychological autopsy of physician suicides, only had previous suicide attempts, suggesting that physicians may be more likely to cause self-harm leading to death by suicide; in other words, physicians who die by suicide may not have had any prior suicide attempt, which is risk factor in the general population ( ). self-treatment or self-prescription is also a concerning contributor due to specialized access to controlled substances, as "medical doctors exploit their profession for purpose of self-treatment" ( ). personality and life experience prior to medical training might also contribute to physician suicide risk, although are poorly studied. in a prospective study of norwegian medical students, the control personality trait, or the degree of compulsiveness, was independently linked to suicidal ideation; and more neuroticism, or the vulnerability personality trait, as a medical student independently predicted more serious suicidal ideations and planning in the first two postgraduate years ( , , ). reality weakness, more commonly associated with serious psychiatric pathology, predicted a transition from suicide ideation to planning ( ). neuroticism and tendency towards perfection may even be traits sought in potential medical trainees. one editorial speculates about the influence of adverse childhood experiences on risk for physician suicide ( ). one south korean study found a more than threefold risk of lifetime suicidal ideation, planning, and attempts among medical students who experienced emotional abuse early in life, characterized by "a continuously cold and uncaring parental attitude" ( ). in china, medical students were surveyed about parental relationships and parenting communication styles, hypothesizing that this could be a highly influential aspect of the student's character ( ). the study found that for chinese medical students, a good relationship with parents was statistically significantly associated with less suicidal ideation, plans and attempts. some studies sought to identify risk factors unique to their cultural context. researchers in the united arab emirates collaborated with eskin ( ) to again assess medical students' religiosity and their attitudes towards suicide ( ). in this study, investigators suggested that the islamic faith may serve as a protective factor against suicidal thoughts and death by suicide among their medical students. in pakistan, a study of suicidal ideation among medical students included religion as a demographic characteristic but did not otherwise include religion in further analyses ( ). among opinion articles, certain conceptualizations of suicide also varied. for example, in one commentary from japan, two concepts directly link overwork with death: karoshi (death due to overwork) and karojisatsu (suicide due to overwork) are considered causes of death in the japanese culture ( ) but nowhere else in the world. early studies in the s and earlier explicitly excluded female and minority physicians ( , , ). results published in from the women physicians' health study, which surveyed a sample of women physicians from the american medical association masterfile ( ), described prior studies that reported an odds ratio as high as for women physician suicides compared to other categories of women but that such studies were based on small numbers of suicide deaths. in , schernhammer performed a systematic review and meta-analysis, concluding that the suicide rate ratio for women physicians was . compared to the general women population, and . for male physicians all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / doi: medrxiv preprint gender difference in terms of rates of suicidal ideation among medical students ( ). little data is known on suicide among physicians of gender minorities, as only one perspective piece written by a nurse who wrote about her transgender child who died by suicide ( ) . health initiatives for surveillance and prevention. this imbalance quickly identifies potential areas for further work. themes such as suicide as a public health issue, postvention, and legal issues were far less studied than others. in the second level of the framework are epidemiologic and demographic focuses for study, which were previously described in section . . along with these subjects are a relatively lesser focus on substance use, compared to other risk factors such as mental health, and on developing educational curricula to address knowledge gaps about suicide and its prevention. wellness is an increasingly popular topic for publication on this level of the framework. in the third level of the framework are prevention approaches. thirteen articles described interventions with a primary aim of preventing physician suicides. frequently, programs were implemented as an organizational response to a physician suicide. five interventions implemented the afsp's interactive screening program for suicide prevention ( , ) as a component of a larger initiative to promote physician well-being; three of the five came from the same institution ( - ). only one of was a randomized controlled trial of a web-based screening program ( ). treatments and responses to physician suicide were discussed as frequently as prevention; treatments and responses differed because treatments tended to focus on individual physician treatment for suicidal behaviors or related mental health disorders, and responses tended to focus on small group, physician health program, or medical board responses to individual circumstances. for example, young et al described the difficult grieving process for a healthcare team after the death a physician ( ). patients were told that the physician who had died "was not available," trust between staff and administrators eroded, morale decreased, and grieving, guilt and other negative consequences remained unaddressed until six weeks after the physicians' death ( ). the process of facilitating a normal grieving process after a suicide death has occurred requires active planning, called postvention. in another study by kaltreider et al, medical students were interviewed after the death of a classmate by suicide ( ). this qualitative study found that students experienced intense, all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / doi: medrxiv preprint intrusive, and persistent thoughts and emotions relating to their classmate's death, sometimes lasting for months. proximity to the site of the death, prior exposure to another suicide previously, or familiarity with depression in themselves or family seemed to increase these effects. legal and policy issues are increasingly recognized as affecting physician health and well-being, and appear in the third and fourth levels of figure . physicians may avoid seeking mental health treatment, avoid self-report, and self-treat mental health and other health issues ( , ) , out of fear of reputational damage or concern about perceived weakness or impairment ( ). licensure application questions could be improved ( ) and compassionate, confidential assistance programs, such as one implemented in the united kingdom in , could offer bespoke services to physicians who might be considered at risk of suicide ( ). in , the uk's general medical council was recommended to fund a pilot program that would include developing a national support service for doctors ( ). however, no literature was found to describe such policies in other countries. only three articles describe suicide clustering and suicide contagion, which are unique public health concepts pertaining to suicide ( - ). however, no further follow-up or other literature regarding these suicide clusters were found in this scoping review. as a result, the themes of postvention and public health approaches to physician suicide both were at the vertex (bottom) of the inverted pyramid. discussion as a scoping literature review, this review did not include a quality assessment of quantitative evidence from included papers nor did it perform a meta-analysis or any statistical comparisons of published evidence, which would be characteristic of a systematic review ( ). more papers on physician suicide have been published over the last two decades than before. this follows an overall trend in published literature on physician well-being and may also reflect growing public recognition of and decreasing stigma relating to suicide. furthermore, the proportion of opinion articles published is lower in the recent past compared to previously, indicating growing application of contemporary statistical and scientific methods to the study of physician suicide. nonetheless, themes identified reveal opportunities for further work. the inverted pyramid framework ( figure ) suggests that there is a dearth of work on examining physician suicide using typical public health approaches to investigate suicide clusters. figure offers a proposed framework that highlights priorities for continued work on physician suicide. this framework highlights the potential for developing an organized public health approach to preventing physician suicide. the pyramid's base identifies areas for immediate, targeted actions, forming the foundation for the following recommendations for action: • mandatory reporting, an approach used for workplace injury reporting, which could improve surveillance of physician deaths by suicide (prevention); • identification of minimum acceptable community standards of validated instruments to assess suicidal ideation and attempts (prevention, public health); • development and implementation of evidence-based screening and service provision for suicide risk factors ( - ) (prevention); • development of early warning systems, like those used to monitor infectious disease outbreaks, to signal suicide clusters and contagion (public health); • implementation and dissemination of best practices, especially regarding postvention or additional directed interventions (postvention); all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / doi: medrxiv preprint • increase education in the healthcare community about suicide warning signs and prevention (education, wellness); • standardized education in the medical community about appropriate reporting on suicides to prevent suicide contagion (education); • reduction of barriers to help-seeking behavior, reduce systematic stigma in licensing and investigative processes (policy issues, legal issues). higher levels in figure interpreted the data. til drafted the manuscript, except for the methods section which was drafted by all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / doi: medrxiv preprint rs, and the entire manuscript was produced with critical revisions and important intellectual content from all authors. acknowledgments the authors wish to acknowledge dr. christine moutier - - - - - - - - - no. (%) of non-opinion articles ( . %) ( . %) ( . %) n/a †publications about an intervention at minimum describe the intervention. † †other study designs included: qualitative studies or psychological autopsy (n= ), validation or evaluation of a survey instrument or screening question ( ), news coverage ( ), poetry ( ), a discussion guide ( ), or a correction ( ). suicide rates among physicians: a quantitative and gender assessment (meta-analysis) details on suicide among us physicians: data from the national violent death reporting system suicide rates by major occupational group - states scoping studies: towards a methodological framework guidance for conducting systematic scoping reviews joanna briggs institute reviewers' manual : methodology for jbi scoping reviews the prevalence of suicidal ideation and suicidal attempts among norwegian physicians suicide rates from to in norwegian physicians compared with other educational groups the process of suicidal planning among medical doctors: predictors in a longitudinal norwegian sample suicide in doctors: a psychological autopsy study building a program on well-being: key design considerations to meet the unique needs of each organization mortality and causes of death among danish medical doctors - aasland og. physician suicide-why? mortality of doctors: do doctors benefit from their medical knowledge? suicide by physicians fifty-two medical student suicides suicide among medical doctors: psychological autopsy data on seven cases chronic suicidality in a physician: an alliance yet to become therapeutic manitoba suicides force consideration of stresses facing medical residents suicides among junior doctors in the nhs an epidemic of suicide among physicians on probation suicide in young doctors cath sh. some human factors in the flying physician physician, heal thyself how healers become killers: physicians as suicide bombers survey on recent suicidal ideation among female university hospital physicians in sweden and italy (the houpe study): cross-sectional associations with work stressors work environment and recent suicidal thoughts among male university hospital physicians in sweden and italy: the health and organization among university hospital why don't academic physicians seek needed professional help for psychological distress changes in the lifetime prevalence of suicidal feelings and thoughts among norwegian doctors from to : a longitudinal study based on national samples suicide whilst under gmc's fitness to practise investigation: were those deaths preventable? re-visioning medicine burnout and suicidal ideation among u.s. medical students suicidal thoughts among medical residents with burnout application of an interpersonal-psychological model of suicidal behavior to physicians and medical trainees an examination of the interpersonal psychological theory of suicidal behavior in physicians suicide and role strain among physicians legha rk. a history of physician suicide in america causes of death among anesthesiologists: a -year survey mortality among male anaesthetists in the united kingdom are anaesthetists prone to suicide? a review of rates and risk factors provocative work experiences predict the acquired capability for suicide in physicians habits of nervous tension and suicide suicidal ideation among medical students and young physicians: a nationwide and prospective study of prevalence and predictors predisposition to emotional distress and psychiatric illness amongst doctors: the role of unconscious and experiential factors early trauma and lifetime suicidal behavior in a nationwide sample of korean medical students suicidal ideation, plans and attempts among medical college students in china: the effect of their parental characteristics a cross-cultural investigation of suicidal behavior and attitudes in austrian and turkish medical students suicidal behavior and attitudes among medical students in the united arab emirates suicidal ideation among medical students of pakistan: a cross-sectional study new occupational threats to japanese physicians: karoshi (death due to overwork) and karojisatsu (suicide due to overwork) all rights reserved. no reuse allowed without permission author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the a mother's story feasibility of a comprehensive wellness and suicide prevention program: a decade of caring for physicians in training and practice depression awareness, and clinical engagement program for faculty and residents at the university of california, davis health system the suicide prevention and depression awareness program at the university of california, san diego school of medicine listening to depression and suicide risk in medical students: the healer education assessment referral (hear) program. acad psychiatry suicidal ideation among students and physicians at a u.s. medical school: a healer education web-based cognitive behavioral therapy intervention for the prevention of suicidal ideation in medical interns a randomized clinical trial consultation to a clinic following suicide author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the the impact of a medical student's suicide. suicide life threat behav dangers of self-prescription practise what you preach: health behaviours and stress among non-consultant hospital doctors our fallen peers: a mandate for change. acad psychiatry doctors' experience of a bespoke physician consultation service: cross-sectional investigation finding the evidence base using citation networks: do to u.s. physicians die by suicide annually? key: cord- -vx rgs r authors: nair, ranjit; patel, krina title: what the intensivists need to know about critically ill myeloma patients date: - - journal: oncologic critical care doi: . / - - - - _ sha: doc_id: cord_uid: vx rgs r multiple myeloma (mm) is a hematological malignancy characterized by an increase in aberrant plasma cells in the bone marrow leading to rising monoclonal protein in serum and urine. with the introduction of novel therapies with manageable side effects, this incurable disease has evolved into a chronic disease with an acceptable quality of life for the majority of patients. accordingly, management of acute complications is fundamental in reducing the morbidity and mortality in mm. mm emergencies include symptoms and signs related directly to the disease and/or to the treatment; many organs may be involved including, but not limited to, renal, cardiovascular, neurologic, hematologic, and infectious complications. this review will focus on the numerous approaches that are aimed at managing these complications. mm is a hematological malignancy characterized by an increase in aberrant plasma cells in the bone marrow leading to rising monoclonal proteins in the serum and urine. in general, it is a disease of older adults; however, mm may effect younger patients as well: % are < years, % are < years, and . % are < years in age [ ] . it accounts for about % of hematological malignancies in the united states, with an estimated , new cases in . patients often present with osteolytic bone lesions, fractures, bone pain, progressive anemia, hypercalcemia, renal insufficiency, recurrent infections, and/or bleeding. if not treated appropriately and urgently, any of these may lead to death. the introduction of novel agents to the traditional arsenal of drugs comprising of steroids, chemotherapy, and autologous stem cell transplantation has shifted the treatment platform from one which often required repeated acute interventions to that of a more chronic manageable disease. as the acute complications can be potentially fatal, early recognition and intensive care management is the key to successful outcomes. these patients who present with complications are often excluded from mm trials, and their prognosis in the modern era is uncertain. reports suggest that in-hospital mortality from acute complications of mm has decreased significantly over the past decade. continued risk factors for death in this group of patients are organ failure, poor chronic health status, and delayed intensive care management [ ] . despite having increased life expectancy now, the majority of mm patients ultimately develop resistant subclones, leading to disease progression and disease-related complications. with increasing complexity of mm treatments, patients are at risk for more challenging drug toxicities. recognizing and managing the potential side effects of present-day novel regimens is therefore a cornerstone in mm care. in this chapter, we will provide a systematic review of the key critical clinical events which cause medical emergencies in mm. this crucial, fundamental knowledge for all clinicians who routinely care for mm including intensivists will allow for optimal treatment outcomes and survival in mm. complications resulting from stem cell transplant or newer therapies including chimeric antigen receptor t cell therapy are beyond the scope of this review and will not be discussed here. renal insufficiency in mm is a medical emergency that needs immediate work-up and management. there is a wide variation in data reporting on the incidence and the degree of renal failure due to differences in the definitions of renal dysfunction, sample sizes of studies, and populations assessed. renal dysfunction occurs in more than a third to half of newly diagnosed patients (serum creatinine (scr) above the upper normal limit or mg/dl or egfr < ml/min/ . m ). table details the list of common causes of renal dysfunction encountered in clinical practice. as per the rifle risk category, approximately - % of patients develop renal failure, and half become dialysis dependent [ , ] . even after an initial renal recovery with appropriate myeloma-directed therapy, more than half of patients can still develop renal failure during the disease course. in the absence of renal recovery, mortality risk is reported to be higher in this population with shortened overall survival. with novel therapies in myeloma, reversibility of renal insufficiency (ri) is associated with improved survival with some studies demonstrating similar survival in patients who recover from acute kidney injury (aki) and those without renal impairment. therefore, achieving renal recovery is of priority and the main goal in these patients [ , , , , ] . mm kidney: in many cases (approximately - % of cases), renal damage is caused by the disproportionate free light chain (flc) excretion through kidney glomeruli, buildup of light chains in distal tubules, and cast formation which further leads to renal tubular obstruction, an entity referred to as mm kidney. the tubules eventually rupture and flc extravasates, causing interstitial inflammation and damage. this is usually compounded by renal injury from hypercalcemia, dehydration, poor intake, use of nephrotoxic analgesics, iv contrast, use of ace/arb blockade, underlying chronic kidney disorder, and/or underlying infection. early diagnosis and timely therapeutic intervention at the time of presentation to the emergency center are critical. therapy should include aggressive resuscitation with iv fluids in efforts to maintain normal urine output, treating concomitant hypercalcemia and other electrolyte imbalances, initiating treatment of underlying infection, and avoiding nephrotoxins. a multidisciplinary approach with hematology and nephrology consults is paramount to optimize care in these patients. suspicion of cast nephropathy in newly diagnosed myeloma patients with serum flc above mg/l should be high, especially if associated selective proteinuria composed of flc/bjp exists, as the degree of renal injury is usually related to tumor load [ ] . a percutaneous kidney biopsy with histology is not required to initiate treatment if myeloma has been confirmed but can be helpful to confirm the diagnosis, assess concomitant light chain or amyloid deposition disease, and predict prognosis. the need for a renal biopsy should be discussed for patients with lower levels of toxic flc, as these patients are more likely to have alternate etiologies for their ri. in patients with significant light chain burden, it is critical to begin treatment immediately with renal recovery as the major immediate goal. the only proven modality with the best chance of renal recovery is rapid cytoreduction to decrease light chain production. newer chemotherapy agents lower flcs quickly, especially with a combinatorial approach. many clinical trials have shown that a bortezomib-dexamethasone combination regimen achieves this goal [ ] . attaining a pre-dialysis serum-free light chain level below mg/l and a % reduction after the first cycle of chemotherapy are independent predictive factors of renal recovery and hemodialysis independence at year [ , ] . hemodialysis or tpe for the sole purpose of offloading tumor burden is currently investigational, as none of the randomized trials have shown a significant benefit. though it takes a few days to weeks to observe the impact of chemotherapy on light chain production, current evidence suggests using hemodialysis only for clinical indications of renal replacement therapy which may exist and not solely for reduction of tumor load. based on two randomized clinical trials, the use of high cutoff hemodialysis to rapidly reduce the load of nephrotoxic light chains may be an option in patients requiring dialysis when treated with bortezomib-based therapies, but the results may be stochastic at best [ , , ] . tpe can rapidly reduce flc loads; however its role in cast nephropathy comes from small retrospective trials which provide no data on the renal histology or sflc levels. in studies that demonstrated benefit, obtaining a biopsy and using a proteasome inhibitor (pi)-based treatment were not uniformly applied, making interpretation of the data difficult. especially in the era of novel drugs, the evidence in favor of tpe is limited [ , , ] unless there is evidence of igm or iga paraproteinemia-associated hyperviscosity syndrome. the utility of urinary alkalization is debated as this theoretically decreases tubular cast formation and, however, can cause risk of renal calcium precipitation, especially if the patient presents with concurrent hypercalcemia. acute tubular necrosis: atn is a consequence of an already compromised renal function in patients with cast nephropathy. hypercalcemiarelated vasoconstriction with subsequent reduced renovascular flow, dehydration, and use of loop diuretics can speed up cast formation. there are some reports of carfilzomib causing direct proximal tubular injury; however, the risk appears to be very small [ ] . monoclonal deposition disease: in contrast to myeloma kidney, midd (heavy chain, light chain, or heavy chain-light chain) and al amyloidosis result from gradual non-fibrillar (congo red negative) and fibrillar (congo red positive) protein deposition, respectively, in the glomeruli. midd is most commonly kappa subtype, while al amyloidosis tends to be lambda; both tend to be associated with predominant albuminuria unlike cast nephropathy. though the most characteristic presentation is nephrotic syndrome due to glomerular involvement, patients with midd can have rapid deterioration of renal function which could be fatal if diagnosis is not reached in a patient presenting with new-onset renal failure. life-threatening complications which need to be closely monitored include arrhythmias, congestive heart failure, and bleeding diathesis. to confirm the diagnosis, subcutaneous fat pad biopsy can be pursued initially, as it is less invasive and can potentially diagnose an al amyloid deposition disease. if negative, proceed with kidney biopsy to look for al amyloid, midd, or other potential renal glomerulopathy. fanconi syndrome: the presence of hypophosphatemia, hypokalemia, hypouricemia, renal tubular acidosis, and glycosuria should raise the suspicion of fanconi syndrome in mm. it is a rare wasting syndrome due to proximal renal tubule dysfunction more commonly seen in patients with mgus leading to loss of glucose, phosphate, amino acids, and uric acid. though it runs a benign course, it can complicate the concomitant electrolyte imbalance and renal failure. ironically, despite the glycosuria, the serum glucose is usually normal, which goes along with the defect in the proximal tubular transport defect [ ] . mm patients are at high risk for cardiac complications secondary multiple factors including an older age group of patients with underlying comorbidities, concurrent kidney involvement, mm-associated deposition disease, and/or anti-mm drug-related side effects. cardiac amyloid: approximately % of patients with mm can have concomitant light chain (al) amyloidosis, the most common type of systemic amyloidosis associated with plasma cell dyscrasias; often this association is missed at the time of mm diagnosis. in amyloidosis, heart involvement is seen in approximately half of the cases [ ] . cardiac amyloidosis can be clinically silent initially, and a mm patient presenting with progressive dyspnea, worsening edema with evidence of heart failure, or dysrhythmia presenting as syncope or hypotensive event requires a thorough work-up to rule out coexisting amyloid disease. detection of amyloid in any organ requires ruling out cardiac involvement. other symptoms which warrant amyloid work-up include easy bruising, periorbital purpura, macroglossia (which is better appreciated when associated with tooth indentation), and carpal tunnel syndrome. once significant symptoms of cardiac amyloid appear, most commonly related to heart failure, median survival shortens, especially in the event of a late diagnosis. amyloid deposition takes place extracellularly throughout the ventricles, atria, valves, conduction system, and in the perivascular regions of small vessels. this eventually leads to thickened, non-dilated ventricles as well as atrial thickening that eventually progresses to thinned dilated atria. the serum concentration of free light chains and cardiac enzymes (n-terminal pro-brain natriuretic peptide and cardiac troponins) are sensitive markers for systemic and cardiac involvement, respectively, in al amyloidosis [ ] . n-terminal pro-b-type natriuretic peptide levels are elevated in cardiac amyloidosis, even in the absence of heart failure, due to regional myocardial stress due to fibrillar extracellular deposition [ ] . cardiac troponins can be elevated, even with an unremarkable coronary angiography, due to predominant deposition in the small intramural vessels sparing the epicardial arteries, but associated with nonischemic destruction of cardiomyocytes. ecg findings suggestive of cardiac involvement include a pattern of low voltage (cardiac changes from amyloid infiltration rather than myocyte hypertrophy) defined as a qrs voltage amplitude of . mv in all limb leads or mv in all precordial leads (sensitivity - %). this may be described as a pseudo-infarct pattern ( - %). atrioventricular right-or left-bundle branch blocks may also suggest cardiac amyloid. echocardiography in advanced stages usually demonstrates a restrictive pattern due to the structural changes and increased echogenicity of the myocardium with a "granular speckling" (low sensitivity of - % but with a high specificity ( - %)). cardiac mri in advanced cardiac amyloidosis shows diffuse subendocardial heterogeneous enhancement in a nonvascular distribution on delayed contrast-enhanced mr images using inversion recovery technique. this, along with diffuse multichambered wall thickening, posterior right atrial wall thickening, or interatrial septal thickening > mm, is specific to amyloidosis [ , , ] . cardiac catheterization is not always required but used to obtain endomyocardial biopsy. it may help to provide important information for cardiac hemodynamics, which generally shows impaired ventricular filling with an elevated left ventricular end-diastolic pressure. demonstration of left ventricular diastolic pressure greater than right remains to distinguish restrictive cardiomyopathy from its differential diagnosis of constrictive pericarditis. fine-needle aspiration of abdominal fat is relatively sensitive for amyloid deposits (> % of patients with al amyloidosis); other sites of amyloid deposition include minor salivary glands, gingiva, and rectum [ , ] . in a patient with clinical suspicion of cardiac involvement, the presence of physical exam findings, blood markers, imaging, and a cardiac biopsy consistent with amyloid are adequate for diagnosis. patients presenting with acute heart failure in al amyloidosis require intensive care monitoring and aggressive supportive care. diuresis is the key and is probably the only treatment modality that can improve hemodynamics. optimal therapy usually requires using higher doses of diuretics or in combinations (furosemide, torsemide, spironolactone, and metolazone) to maintain adequate diuresis, especially in the setting of nephrotic syndrome. an intravenous route is likely preferred in the acute stage, due to the often-present gut edema and better bioavailability. aggressive supportive care includes maintaining strict intake output measurements, dietary salt restriction, measuring daily weights, and monitoring for arrhythmias. the uses of cardiac protective drugs such as beta-blockers should be reserved for cases of atrial fibrillation to maintain adequate filling pressure. in patients without af, its use should be discussed on a case-by-case basis, considering the significant risks including labile blood pressure due to autonomic neuropathy and risk of bradycardia due to coexistent conduction defects. caution should be maintained with the use of angiotensin-converting enzyme inhibitors and angiotensin ii inhibitors, due to similar risk of exacerbating hypotension. both digoxin and calcium blockers, especially nifedipine, have the tendency to bind to amyloid fibrils. this can precipitate congestive heart failure due to increased susceptibility to digoxin toxicity and enhance the negative ionotropic effect with calcium channel blockers [ , , ] . syncope can result from af with rapid heart rate, embolic phenomenon, conduction defects, bradycardia, and ventricular arrhythmias. the most common atrial arrhythmia ( - % of patients) is af and is associated with a very high incidence of thromboembolism. in a mayo clinic study, cardiac amyloidosis patients undergoing transesophageal echocardiography had a higher frequency of intracardiac thrombus ( %) irrespective of the presence of atrial fibrillation. factors associated with increased risk of intracardiac thrombosis include atrial fibrillation, poor left ventricular diastolic function (grade or ), lower left atrial appendage emptying velocity ( cm/s), increased rv wall thickness, low systolic blood pressure, and increased heart rate. this group of patients should be considered high risk for thromboembolic events and merits screening with tee. anticoagulation should be strongly considered in the presence of any of these risk factors due to the heightened risk for mural thrombus development and embolic events. this should be weighed against the risk of bleeding in these patients due to the potential for concomitant coagulopathy and fragile vasculature [ ] . pacemakers are a consideration in the presence of heart block or symptomatic bradycardia. the role of intracardiac defibrillators is controversial due to limited evidence showing a survival benefit both for primary and secondary prevention. several studies report ventricular arrhythmias in about a third of patients; however, this does not appear to be the leading cause of mortality in this patient group. once congestive heart failure occurs, the clinical course for patients rapidly deteriorates, with a median survival of - months from diagnosis. death in severe cardiac amyloid is often heralded by agonal bradycardia, followed by electromechanical dissociation or pea. nonetheless consensus guidelines do not support icd placement for primary prevention of scd in patients if life expectancy is < year. hence these devices have rarely been used in patients with cardiac al amyloidosis. with improvement in the therapeutic platform of cardiac amyloidosis and improved survival, it becomes a priority to be able to select the patients that benefit from antiarrhythmic prophylaxis and primary prevention icds. with the available data, icds should be considered in cardiac amyloid patients with recurrent episodes of non-sustained vt, unexplained syncope, young age at diagnosis, early stages of disease at diagnosis with low levels of cardiac biomarkers, and prognosis > year [ , , , ] . anti-myeloma drug-related cardiotoxicity: proteasome inhibitors are currently the main class of drugs used in front line and in the relapsed setting of mm. fda-approved therapies in this class include bortezomib, carfilzomib, and ixazomib [ ] . among them, carfilzomib has shown the highest rates of cardiotoxicity. in a meta-analysis looking at the cardiotoxicity of carfilzomib, all-grade and high-grade cardiotoxicity were seen in % and % of patients, respectively, higher than the control arm. the most significant events included heart failure ( %), arrhythmias ( . %), and ischemic events ( %). rare reports of carfilzomibassociated cardiac arrests also exist. risk can be higher in patients with comorbidities such as older age, obesity, anemia, kidney dysfunction, previous mediastinal radiation, high-dose corticosteroids, prior or concurrent anthracycline exposure, use of higher carfilzomib doses, short infusion times, and undiagnosed amyloidosis [ , , , , ] . anecdotal reports suggest bortezomib may lead to cardiotoxicity as well, although a recent meta-analysis did not find a significantly increased risk compared to the control arm [ , ] . the first intervention in suspected cardiotoxicity is to hold further therapy and follow expectant management. in cases of congestive heart failure, once the heart failure symptoms improve, it is reasonable to restart the chemotherapy at a lower dose, with a longer infusion time and limiting peri-treatment iv fluids. arrhythmic events would require careful consideration regarding rechallenging patients after considering the risk-benefit ratio. hyperviscosity syndrome: in a patient undergoing work-up for mm or with an established diagnosis, symptoms of headache, nose bleeds, mental status or visual changes, auditory symptoms, focal weakness, excessive lethargy, seizures, or coma should raise suspicion for hyperviscosity syndrome. though extremely uncommon in mm ( %) in comparison to waldenström's macroglobulinemia ( - %), a low threshold should be kept to work up a patient for hyperviscosity as this can be fatal if not recognized early. the viscosity tends to increase with increase in molecular size, shape, and weight of the involved paraprotein and abnormal polymerization. patients tend to develop symptoms when the serum viscosity reaches centi-poise (cp) (normal serum viscosity, . - . cp). this usually corresponds to an m spike of g/dl if igm, g/dl for igg, and - g/dl for iga paraproteinemia. however, these levels are arbitrary, and the relationship is nonlinear as patients can have symptoms even with a level lower or could be asymptomatic with a higher level [ , ] . in mm, igg (mostly igg ) or iga paraprotein tends to be reported more commonly with hyperviscosity than igm most likely due to the inherent rarity of igm mm [ , ] . due to increased serum viscosity, there is impaired blood flow and sludging throughout the microvasculature, leading to tissue ischemia, placing the patient at risk for thrombosis to major organs leading to myocardial, cerebrovascular, and retinal ischemia. with passive venous congestion, patients can often present with bleeding symptoms. thorough physical exam is paramount including a fundoscopic exam, which may reveal dilated, tortuous, engorged, "sausage" veins with associated hemorrhages. bleeding at sites involving mucosa is common which can include oral and nasal cavity, the uterus, and the gastrointestinal tract. purpura and skin bruising are also common. consensus mm guidelines do not recommend plasmapheresis in the absence of signs or symptoms for hyperviscosity. however, a low threshold should be maintained to initiate treatment at the earliest suspicion of symptoms or signs; it follows a two-prong strategy consisting of immediate plasmapheresis to lower the excess paraproteins and chemotherapy directed at the primary process of mm. unlike the response of igm-related paraproteinemia (> % intravascular), where a - % reduction of viscosity is expected with one treatment, iga or igg (< % intravascular) hyperviscosity can take several sessions to achieve the same result [ ] . if plasmapheresis is not readily available and the patient is having lifethreatening symptoms, phlebotomy may reduce hyperviscosity-related acute symptoms. in mm patients, who are often anemic at presentation, blood transfusions should be done only in emergent situations and with caution until after the initiation of plasmapheresis to prevent rise in serum viscosity [ , ] . venous thromboembolism: patients with mm are at an increased risk of venous thrombosis due to patient-related, disease-related, and treatment-related factors. patient-related factors include older age, recent surgery or immobilization, and decreased functional status; the major disease-related factor is increased pro-thrombotic status from heightened inflammatory markers, especially at diagnosis. there is a high level of factor viii and von willebrand factor as well as acquired resistance to activated protein c [ ] . in addition, mm patients treated with immunomodulators especially thalidomide or lenalidomide used in combination with steroids or other chemotherapeutic agents increase the risk further [ , ] . management of acute dvt or pe involves withholding the offending drug if any and starting immediate systemic anticoagulation with low-molecular-weight heparin or unfractionated heparin. lmwh is the preferred anticoagulation at the time of transition to outpatient therapy as evidence suggests better safety and a superior efficacy in stabilizing the clot in comparison to other anticoagulants [ ] . factors that make oral anticoagulation less ideal is drugdrug interaction and unreliable therapeutic levels resulting from patient-related malnutrition, infection, and drug holds from cytopenias. once full anticoagulation is attained, the immunomodulator can be reintroduced. in the presence of thrombocytopenia, which is commonly encountered in mm patients, the dose of lmwh needs adjustment and in general a recommendation of % dose reduction if platelet counts below , /μl, and discontinuation if platelet count < , /μl is followed. duration of anticoagulation should be a minimum of months; however in the presence of the offending drug, systemic anticoagulation should be continued for the total duration of therapy [ ] . arterial thromboembolic events, though rare, have also been reported with thalidomide, lenalidomide, and pomalidomide; rarely, this may present as myocardial infarction or stroke. the role of newer oral anticoagulants including factor xa inhibitors and direct thrombin inhibitors is currently investigational in the treatment of malignancy-associated thromboembolic events; however, they are a good alternative for patients with renal failure [ ] . sepsis/invasive infections: - % of newly diagnosed mm patients succumb to the disease in the first few months with infection contributing to more than half the cases [ , , ] . high incidence of early infection in mm is due to the suppressed humoral and t cell-mediated immunity by the disease itself. immunosuppression is mediated by disease-and treatment-related factors including decreased ratio of functional to dysfunctional immunoglobulins, defects in antibody opsonization, steroid-related t cell defects, secondary immunodeficiency related to chemotherapy, restricted pulmonary reserve from thoracic rib fractures and opiate use, mucosal damage, indwelling catheters, and presence of renal failure [ , , ] . bacterial infections, especially streptococcus pneumoniae, haemophilus influenzae, and escherichia coli, tend to be the predominant organisms causing infection at diagnosis, while staphylococcus aureus and other gram-negative infections are more common in the relapsed refractory setting [ , ] . the use of novel drugs combined with steroids has increased the rate of viral and fungal infections which can occur any time during the disease course [ , , ] . viral infections, especially herpes simplex virus (hsv) and varicella-zoster virus (vzv), have increased in incidence with the introduction of proteasome inhibitors such as bortezomib. viral prophylaxis with acyclovir or valacyclovir is recommended in all patients undergoing therapy with proteasome inhibitors or monoclonal antibodies. although invasive fungal infection (approximately - %) incidence is low, the mortality rate is high with approximately % of patients requiring icu management. the introduction of novel agents tends to be associated with later-onset invasive fungal infections compared to studies from the conventional chemotherapy era where an earlier onset was noted [ ] . furthermore the use of long-term and high-dose dexamethasone can cause persistent defect in t cell-mediated immunity which predisposes patients to mucosal candidiasis, p. jirovecii, hsv, and vzv infections. following sepsis guidelines with aggressive fluid resuscitation and broad-spectrum antibiotic coverage as well as growth factor support if neutropenic is warranted in a patient presenting with suspected sepsis/infection. in case of clinical deterioration with unclear source of infection, persistent febrile neutropenia, or hemodynamic compromise, consider computer tomography (ct) scan including sinus imaging. other investigative modalities for diagnosis should include bronchoscopy/bal with microscopy and bacteria, viral, and fungal culture. in suspected cases of fungal infection, aspergillus pcr and galactomannan testing on serum and bal should be performed. due to concomitant immunodeficiency, treatment with intravenous immunoglobulins is considered in patients with recurrent bacterial or viral infections; however its role in treating an acute infection is limited. direct involvement of pulmonary parenchyma in mm is extremely rare. pulmonary complications can arise from a broad range of different mechanisms both infectious and noninfectious. in majority of the cases presenting with respiratory failure, the etiology is infectious. patients with myeloma can present with lung infiltrates, ground glass opacities, nodules, masses, effusion, and lymphadenopathy. noninfectious causes that need to be ruled out include congestive heart failure, pulmonary embolism, pulmonary hemorrhage, and drug-induced pneumonitis. myeloma lung: case reports and case series describe the various ways mm can involve the lung. presentation of lung disease is variable and includes consolidative or interstitial pattern infiltrates on imaging, intraparenchymal plasmacytomas, intrapulmonary calcifications, or pleural effusions [ , , ] . presentations similar to ards have also been reported [ , , , , ] . a high index of suspicion is required as there is a broad differential of diagnoses with similar radiographic findings. patients with extramedullary mm tend to have more aggressive disease and can deteriorate rapidly due to disease progression if not recognized and treated early. if there is evidence of mm progression on blood tests or bone marrow, a biopsy (surgical, transbronchial) or bronchoscopy with aspiration and/or bronchoalveolar lavage (bal) should be strongly considered with microbiologic and flow cytometric analysis. once infection is excluded and evidence of progression is confirmed, prompt initiation of mm directed therapy is warranted [ ] . diffuse alveolar hemorrhage: in patients presenting with acute onset hypoxia, pulmonary opacities, and fevers, especially in the setting of thrombocytopenia and anemia, pulmonary hemorrhage is a possibility. diffuse alveolar hemorrhage (dah) has been rarely reported in mm patients, but there have been cases associated with bortezomib and in the posttransplant setting [ , , , ] . though rare, this is potentially a fatal event. a thorough microbiology screen, echocardiogram, ct imaging, autoimmune work-up, and coagulopathy work-up are warranted [ ] . bal showing presence of gross blood and the percentage of hemosiderin-laden macrophages (siderophages) > % at bal are suggestive of a diagnosis of dah [ , , ] . aggressive supportive care and empiric broad-spectrum antimicrobial therapy should be considered. the role of early initiation of steroids is currently based on retrospective series and case reports. an md anderson prospective study examined critically ill adult hematopoietic transplant patients treated for dah, and on multivariate analysis, an initial medium ( - mg/day) and high-dose ( mg/day) steroids were associated with a higher icu mortality (p = . ) as compared with the low dose (< mg/day). adjunctive treatment with aminocaproic acid in this study, which is commonly used in dah, did not produce differences in outcomes [ , ] . drug-induced interstitial pneumonitis (dip): dip manifesting as interstitial lung disease has been reported with lenalidomide, thalidomide, and bortezomib [ , , , ] . though mechanisms are not fully elucidated, oxidative stress, prostaglandin e (pge ) inhibition, fibroblast proliferation, and impairment of cell repair by epidermal growth factor receptor blocking may play a role. bal with culture and microscopy should be able to help rule out infectious causes. the lymphocytic preponderance typically seen in bronchoalveolar lavage (bal) is supportive of a hypersensitivity mechanism; however, this finding is nondiagnostic and requires meticulous exclusion of other causes. invasive procedures like biopsy usually do not provide any specific findings diagnostic for dip and, therefore, should be obtained only on a case-by-case basis. once dip is suspected, stopping the offending agent is the main therapeutic approach; steroids may be used in severe cases [ , ] . although no guidelines exist on the duration of steroids, in general, a slow taper is recommended [ ] . caution should be maintained on rechallenging the patients with the offending medication as the risk of recurrent reaction is extremely high. hypercalcemia: being one of the four diagnostic criteria for mm (crab criteriacalcium, renal insufficiency, anemia, and bone disease), hypercalcemia is defined as calcium level corrected for albumin mg/dl. almost a third of mm patients are diagnosed with hypercalcemia at diagnosis or during the disease course [ , ] . the mechanism is due to local osteolysis; at the cellular level, it is mediated by interleukin , interleukin , and tumor necrosis factor α with rankl being the final common mediator. most patients presenting with hypercalcemia have associated anemia, thrombocytopenia, kidney dysfunction, lytic lesions, and advancedstage disease. hypercalcemia is associated with aggressive disease biology and heavy disease burden and remains a poor prognostic feature even in the era of novel agents [ ] . clinically, hypercalcemia can be asymptomatic; however, patients often have symptoms which range from subtle signs of malaise or fatigue to severe consequences including renal failure, altered mentation, seizures, shortened qt, and risk of arrhythmias. gastrointestinal manifestations such as nausea, vomiting, ileus, anorexia, and episodes of pancreatitis have been reported [ ] . if not treated promptly, hypercalcemia is lethal. increased calcium promotes natriuresis which can further worsen an already compromised kidney function. this dehydration then leads to worsening hypercalcemia and renal function. though the threshold levels are arbitrary, patients with serum calcium level < mg/dl or minimal symptoms can be managed with fluid resuscitation which reverses the hypovolemia, repletes the intravascular volume deficit, and improves urinary calcium excretion. though the definitive approach is prompt mm treatment initiation, often due to patient frailty and concomitant organ damage, chemotherapy cannot be initiated until the patient is more stable. if the patient is on calcium, vitamin d supplements, thiazide diuretics, or any other drugs which promote hypercalcemia, they should be stopped immediately. in patients with levels > mg/dl or with significant symptoms, other adjunct therapies should be used as a temporizing measure. loop diuretics, which were previously commonly used for hypercalcemia, should not be routinely used. the utility of forced saline diuresis is not consistent; with lower doses, calciuresis is an indirect effect of the accompanying natriuresis. to cause a direct calciuretic effect, doses as high as mg/h is required, which can cause unwanted metabolic derangements and worsening renal cast formation with risk of worsening renal dysfunction [ ] . the use of calcitonin to treat patients with hypercalcemia is limited owing to its inability to dramatically lower serum calcium levels. however, it can work synergistically with other therapies without significant adverse side effects until chemotherapy can be initiated. calcitonin, by inhibition of bone resorption and increased renal calcium excretion, has rapid onset of action and can lower calcium levels by - mg/dl ( . mmol/l). the dose is iu/kg given subcutaneously or intramuscularly every - h. the efficacy in general wears off after - days due to the escape phenomenon (tachyphylaxis) of the calcitonin sensing receptors [ ] . preclinical studies suggest tachyphylaxis can be prevented with the addition of corticosteroids [ , , ] . with its anti-mm effect, adding corticosteroids can add to the synergy to this combination in the treatment of hypercalcemia. the role of steroids in hypercalcemia of malignancy comes from case reports and case series. corticosteroids decrease calcium absorption from the gut, decrease renal tubular reabsorption, and promote excretion [ , ] . prednisone (dose of - mg/day) or iv hydrocortisone (dose of - mg/day) for - days is usually used in this setting. this dose, which is effective in patients with chronic granulomatous diseases (e.g., sarcoidosis) and malignancies such as lymphoma, may be suboptimal for patients with mm due to the direct osteolytic action of mm cells. if clinical status permits, dexamethasone should be considered at mm treatment doses ranging from to mg weekly or to mg daily on a days on, days off schedule. intravenous bisphosphonates (pamidronate, zoledronic acid, ibandronate) work by blocking osteoclast-mediated bone resorption. intravenous zoledronic acid is more routinely used than other drugs in this category due to its higher potency, efficacy, and shorter infusion time ( min) , when compared to ibandronate and pamidronate [ , , , ] . bisphosphonates have an established role in severe or symptomatic hypercalcemia, but often it is challenging to administer them upfront for mm patients due to the renal function-based dose limitation [ ] . it also has slow onset of action and reaches peak in about - days; therefore it needs to be used in a combinatorial approach. denosumab, on the other hand, has a renally independent clearance mechanism and hence does not require kidney functionbased dosing adjustments. it is currently fda approved to treat hypercalcemia of malignancy refractory to bisphosphonate therapy and is dosed at mg subcutaneous injection every weeks with additional doses of mg on days and of the first month of therapy [ , ] . mithramycin and gallium nitrate are drugs of historical interest and are not currently used in the standard setting. in refractory cases of hypercalcemia seen in advanced mm and kidney involvement, with impending cardiovascular or cerebrovascular deterioration, hemodialysis ca + -free dialysate should be strongly considered. tumor lysis syndrome: due to the low proliferative activity of plasma cells which is < % in majority of patients, tls is extremely uncommon in mm [ , ] . mm patients who are likely to be at risk are patients with high tumor burden (as noted by an increased serum lactate dehydrogenase and beta- microglobulin), diffuse bone marrow disease (plasma cells > % of all nucleated cells as per a case series), extensive skeletal involvement, high-risk cytogenetics, and plasmablastic morphology [ , ] . there are reports of bortezomib, thalidomide, and steroid associated tls [ , ] . tumor lysis syndrome manifests due to lysis of massive numbers of tumor cells releasing intracellular potassium and phosphorous causing hyperkalemia and hyperphosphatemia. this causes calcium to precipitate as calcium phosphate in tissues causing secondary hypocalcemia. hyperuricemia results from the breakdown of nucleic acids in the tumor cells. the resultant metabolic derangement, if left untreated, can progress to kidney failure, mental status changes, seizures, arrhythmias, and even death. diagnosing tls can be challenging in patients with mm due to its uncommon association and often seen associated renal insufficiency [ ] . it is imperative for clinicians to be aware of this complication in mm patients undergoing treatment with novel combinations. tls is managed with aggressive intravenous hydration, rasburicase, allopurinol, and at times sodium bicarbonate. timely initiation of renal replacement therapy is paramount in patients with lifethreatening electrolyte disturbances. epidural spinal cord compression (escc): acute emergencies related to the nervous system in mm are mostly related to escc. vertebral involvement is prevalent in - % of mm patients, and escc is encountered in approximately - % of patients either at presentation or during the course of disease [ , ] . the compression usually results from vertebral fractures caused by osteolytic tumors or malignant osteoporosis. very rarely, it has been reported due to extraosseous epidural mm or from extradural extension of plasmacytoma [ ] . in majority of the cases, thoracic or lumbar spinal cord is involved (> %) [ , ] . it is vital to recognize the symptoms early, with prompt imaging since the strongest predictor of neurologic outcome with treatment is the neurologic status when treatment is initiated [ ] . clinical features depend on the site and degree of involvement. a new-onset back pain or a worsening of an existing pain in a mm patient should prompt a work-up to rule this out. pain can be localized or radiating which usually worsens with lying down. associated bowel and bladder dysfunction is present in about half of the cases [ ] . if treatment is delayed, myelopathy progresses to loss of sensory and motor function leading to paraplegia. spinal cord injury at the cervical level is uncommon; however, if affected above c -c , it results in respiratory, cardiac, and autonomic decompensation [ ] . gadolinium-enhanced mri is the initial gold standard imaging modality as it can aid in the thorough evaluation of spine, degree of tumor or fracture involvement, and for radiation planning [ ] . the safety of mri should be discussed in the presence of pacemakers and defibrillators in a multidisciplinary fashion, and if contraindicated, ct myelography should be performed. there is a lack of consensus guidelines for treatment and hence warrants an emergent multidisciplinary team consult involving the oncologist, neurosurgeon, and radiation oncologist. the optimal management depends on the extent of disease, performance status of the patient, and presence of spine instability. immediate therapeutic strategies involve achieving minimal mobility until stability of the spine is achieved. steroids should be initiated even while awaiting diagnostic study results (dexamethasone at doses mg iv followed by mg daily in divided doses with a taper over weeks) [ ] . doses as high as mg initial bolus were used in the past, but are not used any more due to risk of significant side effects and limited evidence to suggest benefit [ ] . spine stabilization by reducing the mechanical load can be achieved with the use of a neck collar (e.g., philadelphia collar) in case of cervical spine or braces at other sites and is not generally recommended for longer than months [ ] . a thorough baseline neurological exam followed by serial neurological exams should be done to assess clinical status. radiotherapy should be started as soon as possible as mm is extremely radiosensitive, unless in very late in the disease. in a retrospective study which included patients with mm/lymphoma treated with rt alone, % of the patients experienced an improvement in motor function, % had no change, while % deteriorated. in this group, patients who experienced slowly progressive motor deficit defined as time of onset > days had better response rates than patients who had fast onset deficit defined as time of onset < days. the improvements in motor function in these groups were % and %, respectively. no patients who received radiotherapy progressed in the slow onset motor weakness group [ , ] . current evidence does not support resorting to surgery in patients with escc, as most mm cases respond to radiation. the need for surgical stabilization with fixation or by percutaneous repair is in general reserved for cases with spine instability or spine compression from a vertebral body. treatment of the underlying malignancy with systemic chemotherapy or novel agents, bone-modifying agents, and neurorehabilitation can help in recovery of the vertebral and spinal damage [ , ] . cns myelomatosis: neurological symptoms in mm are most commonly due to hypercalcemia, cord compression, anti-mm drug related, hyperviscosity, or amyloid-related neuropathy. direct involvement by malignant cells of the central nervous system (cns) or peripheral nervous system, unlike lymphoma, is uncommon in mm. direct cns involvement in mm often presents as leptomeningeal myelomatosis (lmm), intraparenchymal metastasis, cranial nerve involvement, or radiculopathy [ , , ] . though conventional wisdom would suggest cns involvement is seen later during the disease, most patients present relatively early with median time of diagnosis of lmd around months from mm diagnosis. hence the risk is unlikely only related to improved survival in mm patients but also due to an aggressive early biology. supporting this contention, cns involvement is reported mostly in patients with high-risk mm with heavy tumor burden, plasmablastic morphology, or plasma cell leukemia [ ] . patients often present with focal weakness, cranial nerve palsies, mental status changes, speech and gait disturbances, symptoms of increased intracranial pressure, and/or seizures. csf examination reveals pleocytosis, increased protein levels higher than g/dl, presence of monoclonal plasma cells, and in some cases an increased opening pressure. the diagnosis is usually reached with the first analysis of csf [ ] . mri imaging with and without contrast would aid in identifying the presence and extent of the disease. in the presence of symptoms, steroids can help in alleviating the symptoms and reducing intracranial pressure. therapy involves strategies similar to that used for other hematological malignancies with cns involvement, including intrathecal chemotherapy and cranial irradiation with systemic chemotherapy. despite majority of patients achieving csf clearance with cns-directed therapy showing significant potential for symptom improvement, the overall prognosis is grim with the currently approved therapies in mm. the median survival ranges from to months, with a small fraction surviving past years [ ] . hence it is paramount to have a thorough discussion on the goals of therapy once cns involvement is diagnosed [ , ] . anemia: hematological emergencies in mm most commonly arise due to disease-or treatment-related myelosuppression. anemia (hb > g/dl below the lower limit of normal or a hemoglobin value < g/ dl) is a common complication seen in - % mm patients at presentation and in nearly all patients at some point in the disease course [ ] . several factors drive anemia in mm patients which include bone marrow involvement, low serum erythropoietin level due to kidney dysfunction, hemolysis, impaired availability of storage iron, and anti-mm treatment-related bone marrow suppression. an aggressive anti-mm treatment approach despite the severity of anemia is warranted in patients with anemia suspected to be from mm. this is likely encountered in most cases of newly diagnosed mm. myeloma therapy often worsens the anemia, before it improves once myeloma responds. these patients should be closely monitored for transfusion requirement. no universal guidelines exist for transfusion goals. in general, leukoreduced and irradiated blood is used for transfusion for hemoglobin level < - g/dl or symptomatic anemia. the transfusion goal can vary in the presence of other comorbidities such as cardiac ischemia, active bleeding, or if symptoms due to hypoperfusion are present. a major proportion of patients with mm who have chronic anemia are those with long-standing disease in partial remission or stable response. erythropoiesis-stimulating agents are not routinely used, except in this population after careful selection, to improve the ongoing anemia despite chemotherapy. anti-mm drug-induced tma: in patients who present with new-onset anemia and thrombocytopenia, more than often, it is ascribed to myelosuppressive treatment or for mm progression. in the setting of worsening aki, it is imperative to check for evidence of hemolysis with a haptoglobin level, ldh level, and peripheral blood smear looking for schistocytes. one should strongly consider ruling out tma as it has been reported with novel drugs and with older chemotherapeutic agents; two notable novel drugs are bortezomib and carfilzomib, and a chemotherapeutic agent is cisplatin. prompt discontinuation of these medications is warranted if the medication is suspected to be the likely culprit. plasmapheresis is initiated in most of patients, and eculizumab has been tried in a small number of patients. the role of these two therapies in malignancy-associated tma is controversial and should be discussed on a case-by-case basis [ , , , ] 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incidence and risk of cardiotoxicity associated with bortezomib in the treatment of cancer: a systematic review and metaanalysis acute spinal cord dysfunction. in: the washington manual of medical therapeutics retracted: overview of recent trends in diagnosis and management of leptomeningeal multiple myeloma multiple myeloma involving skin and pulmonary parenchyma after autologous stem cell transplantation proteasome inhibitor associated thrombotic microangiopathy hypercalcemia remains an adverse prognostic factor for newly diagnosed multiple myeloma patients in the era of novel antimyeloma therapies conflicts of interest kp = research funds from poseida, intrexon/ziopharm, takeda. advisory board/consultancy in takeda, dava oncology, celgene, janssen, amgen, bms, and oncopeptides. key: cord- - jrf ec authors: henze, g.; klingebiel, t.; rutkowski, s.; schlegel, p.-g. title: onkologie date: journal: p&#x e ;diatrie doi: . / - - - - _ sha: doc_id: cord_uid: jrf ec die heilungsaussichten für krebskranke kinder haben sich durch die chemotherapie und die modernen diagnostischen verfahren deutlich verbessert. eine rasante entwicklung hat in den letzten jahren auch die stammzelltransplantation genommen, sodass diese heute bei zahlreichen systemischen malignen und nichtmalignen erkrankungen als kurative therapieoption eingesetzt werden kann. heute überleben etwa % der krebskranken kinder und jugendlichen, und die meisten von ihnen führen ein weitgehend normales leben von guter qualität. galten noch um viele der soliden tumoren lediglich durch radikale chirurgische maßnahmen als behandelbar, hat sich das bild heute gänzlich gewandelt. nach dem vorbild der leukämien wurden von der gesellschaft für pädiatrische onkologie (gpo; heute gesellschaft für pädiatrische onkologie und hämatologie, gpoh) kooperative arzneimittelstudien und register organisiert, die mittlerweile alle wichtigen soliden tumoren erfassen und den stellenwert von chemotherapie, strahlentherapie und chirurgie systematisch erforscht und definiert haben. zum einen ist es gelungen, aus unheilbaren erkrankungen heilbare zu machen, zum anderen, nebenwirkungen und folgen der therapien zu reduzieren. hirntumoren im kindesalter weisen in bezug auf ihr histologisches spektrum und ihre häufigkeit, aber auch aufgrund ihrer teils günstigeren prognose erhebliche unterschiede im vergleich zu denen erwachsener auf. die lebensqualität, die intellektuelle leistungsfähigkeit und das psychosoziale verhalten von kindern mit hirntumoren werden häufig durch folgen der tumorerkrankung, aber auch durch die therapie beeinträchtigt. ziel der therapie ist neben der heilung des kindes auch eine weit möglichst normale entwicklung. eltern tumorkranker kinder fällt es oft schwer, ihre handicaps zu akzeptieren und damit umzugehen. nur durch eine optimale erkennung von defiziten und deren rehabilitation kann es gelingen, dass überlebende kinder trotz ihrer defizite einen platz in der mitte unserer gesellschaft finden. unbehandelt sterben kinder mit einer akuten leukämie innerhalb weniger wochen bis monate. patienten mit einer chronischen leukämie können auch ohne behandlung monate bis jahre überleben. j prädisponierende faktoren prädisponierende faktoren für das auftreten einer leukämie sind angeborene oder erworbene störungen des immunsystems, wie z. b. ataxia teleangiectatica (louis-bar-syndrom), bloom-syndrom, agammaglobulinämie, schwerer kombinierter immundefekt, common-variable-immundefekt und wiskott-aldrich-syndrom. deutlich häufiger als andere kinder erkranken auch solche mit chromosomalen störungen, wie z. b. mit down-syndrom, fanconi-anämie und krankheiten, bei denen dna-reparaturprozesse beeinträchtigt sind. für eine genetische komponente bei der leukämieentstehung spricht auch die tatsache, dass das relative erkrankungsrisiko bei monozygoten zwillingen wesentlich höher ist als bei geschwistern eines leukämiekranken kindes. die wahrscheinlichkeit einer leukämieerkrankung beträgt bei unter jahre alten eineiigen zwillingen etwa %. j pathogenese leukämien entstehen durch ein zusammenwirken von genetischen, immunologischen und wachstumsregulierenden vorgängen, beeinflusst von umgebungsfaktoren. unklar ist aber, wie der prozess der klonalen proliferation der leukämiezellen zustande kommt. j klassifizierung die einfachste methode zur klassifizierung der leukämien ist die zytomorphologie. blut-oder knochenmarkausstriche werden nach panoptischer färbung (zumeist nach wright) im lichtmikroskop beurteilt. nach der french-american-british (fab)-klassifizierung wird die all in die typen fab l -l eingeteilt. beim typ fab l handelt es sich um eine "reife" b-zellneoplsie, die nach der neuen klassifikation nicht mehr als lymphoblastisch sondern als akute leukämie vom burkitt-typ bezeichnet wird und einer vollkommen anderen behandlung bedarf (. abb. . und . abb. . ). die aml wird in die typen fab m -m eingeteilt. diese einteilung orientiert sich an den reifungsstufen der normalen knochenmarkzellen (. abb. . , . abb. . und . abb. . ). phosphatase-reaktion, bei der aml die esterase-und peroxidasereaktion von bedeutung. sich spezifische translokationen, durch die fusionsgene entstehen. typische befunde sind in . tab. . dargestellt. bei der cml findet man fast immer ein philadelphia-chromosom, entsprechend einer translokation t( ; ), mit einem fusionsgen, dem bcr-abl-gen, kd (m-bcr-abl) lang. selten gibt es diese translokation auch bei der all, wobei das fusionsgen nur kd lang (m-bcr-abl) ist. die genprodukte solcher fusionsgene haben häufig wachstumsregulierende funktionen. es können dadurch aber auch gene aktiviert, deaktiviert oder dysreguliert werden und so der zelle ein abnormes wachstumsverhalten verleihen. neben diesen spezifischen translokationen kommen in leukämiezellen auch andere aberrationen, wie z. b. inversionen, deletionen, oder auch in etwa % der fälle numerische anomalien, zum größten teil hyperdiploidien durch überzählige chromosomen , x, und , gelegentlich auch hypodiploidien vor, deren bedeutung bisher nicht eindeutig definiert ist. zur charakterisierung der klonalität der leukämiezellen dienen bei der all weiterhin umordnungen (rearrangements) von immunglobulin-und t-zellrezeptorgenen. weil leukämiezellen nicht normal sind, sind diese aber nicht linienspezifisch. mithilfe von amplifikationsverfahren (polymerasekettenreaktion) können sie dazu verwendet werden, residuelle leukämiezellen bis zu einer verdünnung - - - nachzuweisen ("minimal residual disease", mrd). in den letzten jahren sind neue genetische veränderungen, z. b. mutationen des ikaros-gens oder auch andere prognostisch ungünstige aberrationen beschrieben worden, die u. u. strukturen für "gezielte therapien" sind. j pathophysiologie die ungehemmte vermehrung der leukämiezellen führt zu einer knochenmarkmetaplasie und damit zu einer verdrängung der normalen hämatopoese, also einer funktionellen knochenmarkinsuffizienz. aber auch andere organe werden von leukämiezellen infiltriert. j klinik erste, uncharakteristische symptome, wie mattigkeit und spielunlust treten bei akuten leukämien nach einer kurzen, etwa - wochen dauernden anamnese auf. die leitsymptome sind: blässe, blutungsneigung (hämatome und petechien) und fieber. sie entstehen durch die verdrängung der normalen blutbildung im knochenmark. das ausmaß der durch die beeinträchtigung der blutbildung verursachten symptome ist variabel. ein weiteres wichtiges und oft übersehenes oder fehlgedeutetes symptom sind bei der körperlichen untersuchung findet sich eine deutliche schwellung der lymphknoten (> cm) bei knapp %, eine hepatosplenomegalie bei etwa % und eine vergrößerung des thymus bei etwa % der kinder. eine durch leukämiezellen verursachte liquorpleozytose, anfangs meist ohne symptome, ist bei ca. % aller kinder mit einer all nachweisbar. leukämische haut-oder gingivainfiltrate sind selten, man findet sie eher bei myeloischen leukämien. bei sehr hohen leukozytenzahlen, wie z. b. bei der cml, kann bei jungen einmal ein priapismus auftreten. bei der cml findet sich auch meist eine ausgeprägte splenomegalie. tro-und thrombozytopenie, seltener dagegen eine hohe leukozytose. im median liegt die initiale leukozytenzahl bei . /µl. anhand der leukozytenzahl allein lässt sich also eine leukämie beim kind nicht ausschließen. auch die automatisierte differenzialblutbilddiagnostik ist nicht ausreichend, es muss eine manuelle, mikroskopische beurteilung erfolgen. bei kindern mit einer cml findet man in der chronischen phase eine erhöhte proliferation aller zellstränge mit einer vermehrung von eosinophilen und basophilen granulozyten. das blutbild sieht ähnlich aus wie das knochenmark. beim Übergang in einen blastenschub steigt der anteil von blasten (unreife leukämiezellen) an, und es kommt schließlich zu einer hämatopoetischen insuffizienz wie bei den akuten leukämien. die blasten können dabei einen myeloischen oder auch einen lymphatischen phänotyp aufweisen. die abgrenzung gegen die all ist nur durch die kenntnis der anamnese und ggf. durch den nachweis des philadelphia-chromosoms, bzw. des m-bcr-abl-fusionsgens, möglich. knochenmarkpunktion beweisend für die diagnose ist die knochenmarkpunktion. sie wird bei kindern meist am hinteren beckenkamm durchgeführt. außer für die morphologische diagnostik wird knochenmark für andere spezielle untersuchungen entnommen. bei kindern mit einer all findet sich eine über %ige metaplasie mit leukämiezellen bei einer funktionell weitgehend erloschenen normalen hämatopoese. schwieriger ist die beurteilung des knochenmarks bei myeloischen leukämien, insbesondere bei den differenzierteren formen, da die leukämiezellen morphologisch nicht eindeutig von normalen hämatopoetischen vorläuferzellen zu unterscheiden sind. typisch sind aber die monomorphie des zellbilds und das fehlen oder die verminderung von erythro-und megakaryozytopoese. bei erythroleukämien ist die erythropoese dysplastisch und deutlich von der normalen roten blutbildung zu unterscheiden. zur initialdiagnostik gehört weiterhin eine lumbalpunktion, die nur durch einen erfahrenen untersucher erfolgen darf. ein befall des zns wird diagnostiziert, wenn im liquor mindestens zellen/ mm nachweisbar sind, die mikroskopisch als leukämische blasten imponieren. > die diagnose wird durch die knochenmarkpunktion gestellt; auch eine lumbalpunktion ist stets erforderlich. j differenzialdiagnose reaktive leukozytosen z. b. bei infektionen können das bild einer leukämie vortäuschen. besonders ist die infektiöse mononukleose zu erwähnen, bei der neben vergrößerten lymphknoten und einer hepatosplenomegalie auch eine immunthrombozytopenie mit hämorrhagischer diathese bestehen kann. lymphozytosen bis zu . /µl, allerdings mit reifen lymphozyten und ohne zeichen einer hämatopoetischen insuffizienz, gibt es auch bei der pertussis und bei der infektiösen lymphozytose. bei leukämien mit besonders niedriger leukozytenzahl ist immer eine knochenmarkpunktion zur abgrenzung von der schweren aplastischen anämie erforderlich. knochenschmerzen, die oft ein wesentliches symptom der leukämie darstellen, führen häufig zunächst zur fehldiagnose einer rheumatoiden arthritis. eine rasche klärung ist durch eine sorgfältige körperliche untersuchung und ein blutbild möglich. > knochenschmerzen sind häufig und werden oft als "rheuma" fehlgedeutet. weitere klinische differenzialdiagnosen sind infektionskrank heiten, wie die toxoplasmose, zytomegalie oder auch adenovirusinfektio- an die intensiveren behandlungsphasen während etwa des ersten halben jahres schließt sich die remissionserhaltende dauertherapie, bei der all bis zu einer gesamtdauer von mindestens jahren an. wesentliche medikamente während dieser behandlungsphase sind antimetabolite, bei der all -mercaptopurin täglich und mtx -mal wöchentlich oral. bei der aml werden nach dem erreichen der remission - weitere intensive chemotherapieblöcke verab-reicht. die dauertherapie wird diskutiert, ist aber in den meisten internationalen protokollen nicht vorgesehen. bei der aml fab-m ist der aktuelle standard die kombination von chemotherapie mit all-trans-retinsäure (atra). diskutiert wird bei patienten mit niedrigem risiko der verzicht auf chemotherapie zugunsten einer kombination von atra mit arsentrioxid. da l-asparaginase auch die endokrine pankreasfunktion beeinträchtigt, kommt es infolge der gleichzeitigen gabe von glukokortikoiden nicht selten zu einer insulinpflichtigen diabetischen stoffwechsellage. eine weitere nebenwirkung von l-asparaginase ist eine synthesestörung von gerinnungsfaktoren, die entweder zu blutungskomplikationen (hypofibrinogenämie) oder auch zu thrombembolischen komplikationen (at-iii-mangel) führen kann, besonders dann, wenn außerdem eine thrombophilie besteht. das auftreten von blutungskomplikationen wird durch eine gleichzeitig bestehende thrombozytopenie begünstigt. mit zunehmender immunsuppression steigt das risiko für das auftreten von infektionen mit opportunistischen erregern, wie z. b. pneumocystis jiroveci (früher: carinii) oder auch viren der herpesgruppe. zu den schweren, durch die langfristige immunsuppression bedingten komplikationen gehören auch pilzinfektionen. im rahmen der hochdosierten methotrexattherapie kann es zu nierenfunktionsstörungen mit verzögerter mtx-ausscheidung kommen. diese behandlung muss daher genau überwacht werden. insbesondere ist auf die zeitgerechte gabe des antidots, kalziumfolinat, zu achten, weil es sonst bei den verabreichten dosen zu einer letalen mtx-toxizität kommen würde. sowohl während der mtxals auch während der l-asparaginasetherapie können zerebrale krampfanfälle auftreten, die aber meist ohne bleibende folgen sind. > nur durch eine parallel zur zytostatischen therapie durchgeführte supportivtherapie und durch ein gut ausgebildetes und erfahrenes behandlungsteam lässt sich das risiko für das auftreten von schweren oder gar lebensbedrohlichen komplikationen angemessen begrenzen. mit adäquater unterstützender behandlung, insbesondere auch der rechtzeitigen präventiven gabe von gut wirksamen antiemetika und ggf. auch analgetika, ist die therapie aber relativ gut durchführbar und für die kinder erträglich. die früher hohe therapieassoziierte letalität ließ sich trotz zunehmender intensität der behandlung bei der all auf etwa - % und bei der aml auf - % senken. j therapie der chronischen leukämien juvenile myelomonozytäre leukämie (jmml) die einzige kurative therapie ist derzeit die allogene stammzelltransplantation. chronische myeloische leukämie vom erwachsenentyp mit der chemotherapie lässt sich lediglich eine reduktion der zellzahl erreichen. verwendet werden hydroxyharnstoff, busulfan, melphalan oder triethylenmelamin. eine neue dimension hat die einführung der tyrosinkinaseinhibitoren (tki) eröffnet, die die bcr-ablkodierte tyrosinkinase hemmen und mit denen sogar molekulare remissionen erreichbar sind. bereits initial wird der relativ nebenwirkungsarme tki imatinib eingesetzt. bei einem blastenschub mit myeloischem phänotyp sollte eine behandlung wie bei der aml durchgeführt werden, bei lymphatischem phänotyp eine all-therapie. die indikation zur allogenen stammzelltransplantation, früher der einzigen kurativen therapie, wird heute durch die therapiemöglichkeit mit tki zurückhaltend gestellt. j prognose Überlebenswahrscheinlichkeit mit den heute verfügbaren behandlungsmöglichkeiten bleiben nach einmaliger therapie etwa - % aller kinder mit einer all in anhaltender remission; bei der aml sind es etwa - %. rezidiv bei etwa % der all-und % der aml-patienten kommt es zu einem rezidiv, d. h. zum wiederauftreten der leukämie an irgendeinem ort. am häufigsten treten rezidive im knochenmark auf. sie können sich aber auch an anderen organen, wie im zns, bei jungen im hoden oder auch an der haut oder in lymphknoten manifestieren. auch nach einem rezidiv ist oft noch eine kurative behandlung möglich. einige kinder sind mit erneuter chemotherapie heilbar, bei anderen, insbesondere bei früh auftretenden all-knochenmark-und aml-rezidiven, ist eine allogene stammzelltransplantation erforderlich. immerhin überleben nach einem all-rezidiv etwa %, bei der aml etwa - % der kinder, sodass mit erfolgreicher erst-und zweitbehandlung insgesamt etwa - % aller all-und % der aml-patienten langzeitüberlebende sind. kinder mit einer jmml haben mit einer stammzelltransplantation eine Überlebenschance von etwa %. kinder mit einer cml bleiben mit der tki-therapie jahrelang in remission. spätfolgen der behandlung möglich sind organbezogene spätfolgen (kardiologische, endokrinologische, hepatische und zentralnervöse, letztere nach dem wegfall der strahlentherapie selten). eine gravierende und besonders bei über -jährigen mädchen häufige spätfolge ist die avaskuläre knochennekrose, meist des hüftkopfs. nach einer aml-behandlung kann es infolge der hohen kumulativ verabreichten anthrazyklindosen zu einer dilatativen kardiomyopathie kommen, die u. u. zu einer akuten myokardinsuffizienz führt. latente kardiomyopathien sind bei einem teil der aml-patienten nachweisbar, klinisch manifeste aber selten. das risiko für das auftreten einer sekundärneoplasie ist gegenüber der normalbevölkerung erhöht. kumulativ liegt es nach jahren bei einer medianen beobachtungsdauer der patienten von , jahren ( , - jahre) bei , %. die erhöhte inzidenz von sekundärneoplasien ist partiell auf die behandlung aber wohl auch auf andere faktoren, wie z. b. eine erhöhte genetisch bedingte prädisposition, zurückzuführen. im allgemeinen haben kinder, die von ihrer leukämie geheilt sind, eine erfreulich normale lebensqualität. im täglichen leben sind sie meist nicht erkennbar beeinträchtigt. zahlreiche patienten befinden sich bereits im erwachsenenalter und sind in der lage, ein normales berufsleben zu führen. zum teil sind diese ehemaligen patienten auch bereits eltern gesunder eigener kinder. maligne lymphome sind bösartige erkrankungen des lymphatischen systems. etwa % sind non-hodgkin-lymphome (nhl), bei kindern fast immer von hoher malignität, und % hodgkin-lymphome (m. hodgkin, ältere bezeichnung auch lymphogranulomatose). insgesamt stellen sie etwa % aller krebserkrankungen bei kindern und jugendlichen dar. j klassifizierung die einteilung der nhl erfolgt nach kriterien, die die analogie zwischen der lymphomzelle und der korrespondierenden normalen zelle des immunsystems (b-, t-und großzellig-anaplastische nhl) und den reifegrad (unreif oder präkursor und reif oder periphere vorläufer) berücksichtigen. b-nhl sind überwiegend reife neoplasien, die bereits zur immunglobulinsynthese und -sekretion befähigt sind, während t-nhl in der mehrzahl lymphoblastische, also präkursor-nhl darstellen. großzellig-anaplastische nhl ("anaplastic large cell lymphoma", alcl) sind relativ selten, exprimieren meist neben t-zellmarkern das oberflächenantigen cd (syn. ki- ), und es ist häufig eine translokation t( ; ), alk/npm-fusion ("anaplastic lymphoma kinase/nucleophosmin") nachweisbar. j therapie die behandlung der hoch malignen lymphatischen neoplasien besteht aus einer intensiven kombinationschemotherapie. sie erfolgt entsprechend dem immunologischen subtyp und dem ausbreitungsstadium, entweder nach murphy oder aktueller dem internationalen pädiatrischen nhl staging system (ipnhlss). die behandlung der lymphoblastischen t-und b-nhl ist weitgehend identisch mit der der all. reife b-nhl bedürfen einer völlig anderen, sehr intensiven und toxischen chemotherapie. alcl werden ähnlich wie b-nhl behandelt. eine strahlentherapie ist meist nicht erforderlich, eine präventive zns-behandlung aber obligat. die behandlung muss daher in spezialisierten zentren erfolgen, in denen man diese komplikationen kennt und darauf eingerichtet ist. j prognose mit entsprechender therapie können etwa % aller kinder mit b-und t-nhl geheilt werden. wenn rezidive auftreten, so geschieht dies früh, bei b-nhl meist innerhalb des ersten jahres nach diagnosestellung. die erfolgsaussichten einer kurativen behandlung nach einem rezidiv sind ausgesprochen schlecht. j epidemiologie , % der malignen erkrankungen im kindesalter (< jahre) sind neuroblastome (das sind neuerkrankte kinder/jahr), % sind jünger als jahre und ⅓ erkranken im . lebensjahr. die inzidenz beträgt ca. , / . . kinder < , das verhältnis jungen zu mädchen , : (alle epidemiologischen daten des kapitels aus dem jahresbericht des kinderkrebsregisters; www.kinderkrebsregister.de). j pathologie das neuroblastom ist ein maligner embryonaler tumor und entsteht aus den zellen der neuralleiste. da aus diesen zellen die ganglien des sympathischen nervensystems und das nebennierenmark hervorgehen, findet man neuroblastome entlang des grenzstrangs (zervikal, thorakal, abdominal) und in der nebenniere (. abb. . ). eine besonderheit ist das einwachsen nach intraspinal als sog. sanduhrtumor. das neuroblastom gehört zur gruppe der tumoren, die sich durch "kleine, blaue und runde" zellen auszeichnen; typischerweise können sie rosettenförmig angeordnet sein (homer-wright-rosetten). die histopathologische einteilung erfolgt in deutschland nach hughes in malignitätsgrade entsprechend dem ausreifungsgrad. die "international neuroblastoma pathology classification" (inpc) hat die shimada-klassifikation international abgelöst und unterscheidet je nach anteil des schwann-stromas neuroblastome (arm an schwannschem stroma), ganglioneuroblastome (reich) und ganglioneurome (dominant). unerlässlich zur einschätzung der prognose und damit zur therapiesteuerung sind molekulargeneti- bei der behandlung von patienten mit stadium-iv-tumoren haben sich die hochdosistherapie und die allogene stammzelltransplantation nicht durchsetzen können. die cws-gruppe empfiehlt daher gegenwärtig für patienten jenseits des höchstrisikos (für die neue wege gesucht werden müssen) eine metronomische therapie mit oral einzunehmenden medikamenten. j prognose die prognose ist abhängig von histologie, lokalisation, ansprechen und stadium. in der studie cws betrug die rezidivfreie Überlebensrate nach jahren für alle patienten mit lokalisierten tumoren zwischen und %. j nachsorge Ähnlich wie bei anderen tumoren erfolgt eine krankheits-und ein spätfolgen bezogene nachsorge, um langzeitfolgen ebenso frühzeitig zu erkennen wie rezidive und zweitmalignome (kumulative inzidenz , % nach jahren). die behandlung von rezidiven ist sinnvoll und umso erfolgreicher, je später der rückfall auftritt und je kleiner der primärtumor war. j definition das nephroblastom ist ein maligner embryonaler tumor der niere. j epidemiologie mit einer inzidenz von , / . . kindern < jahren ist das nephroblastom der häufigste nierentumor im kindesalter. , % aller malignen tumoren im kindesalter sind nephroblastome, das entspricht neuerkrankten kindern/jahr (www.kinderkrebsregister.de). der häufigkeitsgipfel liegt zwischen dem . und . lebensjahr; % der betroffenen kinder sind jünger als jahre. mädchen erkranken ein einem verhältnis von , : etwa häufiger als jungen. j pathologie es handelt sich um einen embryonalen tumor, der histologisch in die subtypen niedrigmaligne ( %), intermediär ( - %) und hochmaligne ( - %) unterteilt wird. bei der pathologisch-anatomischen klassifikation muss beachtet werden, ob sie an einer niere nach erfolgter chemotherapie oder nach primärer operation erfolgt. so zählt der blastemreiche typ nach primärer operation zur niedrigen malignität, nach präoperativer chemotherapie jedoch zur hohen malignitätsgruppe. die stadieneinteilung nach siop (. tab ein gutes ansprechen auf eine präoperative chemotherapie liegt vor, wenn der pathologe ein histopathologisches ansprechen grad (kein vitaler resttumor), grad (vitaler resttumor < %) oder grad (vitaler resttumor < %) findet. ein schlechtes ansprechen muss diagnostiziert werden, wenn der pathologe zwischen und % vitalen resttumor (grad ), > % (grad ) oder gar kein ansprechen auf die chemotherapie findet. leider ist es bisher nicht gelungen, patienten, die schlecht auf die initiale therapie ansprechen durch eine intensivierung der therapie eine bessere chance auf rezidivfreiheit zu sichern. zur heilung ist eine komplette tumorentfernung im gesunden erforderlich. wenn immer möglich wird die operation extremitä-tenerhaltend durchgeführt. der knochendefekt kann dabei mit einer endoprothese überbrückt werden. möglich sind auch rekonstruktive verfahren mit verwendung von eigenen skelettmaterial oder auch sog. umkehrplastiken bei kniegelenknahen tumoren. j prognose die Überlebenswahrscheinlichkeit liegt nach jahren bei %. j definition die gruppe der ewing-sarkome umfasst morphologisch ähnliche typen, die als klassisches ewing-sarkom (es), atypisches ewing-sarkom und maligner peripherer neuroektodermaler tumor (mpnet) bezeichnet werden. ihre zusammenfassung wird gerechtfertigt durch den nachweis einer einheitlichen molekulargenetischen veränderung bei allen zugehörigen tumoren. j epidemiologie ewing-sarkome sind die zweithäufigsten malignen knochentumoren im kindes-und jugendalter; , % aller bösartigen erkranken sind ewingsarkome ( /jahr). der häufigkeitsgipfel liegt zwischen . und . lebensjahr. es erkranken , / . . < jahren und / . . in der bevölkerung mit männlicher prädisposition ( , : ). j pathologie es handelt sich um einen tumor, der sich, ähnlich wie andere hochmaligne tumoren im kindesalter, durch "kleine, runde blaue" zellen auszeichnet. in der abgrenzung zu anderen tumoren ist es wesentlich, glykoproteinen durch die pas-reaktion und ggf. neuronale marker (nse, s- ) nachzuweisen. von großer bedeutung sind zytogenetische untersuchungen, mit deren hilfe in % die translokation t( ; ) (q ;q ) (betrifft die gene ewsr und fli ), und in % die translokation t( ; ) (q ;q ) (betrifft die gene ewsr und erg) gefunden wird. resultat ist ein onkoprotein, das als aberranter transkriptionsfaktor zur gendysregulation beiträgt. diese untersuchungen sind nicht nur für die exakte diagnose selbst, sondern auch für die beurteilung des verlaufs (nachweis von minimaler resterkrankung) hilfreich. aufgabe des pathologen ist es auch, das ansprechen auf die therapie zu beurteilen. ein anteil von ≤ % vitaler tumorzellen nach ende der präoperativen therapie gilt als "good response", > % gelten als "poor response". mit dieser dreifachkombination wurde in philadelphia eine rückfallfreie Überlebensrate von % nach jahren und von % nach jahren erzielt. bei ¼ der kinder können metastasen initial im liquor (m -stadium) und bei ⅓ der kinder mit der mrt supratentoriell (m -stadium) oder spinal (m -stadium) nachgewiesen werden (. tab. . ). zur verbesserung ihrer schlechten prognose ist eine intensivierung der strahlentherapie (hyperfraktioniert, akzeleriert) und der chemotherapie (z. b. hochdosistherapie) erforderlich. in den ersten lebensjahren werden % der medulloblastome des kindes-und jugendalters diagnostiziert. bei diesen kindern verhält sich das medulloblastom teilweise aggressiver als bei jugendlichen oder erwachsenen. sie hatten früher eine sehr schlechte prognose und besonders häufig unter spätfolgen zu leiden, die vom tumor und der strahlentherapie verursacht wurden. daher wurden konzepte zur verzögerung oder vermeidung der strahlentherapie entwickelt, mit denen insbesondere für kinder mit desmoplastischen oder extensiv nodulären medulloblastomen auch ohne bestrahlung deutlich verbesserte Überlebensraten erreicht wurden. ependymom in einer konsekutiven studie am royal marsden hospital lag die -jahres-Überlebensrate nach adjuvanter chemotherapie mit ccnu und vincristin mit % signifikant höher als mit % nach alleiniger bestrahlung. mit einer intensiven chemotherapie nach einer hyperfraktionierten bestrahlung konnte sogar eine Überlebensrate von ca. % nach jahren erzielt werden. daher werden kinder mit anaplastischen ependymomen häufig analog den medulloblastomen auch chemotherapeutisch behandelt. gliome von hoher malignität mit ccnu und vincristin nach der postoperativen bestrahlung konnte die -jahre-rückfallfreie Überlebensrate von kindern mit einem anaplastischen astrozytom oder glioblastom mehr als verdoppelt werden. in der folgestudie wurden mit kombinierter postoperativer strahlen-und chemotherapie -jahre-rückfallfreie Überlebensraten von % beim anaplastischen astrozytom, % beim glioblastom und % bei anderen malignen gliomen wie z. b. dem anaplastischen oligodendrogliom erzielt. derzeit besteht die hoffnung, dass mit postoperativer bestrahlung und verbesserter chemotherapie oder mit experimentellen therapieansätzen eine verbesserung der prognose erreicht werden kann. die infauste prognose von kindern mit diffus intrinsisch wachsen- dieser hochdosistherapieeffekt verbesserte die -jahresüberlebenswahrscheinlichkeit von kindern mit neuroblastom (stadium iv, > jahr) von % im chemotherapieerhaltungsarm auf % nach autologer szt. kinder mit stadium iv und kinder, deren tumor die amplifikation des n-myc-gens aufweist, profitieren besonders. kinder mit regional begrenztem ewing-sarkom haben mit chemotherapie eine rezidivfreie Überlebenswahrscheinlichkeit von bis zu % und sind keine kandidaten für eine autologe szt. eine untergruppe von kindern mit knochen-oder knochenmarkmetastasen bei diagnose verfügen über eine wesentlich schlechtere prognose und sollten einer hochdosistherapie zugeführt werden. junge kinder mit primärer metastasierung eines medulloblastoms bei diagnosestellung profitieren von der hochdosistherapie. primitive neuroektodermale tumoren (pnet), die histologisch dem medulloblastom ähneln, sind tumoren mit supratentorieller lokalisation. obgleich primär chemotherapiesensibel, weisen diese tumoren nach einem rezidiv meist einen letalen verlauf auf. nach neuesten untersuchungen können möglicherweise junge kinder durch die tandemhochdosischemotherapie eine remission erfahren. nichtmaligne erkrankungen für eine reihe pädiatrischer patienten mit knochenmarkinsuffizienz ("bone marrow failure syndromes"), hämoglobinopathien und immundefekten stellt die allogene stz z. z. die einzige kurative behandlungsmöglichkeit dar. sie wird in spezialisierten behandlungszentren durchgeführt. abstoßungsreaktion j grundlagen in seltenen fällen kann das neue knochenmark von wenigen überlebenden immunologischen effektorzellen des empfängers als fremd erkannt werden und abgestoßen werden, sodass entweder primär kein anwachsen der neuen stammzellen erfolgt oder nach vorübergehendem anwachsen der neuen zellen eine abstoßung im zeitlichen intervall eintritt. verantwortlich für beide prozesse sind sowohl t-lymphozyten als auch nk-zellen des empfängers. diese komplikation ist relativ selten ( - %). häufiger wird sie bei polytransfundierten hla-sensibilisierten patienten beobachtet (patienten mit schwerer aplastischer anämie oder β-thalassaemia major). j klinik bei primärem nichtangehen persistiert die phase der aplasie. bei sekundärer abstoßung kommt es zum vorübergehenden ansteigen der granulozyten, gefolgt von einem erneuten absinken der leukozytenzahlen mehrere tage nach zunächst erfolgreicher hämatopoetischer regeneration. begleitet wird diese episode oft von einer vorübergehenden relativen lymphozytose und fieber. j therapie immunologische rekonditionierung und erneute transplantation. j prognose erfolgsquote ca. % rezidiv > das leukämische rezidiv ist die häufigste todesursache nach zunächst erfolgreicher stz. j grundlagen das rezidiv leitet sich von wenigen leukämischen blasten mit extensiver teilungs-und proliferationskapazität ab, die die vorausgehende konditionierung durch chemotherapeutika und/oder tbi überlebt haben. für die immunologische Überwachung residualer blasten nach transplantation ist der gehalt an immunologischen effektorzellen von großer bedeutung. so weisen transplantationen mit klassischer t-zelldepletion eine höhere rate an rezidiven auf. j häufigkeit - %. j klinik häufig kündigt ein abfall der thrombozytenzahl ein drohendes hämatologisches rezidiv an. im knochenmark finden sich dabei > % blasten, meist verbunden mit dem ausschwemmen unreifer vorstufen in das periphere blut. j therapie verschiedene therapieansätze kommen in unterschiedlichen situationen zum einsatz: absetzen der immunsuppression: eine immunsuppressive therapie ist während der ersten - tage nach allogener nicht t-zelldepletierter stz erforderlich, um eine graftversus-host-erkrankung ( abschn. . . ) möglichst zu vermeiden. in frühen stadien eines rezidivs kann -in einzelnen fällen -durch absetzen der immunsuppression eine remission erzielt werden. die gefahr der induktion einer überschießenden akuten gvhd ist relativ hoch. chemotherapie: durch chemotherapie wird versucht, eine erneute remission zu erreichen. je später das rezidiv nach transplantation auftritt, desto günstiger ist dabei dieser versuch. patienten mit einem rezidiv innerhalb der ersten tage nach transplantation können nur äußerst selten in eine erneute remission gebracht werden. alternative donor" (fremdspender oder hla-nichtidentischer familienspender); c bei vorliegen bestimmter mutationen; d zurzeit in behandlungsprotokollen spezialisierter zentren schwere aplastische anämie; g schwerer kombinierter immundefekt den ponsgliomen konnte durch eine zusätzliche chemotherapie bislang nicht verbessert werden.benigne und niedrigmaligne gliome bei kindern mit einem gliom who i oder ii, das nicht vollständig reseziert werden konnte, gelten folgende gründe als indikationen für eine postoperative nachbehandlung (. abb bei % der kinder wurde eine tumorverkleinerung und bei % eine besserung der beschwerden beobachtet. so gelang es bei kindern unter jahren, den zeitpunkt der strahlentherapie in einen höheren altersbereich hinauszuschieben und die störungen der kognitiven und neuroendokrinen funktionen zu vermindern. dies ist von großer bedeutung, um das risiko einer transplantatgegen-empfänger-erkrankung ( abschn. . . , gvhd) möglichst gering zu halten. bei fehlen eines hla-identischen spenders, kann in bestimmten situationen auf ein elternteil als stammzellspender zurückgegriffen werden. in diesem fall darf jedoch -wegen der gefahr einer massiven gvhd -lediglich die hochaufgereinigte stammzellfraktion transplantiert werden. das hla-muster eines elternspenders stimmt meist lediglich zur hälfte mit dem des kindes überein ("haploidentisch"). der zeitliche verlauf einer transplantation gliedert sich in phasen (. abb. . ): konditionierung, transplantation, aplasie.konditionierung die phase der konditionierungsbehandlung dauert in der regel - tage und umfasst die sequenzielle gabe verschiedener chemotherapeutika mit oder ohne fraktionierte ganzkörperbestrahlung (tbi, "total body irradiation"). ziele dieser inten-siven behandlung sind zum einen die zerstörung residualer tumor-/leukämiezellen, zum anderen die ausschaltung der körpereigenen immunabwehr des empfängers, sodass neues knochenmark nicht mehr abgestoßen werden kann. chemotherapeu tika beinhalten vornehmlich substanzen der gruppe der alkylan zien. der einsatz fraktionierter ganzkörperbestrahlung im kindesalter wird wegen ihrer negativen auswirkungen auf wachstum, endokrine organe, fertilität und wegen der späten induktion von zweittumoren im kindesalter (≥ jahren) nur bei hochrisikopatienten eingesetzt. bei fremdspendertransplantationen und bei haploidentischen transplantationen wird zur konditionierung antilymphozytenglobulin (atg) als zusätzliche prophylaxe gegen eine mögliche abstoßungsreaktion eingesetzt.transplantation die transplantation selbst beinhaltet die i.v.-infusion entweder des gesamten knochenmarks oder der aufgereinigten stammzellfraktion über einen zentralvenösen katheter. die infundierten spenderstammzellen finden über eine kaskade bestimmter adhäsions-und homing-rezeptoren den weg in den nun "leeren" knochenmarkraum, siedeln sich im stroma des knochenmarkraumes an und beginnen -vermittelt durch signale des lokalen stroma -sich zu teilen und zu differenzieren (. abb. . ).aplasie gefolgt werden die beiden vorangegangenen zeitabschnitte durch die phase der aplasie. in dieser phase teilen sich die infundierten stammzellen in tochterstammzellen und in progenitorzellen, die sich sowohl in die verschiedenen blut bildenden linien aufteilen als auch zu antigenpräsentierenden zellen differenzieren. insgesamt ist ein zeitraum von ca. - tagen (autologe szt) bzw. - tagen (allogene szt) nötig, bis eine leukozytenzahl > . / µl bzw. eine granulozytenzahl > /µl im peripheren blut erreicht wird.während der aplasiephase kommen die toxischen nebenwirkungen der konditionierungstherapie zeitverzögert zur ausprägung. diese können beinhalten: aus diesen gründen wird in den meisten transplantationszentren eine engmaschige molekularbiologische Überwachung während des ersten jahres nach transplantation durchgeführt, um patienten mit einem drohenden rezidiv frühzeitig zu erkennen und einer therapie ( abschn. . . und abschn. . . ) zuzuführen.diese molekularbiologische Überwachung erfolgt mittels: chimärismus-analyse: mit dieser technik kann der prozentuale anteil von spender-zu empfängerzellen im peripheren blut in engmaschigen abständen (zunächst wöchentlich, später monatlich) kontrolliert werden. spender-und empfängerzellen unterscheiden sich dabei in der länge sog. minisatellitenregionen (engl. vntr, "variable number of tandem repeats"). ein zunehmender gemischter chimärismus identifiziert patienten mit einem hohen rezidivrisiko. analyse der minimalen resterkrankung: mit dieser technik kann die spezifische restlast residualer leukämiezellen (z. b. bcr-abl-positiver cml-blasten, nachweis des spezifischen klonalen rearrangements der variablen (v), diversen (d), joining (j) und konstanten (k) regionen des leukämischen klons) in der regel im knochenmark erfasst werden. die restlast (mrd, "minimal residual disease") sollte im verlauf der ersten monate nach szt durch die graft-versus-leukemia-reaktion ( abschn. . . ) verschwinden. die indikationen zur knochenmark-bzw. stammzelltransplantation umfassen eine reihe maligner und nichtmaligner systemerkrankungen im kindesalter (. tab. . ). j therapie ganciclovir, foscarnet; ggf. cidofovir. j grundlagenreife t-lymphozyten im transplantat erkennen neben antigenstrukturen residualer maligner zellen auch antigenstrukturen gesunder gewebe des empfängers als "fremd". durch den kontakt mit den antigenen des transplantatempfängers werden diese t-zellen aktiviert, proliferieren und führen über multiple primäre und sekundäre mechanismen zum immunologischen angriff auf gesundes gewebe, der sog. graft-versus-host-erkrankung (syn. transplantatgegen-empfänger-erkrankung).j klinik die akute gvhd stellt eine der wichtigsten komplikationen nach allogener knochenmark-bzw. stammzelltransplantation dar, deren häufigkeit trotz immunsuppressiver therapie und trotz hla-identität in verschiedenen studien zwischen und % liegt und deren letalität bis zu % betragen kann. die gvhd tritt klinisch in unterschiedlichen manifestationsformen auf: die akute gvhd stellt eine lebensbedrohliche erkrankung dar, die die zielorgane haut, intestinum und leber betrifft und während der ersten tage nach szt auftritt. die chronische gvhd tritt gewöhnlich nach den ersten tagen auf und beschreibt ein autoimmunartiges krankheitsbild mit sklerodermieartigen hautveränderungen, atrophie und chronischer entzündungen der mukosa v. a. im bereich der schleimhäute und der konjunktiven.akute gvhd sie tritt innerhalb der ersten tage nach transplantation auf. j prophylaxe die wahl der optimalen gvhd-prophylaxe ist in zahlreichen studien intensiv untersucht worden und wird bislang von verschiedenen zentren unterschiedlich gehandhabt. agenzien, die in der prävention z. z. eingesetzt werden, sind ciclosporin a, fk , prednison, methotrexat und mycophenolat mofetil. die inzidenz der akuten gvhd bei hla-identischen geschwisterspendern beträgt bei der mehrzahl dieser pharmakologischen regime - %. entscheidender nachteil aller dieser kombinationen ist ihr generalisierter effekt der immunsuppression, der zu einer erhöhten rate an lebensbedrohlichen infektionen nach allogener transplantation führt und möglicherweise auch die immunologische erkennung und Überwachung residualer leukämiezellen durch das transplantat negativ beeinflusst.j therapie tritt trotz gvhd-prophylaxe eine akute gvhd auf, so umfasst die therapie -je nach schweregrad der erkrankung -die gabe von decortin in steigender dosierung ( - mg/kgkg/tag) sowie ggf. die verabreichung eines antikörpers, der gegen t-zellen, gegen tnf-α oder gegen den il- -rezeptor gerichtet ist. neue ansätze wie z. b. immunmodulatorisch wirkende mesenchymale stromazellen (msc) oder die extrakorporale photopherese werden derzeit ge testet. therapie: ebv-spezifische t-zellklone; solide tumoren ( - %), zweitleukämien (nach - jahren). die szt ist ein sich rasch entwickelndes feld. neue therapieansätze, hervorgegangen aus der grundlagenforschung, finden mit rascher geschwindigkeit ihren einzug in die klinische anwendung. einige der wichtigsten neuen entwicklungen werden im folgenden kurz dargestellt. ziel dieses ansatzes ist die hochselektive anreicherung humaner hämatopoetischer stammzellen. diese stammzellpopulationen sind positiv für die marker cd und negativ für linienspezifische marker reiferer zellpopulationen. im autologen setting dient diese aufreinigung der entfernung kontaminierender tumorzellen. im allogenen setting wird diese methode durchgeführt, um reife immunkompetente t-zellen und b-zellen aus dem transplantat zu entfernen. die entfernung der t-zellen führt zu einer reduktion der gvhd-inzidenz auf %, während die b-zelldepletion entscheidend zur verringerung ebv-assoziierter lymphome im ersten jahr nach transplantation beiträgt. neben diesen positiven selektionsverfahren werden in jüngster zeit negative depletionsverfahren weiterentwickelt, mit deren hilfe aus einem haploidentischen stammzelltransplantat lediglich reife t-und b-zellen entfernt werden, während neben stammzellen weitere lymphatische progenitoren und dendritische zellen im transplantat erhalten bleiben. dieses verfahren trägt mit dazu bei, die regeneration des immunsystems nach transplantation zu beschleunigen. es findet anwendung bei kindern und jugendlichen, die über keinen konventionellen hla-identischen spender verfügen. durch engmaschiges molekularbiologisches monitoring (serielle chimärismusanalyse des bluts) können diejenigen patienten prospektiv identifiziert werden, bei denen ein rückfall unmittelbar bevorsteht. dabei kommt ein pcr-gesteuertes stufenkonzept zum einsatz, in dem in der ersten stufe zunächst auf die immunsuppression verzichtet wird und bei zunehmenden positiven empfängersignal spenderlymphozyten in steigender dosierung verabreicht werden. durch diesen ansatz kann es bei bis zu % der patienten gelingen, ein drohendes rezidiv zu verhindern. autoimmunerkrankungen werden durch ein repertoire autoreaktiver t-und b-zellklone unterhalten, die -möglicherweise getriggert durch molekulare mimikry zwischen viralen und körpereigenen epitopen -initial einige wenige körpereigene antigene als fremd erkennen und dagegen reagieren. in der weiteren folge einer erkrankung kommt es zum "epitope spreading", der ausweitung des spektrums der erkrankung auf andere autoantigene. in pilotstudien für patienten mit multipler sklerose (ms) oder mit therapierefraktärer juveniler rheumatoider arthritis konnte durch transplantation aufgereinigter autologer stammzellen eine langandauernde remission induziert werden. key: cord- -khhzlt y authors: jain, aditya; leka, stavroula; zwetsloot, gerard i. j. m. title: work, health, safety and well-being: current state of the art date: - - journal: managing health, safety and well-being doi: . / - - - - _ sha: doc_id: cord_uid: khhzlt y this introductory chapter will present a review of the current state of the art in relation to employee health, safety and well-being (hsw). the work environment and the nature of work itself are both important influences on hsw. a substantial part of the general morbidity of the population is related to work. it is estimated that workers suffer million occupational accidents and million occupational diseases each year. the chapter will first define hsw. it will then review the current state of the art by outlining key hsw issues in the contemporary world of work, identifying key needs. it will then discuss the evolution of key theoretical perspectives in this area by linking theory to practice and highlighting the need for aligning perspectives and integrating approaches to managing hsw in the workplace. this chapter focuses on the relationship between work, health, safety and wellbeing. the work environment and the nature of work itself are both important influences on health, safety and well-being (hsw). as a result, workplace health and safety or occupational health and safety have been key areas of concern for many years. traditionally, more focus has been placed on safety concerns in the workplace while health concerns became more prominent with the changing nature of work. well-being on the other hand, is increasingly being considered in relation to work and the workplace in recent years. a good starting point in understanding this evolution in focus and thinking is definitions. according to the oxford dictionary, safety is defined as the condition of being safe; freedom from danger, risk, or injury. safety can also refer to the control of recognized hazards in order to achieve an acceptable level of risk. in terms of work, this mainly concerns physical aspects of the work environment. however, the changing nature of work was associated with the emergence of new types of risk relating to psychological and social aspects of the work environment. this brought about greater focus on health at work. a very influential definition that shaped thinking and action in subsequent years was the world health organization definition of health as a state of complete physical, mental and social well-being and not merely the absence of disease or infirmity (world health organization [who], ) . this definition promoted a more holistic view of health away from a mere focus on physical aspects towards considering social and mental health aspects. although the who definition already referred to a state of well-being, definitions of well-being include additional dimensions to health, such as social, economic, psychological, and spiritual. well-being refers to a good or satisfactory condition of existence; a state characterized by health, happiness, and prosperity. obviously achieving this state is not relevant to the workplace or work alone but rather an overall evaluation of one's life across many areas. as such, actions to improve hsw can be taken within the work context and outside of it. actions taken in the workplace represent workplace interventions that are implemented in the work setting and consider the characteristics of work environments and workers. on the other hand, actions taken outside the workplace represent public health interventions that are implemented in various settings (for example, in schools, communities or countries) and take into consideration the characteristics of particular populations. a key question in terms of hsw interventions when it comes to the workplace concerns responsibility. while every individual is responsible for their own actions in various contexts of life, in a specific setting like the work environment, additional responsibility lies with the employer since the work environment will expose workers to particular work characteristics that might in turn pose a certain level of risk to their hsw. while employer responsibility might be formalized under law, this is not the case across countries or in relation to all possible types of risks to workers' hsw, and in particular new and emerging risks, or risks that are either new or gain in prevalence with the changing nature of work. accordingly, it is important to consider not only legal duties that employers have towards their workforce but also ethical duties that will extend beyond legal compliance. in addition, while employers bear a legal responsibility towards their workforce, they also bear responsibility towards society. this has meant that enterprises have increasingly been held accountable towards society and that interventions in the workplace, whether legally required or not, are now being increasingly considered in terms of their impact beyond the workforce alone but rather society as a whole (see chapters , , and ). this represents a blurring of boundaries between traditional occupational safety and health and public health initiatives that have also resulted in greater emphasis on the concept of well-being in addition to health and safety. at its first session in , the joint international labour organization (ilo)/ world health organization (who) committee on occupational health defined the purpose of occupational health. it revised the definition at its th session in to read as follows: occupational safety and health should aim at: the promotion and maintenance of the highest degree of physical, mental and social well-being of workers in all occupations; the prevention amongst workers of departures from health caused by their working conditions; the protection of workers in their employment from risks resulting from factors adverse to health; the placing and maintenance of the worker in an occupational environment adapted to his physiological and psychological capabilities; and, to summarize, the adaptation of work to man and of each man to his job. almost years later, the target set through this declaration seems ambitious in many parts of the world, both in developed and developing countries. to understand why, it is worth understanding the context underpinning developments in this area as well as current priorities and needs. in recent years, globalization of the world's economies and its repercussions have been perceived as the greatest force for change in the world of work, and consequently in the scope of occupational safety and health, in both positive and negative ways. liberalization of world trade, rapid technological progress, significant developments in transport and communication, shifting patterns of employment, changes in work organization practices, the different employment patterns of men and women, and the size, structure and life cycles of enterprises and of new technologies can all generate new types and patterns of hazards, exposures and risks. demographic changes and population movements, and the consequent pressures on the global environment, can also affect safety and health in the world of work. let us first consider key impacts on the changing nature of the work environment. different types of products and services, organizational structures and work processes, and tools and resources are used in the modern workplace. three main drivers have been proposed in relation to these changes. the first is globalization, a term which refers to the integration of national and regional economies, which became more prevalent since the nineteenth century. according to the organization for economic co-operation and development (oecd, ) , the rapid integration into world markets by six economies (brazil, russia, india, indonesia, china and south africa) was an important component of globalization during the past decades. globalization has led to increased competition across organizations, to a shift in the type of business operations in which companies are engaged, and to extensive outsourcing of activities, primarily to low-wage countries. flanagan ( ) examined the effects of globalization on working conditions (hours, remuneration and safety) and concluded that globalization has led to greater flexibility of the work process, with more part-time employment, temporary employment and independent contracting of staff (european agency for safety & health at work [eu-osha], ; kawachi, ) . houtman and van den bossche ( ) confirmed these conclusions on the basis of eurostat data, reporting that more employees in europe hold a temporary employment contract and yet more people will work 'on call'. oecd reports also confirm these trends. they also highlight that average wage growth has not been equivalent to growth in labour productivity, which is also an outcome of the erosions of the bargaining power of workers in the process of globalization (oecd, ). organizational restructuring which has been on the increase due to economic crises in different parts of the world may have been partly a cause of this. organizational restructuring is accompanied by job insecurity and can result in unemployment with subsequent negative impacts on hsw. however, restructuring should not only be considered a serious threat to individual hsw for those who lose their job (the 'direct victims') but also to their immediate environment (e.g. kieselbach et al., ). in addition, evidence during the past two decades showcases the impact of restructuring on the so-called 'survivors' as concerns health, well-being, productivity, and organizational commitment (kieselbach et al., ) . the second key development is the tertiarization of the labour market, manifested in increased demand for staff in the services sector and reduced employment opportunities in industry and agriculture. this became apparent in the early years of the twentieth century but in recent decades may have been reinforced by globalization, since the outsourcing of manual labour to low-wage countries left predominantly the service economy elsewhere (eu-osha, ; peña-casas & pochet, ) . the third key development relates to technological advancement and the emergence of the internet, which has led to many changes and innovations in work processes. many forms of manual work have become obsolete and staff must offer different skills and qualifications (joling & kraan, ) . moreover, 'new work', a term which amongst others refers to telework, i.e. working from home or a location other than the traditional office, is now more widespread. this can result in blurring the borders between working and private life. work can take place outside the traditional working hours as well as at home or when travelling. hence, it may impinge on the need for rest and recuperation, or interfere with personal commitments. also new forms of working methods such as lean production (a production practice according to which the expenditure of resources other than for the creation of value for the end customer is wasteful and should be eliminated, womack & jones, ) , and just-in-time production (a production strategy that strives to improve a business' return on investment by reducing in-process inventory and associated costs, womack & jones, ) have been introduced (eu- osha, ; kompier, ) . overall there has been concern of the effects new forms of work may have on the hsw of workers, organizations and communities (e.g. benach, amable, muntaner, & benavides, ; benavides, benach, diez-roux, & roman, ; quinlan, ; quinlan, mayhew, & bohle, ; sauter et al., ; virtanen et al., ) . it is also important to mention the prevalence of small and medium-sized enterprises (smes) that are believed to be responsible for over % of new jobs created globally. moreover, in most developing and emerging countries, they also employ more people than large enterprises do. however, occupational safety and health (osh) is often less well managed in smes, creating working conditions that are less safe and posing greater risks to the health of workers than larger enterprises (croucher, stumbitz, quinlan, & vickers, ) . in particular, smes have less time to devote to providing osh training and information due to economies of scale, and have less expertise in hsw. research also confirms a common lack of awareness of the cost implications of occupational accidents and diseases amongst sme owners and managers, as well as a tendency for smes to be reactive, rather than adopting proactive and preventive strategies towards osh (croucher et al., ) . however, there are also changes in the workforce that are associated with hsw in the workplace. the next section considers the most important of these. alongside the factors changing the nature of work itself, changes can also be seen in the working population, with noteworthy trends being: (a) the ageing workforce; (b) the feminization of the workforce; and (c) increased immigration (leka, cox, & zwetsloot, ) . let us now consider these issues in more detail. in industrialized countries, the share of people aged -plus has risen from % in to % and is expected to reach % ( million) by . in developing countries, the share of people aged -plus has risen from % in to % and is expected to reach % ( . billion) by (world economic forum [wef], ). the global population is projected to increase . times from to , but the number of -plus will increase by nearly %, and the -plus by about %. women have a life expectancy of . years more than men and account for about % of the -plus group, rising to % of the -plus group, and % of the -plus group (wef, ) . in response to these global trends, four strategies have been proposed: raising the normal legal retirement age; using international migration to ameliorate the economic effects of population ageing; reforming health systems to have more emphasiz on disease prevention and health promotion; and rethinking business practices, encouraging businesses to employ more older workers, even on a part-time basis (wef, ) . according to the oecd ( ) most countries will have a retirement age for both men and women of at least years by , and this has already been implemented in many countries. this represents an increase from current levels of around . years on average for men and . years on average for women. the same report stresses that high levels of youth unemployment will lead to widespread poverty in old age as young people struggle to save for retirement. since population ageing in industrialized nations has been a prevalent trend in the past decades (ilmarinen, ) , lessons can be learned from it in relation to the workforce. most reviews and meta-analyses in the scientific literature make clear that there is no consistent effect of age on work performance (e.g., benjamin & wilson, ; griffiths, ; salthouse & maurer, ) . overall, older workers perform as well as younger workers. furthermore, there are many positive findings with regard to older workers. for example, older workers demonstrate less turnover and more positive work values than younger workers (warr, ) . they also exhibit more positive attitudes to safety and fewer occupational injuries (siu, phillips, & leung, ) although there is some evidence that it is tenure (time on the job) that should be examined rather than age per se (breslin & smith, ) . however, the evidence from epidemiological and laboratory-based studies paints a less favourable picture of older people's performance. such studies reveal age-related declines in cognitive abilities such as working memory capacity, attention capacity, novel problem-solving, and information processing speed. agerelated deterioration is also documented in motor-response generation, selecting target information from complex displays, visual and auditory abilities, balance, joint mobility, aerobic capacity and endurance (kowalski-trakofler, steiner, & schwerha, ) . as workers get older, they suffer from more musculoskeletal disorders (eurostat, ) , and they are more likely to report work-related stress (griffiths, ) . recent models of ageing and work propose that certain mediating factors underpin the relationship between chronological age, work performance and behaviour and might function at three levels: individual, organizational and societal. at the individual level, for example, experience, job knowledge, abilities, skills, disposition, and motivation may operate (kanfer & ackerman, ) . other mediating variables may reflect organizational policies and practices: for example, age awareness programmes, supervisor and peer attitudes, management style, the physical work environment and equipment, health promotion, workplace adjustments, and learning and development opportunities (griffiths, ) . however, policies and systems implemented so far have, in most countries, not been adequately successful in keeping people healthier and in employment for longer (oecd, ) . a further level of exploration for the relationship between age and work performance might be provided by examining global markets, the wider employment context and worker protection (johnstone, quinlan, & walters, ; quinlan, ) . as discussed, in developed countries there has been a decline in manufacturing and a recent export of some service sector work to developing countries. the way work is designed and organized has changed substantially with a growth in contingent or 'precarious' work and an increase in part-time work, home-based work, telework, multiple job-holding and unpaid overtime. these changes might make it increasingly difficult for older workers to gain or maintain employment, and such employment may entail inferior and unhealthy working conditions. these changes in work design and management have also been accompanied by changes in worker protection; for example, a decline in union density and collective bargaining, some erosion in workers' compensation and public health infrastructure and cutbacks in both disability and unemployment benefits -again contexts which are unlikely to favour vulnerable workers, such as older workers. as such older workers may be affected by increased exposure to certain occupational hazards; decreased opportunities to gain new knowledge and develop new skills; less support from supervisors, and discrimination in terms of selection, career development, learning opportunities and redundancy (chiu, chan, snape, & redman, ; maurer, ; molinie, ) . pronounced gender differences in employment patterns can be observed as a result of a highly segregated labour market based on gender (burchell, fagan, o'brien, & smith, ; fagan & burchell, ; vogel, ) . gender segregation refers to the pattern in which one gender is under-represented in some jobs and overrepresented in others, relative to their percentage share of total employment (fagan & burchell, ) . a growing body of evidence indicates that a high level of gender segregation is a persistent feature of the employment structure globally (e.g. anker, ; burchell et al., ; rubery, smith, & fagan, ) . some scholars have argued that estimates suggest that gender segregation in the labour market is so pervasive, that in order to rectify this imbalance approximately % of women would have to change jobs or professions (messing, ) . considering differences in employment patterns according to gender (and without taking into account sectors where both genders are represented, e.g. agriculture), women's jobs typically involve caring, nurturing and service activities for people, whilst men tend to be concentrated in managerial positions and in manual and technical jobs associated with machinery or physical products. since men and women are differently concentrated in certain occupations and sectors, with different aspects of job content and associated tasks, they are exposed to a different taxonomy of work-related risks (burchell et al., ; eu-osha, ) . for example, women are more frequently exposed to emotionally demanding work, and work in low-status occupations with often restricted autonomy, as compared to men. this differential exposure can result in differential impacts on occupational ill health for men and women (eu-osha, ; oecd, ) . furthermore, due to the gender division of labour, women and men play different roles in relation to children, families and communities with implications for their health (premji, ) . even though women are increasingly joining the paid workforce, in most societies they continue to be mainly responsible for domestic, unpaid work such as cooking, cleaning and caring for children, and so they carry a triple burden (e.g. loewenson, ) . women are also largely represented among unpaid contributing family workers, those who work in a business establishment for a relative who lives in the same household as they do (ilo, ) . balancing responsibilities for paid and unpaid work often leads to stress, depression and fatigue (duxbury & higgins, ; manuh, ) , and can be particularly problematic when income is low and social services and support are lacking. the lack of availability of child care may also mean that women must take their children to work where they may be exposed to hazardous environments. increased migration of workers from developing countries to developed countries or from poorer to more affluent developed countries is still the norm and increasing. migrant workers can be divided into highly-educated and skilled workers, both from developing and industrialized countries, and unskilled workers from developing countries (takala & hämäläinen, ) . they can also be classified as legal and illegal (or regular and irregular) migrant workers who have a different status and, therefore, varying levels of access to basic social services (who, ) . often lowskilled and seasonal workers are concentrated in sectors and occupations with a high level of occupational health and safety risks (who, ) . ethnic minority migrants have been found to have different conditions in comparison to other migrants, and to report lower levels of psychological well-being (shields & price, ) . women migrants represent nearly half of the total migrants in the world and their proportion is growing, especially in asia. they often work as domestic workers or caregivers while men often work as agricultural or construction workers (ilo, ) . in general, migrant workers tend to be employed in high risk sectors, receive little work-related training and information, face language and cultural barriers, lack protection under the destination country's labour laws and experience difficulties in adequately accessing and using health services. common stressors include being away from friends and family, rigid work demands, unpredictable work and having to put up with existing conditions (magana & hovey, ) . in addition, migrant workers' cultural background, anthropometrics and training may differ from those of nationals of host countries, which may have implications in relation to their understanding and use of equipment (kogi, ; o'neill, ) . as can be understood so far, both the nature of work and of workplaces as well as workforce characteristics depend on wider socioeconomic and political influences. a large body of literature has summarized and examined these influences under the area of the social determinants of health. the following section briefly considers these determinants. new forms of work organization and employment have to be considered within the wider picture of employment and working conditions across the world. labour markets and social policies determine employment conditions such as precarious or informal jobs, child labour or slavery, or problems such as having high insecurity, low paid jobs, or working in hazardous conditions, all of which heavily influence health inequalities. figure . shows various interrelationships between employment, working conditions and health inequalities. let us consider unemployment and associated job insecurity as social determinants of health. in the ilo estimated that there were almost million unemployed people in the eu, million of whom were from eu- countries. overall, million people were unemployed in with a quarter of the increase of four million in global unemployment being in the advanced economies, and three quarters being in other regions, with marked effects in east asia, south asia and sub-saharan africa (ilo, a). the same report also highlighted that in those regions where unemployment did not increase further, job quality worsened as vulnerable employment and the number of workers living below or very near the poverty line increased. in the eu, the financial crisis resulted in unprecedented levels of youth unemployment, averaging % for the eu as a whole. the rates for young people (aged - ) not in employment, education or training are . % in the south and peripheral eu countries, and . % in the north and core of the eu (european commission [ec], ). in a pattern intensified by the financial crisis, structural unemployment has been growing and unemployment varies from . % in the south of the eu and peripheries in , to . % in the north and central countries (ec, ) . a large proportion of jobs destroyed were in mid-paid manufacturing and construction occupations (european foundation for the improvement of living & working conditions [eurofound] , ). as a consequence of reduced employment opportunities, poverty has increased in the eu since . household incomes are declining and . % of the eu population is now at risk of poverty or exclusion. children are particularly affected as unemployment and jobless households have increased, together with in-work poverty (ec, ) . this has implications for quality of life and general population health beyond workplace health and safety due to the impact on personal finances. an ilo report summarized the potential impact of financial crises on organizations and health and safety as shown in table . . the surge of unemployment creates tension and negatively impacts public perceptions for social welfare, job security, and financial stability. increased job insecurity reflects the fear of job loss or the loss of the benefits associated with the job (e.g. health insurance benefits, salary reductions, not being promoted, changes in workload or work schedule). it is one of the major consequences of today's turbulent economies and is common across occupations, and both private and publicsector employees (ashford, lee, & bobko, ; ferrie et al., ; sverke, hellgren, & naswall, ) . several studies have shown that job insecurity has detrimental effects on the physical and mental health of employees, and on many organizational outcomes, including performance, job satisfaction, counterproductive behaviours, and commitment (e.g. ferrie et al., ; sverke et al., ) . increased unemployment has given rise to different forms of flexible and temporary employment, also through the introduction of relevant policies such as flexicurity. flexicurity is an integrated strategy for enhancing flexibility and security in the labour market. it attempts to reconcile employers' need for a flexible workforce with workers' need for security (ec, ) . however, several studies have warned of the possible negative outcomes of new types of work arrangements, highlighting that they could be as dangerous as unemployment for workers' health (benach & muntaner, ) . for example, workers on fixed-term contracts are commonly found to have inadequate working conditions by comparison with permanent employees. new forms of work organization and patterns of employment can be summarized in terms of flexible working practices including temporary and part-time employment, tele-working, precarious employment, and home working. although these new practices can result in positive outcomes such as more flexibility, a better worklife balance, and increased productivity, research has also identified several potential negative outcomes. for example, teleworkers may feel isolated, lacking support and career progression (e.g. ertel, pech, & ullsperger, ; schultz & edington, ) . in addition, temporary, part-time and precarious employment can result in higher job demands, job insecurity, lower control and an increased likelihood of labour force exit (benach et al., ; quinlan, ; quinlan et al., ) . workers engaged in insecure and flexible contracts with unpredictable hours and volumes of work are more likely to suffer occupational injuries (ilo, a (ilo, , b . although awareness and evidence in developing countries lags far behind those in the industrialized world, evidence has started to accumulate showing similar findings in developing countries (kortum, leka, & cox, ) . these various complex relationships between the wider socio-economic context, employment and working conditions have resulted in a more complex profile of risk factors that may affect hsw in the workplace. new forms of work organization and the move towards a service based economy have also resulted in new and emerging risks affecting the workforce, organizations and society. these will be considered next. an 'emerging osh risk' is often defined as any occupational risk that is both new and increasing (eu-osha, ). new means that the risk was previously unknown and is caused by new processes, new technologies, new types of workplaces, or social or organizational change; or, a long-standing issue is newly considered to be a risk due to changes in social or public perceptions; or, new scientific knowledge allows a long standing issue to be identified as a risk. a risk is increasing if the number of hazards leading to the risk is growing; or, the likelihood of exposure to the hazard leading to the risk is increasing (exposure level and/or the number of people exposed); or the effect of the hazard on workers' health is getting worse (seriousness of health effects and/or the number of people affected) (houtman, douwes, zondervan, & jongen, ). an article published on eu-osha's osh wiki on new and emerging risks summarizes them as follows (houtman et al., ) : • emerging physical risks: ( ) physical inactivity and ( ) the combined exposure to a mixture of environmental stressors that increase the risks of musculoskeletal disorders (msds), the leading cause of sickness absence and work disability. • emerging psychosocial risks: ( ) job insecurity, ( ) work intensification, high demands at work, and ( ) emotional demands, including violence, harassment and bullying. • emerging dangerous substances due to technological innovation: ( ) chemicals, with specific attention to nanomaterials, and ( ) biological agents. the growing use of computers and automated systems, aimed at optimizing productivity, has caused an increase in sedentary work or prolonged standing at work, resulting in an increase in physical inactivity. work demands are also commonly cited as reasons for physical inactivity (e.g. trost, owen, bauman, sallis, & brown, ) as well as an increase in travelling time to work (houtman et al., ) . physical inactivity is associated with increased health risks such as coronary heart disease, type ii diabetes, and certain types of cancers and psychological disorders (depression and anxiety) (department of health, ; who, ; zhang, xie, lee, & binns, ) . another important result of inactivity is obesity which can lead to several adverse health effects, such as back pain, high blood pressure, cardiovascular disorders, and diabetes (houtman et al., ) . in addition, sedentary jobs are associated with an increased prevalence of musculoskeletal complaints or disorders, e.g. neck and shoulder disorders (e.g. korhonen et al., ) , and upper and lower back disorders (e.g. chen, mcdonald, & cherry, ) . such disorders may lead to sick leave and work disability (e.g. steensma, verbeek, heymans, & bongers, ) . the established health risks associated with sedentary work are premature death in general, type ii diabetes and obesity (van uffelen et al., ) . as concerns msds, there is a considerable body of research indicating that biomechanical or ergonomic risks in combination with psychosocial risks can generate work-related msds (e.g. bongers, ijmker, & van den heuvel, ; briggs, bragge, smith, govil, & straker, ; eu-osha, ) . psychosocial risk factors at work have a greater effect on the prevalence of musculoskeletal complaints when exposure to physical risk factors at work is high rather than when it is low. in addition, factors such as low job control, high job demands, poor management support or little support from colleagues, as well as restructuring, job redesign, outsourcing and downsizing have been shown to be causally related to increased risks in msds (houtman et al., ) . job insecurity has been discussed earlier and is an important stressor resulting in reduced well-being (psychological distress, anxiety, depression, and burnout), reduced job satisfaction (e.g. withdrawal from the job and the organization) and increased psychosomatic complaints as well as physical strains (e.g. wagenaar et al., ) . all these effects are negatively related to personal growth as well as to recognition and participation in social life (de cuyper et al., ) . additionally, decreased well-being and reduced job satisfaction of employees negatively affects the effectiveness of the organization (houtman et al., ) . there are several increasing demands workers are exposed to in the modern workplace including: quantitative (high speed, no time to finish work in regular working hours), qualitative (increased complexity), emotional (emotional load due to direct contact with customers i.e. service relationship situations), and often physical loads as well (houtman et al., ) . the widespread use of information and communication technology (ict) has led to work intensification. developments in technology use in terms of mechanization, automation, and computerization, has led to the substitution of human activities by machines. on the other hand, the use of computers and smart phones with internet access provides easy access to all kinds of information but may also lead to the expectation from colleagues, supervisors and clients that one is always available and can be contacted (e.g. by email). ict work may then lead to stress symptoms due to excessive working hours, workload and increasing complexity of tasks or isolation in home workers; information overload; pressure of having to constantly upgrade skills; human relationships replaced by virtual contacts; and physical impairments such as repetitive strain injuries and other msds due to using inadequate or ergonomically unadapted equipment (houtman et al., ) . psychosocial hazards such as high job demands and low control have been systematically found to be causally linked to cardiovascular heart disease (e.g. backé, seidler, latza, rossnagel, & schumann, ; eller et al., ) , msds (e.g. da costa & vieira, ) as well as mental health problems such as depression and anxiety (e.g. bonde, ; netterstrom et al., ) . in addition, long term absence and disability are causally related to these types of risks (e.g. duijts, kant, swaen, brandt, & van den zeegers, ) . furthermore, as the labour market shifts towards the service industry, emotional demands at work increase with harassment or bullying and violence contributing to this increase (houtman et al., ) . those affected by violence and harassment in the workplace tend to report higher levels of work-related ill health. the proportion of workers reporting symptoms such as sleeping problems, anxiety and irritability is nearly four times greater among those who have experienced violence, bullying and harassment than amongst those who have not (houtman et al., ) . nanotechnology has been defined as the design, characterization, production and application of structures, devices and systems by controlling shape and size at nanometre scale (eu-osha, ). due to their small size, engineered nanomaterials (enms) have unique properties that improve the performance of many products. nanomaterials have applications in many industrial sectors (currently the main areas are materials and manufacturing industry including automotive, construction and chemical industry, electronics and it, health and life sciences, and energy and environment). a key issue of enms is the unknown human risks of the applied nanomaterials during their life cycle, especially for workers exposed to enms at the workplace. workers in nanotechnology may be exposed to novel properties of materials and products causing health effects that have not yet been fully explored. the manufacture, use, maintenance and disposal of nanomaterials may have potential adverse effects on internal organs (eu-osha, ). although there is a considerable lack of knowledge, there are indications that because of their size, enms can enter the body via the digestive system, respiratory system or the skin. once in the body, enms can translocate to organs or tissue distant from the portal of entry. such translocation is facilitated by the propensity of the nanoparticles to enter cells, to cross membranes and to move along the nerves (iavicoli & boccuni, ) . the enms may accumulate in the body, particularly in the lungs, the brain and the liver. the basis for the toxicity appears to be primarily expressed through an ability to cause inflammation and to raise potential for autoimmune deficits, and may induce diseases such as cancer (houtman et al., ) . other dangerous substances concerns include diesel exposure and its link to lung cancer and non-cancer damage to the lung; and man-made mineral fiber exposure (classified as being siliceous or non-siliceous) and the link of their structure to inflammatory, cytotoxic and carcinogenic potential (houtman et al., ) . another three chemical risks have been identified as emerging with a view to allergies and sensitizing effects. they are epoxy resins, isocyanates and dermal exposure (eu-osha, ). epoxy resins have become one of the main causes of occupational allergic contact dermatitis. skin sensitization of the hands, arms, face, and throat as well as photosensitization have also been reported. isocyanates are powerful irritants to the mucous membranes of the eyes and of the gastrointestinal and respiratory tracts. direct skin contact can cause serious inflammation and dermatitis. isocyanates are also powerful asthmatic sensitizing agents (houtman et al., ) . finally, risks related to global epidemics are the most important biological risk issue. pathogens such as the severe acute respiratory syndrome (sars), ebola, and marburg viruses are new or newly recognized. in addition, new outbreaks of wellcharacterized outbreak-prone diseases such as cholera, dengue, measles, meningitis, and yellow fever still emerge (houtman et al., ) . it should be stressed that the profile of risks in the workplace constantly changes and there are additive effects that exacerbate negative impacts. the following section provides an overview of key challenges in relation to hsw in the modern workplace while also acknowledging the lack of research in relation to some of the new and emerging risks identified earlier. the ilo has published global estimates of fatal and non-fatal occupational (ilo, ) and fatal work-related diseases (ilo, b). . million deaths occur annually across countries for reasons attributed to work. over , are caused by occupational accidents while the biggest mortality burden comes from work-related diseases, accounting for about million deaths. globally, cardiovascular and circulatory diseases at % and cancers at % were the top illnesses responsible for / of deaths from work-related diseases, followed by occupational injuries at % and infectious diseases at %. as a result, approximately people die every day due to these causes: occupational accidents kill nearly people every day and work-related diseases provoke the death of approximately more individuals. there were also over million non-fatal occupational accidents (requiring at least four days of absence from work) in , meaning that occupational accidents provoke injury or ill health for approximately , people every day (ilo, b). major industrial accidents are stark reminders of the unsafe conditions still faced by many. for example, the april collapse of the rana plaza building in bangladesh resulted in the death of individuals and injured more, mostly factory workers making garments for overseas retail chains. the international community has since expressed concerns about market pressures which strive to keep basic production costs low, the role of national authorities, and the responsibilities of multinational enterprises and other stakeholders in supply chains towards the health and safety of workers. hazardous sectors such as mining, construction, shipping, and in particular fishing continue to take a heavy toll on human lives and health. meanwhile, the nuclear industry continues to pose serious problems regarding the radiological protection of site workers and the environment. in particular, the protection of emergency workers at the fukushima daiichi power plant in japan has become a focus of international attention since the east japan earthquake. occupational health has recently become a much higher priority, in light of the growing evidence of the enormous loss and suffering caused by occupational diseases and ill health across many different employment sectors. even though it is estimated that fatal diseases account for about % of all work-related fatalities, more than half of all countries do not provide official statistics for occupational diseases (ilo, b). these therefore remain largely invisible, compared to fatal accidents. moreover and as discussed previously, the nature of occupational diseases is changing rapidly, as new technologies and global social changes aggravate existing health risks and create new ones. for example, long-latency diseases include illnesses such as silicosis and other pneumoconioses, asbestos-related diseases and occupational cancers that may take decades to manifest. such diseases remain widespread, as they are often undiagnosed until they result in permanent disability or premature death. pneumoconioses account for a high percentage of all occupational diseases. for example, in latin america, there is a % prevalence rate of silicosis amongst miners, and this figure reaches % among miners over the age of . in vietnam, pneumoconioses account for . % of all compensated occupational diseases (ilo, b) . the use of asbestos has been banned in more than counties, including all eu member states, but the number of deaths from asbestos-related diseases is increasing in many industrialized countries because of exposure that occurred during the s and later. in germany and the uk, for example, the number of deaths from asbestos-induced mesothelioma has been increasing for some years and was expected to peak in - (health & the number of cases of work-related stress, violence and psychosocial disorders has also been increasing. these have often been attributed at least in part to recession-driven enterprise restructuring and redundancies which can be very damaging psychologically. european studies have shown that a large and rapid rise in unemployment has been associated with a significant increase in suicide rates (e.g. lundin & hemmingsson, ). meanwhile, a review of mortality studies in countries across the world has also shown an increase in cardiovascular mortality rates by an average of . % in periods of crisis (falagas, vouloumanou, mavros, & karageorgopoulos, ) . the impact of the issues discussed in this section is presented in chapter . on the basis of the available evidence, it is now recognized that a new paradigm of prevention is required, one that focuses on work-related diseases and not only on occupational injuries. recognition, prevention and treatment of both occupational diseases and accidents, as well as the improvement of recording and notification systems are high priorities for improving the health of individuals and the societies they live in. several perspectives and associated approaches have been taken to promote hsw in the workplace over the years as priorities change and new issues and knowledge emerge. the following section will provide an overview of some key perspectives that have led to the development of modern holistic models to promote hsw in the workplace. the field of occupational health and safety has been defined as the science of the anticipation, recognition, evaluation and control of hazards arising in or from the workplace that could impair the hsw of workers, taking into account the possible impact on the surrounding communities and the general environment (alli, ) . given the broad scope of this definition, several disciplines are relevant to osh that relate to control of the multitude of hazards in the workplace. furthermore, since social, political, technological and economic changes are constantly impacting upon the workplace, the field of osh has been evolving to address new and emerging issues in line with different perspectives. some disciplines of relevance to osh include engineering, ergonomics, toxicology, hygiene, medicine, epidemiology, psychology, sociology, education, and policy. these disciplines often diverge in terms of theoretical foundation and as a result emphasize different aspects in terms of understanding and dealing with osh issues. however, in recent years there has been convergence in thinking about the work environment and a trend towards more holistic perspectives and approaches when considering hsw. indeed, while hsw issues were in the past approached from a mono-disciplinary perspective, multi-disciplinarity is now advocated as the necessary way forward. however, in practice osh professionals often still employ mono-disciplinary perspectives in dealing with accidents and diseases in the workplace, seeking to protect individual workers rather than preventing negative impacts of the work environment and promoting positive outcomes. solely focusing on ameliorating harm rather than promoting hsw has also been criticized in recent years by scholars emphasizing a salutogenic (health promoting) instead of a pathogenic (disease preventing) perspective. let us now consider some of these approaches further in relation to safety, health and well-being. it has been argued that occupational safety has developed and evolved through three ages: . a technical age, . a human factors age, and . a management and culture age (hale & hovden, ) (or as hudson, described them through a technical wave, a systems wave and a culture wave). several authors have since then suggested new ages in safety science. the first age of safety concerned itself with the technical measures to guard machinery, stop explosions and prevent structures collapsing. it lasted from the nineteenth century through until after the second world war and was interested in accidents having technical causes (hale & hovden, ) . the period between the world wars saw the development of research into personnel selection, training and motivation as prevention measures, often based on theories of accident proneness (see hale & glendon, for a review; burnham, for the accident-prone theory). this brought about the second age of safety, which developed separately to technical measures until the period of the s and s, when developments in probabilistic risk analysis and the rise and influence of ergonomics led to a merger of the two approaches in health and safety. there was a move away from an exclusive dominance of the technical view of safety in risk analysis and prevention, and the study of human error and human recovery or prevention came into its own (hale & hovden, ) . just as the second age of human factors was ushered in by increasing realizations that technical risk assessment and prevention measures could not solve all problems, so were the s characterized by an increasing dissatisfaction with the idea that health and safety could be captured simply by matching the individual to technology. in the s management and culture were the focus of development and research, based on many influential thinkers such as heinrich who published his ground-breaking safety management textbook in heinrich, , the sociotechnical management literature (e.g. elden, ; thorsrud, ; trist & bamforth, ) , the social organizational theory of lewin ( ) , the loss prevention approach (bird, ) , and the introduction of participative management in safety (e.g. simard & marchand, ) . however, reason ( ) contended that an over-reliance on osh management systems and insufficient understanding of, and insufficient emphasis on, workplace culture, can lead to failure because "it is the latter that ultimately determines the success or failure of such systems" (p. ). criticism of overreliance on systems was also influenced by the resilience engineering school that posited that instead of focusing on failures, error counting and decomposition, we should address the capabilities to cope with the unforeseen. the ambition is to 'engineer' tools or processes that help organizations to increase their ability to operate in a robust and flexible way. hopkins ( ) views safety culture as one aspect of organizational culture, or more particularly an organizational culture that is focused on safety. further, culture is viewed as a group, not an individual, phenomenon; efforts to change culture, should, in the first instance, focus on changing collective practices (the practices of both managers and workers) and the dominant source of culture is what leaders pay attention to. much of hopkins' work draws on reason's ( ) notion that a safe culture is an informed culture and sutcliffe's ( , ) principles of collective mindfulness and high reliability organizations (i.e. organizations that are able to manage and sustain almost error-free performance despite operating in hazardous conditions where the consequences of errors could be catastrophic). collective mindfulness is based on the premise that variability in human performance enhances safety whilst unvarying performance can undermine safety, particularly in complex socio-technical systems. glendon, clarke, and mckenna ( ) argued that each of the first three periods of development build on one another and refer to this process of development as the fourth age of safety or the integration age where previous ways of thinking are not lost, but remain available to be reflected upon as multiple, more complex perspectives develop and evolve. however, as the limitations of osh management systems and safety rules that attempt to control behaviour have become evident, it has also been proposed that a fifth age of safety has emerged, the adaptive age; an age which transcends the other ages of safety. the adaptive age challenges the view of an organizational safety culture and instead recognizes the existence of socially constructed sub-cultures. the adaptive age embraces adaptive cultures and resilience engineering and requires a change in perspective from human variability as a liability and in need of control, to human variability as an asset and important for safety (borys, else, & leggett, ) . resilience engineering is similar to collective mindfulness since it also focuses on the importance of performance variability for safety. however, what sets resilience engineering apart from collective mindfulness is the focus on learning from successful performance (hollnagel, ) , i.e. why things go right as well as why things go wrong (also called the safety approach (hollnagel, ) . one particular major development in the safety evolution was the move towards managing risks in the work environment. this implied that it is impossible to completely control all aspects of work to avoid negative outcomes, risks always remain. in an ever-changing work environment, a continuous assessment of risks is needed that will point to key risks that may pose a threat to workers' hsw. these then need to be managed following appropriate actions at various levels with the focus being on prevention. the risk management paradigm has been hugely influential not only in terms of managing safety but also managing health as will be discussed in the following sections. let us then consider it further next. in the wake of the chernobyl disaster in , sociologist ulrich beck published 'risikogesellschaft', later published in english as 'risk society: towards a new modernity' in . beck argued that environmental risks had become the predominant product of industrial society. he defined a risk society as "a systematic way of dealing with hazards and insecurities induced and introduced by modernization itself" (beck, , p. ) . while according to british sociologist anthony giddens ( ) , a risk society is a society that is increasingly preoccupied with the future (and also with safety), which generates the notion of risk. giddens ( ) defined two types of risks as external risks (for example natural disasters) and manufactured risks (for example, those derived from industrial processes. as manufactured risks are the product of human activity, authors like giddens and beck argue that it is possible for societies to assess the level of risk that is being produced, or that is about to be produced, in order to mitigate negative outcomes (i.e. responsibility with managing these risks lies with society and more precisely with experts able to do so). one such area is osh risk management. hazard, something that can cause harm if not controlled, is a key term in osh risk management. the outcome is the harm that results from an uncontrolled hazard. in the context of osh, harm describes the direct or indirect degradation, temporary or permanent, of the physical, mental, or social well-being of workers. a risk is a combination of the probability that a particular outcome will occur and the severity of the harm involved (nunes, ) . hazard identification or assessment is an important step in the overall risk assessment and risk management process. through this, hazards are identified, assessed and controlled/eliminated as close to source as reasonably as possible. as technology, resources, social expectations or regulatory requirements change, hazard analysis focuses control measures more closely towards the source of the hazard aiming at prevention. hazard-based programmes may not be able to eliminate all risks to hsw but they avoid implying that there are 'acceptable risks' in the workplace (nunes, ) . a risk assessment needs to be carried out prior to making an intervention. this assessment should identify hazards, identify all affected by the hazard and how, evaluate the risk, and identify and prioritize appropriate control measures. the calculation of risk is based on the likelihood or probability of the harm being realized and the severity of the consequences. the assessment should be recorded and reviewed periodically and whenever there is a significant change to work practices. the assessment should include practical recommendations to control the risk. once recommended controls are implemented, the risk should be re-calculated to determine if it has been lowered to an acceptable level (nunes, ) . risk assessment and calculation is usually easier as regards physical risks but more complex as regards biological, and even more so psychosocial, risks. despite this, the risk management paradigm has been applied to all these types of risks to hsw, and is used extensively both as concerns occupational injury and occupational health. it also represents the cornerstone of osh legislation across countries. osh management systems are based on this paradigm (see chapter for more details). following the pdca (plan-do-check-act) cycle methodology (deming, ) , risk management is a systematic process that includes the examination of all characteristics of the work system where the worker operates, namely, the workplace, the equipment/machinery, materials, work methods/practices and work environment. the main goal of risk management is to eliminate or at least to reduce the risks that cannot be avoided or eliminated to an acceptable level. risk management measures should follow the hierarchy of control principles of prevention, protection and mitigation. worker participation is key in the process of risk management. the risk management process should be reviewed and updated regularly, for instance every year, to ensure that the measures implemented are adequate and effective. additional measures might be necessary if the improvements do not show the expected results (nunes, ) . periodic risk management is also important since workplaces are dynamic due to changes in equipment, substances or work procedures, and new hazards might emerge. another reason is that new knowledge regarding risks can become available, either leading to the need of an intervention or offering new ways of controlling the risk. the review of the risk management process should consider a variety of types of information and draw them from a number of relevant perspectives (e.g. staff, management, stakeholders). however, risk management has been criticized for focusing too heavily on avoiding (controlling) possible negative outcomes and not promoting positive and healthy work environments. this development in thinking has stemmed from a parallel move from pathogenic to salutogenic approaches in health and its management. this evolution in thinking about health and well-being will be considered next. approaches in occupational health and occupational hygiene have evolved in line with developments in several disciplines, including safety engineering, medicine and psychology. the risk management perspective is the cornerstone of occupational hygiene as is evident by its definition. the international occupational hygiene association (ioha, n.d.) refers to occupational hygiene as the discipline of anticipating, recognizing, evaluating and controlling health hazards in the working environment with the objective of protecting worker health and well-being and safeguarding the community at large. although occupational health definitions similarly place great focus on managing risk factors, they overall refer to the promotion and maintenance of health and well-being of employees. similarly to the evolution of perspectives in safety, these definitions have been influenced by the evolution of thinking on health and well-being over the years (schulte & vainio, ) . perspectives on health and illness started with a focus on pathogenesis, as pioneered and developed by williamson and pearse ( ) which is the study of disease origins and causes. pathogenesis starts by considering disease and infirmity and then works retrospectively to determine how individuals can avoid, manage, and/or eliminate that disease or infirmity. the dose-response relationship of the change in effect on an organizm caused by differing levels of exposure (or doses) to a stressor after a certain exposure time was influential in treating disease and illness (as was in chemical safety). this leads professionals using pathogenesis to be reactive because they respond to situations that are currently causing or threatening to cause disease or infirmity (becker, glascoff, & felts, ) . a major shift came in with antonovsky's concept of salutogenesis, the study of health origins and causes, which starts by considering health and looks prospectively at how to create, enhance, and improve physical, mental and social well-being (antonovsky, ) . the assumption of salutogenesis that action needs to occur to move the individual towards optimum health, prompts professionals to be proactive because their focus is on creating a new higher state of health than is currently being experienced (antonovsky, ) . the difference between the biomedical model (based on pathogenesis) and health promotion which is now the cornerstone of public health (based on salutogenesis) is a move away from risk and disease towards resources for health and life (eriksson & lindström, ) , initiating processes not only for health but wellbeing and quality of life. perceived good health is a determinant of quality of life. according to breslow ( ) , the first era of public health involved combating communicable diseases while the second dealt with chronic diseases. their focus was on developing and maintaining health since health provides a person the potential to have the opportunity and ability to move towards the life they want. to facilitate management of health in the first two eras, measurement of the signs, symptoms and associated risks of disease and infirmity were of paramount importance. in the third era of public health most people expect a state of health that enables them to do what they want in life. to facilitate management of an evolved health status, it is necessary to develop new health measures that must go beyond detecting pathogenesis and its precursors to measuring those qualities associated with better health (breslow, ) . however, salutogenesis also presumes that disease and infirmity are not only possible but likely because humans are flawed and subject to entropy (antonovsky, ) . according to a salutogenic perspective, each person should engage in health promoting actions to cause health while they secondarily benefit from the prevention of disease and infirmity. pathogenesis, on the other hand in a complementary fashion primarily focuses on prevention of disease and infirmity, with a secondary benefit of health promotion. both approaches are needed to facilitate the goal of better health and a safer and more health enhancing environment. pathogenesis improves health by decreasing disease and infirmity and salutogenesis enhances health by improving physical, mental, and social well-being. together, these strategies will work to create an environment that nurtures, supports, and facilitates optimal well-being (becker et al., ) . around the same time when salutogenesis was introduced, in a article in science, psychiatrist george l. engel introduced a new medical model, the biopsychosocial model. the biopsychosocial model is a broad view that attributes disease outcome to the intricate, variable interaction of biological factors (genetic, biochemical, etc.), psychological factors (mood, personality, behaviour, etc.), and social factors (cultural, familial, socioeconomic, medical, etc.) . it holds to the idea that biological, psychological, and social processes are integrally and interactively involved in physical health and illness. it was pioneering in advocating the premise that people's psychological experiences and social behaviours are reciprocally related to biological processes. as a result, interventions should address all these dimensions and not narrowly focus on limited perspectives (such as only the biological perspective for example). more focus was now placed on psychological and social factors in the understanding of health and illness. indeed, the traditional medical model of ill health was increasingly recognized as having achieved limited success in tackling occupational health conditions such as stress, anxiety, depression and msds (white, ) . these challenges which have been shown to now have an increasing prevalence in the workplace (as discussed earlier), do not have a clear underlying physical basis nor do they demonstrate a linear relationship between injury, pain and disability. instead, they appear to be strongly mediated by psychological and social factors. accordingly, waddell ( ) categorized such conditions as 'common health problems'. the challenges presented by common health problems contrasts with the past success of occupational medicine in dealing with conditions that have an identifiable cause and a clear relationship between dose and response (waddell & burton, ) . the psychological models that were developed within the fields of occupational, and occupational health psychology, mainly to make sense of the concept of stress, were similarly influenced by conceptualizations of health, illness and safety. early models viewed stress either as a noxious stimulus in the environment (engineering models, derived from engineering) or a response to exposure to aversive of noxious characteristics of the environment (physiological models, derived from medicine). contemporary models focus on the interaction between the environment and the individual and emphasize either explicitly or implicitly the role of psychological processes, such as perception, cognition, and emotion (psychological models). these appear to determine how the individual recognizes, experiences, and responds to stressful situations, how they attempt to cope with that experience and how it might affect their physical, psychological, and social health (cox & griffiths, ) . the risk management paradigm remains an influential perspective in dealing with new and emerging risks in the psychosocial work environment. however, while we are a long way from the challenge of work-related stress being tackled effectively, there has started to be a shift towards promoting well-being at work and not only preventing stress and its associated negative outcomes in terms of both health and safety. this shift has followed trends in public health (discussed earlier) and also psychology towards more positive concepts. the positive psychology movement, championed by seligman and csikszentmihalyi ( ) , is an attempt to shift the emphasis in psychology away from a preoccupation with the pathological, adverse and abnormal aspects of human behaviour and experience. the positive psychology literature offers a number of perspectives that help with understanding how well-being can arise in work situations (lunt et al., ) . for example, the concept of flow was introduced by csikszentmihalyi ( ) which can be defined as a subjective condition where an individual is fully absorbed in, and engaged with, the task he or she is carrying out, promoting an experience of competence and fulfillment. as is evident from our discussion on perspectives on hsw so far, several useful models have been proposed from various disciplines with parallel developments can be observed across these disciplines. however, it should also be noted that often scholars and practitioners operate in silos, ignoring the interplay among the various approaches, and lessons that can be learned from one another. the recent focus on well-being has brought about the question of whether approaches in the workplace should focus only on factors influencing the individual's experience in the work environment or wider influences, considering more the social determinants of health discussed at the beginning of this chapter. in line with this thinking, some holistic models have emerged that recognize the interplay between workplace and non-workplace factors in determining hsw that will be discussed next. the starting point in the development of holistic models of hsw is the recognition that safety and health are different to well-being. as discussed at the beginning of this chapter, well-being refers to a good or satisfactory condition of existence; a state characterized by health, happiness, and prosperity. in particular, three key concepts have been discussed as relevant to well-being: happiness, quality of life and resilience (lunt et al., ) . layard ( ) defined happiness as feeling good; its inverse is feeling bad and wishing for a different experience. factors that affect our levels of happiness include among others family relationships, our financial situation, work, community and friends, our health, personal freedom and personal values. quality of life overlaps with contemporary interpretations of happiness. quality of life is a subjective state that encompasses physical, psychological, and social functioning. a defining feature of quality of life is its basis on the perceived gap between actual and desired living standards. resilience of individuals has been described as partly a context dependent characteristic, in that what enables resilience in one environment may be less adaptive in another (lunt et al., ) . increasingly it is recognized that resilience is important at different organizational levels (teams, organizations) and that these different levels are to some degree interacting (e.g. schelvis, zwetsloot, bos, & wiezer, ) . it is also important to recognize that even though well-being at work may be primarily an employer's responsibility (as well as the worker's), well-being of the worker or workforce is also the responsibility of others in society (e.g. governments, insurance companies, unions, faith-based and non-profit organizations) or may be affected by non-work domains (schulte et al., -see also chapter ). indeed, the well-being of the workforce extends beyond the workplace, and public policy should consider social, economic, and political contexts. schulte et al. ( ) also provide examples of holistic policy models aiming at the promotion of well-being in the workplace that include the who healthy workplace model and the niosh total worker health model (discussed in the next chapter). to promote hsw holistically, there needs to be synergy and integration among the various perspectives. to achieve this, these perspectives need to be aligned considering current knowledge and existing needs, developing capabilities, and mainstreaming a strategic approach in policy and practice. the following chapter considers key policy approaches to managing hsw at the macro level (international, regional, national), meso level (sectoral), and micro level (organizational). subsequent chapters further consider how alignment across perspectives can be achieved in policy and practice. this chapter has provided an overview of the current state of the art in relation to hsw in the workplace as regards key determinants, outcomes and perspectives. with the changing nature of work and new characteristics of the workforce, new challenges are emerging in the workplace. perspectives on how to address these challenges have changed in line with these developments as well as the evolution of knowledge and the impact of wider socio-economic and political factors. emerging issues such as psychosocial factors, the increasing prevalence of non-communicable diseases, and the shift towards well-being (and not merely safety and health) demand new ways of thinking in addressing hsw in the workplace. continuing to work in silos and adopting mono-disciplinary perspectives will not allow us to move forward in this complex landscape. a strategic alignment of perspectives and integrated approaches are needed. this book aims to promote a way forward by outlining and critically evaluating developments in hsw in the workplace, and providing a framework for action in policy and practice. fundamental principles of occupational health and safety gender and jobs: sex 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conditions: working conditions and worker rights in a global economy consequences of modernity risk and responsibility human safety and risk management ageing, health and productivity: a challenge for the new millennium healthy work for older workers: work design and management factors individual behaviour and the control of danger management and culture: the third age of safety. a review of approaches to organizational aspects of safety, health and environment rr -projection of mesothelioma mortality in great britain industrial accident prevention: a scientific approach resilience: the challenge of the unstable safety i and safety ii: the past and future of safety management lessons from gretley: mindful leadership and the law monitoring new and emerging risks trends in quality of work in the netherlands implementing safety culture in a major multi-national challenges and perspectives of occupational health and safety research in nanotechnologies in nanotechnologies in europe the ageing workforce-challenges for occupational health protecting workplace safety and health in difficult economic times -the effect of the financial crisis and economic recession on occupational safety and health statutory occupational health and safety workplace arrangements for the modern labour market use of technology and working conditions in the european union aging, adult development, and work motivation globalization and workers' health health in restructuring: innovative approaches and policy recommendations ergonomics and technology transfer into small and medium sized enterprises new systems of work organization and workers' health work related and individual predictors for incident neck pain among office employees working with video display units perceptions of psychosocial hazards, work-related stress and workplace priority risks in developing countries safety considerations for the aging workforce happiness: lessons from a new science the european framework for 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economies. paris: organization for economic co-operation and development. organization for economic co-operation and development a good life in old age? monitoring and improving quality in long-term care. paris: organization for economic co-operation and development convergence and divergence of working conditions in europe building healthy and equitable workplaces for women and men: a resource for employers and workers representatives workers' compensation and the challenges posed by changing patterns of work: evidence from australia. policy and practice in health and safety the global expansion of precarious employment, work disorganization, and consequences for occupational health: a review of recent research managing the risks of organizational accidents women's employment in europe: trends and prospects aging, job performance, and career development the changing organization of work and the safety and health of working people: knowledge gaps and research directions exploring 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organization, , . world health organization (who) raising awareness of stress at work in developing countries: a modern hazard in a traditional working environment: advice to employers and worker representatives world health organization (who) sedentary behaviours and epithelial ovarian cancer risk key: cord- -uovhtaxb authors: eppstein, david; goodrich, michael t.; liu, james a.; matias, pedro a. title: tracking paths in planar graphs date: - - journal: nan doi: nan sha: doc_id: cord_uid: uovhtaxb we consider the np-complete problem of tracking paths in a graph, first introduced by banik et. al. [ ]. given an undirected graph with a source $s$ and a destination $t$, find the smallest subset of vertices whose intersection with any $s-t$ path results in a unique sequence. in this paper, we show that this problem remains np-complete when the graph is planar and we give a -approximation algorithm in this setting. we also show, via courcelle's theorem, that it can be solved in linear time for graphs of bounded-clique width, when its clique decomposition is given in advance. motivated by applications in surveillance and monitoring, banik et. al. [ , ] introduced the problem of tracking paths in a graph. in essence, the goal is to uniquely determine the simple path traversed by a moving subject or object, based on a sequence of vertices sampled from that path. examples of surveillance applications include the following: (i) vehicle tracking in road networks; (ii) habitat monitoring; (iii) intruder tracking and securing large infrastructures; (iv) tracing back of illicit internet activities by tracking data packets. another application would be to determine the nodes in a network that have been compromised by a spreading infection, given an incomplete transmission history of a pathogen. this information can be helpful in identifying and attenuating the negative impact caused by biological and non-biological infectious agents, such as: highly-contagious diseases (e.g. severe acute respiratory syndrome) which could lead to epidemics [ ] . fake news and hate speech being disseminated in social networks, as well as violations of privacy (e.g. sharing without permission highly sensitive content owned by a user, such as intimate pictures). computer viruses, which spread throughout servers scattered across the internet essentially, an entry-exit pair (s , t ) (see figure ) ) with respect to a cycle c represents two alternative s − t paths and, thus, requires tracking at least one of its branches. we say that (s , t ) is tracked with respect to c if and only if c \{s , t } contains a tracker. in addition, c is tracked if and only if there is no entry-exit pair with respect to c that is untracked. if a cycle contains either (i) trackers or (ii) s or t and tracker in a non-entry/non-exit vertex, then it must be tracked. we say that these cycles are trivially tracked. an alternative characterization of a tracking set, first given by banik et al [ ] , is the following. the overall idea for the approximation algorithm builds on the following two ideas: . the cardinality of the optimal solution cannot be much smaller than the number of faces in the graph. . the average number of trackers per face does not need to be very large. the first idea gives us a lower bound on opt , the cardinality of an optimal solution. the second gives us an upper bound on alg, the cardinality of our approximation algorithm. we consider the following reduction, which takes care of disconnected components, or components that are "attached" to the graph by a cut vertex that is not in an s − t path. while there exists an edge or vertex that does not participate in any s − t path, remove it from the graph. lemma . after reduction , every simple cycle in the graph contains at least one entry-exit pair. this holds for non-planar graphs as well. proof. let c by some simple cycle in the graph. after reduction , there must exist an s − t path that shares an edge with c. the first and last vertices on this path that belong to c correspond to an entry-exit pair. in an embedded undirected planar graph g that results from reduction , opt ≥ (|f | − )/ , where f is the set of faces of g. (a) tracker on x (red) is inactive due to the violation of condition (i). tracker on x (red) is inactive due to the violation of condition (ii). notice that x is entry for exit vertices u and v (with respect to c), therefore c needs another tracker. on a high level, the proof of lemma is done by keeping a set of "active" trackers while reconstructing a planar embedding e of g: we start, as a base case, with any simple s − t path in e and iteratively add faces to it until it matches e. given a fixed, optimal tracking set t * , the addition of each face requires either (i) adding a new tracker from t * to the active set, or (ii) deactivating an active tracker, rendering it useless for distinguishing paths on future faces. as a consequence, each tracker charges at most two faces: the one adding the tracker and the one deactivating it. this demonstrates that |t * | ≥ (|f | − )/ . let outer(e τ ) be the set of outer-edges of our planar reconstructing embedding e τ at time τ . at time τ = , our embedding corresponds to an s−t path and, for all ≤ τ ≤ |f |− , we add exactly one face c from e to e τ , by connecting two vertices u and v in outer(e τ ) with a simple path p (see figure in the appendix). in doing so, we erase an u − v path p in outer(e τ ), so we have that outer(e τ + ) = outer(e τ ) \ p ∪ p. in the end, e |f | = e. by lemma , there is at least one entry-exit pair in e with respect to face c, so any tracking set must contain a tracker on some vertex of c. during the reconstruction process, we maintain a list of trackers in sets a and a , such that (a ∪ a ) ⊆ t * , where a contains active trackers and a contains inactive ones. a tracker in vertex v is active at time τ if and only if it meets both of the following conditions: intuitively, an active tracker can be used to track future faces, although no more than one (see below). an inactive tracker, on the other hand, either cannot be used to track future faces (condition (i)), or its corresponding vertex is entry/exit for some future face (condition (ii)), in which case we require yet another tracker on that face (see figure ). condition (ii) is necessary for dealing with embeddings of g where at least one of s and t is not in the outer face. first, we argue that each time we add a face during the reconstruction process described above, we either (i) need to increase the number of active trackers (by adding it to either a or a ), or (ii) we can get away by re-using and, therefore, deactivating an active tracker. we assume for the rest of the argument that t is on the outer face, because such a planar embedding is always possible to construct. let c be the face added a time τ by connecting vertices u and v in outer(e τ ), as specified above. in addition, let t * be any optimal solution (i.e. |t * | = opt ). we consider two cases, depending on the existence of a tracker in c at time τ : by lemma , there exists a vertex x ∈ c such that x ∈ t * . we place a tracker on x. if x ∈ outer(e τ + ) we add x to a, otherwise we add it to a . let y ∈ c ∩ a be a vertex of c with a tracker. we again consider two cases: (i) y / ∈ {u, v}. then, y ∈ outer(e τ ) but y / ∈ outer(e τ + ), which amounts to moving y from a to a . (ii) y ∈ {u, v}. if (u, v) is an entry-exit pair with respect to c, or if the tracker in y is not active, then there exists x ∈ c \ {u, v} such that x ∈ t * . similarly to case , we place a tracker on x , which corresponds to adding x either to a or a . otherwise, the tracker in y is active and (u, v) is not an entry-exit pair with respect to c. let us assume without loss of generality that y = u. then, the addition of c deactivates the tracker in u by definition of active tracker (condition (ii) is now violated), so we move u from a to a . every tracker in a is charged by at most two faces: one for adding an active tracker to a and another for deactivating it and moving it to a . therefore, |f |− ≤ |a|+ |a |. since |a| + |a | ≤ |t * |, it follows that |f | − ≤ opt . a tight example for the lower bound on op t is illustrated in section . . we say that an undirected planar graph is reduced if it cannot be further reduced by reduction or any of the following reductions. while there exist two adjacent vertices of degree , remove one of them (and its edges) and add an edge connecting its neighbors. reduction . while there exists vertex v / ∈ {s, t} of degree in a -cycle, place a tracker on v and remove it and its edges from the graph. reduction . while there exist non-adjacent vertices u, v / ∈ {s, t} of degree in a -cycle, place a tracker on either u or v and remove it and its edges from the graph. reductions , and can be applied interchangeably and in any order until none of them is applicable, but we will see that they need to be carried out after reduction . fortunately, we will not be required to re-apply reduction after performing the remaining reductions. we denote the degree of a vertex v by deg(v), where the underlying graph can be determined from its context. proof. trivial by definition . claim . reductions , and maintain the property that every cycle in g contains at least one entry-exit pair (see lemma ) . proof. reductions , and only erase faces and vertices of degree , which cannot be in entry-exit pairs (by claim ), so every simple cycle of the graph still contains an entry-exit pair. reduction is safe and can be done in polynomial time, if done after reduction . proof. by lemmas , , and , reductions - are safe, so let us assume without loss of generality that g is reduced. then, every cycle of g of or more vertices is trivially tracked, because it must contain at least vertices of degree at least (by reduction ). similarly, every -or -cycle must contain at least vertices of degree at least by reduction and reductions and (respectively). proof. notice that each tracker added during reductions and is associated with the removal of one face from g. therefore, it is enough to show that the lemma holds with respect to a reduced graph g. let us partition consist of the vertices of degree and degree at least , respectively (notice that there cannot be vertices of degree ). the lemma statement follows from lemma and the fact that |f | ≥ |v ≥ | + . this inequality can be derived by plugging in the inequality |e| ≥ |v ≥ | + |v | in euler's formula for planar graphs: |v | − |e| + |f | = , where e is the set of edges of g. a tight example is illustrated in section . . proof of theorem . by lemmas and , algorithm a is a -approximation to planar-tracking. we show that planar-tracking is np-hard, by reducing from planar- -sat, a special version of the satisfiability problem, shown to be np-complete by lichtenstein [ ]. in -sat, we are given a set x = {x , . . . , x p } of variables and a -cnf formula φ, where each clause in φ is a disjunction of exactly three distinct literals with respect to x . the goal is to find a boolean assignment to all variables in x that satisfies φ. consider the bipartite graph with a vertex for each clause c in φ and each variable x i ∈ x , and edges (x i , c) if and only if c contains x i or its negation x i . lichtenstein [ ] showed that planar- -sat, the subset of instances of -sat whose underlying bipartite graph is planar, remains np-complete. in particular, the definition of planar- -sat requires that a cycle can be drawn connecting all of the variables while maintaining planarity. later, knuth and raghunatan [ ] exploited this condition to show that we can always draw the underlying bipartite graph of a planar- -sat instance in a rectilinear fashion without crossings (example in figure a ): variables are arranged in a horizontal line and clauses are horizontal line segments with vertical legs to represent the literals present in the clause. vertical legs attach to the appropriate variables and are labeled red for negated literals and blue, otherwise. in particular, a given clause is drawn completely above or below the line of variables. we convert a planar rectilinear drawing d of an instance of planar- -sat, with formula φ and a set x of variables, into a planar drawing g corresponding to the instance of planar-tracking. the reduction is straightforward: the union of all the variable and clause gadgets constitutes g (see example in figure in the appendix). details of each gadget are given below. for simplicity, we avoid introducing too many subscripts and we rely on pictures to describe the gadgets. each variable gadget is linked with the next one by setting t i = s i+ , to form a horizontal chain of gadgets, where s = s and t = t p . for convenience, we force trackers in all the s i (except s), by drawing edges between the α (β ) of a variable gadget and the α (β) of the next variable gadget in the chain. we refer to the resulting drawing as the spine. there are exactly two minimum tracking sets associated with the variable gadget with source s i and destination t i . one of them corresponds to a true assignment of x i and the other one to a false assignment. both of them require tracking the vertices in r = {α, α , β, β , µ , µ mi }, as well as the remaining µ k . in addition, the true assignment tracks the even-indexed h k and odd-indexed l k , while the false assignment tracks the odd-indexed h k and even-indexed l k . this requires m i + trackers in total. lemma . the true and false assignments are the only minimum tracking sets. clause gadget. let c = ( a ∨ b ∨ c ) be a clause in φ with literals corresponding to variables x a , x b , x c ∈ x . its gadget, depicted in figure , is a face f c consisting of: literal vertex α from x a 's gadget, corresponding to literal a ; adjacent literal vertices β , β , β , β , β from x b 's gadget, corresponding to literals alternating between b and b ; literal vertex γ from x c 's gadget, corresponding to literal c ; edges (α, γ), (α, β ), (β , γ) and the edges from x b 's gadget connecting all of the β k and β k . we can increase the lengths of the variable gadgets to any polynomial that provides enough literal vertices for all clauses. since d is planar, there are no crossings between clauses. we also impose the following restrictions: . α cannot be one of {h , l ma }; this ensures that the only faces in x a 's gadget that do not require trackers do not become untracked. we apply the equivalent restriction to β , β and γ. . the α k /α k , (see figure ) cannot belong to any other clause gadget; these correspond to the literal vertices following α in x a 's gadget and reserving them ensures that non-clause faces, between nested clauses, are tracked. we apply the equivalent restriction to the γ k /γ k . . all literal vertices in a clause need to be on the same side of the spine; this restriction is trivial because d is rectilinear, but it simplifies the analysis. theorem . there exists a polynomial time reduction from planar- -sat to planar-tracking. it remains to show that planar-tracking is in np; banik et. al. [ ] prove this in the more general case of tracking. we show that tracking can be solved in linear time when the input graph has bounded clique-width, by applying courcelle's theorem [ , , ], a powerful meta-theorem that establishes fixed-parameter tractability of any graph property that is expressible in monadic second order logic. clique-width, first introduced by courcelle et. al. [ ] and revisited by courcelle and olariu [ ] , is an important graph parameter that, intuitively, measures the closeness of a graph to a cograph -a graph with no induced -vertex paths. it is closely related to tree-width, another influential graph parameter that measures closeness of a graph to a tree and that was first introduced by bertelé and brioschi [ ] and later rediscovered by halin [ ] and robertson and seymour [ ] . while both parameters are determined based on specific hierarchical decompositions of a graph, the clique-width is strictly more powerful in the sense that the class of graphs of bounded clique-width includes all graphs of bounded tree-width, but not vice-versa. details on the relationship between these parameters can be bound in courcelle and engelfriet [ ] . graphs of bounded clique-width include series-parallel graphs, outerplanar graphs, pseudoforests, cographs, distance-hereditary graphs, etc. mso vs mso . second order logic extends first order logic, by allowing quantification over relations (of any fixed arity) on the elements of the domain of discourse. monadic second order logic itself only allows quantification over unary relations (subsets of the domain of discourse) and, in the logic of graphs, it comes in two flavors: mso and mso . the only distinction between these is that the latter allows edges to be elements of the domain of discourse (and thus be quantified over), while the former does not. besides the quantifiers (∀ and ∃) and the standard logic operations ¬, ∧, ∨, →, both logics include predicates for equality (=) and relation membership (∈). in addition, mso includes a predicate (∼) that determines vertex adjacency and mso includes a predicate for vertex-edge incidence. mso is, by definition, strictly more expressive, for example: hamiltonicity can be expressed using mso , but not using mso . details on the distinction between the two logics can be found in [ ] . courcelle's theorem. courcelle et. al. [ , ] showed that any graph property expressed in mso and mso is fpt under clique-width and tree-width (respectively). more specifically, they showed that any mso -, mso -expressible property can be tested in f (k, l) · n and g(k , l ) · (n + m) time (respectively) for graphs of clique-width k and tree-width k , where: f and g are computable functions, l and l are the lengths of the logic formulas, n is the number of vertices and m is the number of edges. the result for mso became known as courcelle's theorem and it is also valid in optimization problems with linear evaluation functions [ ] . later, courcelle, makowsky and rotics [ ] extended these results for mso . however, while it is possible to construct tree decompositions of width k in linear time [ ] , there is no fpt algorithm for finding clique decompositions of clique-width k > . fortunately, it is possible to construct a clique decomposition of width exponential in k in cubic time [ ] . in this section, we will take advantage of the latter. we give an alternative definition for tracking set that is easier to express using the logic of graphs. tracking set if and only if there is no s − t simple path p st = p ss ∪ p s t ∪ p t t for s , t ∈ v and corresponding s − s , s − t and t − t paths, such that: . there exists an alternative s − t simple path p s t = p s t and . t ∩ (p s t ∪ p s t ) ⊆ {s , t }. remark . definitions and are equivalent. in the logic formulas presented below, we use lowercase letters to quantify over vertices and uppercase letters to quantify over sets of vertices. we use s and t as free variables and, for convenience, we also use set intersection (∩), union (∪) and containment (⊆), without explicitly expressing these operations using mso . the first two lines of the above equivalence establish that p , q and r form an s − t path. the last line restricts s and t to be an entry-exit pair with respect to the cycle q ∪ q (see figure ) and, in addition, establishes that the cycle q ∪ q is not tracked. the primitive haspath(x, a, b) , whose input consists of a set x ⊆ v and vertices a, b ∈ v , verifies the existence of a simple path between a and b that only uses vertices in x. we define it as follows: remark . haspath(x, a, b) is correctly expressed under mso and it correctly verifies that there exists an a − b path using only vertices in x. remark . istrackingset is correctly expressed under mso and it correctly verifies that the given subset of vertices is a tracking set. theorem . tracking (g, s, t) can be solved in polynomial time if g has bounded cliquewidth. moreover, if a clique decomposition of bounded width is given, it can be solved in linear time. proof. this follows directly from remarks to and the linear time algorithm given by courcelle [ ] for any optimization problem on graphs of bounded clique-width, whose decomposition is given in advance. we showed that planar-tracking is np-complete and we give a -approximation algorithm. we also show that, for graphs of bounded-clique width, tracking can be solved in linear time by applying courcelle's theorem, as long as its clique decomposition is given in advance. a natural direction of future study would be to improve the approximation ratio of planar-tracking or establish constant approximation factors for graphs of larger genus or, more generally, for tracking. another open question is to establish the difficulty of tracking on directed graphs: on one side planar directed acyclic graphs are not known to be np-hard and, on the other side, it is not known whether its general or planar versions are in np. finally, it would be interesting to find efficient algorithms for graphs of bounded tree-width or clique-width without resorting to the finite automaton approach used in courcelle's theorem. proof. see proof in [ ] . proof. banik et. al. [ , lemma ] showed that having three adjacent vertices of degree is redundant. here, we extend their proof and show that we can also get rid of the second adjacent vertex of degree . let u and v be the two adjacent vertices of degree , and let u be the other neighbor of u. notice that v, u are not adjacent, otherwise the u, v edge would then not belong to an s − t path and thus be removed by reduction . hence, no parallel edges are added to the graph after this reduction. we argue that this reduction is safe by showing that there exists a minimum tracking set that does not include u and v simultaneously. take any minimum tracking set t that includes u and v. we can always move the tracker from u to u ; this remains a tracking set, because u immediately follows or precedes u on any s − t path. this can only decrease t 's cardinality. this reduction can be done in o(poly(n)) time, by repeatedly checking for the existence of adjacent vertices of degree . ∈ {s, t} be the vertex of degree in the triangle ∆vs t (see figure b ). then, it must be the case that s and t form an entry-exit pair. this follows from (i) the fact that there exists an entry-exit pair in ∆vs t (by lemma ) and (ii) the fact that v cannot be in an entry-exit pair (by claim ). then, by definition , any feasible solution must place a tracker on v. therefore v and its edges can be removed, since it is not a cut-vertex, and it is not in any entry-exit pair, so that its removal does not eliminate an untracked cycle. clearly, this reduction can be done in o(poly(n)) time. lemma . reduction is safe and can be done in polynomial time, if done after reduction . ∈ {s, t} be the two vertices of degree that connect the same pair of vertices s and t . similarly to the proof of lemma , s and t must form an entry-exit pair. so, by definition , any feasible solution must place a tracker in either u or v. by symmetry, we can track and remove u and its edges. therefore, reduction is safe by claim and the facts that u is neither a cut-vertex nor in an entry-exit pair. to prove lemma , we first prove a couple of helpful lemmas and make a few observations: each vertex in r belongs to a triangle, such that the other two vertices form an entry-exit pair, so must be tracked. each square face, besides the two including h and l mi− , requires tracked vertices. two adjacent vertices cannot both be untracked. lemma . the true/false assignments of x i correspond to tracking sets with respect to x i 's gadget with source s i and destination t i . proof. in a true/false assignment of x i , every face of the gadget is trivially tracked, except the faces including s i /t i (which are clearly tracked) and the faces including h and l mi− . though the latter faces may only contain trackers (depending on x i 's truth value and/or m i 's parity), they are nevertheless tracked because one of these trackers cannot be in an entry-exit pair. the remaining cycles, i.e. the ones which are not faces, are also trivially tracked: since these cycles must have size at least , they must contain trackers given the observation that no adjacent vertices are untracked. lemma . in a minimum tracking set, each column has exactly trackers. proof. since the true/false assignments achieve this property, we only have to show that each column requires at least two trackers. assume that a minimum tracking set has only one tracker in column < k < m i ; it must be on µ k . then all vertices on columns k − , k + must be tracked. for the average number of trackers per column to be at most , the number of trackers per column must be an alternating sequence of . . . , , , , . . . , with column and/or column m i only having tracker, which contradicts the square face property. lemma . the true and false assignments are the only minimum tracking sets. proof. assume that some µ k is not tracked in a minimum tracking set, for < k < m i . by lemma , we only have to show that this causes a column to have trackers. if k = m i − then column m i must have three trackers. otherwise, µ k+ , h k+ , h k+ must be tracked, since they share a square face with µ k . if l k+ is tracked we are done, otherwise, l k+ must be tracked. additionally, µ k+ must be tracked, either by the square face property, or in the case where k = m i − , because it is in r. then, column k + has trackers. now, if all the µ k are tracked, then the only minimum tracking sets are the true and false assignments, by the observation that two adjacent vertices cannot both be untracked and lemma . lemma . clause c is satisfied if and only if its corresponding gadget face f c is tracked. proof. the entry-exit (β , β ) is the only one, with respect to f c , that is tracked if and only if c is satisfied. the remaining entry-exit pairs are tracked by either a β k or a β k . theorem . there exists a polynomial time reduction from planar- -sat to planar-tracking. proof. let (g, g) be an instance of planar-tracking that results from applying the transformation described above to an instance (φ, d) of planar- -sat, where g and d are the underlying planar embeddings. we show that φ is satisfiable if and only if g has a tracking set of size t = ( (⇐) choose the truth assignment of each variable according to the given tracking set. the implication follows from lemma and the fact that if some clause in φ was not satisfied, then its gadget face would have been untracked, a contradiction. (⇒) place t trackers on the literal vertices that correspond to the satisfiable truth assignment of every variable and on all non-literal vertices (except s and t). we show that this corresponds to a tracking set by arguing that every cycle c in g is tracked. we distinguish between two cases: case : c contains no clause edges. then c is tracked by (almost) the same argument given in lemma that shows that a truth assignment of x i corresponds to a tracking set with respect to x i 's gadget. notice that, because of restriction , clause edges are only added to faces that require trackers, so this does not change the argument for the faces which do not require trackers. the only differences are: (i) the addition of the edges that force trackers in every s i , which only helps the argument, and (ii) the fact that c may span multiple variable gadgets, in which case c must traverse at least trackers on the non-literal vertices between two variable gadgets. case : c contains clause edges. notice that, by construction of g, c must have at least one spine edge. if c corresponds to a clause face, then it must be tracked by lemma . otherwise, we show that c contains at least trackers and, thus, is trivially tracked. let us think of c as alternating non-empty paths of two types: clause paths, which only contain clause edges and spine paths, which only contain spine edges. to avoid dealing with complex cycles, we observe that each spine path in c must contain at least tracker; this follows from the fact at least one of the vertices sharing a spine edge must have a tracker (see variable gadget). thus, let us assume that c contains no more than spine/clause paths, or otherwise c immediately contains trackers. notice that if one of the spine paths spans or more variable gadgets, then it must traverse at least trackers on the non-literal vertices between two variable gadgets. since every clause in φ contains exactly distinct literals and c is simple, the only cases where none of the spine paths span multiple variable gadgets are the following: (a) example of a planar- -sat instance drawn in a rectilinear fashion with clauses a = (x ∨ x ∨ x ), b = (x ∨ x ∨ x ), c = (x ∨ x ∨ x ), d = (x ∨ x ∨ x ). example of a planar-tracking instance. the orange vertices ensure that no cycle is untracked (see figure ). illustration of a planar- -sat instance (above) and the corresponding planar-tracking instance associated with the reduction described in section (below). (i) c contains exactly clause paths in different sides of the spine. then, the spine paths connecting the two sides of the spine must traverse trackers each (see variable gadget). therefore, c contains at least trackers. (ii) c contains exactly clause paths on the same side of the spine, which both start or both end at the same variable gadget, one nested in the other. then, by restriction one of the spine paths contains at least vertices, half of which must be tracked. the mathematical theory of infectious diseases and its applications. charles griffin & company ltd, high wycombe a polynomial sized kernel for tracking paths problem tracking paths nonserial dynamic programming survey of target tracking protocols using wireless sensor network a linear-time algorithm for finding tree-decompositions of small treewidth the monadic second-order logic of graphs. i. recognizable sets of finite graphs. information and computation graph structure and monadic second-order logic: a language-theoretic approach handle-rewriting hypergraph grammars key: cord- - ldj j authors: parisotto, simone; calatroni, luca; caliari, marco; schonlieb, carola-bibiane; weickert, joachim title: anisotropic osmosis filtering for shadow removal in images date: - - journal: nan doi: . / - /ab d sha: doc_id: cord_uid: ldj j we present an anisotropic extension of the isotropic osmosis model that has been introduced by weickert et al.~(weickert, ) for visual computing applications, and we adapt it specifically to shadow removal applications. we show that in the integrable setting, linear anisotropic osmosis minimises an energy that involves a suitable quadratic form which models local directional structures. in our shadow removal applications we estimate the local structure via a modified tensor voting approach (moreno, ) and use this information within an anisotropic diffusion inpainting that resembles edge-enhancing anisotropic diffusion inpainting (weickert, , gali'c, ). our numerical scheme combines the nonnegativity preserving stencil of fehrenbach and mirebeau (fehrenbach, ) with an exact time stepping based on highly accurate polynomial approximations of the matrix exponential. the resulting anisotropic model is tested on several synthetic and natural images corrupted by constant shadows. we show that it outperforms isotropic osmosis, since it does not suffer from blurring artefacts at the shadow boundaries. the use of partial differential equations (pdes) has a long tradition in mathematical image processing. in particular, pdes based on transport and diffusion mechanisms have been considered to model several image reconstruction models suitable for image enhancement, denoising, deblurring, inpainting and segmentation. we refer the reader to the review [ ] and the monographs [ , , , , ] for further references. among these models, a very special place is occupied by diffusive pdes encoding anisotropy, i.e. favouring diffusion along some specific directions only. in [ ] nonlinear arxiv: . v [math.ap] sep diffusion pdes with space-variant diffusion tensors are studied. for a regular image domain Ω ⊂ r and a stopping time t > , given a degraded image f ∈ l ∞ (Ω; r) and two smoothing parameters ρ, σ > , the anisotropic diffusion model in [ ] looks for a solution u in a suitable function space satisfying the following initial value problem: on Ω × ( , t ], on Ω, where n is the outward normal unitary vector on ∂Ω and d is a non-constant diffusion tensor satisfying suitable regularity conditions, with eigenvectors inherited from the socalled structure tensor j ρ (∇u σ ). it encodes local directional information of u σ (that is, the image u convolved with a gaussian kernel of standard deviation σ). more precisely, its eigenvectors point in the directions of largest and smallest contrast averaged over a gaussian smoothing scale ρ, and the corresponding eigenvalues measure this contrast. the diffusion tensor uses the same eigenvectors, and its eigenvalues are functions of the eigenvalues of the structure tensor. depending on the application, different models such as edge-enhancing anisotropic diffusion or coherence-enhancing anisotropic diffusion have been proposed [ ] edge-enhancing anisotropic diffusion has been adapted to inpainting problems in [ ] . it is particularly useful for sparse inpainting problems encoutered e.g. in inpainting-based compression applications where it outperforms other pde approaches [ , ] . beyond anisotropic pdes, non-smooth anisotropic regularisers for variational imaging models are also considered. in [ , , , , ] , for instance, directionality is used to define the anisotropic total-variation and total-generalised-variation functionals. this is classically done by considering a re-parametrised version of the gradient operator depending on the local orientation of the image, which allows to enforce diffusion along certain directions only. in terms of variational models, one replaces the squared norm ∇ u∇u by a quadratic form of type ∇ ud∇u. this has a very long tradition in image analysis [ ] . the explicit dependence of these anisotropic models on local terms such as position and local directions of the image makes the analysis of these models more challenging [ , , , ] . also from a numerical point of view, the design of suitable schemes enforcing anisotropy is a non-trivial task since it requires the use of appropriate stencils that perfectly adapt to the local image structure. many methods have been proposed; see e.g. the unifying framework in [ ] and the references therein. while most stencils lead to l -stable schemes, only a few of them allow to preserve nonnegativity and l ∞ stabilty [ , , ] . a rather sophisticated representative among them is the stencil of fehrenbach and mirebeau [ ] which relies on lattice basis reduction ideas. in this work we consider a transport-diffusion pde describing the physical phenomenon of osmosis for imaging applications. compared to standard plain diffusion models, the model considered therein considers an additional drift term, making the process not symmetric (see [ ] for the physical interpretation). for a regular domain Ω ⊂ r , a given vector field d : Ω → r and a given image f ∈ l ∞ (Ω; r), the isotropic osmosis model reads [ ]      u t = ∆u − div(du) on Ω × ( , t ], differently from plain diffusion models, osmosis steady states are non-constant. in particular, if d is defined in terms of a given image v > as d := ∇ log v, convergence to a rescaled version of v can be proven [ ] . this is called the integrable or compatible case. in [ , ] several imaging applications based on ( ) or a slight modification thereof are studied. one of them is the shadow removal problem, see [ , section . ] , as considered in this paper. shadow removal. the problem of shadow removal from a given image f : Ω → r + consists in removing the shadow appearing in f while preserving the image geometry and texture underneath. we will assume in the following constant shadows, i.e. where image intensity values inside and outside the shadow region are in relation with each other up to an (unknown) multiplicative constant. this problem is of great interest in computer vision as it often represents a preprocessing step in several segmentation, tracking and face recognition tasks where shadows are removed to avoid false detections/artefacts in the subsequent image processing. we refer the reader to [ ] for a review on the existing models for shadow removal in images. for our purposes, a mathematical formulation of the shadow removal problem can be obtained decomposing the image domain Ω as where Ω out , Ω sb and Ω in are the unshadowed region, the shadow boundaries and the shadowed region of the image, respectively, see figure for an example. provided that a decomposition as in ( ) is given, which for real images may a challenging problem on its own regard [ ] , the osmosis model ( ) can be easily adapted to solve the shadow removal problem by simply defining the vector field d in ( ) in terms of a shadowed image f as d := ∇ log f on Ω in ∪ Ω out and d = on Ω sb . the continuous figure : decomposition of Ω as in ( ) into (b) unshadowed region, (c) shadow boundaries and (d) shadowed region for a discrete shadowed image f . osmosis model adapted to shadow removal then reads the evolution on the shadow boundary Ω sb can be interpreted as an inpainting step where information is propagated from Ω out to Ω in over Ω sb . due to the action of the laplace operator on Ω sb , image structures in Ω in ∪ Ω out are isotropically diffused on Ω sb , resulting in a shadowless, but blurred inpainting result on Ω sb . to overcome this a post-processing inpainting step is commonly applied, as for instance in figure . (a) isotropic osmosis [ ] (b) post-processing inpainting step [ ] (c) zoom of figure a figure b to remove the blurring artefacts due to laplace inpainting on Ω sb in ( ). note that in natural images several acquisition and/or compression artefacts may render the automatic segmentation of the shadow boundary very challenging. on the other hand, its accurate manual selection may be very tedious. in many practical examples, a rough selection of Ω sb is therefore performed manually by using a brush whose possibly large thickness may badly affect the result of the model ( ) (see figure ) due to the laplace blurring artefacts discussed above. vogel et al. [ ] have presented a discrete osmosis theory and have proven that explicit and implicit finite difference discretisations satisfy its requirements. different splitting schemes have been considered in [ , ] and have been applied to imagery for cultural heritage conservation. in this paper we extend the isotropic osmosis model ( ) and its shadow removal application ( ) to a model that features anisotropic diffusion in the flavour of ( ) and adapts concepts from anisotropic diffusion inpainting [ , ] . this will be implemented by incorporating local directionality depending on image orientations. we show that with this modification we can improve the solution of the shadow removal problem, in particular overcoming blurring artefacts in the region around the shadow boundary, without additional post-processing steps. in the integrable case, the resulting driftdiffusion pde can be derived as the gradient flow of a suitable energy depending on local gradient information. to estimate such local directionality, we adapt the tensor voting framework proposed in [ ] . for the numerical solution we combine a numerical time-stepping method based on exponential integrator techniques with the nonnegative space discretisation of fehrenbach and mirebeau [ ] . our model is validated on several synthetic and natural images affected by constant shadows. results show good lightbalance properties and, compared to plain isotropic osmosis models, avoid the smoothing artefacts on the shadow boundary. an illustrative example of the performance of our model is reported in figure . [ , ] and our anisotropic one to solve the shadow removal problem. organisation of the paper. in section we introduce the anisotropic osmosis model and study analytically its properties. then, in section we study space and time discretisation schemes for the anisotropic model. finally, in section we show the application of the anisotropic model to solve the shadow removal problem. we present in this section a variation of the classical osmosis model ( ) encoding local directional information of the image in the diffusion term, propagating geometric structures dominantly along locally preferred directions. for this reason we call our model anisotropic osmosis model in contrast to the model ( ) which we refer to as isotropic osmosis model. in what follows we introduce the general form of our anisotropic osmosis model and state some properties of solutions that are inherited from the isotropic model. for specific choices of anisotropy we also show connections of the anisotropic osmosis model to anisotropic diffusion-based inpainting methods such as edge-enhancing anisotropic diffusion [ ] . out of these specific instances we derive our proposed anisotropic osmosisinpainting model for shadow removal. let us define an anisotropic osmosis energy as follows. definition . (anisotropic osmosis energy). let Ω ⊂ r , u, v ∈ h (Ω; r + ) be two positive images and let w : Ω → r be a positive semi-definite symmetric matrix field. we define the anisotropic osmosis energy of u with respect to w and the reference image v as we will also use the following alternative notation for e: where e w := e, we . remark . (isotropic case). if w is the identity matrix, then ( ) corresponds to the isotropic osmosis energy considered in [ ] . next we define an anisotropic osmosis evolution whose steady state minimises our anisotropic osmosis energy. proof. for any test function ϕ ∈ c ∞ c (Ω), we compute the optimality condition holding for any critical point u of e. we get: where we have applied the divergence theorem and the neumann boundary conditions in ( ) . due to the positivity of v and since ϕ is compactly supported in Ω, we have that for any x ∈ Ω: by definition of d = ∇v v , we note that the above corresponds to the following pde: div (w (∇u − du)) = , which is the steady state of ( ). similar to the isotropic osmosis pde ( ), the anisotropic model ( ) enjoys some properties which makes it amenable for imaging applications. theorem . . the solution u : Ω → r of the anisotropic osmosis model ( ) satisfies the following properties: (i) conservation of the average grey value: (ii) preservation of non-negativity: u(x, t) ≥ , for all x ∈ Ω and t > ; (iii) non-constant steady states: the steady state of ( ) is given by proof. we follow [ ] and prove statements (i)-(iii) in turn. (i) let µ u (t) := |Ω| Ω u(x, t) dx be the average grey value of the image u at time t ≥ . applying the divergence theorem and the homogeneous neumann boundary conditions in ( ) we obtain: and the statement follows. (ii) assume that t > is the smallest time such that min x,t u(x, t) = , and that this minimum is attained in some inner point µ ∈ Ω. since we have ∇u(µ, t ) = u(µ, t ) = , we deduce: that is in correspondence to the point (µ, t ), the anisotropic osmosis equation behaves like the diffusion equation with non-constant positive semidefinite diffusivity matrix w. for such equations a generalisation of the weak minimum/maximum principle (which holds in its classical form only for positive definite, i.e. elliptic, operators) holds true, see, e.g. [ ] . therefore, for any t ≥ t the solution of the anisotropic model remains non-negative and since the solution has been further assumed to be strictly positive for all t < t , including t = due to the positivity of f , we have that it stays actually non-negative for any t ≥ . (iii) for any c ∈ r, the function w := cv endowed with the neumann-type boundary conditions in ( ) solves the steady state equation of the system anisotropic pde, since there trivially holds: due to mass and non-negativity conservation, the process is then forced to a nonnegative steady state solution w of such a form. furthermore, the constant c ∈ r can be easily found by noticing anisotropic diffusion inpainting with a diffusion tensor has been introduced in [ ] and applied successfully for inpainting-based compression [ , ] . it exploits the edgeenhancing anisotropic diffusion filter that has been proposed for image denoising [ ] . in order to propagate structures from specified image regions into inpainting regions, one uses the differential operator div(d(∇u σ )∇u), where u σ denotes the convolution of u with a gaussian of standard deviation σ. the diffusion tensor d has eigenvectors perpendicular and parallel to ∇u σ . its corresponding eigenvalues are given by with some contrast parameter λ > . thus, the goal is to inpaint fully in the direction of an oriented structure, and to reduce the inpainting perpendicular to a structures, if its contrast is large. processes of this type can inpaint edge-like structures even when the specified data are sparse and the gaps to be bridged are large [ ] . however, they are not well-suited to shadow removal problems, since the shadow boundaries create unphysical edges. therefore, we will have to modify these ideas such that the local structure directions become more robust w.r.t. shadow boundaries. to this end, we will consider and modify more refined structure descriptors such as tensor voting. this will be done next. in this section we present a work-flow which is locally insensitive to the light jump produced by the shadow. this will serve us to force anisotropy on Ω sb along suitable directions. a standard way to provide an estimate of the local structure orientation in an image u consists in computing the eigenvector e associated to the leading eigenvalue λ of the structure tensor j ρ (u) [ ] associated to u. by fixing σ, ρ > to be the pre-and post-smoothing parameters, we recall that the structure tensor j ρ (u) of an image u is defined as: where u σ := k σ * u is a smoothed version of the image u and k σ , k ρ are gaussian convolution kernels. usually, σ and ρ are chosen so that σ ρ, where σ is associated to the noise scale and ρ integrates the orientation information. denoting by λ ≥ λ the eigenvalues of j ρ (u) and by e , e the associated eigenvectors, a classification of the different structural image information in terms of the size of λ and λ can be made following, e.g., [ , , ] . for any x ∈ Ω we thus have: , then x is likely to belong to a homogeneous region; , then x is likely to lie on an edge; , then x is likely to be a corner point. tensor voting has been originally introduced in [ ] for extracting curves in noisy images by means of the grouping of local features consistent in a neighbourhood of the measurements. such framework improves the robustness of structure tensor estimation in presence of noise and image artefacts [ ] . assuming that a generic -tensor b in r has the following matrix representation: where λ , λ are the eigenvalues associated to the eigenvectors e and e , respectively, we have that ( ) can be equivalently rewritten as we can now distinguish the two following quantities: is called saliency or stickness. it provides an estimation of the confidence on the direction e and it is also called orientation certainty or anisotropy measure; • λ is called ballness and it measures the size of the minor-axis of the anisotropy ellipse. since it is in some sense a measure of how the estimation of the main estimated direction is contradicted, it is often called orientation uncertainty or junctionness. the tensor voting operation consists in adding, at each iteration, the contribution of neighbourhood tensors for each point in the domain, resulting in an enhanced tensor field due to the presence of saliency parameter. in [ ] the authors show that the complexity of the original approach may be time-consuming, even for small images. thus, they propose an efficient computation of the tensor voting framework based on steerable filters theory, i.e. complex-valued convolutions. for the shadow removal application we are considering, the estimation of the structure direction e may be affected by the shadow edges, which do not correspond to the actual underlying image structures. the presence of such edges make the use of either the structure tensor or the tensor voting very challenging for estimating the local directions. to circumvent this problem, we propose a modification of the tensor voting framework by means of interpreting shadow edges as bias in the estimation of the structure. from the given initial blurred image u σ , we firstly compute the local orientation θ of the gradient from the eigenvector e . secondly, we compute the saliency of the given shadowed image. finally we apply the tensor voting framework with a modified saliency and orientation on Ω sb so as to mark the shadow boundaries as bias: there, the saliency is zeroed and the orientation is initialised at random. details on the algorithm and results are presented in section . . . let f : Ω → r + be a positive greyscale image with a constant shadow and let Ω be decomposed as in ( ) . we propose the following structure-preserving osmosis model for solving the shadow removal problem: on Ω, here we define the discontinuous vector field d and the discontinuous matrix field w as where i denotes the × identity matrix, and e and e are the directions from our modified tensor voting applied to the initial image f . this means, an isotropic osmosis evolution is performed in Ω c sb , while an anisotropic inpainting is performed on Ω sb . in other words, classical osmosis balances image intensity in the shadowed region with respect to the unshadowed regions, while the nonlinear interpolation preserves structures and avoids blurring on the shadow boundary. the proposed model performs the osmosis and the inpainting step jointly and avoids any post-processing step. we now discuss appropriate discretisations of the anisotropic osmosis model that are consistent with the continuous model ( ) and preserve some of its properties as stated in theorem . . to do so, we consider a discrete rectangular image domain with m × n pixels. let s := m n . the given positive initial image f is then defined as a vector in r s + . for a given grid step size h > , we denote by u = (u i,j ) i,j the approximation of the function u and with u i,j its approximated value in suitable discretisation nodes ((i − )h, (j − )h) with i = , . . . , m and j = , . . . , n . similarly, for k ≥ we denote by u k i,j the value of u i,j at the time node t k = kτ , where τ is the time step size. also, for x ∈ Ω, we denote by λ , λ ∈ r s + the discretised eigenvalues λ (x) and λ (x), while by θ ∈ [ , π) s the discretised orientation θ(x). a discrete theory for osmosis models has been established in [ ] . since it is also applicable to the anisotropic setting, we report here the general result [ , proposition ] and list in the following some discrete solvers fulfilling its assumptions. . for a given f ∈ r s + , consider the fully-discretised problem: where the (unsymmetric) matrix p ∈ r s×s is an irreducible, non-negative matrix with strictly positive diagonal entries and unitary column sum . then the following properties hold true: (i) the evolution preserves positivity and the average grey value of f ; (ii) the eigenvector of p associated to eigenvalue is the unique steady state for k → ∞. as shown in [ ] for the isotropic osmosis model, standard explicit and implicit time finite difference schemes fit this framework, the former being subject to time step size restrictions, the latter being unconditionally stable. furthermore, both schemes converge to the space discretisation of the elliptic steady state for every stable time step size. for the implicit scheme with a bicgstab solver, vogel et al. [ ] report speed-ups of two orders of magnitude compared to the explicit method. in [ ] a peaceman-rachford splitting is shown to satisfy theorem . under a time step size restriction, while the additive operator splitting (aos) considered in [ ] fulfils theorem . for all time step sizes. splitting schemes such as the aos method, however, do not converge to the space-discrete elliptic steady state solution unless the time step size goes to zero. in practice, keeping this time splitting error under control imposes bounds on the time step size. note that in contrast to the theory for fully discrete diffusion filters [ ] , the discrete osmosis theory in theorem . does not require a symmetric matrix. since it does also not involve any isotropy assumption, it is basically also applicable to anisotropic osmosis processes, if suitable space discretisations are employed. this, however, is not straightforward and will be discussed in the sequel. in what follows, we describe the discretisation of the weighting matrix w and the differential operators div and ∇ for a grey-scale image of height m and width n unrolled as u ∈ r s , with s = m n , that defines the spatial discretisation matrix a for the semi-discrete osmosis problem u (t) = au(t), for t ∈ ( , t ], where t > is a positive final time. while diffusion processes are stable in many aspects, e.g. in terms of decreasing l norms and decreasing l ∞ norms, the stability of osmosis processes is restricted to essentially one key property: the preservation of nonnegativity. thus, any suitable space discretisation for osmosis filters should guarantee that it is nonnegativity preserving. for the matrix a this implies that all off-diagonal elements must be nonnegative. while this is easily satisfied for standard discretisations of isotropic processes [ ] , it becomes much more challenging for anisotropic approaches: the drift term is fairly unproblematic and can be handled e.g. with classical upwind discretisations. however, most discretisations of the diffusion term div(w∇u) are only stable in the l norm [ ] . thus, they cannot guarantee preservation of nonnegativity. nonnegativity-preserving discretisations can be found in [ , , ] . in our paper, we use the stencil of fehrenbach and mirebeau [ ] , since it is a fairly sophisticated nonnegativity-preserving method that has been reported to give good results. this anisotropic diffusion discretisation relies on lattice basis reduction ideas and is called ad-lbr. let us sketch its underlying ideas. in [ ] , the authors tackle the minimisation of an anisotropic energy which in our notation reads: where e w := e, we , for any e ∈ r d and w is symmetric and positive definite. the idea is to introduce a discretisation e h of the energy above on the discretised domain Ω h , h > via a sum of weighted squared differences of u ∈ l (Ω h ; r), i.e.: where v (x) ⊂ z d is the stencil and γ x (e) ≥ are the associated weights. the key step is the linearisation of u(x + he) as u(x) + ∇u(x), he , which shows that for each x ∈ Ω h and smooth u one can write: which turns out to be equivalent to require the condition w = e∈v (x) γ x (e)ee t . via the following lemma [ , lemma ]. lemma . . let e , e , e ∈ r be such that e + e + e = and |det(e , e )| = . then for any symmetric positive definite matrix w, there holds: under the convention e +i := e i actually, for any dimension d ≤ and any symmetric positive definite d × d matrix m, there always exists a family (e i ) i∈i of vectors in r d such that e i , me j ≤ for any i = j. such a family is called m-obtuse [ ] . thus, by taking (e , e , e ) as a w-obtuse superbase of z (i.e. a basis of z with |det(e , e , e )| = such that e + e + e = ), a stencil v (x) := {e , e , e , −e , −e , e } can be used to write explicitly the coefficients γ x in ( ) for ≤ i ≤ as done in [ , equation ( )]: the resulting stencil is shown to be independent of the choice of the superbase [ , lemma ] and it is orientated along the preferred diffusion direction given by w. also, the ad-lbr scheme is sparse, i.e. it has a limited support of points for twodimensional images. in the ad-lbr discretisation, an important role is played by the anisotropic ratio κ ∈ [ , ∞), which measures the geometrical shape of the ellipse associated to the eigendecomposition of the anisotropic matrix w: the closer κ is to , the more w is similar to the identity matrix i. the computation of the stencil has a logarithmic cost in the anisotropy ratio κ of the diffusion tensor, making ad-lbr appealing for applications. also, by fixing a direction θ all over the domain Ω for w, the anisotropy ratio κ can be related to the eigenvalues of w; see [ , equation ]. for instance, if w has eigenvalues ε and with < ε , then ε = /κ . for completeness, we report in table the ad-lbr stencil for different choices of ε and a fixed angle θ = π/ that characterises the second eigenvector e = (cos θ, sin θ) of w; cf. also [ , table , table ]. table : ad-lbr stencil for the discretisation of div(w∇( · )): different choices of ε are presented with fixed angle θ = π/ for the first eigenvector. in bold we denote the (i, j) entry. as expected, we observe that in the case of strong anisotropy, the stencil becomes aligned in θ direction. remark . . by construction, the ad-lbr space discretisation matrix a has column sum and non-negative off-diagonal entries. moreover, our numerical experiments give strong evidence that a is also irreducible †. however, a formal proof of the irreducibility of a is left for future research. in this section we see how it is possible to solve the dynamical system ( ), through a highly accurate approximation of the exact solution first of all, we notice that it is not necessary to compute the large and dense matrix exp(t a) explicitly. it is sufficient to compute only its action on the initial solution f . polynomial methods (see, for instance, [ , , ] , approximate the action of the exponential by a polynomial of a certain degree applied to the initial vector. they do not require to solve a linear system of equations: usually they scale the matrix and approximate the solution by an iterative procedure like u k+ = p m k (α k τ a)u k , k = , , . . . , k − , u = f †we applied the tarjan's algorithm finding the strongly connected components of a directed graph [ ] . code is freely available at matlab central: mathworks.com/matlabcentral/fileexchange/ . where p m k is a polynomial of degree m k which approximates the exponential function and after the last iteration, u k ≈ u(t ). such an iterative scheme is usually needed in order to achieve a sufficiently accurate result. for krylov methods (like [ ] ), it is also necessary in order to keep the computational cost as low as possible, since the cost to produce p m k requires o(m k ) scalar products with vectors of the size of f . among the polynomial methods, the truncated taylor series [ ] and the interpolation in the newton form at leja points [ ] are able to bound the relative backward error. this means that they construct an approximation in k iterations with time step size τ = t /k: the tolerance tol can be chosen as small as desidered. the typical value for double precision arithmetic is − and in this sense the time integration is said "exact". the polynomial p m (z) is either the truncated taylor series of e z about a point which depends on the spectrum of the matrix or the interpolation polynomial of e z at real leja points on an interval related to the spectrum of the matrix. the choice of the parameters k and m can be done by simply considering the norm of t a, or estimates of (t a) q /q for small values of q, or, only for interpolation at leja points, estimates on the -pseudospectra of t a. from the implementation point of view, both algorithms simply require one matrix-vector product and one vector update at each degree elevation, and therefore the cost is o(m). when the spectrum of t a has a skinny shape, either horizontal or vertical, usually interpolation at leja points performs better (see [ ] ). since this method can be configured to be exact in time up to machine precision, it is basically possible to reach the final time t in a single time step of size τ = t . however, we prefer to use multiple time steps, since the steady state t is in general not known a priori. on the other hand, there is no restriction on the time step τ and it is therefore possible to implement any desired strategy for steady state detection (based, for instance, on the comparison of the solutions at two successive time steps). in particular, also variable step size τ k implementations are possible without any restriction given by the stability or the computational cost. the approximation of the action of the matrix exponential to a vector can be used also in the so called exponential integrators (we refer to the survey paper [ ] ) when solving general (non-linear) stiff ordinary differential equations. a natural question in this context is whether the matrix p := exp(τ a) satisfies the properties of theorem . for suitable matrices a. we answer this question with the following proposition, for which a simple lemma is required. proof. for every i, j = , . . . , n , we write each element b i,j in terms of the elements of c and d. for every column j we have: proposition . . let a be an irreducible matrix, with column sum and non-negative off-diagonal entries. then the (non-symmetric) matrix p := exp(τ a) is an irreducible positive matrix with column sum . proof. we firstly show that p has column sum . to this end, we use the expansion by hypothesis, a has column sum . then the column sum of a k is for every k ≥ by lemma . . thus, the matrix p = exp(τ a) has column sum . in order to show that all elements of p are positive, we rewrite a as where d = diag(a), i is the identity matrix, n = a − diag(a) is a non-negative matrix and α := min i a i,i − γ, for any γ > . we have that p can be expressed as the product where τ (d − αi + n) is a non-negative matrix with positive diagonal and which is also irreducible. in fact, being that false, there would exist an extra-diagonal element in position (i, j) such that (d − αi + n) k i,j = for all k > . but then, (a) k ij = (αi + (d − αi + n)) k i,j = for all k > , meaning that a is reducible, which is false by hypothesis. so, for any pair (i, j) there exists a k such that (τ (d − αi + n)) k i,j > . since all the powers of τ (d − αi + n) appear in the series which defines exp(τ (d − αi + n)), we conclude that all its entries are positive. finally, exp(τ a) is obtained by scaling with positive scalars the rows of exp(τ (d − αi + n)). therefore, exp(τ a) is a positive (and thus irreducible) matrix. recalling remark . , proposition . shows that the ad-lbr space discretisation in combination with our "exact" polynomial time discretisation leads to a fully discrete anisotropic osmosis scheme that satisfies all assumptions of the discrete theory (subject to the missing formal irreducibility proof). in this section we present several numerical examples showing the application of the isotropic and anisotropic osmosis model to solve the shadow removal problem in synthetic and real-world images. we apply the anisotropic model ( ) to images affected by almost constant shadows, in order to perform jointly the shadow removal and the inpainting procedure on the shadow edges. since in general the ground truth is not available for this problem, the quality of the reconstruction of the anisotropic model in comparison the isotropic approach is assessed by visual inspection. on the thickness of the shadow boundary. in the following numerical experiments we will assume for simplicity that a rough segmentation of the shadow boundary is provided beforehand. for natural real images, this may be a quite challenging task since, due to the possible presence of noise, blur and/or compression artefacts, such region may be not sharp and presents a blurred transition zone from the outside to the inside area of the shadow. as a consequence, standard segmentation methods based, for instance, on edge detection methods may fail. in fact, the task of shadow segmentation has been addressed on its own regard in previous works where brightness-based [ ] or clustering [ ] methods have been applied. however, in many practical situations, the shadow is often roughly detected manually by the user using a brush including pixels both from the inside and the outside of the shadowed area. such region corresponds to Ω sb in ( ) . in our experiments we have observed that if this region is chosen to be too small (i.e. smaller than the whole transition area between non-shadowed and shadowed area), then the shadow removal result is fairly poor. on the contrary, in general, selecting a thicker shadow boundary produces better results. thick boundaries favour the use of the anisotropic model ( )-( ) over the isotropic one ( ): as we seen above, the action of the homogeneous diffusion smoothing on a large region Ω sb is not able to preserve the underlying image structures. figure illustrates these findings. we compare the result obtained applying the isotropic model ( ) on a real image where the thickness of the shadow boundary is chosen differently. we clearly observe that a thicker Ω sb corresponds to a better removal of the shadow for the same large final time t . we will now show the numerical results obtained when the anisotropic model ( )- ( ) was applied to a variety of both synthetic and natural images. pseudocode. algorithm describes the main steps required to solve the joint anisotropic osmosis problem after the estimation of local structures in the given image. for the time integration we used the expleja solver ‡, which is the companion software of [ ] for computing the action of the exponential matrix on a vector. ‡freely available at: bitbucket.org/expleja/expleja firts we apply the anisotropic osmosis model to noise-free synthetic images, where the direction of the gradient z is known a priori. the purpose of this synthetic experiment is to check if our approach is able to effectively remove constant shadows. in figure we show the results obtained for an image with parallel greyscale stripes with θ = • orientation and for colour concentric circles, whose θ is chosen to be as the angle drawn with tangent to the circumferences. both images are corrupted with an almost constant shadow but a transition zone on the shadow borders. we compare the anisotropic model described in section . with the isotropic osmosis model ( ) , which results in an homogenous diffusion inpainting at the shadow boundary. the time discretisation is performed as described in section . . in our visual comparison of both methods, we choose the final time t = and the time-step τ = , and we proceeded using algorithm . in this experiment we also provide a visual representation of the local orientation angle θ inside the shadow boundary Ω sb . it becomes obvious that the anisotropic shadow removal method shows clear advantages at the shadow boundaries, since it does not suffer from blurring artefacts. in order to apply the anisotropic model to natural real-world images, the estimation of the discrete local orientation θ(x) becomes crucial. therefore, we show in section . for the tensor voting, we used the matlab implementation of [ ] from the companion software § of [ ] , a literature review on tensor voting. also, since tensor voting depends on local neighbourhoods, we use a multi-resolution strategy as is described in algorithm . // zeroing and randomizing the data on the shadow edges Ω sb saliency loc = saliency loc.*mask; orientation loc = orientation loc.*mask + π.* rand(size(f (:, :, c))).*( -mask); foreach k in scales do [ saliency, ballness, orientation ] = vote ( saliency loc, orientation loc, s k ); λ = saliency +ballness; λ = ballness; e = ( cos(orientation), sin(orientation)); e = (-sin(orientation), cos(orientation)); tvf = tvf + eigen to tensor ( e , e , λ , λ ); end end [ e , e , λ , λ ] = tensor to eigen ( tvf ); θ = xy ij (e ); // return the local orientation in (i, j) coordinates return θ in figure we compare the local direction estimation by means of the structure §freely available at matlab central: mathworks.com/matlabcentral/fileexchange/ tensor and the tensor voting approach applied on the shadowed image in figure a . we plot the magnitude of the leading eigenvalue of the structure tensor in figure b and of the one computed for the tensor voting algorithm in figure c . these images clearly show that the estimation via tensor voting is less sensitive to the false edges introduced by the shadow boundaries. this is reflected in the plot of the leading directions, too: the directions computed via the structure tensor in figure d are visibly affected by the light jumps while the ones computed with tensor voting in figure e can still connect the structures from outside to inside the shadow with no additional edges. (a) input f (b) stf, λ (c) tvf, λ (d) stf, e (e) tvf, e figure : comparison: structure tensor framework (stf) with (σ, ρ) = ( . , ) versus tensor voting framework (tvf) with multi-scales ( , , ) and σ = . . we plot the main direction e and its associated eigenvalue λ . results on real images. we now combine the proposed tensor voting framework with the anisotropic osmosis model to remove constant shadows by means of an anisotropic drift-diffusion model. in the following experiments we use the final time t = and the time step size τ = . in practice the shadow removal is accomplished almost completely already for t t . however, for a better approximation of the steady state, we use the final time t for comparison. in figure we show a zoom of the results from figure ¶, presented as motivation for this work. note that in this case the shadow is artificially added as a multiplicative rescaling factor c ∈ ( , ). the estimation of the direction on the shadow boundary is computed via the proposed algorithm . (a) shadowed f orientation angle θ isotropic anisotropic figure : zoom of the results for the shadowed image in figure . ¶figure a (zoomed in figure a ) courtesy of r. d. kongskov. in figure we apply the isotropic and the anisotropic model to a several shadowed images affected by natural constant shadows . zoomed details can be found in figure . here the complexity of local image structures makes the application of the anisotropic osmosis model harder. thus, we use the tensor voting algorithm to estimate the local directions connecting the structures from inside to the outside of the shadow region. also in these real-world scenarios, we observe the superiority of the anisotropic approach: structures are interpolated reliably across the light jump introduced by the shadow. we note that our anisotropic approach removes shadows effectively both for synthetic and real-world images. structures are propagated correctly over the shadow boundary. since the ad-lbr stencil requires a positive definite matrix w as input, we need to choose ε > , which may lead to some over-smoothing in the orthogonal direction. also, we noticed some over-smoothing effect in Ω out and Ω in for the ad-lbr scheme, e.g. in figure f , whose understanding is a matter of future research. in terms of efficiency, the computational time needed for computing the action of the exponential matrix onto a vector, that is the product exp(τ a)u , largely depends on the choice of the time-step τ , the final time t and the size of the images. although it is possible to directly choose τ = t and proceed in a single step, we prefer multiple steps. in this way, by comparing two successive solutions u k+ and u k , it is possible to detect whether the evolution is sufficiently close to its steady state. our solvers that are exact in time offer additional advantages over classical inexact methods such as explicit and implicit schemes when one is also interested in good approximations of intermediate results. in this work, we have generalised isotropic osmosis filtering introduced in [ , ] to the anisotropic setting. this was achieved by introducing a weight matrix whose directional information was extracted from a modified tensor voting approach [ ] . when applied to the shadow removal problem, the anisotropic model acts as an inpainting interpolator on the shadow boundary. it is close in spirit to inpainting with edge-enhancing anisotropic diffusion [ , , ] . from a numerical point of view, we have combined the nonnegativity preserving anisotropic diffusion stencil of fehrenbach and mirebeau [ ] with techniques based on exponential integration [ ] : we argued that the fully discrete model satisfies the discrete properties studied in [ ] in a general setting for osmosis. we tested the proposed model for synthetic and real examples, showing that the generalised model acts as a combined osmosis-inpainting model for shadow removal problems, thus avoiding any undesirable post-processing inpainting step. future work will address the investigation on the applicability of the proposed anisotropic model to more general imaging applications. computing the action of the matrix exponential with an application to exponential integrators a variational framework for exemplar-based image inpainting mathematical problems in image processing: partial differential equations and the calculus of variations shadow removal from a real picture sketches & applications, siggraph ' alternating direction implicit (adi) schemes for a pde-based image osmosis model the leja method revisited: backward error analysis for the matrix exponential image processing and analysis: variational, pde, wavelet, and stochastic methods fast shadow removal using adaptive multi-scale illumination transfer low-dimensional lattices. vi. voronoi reduction of three-dimensional lattices sparse non-negative stencils for anisotropic diffusion a feature based correspondence algorithm for image matching an efficient method for tensor voting using steerable filters image compression with anisotropic diffusion anisotropic total variation filtering a review of pde models in image processing and image analysis inferring global perceptual contours from local features novel schemes for hyperbolic pdes using osmosis filters from visual computing a combined corner and edge detector exponential integrators analyzing oriented patterns directional total generalized variation regularization for impulse noise removal directional total generalized variation regularization perceptual grouping by tensor voting: a comparative survey of recent approaches adaptation of tensor voting to image structure estimation consistent positive directional splitting of anisotropic diffusion an investigation of smoothness constraints for the estimation of displacement vector fields from image sequences digital cultural heritage imaging via osmosis filtering higher order total directional variation. part i: imaging applications (forthcoming) higher order total directional variation maximum principles in differential equations analysis of some krylov subspace approximations to the matrix exponential operator geometric partial differential equations and image analysis understanding, optimising, and extending data compression with anisotropic diffusion partial differential equation methods for image inpainting. cambridge monographs on applied and computational mathematics depth-first search and linear graph algorithms scale space and variational methods in computer vision anisotropic diffusion in image processing linear osmosis models for visual computing tensor field interpolation with pdes l -stable nonstandard finite differences for anisotropic diffusion the authors thank j.m. mirebeau for the insightful discussions and comments on the adaptation of the code from [ ] to our problem and the isaac newton institute for mathematical sciences for support and hospitality during the program "variational methods and effective algorithms for imaging and vision" when work on this paper was undertaken. this work was supported by epsrc grant number ep/k / . key: cord- -n jvt authors: vandegrift, jillian; hooper, jennifer; da silva, allegra; bell, kati; snyder, shane; rock, channah m. title: overview of monitoring techniques for evaluating water quality at potable reuse treatment facilities date: - - journal: j am water works assoc doi: . /awwa. sha: doc_id: cord_uid: n jvt needless to say, the safety of treated water for potable reuse must be definitively ensured. numerous methods are available for assessing water quality; it's important to understand their challenges and limitations. i ncreased demand on water supplies, associated with population growth and changes in climatic patterns, has made it necessary for many utilities to consider augmenting their current drinking water sources with recycled water-i.e., potable reuse. for regions with imminent water supply shortages due to uncharacteristically long periods of drought or significant population shifts, the primary options for increasing water supply are water importation, saltwater desalination, and water reuse (snyder ) . water reuse may include a combination of potable and nonpotable uses to increase water supply resiliency. nonpotable reuse is water not intended for direct human consumption, such as irrigation or coolingtower water. potable reuse involves integrating highly treated municipal wastewater directly into drinking water systems, which may or may not include retention in an engineered storage buffer before its introduction, such as direct versus indirect potable reuse. in the united states, approximately - % of the bil gal of treated municipal wastewater effluent is recycled (usepa ). potable reuse was implemented in the united states more than five decades ago, with the first planned project at the los angeles county sanitation district's montebello forebay aquiferrecharge spreading grounds in (usepa ) . the us environmental protection agency (usepa) developed the first national policy statement on water reuse in (usepa ) , and the national research council developed the first set of water quality criteria for reuse in (nrc ) . usepa developed the guidelines for water reuse in , which went through three revisions (usepa (usepa , (usepa , (usepa , . the agency recently published a supplement titled potable reuse compendium (usepa ), appending the guidelines for water reuse to include more details for potable reuse. while there are no federal regulations specific to potable reuse in the united states, the safe drinking water act (sdwa) and the clean water act (cwa) provide the statutory requirements for water quality that apply to potable reuse scenarios. wastewater effluent must meet requirements set forth by the cwa and the national pollutant discharge elimination system. subsequently, advanced treated water must meet the requirements of the sdwa and national primary drinking water regulation (npdwr) maximum contaminant levels (mcls) and abide by nonregulatory water quality standards for aesthetics in the national secondary drinking water regulation mcls. specific regulations, policies, provisions, and/or guidance for potable reuse have been developed in states: arizona, california, florida, hawaii, idaho, massachusetts, nevada, new mexico, north carolina, oklahoma, oregon, pennsylvania, texas, virginia, and washington (usepa while several recent pilot and demonstration-scale studies have been conducted, two notable full-scale potable reuse facilities have provided water directly into the distribution system: big spring colorado river municipal water district ( . mgd) in and wichita falls ( mgd) in , both of which are in texas. there are two additional potable reuse facilities in the process of construction and permitting: village of cloudcroft, n.m., and the el paso advanced water purification facility in texas. figure shows a map of potable reuse projects in the united states. from a global perspective, potable reuse is expanding and will continue to grow as a means to meet drinking water supply needs in the decades to come (khan ) . probably the most well-known international project is the goreangab water reclamation plant in windhoek, namibia, which began in and was expanded in to produce . mgd of potable water (usepa ). this facility supplies approximately % of the city's potable water demand (nrc ) . australia was the first country to develop federal potable reuse guidelines when phase (which included augmentation of drinking water supplies) of the australian guidelines for water recycling was released in (ephc et al. ). singapore's newater facilities can provide up to % of the nation's water supply needs with water that meets or exceeds usepa's drinking water standards and the world health organization's guidelines (usepa (usepa , who ) . potable reuse requires careful monitoring of pathogens and chemical contaminants because of their potentially higher concentrations in the source water. ultimately, monitoring systems need to be accurate, technically accessible, and cost-effective to be successful (mosher et al. ). there has been great interest in further developing and adopting new monitoring techniques to incorporate indicator and surrogate three column figure max width = p (actual column width = p ) figure potable reuse projects in the united states a includes pilots, studies, and prospective projects compounds, optical sensors and biosensors, and bioassays. an indicator compound is an individual chemical or microorganism occurring at a measurable level that represents certain physiochemical and biodegradable characteristics of either a group of trace chemical constituents or microorganisms of interest (nrc , usepa , drewes et al. , who . for example, escherichia coli (e. coli) or coliphages are used as microbial indicators for evaluating disinfection log-reduction credits. a surrogate parameter is a measurable change of a bulk parameter that can assess the performance of a treatment barrier and can be consistently monitored (nrc , usepa . turbidity is often monitored for overall treatment performance and for demonstrating pathogen removal across filtration. indicators and surrogates must be selected on the basis of knowledge of suspected target contaminants. these must also be present at sufficiently high concentrations that are above their relative detection limits (crook et al. ) . drewes et al. ( ) performed an extensive evaluation of indicator and surrogate compounds for wastewater and water reclamation. they noted that a suite of indicator compounds and surrogates should be used to encompass a variety of physical-chemical properties and behaviors to help determine the removal efficacy for specific treatment barriers in instances where actual concentrations cannot be measured. in the united states, use or adoption of a monitoring tool or technique must demonstrate pathogen log-reduction credits required by state regulatory agencies. the riskbased approach used by the sdwa surface water treatment rule can apply to potable reuse scenarios to demonstrate that the appropriate level of protection of human health has been achieved (mosher et al. ) . monitoring schemes must also incorporate verification of water quality at critical control points (walker et al. , halliwell et al. , salveson et al. . figure shows examples of grab samples at critical control points through an advanced treatment scheme at a potable reuse facility. for direct potable reuse, the absence of an environmental buffer reduces failure response times, so the overall treatment process and associated monitoring program must be resilient enough to respond quickly and effectively if off-specification water is encountered (pepper & snyder . pathogens are characterized into three groups: bacteria, protozoa, and viruses. in general, bacteria can be destroyed, whereas protozoa and viruses are typically removed or inactivated. virus removal is particularly important because of the occurrence of viruses in municipal wastewater and acute health impacts (khan ) . bacteria. in general, bacteria range in size from . to μm in length, so they are large enough to be effectively removed via filtration, while they are also susceptible to chemical or ultraviolet (uv) disinfection. enteric pathogenic bacteria tend to be present in lower concentrations in wastewater than indicator species. e. coli, enterococcus spp., and fecal coliforms are often used as indicators of fecal bacteria. e. coli and enterococci are recommended by usepa as an indicator of fecal pollution in freshwater recreational waters because levels of these organisms were shown to be more accurate than fecal coliforms at predicting gastrointestinal illness (usepa (usepa , . fecal coliforms may also be selected for monitoring simply because many utilities already use fecal coliforms for routine testing. table shows typical indicator organisms for monitoring pathogenic bacteria, protozoa and helminths, and viruses (also see the photograph on page ). protozoa and helminths. most protozoan spores, cysts, oocysts, and eggs range in size from μm to more than μm and are thus substantially larger than most bacteria. protozoa and helminths can be eliminated from wastewater using either a physical removal or an inactivation process, such as ozone oxidation or uv disinfection (khan ) . clostridium perfringens has been used as an indicator for protozoa such as giardia lamblia and cryptosporidium parvum. additionally, because of c. perfringens' ability to form spores, this bacterium can be used as an indicator of pathogenic sporeforming protozoa. the methods to detect giardia and cryptosporidium are intensive, relying heavily on laboratory sample preparation and technician skill, whereas the detection method for c. perfringens is straightforward (though not usepa approved) and can distinguish between viable and nonviable spores (mosher et al. , usepa . in europe since the s, c. perfringens has been used as an indicator of fecal contamination in water (nrc ) . north carolina uses c. perfringens as an indicator in its water reuse regulations; to date it is the only state in the country that uses c. perfringens as a protozoan indicator for water reuse monitoring. florida uses cryptosporidium. viruses. ranging in size from . to . μm, viruses are far smaller than bacteria or parasites. enteric viruses replicate in the intestinal tracts of their hosts and are found in the fecal matter of those infected. monitoring for pathogenic viruses is challenging because not only are they notoriously difficult to detect, there are more than pathogenic enteric viruses and viruses (usepa ). however, viruses cannot replicate outside of their host and thus cannot multiply during wastewater or reclaimed water treatment. virus removal and inactivation is complex and species-specific, yet it can be achieved through coagulation/ flocculation/sedimentation, chemical disinfection, uv disinfection, ozone oxidation, and media or membrane filtration (myrmel et al. ) . because enteric viruses are difficult to detect, indicator viruses-most often bacteriophages (also known as phages)-are used. bacteriophages are viruses that infect bacteria; they do not infect humans. three main groups of bacteriophages are frequently used as surrogates for pathogenic waterborne viruses: somatic coliphages, male-specific or f+ ribonucleic acid (rna) phages, and bacteroides fragilis phages (lucena & jofre , who , iawprc study group . there is a lack of consensus about which group serves as the best indicator of enteric viruses. advantages and disadvantages of each category are presented in table . pathogen detection methods. the best methods for monitoring pathogens are rapid, highly sensitive, selective, and can distinguish between viable and nonviable organisms. no single method achieves all of these goals, so a suite of methods is recommended (mosher et al. ). indicator organisms must be present in sufficient concentrations to assess removal and must either strongly correlate to or be conservative with respect to the pathogen of interest. samples are typically concentrated to improve sensitivity using membrane filters, magnetic beads, or microfluidic devices that discriminate particles on the basis of charge or size. molecular biological methods are highly sensitive-i.e., able to detect single copies of a genome. one concern with molecular methods is that they do not distinguish viable from nonviable cells or infectivity of viruses. immunological methods tend to be more rapid than molecular biology methods because they don't require as much sample pretreatment, but they are less sensitive (connelly & baeumner ) . biological molecule assays bioenergy molecules such as adenosine triphosphate (atp) or nicotinamide adenine dinucleotide phosphate (nadp + ) are present in all living organisms. atp and nadp + are not present in viruses. hydrolase enzymes, which represent a large class of microbiological household enzymes, may also be used for quantifying biomass. immunological assays these assays are highly selective but not highly sensitive. examples include the enzyme linked immunosorbent assay and serum neutralization tests. polymerase chain reaction (pcr), quantitative pcr (qpcr) pcr allows for the identification of the deoxyribonucleic acid (dna) or ribonucleic acid (rna) of pathogens or indicators present in a water sample. qpcr allows for the dna or rna to be quantified and is relatively rapid (less than hours). nucleic acid sequence-based amplification (nasba) nasba uses isothermic conditions for dna or rna amplification (rather than the temperature cycles used in pcr and qpcr), and therefore may be somewhat faster than pcr-based methods. droplet digital pcr samples are partitioned into thousands to millions of nanoliter or picolitre volumes inside small chambers on a chip (i.e., chamber digital pcr) or within a water-in-oil droplet (i.e., droplet digital pcr) before pcr amplification. the frequency of positive partitions is used with poisson statistics to estimate the number of target copies in the original sample. this technology starts with a single-stranded pcr product to which a primer is added to initiate a dna-sequencing reaction. turbidity higher-turbidity water may have a higher likelihood of having pathogens present, though correlations are not straightforward or ensured. light scattering suspended particles can be detected because of their ability to scatter light in water. multi-angle lightscattering technology uses lasers and light-scattering detectors to determine the shape and size of particles. microscopic identification an example microscopic identification method is usepa method for detecting cryptosporidium parvum and giardia lamblia. a sample is filtered, and then the oocysts and cysts are separated from the material captured using magnetic beads conjugated to anti-c. parvum and anti-g. lamblia antibodies, then eluted, stained, and counted with differential interference contrast microscopy. biosentry is another tool for microscopic identification and can detect between and spores/ml. raman spectroscopy involves ultraviolet through visible to near infrared light excitation. surfaceenhanced raman spectroscopy (sers) can identify specific microorganisms. a optical tweezers can isolate bacteria, allowing discrimination between different strains of bacteria. b fourier transform infrared (ftir) spectroscopy involves the excitation of molecules by infrared light and the unique signature spectra is measured. matrix-assisted laser desorption/ionization time-of-flight mass spectrometry detects the mass to charge ratio of particles to provide a "fingerprint" of specific microorganisms. c flow cytometry (fc) fc can be combined with the use of nucleic acid probes or fluorescent antibodies to rapidly identify and quantify specific microorganisms. researchers have presented methods to identify pathogenic e. coli o :h , c. parvum, and nonpathogenic e. coli in water. d-f sensitivity has improved through the amplification of viral signals as a result of the use of a novel nucleic acid dye, sybr-gold. these recent advancements make near-real-time measurements for total bacteria and viruses in water a practical possibility. ware et al. processes and their ongoing performance for removing pathogens and other chemicals (crook et al. ) . though each unit treatment process has its own bulk surrogate parameters that lend information on treatment performance, some are applicable across multiple methods. such surrogates include conductivity, boron, calcium, magnesium, uv absorbance, total organic carbon (toc) or dissolved organic carbon, specific uv absorbance, fluorescence excitation/emission matrix spectroscopy, total nitrogen, ammonia, nitrate, and concentration times time (crook et al. , drewes et al. . table shows a list of conventional parameters used to assess performance. solid-state instruments, including those for conductivity, toc, ph, turbidity, and temperature, continue to be the most reliable methods for measuring changes in drinking water quality in real time (storey et al. ) . for potable reuse scenarios that include an environmental buffer, a wastewater tracer test can assess dilution and contaminant degradation in receiving water bodies. wastewater tracers must be recalcitrant and not easily removed during treatment. for example, primidone and carbamazepine are not easily biodegraded and exhibit poor removal (< %) during soil-aquifer treatment (sat) (drewes et al. ) . these compounds can be used to track wastewater contributions to groundwater during sat. artificial sweeteners, such as sucralose and acesulfame-k, are also relatively nondegradable in nature yet abundantly present at levels above their detection limits, and they serve as conservative tracers for the presence of wastewater in surface water and groundwater (anderson et al. ) . sucralose is useful for evaluating the removal of water-soluble, uncharged chemicals with moderate molecular weights (crook et al. ) . trace chemical constituents. trace chemical constituents encompass an array of compounds, including industrial, manufacturing, and consumer products, as well as personal care products and pharmaceuticals; example constituents are described in table . their concentrations in raw, secondary, and tertiary treated wastewater are wide-ranging (drewes et al. ) . a survey of drinking water treatments in the united states demonstrated maximum finished-water constituents of emerging concern (cecs) at concentrations below ng/l, except for the flame retardant tris ( -chloro- -propyl) phosphate (commonly known as tcpp), which had a maximum concentration of ng/l . in california, the water quality control for recycled water, also known as the recycled water policy, was adopted by the state water board in and amended in to include cec monitoring recommended by its science advisory panel (anderson et al. ) . the final list of recommended compounds included cecs (drewes et al. ) . four cec indicator compounds were recommended for potable reuse monitoring by the national water research institute panelnamely, cotinine, meprobamate, carbamazepine, and estrone, because of the following characteristics (crook et al. ): • cotinine: low molecular weight that is likely charged to some degree at ph values employed in water treatment • meprobamate: low molecular weight with a basic structure similar to alkanes and occurs abundantly and in sufficient concentrations • carbamazepine: low molecular weight, unique structure, recalcitrant, abundant in wastewater and occurs at adequate concentrations • estrone: natural steroidal hormone that is present at higher levels than β-estradiol or ethinyl estradiol chemical constituent detection methods. as shown in table , different analytical methods are needed to quantify trace chemical constituents because of their diverse physicalchemical properties. despite considerable progress in detection methods, there are no standard methods for quantification. when interpreting (drewes et al. ) . bioanalytical tools. through drinking water, human exposure to chemicals does not happen singly but rather occurs as a complex mixture of dissolved chemicals and particulate matter. often chemical toxicity is nonadditive and may be synergistic or antagonistic. scientific studies have demonstrated that combinations of chemicals that would not elicit an effect on their own can induce significant toxicity when combined (margiotta-casaluci et al. ) . conversely, antagonistic effects between suites of chemical constituents may occur. because of the complex nature of constituents in wastewater, in addition to potential synergistic effects of mixtures of compounds, biological assays that can rapidly and comprehensively screen water for a suite of toxicological end points may be useful. however, biological assays tend to be subjective and thus should be used appropriately. a comprehensive survey of bioassays indicative of a wide range of responses has been published (escher et al. ) . these include assays that were sensitive to induction of specific modes of toxicity, such as mutagenicity and genotoxicity, xenobiotic toxicity, reactive toxicity, cytotoxicity, and endocrine disruption, among other modes of action. the conclusions of the study demonstrated that while there are limitations to bioassay techniques, they are a valid tool for water quality assessment that complement chemical analyses. a recent study incorporating bioassays covering biological endpoints indicated that a suite of bioassays can be useful in characterizing multiple toxicological pathways relevant to human health and can guide decision-making with regard to the treatment processes (jia et al. ) . the science advisory panel, which convened in to evaluate risks from cecs in water treated for reuse, recommended that estrogen receptor alpha and the aryl hydrocarbon receptor bioassays be used to respectively assess estrogenic and dioxin-like biological activities in recycled water (drewes et al. ). however, these technologies are relatively immature and require further development in order to identify, create, and standardize bioanalytical tools. advanced-treated recycled water can be beneficially reused for a variety of purposes, including augmenting drinking water supplies. multibarrier approaches, including advanced treatment, have been demonstrated to achieve highquality water, even when the source water is significantly degraded (nrc ). however, appropriate monitoring schemes are needed to ensure public health protection. a challenge for potable reuse monitoring is how to effectively characterize pathogens, chemical constituents, and emerging contaminants in advanced treated water in an appropriate time frame. water quality monitoring must incorporate conventional monitoring schemes that apply to wastewater and drinking water treatment; it must also incorporate high-frequency indicator and surrogate compounds to assess pathogen and chemical removal efficacy. complex monitoring programs tracking trace chemical constituents aren't required, but indicator compounds such as sucralose or cotinine can be useful to characterize removal of more recalcitrant compounds. commonly used bulk indicators, such as turbidity and conductivity, remain useful to assess removal of pathogenic organisms, but they have limitations. additional characterization of pathogen removal using indicator organisms (e.g., total coliform bacteria, clostridium perfringens, viral indicators, bacteriophages), incorporating a combination of physical detection, cell culture, and/or molecular biological assays, gas chromatography (gc) and mass spectrometry (ms) gc/ms is one of the most widely used combined techniques because of its sensitivity and selectively. ms measures an analyte dependent on its mass to charge ratio. the compound must be charged before it enters the machine. gc/ms employs two methods to charge or ionize analyte, electron ionization (ei) and chemical ionization (ci). ei results in molecular fragmentation, which gives each compound its own "fingerprint" and allows for individual detection; however, sensitivity is lost. ci involves ionization of a gas and results in less fragmentation than ei. liquid chromatography (lc) and tandem ms lc/ms differs from gc/ms in that the separation of analytes occurs in the liquid phase. lc/ms uses three ionization techniques: electrospray ionization (esi), atmospheric pressure chemical ionization, and atmospheric pressure photoionization. esi is the most common technique. isotope dilution lc and tandem ms lc/ms/ms involves solid-phase extraction and lc/ms/ms using esi in positive and negative modes. urobilin is a metabolic byproduct of heme metabolism that is excreted through human and animal feces in water. urobilin has been shown to be a beneficial biomarker for detection of fecal contamination. fluorescence detection of urobilin is achieved when urobilinogen-zinc, a chelation complex, is excited by blue light and exudes a green fluorescence. may provide useful information to enhance characterization of water quality and subsequent advanced water treatment. monitoring strategies for chemicals of emerging concern (cecs) in recycled water: recommendations of a science advisory panel. california state water resources control board comparative survival of indicator viruses and enteric viruses in seawater and sediment bio-sensors for the detection of waterborne pathogens examining the criteria for direct potable reuse, recommendations of an nwri independent advisory panel (wrrf - ) development of an immunomagnetic bead-immunoliposome fluorescence assay for rapid detection of escherichia coli :h in aqueous samples and comparison of the assay with a standard microbiological method monitoring strategies for constituents of emerging concern (cecs) in recycled water: recommendations of a science advisory panel development of indicators and surrogates for chemical contaminant removal during wastewater treatment and reclamation the application of a recently isolated strain of bacteroides (gb- ) to identify human sources of faecal pollution in a temperate river catchment benchmarking organic micropollutants in wastewater, recycled water and drinking water with national health and medical research council, & natural resource management ministerial council bacteriophage as pollution indicators utilization of hazard analysis and critical control points approach for evaluating integrity of treatment barriers for reuse (wrrf - ) bacteriophages as model organisms in water treatment f-specific rna bacteriophages are adequate model organisms for enteric viruses in freshwater f-specific rna bacteriophages and sensitive host strains in faeces and wastewater of human and animal origin bacteriophages and indicator bacteria in human and animal faeces distinguishing human from animal fecal contamination in water by typing male specific rna coliphages genotyping male-specific rna coliphages by hybridization with oligonucleotide probes bacteriophages as model viruses in water quality control in vitro bioassays to evaluate complex chemical mixtures in recycled water is the replication of somatic coliphages in water environments significant phage methods drinking water through recycling: the benefits and costs of supplying direct to the distribution system simple and rapid f+ coliphage culture, latex agglutination, and typing assay to detect and source track fecal contamination potential use of bacteriophages as indicators of water quality and wastewater treatment processes mode of action of human pharmaceuticals in fish: the effect of the -alpha-reductase inhibitor, dutasteride, on reproduction as a case study enteroviruses and bacteriophages in bathing waters potable reuse research compilation: synthesis of findings (wrf reuse - ). water environment & reuse foundation enteric viruses in inlet and outlet samples from sewage treatment plants water reuse: potential for expanding the nation's water supply through reuse of municipal wastewater indicators for waterborne pathogens. the national academies press quality criteria for water reuse. the national academies press pathogens in water: value and limits of correlation with microbial indicators monitoring for reliability and process control of potable reuse applications (wrrf - ) achieving reliability in potable reuse: the four rs detection of viruses: atomic force microscopy and surface enhanced raman spectroscopy assessment of techniques to evaluate water quality from direct and indirect potable facilities. project . the water research foundation assessment of techniques to evaluate and demonstrate the safety of water from direct potable reuse treatment facilities risk reduction for direct potable reuse (wrrf - ) microbial source tracking: current methodology and future directions emerging chemical contaminants: looking for greater harmony toxicological relevance of edcs and pharmaceuticals in drinking water (wrf ) advances in on-line drinking water quality monitoring and early warning systems. water research swrcb (state water resources control board), . investigation on the feasibility of developing uniform water recycling criteria for direct potable reuse. swrcb bacteriophages active against bacteroides fragilis in sewage-polluted waters human origin of bacteroides fragilis bacteriophages present in the environment potable reuse compendium guidelines for water reuse. epa/ /r- / guidelines for water reuse guidelines for water reuse guidelines for water reuse epa policy statement on water reuse and water reuse background statement critical control point assessment to quantify robustness and reliability of multiple treatment barriers of a dpr scheme evaluation of an alternative ims dissociation procedure for use with method : detection of cryptosporidium in water applications of whole-cell matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry in systematic microbiology potable reuse: guidance for producing safe drinking-water water, sanitation and hygiene links to health: facts and figures. who water quality: guidelines, standards, and health detection of escherichia coli o : h , salmonella typhimurium, and legionella pneumophila in water using a flow-through chemiluminescence microarray readout system spectroscopic differentiation and quantification of micro-organisms in apple juice • planning for direct potable reuse: operational aspects of an integrated drinking water system these resources have been supplied by journal awwa staff. for information on these and other awwa resources key: cord- -aw wirmb authors: wohrley, julie d.; bartlett, allison h. title: the role of the environment and colonization in healthcare-associated infections date: - - journal: healthcare-associated infections in children doi: . / - - - - _ sha: doc_id: cord_uid: aw wirmb healthcare-associated infections (hais) can be caused by endogenous host microbial flora or by exogenous microbes, including those found in the hospital environment. efforts to decrease endogenous pathogens via decolonization and skin antisepsis may decrease the risk of infection in some settings. controlling the spread of potential pathogens from the environment requires meticulous attention to cleaning and disinfection practices. in addition to selection of the appropriate cleaning agent, use of tools that assess the adequacy of cleaning and addition of no-touch cleaning technology may decrease environmental contamination. hand hygiene is also a critical component of preventing transmission of pathogens from the environment to patients via healthcare worker hands. resulting from invasive devices or surgical procedures may be associated with a shift from colonization to infection. a complex interaction occurs throughout the body between commensal organisms and the barriers they colonize, with research on the skin and gastrointestinal tract microbiome shedding light on these interactions. the skin is a protective barrier with large numbers of colonizing bacteria. the skin microbiome is inhabited by bacteria, fungi, viruses, archaea, and mites; however most research has focused on bacteria. the presence of various microbes may influence disease as evidenced by the shift from staphylococcus epidermidis cultured from healthy skin in young children [ ] to propionibacterium acnes in teenagers with acne [ , ] . children with atopic dermatitis have a propensity to infection with staphylococcus aureus [ ] . loss of skin integrity, as with wounds, burns, inflammation, or invasive devices, allows pathogens to enter. the balance between host and flora is also important in the gut and is influenced by antibiotic usage, diarrheal diseases, and critical illness [ ] [ ] [ ] . the gut provides both an essential immune response in maintaining health with normal flora stimulating proliferation of epithelial cells in small and large intestines, participating in development of competent gut-associated immune responses, as well as providing a physical barrier function against pathogen invasion through colonization resistance [ ] [ ] [ ] . inflammatory bowel disease is just one example of altered gut flora associated with a disease state. the secretory antibody system is important in the defense against mucosal infections. specific secretory immunoglobulin a (iga), transported through secretory epithelia to the mucosal surface, inhibits pathogen colonization through microorganism entrapment in mucus and promotion of clearance of entrapped microbes via peristalsis or mucociliary movement [ ] . iga also plays a role in mucosal protection of the gut by binding to a mucous layer that separates commensal bacteria from the apical surface of intestinal epithelial cells [ ] . patients admitted to the hospital bring with them their "normal" flora which may be very different in a previously healthy child than in a technology-dependent child who resides in a long-term care facility. colonization of children with organisms specific to their individual clinical conditions, such as pseudomonas aeruginosa in a tracheostomy-and ventilator-dependent child, or multidrug-resistant enterobacteriaceae in the gi tract of a neurologically impaired adolescent with neurogenic bladder and a history of frequent urinary tract infections, may lead to infection with these pathogens. these resident organisms may be transferred to the environment where they can be acquired directly by other patients or transmitted indirectly by hcw hands. high-risk populations for acquisition of multidrug-resistant organisms include those who are critically ill, who are immunocompromised, or who have been hospitalized for long periods of time, either in acute care or long-term care settings. additional risk factors include prolonged use of antibiotics and contact with colonized patients or the colonized/contaminated hands of hcw. colonizing organisms may produce invasive infection whether or not colonization is acquired in the hospital or the community. colonization with methicillinresistant staphylococcus aureus (mrsa) is a risk factor for subsequent invasive mrsa infection [ ] . a study of relatedness between colonizing strains of s. aureus and those associated with invasive disease in adults found that more than % of s. aureus blood isolates were identical to those colonizing the patient's anterior nares [ ] . genotypes of s. aureus strains from surgical site infections were also noted to be identical to colonizing strains in more than % of surgical patients [ ] . the hospital environment represents a reservoir of organisms such as mrsa, vancomycin-resistant enterococcus (vre), and multidrug-resistant gram-negative pathogens as well as clostridium difficile. notably, these same drug-resistant organisms found on surfaces in acute care hospital settings can be found in outpatient settings [ ] [ ] [ ] [ ] . transmission between patients and the environment may occur directly from contaminated fomites, indirectly from fomites on hcw hands, or indirectly from patient to patient on hcw hands. c. difficile may be spread in this indirect fashion. for example, this organism may be first identified in the stool of hospitalized patients and then later found to be contaminating the hospital room and its contents, with spread to hcw hands and the hospital environment. hospital surfaces may serve as both a reservoir and a vehicle of transmission for pathogens. specific pathogens such as mrsa [ ] , pseudomonas aeruginosa [ ] , acinetobacter [ ] , and c. difficile can contaminate hospital surfaces because of their ability to survive in the environment. the amount of hospital surface contamination varies depending on body site of infection/colonization, patient type, and cleaning practices. vre is commonly associated with environmental contamination, especially in the presence of diarrhea [ ] . in a study of icu patients, the rates of environmental contamination were higher for patients with more than one body site positive for vre [ ] . c. difficile is often identified from rooms of colonized and infected patients, proving difficult to eradicate due to resilience of the spores [ ] . the frequency of positive environmental cultures for c. difficile is high; in one study % ( of ) of environmental cultures in rooms occupied by asymptomatic patients had positive cultures for c. difficile, and % ( of ) of cultures in rooms occupied by patients had c. difficile-associated diarrhea (p = . ) [ , ] . over % of the environmental isolates characterized in this study had an immunoblot type identical to that of the patient [ ] . during an outbreak investigation in an adult long-term care facility, c. difficile skin isolates from asymptomatic patients and from environmental surfaces matched the source patient's isolate in / ( %) and / ( %) cases, respectively [ ] . c. difficile has also been recovered from physician and nurse work areas [ , ] . given the potential for hospital surfaces to be contaminated with pathogens, it stands to reason that the hands, and even the gloves, of hcws can become contaminated as well. contamination of hands of hcws occurs after direct patient care or contact with contaminated surfaces [ ] [ ] [ ] . positive environmental cultures were found to be a risk factor for development of hand/glove contamination [ ] . not surprisingly, the level of hand contamination has been shown to correlate with level of environmental contamination [ ] . prior room occupants infected with healthcare-associated (hca) pathogens may provide a source of exposure to other patients [ , ] . admission to a room in which the prior occupant was infected or colonized with mrsa, vre, acinetobacter, or c. difficile is a risk factor for subsequent colonization or infection with these organisms [ ] [ ] [ ] [ ] . special terminal cleaning (after the patient has been discharged) of rooms previously occupied by patients with c. difficile infection, including the use of hydrogen peroxide vapor, has been implemented to reduce rates of subsequent infection [ ] . these procedures have led to reduced rates of infection in patients subsequently admitted to a room where a prior room occupant was infected or colonized with c. difficile [ , ] . since colonization may lead to infection, two basic strategies -horizontal and vertical -are employed to reduce hais. horizontal strategies seek to broadly reduce the burden of common healthcare-associated pathogens including s. aureus, enterococcus, gram-negative bacteria, and candida through interventions such as hand hygiene and environmental cleaning. vertical strategies target specific pathogens known to cause hais and utilize active surveillance testing as well as directed approaches to decrease colonization and prevent transmission and subsequent infection [ , ] . general horizontal prevention strategies are approached elsewhere in this text and include hand hygiene, contact precautions, isolation, and ppe use (see chap. , principles of infection control). strategies applied to patients known or at risk for pathogen colonization when viewed from a vertical approach fall into three broad categories: active surveillance testing (ast), pathogen-specific isolation, and decolonization. screening (active surveillance testing, or ast) involves detection of colonized patients using culture or molecular methods and typically focuses on high-risk pathogens, including s. aureus (mrsa), enterococcus (vre), and c. difficile, that are transmitted from person-to-person from colonized or infected patients [ , , ] . some of the principles, strategies, challenges, and controversies of ast will be discussed below. optimal samples for ast vary by pathogen. specimens for mrsa testing are most frequently obtained from nares; however s. aureus also colonizes the skin, perineum, pharynx [ ] [ ] [ ] [ ] , gi tract [ ] , vagina [ ] , and axillae [ , ] , and additional sites of screening may be indicated depending on the clinical scenario and potential consequences of infection. specimens may undergo culture-based or molecular methods for detection of s. aureus/mrsa. vre colonization is based on samples from stool, rectal, and perirectal swabs, using both molecular methods and culture-based methods [ ] . rectal or stool samples are also used for detection of multidrug-resistant gram-negative organisms such as extended-spectrum β-lactamase-producing enterobacteriaceae, as well as carbapenemase-producing enterobacteriaceae. pseudomonas and acinetobacter may be detected in multiple sites, depending on clinical situation, including the rectum, skin, nares, pharynx, wounds, urine, and trachea if the patient is mechanically ventilated. the use of ast without additional interventions to reduce risk for transmission has not proven effective. universal screening effort for pathogens has been most widely studied for mrsa and is considered to be controversial due to questions regarding its effectiveness in controlling spread, as well as cost [ ] . screening alone has not shown to be effective in reducing colonization and infection for mrsa [ , ] . studies have failed to show benefit for a combination of ast and isolation in reducing vre infection or colonization; however, outbreaks of vre have been successfully controlled in hospital settings with use of active surveillance, contact precautions, patient isolation, and cohorting [ ] . similarly, active surveillance is most useful following outbreaks of mrsa [ ] . a cluster randomized trial in intensive care units found that universal gown and glove use did not reduce overall acquisition of multidrug-resistant organisms (mdro); there was, however, a small reduction in mrsa transmission noted as a secondary outcome [ ] . another prospective study of icu patients failed to show a difference in mrsa transmission [ ] , with additional concerns for the psychosocial effect that isolation places on patients [ ] . in observational studies, single room isolation was shown to reduce mrsa acquisition and infection among hospitalized patients [ , ] . current recommendations for mrsa colonized and infected patients include isolation in single rooms or cohorting [ , ] . however, experts have called for a review of the current recommendations for contact precautions and isolation for mrsa colonization in view of the above stated concerns [ ] . the addition of targeted decolonization strategies to ast and isolation for control of spread of healthcare-associated pathogens has been most extensively studied to prevent mrsa spread in the hospital setting. patients who are nasally colonized with s. aureus are more than twice as likely as non-colonized patients to develop s. aureus infection [ , , ] . carriage may be classified as persistent, intermittent, or noncarriage [ ] . persistent colonization is associated with an increased risk of infection compared with intermittent or non-carriers [ ] . carriers with high bacterial loads have a higher risk of infection and may be more likely to transmit the bacteria to their environment [ , ] . greater quantities of s. aureus are found in the nares of persistent s. aureus carriers compared with intermittent carriers [ , ] . much research exists regarding the efficacy of active surveillance cultures combined with decolonization to decrease s. aureus transmission and infection in adults, with growing literature in neonatal icus [ ] [ ] [ ] . intranasal antibiotics (mupirocin), with or without antibacterial skin washes (chlorhexidine), have been used in order to decrease the bacterial burden and prevent transmission and infection. shortterm nasal mupirocin has been demonstrated to be effective in eradicating mrsa nasal carriage, with up to % success after week of treatment, and - % efficacy for longer duration of follow-up, depending on patient profile and body sites colonized [ , ] . nasal mupirocin use in high-risk settings has been demonstrated to be effective in eradicating s. aureus nasal colonization and reducing the number of infections in icu, hemodialysis, surgical, and long-term care settings [ ] [ ] [ ] [ ] . in a study of nearly two million adult admissions, a significant reduction in the rate of mrsa transmission and infection was noted after introduction of an infection control bundle, which included decolonization of mrsa carriers and isolation [ , ] as well as a hand hygiene program [ ] . however, a crossover study of universal screening on surgical wards combined with targeted decolonization and contact precautions was unable to demonstrate reduction in mrsa infections despite high compliance with screening [ ] . nasal mupirocin decolonization of nicu infants with mrsa colonization in two units with high prevalence (> %) of mrsa colonization decreased the rate of mrsa infections [ ] . however, a retrospective study in the usa failed to demonstrate benefit when nasal mupirocin was used for days in colonized neonates in a unit with a baseline prevalence of around % [ ] . this study showed that some nicu infants develop infection prior to detection of colonization and infants who remain in the nicu can become recolonized over time [ ] . taken together, these data suggest that decolonization measures may be most beneficial when the baseline rate of colonization is high. additional nicu studies have found a high correlation between colonizing strains and infecting strains and confirmed high rates ( %) of infections occurring before colonization is detected suggesting universal, rather than targeted, decolonization should be used to control the spread of mrsa [ ] . current recommendations suggest that decolonization may be considered in highrisk neonates during an mrsa outbreak or in cases of endemic mrsa when other measures are failing [ ] . a recent society for hospital epidemiology association (shea) survey regarding practices for mrsa identification and eradication in nicus noted that most ( %) performed surveillance screening (ast) for mrsa in neonates with variability in timing of samples, sites sampled, isolation protocols, and decolonization strategies employed [ ] . recommendations for mssa are less clear. invasive mssa infections occur . times more frequently than invasive mrsa infections in neonates, leading to significant morbidity and mortality [ ] . targeted screening followed by mssa decolonization in a single nicu reduced incidence rates of mssa-positive clinical cultures and mssa infections by more than % [ ] . mupirocin resistance among s. aureus isolates has been demonstrated in multiple studies, especially associated with prolonged use. high-level resistance has been associated with decolonization failure, and low-level resistance may be associated with early recolonization [ , ] . therefore, the long-term use of mupirocin is questioned, and alternatives to mupirocin for decolonization in those with mupirocin-resistant strains of mrsa are needed. however, in a long-term study examining use of mupirocin prophylaxis in the nicu over a -year period, the rate of s. aureus (mssa and mrsa) infections decreased from . to . per patient days without any mupirocin-resistant isolates identified [ ] . this finding is consistent with previous reports of low prevalence of resistance among s. aureus isolates from mupirocin-treated neonates [ ] . the use of decolonization may be most effective for patients at risk of infection for short periods of time such as surgical patients, whose risk of infection may be less once the surgical site is closed, as well as icu patients, whose risk may lower once they are discharged from the icu [ , ] . this is of import given that patients are recolonized within weeks or months following decolonization, and thus the effect is often shortlived [ , ] . mupirocin decolonization has been used specifically to reduce the risk of surgical site infections (ssis) associated with gram-positive organisms. in a metaanalysis of rcts or quasi-experimental studies including adult cardiac and orthopedic surgery patients, mupirocin decolonization was found to be significantly protective against gram-positive ssis, specifically s. aureus ssis [ , , ] . preoperative s. aureus decolonization is not routinely recommended for most pediatric patients undergoing surgery, however the impact of preoperative colonization on risk of ssi in children has been examined in many small studies. risk of ssi was not elevated in s. aureus-colonized children undergoing cardiac surgery [ ] . studies in adult cardiac surgery patients, however, suggest a benefit to mupirocinbased decolonization in prevention of ssi [ ] ; this topic as it pertains to cardiac surgery is discussed further in chap. . chlorhexidine (chg) is a widely used broad-spectrum topical antimicrobial agent [ ] . the centers for disease control and prevention recommend its use as a skin cleanser prior to insertion of central venous catheters (cvc) in both children and adults but do not recommend its use in infants less than months of age due to lack of safety and efficacy data in this population [ ] . in spite of these cautions, a national survey of neonatology training program directors indicates that most nicus use chlorhexidine for cvc site prep and maintenance but restrict use based on gestational age, chronological age, and birth weight [ ] . risks to premature infants relate to the increased potential for chemical burns and contact dermatitis in the setting of underdeveloped skin [ ] and the possibility of systemic absorption of chg, although no adverse events have been reported despite demonstrable blood chg levels [ , [ ] [ ] [ ] . chlorhexidine bathing has been suggested as another adjunct to decrease colonization and has been studied in adults and children, including neonates. an adult randomized controlled trial demonstrated that daily chlorhexidine bathing did not reduce hai including central line-associated bloodstream infection (clabsi), catheter-associated urinary tract infection (cauti), or ventilator-associated pneumonia (vap) [ ] . a number of other studies (including clinical trials) in adults, however, have shown positive benefits of chlorhexidine-containing products when used as part of a bundle approach for hai prevention [ ] [ ] [ ] . in the pediatric scrub trial, daily chlorhexidine bathing was compared with standard bathing practices to evaluate effect on incidence of bacteremia in critically ill children [ ] . there was a non-statistically significant reduction in bacteremia in the chg group in the intention-to-treat analysis and a % decrease in bacteremia in the per protocol arm [ ] . the use of universal decolonization raises concerns about the possibility of chlorhexidine resistance. a study from texas children's hospital found that nearly half of nosocomial s. aureus carried one or both genes associated with chlorhexidine tolerance (qaca/qacb and smr), noting that smr-positive isolates were more often resistant to methicillin, ciprofloxacin, or clindamycin as well [ ] . mupirocin resistance was also noted in . % of the isolates in this study [ ] . vertical and horizontal approaches to infection prevention have been compared in two studies: huang et al. compared three approaches to mrsa prevention among adult icu patients in the reduce-mrsa study [ ] . vertical approaches consisted of ast with and without targeted decolonization of mrsa carriers with chg bathing and intranasal mupirocin compared with a horizontal approach involving universal decolonization of all icu patients regardless of mrsa status. universal decolonization was found to be associated with the largest reduction in all-cause bsi ( %) and mrsa clinical culture rates ( %) [ ] . another group showed that improved hand hygiene in addition to universal chg bathing reduced overall infection rate and specific rates of candida cauti and acinetobacter vap [ ] . additionally, when there is high adherence to chg bathing and hand hygiene, there is no additional benefit to ast and isolation to reduce mdro acquisition rates [ ] . selective digestive decontamination (sdd) and selective oral decontamination (sod) are additional methods of universal decolonization employed in an effort to reduce colonization with gram-negative organisms, particularly in critically ill patients. both methods use a polymyxin, an aminoglycoside, and an antifungal, applied to the oropharynx as a paste or gel (sod) or in a liquid form administered per nasogastric or orogastric tube (sdd), paired with systemic antimicrobials, usually an intravenous third-generation cephalosporin. these two strategies have been studied in more than rcts and have been examined in metaanalyses with demonstrated efficacy in reduction of colonization, morbidity, and mortality in adult icu patients [ ] [ ] [ ] . widespread acceptance has been limited by concern over selecting for resistant organisms in universal applications, although long-term follow-up in units employing these strategies have not demonstrated an increase in resistant organisms [ ] . microbiome studies of adults undergoing sdd compared to healthy adults revealed dramatic shifts in the gastrointestinal microbiome of sdd recipients (as would be expected) as well as an increase in the relative abundance of organisms expressing antimicrobial resistance genes [ ] . the pediatric experience with these strategies is limited. in a single meta-analysis of rcts including children, ventilator-associated pneumonia rates were % lower in in those children receiving sdd [ ] . the use in neonatal populations has not been studied. the evidence for perioperative antimicrobial prophylaxis is well established, and the use of antimicrobials prior to incision reduces rates of ssi [ ] by reducing the concentration of potential pathogens within or near the surgical incision. the basic tenets of antimicrobial use to prevent ssi include use of prophylaxis for all elective operations requiring entry into a hollow viscus, involving insertion of intravascular or orthopedic prosthetic devices or implants, or operations in which occurrence of ssi would pose catastrophic risk to the patient (e.g., sternotomy). the choice of antimicrobial is based upon a need for bactericidal activity against the expected pathogens for specific surgical procedures as well as agents which are known to be safe and cost-effective. the goal is to provide bactericidal concentrations in tissues and serum at the time of incision and to be continued throughout the entire operation until the wound is closed. re-dosing of the antimicrobial agent may be required should the procedure last several hours or if there is significant blood loss. an important risk factor contributing to ssi risk is the number of organisms which gain entry into the wound intraoperatively. the greater the burden, the greater the risk of infection. when appropriate antimicrobial prophylaxis has been administered, a bacterial burden of is required to cause ssi; however if a foreign body is present, the threshold to cause infection may be significantly reduced. virulence of the organism also contributes to ssi risk. pre-and perioperative antiseptics are utilized in order to decrease organism burden and thereby reduce the risk of ssi. preoperative bathing with agents such as chlorhexidine has been shown to decrease the amount of endogenous flora on the skin but has not been shown to reduce rates of ssi in pooled analyses of adult surgical patients [ ] . in certain very high-risk populations, however, such as cardiac and orthopedic surgery patients, preoperative chlorhexidine bathing has been associated with reduced rates of ssi (especially those due to s. aureus or mrsa) [ ] [ ] [ ] . it is likely that the benefits of chlorhexidine bathing are influenced by the type of surgical procedure (i.e., high-risk vs. low-risk) as well as the baseline rate of ssi at a given institution. in spite of these controversies, the use of chlorhexidine body wash prior to surgery is routine. there are several options for preoperative skin antisepsis with either chlorhexidine-alcohol or povidone-iodine as the active agent. the authors of a cochrane review conclude that other characteristics of skin prep agents such as potential side effects and cost should be taken into consideration as well until there are definitive data showing clinical superiority of one agent over another [ ] . new cdc guidelines for prevention of ssi recommend an alcohol-based skin antiseptic, such as either chlorhexidine-alcohol or iodophor-alcohol products [ ] [ ] [ ] . surgical site infections generally arise from endogenous sources such as bacteria present on skin surfaces or in a viscus, with greatest risk occurring while the wound is open. in addition to skin surface, bacteria may be found in skin appendages, including sebaceous glands, hair follicles, and sweat glands [ ] . when infections related to exogenous sources occur, they may be sporadic or related to an outbreak. exogenous sources include contamination of the operating room environment, surgical instruments, equipment, or colonized or infected personnel [ , ] . common nosocomial pathogens can persist for months on surfaces, contributing to transmission risk in the absence of regular and thorough cleaning and disinfection [ ] . these pathogens importantly include gram-positive (enterococcus, including vre; s. aureus, including mrsa; and streptococcus pyogenes) as well as gramnegative (acinetobacter spp., e. coli, klebsiella spp., p. aeruginosa, serratia marcescens, or shigella) organisms. spore-forming bacteria, such as clostridium difficile, can survive for months as can fungi and yeast. viruses from the respiratory tract, such as coronavirus, influenza, coxsackie, and rhinovirus, survive a relatively short period of days, whereas viruses from the gastrointestinal tract, such as norovirus or rotavirus, may persist for up to months [ ] . surfaces in rooms of patients infected or colonized with pathogens may (and frequently do) become contaminated. mrsa, vre, acinetobacter spp., norovirus, and c. difficile have been detected on environmental surfaces in rooms of infected or colonized patients, can colonize healthcare workers' hands, and can then be transmitted to others [ ] . contact with the environment is as likely as contact with the affected patient to result in contamination of hcw hands [ ] . the presence of environmental contamination is a risk factor for hcw hand/glove contamination [ ] . admission to a room previously occupied by a patient colonized or infected with mrsa, vre, acinetobacter spp., or c. difficile has been shown to be a risk factor for subsequent development of colonization or infection by these pathogens [ , , ] . multiple studies have demonstrated that a lack of thorough cleaning [ , ] contributes to persistence of environmental contamination. assessing adequacy of cleaning can be performed using various methods including observation for visible soiling, culture-based colony counts, fluorescent dye, and atp detection. for example, fluorescent dye can be applied as a dot to surfaces where it dries clear. if a surface was inadequately wiped, the area fluoresces when exposed to black light. atp bioluminescence systems measure atp, a marker for presence of residual organic material (e.g., human secretions or excretions and food). atp, however, does not indicate presence of viable pathogens, and its absence does not rule out the presence of contamination, and as such, use of fluorescent dye correlates more closely with colony counts than does atp bioluminescence [ ] . focused efforts to eradicate pathogens can improve cleaning efficacy, which may involve specialized teams [ ] or through use of improved monitoring of cleaning practices with markers such as atp and fluorescent dye [ , ] . feedback to environmental services (evs) staff following use of enhanced methods has also been demonstrated to improve the frequency of achieving adequate cleaning [ , ] . in a study of acute care hospitals, only % ( / , ) of environmental surfaces were cleaned at baseline. after educational and procedural interventions combined with provision of objective performance feedback to evs staff, % ( / ) of surfaces were cleaned (p < . ) [ ] . in addition to ensuring each surface is cleaned, it is important to select the correct cleaning product as microorganisms vary in their resistance to disinfectants. for example, disinfection of a room potentially contaminated by c. difficile requires use of hypochlorite-based solutions [ ] rather than phenols or quaternary ammonium compounds generally used for general hospital-based cleaning. in spite of enhanced cleaning methods aimed at improving cleaning thoroughness and monitoring of cleaning practices, many surfaces remain inadequately cleaned. for this reason, no-touch room disinfection units that decontaminate environmental surfaces and objects utilizing either ultraviolet (uv) light or hydrogen peroxide (hp) vapor have been developed [ , ] . these technologies are considered an adjunct to standard cleaning and disinfection since surfaces must be physically clean and the room must be emptied of people prior to use. uv irradiation with certain wavelengths breaks the molecular bond in dna, thereby destroying the organism. this has been shown to be effective against mrsa, vre, and acinetobacter baumannii, in experimentally contaminated rooms [ ] . systems utilizing hp vapor have been found to be effective in eradicating pathogens such as mrsa, mycobacterium tuberculosis, serratia, and c. difficile spores from rooms and equipment [ ] . both of these methods have been found to be effective at reducing hais [ ] . their advantages include ability to substantially reduce c. difficile spores [ ] as well as achieve substantial reductions in vegetative bacteria. failure to adequately clean and sterilize equipment may lead to transmission via contaminated equipment [ ] . the level of disinfection or sterilization considered acceptable depends on the intended use of the object and is categorized as critical (items that come into contact with sterile tissue), semicritical (items contacting mucous membranes, such as endoscopes), and noncritical (items contacting skin, such as stethoscopes). these each require sterilization, high-level disinfection, or low-level disinfection, respectively. cleaning should precede sterilization or disinfection. among the many sources of infection within hospital environments, water remains of significant concern secondary to opportunity for exposure. water is ubiquitous in its use throughout the hospital, not only for routine sanitation but also for air conditioning, mechanical ventilation, bathing, as well as the cleaning and processing of equipment. certain organisms have special predilection for moist environments and include gram-negative bacilli, nontuberculous mycobacteria, fungi, and some viruses. in a recent review of waterborne healthcare-associated infections, of reports described hospitalized children [ ] . the organisms primarily responsible included legionella (hot water 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legionella infection in term neonates caused by a cold mist ultrasonic humidifier key: cord- -emsvxxs authors: tortorici, m. alejandra; veesler, david title: structural insights into coronavirus entry date: - - journal: adv virus res doi: . /bs.aivir. . . sha: doc_id: cord_uid: emsvxxs coronaviruses (covs) have caused outbreaks of deadly pneumonia in humans since the beginning of the st century. the severe acute respiratory syndrome coronavirus (sars-cov) emerged in and was responsible for an epidemic that spread to five continents with a fatality rate of % before being contained in (with additional cases reported in ). the middle-east respiratory syndrome coronavirus (mers-cov) emerged in the arabian peninsula in and has caused recurrent outbreaks in humans with a fatality rate of %. sars-cov and mers-cov are zoonotic viruses that crossed the species barrier using bats/palm civets and dromedary camels, respectively. no specific treatments or vaccines have been approved against any of the six human coronaviruses, highlighting the need to investigate the principles governing viral entry and cross-species transmission as well as to prepare for zoonotic outbreaks which are likely to occur due to the large reservoir of covs found in mammals and birds. here, we review our understanding of the infection mechanism used by coronaviruses derived from recent structural and biochemical studies. three dimensional -o-ac-sia -n-acetyl- -o-acetyl-sialosides ace angiotensin-converting enzyme apn amino-peptidase n cov coronavirus cryoem cryo-electron microscopy dpp dipeptidyl peptidase fab antigen-binding fragment of an antibody mers-cov middle-east respiratory syndrome coronavirus mhv mouse hepatitis virus pdcov porcine δ-cov pedv porcine epidemic diarrhea virus sars-cov severe acute respiratory syndrome coronavirus tgev transmissible gastroenteritis virus coronaviruses (covs) are enveloped viruses with a positive sense rna genome, that belong to the subfamily coronavirinae within the family coronaviridae, which is part of the nidovirales order. they are classified in four genera (α, β, γ, and δ) and four lineages are recognized whithin the β-cov genus (a, b, c and d). covs cause a variety of respiratory and enteric diseases in mammalian and avian species. until recently, covs were considered to be pathogens with a largely veterinary relevance but with limited impact on human health. however, outbreaks of severe acute respiratory syndrome (sars) in - and of middle-east respiratory syndrome (mers) starting in , with fatality rates of % and %, respectively, led covs to be recognized as zoonotic threats with pandemic potential. four other covs are endemic in the human population and cause up to % of mild respiratory tract infections as well as occasional severe disease in young children, the elderly or immunocompromised individuals (isaacs et al., ; su et al., ) . these viruses are hcov-nl and hcov- e (α-covs) as well as hcov-oc and hcov-hku (β-covs). numerous sars-cov and mers-cov-like viruses currently circulate in bats and dromedaries making outbreaks of highly pathogenic human covs a global health threat (ge et al., ; haagmans et al., ; hu et al., ; menachery et al., menachery et al., , sabir et al., ) . the cov virion contains at least four structural proteins: spike (s), envelope (e), membrane (m) and nucleocapsid (n) . in contrast to other β-cov lineages, lineage a covs also encode a hemagglutinin-esterase which serves as receptor-destroying enzyme to facilitate release of viral progeny from infected cells and escape from attachment to non-permissive host cells or decoys (bakkers et al., (bakkers et al., , . s is a class viral fusion protein that promotes host attachment and fusion of the viral and cellular membranes during entry (bosch et al., ) . as a consequence, s determines host range and cell tropism. s is also the main target of neutralizing antibodies elicited during infection and the focus of vaccine design. s is trimeric and each protomer is synthesized as a single polypeptide chain of - residues, depending on the cov species. for many covs, s is processed by host proteases to generate two functional subunits, designated s and s , which remain non-covalently bound in the prefusion conformation (bosch et al., ) . the s subunit comprises the apex of the s trimer, including the receptor-binding domains, and stabilizes the prefusion state of the s fusion machinery, which is anchored in the viral membrane. for all covs, s is further cleaved by host proteases at the so-called s ' site located immediately upstream of the fusion peptide. this cleavage has been proposed to activate the protein for membrane fusion via large-scale, irreversible conformational changes (heald-sargent and gallagher, ; millet and whittaker, ) . although several class viral fusion proteins have been extensively studied, cov s proteins have proven reluctant to structural characterization until recently. structural studies were largely limited to x-ray crystallographic analysis of isolated receptor-binding domains in complex with viral receptor ectodomains or neutralizing antibodies (li et al., a; lu et al., ; peng et al., ; prabakaran et al., ; reguera et al., ; wang et al., ; wong et al., ; wu et al., ; yu et al., ) and of the s postfusion core (duquerroy et al., ; gao et al., ; supekar et al., ; xu et al., a, b; zheng et al., ) with the exception of two low-resolution electron microscopy reports (beniac et al., (beniac et al., , . in the past few years, however, technical advances in single-particle cryo-electron microscopy (cryoem) (bai et al., ; brilot et al., ; campbell et al., campbell et al., , li et al., ; punjani et al., ; scheres, ) together with the implementation of strategies for the stabilization of cov s proteins in prefusion conformation (pallesen et al., ; walls et al., a) led to a surge of structural data for multiple s ectodomain trimers. we review here our current understanding of the mechanism used by covs to infect host cells based on recent structural and biochemical studies of s glycoprotein ectodomains in prefusion and postfusion states as well as complexes with known receptors or neutralizing antibodies. cryoem studies of the s glycoproteins of mouse hepatitis virus (mhv) and hku led to the first structures at high-enough resolution to obtain an atomic model of the prefusion state (kirchdoerfer et al., ; walls et al., a) . these structures revealed that prefusion s ectodomains are Å -long trimers with a triangular cross-section ( fig. a and b) . the s subunit adopts a "v" shaped architecture for β and γ covs (gui et al., ; kirchdoerfer et al., ; shang et al., a; tortorici et al., ; walls et al., a; yuan et al., ) (fig. c) , or a squareshaped organization for αand δ-covs (shang et al., b; walls et al., b; xiong et al., ) . the s subunit folds as β-rich domains designated a, b, c, d. several α-covs harbor a likely duplication of their domain a at the n-terminus of the s glycoprotein walls et al., b) . this additional domain, designated domain , was visualized in the nl s structure and hypothesized to interact with heparan sulfate present at the host cell surface during viral entry (milewska et al., ; walls et al., b) . domain a and domain adopt a galectin-like β-sandwich fold conserved across all cov genera (kirchdoerfer et al., ; peng et al., peng et al., , walls et al., a) (fig. d) . domain b, which shows the highest sequence variability within cov s subunits, has a markedly different architecture between α-, β-, γand δ-covs. b domains of β-covs contain a β-sheet core subdomain decorated with a highly variable external subdomain mediating receptor engagement kirchdoerfer et al., ; li et al., b; lu et al., ; tortorici et al., ; walls et al., a; wang et al., ) (fig. e) . b domains of α-, γand δ-cov form a β-sandwich decorated with loops mediating receptor attachment (reguera et al., ; shang et al., a, b; walls et al., b; wong et al., ; xiong et al., ) . in the context of the s trimer, β/γ cov b domains interact with the a and b domains of another protomer, whereas they pack against the a domain of the same protomer in α/δ-covs (shang et al., a; walls et al., a, b; xiong et al., ) . the s subunit, which is more conserved than s , comprises the fusion machinery and connects to the viral membrane. it is assembled from a large number of α-helices, an antiparallel core β-sheet, a β-rich connector domain and a stem helix leading to the heptad-repeat (hr ) and the transmembrane region (fig. f ) (gui et al., ; kirchdoerfer et al., kirchdoerfer et al., , pallesen et al., ; shang et al., a, b; tortorici et al., ; walls et al., a walls et al., , b, b xiong et al., ; yuan et al., ) . key s features facilitating virus-cell fusion include the fusion peptide, two heptad repeat regions (named hr and hr ) and the transmembrane domain. in the prefusion s conformation, a central helix stretches along the threefold axis, perpendicular to the viral membrane, and is located downstream the hr motif, which folds as four consecutive α-helices (kirchdoerfer et al., ; walls et al., a) . moreover, an upstream helix runs parallel to and is zipped against the central helix via hydrophobic contacts (fig. f ). the cov s subunit shares similarity with the pneumovirus/paramyxovirus f proteins-including a comparable d organization of the core β-sheet, the upstream helix and the central helix-suggesting an evolutionary relatedness between the viral fusion proteins of these different viruses and a conservation of their fusion mechanism (mclellan et al., ; walls et al., a walls et al., , b wong et al., ; xu et al., ; yin et al., ) . a conserved tryptophan-rich segment (y(v/i)kwpw(y/w)vwl) directly preceding the cov s transmembrane region is crucial for proper trimerization. this segment is also required functionally for formation of a fusion pore (schroth-diez et al., ) . furthermore, transmembrane domain interactions within and possibly between s trimers have been proposed to be essential to complete the membrane fusion process (schroth-diez et al., ) . the transmembrane domain is followed by an intraviral/cytoplasmic tail of variable length ( - residues) depending on the coronavirus species, which contains a palmitoylated cysteine-rich region (of about - residues with - cysteines) and a variable c-terminal end (thorp et al., ) . the cytoplasmic tail is involved in assembly, intracellular transport, cell-surface expression and cell-cell fusion (bos et al., ; bosch et al., ; chang et al., ; lontok et al., ; petit et al., ; ye et al., ; youn et al., ) . currently, no structural information is available for any cov full-length s, hindering our understanding of the influence of the transmembrane and cytoplasmic domains on the conformation of exposed antigenic sites, as previously studied for hiv- envelope (chen et al., ; dev et al., ) . cov entry into susceptible cells is a complex process that requires the concerted action of receptor-binding and proteolytic processing of the s protein to promote virus-cell fusion (heald-sargent and gallagher, ; millet and whittaker, ) . domain , domain a and/or domain b can act as receptor-binding domains and both attachment and entry receptors have been described, depending on the cov species. lineage a β-covs attach via their s domain a to -n-acetyl- -oacetyl-sialosides ( -o-ac-sia) found on glycoproteins and glycolipids at the host cell surface to promote entry into susceptible cells (vlasak et al., ) . these include human covs oc and hku , bovine cov (bcov) and porcine hemagglutinating encephalomyelitis virus. we recently identified and visualized by cryoem the hcov-oc s sialoside-binding site, which is located in a groove at the surface of domain a ( fig. a ) (hulswit et al., ; tortorici et al., ) . this site is conserved in all other covs known to attach to -o-ac-sia (β-covs, lineage a) and shares architectural similarity with the ligand-binding pockets of cov hemagglutinin-esterases and influenza virus c/d hemagglutinin-esterase-fusion glycoproteins, highlighting common structural principles of recognition (bakkers et al., (bakkers et al., , hulswit et al., ; rosenthal et al., ; tortorici et al., ) . the current consensus in the field is that hcov-oc only utilizes -o-ac-sialosides as host receptors. in line with this statement, ligand-interacting residues were shown to be essential for s-mediated viral entry (hulswit et al., ; tortorici et al., ) and -o-ac-sia depletion from target cells resulted in severe decrease in virus infectivity (krempl et al., ; vlasak et al., ) . free -o-ac-sia, however, did not trigger s conformational changes associated with membrane fusion . this observation contrasts with data for sars-cov s, for which addition of the human angiotensin-converting enzyme (ace ) ectodomain (the proteinaceous receptor) promoted s refolding to the postfusion state (song et al., ; walls et al., ) . these findings suggested that either -o-ac-sia-containing receptors differ from proteinaceous receptors in their mode of action, or that an interaction with a yet unidentified proteinaceous receptor is required before or after virus internalization for hcov-oc entry into target cells. the sialoside-binding site identified in hcov-oc s is not conserved among covs which are also known to interact with sialoglycans to initiate host cell infection but are outside of the lineage a of β-covs, such as mers-cov (β-cov, lineage c) or infectious bronchitis virus (ibv, δ-cov) wickramasinghe et al., ) . some α-covs such as transmissible gastroenteritis virus (tgev) and porcine epidemic diarrhea virus (pedv) use domain to attach to sialoglycans, presumably to increase virus concentration at the cell surface and enhance subsequent attachment to proteinaceous receptors schwegmann-wessels et al., ) . carbohydrate binding via this domain has been proposed to be a determinant of the tgev enteric tropism since loss of domain appears to correlate with a loss of enteric tropism for porcine respiratory coronavirus (prcov), the latter virus being a naturally occurring tgev variant (krempl et al., ) . covs exploit a limited variety of proteinaceous receptors compared with the large number and diversity of viral species. all covs known to engage proteinaceous receptors do so using domain b with the exception of mhv, which binds ceacam a using domain a (dveksler et al., ; peng et al., ; williams et al., ) . remarkably, viruses from different genera, such as hcov-nl (α-cov) and sars-cov (β-cov), can recognize the same region of ace (entry receptor) using structurally distinct b domains (hofmann et al., ; li et al., a li et al., , wu et al., ). many α-covs, including hcov- e, tgev and prcv, as well as porcine δ-cov (pdcov) utilize aminopeptidase n (apn) as entry receptor (delmas et al., ; delmas et al., ; li et al., ; reguera et al., ; wong et al., ; yeager et al., ) whereas mers-cov uses dipeptidyl peptidase (dpp ) raj et al., ; wang et al., ) . crystal structures of sars-cov, hcov-nl , mers-cov, hcov- e b domains in complex with their cognate receptors provided atomic details of the interacting-interface and identified key residues for cross-species transmission and infection (fig. ) (li et al., a; lu et al., ; wang et al., ; wong et al., ; wu et al., ) . this information will be useful to guide the development of therapeutics and vaccines against human covs. recent cryoem studies revealed that mers-cov s and sars-cov s can adopt open and closed conformations in which the receptor binding site of domain b is exposed and occluded, respectively (gui et al., ; kirchdoerfer et al., ; pallesen et al., ; song et al., ; walls et al., ; yuan et al., ) . in contrast, the mhv, hcov-nl , hcov-hku , pdcov, ibv and hcov-oc s glycoproteins appear to only adopt a closed conformation (kirchdoerfer et al., ; shang et al., a, b; tortorici et al., ; walls et al., a, b; xiong et al., ) and unknown trigger(s), besides proteolytic activation, might be necessary for these viruses to expose their receptor-binding motifs for recognition to occur. these findings suggest that covs have evolved a fine-tuned mechanism to balance masking of the receptor-binding motifs, putatively to avoid neutralization by the host humoral immune response, and their necessary exposure to enable receptor recognition and infection of host cells (walls et al., b wong et al., ) . upon host recognition, covs are internalized via receptor-mediated clathrin-dependent, caveolin-dependent or other uptake pathways (burkard et al., ; eifart et al., ; inoue et al., ; nomura et al., ) . for instance, both clathrin-dependent and clathrin/caveolae-independent entry pathways have been reported for sars-cov (inoue et al., ; wang et al., ) . feline infectious peritonitis virus was suggested to enter host cells via a clathrin/caveolin-independent internalization route (regan et al., ; van hamme et al., ) whereas a caveolin-dependent endocytic uptake has been suggested for hcov- e and hcov-oc (nomura et al., ; owczarek et al., ) . several reports have demonstrated the key role of proteolytic processing of cov s for cell-cell fusion activity and/or virus entry into host cells using experiments of inhibition of intracellular proteases (burkard et al., ; frana et al., ; simmons et al., ; yamada and liu, ) and/or substitutions of residues at the s /s or s ' cleavage sites (belouzard et al., ; millet and whittaker, ; wicht et al., ; yang et al., ) . prior to and/or after uptake of the virion by a host cell, the s protein is proteolytically processed by host proteases at one or two cleavage sites and both receptor-binding and proteolytic processing act in synergy to induce large-scale s conformational changes promoting cov entry. one of the cleavage sites is located at the boundary between the s and s subunits (s /s cleavage site), whereas the other is located immediately upstream of the fusion peptide (s ' cleavage site), reviewed in (millet and whittaker, ) . cleavage at the s /s site can occur upon viral egress, such as for mhv (frana et al., ) , or upon encounter with a target cell, such as for sars-cov (belouzard et al., ; bosch et al., ; shulla et al., ) , to yield two non-covalently associated subunits. this first cleavage event, along with binding to the host receptor, promotes further cleavage at the s ' site for sars-cov s (belouzard et al., ) and mers-cov s (millet and whittaker, ; park et al., ) . proteolysis at the conserved s ' site is essential for fusion activation of all characterized cov s proteins, and it can occur at the host membrane or in internal cellular compartments of the target cell (belouzard et al., ; burkard et al., ; millet and whittaker, ; park et al., ) . cleavage at the mers-cov s /s site by furin during viral egress enables subsequent exposure of the s ' site upon binding to the host receptor and a second cleavage step by serine proteases anchored in the membrane of the target cells, eventually leading to fusion at the cytoplasmic membrane (early entry) . conversely, mers-cov budding with uncleaved s glycoproteins traffic to the endosomes of target cells where cathepsin l or other proteases promote membrane fusion (late entry) . the former mechanism has been proposed to be the route of mers-cov entry into cell types relevant to lung infection, and therefore a significant determinant of mers-cov virulence . moreover, tetraspanin cd has been implicated in clustering dpp and transmembrane serine proteases to promote early entry of mers-cov (earnest et al., (earnest et al., , . pedv, which replicates in the epithelial cells of the small intestine, undergoes s proteolytic activation by trypsin, which is highly abundant in the intestinal lumen . the critical importance of cleavage at the s /s site was also exemplified in studies with the mers-cov-related bat coronavirus hku . although hku s recognizes human dpp , in vitro infectivity assays revealed that entry into human cells required addition of exogenous trypsin, suggesting proteolytic activation of this bat virus did not occur in human cells (wang et al., ) . in line with these findings, various dpp mammalian orthologues, with variable binding affinities for the mers-cov s receptor-binding domain, were shown to support virus or pseudovirus entry into target cells in the presence of an activating protease (barlan et al., ) . these results collectively illustrate how specific s proteolytic cleavage participates in determining the intracellular site of fusion and also viral tropism and pathogenesis of covs. therefore, the zoonotic potential of covs is not only determined by receptor engagement, but also by proteolytic processing of the s protein required for fusion activation. we showed that in vitro trypsin cleavage of mhv, sars-cov and mers-cov s, under limited proteolysis conditions, recapitulated fusion activation by inducing the pre-to postfusion transition (walls et al., b) . the cryoem structure of the mhv s subunit ectodomain trimer revealed that membrane fusion involves large-scale s conformational changes that are reminiscent of the ones described for other class fusion proteins, including the pneumovirus/paramyxovirus f glycoproteins (fig. ) (mclellan et al., ; swanson et al., swanson et al., , walls et al., b; yin et al., ) . these experiments also demonstrated that (i) the s subunits stabilize the s fusion machinery in the spring-loaded, metastable prefusion state before initiation of infection; and (ii) postfusion s is the ground state of the fusion reaction. similarly to the organization of influenza virus hemagglutinins (xiong et al., ) , domain b interacts with the hr -central helix hairpin in prefusion closed s structures likely to stabilize s in the spring-loaded prefusion state. this interaction appears to coordinate receptor engagement with fusion. upon receptor binding and proteolytic cleavage at the s /s and s sites, the s crown is likely shed (as observed for mers-cov s by yuan et al., ) to facilitate a conformational change of s , which involves projection of the fusion peptide to a distance of Å and its insertion into the target membrane (fig. ) (walls et al., a (walls et al., , b . the free energy released upon s refolding from the prefusion to the postfusion state is believed to bring the viral and host membranes in close proximity and promote membrane merger (harrison, ) . recent structural work comparing recombinant s proteins from sars-cov and mers-cov in isolation and in complex with their cognate receptors or neutralizing antibodies suggested an activation mechanism for coronavirus fusion (gui et al., ; kirchdoerfer et al., ; song et al., ; walls et al., ; yuan et al., ) . specifically, sars-cov and mers-cov s structures in complex with neutralizing antibodies isolated from survivors showed both antibodies competitively blocked receptor interaction, in agreement with previous surface plasmon resonance data (corti et al., ; rockx et al., ; traggiai et al., ; walls et al., ) . the anti-sars-cov s antibody, however, functionally mimicked the receptor by promoting s fusogenic conformational rearrangements through a molecular ratcheting mechanism (fig. ) . these observations suggested that upon receptor recognition, bound b domains are locked in the open state, thereby releasing the constraints imposed on the hr -central helix hairpin, allowing refolding of the s fusion machinery and membrane fusion to occur (pallesen et al., ; song et al., ; walls et al., ; yuan et al., ) (fig. ) . proteolytic activation is likely required to ensure that s glycoproteins will work in synergy, with proper spatial and temporal coordination, to drive fusion of the viral and host membranes. a deep knowledge of the organization and chemical composition of carbohydrates obstructing the surface of cov s glycoproteins is key for understanding accessibility to neutralizing antibodies and for guiding the rational development of subunit vaccines and therapeutics. s glycoproteins feature - predicted n-linked oligosaccharides per protomer. a cryoem structure of the hcov-nl s ectodomain allowed to visualize for the first time the extensive n-linked glycans covering the surface of a cov s trimer (walls et al., b) (fig. ) . a subsequent study revealed that numerous glycosylation sites are strictly or topologically conserved between pdcov s and hcov-nl s although the two glycoproteins share only % amino acid sequence identity and the two viruses belong to different genera infecting different hosts (xiong et al., ) . this observation suggested that all covs face similar immune pressure in their respective hosts, and that the areas that are masked by the conserved glycans might be key to the function of s. based on the information gained from the hcov-nl s structure, in which a glycan participates to masking the receptor-binding loops, it was proposed that the s glycan shield is involved in immune evasion, similarly to the well-characterized hiv- envelope trimer (walls et al., b) . comparison of the n-linked oligosaccharides of full-length mers-cov s derived from virions produced in african green monkey veroe cells, or of a purified mers-cov s ectodomain recombinantly produced in hek f cells, revealed an extensive overlap of glycan composition, including the presence of hybrid and complex glycans . processed oligosaccharides were also observed decorating s trimers at the surface of authentic sars-cov virions (krokhin et al., ; ritchie et al., ) . these data indicated that at least a fraction of the mers-cov and sars-cov virions produced in a cell are exposed to the glycan-processing enzymes residing in the golgi apparatus during assembly and budding, in contrast with previous models of cov budding (ng et al., ; stertz et al., ) . a common feature observed in the glycosylation patterns of s glycoproteins is the presence of less densely glycosylated regions surrounding the s /s cleavage site and the conserved fusion peptide, near the s ' cleavage site, probably to allow access to activating host proteases and for membrane fusion to take place (walls et al., b; walls et al., ) (fig. ) . these "glycan holes" could be targeted for epitope-focused immunogen design or new therapeutic development against cov, as supported by the identification of a neutralization epitope within a comparable breach of the hiv- envelope glycan shield (mccoy et al., ) . recent structural and functional characterization of cov s glycoproteins provided insights into the mechanism used by these viruses to infect host cells and suggested possible strategies for rational design of vaccines and therapeutics. introducing stabilizing mutations, which prevent the prefusion to postfusion s transition, led to the elicitation of improved neutralization titers in mice and will be a key tool for the design of subunit vaccines against covs pallesen et al., ) . furthermore, the exposure of the fusion peptide at the surface of prefusion s trimers (walls et al., a) and its conservation among covs indicate it might be an attractive target for broad inhibition of cov entry. major antigenic determinants of mhv and sars-cov s overlap with the fusion peptide region (daniel et al., ; zhang et al., ) and binding of neutralizing antibodies to this site could putatively prevent fusogenic conformational changes, as proposed for influenza virus hemagglutinin or hiv envelope (corti et al., ; kong et al., ; lang et al., ) . finally, masking strain-specific antigenic regions via engineering of additional n-linked glycosylation sites, as implemented for the mers-cov domain b (du et al., ) , bears the promise of focusing the immune response on highly conserved epitopes and eliciting broadly neutralizing antibodies against covs. ribosome structures to near-atomic resolution from thirty thousand cryo-em particles betacoronavirus adaptation to humans involved progressive loss of hemagglutinin-esterase 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structure and stabilization of the hendra virus f glycoprotein in its prefusion form crystal structure of nl respiratory coronavirus receptor-binding domain complexed with its human receptor receptor binding by a ferret-transmissible h avian influenza virus glycan shield and fusion activation of a deltacoronavirus spike glycoprotein fine-tuned for enteric infections crystal structure of the pre-fusion nipah virus fusion glycoprotein reveals a novel hexamer-of-trimers assembly structural basis for coronavirus-mediated membrane fusion. crystal structure of mouse hepatitis virus spike protein fusion core crystallization and preliminary crystallographic analysis of the heptad-repeat complex of sars coronavirus spike protein proteolytic activation of the spike protein at a novel rrrr/s motif is implicated in furin-dependent entry, syncytium formation, and infectivity of coronavirus infectious bronchitis virus in cultured cells two mutations were critical for bat-to-human transmission of 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spike glycoprotein we acknowledge support from the national institute of general medical sciences key: cord- -keh s authors: gong, danyang; dai, xinghong; jih, jonathan; liu, yun-tao; bi, guo-qiang; sun, ren; zhou, z. hong title: dna-packing portal and capsid-associated tegument complexes in the tumor herpesvirus kshv date: - - journal: cell doi: . /j.cell. . . sha: doc_id: cord_uid: keh s assembly of kaposi’s sarcoma-associated herpesvirus (kshv) begins at a bacteriophage-like portal complex that nucleates formation of an icosahedral capsid with capsid-associated tegument complexes (catcs) and facilitates translocation of an ∼ -kb dsdna genome, followed by acquisition of a pleomorphic tegument and envelope. because of deviation from icosahedral symmetry, kshv portal and tegument structures have largely been obscured in previous studies. using symmetry-relaxed cryo-em, we determined the in situ structure of the kshv portal and its interactions with surrounding capsid proteins, catcs, and the terminal end of kshv’s dsdna genome. our atomic models of the portal and capsid/catc, together with visualization of catcs’ variable occupancy and alternate orientation of catc-interacting vertex triplexes, suggest a mechanism whereby the portal orchestrates procapsid formation and asymmetric long-range determination of catc attachment during dna packaging prior to pleomorphic tegumentation/envelopment. structure-based mutageneses confirm that a triplex deep binding groove for catcs is a hotspot that holds promise for antiviral development. resolution of kshv's asymmetric icosahedral structure is achieved via symmetry-relaxed cryo-em using sequential localized classification. interactions between the dnatranslocating portal protein, associated capsid and tegument proteins, and the viral genome reveal surprising variability in tegument protein occupancy and orientation plasticity. first discovered in associated with tumor lesions in aids patients in los angeles (chang et al., ) , kaposi's sarcoma-associated herpesvirus (kshv) has since been shown to cause endemic cancers in sub-saharan africa, the greater mediterranean, and the xinjiang region of china (ganem, ; giffin and damania, ) . kshv is a member of the herpesvirus subfamily gammaherpesvirinae, which also includes epstein-barr virus (ebv), the first identified human oncovirus. like all herpesviruses, assembly of an infectious kshv virion starts at a bacte-riophage-like portal complex that putatively nucleates the formation of a t = icosahedral capsid, which, at maturation, is composed of major capsid protein (mcp), small capsid protein (scp), ab heterotrimers of the tri monomer and tri dimer and is decorated by capsid-associated tegument complexes (catcs) (cardone et al., ) . upon establishment of an initial procapsid, the portal facilitates the translocation of kshv's $ -kb genome. this key process involves recruitment of an atp-driven terminase (yang et al., ; heming et al., ) to the unique portal vertex to recognize, package, and cleave concatemeric viral double-stranded (ds)dna, which, in conjunction with catc's critical supporting roles (heming et al., ) , give rise to viable genome-containing nucleocapsids (adelman et al., ; beard et al., ) . unlike the comparatively high occupancies of capsid-associated tegument proteins in alphaherpesviruses wang et al., ) and betaherpesviruses yu et al., ) , kshv catc binding sites are markedly partially and/or more flexibly occupied, leading to poorly resolved catc structures in prior icosahedral reconstructions of kshv (dai et al., . the catc nonetheless plays a critical role in the release of a pleomorphic virion because the recruitment of outer tegument proteins and a glycoprotein-sporting envelope leading to virion egress depend on interactions of various viral proteins with constituents of the catc (owen et al., ; sathish et al., ) . in the absence of an in situ kshv portal and catc structures, how a single portal protein (porf ) orchestrates the rise of a robust capsid and how order is maintained in the subsequently complex and variable processes of tegumentation and envelopment remains unknown. an invaluable body of pioneering work has been accomplished so far on the prototypical herpesvirus herpes simplex virus type (hsv- ) portal, including microscopy studies confirming its localization at a capsid vertex (cardone et al., ) , its dodecameric stoichiometry (rochat et al., ) , and, more recently, the impressive identification and reconstruction of the -fold vertex region surrounding the portal (mcelwee et al., ) . here we present the first atomic structures of a gammaherpesvirus portal vertex in kshv, which allowed us to use structure-guided mutageneses to identify a viable drug target. simultaneously, our method of symmetry relaxation and sequential localized classification enabled us to dissect a heretofore puzzling schema of variable catc occupancy at capsid vertices, providing insights into portal-seeded capsid assembly and how structural plasticity arises from a well-defined and highly ordered capsid. to determine high-resolution structures of kshv's unique portal vertex, we imaged frozen-hydrated kshv virions, obtaining , virion particle images. prior kshv capsid reconstructions, although informative, have been calculated with icosahedral symmetry applied, obscuring non-icosahedrally related structures trus et al., ; wu et al., ) . here we developed a workflow (figures s and s ) to determine structures of the non-icosahedrally arranged components. this sub-particle data processing procedure, employing sequential localized classification with symmetry relaxation, allows stepwise reconstruction of selected regions of a viral particle. briefly, two rounds of sub-particle classification were performed to relax icosahedral and local symmetries. in the first round, ''subparticles'' of capsid vertices were extracted from each virion image using coordinates calculated from an initial icosahedral reconstruction of the virion particle. we then performed d classification with -fold symmetry applied to sort out the unique portal vertex sub-particle from the penton vertex sub-particles for each virion image. subsequent refinement yielded a . -Å resolution reconstruction of the portal vertex with -fold (c ) symmetry (first visualized in hsv- ; mcelwee et al., ) , revealing high-resolution structures of the capsid components surrounding the portal (table ; figures s , s a, and s d). a second round of sub-particle classification further relaxed c symmetry at the portal vertex, and subsequent refinement with c symmetry resulted in a . -Å reconstruction, revealing high-resolution features of the dodecameric portal (table ; fig- ures s , s a, and s e). using a specific orientation determined in our c classification, we then calculated an asymmetric (c ) reconstruction of the entire dsdna-containing capsid at . Å (table ; figures a, s , s b, and s g; video s ) and a c reconstruction of the portal vertex at . Å (table ; figures b, s , s a, and s f). our c structures reveal concentrically packed layers of dna with an inter-duplex distance of $ Å , quasi- -fold-organized capsid and tegument densities, a quasi- -fold-symmetric portal dodecamer, and asymmetric terminal dna within a dna translocation channel capped by a distinctive density visible at lower thresholds (figures a and b) . this stepped implementation of symmetry expansion and sub-particle classification thus proved effective in teasing apart the multiple symmetry mismatches present in herpesvirus capsids. structure of the dna-translocating portal using our c portal reconstruction, we atomically modeled kshv's -amino acid (aa) portal protein porf (table ; figure c; video s ) . despite a lack of sequence homology, our porf model showed striking similarities with structures of phage portal proteins in domain organization and topology (lebedev et al., ; lokareddy et al., ; sun et al., ) . we thus named the five domains of our porf model in a fashion analogous to phage portal proteins: wing (aa - and - ), wall (aa - and - ), stem (aa - and - ), clip (aa - [aa - unmodeled] ), and b-hairpin (aa - ) ( figure d ). the n-terminal eight residues (aa - ) and c-terminal residues (aa - ) were disordered and also not modeled. also similar to phages, copies of porf arrange in a vaguely flying saucer-like ring (lebedev et al., ; lokareddy et al., ; sun et al., ) . in agreement with previous tomographic (cardone et al., ; chang et al., ) and intermediate-resolution (mcelwee et al., ; rochat et al., ) visualizations of herpesvirus portals, we observe porf portal docking in the capsid through interactions between the portal's wing domains and the surrounding mcp floor and, additionally, between the base of the portal clip and the tri subunit of periportal triplexes ( figure b ). clip, stem, b-hairpin, and wall domains largely form the interior of the portal channel (i.e., dna translocation channel) through which dna is threaded into the capsid during genome packaging ( figures d and e ). of the channel-lining domains, b-hairpin and clip domains form the most constricted regions of the channel at $ Å and $ Å in diameter, respectively ( figures d and e) . notably, both regions contain characteristic b sheets and interact with dna in our mature virion state. arranged radially, b-hairpins comprise an apertured disk perpendicular to the portal channel axis ( figure e ). interestingly, residues that line the channel are markedly positively charged above this aperture, likely facilitating interactions with negatively charged dna ( figure f ). twelve sets of three-stranded b sheets roughly parallel to the channel axis form the base of the clip region ( figure g ). each b sheet is the result of a + augmentation motif, where two parallel clip b strands from each porf subunit are augmented in anti-parallel fashion by a single clip b strand from its clockwise neighbor (when viewing toward the capsid interior) ( figure g , insets); this results in a ''daisy-chained'' ring structure, conceivably ideal for propagating and coordinating conformation changes among subunits in the dodecameric complex during dsdna translocation. in contrast to phage portals, a turret-like density arises from our kshv clip, extending distally toward the portal-capping density ( figures b and g ). although we were unable to model this clip turret, we identified what appeared to be helical structures comprising the turret walls ( figure g ), consistent with porf secondary structure and disorder predictions, which show strong helix propensity in this region (aa - ) (figure s a) . during dna packaging, the distal end of this turret is the putative docking site of terminase (yang et al., ; heming et al., ) . the lower resolution of the clip turret is thus likely a result of inherent plasticity in the structure to accommodate interactions with various partners during different stages of viral assembly (e.g., terminase during active dna packaging and the portal cap after packaging). a portal-effected global distribution of catc our c portal vertex reconstruction revealed tegument densities sitting atop the periportal triplexes ta and tc (figures b and s f) . the morphology of these densities-a helix bundle supported by a triplex-bridging base-resemble that of catcs (c) porf model, shown as rainbow-colored ribbons (blue, n terminus / red, c terminus). inset depictions are ribbon-and-stick in c mesh density. (legend continued on next page) identified around penton vertices in previous icosahedral reconstructions of kshv (dai et al., ) and of neurotropic hsv- and hsv- . however, peripenton catc densities in the kshv icosahedral reconstruction were distinctively weaker than surrounding capsid proteins and only discernible when low-pass-filtered to $ -Å , suggesting low catc occupancy and/or flexibility (dai et al., ) ; in contrast, the hsv- icosahedral reconstruction indicated full catc occupancy . interestingly, our c reconstruction of the kshv portal vertex reveals the presence of five catc densities with a strength comparable with that of underlying capsid elements, indicating full occupancy of the five catc registers surrounding the portal vertex. this preferred association of catc with the portal vertex over the remaining (penton) vertices suggests an important role of catc at the portal vertex, to be discussed later. our c reconstruction of the genome-containing capsid also indicates a curious binding pattern of catcs to penton vertices in a manner dictated by the portal vertex. when displayed at a density threshold appropriate for capsid proteins, our c capsid reconstruction shows two adjacent (i.e., ortho) catcs bound to each portal-proximal penton vertex, one catc to each portaldistal penton vertex, and no visible catcs at the portal-opposite penton vertex (figure a ). incrementally decreasing the density display threshold reveals additional catcs of progressively weaker density (indicating progressively lower occupancies at those registers) manifesting at penton vertices in a ''portal-outward'' manner ( figures b and s ). importantly, within each penton vertex, catcs appear to selectively bind registers on the portal side of a hypothetical ''equatorial'' bisecting the penton vertex, with a clear preference for the most portal-side register ( figure s ). adherence to this ''portal-side equatorial rule'' is exemplified by the observation that portal-proximal penton vertices (with two registers portal-side of the equatorial) max out at two catc copies as the density display threshold is decreased, whereas portal-distal penton vertices (with three portal-side registers) max out at three catc copies. portalopposite vertices can bind the catc at any register (i.e., no preferred register), up to five catcs in total, because every register is portal-side ( figures b and s ) . given that catc occupancy varies among vertices in a nonrandom fashion, the existence of catcs of varying densities indicates that catc occupancy varies even among capsids and is thus not fully determined in our c reconstruction (i.e., the averaging of capsids with differing occupancies/binding patterns inherently obscures information on specific occupancy). thus, to accurately assess the specific occupancy of penton vertex catcs and to understand the structural basis of a catc's discriminatory association with portal and penton vertices, we relaxed -fold symmetry for penton vertex sub-particles and performed d focused classification of their catc-binding registers ( figure s ). using a mask encompassing the region surrounding one catc, four resulting classes were obtained. although three classes clearly lacked catc densities, one class contained a catc density of a quality comparable with surrounding capsid protein densities ( figure s ). . % of masked sub-particles (i.e., . % of penton vertex registers) were assigned to this catc-binding class, slightly higher than the $ % occupancy estimated in our previous kshv icosahedral study (dai et al., ) . we further distinguished between possible catc binding occupancies and permutations at the five registers of a single penton vertex using geometry-based sub-particle classification. in all, eight possible permutations of zero to five catcs can bind a penton vertex, all of which were observed and reconstructed in our analyses ( figure a ). in agreement with trends observed in our c capsid reconstruction, penton vertices with two adjacent catcs bound were most abundant (ortho-catc-binding, at . % of penton vertex sub-particles), followed by penton vertices with a single catc bound (one-catc-binding, at . % of sub-particles). five-catc-binding penton vertices were rarest, as expected, at . % of sub-particles because, barring deviations to the c capsid-observed consensus binding pattern, these should be limited to the portal-opposite vertices of highly bound capsids. finally, we tallied the total number of penton catcs in each capsid from our classified penton vertex sub-particles. the resulting histogram follows a sharp, slightly left-skewed gaussian distribution peaking at approximately penton catcs per capsid ( figure b ). intriguingly, additional catc binding falls off sharply after capsids have bound penton catcs, which happens to be the theoretical maximum of a capsid with ''full'' penton vertex catc binding in compliance with the portal-side equatorial rule. in all, these results-the full occupancy of portal catc registers, the portal vertex-referenced directional binding of catc within penton vertices, and the portal-dictated maximum of allowed binding registersstrongly suggest that the nucleating portal effects long-range (allosteric) structural influence on the penton vertices of each capsid. further d refinement of classes obtained from our masked classification of penton vertex sub-particles yielded catc-binding and catc-absent reconstructions of penton vertex registers at . Å and . Å , respectively (table ; figures s , s c, s h, and s i). from the catc binding reconstruction, we identified (d) two opposing porf portal subunits, colored by domains assigned according to dsdna phage portal structures (lebedev et al., ; lokareddy et al., ; sun et al., ) , shown superimposed with gaussian-filtered c portal vertex density. (e) clip (top) and b-hairpin (bottom) slices define two narrow constrictions within the dna translocation channel. (f) electrostatic surface potential rendering of the portal's dna translocation channel. (g) c density shaded by the corresponding porf domain (gaussian-filtered c density shown for lower-resolution clip turret). mint green cylinders represent helix-like structures observed to extend from the clip in c reconstructions as in (b) but disordered in the c density. three consecutive porf subunits illustrate the daisy-chained + b sheet augmentation facilitated by the modeled clip's three b strands (insets). unmodeled turret densities extend from the middle ring of b strands (dotted black circles). see also figure s and videos s and s . and modeled the constituents of catc, which include two copies of porf , two copies of porf , and one copy of porf (table ; figures a, b, and s a) ; this is in one-toone correspondence with pul , pul , and pul , respectively, of hsv- catc . comparing reconstructions of the portal vertex and a catcbinding penton vertex reveals nearly identical structures of their associated catcs ( figures c and d ). at both vertices, porf bridges the triplexes ta and tc and supports a fourmembered helix bundle composed of the n-terminal segments of two porf subunits (aa - ) and the very c-terminal segments of two porf subunits (aa , - , ) ( figures b- d ). obvious differences aside-e.g., a portal in the portal vertex, an scp-decorated penton in the penton vertex, or catc occupancies-the two vertices also differ in the presence of a disk-like portal cap at the distal ends of the portal vertex's catc helix bundles ( figure c , magenta) versus a globular density cocked to the right of the penton vertex's catc helix bundle ( figure d , magenta). because this globular density's size and proximity to catc ( figure e ) are reminiscent of the flexibly tethered head domain of pul in hsv- (a homolog of kshv porf ), we interpret this density as the head domain of one of the porf copies in the catc. indeed, the crystal structure of an hsv- pul head domain (pdb: f u) (bowman et al., ) satisfactorily fits into the globular density ( figure f ), lending support to its assignment as porf . we then proceeded to construct a homology model of the porf head domain (aa - ) using pdb: f u as a template (figures e and f) . from the homology model, we estimate that aa - map to a finger-like density that inserts between an adjacent penton mcp and scp, serving as the sole interacting residues between catc and the penton protrusion ( figure e ). of note, because the globular density accommodates only one copy of porf /pul head domain (whereas the corresponding region in hsv- catc accommodates two), we previously asserted-based on a -Å icosahedral reconstructionthat the kshv catc had a different stoichiometry than the hsv- catc (dai et al., ) . but, as noted in a subsequent study demonstrating differences in globular head domain arrangement between kshv and neurotropic alphaherpesviruses (dai et al., ; liu et al., ) , and as our present sub-particle reconstructions reveal, the kshv catc does, in fact, bear the same stoichiometry/architecture as the hsv- catc. these findings indicate that the second head domain of porf is present but perhaps flexibly tethered elsewhere. indeed, a second globular density gradually appears to the left of the penton vertex's catc helix bundle at lower resolutions/ density thresholds (figures d, green circle, s b, and s c). in the kshv portal vertex, secondary structures in the portal cap beyond the catc helix bundles are not resolved as in the penton vertex's globular density. nonetheless, several lines of evidence support interpretation of the portal cap as porf . first, catc helix bundles clearly connect with the portal cap at lower resolutions/density thresholds ( figure c , black circles). (legend continued on next page) second, the portal cap density is about the size of five porf head domains and further exhibits five weaker globular densities attached at its periphery ( figure c , green circle), analogous to the globular density assigned to the second porf head domain at the penton vertex (figures d, green circle, s b, and s c). third, functional data indicate that hsv- pul (porf 's homolog), but not hsv- pul (porf 's homolog), is critical for viral genome encapsidation, especially near the termination of genome packaging (mcnab et al., ; ogasawara et al., ) . moreover, pul plays an important role in docking the incoming capsid at host cell nuclear pores and gating the release of the viral genome (huffman et al., ; pasdeloup et al., ) . it is therefore plausible that porf could be intimately associated with the portal channel, and the portal cap is certainly poised in a prime position to execute porf 's roles in regulating the retention and release of the viral genome. despite similar architecture, kshv and hsv- catcs bind their underlying triplexes in somewhat different fashions. in both viruses, the porf -equivalent catc subunit (pul in hsv- ) functions as a structural framework facilitating capsid association of the other four subunits that make up the catc helix bundle. side-by-side comparison of porf and pul models reveals similar core structures of separate n-and c-terminal b sheet-rich domains positioned beneath a central a helix-rich arch ( figures g and h , orange). however, several prominent features visible in pul are missing in porf , accounting for porf 's fewer residues: a ''hump,'' characterized by four short helices at the top of the arch that constrain and orient catc's helix bundle in hsv- ; an extended helix; and a short helix bundle at the vertex-proximal end of pul . notably, pul 's short helix bundle makes direct contact with the underlying triplex ta, almost solely mediating catc-ta binding in hsv- . in porf , nearly the entirety of this region is disordered and/or invisible in both portal and penton vertex reconstructions save for a -aa ''anchoring loop'' (al; aa - ) ( figure g , yellow). although porf 's unmodeled residues in this region may potentially account for the critical ta-interacting helix bundle, a structure-based sequence alignment of porf with pul suggests that the ta-binding short helix bundle in pul is missing, even in the porf sequence ( figure s b ). the importance of porf 's al for ta binding becomes apparent in light of an alternate morphology of catc-decorated kshv triplexes not observed at the catc-decorated penton vertices of hsv- . a comparison of our models of kshv triplexes with and without the catc (table ; figures i and j ) reveals that all ta triplexes underlying catcs experience an $ counterclockwise axial rotation relative to canonical undecorated triplex ta (cf. figures i and j ). our models suggest that this rotation may be due to the steric specificity of porf 's ta-binding al. specifically, a counterclockwise rotation of ta exposes a deep groove running between the molecular boundaries of tri and the tri a/b dimer that facilitates docking of porf 's al and, thus, catc. strikingly, despite catcdecorated ta's large degree of apical (i.e., main body) rotation, our structures show that tri 's n-anchor-the characteristic n terminus of tri subunits that penetrates the capsid floor-maintains a conserved orientation in the capsid interior at penton vertices regardless of ta's apical orientation (and, by extension, regardless of catc decoration) ( figures i and j , respective insets). (periportal ta triplexes, which are all catc-decorated, exhibit counterclockwise apical rotation, but their tri n-anchors are disordered and appear to adopt a unique configuration.) in the context of capsid assembly, our observations allow several structure-based inferences. first, the presence of a uniform tri n-anchor orientation (barring periportal triplexes) supports the notion that initial triplex attachment to the procapsid, putatively through the tri n-anchor, precedes the stage of catc binding (reviewed in heming et al., ) . second, whatever the means by which the portal effects triplex orientation and, thus, catc binding (or perhaps vice versa) presumably occurs at a stage of procapsid maturation before triplexes are fully ''stapled'' to the mcp floor by main-body interactions . this is predicated upon the necessity for triplex ta to have sufficient rotational freedom to permit adoption of a catc-bound orientation. third, for reasons one and two, catc binding occupancy is highly unlikely to be predetermined but, rather, determined with the simultaneous maturation of the procapsid, although this is subject to influence by the portal, as demonstrated previously. at the vertex-proximal end of catc, interactions with triplex ta occur exclusively through porf . an n-terminal helix (nh; aa - ) and two strands from porf 's n-terminal b-barrel domain directly contact the apical surfaces of ta's tri a/b dimer (figures a- c; video s ). additionally, the aforementioned al binds in ta's deep hydrophobic cleft between tri and the tri a/b dimer (figures b- d; video s ). al binding involves three porf hydrophobic residues-val , leu , and phe ( figure d )-as well as b sheet-like hydrogen bonding between the al and an adjacent ta tri strand ( figure e ). (legend continued on next page) at the vertex-distal end of catc, porf sits atop, but does not directly bind to, triplex tc. instead, catc-tc interactions rely on the n-terminal tails of catc's two porf subunits, which extend distally from the helix bundle and descend beneath porf to contact tc (figures a and f ). contact with tc occurs mainly via a short helical motif (aa - ) from the n-terminal tail of the ''upper'' (magenta) porf subunit. an adjacent helix belonging to tc tri (aa - ) orthogonal to the porf helical motif fits within the motif's helical groove (figure f ), with tri 's thr and arg and porf 's arg contributing hydrogen bonds to this intermolecular interaction ( figure g ). in contrast to upper porf , ''lower'' (green) porf exhibits no apparent direct contacts with tc in our visible structure. nonetheless, lower porf plays an important role in lashing catc's constituents as a collective unit. chiefly, lower porf 's n-terminal tail contributes two b strands (aa - and - ) to form two sets of b sheet interactions-the first with one b strand from porf (aa - ) and upper porf (aa - ) each ( figure f , starred) and the second with a b strand from porf (aa - ) ( figure h , starred). these two sets of b sheets fasten the vertex-distal end of catc in a quasi b-barrel. the first and residues of upper and lower porf 's n-terminal tails, respectively, are flexible and, thus, unmodeled ( figure h ). however, we speculate that some of these unmodeled residues may also bind tc, augmenting previously described catc-tc interactions at upper porf 's short helical motif. particularly, strong unassigned densities roughly three aa in length occupy the tri -tri a/b surface groove of tc ( figure i ), analogous to porf 's al occupying triplex ta's groove ( figures b- d ). given that the n-terminal tail of upper porf appears to extend away from tc, and given the proximity of lower porf 's n th -most residue to tc's groove, the unassigned densities likely belong to the unmodeled n-terminal residues of lower porf . if interactions here are also similar to al binding at ta (i.e., in part involving b sheet-like hydrogen bonds between adjacent backbones), then porf binding at this site may not necessarily be sequence-specific, thus accounting for the unresolved sidechain densities because of averaging. to validate our structural interpretation of the roles of the porf nh and al in catc-ta binding, we constructed three porf mutants with deletions in either the nh or al or both, named dnh, dal, and dnh-dal. we posited that these ''loss-of-ta-interaction'' porf mutants might serve as dominant negatives if nh and/or al are critical for catc-ta binding. essentially, porf mutants expressed in kshv-replicating cells should compete against wild-type (wt) porf ( wt) for incorporation into catc so that mutant-incorporated catc would be deficient in capsid association, inhibiting kshv virion formation. indeed, expression of dnh and dal reduced virion production to . % and . % of that of the control, respectively, whereas expression of dnh-dal inhibited levels to . % (figures j and k) . importantly, viral dna replication and viral rna transcription were not significantly affected (figures s a and s c) . these results confirm that the porf nh and al are both important for kshv virion production, supporting our interpretation that these aa stretches mediate catc-ta binding. similarly, to test our hypothesis that porf 's n-terminal tail is important for catc capsid association and, hence, function, we generated two porf mutants: dn , which deletes the first n-terminal residues of porf (i.e., all porf residues in contact with triplex tc), and dn , which deletes porf 's first residues (i.e., retains all porf residues visible in our structure but deletes the disordered, unmodeled n-terminal tails). compared with wt porf ( wt), dn expression reduced virion production to . % of that of the control without significantly affecting viral dna replication or rna transcription ( figures l, m , s b, and s d), indicating that porf n-terminal interactions with tc ( figures f- i ) are important for viral efficacy. remarkably, expression of dn also inhibited virion production to . % of that of the control ( figures l, m , s b, and s d), suggesting that porf 's first residues of porf are required for optimal porf -tc binding. these findings are therefore consistent with our speculation that lower porf 's unmodeled n-terminal residues (aa - ) bind tc's hydrophobic groove, enhancing catc-tc binding. finally, we mutated residues in the hydrophobic triplex groove, creating six mutants: tri -i r, tri -l r/i r/ l e, tri -a r, tri -l r, tri -a r/l r, and tri -v r ( figure i ). these mutants affect binding grooves indiscriminately at both ta and tc triplexes (and, therefore, catc binding at both its vertex-proximal and vertex-distal ends). all mutants yielded decreased virion production ( figure n ), (d) the al (ribbon-and-stick superimposed with mesh density) binds a hydrophobic groove between the tri and tri dimer (cyan, hydrophilic / maroon, hydrophobic). (e) the al forms b sheet-like hydrogen bonds (dotted lines) with an adjacent tri strand (some side chains are hidden for clarity). (f and g) consecutive enlarged views showing catc-tc interaction via upper porf , which interacts with tc tri via a helical motif (g). dotted lines represent hydrogen bonds. lower porf facilitates two sets of b sheets (starred in f and h) at the catc's vertex-distal end. (h and i) the flexible n-terminal residues of upper and lower porf could not be modeled, although the disordered density from the two porf copies can be traced (h, dashed lines) and observed (i, gray density). (j-m) structure-guided mutageneses of porf (j and k) and porf (l and m), confirming the importance of their respective triplex-binding segments in infectious virion production. overexpressing porf mutants with deletions of n-terminal helix (dnh) and/or anchoring loop (dal) in kshv lytic-replicating cells reduced virion production normalized to the control (vector) (j). likewise, overexpressing porf with truncated n-terminal or residues reduced virion production (l). expression of mutant porf (k) and porf (m) was verified by western blotting with an anti-flag antibody. expression of cellular gapdh was examined as an internal control. (n-p) mutageneses of triplex protein residues at the catc-binding groove. measures of viral titer (n), genome replication (o), and rna transcription (p) are shown. the dashed line in (n) indicates the detection limit. data are mean ± sem (n = biologically independent samples). see also figure s and video s . whereas neither viral dna replication nor rna transcription were significantly affected ( figures o and p) . impressively, virion production of mutant tri -a r/l r was under the detection limit ( figure n, ud) . the visualization of porf and porf peptide fragments binding in the respective deep grooves of triplex ta and tc (figures a - i) and our demonstration of their importance in viral replication ( figures j- p ) open exciting prospects for future kshv inhibitor design and drug development. currently, most available drugs countering herpesvirus infections are nucleic acid analogs that target viral dna synthesis. unfortunately, these often elicit negative side effects, including frequent induction of drug-resistant mutant viruses and varying kinds of toxicity (gilbert et al., ; reusser, ; walling et al., ) . that the dna-packaging portal vertex is critically involved in the production of virus progeny and that the five portal vertex catc registers exhibit full catc occupancy-which suggests that disrupting a single portal-associated catc might abolish the assembly of infectious virion-presents the possibility of a novel antiviral target without homology with cellular proteins. therefore, a catc-specific inhibitor would be very potent, and the deep groove on triplexes can serve as a structurally informed target for focused antiviral development. the portal is widely believed to serve as the nucleating nexus of herpesvirus procapsid formation newcomb et al., )-although capsid-like particles have been observed in capsid protein-expressing insect cells and cell-free systems in the absence of portal protein (newcomb et al., ; perkins et al., ; tatman et al., )-by facilitating initial interactions with scaffold-tethered mcp subunits and portal-adjacent triplexes (deng et al., ; zhou et al., ) . a metastable spherical procapsid then forms as follows. tethering by the scaffolding protein brings together additional mcp molecules. the scaffolding core ensures proper curvature and size of the capsid in what is known as the ''rope'' mechanism (deng et al., ) , and heterotrimeric triplexes, with the n-terminal anchor of tri (porf ) traversing the capsid shell, ''plug'' every -fold hole between mcp capsomers. our data showing that catc-triplex ta association correlates with a apical rotation of ta ( figure i and j)-but not the -fold-related , eliminating the possibility of this being a stochastic event-indicate that the determination of catc binding (and, likely, binding of catc itself or at least a subunit of catc; thurlow et al., ) occurs not long after procapsid formation, when the triplex's apical orientation has yet to be fixed by procapsid maturation. procapsid maturation is concomitant with dna packaging; head-full is likely sensed through capsid elements and relayed by the portal through the turret-like structure to the externally located terminase for cleavage of the concatemeric genome (yang et al., ) , and the spring-loaded dsdna genome is corked inside the capsid by the portal cap, putatively formed by five portal-adjacent catcs ( figure b) . that capsid structures are rigidly symmetric reflects the capsid's well-defined and conserved role in packaging and protecting the viral genome, in marked contrast to both form and function of the largely pleomorphic tegument layer. accordingly, all three sub-families of herpesviruses have highly conserved capsid proteins and structures but only partially conserved tegument and envelope proteins because these more often reflect specific host cell adaptations. among tegument proteins, catc is unique in possessing a structural role and, thus, being relatively conserved, but even so, a surprising finding here is that, unlike the full occupancy of catc at capsid vertices in neurotropic hsv- and hsv- (dai et al., ; wang et al., ) , catc occupancy in kshv at penton vertices is only partial and varies even among kshv capsids (figures and ) . that kshv catc occupancy appears to be dictated at least in part by the portal not only underscores the portal's aforementioned nucleation role but further spotlights its allosteric effect in defining an emergent first level of variability that, importantly, demarcates a departure from symmetry in kshv metastructure. a second constructive level of variability is facilitated by porf , which accounts for two of the five subunits in catc and is the largest tegument protein, with , residues folded into multiple domains joined by presumably flexible linkers (the vast majority of porf is thus invisible in our structure). some of these domains recruit other tegument proteins and bind the endodomains of envelope glycoproteins (rozen et al., ; sathish et al., ) , introducing pleomorphic variability. last, the majority of tegument proteins, including porf , have been shown to be capable of being packaged into non-infectious virus-like vesicles in the absence of capsids (gong et al., ) , suggesting an alternate pathway of tegument incorporation into virions independent of the capsid/catc, implicating yet another level of assembly-driven variability. in light of the tegument's primary role in manipulating host cells to facilitate virus replication, the multiple layers of structural variability at play in the tegument provide unique opportunities in the context of herpesvirus adaptability. in much the same way that more genetically fluid rna viruses such as influenza viruses (harris et al., ; vahey and fletcher, ) , filoviruses (bharat et al., ) , and coronaviruses (goldsmith et al., ) benefit from heterogeneous compositions to rapidly respond to selective pressures, structural pleomorphism may provide a crucial avenue of diversity in more genetically constrained dsdna viruses (because of lower mutation rates) like kshv, resulting in an increase in evolutionary bandwidth. in essence, facing a relative lack of intraspecies genetic diversity (the tradeoff is more viable progeny), adaptability regarding the ability to manipulate hosts is, instead, implemented at a structural level. our findings regarding kshv catc occupancy offer a pertinent example: given that hsv pul (kshv porf 's homolog) is involved in axonic transport of alphaherpesvirus capsids luxton et al., ) , the absence of full catc occupancy in non-neurotropic kshv perhaps reflects its lack of need for long-range neuronal transport. the emergent transition from rigidly symmetric structures to less-structured compartments described here thus sheds light on a delicate balance of conservation and adaptation that delineates the genetic arms race between herpesvirus and host. detailed methods are provided in the online version of this paper and include the following: we thank the bioinformatics center of the university of science and technology of china, school of life science, for providing supercomputing resources for this project. this research has been supported in part by grants from the national key r&d program of china ( yfa and yfa and nih (gm , de , de , de , and ai to z.h.z and ca , ca , and de to r.s.). we acknowledge the use of resources at the electron imaging center for nanomachines supported by ucla and by instrumentation grants from the nih ( s rr and u gm ) and nsf (dbi- and dmr- ) . further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, z. hong zhou (hong.zhou@ucla.edu). viruses and cell lines kshv virions were isolated from islk-kshv-bac cells, received as a gift from dr. jae u. jung of the university of southern california. dr. jung's group previously established the islk-kshv-bac cell line, which supports robust kshv lytic replication and the production of kshv virions as previously described (brulois et al., ; dai et al., . islk-kshv-bac cells were cultured in dulbecco's modified eagle medium (dmem) supplemented with % fetal bovine serum, % penicillin streptomycin, mg/ml puromycin, mg/ml g , and , mg/ml hygromycin b. to induce kshv lytic replication and thus kshv virion production, cells were treated with mg/ml doxycycline plus mm sodium butyrate (nab) for three days, after which tissue culture supernatant was collected. kshv virions were pelleted by ultracentrifugation at , g for hour, then resuspended in phosphate buffered saline (pbs) and further purified in %- % (w/v) sucrose density gradient sedimented at , g for hour. in addition to cryoem studies, these kshv virions were also used to infect human t cells at a moi of . t cells infected with wild-type kshv virions (termed t-kshv) or transfected with triplex mutant kshv bacs were selected and maintained in dmem supplemented with % fetal bovine serum, % penicillin-streptomycin, and mg/ml hygromycin b . these t cells harboring kshv wild-type or triplex protein mutants were used for all functional analyses as described below in method details. cryoem and icosahedral reconstruction aliquots of . ml purified virion sample were applied onto quantifoil r / cu grids, manually blotted with filter paper and plungefrozen in liquid ethane. super-resolution movies of purified wild-type kshv intact virions were recorded on a gatan k direct electron detection camera in counting mode with a pixel size of . -Å /pixel at the specimen scale. the frames in each movie were subjected to drift correction using motioncorr (li et al., ) and averaged to produce one micrograph. defocus for each micrograph was determined by ctffind (mindell and grigorieff, ) , and a total of , viral particles were picked manually. because the size of boxed particles ( , , pixels) was so large that the cumulative dataset required an unrealistic amount of computer memory for computation, boxed particles were normalized and binned four times prior to implementing standard icosahedral reconstruction procedures in relion (scheres, ) . using a gaussian ball as the initial reference, auto-refinement for icosahedral reconstruction was performed imposing i symmetry, generating an icosahedral map with one of the twelve -fold axes aligned on the z axis. sub-particle extraction from icosahedron vertices and local focus value calculation as illustrated in figure s , we extracted twelve sub-particles corresponding to the twelve capsid vertices for each kshv virion based on the icosahedral orientation described above. to do so, we first expanded the icosahedral symmetry of the particles using relion_particle_symmetry_expand, generating icosahedrally-related orientations for each particle. each orientation has three euler angles denoted as parameters within the relion star files: rot (_rlnanglerot), tilt (_rlnangletilt), and psi (_rlnanglepsi). we then defined the orientation for each of the twelve vertices from the icosahedrally-related orientations as follows. first, we noted that because our icosahedral reconstruction was performed using i symmetry, there are redundant orientations relative to each vertex that differ only in their rot angles (i.e., the in-plane rotation angle about the z axis). for this reason, we classified the orientations into twelve groups each, with five orientations per group that differ only in their rot angles. we then randomly chose one of the five orientations in each group as the orientation of that vertex, thereby defining the orientations for all twelve vertices of each capsid. next, we determined the location of each vertex sub-particle on the viral particle image. the two-dimensional cartesian positions (x, y) of each sub-particle on their respective particle image were calculated using the following formula: x = cosðpsiÞsinðtiltÞd + c À ox y = À sinðpsiÞsinðtiltÞd + c À oy ( ) where d is the distance from the center of the reconstructed capsid to the vertex in pixels and c is the center of the d projection image (in our case, the projection center is at [ , ], so c = pixels). because icosahedral reconstruction was performed with four times-binned particles, ox and oy are four times the offset distance (_rlnoriginx and _rlnoriginy in relion) of each particle image relative to the projection center of the icosahedral reconstruction. finally, sub-particles ( pixels) containing only vertices, henceforth termed ''vertex sub-particles,'' were extracted from original unbinned particle images based on their calculated positions using relion_preprocess without further normalization. our sub-particle reconstruction method also enabled a more accurate determination of the defocus for each sub-particle, thus alleviating the well-documented depth-of-focus problem (derosier, ; zhang and zhou, ) . the defocus value of each vertex sub-particle was calculated based on its location with the following formula: where dz is the original defocus and dz is the new defocus for each vertex. classification and refinement of vertex sub-particles with -fold symmetry to identify the unique portal vertex from among the icosahedral vertices for each virus, we classified all vertex sub-particles with -fold symmetry ( figure s ). no rotational orientation search was allowed during classification (using the -skip_rotate parameter in relion), though the center for each vertex sub-particle was refined with a ± pixel offset search. the initial reference for classification was a -Å reconstruction of the vertex sub-particles using relion_reconstruct. although the portal vertex lacks true -fold symmetry, this classification with -fold symmetry imposed successfully distinguished between penton and portal vertices. through iterations of d classification, four classes were ultimately generated, with one class in particular exhibiting markedly different structures (i.e., a blurry central channel with a rod-like density) ( figure s ). additionally, this class contained . %, or approximately / th of the vertex sub-particles, consistent with the expectation that one out of twelve capsid vertices in each particle is a portal vertex. we thus considered this class the portal vertex class. in rare instances, two or more vertices from a capsid were classified into the portal vertex class, likely due to the low quality of these individual particles and/or errors in classification. these redundant sub-particles were removed as follows: if two or more vertices from the same virus particle were assigned to the portal vertex class, only the vertex subparticle with the highest _rlnmaxvalueprobdistribution score was retained. upon removing all redundant particles, , vertex sub-particles remained and were deemed sub-particles of the portal vertex, henceforth referred to as ''portal vertex sub-particles.'' d auto-refinement with -fold symmetry imposed was then performed on these portal vertex sub-particles with only a local search for orientation determination. using relion_postprocess, the final resolution of this c reconstruction was calculated with two independently refined maps from halves of the dataset with gold-standard fsc at the . criterion (rosenthal and henderson, ) , and determined to be . -Å ( figures s a and s d ). this reconstruction of the portal vertex contains a well-resolved -fold-arranged capsid and tegument, but a smeared portal dodecamer density due to symmetry mismatch. reconstructing the portal dodecamer with -fold symmetry from the portal vertex sub-particles, we further extracted sub-particles containing only the portal dodecamer in order to reconstruct the -fold symmetric portal. the positions of portal dodecamers on portal vertex sub-particles were determined using the above formula ( ). the euler angles (rot, tilt, and psi), ox, and oy are the orientation parameters of the portal vertex sub-particles; d is the distance from the center of the dodecamer to the center of the portal vertex sub-particle reconstruction (- pixels); and c is the center of the d projection image of the portal vertex sub-particle ( pixels). the sub-particles of portal dodecamers ( x pixels), henceforth referred to as ''dodecamer sub-particles'' were then extracted with relion_preprocess using these parameters. to reconstruct the portal dodecamer, we expanded the -fold symmetry of the dodecamer sub-particles using relion_particle_-symmetry_expand, generating five unique orientations for each dodecamer sub-particle. we then applied d classification with -fold symmetry imposed and without rotational orientation search, which, after iterations, yielded five classes of similar structures with a rotational difference of approximately between classes. ideally, each of the five expanded orientations of a dodecamer sub-particle should be assigned to exactly one of the five classes, such that each class should contain % of the symmetry-expanded sub-particles. after removing redundant particles as previously described-only particles with the highest _rlnmaxvalueprobdistribution score was retained-the five classes contained , , , , , , , , , particles, respectively. as the five reconstructed classes were of the same quality upon visual inspection, we chose the class with the most abundant particles for d refinement with -fold symmetry imposed and limited to local orientation search. as with the previous reconstruction, the resolution of this c portal dodecamer reconstruction was calculated with relion_postprocess using gold-standard fsc at the . criterion (rosenthal and henderson, ) , and determined to be $ . -Å . however, both a visual assessment of the portal's density and local resolution estimate derived from resmap (kucukelbir et al., ) indicate the majority of the portal itself has a resolution of $ . -Å ( figures s a and s e ), thereby permitting ab initio modeling. the lower resolution estimate obtained from fsc calculation likely results from the unresolved, flexible regions of the portal and/or the surrounding dna and protein densities that deviate from -fold symmetry, which are present in the map and therefore factored into the estimation. since each portal orientation determined from the previous round of portal dodecamer classification was selected from one of the five expanded orientations of each portal vertex sub-particle, these orientations can be used for d refinement of the portal vertex and whole virion without symmetry. the asymmetric auto-refinement for both portal vertex sub-particles and virion particles was thus performed with a local search for orientations determined from the classification of the portal dodecamer. due to the large computational requirement for refinement of the whole virion, we performed this refinement using two times-binned particles. the resolution of the portal vertex and whole virion c reconstructions was determined by relion_postprocess to be . -Å and . -Å , respectively ( figures s a, s b , s f, and s g), according to gold-standard fsc at the . criterion (rosenthal and henderson, ) . focused classification of symmetry-relaxed penton vertex sub-particles our classification of vertex sub-particles identified not only the portal vertex class, but also generated three classes of penton vertex ( figure s ). the sub-particles of these three classes were combined and deemed ''penton vertex sub-particles.'' due to the -fold symmetry surrounding capsid vertices, catc can bind to any of five registers at the penton vertex. to determine the structures of both catc-bound and catc-absent registers, we expanded the -fold symmetry of penton vertex sub-particles by relion_particle_symmetry_expand, producing , , symmetry-expanded sub-particles of penton vertex. to create a mask for focused classification of catc, one catc-containing region with its corresponding triplex ta was manually traced using volume_ tracer in chimera , after which a mask encompassing the traced region was created by relion_mask_create in relion. focused classification of the masked region was then performed on symmetry-expanded penton vertex sub-particles with neither angular nor offset search (using the -skip_align parameter in relion). since classification was performed on such a small area relative to the whole reconstruction, we specified a tau factor of during classification (scheres, ) . after iterations, four classes were generated, among which only one class had apparent catc density corresponding to . % of symmetry-expanded masked regions, consistent with the occupancy calculated from a previous study (dai et al., ) . we therefore considered this a catc-binding class. the other three classes, though of slightly differing map quality, clearly lacked catc density while sharing the same triplex ta orientation. we therefore regarded these three classes as catc-absent classes. gag gac cac- , and also cloned into redtrackcmv vector to generate an n-terminal flag-tagged expression plasmid ( wt). porf mutants and porf mutants were generated by pcr-based deletion mutagenesis from wt and wt, respectively. sequences of the pcr-amplified fragments and their correct insertion in the plasmid were verified by sequencing. the concentration of infectious kshv virions was determined as previously described (gong et al., ) . briefly, t-kshv cells were transfected with tegument protein expression plasmids or the empty vector as control. at h post-transfection, cells were treated with . mm nab plus ng/ml -o-tetradecanoylphorbol- -acetate (tpa) to induce kshv lytic replication. three days later, supernatants were collected, centrifuged at , g for min at c to remove cellular debris, serially diluted in dmem with % fbs, and then used to infect t cells in -well plates by spinoculation ( , g for h at c). two days post-infection, gfp-positive cell clusters containing two or more cells were counted under a fluorescence microscope to determine the titer of kshv virion. infectious units (iu) are expressed as the number of gfp-positive cell clusters in each well at a specific dilution of the viral stock. measuring viral dna replication and rna transcription by real-time pcr total dna was isolated from t-kshv cells induced with nab plus tpa, and viral genome copy numbers were determined by realtime pcr using primers for the essential viral gene orf . total rna was extracted from cells with purelink rna mini kit (thermo fisher scientific), treated with dnase i, then reverse-transcribed using superscript iii reverse transcriptase (thermo fisher scientific) and random hexamers. real-time pcr was then performed with the following primers to detect the corresponding dna or rna transcripts. host house-keeping gene gapdh: western blotting and antibodies cells were lysed in x western blotting loading buffer, resolved by sds-page gel electrophoresis, and transferred onto pvdf membrane. proteins were detected with antibodies against flag-epitope (sigma-aldrich) or gapdh (abcam). hrp-conjugated secondary antibodies (cell signaling technology) were used for detection with supersignal west femto maximum sensitivity substrate (thermo fisher scientific). construction of tri and tri kshv mutants kshv-bac plasmid was modified according to a previously described method (brulois et al., ; gong et al., ) . briefly, dna fragments of kshv orf (tri ) and orf (tri ) with defined mutations were used to replace the wild-type sequence in kshv-bac plasmid by homologous recombination. restriction patterns of mutated kshv bac plasmids were verified by comparison to that of wild-type kshv-bac plasmid to ensure overall genome integrity. fragments containing mutations were pcr-amplified from bac plasmids and sequenced to confirm that all mutations were correct. mutant bac plasmids were transfected into t cells individually, followed by selection with mg/ml hygromycin b for one month to generate cell lines latently infected by a specific kshv mutant virus. as described above, kshv lytic replication was induced by treatment of cells with . mm nab plus ng/ml tpa. three days later, supernatants were collected for determining titers of infectious kshv virions, while cells were harvested for measuring viral dna replication and rna transcription as described above. statistical comparisons between groups were made using student's t test calculated in microsoft excel. data and error bars displayed for measured relative virion production, viral titer, genome replication levels, and rna transcription levels (shown in figures j, l, n - p, and s a-s d) are presented as mean ± sem (n = biologically independent samples). six cryoem maps generated during this study have been deposited in the electron microscopy data bank (emdb) and are available under accession numbers emd- (c virion capsid reconstruction), emd- (c portal vertex reconstruction), emd- (c portal vertex reconstruction), emd- (c portal reconstruction), emd- (c penton vertex register, catc-absent reconstruction), and emd- (c penton vertex register, catc-binding reconstruction). atomic models corresponding to emd- , emd- , emd- , and emd- have been deposited in the protein data bank (pdb) and are available under accession numbers pdb- ppb, pdb- ppi, pdb- ppd, and pdb- pph, respectively. sub-particle reconstruction scripts used in our workflow have been deposited on github and can be accessed here: https://github.com/procyontao/herpesportal. figure s . sequential localized classification and sub-particle reconstruction, related to table flowchart illustrates the application of sequential localized classification and reconstruction to resolve symmetry-mismatched structures of portal and penton vertices. table and star methods (a-c) gold-standard fsc curves of all cryoem reconstructions. based on the . criterion, the resolutions of our c portal vertex, c portal vertex, and c portal reconstructions are . -Å , . -Å , and . -Å , respectively. the resolution of our c capsid reconstruction is . -Å , and the resolution of our penton vertex reconstructions with and without catc are . -Å and . -Å , respectively. (d-i) density maps colored by local resolution estimated from resmap (kucukelbir et al., ) . note that despite an fsc estimated resolution of . -Å , the vast majority of our c map reached a resolution of . -Å or better, thus enabling atomic model building. figure s . bioinformatics predictions and analyses, related to figures and (a) secondary structure and disorder prediction for porf obtained from phyre (kelley et al., ) , annotated by porf atomic model domains for reference as per key. (b) structure-based pairwise alignment of hsv- pul and kshv porf , performed using matchmaker in chimera . porf is only visible in the unsharpened map. boxed regions correspond to colored inset boxes illustrating residue features (ribbon-and-stick) in density (mesh). (b) c reconstruction of penton vertex with one catc bound, colored as per key. dotted black circle denotes density putatively assigned to the second porf head domain (density shown at a lower threshold than that of surrounding features). inset displays the c reconstruction in (a) fitted within the same reconstruction gaussian-filtered at . s to showcase connectivity between the weaker (green) putative porf head domain and catc helix bundle. (c) catc density shown with both porf head domains (at lower thresholds). note that a helical density feature can be observed in the putative (green) porf head domain density. figure s . overexpressing porf /porf mutants does not significantly affect kshv dna replication or gene expression, related to figure (a-b) viral genome replication in cells overexpressing wild-type or mutant forms of porf (a) or porf (b). t-kshv cells were transfected with corresponding expression plasmids or empty vector as control, and then induced with nab and tpa to facilitate kshv lytic replication. total dna was extracted from cells, and genome replication was determined by qpcr. (c-d) viral rna transcription in cells transfected with wild-type or mutant forms of porf (c) or porf (d). total rna was extracted from the same cells as (a-b). viral rna transcripts were quantified by rt-qpcr and presented as x-fold changes over rna level of empty vector transfected cells. data is mean ± sem (n = biologically independent samples). phenix: a comprehensive python-based system for macromolecular structure solution herpes simplex virus dna packaging sequences adopt novel structures that are specifically recognized by a component of the cleavage and packaging machinery dna cleavage and packaging proteins encoded by genes u(l) cryo-electron tomography of marburg virus particles and their morphogenesis within infected cells structural characterization of the ul dna-packaging protein from herpes simplex virus type construction and manipulation of a new kaposi's sarcoma-associated herpesvirus bacterial artificial chromosome clone visualization of the herpes simplex virus portal in situ by cryo-electron tomography procapsid assembly, maturation, nuclear exit: dynamic steps in the production of infectious herpesvirions identification of herpesvirus-like dna sequences in aids-associated kaposi's sarcoma electron cryotomography reveals the portal in the herpesvirus capsid organization of capsid-associated tegument components in kaposi's sarcoma-associated herpesvirus structure and mutagenesis reveal essential capsid protein interactions for kshv replication cryo-electron tomography of kaposi's sarcoma-associated herpesvirus capsids reveals dynamic scaffolding structures essential to capsid assembly and maturation correction of high-resolution data for curvature of the ewald sphere features and development of coot kshv and the pathogenesis of kaposi sarcoma: listening to human biology and medicine kshv: pathways to tumorigenesis and persistent infection resistance of herpesviruses to antiviral drugs: clinical impacts and molecular mechanisms ultrastructural characterization of sars coronavirus a herpesvirus protein selectively inhibits cellular mrna nuclear export virus-like vesicles of kaposi's sarcoma-associated herpesvirus activate lytic replication by triggering differentiation signaling influenza virus pleiomorphy characterized by cryoelectron tomography herpesvirus capsid assembly and dna packaging the c terminus of the herpes simplex virus ul protein is required for release of viral genomes from capsids bound to nuclear pores the phyre web portal for protein modeling, prediction and analysis quantifying the local resolution of cryo-em density maps structural framework for dna translocation via the viral portal protein electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-em a pul dimer interfaces the pseudorabies virus capsid and tegument different capsid-binding patterns of the b-herpesvirusspecific tegument protein pp (pm /pul ) in murine and human cytomegaloviruses portal protein functions akin to a dna-sensor that couples genome-packaging to icosahedral capsid maturation targeting of herpesvirus capsid transport in axons is coupled to association with specific sets of tegument proteins structure of the herpes simplex virus portal-vertex accurate determination of local defocus and specimen tilt in electron microscopy a viral scaffolding protein triggers portal ring oligomerization and incorporation during procapsid assembly cell-free assembly of the herpes simplex virus capsid involvement of the portal at an early step in herpes simplex virus capsid assembly role of the ul gene product in packaging dna into the herpes simplex virus capsid: location of ul product in the capsid and demonstration that it binds dna tegument assembly and secondary envelopment of alphaherpesviruses herpesvirus capsid association with the nuclear pore complex and viral dna release involve the nucleoporin can/nup and the capsid protein pul small capsid protein porf is essential for assembly of kaposi's sarcoma-associated herpesvirus capsids ucsf chimera-a visualization system for exploratory research and analysis herpesvirus resistance to antiviral drugs: a review of the mechanisms, clinical importance and therapeutic options seeing the portal in herpes simplex virus type b capsids optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy virion-wide protein interactions of kaposi's sarcoma-associated herpesvirus tegument proteins of kaposi's sarcoma-associated herpesvirus and related gamma-herpesviruses relion: implementation of a bayesian approach to cryo-em structure determination processing of structurally heterogeneous cryo-em data in relion cryo-em structure of the bacteriophage t portal protein assembly at near-atomic resolution assembly of herpes simplex virus type capsids using a panel of recombinant baculoviruses herpes simplex virus type dna-packaging protein ul is required for efficient binding of ul to capsids capsid structure of kaposi's sarcoma-associated herpesvirus, a gammaherpesvirus, compared to those of an alphaherpesvirus, herpes simplex virus type , and a betaherpesvirus, cytomegalovirus low-fidelity assembly of influenza a virus promotes escape from host cells epstein-barr virus replication in oral hairy leukoplakia: response, persistence, and resistance to treatment with valacyclovir structure of the herpes simplex virus type c-capsid with capsid-vertex-specific component protein structure modeling with modeller threedimensional structure of the human herpesvirus capsid atomic structure of the human cytomegalovirus capsid with its securing tegument layer of pp building atomic models based on near atomic resolution cryoem maps with existing tools limiting factors in atomic resolution cryo electron microscopy: no simple tricks identification of the sites of interaction between the scaffold and outer shell in herpes simplex virus- capsids by difference electron imaging four levels of hierarchical organization, including noncovalent chainmail, brace the mature tumor herpesvirus capsid against pressurization to reiterate, capsid vertices can theoretically be bound by any number of catcs from zero up to five. moreover, in cases of two catcs bound to a penton vertex, the two catcs can either occupy registers adjacent to each other or registers with a gap (i.e., an empty register) in between. we named these two binding patterns ''ortho-catc-binding'' and ''meta-catc-binding,'' respectively. in instances of three catcs bound to a penton vertex, the two vacant registers can either be adjacent or have a gap (i.e., an occupied register) between them, analogous to the case of two vertex-bound catcs. we thus named these two permutations ''ortho-catc-absent'' and ''meta-catc-absent,'' respectively. altogether, eight classes of penton vertices are possible. these are: zero-, one-, four-, and five-catc-binding vertices; ortho-and meta-catc-bind classified penton vertex sub-particles were then used for subsequent auto-refinement. given that five expanded orientations for each vertex were already each assigned to either a catc-binding or catc-absent class (due to masked classification of the catcbinding region), orientations for vertex sub-particles were determined as follows. the orientations for zero-and five-catc-binding vertex sub-particles were randomly selected from their five orientations in the catc-absent and catc-binding classes, respectively. orientations of one-catc-binding and four-catc-binding vertices were chosen as their single orientation in the catc-binding and catc-absent classes, respectively. where each vertex bound two catcs, orientations were chosen as one of the two orientations in the catc-binding class, such that the second (i.e., non-chosen) orientation was a and counterclockwise rotation from the chosen orientation, for ortho-and meta-catc-binding vertices, respectively. similarly, the orientations for ortho-and meta-catcabsent classes were chosen such that the non-chosen catc-absent orientation was a and counterclockwise rotation from the assigned orientation, respectively. for all auto-refinements, sub-particle orientations were determined by local angular and offset searches about these selected orientations, and initial references were -Å reconstructions of the sub-particles generated by re-lion_reconstruct. -fold symmetry was imposed during refinements of zero-and five-catc-binding vertices, while refinements of all other binding occupancies were carried out without symmetry. the resolutions of the resulting eight reconstructions determined by relion_postprocess according to gold-standard fsc at the . criterion were . -Å , . -Å , . -Å , and . -Å for zero-, one-, four-, and five-catc-binding vertices, respectively; . -Å and . -Å for ortho-and meta-catc-binding vertices, respectively; and . -Å and . -Å for ortho-and meta-catc-absent vertices, respectively. as the orientation information of all catc-binding and catc-absent registers and vertex sub-particles were conserved in their respective header files in our workflow, we were able to trace each register and vertex back to their original particle image. this enabled us to conduct a survey of the global number of catcs in each capsid and generate a histogram tallying this data (figure b) all authors reviewed and approved the paper. the authors declare no competing interests. refinement of the catc-binding and catc-absent structures without symmetry we next performed separate d auto-refinements without symmetry on both penton vertex sub-particles in the catc-binding class and in the catc-absent classes. orientations for sub-particles were determined by local search around each sub-particle's predetermined orientation from the mask refinement. a -Å reconstruction of penton vertex obtained through relion_reconstruct was used as the initial reference for refinements. the final resolutions of the catc-binding and catc-absent reconstructions determined by relion_postprocess were . -Å and . -Å , respectively, according to gold-standard fsc at the . criterion (rosenthal and henderson, ; figure s c ). of note, because focused classification was performed with respect to the masked area, only structural features within the masked area (which encompasses one catc and an associated triplex ta) of the refined structure are genuine, while the other registers of catc and triplex ta are of mixed occupancy and/or conformations (figure s ) (these will be fully separated in the following procedure). local resolution estimation by resmap (kucukelbir et al., ) indicates the masked area of both catc-binding and catc-absent reconstructions reached a resolution of $ . -Å ( figures s h and s i ). key: cord- -isbc r o authors: munjal, geetika; hanmandlu, madasu; srivastava, sangeet title: phylogenetics algorithms and applications date: - - journal: ambient communications and computer systems doi: . / - - - - _ sha: doc_id: cord_uid: isbc r o phylogenetics is a powerful approach in finding evolution of current day species. by studying phylogenetic trees, scientists gain a better understanding of how species have evolved while explaining the similarities and differences among species. the phylogenetic study can help in analysing the evolution and the similarities among diseases and viruses, and further help in prescribing their vaccines against them. this paper explores computational solutions for building phylogeny of species along with highlighting benefits of alignment-free methods of phylogenetics. the paper has also discussed the application of phylogenetic study in disease diagnosis and evolution. phylogenetics can be considered as one of the best tools for understanding the spread of contagious disease, for example, transmission of the human immunodeficiency virus (hiv) and the origin and subsequent evolution of the severe acute respiratory syndrome (sars) associated coronavirus (scov) [ ] . earlier, morphological traits were used for assessing similarities between species and building phylogenetic trees. presently, phylogenetics relies on information extracted from genetic material such as deoxyribonucleic acid (dna), ribonucleic acid (rna) or protein sequences [ ] . methods used for phylogenetic inference have changed drastically during the past two decades: from alignment-based to alignment-free methods [ ] . this paper has reviewed various methods under phylogenetic tree construction from character to distance methods and alignment-based to alignment-free methods. a brief review of phylogenetic tree applications is also given in cancer studies. a phylogenetic tree can be unrooted or rooted, implying directions corresponding to evolutionary time, i.e. the species at the leaves of a tree relate to the current day species. the species can be expressed as dna strings which are formed by combining four nucleotides a, t, c and g (a-adenine, t-thymine, c-cytosine and g-guanine). in literature, various string processing algorithms are reported which can quickly analyse these dna and rna sequences and build a phylogeny of sequences or species based on their similarity and dissimilarity. a high similarity among two sequences usually implies significant functional or structural likeliness, and these sequences are closely related in the phylogenetic tree. to get more precise information about the extent of similarity to some other sequence stored in a database, we must be able to compare sequences quickly with a set of sequences. for this, we need to perform the multiple sequence comparison. dynamic programming concepts facilitate this comparison using alignment methods, but it involves more computation. moreover, the iterative computational steps limit its utility for long length sequences [ ] . alignment-free methods overcome this limitation as they follow alternative metrics like word frequency or sequence entropy for finding similarity between sequences. phylogenetic tree generation consists of sequence alignment where the resulting tree reveals how alignment can influence the tree formation. alignment-based methodologies are probably the most widely used tools in sequence analysis problems [ ] . they consist of arranging two sequences: one on the top of another to highlight their common symbols and substrings. an alignment method is based on alignment parameters including insertion, deletions and gaps which play a pivotal role in the construction of the phylogenetic tree. a phylogenetic tree is formed as an outcome of sequence analysis performed on the dna or rna strings [ ] . sequence comparison reveals the patterns of shared history between species, helping in the prediction of ancestral states. the comparison of sequences also helps in understanding the biology of living organisms which is required to find similarity and relationship among species. for sequence comparison, we can follow alignment-based or alignment-free methods [ , , ] . sequence alignment is a method to identify homologous sequences. it is categorized as pairwise alignment in which only two sequences are compared at a time whereas in multiple sequence alignment more than two sequences are compared. alignmentbased can be global or local [ , ] . these alignment-based algorithms can also be used with distance methods to express the similarity between two sequences, reflecting the number of changes in each sequence. figure gives a hierarchical view of various methods for phylogenetic tree building. the character-based methods compare all sequences simultaneously considering one character/site at a time. these are maximum parsimony and maximum likelihood. these methods use probability and consider variation in a set of sequences [ ] . both approaches consider the tree with the best score tree, which requires the smallest number of changes to perform alignment. maximum parsimony method suffers badly from the long-branch attraction and gives the least information about the branch lengths [ ] . in such cases, if two external branches are separated by short internal branches, it leads to the incorrect tree. some of the salient features of character-based methods are mentioned in table . distance-based methods use the dissimilarity (the distance) between the two sequences to construct trees. they are much less computationally intensive than the character based methods are mostly accurate as they take mutations into count. for tree generation, generally, hierarchical clustering is used in which dendrograms (clusters) are created. table briefly compares various phylogenetic tree construction methods. multiple alignments of related sequences may often yield the most helpful information on its phylogeny. however, it can produce incorrect results when applied to more divergent sequence rearrangements [ ] . some computationally intensive multiple alignment methods align sequences strictly based on the order in which they receive them. multiple sequence alignment methods emphasize that more closely related sequences should be aligned first. in cases of sequences being less related to one another, however, sharing a common ancestor may be clustered separately [ ] . this implies that they can be more accurately aligned, but may result in incorrect phylogeny. alignment can provide an optimized tree if a recursive approach is followed; however, this will increase the complexity of the problem. if the differences among the lengths of sequences are very high, the alignment performance significantly impacts tree generation. the use of dynamic programming in alignment makes computation more complicated, and iterative steps limit their utility for large datasets. therefore, consistent efforts have been made in developing and improving multiple sequence alignment methods for supporting variable length sequences with high accuracy and also for aligning a larger number of sequences simultaneously. because of the problems associated with alignment-based phylogeny the importance of alignment-free methods is apparent [ ] . hence, the alignment quality affects the relationship created in a phylogenetic tree based on the consideration discussed above. alignment-free methods proposed in recent years can be classified into various categories as shown in fig. . these include k-tuple based on the word frequencies, methods that represent the sequence without using the word frequencies, i.e. compression algorithms probabilistic methods and information theory-based method. in the k-tuple method, a genetic sequence is represented by a frequency vector of fixed length subsequence and the similarity or dissimilarity measures are found based on the frequency vector of subsequence. the probabilistic methods represent the sequences using the transition matrix of a markov chain [ ] of a pre-specified order, and comparison of two sequences is done by finding the distance between two transition matrices. graphical representation comprising d or d or even d methods provides an easy way to view, sort and compare various sequences. graphical representation further helps in recognizing major characteristics among similar biological sequences. as discussed k-tuple method uses k-words to characterize the compositional features of a sequence numerically. a biological sequence is numerically converted into a vector or a matrix composed of the word frequency. the k-word frequency pro-vides a fast arithmetic speed and can be applied to full sequences. the problem with k-tuple is a big value of k that poses a challenge in the computing time and space, and k-word methods underestimate or even ignore the importance of its location. the string-based distance measure uses substring matches with k mismatches. cancer research is considered one of the most significant areas in the medical community. mutations in genomic sequences are responsible for cancer development and increased aggressiveness in patients [ , ] . the combination of all such genes mutations, or progression pathways, across a population can be summarized in a phylogeny describing the different evolutionary pathways [ ] . application of the phylogenetic tree can be explored for finding similarities among breast cancer subtypes based on gene data [ , ] . discovery of genes associated in cancer subtype help researchers to map different pathways to classify cancer subtypes according to their mutations. methods of phylogenetic tree inference have proliferated in cancer genome studies such as breast cancer [ ] . phylogenetic can capture important mutational events among different cancer types; a network approach can also capture tumour similarities. it has been observed from the literature that in cancer disease, the driver genes change the cancer progression, and it even affects the participation of other genes thus generating gene interaction network. phylogenetic methods can solve the problem of class prediction by using a classification tree. phylogenetic methods give us a deeper understanding of biological heterogeneity among cancer subtype. the research focuses on the various methods of sequence analysis to generate phylogenetic trees. the limitations associated with sequence alignment methods lead to the development of alignment-free sequence analysis. however, most of the existing alignment-free methods are unable to build an accurate tree so more refinement is required in alignment-free methods. the phylogenetic study is not limited to species evolution, but disease evolution as well. extending phylogenetic to disease diagnosis can give birth to new treatment options and understanding its progression. use of phylogenetics in the molecular epidemiology and evolutionary studies of viral infections reconstructing optimal phylogenetic trees : a challenge in experimental algorithmics editorial: alignment-free methods in computational biology analyzing dna strings using information theory concepts modified k-tuple method for the construction of phylogenetic trees the evolution of tumour phylogenetics: principles and practice on maximum entropy principle for sequence comparison a general method applicable to the search for similarity in the amino acid sequence of two proteins comparison of biosequences approximate maximum parsimony and ancestral maximum likelihood phylogenetic trees in bioinformatics weighted relative entropy for phylogenetic tree based on -step markov model phylooncology: understanding cancer through phylogenetic analysis tumor classification using phylogenetic methods on expression data novel gene selection method for breast cancer classification sequence analysis integrating alignment-based and alignmentfree sequence similarity measures for biological sequence classification hidden markov models with applications to dna sequence analysis is multiple-sequence alignment required for accurate inference of phylogeny? constructing phylogenetic trees using multiple sequence alignment phylogenetic trees in bioinformatics constructing phylogenetic trees using maximum likelihood applications and algorithms for inference of huge phylogenetic trees: a review upgma clustering revisited: a weight-driven approach to transitive approximation an improved phylogenetic tree comparison method neighbor-net: an agglomerative method for the construction of phylogenetic networks bioinformatics: a practical guide to the analysis of genes and proteins sequence similarity using composition method sequence analysis kmacs: the k-mismatch average common substring approach to alignment-free sequence comparison key: cord- -z zx gd authors: ljubin-sternak, sunčanica; meštrović, tomislav; ivković-jureković, irena; kolarić, branko; slović, anamarija; forčić, dubravko; tot, tatjana; mijač, maja; vraneš, jasmina title: the emerging role of rhinoviruses in lower respiratory tract infections in children – clinical and molecular epidemiological study from croatia, – date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: z zx gd rhinoviruses (rvs) are increasingly implicated not only in mild upper respiratory tract infections, but also in more severe lower respiratory tract infections; however, little is known about species diversity and viral epidemiology of rvs among the infected children. therefore, we investigated the rhinovirus (rv) infection prevalence over a -year period, compared it with prevalence patterns of other common respiratory viruses, and explored clinical and molecular epidemiology of rv infections among children hospitalized with acute respiratory infection in north-western and central parts of croatia. for respiratory virus detection, nasopharyngeal and pharyngeal flocked swabs were taken from each patient and subsequently analyzed with multiplex rt-pcr. to determine the rv species in a subset of positive children, ′utr in rv-positive samples has been sequenced. nucleotide sequences of referent rv strains were retrieved by searching the database with basic local alignment tool, and used to construct alignments and phylogenetic trees using mafft multiple sequence alignment tool and the maximum likelihood method, respectively. in our study population rv was the most frequently detected virus, diagnosed in patients ( . %), of which . % was detected as a monoinfection. median age of rv-infected children was . years, and more than half of children infected with rv ( . %) presented with lower respiratory tract infections. most rv cases were detected from september to december, and all three species co-circulated during the analyzed period ( – ). sequence analysis based on ′utr region yielded distinct strains; the most prevalent was rv-c ( . %) followed by rv-a ( . %) and rv-b ( . %). most of rv-a sequences formed a distinct phylogenetic group; only strains ri/hr - (along with a reference strain mf ) clustered with rv-c strains. strains belonging to the group c were the most diverse ( . % identity among strains), while group b was the most conserved ( . % identity among strains). despite such differences in strain groups (hitherto undescribed in croatia), clinical presentation of infected children was rather similar. our results are consistent with newer studies that investigated the etiology of acute respiratory infections, especially those focused on children with lower respiratory tract infections, where rvs should always be considered as potentially serious pathogens. rhinoviruses (rvs) are increasingly implicated not only in mild upper respiratory tract infections, but also in more severe lower respiratory tract infections; however, little is known about species diversity and viral epidemiology of rvs among the infected children. therefore, we investigated the rhinovirus (rv) infection prevalence over a -year period, compared it with prevalence patterns of other common respiratory viruses, and explored clinical and molecular epidemiology of rv infections among children hospitalized with acute respiratory infection in north-western and central parts of croatia. for respiratory virus detection, nasopharyngeal and pharyngeal flocked swabs were taken from each patient and subsequently analyzed with multiplex rt-pcr. to determine the rv species in a subset of positive children, utr in rv-positive samples has been sequenced. nucleotide sequences of referent rv strains were retrieved by searching the database with basic local alignment tool, and used to construct alignments and phylogenetic trees using mafft multiple sequence alignment tool and the maximum likelihood method, respectively. in our study population rv was the most frequently detected virus, diagnosed in patients ( . %), of which . % was detected as a monoinfection. median age of rv-infected children was . years, and more than half of children infected with rv ( . %) presented with lower respiratory tract infections. most rv cases were detected from september to december, and all three species co-circulated during the analyzed period ( ) ( ) ( ) . sequence analysis based on utr region yielded distinct strains; the most prevalent was rv-c ( . %) followed by rv-a ( . %) and rv-b ( . %). most of rv-a sequences formed introduction rhinoviruses (rvs) are small, non-enveloped viruses that belong to the family picornaviridae, genus enterovirus. to date, there are rhinovirus (rv) genotypes recognized and classified into the three species as rv-a ( types), rv-b ( types), and rv-c ( types) (royston and tapparel, ; pan et al., ) . rv-a and rv-b were discovered by isolation on monkey kidney cells in s (price, ) while rv-c genotypes, which are not cultivable using ordinary culture methods, have been identified decades after following the rise of molecular techniques (lamson et al., ; lau et al., ) . there are also differences between rv species in utilization of cell entry receptor: a majority of rv-a and rv-b attach to the intercellular adhesion molecule (icam)- (classified as the major receptor group) and the others alternatively bind low density lipoprotein receptor (ldl-r) (minor receptor group), whereas rv-c utilizes human cadherin-related family member (cdhr ) (bochkov et al., ; royston and tapparel, ) . rhinovirus genome is a . -kb single-stranded, positive-sense rna with a single open reading frame (orf) joined to a untranslated region ( utr) and a short viral priming protein (vpg) (jacobs et al., ) . orf encodes a poly-protein which is cleaved by virally encoded proteases in proteins. four proteins -vp , vp , vp , and vp -make up the viral capsid and account for the virus' antigenic diversity, while the remaining non-structural proteins are involved in viral genome replication and assembly. a rather conserved utr region that harbors internal ribosomal entry site (ires) is usually utilized for rv detection from clinical samples, while more precise genotyping is based on vp /vp or vp sequence analysis. rhinovirus have been neglected for decades, primarily because they were considered less virulent and only capable of causing mild common cold, and were not recognized, until recently, as important causative agents of lower respiratory tract infections (lrtis) and severe respiratory disease . introduction of sensitive and technically simple molecular detection assays, especially multiplex pcr, enabled affordable rv detection in line with other common respiratory viruses in clinical samples. together with coronaviruses, rvs are indeed responsible for majority of upper respiratory tract infection (urti), but also for substantial rate of lrtis in all age groups (lauinger et al., ; zhao et al., zhao et al., ,Čivljak et al., . many studies showed that rv is one of the leading causes of pneumonia, bronchiolitis and other form of severe respiratory disease (zhao et al., ) , standing side by side with respiratory syncytial virus (rsv) in children and influenza in elderly (esposito et al., ; ning et al., ; Čivljak et al., ) . there are several studies that report increased rates of asthma exacerbations and lrtis among children with rv-c when compared to those infected with rv-a and rv-b (bizzintino et al., ; lauinger et al., ) ; however, recent studies did not find any relationship between a specific rv species and the severity of clinical presentation (jacobs et al., ; van der linden et al., ; zhao et al., ) . data on rv prevalence in croatia are scarce, and molecular epidemiology was thus far not described. this study aims to determine the rv prevalence, compare it with prevalence patterns of other common respiratory viruses, as well as to explore clinical and molecular epidemiological features of rv infections among hospitalized children with acute respiratory infection. the research was performed in accordance with relevant guidelines/regulations and in line with the declaration of helsinki, as revised in . written informed consent was obtained from all participants (children's parents or their legal guardians). the study was approved by the ethics committee of the dr. andrija Štampar teaching institute of public health and conducted as part of the croatian science foundation project entitled "new and neglected respiratory viruses in vulnerable groups of patients" (no. ip- - - ). all standard biosecurity and institutional safety procedures have been adhered to. from may to april , a total of patients were included from hospitals located in north-western and central part of croatia: clinical hospital zagreb and general hospital karlovac, respectively. inclusion criteria were: age lower than years, a clinical diagnosis of acute respiratory tract infection (ari), and need for hospitalization [on ward (≥ day) or day hospital (for more than but less than h)]. exclusion criteria were presumed bacterial respiratory infection -including otitis, sinusitis and bacterial pneumonia, healthcare-associated infection, and ambulatory treated patients. patients were categorized into the four groups according to their respective age (i.e., < , - . , - . and ≥ years of age), and two groups according to the localization of infection in those with upper respiratory tract infection (urti) and lower respiratory tract infection (lrti). urti was defined by symptoms of the common cold, coryza, cough, and hoarseness with or without fever, so clinical syndromes of respiratory catarrh, rhinitis and/or pharyngitis represented urti category. lrti was defined by symptoms of tachypnea, wheezing, severe cough, breathlessness, and by specific clinical signs such as nasal flaring, jugular and intercostal retractions, cyanosis (in rare instances), as well as wheezing, crackles and inspiratory rhonchi or generally reduced breath sounds during auscultation. clinical syndromes of bronchitis, bronchiolitis and pneumonia were included in lrti category (tregoning and schwarze, ; ljubin-sternak et al., ) . to avoid unnecessary x-ray exposure, chest radiographs were taken only for some of the patients in order to exclude or confirm bacterial pneumonia. for respiratory virus detection, nasopharyngeal and pharyngeal flocked swabs from each patient were collected, combined, and placed in viral transport medium (utm tm , copan, italy). specimens were immediately transported to the molecular microbiology laboratory at the public health institute where they were stored at − • c until tested. the results of virology testing were released to the physicians periodically, approximately once per week. as a part of routine care, nasopharyngeal, pharyngeal swabs, and blood cultures were taken from hospitalized patients and submitted for bacterial diagnostics using standard cultivation methods. demographic and clinical data, antimicrobial use records, and the results of routine bacterial studies were collected by a retrospective review of patient charts. to isolate viral dna and rna from viral transport medium, µl has been extracted according to the manufacturer's protocol using ribospin tm vrd kit (geneall biotechnology, seoul, korea). multiplex rt-pcr for respiratory viruses using seeplex r rv detection kit (seegene inc., seoul, korea) was performed. briefly, multiplex pcr and cdna synthesis as onestep reaction was performed and set up in three different tubes with three sets of primers. more specifically, "a set" contained primers for simultaneous amplification of target sequences of adenovirus (adv), human coronavirus (hcov) e/nl , parainfluenza virus (piv) types - , and pcr internal control to check for the presence of substances that may interfere with amplification; "b set" contained primers for hcov oc , rv groups a/b/c, rsv type a and b, influenza (flu) type a, and pcr internal control; and "c set" contained primers for human bocavirus (hbov), flu type b, human metapneumovirus (hmpv), piv type , human enterovirus (hev), and the overall process control (human rnase p was included throughout the entire process as a control from nucleic acid extraction to amplification). amplification was performed on thermal cycler geneamp r pcr system (applied biosystems, foster city, united states). detection of pcr products was done by microchip electrophoresis on the mce r - multina device (shimadzu, kyoto, japan) -including software analysis showing results in the form of electropherograms and virtual image gels. to determine rv species, utr of the genome in rv-positive samples was sequenced. total rna was extracted from µl of rv-positive samples by the method reported by chomczynski and mackey ( ) . reverse transcription was performed at • c for min, in a reaction mix containing µl of isolated rna, × pcr buffer (ge healthcare, united kingdom), . mm of each dntp, u of rnase inhibitor (thermo fisher scientific, united states), . mm mgcl , . mm of random hexanucleotide primers and u of mulv reverse transcriptase (thermo fisher scientific, united states) in a final volume of µl. pcr reaction was performed by amplifying utr, using ntr + ( caa gya ctt ctg tyt ccc ) and ntr-( cac gga cac cca aag tag t ) primer pair, modified from wisdom et al. ( ) , corresponding to positions - and - of strain a (acc. no. x . ), respectively. final pcr mixtures contained × onetaq pcr buffer (new england biolabs, united states), mm dntp, . mm mgcl , . mm of each primer, and . u of onetaq dna polymerase (new england biolabs, united states). the amplified products were separated on a . % agarose gel, excised and purified by centrifugation through glass wool (sun et al., ) . sequencing reactions were set up with purified dna, one of the specific primers used for amplification, and a bigdye terminator v . cycle sequencing kit (thermo fisher scientific, united states) according to the manufacturer's protocol. sequencing and sequence analysis were performed on a genetic analyzer (thermo fisher scientific, united states). nucleotide sequences of referent rv strains were retrieved by searching the database with blast (basic local alignment tool) and used to construct alignments and phylogenetic trees. alignments were performed using mafft multiple sequence alignment tool available at the embl-ebi website , and edited in aliview v . (larsson, ) . phylogenetic trees were generated using the maximum likelihood method with molecular evolutionary genetics analyses (mega) software v . (tamura et al., ) , under the most appropriate model of nt substitution determined with jmodeltest v . . (darriba et al., ) . bootstrap probabilities for , iterations were calculated to evaluate confidence estimates. sequence conservation (defined as percentage of genomic positions identical in all strains; gaps were ignored during computing) and evolutionary distances (p-distances) within and between groups have been calculated using mega . . the sequences of hrsv strains obtained in this study were deposited in the genbank under acc. nos. mn -mn . data analysis was performed using stata/mp (ver. . ; statacorp llc, college station, united states). age was presented using medians with stated interquartile range (iqr). demographic and clinical parameters were compared by χ -test or fisher's exact test for categorical variables and by kruskal-wallis test for continuous variables. to assess the strength of association between dependent variable (rv positive patients) and age we used univariate logistic regression. p-value was set to < . . in total, patients were screened for respiratory viruses. the exact prevalence pattern of detected viruses can be seen in table . rv was the most frequently detected virus, diagnosed in patients ( . %); . % as monoinfection, and . % as coinfection with other respiratory viruses. this was followed by rsv ( . %), adv ( . %), pivs ( . %), flu types a and b ( . %), hcov /nl and oc ( . %), hbov ( . ), hev ( . %), and hmpv ( . %). there was a significant difference in age according to the specific virus (p < . ) (figure ) . median age in years of rv infected children ( . , iqr . ) was higher than in children infected with rsv ( . , iqr . ), pivs ( . , iqr . ), hcov ( . , iqr . ), hmpv ( . , iqr . ), and hbov ( . , iqr . ), but lower than in those infected with flu ( . , iqr . ), adv ( . , iqr . ), and hev ( . , iqr . ) (figure ) . there was no statistically significant difference in the prevalence of rv infection between age groups (p = . ). additionally, age ( year increase) was not significant predictor for rv positivity (or = . , % ci = . - . ; p = . ). according to the clinical presentation, there was a significant difference in the proportion of lrtis between the type of the respiratory viruses (p = . ) (figure ) . more than half of children infected with rv ( ; . %) presented with lrti; nonetheless, this was not significantly different from the proportion of rv positive children with urti ( ; . %) (p = . ), while children with rsv infection significantly more often presented with lrti (p < . ). out of the rv-positive samples by multiplex pcr, we were able to sequence samples ( %). sequence analysis based on bp of ' utr region of samples yielded viral types (seven strains had identical sequence) (figure ) . the most prevalent was rv-c ( / ; . %) followed by rv-a ( / ; . %) and rv-b ( / ; . %). most of rv-a sequences formed a distinct phylogenetic group; only strain ri/hr - (along with a reference strain mf ) clustered with rv-c strains (figure ) . of the three respective groups, strains belonging to the group c were the most diverse, with identity of . % ( of identical positions), while group b was the most conserved with . % identity among strains ( / ). group a comprised strains which shared an overall . % identical positions ( / ). calculated p-distances between groups showed group a and c are more closely related (p-distance . ) than to group b. similar p-value was calculated between group b and group a (p-distance . ) or group c strains (p-distance . ), respectively. no significant difference has been demonstrated in clinical symptoms based on the rv species, with the exception of increased frequency of antibiotic treatment in those infected with rv-a species (p = . ) ( table ) . most rv cases were detected from september to december, and all three species co-circulated during the analyzed period (figure ) . in this study we initially evaluated the prevalence of rv and other common respiratory viruses, as well as rv species distribution in hospitalized children with symptoms of ari over a period of years. our results are consistent with previously published studies that investigated the etiology of ari, especially those focused on children with lrtis (chen et al., ; ning et al., ) . indeed, rv is right next to rsv when addressing the most common causes of bronchiolitis in hospitalized children, as demonstrated in very low-birth-weight infants from argentina and in one multicentre prospective study from the united states, respectively (mansbach et al., ; miller et al., ) . although rv can be found both in upper and lower respiratory tract, the potential for spread and the pathophysiology of infection in those two regions differs (as evidenced by studies conducted on rsv) (kim et al., ; gonzález-parra and dobrovolny, ) . possible causes of such disparity are fundamental differences in the immune responses and virus-cell interactions between these two anatomical regions, resulting in altered disease manifestation and spread (gonzález-parra and dobrovolny, ). there is also evidence that mucus velocity is decreased in small children (akin to elderly individuals) (grubb et al., ) , making them more susceptible to lrti and creating in turn a potential niche for more detrimental effect of rv infection. when age is concerned, our study has showed that rv holds a middle ground, not affecting very young children as is the case with rsv. this is comparable with the results found in the study by cebey-lópez et al. ( ) , where rsv infection was also seen in the youngest age groups (less than year of age), rv was present in somewhat older children (mean between . and . months), while adv predominated in patients older than months. conversely, some other author groups pointed out how rv can predominate in practically all age groups (tsagarakis et al., ) . in order to determine the rv species, we decided to sequence and analyze utr, which has been established as a relatively simple and rapid technique for identifying the rv serotypes in clinical samples. moreover, it has been shown that utr rt-pcr demonstrated greater sensitivity than vp -vp pcr, as reflected by the higher positivity rate in amplification of clinical isolates, and further, no need for a nested pcr or multiple primer pairs -reducing in turn the contamination rate, cost and turnaround time (kiang et al., ) . despite using primers which target the region widely used for typing purposes, more than half of the samples produced no amplicons suitable for sequencing. such low rate of successful rv sequencing from clinical samples may be a consequence of mutations in primer regions or low viral load in original sample, but most probably arises due to rna degradation during freezing/thawing cycles. phylogenetic groups were readily distinguished and all three rv groups were detected, with group c and a predominating. the proportion of the three rv species revealed in this study (rv-a . %, rv-b . %, and rv-c, . %) is consistent with prior studies worldwide (rv-a, . - . %; rv-b, . - %; rv-c, - . %) (lauinger et al., ; rahamat-langendoen et al., ; jacobs et al., ; tsatsral et al., ; ratnamohan et al., ; van der linden et al., ; zhao et al., ) . intermixing of groups was not observed, except for a single group a sequence (ri/hr - ) which clustered with group c sequences, indicating a recombination event or co-infection as was proposed by richter et al. ( ) . furthermore, group b strains were detected sporadically during the analyzed period, which is in accordance with other studies (lauinger et al., ; launes et al., ; richter et al., ) . these results represent the first report on rv diversity in croatia. in previous reports rv-c (and in lesser extent rv-a) have been associated with more severe illness (bizzintino et al., ; lauinger et al., ; linder et al., ; chen et al., ) ; however, more recent studies failed to report the connections between species and disease severity (rahamat-langendoen et al., ; jacobs et al., ; van der linden et al., ; zhao et al., ) . in this study there were no significant differences observed in clinical symptoms among three species, except more frequent utilization of antibiotic therapy in patients with rv-a species which can be result of subjective clinical assessment of more severe disease and empirical introduction of therapy. rv circulated throughout the -year period covered by this study with peaks in autumn and winter months. previous studies reported that rv infections occur all year round, with peaks of infection usually in spring and autumn months (Čivljak et al., ) . however, recent research endeavors also report peaks in autumn and winter months (zhao et al., ) , and some of them specifically note that rv-c demonstrate peak in winter months (linder et al., ) . our study also observed no rv-c detection in spring and summer season. there are several limitations of the study. due to crosssectional study approach, a control group of asymptomatic patients could not have been included (which would facilitate assessment of rv infection severity). furthermore, we performed only utr targeted rt-pcr assay and did not confirm our results with vp /vp or vp sequences analysis. some authors report discordance between proposed phylogeny groups when sequences from the 'utr and vp /vp coding regions were analyzed, which can result in imprecise classification (ratnamohan et al., ) . more specifically, samples that clustered as rv a using utr analysis can be revealed as rv-c when vp /vp region is analyzed (ratnamohan et al., ) . also, complicated rhinovirus infections were excluded from the study. notwithstanding the aforementioned limitations, this is the first study endeavor cataloging a circulation of rv species in croatia over years. in conclusion, in our study more than half of children infected with rv presented with lrti, which (together with other newer studies in the field) underlines the need for paradigm shift where rvs will not be merely associated with urti, but also considered in cases of lrti. regardless of the diversity of rv found in this study and the purported heterogeneity of the rv strains infecting the children, the similarity of clinical presentation negates the notion that certain rv species might be more virulent, at least in our case. more clinical and epidemiological studies are warranted to further elucidate this issue, with inevitable use of animal models to study pathogenic specificities of rv infection. the datasets generated for this study can be found in the genbank-accession numbers from mn to mn . the studies involving human participants were reviewed and approved by the ethics committee of the dr. andrija Štampar teaching institute of public health and conducted as part of the croatian science foundation project entitled "new and neglected respiratory viruses in vulnerable groups of patients" (no. ip- - - ) . written informed consent to participate in this study was provided by the participants' legal guardian/ next of kin. slj-s, ii-j, and jv designed the research. slj-s, as, and df performed the experiments. mm and tt collected the data. bk, as, and df analyzed the data. slj-s, tm, mm, tt, ii-j, as, and df interpreted the data and prepared the draft of the manuscript. jv and tm critically reviewed the draft. slj-s and tm wrote the final version of the manuscript. all authors reviewed and approved the final version of the manuscript. this work has been fully supported by the croatian science foundation under the project no. ip- - - titled "new and neglected respiratory viruses in vulnerable groups of patients" (principal investigator slj-s). the funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript. association between human rhinovirus c and severity of asthma in children cadherin-related family member , a childhood asthma susceptibility gene product, mediates rhinovirus c binding and replication viral co-infections in pediatric patients hospitalized with lower tract acute respiratory infections epidemiologic, clinical, and virologic characteristics of human rhinovirus infection among otherwise healthy children and adults: rhinovirus among adults and children single-step method of total rna isolated by acid guanidine phenol extraction viral pathogens associated with acute respiratory illness in hospitalized adults and elderly from jmodeltest : more models, new heuristics and parallel computing impact of viral infections in children with community-acquired pneumoniae: results of a study of respiratory viruses prophylactic administration of respiratory syncytial virus immune globulin to high-risk infants and young children human rhinoviruses clinical and molecular epidemiology of human rhinovirus infections in patients with hematologic malignancy assay for noncoding region analysis of all human rhinovirus prototype strains ct findings in viral lower respiratory tract infections caused by parainfluenza virus, influenza virus and respiratory syncytial virus masstag polymerase-chain-reaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused influenza-like illness in new york state during aliview: a fast and lightweight alignment viewer and editor for large datasets clinical features and complete genome characterization of a distinct human rhinovirus (rv) genetic cluster, probably representing a previously undetected rv species rv-c, associated with acute respiratory illness in children patient characteristics and severity of human rhinovirus infections in children molecular epidemiology of severe respiratory disease by human rhinoviruses and enteroviruses at a tertiary paediatric hospital in human rhinovirus c: age, season, and lower respiratory illness over the past decades etiology and clinical characteristics of single and multiple respiratory virus infections diagnosed in croatian children in two respiratory seasons prospective multicenter study of viral etiology and hospital length of stay in children with severe bronchiolitis human rhinoviruses in severe respiratory disease in very low birth weight infants the etiology of community-acquired pneumonia among children under years of age in mainland china genome sequences of rhinovirus genotype c detected in three patients with acute respiratory illness the isolation of a new virus associated with respiratory clinical disease in humans the significance of rhinovirus detection in hospitalized children: clinical, epidemiological and virological features phylogenetic analysis of human rhinoviruses collected over four successive years in sydney molecular epidemiology of rhinoviruses in cyprus over three consecutive seasons rhinoviruses and respiratory enteroviruses: not as simple as abc a quick, cost-free method of purification of dna fragments from agarose gel mega : molecular evolutionary genetics analysis version . rhinovirus-from bench to bedside respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology age-related prevalence of common upper respiratory pathogens, based on the application of the filmarray respiratory panel in a tertiary hospital in greece molecular epidemiology of the human rhinovirus infection in mongolia during - a molecular epidemiological perspective of rhinovirus types circulating in amsterdam from screening respiratory samples for detection of human rhinoviruses (rvs) and enteroviruses: comprehensive vp -vp typing reveals high incidence and genetic diversity of rv species c genotypic diversity and epidemiology of human rhinovirus among children with severe acute respiratory tract infection in shanghai the authors wish to thank matea kvaternik celjak for her technical assistance. key: cord- - iqhf o authors: macraild, christopher a.; seow, jeffrey; das, sreedam c.; norton, raymond s. title: disordered epitopes as peptide vaccines date: - - journal: pept sci (hoboken) doi: . /pep . sha: doc_id: cord_uid: iqhf o the development of clinically useful peptide‐based vaccines remains a long‐standing goal. this review highlights that intrinsically disordered protein antigens, which lack an ordered three‐dimensional structure, represent excellent starting points for the development of such vaccines. disordered proteins represent an important class of antigen in a wide range of human pathogens, and, contrary to widespread belief, they are frequently targets of protective antibody responses. importantly, disordered epitopes appear invariably to be linear epitopes, rendering them ideally suited to incorporation into a peptide vaccine. nonetheless, the conformational properties of disordered antigens, and hence their recognition by antibodies, frequently depend on the interactions they make and the context in which they are presented to the immune system. these effects must be considered in the design of an effective vaccine. here we discuss these issues and propose design principles that may facilitate the development of peptide vaccines targeting disordered antigens. vaccines are an indispensable tool in the fight against disease and have had a significant impact on public health over several decades. [ ] traditionally, vaccines have relied on the use of live-attenuated or inactivated forms of the pathogen to induce a protective immune response. however, in some cases, such as malaria, the pathogen is difficult to culture in vitro at large scale. moreover, the use of whole organisms as vaccines introduces a high antigenic load when often only a small subset of antigens is driving protection. [ ] there is also the possibility of adverse allergenic reactions to certain antigens in these preparations. these obstacles have led to an increased interest in subunit vaccines, in which single, or a select few, proteins are used in a vaccine formulation to induce protective immunity. [ ] the use of peptides as vaccines takes this rationale further, as even a single protein antigen may have many epitopes, not all of which contribute to protective immunity. peptide vaccines offer a means to formulate vaccines containing only epitopes that are capable of inducing a positive and efficient immune response. [ , ] the ease of peptide synthesis makes large scale production feasible whilst also offering a cleaner vaccine preparation lacking biological contaminations commonly associated with recombinant expression or whole organism vaccines. the specificity of peptides also allows for improved customisability, facilitating, for example, a multiepitope approach to target different strains or stages in the life cycle of the pathogen. for these reasons, there has been long-standing interest in this area of vaccine design, with over peptide vaccines reaching clinical trials, targeting a wide range of indications ( table ) . seven of these vaccines have reached phase iii trial ( table ). much of this recent interest has focussed on t-cell epitopes, with all of the vaccines to reach phase iii being t-cell based. for many diseases, however, b-cell responses also play an important role in immune protection, but peptide-based vaccines based on b-cell epitopes have proved more challenging for a number of reasons. owing to the small size of peptides, they are generally poorly immunogenic. in addition, t-cell epitopes required for the establishment of a robust immune response may also be absent in smaller peptides. to address these issues, carrier proteins or adjuvants are typically included in the vaccine formulation. peptides are also prone to enzymatic degradation; to combat this, the epitopes can be modified by conformational stabilization or cyclisation, or peptide mimetics that are resistant to proteolysis can be used. [ ] finally, and perhaps most importantly, peptidebased approaches are limited because the antibody response to many protein antigens is dominated by conformational epitopes, which are difficult or impossible to capture effectively in a peptide-based design. in this review, we argue that intrinsically disordered protein antigens, which lack an ordered three-dimensional structure, represent excellent starting points for the development of peptide vaccines. our argument is structured as follows: first, we establish that disordered proteins represent an important class of antigen in a wide range of human pathogens. second, we describe the unique features of these antigens and their interactions with antibodies, highlighting those properties that render them particularly suited to the development of peptide vaccines. in particular, we note that disordered epitopes appear invariably to be linear epitopes, so the challenge of capturing conformational epitopes in a peptide vaccine may not apply. we then outline some of the opportunities and challenges presented by the peptidebased approach to vaccine development, as exemplified by efforts to develop a vaccine based on the disordered malaria antigen msp . finally, we speculate on future developments, within peptide science and beyond, that will more fully enable the promise of this approach to be realized. intrinsically disordered proteins are found throughout biology, [ ] and are particularly enriched in the context of molecular signaling and regulatory functions. [ ] consistent with this regulatory role, the extent of disorder within the proteome of individual species tends to increase with the environmental complexity of the organisms life-cycle. [ ] thus, organisms that live in diverse or highly variable environments tend to have highly disordered proteomes, reflecting the need for sophisticated regulatory mechanisms to survive such environmental diversity. as a result, disordered proteins are particularly abundant in a range of pathogenic organisms, and in some viruses, [ ] [ ] [ ] [ ] [ ] which must survive diverse and often hostile host environments. in these contexts, disordered proteins play important roles in host-pathogen interactions, [ ] and accordingly, disordered proteins are often exposed to the host immune system. [ ] even amongst bacteria, which have significantly more ordered proteomes than eukaryotic species, many proteins involved in pathogenic effector mechanisms appear to be disordered. [ ] it is often assumed that disordered antigens impede the development of an effective immune response, and hence that their abundance represents an adaptive mechanism enabling the pathogen to evade the immune response. [ ] [ ] [ ] [ ] [ ] mechanisms proposed to account for this immune evasion are diverse and contested, and we critically assess several of these mechanisms in section below. despite the fact that these hypotheses have received limited direct experimental support, the underlying assumption that repetitive or disordered antigens will fail to elicit an effective antibody response continues to enjoy significant currency. [ ] [ ] [ ] however, recent sequencing of the genomes of several species of parasite has demonstrated that low-complexity and disordered sequences are unusually abundant in a wide range of genes, including many that do not appear to be antigenically important. [ ] this suggests that the presence of such sequences in these parasites is not primarily a strategy to evade the host immune response. this has prompted significant debate surrounding the mechanisms responsible for generating and maintaining this abundance of low-complexity sequence. [ ] [ ] [ ] in fact, it seems likely that distinct classes of low-complexity sequences exist within parasite genomes, suggesting a diversity of contributory mechanisms. [ ] . | disordered antigens are important targets in vaccine development as noted above, the abundance of disordered and low-complexity sequences in the plasmodium species that are responsible for malaria and in related parasites was first identified through analysis of antigenically important proteins. it is not surprising, then, that a substantial fraction of malaria vaccine candidates contain regions of disorder, and that these regions are often important targets of antibodies. the most prominent example is the circumsporozoite protein (csp), the dominant antigen on the surface of the sporozoite, the parasite form that is injected into the host by the mosquito and which must replicate within host hepatocytes prior to the establishment of the symptomatic blood-stage infection. [ ] csp has been extensively studied as an antigen for inclusion in a malaria vaccine, and the recently-approved vaccine rts,s includes the c-terminal half of csp, fused to the hepatitis b s-antigen and assembled as a viruslike particle. [ ] the rts,s antigen includes the thrombospondin repeat domain at the c-terminus of csp, although it is not clear that this domain is correctly folded in the vaccine formulation. [ ] the rest of csp is predicted to be disordered, and these predictions have been confirmed experimentally for the tetra-peptide repeats of csp that appear to be immunodominant and are targets of protective antibodies induced by rts,s. [ ] [ ] [ ] [ ] a second important malaria vaccine candidate that is entirely disordered is merozoite surface protein (msp ). antibodies targeting msp arise in the course of malaria infection, and the presence of these antibodies correlates with protection from subsequent infection. [ ] [ ] [ ] msp also induces robust antibody responses when used as a vaccine, and these responses were shown to mediate protection in a phase iib trial in papua new guinea. [ , ] the conformational and antigenic properties of msp have been the subject of extensive study, [ ] [ ] [ ] [ ] providing important insights into the interplay between antigenicity and disorder, to which we will return in section below. many additional antigens of significant interest as vaccine candidates against malaria and other diseases are known or predicted to be disordered, and we highlight a number of examples in table . it is apparent, however, that in many cases the disordered nature of these antigens is not widely appreciated, raising the possibility that many important antigens are yet to be recognized as disordered. the preceding discussion has established that disordered proteins are important antigens in a broad range of immunological contexts. it would be natural to suppose, then, that antibodies are able to effectively recognize disordered antigens. on the other hand, we have also alluded to a number of arguments which suggest that the antibody response to disordered antigens will be dysfunctional or even absent. [ , , [ ] [ ] [ ] ] to address this apparent contradiction, we recently assembled a large dataset [ ] of well-characterized protein antigens from the immune epitope database (iedb). [ ] epitopes arising from protein regions predicted to be disordered are well represented in this dataset. in fact, we found a positive correlation between predicted disorder and the likelihood that any putative epitope will test positive in an antibody binding assay reported in the iedb, establishing that disorder is no impediment to effective antibody recognition ( figure a ). others have recently made the same observation, and extended it to show that predictors of protein disorder out-perform dedicated sequence-based predictors for the identification of b-cell epitopes. [ ] this may reflect the fact that residues that score highly in disorder predictors are more likely to be solvent-exposed, [ ] as solvent exposure is both a prerequisite for, and established predictor of, antigenicity. [ ] . | affinity maturation of antibodies to disordered antigens it has been argued that disordered antigens may impede the development of an effective immune response by interfering with the process by which b-cells mature and differentiate into memory or antibody producing cells. [ , , , ] in the process of this maturation, antibodies undergo somatic hypermutation and selection for high-affinity antigen binding. this affinity maturation is essential to the development of a high-affinity and long-lived antibody response. if the affinity maturation of antibodies to disordered antigens were significantly impeded, we would expect such antibodies to have fewer mutations with respect to germline antibody sequences than do antibodies to ordered antigens. in fact, when we compared the available antibody sequences to the corresponding germline sequences, we detected no difference in the number of v-gene mutations between the two classes of antibodies ( figure b) , implying that affinity maturation proceeds with equivalent efficacy for antibodies to ordered and disordered antigens. [ ] highly repetitive antigens are capable of eliciting b-cell responses that are independent of the t-cell help normally required for b-cell maturation. such responses are characterized by defective affinity maturation and a failure to establish a conventional memory response. [ ] as such, this has been proposed as one reason that b-cells to repetitive and disordered antigens may fail to mature. [ ] indeed, the repetitive malaria antigen csp can induce a b-cell response in the absence of tcell help, but this response is much weaker and more transient than that in the presence of t-cells. thus, the normal response to this prototypical repetitive antigen is t-cell dependent. [ ] fig ure disordered antigens are bona fide targets of antibody recognition. a, epitopes within disordered protein regions are more likely to be targets of positive antibody binding assays. b, antibodies to disordered epitopes (purple) and ordered epitopes (green) are subject to similar levels of somatic hypermutation. c, disorder accounts for only a small fraction of the variability in antibody affinity. d, disordered epitopes (purple) are exclusively short linear epitopes, while ordered epitopes (green) are predominantly conformational epitopes spanning many residues in the primary sequence. modified with permission from ref. additional specific evidence for affinity maturation in antibodies targeting this epitope comes from recent studies of the antibody responses induced in clinical trials of the rts,s vaccine, [ ] and in the context of natural infection. [ ] these studies have a further basis for the misconception that disordered proteins will be poorly recognized by antibodies arises from the fact that disordered epitopes, by definition, fail to adopt a single well-defined conformation in the context of the native protein that might be recognized by an antibody. it is clear, however, that disordered proteins are capable of being recognized by protein binding partners, antibodies or otherwise. to achieve this, disordered proteins often undergo a process of coupled folding and binding in which the binding partner acts effectively as a template into which the disordered protein can fold into a relatively well-defined bound conformation. [ ] nonetheless, coupled folding and binding places both kinetic and thermodynamic constraints on the interaction. [ ] in particular, the significant entropic cost of folding reduces the overall binding affinity of these interactions, unless this cost can be offset by other means. [ ] thus, the interactions of disordered proteins are often of relatively low affinity, despite maintaining high specificity. these properties are ideally suited to mediators of signals that must be switched on or off rapidly, accounting for the enrichment of disorder in proteins involved in signal transduction and regulation. [ ] these interactions are quite distinct, however, from typical antibody-antigen interactions, which are selected for high affinity and slow dissociation. these considerations raise the possibility that antibodies that recognize disordered antigens may do so with reduced affinity, reflecting the substantial entropic cost that must accompany the disordered antigen adopting a single antibody-bound conformation. [ ] [ ] [ ] in fact, using our dataset of ordered and disordered epitopes from the iedb, we have shown that disorder accounts for very little of the wide variation observed in the affinities of antibodies for protein antigens. [ ] specifically, we observed an approximately seven-fold difference in median affinity between antibodies that recognize ordered epitopes and those that recognize disordered epitopes, a loss of affinity that is very much smaller than might be expected on the basis of the expected entropic cost of coupled folding and binding ( figure c ). [ , ] to investigate the structural basis for the relatively high affinity with which antibodies recognize disordered antigens, we crossreferenced our iedb-derived dataset of ordered and disordered antigens with the protein databank (pdb), [ ] resulting in a set of structures of antibody-protein complexes, of which represent disordered epitopes. [ ] comparison of these structures revealed that disordered epitopes involve substantially fewer residues than ordered epitopes, perhaps as a means to minimize the entropic cost of binding ( figure d ). thus, disordered epitopes rarely span more than residues of the antigen primary sequence, and the majority of these residues make direct interactions with antibody. these structural observations are consistent with epitope mapping experiments with a large number of antibodies against several disordered antigens, [ ] which imply that disordered epitopes are overwhelmingly linear epitopes, and that antibody recognition is determined largely by the local antigen sequence. this stands in contrast to more conventional ordered antigens, in which a large fraction of the antibody response is typically directed to conformational epitopes. a further implication of the reduction in size of disordered epitopes is that disordered epitopes are much more efficient than ordered epitopes in their interactions with antibody, in the sense that they achieve more binding free energy per unit of interaction surface area, or per epitope residue. [ ] comparison of the structures of antibodybound ordered and disordered antigens reveals a number of features of the antibody-antigen interaction that may contribute to this efficiency, including a more concave antigen binding site with better complementarity to the shape of the antigen, as well as a two-fold increase in the number of inter-molecular h-bonds per epitope residue. these structural features imply that antibodies to disordered epitopes are better matched to the bound conformation of that epitope than are antibodies to ordered epitopes. it is likely that this reflects to some extent the ability of the disordered epitope to adapt to its binding site on the antibody. indeed, this flexibility permits some disordered epitopes to adopt distinct conformations bound to different antibodies. [ , , ] nonetheless, this exquisite complementarity is also likely to require adaptation on the part of the antibody. [ ] as such, it may also be seen as evidence to support the conclusion that affinity maturation of antibodies to disordered epitopes is at least as effective as is affinity maturation targeting ordered epitopes. we now place the preceding general considerations on a more specific footing, by outlining their implications in the context of vaccine development efforts against the disordered malaria antigen msp . [ , ] msp is a highly abundant, c-terminally glycosylphosphatidylinositol (gpi)-anchored blood-stage surface antigen of the malaria parasite p. falciparum and a potential component of a malaria vaccine. [ , [ ] [ ] [ ] msp has conserved n-and c-terminal regions, flanking a central region comprising polymorphic repetitive sequences and non-repetitive sequences that are predominantly dimorphic (figure ). these dimorphic sequences within the central variable region differentiate msp variants into two allelic families, d and fc msp . [ , ] the combination b vaccine, which included d msp as one of three protein antigens, yielded evidence for protection in a clinical trial in papua new guinea, but this protection was strain-specific, with vaccines not being protected from malarial strains expressing fc -family msp sequences. [ ] thus, a primary concern for vaccine development against msp , like many other malaria antigens, has been to address the polymorphism in the antigen. [ ] although the studies described above have established that disorder is no impediment to the development of a robust antibody response, the interplay between disorder and antigenicity may be more complicated at the level of a single antigen. in our studies of msp , we have compared experimental measures of conformational disorder derived from nmr relaxation rates with observed patterns of antigenicity inferred from experimental immunizations of animals and humans. [ ] the result of that study, in contrast to the bioinformatic analysis, [ , ] a key advantage afforded by peptide-based approaches to vaccine development is the ability to omit ineffective regions such as these polymorphic regions entirely, ensuring that the response is focused on conserved epitopes that are more likely to elicit broadly protective responses. this is exemplified by the long synthetic peptides derived from the c-terminal half of msp , which have been considered potential components of a malaria vaccine. [ , ] these peptides include both conserved and dimorphic family-specific sequences, but exclude the highly polymorphic regions of msp . beyond the specificity of the immediate epitope-antibody interaction, the broader context in which the antibody encounters the antigen is likely to influence the way in which antibodies are able to recognize disordered antigens. such effects are well-known in the case of ordered antigens, and can arise when, for example, molecular interactions lead to conformational changes that mask or disrupt epitopes or otherwise render them cryptic. [ , ] similar effects are likely in disordered antigens; indeed, the conformational properties of disordered proteins are uniquely sensitive to modulation by interactions, posttranslational modifications and other mechanisms. as such, antibody recognition of disordered epitopes may be expected to be particularly sensitive to the context in which the antibody is recognized. an example of this is seen in the masking of conserved epitopes in msp . amongst a panel of monoclonal mouse antibodies raised against recombinant msp , all of those targeting variable epitopes also recognized msp on the parasite surface, whereas many antibodies targeting conserved epitopes recognized the parasites weakly or not at all ( figure ). [ ] together, these data imply that at least some conserved epitopes within msp may be masked by the conformations or interactions adopted by the antigen on the parasite surface. the structural basis of this masking has recently been characterized in the case of one such epitope. d , a murine monoclonal antibody (mab) recognizing an epitope in the conserved n-terminal region binds recombinant msp and epitope-derived peptides with high affinity, yet it fails to recognize the parasite surface. [ ] the n-terminal region of msp , including the d epitope, binds lipids, adopting an amphipathic helical conformation that is expected to be heavily populated in parasite msp given that it is gpi-anchored to the parasite membrane. [ ] this helical conformation is incompatible with the conformation that is recognized by d , as revealed by x-ray crystallography ( figure a,b) . [ ] moreover, the d epitope is lost in recombinant msp when the protein is c-terminally tethered to lipid vesicles, [ ] indicating that the n-terminal interaction with lipid and the accompanying conformational change are sufficient to account for the observed masking of the d epitope on the parasite surface. to explore the impact of lipid conjugation on the c-terminal domain of msp , a shorter construct of residues, msp - , consisting of only the conserved c-terminal region, was generated. [ ] our the epitopes of a panel of monoclonal antibodies are also shown, with antibodies that strongly recognize the parasite antigen in blue text, and those that do so only weakly or not at all in red that many residues in msp - interact with lipid, including some in epitopes recognized by c-terminal region specific monoclonal antibodies (mabs). in contrast to the n-terminal region however, there is no indication of stable helical conformation within lipid-bound msp - . [ , ] nonetheless, the antigenicity of liposome-conjugated msp - and full-length msp are altered in comparison to the lipid-free recombinant peptides ( figure c ). importantly, it also appears that the binding pattern of c-terminal specific mabs for liposome-conjugated antigens ( figure d) is consistent with that of the mabs for parasite lysate. thus, lipid tethering appears to alter the antigenic state of these msp constructs, rendering it more similar to that of the antigen on the parasite surface. lipidconjugation of msp based vaccines may therefore provide a more effective antibody response. similar masking of potentially protective epitopes is seen in the preerythrocytic malaria antigen csp. although the repetitive sequences in csp have long been the focus of vaccine development efforts, recent studies have identified epitopes within the conserved n-terminal region that appear to contribute to the protective response to csp in the context of natural immunity and vaccination. [ ] [ ] [ ] these antibodies act synergistically with antibodies targeting the csp repeats. [ ] in the course of parasite development within the mosquito and subsequent migration through the human host to the liver, csp undergoes proteolytic processing, which is essential for the establishment of a liver-stage infection. this proteolysis exposes otherwise cryptic epitopes in both the n-and c-terminal regions of csp, indicating conformational changes that appear to involve long-range interactions within csp, which in turn determine the accessibility of some of these n-terminal epitopes. [ , , ] the nature of these interactions and the associated conformational changes remain largely obscure. in light of the efficacy of antibodies targeting n-terminal and possibly also c-terminal epitopes, it is likely that a better understanding of these interactions will facilitate the design of improved csp-based vaccine constructs. disordered proteins typically adopt a well-defined structure when interacting with binding partners, as described above. nonetheless, in some cases significant disorder persists in these interactions, giving rise to what are known as "fuzzy interactions." [ ] it is becoming increas- [ ] and b, d mab-bound msp [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (pdb id qyo); [ ] key d paratope residues involved in binding are shown in white. the a-helical configuration of the lipid-bound peptide removes the backbone flexibility required for arg to access tyr , which provides a structural rationale for d epitope masking at the parasite membrane. c, schematic of lipid tethering of c-terminal region of msp (msp - ) where msp - was synthesized with a c-terminal his -tag to immobilize on nickel bound to nickel-chelating lipid. d, elisa showing the effects of lipid tethering on the binding of four c-terminal region-specific mouse mabs for msp - . [ ] five fold. [ ] nmr experiments provide evidence of transient interactions that are not resolved in the crystal structure and which involve variable residues that are distal to the structurally defined epitope. these transient or fuzzy interactions thus appear to modulate the specificity of d , rendering the antibody somewhat strain-specific in spite of the conserved nature of its epitope. [ , ] although the generality of this effect remains to be established, it appears to originate from largely non-specific interactions that occur, almost inevitably, as a result of the high effective concentration of the disordered regions that immediately flank the structurally defined epitope. [ ] as such, it is expected that similar fuzzy interactions may contribute to antibody recognition of many disordered antigens. the conserved c-terminal region of msp is particularly immunodominant, with a total of five mouse mabs able to recognize an overlapping epitope spanning msp - . [ ] as discussed above, antibodies targeting the conserved regions have shown differing ability to recognize parasite msp (figure [ ] upon antibody binding, the -residue peptide epitope was found to adopt a b-bend ribbon conformation, characterized by consecutive overlapping b-turns ( figure ). the two b-turns in the epitope were stabilized by hydrogen bonds involving asn -asn and glu -gly , and allowed key interactions of residues lys , glu , and asn with the d paratope. this structural information suggests strategies by which stabilized analogues of the epitope could be optimized for use as a peptide vaccine, with a view to guiding the specificity of the antibody response towards d -like epitopes that are both conserved and accessible on the parasite surface, and thus are more likely to contribute to a broadly protective immune response. disordered proteins are highly abundant in infectious organisms across all kingdoms of life, and these proteins are bona fide targets of antibody responses in the context of natural infection and vaccination. [ ] in contrast to widely-held views that disordered antigens will fail to elicit an effective antibody response, [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] it is clear that they play an important role as protective antigens in a range of diseases (table ) . epitopes from disordered antigens are typically short linear epitopes, not the more complex conformational epitopes dominant in ordered antigens. as such, disordered antigens are well suited as targets of peptide vaccines. despite the potential promise of this approach, careful consideration must be paid to the context in which target epitopes are recognized by the immune system. the conformational and antigenic properties of disordered antigens can be strongly modulated by their interactions, both in their native parasite context and in any vaccine formulation. mismatch between the parasite and vaccine context has probably contributed to the failure of a number of peptide-based vaccine strategies. [ ] the use of long peptides in a vaccine formulation, potentially incorporating several protective epitopes within their native sequence, may offer one strategy to address this issue. [ , ] potentially a more effective approach will be to define the structures of disordered peptide epitopes bound to their cognate antibodies, and to use this structural information as a basis for design of peptides in which these structures are stabilized. to this end, the range of available strategies for stabilizing the conformation of synthetic peptides represents a clear advantage to the peptide vaccine approach. future developments in this area, enabling the stabilization of a wider range of peptide conformations while limiting perturbation of residues essential for antibody binding, will serve to broaden the scope of the strategy further. it is clear that the targeted vaccine development strategy described here requires a detailed understanding of the functional determinants of immune protection, and of the specific antigens and epitopes responsible for mediating these functions. while this is well understood for some pathogens, for other less studied or immunologically complex pathogens, such information may still be emerging. in this context, new strategies for high-throughput profiling of immunological responses and identification of correlates of immune protection [ ] represent a welcome advance that promises to allow peptidebased vaccine development against diseases for which no effective vaccine currently exists. proc. natl. acad. sci proc. natl. acad. sci sosnick proc. natl. acad. sci proc. natl. acad. sci key: cord- - asegs authors: yang, wenmian; gao, wenyuan; zhou, xiaojie; jia, weijia; zhang, shaohua; luo, yutao title: herding effect based attention for personalized time-sync video recommendation date: - - journal: nan doi: . /icme. . sha: doc_id: cord_uid: asegs time-sync comment (tsc) is a new form of user-interaction review associated with real-time video contents, which contains a user's preferences for videos and therefore well suited as the data source for video recommendations. however, existing review-based recommendation methods ignore the context-dependent (generated by user-interaction), real-time, and time-sensitive properties of tsc data. to bridge the above gaps, in this paper, we use video images and users' tscs to design an image-text fusion model with a novel herding effect attention mechanism (called itf-hea), which can predict users' favorite videos with model-based collaborative filtering. specifically, in the hea mechanism, we weight the context information based on the semantic similarities and time intervals between each tsc and its context, thereby considering influences of the herding effect in the model. experiments show that itf-hea is on average . % higher than the state-of-the-art method upon f -score in baselines. recently, watching online videos of news and amusement has become mainstream entertainment during people's leisure time. therefore, efficient and accurate personalized video recommendation methods bring significant convenience to their life. most of the video recommendation methods focus on users' behaviors such as their browsing history [ , ] and reviews [ , ] . in real scenarios, however, most people are unwilling to do high-quality reviews after watching videos, which causes the scarcity of valuable video reviews. furthermore, some multi-feature based methods [ , ] combine image information with review information to generate users' interests from a more comprehensive perspective. however, their methods have only achieved limited improvement because the images and reviews usually contain unequal information [ ] . that is, the text information and image information generally describe the different amount of contents. an image in a video only describes one moment of the video content, while a review usually describes the overall contents of the video. the information gap causes the fusion of reviews and images to lose great information. meanwhile, a new form of user-interactive review -timesync comment (tsc) (first introduced by wu et al. [ ] , see fig. ) has become increasingly popular in china and japan, especially among young people. nowadays, many popular chinese video websites such as youku (http://www.youku.com), bilibili (http://bilibili.tv) and the japanese video website niconico (http://www.nicovideo.jp) support the tsc. tscs convey information involving the content of the current video frame, feelings of users or replies to other tscs, which can accurately express the users' preferences for the video. moreover, each tsc has a corresponding timestamp to record the posted time. compared with traditional video reviews, tscs are much easier to obtain their corresponding images by timestamps. the users' real-time feedbacks and the vast amount make tscs valuable and accessible sources for personalized video recommendations. in this paper, we focus on mining the users' preferences and videos' features from tscs and corresponding images to recommend videos towards users through model-based collaborative filtering (cf). tscs have several features distinguished from the traditional video reviews: ( )context-dependent. tscs are usually context-dependent, i.e., the latter comments often depend on the former ones. this phenomenon is known as the herding effect in social science [ , ] . an example of the herding effect is shown in fig. . user a said "i love the male commander!" to express his love to the role male commander when he appears in the video. after a few seconds, user b and user c followed up by saying "i like the male commander too..." and "i am so sad when he died." in this case, user b and user c may not make their comments if user a has not. that is, the emergence of a tsc is usually not independent, but a probability event influenced by other preorder comments. ( )real-time. each tsc has a timestamp synchronous to the playback time of the video. the coverage of a tsc is usually only a short time before its timestamp. therefore, the content of each tsc is closely related to the video content corresponding to its timestamp, which makes it easy to sample corresponding image information by timestamp. ( )timesensitive. according to our observation, tscs with a large interval of timestamps are unlikely to discuss the same topic, even if their semantics are similar. users are more likely to follow those newer tscs than older ones. as a result, the herding effect mentioned before will not last long. these features make tsc a particular review. however, most of the current tsc-based recommendations [ , ] assume that tscs are independent of each other and ignore the time information. such assumption ignores the above features of tscs which causes the loss of crucial semantic information and affects the accuracy of results. therefore, how to take tscs' features of context-dependent (herding effect), real-time and time-sensitive into account to extract the textual information and fuse it with visual information accurately and effectively are the central challenges. based on the above motivations and challenges, we propose an image-text fusion model with a novel herding effect attention mechanism (called itf-hea). in itf-hea, we generate users' preferences and summarize video contents through model-based cf. to analyze the influence of text information, image information and contextual information separately, we split itf-hea into two models: text-based model(tm) and image-text fusion model (itf), and one attention mechanism: herding effect attention (hea) mechanism. specifically, in tm, we sample and embed the tscs to obtain the sentence vectors (tsc features) by bidirectional long short-term memory (lstm) at first, and then combine tsc features with the hidden (embedding) features of the users and videos respectively to predict the likeness of the user to the video. in itf, we sample the corresponding video frame (image) features and incorporate them with tsc features to replace single tsc features in tm. finally, we design the hea mechanism which is based on contextual semantic similarity and time interval of tscs to incorporate contextual information into tsc features to replace the features in tm and itf. the main contributions of this paper are as follows: we propose a novel hea mechanism, which takes tscs' features of contextual relevance, real time and timeliness into account, to extract the textual features of the tscs more accurately and effectively. we design an image-text fusion model using model-based cf, combine it with hea mechanism and get the itf-hea, which can predict the likeness of the user to the video more accurately and sufficiently. we evaluate the itf-hea with real-world datasets on mainstream video-sharing websites and compare it with state-ofthe-art video recommendation methods. the results show that our model outperforms baselines in both precision and f score on average . % and . % respectively. in this section, we discuss the related work in three aspects. time-sync video comments are first introduced by wu et al. [ ] . then, yang et al. [ ] sum up the features of tscs, which inspire our work. lv et al. [ ] propose a video understanding framework to assign temporal labels to highlighted video shots. they are the first to analyze the tscs using the neural network. recently, liao et al. [ ] present a larger-scale tsc dataset with four-level structures and rich self-labeled attributes, which brings convenience for future research on tscs. the above methods show that tsc is a kind of data with great potential and development value. video recommendation has attracted great attention from both the industry and academia. most of the current state-of-the-art methods are based on cf. mcauley et al. [ ] combine latent rating dimensions with latent review topics, which is a review-based method. diao et al. [ ] propose a probabilistic model based on cf and topic modeling, which is an lda [ ] based method and allows capturing the interest distribution of users and the content distribution of movies. he et al. [ ] propose a scalable factorization model to incorporate visual signals into predictors of people's opinions, which is the state-of-the-art visual-based model. however, the above recommendation methods are not well-designed for tscs as they ignore the interactive, real-time, and timeliness properties of tsc data. attention mechanism has been shown effective in natural language processing [ , , ] . recently, attention models have been used increasingly in recommendation systems to assign weights to user-item pairs. chen et al. [ ] introduce a novel attention mechanism in cf to address the challenging item and component-level implicit feedback in the multimedia recommendation, which can be seamlessly incorporated into classic cf models with implicit feedback. seo et al. [ ] propose to model user preferences and item properties using convolutional neural networks (cnn) with dual local and global attention. our herding effect attention mechanism adopts the soft attention [ ] , which learns the attentive weights based on the importance to the final task. in this section, we describe two cf models and an attention mechanism. first, the problem formulation is provided in section . . then, we propose a text-based model by using textual features of tscs in section . . next, we design an image-text fusion model to jointly model video images as well as tscs in section . . finally, to take full consideration of the features of tscs, we implement herding effect attention mechanism and give the complete neural network structure of the image-text fusion model with herding effect attention in section . . suppose there are n tscs, t sc = {tsc , tsc , ..., tscn }. for tsci, we define the corresponding visual feature as vsli (see section . for details) and sentiment polarity poli which is determined by the stanford sentiment analysis toolkit (http://nlp.stanford.edu/sentiment). besides, we define ui to represent the user id and vi to express the video id of tsci. as mentioned in section , tscs are easily affected by previous comments. therefore, for tsci, we continuously sample m preorder tscs contexti = {prei, , prei, , ..., prei,m } as context information (prei,m is tsci itself). for each prei,j ∈ contexti, we define the time-stamp ti,j to represent its posted video time. the word list of tsci and its context information prei,j are defined as intuitively, the preference of users is extracted from their published tscs in corresponding videos, while the textual features of the videos are summed up from the tscs published in the video. based on above, in this section, we first extract the features of tscs by bidirectional lstm. then, features of tscs are merged with the latent factors of users and videos. finally the likenesses of the users to videos are predicted by cf. the general framework of text-based model (tm) is shown in fig. . more concretely, to capture the word sequential information from tscs, we use the bidirectional long short-term memory (bi-lstm) network [ ] to convert word features into tsc features. for each tsci, we have and where seqi ∈ r d and ⊕ denotes vector concatenation. after lstm layer, we get the sequence feature seqi as the output. then, we define guu i as the latent factor of user ui, which is the feature based on user's historical preference. likewise, the feature of video vi is defined as gvv i . afterward, we design ⊗ function to merge guu i and gvv i with seqi respectively, and obtain pi = gu i ⊗ seqi ( ) and where ⊗: in our framework, we take the prediction of a user's favor to a video (or video clip) as a binary classification problem, where means a user likes the video, and otherwise. therefore, we define the likeness of user ui to video vi though tsci in the training data aŝ where sigmoid(x) = +e −x and " " denotes inner product. generally, users comment on their favorite videos with positive sentiment. therefore, we determine the polarity of each tsc by the stanford sentiment analysis toolkit [ ] . for simplicity, we set the polarity of each tsc as if the result is positive or neutral, and otherwise. we define yi = poli as the ground truth of the likeness of user ui for video vi through tsci, where poli is the polarity of tsci. at last, we use the binary cross-entropy as our loss function to model user preference. the final objective function is maximized as: in the training phase, the parameters can be learned via adam [ ] . after trainning, we usê to express the predicted likeness of user u on video v, and to express the real likeness of user u for video v, where listu,v expresses the total tscs that user u has commented on video v. then, the ground truth of testing data is defined as yu,v = p ou,v < . p ou,v ≥ . ( ) in the time-sync video, each tsc has a timestamp that records the corresponding video time when the tsc is published. so that, we can easily obtain the corresponding image information for better feature extraction. in this section, we focus on merging tsc text features with corresponding visual features to obtain more comprehensive features. the general framework of image-text fusion model (itf) is shown in fig. . for tsci, we use vsli to indicate the visual feature when tsci is posted. the visual features are with the output of -way obtained from a public tsc data set extracted by chen et al. [ ] , which are trained by the caffe reference model with convolutional layers followed by fully-connected layers that have been pre-trained on . million imagenet (ilsvrc ) images. since the dimension of vsli is , we reduce its dimension to d and get vsl i by where dense is the fully-connected layer with the activation function elu [ ] . to combine the image features and textual features, we first concatenate the sequence feature seqi with the visual feature vsl i , and obtain the × d-dimensional vector comi: then, we reduce the demension of comi to d and get the com i by finally, we use com i instead of seqi to merge with guu i and gvv i by eq.( ) and eq.( ) and predict the likeness by eq.( ). existing review-based recommendation methods usually handle each comment separately without considering the context associations between the comments. however, tscs are highly semantic relevant and time-related, which is so-called the herding effect. that is, tscs may be affected by other preorder tscs on the similar topic. also, tscs with similar semantics and the short interval of the time-stamp are more likely to influence each other. based fig. . herding effect attention on above, we design an hea mechanism, which calculates the influence weights of tsc contexts by their semantic similarities and timestamp intervals in an lstm-based encoder-decoder framework. the framework of hea is shown in fig. . we formalize the hea into an encoder-decoder framework. given context features seqi as the input to the lstm, the output are obtained as: we use h = (h , h , ..., hm ) to express the hidden status vectors of the encoder output. to calculate the influence weights of contexts of tscs, for prei,j, we define the semantic similarity vector and time delay vector as simj = (sim(j, ), sim(j, ), ..., sim(j, m )) and t dj = (delay(j, ), delay(j, ), ..., delay(j, m )) , where sim(k, j) = seqi,k seqi,j |seqi,k||seqi,j| ( ) represents the semantic similarity between prei,j and pre i,k , and represents the influence of pre i,k on prei,j decreases with the increasing time interval (β is a hyper-parameter that will be discussed in section ). since the semantic similarity may have negative numbers, we first normalize simj by softmax as: next, we calculate the attention score vector of prei,j as: the final attention score distribution aj is obtained by normalizing the attention score vector aj by softmax function. we compute the input of decoder as: and get the output as: finally, we use hm instead of seqi that we used in section . and . as the textual feature. we integrate the context-dependent, real-time and time-sensitive properties of the tscs into the model by the hea mechanism, which can be applied in both tm and itf to improve the tsc feature extraction. the complete network structure of the itf-hea is shown in fig. in this section, we demonstrate the effectiveness of our proposed method by comparing with well-known methods of video recommendation. we provide necessary parameters of our model at first and then analyze the performance of our model on time-sync video recommendation. finally, we analyze the effect of the hyperparameters on the experimental results. the data used in this paper are crawled from a chinese time-sync video site bilibili by chen et al. [ ] , which are obtained from the movie category till december th, . in this paper, we select users who have posted the most tscs and commented on more than videos. these users have commented on a total of videos, and we select all the comments in those videos as a subdataset. in the sub-dataset, , users have published , , tscs in total. for each of the users, we select half of the videos where they have commented as the training set, and the other half as the test set. we make sure that at least videos per user can be recommended (to ensure the effectiveness of top ). in the test set, we get , (user, video) pairs, where , pairs are positive, and , pairs are negative in sentiment polarity. in the training set, we obtain , (user, video) pairs with , tscs (a user may make more than one tsc in a video), where , tscs are positive and , tscs are negative. in our model, hyper-parameter β and number of contextual tscs m need to be decided. we select % data of the test set (actually , (user, video) pairs) as the validation set to tune β. the initial learning rate of adam [ ] is . and the vector dimension d is set as . we get the best results when β = . , and m = , which are discussed in section . . in this section, we use the test set described in section . to compare our complete model with existing methods. to evaluate the performance of the proposed models, we compare our model with the following methods as baselines: • hft: a state-of-the-art method regarding making rating prediction with textual reviews [ ] . in the experiments, we set the ratings of positively commented videos as , and otherwise. • jmars: a latent dirichlet allocation (lda) based method to make rating prediction with textual reviews [ ] . • vbpr: a visual-based recommendation method [ ] . • kfrci: a novel key frame recommender by modeling user tscs and keyframe images simultaneously [ ] . in the experiments, the likeness score of the video is the average score of all the frames the users have commented on. • itf-hea: the image-text fusion model proposed in section . with herding effect attention mechanism proposed in section . . for each method in the baselines, we select a set of the best experimental parameters according to the range of the parameters given in their experiments and calculate the likenesses/ratings between users and videos. our experiments are conducted by predicting top , , and favorite videos respectively. the top x is the top prediction of user's likeness to the videos in test set calculated by eq. ( ) . we recommend all x videos to each user and consider these are the user's favorite videos. we adopt f -score and precision to evaluate the performance of the baselines and our models. all the models are repeated for times, and we report the average values as the final results for clear illustration. the results of f -score and precision are shown in table . from table , we can see: itf-hea achieves the best performance on f and precision (f is proportional to precision in the top , and ). it has enhanced the performance by about . %, . % and . % (on average . %) upon f -score and . %, . % and . % (on average . %) upon precision on top , and respectively compared with kfrci, which performs best among baselines. in other methods of baselines, the vision-based method vbpr has better performance than the others; the text-based method hft and jmars have similar performance, while the pmf method has the worst. next, we compare the models proposed in section : • tm: the text-based model proposed in section . . • t-hea: the text-based model proposed in section . with herding effect attention mechanism proposed in section . . • itf: the image-text fusion model proposed in section . . • itf-hea: the image-text fusion model proposed in section . with herding effect attention mechanism proposed in section . . to analyze the effects of text features, image features and the attention mechanism in our model. the results of f -score and precision are shown in table . the results show that although t-hea only uses the textual information, the experimental results are still better than itf. t-hea even has better performance than the state-of-the-art method kfrci, which shows that our hea mechanism can effectively offset the effects of the herding effect and improve the performance of the model. the results also show that the context and timestamp of the tscs are vital information and need to be considered. finally, we discuss the influence of hyper-parameter β and the number of contextual tscs m on the experimental results. we fix m = , changing the value of β from to . (the step size is . ), and calculate the f -score of top , and users' favorite videos in the validation set at first. the best results are obtained when β = . . we also calculate the f -score for the different hyper-parameters in the test set, and the results are shown in fig. . the hyper-parameter β gains the best performance when β = . in any case, which is the same with the validation set. when β is bigger, the result of the experiment is worse because it weakens the weight of other tscs in the attention layer. when β = , it has the worst performance, because the time information is not considered. for the number of context length m , we fix β = . , and set m as , , and , respectively. the results are shown in fig. . ift-hea gets the best performance when m = and the worst when m = . this result confirms that the herding effect of tscs is time-sensitive and will not last long, which meets our observation in section . in this paper, we proposed a novel personalized online video recommendation with the dataset of both tscs and its corresponding images through model-based cf method. to extract the textual features of the tscs more accurately and effectively, we designed the hea mechanism to add influence weight to each tsc based on their semantic similarity and time interval. in this way, we integrated the context-dependent, real-time and time-sensitive properties of tscs in the neural network framework and predicted the users' preferences for online videos accurately and effectively. extensive experiments on real-world dataset proved that our model could recommend videos to users more precisely than the state-of-the-art method with the hea mechanism. this is the first step towards our goal in personalized video recommendation, and there is much space for further improvements. for example, to design a more accurate fusion model to capture comprehensive user preference is a challenging problem. besides, how to measure the weight of each video to the user's preference is also a challenging problem. when recurrent neural networks meet the neighborhood for session-based recommendation exploring the use of time-dependent cross-network information for personalized recommendations personalized key frame recommendation aspect based recommendations: recommending items with the most valuable aspects based on user reviews a unified personalized video recommendation via dynamic recurrent neural networks contextual video recommendation by multimodal relevance and user feedback multimodal neural language models crowdsourced time-sync video tagging using temporal and personalized topic modeling predicting the popularity of danmu-enabled videos: a multifactor view tradeoff between distributed social learning and herding effect in online rating systems: evidence from a real-world intervention video recommendation using crowdsourced timesync comments crowdsourced time-sync video tagging using semantic association graph reading the videos: temporal labeling for crowdsourced time-sync videos based on semantic embedding tscset: a crowdsourced time-sync comment dataset for exploration of user experience improvement hidden factors and hidden topics: understanding rating dimensions with review text jointly modeling aspects, ratings and sentiments for movie recommendation (jmars) latent dirichlet allocation vbpr: visual bayesian personalized ranking from implicit feedback attention-fused deep matching network for natural language inference translating embeddings for knowledge graph completion with relation attention mechanism neural relation extraction via inner-sentence noise reduction and transfer learning attentive collaborative filtering: multimedia recommendation with item-and component-level attention interpretable convolutional neural networks with dual local and global attention for review rating prediction attention is all you need bidirectional recurrent neural networks the stanford corenlp natural language processing toolkit adam: a method for stochastic optimization fast and accurate deep network learning by exponential linear units (elus) key: cord- -zhlvvuj authors: nordén, rickard; magnusson, jesper; lundin, anna; tang, ka-wei; nilsson, staffan; lindh, magnus; andersson, lars-magnus; riise, gerdt c; westin, johan title: quantification of torque teno virus and epstein-barr virus is of limited value for predicting the net state of immunosuppression after lung transplantation date: - - journal: open forum infect dis doi: . /ofid/ofy sha: doc_id: cord_uid: zhlvvuj background: major hurdles for survival after lung transplantation are rejections and infectious complications. adequate methods for monitoring immune suppression status are lacking. here, we evaluated quantification of torque teno virus (ttv) and epstein-barr virus (ebv) as biomarkers for defining the net state of immunosuppression in lung-transplanted patients. methods: this prospective single-center study included patients followed for years after transplantation. bacterial infections, fungal infections, viral respiratory infections (vrti), cytomegalovirus (cmv) viremia, and acute rejections, as well as ttv and ebv levels, were monitored. results: the levels of torque teno virus dna increased rapidly after transplantation, likely due to immunosuppressive treatment. a modest increase in levels of epstein-barr virus dna was also observed after transplantation. there were no associations between either ttv or ebv and infectious events or acute rejection, respectively, during follow-up. when tacrolimus was the main immunosuppressive treatment, ttv dna levels were significantly elevated – months after transplantation as compared with cyclosporine treatment. conclusions: although replication of ttv, but not ebv, appears to reflect the functionality of the immune system, depending on the type of immunosuppressive treatment, quantification of ttv or ebv as biomarkers has limited potential for defining the net state of immune suppression. for patients with end-stage lung disease, limited life expectancy, and otherwise acceptable physical condition, lung transplantation may be a life-saving therapeutic option. the -year survival rate is only approximately % in most centers, and major limiting factors for the outcome of lung transplantation are the frequent occurrence of infectious complications and rejection of the transplanted organ [ ] . standard immunosuppression, to prevent graft rejection, consists of a combination of immunosuppressive drugs that subvert b and t cells [ ] [ ] [ ] . currently, there is a lack of an easy-to-use marker that accurately predicts the net effect of the combined immunosuppression. such a biomarker would be an important tool in the management of patients with compromised immune function to identify overor underimmunosuppression and evaluate the risk of infectious complications and acute rejections. viruses are often only viewed as pathogenic entities, but there are several examples of viruses that are frequently found in healthy individuals without causing obvious disease [ , ] . torque teno virus (ttv) is a human dna virus that causes asymptomatic viremia with a high prevalence in the general population [ ] [ ] [ ] [ ] [ ] [ ] [ ] . cell-mediated immunity is considered important in controlling ttv infection [ ] [ ] [ ] [ ] . accordingly, serum levels of ttv-dna increase dramatically in patients who have undergone solid organ transplantation, presumably as a result of immunosuppression [ , , ] . epstein-barr virus (ebv) is another dna virus, commonly acquired early in life, with a high prevalence in us adults [ ] , that establishes latent infection in memory b cells [ , ] . the levels of ebv often increase in blood after immunosuppressive treatment, and ebv can cause post-transplant lymphoproliferative disorder (ptld) when the immune system is repressed. hence, both ttv and ebv have previously been suggested as surrogate markers of the net state of immunosuppression [ , , ] . the aim of the present study was to evaluate levels of ttv and ebv in relation to the frequency of infectious events and acute rejections over time in a prospective manner in a single-center cohort of lung-transplanted patients. all patients over the age of years who received a single or double lung transplant, who survived the initial postoperative intensive care period and remained in gothenburg during the follow-up period, between january , , and april , , at the sahlgrenska university hospital transplant centre, gothenburg, sweden, were asked to participate in this prospective single-center cohort study. all included subjects underwent follow-up (fu) visits at , , , . , , , , , and months after transplantation. at every fu visit, whole blood, serum, and nasopharyngeal samples (nph) were collected. protocol bronchoscopies were performed at , , and months post-transplantation and when indicated based on clinical presentation. bronchoalveolar lavage (bal) samples were collected at every bronchoscopy. all bal samples underwent direct microscopy for early detection of aspergillus hyphae and were cultured for bacteria and fungi and tested for legionella pneumophila, pneumocystis jirovecii, and human cytomegalovirus (cmv) using real-time polymerase chain reaction (pcr). bal samples from patients with cystic fibrosis were routinely cultured for mycobacterium tuberculosis and atypical mycobacteria. bacterial and fungal culture testing from blood, urine, and sputum was performed upon suspicion of infection. nph and bal samples from every fu visit, as well as additional nph samples obtained upon suspicion of airway infection between scheduled fu visits, were analyzed by multiplex real-time pcr for detection of airway pathogens. serum samples obtained at pretransplant evaluation were used for ttv-dna and ebv-dna quantification. for quantification of ttv and ebv during fu, serum and whole blood samples were used, respectively. induction therapy consisted of rabbit antithymocyte globulin, which was given for - consecutive days together with methylprednisolone intravenously. post-transplantation immunosuppression included prednisone . mg/kg/d and mycophenolate mofetil (mmf) g/d. the patients then received either oral cyclosporine (csa; - mg/kg), adjusted to maintain a serum level of - ng/ml, or tacrolimus (tac; . mg/kg) given orally divided in doses daily, adjusted to maintain a serum level of - ng/ml. the choice between csa and tac was made based on clinical presentation. patients previously treated with tac and patients with cystic fibrosis were given tac. there was also a preference for tac if the recipient was younger. the dosage of immunosuppression was gradually lowered during fu. further changes in immunosuppressive therapy were based on clinical presentation. four patients, who either underwent retransplantation or had previous immunosuppressive treatment due to autoimmune conditions, were given nonstandard immunosuppression therapy based on previous exposure to immunosuppressive drugs. all patients received a combination of mg of sulfametoxazol and mg of trimetoprim thrice weekly to prevent pneumocystis jirovecii infection. patients seropositive for cmv at the pretransplant evaluation were given valganciclovir at a dose of mg daily for months. patients seronegative for cmv pretransplant while the organ donor tested positive (ie, cmv mismatches) were given the same treatment for months. bacterial infection was defined as a positive bacterial culture in conjunction with symptoms of clinical infection or, in the absence of positive culture, symptoms consistent with bacterial infection that was treated with antibiotics. fungal infection was defined as significant presence of fungi in culture from a sterile location in conjunction with symptoms of infection according to the european organization for research and treatment of cancer (eortc) criteria [ ] . vrti or mycoplasma pneumoniae infection was defined as detection of a pathogen by multiplex real-time pcr in nph or bal. cmv viremia was defined as elevated levels of cmv-dna that prompted antiviral therapy. cmv-seronegative recipients were considered to have viremia if cmv-dna was detected, whereas recipients seropositive for cmv prior to transplantation where considered to have cmv viremia only if the level was above . log ( ) copies/ml. to distinguish milder infections from more severe infectious events, a subgroup of events requiring initial intravenous antimicrobial (antibacterial, antiviral, or antifungal) therapy were defined as severe. acute rejection was defined as either a lung biopsy showing rejection of ishlt grade a or higher [ ] or, in the absence of a biopsy, physical (increased need for oxygen) and radiological findings (progressive infiltrate without signs of infection) consistent with acute rejection followed by a prompt response to high-dose ( g/d for consecutive days) corticosteroid therapy (methylprednisolone). nucleic acid isolation was performed with a magna pure lc total nucleic acid or dna isolation kit for serum (ttv) or whole blood (ebv and cmv) using a standardized protocol, according to the instructions (roche diagnostics, mannheim, germany). input/output volume was set to / µl, and the sample was eluted in extraction buffer. the ttv-dna levels were determined using a real-time pcr system (applied biosystems, foster city, ca). each pcr reaction contained µl of extracted total nucleic acid, µl of x universal master mix (applied biosystems, foster city, ca), . µl and µm of forward and reverse primer, respectively, . µl and µm of bhq hydrolysis probe, and µl of rnase-free h o. the reaction conditions were °c for minutes and °c for minutes prior to cycles at °c for seconds and °c for seconds. the assay range was determined by serial dilution of plasmids with an insert of a synthesized sequence matching the ttv pcr product, and quantification was obtained from a plot of ct values. cmv-and ebv-dna levels were determined using a method described previously with minor modifications [ ] . all primer and probe sequences are listed in supplementary table . for ebv and cmv quantification, a control sample consisting of unrelated phocine herpesvirus (phhv- ) was included prior to nucleic acid extraction. ttv load was quantified on the qx droplet digital pcr system using the ddpcr supermix for probes (no dutp; bio-rad, hercules, ca). primer and probe concentrations in the -µl final reaction were and nm, respectively. the pcr conditions were °c for minutes followed by °c for minutes, then seconds at °c and minute at °c for a total of cycles, and finally a step at °c for minutes. after overnight incubation at °c, the droplet fluorescence signal was determined. data analysis was done using bio-rad quanta soft analysis software. isolated dna and rna were analyzed using a multiplex real-time pcr system designed to detect adenovirus, bocavirus, chlamydophila pneumoniae, human coronavirus (nl , hku , oc and e), human enterovirus, human metapneumovirus, human rhinovirus, influenza a virus, influenza b virus, mycoplasma pneumoniae, parainfluenzavirus ( , , and ), and respiratory syncytial virus. the pcr settings and a list of primers and probes have been described previously [ ] . comparisons on group level for numerical variables were performed using the mann-whitney u test. pearson's correlation and linear regression were used to evaluate the relationship between the real-time pcr and ddpcr measurements. cox regression with ttv, ebv, and both as time-dependent covariates was used to analyze the relationship between the biomarkers and the outcomes vrti, fungal infection, bacterial infection, cmv viremia, or any infectious event. each patient was treated as a cluster to handle multiple infections occurring in a single individual. univariable logistic regression was used in each of periods, q ( - months), q ( - ), q ( ) ( ) ( ) ( ) ( ) ( ) ( ) , and q ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , with the outcomes vrti, fungal infection, bacterial infection, cmv viremia infection, or acute rejection and the predictors age, treatment, cmv mismatch, log ttv (beginning of period), or log ebv (beginning of the period). a p value <. was considered significant. r software, version . . (r core team, , r foundation for statistical computing, vienna, austria; http://www.r-project.org/), was used for all statistical analyses except for pearson's correlation analysis, which was performed using graphpad prism software (graphpad software inc., san diego, ca). the study was approved by the regional ethical review board in gothenburg (dnr: - ), and all subjects provided written and oral consent to participate. in total, lung transplant recipients were included in the study, of which ( %) were still alive months after transplantation. detailed patient description is outlined in table . blood-, bal-, and nasopharyngeal samples (nph) were collected longitudinally at follow-up (fu) visits until months post-lung transplantation (ltx; n = ), including pretransplant samples. the total nucleic acid content was isolated from serum or whole blood samples and analyzed for ttv-, ebv-, and cmv-dna load by real-time pcr. the total nucleic acid content was isolated from the nph and bal samples and analyzed for respiratory pathogens using multiplex real-time pcr. a majority of the patients had detectable ttv-dna levels before transplantation (pre-ltx), and the levels subsequently increased until a peak was reached at months post-ltx, after which the levels gradually decreased (figure ). the mean ttv-dna levels for the respective time periods were . log - months post-ltx, . log - months post-ltx, . log - months post-ltx, and . log - months post-ltx, respectively. conversely, a majority of the patients had no detectable ebv-dna prior to transplantation as determined in serum. at month post-ltx, a peak in mean ebv-dna levels was observed, which decreased already months post-ltx and thereafter remained at a relatively constant level, determined in whole blood (figure ). the mean ebv-dna levels for the respective time periods were . log - months post-ltx, . log - months post-ltx, . log - months post-ltx, and . log - months post-ltx, respectively. overall, fewer patients had detectable levels of ebv-dna compared with ttv-dna ( figure ). although the individual ttv-dna levels varied, a majority of the patients had detectable levels across the entire fu period. in comparison, only a minority of the patients had detectable levels of ebv-dna over a longer consecutive period, and a large fraction had ebv-dna levels below the level of quantification (loq) at any given time point during fu (figure ). three patients developed suspected ptld. no case was biopsy-verified, and all were reversed upon modification of immunosuppression. detailed individual ttvand ebv-dna level kinetics for each patient, including information regarding all infectious events and acute rejections, are displayed in supplementary figure . comparison of ttv-and ebv-dna levels in lung transplant recipients who received either tacrolimus-or cyclosporinebased therapy revealed that cyclosporine-treated patients had significantly lower ttv-dna levels in serum at month post-ltx and onwards, compared with the tacrolimustreated patients (figure ). there was no significant difference in ebv-dna levels in whole blood between tacrolimus-and cyclosporine-treated patients at any time point during fu (figure ). the fu was divided into periods, denoted q through q , defined as - (q ), - (q ), - (q ), and - (q ) months after transplantation. the periodic intervals were chosen to distinguish the initial phase of intensive immunosuppression from the later periods when immunosuppressive therapy was gradually lowered. viral infections (vrti) were more common during the first months, while bacterial and fungal infections were evenly distributed throughout the period and cmv viremia occurred at a higher frequency - months after ltx after discontinuation of prophylactic ganciclovir treatment ( table ) . events of acute rejections were most common during the first postoperative months and then decreased over time ( figure ). all but episodes of acute rejections were biopsy-verified. the frequency of infectious events ( figure and table ) was compared with the mean ttv-dna or ebv-dna levels during each period (q -q ) and the during the total fu period, respectively. no statistically significant association was found. with logistic regression, log ttv-dna or log ebv-dna levels in the beginning of the period did not predict any infectious event in any of the periods (supplementary table ). next, we performed a cox regression analysis to establish if ttv-or ebv-dna levels were associated with either viral table ). no significant associations were found using this criterion. when comparing other factors relevant to infections and acute rejection, using logistic regression, we found that age, cmv mismatch, and dominant immunosuppressive treatment did not predict outcome in any of the periods denoted q through q (supplementary table ). most acute rejection events (ars) occurred within the first months post-ltx (figure ). logistic regression with initial ttv-dna or ebv-dna for each period, respectively, did not predict whether an acute rejection event would occur during that period (data not shown). previous studies [ , ] have described higher mean ttv-dna levels in lung transplant patients than reported here. in order to verify the real-time pcr results, ttv-dna levels of patients with mean ttv-dna levels above log in period q were assessed by digital droplet pcr (ddpcr). the ttv-dna level was underestimated by more than log by real-time pcr in samples, and overall the levels obtained by realtime pcr correlated well with the results obtained by ddpcr (pearson r = . , p = . ) (supplementary table ). ttv and ebv has been proposed as biomarkers reflecting the functionality of the immune system after ltx. in this prospective study, evaluating serial samples for ttv and ebv and carefully recording infectious and rejection events during an fu period of months, we found no associations between the biomarkers and the risk of complications. we found that ttv-dna levels increased rapidly; month after ltx, the levels were already markedly elevated, presumably due to ablation of the functional effector cells of the immune system that normally control the ttv infection [ , ] . at months post-ltx, the mean ttv-dna level peaked and then gradually declined, reflecting the gradual moderation of immunosuppressive therapy. interestingly, the ttv-dna level was lower in patients who received cyclosporine treatment, possibly mirroring a different immune modulatory mechanism for cyclosporine compared with tacrolimus. görzer et al. [ ] previously suggested that this might be due to cyclosporine being less efficient as an immunosuppressant. however, we found no association between type of immunosuppressive regimen and acute rejection or other events that would indicate a difference in net immunosuppressive effect. recently, ttv-dna has been associated with chronic lung allograft dysfunction after lung transplantation [ ] , but in our study this was not included as an outcome. it is possible that the observed higher mean ttv-dna levels in other studies could be explained by differences in induction therapy and distribution of respective immunosuppressive regimen. for example, in the study by görzer et al. [ ] , the type of induction therapy was not defined and only % of the patients were treated with cyclosporine. there is some evidence that ttv replication is dependent on the type of induction therapy that appears to reflect changes in lymphocyte concentration; however, the duration is brief, after which the levels of ttv recover [ ] . in the present study, ttv viral load was determined in serum samples, whereas plasma was used in previous studies [ , ] . however, to our knowledge, there are no apparent reasons for variations in ttv-dna load, depending on whether plasma or serum is being used for analysis. to compare results regarding ttv levels between different transplantation centers, it is vital to reliably quantify the concentrations of ttv-dna in a standardized manner. here it was shown that the real-time pcr method was adequate as compared with the ddpcr method, which is considered a more reliable method for accurate quantification [ ] . nonetheless, real-time pcr is prone to interlaboratory differences, and utilization of ddpcr for quantification has been suggested to circumvent this problem, facilitating direct comparison of results between centers [ , ] . digital droplet pcr depends on partitioning of each master mix followed by end point pcr. quantitation is determined using poisson statistics to generate a result that is not dependent on a relation to a standard curve and should therefore exhibit lower variability between laboratories. however, the ddpcr assays still depend on amplification of a specific target sequence, and depending on the design of the primer and probe, the targeted sequence can vary. recent work has shown that viral dna in plasma samples is to a large extent free and not associated with viral particles and is also subjected to various degrees of degradation, influencing the results, depending on the amplicon length determined by the pcr-assay design [ ] . thus, standardization of the entire assay including primer and probe design, use of international standards, and assessment of the commutability of reference material is needed before a direct comparison of interlaboratory results can be made with absolute confidence, even with the use of ddpcr [ , ] . it remains to be clarified in which cell types ttv replication occurs, but cd + t cells and cd + + t cells appear to be important for controlling the infection, whereas ebv resides within the b-cell pool and is controlled by cd + and cd + t cells [ , , , , , [ ] [ ] [ ] [ ] . in this work, we show that the choice of calcineurin inhibitor affects the ttv-but not the ebv-dna levels, possibly reflecting separate mechanisms for viral replication rather than merely ablation of the t-cell pool. as the choice of immunosuppressive regimen was made at the discretion of the treating physician, these findings should be interpreted with caution but warrant further investigation. in this study, infectious events were defined as symptoms prompting antimicrobial therapy also in cases without a positive culture (bacterial and fungal infections) or simply detection of a potential pathogen by pcr (vrti, cmv). attempting to focus on more severe infectious events, subgroup analysis of events requiring intravenous therapy was made. also, this analysis failed to reveal any significant association between ttv-or ebv-dna levels and infectious events. however, no proper validated scoring system for grading of the severity of infectious complications was used in this study, and we cannot exclude that our definitions cause underestimation of severe complications. further studies using proper grading of events are needed. ebv has previously been suggested as a surrogate marker for immunosuppression as an association between elevated ebv-dna levels and reduced incidence of acute rejection of lung transplants was observed [ ] . in addition, ebv-dna was safely used as a trigger for reduction of immunosuppression late after lung transplantation and was found to be associated with a shorter time to development of infectious complications or solid tumors, but not ptld [ , ] . we found no association between ebv-dna and acute rejection at any time during fu. a direct comparison could, however, be hampered by the fact that ebv-dna was analyzed in this study, and ebv-encoded rna in an earlier study [ ] . in the present study, mean ebv-dna levels did not increase as markedly as ttv-dna levels after transplantation, even though ebv-specific t cells most likely are subverted by immunosuppressive therapy [ ] . the seroprevalence pre-ltx and number of patients positive for ebv-dna at any time during fu were comparable to previous studies [ , , ] . one possible caveat when determining ebv dna load is the use of valgangciclovir for preventing reactivation or primary infection of cmv. all patients received months of valgangciclovir treatment initially after ltx; there are a few studies showing that valgangciclovir treatment reduces ebv load, which may have affected the results in this study [ , ] . in conclusion, ttv-but not ebv-dna load appears to reflect the function of the immune system after lung transplantation, depending on the type of immunosuppressive treatment. however, we found no association between either ttv-or ebv-dna load and infectious events or acute rejections, which suggests a limited clinical applicability as biomarkers predicting short-term outcomes related to the net state of immunosuppression. further studies are warranted to define the effect of immunomodulation on ttv and ebv replication in relation to various immunosuppressive regimens. supplementary materials are available at open forum infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. international society for heart and lung transplantation. the registry of the 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for the suppression of epstein-barr virus replication financial support. this project was supported by funding from stiftelsen professor lars-erik gelins minnesfond, fou laboratoriemedicin, sahlgrenska university hospital, the swedish heart-lung foundation, the region västra götaland research funds (vgfoureg- , vgfoureg- ), regional alf funds (alfgbg- , alfgbg- ), and region halland. potential conflicts of interest. all authors: no reported conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord- - tp ig o authors: hayman, david t s title: african primates: likely victims, not reservoirs, of ebolaviruses date: - - journal: j infect dis doi: . /infdis/jiz sha: doc_id: cord_uid: tp ig o nan ebola virus disease (evd) kills almost half those people infected. four different viruses from ebolavirus species have caused evd in africa: zaire ebolavirus (ebov), sudan ebolavirus (sudv), bundibugyo ebolavirus (bdbv), and tai forest ebolavirus (tafv), with all but tafv causing fatal human disease. outbreaks have been sporadic and unpredictable, but the frequency and size of outbreaks appear to be increasing [ , ] . the ongoing outbreak in the democratic republic of the congo (drc) is the second largest on record, with > cases and > deaths reported from health zones [ ] . the largest evd outbreak, originating in guinea, west africa, in [ ] , ended in after cases and deaths [ ] . preventing evd outbreaks is challenging because the "reservoir hosts" of the viruses that cause the disease are not known. the weight of evidence suggests that fruit bats are the natural hosts, but this is uncertain [ ] . the uncertainty is partially because most outbreaks in people are not directly linked to bats. circumstantial evidence linked bats to the west african outbreak [ ] , but index case exposure to bats has only once been reported with any confidence [ ] . in contrast, hunting or butchering primates has been linked to several evd index cases. in particular, africa's great apes, gorillas and chimpanzees, have been sources of human infection, and human evd outbreaks have occurred concurrently with outbreaks in apes in central and west africa [ , ] . high case fatality rates among apes [ ] [ ] [ ] , however, suggest they are not maintenance reservoir hosts [ ] . wildlife mortality events during evd outbreaks have involved other mammals, including monkeys, pigs, and antelope [ ] . contact with monkeys has been reported in human outbreaks in central africa [ , ] and chimpanzees in ivory coast [ ] . monkeys themselves appear to be susceptible to ebov infection, at least experimentally [ ] . outside of africa, reston ebolavirus (restv) has been linked to monkeys, with macaques imported to the united states from the philippines infected [ ] , but the mammals linked to restv in asia are similar to africa, with pigs, monkeys, and bats all implicated as hosts [ ] [ ] [ ] . serological data may be well suited for surveillance studies, because antibodies are longer lasting than viral infection and provide evidence of survival. experimental evidence suggests that ebov infection in bats may be acute, nonfatal, and short-lived, but induces antibodies [ ] . this experimental work is supported by field data from related marburg viruses, first identified after african monkeys infected people in europe [ ] , which apparently persist within large colonies of cave-dwelling egyptian fruit bats, and restv in asian bats. in both cases, viruses or viral rna and antibodies were detected in apparently healthy bats [ , ] . just study has detected ebov rna in bats, but anti-ebov antibodies are widespread in african bats and the rna-positive bats were, again, apparently healthy [ , [ ] [ ] [ ] [ ] . in contrast, while anti-ebov antibodies have been observed in african apes and monkeys [ , ] , suggesting that nonlethal infections might occur, the prevalence of antibodies is low (similar to those reported for restv in asian macaques [ ] ), and ebov rna has been isolated from dead apes [ ] . thus, together the evidence for bats being the true reservoir host for evd causing viruses is convincing, but relies on serological evidence of infection rather than virus detection, and the role of nonhuman primates as reservoirs remains uncertain. the role of primates in evd epidemiology has been unclear largely because study sample sizes have been small. serology is further complicated by different methodologies and antibody-positive sera cross-reacting among different evd-causing viruses. a report by ayouba et al, in this issue of the journal of infectious diseases, has taken a significant step toward addressing these problems [ ] . the team utilized a large sample (n = ) of tissues from multiple species of african primates, collected from to from ivory coast in west africa and drc and cameroon in central africa. the study more than triples the number of all previous primate samples reported and is similarly powered to some studies showing high seroprevalence of anti-ebov antibodies in certain african fruit bat species [ , ] . a single luminex-based serological assay that included antigens from viruses (ebov, sudv, bdbv, and restv) was used, and the team discovered that none of ape samples and only of monkey samples met their seropositive criteria. the data strongly suggest that the primates sampled are unlikely reservoir hosts. the work highlights the importance of multiyear, multisite empirical studies and archiving samples. specimen collection in general has created some controversy in areas such as conservation biology [ ] , but for epidemiologists tissue archives may enable us to better understand the epidemiology of infectious diseases. here, the primate samples were collected for lentivirus research (eg, human immunodeficiency virus [hiv] and its relatives), then repurposed for evd research. in other systems, archived sample banks have helped identify middle east respiratory syndrome coronavirus-seropositive camels in east africa over -year (kenya) and -year (sudan and somalia) periods, suggesting extensive virus circulation in camels prior to the first human outbreaks [ ] [ ] [ ] [ ] . some impressive examples of using archaeological samples have led to the sequencing of yersinia pestis genomes from black death victims in london, england, dated to - [ ] , and bronze age hepatitis b viral dna [ ] . the instability of rna viruses will prevent paleovirological studies on these timeframes, though gene sequencing from archived samples has helped identify hiv type (hiv- ) sequences predating the first aids diagnosis, with hiv sequences from and in drc informing our understanding of pandemic hiv- origins and evolution [ , ] . ideally, evd-causing viruses themselves will be isolated in space and time through wildlife surveillance to understand viral transmission dynamics. phylogenetic models that estimate the relationship between genetic sequences have been used with sample location data to place the first case from drc near the root of the ebov phylogenetic tree, suggesting that all other known outbreaks descended from a closely related virus [ ] . although the analysis contained just a few viral fragments, it suggested that later outbreaks were epidemiologically linked and occurred in a wave-like pattern, spreading at approximately km per year. once ebov rna fragments were discovered in bats, the same team used similar models to reconstruct the ancestry of ebov, including fragments of viral rna from bats [ ] . their analyses suggested that all of the genetic variation present in ebov, including from fruit bats, was the product of mutations accumulated within a -year time period, supporting the ancestry of ebov in bat reservoirs and the role of bats in ebov epidemiology. the absence of robust data on ebola virus reservoirs makes forecasting when and where outbreaks may occur difficult, limiting preventive measures [ , ] . the lack of data relating to bats themselves led researchers to characterize the traits of all filovirus-seropositive and virus-positive bat species to predict potential undetected bat species [ ] . putative bat hosts have been included in models to predict the spatial risk of human outbreaks [ ] . similar modeling approaches have been used to model the spatial and temporal risk of human and ape evd, finding the greatest risk during wet to dry season transitions in sparsely populated regions of tropical africa [ ] , supporting previous work [ ] . all of these studies are limited by data, but ayouba et al's comprehensive study supports the assumption that bats, not primates, are likely reservoir hosts and that nonhuman primates may be viewed as both sentinels for human infection and victims of evd [ , , , ] . these are important findings because they can inform field and surveillance studies, which are costly and difficult in most areas where evd outbreaks occur and for the species linked to evd. to really manage and prevent evd, however, we also need to understand why outbreaks appear to be increasing in frequency. recent analyses of forest fragmentation and evd emergence suggest there may be links [ , ] . if so, there may be management options that can be implemented alongside human and wildlife surveillance and public health interventions to reduce the risk of human and, potentially, primate evd emergence in the first place. financial support. the author was supported by the rutherford discovery fellowship (award number rdf-mau ) and the marsden fund (award number mau ). potential conflicts of interest. author certifies no potential conflicts of interest. the author has submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. temporal and spatial analysis 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direct evidence of extensive diversity of hiv- in kinshasa by an african hiv- sequence from and implications for the origin of the epidemic wavelike spread of ebola zaire recent common ancestry of ebola zaire virus found in a bat reservoir field investigations of an outbreak of ebola hemorrhagic fever, kikwit, democratic republic of the congo, : arthropod studies search for the ebola virus reservoir in kikwit, democratic republic of the congo: reflections on a vertebrate collection undiscovered bat hosts of filoviruses mapping the zoonotic niche of ebola virus disease in africa spatiotemporal fluctuations and triggers of ebola virus spillover trigger events: enviroclimatic coupling of ebola hemorrhagic fever outbreaks multiple ebola virus transmission events and rapid decline of central african wildlife habitat fragmentation, biodiversity loss and the risk of novel infectious disease emergence the nexus between forest fragmentation in africa and ebola virus disease outbreaks key: cord- -tcr xlf authors: nambiar, puja; silibovsky, randi; belden, katherine a. title: infection in kidney transplantation date: - - journal: contemporary kidney transplantation doi: . / - - - - _ sha: doc_id: cord_uid: tcr xlf infection is an important cause of morbidity and mortality after kidney transplantation. it has been estimated that % of kidney transplant recipients will experience an infection episode within the first years after transplantation (dharnidharka et al. ). after cardiovascular disease, infection is the second leading cause of death in recipients with allograft function (snyder et al. ). the immunosuppressive therapy required to prevent organ rejection places the kidney transplant recipient at increased risk for donor-derived, nosocomial, and community-acquired infections as well as reactivation of latent pathogens. pretransplant screening, immunizations, and optimal antibacterial and antiviral prophylaxis can help to reduce the impact of infection. awareness of the approach to infection in the transplant recipient including diagnostic and management strategies is essential to optimizing outcomes. a total of , kidney transplants were performed in the united states in . as the incidence of acute rejection has declined, the probability of graft and patient survival continues to improve (usrds ) . infection, however, remains an important cause of morbidity and mortality after kidney transplantation. it has been estimated that % of kidney transplant recipients will experience an infection episode within the first years after transplantation (dharnidharka et al. ). after cardiovascular disease, infection is the second leading cause of death in recipients with allograft function (snyder et al. ). the immunosuppressive therapy required to prevent organ rejection places the kidney transplant recipient at increased risk for donor-derived, nosocomial, and community-acquired infections as well as reactivation of latent pathogens. the kidney transplant recipient's net state of immune suppression and epidemiologic exposures determine the risk for infection at a given time. a traditional timeline has been used to predict patterns of infection after organ transplantation. this timeline has been altered in recent years with changes in immunosuppressive therapy and the routine use of antibacterial and antiviral prophylaxis. treatment for acute rejection and coinfection with viruses such as cytomegalovirus (cmv) and epstein-barr virus (ebv) may also alter predictable patterns of infection (fishman ) . the basic concepts of the traditional timeline, however, are still used to establish a differential diagnosis for infection at varied intervals posttransplantation (fig. ) . within the first month, infections are noted to include those related to surgical complications, nosocomial exposures, and donor-derived pathogens. multidrug-resistant organisms including methicillin-resistant staphylococcus aureus (mrsa), vancomycin-resistant enterococcus (vre), and carbapenem-resistant enterobacteriaceae (cre) are important considerations, as is clostridium difficile. urinary tract infections are common within the first months. opportunistic infections are more likely to occur - months after transplantation, reflecting the greater impact of immune suppression during this time. reactivation of latent pathogens such as polyoma virus bk, hepatitis c virus (hcv), and mycobacterium tuberculosis may also occur. prophylaxis for pneumocystis jiroveci, herpes viruses including cmv, and hepatitis b virus (hbv) makes these infections less common during this time period. beyond months, the degree of immune suppression for most patients decreases. risk remains, however, for community-acquired infection, environmental exposures, recurrent infection, and the late presentation of viral infection, in particular cmv, once prophylaxis has been discontinued (fishman ; karuthu and blumberg ) . interventions can be undertaken to reduce the impact of infection after kidney transplantation. pretransplant screening of donors and recipients for infection that can be transmitted with organ donation or reactivated in an immune suppressed recipient is essential for optimizing transplant outcomes. guidelines for pretransplant screening are available from the american society for transplantation (fischer et al. ) , kidney disease: improving global outcomes (kdigo ) and the us public health service (seem et al. ) . recommended screening tests for donors and recipients are listed in table . screening of living donors is performed prior to transplantation with varied timing. if there is a hiv(þ) consider if hiv is well controlled hcv: anti-hcv and hcv nat hcv(þ) hcv(À) reject, may be a consideration in the future hcv(À) hcv(þ) consider, hcv(þ) candidates should have a liver biopsy, improved outcomes if hcv is treated pretransplant hcv(þ) hcv(þ) consider (as for dÀ/rþ) hbv: hbsag, hbsab and hbcab (igm/igg); hbv nat (center dependent) sag(À), cab(À) sag(À), cab(þ), sab(þ/À) accept, vaccinate sab(À) candidates consider, with prophylaxis posttransplant sag(À), cab(þ) sag(À), cab (þ/À), sab (þ/À) accept if donor is cigm(À) and vaccinate sab(À) candidates, offer prophylaxis posttransplant if sab(À) or lost; reject if donor is cigm(þ) sag(þ), cab(þ) sag(À), cab (þ/À), sab (þ/À) reject cmv igg cmv(þ) or (À) cmv(þ) accept; will need posttransplant prophylaxis or preemptive therapy cmv(þ) cmv(À) accept; high risk for cmv infection, will need posttransplant prophylaxis ebv igg ebv(þ) or (À) ebv(þ) accept ebv(þ) ebv(À) accept; at risk for primary ebv and ptld, monitor posttransplant hsv / igg hsv(þ) hsv(þ) or (À) (fischer et al. ) . deceased donor screening, in contrast, is under time constraints and is usually performed within hours of transplantation in coordination with organ procurement organizations. infection with hiv, hbv, and hcv may not be detected in the early stages of infection. many transplant centers now perform more sensitive rapid molecular testing on potential organ donors including nucleic acid amplification (nat) testing for hiv, hbv, and hcv. a comprehensive medical and social history on potential organ donors is required in order to identify risk factors for blood borne pathogens. in efforts to expand the pool of available organs, recipients may consent to receipt of a kidney from a nat negative donor who is deemed "high risk" for blood borne infection based on identified risk factors. recipients of such organs are monitored posttransplantation with testing for hiv, hbv, and hcv between and months and for hbvagain at months (fischer et al. ; seem et al. ; kovacs et al. ; len et al. ). use of hcv-and hbv-positive organs can be considered in respective positive recipients. furthermore, in the hiv organ policy equity act lifted a long-standing ban on allowing hivpositive organs to be donated to hiv-positive recipients (mgbako et al. ; muller et al. ) . donors who have active bacterial infection at the time of kidney procurement may transmit infection to the recipient. screening for bacterial infection in kidney donors includes assessing for urinary tract infection and bacteremia. urine and blood culture data are reviewed. if a kidney donor is known to have a urinary tract or systemic infection with a virulent organism such as staphylococcus aureus, pseudomonas aeruginosa, or candida species, the organ recipient is usually treated with a - day course of targeted antimicrobial therapy since these bacteria can compromise vascular and urinary anastomoses leading to mycotic aneurysms, anastomotic, and organ failure (fischer et al. ). allograft contamination can occur during organ procurement or processing. interpretation of organ preservation fluid cultures is challenging. the risk of transmission of infection to the organ recipient from contaminated preservation fluid, however, is low (fischer et al. ; len et al. ). candidates for kidney transplantation should have their vaccine status reviewed and updated in accordance with recommendations issued by the advisory committee on immunization practices with the centers for disease control and prevention (cdc ). while vaccinations in end stage renal disease patients may be less effective and durable than in healthy patients, a better response can be anticipated prior to transplantation than after (janus et al. ; kausz and pahari ) . special consideration should be given to vaccination for pneumococcus, influenza, and hbv. two pneumococcal vaccines are currently licensed for use in the united states: the -valent pneumococcal conjugate vaccine (pcv , prevnar ) and the -valent-pneumococcal-polysaccharide vaccine (ppsv , pneumovax ). current guidelines recommend that unvaccinated patients with chronic renal failure receive pcv followed at least weeks later by ppsv (kobayashi et al. ) . a second dose of ppsv is recommended years after the first dose (cdc ). influenza vaccination should be administered annually. there are a number of influenza vaccine formulations available. live attenuated influenza vaccination (flumist) is not recommended in chronic kidney disease patients. an inactivated vaccine option should be used (cdc ) . a high dose inactivated influenza vaccine is now available and was shown to induce a higher antibody response than traditional vaccines in adults over the age of (diaz-granados et al. ) . the use of this vaccine in transplant candidates and recipients is currently under investigation. transplant candidates not immune to hbv should receive high dose hbv vaccination ( micrograms antigen per dose) due to decreased response rates with standard dosing (huprikar et al. ) . human cytomegalovirus-human herpes (cmv), a member of the family herpesviridae, is an opportunistic pathogen occurring in - % of solid organ transplant recipients (brennan ) . cmv is a cause of significant morbidity and mortality in this population (mwintshi and brennan ) . the incidence of cmv in the renal transplant population is estimated to be between % and % (patel and paya ) . renal transplant patients are at lower risk for primary cmv compared with other organ transplant recipients owing to a lower burden of latent virus in renal allograft tissue. the risk factors for development of cmv disease include donor seropositivity/recipient seronegativity(dþ/rÀ), use of induction immunosuppression (antilymphocyte antibodies), donor age > years, simultaneous kidney-pancreas transplantation, treatment for acute rejection, impaired transplant function, and concurrent infection from other viruses (like ebv and hhv- and ) (de keyzer et al. ) . cmv-seronegative recipients of cmv-seropositive donors (d + /r À ) are at the highest risk, whereas d + /r + or d À /r + transplantations are considered to be moderate risk with d À /r À being lowest risk, with an incidence of cmv disease < % (de keyzer et al. ) . immunosuppressive drugs also influence the incidence and severity of cmv disease. for instance, cyclosporine increases the risk of cmv disease, whereas use of sirolimus seems to have a protective effect (san juan et al. ) the use of antilymphocyte antibody (antithymocyte globulin or muromonab-cd ) is associated with a two to fivefold increase in the rate of cmv, but basiliximab and daclizumab do not seem to increase its incidence (de keyzer et al. ) . cmv infection may occur in solid organ transplantation recipients as primary infection when a cmv seronegative individual receives cells latently infected with cmv from a seropositive donor, donor-derived reinfection, or reactivation of latent recipient infection (patel and paya ) . the following definitions are commonly used in the transplant literature to differentiate cmv infection from cmv disease. cmv infection is evidence of cmv replication regardless of symptoms, and cmv disease is evidence of cmv infection with symptoms, such as viral syndrome, leukopenia, thrombocytopenia, or invasive tissue disease (e.g., pneumonitis, hepatitis, retinitis, gastrointestinal disease) (humar and snydman ) . cmv disease and even asymptomatic cmv infection have been shown to be independent risk factors for reduced graft survival and overall mortality beyond days posttransplantation . infection with cmv has been implicated in acute allograft dysfunction and chronic allograft nephropathy (mclaughlin et al. ; tong et al. ) . cmv disease is also associated with posttransplant lymphoproliferative disorder (ptld), posttransplant diabetes mellitus, and transplant artery stenosis (pouria et al. ; hjelmesaeth et al. ; manez et al. ) . cmv infection can occur as acute infection between the first and months following transplant, when immunosuppression is at its maximum or as delayed infection from reactivation of latent virus after antiviral prophylaxis has completed, later in the first year. given the significant effect of cmv on patient outcomes, prevention plays an important role. serologic screening for cmv should be performed on both donor and recipient prior to transplant to categorize high risk patients. several cmv vaccine candidates are under investigation although none are currently available. universal prophylaxis involves giving antivirals to those recipients at risk posttransplant before the onset of infection, whereas in preemptive therapy patients are monitored at regular intervals and started on antivirals when there is early evidence of replication prior to onset of clinical disease. chemoprophylaxis in high risk patients (dþ/rÀ) has shown to reduce the incidence of cmv disease by % and has decreased cmv associated mortality and opportunistic infection (hodson et al. ) . preemptive therapy in high risk patients based on cmv viral load monitoring has not shown reduction in acute rejection or all-cause mortality (strippoli et al. ) . a randomized controlled trial by kliem et al. in comparing oral ganciclovir chemoprophylaxis with viral load monitoring revealed improved graft survival in those who received ganciclovir chemoprophylaxis (kliem et al. ) . a recent cochrane review from concluded that the efficacy of preemptive therapy compared with prophylaxis to prevent cmv disease remains unclear due to significant heterogeneity between studies and that additional head-tohead studies are required to determine the relative benefits and harms of preemptive therapy and prophylaxis to prevent cmv disease in solid organ transplant recipients (owers et al. ) . standard prophylactic guidelines recommend therapy in dþ/rÀ, dþ/rþ, and dÀ/rþ using oral ganciclovir or valganciclovir for a minimum of months posttransplant and - months after treatment of rejection with antilymphocyte therapy (humar and snydman ; kotton et al. ). valganciclovir has replaced ganciclovir because of better bioavailability, lower pill burden, and reduced availability of oral ganciclovir (paya et al. ). the optimal length of prophylaxis is unknown, but recent trials have shown that months of prophylaxis is more effective in decreasing the incidence of cmv disease in dþ/ rÀ kidney transplant recipients (humar et al. ; doyle et al. ) . current guidelines recommend dosing valganciclovir at mg daily (adjusted for renal dysfunction) if tolerated in dþ/ rÀ recipients. some centers have successfully treated patients with half of this dose ( mg daily) with less drug toxicity. however, dþ/rÀ recipients may be at higher risk of breakthrough infection and the development of resistance with this lower dosing strategy (kotton et al. ) . the diagnosis of cmv disease can be made by several techniques including cmv antigenemia assay, nucleic acid testing (nat), serology, antibody testing, viral culture, and histopathology. nat is generally more sensitive than antibody testing or culture. higher values by nat are suggestive of cmv disease and weekly viremia testing can be used to monitor response to therapy. the interlaboratory variability of nat is expected to be reduced with the recent establishment of international standards, intended to be used in the standardization of nucleic acid amplification technique (nat)-based assays for hcmv (karuthu and blumberg ) . patients with gastrointestinal and neurologic cmv disease often fail to exhibit cmv viremia and histopathology is necessary to establish diagnosis in these instances. treatment of active cmv disease requires a combination of immunomodulation, antiviral therapy with or without adjuvant therapy and if possible, reduction of immunosuppression (kotton et al. ; green et al. ). the mainstay of therapy is intravenous ganciclovir. the victor trial (valcyte in cmv disease treatment of solid organ recipients) demonstrated oral valganciclovir was not inferior to intravenous ganciclovir in mild to moderate cmv disease in solid organ transplant recipients (asberg et al. ). the current guidelines recommend renally adjusted intravenous ganciclovir mg/kg twice daily or oral valganciclovir, mg twice daily for mild cmv disease (kotton et al. ) . in severe cmv disease, intravenous ganciclovir is preferred with reduction of immunosupression despite the increased risk of rejection (de keyzer et al. ) . the use of adjuvant therapy with cmv-specific hyperimmune globulin or standard intravenous immunoglobulin may be considered in individuals with hypogammaglobulinemia, severe systemic infection, or in failure to respond to standard therapy (humar et al. ) . cmv resistance to ganciclovir has been noted in renal transplant recipients due to mutations in ul , the gene responsible for the first phosphorylation step in ganciclovir activation and ul , the gene responsible for dna polymerase (limaye et al. ) . cmv resistance should be considered when patients have worsening disease or persistent, unchanged viremia at weeks of therapy and in such cases, genotype testing for mutations of the genes encoding ul and ul should be performed (weikert and blumberg ). treatment options for drug resistant cmv include the use of high dose ganciclovir, foscarnet, and cidofovir; however, no clinical trial data are available regarding optimal therapy options for resistant cmv. the use of novel agents including leflunomide and artesunate has been attempted as salvage therapy with varying success. several new antiviral treatment options are currently under investigation including maribavir and brincidofovir (an oral prodrug of cidofovir with less nephrotoxicity) for use in the treatment of drug resistant cmv (limaye et al. ) . epstein barr virus -human herpesvirus (ebv) is a ubiquitous gamma herpes virus that remains latent in lymphocytes following primary infection. it is responsible for posttransplant lymphoproliferative disorder (ptld) which increases morbidity and mortality in the transplant population. approximately - % of ptld cases have been associated with ebv (karuthu and blumberg ) . ptld most commonly occurs in the first year posttransplant (cockfield et al. ) . the risk factors for ptld include ebv naïve recipients who receive ebv seropositive organs, active primary ebv infection, younger recipient, coinfection by cmv and other viruses, prior splenectomy, second transplant, acute or chronic graft versus host disease, immunosuppressive drug regimen (okt or polyclonal antilymphocyte antibody), and the type of organ transplanted. kidney transplant recipients are at lower risk compared with other types of transplants and have an incidence of approximately - % (gulley and tang ; allen et al. ; taylor et al. ) . the majority of symptomatic ebv infections in renal transplant recipients are primary infection likely related to transmission of donor virus. ebv disease can be asymptomatic or presents with a nonspecific febrile syndrome, lymphadenopathy, hepatosplenomegaly, atypicalþ lymphocytosis, hematologic disorders including anemia, leukopenia, thrombocytopenia, and organ-specific diseases like gastroenteritis, hepatitis, or pneumonitis (allen et al. ). ptld typically follows primary infection and frequently presents as a rapidly enlarging mass in the grafted organ, lymph nodes, bone marrow, or extranodal sites (manez et al. ) . ptld is divided into four major histopathologic subtypes as per the world health organization (who): early lesions, polymorphic ptld, monomorphic ptld, and classical hodgkin lymphoma type ptld. definitive diagnosis of ptld requires histopathologic confirmation by tissue excision biopsy with immunologic cell typing, cytogenetics, immunoglobulin gene rearrangements, and ebvspecific staining (allen et al. ). staging is performed by histologic types (monoclonal versus polyclonal, t cell versus b cell) and location (allograft, other organs, metastasis) (weikert and blumberg ) . clinical management of ptld typically involves reduction of immunosuppression which can lead to remission in - % of the patients (weikert and blumberg ). antiviral therapy with acyclovir or ganciclovir is controversial and no evidence supports its efficacy (taylor et al. ) . rituximab (monoclonal antibody to cd ) is commonly used for treatment of ptld in recipients who failed reduction of immunosuppression alone (allen et al. ). in isolated graft ptld, surgical resection is an option (weikert and blumberg ) . in patients that fail the above strategies, ifn and ivig have been used with varying success and cytotoxic chemotherapy with radiation remains salvage therapy (green et al. ). there is no standardized therapy to prevent ptld. kdigo guidelines recommend monitoring ebv viral load in high risk renal transplant patients within the first week after transplant, then at least monthly for - months and then every months for the rest of the first posttransplant year. additional viral load monitoring is recommended after treatment for acute rejection in high risk groups (children, ebv dþ/rÀ). outcomes with ptld in renal transplant patients vary according to the site involved. patients with isolated graft involvement have a -year survival of % compared with those patients with ptld extending beyond the allograft whose survival varied between % and % (weikert and blumberg ). human herpesvirus herpes simplex virus types and (hsv)and human herpesvirus varicella zoster virus (vzv)are alpha herpes viruses. hsv has a seroprevalence of % in the adult population, while hsv has a seroprevalence of % and vzv rates can be as high as % (green et al. ). the incidence of hsv disease in renal transplant recipients is approximately % and vzv - % (patel and paya ) . hsv may cause primary infection following which the virus remains latent in the sensory nerve ganglia or more commonly causes reactivation infection. hsv may be seen as early as in the first posttransplant month in the absence of prophylaxis. hsv infection usually presents with oral or genital mucocutaneous lesions, occasionally pneumonitis, tracheobronchitis, esophagitis, hepatitis, encephalitis, or disseminated infection (green et al. ) . vzv causes localized dermatomal or multidermatomal or disseminated zoster with or without visceral involvement (pneumonitis, hepatitis, pancreatitis, encephalitis). pretransplant screening for prior vzv infection should be performed, and naïve patients should be vaccinated with live attenuated varicella vaccine before transplant whenever possible in order to avoid primary vzv infection posttransplantation (fehr et al. ) . since vzv is a live vaccine, it should not be given if transplant is expected within - weeks in order to avoid active shedding of virus at the time of transplant. posttransplant prophylaxis is recommended with acyclovir, valacyclovir, or ganciclovir (in those who need cmv prophylaxis) for approximately - months posttransplant in order to avoid hsv and vzv reactivation (green et al. ) . diagnosis of hsv and vzv infection can be made with pcr or direct fluorescence antibody for hsv from vesicular lesions, csf, or visceral tissue samples. serologies are rarely helpful in active infection owing to high seroprevalence. kdigo guidelines recommend that renal transplant recipients who develop less severe hsv or vzv infections can be treated with an appropriate oral antiviral agent (e.g., acyclovir, valacyclovir, or famciclovir), and those with systemic infection should be treated with intravenous acyclovir and a reduction in immunosuppressive medication and subsequently switched to an appropriate oral antiviral agent (green et al. ). the use of foscarnet, cidofovir, or topical trifluridine may be considered in patients with acyclovir resistant virus with careful monitoring of renal functions (kotton and fishman ; tan and goh ) . human herpesvirus and human herpesvirus (hhv and hhv ) are ubiquitous with high seroprevalence in adults. these viruses are common causes of fever in children and remain latent in lymphocytes following primary infection. hhv uses the cd molecule as its receptor but may also infect other cell types, such as monocytes, and epithelial and endothelial cells. hhv uses the cd molecule as its receptor and is more strictly lymphotropic. infection occurs as a result of reactivation in the first weeks following transplant often in recipients not on cmv prophylaxis (singh and carrigan ) . clinical manifestations include fever, rash, hepatitis, interstitial pneumonitis, encephalitis, leukopenia, and myelosuppression. owing to its immunomodulatory effects, it is hypothesized that hhv may act as a cofactor for hhv and cmv reactivation, while both hhv and hhv may act as cofactors in the pathogenesis of cmv disease and acute rejection (kidd et al. ; chapenko et al. ; dockell and paya ) . the diagnosis of hhv and hhv is made by tissue immunohistochemistry or nat testing of peripheral blood lymphocytes. treatment includes reduction in immunosuppression and ganciclovir, but cidofovir and foscarnet have also been utilized (green et al. ; kotton and fishman ; dockell and paya ) . hhv is associated with primary effusion lymphoma, kaposi's sarcoma, and multicentric castleman's disease. infection can be acquired as primary through the allograft or through reactivation of latent virus (diociaiuti et al. ; regamy et al. ). hhv causes kaposi's sarcoma, the most common presentation in renal transplant recipients, through upregulation of vascular endothelial growth factor (vegf) receptor f k /kdr in endothelial cells (stallone et al. ) . treatment includes reduction in immunosuppression and cytotoxic chemotherapy. sirolimus, an immunosuppressive drug used in renal transplant patients is thought to inhibit not only the production of vegf but also dampens its effect on endothelial cells (stallone et al. ). bk polyomavirus (bkv) and jc polyomavirus (jcv) belong to the family polyomaviridae. bkv is responsible for causing polyomavirus associated nephropathy (pvan) in % of cases and jcv in less than % of the cases. pvan occurs in - % of patients with renal transplantation and causes renal allograft loss in - % of cases dadhania et al. ) . the risk factors for bkv associated pvan include the use of potent immunosuppressive regimens, caucasian race, older age, diabetes mellitus, cadaveric renal transplant, and combined kidney and pancreas transplant trofe et al. ) . bkv is known to cause interstitial nephritis, ureteral stenosis, and ureteral stricture of the allograft kidney most commonly occurring within the first - months after renal transplant patients when immunosuppression is at its highest (randhawa and brennan ) . jcv less commonly causes pvan and is more frequently associated with progressive multifocal leukoencephalopathy (pml), a demyelinating disorder of the white matter presenting as neurologic impairment and dementia (phillips et al. ) . diagnosis of bkv includes the use of viral load assays (blood, urine), detection of viral cytopathic effect (decoy cells), nat, bkv-specific antibody, or histopathology (hariharan ) . kdigo guidelines recommend screening all renal transplant recipients for bkv with quantitative plasma nat at least monthly for the first - months after transplantation, then every months until the end of the first posttransplant year, whenever there is an unexplained rise in serum creatinine, and after treatment for acute rejection. the guidelines suggest reducing immunosuppressive medications when bkv plasma nat is persistently greater than , copies/ ml ( copies/l) (kdigo ). sustained high bk viremia in spite of reduction in immunosuppression may need additional antiviral therapy, although data regarding optimal treatment options are unknown. there are limited data regarding the effectiveness of leflunomide and/or cidofovir or the use of fluoroquinolones or ivig for treatment of bkv infection (randhawa and brennan ; josephson et al. ) . to date there is no effective treatment for pml. patients with allograft loss due to pvan have undergone successful retransplantation (hariharan ) . patients with chronic renal failure, in particular those receiving hemodialysis, are at increased risk for contracting hepatitis b virus (hbv). the prevalence of hepatitis b surface antigen (hbsag)positive patients has declined because of hbv vaccination, strict segregation of hbsag-positive patients in dialysis units, improved screening of blood products, and the use of erythropoiesis stimulating agents (karuthu and blumberg ) . approximately - % of patients with a history of hbv prior to transplant will reactivate posttransplant (weikert and blumberg ) . serial monitoring of hbv dna every - months is required after transplantation as liver enzyme levels do not reflect infection status and elevated viral loads suggest resistance to therapy (levitsky et al. ) . in a meta-analysis conducted by fabrizi and his colleagues, hbsag seropositivity was an independent risk factor for allograft loss and posttransplant death (fabrizi et al. ) . the treatment options currently approved for chronic hbv include: ifn alpha, pegylated ifn, lamivudine, entecavir, telbivudine, tenofovir, and adefovir (fabrizi et al. ; chan et al. ; chang et al. ) . kdigo recommends that interferon treatment generally be avoided because of the high associated incidence of rejection. tenofovir or entecavir are preferable to lamivudine, to minimize the development of drug resistance, unless medication cost requires that lamivudine be used. during therapy with antivirals, hbv dna and alt levels should be measured every months to monitor efficacy and to detect drug resistance. all hbsagpositive renal transplant recipients should receive prophylaxis with tenofovir, entecavir, or lamivudine. hbsag-positive patients with cirrhosis should be screened for hepatocellular carcinoma every months with liver ultrasound and alpha feto-protein. patients who are negative for hbsag and have hbsab titer < miu/ml should receive booster vaccination to raise the titer to > miu/ ml (kdigo ). hepatitis c virus (hcv) infection has been increasingly recognized in end stage renal disease patients (esrd). donor-derived hcv may uncommonly occur after transplantation. screening of patients with esrd and testing renal transplant patients for newly acquired hcv should include nat (levitsky et al. ) . hcv-positive donors can be considered for hcv-positive recipients and possibly will be considered for hcv-negative recipients in the future given improved treatment options for cure of hcv that could be administered post transplant. hcv-infected renal transplant recipients have decreased survival and increased complication rates. posttransplant complications include glomerulonephritis (gn), posttransplant diabetes mellitus, and accelerated progression to cirrhosis with fibrosing cholestatic hepatitis (morales et al. ) . liver biopsy within - months of transplantation and subsequent biopsies are required for evaluation of liver disease posttransplant as - % of patients may have normal liver enzyme levels with abnormal histologic features (ashry ahmed gheith ). hcv-infected recipients should be tested for proteinuria every - months, and patients with new onset proteinuria should undergo allograft biopsy (kdigo ) . the effect of immunosuppression on the progression of hcv-related liver injury and the management of immunosuppression in the hcvinfected renal transplant recipient remain uncertain. thus, it is preferable to treat hcv in transplant candidates prior to transplantation given the potential for improved outcomes with successful hcv treatment and the complications associated with treatment posttransplant. patients with a sustained virologic response to pretransplant treatment have a reduced risk for hcv recurrence and decreased posttransplant gn (domınguez-gil and morales ). options for treatment include interferon/ peginterferon alone or in combination with ribavirin. the risk of toxicity with the addition of ribavirin has limited the use of combination therapy in chronic kidney disease (ckd) patients. the availability of direct acting hcv protease and polymerase inhibitors has sparked new enthusiasm for treating hcv-infected ckd patients and studies are ongoing evaluating the use of these agents in ckd. if treatment cannot be given prior to transplant, kdigo recommends monotherapy with standard interferon for hcv-infected renal transplant recipients in whom the benefits of antiviral treatment clearly outweigh the risks (kdigo ). the use of direct acting hcv antivirals posttransplantation can also be considered and will likely be preferred in the future given improved tolerance and efficacy with these agents with an understanding that drug interactions with calcineurin inhibitors may occur.a study looking at hcv-positive kidney transplant recipients ( % treated pre-transplant with interferon unsuccessfully) treated with direct acting antivirals posttransplant found that % cleared the virus and had a sustained virologic response at weeks. the most common agents used were sofosbuvir and simeprevir (sawinski et al. ). human immunodeficiency virus (hiv) belongs to the family of retroviridae. with the introduction of antiretroviral therapy (art) in the mid- s, the incidence of hiv related deaths has been reduced. renal diseases related to hiv infection include hiv associated nephropathy (hivan), immune complex diseases, and thrombotic microangiopathy (frassetto et al. ). a total of % of patients with hiv develop hivan and it remains an important complication of hiv infection, progressing rapidly to end stage renal disease (esrd) (shahinian et al. ) . a large prospective clinical trial examining outcomes among hiv + kidney transplant recipients reported -year patient and graft survival rates of . % and . %, respectively, which were similar to survival rates among a cohort of unmatched elderly (> years) hivnegative (hiv À ) kidney recipients (stock et al. ). the candidates for transplantation include those with well-controlled hiv infection with undetectable viral loads, cd > cells per microliter, and absence of untreatable infections or malignancies (blumberg et al. ). the most significant complications in this patient population posttransplant include increased rejection rates (up to %), managing drug interactions between art and immunosuppressive therapy and complications related to cardiovascular risk factors and hepatitis coinfection (blumberg et al. ). the choice of art should be based on susceptibility results and if possible, the use of protease inhibitors should be avoided owing to significant drug interactions with this class of art. with regards to immunosuppressive therapy, the use of thymoglobulin may result in prolonged depression of cd counts, whereas monocloncal anti-il receptor antibodies, such as basiliximab/daclizumab, have been shown to increase cd cell counts (ciuffreda et al. ; carter et al. ) . the risks of antilymphocyte therapy should be balanced with the risks of rejection in hiv-infected recipients. of note, hiv-positive donors can now be considered in hivpositive recipients. the various respiratory viruses that cause infection affecting the renal transplant patient population include adenovirus, respiratory syncytial virus (rsv), influenza, parainfluenza, human metapneumovirus, rhinovirus, and coronavirus (green et al. ) . clinical manifestations include upper respiratory tract infection, bronchitis, and pneumonia. in addition to respiratory illness, adenovirus is known to cause gastroenteritis, hemorrhagic cystitis, pancreatitis, meningoencephalitis, necrotizing hepatitis, and nephritis/renal dysfunction in renal transplant recipients (pham et al. ; alsaad et al. ) . infection with these viruses may also be associated with rejection (weikert and blumberg ) . prevention involves hand hygiene and the use of droplet precautions for those suspected of having infection. influenza vaccination is recommended prior to transplant and yearly following transplant. treatment of respiratory viral infection involves supportive care and antiviral medications. influenza can be treated with oseltamivir or zanamavir. ribavirin is approved for the treatment of rsv. adenovirus infection is treated with reduction of immunosuppression with consideration of cidofovir (ison ) . emerging viral pathogens include newly recognized viruses or previously known viruses that are either increasing or threatening to increase in incidence. some of the emerging viruses causing infections in renal transplant population include human t-cell leukemia virus type (htlv- ), hepatitis e virus (hev), measles virus, rabies virus, lymphocytic choriomeningitis virus (lcmv), dengue virus (denv), west nile virus, and zika virus. case reports of adult t-cell leukemia (atl) following renal transplantation in htlv- -positive patients have been documented, though in a case series of renal transplant recipients with long-term follow-up, no cases of atl or htlv- -associated myelopathy (ham) developed (nakamura et al. ; tanabe et al. ) . hev may induce kidney injury with significant reduction in glomerular filtration rate. glomerular injuries such as membranoproliferative glomerulonephritis have been described in kidney transplant patients with acute and chronic hev infections (kamar et al. ) . the incidence of measles in transplant recipients is unclear. cases of subacute measles encephalitis (sme) have developed in renal transplant recipients. the clinical course is one of deteriorating mental status and treatment refractory seizures (waggoner and deresinski ) . worldwide, vector-borne viral disease is increasing in incidence and can be transmitted with blood products and organ transplantation. fatal cases of dengue have been reported within the first month following renal transplant (waggoner and deresinski ) . west nile virus has also been reported in transplant recipients with a high incidence of neuroinvasive disease and poor outcomes. zika virus is also now a concern. cases of donor-derived rabies in the sot population have been reported. patients typically developed encephalitis between and months posttransplant, and all symptomatic reported patients died (srinivasan et al. ) . cases of lcmv causing severe disease in organ transplant patients have been documented. the clusters of lcmv infection occurred in the united states and involved kidney, liver, and lung transplants; symptoms included fever, abdominal pain, nausea, diarrhea, and altered mental status (srinivasan et al. ; barry et al. ; fischer et al. ) . two renal transplant recipients survived lcmv infection. ribavirin has been employed in some cases, though the benefits remain unclear (waggoner and deresinski ) . data regarding the incidence, screening and treatment options of the above-mentioned emerging viruses are limited. given the risk of donor-derived viral transmission, organs should not be accepted from donors with unexplained febrile or neurologic illness. in unclear cases, the risk of donor-derived infection should be balanced with the benefit of the transplant. bacterial infections after renal transplantation can be due to surgical complications at the time of transplantation, nosocomial infection, immunosuppression, or community-acquired infection. donorderived bacterial infections from the transplanted kidney or blood stream can occur as well. about % of kidney transplant recipients develop bacterial infections (patel and paya ) . occurring any time posttransplantation, urinary tract infections account for the overwhelming majority of these infections and are the most common bacterial infections prolonging or leading to re-hospitalization (wyner ) . enterococci, staphylococci, enteric gram-negative organisms, and p. aeruginosa are the most common bacteria isolated (wyner ) . bacterial pneumonia, postoperative wound infections, and bacteremia or sepsis, although less common, also prolong or lead to rehospitalizations after transplantation (karuthu and blumberg ) . common bacterial pathogens for these infections are gram-negative organisms, including multidrug resistant bacteria; gram positive organisms, including methicillin-resistant staphylococcus aureus (mrsa), and vancomycin-resistant entercococci (vre), as well as organisms more typically seen in immunocompromised patients such as listeria. months after the operation, bacterial pathogens include streptococcus species, mycoplasma, legionella, listeria, salmonella, and nocardia. trimethoprim-sulfamethoxazole (tmp-smx) prophylaxis has been shown to reduce the incidence of some of these infections. increased antimicrobial resistance, urgency of treatment, drug interactions, and toxicities, as well as the risk for clostridium difficile colitis all contribute to the complex decision making required for antimicrobial management. risk factors for urinary tract infection after transplantation are a prolonged period of hemodialysis before transplant, prolonged bladder catheterization, female sex, deceased donor transplant, kidney-pancreas transplant with bladder drainage, uretero-vesical stents, and an increased immunosuppressed state (karuthu and blumberg ; lapchik et al. ) . prophylaxis to lower the risk of infection after transplant with trimethoprim-sulfamethoxazole is routine (karuthu and blumberg ) . controversy regarding the exact dosing and duration of prophylaxis exists. typically it is given at a dose of mg trimethoprim and mg of sulfamethoxazole daily for - months (kdigo ). trimethoprim-sulfamethoxazole reduces the risk of uti and bacteremia (karuthu and blumberg ; patel and paya ) . symptoms of uti include frequency, urgency, and dysuria as well as nausea and vague abdominal complaints. some patients are asymptomatic. escherichia coli is the most common pathogen and an increasing number of pathogens are multidrug resistant. sensitivity testing is required. treatment of asymptomatic bacteriuria in the renal transplant recipient is controversial and is not routinely recommended (coussement and abramowicz ) . although not well studied, since utis in renal transplant patients are complicated, - days of antibiotics is a typical duration. removal of stents and catheters as well as drainage of abscesses are frequently required to prevent relapse and for cure. surgical wound infections, occurring at a rate of - %, usually present within the first weeks after transplant (ramos et al. ) . obesity, urine leaks, re-operation through the original incision, diabetes, high creatinine levels in plasma, and prolonged bladder catheterization are risk factors for wound infections (humar et al. ; khoury and brennan ) . improved organ procurement, preservation, and surgical techniques along with preoperative antibiotics all reduce the risk of subsequent postoperative wound infection. bacterial organisms causing these types of infections may be nosocomial and multidrug-resistant making antibiotic treatment difficult due to limited options or toxicities. source control with good wound care is critical in the management of these types of infections. although pneumonia is the most common bacterial infection in all solid organ transplant recipients, its incidence is lowest in those who have received a kidney (khoury and brennan ) . occurring early in the posttransplant period, cmv infection and rejection treatment with antilymphocyte preparations increase the pneumonia risk. hospital-acquired pneumonia due to resistant pathogens, such as mrsa, and extended spectrum beta lactamase (esbl) or carbapenemresistant (cre) gram-negative organisms are increasing in incidence and sometimes require nephrotoxic agents for treatment. communityacquired pneumonia can occur any time after transplantation and the incidence of communityacquired pneumonia specifically due to streptococcus pneumoniae can be lowered with vaccination. bacteremia and sepsis are most commonly due to a urinary source, followed by lung, wound, and abdomen (khoury and brennan ) . intravenous catheters also play a role. diabetes mellitus and posttransplant dialysis increase the incidence of sepsis which decreases the survival rate in these patients (abbott et al. ) . prompt treatment with broad spectrum antibiotics followed by rapid de-escalation to pathogen-specific therapy based on sensitivities is required. removal of foreign bodies such as intravenous catheters and stents is also necessary for cure. nocardia is a rare infection seen in the renal transplant recipient occurring in less than % of patients (wilson et al. ). trimethoprim-sulfamethoxazole prophylaxis used after transplant to prevent pneumocystis jiroveci pneumonia (pcp) likely prevents nocardia infection as well. nocardia asteroides is the most common species and causes pulmonary infections, including cavitary lesions and pleural effusions (patel and paya ) . other common sites of infection, due to dissemination, are central nervous system (cns) and cutaneous. all patients with nocardia should be evaluated for cns disease. allograft rejection, high-dose prednisone, azathioprine, instead of cyclosporine based immunosuppression, and neutropenia are risk factors for this infection (patel and paya ) . diagnosis is made by the identification of branching and beading rods on gram and modified acid fast staining and cultures of infected sites. antimicrobial susceptibility testing should be performed on all isolates. high dose trimethoprim-sulfamethoxazole sometimes in combination with amikacin is the treatment of choice, but allergic reactions and other side effects sometimes limit their use. alternatives include imipenem, minocycline, and ceftriaxone, but choices should be based on susceptibilities and site of infection (spelman ) . nocardia infections can relapse and prolonged therapy up to a year is recommended followed by chronic suppressive therapy (spelman ; arduino et al. ). listeria monocytogenes is a bacterial organism that is transmitted most commonly during summer and early fall to humans via the gastrointestinal tract from contaminated dairy products, raw vegetables, and meat. although more common during the first months after transplantation, infection may occur at any point, and risk is increased with rejection therapy (patel and paya ) . infections involving the central nervous system, such as meningitis and meningoencephalitis, are most common and present with headaches, fever, meningismus, altered mental status, and possibly focal neurologic deficits including cranial nerve palsies and seizures (patel and paya ) . cerebrospinal fluid examination typically reveals a pleocytosis, mostly polymorphonuclear leukocytes, decreased glucose, and elevated protein, but as the name implies, a mononuclear predominance may occur instead. gram staining has a low sensitivity and may be negative or reveal gram positive bacilli which may be confused with diphtheroids. other sites of infection include bacteremia, pneumonia, endophthalmitis, and septic arthritis. while trimethoprim-sulfamethoxazole, used for p. carinii prophylaxis, may also prevent infection with listeria, the treatment of choice is intravenous ampicillin and gentamicin for up to weeks in those with cns infections to prevent relapses. gentamicin is usually continued for a shorter duration, about weeks if kidney function is stable. (gelfand ) . trimethoprim-sulfamethoxazole is an alternative treatment for those who are allergic to penicillin. decreasing immunosuppressive agents is sometimes, but not always necessary. legionella infections in renal transplant recipients most commonly occur early in the posttransplantation period, but can be seen any time, especially during episodes of rejection. legionella pneumophila is the most common species to infect humans, and although more commonly community-acquired, nosocomial transmission occurs (patel and paya ) . most infections are pulmonary including pneumonia, and abscess with cavitation. symptoms are typical of lung infections but also may include headache and diarrhea. a legionella urinary antigen test and culture of lower respiratory secretions on selective media are used for diagnosis. empiric treatment for legionella is appropriate while waiting for results. quinolone antibiotics, such as levofloxacin, are preferred over macrolides in renal transplant patients because of drug interactions between macrolides and immunosuppressive medications. initially given intravenously, quinolone antibiotics can be quickly deescalated to oral treatment when the patient has defervesced. renal transplant patients, especially those who are severely ill at presentation, should receive days of treatment (yu ) . along with pcp and listeria, as noted above, prophylaxis with trimethoprim-sulfamethoxazole may also prevent legionella infection. immunosuppression increases the risk of developing mycobacterium tuberculosis (tb) disease. although the majority of tuberculosis infections in renal transplant recipients occur in the first months, tb can occur any time after transplantation (khoury and brennan ) . its overall incidence is lower in the united states when compared to the rest of the world, and foreign-born recipients are at greatest risk. having a high index of suspicion is important in renal transplant patients because presentation can be atypical and pretransplant screening with tuberculin skin tests or ifn-gamma release assays are unreliable in chronic kidney disease patients due to anergy. extra-pulmonary sites of infection and disseminated disease occur in about a third of cases (karuthu and blumberg ). laryngeal, meningeal, skeletal, cutaneous, intestinal, and renal infections are examples of extra-pulmonary disease. fevers are common, but sweats and weight loss may be absent (patel and paya ) . screening prior to transplant should include a history regarding prior exposures, and treatment for tb, as well as a chest x-ray and urine afb culture. prophylaxis with isoniazid or rifampin should be offered to patients prior to transplantation with a history of inadequately treated tb, an abnormal chest x-ray suggestive of prior tb exposure, a positive ppd or ifn gamma assay, contact with someone with active tb, or a kidney from a ppd-positive donor in order to minimize reactivation disease after transplantation (khoury and brennan ) . patients receiving treatment for latent tb may undergo renal transplantation and complete their defined course afterwards with special attention to potential drug interactions and toxicities (karuthu and blumberg ) . diagnosis of tb after renal transplantation often requires a biopsy of the infected site with stains for acid fast bacilli and cultures for sensitivity testing. treatment of active disease after transplantation requires multiple drugs and should follow the american thoracic society, center for disease control, and infectious disease society of america guidelines (mmwr ) . special attention to drug toxicities and interactions with immunosuppressive agents is required. rifampin, in particular, decreases cyclosporine levels and increases the risk for rejection. fungal infections in kidney transplant recipients occur less frequently than in other solid organ transplant recipients. most present within the first months after transplantation (hagerty et al. ) and can represent primary, reactivated, or donor-derived infection. those associated with geographic and environmental exposures include histoplasmosis, coccidioidomycosis, blastomycosis, and paracoccidioidomycosis. others are considered opportunistic and include infections such as candida, aspergillus, and cryptococcus (karuthu and blumberg ) . broad spectrum antibiotics, corticosteroids, diabetes mellitus, rejection therapy, cmv infection, and duration of pretransplant dialysis are risk factors (khoury and brennan ) . esophageal candidiasis, urogenital candidiasis, and pneumonia are the three most common sites of fungal infections in these patients (abbott et al. ) . clinical presentation may be nonspecific and diagnosis difficult due to testing limitations. positive cultures may represent colonization rather than infection with pathogens such as candida and aspergillus. cultures, antigen assays, serum galactomannan assays, and radiography may be helpful, but are not always diagnostic. subsequently, biopsy with pathology and cultures is considered the gold standard for diagnosing fungal infections (karuthu and blumberg ) . drug interactions and toxicities as well as immune reconstitution, due to lowering of immunosuppressive medications, further complicate the management of fungal disease in these patients and require expert advice (karuthu and blumberg ) . pneumocystis jiroveci (formerly pneumocystis carinii (pcp), protozoa) is a pathogen currently considered a fungus based on nucleic acid and biochemical analysis. presenting as pneumonia with interstitial infiltrates on chest x-ray within the first year after transplantation in those not receiving prophylaxis, mortality may be high. nonproductive cough and shortness of breath with rapid progression to hypoxia is a classic presentation. diagnosis is based on silver staining of deep respiratory specimens from induced sputum, bronchoalveolar lavage, or transbronchial biopsy (martin and fishman ) . the treatment of choice is high dose trimethoprim-sulfamethoxazole for days with corticosteroids in hypoxic patients (partial pressure of oxygen of < mmhg on room air) tapered over days. atovaquone or clindamycin plus pyrimethamine are alternative agents (martin and fishman ) . trimethoprim-sulfamethoxazole prophylaxis for - months after transplantation is highly effective in preventing this infection and should be administered to all renal transplant patients if tolerated. frequently used alternatives for prophylaxis in allergic patients include dapsone (if glucose- phosphate dehydrogenase levels are normal) and atovaquone. infection remains an important concern in patients undergoing kidney transplantation. attention to pretransplant screening of the potential organ donor and recipient is essential to optimizing transplant outcomes. advances in the management of transplant-related infections include the increasing use of rapid molecular diagnostic testing as well as improvements in the approach to prophylaxis and treatment. ongoing challenges include the need for prolonged immunosuppression to prevent organ rejection, drug-drug interactions, and the management of resistant and emerging pathogens. continued awareness of the risks, timing, and presentation of infection posttransplant and strategies to reduce its impact will contribute further to progress in the field of kidney transplantation. hospitalizations for bacterial septicemia after renal transplantation in the united states epstein-barr virus and posttransplant lymphoproliferative disorder in solid organ transplant recipients late-onset acute haemorrhagic necrotizing granulomatous adenovirus tubulointerstitial nephritis in a renal allograft nocardiosis in renal transplant recipients undergoing immunosuppression with cyclosporine long-term outcomes of cmv disease treatment with valganciclovir versus iv ganciclovir in solid organ transplant recipients dilemma of hcv infection in renal transplant recipients lymphocytic choriomeningitis 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on the development of cytomegalovirus disease after renal transplantation successful treatment of hepatitis c in renal transplant recipients with direct-acting antiviral agents phs guideline for reducing human immunodeficiency virus, hepatitis b virus, and hepatitis c virus transmission through organ transplantation prevalence of hiv-associated nephropathy in autopsies of hiv-infected patients human herpesvirus- in transplantation: an emerging pathogen rates of first infection following kidney transplant in the united states up to date transmission of rabies virus from an organ donor to four transplant recipients sirolimus for kaposi's sarcoma in renal transplant recipients outcomes of kidney transplantation in hiv-infected recipients preemptive treatment for cytomegalovirus viremia to prevent cytomegalovirus disease in solid organ transplant recipients viral infections affecting the skin in organ transplant recipients: epidemiology and current management strategies long-term results in human t-cell leukemia virus type -positive renal transplant recipients post-transplant lymphoproliferative disorders (ptld) after solid organ transplantation the association of viral infection and chronic allograft nephropathy with graft dysfunction after renal transplantation polyomavirus in kidney and kidney-pancreas transplant recipients epidemiology of kidney disease in the unites states. national institutes of health, national institute of diabetes and digestive and kidney diseases in: safdar a (ed) principles and practice of transplant infectious diseases nocardial infections in renal transplant recipients the evaluation and management of urinary tract infections in recipients of solid organ transplants treatment and prevention of legionella infection key: cord- -lq yr authors: cunningham, steve title: bronchiolitis date: - - journal: kendig's disorders of the respiratory tract in children doi: . /b - - - - . - sha: doc_id: cord_uid: lq yr acute viral bronchiolitis is a common viral lower respiratory tract infection in young children. most typically caused by respiratory syncytial virus in % of cases, the condition lasts for to days, with a prolonged cough in many. children with comorbidity, particularly those born prematurely or with significant congenital heart disease, are at risk of more severe disease. nasal obstruction progresses over to days to difficulty with feeding and increased work of breathing with hypoxemia. crackles and/or wheeze may be auscultated. apnoea may be a presenting sign in those less than months of age. viral load is highest at peak of symptoms and in those with more severe disease. approximately % to % of all children are admitted to hospital with bronchiolitis. the differential diagnosis may include bacterial pneumonia, congenital lesions of the lung or heart, or an interstitial lung disease. there are no effective treatments, and admission is for feeding support (by nasogastric or intravenous fluids) or treatment of hypoxemia. critical care support is required for some infants experiencing respiratory failure, though mortality rates remain unchanged. practice within and between countries varies significantly and alignment of practice is a common goal of guidelines. vaccines for rsv are in advanced development, as are several antiviral therapies for rsv. in most children, acute symptoms improve within to days and cough by weeks. recurrent wheeze is common following acute bronchiolitis and a good association with a diagnosis of asthma in childhood. rsv infects % to % of infants in the first year of life. , the rapid development of vaccines and treatment therapies for rsv has added impetus to the need to better define the burden of rsv disease. globally there are an estimated . million cases of rsv lower respiratory tract infection each year in children under years of age, resulting in . million admissions to the hospital and to thousand deaths (with the majority in low-and middle-income countries). in the united states an estimated % of children will attend primary care each year with rsv bronchiolitis, and up to % attend an emergency department (ed). admission to hospital with rsv bronchiolitis is typically around . % of all infants, , though in previously healthy term infants, the admission rate to hospital with rsv bronchiolitis can be as low as . %. in infants who are born preterm at to weeks' gestation, % will develop bronchiolitis and % require admission to the hospital. the risk of bronchiolitis is increased in a range of conditions compared with term infants, including preterm birth (respiratory rate [rr] . ) , cystic fibrosis (rr . ), congenital heart disease (rr . ), chronic lung disease (rr . ), immunodeficiency (rr . ), down syndrome (rr . ), and cerebral palsy (rr . ). , epidemiology bronchiolitis is the most common lower respiratory tract infection in children. the condition forms part of the spectrum of viral lower respiratory tract infection that includes bronchiolitis, viral pneumonia, and viral-induced wheeze. in polar hemispheres (north and south), bronchiolitis is a seasonal disease, dominating winter months, with a peak over to weeks around the winter solstice. in tropical climates, the disease is associated with rainy months and is seasonally more dispersed. climate and environment appear to influence both season and severity. , bronchiolitis is diagnosed clinically by integrating characteristic but variable signs and symptoms across a broad age range, though the majority of cases occur in children under year of age. the condition can be caused by any respiratory virus and has a wide spectrum of disease severity. a "classic" case would be an infant aged to months of age who develops coryza and over the subsequent to days has increased difficulty with breathing, and consequent inability to maintain adequate oral feeding. wheeze or crackles can be heard on auscultation. improvement occurs by days to , though a characteristic harsh cough may persist for days or more. , while the diagnosis often appears straightforward, the wide range of disease severity across a skewed but broad age range and the need for clinical diagnosis (with associated inconsistency) creates difficulty in establishing precise data. in addition, while bronchiolitis is a clinical diagnosis applied to any infecting agent, the majority of data available relate to bronchiolitis caused by respiratory syncytial virus (rsv) infection; and within rsv bronchiolitis is a focus on those at high risk, in particular, those born prematurely. reference to these groups synonymously with bronchiolitis can make interpretation of epidemiological data difficult and may reduce the understanding of bronchiolitis caused by non-rsv and in lower risk patients (particularly children born at term). there are only limited estimates of population risk for bronchiolitis associated with all respiratory virus infections, but approximately % of infants are affected by bronchiolitis in the first year of life. in the united kingdom, using primary care databases, the year incidence of children given a specific diagnosis of bronchiolitis is to per children, , rising to per when a broader definition of bronchiolitis was used to capture potential cases. this study highlights that in children with typical lower respiratory tract signs and symptoms, clinicians may not ascribe the discrete diagnosis of bronchiolitis; a finding also found keywords bronchiolitis viral lower respiratory tract infection wheeze respiratory syncytial virus abstract acute viral bronchiolitis is a common viral lower respiratory tract infection in young children. most typically caused by respiratory syncytial virus in % of cases, the condition lasts for to days, with a prolonged cough in many. children with comorbidity, particularly those born prematurely or with significant congenital heart disease, are at risk of more severe disease. nasal obstruction progresses over to days to difficulty with feeding and increased work of breathing with hypoxemia. crackles and/or wheeze may be auscultated. apnoea may be a presenting sign in those less than months of age. viral load is highest at peak of symptoms and in those with more severe disease. approximately % to % of all children are admitted to hospital with bronchiolitis. the differential diagnosis may include bacterial pneumonia, congenital lesions of the lung or heart, or an interstitial lung disease. there are no effective treatments, and admission is for feeding support (by nasogastric or intravenous fluids) or treatment of hypoxemia. critical care support is required for some infants experiencing respiratory failure, though mortality rates remain unchanged. practice within and between countries varies significantly and alignment of practice is a common goal of guidelines. vaccines for rsv are in advanced development, as are several antiviral therapies for rsv. in most children, acute symptoms improve within to days and cough by weeks. recurrent wheeze is common following acute bronchiolitis and a good association with a diagnosis of asthma in childhood. for infants born ≤ weeks gestation, % of infants will have a lower respiratory tract infection in the first year of life, with % rsv positive and % rsv negative; of these infants, % of rsv positive will be admitted to the hospital versus % of rsv negative. recent studies suggest that hospitalization rates for high-risk infants due to rsv are reducing over time and are now similar to those for rsv negative, possibly as a result of improvements in neonatal care or immunoprophylaxis in high-risk groups ( fig. . ). , risk of death is much higher amongst high-risk groups who are rsv positive, including preterm ( . %), congenital heart disease ( . %), and bronchopulmonary dysplasia ( . %). bronchiolitis has a viral etiology, with rsv the most common cause, reported in % to % , of cases. other viruses associated with bronchiolitis are human rhinovirus ( %), influenza, coronavirus, human metapneumovirus, adenovirus, parainfluenza virus and human boca virus , ; that is, "any respiratory virus." rsv has two strains, a and b, with rsv a associated with more severe disease. , reinfection in the same season with the same or different strain is possible. as a sole infecting agent, rsv is associated with more severe bronchiolitis than other single respiratory virus infections. coinfection of rsv with rhinovirus can produce even more severe disease. rsv is the most common infectious agent in children admitted to the hospital with radiological features consistent with pneumonia (occurring in % of children-most commonly those under years of age). in young children who are well immunized, rsv represents the most common cause of lower respiratory tract infection. what commences as an upper respiratory tract infection becomes a lower respiratory tract infection over the course of to days. infants are particularly susceptible as they have small bronchi that are more likely to become blocked by secretions and edema, and a less well-developed ability to respond to and clear viral infection. histopathology is naturally limited to the most severe cases who have died, where the bronchioles are edematous and blocked by necrotic epithelium and neutrophils, with some mucus binding this debris together. airway obstruction is intensified by poor airway clearance associated with loss of cilia function occurring within hours and persisting for up to months after the illness. destruction of cilia is considered to be caused by virus replication and not mediated by inflammation. rsv is associated with more severe airway pathology than that found in children dying from other respiratory viruses, even in those not mechanically ventilated. viral shedding is higher and more prolonged in younger infants and those with more severe disease. increased disease severity, longer hospital stay and use of intensive care is associated with higher viral load for rsv in nasopharyngeal secretions. , severity of disease is associated with both infant risk factors (including lack of adaptive t cell response), , but also rsv virus specific factors (viral antigen load and direct cytotoxic effects). determining the relative contribution of both of these to disease severity will be important; if the latter is dominant, antiviral agents provided early in the course of the disease may reduce severity, whereas dominance of the former might need additional immunomodulators. biomarkers are now sought to better characterize those at risk of greater disease severity and to indicate recovery. infants hospitalized with rsv bronchiolitis have increased interleukin (il)- and il- in secretions. polymorphisms of surfactant protein a are associated with increased risk of intensive care admission. cysteinyl leukotrienes are increased in infants with rsv bronchiolitis and are still increased month following infection. more severe disease is also associated with increased serum cathelicidin, lactate dehydrogenase, caspase and il- . there is some evidence that more severe disease may be associated with an insufficient inflammatory response. the interrelationship of the microbiome in bronchiolitis is also being actively explored. bronchiolitis is diagnosed clinically. variance in the clinical interpretation of symptoms and physical findings lead to inconsistency in diagnosis, particularly in milder cases and children over year of age. typical symptoms are rhinorrhea, proceeding over to days to a characteristic harsh moist cough with pyrexia that is typically below °c, although fever above . °c is seen in % of infants. ability to achieve adequate oral feeding declines as nasal obstruction with secretions develops and work of breathing increases. the time to peak symptoms of days is associated with the peak in viral load, , varying from infant to infant. in younger children (particularly < weeks of age), apnea may be a presenting sign, sometimes in the absence of other features of bronchiolitis. apnea may be temporarily improved by nasal suctioning, but it is most likely a direct viral effect in young infants. apnea is a "red flag" sign in bronchiolitis that warrants a period of review in a supervised clinical setting to ensure that it has resolved. patients more likely to require intensive care include preterm infants and those with apnea, low birth weight, or a respiratory rate greater than /min. , children tend not to relapse during the improving phase of the illness, which should give confidence to clinicians when considering discharge from ed or hospital. , physical findings include an increased respiratory rate, chest recession, use of accessory muscles, hyperinflation, wheezing, crackles, and reduced arterial oxygen saturations. physical findings vary depending on sleep state (and associated changes in tidal volume). respiratory rate is a key marker of disease severity, with ≥ /min considered severe and ≥ / min critical. , oxygen saturation may be improved (at least temporarily) by removal of nasal secretions. bronchiolitis is a highly variable disease that requires assessment of disease severity by clinicians for decision making, some of which is subjective. clinical scoring systems have been developed in an attempt to standardize care and minimize variance. many early scores derived from asthma scores. the most commonly applied scores for bronchiolitis are outlined in ) and to improve the ability to identify those at risk of deterioration. the ability of clinical scores to retain precision and reliability, when scoring is performed by larger numbers of health care professionals in the context of multicenter phase iii trials, is of current interest. symptoms in bronchiolitis vary across a wide but skewed continuum from mildly increased work of breathing with cough to respiratory failure and death. often divided into mild, moderate, and severe disease, the perspective on these gradations varies across health care systems. a world health organization (who) workshop has provided candidate definitions differentiating a diagnosis of rsv lower respiratory tract infection (spo < %) from severe (< %) and very severe rsv disease (spo < %, inability to feed orally, or reduced level of consciousness). infants can display variance in spo within this range ( % to %) over short periods of observation without significant change in clinical status, , , which may limit the discriminatory reliability of these definitions. from a secondary care perspective, moderate severity is often considered a need for admission to hospital and severe by need for critical care (positive pressure support). clinical scores are often designed to identify transition points in the level of care required. the currently available evidence concerning transition points in level of care is poor. treatment guidance, particularly benefit from use of interventions at the ed/ward (i.e., spo ) and ward/critical care floor interface (i.e., highflow nasal cannula [hfnc] oxygen and continuous positive airway pressure [cpap]) is much needed. guidelines have provided signs and symptoms that should alert clinicians to a child at risk of deterioration and suggested criteria for admission to the hospital. in hospitals, those most likely to deteriorate to the extent of being provided with critical care support are of lower birth weight (< lbs, . kg) and/or have a respiratory rate ≥ /min on day of admission. chest radiography is not required to confirm a diagnosis of bronchiolitis. a chest radiograph often leads to increased diagnostic uncertainty as the features may be similar to those of pneumonia (atelectasis, mucous plugging, and loss of volume) and consequently lead to greater inappropriate use of antibiotics. chest radiography should be reserved for a child who is atypical, for example, showing persistently focal crackles, a temperature remaining above °c despite antipyretics, or respiratory failure requiring critical care support. , laboratory tests do not aid in the clinical diagnosis of bronchiolitis. serious bacterial infection is unusual and complete blood counts and blood cultures are unhelpful (though recent evidence suggests that although still uncommon, it may be more frequent than previously considered). dehydration is usually mild and best assessed clinically without electrolyte measurement. approximately % of infants with bronchiolitis can have concurrent urinary tract infection, so urine culture may be of value in persistently febrile infants, particularly those under months of age. measurement of arterial/capillary carbon dioxide is commonly performed, but can be restricted to those children with increased respiratory rate and work of breathing despite oxygen supplementation. the clinical interpretation of signs and symptoms is difficult in a condition where age boundaries are loose (and skew to older ages in those with comorbidity) and symptoms vary from patient to patient and time to time. this naturally leads to variation in diagnosis and differential diagnosis. there is a common understanding that a clearer diagnosis is possible in those under year of age and most guidelines reflect this. however, constraining a diagnosis of bronchiolitis to those less than year of age may reduce the ability to identify the whole population of children with bronchiolitis who could benefit from potential interventions. in general, a broader bronchiolitis) is often performed to aid cohorting of patients within hospitals. the increasing recognition that multiple viruses may be identified in those with acute bronchiolitis has called into question the benefit of cohorting based on rsv status. pcr diagnostics may sometimes be considered oversensitive to the detection of virus fragments postinfection, and multiplex pcr results should be interpreted with this understanding. differential diagnosis includes bacterial pneumonia or an alternative cause of crackles, wheeze, and increased work of breathing in a young child. persisting crackles (crepitations) in one lung zone, fixed focal wheeze, persistent pyrexia (> °c) or persistently increased work of breathing in a child who appears otherwise recovered warrant further evaluation. prescient in the mind of most clinicians is that a bacterial pneumonia may be missed. chest radiographs have similar appearances and are poor discriminators. we can assume that bacterial coinfection risk is low, as the use of antibiotics in bronchiolitis is not associated with faster recovery. further investigation could be limited to those with the persisting clinical features noted above. in children with more severe disease, there may be a role for antibiotics as bacteria are isolated in % to % of lavage samples in children with severe bronchiolitis who are intubated and ventilated. [ ] [ ] [ ] definition of bronchiolitis is used in north america and asia that captures a higher percentage of older children with wheezing, where rhinovirus is the dominant infecting agent. such children may be given a diagnosis of viral induced wheeze in other countries. there is most likely a continuum of viral lower respiratory tract infection across age ranges that moves from current diagnoses of viral bronchiolitis to viral pneumonia and viral-induced wheeze/wheezy bronchitis. , the clinical features consistent with a diagnosis of bronchiolitis across different guidelines are presented in table . . diagnosis has a typical onset of a viral respiratory tract prodrome proceeding to lower respiratory symptoms over to days. the south african guideline ( ) considers hyperinflation the most reliable clinical sign in bronchiolitis. the uk guideline ( ) provides a more proscriptive definition. testing of nasal secretions for virus may help consolidate the clinical diagnosis of bronchiolitis and inform health care logistics. most commonly used, and with highest precision, are polymerase chain reaction (pcr) diagnostics for a range of respiratory viruses, but point of care (poc) testing for a more limited range of viruses (most often rsv) is increasingly precise and cost effective. testing for rsv (as the most common infecting agent in therapies in addition to supplemental oxygen and hydration are poorly supported by current evidence. there is some evidence that infants handled less get better quicker, and the use of additional therapies should be considered with that in mind. there is widespread variation across hospitals and countries in the management and treatment of bronchiolitis reflecting local custom and individual clinician practice. reducing variation and associated health care costs is a key aim of bronchiolitis management presented through guidelines. guidelines for the care of infants with bronchiolitis based on systematic review and published in english are available from the united kingdom ( ), and south africa ( ), which has been updated as a critical review . no therapies receive support across all guidelines for use with the exception of supplemental oxygen. chest physiotherapy does not speed recovery. antibiotics, though still widely used, are of no benefit in bronchiolitis. in addition, bronchodilators are less likely to be recommended in more recent guidelines, and the theory that they may be of greater benefit in infants more likely to develop asthma has been refuted. nebulized hypertonic saline has been of benefit in cystic fibrosis and in early trials in bronchiolitis, but larger well-designed trials have not demonstrated a persuasive benefit. [ ] [ ] [ ] [ ] [ ] recent years have seen the increasing use of hfnc oxygen in acute bronchiolitis. though clinical trials have not yet demonstrated important clinical or physiological benefits, large well-designed trials are in progress and are beginning to report. a, cpap has some benefit in bronchiolitis, and may prevent deterioration when used early. as with all management in bronchiolitis, the use of hfnc oxygen, cpap, and intubation varies across sites irrespective of disease severity, and better understanding of the risks and benefits of these interventions is required. there are no current effective pharmacological treatments for rsv. while ribavirin was previously used as an antiviral treatment for rsv, it is now considered ineffective. novel treatments for acute infection are in development: antivirals and nebulized immunoglobulin. in this rapidly moving field, it seems probable that a treatment for rsv will become available in the next years. reduction in viral load has been demonstrated in adult challenge models of rsv treated with the antivirals als- and gs- prevention of spread of rsv depends on good hygiene, in particular, hand washing, as rsv may survive for up to hours on surfaces contaminated by droplets. similar precautions are appropriate for other respiratory virus infections associated with bronchiolitis. many hospitals use poc testing for rsv to determine cohorting of infants as inpatients. while this is still common, the practice is called into question by the range of coinfection with other respiratory viruses revealed by pcr panel testing; up to % of children with viral respiratory tract infection have more than one virus detected. prevention of rsv (as the most common cause of bronchiolitis) has been a long-term goal. early formalin inactivated there are no trials of outcome for antibiotic use in children with bronchiolitis receiving intensive care. though uncommon, congenital lesions may masquerade as bronchiolitis, and this should be borne in mind for children with atypical clinical features or those slow to recover. congenital heart disease may present as bronchiolitis when pulmonary vascular resistance falls increasing left to right shunt. more difficult to differentiate are children with congenital (or less commonly acquired) pulmonary malformations. fixed focal wheeze may be a sign of tracheomalacia or bronchomalacia, stenosis, or compression from lobar emphysema or a bronchogenic cyst and would warrant a chest radiograph. a slow recovering course with persistent chest signs could be an infected congenital pulmonary malformation (such as a congenital cystic adenomatoid malformation [ccam] or sequestration). children with persistent fine crackles, tachypnea, and low (often borderline) oxygen saturation may have an interstitial lung disease, particularly neuroendocrine cell hyperplasia (nehi) presenting as recurrent "bronchiolitis." young children with persistent, sometimes focal, crackles postadenovirus (though may also be other respiratory viruses and mycoplasma pneumonia) should be evaluated for postinfectious bronchiolitis obliterans (pibo); see section of the book. management of bronchiolitis is supportive, assisting hydration and hypoxemia until improvement. with increased respiratory rate and nasal secretions, oral feeding is challenged, and those with severe disease require assistance with feeding by enteral or parenteral means. the threshold for supporting hydration is typically when an infant's intake is reduced to % to % of usual volume. the chosen percentage of intake depends on the child's status: an expreterm -week-old infant on day of illness may be supported at % understanding that they most likely will deteriorate, whereas a robust -month-old term infant may be able to tolerate % feed volume for a couple of days until disease resolution. nasogastric feeding is easier to administer than intravenous fluids but has no advantage in recovery from acute disease. oxygen may be used to treat hypoxemia. the threshold oxygen saturation at which to use supplemental oxygen varies across guidelines and is typically set between % and % at sea level. in children admitted to hospital with bronchiolitis, management at a threshold of % spo is safe and as clinically effective as a % target. the threshold oxygen saturation for admission to hospital is often %, as some data suggest that infants have a higher risk of desaturating further at this oxygen saturation. oxygen desaturation may however have a disproportionate influence on decisions to admit children to the hospital, and much like hydration status (previously mentioned), the context of the measurement should be considered. many infants discharged home from ed with bronchiolitis experience desaturation events subsequently that are not associated with clinical deterioration. in hospitals, the use of intermittent oxygen saturation monitoring is much discussed, and though the benefit below % spo is not established, once stable above % spo , oxygen saturation monitoring should be stopped. use of bronchiolitis occurs in % of those who are rsv positive and % of those who are rsv negative. recurrent postinfectious wheeze is not reduced by montelukast or inhaled corticosteroids, , but there is good evidence of benefit from palivizumab, and potentially azithromycin, though the latter requires further study. in the longer term, there is good evidence that children who have had an admission to the hospital for rsv bronchiolitis are times more likely to have a diagnosis of asthma and lower lung function at age years and a higher incidence of asthma at age and years. the question remains whether such children are predisposed to bronchiolitis because of premorbid anatomy and the consequent interrelationship between host and virus specific effects on the development of asthma. vaccines were associated with more severe enhanced rsv disease and deaths, possibly resulting from inadequate t cell priming. subsequent vaccine development has been cautious in view of this experience. in the s, rsv intravenous immunoglobulin was developed , but was rapidly superseded by palivizumab, a monoclonal antibody delivered by monthly intramuscular injection. when administered over the rsv season, it reduces hospital admission in high-risk infants. palivizumab's monthly injections and limited efficacy have prompted the development of extended life monoclonal antibodies that are undergoing licensing trials in preterm infants. they will hopefully be evaluated in the future for high-risk infants born at term. rsv vaccine development has gained significant impetus over the last years with a wide range of candidate vaccines in development both for pediatric and maternal use; maternal immunization could provide passive transplacental protection to infants in the first to months of life (http:// www.path.org). a phase iii trial of maternal immunization by novovax is expected to conclude in . for most children, 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children with bronchiolitis mechanical ventilation drives inflammation in severe viral bronchiolitis bronchiolitis in children (sign ). nhs quality improvement: scotland respiratory syncytial virus, an ongoing medical dilemma: an expert commentary on respiratory syncytial virus prophylactic and therapeutic pharmaceuticals currently in clinical trials. influenza other respir viruses activity of oral als- in a respiratory syncytial virus challenge study oral gs- activity in a respiratory syncytial virus challenge study possible transmission by fomites of respiratory syncytial virus viral coinfection in childhood respiratory tract infections respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine key: cord- -yd p buu authors: acevedo, orlando a.; díaz, fabián e.; beals, tomas e.; benavente, felipe m.; soto, jorge a.; escobar-vera, jorge; gonzález, pablo a.; kalergis, alexis m. title: contribution of fcγ receptor-mediated immunity to the pathogenesis caused by the human respiratory syncytial virus date: - - journal: front cell infect microbiol doi: . /fcimb. . sha: doc_id: cord_uid: yd p buu the human respiratory syncytial virus (hrsv) is the leading cause of severe acute lower respiratory tract infections (alrtis) in humans at all ages and is the main cause of hospitalization due to pneumonia, asthma, and bronchiolitis in infants. hrsv symptoms mainly develop due to an excessive host immune and inflammatory response in the respiratory tissue. hrsv infection during life is frequent and likely because of non-optimal immunological memory is developed against this virus. vaccine development against this pathogen has been delayed after the detrimental effects produced in children by vaccination with a formalin-inactivated hrsv preparation (fi-hrsv), which caused enhanced disease upon natural viral infection. since then, several studies have focused on understanding the mechanisms underlying such disease exacerbation. along these lines, several studies have suggested that antibodies elicited by immunization with fi-hrsv show low neutralizing capacity and promote the formation of immune complexes containing hrsv (hrsv-ics), which contribute to hrsv pathogenesis through the engagement of fc gamma receptors (fcγrs) expressed on the surface of immune cells. furthermore, a role for fcγrs is supported by studies evaluating the contribution of these molecules to hrsv-induced disease. these studies have shown that fcγrs can modulate viral clearance by the host and the inflammatory response triggered by hrsv infection. in addition, ics can facilitate viral entry into host cells expressing fcγrs, thus extending hrsv infectivity. in this article, we discuss current knowledge relative to the contribution of hrsv-ics and fcγrs to the pathogenesis caused by hrsv and their putative role in the exacerbation of the disease caused by this virus after fi-hrsv vaccination. a better understanding fcγrs involvement in the immune response against hrsv will contribute to the development of new prophylactic or therapeutic tools to promote virus clearance with limited inflammatory damage to the airways. the human respiratory syncytial virus (hrsv) is the leading cause of severe acute lower respiratory tract infections (alrtis) in humans at all ages and is the main cause of hospitalization due to pneumonia, asthma, and bronchiolitis in infants. hrsv symptoms mainly develop due to an excessive host immune and inflammatory response in the respiratory tissue. hrsv infection during life is frequent and likely because of nonoptimal immunological memory is developed against this virus. vaccine development against this pathogen has been delayed after the detrimental effects produced in children by vaccination with a formalin-inactivated hrsv preparation (fi-hrsv), which caused enhanced disease upon natural viral infection. since then, several studies have focused on understanding the mechanisms underlying such disease exacerbation. along these lines, several studies have suggested that antibodies elicited by immunization with fi-hrsv show low neutralizing capacity and promote the formation of immune complexes containing hrsv (hrsv-ics), which contribute to hrsv pathogenesis through the engagement of fc gamma receptors (fcγrs) expressed on the surface of immune cells. furthermore, a role for fcγrs is supported by studies evaluating the contribution of these molecules to hrsv-induced disease. these studies have shown that fcγrs can modulate viral clearance by the host and the inflammatory response triggered by hrsv infection. in addition, ics can facilitate viral entry into host cells expressing fcγrs, thus extending hrsv infectivity. in this article, we discuss current knowledge relative to the contribution of hrsv-ics and fcγrs to the pathogenesis caused by hrsv and their putative role in the exacerbation of the disease caused by this virus after fi-hrsv vaccination. a better understanding fcγrs involvement in the immune response against hrsv will contribute to the development of new prophylactic or therapeutic tools to promote virus clearance with limited inflammatory damage to the airways. the human respiratory syncytial virus (hrsv) is a singlestranded rna enveloped virus belonging to the pneumoviridae family (amarasinghe et al., ) . the viral particle has a filamentous structure, which consists in a nucleocapsid surrounded by a lipid bilayer envelope obtained from the plasma membrane of the host cell (el omari et al., ) . importantly, infection by hrsv is the most frequent cause of severe acute lower respiratory tract infections (alrtis) in children younger than years old (scheltema et al., ) and infection during the first year of life is the main cause of hospitalization in infants (song et al., ) . according to epidemiological studies, during the past decade, nearly million cases of new alrtis episodes affect children during the first months of life are due to hrsv infection each year (shi et al., ) . therefore, infection by this virus represents a major health and socio-economic burden worldwide (diez-domingo et al., ; amand et al., ) . clinical manifestations caused by hrsv infection range from mild symptoms, such as rhinitis, to more severe consequences, which include bronchiolitis, and pneumonia (pickles and devincenzo, ) . besides, extra-pulmonary manifestations of hrsv infection have also been reported to occur, such as acute neurological symptoms with seizures and ataxia observed in hrsv-infected children (eisenhut, ; bohmwald et al., ) and long-term behavioral and cognitive impairments in animal models (espinoza et al., ) . remarkably, it is known that most children become infected with hrsv during the first years of life (domachowske and rosenberg, ) , likely because hrsv can efficiently pass on from one individual to another, but also because of the capacity of this virus to negatively modulate both, t cell and b cell responses upon infection allowing frequent re-infections (prabhudas et al., ; cespedes et al., ; zhivaki et al., ) . these features are thought to be mediated by host and viral factors. for instance, it is known that infants show reduced capacity to produce neutralizing antibodies against hrsv, as compared to adults making the former more susceptible to recurrent infections (siegrist and aspinall, ) . although maternally-delivered antibodies (matabs) are reported to delay the onset of primary hrsv infection, their presence in the blood of infants is not associated with the development of less severe disease symptoms (jans et al., ) . these observations suggest that antibody-mediated neutralization of hrsv may not be sufficient by itself to limit hrsv infection and disease severity. furthermore, hrsv encodes several proteins that have the ability to negatively modulate or impair the host antiviral immune response, therefore contributing to re-infections (mason et al., ; cespedes et al., ; saint et al., ; bohmwald et al., ; gomez et al., ; canedo-marroquin et al., ; ward et al., ) . such knowledge is relevant for designing novel vaccines and therapeutic approaches that can prevent the pathology caused by hrsv. as a result, several clinical trials are currently in progress to assess the safety and effectiveness of different hrsv vaccine candidates (cautivo et al., ; rey-jurado and kalergis, ; rezaee et al., ) . among them, we have developed a unique approach to be administered to newborns and young infants. immunization in the mouse model with a recombinant bacillus of calmette-guérin (bcg) that expresses the nucleoprotein (n) of hrsv (rbcg-n-hrsv) induce the production of neutralizing antibodies against hrsv and a t helper (th ) cellular immunity that protects from hrsv associated-lung pathology by decreasing the infiltration of inflammatory immune cells into the lungs and reduce viral loads in the airways of hrsv-infected mice (bueno et al., ; cautivo et al., ; leyrat et al., ) furthermore, a single low dose of this vaccine produced using current good manufacturing practices (cgmp), conferred protection against hrsv infection in the mouse model (cespedes et al., ) . given these results, this recombinant-based vaccine arises as a promising candidate to prevent lung damage caused by this virus (cespedes et al., ) . in this context, it is possible that a mechanism that contributes to the prevention of hrsv pathology following rbcg-n-hrsv vaccination is the induction of antibodies that recognize the hrsv n protein, which is necessary for viral replication and the inhibition of the immunological synapse (is) between dcs and t cells that promote t-cell activation (cespedes et al., ) . therefore, if the hrsv n protein becomes neutralized by antibodies during infection it cannot contribute to viral replication, but also will fail in its ability to impair the formation of the is between dcs and t cells, thus hampering a proper immune response against hrsv. furthermore, a recent publication from our group shows that immunization with rbcg-n-hrsv can induce the production of antibodies against other hrsv proteins, such as f and g which can serve to neutralize infection, therefore reducing hrsv associated pathology (soto et al., ) . in contrast, vaccine candidates from other groups use the f protein as a target antigen to confer immunity. for example, novavax inc. is currently performing a clinical trial based on the use of nanoparticles linked with hrsv f protein to induce the production of neutralizing antibodies against hrsv (mazur et al., ) . similarly, janssen is currently testing adenovirus based vector vaccines, encoding pre-fusion forms of the hrsv f protein that also induce the production of anti-hrsv neutralizing antibodies (mazur et al., ) . finally, other live attenuated vaccines as is the case of rbcg-n-hrsv are based in attenuated hrsv that lack some particular proteins such as m - , ns , or both (mazur et al., ) . together, these data indicate that it is of vital importance to delineate the mechanisms contributing to hrsv induced pathology in order to prevent or treat infection. at this latter point, recurrent hrsv re-infection episodes which are common thorough life have encouraged the generation of studies that seek to define the mechanisms responsible for what is considered an impaired or non-optimal immune response elicited against hrsv upon infection to account for re-infection episodes (openshaw and chiu, ; cespedes et al., ; shao et al., ) . along these lines, a role for the interaction between immune complexes consisting of iggs and hrsv (ics) with fc gamma receptors (fcγrs) could be a process contributing to both, re-infection episodes, and enhancement of hrsv-disease elicited by vaccination with formalin-inactivated hrsv (fi-hrsv) and later hrsv natural infection (kim et al., ) . this hypothesis is supported by the fact that high amounts of antibodies with low neutralizing activity can be induced by immunization with fi-hrsv, which correlates with enhancement of the hrsv-induced disease (kapikian et al., ; kim et al., ) . therefore, it is possible that these low affinity antibodies promote the infection of fcγr-bearing cells through a phenomena called antibody dependent enhancement (ade), as previously observed for other viruses (yip et al., ; gu et al., ; flipse et al., ) . furthermore, in vitro and in vivo studies have shown that the blockade or absence of particular fcγrs expressed on the surface of immune cells can modulate the immune response against this virus and the onset of hrsvinduced disease (osiowy et al., ; kruijsen et al., ; gomez et al., ; van erp et al., ) . in this article, we review and discuss the current understanding on the contribution of fcγrs to infection and the modulation of the immune response against hrsv both, in vitro and in vivo and their impact on hrsv-induced pathology. fc-gamma receptors (fcγrs) bind to immunoglobulin g (igg) antibodies (ab), by recognizing the fc region of the igg, which promotes receptor clustering on the cell surface and the phosphorylation of tyrosine residues present on signaling motifs within the intracellular region of these receptors. fcγrs engagement ultimately leads to signaling cascades in the cell that can result in the expression of surface molecules and secretion of soluble mediators to modulate the host immune responses (getahun and cambier, ; renner et al., ) ; (soto et al., ) . importantly, these types of receptors are expressed on the surface of immune cells, such as neutrophils, dendritic cells (dcs) and macrophages, among others (zhang et al., ) . in general, classic members of this family of proteins were classified according to their immune-modulatory properties, which either promote or inhibit inflammatory responses (nimmerjahn and ravetch, ; guilliams et al., ) . however, fcγrs can also be classified as type-i or type-ii, based on their capacity to interact with the two (open or closed) conformational states of the igg fc domain (banegas banegas et al., ) . type-i fcγrs include the classic fcγrs and can only be engaged by the igg fc domain in the open conformation state (banegas banegas et al., ) . in contrast, type-ii (non-canonical fcγrs), include c-type lectin receptors cd and dendritic cell-specific intercellular adhesion molecule- -grabbing nonintegrin (dc-sign), which preferentially bind igg fcs in a closed conformation (banegas banegas et al., ) . in humans, the so-called classic fcγrs are known as: fcγri (cd ), fcγriia (cd a), fcγriib (cd b), fcγriic (cd c), fcγriiia (cd a), and fcγriiib (cd b) ( table ) (tripp et al., ; guilliams et al., ) . among them, a study performed during indicates that the expression of fcγriiia is increased in natural killer cells (nk cells) from patients with severe hrsv associated pathology. thus, suggesting that this receptor and this particular cell population could be contributing to hrsv disease ( table , tripp et al., ) . nevertheless, there are two more non-classic human fc-gamma receptors: neonatal fc-receptor (fcrn) and cytosolic tripartite motif (trim) that bind igg once internalized into the cells (guilliams et al., ) . however, there is no study about it contribution to hrsv induced pathology in hrsv positive patients (table ) . importantly, all canonical fcγrs with the exception of fcγriib are involved in activating functions, such as phagocytosis, antibody-dependent cellular cytotoxicity (adcc) and the release of inflammatory cytokines following fcγrcrosslinking by igg-opsonized complexes (guilliams et al., ) . the activation of such processes relies on the src-family kinasemediated phosphorylation of an immunoreceptor tyrosinebased activating motif (itam) that is located in the cytoplasmic portion of these activating fc-receptors (nimmerjahn and ravetch, ) . subsequently, phosphoinositide -kinase (pi k) is activated, which generates phosphatidylinositol trisphosphates (pip s), leading to the recruitment of bruton's tyrosine kinase (btk) and the activation of phospholipase cγ (plcγ), which promotes the release of calcium (ca + ) from the endoplasmic reticulum (er) that in turn activates cell effector functions (nimmerjahn and ravetch, ) . in contrast, fcγriib which is able to terminate the activation cascades associated with the engagement of activating fcγrs (malbec et al., ) , is also known as the inhibitory fcγr. during this process fcγriib becomes engaged by ics and it co-aggregates with activating receptors. following that, different recruited kinases phosphorylate a conserved tyrosine within an immunoreceptor tyrosine-based inhibitory motif (itim) located in the cytoplasmic tail of fcγriib (malbec et al., ) . this phosphorylation step leads to the recruitment of tyrosine phosphatases shp- and shp- , as well as the inositol phosphatases ship- and ship- that suppress the activating signals derived from activating fcγrs (d'ambrosio et al., ; ono et al., ) . in the mouse, there are four different canonical fcγrs expressed on the cell surface: fcγri, fcγriib, fcγriii, and fcγriv ( table ) (nimmerjahn and ravetch, ) . among them, fcγri, fcγriii, and fcγriv are activating, whereas fcγriib is the only one that is inhibitory. of interest, a proinflammatory role for the fcγriii receptor has been reported during hrsv infection in the mouse model , whereas the inhibitory fcγriib has been shown to hamper inflammatory reactions during allergic-like rhinitis (malbec et al., ) , allergic asthma (d'ambrosio et al., ) , and hrsv infection . therefore, such receptors appear as attractive targets for novel therapeutic approaches against this kind of diseases. based on animal studies, neutrophils have been described to promote inflammation and tissue damage during hrsv infection (yasui et al., ) . in addition, other studies in mice that evaluated the role of fcγrs on the lung damage produced by neutrophils in models of acute lung injury (ali), which resembles those caused by hrsv infection , have suggested that animals lacking activating fcγrs (fcrγ −/− mice) can be protected from ali triggered by administration of igg mabs that recognize self-antigens, such as mhc-i molecules (looney et al., ) . supporting a role for neutrophils and activating fcγrs in this model of lung injury, the same study showed that ali was observed when fcrγ −/− mice were adoptively transferred with wild-type neutrophils followed by the administration anti-mhc-i mabs (looney et al., ) . taken together, these results suggest that lung disease in this model is dependent on the expression of activating fcγrs by neutrophils. for the case of hrsv infection, it has been shown that the recruitment of neutrophils to the lungs of infected mice is modulated by the presence of different fcγrs (figure ) . for instance, it was reported that animals lacking the activating fcγriii (fcγriii −/− ) showed decreased neutrophil recruitment and higher viral loads , suggesting that fcγriii could play a pro-inflammatory role during hrsv primary infection and promotes viral clearance. consistent with the results described above, mice lacking the inhibitory fcγriib (fcγriib −/− ) showed increased neutrophil infiltration in lungs due to hrsv infection but decreased viral loads , thus suggesting that this receptor can play an anti-inflammatory role during hrsv-induced disease despite it contributes to viral replication ). an in vitro study using human neutrophils showed that hrsv-ics, established with hrsv and anti-hrsv autologous serum, but not free hrsv or antibodies alone, could promote the release of reactive oxygen species (ros) by neutrophils, which could contribute to lung tissue damage (figure ) (kaul et al., ; winterbourn et al., ) . therefore, it is possible that the activation of neutrophils, mediated by the engagement of fcγrs likely occurs under physiological conditions, when individuals become infected. this notion, is further supported by a study showing increased release of il- by human neutrophils challenged with opsonized hrsv (arnold et al., ) . this cytokine is relevant, as it has been described that secreted il- works as a chemotactic signal for neutrophils that induces their activation leading to pro-inflammatory responses (henkels et al., ) . this in vitro evidence suggests that the engagement of fcγrs can activate neutrophils and therefore contribute to lung inflammation and the progression of hrsv disease (figure ) . dendritic cells (dcs) can modulate the immune response during viral infections after capturing ics through either, activating or inhibitory fcγrs (guilliams et al., ) . along these lines, iggantigen complexes can trigger activating signals in human dcs (hdcs) after binding to fcγriii and promote an inflammatory response (bandukwala et al., ) . in contrast, binding of ics to the inhibitory fcγriib trigger inhibiting signals that can lead to reduced inflammation (boruchov et al., ) . in addition, it has been described that hrsv-ics containing either, neutralizing or non-neutralizing antibodies can modulate dc function and subsequent t cell responses elicited by the antigen presentation of these cells (kruijsen et al., ; gomez et al., ) . in the context of hrsv infection, it has been reported that dc-mediated t cell activation and ifn-γ production by these cells, is modulated by the presence of activating fcγrs on the dc surface (kruijsen et al., ) . in this case, it was observed that dcs derived from wt adult mice were able to induce the production of ifn-γ by cd + t cells in the presence of anti-hrsv immune serum obtained from mice being challenged with hrsv (kruijsen et al., ) . nevertheless, this observed increase in the ifn-γ response by cd + t cells was reduced when the dcs were derived from fcrγ −/− mice. therefore, the expression of all activating fcγrs on the dc surface is required to promote the production of this cytokine by cd + t cells. remarkably, unaltered secretion of ifn-γ by cd + t cells was observed in dcs derived from fcγriib −/− mice, when compared to wt mice (kruijsen et al., ) , indicating that dc-mediated stimulation of ifn-γ secretion by cd + t cells does not depend on the presence of the inhibitory fcγriib (kruijsen et al., ) . interestingly, another report indicates that cd + t cells represent an important source of ifn-γ during neonatal hrsv infection in the murine model, which is required to prevent reinfection and disease severity in adult mice (lee et al., ) . figure | putative mechanisms explaining hrsv-induced inflammation due to hrsv-ic interaction with fc-gamma receptors expressed on the surface of neutrophils. during a primary infection ( ) hrsv induces the secretion of pro-inflammatory cytokines ( ) and chemokines that promote neutrophil recruitment to the lungs and the airways ( ). during infection, hrsv is phagocyted by dcs and impair its maturation ( ). infected dcs migrate to lymph nodes ( ) but fail to activate t cells ( ). by a poorly understood mechanism, t cells fail to help naïve b cells ( ) and promote the proliferation of plasma cells that produce anti-hrsv antibodies with a low neutralizing capacity ( ). serum antibodies produced after a primary hrsv infection can opsonize hrsv during secondary infection ( ). opsonized hrsv is then phagocyted by neutrophils through fcγrs ( ). the infection of these cells triggers the release of cytokines such as il- that promote the activation and the recruitment of neutrophils ( ). activated neutrophils then release metabolic products, i.e., reactive oxygen species (ros) that promote lung damage and inflammation ( ). thus, it is possible that activating fcγrs contribute to prevent re-infection during adulthood, by promoting ifn-γ production by cd + t cells through dc-mediated antigen presentation. however, it is necessary to determine whether activating fcγrs on the dc surface also modulate the production of this cytokine by neonatal cd + t-cells to prevent re-infection. recent studies have shown that another igg fc receptor, particularly the neonatal fc receptor for igg (fcrn), which is a non-classical fc receptor that binds igg at acidic ph (< , ) (qiao et al., ) , does not contribute to the activation of cd + t cells when dcs are loaded with hrsv-ics (kruijsen et al., ) . moreover, bone marrow-derived dcs (bmdcs) from fcγrn −/− mice exhibit unaltered capacity to induce the production of ifn-γ by cd + t-cells (kruijsen et al., ) . these results were validated in vivo, as fcγrn −/− mice also displayed unaltered ifn-γ production by cd + t-cells after being intranasally challenged with hrsv-ics (kruijsen et al., ) . results from our group indicate that bmdcs display a reduced capacity to induce il- production by cd + t cells after being loaded with hrsv-ics that had the neutralizing antibody palivizumab (synagis tm ) . in contrast, when the assay was performed with bmdcs derived from either, fcγriii −/− or fcγriib −/− mice il- secretion by cd + t cells was restored. this results prompts that when present, these receptors impair the capacity of dcs to induce the secretion of il- by cd + t cells. it should be noted that, the production of this cytokine is required for the generation of memory regulatory cd + t cells (tregs), which perform anti-inflammatory functions during hrsv infection and protect against re-infections (durant et al., ) . thus, it is possible that both, fcγriii and fcγriib contribute to hrsv pathogenesis and re-infection by impairing the capacity of dcs to promote the production of il- by cd + t cells. in humans, the presence of two types of fcγrs has been recognized (banegas banegas et al., ) . type-i fcγrs are members of the immunoglobulin superfamily and can be either activating or inhibitory ravetch, , ) . in contrast, type-ii fcγrs are members of the c-type lectin receptor family and comprise two different members: the ige receptor and the surface protein dc-sign (banegas banegas et al., ; miettinen, ) , which is able to recognize the fc portion of igg (kaneko et al., ; svajger et al., ) , but also the g protein expressed by hrsv (johnson et al., ) . of interest, studies evaluating the role of dc-sign in hdcs during hrsv infection, showed that the blockade of this receptor with specific mabs led to an increase in the expression of maturation markers, such as cd and cd following hrsv infection (johnson et al., ) . this suggests that the interaction between hrsv surface proteins and dc-sign can suppress some aspects of dc activation in humans, thus contributing to an impaired protective immunity following hrsv infection. however, further studies are required to study the influence of this receptor during infection in vivo and hrsvinduced pathology, as well as its consequences on dc mediated t-cell activation. in a recent study, it was shown that young infants (i.e., < months old) generate a highly neutralizing antibody response that is biased from the post-fusion to the pre-fusion form of hrsv f protein. however, as children become older (i.e., from < months old to > months old), this response is re-directed against post-fusion conformation antigens (goodwin et al., ) . thus, the antibodies generated display a weak neutralizing capacity that fail to prevent hrsv infection. therefore, it is possible that the generation of a pool of low-neutralizing figure | proposed mechanism to explain enhancement of hrsv-induced disease following fi-hrsv vaccination. formalin hrsv inactivation produces a non-infectious virus with a high proportion of post-fusion conformation epitopes in the f protein (post-f) ( ). the inactivated virus is then phagocyted by b cells ( ) that can present hrsv antigens to t cells in the context of mhc molecules ( ). the interaction between b and t cells allows the differentiation of b cells into plasma cells that generate antibodies against the post-fusion conformation of the hrsv f protein ( ). such antibodies failed to neutralize hrsv infection but also may enhance the infection of fcγr bearing cells such as dcs. when infection by hrsv occurs, the low neutralizing antibodies induced by the fi-hrsv vaccine can form immune-complexes (ics) with hrsv ( ) that leads to the activation of fc-gamma receptors expressed on the surface of dcs ( ). subsequently, an impaired dc-mediated t cell activation ( ) can induced cd + t cells with a th -biased phenotype that promotes lung damage ( ). furthermore, low secretion of il- by cd + cells activated by hrsv-ic-loaded dcs can lead to a poor memory response that contributes to hrsv re-infection ( ). antibodies during infancy can facilitate infection of immune cells that express fcγrs, a phenomenon called ade of infection that has been observed for other viruses such as dengue virus (flipse et al., ) , acute respiratory syndrome coronavirus (yip et al., ) and porcine reproductive and respiratory syndrome virus infection (gu et al., ) . in this context, antibodies might exert different effector functions through their fc regions and for hrsv, ade during infection is an effect that has been reported in in vitro studies (gimenez et al., ; krilov et al., ; osiowy et al., ) . however, a role of ade during hrsv pathogenesis in vivo has been proposed, but remains to be confirmed during re-infection. to date, in vitro enhancement of infection of monocytederived cell lines due by fcγr binding by mabs and patient sera has been reported (gimenez et al., ; krilov et al., ; osiowy et al., ) , demonstrating that non-neutralizing mabs can enhance the infection of phagocytic cell lines expressing these receptors (gimenez et al., ) . further, when neutralizing antibodies were applied at sub-neutralizing concentrations (i.e., diluted), they induced ade in phagocytic cells bearing fcγrs. this was also observed using human sera and purified human immunoglobulin (ivig) (van erp et al., ) . together, these results suggest that the interaction of hrsv-ics generated with low neutralizing antibodies can promote the infection of immune cells in vitro, therefore contributing to hrsv pathogenesis under physiological conditions. the administration of a formalin-inactivated hrsv vaccine to children nearly years ago, which was aimed at preventing severe respiratory disease elicited by hrsv infection was unable to produce protective immunity against hrsv. contrarily to what was expected, its administration resulted in increased morbidity and mortality in vaccinated infants when they were later infected by the virus (kim et al., ) . although the mechanisms underlying the pathological effects of fi-rsv vaccine have not been totally elucidated, this episode revealed complexities associated to vaccine development, which has been hampered, and raised hypothesis about the pathologic roles of hrsv-ics (kim et al., ; polack et al., ; delgado et al., ) . enhanced hrsv disease (erd) after fi-rsv immunization of balb/c mice has been associated with alveolar deposition of ics, which was observed dpi of hrsv by means of co-localization of igg with the complement component (c protein). the role of complement fixing ics in erd was supported by experiments in c −/− mice, which showed significantly less airway hyperresponsiveness (ahr) in comparison to wt counterparts, after fi-hrsv vaccination and hrsv challenge, arguing for a role of complement in bronchoconstriction observed in erd (polack et al., ) . these experimental studies were supported by histological analysis of lung sections from two infants that suffered fatal erd, in which ic-mediated complement activation was observed through extensive peribronchiolar complement component d (c d) deposition in the airway tissue (polack et al., ) . furthermore, a sub-optimal, nonprotective antibody response in mice, characterized by high levels of non-neutralizing anti-f and anti-g igg antibodies, was observed after immunization with fi-hrsv, but not infectious hrsv (polack et al., ) . the lack of affinity maturation in abs elicited by fi-hrsv was associated with enhanced lung histopathology and ahr, whereas the supplementation of tolllike receptor (tlr) agonists, performed during immunization promoted proper affinity maturation that prevented erd after hrsv challenge, showing that a deficient tlr stimulation in b cells is likely responsible for the lack of ab affinity maturation after fi-hrsv vaccination (delgado et al., ) . furthermore, cotton rats vaccinated with fi-rsv elicited high levels of hrsvspecific antibodies, which displayed low neutralizing titers in vero cells (piedra et al., ) . these antibodies were also able to cause ade in in vitro assays. these studies suggest that sub-optimal antibody production and the generation of ics play a role in erd development (figure ) . furthermore, recent studies suggest that cd + subsets and a th -biased immune response are key for ahr and erd (knudson et al., ) . in this context, tam (tyro , axl, and mertk) receptors, which are expressed in various cells and tissues, and their ligand growth arrest-specific (gas ) could be involved in the production of a th -biased immune responses that reduce the production of type igg a subclass antibodies (shibata and ato, ) . these antibodies could have an effective neutralizing capacity against hrsv and therefore prevent hrsv induced disease, but their production is lowered as a consequence of fi-hrsv immunization followed by hrsv infection. therefore, it is possible that the tam/gas signaling axis can contribute to the generation of low neutralizing antibodies that failed to neutralize hrsv infection and instead contributes to the pathology caused by hrsv infection through the engagement of fcγrs. the hrsv is a leading cause of respiratory illness in infants and a major health burden worldwide. re-infections with this virus are common and can contribute to additional clinical manifestations, such as asthma and allergies. for this reason, several studies have focused on understanding the mechanisms that can contribute to hrsv induced pathology, but also to elucidate the factors that contribute to re-infection episodes throughout life. in this context, some studies suggested that low number of memory hrsv-specific cd + t cells could be associated with re-infection episodes and that the levels of such cells could be regulated by virus-specific antibodies, by modulating the function of antigen presenting cells, such as dcs. furthermore, recent studies suggest that the generation of regulatory memory t cells could be impaired by the interaction of hrsv-ics with dcs, pointing out these phenomena as an interesting research topic that deserves analysis. in this review, and based on several studies, we discussed the role of fcγrs during hrsv infection and their immunemodulatory properties that can account for recurrent hrsv infection episodes and the enhancement of the disease caused by fi-hrsv vaccination. however, further research is needed to understand how hrsv induces the production of antibodies that fail to prevent re-infections. knowledge of such mechanisms would certainly be appreciated for vaccine and therapy development against hrsv, which represents a major global health problem. oa and fd are responsible for the writing of this review article. tb, fb, je-v, js, and pg reviewed the manuscript. ak is the leading investigator and assisted in the organization and revision of this article. all authors listed approved the version to be published and have made a substantial and intellectual contribution to the work. this work was supported by comisión nacional de investigación científica y tecnológica (conicyt) programa formación de capital humano avanzado-beca de doctorado en chile n • and n • . fondecyt (n • and ). ak is a helen c. levitt visiting professor at the department of microbiology and immunology university of iowa; programa semillero de investigación, dgi, universidad de antofagasta (grant n • ) and the millennium 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trafficking reactive oxygen species and neutrophil function neutrophil-mediated inflammation in respiratory syncytial viral bronchiolitis antibody-dependent infection of human macrophages by severe acute respiratory syndrome coronavirus evolution of epidemiologic methods and concepts in selected textbooks of the th century mechanism of p phox-induced increase of reactive oxygen species in peripheral blood mononuclear cells from premature infants on oxygen therapy respiratory syncytial virus infects regulatory b cells in human neonates via chemokine receptor cx cr and promotes lung disease severity key: cord- -n knqj z authors: su, yunfang; hou, yixuan; wang, qiuhong title: the enhanced replication of an s-intact pedv during coinfection with an s ntd-del pedv in piglets date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: n knqj z porcine epidemic diarrhea virus (pedv) variants having a large deletion in the n-terminal domain of the s subunit of spike (s) protein were designated as s ntd-del pedvs. they replicate well in experimentally infected pigs. however, on farms they often co-infect pigs with the pedv containing an intact s protein (s-intact pedv). we aimed to characterize viral replication and pathogenesis in neonatal gnotobiotic pigs infected simultaneously with the two types of pedv using two recombinant pedvs: icpc a and its s ntd-del form icpc a-s Δ . additionally, viral replication was compared in vero and ipec-dq cells at the presence of bovine mucin (bm), porcine gastric mucin (pgm), swine bile and bile acids during inoculation. in the pigs coinfected with icpc a and icpc a-s Δ , icpc a replicated to a higher peak titer than its infection of pigs without the presence of icpc a-s Δ . the severity of diarrhea and intestinal atrophy were similar between icpc a and the coinfection groups, but were significantly higher than icpc a-s Δ group. in vero and ipec-dq cells, certain concentrations of bm, pgm, bile and bile acids increased significantly the infectivity of icpc a but had no or negative effects on icpc a-s Δ . these results indicated that the replication of the s-intact pedv was enhanced during coinfection in piglets. this observation may be explained partially by the fact that mucin, bile and bile acids in gastrointestinal tract had facilitating effects on the infection of s-intact pedv, but no/inhibition effects on s ntd-del pedv. porcine epidemic diarrhea virus (pedv) belongs to the genus alphacoronavirus in the family coronaviridae. it causes severe gastroenteritis in neonatal pigs worldwide. the genome size of pedv is kb. it contains orf a and orf b encoding - non-structural proteins, an orf encoding an accessory protein, and four orfs encoding structural proteins: spike (s), membrane (m), envelop (e), and nucleocapsid (n) proteins. pedv s protein mediates the essential functions of receptor binding (via s subunit) and subsequent fusion of the viral and cellular membranes (via s subunit) (li, ) . s subunit contains two domains, the n-terminal domain (s ntd, residues - based on pedv cv ) and the c-terminal domain (s ctd, residues - ). however, the cellular receptor of pedv is still unknown (li et al., b; shirato et al., ) . although the sialic acid binding activity of s -ntd was observed in many coronaviuses and facilitated virus replication (schwegmann-wessels et al., ; li et al., ; desmarets et al., ) , s -ntd is dispensable for some mutants of transmissible gastroenteritis coronavirus (tgev) and pedv (schwegmann-wessels et al., ; oka et al., ; suzuki et al., ; hou et al., ) . pedv variants with a big deletion in the s -ntd have been designated as s ntd-del pedvs (hou et al., ) . they have been detected in field pig samples, indicating sufficient replication of these s ntd-del pedvs in vivo (suzuki et al., ; diep et al., ; zhang et al., a; su et al., ) . interestingly, most field s ntd-del pedvs exist as a member of coinfection of pigs with the s-intact pedv (diep et al., ; su et al., ) . however, the mechanisms and consequences of the coinfection have not been investigated. studies showed that under unfavorable conditions as encountered in the intestinal tract, sialic acid-binding of tgev played an important role in viral replication. when absorption time was reduced, tgev infectivity was also reduced in the cells whose sialic acids were removed (schwegmann-wessels et al., ) . consequently, sialic acid binding activity may facilitate the infection of tgev by helping the virus resist detergent-like substances encountered during passing through the gastrointestinal tract (krempl et al., ) . therefore, we hypothesized that mucin, rich in sialic acid, may enhance the infection of pedv. interestingly, bile acids can induce or promote the replication of some enteric viruses like a porcine sapovirus (chang et al., ) and human noroviruses (ettayebi et al., ) . since intestinal epithelial cells are exposed to bile acids, we hypothesized that bile acids promote pedv infection. previously, we generated two recombinant pedvs, pedv icpc a and icpc a-s Δ (hou et al., ) . pedv icpc a is an infectious cdna clone-derived virus of the emerging highly virulent pedv pc a strain. on the other hand, pedv icpc a-s Δ is a mutant version of icpc a bearing a -aa deletion (residues - ) in the s ntd region, corresponding to the s domain (s °) of the spike protein (li et al., a; hou et al., ) . in this study, we investigated the replication and pathogenesis of the two viruses during coinfection in neonatal gnotobiotic (gn) piglets. some intestinal substances (mucin, bile and bile acids) were tested for their effects on the infection of the individual viruses in vitro. we selected two cell lines, vero and ipec-dq cells. vero cells are the most commonly used cell line for pedv isolation and propagation (teeravechyan et al., ) . ipec-dq cell is a subclone of porcine small intestinal cell line ipec-j (zhang et al., b) . although pedv grows less efficiently in ipec-dq cells than in vero cells, pedv infection of ipec-dq cells is more relevant physiologically to pedv natural infection in the intestines of pigs. vero cells (atcc number ccl ) were cultured in dulbecco's modified eagle's medium (dmem, gibco, carlsbad, ca, usa) supplemented with % fetal bovine serum (fbs, hyclone, logan, ut, usa) and % penicillin/streptomycin (gibco). for virus propagation in vero cells, the maintenance medium was dmem supplemented with . % tryptose phosphate broth solution (tpb, sigma, st. louis, mo, usa), % penicillin/streptomycin (gibco) and μg/ml trypsin ( . % trypsin, gibco). ipec-dq cell line was a gift from dr. dongwan yoo, university of illinois at urbana-champaign, urbana il, usa. it was a subclone of ipec-j cells, a porcine small intestinal cell line, and maintained in roswell park memorial institute medium (rpmi- , gibco) supplemented with % fbs and % penicillin/streptomycin (zhang et al., b) . for virus propagation in ipec-dq cells, maintenance medium was rpmi- containing . % tpb, % penicillin/streptomycin and μg/ml trypsin. two recombinant pedv, icpc a and icpc a-s Δ , were generated previously by our lab using the infectious clone of pedv pc a strain (hou et al., ) . neuraminidase (na, from clostridium welchii), porcine gastric mucin (pgm) and several bile acids [(glycochenodeoxycholic acid (gcdca), chenodeoxycholic acid (cdca), deoxycholic acid (dca), and ursodeoxycholic acid (udca)] were all purchased from sigma. bovine mucin (bm) was purchased from worthington (worthington biochemical corp., lakewood, nj, usa). swine bile was collected from a pregnant sow after c-section surgery. the mouse monoclonal antibodies (mab) h , f and a targeting the s °domain of pedv s protein (fig. s a ) was provided by dr. berend jan bosch, utrecht university, utrecht, netherlands (li et al., a) . the mab sd - targeting pedv nucleocapsid (n) protein was provided by drs. eric nelson and steven lawson, south dakota state university. the guinea pig polyclonal antibody (pab) gp targeting the s subunit of the s protein of pedv pc strain (s Δ ) (fig. s a) , rabbit pab rb targeting pedv n protein and swine pedv-positive serum (virus neutralization titer : ) were generated by our laboratory previously (hou et al., ) . . . duplex rt-qpcr method for the detection and differentiation of pedv icpc a and icpc a-s Δ based on the nucleotide sequence differences in the s ntd coding region between the icpc a and icpc a-s Δ , a duplex taqman real-time reverse-transcription quantitative pcr (rt-qpcr) was developed. we designed the primers and probes (table , fig. s b ) and had them synthesized by integrated dna technologies (skokie, il, usa). the rna of the two viruses was extracted using rneasy mini kit (qiagen, valencia, ca, usa). to generate a standard dna, a kit of superscript™ iii first-strand synthesis supermix (thermo fisher scientific, carlsbad, ca, usa) was used for cdna synthesis following manufacturer's instructions. primer set pedv- f and pedv- r (oka et al., ) was used to amplify a . kb or a . kb fragment covering pedv partial nsp gene and s gene of icpc a and icpc a-s Δ , respectively. the pcr products were purified using gel extraction kit (qiagen) and used as standard dna. the reaction consisted with μl × pcr buffer, . μl mm dntps (qiagen), . μl each primer ( μm), . μl each probe ( μm), . μl rnasin (promega, wi, usa), . μl enzyme mix (qiagen), . μl each dna/rna and . μl sterilized water. the rt-qpcr procedure was: °c for min, °c for min and cycles of °c for s and °c for s. we generated two standard curves using light-cycler® system (roche life science, indianapolis, in, usa). the two standard curves for the serially diluted dna fragments of icpc a and icpc a-s Δ showed regressions with coefficients of - . and - . , respectively, and the corresponding correlation coefficient (r ) of . and . , respectively. the primers and probes were further tested using individual and the mixture of two viruses. we tested the correlation between s gene titers and infectious titers of pedv icpc a and icpc a-s Δ using serially diluted, cell cultured viruses (table s and s ). for the samples of mixed viruses, the duplex rt-qpcr was performed to differentiate the s gene of each virus. the detection limit was log s gene copies/ml for both icpc a and icpc a-s Δ . the gn piglets were derived and raised as described previously (saif et al., ) . thirty-one gn piglets were assigned to five experimental groups: ( ) icpc a (n = ; pfu/pig); ( ) icpc a-s Δ (n = ; pfu/pig); ( ) coinfection of icpc a and icpc a-s Δ (n = ; pfu/pig of each virus); ( ) mock (n = ; pbs); ( ) second passage of coinfection [n = ; small intestinal contents (sic) (sample #pe ) of one piglet in group collected at day post inoculation (dpi), at a dose of log copies/ml based on the n gene, log copies/ml and log copies/ml based on the icpc a-specific s gene and icpc a-s Δ -specific s gene, respectively, corresponding to table primers and probes of the duplex rt-qpcr. log pfu/ml of infectious pedv] . at days of age, piglets in groups - were orally inoculated with individual or the mixture of the viruses. the group pig was inoculated at days of age and was euthanized at dpi. to examine histopathological changes, one or two piglets of groups - were euthanized at . dpi and - dpi, respectively. after inoculation, all piglets were evaluated daily for clinical signs, such as diarrhea, vomiting, anorexia, and depression. fecal consistency was examined by collecting rectal swabs and scored as follows: , solid; , pasty; , semiliquid (mild diarrhea); and , liquid (severe diarrhea). total fecal rna was extracted using magmax- rna isolation kit (thermo fisher scientific), and the viral rna was titrated by the rt-qpcr targeting the n gene and the duplex rt-qpcr targeting the s gene of each virus. fecal infectious pedv shedding titers were tested using vero cells in -well plates and determined as % tissue culture infective doses (tcid ) (reed and muench, ) . at necropsy, three sections of the jejunum (proximal, middle, and distal) and one section of the ileum were collected and fixed in . % formalin (fisher scientific co llc, florence, ky, usa). all tissues were trimmed, processed, embedded in paraffin, and sectioned using routine procedures . to compare the pedv-specific histopathological changes among different groups of pigs, ihc staining was performed as described previously for the detection of pedv n proteins using mab sd - as the primary antibody (hou et al., ) . the ihc staining was carried out using a nonbiotin polymerized horseradish peroxidase system (biogenex laboratories, san ramon, ca, usa). finally, tissues were counterstained with hematoxylin. images of tissues were observed and captured by olympus ix- fluorescent microscope (center valley, pa, usa). ratios of villous height to crypt depth (vh/ cd) of the jejunum and ileum of individual piglets were measured using a computerized imaging system (metamorph, olympus, japan) as described previously . for each intestinal section, villi and crypts were measured. to detect and differentiate icpc a-and icpc a-s Δ -infected cells in the coinfected pigs, if staining was performed. the slide preparation steps were as same as those of the above ihc procedure. the mixture of mouse mabs h , f and a ( : in pbs) (li et al., a) targeting the s °domain of pedv s protein was used as the primary antibody for the staining of icpc a-infected cells only. tissues were incubated at ℃ overnight, after three washings with pbst (pbs with . % tween ), alexa fluor -conjugated goat antimouse igg (h + l) (thermo fisher scientific) ( : diluted in pbs) was used as the second antibody and tissues were incubated at room temperature for h. after washing with pbst, guinea pig pab gp ( : diluted in pbs), targeting the s Δ and was for the staining of both viruses, was added. then, the slides were incubated at room temperature for h. after washing three times with pbst, alexa fluor -conjugated goat anti-guinea pig igg (h + l) (thermo fisher scientific) ( : diluted in pbs) was used as the nd antibody and the slides were incubated at room temperature for h. after washing with pbst, mg/ml dapi ( ′, -diamidino- -phenylindole, dihydrochloride, roche life science, indianapolis, in, usa) was added for nucleic acid staining. finally, an autofluorescence quenching kit (vector® trueview™, burlingame, ca, usa) was used to quench the autofluorescence of the tissues. images of stained tissues were captured by olympus ix- fluorescent microscope. red, green and blue fluorescence staining was merged by imagej software (https://imagej.nih. gov). . . isolation of the pig-passaged icpc a and icpc a-s Δ from the sic of a pig coinfected with the viruses the sic (sample #pe ) of a coinfected piglet in group was positive for both viruses by the duplex rt-qpcr. virus isolation was performed using plague assays (oka et al., ) . the sic was diluted in dmem and the % suspension was vortexed briefly followed by centrifugation at , ×g for min at ℃. a series of -fold dilutions ( − - − ) of the supernatants were prepared in dmem (containing . μg/ml trypsin) and were used immediately for inoculation of vero cell monolayers in -well plates ( μl per well). after incubating at ℃ for h, the inoculum was removed and the monolayers were washed once with pbs. then ml/well of agarose overlay was added. the plate was kept for days before picking up clones. for propagating each clone, vero cell monolayers in -well plates were prepared and individual plagues were picked and added into individual wells. at dpi, rna was extracted and the duplex rt-qpcr method specific for both viruses was performed. . . multi-step growth kinetics and if staining of coinfection of pedv icpc a and icpc a-s Δ in vero and ipec-dq cells the growth kinetics of icpc a-s Δ and icpc a during the coinfection of the two viruses were investigated in vero and ipec-dq cells. cell monolayers on -well plates were inoculated with one of the three inocula: ( ) . multiplicity of infection (moi) of icpc a; ( ) . moi of icpc a-s Δ ; and ( ) . moi of icpc a and . moi of icpc a-s Δ . viruses were diluted in dmem containing μg/ml trypsin and were added to cell monolayers. after incubating at ℃ for h, the inoculum was removed and the cell monolayers were washed for three times. then the plates were added with the maintenance medium and incubated at ℃. the samples were collected at different time points ( h, h, h, h, h) and frozen (- ℃) and thawed once before testing. rna was extracted using the magmax- rna isolation kit (thermo fisher scientific) and virus titers were tested by the duplex rt-qpcr specific for both viruses. for if staining of s proteins at h post infection (hpi), the procedure in . section was performed. . . infection of pedv icpc a and icpc a-s Δ in na-treated vero and ipec-dq cells vero/ipec-dq cell monolayers in -well plates were treated with mu na in dmem/rpmi- or mock dmem/rpmi- at ℃ for h. after three washings with dmem/rpmi- , cells were inoculated with icpc a or icpc a-s Δ at a moi of . with μg/ml trypsin for h. next, the inoculum was removed followed by three washings. dmem/rpmi- containing μg/ml soybean trypsin inhibitor (sbti) and swine pedv positive serum (virus neutralization titer : , : dilution) was added to prevent virus spreading. cells were fixed with acetone-methanol ( : ) at hpi. the numbers of pedv-infected cell foci were determined by if assays as described for the staining of pedv n proteins (hou et al., ) . briefly, rabbit pab rb ( : ) was used as the primary antibody. after incubating at ℃ overnight, cells were washed three times. then alexa fluor -conjugated goat anti-rabbit igg (h + l) (thermo fisher scientific) ( : ) was used as the nd antibody and the cells were incubated at room temperature for h. after washing and adding mounting medium, cells were observed and picture were captured by using olympus ix- fluorescent microscope. the numbers of fluorescent focus units (ffu) were counted by imagej software. the percentages of ffu were calculated by the value of mock group as %. . . effect of mucin, bile and bile acids on the infection of pedv icpc a and icpc a-s Δ in vero and ipec-dq cells viruses (icpc a or icpc a-s Δ ) were mixed with different concentrations of bm ( , . , . , . mg/ml) or pgm ( , . , . , . , . mg/ml). then μg/ml trypsin were added to the mixture. monolayer of vero/ipec-dq cells in -well plates were inoculated with each virus mixture at a moi of . . the plates were incubated at ℃ for h, then the inoculum was removed. after three washings, dmem/rpmi- containing μg/ml sbti and swine pedv positive serum ( : dilution) was added. cells were fixed at hpi. the numbers of ffu were determined by if assay for the staining of pedv n proteins (see section . ). the percentages of ffu were calculated by the value of mock group as %. similarly, different concentration of swine bile ( , . , . , . , . %) and bile acids (gcdca, cdca, dca, and udca) at , , , , μm were tested. statistical analysis was performed using graphpad prism software (graphpad software, la jolla, ca, usa). the experimental data were analyzed by one-way anova and student`s t-test. data are shown as m ± sd, and a p < . was considered as significant. . . the replication of icpc a was eventually enhanced during coinfection with icpc a-s Δ in neonatal gn piglets as shown in fig. , all pedv-inoculated pigs but no pigs in the mock group had diarrhea and shed pedv. the fecal consistency scores and peak fecal pedv n and s rna shedding titers of piglets in the coinfection group reached the peak at . dpi, which was delayed half a day compared with the icpc a group ( dpi) but earlier than the icpc a-s Δ group (> dpi). by dpi, all pigs in the icpc a and coinfection groups but no pigs in the icpc a-s Δ group died or showed moribund. compared with the peak fecal pedv n gene shedding titer ( . ± . log copies/ml) of piglets in the icpc a group ( dpi), pigs in the coinfection group had a significantly higher peak titer ( . ± . log copies/ml) ( fig. b and table ) at a delayed time point ( . dpi). in addition, the peak fecal infectious pedv shedding titer of the coinfection group ( . ± . log tcid /ml) were significantly higher than that of the single infection groups ( . ± . and . ± . log tcid /ml of icpc a and icpc a-s Δ groups, respectively) ( table ) . interestingly, the peak s gene shedding titers of icpc a ( . ± . log copies/ml) in the coinfection group at . dpi was significantly higher than that of icpc a group ( . ± . log copies/ml) at dpi (fig. c, table ). however, the peak s gene shedding titers ( . ± . log copies/ml) of icpc a-s Δ in the coinfection group was significantly lower than that of the icpc a-s Δ group ( . ± . log copies/ml) (fig. c , table ). in the second passage of coinfection, only icpc a but not icpc a-s Δ s gene was detected in the large intestinal contents (lic) of the piglet, which was orally inoculated with the sic (sample #pe ) of one pig in the coinfection group and was positive for both viruses. the lic had a virus titer of . log tcid /ml for infectious pedv, . log s gene copies/ml and . log n gene copies/ml for icpc a. histopathological examination was performed and villous atrophy was observed in the jejunum and ileum of piglets in all pedv-inoculated groups ( fig. a and b ). the small intestinal villi in the icpc a-s Δ group had a milder villous atrophy than those in the icpc a and coinfection groups. the vh/cd ratios of jejunum and ileum of all the pedv-inoculated groups were significantly lower than those of the mock group (fig. b) . the ratios of the icpc a and coinfection groups were similar and were significantly lower than that of the icpc a-s Δ group. ihc staining showed that no pedv-positive intestinal epithelial cells were observed in all pedv-inoculated pigs fig. . evaluation of the replication of icpc a and icpc a-s Δ in gn pigs infected with individual or both viruses. (a) fecal consistency scores of individual gn piglets and the mean of each group were shown. the scores were named as follows: , solid (normal); , pasty (normal); , semiliquid (mild diarrhea); and , liquid (severe diarrhea). two piglets from icpc a, icpc a-s Δ and coinfection groups, and one pig from mock group were euthanized at hpi and - dpi, respectively, for histopathological examination. (b) fecal pedv n gene rna shedding titers (for both icpc a and icpc a-s Δ ) of each group. data are shown as mean (m) ± standard deviations (sd) of pigs in each group. (c) fecal pedv s gene rna shedding titles of each virus. data are shown as m ± sd. at . dpi (data not shown). at - dpi, a few pedv n antigens were observed in the small intestine of the icpc a-s Δ -infected piglets, while extensive antigens were stained in those of the icpc a and coinfection piglets ( fig. a) . by if staining for the pedv s proteins, a few pedv antigens were observed in the jejunum of the icpc a-s Δ -inoculated piglets (green alone, positive for s Δ only), while many antigens were observed in those of the icpc a-inoculated piglets [yellow, representing positive for both s Δ (green) and s °d omain (red)] and the coinfection piglets (yellow) (fig. c) . . . icpc a alone, but not icpc a-s Δ alone was isolated from the sic of one of the pigs infected with the two viruses simultaneously the sic of the one piglet in the coinfection group, which was positive for both viruses by the duplex rt-qpcr were used for virus isolation in vero cells using plague assays. we picked up plagues and found that plagues were positive for icpc a and plagues were positive for both viruses by the duplex rt-qpcr. no plagues were positive for s ntd-del pedv alone. during coinfection in vero cells, icpc a replicated to a titer of . ± . log copies/ml at hpi, which was similar to that of the single infection ( . ± . log copies/ml) (fig. a) . however, icpc a-s Δ s gene peak titer was . ± . log copies/ ml, which was significantly lower than that of the single infection ( . ± . log copies/ml). in vero cells, icpc a alone and icpc a-s Δ alone replicated to similar titers. similarly, during coinfection in ipec-dq cells, icpc a replicated to a similar titer to its single infection ( . ± . vs. . ± . log copies/ml at hpi (fig. c ). icpc a-s Δ s gene peak titer was . ± . log copies/ml, which was significantly lower than that of the single infection ( . ± . log copies/ml). in contrast to those results in vero cells, icpc a alone replicated to significantly higher titers than icpc a-s Δ alone in ipec-dq cells. by if staining, more icpc a-s Δ -infected cells (green) were observed in vero cells than in iepc-dq cells in both icpc a-s Δ alone and co-infection conditions ( fig. b and d) . at the second passage of the coinfection, icpc a rna and antigens were detected in both cells; however, icpc a-s Δ rna and antigens were detected exclusively in vero cells but not in ipec-dq cells (data not shown). . . the percentage of infectivity of icpc a was significantly lower than that of icpc a-s Δ in na-treated vero and ipec-dq cells after removing sialic acids from the cell surface using mu na in vero and ipec-dq cells, the percentages of infectivity of icpc a and icpc a-s Δ were reduced significantly (fig. ) . however, in both cells treated with na, the percentages of infectivity of icpc a were significantly lower than those of icpc a-s Δ . . . mucin enhanced the infectivity of icpc a but had no or inhibition effects on icpc a-s Δ in both vero and ipec-dq cells in vero cells, . - . , . and . mg/ml bm increased, had no effects and decreased the infectivity of icpc a, respectively (fig. a ). . - . mg/ml pgm increased the infectivity of icpc a . - . fold (fig. b) . however, the infectivity of icpc a-s Δ was unaffected or inhibited significantly by bm and pgm at the tested concentration ( fig. a and b) . in ipec-dq cells, the infectivity of icpc a was increased . - . fold and . - . fold by bm ( . - . mg/ml) and pgm ( . - . mg/ ml), respectively. however, there was no effects at the highest pgm concentration tested ( . mg/ml). on the other hand, low to medium concentrations of bm ( . - . mg/ml) and pgm ( . - . mg/ml) had no effects on the infectivity of icpc a-s Δ . only high concentrations of bm ( . mg/ml) and pgm ( . - . mg/ml) reduced the infectivity of icpc a-s Δ significantly ( fig. c and d ). . . bile and bile acids enhanced the infectivity of icpc a but had no or inhibition effects on icpc a-s Δ in vero cells, low to medium concentrations of swine bile ( . - . %) increased . - . fold of the infectivity of icpc a (fig. a) . (fig. b-e) . however, the infectivity of icpc a-s Δ was unaffected ( . - . %) or inhibited significantly by bile ( . - . %) by bile (fig. a) . bile acids a piglets in group were inoculated with pfu/pig of icpc a; piglets in group were inoculated with pfu/pig of icpc a-s Δ ; piglets in group were inoculated with pfu/pig of each virus; piglets in group were inoculated with pbs; piglets in group were inoculated with the sic of piglet in group with a dose of log pfu/ml targeting n gene of pedv. b fecal consistency (fc) was scored as follows: , solid, , pasty, , semiliquid; and , liquid. an fc score of and were considered diarrhea and severe diarrhea, respectively. c values in parentheses are the number of positive results/number of animals tested. d the detection limit of pedv rt-qpcr targeting the s gene of icpc a or icpc a-s Δ is log copies/ml. e the detection limit of pedv rt-qpcr targeting the n gene of both icpc a and icpc a-s Δ is . log copies/ml . f the pig number for diarrhea observation is or less than the total pig number because or pigs of each group were euthanized at hpi. g the experimental data were analyzed by student`s t-test. letters 'a, b and c' indicate a mean significant difference between groups (p < . ). '-', not detected. y. su et al. veterinary microbiology ( ) - at the tested concentrations had no effects on the infection of icpc a-s Δ (fig. b-e) . similarly, in ipec-dq cells, the infectivity of icpc a was increased significantly by . - . % bile but was decreased significantly by the concentration of . % (fig. f) . however, . - . % bile decreased the infectivity of icpc a-s Δ significantly. bile acids gcdca (fig. g-j) . no significant effects of bile acids on icpc a-s Δ were observed except for that the highest concentration ( μm) of gcdca and dca decreased its infectivity significantly (fig. g-j) . fig. . histopathological examination of the gn piglets infected with individual or both icpc a and icpc a-s Δ . (a) ihc staining of pedv n proteins in the jejunal and ileal sections of piglets that died or were euthanized at - dpi (magnification, ×) . the brown signals represented the pedv n antigens in enterocytes. (b) villous height to crypt depth (vh/cd) ratios of jejunum and ileum of the gn piglets euthanized at - dpi. for each intestinal section, villi and crypts were measured. data are shown as the m ± sd. number of asterisks indicate significant difference between groups (*, p < . ; **, p < . ; ***, p < . ). 'ns', not significant. (c) if staining of pedv s proteins in the jejunal sections of piglets that died or were euthanized at - dpi (magnification, x). tissue sections were stained for the detection of icpc a and icpc a-s Δ antigens targeting the s Δ protein (green), and for icpc a antigens targeting the s °domain (red), and counterstained for cell nuclei (blue). in the merged images, yellow dots represented icpc a-infected (or co-infected in coinfection group) cells (merged from red and green) and the green dots represented icpc a-s Δ infection alone. (magnification, x) . cells were stained for the detection of icpc a and icpc a-s Δ antigens targeting the s Δ protein (green), and for icpc a antigens targeting the s °domain (red), and counterstained for cell nuclei (blue). in the merged images, yellow dots represented icpc a-infected (or co-infected in coinfection condition) cells and the green dots represented icpc a-s Δ infection alone. the experimental data were analyzed by student`s t-test. letters 'a, b and c' indicate a mean significant difference between groups (p < . ). previously, our laboratory isolated the first s ntd-del pedv strain, pc , from vero cell culture (oka et al., ) . recent studies revealed that s ntd-del pedvs naturally evolved in the field and often coinfected pigs with the s-intact pedv (suzuki et al., ; diep et al., ; zhang et al., a; su et al., ) . the s ntd-del pedv had no tissue tropism change compared with the s-intact pedv (suzuki et al., ; hou et al., ) . this differs from porcine respiratory coronavirus (prcv) that is a s ntd-del version of tgev and changes the major tissue tropism from intestines to respiratory tract (zhang et al., ) . in this study, we investigated the replication of s ntd- fig. . infection of pedv icpc a and icpc a-s Δ in neuraminidase (na)-treated vero and ipec-dq cells. vero or ipec-dq cell monolayers in -well plates were treated with mu na or mock at ℃ for h. after three washings with dmem/rpmi- , cells were inoculated with equal amounts of virus at a moi of . diluted in the medium containing μg/ml trypsin. after incubation at ℃ for h, the inoculum was removed followed by three washings. dmem/rpmi- containing μg/ml soybean trypsin inhibitor (sbti) and swine pedv positive serum (virus neutralization titer : , : dilution) was added. at hpi, cells were fixed with acetone-methanol ( : ) and infectivity was tested by if assay. each experiment was performed three times. data are shown as the m ± sd, and number of asterisks indicate significant difference between groups (*, p < . ; **, p < . ; ***, p < . ). fig. . effect of bovine mucin (bm) and porcine gastric mucin (pgm) on the infection of pedv icpc a and icpc a-s Δ in vero (a and b) and ipec-dq cells (c and d). monolayers of vero or ipec-dq cells in -well plates were inoculated with a moi of . icpc a or icpc a-s Δ in medium containing μg/ml trypsin and different concentrations of bm or pgm. after incubation at ℃ for h, the inoculum was removed followed by three washings. dmem/rpmi- containing μg/ml soybean trypsin inhibitor (sbti) and swine pedv positive serum (virus neutralization titer : , : dilution) was added. at hpi, cells were fixed with acetone-methanol ( : ) and infectivity was tested by if assay. each experiment was performed three times. data are shown as m ± sd, and number of asterisks indicate significant difference between groups (*, p < . ; **, p < . ; ***, p < . ). del and s-intact pedvs during coinfection in pigs. we developed a duplex rt-qpcr targeting the s gene of pedv to differentiate the two viruses. we found that the duplex rt-qpcr assay had different sensitivities for the detection of infectious icpc a and icpc a-s Δ (table s ) although it had a similar detection limit ( log copies/ml) for both viruses. for example, log s gene copies/ ml corresponded to . and . log ffu/ml for icpc a and icpc a-s Δ , respectively. it may reflect the fact that icpc a-s Δ replicates to lower peak infectious titers (∼ . log ffu/ml) than that (∼ . log ffu/ml) of icpc a in vero cells although both viruses replicated to similar peak titers based on the s gene (∼ . log copies/ml) (fig. a) . in the mixture of two viruses, the detection fig. . effect of bile and bile acids on the infection of pedv icpc a and icpc a-s Δ in vero (a-e) and ipec-dq cells (f-j). monolayers of vero or ipec-dq cells in -well plates were inoculated with a moi of . icpc a or icpc a-s Δ in medium containing μg/ml trypsin and different concentrations of bile or bile acids. after incubation at ℃ for h, the inoculum was removed followed by three washings. dmem/rpmi- containing μg/ml sbti and swine pedv positive serum (virus neutralization titer : , : dilution) was added. at hpi, cells were fixed with acetone-methanol ( : ) and infectivity was tested by if assay. each experiment was performed three times. data are shown as m ± sd, and number of asterisks indicate significant difference between groups (*, p < . ; **, p < . ; ***, p < . ). sensitivity of each virus differed significantly (tables s ). for the detection of icpc a at high titers ( . log ffu/ml) and as the predominant virus in the mixture of two viruses (icpc a: icpc a-s Δ = - : ), the s gene titers (∼ . - . log s gene copies/ ml) (table s ) were similar to that (∼ . log s gene copies/ml) in the single virus alone (table s ). because icpc a replicated to high titers and was the predominant virus during the coinfection in pigs, we can predict that the peak infectious titer of icpc a in coinfection was ∼ . log -higher than that in its single infection based on the duplex rt-qpcr results (∼ . vs ∼ . log s gene copies/ml) (table ) . so, we conclude that the replication of icpc a was interfered at the beginning based on delayed timing ( . vs . dpi) to reach peak titers but enhanced eventually in coinfection compared with its single infection in pigs. on the other hand, the sensitivity for the detection of icpc a-s Δ decreased significantly and icpc a-s Δ became undetectable when icpc a was high ( . log ffu/ml) and icpc a-s Δ infectious titers were lower than . log ffu/ml in the mixture of two viruses (table s ). therefore, the decreased peak s gene titer (∼ . log copies/ml) of icpc a-s Δ in coinfection of pigs compared with that (∼ . log copies/ml) in its single infection may be due to inhibition of replication or the decreased detection sensitivity of the duplex rt-qpcr for the detection of icpc a-s Δ during coinfection (table ). in the first passage of coinfection, we observed much lower numbers of s ntd-del pedv-infected cells than the s-intact pedv-infected cells in the small intestines of pigs infected with the same dose of both viruses simultaneously. in addition, plaque assays were performed to isolate and purify individual viruses from the sic of one of the coinfection pigs. however, only s-intact pedv was isolated alone. in contrast, s ntd-del pedv was exclusively detected together with the sintact pedv from the plagues. in the second passage of the coinfection in pigs, s-intact pedv but not s ntd-del pedv rna was detected in the lic of the pig inoculated with the sic of the pig inoculated with both viruses. this finding suggests that s ntd-del pedv had no replication advantage and was either outcompeted or coexisted with sintact pedv in pigs. this conclusion is in agreement with what were observed in the field (diep et al., ; su et al., ) . s ntd-del pedv replicated to a lower peak titer in coinfection than that in single virus infection in both vero cells and ipec-dq cells. these in vitro results were similar to those in the pig studies. in the second passage of coinfection, s ntd-del pedv rna was detected in vero cells but not in ipec-dq cells, probably due to the lower replication efficiency of the s ntd-del pedv in ipec-dq cells than in vero cells ( fig. a and c) . similar to other sialic acid-binding coronaviruses, the s ntd of pedv has a sialic acid binding activity, and the sugar-binding activities of a field isolate pedv variant chgd- was stronger than that of the prototype strain pedv cv using bm (deng et al., ) . the sialic acid binding activity occurred within the n-terminal residues and the capacity of sialic acid binding differs among pedv strains using an hemagglutination assay . the recombinant virus used in this study, pedv icpc a-s Δ , is a s ntd-del version of icpc a. it lost most sialic acid binding activity tested in vero cells (hou et al., ) . in this study, we comparatively tested icpc a-s Δ and icpc a in vero and ipec-dq cells treated with na. similar to the previous reports, our results confirmed that the binding of s ntd of pedv to sialic acids on cell surface enhanced virus infectivity. bm is a mixture of highly glycosylated proteins containing sugar moieties, such as -n-acetyl- -o-acetylneuraminic acid (neu , ac ), -n-glycolylneuraminic acid (neu gc), and -n-acetylneuraminic acid (neu ac). among them, neu gc and neu ac can serve as receptors or co-receptors for some alphacoronaviruses (e.g., tgev) and gammacoronaviruses [e.g., infectious bronchitis virus (ibv)] (cavanagh and davis, ; krempl et al., ) . similar to tgev, which uses neu gc and neu ac as co-receptors (krempl et al., ; schultze et al., ; schwegmann-wessels and herrler, ) , the s ntd of pedv interacted with sugar (deng et al., ) . pgm contains . - . % n-acetylneuraminic acid (neuac). our data indicated that low-medium concentrations of bm or pgm enhanced the infectivity of s-intact pedv in both vero and ipec-dq cells. in contrast, the s ntd-del pedv was not affected or inhibited, probably due to the covered mucin that blocked virus binding to the receptors. the intestinal epithelial cells are covered by a layer of mucus that is rich in sialic acids. these results suggest that low-medium amount of mucin binding to s-intact pedv may help the virus particles approach the receptors via sticking to the sialic acids on the cell surface. however, the receptor binding domain (rbd) on the s protein of s ntd-del pedv, which lacks most sialic acid binding activity, is probably covered with mucin and blocked its binding to the receptors. when high concentration of mucin saturates the s ntd of s-intact pedv, it can also cover viral rbd and block its binding to the receptors, resulting in decreased infectivity as observed in . mg/ml bm effects on s-intact pedv in vero cells (fig. a) . the major components of bile include bile acids, cholesterol, lipids, bilirubin, proteins and carbohydrates. the bile acids are stored in the gall bladder (at ∼ mm) and released into the small intestine (mcleod and wiggins, ) . in the small intestine, the total bile acids have a concentration of - mm (dowling, ) . we selected two primary bile acids (gcdca and cdca) and two secondary bile acids (dca and udca) to test the effects of bile acids on pedv infection. the secondary bile acids are dehydroxylated ones from the primary bile acids by intestinal bacteria (björkhem, ) . our data indicated that a broad range of concentrations of bile and bile acids enhanced the infectivity of s-intact pedv in both vero and ipec-dq cells. however, bile and bile acids cannot increase the infectivity of s ntd-del pedv. these results suggest that the bile-and bile acid-mediated promotion of pedv infection is related to the s ntd. it was reported that gcdca may facilitate the adaptation of a s-intact pedv to trypsin-free growth in vero cells at the early passages (< p ) (kim et al., ) . however, the mechanisms of the bile acid function on the pedv infection is unknown and consequently need to be further investigated. the inability of the s ntd-del pedv to outcompete the s-intact pedv may account for the fact that s ntd-del pedvs were exclusively detected from coinfection with the s-intact pedv (diep et al., ; su et al., ) . consequently, the disease caused by the coinfection was as severe as that by the highly virulent s-intact pedv alone. this situation is quite different from what occurred for prcv and tgev: the wild spread of clinically mild prcv, which often infects pigs asymptomatically and repeatedly, inducing protective herd immunity against tgev outbreaks (vancott et al., ; brim et al., ) . prcv functions as a natural effective vaccine against tgev. however, for pedv, the clinically mild s ntd-del pedv variants do not infect pigs alone. therefore, safe and effective vaccines are still desired to control the deadly pedv infection in neonatal piglets. due to the % mortality rates of piglets in the icpc a-infected pigs (hou et al., ) and coinfection groups, we currently cannot determine whether the pathogenicity of pedv icpc a is enhanced during the coinfection. that should be investigated in older pigs (weaned pigs, sows and boars) which are more resistant to pedv infection and diseases (niederwerder and hesse, ) . in addition, the emergence of s ntd-del pedv variants may complicate pedv disease pattern on farms, which needs to be further investigated. in this study, we showed that the replication of the s-intact pedv was enhanced during coinfection with an s ntd-del pedv in pigs. we found that mucin, bile and bile acids can all increase the infection of sintact pedv but not the s ntd-del pedv. this feature may help explain why s-intact pedv outcompetes s ntd-del pedv in vivo. further studies are needed to understand the mechanisms of mucin-, bile-and bile acid-mediated enhancement or inhibition effects on the infection of different pedv variants. all animal experiments were performed according to the protocols approved by institutional animal care and use committee (iacuc) of the ohio state university (osu). mechanism of bile acid biosynthesis in mammalian liver cellular immune responses of pigs after primary inoculation with porcine respiratory coronavirus or transmissible gastroenteritis virus and challenge with transmissible gastroenteritis virus coronavirus ibv: removal of spike glycopolypeptide s by urea abolishes infectivity and haemagglutination but not attachment to cells bile acids are essential for porcine enteric calicivirus replication in association with downregulation of signal transducer and activator of transcription identification and comparison of receptor binding characteristics of the spike protein of two porcine epidemic diarrhea virus strains role of sialic acids in feline enteric coronavirus infections novel porcine epidemic diarrhea virus (pedv) variants with large deletions in the spike (s) gene coexist with pedv strains possessing an intact s gene in 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sequences, a key residue in the spike protein and deletions in nonstructural protein b of us strains of the virulent and attenuated coronaviruses, transmissible gastroenteritis virus and porcine respiratory coronavirus identification of porcine epidemic diarrhea virus variant with a large spike gene deletion from a clinical swine sample in the united states type iii interferon restriction by porcine epidemic diarrhea virus and the role of viral protein nsp in irf signaling the authors declare no conflict of interest. none. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.vetmic. . . . key: cord- -wig wdno authors: xu, qi; shan, yuanyuan; wang, ning; liu, yaping; zhang, maojie; ma, meihu title: sialic acid involves in the interaction between ovomucin and hemagglutinin and influences the antiviral activity of ovomucin date: - - journal: int j biol macromol doi: . /j.ijbiomac. . . sha: doc_id: cord_uid: wig wdno ovomucin (ovm) plays an important role in inhibiting infection of various pathogens. however, this bioactivity mechanism is not much known. here, the role of sialic acid in ovm anti-virus activity has been studied by elisa with lectin or ligand. structural changes of ovm after removing sialic acid were analyzed by circular dichroism and fluorescence spectroscopy. ovm could be binding to the hemagglutinin (ha) of avian influenza viruses h( )n( ) and h( )n( ), this binding was specific and required the involvement of sialic acid. when sialic acid was removed, the binding was significantly reduced . % and . %, respectively. therefore, sialic acid was proved as a recognition site which avian influenza virus bound to. meanwhile, the endogenous fluorescence and surface hydrophobicity of ovm removing sialic acid were increased and the secondary structure tended to shift to random coil. this indicated that ovm molecules were in an unfolded state and spatial conformation disorder raising weakly. remarkably, free sialic acid strongly promoted ovm binding to ha and thereby enhanced the interaction. it may contribute to the inhibition of host cell infection, agglutinate viruses. this study can be extended to the deepening of passive immunization field. ovomucin (ovm) has a unique antiviral activity, the mechanism of which is not entirely known. ovm is a highly glycosylated protein containing sialic acid (sa), which belongs to the mucin family [ ] . mucins are a major component of mucus, which are widely distributed in the body's internal surface and mucosal tissues, such as the respiratory tract and intestines. they provide an important innate immune barrier to potential toxins, particles, and pathogens [ ] . it can prevent pathogens from being in contact with susceptible cells. it is thought that the adhesion of mucins to pathogens is an important mechanism with a potentially significant effect. ovm has antiviral properties, that is similar in structure and composition to the influenza virus receptor and early findings suggest that ovm has an inhibitory effect on swine influenza virus-induced hemagglutination [ ] . the interaction of ha on the surface of the virus with ovm leads to the release of glycopeptide complexes. hemagglutination inhibition assays and enzyme-linked immunosorbent assays have revealed the high affinity of ovm for bovine rotavirus, chicken new castle disease virus and human influenza virus [ ] [ ] [ ] . it was found that nacetylneuraminic acid (neuac, a specific subtype of sialic acid in ovm) in the β-subunit could greatly facilitate the interaction between ovm and chicken new castle disease virus. the alteration of the conformation of ovm by the alkylation of disulfide bonds leads to loss of binding to ovm antibody [ ] . however, evaluations of antiviral activity in these studies focused primarily on the inhibition of viral-induced hemagglutination and did not include other more accurate and intuitive antiviral methods. structurally, ovm is a highly glycosylated protein whose monosaccharides are predominantly in the forms of oligosaccharides and glycosides consisting of fewer than monosaccharides [ , ] , including nglycosidic bonds and o-glycosidic bonds [ ] . n-glycans are linked to the aspartic acid (asp) residues of the polypeptide sequence asn-x-ser/thr, where x represents any amino acid except proline, and oglycans are predominantly linked to the serine (ser) and threonine (thr) residues [ , ] . these oligosaccharides mainly include mannose (man), galactose (gal), n-acetylgalactosamine (galnac), nacetylglucosamine (glcnac), sa, fuctose (fuc) [ ] and sulfuric acid esters [ ] . sialic acid of ovm can promote interaction with chicken new castle disease virus. meanwhile, various viruses such as influenza virus, coronavirus and rotavirus utilize glycoproteins containing sialic acid on the surface of host cells as recognition receptors [ ] [ ] [ ] . sialic acid can be recognized by the epitope on the globular head of the influenza virus ha, thereby inducing interaction with the corresponding ha receptor binding sites and interfering with or blocking the adsorption of the virus to the cells [ ] . sialic acid residues are generally located at the terminus of the n-linked oligosaccharide chain and the o-linked oligosaccharide chain with α , -, α , and α , -linkages [ ] . different influenza viruses are capable of specifically recognizing different linked types of sialic acids. human influenza viruses are more likely to bind to the α , -linkage sialic acid receptor, and avian influenza viruses preferentially recognize α , -linkage sialic acids [ , ] . ovm is a glycoprotein containing a large amount of sialic acid. its antiviral activity has not been studied deeply, and its anti-infective mechanism is barely understood. the role that sialic acid plays and whether it is recognized as the same receptor of the influenza virus and binds to ha remain to be revealed. therefore, this study aims to verify the interaction between ovm and ha and demonstrate the function of sialic acid in this interaction to explain the possible mechanism of ovm for satisfactory antiviral activity and increase the knowledge of the role of ovm in passive immunity. ovm was crude extracted according to a previously reported method [ ] with modifications. in brief, ml of fresh whole egg white was stirred at °c for min and subsequently diluted with ml of mm nacl. the ph was adjusted to . with m hcl, and the solution was incubated overnight at °c. the egg white solution was centrifuged at , g for min at °c, and the precipitate was resuspended with mm nacl for h and then centrifuged under the same conditions. after the precipitate, which was crude ovm, was washed several times with distilled water, the above procedure was repeated. the extract was freeze-dried and stored at − °c. ovm crude extract was purified by gel filtration chromatography (sephacryl s- hr, mm × cm) using the akta purification system (ge, usa). the target elution peak was dialyzed by distilled water, and the product was purified ovm. purified ovm ( mg) was mixed with μl of ph . sodium acetate buffer and μl of na enzyme and incubated in a °c water bath for h. desialylated ovm (dsa-ovm) was dialyzed against kd dialysis bags for h with ph . borate buffer. the solution was removed, stirred for h and centrifuged for min at r/min; the supernatant was saved for subsequent experiments. the dsa-ovm supernatant was centrifuged for min in a kd ultrafiltration tube at g and concentrated to . ml. a standard curve was used to quantify the concentration of ovm. the effect of enzymatic hydrolysis and the chemical bond of sialic acid in oligosaccharide chains was evaluated by elisa using lectins sambucus nigra (sna) and maackia amurensis (maa). a : dilution of sna was added to the elisa plate at μl/well, and the plate was incubated overnight at °c. to the control group was added pbs buffer without sna. the plates were washed times for min each with pbst and then again with pbst dissolved in % skim milk at μl/well and incubated at °c for h for blocking. after the plates were washed, dsa-ovm and ovm diluted to μg/ml were added to the experimental and control wells, respectively, at μl/well, and the plates were incubated at °c for h. after the plates were washed, ovm antibody diluted : was added at μl/well, and the plates were incubated at °c for . h. after the plates were washed, hrp-labeled goat anti-mouse igg diluted : was added at μl/well, and the plates were incubated at °c for min, washed and stained. the function of sialic acid was assessed by the change in the binding of ha to ovm with the removal of sialic acid. the ha proteins of the influenza viruses h n (ha ) and h n (ha ) were added to the elisa plate at a dilution of : at μl/well, and the plate was incubated overnight at °c. to the control group was added pbs buffer without ha. other operations were the same as in section . . the common components of oligosaccharide chains (gal, fuc, man and sialic acid) were separately mixed with ovm for competitive binding analysis with ha. subsequently, the effect of free sialic acid was analyzed by different additional sequences. the additional sequences were the addition of sialic acid followed by the addition of ovm, the addition of ovm followed by the addition of sialic acid, and the addition of preincubation mixture. the elisa procedure was the same as previously described. the protein solution was diluted -fold, the final protein concentration was . mg/ml, the optical path of the quartz cell was . cm, the sensitivity was mdeg/m, the wavelength scanning range was - nm, the speed was nm/s, and the resolution was . nm measured at room temperature. the optical path of the quartz cell was . cm. using tyrosine (tyr) as an intrinsic probe, the excitation wavelength was nm, and the emission spectrum was scanned at - nm. using tryptophan (trp), the excitation wavelength was nm, and the emission spectrum was scanned at - nm. the excitation and emission monochromators each had a bandwidth of nm. ovm was incubated at room temperature for h, and its concentration was diluted to μg/ml. blank samples were measured under the same conditions. using l-anilinonaphthalene- -sulphonate (ans) as a fluorescence probe, the surface hydrophobicity of ovm was measured by the fluorescence method. ovm was diluted to . - . mg/ml with ph . borate buffer. fluorescence spectra of ans were scanned by adding μl of diluted sample to μl of mmol/l ans solution. the excitation wavelength was nm, and the scan range was - nm. the fluorescence intensity at nm excitation and nm emission was used to making a standard curve for protein concentration. the control was blank ans solution with addition of the corresponding sample buffer. all values were expressed as mean ± s.e.m. anova with bonferroni's multiple-comparison test when more than two groups were compared. all the assays were run in triplicate and were representative of at least independent experiments. a p value b . was considered statistically significant and the asterisks in all figures are defined, *p b . , **p b . , ***p b . . in the elisa reaction, a variety of factors together affected the final test results, and the antigenic epitope of the sample had a significant impact. to ensure the accuracy of the experiment, we needed to verify whether sialidase enzymolysis would affect the other functions of ovm. in this experiment, elisa reaction of ovm was the key to analyzing physiological immune activity. the purified ovm was digested with sialidase to obtain dsa-ovm. the concentration of natural ovm was . mg/ml, and that of dsa-ovm was . mg/ml. for a concentration of μg/ml, the binding changes in ovm and dsa-ovm to antibody were analyzed (fig. a) . compared with that of natural ovomucin, the binding of dsa-ovm and antibody did not change significantly after enzymolysis, exhibiting a good consistency and no significant effect on the antibody binding activity. lectins sna binds specifically to sialic acid linked by an α - linkage, while maa specifically recognizes an α - -linkage. as shown in fig. b , the terminal sialic acid was effectively removed in dsa-ovm after enzymatic hydrolysis, and its binding activity was obviously lower than that in the natural ovm. the binding activity of natural ovm to lectin sna was higher than that to lectin maa, indicating that the terminal sialic acid glycosidic linkage in the ovm oligosaccharide chain was mainly α - and that less α - was present. after enzymolysis, all sialic acids in dsa-ovm significantly decreased (p b . ). the surface antigen ha of the influenza virus could bind to related glycoproteins via protein-protein or protein-carbohydrate chain interactions. to verify whether sialic acid is an important site of the interaction between ovm and ha, we designed a deglycosylation experiment. as shown in fig. , ha of both h n and h n bound to ovm and did not react with bsa (negative control). after sialic acid was removed, the binding of dsa-ovm to ha was significantly lower than that of natural ovomucin. at the same dose, the binding capacity of dsa-ovm to ha decreased . %, and the binding capacity to ha also decreased by . %. this indicated that ha recognizes sialic acid on ovm. sialic acid in ovm is distributed on the oligosaccharide chain terminus. therefore, it can be considered that this recognition involves the participation of sialic acid and oligosaccharide chains. based on the above results, it has been demonstrated that the carbohydrate chain is one of the recognition regions for the interaction between ovm and ha. sialic acid is the critical target. to determine whether this interaction occurred with the participation of other sugar chain components, a sugar competition experiment was conducted. different kinds of sugar were added to the plate coated with ha, followed by washing off and then adding ovm to measure the binding changes. the results displayed in fig. indicate that no monosaccharide had an inhibitory effect on ovm binding ha. there was no significant difference in binding after pre-incubation of carbohydrates and ha compared to the negative control, and the addition of free sialic acid did not cause a decrease in binding. this binding trend was consistent with both ha and ha . according to the results in fig. , when the reaction sequence in the experiment was the addition of sialic acid firstly and then the addition of ovm, it was unexpectedly found that free sialic acid promoted the interaction of ovm and ha. to analyze the role played by free sialic acid in the binding of ovm to ha, further experiments were carried out with different sequences of additions. adding ovm at first and then adding sa or adding the two components together were analyzed (fig. ) . when ovm was first added, followed by sa, neither ovm nor dsa-ovm showed any significant change compared with the negative control, and the binding intensity tended to be similar in ha and ha . when ovm was mixed with sa and added at the same time, it is interesting found that the binding of ovm to ha was significantly enhanced far more than the negative control and other groups. this change in ha was as significant as in ha . based on the above results, it was found that free sialic acid enhances the binding of ovm to influenza virus ha. the changes in the secondary structure of dsa-ovm and ovm were analyzed by circular dichroism spectroscopy (cd), and the results were shown in fig. a . the cd spectra of ovm showed weak negative peaks at nm and nm and a positive peak approximately nm, characteristic of α-helical and β-sheet hybrids [ ] . the negative peak of the dsa-ovm spectrum near nm was weaker and closer to the short wavelength direction than ovm. data on specific changes are shown in table . after removing sialic acid, ovomucin α-helix decreased, while the random coil increased, indicating that the degree of disorder increased [ ] . fluorescence spectroscopy is a powerful tool for studying the changes in the protein microenvironment in solution. aromatic amino acid (tryptophan (trp), tyrosine (tyr), and phenylalanine (phe)) residues in ovm molecules can fluoresce. therefore, the changes in the ovm molecule before and after removing the terminal sialic acid can be reflected according to the change in endogenous fluorescence [ , ] . the endogenous fluorescence spectra of ovm before and after removing the terminal sialic acid at excitation wavelengths of nm and nm were shown in fig. b and c, respectively. as shown in fig. . effects of sa different additional sequences on binding. (a) dsa-ovm binding with ha was reduced. adding sa after adding dsa-ovm or ovm was not able to improve the binding. however, the addition of ovm premixed with sa greatly enhanced the interaction with ha , and pre-incubation the dsa-ovm with sa had a significant improvement in its combination with ha . (b) adding sa after adding ovm or dsa-ovm did not influence its binding ability with ha . the addition of dsa-ovm premixed with sa strengthened the interaction with ha significantly, and as much as pre-incubation the ovm with sa prior to addition to the ha . ovm/sa represents the addition of natural ovomucin first then the addition of sa; dsa-ovm/sa represents the addition of dsa-ovm first and then the addition of sa; ovm + sa represents a pre-incubated mixture of natural ovomucin and sa being added at the same time; dsa-ovm + sa represents a pre-incubated mixture of dsa-ovm and sa being added at the same time; ovm, natural ovomucin; dsa-ovm, ovomucin with the removal of the sialic acid residue; sa, sialic acid, here n-acetylneuraminic acid was used as specific subtype of sialic acid in ovomucin. pre-incubation free monosaccharide with ha also did not interfere with the binding. even the addition of free sialic acid induced increase slightly. (−), negative control, without adding monosaccharide; gal, galactose; fuc, fucose; man, mannose; sa, sialic acid, here n-acetylneuraminic acid was used as specific subtype of sialic acid in ovomucin. the data are presented as mean ± s.e.m. the tyr fluorescence spectrum of fig. b , removal of terminal sialic acid results in enhancement of the endogenous fluorescence intensity of ovm, but no obvious migration of the fluorescence peak occurs. the fluorescence spectra of trp was in fig. c . the overall trend in fluorescence intensity agrees with the result of tyr being a fluorescent probe. however, the λ max of the trp spectrum was blueshifted after removing the terminal sialic acid, suggesting that after removal of sialic acid, the ovm microenvironment hydrophobicity is enhanced. in order to verify this result, the changes in the ans fluorescence spectra of ovm before and after the removal of terminal sialic acid were investigated by an ans fluorescence probe. according to the result, the fluorescence intensity of ans was enhanced after ovm removal of the terminal sialic acid. the value of ovm surface hydrophobicity was . and the value of dsa-ovm was . . this shows that ovm is located in a microenvironment with a non-polar increase and enhanced surface hydrophobicity. this is related to the unfolding of the ovm molecule and the increase in disorder, resulting in the exposure of the hydrophobic region, strengthening the binding of ans to the ovm hydrophobic region, and enhancing the fluorescence intensity [ ] . the exact mechanism through which ovm exerts its antiviral function is not entirely clear at present. this research studied the interaction between ovm and the influenza virus ha and the role of sialic acid in this interaction. it was found that the ovm carbohydrate chain contains terminal sialic acid with both α- - and α - -linkages (fig. b) . has cannot bind to other proteins such as bsa but can interact with ovm. this interaction is dependent on the presence of sialic acid (fig. ) . monosaccharides have no influence on the binding of ovm with ha. it is noteworthy that the binding of ha with ovm was strongly promoted by free sialic acid (figs. and ) . it is indicating that sialic acid is involved in the binding of ovm to influenza virus, and additional free sialic acid could enhance the ovm antiviral process. mucin, a natural barrier widely found in animals, always plays critical role in antiviral and antibacterial activities [ ] [ ] [ ] [ ] . ovm is a mucin protein in egg. according to our previous research, ovm contains plenty of sialic acid, and its subtype is neuac. in this study, we demonstrated the glycosidic bond type of sialic acid linking the ovm carbohydrate chain terminus, which contains both α - and α - -linkages. the content of sialic acid with the α - -linkage was higher than that with the α - -linkage (fig. b) . in the process through which the influenza virus infects the body, the first step is to recognize and bind to the cell surface receptor through the viral surface protein ha [ ] . the main receptors are cell surface glycoconjugates, and sialic acid is required for this process. human influenza viruses mainly recognize α - -linked sialic acids, whereas avian influenza viruses prefer α - -linked sialic acids [ , ] . therefore, due to the type of sialic acid linkages in the ovm oligosaccharide chain, we believe that ovm has anti-infective activity against both human and avian influenza viruses. furthermore, the anti-avian influenza virus function is closely related to α - linked sialic acid. to confirm this hypothesis, this study was carried out to enzymatically hydrolyze sialic acid. using the ha of influenza viruses h n and h n as the binding protein, the changes in ovm as an acceptor with or without sialic acid binding ha were measured. firstly, the interaction of ovm before and after removing sialic acid with its antibody was analyzed, and it was found that sialic acid was not the binding site. no significant difference was found in the binding activity before and after the removal of sialic acid. this demonstrates that sialic acid does not participate in all the functions of ovm but also that follow-up experiments can use an elisa reaction between ovm and an antibody. this ensures that the results are real and effective. subsequently, the interaction of natural ovm and dsa-ovm with ha was investigated, and no binding reaction between ha and bsa was found, which indicated that the recognition of glycoprotein was specific (fig. ) . however, the obvious interaction between ha and natural ovm indicates that ha can specifically recognize ovm and associate with it. on the other hand, the binding of ovm to ha was significantly reduced after sialic acid removal to only % to % of the original (fig. ) . these results showed the same tendency in h n and h n , which proves that sialic acid is involved in the recognition of ha and ovm. after sialic acid was removed, this binding could be inhibited. thus, we demonstrate that one of the antiviral activity mechanisms of ovm is associated with sialic acid in the ovm oligosaccharide chain competing with the cellular receptor for binding ha. after removing sialic acid, ovm still retains some ability to bind ha, presumably through other recognition mechanisms. in fact, sialic acid was not completely removed after enzymatic hydrolysis (fig. b) . therefore, this binding may be associated with sialic acid residues, and the role of sialic acid in this mechanism may be underestimated. to further demonstrate the influence of the terminal monosaccharide of carbohydrate chain in the binding of ovm with ha, several monosaccharides that are commonly found near the end of the oligosaccharide chain were selected and tested. to determine whether gal, fuc, man and free sialic acid could bind with ha, we conducted competition experiment. the results show that after adding of these free sugars first, the binding of ovm and ha was not significantly different and basically maintained at the same level. it suggests that these carbohydrates are not involved in binding and cannot prevent this interaction. monomeric sialic acid is thought to be unable to compete effectively with the receptor on target cells for binding to influenza virus, while dendritic sialic acid structure is more effective against influenza virus [ ] . the process by which influenza virus recognizes sialic acid is strongly related to its linked pattern [ , , , ] . this is similar to the finding that free sialic acid does not effectively bind with ha in this study (fig. ) . sialic acid is a highly electronegative sugar molecule, and the role in ovm is worth in-depth exploration. for this reason, we designed the influence of free sialic acid on the interaction between ovm and ha under different sequences of addition. when ovm was added firstly and then free sialic acid, there was no significant change in the binding of ovm with ha, and the interaction remained at the same level as the control. however, it was exciting to note that when ovm was preincubated with free sialic acid, its binding to ha was significantly enhanced compared to the control. it had the same effect on h n and h n (fig. ) . sialic acid in ovm was involved in the recognition of ha. free sialic acid does not directly bind to ha but mixed with ovm can effectively improve the interaction. sialic acid is electronegative and carries multiple o atoms and n atoms that can have many unique physicochemical effects [ , ] . taken together, this study proposes a novel mechanism hypothesis that sialic acid plays an active role in the antiviral activity of ovm (fig. ) . first, sialic acid in the ovm carbohydrate chain is recognized by the influenza virus ha as a receptor [ ] . on the other hand, free sialic acid is not directly involved in ha binding, but it can bind ovm to form an eupolymer by electrostatic interaction or hydrogen bond [ ] , promote ovm to bind and co-precipitate with ha. therefore, ovm maintain effective defense against virus, and enhance its antiviral activity. in a future study, this hypothesis will be explored in vitro and in cell experiments. in addition, we also analyzed the influence of ovm structure after removing sialic acid. the results exhibited that the secondary structure does not change too much but can cause ovm tertiary structural changes and that increasing the surface hydrophobicity causes molecular expansion [ , ] . this may increase the hydrogen bond binding sites of sialic acid to ovm. therefore, the structural interpretation of ovm antiviral activity is worth further study. ovomucin contains sialic acids with both α - and α - linkages, and the α - bond is higher than the α - bond, which helps it against a wide variety of influenza viruses. the hemagglutinin of the influenza virus recognizes and binds to the ovomucin carbohydrate chain terminal sialic acid, and the interaction is greatly diminished after the sialic acid is removed. therefore, sialic acid is an important recognition site for ovomucin to play a role in the binding of hemagglutinin competing with host cell surface receptors. at the same time, the removal of ovomucin sialic acid lead to a slight increase in the random coil of secondary structure and enhance surface hydrophobicity. we also found a unique role for free sialic acid. the addition of free sialic acid can promote the binding of ovomucin to hemagglutinin and enhance ovomucin anti-influenza virus activity. this in-depth study and exposition can complement the mechanism of ovomucin involved in innate immunity and provide new ideas and perspectives for the development of antivirus agents. an inventory of mucin genes in the chicken genome shows that the mucin domain of muc is encoded by multiple exons and that ovomucin is part of a locus of related gel-forming mucins understanding the adhesion mechanism of a mucin binding domain from lactobacillus fermentum and its role 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at nonconsensus sites chemical and physical characterization of ovomucin, a sulfated glycoprotein complex from chicken eggs determination of the structure of sulfated tetra-and pentasaccharides obtained by alkaline borohydride degradation of hen ovomucin. a fast atom bombardment-mass spectrometric and h-nmr spectroscopic study natural and synthetic sialic acid-containing inhibitors of influenza virus receptor binding early detection of influenza a(h ) viruses with affinity for the human sialic acid receptor by maldi-tof mass spectrometry based mutation detection the receptor preference of influenza viruses the biology of influenza viruses inflammation-dependent changes in α , -, α , -, and α , -sialic acid glycotopes on serum glycoproteins in mice investigating the interaction between influenza and sialic acid: making and breaking the link, influenza virus sialidase-a drug discovery target influenza virus sialidase-a drug discovery target a new method of separating ovomucin from egg white the use of circular dichroism in the investigation of protein structure and function simultaneous quantification of protein order and disorder binding interaction of atorvastatin with bovine serum albumin: spectroscopic methods and molecular docking mg + binding affects the structure and activity of ovomucin protein unfolding as a switch from selfrecognition to high-affinity client binding sialic acids: fascinating sugars in higher animals and man evaluation of the porcine gastric mucin binding assay for highpressure-inactivation studies using murine norovirus and tulane virus impact of mucin, bile salts and cholesterol on the virulence of vibrio anguillarum towards gnotobiotic sea bass (dicentrarchus labrax) larvae mucins: a biologically relevant glycan barrier in mucosal protection receptor specificity in human, avian, and equine h and h influenza virus isolates alpha - -and alpha - -n-linked sialic acids allow efficient interaction of newcastle disease virus with target cells inhibition of viral adhesion and infection by sialic-acidconjugated dendritic polymers binding kinetics of influenza viruses to sialic acid-containing carbohydrates identification of the sialic acid structures recognized by minute virus of mice and the role of binding affinity in virulence adaptation sialic acids in molecular and cellular interactions glycosylation of human iga directly inhibits influenza a and other sialic-acid-binding viruses sialic acid-triggered macroscopic properties switching on a smart polymer surface oxidation of c-reactive protein by hypochlorous acid leads to the formation of potent platelet activator glyoxal-induced modification enhances stability of hemoglobin and lowers iron-mediated oxidation reactions of the heme protein: an in vitro study this work was financially supported by national natural science foundation of china [grant no. ]. key: cord- -b yuh y authors: shi, yanhong; zheng, cheng; li, jinhang; yang, li; wang, zhengtao; wang, rui title: separation and quantification of four main chiral glucosinolates in radix isatidis and its granules using high-performance liquid chromatography/diode array detector coupled with circular dichroism detection date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: b yuh y as chemical drugs, separation and quantification of the specific enantiomer from the chiral compounds in herbal medicines are becoming more important. to clarify the chemical characterization of chiral glucosinolates—the antiviral active ingredients of radix isatidis, an optimized efficient method of hplc-uv-cd was developed to simultaneously separate and quantify the four main chiral glucosinolates: progoitrin, epiprogoitrin, and r,s-goitrin. the first step was to determine progoitrin, epiprogoitrin, and r,s-goitrin using hplc-uv, and then determine the r-goitrin and s-goitrin by coupling with cd detection. subsequently, through the linear relations between anisotropy factor (g factor) and the percent optical purity of r-goitrin, the contents of r-goitrin and s-goitrin from the r,s-goitrin mixture were calculated separately. furthermore, the chemical composition features of the four chiral glucosinolates in samples from crude drugs, decoction pieces, and granules of r. isatidis were conducted. the total content of the four glucosinolates was obviously higher in crude drugs, and the variance character of each glucosinolate contents was different. in summary, the accurate measurement method reported here allows for better control of the internal quality of r. isatidis and its granules and provides a powerful approach for the analysis of other chiral components in traditional chinese medicines. natural products from traditional chinese medicines (tcms) represent a major part of today's pharmaceutical market as they possess a number of biological activities, such as antimicrobial, antitumor, anti-inflammatory, antiviral, cardiovascular activities, and so on [ ] . since chirality is a fundamental characteristic of nature, a large number of well-known therapeutic ingredients from elution were compared. comparison with different compositions of acetonitrile/methanol-water, methanol-water with different modifiers including formic acid and ammonium acetate showed the combination of methanol and mm ammonium acetate (adjusted ph for . with formic acid) gave an optimal mobile phase system for glucosinolates separation. as shown in figure s , in order to acquire better peak intensity, five uv wavelengths- , , , , nm-were detected and nm gave the best results. in addition, the maximum absorption cd wavelength of rand s-goitrin was focused on and nm, respectively, whereas the detection range of the present cd- detector was - nm. therefore, the cd wavelength was set at nm. as shown in table , all calibration curves of the glucosinolates showed good linearity (r > . ) within a relatively wide range of concentration ( . - . mg/ml). the limit of detection (lod) and the limit of quantification (loq) were lower than . and . µg/ml, respectively. table . regression equations, linear range, limit of detection (lod) and limit of quantification (loq) for three analytes. the known purities of r-goitrin (%) and s-goitrin (%) were mixed and simultaneously determined by uv and cd to calculate the g factors ( table ). the relative purities of r-goitrin (%) and g factors made calibration curves. the calibration curve between g factors (x ) and the different relative purities of r-goitrin (y ) was y = − . x + . (r = . ), which showed a good linearity with a wide range of r-goitrin purities ( ~ %). the precision, repeatability, and stability of the developed method were also validated for each analyte, and the rsd values were less than . %. the recoveries of five glucosinolates were measured from . to . %, and the rsd values were less than . % (table ) . compared to previous published methods [ ] [ ] [ ] [ ] , glucosinolates profiles of crude drug, decoction pieces, and granules of r. isatidis were performed using hplc-uv-cd, which improved the limitation of high-cost chiral columns, complex operation of sample preparation, and simultaneous quantification of the characteristic glucosinolates. in the typical uv and cd chromatograms of r. isatidis samples (figure ), r-goitrin and s-goitrin showed the same retention time, uv absorption feature, and overlapping peak. they could not be separated by uv detection without using the specific chiral columns ( figure a ,b; upper). in contrast to that, r-goitrin and s-goitrin had completely opposite chromatographic profiles at the same retention time as shown in cd chromatograms ( figure a ,b; lower). notably, in the typical cd chromatograms, only r-goitrin could be detected in the mixed r,s-goitrin, crude drugs, decoction pieces, and granules of r. isatidis. therefore, the r-goitrin content could be quantified using cd detector, while the s-goitrin content was calculated using the mixed r,s-goitrin and the determined r-goitrin content. combining the cd chromatographic properties with total content of the mixed r,s-goitrin, it provided an efficient method to determine the content of r-goitrin in the mixed r,s-goitrin without chiral separation. compared to previous published methods [ ] [ ] [ ] [ ] , glucosinolates profiles of crude drug, decoction pieces, and granules of r. isatidis were performed using hplc-uv-cd, which improved the limitation of high-cost chiral columns, complex operation of sample preparation, and simultaneous quantification of the characteristic glucosinolates. in the typical uv and cd chromatograms of r. isatidis samples (figure ), r-goitrin and s-goitrin showed the same retention time, uv absorption feature, and overlapping peak. they could not be separated by uv detection without using the specific chiral columns ( figure a ,b; upper). in contrast to that, r-goitrin and s-goitrin had completely opposite chromatographic profiles at the same retention time as shown in cd chromatograms ( figure a ,b; lower). notably, in the typical cd chromatograms, only r-goitrin could be detected in the mixed r,s-goitrin, crude drugs, decoction pieces, and granules of r. isatidis. therefore, the r-goitrin content could be quantified using cd detector, while the s-goitrin content was calculated using the mixed r,s-goitrin and the determined r-goitrin content. combining the cd chromatographic properties with total content of the mixed r,s-goitrin, it provided an efficient method to determine the content of r-goitrin in the mixed r,s-goitrin without chiral separation. as shown in figure , the chemical composition of progoitrin, epiprogoitrin, and r,s-goitrin in crude drugs, decoction pieces, and granules of r. isatidis showed that the chromatographic patterns were found to be consistent between all samples but their contents obviously varied. the three analytes could be easily detected in the uv chromatograms, while only r-goitrin was determined in cd chromatograms. we think the different absorption intensity between uv and cd detection might be due to the differences in chemical structures. therefore, the content variations of these characteristic glucosinolates should be conducted. the contents of four glucosinolates-progoitrin, epiprogoitrin, r-goitrin, and s-goitrin-were simultaneously quantified in samples using hplc-uv coupled with cd detection. as shown in figure and table s , the total contents of four glucosinolates in three types of r. isatidis were significantly different. it was obviously higher in crude drugs ( . - . mg/g, mean ± sd: . ± . ) and decoction pieces ( . - . mg/g, mean ± sd: . ± . ), while the glucosinolates occupied relatively lower contents in its granules ( . - . mg/g, mean ± sd: . ± . ). as shown in figure , the chemical composition of progoitrin, epiprogoitrin, and r,s-goitrin in crude drugs, decoction pieces, and granules of r. isatidis showed that the chromatographic patterns were found to be consistent between all samples but their contents obviously varied. the three analytes could be easily detected in the uv chromatograms, while only r-goitrin was determined in cd chromatograms. we think the different absorption intensity between uv and cd detection might be due to the differences in chemical structures. therefore, the content variations of these characteristic glucosinolates should be conducted. the contents of four glucosinolates-progoitrin, epiprogoitrin, r-goitrin, and s-goitrin-were simultaneously quantified in samples using hplc-uv coupled with cd detection. as shown in figure and table s , the total contents of four glucosinolates in three types of r. isatidis were significantly different. it was obviously higher in crude drugs ( . - . mg/g, mean ± sd: . ± . ) and decoction pieces ( . - . mg/g, mean ± sd: . ± . ), while the glucosinolates occupied relatively lower contents in its granules ( . - . mg/g, mean ± sd: . ± . ). most previous studies have focused only on the content of r,s-goitrin mixture in r. isatidis and its related products. in the present study, r-goitrin accounted for a majority of the r,s-goitrin component in crude drugs, decoction pieces, and granules of r. isatidis; the r-goitrin content was twice as much as that of s-goitrin. the results might provide positive evidence that r-goitrin is responsible for the pharmacological properties of r. isatidis. it was noticed that progoitrin and epiprogoitrin contents showed an obvious declining trend in crude drugs and decoction pieces of r. isatidis. in particular, they could not be detected in most of the granule samples. compared with crude drugs, r-goitrin and s-goitrin contents in decoction most previous studies have focused only on the content of r,s-goitrin mixture in r. isatidis and its related products. in the present study, r-goitrin accounted for a majority of the r,s-goitrin component in crude drugs, decoction pieces, and granules of r. isatidis; the r-goitrin content was twice as much as that of s-goitrin. the results might provide positive evidence that r-goitrin is responsible for the pharmacological properties of r. isatidis. it was noticed that progoitrin and epiprogoitrin contents showed an obvious declining trend in crude drugs and decoction pieces of r. isatidis. in particular, they could not be detected in most of the granule samples. compared with crude drugs, r-goitrin and s-goitrin contents in decoction pieces increased slightly. the traditional processing and extraction methods could improve the biotransformation of progoitrin and epiprogoitrin, and then increase the content of the degradation products (r-and s-goitrin). this is consistent with our previous report [ , ] . however, the content dynamic change features of the glucosinolates in crude drugs, decoction pieces, and granules as well as the main influencing parameters for biotransformation among these glucosinolates need to be further investigated. fifteen crude drugs (b -b ), nine decoction pieces (b -b ), and thirteen granules (b -b ) of r. isatidis were collected from the main commercial herbal markets and different herbal manufactories in china (table s ). the vouchers have been deposited in shanghai r&d centre for standardization of chinese medicines, shanghai university of traditional chinese medicine. the four glucosinolates-epiprogoitrin, progoitrin, and r,s-goitrin-were isolated and purified from the root of i. indigotica fort. in our laboratory. r-goitrin and s-goitrin were prepared from r,s-goitrin using the shiseido ® cd-ph chiral column ( µm, × . mm) with acetonitrile-water ( : , v/v). their chemical structures ( figure ) were elucidated by a series of spectroscopic and chemical analyses [ ] . the purities of five glucosinolates were determined to be higher than . % through hplc-dad analysis. hplc-grade acetonitrile, methanol, formic acid (≥ . %, hplc grade), and ammonium acetate (≥ . %, hplc grade) for hplc analysis were purchased from thermo fisher scientific (swedesboro, nj, usa). water was prepared by a milli-q ® system (millipore, ma, usa). all other reagents of analytical grade for extraction were purchased from tianjin damao chemical reagent factory (tianjin, china). molecules , , x for peer review of pieces increased slightly. the traditional processing and extraction methods could improve the biotransformation of progoitrin and epiprogoitrin, and then increase the content of the degradation products (r-and s-goitrin). this is consistent with our previous report [ , ] . however, the content dynamic change features of the glucosinolates in crude drugs, decoction pieces, and granules as well as the main influencing parameters for biotransformation among these glucosinolates need to be further investigated. fifteen crude drugs (b -b ), nine decoction pieces (b -b ), and thirteen granules (b -b ) of r. isatidis were collected from the main commercial herbal markets and different herbal manufactories in china (table s ). the vouchers have been deposited in shanghai r&d centre for standardization of chinese medicines, shanghai university of traditional chinese medicine. the four glucosinolates-epiprogoitrin, progoitrin, and r,s-goitrin-were isolated and purified from the root of i. indigotica fort. in our laboratory. r-goitrin and s-goitrin were prepared from r,s-goitrin using the shiseido ® cd-ph chiral column ( μm, × . mm) with acetonitrile-water ( : , v/v). their chemical structures ( figure ) were elucidated by a series of spectroscopic and chemical analyses [ ] . the purities of five glucosinolates were determined to be higher than . % through hplc-dad analysis. hplc-grade acetonitrile, methanol, formic acid (≥ . %, hplc grade), and ammonium acetate (≥ . %, hplc grade) for hplc analysis were purchased from thermo fisher scientific (swedesboro, nj, usa). water was prepared by a milli-q ® system (millipore, ma, usa). all other reagents of analytical grade for extraction were purchased from tianjin damao chemical reagent factory (tianjin, china). hplc system (agilent , santa clara, ca, usa) was equipped with a g dad, a quaternary pump and an online degasser. a cd detector (cd- , jasco, tokyo, japan) was epiprogoitrin ( ) progoitrin ( ) s-goitrin ( b) r-goitrin ( a) detector for the chiral analysis. the mobile phase consisted of methanol (a) and mm ammonium acetate (adjusted ph for . with formic acid b). the sample solutions were analyzed on a ultimate aq-c column ( µm, × . mm) with a gradient elution system as follows: - min, % a; - min, - % a; - min, % a. the flow rate was . ml/min, and the column temperature was set at • c. uv and cd detective wavelengths were set at nm and nm, respectively. each of the five reference compounds was accurately weighed and then dissolved in water to prepare the standard solutions of . mg/ml for progoitrin, . mg/ml for epiprogoitrin, . mg/ml for r,s-goitrin, . mg/ml for r-goitrin, and . mg/ml for s-goitrin. for uv detection, a series of standard solutions were prepared by appropriate dilution of the stock solutions to make calibration curves. the calibration curve was obtained by plotting peak area (y ) of each reference glucosinolate against the concentration of the corresponding compound (x ). furthermore, the known purities of r-goitrin (%) were determined by uv and cd to calculate the anisotropy factor (g = ∆a/a). the calibration curve depended on g factors (y ) of each reference r-goitrin against the relative purities of r-goitrin (x ). crude drugs and decoction pieces: the respective sample was pulverized to obtain homogeneous fine powder. . g of the fine powder was accurately weighed and extracted with ml water by refluxing in a water bath for h, then cooled and filtered. ml of the filtrate was dissolved with ml water containing % formic acid (v/v). the mixed solution was packed into a spe column (waters oasis wax cc cartridge/ mg, waters, usa), which was activated with ml methanol and then washed with ml water before adding sample solution. the spe column was orderly eluted with ml water containing % formic acid, ml methanol (a) and ml methanol containing % ammonium hydroxide (v/v, b). subsequently, the eluted solutions of a and b was dried by flushing with nitrogen (n ). the residue was dissolved with . ml methanol for hplc analysis. granules: the granules were pulverized and screened through a µm sieve to obtain homogeneous fine powder. . g (containing sugar) and . g (no sugar) of the fine powder was accurately weighed and extracted with ml water by ultrasonication at room temperature for min, then cooled and filtered. ml of the filtrate was dissolved with ml water containing % formic acid (v/v). the mixed solution was packed into a spe column. subsequently, the sample solution was prepared through the same operation procedure with sample solutions of crude drugs and decoction pieces. linearity was assessed by generating six-point calibration curves for each reference compound. the precision was evaluated by replicate injections of a mixture solution containing four glucosinolates and a sample solution of r. isatidis (b ). six injections of the same preparation solutions per day were investigated for three days. six preparation solutions of a sample (b ) were analyzed for repeatability. to determine the recovery rate of extraction, the four reference components (approximately %, % and % of the original amount) were added to the weighed powder of r. isatidis that was extracted and analyzed. as the cd signal depends only on the enantiomeric composition of the chiral molecule whereas uv absorbance is related to the analyte concentration, the measurement method of the enantiomeric content from the chiral sample was referred to the reported literatures [ , ] . firstly, the cd detector recorded both dichroic (∆ε or ∆a) and uv (ε or a) signals at the optimized wavelength and calculated the anisotropy factor (g = ∆ε/ε or ∆a/a), which is dependent of the enantiomeric purity. secondly, screening for the bioactive constituents of traditional chinese medicine-progress and challenges the market of chiral drugs: chiral switches versus de novo enantiomerically pure compounds putting chirality to work: the strategy of chiral switches fda drug approvals recent progress in the studies of chemical consituents, pharmacological effects and quality control methods on the roots of isatis indigotica english), part i; china medical science and technology press progress of studies on chemical constituents and pharmacological effects of radix isatidis. chin. wild plant reour efficacy of treatment if influenza a (h n ) with oseltamivir phosphate and isatis root granules antiviral activity of isatis indigotica extract and its derived indirubin against japanese encephalitis virus. evid. based complement. altern in vitro inhibition of influenza virus infection by a crude extract from isatis indigotica root resulting in the prevention of viral attachment progress of studies on antiviral effective constituents from radix isatidis antivirus constituents of radix of isatis indigotica the in vitro anti-virus effects and dose-effect relationship of epigoitrin and fructopyrano-( → )-glucopyranose based on "deletion/increment" strategy determination of r,s-goitrin of banlangen preparation by rp-hplc biotransformation of glucosinolates epiprogoitrin and progoitrin to (r)-and (s)-goitrin in radix isatidis quantitative and chemical fingerprint analysis for the quality evaluation of isatis indigotica based on ultra-performace liquid chromatography with photodiode array detector combined with chemometric methods - -vinyl- -thiooxazolidone, an antithyroid compound from yellow turip and from brassica seeds determination of epigoitrin and goitrin in isatidis radix by chiral high performance liquid chromatography. chin stereospecific assay of (r)-and (s)-goitrin in commercial formulation of radix isatidis by reversed phase high-performance liqiud chromatography qualitative and quantitative analyses of goitrin-epigoitrin in isatis indigotica using supercritical fluid chromatography-photodiode array detector-mass spectrometry improved chiral separation of (r,s)-goitrin by sfc: an application in traditional chinese medicine an efficient method for separation and purification of glucosinolate stereoisomers from radix isatidis chiral analysis of milnacipran by a nonchrial hplc-circular dichroism: improvement of the linearity of dichroic response by temperature control reliable assay of extreme enantiomeric purity values by a new circular dichroism based on hplc detection system author contributions: l.y., z.w., and r.w. designed the research; y.s., c.z., and j.l. performed the experiments and analyzed the data; y.s. and c.z. wrote the paper. all of the authors have read and approved the content of the manuscript. the authors declare no conflict of interest. of through the linear relations between g factor and the percent optical purity of the enantiomeric, the relative proportion of the enantiomeric in the chiral molecule were calculated. finally, the absolute content of enantiomeric in the chiral molecule was measured by its relative purity and uv absorbance. the present study proposed an efficient method of hplc-uv-cd for simultaneous separation and quantification of the main chiral glucosinolates in r. isatidis. progoitrin, epiprogoitrin and r,s-goitrin were easily determined by uv detection, whereas r-goitrin and s-goitrin were separated by coupling with cd detection. through the linear relations between g factor and the percent optical purities of r-goitrin, the contents of r-goitrin and s-goitrin were successfully calculated. subsequently, glucosinolate profiles in crude drugs, decoction pieces and granules of r. isatidis were conducted and found that the variance character of each of the glucosinolate contents was different. in summary, the new method allows better control of the internal quality of r. isatidis and its granules and provides a powerful approach and foundation for further investigation of dynamic change features of the contents and the main influencing parameters for biotransformation of glucosinolates in r. isatidis. the following are available online. figure s : the cd absorbance features of rand s-goitrin (a), maximum uv absorbance spectrum of the four glucosinolates (b), and the chemical profiling of the four glucosinolates in five different wavelength (c); table s : crude drugs, decoction pieces and granules of radix isatidis used in this study; table s : the contents of four glucosinolates in samples from crude drugs, decoction pieces and granules of radix isatidis (n = ). key: cord- - hn authors: shaw, dario r.; ali, muhammad; katuri, krishna p.; gralnick, jeffrey a.; reimann, joachim; mesman, rob; van niftrik, laura; jetten, mike s. m.; saikaly, pascal e. title: extracellular electron transfer-dependent anaerobic oxidation of ammonium by anammox bacteria date: - - journal: biorxiv doi: . / sha: doc_id: cord_uid: hn anaerobic ammonium oxidation (anammox) by anammox bacteria contributes significantly to the global nitrogen cycle, and plays a major role in sustainable wastewater treatment. anammox bacteria convert ammonium (nh +) to dinitrogen gas (n ) using nitrite (no −) or nitric oxide (no) as the electron acceptor. in the absence of no − or no, anammox bacteria can couple formate oxidation to the reduction of metal oxides such as fe(iii) or mn(iv). their genomes contain homologs of geobacter and shewanella cytochromes involved in extracellular electron transfer (eet). however, it is still unknown whether anammox bacteria have eet capability and can couple the oxidation of nh + with transfer of electrons to carbon-based insoluble extracellular electron acceptors. here we show using complementary approaches that in the absence of no −, freshwater and marine anammox bacteria couple the oxidation of nh + with transfer of electrons to carbon-based insoluble extracellular electron acceptors such as graphene oxide (go) or electrodes poised at a certain potential in microbial electrolysis cells (mecs). metagenomics, fluorescence in-situ hybridization and electrochemical analyses coupled with mec performance confirmed that anammox electrode biofilms were responsible for current generation through eet-dependent oxidation of nh +. n-labelling experiments revealed the molecular mechanism of the eet-dependent anammox process. nh + was oxidized to n via hydroxylamine (nh oh) as intermediate when electrode was the terminal electron acceptor. comparative transcriptomics analysis supported isotope labelling experiments and revealed an alternative pathway for nh + oxidation coupled to eet when electrode is used as electron acceptor compared to no −as electron acceptor. to our knowledge, our results provide the first experimental evidence that marine and freshwater anammox bacteria can couple nh + oxidation with eet, which is a significant finding, and challenges our perception of a key player of anaerobic oxidation of nh + in natural environments and engineered systems. abstract: anaerobic ammonium oxidation (anammox) by anammox bacteria contributes significantly to the global nitrogen cycle, and plays a major role in sustainable wastewater treatment. anammox bacteria convert ammonium (nh + ) to dinitrogen gas (n ) using nitrite (no - ) or nitric oxide (no) as the electron acceptor. in the absence of no or no, anammox bacteria can couple formate oxidation to the reduction of metal oxides such as fe(iii) or mn(iv). their genomes contain homologs of geobacter and shewanella cytochromes involved in extracellular electron transfer (eet). however, it is still unknown whether anammox bacteria have eet capability and can couple the oxidation of nh + with transfer of electrons to carbon-based insoluble extracellular electron acceptors. here we show using complementary approaches that in the absence of no -, freshwater and marine anammox bacteria couple the oxidation of nh + with transfer of electrons to carbon-based insoluble extracellular electron acceptors such as graphene oxide (go) or electrodes poised at a certain potential in microbial electrolysis cells (mecs). metagenomics, fluorescence in-situ hybridization and electrochemical analyses coupled with mec performance confirmed that anammox electrode biofilms were responsible for current generation through eet-dependent oxidation of nh + . n-labelling experiments revealed the molecular mechanism of the eet-dependent anammox process. nh + was oxidized to n via hydroxylamine (nh oh) as intermediate when electrode was the terminal electron acceptor. comparative transcriptomics analysis supported isotope labelling experiments and revealed an alternative pathway for nh + oxidation coupled to eet when electrode is used as electron acceptor compared to no as electron acceptor. to our knowledge, our results provide the first experimental evidence that marine and freshwater anammox bacteria can couple nh + oxidation with eet, which is a significant finding, and challenges our perception of a key player of anaerobic oxidation of nh + in natural environments and engineered systems. main text: anaerobic ammonium oxidation (anammox) by anammox bacteria contributes up to % of n emitted into earth's atmosphere from the oceans ( , ). also, anammox bacteria has been extensively investigated for energy-efficient removal of nh + from wastewater ( ). initially, anammox bacteria were assumed to be restricted to nh + as electron donor and no or no as electron acceptor ( , ). more than a decade ago, preliminary experiments showed that kuenenia stuttgartiensis and scalindua could couple the oxidation of formate to the reduction of insoluble extracellular electron acceptors such as fe(iii) or mn(iv) oxides ( , ). however, the mechanism of how anammox bacteria reduce insoluble extracellular electron acceptors has remained unexplored to date. also, growth or electrochemical activity was not quantified in these experiments. further, these experiments could not discriminate between fe(iii) oxide reduction for nutritional acquisition (i.e., via siderophores) versus respiration through extracellular electron transfer (eet) ( ). therefore, with these preliminary experiments it could not be determined if anammox bacteria have eet capability or not. although preliminary work showed that k. stuttgartiensis could not reduce mn(iv) or fe(iii) with nh + as electron donor ( ), the possibility of anammox bacteria to oxidize nh + coupled to eet to other insoluble extracellular electron acceptors cannot be ruled out. in fact eet (and set of genes involved with eet) is not uniformly applied to all insoluble extracellular electron acceptors; some electroactive bacteria are not able to transfer electrons to carbon-based insoluble extracellular electron acceptors such as electrodes in bioelectrochemical systems but could reduce metal oxides and vice versa ( ). it is known for more than two decades that carbon-based high- molecular-weight organic materials, which are ubiquitous in terrestrial and aquatic environments and that are not involved in microbial metabolism (i.e., humic substances) can be used as external electron acceptor for the anaerobic oxidation of compounds ( ). also, it has been reported that anaerobic nh + oxidation linked to microbial reduction of natural organic matter fuels nitrogen loss in marine sediments ( ). a literature survey of more than eet-capable species indicated that there are many ecological niches for microorganisms able to perform eet ( ) . this resonates with a recent finding where listeria monocytogenes, a host-associated pathogen and fermentative gram-positive bacterium, was able to respire through a flavin-based eet process and behaved as an electrochemically active microorganism (i.e., able to transfer electrons from oxidized fuel (substrate) to a working electrode via eet process) ( ). further it was reported that anammox bacteria seem to have homologs of geobacter and shewanella multi-heme cytochromes that are responsible for eet ( ). these observations stimulated us to investigate whether anammox bacteria can couple nh + oxidation with eet to carbon-based insoluble extracellular electron acceptor and can behave as electrochemically active bacteria. (fig. s b-g) . also, a previously enriched k. stuttgartiensis (freshwater anammox species) culture was used ( ). the anammox cells were incubated anoxically for hours in the presence of nh + ( mm) and graphene oxide (go) as a proxy for insoluble electron acceptor. no no or no were added to the incubations. go particles are bigger than bacterial cells and cannot be internalized, and thus go can only be reduced by eet ( ). indeed, go was reduced by anammox bacteria as shown by the formation of suspended reduced go (rgo), which is black in color and insoluble (fig. a) ( ). in contrast, abiotic controls did not form insoluble black precipitates. reduction of go to rgo by anammox bacteria was further confirmed by raman spectroscopy, where the formation of the characteristic d and d+d¢ peaks of rgo ( ) were detected in the vials with anammox cells (fig. b) , whereas no peaks were detected in the abiotic control. further, isotope analysis of the produced n gas showed that anammox cells were capable of n formation (fig. c ). in contrast, n production was not significant in any of the tested anammox species or controls, suggesting that unlabeled no or no were not involved. the production of n indicated that the anammox cultures use a different mechanism for nh + oxidation in the presence of an insoluble extracellular electron acceptor (further explained below). gas production was not observed in the abiotic control (fig. c) . to determine if anammox bacteria are still dominant after incubation with go, we extracted and sequenced total dna from the brocadia and scalindua vials at the end of the experiment. differential coverage showed that the metagenomes were dominated by anammox bacteria (fig. s a removal were observed in any of the abiotic controls. subsequently, the ca. brocadia culture was inoculated into the mec and operated under optimal conditions for anammox (i.e., addition of nh + and no -). under this scenario, nh + and no were completely removed from the medium without any current generation ( fig. a) . stoichiometric ratios of consumed no − to consumed nh + (∆no − /∆nh + ) and produced no − to consumed nh + (∆no − /∆nh + ) were in the range of . - . and . - . , respectively, which are close to the theoretical ratios of the anammox reaction ( ). these ratios indicated that anammox bacteria were responsible for nh + removal in the mec. subsequently, no was gradually decreased to mm leaving the electrodes as the sole electron acceptor. when the exogenous electron acceptor (i.e., no -) was completely removed from the feed, anammox cells began to form a biofilm on the surface of the electrodes (fig. s i ) and current generation coupled to nh + oxidation was observed in the absence of no -( fig. a) . further, no and no − were below the detection limit at all time points when the working electrode was used as the sole electron acceptor. the magnitude of current generation was proportional to the nh + concentration ( fig. a) and maximum current density was observed at set potential of . v vs ag/agcl. there was no visible biofilm growth and current generation at the mole of electrons transferred to the electrode per mole of nh + oxidized to n (table s ) was stoichiometrically close to equation (eq. ). also, electron balance calculations showed that coulombic efficiency (ce) was > % for all nh + concentrations and anammox cultures tested in the experiments with electrodes as the sole electron acceptor (table s ). (table s ). in addition, nh + oxidation and current production were not affected by the addition of penicillin g (fig. s ) (table s ) . this observation agrees with the nh + removal and oxidation to n observed in the mecs and isotope labeling experiments ( fig. a, fig a) . the genes encoding for no and no reductases (nir genes) and their redox couples were significantly downregulated when electrode was used as the electron acceptor (table s ). this is expected as no was not added in the electrode-dependent anammox process. also, this supports the hypothesis that no is not an intermediate of the electrode-dependent anammox process and that there was no effect of ptio when no was replaced by the electrode as electron acceptor ( suggest an alternative pathway for nh + oxidation coupled to eet when working electrode is used as electron acceptor compared to no as electron acceptor. in conclusion, our study provides the first experimental evidence that phylogenetically and microbial nitrogen cycling processes in oxygen minimum molecular mechanism of anaerobic ammonium oxidation nitric oxide-dependent anaerobic ammonium oxidation deciphering the evolution and metabolism of an anammox bacterium from a community genome enrichment and characterization of marine anammox bacteria associated with global nitrogen gas production our analysis showed upregulation under electrode-dependent anammox process of the genes in the wood-ljungdahl pathway for co fixation and acetyl-coa synthesis glts catalyzes the binding of the ammonium- nitrogen to -oxoglutarate with the oxidation of fdred ( ). the -oxoglutarate used for this reaction can be provided by the key enzyme of the rtca cycle -oxoglutarate:ferredoxin oxidoreductase (ogor) ( ) iron assimilation in anammox bacteria in eet-dependent anammox process surprisingly the proteins involved in iron transport and assimilation are still unknown. our analysis revealed that in the absence of soluble electron acceptors (i.e., no -, no -), ca. brocadia electricigens expressed two gene clusters encoding a siderophore-mediated iron uptake system (fig. s , table s and ). the expressed siderophore-mediated transport system fe(iii) uptake relies on beta-barrel tonb-dependent receptors in the outer membrane ( ) and an energy-transducing protein complex tonb-exbb-exbd that links the outer with the inner membrane and generate a proton motive force ( ). a periplasmic iron-binding protein and an atp-dependent abc fe(iii) reduction in the cytoplasm can be carried out by ferric-chelate reductases/rubredoxins, from which multiple genes were found to be expressed (fig. s , table s ) this finding is in agreement with a previous study using the eet-capable model bacteria geobacter sulfurreducens ( ), in which it was shown that the pathways required for eet and metal oxide reduction are distinct. : rieske/cytochrome b complex; bc- : rieske/cytochrome b complex; bc- : rieske/cytochrome b complex; cyt c ( heme): periplasmic mono-heme c-type cytochrome membrane-anchored tetraheme c-type cytochrome; cyt c ( hemes): outer membrane penta-heme c-type cytochrome cytochrome c redox partner of the etm; cyt nir: cytochrome c; etm: electron transfer module for hydrazine synthesis; exbb: biopolymer transport protein exbb/tolq; exbd: biopolymer transport protein exbd/tolr; fdh: membrane-bound formate dehydrogenase foca: formate/nitrite transporter; glts: glutamate synthase; hao brosi_a : hydroxylamine oxidoreductase; hao brosi_a : hydroxylamine oxidoreductase; hao ex _ : hydroxylamine oxidoreductase; hdh: hydrazine dehydrogenase; hzs: hydrazine synthase; membnxr: membrane-bound complex of the nxr gene cluster; mscl: large mechanosensitive channel; mscs: pore-forming small mechanosensitive channel; nadh-dh: nadh dehydrogenase; nir brosi_a : nitrite reductase; norvw: flavodoxin nitric oxide reductase; nrfa: ammonium-forming nitrite reductase heme): outer membrane lipoprotein mono-heme c-type cytochrome ompa: ompa-like outer membrane protein, porin; pfdo: pyruvate ferredoxin oxidoreductase; pfl: pyruvate formate lyase; pp binding protein: iron abc transporter periplasmic substrate-binding protein; rnf: rnfabcdge type electron transport complex rubredoxin/ferric-chelate reductase; s-layer: s-layer protein; tonb: energy transducer tonb acetoxime (c h no), and the molecular ion peaks were detected at mass to charge (m/z) = and for nh oh and nh oh, respectively. m of nh oh and nh oh were used as standards. to determine the source of the oxygen used in the electrode-dependent nh + oxidation to nh oh, mecs were incubated with nh cl ( mm, cambridge isotope laboratories) in presence of % d o for hours. stable isotopes of nh oh were determined by gc/ms analysis after derivatization using acetone as described above. activity and electron balance calculations activities of specific anammox ( n ) with nitrite as the preferred electron acceptor and electrode- dependent anammox ( n ) with working electrode ( . v vs ag/agcl) as sole electron acceptor were calculated based on the changes in gas concentrations in single-chamber mec batch incubations. the activity was normalized against protein content of the biofilm on the electrodes. protein content was measured as described below (see analytical methods section). the moles of electrons recovered as current per mole of nh + oxidized were calculated using: analytical methods all samples were filtered through a . µm pore-size syringe filters (pall corporation) prior to chemical analysis. nh + concentration was determined photometrically using the indophenol method ( ) (lower detection limit = μm). absorbance at a wavelength of nm was determined using multi-label plate readers (spectramax plus ; molecular devices, ca, usa). no concentration was determined by the naphthylethylenediamine method ( ) (lower detection limit = μm). samples were mixed with . mm naphthylethylenediamine solution, and the absorbance was measured at a wavelength of nm. no concentration was measured by hach kits (hach, co, usa; lower detection limit = . mg l - no --n). user's guide was followed for these kits and concentrations were measured by spectrophotometer (d , hach, co, usa). concentrations of nh oh and hydrazine (n h ) were determined colorimetrically as previously described ( ). for nh oh, liquid samples were mixed with -quinolinol solution ( . % (w/v) trichloroacetic acid, . % (w/v) -hydroxyquinoline and . m na co ) and heated at °c for min. after cooling down for min, absorbance was measured at nm ( ). n h was derivatized with % (w/v) p-dimethylaminobenzaldehyde and absorbance at nm was measured ( ). the concentration of biomass on the working electrodes was determined as protein concentration using dc protein assay kit (bio-rad, tokyo, japan) according to manufacturer's instructions. bovine serum albumin was used as the protein standard. putative eet-dependent anammox pathway we provided evidence that phylogenetically distant anammox bacteria can perform eet and are electrochemically active, and we elucidated the molecular mechanism of nh + oxidation, which by itself are significant findings that changes our perception of a key player in the global nitrogen (table s and table s ). this result is consistent with the nh + uptake, oxidation and final conversion to n observed in the mecs and isotope labeling experiments ( fig. a, fig a) . the requirement of more moles of nh + when anammox growth is based on eet compared to no as electron acceptor (eq. ), increases the demand of nh + import into the cell, which can explain the upregulation of the ammonium transporters. in contrast, the genes encoding for no and no reductases (nir genes) and their redox couples were significantly downregulated ( fig. s , table s ). this agrees, with the fact that no and no were below the detection limit in the mecs (fig. a, fig. s a and b) and there was no effect of ptio when no was replaced by electrode as electron acceptor (fig. s ) (fig. ) . the four low-potential electrons released from this reaction must be stored until they are transferred to a redox partner (fig. e, fig. s c and d) exhibited oxidation/reduction peaks, which suggests that additional cytochrome(s) that transfer electrons directly to the electrode via solvent exposed hemes may be involved. also, no cytochromes for long-range electron transport were detected in the analysis (table s and ), suggesting that eet to electrodes by anammox bacteria rely on a direct eet mechanism. homology detection and structure prediction by hidden markov model comparison (hmm-hmm) of the highly upregulated penta-heme cytochrome ex _ (fig. s , table s ) gave high probability hits to proteins associated to the extracellular matrix and outer membrane iron respiratory proteins such as mtrf, omca and mtrc. also, it is worth mentioning that the gene cluster ex _ - was one of the most upregulated under set-potential conditions. therefore, future work should focus on determining the role of ex _ - in the eet- dependent anammox process. likewise, we also found the expression of outer membrane mono- heme c-type cytochromes (om cyt c ( heme) (fig. s , table s ) homologs to g. key: cord- -oununr g authors: he, wei; wang, ningning; tan, jimin; wang, ruyi; yang, yichen; li, gairu; guan, haifei; zheng, yuna; shi, xinze; ye, rui; su, shuo; zhou, jiyong title: comprehensive codon usage analysis of porcine deltacoronavirus date: - - journal: mol phylogenet evol doi: . /j.ympev. . sha: doc_id: cord_uid: oununr g porcine deltacoronavirus (pdcov) is a newly identified coronavirus of pigs that was first reported in hong kong in . since then, many pdcov isolates have been identified worldwide. in this study, we analyzed the codon usage pattern of the s gene using complete coding sequences and complete pdcov genomes to gain a deeper understanding of their genetic relationships and evolutionary history. we found that during evolution three groups evolved with a relatively low codon usage bias (effective number of codons (enc) of ). the factors driving bias were complex. however, the primary element influencing the codon bias of pdcovs was natural selection. our results revealed that different natural environments may have a significant impact on the genetic characteristics of the strains. in the future, more epidemiological surveys are required to examine the factors that resulted in the emergence and outbreak of this virus. coronaviruses (covs) are the causative agents of major diseases in a variety of avian and mammalian species including humans. covs belong to the subfamily orthocoronavirinae of the coronaviridae, order nidovirales. the orthocoronavirinae subfamily is further divided into four genera including, alphacoronavirus, betacoronavirus, gammacoronavirus, and the recently identified deltacoronavirus (chan et al., ; king et al., ) . to date, six covs have been reported in pigs: transmissible gastroenteritis virus (tgev), porcine respiratory coronavirus (prcv), swine enteric alphacoronavirus (seacov), porcine epidemic diarrhea virus (pedv), porcine hemagglutinating encephalomyelitis virus (phev), and porcine deltacoronavirus (pdcov) (pan et al., ; homwong et al., ) . pdcov was first recorded as an emerging enteropathogenic coronavirus in pigs in hong kong in (chan et al., ; woo et al., ) , and thereafter was isolated from a swine farm in ohio, usa in (wang et al., a) . since then, pdcov has been reported in many countries and regions, including usa, canada, south korea, mainland china, mexico, japan, thailand, viet nam, and lao pdr (lee and lee, ; suzuki et al., ; saeng-chuto et al., ; wang et al., b; ajayi et al., ; perez-rivera et al., ) . a previous study showed that the global pdcovs consist of the china lineage, the usa/japan/south korea lineage, and the viet nam/laos/thailand lineage . pdcov is an enveloped, positive-sense, and single-stranded rna virus with a genome size of approximately . kb. the genome includes a ′utr, orf a/ b, the spike (s), the envelope (e), the membrane (m), nonstructural protein (ns ), the nucleocapsid (n), the nonstructural protein (ns ), and a ′utr (lee and lee, ) . the codon usage pattern is an important indicator of genome evolution. except for methionine and tryptophan, more than one codon can encode an amino acid due to the redundancy of the genetic code. codons encoding the same amino acid also are known as synonymous codons. interestingly, the codon usage is not random and some codons are used more than others, a phenomenon referred to as codon usage bias (marin et al., ) . codon usage bias has been reported for some rna viruses. however, the degree of bias varies depending on the identity of the specific virus. for instance, rubella virus and rotavirus show strong codon usage biases, whereas equine infectious anemia virus (eiav), ebola virus (ebov), the n gene of rabies virus (rabv), and porcine epidemic diarrhea virus (pedv) show weak codon usage bias (belalov and lukashev, ; yin et al., ; cristina et al., ; he et al., ) . natural selection, mutation pressure, the abundance of trna, rna structure, and gene length all contribute to the codon usage bias (jenkins and holmes, ; parmley and hurst, ; hershberg and petrov, ; plotkin and kudla, ) . the virus and host can both influence codon usage, which likely affects the survival, evolution, fitness, and immune evasion of the virus from host defenses (li et al., b he et al., ) . indeed, synonymous triplets are not used randomly, and factors such as natural selection and saltatorial bias can cause synonymous codon usage to diverge (sharp and li, ) . investigating the codon usage patterns of viruses could provide insights into their molecular evolution and viral gene expression regulation, assisting vaccine design, in which high levels of viral antigen expression are likely to be needed to produce immunity (butt et al., ) . given the recent increase in pdcov epidemics and the threat to pork production, in the present study, we reported an exhaustive genome-wide investigation of pdcov codon usage and evaluated the possible influencing factors. we retrieved all pdcovs sequences from the national center for biotechnology information (ncbi) nucleotide database (http://www. ncbi.nlm.nih.gov) available up to april . the detailed sequence information (serial number, strain name, accession number, location, and isolation year) for all complete coding sequences of the s gene and complete coding sequences (with the following concatenated order: orf ab-s-e-m-ns -n-ns ) of pdcov are displayed in supplementary materials (table s ). potential recombination signals were detected using rdp (recombination detection program version ) (martin et al., ) with default settings. seven methods were chosen for the analysis, including rdp, geneconv, chimaera, maxchi, bootscan, siscan, and seq. in particular, four methods were firstly applied. thereafter, the remaining sequences were run again with at least two methods until there was no recombination signal. phylogenetic trees were reconstructed in raxml (v . . ) to study the relationship between the multivariate and sample, a multidimensional statistical method, pca, was applied. pca is mainly a mathematical transformation process that converts the relevant variables (dependent on the relative synonymous codon usage (rscu) values) into a smaller number of irrelevant variables (called the principal components). every coding sequence was split into a -dimensional vector, and each dimension represented the matching dedication of the rscu values of different synonymous codons, which included only a specific amino group, without aug, ugg and the three stop codons. the parameters used for the pca were calculated in program codon w (http://codonw.sourceforge.net/). the compositional characteristics of the pdcov coding sequences of the s gene and complete genomes, were calculated. the frequency of all nucleotides (gc%, au%, a%, u%, g% and c%) was estimated using bioedit (http://www.softpedia.com/get/science-cad/bioedit.shtml). the a, c, g, and u frequencies in synonymous codons at different sites (gc %, gc %, gc %, gc %, a %, u %, g %, c %, au %) of each sequence were computed using cusp (http://emboss.toulouse.inra.fr/ cgi-bin/emboss/cusp) and codon w (http://codonw.sourceforge.net/). the relative dinucleotides abundances were computed according to a previously reported method (karlin and burge, ) . the odds ratio of the ability of the observed frequencies of the dinucleotides was computed using the equation below: = p f f f xy xy y x where the frequency of nucleotide x is represented by f x , the frequency of nucleotide y is represented by f y , the expected frequency of the dinucleotide xy is represented by f y f x , and the frequency of the dinucleotide xy is represented by f xy . as an universal standard, for < . or xy > . , we considered that the xy pair was under-represented or over-represented respectively, compared with the random association of single nucleotides and according to its relative abundance (butt et al., ) . rscu refers to the relative probability of a specific synonymous codon, which indicates whether the codon usage is influenced by the amino acid composition. in the case where all synonymous codons of a particular amino acid are assumed to be used equally, the rscu value of a sequence is the ratio of the frequency at which the codon is actually observed at its expected frequency . the rscu is calculated as: where g ij is the derived value of the ith codon for the jth amino acid with n i kinds of synonymous codons. rscu values = . , > . , and < . , represent no bias, positive codon usage bias, and negative codon usage bias, respectively. the rscu was calculated using mega (https://www.megasoftware.net/). the degree of codon usage bias, measured by the enc, was estimated taking into account the number of amino acids and the gene length. the enc values vary between and , with values closer to indicating a high codon usage bias and values closer to indicating a low codon usage bias. the enc value can reflect the preference of a synonymous codon in a family of codons. highly expressed genes often show a high codon usage bias, whereas poorly expressed genes contain more rare codons and thus a lower codon usage bias. generally, the codon usage is considered to show strong bias when the enc value is less than or equal to (comeron and aguade, ) . we used the following equation to calculate the enc (fuglsang, ) : where the average value of f i (i = , , , ) for the i-fold degenerate amino acids is represented by f. the following equation was used to calculate f i values: where the total number of appearances of the codons for that amino acid is represented by n and the total number of appearances of the j th codon for that amino acid is represented by n j . relative dinucleotides frequencies among different groups of s gene and complete genomes of pdcov strains. enc-plot analysis is commonly used to determine the factors influencing the codon usage bias (i.e. mutation pressure). the enc values relative to the gc values (the frequency of guanine or cytosine at the third codon position of synonymous codons excluding met, trp and stop codons) were plotted (karlin and burge, ) . when the codon usage is limited only to the gc mutation, the expected enc value falls on a theoretical curve (the functional relationship between the enc expectation curve and the gc value). when the actual enc-plot values of these sequences are lower than the standard curve, it is suggestive of natural selection playing a role in driving codon usage bias (fuglsang, ) . the theoretical enc values in enc-plot analysis were calculated as follows. where s denotes the frequency of c or g at the synonymous codons third position (i.e. gc ). neutrality analysis or neutrality evolution analysis was carried out to compare and define the effect of natural selection and mutation pressure on the pdcov codon usage patterns by comparing the value of gc s of synonymous codons with the gc s value using diagonal analysis. in the graph, the plot regression coefficient is considered as the mutation selection balance coefficient, and the evolutionary rates caused by natural selection pressure and mutation pressure are represented by the slope of the regression line. if all points are distributed along the diagonal and there is no significant difference in the three codon positions, this indicates that there is only weak or no external selection pressure. however, if the regression curve is parallel or tilted to the horizontal axis, this would indicate that the correlation between the changes of gc and gc is very low. thus, the regression curve shows that the effect of natural selection evolution effectively balances the degree of neutrality (kumar et al., ) . pr analysis was used to investigate the effect of selection and mutation pressure on gene codon usage. pr is a gene map with au deviation [a / (a + u )] as the ordinate and gc deviation [g / (g + c )] as the abscissa. at the center of the graph, the values of the two coordinates are . , which means that g = c and a = u (pr ), and there is no deviation between the mutation effect and selectivity (substitution rate) (sueoka, ) . after removal of recombinant sequences, s gene and complete genomes were left for further analysis. phylogenetic analysis of s gene based on ml (fig. a) and bi (fig. b) trees revealed three individual pdcov groups including, china, usa-japan-korea, and thailand-early-china-vietnam groups. we then used these three groups to investigate into codon usage and associations. pca showed that the three groups clustered separately, especially the usa-japan-korea group, although several overlaps existed between the usa-japan-korea and thailand-early china-vietnam groups (fig. ) . for whole genomes, the three groups clustered separately too, except for several overlaps between the usa-japan-korea and the thailand-early china-vietnam groups. the nucleotide u was the most abundant in the s gene, followed by a, c and g, regardless of the individual phylogenetic group (table ) . the detailed information of the nucleotide composition is shown in table s . the nucleotide composition of synonymous codons at the third position of (a , c , g , u ) showed that the frequencies of u and a were higher than c and g . the percentage content of au and gc were indicative of au-rich component in the coding sequences of pdcov. analysis of the synonymous codons at the first, second and third position showed that the values of gc were the highest, followed by gc and gc (table s ). the same pattern was identified for whole genomes. overall, these results illustrated that a relatively large part of the pdcov coding sequence comprises a and u nucleotides. all of the pdcov optimal synonymous codons for the corresponding amino acids of the s gene ended with u (perez-rivera et al., ) ( table ) . a total of of the priority codons had rscu values greater than . (cuu (l), guu (v), ucu (s), ccu (p), acu (t), aga (r), and ggc (g)). however, the remaining codons had rscu values less than . , with no underrepresented codons observed within the preferred codons. for whole genomes, u-ended codons were also the preferred codons among the most abundant synonymous codons ( table ). the rscu analyses and the nucleotide composition revealed that the compositional constraints (the nucleotides u in this case) had the most influence on the selection of the preferred codons. the relative abundances of the dinucleotides of pdcov coding sequences were calculated. we found that dinucleotides were not present randomly. none of the dinucleotide relative abundance values corresponded to the theoretical frequency (i.e., . ) (fig. , table ). furthermore, in the s gene, cpa ( . ± . ) and upg ( . ± . ) showed different degrees (marginal or peripheral) of overrepresentation. only cpg ( . ± . ) was underrepresented. for whole genomes, the overrepresented and underrepresented dinucleotides were upg ( . ± . ) and cpg ( . ± . ), respectively. enc values were estimated to evaluate the extent of codon usage deviation within coding sequences of different pdcov isolates. this analysis showed that pdcov coding sequences were relatively conserved and stable in terms of the s coding sequences or whole genomes with a low codon usage bias. the enc values of the s coding sequences ranged from . to . , with an average of . (enc > ) ( table ). the enc values of complete genome coding sequences were also within the range of the s gene, with no obvious difference in relation to phylogenetic groups. . . influence of mutation pressure on the pdcov codon usage pattern enc-plot analysis was carried out to reveal the constraint of mutation pressure on the pdcov codon usage pattern. the values of gc were plotted against the enc values according to individual phylogenetic group. we found that all points regardless of group concentrated on the left side and near to the expected curve for the s gene (fig. a) . for whole genome coding sequences, all the points were also under but close to the standard curve (fig. b) . here, neutrality analysis or diagonal analysis was used, between the gc s and gc s values, to judge the effects of natural selection and mutation pressure (fig. ). in the s gene, the relationships between gc s and gc s were calculated based on the three phylogenetic groups. the correlation coefficient in the usa-japan-korea group, china group, and thailand-early china-vietnam group were the . ± . , . ± . , and . ± . , respectively. thus, the percentages of constrain of natural selection were . %, . %, and . % for the s gene (fig. a) . for whole genomes, gc s and gc s significantly correlated, with a correlation coefficient of . ± . according to the usa-japan-korea group, indicating an . % limit for natural selection or . % of gc relative binding ( % neutral or % constraint) (fig. b) . overall, the above results indicate that the effect of mutation pressure is in all codon positions, but natural selection plays a major role driving the codon usage bias of pdcov. considering the limited number of sequences in the china and thailand-early china-vietnam groups, they were excluded from the results. in addition, pr analysis was carried out (fig. ) . we found that the a ≠ u, c ≠ g, for both the s gene and whole genomes, which indicates the inequivalent role of mutation pressure and natural selection in shaping the codon usage of pdcov. pdcov is an emerging coronavirus that infects the whole of the small intestine, especially the jejunum and ileum, causing severe enteritis, diarrhea, and vomiting in piglets. pdcov was first discovered in hong kong, china in (woo et al., ) . at the beginning of , pdcov was first reported in the usa, after which at least usa states confirmed its presence as of december . in recent years, china, south korea, thailand, and other asian countries have suffered from recurrent outbreaks (lorsirigool et al., ; janetanakit et al., ; dong et al., ; lee et al., ) . phylogenetic analysis is well studied to demonstrate the evolution of virus (he et al., ; li et al., a; su et al., su et al., , here, we first analyzed the codon usage patterns of the s coding sequences, as well as whole genome coding sequences of pdcovs isolated from around the world to determine the factors driving codon usage, and provided a comprehensive understanding of the characteristics and evolution of pdcov whole coding genes. phylogenetic analysis of the s gene revealed that sequences clustered into three different groups similarly to a previous study , but with more accuracy since more methods were applied and recombinant sequences were excluded. additionally, pca analysis also indicated three potential evolutionary groups. based on the s coding gene and complete coding genomes, we found a significant preference for a and u nucleotides, rather than g and c. the contents of au and gc were not equal and were more inclined towards the usage of au nucleotides. if the use of a synonymous codon was affected only by mutation pressure, the frequency of u and a nucleotides in the third codon position should be equal to the frequency of g and c ( van hemert et al., ) . thus, we can conclude that there was a low bias in the usage of nucleotides in all pdcov strains. rscu analysis revealed that pdcov genomes have a tendency towards uending codons. in addition, the relative probability distribution of dinucleotides showed that codons and dinucleotides were used unequal and followed certain rules. dinucleotide abundance influences the codon usage bias in certain organisms, including rna and dna viruses (rothberg and wimmer, ) . dinucleotide sequences may be derived from odd partial of amino acid changes or codon usage bias; therefore, we analyzed dinucleotide composition distribution (plotkin et al., ; cristina et al., ) . the translational selection pressure on a dinucleotide is the entropy cost of a given set of constraints that alter the number of dinucleotide occurrences, in this case the amino acid sequence of the given protein sequence and the cost of the codon usage bias (cristina et al., ) . analyses of the frequencies of codons and dinucleotides revealed that translation selection also played a part in the codon usage of pdcovs. these initial observations prompted further investigation to assess the extent of codon usage bias using enc analyses. for pdcov, the enc value based on the s gene or complete coding genomes was , indicative of slight bias and that different pdcovs are relatively conserved and stable. previous studies indicated that enc values correlate negatively with gene expression . thus, a higher enc value indicates lower gene expression and lower codon preference. a low codon bias could be explained by the need to better adapt towards efficient replication and survival in the host, and to reduce the energy required for virus biosynthesis while avoiding competition with host protein synthesis . when the enc and gc values of pdcovs were plotted, mutation pressure was revealed as a moderate factor influencing the pdcov codon usage pattern. according to previous reports, both natural selection and mutation pressure can affect the enc value, which indicates that the relative contribution of selection and mutation on the codon usage pattern are not robust gu et al., ) . it is worth mentioning that the codon usage bias of species with a/u biased genomes is different from that of genomes with a g/c bias. therefore, simple enc-gc map analysis might be misleading. generally, mutation pressure will always have a role in driving the codon usage of viruses. here, using neutrality plots we found that natural selection was a more dominant factor compared with mutation pressure (shi et al., ) . natural selection can lead to weak codon usage bias while the virus is trying to adapt to the host cells (matsumoto et al., ) . pr bias plot analysis showed that both natural selection and mutation pressure contributed to the observed codon bias consistent with the neutrality analysis. in summary, we found that the codon usage of the s gene was similar to the complete coding genome. to open new perspectives, a further exploration of the function and features of functional genes is worth studying. here, we found that, to a large extent, the codon usage pattern and the sequences characteristics of pdcovs 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deltacoronavirus comprehensive analysis of the overall codon usage patterns in equine infectious anemia virus detection and spike gene characterization in porcine deltacoronavirus in china during the authors declare no competing financial interest. supplementary data to this article can be found online at https:// doi.org/ . /j.ympev. . . key: cord- -nvgz su authors: li, kun; mccray, paul b. title: development of a mouse-adapted mers coronavirus date: - - journal: mers coronavirus doi: . / - - - - _ sha: doc_id: cord_uid: nvgz su first identified in , middle east respiratory syndrome coronavirus (mers-cov) is a novel virus that can cause acute respiratory distress syndrome (ards), multiorgan failure, and death, with a case fatality rate of ~ %. an animal model that supports mers-cov infection and causes severe lung disease is useful to study pathogenesis and evaluate therapies and vaccines. the murine dipeptidyl peptidase (dpp ) protein is not a functional receptor for mers-cov; thus, mice are resistant to mers-cov infection. we generated human dpp knock-in (hdpp ki) mice by replacing exons – at the mouse dpp locus with exons – from the human dpp gene. the resultant human dpp ki mice are permissive to mers-cov (hcov-emc/ strain) infection but develop no disease. to generate a mouse model with associated morbidity and mortality from respiratory disease, we serially passaged hcov-emc/ strain in the lungs of young hdpp ki mice. after in vivo passages, an adapted virus clone was isolated and designated mers(ma) . . . this virus clone produced significantly higher titers than the parental clone in the lungs of hdpp ki mice and caused diffuse lung injury and a fatal respiratory infection. in this chapter, we will describe in detail the procedures used to mouse adapt mers-cov by serial passage of the virus in lungs. we also describe the methods used to isolate virus clones and characterize virus infection. middle east respiratory syndrome (mers) is a fatal respiratory illness that first appeared on the saudi arabian peninsula in mid- . it is caused by a novel betacoronavirus, mers coronavirus (mers-cov) [ ] . shortly after the identification of the virus, its receptor dipeptidyl peptidase (dpp ) was discovered [ ] . as of september , the world health organization (who) has reported laboratory-confirmed cases of mers in countries, including associated deaths (fatality rate: %). mers-cov does not currently have pandemic potential [ ] [ ] [ ] . however, mers-cov is still epidemic in the middle east and remains a cause for significant concern due to the potential spread by global travel as demonstrated by the outbreak in south korea in [ ] [ ] [ ] . to date, there are only two published reports on autopsy findings from subjects who died from mers [ , ] and our understanding of mers-cov pathogenesis in humans is still limited. animal models are useful for the study of viral diseases and play an important role in the investigation of pathogenesis and evaluation of antiviral therapies and vaccines. an ideal animal model should be permissive to the viral infection and develop disease and pathology with similarities to that observed in humans. mers-cov infection has been evaluated in two nonhuman primate (nhp) models, the rhesus macaque and common marmoset [ ] [ ] [ ] [ ] [ ] . both species are susceptible to mers-cov infection. however, mers-cov caused only a transient lower respiratory tract infection without mortality in rhesus macaques [ ] [ ] [ ] . in common marmosets, the consequences of mers-cov infection are controversial. falzarano et al. reported the common marmoset reproduced several features of mers-cov infection in humans including progressive severe pneumonia [ ] , while another group observed only mild to moderate nonlethal respiratory disease following mers-cov infection [ ] . thus, the common marmoset is potentially a model to study pathogenesis and evaluate antiviral therapies and vaccines. however, nhps are expensive, their availability limited, and their use may raise ethical concerns. in contrast, small animal models provide advantages over nhps, including reduced cost, availability in large numbers, ease of handling, and species-specific reagents, especially for the studies of highly pathogenic viruses like mers-cov in the biosafety level (bsl- ) laboratory. unfortunately, common small laboratory animals like mice [ , ] , ferrets [ ] , guinea pigs [ ] , and hamsters [ ] are not susceptible to mers-cov infection because their homologous dpp cannot be bound and utilized by mers-cov as a host receptor for entry [ , ] . haagmans et al. detected mers-cov rna in the respiratory tract of new zealand white rabbits following inoculation but found no clinical signs of disease [ ] . several strategies have been used to overcome this receptor incompatibility and develop mouse models of mers-cov infection. in , we developed the first mouse model of mers-cov infection [ ] . we delivered a recombinant adenovirus encoding human dpp (hdpp ) to the lungs of mice. transient expression of hdpp by adenovirus transduction made the mice temporarily permissive for mers-cov infection, but animals developed only mild lung disease. generation of transgenic mice expressing a virus receptor is a common strategy to make mice permissive to infection. several groups in addition to ours developed mice with transgenic expression of hdpp using different promoters [ ] [ ] [ ] [ ] . the disease severity following mers-cov infection in transgenic mice correlated with the cellular distribution and expression level of hdpp . in transgenic mice expressing hdpp driven by the cytokeratin promoter [ ] or a ubiquitous promoter [ , , ] , mers-cov replicates and causes respiratory disease and mortality. however, the lethality was found to be secondary to overwhelming central nervous system (cns) disease or multiorgan damage. this was also observed in mice transgenic for human ace driven by the cytokeratin promoter and infected with sars-cov [ ] . in transgenic mice expressing hdpp under the human surfactant protein c (spc) promoter, which restricts expression to bronchiolar and alveolar epithelia, mers-cov infection caused only mild disease [ ] . thus, these transgenic mice do not reproduce a severe lung disease phenotype that resembles mers. alternative strategies for the creation of mouse models of mers-cov infection are generation of dpp humanized mice and adaptation of the virus to the animals. pascal et al. reported a model in which all of the mouse dpp exons had been humanized and also generated humanized monoclonal antibodies against the mers-cov s protein using a novel strategy [ ] . mers-cov infection in this model caused pulmonary edema, vascular cuffing, and alveolar septal thickening with an associated~ % weight loss, necessitating euthanasia [ ] . another mers mouse model was engineered by changing two amino acids in the mouse dpp locus using crispr-cas technology [ ] . this model supported mers-cov replication without severe disease. similarly, our human dpp knock-in mouse model supported mers-cov replication but did not lead to a severe lung disease phenotype [ ] . two mouse-adapted (ma) strains of mers-cov were subsequently developed independently by serial passage of the hcov-emc/ strain [ ] in the lungs of the two humanized mouse models [ , ] . the resultant mers- and mers ma . . mouse-adapted mers-cov strains replicated to high titers in the lungs of the crispr-cas genetically engineered mouse model and the hdpp knock-in mouse model, respectively. the respiratory disease that developed in both mouse models and the associated mortality shared similarities with severe cases of mers [ , ] . mouse adaptation was also successfully used to generate several sars-cov strains capable of modeling severe sars-cov lung disease in mice [ ] [ ] [ ] . thus, the adaption of the virus to enhance virulence in the mouse is a very useful approach to generate mouse models for coronavirus-associated lung disease. these materials can be altered to fit the requirements for other viruses of interest. . hcov-emc/ strain. . hdpp knock-in mouse (c bl/ strain with mouse dpp exons - replaced with the human codons). all procedures are performed under bsl- laboratory conditions and must follow the standard operating protocol of a bsl- facility and regulatory agencies. all manipulations of infectious specimens, samples, and mice must be performed within a biosafety cabinet or within a contained device such as a centrifuge. all tissue culture media and waste must be bleached and autoclaved prior to disposal. all instruments must be disinfected with virex plus or % bleach. . insert surgical scissors under the sternum and cut the diaphragm following the costal arch. remove the rib cage using scissors and forceps, exposing the lungs and heart. . fill a ml syringe with cold sterile dpbs using a g  / inch needle. insert the needle into the apex of the left ventricle and make a small incision in the right atrium. slowly perfuse ! ml cold sterile dpbs into the left ventricle. next, insert the needle into the apex of the right ventricle and perfuse ! ml cold sterile dpbs (see note ). . remove the lungs and heart from the thoracic cavity. remove the liver, kidney, spleen, and small intestine. place organs in a small polystyrene weighing dish. remove remaining connective tissue. . to harvest the brain, turn the mouse over and wet the fur of the head with % ethanol. grasp the ears with forceps and cut off the skin and fur to expose the skull. remove remaining skin at the base of the neck to further expose the skull. immobilize the mouse and make an incision along the sagittal suture of the skull using a single edge razor blade (see note ) . wedge one prong of the curved serrated forceps into the now opened sagittal suture. slowly pry up the skull, grasp the piece of skull with forceps and peel outward to remove. repeat on the other side. use curved forceps to lift the brain from the skull. place brain tissue in the small polystyrene weighing dish. . carefully place each organ into a ml disposable tissue grinder filled with ml sterile cold dpbs for homogenization or into ml of trizol for rna extraction (see note ). . grind the lung tissue and transfer the homogenates or rna samples into . ml sterile screw-top tubes with o-ring cap (see note ) . store samples at À c. thaw and spin down the cell debris in lung homogenates before use. the virulence of the virus should be evaluated in groups of mice by weight loss and survival after every - in vivo passages. after the virulence of the virus has been significantly enhanced, single plaques of the adapted virus should be purified and evaluated. . plate vero cells in d media in -well plates one day before infection. . rapidly thaw lung homogenates from the selected passage. mix the lung homogenates from two mice in a . ml sterile screwtop tube with o-ring cap on ice. . serially dilute the mixed lung homogenates tenfold in . ml sterile screw-top tubes with o-ring caps, using ice-cold serumfree dmem (see note ) . keep the dilutions on ice. . remove the medium from each well and add diluted samples (in a volume of μl) to each well. . place the plates in the c incubator for h and rotate gently every min. . melt % low melting point agarose and maintain in a c water bath. . mix overlay media and % low melting point agarose at a volume ratio of : . rotate the tube several times to fully mix. overlay cells with . ml of mixed media using ml stripettes. . let the plates sit in the hood for~ min at rt or until the agarose overlay turns solid. add . ml d medium on the top of solidified agarose. . place the plates in the c incubator. . after days, plaques should be visible. remove the liquid on the top of the agarose. circle the visible plaques on the underside of the plates using a permanent marker (see note ). . vertically penetrate the agarose and pipette the circled plaque several times with a ml graduated transfer pipette. the agarose above the plaque will be pulled into the pipette. . transfer the agarose above the plaque into a ml conical tube filled with μl dmem by pipetting up and down several times. . transfer the μl dmem containing the agarose into a . ml sterile screw-top tube with o-ring cap. . repeat the procedure and pick six single plaques. store the tubes at À c. . animal euthanasia should follow the institution's animal care and use guidelines. there may be differences in institutional requirements regarding when euthanasia is required based on weight loss. . use the lid of an insulated foam shipping box. cut absorbent bench underpad (  cm) into small pieces that fit the lid. . carefully keep the tip of the needle in the lumen of the ventricle. be sure to puncture the right atrium as this will help to drain blood during the perfusion. the lung will turn white after perfusion. . make sure that the incision does not exceed the thickness of the skull; avoid cutting into the brain tissue. . the individual organs can be divided into pieces and transferred to grinders with pbs or trizol separately. . lung homogenates are immediately transferred to tubes on ice. rna samples are transferred to a tube and incubated for at least min at room temperature per our bsl- specific standard operating procedures. . at days post infection with pfu/mouse, hcov-emc/ strain replicates in the lung of the hdpp ki mice to a titer of around  pfu/ml, which equals  pfu in μl. we chose the pfu/mouse inoculum to begin because we wanted to use a similar dose range during in vivo serial passage while skipping the titration step. . titrate the homogenates of the passage of interest and select a virus dilution that produces plaques in a well. this allows identification of individual clear single plaques. . only circle the unambiguous clear single plaques. . compared to vero cells, we found that the mers-cov rna genome is more stable when propagated in huh cells (less likely to introduce genomic deletions, insertions, or point mutations). isolation of a novel coronavirus from a man with pneumonia in saudi arabia dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk person-toperson spread of the mers coronavirus-an evolving picture the role of superspreading in middle east respiratory syndrome coronavirus (mers-cov) transmission spread of mers to south korea and china infectious diseases epidemic threats and mass gatherings: refocusing global attention on the continuing spread of the middle east respiratory syndrome coronavirus (mers-cov) comparative epidemiology of middle east respiratory syndrome coronavirus (mers-cov) in saudi arabia and south korea clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates histopathology of middle east respiratory syndrome coronovirus (mers-cov) infection -clinicopathological and ultrastructural study middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques an animal model of mers produced by infection of rhesus macaques with mers coronavirus pathogenicity and viral shedding of mers-cov in immunocompromised rhesus macaques intratracheal exposure of common marmosets to mers-cov jordan-n / or mers-cov emc/ isolates does not result in lethal disease infection with mers-cov causes lethal pneumonia in the common marmoset mouse dipeptidyl peptidase is not a functional receptor for middle east respiratory syndrome coronavirus infection wild-type and innate immune-deficient mice are not susceptible to the middle east respiratory syndrome coronavirus adenosine deaminase acts as a natural antagonist for dipeptidyl peptidase -mediated entry of the middle east respiratory syndrome coronavirus domestic pig unlikely reservoir for mers-cov the middle east respiratory syndrome coronavirus (mers-cov) does not replicate in syrian hamsters host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase receptor variation and susceptibility to middle east respiratory syndrome coronavirus infection asymptomatic middle east respiratory syndrome coronavirus infection in rabbits rapid generation of a mouse model for middle east respiratory syndrome generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease middle east respiratory syndrome coronavirus causes multiple organ damage and lethal disease in mice transgenic for human dipeptidyl peptidase multi-organ damage in human dipeptidyl peptidase transgenic mice infected with middle east respiratory syndrome-coronavirus a human dpp -knockin mouse's susceptibility to infection by authentic and pseudotyped mers-cov lethal infection of k -hace mice infected with severe acute respiratory syndrome coronavirus pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection cd + t cells and macrophages regulate pathogenesis in a mouse model of middle east respiratory syndrome a mouse model for mers coronavirus-induced acute respiratory distress syndrome mouse-adapted mers coronavirus causes lethal lung disease in human dpp knockin mice a mouse-adapted sars-coronavirus causes disease and mortality in balb/c mice a new mouse-adapted strain of sars-cov as a lethal model for evaluating antiviral agents in vitro and in vivo molecular determinants of severe acute respiratory syndrome coronavirus pathogenesis and virulence in young and aged mouse models of human disease we thank chris wohlford-lenane and jennifer bartlett for careful review of the manuscript. this work is supported by the national institutes of health p ai . we also acknowledge the support of the cell morphology core and pathology core, partially supported by the center for gene therapy for cystic fibrosis (national institutes of health p dk- ) and the cystic fibrosis foundation. p.b.m. is supported by the roy j. carver charitable trust. key: cord- -yqqqe authors: ning, qin; wu, ting; su, hai-bin; ma, ke; qi, jun-ying; ni, ming; wu, di title: antiviral therapy for aechb and severe hepatitis b (liver failure) date: - - journal: acute exacerbation of chronic hepatitis b doi: . / - - - - _ sha: doc_id: cord_uid: yqqqe this chapter describes the principles of antiviral therapy, treatment strategies, medications and recommendations for aechb, hbv-aclf, hbv-related liver cirrhosis, hbv-related hcc, and liver transplantation. . severe exacerbation of chronic hepatitis b is closely related to continuous hbv replication. therefore, inhibiting hbv replication to reduce viral load may block disease progression and improve the quality of life of these patients. etv or tdf has been recommend first-line drug for the treatment of aechb. . a hyperactive immune response due to continuous hbv replication is the main mechanism for development of severe hepatitis b. in addition to comprehensive treatment, early administration of potent nucleoside analogs can rapidly reduce hbv dna concentration, relieve immune injury induced by hbv, and reduce liver inflammation and patient mortality. antiviral agents have become important in the treatment of severe exacerbation of chronic hepatitis b. . long-term antiviral treatment with nucleoside analogs can delay or reverse the progress of liver cirrhosis. virologic response, viral resistance and adverse drug reactions should be closely monitored during treatment. the treatment should be optimized for maximum effect based on each patient’s responses. . effective antiviral therapy can suppress hbv replication and reduce the incidence of hbv-related hcc. patients with hbv-related hcc should receive individualized and optimal multidisciplinary comprehensive treatment. anti-viral drugs with high efficacy, low resistance and low adverse drug reactions should be selected to improve the patient’s quality of life and prolong survival time. . methods to prevent hbv reinfection after liver transplantation include passive immunization (hbig), antiviral treatment (nucleoside analogs) and active immunization (hepatitis b vaccine). . clinical trials involving sequential combination therapy with nuc and peg-ifn have shown statistically significant decline in hbsag levels on treatment and high rates of sustained post-treatment serologic response. combination therapy with novel daa and immunotherapeutic approach may hold promise to overcome both cccdna persistence and immune escape, representing a critical step towards hbv cure. dence of hbv-related hcc. patients with hbv-related hcc should receive individualized and optimal multidisciplinary comprehensive treatment. anti-viral drugs with high efficacy, low resistance and low adverse drug reactions should be selected to improve the patient's quality of life and prolong survival time. . methods to prevent hbv reinfection after liver transplantation include passive immunization (hbig), antiviral treatment (nucleoside analogs) and active immunization (hepatitis b vaccine). . clinical trials involving sequential combination therapy with nuc and peg-ifn have shown statistically significant decline in hbsag levels on treatment and high rates of sustained post-treatment serologic response. combination therapy with novel daa and immunotherapeutic approach may hold promise to overcome both cccdna persistence and immune escape, representing a critical step towards hbv cure. ting wu and qin ning chb is a progressive disease, and its development is the interaction between hbv and the host's immune response. host's immune system eliminates hbv through cytolysis and non-cytolysis mechanism, which leads to hepatic inflammation, hepatocyte necrosis and apoptosis. continuous hepatic inflammation can lead to liver fibrosis, liver cirrhosis, liver failure, and even liver cancer gradually. hbv replication plays a key role in the disease evolution. therefore, suppressing hbv replication is crucial in treatment of chb. the development of continuous liver inflammation, cirrhosis and liver cancer is closely related to the sustainable hbv replication in chb patients. hence, inhibiting hbv replication and reducing hbv dna load is the key to block the disease progression and improve the survival quality in the treatment of chb [ ] . the fundamental goal of chb treatment is to eliminate hbv or suppress hbv permanently, thus to reduce viral pathogenicity and infectivity, relieve or inhibit hepatic necroinflammation. however, the existing antiviral drugs cannot remove the intercellular covalently closed circular dna (cccdna). therefore, the goal of antiviral treatment is to sustainably suppress the virus, reduce or prevent hepatic injury and disease progression. the short-term goal of clinical practice is sustainable hbv suppression, alt normalization and prevention from decompensated liver cirrhosis (initial response), releasing the hepatic necroinflammation and liver fibrosis during and after treatment. the long-term and ultimate goal is prevention from liver decompensation, relief or halt of the progression to liver cirrhosis or hcc, and prolonged survival period (sustained response) [ ] [ ] [ ] [ ] [ ] [ ] . hbv is difficultly to be eliminated, and there is no drug can absolutely eradicate hbv infection so far. many drugs can function against hbv, but only two classes are internationally recognized as antiviral drugs: interferon and nucleoside analogues (fig. . ). common guidelines for management of chb (table the recommendations for the treatment of hbeag positive patients with chb in guidelines (table . ) [ ] [ ] [ ] [ ] ] . the recommendations for the treatment of hbeag negative patients with chb in guidelines (table . ) [ ] [ ] [ ] [ ] ] . the recommendations for the treatment of cirrhotic patients with chb in guidelines (table . ) [ ] [ ] [ ] [ ] . the recommendations for liver biopsy in guidelines (table . ) [ ] [ ] [ ] [ ] . the recommendations for chb initial treatment options in guidelines (table . ) [ ] [ ] [ ] [ ] ] . the recommendations for chb cirrhosis initial treatment options in guidelines (table . ) [ ] [ ] [ ] [ ] . the recommendations for chb treatment duration in guidelines (table . ) [ ] [ ] [ ] [ ] ] . . antiviral treatment strategies for hbeag positive patients ( fig. . ). . antiviral treatment strategies for hbeag negative patients (fig. . ). the recommendations for management of lamivudine drug-resistance in guidelines (table . ) [ ] [ ] [ ] [ ] ] . start antiviral treatment to avoid liver transplantation entecavir and tenofovir as the first-line choice, or pegylated interferons aasld pegylated interferon-α, entecavir or tenofovir are preferred; interferon-α/pegylated interferon-α, lamivudine, adefovir, entecavir, tenofovir or telbivudine can be used; adefovir or entecavir for interferon nonresponders or patients with interferon contraindication. csld/csid pegylated interferon-α, entecavir who tenofovir and entecavir as the first-line choice ifn may be considered when hbv dna viral load and genotyping are available, or co-infection with hdv nas with low resistance first; use interferon with caution, start from small doses decompensation nas with low resistance first; interferon is forbidden the recommendations for management of adefovir drug-resistance in guidelines (table . ) [ ] [ ] [ ] [ ] ] . consensus of antiviral treatment in special populations with chronic hepatitis b was put forward by china expert committee of antiviral treatment in special population with chronic hepatitis b in . in china, hbv infection is one of the main causes of liver failure. hbv related liver failure can be further divided into acute liver failure, subacute liver failure, acuteon-chronic liver failure and chronic liver failure. nucleoside analogues can be safely used in the treatment of hbv related liver failure, and improve the prognosis of patients. nucleoside analogues treatment can improve the survival rate and reduce the incidence of complications in hbv related acute and subacute liver failure patients. therefore, nucleoside analogues treatment should be early applied in hbsag positive and hbv dna detectable patients with acute and subacute liver failure, and nucleoside analogues can quickly inhibit virus, including lamivudine, entecavir and tenofovir, are recommended for these patients. drug resistance should be monitored in long-term nucleoside analogues treatment. remaining hbv cannot be completely excluded even when hbsag and hbv dna are undetectable during the treatment, therefore the antiviral treatment should continue to hbsag seroconversion. antiviral treatment is unnecessary for anti-hbs positive patients at first visit [ ] . for patients with hbv related primary liver cancer, liver cancer resection, radiofrequency ablation and interventional therapy all can lead to hbv reactivation and liver function damage aggravation, antiviral treatment is decided depending d on liver compensatory situation. ifn-α can exhibit both anti-virus and anti-cancer effect, delay the tumor recurrence and prolong the median survival period. ifn-α should be the preference if the patients can tolerate ifn-α. if the patients have contraindications to ifn-α, lamivudine, adefovir or entecavir can be chosen depending on hbv dna loads, cirrhosis compensatory situation and kidney function. for patients undergoing hepatic arterial chemotherapy, prophylactic nucleoside analogues treatment should be given before chemotherapy. for patients with advanced liver cancer, portal vein branch thrombosis, but without contraindications to ifn-α, ifn-α treatment can extend survival period [ ] [ ] [ ] [ ] [ ] [ ] [ ] . patients awaiting liver transplantation because of hbv-related end-stage liver disease or liver cancer should be given nucleoside analogues with strong hbv inhibition and low drug-resistance, or nucleotides analogues combination treatment, in order to reduce viral load and prevent graft re-infection. lamivudine and (or) adefovir combination with hbig can be safely and effectively prevent graft re-infection, and reduce the re-infection rate to below %. hbv-associated liver transplant patients require lifelong treatment of antiviral drugs for the prevention of hepatitis b recurrence. hbsag-negative patients receiving anti-hbs positive donor liver should also receive long-term treatment of lamivudine or preventive treatment of hbig [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . according to who, elderly chb patients refers to chb patients aged ≥ years old. generally speaking, according to current guidelines, ≥ years of age is not a contraindication to antiviral therapy, so their treatment can refer to the relevant guidelines, but their desire, risks and benefits of treatment should be comprehensive evaluated. especially for the patients using ifn-α, the expected survival and liver function compensatory situation, possible side effects, underlying hypertension, diabetes, coronary heart disease, and the improvement of liver function should be comprehensive evaluated. additionally, the treatment response, side effects, blood sugar, kidney function and occurrence of liver cancer should be closely monitored during and after treatment [ ] . children chb patients are usually in the immune tolerance phase of hbv infection, hence they could not receive antiviral treatment, but should be closely followed up. fda has approved of ifn-α ( - years of age), lamivudine ( - years of age), and adefovir ( - years of age) for use in children. recommended ifn-α dose is miu/m of body surface area three times per week, and the maximum dose is miu/m total body surface area. it is showed that ifn-α effects the same in children as in adult. recommended lamivudine dose is mg/(kg day) with a maximum dose of mg/day. recommended adefovir dosage and usage are the same with adult patients [ , [ ] [ ] [ ] [ ] [ ] [ ] . mother to child transmission is the main route of transmission of hbv infection in china, in order to block the transmission of hbv, antiviral therapy in pregnant chb patients is very important. firstly, antiviral treatment should be completed before pregnancy as possible. it is recommended to consider pregnant months later after interferon or nucleoside analogs treatment. secondly, unwanted pregnancies should be terminated during ifn-α treatment because of its pregnancy toxicity. pregnancy safety of nucleosides analogs has not been proved by any clinical trials, but a large number of studies have shown that lamivudine and tenofovir were safe, so the treatment with lamivudine could continue under the premise of full communication with patients. telbivudine, adefovir or entecavir treatment may switch to lamivudine treatment. pregnant chb patients with slight alt elevation, should be monitored closely or given liver protection and symptomatic treatment, and given antiviral treatment after delivery. pregnant chb patients with poor liver function could be given lamivudine treatment after full consultations with patients and signed informed consent forms. serum hbv dna load in pregnant chb patients is the key factors of mother to child transmission, and effective antiviral treatment can significantly reduce the transmission incidence. according to the findings, lamivudine or telbivudine treatment could start in [ ] [ ] [ ] [ ] [ ] [ ] [ ] week of pregnancy to block transmission, and the withdrawal can refer to the patients with immunosuppressive agents or chemotherapy. in addition, women with husbands receiving ifn-α antiviral treatment, should only consider pregnancy months later after withdrawal. there is no evidence at present that nucleosides analogs have a negative impact on sperm and embryo, pregnancy could be taken into account under the premise of full communication with patient [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . hbv and hcv co-infection increases the incidence of severe hepatitis, liver cirrhosis and liver cancer. when co-infection, hcv may inhibit hbv generally, and different treatment should be given depending on hbv and hcv viral load and alt level (table . ) [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . hbv and hiv co-infection increases hbv dna load, reduces spontaneous hbeag seroconversion rate, aggravates liver damage and increases mortality in patients with liver disease. anti-hbv regimens should combine with highly active antiretroviral therapy (haart). if anti-hbv and hiv treatment is needed, anti-hbv drugs such as tenofovir + lamivudine or tenofovir + truvada could be used in haart. if haart only contains lamivudine as anti-hbv drugs, hbv drug resistance should be closely monitored and treatment should be adjusted in time. if haart is unnecessary, adefovir, telbivudine and ifn-α can be used for anti-hbv treatment. because lamivudine, tenofovir and entecavir monotherapy induced risk of hiv drug-resistance, lamivudine, tenofovir and entecavir treatment are not recommend to these patients [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . anti-hbv treatment is critical for hepatitis b virus associated glomerulonephritis (hbv-ag). patient diagnosed with hbv-ag must start anti-viral therapy as long as hbv dna is detectable. a number of studies show that kidney disease alleviated significantly after lamivudine treatment, along with hbv dna decline and hbeag clearance. adefovir has been shown to increase serum creatinine level in some patients in clinical trials, therefore should be carefully chosen. there is not enough clinical evidence of telbivudine and entecavir treatment for hbv-ag. there is no consensus of nucleoside analogue treatment for hbv-ag currently. safety and efficacy of ifn-α and pegylated ifn-α treatment for hbv-ag have not been proved [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . elevation of hbv dna can be observed in - % of the hbsag-positive patients receiving immunosuppressive agents or cytotoxic therapy, including corticosteroids, anti-cd and anti-tnf. some patients suffer from transaminase elevation and jaundice, and severe patients develop to fulminant liver failure even death. nucleoside analogues prophylactic treatment can decrease hbv reactivation. regardless of the hbv dna level, hbsag carriers should receive nucleoside analogues antiviral treatment - weeks before immunosuppressive or cytotoxic therapy. antiviral drugs inhibit hbv dna rapid, such as lamivudine, telbivudine and entecavir, are preferred for prophylaxis. most patients cannot tolerate the recurrent aggravations induced by drug-resistance. prophylaxis decision should be made depending on baseline hbv dna load and duration of immunosuppressive therapy or cytotoxic therapy. antiviral drugs with low resistance are recommended if prophylaxis will last more than months. treatment duration: if baseline hbv dna ≤ copies/ml, treatment should be continued to months after immunosuppressive therapy or cytotoxic therapy completed; if baseline hbv dna > copies/ ml, treatment should be continued to the referring withdrawal standard of ordinary chb patients. however, ifn-α is not recommended because of the bone marrow suppression. there is no consensus of prophylactic antiviral treatment for hbsag-negative and anti-hbc-positive patients during the immunosuppressive or cytotoxic therapy. serum hbv marker and hbv dna level should be monitored [ , [ ] [ ] [ ] [ ] [ ] [ ] . hbv infection itself is not correlated with thyroid dysfunction. ifn-α can aggravate underlying autoimmune thyroid disease or induce emerging thyroid disease in some patients because of its immunomodulatory activity and direct thyroid toxicity. uncontrolled thyroid dysfunction contraindicates ifn-α antiviral therapy. patients with previous thyroid dysfunction or high titer of thyroid autoantibody (tpo-ab > iu/ml) before treatment should be monitored for thyroid function during ifn-α antiviral treatment. patients with emerging thyroid dysfunction during treatment should terminate antiviral treatment. majority of thyroid dysfunction emerged during treatment in patients without history of thyroid dysfunction is reversible, and can restore after ifn-α withdrawal [ ] [ ] [ ] [ ] . resistance to nucleoside analogues is a serious problem in chb treatment, which does make the long-term treatment strategies become a difficulty. a variety of factors may be associated with resistance to nucleoside analogues, including nucleoside analogue type, hbv dna load at initial therapy, liver fibrosis/ cirrhosis, and previous nucleoside analogues treatment. in addition, male gender, high body mass index and alcohol abuse are also the risk factors of resistance mutations in antiviral therapy. however, a growing number of studies suggest that early virological response is an important indicator to predict drug resistance [ , ] . select nucleoside analogues treatment indications reasonably. nucleoside analogues treatment is not recommended for the hbv infected patients in immune tolerant phase or non-active phase, especially those who are younger, if they do not receive immunosuppressive therapy or chemotherapy. for the chb patients with first flare, especially those who are younger, nucleoside analogues should be given with caution after fully analysis of inductions. select nucleoside analogues treatment strategies reasonably. treatment should be consulted the guideline on prevention and treatment of chronic hepatitis b in china. for patients with antiviral therapy indications, drugs with strong antiviral activity and low resistance are recommended if nucleoside analogues are chosen. the previous antiretroviral therapy, including drugs, treatment response and resistance mutations, should be understood in order to avoid nucleoside analogues with cross-resistance. furthermore, sequential monotherapy treatment should be avoided for multi-drug resistance. improve patients' compliance. prescribed time and adequate medication should be repeatedly emphasized to the patients during nucleoside analogues treatment. according to the clinical trial, more than % of cases with virologic breakthrough are resulting from poor compliance. gradual dose reduction will significantly increase the risk of resistance and is forbidden. regularly detect hbv dna load and genotypic resistance and timely adjust treatment. hbv dna load is the most important indicators of drug resistance in nucleoside analogue antiviral therapy. hbv dna levels should be monitored regularly during treatment. numerous clinical trial data show that early virological response is an important predictor of drug-resistance. therefore, aasld and easl guidelines both recommend adjusting treatment plan based on evr to improve the efficacy and reduce the incidence of drug resistance [ , [ ] [ ] [ ] [ ] . patients with normal alt and mild inflammation or fibrosis (<g s ) before treatment can stop the anti-viral treatment, and need to be monitored closely for antiretroviral therapy again promptly if aura symptoms happened. most patients with nucleoside analogues resistant, especially the decompensated cirrhotic patients, need rescue therapy timely. virologic breakthrough usually precede biochemical breakthrough, and rescue therapy before biochemical breakthrough can avoid sudden hepatitis flare and aggravation. management of lamivudine and adefovir resistance refers to guide recommendations above. add adefovir or tenofovir (the latter has not been approved by sfda, and the safety of combination of entecavir and tenofovir needs further study), or switch to ifn-α or pegylated ifn-α for patients with entecavir resistance. add adefovir or tenofovir for patients with telbivudine resistance. tenofovir resistance has not detected, if happened, entecavir, telbivudine, lamivudine or emtricitabine can be added theoretically. for lamivudine and adefovir multidrug resistance, switch to emtricitabine or combination of entecavir and tenofovir (but not been approved by sfda), or switch to ifn-α or pegylated ifn-α treatment [ , , ] . definition of refractory chb: chb patients with treatment failure or poor efficacy and chb patients have been confirmed poor efficacy by evidence-based medicine, using anti-hbv drugs, including nucleoside analogues and (or) interferon, under the existing guidelines or recommendations for various reasons and (or) factors. refractory chb patients include: ( ) patients with primary non-response, partial virological response and (or) virologic breakthrough, drug-resistant hbv mutations and clinical drug-resistant. ( ) patients with serological (hbeag) no response or partial response, namely hbeag-positive patients without hbeag loss or hbeag seroconversion after initial treatment of more than year. ( ) high baseline hbv viral load: hbeag-positive patients with hbv dna > copies/ml, hbeagnegative patients with hbv dna > copies/ml. ( ) cirrhotic (compensated and decompensated) patients and aechb patients. ( ) other patients with hbv reactivation after transplantation, immunosuppressive therapy, combination of other viral infections such as hcv and hiv, combination of metabolic/autoimmune diseases such as insulin resistance, hyperlipidemia and (or) non-alcoholic steatohepatitis and fiber cholestatic hepatitis. the present guidelines for the treatment of refractory chb involve virus strains resistant mutations and virologic breakthrough, partial virological response, cirrhosis, virus reactivation after liver transplantation, receiving immunosuppressive therapy, and combination of other viral infections (hcv and hiv). majority of the treatment recommendations are based on expert consensus, clinical experience, or clinical studies of small sample. however, for patients with serological no-response or partial response, high baseline hbv viral load, aechb, insulin resistance and non-alcoholic steatohepatitis, and fiber cholestatic hepatitis, there is no clear guidance. currently ongoing hepatitis b related clinical researches more concentrated in drug selection and optimization of treatment-naïve patients, and only a small portion and small-scale studies involve refractory chb. anti-hbv treatment was not taken into account in aecdhb in the past. it is thought that immune pathological damage is the key in the development of aechb, and hbv is just a promoter. the role of hepatitis virus in development of aechb has not been paid enough attention. with the study of aechb mechanism deep going, more and more scholars have realized that constant hbv replication induced hyperactive immune response is a major factor in exacerbation. when hbv induces hypersensitivity, a large amount of immune complexes generate and activate the immune network, leading to serious hepatocyte damage through the following mechanisms: ( ) th cells activate, release interleukin- (il- ), and mediate cytotoxicity of cytotoxic t cell (ctl), macrophages and natural killer (nk) cells; ( ) macrophages activated by hbv and endotoxin release cytokines (including tumor necrosis factor tnf-α and fgl ), inducing direct hepatocyte damage or secondary damage by microcirculation disturbance; ( ) fas ligands (fas-l) express increasingly on the surface of infected hepatocytes, conjunct to fas expressed by ctl, and induced apoptosis. antiviral therapy can quickly suppress hbv replication, reduce intercellular viral spread, and inhibit the membrane target antigen expression, so as to inhibit ctl attack and relieve hepatocyte injury and necrosis. antiviral treatment at early stage in disease is the pivot to terminate intense cellular and humoral immunity. therefore, it is advocated to start antiviral treatment for severe hepatitis patients with hbv replication. some scholars believe that viral load is an important indicator for aechb, although it does not directly related to liver damage. hbeag and hbv dna seroclearance is important for remission and positive for improving cure rate of severe hepatitis. therefore, early effective anti-viral therapy can reduce the viral load, suppress virus generation by infected hepatocytes, decrease infection of newborn hepatocytes, alleviate liver inflammation and is beneficial to liver recovery. antiviral therapy has become an effective treatment for aechb [ ] . whether antiviral therapy increase or alleviate immune response in the advanced stage of severe hepatitis had been controversial. however, recent studies reach a consensus that antiviral treatment can slow disease progression and improve recent survival rate. in most early studies, lamivudine failed to improve liver function and survival in aechb compared to placebo [ ] [ ] [ ] [ ] [ ] . however, a study from taiwan showed that lamivudine could improve survival in patients with baseline total bilirubin less than mmol/l ( mg/dl) [ ] . wong and chan reported that antiviral therapy in aechb cannot improve resent survival, but could prevent further deterioration [ ] . in early stage, there is intense immune response, high viral load, severe inflammation, and ongoing immune liver damage. viral load can affect the progression and prognosis. patients with hbv dna positive have a relative poor prognosis because of more virus antigen on hepatocyte surface activating immune injury. in middle-advanced stage, the impact of hbv dna on progression and prognosis would weaken because immune response has alleviated after self-regulation. therefore, hbv dna level in early severe chb is a significant indicator for prognosis, and antiviral therapy is essential. in middle-advanced stage, hbv dna level has little influence on the prognosis, and antiviral therapy is meaningful to prevent recurrence. in addition, lamivudine can obtain sustained virologic response, but long-term treatment may induce viral resistance and virologic breakthrough, which reduce the efficacy of antiviral therapy [ ] . most recent studies suggest that antiviral therapy for hbv dna positive patients in early severe hepatitis can postpone disease progression and improve recent survival rate [ ] . ma et al. analyzed cases of hbv-aclf retrospectively. patients added entecavir on the basis of standard medical treatment, another patients only received standard medical treatment without nucleoside analogues. the -month and -month survival rate of entecavir-treated patients were . % ( / ) and . % ( / ) respectively, and significantly higher than those of control with . % ( / ) and . % ( / ). entecavir-treated patients get a significant improved meld scores compared to control post treatment, suggesting that entecavir can postpone progression of hbv-aclf and improve recent survival [ ] . lin et al. investigated hbv-aclf cases with entecavir treatment, and concluded that entecavir can significantly increase the survival rate [ ] . hu et al. investigated the efficacy of lamivudine and entecavir on hbv-aclf. after -month treatment, survival rates are similar, but clinical improvement rate in lamivudine and entecavir group were significantly higher than basic treatment group. after -month treatment, the cumulative survival rates of lamivudine and entecavir group were . %, . % respectively and significantly higher than the basic treatment group ( %). in patients with baseline hbv dna > iu/ml, cumulative survival rate in antiviral treatment group were higher than basic treatment group. patients with pretreatment meld scores > had a lower cumulative survival rate than patients with meld scores ≤ , but obtained better response to antiviral therapy. it also showed that there was no significant difference in efficacy using entecavir and lamivudine treatment for hbv-aclf [ ] . tillmann et al. summarized cases of aechb with lamivudine treatment. it suggested that patients with lamivudine treatment had an overall survival rate without transplant of . %, but patients without lamivudine only . % [ ] . garg et al. found that tenofovir can significantly reduce hbv dna levels in hbv-aclf, improve ctp and meld score, and decrease mortality. hbv dna decrease of more than lg copies/ml after -week treatment is a predictor of good prognosis. liver failure is a common syndrome, and its incidence is increasing with the use of alcohol and growing epidemic of obesity and diabetes. liver failure is defined as inability of the liver to perform its normal, metabolic, excretory and biotransformation functions by chinese medical association [ ] . its manifestation includes coagulopathy, jaundice, hepatic encephalopathy (he) and ascites. liver failure can be divided into four classes: acute liver failure (alf), sub-acute liver failure (salf), acute on chronic liver failure (aclf), and chronic liver failure (clf) according to histopathological characteristics and the progression of disease. alf is a syndrome with liver function deterioration rapidly accompanied with a grade ii or higher he within weeks illness duration. salf onset is slowly than alf that symptoms occur within - weeks. liver failure occurred with known or unknown chronic liver disease refer to aclf. clf is defined as progressive deterioration and decompensation of liver function in patients with liver cirrhosis, mainly manifested with complications of portal hypertension. based on the severity of clinical manifestations, sub-acute and acute-on-chronic liver failure can be divided into early, middle, and end stages. early stage has severe fatigue and gastrointestinal symptoms, total bilirubin level is more than μmol/l or daily increase of total bilirubin is more than μmol/l and prothrombin activity (pta) is less than %. middle stage has stage ii he and/or ascites and pta ≤ %. end stage has refractory complications such as stage iii or higher he, hepatorenal syndrome, massive hemorrhage of the upper alimentary tract, severe infection and refractory fluid and electrolyte imbalance, pta ≤ %. otherwise, asian pacific association for the study of the liver (apasl) define aclf as a severe liver injury, leading to coagulation abnormality usually with an inr ≥ . , and any degree of mental alteration (he) in a patient with pre-existing liver disease and with an illness duration less than weeks [ ] . etiology, epidemic and precipitating factors of liver failure are different between western countries and china. non-infection factors such as alcohol and drug induced liver failure are predominant in western countries [ ] . however, hepatitis b virus (hbv) infection is the main reason to induce liver failure [ ] . we retrospective analysis the etiology of cases of liver failure came from provinces in china northern area from to and found hbv infection induced liver failure was about . % [ ] . so far, liver transplantation is the most effective way to treat hbv induced liver failure. but due to the cost and shortage of organ donor, liver transplantation can't be used widely. comprehensive treatment including supportive therapy, antiviral therapy and immunoregulation therapy is the main way to treat hbv related liver failure in china. but mortality of liver failure is high and total curative rate is only about . % because of the complicate pathophysiology in liver failure [ ] . the precise mechanism underlying the liver injury caused by hbv-aclf and the factors contributing to the progression of liver failure remain unknown. generally, virus factors, host factors, and their interaction determine the outcome of aclf. hbv dna replication is one of the key factors causing the progression from liver damage to liver failure. the hbv dna level is closely associated with the risk of hepatocellular carcinoma development, and hbv dna suppression significantly improves the prognosis of cirrhosis. current clinical guidelines advocate oral antiviral treatment in decompensated cirrhosis and sustained hbv dna suppression to reduce sequelae [ , , , ] . (liver failure) eight different hbv genotypes (a-h) have been described based on their genomic heterogeneity. many studies showed that the severity of hbv infection correlated with hbv genetype. in the asia-pacific countries, genotype b and c hbv are predominant with genotype c hbv associated with delayed hbeag seroconversion and more aggressive disease activity as compared to other hbv genotypes [ ] . patients infected with hbv genetype c have high hbv dna level and high hbeag positive rate than people infected with other hbv genetypes. these patients have low response to anti-viral therapy and progress to liver failure, particular in patients infected with genetype c . zhang et al. analyses hepatitis b patients and found that the most common hbv genetype was b and c in chronic hepatitis b patients [ ] . patients infected with genetype c was more predisposed to chronic and cirrhosis and hepatic carcinoma. genetype b and c had no influence on illness progression in acute and mild patients, but hbeag positive rate and hbv dna level were high in patients infected with genetype c. further studies showed that hbv bcp/pre c mutation was associated with hbv genetype. a t and c mutation probably associated with aclf. c was an independent prognostic factor in aclf patients [ ] . hbv dna polymerase lack proof function which can lead to viral mutation during replication. in addition, hbv exist in many quasispecies. under select press, quasispecies variance can cause the change of hbv replication, pathogenicity, antigen epitope which lead to influence immune response and viral resistance. over immune response can result in severe liver injures. hbv pro core/core, pre s, p gene and multi gene mutation were found to be associated with liver failure [ ] . one of the functions of hbeag is to induce immune tolerance. in the absence of hbeag, patients harboring pre-core mutant hbv may have a more rigorous immunological response. chronic infection with pre-core mutants has been often associated with multiple flares with interspersed asymptomatic periods. mutations at the basal core promoter (bcp) regions lead to decreased hbeag synthesis, active liver histology, and increased viral replication. these exacerbations are seen to lead to fulminant hepatic failure [ , , ] . we investigated hbv bcp a t/g a double mutation in liver failure patients [ ] . longitudinal study showed that nucleotide mutation sites were occurred more in hbv-aclf than cirrhosis patients among which nt , nt , nt and nt were associated with hbv-aclf. t mutation was found exist more in genetype b than genetype c ( . % vs . %), a/g mutation is found frequently in hbeag negative patient than positive patients ( % vs . %). these indicated that pre core/bcp mutation associated with the occurrence of liver failure and influence patients outcome. hbx is a multifunction regulatory protein which can influence gene transcription, activate signal transduction, enhance viral replication, accelerate protein degradation and regulate cell apoptosis. hbx can participate in the process of hbv pre s and bcp/pre core mutation liver failure caused by chronic hbv infection (i.e., chronic severe hepatitis b) is a common life-threatening disease in china. the pathogenesis of chronic severe hepatitis b is complex and is currently not completely understood. however, one widely accepted mechanism is the induction of cellular immune responses mediated primarily by cytotoxic t lymphocytes (ctls) and delayed-type hypersensitivity t cells. these immune responses are induced by viral protein antigens expressed in the target cell surface due to the active replication of hbv and eventually result in large areas of liver cell necrosis [ ] . the specific mechanisms may involve several factors [ ] . using a hybridization assay for hbv dna and a conventional enzyme immunoassay to measure the hbeag level, an earlier study showed significant parallel increases in serum hbeag and hbv dna levels and accumulation of intracellular viral proteins several weeks before the hepatitis flare. in addition, there was a subsequent increase in anti-hbe production and hbeag/anti-hbe immune complex formation, implicating the important role of the immune response to hbv in initiating the hepatitis flare [ ] . immunohistologic studies during the hepatitis flares have shown cd + t cells in the mononuclear cell infiltrates, strong membranous expression of human leukocyte antigen class i (hla-i), and cytoplasmic or membranous/submembranous hepatitis b core antigen (hbcag) expression [ , ] . some findings suggest that hepatitis b flares are the results of dynamic changes of the innate and adaptive immune responses with hla-i restricted, ctl mediated immune cytolysis of hbv antigen(s) expressing hepatocytes and its downstream apoptotic mechanisms [ , ] . the activated th cells release interleukin- and excite cytotoxic effects of ctls, macrophages, natural killer cells, and lymphokines [ ] . macrophages, activated by hbv and endotoxins, release various cytokines mainly with tumor necrosis factor (tnf)-a, which directly damage liver cells and also result in secondary injury of liver cells through disturbances in microcirculation. fas ligands (fas l) are highly expressed in the surface of hbv-infected liver cells and combine with fas expressed by ctls, together inducing hepatocellular apoptosis. therefore, antiviral therapy early in chronic severe hepatitis b is beneficial for suppressing intense cellular immune responses induced by hbv replication. if hbv replication is suppressed rapidly, the immune pathological responses of chronic severe hepatitis b may be reduced, thus effectively blocking the disease progression. the administration of anti-hbv therapy in chronic severe hepatitis b (acute-or subacute-on-chronic liver failure) is still undergoing research, and limited data are presently available. so far, anti hbv treatment drugs include interferon and oral antiviral drugs. interferon application is contraindicated in the treatment of liver failure due to its limited anti-viral efficacy, significant adverse drug effects, and induction of immune enhancement, which can further result in aggravation of liver damage. oral anti hbv drugs including adefovir dipivoxil (adv), lamivudine (lam), telbivudine (tbv), entecavir (etv) and tenofovir (tdf) have few adverse effects. antiviral therapy may have the advantage of shortening the replication and thereby reduce disease duration without the side effects of interferon. therefore, many studies have been carried out to find the efficacy of oral antiviral drugs on patients with liver failure. the effect of antiviral therapy in hbv related acute liver failure is controversial. some studies reported that antiviral therapy can't improve outcome because hbvdna can be eradicated spontaneously due to enhanced immune response in liver failure patients. but hbv infection is the initial factor to induce over immune response that can lead to liver damage. early treatment with antiviral therapy can inhibit hbv dna replication and attenuate immune reaction which can reduce liver damage, hepatocyte apoptosis and necrosis. tillmann et al. [ ] reported that acute hbv related liver failure patients were treated with lam and patients were treated with placebo. encephalopathy occurred in ( . %) and ( . %) patients, respectively (p = . ). it demonstrated that early use of antiviral drugs can reduce the rate of encephalopathy and mortality. in addition, a prospective study about etv on acute liver failure patients demonstrated that etv can reduce hbv dna load significantly and patients achieved anti hbsag conversion [ ] . therefore, monitor closely and early use antiviral drugs to reduce hepatocyte apoptosis and necrosis on patients with hbv related acute liver failure are very important. the objective of antiviral treatment for hbv-aclf is to reduce viral load at an appreciably high rate, thereby promoting reduction in hepatocyte cell death and improved survival outcomes by prevention of decompensation related multiorgan complications in this group of severely ill patients. several studies have delineated the fact that low pretreatment hbv dna load and a rapid decrement in viral load improves outcomes in hbv-aclf [ ] , whereas a study from india reported that a log decrease in hbv dna at week improved survival benefit in patients with hbv-aclf [ ] . antiviral therapy also promotes chances of stabilization to liver transplant time and improves transplant outcomes. studies have debated on the issue of antiviral therapy related improvement in the long term [ ] . lam decreased viral load significantly, but did not result in significant biochemical or clinical improvement compared with those patients given placebo. mortality of patients receiving nucleoside analog therapy was significantly lower than the placebo group, which indicated that antiviral therapy improved prognosis of patients with hbv-aclf if implemented as soon as possible [ ] . even in the age of effective antiviral therapy, early transfer to a transplantation facility should be considered before managing conservatively by medical means. the apasl consensus guidelines on aclf describe the value of early and prompt institution of antiviral therapy in hbv-aclf. hbv dna levels are now not an indication for commencement of antivirals in hbv-aclf reactivation, as earlier starting of such therapy, even prophylactic, has been found to have great survival benefit in the long run. from to , early and middle stage hbv-aclf patients in our hospital were recruited in a prospective study to evaluate the efficacy and safety of lam and etv [ ] . no antiviral therapy was used in control group. this study showed that lam or etv can reduce and months mortality, improve survival rate on patients with aclf-hbv. three and months cumulative survival rate of lam and etv therapy were . % and . %, % and . %, respectively, which was higher than control group ( % and %, p = . and . ). no significant different on survival rate between lam and etv (p = . ). this study also showed that meld score was an effective prognostic predict factor on patients with aclf-hbv. patients with meld score less than had a good outcome. another study also demonstrated that for hbeag-negative patients with hbv-aclf, when entecavir was added to comprehensive therapy, a meld score ≥ predicted very poor prognosis due to fatal liver failure [ ] . hu et al. made a survival analysis on hbv-aclf patients and results indicated that nucleoside analog application in early and middle stage hbv-aclf can improve survival rate and prolong patients' life. median survival time was . and . months in patients treated with and without antiviral therapy. another study indicated that patients with meld score - and hbv dna load decrease log had a better outcome than patients with hbv dna load decrease less than log within weeks treatment [ ] . in addition, mortality had no relationship with hbv dna load if meld score > . xiao et al. also demonstrated that nucleoside analog treatment is an independent prognosis predict factor in hbv induced liver failure. lam and etv had no difference. although antiviral therapy can improve patients survival rate, treatment with and without nucleoside analog had no difference in late stage liver failure patients. currently, liver transplantation is the ultimate therapeutic option for decompensated cirrhosis patients. however, liver transplantation can't be used in all patients because of the shortage of donor organs. therefore, the aim to treat decompensated cirrhosis is to decrease the occurrence of disease associated complications and the liver associated mortality rate [ ] . the natural history of decompensated hbv-related cirrhosis is affected by high hbv replication which may exist in some decompensated hbv-related cirrhosis patients [ ] . hepatic necroinflammation and fibrosis progression are improved after sustained viral suppression is achieved which can prevent decompensation in cirrhosis [ ] . oral nas treatment are strongly recommended in most clinical guidelines for decompensated hbv-related cirrhosis patients no matter what hbv dna replication level is [ , ] . yao et al. [ ] found that ctp scores was reduced more than three points in % lam treated patients with severely decompensated cirrhosis. furthermore, ctp scores was decreased to less than seven point in % of these patients, and their statuses on the united network of organ sharing waiting list changed to inactive. a randomized controlled trial in asia demonstrated less liver-related morbidity in the lam-treated patients with hbv associated advanced compensated cirrhosis compared to the untreated controls because of the reduced incidence of hepatic decompensation and lower risk of hcc. increased ctp scores were noted in . % of the patients in the lam group compared to . % of the patients in the placebo group (p = . ) [ ] . fontana et al. showed most deaths caused by liver related complications occurred within the first months in patients with decompensated hbv-related cirrhosis treated with lam. pretreatment high hbv dna replication level, serum bilirubin and creatinine were associated with -months mortality rates significantly [ ] . this finding indicates early antiviral therapy might be important. lamivudine lam is a nucleoside analogue that inhibits hbv dna synthesis which was the first oral drug to treat chronic hbv infection in . its mechanism is to compete with nature cytidine to inhibit hbv polymerase, then terminate hbv replication. chan et al. [ ] studied the effect of lamivudine in treatment of severe hepatitis-b-related acute exacerbations leading to aclf in patients as against controls. it was concluded that lamivudine had no survival benefit compared with conventional treatment in severe aggravations of chronic hepatitis b and that liver transplantation should be considered in these patients with thrombocytopenia and high bilirubin. however, another meta study [ ] analysis studies to evaluate the short-term effect of lamivudine (lmv) treatment for severe chronic hepatitis b. they found that the survival rates and pta of the test group were distinctively higher than those of the control group at weeks , , and of the treatment course. the hbv-dna negative change rate was distinctively higher throughout the weeks of lmv treatment. for patients who started lmv treatment in the middle stage, the mortality rate of the test group was lower. they concluded that lmv decreased hbv-dna levels in the serum, improved liver function in patients, and enhanced survival rate during the early and medium stages of severe chronic hepatitis b. tsubota et al. [ ] studied patients with spontaneous severe acute exacerbation treated with lamivudine, and found that lamivudine monotherapy did not prevent hepatic failure deterioration significantly, but it resulted in long-term benefits. baseline serum bilirubin, pre-existing cirrhosis and baseline pt were independent determinants of prognosis. adv is an acyclic nucleotide analogue of adenosine monophosphate. use of adefovir for hbv-aclf has been rare. in two case reports, adefovir dipivoxil failed to salvage cases of lamivudine resistance after jaundice and liver failure developed. a lower antiviral potency and the potential risk of nephrotoxicity of adv remain a problem for routine use as a first-line treatment. it is hence not advisable to use adefovir as a first-line drug in the treatment of acute severe exacerbation. but considered to the high viral resistant to lam, adv plus lam can reduce the incidence of lam resistance. the combination of adv and lam can be used in liver failure patients. patients with decompensated cirrhosis caused by lam-resistant hbv were treated with adv and hbv dna level become undetectable occurred in % of patients and ctp scores were improved [ ] . but another study focused on long time outcome found that resistance to adv was % and renal toxicity was confirmed in % of patients [ ] . etv is a cyclopentyl guanosine analogue that can inhibit hbv polymerase's function. compared with lam and adv, etv has a more potent activity against wild type hbv [ , ] . etv has been studied in in cirrhotic patients. one korea study showed ctp and meld scores were improved in patients treated with etv for month. the -year cumulative incidence of hcc was . %, and the cumulative death rate was % [ ] . many studies have been carried out to compared the efficacy of etv and lam to treat hbv related aclf. the short-term efficacy of entecavir vs lamivudine was similar and the degree of pretreatment liver failure significantly affected the outcome of treatment [ , ] . in summary, the pros and cons of lam vs etv in decompensated or severe acute exacerbation of chronic hepatitis b were etv being more effective in promoting faster viral load decrement. also the available clinical evidence suggests that clinicians treating chronic hepatitis b patients with acute hbv exacerbation or decompensated liver disease should use the most potent nucleoside analogs available [ ] . tbv is a synthetic thymidine nucleoside analogue that has potent antiviral activity against hbv. one study investigated the short-term efficacy and safety of tbv therapy in liver failure patients caused by chronic hepatitis b virus (hbv) infection [ ] . in this study, patients were treated with tbv, and the other patients were treated with lam. hbv dna levels in the tbv group fell to the lower limit of detection after the fifth week, which was more rapid than in the lam group. in addition, the total bilirubin and prothrombin time activity of the patients with tbv treatment showed a more significant improvement as compared to the patients treated with lam from the start of the fifth week. they concluded that tbv treatment is superior to lam treatment in improving the condition of patients with liver failure as a result of chronic hbv infection in the short term. but viral resistance is also a major concern. a study in decompensated cirrhosis patients with hbv infection showed genotypic resistance was developed in % of the tbv patients during the -year period [ ] . therefore, tbv used as a first-line treatment has limitations in patients with hbv-related decompensated cirrhosis. tdf is an acyclic nucleotide analogue with potent inhibition of hbv polymerase/ reverse transcriptase. in a seminal study by garg et al. [ ] , consecutive patients with aclf due to spontaneous reactivation of chronic hepatitis b were randomized to receive either tdf or placebo. the primary endpoint was survival at month. more than log reduction in hbv dna levels at weeks was associated with survival rate. the authors concluded that tdf had potent activity to reduce hbv-dna levels, improve ctp and meld scores, and reduce mortality in patients with severe spontaneous reactivation of chronic hepatitis b presenting as aclf, and that reduction in hbv-dna levels at week should be considered a desirable goal and a good predictor of survival. until now, no studies have reported viral resistance to tdf. therefore, tdf and etv can be considered to be the first-line therapy because their potent activity against hbv replication and high resistance barrier. in addition, the data about tdf to treat hbv-aclf is limited. thus, larger prospective and multicenter studies are encouraged to evaluate further the effect of tdf on shortterm mortality of patients with hbv associated aclf. there still lack multicenter, larger samples, prospective and randomized clinical trial to test the efficacy of antiviral therapy. but it is likely that antiviral therapy with nucleos(t)ide can improve patients survival rate in patients with hbv-related liver failure [ ] . therefore, antiviral therapy is reasonable to try in patients with high hbv dna replication. in recent year, lam and other nucleoside analogue have been used in severe hepatitis b wildly. the efficacy of antiviral therapy is correlated with the time to start. many clinical trials demonstrated that early antiviral treatment is important for patients with liver failure [ ] . antiviral therapy used in early and middle stage liver failure can improve patients survival rate. in patients with late stage, such as serum total bilirubin is more than μmol/l, it's rare that patients can get benefit from antiviral therapy. therefore, early use antiviral therapy can reduce viral load, inhibit viral replication, reduce new hepatocyte be infected by hbv again and alleviate liver inflammation and all of that are benefit to hepatocyte regeneration. the criteria for antiviral therapy dependent on hbv dna level in serum. some people suggested antiviral therapy should be used in patients with hbeag positive and hbv dna > copies/ml or hbeag negative and hbv dna > copies/ ml. however, take into account over immune response in liver failure patients that associated with viral eradication, even patients with hbsag positive and hbv dna undetectable should be considered for antiviral therapy. in our opinion, antiviral therapy should be used in all liver failure patients with hbv replication immediately. the feature of oral antiviral drugs should be considered when used in liver failure patients. side effects of nucleos(t)ide, including elevate ck, myopathies and lactate acidosis, can occurred during treatment. we observed the change and effect of elevated ck during the treatment of etv and lam in liver failure patients. we found that no different of ck elevation in both drugs. ck elevation was consistence with infection and hepatic renal syndrome. but long term safety of nas has not been confirmed in liver failure patients. antiviral therapy should be used for life because the nas treatment can't eradicate cccdna in the hepatocyte. some cirrhotic patients developed viral resistance to lam during long-term lam therapy which cause virologic response loss [ , ] . in antiviral treatment naïve patient, the lam resistance rate is up to % after years of continuous therapy and the annual resistance rate is up to % [ ] , compared with that etv resistance rate is less than . % in patients treated with etv at years [ ] . therefore, lam should be used with careful monitoring for the development of resistance. and etv or tdf rather than lam is recommended to be used as the first-line therapy in patients with hbv infection because of its high genetic barrier and potent activity against hbv replication [ ] . multiorgan failure occurred rapidly frequently in liver failure patients. although some patients can recover treated by comprehensive and antiviral therapy, all patients should be evaluated for liver transplantation. it's challenge to study the effect of na, how and when to use na and how to treat viral resistance to na in liver failure patients. further studies are needed to evaluate patient outcomes after antiviral therapy with na in liver failure patients. for patients with chronic hepatitis b (chb), hbv continue replication caused progression of liver disease, eventually lead to cirrhosis or hepatocellular carcinoma. the principle of treatment for hbv-related cirrhosis is a comprehensive treatment, including antiviral, anti-inflammatory, hepatoprotective treatment and anti-fibrosis, which antiviral treatment is the key point. since , listed lamivudine, there were many new ideas and concepts on treatment for hbv-related cirrhosis, liver failure. a large number of evidence-based medicine evidence suggests that sustained suppression of hbv by anti-viral treatment can reduce liver inflammation and fibrosis, reduce or delay disease progression, and ultimately improve survival rates and quality of life. internationally there are many chb antiviral therapy management guidelines, but antiviral therapy for hbv-related cirrhosis was still a hot and difficult issue in clinic. this article reviews some of the new progress and new perspectives of antiviral therapy liver cirrhosis based on the recent research. management is guided by recommendations from the american association for the society of liver disease (aasld) [ ] , european association for the study of liver (easl) [ ] , asian pacific association for the study of liver (apasl) [ ] , and society of hepatology and infectious diseases of chinese medical association (cma) have formed a consensus [ ] . guideline edition issued by the chinese medical association clearly stated that the overall goal of treatment is to chb was to suppress hbv as long as possible, relieve inflammation or necrosis of liver cells and liver fibrosis, prevent the progression of cirrhosis, reduce and prevent the occurrence of hepatic decompensation, hcc and its complications, thereby improving the quality of life and survival rate. for patients with cirrhosis, whether compensated or non-compensated, antiviral treatment can delay or reduce hepatic decompensation and hcc occurs, does not change the final outcome of end-stage liver cirrhosis. in the guideline edition issued by the chinese medical association, the goal of antiviral therapy in hbv compensated cirrhotic patients is to prevent progression of the disease to decompensated cirrhosis, end-stage liver disease, hepatocellular carcinoma. the goal of antiviral therapy in hbv-related decompensated cirrhosis is to improve the hepatic disease severity, improve the clinical symptoms and quality of life, and prolong patient's survival. for the endpoint of the antiviral treatment, the definition of each guide is slightly different. guideline edition issued by aasld explicitly mentioned these patients with compensated cirrhosis should receive long-term treatment. however, treatment maybe stopped in hbeag positive patients if they have confirmed hbeag seroconversion and have completed at least months of consolidation therapy and in hbeag negative patients if they have confirmed hbsag clearance. for these patients with decompensated cirrhosis life-long treatment is recommended. guidelines issued by american society of digestive disease [ ] recommend long-term treatment until negative hbv dna and hbsag disappears. in the guideline edition issued by easl, endpoint of antiviral treatment has even been further subdivided into "ideal endpoint", "satisfactory endpoint" and "basic endpoint", where "ideal endpoint" means to achieve hbsag clearance with/without hbsag seroconversion. patients with liver cirrhosis generally have characteristics of longer course of disease, most of mother to child transmission, the large proportion of treated patients, high complexity of quasispecies and higher risk of resistance. the clinical treatment decisions in these patients need to consider a variety of factors, including long-term treatment, delay disease progression, improve histology, low resistance rates, good safety and patient tolerance, etc. there are differences in current guidelines of countries in antiviral therapy indications for liver cirrhosis, mainly cutoff viral load. for compensated cirrhosis, apasl guideline pointed hbv dna > iu/ml, aasld guideline that the hbv dna > iu/ml or hbv dna < iu/ml but elevated alt should be treated with antiviral treatment, and easl guidelines as long as detectable hbv dna should be treated. chinese guideline point that whether normal or elevated alt, hbeag(+) and hbv dna ≥ copies/ml, hbeag negative and hbv dna ≥ copies/ml should be antiviral treated. for decompensated cirrhosis, the recommendations of the guidelines is basically the same: as long as hbv dna can be detected, even if the viral load is low, it should be treated. currently there are two types of anti-hbv drugs, including interferons and nucleos(t)ide analogues. among them, decompensated cirrhosis is a contraindication to interferon. even compensated cirrhosis, due to the risk of hepatic decompensation, the use of interferon should be very careful. patients need to be closely monitored in the clinical application process, dose adjustments, the injection interval prolonged, and assistant drug to reduce adverse reactions and other measures so that the patient can safely complete drug treatment. on the contrary, the nucleos(t) ide analogues with strong antiviral effect, ease of use, safety, and well tolerance were recommended as the preferred treatment of liver cirrhosis by the major guidelines. currently, lamivudine (lam), adefovir dipivoxil (adv), telbivudine (ldt), entecavir (etv), and tenofovir disoproxil fumarate (tdf) have been approved for chb therapy. different nucleoside drug efficacy and safety varies. cirrhotic patients should be treated with potent, fast, direct inhibition of viral replication drugs. in aasld easl and apasl guidelines, for decompensated cirrhosis nucleoside (acid) analogues with potent effect and low resistance, such as entecavir or tenofovir should be selected. the reveal-hbv study of chen [ ] has established an hbv viral load paradigm in the natural history of chb. serum hbv dna level has been shown to be significantly and independently associated with incidence of hepatocellular carcinoma (hcc) and cirrhosis. liaw et al. [ ] evaluated the effectiveness of antiviral therapy in preventing disease progression in patients with chb and advanced fibrosis or cirrhosis. this is a large-scale, multi-center, randomized, double-blind, placebo-controlled prospective study. the study has changed the concept of the world, especially the chinese doctors to treat chb and liver cirrhosis, and promote the development of antiviral treatment for chb and liver cirrhosis. results showed that -year follow-up hepatocellular carcinoma occurred in patients ( . %) who received lam and patients ( . %) who received placebo (p = . ). this study also found that lam therapy reduced the risk of hcc by %, the risk of disease progression to fibrosis/cirrhosis by %. it first confirmed that antiviral therapy with lam could delay disease progression, reduce the incidence of hcc. in the study of patients with decompensated cirrhosis has also been confirmed lam was well tolerated, could effectively stabilize or improve liver function, and delay progression of liver disease, reduce liver transplantation. hann et al. [ ] conducted a prospective, multicenter study evaluated lam in decompensated cirrhosis patients, % of whom were not waiting for liver transplantation. all patients tested hbsag(+) and % tested positive for hbeag(+) at baseline. in % of patients hbv dna levels were detectable by the branched chain dna assay. the virus in % of these patients after months treatment and in % overall became undetectable by the bdna assay. alt, bilirubin and albumin level improved throughout treatment. from at baseline to at last visit the median ctp score also improved. after a median of . months of treatment, a virologic breakthrough occurred in only % patients. treatment of lam can improve liver function in nontransplantation candidates with decompensated cirrhosis. a meta-analysis from huang et al. [ ] indicated that lam and ldt significantly decrease the mortality rate and disease severity in decompensated cirrhosis patients. eight studies (total patients) were included. data showed that lam and ldt significantly decreased the mortality rate (rr . , % ci . - . ), improved the ctp scores (mean difference − . , % ci − . to − . ). in the study of patients with liver cirrhosis showed clinical improvement after treatment with lam - months. and even in patients with clinical improvement, they may develop to hcc, therefore such patients still need early treatment, and close monitoring of hcc. it is showed good efficacy and safety in the lam treatment of hbv related compensated or decompensated liver cirrhosis. however, in the long course of lam treatment viral resistance could not be ignored. more importantly, if these patients with chronic liver disease for a long time, poor liver reserve function, not promptly be treated, condition will deteriorate or even lead to death due to viral resistance. in clinical use of lam, the proportion of viral resistance increased year by year, %, %, %, %, the first year, second year, third year, fourth year, respectively. after the occurrence of viral resistance, some patients will be worsening, and even hepatic decompensation. therefore it is emphasized that patients with compensated or decompensated liver cirrhosis by lam therapy need to be improved compliance, closely monitoring and follow-up, to be adjusted treatment based on serum hbv dna response situation. some patients with decompensated cirrhosis or high viral load should be considered an initial combined with adefovir dipivoxil. the rates of adv resistance in lam-resistant subjects with hbv chronic hepatitis and cirrhosis are reported to be . % and . % after and years, respectively [ ] . nevertheless, adv use as a rescue therapy is affected by a primary nonresponse in - % of patients. in a study of adv add-on lam rescue therapy in lamivudine-resistant patients, kim et al. [ ] reviewed patients with adv add-on rescue treatment for years. after years treatment . % patients had complete virological response. alt in . % patients normalized, hbeag seroconversion occurred in . % patients. a study from woo et al. [ ] was to determine the long-term clinical outcomes after adv rescue therapy in decompensated patients infected with lamivudine-resistant hbv. in total, patients with a decompensated state and lamivudine-resistant hbv were treated with adv at a dosage of mg/day for a median of months in this multicenter cohort study. following adv treatment, ( . %) of patients experienced a decrease in child-pugh score of at least points, and the overall end-stage liver disease score decreased from ± to ± (mean ± sd, p < . ) during the follow-up period. with adv treatment, patients ( . %) had undetectable serum hbv dna (detection limit, . pg/ml). virologic breakthrough occurred in patients ( . %) and patients had a suboptimal adv response. the overall survival rate was . % ( / ), and a suboptimal response to adv treatment was associated with both no improvement in child-pugh score (≥ points; p = . ) and high mortality following adv rescue therapy (p = . ). three years of adv treatment was effective and safe in decompensated patients with lamivudine-resistant hbv. the globe [ ] trial was one of the largest international multi-center clinical trials of ldt treatment of chb. the safety and efficacy of ldt versus lam monotherapy has been compared for years in chb patients. hbeag(+) and hbeag(−) patients were treated weeks with ldt or lam once daily. the patients treated by ldt achieved superior therapeutic response versus these treated by lam in hbeag positive ( % vs %; p < . ) and hbeag negative ( % vs %; p = . ) patients. in both the hbeag positive and the hbeag negative groups, greater hbv dna suppression and less resistance was observed in patients treated by ldt than lam. after weeks of therapy in the phase iii globe study, hbv resistance (breakthrough and resistance mutations) to ldt occurred in % of patients with hbeag(+) and % of patients with hbeag(−). after weeks of therapy, hbv dna rebound in . - . % of hbeag(+) and . - . % of hbeag(−) ldt-treated patients associated with breakthrough and resistance mutations [ ] . ldt is not active against lamivudine-resistant hbv. in the globe study, patients who failed lam therapy showed cross-resistance to ldt. ldt was relegated to second-line status in the management of chronic hbv infection due to increasing resistance over time. in aasld and easl guidelines ldt is not recommended as first-line drug for monotherapy. when necessary the combined adv or tdf is recommended. the "roadmap concept" was derived primarily from a phase iii global registration study of ldt. an optimized strategy based on the roadmap concept is supposed to improve the clinical outcomes of patients with suboptimal antiviral response. sun et al. [ ] conducted the efficacy optimization of response to telbivudine (effort) study to investigate the efficacy and safety of the roadmap strategy by adding adv to ldt for suboptimal responders. in all, hbeag(+), na naive patients with chb were randomized to the mono or optimize group. patients in the optimize group were treated with ldt for weeks. subsequently, patients with hbv dna ≥ copies/ml at weeks were added adv to weeks, while those with hbv dna < copies/ml continued monotherapy. mono group received ldt monotherapy and added adv if development of viral breakthrough. due to suboptimal response % patients in the optimize group had been added adv. in the optimize group, more patients at weeks achieved hbv dna < copies/ml versus the mono group ( . % vs . %, p < . ), and with less genotypic resistance ( . % vs . %, p < . ). combination therapy showed an additive antiviral and low resistance potency. in two groups all patients were well tolerated. patients with ldt which are suboptimal virological responders at weeks are recommended to add adv. these patients with ldt monotherapy can be benefited from combination therapy without increased side effects. chan et al. [ ] studied the safety and efficacy of ldt and lam in hbv-related decompensated cirrhosis patients. in this double-blind trial, treatment-naive patients with decompensated cirrhosis in academic hospitals were randomized ( : ) to receive ldt or lam for weeks. a modified endpoint was used hbv dna < copies/ml and alt normalization. in intent-to-treat analysis (missing = failure) response rates were . % vs . % after weeks (p = . ) and . % vs . % after weeks (p = . ) for ldt vs lam. cumulative death and hcc rates were % and % in patients with ldt, and % and % compared to lam, respectively. cumulative genotypic resistance rate were % in patients with ldt, and % compared to lam during a -year period. comparable to lam, ldt can effectively stabilize liver function and is well tolerated. however, these two drugs are not recommended as first-line drugs due to high virological breakthrough rates. international clinical trials [ , ] and independent registrational studies in china have shown that etv achieved statistically superior virological and biochemical responses compared with lam. shim et al. [ ] evaluated etv as first-line monotherapy in patients with hbv-related decompensated cirrhosis primarily treated with . mg/day etv. there was no significant differences between groups in mean hbv dna changes, rates of hbeag seroconversion or hbeag loss, the proportion of patients with alt normalization after treatment. after months of treatment, the meld score and the ctp score in decompensated patients improved significantly. in these patients, % achieved ctp class a status and % showing a decrease of ctp score p points. the cumulative death rates and hcc rates in years were % and . %, respectively. zoutendijk et al. [ ] conducted a study to investigate the effect of etv on disease progression in patients treated with etv. all patients were divided into three groups, chronic hepatitis b without cirrhosis group (n = ), decompensated cirrhosis group (n = ) and cirrhosis group (n = ). the virological response (vr) was not influenced by the severity of liver disease (p = . ). in cirrhosis group, the probability of developing clinical events was higher (hr . ( % ci . - . ), p < . ) compared to two other groups during a median follow-up of months. vr was associated with a lower probability of disease progression (hr . ( % ci . - . ), p = . ). patients with compensated and decompensated cirrhosis who have achieved vr can achieve significant clinical benefits (hr . ( % ci . - . ), p = . ). in patients with cirrhosis, virological response to etv treatment lead to a lower probability of disease progression. the association between disease progression and viral replication was reduced with a threshold of iu/ml. it suggested that complete viral suppression was essential for patients with na treatment, especially in cirrhosis patients. tenofovir disoproxil fumarate (tdf) is an acyclic adenine nucleotide analogue. tdf as a potent, oral antiviral with low resistance rates, has been recommend firstline drug for the treatment of chb in a number of international guidelines. a study [ ] evaluated the effects on chb patients with fibrosis and cirrhosis treated with tdf of at least years. ( %) patients of completed weeks treatment. % patients ( / ) had biopsy results at both baseline and weeks. % patients ( / ) had histological improvement, and % ( / ) had regression of fibrosis at weeks (p < . ). % ( / ) cirrhosis patients (ishak score or ) at baseline, % ( / ) no longer had cirrhosis (≥ unit decrease in score). the histological response and regression of fibrosis seen in this study are probably due to the potent viral suppression achieved with long-term use of tdf. liaw et al. [ ] conducted a clinical trial to observe chb patients with decompensated liver disease received either etv (n = ), emtricitabine (ftc)/ tdf (n = ), or tdf (n = ). after weeks treatment, hbv dna was < iu/ ml ( copies/ml) occurred in . % (etv), . % (ftc/tdf), and . % (tdf) of patients. alt normalization occurred in % (etv), % (ftc/tdf), and % (tdf). hbeag loss/seroconversion rate were: / % (etv), / % (ftc/ tdf), and / % (tdf). in three groups meld scores and ctp scores both improved. this study demonstrated that all nas were well tolerated in chb patients with decompensated liver disease and can effectively improve virologic and biochemical parameters. response-guided therapy study [ ] suggested continuous treatment with lam ( years) delayed clinical progression in patients with chronic hepatitis and advanced fibrosis by significantly reducing the incidence of the risk of hepatocellular carcinoma and hepatic decompensation. the biggest risk is the occurrence of viral resistance in the longterm antiviral therapy. although rescue therapy can save some patients regain virologic response, but improper treatment or less often sicker or even canceling the original clinical benefit. although treatment can save some patients regain virologic response, but improper or untimely treatment often leads to deterioration or even canceling the existing clinical benefit. therefore, in the long-term antiviral therapy in patients with cirrhosis, how to overcome and prevent resistance, maximize the clinical benefit of antiviral therapy (including histological improvement and prevent and delay disease progression) is required by clinicians to consider. response-guided therapy refers to select appropriate medication according to the baseline characteristics of patients, and based on the patient's response to treatment, especially for those who did not achieve an early virological response, timely to adjust treatment to achieve a better long-term results. response-guided therapy is a hot point in the current study of antiviral treatment for chb, it is also an important strategy and important measure for the prevention of viral resistance. some experts even believe that all of antiviral therapy will need to be optimized. response-guided therapy means optimization based on baseline characteristics and early virologic response. numerous studies have demonstrated that baseline parameters such as low viral load, high serum alt level, high inflammation activity score prompted by liver biopsy and early virological response predict better longterm effect. in keeffe's [ ] "road map concept", response-guided therapy according to virologic response at weeks has been recommended. combination therapy is an important part of response-guided therapy. combination therapy includes an initial stage combination, the combination in the course of treatment (poor response or resistance), the combination treatment for treated patients with relapse. in easl guideline it was referred that adv or tdf with lam combination treatment need to consider in patients with liver cirrhosis. in aasld guideline lam or adv was recommend initial treatment for patients with decompensated cirrhosis, but their combination is recommended to reduce the risk of resistance and rapid inhibition of virus. the combined treatment for lam+adv is the most clinically used and studied. in first-generation na lam, ymdd mutation (rtm v/i) developed in up to % of patients in years [ ] . the combination of adv dipivoxil and lam was found to lower the rates of resistance to lam and serum hbv dna levels, and fasten the rates of alt normalization in hbeag(+) patients, with similar rates of hbeag seroconversion [ ] . in china a prospective cohort study [ ] from eight medical centers was conducted to observe the effect of response-guided combination therapy with lam and adv in patients with chb. according to hbv dna level at weeks, a total of patients with chb and cirrhosis treated lam were divided into three groups: complete response group (arm a, n = , hbv dna ≤ iu/ ml), partial response group (arm b, n = , hbv dna: - iu/ml) and inadequate response group (arm c, n = , hbv dna > iu/ml). patients was added adv at week in arm c, but at week in arms a and b. at weeks undetectable rate of hbv dna and ymdd mutation rate in three arms was . %, . %, . % (p = . ) and %, . %, % (p = . ), respectively. the data showed that early complete virologic response at weeks was associated with maintained viral suppression. hbv dna level of these patients without complete virological response at week , adding adv therapy further decreased by log iu/ml. all patients achieved biochemical improvement including alt/ast decline and alb elevation. in patients with hbv dna breakthrough due to ymdd mutations, adv and lam combination therapy did not lead to further multiple drug resistance. in chb patients with compensated liver cirrhosis, continuous hbv suppression for longterm and liver function improvement could be obtained by optimized responseguided add-on therapy of lam and adv. in recent years, some new antiviral drugs have gradually entered people's field of vision. truvada is a fixed-dose combination of two antiretroviral drugs (emtricitabine and tdf) approved by the fda for anti hiv therapy. the molecular structure of ftc was similar to that of lam, and the drug resistance was also similar to lam. in a double-blind study [ ] evaluated weeks treatment of , or mg once daily doses of emtricitabine in patients with chronic hepatitis b. then these patients were followed mg emtricitabine treatment for an additional weeks. after years, % of the patients had normal alt, % seroconverted to anti-hbe and % had serum hbv dna ≤ copies/ml. eighteen percent of the patients treated with mg emtricitabine developed resistance mutations after years. emtricitabine mg once daily was chosen as the optimal dose for chb based on these data. emtricitabine was well tolerated and confirmed a potent antiviral response for up to years. in a randomized double-blind, -week trial [ ] , patients were divided ( : ) to groups given a combination of emtricitabine (ftc, mg; n = ) and tdf ( mg, ftc/tdf) or monotherapy of tdf ( mg, n = ). patients were hbeag(+) or hbeag(−), with levels of hbv dna ≥ log iu/ml and lam resistance mutations (rtm i/v ± rtl m). after weeks of treatment, . % in the ftc/tdf group and . % of patients in the tdf group had levels of hbv dna < iu/ml (p = . ). hbeag loss and seroconversion was not significant difference between groups; only patient ( . %) in the ftc/tdf group lost hbsag. no additional benefit was observed with the combination therapy of emtricitabine and tdf vs tdf monotherapy. prolonged therapy with an oral nucleoside or nucleotide can lead to the development of antiviral resistance. loss of initial response and hbv dna rebound induce the development of resistance. these patients with resistance may develop biochemical breakthrough and histologic deterioration. sometimes severe exacerbations occur due to virus resistance in patients with cirrhosis. there are many risk factors for resistance development, such as potency of the antiviral agent, pretreatment hbv dna titer, period of treatment, nucleotide antiviral therapy or oral nucleoside history, and the degree of genetic barrier to resistance to the individual drug. thus, either etv or tdf, which possess the lowest genotypic resistance, should be used as the initial therapy. patients should be evaluated in the course of treatment, and these patients with poor response should be treated combination therapy early. managing resistance recommendations vary but generally involve either adding a drug in a separate class or switching to a more potent drug within the same class. in clinical practice, most members of the panel generally avoid monotherapy in patients with resistance and either use add-on therapy with tdf or etv or switch to tenofovir/emtricitabine. however, in patients with lam resistance, there are data providing compelling reassurance that tdf monotherapy is sufficient [ ] . data suggest that tdf may also be sufficient for patients with adefovir resistance [ ] was limited. however, with newer anti-hbv agents such as etv and tdf, viral resistance in previously treatment-naïve patients is very rare and the vast majority of cases of virologic breakthrough in clinical practice are due to nonadherence [ , ] . to see treatment measures in sect. . . clinical trials and cohorts from clinical practice have shown that nas are generally well-tolerated and safe [ ] . rare serious adverse reactions includes renal insufficiency, myositis, rhabdomyolysis, lactic acidosis, etc. some of these drugs can induce impairment of mitochondrial replication with mitochondrial dysfunction or loss due to a low level of activity against the human mitochondrial dna polymerase gamma. lam has been well-tolerated and effective in patients with hbv related decompensated cirrhosis [ , ] . adefovir dipivoxil and tdf are associated with a dose-dependent renal toxicity, except for ldt, a drug that may even improve creatinine clearance [ ] . . % patients had elevation of serum creatinine ≥ . mg/dl above baseline have been reported after years of tdf therapy in chb patients [ ] . hadziyannis et al. [ ] conducted a cohort study to investigate the efficacy, safety, and resistance profile of adefovir dipivoxil treatment for up to weeks in patients with hbeag(−) chb that was lost when weeks adv treatment was discontinued. a total of hbeag(−) chb patients treated with adv for years. serum creatinine elevations ( . mg/dl above baseline) occurs in % of these patients. similarly, % of the hbeag(+) chb patients treated adv for years had reversible creatinine elevations, % developed hypophosphatemia, and % had albuminuria [ ] . there were a growing number of studies of lam and adv combination therapy in both treatment-experienced and treatment-naïve patients with lamivudine-resistant hbv. lampertico et al. [ ] conducted a study to investigate the risk of resistance in the long term of lam and adv combination therapy in lamivudine-experienced chb patients. the results showed that % of patients with lamivudine resistance developed mild nephrotoxicity. after increasing the adv-dosing interval, all these patients were able to continue combination therapy. before treatment estimated creatinine clearance and serum creatinine levels should be tested in all chb patients treated with nas. in patients with creatinine clearance < ml dosing adjustments are needed, regardless of the type of nas. etv preclinical research data showed that a higher incidence of solid tumors in animals was associated with prolonged administration of high-dose compared to placebo. however, in clinical trials prolonged administration of etv was not associated with an increased incidence of malignancy. in a clinical trials conducted by lai et al. [ ] the severity and frequency of laboratory and clinical adverse events were similar among lam-treated and etv-treated patients. furthermore no evidence of serious adverse events and mitochondrial were observed in patients with etv treatment for up to years [ ] . in recent years, in patients with liver cirrhosis in high meld score (> ) taking etv, lactic acidosis, and even death cases were reported. although rare happening, it still has to be pay more attention by the clinician, and needs for future research. ldt may cause myopathy and peripheral neuropathy. in treatment of ldt combined with peg-ifn- a, moderate-severe peripheral neuropathy may occur in patients. therefore ldt was forbidden in combination with peg-ifn. in these studies [ , ] , patients treated with ldt at years had a significantly higher incidence of severe elevations of serum creatine phosphokinase (i.e., seven times upper limit of normal) compared to patients treated with lam, . % and . %, respectively. although most of them are asymptomatic, there are still two cases of patients with ldt-induced symptomatic myopathy had to be terminated treatment. in chinese guideline, it has been stated that careful medical history investigation before treatment was needed to reduce the risk. in the course of treatment, patients with serum creatinine, ck or lactate dehydrogenase significantly increased, and accompanied by myalgia or weakness, should be immediately tested. once diagnosed with uremia, myositis, rhabdomyolysis or lactic acidosis, patients should be promptly discontinued treatment or switched to other drugs, and given the appropriate treatment. antiviral therapy with nucleos(t)ide analogue is an important means to delay or reverse progression of liver fibrosis and cirrhosis. cirrhotic patients need long-term or even lifelong antiviral treatment. all of the five antiviral agents could effectively inhibit virus replication, improve biochemical and pathological parameters in chb cirrhotic patients with good tolerance. patients should be fully assessed baseline characteristics before treatment, and closely monitored therapeutic response and adverse reactions during treatment, then to optimized treatment. according to both drug resistance and efficacy profile, etv and tdf are superior to ldt, adv, and lam, and can be recommended as the first-line drug for nuc-naïve patients with hbv related decompensated cirrhosis. finally, hbv related cirrhosis patients treated oral nuc(s) must be frequently laboratory and clinical assessed to insure medication compliance and surveillance for clinical and virological response as well as drug resistance, drug side effects, and hepatocellular carcinoma. in the world hepatocellular carcinoma is one of the most frequent malignancies: estimated , new cancer cases worldwide occurred in ( % in china alone). it is the fifth most common cancer in men ( , cases, . % of the total) and the ninth in women ( , cases, . %) [ ] . hepatocellular carcinoma is the second most common cause of cancer death worldwide, estimated to be responsible for nearly , deaths in ( . % of the total). given the very poor prognosis for liver cancer (the ratio of mortality to incidence is . ), the geographical patterns in incidence and mortality are quite similar [ ] . chronic infections with hepatitis c virus (hcv) and hepatitis b virus (hbv) are the major recognized risk factors for hcc worldwide [ ] , hbv being most common in eastern asia and hcv in mediterranean countries [ ] . current hcc treatment is the comprehensive treatment based on resection, liver transplantation, or percutaneous local ablative treatment. more and more studies indicated that after resection antiviral therapy effectively inhibit hbv replication and sequentially decrease the rate recurrence of hcc. chronic hepatitis b is the most frequent etiology of hcc. chen [ ] conducted a prospective cohort study to evaluate the relationship between mortality and past hbv dna level for years of follow-up. hbv dna level had been measured on stored samples in hbsag(+) adults from cohort entry ( - ). there was a significant increase in mortality in patients with hcc across viral load categories (p < . ). compared to the hbv undetected category, the relative risk for hcc mortality in the high viral load group was . ( % ci . - . ) and . ( % ci . - . ) in the low viral load group. the relative risk for chronic liver disease mortality were . ( % ci . - . ) and . ( % ci . - . ), respectively (p < . ). with increased follow-up time, the rr associated with high viral load did not change. in surviving cohort patients evaluated for liver disease in , the disease significantly associated with viral load. the data showed that increased mortality from hcc and cld was associated with viral load in patients infected hbv. hbv dna level may be a useful prognostic indicator in chb patients, and treatment interventions to inhibition of virus replication should be explored. the reveal-hbv study of chen [ ] assessed the relationship between risk of serum hbv dna level and hcc. from to , this prospective cohort study in taiwan enrolled participants who were hbsag(+) and - years from a community-based cancer screening program. during , person-years of follow-up and a mean follow-up of . years, there were incident cases of liver cancer and deaths. the incidence of liver cancer grew in patients with hbv dna level at baseline in a dose-response relationship ranging from . % personyears for an hbv dna < copies/ml to . % person-years for an hbv dna × copies/ml or greater. the cumulative incidence rates of liver cancer in these patients were . % and . %, respectively. after adjustment for age, sex, alcohol consumption, cigarette smoking, serum alt level, hbeag, and liver cirrhosis at baseline, the biological gradient of liver cancer by hbv dna levels were significant different (p < . ). the dose-response relationship was most prominent for hbeag(−) patients without liver cirrhosis and with normal serum alt levels at baseline. chb patients with persistent elevation of viral load had the highest liver cancer risk during follow-up. these data proved that high level of hbv dna (≥ , copies/ml) was a strong risk factor of liver cancer independent of liver cirrhosis, hbeag, and serum alt level. these data showed that the correlation between hcc and hbv dna level was more closely than alt. the current guidelines [ ] [ ] [ ] for management of chb are of the view that: for patients with chronic hepatitis b the primary goal of treatment is to permanently suppress or eliminate hepatitis b virus replication. thus hepatic infectivity and pathogenicity could be decreased, and thereby necroinflammation could be stopped or reduced. the short-term goal in clinical terms is to reduce hepatic inflammation, to prevent the development of hepatic fibrosis and decompensation, to ensure a sustained loss of hbv-dna and alt normalization. the long-term goal is to prevent progression to cirrhosis and hcc, to prevent alt flares that may lead to hepatic decompensation, and finally prolong the survival time. therefore, the ideal treatment for patients with chb should be able to effectively reduce the hbv dna level, thereby inhibit or stop the deterioration of liver disease, reduce the incidence of severe exacerbation and hcc. hbv chronic infection is a major risk predictor for the development of hepatocellular carcinoma. the hepatocarcinogenesis in chb patients has been extensively investigated, and a number of predictors relate to occurrence of hepatocellular carcinoma. the most significant predictors associated with hepatocellular carcinoma include chronic hcv and hbv infection, aflatoxin b , chronic alcohol consumption and virtually all cirrhosis-inducing conditions [ ] . for hepatocellular carcinoma in human, chronic infection of hbv was considered as the major environmental etiological factor [ ] . hbv-induced hepatocarcinogenesis can involve an array of processes, including host-viral interactions, sustained cycles of necrosis-inflammation-regeneration, viral-endoplasmic-reticulum interactions (induction of oxidative stress), viral integration into the host genome (and associated host dna deletions) and the targeted activation of oncogenic pathways by various viral proteins. a predominant risk factor for hcc is chronic active hepatitis. the mechanisms of chronic active hepatitis consist of a combination of complementary, effects, several involved in liver cell inflammation, and necrosis thus fibrosis and cytokine synthesis. the underlying chronic active hepatitis inflammatory is a major risk factor for the higher hcc occurrence in patients with progressive cirrhosis. fundamentally important information on these issues have been provided in animal models for hepadna virus infection [ ] . integration of hbv dna results continuous replication of the virus, which induces occurrence of genetic alterations. the hbv-dna sequences are integrated into cellular dna in most (approximately %) liver-tumor samples from hbsagpositive patients [ ] . hbv genome integration should be viewed as a "driver" of liver carcinogenesis. in the impact of genes, some of the other genes involved, and play an important role in the carcinogenesis process. in addition, hbv dna integration effects on host cells including cell gene deletion, chromosomal rearrangements, genomic dna copy number variation, loss of chromosomal heterozygosity, etc. [ , ] . in the host cells, the integration of hbv damage mechanisms to protect the integrity of the chromosome. hepatitis b virus x protein (hbx) exhibits pleiotropic effects on different pathways involved in intracellular signaling and transcriptional activation that modulate cell responses to protein degradation, genotoxic stress, apoptosis and cell division; these biological effects might contribute to the potential transforming activities of hbx. hbx has been confirmed to interact with p , accordingly inactivating several essential p -dependent activities, including p -mediated apoptosis transactivation properties of p , regulation of cell cycle dna repair genes and tumor suppressor genes. hbx may also play a role in tumorigenesis of hepatocellular carcinoma through modulation of angiogenesis pathway. indeed, hbx expression induces upregulation of the vascular endothelial growth factor (vegf) transcription and stabilizes hypoxia inducible factor (hif)- [ , ] . moreover, hbx causes multipolar spindle formation, chromosome segregation defects, and appearance of multinucleate cells by inducing aberrant centrosome duplication; these biological actions might be due to sequestration of the nuclear transport receptor crm in the cytoplasm [ ] . it has been frequently reported that hbx with mutations in amino acids and may be associated with the severity of chb. studies indicated that these mutations had also been detected in hcc tissue and arose before the development of hcc [ ] . other studies [ ] have pointed out that many signaling pathways have been outlined as common targets deregulated during hepatocarcinogenesis, including the wnt/β-catenin, tgf-β pathway, ras/mapk pathway, pi k/akt/mtor pathway, jak/stat pathway, pkc pathway, etc. in addition, fibrinogen-like protein (fgl ), hgf, igfs and other coagulation factors, growth factors and other angiogenesis gene may also be involved in the occurrence and development of hcc [ ] . . wnt/β-catenin pathway: the wnt signaling pathway is an evolutionary highly conserved pathway and involved in the regulation of proliferation, motility, cell/ cell interaction, organogenesis and axis formation. the accumulation and expression of β-catenin in the nucleus were decreased, and cell proliferation was suppressed followed by up-regulated gsk- β activity due to hbx induction [ ] . hbx mutants may participate in the development and progression of hcc, at least in part through the wnt- a pathway [ ] . . tgf-β pathway: tgf-β is a central regulatory factor in control of hepatocyte proliferation and death. paradoxically, either under-or overexpression seem to have deleterious consequences resulting in an increased turnover of liver cells and thereby predisposing to cancer progression [ ] . in both cases the escape from the antiproliferative, proapoptotic action of tgf-β would be a prerequisite for tumor progression. at the stage of tumor occurrence, tgf-β can promote tumor cell invasion, metastasis, but suppresses tumor growth in liver damage stage. tgf-β receptor i kinase inhibitors, blocking the tgf-β signaling pathway, show anti-tumor effect [ ] . . ras/mapk pathway: ras/mapk signaling pathway is a signal cascade waterfall reaction caused by external signal activated receptor in the cytoplasm, involving a variety of connectors, nucleotide exchange factor, small gtp binding protein. hbx retains the ability to overcome ras-induced senescence in human cells immortalized by htert, although hbx alone could neither immortalize nor transform human cells. the ability of hbx to collaborate with active ras in cell transformation may explain its role in hcc [ ] . . pi k/akt/mtor pathway: the pi k/akt/mtor protein cascade is one of major signaling pathways associated with receptor tyrosine kinases (rtks) [ ] . in nontransformed cells, a tumor suppressor pten (phosphatase and tensin homolog deleted from chromosome ), which inhibits this pathway by blocking akt activation and reversing the pi k reaction, control the pi k/akt/ mtor pathway. in almost half of the studied hccs, pten expression was reduced or absent, and hepatocyte-specific abrogation of pten expression in mice results in the development of hcc [ ] . . jak/stat pathway: the jak/stat pathway is activated by more than cytokines and growth factors and involves in multiple cell functions such as differentiation, proliferation, and apoptosis [ ] . in this pathway, the cytokines induce phosphorylation of the janus tyrosine kinases (jak , and , tyk ), followed by activation of stat - [ ] . both hbv and hcv are able to induce jak/stat pathways [ ] . in hcc, phosphorylation of jak , jak , and tyk tyrosine kinases was not detected in normal livers but increased significantly from surrounding non-neoplastic livers to hccs. activation of stat , stat , and stat was statistically higher in tumors than in respective surrounding livers, with pstat being higher in hcc with poor prognosis than hcc with better prognosis. the levels of jak/stat targets, including bcl-xl, mcl- , cyclin d , and c-myc were markedly increased in the majority of hccs [ ] . . pkc pathway: pkc isozymes have a central role in cellular signaling transduction involved in cell proliferation, differentiation, apoptosis and angiogenesis [ ] . pkc-α, pkc-δ, and pkc-ι have been found to be over-expressed in human hcc cell lines. the focus of pkc research in hcc has predominantly been on pkc-α. its expression is significantly increased in cancerous tissue and is correlated with tumor size and tnm stage. in addition, over-expression of the mrna of this isozyme has been associated with a shorter survival rate, and thus may be a marker for disease prognosis in cancer patients [ ] . . fgl : fgl could directly generate thrombin from prothrombin without activation of the conventional coagulation cascade. it was confirmed to be overexpressed in various human malignant tumors [ ] . the hfg was associated to the hypercoagulability in cancer and may induce tumor metastasis and angiogenesis via cytokine induction [ ] . fgl was overexpressed in hcc tissues and co-localized with fibrin deposition. fgl contributed to hcc tumor angiogenesis and growth in a thrombin-dependent manner, and down regulation of its expression might be of therapeutic significance in hcc [ ] . to investigate whether prevention of hcc by the hbv vaccine and to identify the predictors of hcc for vaccinated birth cohorts, a population-based study [ ] in taiwan the study shows strong evidence that the hbv vaccine reduce the incidence of hcc. those who received incomplete hbv vaccination (i.e., less than three administrations of the vaccine) during infancy and infants of hbeag-and hbsagseropositive mothers without hbig injection at birth had higher risk of developing hcc. approximately % of children with hcc born to hbeag and hbsag carrier mothers did not receive hbig at birth. improvements of the hbig injection rate within h after birth in infants of high-risk mothers should be implemented. this study has limitations in that the role of host factors, such as genetic polymorphisms, hbv genotype, virus mutation, were not studied, which could influence the interpretation of the data. a number of studies on the long-term treatment of interferon (ifn) or na for patients with hbv showed the prevention of hcc. a meta-analysis [ ] compared risk of hcc in chb patients who received ifn or na. a total of studies were included in this review. ifn treatment ( studies; n = ) showed a significantly reduced risk of hcc for patients treated by ifn compared to controls (rr . , % ci . - . ; studies) and for compensated cirrhotic patients (rr . , % ci . - . ; studies). there was no statistical heterogeneity for these comparisons. na treatment ( studies; n = ) showed a significantly reduced risk of hcc for patients treated by nas compared with controls (rr . , % ci . - . ; studies). na treatment demonstrated a more profound reduction in hcc risk of % compared to ifn which produced only a modest effect of %. this is perhaps not a surprising finding, as the viral load is found to be the most important factor leading to cirrhosis and cancer development in the liver. the more effective reduction in hcc risk may be related to the more profound effects of viral suppression of oral anti-viral agents than ifn [ ] . across subgroups there was a significantly reduced risk of hcc: hbeag(+) patients (rr . , % ci . - . ; five studies); compensated cirrhotic patients (rr . , % ci . - . ; three studies); non-cirrhotic patients (rr . , % ci . - . ; two studies); patients with drug resistance (rr . , % ci . - . ; three studies); and those without drug resistance (rr . , % ci . - . ; three studies). in subgroup analysis of ifn studies, a more significant reduction in hcc risk among those with early cirrhosis was found. the effects of ifn could be beyond its viral suppressive activities. previous studies have shown that at least ifn-α b has inhibitory activities on hepatic stellate cells (hscs) activation by suppressing the effects of il- β, tnf-α and probably inducing apoptosis of hsc [ , ] . as hscs play a central role in fibrogenesis, the effects of ifn-α on hscs are worthy of further investigation. on the other hand, the anti-cancer effect of nas, and probably ifn, was more prominent among hbeag-positive than among hbeag-negative patients. this discrepant results based on hbeag status is consistent with the fact that while hbeag(+) patients usually have a higher hbv dna level, treatment of hbeag(−) patients is more difficult and sustained virological responses are uncommon [ , ] . high risk population hbv-hcc refers to patients who are the middle-aged men with high hbv load, with hbv and hcv co-infection, with family history of liver cancer, alcoholics, and with diabetes mellitus. the long-term effect of antiviral therapy for patients with hbv showed the prevention of hcc. however, according to current national management guidelines for chb, there are still some patients without antiviral treatment. thus, some of the high risk population of hbv-hcc may lost the opportunity of early interventional treatment. current guidelines recommend liver biopsy to assess the degree of necroinflammation and liver fibrosis prior to treatment initiation in patients with increased hbv dna and/or mild elevated alt levels ( - times the uln). for patients older than years, liver biopsy should be considered. in those with "high normal" alt levels liver biopsy is strongly recommended [ ] . although liver biopsy remains the gold standard for assessing hepatic fibrosis, its use has several limitations including sampling error and intra-or inter-observer sampling variability [ ] . inadequate liver biopsy may further pose misleading histological information that precludes cirrhotic patients from antiviral treatment [ ] . in the report by tong et al. [ ] , of patients who developed hcc were diagnosed as having chronic hepatitis by histology and therefore did not fulfill the recommended treatment criteria. these patients probably had normal alt and/or intermediate hbv dna levels (between , and , copies/ml). in this report, of patients with cirrhosis who developed hcc could not be identified by the treatment recommendations. in other words, patients with cirrhosis have a significant risk of developing hcc even when their hbv dna levels are not high. patients with elevated alt between . and times the uln also was a strong risk predictor of hcc or complications [ , ] , a claim supported by a korean population study. the reveal study suggested that high hbv dna level significantly increased risk of hcc independent of liver cirrhosis, hbeag, and serum alt level. hbv dna consistently replicates and is integrated into the host genome, adding to the coexistence of metabolic disorders, inflammatory responses and oxidative injuries, which induce genetic instability and an imbalance of cell growth and apoptotic tolerance signals. these are all biologic driving forces for hcc development in chb patients. therefore, we must pay more attention to the effect of continuous hbv replication on the prognosis of patients. any antiviral drugs, if not completely clear the virus but can reduce the viral load, it may reduce the risk of patients with hcc. the risk factors for hcc include progression to cirrhosis, longer duration of hbv infection, higher serum viral load (≥ copies/ml), males, age > years, alcohol, ethnic groups native to regions of east asia and sub-saharan africa, the virus genotype (genotype a in african population or genotype c in asian population), co-infection with human immunodeficiency viruses (hiv) or hepatitis c, d, decompensated liver cirrhosis, persistent inflammation of the liver, continuous hbeag positive, and a family history of liver cancer [ ] . cirrhosis is the most important independent risk factor for hcc. up to - % of hcc occur in patients with cirrhosis. effective antiviral therapy inhibits hbv replication, reduces serum viral load and accelerates serum conversion of hbeag, which may therefore alleviate liver damage and reduce the development of cirrhosis. all the patients with the above risk factors should be received antiviral therapy. ifn-α is an immune modulator inducing antiviral, immune regulation, anti-tumor and anti-fibrosis effects. its antiviral mechanism is a complex mode of action including the destabilization of viral nucleocapsid, inhibition of viral genome transcription, activation of natural killer (nk)/nkt cells. however, disadvantages of ifn shortcomings are prominent, such as the high cost of peg-ifn, intolerance to ifn therapy in patients with cirrhosis. compared with ifn, na is safer, better tolerance for these patients. current guidelines from apasl, easl and aasld, do not provide treatment recommendations for patients with hbv-hcc. the chinese expert consensus [ ] on antiviral therapy to treat hbv/hcv-related hcc has been published in . this expert consensus indicated that promptly initiation of antiviral therapy is not only important for preventing the incidence of hcc in patients chb, but also essential for reducing hbv reactivation, improving liver function, delaying or reducing recurrence of hcc, and prolonging survival of patients with hbv-hcc after palliative and curative therapies. it puts forward the overall principle and target of antiviral therapy of hbv-hcc, and emphasizes the antiviral therapy is a part of comprehensive treatment. at present, suitable treatment for hbv-hcc is multidisciplinary comprehensive treatment. a large number of evidence-based medical evidence suggested, standard anti-hbv treatment for these patients help to improve the overall curative effect, prevent the recurrence of the tumor, and improve the os. therefore, anti-hbv therapy should be taken as a very important part of comprehensive treatment of hbv-hcc (fig. . ) . following curative liver resection for hcc, - % of postoperative death is caused by recurrent disease [ ] . high serum hbv dna levels were a strong predictor of hcc. effective control of hbv replication with antiviral therapy may lower its recurrence after liver resection. in , kubo et al. [ ] first reported the relationship between recurrence of hbv-hcc after resection and hbv dna level. later in another study [ ] he pointed out that patients with high hbv dna levels (more than meq/ml) have high risk with recurrence and poor prognosis. on the contrary, kim et al. [ ] included consecutive patients undergoing curative resection and found that, sustained hbv viremia (> log copies/ml) increased recurrence, but did not have a marked effect on survival. an et al. [ ] investigated the hbv dna changing patterns and their effects on outcome in hbv-hcc patients with resection. all patients were followed up. data from alive patients without recurrence at months were collected. the mean period of follow-up was . months and the mean age was years. for the entire population with multivariate analysis, tumor size > cm (p = . ), hbv dna > copies/ml, child-pugh class b (p = . ) at the time of resection (p = . ), and vascular invasion (p = . ) were independently risk factors of hcc recurrence. on multivariate analysis for patients, sustained hbv dna level < copies/ml was the only risk predictor for both longer survival (or . ; % ci . - . ; p = . ) and low recurrence (or . ; % ci . - . ; p = . ). that clarified that a sustained high hbv dna is among the most important risk factors of adverse outcome after liver resection of hbv related hcc. the sustained suppression of hbv dna < copies/ml strongly benefit to long-term survival and recurrence-free. kim et al. failed to show the difference in survival between the sustained viremia (> log copies/ml) and non-viremia groups despite the high recurrence rate in the sustained viremia group. the reason may be that researchers have used a higher hbv dna cut-off value (> copies/ml) to differentiate between patients with high and low viremia. in an's results, they found that a lower hbv dna level cutoff value of copies/ml is superior to > copies/ml in predicting outcomes after resection. it is therefore needed to suppress further hbv dna to a lesser level in order to obtain better clinical outcomes after surgery. studies have shown that positive hbeag was a risk predictor for recurrence of patients after resection of hcc. sun et al. [ ] evaluated the impact of hbeag on patients' survival and tumor recurrence after curative resection of hbv-hcc. all patients undergone curative resection with small hcc (⩽ cm) were divided into hbeag(+) and hbeag(−) groups. postoperative outcomes and clinicopathological factors of two groups were compared, and risk predictors for recurrence and survival were investigated. hbeag(−) patients had higher -year disease free survival rates ( . % vs . %, p = . ) and -year overall survival rates ( % vs . %, p = . ). there was no significant difference in tumor factors and operative morbidity between hbeag(+) and hbeag(−) groups, but more macronodular cirrhosis, higher serum alanine aminotransferase levels, and younger age were found in the hbeag(+) group. in patients for multivariate analysis, macronodular cirrhosis, hbeag(+) and age > years were independent risk predictors for overall survival, and multiple tumor nodules and hbeag(+) were independent predictors for disease free survival. in patients with small hcc after curative resection, hbeag(+) was a significant risk factor of early recurrence (within year). because of the adverse effects, the impact of ifn-α therapy after curative resection on recurrence of hcc and the os among patients with hbv infection are controversial. theoretically, the effect of postoperative ifn-α therapy on recurrence might be related with the direct suppression of tumor growth and metastasis, the inhibition of hbv replication, down-regulating expression of vascular endovascular growth factor (vegf), antiangiogenesis effect, and modulating some factors in tumor microenvironment. however the results of clinical trials are not the same. in recent years, more and more studies show that reasonable application of ifn-α can prevent the recurrence of the tumor and prolong the survival time of the patients. qu et al. [ ] conducted a retrospective study to investigate the impact of ifn-α therapy on survival and recurrence after curative resection in patients with hbv-hcc. of hbv-hcc patients with curative resection, patients were treated postoperative by ifn-α therapy ( million units three times every week for months). patients with postoperative ifn-α therapy had higher os rates (p = . , hr: . , % ci: . - . ). there was no significant difference in dfs rates between the two groups (p = . , hr: . , % ci: . - . ). on multivariate analysis, postoperative ifn-α therapy was an independent factor for os (p = . , hr: . , % ci: . - . ) and significantly reduced early recurrence (p = . , hr: . , % ci: . - . ). however, patients with or without postoperative ifn-α therapy had similar cumulative late recurrence rates (hr: . , % ci: . - . , p = . ). sun et al. [ ] evaluated the effects of postoperative ifn-α treatment on survival and recurrence in patients with hbv related hcc. all patients were randomized after curative resection into ifn-α treatment (n = , μg i.m. tiw for months) and control groups (n = ). if recurrence was diagnosed, treatment was terminated, these recurrence patients was managed in the same way in both groups. all clinicopathological parameters in two groups were analyzed. the median os was . months in the treatment group and . months in the control group (p = . ); the median dfs period was . versus . months (p = . ). that concluded that ifn-α therapy improved the os of hbv-hcc patients after curative resection, probably by postponing recurrence. someya et al. [ ] investigated consecutive patients with hbv cirrhosis and hcc who underwent potentially curative ablation for hcc. eleven patients received long-term ifn therapy. initial dna was high (> . log copies/ml) in patients and low (< . log copies/m) in . hcc recurrence rates in the high dna group and low dna group were . % and . % at the fifth year, and . % and . % at the tenth year, respectively (p = . ). similarly, recurrence rates after treatment of hcc in the abnormal ast group (n = ) and normal ast group (< iu/l, n = ) were . % and . % at the fifth year, and % and . % at the tenth year, respectively (p = . ). five of patients with normal ast, and of the patients with abnormal ast, received ifn-α after confirmation of tumor ablation. in the subgroup of abnormal ast, tumor recurrence rates in the untreated and ifn-α groups were . % and . % at the end of the first year, . % and . % at the second year, and . % and . % at the third year, respectively (p = . ). on multivariate analysis, ifn-α significantly reduced the recurrence rate (p = . , hr = . ) even after adjusting for background characteristics. pathogenic mechanism of hbv-hcc mainly related with the integration of hbv dna into host hepatocytes. therefore, inhibition of inflammation and viral replication can reduce the hbv dna level and the risk of hcc. after the resection the residual liver is still cirrhosis, still have a high risk of new cancer. hbv-hcc occurrence seems to have the relationship with the hbv greater than the liver repeatedly inflammation [ ] . tang et al. [ ] reported that high hbv dna levels is associated with increased risk for development of hcc. ifn has a dual role of antiviral and immune regulation. ifn as immune regulator can not only activate or mediated macrophages, nk cells and cytotoxic t lymphocyte, but also adjust the antibody. its antiviral activity includes induction of , oligonucleotide adenosine monophosphate synthetase and protein kinase. moreover, ifn also has the anti-fibrosis, anti-proliferation and anti-tumor effects. the experimental study [ ] confirmed that ifn exerts potent growth inhibitory effects on the hcc cell line plc/prf/ both in vitro and in vivo and its mode of action in this animal model system appears to be predominantly mediated by a direct antiproliferative effect on tumor cells. breitenstein et al. [ ] searched cochrane central register of controlled trials between january to october and evaluated the effects of ifn-α or -β in patients after surgical resection or ablation of hbv-hcc. seven rcts (n = patients) were included in a meta-analysis review. patients treated with ifn had a significantly decreased mortality rate than control group (rr . , % ci . - . , p < . ) and significantly reduced risk of tumor recurrence (rr . , % ci . - . , p = . ). as of the trials used ifn-α, it is interesting that the only study [ ] on ifn-β showed the largest beneficial effect on tumor recurrence. due to the small number of cases in this study, further clinical evaluation of ifn-β in the adjuvant setting of hcc treatment seems to be indicated. the rate of treatment discontinuation ranged from % to % because of the side-effects of ifn which were dose dependent and often serious. severe adverse effects of the adjuvant ifn treatment leading either to treatment disruption or dose reduction occurred in up to a quarter of the patients. in particular, work is needed to optimize the type and dosage of ifn to minimize side-effects, and to study the combination of ifn treatment with other (neo)adjuvant agents. reasonable application of nas can prevent the recurrence of hcc and prolong the survival time of the patients. a comparative nonrandomized study [ ] of postoperative antiviral treatment was conducted on hcc patients who underwent curative hepatectomy. the authors assessed the impact of antiviral therapy for patients who underwent partial hepatectomy for hbv-hcc in the immuneactive phase of hbv infection. all patients in the treatment group treated by lamivudine (lam) with or without adefovir dipivoxil (adv), while patients in control group received no antiviral treatment. at -month postoperation, the treatment group also had a significantly greater increase in residual liver volume per unit surface area following hepatectomy ( . ± . cm /m vs. . ± . cm / m ). the os rate was a significant difference between two groups. the os rates of -and -year were . % and %, respectively, for the control group, and . % and . %, respectively, for the treatment group. these results revealed that antiviral therapy with nas effectively relieved hbv-induced liver damage, improved liver function, promoted hepatocyte regeneration, and increased volume of residual liver, thus enhancing tolerance to subsequent therapy. there were no serious adverse effects to lam therapy in this study. however, the most significant problem associated with long-term therapy with lam is emergence of resistance. in this study, the emergence of ymdd mutants was observed in of patients in the lam group. administration of adv successfully controlled the virus. in a meta-analysis, wong et al. [ ] assessed whether anti-viral therapy with nas could prevent hbv-hcc patients from tumor recurrence after curative treatment. a total number of patients from cohort studies were included: patients without antiviral treatment (control group) and patients with antiviral treatment group. lam was the primary antiviral therapy in the majority of patients. patients with lam resistance was treated by either switching to entecavir (etv) or adding adv therapy. thirteen patients received etv as primary antiviral therapy. most of the antiviral therapies were started after the curative treatment of hcc. the recurrence rate of hcc in the antiviral treatment group was significant lower compared to control group ( % and %; p = . ). in the antiviral treatment group the risk of hcc was reduced by %. there were also significant differences in favour of antiviral treatment group in terms of overall mortality ( % vs. %; p < . ) and liver-related mortality ( % vs. %; p = . ). hcc patients with anti-viral therapy after curative treatment may be reduced the risk of hcc recurrence for %. besides, antiviral therapy significantly improved os, as overall mortality was reduced by %. after curative treatment of hcc, patients should be monitored regularly concerning their viral status for consideration of antiviral therapy. antiviral therapy was beneficial as it not only might reduce hcc recurrence and liver failure secondary to reactivation of hbv due to viral suppression ( % reduction in the mortality secondary to liver failure in the antiviral therapy group). after ablation, the use of ifn or nas can reduce the recurrence of hcc, improve liver function, thus enhancing tolerance to subsequent therapy and prolong the survival time of the patients. recurrence in patients with hbv-hcc after ablation was common. chung et al. [ ] assessed the correlation between viral load and intrahepatic recurrence and predictors of intrahepatic recurrence after percutaneous ablation. hbv-hcc patients undergoing percutaneous ethanol injection (pei) or radiofrequency ablation (rfa), between . and . were prospectively enrolled. a total of patients (mean age, . years; male, . %) were included. ninety patients ( . %) had serum hbv dna ≥ iu/ml. the median followup duration was . months (range, . - . ) and . % patients (n = ) experienced intrahepatic tumor recurrence. on multivariate analysis, hbeag(+) was an independent risk predictor of late recurrence (≥ year) (p = . ; hr . ) and intrahepatic recurrence (p = . ; hr . ). the afp level also significantly predicted late recurrence (p = . ; hr, . ). however, neither serum hbv dna titers nor the ablation method were correlated with intrahepatic recurrence. xia et al. [ ] conducted a study to investigate the risk factors of recurrence in patients with hbv-hcc after rfa. all patients with small hcc enrolled in this study. in patients the intrahepatic recurrence rate was . % by median follow-up of months. on univariate analysis, meld score, afp, hbv dna, precollagen iii, and hyaluronic acid were independent risk factors for recurrence. on multivariate analysis, hyaluronic acid and hbv dna were independent risk factors for recurrence. the cumulative -, -, and -year dfs rates were . %, . %, and . % in the high hbv dna group (> × copies/ml) and . %, . %, and . % in the low hbv dna group (≤ × copies/ml), respectively. that showed significant difference between the two groups (p = . ). goto et al. [ ] reported the similar results that serum hbv dna load (> . log copies/ml) were associated with the recurrence. thus reasonable antiviral therapy can improve liver function and prevent the recurrence of the tumor. lin et al. [ ] assessed the impact of ifn-α in preventing hcc recurrence. thirty eligible patients were randomized into three groups: ifn-α-continuous group (n = , ifn-α mu tiw for months), ifn-α-intermittent group (n = , ifn-α mu daily for days every month for months followed by mu daily for days every months for a further months), and control group (n = , no ifn-α therapy). the three groups were comparable in terms of demographics, laboratory data, and etiology at entry and hcc characteristics. after a median follow-up of months, % patients (n = ) in the control group and % patients (n = ) in treatment groups ( patients in the ifn-α-intermittent group and patients in the ifn-α-continuous group) developed an hcc recurrence (p = . ). cumulative hcc recurrence rates in the control groups ifn-α-intermittent, ifn-α-continuous, and were %, . %, and . % at the end of year and %, . %, and . % at the end of years (p = . ), respectively (control vs. ifn-α-continuous group, p = . ; vs. ifn-α-intermittent group, p = . ). the cumulative hcc recurrence rate of the patients treated with ifn-α and the control group was % and % at the end of year and % and % at the end of years, respectively (p = . ) if both ifn-α groups were combined. the conclusion was that hcc recurrence may be reduced by ifn-α therapy after medical ablation therapy for primary tumors. antiviral, anti-tumor and anti-angiogenesis effect of inf can effectively resist the risk factors of recurrence after ablation. some patients do not tolerate the adverse reaction of ifn, still should be treated with nas to inhibit viral replication, relieve the liver inflammation, improve liver function, enhancing tolerance to subsequent repeated ablation. yoshida et al. [ ] evaluated the efficacy of lam in hbv-hcc patients who were treated with rfa. complete ablation of hcc was achieved in patients in this study. after rfa, patients was treated by lam. there were ( %) patients with serum hbv-dna negative conversion. four patients showed alt elevation and redetection of hbv-dna. there was no difference in recurrence-free survival and overall survival between the two groups. in the lam group no specific adverse effect was observed. the conclusion was that lam for patients with hbv-hcc after rfa was safe and liver function was improved. kuzuya et al. [ ] evaluated the impact of antiviral therapy with lam on patients after initial treatment for hbv-hcc. comparison was made between patients who did not received lam therapy after treatment for hcc (control group) and patients who received at a dose of mg/day (lam group) in terms of changes in survival, hcc recurrence, and remnant liver function. there was no significant difference in cumulative recurrence rates of hcc between the two groups (p = . ). however, median ctp score at the time of hcc recurrence was significantly different; (range - ) in the control group versus (range - ) in the lam group (p = . ). in the lam group, all patients were able to receive curative treatment for recurrent hcc. in contrast in the control group, of patients were unable to receive curative optimal therapy for recurrent hcc due to deterioration of remnant liver function. in the lam group, the cumulative survival rates of patients tended to be higher than those of patients in the control group (p = . ). the conclusion is that lam therapy is beneficial for hbv-hcc patients after initial treatment because it contributes to improving remnant liver function, accordingly decreasing the probability of liver failure and increasing the possibility to receive available treatment for recurrent hcc. clinical evidence showed hbv reactivation may occur in chronic hbv carriers with tumor during chemotherapy, then followed by hepatic decompensation and various complications. hbv reactivation occurs in nearly between % and % [ ] [ ] [ ] . similarly, hbv reactivation may occur in patients with hbv-hcc after tace. some of these patients even treated with lam still occur hepatic decompensation or liver failure and eventually death, because of the delay of lam antiviral therapy, suggesting that these patients need to be treated by antiviral drugs before tace. moreover, more studies [ ] [ ] [ ] also suggested that before chemotherapy early antiviral therapy can significantly reduce the chemotherapy-induced reactivation of hbv. tace is local treatment, different from systemic chemotherapy. therefore, early antiviral treatment before tace in patients with hbv-hcc can reduce the occurrence of postoperative virus reactivation, reduce the hepatitis flare caused by hbv, and reduce the mortality of acute exacerbation of chb. in , a study about reactivation of hbv in patients with hbv-hcc undergoing tace of jang et al. [ ] was published in hepatology. in a prospective and randomized study, consecutive patients with hbv-hcc undergoing tace (cisplatin mg/m and epirubicin mg/m ) at monthly intervals were assigned to receive lam mg daily from the start of tace (preemptive group) or not (control group). during the study, . % patients ( / ) in the preemptive group and . % patients ( / ) in the control group developed hepatitis due to hbv reactivation (p = . ). in addition, there were significantly more incidences of severe grade of hepatitis (p = . ) and overall hepatitis (p = . ) in the control group. on multivariate analysis, hbv dna level > copies/ml in baseline was the only independent predictor of hepatitis due to hbv reactivation during chemo-lipiodolization (p = . ). these data demonstrated preemptive lam therapy effectively reduced hepatitis due to hbv reactivation and hepatic morbidity during tace. preemptive therapy should be considered in hcc patients with an hbv dna level > copies/ml. preemptive antiviral therapy would effectively reduce liver-related morbidity attributable to hbv reactivation and would allow more prolonged chemotherapy. this study also suggested that preemptive lam therapy decreases the severity of clinical hepatitis if it develops during tace. zhu et al. [ ] investigated the efficacy of adjuvant tace with or without antiviral therapy for hbv-hcc patients after radical hepatectomy. this study enrolled patients, of whom were treated using tace combined with antiviral therapy and using tace alone. analysis of all patients suggested that overall survival of the combination therapy group was better compared to the tace-only group (p = . ), while disease free survival was similar between the two groups (p = . ). analysis of only propensity score-matched pairs proved -year overall survival in the combination therapy group was significantly better ( . % vs. . %, p = . ) and also suggested better -year disease free survival ( . % vs. . %, p = . ). for patients after hcc recurrence, radical hepatectomy was the treatment choice for a significantly larger proportion of patients from the combination therapy group than from the tace-only group (p = . ). these data suggested that combining tace with antiviral therapy significantly improved overall survival and potentially disease free survival relative to tace alone in hbv-hcc patients. combination tace with antiviral therapy also improves remnant liver function, increasing the chance of curative resection in case of tumor recurrence. combination tace with antiviral therapy may benefit to prevent recurrence of hcc after radical hepatectomy. it has been observed that hbv reactivation occurs after the end of radiotherapy in a way similar to that after cytotoxic chemotherapy [ ] . radiotherapy to hcc can damage immune system, and cause leukocytes decreased, following by hbv reactivation. so antiviral therapy before radiotherapy for hbv dna positive patients is necessary. kim et al. [ ] evaluated the impact of three-dimensional conformal radiotherapy ( d-crt) on hbv reactivation and hepatitis exacerbation in hbv-hcc patients. this study enrolled hbv-hcc patients who underwent d-crt to the liver. group (n = ) treated lam before and during d-crt and group (n = ) did not treat with nas before d-crt. to investigate spontaneous hbv reactivation, hcc patients received no specific treatment for chb or hcc were included as a control group. the cumulative rate of radiation-induced liver disease in groups was higher than group ( . % vs. . %, p > . ). the cumulative rate of hbv reactivation was significantly higher in group ( . %) than in group ( %) or the control group ( . %) (p < . each). there was no significant difference in cumulative rate of chb exacerbation between groups ( %) and ( . %) or the control group ( . %) (p > . each). that demonstrated that hbv reactivation and consequent chb exacerbation should be considered in hbv-hcc patients after d-crt and antiviral therapy should be recommended to prevent liver function after rt. in study of huang et al. [ ] , the rate of hbv reactivation and chb exacerbation was . % ( / ) and . % ( / ), respectively. there was a relatively high rate of hbv reactivation in those patients and whose prognosis was unfavorable. the serum hbv dna level and some dosimetric parameters (normal liver volume, v , and dmean) were the prognosis factors for hbv reactivation and should been considered carefully before crt. the goal of anti-hbv therapy is to effectively reduce the hbv dna level, thereby reduce the incidence of cirrhosis and hcc. although antiviral therapy is recommended in guidelines from apasl, easl and aasld, the specific procedures are not the same. and these guidelines do not give treatment recommendations for patients with hbv-hcc. current clinical studies have confirmed that early antiviral therapy is necessary for the prevention of liver function and reduce the integration of the viral dna. although antiviral therapy inhibits viral replication, the integration of viral dna continued. there are two distinct types of hcc recurrence: tumors grown from dissemination of the primary tumor and de novo tumors arising from the "field effect" in diseased liver [ , ] . this may argue for an earlier antiviral intervention, as adjuvant therapy after the resection for the hcc patients with a high hbv dna level to prevent recurrence. anti-hbv therapy were performed in the light of the recently updated hbv treatment guidelines, on the recurrence and prognosis of hcc. to substantiate the beneficial effects of antiviral therapies, future randomized clinical trials (rcts) with longer follow-up, larger sample size, and regular hbv dna level monitoring will be needed to conduct. the molecular mechanisms of preventing tumor recurrence also need to be further studied. jun-ying qi and ming ni liver transplant is an effective treatment for hbv-related end-stage liver disease. the risk of hbv reinfection after liver transplant is the main limiting factor for long-term survival rate. combination therapy of lamivudine and hepatitis b immunoglobulin (hbig) reduced the rate of recurrence. however, considering the disadvantages of high dose hbig and high rate of lamivudine resistance, other therapies that composed by entecavir, tenofovir, or lamivudine plus adefovir, with or without hbig have been used in several liver transplant centers. other researchers have used posttransplant hbv vaccination for achieving a lasting endogenous anti-hbs antibody, yet the efficacy is still controversial. the combination hbig/nucleotide (acid) prophylaxis should be converted to oral prophylaxis within or years after liver transplantation. recently, the discovery of sodium taurocholate co-transporting polypeptide (ntcp) as the cellular receptor for hbv entry has opened up many channels of investigation, which indicate the possibility of using ntcp inhibitor in the prophylaxis of hepatitis b recurrence post lt. liver transplant (lt) is an effective treatment for hepatitis b virus (hbv)-related end-stage liver disease (such as acute or chronic liver failure, cirrhosis, hepatocellular carcinoma and so on). china has become the world's second-largest lt country as there are nearly operations annually. until the end of , there were , cases of liver transplant had been completed in china, % of which were due to hepatitis b-related liver disease, and nearly % recipients with detectable hbv dna. the risk of hbv reinfection after lt is the main limiting factor for long-term survival rate. the rate of hbv reinfection is as high as % without antiviral prophylaxis [ ] . lt recipients with recurrent hepatitis b develop an aggressive form of fibrosing cholestatic hepatitis, cirrhosis or graft failure within years [ , ] , which lead to death or re-lt. combined treatment of hepatitis b immunoglobulin (hbig) and nucleos(t)ide analogues (nas) reduced the hbv recurrence rate to - % after years of liver transplantation [ ] [ ] [ ] [ ] [ ] . [ ] . another problem in patients with hbv disease post lt is the presence of extrahepatic reservoirs of the virus that are continuous latent source of hbv recurrence [ ] . on the other hand, late recurrence is related to low anti-hbs titer or the development of hbs viral escape mutations or ymdd mutations [ ] . the strategies to prevent hbv reinfection after lt involve three stages: pre-, at and post-transplant. currently, the strategies include passive immunization (hbig), antiviral therapy (nas) and active immunization (hepatitis b vaccine). hbig was the first drug to effectively prevent hbv recurrence. limited duration of hbig therapy (< months) [ , iu iv at lt, , iu iv daily for days after lt, then iv at different intervals to maintain anti-hbs titers > iu/l] delayed but did not prevent hbv reinfection [ ] . the efficacy of this treatment seemed to be dependent on the viral load pretransplant. there was % developed recurrent hepatitis b years after transplant in patients with detectable hbv dna in serum. the recurrent rate were % in patients who were hbv dna negative pretransplant [ ] . this problem was partially resolved by using higher doses of hbig. monthly fixed doses of , iu of hbig (to keep anti-hbs levels > iu/l) or different hbig doses adjusted to maintain anti-hbs > iu/l for the first months after liver transplant significantly reduced the rate of recurrence in patients with detectable hbv dna pretransplant [ ] [ ] [ ] . however, using high doses of hbig was very expensive. lamivudine had apparent effect on hbv dna replication, decreasing the positive rate of hbv dna in patients undergoing or waiting for liver transplantation. data from north american transplant centers showed that treatment with lamivudine improved pre-liver transplantation and liver transplantation-free survival of patients awaiting liver transplantation for hbv-related cirrhosis [ ] . the early results of monotherapy of using lamivudine to prevent hbv recurrence post-lt were promising. in nine of ten survivors, hbsag and hbv dna were negative, and liver biopsy showed no evidence of recurrent by week [ ] . however, % patients re-infected with lamivudine-resistant hbv by - months post-transplant [ ] . hbig and lamivudine are different in action mechanisms. hbig is a specific passive immune agents prepared from individual plasma who has been infected by hbv or injected hepatitis b vaccine. high concentration of hbig can neutralize hbv and block its infection of hepatocytes. lamivudine is a potent inhibitor of hbv-associated dna polymerase to block hbv replication. therefore hbig and lamivudine play a complementary role to each other. combined treatment of high dose iv hbig and lamivudine had been the accepted standard prophylaxis for post-lt hbv recurrence. lamivudine was used pre-lt for reducing the viral load in the peri-lt period in most centers. hbig was used at a dose of , iu daily for the first week post-operative and then either at a fixed dose of , iu/month or with different doses to keep anti-hbs titers > iu/l [ , , [ ] [ ] [ ] . some centers had targeted anti-hbs titers > iu/l in hbv dna positive patients for - months post-lt. compared to the monotherapy of hbig or lamivudine, these combined treatments are highly effective [ , ] . however, the long-term use of hbig has many disadvantages, such as high cost, the need for injection, headache, flushing, and chest pain [ , ] . moreover, the long-term use of lamivudine induces viral resistance, which leads to a high rate of recurrence post-lt [ ] . a number of studies have shown that im hbig has similar kinetics and produces roughly equivalent trough concentrations of anti-hbs compared to iv equivalent doses of hbig but less expensive [ ] . the largest reported data of prophylaxis with using of im hbig comes from investigators in australia and new zealand [ ] . im or iu hbig daily for week from transplantation and monthly thereafter. lamivudine, mg/day, was administered to candidates waiting for transplantation with hepatitis b surface antigen (hbsag)-positive and continued posttransplantation. thirty-seven patients with positive hbsag ( patients had hepatitis b, patients had hepatitis b and c, and patient had hepatitis b, c, and d) underwent liver transplantation using this protocol. thirty-six patients were hbv dna positive. the therapy had no significant adverse events and was well tolerated. all patients were hbv dna negative and patients were hbsag negative at latest follow-up. this investigation suggested that low-dose hbig combined with lamivudine prevented recurrence of hepatitis b posttransplantation is more cost-effective. long-term results of this protocol showed that the actual rate of hbv recurrence at years was % in hbsag-positive patients underwent liver transplantation [ ] . recently, entecavir and tenofovir have been approved as the first-line regimen for the treatment of chronic hepatitis b. these new nas have replaced lamivudine as the prophylaxis of hbv recurrence post lt. according to easl clinical practice guidelines, to achieve the lowest level of hbv dna pre-lt, nas with high barrier to resistance is recommended as pre-transplant antiviral therapy for all hbsag positive patients undergoing liver transplantation [ ] . hu et al [ ] reported a lower hepatitis b recurrence rate in patients received entecavir than those received lamivudine. a total of patients were administered entecavir combined with lowdose hbig, and patients received lamivudine plus low-dose hbig in the control group. two patients in the entecavir group developed hbv recurrence with no evidence of viral resistance in the median months follow-up time. eleven patients in the lamivudine group developed hbv recurrence, three of whom were proved hbv resistance in the median months follow-up period. further analysis demonstrated that hcc at the time of liver transplantation and low anti-hbs titer post-liver transplantation were independent risk factors for hbv recurrence. perrillo et al. [ ] investigated the efficacy of entecavir combined with various hbig regimens after liver transplantation. sixty-one patients with hbv-related liver disease took . mg/day of entecavir plus various hbig regimens. in the median weeks follow-up period, only patients showed positivity hbsag but hbv dna remained undetected. na et al. [ ] showed that of recipients who received entecavir plus hbig experienced hbv recurrence after liver transplantation in the median months follow-up time. among these patients, had received lamivudine followed by entecavir. studies concerning the efficacy of tenofovir in the prophylaxis of hbv reinfection post-lt are limited. jiménez-pérez et al. [ ] reported that four patients received tenofovir plus hbig with or without entecavir for the prophylaxis of hepatitis b recurrence. no hepatitis b recurrence was observed in these four patients at months. several researchers have investigated if it was possible to stop hbig after an initial successful prophylaxis with combined hbig/lamivudine. in one largest prospective study, patients who were hbvdna negative before liver transplantation received prophylaxis with hbig/lamivudine for month after transplantation, then they were randomized to continue combination prophylaxis or lamivudine monotherapy [ ] . the early results showed that there was no recurrence case in either group by months. however, - % of the patients who were converted to lamivudine monotherapy had viral breakthrough because of lamivudine resistance in longer follow-up [ ] . an alternative choice was to change from hbig/lamivudine to a combination of antiviral drugs had a higher barrier of resistance than lamivudine. several studies indicated that this method may be more effective [ , ] . in a prospective study, of patients receiving prophylaxis with low-dose im hbig/ lamivudine months post-lt were changed to adefovir/lamivudine combination therapy, whereas the other patients continued previous prophylaxis [ ] . at a median of months post-switch, patients in both group had no recurrence. one a low titer of hbsag in serum was detected in patient in the adefovir/lamivudine group but hbv dna was negative. this change in therapy improved the life quality of patients and significantly saved the cost. more recently, a multicenter, prospective study demonstrated the results of hbig-sparing regimen combined with lamivudine plus adefovir dipivoxil initiated at the time of waiting for liver transplantation and continued post-transplantation [ ] . twenty-six patients were recruited into this study at the time of listing for transplantation. twelve patients had lam exposure before the study, but none had lamivudine resistance. to the patients who underwent transplantation, iu/day of intramuscular hbig was given immediately after transplantation for days. all transplanted patients remained alive without hbv recurrence at a median of months after transplantation. after the completion of this prospective study, the regimen was modified that no perioperative hbig was administered if the pretransplant hbv dna level < log( ) iu/ml. another patients with hbv-related liver disease underwent transplantation ( without hbig). all the patients remained alive without hbv recurrence at a median of months post-transplantation. this study indicated that combination of lamivudine and adefovir initiated at the time of listing for transplantation was safe and effective prevention of hbv recurrent without high costs and long-term hbig therapy. other researchers used posttransplant hbv vaccination for achieving a durable endogenous anti-hbs antibody. two studies reported that - % of patients achieved an anti-hbs titer > iu/l following cessation of hbig and immunization with - courses of recombinant im hbv vaccine [ , ] . however, other investigations using the same protocol have failed to replicate these results [ , ] . moreover, the low response ( - %) was reported in cirrhotic patients awaiting for lt [ ] . more recently, di paolo et al investigated the efficacy of year, monthly vaccination together with hbig plus lamivudine in lt patients. one year after vaccination, . % patients maintained anti-hbs titers more than iu/l [ ] . these results suggested that hbig can be considered as an additional strategy in the prophylaxis against hbv recurrence post lt: ( ) vaccine administration should be long-lasting (e.g. year); ( ) passive prophylaxis with hbig should preferably be maintained during the initial phase of vaccination and nas should be maintained during the entire vaccination period. lamivudine is the most widely used na to prevent hepatitis b recurrence. however, lamivudine resistance can result in hepatic decompensation and increases the rate of post-transplant recurrence. newer nas with lower resistance rates should therefore replace lamivudine in hepatitis b prophylaxis. schiff et al [ ] investigated the effect of adefovir dipivoxil as the rescue therapy in listing or post-lt patients with chronic hepatitis b and lamivudine-resistance. among listing patients, the percentage of hbv dna levels undetectable at weeks and was % and %, respectively. after weeks, liver function normalized in % and % of these patients respectively. and coagulation function normalized in % and % of these patients respectively. among post-transplantation patients, the percentage of serum hbv dna levels undetectable at weeks and was % and %, respectively. after weeks, liver function and coagulation function normalized in %, %, %, and % of these patients, respectively. among listing patients who underwent liver transplantation, prevention of graft reinfection over a median of weeks was similar among patients who did or did not receive hbig. hbsag was detected on the first test only in % and % of patients who did or did not receive hbig, respectively. serum hbv dna was detected on follow-up in % and % of patients who did or did not receive hbig, respectively. adefovir dipivoxilrelated adverse events occurred in % of patients and led to drug withdrawal. cumulative resistance rate were %, %, and % at , , and weeks, respectively. in conclusion, adefovir dipivoxil is safe and effective in prevention of graft reinfection with or without hbig for listing or post-transplant chb patients with lamivudine-resistance. more recently, one study indicated that late hbig replaced by adefovir dipivoxil (at least months post-transplant) can prevent late hbv recurrence at less cost [ ] . in a prospective open-labeled study, lamivudine plus adefovir dipivoxil given from the time of listing was well tolerated, prevented lamivudine resistance pre-transplantation and post-transplantation, regardless of the baseline hbv-dna level [ ] . the rescue therapy for patients with lamivudine or telbivudine resistance is to add adefovir or tenofovir, or change to tenofovir + emtricitabine. for patients with adefovir resistance, the approach is to add lamivudine or entecavir, or switch to tenofovir + emtricitabine. for patients with entecavir resistance, the approach is to add adefovir or tenofovir. combination therapy is still effective for some patients with multi-antiviral drugs-resistance according to evidence based medicine and clinical practice [ ] . regular monitoring and follow-up for patients post lt is also very important. items include liver function, hepatitis b markers, hbv dna quantitative, mutant, blood concentration of immunosuppressive drug and ultrasound examination. for hepatitis b recurrence patients, therapy include: support treatment, hepatocyte protection, anti hbv therapy, immunosuppressant regimen adjustment (withdrawal, reduction or change immunosuppressive agents) and liver retransplant. hepatitis b is a major cause of liver failure in asia, although the use of hbig plus lamivudine can effectively prevent hbv reinfection in liver transplantation, but the cost is high. active immunization approach is still controversial. combined hbig/ nucleos(t)ide prophylaxis should be considered to switch to oral prophylaxis at or years post-lt, particularly in patients with low hbv dna loads before antiviral therapy or hbv dna negative at lt, and in patients with liver failure due to hbv or hdv coinfection, since these patients are at lower risk of recurrence once hbig is ceased. recently, the seminal discovery of sodium taurocholate co-transporting polypeptide (ntcp) as the cellular receptor for hbv entry has opened up many channels of investigation, making hbv entry amenable to therapeutic intervention. several fda approved drugs with ntcp inhibiting activity were tested for their ability to inhibit hbv infection of the cell line [ ] [ ] [ ] . these investigations indicate the possibility of using ntcp inhibitor in the prophylaxis of hepatitis b recurrence post lt. di wu and qin ning both host and viral factors are associated with the chronicity of hbv infection. hbv has a capability of escaping the host immune responses. more importantly, the hbv genome forms a stable minichromosome, namely covalently closed circular dna (cccdna), in the nuclei of infected hepatocytes, enabling hbv to persist its infection [ ] . the goal of anti-hbv therapy is to prevent the progression of hbv-related liver disease, which may be achieved initially through sustained immunologic control over hbv, and ultimately, by completely eliminating the virus [ , , ] . however, due to the fact that hbv cccdna persists stably at a very low level even after the loss of hbsag in chronic infected patients, elimination of hbv (complete cure) is rarely achieved. it is suggested that serum hbsag could represent a surrogate marker of intrahepatic cccdna and a marker of host immune control of hbv infection. seroclearance of hbsag is found to be associated with functional remission and improved long-term clinical outcomes in patients with chronic hepatitis b, under this circumstance, even though hbv genome cannot be cleared, the host immune system is in general sufficient to control the few persisting infected hepatocytes [ ] [ ] [ ] . therefore, hbsag seroclearance with or without the appearance of hbsab (functional cure) is considered the ideal endpoint for anti-hbv therapy, representing durable immunologic control over the virus and complete suppression of hbv replication, which is the critical step towards complete cure for hepatitis b [ , , ] . nuc and ifn or its pegylated form, peg-ifn, are the only two types of approved antiviral therapeutics. as the ideal endpoint for anti-hbv treatment, hbsag loss is achieved in very few patients after long-term nuc or -week courses of peg-ifn therapy [ ] [ ] [ ] . these current standard antiviral therapies can only suppress the hbv replication and viral protein production, but cannot eliminate hbv cccdna and cure chronic hbv infection. therefore, new treatment approaches such as optimal combination therapy with the approved antivirals or emerging novel therapeutics are needed to improve rates of hbsag loss and, ideally, hbsag seroconversion. different characteristics, mechanisms of action and antiviral activities of nuc and ifn provide the possibility of combining these two types of agents for improving chances of sustained post-treatment response, thereby allowing the discontinuation of nuc without virus relapse, through harnessing both immunomodulatory and direct antiviral mechanisms [ , ] . according to the updated chinese guidelines, asian-pacific guidelines, as well as european guidelines for the treatment of chronic hepatitis b, sequential therapy with additional peg-ifn or switching to peg-ifn can be considered in chb patients who have achieved virological remission by long-term nuc treatment [ ] , though clinical trials evaluating either simultaneous or sequential combination therapy with nuc and ifn for chb patients drew different conclusions. several previous studies exploring the efficacy of simultaneous combination with peg-ifn and lam or adv have demonstrated that the therapeutic strategy led to higher rates of virological response during treatment, but did not improve durable post-treatment responses [ , , , ]. an exploratory study showed that combination treatment with peg-ifn plus adv for weeks led to remarkable decline in serum hbv dna level and intrahepatic cccdna, which was significantly correlated with reduced serum hbsag [ ] . a recent study evaluating the efficacy of combination therapy with ldt and peg-ifn in hbeag-positive chb patients have demonstrated that the combination therapy led to greater reductions in hbv dna and hbsag levels, however, it may contribute to an increased risk of unexpected severe peripheral neuropathy, combination therapy with ldt and peg-ifn should not be used [ ] . in a prospective, active-controlled randomized trial evaluated loss of hbsag in patients receiving the combination of tdf and peg-ifn for a finite duration, chb patients were randomly assigned to receive combination therapy for weeks, combination therapy for weeks followed by tdf for weeks, tdf for weeks, or peg-ifn for weeks. the study demonstrated that, . % of patients receiving -week course of combination therapy with tdf and peg-ifn had hbsag loss, which was significantly higher than those receiving tdf or peg-ifn given alone [ ] . however, it is worth noting that a prolonged followup of these subgroups of patients is required to determine the durability of treatment response and long-term benefits. although simultaneous combination of peg-ifn and nuc other than tdf may not improve sustained response rate, the optimal approach for combination treatment remains to be determine and should take into consideration the time schedule of drug administration. late breaking clinical trials have demonstrated that sequential combination therapy with nuc and ifn, either "switch" or "add-on", showed more promising results, with higher probabilities of hbeag seroconversion and hbsag loss than nuc monotherapy. an observation study has shown that the add-on of peg-ifn to a stable nuc therapy in chb patients with suppression of hbv dna, induced hbsag seroconversion in out of patients [ ] . a prospective study demonstrated that additional of peg-ifn to a long-term nuc treatment in hbeag-negative patients with undetectable hbv dna, led to a durable hbsag loss in out of patients [ ] . a global multicentered, randomized controlled trial (ares study) assessed the effectiveness of add-on peg-ifn to etv therapy in hbeag positive patients, compared to etv monotherapy, weeks of peg-ifn add-on therapy did not improve response rates (defined as hbeag loss with hbv dna < iu/ml at week ), but led to greater viral decline and appeared to prevent relapse after stopping etv, which may facilitate the discontinuation of nuc treatment [ ] . another randomized controlled trial has shown that neither etv pretreatment nor etv add-on to peg-ifn demonstrated superiority in sustained posttreatment response compared with weeks of peg-ifn alfa- a monotherapy in treatmentnaive hbeag-positive patients [ ] . a prospective, randomized controlled trial (osst study) reported that switching to -week course of peg-ifn in hbeag positive chb patients who achieved virologic remission after long-term etv treatment led to significantly increased rates of hbeag seroconversion and hbsag loss ( . %) [ ] . data from -year follow-up of these patients who received sequential therapy showed that rates of hbeag seroconversion increased from . % at the end of treatment to . % at -year post-treatment, besides, hbsag loss was durable in of patients [ ] . these results are in consistent with findings from earlier studies exploring sequential combination therapy with nuc and ifn but only tested in a small number of patients [ , ] . an exploratory study assessed the efficacy of sequential therapy in genotypes a, b, c and e chb patients with high hbv viremia, patients receiving etv alone for weeks, followed by etv plus peg-ifn for weeks, lastly only peg-ifn for weeks achieved significantly higher rates of hbeag and hbsag seroconversion than those receiving peg-ifn monotherapy for weeks [ ] . an interim analysis from new switch study demonstrated that sequential combination therapy with etv and peg-ifn for weeks in nuc-experienced hbeag positive chb patients who achieved partial responses, with hbv dna suppression and hbeag loss, led to a high rate of hbsag loss ( . %) [ ] . accumulating evidences suggest that quantitative hbsag (qhbsag) is a useful marker for guiding treatment decision, e.g. individualizing the treatment, implementing stopping rules for ending or extending ifn treatment [ ] . recently, several studies identified that hbsag loss occurred in patients with a low baseline qhbsag and high on-treatment reduction, therefore, a baseline or response-guided approach based on hbsag kinetics may help identify chb patients with the greatest chance of benefit. the osst study has demonstrated that patients undergoing long-term etv treatment with low hbsag titers (< iu/ml) and hbeag loss were suitable for sequential therapy with peg-ifn as they had a good chance of both hbsag loss ( . %) and hbeag seroconversion ( . %). patients whose hbsag levels declined to iu/ml at week of sequential therapy have the greatest chance of achieving hbsag loss. while, patients whose hbsag levels were > iu/ml at week might consider stopping peg-ifn treatment as they had a minimal chance of achieving hbeag seroconversion and hbsag loss [ ] . these findings are consistent with results from previous studies and interim analyses from the new switch study, suggesting that qhbsag identifies nuc-treated patients who are the best candidates for sequential therapy, and allows response-guided treatment [ ] [ ] [ ] . however, given the small number of patients included in the exploratory analyses, these results need to be interpreted cautiously and warrant further investigation and validation. differences in study designs and characteristics of patients make it difficult to determine the optimal combination therapy with nuc and peg-ifn for chb patients at this stage. nevertheless, we could speculate that once suppression of hbv viremia has been achieved by pretreatment with nuc, the additional use of peg-ifn would be more beneficial. these new therapeutic strategies require further investigation before being introduced into routine clinical practice. complete cure of hbv infection depends not only on the deep suppression of hbv replication, but also on the induction of durable antiviral immune response [ ] . besides the approved therapeutics, several novel therapeutic approaches including direct acting antivirals (daa) targeting different stages of the life cycle of hbv (including hbv entry, hbv genome processing, virus protein assembly, etc.) as well as immunological approaches are currently under early stage of preclinical or clinical investigation, these promising therapeutics may act in a synergistic way with currently available antiviral agents and have the potential to achieve a cure of hbv infection. specific inhibition of hbv entry may be a promising therapeutic concept to control hbv infection. a currently identified cellular receptor for hbv entry, the sodium taurocholate co-transporting polypeptide (ntcp), is an emerging target providing new research possibilities and allowing the development of hbv entry inhibitors [ ] . cyclosporine a (csa) can interfere with the binding between large envelope protein and ntcp, and thus prevent hbv entry into cultured hepatocytes [ , ] . myrcludex-b also can inhibit the binding of the hbv envelope proteins to ntcp, blocking the hbv/hepatitis d virus (hdv)'s entry, which is now under clinical development [ ] . however, these entry inhibitors can prevent new hbv infection [ , ] , but do not directly target on cccdna or eliminate the preexisting hbv infection. therefore, antiviral strategies combining entry inhibitors with anti-hbv agents might be superior to their use as monotherapy by taking advantage of synergy. therapeutic approaches targeting cccdna for hbv cure aim to directly degrade or alternatively block cccdna formation, or silence cccdna transcription. rnaguided nucleases, such as the clustered regularly interspaced short palindromic repeats (crispr)/cas [ , ] , might be the most promising strategy to target cccdna. however, the risk of undesired off-target mutagenesis and delivery constitute the major limits. histone modifications, e.g. inhibitors of histone acetyltransferase, offer great potential as therapeutic candidates for chb patients through transcriptional silencing of cccdna [ , ] . activation of ifn-a and lymphotoxin beta receptor (ltβr) has been shown to induce cytidine deaminases of the apobec family, triggering degradation of cccdna in hbv cell culture model systems. these novel strategies will make elimination of hbv a real possibility [ ] . several attempts have been made to develop capsid assembly modulators/core inhibitors, which can be divided into two main classes. the first class, including phenylpropenamides (ppas) and sulfamoylbenzamide derivatives, e.g. at- and at- , can inhibit the entry of pregenomic rna (pgrna) into the immature nucleocapsid [ , ] resulting in nucleocapsid with normal size and geometry but empty of nucleic acid. the other class, including heteroaryldihydropyrimidines (haps) antiviral compounds, e.g. bay - [ ] and nvr - , can directly inhibit the nucleocapsid formation, resulting in virus particles with abnormal size and structure. ppas and haps show synergy in vitro with nucleoside reverse transcriptase inhibitors (nrtis) and overcome resistance to nrti [ , ] , highlighting the value for developing combination therapy. post-transcriptional gene silencing by rna interference (rnai), is a new therapeutic approach to hepatitis b [ , ] . inhibiting protein production by targeting hbv messenger rna (mrna) for translational repression or degradation can impair virus replication and augment the hbv-specific immune response. several rnai-based drug candidates have currently entered early-phase clinical development for the treatment of chb, including aln-hbv and tkm-hbv [ , ] , showing clinical efficacy significant declines in hbv dna, hbsag, and cccdna levels. despite lingering concerns about delivery, the risk of resistance and safety, the rnai-based therapeutics might be promising when combined with other antiviral agents. future trials with rnai-based therapeutics in combination with ifn or other antivirals are required to determine whether these agents would act synergistically to reduce viral antigen production, activate and restore the host immune responses, and subsequently eliminate hbv infection. hbsag production and secretion is capable of altering the host immune response by inducing t cell exhaustion and tolerance to hbv, which partially mediate hbv persistence. control of hbsag secretion may help restore t cell function, suggesting the possibility of developing anti-hbv treatments targeting hbsag production and release. nucleic acid polymer (nap) could prevent hbsag release from infected hepatocytes, leading to a restoration of the immune response. newly developed hbsag release inhibitors, e.g., rep and rep [ ] [ ] [ ] , appear potent in preventing the release of hbsag in humans and thereby reducing serum hbsag levels and also potentially promoting surface antibody seroconversion. however, it remains to be seen whether these compounds may cause detrimental intrahepatocyte accumulation of hbsag. emerging exciting advances have also led to new promising approaches to attenuate hbv-induced immune impairment, such as toll like receptor (tlr) agonists [ , ] , pleiotropic cytokines [ ] [ ] [ ] , programmed cell death- (pd- ) and its ligand pd-l blockages [ ] , therapeutic vaccines [ , ] , etc. tlr agonists can activate intracellular innate pathways and stimulate both innate and adaptive immune responses. a recently developed oral active agonist of tlr- , gs- , has been shown to enhance ifn-a and isg expression and activate nk cells, t cells and b cells in animal studies [ , ] , however, early human studies have shown limited efficacy of the tlr- agonist at tolerated doses, and further research into this tlr agonist was subsequently discontinued. therapeutic cytokines play critical roles in the control of hbv infection and mediate a non-cytolytic clearance of the virus [ ] [ ] [ ] . several studies investigated the antiviral activities and therapeutic potential of cytokines including granulocyte-macrophage colony-stimulating factor (gmcsf), il- , il- , etc. a previous study has shown that hbsag vaccine in combination with lam or il- could induce antiviral immune response and consequently elimination of hbv may be achieved in chb patients [ ] . a prospective study investigated whether additional gmcsf could enhanced the immunomodulatory effect of ifn, demonstrating that the combination treatment with gmcsf and ifn was effective in patients who had previously failed ifn monotherapy [ ] . a recent prospective, randomized controlled trial (anchor study) evaluated whether sequential combination therapy with nuc, peg-ifn and gmcsf could induce hbsag loss in chb patients treated with long-term nuc and demonstrated that for patients who achieved virological suppression with nuc, this sequential combination therapy significantly increases rates of hbsag loss and hbsab appearance [ ] . the difficulties in eliminating cccdna and breaking the immune tolerance constitute the major obstacles for a cure of hbv infection. 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hepatocytes through targeting a membrane transporter, sodium taurocholate cotransporting polypeptide (ntcp) the entry inhibitor myrcludex-b efficiently blocks intrahepatic virus spreading in humanized mice previously infected with hepatitis b virus a novel tricyclic polyketide, vanitaracin a, specifically inhibits the entry of hepatitis b and d viruses by targeting sodium taurocholate cotransporting polypeptide ntcp opens the door for hepatitis b virus infection targeting hepatitis b virus cccdna using crispr/ cas crispr/cas cleavage of viral dna efficiently suppresses hepatitis b virus hepatitis b virus replication is regulated by the acetylation status of hepatitis b virus cccdna-bound h and h histones specific and nonhepatotoxic degradation of nuclear hepatitis b virus cccdna phenylpropenamide derivatives at- and at- inhibit replication of wild-type and lamivudine-resistant strains of hepatitis b virus in vitro the phenylpropenamide derivative at- blocks hbv replication at the level 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receptor- , induces prolonged suppression of hepatitis b virus in chronically infected chimpanzees functional activation of nk and cd + t cells in vitro by the toll-like receptor agonist gs- clinical and immunological efficacy of intradermal vaccine plus lamivudine with or without interleukin- in patients with chronic hepatitis b efficacy of granulocyte-macrophage colonystimulating factor or lamivudine combination with recombinant interferon in non-responders to interferon in hepatitis b virus-related chronic liver disease patients control and eradication strategies of hepatitis b virus enhancing virus-specific immunity in vivo by combining therapeutic vaccination and pd-l blockade in chronic hepadnaviral infection safety, tolerability and immunogenicity of gs- , a hepatitis b virus-specific therapeutic vaccine, in healthy subjects: a randomized study tg , an immunotherapeutic to treat chronic hepatitis b, induces robust t cells and exerts an antiviral effect in hbv-persistent mice combination/ sequential therapy with etv, peg-ifn alpha- b and gmcsf enhanced hbsag loss and appearance of hbsab in na suppressed chb patients (the anchor a study): an interim analysis toward a cure for hepatitis b virus infection: combination therapy involving viral suppression and immune modulation and long-term outcome viral hepatitis. hbv cure-can we pin our hopes on immunotherapy? key: cord- - fcw ol authors: tetro, jason a. title: from hidden outbreaks to epidemic emergencies: the threat associated with neglecting emerging pathogens date: - - journal: microbes infect doi: . /j.micinf. . . sha: doc_id: cord_uid: fcw ol not all infectious disease outbreaks undergo full epidemiological investigations. in certain situations, the resultant lack of knowledge has led to the development of epidemics and public health emergencies. this review will examine six emerging pathogens including their history, present status, and potential to expand to epidemics. recommendations to improve our understanding of these hidden outbreaks and others also will be provided in the context of health systems policy. infectious disease outbreaks are common worldwide. while every occurrence deserves a proper epidemiological investigation, several constraints such as limited resources, political considerations, and an assumed familiarity with the pathogen may hinder the process [ , ] . this may result in what are known as "hidden outbreaks" in which spread is known to transpire in a localized environment (such as an endemic pathogen) but inquiries are not considered to be worthwhile in the larger context of global human health. this practice may be considered sound, however there is the potential an outbreak may expand to become an epidemic such that both caseload and costs are significantly increased [ , ] . the hidden outbreak begins no differently than isolated outbreaks with a single point-source such as a household, hospital, or restaurant. while several to dozens of people may be infected, the overall impact on society is considered to be low. yet, as seen in several instances over the last decade, certain pathogens have become international juggernauts (fig. ) . public health authorities are caught off guard, calls for alarm are made by the scientific and medical communities, and the media must find a balance between objective reporting and the inevitable contagion of fear [ ] . the quintessential example of such expansion is the ebola virus epidemic in [ ] . a small outbreak occurred in the small village of meliandou in guinea at the end of the previous year and eventually turned into a crisis in three west african nations and led to domestic cases in europe and the united states. the spread was so troubling, the world health organization declared a public health emergency of international concern (pheic) [ ] . the event eventually was controlled yet the virus was considered to be too volatile to ignore any further. any sign of ebola in the human population regardless of population size was deemed serious enough to be met with a significant response [ ] to control spread. in addition to understanding the biological nature of a pathogen as the etiology of an outbreak, an examination of the anthropogenic effects facilitating amplification to epidemic status is needed. these factors have been reviewed elsewhere [ ] and include the grouping of susceptibles in both the healthcare environment as well as the community, changes in human consumption of natural habitats, territories, and food sources, increased amount and ease of international travel, globalized trade, and political strife. in the context of expanding hidden outbreaks, two factors are considered the most important, travel and trade [ , ] . one of the best known examples of travel-related spread of a hidden outbreak into an epidemic is the sars coronavirus [ ] . the outbreak began in the small village of foshan in china and expanded to various areas of the country as well as hong kong, singapore, vietnam, and canada. although a lack of proper containment of the virus at the source may have facilitated the spread of the virus into the community [ ] , travel was deemed as the most influential anthropogenic factor in the development of this epidemic [ ] . another travel-related expansion began in the same year as sars in the province of quebec, canada. known as the toxinotype iii, north american pfge type , and pcr-ribotype (nap / ) strain of clostridium difficile [ ] , the bacterium was found to produce up to times the amount of toxin of other known subtypes and led to over , deaths [ ] . this should have sounded alarms, yet the emergency was considered to be localized with no other regions reporting such activity [ ] . eventually, the outbreak was found to be part of a larger, unrecognized community-based spread of the pathogen. by , nap / was found to be prevalent worldwide [ ] . travel of animals through trade has also proven to be a contributing factor. escherichia coli o :h was originally identified as a human pathogen in [ ] but for many years thought to be a sporadic illness [ e ] . however, the strain spread throughout canada and the united states and over time into other parts of the world due in part to travel as well as livestock movement through commercial routes [ , ] . trade in general also can lead to inadvertent spread of a pathogen via arthropod vectors such as mosquitoes. this route has been seen as the means for the expansion of the zika virus [ , ] , which was originally found in uganda in . due to international trade [ ] , the virus found its way to the pacific and eventually the americas leading to the now infamous epidemic in brazil. while the exact vehicle has not identified, a possible route may have involved the importation of mosquitoes in high enough numbers to meet the minimum requirements for establishment in the new environment [ ] . due to lessons learned, increased surveillance for these infectious agents and other well-known species, such as avian influenzaviruses [ ] , polioviruses [ ] , and measles [ ] are now in place. yet, several pathogens given little attention have begun to show signs of expansion from hidden outbreaks into large scale epidemics. although none currently poses a significant global threat, increased vigilance is needed to ensure history does not repeat itself. this review will examine six hidden outbreaks that have for the most part eluded widespread attention and will explore their potential to form epidemics. these analyses will include a list of questions that require answering to gain a better understanding of the potential of each pathogen to expand into significant pathogens of concern. . current hidden outbreaks in , satoh et al. [ ] discovered a strain of yeast in the ear canal of a japanese individual. genetic analysis revealed the isolate was a new species which was named c. auris. the species was closely associated with c. ruelliae and c. haemulonii, the latter of which had been known to cause fungemia [ ] . c. auris was later isolated in other south korean patients with otitis media [ ] . the discovery was concerning yet assessing the significance in terms of risk for an epidemic was difficult at best. two years later, the first cases of c. auris fungemia in south korea [ ] were detected, including one from an isolate taken years earlier in . the findings suggested the species already could possibly be endemic in the country. moreover, an increased tolerance to fluconazole was identified suggesting future cases could be more difficult to control. over the next three years, antimicrobial resistant strains of c. auris healthcare-acquired infections were observed in kuwait [ ] india [ ] , and south africa [ ] . in , the yeast was discovered in the united kingdom [ ] , south america [ ] , europe [ ] and canada [ ] . genetic rdna analysis of isolates in the uk demonstrated several geographic origins [ ] highlighting the importance of human travel in the spread of the species. to date, c. auris remains for the most part a health-care associated infection affecting immunocompromised individuals. while there is increasing attention in the healthcare field to the risk of this pathogen [ ] , the international surveillance program sentry has demonstrated the contribution of c. auris to the overall burden of candida species remains small and limited to healthcare facilities. however, the nearly simultaneous emergence of the species across several continents suggests the yeast is ubiquitous in the environment and may pose a threat to an increasingly immunocompromised population [ ] . additionally, the increase in antimicrobial resistance seen in numerous isolates confers significant hurdles to combat fungemia [ ] . guidelines to deal with c. auris are either in place [ ] or in development [ ] but as seen in the case of c. difficile, this may only have a limited effect in controlling its spread in the community and through travel. public surveillance is necessary to determine the as of yet unknown prevalence of the yeast in the community and potential routes of transmission should also be examined to determine whether preventative strategies such as hygiene measures in the community may help to reduce the burden. coccidioidomycosis has been a recognized disease since [ ] although it is better known as valley fever [ ] due to the geographic location of the first american cases, the san joaquin valley in california. the disease is caused by the fungal pathogen coccidioides immitis [ ] and is marked by skin lesions and the potentially fatal formation of granulomas in numerous organs. at first, the pathogen was considered to be limited to arid desert regions in the united states and argentina [ ] , however, wider surveillance has detected the fungus in other geographic regions of the united states including arkansas [ ] , utah [ ] , arizona [ ] , texas [ ] as well as in canada [ , ] , central and south america [ ] . in addition, a retrospective analysis of coccidioidomycosis in china revealed cases involving no history or travel to endemic areas [ ] suggesting the fungus already may be spreading globally with no defined routes identified. although the mortality of this pathogen is quite low at less than one case per million person-years [ ] , its expansion over the last half century highlights the potential for an increase in prevalence not only in the americas but also asia. should the species become endemic in china, there is a greater potential for a rapid increase in outbreaks, and possible epidemics as evidenced by sars. moreover, the ability of the fungus to infect animals, such as dogs, cats, bats, rodents, and armadillos [ ] , potentiates even greater spread via domesticated and possibly feral reservoirs. combined with a noted increase in planetary temperature and resultant increases in desertification [ ] , there is a need to increase surveillance and explore the modes of transmission including environmental dissemination of dust through wind currents [ ] . human t-lymphotrophic virus was first isolated in [ ] from skin cancer and was considered an atypical oncovirus. other diseases were eventually associated with this virus including adult t-cell leukemia, htlv- associated myelopathy, tropical spastic paraparesis, uveitis, dermatitis, psychological disorders, and general immunosuppression [ ] . though the virus was detected worldwide, until recently, it was considered sporadic. that view has changed due to epidemics in several regions including brazil, spain, sub-saharan west africa, the middle east, the caribbean, japan, and australia [ ] . due to the bloodborne nature of the virus, which can only be transmitted efficiently through unprotected sexual contact [ e ], the virus typically remains endemic to a region without significant geographic spread. however, historical analyses of htlv- reveal the virus can be introduced into another area through large scale population movements from endemic regions [ , ] . for example, trevino et al. [ ] examined htlv in spain and discovered htlv- has entered the country as a result of migration. while the virus has yet to become endemic in the spanish population, the authors suggest this will occur in time. due to the continued effect of mass migrations from endemic areas into naïve ones, the risk for an increase in the prevalence of htlv- exists. surveillance for this virus needs to be recognized as a priority in countries receiving these individuals. while halting the spread of the virus requires a focus on safe sexual practices, there is a need to identify possible warning signs such as those in spain prior to expansion of the virus. discovered in as an atypical mycobacterium species, m. ulcerans has been widely seen as a rare infectious agent in comparison to other species such as m. tuberculosis and m. leprae. according to k€ aser et al. [ ] , two different lineages of the bacterium exist with one being far more virulent than the other. the virulent strain is known to cause painful skin lesions known as buruli ulcers, named after the ugandan region where this infection was first described [ ] . the bacterium and associated disease has subsequently been found in several other areas of africa including angola [ ] , benin [ ] , the democratic republic of the congo [ ] , côte d'ivoire [ ] , ghana [ ] , nigeria [ ] , and togo [ ] . the infection also has been identified in australia [ ] , and most recently jordan [ ] . while the association between the bacterium and the symptoms are well established, the mode of transmission has yet to be elucidated. the infection is self-limiting and several reports suggest the highest risk is associated with living in a riverine region [ e ]. as to the potential route for inoculation, several theories have been suggested such as bites from water bugs [ , ] and the potential for colonization with the bacterium through contact with soil [ ] and watersheds [ , ] . without knowing the exact route of transmission, determining the risk of spread from one environment to another is difficult at best. in a genetic analysis of the spread of the pathogenic branch in africa, vandelannoote et al. [ ] suggested the bacterium spread at the same time as populations were divided by european colonial rule. infected individuals contaminated pristine water sources allowing for the growth and dissemination of the species. in this regard, the potential for spread of the bacterium relies on migration of those who are infected. while the morbidity associated with this disease should prevent such movements by those infected, political and socioeconomic strife may lead to forced travel during infection. surveillance for ulcers should be conducted in healthcare facilities to identify new cases in non-endemic regions. in the event a case is discovered, efforts to minimize the potential spread to watersheds need to be in place to prevent the development of endemicity. s. pyogenes has been a significant pathogen for centuries in the form of scarlet fever [ ] . however, the bacterium has the ability to invade systemically causing bacteremia, endocarditis, toxic shock syndrome, necrotizing fasciitis and endometritis [ , ] . several contributing factors are associated with these complications [ , ] including fibronectin binding proteins to facilitate invasion, cysteine proteases to escape immune attack, and superantigens that trigger a massive immune response leading to the potential for tissue damage and organ failure. for the last two decades, a significant increase in the number of invasive infections has been seen in certain regions of north america and europe. gheradi et al. [ ] have shown several serotypes have increased their circulation in these areas. while between and % have been associated with one particular serotype, emm , the risk of complications associated with the other serotypes, including known invasive members such as emm , emm and emm [ ] as well as emm [ ] and emm [ ] . the inability to identify a single serogroup to explain the rise in cases suggests the risk factors associated with the rise of this infection lies in the nature of the susceptible population. nelson et al. [ ] examined cases of invasive group a streptococcus infection and found several human factors associated with invasion and mortality including early childhood, advanced age, underlying chronic illness, and immunosuppression. given the population-based obstacles regarding prevention of invasive infection, emphasis needs to be placed on surveillance of all s. pyogenes infections. while this does occur in many regions of the developed world, more needs to be done to prepare for the inevitable introduction of an invasive serotype into a community. although this likely will not prevent illnesses and small outbreaks, public health authorities will have sufficient information to warn the public of a new invader and emphasize hygiene guidelines aimed at prevention of spread. yellow fever has been known for over four centuries as a serious illness with the potential to cause death [ ] . the virus is present in both africa and south america although limited to a few regions on these continents. however, due to the ubiquity of its mosquito vectors, aedes and haemagogus species, there is potential for escape from these regions. shearer et al. [ ] performed a modeling analysis of this virus and determined numerous areas of the world including southeast asia, and central america may be receptive to the virus. in addition, the virus may be transported to non-traditional regions such as the southern regions of china and the united states. in another example, ibañez-justicia et al. [ ] reported on the introduction of aedes aegypti into a netherlands airport. while only six insects were identified, as saarman et al. [ ] point out, the number of mosquitoes required to develop a potentially endemic population may be as little as . moreover, yellow fever can develop an urban transmission cycle in an unvaccinated population [ ] . historically, yellow fever has not caused significant widespread concern due to the usually remote nature of endemicity both in africa and south america [ ] and the availability of a vaccine. however, a recent outbreak in brazil has raised the concerns for those traveling to the region [ , ] . hamer et al. [ ] describe cases of yellow fever in travelers of whom died. none were vaccinated. moreover, due to the precedent of zika virus [ ] , concerns have been raised regarding the potential of the virus to move north into central and north america where populations are not vaccinated against the virus. the resultant scare has led to a concern for vaccine supply [ ] as there is not enough to cover the entire american population living within zones where aedes may thrive. the most important tool in determining the risk of yellow fever expansion is surveillance. the identification of mosquitoes in amsterdam [ ] is one example of how proper environmental screening of packages from endemic areas may serve to prevent the introduction and eventual reception of a pathogen. in addition, promotion of vaccination will help to reduce the likelihood of infection in travelers. while human to human transmission has not been demonstrated for this virus, the virus may be brought into a region where competent mosquitoes thrive allowing for the initiation of the sylvatic and/or urban transmission cycles. the abundance of infectious disease outbreaks worldwide outnumbers the resources available to perform extensive investigations, mitigate morbidity, and determine prevention strategies. decisions are not easy to make in light of the potential for misjudging a potential epidemic-causing strain as an isolated or sporadic case. though these occurrences are rare, as demonstrated by previous epidemic of worldwide concern, the consequences may be drastic. moving forward, each outbreak associated with these six pathogens needs to be seen as a potential epidemic and algorithms for management need to be in place. developing these decisionbased policies require the use of advanced methods to improve confidence in results. the use of advanced molecular analysis techniques such as whole genome sequencing may provide a wider perspective on the nature of the outbreak and whether it truly is isolated or is a part of a larger problem. molecular and syndromic surveillance in at-risk areas can provide real time assessments and provide predictive information. the latter may be aided through the use of social media, which is currently being examined for its power in identifying and predicting outbreaks [ e ]. mathematical modeling of anthropogenic effects apart from travel and trade, such as climate change, urbanization, and food supply and demand can enhance the fidelity and accuracy of these calculations. finally, the appreciation of social and cultural practices can offer valuable insight into how healthcare workers and the community may react in the event of pathogen detection. this information also can offer best practices for collaborative efforts to reduce the chances for amplification and spread to wider areas. in the case of the six pathogens listed in this review, there has yet to be an expansion of cases to epidemic status. however, this situation quickly can change 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detection and prediction through social networking sites social media and outbreaks of emerging infectious diseases: a systematic review of literature. pii: s e ( ) - using social media for actionable disease surveillance and outbreak management: a systematic literature review combining search, social media, and traditional data sources to improve influenza surveillance none. key: cord- - x ann authors: harvey, ruth; mattiuzzo, giada; hassall, mark; sieberg, andrea; müller, marcel a.; drosten, christian; rigsby, peter; oxenford, christopher j. title: comparison of serologic assays for middle east respiratory syndrome coronavirus date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: x ann middle east respiratory syndrome coronavirus (mers-cov) was detected in humans in . since then, sporadic outbreaks with primary transmission through dromedary camels to humans and outbreaks in healthcare settings have shown that mers-cov continues to pose a threat to human health. several serologic assays for mers-cov have been developed globally. we describe a collaborative study to investigate the comparability of serologic assays for mers-cov and assess any benefit associated with the introduction of a standard reference reagent for mers-cov serology. our study findings indicate that, when possible, laboratories should use a testing algorithm including > tests to ensure correct diagnosis of mers-cov. we also demonstrate that the use of a reference reagent greatly improves the agreement between assays, enabling more consistent and therefore more meaningful comparisons between results. middle east respiratory syndrome coronavirus (mers-cov) was detected in humans in . since then, sporadic outbreaks with primary transmission through dromedary camels to humans and outbreaks in healthcare settings have shown that mers-cov continues to pose a threat to human health. several serologic assays for mers-cov have been developed globally. we describe a collaborative study to investigate the comparability of serologic assays for mers-cov and assess any benefit associated with the introduction of a standard reference reagent for mers-cov serology. our study findings indicate that, when possible, laboratories should use a testing algorithm including > tests to ensure correct diagnosis of mers-cov. we also demonstrate that the use of a reference reagent greatly improves the agreement between assays, enabling more consistent and therefore more meaningful comparisons between results. s ince the emergence of middle east respiratory syndrome coronavirus (mers-cov) in ( ), more than , laboratory-confirmed cases have been reported to the world health organization (who); approximately one third of these cases were fatal. a large proportion of mers-cov cases have been the result of human-to-human transmission in healthcare settings ( , ) ; outbreaks have occurred in several countries, with the largest outbreaks seen in saudi arabia, united arab emirates, and south korea ( ). dromedary camels are the putative reservoir hosts for mers-cov; they experience no or mild symptoms upon infection ( ) . primary infection can occur from dromedary camels to humans, and new cases with evidence of camel contact continue to occur sporadically ( ) . mers-cov is of the high-threat pathogens on the who research and development blueprint ( ) , a document that sets out a roadmap for research and development of diagnostics, preventive and therapeutic products for prevention, and early detection and response to these high-priority pathogens. the who roadmap for mers-cov lists several priority activities, including improved diagnostics and vaccines for humans and camels as well as basic and translational research ( ) . serologic assays are critical for the evaluation of the efficacy of new vaccines and patient treatment, as are diagnostic tools to confirm infections and perform serosurveillance. a variety of serologic assays have been developed globally, both commercially and in-house; however, there is no evidence supporting the quality of performance of these assays and their consistency with one another. participants at the who intercountry meeting on mers-cov in cairo, egypt, june - , , recognized this issue as a public health priority and called for a study to compare currently available serologic assays ( ) . therefore, we assembled a panel of human serum or plasma and polyclonal antibodies to compare the performance of serologic assays for mers-cov. we invited participants to use their testing algorithms to diagnose each sample as if it were a real patient sample. the assays were evaluated for sensitivity and specificity. pas et al. described in the impact that a single international standard would have on reducing interlaboratory variability for mers-cov diagnostics (albeit in this case for nat assays) ( ) . to this end, we included samples in the panel as examples of potential who international standard material, and we assessed their effectiveness in harmonization of the data from the participant laboratories. the national institute for biological standards and control (nibsc) human material advisory committee (project / mp) approved this project. the ministry of health, oman; ministry of health, saudi arabia; and korea national institute of health, south korea, donated convalescent serum and plasma samples from pcr-confirmed mers-cov-infected patients. all patient donors gave informed consent for the use of their serum or plasma, and samples were anonymized. we treated all mers-cov convalescent-phase serum and plasma with solvent detergent using a validated method ( ) to ensure there was no residual infectious virus. we stored all study samples at - °c until dispatched on ice packs to participants. we blind coded the study samples provided to the participants (table ) . we included plasma samples (samples , , , , and ) as individual patient samples. other smaller serum donations were pooled in samples (samples , , and ) based on their antibody titer, which we determined using the recombivirus human mers-cov nucleoprotein (np) antibody (igg) elisa kit (alpha diagnostic international, https:// adi.com). we categorized samples into high-, medium-, or low-positive pools ( figure ). we included mers-cov-negative serum with antibodies against other human coronavirus hcov- e, hcov-nl , hcov-oc , and hcov-hku (samples , , , , , , and ) to test specificity of the assays ( table ). co-authors a.s., m.a.m., and c.d. precharacterized and donated these samples. purified human mers polyclonal antibodies from transchromosomal (tc) cattle ( ) immunized with either recombinant spike protein or whole inactivated virus (samples , , and ) were donated by eddie j. sullivan (sab biotherapeutics, inc., sioux falls, sd, usa). we diluted the material in universal buffer ( mmol/l tris-hcl, ph . , . % human serum albumin, % trehalose) at a concentration of mg/ml. the participating laboratories (listed at the end of this article) were located in australia, china ( mainland, hong kong), germany, south korea, the netherlands, united states, and the united kingdom ( laboratories). participating organizations included national control laboratories, diagnostic laboratories, and research laboratories. participants tested the sample panel using their routine assays for mers-cov serology. we asked participants to perform independent assays on different days. we provided an excel spreadsheet (microsoft, http://www.microsoft.com) for reporting the raw data for each assay and any interpretation of the results, such as positive or negative diagnosis of the samples. we combined titers and relative potency (relative titer) estimates as unweighted geometric means (gms) for each sample and laboratory and used these laboratory gms to calculate overall unweighted gms for each sample. we expressed variability between laboratories using geometric coefficients of variation (gcv = [ s − ] × %, where s is the sd of log transformed estimates). to mitigate the effect of any potential outliers, we calculated robust estimates of s using the r package wrs ( ). we referred to all participating laboratories by code numbers, which were randomly allocated. if a laboratory returned data using different assay methods, we assayed the results separately for each method and referred to them according to their laboratory number and assay code; for example, ppnt (pseudoparticle neutralization test) and tcid ( % tissue culture infectious dose). a total of datasets were returned (table , https://wwwnc. cdc.gov/eid/article/ / / - -t .htm). data covered a range of different assay formats: neutralization assays, eli-sa, immunofluorescence tests, and microarray. in general, there was good agreement between all the assays tested in this study. in assays with a quantitative measurement, the limit of detection and titer of samples varied greatly, but overall determination of positive or negative agreed between all assays except for (laboratory tcid mn [microneutralization]), which failed to detect positive samples (samples and ) that all other tests detected as positive. the panel of negative control samples was deemed to be negative in all quantitative assays. there were instances of laboratories reporting a result above cutoff for samples in assay, but these samples were correctly diagnosed as negative overall by their testing algorithms: laboratory detected samples and as above cutoff at : dilution in assay only; laboratory detected sample as above cutoff at : and : dilutions in assay; and laboratory detected sample as above cutoff at dilution tested. participants detected pool a, the high-titer mers-cov antibody pool (sample ) in all assays (table ) . they detected pool b, the medium-titer pool (sample ), in all but of the quantitative assays, a tcid mn assay from laboratory . in all other quantitative assays, participants detected the high pool at a higher titer than the medium pool. in the qualitative assays, assays gave borderline positive or equivocal results for the medium pool; these assays were a euroimmun s elisa (https:// www.euroimmun.com) in laboratories and and an in-house immunofluorescence assay in laboratory . the low-positive pool (pool c, sample ) was only detected as positive in a single assay in the study, the alpha diagnostic international mers np elisa performed in laboratory . participants scored positive the purified igg samples from mers-cov-immunized transchromosomal bovine samples (samples and ) in all the qualitative assays, but of the quantitative assays, n elisa from laboratory and the alpha diagnostic international mers np elisa from laboratory , were unable to detect these samples. we expected these np assays to fail to detect sample because this antibody was raised against recombinant emerging infectious diseases • www.cdc.gov/eid • vol. , no. , october mers spike protein only; however, it was surprising that the assays did not detect sample , which was an antibody raised against whole inactivated virus. for the individual convalescent-phase plasma samples, we saw more variability in the results when compared with the pooled material, but despite this, of the samples (samples , , and ) were correctly identified as positive in all tests. sample was correctly diagnosed as positive in all tests but was identified as borderline positive by laboratory in a euroimmun elisa assay. sample was the most difficult individual patient plasma to detect as positive; it was negative in the tcid mn assay in laboratory ; equivocal/positive in the in-house immunofluorescence assay in laboratory ; and, in the euroimmun elisa, borderline/negative in lab , equivocal in lab , and weak positive in lab . sample was the weakest positive of the individual samples tested in the panel, as we saw from the titers in the quantitative assays that detected it as positive. we compiled the results of quantitative assays for the individual positive plasma samples ( figure ). to simplify comparison of the assays, we reported results from the different laboratories as relative to either the transchromosomal bovine igg sample raised against whole inactivated virus (sample ) or the high-positive pooled human serum (pool a, sample ). when we expressed data as a potency relative to either of the chosen potential reference preparations with an assigned arbitrary value of , , we observed improvement in the agreement between tests (table ; figure ). we saw the greatest reduction in the variation between laboratories (smaller sem in figure and smaller percentage geometric coefficient of variation [gcv] in table ) when we used pooled human serum (sample ) as standard. the transchromosomal bovine igg raised against whole virus (sample ) showed a substantial improvement in the agreement between laboratories; however, elisa methods included in this study could not identify this sample as positive. as detection of sporadic cases of mers-cov continues, development of new vaccines to combat the disease remains important, as does serosurveillance to understand exposure to the disease and the severity of illness in persons exposed to the virus ( ) . the importance of serologic assays for the diagnosis of mers cases should also be considered; the who guideline for laboratory testing for mers-cov, updated in january , specifically includes sample collection from suspected mers-cov cases for serology ( ) . the guideline lists situations in which laboratories should conduct serologic testing for mers-cov: for defining a sporadic mers-cov case if molecular testing, such as nucleic acid amplification methods, is not possible; as part of an investigation of an ongoing outbreak; or serologic surveys, such as retrospective analysis of the extent of an outbreak. in this collaborative study, we evaluated the performance of assays to detect mers-cov antibodies using a panel of serologic samples. all laboratories correctly identified the negative samples in the panel, including those containing antibodies against other coronaviruses, implying good specificity of the assays. laboratories reported the positive results correctly except for sample , which tested negative by all but assay; however, these results demonstrated the importance of a testing algorithm. we observed instances of a sample being incorrectly determined as negative or borderline/equivocal in a single test in a single laboratory (table ) . however, because each laboratory used a testing algorithm involving > method of analysis, all the samples with sporadic spurious results were correctly diagnosed as positive or negative; if they had used a single assay type, we would have found a higher proportion of incorrect results. the results for sample , which was a pool of serum samples from patients with confirmed mers-cov, highlight a lack of sensitivity in most of the assays in this study; further investigation would be needed to determine why the antibody titers in this pool were below the limit of detection in all but assay targeting n protein. it is important to understand whether there is a specific window of time in which clinical samples for serology should be taken or whether some patients do not mount a detectable antibody response against the spike protein during infection. such information may lead to further updates of who guidelines on laboratory testing for mers-cov. the raw titers reported from the laboratories performing quantitative assays varied greatly, for some samples > , -fold, between laboratories ( table ). the use of a reference material in the assays tightened the values from the laboratories for all the samples, enhanced comparability (figure ) , and reduced the gcv percentage between all laboratories (table ) , perhaps unsurprisingly, but the magnitude of reduction in gcv percentage was noteworthy. mers-cov is an example of an important emerging pathogen with potential to cause outbreaks; diagnostic tests for emerging pathogens are often developed during outbreaks without proper validation or calibration. this study showed the importance of using a standard reagent to allow better comparison of results from different laboratories or interpretation of results from different studies or clinical trials. as we continue to face emerging pathogens, which pose significant risks to human health, it is important to use the experience gained from studies such as this to improve our response to the next threat. standardizing assays is a key issue when different groups around the world are working to develop and produce novel assays and vaccine products. the need for a standard for mers-cov serology was discussed and was widely supported at the who-international vaccine institute joint symposium for mers-cov vaccine development in seoul, south korea, june - , . several potential vaccines are in development, and the immune response elicited, their efficacy, and correlates of protection must be assessed. the use of a reference such as who international standards ( ) will enable worldwide harmonization of assays and comparability of the results from different preclinical and clinical studies. assessing the specificity and sensitivity of methods is crucial to improve our understanding of the use and limitations of serologic assays for emerging diseases for which we have little knowledge of disease progression, antibody profiles, and other key information that is available for other infectious diseases. national institute for viral disease control and prevention department of viroscience high-containment microbiology isolation of a novel coronavirus from a man with pneumonia in saudi arabia world health organization. who mers-cov global summary and assessment of risk scope and extent of healthcare-associated middle east respiratory syndrome coronavirus transmission during two contemporaneous outbreaks in riyadh, saudi arabia middle east respiratory syndrome coronavirus (mers-cov) mers-cov in upper respiratory tract and lungs of dromedary camels, saudi arabia reported direct and indirect contact with dromedary camels among laboratory-confirmed mers-cov cases annual review of diseases prioritized under the research and development blueprint a roadmap for mers-cov research and product development: report from a world health organization consultation report on the intercountry meeting on the middle east respiratory syndrome coronavirus (merscov) outbreak in the eastern mediterranean region geneva: the organization first international external quality assessment of molecular diagnostics for mers-cov robustness of solvent/detergent treatment of plasma derivatives: a data collection from plasma protein therapeutics association member companies triple immunoglobulin gene knockout transchromosomic cattle: bovine lambda cluster deletion and its effect on fully human polyclonal antibody production wrs : wilcox robust estimation and testing mers: progress on the global response, remaining challenges and the way forward world health organization. laboratory testing for middle east respiratory syndrome coronavirus interim guidance recommendations for the preparation, characterization, and establishment of international and other biological reference standards (revised we thank the following institutions and organizations: universitätsklinikum, bonn, germany; charité-universitätsmedizin, berlin, germany; korea national institute of health; the central public health laboratory, ministry of health, sultanate of oman; public health department, supreme council of health, qatar; the ministry of health, public health directorate, kingdom of saudi arabia; sab biotherapeutics, south dakota, usa. we also thank humayun asghar from the who eastern mediterranean regional office for assistance obtaining the serum for the study. dr. harvey is a principal scientist at the national institute for biological standards and control, united kingdom. her research interests include influenza virus vaccines and respiratory viruses including mers coronavirus. key: cord- -w sh f h authors: xue, lan; zeng, guang title: china’s institutional mechanisms for influenza a (h n ) prevention and control date: - - journal: a comprehensive evaluation on emergency response in china doi: . / - - - - _ sha: doc_id: cord_uid: w sh f h innovation in institutional mechanisms is a fundamental issue in effectively dealing with public health emergencies. in the wake of the sars epidemic, china initially established a public health emergency management system and an emergency organization and management network, placing emphasis on “government leading, unified command, local management, responsibility on all levels, management by classifications, and inter-departmental coordination,” which strengthened the existing health emergency preparation system. and insufficient preparedness. china's fight against sars, as it were, not only posed a great challenge to the nation's socialist modernization, but at the same time offered an important opportunity for the country to improve emergency management, especially in the field of public health. in the wake of the sars epidemic, the chinese government began pushing for a national emergency management system in a systematic, planned and gradual manner, and made remarkable progress in emergency management structured on the "one plan three systems (contingency plans, institutions, mechanisms and legislation)." in regards to contingency planning, the country formed a system consisting of contingency plans at central, local, departmental, and enterprise levels as well as plans for major events, and this system played an important role in dealing with public emergencies. in regards to institution building, a national emergency management system consisting of general emergency management offices as well as of special emergency management bodies were established. the emergency management office of the state council and emergency management bodies of provincial (regional and municipal) governments were set up in succession, in addition to emergency management systems in specific fields such as health. in comparison with the pre-sars environment where "departments played a dominant role, and coordination was inadequate," these new institutions showed the permanent, comprehensive, and specialized nature of emergency management, and laid an organizational foundation for the future. in addition, society as a whole began getting involved with the emergency management process, including: further strengthening military emergency system construction and local assistance ; giving full rein to experts in in march , the third session of the th national people's congress (npc) reviewed and adopted the report on the work of the government, which stated that "… we have formulated the national contingency plan for public health emergencies, as well as special-purpose and department-specific contingency plans concerning natural disasters, accidents, public health, social security, etc.; provinces (regions and cities) also have completed their own work on the development of overall contingency plans. breakthroughs have been made in building a government by the rule of law and in fully fulfilling government functions." see: report on the work of the government, delivered by wen jiabao at the third session of the th national people's congress on march - , . emergency management ; and developing and implementing local emergency management plans that targeted "communities, rural areas, enterprises and schools." looking at mechanism construction, progress was also made in research on a science-based emergency management system, and an emergency management mechanism characterized by "unified leadership, responsiveness, orderly coordination, and efficient operation"-which enabled the interconnection of early warning, mass mobilization, quick response, and emergency handling-was gradually established to effectively mitigated public health emergencies. in regards to legislation, the emergency response law was took effect on november st, . health emergency system long term. efforts would be made also to strengthen the disease control and prevention system, increase public health emergency management capabilities, boost rural health development, improve healthcare for the rural population, strengthen environmental health system, and implement national health campaigns. in february , the moh outline the following overall goals for public health emergency work in the th five-year plan: establish and improve health emergency management legislation and the health emergency contingency planning system; build an emergency management mechanism characterized by "unified leadership, responsiveness, orderly coordination, and efficient operation" with "predominantly local management, hierarchical responsibility, and comprehensive coordination;" bolster health emergency management recruitment; improve the public health emergency monitoring and warning system; strengthen capacity for quick and effective response to health emergencies; and shape an environment of health emergency management characterized by inter-departmental coordination, collaboration, and social participation under the leadership of central and local governments. during and after the sars epidemic, china continued to establish and improve public health emergency legislation, regulations and contingency plans, and initially formed a national system for public health emergencies contingency planning (see fig. the moh established a public health emergency coordination mechanism with thirty one central and national departments to deal with inter-departmental collaboration, which effectively strengthened communication and coordination between departments dealing with public health emergencies. the national government and the special administrative regions of hong kong and macau entered into a three-party emergency response collaboration agreement and decided upon implementation regulations, and established a linkage mechanism for information communication and health emergency response. additionally, the moh established a joint prevention and control mechanism with the ministry of agriculture (moa) to protect against highly pathogenic zoonotic viruses such as avian influenza and streptococcus suis; the moh established a coordination mechanism for joint prevention and control of public health emergencies at ports with the general administration of quality supervision, inspection and quarantine (aqsiq). in collaboration with the ministry of railway (mor), the ministry of transport (mot), and the aqsiq, the moh issued notices on the prevention and control of the importation of infectious diseases from abroad; and together with the moe, issued a document requiring schools to appoint part-time or full-time teachers to identify and report infectious diseases or other health emergencies at the school. this time marked the initial formation of a working inter-departmental mechanism positioned to combat health emergencies through "paying equal attention to both prevention and response, and instilling continued collaboration for any event." on april th, health minister chen zhu convened a meeting of the moh leading group and expert panel for influenza pandemic prevention and control, at which the attendees analyzed swine influenza situations in the united states and mexico, predicted epidemic trends, and deliberated on domestic strategies and measures to cope with a swine flu pandemic. health minister chen also held an inter-departmental meeting with the moa, the aqsiq, and other ministries to analyze epidemic trends and discuss response strategies and measures. immediately after the meeting was over, the moh reported in writing that very night to the state council on the progress of epidemic prevention and control work. on april th, following the emergency meeting held in geneva, the who elevated the pandemic alert level from phase to phase , stating that the "swine flu" was widespread and was being transmitted by humans in different ways. general secretary hu jintao issued instructions to place prevention and control against this virus as the nation's top priority. on the same day, vice premier li keqiang convened the state council meeting regarding the human-swine influenza prevention working mechanism, resulting in the decision to establish a multi-departmental working mechanism for joint prevention and control of the human-swine influenza. as required by the state council meeting, the moh called together the publicity department of the communist party of china (ccppd), the ministry of foreign affairs (mfa), the ndrc, the miit, the mof, the ministry of transport (mot), the moa, the moc, the aqsiq, the china national tourism administration (cnta), the civil aviation administration of china (caac) among other departments on that very night for a meeting to deliberate on influenza a (h n ) prevention and control; the meeting established the multi-departmental working mechanism for joint prevention and control of human-swine influenza and the notice on strengthening human-swine influenza prevention and control was drafted and published on the night of april th after state council review. the moh issued the notice of the moh general office on strengthening preparedness for and response to human swine influenza. on april th, premier wen jiabao convened a state council executive meeting which deliberated on how to strengthen national response to human-swine influenza; at the meeting they defined the overall prevention and control principles and strategies of "taking threats to public health seriously, responding actively, and coping with the epidemic in a scientific manner according to law through joint prevention and control efforts." on april th, the who raised its pandemic alert level from phase to phase . on april th, at a press conference held at the state council information office, the moh declared the establishment of a multi-departmental working mechanism for joint prevention and control against the human-swine influenza, which would be spearheaded by the moh. under this mechanism, departments and institutions (which later increased to ) constituted work groups-general office, ports, healthcare, support, dissemination and communication, foreign collaboration, science and technology, and animal husbandry and veterinary-and an expert committee, forming a " + " pattern for joint prevention and control efforts. on the afternoon of may st, the joint prevention and control mechanism held its second joint conference, renaming the human-swine influenza which was occurring in mexico and the united states to "influenza a (h n )." the former wording of "multi-departmental work mechanism for joint prevention and control of human-swine influenza" was changed to the "joint national influenza a (h n ) prevention and control mechanism" and roles and responsibilities were outlined for the mechanism, work groups, and the expert committee. at the same time, a meeting system for all members and liaisons was established. problems in principle which a work group encountered would be solved by the work group itself, and those which the work group struggled with would be settled through coordination under the joint prevention and control mechanism-general affairs would be solved by regular liaison meetings, and major issues decided by plenary meetings. health minister chen zhu and moh party group secretary and vice health minister zhang mao chaired the joint national influenza a (h n ) prevention and control mechanism, and the mechanism was comprised of eight working groups-general office, ports, healthcare, support, dissemination and communication, foreign collaboration, science and technology, and animal husbandry and veterinary-and an expert advisory committee. the heads of the work groups, the leaders of the health department of the people's liberation army general logistics department (gld), the logistics department of the chinese people's armed police force (papf), and the chairman of the expert advisory committee, served as members of the joint prevention and control mechanism. the work groups, the gld health department and the papf logistics department each designated one or two departmental-level officials as liaisons for routine communication purposes. the main duties of the joint national influenza a (h n ) prevention and control mechanism included: meet regularly to evaluate epidemic trends and determine prevention and control strategies; formulate prevention and control policies, response plans and major measures; coordinate and provide guidance on the implementation by all related departments and in various regions of prevention and control measures; and organize supervision and inspection activities concerning the implementation of prevention and control measures. see table . for the main responsibilities of the work groups and the expert advisory committee. thirty three meetings were convened under the joint national prevention and control mechanism, in which regulations were formulated, signed, and issued for local implementation. implementation issues would be reported in real time to related state council departments for instructions. the establishment of the joint national prevention and control mechanism played a crucial role in the scientific and orderly response to influenza a (h n ), in that ( ) the mechanism raised the priority level for influenza a (h n ) prevention and control for related departments and local governments, ( ) clarified and divided responsibilities, ( ) addressed investment issues, and ( ) enhanced interdepartmental cooperation. a consultation system and a liaison meeting system were established under the joint national prevention and control mechanism, which were designed to ensure effective implementation of prevention and control measures. specific issues in prevention and control work would be resolved through consultation at liaisons meetings, and major issues decided by plenary meetings. each working group established a fixed meeting system where they could provide timely progress comprehensively coordinate routine affairs among departments of the joint prevention and control mechanism; organize regular meetings of the joint prevention and control mechanism and oversee the handling of top agendas; collect, sort out, and report to higher-ups about progress in prevention and control efforts; prepare progress reports on the joint prevention and control mechanism; publish information on epidemic situations and response efforts; ensure the consistency in style of writing for documents intended for outside use; and shoulder other assignments from leaders report in a timely fashion on influenza a (h n ) situations and on progress made in major efforts under the joint prevention and control mechanism, and guide public opinion positively and correctly; arrange news releases on the joint prevention and control mechanism, and where necessary, organize press conferences; track public opinion at home and abroad, and clarify facts in a timely fashion; strengthen management and guidance on the release of (continued) . the establishment, composition and operations … reports, discuss and address problems, and push ahead with prevention and control work within their fields. the establishment and improvement of the coordination mechanism remarkably increased the efficiency of inter-departmental coordination and response efforts. this success evinces the importance of a multi-departmental coordination and collaboration mechanism based on risk communication for effective epidemic prevention and control. thirdly, horizontal collaboration between related departments was strengthened. for example, on april th, , the moh office of health emergency sent a letter to the miit department of consumer goods industry recommending an increase in the national stockpile of supplies necessary for influenza a (h n ) prevention and control including medical supplies and response gear. in another example, the office of health emergency, department of medical affairs, and bureau of disease control and prevention of the moh, the china cdc, and the chinese medical association, jointly formulated the technical guidance on prevention and control of human-swine influenza, and the plan for diagnosis and treatment of human-swine influenza ( ). one more example occurred on july th, when the foreign collaboration group for the joint prevention and control mechanism, along with the ndrc, the miit, and the sfda met to discuss donating influenza a (h n ) prevention and control materials to the who and developing countries affected by the pandemic. during the course of the epidemic response efforts, risk evaluation, and risk management, it was necessary to build unobstructed information exchange channels to ensure the accurate transfer of data and information. under the joint prevention and control mechanism, each of the lead departments for the work groups and the expert advisory committee appointed people to collect, sort out, tabulate and analyze their work groups' epidemic information and latest progress on a daily basis, and to report in writing daily data collected by : to the general group prior to : p.m. the general group would then prioritize epidemic information and progress reports, and submit it representing the entire joint prevention and control mechanism to the general duty office of the state council. strengthened coordination and communication between the work groups and their members ensured an unobstructed flow of information as the working groups were informed of major issues as they happened. the general group was also charged with publishing information crucial for the public's knowledge on epidemic prevention and control. updates on influenza a (h n ) prevention and control were submitted via four reporting systems: the disease monitoring information reporting management subsystem of the china information system for disease control and prevention, the public health emergency reported information management system, the china influenza information monitoring system, and administrative reporting system for health departments. reported information mainly included: ongoing epidemic situations, monitoring results from sentinel hospitals and network laboratories, progress in vaccination and results of side effects monitoring, and ongoing regional and departmental response efforts. with influenza a (h n ) cases rising rapidly, issues with epidemic information reporting began to occur, such as overlapping reports, large discrepancies between confirmed reported cases and actual cases, and the circulation of ambiguous epidemic information. to better and more accurately reflect national epidemic situations and trends, the moh general office issued the notice on strengthening work of reporting deaths from influenza a (h n ) on november th, , and the notice on adjusting the work of reporting information on the influenza a (h n ) epidemic on november th, . by april th, , the general group had submitted more than work reports regarding influenza a (h n ), and compiled and published over response progress to influenza a (h n ) under the joint prevention and control mechanism reports. information on epidemic trends and response efforts was released in a timely, open, and transparent manner, and by this time eight news conferences and nine press briefings had been held regarding the latest progress in influenza a (h n ) prevention and control. to ensure that prevention and control measures were implemented effectively and efficiently, the work groups each established a supervision and inspection system by which to examine routine work on a regular basis, identify existing deficiencies and problems, and supervise and inspect response contingency plans and procedures, operational capacities, epidemic monitoring, epidemiological investigations, designated hospitals and their isolated areas, medical observation, material supplies, staff training, and information dissemination on prevention and control. in the course of influenza a(h n ) prevention and control efforts, local governments, as instructed by the central government, examined their own conditions and established local bodies to command and coordinate response measures. the local departments worked together to implement disease prevention and control measures in priority areas and among targeted groups, and ensured continued epidemic monitoring and treatment. the following three modes mainly represent actual prevention and control measures adopted by local governments. the first work mode was similar to the national joint prevention and control mechanism. for example, shaanxi set up a leading group for influenza a (h n ) prevention and control, whose office was located inside the provincial department of health and their local structure followed the " + " joint prevention and control mechanism model. guangdong established a joint prevention and control mechanism with the participation of thirty two departments, and nine work groups and three panels of clinical, disease prevention and control, and etiological experts functioned under the mechanism. on april th, fujian established an influenza a (h n ) prevention and control work group, headed by a provincial government official; the office was located inside the building of the provincial department of health whose emergency management office was charged with performing routine work for the group. the emergency guidance on influenza a (h n ) prevention and control in fujian (tentative), issued on may th, , outlined response guidance as "prevention first through joint prevention and control, timely management, and level-by-level responsibility." the second work mode was the emergency operations center or leading group. after discovering their first confirmed influenza a (h n ) case, some provinces and cities upgraded their existing disease prevention and control mechanisms and established an influenza a (h n ) response leading group or emergency operations center. beijing was the first in the country to establish a municipal-level public health emergency operations center in may , and had earlier (april th, ) launched a public health emergency response mechanism after the who declared the outbreak of swine influenza in mexico; an influenza prevention and control office (at the general office of the municipal government before it relocated to the municipal bureau of health) was established under the emergency operations center, whose members included twenty two committees, eighteen district and county governments, and the gld health department. this control office established a public health emergency response and medical rescue collaboration mechanism with the china cdc, the academy of military medical sciences, and other institutions. it established a mutual fixed epidemic communication system with local agricultural, educational, industrial, and commercial departments. a joint command response mechanism was also created with other special operations centers in beijing. on may th, shandong established a provincial public health emergency leading group and started level-ii response measures after discovering its first-and the country's second-confirmed imported case of influenza a (h n ). on june nd, hubei established an influenza a (h n ) emergency operations center after the province's first case was confirmed. immediately after the country's first influenza a (h n ) case was confirmed on may th in sichuan, sichuan initiated level-ii public health emergency response measures and set up a provincial response leading group as per the state council's requirement of handling the virus as a category b infectious disease. the third work mode was the joint conference system. some provinces and cities established a joint conference system in response to the influenza a (h n ) outbreak. for example, henan established an influenza a (h n ) joint conference system on april th, and on the same day guangxi established a -department joint conference system for its own prevention and control efforts. while establishing provincial-level joint prevention and control mechanisms, health departments also set up internal expert panels. for example, fujian provincial department of health set up a provincial-level influenza a (h n ) prevention and control expert supervision panel; guangdong provincial department of health established three expert panels for clinics, disease prevention and control, and etiology; sichuan provincial department of health established a leading group, a technical guidance expert panel, and a medical rescue panel on april th. the composition of local mechanisms for influenza a (h n ) prevention and control basically followed the framework of the national joint prevention and control mechanism, with an office and several work groups collaborating under a leading group or operations center. for example, beijing's public health emergency operations center was responsible for the city's influenza prevention and control, and it was comprised of eight work groups plus an office, these groups included: immigration inspection, healthcare, epidemiological survey, material security, dissemination and communication, information, animal husbandry and veterinary, and social prevention and control supervision (referred to as "one office and eight groups"). fujian's influenza a (h n ) prevention and control leading group consisted of thirty one departments and organizations, including the provincial department of health, a press office, and a development and reform commission. sichuan's influenza a (h n ) prevention and control leading group (operations center) included departments from emergency management, public security, development and reform, transportation, immigration inspection and quarantine, tourism, civil aviation, foreign affairs, and publicity. the office of sichuan's influenza a (h n ) prevention and control leading group was originally located in the provincial department of health, which was then moved to the provincial government's general office building as the epidemic worsened. its work groups consisted of emergency coordination, general support, information secretaries, epidemic prevention and control, medical rescue, supervision and inspection, press and communication, and health education. local influenza a (h n ) prevention and control mechanisms adopted a similar communication and coordination mechanism to the joint national prevention and control mechanism, and operated in with joint offices, conferences, etc. sichuan is one example of this. the provincial operations center and the provincial leading group (headquarters) shared offices in order to strengthen the province's joint prevention and control against influenza a (h n ), and its seven work groups were comprised of highly capable professionals from the emergency management office, third secretariat office of the general office, and the provincial health department. the health department met regularly with the departments of public security, civil aviation, immigration quarantine, economy and trade, animal husbandry, as well as pla and people's armed police troops stationed in the province. adjustments were made in real time in according with latest local epidemic situations. latest information on epidemic updates across the province were reported daily to members and related departments, and where cases were discovered, the departments of health, public security, foreign affairs, railway, transport, and others worked closely to track close contacts and ensure they were medically observed. through close collaboration between departments at various levels on joint prevention and control, sichuan ensured that cases were discovered, reported, isolated and treated at the earliest possible time, which delayed the spread of the virus and lowered epidemic intensity. government departments, enterprises, institutions, communities, and nonprofit organizations (npos) all play important roles in prevention and control of an infectious disease. with the country's response efforts entering its second phase, the th meeting of the national joint prevention and control mechanism, held on june th, , proposed further improvements of existing mechanisms, in particular establishing accountability systems and mass prevention and control mechanisms with participation from urban communities, schools, enterprises and villages. these mechanisms could better implement tailored measures, disseminate self-protection knowledge for families and individuals, and improve measures that maintain the status quo and normal economic operations. when confronted with a public health emergency, under the guidance of the government, the society can effectively avoid or reduce potential damage by achieving preliminary prevention and control targets at local levels through community involvement and solidarity along with raising public awareness in self-protection. in its work report, the who mentioned two types of community participation, i.e. participation as a means, and participation as a goal, and analyzed effects of the two. in the course of china's influenza a (h n ) prevention and control efforts, communities, the most basic social units, played an important role in knowledge dissemination and health education, the tracking and isolation of close contacts, and epidemic supervision. when uncertainties still surrounded the pathology and virulence of influenza a (h n ) in the early days of the epidemic, local communities launched information dissemination and health education campaigns, playing a crucial role in stabilizing public opinion and raising awareness of scientific disease prevention and treatment methods. in the case of community-level outbreaks, affected communities generally adopted comprehensive response measures, which emphasized managing the sources of infection in order to contain and control the transmission of the influenza virus. measures mainly included the following: ( ) sub-district offices or town governments mobilized social forces-as per laws, rules, and regulations-to provide support for isolated cases, including logistical service to personnel engaging in medical observation; ( ) close contacts were medically observed centrally or at home, and healthcare workers reported daily on patients' progress; ( ) patients with influenza-like symptoms were recommended to rest at home and not participate in unnecessary public gatherings or travel; ( ) schools, nurseries and kindergartens, nursing homes, and construction sites were required to conduct health inspections, and enterprises with a concentrated amount of personnel or those who provided social services were required to perform morning health inspections; ( ) information on epidemic trends and response measures were published in real time, and efforts were made to strengthen information disclosure within communities; and ( ) health education and risk communication were carried out through multiple channels. when the virus broke out in communities, healthcare departments managed cases categorically and adopted comprehensive measures for strengthening the treatment of severely ill cases, lowering case fatality rates, and mitigating epidemic damage. with prevention and control measures in place against community-level outbreaks, response measures for priority areas mainly included the following: ( ) as per related laws, rules, and regulations, local governments mobilized social forces to ensure the logistical support of measures like home-based treatment of cases with influenza-like symptoms; ( ) migration was cut or restricted, recreational areas were temporarily shut down, and large-scale gatherings were canceled or postponed. enterprises and institutions within communities were permitted to grant time off for all or some of their employees; ( ) schools, nurseries, and kindergartens were closed per related regulations; ( ) enterprises and institutions as social services providers with large workforces implemented a health reporting system, management was enhanced where there were large flows of people, and people with influenza-like symptoms were recommended to rest and receive treatment at home; ( ) when necessary, outbreak points were put under isolated control, and quarantine measures were taken in epidemic areas. at the same time, measures were taken to organize and encourage volunteers to participate in prevention and control activities, to help maintain the normal operation within communities, and provide mental health interventions to avoid adverse effects on public health. these measures, which were designed based on real community conditions, effectively guaranteed the protection of the status quo, and laid a strong foundation for local influenza a (h n ) prevention and control efforts. in the course of influenza a (h n ) prevention and control efforts, drug stockpiling enterprises responded actively to the government's call for material reserves and production. because influenza drugs weren't prevalent in clinical use, they were traditionally stockpiled through loans, government subsidies, business opportunities and moderate enterprise compensation. problems arose during the implementation of this mechanism such as subsidy inaccessibility and unreasonable compensation. for example, the current % subsidy policies regarding corporate loans and government subsidies hardly met the needs of enterprises, and the problem of unreasonable compensation to pharmaceutical enterprises still existed, partly because specific mechanisms were lacking. at the same time, more than % of emergency response drugs were not on the standing list of medications, and so it was necessary to build a long-term relationship between the government and enterprises to specify respective duties, and link stockpile funding with corporate social responsibility to balance compensation. reagent manufacturing was one of the government's top priorities during the entire course of its influenza a (h n ) prevention and control efforts. some reagent manufacturers which had developed and produced reagents for biological agents such as anthrax during the olympic games already had experience in emergency response. for example, beijing kinghawk pharmaceutical co., ltd., the country's first to obtain approval for an influenza a (h n ) testing kit, signed a strategic alliance agreement with the china cdc during the influenza a (h n ) epidemic. the enterprise also provided its laboratories voluntarily when there was no clear policy on state funding, doing its best for society as a corporate citizen. with its technology reserve, seven production platforms and ninety approved products, kinghawk was able to perform research and development on product standardization during a critical time of the epidemic. the china cdc had access to international resources for preliminary research and development and successfully obtained information and strains from the who. this collaboration between the two parties made it possible to develop preliminary products in h and thus ensured that considerable demand for clinical diagnosis was met. this played a positive role in case diagnosis during the early phases of the influenza a (h n ) epidemic and was also quite meaningful in terms of drug use guidance. the government provided active support to research and development efforts. take kinghawk as an example. after kinghawk signed the agreement with the government on may th, , both the beijing economic-technological development food and drug administration and the beijing food and drug administration provided recommendations. kinghawk had developed a rapid test kit by may th, received approval from the cfda on june th to launch the emergency response system, and got approved for manufacturing the reagent for , people on september th. kinghawk had collaborated with the china cdc in the past and had experience in reagent development and manufacturing. the development of the reagent fully demonstrated the efficiency in collaboration between the government and a commercial enterprise, and also guaranteed the timeliness of preliminary disease diagnosis. in the containment phase, hotels and other requisitioned enterprises across the country showed full support for the response measures by providing isolation zones of influenza a (h n ) cases. many hotels suitable for isolation purposes were private firms and thus could not be requisitioned through administrative orders, which put a certain amount of pressure on local governments. however, coordination efforts by local governments did earn support and assistance from these hotels. transport enterprises shouldered the heavy task of implementing disease prevention and control for the floating population. civil aviation, railways, road and related enterprises implemented strict prevention and control measures, including disseminating knowledge about disease prevention and control, and providing necessary infrastructure support for emergency response efforts targeting the floating population. all in all, a healthy transportation environment helped lower the transmission of the disease. facing the unexpected onset of influenza a (h n ), these transportation enterprises all established prevention and control groups. for example, the caac north china regional administration established a capital airport influenza a (h n ) prevention and control leading group, which was based on the former capital airport public health emergency leading group; beijing capital international airport company limited, air china limited, china southern beijing company, china eastern beijing company, hainan airlines beijing company, caac air traffic management bureau all set up their own epidemic response teams to ensure the orderly implementation of prevention and control measures. telecommunications enterprises did their duties as corporate citizens and actively cooperated in influenza a (h n ) prevention and control efforts. china mobile group beijing company limited, china telecom group beijing company limited, china unicom beijing company limited, among other telecommunications operators, suspended their normal user notification group-messaging services and mustered network resources to send messages on epidemic updates while increasing maintenance staff, strengthening network monitoring, and closely watching the impact of group messaging on their systems. nonprofit organizations, or npos, are organizations that fulfill particular social causes or missions without seeking profit for their efforts, and npos represented a crucial social force in the course of the nation's influenza a (h n ) prevention and control. for example, the beijing red cross established a public health emergency operations center which consisted of a general information group, a rescue response group, a fundraising and aid group, a publicity group, and a public relations group. this organization actively engaged in response efforts in accordance with the beijing red cross emergency contingency plans for public emergencies. the entire nation was involved in epidemic prevention and control, including its citizens. during the response to the virus, volunteers played an important role when multiple departments suffered emergency manpower shortages. for example, medical and healthcare students in colleges and universities volunteered to work on the front lines of epidemic prevention and control. in beijing, student volunteers from capital medical university assisted with response efforts at capital airport, and similar volunteering also occurred in fujian. the public actively supported the government's influenza a (h n ) prevention and control measures, and voluntarily took part in the process via the internet and other media channels; for example volunteers called those who had just returned from abroad and informed them of potential isolation measures. at the same time, increased public health awareness was also instrumental in successfully dealing with the disease. prevention and control mechanisms at the beginning of the influenza a (h n ) epidemic, china established a national level emergency management mechanism directly under the leadership of the state council that enabled cross-departmental joint prevention and control collaboration, which provided an effective organizational support and operation mechanism for the response efforts. though the moh had formulated the ministry of health's influenza pandemic preparedness and response plan (tentative) before the epidemic broke out, this document focused only on the duties of the moh and didn't encompass more complex coordination and collaboration with related government departments. the joint prevention and control mechanism remedied this flaw by providing a platform for coordination and collaboration between the moh and other related departments. also, because this mechanism was not like the state council's operations center, it allowed some space for strengthening the state council's leadership and collaboration once the epidemic worsened. during the prevention and control efforts, local governments adapted and innovated central policies and their implementation in light of local epidemic situations, public health trends, and demographic and economic conditions. some areas established prevention and control mechanisms with local characteristics. the main features of these mechanisms are as follows: the first was the establishment of a strong leadership system. in the process of prevention and control, local governments established their respective public health emergency leadership systems based on local epidemic situations, geographic features, and public health resources. the second was the innovation in ideas and methods. local epidemic prevention and control bodies closely monitored trends and reengineered their methods based on existing departmental systems in order to better target obstacles encountered in operations. for example, in the early days of the epidemic, the beijing government issued a notice on further specifying duties and prioritizing operations to strengthen influenza a (h n ) prevention and control, which articulated the new public health notion of "responsibility of four sides" (government, departments, enterprises, and individuals) . this clarification brought about effective collaboration between the government and the society in public health emergency management. the beijing immigration inspection and quarantine bureau employed risk analysis methods in its prevention and control efforts and ensured electronic transfer of information on inbound passengers, which not only increased quarantine and inspection efficiency but also scientifically and efficiently improved response measures. fujian was the country's first province to implement temporary isolation measures through its local health department. henan created an epidemic prevention and control network of "three horizontal fronts"-arrangements at a government level, measures at enterprise (institution) level, and protection at a local level; and "three vertical fronts"-government supervision, inter-departmental collaboration, and public opinion guidance. these institutional innovations proved very effective in the response efforts. the third was the establishment of an inter-provincial support mechanism. on november th, , the moh general office issued the notice on strengthening medical treatment of influenza a (h n ) patients (no. , ) , announcing the decision to establish an inter-provincial support mechanism for medical treatment of influenza a (h n ) patients as per the notice of the state council on strengthening the ongoing work on influenza a (h n ) prevention and control (no. , ) and as needed for patients. the form of assistance was technical support, especially in regards to medical treatment of seriously and critically ill patients. throughout the entire duration of the prevention and control efforts, governments greatly heeded experts in various fields, which aided governments in creating more scientific policy adjustments and technical plans, and consequently reduced blindness and uncertainty in policy implementation. experts from cdcs, hospitals, publicity departments, and other departments took part in the decision-making process, and their input was adopted in real time. some experts even took the initiative to provide police recommendations directly to decision makers. at the same time, governments sought out expert opinions through different methods and channels, i.e., consultation at joint prevention and control meetings or direct consultation with the experts. expert recommendations ensured scientific policies and more targeted and effective policy formulation. in regards to policymaking, some local governments formulated policies and adjustments based on local conditions and epidemic trends. for example, jiangmen experimented with a home-based isolation policy, while shenzhen created corridors at ports specifically for foreigners and a separate one for students commuting between shenzhen and hong kong for school. also, in terms of policy adjustment, some local departments were able to adjust related policies in time to better suit local epidemic situations. as for issues that necessitated policy coordination, local departments also made strategic adjustments as early as possible. for example, the guangdong immigration inspection and quarantine bureau, at experts' suggestion, transferred persons who required isolation and medical observation to health departments for categorical management, which thus ensured the efficient use of epidemic prevention and control resources. over the course of influenza a (h n ) prevention and control efforts, the government cultivated an environment of widespread social participation under the leadership of the party and government, with enterprises, communities, volunteers and other social actors playing crucial roles in the response efforts. the joint national prevention and control mechanism was essentially a command and decision-making mechanism established according to the potential amount of damage influenza a (h n ) could inflict upon society. on the one hand, influenza a (h n ) response required inter-departmental collaboration, and relying solely upon health departments for countermeasures wouldn't be enough; on the other hand, because the virus was not as virulent as to merit the establishment of a state council operations center (or headquarters), the state council instead instructed the moh to establish a multi-departmental joint prevention and control mechanism; and this new organization represented a relatively flexible and effective response mechanism. although local governments were already aware of the epidemic at its onset and were actively engaging with different departments in their response efforts, because there were no explicit provisions in related laws and contingency plans for the joint national prevention and control mechanism at the central level, no corresponding normative documents were available for its implementation at a local level. no unified standards on the name, content, form of establishment, and system structure for local governments' prevention and control bodies existed. although local governments adapted as they went, it was still an environment that incited disorder and confusion. on the one hand, participating departments fully endorsed the joint national prevention and control mechanism. this mechanism, they thought, possessed several advantages: firstly, the joint consultation system made it possible to directly formulate and sign policies at joint prevention and control conferences, which saved time for everyone; secondly, the joint briefing system required the work groups to send daily reports to other units and departments, thus facilitating both inter-group and inter-departmental communication; and finally, internal collaboration within groups was solid, and the briefing system allowed an unobstructed flow of information. however, the joint national prevention and control mechanism based upon consultation and communication had its limitations. on issues involving departmental interest, division of duty, and so on, this horizontal collaboration was less efficient than regulation and control by a single, high level leadership department. one contested issue dealt with the location of the local joint prevention and control office: should it be set up in the comprehensive emergency management office of the local government or in the emergency management office of a local specialized department. some provincial emergency management offices insisted that for an emergency event like the ongoing influenza a (h n ) epidemic, a joint prevention and control office should be located in a specialized department so as to leverage the department's expertise and increase response flexibility, convenience and efficiency. in this scenario, the provincial emergency management office would be tasked with solving issues that the specialized department could not. on the other hand, some provincial health department's emergency offices argued that if the office was located in the local government, the joint prevention and control office would enjoy greater authority and more efficient collaboration. achieving a smooth and effective transition between peacetime and public emergency, and establishing mechanisms that combined crucial components from both systems, was a new challenge that arose in the influenza a (h n ) epidemic. after the sars epidemic, local governments established permanent public health emergency response departments and corresponding working mechanisms to deal with future public health emergencies. these departments and mechanisms should have been employed upon the onset of the influenza a (h n ) epidemic. however, most provinces established completely new leading groups only after the central government established joint national prevention and control mechanism. in one example, a provincial health department already had a permanent public health emergency operations center, but, after the central government established the influenza a (h n ) joint national prevention and control mechanism, this province created an entirely new prevention and control leading group and a port leading group. at the same time, the health department also established new eternal mechanisms, including: the provincial cdc established an emergency response department with leaders from major sections like emergency management and vaccination planning (starting in , this provincial cdc implemented a " in " meeting system with participation from emergency management, disease control, and the disease monitoring department). the main reason for this redundancy was because the central government did not provide specific conditions or qualifications for contingency planning and management for the transition period between peacetime to emergency. thus, local governments lacked a clear transition mechanism that they could utilize. it was the reason that many local governments chose to re-establish emergency management bodies when influenza a (h n ) broke out. as public health emergency management involved multiple collaboration systems from the central government down to local governments, regions, and departments, inter-departmental collaboration in the response efforts was intrinsically complicated. the response to this epidemic revealed problems that existed both in horizontal and vertical coordination. in regards to horizontal coordination between central departments, the health, education, security, transportation and many other departments were involved in the influenza a (h n ) response efforts, which created an environment where responsibilities could easily overlap and grey areas would occur in management. there was also a lack of coordination and standardization between central-level ministries' policy documents for influenza a (h n ) countermeasures. for example, in regards to content standardization, the health authorities felt that using the temperature of . °c as the sole standard for sending people to the hospital was unreasonable and would cause an unnecessary burden on hospitals. in regards to time standardization, on december nd, , one province stipulated that only patients with a temperature of °c or higher must be sent to a hospital, and it took the country two more weeks to follow suit. in regards to inter-departmental work, port laboratories in some provinces had begun testing in the early days of epidemic, but stopped after provincial health departments decided that ports were not fit for such work. obstacles also arose in horizontal coordination and collaboration between local departments. a lack of information communication between local departments due to the unavailability of complete information in the early stages of prevention and control made it nearly impossible for effective collaboration. in regards to the division of labor and coordination between the central and local governments for disease prevention and control, some local governments held that the central government should have presented broader goals and authorized provinces and cities greater autonomy in their response measures. some felt that the central government should not have made influenza a (h n ) prevention and control an issue of political significance but should have been objective in understanding the differences between executive leadership and scientists' opinions. while the main duty of administrative leaders should have been to organize and mobilize social resources needed to cope with the epidemic, scientists should have been the ones to handle technical issues such as epidemic analysis and response measures. at the same time, more efficient communication should have been present between central and local departments tasked with specific operations. for example, some local management departments felt that the entire process was quite political, making some documents difficult to fully implement; in the two most volatile months that lasted from april th to june, documents were issued frequently, and in some cases were in conflict with one another and lacked integrity and continuity. in regards to adjustment of prevention and control strategies, some regions' health departments reported the following issues: higher-level departments frequently adjusted technical guidance and strategies for prevention and control, there was a wide variety of information reporting methods and they were constantly in flux, different departments formulated their own response requirements, and differences occurred in measures and standards; all of which greatly complicated local response operations. certain communication and coordination issues also existed within the health department's internal system. the moh internal horizontal collaboration needs to be strengthened epidemiological investigations, clinical diagnoses, and laboratory testing to combine the medical treatment and disease prevention. for example, the china cdc played a crucial role as a central technical support body of influenza a (h n ) prevention and control in epidemiological information collection, monitoring, analysis, and judgment, but at the same time it also had a lot of administrative duties, and its services and duties overlapped with those of the moh's bureau of disease control and prevention. there should be unified leadership and coordination between higher and lower-level health departments within the national epidemic prevention and control system. a certain degree of flexibility is also necessary as provinces differ in epidemic situations, medical resources, geographic features, and so on. in regards to information reporting within the health system, though the china cdc and the moh had established information systems relating to epidemic surveillance, including an epidemic direct reporting system, no information sharing mechanism was created between the china cdc and medical institutions; in particular, some county level medical institutions didn't even have sound data collection and reporting systems. this resulted in a single point of decision making and command, and their lack of network and information technology weakened the support they could've had in implementing response measures. npos such as the red cross society of china played important roles during the influenza a (h n ) prevention and control efforts. however, by comparison with developed countries, china still lags behind in terms of public participation in public health emergencies. there still remain limitations in skill and knowledge, as no emergency volunteer systems or working mechanisms were formed, and no leveraging of npo resources really occurred. on public emergency management bodies of the chinese government key: cord- -kc thr authors: bradt, david a.; drummond, christina m. title: technical annexes date: - - journal: pocket field guide for disaster health professionals doi: . / - - - - _ sha: doc_id: cord_uid: kc thr . humanitarian programs ; . security sector ; . health sector : core disciplines in disaster health . primary health care programs . disease prevention . clinical facilities . reproductive health . water and sanitation . food and nutrition . chemical weapons . epi methods ; . tropical medicine : tropical infectious diseases—vector-borne and zoonotic . tropical infectious diseases—non-vector-borne ; . epidemic preparedness and response ; . communicable disease control : diarrhea . influenza . malaria . measles . meningitis . viral hemorrhagic fever ; . diagnostic laboratory : indications, laboratory tests, and expected availability . specimen handling ; . acronyms ; this section provides guidance on technical issues in the health sector. the annexes contain compilations of frequently used reference information. • humanitarian programs-contains conceptual frameworks on global clusters, relief programs, humanitarian financing, and early recovery. • security sector-contains key definitions from the rome statute of the international criminal court • health sector-contains a broad range of core health technical information including environmental classification of water and excreta-related diseases, disease prevention measures, water treatment end points, anthropometric classifications, micronutrient deficiency states, management of chemical weapon exposures, and epi methods. • tropical medicine-contains clinical summaries of tropical infectious diseases with details on disease vector and host, clinical presentation, diagnostic lab tests, clinical epidemiology, and therapy. • epidemic preparedness and response-contains core principles of epidemic preparedness and response. • communicable disease control-contains an overview of selected communicable diseases of epidemic potential including diarrhea, influenza, malaria, measles, meningitis, and viral hemorrhagic fever. • diagnostic laboratory-contains guidance on lab specimen handling and testing. • acronyms-contains acronyms commonly used in disaster management and humanitarian assistance. a. in-kind donations (eg food, seeds, tools, fishing nets, etc) b. types of community projects in food-for-assets programs ( ) natural resources development (a) water harvesting (b) soil conservation ( ) restoration of agri(aqua)culture potential (a) irrigation systems (b) seed systems ( ) infrastructure rehabilitation (a) schools (b) market places (c) community granaries (d) warehouses (e) roads (f) bridges ( ) diversification of livelihoods (a) training and experience sharing . increase individual purchasing power a. cash distribution b. cash for work (cash for assets) c. vouchers d. micro-credit e. job fairs f . artisanal production g. livelihoods/income generation . support market resumption a. market rehabilitation b. infrastructure rehabilitation c. micro-finance institutions goals-protect what's left ( month), restore the system ( months), improve the system ( . promote transformational development support far-reaching, fundamental changes in relatively stable developing countries, with emphasis on improvements in governance and institutions, human capacity, and economic structure, so that countries can sustain further economic and social progress without depending on foreign aid. focus on those countries with significant need for assistance and with adequate (or better) commitment to ruling justly, promoting economic freedom, and investing in people. reduce fragility and establish the foundation for development progress by supporting stabilization, reform, and capacity development in fragile states when and where u.s. assistance can make a significant difference. . support strategic states help achieve major u.s. foreign policy goals in specific countries of high priority from a strategic standpoint. . international cooperation to protect lives and health . timely and sustained high-level political leadership to the disease . transparency in reporting of cases of disease in humans and in animals caused by strains that have pandemic potential to increase understanding, enhance preparedness, and ensure rapid and timely response to potential outbreaks . immediate sharing of epidemiological data and clinical samples with the world health organization (who) and the international community to characterize the nature and evolution of any outbreaks as quickly as possible . prevention and containment of an incipient epidemic through capacity building and in-country collaboration with international partners . rapid response to the first signs of accelerated disease transmission . work in a manner supportive of key multilateral organizations (who, fao, oie) . timely coordination of bilateral and multilateral resource allocations; dedication of domestic resources (human and financial); improvements in public awareness; and development of economic and trade contingency plans . increased coordination and harmonization of preparedness, prevention, response and containment activities among nations . actions based on the best available science . genocide (article )-acts committed with intent to destroy, in whole or in part, a national, ethnic, racial, or religious group a. killing members of the group b. causing serious bodily or mental harm to members of the group c. inflicting on the group conditions of life calculated to bring about its physical destruction in whole or in part d. imposing measures intended to prevent births within the group e. forcibly transferring children of the group to another group . crimes against humanity (article )-acts committed as part of a widespread or systematic attack against any civilian population, with knowledge of the attack a. murder b. extermination c. enslavement d. deportation e. imprisonment in violation of international law f. torture g. rape, sexual slavery, enforced prostitution, forced pregnancy, enforced sterilization, or other comparable form of sexual violence h. persecution on political, racial, national, ethnic, cultural, religious, gender, or other grounds universally recognized as impermissible under international law i. enforced disappearance j. apartheid k. other inhumane acts intentionally causing great suffering or serious injury to body or to mental or physical health . war crimes (article ) a. grave breaches of the geneva conventions of aug ( ) willful killing ( ) torture or inhumane treatment including biological experiments ( ) willfully causing great suffering ( ) extensive destruction and appropriation of property ( ) compelling a pow to serve in the armed forces of a hostile power ( ) willfully depriving a pow of the right to a fair trial ( ) unlawful deportation ( ) taking of hostages b. serious violations of laws and customs applicable in international armed conflict ( ) intentionally directing attacks against the civilian population or against civilians not taking direct part in hostilities ( ) intentionally directing attacks against civilian objects ( ) intentionally directing attacks against personnel, installations, material, units, or vehicles involved in humanitarian assistance or peacekeeping mission ( ) intentionally launching an attack in the knowledge that it will cause incidental civilian loss of life or severe damage to the natural environment ( ) attacking undefended towns, villages, dwellings, or buildings which are not military targets ( ) killing or wounding a combatant who has surrendered ( ) improper use of a flag of truce, flag or insignia or uniform of the enemy or of the un, or emblems of the geneva conventions resulting in death or serious personal injury ( ) transfer by the occupying power of parts of its own civilian population into the territory it occupies, or the deportation or transfer of all or parts of the population of the occupied territory within or outside the territory ( ) intentionally directing attacks against buildings dedicated to religion, education, art, science, charitable purposes, historic monuments, hospitals, and places where sick are collected, provided they are not military objectives ( ) subjecting persons to physical mutilation or to medical or scientific experiments which are not justified by the medical treatment nor carried out in his/her interest ( ) killing or wounding treacherously individuals belonging to the hostile nation or army ( ) declaring that no quarter will be given ( ) destroying or seizing the enemy's property unless such be imperatively demanded by the necessities of war ( ) declaring abolished, suspended, or inadmissible in a court of law the rights and actions of the nationals of the hostile party ( ) compelling the nationals of the hostile party to take part in the operations of war directed against their own country ( ) pillaging a town or place, even when taken by assault ( ) a range of generic prevention measures should be considered for its impact on diseases in a biological "all-hazards" environment. overall, excreta disposal, water quantity, personal hygiene, and food hygiene commonly contribute more to environmental health than do other listed measures. epidemic threats will oblige heightened consideration of disease-specific strategies for prevention and control. c. water treatment (bold text of particular relevance in clinical facilities) ppm = mg/kg (solids) = mg/l (liquids) = ug/ml (liquids) = basic unit of measure for chloroscopes : , ppm = % • sam = whz < − , muac < . cm, or bilateral pitting edema (who). whm not in definition. • sam prevalence worldwide ≈ , , . • sam mortality ≈ x mortality of normally nourished child and its cfr can be - %. • gam = mam + sam • gam = moderate wasting cases, severe wasting cases, or bilateral pitting edema cases (where due to malnutrition) • underweight is not used for screening or surveys in nutritional emergencies. it reflects past (chronic) and present (acute) undernutrition and is unable to distinguish between them. it encompasses children who are wasted and/or stunted. however, weight gain over time can be a sensitive indicator of growth faltering which is easily tracked on road to health charts. • stunting generally occurs before age . it is irreversible. • stunting prevalence worldwide ≈ , , . • stunting is not a good predictor of mortality, but the cfr from ids in cases of severe stunting ≈ x the cfr from ids in cases without stunting. reference standards can be absolute muac, centile, % of median reference, or z scores: • muac easy to understand. an excellent predictor of mortality. permits comparisons between age groups insofar as the low growth velocity of muac in the u age group makes data roughly comparable. may be used alone in "quick-and-dirty" convenience samples to estimate local prevalence of wasting. however, not used alone in authoritative anthropometric surveys, and is commonly part of a two stage screening process to determine eligibility for feeding programs. • overall whz gives higher prevalence of malnutrition than whm for the same population. this is most marked where there is low baseline prevalence of disease, and especially for adolescents (who get subsequently over-referred). whz is more statistically valid, but whm is better predictor of mortality and is used for admission to tfcs. weight-for-age is influenced by weight-for-height and height-for-age. it can be difficult to interpret. b. adults and adolescents (o ) anthropometrics: bmi = weight (kg) / height (m) . death rates-calculated incidence of death expressed per , p/d or per p/mo; data collected by retrospective surveys (eg month period) to gauge severity of public health emergency particularly where sudden events lead to spike in mortality a. cdr-crude death rate b. asdr-age-specific death rate (eg u dr or death rate of children - yr) during a studied time interval (written as . mortality rates-calculated probability of dying before a specified age expressed per live births; data collected by national health authorities in periodic (annual) demographic surveys to reflect ongoing health status a. cmr-calculated probability of mortality in given population for specific time b. imr-calculated probability of a live borne child dying before yr c. u mr-calculated probability of a live borne child dying before yr nb mr ≠ dr. eg cmr ≠ cdr, u mr ≠ u dr. different rates measure different things and are not directly comparable. however, mrs may be converted into drs by the following: cdr or u dr (deaths/ , /d) = − ln( −p/ ) × . where p = cmr or u mr (deaths/ live births). however, this has little field utility. nb mmr-maternal mortality ratio has different units in numerators (maternal deaths) and denominators (live births), thus is a ratio, not a rate the application of study findings to an entire population from which the sample was drawn. if the survey was well-conducted, the results may be considered representative of the entire population. this is scientifically justified. however a confidence interval should accompany any parameter estimate of that population. extrapolation the extension of study findings to a population or period which was not represented in the sample. it works by association-if populations appear to be experiencing similar conditions, the morbidity/mortality experience of one may be imputed to the other. this is not scientifically justified, but is often done where data are insufficient or impossible to collect. s/sx think differential diagnosis (below). . severe muscle pain may be a symptom of sepsis even without fever. . elderly patients with sepsis may be afebrile. in elderly patients, fever is rarely caused by a viral infection. . septic patients who are hypothermic have a worse prognosis than those with high fever. treat as a medical emergency. . fever in a postoperative patient is usually related to the surgical procedure (eg pneumonia, uti, wound, or deep infection). . fever with jaundice is rarely due to viral hepatitis. think liver abscess, cholangitis, etc. . the rash of early meningococcal infection may resemble a viral rash. . generalized rashes involving the palms and soles may be due to drugs, viral infections, rickettsial infections, or syphilis. . all febrile travelers in or returned from a malaria infected area must have malaria excluded. . disseminated tb must be suspected in all elderly patients with fever and multisystem disease who have been in an area with endemic tb. . septic arthritis may be present even in a joint which is mobile. . back pain with fever may be caused by vertebral osteomyelitis or an epidural abscess. . a patient may have more than one infection requiring treatment (eg malaria and typhoid), especially if they are elderly, immunosuppressed, or have travelled. . always remember common infections, not just opportunistic infections, in aids patients with a fever. understand morbidity multipliers-measles, malnutrition, and tb/hiv. understand occult co-morbidities. for any undifferentiated illness, even in infants, think of hiv, tb, syphilis, and sarcoid. for any child, think of malaria, hookworm, and anemia. malarial anemia usually in pedes < year-old; hookworm anemia usually in pedes > year-old. for any icp, think of tb, vl, histoplasmosis, and strongyloides. must treat early. watch for clinical mimics-malaria presenting as pneumonia or diarrhea in pedes; vl presenting as malaria in adults; lepto presenting as mild df (esp in df endemic areas where the pt has mild onset of illness, worsening course, and no rash but jaundice). tx do basic things well, use equipment you understand, teach others, delegate. this annex profiles selected communicable diseases of epidemic potential whose incidence, management complexity, or mortality obliges particular attention. • if (+) agglutination to o antisera, then the strain is further tested for agglutination to antiserum of ogawa and inaba serotypes. • if (+) agglutination to o antisera, then the strain is not further subdivided (except as producer or non-producer of ct as noted below). • if (−) agglutination to o and o antisera, then the strain is known as non-o , non-o v. cholerae. a strain is further identified as a producer or non-producer of cholera toxin (ct). ct production is a major determinant of disease development. strains lacking ct do not produce epidemics even if from the o or o serogroup. • serogroup o exists as main biotypes-classical and el tor-though hybrids also exist. each biotype occurs as two serotypes-ogawa and inaba. classic biotype caused the th and th pandemics but little epidemic disease since the s though it still causes cases in india. el tor biotype caused the th (current) pandemic and almost all recent outbreaks. el tor was first isolated in in el tor, egypt after importation by indonesian pilgrims travelling to mecca. it survives longer in the environment and produces ct similar to the classical biotype. presumably because of ct pathogenicity, the % of cholera patients with severe disease has doubled over the past yrs. these patients tend to require iv fluid therapy. • serogroup o may have evolved from strains of o el tor as they share many properties though not agglutination. in spring of in dhaka, o cases exceeded o el tor cases for the first time, and it was postulated that o may become the cause of an th pandemic. however, since then, o has again become dominant. infective dose depends on individual susceptibility. relevant host factors include immunity produced by prior infection with serogroup o as well as stomach acidity. id may be , orgs, so personal hygiene plays a lesser role than in shigellosis where the id is much lower. shigella has species. • s. dysenteriae type (sd or shiga bacillus) causes the severest disease of all shigella sp because of its neurotoxin (shiga toxin), longer duration of illness, higher abx resistance, higher cfr thru invasive complications, and great epidemic potential. • s. flexneri is the most common, and is generally endemic, in developing countries • s. sonnei is the most common in industrial countries • s. boydii and s. sonnei give mild disease. some kinds of e. coli produce a shiga toxin. shiga toxin genes reside in a bacteriophage genome integrated into the bacterial chromosome. some abx, eg fluoroquinolones, induce expression of phage genes. the bacteria that make these toxins are variously called "shiga toxin-producing e. coli" (stec), "enterohemorrhagic e. coli" (ehec), or "verocytotoxic e. coli" (vtec). all terms refer to the same group of bacteria. • e. coli o :h (often called "e. coli o " or "o ") is the most commonly identified stec in north america, and it causes most e. coli outbreaks. approximately - % of ehec infections result in hus. • non-o stec serogroups also cause disease. in the usa, serogroups o , o , and o are the most commonly identified e. coli pathogens overall. weather (esp weeks - in apr-may) creating increased biological activity; post-monsoon (esp weeks - in aug-sep) with contamination of water sources. pre-monsoon epidemics are generally worse than postmonsoon ones. dysentery has low level year-round incidence, but epidemics occur roughly each decade. epidemic strains display new, additive antibiotic resistance which probably triggers the epidemic. once resistant strains have become endemic, antibiotic susceptibility rarely reappears. sd acquires resistance quickly. sf acquires it more slowly, and that resistance may wane with decreasing abx pressure. at icddr, annual proportional incidence approximates the following: clean water and waste management especially for cholera. personal hygiene (hand washing with soap and clean towels) especially for shigella. water safe drinking water (boiled, chlorinated) nb sphere standards are not enough-you need increased quantities of chlorinated water at household level. san clean latrines for safe disposal of excreta hand washing with soap food safe food (cooked, stored) breast feeding fomites safe disposal of dead bodies with disinfection of clothing nb after outbreak of a fecal-oral pathogen, food hygiene and funereal practices may influence human-to-human transmission more than water quality. health education to affected population wash hands with soap: after using toilets/latrines. after disposing of children's feces. before preparing food. before eating. before feeding children. dukoral has been the main vaccine considered for use in high-risk populations. • morc-vax and shanchol-similar to dukoral except they do not contain the rbs, hence do not require a buffer, and are / the cost to produce. morc-vax, produced in vietnam, is derived from a vaccine administered to millions of people since , but is not who pre-qualified, and is not expected to have international distribution. • shanchol, produced in india, has international distribution (eg used in the haiti cholera vaccination campaign of ), and is now the agent of choice for who. it confers immunity d p nd dose, effectiveness > % at mo, and protection > % at yr. also confers short-term protection vs etec. dose: . cc vaccine followed by water ingestion but no fasting needed; doses, wks apart; cold chain required except for day of use. • orochol-bivalent formulation as in dukoral without rbs of ct. dose: single dose. no longer manufactured. who recommendations: "vaccination should not disrupt the provision of other high-priority health interventions to control or prevent cholera outbreaks. vaccines provide a short-term effect that can be implemented to bring about an immediate response while the longer term interventions of improving water and sanitation, which involve large investments, are put into place." [ ] icddr recommendations: "because of limitations in terms of transport, formulation, and cost of the current dukoral vaccine, the cots program does not require the utilization of the vaccine during an outbreak; it is not necessary to vaccinate to overcome an outbreak. however, if dukoral is readily available and staff are properly trained in its use according to the guidelines that come with the vaccine, the cots program permits dukoral's use (ideally before an outbreak) in the following high-risk populations: refugee populations in which cholera is present, health care workers managing cholera cases, and communities in which the incidence rate is greater than in annually." [ ] epidemiological surveillance (specific to cholera) epidemiological assumptions (who, cots): estimated attack rates: - % extremely vulnerable hosts and poor environmental health (who) % (refugee camps with malnutrition) (cots) % (rural communities of < p) (cots) % (severe epidemic-good estimate of ultimate disease burden) (who) . % (endemic areas with bad sanitation) (cots) . % (endemic areas in open settings-suitable for initial calculations of early resource requirements) nb overall, % of cases are mild and difficult to distinguish from other types of d. nb asymptomatic carriers are very common ( x # of cases). referral rates for ivs % of cases (much higher- % at icddr as it shortens recovery time) case fatality ratios % (with good care) the following catchment populations will yield acute pts of whom will be severely dehydrated: refugee camp of people (ar of % = pts) open settings in endemic area with , people (ar . % = pts) a population of , infected individuals in an epidemic area will yield the following (who): population infected , clinical cases , ( % of infected population) cases needing early resources ( % of cases) cases needing iv therapy ( % of cases) anticipated deaths ( % cfr) nb in non-endemic areas, ar adults > ar pedes because adults have higher exposure risks. in endemic areas, ar pedes > ar adults because adults have been exposed since childhood delivery of health services shigella are fragile and difficult to recover if transport time > d. - isolates initially to confirm outbreak - isolates initially to create abx use policy (bacterial resistance renders cotrimoxazole, amp/amox, nalidixic acid, and tetracycline unusable) - isolates monthly from ipd and opd before abx therapy to assess evolving abx resistance - isolates periodically to reference laboratory to confirm abx resistance patterns and undertake molecular studies isolates at end of the outbreak to confirm that new diarrheas are not epidemic pathogens nb systematic sampling is most representative-eg every th pt or all pts q weeks adjusted as needed to collect the necessary specs. sensitivity > > important than specificity in rdt screening during an epidemic. pts from one geographic area are more likely to constitute a cluster involving a new pathogen. an area may be considered cholera-free after incubation periods (total of d) have passed without cholera disease. however, hospital monitoring should continue for a year due to tendency of enteric pathogens to re-emerge long after they are declared gone. cholera may be viable but nonculturable from the environment; environmental monitoring has many false negatives. consider improvements to existing diagnostic labs • hotline set up for reporting of rumor this often translates into a hastily conceived vaccination campaign that distracts from core principles of cholera management. for every symptomatic pt, there may be asymptomatic carriers. in an established epidemic, the affected community is already extensively infected. cholera vaccination, under these circumstances, has little public health benefit for the resource investment. if undertaken, the following will apply: • vaccination campaign requires numerous staff. community mobilizers are key. clinical staff should not be poached from their clinical duties. supervisors must be free to move at will. • logistics is key-if the st day goes badly, the campaign goes badly. • mark the domiciles which are done. • hold after-action meetings each day. • last day, use mobilizers with mobile broadcasting to attract those who missed out. • second phase vaccination should include chws with multi-purpose messages on water and sanitation. avoid: press exaggeration abx prophylaxis reliance on ivf and insufficient ors lab investigation of cases once epidemic etiology is ascertained prolonged hospitalization hospital discharge criteria requiring multiple negative stool cultures enthusiasm for ocv during epidemic exaggerated water purification objectives concentration of technical competencies in moh at expense of districts failure to share information with stakeholders influenza viruses comprise genera-influenza types a, b, and c-each with species. • influenza type a is divided into subtypes based upon serological response to hemagglutinin (ha) and neuraminidase (na) glycoproteins. there are different ha subtypes and different na subtypes. h n , h n , and h n are responsible for the major human pandemics in the last century. h n virus circulated between and but currently does not. only influenza a subtypes infect birds, and all subtypes can do so. bird flu viruses do not usually infect humans. but, in , an outbreak of h n avian influenza in poultry in hong kong marked the first known direct human transmission of avian influenza virus from birds to humans. since then, h , h , and h avian influenza subtypes have been shown to infect humans. • influenza type b is morphologically similar to a and also creates seasonal and epidemic disease. • influenza type c is rare but can cause local epidemics. seasonal human influenza vaccine currently has strains-h n /h n /b. influenza disease in humans has a short incubation period ( - d). early symptoms are non-specific. it is highly infectious, especially early in the course of the disease, with a large # of asymptomatic carriers. transmission potential (r ) is a function of infectivity, period of contagiousness, daily contact rate, and host immunity. in general, the faster the transmission, the less feasible is interrupting transmission thru usual disease control tools of case finding, isolation, contact tracing, and ring vaccination. • specific groups of exposed or at risk in the community-most likely to work when there is limited disease transmission in the area, most cases can be traced to a specific contact or setting, and intervention is considered likely to slow the spread of disease eg quarantine of groups of people at known common source exposure (airplane, school, workplace, hospital, public gathering; ensure delivery of medical care, food, and social services to persons in quarantine with special attention to vulnerable groups) (useless once there is community-based spread) eg containment measures at specific sites or buildings of disease exposure (focused measures to > social distance) cancel public events (concerts, sports, movies) close buildings (recreational facilities, youth clubs) restrict access to certain sites or buildings • community-wide measures (affecting exposed and non-exposed)most likely to work where there is moderate to extensive disease transmission in the area, many cases cannot be traced, cases are increasing, and there is delay between sx onset and case isolation. infection control measures ari etiquette-cover nose/mouth during cough or sneeze, use tissues, wash hands avoidance of public gatherings by persons at high risk of complications nb use of masks by well persons is not recommended "snow" (stay-at-home) days and self-shielding (reverse quarantine) for initial d period of community outbreak-may reduce transmission without explicit activity restrictions closure of schools, offices, large group gatherings, public transport (pedes more likely to transmit disease than adults) nb community quarantine (cordon sanitaire)-restriction of travel in and out of an area is unlikely to prevent introduction or spread of disease anopheles vector biology egg becomes adult mosquito in d adult mosquito becomes infective in d after bite on infected host susceptible human host becomes infective in d after bite from infected mosquito :. earliest human clinical disease in d after eggs are laid follow the -d rule: dusk and dawn stay indoors as much as possible with window screens in good repair dress in light colored long sleeve shirts and long pants when outside identify cause of the outbreak undertake vaccination campaign strengthen routine immunization and surveillance meningitis is a disease with significant mortality. meningococcus (neisseria meningitides) is renown for its rapid onset, rapid progression (death sometimes within hours), and high mortality ( % untreated). there are serogroups of neisseria meningitides but only (a, b, c, w, x, y) are known to cause epidemics. the bacteria spread from person to person via respiratory and nasal secretions. polysaccharide vaccines are available with serotypes (a and c), serotypes (a, c, and w) or serotypes (a, c, w, and y). duration of immunity is approximately years. meningococcal protein conjugate vaccines confer longer immunity but at higher cost than polysaccharide vaccines. monovalent conjugate vaccine against group c dates from , and tetravalent (a, c, w, and y) conjugate vaccine dates from . group b vaccine made from bacterial proteins has been licensed since but is not readily available. meningococcal vaccines have a very low incidence of side effects. regular disease surveillance is necessary to detect outbreaks. the epidemic threshold is suspected cases/ , population in any given week. two suspected cases of meningitis in the same settlement should trigger an outbreak investigation. nasopharyngeal carriage rates do not predict epidemics. - % of meningococcal disease presents with meningitis. % of cases occur in patients < y/o. peak incidence in meningitis belt is ages - yrs. diagnosis is straightforward when patient presents with signs of meningitis-fever, headache, vomiting, changes in mental status. however, most patients have non-specific illness - days before onset of meningitis. cfr of untreated meningococcal meningitis can be %. cfr of properly treated meningococcal meningitis is < %. - % of meningococcal disease presents with septicemia unaccompanied by meningitis or other focal features. it is a dramatic illness which affects previously healthy children and young adults. it presents with acute fever leading to purpura fulminans (hemorrhagic or purpuric rash), shock, and waterhouse-friderichsen syndrome (acute adrenal failure). etiologic diagnosis can be easily missed. cfr of meningococcal septicemia is % and may be % even with proper treatment. diagnosis may be confirmed by agglutination tests, polymerase chain reaction, culture and sensitivity testing of spinal fluid and blood. in many situations, these tests are not available. throat swabs may be helpful on occasions. do not delay treatment for tests or test results. minutes count. it is more important to have a live patient without a confirmed diagnosis than a dead one with a diagnosis. differential diagnosis in a tropical patient with fever and altered mental status, but without purpura or shock, includes cerebral malaria. co-infection may occur. standardized case management of bacterial meningitis in developed countries involves - days of parenteral antibiotic therapy. drug of choice in adults and older children is ceftriaxone which also rapidly eliminates the carrier state. alternate drugs include ampicillin and benzylpenicillin which do not eliminate the carrier state. in developing countries, days of parenteral antibiotic therapy are empirically shown to be effective. in large epidemics in resource-poor settings, a single im dose of chloramphenicol in oil is the drug of choice. for patients who do not improve in hours, a repeat dose may be given. viral meningitis is rarely serious and requires only supportive care, recovery is usually complete. patient isolation and disinfection of the room, clothing, or bedding are not necessary. respiratory precautions are advised particularly early in the course of treatment. chemoprophylaxis of contacts is available in some settings but rarely in the disaster setting. vigilance and education of close contacts is mandatory. epidemic preparedness and early detection of outbreaks are key. vaccines against n. meningitides serogroups a, c, y and w are very effective in controlling epidemics. in epidemic settings, children - are the priority target with serogroups a and c typically the priority antigens. rapid mass vaccination campaigns can contain outbreaks in - weeks. for immunocompetent patients over years, vaccine efficacy rate is % one week after injection. however, duration of immunity may be as little as years in younger children. in some countries, vaccine may also be used with close contacts of sporadic disease cases to prevent secondary cases. chemoprophylaxis of contacts is not recommended in epidemics, but community education and ready access to health care are essential. preventive medicine [ ] source control/reduction/elimination undertake quarantine and culling of sick reservoir animals and known disease carrier species. avoid unnecessary contact with or consumption of dead reservoir animals or known disease carrier species. avoid unnecessary contact with suspected reservoir animals and known disease carrier species (eg primates). avoid direct or close contact with symptomatic patients. establish appropriate communicable disease controls for burial of the dead. administrative controls (improve people's work practices) environmental and engineering controls (isolate people from the hazard) avoid needle stick exposure to blood specimens thru automated machine handling. ppe (protect people with ppe) use standard precautions-gloves, masks, and protective clothing-if handling infected animals or patients. wash hands after visiting sick patients. active surveillance and contact tracing (enhanced surveillance) through community-based mobile teams active case finding (screening and triage) and contact tracing dedicated isolation facility food provision to isolated patients so they are not dependent on family case definition treatment protocols emphasizing supportive care and treatment of complications essential drugs referral guidelines secondary prevention barrier nursing strictly enforced family and community education ministerial task force to address policy local health authority task force to address procedures national level task forces to comprise if a lab is not available, then you need a sampling strategy that addresses specimen acquisition, preparation, and transportation in compliance with international regulations on the transport of infectious substances. guidance note on using the cluster approach to strengthen humanitarian response international conference on primary health care selective primary health care-an interim strategy for disease control in developing countries water and excreta-related diseases: unitary environmental classification infections related to water and excreta: the health dimension of the decade world health organization. cholera vaccines: who position paper available from: international centre for diarrhoeal disease research history and epidemiology of global smallpox eradication available from: us department of health and human services communicable disease control in emergencies-a field manual. geneva: world health organization ebola: technical guidance documents for medical staff world health organization. manual for the care and management of patients in ebola care units/community care centers-interim emergency guidance. who/ evd/manual/ecu/ . . geneva: world health organization what tests does it perform? is there transport to and from the laboratory? who prepares transport media? who provides specimen collection material and supplies? how can these supplies be obtained? who provides cool packs, transport boxes, car, driver …? • refrigerate other vials for cytology, chemistry ( °c) leak-proof specimen container wrapped with enough absorbent material to absorb the entire content of the st container . leak-proof secondary container usually plastic or metal . outer shipping container whose smallest dimension is mm diagnostic specimens use iata packing instruction without biohazard label. infectious materials use iata packing instruction with biohazard label. what to send with the sample? lab request form with: • sender's name and contact info • patient name, age, sex • sample date, time • suspected clinical diagnosis with main signs and symptoms • sample macroscopic description • context-outbreak confirmation, ongoing verification, outbreak end, etc • epidemiological or demographic data where to send the sample? • reference lab • contact person what and when to expect results? source: world health organization world health organization department of communicable disease surveillance and response. highlights of specimen collection in emergency situations. undated . designate a lead official in the lcc. . anticipate roles for partner agencies (eg inter-agency and team coordination, disease surveillance, field epidemiological investigation, laboratory identification, case management guideline development, outbreak logistics, public information, and social mobilization). . identify sources of funds. . intensify disease surveillance. . identify reference lab(s) for communicable diseases of epidemic potential. . ensure mechanism for specimen transport. a. initial response to suspected outbreak . form an emergency team to investigate and manage the outbreak a. identify key roles on the outbreak investigation team(s) ( ) epidemiology and surveillance ( ) case management ( ) water and sanitation ( ) laboratory services ( ) communication b. staff those roles ( ) epidemiologist-to monitor proper data collection and surveillance procedures ( ) physician-to confirm clinical s/sx and train health workers in case management ( ) water and sanitation expert-to develop a plan for reducing sources of contamination ( ) microbiologist-to take environmental/biological samples for laboratory confirmation, train health workers in proper sampling techniques, and confirm use of appropriate methods in the diagnostic laboratory ( ) key: cord- -s qd x b authors: cadwell, ken; debnath, jayanta title: beyond self-eating: the control of nonautophagic functions and signaling pathways by autophagy-related proteins date: - - journal: j cell biol doi: . /jcb. sha: doc_id: cord_uid: s qd x b the identification of conserved autophagy-related proteins (atgs) that mediate bulk degradation of cytosolic material laid the foundation for breakthroughs linking autophagy to a litany of physiological processes and disease conditions. recent discoveries are revealing that these same atgs orchestrate processes that are related to, and yet clearly distinct from, classic autophagy. autophagy-related functions include secretion, trafficking of phagocytosed material, replication and egress of viral particles, and regulation of inflammatory and immune signaling cascades. here, we define common processes dependent on atgs, and discuss the challenges in mechanistically separating autophagy from these related pathways. elucidating the molecular events that distinguish how individual atgs function promises to improve our understanding of the origin of diseases ranging from autoimmunity to cancer. twenty five years ago in the journal of cell biology, professor yoshinori ohsumi and colleagues published the first of several landmark papers demonstrating molecular control of macroautophagy in response to nutrient starvation in saccharomyces cerevisiae (takeshige et al., ) . thereafter, several groups identified autophagy-related proteins (atgs), evolutionarily conserved molecules that control fundamental aspects of the macroautophagy pathway, including the formation of autophagosomes, double membrane vesicles that capture cellular cargo and subsequently deliver them to the lysosome for degradation (tsukada and ohsumi, ; thumm et al., ; harding et al., ) . since the discovery of atgs, an explosion of research on autophagy has led to seminal advances in understanding the molecular regulation of the autophagy trafficking process, dissecting how autophagy controls cell survival and metabolic fitness in response to countless stressors, and illuminating the diverse functions of the autophagy pathway in both normal physiology and disease (choi et al., ; kaur and debnath, ) . at the same time, we have begun to appreciate that various atgs and other autophagy regulators are deployed in assorted fundamental processes that are distinct and separable from their wellestablished roles in mediating autodigestion via the lysosome. this review highlights this exciting new facet of autophagy research and summarizes our current understanding of these autophagy-related functions and signaling pathways mediated by individual atgs as well as entire cell biological subroutines using multiple components of the autophagy machinery. autophagy consists of three cellular self-eating mechanisms that converge on the lysosome: microautophagy, chaperonemediated autophagy, and macroautophagy. among these, macroautophagy (hereafter called autophagy) is the most well studied and genetically controlled by atgs. classic autophagy proceeds through multiple "canonical" steps that include ( ) initiation by an autophagy-inducing signal, ( ) nucleation of an isolation membrane or phagophore assembly site, ( ) elongation and sealing of this double membrane around the cargo to be sequestered to form an autophagosome, ( ) docking and fusion of the autophagosome with the lysosome to form an autolysosome, and ( ) degradation of the vesicle contents by lysosomal enzymes ( fig. a) . initiation, nucleation, and elongation require the hierarchical recruitment and activity of ∼ atgs and other proteins to the phagophore assembly site to construct the autophagosome (codogno et al., ; mizushima et al., ) . in this context, the term "noncanonical autophagy" refers to the formation of classic double membrane autophagosomes that does not require the activity of one or more key atgs. nonetheless, both canonical and noncanonical autophagy are fundamentally autodigestive pathways requiring autophagosome formation, followed by fusion with the lysosome (codogno et al., ). adding to this complexity, atg proteins, both individually and as part of larger networks, control pathways that either do not involve the formation of a classic autophagosome or do not terminate in lysosomal degradation and nutrient recycling (subramani and malhotra, ) . although not inclusive, the list of autophagy-related processes includes secretion and exocytosis, lc -associated phagocytosis (lap), viral replication the identification of conserved autophagy-related proteins (atgs) that mediate bulk degradation of cytosolic material laid the foundation for breakthroughs linking autophagy to a litany of physiological processes and disease conditions. recent discoveries are revealing that these same atgs orchestrate processes that are related to, and yet clearly distinct from, classic autophagy. autophagy-related functions include secretion, trafficking of phagocytosed material, replication and egress of viral particles, and regulation of inflammatory and immune signaling cascades. here, we define common processes dependent on atgs, and discuss the challenges in mechanistically separating autophagy from these related pathways. elucidating the molecular events that distinguish how individual atgs function promises to improve our understanding of the origin of diseases ranging from autoimmunity to cancer. and exit, antigen presentation, and atg-mediated regulation of inflammatory and immune signaling. importantly, these processes are fundamentally distinct from classic autophagy, and certainly, many can be construed as "noncanonical" functions for the individual atg proteins that are involved. however, they are not by definition "noncanonical autophagy" in its strictest sense. hence, to avoid confusion, the term "noncanonical" should be avoided when referring to these autophagy-related processes. in the following sections, we overview our current understanding of this diverse collection of autophagy-related processes that are distinct from classic autophagy. in addition to its established role in lysosomal degradation, the autophagy machinery controls extracellular secretion. evidence to date most notably implicates atgs in unconventional secretion of proteins lacking an n-terminal signal sequence (dupont et al., ; deretic et al., ; malhotra, ; subramani and malhotra, ) . whereas the majority of eukaryotic secretory proteins classically transit to the surface via the er and golgi apparatus, a growing list of proteins traffic through unconventional mechanisms that do not require insertion into the er and/or bypass the golgi (rabouille et al., ; malhotra, ) . in addition, some classically secreted proteins appear to be preferentially rerouted through unconventional pathways to facilitate trafficking during stress (gee et al., ) . studies to date have uncovered clear genetic requirements for two proteins originally implicated in the stacking of the golgi apparatus, gra sp and gra sp , as well as atgs in mediating these alternative secretory pathways (rabouille et al., ; malhotra, ; zhang and schekman, ) . atgs have been genetically linked to the unconventional secretion of acyl-coa-binding protein acb in yeast (acba in dictyostelium discoideum; duran et al., ; manjithaya et al., ) , several inflammatory mediators in mammalian cells, including il- β and il- ; the high mobility group protein b (hmgb ); and finally, the plasma membrane trafficking of the integral membrane protein Δf cftr (dupont et al., ; figure . classic autophagy compared with related trafficking pathways. (a) classic autophagy: diverse stimuli elicit the hierarchical recruitment and activity of multiple atgs (yellow) and other regulatory proteins (blue) to construct the double membrane autophagosome. the lipidation of lc (lc -ii) is crucial for the capture of autophagic cargo and to stabilize of the inner autophagosomal membrane. the autophagosome subsequently fuses with the lysosome in a stx -dependent manner, resulting in degradation of the vesicle contents by lysosomal enzymes. (b) secretory autophagy: atgs mediate the unconventional secretion of multiple proteins (e.g., acb in yeast, and il- β, il- , and hmgb in mammalian cells) that lack an n-terminal signal sequence. these targets are postulated to be released via several putative mechanisms. first, the atg conjugation machinery promotes the formation of an lc + autophagosome-like intermediate, and the contents enwrapped within the inner membrane of autophagosome are released extracellularly instead of degraded in lysosomes. second, targets of secretory autophagy, such as il- β, are translocated into the intramembrane space of an lc + double membrane vesicular intermediate that fuses directly with the plasma membrane or fuses with a mvb intermediate that is secreted. last, although formal experimental evidence is lacking, secretory autophagy may involve an mvb/amphisome intermediate and the exocytic release of small extracellular microvesicles. regardless of the exact pathway, recent work indicates that secretory autophagy proceeds through a dedicated sna re machinery, which diverts secreted targets away from the lysosome and toward the plasma membrane (pm). (c) lap: the phagocytosis of pathogens and other prey in certain cell types (e.g., macrophages and dendritic cells) recruits uvr ag and rubicon (rub cn), thereby activating the beclin- -vps complex to generate phosphatidylinositol -phosphate and nox , an nad ph oxidase that generates ros inside the phagosome. this subsequently triggers the recruitment and activation of the atg conjugation machinery, which mediates lc -ii at the single membrane phagosome. lc -ii expedites fusion to lysosomes and degradation of the offending pathogen. gee et al., ) . to date, apart from these limited targets, the broader autophagy-dependent secretome remains uncharacterized. accordingly, recent quantitative proteomic analysis of the secretome from atg -deficient macrophages has uncovered new leaderless proteins that may be secreted in an autophagydependent manner (kimura et al., ) . however, it remains unclear from these genetic loss-of-function studies whether the observed secretory defects represent a direct versus indirect consequence of impaired autophagy. the mechanistic underpinnings of secretory autophagy are only beginning to emerge, and numerous questions remain unaddressed. first, despite the genetic interconnections between atgs and grh , the yeast gra sp orthologue, in s. cerevisiae, recent work questions whether autophagy and grh truly converge on a common secretory pathway (cruz-garcia et al., ) . second, although atgs that promote early autophagosome formation are genetically required for unconventional secretion, it is unclear whether secretory autophagy targets are actually captured into the autophagosomal lumen ( fig. b) . in fact, recent work demonstrates that il- β secretion requires atgs but proceeds via translocation into the intermembrane space of the autophagosome (zhang et al., ; fig. b) . third, it remains unclear how secreted targets are transported to the cell surface. because evidence supports that contents of multivesicular bodies (mvbs) can be directly exported to the cell surface, most notably the release of small extracellular microvesicles (exosomes), a role for the late endocytic pathway seems attractive (colombo et al., ; fig. b) . at the same time, recent evidence demonstrates interconnections between autophagy and the retromer complex, a protein assembly implicated in plasma membrane exocytosis of diverse molecules from early endosomes (steinberg et al., ) . during metabolic stress, the induction of autophagy elicits lc + autophagic compartments that bind and sequester a key inhibitor of retromer complex, the rabgap protein tbc d ; as a result, autophagy activates the retromer-driven translocation of proteins to the plasma membrane surface, most notably the glucose transporter glut / slc a (roy et al., ) . last, defining the mechanisms by which targets of secretory autophagy are diverted away from lysosomal degradation remains an important question for further study. indeed, recent work indicates that secretory autophagy involves autophagosome-like vesicles that bypass stx dependent fusion with lysosomes; rather, they use the sna re protein sec b in combination with plasma membrane syntaxins to complete cargo secretion (kimura et al., ; fig. ). in addition to unconventional secretion, atgs promotes the efficient egress of secretory lysosomes in osteoclasts (de-selm et al., ) and the conventional secretion of cytokines during oncogene-driven cancer cell invasion and senescence (narita et al., ; lock et al., ) . further dissecting the cellular mechanisms through which autophagy mediators facilitate these diverse secretory processes remains an important topic for future study. studies of lap poignantly illustrate how key elements of the core autophagy machinery can be redirected toward lysosomal pathways that are distinct from the canonical autophagosome-to-lysosome cascade. lap represents a process akin to macroautophagy in which phagosomes engulf extracellular contents, such as microorganisms or dying cells, which are subsequently trafficked to the lysosome (sanjuan et al., ) . during lap, elements of the autophagy machinery are recruited to phagosomes, upon which they facilitate maturation and the digestion of phagosomal contents (fig. c) . originally discovered during phagocytosis of particles containing toll-like receptor (tlr) ligands, lap involves the recruitment of the beclin- (becn )-vps complex to the phagosome, resulting in production of phosphatidylinositol -phosphate and the subsequent formation of phosphatidylethanolamine lipidated, membrane-bound lc (lc -ii; (sanjuan et al., ) . lc -ii facilitates phagosome maturation in lap, probably by recruiting molecules that enhances fusion to the endolysosomal compartment, such as the homotypic fusion and protein sorting complex involved in rab -mediated lysosomal fusion (mcewan et al., ) . in addition to beclin- , multiple atgs are required for lap, including all key elements of the lc conjugation machinery (atg , atg , atg , atg , and atg l; martinez et al., ) . despite this common utilization of multiple atgs, lap is a mechanistically distinct process. during classic autophagy, lc lipidation occurs on early double membrane structures, called phagophores, and functions in the capture of autophagic cargo and to enhance the stability of the inner autophagosomal membrane (stolz et al., ; tsuboyama et al., ) . in contrast, during lap, components of the autophagy conjugation machinery recruit lc -ii directly onto phagosomes, which are single membrane organelles. as a result, lap does not use the ulk (unc- -like autophagy activating kinase) complex that initiates canonical autophagy (martinez et al., ) . notably, similar pathways direct lc conjugation onto single-membrane organelles during entosis and macropinocytosis, two processes that also converge on the lysosome (florey et al., ) . furthermore, lap uses a unique vps complex, composed of beclin- , uvr ag, and rubicon (rub cn), a protein that is inhibitory to classic autophagy (martinez et al., ) . last, generation of reactive oxygen species (ros) by nad ph oxidase- (nox ) has also been critically implicated in the control of lap (martinez et al., ) . lap serves as a host defense system targeting several pathogens, including aspergillus fumigatus and salmonella typhimurium (huang et al., ; martinez et al., ) and has emerged as a key regulator of inflammation and immunity. previous studies have implicated autophagy in antigen presentation to t cells by major histocompatibility complex (mhc) molecules (dengjel et al., ; schmid et al., ) . however, optimal mhc-ii presentation of peptides from phagocytosed pathogens requires lc targeting to phagosomes by atgs and nox , suggesting that lap mediates presentation of extracellularly derived antigens (romao et al., ) . importantly, lap directs the production of type i ifn (ifn-i), namely ifn-α, in response to host-dna containing immune complexes by plasmacytoid dendritic cells (henault et al., ) . in addition to controlling the immune response, lap is required for the phagocytosis and degradation of photoreceptor outer segments by retinal pigment epithelial cells in mice, which is essential for proper vision (kim et al., ) . overall, these studies reinforce the physiological importance of lap as an autophagy-related pathway. in addition to lap, studies have uncovered a growing list of nonautophagic functions mediated by atgs that modulate the infection of host cells and tissues. in this section, we focus on how individual atgs can either augment or diminish the propagation of assorted viruses as well as how atgs restrict intracellular pathogens via pathways that are distinct from both classic autophagy and lap. subversion during viral replication and transmission. growing evidence supports that viruses use lc + membranes to exit host cells via exocytic pathways analogous to secretory autophagy (fig. ) . infections by poliovirus and coxsackievirus b (cvb), two nonenveloped rna viruses, result in the formation of lc + double membrane vesicles in an atg-dependent manner, which serve as scaffolds for viral replication complexes (jackson et al., ; wong et al., ) . instead of degradation in the lysosome, sequestered virions are released from cells within a membrane coat through a process termed autophagosome-mediated exit without lysis (fig. a; taylor et al., ) . the "envelope" acquired during egress shields virion clusters from immune recognition and aids entry into neighboring cells (bird et al., ; chen et al., b) . consistent with these studies that an autophagy-like process is required for viral replication and spread, atg deletion in pancreatic acinar cells of mice leads to a , -fold reduction in cvb replication and protection from pancreatitis (alirezaei et al., ) . in addition to these picornaviruses, lipidated lc also contributes to the envelopment and exocytosis of certain herpesviruses during lytic infection. epstein-barr virus and varicella-zoster virus acquire lc -conjugated membranes during envelope acquisition in the cytosol, which can be detected in purified virions (fig. b; nowag et al., ; buckingham et al., ) . accordingly, inhibiting lc lipidation via atg or atg l knockdown impairs viral exit and results in the accumulation of viral dna in the cytosol (nowag et al., ) . finally, influenza a virus (iav) encodes an ion channel protein, matrix protein , that blocks lysosomal degradation of the virion and facilitates viral egress through binding and redirecting lc -ii to the plasma membrane. notably, although the improved filamentous budding of iav is dependent on atg conjugation pathways that lipidate lc , the overall infectious virus production remains intact (fig. c; gannagé et al., ; beale et al., ) . in addition to these effects on viral exocytosis, interactions with lc are also required for the processing and inclusion of hiv gag into the virion, whereas hiv nef inhibits beclin- to prevent virion degradation through autophagy (kyei et al., ) . in contrast, the multilamellar membranes that harbor coronavirus replication complexes are decorated with the nonlipidated form of lc ; down-regulation of lc impairs viral replication, but atg deletion has no effect (reggiori et al., ) . thus, viruses subvert lc -mediated membrane trafficking events through different ways. although we focus on strategies by which viruses coopt atgs for their own benefit, some viruses are restricted by autophagy. for example, autophagic degradation of er, reticulophagy, restricts dengue and zika virus replication on er-derived membranes. the ns protease encoded by these and other flaviviruses counteract this inhibition by cleaving the reticulophagy receptor fam b (lennemann and coyne, ) . also, multiple herpesviruses encode proteins that bind and inhibit beclin- to avoid autophagy-mediated targeting to the lysosome and antigen presentation to t cells (deretic and levine, ). in other situations, it may be nonautophagic functions of atgs that determine virulence, as suggested by an sirna screen comparing the requirement of diverse atgs during infection by six viruses (mauthe et al., ) . this screen revealed that many of these proteins augment or diminish the replication of individual viruses independent of other atgs, consistent with the conclusion that nonautophagic functions of atgs are pervasive during viral infection. when antimicrobial immunity is not xenophagy or lap. xenophagy, in which an internalized microbe is sequestered in an autophagosome and targeted to the lysosome, is an established form of cell-autonomous defense (cadwell, ) . however, mechanisms by which atgs restrict intracellular pathogens are sometimes radically different from classic autophagy or lap. the cytokine ifn-γ inhibits toxoplasma gondii replication in macrophages by triggering a process referred to as targeting by autophagy proteins (tag), which requires atgs involved in lc conjugation, but not the lysosome. instead of promoting acidification, the atg orthologue gab ara pl (gate- ) recruits ifn-inducible gtpases to the parasitophorous vacuole where they disrupt the membrane and destroy the replicative niche of t. gondii park et al., b; sasai et al., ) . a similar mechanism disrupts the membranous structures on which noroviruses replicate (biering et al., ) . additionally, though xenophagy inhibits mycobacterium tuberculosis replication in cultured macrophages, experiments using cell type-specific knockout mice revealed a surprising autophagy-independent function of atg in neutrophils. deletion of atg , but not the other atgs involved in lc conjugation, increases the amount of neutrophils that infiltrate and damage the lung during mycobacterium tuberculosis infection (kimmey et al., ) . as with viruses, bacteria can benefit from selective components of the autophagy machinery. the multimembrane vacuoles that support brucella abortis replication are generated by atgs that control pi kinase activity (ulk , beclin- , and atg ), but not atgs required for lc conjugation (atg , atg l , atg , lc b, and atg b) (starr et al., ) . in addition to these cellautonomous functions of atgs during infection, nonautophagic atg functions are important for immune signaling pathways that contribute to multi-cellular immunity, which is discussed in the following section. atgs have a fundamental role in suppressing immune signaling (cadwell, ) . among the first indications that autophagy exacts inhibitory functions came from population genetic studies implicating a coding variant of atg l in crohn's disease, a type of inflammatory bowel disease (ibd) associated with an abnormal immune response to the gut microbiota. consistent with this genetic association, atg l deletion causes macrophages to overproduce the cytokine il- β, which mediates intestinal inflammation (saitoh et al., ) . although increased production of immune effectors is pathological in this setting, atg l mutation unexpectedly enhances innate immune resistance to oral infection by the gram-negative bacterium citrobacter rodentium (marchiando et al., ) . additionally, deletion of other atgs throughout the autophagy pathway leads to a general increase in cytokine levels that promote resistance to influenza virus infection and inhibition of herpesvirus reactivation from latency (park et al., a) . in this section, we provide examples highlighting different mechanisms by which atgs reduce signaling downstream of cytosolic pathogen sensors. (rig-i) antiviral signaling. the presence of nucleic acid in the cytoplasm derived from intracellular pathogens induces transcription of ifn-i. the magnitude of this response is regulated by atgs through both autophagy-dependent and -independent mechanisms. rig-i binds short double-stranded rna with a ′ppp moiety, which exposes the caspase activation and recruitment domain (card) to allow interaction with the card of mitochondrial antiviral signaling protein (mavs; yoneyama et al., ; kawai et al., ; meylan et al., ; seth et al., ) . after this card-card interaction, mavs oligomerizes on the mitochondrial outer surface to activate tbk- , which in turn leads to the phosphorylation and nuclear translocation of the transcription factors ifn regulatory factor (irf ), irf , and nfκb (honda et al., ; seth et al., ) . the atg -atg conjugate inhibits this rna recognition pathway by binding the cards of rig-i and mavs (fig. ; jounai et al., ) . although it is unclear how these interactions prevent mavs function, the direct binding of atg -atg to cards suggests that the mechanism is autophagy-independent. an alternative mechanism involves increased ifn-i production upon disruption of mitophagy. ros from damaged mitochondria accumulate upon atg deletion, which enhances rig-i signaling ( fig. ; tal et al., ) . mitophagy also mediates the concurrent degradation of mavs upon its translocation to mitochondria. the recruitment of the atg l -atg -atg complex to mitochondria by nlrx and elongation factor tu (tufm) decreases mavs activity ( fig. ; lei et al., ) . the importance of this pathway is highlighted by the observation that the human parainfluenza virus type matrix protein induces mitophagy through interaction with tufm, leading to decreased immune signaling and increased viral replication (ding et al., ) . also, a single base substitution that confers the ability of the viral polymerase basic protein to bind tufm and induce autophagy allows avian influenza virus to infect human cells (kuo et al., ) . most likely through a similar process, the mitochondrial protein cox b binds atg and mavs, and reduces ros and ifn-i production during viral infection (zhao et al., ) . the commonality in these studies is that inhibiting the atg -atg conjugate increases ifn-i and decreases replication of rna viruses. . atgs in inflammatory and immune signaling. atgs regulate immune signaling cascades through autophagy-dependent and -independent mechanisms. the mitochondrial protein tufm recruits the atg l -atg -atg complex to mediate the autophagic removal of mitochondria that produce ros, an activator of rig-i signaling and the nlrp inflammasome. by targeting mitochondria, autophagy further inhibits ifn-i production by removing the signaling intermediate mavs, which aggregates on mitochondrial surfaces downstream of viral rna recognition by rig-i. also, the atg -atg complex inhibits rig-i and mavs through an inhibitory binding event. beclin- prevents sustained signaling by inducing the autophagic removal of cytosolic dna and inhibiting cgas through direct binding. cgamp generated by cgas activates ulk to inhibit sti ng in a negative feedback loop. atg l interferes with the trafficking of sti ng to prevent continuous tbk- activation. ). similar to rna sensing by rig-i, sti ng (stimulator of interferon genes) mediates a tbk- dependent signaling cascade in the presence of cgamp (chen et al., ) . deletion of atg l , but not other atgs, increases the colocalization between sti ng and tbk- to enhance ifn-i production in response to cytosolic dna ( fig. ; saitoh et al., ) . also, generation of cgamp by cgas activates ulk , which phosphorylates and inhibits sti ng while also inducing autophagy ( fig. ; konno et al., ) . similarly, beclin- can bind and inhibit cgas and simultaneously mediate the degradation of cytoplasmic dna through autophagy ( fig. ; liang et al., ) . thus, beclin- prevents sustained cgas activation through both direct inhibition and depriving the enzyme of its substrate. in these examples, conserved autophagy proteins (atg l , ulk , and beclin- ) display functions independent of autophagy-related processes, although these functions do not necessarily preclude their role in classic autophagy. atgs and inflammasome activation. inflammasomes are multiprotein complexes that induce the cleavage of pro-il- β and pro-il- by caspase- to generate the activate forms of these cytokines (sharma and kanneganti, ) . the nlrp inflammasome responds to a variety of stimuli that directly or indirectly cause the release of ros and dna from leaky mitochondria (e.g., bacterial toxins and microcrystalline substances). inhibiting mitophagy, therefore, leads to accumulation of damaged mitochondria that induce pathological cytokine production downstream of nlrp inflammasome activation ( fig. ; saitoh et al., ; zhong et al., ) . atgs also inhibit the caspase- inflammasome, which is activated by the bacterial cell wall component lipopolysaccharide in the cytosol. when ifn-inducible gtpases attack the salmonellacontaining vacuole as part of the host defense mechanism, lc is recruited to the damaged membrane by the linker protein ndp to mediate the autophagic sequestration of bacteria and lipopolysaccharide, which inhibits inflammasome activation (meunier et al., ) . hence, it is likely that removal of damaged vesicles and organelles (or their contents) through classic autophagy restrains inflammasome activity, thereby explaining why inhibiting autophagy proteins such as atg l enhance il- β and il- production. here, we use ibd, rheumatoid arthritis (ra), and systemic lupus erythematosus (sle) as examples of how atgs contribute to complex inflammatory diseases. ibd includes crohn's disease and ulcerative colitis, and is frequently a debilitating condition that involves chronic inflammation in the small intestine or colon, although any part of the gastrointestinal tract can be affected. many genetic variants that increase risk of ibd, including several known to affect autophagy, are also found in individuals without disease (lassen and xavier, ) . a particularly common polymorphism in atg l (up to % heterozygosity in certain populations) linked to crohn's disease introduces a caspase- cleavage site that destabilizes the protein product (atg l t a ), thereby causing a reduction in autophagy (lassen et al., ; murthy et al., ) . atg l t a is associated with structural defects in paneth cells, intestinal epithelial cells that produce antimicrobial granules (cadwell et al., ) . additional inflammatory pathologies in the intestinal epithelium of atg l mutant mice is triggered by a commensal enteric virus (cadwell et al., ; kernbauer et al., ) , or by dual deletion of atg l and the er stress transcription factor xbp- in paneth cells (adolph et al., ) . these findings are consistent with epidemiological observations suggesting that ibd is caused by the confluence of multiple genetic and environmental susceptibility factors. several findings support the idea that the classic autophagy function of atg l is critical for protecting the epithelial barrier and preventing a sustained immune reaction. atg-deficient paneth cells display unresolved er stress and mitochondrial damage that contribute to necroptosis, a type of programmed necrotic cell death (diamanti et al., ; matsuzawa-ishimoto et al., ; tschurtschenthaler et al., ) . these observations suggest that the organelle homeostasis function of autophagy is important to counteract the secretory burden of this highly differentiated cell type and prevent inflammatory sequelae. also, atgs are generally required to protect the epithelial barrier, potentially through xenophagy or mediating mucus production by goblet cells (benjamin et al., ; conway et al., b; patel et al., ) . an important role for autophagy in immune cells should also be considered. increased inflammasome activity in macrophages and decreased differentiation of antiinflammatory t cells are both consequences of atg l inhibition, and can cause intestinal inflammation (saitoh et al., ; chu et al., ; kabat et al., ) . however, many of these studies rely on animal models in which classic autophagy and related processes are difficult to distinguish. atg l t a disrupts secretory autophagy, leading to impaired exocytosis of lysozyme from paneth cells during salmonella infection (bel et al., ) , and the effect of atg l t a on the necroptosis signaling complex likely involves disruption in atg-mediating immune signaling (matsuzawa-ishimoto et al., ) . in addition to being an unstable protein, atg l t a displays impaired binding with tmem , a transmembrane protein that mediates the trafficking of lc + vesicles through a process distinct from classic autophagy (boada-romero et al., ) . also, with the exception of graft-versus-host disease (hubbard-lucey et al., ) , the ibd variant of atg l is not linked to other inflammatory disorders associated with autophagy dysfunction, such as vici syndrome . thus, investigating the potential nonautophagic functions of atg l in maintaining the intestinal barrier remains a critically important future direction. ra is an autoimmune disease that primarily affects the joints and is associated with the presence of autoantibodies that are reactive to citrullinated peptides that are presented by mhc-ii molecules. in contrast to ibd, atg function may promote ra. atg is required for generation of citrullinated antigens by peptidylarginine deiminases, potentially by mediating the trafficking of these enzymes to mhc-ii antigen loading compartments (ireland and unanue, ) . ctla on anti-inflammatory t cells binds b molecules on dendritic cells to inhibit lc expression and autophagy, which dampens the capacity to present antigens (alissafi et al., ) . the ctla -ig fusion molecule abatacept, which is used to treat ra, also inhibits atg-mediated antigen presentation by dendritic cells, suggesting that dampening autophagy is part of the mechanism of action of this drug (alissafi et al., ) . because it is currently unclear when autophagy or an autophagy-related process such as lap mediates antigen presentation, an important future direction will be to determine which atgs are necessary for autoantigen presentation, and whether this differs among cell types. polymorphisms in a noncoding region of atg is associated with sle, a multiorgan autoimmune disease characterized by antinuclear antibodies, indicative of improper immune activation toward cellular contents (harley et al., ) . atg in b cells is required for autoantibody generation in the lpr and tlr transgenic mouse models of sle (weindel et al., ; arnold et al., ) , consistent with the role of autophagy in supporting the secretory burden and organelle homeostasis in differentiated b cells (conway et al., a; pengo et al., ; chen et al., chen et al., , a . in contrast to this proposed role of autophagy in b cells that facilitates sle pathogenesis, the genetic ablation of lap-specific atgs in phagocytic cells results in inefficient clearance of dead cells and their immunogenic contents, leading to a sle-like disease in mice (martinez et al., ) . thus, it is possible that inhibiting autophagy may ameliorate, whereas inhibiting lap may exacerbate disease. developing drugs that can exclusively target classic autophagy without effecting autophagy-related processes, or vice versa, may be necessary to treat certain disorders. overall, these studies illustrate the wide array of nonautophagic functions mediated by the atg machinery. these diverse functions deepen our understanding of atgs in enacting cellautonomous and non-cell-autonomous biological functions beyond self-eating in both normal and disease states. they also broach the importance of revisiting phenotypes and functions, both in vitro and in vivo, that to date have been attributed to classic autophagy based on the genetic analysis of a single individual atg. going forward, it will be critical for future researchers to keep in mind alternative pathways, not just autophagy, upon discovering new atg-dependent phenotypes. importantly, in future studies to delineate how autophagy influences both normal physiology and disease, it will be incumbent on researchers to interrogate multiple atgs controlling distinct elements of the autophagy trafficking pathway to definitively attribute a role for classic autophagy on cell fate and function. in a similar vein, as we develop more precise methods to therapeutically target specific autophagy machinery components, the role of alternative autophagy-related processes and signaling pathways merit thoughtful and rigorous consideration. paneth cells as a site of origin for intestinal inflammation pancreatic acinar cell-specific autophagy disruption reduces coxsackievirus replication and pathogenesis in vivo tregs restrain dendritic cell autophagy to ameliorate autoimmunity autophagy is dispensable for b-cell development but essential for humoral autoimmune responses a lc -interacting motif in the influenza a virus m protein is required to subvert autophagy and maintain virion stability paneth cells secrete lysozyme via secretory autophagy during bacterial infection of the intestine intestinal epithelial autophagy is essential for host defense against invasive bacteria viral replication complexes are targeted by lc -guided interferon-inducible gtpases nonlytic viral spread enhanced by autophagy components the t a crohn's disease risk polymorphism impairs function of the wd domain of atg l exocytosis of varicella-zoster virus virions involves a convergence of endosomal and autophagy pathways crosstalk between autophagy and inflammatory signalling pathways: balancing defence and homeostasis a key role for autophagy and the autophagy gene atg l in mouse and human intestinal paneth cells virusplus-susceptibility gene interaction determines crohn's disease gene atg l phenotypes in intestine essential role for autophagy in the maintenance of immunological memory against influenza infection requirement for autophagy in the long-term persistence but not initial formation of memory b cells regulation and function of the cgas-sti ng pathway of cytosolic dna sensing phosphatidylserine vesicles enable efficient en bloc transmission of enteroviruses autophagy in human health and disease the parasitophorous vacuole membrane of toxoplasma gondii is targeted for disruption by ubiquitin-like conjugation systems of autophagy genemicrobiota interactions contribute to the pathogenesis of inflammatory bowel disease canonical and noncanonical autophagy: variations on a common theme of self-eating? biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles atg regulates plasma cell differentiation atg l is required for autophagy in intestinal epithelial cells and protection of mice from salmonella infection remodeling of secretory compartments creates cups during nutrient starvation autophagy promotes mhc class ii presentation of peptides from intracellular source proteins autophagy, immunity, and microbial adaptations autophagy intersections with conventional and unconventional secretion in tissue development, remodeling and inflammation autophagy proteins regulate the secretory component of osteoclastic bone resorption ikkα controls atg l degradation to prevent er stress during inflammation the matrix protein of human parainfluenza virus type induces mitophagy that suppresses interferon responses autophagy-based unconventional secretory pathway for extracellular delivery of il- β unconventional secretion of acb is mediated by autophagosomes autophagy machinery mediates macroendocytic processing and entotic cell death by targeting single membranes matrix protein of influenza a virus blocks autophagosome fusion with lysosomes rescue of Δf -cftr trafficking via a gra sp-dependent unconventional secretion pathway genetic and phenotypic overlap between autophagy and the cytoplasm to vacuole protein targeting pathway genome-wide association scan in women with systemic lupus erythematosus identifies susceptibility variants in itg am, pxk, kiaa and other loci noncanonical autophagy is required for type i interferon secretion in response to dnaimmune complexes irf- is the master regulator of type-i interferon-dependent immune responses activation of antibacterial autophagy by nad ph oxidases autophagy gene atg l prevents lethal t cell alloreactivity mediated by dendritic cells autophagy in antigen-presenting cells results in presentation of citrullinated peptides to cd t cells subversion of cellular autophagosomal machinery by rna viruses the atg atg conjugate associates with innate antiviral immune responses the autophagy gene atg l differentially regulates treg and th cells to control intestinal inflammation. elife. :e autophagy at the crossroads of catabolism and anabolism ips- , an adaptor triggering rig-i-and mda -mediated type i interferon induction an enteric virus can replace the beneficial function of commensal bacteria noncanonical autophagy promotes the visual cycle unique role for atg in neutrophil-mediated immunopathology during m. tuberculosis infection dedicated sna res and specialized trim cargo receptors mediate secretory autophagy cyclic dinucleotides trigger ulk (atg ) phosphorylation of sti ng to prevent sustained innate immune signaling inhibition of avian influenza a virus replication in human cells by host restriction factor tufm is correlated with autophagy autophagy pathway intersects with hiv- biosynthesis and regulates viral yields in macrophages genetic control of autophagy underlies pathogenesis of inflammatory bowel disease atg l t a variant decreases selective autophagy resulting in altered cytokine signaling and decreased antibacterial defense the mitochondrial proteins nlrx and tufm form a complex that regulates type i interferon and autophagy dengue and zika viruses subvert reticulophagy by ns b -mediated cleavage of fam b crosstalk between the cgas dna sensor and beclin- autophagy protein shapes innate antimicrobial immune responses autophagydependent production of secreted factors facilitates oncogenic rasdriven invasion homeostatic control of innate lung inflammation by vici syndrome gene epg and additional autophagy genes promotes influenza pathogenesis unconventional protein secretion: an evolving mechanism unconventional secretion of pichia pastoris acb is dependent on gra sp protein, peroxisomal functions, and autophagosome formation a deficiency in the autophagy gene atg l enhances resistance to enteric bacterial infection microtubule-associated protein light chain alpha (lc )-associated phagocytosis is required for the efficient clearance of dead cells molecular characterization of lc -associated phagocytosis reveals distinct roles for rubicon, nox and autophagy proteins noncanonical autophagy inhibits the autoinflammatory, lupus-like response to dying cells autophagy protein atg l prevents necroptosis in the intestinal epithelium an sirna screen for atg protein depletion reveals the extent of the unconventional functions of the autophagy proteome in virus replication ple khm regulates autophagosome-lysosome fusion through hops complex and lc /gab arap proteins caspase- activation requires lysis of pathogen-containing vacuoles by ifn-induced gtpases cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus the role of atg proteins in autophagosome formation a crohn's disease variant in atg l enhances its degradation by caspase spatial coupling of mtor and autophagy augments secretory phenotypes macroautophagy proteins assist epstein barr virus production and get incorporated into the virus particles. ebiomedicine autophagy genes enhance murine gammaherpesvirus reactivation from latency by preventing virus-induced systemic inflammation targeting by autophagy proteins (tag): targeting of ifnginducible gtpases to membranes by the lc conjugation system of autophagy autophagy proteins control goblet cell function by potentiating reactive oxygen species production plasma cells require autophagy for sustainable immunoglobulin production diversity in unconventional protein secretion coronaviruses hijack the lc -i-positive edemosomes, er-derived vesicles exporting short-lived erad regulators, for replication autophagy proteins stabilize pathogen-containing phagosomes for prolonged mhc ii antigen processing autophagy-dependent shuttling of tbc d controls plasma membrane translocation of glut and glucose uptake loss of the autophagy protein atg l enhances endotoxin-induced il- beta production atg a controls dsdnadriven dynamic translocation of sti ng and the innate immune response toll-like receptor signalling in macrophages links the autophagy pathway to phagocytosis essential role for gab arap autophagy proteins in interferon-inducible gtpase-mediated host defense antigen-loading compartments for major histocompatibility complex class ii molecules continuously receive input from autophagosomes identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf the cell biology of inflammasomes: mechanisms of inflammasome activation and regulation selective subversion of autophagy complexes facilitates completion of the brucella intracellular cycle a global analysis of snx -retromer assembly and cargo specificity reveals a function in glucose and metal ion transport cargo recognition and trafficking in selective autophagy non-autophagic roles of autophagyrelated proteins cyclic gmp-amp synthase is a cytosolic dna sensor that activates the type i interferon pathway autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction absence of autophagy results in reactive oxygen species-dependent amplification of rlr signaling role of microtubules in extracellular release of poliovirus isolation of autophagocytosis mutants of saccharomyces cerevisiae defective atg l -mediated removal of ire α drives crohn's disease-like ileitis the atg conjugation systems are important for degradation of the inner autophagosomal membrane isolation and characterization of autophagydefective mutants of saccharomyces cerevisiae b cell autophagy mediates tlr -dependent autoimmunity and inflammation autophagosome supports coxsackievirus b replication in host cells cyclic gmp-amp is an endogenous second messenger in innate immune signaling by cytosolic dna the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses unconventional secretion, unconventional solutions translocation of interleukin- β into a vesicle intermediate in autophagy-mediated secretion cox b regulates mavs-mediated antiviral signaling through interaction with atg and repressing ros production nf-κb restricts inflammasome activation via elimination of damaged mitochondria grant support includes the national institutes of health (ca , ca , ca , and ag to j. debnath, and hl , dk , dk , and ai to k. cadwell key: cord- -gl lozn authors: jeanes, annette; coen, pietro g.; drey, nicolas s.; gould, dinah j. title: moving beyond hand hygiene monitoring as a marker of infection prevention performance: development of a tailored infection control continuous quality improvement tool date: - - journal: am j infect control doi: . /j.ajic. . . sha: doc_id: cord_uid: gl lozn background: infection control practice compliance is commonly monitored by measuring hand hygiene compliance. the limitations of this approach were recognized in acute health care organization that led to the development of an infection control continuous quality improvement tool. methods: the pronovost cycle, barriers and mitigation tool, and hexagon framework were used to review the existing monitoring system and develop a quality improvement data collection tool that considered the context of care delivery. results: barriers and opportunities for improvement including ambiguity, consistency and feasibility of expectations, the environment, knowledge, and education were combined in a monitoring tool that was piloted and modified in response to feedback. local adaptations enabled staff to prioritize and monitor issues important in their own workplace. the tool replaced the previous system and was positively evaluated by auditors. challenges included ensuring staff had time to train in use of the tool, time to collect the audit, and the reporting of low scores that conflicted with a target-based performance system. conclusions: hand hygiene compliance monitoring alone misses other important aspects of infection control compliance. a continuous quality improvement tool was developed reflecting specific organizational needs that could be transferred or adapted to other organizations. preventing infection in health care settings depends on the practices and behaviors of health care workers (hcw) and organizational factors that influence practice. hand hygiene has long been considered the most important infection prevention precaution, and hand hygiene audit data are reported at a senior level as part of quality assurance, overlooking the contribution of other important practices, for example isolation of infectious patients and sterilization and disinfection of equipment. the value of hand hygiene remains undisputed, but despite regular monitoring and performance feedback, compliance is suboptimal. , hand hygiene forms only part of an overall infection prevention program, and its use as the overall indicator of infection prevention excellence is questionable, especially as monitoring is fraught with pitfalls. hand hygiene compliance monitoring is commonly undertaken by directly observing practice, but this is flawed because hcws are aware of scrutiny and transient improvement in performance may occur, generating inflated scores and not reflecting usual behavior. [ ] [ ] [ ] a review of the hand hygiene monitoring throughout organization found the data that were based on observation of practice did not accurately reflect infection control compliance, contributed little to improving practice, were not considered the best use of time, and lacked local credibility. these factors are likely to influence the value of these data in practice improvement. achieving and maintaining high levels of infection control compliance is challenging, and few studies consider the context of care or barriers and opportunities to improve compliance. recognition of the context of and constraints on practice can provide insights into the opportunities for practice improvements. the collection of related data , includes the variability of activities, associated infection risks, and the importance of clarifying expectations of compliance. a data collection tool was required to provide credible information relating to a range of infection prevention practices, reflecting the operational risks and constraints encountered in different clinical settings and generating data of value for practice improvement. the aim of this work was to develop and implement an infection control performance and quality improvement data collection tool to meet the needs of a large, acute health care provider and to improve the credibility and use of infection control performance monitoring. the infection control continuous quality improvement (ic-cqi) tool was developed in an acute teaching hospital in london, with over , inpatient beds and , staff spread across hospitals on separate sites, providing emergency, general medicine, surgery, critical care, maternity, neonatal, and cancer services. hand hygiene compliance monitoring was established, but other aspects of infection control practice were not systematically monitored. to create an ic-cqi tool and reporting framework the infection prevention team used the pronovost knowledge translation cycle to review the current hand hygiene monitoring tool, and to develop a quality improvement data collection tool. the barriers and mitigation tool was used to identify workplace improvement barriers and potential solutions that involved "walking the process": observing clinical processes and compliance measurement as they occurred in different clinical areas. a double loop learning cycle was used to ensure that the context, values, assumptions, and culture of the whole organization were included in proposed quality improvement intervention using the hexagon tool framework to assess feasibility and how to engage with stakeholders. a variety of arrangements including questionnaires, day to day contacts with auditors, feedback from users via the ic-cqi data input system, discussion groups, and ic-cqi training sessions were made for providing feedback on the data collection tool and process, to meet the operational needs, including time constraints, of different practitioners and clinical areas. modifications to the tool and implementation of the change were made in response to feedback. no other routine infection control performance data were collected apart from monthly hand hygiene compliance data collection and reporting that took place continuously across all clinical areas until this was replaced by the ic-cqi system. intermittent validation was then undertaken of ic-cqi results including hand hygiene product availability, isolation practices, appropriateness of use of personal protective equipment (ppe), and compliance with standards of invasive devices insertion and management throughout the implementation period. results are reported using the pronovost knowledge translation cycle. summarize the science a literature review focused on the current evidence for opportunities and barriers to compliance in infection control, including hand hygiene that is the most researched infection control intervention, and in addition evidence from related fields such as psychology. five themes emerged that are summarized. a) knowledge, education, and training improving infection control knowledge, education, and training can enhance compliance and potentially reduce infection acquisition, [ ] [ ] [ ] although its impact and value is disputed. , the effect of education is difficult to gauge as it is one of a bundle of interventions in studies in which it has been judged to be beneficial. [ ] [ ] [ ] [ ] further, improvements after education may not be sustained, [ ] [ ] [ ] and may require continuous renewal to sustain reported effectiveness, although a limited ongoing residual effect has been reported. the "stickiness" and memorability of ideas have influenced key concepts such as the "my five moments of hand hygiene" concept promoted by the world health organization, which has been widely adopted as an approach to indicate when hands should be cleaned. lack of understanding of expectations and ambiguity is a significant barrier to compliance, particularly with guidelines. this is compounded by the considerable variation in the scope, approach, content, expectations, and terminology in infection control guidance and recommendations. , improving and clarifying infection control information and expectations may improve compliance, knowledge and high levels of self-efficacy are recognized to improve performance, whereas poor self-efficacy, despite good theoretical knowledge, is more likely to be associated with lower compliance. the promotion of infection control compliance has been used widely in acute care settings with variable outcomes, and has included marketing, [ ] [ ] [ ] campaigns, [ ] [ ] [ ] stimulating, [ ] [ ] [ ] and reminding staff of infection control requirements. , motivation associated with infection control is complex and appears to be related to culture, beliefs, and values. emotion, habit/routine, and incentives affect behavior, and whereas pride in work, empathy, automatic habits, and rewards may have a positive impact on compliance, sanctions could have a negative effect. other factors such as the protection of patients or self [ ] [ ] [ ] [ ] or the perception of risk may also affect performance. , [ ] [ ] [ ] c) environmental and human factors the health care environment influences infection control compliance including equipment design, position, and workflow. [ ] [ ] [ ] provision, availability, and accessibility of hand hygiene facilities and products are important factors in hand hygiene compliance. [ ] [ ] [ ] [ ] [ ] [ ] inadequate hand hygiene may be mitigated by reducing the environmental contamination of the patient environment including computer keyboards and telephones. improving cleaning efficacy may reduce hand contamination. , however, this requires an environment designed to expedite cleaning, competent cleaning staff, and sufficient time and opportunity to clean in busy clinical environments. , other significant barriers to infection control compliance include pressure of work, understaffing, overcrowding, high bed occupancy and patient turnover, high patient-to-nurse ratio, and lack of time for education, , , [ ] [ ] [ ] [ ] which may contribute to infection transmission and outbreaks. [ ] [ ] [ ] d) organizational priorities and culture in the united kingdom, health care organizational performance is closely monitored, and achievement of quality standards and performance targets are important. health care infection acquisitions are an important marker for the quality of care delivered and organizational management. , effective infection prevention is typically a bundle of interventions including isolation provision and practice, the use of ppe, cleanliness including environment, and hand hygiene. culture, commitment, and leadership at unit or ward level affect the implementation of best practice, , , whereas organizational culture can influence, improve, and sustain infection control compliance. , , strong leadership, good role models, local ownership, champions, empowerment, commitment, and role have been found to be important. , , e) feedback of observation of practice performance feedback is widely used to improve hand hygiene performance, , - but has been found in other settings to be a potentially destructive process that demotivates and diminishes performance if not done well. behavior changes related to observation may promote "good" behavior such as increasing hand hygiene compliance, with more pronounced effects when observers are known to the staff. , another benefit of observation is the opportunity to check technique, , , praise, recognize constraints, or offer advice and support. clarifying and standardizing expectations prior to observation may reduce inconsistency in expectations. observing sequences of care has been used as an alternative approach to simply observing hand hygiene practice. [ ] [ ] [ ] the hcw is observed for the duration of the care activity, such as mobilizing a patient or measuring vital signs, to enable the observer to put the actions observed into the context and constraints of practice. the review of organizational data in an acute nhs trust from to , established that although reported levels of compliance were high (fig ) and met the performance target of > % compliance, the data collection method lacked validity and relaibility. nurses were responsible for data collection, and it was perceived by some as "ticking boxes" with no expectation that improvement would occur. many nurses had not received training in auditing hand hygiene, no time was allocated to undertake it, and providing feedback was difficult particularly if the results were poor. noncompliant staff were seldom challenged because of fear of a negative response. factors impeding compliance, such as empty soap dispensers or ambiguity of expectations, were not resolved when observed as they should have been. other issues reported by staff were inconsistent feedback about performance, inadequate facilities, dissatisfaction with products and supplies, lack of appropriate knowledge, ambiguity related to definitions, expectations and standards, and dif-ficulty in observing single rooms without causing disruption. infection control risks varied across the organization and specialties. misconceptions such as when to wear disposable gloves were not managed or monitored, and there was a focus on identifying failures in compliance, whereas good practice was unrecognized. some staff, including medical staff, were largely disengaged from the process of monitoring and improving infection control practice. the context is summarized in categories of the hexagon framework that examines the current process and context of the proposed change. ( ) needs infection control performance data provides assurance of compliance with national standards. related audits including cleaning and environmental monitoring were not collated or widely disseminated. the organization was often blind to infection control issues until a significant problem emerged. ( ) fit the proposal reflected the core values of the organization. the organization supported quality improvement and this initiative created opportunities for continuous improvement based on known and locally identified opportunities and barriers. it was an organizational priority that staff were competent and regularly updated in infection prevention and control. some managers perceived that reporting low scores was an admission of failure rather than an opportunity for improvement which was a potential barrier. the potential to save time auditing aligned with an organizational strategy to reduce costs and improve efficacy. ( ) resource no additional resource was required as this replaced the established data collection, reporting, and dissemination infrastructure. although it was recognized that in the existing system staff were often untrained and given no additional time to undertake this work. there was potential for a decreased dependence on nurses as data collection could be shared with other team members and undertaken throughout the -hour period. ( ) evidence there was evidence that the current system lacked credibility and had little effect on improving reported compliance. there was evidence of systematic defects and barriers to infection control compliance including ambiguity, disengagement, and unreliable hand hygiene product provision. issues such as isolation were increasingly problematic and required improvement, particularly with the emergence of ebola, severe acute respiratory syndrome, and middle east respiratory syndrome, when considerable resource was required to ensure staff were educated and resourced to be able to manage these emerging viruses safely. ( ) capacity there was capacity within the infection control department to support the changes in the data collection and reporting, training, communication, and validation of the data collected. a number of other competing changes and initiatives prevalent in the organization including building, reorganization of services, and staffing structure were potential barriers. five key themes emerged from the literature review, barriers and mitigation work, , feedback from auditors, and observations from stakeholders suggesting barriers and opportunities for improvement that were combined to produce a draft ic-cqi tool. this was pilot tested in several areas while the existing hand hygiene monitoring arrangements continued throughout the remainder of the organization (fig ) . the draft ic-cqi tool was clarified and simplified in response to feedback. the rationale for including criteria in the final ic-cqi tool is summarized in table . results of the pilot studies and the final ic-cqi tool was presented to senior managers, infection control staff, and auditors who agreed that the results would be used to provide a monthly score of infection control performance (table ) . it took more than years to develop the ic-cqi tool, educate staff in the purpose, methods, and implications for practice and integrate the data into the established quality measurement system in the organization. over hours of training in the final tool was delivered across the organization, and more than people attended training in - . the use of the ic-cqi tool was finally established throughout the organization in august , and the existing hand hygiene audit discontinued. all areas were expected to report using the new tool from september (fig ) . progress developing and establishing the process is summarized in the following text. basic infection control training including hand hygiene is mandatory in the first month at work, with online updating every years. initially, externally employed staff were excluded from this training and senior medical staff often opted out, but during the development of the new tool this training became mandatory for all staff. electronic training records were reported monthly to managers and the executive team. a list of common infection control questions was developed to assess the knowledge of hcws and identify education requirements. each ward or department were required to ask a representative sample of staff working in the area monthly either standard or locally developed and agreed questions. examples could include: when do you need to isolate a patient with diarrhea? describe how a spillage of blood should be cleaned up? the infection control knowledge of auditors was a limiting factor, and initially some auditors restricted questions to the ones they could answer. the range of questions and potential for improving infection control knowledge increased when answer sheets were provided. an infection control link personnel system was already established to provide local induction and refresh basic skills and knowledge in the workplace, which included hand hygiene techniques, cleaning equipment, and the use of ppe. staff turnover was high particularly in junior doctors and the burden of local induction and support was onerous in some areas. delivery varied across the organization and reflected local commitment to the induction of new team members and the energy and commitment of the link staff. it was envisaged that prompts and reminders would increase awareness and that posters and screen savers could provide useful information such as the actions to take after a needle stick injury or how to clean equipment. however, audible reminders confused some patients, irritated some staff, and were rapidly removed or sabotaged by detractors. some senior managers removed infection control notice boards as they found them "untidy," and plans to install monitors failed as there was no space or electrical supply or funds. the most enduring promotion was hand hygiene technique stickers on hand hygiene product dispensers. in addition, regular supplies of posters were delivered to wards and departments. local managers were responsible for arranging maintenance, but the process could be onerous and protracted. metrics relating to minor repairs were not collated and recurring problems were largely invisible. an issue mentioned frequently was empty or broken soap and alcohol hand gel dispensers. organization-wide audits in found % of soap dispensers were broken or empty and there were examples of delays of several days before they were repaired. in - , a project was undertaken to replace all soap and alcohol hand gel dispensers with standardized products and dispensers that staff had positively evaluated. the condition of soap and gel dispensers was subsequently included in the ic-cqi tool, and subsequent validated scores indicated a sustained improvement across the organization. the range of specialties, workflows, client groups, facilities, and infection risks provided a wide variety of area-specific issues that emerged from staff observations, audits, feedback, complaints, and root cause analysis. these included correcting air pressure in isolation rooms, staff refusing to remove wrist watches, parents visiting neonates while contagious, inappropriate disposable glove use, and poor patient hand hygiene. staff identified local issues requiring improvement and agreed on expectations and actions. these were included in the daily handover, local education, knowledge assessment, and the progress audited. once improvement was demonstrated, monitoring could stop and switch to other issues of concern. is there alcohol gel at each bed end that is filled and working? are soap and hand towel dispensers filled, clean, and working at each sink? are the alcohol gel dispensers at entrance and wall-mounted dispensers filled and working? check taps−are they correctly adjusted for elbow operation? is gel available on desks, next to keyboards, and by notes trolleys? check keyboards−are they being cleaned regularly? area specific criteria examples include: are patients provided with hand wipes at mealtimes? is the environment clean and cleaned to a high standard? is glove use appropriate? observation single room/sequence of care observation. some areas readily used this opportunity to identify, improve, and monitor issues, whereas others were reluctant to highlight problems as there was anxiety about producing a low score even for a short period. sometimes, encouragement was required to tackle an area of practice that was recognized as requiring improvement. e. observation of single room practice and sequences of care feedback indicated that observing practice was valued by staff as an opportunity to look at practice delivery and the environment. there was also a recognition that staff disliked covert observation and wanted to understand what was expected and how they could improve practice. two types of observation: observing sequences of care and single room isolation practice were identified in pilots as potentially useful and acceptable to hcws and were subsequently adopted. both required training auditors and clarification and agreement of expectations with practitioners and subsequent inclusion in local education. sequence of care monitoring requires the observer to compare the infection control expectation with performance, records, data, and offers an opportunity to provide feedback. examples include observing doctors on a ward round or a nurse preparing and administering intravenous drugs. sometimes, it was difficult to understand what was monitored as documentation was often limited and it was not always possible to validate these observations. single room observation monitored isolation practice using an audit tool. an observer records the infection control practice expectations, and then while positioned outside the room, observes, records, and offers feedback of performance. this may include infection control precautions taken by people entering and leaving the room, if the isolation sign was accurate, and if adequate ppe was available. these data were simple to validate, and the tool was also used to check isolation practice compliance ad hoc to clarify expectations of new patients requiring isolation. the ic-cqi tool ( table ) was evaluated to assess acceptability to local auditors and managers and if the results were perceived to be a fair reflection of performance. at this stage, the assessment focused on the perspective of the auditors and those using the information rather than those being assessed. in december , % ( of ) of data collectors responding to online questionnaires had time allocated to undertake data collection. this had increased to % ( of ) by june . those trained in the use of the tool had also increased slightly from % ( of ) in to % ( of ) in . use of the tool had increased to % ( of wards sampled) in , and % ( of ) believed the tool had helped improve infection control practice in their area. a total of % ( ) did not believe the tool had led to a decline in infection control practice standards, and % ( of ) believed the data were an accurate reflection of practice. in addition, auditors assessed observation, knowledge, education, and promotion of awareness as the most valuable components of the tool, while at the same time the most difficult to collect (fig ) . data collectors/auditors consistently requested more training in the use of the tool, more prepared questions rather than locally developed questions, and simplification of the data collection process. validation of the scores obtained were undertaken by the infection control team, although some observations were difficult to validate particularly in the presence of local interpretations. the use of the tool and validity of the data collected continue to be evaluated. infection reduction data are not reported here as it is unlikely that outcomes will be directly attributable to the use of the tool as other improvements and changes in care occur frequently such as increased isolation provision and increases in robotic surgery. broadening the scope of monitoring to include other aspects of infection control practice beyond hand hygiene was hampered by a lack of a robust evidence base for some common infection control practices and inconsistent opinions from subject matter experts. this created some ambiguity of expectations of infection control practice, but consensus was often achieved when the rationale for practice was examined and options explored. the approach was sometimes uncomfortable for senior infection control staff but liberating for junior staff who were empowered to question entrenched habitual practice. changes in the focus of monitoring considered the value of knowledge, education, training, and human factors and were readily accepted, but, despite evidence that local ownership and participation is beneficial, this was difficult to achieve. staff were seldom allocated time to attend training or collect information and sometimes lost the momentum to identify new areas for local improvement. lack of energy and resilience has been recognized previously in nhs staff, and it has been suggested that this may be related to an underlying lack of engagement and absence of positive reinforcement for previous efforts. these issues were particularly problematic when there was potential for reporting lower scores conflicting with the established organizational aspiration of reporting high performance. resistance to acknowledging and monitoring areas that required improvement was a recurring issue during the development and implementation of this tool. although the fallibility of the previous monitoring system was recognized within the organization, the use of soft intelligence provided by local observations and feedback was a major departure from the normal practice of collecting data for assurance to one of improving practice. consequently, the assimilation of this change within the organization was slow, and auditors reported persistent pressure from managers to achieve targets and avoid highlighting areas for improvement. at times this perpetuated the organizational blindness to problems and it was unclear if this influenced the priority given for time for training and data collection. establishing rational and feasible expectations of hcws within the context of care delivery and providing information including data that was useful locally had a positive impact on engagement and acceptance of the changes introduced. however, the flexibility and adaptability of the tool created inconsistencies and anomalies that hampered standardization of practice and validation of results. the use of observation of practice was valued by auditors but standardization of practice was simpler to achieve and validate when an unambiguous audit tool was provided, for example, the single room audit tool as this was less reliant of staff knowledge and local variations. a widely used hand hygiene compliance monitoring system produced data that did not contribute to quality and safety improvement in organization, and an alternative quality improvement tool was developed and implemented. consideration of the context of care delivery led to the creation of a flexible and pragmatic tool that could be adapted. the previous focus on hand hygiene compliance was replaced by monitoring performance in a range of infection controlrelated factors. this work was undertaken in organization that may limit generalizability. the barriers and opportunities identified may vary in other organizations and facilities that may affect replication, , although issues such as ambiguity, poor role models, and knowledge are likely to be common. the tool is now established as a performance metric in the organization and has been adopted and adapted in other health care organizations. the removal of 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respiratory syndrome in the middle east date: - - journal: fowler's zoo and wild animal medicine current therapy, volume doi: . /b - - - - . - sha: doc_id: cord_uid: q exf s nan middle east respiratory syndrome (mers) is an emerging infectious zoonotic disease caused by a novel coronavirus (cov). mers was first reported in in jeddah, kingdom of saudi arabia (ksa), and in jordan, respectively. the disease was considered a potential pandemic threat to public health in the persian gulf region. (see also chapter .) most known covs infect and circulate in animals, mainly bats, but a number of covs are known to cause human disease (see also chapter ). [ ] [ ] [ ] the rapid emergence of mers-cov coupled with its limited geographic distribution has led to the suspicion that this is a zoonotic disease with an animal reservoir, and the evidence supports the hypothesis that dromedary camels (dcs) are the reservoir host. in dcs mers-cov causes a mild, transient upper respiratory tract (urt) infection. [ ] [ ] [ ] a mild or asymptomatic disease has also been reported in humans, but this is not always the case. mers-cov infection in humans often results in a severe, life-threatening disease of the lower respiratory tract (lrt), with high mortality. , , immunocompromised, elderly people and those with comorbidities, usually with a history of close contact with infected dcs, are particularly susceptible. is a positive-sense enveloped single-stranded rna virus and is the first lineage of c betacoronavirus known to infect humans. , it is more closely related to bat covs hku and hku (lineage c) than to the severe acute respiratory syndrome cov (sars-cov, (lineage b). , recent genome sequencing analysis reported the genomic evolution rate ( . × substitutions per site), suggesting that mers-cov diverged from its viral ancestor in march . analysis of human mers-cov sequences has identified several circulating genotypes. these distinct genotypes are phylogenetically classified into clades a, b, and, most recently, c, which correlate with outbreaks of mers among humans. , , , the emergence of divergent mers-cov clades in humans since is consistent with several independent sporadic introductions into the human population from an animal reservoir, of which the camel was unquestionably the source. , , , pathogenesis host cell entry of mers-cov is mediated by the binding of mers spike (s) proteins to a specific cellular receptor known as dipeptidyl peptidase (dpp ). dpp is expressed on the epithelial and endothelial cells of most human organ tissues in ex vivo studies using human tissue culture lines; this may account for the multisystem clinical spectrum of the mers-cov infection. , a strain cultured from a fatal human case was experimentally inoculated into three dcs using intratracheal, intranasal, and conjunctival routes. a mild transient disease resulted in submucosal inflammation and necrosis in the urt and lrt, but the alveoli remained unaffected. experimental inoculation of rhesus macaques (macaca mulatta) and common marmosets (callithrix jacchus) resulted in mild to severe lrt disease causing multifocal interstitial pneumonia in the macaques and extensive fatal pneumonia in the marmosets. , , since at least . several mers-cov serologic surveys confirm that the disease is not present in domesticated livestock (namely, horses, sheep, goats, and cattle) and is enzootic in the dc population across the arabian peninsula as well as in north and east africa. , , , , [ ] [ ] [ ] [ ] [ ] historically, the camel was the mainstay of land-based trade transportation and was used extensively as a food source across the entire region prior to industrialization during the latter part of the th century. , the free movement of humans and animals across the region supports the widespread prevalence and genetic diversity of mers-cov in the dc populations of arabia and east africa today. , the temporal dynamics of mers infection in dcs in al-ahsa, ksa, was examined by collecting nasal swabs and lung tissue during postmortem examination from two independent groups of animals over the course of a year and testing these for mers-cov rna by real-time reverse-transcriptase polymerase chain reaction (rt-rtpcr). positive samples were typically associated with young immunologically naive animals (< years of age) rather than adults (> years of age). seasonal peaks were detected during the winter months and coincided with the calving season, less extreme environmental conditions, cooler ambient temperatures, and higher relative humidity, for the transmission of infection amongst susceptible individuals. this seasonal peak has also been described in epidemic nosocomial outbreaks in humans that occur more frequently during the winter months. , extensive virologic evidence has been accumulated since supporting the epidemiologic link between dcs and humans in the transmission of mers-cov, although epidemiology mers-cov belongs to a lineage commonly associated with bats, the closest relatives of which lineage were recently identified in vesper bats (i.e., various species of the family vespertilionidae) from europe, asia, and south africa. initial research efforts have focused on establishing an epidemiologic link between bats and humans. , , , there is no conclusive evidence to support the theory that bats are the source of human infection, although there is consensus that bats are the ancestral hosts of the disease. , , a related mers-like cov virus, isolated from an african pipistrelle bat (pipistrellus hesperidus) in uganda, has shown high divergence of the s protein nucleotide sequence compared with an index mers-cov s protein sequence ( % amino acid identity divergence). this suggests that the two viruses differ significantly in receptor binding properties, implying that the mers-like cov virus is not a zoonotic threat and supporting the theory of a common ancestry. to date the only direct link between bats and human disease was a single instance when an rna sequence from a fatal human mers index case showed a % nucleotide match of a polymerase chain reaction (pcr)-amplified sequence of a fecal pellet from an egyptian tomb bat (taphozous perforates) collected in the same area of bisha, ksa. the human fatality was an owner of four dcs, which also tested positive for the same strain of mers-cov. at present, bat-to-human infection by mers-cov is considered to be purely speculative. surveillance of dcs in ksa has shown that mers-cov clade b has been enzootic in the camel population in arabia genetic deep sequencing methods (i.e., high-throughput sequencing) have been readily available to researchers since the disease was first reported. sequenced data have been used in these cases to successfully construct the phylogenetic tree between related viruses and hosts. , , , direct mers-cov antigen detection is possible but has been rarely performed. immunochromatography assays and monoclonal antibody-based capture elisas targeting the mers-cov nucleocapsid protein have been described. since the virus was first reported in , a range of comprehensive laboratory tests has been developed. , to better understand the disease, it has been important to collate sampling methodology data, laboratory results, and analyses in combination with clinical and epidemiologic data. until laboratory assays are fully validated, a combination of molecular and serologic laboratory tests is required to improve confidence in laboratory diagnosis during outbreaks. in cases of mild or asymptomatic infection, full validation of serologic assays is required to rule out false-negative results. validation is also required to successfully apply newly developed diagnostic serology algorithms to inform public health decisions. , , treatment therapeutic options for mers-cov are limited. supportive treatment is indicated for hospitalized patients, but vigilance for complications is essential. empirical use of antimicrobial agents or steroids has not succeeded in reversing the progression of severe disease. , , no specific drug or vaccine is currently available to treat mers. indeed, it has been stated that the complexity and time required for the development and registration process of drugs for human use impedes the ability to counter the rapid threat against an emerging infectious agent. for example, there is no vaccine available against sars-cov because of the brevity of the threat to the public health. , it is likely that a mers-cov vaccine for human use may not be developed due to a lack of commercial interest, or if the threat posed by mers-cov declines in the meantime. nevertheless, given the prevalence of mers-cov infection in the middle east's dc population and due to the potential for spillover to the human population in direct contact with dcs, the development of a vaccine for use in dcs may be feasible. , , a recent successful trial of a mers orthopoxvirus vaccine has conferred mucosal immunity in the urts of dcs. eradication of mers-cov from herds may be possible, if vaccines are administered to young, immunologically naive camels prior to exposure. , , identification of the zoonotic source of mers guides control strategies at the human-animal interface. , by preventing spillover of mers-cov from animals to humans, the risk of nosocomial and familial outbreaks in the middle east could be eliminated. strategic serosurveys of humans using samples collected after have been infrequent. , , there is a paucity of baseline data to describe the proportion of the potentially infected human population for much of the arabian peninsula and all of east africa, including the horn of africa. the exact mechanism of transmission from camels to human remains uncertain. sustained close contact is most probably necessary for transmission by aerosolized droplets, as mers-cov viral rna has been detected in air samples from a barn housing infected dcs in qatar, and the virus may remain viable in aerosol for up to minutes. , [ ] [ ] [ ] the potential public health risk resulting from aerosol-generating activities ranges from contamination of a room occupied by a symptomatic patient to slaughter practices. , , aerosolized transmission of mers-cov has been attributed to hospital outbreaks in ksa and south korea. , , mers-cov spreads inefficiently from human to human, but transmission is effective in a hospital environment, where susceptible individuals are concentrated and the risks are amplified by poor infection prevention and control (ipc) protocols. in some reported cases of mers, direct contact with camels was not apparent. , camel-to-human transmission through other routes is, however, possible owing to the consumption of unpasteurized camel milk or raw camel meat and in traditional medicine, when camel urine is consumed as a natural remedy for a variety of ailments. , a recent survey has found that infected camels may shed mers-cov virus in milk and urine, and the virus has been shown to remain infectious for days in milk stored at °c. , the transmission risks associated with the handling of camel products, raw milk, urine, and meat during animal slaughter are yet to be fully elucidated. further studies are needed to demonstrate the potential of camel-to-human transmission. serologic methods with high sensitivity and specificity to detect mers-cov antibodies have been developed for use in seroepidemiologic studies. methods include indirect immunofluorescence assays, enzyme-linked immunosorbent assays (elisas), protein microarray technology, and microneutralization (mn) assays. , [ ] [ ] [ ] pseudoparticle virus neutralization tests (ppnts) and conventional mn assays have also been used to detect neutralizing antibodies to mers-cov. validated molecular assays have been developed. , , rt-rtpcr is the preferred diagnostic method for the detection of mers-cov and has been endorsed by the who. confirmation of mers in suspected cases requires the screening of samples targeting a number of genes specific to mers-cov, namely up e, orf a, orf b, and n genes. , , , its infancy. aside from bats, the role that other wildlife may play in the ecology of mers-cov in east africa and arabia is yet to be elucidated. at present the implementation of intensive ipc measures in human health care is vital, including improving education and awareness among healthcare workers. , most human cases have been linked to lapses in ipc, as one-fifth of viral infections have been reported among healthcare workers. , stringent precautions while handling suspected mers-cov patients include the use of personal protective equipment (ppe) (i.e., disposable gloves, gowns, respiratory protection, and eye protection). , , immunocompromised individuals and those with preexisting medical conditions should avoid close contact with dcs. , public health authorities should adopt a standardized public health response protocol to include standardized case reporting methodology as defined by the who. , standardization of case definitions aids accurate calculation of a case fatality ratio by including mild or asymptomatic cases. the health authority of abu dhabi in the united arab emirates recently implemented a standardized reporting option for mers, successfully incorporating it into existing epidemiologic surveillance systems with the aim of enhancing surveillance, educating healthcare workers, and ensuring laboratory capacity. in countries where mers-cov is enzootic in dcs, mers control at the animal-human interface is unlikely to succeed unless appropriate preventive strategies are implemented. these should include the following: • strict regulation of camel movement with imposition of a requirement for mers clearance prior to the importation and transport of camels, including animals presented for slaughter. • camels with detectable mers-cov rna should be quarantined and tested at regular intervals. • use of appropriate ppes while handling dcs. • increased awareness among camel owners and the general public of the risks of consuming unpasteurized camel milk and urine. this may prove challenging given the depth of customs and beliefs in some areas. • accelerated development of safe and effective mers vaccines for animal and human use. , conclusions mers-cov has been observed for only years, and vigilance is vital for the containment of the disease due to the high case fatality rate in humans and possible genetic instability of the virus. continued laboratory testing, genetic sequencing, analysis, timely data sharing, and clear communication are essential if such vigilance is to be effective. nonetheless, despite the potential for a pandemic outbreak at multiple mass gatherings during the islamic calendar (hajj, eid, and umrah) there were no reported outbreaks of mers during or immediately after these events. as such mers-cov is not a virus of pandemic concern. since our understanding of mers has increased greatly although gaps in knowledge still exist. the understanding of the disease's ecology-especially the interplay between camels, humans, and the environment-is still in who mers-cov global summary and risk assessment middle east respiratory syndrome coronavirus "mers-cov": current knowledge gaps evidence for zoonotic origins of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus (mers-cov) origin and animal reservoir middle east respiratory syndrome coronavirus (mers-cov): animal to human interaction middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation evidence for camel-to-human transmission of mers coronavirus middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd who emergencies: mers-cov. available at mers coronavirus: diagnostics, epidemiology and transmission dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia pathogenesis of middle east respiratory syndrome coronavirus replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels response to emergence of middle east respiratory syndrome coronavirus transmission of middle east respiratory syndrome coronavirus infections in healthcare settings hospital outbreak of middle east respiratory syndrome coronavirus detection of the middle east respiratory syndrome coronavirus genome in an air sample originating from a camel barn owned by an infected patient mers-cov outbreak in jeddah-a link to health care facilities mers coronavirus: data gaps for laboratory preparedness asymptomatic mers-cov infection in humans possibly linked to infected camels imported from oman to united arab emirates an orthopoxvirusbased vaccine reduces virus excretion after mers-cov infection in dromedary camels centers for disease control and prevention: interim prevention and control recommendations for hospitalized patients with middle east respiratory syndrome coronavirus (mers-cov). available at an animal model of mers produced by infection of rhesus macaques with mers coronavirus further evidence for bats as the evolutionary source of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus in bats, saudi arabia mers coronaviruses in dromedary camels middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia antibodies against mers coronavirus in dromedaries middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan isolation of mers coronavirus from a dromedary camel mers-cov in upper respiratory tract and lungs of dromedary camels, saudi arabia the authors wish to thank the following: h. e. ghanim mubarak al hajeri and the senior management of al ain zoo for their encouragement and support, dr. ahsan ul haq of dubai camel hospital for his technical insight, and dr. andrew higgins, fellow of the zoological society of london and honorary editor in chief of the veterinary journal, who reviewed the manuscript. key: cord- -fnm im authors: lee, brian r.; hassan, ferdaus; jackson, mary anne; selvarangan, rangaraj title: impact of multiplex molecular assay turn-around-time on antibiotic utilization and clinical management of hospitalized children with acute respiratory tract infections date: - - journal: j clin virol doi: . /j.jcv. . . sha: doc_id: cord_uid: fnm im background: empiric antibiotic treatment is common among children with acute respiratory tract infections (arti), despite infections being predominately viral. the use of molecular respiratory panel assays has become increasingly common for medical care of patients with artis. study design: this was a -year retrospective, single-centered study of pediatric inpatients who tested positive for an arti respiratory pathogen. we examined the relationship between clinical outcomes and whether the patient was tested using the luminex respiratory viral panel ([rvp]; in-use: dec. – jul. ) or biofire respiratory pathogen panel ([rp]; in-use aug. – jun. ). the prevalence and duration of pre-test empiric antibiotics, post-test oseltamivir administration to influenza patients, chest x-rays and length of stay between the two assays was compared. results: a total of patients ( rvp; rp) were included. the median laboratory turn-around-time for rp was significantly shorter than rvp ( . vs. . h, respectively; p < . ). patients tested with rp were less likely to receive empiric antibiotics (or: . ; p < . ; % ci: . , . ) and had a shorter duration of empiric broad-spectrum antibiotics ( . h vs. . h; p < . ) compared to rvp patients. rp influenza patients had increased oseltamivir use post- test compared to rvp influenza patients (or: . ; p < . ; % ci: . , . ). conclusions: rapid molecular testing positively impacts patient management of artis. adopting assays with a shorter turn-around-time improves decision making by decreasing empirical antibiotic use and duration, decreasing chest x-rays, increasing timely oseltamivir administration, and reducing length of stay. acute respiratory tract infections (arti) -including the common cold, otitis media, pharyngitis, acute bronchiolitis, and pneumonia-are the most common diagnoses among patients seeking medical care in the us, and account for the majority of all antibiotic prescriptions [ , ] . donnelly et al. estimated that each year there are million emergency department (ed) visits for patients < years of age with a diagnosis of arti (rate: per ed visits). [ ] . antimicrobials are not indicated for the common cold, bronchitis/bronchiolitis and the vast majority of pharyngitis cases, and specific criteria exists to target appropriate antibiotic use for otitis media, sinusitis and streptococcal pharyngitis. yet, antibiotic prescribing is very common among children with arti, for bronchitis ( %), sinusitis ( %), and acute otitis media ( %) [ ] . a study evaluating bronchiolitis management before and after the aap guidelines found a significant reduction in rsv testing (p < . ) and decreased corticosteroids and bronchodilators use (p < . ) [ ] . however, the trend in antibiotic use did not change (p = . ) in the post-aap guideline period further highlighting the significant problem of overuse of antibiotics for arti. while the use of stringent diagnostic criteria to confirm the diagnosis of arti is key in optimizing antibiotic prescribing, empiric treatment for arti remains common because viral symptoms are often clinically similar and difficult to distinguish from those caused by bacteria. therefore, laboratory testing that provides accurate and prompt detection of pathogens associated with respiratory infections is https://doi.org/ . /j.jcv. . . received august ; received in revised form november ; accepted november important for proper patient care management. molecular respiratory panel (mrp) assays are becoming increasingly popular due to their ability to detect multiple pathogens with high sensitivity and specificity and documented cost savings [ ] [ ] [ ] . while research has demonstrated that mrps may have a positive impact on patient outcomes such as decreasing empiric antibiotic exposures, length of hospital stay (los), and improving timely oseltamivir treatment for influenza patients [ ] [ ] [ ] [ ] [ ] [ ] , there is a dearth of information on whether this clinical impact is conditional on the turn-around-time (tat) of the mrp assay. both xtag respiratory viral panel (rvp) and biofire respiratory panel (rp) are fda-cleared mrp assays that can detect and respiratory pathogens, respectively. the objective of this study was to compare the impact of rvp and rp on clinical outcomes for patients that were admitted in our hospital from to . we hypothesized that the rapid detection of respiratory pathogens by rp compared to rvp would be positively associated with changes in antibiotic treatment, initiation of oseltamivir and los on pediatric patients < years old. we performed a retrospective cohort study of pediatric patients who were admitted to our -bed free-standing children's hospital between december -june and who tested positive for at least one of the respiratory pathogens on either the rvp (luminex inc., texas) or rp (biofire llc, idaho) mrp assay. data were abstracted from the electronic medical record for patients where the rvp or rp assay occurred either while admitted as an inpatient or during the ed encounter. for patients initially seen at the ed and subsequently admitted, all medical services were considered one clinical episode. patients were excluded for all who were either ) undergoing immune suppressive therapy; ) were admitted to the nicu; ) had a los greater than days; or ) rvp or rp assay was not ordered within the first h of hospitalization. this study was reviewed and approved by the children's mercy hospital institutional review board. in our hospital the rvp assay was introduced in december and was replaced by the rp assay in august . patients' respiratory samples were tested by either the rvp or rp based on test in use date. at our institution rvp tests were run in batch mode once daily, days a week, whereas rp tests were run as the samples arrived ( / ) in the clinical lab. the primary outcome of this study was appropriate antimicrobial therapy. binary treatment indicators were based on antimicrobial use during a -hour period both before and after the mrp assay results were reported in the laboratory information system (fig. ) , including: ) use of empiric systemic antibiotic treatment between time of admission and result availability and ) administering oseltamivir to influenza positive patients during the h following result availability. the prevalence of narrow-and broad-spectrum empiric antibiotic treatment was also calculated, including the duration of empiric therapy, which was defined as the time difference, in hours, between the first and last antibiotic administration. we considered penicillin, ampicillin and clindamycin as narrow-spectrum antibiotics; all other antibiotics were considered broad-spectrum. we also evaluated use of chest radiographs within the first h of admission and los (in hours) as secondary outcomes. lastly, we calculated the turn-around-time ([tat]; i.e., time from when the clinical laboratory received the specimen to when the test was completed and results reported in the laboratory information system) for each specimen tested. we dichotomized our study time period into either rvp patients or rp patients. the patient's age at the time of admission was categorized into either < days (infant sepsis work-up), - months (upper age range from aap guideline on bronchiolitis management [ ] ) or years and older. a mutually exclusive pathogen indicator was created based on the organism(s) detected in the rvp/rp assay (rhinovirus/ enterovirus, influenza virus, respiratory syncytial virus (rsv), adenovirus, human metapneumovirus, parainfluenza, co-detection [ + viruses], or bacterial pathogen). for example, a patient who was positive for rsv only was categorized as 'rsv' whereas a patient who tested positive for both rsv and rhinovirus/enterovirus was categorized as 'co-detection'. any patient with a bacterial pathogen, regardless of concurrent viral infection, was categorized as 'bacterial pathogen'. in order to further depict clinical presentation and medical decision making, we included the location where the mrp assay was ordered (picu, ed, medical/surgical, or other) and the calendar quarter. the frequency distribution of our categorical outcomes was calculated for both rvp/rp time periods, with pearson's chi-square used to compare proportions. hospital los and tat were treated as continuous outcomes with the mann-whitney u test used to compare distributions. multivariable logistic models were used for each of our categorical outcomes to examine the relationship with rvp/rp time period, after adjusting to medical service, patient age, pathogen, and seasonality. all analysis were completed using sas (sas . ; sas institute, cary, nc). a total of , patients in whom either one of the mrp assays (luminex rvp or biofire rp) was obtained during the study time period were initially identified. we excluded ( . %) patients who tested negative for any pathogens since the focus of the current study was to describe the impact of rapid detection of respiratory pathogens in management of hospitalized patients. we further excluded ( . %) who were admitted to either the nicu or hematology/oncology ward, ( . %) who did not have the rvp/rp ordered within the first h of admission, ( . %) who had a los > days, ( . %) who were seen in the ed but were not admitted to the hospital, and (. %) who were ≥ years old at the time of the admission. our final study group included patients a total of ( . %) patients were tested with the rvp assay (table ) . those patients who were ≥ years old represented the largest age group in our analysis (n = ; . %). nearly half of our patients tested positive for rhinovirus/enterovirus only whereas ( . %) patients were positive for ≥ bacterial organisms. the frequency distribution of respiratory pathogens between the two rp/rvp time periods were comparable (fig. ) utilizing mrp assays may increase the likelihood of optimal treatment of viral arti [ ] [ ] [ ] [ ] . however, since some of these studies have been restricted to adult patients [ ] , examined clinical impact for influenza patients only [ ] or only included data from peak respiratory seasons [ , ] more studies are needed to provide a comprehensive assessment of the clinical impact for pediatric arti patients. our study included pediatric patients with positive respiratory pathogens that were collected over years. we observed that relying on rapid diagnostic testing for arti cases may have a direct impact for the patient management, including reducing unnecessary antibiotic and radiation exposure, and reducing los. despite artis being predominately viral in nature, empiric antibiotic treatment for arti remains prevalent. kronman et al. found that antibiotics were frequently prescribed for bronchitis, sinusitis, and acute otitis media patients. [ ] byington et al. examined patients who received a direct fluorescent assay for respiratory pathogens and found that only . % of patients who also received bacterial cultures tested positive for a bacterial infections [ ] . we propose that use of mrp assays significantly improves diagnostic yield and accurately identifies viral etiology in majority of patients (∼ %; internal data monitoring over past years), providing clinicians added confidence in antibiotic treatment decisions. our findings provide much needed data to federal programs and insurance agencies that question the value of mrp assays in management of arti patients [ ] . we feel our study contributes to existing evidence [ ] on how rapid molecular diagnostics may further optimize appropriate care and management of arti patients. our study found that patients tested with rp, which had a shorter tat, were less likely to receive empiric antibiotics. we hypothesized that having a shorter tat may influence the clinician's decision making to delay empiric antibiotic therapy knowing that assay results would be available within a few hours. a study among patients who tested positive using a rapid influenza test found that when the clinician was aware of the results they were significantly less likely to prescribe antibiotics ( . % vs. . %) and more likely to give an antiviral [ ] [ ] [ ] [ ] [ ] [ ] prescription ( . % vs. . %) relative to when the clinician was unaware the patient was positive for influenza. [ ] in our study, patients with rp test results available approximately h faster than rvp were less likely to have empiric antibiotic therapy ( . % vs. . %) as well as a significantly shorter duration of empiric broad-spectrum ( . vs. . h) and narrow-spectrum therapy ( . vs. . h). according to cdc guidelines, anti-influenza therapy is recommended for patients < years with signs and symptoms of flu-like illness. [ ] in addition, multiple observational studies have demonstrated that receipt of oseltamivir significantly improves patient outcomes for patients with influenza [ ] [ ] [ ] [ ] [ ] [ ] , although clinical improvement is more likely if oseltamivir is initiated shortly after symptom onset [ , [ ] [ ] [ ] [ ] [ ] . a prospective study of icu influenza patients compared early treatment vs. late treatment oseltamivir administration and found that early treatment had shorter icu los ( . vs. . days), shorter hospital los ( . vs. . days), and decreased likelihood of mortality ( . % vs. . %) [ ] . in our study, nearly % of rp influenza patients received oseltamivir within h of result availability, compared with . % for rvp influenza patients. utilization of tests with shorter tat may ensure that optimal treatment for influenza infections is given. determination of cost effectiveness is important when hospitals decide on adopting rapid diagnostic testing. an increasing number of studies have concluded that rapid diagnostics that test for arti viruses provide a cost savings. [ , ] in our study, rp patients had decreased empirical antibiotic use, decreased likelihood of chest x-rays, and shorter los which could translate into decreased hospital cost. further cost effectiveness research is needed that also considers the cost of the rapid test for all patients, not just those who test positive. there were limitations with this study. we excluded patients seen in the either the nicu or hematology/oncology ward as well as patients who had a los > days. however, we purposely excluded these patients in an attempt to have a sample of otherwise healthy children. second, this was a single-center study at a freestanding pediatric hospital, therefore our results may not generalize to all centers that provide care for arti. third, we did not examine concurrent bacterial infections, which could partly explain empiric antibiotic treatment. since the prevalence of concurrent bacterial infection is unlikely to be influenced by the assay type (i.e., rvp/rp), we would argue the potential of this biasing our results is minimal. we observed less human metapneumovirus and influenza cases during the rp phase which may have influenced treatment strategies, especially oseltamivir use. patients tested but never admitted were excluded, thus our inpatient sample may represent patients with more severe arti symptoms. lastly, we did exclude patients who tested negative, thus our study does not provide a complete representation of arti patients but rather addresses the importance of rapid respiratory pathogen detection on management of hospitalized patients. we intend to conduct future research on mrp assays that includes both negative and positive patients. this study demonstrates that adopting mrp assays with a shorter tat can have a significant improvement for pediatric inpatients with viral arti, including decreased exposure to empirical antibiotics, decreased exposure to chest x-rays, increased optimization of timely oseltamivir administration, and shorter los. 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participant data key: cord- -zvwy bgt authors: chapman, christie; delahanty, kim title: convergencia mundial de las enfermedades infecciosas emergentes: a tan solo un viaje en avión de distancia date: - - journal: nan doi: . /j.nursi. . . sha: doc_id: cord_uid: zvwy bgt nan el de abril de , un macaco de la india, utilizado con fines de investigación en un estudio sobre la fiebre amarilla en la selva zika de uganda, enfermó con fiebre de , °c. los científicos tomaron muestras de sangre y se le realizaron análisis. el macacus rhesus n.º estaba aquejado de algo desconocido. con el tiempo, el virus emergente se bautizaría así por el lugar donde se descubrió por primera vez. a principios de , los científicos aislaron también el virus del zika en mosquitos aedes africanus capturados en la misma selva. a pesar de que el virus del zika despertaría una atención científica esporádica y limitada durante las siguientes décadas, había surgido una nueva amenaza microbiana para la salud humana . aunque hoy día estamos familiarizados con la neumonía atípica asiática (o síndrome respiratorio agudo grave [sars, severe acute respiratory syndrome]), la enfermedad del Ébola y la infección por el virus de la inmunodeficiencia humana (vih), no hace mucho eran consideradas enfermedades infecciosas emergentes. según los centers for disease control and prevention (cdc), una enfermedad infecciosa emergente es aquella cuya incidencia ha aumentado en los últimos años y podría aumentar en un futuro próximo . curiosamente, en la bibliografía se informa de que el % de las enfermedades que en la actualidad se consideran "emergentes" son zoonóticas, lo que significa que se originaron en animales . ningún factor por sí solo desencadena la aparición de enfermedades. los seres humanos y los microbios se hallan en el centro de un conjunto complejo de elementos en continua evolución, los cuales originan esta tormenta perfecta. el modelo de convergencia elaborado por el institute of medicine es una forma útil de visualizar la aparición de enfermedades (v. el cuadro visualización de una enfermedad emergente según el modelo de convergencia). esta ilustración muestra cómo varios factores en continua evolución influyen en la interacción entre el ser humano y los microbios. de acuerdo con este modelo, la aparición de una amenaza microbiana para la salud del ser humano se origina a partir de la convergencia de cuatro ámbitos: factores genéticos y biológicos. factores medioambientales físicos factores ecológicos. factores sociales, políticos y económicos. en la intersección entre el ser humano y los microbios hay un cuadrado que representa la convergencia de los factores que conducen a la aparición de una enfermedad infecciosa. el interior de esta caja es un gradiente en movimiento hacia el interior que pasa del color blanco al negro. los bordes exteriores blancos representan lo que sabemos acerca de los factores en la aparición. el centro negro representa lo desconocido. al igual que ocurre con la búsqueda de la "caja negra" de un avión después de un accidente para tratar de entender por qué se produjo, los investigadores trabajan continuamente para arrojar luz en la "caja negra" de la aparición de nuevas enfermedades. este conocimiento podría ayudar a evitar la próxima amenaza microbiana global. reproducido con el permiso de national academies press, copyright , national academy of sciences. los rinovirus, causantes del resfriado común. sin embargo, de vez en cuando descubrimos un microbio patógeno que puede hacernos mucho daño o incluso puede poner en peligro nuestra existencia. a pesar de la tecnología de que se dispone, podemos identificar solo una pequeña parte de los microbios que habitan en la tierra y en la biosfera humana. todavía no entendemos muchas cosas sobre el mundo invisible de los microbios y sus efectos en el cuerpo humano. los microbios son la forma de vida más abundante y se han desarrollado durante millones de años. durante este largo tiempo de vida, se han vuelto muy hábiles en adaptarse para así evitar la aniquilación por el sistema inmunológico humano . desafortunadamente, los seres humanos les ayudan, sin saberlo, en sus esfuerzos por imponerse. hemos sido tan generosos en el uso de antibióticos que los microbios han aprendido a crear versiones resistentes de sí mismos y a propagar esta resistencia a otros microbios. mediante la creación de entornos para que estos "súper" microbios proliferen, los seres humanos les están quitando el poder a las mejores armas que tenemos en nuestro arsenal para luchar contra las peligrosas infecciones bacterianas . el acto de viajar es una fuerza poderosa en la aparición y transmisión de enfermedades. hoy día, las personas podemos viajar hasta el otro extremo de nuestro planeta en menos tiempo del que se necesita para que se manifiesten los síntomas y signos de una enfermedad en personas infectadas, las cuales, a su vez, pueden infectar a cualquier otra persona que entre en contacto con ellas durante su recorrido . por ejemplo, se ha descrito el efecto de los viajes en la transmisión de una enfermedad. en , los investigadores publicaron datos empíricos que sugerían que la propagación de la gripe en estados unidos en se retrasó debido al descenso de los viajes en avión después de los ataques del de septiembre de aquel año . las repercusiones de los desplazamientos en la transmisión del virus del zika después de los juegos olímpicos de en brasil es un ejemplo similar. el período de incubación estimado del virus del zika desde la exposición hasta la aparición de los primeros síntomas dura entre y días , . el deportista olímpico o el espectador desprevenido que viaja de vuelta de río dispone de mucho tiempo para propagar el contagio. muchas personas viajan por negocios o por placer, pero otras se ven obligadas a abandonar su hogar para escapar de la guerra, el hambre, la opresión o la pobreza. este tipo de emigración trae consigo muchos más elementos que pueden contribuir a la aparición de enfermedades infecciosas: hacinamiento, condiciones de vida y fuentes de agua insalubres, así como prácticas nocivas de obtención y preparación de alimentos . millones de personas en aldeas y campamentos de explotación forestal de África oriental y meridional carecen de suficientes animales domesticados para el consumo de carne y deben depender de la caza de animales salvajes para satisfacer esta necesidad. aproximadamente, un millón de toneladas métricas de carne de animales salvajes se consume cada año en África central. también es alarmante la entrada anual de miles de libras de carne de primates, antílopes y otros animales salvajes en estados unidos y europa como objeto de contrabando para actos culturales . su caza, preparación y consumo están vinculados con varias pandemias y epidemias, sobre todo con el vih, la enfermedad del Ébola y el sars . aunque el aumento del riesgo de transmisión de enfermedades debido al comercio internacional es un fenómeno no muy bien entendido, podría ser un peligro potencial para las personas. los cambios en la forma en que los seres humanos utilizan la tierra y el medio ambiente son motivos habituales de la aparición de enfermedades (v. el cuadro motivos de la aparición de enfermedades en los seres humanos). el desarrollo territorial para vivienda o para uso agrícola y la construcción de embalses y presas para crear fuentes de agua para uso agrícola y público en áreas geográficas previamente no desarrolladas aumentan las posibilidades de que los seres humanos entren en contacto con mosquitos, garrapatas, roedores y otros animales que pueden ser portadores de una enfermedad infecciosa . la incursión humana en estas áreas vírgenes aumenta el acceso de los seres humanos a zonas remotas e incorpora más vectores y reservorios de infección a nuevos huéspedes . en respuesta a cada amenaza de enfermedad emergente, por lo general, empleamos recursos sanitarios a nivel mundial en la búsqueda de vacunas para eludir la capacidad de estos microbios para hacer daño a gran escala. las vacunas han evitado un número aproximado de millones de muertes solo en estados unidos y continúan salvando , millones de vidas en todo el mundo cada año . un avance reciente y apasionante en el desarrollo de vacunas es la finalización de la fase de los ensayos clínicos de la rts,s/as , una vacuna contra la malaria que se experimentó en los países subsaharianos, donde el número de muertes por malaria en apenas pasó de . . el trabajo va avanzando rápidamente en vacunas para otras amenazas que se plantean actualmente, como la enfermedad del Ébola, el dengue y el zika. las vacunas son un arma potente contra la amenaza de una enfermedad conocida, pero ¿no sería maravilloso que pudiéramos contar con la tecnología necesaria para localizar y prevenir estas enfermedades emergentes antes que tuvieran la oportunidad de causar estragos? esto todavía no es realidad, pero grupos como el global disease detection program (gdd) de los cdc y la global outbreak alert and response network de la oms están vigilando sin descanso la evolución de la interacción entre microbios y seres humanos en todo el planeta, creando tecnología de seguimiento y respondiendo a los informes sobre el aumento de enfermedades y otras emergencias sanitarias . cada uno de nosotros puede colaborar para disminuir la amenaza de las enfermedades infecciosas saliendo de nuestro refugio doméstico y adoptando una mentalidad global. tenemos que ver la realidad de nuestra interrelación y ser conscientes de que lo que está sucediendo en otras partes de nuestro mundo puede encontrar la manera de que ocurra en nuestra ciudad. podemos hacernos menos vulnerables a las amenazas de enfermedades (conocidas y desconocidas) mediante la comprensión de los hechos básicos acerca de cómo se transmiten las enfermedades, la importancia de la higiene de las manos y de mantenerse al día de las vacunas que corresponden a cada edad. las enfermedades infecciosas emergentes son una amenaza para todos y cada uno de los habitantes del planeta y todo el mundo puede desempeñar un papel, ya sea en el retraso de la transmisión o la prevención. después de todo, la próxima enfermedad emergente podría estar a tan solo un viaje en avión de distancia. ■ bibliografÍa zika virus outside africa what we do microbial threats to health: emergence, detection, and response. institute of medicine of the national academies about antimicrobial resistance empirical evidence for the effect of airline travel on interregional influenza spread in the united states zika virus: symptoms, testing and treatment zika virus infection: an overview tourism and the health effects of infectious diseases targeting transmission pathways for emerging zoonotic disease surveillance and control. vector borne zoonotic dis breakthroughs in bioscience: vaccinesessential weapons in the fight against disease world health organization. malaria vaccine development the viral storm christie chapman es especialista en prevención de infecciones y kim delahanty es directora administrativa de epidemiología clínica de prevención de infecciones las autoras declaran no tener ningún conflicto de intereses económicos relacionado con este artículo key: cord- -hc akd authors: liu, quan-hui; xiong, xinyue; zhang, qian; perra, nicola title: epidemic spreading on time-varying multiplex networks date: - - journal: nan doi: . /physreve. . sha: doc_id: cord_uid: hc akd social interactions are stratified in multiple contexts and are subject to complex temporal dynamics. the systematic study of these two features of social systems has started only very recently, mainly thanks to the development of multiplex and time-varying networks. however, these two advancements have progressed almost in parallel with very little overlap. thus, the interplay between multiplexity and the temporal nature of connectivity patterns is poorly understood. here, we aim to tackle this limitation by introducing a time-varying model of multiplex networks. we are interested in characterizing how these two properties affect contagion processes. to this end, we study susceptible-infected-susceptible epidemic models unfolding at comparable timescale with respect to the evolution of the multiplex network. we study both analytically and numerically the epidemic threshold as a function of the multiplexity and the features of each layer. we found that higher values of multiplexity significantly reduce the epidemic threshold especially when the temporal activation patterns of nodes present on multiple layers are positively correlated. furthermore, when the average connectivity across layers is very different, the contagion dynamics is driven by the features of the more densely connected layer. here, the epidemic threshold is equivalent to that of a single layered graph and the impact of the disease, in the layer driving the contagion, is independent of the multiplexity. however, this is not the case in the other layers where the spreading dynamics is sharply influenced by it. the results presented provide another step towards the characterization of the properties of real networks and their effects on contagion phenomena. social interactions take place in different contexts and modes of communication. on a daily basis we interact at work, in the family, and across a wide range of online platforms or tools, e.g., facebook, twitter, emails, mobile phones, etc. in the language of modern network science, social networks can be conveniently modeled and described as multilayer networks [ ] [ ] [ ] [ ] [ ] . this is not a new idea. indeed, the intuition that social interactions are stratified in different layers dates back several decades [ ] [ ] [ ] . however, the digitalization of our communications and the miniaturization of devices has just recently provided the data necessary to observe, at scale, and characterize the multilayer nature of social interactions. as in the study of single layered networks, the research on multilayer graphs is divided in two interconnected areas. the first deals with the characterization of the structural properties of such entities [ , ] . one of the central observations is that the complex topology describing each type of interaction (i.e., each layer) might be different. indeed, the set and intensity of interactions in different contexts (e.g., work, family, etc.) or platforms (e.g., facebook, twitter, etc.) is not the same. nevertheless, layers are coupled by individuals active across two or more of them. the presence of such coupling as well as its * n.perra@greenwich.ac.uk degree is often referred to as multiplexity. another interesting feature of multilayer graphs is that the connectivity patterns in different layers might be topologically and temporally correlated [ ] [ ] [ ] . the second area of research instead considers the function, such as sustaining diffusion or contagion processes, of multilayer networks [ , , ] . a large fraction of this research aims at characterizing how the complex structural properties of multilayer graphs affect dynamical processes unfolding on their fabric. the first important observation is that disentangling connections in different layers gives rise to complex and highly nontrivial dynamics function of the interplay between interlayer and intralayer connections [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . a complete summary of the main results in the literature is beyond the scope of the paper. we refer the interested reader to these recent resources for details [ ] [ ] [ ] [ ] [ ] . despite the incredible growth of this area of network science over the last years, one particular aspect of multilayer networks is still largely unexplored: the interplay between multiplexity and the temporal nature of the connectivity patterns especially when dynamical processes unfolding on their fabric are concerned [ ] . this should not come as a surprise. indeed, the systematic study of the temporal dynamics even in single layered graphs is very recent. in fact, the literature has been mostly focused on time-integrated properties of networks [ , ] . as result, complex temporal dynamics acting at shorter timescales have been traditionally discarded. however, the recent technological advances in data storing and collection are providing unprecedented means to probe also the temporal dimension of real systems. the access to this feature is allowing to discover properties of social acts invisible in time aggregated data sets, and is helping characterize the microscopic mechanisms driving their dynamics at all timescales [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the advances in this arena are allowing to investigate the effects such temporal dynamics have on dynamical processes unfolding on time-varying networks. the study of the propagation of infectious diseases, ideas, rumors, or memes, etc., on temporal graphs shows a rich and nontrivial phenomenology radically different than what is observed on their static or annealed counterparts [ , . before going any further, it is important to notice how in their more general form, multilayer networks might be characterized by different types of nodes in each layer. for example, modern transportation systems in cities can be characterized as a multilayer network in which each layer captures a different transportation mode (tube, bus, public bikes, etc.) and the links between layers connect stations (nodes) where people can switch mode of transport [ , ] . a particular version of multilayer networks, called multiplex, is typically used in social networks. here, the entities in each layer are of the same type (i.e., people). the interlayer links are drawn only to connect the same person in different layers. in this context, we introduce a model of time-varying multiplex networks. we aim to characterize the effects of temporal connectivity patterns and multiplexity on contagion processes. we model the intralayer evolution of connections using the activity-driven framework [ ] . in this model of time-varying networks, nodes are assigned with an activity describing their propensity to engage in social interactions per unit time [ ] . once active, a node selects a partner to interact. several selection mechanisms have been proposed, capturing different features of real social networks [ , , [ ] [ ] [ ] . the simplest, that will be used here, is memoryless and random [ ] . in these settings, active nodes do not have a preference towards individuals previously connected. despite such simplification, the mechanism allows capturing the heterogeneity of time-integrated connectivity patterns observed in real networks while guaranteeing mathematical tractability [ ] . the multiplexity or coupling between layers is modulated by a probability p. if p = all nodes are present in all layers. if p = , the multiplex is formed by m disconnected graphs. we consider p as a parameter and explore different regime of coupling between layers. furthermore, each layer is characterized by an activity distribution. we consider different scenarios in which the activity of coupling nodes, which are present in different layers (regulated by p), is uncorrelated as well as others in which it is instead positively or negatively correlated. in these settings, we study the unfolding of susceptible-infected-susceptible (sis) epidemic processes [ ] [ ] [ ] . we derive analytically the epidemic threshold for two layers for any p and any distributions of activities. in the limit of p = we find analytically the epidemic threshold for any number of layers. interestingly, the threshold is a not trivial generalization of the correspondent quantity in the monoplex (single layer network). in the general case < p < we found that the threshold is a decreasing function of p. positive correlations of coupling nodes push the threshold to smaller values with respect to the uncorrelated and negatively correlated cases. furthermore, when the average connectivity of two layers is very different, the critical behavior of the system is driven by the more densely connected layer. in such a scenario the epidemic threshold is not affected by the multiplexity, its value is equivalent to the case of a monoplex, and the coupling affects only the layer featuring the smaller average connectivity. the paper is organized as follows. in sec. ii we introduce the multiplex model. in sec. iii we study first both analytically and numerically the spreading of sis processes. finally, in sec. iv we discuss our conclusions. we first introduce the multiplex model. for simplicity of discussion, we consider the case in which the system is characterized by m = layers a and b. however, the same approach can be used to create a multiplex with any number of layers. let us define n as the number of nodes in each layer. in general, we have three different categories of nodes: n a , n b , and n o . they describe, respectively, the number of nodes that are present only in layer a, b, or in both. the last category is defined by a parameter p: the coupling between layers (multiplexity). thus, on average, we have n a = n b = ( − p)n and n o = pn. as mentioned in the introduction, the temporal dynamics in each layer is defined by the activity-driven framework [ ] . thus, each noncoupling node is characterized by an activity extracted from a distribution f a (a) or f b (a) which captures its propensity to be engaged in a social interaction per unit time. observations in real networks show that the activity typically follows a heavy-tailed distribution [ ] [ ] [ ] , , ] . here, we assume that activities follow power laws, thus, f x (a) = c x a −γ x with x = [a, b] and a to avoid divergences. coupling nodes instead are characterized by a joint activity distribution h(a a , a b ). as mentioned in the introduction, real multiplex networks are characterized by correlations across layers. in particular, the study of a wide range of real systems shows a complex and case dependent phenomenology in which the topological features (i.e., static connectivity patterns) of coupling nodes can be either positively or negatively correlated [ ] . furthermore, researchers found evidence of positive temporal correlations between the activation patterns across layers [ , ] . to account for such observations and explore their effects on spreading processes, we consider three simple prototypical cases in which the activities of coupling nodes in the two layers are (i) uncorrelated, or (ii) positively and (iii) negatively correlated. to simplify the formulation and to avoid adding other parameters, in case of positive and negative correlations we adopt the following steps. we first extract n o activities from the two distributions f x (a). then, we order them. in the case of positive correlation, a node that has the rth activity in a will be assigned to the correspondent activity in b. in other words, the first node will be assigned to the highest activity extracted from f a (a) and the highest value extracted from f b (a). the second will be assigned to the second highest activity extracted from both distributions, etc. in the case of negative correlations instead, a node that has the rth activity in a will be assigned the (pn − r + )th in b. in other words, the first node will be assigned to the highest activity in a and to the lowest activity in b. the second node will be assigned to the second highest activity in a and to the second lowest in b, etc. in these settings, the temporal evolution of the multiplex network is defined as follows. for each realization, we randomly select pn nodes as coupling nodes between layers. at each time step t, note the following: (i) each node is active with a probability defined by its activity. (ii) each active node creates m x links with randomly selected nodes. multiple connections with the same node in the same layer within each time step are not allowed. (iii) coupling nodes can be active and create connections in both layers. (iv) at time step t + t all connections are deleted and the process restarts from the first point. all connections have the same duration of t. in the following, we set, without lack of generality, t = . at each time the topology within each layer is characterized, mostly, by a set of disconnected stars of size m x + . thus, at the minimal temporal resolution each network looks very different than the static or annealed graphs we are used to seeing in the literature [ ] . however, it is possible to show that, integrating links over t time steps in the limit in which t n, the resulting network has a degree distribution that follows the activity [ , , ] . in other words, the heterogeneities in the activity distribution translate in heterogenous timeaggregated connectivity patterns typically of real networks. thus, as observed in real temporal networks the topological features at different timescales are very different than the late (or time-integrated) characteristics [ ] . at each time step the average degree in each layer can be computed as where e x t is the number of links generated in each layer at each time step. furthermore, a x = da f x (a)a and a x o = da a da b h(a a , a b )a x are the average activity of noncoupling and coupling nodes in each layer, respectively. similarly, the total average degree (often called overlapping degree [ ] ), at each time step, is thus, the average connectivity, at each time step, is determined by the number of links created in each layer, and by the interplay between the average activity of coupling and noncoupling nodes. as shown in fig. (a), eq. ( ) describes quite well the behavior of the average overlapping degree which is an increasing function of the multiplexity p. indeed, the larger the fraction of coupling nodes, the larger the connectivity of such nodes across layers. as we will see in the next section, this feature affects significantly the unfolding on contagion processes. in fig. (b) we show the integrated degree distribution of the overlapping degree for different p. the plot clearly shows how the functional form is defined by the activity distributions of the two layers which in this case are equal. an increase in the fraction of coupling nodes does not change the distribution of the overlapping degree; it introduces a vertical shift which, however, is more visible for certain values of k. in order to understand how the interplay between multiplexity and temporal connectivity patterns affects dynamical processes, we consider sis contagion phenomena spreading on the multiplex model introduced in the previous section. in this prototypical epidemic model each node can be in one of two compartments. healthy nodes are susceptible to the disease and thus in the compartment s. infectious nodes instead join the compartment i . the natural history of the disease is defined as follows. a susceptible, in contact with an infected node, might get sick and infectious with probability λ. each infected node spontaneously recovers with rate μ, thus staying infectious for μ − time steps, on average. one crucial feature of epidemic models is the threshold which determines the conditions above which a disease is able to affect a macroscopic fraction of the population [ ] [ ] [ ] . in case of sis models, below the threshold the disease dies out, reaching the so called disease-free equilibrium. above threshold instead, the epidemic reaches an endemic stationary state. this can be captured running the simulations for longer times and thus estimating the fraction of infected nodes for t → ∞: i ∞ . in general, in a multiplex network, such fraction might be different across layers. thus, we can define i x ∞ . to characterize the threshold we could study the behavior of such fraction(s) as function of λ/μ. indeed, the final number of infected nodes acts as order parameter in a second order phase transition, thus defining the critical conditions for the spreading [ ] . however, due to the stochastic nature of the process, the numerical estimation of the endemic state, especially in proximity of the threshold, is not easy. thus, we adopt another method measuring the lifetime of the process l [ ] . this quantity is defined as the average time the disease needs either to die out or to infect a macroscopic fraction y of the population. the lifetime acts as the susceptibility in phase transitions thus allows a more precise numerical estimation [ ] . in the case of single layer activity-driven networks, in which partners of interactions are chosen at random and without memory of past connections, the threshold can be written as (see ref. [ ] for details) thus, the conditions necessary for the spread of the disease are set by the interplay between the features of the disease (left side) and the dynamical properties of the time-varying networks where the contagion unfolds (right side). the latter are regulated by first and second moments of the activity distribution and by the number of connections created by each active node (i.e., m). it is important to notice that eq. ( ) considers the case in which the timescale describing the evolution of the connectivity patterns and the epidemic process is comparable. the contagion process is unfolding on a time-varying network. in the case when links are integrated over time and the sis process spreads on a static or annealed version of the graph, the epidemic threshold will be much smaller [ , , ] . this is due to the concurrency of connections which favors the spreading. in fact, by aggregating the connections over time, the degree of each node increases thus facilitating the unfolding of the disease. in this limit of timescale separation between the dynamics of and on networks, the evolution of the connectivity patterns is considered either much slower (static case) or much faster (annealed case) with respect to the epidemic process. in the following, we will only consider the case of comparable timescales. this is the regime of time-varying networks which is extremely relevant for a variety of spreading processes ranging from sexual transmitted diseases and influenzalike illnesses to rumors and information propagation [ ] . what is the threshold in the case of our multiplex and timevarying network model? in the limit p = the number of coupling nodes is zero. the two layers are disconnected, thus, the system is characterized by two independent thresholds regulated by the activity distributions of the two layers. the most interesting question is then what happens for p > . to find an answer to this conundrum, let us define i , respectively, as the number of noncoupling nodes of activity a and as the number of coupling nodes of activity a a and a b . the implicit assumption we are making by dividing nodes according to their activities is that of statistical equivalence within activity classes [ , ] . in these settings, we can write the variation of the number of infected noncoupling nodes as function of time as where we omitted the dependence of time. the first term on the right-hand side considers nodes recovering, thus leaving the infectious compartment. the second and third terms account for the activation of susceptibles in activity class a (s x a = n x a − i x a ) that select infected nodes (noncoupling and coupling) as partners and get infected. the last two terms instead consider the opposite: infected nodes activate, select as partners noncoupling and coupling nodes in the activity class a infecting them as a result. similarly, we can write the expression for the variation of coupling nodes of activity classes a a and a b as the general structure of the equation is similar to the one we wrote above. the main difference is, however, that coupling nodes can be infected and can infect in both layers. the first term in the right-hand side accounts for the recovery process. the next four (two for each element in the sum in y) consider the activation of susceptible nodes that select as partners both noncoupling and coupling infected nodes and get infected. the last four terms account for the reverse process. in order to compute the epidemic threshold we need to define four auxiliary functions, thus defining a closed system of differential equations. in particular, we define x = da i x a a and o x = da a da b i o a a ,a b a x . for simplicity, we will skip the detailed derivation here (see the appendix for the details). by manipulating the previous three differential equations we can obtain four more, one for each auxiliary function. the condition for the spreading of the disease can be obtained by studying the spectral properties of the jacobian matrix of such system of seven differential equations. in particular, if the largest eigenvalue of the jacobian matrix is larger than zero, the system of equations will not be stable and, consequently, the number of infected nodes will increase. thus, the epidemic threshold can be obtained by studying the conditions for which this holds. as sanity check, let us consider first the limit p = . in this case, each layer acts independently and we expect the threshold of each to follow eq. ( ). this is exactly what we find. in particular, the system of equations can be de-coupled in two different subsets (one per layer) which are governed by two jacobian matrices whose largest eigenvalues are where a n x = da f x (a)a n . thus, the spreading process will be able to affect a finite fraction of the total population in case either of these two eigenvalues is larger than zero, which implies λ μ > (m x a x + m x a x ) − as expected. it is important to notice that in case of a multiplex network the disease might be able to spread in one layer but not in the other. however, in case the condition for the spreading is respected in both layers, they will experience the disease. let us consider the opposite limit: p = . as described in details in the appendix, the condition for the spreading of the disease reads as where a n interestingly, the threshold is a function of the first and second moments of the activity distributions of the coupling nodes which are modulated by the number of links each active node creates, plus a term which encodes the correlation of the activities of such nodes in the two layers. before showing the numerical simulations to validate the mathematical formulation, an important observation is in order. in this limit, effectively, we could think the multiplex as a multigraph: a single layer network with two types of edges. in case the joint probability distribution of activity is h(a a , a b ) = f (a a )δ(a b − a a ), thus two activities are exactly the same, and m a = m b the threshold reduces to eq. ( ) (valid for a single layer network) in which the number of links created by active nodes is m. however, for a general form of the joint distribution and in case of different number of links created by each active node in different layers, this correspondence breaks down. in all the following simulations, we set n = , = − , μ = . , y = . , start the epidemic process from a % of nodes selected randomly as initial seeds, and show the averages of independent simulations. in fig. we show the first results considering a simple scenario in which m a = m b = and the exponents for the distributions of activities are the same γ x = . . the first observation is that in all three cases the analytical solutions (vertical dotted lines) agree with the results from simulations. the second observation is that in case of positive correlation between the activities of nodes in two layers, the threshold is significantly smaller than in the other two cases. this is not surprising as the nodes sustaining the spreading in both layers are the same. thus, effectively, active nodes are capable to infect the double number of other nodes. as we mentioned above, many real multiplexes are characterized by different types of positive correlations. when thinking at real outbreaks, the effect of such feature on the spreading process suggests quite a worrying scenario. however, it is important to remember that real multiplexes are sparse, thus characterized by values of multiplexity which are far from the limit p = [ ] . as we will see below, this aspect plays a crucial role in the more realistic cases of < p < . the thresholds of the uncorrelated and negatively correlated cases are very similar. in fact, due to the heterogeneous nature of the activity distributions, except for few nodes in the tails, the effective difference between the activities matched in reverse or random order is not large, for the majority of nodes. in figs. (b) and (c) we show the behavior of the threshold as function of the activity exponents and the number of links created by active nodes in the two layers. for a given distribution of activity in a layer, increasing the exponent in the other (thus reducing the heterogeneity in the activity distribution) results in an increase of the threshold. this is due to the change of the first and second moments which decrease as a result of the reduced heterogeneity. in the settings considered here, if both exponents of activity distributions are larger than . , the critical value of λ becomes larger than , as shown in fig. (b) . thus, in such region of parameters, the disease will not be able to spread. for a given number of links created in a layer by each active node, increasing the links created in the other layer results in a quite rapid reduction of the threshold. this is due to the increase of the connectivity and thus the spreading potential of active nodes. in the limit p = , we are able to obtain an expression for the threshold of an sis process unfolding on m layers. it is important to stress that this scenario is rather unrealistic. indeed, in real multiplexes the majority of nodes is present only in one or two layers [ ] . nevertheless, we can argue that understanding the behavior of the threshold also in this case is of theoretical interest. with this observation in mind, the analytical condition for the spreading of the disease can be written as (see the appendix for details) where x = [a, b, . . . , z] and z > y implies an alphabetical ordering. the first observation is that in case h(a y , a z ) = f (a y )δ(a z − a y ) ∀ y, z ∈ x, thus the activity is the same for each node across each layer, eq. ( ) reduces to which is the threshold for a single layer activity-driven network in which m → mm. this is the generalization of the correspondence between the two thresholds we discussed above for two layers. the second observation is that, in general, increasing the number of layers decreases the epidemic threshold. indeed, each new layer increases the connectivity potential of each node and thus the fragility of the system to the contagion process. figure (a) shows the analytical behavior of the epidemic threshold up to m = for the simplest case of uncorrelated and positively correlated activities between layers confirming this result. in fig. (b) we show the comparison between the analytical results and the numerical simulations. the plot shows a perfect match between the two. furthermore, the two plots confirm the effects of positive correlations which facilitate the spreading of the disease. we now turn the attention to the most interesting and realistic cases which are different from the two limits of null and total coupling of nodes considered above. for a general value of p, we could not find a general closed expression for the epidemic threshold. however, the condition for the spreading can be obtained by investigating, numerically, the spectral properties of the jacobian (see the appendix for details). in fig. we show the lifetime of sis spreading processes unfolding on a multiplex network for three different values of p. figure (a) shows the uncorrelated case and the dashed vertical lines describe the analytical predictions. the first observation is that the larger the multiplexity between two layers, the smaller the threshold. this should not come as a surprise. in fact, as shown previously in fig. , the average connectivity in the system increases as a function of p. thus, increasing the fraction of nodes active in both layers increases the spreading power of such nodes when they get infected. the second observation is that the analytical predictions match remarkably well the simulations. the bottom panel shows instead the case of positive correlation between the activities of coupling nodes in the two layers. also in this case, the larger the multiplexity the smaller the epidemic threshold. the comparison between the two panels highlights it is important to notice that also here our analytical predictions match remarkably well the numerical simulations. in fig. we show the behavior of the (analytical) epidemic threshold as function of p for three types of correlations. the results confirm what is discussed above. the larger the multiplexity, the smaller the threshold. negative and null correlations of coupling nodes exhibit very similar thresholds. instead, positive correlations push the critical value to smaller values. furthermore, the smaller the multiplexity, the smaller the effect of positive correlations as the difference between the thresholds increases as function of p. this is a reassuring result. indeed, as mentioned above, among the properties of real multiplex systems we find the presence of positive topological and temporal correlations as well as low values of multiplexity. the first feature favors the spreading of diseases. luckily instead, the second property reduces the advantage of positive correlations pushing the threshold to higher values which are closer to the case of negative and null correlations. it is also important to notice how the threshold of a multiplex network (p > ) is always smaller than the threshold of a monoplex (p = ) with the same features. indeed, the presence of coupling nodes effectively increases the spreading potential of the disease, thus reducing the threshold. however, the presence of few coupling nodes (p ∼ ) does not significantly change the threshold; this result and the effect of multiplexity on the spreading power of diseases is in line with what was already discussed in the literature for static multiplexes [ , ] . in fig. , we show how the epidemic threshold varies when the average connectivity of the two layers is progressively different and asymmetric. in other words, we investigate what happens when one layer has a much larger average connectivity than the other. this situation simulates individuals engaged in two different social contexts, one characterized by fewer interactions (e.g., close family interactions) and one instead by many more connections (e.g., work environment). in the figure, we consider a multiplex network in which the layer a is characterized by m a = . we then let m b vary from to and measure the impact of this variation on the epidemic threshold for different values of p. for simplicity, we considered the case of uncorrelated activities in the two layers, but the results qualitatively hold also for the other types of correlations. few observations are in order. as expected, the case p = is the upper bound of the epidemic threshold. however, the larger the asymmetry between the two layers, thus, the larger the average connectivity in the layer b, the smaller the effect of the multiplexity on the threshold. indeed, while systems characterized by m b = and higher multiplexity feature a significantly smaller threshold with respect to the monoplex, for m b such differences become progressively negligible and the effects of multiplexity vanish. in this regime, the layer with the largest average connectivity drives the spreading of the disease. the connectivity of layer b effectively determines the dynamics of the contagion, and thus the critical behavior is not influenced by coupling nodes. interestingly, this result generalizes to the case of time-varying systems which have been found in the case of static multiplexes. indeed, cozzo et al. [ ] showed how the threshold of contact based processes is driven by the dominant layer which roughly corresponds to the layer featuring higher connectivity. in order to get a deeper understanding on this phenomenon, we show the asymptotic number of infected nodes in each layer for m a = and m b = in fig. . for any λ above the threshold the fraction of infected nodes in layer b [ fig. (b) ] is larger than in layer a [ fig. (a) ] and is independent of the fraction of coupling nodes. as discussed above, in these settings the layer b is driving the contagion process and the imbalance between the connectivity patterns is large enough to behave as a monoplex. however, for layer a the contagion process is still highly influenced by p. indeed, as the fraction of coupling nodes increases, layer a is more and more influenced by the contagion process unfolding in b. overall, these results are qualitatively similar to the literature of spreading phenomena in static multilayer networks [ ] . we presented a time-varying model of multiplex networks. the intralayer temporal dynamics follows the activity-driven framework which was developed for single layered networks (i.e., monoplexes). thus, nodes are endowed with an activity that describes their propensity, per unit time, to initiate social interactions. we define the multiplexity as a free parameter p regulating the fraction of coupling nodes between layers. the activities of such nodes are considered, in general, to be different but potentially correlated. in these settings, we studied how multiplexity and temporal connectivity patterns affect dynamical processes unfolding on such systems. to this end, we considered a prototypical model of infectious diseases: the sis model. we derived analytically the epidemic threshold of the process as function of p. in the limit p = , the system is constituted by disconnected networks that behave as monoplexes. in the opposite limit instead (i.e., p = ) the epidemic threshold is a function of the first and second moments of the activity distributions as well as by their correlations across layers. we found that systems characterized by positive correlations are much more fragile to the spreading of the contagion process with respect to negative and null correlations. as several real multiplex systems feature positive topological and temporal correlations [ ] [ ] [ ] , this result depicts a worrying scenario. luckily, real multiplexes are also sparse, thus characterized by multiplexity values far from limit p = [ ] . the threshold also varies as a function of the number of layers m. indeed, with perfect coupling each node is present and potentially active in each layer. thus, the larger m, the smaller the epidemic threshold as the spreading potential of each node increases. in the general and more realistic case < p < , we could not find a closed expression for the epidemic threshold. however, the critical conditions for the spreading can be calculated from the theory by investigating numerically the spectral properties of the jacobian matrix describing the contagion dynamics. also in this case, positive correlations of activities across layers help the spreading by lowering the epidemic threshold, while negative and null correlations result in very similar thresholds. moreover, the lower the multiplexity, the larger the epidemic threshold. indeed, the case of disconnected monoplexes (i.e., p = ) is the upper bound for the threshold. furthermore, the difference between the thresholds in the case of positive and the other two types of correlations decreases by lowering the multiplexity. considering the features of real multiplexes, this is a rather reassuring result. in fact, on one side the spreading is favored by positive correlations. on the other, the effect of such correlations is far less important for small values of multiplexity, which are typical of real systems. interestingly, the role of the multiplexity is drastically reduced in case the average connectivity in one layer is much larger than the other. in this scenario, which mimics the possible asymmetry in the contact patterns typical of different social contexts (e.g., family or work environment), one layer drives the contagion dynamics and the epidemic threshold is indistinguishable from the monoplex. however, the multiplexity is still significantly important in the other layer as the fraction of nodes present in both layers largely determines the spreading dynamics. some of these results are qualitatively in line with the literature of contagion processes unfolding on static and annealed multiplexes. however, as known in the case of single layered graphs, time-varying dynamics induces large quantitative differences [ , ] . indeed, the concurrency and order of connections are crucial ingredients for the spreading and neglecting them, in favor of static and annealed representations, generally results in smaller thresholds. while the limits of timescale separation might be relevant to describe certain types of processes, they might lead to large overestimation of the spreading potential of contagion phenomena. the model presented here comes with several limitations. in fact, we considered the simplest version of the activitydriven framework in which, at each time step, links are created randomly. future work could explore the role of more realistic connectivity patterns in which nodes activate more likely a subset of (strong) ties and/or nodes are part of communities of tightly linked individuals. furthermore, we assumed that the activation process is poissonian and the activity of each node is not a function of time. future work could explore more realistic dynamics considering bursty activation and aging processes. all these features of real time-varying networks have been studied at length in the literature of single layered networks but their interplay with multiplexity when dynamical processes are concerned is still unexplored. thus, the results presented here are a step towards the understanding of the temporal properties of multiplex networks and their impact on contagion processes unfolding on their fabric. integrating over all activity spectrum of eq. ( ), it obtains the following equation: thus, eq. (a ) can be further simplified as and integrating over all activity spectrum of eq. ( ), it obtains the following equation: multiplying both sides of eq. ( ) by a x , and integrating over all activity spectrum, we get the following equation: replacing x with a and b in eq. (a ), respectively, we have and in the same way, multiplying both sides of eq. ( ) by a x and integrating over all activity spectrum, it obtains the following two equations: when x is replaced with a and b, respectively. when the system enters the steady state, we have . thus, the critical condition is determined by the following jacobian matrix: if the largest eigenvalue of j is larger than zero, the epidemic will outbreak. otherwise, the epidemic will die out. specifically, if p = , two layers are independent, thus regulated by two independent jacobians, and we can get the following two eigenvalues: for < p < , we could not find a general analytical expression for the eigenvalues of j . however, the critical transmission rate can be determined by finding the value of λ leading the largest eigenvalue of j to zero. in other words, rather than solving explicitly the characteristic polynomial |j − i | = and defining the condition for the spreading max > as done above, we can determine the critical value of λ as the value corresponding to the largest eigenvalue to be zero [ , ] . · · · · · · · · · · · · multilayer networks. structure and function multilayer networks mathematical formulation of multilayer networks the structure and dynamics of multilayer networks multiplex networks: basic formalism and structural properties social network analysis: methods and applications multiplexity in adult friendships social capital in the creation of human capital measuring and modeling correlations in multiplex networks quantifying dynamical spillover in co-evolving multiplex networks effects of temporal correlations in social multiplex networks the physics of spreading processes in multilayer networks spreading processes in multilayer networks diffusion dynamics on multiplex networks global stability for epidemic models on multiplex networks epidemic spreading and bond percolation on multilayer networks optimal percolation on multiplex networks resource control of epidemic spreading through a multilayer network dynamical interplay between awareness and epidemic spreading in multiplex networks epidemic spreading and risk perception in multiplex networks: a self-organized percolation method multiple routes transmitted epidemics on multiplex networks epidemics in partially overlapped multiplex networks immunization of epidemics in multiplex networks asymmetrically interacting spreading dynamics on complex layered networks conditions for viral influence spreading through multiplex correlated social networks contactbased social contagion in multiplex networks temporal networks modern temporal network theory: a colloquium activity driven modeling of time-varying networks time varying networks and the weakness of strong ties asymptotic theory of time-varying social networks with heterogeneous activity and tie allocation from calls to communities: a model for time-varying social networks the dynamical strength of social ties in information spreading persistence and periodicity in a dynamic proximity network what's in a crowd? analysis of face-to-face behavioral networks from seconds to months: an overview of multi-scale dynamics of mobile telephone calls persistence of social signatures in human communication fundamental structures of dynamic social networks face-to-face interactions random walks and search in time varying networks quantifying the effect of temporal resolution on time-varying networks controlling contagion processes in activity driven networks contagion dynamics in time-varying metapopulation networks epidemic spreading in time-varying community networks immunization strategies for epidemic processes in time-varying contact networks random walks on temporal networks analytical computation of the epidemic threshold on temporal networks causality driven slow-down and speed-up of diffusion in non-markovian temporal networks spatio-temporal networks: reachability, centrality and robustness random walk centrality for temporal networks importance of individual events in temporal networks bursts of vertex activation and epidemics in evolving networks attractiveness and activity in internet communities contrasting effects of strong ties on sir and sis processes in temporal networks committed activists and the reshaping of status-quo social consensus betweenness preference: quantifying correlations in the topological dynamics of temporal networks bursty communication patterns facilitate spreading in a threshold-based epidemic dynamics birth and death of links control disease spreading in empirical contact networks the basic reproduction number as a predictor for epidemic outbreaks in temporal networks statistical physics of vaccination social phenomena: from data analysis to models burstiness and tie reinforcement in time-varying social networks random walks on activity-driven networks with attractiveness epidemic spreading in modular time-varying networks modeling infectious disease in humans and animals modeling dynamical processes in complex socio-technical systems epidemic processes in complex networks the role of endogenous and exogenous mechanisms in the formation of r&d networks networks. an introduction topological properties of a time-integrated activity-driven network structural measures for multiplex networks nature of the epidemic threshold for the susceptible-infected-susceptible dynamics in networks telling tails explain the discrepancy in sexual partner reports concurrent partnerships and transmission dynamics in networks dynamical processes on complex networks a unified approach to percolation processes on multiplex networks clustering determines the dynamics of complex contagions in multiplex networks further, the maximum eigenvalue of matrix j m can be calculated asthus, the critical transmission rate isfurther, if the activities of the same node in each layer are the same, the above equation can be simplified as follows:(a ) key: cord- -gptjqti authors: ball, christopher; forrester, anne; herrmann, andreas; lemiere, stephane; ganapathy, kannan title: comparative protective immunity provided by live vaccines of newcastle disease virus or avian metapneumovirus when co-administered alongside classical and variant strains of infectious bronchitis virus in day-old broiler chicks date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: gptjqti abstract this study reports on the simultaneous administration of live ndv or ampv subtype b vaccines alongside two live ibv (massachusetts-h and b-cr ) vaccines in day-old maternal-antibody positive commercial broiler chicks. in the first experiment, chicks were divided into four groups; one unvaccinated and three groups vaccinated with live ndv vg/ga-avinew, live h + cr , or vg/ga-avinew + h + cr . in the second experiment, live ampv subtype b vaccine was used in place of ndv. clinical signs were monitored daily and oropharyngeal swabs were taken at regular intervals for vaccine virus detection. blood was collected at dpv for serology. chicks from each group were challenged with virulent strains of m or qx or ampv subtype b. for ibv, after days post challenge (dpc), tracheal ciliary protection was assessed. for ampv, clinical scores were recorded up to dpc. for ndv, haemagglutination inhibition (hi) antibody titres were assayed as an indicator of protective immunity. in both experiments, ciliary protection for ibv vaccinated groups was maintained above %. the protection against virulent ampv challenge was not compromised when ampv, h and cr were co-administered. ndv hi mean titres in single and combined ndv-vaccinated groups remained above the protective titre (> log ). both experiments demonstrated that simultaneous administration of live ndv vg/ga-avinew or ampv subtype b alongside h and cr vaccines does not interfere with protection conferred against ndv, ibv or ampv. newcastle disease (nd), caused by an avian paramyxovirus serotype (now designated as orthoavulavirus ) [ ] , and infectious bronchitis (ib) caused by an avian coronavirus [ ] , are known to give rise to severe diseases in chickens, with disastrous economic losses and welfare concerns [ , ] . nd and ib have been important and common chicken diseases worldwide since and respectively. in the last three decades, publications from both our and other laboratories have highlighted the importance of ampv [ ] , which causes a drop in egg production and quality, and swollen head syndrome in chickens [ , ] . infectious bronchitis virus (ibv), avian metapneumovirus (ampv) and newcastle disease virus (ndv) are respiratory rna viruses that primarily infect the tracheal epithelium of chickens [ ] . a number of countries have introduced the use of live and inactivated ndv, ibv and ampv vaccines [ ] . simultaneous vaccination with multiple live viral vaccines is carried out in poultry for a number of reasons. these include the avoidance of inducing stress in chicks through excessive handling, and to reduce vaccination costs [ ] . traditionally, ndv and single ibv massachusetts live vaccines are given to day-old chicks at the hatchery or in the farms, mostly by spray. however, as the disease and losses caused by variant ibvs increase [ ] [ ] [ ] , growing numbers of producers have been including a b vaccine in the day-old vaccination programme. all three diseases (ibv, ampv and ndv) are now routinely controlled in the poultry industry through the use of live attenuated vaccines [ , ] , with long lasting antibody titres recorded for inactivated ndv vaccinations [ ] . reports since the s have described how ibv vaccines can impair the efficacy of ndv vaccines [ , ] , potentially leading to production losses [ ] . gelb vaccine in broiler chickens [ ] . with the introduction of ampv in s, the possibility of heterologous co-vaccination of ndv + ampv and ibv + ampv at day-old was investigated [ ] [ ] [ ] [ ] [ ] . these publications showed that simultaneous administration of ampv vaccine alongside ndv or ibv vaccine were compatible, with protection against virulent ibv, ndv or ampv remaining uncompromised. however, in these studies, only a single vaccine strain of ibv (massachusetts) was used. with the emergence of variant ibvs, inclusion of a variant ibv alongside massachusetts for dayold vaccination has become a common practice [ , , ] . recent work has shown that ibv vaccines can undergo genetic mutations following inoculation into the chicken host [ , ] . as persistent mutations can alter strain characteristics [ ] , it is preferable to monitor potential changes. furthermore, it will be beneficial to establish if simultaneous vaccine administration has any role in nucleotide mutations occurring in the ibv s gene and subsequent amino acid changes. in contrast to ibv vaccine strains, prolonged persistence or genetic mutations in ndv or ampv vaccine strains have not been reported in chickens. to date, the impact of co-administration of live ndv or ampv alongside classical and variant live ibv vaccines in ibv-ampv-ndv maternal antibody positive birds has not been investigated. with the increased use of co-vaccination strategies in commercial broiler chicks, it is important to understand how such vaccinecombinations may impact protection against ibv, ampv or ndv challenges. this study reports the results of two experiments where live ibv vaccines of massachusetts and b were coadministered alongside a live ampv subtype b or live newcastle disease vaccine in commercial broiler chicks. chicks: day-old commercial broiler chicks were obtained from a commercial hatchery, and raised in the university of liverpool isolation facilities. chicks were reared on deep litter with water and feed provided ad libitum. no in-feed or in-water antibiotics were used throughout the study. all experimental procedures were conducted according to uk legislation on the use of animals for experiments, and were granted ethical approval by the university of liverpool ethics committee. vaccine strains: commercially available live ibv mass type strain h (bioral, boehringer ingelheim, lyon, france) and ibv variant b type strain cr (gallivac Ò ib , boehringer ingelheim, lyon, france), ndv vg/ga-avinew strain (avinew Ò neo, boehringer ingelheim, lyon, france) and ampv subtype b (nemovac Ò , boehringer ingelheim, lyon, france) vaccines were administered during this study. all vaccines were prepared prior to administration using sterile distilled water, to allow for ml of vaccine per chick to be administered according to manufacturer's instructions [ , ] . challenge strains: virulent virus strains of ibv (m , . ciliostatic dose (cd) /ml; qx, . cd /ml) and ampv (subtype b; . cd /ml) were used for challenge. all challenge strains have been propagated in our laboratory and used in several previous studies [ , , ] . pcr and bacterial culturing confirmed an absence of ndv, ampv, avian influenza virus (aiv), infectious laryngotracheitis virus (iltv), infectious bursal disease virus (ibdv), fowl adenovirus (fadv) and mycoplasmas in all inocula [ ] [ ] [ ] [ ] [ ] [ ] . experiment : chicks were randomly divided into four groups ( chicks per group) and kept in separate isolation units. at day-old, each chick was inoculated via oculo ( ml) and nasal ( ml) routes with the following; vg/ga-avinew (group a), mass + b (group b), vg/ga-avinew + mass + b (group c) and sterile h o (group d). chicks were observed daily for clinical signs [ ] ; mild = coughing, head shaking and depression of short duration, severe = gasping, coughing and depression, accompanied by ruffled feathers. mortality rates and lesions following necropsy were recorded. at dpv, chicks from each group were separated into two further groups (a , a , b , b , c , c , d and d ), with chicks per sub-group. chicks from a , b , c and d were challenged with ml of virulent m , and chicks from a , b , c and d were challenged with ml of virulent qx via oculo-nasal route. chicks were observed daily for clinical signs as previously described, with mortality rates and post mortem lesions recorded. at , , , , and dpv, oropharyngeal (op) swabs were collected from five chicks in each group. at , , dpv, five trachea and kidney samples were collected from each group. at dpc, five trachea and kidney samples were collected and processed as previously described [ ] . blood was collected from ten chicks at dpv and eight chicks at dpv for the detection of ibv and ndv antibodies. experiment : day-old broiler chicks were randomly divided into four groups and kept in separate isolation units, with chicks per group. at day-old, each chick was inoculated via oculo ( ml) and nasal ( ml) routes with the following; ampv subtype b (group a), mass + b (group b), ampv subtype b + mass + b (group c) and sterile h o (group d). chicks were observed daily for clinical signs as described above. at days post vaccination (dpv), chicks from each group (a-d) were further split into twelve groups (a -a , b -b , c-c , d -d ), with five chicks per group. chicks from a , b , c and d were challenged with ml of virulent m , and chicks in the a , b , c and d groups were challenged with ml of virulent ampv b via oculo-nasal route. chicks from a , b , c and d were sham-inoculated with sterile distilled water. all birds were observed daily for clinical signs, with ibv challenge groups scored as above, and ampv challenge groups scored as follows: = no nasal exudate, = mild exudate, = unilateral excessive exudate, = bilateral excessive exudate [ ] . at , , , , and dpv, oropharyngeal (op) swabs were collected from five chicks in each group. at , , dpv five trachea and kidney samples were collected. at dpc, five trachea and kidney samples were collected and processed as previously described [ ] . blood was collected from ten chicks at dpv and eight chicks at dpv for the detection of ibv and ampv antibodies. evaluation of tracheal protection following m challenge: at days post challenge (dpc), five chicks from each group were humanely killed by wing vein injection of sodium pentobarbital. tracheas were removed from each chick and processed for ciliary and percentage protection assessment as previously described [ , ] . briefly, rings from each bird were examined, with a maximum possible ciliary score of indicating no protection (no cilia beating). the mean ciliary score for each bird was calculated and percentage protection for each group was calculated. measurement of antibody levels: commercial elisa kits [ibv (idexx, maine, united states) and ampv (biochek, er reeuwijk, netherlands)] were used according to manufacturer's instructions, with a titre result higher than (ibv) or (ampv) indicating a positive protection titre. haemagluttination inhibition (hi) was utilised to determine ibv m and ndv protection status as previously described [ ] . ibv, ndv and ampv detection by rt-pcr: swabs were pooled and dipped as a single sample per group, per day in a sterile bijou containing . ml of eagles serum-free minimum essential medium with glutamine, streptomycin [ mg/ml] and penicillin [ iu/ml], which was stored at À °c until required. rna was extracted from the swab and tissue samples using the phenol chloroform method [ ] and subjected to rt-pcr for ibv, ndv and ampv [ , , ] . positive amplicons were purified and sent for bi-directional commercial sanger sequencing (source bioscience, nottingham, uk). ibv and ampv viral load in trachea and kidney: total rna was extracted from each group's five trachea and kidney samples using the rneasy plus mini kit (qiagen, manchester, uk). quantification of viral rna was determined by quantitative rt-pcr (qrt-pcr) for both ibv and ampv [ , ] . the ibv qrt-pcr was performed using the qiagen onestep rt-pcr kit and ng of total rna per reaction. the ampv assay was performed using the qiagen quantitect xrt-pcr no rox master mix and ng of total rna per reaction. ct values were converted to log of relative equivalent units (reu) of viral rna, using previously determined standard curves for ibv and ampv [ ] . sequencing analysis: ibv partial-s gene sequences were trimmed to bp, analysed in chromaspro v . . (http://technelysium.com.au/), and blast searches were conducted to confirm the isolate identity. alignments were carried out in mega [ ] , using clustal w [ ] . single nucleotide polymorphism (snp) and insertion and deletion (indel) detection was carried out in mega , following alignment to sequenced vaccine strains. snps were characterised as non-synonymous (ns) if it led to an amino acid change, and synonymous (s) if it led to no amino acid changes. insertions and deletions (indels) were defined as the insertion or deletion of a nucleotide that altered the sequence length. the d s /d ns ratio, to identify positive or purifying selection pressure for each group was calculated per day. this was done using the nei-gojobori method [ ] where < . indicated the recovered strains to be under positive selection pressure, and > . indicated strains to be under purifying selection [ , ] . positive or purifying selection was considered significant at p < . . translated amino acid variations were also identified and variations that resulted in a change in hydrophobicity were noted according to the kyte and doolittle scale [ ] . statistical analysis: statistical analysis was conducted using one-way analysis of variance (anova), followed by turkey's test to examine the differences between pairs of means. differences were considered to be significant when p < . . analysis was carried out using spss statistics . following day-old vaccination, in both ibv-vaccinated groups, mild respiratory signs were recorded from to dpv. after m or qx challenge, groups that received no ibv vaccination had mild signs throughout the experiment. the ibv-vaccinated groups, which were m -challenged or qx-challenged showed mild signs for a total of two days, at - dpc. ciliostasis results showed that all ibv-vaccinated groups (b , b , c , c ) have high protection against both ibv challenge strains, ranging from to % (c - %; c - . %; b - %; b - . %). the ndv hi titres in groups that had not received an ndv vaccine were below log (table ) , whereas the groups that received ndv vaccine singly or in combination with ibv were . and . respectively. a significant increase (p < . ) in titre was seen for group a (ndv vaccinated) when compared to group c (combined vaccination) at dpv. when the same sera were assayed against ibv hi antibodies, ibv-vaccinated groups showed an increase in hi log titre at dpv when compared to dpv (table ) . furthermore, there was a significant increase (p < . ) in titre for both groups a and b when compared to group c for the ibv ( / ) antigen. there was a stronger response towards the / compared to the m hi antigen for all ibv vaccinated groups (group b - . and . ; group c - . and . ). the mean ibv elisa antibody titre at day-old was . (± ), indicating the presence of maternally derived antibodies (mda). by dpv, antibody levels declined to below the detectable titre ( ) in all groups (group a - . , group b - , group c - . and group d - . ). despite being below detectable levels, the two groups receiving ibv vaccines (b and c) had higher antibody titres compared to the ibvunvaccinated groups (a and d). ibv rna was detected at all sampling time points ( , , , and dpv) in groups b and c, and was absent from the ibvunvaccinated groups (a and d). the mass-type vaccine was detected up until dpv in group b and dpv in group c. after this time, only the b-type vaccine was identified in all subsequent samples in both ibv-vaccinated groups. all mass-type identified strains had partial-s nucleotide similarity to the original vaccine, ranging from . to . %, whereas the b-type had . - . % relatedness to the cr vaccine strain. ndv was not detected at any time. total snp counts remained low, with the exception of samples from groups b and c at dpv, which contained greater total snp counts (n = ) ( table ). this translated to amino acid (aa) changes in group b and in group c. all changes in blike samples were from hydrophobic to hydrophilic properties, and the majority ( %) of changes in the mass-like samples were hydrophilic to hydrophobic. the average d s /d ns ratio was . for b strains and . for mass strains, indicating that the mass genotype was significantly under purifying selection pressure (p < . ). no indels were present for any of the sequences analysed. . . . molecular detection of ibv and ndv from trachea and kidney tissues up to dpv: ibv vaccinal strains were recovered from both trachea and kidney tissue in group b at and dpv, whereas all sampling days were positive in group c (table ) . no trachea samples were ibv-positive at dpv and no mass-type strains were recovered in the kidney past dpv. we did not detect ndv in any samples. sequence similarity to the ibv vaccine strains were between and %, with a single b exception of % at dpv. the majority of strains recovered from the kidney and trachea ( . %) had a d s /d ns ratio of over . , demonstrating that the majority of partial s nucleotide variations had no effect on the translated amino acid composition. the exceptions were the two b-like strains from group c kidney samples at dpv. this group had an average of five non-synonymous changes per strain, which translated to three hydrophobicity changes within all recovered strains. ibv m challenge at dpc: ibv was detected in the trachea of group d (non-vaccinated), and in the trachea and kidney of group a (ndv vaccinated) ( table ). ibv vaccine strains were detected in groups b (mass vaccinated À % similarity) and c ( b vaccinated À % similarity). the majority of variations led to changes in the translated amino acids (n = ; average d s /d ns ratio = . ), however only one change in hydrophobic properties (hydrophilic to hydrophobic) was identified from a sample identified as highly similar to the virulent strain (group a ; trachea). ibv qx challenge at dpc: virulent qx strains were detected in the trachea and kidney of group d (non-vaccinated), with - % nucleotide similarity to the virulent strain. however, only tracheal samples were ibv-positive for group a (ndv-vaccinated) ( table ). vaccinal strains were detected in groups b ( b; - % similarity) and c ( b and mass; % similarity). the minority of nucleotide variations caused an amino acid change (n = ; average d s /d ns ratio = . ), which resulted in seven hydrophobicity changes (hydrophilic to hydrophobic = ; hydrophobic to hydrophilic = ). vaccine strains were recovered from kidney samples in groups b and c, with the majority of changes in b-like samples being non-synonymous. from a total of amino acid changes, only six caused a change in hydrophobicity. in the ibv vaccinated tracheal samples, viral load reduced from dpv to dpv (group b = . to log reu; group c = . to . log reu), however ibv presence were significantly higher (p < . ) at dpv for both groups (group b = . ; group c = . ) when compared to dpv (fig. ) . at both and dpv, there was a significantly higher tracheal viral load in the ibvvaccination group when compared to the ndv + ibv vaccine group. post challenge, high viral loads were present in the trachea (fig. for the kidney samples at and dpv, groups b (ibv vaccinated) and c (ndv + ibv vaccinated) increased from to dpv ( . to . log reu and . to . log reu respectively) (fig. ) . post challenge (fig. ) , significantly lower viral loads were present in non-ibv vaccinated groups (a - . log reu; a - . log reu; c - . log reu; d - . log reu) compared to ibv vaccinated groups (b - . log reu; b - . log reu; c - . log reu). following m challenge, viral load did not change in group b (ibv vaccinated). the virulent qx strain was detected at a greater level in the control group (group d) when compared to the m challenge control group. however, the viral load in both ndv vaccinated groups (a and a ) remained low (< . log reu) following challenge. up to dpv: all ibv vaccinated groups demonstrated mild clinical signs (prominent snicking and sneezing), starting at and dpv (group b -mass + b; group c -ampv + mass + b respectively), and continuing up to dpv. group a (ampv vaccinated) and group d (control) showed no clinical signs. no groups presented with moderate clinical signs at any point. up to dpc: following ibv m challenge, groups a (ampv vaccinated) and d (control) showed signs from dpc to and dpc respectively. mild signs in groups b and c subsided after dpc. for ampv challenge, greater clinical signs were observed in non-vaccinated birds (group d ), or birds only receiving the ibv vaccines (group b ), compared to the group receiving the combined vaccination (group c ). all signs were cleared from ampv challenge groups by dpc. groups receiving no challenge virus (a , b , c and d ) were absent of clinical signs. all ibv vaccinated groups showed high protection against the m challenge strains ( . - . % protection score). the combined groups (c and c ) demonstrated a similar level of protection percentage ( . and . respectively) when compared to the single vaccination groups (b and b , both . %). the mean serum ampv antibody titre at dpv was . (± . ), indicating the presence of maternally derived antibodies (mda) against ampv (fig. ) . by dpv, antibody levels declined to below the detectable titre for the ibv vaccinated group (group b). however, the ampv vaccinated and combined groups remained at positive titres ( and . respectively). by dpc, only groups b , b , d and d showed antibody levels below the detectable limit. following ampv challenge, groups a (ampv vaccinated) and b (ibv vaccinated) presented with higher titres ( . and . respectively) when compared with group c , which received the combined ampv + ibv vaccination ( . ). the ampv vaccinated-ampv challenged group (a ), and the combined vaccinated-ampv challenged group (c ) had significantly higher (p < . ) titres compared to the non-challenged sub groups a and c . h ( ) we were able to detect both ibv and ampv as early as dpv. however, while ibv was identified until dpv, we did not detect ampv beyond dpv in either single or combined vaccine groups. partial s sequencing of ibv positive samples demonstrated a high similarity to the original inoculum, with identities ranging from . to . % in group b to . - . % in group c. no mass-types were identified after dpv in either group. on aver- fig. . experiment : quantification of ibv viral load in the trachea and kidney at dpv following vaccination with either (group b) h + cr or (group c) vg/ga-avinew + h + cr . data is presented ± standard error margins (sem). significance testing carried out within sampling point of the same tissue type, with differences between groups (p < . ) labelled with different letters. data is presented ± standard error margins (sem). significance testing carried out between groups of the same tissue type, with differences between groups (p < . ) labelled with different letters. the d s /d ns ratio was an average of . for the mass-like strains and . for the b samples ( table ). the two b-positive samples with greater than average snp counts also showed an increase in amino acid changes, and were the only samples with hydrophobicity changes, with the majority (n = / ) being a change from hydrophilic to hydrophobic. up to dpv: ibv vaccinal strains were recovered from groups b and c at all sampling points, with the exception of the kidney at dpv (table ). all detected ibvs had a high partial-s nucleotide similarity with the initial vaccine strain (over . %), with only a single non-synonymous change at all time points for groups b and c. the tracheal sample from group c at dpv had snps, which translated into nine amino acid variations (containing a single hydrophobic to hydrophilic change). the ampv vaccine was detected up until dpv in tracheal samples, with the kidney remaining negative throughout. ibv m challenge at dpc: ibv was detected in the kidney of group b (ibv-vaccinated) and group c (ampv + ibvvaccinated), with samples having a high similarity with the vaccine strain (over %) ( table ). all samples from groups a (ampv vaccinated) and d (non-vaccinated) were positive for ibv, with % identity to the challenge strain. groups not receiving an m challenge were ibv negative and no ampv was detected in birds challenged with m . ampv subtype b challenge at dpc: no ampv was detected in group a (ampv vaccinated) or group c (ampv + ibv vaccinated). the virus was detected in the trachea of birds in group b (ibv vaccinated) and both trachea and kidneys of group d (nonvaccinated). post vaccination in the ibv vaccinated tracheal samples, viral load increased from dpv to dpv (group b = . to . log reu; group c = . to . log reu) (fig. ) . however, ibv presence was significantly lower (p < . ) at dpv for group b when compared to both and dpv. the ibv vaccination group (group b) had a significantly higher tracheal viral load when compared to the combined (group c) vaccine group at both and dpv. while both groups were negative at dpv, viral load in the kidney was significantly higher at and dpv (p < . ) in group b compared to the combined group c. post challenge with ibv m , groups vaccinated with ibv were showed to be negative by qrt-pcr, whereas group a (ampv vaccinated) and group d (non-vaccinated) showed readings of . and . log reu respectively (fig. ) . post vaccination, kidney samples were negative for ibv from all groups at dpv. however, viral load increased from to dpv in all ibv vaccinated groups (group b - . to . log reu; group c - . to . log reu). post challenge with ibv m , groups not receiving an ibv vaccine showed virus presence (group a - . log reu; group d - . log reu), with vaccinated groups having low detection levels (group b - . log reu; group c - . log reu). co-vaccination of day-old broiler chicks with ndv or ampv alongside two different live ibv (mass and b) vaccines does not impair protection conferred against either ibv or ampv challenge. in both experiments, following ibv challenge, mild respiratory signs were observed for the first two days and none thereafter, demonstrating clinical protection conferred by ibv mass h + b cr , with or without ndv or ampv. ciliostasis analysis was also used to confirm ibv protection [ ] , where a higher protection percentage suggests better protection against a virulent ibv challenge. ciliostasis results in the current study highlighted excellent ibv protection against m and qx challenges in groups receiving h + b, or the same ibv vaccines given along with ampv or ndv vaccines. previous work has shown that the combination of mass + b conferred excellent ciliary protection against asian (qx and q ) and middle east (is/ / and is/ / ) ibv strains [ , [ ] [ ] [ ] [ ] . when ibv h and cr vaccine strains are combined with the ndv or ampv vaccine, protection levels remained at or above %. this reconfirms that such combined vaccination did not compromise the protection conferred against virulent ibvs. in addition to the cilia-stopping test, detection of virus and viral load in the trachea and kidney of challenged groups was attempted to demonstrate protection against ibv m and qx. no vaccine viruses were detected in the trachea at dpc. however, both virulent challenge viruses were present, suggesting host clearance of ibv vaccines by at least dpv [ , ] . the co-administration of either ndv or ampv alongside the ibv vaccine caused a lower ibv vaccine virus load in both the trachea and kidney at dpv. however, post-challenge with virulent ibv, the combined ibv with ndv or ampv vaccination had no effect on viral load when compared with ibv vaccination alone. for both experiments, despite relatively higher tracheal viral load, a low reu value was shown in kidney tissue from non-ibv vaccinated groups following m and qx challenge. these results, along with the cilia-stopping test, demonstrated that comparable levels of protection are found when ibv h and cr vaccine strains are given either alone or alongside an ndv or ampv vaccine. control of newcastle disease is of paramount importance in endemic countries. control is normally achieved by the use of live and inactivated vaccines [ ] . haemagluttination inhibition titres were traditionally used to confirm protection against ndv challenge [ , ] , and the mean ndv hi titre in both single and combined vaccinated groups described here were > . log , which is above the protective titre against virulent ndvs [ , , , ] . current work showed that simultaneous vaccination of ndv + h + cr in commercial broiler chicks does not interfere with the induction of protective immunity against ndv. in this study, following challenge with virulent ampv, no clinical signs were found in the single (ampv) or combined (ampv + h + cr ) vaccinated groups, confirming clinical protection against the challenge virus [ , , ] . furthermore, following challenge with the virulent strain, no antigen was detected by rt-pcr from tissue samples in either of the vaccinated groups. the ampv elisa antibody titres in the triple-vaccinated group was lower than the single vaccinated group, echoing findings from previous work [ ] . however, it has been reported that levels of humoral antibodies have no association with clinical protection against ampv [ , ] . it is evident that experimental vaccination of commercial broilers (with ampv and ibv mdas) with ampv subtype b and two ibv strains (h and cr ) had no adverse effects on clinical protection conferred against a virulent challenge. in both experiments, the persistence of vaccinal strains following individual ibv, ndv, ampv and combined (h + cr + ndv; h + cr + ampv) vaccination were monitored at intervals. for ndv and ampv, classical rt-pcrs [ , ] were applied to rna extracted from oropharyngeal swabs, with no detection of the ndv vaccine at any sampling points. previous work has shown detection of ndv vaccine virus in spf chickens for up to four days post vaccination [ , ] . in this experiment, first sampling was carried out at dpv, and the failure to detect ndv may have been due to rapid clearance of the vaccine [ ] . for ampv, it was possible to detect the virus up to dpv using dry oropharyngeal swabs and until dpv from tracheal tissue. while identified the ampv vaccine at dpv in a combined ibv + ampv vaccinated group [ ] , there was no difference between single and combined vaccinated groups in the current study, suggesting that combined vaccination does not impair a host's ability to rapidly clear the ampv vaccine strain. ibv vaccinal strains have previously been shown to persist in chickens for a longer period [ , ] . to further investigate this, the growth kinetics and genetic characteristics of live ibv vaccines used in this study were cross compared with previous reports [ , ] . vaccinal strains were detected in op swabs throughout the current experiment, with the variant b strain dominating at the later ages of the birds. with both experiments, sanger sequencing showed that the genotype of strains recovered using op swabs altered from mass to b after dpv. as reported previously [ , ] , while the mass genotype made up the majority of detections during early sampling days ( - dpv), the b strain became the dominant strain by dpv. the reason for the shifting of genotypes from mass to b is not known, however, previous work has highlighted that different ibv strains can differentially induce host expression pathways, in particular tlr [ ] and ifn-b [ ] . such differences may have played a role in a host's ability to clear the mass vaccine virus at an earlier time point when compared to the b vaccine. further work is required to understand potential underlying mechanisms. at no point did the nucleotide similarity for recovered mass vaccine strains drop below % when compared to the original vaccine sequence. this suggests that under experimental conditions, the vaccine strain underwent minimal alteration following inoculation [ ] . similarly, the b vaccine strain remained high for all but one sampling point ( - % with the exception of % at dpv in experiment ). limited genetic changes were noted in the tissues samples, with - and - . snps per sequence in the mass and b vaccine strains respectively. a higher number of polymorphisms were seen in the b sequences, compared to massachusetts, indicating that the persistence of this particular genotype may contribute to greater genetic variations within local virus populations [ , , ] . however, the d s /d ns ratios suggest purifying selection for both genotypes [ ] , and therefore evolutionary constraint [ ] . previous work implied b amino acid variations do not follow a consistent pattern over time [ ] , which was also witnessed in the current study. no persistent alteration was witnessed from any tissues in any of the vaccination programs. in conclusion, this study indicates that the combined vaccination consisting of ndv (vg/ga-avinew) or ampv (subtype b) alongside two strains ibv (massachusetts -h and b -cr ) does not impair protective immunity against the globally important ibv, ndv or ampv strains that were 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arkdpi-derived infectious bronchitis virus vaccines likelihood models for detecting positively selected amino acid sites and applications to the hiv- envelope gene genetic diversity and purifying selection in west nile virus populations are maintained during host switching variation in the spike protein of the /b type of infectious bronchitis virus, in the field and during alternate passage in chickens and embryonated eggs the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. key: cord- -wrueslce authors: stecz, patryk; slezáčková, alena; millová, katarína; nowakowska-domagała, katarzyna title: the predictive role of positive mental health for attitudes towards suicide and suicide prevention: is the well-being of students of the helping professions a worthwhile goal for suicide prevention? date: - - journal: j happiness stud doi: . /s - - - sha: doc_id: cord_uid: wrueslce this study evaluates the potential value of eudaimonic well-being in assessing pro-preventive orientation towards suicide and recognizing suicide as a solution. the aim was to integrate positive and negative conceptualizations of mental health for predicting attitudes towards suicide, and towards suicide prevention, among students of the helping professions. the study participants ( women and men, mean age . ± . ) answered a set of questionnaires, including a questionnaire on attitudes towards suicide, goldberg health questionnaire (ghq- ), psychological well-being scale (pwb- ) and centrality of religiosity scale. multiple regression analysis showed that environmental mastery, purpose in life and positive relationships, controlled for religiousness and psychological problems related to general mental health, predicted the variability of attitudes towards suicide and pro-preventive orientation. sociodemographic variables were not related to attitudes towards suicide. our findings suggest that positive mental health, represented jointly by low mental health problems and eudaimonic components of happiness, plays a role in predicting pro-preventive attitudes. therefore, improving positive mental health among students in the helping professions, these being the future gatekeepers, could be considered an auxiliary strategy for suicide prevention. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. the understanding of suicidal behavior has been recently influenced by concepts originating from positive psychology, which served to synthesize theories organized around the topic of prevention (barnes ; hirsch et al. ) . positive suicidology aims at identifying protective factors for suicidal behavior and examining useful constructs from positive psychology to increase the robustness of suicide prevention (wingate et al. ) . identifying those at risk is an important goal and suicide prevention is a challenging task faced by the entire community, including groups of gatekeepers. together, their attitudes towards suicide (ats), suicide prevention (atsp) and mental health constitute a personal background that can be brought to the intervention, and as such, merit more research interest within the area of positive suicidology. professional gatekeepers comprise healthcare providers (designated gatekeepers) and specialists from outside the health sector (emergent groups such as counselors, clergy, teachers) who are likely to interact with people at higher risk of suicide (goldsmith et al. ) . the term gatekeepers also refers to the members of the community, including significant others, peers and co-workers, who are able to act in different ways in order to prevent suicide, e.g. by facilitating referral (isaac et al. ). for example, a study of gatekeeper training for college students by rallis et al. ( ) suggests that this particular population may hold implications for suicide prevention. it seems necessary to clarify the extent to which the student population plays a role in gatekeeping. in many countries, the college population, and young adults in general, are considered to have relatively high exposure to suicide (aldrich ; makaruk et al. ; rotenstein et al. ) . students interact regularly with each other and research shows that when facing a crisis, they are more likely to seek support from friends than the mental health services (drum et al. ). vogel et al. ( ) report examples of students encouraging those at risk to seek professional help, which, in fact, could be regarded as gatekeeping by early intervention. on graduation, psychology, law and medicine students can be regarded as being part of the way to developing their capability to provide emotional, general health and mental health support, or legal assistance; however, it is not easy to predict how many would have a high level of exposure to suicide at their future workplaces. in general, while university students may be regarded as playing the role of lay gatekeepers, students of the helping professions could be argued to have particular importance in suicide prevention. this subgroup is extensively educated to provide different types of professional support, and many of their graduates may go on to work in professions which are recognized to have substantial or high priority for gatekeeping. mental health, ats and suicidal behavior are known to be interrelated (e.g., cavanagh et al. ; gibb et al. ) . psychological well-being is associated with mental health, and both aspects should be included in any holistic approach towards preventive health and positive functioning. both factors could influence suicide prevention within both the general population and that of potential gatekeepers, although this phenomenon has been only lightly discussed (burnette et al. ; hirsch et al. ; lygnugaryte-griksiene et al. ; quinnett ) . it has been suggested that the well-being of providers and their attitudes towards the end-of-life decision are important factors in bringing hope and preventing suicide (burnette et al. ; o'hara ) . this view is supported by foulds ( ) and porter ( ) who consider that the well-being of providers achieved by selfactualization empowers client growth. surprisingly, no previous studies have evaluated the prediction of ats and atsp based on a comprehensive mental health model incorporating indicators of positive and negative functioning. however, such an approach has been proposed by the dual-factor model of positive mental health (dfm) (greenspoon and saklofske ) , which was originally introduced to address the limitations of traditional health models. the dfm characterizes complete mental health as a combination of high psychological, or subjective, well-being with a low level of psychopathology. indeed, joseph and wood ( ) argue that positive and negative functioning could be seen as interrelated constructs, constituting a higherorder continuum of holistic well-being. although there is no consensus regarding the list of indicators that can be used to determine psychopathology and well-being for the dfm, the positive and negative indicators of mental health can be seen as two continua, with the upper end often conceptualized as flourishing, and the lower end defined as languishing (keyes ; wang et al. ) . regarding the indicators that can be included in the positive continuum, keyes ( ) identifies emotional, psychological and social well-being, which he named the "complete state model of mental health" (keyes , p. ) . the way the dfm integrates symptoms with personal resources makes it a comprehensive form of assessment. assessments of personal resources have been empirically confirmed to play an important role in predicting states of impaired functioning, such as depression, even when controlled for negative functioning . hence, there is a need to evaluate the potential of dfm with regard to predicting ats and atsp, since previous analyses have focused mainly on mental health problems (eskin et al. ; gibb et al. ; sallander renberg and jacobsson ) . in addition, little attention has been also paid to assessing the role of positive indicators for predicting ats and atsp among the potential recipients of gatekeeping training. furthermore, few studies have examined the relationship between positive mental health and ats among different groups of gatekeepers, and this may have cast a shadow across many training programs aimed at this population. most studies demonstrate a narrow focus on increasing knowledge on risk identification or teaching how to develop an intervention plan which could increase self-efficacy, but fail to promote optimal functioning (isaac et al. ). for the purpose of this study, it is important to distinguish two terms which are applicable to dfm and are related to well-being and suicide theories. life meaningfulness and meaninglessness are referred to in diverse psychological concepts within both positive and pathogenic paradigms, including ryff's eudaimonic approach to psychological well-being, models of hope, pro-preventive orientation towards suicide vs. beck's negative triad, shneidman's cognitive constriction or orientation towards suicide as a solution, respectively (delle fave et al. ; gibb et al. ; ringel ; ryff ; sallander renberg and jacobsson ; shneidman ; snyder et al. ; wenzel and beck ) . the interdependence of life meaningfulness (related to eudaimonia) and meaninglessness (related to suicidality) could be framed in two ways: (a) they are organized within a single continuum ranging from positive to negative functioning, or (b) they form a hierarchicallyorganized structure, where negative functioning influences well-being, i.e. the highest level of the hierarchy (see . the latter approach suggests that to investigate their relationship with ats and atsp, measures of both negative and positive functioning, i.e. those fitting the assumptions of the dfm, should be integrated. for gatekeepers, finding meaning in life plays an important role in predicting their helping behavior and valuing the end-of life decision. meaningfulness is an important aspect of eudaimonia, which defines happiness as personal growth, finding meaning in life and maintaining self-actualization (ryff ) . maintaining good eudaimonic well-being among providers ensures that their hope and authenticity may overcome the difficulties presented by a client (foulds ; o'hara ) ; such progress can be achieved by the client as a result of internalization, behavioral modeling and positive effect of self-disclosure (knox and hill ; kottler ) . these findings suggest that by focusing on the self-actualization of providers, it is possible to increase positive functioning, which is desirable for communicating emphatic understanding and facilitating genuineness. facilitating the personal resources of those who intervene with a suicidal or dying person is a particularly important goal, as helping behavior can entail a number of undesirable outcomes such as emotional exhaustion, compassion fatigue and burnout (benson and magraith ; marquis ). this may contribute to the development of negative attitudes towards those who are likely to die, manifested by avoidance or low collaboration (nia et al. ) . if life meaningfulness, represented by eudaimonia, could be at one extreme, meaninglessness would be at the other. for example, according to the escape theory of suicide, the experience of meaninglessness could be characterized as a stage in a suicidal crisis, resulting from dissociating from an unhappy state into cognitive deconstruction (baumeister ) . regarding the existential approach towards suicidality, meaninglessness is experienced more consciously as an individual's choice and a solution to existential dilemmas (orbach ; rogers ) . in fact, from an existentialist perspective, meaningfulness could be regarded as the outcome of dealing with the universal fear of death by transcending it into focusing on a meaningful life; this can be achieved, for example, by embracing religious systems (becker ) or engaging in self-actualization attained by fulfilling intrinsic psychological needs (ryff ) . similarly, meaninglessness could arise through the transcendence of fear of death into death fearlessness (chu et al. ; grewal and porter ) , which has implications for ats and atsp. the absence of mental health concerns among providers does not give a comprehensive view on their functioning; such an approach may fail to identify those who are languishing in terms of positive indicators of well-being and are likely to develop mental illness (suldo and shaffer ) . for example, low hopers are more prone to developing suicide risk (davidson et al. ), suggesting that permissive ats plays a mediating role. furthermore, providers with low positive functioning are thought to harbor more negative attainable expectations towards themselves and others, and to be less likely to discover and pursue purposeful goals (snyder et al. ) which impedes the establishment of a supportive relationship (brossart et al. ; o'hara ) , and possibly lessen the preparedness and willingness of the gatekeeper to prevent suicide. the working alliance with the client is determined by the ability of the provider to formulate realizable goals and tasks, as well as by their beliefs about the expected outcomes. the personal development of the providers affects their ability to see possibilities beyond the present struggle, as well as their belief in the capacity for the client to change and their belief in the power of prevention and intervention (o'hara ; snyder et al. ) . ats and atsp reflect the beliefs held by the provider that intervention is manageable and meaningful (sallander renberg and jacobsson ), which suggests that hopefulness and life meaningfulness serve as related themes to pro-preventive orientation. propreventive orientation is a construct derived from research on ats and atsp (sallander renberg and jacobsson ), which could be defined as the subjective preparedness and willingness to intervene personally; it also involves positive cognitive expectations towards prevention. low pro-preventive orientation may therefore indicate individual disengagement from initiating the intervention (sallander renberg and jacobsson ; stecz ). a number of important sources of attitudes towards suicide exist in the literature. they are framed by cultural norms, religious beliefs and the job environment, which differ across societies and particular communities (gearing and lizardi ; hjelmeland et al. ; lee et al. ; park et al. ; poreddi et al. ; sisask et al. ) . several studies suggest that sociodemographic variables such as male gender, higher economic status or being married were associated with more a pro-preventive orientation towards suicide (poreddi et al. ) ; however, the effect of gender and marital status vary between countries (lee et al. ; park et al. ) . secondly, ats is argued to be associated with psychological problems related to general mental health. psychological distress and decreased subjective well-being have been found to be associated with more accepting ats but more rejecting approach towards suicidal persons (eskin et al. ).in addition, permissive ats are thought to be potential risk factors for suicidal behavior and to be related to mental disorders: the prevalence of mental disorders at the time of death among those who commit suicide is considered to exceed % (herrera ) . finally, psychological wellbeing, comprising purpose in life and positive expectations towards life, could be argued to affect the orientation towards suicide prevention. the clinical implications of the model could be applicable for a wide range of helping situations. the variability of ats could be explained by the variation in the aspects of happiness, which are acknowledged to have a complex framework (delle fave et al. ) . bringing psychological symptoms and psychological well-being together integrates two approaches towards positive mental health, as demonstrated by the dfm. this corresponds well with the debate taking place within positive psychology, which integrates different views on happiness and embraces both the negative and positive aspects of human life (ivtzan et al. ) . previous research has linked suicidality and permissive ats with low hedonistic wellbeing and hopelessness in both general and clinical populations (cheavens et al. ; davidson et al. ; joiner et al. ; yamokoski et al. ): findings which consolidate theories in suicidology and positive psychology in some regard. however, the relationship between eudaimonic well-being and ats/atsp seems to be underinvestigated, and our understanding of the relevance of the happiness felt by potential and future gatekeepers toward their effectiveness at suicide prevention has hence evolved in a fragmented way. our research question concerns whether it is possible for psychological well-being to predict ats and atsp among students of the helping professions, controlling for sociodemographic characteristics, religiousness and psychological problems related to their general mental health. it was hypothesized that higher level of eudaimonic well-being would have predictive validity for stronger orientation towards suicide prevention when controlling for sociodemographic variables (gender, economic situation, marital status), religious beliefs and psychological problems related to mental health. we argue that the hierarchical view of ats and atsp among the students of the helping professions, reflecting future gatekeepers, should be awarded further consideration as a framework. first, ats and atsp might be associated with sociodemographic and personal variables (e.g. gender, marital status, economic status, religiousness), as shown in previous research (bagley and ramsay ; lee et al. ; park et al. ; poreddi et al. ) . secondly, permissive ats and low orientation towards suicide prevention are expected to demonstrate a relationship with higher levels of emotional distress, somatic complaints, anxiety and depression, comprising decreased general mental health and lower subjective well-being as indicated by general health questionnaire (ghq). finally, ats and atsp would be predicted by eudaimonic happiness, which should serve a preventative function even when risk factors occur. a hierarchical model might demonstrate the effect of psychological well-being on pro-preventive orientation, controlled for confounding variables. the study was approved by the university of Łódź bioethics committee and all participants gave their informed consent to participate. participation was fully voluntary and was not supported financially nor with the offer of course credits; similarly, those who declined to take part were not placed under any pressure to do so. the sample (n = ) consisted of students of the helping professions (law, medicine, psychology) from two public universities in central poland. the rationale for considering law, medicine and psychology students as study participants was that gatekeeping involves different agents and employs a wide range of interventions comprising health, psychological and legal support. in addition, a more heterogeneous group was formed in order to increase external validity of the results. all students enrolled in the obligatory course were invited to take part in the study, with . % agreeing to do so. women (n = ) comprised . % of the sample, which moderately affected sample specificity (one sample χ ( ) = . , φ = . , p < . ). the ethnicity of the sample was caucasian. the demographic characteristics of the participants are outlined in table . the psychological wellbeing scale, developed by ryff ( ) and adapted in poland by krok ( ) is a statistically-sound and theoretically-driven questionnaire based on the eudaimonic concept of happiness. respondents use a seven-point likert scale to rate how much they agree or disagree with statements measuring six aspects of well-being and happiness: self-acceptance, purpose in life, positive relations with others, personal growth, environmental mastery and autonomy (i.e. "i have confidence in my opinions, even if they are contrary to the general consensus"). cronbach's internal consistency coefficients for psychological well-being subscales range from α = . (personal growth) to α = . (selfacceptance), with values of α = . (purpose in life), α = . (positive relations with others), α = . (autonomy) and α = . (environmental mastery) observed in our sample. the atts developed by sallander renberg and jacobsson ( ) is a self-report scale used to assess ats and atsp. the polish atts version with items has been demonstrated to have a unique five-factor structure, with mcdonald's omega coefficients varying from . (suicide preventability-cognitive) to . (suicide as a right) and has shown generally adequate reliability and acceptable construct validity (stecz ). in our sample, most internal consistency reliabilities were acceptable, with suicide as a right (cronbach's α = . ), suicide as a solution (α = . ), suicide (in)comprehensibility (α = . ), suicide preventability-cognitive (α = . ) and orientation towards suicide prevention (α = . ). scores range from to , except for the six-item subscale suicide as a right, which ranges from to . we analyzed four domains (suicide preventability-cognitive, orientation towards suicide prevention, suicide (in)comprehensibility, suicide as a solution) which were considered to be the most central for the purpose of this study. the ghq- developed by goldberg ( ) is intended for use as a self-report screening instrument for detecting the possibility of psychological disorder. the ghq- consists of statements divided into four subscales: somatic symptoms (hypochondria), anxiety and insomnia, social dysfunction (understood as inability to pursue activities), and severe depression (e.g., "found yourself wishing you were dead and away from it all"). the overall score comprising items ranges from to . high scores suggest emotional distress and low subjective well-being associated with risk of general and mental health problems. the ghq- contains several positively-worded items (e.g. "have been feeling reasonably happy") with the response option "more so than usual" so that lower scores could be interpreted as higher level of functioning in positive categories , providing a measure of subjective (emotional) well-being. the polish validation demonstrated adequate psychometric properties and reproduced its factorability (goldberg et al. ) ; similarly, the ghq- demonstrated acceptable reliability in our sample (α = . ), with satisfactory internal consistency values for the somatic symptoms (α = . ), anxiety (α = . ), dysfunction (α = . ), and depression (α = . ) subscales. although all ghq- subscales represent operational variables related to psychological and mental health problems, they are closely correlated with each other (r ranged from . to . ; p < . ); therefore, the general ghq- score was used in the analyses in the present study to provide a more holistic approach. the scale was developed by huber (huber ; huber and huber ) for measuring the salience of religious meanings in the individual. the five core dimensions, considered to be representative for the total of religious life, were: worship (public practice), religious or spiritual experience, prayer, religious beliefs and religious interests. the instrument also allows the measurement of the general importance of religion for the individual. polish version, provided with items, demonstrated adequate psychometric properties (zarzycka ) , as reported in our sample by cronbach's alpha for overall centrality of religiosity (α = . ). general religiousness score range from to with higher scores reflecting higher levels of religious centrality in the individual's life. preliminary analyses, independent samples t-tests, spearman and kendall's rank correlations, pearson correlations (when the assumptions were not violated) and three-step hierarchical regression analyses were conducted. robust % cis were computed for all correlation and regression coefficients using bootstrapping with samples. bonferroni's correction was applied for familywise type i error in correlation analysis. all calculations were performed using ibm spss statistics . prior to conducting the analysis, a sample size of subjects was considered to be adequate, given the originally planned independent variables (including demographic variables, religiousness, general mental health problems and dimensions of pwb) used in the hierarchical multiple regression (tabachnick and fidell ) . data were tested for violations in distribution and the presence of multivariate outliers for testing the relevant assumptions of multiple regression. none of the absolute values of skewness (all above − . ) or kurtosis (all above − . ) indicated substantial violations of normality (e.g., kline ) . no extreme multivariate outliers were detected in data sets for prediction of ats/atsp. no significant differences were found in ats and atsp with regard to gender or marital status (tables and ) . no significant relationships were found between ats/atsp and socioeconomic status (τ b = − . with suicide as a solution; τ b = − . with preventability-cognitive; τ b = . with suicide (in)comprehensibility and τ b = . with orientation towards suicide prevention; p > . ) and age (r s = . with suicide as a solution; r s = . with preventability-cognitive; r s = − . with suicide (in)comprehensibility and r s = − . with orientation towards suicide prevention; p > . ). therefore, these variables were not included in further analyses. descriptive statistics for ats/atsp, religiousness, general mental health problems and dimensions of psychological well-being are shown in table , together with correlations between the variables. significant correlations were found between suicide (in)comprehensibility and religiousness; preventability-cognitive/suicide (in)comprehensibility and general health problems. regarding dimensions of psychological wellbeing, significant correlations were observed between all dimensions and preventability-cognitive; suicide (in)comprehensibility correlated with environmental mastery (table ) . four sets of hierarchical multiple regression analyses were conducted to determine the role of psychological well-being in predicting ats and atsp. each regression analysis included religiousness (stage ), general mental health problems (stage ) and six dimensions of psychological well-being (stage ). the results of the multiple regression analyses are given in tables , , and . no significant predictor was found for suicide as a solution. moreover, whole models to were also not significant (table ) . as seen in table , religiousness and general mental health problems significantly predicted the preventability-cognitive attitude towards suicide (stage one and two). religiousness remained significant even after adding general mental health problems (step two) and psychological well-being dimensions (stage three). the only significant predictor from psychological well-being dimensions was environmental mastery. psychological wellbeing accounted for additional . % of the variance in suicide preventability-cognitive and was the most important predictor in the regression model. religiousness and general mental health problems significantly predicted suicide (in) comprehensibility (table ) . both variables remained significant even after adding psychological well-being dimensions into the regression model (stage two and three). in contrast, psychological well-being was not a significant predictor of suicide (in)comprehensibility when introduced at stage three. the significant increase of explained variance in the model was observed only at stage two after adding general mental health problems (stage two). orientation towards suicide prevention (table ) was significantly predicted by religiousness (stage one) and two dimensions of well-being (introduced at stage three): positive relations with others and purpose in life. religiousness remained significant when controlled for general mental health problems (stage two), but was not significant when table results of multiple regression analysis (dependent variable: suicide as a solution) *significance testing based on bootstrapped se. bootstrap based on samples step : r = . , f( , ) = . , p > . step : r = . , f( , ) = . , p > . , Δr = . , f( , ) = . , p > . step : r = . , f( , ) = . , p > . , Δr = . , f( , ) = . , p > . step predictors ( all independent variables were included in stage three. contrary to the previously-examined atsp, the presence of general mental health problems was not a significant predictor. moreover, the addition of psychological well-being variables to the regression model at stage three explained an additional % of the variation in orientation towards suicide prevention, and this change was significant. step : r = . , f( , ) = . , p = . step : r = . , f( , ) = . , p < . , Δr = . , f( , ) = . , p < . step : r = . , f( , ) = . , p < . , Δr = . , f( , ) = . , p < . step table results of multiple regression analysis (dependent variable: suicide (in)comprehensibility) *significance testing based on bootstrapped se. bootstrap based on samples step : r = . , f( , ) = . , p < . step : r = . , f( , ) = . , p < . , Δr = . , f( , ) = . , p < . step : r = . , f( , ) = . , p < . , Δr = . , f( , ) = . , p > . step the role of gatekeepers is not restricted to identifying suicide risk or making referrals to crisis intervention services. from a positive psychology perspective, providers, also known as designated gatekeepers, serve as significant others to those in crisis, and thus may facilitate personal growth and rehabilitation of those at suicide risk. authenticity and positive regard towards a person experiencing life meaninglessness are thought to work effectively if they correspond with provider's own self-actualization and positive functioning (foulds ; o'hara ) . the central assumption of the present study was that the positive mental health of future gatekeepers could be related to a pro-preventive orientation towards suicide, which would have consequences for social policy. the study was directed towards students of the helping professions. these participants are future gatekeepers, and have been found to score lower on eudaimonic wellbeing, controlled for religiousness and symptoms of psychological disorders; as hypothesized, they were found to be oriented less strongly towards suicide prevention. also, as hypothesized, low eudaimonic well-being predicted more acceptable ats; however, this result was not significant when controlled for confounding variables. it is somewhat surprising that neither age, gender nor economic status were associated with ats and atsp: other findings suggest that cultural differences or sociodemographic variables may influence ats (lee et al. ; poreddi et al. ; sallander renberg and jacobsson ) . although this result may appear confusing, religion is known to play an important role within the polish community (storm ) . our present findings indicate that religiousness may have a stronger influence on ats variability than other demographic characteristics (cf. zou et al. ) . due to the structure of the sample, the student population does not provide enough generalizability for making unbiased inferences related to demographics. step : r = . , f( , ) = . , p = . step : r = . , f( , ) = . , p = . , Δr < . , f( , ) = . , p > . step : r = . , f( , ) = . , p < . , Δr = . , f( , ) = . , p < . step predictors ( our findings suggest that positive relations with others may be a significant predictor of orientation towards suicide prevention. an orientation towards prevention involves an instrumental component, defined as readiness to prevent suicide when approaching a person in crisis (sallander renberg and jacobsson ; stecz ) . the achievement of close relationships with others then could be seen as the ability to empathize and care. our findings can be better understood when viewed from the perspective of ryff's ( ) arguments linking positive relations with others with the concepts of maturity and generativity, described as providing care for others and feeling warmth for human beings. according to the multidimensional model of psychological well-being, a decreased quality of relationships could be attributed to difficulties with being concerned about others (ryff ) ; thus, individuals with unsatisfying relationships are less likely to become involved in suicide prevention, which could be explained by high demands for empathy, care and positive regard in the helping professions. considering the multidimensionality of psychological well-being and its relevance to suicide theories, it is interesting to note that our results confirm that purpose in life is only significantly related to orientation towards suicide prevention when controlled by other variables in the proposed model. other attitude dimensions (suicide preventabilitycognitive, suicide as a solution and suicide (in)comprehensibility) were not found to be related to purpose in life when controlled for confounders. at the same time, this study demonstrated that orientation towards prevention is predicted by environmental mastery. this result extends previous studies and deserves further discussion. to clarify the role of purpose in life for understanding the formation of schemata related to suicide and suicide intervention, it is necessary to more precisely define life meaningfulness. meaning-making should be then considered as a broader construct and a process referring to human beliefs, subjective feelings and goals, comprised by different subconstructs (park ) . ryff and keyes ( ) proposes that purpose in life is influenced by at least two aspects: the realization of one's goals and the belief that one's life is meaningful. it is possible that only some of the subcomponents of purpose in life are relevant to ats in general and to pro-preventive orientation. furthermore, situational level of purpose in life varies according to stressful events among providers, suggesting that reduced purpose in life could be interpreted as the initiation of change or restoration of meaning-making processes (skaggs and barron ) . our findings provide evidence for the generalizability of the dfm, comprising both symptoms of psychological disorders (also interpreted as decreased subjective well-being) and eudaimonic well-being, for studying the predictors of ats among future gatekeepers, i.e. students of the helping professions. a pro-preventive orientation towards suicide is better explained by a joint model than by any fragmentary model emphasizing either psychological well-being or psychological complaints. current model is also controlled for centrality of religiousness, which is known to be of high importance in predicting ats. a study based on a turkish sample found religiousness to be associated with stigmatizing ats (vatan et al. ). in a sample on christian adults, domino and miller ( ) report greater religiousness to be related to perceiving suicide as a unforgivable moral evil. our study revealed that religious beliefs and faith predicted suicide incomprehensibility, which highlights the importance of negative emotions and evaluations associated with suicide perception among more religious potential gatekeepers and confirms that our results are consistent with those of earlier studies. although our hypotheses were supported, the relationship between psychological well-being domains and ats/atsp is not as strong as expected. the findings might indicate that the constructs of well-being and pro-preventive orientation are linked by different mechanisms than assumed. a possible explanation for identifying environmental mastery as a significant predictor of pro-preventive orientation could be that helping behavior and the preparedness to help in difficult situations have been demonstrated to be dependent on specific self-evaluations, such as self-efficacy (bandura ; jerusalem and schwarzer ; lipson ) . environmental mastery has been defined by ryff ( ) as a sense of competence in managing activities in accordance with personal needs and values. negative expectations about managing and improving the surrounding context refer to pessimistic appraisals and predict negative interaction regulation if self-efficacy is deficient (knopp and delle-fave ) . the underlying causes for negative expectations and pessimistic attitudes have been described extensively in literature through the lenses of a fixed versus open mindset (dweck ) , attribution hypothesis (abramson et al. ) , expectation bias (coats et al. ; rosenthal and jacobson ) or nocebo effect (corsi and colloca ) . due to certain limitations of the study design, these findings cannot be extrapolated to all groups of the gatekeepers and need to be interpreted with caution. the crosssectional design and convenient sampling method preclude a definitive discussion of the causal relationships between well-being and pro-preventive orientation. in addition, the sample consisted of undergraduate students, who play a role in gatekeeping but have limited experience in prevention, and data collection was performed during their educational training, before establishing professional career. our results, could not be therefore generalized on the population of actual gatekeepers. our attempt to recruit a wide range of potential future gatekeepers (including law students) could be also regarded as a trade-off between external and internal validity. finally, some caution should be observed when interpreting the results, as the internal consistencies of the atts subscales were a little lower; in addition, the available questionnaires on ats and atsp rely on self-report, and as they generally have rather limited psychometric characteristics, they can be exposed to measurement bias (kodaka et al. ; stecz ) . furthermore, the data demonstrated significant results, but the described models did not account for a large proportion of the variance observed in ats/atsp. hence, there is abundant room for conceptualizing a more complex theoretical model related to dfm for predicting ats and atsp. for example, helping behavior has been previously explained within the context of hope theory (o'hara ), compassion and self-compassion effect on increased effectiveness (raab ) or by self-efficacy theory, considering gatekeeper training programs focused on knowledge and skills improvement (lipson ) . a number of other variables, including cultural norms (range et al. ) , value system (gagnon and hasking ) or 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behavior centrality of religiosity scale attitudes towards suicide in urban and rural china: a population based, cross-sectional study the authors would like to thank anna stecz ma for her kind assistance. key: cord- -mabrmzd authors: jacomin, anne-claire; taillebourg, emmanuel; fauvarque, marie-odile title: deubiquitinating enzymes related to autophagy: new therapeutic opportunities? date: - - journal: cells doi: . /cells sha: doc_id: cord_uid: mabrmzd autophagy is an evolutionary conserved catabolic process that allows for the degradation of intracellular components by lysosomes. this process can be triggered by nutrient deprivation, microbial infections or other challenges to promote cell survival under these stressed conditions. however, basal levels of autophagy are also crucial for the maintenance of proper cellular homeostasis by ensuring the selective removal of protein aggregates and dysfunctional organelles. a tight regulation of this process is essential for cellular survival and organismal health. indeed, deregulation of autophagy is associated with a broad range of pathologies such as neuronal degeneration, inflammatory diseases, and cancer progression. ubiquitination and deubiquitination of autophagy substrates, as well as components of the autophagic machinery, are critical regulatory mechanisms of autophagy. here, we review the main evidence implicating deubiquitinating enzymes (dubs) in the regulation of autophagy. we also discuss how they may constitute new therapeutic opportunities in the treatment of pathologies such as cancers, neurodegenerative diseases or infections. autophagy is a lysosomal catabolic process that ensures the degradation and recycling of intra-cytoplasmic components and, therefore, highly contributes to the maintenance of cellular homeostasis. in order to adapt to various stresses and promote its survival under challenging conditions, the cell also uses autophagy to degrade a broad range of endogenous or exogenous substrates [ ] . the best-studied endogenous substrates of autophagy are mitochondria and protein aggregates [ ] [ ] [ ] . defects in the elimination of such substrates are often associated with human pathologies, including neurodegenerative diseases or cancers [ ] [ ] [ ] . autophagy is a very dynamic process and has to be tightly regulated to provide a timed and efficient response to a multitude of signals. posttranslational modifications play a significant role in the induction and regulation of autophagy [ , ] . ubiquitination of substrates and protein of the autophagy machinery has notably emerged as a central regulatory mechanism of autophagy acting at various levels to promote autophagy induction or shutdown [ , ] . ubiquitination is a reversible protein modification that consists of the covalent attachment of one or several ubiquitin moieties to protein substrates [ ] [ ] [ ] . while ubiquitination is achieved by the sequential action of e ubiquitin-activating enzyme, e ubiquitin-conjugating enzyme, and an e ubiquitin ligase, the removal of ubiquitin from a protein is catalyzed by deubiquitinating enzymes (dubs) [ ] . ubiquitination can promote or interfere with protein-protein interactions, modifying the the main regulators of autophagy are ampk (amp-activated kinase) and mtorc (mtor complex ( ) which act as autophagy activator and inhibitor, respectively. ampk and mtorc regulate the activation of the ulk complex which, together with the pi k-Ш complex, initiates the autophagosome formation. the formation of an autophagosome starts with the generation of a phagophore which elongates into an isolation membrane where the cargos to be degraded are gathered. the enclosure of the isolation membrane forms a double-membraned autophagosome that matures and eventually fuses with the lysosome, forming an autolysosome where lysosomal hydrolases degrade its content. the atg and lc conjugation systems are essential for the autophagy process and formation of the autophagosome. the atg conjugation system consists in the formation of the atg -atg -atg l complex that then promotes the conjugation of lc ; pro-lc is cleaved by atg (lc -i) which is then conjugated with phosphatidylethanolamine (pe) (lc -ii) before being anchored to the nascent autophagosomal membrane. ubiquitin is a small globular protein with a -grasp superfold conformation [ , ] . ubiquitin is covalently conjugated by its terminal glycine (g ) onto a lysine residue of a substrate protein. protein ubiquitination is a complex process requiring the successive activity of three types of enzymes: an e ubiquitin-activating enzyme, an e ubiquitin-conjugating enzyme, and an e ubiquitin-ligase enzyme. the ubiquitination process can be broken down in two main steps: ( ) the atp-dependent activation of a ubiquitin molecule by conformational modification of its c-terminus extremity by an e enzyme followed by its transfer onto an e enzyme, and ( ) the conjugation of the activated ubiquitin onto a substrate protein, mediated by an e enzyme that bridges the e to a the main regulators of autophagy are ampk (amp-activated kinase) and mtorc (mtor complex ( ) which act as autophagy activator and inhibitor, respectively. ampk and mtorc regulate the activation of the ulk complex which, together with the pi k-iii complex, initiates the autophagosome formation. the formation of an autophagosome starts with the generation of a phagophore which elongates into an isolation membrane where the cargos to be degraded are gathered. the enclosure of the isolation membrane forms a double-membraned autophagosome that matures and eventually fuses with the lysosome, forming an autolysosome where lysosomal hydrolases degrade its content. the atg and lc conjugation systems are essential for the autophagy process and formation of the autophagosome. the atg conjugation system consists in the formation of the atg -atg -atg l complex that then promotes the conjugation of lc ; pro-lc is cleaved by atg (lc -i) which is then conjugated with phosphatidylethanolamine (pe) (lc -ii) before being anchored to the nascent autophagosomal membrane. ubiquitin is a small globular protein with a β-grasp superfold conformation [ , ] . ubiquitin is covalently conjugated by its terminal glycine (g ) onto a lysine residue of a substrate protein. protein ubiquitination is a complex process requiring the successive activity of three types of enzymes: an e ubiquitin-activating enzyme, an e ubiquitin-conjugating enzyme, and an e ubiquitin-ligase enzyme. the ubiquitination process can be broken down in two main steps: ( ) the atp-dependent activation of a ubiquitin molecule by conformational modification of its c-terminus extremity by an e enzyme followed by its transfer onto an e enzyme, and ( ) the conjugation of the activated ubiquitin onto a substrate protein, mediated by an e enzyme that bridges the e to a specific substrate allowing for the subsequent transfer of ubiquitin from the e enzyme to the substrate through the formation of an isopeptide bond [ , ] . alternatively, e ligases of the hect family possess e and e activities [ , ] . there is also evidence for the addition of ubiquitin moieties onto non-lysine residues. the ubiquitination of cysteine or serine and threonine residues requires the formation of thiol-or oxy-ester bonds respectively [ ] . ubiquitination was discovered in and first described for its essential role in targeting proteins to the proteasome for degradation [ ] . it is now widely recognized that ubiquitination also acts in other cellular processes. indeed, a broad range of types of ubiquitination have been reported. substrates can be modified by the attachment of a single ubiquitin molecule (mono-ubiquitination) or several single ubiquitin molecules on different lysine residues of the substrate (multi-mono-ubiquitination). mono-ubiquitination is primarily described for its function in endocytosis of plasma membrane receptors [ ] . alternatively, several ubiquitin molecules can be ligated to one another using ubiquitin internal lysine residues, forming chains of ubiquitin moieties that can elongate on the substrate or directly be attached to a target protein (poly-ubiquitination). because ubiquitin has seven lysine residues, there can be at least as many types of ubiquitin chains that can be generated (k , k , k , k , k -linked ubiquitin chains) [ , ] . poly-ubiquitin chains can also be assembled through n-to c-terminal interaction to form linear chains (m ) [ , ] . the tridimensional structures of ubiquitin chains vary depending on the lysine in the ubiquitin used to generate the chain and affect the function or stability of the substrate. for instance, k -linked ubiquitin chains have a compact conformation and target substrates for proteasomal degradation, while k -linked ubiquitin chains display an open conformation and are mostly described to promote signal transduction through the assembly of large protein complexes [ , ] . protein ubiquitination is a reversible posttranslational modification. the hydrolysis of ubiquitin linkages is conducted by a specific family of proteases: the dubs. these enzymes can act at different stages of the protein ubiquitination process: ( ) at the "initial" stage, by cleaving the ubiquitin precursors to supply ubiquitin monomers to the ubiquitination enzymes; ( ) at the "intermediate" stage, by the regulated removal of ubiquitin moieties from proteins to alter their fate (stabilization, conformational change); and ( ) at the "final" stage by the removal of ubiquitin chains from substrates addressed to the proteasome to facilitate their degradation and processing into ubiquitin monomers, free to enter a new ubiquitination cycle ( figure ) [ ] [ ] [ ] . the hydrolysis of k -linked ubiquitin chains, most well-known to induce the proteasomal degradation, can affect the fate of the protein they are added to either by protecting substrates from degradation or by supporting proteasomal degradation, as the removal of k -linked ubiquitin chains, mostly by proteasomal dubs, is also required for protein entry into the proteasome [ , ] . there are approximately dubs encoded by the human genome [ , ] , which are divided into two main families: the cysteine proteases and the metalloproteases. there are dubs from the metalloprotease family characterized by a jamm (jab /pab /mpn domain-containing metalloenzymes) domain that catalyzes the hydrolysis of isopeptide bonds in the presence of zn + . cysteine proteases are divided into five sub-families according to the sequence and structure of their catalytic domain: ubiquitin-specific proteases (usps), ubiquitin c-hydrolases (uchs), otubain proteases (otus), machado joseph disease proteases (mjds), and the most recently identified sub-family miu-containing novel dub family (mindys). the most abundant sub-family of dubs is the usps with over members, come after the otus ( members), uch, mjds and mindys (each with four members) [ ] . protein posttranslational modifications are crucial in the dynamic regulation of the autophagy process. modifications of core components of the autophagic machinery are notably essential for the induction of autophagy. autophagy proteins that are ubiquitinated constitute substrates for dubs, therefore regulating their function and/or stability [ ] . the mechanistic target of rapamycin complex (mtorc ) plays a central role in the integration of various environmental signals to regulate cell metabolism, growth, proliferation, and survival. in nutrient-rich conditions, mtorc is active and promotes cell growth while down-regulating autophagy. conversely, down-regulation of mtorc activity during nutrient deprivation activates autophagy. the protease otub was recently reported to inhibit mtorc activity by deubiquitinating and stabilizing the inhibitor deptor in response to amino acid deprivation [ ] ( figure a ). deptor stabilization by otub depends on its catalytic activity as the catalytically inactive mutant otub -c a fails to both remove ubiquitin moieties from deptor and protect it from proteasomal degradation. consistent with these observations, otub overexpression induces autophagy while its knockdown represses autophagy induction [ ] . protein posttranslational modifications are crucial in the dynamic regulation of the autophagy process. modifications of core components of the autophagic machinery are notably essential for the induction of autophagy. autophagy proteins that are ubiquitinated constitute substrates for dubs, therefore regulating their function and/or stability [ ] . the mechanistic target of rapamycin complex (mtorc ) plays a central role in the integration of various environmental signals to regulate cell metabolism, growth, proliferation, and survival. in nutrient-rich conditions, mtorc is active and promotes cell growth while down-regulating autophagy. conversely, down-regulation of mtorc activity during nutrient deprivation activates autophagy. the protease otub was recently reported to inhibit mtorc activity by deubiquitinating and stabilizing the inhibitor deptor in response to amino acid deprivation [ ] ( figure a ). deptor stabilization by otub depends on its catalytic activity as the catalytically inactive mutant otub -c a fails to both remove ubiquitin moieties from deptor and protect it from proteasomal degradation. consistent with these observations, otub overexpression induces autophagy while its knockdown represses autophagy induction [ ] . the serine/threonine protein kinase ulk (unc -like kinase ) is a critical inducer of autophagy (see above and figure ). dynamic phosphorylation and polyubiquitination regulate ulk activity. notably, the ubiquitination of ulk with k -linked ubiquitin chains contributes to its stabilization and activity [ ] . in contrast, the linkage of k -linked ubiquitin chains leads to proteasomal degradation of ulk , resulting in a blockade of starvation-induced autophagy [ , ] . a loss-of-function screen of dubs in hela cells identified usp as the first dub to be involved in regulating ulk ubiquitination and stability. usp interacts with and deubiquitinates ulk , thus protecting it from degradation. the maintenance of basal levels of ulk by usp contributes to rapid induction of autophagy under stress condition. indeed, silencing of usp encoding gene inhibits autophagosomes and autolysosomes formation in response to starvation ( figure b ). however, the interaction between ulk and usp is weakened upon prolonged induction of autophagy ( to h), leading to a reduction of the level of ulk protein while its ubiquitinated form accumulates [ ] . the molecular mechanisms regulating the dissociation of usp and ulk are not known yet but could depend on posttranslational or allosteric modifications on usp as its stability is not affected by starvation; alternatively, the e ubiquitin ligase nedd l, may compete with usp to interact with ulk and promotes its proteasomal degradation [ ] . beclin is a multivalent adaptor protein and forms, with vps and vps , the core components of the pi k-iii signaling complex, which is essential for the maturation of the autophagosome (see above and figure ). beclin also interacts transiently with accessory factors, such as atg l, ambra , uvrag or bcl- [ ] . moreover, beclin versatile ubiquitination is tightly linked to its function and activation [ ] . different types of ubiquitin chains, including k -and k -linked chains, were found on beclin , and several dubs control beclin ubiquitination status and activity ( figure c ). usp is a ubiquitin-specific protease tightly associated with the proteasome which has been shown to cleave k -linked ubiquitin chains [ , ] ; however, other studies have shown that usp is also able to cleave k -linked ubiquitin chains [ , ] . knockdown of usp or its inhibition with the inhibitor iu (see below section . ) induces the activation of autophagy, indicating that usp is a negative regulator of autophagy in h (neuroglioma) cells. because silencing the usp encoding gene does not affect the stability of beclin or other components of the complex, this suggests that its ability to regulate autophagy is independent of its proteasomal function in h (neuroglioma) cells. according to this observation, it was shown that usp suppresses the activity of beclin complex and induction of autophagy by interacting with and controlling k -rather than k -linked ubiquitin chains of beclin [ ] . autophagy is highly interconnected with immune processes and can be triggered by activated tlr (toll-like receptor ). activation of tlr by microbial components contributes to the recruitment of adaptor proteins and enzymes required for the signal transduction and induction of the immune response by nf-κb (nuclear factor kappa b) transcription factors, such as the e ubiquitin ligase and scaffold protein traf (tnfr-associated factor ). traf is then responsible for the induction of autophagy following the activation of the tlr in macrophages through the ubiquitination of beclin with k -ubiquitin chains in murine macrophages raw . [ ] . indeed, ubiquitination of the lysine residue within the bh domain of beclin prevents its interaction with the inhibitor bcl- [ , ] . in addition, min and colleagues showed that traf and usp compete for the interaction with beclin in hek t (human embryonic kidney t) cells and that usp negatively regulates autophagy in thp- monocyte cells [ ] . traf and beclin interact through their coiled-coil domains in the absence of usp , whereas the interaction is gradually attenuated when the cells are co-transfected with increasing quantity of usp . however, the study does not provide any evidence for the catalytic role of usp and instead suggests that the interaction of beclin with usp inhibit traf -mediated ubiquitination of beclin [ ] . additionally, the dub a -a downstream target of nf-κb-is responsible for the catalytic removal of k -linked ubiquitin chains on the lysine of beclin to limit the induction of autophagy in the murine macrophage cell line raw . [ ] . interestingly, beclin and the bcl- family member, mcl- , compete for their interaction with usp x, which contributes to their stabilization by protecting them from proteasomal degradation in hek t cells [ ] . in the same study, it was observed that mcl- levels were increased in malignant tissues from melanoma patients while beclin was destabilized, suggesting that usp x promotes tumor progression. however, usp x function in tumorigenesis appears to be more complex and context-dependent as independent studies have shown that usp x can be either a tumor promoter or suppressor depending on the origin of the cells [ , ] . the addition of k -linked ubiquitin chains to beclin by the e ligase nedd triggers its degradation by the proteasome [ ] . the enzymes usp and usp are known to process k -linked ubiquitin chains resulting in the stabilization of beclin in hek t cells (for both usp and usp roles), as well as in mef, hela and bcap- cells (role of usp ) [ , ] . although several lysine residues in beclin may be ubiquitinated with k -linked chains, usp seems to mainly target ubiquitin moieties bound to the lysine of beclin in hek t cells, suggesting that its function may not be redundant, but instead complementary to usp [ ] . finally, the dub usp was found to be a regulator of early steps of starvation-induced autophagy activation by promoting the interaction between beclin and the ras-like gtpase ralb. indeed, the assembly of the complex ralb-exo -beclin is made possible by the deubiquitination of ralb at its lysine residue in hek t and hela cells [ ] . the identification of usp as indirectly regulating beclin activity through the deubiquitination of one of its partners reinforces the hypothesis that the modification and activation of autophagy machinery components are context-specific. the formation of protein aggregates is a continuous process in the cell, and their degradation by autophagy, referred to as aggrephagy, is one of the first types of selective autophagy that has been described. aggrephagy involves the autophagy receptors p /sqstm and nbr which are recruited to ubiquitinated protein aggregates [ ] [ ] [ ] [ ] . the accumulation of protein aggregates that fail to be degraded in neuronal or glial cells is a hallmark of various neurodegenerative diseases. aggregation of α-synuclein is characteristic of the pathogenesis of parkinson's disease in which mono-and poly-ubiquitinated α-synuclein are major constituents of the lewy bodies [ , ] . mono-ubiquitination of α-synuclein seems to be required for its targeting of proteasomal degradation and thus negatively regulates its autophagic degradation. usp x interacts with α-synuclein in vitro and in vivo and contributes to the removal of mono-ubiquitin moieties and favors its targeting for degradation by autophagy rather than the proteasome [ ] . conversely, uch-l -a dub associated with parkinson's disease and whose gene is frequently mutated in familial forms of the pathology [ ] -promotes the accumulation of α-synuclein aggregates in oligodendrocytes [ , ] . another independent study also reported that membrane-associated uch-l contributes to α-synuclein neurotoxicity, possibly by negatively regulating its lysosomal degradation [ ] ( figure d ). finally, a recent study demonstrates the prevalence of k -linked ubiquitin chain conjugates in lewy bodies, suggesting that their elimination can be primarily ensured by the lysosomal route rather than the s proteasome. in addition, this study identifies usp as one of the best markers of lewy bodies in human pigmented neurons in sporadic cases of parkinson's disease and demonstrates the ability of usp to hydrolyze k -linked ubiquitin chains from α-synuclein in vitro [ ] . moreover, usp gene extinction significantly reduces the total level of α-synuclein both in a drosophila model of parkinson's disease and in human embryonic kidney (hek t) cultured cells [ ] . thus, the presence of usp on endosomal membranes (see below) and in lewy bodies, as well as its ability to deubiquitinate α-synuclein, makes it a preferred therapeutic target according to the assumption that inhibition of usp could directly promote the elimination of amyloid fibers by the endocytic and lysosomal pathways. it was recently shown that the selective autophagy receptor p binds to protein aggregates modified not only with k -linked ubiquitin chains but also with k -linked ubiquitin chains [ , ] . zranb /trabid is a k -and k -specific dub [ ] . knockdown of zranb enhances the recruitment of p to k -associated protein aggregates, suggesting that zranb is a negative regulator of aggrephagy; yet, the physiological function of k -linked ubiquitin chains and the role of zranb are not entirely understood [ ] . in drosophila, the p homolog ref ( )p is also implicated in the clearance of ubiquitinated protein aggregates [ , ] . however, little is known about the regulation of autophagy-associated ubiquitination processes in flies. the only dub known to regulate ubiquitin-dependent autophagy negatively is dusp [ ] . this protein negatively regulates the formation of ubiquitinated nuclear aggregates, while promoting cell growth. indeed, deletion of the dusp encoding gene results in the robust accumulation of ubiquitinated protein aggregates, which include the histone protein h b, and in the activation of autophagy, independently of the tor pathway [ ] (figure d ). protein aggregates clearance requires the action of selective receptors to be adequately targeted for autophagic degradation [ ] . ndp is an autophagy receptor mostly described for its role in addressing ubiquitin-decorated bacteria for degradation by autophagy [ , ] . however, a new role for ndp in the formation of traf aggregates was unveiled [ ] . indeed, ndp mediates the aggregation and selective autophagic degradation of the tlr adaptor molecule trif and the signaling molecule traf in response to tlr stimulation. ubiquitination of ndp , mediated by traf , is necessary for its activity and is counteracted by a [ ] ( figure d ). with the variety of proteins prone to aggregations, it is not surprising that different dubs are involved in the regulation of the formation and degradation of protein aggregates. protein aggregation is a hallmark of various pathologies, notably neurodegeneration and infection, and a better understanding of the regulatory mechanisms associated with each pathology will greatly benefit the development of appropriate and targeted treatments. mitophagy refers to the clearance of exhausted mitochondria by autophagy. the mitochondrial kinase pink and the e -ubiquitin ligase parkin play a central role in the mitochondrial quality control. upon mitochondria damage and loss of membrane potential, parkin is translocated to the outer mitochondrial membrane (omm) and activated by stabilized pink [ ] . active parkin ubiquitinates a myriad of substrates on the omm that can be recognized by ubiquitin-binding selective autophagy receptors [ , ] . so far, three dubs-usp , s-usp , and usp -have been reported to counteract parkin activity following acute mitochondrial depolarization, thus acting as a negative regulator of mitophagy. usp is the only dub identified so far as a positive regulator of mitophagy ( figure e ). usp is a mitochondrial enzyme, tethered to the outer membrane of the mitochondria with its catalytic domain facing the cytoplasm [ ] . several independent studies point out that usp is one of the major dub regulating mitophagy. overexpression of usp reverses the ubiquitination of parkin substrates, such as tom , and impairs mitophagy [ ] [ ] [ ] [ ] . usp function in autophagy is dependent on its catalytic activity as a catalytically inactive mutant usp -c a is ineffective at inhibiting mitophagy [ ] . the depletion of usp was shown to enhance the degradation of mitochondria in neuronal and hela cell cultures [ , ] . usp knockdown also increases the ubiquitination level on multiple parkin substrates, thus confirming that usp antagonizes parkin function. usp proteolytic activity is more efficient on k -linked ubiquitin chains, even though it can also process k , k and k chains [ , ] . the ubiquitin chains targeted by usp are similar to the ones parkin adds to its substrates, suggesting that these two enzymes act as antagonists on shared substrates. the majority of the work carried out on the regulation of mitophagy have relied on cells overexpressing parkin along with the use of mitochondrial-depolarizing agents. such experiments simulate an extreme scenario of mitochondrial stress and interpretations may not be relevant to basal conditions of mitochondrial clearance [ ] . however, recently published work by marcassa and colleagues describes the investigation into the role of usp in more physiological conditions [ ] . the authors propose a new model in which usp acts upstream pink as the depletion of pink in cells lacking usp abrogated the increased mitophagy induced by usp knockdown in u os cells [ ] . in the same study, usp is revealed to regulate the degradation of peroxisome by autophagy (pexophagy) in a similar way to its role in mitophagy. like mitochondria, peroxisomes are the main source of reactive oxygen species (ros) that can be damaging to the cells if produced in high quantities [ ] . moreover, contact sites between mitochondria and peroxisomes exist, and mitochondria were shown to play a role in the generation of peroxisomes [ ] ; thus reinforcing the hypothesis of an intrinsic relationship between both organelles. the depletion of usp increases both pexophagy and mitophagy. however, the localization of usp on mitochondria and peroxisomes relies on distinct sequences, suggesting that the role of usp in pexophagy is independent to its mitochondrial function [ ] . it is possible that usp acts at different levels during the mitophagy or pexophagy processes depending on the conditions. in basal condition, usp could serve as a safety check-point to avoid mitophagy or pexophagy to be triggered inappropriately. besides, other studies identified additional dubs which may also contribute to the regulation of mitophagy. usp short form (s-usp ) is another dub that is localized at the mitochondria [ ] . in a similar manner to usp , overexpression of s-usp impairs mitophagy while s-usp knockdown enhances mitochondrial degradation [ ] . usp is the third dub identified to antagonize parkin-mediated mitophagy. overexpression of usp inhibits mitophagy dependently of its catalytic activity, while depletion of usp enhances mitophagy. unlike usp and s-usp , usp is only rarely localized at the mitochondria [ ] . only one dub, usp , may act as a positive regulator of mitophagy through parkin regulation. usp knockdown impairs parkin-mediated mitophagy by preventing parkin recruitment of depolarized mitochondria. in this process, usp selectively removes k -linked ubiquitin chains on parkin and counteracts parkin auto-ubiquitination and auto-catalytic activation [ ] . whether and how these dubs act in concert or within different organs or situations remains to be determined to fully understand their specific requirements in physiological or stressed conditions. proteins can be degraded by autophagy independently of their aggregation in a way that can be either dependent or independent of their ubiquitination state. to date, only a few substrates, known to be directly targeted for degradation by autophagy in response to ubiquitination, have been shown to be regulated by a specific dub ( figure f ). the hypoxia-inducible factor , α subunit (hif- α) is a transcription factor essential for cells to adapt rapidly to low oxygen levels (hypoxia). when oxygen is available, hif- α is polyubiquitinated and rapidly degraded either by the proteasome or directly by the lysosome through chaperone-mediated autophagy [ , ] . the dub cezanne/otud b is itself induced by oxygen deprivation in cultured endothelial cells. moreover, loss of cezanne reduces the amount of hif- α protein while cezanne overexpression stabilizes hif- α and protects it from autophagic degradation in a catalytic-dependent manner by specifically processing k -linked ubiquitin chains [ ] . mutation of the cma-targeting motif (kferq motif) of hif- α makes it insensitive to cezanne knockdown, thus suggesting that cezanne specifically regulates the degradation of hif- α mediated by cma. cezanne is not the only dub to regulate the ubiquitination status of hif- α. indeed, usp is essential for the removal of ubiquitin moieties on hif- α in normoxia, contributing to the maintenance of a basal level of hif- α. in this case, however, usp appears to protect hif- α from proteasomal degradation [ ] . these two studies show that different dubs can regulate the fate of a shared substrate depending on the physiology of the cell. connexin- /cx is a member of the connexin family that forms the gap junction channels between adjacent cells, enabling direct intercellular exchanges between cells, which is another example of a substrate for at least two different degradative pathways. indeed, cx polyubiquitination triggers its degradation through either the proteasome or the lysosome via endocytosis and autophagy [ ] [ ] [ ] [ ] . usp interacts with and deubiquitinates cx , removing monoubiquitin moieties as well as k -and k -linked ubiquitin chains. cx ubiquitination and degradation by autophagy are increased in usp knockdown cells [ ] . even though usp regulates autophagic degradation of cx in basal condition, one cannot exclude that usp may also affect cx through the endolysosomal pathways in different conditions as usp is well described for its implication in the endocytosis of various plasma membrane receptors [ ] [ ] [ ] . thus, there are several cases of proteins being degraded through different processes notably as a result of the linkage of different kinds of ubiquitin moieties and undergoing tight regulation by specific dubs. autophagy and endocytosis are two conserved and interconnected degradative pathways among eukaryotes. moreover, fusion events between autophagosomes and endocytic compartments have been observed and investigated [ , ] . endocytosis and autophagy converge not only at the level of lysosomes but also at the level of early and late endosomes, forming another type of vesicle called amphisomes. several dubs, known for their role in endocytosis, also impact directly or indirectly the autophagic flux ( figure g ). amsh is a metalloprotease of the jamm type involved in the sorting of cell-surface receptors at endosomes [ ] [ ] [ ] [ ] . amsh localizes at the endosomes and promotes the recycling of internalized receptors [ , ] . disruption of amsh in mice results in the loss of neurons in the hippocampus and severe atrophy of the cerebral cortex [ ] . an independent study observed that the loss of amsh in neurons results in the accumulation of ubiquitinated protein aggregates associated with the autophagy receptor p , indicating that the autophagy flux is impaired [ ] ( figure d) . however, at this stage of the study, it is not possible to discriminate whether the blockade of the autophagy flux results from an impairment in the endocytic process or a lack of targeting of cargoes to autophagosome, independently of endocytosis. in the plant model arabidopsis thaliana, the ortholog amsh interacts with the escrt-iii protein vps . and contributes to autophagic degradation [ ] (figure g ). usp is a second dub playing a major role in endocytosis by regulating both the ubiquitination status of cargoes and members of the escrt machinery regulating membrane deformation and scission events [ ] [ ] [ ] [ ] [ ] [ ] . usp has been extensively studied for its role in the regulation of the trafficking and lysosomal degradation of receptors, such as egfr, through the endocytic process [ , ] . in addition to its role in endocytosis, loss of dusp in drosophila blocks the progression of autophagy, resulting in the accumulation of ref( )p/p and ubiquitinated proteins [ ] . as for dusp , dusp depletion affects both the autophagic flux and endocytosis process. indeed, silencing dusp encoding gene results in the accumulation of autophagosomes in drosophila [ ] , and usp negatively regulates the endocytosis and translocation of the notch receptor to the lysosomes in both drosophila and mammalian cells [ ] . although these studies cannot exclude a direct role of these dubs in the regulation of autophagy, they support a close imbrication of endocytosis and autophagy and suggest interdependence of the two processes. this hypothesis is reinforced by the fact that disruption of the endocytic process using dominant-negative rab or by their knockdown also results in impaired autophagy [ ] . the particular case of the β -adrenergic receptors (β -ars) also illustrates a reliable interconnection between the endocytic and autophagic processes and their tight regulation by ubiquitination. β -adrenergic receptors (β -ars) availability on the plasma membrane is tightly regulated by balancing their internalization and recycling rates. misregulation of β -ars trafficking has been associated in various pathologies, including heart failure and asthma [ ] . β -ars take an unconventional route to the lysosomes; indeed, after their endocytic internalization, ubiquitinated β -ars are directed to the autophagosomes rather than the lysosomes [ ] . the post-endocytic sorting of the receptor from the endosomes to the autophagosomes is modulated by the proteases usp and usp [ ] . however, solely usp was shown to promote the deubiquitination of β -ars and their post-endocytic trafficking to autophagosomes. in this process, phosphorylation of usp at serine residue is required for its activity providing an additional level of regulation [ ] . recently, the protein chmp a of the escrt-iii complex was identified to be crucial for the closure of the autophagosome [ ] . therefore, endosomes-associated dubs such as usp and amsh, or other dubs that remain to be identified, may also play a direct regulatory role in this process through the regulation of escrt-iii components activity, as recently shown for chmp b during endocytosis [ ] . the expression of a number of genes related to autophagy is activated upon starvation in mammalian cell culture. in this process, histone protein h b monoubiquitination (h bub ) is an essential modification for the regulation of gene transcription. the level of h bub is controlled by the protease usp which is upregulated after starvation. knockdown of usp results in the maintenance of h bub upon starvation and abolishes the change in expression of starvation-induced autophagy-related genes. moreover, downregulation of usp encoding gene blocks the induction of autophagy in mescs (mouse embryonic stem cells) ( figure h ). this study thus unveils a new role for dub in the transcriptional regulation of autophagy through the modulation of h b monoubiquitination [ ] . ubiquitination of microbial molecular patterns is used by eukaryotic cells to tag invasive pathogens and target them for autophagic degradation. this reaction leads to the accumulation of ubiquitinated protein aggregate known as ubiquitinated aggresome-like induced structures (alis). such aggregates contribute to the upregulation of autophagy and the removal of intracellular pathogens. in response to this host defense mechanism, intracellular pathogens, such as bacteria or viruses, have developed strategies to hijack the host ubiquitin pathway by expressing dub-like enzymes able to counteract ubiquitination and permit them to escape their elimination by autophagy. for instance, the intracellular pathogenic bacteria salmonella enterica serovar typhimurium (s. typhimurium) counteracts the alis-induced autophagy by translocating a dub-like enzyme, ssel (salmonella-secreted factor l), into the cytosol. lysates from mouse macrophages infected with ∆ssel mutant bacteria are enriched in ubiquitinated proteins, and immunofluorescence experiments revealed that these bacteria are more prone to ubiquitination and recognition by autophagy markers such as lc or p [ , ] . secreted ssel deubiquitinates alis and the salmonella-containing vacuoles, reducing the induction of autophagy, further promoting the survival and replication of s. typhimurium [ ] ( figure d ). like salmonella, legionella is an intracellular bacterium that can establish niches in cytoplasmic vacuoles which allows for the survival and replication of the bacteria. the legionella pneumophila effector protein ravz is a secreted cysteine protease that interferes with the autophagy machinery by irreversibly deconjugating lc from the autophagosome membrane [ ] . lc is an autophagy-related ubiquitin-like protein anchored to the autophagosome membrane. the process leading to lc lipidation and association to the membrane is similar to the ubiquitination process and requires a ubiquitin-like conjugation system. ravz deconjugates lc from the autophagosome membrane by hydrolyzing the amide bond between the c-terminal glycine residue and an adjacent aromatic residue; the lack of terminal glycine residue prevents the conjugation of lc to the membrane [ , ] . ravz can also process conventional ubiquitin chains and prevent the targeting of intracellular bacteria for autophagic degradation [ ] . indeed, using a co-infection system with salmonella and legionella, kubori and colleagues showed that legionella ravz protease prevents the recruitment of the autophagy receptors p and ndp to the salmonella-containing vacuoles. the lack of autophagy receptors at the scvs is due to the removal of ubiquitin moieties from the scvs by ravz [ ] . it was recently shown that ravz specificity toward lc anchored to the autophagosomal membrane depends on its interaction with pi p [ ] ; this observation suggests that ravz activity as dub on ubiquitin coats depends on the lipid structure of the nearby vacuole. autophagy also targets viruses; yet, many viruses exploit autophagy for their replication [ ] . coronaviruses induce the formation of double-membrane vesicles that allow for their replication and are often decorated with lc and cell infection with coronaviruses is often accompanied with induction of autophagy [ ] . the non-structural protein plp -tm, which is a transmembrane papain-like protease with deubiquitinating activity [ ] , is sufficient for the accumulation of autophagosomes in different cell lines. however, its role in the regulation of autophagy is independent of its protease activity [ ] . plp -tm interacts with lc and promotes the accumulation of autophagosomes by blocking their fusion with the lysosomes. in their study, chen and colleagues suggest that plp -tm blocks autophagosome-lysosome fusion through its interaction with beclin , a prime target for viruses that manipulate the autophagy pathway [ ] . plp -tm also promotes the interaction of sting (stimulator of interferon genes) with beclin , possibly to impede the activation of downstream antiviral responses, accentuated by the deubiquitination of components of the signaling cascade such as rig- or traf [ , ] . these studies provide fascinating examples of possible coevolution and adaptation of the pathogens with their host, where pathogens managed to bypass the host's defense to their own benefit. ubiquitination regulates major cellular functions by controlling protein stability and activity, and defects in this process contribute to the development of many diseases. in some cases, ubiquitin-dependent autophagic processes constitute entry points to design new treatments. depending on the context, however, autophagy can either be beneficial and contribute to survival and recovery or have adverse effects. as such, there is a need for in-depth understanding of autophagy function and regulation in pathological or physiological situations to define in which situation the inhibition of a particular dub will be beneficial or detrimental. interestingly, the design of chemical tools is also a powerful strategy to probe the effect of dubs inhibition to help both the understanding of their role in the regulation of autophagy and the design of future treatments to modulate autophagy in the corresponding pathologies. efficiency and usability of an inhibitor depends on its specificity. as such, the discovery of dub-focused drugs has been challenging [ ] . indeed, although dubs have a catalytic pocket that is suitable for drug development, their sequence and structure are very similar. moreover, dubs are flexible enzymes, and the regulation of their activity can involve allosteric effects as described for several dubs that alternate between active and inactive conformations [ ] [ ] [ ] [ ] [ ] [ ] . for instance, the free catalytic domain of usp undergoes significant structural modifications when it is complexed to ubal (ubiquitin aldehyde, an irreversible dub inhibitor) [ , ] . recent publications of the dynamic interaction of usp with specific small-molecule inhibitors demonstrated that the binding of the molecules into the active site of usp modifies the catalytic residue c . this modification of the active site of usp results in its inability to change conformation and perform the cleavage of ubiquitin chains. these studies show that the development of specific inhibitors binding to the active site of dubs is a realistic approach, opening new avenues in the field [ , ] . in addition to intrinsic modulation of their activity and substrate specificity, some dubs require cofactors. for instance, the full activation of usp requires its interaction with hsp , which promotes the binding of ubiquitin to the catalytic domain of usp [ ] . another example is the proteasome-associated enzyme usp , whose activity is strongly enhanced when in association with the proteasome [ ] . despite the complexity of the regulation of dubs activity, much effort has been placed in the identification and development of small-molecule regulating dubs catalytic activity. some of the most successful small-molecules affecting autophagy the process, through the inhibition of the activity of autophagy-associated dubs, are shortly introduced hereafter. screens for inhibitors of dubs sought to identify new small-molecules with potential in two main therapeutic fields, for cancer treatment and neurodegeneration (reviewed in [ ] ). in both fields, some of the inhibitors' targets play a role in the regulation of autophagy that possibly contributes to pathogenesis. usp is an enzyme associated with the proteasome, which plays an essential role in the regulation of protein turnover. the role of usp is particularly important in neurons to maintain synaptic functions and constitutes an appealing target for drug development in order to modulate the activity of the proteasome [ ] . a screen of , compounds using proteasome reconstituted with usp , led to the identification of the first inhibitor of usp . the small-molecule iu inhibits specifically usp with an ic of - µm [ ] . the inhibitor iu blocks the activity of usp only in the presence of the proteasome, suggesting that it binds only to the activated enzyme. moreover, the compound iu abrogates the catalytic activity of usp without affecting its noncatalytic regulatory function [ ] . however, with the growing number of usp substrates identified, there was a need for the development of iu analogs with improved selectivity over the usp -substrate complexes. a curated screen of variants of iu led to the identification of iu - as a new potent inhibitor of usp . iu - treatment of murine primary neuron cultures and in neurons derived from human-induced pluripotent stem cells (ipsc) accelerates the degradation of the microtubule-associated protein tau, which is implicated in many neurodegenerative diseases. besides, the inhibition of usp by iu - induced an increase of the autophagy flux, consistent with the increased degradation rate of tau [ ] . as mentioned above, uch-l is a negative regulator of autophagy widely studied for its implication in parkinson's disease and its contribution to the aggregation of α-synuclein as a result of autophagy blockade [ , ] . uch-l is also expressed in various primary lung tumors while not detectable in normal, healthy lung tissue, suggesting a possible contribution to cancer [ ] . because of the correlation between uch-l and tumor progression, as well as its implication in neurodegenerative disease, uch-l is a recognized target for the development of therapeutic inhibitors. as such, the compound ldn- was identified in a high throughput drug screening as a specific uch-l inhibitor. treating h lung cancer cell line with this compound significantly reduces the cell proliferation rate [ ] . nsc is another inhibitor that affects uch-l activity. however, nsc activity is not specific to uch-l , and it is already known to inhibit usp and usp [ , ] . the amount of p in cells is reduced after treatment with both ldn- and ncs , suggesting that these drugs could prevent the accumulation of protein aggregates [ ] . therefore, these inhibitors may constitute new tools to investigate further the implication of uch-l in parkinson's disease and evaluate whether uch-l inhibition favors the clearance of α-synuclein aggregates in neurons. inhibition of early regulators is another strategy to inhibit autophagy in some situations. the inhibitor wp , which targets usp x, was reported to lead to an increase in ulk ubiquitination, inducing its transfer to the aggresomes and its inhibition, further resulting in the blockade of autophagy in several cultured cell lines, including the bone osteosarcoma u os cell line [ ] . it was speculated that the inhibition of usp x could be responsible for the accumulation and subsequent aggregation of ubiquitinated ulk . however, silencing usp x did not result in changes in ulk expression level when cells were treated with wp . this could be because wp is only partially specific and could have other targets in vivo that remain to be discovered [ , ] . in order to screen and select new small-molecules interfering with autophagy in mammalian cells, an imaging-based assay has been optimized by liu and colleagues that makes use of cells expressing the autophagy marker gfp-lc to quantify the accumulation of autophagosomes [ ] . using this assay, they screened the iccb known bioactives library, a collection of compounds, and they identified the inhibitor spautin- (specific and potent autophagy inhibitor ). spautin- inhibits the catalytic activity of both usp and usp with an ic of~ . - . µm. these two dubs are involved in the regulation of beclin ubiquitination in the vps complex and, therefore, constitute an entry point to modulate the initiation of autophagy. cancer cell lines treated with spautin- demonstrated an increased cell death rate under starvation conditions. as such, spautin- constitutes a potential lead for the development of autophagy inhibitors for anti-cancer therapies [ ] . because of its essential function in mitophagy, which is crucial to clear damaged mitochondria notably in neuronal cells, several small-molecule inhibitors of usp have been developed in the past few years. for example, based on a phenotypic screening, it was shown that the inhibition of usp by the compound -oxospiramilactone enhances the activity of usp 's targets mfn and mfn -two gtpases anchored at the omm and essential for tethering adjacent mitochondria-and promotes mitochondrial fusion, thus contributing to the restoration of the mitochondria network [ ] . more recently, an in vitro study identified a new small-molecule mf- , as a potent and selective inhibitor of usp . this compound has the opposite effect of -oxospiramilactone, as mf- -mediated inhibition of usp accelerates mitophagy [ ] . these two studies highlight the fact that the same dubs can be involved in different processes, dependent on the signal they may receive and the interaction within different protein complexes. there is no doubt that new inhibitors of dubs will arise with problems related to the existence of several substrates or to poor selectivity, requiring in-depth analysis of the selected compounds in different cell types and stress situations before any preclinical assays. interestingly, these investigations may tell a lot about how dubs regulate autophagy and other cell processes, and may be used as molecular tools to unveil regulatory mechanisms. protein ubiquitination is an essential, reversible, posttranslational modification involved in virtually every cellular process. the past decades have seen remarkable progress in the understanding of the function of dubs, their mechanism of action and regulation. recently, there has been an increasing body of evidence that ubiquitination plays a crucial role in regulating autophagy, and dubs intervene at multiple steps in autophagy. deregulation in both autophagy and ubiquitination/deubiquitination processes have been linked to many pathologies such as neurodegenerative diseases, cancer onset and progression, and different kinds of viral or bacterial infections. also, considerable effort was placed on the development and optimization of small-molecules acting as dubs inhibitors. such molecules can serve not only as leads for the development of drug-like molecules but also as tremendous useful tools to investigate the molecular mechanism of autophagy and its regulation by the ubiquitin system. by the development of molecules targeting protein-protein interaction instead of the catalytic activity, it could be possible to manipulate and orientate precisely the function of a dub towards a given process and/or target to avoid pleiotropic effects. autophagy at the crossroads of catabolism and anabolism cargo recognition and degradation by selective autophagy aggrephagy: selective disposal of protein aggregates by macroautophagy mitophagy and quality control mechanisms in mitochondrial maintenance clearance of misfolded and aggregated proteins by aggrephagy and implications for aggregation diseases the role for autophagy in cancer compromised autophagy and neurodegenerative diseases regulation of autophagy by protein post-translational modification posttranslational modification of autophagy-related proteins in macroautophagy the role of ubiquitin system in autophagy ubiquitin signaling and autophagy ubiquitin: structures, functions, mechanisms the demographics of the ubiquitin system the emerging complexity of protein ubiquitination mechanisms of deubiquitinase specificity and regulation proteasomal and autophagic degradation systems ubiquitin modifications the many roles of ubiquitin in nf-κb signaling ubiquitination: friend and foe in cancer deubiquitylating enzymes and drug discovery: emerging opportunities functions of lysosomes autophagy as a stress-response and quality-control mechanism: implications for cell injury and human disease the machinery of macroautophagy autophagosome formation from membrane compartments enriched in phosphatidylinositol -phosphate and dynamically connected to the endoplasmic reticulum the chaperone-mediated autophagy receptor organizes in dynamic protein complexes at the lysosomal membrane the coming of age of chaperone-mediated autophagy an intralysosomal hsp is required for a selective pathway of lysosomal protein degradation selective endosomal microautophagy is starvation-inducible in drosophila microautophagy of cytosolic proteins by late endosomes hsc - deforms membranes to promote synaptic protein turnover by endosomal microautophagy chaperone-mediated autophagy and endosomal microautophagy: joint by a chaperone making new contacts: the mtor network in metabolism and signalling crosstalk two distinct vps phosphatidylinositol -kinase complexes function in autophagy and carboxypeptidase y sorting in saccharomyces cerevisiae a ubiquitin-like system mediates protein lipidation atg , a ubiquitin-like protein required for autophagosome formation, mediates membrane tethering and hemifusion gabarap and gate localize to autophagosomal membrane depending on form-ii formation regulation of the tumor-suppressor beclin by distinct ubiquitination cascades mtor inhibits autophagy by controlling ulk ubiquitylation, self-association and function through ambra and traf structure of ubiquitin refined at . Å resolution the ubiquitin domain superfold: structure-based sequence alignments and characterization of binding epitopes the ubiquitin system getting into position: the catalytic mechanisms of protein ubiquitylation ubiquitin ligases: structure, function, and regulation hect and ring finger families of e ubiquitin ligases at a glance ubiquitination of substrates by esterification ubiquitin dependence of selective protein degradation demonstrated in the mammalian cell cycle mutant ts distinct monoubiquitin signals in receptor endocytosis the ubiquitin code a proteomics approach to understanding protein ubiquitination a ubiquitin ligase complex assembles linear polyubiquitin chains linear ubiquitin chains: enzymes, mechanisms and biology crystal structure and solution nmr studies of lys -linked tetraubiquitin at neutral ph solution conformation of lys -linked di-ubiquitin chain provides clues to functional diversity of polyubiquitin signaling deubiquitylation and regulation of the immune response regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes deubiquitylases from genes to organism role of rpn metalloprotease in deubiquitination and degradation by the s proteasome identifying usps regulating immune signals in drosophila: usp deubiquitinates imd and promotes its degradation by interacting with the proteasome a genomic and functional inventory of deubiquitinating enzymes emerging roles of e ubiquitin ligases in autophagy otub protein suppresses mtor complex (mtorc ) activity by deubiquitinating the mtorc inhibitor deptor chaperone-like protein p regulates ulk stability and autophagy fine-tuning of ulk mrna and protein levels is required for autophagy oscillation the deubiquitinating enzyme usp stabilizes ulk and promotes autophagy initiation conformational flexibility of becn : essential to its key role in autophagy and beyond enhancement of proteasome activity by a small-molecule inhibitor of usp deubiquitinating enzyme ubp functions noncatalytically to delay proteasomal degradation deubiquitination of dishevelled by usp is required for wnt signaling phosphorylation and activation of ubiquitin-specific protease- by akt regulates the ubiquitin-proteasome system usp regulates autophagy by suppressing k ubiquitination of beclin traf and a regulate lysine -linked ubiquitination of beclin- to control tlr -induced autophagy disruption of the beclin -bcl autophagy regulatory complex promotes longevity in mice ubiquitin-specific protease negatively regulates toll-like receptor -mediated signaling and autophagy induction by inhibiting ubiquitination of tak -binding protein and beclin beclin restrains tumorigenesis through mcl- destabilization in an autophagy-independent reciprocal manner deubiquitinase usp x stabilizes mcl and promotes tumour cell survival the deubiquitinase usp x suppresses pancreatic ductal adenocarcinoma nedd -dependent lysine- -linked polyubiquitination of the tumour suppressor beclin usp modulates autophagy and antiviral immune responses by deubiquitinating beclin- beclin controls the levels of p by regulating the deubiquitination activity of usp and usp the deubiquitylase usp discriminates between ralb functions in autophagy and innate immune response essential role of sequestosome /p in regulating accumulation of lys -ubiquitinated proteins p is a common component of cytoplasmic inclusions in protein aggregation diseases a role for nbr in autophagosomal degradation of ubiquitinated substrates regulation of selective autophagy: the p /sqstm paradigm the lewy body in parkinson's disease and related neurodegenerative disorders ubiquitination of α-synuclein and autophagy in parkinson's disease engelender, s. α-synuclein fate is determined by usp x-regulated monoubiquitination familial mutations and post-translational modifications of uch-l in parkinson's disease and neurodegenerative disorders inhibition of uch-l in oligodendroglial cells results in microtubule stabilization and prevents α-synuclein aggregate formation by activating the autophagic pathway: implications for multiple system atrophy the functions of uch-l and its relation to neurodegenerative diseases membrane-associated farnesylated uch-l promotes α-synuclein neurotoxicity and is a therapeutic target for parkinson's disease deubiquitinase usp regulates α-synuclein clearance and modifies its toxicity in lewy body disease lysine -linked polyubiquitin potentially partners with p to promote the clearance of protein inclusions by autophagy novel polyubiquitin imaging system, polyub-fc, reveals that k -linked polyubiquitin is recruited by sqstm /p an ankyrin-repeat ubiquitin-binding domain determines trabid's specificity for atypical ubiquitin chains ref( )p, the drosophila melanogaster homologue of mammalian p , is required for the formation of protein aggregates in adult brain ref( )p and ubiquitinated proteins are conserved markers of neuronal aging, aggregate formation and progressive autophagic defects the deubiquitinating enzyme usp controls selective autophagy activation by ubiquitinated proteins lc c, bound selectively by a noncanonical lir motif in ndp , is required for antibacterial autophagy the tbk adaptor and autophagy receptor ndp restricts the proliferation of ubiquitin-coated bacteria regulation of toll-like receptor signaling by ndp -mediated selective autophagy is normally inactivated by a parkin is activated by pink -dependent phosphorylation of ubiquitin at ser emerging themes in mitochondrial homeostasis the three 'p's of mitophagy: parkin, pink , and post-translational modifications regulation of mitochondrial morphology by usp , a deubiquitinating enzyme present in the mitochondrial outer membrane the mitochondrial deubiquitinase usp opposes parkin-mediated mitophagy usp deubiquitylates mitochondrial parkin substrates and restricts apoptotic cell death usp and parkin homeostatically regulate atypical ubiquitin chains on mitochondria deubiquitinating enzymes regulate park -mediated mitophagy ubiquitin linkage-specific affimers reveal insights into k -linked ubiquitin signaling organelle turnover: a usp safety catch restrains the trigger for mitophagy and pexophagy dual role of usp in controlling basal pexophagy and mitophagy peroxisomal dysfunction in age-related diseases newly born peroxisomes are a hybrid of mitochondrial and er-derived pre-peroxisomes the deubiquitinase usp antagonizes parkin-mediated mitochondrial ubiquitination and mitophagy usp regulates mitophagy by removing k -linked ubiquitin conjugates from parkin chaperone-mediated autophagy targets hypoxia-inducible factor- α (hif- α) for lysosomal degradation stub /chip is required for hif a degradation by chaperone-mediated autophagy cezanne (otud b) regulates hif- α homeostasis in a proteasome-independent manner hif α deubiquitination by usp is essential for ciliogenesis in normoxia regulation of connexins by the ubiquitin system: implications for intercellular communication and cancer epidermal growth factor regulates ubiquitination, internalization and proteasome-dependent degradation of connexin gap junction turnover is achieved by the internalization of small endocytic double-membrane vesicles internalized gap junctions are degraded by autophagy the ubiquitin-specific protease usp deubiquitinates and stabilizes cx regulation of epidermal growth factor receptor ubiquitination and trafficking by the usp .stam complex essential role of ubiquitin-specific protease for receptor tyrosine kinase stability and endocytic trafficking in vivo the mit domain of ubpy constitutes a chmp binding and endosomal localization signal required for efficient epidermal growth factor receptor degradation induction of autophagy promotes fusion of multivesicular bodies with autophagic vacuoles in k cells new insights into autophagosome-lysosome fusion amsh, an escrt-iii associated enzyme, deubiquitinates cargo on mvb/late endosomes targeting of amsh to endosomes is required for epidermal growth factor receptor degradation activation of the endosome-associated ubiquitin isopeptidase amsh by stam, a component of the multivesicular body-sorting machinery amsh interacts with escrt- to regulate the stability and trafficking of cxcr endocytosis: the dub version amsh is an endosome-associated ubiquitin isopeptidase loss of neurons in the hippocampus and cerebral cortex of amsh-deficient mice amsh is required to degrade ubiquitinated proteins in the central nervous system the deubiquitinating enzyme amsh and the escrt-iii subunit vsp . are required for autophagic degradation in arabidopsis chmp b is a target of usp /ubpy regulated by ubiquitin during endocytosis regulation of endocytic sorting by escrt-dub-mediated deubiquitination ubpy controls the stability of the escrt- subunit hrs in development the ubiquitin isopeptidase ubpy regulates endosomal ubiquitin dynamics and is essential for receptor down-regulation the deubiquitinating enzyme ubpy is required for lysosomal biogenesis and productive autophagy in drosophila the ubiquitin-specific protease (usp ) is a negative regulator of notch signaling acting on notch receptor trafficking toward degradation a functional endosomal pathway is necessary for lysosome biogenesis in drosophila trafficking of β-adrenergic receptors: implications in intracellular receptor signaling phosphorylation of the deubiquitinase usp by protein kinase a regulates post-endocytic trafficking of β adrenergic receptors to autophagosomes during physiological stress the deubiquitinases usp and usp coordinate β adrenergic receptor recycling and resensitization an autophagy assay reveals the escrt-iii component chmp a as a regulator of phagophore closure histone h b monoubiquitination is a critical epigenetic switch for the regulation of autophagy the salmonella deubiquitinase ssel inhibits selective autophagy of cytosolic aggregates ssel, a salmonella deubiquitinase required for macrophage killing and virulence the legionella effector ravz inhibits host autophagy through irreversible atg deconjugation elucidation of the anti-autophagy mechanism of the legionella effector ravz using semisynthetic lc proteins legionella ravz plays a role in preventing ubiquitin recruitment to bacteria-containing vacuoles the legionella anti-autophagy effector ravz targets the autophagosome via pi p-and curvature-sensing motifs viruses and the autophagy pathway involvement of autophagy in coronavirus replication deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases coronavirus membrane-associated papain-like proteases induce autophagy through interacting with beclin to negatively regulate antiviral innate immunity beclin- targeting for viral immune escape coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of sting-mediated signaling crystal structure of a ubp-family deubiquitinating enzyme in isolation and in complex with ubiquitin aldehyde structure and mechanisms of the proteasome-associated deubiquitinating enzyme usp amino-terminal dimerization, nrdp -rhodanese interaction, and inhibited catalytic domain conformation of the ubiquitin-specific protease (usp ) structural basis for conformational plasticity of the parkinson's disease-associated ubiquitin hydrolase uch-l structural basis and specificity of human otubain -mediated deubiquitination structure validation of the josephin domain of ataxin- : conclusive evidence for an open conformation mechanism of usp /hausp activation by its c-terminal ubiquitin-like domain and allosteric regulation by gmp-synthetase usp -specific inhibitors target and modify the enzyme's active site via distinct chemical mechanisms active site-targeted covalent irreversible inhibitors of usp impair the functions of foxp + t-regulatory cells by promoting ubiquitination of tip characterization of the deubiquitinating activity of usp and its role in endoplasmic reticulum-associated degradation synaptic defects in ataxia mice result from a mutation in usp , encoding a ubiquitin-specific protease an inhibitor of the proteasomal deubiquitinating enzyme usp induces tau elimination in cultured neurons expression of the protein gene product . , pgp . , is correlated with t-status in non-small cell lung cancer discovery of inhibitors that elucidate the role of uch-l activity in the h lung cancer cell line ubiquitin c-terminal hydrolase l regulates autophagy by inhibiting autophagosome formation through its deubiquitinating enzyme activity characterization of ubiquitin and ubiquitin-like-protein isopeptidase activities deubiquitinase inhibition by wp leads to ulk aggregation and blockade of autophagy deubiquitinase inhibition by small-molecule wp triggers aggresome formation and tumor cell apoptosis a small natural molecule promotes mitochondrial fusion through inhibition of the deubiquitinase usp novel highly selective inhibitors of ubiquitin specific protease (usp ) accelerate mitophagy key: cord- -ol ofj authors: abdul-cader, mohamed sarjoon; ehiremen, godsent; nagy, eva; abdul-careem, mohamed faizal title: low pathogenic avian influenza virus infection increases the staining intensity of kul + cells including macrophages yet decrease of the staining intensity of kul + cells using clodronate liposomes did not affect the viral genome loads in chickens date: - - journal: vet immunol immunopathol doi: . /j.vetimm. . . sha: doc_id: cord_uid: ol ofj the effect of depletion of macrophages using clodronate liposomes as well as macrophage response following viral infections have been studied in various mouse-virus infection models, but they have not been extensively studied in chickens relevant to virus infections. when we infected day chickens with h n low pathogenic avian influenza virus (lpaiv), we observed that h n lpaiv infection increased the staining intensity of kul + cells in trachea, lungs and duodenum of chickens at days post-infection. then, we used clodronate liposomes intra-abdominally in day-old chickens and found significant reduction of staining intensity of kul + cells in trachea and duodenum but not in lungs at days post-treatment. when we infected the clodronate liposome and pbs liposome treated chickens with h n lpaiv intra-nasally at day , we found no effect on h n lpaiv genome loads in trachea, lungs and duodenum of chickens. this study indicates that although kul + cell intensity are increased in respiratory and gastrointestinal tissues in chickens following h n lpaiv infection, the decrease of kul + cell intensity using clodronate liposomes did not change the h n lpaiv genome loads in any of the examined tissues suggesting that kul + cells may not be critical during h n lpaiv infection in chicken. the innate immune system, which mounts potent, nonspecific and broadly effective host responses, is equipped with an array of immune cells. one of the key immune cells indispensable in this regard is the macrophages. in addition to phagocytic activities of macrophages against various microbes and harmful substances, they also act as antigen presenting cells and a source of cytokines and chemokines facilitating the induction of antigen specific adaptive immune responses (arango duque and descoteaux, ) . in avian species, mobilization of macrophages into the site of infection contribute more than resident macrophages in phagocytic activity (maina, ) . liposome encapsulated clodronate (dichloromethylene diphosphonate or cl -mdp) has been widely used to deplete macrophages (benoit et al., ; kameka et al., a; leemans et al., ) . once phagocytosed by macrophages, clodronate liposomes get accumulated in the cytosol, resulting in apoptosis and depletion of macrophages (van rooijen et al., ) . it has been shown that the use of clodronate liposomes significantly depletes macrophages in chickens (jeurissen et al., ) . the effect of depletion of macrophages using clodronate liposomes has been studied against various virus infections in mouse models, such as measles (roscic-mrkic et al., ) and influenza (tate et al., ) . in chickens, increased marek's disease virus genome load in the blood and spleen following treatment with clodronate liposomes has been shown (rivas et al., ) . however, macrophage depletion using clodronate liposomes has not been extensively studied during other viral infections in chickens. macrophage recruitments following viral infections in chickens such as marek's disease virus, infectious bursal disease virus, adenovirus and infectious bronchitis virus infections have been shown previously (abdul-careem et al., ; abdul-careem et al., ; fulton et al., ; kameka et al., b; nakamura et al., ) . however, studies that investigated macrophage response in chickens following avian influenza virus infection are scarce (cornelissen et al., ; rebel et al., ) . avian macrophages have been shown to be targeted for viral replication during infectious bronchitis corona virus , marek's disease virus (barrow et al., ; chakraborty et al., ) , avian influenza virus (barjesteh et al., ) , infectious laryngotrcaheitis virus (calnek et al., ) and reovirus (swanson et al., ) infections. avian macrophages are also have been shown to elicit antiviral response depending on nitric oxide (no) production against avian influenza virus , infectious laryngotracheitis virus (haddadi et al., ) and marek's disease virus (xing and schat, ) . following avian influenza virus infection, macrophages secrete pro-inflammatory cytokines and chemokines, facilitating the development of immune response, which further reduce the replication and spread of avian influenza virus in the host (herold et al., ; peschke et al., ; seo et al., ) . the effect of macrophage depletion on avian influenza virus replication in chickens has not been studied. kim et al. reported that the depletion of macrophages using clodronate liposomes in pigs, results % mortality after infection with h n influenza virus while zero mortality in infected control pigs (kim et al., ) . similarly, tate et al. have shown that the depletion of macrophages in a mouse model of influenza virus infection leads to severe viral pneumonia in h n influenza virus infected mice (tate et al., ) . we hypothesized that lpaiv infection will increase the staining intensity of kul + cells, which include macrophages, monocytes and interdigitating cells (mast et al., ) in respiratory and gastrointestinal tracts. we also hypothesized that the decrease of the staining intensity of kul marker + cells following intra-abdominal administration of clodronate liposomes will augment replication of low pathogenic avian influenza virus (lpaiv) in respiratory and intestinal tracts of chickens. the veterinary science animal care committee (vsacc) and health science animal care committee (hsacc) have approved the use of specific pathogen free (spf) eggs, embryos, and chickens used in all our experimental procedures. the eggs were purchased from the canadian food inspection agency (cfia), ottawa, canada and incubated ( - % relative humidity and . - . °c temperature depending on the stage of the incubation) at health research innovation center (hric), university of calgary. a lpaiv, a/duck/czech/ (h n ), propagated in the embryonated chicken eggs, was used in the studies. the titer of h n was determined by plaque assay using madin-darby canine kidney (mdck) cells. clodronate liposomes (foundation clodronate liposomes, amsterdam, netherland) were used for decreasing the staining intensity of kul + cells in chickens as described earlier (kameka et al., a) and phosphate buffered saline (pbs) liposomes were used as a control. six day-old chickens (n = ) were infected with . × pfu of h n lpaiv per chicken intra-nasally with uninfected controls (n = ). at days post-infection, the chickens were euthanized and the trachea, lungs and duodenum were collected. the collected samples were preserved in optimum cutting temperature (oct, vwr international, mississauga on, canada) and immunofluorescent assay was performed to quantify the staining intensity of kul + cells as described earlier (abdul-cader et al., ). we delivered . ml ( mg/ml) of clodronate liposomes (n = ) or pbs liposomes (n = ) intra-abdominally to each day-old chicken. at day , subsets of chickens (clodronate liposome, n = and pbs liposome, n = treated groups) of both groups were infected with . × pfu of h n lpaiv per chicken intra-nasally with uninfected controls (n = per group). at days post-infection, the chickens were euthanized and trachea, lung and duodenum were collected. previously, it has been shown that lower gastrointestinal tract is also a site of lpaiv replication (slemons and swayne, ) as has been the duodenum (wang et al., ) . from the tissue samples collected, rna extraction was done to quantify the genome loads of h n lpaiv. a portion of samples were preserved in oct and immunofluorescent assay was performed to quantify the staining intensity of kul + cells as described earlier (abdul-cader et al., ). for the quantification of staining intensity of kul + cells in the tissues, five areas with highest dylight ® fluorescent signals and corresponding nuclear stained , -diamidino- -phenylindole (dapi) areas were captured under x magnification from each tissue section. then, these images were subjected to fluorescent intensity quantification using image-j software (national institute of health, bethesda, maryland, usa). the resultant fluorescent intensities for dylight ® positive signals were expressed relative to the total nuclear stained areas as a percentage. to identify group differences, we analyzed all the data (graphpad prism software , la jolla, ca, usa) using non-parametric test, mann-whitney u test due to low number of animals per group. before being analyzed each set of data, the outlier test was conducted using the grubbs' test (graphpad software inc., la jolla, ca, usa). the differences between groups were considered significant at p ≤ . . first, we questioned whether h n lpaiv infection contributes to the increase of staining intensity of kul + cells including macrophage populations in trachea, lungs and duodenum. when we infected day-old chickens with h n lpaiv intra-nasally, we found that, at days post-infection, h n lpaiv infection significantly increased the staining intensity of kul + cells in trachea (fig. a , p < . ), lungs (fig. b , p < . ) and duodenum (fig. c , p < . ) compared to the uninfected control chickens. in trachea, the staining intensity of kul + cells distributed mainly mucosal and submucosal areas with some distribution between cartilages and serosal surface. in the lungs and duodenum, the kul + cells mainly distributed throughout the lung parenchyma and mucosa respectively (fig. a-c) . increased recruitment of kul + cells following viral infections other than avian influenza virus in chickens are shown (abdul-careem et al., ; abdul-careem et al., ; fulton et al., ; kameka et al., b; nakamura et al., ) . similarly, it has been shown that avian influenza virus infection also recruit kul + cells in chickens (cornelissen et al., ; rebel et al., ) . the recruitment of kul + cells following viral infections may potentially be due to the availability of pathogen associated molecular patterns (pamps) of h n lpaiv during the infection, increasing the recruitment of these cells. it has been previously shown that toll-like receptor (tlr) of the innate immune system recognizes pamps of influenza virus and fig. . h n lpaiv infection increases the staining intensity of kul + cells in trachea, lungs and duodenum of chicken. at days of age, a group of chickens (n = ) were infected with h n lpaiv intra-nasally while another group was left as uninfected controls (n = ). at days post-infection, trachea, lung and duodenum were collected for immunofluorescent assay to quantify the staining intensity of kul + cells. representative images from trachea (a), lung (b) and duodenum (b) are shown. the mann-whitney u test was performed to identify group differences and the differences were considered significant at p < . . fig. . clodronate liposomes decrease the staining intensity of kul + cells in trachea and duodenum of chicken. at days of age, the chickens were treated with clodronate liposomes (n = ) or pbs liposomes (n = ) intra-abdominally. at days post-treatment, trachea, lung and duodenum were collected. the immunofluorescent assay was performed for the quantification of the staining intensity of kul + cells. representative images obtained following the immunofluorescent assay for the quantification of the staining intensity of kul + cells in trachea (a), lung (b) and duodenum (c) are shown along with quantitative data. the mann-whitney u test was performed to identify group differences and the differences were considered significant at p < . . activate mouse mononuclear cells (lund et al., ) . furthermore, lee et al. have shown that activation of tlr induces differentiation of myeloid-derived suppressor cells into one of the kul + cells, macrophages (lee et al., ) . we observed that intra-abdominally delivered clodronate liposomes in day-old chickens significantly reduced the staining intensity of kul + cell populations in trachea and duodenum but not in lung at days post-treatment when compared to the controls that received pbs liposomes ( fig. a and c, p < . ; fig. b , p > . ). in previous studies, it has been found that treatment of clodronate liposomes significantly decrease staining intensity of cv -chnl- . antibody specific cells populations in chickens (jeurissen et al., ) . cv -chnl- . antibody specific cells include macrophages, monocytes and interdigitating cells which are essentially similar to kul + cells. in agreement with this finding, we observed decreased staining intensity of kul + cells in this study in trachea and duodenum following intra-abdominal clodronate liposome treatment. since we observed that the clodronate liposomes significantly decreased the staining intensity of kul + cells in trachea and duodenum, then we investigated to see whether this reduction in the staining intensity of kul + cells influences the h n lpaiv infection. we found that the clodronate liposome-mediated reduction of the staining intensity of kul + cells was associated with no change of h n lpaiv infection at days post-infection ( days post-treatment) in trachea (fig. a , p > . ), lung (fig. b , p > . ) and duodenum (fig. c , p > . ). kul + cells including macrophages are important immune cells that can be beneficial in innate antiviral response against h n lpaiv infection via several mechanisms. first, it is known that macrophages can lead to the production of a number of antiviral cytokines including ifnγ (barjesteh et al., ; dimier et al., ) , il- β (lavric et al., ) and release of reactive nitrogen species such as no (setta et al., ) . previously, it has been shown that no originated from avian macrophages are inhibiting infectious laryngotracheitis virus (haddadi et al., ) , reo virus (pertile et al., ) and marek's disease virus (xing and schat, ) infections in vitro. we have previously observed that inhibition of h n lpaiv replication is attributable to no produced from kul + cells . second, tracheal fig. . reduction of the staining intensity of kul + cells with clodronate liposomes did not change h n infection in trachea, lungs and duodenum. at days of age, the chickens were treated with clodronate liposomes (n = ) or pbs liposomes (n = ) intra-abdominally. after h, all chickens were infected with h n lpaiv intra-nasally. at days post-infection, trachea (a), lung (b) and duodenum (c) were collected. real-time pcr assay was performed for h n lpaiv genome quantification. the mann-whitney u test was performed to identify group differences and the differences were considered significant at p < . . macrophages can play a role in clearing h n lpaiv infected cells as has been shown in mouse model of influenza infection (hashimoto et al., ) . however, the lack of significant differences in h n lpaiv genome loads between clodronate liposome treated and controls could be two-fold. first, immune cell functions are redundant (hamada et al., ) and it is possible that even the kul + cell staining was decreased following clodronate liposome treatment, the functions of these cells may have been compensated by other intact immune cells in chicken. second, it is possible that the number of animals used per group (n = ) may not have provided adequate power in our experiments to show differences in h n lpaiv genome loads in all the examined tissues in chickens although number of animals used are adequate for differentiating of kul + cells in trachea and duodenum. although our investigations led to generation of significant new knowledge, there were some limitations. first, the monoclonal antibody used for the quantification of macrophages detects kul mannose receptor and these receptors are also expressed by monocytes and dendritic cells of chicken (mast et al., ) as such it is possible that the quantified macrophages may also include monocytes and dendritic cells. however, only macrophages and their precursor, blood monocyte will uptake the liposomes; since it has been shown that liposomes of the size > nm will not be internalized by non-phagocytic cells and other immune cells such as granulocytes (claassen et al., ) . dendritic cells are also unaffected upon administration of clodronate encapsulated-liposomes (van rooijen and hendrikx, ). however, we believe that kul cell staining alone is not adequate for characterizing cells reduced by clodronate liposomes and further immune markers should have been included in the experiments. second, we did not observe that intra-abdominal clodronate liposome treatment completely decrease the staining intensity of kul + cells. third, in our experiments we used a lpaiv strain h n which does not lead to clinical signs due to the fact that it establishes low grade infection. consequently, we resorted to real-time pcr technique to quantify this low grade h n lpaiv infection in the observed tissues, which is more sensitive than the immunofluorescent assay (landry et al., ; perosa et al., ) , although immunofluorescent assay would have given more meaningful data relevant to h n lpaiv infection in the observed respiratory and gastrointestinal tissues. in conclusion, we found that h n lpaiv infection contributes to increase of the staining intensity of kul + cell populations in trachea, lungs and duodenum of chickens. although, we observed that intra-abdominal treatment of clodronate liposomes reduced the staining intensity of kul + cells in trachea and duodenum of chickens, the decrease of the staining intensity of kul + cells did not alter the h n lpaiv genome load in examined tissues. further investigations are warranted to investigate other roles of kul + cells in the host responses elicited against h n lpaiv infection. the authors declare that there is no conflict of interest. toll-like receptor (tlr) signalling-mediated antiviral response against avian influenza virus infection correlates with macrophage recruitment and nitric oxide production host responses in the bursa of fabricius of chickens infected with virulent marek's disease virus induction of innate host responses in the lungs of chickens following infection with a very virulent strain of marek's disease virus infectious bronchitis corona virus establishes productive infection in avian macrophages interfering with selected antimicrobial functions macrophage cytokines: involvement in immunity and infectious diseases tlr ligands induce antiviral responses in chicken macrophages infection of macrophages by a lymphotropic herpesvirus: a new tropism for marek's disease virus effects of alveolar macrophage depletion on liposomal vaccine protection against respiratory syncytial virus (rsv) in vitro infection studies with infectious laryngotracheitis virus marek's disease virus infection of phagocytes: a de novo in vitro infection model a new method for removal of mononuclear phagocytes from heterogeneous cell populations in vitro, using the liposomemediated macrophage 'suicide' technique differences in highly pathogenic avian influenza viral pathogenesis and associated early inflammatory response in chickens and ducks inhibition of eimeria tenella development in vitro mediated by chicken macrophages and fibroblasts treated with chicken cell supernatants with ifn-gamma activity cellular response of the respiratory tract of chickens to infection with massachusetts and australian t infectious bronchitis viruses induction of toll-like receptor signaling in avian macrophages inhibits infectious laryngotracheitis virus replication in a nitric oxide dependent way multiple redundant effector mechanisms of cd + t cells protect against influenza infection evidence for phagocytosis of influenza virus-infected, apoptotic cells by neutrophils and macrophages in mice alveolar epithelial cells direct monocyte transepithelial migration upon influenza virus infection: impact of chemokines and adhesion molecules inadequate anti-polysaccharide antibody responses in the chicken clodronate treatment significantly depletes macrophages in chickens induction of innate immune response following infectious bronchitis corona virus infection in the respiratory tract of chickens alveolar macrophages are indispensable for controlling influenza viruses in lungs of pigs real-time pcr compared to binax now and cytospin-immunofluorescence for detection of influenza in hospitalized patients mycoplasma synoviae lipoprotein mspb, the n-terminal part of vlha haemagglutinin, induces secretion of nitric oxide, il- and il- beta in chicken macrophages resiquimod, a tlr / agonist, promotes differentiation of myeloid-derived suppressor cells into macrophages and dendritic cells depletion of alveolar macrophages exerts protective effects in pulmonary tuberculosis in mice recognition of single-stranded rna viruses by toll-like receptor some recent advances on the study and understanding of the functional design of the avian lung: morphological and morphometric perspectives characterisation of chicken monocytes, macrophages and interdigitating cells by the monoclonal antibody kul proliferation of lung macrophages in acute fatal viral infections in chickens comparison of the direct fluorescence assay and real-time polymerase chain reaction for the detection of influenza virus a and b in immunocompromised patients an antiviral effect of nitric oxide: inhibition of reovirus replication role of macrophage cytokines in influenza a virus infections highly pathogenic or low pathogenic avian influenza virus subtype h n infection in chicken lungs: small differences in general acute responses intravenous treatment with liposome-encapsulated dichloromethylene bisphosphonate (cl mbp) suppresses nitric oxide production and reduces genetic resistance to marek's disease roles of macrophages in measles virus infection of genetically modified mice no apoptotic deaths and different levels of inductions of inflammatory cytokines in alveolar macrophages infected with influenza viruses immune dynamics following infection of avian macrophages and epithelial cells with typhoidal and non-typhoidal salmonella enterica serovars; bacterial invasion and persistence, nitric oxide and oxygen production, differential host gene expression, nf-kappab signalling and cell cytotoxicity replication of a waterfowl-origin influenza virus in the kidney and intestine of chickens restricted growth of avirulent avian reovirus strain in macrophage derived hd cells critical role of airway macrophages in modulating disease severity during influenza virus infection of mice liposomes for specific depletion of macrophages from organs and tissues apoptosis of macrophages induced by liposome-mediated intracellular delivery of clodronate and propamidine cytokine expression in three chicken host systems infected with h n influenza viruses with different pathogenicities inhibitory effects of nitric oxide and gamma interferon on in vitro and in vivo replication of marek's disease virus we acknowledge funding from margaret gunn endowment for animal research (grant #, , university of calgary). we acknowledge the routine animal management by the staff at prion-virology animal facility of the university of calgary. key: cord- -k vdqlgs authors: eisenberg, ronald l. title: pneumonia date: - - journal: what radiology residents need to know: chest radiology doi: . / - - - - _ sha: doc_id: cord_uid: k vdqlgs this chapter describes the imaging patterns of pneumonia (lobar, lobular, interstitial, round) and its complications (abscess, empyema, pneumatocele); bacterial, fungal, and viral infections; and the many manifestations of pulmonary tuberculosis. electronic supplementary material : the online version of this chapter ( . / - - - - _ ) contains supplementary material, which is available to authorized users. • any pneumonia developing in a patient at least hours after being hospitalized • second most common nosocomial infection (after urinary tract infections) • unlike community-acquired pneumonia, hap is usually caused by a bacterial infection, rather than a virus • high morbidity and mortality rates and the primary cause of death in intensive care units • any pneumonia developing in a patient at least - hours after endotracheal tube intubation • typically affecting critically ill patients in an intensive care unit, vap develops in up to % of ventilated individuals and is associated with a mortality rate of up to % lobar pneumonia • homogeneous consolidation of all or a substantial percentage of a single lobe • sharply marginated by one or more fissures ( [ ] • silhouette sign when the consolidation is adjacent to the heart, aorta, or hemidiaphragm ( fig. • foreign material entering into the tracheobronchial tree secondary to gastroesophageal reflux, altered mental status (drug overdose, anesthesia), or neurologic disorder (stroke, traumatic brain injury) • often develops in intubated patients, despite the presence of an inflatable cuff • usually occurs in the most dependent portions of the lung ○ supine -superior and posterior basal segments of the lower lobes or posterior segment of the upper lobes ( fig. . ) ○ upright -lower lobes (typically on the right, because the right main bronchus runs more vertically and is wider) • rapid appearance of air-space consolidation, especially in bedridden patients. • noninfectious aspiration -usually clearance in less than week ○ mendelson syndrome -large-volume aspiration of gastric acid, which produces a chemical pneumonitis and acute lung injury (even ards) and a diffuse radiographic pattern simulating pulmonary edema ( • radiographic clearance of pneumonia usually lags well behind clinical improvement • follow-up chest radiographs are recommended in approximately - weeks to ensure complete resolution of the consolidation and to assess persistent abnormality of the lung parenchyma (scarring, bronchiectasis) • failure of a pneumonia to resolve by weeks suggests an inaccurate diagnosis or an endobronchial obstruction as a cause of postobstructive pneumonia ( fig. . ) • especially in patients with smoking history or over age , ct should be considered to exclude an underlying bronchial lesion (see fig. e . ) • irregular infectious cavity containing necrotic debris or fluid. • often an air-fluid level, which usually has the same extent on both frontal and lateral views thicker, often irregular wall disparity in length of air-fluid levels air-fluid levels of relatively equal lengths split pleura sign on ct empyema necessitans (fig. . ) • chronic empyema draining via a sinus tract into the subcutaneous tissues of the chest wall, most commonly related to tuberculosis or fungal infection (actinomycosis, aspergillosis, blastomycosis, mucormycosis) • loculated pleural fluid collection or mass with associated rib destruction and often bubbles of loculated gas in soft tissues • gram-negative bacterial pneumonia that is most common in debilitated middle-aged and older men with alcoholism (about two-thirds of cases); high mortality rate • tends to form a voluminous exudate that produces a homogeneous parenchymal consolidation containing an air bronchogram • lobar enlargement (especially the right upper) with the characteristic bulging fissure sign (fig. . ) ○ bulging fissure sign also in haemophilus influenzae pneumonia (predominantly in compromised hosts, such as chronic pulmonary disease, immune deficiency, alcoholism, diabetes) (see fig. e . ) • most frequently result from infectious particles reaching the lung from an infected heart valve (especially the tricuspid), intravenous catheter, or injected debris • persons at risk include drug abusers, immunocompromised patients, individuals with septal defects, and those with indwelling venous catheters, pacemakers, or prosthetic heart valves • initially, multiple ill-defined round or wedge-shaped opacities with a swirling pattern that are usually peripheral and tend to involve the lower lobes (starry night sign -mimicking the brush strokes in van gogh's painting of that name) • cavitary pulmonary nodules tend to develop rapidly ( - days) • transient, rapidly changing, migrating, nonsegmental areas of parenchymal consolidation, which are associated with blood eosinophilia and minimal (or no) pulmonary symptoms • bilateral patchy consolidations with ill-defined margins that are predominantly located in the periphery of the lung • may produce single or multiple air-space nodules with surrounding ground-glass opacities (fig. . ) • unlike chronic eosinophilic pneumonia (see below), the transient air-space abnormalities resolve in some areas and reappear in others over days • loeffler's syndrome (also known as simple pulmonary eosinophilia) is applied to idiopathic cases; a similar imaging pattern can occur in response to parasitic infection or be drug-induced chronic eosinophilic pneumonia (fig. . ; see fig. e . ) • classic appearance of multifocal areas of consolidation in both lungs, especially the upper lobes, reflecting inflammatory eosinophils filling alveoli and infiltrating the interstitium • characteristic peripheral predominance ("reverse pulmonary edema pattern") • rapid response to steroid therapy (clinical improvement within hours, radiographic clearing within a few days) fig. . loeffler's syndrome. peripheral air-space nodule with surrounding ground-glass opacity in the right lower lobe (arrow). follow-up study showed that the nodule had disappeared [ ] fungal pneumonia aspergillosis • common fungus found in soil, on plants, and in decaying matter, as well as in household dust and building materials, which does not harm persons with normal immune systems and no allergic hypersensitivity • invasive aspergillosis (most aggressive form) is essentially limited to debilitated patients, diabetics, and neutropenic individuals with severely compromised immune systems (organ or bone marrow transplants, high-dose steroids or chemotherapy, lymphoma, leukemia) • central mass within a cavity in invasive aspergillosis is almost always necrotic lung (fig. . ; see figs. e . -e . ) • ct halo sign -early finding of a zone of ground-glass opacity (usually related to hemorrhage) surrounding a nodule or mass is strongly suggestive of invasive aspergillosis in an immunocompromised patient (fig. . ; see fig. e . ) • aspergilloma -solid homogeneous, rounded, mobile mycetoma that develops in a pre-existing cyst or cavity (primarily upper lobe) in a patient with underlying lung disease and is separated from its wall by a crescentic air space (air crescent sign) (see fig. e . ) • histoplasmosis -central united states; nodules often calcify ( fig. . ) • coccidioidomycosis -southwestern united states (also northern mexico and central and south america) • actinomycosis, nocardia -pleural effusion and extension to the chest wall are common (may develop empyema) (see fig. e . ) • candidiasis, aspergillosis, sporotrichosis, and mucormycosisessentially limited to debilitated patients and those with underlying diseases (diabetes mellitus, lymphoma, leukemia) (see fig. e . ) • caused by a yeast-like fungus and almost exclusively seen in immunosuppressed patients (especially aids, lymphoproliferative diseases, or renal transplants) • initially, bilateral diffuse interstitial opacities spreading outward from the hila • if untreated, this soon progresses to a homogeneous diffuse alveolar consolidation that may simulate pulmonary edema • thin-walled, air-filled lung cysts (especially apical and subpleural) occur in about % of patients and may cause a pneumothorax • thick-walled cavities usually indicate superinfection • hilar adenopathy and significant pleural effusions are rare (their presence should raise the possibility of an alternate diagnosis) • although a clinical diagnosis, may appear on chest radiographs as bilateral hilar enlargement due to lymphadenopathy (see fig. e . ) • important to look for medial displacement of gas within the stomach and splenic flexure caused by splenomegaly varicella (chickenpox) pneumonia (fig. . ; see fig. e . ) • diffuse distribution of small ( - mm), poorly defined nodules, which may coalesce to produce extensive bilateral fluffy infiltrates that tend to develop near the hilum and lung bases • healed varicella pneumonia classically appears as tiny military calcifications scattered widely throughout both lungs (develops several years after the pulmonary infection) • no calcification of hilar lymph nodes (unlike histoplasmosis or tuberculosis, the two other major causes of diffuse pulmonary calcifications) • cytomegalovirus, which typically occurs in immunosuppressed individuals (especially after transplantation) (fig. . ) • respiratory syncytial virus in infants and young children (see fig. e . ) • although traditionally considered a disease of children and young adults, with the dramatic decrease in the prevalence of tuberculosis (especially in children and young adults), primary pulmonary disease can develop at any age • primary tuberculosis may affect any lobe, so that the diagnosis cannot be excluded because the infection is not in the upper lobe fig. . tree-in-bud pattern (cytomegalovirus). centrilobular groundglass opacities in addition to nodules and "tree-in-bud" opacities in a patient with chronic myelogenous leukemia who underwent bone marrow transplantation [ ] • "latent" tb refers to someone who has a positive tst with no history of tuberculous infection or imaging evidence of active or old disease (most often detected when undergoing routine screening for employment or school) • "inactive" tb refers to someone with imaging evidence of prior tuberculosis but no sign of active disease imaging • one or more foci of lobar or segmental air-space consolidation that is usually homogeneous, dense, and well defined (fig. . ) • if multiple, randomly distributed throughout the lungs • cavitation is infrequent. • characteristic apical pleural thickening and fibronodular appearance in one or both upper lungs (fig. . ; see fig. e . ) • ct -enlarged lymph nodes typically have a low-density center (due to caseous necrosis) with rim enhancement (reflecting granulomatous tissue); may demonstrate subtle cavitation that is not visible on chest radiographs (see fig. e and is almost diagnostic of postprimary tuberculosis • because an apical lesion may be obscured by overlying clavicle or ribs, an apical lordotic view is often of value • cavitation is common (about %) and characteristic of postprimary disease (figs. . and . ; see fig. e . ) • the presence of cavitation indicates that the disease is highly contagious, and this finding alone warrants putting the patient in respiratory isolation • air-fluid levels in cavities are uncommon and usually a manifestation of superinfection (see fig. e . ) • tuberculous cavities may result in endobronchial spread and the classic "tree-in-bud" pattern of centrilobular bronchial dilatation and filling by mucus, pus, or fluid, associated with a linear a b note the thickening of bronchial walls (white arrow) [ ] branching pattern that resembles a budding tree and is generally more pronounced in the lung periphery (see figs. e . -e . ) • other complications of cavitation include rupture into the pleural space (leading to empyema or bronchopleural fistula) and the development of a pseudoaneurysm of the pulmonary artery (rasmussen aneurysm) • pleural effusion and lymph node enlargement are rare in postprimary tuberculous disease (though common and sometimes the only finding in primary disease) • as the disease heals, fibrotic changes develop in the surrounding lung, which cause volume loss in the affected segment with displacement of the fissures and hilar structures (see fig. e . ) • ct -because the diagnosis of typical postprimary tuberculosis is generally evident on chest radiographs, ct is primarily used to assess the extent and nature of the disease (more sensitive for demonstrating cavitation and such complications as vascular erosion, rupture into the pleural space, and miliary and endobronchial spread) • hematogenous dissemination that usually occurs in patients with altered host resistance to the primary infection • almost invariably leads to a dramatic febrile response with night sweats and chills • there may be minimal symptoms in severely debilitated patients, especially elderly persons and those receiving steroids imaging • diffuse pattern of innumerable tiny ( - mm), discrete, relatively well-defined pulmonary nodules distributed uniformly throughout both lungs (fig. . ; see figs. e . and e . ) • ct -may detect the presence of diffuse lung involvement when corresponding chest radiographs are normal or show only minimal or limited disease clinical imaging: an atlas of differential diagnosis lower lobe-predominant diseases of the lung mosaic attenuation multiple cystlike lung lesions in the adult pulmonary tuberculosis: role of radiology in diagnosis and management eosinophilic lung diseases: a clinical, radiologic, and pathologic overview upper lobe-predominant diseases of the lung tree-in-bud pattern key: cord- -gsvswr v authors: hedblom, grant a.; reiland, holly a.; sylte, matthew j.; johnson, timothy j.; baumler, david j. title: segmented filamentous bacteria – metabolism meets immunity date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: gsvswr v segmented filamentous bacteria (sfb) are a group of host-adapted, commensal organisms that attach to the ileal epithelium of vertebrate and invertebrate hosts. a genetic relative of the genus clostridium, these morphologically unique bacteria display a replication and differentiation lifecycle initiated by epithelial tissue binding and filamentation. sfb intimately bind to the surface of absorptive intestinal epithelium without inducing an inflammatory response. rather, their presence impacts the generation of innate and differentiation of acquired immunity, which impact the clearance of extracellular bacterial or fungal pathogens in the gastrointestinal and respiratory tracts. sfb have recently garnered attention due to their role in promoting adaptive and innate immunity in mice and rats through the differentiation and maturation of th cells in the intestinal tract and production of immunoglobulin a (iga). sfb are the first commensal bacteria identified that impact the maturation and development of th cells in mice. recently, microbiome studies have revealed the presence of candidatus arthromitus (occasionally designated as candidatus savagella), a proposed candidate species of sfb, in higher proportions in higher-performing flocks as compared to matched lower-performing flocks, suggesting that sfb may serve to establish a healthy gut and protect commercial turkeys from pathogens resulting in morbidity and decreased performance. in this review we seek to describe the life cycle, host specificity, and genetic capabilities of sfb, such as bacterial metabolism, and how these factors influence the host immunity and microbiome. although the role of sfb to induce antigen-specific th cells in poultry is unknown, they may play an important role in modulating the immune response in the intestinal tract to promote resistance against some infectious diseases and promote food-safety. this review demonstrates the importance of studying and further characterizing commensal, host-specific bacteria in food-producing animals and their importance to animal health. the distal gastrointestinal tract of all animals is colonized by a diverse array of bacterial, fungal, and protozoan species. an animal host maintains a mutualistic relationship with its microbial inhabitants in which the microbes provide protection from pathogenic bacteria through competing for ecological niches and fostering a stable environment for the development of host immunity (bäckhed et al., ) . many vertebrate intestines (such as mice, rats, chickens, humans, and turkeys) harbor commensal organisms named segmented filamentous bacteria (sfb) that bind specifically to the host intestinal epithelium. these organisms are closely related to the genus clostridium, and appear as long, segmented filaments that bind tightly to the host epithelium via a specialized structure (chase and erlandsen, ) . these bacteria were initially detected through microscopic examination of the gastrointestinal epithelium of mice (davis and savage, ) . sfb drew the attention of researchers due to their unique morphology, life cycle and binding location (schnupf et al., ) . since their discovery, a large body of research has been generated to characterize these bacteria and understand their role in the host-microbiome relationship. through examining mouse and rat models, it has been revealed that these bacteria play an important role in adaptive and innate immunity of the host. segmented filamentous bacteria are gram-positive, sporeforming bacteria that possess the capability to develop into long filaments, which are divided via the production of transverse septa (ericsson et al., ) . these bacteria exist in a developmental or vegetative form, which are characterized by the presence of intrasegmental bodies and spores, respectively (figure ) . sfb produce intrasegmental bodies, which can appear as either a spore or a holdfast. the distinction between these morphologies indicates that the holdfast form serves as the vegetative form, while the spore serves as the dormant form (chase and erlandsen, ) . segmented filamentous bacteria selectively attach to the ileal epithelium of the host species via the production of rounded, nipple-like projections called holdfasts. the holdfast serves as an anchoring mechanism and point of filament elongation, attaching to host enterocytes in the mucous membrane of the figure | gram stain of candidatus arthromitus from turkey ileum containing intrasegmental bodies ( , × magnification) (reiland, ) . epithelium, without penetrating the host cell wall (sanford, ) . this structure leads to the displacement and destruction of the intestinal microvilli surrounding the point of attachment (snellen and savage, ) , and leads to alterations in the electron density of both the host cell plasma membrane and apical cytoplasm (ericsson et al., ) . once attached, actin polymerization occurs directly underneath the holdfast structure and creates a pedestal-like formation similar to the adherence mechanism of salmonella enterica serovar typhimurium (jepson et al., ; ericsson et al., ) . though this attachment mechanism resembles that of pathogenic bacteria, it does not damage the host cell and causes no inflammatory response in the lamina propria (caselli et al., ) . once attached to the host epithelium, the filament begins to extend from the distal end, releasing additional holdfasts and spores from the maturing filament (chase and erlandsen, ) . the life cycle of an sfb filament is assumed to be about - days based on the rapid shedding of the intestinal epithelium of rodents, and longitudinal studies have shown that sfb appear in juvenile mice that are around days in age (davis and savage, ) . at this stage of development, sfb proliferate to become a dominant gut microbe and then recede in mature vertebrates to lower levels. during the early stages of colonization, sfb are transiently colonized with rod-shaped bacteria (schnupf et al., ) . it is believed that the spread of sfb then occurs via vertical transmission of spores from parents to offspring, as these bacteria are widely considered to be obligate anaerobes and have also appeared in the intestinal tissue of weaning mice (schnupf et al., ) . colonization occurs in mouse and rat hosts at the onset of the weaning process and has been found to be the same in arbitrary human microbiome studies (ericsson et al., ) . studies performed on human subjects ages months to years old revealed that % of individuals carry sfb in their gut from ages - months, % carry sfb from ages - months, and only . % carry sfb from ages - (yin et al., ) . the age-related drop in sfb intestinal carriage may be pharmaceutically reversed. for example, transient feeding mice rapamycin enhanced their lifespan, while dramatically increasing the prevalence of sfb in the small intestine (bitto et al., ) . in chickens, sfb colonization peaked at approximately weeks of age, and decreased as they aged to weeks of age. the decrease was inversely proportional to the amount of intestinal iga present (liao et al., ) . it is unknown whether increasing the prevalence of sfb in the intestinal tracts of adults is beneficial, or may result in autoimmunity (e.g., rheumatoid arthritis) (wu et al., ) . segmented filamentous bacteria spores germinate in the host's gut to produce teardrop-shaped, single-celled bacteria referred to as intracellular offspring (schnupf et al., ) . amongst intestinal commensals and symbionts, sfb are unique because they penetrate the intestinal mucus layer and intimately associate with host cells without invading the host (chase and erlandsen, ; sanford, ) . it is assumed that the intracellular offspring use flagella to reach the apical surface of polarized epithelium (kuwahara et al., ) . though cellular flagella have not been observed microscopically, an analysis of a sfb genome revealed a full set of chemotaxis and flagellin biosynthesis genes, strongly suggesting the presence of flagella in the early stages of spore maturation (kuwahara et al., ) . intracellular offspring attach to absorptive epithelial cells via their holdfast and induce condensed actin rearrangements underneath the point of attachment while displacing some of the neighboring microvilli structures (chase and erlandsen, ) . before luminal attachment, the nucleoid region of the intracellular offspring appears condensed, suggesting reduced amounts of transcription. however, when the intracellular offspring attaches to the host, the nucleoid region decondenses and allows for genomic transcription (chase and erlandsen, ) . holdfast attachment to enterocytes in the terminal ilea causes the intracellular offspring to increase in size, reaching up to µm in length before bacterial division commences through transverse septum formation (chase and erlandsen, ) . filaments continue to grow and divide from their distal end, reaching their maximum length of around - µm (martin et al., ; schnupf et al., ) . once the filament reaches its maximum length, a second round of symmetric division begins from the distal end to divide each original segment in half into secondary undifferentiated cells. these secondary cells range in length from to . µm, forming segments containing - cells (chase and erlandsen, ) . after elongation, the filament will often separate from the holdfast segment and enter into the ileum. these secondary segments then undergo differentiation form a mother cell and a daughter cell (klaasen et al., ) . differentiation of these filaments appears to be more pronounced in the presence of slightly aerobic conditions, when oxygen concentrations range from to . % environmental oxygen (schupf et al., ) . once differentiated, the mother cell engulfs and houses the daughter cell, where the daughter cell undergoes division into two intracellular offspring (klaasen et al., ) . the intracellular offspring contained within the mother cell are then subject to two fates, dispersal from the filament or sporulation (davis and savage, ; klaasen et al., ) . if favorable growth conditions are present, the septa that separate individual mother cells are degraded to form a tube in which the intracellular offspring are dispersed into the host's intestinal tract. these released offspring are then allowed to colonize additional host tissue and undergo filamentation and differentiation, thus completing the sfb lifecycle (martin et al., ; schnupf et al., ) . when an unfavorable or hostile environment is presented, the two intracellular offspring produce a single spore coat that covers both of the cells. once coated with the layer of peptidoglycan, the spore matures into a complete endospore inside of the mother cell, and is released from the filament (martin et al., ; schnupf et al., ) . these spores lack the ability to colonize the host until favorable environmental conditions, such as appropriate concentrations of oxygen are presented, and shed in the host's feces. once shed, the spores can be transmitted to another host via horizontal transmission (davis and savage, ) . though it was previously proposed that the entire sfb lifecycle occurred while attached to the host tissue (chase and erlandsen, ) , filaments containing intracellular offspring have not been observed in published tem and sem images of filaments attached to enterocytes (caselli et al., ) . alternatively, filaments containing differentiated intracellular offspring only appeared as detached and free-floating, indicating that maturation and differentiation of segments occur independently from host tissues. these filaments are separated from the holdfast segment, which then penetrates the epithelium until it undergoes endocytosis, phagocytosis, or transcytosis (caselli et al., ) . the ingestion of the holdfast segment presents a great number of bacterial antigens to antigen presenting cells and lymphocytes contained within the ileal epithelium (caselli et al., ) . in rare cases, a small number of segments may remain attached to the holdfast, but these segments often exhibit irregular morphologies and present enlarged intrasegmental junctions (caselli et al., ) . the genome of a rat-isolated sfb and a number of mice sfb isolates have recently been sequenced and published (kuwahara et al., ; prakash et al., ; sczesnak et al., ; pamp et al., ) . these sfb genomes are highly similar but do contain several species-specific genes of unknown function that may be involved in the species-specificity of sfb colonization (prakash et al., ) . all sequenced sfb have displayed a small genome size of . - . mbp, low g+c content ( . %), and around , - , protein encoding genes (prakash et al., ) . sfb possess a highly reduced genome similar to their genetic relatives within the genus clostridium (ericsson et al., ) . the biosynthetic pathways of most amino acids, vitamins and cofactors (such as b , b , and b , pyridoxine, nicotinamide, pantothenate, and biotin) are incomplete or absent altogether in sfb genomes (kuwahara et al., ; sczesnak et al., ; pamp et al., ) . sfb are also unable to synthesize nucleotides independently; instead they utilize alternative pathways that rely on the uptake of nucleotide bases (prakash et al., ) . to obtain nucleotides, amino acids, and peptides from the environment, sfb genomes contains genes encoding two extracellular nucleases as well as a list of proteases and peptidases, of which are membrane associated and to that are thought to be secreted (kuwahara et al., ; sczesnak et al., ) . in addition, sfb genomes contain numerous open reading frames (orfs) thought to encode a large number of transporters and permeases for small molecules and ions (such as amino acids, oligopeptide, dipeptides, manganese, zinc, iron, and phosphate) compared to other organisms with small genomes (sczesnak et al., ) . a particularly strong requirement for iron uptake was noted by sczesnak et al., since six different orfs for iron transporters are found in the mouse genome as well as three orfs for ferric iron regulator family proteins (sczesnak et al., ) . sfb also have several orfs for phosphotransferase systems predicted for uptake of sugars such as mannose, cellobiose, mannitol, and fructose (prakash et al., ; sczesnak et al., ) . finally, the sfb genomes contain genes for the non-oxidative pentose phosphate pathway and a complete glycolysis pathway to convert glucose to pyruvate, but are deficient for genes encoding almost all components of the krebs cycle, which is required for aerobic respiration (prakash et al., ; sczesnak et al., ) . however, sfb can tolerate small concentrations of oxygen and counteract oxidative stress, as sfb genomes contain genes predicted for two catalases, a peroxidase (rubrerythrin), and an arginase, which might limit nitric oxide production through catabolism of arginine (kuwahara et al., ; pamp et al., ) . these protective mechanisms are likely essential, given the replicative niche of sfb at the surface of the small intestinal epithelium where the oxygen tension is estimated to be around . % (he et al., ) . there are several factors that have been discovered about sfb that explain their auxotrophic nature. the genome of sfb isolated from a rat host (rat-yit) contains putative genes predicted to encode for proteases and for peptidases along with many other genes through to be involved with sporulation and germination (prakash et al., ) . peroxidase and catalase genes were also found, which explains the potential for sfb to exist in microaerophilic environments (prakash et al., ) . the genomes of sfb sequenced from mice and rat hosts revealed several clustered regularly interspaced palindromic repeat (crispr) loci, which serve as a prokaryotic defense mechanism, indicating that sfb genomes may have had exposure to invading dna throughout their evolutionary history (prakash et al., ) . flagellar, pilus, and chemotactic genes have been found in sfb genomes that suggest motility, which explains the organism's ability to penetrate the mucus layer lining of intestinal epithelial cells (prakash et al., ; schnupf et al., ) we have generated a representation of the metabolic and physiological predicted capabilities inferred from the genome contents of publicly sequenced genomes of sfbs (figure ). an extensive body of work has evidenced the presence of sfb in a large number of animal species, such as horses, cattle, pigs, turkeys, chickens and even humans (ericsson et al., ) . in nearly all of the mammalian species studied for the presence of sfb, the bacteria selectively colonize the ileum of the host, with the exception of fish species that lack a welldefined ileum (ericsson et al., ) . in poultry, sfb colonizes ileum (figure ) , the cecal tonsil or cecum (figure ; rahimi et al., ; bohorquez et al., ; liao et al., ) . attempts to colonize an animal species with sfb from another species have been unsuccessful. when germ-free mice and rats were inoculated with ileal homogenates containing sfb from both species, animals became colonized with sfb from their own species, indicating that sfb are host-specific and host selective (tannock et al., ) . host-specificity of sfb may be due to differences in the sequences of flagella genes flic and flicc , which show greater variability than flic and flic (chen et al., ) . it is not known with which host proteins the sfb flagella proteins interact to become adherent, however, sfb flagellar proteins may induce th cells by signaling through toll-like receptor (tlr ) in a subset of cd c hi cd b hi intestinal dendritic cells (uematsu and akira, ). for most animal species, holdfast cells have the capability to attach to goblet cells, m-cells, absorptive enterocytes, and cellular junctions of the ileal epithelium (meyerholtz et al., ) , whereas less is known for poultry. species to species variance exists in the preferred cell of attachment. for example, sfb in rats and figure | metabolic features of candidatus arthromitus through inference of genome contents from annotated genomes through use of rapid annotation using subsystem technology (reiland, ) . frontiers in microbiology | www.frontiersin.org pigs attach to follicle-associated epithelial cells (over peyer's patches) and absorptive ileal villi (tannock et al., ) . in mice and horse, attachment occurs primarily to follicle-associated epithelial cells and mainly to absorptive ileal villi for rabbits, cattle and canines . sfb are unique amongst intestinal commensals and symbionts because they penetrate the intestinal mucus layer and intimately associate with host cells, but do not invade the host (sanford, ) . the exact mechanism of host specificity remains unclear, but it is believed that initial binding of the holdfast segment to the host epithelium serves as a ligand-receptor interaction, triggering a response from the host (kuwahara et al., ) . sfb binding elicits actin polymerization and condensation at the point of attachment (chase and erlandsen, ) , suggesting a specific host response. host specificity of sfb suggests that these bacteria have coevolved with their hosts to promote the commensal relationship that exists between the two species. though initially considered to be common commensal members of the host microbiota, recent research suggests that sfb serve an important role in modulating host microbiome and immunity. sfb are unique amongst intestinal commensals and symbionts because they penetrate the intestinal mucus layer and intimately associate with host cells without invading the host (chase and erlandsen, ; lee et al., ; mortha et al., ; atarashi et al., ; bunker et al., ; farkas et al., ; furusawa et al., ; sadler et al., ) . the holdfast binding of sfb to the ileal mucosa does not elicit a strong inflammatory response (klaasen et al., ) . several interactions between the host and sfb have been investigated, indicating almost entirely positive associations between the sfb and the host, with rare exceptions. intestinal colonization of sfb in rainbow trout may produce a fatal disease called rainbow trout gastroenteritis (rtge), where sfb expand to large numbers in the intestinal tract and cause enterocytes to detach (del-pozo et al., ) . sfb were not always present near to all intestinal rtge lesions, suggesting that sfb affects intestinal barrier function and other bacteria may not be crucial for producing rtge lesions. experimental infection of susceptible rainbow trout stock with feces from rtge animals induced colonization and characteristic histopathological lesions containing sfb and an unidentified gram-negative coccus, suggesting that sfb, in part, may be the etiological cause of rtge (mccarthy et al., ) . recent advances have identified different methods to culture sfb in vitro, accelerating research to study the interaction of sfb with different members of the intestinal microbiota (ericsson et al., ; schupf et al., ) . a large amount of research has demonstrated the indispensable role that sfb play in the maturation of the host gut immune barrier, inducing both innate and adaptive immune responses (ivanov et al., ; sonnenberg et al., ; mortha et al., ; atarashi et al., ; bunker et al., ; farkas et al., ; furusawa et al., ; edelblum et al., ; schnupf et al., ) . immune modulation of sfb may extend beyond the intestinal tract in the blood, or to other mucosal barriers (mcaleer et al., ) . mice colonized with sfb were more resistant to sepsis secondary to experimental cecal ligation and puncture injury (cabrera-perez et al., ) . oral vancomycin treatment to mice diminished the number of intestinal grampositive bacteria (including sfb), which negatively impacted anti-fungal th immunity in the respiratory tract (mcaleer et al., ) . these data suggest that the composition of intestinal microbiota, especially sfb, is vital for impacting immunity to bacterial and fungal pathogens beyond the intestinal tract. segmented filamentous bacteria are best known for their ability to induce the differentiation of naïve cd + t cells to form antigen-specific th cd + cells (schnupf et al., ) in the terminal ileum of mice . ivanov et al. first demonstrated that conventionally raised b mice purchased form taconic farms were highly colonized with sfb; these bacteria were absent from conventional raised mice purchased from the jackson laboratory (ivanov et al., ) . introduction of sfb to b mice from the jackson laboratory induced il- a and il- production from intestinal cd + t cells, which became refractory to colitis induced by the intestinal pathogen citrobacter rodentium (ivanov et al., ) . il- is a cytokine that enhances production of antimicrobial peptides from intestinal epithelial cell and prevent bacterial pathogens from inducing attaching and effacing lesions (schupf et al., ) . sfb are not the only bacteria capable of inducing th cd + t cells in the intestinal tract of animals. virulent shiga-toxin producing e. coli (o ) and citrobacter rodentium induced a th response in the murine intestinal tract, and was dependent on bacterial adherence to host cells (atarashi et al., ) , as well as the commensal bifidobacterium adolescentis (tan et al., ) . th cells are a subset of cd + t cells that are distinguished by the expression of t cell receptor cd , nuclear transcription factor rar-related orphan receptor gamma t (rorγt) and production of interleukins il- a, il- f, il- , and il- (schnupf et al., ) . other immune cells present in the intestinal lamina propria are capable of secreting il- a and il- or express rorγt [e.g., innate lymphoid cells type (ilc ) and lymphoid tissue inducer-like cells (lti)], but these cells lack expression of cd (sonnenberg et al., ; mortha et al., ) . however, ilc and lti are not dependent on sfb for their induction. in the intestinal epithelium, il- a and il- f help to modulate neutrophil chemotaxis through producing cxcl chemokines via binding interactions with il- receptors il- ra and il- rc. il- a and il- f additionally aid in regulating the activation and differentiation of host neutrophils, and stimulate the production of host-defense peptides (schnupf et al., ) . systemic depletion of neutrophils in mice caused increased production of il- a and ileal sfb colonization (flannigan et al., ) . thus, neutrophil recruitment may lessen il- a and chemokine production, and serve as a negative feedback loop to limit sfb colonization. the regulation of these immunestimulatory compounds and cell types is essential in combatting intestinal colonization and infection from microorganisms. th cells provide colonization resistance to other pathogenic bacteria present at mucosal barriers, such as escherichia coli in the intestinal tract (zheng et al., ; edelblum et al., ) and respiratory fungi (mcaleer et al., ) . in newborn or germ-free mice, the presence of th cells in the lamina propria is rare, appearing only after colonization by microbes (gaboriau-routhiau et al., ) . the role of sfb in th cell production was initially demonstrated when mice were inoculated with mouse, rat, and human microbiota containing bacterial spores similar to that of the genus clostridium. only the experimental mice inoculated with a mouse-derived bacteria were shown to produce th cells in response to colonization. mice colonized with rat-and human-derived bacteria produced much less of a th response when compared to the mousederived microbiome treatment, indicating host-specific bacteria (such as sfb) as the causative agent of the immune response (gaboriau-routhiau et al., ; chung et al., ) . this association was also confirmed when s rrna sequencing was performed on the gut microbiome of mice presenting ileal th cells, revealing the presence of sfb (ivanov et al., ) . in experiments testing the reactivity of mouse lamina propria against a sfb expression library, two proteins of unknown function elicited a th cell response (yang et al., ) . it was predicted that these unknown proteins may serve as cell surface proteins, potentially elucidating the role that sfb attachment may serve in stimulating host immunity (yang et al., ) . proteins from sfb, secreted or bacterial-associated, are believed to interact with host cells and modulate immunity include adpribosyltransferases and a myosin-cross reactive antigen . the exact antigen presenting cell responsible for immune modulation by sbf is controversial, but it appears that sfb antigens presented to both intestinal macrophages or cd + intestinal dendritic cells (goto et al., ) are involved. because of the intimal relationship of sfb with intestinal epithelium, it is possible that metabolites from sfb may also impact the differentiation of th cells. intestinal macrophages, and not intestinal dendritic cells, appear to be vital for generating sfb-specific th responses in the murine ileum . analysis of the t cell receptor repertoire of th cells recognize peptide antigens produced by sfb (yang et al., ) . the addition of the th -indcing bacterial pathogen listeria monocytogenes failed to impact induction of th cells in sfb colonized mice (yang et al., ) , suggesting that the match of t-cell effector function with antigen specificity is driven by the type of bacteria that produce the antigen. th cell differentiation is additionally mediated through the production of serum amyloid a (saa) and reactive oxygen species (ros) produced in response to sfb binding. production of saa in the host epithelium is initiated by sfb binding and subsequent actin rearrangements, leading to a signal amplification via il- and ilc , both of which aid in th cell differentiation (schnupf et al., ) . saa also stimulates intestinal antigen presenting cells to secrete il- , which assists in th activation and survival (schnupf et al., ) , but il- also has as an antagonistic effect on development of th immunity (shih et al., ) . ros produced as a consequence of sfb binding to enterocytes helps to create a chemical environment that promotes th differentiation, as demonstrated in mice treated with ros scavenging compounds having lower amounts of th cells in vivo (atarashi et al., ) . sfb flagellar proteins may induce th cells by signaling through tlr in a subset of cd c hi cd b hi intestinal dendritic cells (uematsu and akira, ). the flagellar binding motifs that are targeted by tlr appear to be highly conserved in sfb and is nearly absent from other similar clostridium species, suggesting a specific role for sfb to modulate th immunity (prakash et al., ) . although sfb-induced th immunity may benefit the animal host, there are long-term consequences. sfb-induced th immunity is linked to the development of autoimmunity in susceptible breeds of mice (lee et al., ; yang et al., ; teng et al., ) by inducing differentiation and egress of t follicular cells from peyer's patches . it is unknown whether natural colonization by sfb in poultry is capable of promoting autoimmunity, but it must be considered if sfb, or its antigens are utilized as immunomodulators for food-producing animals. segmented filamentous bacteria mono-associated mice display rapid growth and development of peyer's patches, and sfb can also stimulate the formation of lymphoid follicles and tertiary lymphoid tissues in the host (lecuyer et al., ) . this activation of the host's intestinal immunity causes a drastic increase in fecal concentrations of secretory immunoglobulin a (siga), as the number and activity of iga secreting b-cells rises (klaasen et al., ) . germ-free mice monoassociated with sfb triggers the production of iga serum levels equivalent to that of specific pathogen free, sfb-negative mice (klaasen et al., ) . the expansion and stimulation of germinal centers present in peyer's patches is not entirely unique to sfb and has been seen to occur in other commensal bacteria such as morganella morganii and bacteroides distasonis, however, the response from sfb is much greater than these other organisms (schnupf et al., ) . both t-cell dependent (b- cell) and t-cell independent (b- cell) production of siga occurs in sfb mono-associated mice (schupf et al., ) . the amount of sfb-specific iga produced by the host in response to bacterial colonization is as high as . % of the total iga of the organism (talham et al., ) . iga transmitted by nursing mice to suckling pups has been shown to inhibit sfb colonization, and only after weaning do sfb populations begin to increase, coinciding with the time in which sfb colonization is typically recognized in mice (jiang et al., ) . the induction of siga by sfb may serve as a negative feedback mechanism to prevent overcolonization by sfb and dysbiosis in older animals (ohashi et al., ; liao et al., ) . light turkey syndrome (lts) is growing problem facing commercial turkey production in the united states. lts is a condition in which turkey flocks fail to meet their genetic potential weight, yielding birds that are - pounds below the industry standard for average flock weight (danzeisen et al., ) . birds affected by lts display symptoms similar to poult enteritis complex (pec), a disease in which birds experience weight loss, diarrhea, lethargy, and depression (mor et al., ) . however, lts is dissimilar to pec in that birds do not experience watery and pale intestinal contents or distended ceca, indicating differences between the syndromes (morishita et al., ) . the causative agent of pec is suspected to be microbial in nature, as inoculation of healthy birds with fecal homogenates derived from birds experiencing pec produced light weight poults when compared to un-inoculated birds, but a single responsible microbe has yet to be determined (mor et al., ) . similarly, inoculating healthy birds with fecal homogenates derived from turkeys with lts produced birds that were lighter than the control groups (mor et al., ) . the two conditions are not dependent on each other, as lts can occur in the absence of pec (danzeisen et al., ) . there exist a number of potential factors that may lead to the development of lts, such as colonization by pathogenic bacteria, viral infection, stunting of immune system development, inhibited nutrient absorption, and alterations to gut microbiome (danzeisen et al., ) . typically, lts/pes affects birds less than weeks of age (morishita et al., ) . a higher number of different pathogenic organisms are found in these younger birds than in birds aged - weeks. virus strains such as astrovirus, reovirus, and rotavirus types were detected in the host, and are not associated with poult mortality. coronavirus, which is commonly associated with mortality, was not detected in lts/pes poults (morishita et al., ) . in an attempt to understand the microbial basis of lts, danzeisen et al. performed s rrna microbiome analysis of low-performing and high-performing (based upon flock weights) flocks to determine the role of microbial succession in promoting digestive health samples were sequenced to discern the presence and abundance of dominant otus present in higher-performing flocks as compared to lowerperforming flocks (danzeisen et al., ) . after analysis it was determined that at the age of - weeks, higher-performing turkey flocks harbored significantly higher proportions of clostridium bartlettii and candidatus division arthromitus, a sfb (danzeisen et al., ) . previous studies regarding sfb colonization of poultry have identified sfb as a causative agent in intestinal disease (goodwin et al., ) , but sfb was later ruled out as a causative agent (sell et al., ) . also, sfb belong to several microbial taxa and are not considered a homogeneous group (thompson et al., ) . as demonstrated in mice and rat models, sfb have been proven to be potent stimulators of host immunity and ileal health. though the role of sfb (in particular candidatus arthromitus) in the digestive health of turkeys is not quite understood, the evidence provided by danzeisen et al. suggests that epithelial binding of these bacteria may promote early digestive health (danzeisen et al., ) . the potential for candidatus arthromitus to serve as an immunostimulatory probiotic makes it an organism of great interest to poultry researchers, as the turkey production industry is in need of alternatives to promote animal health in this age of restricted use of antibiotics in food-producing animals and increasing antimicrobial resistance. since their initial characterization in the s, sfb have transitioned from being considered an interesting and unique member of the gut microbiome with a unique morphology, to serving as a model organism to study immunomodulatory symbiotic bacteria and their effects on the host. the hostspecific binding mechanism employed by these bacteria to attach to ileal epithelium is similar to that of enteric pathogens. unlike enteric pathogens, sfb do not harm the host epithelium and instead live in a commensal, if not mutualistic manner. intimate binding to the host mucosal epithelium allows sfb to receive nutrients from the host, satisfying their auxotrophic requirements, while delivering antigens to the host. epithelial binding also initiates several immune responses from the host. as demonstrated in mice and rat models, sfb have been shown to stimulate the maturation of the host's th and iga responses, improving the ability of the host to protect against invading pathogens. additionally, sfb compete with other members of the intestinal microbiota by modulating access to nutrients and occupying available ecological niches. the fitness-bolstering effects produced by sfb in mouse models are well-understood, but little is known about the roles these bacteria play in the other vertebrate animals. it has been suggested through microbiome analyses of turkeys that sfb, specifically candidatus arthromitus, may provide a protective role in preventing the onset of the enteric condition lts, the cause of which is not well understood. the role of sfb in turkeys must be better elucidated to determine the beneficial effects these bacteria have in disease prevention and in ileal health. developing an understanding the role that commensal microorganisms, like sfb, play in the overall function of the gut microbiome will aid in our understanding the interplay between microbiome and host, providing insights into digestive health and the development of immunity. hr and gh contributed equally to the writing of the manuscript. ms, tj, and db also helped to write the manuscript. we would like to thank usda-afri award # - - and also the department of food science and nutrition and the college of food, agricultural and natural resource sciences at the university of minnesota for providing the funds needed to perform and complete this study. th cell induction by adhesion of microbes to intestinal epithelial cells host-bacterial mutualism in the human intestine transient rapamycin treatment can increase lifespan and healthspan in middle-aged mice ultrastructural development of the small intestinal mucosa in the embryo and turkey poult: a light and electron microscopy study innate and adaptive humoral responses coat 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syndrome in poults homeostatic il- receptor signaling limits th response through il- -mediated containment of commensal microbiota freeze-fracture study of the filametous, segmented microorganism attached to the murine small bowel cd (+) lymphoid tissue-inducer cells promote innate immunity in the gut segmented filamentous bacteria are potent stimuli of a physiologically normal state of the murine gut mucosal immune system identifying species of symbiont bacteria from the human gut that, alone, can induce intestinal th cells in mice host specificity of filamentous, segmented microorganisms adherent to the small bowel epithelium in mice and rats gut microbiota drive autoimmune arthritis by promoting differentiation and migration of peyer's patch t follicular helper cells immune-modulating gut symbionts are not "candidatus arthromitus immune responses of tlr + lamina propria dendritic cells in enterobacterial infection gut-residing segmented filamentous bacteria drive autoimmune arthritis via t helper cells focused specificity of intestinal th cells towards commensal bacterial antigens comparative analysis of the distribution of segmented filamentous bacteria in humans, mice and chickens interleukin mediates early host defense against attaching and effacing bacterial pathogens we thank judi stasko (usda ars national animal disease center) for technical assistance with scanning electron microscopy. key: cord- -g os m authors: martins-da-silva, andrea; telleria, erich loza; batista, michel; marchini, fabricio klerynton; traub-csekö, yara maria; tempone, antonio jorge title: identification of secreted proteins involved in nonspecific dsrna-mediated lutzomyia longipalpis ll cell antiviral response date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: g os m hematophagous insects transmit infectious diseases. sand flies are vectors of leishmaniasis, but can also transmit viruses. we have been studying immune responses of lutzomyia longipalpis, the main vector of visceral leishmaniasis in the americas. we identified a non-specific antiviral response in l. longipalpis ll embryonic cells when treated with non-specific double-stranded rnas (dsrnas). this response is reminiscent of interferon response in mammals. we are investigating putative effectors for this antiviral response. secreted molecules have been implicated in immune responses, including interferon-related responses. we conducted a mass spectrometry analysis of conditioned medium from ll cells and h after dsrna or mock treatment. we identified proteins. at h, proteins had an abundance equal or greater than -fold change, while the levels of proteins were reduced when compared to control cells. at the h time point, these numbers were and , respectively. the two most abundant secreted peptides at h in the dsrna-transfected group were phospholipid scramblase, an interferon-inducible protein that mediates antiviral activity, and forskolin-binding protein (fkbp), a member of the immunophilin family, which mediates the effect of immunosuppressive drugs. the transcription profile of most candidates did not follow the pattern of secreted protein abundance. insect-borne diseases afflict billions of people throughout the world. these illnesses are caused by different pathogens such as helminths, protists, bacteria, and viruses, which are the etiologic agents of diseases such as filariasis, leishmaniasis, babesiosis and dengue. among the most important insect vectors are mosquitos and sand flies [ , ] . phlebotomine sandflies from the genera phlebotomus and lutzomyia are the main vectors of leishmania in the old and new world, respectively. in the old world, these dipterans are also proven virus vectors of phlebovirus, vesiculovirus and orbivirus [ ] [ ] [ ] . despite the lack of evidence proving the vectorial capacity of new world sandflies in the transmission of viruses to humans, the occurrence of diverse virus species naturally infecting lutzomyia spp. populations has been demonstrated [ ] [ ] [ ] [ ] . among insect transmitted diseases, the arboviruses are among the most devastating infectious illnesses in our planet. chikungunya virus (chikv), west nile virus (wnv) and dengue virus (denv) are three of a large number of human pathogenic arthropod-borne viruses [ ] . also, the world recently witnessed the reemergence of zika virus (zikv), which is associated with severe human neurological pathologies in adults and fetuses [ ] . the predominant strategy for controlling vector-borne diseases consists of targeting the vectors, mostly using insecticides. the deleterious side effects of insecticide usage are widely known. among these are consequences to ecosystems, animal and human health, and development of resistance, pointing to the urgency of developing new control strategies. the understanding of biological mechanisms involved in the relationship between pathogens and vectors can provide new approaches for the development of control strategies. the infection of aedes aegypti with wolbachia [ ] is an excellent example of a novel arbovirus control strategy. our group has been studying the interaction and immune responses of the sand fly lutzomyia longipalpis (diptera: psychodidae: phlebotominae), the most important vector of american visceral leishmaniasis [ ] , when infected with different pathogens [ ] [ ] [ ] . the establishment of continuous insect cell lines has been an important tool for the study of insect biology. cells from drosophila melanogaster (s ), aedes albopictus (c / ), and anopheles gambie (sua b), among other insect cell lines, have been employed to scrutinize diverse features of insect immunity [ ] [ ] [ ] . we have detected complex immune responses in the lutzomyia longipalpis ll embryonic cell line [ ] when challenged by different pathogens [ ] . we have also identified a non-specific antiviral response in these cells when transfected with non-specific double-stranded rna (dsrna) [ ] , in a way similar to interferon response in mammals [ ] . after dsrna transfection, regardless of nucleotide sequence, there was no replication of a west nile reporter virus that expresses luciferase and lacks structural genes, as indicated by a lack of luciferase production [ ] . we are presently searching for molecules responsible for this response. secreted proteins have a key role in biological processes involving cell signaling. naïve cells can be modulated by viral or host components transferred from neighboring infected cells via the release of extracellular vesicles and secretion of soluble molecules, leading to activation of host immune response and changes in the secreted protein repertoire [ ] [ ] [ ] . in order to investigate the effect of polyinosinic:polycytidylic acid (poly i:c) transfection on the protein secretion profile of l. longipalpis ll cells, we carried out mass spectrometry (ms) analyses comparing the protein composition of conditioned medium from poly (i:c) and mock-treated ll cells. l. longipalpis embryonic ll cells obtained from dr. robert b. tesh (university of texas medical branch, galveston, tx, usa) were grown at • c in l medium (sigma, mendota heights, mi, usa) supplemented with % fetal bovine serum (fbs; hyclone, logan, ut, usa), % tryptose phosphate broth, and % antibiotics (penicillin u/ml and streptomycin mg/ml, sigma). for mock transfection, ng/ml lipofectin transfection reagent (invitrogen, carlsbad, ca, usa) were added to l medium (leibovitz) containing % tryptose phosphate broth (tpb). the transfection mix contained the mock transfection mix added with ng/ml of polyinosinic:polycytidilic acid (poly i:c), a synthetic analog of double-stranded rna (invitrogen). these mixtures were added to × ll cells for h and supernatant was aspirated and stored. new medium was added and supernatant was aspirated and stored after h. for rna preparation, transfected and mock-transfected cells were collected after , , , and h incubations, as described above. cell viability was verified by trypan blue staining. more than % of cells were viable in all experiments. protease inhibitor cocktail × (sigma) was added to the media. dead cells and large debris were pelleted by centrifugation at × g for min. supernatants were centrifuged again at , × g for min to exclude remaining cell debris and microvesicles. the proteins in the supernatant were then precipitated using trichloroacetic acid (tca). one volume of tca % (sigma) was added to four volumes of conditioned medium and incubated for min at − • c. the samples were centrifuged at , × g for min. the supernatant was removed and the protein pellet was washed with cold acetone, vortexed and centrifuged at , × g for min. the material was submitted to mass spectrometry. the pellets were suspended in m urea, m thiourea, and mm -( -hydroxyethyl)- piperazineethanesulfonic acid (hepes). the proteins were reduced with mm dithiothreitol (dtt) and mm ammonium bicarbonate (abc), and alkylated with . mm iodacetamide and mm abc. before trypsinization, the samples were purified with detergent removal spin columns (thermo scientific). then, the samples were digested in mm abc with trypsin (promega, cat. v , madison, wi, usa) in a : trypsin to protein mass ratio by incubation at • c for h. after trypsinization, trifluoroacetic acid (tfa) was added to a final concentration of . %. peptides were desalted with homemade c spin columns. the peptides were analyzed in triplicate by liquid chromatography tandem-mass spectrometry (lc-ms/ms) in a thermo scientific easy-nlc system coupled to a ltq orbitrap xl etd (mass spectrometry facility rpt h/carlos chagas institute-fiocruz, curitiba, pr, brazil). peptide separation was carried out in -cm ( -µm inner diameter) fused silica, in-house packed with reversed-phase reprosil-pur c -aq -µm resin (dr. maisch gmbh, ammerbuch-entringen). chromatography runs were performed in a flow rate of nl/min from to % mecn in . % formic acid in a -min gradient, and a voltage of . kv was applied for peptide ionization. the mass spectrometer operated in a data-dependent acquisition mode. survey full-scan ms spectra (at a - m/z range) were acquired in the orbitrap analyzer with resolution of , at m/z (after accumulation to a target value of , in the c-trap). the ten most intense ions were sequentially isolated and fragmented in the linear ion trap using collision-induced dissociation at a target value of , . the "lock mass" option was enabled at . m/z in all full scans to improve mass accuracy of precursor ions [ ] . protein identification was performed with maxquant algorithm [ , ] version . . . . default parameters of the software were used for all analysis steps, unless stated otherwise. proteins were searched against an l. longipalpis protein sequence database (containing , protein sequences from the vectorbase protein database, downloaded on december ) and common contaminants, besides their respective reverse sequences to estimate the false discovery rate (fdr). carbamidomethylation of cysteine was set as a fixed modification, while methionine oxidation and n-terminal acetylation (protein) were allowed as variable modifications. in addition, an fdr threshold of . was applied at both peptide and protein levels. protein quantification was performed using a label-free approach, where the peptide peaks were detected as three-dimensional features-retention time versus signal intensity (extracted ion chromatogram, xic) versus mass/charge-and were aligned and compared across the runs, as previously described [ ] . the amino acid sequences of identified secreted proteins were subjected to bioinformatic analyses. the protein signal peptide (sp) from proteins was estimated using the software tool prediction of signal peptide "predsi" at http://www.predisi.de./home.html [ ] , under standard configuration. protein-protein interactions were performed using the search tool for the retrieval of interacting genes/proteins (string) database v . at http://string.embl.de/ [ ] . three biological replicates of × cells were transfected. the supernatant was removed , , , and h post-transfection and the cells, after being washed three times with pbs, were resuspended in ml of trizol (ambion, waltham, ma, usa). the rna was extracted following the trizol manufacturer's instructions. total rna was precipitated with isopropanol, resuspended in water and stored at − • c. rna was treated with dnase i (promega) and cdna was synthesized from µg of total rna using superscript iii first-strand synthesis (invitrogen). real-time polymerase chain reaction (qpcr) was performed using sybr green pcr master mix (applied biosystems, foster city, ca, usa) and the primers listed in table . expression levels were determined through (-delta delta c(t)) method (ddct) [ ] , normalized using rp gene expression [ ] , yielding the relative expression value. statistical analyses were done using graphpad prism software, version . (graphpad software, inc., san diego, ca, usa). for comparison of the transfected and mock samples at different times after transfection, an unpaired two-tailed student's t test was performed. * p ≤ . , ** p ≤ . , *** p ≤ . . table . list of primers used for qpcr. hemocytin basic transcription factor btf -f agtgcataagcaggcaacacca btf -r tacgtcgccaccgaaatgagtt juvenile hormone-inducible protein jhip-f ccttgctgagctccttgagaaact jhip-r tacacatggccattcccatcttcc eukaryotic translation initiation factor etif -f tcgataggcatctcacgtttccac etif -r tcttccccactgtatccaggatgt transmembrane protease serine -like repressor splicing factor rrm-f aatttgggaagctcaata rrm-r gaggatgtcgcaagccttct forskolin-binding protein proliferating cell nuclear antigen pcna-f catgaatctcgaccaggagcactt pcna-r tcacggcaaatgcgtgcaaa tubulin-specific chaperone a tbca-f cgtacgaaaaggaagcagatcagca tbca-r cttccttccggatcacgtgttcat ribosomal protein rp -f gaccgatatgccaagctaaagca rp -r ggggagcatgtggcgtgtctt comparative analysis of expression values was performed to verify the correlation between mrna levels of ll cells (samples collected , , and h post transfection) evaluated by qpcr, and secreted protein levels (samples collected or h post transfection) evaluated by mass spectrometry. linear regression, pearson's correlation coefficient (r), and goodness of fit (r ) were calculated using graphpad prism software (version . ). immune responses are triggered through the recognition of pathogen-associated molecular patterns (pamps) by host pattern recognition receptors (prrs). binding of pamps leads to the activation of signaling pathways: rnai, janus kinase/signal transducers and activators of transcription (jak/stat), immune deficiency (imd) and toll [ ] [ ] [ ] [ ] . the major insect innate immune response triggered by virus infection is normally the small interfering rna pathway. rnai is a molecular mechanism in which the presence of intracellular dsrna leads to the production of small rnas that suppress the expression of genes with matching sequences [ ] . nevertheless, alternative mechanisms exist. in previous work, our group identified an antiviral response when the ll embryonic l. longipalpis cell lineage was transfected with dsrna, which included the synthetic poly i:c. after dsrna transfection, cells did not support wnv virus like particle (vlp) replication, as evidenced by lack of luciferase reporter gene expression. this block in replication may be due to a cellular antiviral response triggered by dsrna. [ ] . a similar non-specific antiviral response was observed in the marine shrimps litopanaeus vannamei and penaeus monodon [ , ] . more recently, the occurrence of dsrna-mediated non-specific antiviral response was also described in two members of the apidae family: the honey bee apis melifera, and the bumblebee bombus terrestris [ ] [ ] [ ] . this non-specific response differs from what is seen in drosophila, where transfection with virus-specific dsrna triggers an rna interference (rnai)-mediated response against the virus, whereas non-related dsrnas fail to activate the antiviral response [ ] . in vertebrates, intracellular dsrna is recognized by the prr retinoic acid-inducible gene (rig-i) and the melanoma differentiation-associated protein (mda ). these are also known as rig-i-like receptors (rlrs) [ , ] . these receptors recruit the mitochondrial antiviral-signaling (mavs) protein that mobilizes kinases and ubiquitin ligases, leading to the activation of transcription factors, such as interferon receptor factor (irf- ) and nuclear factor κb (nf-κb) [ , ] . the transcription factors induce type i interferon (ifn) and pro-inflammatory cytokine production. dsrna, including poly i:c, is also detected by toll-like receptor , initiating the type-i interferon (ifn-α, β) signaling pathway via a toll/interleukin- receptor (tir)-domain-containing adapter-inducing interferon-β (trif) signal, which activates interferon receptor factor (irf- ) and nuclear factor κb (nf-κb), leading to ifn-β expression [ , ] . to investigate variations in the exoproteome profile of dsrna-transfected l. longipalpis ll cells, we carried out mass spectrometry (ms) analyses comparing the protein composition of conditioned medium from poly i:c and mock-treated cell cultures at two time points ( and h); in total proteins were identified (table s ) . only . % ( ) of all identified secreted proteins presented a signal peptide (sp). among proteins with ≥ -fold positive change variation, this percentage decreased to . %, with only six proteins with sp: at h and at h (table s ). the lack of signal peptide in the majority of secreted proteins suggests that unconventional protein secretion pathways are the main secretory mechanisms in ll cells, as already seen in other systems [ ] . this could be due at least partly to exosomal transport. we identified some of the known exosome markers among our secreted proteins, based on the exo carta exosome marker list (www.exocarta.org/exosome_markers). among these were glyceraldehyde- -phosphate dehydrogenase (gapdh), enolase (eno), fructose-bisphosphate aldolase (aldoa), moesin (msn), phosphoglycerate kinase (pgk), cluster of differentiation (cds) , and , the heat shock proteins (hsps) and , and annexins (table s ). these were not differentially expressed with a greater than -fold change and were present in both mock and poly i:c transfection samples. we performed an interaction analysis of the ll dsrna-transfected exoproteome. the network analysis of proteins presenting different abundance between mock and transfected groups (table s ) predicted that proteins (table s ) were connected by edges ( figure a) forming an intricate network. the prevised protein interaction (ppi) enrichment p-value was . , meaning that secreted proteins have more relations among themselves than what would be expected for a random set of proteins of similar size drawn from the genome. exoproteome functional enrichment analysis identified enhanced interactions related to specific biological functions. these interactions have a significant false discovery rate (fdr) of less than . . among these gene ontology (go) categories and pathways, we can quote: biological process go: (peptide metabolic process) fdr: we also carried out string interaction analysis ( figure b ) of proteins with ≥ -fold change variation detected on mass spectrometry analysis. from the protein sequences (table s ) shown in figure b ,c, were identified in the string database (table s ) and from those, (table s ) ( figure b) were predicted as connected at least to one protein. this interaction analysis displayed a significant ppi enrichment p-value of . × − . in this interactome we can distinguish two major protein groups: the first one with members is related to oxidative stress modulation and includes phospholipid scramblase (plscr ), thioredoxin (txn), glutaredoxin (glrx ), superoxide dismutase (sod ), and forskolin-binding protein (fkbp ), among others. the second group is comprised of proteins linked with peptide synthesis and cytoskeleton organization. among these are the riboproteins rps and rplp , the eukaryotic translation initiation factor (eif d), moesin (msn), cortactin protein (cttn), and others. two biological functions were enhanced in the ≥ -fold change secreted protein cluster: cellular component: go: (extracellular exosome) fdr: . × − and molecular function: go. (rna binding) fdr: . × − . these same biological function enrichments are found when we study the whole exoproteome. the complete fdr-filtered (p < . ) lists are shown in table s . the measurement of relative protein abundance revealed that the secretion of most proteins did not vary significantly between test and control supernatants (figure a ). only . % ( ) of all identified proteins at h presented variation of ≥ -fold change, and . % ( ) presented variation of ≤ -fold change ( figure b ). these percentages change to . % ( ) and . % ( ) , respectively, at h after dsrna transfection ( figure c ). we focused on the proteins with ≥ -fold change variation, which represent . % of the total identified proteins ( figure c ). the proteins with intensified secretion in the first h had their relative amounts decreased at h. most proteins more abundant at h had a basal detection level at h. interestingly only one protein, the enzyme carboxylesterase, presented secretion levels with ≥ -fold change at both time points. in insects this enzyme is related to detoxifying processes, including insecticide resistance [ ] . several proteins with higher secretion at h or h are related to immune responses. among the proteins with higher secretion at h we can highlight eight proteins related to immune response. phospholipid scramblase (plscr ) had a positive variation with a greater than -fold change. scramblases are conserved in all eukaryotic organisms. they are multiple palmitoylated, endofacial membrane proteins originally identified based on their capacity to promote accelerated transbilayer phospholipid movement in response to ca + [ ] . plscr was identified as an interferon and growth factor-inducible protein necessary for the ifn-dependent induction of gene expression and antiviral activity [ ] . in human and mouse plasmacytoid dendritic cells (pdcs), which are professional interferon-producing cells specialized in recognizing viral rna and dna through the endosomal toll-like receptors (tlrs) tlr and tlr , respectively, plscr was described as a tlr -binding protein that plays a significant role in type- interferon responses in pdcs by regulating tlr expression and trafficking [ ] . the direct interaction of plscr with human t-cell leukemia virus type- (htlv- ) leads to a reduction of virus titers [ ] . these reports demonstrate the significant, but not fully understood, role of phospholipid scramblase in antiviral response. another interesting protein with a greater than -fold increased secretion at h was the hexameric chaperone complex prefoldin (pfd). subunits of prefoldin have been found to be associated with cellular response to viral infection. the pfdn expression was positively modulated in both vaccinia virus-infected hek cells and in the rabv-infected n a cells [ , ] . pfdn was found directly bound to the hepatitis c virus (hcv) f protein [ ] . the string interactome maps were generated using default settings (medium confidence of . , with criteria for linkage: neighborhood, genefusion, co-occurrence, co-expression, experimental evidence, existing databases, and textmining). proteins were represented as nodes and their functional links were defined by solid lines. the thickness of the lines signifies the level of confidence of the reported association. the non-connected nodes were suppressed. no additional interplay proteins were added to the networks. the protein symbols and node colors are listed in table s . still within the proteins with higher secretion at h, we found forskolin-binding protein (fkbp ), an immunophilin family member, with a secretion level increase of greater than -fold. this is a highly conserved group of proteins which binds to immunosuppressive drugs such as fk , rapamycin, and cyclosporine [ , ] . fkbps are involved in several biochemical processes and participate in the immune response against viral infection. mitochondrial fkbp was identified as a tumor necrosis factor (tnf) receptor-associated factor (traf ) and tnf receptor-associated factor (traf ) binding protein. binding of fkbp to traf proteins facilitates the type-i interferon response induced by dsrna transfection or newcastle disease virus (ndv) infection in murine fibroblasts. depletion of fkbp reduced the expression of type inf in these cells [ ] . one interesting protein with a greater than -fold enhanced secretion at h is coatomer protein i (copi). copi-coated vesicles are associated with transport from the golgi to the endoplasmic reticulum [ ] , endocytosis [ ] , maturation of autophagic vacuoles [ ] , expression of cell surface receptors, and modulation of the plasma membrane lipid composition [ ] . studies demonstrated the involvement of copi subunits in diverse aspects of virus infection [ ] [ ] [ ] . depletion of the copi β subunit in human /ace cells led to a decrease in the severe acute respiratory syndromecoronavirus (sars-cov) replication [ ] . the string interactome maps were generated using default settings (medium confidence of . , with criteria for linkage: neighborhood, genefusion, co-occurrence, co-expression, experimental evidence, existing databases, and textmining). proteins were represented as nodes and their functional links were defined by solid lines. the thickness of the lines signifies the level of confidence of the reported association. the non-connected nodes were suppressed. no additional interplay proteins were added to the networks. the protein symbols and node colors are listed in table s . still within the proteins with higher secretion at h, we found forskolin-binding protein (fkbp ), an immunophilin family member, with a secretion level increase of greater than -fold. this is a highly conserved group of proteins which binds to immunosuppressive drugs such as fk , rapamycin, and cyclosporine [ , ] . fkbps are involved in several biochemical processes and participate in the immune response against viral infection. mitochondrial fkbp was identified as a tumor necrosis factor (tnf) receptor-associated factor (traf ) and tnf receptor-associated factor (traf ) binding protein. binding of fkbp to traf proteins facilitates the type-i interferon response induced by dsrna transfection or newcastle disease virus (ndv) infection in murine fibroblasts. depletion of fkbp reduced the expression of type inf in these cells [ ] . one interesting protein with a greater than -fold enhanced secretion at h is coatomer protein i (copi). copi-coated vesicles are associated with transport from the golgi to the endoplasmic reticulum [ ] , endocytosis [ ] , maturation of autophagic vacuoles [ ] , expression of cell surface receptors, and modulation of the plasma membrane lipid composition [ ] . studies demonstrated the involvement of copi subunits in diverse aspects of virus infection [ ] [ ] [ ] . depletion of the copi β subunit in human /ace cells led to a decrease in the severe acute respiratory syndrome-coronavirus (sars-cov) replication [ ] . we identified two juvenile hormone-inducible proteins at h with . -and . -fold increases. despite the absence of information on the possible involvement of juvenile hormone-inducible proteins in the insect antiviral response, the juvenile hormone itself and retinoic acid are terpenoids, with similar structure and biological effects in mammalian and insect cells [ ] . retinoic acid-inducible genes are related to type-i ifn response in mammals [ , ] . viruses , , of we identified two juvenile hormone-inducible proteins at h with . -and . -fold increases. despite the absence of information on the possible involvement of juvenile hormone-inducible proteins in the insect antiviral response, the juvenile hormone itself and retinoic acid are terpenoids, with similar structure and biological effects in mammalian and insect cells [ ] . retinoic acidinducible genes are related to type-i ifn response in mammals [ , ] . another virus infection-related protein found was kinesin, an atpase protein belonging to the class of motor proteins. kinesins move along microtubule (mt) filaments, and are power-driven by the hydrolysis of adenosine triphosphate (atp) [ ] . recently this protein has been associated with the intracellular transport of different viruses. hiv- trafficking to the nucleus is kinesin-and dynein motor-dependent [ ] . kinesin was also identified as a chandipura virus matrix binding protein [ ] . kinesin from lepidopteran trichoplusia ni binds directly to nucleocapsid proteins of the autographa californica multiple nucleopolyhedrovirus (acmnpv) [ ] , and the α herpersviral envelope protein pus uses kinesin to promote the anterograde axonal transport of herpes simplex virus (hsv- ) [ ] . these results demonstrate that kinesin protein interacts directly with the virus particle. the insect protein canopy is a homolog of the human protein associated with toll-like receptor (tlr) (prat ). prat is required for cell surface expression of the toll family of receptors and for the intracellular nucleic acid-sensing tlr / . this protein is essential for tlr-dependent immune response [ ] . the . -fold increased secretion of this prat homolog signals the participation of toll-like receptors in the ll antiviral non-specific immune response. the barrier to autointegration factor (baf) dna binding protein had an increase of . -fold at h. this protein is described as a potent antiviral effector against poxyviruses, retroviruses and the herpes virus. the baf antiviral property is regulated by kinases and phosphatases, with dephosphorylation activating its antiviral properties [ ] . notwithstanding the higher number of positively modulated secreted proteins at h when compared with h, the number of proteins described in immune response to viruses in interferon-like responses was smaller than what was seen at h. among them we quote the protein hemocytin, with a secretion increase of . -fold. hemocytin is an insect humoral lectin homologous to mammalian von willebrand factor, which is involved in platelet adhesion to subendothelium [ ] . hemocytin expression is induced by pathogens. lectins are adhesive molecules which bind carbohydrates and among other functions are involved in hemolymph bacterial clearance [ ] . also of interest is the occurrence of glutaredoxin (fold change of . ), thioredoxin (fold change of . ) and superoxide dismutase (fold change of . ), antioxidant enzymes involved in the control of oxidative stress at h. reactive oxygen species (ros) production, generated due to microbial invasion, has long been known to exert an antimicrobial effect. the activation of the antiviral and inflammatory signaling pathways has also been linked with the production of ros [ , ] . due to the high chemical reactivity of ros, cells possess scavenger antioxidant mechanisms that maintain redox homeostasis [ ] . real time pcr (qpcr) analysis of genes coding for significantly positive-or negatively-modulated secreted proteins revealed the existence of some genes with transcriptional profile correlated with the amount of protein detected in the supernatants ( figure a ). there were seven molecules that presented a correlation between mrna and secreted protein levels (represented in figure a ). three of them, hemocytin (hmc), lecithin cholesterol acyltransferase (lcat), and scramblase (scr), showed coordinated mrna and secreted protein levels. when mrna levels changed at or h post transfection (either by up-or down-regulation), we detected a corresponding change in secreted protein levels or h after the mrna level changes. hemocytin (hmc), an insect humoral lectin which plays an important role in a non-specific self-defense mechanism [ ] , had increased mrna and secreted protein levels at h and h. this indicates that the transcription, translation and secretion of this molecule are correlated and the prolonged increase of its expression suggests this molecule may be important for the ll cells response to poly i:c transfection. two other molecules, lecithin cholesterol acyltransferase (lcat) (a plasma enzyme which esterifies cholesterol) [ ] , and scramblase (scr) (a regulator of transbilayer lipid asymmetry in eukaryotic cell membranes) [ ] , were reduced both in mrna and secreted protein levels at or h post-transfection. these two molecules are involved in lipid metabolism or membrane formation and the reduction of their expression may suggest that ll cells reduce vesicle formation h after transfection challenge. three other molecules showed increased mrna levels at or h post transfection and increased secreted protein levels at h, with posterior decrease at h. one was the eukaryotic translation initiation factor (etif ), which in humans is directly recruited by the hepatitis c virus (hcv) internal ribosome entry site (ires) to promote the translation of viral proteins [ ] . in ll cells it showed increased mrna levels at h and h, and increased secreted protein levels h after mrna increase. curiously, h post transfection the secreted protein levels decreased drastically. juvenile hormone-inducible protein (jhip) showed increased mrna levels after h and increased secreted protein levels h after mrna increase. curiously, at h post transfection the secreted protein levels decreased drastically regardless of changes in mrna levels prior to this timepoint. basal transcription factor (btf ) has been reported to play a significant role in the transcriptional regulation related to eukaryote growth and development [ ] . in ll cells it showed increased mrna levels at h and increased secreted protein level h after mrna increase. at h post transfection the secreted protein levels decreased drastically regardless of changes in mrna levels prior to this time point. transketolase mrna levels increased at h post transfection and the secreted protein levels also increased at this same time point, suggesting a late effect. curiously, regardless of the maintenance of mrna levels from h to h, the secreted protein levels were considerably low at h post transfection. changes in transketolase activity are involved in a number of tumors and degenerative diseases [ ] . based on the data presented in figure a , we observed that in the majority of the molecules (six out of seven) there was a correlated change of secreted protein levels h to h after changes in mrna levels. the remaining genes investigated showed no correlation between the mrna levels and the detection of proteins in the supernatants. ( figure b ). the linear regression of mrna levels plotted against secreted protein levels revealed a weak correlation (figure ). the pearson correlation coefficient (r) and goodness of fit (r ) calculated for mrna levels ( h, h, h and h) versus secreted protein ( h and h) are shown in figure and table . the non-correlation between transcription levels and amounts of secreted protein indicates that secretion is regulated by some not-yet disclosed mechanism, involving transcription, translation, storage, sorting, and transport, as already reported for other systems [ ] . this non-correlation was also observed when the exoproteome of wrl- human hepatic cells infected with the chikungunya virus was analyzed [ ] . we observed that the majority of analyzed genes were transcribed in the first h ( figure a,b) . the results obtained with the signal peptide prediction, the secreted protein interaction network, the identified biological functions enriched pathways, and the relatively small number of proteins with significant abundance variation reveal the existence of finely-tuned protein secretion modulation in response to dsrna treatment. there is also a strong indication for the participation of the exosomal secretory pathway as the major ll cell secretion mechanism in this response. the increased secretion of proteins such as phospholipid scramblase, fkbp, juvenile hormone-inducible proteins, and protein canopy in the first h indicate that this non-specific antiviral response is in a way similar to an interferon-like response mediated by the toll pathway. this response occurs preferentially in the first hours after transfection. the increase of antioxidant enzymes at h indicates that the cells are working to control the oxidative stress level in the culture. we are presently in the process of validating these findings by silencing specific genes with subsequent analysis of nonspecific antiviral response. (table ) and samples collected at different time points posttransfection. quantification was normalized relative to the house keeping gene rp , and relative gene expression expressed as fold change was calculated relative to the mock group. bars represent mean with standard error (sem) of two biological replicates. tables represent protein fold change corresponding to each gene name. * p ≤ . , ** p ≤ . , *** p ≤ . . (table ) and samples collected at different time points post-transfection. quantification was normalized relative to the house keeping gene rp , and relative gene expression expressed as fold change was calculated relative to the mock group. bars represent mean with standard error (sem) of two biological replicates. tables represent protein fold change corresponding to each gene name. * p ≤ . , ** p ≤ . , *** p ≤ . . biology of phlebotomine sand flies as vectors of disease agents a new arbovirus isolated in india from patients with febrile illness isolation of chandipura virus from sandflies in aurangabad the genus phlebovirus and its vectors carajas and maraba viruses, two new v esiculoviruses isolated from phlebotomine sand flies in brazil viremia and immune response with sequential phlebovirus infections ecuador paraiso escondido virus, a new flavivirus isolated from new world sand flies in ecuador, is the first representative of a novel clade in the genus flavivirus factors 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transmission pathways in t lymphocytes are inhibited by complexes formed between an immunophilin and either fk or rapamycin calcineurin is a common target of cyclophilin-cyclosporin a and fkbp-fk complexes mitochondria-nucleus shuttling fk -binding protein interacts with traf proteins and facilitates the rig-i-like receptor-mediated expression of type i ifn structure of coatomer cage proteins and the relationship among copi, copii, and clathrin vesicle coats involvement of specific copi subunits in protein sorting from the late endosome to the vacuole in yeast early endosomes and endosomal coatomer are required for rnai screen of salmonella invasion shows role of copi in membrane targeting of cholesterol and cdc copi activity coupled with fatty acid biosynthesis is required for viral replication a role for the host coatomer and kdel receptor in early vaccinia biogenesis. proc. natl. acad. sci human host factors required for influenza virus replication a kinome-wide small interfering rna screen identifies proviral and antiviral host factors in severe acute respiratory syndrome coronavirus replication, including double-stranded rna-activated protein kinase and early secretory pathway proteins activation of mammalian retinoid x receptors by the insect growth regulator methoprene regulation of antiviral innate immune signaling by stress-induced rna granules myosin v motor proteins: marching stepwise towards a mechanism hiv- capsids bind and exploit the kinesin- adaptor fez for inward movement to the nucleus host interactions of chandipura virus matrix protein trichoplusia ni kinesin- associates with autographa californica multiple nucleopolyhedrovirus nucleocapsid proteins and is required for production of budded virus the basic domain of herpes simplex virus pus recruits kinesin- to facilitate egress from neurons a protein associated with toll-like receptor (tlr) (prat a) is required for tlr-dependent immune responses the barrier to autointegration factor: interlocking antiviral defense with genome maintenance cloning and expression of the gene of hemocytin, an insect humoral lectin which is homologous with the mammalian von willebrand factor immune proteins and their gene expression in the silkworm, bombyx mori hsv infection induces production of ros, which potentiate signaling from pattern recognition receptors: role for s-glutathionylation of traf and reactive oxygen species activate nf-κb (p ) and p and induce apoptosis in rvfv infected liver cells the nrf cell defence pathway: keap -dependent and -independent mechanisms of regulation establishment and application of a high throughput screening system targeting the interaction between hcv internal ribosome entry site and human eukaryotic translation initiation factor exploring the roles of basal transcription factor in eukaryotic growth and development structure and functioning mechanism of transketolase control of cystic fibrosis transmembrane conductance regulator membrane trafficking: not just from the endoplasmic reticulum to the golgi differential analysis of the secretome of wrl cells infected with the chikungunya virus the authors declare no conflict of interest. key: cord- -blwguyl authors: guleria, randeep; mathur, vartika; dhanuka, ashutosh title: health effects of changing environment date: - - journal: natural resource management: ecological perspectives doi: . / - - - - _ sha: doc_id: cord_uid: blwguyl environment plays a crucial role in our economic, social and cultural behaviour as well as on health. however, since the beginning of industrialization era, focus on economic development has caused detrimental effects on the environment. last two centuries have witnessed changes in global environmental factors such as rise in temperature leading to global warming, depletion of stratospheric ozone layer, loss of biodiversity and marked degradation in air and water quality due to atmospheric pollution, thereby causing upsurge in infectious and non-infectious diseases. environmental health has emerged as an important part of medicine. the world health organization (who) estimates that % of global disease burden and % of all deaths can be attributed to environmental factors. deaths from heart disease, cancer, respiratory disorders and many vector-borne diseases such as malaria, dengue, chikungunya and cholera have increased due to changes in climate, especially in developing countries. besides limited attention to sanitation, hygiene, as well as quality of food and drinking water, factors such as deforestation, increasing vehicular traffic, migration from rural to urban areas, decreasing water resources and inadequate drainage systems contribute to increase incidence of diseases. the need of the hour is to sensitize ourselves about the way our ecology is being degraded and the health effects it is causing. a holistic view is needed to address the problem of environmental health where agriculture, animal husbandry, public health, water safety and air pollution need to be looked at in a combined manner for education, planning and resource allocation. therefore, a close association between scientists, public health professionals and administrators is needed for integrated design and development of framework to attain harmony between man and nature. an ecosystem is defined as a community of living beings surviving and interacting in mutual and interdependent relationship with their physical environment. for thousands of years, man has lived in harmony with their natural surroundings. environment has played a crucial role in his economic, social and cultural behaviour as well as on his health. the role of environment in various diseases has been well documented, both in communicable and non-communicable diseases. since the dawn of industrialization era in europe years ago and its subsequent spread to the rest of world, economic development and physical comfort for mankind have increased at a tremendous pace. this increase is perhaps the most rapid over the last three decades. often this has been done, knowingly or unknowingly, at the cost of our environment. the last years have witnessed a sharp increase in the global temperature from its levels around years ago, owing to industrialization (mann et al. ; marcott et al. ) . moreover, other concerns such as depletion of carbon fuels at alarming rates, damage to the ozone layer and rise in seawater levels, combined with global warming, have damaged our environment extensively, leading to changes in the aquatic biodiversity and to the extinction of many species of plants and animals (thomas et al. ) . increase in urbanization has led to loss of dense forests. air pollution has risen to the extent that many big cities in the world have a highly toxic air quality. however, very little has been done by various governmental and non-governmental agencies with almost no visible results. the need of the hour is to sensitize the scientific community, as well as the common man, about the way our ecology is being degraded and the health effects it is causing and to suggest ways to get remedies for this situation. environmental health has emerged as an important part of medicine, due to the rapid environmental changes linked to industrialization and urbanization. it is being increasingly recognized that environmental factors play a key role in human health and are linked to many chronic and infectious diseases. deaths from heart disease and respiratory illness are increasing, and many diseases such as malaria, dengue, chikungunya and cholera are sensitive to changes in the climate (mcmichael et al. ; patz et al. ) . according to the world health organization (who), 'in its broadest sense, environmental health comprises those aspects of human health, disease and injuries that are determined or influenced by factors in the environment. this includes the study of both direct pathological effects of various chemical, physical and biological agents, as well as effects on health of the broad physical and social environment, which include housing, urban development, land use and transportation, industry and agriculture'. the who estimates that % of the global disease burden and % of all deaths can be attributed to environmental factors. moreover, environmental factors have a much bigger impact in developing countries than developed ones, and this effect is seen much more in the vulnerable population such as children and elderly. in a developing country like india, the burden of various diseases is increasing due to environmental factors and the changes in our environment. it is estimated that % diarrhoeal disease burden may be attributed to environmental factors such as unsafe food and drinking water, as well as poor sanitation and hygiene. similarly, in india there is strong evidence linking lower respiratory tract infection to indoor air pollution caused by the use of solid fuels in household. almost % of acute lower respiratory tract infections in developing countries are attributable to environmental factors. besides this, a close association of vector-borne diseases and environmental conditions has been established. furthermore, factors such as deforestation, increasing vehicular traffic, migration from rural to urban areas, decreasing water resources and inadequate drainage system are important environmental and ecological factors that contribute to infectious diseases. the temperature of the earth has increased by about . °c over the past years (griggs and noguer ; mccarthy ) . winters are shortening and average temperature is rising. intergovernmental panel on climate change (ipcc) of united nations predicts that the global temperature will rise by . - . °c by the turn of this century, if no remediable actions are taken (houghton et al. ). this will lead to rise in sea level by - cm and drowning of coastal cities, which comprise % of world's major cities (crutzen ; fitzgerald et al. ; nicholls and cazenave ) . higher temperatures will lead to melting of polar ice, melting of glaciers, floods and droughts (patz et al. ) . average temperature shall rise during both summers and winters. heat waves will increase, and average annual precipitation will also increase correspondingly. heat waves, floods and droughts lead to natural calamities, shortage of food supplies, increased risk of infectious diseases and increased human mortality (haines et al. ). clean water is essential for the survival of humans. water pollution due to environmental changes therefore constitutes another serious risk to the health of our planet. water pollution occurs when energy and substances are released and degrades the quality of water for other users. anything that is added to water, which is more than its capacity to break it down, constitutes water pollution. anthropogenic activities such as industrial waste effluents, sewage disposal and agricultural activities are some of the major causes of water pollution (manivasakam ; tilman et al. ) . chemical pollution of surface water causes major health problems as it can be used directly for drinking or it may contaminate shallow wells, used for drinking. ground water, which is much deeper, has very few pathogens as it gets filtered when it passes through many underground layers. it can be polluted by toxic chemicals such as fluoride and arsenic which may be present in the soil or the rock layers. similarly, pollution of coastal water can cause contamination of sea food (guleria ) . changing environment has a serious effect on safe water, affecting not only human health but also changing the ecology of plants. the global effect of water pollution has not been studied in detail and is limited to mainly outbreaks of waterborne infections or certain chemical toxins in limited areas, such as arsenic in drinking water in bangladesh, 'minamata' disease in japan, etc. (argos et al. ; harada ) . the burden of waterborne diseases is grossly underreported in india due to lack of data, poor surveillance and reporting. according to a report from the ministry of health and family welfare, nearly million people are affected by waterborne diseases such as diarrhoea, enteric fever, amoebiasis and helminthic infestations, every year. who estimates > , deaths annually, in india alone, due to contaminated water consumption. moreover, floods and droughts also affect human health. floods lead to physical injury as well as spread of waterborne diseases such as diarrhoea, enteric fever and viral hepatitis. overcrowding occurs and sanitation is affected, leading to respiratory infections. diseases such as malaria and dengue may turn into epidemics. on the other hand, drought leads to lack of sanitation, decreased food production and ultimately malnutrition. another aspect of waterborne diseases is chemical contamination leading to diseases such as fluorosis and methemoglobinemia, due to contamination of soil water owing to fluoride and fertilizers. chronic exposure to contaminated water can cause significant health effects and can lead to liver and kidney damages. this occurs due to chronic exposure to copper, cadmium, arsenic, mercury, chromium and chlorobenzene. endocrine effects have been reported, and problems relating to reproduction, development and behaviour have also been observed. enso is a cycle of seawater temperature and pressure changes occurring over the southern pacific ocean at an interval of - years and lasting for - months. this leads to episodes of floods in the southwest united states, mexico and western coast of latin america and droughts in southeast asia and the pacific islands (kovats et al. ). this may be followed by cold waves called la niña. higher global temperatures are predicted to lead to more frequent and severe ensos, and this will lead to significant effects on human health, in the coming years (bouma et al. ). change in the global climate has led to higher temperatures, humidity and floods, which has made the environment more conducive for parasites such as mosquitoes and fleas (patz et al. ) . malaria, dengue and other vector-borne diseases are expected to increase both in magnitude and their geographical reach (haines et al. ) . people living at higher altitudes may also likely experience resurgence in vector-borne diseases, due to a rise of average temperatures in these regions. moreover, these diseases can spread to any part of the world in a very short time (at times during the incubation period) and cause an outbreak in a community where these diseases do not usually occur, resulting in diagnostic difficulties. this has recently been seen during the ebola and the mers coronavirus outbreaks. other factors such as breakdown of public health infrastructure, shortage of medical supplies and changes in land use also contribute to adversities in health, due to water pollution. air pollutants affect the human body through the inhalational route. environmental changes due to industrialization have drastically altered the quality of the air we breathe. there are hundred substances that pollute the air and may harm human health. pollutants are generally classified as primary or secondary pollutants. chemicals that are directly emitted from a source are known as primary pollutants. these include sulphur dioxide, nitrogen oxides, carbon monoxide, volatile organic compounds, etc. moreover, particulate matter emitted due to combustion from automobile exhaust, heating, cooking and industrial sources are also primary pollutants. secondary pollutants, such as formaldehyde, nitric acid and different aldehydes, on the other hand, are formed from chemical or photochemical reaction in the atmosphere. on exposure to sunlight, volatile organic compounds and nitrogen oxides react photochemically, producing pollutants such as ozone. air pollution and occupational exposure may cause a variety of negative health outcomes, including reduced lung function in children as well as increased susceptibility to infections, airway inflammation and cardiovascular diseases. respiratory disorders due to air pollution are emerging to be a major contributor to mortality, according to recent epidemiologic studies. moreover, low-level air pollution is recently being recognized as a risk factor for lung diseases and death from copd (bosson and blomberg ) . with newer insights into the immunopathogenesis of asthma, the contribution of air pollution to allergen sensitization and airway hyperresponsiveness are being established. for example, increased exposure to nitrogen dioxide during infancy correlates with increased risk for asthma in later childhood. ozone can produce significant adverse effects on human health (gryparis et al. ; teague and bayer ; uysal and schapira ) . moreover, recent research is now linking air pollution to increased risk of respiratory symptoms and duration of respiratory tract symptoms. international agency for research on cancer recently designated diesel exhaust as a human carcinogen. sulphur oxides are produced mainly from industrial activities processing materials that contain sulphur, such as generation of electricity from coal, oil or gas, as well as by combustion of fossil fuels. sulphur dioxide is also present in motor vehicle emission. together with ozone, it is known to cause foliar injury and reduction in plant growth (smith ; tingey and reinert ) . it is mainly absorbed in the upper airways as it is water soluble. its exposure is known to cause symptoms such as nose and throat irritation. it may travel to the lower airways and cause bronchoconstriction and dyspnoea, especially in asthmatic individuals, thus worsening their condition (balmes et al. ; ierodiakonou et al. ) . nitrogen oxides are emitted primarily from motor vehicle exhausts, as well as from stationary sources such as electric utilities and industrial boilers. compounds such as sulphur and nitrogen oxides cause chemical reactions in air and acid rains. although acid rains do not affect humans radically, they may indirectly cause health problems, particularly difficulty in breathing and, in extreme cases, lung problems such as asthma or chronic bronchitis. moreover, nitrogen oxides are the main precursors in the formation of tropospheric ozone. they also form nitrate particles and acid aerosols. exposure to nitrogen dioxide for a short term leads to changes in airway responsiveness and deterioration in pulmonary function in individuals with underlying lung disease. long-term exposure may lead to increased chances of recurrent respiratory tract infections and alter lung mechanics (berglund et al. ) . carbon monoxide is produced mainly due to motor vehicle emission. in urban areas more than % of the carbon monoxide emission may be due to motor vehicles. besides this, the combustion of coal, oil and gas also leads to carbon monoxide production. moreover, tobacco smoke is one of the main sources of indoor pollution of carbon monoxide. high levels of carbon monoxide are extremely dangerous to humans, more so because it is colourless, tasteless and odourless and therefore cannot be detected by humans. early symptoms of carbon monoxide include weakness, headache, nausea, dizziness, confusion, disorientation and visual instability. carbon monoxide quickly enters the blood stream and forms carboxyhaemoglobin which causes more systemic effects. it reduces oxygen delivery to the tissues and may have a serious health threat to those with underlying heart disease (badman and jaffé ) . prolonged or severe exposure may result in lethal arrhythmias, electrocardiographic changes, pulmonary oedema, various neurological symptoms as well as death, most likely due to cardiac failure. carbon monoxide is known to cause foetal development disorders, brain lesions and, in extreme cases, even mortality (raub et al. ) . the atmospheric levels of lead have decreased due to the use of unleaded fuel. however, lead toxicity continues to be a problem, due to the exposure occurring in drinking water. lead exposure leads to adverse effects on the central nervous system, causing neurological symptoms such as sleep disorders, fatigue, trembles in limbs, blurred vision and slurred speech, as well as kidney and liver disorders (kampa and castanas ) . lead toxicity can lead to lower intelligence, learning deficits and behavioural disturbances. ozone is an important secondary pollutant and is a component of photochemical smog. it is a pulmonary irritant and an oxidant. it may produce significant adverse effects on human health. exposure to ozone causes airway inflammation, airway hyperreactivity and a decline in lung functions. ozone exposure causes cough, chest tightness and wheezing. the increase in the levels of tropospheric ozone is associated with reduced baseline lung functional as well as structural abnormalities, exacerbation of asthma and premature mortality. recent studies have shown increased admissions for chest complaints and worsening of asthma on exposure to even low levels of ozone. studies looking at long-term exposure to ozone suggest that a cumulative long-term exposure in childhood may affect lung function, especially that of the small airways of the lung, in adult life (künzli et al. ) . ozone also affects mucous membrane and causes pulmonary inflammation and has both a local and systemic effect on the immune system. patients with underlying respiratory illness such as asthma and chronic obstructive airway disease are more prone to the harmful effect of ozone. high ozone concentrations have been linked with increased hospital admissions for pneumonia, copd and asthma (gryparis et al. ; teague and bayer ; uysal and schapira ) . particulate matter consists of liquid or solid mass contained in an aerosol. it is a mixture of numerous different chemicals, with varying properties. major sources of particulate matter are factories, power plants, incinerators, motor vehicles, construction activities, fire and dust. broadly particulate matters from . to μm in diameter are coarse particulate matter. coarse particulate matter consists mainly of airborne soil dust and elements such as silicon and aluminium. fine particles of less than . μm are composed mainly of sulphate and organic material. particulate matter in air is associated with allergic rhinitis, lung inflammation, pulmonary disorders, cardiac arrhythmia, ischemic cardiovascular events, higher incidences of cancer and shortening of life (carlsten and georas ; dockery et al. ; kampa and castanas ; pope iii et al. ; raaschou-nielsen et al. ) . only recently we began to understand the cardiovascular effects of air pollution. high levels of air pollution worsen underlying heart disease. but now it is becoming clear that persistent exposure to high levels of air pollution may also lead to heart disease. this is especially true for particulate matter. inflammation in lungs also causes inflammation in the blood, leading to atherosclerosis and an increase incidence of coronary artery disease that may be fatal (fig. . ). many well-conducted studies have demonstrated a - % higher risk of coronary artery disease in individuals exposed to high levels of air pollution, for many years (cesaroni et al. ; miller et al. ). this increased risk has been linked to higher levels of pm . in the ambient air. studies have also looked at subclinical atherosclerosis, which is the pathological process associated with coronary artery disease. a positive association between subclinical atherosclerosis in the carotid and the coronary arteries has been observed with long-term exposure to high levels of air pollution (künzli et al. ; künzli et al. ) . there is therefore now a significant body of evidence linking air pollution to cardiovascular diseases and increased mortality. many investigators argue that air pollution should now be considered as a preventable risk factor like smoking and dyslipidemia for the development of coronary artery disease, and steps should be taken to bring down the exposure to air pollution. indoor air pollution and its effect on human health are important as individuals spend more than % of their time indoors. cooking is an integral part of indoor human activity. the who has estimated that about % of the world's population, or about billion people, still uses solid fuel for their household energy needs. of these, about . billion people use biological material (wood, charcoal, crop waste and dung), and the remaining use coal. in india, about % of the population has been estimated to depend upon wood, and about % depend upon dung for energy. although this number is slowly decreasing and moving towards the use of other fuels such as liquefied petroleum gas (lpg) and kerosene, it is still very significant. in india in , of the . billion people, about million still used solid fuel for cooking or heating. many studies over the last three decades have documented the link between solid fuel exposure and different respiratory diseases. lim et al. ( ) estimated more than million premature deaths per year due to indoor air pollution, because of solid fuel used for cooking purposes. exposure to high concentrations of harmful substances in smoke during use of biomass fuel causes significant illness amongst homemakers and young children. it has been shown that biomass fuel is a less efficient means of energy production and a number of carcinogenic constituents are released during biomass combustion (chafe et al. ; smith and sagar ) . inhalation of these particles in high concentration leads to 'lung overloading' and sustained inflammation. this results in the release of reactive oxygen that causes deoxyribonucleic acid (dna) damage. indoor smoke produced due to burning of solid fuel contains many pollutants. particulate matter, nitrogen oxides, carbon monoxide, benzene, . butadiene, polycyclic aromatic hydrocarbons, free radicals and volatile organic compounds are many of the toxic substances that have been found in smoke produced by burning solid fuels. chronic exposure to these harmful substances leads to lung fibrosis and subsequently the development of lung cancer. the evidence for the development of lung cancer due to biomass exposure has been shown in experimental animals, but the evidence in humans is not that strong. indoor air pollution thus accounts for a significant proportion of the global burden of disease in developing countries. the link between solid fuel exposure and chronic obstructive lung disease in women and acute respiratory tract infection in children is strong. the commendable initiative by the government of india called 'give it up' is a step in trying to decrease the effects of indoor air pollution on human health. also steps to improve ventilation in kitchens or use smokeless stoves chulla may also help in reducing the exposure to indoor air pollution (reddy et al. ). waste generated from used electronic devices and household appliances constitutes e-waste. it comprises of a wide range of equipments and devices falling under 'hazardous' and 'non-hazardous' categories such as computers, mobile phones, refrigerators, washing machines, air conditioners, personal stereos, consumer electronics, etc., that are discarded by users (puckett et al. ) . pollution due to electronic and electrical waste has rapidly grown over the last decade due to progressive increase in production of electronics, lack of proper disposal facilities in india and dumping of e-waste from developed countries. in alone, india generated about . million tons of e-waste (double the amount as compared to ), which is progressing rapidly. e-waste may contain many toxic substances which may be harmful to the environment and human health. this can have a significant economic and social impact on society. iron and steel constitute about % of the e-waste followed by plastics ( %), nonferrous metals ( %) and other constituents ( %). others include nonferrous metals like copper, aluminium, silver, gold, platinum, palladium, etc. the presence of elements such as lead, mercury, arsenic, cadmium, selenium and hexavalent chromium, with flame retardants beyond threshold quantities of e-waste, classifies them as hazardous waste. manual recycling of e-waste is done predominantly via the unorganized sector, and the work force involved consists predominantly of individuals with low literacy and hardly any training to protect themselves from ill effects and to identify warning signals of toxicity. accordingly, a significant percentage of health problems due to e-waste results from direct contact with harmful materials and inhalation of toxic fumes. moreover, these materials may get accumulated in the food and water and are consumed. heavy metals such as lead can cause kidney failure, neurologic manifestations and hypertension. mercury toxicity can lead to central and peripheral nervous system damage and hepatic and renal toxicity (guleria ) . furthermore, uncontrolled burning, disposal and dismantling of e-waste can cause a number of problems including air pollution and water pollution. there is a lack of an environmentally effective recycling infrastructure for e-waste, and this leads to pollution of the environment. this is gradually changing our ecology. there is, therefore, a need to increase public awareness about the harmful effects of e-waste and develop an effective recycling and disposal plan, to prevent or minimize air and water pollution. there should be general awareness of how changes in climate and environment lead to significant acute and chronic effects on human health. these effects can be both for infectious and non-infectious illnesses. a holistic view is needed to address the problem of environmental health where agriculture, animal husbandry, public health, water safety and air pollution need to be looked at in a combined manner for education, planning and resource allocation. general population should also be made aware about the ways to reduce harm to our environment. intergovernmental efforts should be made to check climate change, avoid deforestation and use alternative sources of energy like solar energy instead of petroleum products. ultimately, as embedded in its definition, ecosystem is a community, and unless all people in community put efforts to conserve it, no amount of individual effort can suffice. therefore, a close teamwork between scientists, public health professionals and administrators is needed for integrated vertical and horizontal planning. arsenic exposure from drinking water, and all-cause and chronic-disease mortalities in bangladesh (heals): a prospective cohort study blood and air pollution; state of knowledge and research needs symptomatic bronchoconstriction after shortterm inhalation of sulfur dioxide health risk evaluation of nitrogen oxides update in environmental and occupational medicine global assessment of el niño's disaster burden update in environmental and occupational lung diseases long term 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pollution el niño and health association between lifetime ambient ozone exposure and pulmonary function in college freshmen-results of a pilot study ambient air pollution and atherosclerosis in los angeles ambient air pollution and the progression of atherosclerosis in adults a comparative risk assessment of burden of disease and injury attributable to risk factors in regions - : a systematic analysis for the global burden of disease study physico-chemical examination of water sewage and industrial effluents. physico-chemical examination of water sewage and industrial effluents proxy-based reconstructions of hemispheric and global surface temperature variations over the past two millennia a reconstruction of regional and global temperature for the past , years climate change : impacts, adaptation, and vulnerability: contribution of working group ii to the third assessment report of the intergovernmental panel on climate change climate change and human health: present and future risks long-term exposure to air pollution and incidence of cardiovascular events in women sea-level rise and its impact on coastal zones effects of environmental change on emerging parasitic diseases the potential health impacts of climate variability and change for the united states: executive summary of the report of the health sector of the us national assessment impact of regional climate change on human health particulate air pollution as a predictor of mortality in a prospective study of us adults exporting harm: the high-tech trashing of asia. basel action network. e-waste/technotrashfinalcom air pollution and lung cancer incidence in european cohorts: prospective analyses from the european study of cohorts for air pollution effects (escape) carbon monoxide poisoning-a public health perspective domestic cooking fuel and lung functions in healthy non-smoking women air pollution and forests: interactions between air contaminants and forest ecosystems making the clean available: escaping india's chulha trap outdoor air pollution: asthma and other concerns extinction risk from climate change forecasting agriculturally driven global environmental change the effect of ozone and sulphur dioxide singly and in combination on plant growth effects of ozone on lung function and lung diseases key: cord- -rr xua authors: nan title: news date: - - journal: aust vet j doi: . /avj. sha: doc_id: cord_uid: rr xua nan photo: istock adelaide vet speaks out against dog anti-vaxxers an adelaide vet has taken to the air to address a growing number of pet owners who believe that vaccinations cause autism in dogs. speaking to the fiveaa radio station, dr derek mcnair expressed concern that certain areas of adelaide had low vaccination rates as a new trend of using herbal remedies instead of vaccines grows in popularity. "i'm not anti-holistic treatments and helping for some things, but to use it in place of vaccines is totally ridiculous and stupid." the trend comes as "anti-vax" material floods the internet, with the british veterinary association recently forced to publicly deny that dogs can get autism. similar discussions have been across the media throughout australia. the avas president, dr paula parker, has been responding to the media with a clear message that it is dangerous to forgo pet vaccinations. shipments of australian and new zealand dairy cows to sri lanka have been halted after a large number of the imported livestock have died on-farm. the sri lankan government has ordered an inquiry into the high mortality rates, along with reports of poor milk yields, low conception rate, stillbirths, and unrecognised diseases. queensland country live reports that , holstein-jersey heifers have been shipped to sri lanka under a government program to increase the country's fresh milk production. local farmers say the cows are unsuited to sri lanka's hot and humid tropical conditions, despite the same australian-bred cows have been performing well in indonesia. the ava is monitoring the story, particularly additional disclosures on the compliance with the nutritional, veterinary and animal husbandry recommendations made. woolworths over price rise news report a number of pet food brands have recently gone missing from supermarket shelves following a pricing dispute with manufacturer mars petcare. coles and woolworths' customers will have to go elsewhere for their pet's favourite brand after mars pulled the plug on distribution to the two supermarket giants following a price rise disagreement. mars produce different pet food brands globally, including royal canin, pedigree, whiskas, dine and my dog. representatives from the supermarkets said they expected a deal to be brokered soon. you can't be what you can't see i spent much of january and february of this year buried in research and thinking about women, leadership and the future of our profession. social media is littered with quotes and catchphrases about leadership. one that stuck with me through the data and the papers is 'you can't be what you can't see'. if you can't see someone that seems similar to you in a position of leadership, then it can be difficult to believe that that role, or one similar to it, is possible for you, or although it might be possible, the cost of forging a scarcely trodden path may be too great. there is some veterinary specific data that outlines this well. in the november report 'motivation, satisfaction and retention: understanding the importance of vets' day to day work experiences' from the bva and the university of exeter, two of the key findings included 'feeling like one fits in with those who have been successful before you, and having role models' as being important to motivating veterinarians, facilitating their professional satisfaction and retaining them in the profession. of the myriad of issues that our profession faces and cares deeply about, the financial security of veterinarians, and particularly of women, is the one closet to my heart. i have seen several members of our profession profess that as a natural consequence of more women in the profession, there will be increasing gender representation in leadership positions overtime. this is an alluring proposition, but not one supported by the data. vertical segregation describes the phenomenon where there is predominance of men in more senior positions, and a predominance of women in more junior positions. vertical segregation occurs across every aspect of the australian economy, and even in female dominated professions, women are under-represented in leadership roles. the reasons for this are a complex interaction of unconscious bias, structural and cultural factors that require more discussion than can be accommodated in this column. one of the recommended interventions however, is to shed light on diverse role models. one of the greatest pleasures of being the ava president, is the opportunity to work with leaders across our profession, in all of the contexts where we contribute to the community. so, in this column, i wanted to introduce you to some of the women i have had the privilege of seeing in action and whom i admire for their leadership, their integrity and their service. i hope that you can see them too and know that regardless of the leadership role you would like to play, that there is someone who has forged a path. • if you want to be a pillar of your community, meet melissa rogers. • if you want to steward the standards of our education and be proud to promote them to the world, meet julie strous. • if you want to be a young veterinary business owner, meet kat lovell. • if you want to be a part of our leadership of animal health in the world, meet jennifer davis. • if you want to lead a global animal health company, meet stephanie armstrong. • if you want to be excited about nutrition and life, meet penny dobson. • if you want to champion collaboration across our sector, meet janet murray. • if you want to know how to exemplify excellence, compassion and grace, meet liisa ahlstrom. • if you want to be a pioneer in our sector and do so with ethics, charisma and a smile, meet rachel chay. • if you want to meet a leader who understands how looking after your team makes good business sense and puts it into practice, meet jacqui von hoff. • if you want to change the face of dairy medicine, meet gemma chuck. • if you want to feel excited about being a veterinarian and putting that out there into the world, meet zoe vogels. • if you want to lead the education of the next generation of veterinarians, meet cristy secombe. corey snell, ceo achieving better health outcomes for pets as you read this column our flagship ava annual conference in perth will have concluded. this year the theme was "the profession: better working together". here we introduced an innovation symposium and our food security day. the food security day focused on biodiversity and welfare, bringing experts from around the world together to discuss the challenges and opportunities faced to sustainably feed more than . billion people by . both of these broke new ground for the ava and our aim is to continue these inaugural events. breaking new ground is not limited to our conference events. at the ava we have been asking ourselves how the ava could assist you in contributing to the optimum health and welfare outcomes for australian pets. most importantly how could we assist our members in providing better care to ensure happier clients? as i have learned more about the challenges our veterinarians face, i have been particularly concerned by the pressure experienced by veterinarians resulting from the inherent tension between cost of veterinary care and our clients propensity to pay for services. i understand that this is one of the biggest causes of stress for the profession. i imagine it must be heartbreaking when a pet's owner is unable to pay for veterinary treatment and they are forced to make a decision that is not in the best interests of the animal. one of the tools which can help the financial preparedness of our clients is pet insurance. at the ava annual conference, we launched our partnership with guild insurance in the development of an ava endorsed pet insurance offering called 'vets choice'. guild insurance and the ava have collaborated to develop a pet insurance solution that that has been designed to meet the needs of our profession and our customers. it is my hope that by increasing the proportion of pet insurance in australia will lead to a more sustainable veterinary practices and i am particularly excited at the opportunity this provides not only the broader public but our profession and our members as well. we've got something exciting coming… vets choice is a modern pet insurance product, packed with market leading features and benefits, brought to you by the ava and guild insurance. we'd love to tell you what the product is, but it's different now than it was three weeks ago, and we're still working to refine and respond to the needs of what both you as the vet and your patients need most. send an email to vetschoice@guildinsurance.com.au if you want to be among the first to be part of it… is the dingo really just another type of dog? the dingo has been present in australia for at least the past few thousand years, with the earliest dated fossil being around years old. they are recognised native animals, but are also classified as a pest species in many jurisdictions and often defined as 'wild dogs'. since the commencement of livestock farming across australia over the past years, the dingo has had a negative relationship with graziers. early on, many efforts were made to eliminate populations and this has resulted in the dingo being absent from many parts of its historical range. the control of wild dogs is an ongoing issue for graziers because they can cause significant financial losses through predation of sheep, goats and calves. by sight alone, it is often difficult to differentiate dingoes and their hybrids from wild dogs and it is this dilemma that lead to the western australian government to recently consider removing the dingo's status as native wildlife and classify them as 'non-fauna'. this would make them no different to any other dog and allow them to be killed throughout the state. the canidae is a diverse group that includes the domestic dog, wolves, coyotes and dingoes. individuals can interbreed and produce fertile offspring and therefore technically could be classified as one species using the 'biological species' concept. istock photo news n a paper published in the veterinary record last month investigated the presence of bacterial contamination in commercially available raw meat pet food products in sweden. samples were taken from raw meat pet food products sold in supermarkets in sweden and produced by different pet food manufacturers. results showed all samples were contaminated with enterobacteriaceae bacteria, suggesting faecal contamination because these bacteria are found in the gastrointestinal tracts of animals. the presence of e. coli and other enterobacteriaceae bacteria is often used as an indicator of hygiene during processing. in addition to the presence of enterobacteriaceae bacteria being found on the samples, salmonella species were found in four of the samples and campylobacter species in three samples. in two samples, the level of clostridium perfringens bacteria exceeded bacteria/g, which is above the maximum limit for anaerobic bacteria permitted by swedish national guidelines. the authors concluded that raw pet meat food sold commercially in sweden should be handled carefully, with owners needing to implement strict hygiene protocols when storing, handling and feeding raw meat pet food products. they also discussed the potential health risks that raw meat pet food products present to both humans and animals, particularly geriatric, neonatal and immunocompromised individuals. these findings come at a time when raw meat-based diets for pets are becoming increasingly popular. the proliferation of both commercially available raw diets such as 'biologically appropriate raw food (barf)', as well as homemade diets, is leading to growth in the raw pet meat sector. although commercial dry and canned pet foods still dominate the pet food market, the introduction of raw diets and homemade diets is leading to a small but passionate group of owners who promote raw diets for health and philosophical reasons. the pet food industry in australia is currently under review. after a senate inquiry examined regulatory approaches to ensure the safety of pet food in late , the resulting inquiry's report recommended widespread changes to both oversight and safety standards of the sector. the federal department of agriculture and water resources is currently leading a working group comprising representatives from state governments, the australian veterinary association, rspca australia and food standards australia and new zealand. this working group is examining how best to regulate and improve safety standards in the pet food industry. commercially available raw pet meat products will be included in this review, as well as treats and manufactured pet food. it is currently unknown if the results of this swedish study are replicated in the raw pet meat market here in australia. similar results were found when raw pet meat products were tested in the netherlands in , with high levels of bacterial contamination as well as several parasites including sarcocystis species and toxoplasma gondii detected. if owners insist on feeding raw pet meat products to their pets, the authors of the veterinary record paper recommend the following risk reduction tips: keep raw pet meat frozen until use, always handle raw pet meat with separate utensils as well as wash hands thoroughly after contact and do not feed raw pet meat products in households with immunocompromised individuals. in early april the animal health quadrilateral group (quads), senior animal health officials from australia, canada, new zealand and the united states met for their annual meeting. the issue of attracting, developing and retaining government veterinarians was an item of keen interest on the agenda. government veterinarians play critical, unique and diverse roles in a country's veterinary services, which underpin a country's animal health status and trade. the risks and opportunities in animal health will continue to evolve, as will the expectations, interests and demographics of the veterinary profession. the future will bring new information and diagnostic technologies; changes to risk and community expectations; and evolving communication needs. therefore, we need to be proactive, and ensure the skills of future veterinarians reflect this changing context. in the field of government veterinary services there may be a need for increased veterinary public health specialist expertise, such as epidemiology, risk management, and diagnostics. it's also expected that we will need to increase our collaboration with experts from other disciplines such as information technology and data analysis; laboratory, public health, social and environmental scientists; communications professionals; and economists. the world organisation for animal health (oie) is currently developing their th strategic plan ( - ) and they too are looking at their future role, particularly on global issues such as food security, climate change, species conservation and the future of the veterinary profession. as president of the oie, i have been leading discussions in the development of the oie's th strategic plan. in reviewing the external forces that will impact the organisation's future we see the issues of big data, centrality of social media, changing public perceptions and declining trust in governments, and we need to take these issues into account. at home, the australasian veterinary boards council (avbc) has discussed the future of the veterinary profession, and therefore how to educate the veterinarians of the future. similar to the quads group and the oie, the avbc also recognised that the profession's future is inherently linked to broader global issues. veterinarians will be managers of expert knowledge and will need to work in the one health space, where environmental, human and animal health intersect. technology is playing an increasing and positive role in veterinary science education. assisted and virtual reality enhance practical learning and learning is expected to become increasingly collaborative with students study virtually across a range of institutions. it reminds me of the plenary presentation by jordan nguyen at last year's ava conference on 'the boundaries between human and technological evolution and intelligent technologies'. he certainly opened my eyes to what the future might look like! and speaking of the future, i have recently really enjoyed getting around to a few of our veterinary schools during my travels and speaking with the veterinarians of the future. i recently dropped in to the campus in gatton and met with university of queensland staff and students. i really value the perspectives gained from these visits and hearing what's happening at the forefront of our profession. this month, following the ava conference in perth, i'll return to my alma mater, murdoch university, to speak with and meet staff and students about the importance of one health and the broad range of opportunities available to us as veterinarians. vets really do play such an important role in animal and human health and we have the opportunity to truly contribute to the important issues facing the globe. following the ava conference and trip to perth, i will be flying to paris for oie council meetings in mid-may and the general session at the end of the month, my first as president of the oie assembly. i look forward to introducing you to a couple of members of the australian delegation to the oie general session so they can tell you about their veterinary career paths. assisted and virtual reality enhance practical learning and learning is expected to become increasingly collaborative with students study virtually across a range of institutions. veterinarians have been thinking about preventive care protocols in their clinics for some time now offering annual wellness checks. these annual checks may include vaccination based on the patient's individual needs, and may or may not include diagnostics. for some, the decision not to include diagnostics in the wellness check has been due to consideration of whether there is value in adding diagnostic testing when a patient has an unremarkable history and clinical examination, but the evidence to do so is mounting. several peak bodies have produced guidelines on care of dogs and cats at different life stages, including the american association of feline practitioners (aafp) , , the american animal hospital association (aaha) , , and the australian veterinary association . all of these recommend regular health checks (annual for adults, and every months for seniors and older), with diagnostic testing included, especially as animals age. add to this, a series of published studies evaluating the results of diagnostic testing in apparently healthy adult and senior pets, and we can start to draw some evidence-based conclusions around the value of diagnostics in this preventive care setting. in a study of cats and one involving dogs , there were significant numbers of patients with changes on blood and urine screens, and a more recent study undertaken in australia involving dogs and cats showed . % of dogs and . % of cats with significant changes that warranted further investigation . aaha however, were interested in leveraging the huge volumes of data produced by the industry each year to understand the issue on a much larger scale, and took a somewhat different approach to data collection . in their 'big data' study -the aaha research team evaluated over , patients, but not necessarily in the same detail as the previous studies. here are some details from the study that all veterinarians should be aware of: • data was gathered from , consenting practices and harvested from their practice management software • investigators evaluated profiles on patients that were classified as 'wellness examinations' in the practice information management system • the patient visit needed to include a cbc, full chemistry profile including idexx sdma®, and ideally a urinalysis. • for some parameters, the reference interval was widened slightly to a critical threshold before a result was abnormal (for example for liver enzymes the lower range was set to zero, and in dogs the upper end of phosphorus was increased by %). • for the majority of biochemical parameters, the requirement was that at least three or more results had to be abnormal before the findings were considered significant. • a single abnormal sdma result was considered significant. • for haematology, only numerical values were assessed (changes on blood film reviews were not included), and abnormalities identified included: • anaemia • reticulocytosis • leucocytosis • stress leucogram (lymphopaenia and eosinopaenia) • eosinophilia in many regards the approach taken was a conservative one and means that potentially significant changes preventive care were under-reported. this in-built 'conservativism' is because not all the haematological parameters were evaluated, and there was no comparison to previous results. where we now recognise the benefits of evaluating the health status of the pet by using an individual reference interval (comparing current results to previous results obtained for that patient in health) for a number of routine parameters, creatinine being a good example. in the aaha study the results were as follows, with respect to results that warranted further investigation. in this study the addition of sdma made a significant difference to the number of patients warranting further evaluation. in the adult and senior populations there were an extra per patients who required further investigation, whereas it was per for geriatric patients. in conclusion, and as we seek to enhance the health and wellbeing of pets, people and livestock, i would suggest that studies such as the ones discussed here provide supporting evidence that the inclusion of diagnostic testing in discussions on wellness and preventive care is critical. moreover, observation suggests that it is also something that clients have come to expect. the animal medicines australia pet ownership report showed nearly as many clients presented their pets to the vet to keep them well as those presenting for illness . dr graham swinney bvsc, fanzcvs (canine medicine) the eastern bettong is a small marsupial that was once common across the east coast of australia. shortly after the introduction of predators, particularly the fox, their populations crashed and they became extinct on the australian mainland around years ago. fortunately for the eastern bettong, tasmania is free of foxes and as a result is a remaining stronghold for the species. breeding programs for the bettong exist in predator-proof areas in the act and the animals in these protected areas are continuing to flourish. in , eastern bettongs were introduced to the wild near the lower cotter river catchment, kilometres southwest of canberra in an area that had been heavily baited to reduce fox population numbers. eight months after their initial release and through intense monitoring, ecologists involved with the program discovered that every female bettong found showed signs of breeding success. at the time this was a huge success for the program as it was the first time that wild-bred eastern bettongs were present on the mainland in the past years. some concerns were raised at this time, because survival rates of released bettong were found to be around %, but there was hope that the new joeys being born would help boost future populations. act parks and conservation was responsible for controlling feral pests in the release area and at the peak of the project were servicing bait stations on a weekly basis as well as monitoring between and wildlife cameras to identify the animals present in the area. unfortunately, abc news has recently reported that all eastern bettongs released into the wild as part of the project have now died and foxes are being held responsible. although the project was not as great a success as had initially been hoped for, director of act parks and conservation services, daniel iglesias said that the team has still gained valuable information through the project because during the early months the bettongs were coexisting and reproducing in the presence of foxes. however, this only occurred when the fox population was low enough to allow the bettongs to survive, and the methods used to achieve this low level of foxes in the wild are not practical on an ongoing or widespread basis. the introduction of predators to australia has been a contributing factor to the demise and extinction of our native mammals and given us the world's worst rate of mammal extinction. it is not possible to ever eliminate the threat of introduced predators from the wild, but finding ways to successfully manage their populations on a large scale is something that will need to be focussed on and invested in by the government, if we wish to slow down or reverse our rapid extinction rates of native wildlife. if this ever becomes a reality, we may one day find eastern bettongs scurrying around in areas they've been absent from for over years. the complexity of chf means a multitude of treatment approaches are required to ensure a good quality of life and longevity in our pets. in her thesis, mariko examines current treatments of chf, posing vital questions on how they can be improved. a new focus in congestive heart failure? it is well known that pimobendan, in conjunction with other drugs, is key to the treatment of chf. it is currently most commonly used and prescribed in tablet form; however, mariko assesses the use of a pimobendan oral solution in dogs. the pimobendan solution was seen to enhance echocardiographic measures of cardiac systolic function, reaching a maximal effect at - hours following administration. this study is notably the first to use non-invasive imaging of the acute cardiovascular effects of pimobendan. in addition to validating the efficacy of this solution, it raises the possibility of therapeutic monitoring in the future. mariko then examined the use of pimobendan in cats and her findings clearly showed reduced metabolism of pimobendan to its active metabolite in cats compared with dogs. interestingly, the cardiovascular effects of pimobendan could not be readily identified on echocardiography. these interspecies differences highlight the necessity for further research into the role and optimal dosing regimen of this drug in feline cardiovascular disease. in addition to examining existing treatment options for chf, mariko also studied the potential of the natriuretic peptide system as a therapeutic target in dogs with chf. in a recently published article, she identified that the cardiorenal effects of bnp - (a type of natriuretic peptide) were diminished in dogs with chf caused by myxomatous mitral valve disease. this suggested defects in what is essentially a protective mechanism in the context of chf and is a finding that prompts further investigations into therapeutics targeting this system. a recent case involving a fashion industry start up being penalised around $ , for underpaying staff has brought the issue of unpaid work into the spotlight. this came only weeks after a prominent australian company director was slammed for making comments around millennials "not wanting to work for free". these situations -while very different -both raise the question: is unpaid work permissible? the case. it's important to first outline exactly how and why this particular fashion start up fell foul of the fair work commission. over a -year period, the business was reported to have underpaid separate employees around $ , . one particular worker was said to be on an "unpaid internship" for a -month period -even though the nature of their engagement was that of an employee. the commission found that the business had failed to pay the workers their minimum entitlements under relevant employment legislation including minimum hourly rates, penalty rates and leave entitlements. as a result of the underpayment, the company was penalised around $ , and the sole director was individually penalised a further $ , . back pay was also ordered in full to the three workers. the severity of the penalty was largely due to the fact the company were said to have known they were under paying staff, with the commission labelling the underpayments "significant and deliberate". in short, yes, but be careful. as highlighted by the fair work ombudsman, sandra parker in the wake of the case, "unpaid placements are lawful where they are part of a vocational placement related to a course of study. however, the law prohibits the exploitation of workers when they are fulfilling the role of an actual employee." if you have been approached by a student looking for work experience, ensure it is part of a genuine placement through their learning institution. it might also be prudent to contact the institution (tafe, university etc.) directly to discuss the finer details including insurance and whether it can genuinely be unpaid. other than student placements -are there any other situations where unpaid work is permissible? there may be other situations where work can be unpaid, but this is assessed on a case by case basis and employers should check with the ava hr advisory service before providing any unpaid work. when the work is not part of a vocational placement, some of the major factors when considering whether work can be unpaid include; • will the work be for the benefit of the individual, or the business? • if the work primarily benefits the business, it is likely to require payment. • how long did the arrangement last? • the longer the arrangement, the more likely it is to requirement payment. • what was the nature of the work? productive work that is meaningful to the business will almost certainly requirement payment. observing, learning and performing very rudimentary tasks that give the individual a general "feel" for a role or industry may form part of an unpaid working arrangement. considering the above, unpaid work should almost solely benefit the individual (not the business), should only be for a brief period and should be largely observational. an unpaid trial can be permissible if it is purely to demonstrate a skill or skills associated with a particular role. the engagement should be brief (maximum one shift) and the individual should be under direct supervision the entire time. let it be clear -this is unrelated to a probation period which is sometimes referred to as a trial period. a probation period is generally between to months and must be paid as an employment relationship has already commenced. in summary. if you are considering engaging someone to perform unpaid work, ensure what you are doing is lawful and permissible. ask yourself the following; • is it part of a vocational placement related to a course of study? • if so, check with the learning institute to ensure it is unpaid. • -is it an individual wanting to gain experience in the industry (no vocational placement)? • ensure they do not perform any productive work, and their engagement is brief and observational. • is it a trial? it must be purely about the individual demonstrating skill/s relating to the role, it must be brief and the individual must be directly supervised for the entire engagement. if your plan to engage someone doesn't match any of the above, it is likely they will be an employee and will require payment. if you are unsure about anything in this article, contact the ava hr advisory service on or email hrhotline@ava.com.au. the material contained in this publication is general comment and is not intended as advice on any particular matter. no reader should act or fail to act on the basis of any material contained herein. the material contained in this publication should not be relied on as a substitute for legal or professional advice on any particular matter. wentworth advantage pty ltd, expressly disclaim all and any liability to any persons whatsoever in respect of anything done or omitted to be done by any such person in reliance whether in whole or in part upon any of the contents of this publication. without limiting the generality of this disclaimer, no author or editor shall have any responsibility for any other author or editor. for further information please contact wentworth advantage pty ltd. results from the survey revealed there were more than , volunteer wildlife rehabilitators in nsw, with almost half of those belonging to the largest wildlife rehabilitation group wires. on average, , animals go through wildlife rehabilitator hands every year and more than , calls for assistance and education are provided to the public annually. it was estimated it would cost the government, conservatively, more than a$ million to replace the time and resources spent by volunteer wildlife carers every year in nsw alone. most wildlife rehabilitation is conducted in the homes of volunteers, although a small number of wildlife hospitals and centrally based facilities supplement this home care. stemming from surveys and consultation work with peak bodies, the work of private veterinary practices was recognised as an essential element in the wildlife rehabilitation sector. it was estimated around , wild animals are seen by nsw private veterinary practices annually at a cost of more than a$ . million of pro bono services and products. this differs significantly from a previously published survey by orr and tribe, which estimated around , wild animals were seen annually by nsw private practices. it is currently unknown just how many wild animals pass through veterinary practices every year, although the strategy did recognise that "balancing the running of a private practice with the lack of time, facilities and resources for treating free-living wildlife is very challenging." through the findings of the two surveys and stakeholder consultation, several challenges facing the sector were identified. wildlife rehabilitators overwhelmingly identified the finding and keeping of new volunteers as a significant issue, with social demographics, time, financial demands, group politics, burnout and conflict all affecting increased participation rates. other challenges identified in this process were the lack of succession planning in many groups, particularly because of the older demographic of wildlife rehabilitators. this may lead to a loss of skills, less community support and fragmented and inefficient use of volunteer time if unmitigated. three-quarters of wildlife rehabilitators who responded to the survey indicated their support for stronger standards of care for wildlife as well as improved access to mentoring. one of the aims of this draft strategy is to standardise training and introduce a minimum standard of care for the sector. this would include a standard induction for all volunteers, as well as specialised species training to improve volunteers' depth of knowledge. it's important to note that the strategy acknowledges this is an ambitious goal. other challenges identified by volunteers as affecting the sector included a lack of strategic support from a strong, unified peak body. even though more than half of all wildlife carers belong to the group wires, most carers wanted improved advocacy and leadership for the sector and greater access to funding opportunities. less than % of all respondents were satisfied with the current level of support provided by the state government. unsurprisingly, a lack of funds was another key challenge facing volunteers. around % of wildlife carers surveyed reported incurring out-of-pocket expenses in the year prior. specific issues facing private veterinary practices many of the veterinarians and nurses surveyed reported their formal education wasn't very useful for dealing with injured free-living wildlife. additionally, the most common complaint from veterinary respondents was in relation to the behaviour of volunteers and their group leaders, as well as response times for collecting injured wildlife. the role of wildlife rehabilitation in environmental management was recognised as a key strength of the sector. most volunteers considered their efforts as greatly benefiting the environment. increasing the collection and use of data captured by the wildlife rehabilitation sector would improve wildlife and threatened species management, which was identified as an important outcome of this strategic document. additionally, much-needed research into post-release outcomes could be facilitated by better engaging with the wildlife rehabilitation community to improve treatment options and management decisions. several of the changes proposed by this draft strategy could have important outcomes for improving wildlife health and welfare in nsw. the strategy aims to: • develop a peak body for wildlife rehabilitation in nsw by either encouraging the two main bodies to unite or create an advisory board comprised of government, wildlife rehabilitation, animal welfare, veterinary and natural resource representatives • develop standards to be adopted by the sector • develop a charter for volunteer engagement with veterinary practices • commit a$ . million to implement the strategy • introduce minimum standards for volunteer training, with learning outcomes and performance criteria to demonstrate proficiency • commit a$ . million (of the $ . million total) to deliver standard resources for veterinarians and veterinary nurses in the handling, triage and treatment of wildlife; this will be delivered by taronga zoo in partnership with the university of sydney and the office of environment and heritage • streamline data capture via online reporting of wildlife rehabilitation statistics and prepare annual reports for the sector and community • fund post-release monitoring of rehabilitated koalas • fund a single wildlife rescue telephone number for the public. nsw is not the only state that will see changes to the wildlife rehabilitation sector in . at the start of the year, western australia introduced licensing for all wildlife carers to keep track of volunteers and the relevant department in tasmania is currently finalising resources for veterinarians and nurses to aid in the triage and treatment of wildlife. it is expected the final nsw strategy will be implemented from july . news n i've recently been involved in the development of a business plan as part of my role as the ava student president. given my 'young grasshopper' status i found myself thinking, what does it take to be an entrepreneur in the veterinary industry? do people just find themselves taking over the mantle of head vet/ business owner as a rite of passage? is it a matter of luck, just serendipitously having the opportunity present itself, or can a fresh out of school veterinarian manifest it through sheer will power and effort? personally, i'm still holding out for a small loan of $ million from my parents. to find out more i had a chat with zachary lederhose, a recent graduate who has stepped into the world of business ownership. it might be more accurate to describe his current success in the field as 'hitting the ground running' but he won't claim as much. what motivated you to start your own business, zach? well, like just about every new graduate, i started out working as a general practitioner. loved the job and was a fantastic learning experience. that said, the main thing that stood out for me was not being able to have as much say in the direction we took as a team that i wanted. it wasn't that anything was wrong, it was a great atmosphere and we all got along really well. the sense of autonomy was what i sought. did it change much for your income? i wouldn't say it was the main driver behind my ambition to start a business. i was doing well in my previous role and could have very happily continued as such in that space. it all depends on what you, as a professional, value and where you get satisfaction in your job. for me, it felt like my earning potential was capped as an associate and that limited my opportunities. being my own boss makes it easier to pursue some of the things that interest me. for example, being able to purchase certain medical equipment to provide niche services or the flexibility to attend conferences that interest me. how do you find being responsible for a team? i think i was very fortunate that in my first job i had a great mentor in mike mesley at snowy vets. mike taught me a lot and we share similar opinions on practice management. with that behind me, i was confident in my abilities and i think that's a big part of the equation, believing you can actually do it. i also invested a lot of effort into developing my communication skills and bring the same focus to my current clinic. it's such an important skill -not just for interacting with clients and achieving the best outcome but to make sure we're working well together. the feedback i get from my team is important to me. as great as it is to have the constant loop of positives and 'yes men', sometimes i just need to be told i'm being a bit of an idiot. keeping my team comfortable to be that voice of reason helps me take stock of what i'm doing and means they are engaged in the decisions we make for the clinic. do you find your age and experience level makes it harder? to be honest, not a great deal. sure, i've faced some challenges along the way but nothing that anyone else starting out wouldn't come across. i would probably put it down to having a fantastic support network, including people from my private life. i can acknowledge i've been fortunate in that regard and it probably isn't the case for many of my peers. in a lot of ways, it comes down to having someone you can relate to who's been through the process. with a shifting demographic in the profession, i can imagine that's not always easy. that said, i did have a client ask how work experience was going for me the other day… i've certainly drawn some inspiration from my conversation with zach. hopefully for some of you reading it does the same, giving you that extra bit of encouragement to be brave and take a leap. similarly, if you're already a business owner keep an eye out; there are a fair few sharp minds coming through the ranks that could use your help. i'm fascinated by the prospect of more people like zach taking steps forward in business and the impact their innovation has on the profession, even if it's just one workplace at a time. all things considered, i should probably pick up that copy of barefoot investor that's been sitting on my coffee surprisingly, cats of all ages, and with both effusive (wet) and non-effusive (dry) forms, responded equally well to the treatment. the response was dramatic, with fever typically resolving within - hours of commencement and concurrent daily improvements in appetite, activity levels and weight gain were seen. cats with the effusive form had a reduction in abdominal effusion over a - -week period within - days of commencing treatment. after weeks of treatment, all cats that had responded to treatment appeared normal, or near normal, to their owners. the safety profile of gs- was also impressive, with no systemic signs of toxicity based on complete blood counts and serum chemistry values monitored over the treatment periods of - weeks in almost all individuals, with only one possible exception. a single cat showed a mild increase in renal parameters, but month later these abnormalities were not detected and the cat was also in remission. the study suggests using gs- at a dose of . mg/kg by subcutaneous injection every hours for weeks is a feasible way to treat fip. although the number of animals in this field trial study was limited, there were very promising results for commercialisation of this or similar nucleoside analogues in the future and someday fip may no longer be considered an untreatable disease. the studies examined the effects of two manufactured chemicals; diethylhexyl phthalate (dehp) and polychlorinated biphenyl (pcb ). dehp is a common plasticiser found in many household items (e.g. containers, toys, upholstery) and is used as a fragrance carrier in cosmetics, laundry detergents and air fresheners. pcb was used in coolant liquid but is now banned globally. both chemicals are widely detectable in the environment and one of the most common routes of exposure is ingestion of food. the researchers exposed sperm samples from both species to these two chemicals at concentrations that have been previously detected in the male reproductive tract and at levels commonly found in the environment. in both species, the exposure to these chemicals at these levels resulted in reduced sperm motility and increased the fragmentation of dna, which negatively affects overall fertility. as we increasingly share our homes with our pets, they are exposed to the same environmental and household contaminants as us and it is this exposure that researcher associate professor richard lea believes may be responsible for the fall in sperm quality in both dogs and humans in recent years. this finding also highlights the opportunity to use dogs as an effective model for future research into the effects of pollutants on human fertility, as other external influences, such as diet, are much easier to control in dogs than humans. stephen reinisch even more surprisingly, when used to inseminate ewes, both samples had almost the same pregnancy rate, with a rate of % for the -year-old sperm compared with % for the recently frozen sperm. this finding of the long-term viability of frozen semen is significant because it shows that genetics from individuals can be stored and used long after the original male has passed away, which could prove useful for breeding programs for many species in the future. the lambs produced from the study will also provide a useful baseline for the researchers to examine the genetic progress made by the wool industry through selective breeding over the past years. these lambs displayed characteristics that were common in merinos at that time, with large body wrinkles to maximise skin surface area and therefore wool yields. that particular characteristic has since gone out of favour, having been found to increase the risk of fly strike and create difficulties with shearing. stephen reinisch veterinary writer stephen reinisch veterinary writer delivery terms and legal title prices include delivery of print journals to the recipient's address. delivery terms are delivered at place (dap); the recipient is responsible for paying any import duty or taxes. title to all issues transfers free of board (fob) our shipping point, freight prepaid. we will endeavour to fulfil claims for missing or damaged copies within six months of publication, within our reasonable discretion and subject to availability. printing and despatch printed in australia by ligare pty ltd. all journals are normally despatched direct from the 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creating new collective works or for resale. special requests should be addressed to permissions@wiley.com offprints printed offprints may be ordered online for a fee. please click on the following link and fill in the necessary details and ensure that you type information in all of the required fields: www.sheridan.com/wiley/eoc. policy predicaments or precedents i write with concern that the current ava live export policy has serious implications and potentially sets a precedent that is detrimental to the advancement of the profession. through its constitution , the ava seeks to speak on behalf of the profession (members and non-members) to government, authorities, any person, or company. policy and position statements are drafted by the policy council or the board and once approved by the board become official statements of the association. the ava uses policy and position statements to outline the position of the association when talking on behalf of the profession and these form the basis of advice given to government, other authorities, media, etc. the ava live export policy states: "where live export occurs, an australian-registered shipboard veterinarian must accompany each shipment and this veterinarian must be independent and thus not employed by either the exporting company or the shipping company." background information is provided in policy statements and is supposed to explain the rationale and scientific evidence supporting the policy position. the background information in the ava's live export policy does not provide any rationale or evidence why australian-registered shipboard veterinarian must not be employed by the exporter or shipping company. the exporter, in many cases, is the owner of the animals during the export voyage. if ownership has been transferred to another party, the exporter is the owner's representative and responsible for the animals during the export voyage. the rest of the policy is concerned about animal welfare, it is assumed the reason for the non-employment recommendation is to ensure animal welfare. this is concerning for a number of reasons. firstly, it implies that the legislative system regulating veterinarians in australia is broken, as the peak professional body is recommending the only way to ensure animal welfare is for veterinarians to be independent and not employed by the animal owner or owner's representative. it seems, the ava does not believe that the state and territory veterinary surgeons boards or the federal government have effective legislation or are able to effectively regulate against the current legislation. secondly, it implies there is a problem with fee-for-service arrangements between veterinarians and animal owners and achieving acceptable animal welfare outcomes. nearly all veterinarians providing clinical services to any species, either as an owner of a veterinary business or employee veterinarians, operate under this fee-forservice arrangement with the animal owner. how does the ava systematically review and assess policies for possible unintended consequences, implications, misrepresentation of the profession, or the possibility of setting a precedents that is not in the interest of the profession as whole? should the board review the live export policy, will the ava stand by its recommendations to the moss review and ensure that relevant people with leadership skills, deep knowledge and understanding of the industry, animal health and welfare, and epidemiology are sought? dr andrew way face the facts: gender equality corporate-site/documents/ animals-and-plants/native-animals/volunteerwildlife-rehabilitation-sector-strategyconsultation-draft- animal welfare implications of treating wildlife in australian veterinary practices tel: + ( ) japan: for japanese speaking support, email: cs-japan@wiley.com. visit our online customer help available in languages at www.wileycustomerhelp.com/ask production editor sylvia cheong. email: avj@wiley.com information for subscribers australian veterinary journal is published in issues per year. institutional subscription prices for are: print & online: us$ (australia & new zealand), us$ (us), € (europe), £ (uk), us$ (rest of world). prices are exclusive of tax. asia-pacific gst, canadian gst/hst and european vat will be applied at the appropriate rates. for more information on current tax rates, please go to www.wileyonlinelibrary.com/tax-vat. the price includes online access to the current content and all online back files to ava members communication live export update # this will be going to policy council and the chair of the policy council will provide a response following this meeting.access it on the go across any device -where you want, when you want.the avj will be a tap and swipe away from july www.ava.com.au key: cord- -y pzsoqa authors: adalja, amesh a. title: biothreat agents and emerging infectious disease in the emergency department date: - - journal: emerg med clin north am doi: . /j.emc. . . sha: doc_id: cord_uid: y pzsoqa the challenges faced by the emergency physician with recognizing and treating category a biothreat agents and emerging infectious disease are summarized and reviewed. emergency physicians in every location in the world, in developed and developing countries alike, will undoubtedly be confronted with the possibility of an emerging infectious disease in their career. a subset of these physicians may be faced with a patient who has potentially been exposed to biological weapons. of the myriad infectious disease emergencies an emergency physician contends with, these possibilities are the gravest and most impactful. in such scenarios, the emergency department (ed) clinician can be the key in recognizing or containing an outbreak. the challenge inherent with emerging infectious diseases presenting in the ed is that such cases can be camouflaged, lurking amongst innumerable infectious disease clinical syndromes, from common colds to viral rashes. this article provides guidance to emergency physicians as to how to approach this challenging problem as well as familiarizing readers with specific microbial threats of high consequence. a key method for detecting the presence of an emerging infectious disease syndrome or a biological weapons exposure in an ed patient is to develop a general approach that seeks out key historical and physical examination clues. this approach is not different from what is included in a full history and physical examination but requires meticulous attention to certain aspects of the history. travel history becomes a key focus of the history because many infectious diseases, especially of the emerging variety, have delimited borders in which they are prevalent. the travel history must be coupled, however, with situational awareness as to what infections are known to be present in specific parts of the world. such a task is daunting for most physicians and, therefore, it is important that they know where such resources can be found. both the centers for disease control and prevention (cdc) (www.cdc.gov/travel) and promed (program for monitoring emerging diseases) (www.promedmail.org) are such resources that are easy to access and continually updated. using these resources, a busy provider can quickly assess which specific infection risks any given country might confer. an important component of the travel history is understanding the dates of travel and how they relate to the incubation period of specific infections. travel must be contextualized and integrated with incubation period, because domestic infections acquired before or after travel might be mistaken for a travel-related infection. additionally, eds in a given geographic locale (eg, metropolitan area, county, or state) should develop a mechanism to have insight into changes in ed volume, chief complaint mix, and unusual diagnoses at other eds in the region. much of this can be accomplished through leveraging emergency health care coalitions and local or state health departments to develop tools to enhance insight into the vicissitudes of a given region's ed-relevant infectious disease problems through syndromic surveillance programs. an important component of an individual's risk for particular infections is related to exposures. attention must be paid to animal exposures (domestic and wild), eating habits, occupation, and hobbies. additionally, it is essential to determine if a person has had any sick contacts or has attended a mass gathering, because an ed physician might be seeing one of the first formal presentations of a wider outbreak. of the specific biological agents, the category a agents (anthrax, plague, tularemia, and botulism), and certain viral hemorrhagic fevers (vhfs) (eg, ebola, marburg, machupo, and lassa fever) are of the highest priority. table provides salient points regarding the treatment of the category a biothreat agents. in all cases of uncertainty, prompt consultation with an infectious disease physician is recommended. anthrax is caused by the gram-positive bacillus, bacillus anthracis. it is a ubiquitous spore-forming gram-positive bacterium that is found naturally in the soil worldwide. it is a disease of herbivores. humans can contract of forms of the infection: cutaneous, inhalational, injectional, and gastrointestinal. , of these forms, cutaneous is by far the most common and represents a majority of cases. an intentional release of anthrax is expected to result in primarily inhalational cases. anthrax is not contagious from person to person and no special precautions are required. adalja cutaneous anthrax is characterized by a painless black ulceration ( fig. ) that occurs on the site of exposure. infection is more common in those exposed to animal products contaminated with spores, such as meat, drum skins, or wool. after an incubation period of approximately days, the lesion characteristically begins as a papule and progresses to a black eschar. diagnosis is often clinical, although culture, biopsy, polymerase chain reaction (pcr), and serology confirm the diagnosis. mortality is low if the disease is recognized and treated with appropriate antimicrobials. treatment regimens include oral ciprofloxacin or doxycycline (although penicillin may be used if susceptibility is known) for days. if exposure was through a biological attack, treatment duration is extended to days to cover incubating spores that may have been inhaled. injectional anthrax has been exclusively linked to use of contaminated illicit drugs whereas gastrointestinal anthrax is due to ingestion of contaminated food. , inhalational anthrax is the deadliest form of anthrax and occurs on inhalation of as little as spore. anthrax was historically known as wool sorter's disease because of its linkage with the occupation of wool sorting, in which spores on sheep's wool became aerosolized. the disease is characterized not by pneumonia but by mediastinal widening (fig. ) that can progress rapidly to shock. toxin-laden pleural effusions may be present. the disease begins after a week-long incubation period and is typically biphasic with flulike symptoms (with the notable exception of rhinorrhea) occurring before a terminal phase. when anthrax of any form progresses, the grave complication of hemorrhagic meningitis can occur. the treatment of systemic anthrax syndromes (inhalational, gastrointestinal, and injectional) involves first ruling out the presence or absence of meningitis via a lumbar puncture. if meningitis is confirmed or cannot be ruled out, the treatment regimen should include central nervous system penetrating drugs, of which should be a protein synthesis inhibitor (eg, linezolid) and of which should be bactericidal (eg, meropenem). the third drug could be ciprofloxacin. if meningitis has been ruled out, ciprofloxacin and clindamycin or linezolid could be used for treatment (with deescalation of ciprofloxacin to penicillin once drug susceptibility is known). treatment is for weeks to weeks. adjunctive antibody therapies, available from the cdc, such as anthrax immune globulin, raxibacumab, and obiltoxaximab, also should be given. additionally, if present, pleural effusions, pericardial effusions, and ascites should be drained, a factor that has likely improved survival rates from inhalational anthrax in the modern era. postexposure prophylaxis, for those exposed to anthrax spores, includes both an abbreviated -dose regimen of the vaccine coupled with days of oral ciprofloxacin or doxycycline (antibody therapies can be used in this manner when no other prophylaxis method can be used). in a mass event the post-exposure prophylaxis regimen for adults can be shortened to days after the first vaccine dose or weeks after the last vaccine dose, which ever comes later. plague is caused by the gram-negative bacillus yesinia pestis and is endemic in many parts of the world, including the western united states. this zoonotic infection is naturally spread from rodents, such as prairie dogs, to humans via the bite of a flea. there are forms of plague: bubonic (the most common), pneumonic, and septicemic. if used as a bioweapon, plague is expected to present in its pneumonic form. bubonic plague is characterized by marked painful lymphadenopathy (fig. ) that develops after a -day to -day incubation period whereas pneumonic plague (fig. ) may be indistinguishable from ordinary community-acquired pneumonia but has a mortality rate that can reach %. pneumonic plague has a -day to -day incubation period. pneumonic plague is transmissible from person to person through respiratory droplets and requires patients be placed in droplet isolation. , diagnosis of pneumonic plague in an intentional attack requires a high index of suspicion and can be made through pcr, serology, and/or culture. , the treatment of plague is with aminoglycoside antibiotics, such as gentamicin or streptomycin, for days to days, whereas postexposure prophylaxis of those exposed to an aerosol in a bioweapon attack consists of oral ciprofloxacin or doxycycline. , tularemia tularemia is caused by the gram-negative bacillus francisella tularenesis and is naturally a zoonotic infection that is common in many parts of the united states. naturally, tularemia may occur through tick, fly, and mosquito bites or through contact with reservoir animals (eg, rabbits). contaminated uncooked food or water can also be a vehicle for spread. the most common presentation of tularemia is the ulceroglandular cutaneous form whereas a biologic attack likely results in pneumonic tularemia. tularemia is not contagious between humans. tularemia is notable for its low infectious dose in which inhalation of just a small number of bacilli can result in disease. because of this, it is important to notify laboratory personnel about the possibility of tularemia, so they are able to don appropriate personal protective equipment when working with clinical specimens. pneumonic tularemia occurs after a -day to -day incubation period and is essentially indistinguishable from community-acquired pneumonia. even physicians who live in endemic areas often miss the diagnosis of tularemia in its various form, highlighting the need for tularemia to be in the differential diagnosis of compatible syndromes in endemic areas. temperature-pulse disassociation may be present and can serve as a clue to diagnosis. the treatment of tularemia is aminoglycoside antibiotics, such as gentamicin or streptomycin, for days to days. postexposure prophylaxis, to those exposed to an aerosol in a biological weapons attack, is with oral doxycycline or ciprofloxacin. , botulism botulism is caused by the acetylcholine release-blocking neurotoxin released by clostridium botulinum, a ubiquitous spore-forming gram-positive rod. there are several forms of naturally occurring botulism: infant, wound, and gastrointestinal. these forms result from exposure to spores of the bacteria, which then germinate and elaborate toxin. in a biological attack, inhalational botulism is expected, and it manifests similar to gastrointestinal botulism. botulism is not contagious. , after hours to hours postexposure to spores, clinical botulism occurs. it is characterized by a symmetric, flaccid, descending paralysis without sensory symptoms. patients are afebrile and not tachycardic. cranial neuropathies are common. paralysis progresses to involve respiratory muscles and can be prolonged, requiring long durations of mechanical ventilation. diagnosis is largely clinical. confirmatory mouse bioassay testing is used to determine which of the several botulinum toxinotypes is responsible. , heptavalent antitoxin, which neutralizes toxinotypes a to g, is obtainable from the cdc and can neutralize toxin. antibiotics and babybig (california department of public health, a bivalent botulinum antitoxin used in infant botulism) are not indicated. , there was controversy over the existence of an eighth toxinotype (h) but it has been shown to be a hybrid toxin and is neutralized by a-type antitoxin. more recently, a toxinotype x has been described and is unable to be neutralized by any available antitoxin. smallpox is the only human infectious disease that has been eradicated from the planet. as such, there is little current clinical experience with this disease. smallpox is a significantly contagious disease that is spread via airborne, respiratory droplet, or direct contact route. fomites are also known to spread the virus. , the clinical presentation of smallpox begins with flulike symptoms after a -day incubation period which is followed by the characteristic rash. the rash begins in a papular form and then progresses to umbilicated lesions and finally to pustules that crust and scab. a person with smallpox is contagious only from the appearance of the rash until the rash is scabbed, a key factor that led to its control. , the rash of smallpox (fig. ) must be distinguished from similar rashes that can be seen with varicella. several points of distinction are important. the rash of smallpox is centrifugal with more lesions on the face and extremities while the varicella rash is centripetal. the lesions of the smallpox rash are all at identical stages with identical appearances whereas the rash of varicella may have lesions of different stages. the case fatality rate of smallpox was historically %. , a diagnosis of smallpox would be a national security emergency of the highest order because even case represents either a laboratory accident or a biological attack. because smallpox vaccination is no longer routine, there is a sizable amount of the world population that lacks immunity. any suspicion of smallpox should prompt infectious disease consultation, airborne isolation procedures, and notification of local, state, and national public health authorities. the cdc has a telephone consultation service in place to discuss potential cases with experts. a diagnosis of smallpox initially is based on clinical suspicion while confirmatory testing by pcr, viral culture, or electron microscopy is performed under appropriate biosafety conditions. , there is no food and drug administration-approved treatment of smallpox currently, although several experimental antiviral compounds are in late stages of clinical development and might be accessible. the smallpox vaccine is effective as postexposure prophylaxis, even during the incubation period, and should be given to all patient contacts, who also will be placed under public health surveillance. the vaccine is contraindicated in the immunosuppressed and pregnant and those with eczema. experimental attenuated vaccines may be more suitable for these individuals. [ ] [ ] [ ] additionally, the smallpox vaccine carries a risk of myocarditis. vhfs are caused by a diverse group of viruses, each with its own unique microbiological, epidemiologic, and clinical features. of this group, which ranges from yellow fever to ebola, certain are more important as potential biological weapons than others. in the biological weapons context, it is the filoviruses (ebola and marburg) as well as the arenaviruses (lassa fever, machupo, and others) that merit concern. despite their differences, this group of viruses is characterized by a clinical presentation that often includes general malaise, fever, rash, prostration, pharyngitis, nausea, vomiting, and diarrhea. disease can rapidly progress to shock and multiple organ dysfunction syndrome with disseminated intravascular coagulation and hemorrhagic manifestations. diagnosis can be made using molecular tests, but a high index of suspicion is needed to differentiate these infections from ordinary septic shock. in the ed patient, travel to endemic areas, exotic animal exposure, or laboratory work with vhfs might be the only clue to the etiology. in a biological attack, a cluster of patients with similar symptoms may present to several eds in a given region. any suspicion of a vhf should prompt immediate consultation with an infectious disease physician and state and local health authorities. although these viruses are spread via blood and body fluid exposure and do not spread between humans via the airborne route, the experience of the united states during the west africa ebola outbreak has influenced infection control recommendations. the nosocomial infections at a hospital in texas have led to recommendations for strict airborne and body fluid isolation for patients suspected of having a vhf, with transfer to definitive care at specialized units for confirmed cases. treatment is generally supportive and has proved life-saving in the case of ebola. the recent experience with ebola highlighted the fact that simple supportive care with fluids and electrolytes brought fatality rates down from % to less than %. there are several experimental treatments and vaccines (which can be used for postexposure prophylaxis) that are available for filovirus infections and arenavirus infections that would likely be used in any domestic vhf cases caused by these groups of viruses. for ebola exposures, the experimental vaccine would be indicated for postexposure prophylaxis whereas a combination of experimental antiviral agents (eg, favipiravir) and antibody-based therapies, such as zmapp, might be indicated after consultation with cdc. lassa fever can be treated with intravenous ribavirin, which is available via cdc. the possibility of vhf infection should be considered in those with severe illness and travel to areas in which these infections are endemic, such as parts of africa (eg, democratic republic of congo, uganda, and nigeria) or south america (eg, brazil and argentina). consultation of the cdc travel web site (www.cdc.gov/travel) is advised to determine specific vhf travel risks. coronaviruses (covs) are major causes of the common cold and rarely cause severe disease in immunocompetent hosts. there are, however, covs that have the capacity to cause severe disease: severe acute respiratory syndrome (sars)-cov and middle east respiratory syndrome (mers)-cov. although both sars and mers present to the ed as ordinary upper or lower respiratory tract infections, they have distinct geographic and epidemiologic features that should alert an ed physician to the possibility of their presence. sars, which emerged in china in , was a worldwide infectious disease emergency that led to more than cases worldwide, with approximately % of cases fatal-including in the united states. the virus was zoonotic in origin and linked to human consumption of palm civet cats. the spread of the virus was abetted by the presence of superspreading events in which certain individuals infected a disproportionate number of others. the epidemic extinguished once infection control measures were instituted in health care settings and the consumption of palm civet cats ceased. mers is also a zoonotic respiratory cov that emerged in the arabian peninsula in and has been linked to contact with both bats and camels. all cases have an epidemiologic link to the arabian peninsula, including a multiple-ed superspreading event that occurred in south korea. mortality rates are approximately %. in the united states, imported mild cases have been diagnosed in travelers returning from the middle east. mers should be suspected in individuals with upper or lower respiratory infection after travel to the middle east in the prior weeks, and confirmatory molecular testing can be done in conjunction with state and local health authorities. many respiratory viral panels have the capacity to identify the presence of a cov and may be helpful in the work-up. infectious disease consultation and institution of droplet or airborne precautions are advised. there are no antivirals or vaccines available for any cov. influenza is often considered one of the highest pandemic threats. prior influenza pandemics have killed millions and have caused severe societal disruption. each modern pandemic ( , , , and ) has been linked to the emergence of a novel influenza a variant of zoonotic (avian, swine, or a combination) origin. zoonotic influenza viruses, in their first forays into humans, can cause a range of illness, ranging from ordinary influenza to fulminant disease, including pneumonia and acute respiratory distress syndrome. poor to limited nonsustained human-tohuman transmission characterizes these viruses in the prepandemic stage, with most cases linked directly or indirectly to poultry exposure. it is when sustained human-to-human transmission occurs that a pandemic is eminent. because of this threat, monitoring and surveillance efforts exist for avian influenza infections in humans and poultry. currently, of the myriad zoonotic influenza infections, the h n strain of influenza a has been deemed the highest threat amongst these viruses currently, although others (such as h n ) are also important to track. the ed physician should suspect avian influenza in travelers from china and other areas in which avian influenza is known to circulate, who present within approximately week after travel with upper or lower respiratory tract infection. additionally, domestic agricultural workers or those with agricultural contact with flulike symptoms (eg, children at fairs) also may harbor zoonotic influenza infections. diagnosis is similar to ordinary influenza, but a rapid or standard molecular test may or may not be able to detect influenza. confirmatory testing is via health authorities. treatment involves supportive care coupled to antiviral therapy with either oral oseltamivir or, if disease severity is high, intravenous peramivir. infectious disease consultation and institution of droplet precautions is advised. mosquito-borne arboviruses have increasingly taken on importance in the field of emerging infectious disease with the explosion of cases of west nile, chikungunya, and zika in the western hemisphere. additionally, local transmission of dengue fever has occurred in florida, texas, hawaii, and new york. , chikungunya, dengue fever, and zika are all spread by the aedes species of mosquitoes, which have habitats both within and outside the united states and cause clinically indistinguishable syndromes. these syndromes all involve fevers, rash, myalgias, and arthralgias. conjunctivitis has been noted with zika. prolonged debilitating arthralgias can occur with chikungunya whereas severe illness, including shock and hemorrhagic manifestations, can occur with dengue fever (especially with repeat infection with disparate strains). , zika has been linked to guillain-barré syndrome and requires special counseling regarding sexual transmission and pregnancy given its ability to cause a devastating congenital syndrome. , travel history and residence in an endemic area (eg, key west, hawaii, and texas) are important elements of the history. diagnostic testing is commercially available for each infection. consultation with the cdc travel web site is advised to assess specific risks for individual patients' travel history. no vaccines or antiviral therapies are available for these infections. emergency physicians play a crucial and unique role in the defense against emerging infectious disease and are most likely to encounter the first cases of any new infectious disease syndrome. the challenge that the emergency physician faces is that these cases do not announce themselves and are hidden among the sea of chief complaints that any ed sees on a given day. with many clinical scenarios, a busy physician may not be inclined to pursue a specific diagnosis if it "doesn't change treatment," may lengthen ed length of stay, and will not produce a result while a patient is in the ed, creating follow-up logistical problems. failing to diagnose an epidemiologically important emerging infectious disease or biological attack, however, can have tremendous cascading effects involving all sectors of society (health care, government, and economy) that can be minimized or averted with a proactive approach. in the current era, there are several molecular multianalyte tests available, some of which are clinical laboratory improvement amendments (clia) waived and available at point of care for specific infectious disease syndromes, such as gastrointestinal infections, meningitis, and respiratory infections, that can be used to increase the rate of microbial specific diagnoses in ed settings. biodefense cartridges, which probe for select bioagents, are also available. these tests, in the hands of an astute physician, can improve the nation's resiliency to infectious disease emergencies, and negative results in the right clinical context may prompt further investigation for a specific etiology. by having a working knowledge of emerging infectious disease and biothreat landscape, an emergency physician becomes a key component of the infectious disease emergency system. injectional anthrax in heroin users clinical management of potential bioterrorismrelated conditions mandell, douglas, and bennett's principles and practice of infectious diseases centers for disease control and prevention expert panel meetings on prevention and treatment of anthrax in adults systematic review: a century of inhalational anthrax cases from to mandell, douglas, and bennett's principles and practice of infectious diseases s principles and practice of infectious diseases clinical recognition and management of tularemia in missouri: a retrospective records review of cases mandell, douglas, and bennett's principles and practice of infectious diseases a novel botulinum neurotoxin, previously reported as serotype h, has a hybrid-like structure with regions of similarity to the structures of serotypes a and f and is neutralized with serotype a antitoxin identification and characterization of a novel botulinum neurotoxin orthopoxviruses: vaccinia (smallpox vaccine), variola (smallpox), monkeypox, and cowpox acam : the new smallpox vaccine for united states strategic national stockpile safety and immunogenicity of imvamune smallpox vaccine using different strategies for a post event scenario safety and immunogenicity of lc m , an attenuated smallpox vaccine in vaccinia-naive adults acam prescribing information mandell, douglas, and bennett's principles and practice of infectious diseases systems for rapidly detecting and treating persons with ebola virus disease -united states evidence-based guidelines for supportive care of patients with ebola virus disease experimental therapies for ebola virus disease: what have we learned? including severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) summary of influenza risk assessment tool (irat) results outbreak of influenza a (h n ) variant virus infections among persons attending agricultural fairs housing infected swine -michigan and ohio lessons learned during dengue outbreaks in the united states health commissioner reports dengue virus case japanese encephalitis, west nile encephalitis, st. louis encephalitis, tick-borne encephalitis, kyasanur forest disease, alkhurma hemorrhagic fever, zika) mandell, douglas, and bennett's principles and practice of infectious diseases guillain-barre syndrome associated with zika virus infection in columbia zika virus and birth defects-reviewing the evidence for causality key: cord- -luqvw y authors: levinson, julia; kohl, kid; baltag, valentina; ross, david title: investigating the effectiveness of school health services delivered by a health provider: a systematic review of systematic reviews date: - - journal: biorxiv doi: . / sha: doc_id: cord_uid: luqvw y schools are the only institution regularly reaching the majority of school-age children and adolescents across the globe. although at least countries have school health services, there is no rigorous, evidence-based guidance on which school health services are effective and should be implemented in schools. to investigate the effectiveness of school health services for improving the health of school-age children and adolescents, a systematic review of systematic reviews (overview) was conducted. five databases were searched through june . systematic reviews of intervention studies that evaluated school-based or school-linked health services delivered by a health provider were included. review quality was assessed using a modified ballard and montgomery four-item checklist. references were screened and systematic reviews containing primary studies were assessed narratively. interventions with evidence for effectiveness addressed autism, depression, anxiety, obesity, dental caries, visual acuity, asthma, and sleep. no review evaluated the effectiveness of a multi-component school health services intervention addressing multiple health areas. strongest evidence supports implementation of anxiety prevention programs, indicated asthma education, and vision screening with provision of free spectacles. additional systematic reviews are needed that analyze the effectiveness of comprehensive school health services, and specific services for under-researched health areas relevant for this population. and should be implemented in schools. to investigate the effectiveness of school health services for improving the health of school-age children and adolescents, a systematic review of systematic reviews (overview) was conducted. five databases were searched through june . systematic reviews of intervention studies that evaluated school-based or school-linked health services delivered by a health provider were included. review quality was assessed using a modified ballard and montgomery four-item checklist. references were screened and systematic reviews containing primary studies were assessed narratively. interventions with evidence for effectiveness addressed autism, depression, anxiety, obesity, dental caries, visual acuity, asthma, and sleep. no review evaluated the effectiveness of a multi-component school health services intervention addressing multiple health areas. strongest evidence supports implementation of anxiety prevention programs, indicated asthma education, and vision screening with provision of free spectacles. additional systematic reviews are needed that analyze the effectiveness of comprehensive school health services, and specific services for under-researched health areas relevant for this population. the world health organization (who) launched the global school health initiative in with the goal to improve child, adolescent and community health through health promotion and programming in schools [ ] . this initiative is dedicated to promoting development of school health programs and increasing the number of health-promoting schools, characterized by who as "a school constantly strengthening its capacity as a healthy setting for living, learning and working" [ ] . in , who, the united nations educational, scientific and cultural organization (unesco), the united nations children's fund (unicef) and the world bank developed a partnership for focusing resources on effective school health -a fresh start approach [ ] . the fresh framework promotes four pillars: health-related school policies, provision of safe water and sanitation, skills-based health education and school-based health and nutrition services [ ] . while various guidance documents have been published by united nations (un) organizations addressing a range of services from oral health to malaria [ ] [ ] [ ] [ ] [ ] , there is no internationally accepted guideline regarding school health services. this systematic review of systematic reviews, henceforth referred to as an overview, will inform the upcoming development of a who guideline that addresses one pillar of the fresh framework: school health services delivered by a health provider. schools offer a unique platform for health care delivery. in , the global means for the primary and secondary net school enrollment rates were % and %, respectively, thus the potential reach of school health services is wide [ ] . additionally, a recent review found that school-based or school-linked health services already exist in at least countries [ ] . the global accelerated action for the health of adolescents (aa-ha!) implementation guidance calls for the prioritization of school health programs as an important step towards universal health coverage and urges that "every school should be a health promoting school" [ ] . the primary objective of this overview was to explore the effectiveness of school- based or school-linked health services delivered by a health provider for improving the health of school-age children and adolescents. through a comprehensive literature search, the overview aimed to identify health areas and specific school health service interventions that have at least some evidence of effectiveness. it was also designed to suggest further research in areas where recent systematic reviews (srs) exist, but with insufficient evidence. finally, the overview aimed to identify the health areas and specific school health services interventions for which no srs were found, whether because the primary literature does not exist or where there are primary studies but no sr has been conducted. this overview was conducted using the preferred reporting items for systematic reviews and meta-analyses (prisma) [ ] . a protocol was developed a priori that outlined the overview objectives, aims, operational definitions, search strategy, inclusion/exclusion criteria, and quality appraisal methods. this document was followed throughout the review process and is available in s appendix. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] enrolled in schools; (b) interventions were school-based or school-linked health services, involved a health provider (see definitions in s appendix), and were of any duration or length of follow-up; (c) intervention effectiveness was compared to either no intervention, an alternative intervention, the same intervention in a different setting (i.e. not in schools), an active control, or a waitlist control; (d) interventions aimed to improve some aspect of health; and (e) study designs were either randomized controlled trials (rcts), quasi-experimental studies (qes), or other non-randomized intervention studies. there were no date restrictions on publication of included srs. in addition to these criteria for included studies, the srs themselves had to fulfill the following criteria: (a) included the words "systematic review" in the title or abstract; (b) outlined inclusion criteria within the methods section; (c) published in peer-reviewed journals and indexed before june , ; (d) published in the english language. in addition to srs that did not meet these inclusion criteria, srs were excluded if the review was superseded by a newer version. study selection citations identified from the systematic search were uploaded to covidence systematic review software [ ] and duplicates were automatically deleted. two reviewers (kk and jl) screened all titles and abstracts using the inclusion/exclusion criteria and excluded all articles that were definitely ineligible. articles that received conflicting votes (ineligible vs. potentially or probably eligible) were discussed and consensus was reached. the same two reviewers screened the full text of all the potentially or probably eligible articles using a ranked list of the inclusion criteria (s appendix). reasons for exclusion were selected from the ranked list. if consensus was not possible during title/abstract or full text screening, a third reviewer (dr), who had the casting vote, would have been asked to independently screen the article. this was never required as consensus was always reached. data collection one reviewer (jl) extracted summary data from each selected article using a customized standard form with independent data extraction performed for % of included srs by one of the other reviewers (dr or kk). there was % agreement between reviewers for all items within the standard form, with discrepancies only in level of detail . data items included the research design of the sr and primary studies, sample description and setting, intervention characteristics, outcomes, meta-analysis results, quality appraisal, and conclusions. due to the heterogeneity of the srs included in this overview, it was not possible to perform a meta-analysis. outcome measures were collected from included studies. risk of bias within primary studies was recorded in s appendix. risk of bias across srs was determined using ballard and montgomery's four-item checklist for overviews of srs [ ] . these items include: ( ) overlap (see below), ( ) rating of confidence from the amstar checklist [ ] , ( ) date of publication, and ( ) match between the scope of the included srs and the overview itself. an important consideration in overviews is the degree of overlap, or the use of the same primary study in multiple included srs. high overlap can contribute to biased results [ ] . this overview used the corrected covered area (cca), a comprehensive and validated measure, to determine overlap [ ] . the cca is calculated using three variables: the number of "index" publications (r), the number of total publications (n), and the number of srs within the overview (c). an "index" publication is the first appearance of a primary study within an overview. the formula for the cca is: ccas can be interpreted as indicating slight, moderate, high or very high overlap with scores of - , - , - , or > , respectively [ ] . the amstar checklist [ ] was used to appraise quality of included srs. one reviewer (jl) assessed all srs and a second (kk) duplicated appraisal of %, with % agreement and only minor disagreements that did not impact grades of confidence. following the recommendation of the amstar developers [ ] , individual ratings were not combined into an overall score. instead, the authors determined which of the items on the checklist were critical for this overview and which of the items were non-critical. building on a method suggested by shea and colleagues [ ] , grades of confidence in the results of each sr were generated based on critical flaws and non-critical weaknesses. the grading system is available in s appendix. confidence in results ranged from high (three or fewer non-critical weaknesses) to critically low (more than three critical flaws with or without non-critical weaknesses) [ ] . used meta-analysis to combine results [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , whereas the remaining nine srs narratively synthesized results [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . eleven srs included studies located in countries with high- income or upper-middle income economies only [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , , ] . six srs included at least one study from countries with lower-middle income or lower income economies [ , , , , , ] . the final three srs either did not state the locations of included studies [ , ] or provided regions rather than specific country locations [ ] . to be included in this overview, at least % of studies within each sr had to fulfill all inclusion criteria. in four srs, % of included studies fulfilled all inclusion criteria [ , , , ] , although brendel and colleagues [ ] only included one study in total. in another four srs, % to % of included studies fulfilled all the inclusion criteria [ , , , ] . in the remaining twelve srs, % to % of included studies fulfilled all the inclusion criteria [ , , [ ] [ ] [ ] [ ] , , , [ ] [ ] [ ] . all srs primarily examined studies on school-based, rather than school-linked interventions. the srs covered eight health areas: nine on mental health [ , , , , , [ ] [ ] [ ] ] , four on oral health [ , , , ] , two on asthma [ , ] , and one sr each on sleep [ ] ; obesity [ ] ; vision [ ] ; menstrual management [ ] ; and sexual and reproductive health (srh) [ ] . eleven srs included only cluster-and individually- randomized controlled trials [ ] [ ] [ ] , , , , , , ] , seven srs included other types of controlled and uncontrolled experimental studies in addition to rcts [ , , , , , , ] , and two srs included only qes [ ] or controlled clinical trials [ ] . table the corrected covered area (cca) was found to be , indicating only slight overlap between the srs. calculations for the cca can be found in s appendix. table presents the remaining three of the four items of ballard and montgomery's checklist for overviews of reviews: ( ) levels of confidence in results for each included sr, ( ) publication year, and ( ) match in scope to the overview. a majority of the studies ( %) were given low or critically low levels of confidence. only three srs [ , , ] were scored as having moderate levels of confidence and just one [ ] was given a high level of confidence. the details of the quality appraisal of primary studies included in the srs are given in s appendix. [ ] oral health moderate % bastounis [ ] mental health low % brendel [ ] mental health critically low % chung [ ] sleep low % cooper [ ] oral health high % evans [ ] vision moderate % geryk [ ] asthma critically low % gold [ ] mental [ ] obesity critically low % stein [ ] oral health low % sullivan [ ] mental health critically low % walter [ ] asthma low % werner-seidler [ ] mental health low % a the four items of the checklist include: overlap, amstar rating of confidence, up-to-date, and relevance of included studies b see s appendix for amstar rating information c i.e., percentage of studies within the systematic review that clearly fulfill all inclusion criteria srh = sexual and reproductive health none of the srs evaluated comprehensive, multi-component, or multi-health area school health services. two srs found strong evidence for the potential effectiveness of educational interventions for children and adolescents with asthma diagnoses (table a) [ , ] . geryk and colleagues found that education on correct use of an inhaler improved inhaler technique, regardless of deliverer, method, or duration of the intervention [ ] . however, they did not assess risk of bias or appraise the quality of included studies [ ] . walter and colleagues found that family asthma educational programs for children and their parents or caregivers improved quality of life for both caregivers and children, and decreased asthma exacerbations for children [ ] . while results from primary studies were statistically significant in both srs, heterogeneity of interventions precluded meta-analysis by walter and colleagues [ ] and no reason was given for why meta-analysis was not performed by geryk and colleagues [ ] . hennegan and montgomery assessed the effectiveness of "hardware" and "software" menstrual management interventions (table b ) [ ] . hardware interventions included provision of sanitary products and software interventions focused on menstrual management education. a metaanalysis of two studies on sanitary pad provision found a moderate but statistically non-significant effect on school attendance. however, it was unclear whether these studies involved a health provider [ ] . outcomes across studies differed, but the authors noted trends toward improvement in menstruation knowledge, management practices, psychosocial outcomes, and school attendance. hennegan and montgomery found a high level of heterogeneity and substantial risk of bias in the included studies overall, thus they were unable to make conclusions about the effectiveness of menstrual management interventions [ ] . the effectiveness of school-based mental health services was assessed in nine srs (table c ) [ , , , , , [ ] [ ] [ ] , ] . srs addressed various intervention types: universal interventions [ , , , , , ] ; targeted interventions for military-connected children [ ] , children and adolescents at risk for depression and/or anxiety [ , ] , refugee and war-traumatized youth [ ] , and children referred to therapy [ ] ; and indicated interventions for children and/or adolescents diagnosed with autism spectrum disorder [ ] , depression [ , , ] , or anxiety [ , , ] . prevention and treatment of mood disorders was assessed in five srs, all of which targeted children and adolescents using rcts of established programs. higgins and o'sullivan assessed the friends for life program, a manual-based cognitive behavioral anxiety prevention program comprised of ten sessions with developmentally-tailored programs for different age groups [ ] . they found statistically significant improvements in self-reported measures of anxiety for participants who completed the program as compared to those in the control group [ ] . a sr and meta-analysis by bastounis and colleagues on the educational and preventative penn resiliency program (prp) and its derivatives found small, non-significant effect sizes for the prevalence of both depression and anxiety, favoring the intervention in the former and the control in the latter [ ] . the remaining three srs also assessed friends and prp along with additional often-overlapping programs. neil and christensen analyzed unique anxiety prevention and early intervention programs and found reductions in anxiety symptoms in % of the included studies [ ] . kavanagh and colleagues examined depression and anxiety group counseling programs based on cognitive behavioral therapy (cbt) and found statistically significant reductions of depressive symptoms at both four weeks and three months follow-up [ , ] . finally, meta-analysis by werner-seidler and colleagues of rcts on the effectiveness of depression and anxiety prevention and group therapy programs found small yet statistically significant effect sizes in favor of the intervention groups for both depression and anxiety as compared to control groups [ ] . although the overall degree of overlap between all srs within this overview was slight, the overlap between just these five srs targeting mood disorders was high (cca = ). assessments of music [ ] and art therapy [ ] in two srs reported weak evidence of effectiveness. gold and colleagues assessed daily music therapy as an intervention to improve verbal and gestural communicative skills and reduce behavioral problems in children diagnosed with autism spectrum disorder [ ] . meta-analysis found small but statistically significant effect sizes in favor of music therapy for gestural communication, verbal communication, and behavioral problems [ ] . oppositional defiant disorder (odd), separation anxiety disorder (sad), moderate to severe behavior problems, or learning disorders [ ] . the authors found improvements in classroom behavior, symptoms of odd, and symptoms of sad [ ] . however, in the studies included in both gold and colleagues' and mcdonald and drey's srs, the numbers of participants per intervention group were very small: - and - per study, respectively, introducing possibility of bias [ , ] . mostly favorable evidence of effectiveness was found in a sr of social-emotional interventions for refugee and war-traumatized youth from countries [ ] . improvements in trauma-related symptoms and impairment were found through narrative assessment of creative expression interventions, cognitive behavioral interventions, and multifaceted interventions [ ] . in contrast with gold and colleagues [ ] , this sr by sullivan and simonson found negative effects from music therapy interventions [ ] . however, there was no risk of bias assessment in this sr and therefore the results must be interpreted cautiously. the final sr on mental health services examined well-being interventions for children with a parent in the military [ ] . only one quasi-experimental study from the united states in was included in the sr. the study assessed a group counseling intervention and found no statistically significant effects on the prevalence of anxiety, self-esteem, internalizing behavior or externalizing behavior [ ] . schroeder and colleagues reviewed the effectiveness of obesity treatment and prevention interventions that specifically involved a school nurse (table d ) [ ] . most interventions involved school-nurse-delivered nutrition counseling, nutrition and health education, and some parent involvement or physical activity. meta-analysis indicated small, yet statistically significant, reductions in body mass index (bmi), bmi z-score, and bmi percentile for both obesity treatment and prevention [ ] . srs on oral health interventions focused on prevention [ , ] , screening [ ] , and education [ , ] (table e ). strongest evidence in favor of oral health interventions emerged from a sr on universal topical application of fluoride gel for the prevention of dental caries [ ] . meta-analysis results indicated a statistically significant effect on the before-after change in caries prevalence [ ] . a universal educational intervention on oral hygiene and caries produced weaker evidence of effectiveness [ ] . small but statistically significant effect sizes were found in favor of the intervention for mean plaque levels and oral hygiene, but no statistical significance was found for change in gingivitis indices [ ] . two srs on dental health screening [ ] and behavioral interventions for caries prevention [ ] found limited evidence of effectiveness. arora and colleagues did not find any rcts that looked at the effectiveness of dental health screening versus no screening on improving oral health outcomes, but their search did locate six rcts from the united kingdom and india with dental care attendance as the outcome [ ] . the data was too heterogeneous to meta-analyze, and the authors determined that the certainty of the evidence of the benefit of dental screening in increasing dental attendance was very low [ ] . the other sr examined behavioral interventions in the form of education on tooth brushing and the use of fluoride toothpaste in brazil, italy, united kingdom, and iran [ ] . due to the diversity in outcome measures and intervention intensities, the authors felt unable to make any evidence-based recommendations [ ] . a sr on sexual health interventions for prevention of sexually transmitted infections (stis) and human immunodeficiency virus (hiv) in sub-saharan african countries found that educational interventions were successful in increasing knowledge and attitudes for participants (table f ) [ ] . however, the sr suggested that the studies were ineffective in changing self-reported risky behaviors, although follow-up was either immediate or short-term (less than months) [ ] . this sr did not discuss the quality or risk of bias of included studies [ ] . chung and colleagues systematically reviewed and meta-analyzed universal sleep education programs as compared to no additional sleep intervention from australia, new zealand, brazil, and hong kong (table g ) [ ] . five of the included studies examined the same weekly sleep education program from the australian centre for education in sleep. the sixth study assessed a -day program in brazil. meta-analysis of the six studies showed statistically significant short-term benefits for weekday sleep time, weekend sleep time, and mood [ ] . however, these results did not persist at follow-up [ ] . evans and colleagues reviewed seven rcts from china, india, and tanzania on vision screening for correctable visual acuity deficits at or before school entry (table h ) [ ] . through meta-analysis of two rcts, the authors found that school vision screening combined with provision of free spectacles resulted in a statistically significant % increase in the wearing of spectacles at - months follow-up as compared to vision screening combined with prescription for spectacles only [ ] . evans and colleagues found no statistically significant difference in the proportion of students wearing spectacles at - months follow-up between vision screening with provision of ready-made spectacles and vision screening with provision of custom-made spectacles in a meta-analysis of three rcts [ ] . education on the wearing of spectacles in addition to vision screening as compared to vision screening alone did not have a significant effect [ ] . no srs found eligible studies comparing vision screening with no vision screening. this overview found srs covering primary studies. the majority of srs assessed educational, counseling, or preventive interventions, most of which were special research interventions rather than routinely-delivered school health services. no sr examined comprehensive or multi-component school health services, despite the fact that comprehensive services may be more efficient, easier to implement, and more sustainable than single interventions [ ] . results from this overview suggest that certain interventions can be effective in improving child and adolescent health outcomes, and thus may be worthwhile for integration into school health programs. vision screening is one of the most common forms of school health services [ ] , although the majority of programs are concentrated in high-income countries (hic) [ ] . although prevalence of visual impairment varies widely by ethnic group and age [ ] , who estimates that at least million children below age are visually impaired [ ] . evans and colleagues found strong evidence from china, tanzania, and india that school vision screening for correctable visual acuity deficits increased wearing of spectacles when spectacles were provided at no cost [ ] . a recent guideline from the international agency for prevention of blindness (iapb) reiterates the importance of free spectacles and goes further to suggest that low-and middle-income countries (lmic) adopt comprehensive school eye health programs [ ] . vision screening linked with free provision of spectacles, as a component of a comprehensive school eye health program, is an example of a cost-effective form of school health services that may be implemented. five srs covered depression and/or anxiety prevention and early intervention programs, with the friends for life program (friends) and the penn resiliency program (prp) most common [ , , , , ] . given that friends has been endorsed by who [ ] and was found to be effective in decreasing anxiety symptoms in all four srs where it was mentioned in this overview [ , , , ] , policy makers and school health officials may consider incorporating this or similar programs into existing school health services. the four srs that included prp found mixed evidence [ ] or no evidence of effectiveness [ , , , ] , bringing the popularity of this intervention into question. finally, creative therapy interventions seem to be effective for indicated populations of school-age children, such as children with autism spectrum disorder [ , ] . however, this conclusion should be interpreted cautiously due to small effect sizes, small sample sizes, and conflicting evidence on the effectiveness of music therapy between sullivan and simonson [ ] and gold and colleagues [ ] . comprehensive school programs that promote healthy school environments, health and nutrition literacy, and physical activity are one of the six key areas for ending childhood obesity recommended by who [ ] . this overview found only one sr that assessed obesity treatment and prevention delivered by a health professional in schools, despite the fact that over million school-age children and adolescents were overweight or obese in [ ] . schroeder and colleagues found that school nurses are well positioned to deliver nutritional counseling, design and coordinate physical activity interventions, and educate parents, students, and staff on health, nutrition, and fitness [ ] . however, all included primary studies were delivered in hic [ ] . schools are considered to be an ideal platform for oral health promotion through education, services, and the school environment [ ] . the most promising evidence from a sr was on topical application of fluoride gel for the prevention of dental caries [ ] . educational interventions had mixed effects. a sr that focused on behavioral education, such as demonstrating how to correctly brush teeth, found no evidence for reduction in caries [ ] , whereas a sr on oral hygiene and caries education found evidence for decreased plaque and improved hygiene [ ] . more research should be done to identify the content and methods of deliver that make some oral health education interventions more effective than others. it is difficult to determine overall effectiveness of school health services from this overview because the included srs do not sufficiently cover the health areas most relevant for children and adolescents. in , the top five leading causes of death for - year olds were lower respiratory infections, diarrheal diseases, meningitis, drowning, and road injury [ ] . among - year olds, the leading causes of death were lower respiratory infections, drowning, road injury, diarrheal diseases, and meningitis [ ] . finally, for [ ] [ ] [ ] [ ] [ ] year olds, the leading causes of death were road injury, self-harm, interpersonal violence, diarrheal diseases, and lower respiratory infections [ ] . leading causes of disability-adjusted life years (dalys) for - year olds were iron deficiency anemia, road injury, depressive disorders, lower respiratory infections, and diarrheal diseases [ ] . this overview shows that the current sr literature does address mental health, specifically mood disorders. however, the causes of death and disability beyond self-harm and depressive disorders are currently not addressed. although mortality and morbidity statistics vary by region and country, it is clear that the health areas included in this overview reflect a small subset of the global burden of disease for children and adolescents. furthermore, this overview exposes a mismatch between the sr literature on effectiveness of school health services and the actual school health services that are most commonly delivered. vaccinations have been identified as the most common type of intervention in schools in at least countries or territories [ ] , and there is evidence of effectiveness from primary studies regarding feasibility of school-based vaccination programs [ , ] . yet no srs on vaccinations fulfilled the inclusion criteria for this overview, suggesting the need for these srs to be conducted. additionally, at least countries or territories include some form of school health services that are routinely delivered, as opposed to special research interventions [ ] . this overview primarily found evidence for special research interventions, suggesting a need for assessment of routinely-delivered school health services. one of the central questions of this overview was whether the school health services that are regularly delivered across the globe are evidence-based. the mismatch in the sr literature identified by this overview demonstrates that more research must be done before an answer to this question can be determined. another important gap that this overview reveals is a lack of research on interventions carried out in lmic and low-income countries (lic). only one of the srs included in this overview examined studies from a majority of lmic and lic [ , ] . this is problematic given that health disparities for children and adolescents are greater in lmic and lic than in higher income countries [ ] . additionally, resources differ by income level and therefore effective interventions in hic may need to be tailored or changed entirely in order to be feasible in lmic and lic. who reports densities of less than one physician per population in countries and less than three nurses or although three srs mentioned the cost of specialized professionals delivering interventions versus teachers or a school nurse [ , , ] , cost, let alone cost-effectiveness, was not closely analyzed in any of the included srs. for useful recommendations to be made regarding school health services, cost-effectiveness must be more closely examined by primary studies and srs. although overviews offer a comprehensive method for synthesizing evidence, they also come with important methodological limitations. first, an overview is unlikely to include the latest evidence if recent primary studies have not yet been included in srs. this lag may preclude the ability for an overview to truly reflect current knowledge [ ] . while this overview found significant gaps in the evidence for certain health areas, this does not necessarily mean that relevant high quality primary trials have not been conducted. second, the ability of overviews to make valid and accurate conclusions is dependent upon the accuracy, rigor, and inclusiveness of the srs themselves. % (n= ) of srs included in this overview were given ratings of low or critically low confidence using the amstar checklist, although this is not unusual given the stringency of the checklist. nonetheless, it is interesting to note that all four of the srs given moderate or high levels of confidence were cochrane reviews. the remaining cochrane review was given a critically low level of confidence, though this may be because it was published in and standards for both the methods and reporting of cochrane reviews have improved in recent years. third, the scopes of individual srs often differ from the scope of the overview, a problem that ballard and montgomery call a "scope mismatch" [ ] . in this overview, srs with at least % of included studies fulfilling all criteria were included after extensive discussion between the authors and experts in the field. this implies that a narrower range of srs would have been eligible if a stricter cut-off had been selected, and vice versa. it is important to take this into account when interpreting results. finally, overlap of primary trials between srs can bias results of an overview [ ] . there is no definitive guidance on how to correct for overlap, as both including or excluding overlapping srs presents potentially biased results [ , ] . this overview measured overlap using the corrected covered area (cca) [ ] and did not exclude overlapping studies. however, the degree of overlap across all srs was graded as being small (cca = ), while there was high overlap in srs on mood disorders (cca = ). cca values for all health areas and calculations are available in s appendix. a key limitation of this overview is that only publications self-titled "systematic reviews" were included. this decision was made because of the vast numbers of reviews available and the increased rigor associated with the term "systematic". a sensitivity analysis comparing "systematic*" with "systematic review" found that the number of search results increased almost three-fold, but did not reveal any new articles that would eventually have met the subsequent eligibility criteria. another limitation is that this overview only included randomized and non-randomized controlled trials, quasi-experimental studies and other controlled study designs where health professionals delivered the intervention. while this strengthens the rigor of included studies and improves decision-making ability, it also excludes potentially relevant literature. a strength of this overview is that it attempted to answer a question that has not yet been answered regarding the effectiveness of both comprehensive and specific school health services delivered by a health provider. while other pillars of health promoting schools have relevant guidance documents, guidance on school health services is limited and not explicitly evidence-based. given the wide reach of schools and the fact that school health services already exist in most countries, international guidelines are needed to clarify whether school health services can be effective, and if so, which interventions should and should not be included. this overview makes an important first step toward that guideline. this overview presents multiple effective interventions that may be offered as a part of school health services delivered by a health provider. however, it is difficult to formulate an overarching answer about the effectiveness of school health services for improving the health of school-age children and adolescents due to the heterogeneity of srs found and the evident gaps in the sr literature. more than half of included srs analyzed mental health and oral health interventions, and no srs were found that assessed other relevant health areas, such as vaccinations, communicable diseases, injuries, etc. further, no srs evaluated comprehensive or multi-component school health services. if school health services are to truly improve the health of children and adolescents, they must comprehensively address the most pressing problems of this population. in order for policy makers and leaders in school health to make evidence-based recommendations on which services should be available in schools, who should deliver them, and how should they be delivered, more srs must be done. these srs must assess routine, multi-component school health services and the characteristics that make them effective, with special attention to content, quality, intensity, method of delivery, and cost. the gaps in the sr literature identified by this overview will inform the commissioning of new srs by who to feed into evidence-based global recommendations. who | global school health initiative focusing resources on effective school health: a fresh start to enhancing the quality and equity of education who information series on school health: oral health promotion: an essential element of a health-promoting school. geneva: world health organization school-based deworming: a planner's guide to proposal development for national school-based deworming programs deworm the world basic guide for school directors, teachers, students, parents and administrators usaid hygiene improvement project malaria control in schools: a toolkit on effective education sector responses to malaria in africa world bank open data global overview of school health services: data from countries world health organization. global accelerated action for the health of adolescents (aa-ha!): guidance to support country implementation preferred reporting items for systematic reviews and meta-analyses: the prisma statement covidence systematic review software veritas health innovation risk of bias in overviews of reviews: a scoping review of methodological guidance and four-item checklist amstar : a critical appraisal tool for systematic reviews that include randomised or non-randomised studies of healthcare interventions, or both. the bmj overviews of systematic reviews: great promise, greater challenge systematic review finds overlapping reviews were not mentioned in every other overview amstar : a critical appraisal tool for systematic reviews that include randomised or non-randomised studies of healthcare interventions, or both school dental screening programmes for oral health. the cochrane library the effectiveness of the penn resiliency programme (prp) and its adapted versions in reducing depression and anxiety and improving explanatory style: a systematic review and meta-analysis school-based sleep education programs for short sleep duration in adolescents: a systematic review and meta-analysis vision screening for correctable visual acuity deficits in school-age children and adolescents. the cochrane library do menstrual hygiene management interventions improve education and psychosocial outcomes for women and girls in low and middle income countries? a systematic review music therapy for autistic spectrum disorder. the cochrane library school-based cognitivebehavioural interventions: a systematic review of effects and inequalities fluoride gels for preventing dental caries in children and adolescents. the cochrane library are school nurses an overlooked resource in reducing childhood obesity? a systematic review and meta-analysis effectiveness of oral health education on oral hygiene and dental caries in schoolchildren: systematic review and meta-analysis school-based depression and anxiety prevention programs for young people: a systematic review and meta-analysis effects of school-based interventions with u.s. military-connected children: a systematic review primary school-based behavioural interventions for preventing caries a systematic review of school-based interventions that include inhaler technique education what works": systematic review of the "friends for life" programme as a universal school-based intervention programme for the prevention of child and youth anxiety primary-school-based art therapy: a review of controlled studies efficacy and effectiveness of school-based prevention and early intervention programs for anxiety a systematic review of school-based sexual health interventions to prevent sti/hiv in sub-saharan africa a systematic review of school-based social-emotional interventions for refugee and war-traumatized youth effectiveness of school-based family asthma educational programs in quality of life and asthma exacerbations in asthmatic children aged five to : a systematic review world bank country and lending groups -world bank data help desk school-based cognitivebehavioural interventions: a systematic review of effects and inequalities global variations and time trends in the prevalence of childhood myopia, a systematic review and quantitative meta-analysis: implications for aetiology and early prevention vision impairment and blindness. in: world health organization standard school eye health guidelines for low and middle-income countries the international agency for the prevention of blindness prevention of mental disorders: effective interventions and policy options geneva: world health organization obesity and overweight. in: world health organization who [internet human papillomavirus vaccination in tanzanian schoolgirls: cluster-randomized trial comparing vaccine-delivery strategies feasibility of delivering hpv vaccine to girls aged to years in uganda world health statistics : monitoring health for the sdgs, sustainable development goals. geneva: world health organization ensure healthy lives and promote wellbeing for all at all ages we would like to thank tomas allen for his guidance and support in designing the search strategy. we are also grateful for advice from the who school health services guideline steering group and guideline development group members. key: cord- -kgzbd w authors: koo, bonhan; kim, da-eun; kweon, jiyeon; jin, choong eun; kim, sung-han; kim, yongsub; shin, yong title: crispr/dcas -mediated biosensor for detection of tick-borne diseases date: - - journal: sens actuators b chem doi: . /j.snb. . . sha: doc_id: cord_uid: kgzbd w rapid and highly sensitive detection of biomolecules is greatly needed for pathogen diagnosis in clinical samples, but the method needs to be significantly improved in terms of sensitivity and specificity for actual use in clinical settings. here, we report the development of an improved molecular diagnostics tool that utilizes crispr/dcas -mediated biosensor that couples a nuclease inactivated cas (dcas ) and single microring resonator biosensor, enables label-free and real-time detection of pathogenic dna and rna. we addressed the clinical utility of this crispr/dcas -mediated biosensor in tick-borne illnesses including scrub typhus (st) and severe fever with thrombocytopenia syndrome (sfts), whose clinical presentations are too similar to be easily differentiated. by using crispr/dcas -mediated biosensor, we achieved single molecule sensitivity for the detection of st ( . am) and sfts ( . am); this detection sensitivity is times more sensitive than that of rt-pcr assay. finally, crispr/dcas -mediated biosensor was able to clearly distinguish between st and sfts in serum samples within min. we believe that crispr/dcas -mediated biosensor will be useful for rapid and accurate molecular diagnostic tool that is suitable for immediate clinical applications. due to the recent increase in the occurrence of emerging infectious diseases such as zika, ebola, and mers-cov, advances have been made for fast detection of target pathogens [ ] [ ] [ ] . to control the spreading of pathogens from one community to another and to provide accurate treatment to patients, correct diagnosis of pathogen in first-line diagnostic places including clinics is crucial. although many different diagnostic approaches have been introduced, development of a rapid and high sensitive approach for clinical diagnosis still remains a challenge. rapid diagnostic testing (rdt) based on antibody detection for infectious diseases produces results within an hour [ , ] . however, the detection sensitivity of rdt is lower than that of sequence-based nucleic acid amplification methods such as pcr [ ] [ ] [ ] . hence, sequencebased nucleic acid testing has many applications in clinical fields such as diagnosis and epidemiology. however, those approaches are limited in that they are long processing time, high-cost, and relatively less sensitive for clinical use [ ] . over the past decade, biosensors combined with electrical, electrochemical, and optical techniques have been developed to improve detection sensitivity [ ] . recently the nucleic acid amplification and detection technologies that enable rapid, simple, and sensitive detection of biomolecules have been developed. the isothermal solid-phase dna amplification and detection (isad) to simultaneous amplify and detect dna molecules on silicon microring resonator (smr) biosensor, and the isothermal onestep rna amplification and detection (iroad) allows for amplification and detection of rna after complementary dna synthesis on smr biosensor, that offer simple, rapid, label-free, and real-time multiplexed detection of biomolecules near the sensor surface by using refractive index changes [ ] [ ] [ ] . for clinical use, however, the sensitivity and specificity of these technologies need to be further improved. in vivo genome editing technologies have great clinical application potentials [ , ] . zinc-finger nucleases (zfns) or transcription activator-like effectors (tales)-based editing tools have been introduced for dna targeting and regulation, however, these proteins need to be individually designed for dna binding, which remains a hurdle for regulating multiple loci [ , ] . in contrast, rna-guided clustered regularly interspaced short palindromic repeats (crispr) associated cas targets specific genomic loci via a single guide rna (sgrna), which contains a -bp guide sequence followed by a -bp protospacer adjacent motif (pam) and recognizes target dna through watson-crick base pairing [ ] . the system uses an endonuclease cas for nucleotide sequence based dna targeting. cas is guided by the sgrna that specifically bind to and cleave double-stranded dna in a site-specific manner [ ] [ ] [ ] [ ] . recently, cas nuclease can be used to enrich and detect the small amounts of tumor fragments in the circulating tumor dna (ctdna) or zika virus [ , ] . also, other types of cas proteins such as cas a or cas are used for rapid detection of nucleic acids [ ] [ ] [ ] [ ] . in addition to wild-type cas or cas orthologue proteins, catalytically inactivated cas protein (dcas ) has great potential for various biological studies [ , ] . in contrast to the wild-type cas , the nuclease-deficient dcas have the ability to bind to dna using sgrna without cutting. hence, dcas technology is a widely useful tool for in vivo and in vitro diagnostics [ , [ ] [ ] [ ] . in this study, we developed an improved diagnostic tool by combining a crispr/dcas and an isothermal diagnostic approach based on smr biosensor for simultaneous nucleic acid (rna and dna) amplification and detection with speed as well as high sensitivity and specificity. the biosensors transduce the presence of target molecules based on binding-induced changes in the refractive index proximal to the waveguide surface [ ] . in the case of combination of dcas and isothermal based biosensor, dcas can recognize the target dna or cdna but not cut the sequences of target. because of the binding property of dcas , the refractive index is changed based on the binding with target and dcas that could be more enhanced the sensitivity than smr biosensor alone. on the other hand, cas can recognize the target dna or cdna and cut the target sequences. thus, the binding-induced changes are not affected. we demonstrated the clinical usefulness of this technology by using orientia tsutsugamushi, the causative agent of scrub typhus (st), and bunyavirus, the causative agent of severe fever with thrombocytopenia syndrome (sfts). st and sfts are tick-borne infectious diseases that are common in eastern asia, especially in korea, china, and japan [ , ] . moreover, the clinical presentations of these diseases substantially overlap; therefore, a rapid and highly sensitive detection method is greatly needed. our method clearly distinguished between st and sfts within min from serum samples. for purification of recombinant dcas protein, t express bl (de ) e.coli were transformed with pet a-his -dcas plasmid. after culturing e.coli in luria-bertani (lb) broth at °c, protein expression was induced with . mm isopropyl β-d- -thiogalactopyranoside (iptg) for h at °c. cell pellet was collected by centrifugation at g, and then lysed by sonication in lysis buffer (nah po mm, nacl mm, imidazole mm ph . , mm pmsf, mm dtt, mg/ml lysozyme). soluble lysate was obtained by centrifugation at g and incubated with ni-nta agarose beads for - h (qiagen). protein bound ni-nitrilotriacetic acid (nta) agarose beads were washed (nah po mm, nacl mm, imidazole mm ph . ) and dcas protein was eluted with imidazole containing buffer (nah po mm, nacl mm, imidazole mm ph . ). with k amicon centrifugal filter (millipore), the buffer of eluted protein was exchanged, concentrated, and analyzed with - % bis-tris gels (thermofisher). the pcr products ( ng), which contained each orientia tsutsugamushi and bunyavirus dna sequence, were mixed with . μl of rehydration buffer and . μl of mm mgac solution supplied in twistamp basic rt kit. in μl reactions, buffer mixed pcr products were incubated with μg cas protein and ng sgrna for h at °c as previous study [ ] . for the positive control of the cleavage assay, same pcr products were cleaved in × nebuffer . ( mm nacl, mm tris−hcl, mm mgcl , μg/ml bsa, new england biolabs) condition. the rnase a ( μg) was added to samples to remove sgrna and the final samples were analyzed with agarose gel electrophoresis. dsdna templates were prepared by annealing the ′ biotinylated target dna strands and the non-biotinylated non-target dna strands at a : . molar ratio. (ot_ _f_biotin: tataaagatcttgttaaattgca gcgtcatgcaggaattaggaaagc, ot_ _r:gctttcctaattcctgca tgacgctgcaatttaacaagatctttata, sfts_f_biotin:aaaaattag ctgcccaacaagaagaagatgcaaagaatcaaggtgaa, sfts_r: ttc accttgattctttgcatcttcttcttgttgggcagctaattttt) nm dsdna was incubated with nm dcas and μm sgrna in cleavage buffer condition. after min incubation at °c, samples were resolved with % tbe gels using . × tbe buffer supplemented with mm mgcl . then, in vitro binding status was analyzed by using chemiluminescent nucleic acid detection module kit (thermofisher) and biodyne b nylon membrane (thermofisher) according to the manufacturer's protocol. for simultaneous amplification and detection of target dna or rna using smr biosensor alone, we used the rpa and reverse transcription (rt)-rpa solution for dna and rna, respectively. to prepare rpa or rt-rpa solution, . μl rehydration buffer, μl of rnase inhibitor and h o, . μl of μm another primer were mixed. then, reaction mix was added to freeze-dried enzyme. after that, . μl of mm magnesium acetate (mgac) was dissolved into the tube. after mixing, μl of reaction buffer was split into five μl aliquots. to begin the reactions, μl of dna or rna extracted from blood serum of the patients and μl of dcas rnps ( ng of dcas and ng of grna) were added to μl reaction aliquot. the volume of μl mixture was added into a sensor chip with an acrylic well placed surround the microring sensor area and μl mineral oil added to prevent evaporation the mixture during amplification. the chip was then placed on a thermal pad to keep a specific temperature ( °c for dna and °c for rna). the wavelength shift was measured every min to monitor the amplification of target dna or rna. relative resonant wavelength shift was calculated by the equation; ΔΔpm = (target wavelength value, pm) − (non-target wavelength value, pm). st and sfts serum samples were collected from the patients in asan medical center. the study protocol was approved by the institutional review board of asan medical center, and informed consent was obtained from all participants. sfts was confirmed by detecting viral ribonucleic acid (rna) by real-time rt-pcr in serum, using a diastar x onestep rt-pcr pre-mix kit (solgent, daejeon, south korea). a diagnosis of st was established when we observed either a single positive result of an immunofluorescence assay (ifa; sd bioline tsutsugamushi assay; standard diagnostics, yongin, south korea), or a ≥ : or fourfold rise of ifa titer in successive samples. . . crispr/dcas -mediated biosensor as a molecular diagnostic tool fig. a shows the design of crispr/dcas -mediated biosensor that couples smr-based isothermal nucleic acid amplification and dcas rnp. for simultaneous amplification and detection of nucleic acid, sequence specific primer of target was immobilized to the surface of the smr biosensor and dcas rnp was in reaction chamber with single temperature for isothermal reaction with rpa. for dna, a primer binds a recombinase enzyme to extend the dna. for rna, rt-rpa was used for the formation of complex of a primer and a recombinase enzyme to transcribe cdna from rna. the amplified nucleic acid targets were simultaneously detected by monitoring the wavelength shift. during the nucleic acid amplification process with dcas rnp on the smr biosensor, the immobilized primer was hybridized with the target templates; dcas rnp, which allows sequence specific binding to the target nucleic acids (nearby the waveguide surface at immobilized forward primer, supplementary fig. s ), subsequently caused a dramatic increase as a signal enhancer in the proportion of each resonant wavelength. the smr biosensors transduce the presence of target molecules based on binding-induced changes in the refractive index proximal to the waveguide surface. dcas binds to the amplified product on the surface of the sensor and increases the molecular weight of the sensor surface, thereby increasing the detection sensitivity by increasing the refractive index change. therefore, the crispr/dcas -mediated biosensor detected the pathogenic nucleic acids with high sensitivity within min compared to the smr alone (fig. a) . to achieve sensitive detection with dcas rnp on smr biosensor, we constructed guide rnas (grnas) targeting two tick-borne pathogens that have substantially overlapping clinical presentations: orientia tsutsugamushi, the causative agent of scrub typhus (st), and bunyavirus, the causative agent of severe fever with thrombocytopenia syndrome (sfts) (fig. b) . we observed that cas rnps induced targeted dna cleavage and dcas rnps bound target dna in rpa buffer conditions using an in vitro cleavage assay and an electrophoretic mobility shift assay (emsa), respectively ( fig. a, b) . to determine whether dcas rnp could enhance the detection sensitivity of the smr biosensor, we amplified dna fragments from st clinical samples; as a result, we observed that the signal was higher with st + dcas rnps than with st alone or st + cas rnps at all time points (from min to min) (fig. a) . the relative resonant wavelength shift results of the st with dcas rnps showed that it is possible to clearly separate the positive and negative than st alone and st with cas rnps. in the case of st with cas rnps, because of the cutting property of cas rnps, the result of relative resonant wavelength shift was reduced and looked like a negative. this improvement is due to the specific binding of dcas rnps with the target fragment on the biosensor, and not a non-specific binding effect (fig. b) . remarkably, dcas rnps improved both detection sensitivity and specificity. we also optimized the concentration of dcas rnps for sensitive and specific detection of pathogenic nucleic acids (fig. c ). next, we determined the detection sensitivity of crispr/dcas mediated biosensor for detection of pathogenic dna (for st) or rna (for sfts) fragments from orientia tsutsugamushi and bunyavirus. we used a rpa-or rt-rpa based crispr/dca mediated biosensor for double-stranded dna and single-stranded rna amplification, respectively. to test the detection sensitivity of st and sfts alone, and with dcas rnps, we used serially diluted samples containing × °to × copies of the st amplicon and t transcribed sfts rna. as a result, by using dcas rnps, we achieved single-molecule sensitivity for the detection of st ( . am) and sfts ( . am) within min (fig. a, b) . the detection sensitivity of this crispr/dcas -mediated biosensor ( copy) was superior to that of the smr biosensor alone (∼ copies) (fig. a, b ) and real-time pcrs (∼ copies) (fig. c, d) . finally, we investigated the clinical utility of this crispr-mediated biosensor for clinical applications that require speed, high sensitivity, and specificity, such as the diagnosis of incipient tick-borne illnesses. we used the crispr/dcas -mediated biosensor to analyze six clinical samples-three from st patients and three from sfts patients (fig. ) . when we employed the crispr/dcas -mediated biosensor with st primers, we observed elevated signals only in st samples and not in sfts samples (fig. a) . when we employed the crispr-mediated biosensor with sfts primers, we detected elevated signals only in sfts samples. remarkably, the sfts samples were more clearly detected by the crispr/dcas -mediated biosensor than by the smr biosensor alone (fig. b) . thus, we demonstrated that the crispr-mediated biosensor can clearly distinguish the two pathogens from clinical samples. we developed a crispr/dcas -mediated biosensor that combines a catalytically inactive dcas and smr biosensor based isothermal error bars indicate standard deviation from the mean, based on at least independent experiments. (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article). nucleic acid amplification for rapid and high sensitive detection of pathogens in clinical settings. using crispr/dcas -mediated biosensor, we achieved single molecule sensitivity for the detection of st ( . am) and sfts ( . am). also, our method clearly distinguished between st and sfts within min from serum samples. we addressed several features for clinical use of this assay. first, crispr/dcas -mediated biosensor enhances detection sensitivity through triple-targeting (primer, target strand, and dcas ). dcas shows higher sensitivity and specificity than the conventional method because it requires accurate target recognition of nt of grna (supplementary table s ). importantly, the triple-targeting method provides a significant advancement in terms of detection sensitivity from our previous smr biosensor approach that utilized primer to target strand only [ , ] . by using triple-targeting, we were able to detect a single copy target, which translates into times higher sensitivity than that of smr biosensor alone ( copies) and times more sensitive than that of real-time (rt)-pcr methods ( copies). second, the readout system of crispr/dcas -mediated biosensor differs from other crispr based diagnostic assays (i.e., fluorescence dye) in fundamental ways [ ] [ ] [ ] [ ] . smr biosensor based isothermal nucleic acid amplification assay can be simultaneously amplified and detect the target without labeling. in addition, crispr/dcas enhanced the refractive index change on the sensor surface. therefore, the readout of sample using our assay can be carried out within min. third, the results of false-positive or false-negative are minimized by the use of sgrna, primer, and dcas . we have also shown that this assay is able to clearly distinguish the two pathogens in clinical samples. our data on the clinical sensitivity and specificity highlights the utility and practicality of crispr/dcas -mediated biosensor. fourth, gootenberg et al. developed a crispr/cas a (previously known as c c )-based diagnostic assay [ , ] . because cas a is a programmable rna-guided ribonuclease, cas a-based diagnosis is an rna-based detection method that exploits the collateral effect of cas a. our complementary approach using crispr/dcas is a dna-based detection method that is able to detect nucleic acids during isothermal nucleic acid amplification without in vitro rna transcription. finally, our crispr/dcas -mediated biosensor method is being further improved for convenient operation based on the assured criteria (affordable, sensitivity, specificity, user-friendly, rapid, equipment-free, deliverable) in clinical settings [ ] . a microfluidic platform is being developed for sample processing that uses nonchaotropic reagent-based thin film techniques. the platform is devised to allow simple, low-cost, rapid, and high-throughput nucleic acids (dna, 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ultrasensitive detection of target dna using pcr rapid, low-cost detection of zika virus using programmable biomolecular components nucleic acid detection with crispr-cas a/c c two distinct rnase activities of crisrp-c c enable guide-rna processing and rna detection crispr-cas a target binding unleashes indiscriminate single-stranded dnase activity multiplexed and portable nucleic acid detection platform with cas , cas a, and csm use of cellular crispr (clusters of regularly interspaced short palindromic repeats) spacer-based microarrays for detection of viruses in environmental samples rna-guided gene activation by crispr-cas -based transcription factors conditionally stabilized dcas activator for controlling gene expression in human cell reprogramming and differentiation orientia tsutsugamushi infection: overview and immune responses phylogenetic analysis of severe fever with thrombocytopenia syndrome virus in south korea and migratory bird routes between china, south korea, and japan digenome-seq: genome-wide profiling of crispr-cas off-target effects in human cells his research is focused on development of molecular diagnostic platform based on optical biophotonics for detection of disease related biomarkers her research is now focused on study of crispr/cas function in clinical applications she is a postdoctoral fellow at university of ulsan college of medicine and asan medical center her research is focused on development of molecular diagnostic platform based on optical biophotonics for detection of disease related biomarkers he is an associate professor at university of ulsan college of medicine and asan medical center he is an assistant professor at university of ulsan college of medicine and asan medical center his research is now focused on development of molecular diagnostic platform based on optical biophotonics for detection of disease related biomarkers this study was supported by the ministry of science, ict and future planning (msip) and the ministry of education through the national research foundation of korea (nrf) ( r a b , r d a a ). supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.snb. . . . key: cord- -utvy i l authors: tobar vega, pool; erramilli, shruti; lee, eugene title: talaromyces marneffei laboratory cross reactivity with histoplasma and blastomyces urinary antigen date: - - journal: int j infect dis doi: . /j.ijid. . . sha: doc_id: cord_uid: utvy i l talaromyces marneffei is a fungal opportunistic infection usually seen in immunocompromised patients from eastern countries. in the us when examining hiv-patients for suspected fungal infections, laboratory serological tests guide therapy until cultures are available. we present the case of a -year-old hiv patient originally from thailand in which urine lab results were positive for blastomyces and histoplasma antigen, but biopsy showed t. marneffei. concomitantly the patient presented with hyponatremia which was deemed to be from siadh. we present the first case of a patient with t. marneffei cross reactivity with blastomyces, histoplasma and siadh due to pulmonary disease. endemic to southeast asia, east asia and china, talaromyces marneffei is a dimorphic fungus capable of causing systemic fungal infections in immunocompromised patients (supparatpinyo et al., ) . since its discovery in the s, the majority of cases have been documented in hiv patients with low cd counts. in northern thailand, t. marneffei is the fourth most prevalent opportunistic infection in this population (chariyalertsak et al., ) . clinical manifestations include fever, malaise, lymphadenopathy, cough, and hepatosplenomegaly (wu et al., ) . while the frequency of t. marneffei infection has decreased with the advent of retroviral therapy, if left untreated the infection frequently leads to respiratory failure with a poor prognosis. in the u.s. patients with hiv infection usually undergo testing for endemic fungal infections such as blastomyces, histoplasma, coccidioides and paracoccidioides. indirect serological results help to make faster decisions given that cultures take several days or weeks to grow. clinical and geographic context plays a particularly important role because some of these tests have been shown to have cross-reactivities. pulmonary infection either by fungi, bacteria or virus has been observed to cause concomitant hyponatremia, with inappropriate levels of antidiuretic hormone (siadh) often found as the underlying etiology. the exact mechanism is not understood but hypoxemia and hypercapnia are thought to play an important role in the pathophysiology (rose et al., ) . in the following, we describe a t. marneffei infection with unusual laboratory and clinical characteristics. the patient was a -year-old male from thailand who presented with generalized weakness and fever. his past medical history was relevant for hiv infection (since age ) on haart (bictegravir, emtricitabine & tenofovir alafenamide). two weeks prior to his admission, he had travelled to chicago, las vegas and utah. during this time, he developed a productive cough with blood-tinged sputum, subjective fever, chills, and anorexia with associated weight loss. on physical exam, he was noted to have multiple erythematous, raised, scaly/crusted lesions on the face, neck and abdomen (figure hiv viral load was . * copies/ml and cd count was cells/ml. right upper quadrant ultrasound revealed hepatomegaly and a chest x-ray reported bilateral peri-tracheal soft densities up to cm in diameter, interstitial markings, and bilateral pulmonary nodules. chest ct scan without contrast showed patchy pulmonary densities and multiple peri-hilar nodules ( figure ). ct scan with contrast of the head and neck did not reveal acute intracranial abnormalities but did show cervical lymphadenopathy. gram stain smear and culture of the sputum were negative. respiratory viral panel including influenza, parainfluenza, coronavirus and rsv was negative. legionella urine antigen was negative. bacterial blood cultures and gram stain were negative. quantiferon gold and three sputum samples for afb/culture were negative for tuberculosis. serological testing for cryptococci and blastomyces were negative. however, urine antigen testing for both blastomyces and histoplasma were positive. finally, a biopsy of one of the cutaneous lesions demonstrated dermal and subcutaneous neutrophil and histiocyte infiltrate with the presence of intracellular yeast, findings which were consistent with t. marneffei. t. marneffei infections typically manifest in severely immunocompromised patients. current guidelines recommend that in hiv patients from endemic countries with a cd count < cells/ml, primary preventive therapy with itraconazole should be initiated (panel on opportunistic infections in hiv-infected adults and adolescents, ). clinical manifestations appear to vary depending on the severity and underlying etiology of immune compromise in the patient, differing between hiv vs. non-hiv causes such as malignancies or transplant patients. fever, splenomegaly, anemia, transaminitis, and absence of leukocytosis seem to be more frequently found in hiv-positive patients (kawila et al., ) . in our case, clinical findings included fever, neck lymphadenopathy, and respiratory symptoms. laboratory work demonstrated transaminitis along with a cd count of cells/ml. an initial laboratory test for endemic fungi can guide initial treatment towards early antifungal medication. however, crossreactivity between antigens in various fungal infection detection tests is well-documented, and cross reactions between histoplasma and blastomyces antigens are the most common (wheat et al., ) . others have been described, such as that of histoplasma antigen in patients with sporotrichosis (assi et al., ) . in our case, urine antigen testing results for both histoplasma and blastomyces were positive, serum testing was negative. it is of note that the sensitivities of the blastomyces and histoplasma antigen detection test in urine are approximately % and % respectively. specificity is around % for both tests, usually having to rule out each other as the main confounder (cunningham et al., ; frost and novicki, ) . hiv status can affect tests based on antibody detection. since the tests used to guide therapy are based on antigen detection, sensitivity is unlikely to be affected by hiv infection. these infections in their disseminated forms will receive amphotericin-b with itraconazole. however, differentiation is important given that blastomycosis is treated for a year versus talaromyces which is treated for weeks (sirisanthana et al., ; saccente and woods, ) . siadh is an exclusion diagnosis that requires an extensive work up to rule out other etiologies including adrenal insufficiency, thyroid disease, and volume depletion (shu et al., ) . adh is produced on the paraventricular thalamic nucleus and thus classically this syndrome is observed after neurological insults that cause an excess in adh. nevertheless, it has been observed that respiratory tract infections can cause inadequate adh secretion and these are the most common infections in hiv patients. increase in the a-a gradient and hypoxia/hypercapniainduced adh secretion are some of the non-osmotic mechanisms thought to trigger elevations in adh in this population. it has been proposed that hypercapnic acidosis and hypoxemia induce central release of vasopressin through peripheral chemoreceptors and baroreceptors stimulation respectively (rose et al., ; dreyfuss et al., ) . tuberculosis, cryptosporidium, plasmodium infections and pneumocystis pneumonia have been previously reported as pulmonary infections causing siadh. it is of note that hiv by itself could contribute to siadh. but the mechanism underlying this infection is usually mediated by hiv induced thyroid and adrenal insufficiency. in our case, the patient had increased urinary sodium, decreased serum osmolality, normal cortisol and tsh levels and absence of neurological affect, leaving the pulmonary fungal infection as one of the explanations for inadequate adh secretion. in conclusion, laboratory work up for endemic fungal infection can have false positive results with infections such as talaromyces. this cross reactivity is especially important when assessing patients from endemic countries. manifestations of t. marneffei infection are diverse, and disease description is limited due to the small number of cases. a novel manifestation observed in our patient was the presence of siadh likely secondary to the respiratory talaromyces infection. to our knowledge, this is the first case reporting systemic mycosis due totalaromyces marneffei with associated hyponatremia secondary to siadh and cross-reactivity with blastomyces and histoplasma in urine antigen testing. cross-reactivity in the histoplasma antigen enzyme immunoassay caused by sporotrichosis clinical presentation and risk behaviors of patients with acquired immunodeficiency syndrome in thailand, - : regional variation and temporal trends sensitivity and specificity of histoplasma antigen detection by enzyme immunoassay acute infectious pneumonia is accompanied by a latent vasopressin-dependent impairment of renal water excretion blastomyces antigen detection for diagnosis and management of blastomycosis panel on opportunistic infections in hiv-infected adults and adolescents. guidelines for the prevention and treatment of opportunistic infections in hiv-infected adults and adolescents: recommendations from the centers for disease control and prevention, the national institutes of health, and the hiv medicine association of the infectious diseases society of america antidiuresis and vasopressin release with hypoxemia and hypercapnia in conscious dogs clinical and laboratory update on blastomycosis hiv/aids-related hyponatremia: an old but still serious problem amphotericin b and itraconazole for treatment of disseminated penicillium marneffei infection in human immunodeficiency virus-infected patients disseminated penicillium marneffei infection in southeast asia evaluation of cross-reactions in histoplasma capsulatum serologic tests clinical presentations and outcomes of penicillium marneffei infections: a series from aknowledgement is given to dr saad, peguy who proof read this manuscript. consent was obtained from patient for the publication of this manuscript.consent was obtained from patient for the publication of this manuscript. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. the authors declare no conflict of interest. key: cord- -o qbqxhx authors: cavalcante, liliane t. f.; muniz, cláudia p.; jia, hongwei; augusto, anderson m.; troccoli, fernando; medeiros, sheila de o.; dias, carlos g. a.; switzer, william m.; soares, marcelo a.; santos, andré f. title: clinical and molecular features of feline foamy virus and feline leukemia virus co-infection in naturally-infected cats date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: o qbqxhx feline foamy virus (ffv) and feline leukemia virus (felv) belong to the retroviridae family. while disease has not been reported for ffv infection, felv infection can cause anemia and immunosuppression (progressive infection). co-infection with ffv/felv allows evaluation of the pathogenic potential and epidemiology of ffv infection in cats with felv pathology. blood and buccal swab samples from cats were collected in rio de janeiro. plasma was serologically tested for felv. dna extracted from peripheral blood mononuclear cells and buccal swabs was used to pcr detect ffv and felv. a qpcr was developed to detect and measure ffv proviral loads (pvls) in cats. felv qpcr was performed using previous methods. the median log pvl of ffv mono-infected individuals was lower than found in ffv/felv co-infected cats in buccal swabs (p = . ). we found % of cats had detectable buccal ffv dna in ffv mono-infected and ffv co-infected felv-progressive cats, while in felv-regressive cats (those without signs of disease) % of cats had detectable buccal ffv dna (p = . ). our results suggest that regressive felv infection may reduce ffv saliva transmission, the main mode of fv transmission. we did not find evidence of differences in pathogenicity in ffv mono- and -dually infected cats. in summary, we show that fvs may interact with felv within the same host. our study supports the utility of cats naturally co-infected with retroviruses as a model to investigate the impact of fv on immunocompromised mammalian hosts. foamy viruses (fv) are complex retroviruses that belong to the retroviridae family and comprise a unique genus within the spumaretrovirinae subfamily that naturally infect many vertebrates, including feline, simian, bovine, and equine [ ] . fv infection in humans is invariably associated with zoonotic stomatitis. the risk of developing lymphoid malignancy is estimated to be to -fold greater in fiv-infected only cats compared with uninfected cats [ ] . several studies have investigated co-infection in domestic cats [ , , ] . other studies have investigated the virologic factors involved in retrovirus co-infection [ ] . but they mostly focused on fiv/felv co-infections. a study by yamamoto et al. showed that a pre-existent felv infection enhanced the expression of fiv in the body and increased the severity of transient primary and chronic secondary stages of fiv infection [ ] . only a single study has evaluated the presence of all three retroviruses in cats [ ] , but was limited only to prevalence estimates and not their possible interactions or associations with disease. in another study, felv and fiv co-infection was examined and showed a greater risk for lymphoid cancers in dually infected cats [ ] . the combination of susceptibility to multiple retroviral infections and varied possible disease outcomes makes domestic cats an attractive animal model to study the epidemiology and pathogenesis of co-infection by distinct retroviruses. we examined blood and buccal swab specimens of domestic cats in brazil for detection and quantification of each feline virus to evaluate their potential association with disease and transmissibility in animals with single or multiple retroviral infections. this project was approved by the ethics committee on animal use of the center for health sciences at federal university of rio de janeiro (ceua-ccs) (protocol ibo - / ). blood and buccal swab samples from domestic cats were collected between and from three different veterinary clinics in rio de janeiro (n = ) or captured within the grounds of fundação jardim zoológico da cidade do rio de janeiro (riozoo, n = ), an urban area in the north zone of the city. cats coming from veterinary clinics were selected for this study once they were suspected of felv infection, while cats from riozoo were selected randomly, independently whether they looked sick or not. the cats from clinics were not previously vaccinated for felv, while for captured animals we are not sure about their vaccination status although the chances they have been vaccinated are minimal, since brazilian public policies do not include felv vaccination. upon presentation at the clinics or after capture, the cats were weighed and anesthetized with the administration of g of ketamine or . µg of xylazine per kilogram of body weight. after anesthesia, - ml of whole blood was collected from the jugular vein by experienced veterinarians and placed into an edta blood tube and stored at room temperature until processing. buccal swabs were collected from cats by rubbing the upper, bottom and side of the oral cavity with a sterile cotton swab. hemogram tests displaying blood cell counts of animals were performed by a veterinarian diagnostic laboratory. hemogram results were kindly provided by dr. carlos gabriel dias. cats with low erythrocytes (below milions/µl), hemoglobin (below g/dl) or hematocrit (below %) levels were considered anemic. twenty-eight cats were followed three years after the initial collection to determine their life status (alive or dead). for felv-infected cats, the time elapsed from infection was estimated based on the diagnosis date at the veterinary clinics. for cats whose infection status was unknown before sampling, the time elapsed from infection was considered using the sampling date. cats were classified according to gender, age, outdoor access, household type, neuter status and health status. for age, cats were classified as: (a) kitten, up to months old; (b) young, between - months of age and (c) adult, above one year of age. for feral cats without a known birthdate, age was estimated by veterinarian evaluation. household status was classified as: (a) single, cats living alone at home; (b) multicat, cats living with up to cats in the same house; (c) shelter, cats living in a shelter or house with more than cats; and (d) feral, cats that live or have lived in the streets before. cats classified as single, multicat and shelter were also categorized for outdoor access. cats were considered sick when any clinical signs, including respiratory tract disease (rtd), progressive weight loss (pwl), ocular secretion (os), anemia, cachexia, ulcers, hair loss, presence of ectoparasites, tooth loss, prolapses, scabies, pemphigus foliaceous, pyometra, sporotrichosis, diarrhea, neurological symptoms, abortion, kidney disease, stomatitis and neoplasia, or hematological changes were reported. healthy cats were those without any reported clinical signs at the time of specimen collection. blood samples were centrifuged at × g for min to separate plasma from cells. plasma aliquots were stored at − • c for fiv/felv serologic testing. peripheral blood mononuclear cells (pbmcs) were obtained from the cellular fraction by ficoll-paque plus (ge healthcare, waukesha, wi, usa) centrifugation and were stored at − • c until genomic dna (gdna) extraction. gdna from pbmc and buccal swabs was extracted using the purelink ® genomic dna mini kit (invitrogen, carlsbad, ca, usa) and was quantified using a nanovue plus™ nanodrop (ge healthcare). pcr amplification of cytochrome b oxidase gene (cytb) sequences was used to verify dna integrity of the extracted material as previously described [ ] . plasma from domestic cats was tested for fiv/felv by two commercial immunochromatographic tests: the fiv ac/felv ag test alere ® (bionote inc., gyeonggi-do, korea) or quickvet fiv/felv ubio (biotechnology systems, kerala, india) kits at a veterinary clinic and ufrj, respectively. these tests simultaneously detect an felv antigen and an fiv antibody. assay sensitivity of % and . % for felv and % and . % for fiv, and specificity of % and . % for felv and % and . % for fiv were established by alere ® and ubio, respectively. nested or semi-nested pcr was performed to diagnose ffv infection by detection of four different ffv genome sequences, including the long terminal repeat (ltr, -bp) [ ] , integrase (int, -bp) [ ] , gag/polymerase (pol) ( -bp) [ ] and envelope (env, -bp) [ ] . three new pcr primers for this study (ffv ltr out, ffv_f and ffv_r ) were designed using an alignment of the four available complete ffv genomes from domestic cats (genbank accession numbers aj , aj , nc and y ). primer sequences are provided in table . primers used for detection of the fiv reverse transcriptase (rt) portion ( -bp) of the pol gene and of felv env sequences ( -bp) ( table ) were published previously ( [ ] ), respectively. for all published primers, the pcr conditions were followed according to those previous studies [ , [ ] [ ] [ ] [ ] [ ] . pcr conditions using newly designed primers were performed using × pcr buffer, mm mgcl , mm dntps, pmol forward and reverse primers, unit of taq platinum polymerase (invitrogen) and - nanograms of dna in a total reaction volume of µl. amplicons from pcr-positive samples were purified using the pcr dna mini kit (real genomics rbc, banqiao, taiwan) following the manufacturer's protocol and were sequenced in an abi xl platform (thermo scientific, waltham, ma, usa). a new quantitative pcr assay was developed to simultaneously detect and measure ffv proviral loads in infected cats. generic pol primers and probe were designed using an alignment of complete ffv genomes at genbank. the forward and reverse primers ffvpf -cat gtt gtc agc acc aag tat ac- and ffvpr -tgc aag aga gca agt tct tct tc- , respectively, and probe ffvpfp -fam-ttg gaa ttg att gta att tac cat ttgc-bhq - were used to detect a -bp pol sequence. cycling conditions included an initial step of • c for min, followed by cycles of s at • c and s at • c with a final hold step at • c using a bio-rad icycler cfx (bio-rad laboratories, hercules, ca, usa). for assay validation, human blood donor pbmc dnas and five different human cell lines (mcf- , cemx , jurkat, lncap and hela) were used as negative controls to assess assay specificity. specificity and sensitivity were also determined using pbmc lysates from us domestic cats previously found to be ffv-negative (n = ) or positive (n = ) by western blot analysis. these cat specimens were available from a previous study investigating the zoonotic potential of feline retroviruses [ ] . the limit of detection (lod) of the assay was determined by testing of - replicates (depending on the copy number) ten and fivefold serial dilutions of plasmids containing a cloned fragment of the ffv int sequence ranging from to a single copy diluted in µg of negative human gdna. once the potential lod was determined using replicates of the -fold dilutions, we used a larger number of five-fold plasmid dilution replicates to further refine the lod. we tested ten copy replicates, five copy replicates and single copy replicates. standard curves were constructed using - copies of plasmid dna. the number of provirus copies/cell was estimated using the formula: number of copies detected/(dna quantification in picograms/ pgs cell) where the mass for each domestic cat cell genome was considered as six picograms dna per cell [ ] . the amount of dna in nanograms was measured by spectrophotometry. the felv qpcr test was performed as previously described [ ] using a unique region within the u of the felv ltr with a reported detection limit of copies per reaction. specificity of the assay was % by obtaining negative results for pbmc dna from pathogen-free, felv-negative cats. assay sensitivity was also % by confirmation of infection in felv p -positive cats. cats that were experimentally infected and that became persistently antigenaemic as determined by p elisa were considered positive. the primers and probe felv-u -exo-f -aac agc aga agt ttc aag gcc- (forward), felv-u -exo-r -tta tag cag aaa gcg cgc g- (reverse) and felv-u -probe -fam-cca gca gtc tcc agg ctc ccc a-tamra- were used to detect a -bp ltr sequence. standard curves were performed with a plasmid containing the target region in a serial dilution range of - copies. the cycling conditions consisted of an initial step of gotaq (promega, madison, wi, usa) activation at • c for min followed by cycles of s at • c and min at • c using an abi real-time cycler (applied biosystems, foster city, ca, usa). the number of pvl copies/cell was calculated similarly to the ffv pvl, except the pvl was divided by two to compensate for the presence of two ltr copies per integrated provirus. we classified ffv-infected cats that were tested for both oral swab and pbmc tissues by nested pcr and/or qpcr. ffv-positive cats without virus detected in saliva were considered as latent infections, and were classified as non-transmissible. ffv-positive cats with virus detected in oral swabs were considered as potential ffv transmitters to other cats and were classified as transmissible. cats testing qpcr positive for felv were classified into two groups based on vl and serostatus: progressives (those with high pvl, most seropositive) and regressives (those with low vl, most seronegative). the mann-whitney test was used for statistical comparisons of pvl between groups. kaplan-meier survival curves were generated for survival analysis. linear regression was used to evaluate a correlation between the ffv and felv vls. odds ratios (or), % confidence intervals ( % ci) and p-values were determined to analyze risk factors for felv and ffv co-infection, including sex, age, whether neutered, and household status. a multivariate analysis was conducted by adjusting the model for variables that showed significant associations with ffv/felv co-infection, i.e., that showed p-values below . . to test the different proportion of non-and transmissible cats, probabilities (p) were calculated using the chi-square test. all statistics were performed using medcalc v. . . . or in the r environment and p-values ≤ . were considered significant. all domestic cats sampled in this study were from fundação jardim zoológico da cidade do rio de janeiro (riozoo) and from veterinary clinics throughout the city of rio de janeiro. cats were classified according to gender, age, outdoor access, sexual sterilization, household type, and health status ( table ) . we found an approximately equal proportion of male and female cats and a higher number of adult individuals ( %). twenty of male cats ( %) were neutered, while of females ( %) were spayed. most cats ( %) were classified as feral and of the cats residing in any household ( multicats, shelters and singles), the majority ( %) had no outdoor access. the majority of cats ( %) were also sick at the time of sample collection. blood dna specimens from all cats were tested by nested pcr and serology for diagnosis of fiv and felv infection. buccal swab genomic dna (gdna) specimens available from cats were also analyzed using pcr testing. animals were considered pcr-positive when at least one viral sequence was detected in pbmcs and/or buccal swab samples. we found three fiv-positive animals, of which / ( . %) were positive only by serology and / ( . %) was pcr-positive only. of two serologically fiv-positive animals, one was a house cat and the other was a feral cat. the fiv pcr-positive only cat was feral (table ) . we found / ( %) animals reactive in the felv p antigen test in plasma and an equal number of pcr-positive animals but cats did not always have concordant p and pcr results. specimens from five cats were pcr-positive only and five different cats were felv p -reactive only. cats positive using at least one assay (serology or pcr) were classified as infected. among the household cats (single, multi-cat and shelter) and feral cats, felv-positive results in both pcr and serological tests tended to be higher in households, % ( / ) compared to feral cats % ( / ) (p = . ; fisher´s exact test; table ). two cats were dually infected with both fiv and felv. the pbmc and buccal swab gdna samples were screened by nested pcr for amplification of four different ffv genomic sequences (table ). overall, we found a ffv pcr prevalence of % ( / ). we found more cats with gag/pol pcr-positive results ( / , % in pbmcs and / , % in buccal swab gdna) compared to other viral targets. of the ffv-positive animals, had both pbmc and buccal swab gdna specimens available for nested ffv pcr testing. twenty-nine animals were pcr-positive ( %) in the pbmc compartment, while only ( %) were positive in the buccal swab (p = . ). comparing both tissue compartments, animals were positive in both pbmc and buccal swab, while cats were positive only in pbmc and four were positive only in the buccal swab. no significant differences were found when comparing feral to household cats that were ffv pcr-positive in either the pbmc or the buccal swab compartments (p = . and p = . , respectively). ( ) a cats with at least one virus fragment detected. b in three cases, a limited number of samples were tested due to material availability. we also developed a new quantitative real-time pcr (qpcr) assay to simultaneously detect and measure ffv proviral load (pvl) in cats. testing of replicates containing serial dilutions of ffv integrase (int)-containing plasmids showed that / replicates ( %) containing five copies/ug dna tested positive, whereas / ( %) replicates containing a single ffv copy were qpcr positive. hence, we set detection limit of the ffv qpcr assay at five ffv copies/ug gdna or about . × − copies/ cells. ffv int sequences were not detected in pbmc gdna from us human blood donors or in five different human cell lines. the sensitivity of the qpcr assay was also evaluated using pbmc lysates from us domestic cats for which the infection status was previously determined by wb testing [ ] . detection of ffv correlated with the wb and/or nested pcr results in . % ( / ) of the samples. the average and median ffv pvls were . × and . × copies/ cells, respectively. two wb-positive but qpcr-negative samples also tested negative for β-actin, indicating possible dna degradation or pcr inhibitors in those samples. five of the ffv wb-negative animals had low pvls ( - copies/ cells), indicating possible infection during the seroconversion window period or latent infection with a revertant antibody response. one cat with an indeterminate wb result (seroreactivity to a single gag protein) also tested qpcr negative, suggesting possible nonspecific seroreactivity in this animal. pbmc gdna was further available from of brazilian cats testing positive using the nested pcr assays. of these cats, were qpcr-positive with a median pvl of − . log copies/cell ( , copies/ cells) and an average of − . log copies/cell ( , copies/ cells) ( figure a ). of cats with available buccal swab gdna for qpcr testing, were positive with a median pvl of − . log copies/cell ( , copies/ cells). of the cats testing negative by nested pcr, had pbmc gdna available for qpcr testing. of these cats, were qpcr-positive with a median pvl of − . log copies/cell ( , copies/ cells). buccal swab gdna from nested pcr-negative animals identified seven qpcr positive cats with a median pvl of − . log copies/cell ( , copies/ cells). there was no pvl differences between animals that were previously positive or negative for ffv using the nested assays (p > . for both tissues). interestingly, the pvl was higher in buccal swab than in pbmc specimens (p = . ). fifty-three of the pbmc samples tested ( %) were qpcr-positive versus % ( / ) of buccal swab samples. for ffv-monoinfected cats, we did not find differences between pvl in pbmcs (median pvl of − . log copies/cell; n = ) and in buccal swabs (median pvl of − . log copies/cell; n = ) (p = ). by using qpcr, the total number of ffv-positive cats increased from ( %) to ( %). of these qpcr-positive cats, ( . %) were monoinfected with ffv. analysis of cats classified as potentially transmissible and non-transmissible found no statistical difference between ffv pvls. by using qpcr, the total number of ffv-positive cats increased from ( %) to ( %). of these qpcr-positive cats, ( . %) were monoinfected with ffv. analysis of cats classified as potentially transmissible and non-transmissible found no statistical difference between ffv pvls. of felv-positive cats diagnosed by serological and/or molecular assays, with available pbmc gdna were felv qpcr-positive with a median pvl of . log copies/cell ( . × copies/ cells) ( figure b) . buccal swab gdna was available for cats, of which were felv of felv-positive cats diagnosed by serological and/or molecular assays, with available pbmc gdna were felv qpcr-positive with a median pvl of . log copies/cell ( . × copies/ cells) ( figure b) . buccal swab gdna was available for cats, of which were felv qpcr-positive with a median pvl of − . log copies/cell ( . × copies/ cells). the felv pvl was approximately × higher in pbmc than in buccal specimens (p = . ). our pvl data are correlated with felv outcome (progressives of group i, those cats with high vl, mostly seropositive) and regressives (group ii, those with low vl, mostly seronegative). group i (n = ) had a higher pvl with an average of . log felv copies/cell ( . × copies/ cells) and median pvl of . felv log copies/cell ( . × copies/ cells) whereas group ii (n = ) cats had a lower median pvl of − . felv log copies/cell ( . × copies/ cells) (p < . ). in this case, there was no difference between serological status of the two groups: in group i, % (n = ) were felv-seronegatives while in group ii, % (n = ) had that status (p = . ). we also tested the of the cats with negative felv serology and/or molecular assays with available pbmc gdna. we found felv-positive cats by qpcr with a median of − . log copies/cell ( . × copies/ cells). this median pvl is almost × lower than the median pbmc pvl in felv-seropositive cats (p < . ). pvls of felv-seronegative cats were similar to those measured in the group ii felv-positive cats (p = . ), indicating that the felv-seronegative cats with low pvls likely had a regressive infection. testing of buccal swab gdna samples identified four qpcr-positive cats with a median felv pvl of − . log copies/cell ( . × copies/ cells). however, there was no difference between buccal swab pvls in felv-seronegative and seropositive cats (p = . ). cats with progressive and regressive infections differed in the qpcr detection of felv in buccal tissues. of regressive animals, only five ( %) had detectable pvl in buccal cells, while all ( %) of animals with progressive infection had detectable pvl in that compartment (p < . ). nonetheless, buccal swab pvls of progressive and regressive animals were similar (p = . ). by using qpcr, the total number of felv-positive cats increased from ( %) to ( %). of the qpcr-positive cats, eight ( %) were monoinfected with felv, while ( %) were co-infected with both ffv and felv and two ( %) were infected with all three feline retroviruses. we found % ( / ) of the cats in our study to be ffv-monoinfected, % ( / ) with felv monoinfection, % ( / ) with dual ffv/felv co-infection, % ( / ) with dual ffv/fiv co-infection, % ( / ) with triple ffv/felv/fiv co-infection and % ( / ) with no evidence of any feline retrovirus. no fiv monoinfections or dual felv/fiv co-infections were identified. therefore, subsequent statistical analyses were based on the three groups consisting of ffv and felv monoinfections and ffv/felv dual infections, since these infections were more common in our population. we found an approximate : ratio between females and males in all three ffv and felv infection groups (table ) . kittens, young, and adult cats were present in all three groups, except for the felv-monoinfected group which only contained adults. the majority of cats living in multi-cat homes or shelters were ffv/felv-co-infected ( % and %, respectively). the majority of cats with outdoor access and poor health were also co-infected with ffv and felv (table ), but these differences were not statistically significant. in captive animals, monoinfection of ffv was . % ( / ) while in feral animals the prevalence was % ( / ) (p < . ). however, the total prevalence of ffv (mono and dual-infection with felv) was % in captive animals and % in feral animals (p = . ). felv monoinfection prevalence was lower in feral ( %) compared to captive ( %) cats, and similarly the total prevalence of felv was lower in feral cats ( %) than in the captive cats ( %) (p = . ). odds ratios were determined to evaluate whether ffv infection was a risk factor for felv infection. infection with ffv did not increase the chances of having felv infection (or = . , p = . ). among ffv-infected cats, odds ratios were also calculated to evaluate risk factors involved with felv co-infection (table ). neutered ffv-infected cats were more likely to be co-infected than non-neutered counterparts. cats living in shelters showed higher rates of co-infection than those cats in other housing types. non-feral cats living alone, in a shelter or multicat household had a higher risk of co-infection than feral cats (or = . , p = . ). no associations of co-infection were found with either age or sex. we also evaluated risk factors for ffv co-infection of felv-infected cats. no significant differences were observed when considering the same factors analyzed for ffv-infected cats (or = . , p = . ). since the street and neuter variables showed significance at the univariate level, a multivariate analysis was performed with adjustment for those two variables. while the effect of neutering was lost (or = . ; p = . ), street cats remained significantly less prone for co-infection with ffv and felv than non-street cats (or = . ; p = . ). when we compared median log pvls of ffv mono-and ffv/felv co-infected individuals ( figure ) we found higher ffv pvls in buccal swab specimens in co-infected cats (p < . ; figure a ). in contrast, differences in pbmc pvls between mono-and co-infected cats were not significant (p = . ). however, pvls in the buccal specimens were significantly higher than those in pbmc of co-infected cats (p < . ). no differences were found in felv pvls between buccal swabs and pbmcs or between felv mono-and co-infected cats (p = and p = . , respectively; figure b ). we also evaluated pvls of cat samples that were tested for both ffv and felv, but did not observe a correlation between felv and ffv pvls in pbmcs among cats with progressive or regressive infection ( figure a) . similarly, there was no correlation between the ffv and felv pvls in buccal specimens ( figure b ) except for felv-regressive individuals (r = . ; p = . ; figure b ), which showed a negative association. the percentage of potentially transmissible ffv with detectable pvls in buccal swabs in monoinfected cats ( . %) was very similar to that in felv progressive cats ( . %) (figure ). the opposite was found when comparing ffv-monoinfected cats and felv regressives (p = . ). for cats with ffv and felv positive results for both buccal swab and pbmc specimens, we compared the ffv pvls between ffv-monoinfected and ffv/felv-co-infected animals and also the pvls between felv progressive and regressive animals. however, we did not observe a correlation between pvls in buccal swab or pbmc in either situation (data not shown). for cats with ffv and felv positive results for both buccal swab and pbmc specimens, we compared the ffv pvls between ffv-monoinfected and ffv/felv-co-infected animals and also the pvls between felv progressive and regressive animals. however, we did not observe a correlation between pvls in buccal swab or pbmc in either situation (data not shown). for cats with ffv and felv positive results for both buccal swab and pbmc specimens, we compared the ffv pvls between ffv-monoinfected and ffv/felv-co-infected animals and also the pvls between felv progressive and regressive animals. however, we did not observe a correlation between pvls in buccal swab or pbmc in either situation (data not shown). to investigate possible disease associations in cats with mono or dual ffv and felv infection we analyzed clinical signs reported in these animals at the time of sampling. we did not detect any differences among the proportions of major clinical signs (rtd, pwl and os) in ffv-monoinfected, or felv-monoinfected regressive or progressive cats ( table ). five of seven ( %) cats with ffv/felv-progressive infection were anemic at the time of data collection by hemogram testing while none of the ffv/felv-regressive cats were anemic ( / ; p = . ). less common clinical signs detected were cachexia ( / ), ulcers ( / ), hair loss ( / ), presence of ectoparasites ( / ), tooth loss ( / ), prolapses ( / ), scabies ( / ), pemphigus foliaceous ( / ), pyometra ( / ), sporotrichosis ( / ), diarrhea ( / ), neurological ( / ), abortion ( / ), kidney disease ( / ), stomatitis ( / ) and neoplasia ( / ). however, the low frequency of these clinical signs did not permit an analysis of their association with retrovirus infection of these cats. we analyzed ffv pvls in cats with or without each reported major clinical signs, but did not observe differences between pvls in cats with or without pwl (p = . ), rtd (p = . ) or os (p = . ). we also did not find any ffv pvl differences in cats with or without any clinical signs (p = . ), including anemia and death (p = . and p = . , respectively). for felv-positive cats (either by serology or pcr), anemia at the time of sampling was correlated with higher pbmc pvl, with a median of . log felv copies/cell ( , , copies/ cells) compared to − . log felv copies/cell ( , copies/ cells) in cats without anemia (p = . , mann-whitney u test). death was also correlated with higher pbmc pvl, with a median of . log felv copies/cell ( , , copies/ cells) among cats who died during follow-up compared to a median of − log felv copies/cell ( , copies/ cells) at sampling time among those who were still alive at the end of follow-up (p = . , mann-whitney u test). we followed cats from entry into the study until months later. among those, three were ffv-monoinfected, and were felv progressives and five were felv regressives, irrespective of their ffv infection status. after months of follow-up, all three ffv monoinfected cats were alive. after months, . % ( / ) of cats with progressive infection had died (figure ), of which three were feral ( %) and nine were captive ( %), were adults ( %) and one was young ( %) whereas the th cat was lost to follow-up at months. in contrast, all regressive cats were alive after months (p = . ). among regressive cats, two were feral ( %) and three were captive ( %), while four were adults ( %) and one was a kitten ( %). there was no difference in age or household status in these cat groups. similar rates of ffv infection were found in both groups; / ( . %) in the first group and / ( . %) in the second. in this study, we evaluated fv infection in cats co-infected with other retroviruses known to impair the feline host immune system with pathologic consequences. retroviral infection of domestic cats was used as a model to investigate possible outcomes associated with mono or dual infections because biological materials such as blood and buccal swabs are usually collected from domestic cats at veterinary clinics for periodic clinical examination and pets can also be easily followed. moreover, domestic cats can be infected with felv, a retrovirus historically considered to be the causative agent of more clinical syndromes than any other single microbial agent in cats, affording an assessment of dual retroviral infections on disease outcomes in cats [ ] . unfortunately, the low fiv prevalence in our study population did not permit an evaluation of dual or triple infection with that retrovirus. in this study we found that both ffv and felv qpcr testing were more sensitive than their respective nested pcr assays for detecting ffv and felv, respectively. even though the felv and ffv nested pcr assays in our study targeted multiple viral regions, none were able to detect % of the qpcr-positive samples. these results likely reflect viral sequence variation at the primer locations and the target viral genomic fragment size for which detection of smaller amplicon sizes, as those in the qpcr tests, is typically more sensitive. we have obtained similar results when comparing nested pcr and qpcr testing of new world primates (nwp) [ ] . in this study, about one fourth of ffv-infected cats were pcr-positive in only the pbmc compartment. these animals may represent recent infections, in which the virus has not yet disseminated to the oral tissues. this hypothesis is supported by a previous report showing a delay in the appearance of sfv in the saliva of cynomolgus macaques (macaca fascicularis) infected through blood transfusion from an sfv-infected animal [ ] . epstein-barr virus (ebv) shares some features with fv, including a high incidence of infection, a salivary mode of transmission and replication in the oropharyngeal tissues. as it is well established for ebv, it is possible that migratory cells, such as macrophages and other leukocytes initially infected by fv, eventually traffic to the oropharyngeal tissues and spread infection to less differentiated epithelial cells [ ] . a recent study with nwp showed an average of − . log sfv copies/cell in pbmc [ ] whereas in old world primates (owp) the pvl average was − . log sfv copies/cell among rhesus macaques [ ] . we found a × higher ffv pvl (− . log copies/cell) in the same tissue in this study, we evaluated fv infection in cats co-infected with other retroviruses known to impair the feline host immune system with pathologic consequences. retroviral infection of domestic cats was used as a model to investigate possible outcomes associated with mono or dual infections because biological materials such as blood and buccal swabs are usually collected from domestic cats at veterinary clinics for periodic clinical examination and pets can also be easily followed. moreover, domestic cats can be infected with felv, a retrovirus historically considered to be the causative agent of more clinical syndromes than any other single microbial agent in cats, affording an assessment of dual retroviral infections on disease outcomes in cats [ ] . unfortunately, the low fiv prevalence in our study population did not permit an evaluation of dual or triple infection with that retrovirus. in this study we found that both ffv and felv qpcr testing were more sensitive than their respective nested pcr assays for detecting ffv and felv, respectively. even though the felv and ffv nested pcr assays in our study targeted multiple viral regions, none were able to detect % of the qpcr-positive samples. these results likely reflect viral sequence variation at the primer locations and the target viral genomic fragment size for which detection of smaller amplicon sizes, as those in the qpcr tests, is typically more sensitive. we have obtained similar results when comparing nested pcr and qpcr testing of new world primates (nwp) [ ] . in this study, about one fourth of ffv-infected cats were pcr-positive in only the pbmc compartment. these animals may represent recent infections, in which the virus has not yet disseminated to the oral tissues. this hypothesis is supported by a previous report showing a delay in the appearance of sfv in the saliva of cynomolgus macaques (macaca fascicularis) infected through blood transfusion from an sfv-infected animal [ ] . epstein-barr virus (ebv) shares some features with fv, including a high incidence of infection, a salivary mode of transmission and replication in the oropharyngeal tissues. as it is well established for ebv, it is possible that migratory cells, such as macrophages and other leukocytes initially infected by fv, eventually traffic to the oropharyngeal tissues and spread infection to less differentiated epithelial cells [ ] . a recent study with nwp showed an average of − . log sfv copies/cell in pbmc [ ] whereas in old world primates (owp) the pvl average was − . log sfv copies/cell among rhesus macaques [ ] . we found a × higher ffv pvl (− . log copies/cell) in the same tissue compartment in cats. at oral sites, the average nwp sfv pvl was − . log copies/cell [ ] while in owps the pvl ranged from − . to − log sfv dna copies/cell [ ] . we found similar ffv pvls in the cat oral cavity (− . log copies/cell). unlike the previous work with primates [ ] , we did not find significant differences between the pvl in pbmc and in oral tissues of cats. these data suggest that fv pvl distribution at distinct anatomical sites can vary in different natural hosts. we also showed that buccal swabs can be useful as non-invasive biological materials for ffv diagnosis, as recently shown for sfv [ ] . non-feral cats were almost five times more likely to be co-infected with ffv/felv than feral cats. we hypothesize that being confined in a household or shelter environment increases the risk of co-infection since these cats may have more intimate contact during grooming or sharing feeding bowls. we also considered possible recruitment bias since clinic cats were suspected to be ill as explained in methods. these cat behaviors are reported as the most likely routes of felv transmission [ ] , as opposed to biting, which has been more strongly associated with sfv transmission [ ] . in our study the ffv prevalence was not different between captive and feral adult cats, while in sfv studies of owps the prevalence is higher in adult captive (~ %) [ ] than in wild-born primates (~ %) [ ] , whereas the sfv prevalence in nwp were found very similar in captive and wild animals ( - %) [ ] . these differences can likely be explained by some captive cats having outdoor access and in shelters it is not uncommon for the introduction of new individuals from the street, both increasing the likelihood of exposures to potentially infected cats. using qpcr, we identified two distinct cat groups based on felv pvl; a regressive group with low pvls (− . to . log copies/cell) and a progressive group with higher pvls ( . to . log copies/cell). our results support previous studies showing that pvls measured by qpcr can be used to identify a regressive infection outcome in which plasma viremia has been cleared [ ] . moreover, the felv pvl we detected is within the range reported by others for experimentally and naturally infected cats [ ] . however, we show for the first time that naturally-infected cats with progressive felv infection can result in death after months of follow-up, although the cause of death cannot be exclusively attributed to felv infection. other studies showed the same outcomes only for experimentally infected cats [ , ] . in one of those studies, progressive and regressive cats were followed for weeks post transfusion and the authors found that % ( / ) of regressive cats remained healthy while % ( / ) of progressive cats developed non-regenerative anemia and multicentric t-cell lymphoma [ ] . to the best of our knowledge, our study is the first to observe the influence of another retrovirus on fv pvl in buccal swabs. a previous co-infection study conducted in the rhesus macaque model has shown that a pre-existing sfv infection can influence the biology and the outcomes of an siv infection, including higher plasma siv vl, a decreasing trend in the cd + t-cell counts and a greater number of animal deaths [ ] . however, sfv pvls were not assessed in that study. herein, with the determination of pvls of two feline retroviruses (ffv and felv), it was possible to observe that felv progressive or regressive infections were not able to reactivate ffv from latency in pbmc. however, a recent study showed that ffv pvl in blood was positive correlated in cats with status of felv progressive, presence of felv-b subtype and positive status for feline coronavirus (fecov), demonstrating a mechanism of complex interaction that need to be clarified [ ] . furthermore, co-infected cats with progressive felv infection are likely more prone to become ffv-transmissible and vice-versa as evidenced by the higher pvls in these oral compartments. as the proportion of potential-transmissible ffv infections was similar between ffv monoinfections and in cats with progressive felv infections, but lower in regressive felv co-infections, we postulate that the latency mechanism in regressive felv could also contribute to reduction of the establishment of ffv colonization of the oral cavity. these findings may also be a consequence of an altered disease susceptibility caused by the cats' virome composition. the virome influences the phenotype of the host in a combinatorial manner by interacting with other components of the microbiome and by interacting with individual variations in host genetics. together these interactions may influence a range of phenotypes as we observed in the distinct behavior of felv progressive and regressive infections as also in the felv regressive correlation with reduced ffv saliva transmission [ ] . the microbiome composition may also allow the stimulation of specific immune cell subsets, which may carry the ffv proviral dna, thus indirectly increasing the ffv pvl due to the increased number of cells. we also found that ffv pvl was higher in buccal tissues of ffv/felv co-infected cats than among ffv monoinfections. however, we identified a negative correlation between the pvls of both viruses circulating in this tissue. a previous study has reported felv as a cofactor for fiv infection by enhancing the expression and spread of fiv in the host and by increasing the severity of immunodeficiency caused by fiv by an unknown mechanism [ ] . another study evaluated fiv/felv interactions in in vitro and in vivo experiments, but did not find evidence of direct viral interactions [ ] . since felv is mainly transmitted through saliva [ ] and superficial differentiated epithelial cells of the oral mucosa are the major cell types in which ffv replicates [ ] , we hypothesize that both viruses can infect the same compartment. just like the tax protein of htlv- can activate hiv expression in vitro via the ltr [ ] , felv may enhance ffv replication in the oral cavity by a similar mechanism, or related to regulatory protein synergism, or some other means. a simplified model of this hypothesis is presented in figure . any potential interaction mechanism between these two viruses, and their infectiousness, remains to be determined and any contributions of infectious dna-containing viral particles compared to integrated proviral dna sequences was not measured in our study due to the small volumes of materials obtained for analysis. however, we identified a negative correlation between the pvls of both viruses circulating in this tissue. a previous study has reported felv as a cofactor for fiv infection by enhancing the expression and spread of fiv in the host and by increasing the severity of immunodeficiency caused by fiv by an unknown mechanism [ ] . another study evaluated fiv/felv interactions in in vitro and in vivo experiments, but did not find evidence of direct viral interactions [ ] . since felv is mainly transmitted through saliva [ ] and superficial differentiated epithelial cells of the oral mucosa are the major cell types in which ffv replicates [ ] , we hypothesize that both viruses can infect the same compartment. just like the tax protein of htlv- can activate hiv expression in vitro via the ltr [ ] , felv may enhance ffv replication in the oral cavity by a similar mechanism, or related to regulatory protein synergism, or some other means. a simplified model of this hypothesis is presented in figure . any potential interaction mechanism between these two viruses, and their infectiousness, remains to be determined and any contributions of infectious dna-containing viral particles compared to integrated proviral dna sequences was not measured in our study due to the small volumes of materials obtained for analysis. although we found evidence for increased buccal ffv pvls in cats co-infected with felv with the potential for increased transmissibility, we did not observe significant disease associations in these cats. a small number of dually infected cats with felv-progressive infection had anemia but this trend was not highly significant. we also did not find large numbers of fiv-infected cats to evaluate the effect of fv co-infection on disease outcomes. additional studies with larger numbers of naturally or experimentally infected cats and longer periods of follow-up may be needed to determine these potential associations. our study also reinforces the importance of felv qpcr testing to define felv infection outcomes, enabling differential clinical treatment of cats that were negative by serological methods. for example, blood transfusion from regressive cats containing latent feline leukemia provirus caused infection and disease in naïve recipient cats [ ] . moreover, felv regressive cats with undetectable antigenemia, but that are immune to vaccination, are often equivocally vaccinated [ ] . figure . hypothetical pathway to progressive or regressive outcomes via dual infection with feline foamy virus (ffv) and feline leukaemia virus (felv) and potentially active viral replication measured by proviral load (pvl). felv progressive and regressive outcome determined by pbmc tissue (blood) pvls were correlated to different ffv and felv pvl patterns of detection in the oral cavity. figure . hypothetical pathway to progressive or regressive outcomes via dual infection with feline foamy virus (ffv) and feline leukaemia virus (felv) and potentially active viral replication measured by proviral load (pvl). felv progressive and regressive outcome determined by pbmc tissue (blood) pvls were correlated to different ffv and felv pvl patterns of detection in the oral cavity. although we found evidence for increased buccal ffv pvls in cats co-infected with felv with the potential for increased transmissibility, we did not observe significant disease associations in these cats. a small number of dually infected cats with felv-progressive infection had anemia but this trend was not highly significant. we also did not find large numbers of fiv-infected cats to evaluate the effect of fv co-infection on disease outcomes. additional studies with larger numbers of naturally or experimentally infected cats and longer periods of follow-up may be needed to determine these potential associations. our study also reinforces the importance of felv qpcr testing to define felv infection outcomes, enabling differential clinical treatment of cats that were negative by serological methods. for example, blood transfusion from regressive cats containing latent feline leukemia provirus caused infection and disease in naïve recipient cats [ ] . moreover, felv regressive cats with undetectable antigenemia, but that are immune to vaccination, are often equivocally vaccinated [ ] . in summary, we provide evidence that fvs may interact with other retroviruses infecting cats. our study supports that domestic cats naturally infected by ffv and felv can be used as a model to investigate the potential impact of fv on immunocompromised mammalian hosts. the results of these animal model studies may help inform the design of clinical and research investigations into the potential for sfv to cause disease in infected humans. the tools developed in our study will be useful to further explore consequences and interactions of multiple feline retrovirus infections of cats. virus taxonomy identification of a human population infected with simian foamy viruses frequent simian foamy virus 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outcomes in a domestic cat breeding colony: relationship to endogenous felv and other chronic viral infections evaluation of in vivo and in vitro interactions of feline immunodeficiency virus and feline leukemia virus activation of the hiv- ltr by t cell mitogens and the trans-activator protein of htlv-i this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we thank the veterinary clinicians who collected cat specimens and associated clinical data over the study period. this work is part of the requirements for the phd thesis of l.t.f.c. at the department of genetics, universidade federal do rio de janeiro, brazil. use of trade names is for identification only and does not imply endorsement by the u.s. department of health and human services, the public health service, or the cdc. the findings and conclusions in this report are those of the authors and do not necessarily represent the views of the cdc, or any of the authors' affiliated institutions. the authors declare no conflict of interest. key: cord- - vjqrnwh authors: hraber, peter; o’maille, paul e.; silberfarb, andrew; davis-anderson, katie; generous, nicholas; mcmahon, benjamin h.; fair, jeanne m. title: resources to discover and use short linear motifs in viral proteins date: - - journal: trends biotechnol doi: . /j.tibtech. . . sha: doc_id: cord_uid: vjqrnwh viral proteins evade host immune function by molecular mimicry, often achieved by short linear motifs (slims) of three to ten consecutive amino acids (aas). motif mimicry tolerates mutations, evolves quickly to modify interactions with the host, and enables modular interactions with protein complexes. host cells cannot easily coordinate changes to conserved motif recognition and binding interfaces under selective pressure to maintain critical signaling pathways. slims offer potential for use in synthetic biology, such as better immunogens and therapies, but may also present biosecurity challenges. we survey viral uses of slims to mimic host proteins, and information resources available for motif discovery. as the number of examples continues to grow, knowledge management tools are essential to help organize and compare new findings. viruses exploit host cellular processes to replicate, and have developed myriad ways to subvert host immune defenses. molecular mimicry (see glossary) is a common and effective strategy, enabling a pathogen to usurp host protein function by resemblance [ , ] . molecular mimicry varies over a continuum, from one extreme that includes sequence and structural similarity (i.e., orthologs) of entire proteins, to another extreme of chemical similarity at only a few localized sites, as is the case for short linear motifs (slims). the growing body of literature on slims indicates that some important virushost interactions can be attributed to a few well-chosen aas [ ] [ ] [ ] [ ] [ ] . rather than devote entire proteins to one function, slims enable multifunctional viral proteins. interactions between globular virus and host proteins have picomolar affinities, while slims have micromolar binding affinities with globular host proteins [ ] . moderate binding affinity of slims facilitates disruption of signaling interactions, rather than competing for stable formation of persistent protein complexes. synthetic biology practitioners can benefit from an introduction to how slims enable viral interference with host cell functions and computational resources available for slim analysis. viral slims are potentially useful in synthetic biology, to provide a toolkit for new functions, for example, to modulate immune responses or to complement and interact with newly developed adjuvants in a synergistic manner [ ] . research efforts to develop broad-spectrum antiviral compounds or design broadly cross-protective vaccine immunogens benefit directly from knowledge of gene products, protein functions, and motifs involved with viral immune interference. n-linked glycosylation of the png sequon is a well-known example used by viral glycoproteins as camouflage against immune recognition [ , ] . the distribution of n-linked glycosylation sites has recently been recognized as essential for the design of immunogens to induce broadly cross-reactive immune protection against such challenging viruses as hiv- [ , ] . motifs associated with cellular trafficking (localization, transport, secretion, and sequestration) are readily edited to modify where expression products go, and change interaction profiles with other proteins [ ] . in addition to motifs that stabilize the structure of immunogens, such as trimerization ('foldon') [ ] [ ] [ ] [ ] and dimerization [ , ] domains, motifs that interact with cellular processes for innate antiviral pathways could be used to enhance immunogenicity. while slims in eukaryotic proteins have been discussed extensively, slim involvement in viral immunomodulation remains less thoroughly explored, and suggests new opportunities for use in engineered biotechnology applications. the ability to transfer genetic components across species, or to introduce such components de novo, enables new functions. while such functions are generally well intended, some risk also exists for harmful effects. subject to the technical advances of synthetic biology, such effects are not short linear motifs (slims) are patterns of three to ten consecutive aas used by eukaryotic cells for tasks that include: signaling, localization, degradation, and proteolytic cleavage. viruses use slims to their advantage, including interference with antiviral innate immune pathways. viral slims can tolerate mutations, evolve quickly to modify host interactions, and co-occur in a modular manner or involve multiprotein complexes. slims are useful in synthetic biology, where minor edits can alter target specificity, modulate persistence, reprogram interactions with cell-signaling domains, and alter protein function in myriad other ways. aside from possible beneficial uses, for example, to produce better immunogens and develop therapeutic interventions against infectious disease, slims may help characterize new and emerging threats to global health. necessarily a taxonomically relevant property. it may be necessary to evaluate risks of new functions by other means than taxonomy or even protein functional evaluations. instead, new methods are needed that assess functions at a finer resolution than the gene, whether by computational analysis or functional phenotypic assessments [ ] [ ] [ ] . slim analysis might help with such assessments. viral proteins can modulate immunity in several ways, which include: shutdown of host macromolecular synthesis, inhibiting antigen production or apoptosis, and interference with such processes as antigen presentation by mhc, natural killer (nk) cell function, antiviral cytokines, or interferon responses. each of these processes involves coordination among multiple components in host cells. viral interference with these functions is frequently attributed to entire proteins, but in some important cases has been localized to slims. because of their compact size, slims are modular, rapidly evolvable sequence elements. different instances of a given slim can vary in sequence while maintaining the overall functional profile, that is, the regular expression for the sequence motif, where a few positions are invariant while other positions tolerate numerous substitutions. thus, partial sequence matches are sufficient for transient binding interactions with target domains, for example, signal transduction proteins. this observation led to the proposal of ex nihilo slim evolution -the evolution of a novel slim 'from nothing' -the appearance of a new functional module from a previously nonfunctional region of protein sequence [ ] . because hosts' interaction networks are often conserved, slims represent a significant vulnerability for opportunistic exploitation. these properties enable pathogens to acquire host-like slims rapidly through ex nihilo convergent evolution, to rewire host interaction networks, and to acquire tropism and virulence traits needed for successful adaptation and propagation [ ] . over motifs are known, with , validated instances, and many more motifs may await discovery [ , ] . focus on viral motifs may reveal practical utility, to broaden the repertoire of tools available to reprogram molecular function in synthetic biology. one example of how viral proteins use slims to subvert host cell function is illustrated by epstein-barr virus (ebv), which persists in resting memory b cells of nearly all (> %) individuals throughout their adult lives [ ] . latent membrane protein (lmp ) is central to ebv persistence. the cytoplasmic tail of this membrane-bound protein includes pxxpxp and pxqxt motifs that recruit signaling proteins (jak , that is, janus kinase , and several trafs, tumor necrosis factor receptor-associated factors, respectively) [ ] . together, the motifs mimic the cytoplasmic domain of cd to activate nuclear factor-kb via intermediates, including a third motif yyd$ (where $ denotes the c terminus), the tradd (tumor necrosis factor receptor-associated death domain) binding domain. the overall result is that lmp inhibits apoptosis and infected b cell proliferation, to confer viral persistence [ ] . other examples of viral slim contributions to motif mimicry involved with immune function include: protein degradation, transcription, translation, and transport into and out of the nucleus [ ] . given the continued growth of this field [ ] , established frameworks can manage and exploit this knowledge beyond catalogues of currently known motifs [ , , ] or details on contributions of one viral protein (e.g., [ , ] ). work to use slims in bioengineering can benefit from understanding viral protein function. this information is organized in viral knowledge bases, such as viralzone (box ). ontologies describe systematically the many different functional roles of viral proteins, including immune evasion. by promoting use of standard terms for relationships between concepts, an ontology arranges concepts into a framework that can be updated as knowledge grows. protein function is captured broadly in such a framework, though the nuanced details of interactions with other molecules are not localized to domains or motifs. go is an authoritative resource for annotating functions of gene sequences [ , ] . an example of interest is 'evasion or tolerance by virus of host immune response' [ ] (www.ebi.ac.uk/quickgo/ glossary adjuvant: an additive to a vaccine that promotes nonspecific immune responses. when administered together with an antigen, it induces more potent responses than the antigen alone. autophagy: an evolutionarily conserved degradation system for maintaining cellular homeostasis and innate immunity to clear pathogens from cells. domain: structure-based modular subunit of a protein, often with a specific function. domains are generally larger and associated with structural subunits, while motifs are shorter and associated with intrinsically disordered protein regions. elm: the eukaryotic linear motif resource (elm.eu.org), a repository of known slims; includes annotation from primary literature and information about experimental assays used. glycosylation: post-translational modification by host transferases linking sugar molecules to side chains of either nitrogen (nlinked) in asparagine or oxygen (o-linked) in serine or threonine. viral proteins like hiv- envelope can be heavily glycosylated, providing camouflage against immune recognition, shifting as the png sequon mutates. go: gene ontology, an ontology for genetic products of any and all organisms, providing a framework to annotate gene and protein functions in genetic sequence databases and bioinformatics analysis procedures. go includes multiple dimensions to capture biological complexity in adequate depth: molecular function, cellular component, and biological process. go development is conducted by a consortium of research communities and databases, which regularly solicits input and feedback from the broader research community. immune modulation: interference with an immune-related process by a pathogen. intrinsically disordered protein/ domain: regions of a protein sequence that are predicted or experimentally shown not to form consistent structure, e.g., a helices or b sheets. such regions tend to be more accessible for interactions with other proteins. term/go: , figure ). concepts are hierarchically organized, and include a definition, synonyms, and lists of parents and children. functional annotation in go reflects the diverse effects of viral proteins on immune interference. modulating autophagy is an example of recent advances in this research area [ , ] . a growing number of reports describe how virus proteins and slims therein modulate autophagy to promote various aspects of their life cycle [ , ] . both go and viralzone have developed concepts to detail molecular mimicry: structural similarity that enables repurposing or hijacking of molecular function by pathogens, such as viruses. molecular mimicry varies in extent from entire globular proteins, to localized domains, down to short linear motifs. motif class: a regular expression that summarizes known variants to define a sequence motif. motif instance: a particular motif as found in a protein or translated genetic sequence from a specific organism, strain, or isolate. ontology: a representation of domain-specific knowledge organized as concepts, their properties, and their relationships. png sequon: three aa motif n [^p] [st] , that is, asparagine, then any aa except proline, then serine or threonine, recognized by glycosyltransferase as a potential nlinked glycosylation site. regular expression: a string of characters that concisely represents many alternative sequence variants; may include wildcards to represent any character, groupings of possible characters, repetition, negation, start and end of sequence, etc. short linear motif (slim): also known as minimotifs or morfs (molecular recognition features). frequently represented as regular expressions, typically three to te-naas long. viralzone: a knowledge base (https://viralzone.expasy.org) that documents viral families, genome architectures, proteins, hostprotein interactions, and an ontology for the functions of viral proteins. a bridge that links literature reports to go term annotation, viralzone is an online knowledge base that contains 'textbook' information about viral taxonomy, replication, genome organization, and virion structure, and provides links to viral sequence data [ , ] . importantly, viralzone staff collaborate with the go consortium to define entries for virus-specific molecular functions [ ] [ ] [ ] [ ] . viralzone cross-references its keywords with go consortium terms and uniprot [ ] identifiers. this makes it possible to search for viral proteins by their functional role. viralzone staff have developed go concepts specifically for viruses, to represent the diversity of viral replication and processes involved with viral entry, replication, and egress [ ] . viralzone staff have also developed a detailed listing of virus-host interactions, with entries for functions and go terms [ , ] . each entry ('keyword') has a unique identifier. unlike enzyme commission (ec) numbers [ ] , viralzone ids are arbitrary numbers and do not indicate position in the concept hierarchy. instead, organization of the keyword hierarchy is provided online. the web address https://viralzone.expasy.org/ is an entry point into the viralzone concept space ( figure i) . blue text indicates a link to more detail. shown is the vertebrate host-virus interactions page [ ] . also available are summaries for invertebrate, plant, and bacterial host-virus interactions. adapted from www.ebi.ac.uk/quickgo/term/go: [ ] . most arrows indicate 'is a' relations, where the hierarchy is refined by specialization. blue arrows indicate 'part of' relations, which relate to the symbiont process as parts to a whole. autophagy processes, including positive and negative regulation of xenophagy, the selective autophagy of pathogens [ ] [ ] [ ] . understanding the functional roles of slims can help identify related mechanisms or processes, or possibly identify knowledge gaps where slims may be posited but not yet identified. an overview of slim functions may also help to prioritize which are of greatest potential for use or abuse when artificially added to modify protein function. databases and discovery tools are also useful to identify known and new slims. identification of shared structural features across divergent protein families led to analysis and identification of modular protein domains. protein domains are used to categorize protein function, and the interpro database [ ] (www.ebi.ac.uk/interpro) aggregates information at this within-protein level. the identification of protein domains led to recognition of slims as compact, small-scale functional modules [ ] . elm is a database of eukaryotic motifs (box ), though its representation of viral-host interactions is not fully developed. at present, elm entries map to go terms. most motifs map to multiple go terms; the median is seven go terms per motif and the maximum is (mod_plk_ ). immuneassociated function of the lig_irf _lxis_ motif is involved in signal transduction responses to pathogen-associated molecular patterns; this motif maps to go terms, but none occur in the viralzone vocabulary. in total, only three elm entries utilize go terms from viralzone: lig_bh_bh _ , lig_hcf- _hbm_ , and lig_rb_pabgroove_ . these three motifs all map to the most general (ii) deg, degradation sites, part of polyubiquitination; (iii) doc, docking sites, involved in protein recruitment but not directly targeted by an active site; (iv) lig, ligand binding sites, primarily for protein-protein interactions; (v) mod, post-translational modification sites; and (vi) trg, targeting sites for subcellular localization. elm has also spun-off several specialized databases: phospho.elm for phosphorylation sites with experimental evidence [ ] , switches.elm for conditional molecular switches, such as requiring that a site be modified [ ] , and ielm, with an emphasis on protein-protein interactions [ ] . a detailed tutorial provides orientation for elm use [ ] . elm documents each motif class with a concise description of its function. for example, one type of nuclear localization signal (nls), trg_nls_bipartite_ the 'classic bipartite nls', which binds to importin-a for nuclear pore transfer and is utilized by the pb protein of influenza a, is documented here: elm.eu.org/elms/ trg_nls_bipartite_ . the abstract and functional site descriptions summarize what is known about the motif. go term, go: ('modulation by virus of host morphology or physiology', a synonym of 'virushost interaction'). this underscores the prevalent mode of elm motif discovery and annotation does not emphasize host-virus interactions, but rather systems-level interactions within eukaryotic cells. thus, better integration of viralzone-go-term vocabulary with elm or another domain-level representation of viral slims is needed to promote potential utility for biotechnology. despite not using the viralzone ontology, elm documents other motif classes with go terms that refer to 'viral', 'virus', 'immune', or 'immunity' (table ) . because the focus is motif function in the host cell context, elm does not directly indicate how viral immune interference results. further, the relative lack of viral motifs in elm does not indicate their absence in vivo, but rather the evidence-based requirement for elm inclusion. related to the earlier observation, a review of how viruses use slims to interfere with host cells [ ] lists examples that represent viral mimicry of host slims (table ). only % of these have corresponding elm entries, though the slims are known. the remaining % indicate that elm does not fully capture all known viral motifs. this strong requirement by elm for evidence-based motif classes and instances is not strictly a drawback. indeed, the elm creators are very aware that computational analysis alone is error prone and can yield misleading outcomes. in [ ] , they discuss this issue in depth, and recommend a workflow for slim discovery that culminates in experimental validation, whether in vivo or in vitro. working with viral-host systems adds layers of difficulty to experimental motif validation, so it should not be surprising or to the detriment of available information resources that viral slims are less thoroughly documented. searching arbitrary sequences for motif instances is computationally straightforward. box provides an example of motif searching with elm, which might facilitate comparative analysis of two related proteins from different species of human herpesvirus (hsv). resources such as interpro and uniprot are able to perform similar assessments, but give broader, domain-level representations with less functional detail than the slim searches enabled by elm. reports in the primary literature take a different approach, by marking slims in a protein alignment, which includes orthologues to mark conservation (e.g., figure in [ ] ). the elm-generated report combines predicted slims with information from annotated domains and local disorder predictions, for a perspective that complements the other approaches. clearly, false positives are inevitable among slim search results. this makes it necessary to filter for the most significant and informative outcomes. this leads to consideration of in silico (computational) methods for slim evaluation. a highly recommended, authoritative review of slim discovery techniques, from an author of the slimsuite software package, discusses motif identification techniques in depth [ ] . methods for slim discovery can be divided into two broad classes: (i) de novo discovery of new slims, and (ii) instance prediction to find new occurrences of known slims. there are currently at least eight software packages available to discover new slims and packages ( stand-alone programs or servers and two software suites that consist of multiple tools: slimsuite [ ] , which includes ten utilities, and meme [ ] , which consists of five tools for slim instance detection). though meme was developed for discovery of dna sequence motifs, it generates ungapped, profile-based motifs using the expectation-maximization (em) algorithm. no single method is inherently better than the rest, but the choice of which to use depends on several factors, such as the input sequence data, whether one sequence, an alignment, or a collection of nonhomologous sequences [ ] . to illustrate the diversity of motif discovery methods, this section mentions only a handful of the software tools available (table ) . readers seeking to learn more about the full set of alternatives are strongly encouraged to consult [ ] , particularly tables , , and therein. another helpful resource ( table in [ ] ) lists online motif discovery bioinformatics services. several alternative approaches for discovery of new motifs have been advanced. edwards and palopoli [ ] review the alternatives in depth, discussing their merits and drawbacks. briefly, they can be divided into alignment-based and alignment-free methods. an alignment-based approach looks for conserved sites among homologous sequences, but can be misled by high sequence conservation in globular domains. a program called slimprints works around this with a specialized approach to model substitutions [ ] . slimprints uses a statistical model of relative local conservation, which looks for clusters of overly constrained sites in a window of about aas, using iupred scores (intrinsically unordered prediction; see later) to weigh sites in intrinsically disordered protein regions more heavily than sites in globular (ordered) regimes [ ] . in contrast, alignment-free methods look for enrichment of amino acid patterns in proteins that are expected by other means to perform similar motif-related roles, for example, by go category annotations or protein-protein interaction (ppi) data, that is, via databases that capture experimental evidence for protein colocalization and functional interactions. an important caveat is that to assume such sequences are independent could yield spurious enrichment of shared patterns, so alignment-free methods need to compensate for evolutionary constraints at the domain level, rather than for full-length homologous proteins. the development of such corrections and their relative advantages are detailed in [ ] . some programs (e.g., slimdisc [ ] , slimfinder [ , ] , and dilimot [ , ] ) produce regular expressions that compensate for phylogenetic relatedness, while others (meme suite, glam [ ] , and nestedmica [ , ] ) produce probabilistic profiles. for more discussion of these and issues of concern for computational motif discovery, see [ ] . filtering methods control high false positive rates from slim instance detection. structural information, whether known or predicted, can be used for filtering. box illustrates how elm filters results to hsv- and hsv- virulence factor icp . assists in viral immune evasion by molecular mimicry. hsv- neurovirulence protein icp . , encoded by the g . gene, initiates immune interference by binding and sequestering cellular proteins that would stimulate autophagy, translational arrest, and type i interferon responses. hsv- icp . binds tank-binding kinase (tbk ) to prevent type i interferon induction [ ] , beclin- to prevent autophagy [ ] , and both pp a and eif a to overcome translational arrest [ ] . hsv- g . contains an intron not present in hsv- , and up to four isoforms of hsv- icp . are known [ ] . full-length hsv- icp . has conserved pp a and eif a-binding domains, but lacks tbk and beclin- binding domains [ ] . additional hsv- motifs influence intracellular localization [ ] , virion maturation, and egress [ ] , not yet characterized in hsv- . hsv- is recognized as more virulent than hsv- , but both can cause neuropathology, including viral encephalitis and meningitis [ ] . to attenuate virulence, icp . is routinely deleted or inactivated when making hsv- constructs for oncolytic therapy [ ] . both are the same length and share domain structures, and partially share slim compositions ( figure i ). identifying differences in slims from each could provide clues for more detailed experimental investigations to understand icp . virulence determinants and host protein targets. exclude a region predicted to fold as globular protein. these predictions were made by smart (simple modular architecture research tool) [ ] and pfam [ ] domain matches, corroborated by globplot [ ] . another widely used approach is to identify regions of local disorder, where protein structure is not clearly defined, making that region accessible to interact with other proteins. iupred [ ] is commonly used for this task, though the choice of parameter settings and how to interpret results varies. elm results include an iupred disorder score and a simple cutoff of . to define the disorder transition. above this value, local protein structure is considered accessible for interaction with other proteins. scoring schemes filter for statistical enrichment of motif instances. an approach of filtering by homology [ ] seems inappropriate for use to detect virus interactions with host proteins, as it may exclude nonhomologous regions with motifs that do interact, yielding false negatives. regardless, failure to consider evolutionary relatedness among sequences being searched could introduce bias due to common ancestry, rather than independence, among sequences. a simple approach to instance prediction is a stand-alone program called shettimotif [ ] . it was used to scan protein sequences from poxviridae genomes (an average of proteins per poxvirus) for low-complexity regions and regular expressions defined by prosite. the approach compared numbers of proteins per genome that carry each motif, and doubtlessly includes many motif instances that are not functional as slims. also, shorter motifs occur more frequently than longer motifs [ , ] , partly due to chance alone. regardless, systematic error may be considered a source of background noise across the large number of proteins in viral proteomes, each having different host specificities, to enable somewhat meaningful comparisons, in such a 'statistical genomics' approach [ ] . the comparisons could be more meaningful if false positive motif instances were reduced. becerra et al. developed another approach to instance counting [ ] , which involves comparison with a null distribution from permuting primary sequence and testing for presence of the motif in the permuted sequences. a motif is considered rare and therefore significantly unlikely to occur by chance if it is present at or below some cutoff frequency. restricting the sequence region that is used for permutation testing, such as by use of structural considerations, can further focus the search. indeed, such a hybrid filtering approach was described recently and evaluated on the hiv- proteome [ ] . following methods described in an earlier study [ ] , becerra et al. used iupred with a modified, window-based scoring procedure to identify intrinsically disordered protein regions, and tested for statistical rarity below % of shuffled variants. the approach further considered conservation above % in a set of aligned sequences, though combining three filtering criteria was too stringent and excluded all motif candidates [ ] . while algorithmic approaches seek to identify a broad range of slim types, more specialized resources have emerged to track the distribution of a particular slim in viral proteins. for example, ilir@viral is a web resource dedicated to detecting lir motif-containing proteins in viruses [ ] . lc interacting regions (lir motifs) are slims that mediate protein-protein interactions involved in autophagy, as used by influenza a virus m protein to subvert autophagy and maintain virion stability [ ] . using curated text mining analysis and position-specific scoring matrices, ilir@viral analyzed reviewed viral sequences available from uniprot across individual viral species and found that viral sequences contain lir motifs. while many predicted instances may represent false positives, the enrichment of lir motifs in viral sequences is consistent with viral adaptation to host xenophagy [ ] . curiously, elm currently lists the lir motif as a candidate, rather than an accepted motif class. embedding slims into engineered constructs may enable specific effects on cellular immune processes, for applications that include targeted drug delivery, pathogen-specific adjuvants, potent and broadly effective immunogens, transformational medical countermeasures, and improved design of vectors for gene therapy. slim modularity may enable easy ways to reprogram protein function with a few localized modifications. to realize the potential utility of slims in synthetic biology, more research is needed to expand and integrate our collection of knowledge on viral slims (see outstanding questions). detecting slims in variant sequences may help to identify functional innovation or changes in virulence, in a manner that does not rely strictly on functional assessment at the whole-gene level, to identify how sequence-specific variation may interact with host responses. this may be particularly useful and important to understand new variants and assess the risk that they may spread and cause harmful effects on human health or agricultural interests. such knowledge is needed in an era where synthetic biology may introduce new risks for biological error and biological terror. detecting and understanding slim variants can help to reduce such risks and identify newly emerging threats to global health and security because watch lists for harmful organisms to ensure public safety by preventing access to select known risks may be inadequate [ ] [ ] [ ] . slims in viral proteins can interact in many different ways with host proteins to modulate immune responses. a motif may be necessary but not sufficient for any inferred function. the simplest case is where a viral slim interacts directly with a host protein to yield an immunomodulated phenotype. more elaborate cases are known, such as the multifunctional proteins e a (ebv), nef (hiv- ), and icp . (hsv). computational prediction of slim classes and new instances is a process, which involves experimental confirmation and validation. high-throughput methods for experimental assessment of protein interactions are useful to validate computational predictions [ , ] , and more assays are needed to evaluate functional and phenotypic effects of adding or deleting slims. resolved map of human-virus protein-protein interaction networks. plos pathog. , e what specific constraints limit slim evolvability? what strategies are most effective to advance knowledge of viral immunomodulatory slims in the design of vaccines and therapies to promote global health? for example, can some viral peptides be useful as adjuvants? signatures of pleiotropy, economy and convergent evolution in a domain pathogen mimicry of host protein-protein interfaces modulates immunity use of host-like peptide motifs in viral proteins is a prevalent strategy in host-virus interactions slimsearch . : biological context for short linear motifs in proteins how viruses hijack cell regulation attributes of short linear motifs how pathogens use linear motifs to perturb host cell networks short linear motifs: ubiquitous and functionally diverse protein interaction modules directing cell regulation applications of immunomodulatory immune synergies to adjuvant discovery and vaccine development targeting hostderived glycans on enveloped viruses for antibodybased vaccine design structure and immune recognition of the hiv glycan shield completeness of hiv- envelope glycan shield at transmission determines neutralization breadth protein and glycan mimicry in hiv vaccine design hiv- nef: a master manipulator of the membrane trafficking machinery mediating immune evasion immunogenicity and protection efficacy of monomeric and trimeric recombinant sars coronavirus spike protein subunit vaccine candidates a fusion intermediate gp immunogen elicits neutralizing antibodies to hiv- immunosilencing a highly immunogenic protein trimerization domain vaccination with soluble headless hemagglutinin protects mice from challenge with divergent influenza viruses human chemokine mip a increases efficiency of targeted dna fusion vaccines dengue e protein domain iii-based dna immunisation induces strong antibody responses to all four viral serotypes options for synthetic dna order screening, revisited a transatlantic perspective on emerging issues in biological engineering biodefense in the age of synthetic biology short linear motifs -ex nihilo evolution of protein regulation convergent evolution and mimicry of protein linear motifs in host-pathogen interactions the present and the future of motif-mediated protein-protein interactions a million peptide motifs for the molecular biologist a review of functional motifs utilized by viruses hacking the cell: network intrusion and exploitation by adenovirus e a the goa database: gene ontology annotation updates for quickgo: a web-based tool for gene ontology searching exploring autophagy with gene ontology autophagy in negative-strand rna virus infection autophagy during viral infection -a double-edged sword interpro in : improving coverage, classification and access to protein sequence annotations elm server: a new resource for investigating short functional sites in modular eukaryotic proteins experimental detection of short regulatory motifs in eukaryotic proteins: tips for good practice as well as for bad an eif a-binding motif in protein phosphatase subunit gadd and its viral orthologs is required to promote dephosphorylation of eif a computational prediction of short linear motifs from protein sequences meme suite: tools for motif discovery and searching slimprints: conservationbased discovery of functional motif fingerprints in intrinsically disordered protein regions slimdisc: short, linear motif discovery, correcting for common evolutionary descent slimfinder: a probabilistic method for identifying over-represented, convergently evolved, short linear motifs in proteins slimfinder: a web server to find novel, significantly over-represented, short protein motifs systematic discovery of new recognition peptides mediating protein interaction networks dilimot: discovery of linear motifs in proteins discovering sequence motifs with arbitrary insertions and deletions nestedmica: sensitive inference of over-represented motifs in nucleic acid sequence nestedmica as an ab initio protein motif discovery tool smart: recent updates, new developments and status in interpro in -beyond protein family and domain annotations globplot: exploring protein sequences for globularity and disorder iupred: web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content a computational strategy for the prediction of functional linear peptide motifs in proteins a bioinformatics pipeline to search functional motifs within whole-proteome data: a case study of poxviruses prediction of virus-host protein-protein interactions mediated by short linear motifs intrinsic disorder in ubiquitination substrates ilir@viral: a web resource for lir motif-containing proteins in viruses a lc -interacting motif in the influenza a virus m protein is required to subvert autophagy and maintain virion stability high-throughput methods for identification of protein-protein interactions involving short linear motifs viralzone: a knowledge resource to understand virus diversity viralzone: recent updates to the virus knowledge resource an integrated ontology resource to explore and study host-virus relationships representing virus-host interactions and other multi-organism processes in the gene ontology the ins and outs of eukaryotic viruses: knowledge base and ontology of a viral infection uniprot: the universal protein knowledgebase fifty-five years of enzyme classification: advances and difficulties the eukaryotic linear motif resource elm: years and counting elm -data update and new functionality of the eukaryotic linear motif resource elm: a database of phosphorylation sites -update the switches.elm resource: a compendium of conditional regulatory interaction interfaces ielm -a web server to explore short linear motif-mediated interactions exploring short linear motifs using the elm database and tools control of tank-binding kinase -mediated signaling by the g . protein of herpes simplex virus hsv- icp . confers neurovirulence by targeting the beclin autophagy protein a conserved domain of herpes simplex virus icp . regulates protein phosphatase complex in mammalian cells up to four distinct polypeptides are produced from the g . open reading frame of herpes simplex virus herpes simplex virus icp . confers neurovirulence by regulating the type i interferon response an n-terminal arginine-rich cluster and a proline-alanine-threonine repeat region determine the cellular localization of the herpes simplex virus type icp . protein and its ligand, protein phosphatase replication of herpes simplex virus depends on the g . functions that facilitate virus response to interferon and egress in the different stages of productive infection the herpes simplex virus neurovirulence factor g . : revealing virus-host interactions elm: the status of the eukaryotic linear motif resource key: cord- -kwzo puo authors: si, lulu; meng, yu; tian, fang; li, weihua; zou, peng; wang, qian; xu, wei; wang, yuzhu; xia, minjie; hu, jingying; jiang, shibo; lu, lu title: a peptide-based virus inactivator protects male mice against zika virus-induced damage of testicular tissue date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: kwzo puo zika virus (zikv) was a re-emerging arbovirus associated with guillain–barré syndrome in adult and congenital zika syndrome in fetus and infant. although zikv was mainly transmitted by mosquito bites, many sexual transmission cases have been reported since the outbreak in . zikv can persist in testis and semen for a long time, causing testicular tissue damage and reducing sperm quality. however, no drug has been approved for prevention or treatment of zikv infection, especially infection in male testicular tissue. previously reported peptide z could inactivate zikv, inhibiting zikv infection in vitro and in vivo. importantly, z could inhibit vertical transmission of zikv in pregnant mice, reducing zikv infection in fetus. here we showed that intraperitoneally administered z could also be distributed to testis and epididymis, resulting in the reduction of zikv rna copies in testicular tissue and protection of testis and epididymis against zikv-induced pathological damage and poor sperm quality in type i interferon receptor-deficient a mice. thus, z , a zikv inactivator, could serve as an antiviral agent for treatment of zikv infection and attenuation of zikv-induced testicular tissue damage. zika virus was a re-emerging arbovirus (gao, ; pettersson and bohlin, ) , like dengue virus (denv) and japanese encephalitis virus (jev), belonging to flavivirus genus in the flaviviridae family. since the outbreak in brazil in , zikv has rapidly spread to countries and territories (baud et al., a; gorshkov et al., ) , attracting global attention. about % of those infected with zikv presented with asymptomatic, or only mild illness (petersen e. et al., ; pierson and diamond, ) . however, zikv infection has been associated with more severe complications: guillain-barré syndrome in adult (brasil et al., ; peixoto et al., ) and congenital zika syndrome in fetus and infant (baud et al., b; gurung et al., ) . zikv is mainly transmitted through mosquito bites (petersen l.r. et al., ) , while it can also be transmitted via in utero from mother to fetus (besnard et al., ) , blood transfusion (tai et al., ) and intercourse (duggal et al., ; mead et al., ; sakkas et al., ) . it is reported that zikv was transmitted through sexual contact, perhaps up to days after the onset of symptoms (turmel et al., ) , and infective virions were still isolated from semen days after infection (arsuaga et al., ; garcia-bujalance et al., ) . the viral load in semen was , times that in the blood at weeks postinfection (mansuy et al., ) , and viral rna was still detected up to days after illness onset (barzon et al., ) . it is also reported that zikv infection caused patients to have a decreasing total sperm count in the acute phase of infection (joguet et al., ) and abnormal spermogram results year after infection (avelino-silva et al., ) , suggesting zikv was harmful to human spermatozoa production. testis explants from uninfected donors were also proven to be susceptible to zikv infection (matusali et al., ) . as determined from an in vitro human testicular organoid culture system, zikv-infected testicular organoids may lead to multiple kinds of cell death (strange et al., ) . although little was known about zikv infection in human testis and epididymis, except for semen, many murine models were used to study damage to testicular tissue. govero et al. ( ) performed a study in wild-type c bl/ mice in the presence of the anti-ifnar antibody and revealed that zikv preferentially infected spermatogonia and sertoli cells in the testis. this led to cell death and destruction of the seminiferous tubules in association with testis damage and poor sperm quality (govero et al., ) . ma et al. ( ) also established a mouse model using ifnα/β receptor-deficient mice (ifnar −/− knockout mice), and demonstrated that zikv infection induced inflammation in the testis and epididymis, leading to severe damage to testes at days post-infection. taken together, these findings suggested that zikv could persist in testicular tissue for a long time, causing severe damage to testis and epididymis and reducing sperm quality. currently, no approved drug is available to inhibit zikv infection (da silva et al., ) , especially infection in testicular tissue. ebselen (ebs), an antioxidant in clinical trials, was reported to alleviate testicular pathology in zikv-infected mice by reducing the level of oxidative stress and proinflammatory cytokines. however, it only had a weak effect on zikv directly, and its safety for pregnant women was unknown (simanjuntak et al., ) . this calls for the development of safe and effective drugs to prevent zikv-induced testicular damage. the testis is a male reproductive organ, mainly producing spermatozoa and androgen. specifically, spermatogenesis is a complex cellular event taking place in the seminiferous epithelium of seminiferous tubules and protected by sertoli cells that form the blood-testes barrier (btb) by tight junction protein (su et al., ) . the btb provides a specialized microenvironment for spermatogenesis by preventing harmful agents from entering the seminiferous tubule, but this was found to pose a major obstacle to the delivery of therapeutic drugs to the seminiferous epithelium (cheng and mruk, ) . therefore, any drug able to prevent zikvinduced damage in testicular tissue should be able to cross the btb into seminiferous tubules, or reach the testicular tissue, to inhibit zikv from entering into seminiferous tubules. the most promising anti-zikv drugs so far include small-molecule compounds (deng et al., a; chan et al., ; li et al., ) , antibodies (zhang et al., ; wang et al., wang et al., , , and peptides (yu et al., ; jackman et al., ) . compared with small-molecule compounds, peptides were safer, especially for pregnant women. as a macromolecular substance, passing through the btb was challenging for antibodies, and it is reported that the concentration of specific igg entering into the rete testis was . % of that in blood serum (knee et al., ) . therefore, the safer and cheaper peptide drugs, which consisted of dozens of amino acids, began to gain gradual acceptance. we previously demonstrated that an amphipathic peptide z , derived from the stem region of zikv e protein (figure a) , inhibited zikv infection in vivo and in vitro, suggesting its promise as an anti-zikv candidate drug. what's more, z had a protective effect in pregnant mice and their fetuses, suggesting it was able to cross the placental barrier (yu et al., ) . however, whether z could enter the seminiferous tubules and protect testicular tissue against zikv infection remained unknown. in this work, we showed that intraperitoneally injected z could be distributed in the testicular tissue. it then inhibited zikv infection, resulting in significant reduction of viral loads and protection of testis, epididymis, and sperm from zikvinduced pathological damage. these results suggest that z has the potential to be further developed as an anti-zikv agent for treatment and prevention of zikv-induced damage in testicular tissue. (sirohi et al., ) ]; di, dii, diii, stem and transmembrane domain are shown in red, yellow, blue, orange, and wheat, respectively; the fusion peptide is shown in cyan, and z ( - ) is shown in green. tm cells were infected with zikv strain sz (b), mr (c) and flr (d), treated with z at different concentration, and then viral copies in the supernatant at h were measured by qrt-pcr. data were presented as means ± sd. (e) z -treated zikv of different strain lost infectivity on tm cells irreversibly. after incubation with z at • c for h, zikv particles were separated from the unbound z by peg to measure their infectivity on tm cells. each sample was tested in triplicate and the experiments were repeated at least once. the data from two independent experiments were presented as mean ± sd. mixture f- (dmem/f , thermo fisher scientific, waltham, ma, united states) supplemented with % horse serum and . % fbs at • c and % co . zika virus (zikv) strain sz / (genbank no. ku ) was kindly provided by dr. cheng-feng qin (deng et al., b) and preserved in our laboratory. zikv strains mr (#vr ) and flr (#vr ) were obtained from atcc. zikv was propagated in vero-e cells. briefly, vero-e cells were infected with the virus at multiplicity of infection (moi) of . . the supernatants were harvested at days post-infection, centrifuged at , rpm for min to remove cellular debris, and stored at − • c as stock. peptides z (mavlgdtawdfgsvggalnslgkgihqifg aaf), z -cy and scrambled peptide (ldiiaglsagfq ggatfvdahgmvkasflggnw) were synthesized at kangbei bio, co., ltd. (ningbo, china) with % purity. peptides were solubilized in dimethyl sulfoxide (dmso) at mm and stored at − • c. virus titer was detected by plaque forming assay as shown below. bhk- cells were seeded onto a -well plate with × cells per well and cultured overnight at • c and % co . serially fold diluted virus were added to each well and incubated for h at • c. then the supernatant was replaced with ml dmem containing % low melting agarose and % fbs. after agarose solidification, the cells were cultured at • c and % co for days. then cells were fixed with % formalin and stained with % crystal violet overnight. finally, the plaque forming units were counted, and virus titer was calculated. six male a mice ( - weeks old) were randomly divided into two groups. mice in each group (n = ) were injected intraperitoneally with z -cy ( µg in µl pbs) or pbs (vehicle in µl pbs) as control. after anesthetization with pentobarbital sodium, mice were imaged by the ivis lumina k series iii in vivo imaging system from perkinelmer (waltham, ma, united states) for h. to determine the distribution of z -cy in the testicular tissue, mice were sacrificed using pentobarbital sodium, and all testes and epididymides were removed for imaging. the radiant efficiency (ps − cm − sr − ) (µw − cm − ) in mouse body and testis and epididymis was calculated by living image . . software. to determine the protection of z against zikv-induced testis damage, male a mice ( - weeks old) were randomly divided into three groups (n = ): z -treated group, vehicletreated group, and mock-infected group. mice in the z -or vehicle-treated group were intraperitoneally injected (i.p.) with pfu zikv with z ( mg/kg) or vehicle on day , followed by i.p. administration of z ( mg/kg) or vehicle once daily for six consecutive days, respectively. mice in the mock-infected group only received pbs as normal control. the body weight was monitored daily for days, and blood was collected at , , , , and days post-infection (d.p.i.) for detection of zikv copies in sera. at d.p.i., mice were euthanized by co inhalation, and the testes and epididymides were removed. the weight and size of testes were measured as previously described (de la vega et al., ) . after imaging, the left testes and epididymides were immersed in bouin's for hematoxylin-eosin (h&e) staining. the right testes and epididymides were soaked in rnaiso plus reagent at − • c for further detection of zikv copies. for safety analysis of z in testicular tissue, male balb/c mice ( - weeks old) were randomly divided into two groups. five mice in each group were i.v. administered with z at mg/kg of body weight or pbs control for days. the body weight of mice was monitored every other day for days. blood was collected at h, as well as , , and days post-injection and sera were separated from the blood samples for use in elisa to detect the concentration of testosterone and inhibin b as well as the titer of z -specific antibody. at days, mice were sacrificed, and the testes and epididymides were removed for histological examination. mature sperm in the cauda epididymis of the three groups of male a mice were collected and placed in ml pbs (preincubated at • c) immediately after euthanasia. the sperm suspension was analyzed for total sperm count and motility by computer-assisted sperm analysis (casa), as previously described (goodson et al., ) , using hamilton thorne ivos ii (beverly, ma, united states). then, after smear, desiccation and fixation, remaining sperm were stained by the papanicolaou staining method for manual morphological analysis. sperm morphology was observed in each mouse. zikv-infected mice were euthanized at days post-infection. testes and epididymides were homogenized with beads in ml rnaiso plus reagent (takara, japan) using tissuelyser- (jingxin, shanghai, china) after weighing. homogenized tissue were centrifuged for min at , rpm at • c, then total rna in tissues was extracted according to the operating manual and stored at − • c for the next step. sperm collected in pbs were placed in rnaiso to extract total rna under the same procedure. viral rna in sera samples on specific days was extracted using the easypure r viral dna/rna kit (transgen, china) and stored at − • c for the next step. zikv rna was examined by one-step real-time quantitative reverse transcription pcr (qrt-pcr) using the mastercycler r ep realplex real-time pcr system (eppendorf, germany). zikv rna copies were calculated based on the standard curve which was determined by plasmid containing specific sequence. the following primers were used: zikv-f: -ttggtcatgatactgctgattgc- ; zikv-r: -ccttccacaaagtccctattgc- ; zikv-probe: -fam-cggcatacagcatcaggtgcataggag-bhq - . the concentration of testosterone and inhibin b in the sera of balb/c mice was detected by mouse testosterone (ml , mlbio, shanghai, china) and inhibin b elisa kit (ml , mlbio, shanghai, china), respectively. according to the manual, µl standard or testing samples were added to a -well plate, which was coated with purified mouse testosterone or inhibin b antibody combined with hrp labeling. hrp-conjugate reagent was added to each well, except blank well (no sample; hrpconjugate reagent added as background). the plate was closed with closure plate membrane and incubated at • c for min. after washing, chromogen solution was added and incubated for min at • c. stop solution was added to each well, and absorbance was read at nm. peptide z was dissolved in dmso and diluted to different concentration by dmem/f . then µl of different zikv strains were incubated with z for h at • c. the mixture was added to × cells seeded into a -well plate and incubated at • c for h. after the culture supernatant was replaced by dmem/f- with % horse serum, cells were cultured for h at • c. then the culture supernatant was collected to detect zikv rna copies by qrt-pcr, as described above. the ability of z to inactivate different zikv strains was determined as follows. briefly, µl z or z -scr, at graded concentration were added to µl zikv ( × pfu/ml), followed by incubation at • c for h. then, peg- and nacl were added to the treated virus at final concentration of % and . m, respectively. after incubation on ice for h, the mixture was centrifuged at , rpm for h. the supernatants were removed, and the pellet was resuspended in µl dmem with % fbs. the infectivity of the zikv particles in the pellet was determined by cck- on bhk- cells or qrt-pcr on tm cells. the testis and epididymidis of z -or vehicle-treated zikvinfected mice and mock-infected mice were all collected post mortem. tissues were fixed in bouin's overnight, dehydrated, embedded in paraffin and sectioned. then the sections ( µm thick) were stained by h&e. subsequently, observation was made via panoramic scanner ( d histech pannoramic midi, hungary). student's unpaired two-tailed t-test was used to monitor the distribution of z in male a mouse body and testicular tissue and to analyze the difference of viral rna level in sera or tissues between z -and vehicle-treated a mice. one-way anova was used to examine the effect of z on the weight, length and width of testes, as well as sperm count, sperm motility and progressive sperm motility among the three groups. p-value was calculated by graphpad prism software, v. . , and significant difference was achieved with p-value less than . . * p < . ; * * p < . ; * * * p < . ; * * * * p< . . to determine the protective effect of z on zikv infection of testicular tissue, we tested if z could inhibit infection by different zikv strains of asian and african lineages in mouse sertoli tm cells, which are nurse-like cells that support spermatogenesis (wei et al., ) and important target cells for zikv testicular infection. zikv sz (asian lineage), flr (asian lineage), or mr (african lineage) was pretreated with z at different concentration before addition of tm cells and incubation at • c for h. after replacement of culture medium and further incubation for h, the viral copies in the supernatant were examined by qrt-pcr. as shown in figures b-d, z treatment resulted in a decrease of zikv copies in a dosedependent manner. considering that z -mediated inhibition of zikv infection is possibly attributed to its viral inactivation activity (yu et al., ) , different strains of zikv were incubated with z at different concentration for h at • c, followed by separating virions from the unbound free peptide by peg and detecting the infectivity of z -treated zikv in tm cells ( figure e ) and bhk- cells (supplementary figure s ) . we found that z -treated zikv strains lost their infectivity in a dose-dependent manner with % effective concentration (ec ) of . ± . µm (for sz ), . ± . µm (for mr ) and . ± . µm (for flr), respectively, suggesting that z inhibits infection of zikv strains of both asian and african lineages in tm and bhk- cells via inactivation of virions. to determine whether z entered testicular tissue of male mice, we employed cy -conjugated z peptide (z -cy ) to detect the distribution of z in the organs of male mice. as shown in figure a , the bodies of the z -cy -treated mice showed a strong fluorescence signal with average radiant efficiency of about . × (ps − cm − sr − ) (µw − cm − ), which is significantly higher than that in pbs-treated mice (p = . , student's two-tailed t-test; figure b) . then, the testes and epididymides were collected for examination of the fluorescence signal in the testicular tissue. as expected, both testes and epididymides of mice treated with z -cy showed a strong fluorescence signal, while those in the pbs-treated mice displayed no significant fluorescence signal (figure c) . the average radiant efficiency in testes and epididymides of z -cy -treated mice was significantly figure | distribution of z in the testicular tissue of male a mice. (a) imaging of male a mice treated with z -cy or pbs by the ivis lumina k series iii from perkinelmer. male a mice were injected intraperitoneally with µg z -cy (n = ) or pbs (n = ) as control, followed by imaging analysis. (b) the statistical analysis of fluorescence signal intensity in mouse body. data were presented as means ± sd. (c) imaging of the testes and epididymides from male a mice. (d) statistical analysis of fluorescence signal intensity in testis. each sample was tested in triplicate and the data were presented as mean ± sd. (e) statistical analysis of fluorescence signal intensity in epididymis. data were presented as means ± sd. * p < . ; * * * * p < . , student's two-tailed t-test. higher than that of pbs-treated mice (p < . , student's twotailed t-test; figures d,e) . these results suggest that z peptide can be distributed in the testis and epididymis of male mice. to determine the protective effect of z against zikv infection of male mice, pfu zikv were intraperitoneally injected into male a mice (type i interferon receptor-deficient). the infected mice were i.p. administered with z at mg/kg of body weight or vehicle, respectively, daily for days (figure a) . mice in the mock-infected group received pbs as normal control. results showed that the z -treated mice had neither weight loss ( figure b ) nor obvious clinical symptoms, consistent with mice in the mock-infected group (data not shown). however, in the vehicle-treated group, mouse body weight began to decline from the fifth day post-infection (d.p.i.) (figure b) , and some symptoms, like hunched posture and ruffled fur, appeared. we then examined viral copies in sera of z -or vehicle-treated mice at different time points by qrt-pcr. a high level of viral load was detected in sera of vehicle-treated mice, e.g., about copies/ml at d.p.i. (figure c) . however, viral load in sera of z -treated mice (figure c ) was as low as copies/ml at all time points tested, significantly lower than that of vehicle-treated mice. these results suggest that z can exert protection against zikv infection of male mice. to further evaluate the protective effect of z against zikvinduced damage of testicular tissue in male mice, all mice were sacrificed at d.p.i. and their testes were collected for analysis of weight and size. we found that testis weight in vehicle-treated mice was around mg, which was significantly lower than that in z -treated mice (∼ mg) (p < . , one-way anova, figure a ). the length and width of testes in vehicle-treated mice were both significantly decreased compared with those of testes in z -treated mice the change of mouse body weight was monitored daily for days. (c) the change of zikv rna level in mouse sera were detected by qrt-pcr at days , , , , and after zikv infection. male a mice were intraperitoneally injected with pfu zikv with z ( mg/kg) or vehicle on day , followed by daily injection of z ( mg/kg) or vehicle for six consecutive days. each sample was tested in triplicate and the data were presented as mean ± sem, * * * p < . ; * * * * p < . , student's two-tailed t-test. (p = . and p < . , one-way anova, figures b,c) . no significant difference in weight (p > . , one-way anova, figure a ), as well as length (p > . , one-way anova, figure b ), and width (p > . , one-way anova, figure c ) of testes were noted between z -treated zikvinfected mice and mock-infected mice. the representative image of testes from the three groups of mice were shown in figure d . subsequently, we examined the testes and epididymides for histopathological changes. the results of h&e staining of testes in vehicle-treated mice revealed that the normal architecture of the seminiferous tubule was seriously destroyed and replaced with an infiltrate of mixed inflammatory cells and necrotic debris, accompanied by degeneration of the spermatogenic lineage ( figure e, upper panel) . the connective tissue areas surrounding the seminiferous tubule had also been infiltrated by a large number of inflammatory cells (figure e, upper panel) . however, the architecture of the seminiferous tubule in the testes of both z -treated and mock-infected mice was intact and clear. spermatogenic cells at different stages were organized tightly and identified clearly (figure e, upper panel) . histological analysis of epididymis showed that epididymides from mice in the vehicle-treated group were also damaged. sperm in the lumen of caput epididymides decreased precipitously, only to be replaced by secretions and numerous necrotic epithelial cells (figure e, middle panel) . the lumens of cauda epididymis contained degenerating spermatozoa and a small number of normal spermatozoa, accompanied by scattered necrotic epithelial cells and inflammatory cells (figure e , lower panel). however, histological analysis of the caput epididymis and cauda epididymis showed no apparent microscopic differences between z -treated and mock-infected mice (figure e middle, lower panel). the architecture of the caput epididymis and cauda epididymis in these two groups was normal with no obvious morphological damage, suggesting that z protected testicular tissue against zikv-induced pathological damage. we used casa to evaluate the protective effect of z on the count and motility of mouse sperm. as shown in figure , the figure | z effectively attenuated damage to testes and epididymides of zikv-infected male a mice. (a) the weight, (b) length, and (c) width of testes from z -or vehicle-treated zikv-infected and mock-infected male a mice at day . each symbol represents one testis; all horizontal bars indicate mean, and error bars reflect sem. (d) the representative image of testes from z -or vehicle-treated zikv-infected and mock-infected male a mice at day . scale bar, mm. (e) histopathological analyses of testes and epididymides collected from z -or vehicle-treated zikv-infected male a mice and mock-infected mice used as a control. scale bar: µm. upper panel, testes; middle panel, caput epididymides; lower panel, cauda epididymides. * * p < . ; * * * * p < . ; one-way analysis of variance with tukey's multiple comparison post hoc tests. sperm count of z -treated mice was significantly higher than that of the vehicle-treated group (p = . , one-way anova; figure a ). meanwhile, the percentages of total ( figure b ) and progressively ( figure c ) motile sperm in z -treated mice were dramatically higher than those in the vehicle-treated group (p = . and p = . , one-way anova), but similar to that in the mock-infected mouse group (p > . , oneway anova). papanicolaou staining of morphological spermatic features revealed more noticeable teratogenesis of sperm in vehicle-treated mice compared to the other groups ( figure d) . to investigate whether the protective effect of z on testicular tissue results from the reduction of local viral load, we examined the viral copies in different testicular tissues. results showed a high level of viral rna ( - equivalents per g) detected in the testis and epididymis of vehicle-treated mice at d.p.i., much higher than that ( - equivalents per g) in z -treated mice (figures a,b) . notably, zikv rna was also detected (up to equivalents per ml) in the mature sperm collected from cauda epididymis in vehicle-treated mice, which was significantly higher than that in z -treated mice (p = . , student's twotailed t-test; figure c ). finally, to examine the safety of z for male mice, male balb/c mice were injected intravenously with z at mg/kg of body weight (n = ) or pbs (n = ). results showed that body weight change of mice was nearly consistent in the two groups ( figure a) , indicating that z peptide did not cause significant harm to the male mice. since the levels of testosterone and inhibin b reflect testicular function and sperm count, the concentration of these hormones in mouse sera at the indicated time points was measured. we found no significant difference between the z -and pbs-treated groups at all time points tested (figures b,c) , suggesting z may not affect the function of testis or sperm. we then compared the potential histopathological changes between the two groups. as shown in figure d , h&e analysis of testis and epididymis revealed no obvious pathological abnormality in mice treated with z compared with the pbs group. besides, the titer of z -specific antibody in sera of mice at and days post-injection was detected. as shown in figures e,f , no significant level of z -specific antibody was detected in the sera of mice that were intravenously injected with high doses of z peptide, consistent with the finding from our previous report for studying anti-mers-cov peptides (xia et al., ) . this result suggests that z peptide consisting of amino acids is unable to elicit a significant z -specific antibody response after it is intravenously administered in the absence of figure | z inhibited zikv replication in testes, epididymides and sperm. zikv rna copies in (a) testes, (b) epididymides, and (c) sperm of z -or vehicle-treated zikv-infected male a mice at day were detected by qrt-pcr. each symbol represents data from individual mice; all horizontal bars indicate mean, and error bars reflect sem. experiment was repeated at least twice. * * * p < . or * * * * p < . respectively, student's two-tailed t-test. figure | safety analysis of z for male balb/c mice. balb/c mice were injected with z ( mg/kg/day, i.v.) for days (n = ), and another group of mice (n = ) received pbs as a control. (a) body weight change of balb/c mice at different time points. data were presented as means ± sem. concentration of (b) testosterone and (c) inhibin b in sera before and after z injection. data were presented as means ± sem of triplicate experiments. (d) histological analysis of the testis and epididymis collected from z -or pbs-treated male balb/c mice. scale bar: µm. (e) z -specific antibody response in mice days after i.v. administration of z or pbs. (f) z -apecific antibody response in mice days after i.v. administration of z or pbs. each sample was tested in triplicate and the data were presented as mean ± sd. adjuvant. therefore, z is safe for male mice, especially for their testicular tissue. currently, many studies have reported the deleterious effects of zikv on male testicular tissue, causing severe damage of testis and epididymis, even leading to infertility uraki et al., ) . two dna vaccines were reported to reduce zikv persistence in the testicular tissue and zikv-associated pathological lesion (griffin et al., ) , or partially prevent infertility of male mice (de la vega et al., ) . however, no effective and safe antiviral agent has ever been reported to prevent or treat zikv infection in testicular tissue. our previous study has demonstrated that z peptide is highly effective in inhibiting zikv infection in vivo and in vitro (yu et al., ) . noticeably, it can penetrate the placental barrier to inhibit vertical transmission of zikv in pregnant mice. however, whether z could cross the btb and protect testicular tissue against zikv infection remained unknown. several studies have reported that sertoli cells play an important role in the entry of zikv into the seminiferous tubules and support long-term replication of zikv in the testicular tissue (siemann et al., ; kumar et al., ) . we found that z peptide possesses potent antiviral activity against zikv infection in bhk- and vero cells (yu et al., ) . in this study, we found that z was also highly effective in inhibiting infection of divergent zikv strains with asian and african lineages in tm cells, the mouse sertoli cell line. particularly, z treatment via intraperitoneal injection resulted in dramatically decreased zikv rna level in the testis of a mice, suggesting that z can protect testis against zikv infection in sertoli cells. meanwhile, we employed z -cy to examine whether z could enter seminiferous tubule, and we found that intraperitoneally injected z could be distributed in the testicular tissue of male a mice, consistent with the observation in mice intravenously administered with z (yu et al., ) . however, because of the intricate structure of capillary vessel and seminiferous tubule in mouse testis, we could not obtain sufficient evidence to prove that z crossed btb into seminiferous tubule. h&e analysis showed no obvious pathological damage in the testicular tissue of z -treated mice, but it did reveal severe pathological damage in the testis and epididymis of vehicle-treated mice, consistent with the findings of other studies (govero et al., ; ma et al., ) . when combined with evidence that viral load in mature sperm of z -treated a mice was significantly decreased, we speculate that z may, indeed, cross the btb and enter seminiferous tubule to inhibit zikv infection in the sperm. zika virus infection in the testicular tissue not only damages male testicular tissue, resulting in pathological lesion of testes and epididymides, but also produces zikv-infected semen, causing infertility. in addition, zikv in semen of an infected male can be sexually transmitted to his pregnant partner (russell et al., ; nelson et al., ) , who can further pass the virus to her fetus, causing congenital zika syndrome in the newborn (yarrington et al., ) . sexual transmission may also contribute to the spread of zikv in regions where the aedes mosquito is not endemic (rowland et al., ) . here we found that z treatment could significantly reduce viral load in sperm of zikv-infected a mice and improve the number and motility of sperm, implying that application of z can limit the damage to testicular tissue and sperm caused by zikv infection and reduce the risk of sexual transmission of zikv. conclusion z administered via intraperitoneal or intravenous injection could be distributed in mouse testicular tissue, protect the tissue against zikv infection and zikv-induced pathological damage and poor sperm quality, suggesting that z peptide has the potential to be further developed as an anti-zikv therapeutic for treatment of zikv infection and attenuation of zikv-induced damage in the testicular tissue. all datasets generated for this study are included in the manuscript/supplementary files. the animal study was reviewed and approved by shanghai public health clinical center animal welfare and ethics committee institutional laboratory animal care and use committee at fudan university. probable sexual transmission of zika virus from a vasectomised man potential effect of zika virus infection on human male fertility? 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a highly potent antibody against zika virus we are very grateful to the staff at the animal experiment department of shanghai public health clinical center for their contribution to this study. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmicb. . /full#supplementary-material key: cord- - tj eve authors: porter, mason a. title: nonlinearity + networks: a vision date: - - journal: nan doi: nan sha: doc_id: cord_uid: tj eve i briefly survey several fascinating topics in networks and nonlinearity. i highlight a few methods and ideas, including several of personal interest, that i anticipate to be especially important during the next several years. these topics include temporal networks (in which the entities and/or their interactions change in time), stochastic and deterministic dynamical processes on networks, adaptive networks (in which a dynamical process on a network is coupled to dynamics of network structure), and network structure and dynamics that include"higher-order"interactions (which involve three or more entities in a network). i draw examples from a variety of scenarios, including contagion dynamics, opinion models, waves, and coupled oscillators. in its broadest form, a network consists of the connectivity patterns and connection strengths in a complex system of interacting entities [ ] . the most traditional type of network is a graph g = (v, e) (see fig. a) , where v is a set of "nodes" (i.e., "vertices") that encode entities and e ⊆ v × v is a set of "edges" (i.e., "links" or "ties") that encode the interactions between those entities. however, recent uses of the term "network" have focused increasingly on connectivity patterns that are more general than graphs [ ] : a network's nodes and/or edges (or their associated weights) can change in time [ , ] (see section ), nodes and edges can include annotations [ ] , a network can include multiple types of edges and/or multiple types of nodes [ , ] , it can have associated dynamical processes [ ] (see sections , , and ) , it can include memory [ ] , connections can occur between an arbitrary number of entities [ , ] (see section ) , and so on. associated with a graph is an adjacency matrix a with entries a i j . in the simplest scenario, edges either exist or they don't. if edges have directions, a i j = when there is an edge from entity j to entity i and a i j = when there is no such edge. when a i j = , node i is "adjacent" to node j (because we can reach i directly from j), and the associated edge is "incident" from node j and to node i. the edge from j to i is an "out-edge" of j and an "in-edge" of i. the number of out-edges of a node is its "out-degree", and the number of in-edges of a node is its "in-degree". for an undirected network, a i j = a ji , and the number of edges that are attached to a node is the node's "degree". one can assign weights to edges to represent connections with different strengths (e.g., stronger friendships or larger transportation capacity) by defining a function w : e −→ r. in many applications, the weights are nonnegative, although several applications [ ] (such as in international relations) incorporate positive, negative, and zero weights. in some applications, nodes can also have selfedges and multi-edges. the spectral properties of adjacency (and other) matrices give important information about their associated graphs [ , ] . for undirected networks, it is common to exploit the beneficent property that all eigenvalues of symmetric matrices are real. traditional studies of networks consider time-independent structures, but most networks evolve in time. for example, social networks of people and animals change based on their interactions, roads are occasionally closed for repairs and new roads are built, and airline routes change with the seasons and over the years. to study such time-dependent structures, one can analyze "temporal networks". see [ , ] for reviews and [ , ] for edited collections. the key idea of a temporal network is that networks change in time, but there are many ways to model such changes, and the time scales of interactions and other changes play a crucial role in the modeling process. there are also other [i drew this network using tikz-network, by jürgen hackl and available at https://github.com/hackl/tikz-network), which allows one to draw networks (including multilayer networks) directly in a l a t e x file.] . an example of a multilayer network with three layers. we label each layer using di↵erent colours for its state nodes and its edges: black nodes and brown edges (three of which are unidirectional) for layer , purple nodes and green edges for layer , and pink nodes and grey edges for layer . each state node (i.e. nodelayer tuple) has a corresponding physical node and layer, so the tuple (a, ) denotes physical node a on layer , the tuple (d, ) denotes physical node d on layer , and so on. we draw intralayer edges using solid arcs and interlayer edges using broken arcs; an interlayer edge is dashed (and magenta) if it connects corresponding entities and dotted (and blue) if it connects distinct ones. we include arrowheads to represent unidirectional edges. we drew this network using tikz-network (jürgen hackl, https://github.com/hackl/tikz-network), which allows one to draw multilayer networks directly in a l at ex file. , which is by jürgen hackl and is available at https://github.com/hackl/tikz-network. panel (b) is inspired by fig. of [ ] . panel (d), which is in the public domain, was drawn by wikipedia user cflm and is available at https://en.wikipedia.org/wiki/simplicial_complex.] important modeling considerations. to illustrate potential complications, suppose that an edge in a temporal network represents close physical proximity between two people in a short time window (e.g., with a duration of two minutes). it is relevant to consider whether there is an underlying social network (e.g., the friendship network of mathematics ph.d. students at ucla) or if the people in the network do not in general have any other relationships with each other (e.g., two people who happen to be visiting a particular museum on the same day). in both scenarios, edges that represent close physical proximity still appear and disappear over time, but indirect connections (i.e., between people who are on the same connected component, but without an edge between them) in a time window may play different roles in the spread of information. moreover, network structure itself is often influenced by a spreading process or other dynamics, as perhaps one arranges a meeting to discuss a topic (e.g., to give me comments on a draft of this chapter). see my discussion of adaptive networks in section . for convenience, most work on temporal networks employs discrete time (see fig. (b) ). discrete time can arise from the natural discreteness of a setting, dis-cretization of continuous activity over different time windows, data measurement that occurs at discrete times, and so on. one way to represent a discrete-time (or discretized-time) temporal network is to use the formalism of "multilayer networks" [ , ] . one can also use multilayer networks to study networks with multiple types of relations, networks with multiple subsystems, and other complicated networked structures. fig. (c)) has a set v of nodesthese are sometimes called "physical nodes", and each of them corresponds to an entity, such as a person -that have instantiations as "state nodes" (i.e., node-layer tuples, which are elements of the set v m ) on layers in l. one layer in the set l is a combination, through the cartesian product l × · · · × l d , of elementary layers. the number d indicates the number of types of layering; these are called "aspects". a temporal network with one type of relationship has one type of layering, a timeindependent network with multiple types of social relationships also has one type of layering, a multirelational network that changes in time has two types of layering, and so on. the set of state nodes in m is v m ⊆ v × l × · · · × l d , and the set of indicates that there is an edge from node j on layer β to node i on layer α (and vice versa, if m is undirected). for example, in fig. (c) , there is a directed intralayer edge from (a, ) to (b, ) and an undirected interlayer edge between (a, ) and (a, ). the multilayer network in fig. (c) has three layers, |v | = physical nodes, d = aspect, |v m | = state nodes, and |e m | = edges. to consider weighted edges, one proceeds as in ordinary graphs by defining a function w : e m −→ r. as in ordinary graphs, one can also incorporate self-edges and multi-edges. multilayer networks can include both intralayer edges (which have the same meaning as in graphs) and interlayer edges. the multilayer network in fig. (c) has directed intralayer edges, undirected intralayer edges, and undirected interlayer edges. in most studies thus far of multilayer representations of temporal networks, researchers have included interlayer edges only between state nodes in consecutive layers and only between state nodes that are associated with the same entity (see fig. (c)). however, this restriction is not always desirable (see [ ] for an example), and one can envision interlayer couplings that incorporate ideas like time horizons and interlayer edge weights that decay over time. for convenience, many researchers have used undirected interlayer edges in multilayer analyses of temporal networks, but it is often desirable for such edges to be directed to reflect the arrow of time [ ] . the sequence of network layers, which constitute time layers, can represent a discrete-time temporal network at different time instances or a continuous-time network in which one bins (i.e., aggregates) the network's edges to form a sequence of time windows with interactions in each window. each d-aspect multilayer network with the same number of nodes in each layer has an associated adjacency tensor a of order (d + ). for unweighted multilayer networks, each edge in e m is associated with a entry of a, and the other entries (the "missing" edges) are . if a multilayer network does not have the same number of nodes in each layer, one can add empty nodes so that it does, but the edges that are attached to such nodes are "forbidden". there has been some research on tensorial properties of a [ ] (and it is worthwhile to undertake further studies of them), but the most common approach for computations is to flatten a into a "supra-adjacency matrix" a m [ , ] , which is the adjacency matrix of the graph g m that is associated with m. the entries of diagonal blocks of a m correspond to intralayer edges, and the entries of off-diagonal blocks correspond to interlayer edges. following a long line of research in sociology [ ] , two important ingredients in the study of networks are examining ( ) the importances ("centralities") of nodes, edges, and other small network structures and the relationship of measures of importance to dynamical processes on networks and ( ) the large-scale organization of networks [ , ] . studying central nodes in networks is useful for numerous applications, such as ranking web pages, football teams, or physicists [ ] . it can also help reveal the roles of nodes in networks, such as those that experience high traffic or help bridge different parts of a network [ , ] . mesoscale features can impact network function and dynamics in important ways. small subgraphs called "motifs" may appear frequently in some networks [ ] , perhaps indicating fundamental structures such as feedback loops and other building blocks of global behavior [ ] . various types of largerscale network structures, such as dense "communities" of nodes [ , ] and coreperiphery structures [ , ] , are also sometimes related to dynamical modules (e.g., a set of synchronized neurons) or functional modules (e.g., a set of proteins that are important for a certain regulatory process) [ ] . a common way to study large-scale structures is inference using statistical models of random networks, such as through stochastic block models (sbms) [ ] . much recent research has generalized the study of large-scale network structure to temporal and multilayer networks [ , , ] . various types of centrality -including betweenness centrality [ , ] , bonacich and katz centrality [ , ] , communicability [ ] , pagerank [ , ] , and eigenvector centrality [ , ] -have been generalized to temporal networks using a variety of approaches. such generalizations make it possible to examine how node importances change over time as network structure evolves. in recent work, my collaborators and i used multilayer representations of temporal networks to generalize eigenvector-based centralities to temporal networks [ , ] . one computes the eigenvector-based centralities of nodes for a timeindependent network as the entries of the "dominant" eigenvector, which is associated with the largest positive eigenvalue (by the perron-frobenius theorem, the eigenvalue with the largest magnitude is guaranteed to be positive in these situations) of a centrality matrix c(a). examples include eigenvector centrality (by using c(a) = a) [ ] , hub and authority scores (by using c(a) = aa t for hubs and a t a for authorities) [ ] , and pagerank [ ] . given a discrete-time temporal network in the form of a sequence of adjacency matrices i j denotes a directed edge from entity i to entity j in time layer t, we construct a "supracentrality matrix" c(ω), which couples centrality matrices c(a (t) ) of the individual time layers. we then compute the dominant eigenvector of c(ω), where ω is an interlayer coupling strength. in [ , ] , a key example was the ranking of doctoral programs in the mathematical sciences (using data from the mathematics genealogy project [ ] ), where an edge from one institution to another arises when someone with a ph.d. from the first institution supervises a ph.d. student at the second institution. by calculating timedependent centralities, we can study how the rankings of mathematical-sciences doctoral programs change over time and the dependence of such rankings on the value of ω. larger values of ω impose more ranking consistency across time, so centrality trajectories are less volatile for larger ω [ , ] . multilayer representations of temporal networks have been very insightful in the detection of communities and how they split, merge, and otherwise evolve over time. numerous methods for community detection -including inference via sbms [ ] , maximization of objective functions (especially "modularity") [ ] , and methods based on random walks and bottlenecks to their traversal of a network [ , ] -have been generalized from graphs to multilayer networks. they have yielded insights in a diverse variety of applications, including brain networks [ ] , granular materials [ ] , political voting networks [ , ] , disease spreading [ ] , and ecology and animal behavior [ , ] . to assist with such applications, there are efforts to develop and analyze multilayer random-network models that incorporate rich and flexible structures [ ] , such as diverse types of interlayer correlations. activity-driven (ad) models of temporal networks [ ] are a popular family of generative models that encode instantaneous time-dependent descriptions of network dynamics through a function called an "activity potential", which encodes the mechanism to generate connections and characterizes the interactions between enti-ties in a network. an activity potential encapsulates all of the information about the temporal network dynamics of an ad model, making it tractable to study dynamical processes (such as ones from section ) on networks that are generated by such a model. it is also common to compare the properties of networks that are generated by ad models to those of empirical temporal networks [ ] . in the original ad model of perra et al. [ ] , one considers a network with n entities, which we encode by the nodes. we suppose that node i has an activity rate a i = ηx i , which gives the probability per unit time to create new interactions with other nodes. the scaling factor η ensures that the mean number of active nodes per unit time is η we define the activity rates such that x i ∈ [ , ], where > , and we assign each x i from a probability distribution f(x) that can either take a desired functional form or be constructed from empirical data. the model uses the following generative process: • at each discrete time step (of length ∆t), start with a network g t that consists of n isolated nodes. • with a probability a i ∆t that is independent of other nodes, node i is active and generates m edges, each of which attaches to other nodes uniformly (i.e., with the same probability for each node) and independently at random (without replacement). nodes that are not active can still receive edges from active nodes. • at the next time step t + ∆t, we delete all edges from g t , so all interactions have a constant duration of ∆t. we then generate new interactions from scratch. this is convenient, as it allows one to apply techniques from markov chains. because entities in time step t do not have any memory of previous time steps, f(x) encodes the network structure and dynamics. the ad model of perra et al. [ ] is overly simplistic, but it is amenable to analysis and has provided a foundation for many more general ad models, including ones that incorporate memory [ ] . in section . , i discuss a generalization of ad models to simplicial complexes [ ] that allows one to study instantaneous interactions that involve three or more entities in a network. many networked systems evolve continuously in time, but most investigations of time-dependent networks rely on discrete or discretized time. it is important to undertake more analysis of continuous-time temporal networks. researchers have examined continuous-time networks in a variety of scenarios. examples include a compartmental model of biological contagions [ ] , a generalization of katz centrality to continuous time [ ] , generalizations of ad models (see section . . ) to continuous time [ , ] , and rankings in competitive sports [ ] . in a recent paper [ ] , my collaborators and i formulated a notion of "tie-decay networks" for studying networks that evolve in continuous time. they distinguished between interactions, which they modeled as discrete contacts, and ties, which encode relationships and their strength as a function of time. for example, perhaps the strength of a tie decays exponentially after the most recent interaction. more realistically, perhaps the decay rate depends on the weight of a tie, with strong ties decaying more slowly than weak ones. one can also use point-process models like hawkes processes [ ] to examine similar ideas using a node-centric perspective. suppose that there are n interacting entities, and let b(t) be the n × n timedependent, real, non-negative matrix whose entries b i j (t) encode the tie strength between agents i and j at time t. in [ ] , we made the following simplifying assumptions: . as in [ ] , ties decay exponentially when there are no interactions: where α ≥ is the decay rate. . if two entities interact at time t = τ, the strength of the tie between them grows instantaneously by . see [ ] for a comparison of various choices, including those in [ ] and [ ] , for tie evolution over time. in practice (e.g., in data-driven applications), one obtains b(t) by discretizing time, so let's suppose that there is at most one interaction during each time step of length ∆t. this occurs, for example, in a poisson process. such time discretization is common in the simulation of stochastic dynamical systems, such as in gillespie algorithms [ , , ] . consider an n × n matrix a(t) in which a i j (t) = if node i interacts with node j at time t and a i j (t) = otherwise. for a directed network, a(t) has exactly one nonzero entry during each time step when there is an interaction and no nonzero entries when there isn't one. for an undirected network, because of the symmetric nature of interactions, there are exactly two nonzero entries in time steps that include an interaction. we write equivalently, if interactions between entities occur at times τ ( ) such that ≤ τ ( ) < τ ( ) < . . . < τ (t ) , then at time t ≥ τ (t ) , we have in [ ] , my coauthors and i generalized pagerank [ , ] to tie-decay networks. one nice feature of their tie-decay pagerank is that it is applicable not just to data sets, but also to data streams, as one updates the pagerank values as new data arrives. by contrast, one problematic feature of many methods that rely on multilayer representations of temporal networks is that one needs to recompute everything for an entire data set upon acquiring new data, rather than updating prior results in a computationally efficient way. a dynamical process can be discrete, continuous, or some mixture of the two; it can also be either deterministic or stochastic. it can take the form of one or several coupled ordinary differential equations (odes), partial differential equations (pdes), maps, stochastic differential equations, and so on. a dynamical process requires a rule for updating the states of its dependent variables with respect one or more independent variables (e.g., time), and one also has (one or a variety of) initial conditions and/or boundary conditions. to formalize a dynamical process on a network, one needs a rule for how to update the states of the nodes and/or edges. the nodes (of one or more types) of a network are connected to each other in nontrivial ways by one or more types of edges. this leads to a natural question: how does nontrivial connectivity between nodes affect dynamical processes on a network [ ] ? when studying a dynamical process on a network, the network structure encodes which entities (i.e., nodes) of a system interact with each other and which do not. if desired, one can ignore the network structure entirely and just write out a dynamical system. however, keeping track of network structure is often a very useful and insightful form of bookkeeping, which one can exploit to systematically explore how particular structures affect the dynamics of particular dynamical processes. prominent examples of dynamical processes on networks include coupled oscillators [ , ] , games [ ] , and the spread of diseases [ , ] and opinions [ , ] . there is also a large body of research on the control of dynamical processes on networks [ , ] . most studies of dynamics on networks have focused on extending familiar models -such as compartmental models of biological contagions [ ] or kuramoto phase oscillators [ ] -by coupling entities using various types of network structures, but it is also important to formulate new dynamical processes from scratch, rather than only studying more complicated generalizations of our favorite models. when trying to illuminate the effects of network structure on a dynamical process, it is often insightful to provide a baseline comparison by examining the process on a convenient ensemble of random networks [ ] . a simple, but illustrative, dynamical process on a network is the watts threshold model (wtm) of a social contagion [ , ] . it provides a framework for illustrating how network structure can affect state changes, such as the adoption of a product or a behavior, and for exploring which scenarios lead to "virality" (in the form of state changes of a large number of nodes in a network). the original wtm [ ] , a binary-state threshold model that resembles bootstrap percolation [ ] , has a deterministic update rule, so stochasticity can come only from other sources (see section . ). in a binary state model, each node is in one of two states; see [ ] for a tabulation of well-known binary-state dynamics on networks. the wtm is a modification of mark granovetter's threshold model for social influence in a fully-mixed population [ ] . see [ , ] for early work on threshold models on networks that developed independently from investigations of the wtm. threshold contagion models have been developed for many scenarios, including contagions with multiple stages [ ] , models with adoption latency [ ] , models with synergistic interactions [ ] , and situations with hipsters (who may prefer to adopt a minority state) [ ] . in a binary-state threshold model such as the wtm, each node i has a threshold r i that one draws from some distribution. suppose that r i is constant in time, although one can generalize it to be time-dependent. at any time, each node can be in one of two states: (which represents being inactive, not adopted, not infected, and so on) or (active, adopted, infected, and so on). a binary-state model is a drastic oversimplification of reality, but the wtm is able to capture two crucial features of social systems [ ] : interdependence (an entity's behavior depends on the behavior of other entities) and heterogeneity (as nodes with different threshold values behave differently). one can assign a seed number or seed fraction of nodes to the active state, and one can choose the initially active nodes either deterministically or randomly. the states of the nodes change in time according to an update rule, which can either be synchronous (such that it is a map) or asynchronous (e.g., as a discretization of continuous time) [ ] . in the wtm, the update rule is deterministic, so this choice affects only how long it takes to reach a steady state; it does not affect the steady state itself. with a stochastic update rule, the synchronous and asynchronous versions of ostensibly the "same" model can behave in drastically different ways [ ] . in the wtm on an undirected network, to update the state of a node, one compares its fraction s i /k i of active neighbors (where s i is the number of active neighbors and k i is the degree of node i) to the node's threshold r i . an inactive node i becomes active (i.e., it switches from state to state ) if s i /k i ≥ r i ; otherwise, it stays inactive. the states of nodes in the wtm are monotonic, in the sense that a node that becomes active remains active forever. this feature is convenient for deriving accurate approximations for the global behavior of the wtm using branchingprocess approximations [ , ] or when analyzing the behavior of the wtm using tools such as persistent homology [ ] . a dynamical process on a network can take the form of a stochastic process [ , ] . there are several possible sources of stochasticity: ( ) choice of initial condition, ( ) choice of which nodes or edges to update (when considering asynchronous updating), ( ) the rule for updating nodes or edges, ( ) the values of parameters in an update rule, and ( ) selection of particular networks from a random-graph ensemble (i.e., a probability distribution on graphs). some or all of these sources of randomness can be present when studying dynamical processes on networks. it is desirable to compare the sample mean of a stochastic process on a network to an ensemble average (i.e., to an expectation over a suitable probability distribution). prominent examples of stochastic processes on networks include percolation [ ] , random walks [ ] , compartment models of biological contagions [ , ] , bounded-confidence models with continuous-valued opinions [ ] , and other opinion and voter models [ , , , ] . compartmental models of biological contagions are a topic of intense interest in network science [ , , , ] . a compartment represents a possible state of a node; examples include susceptible, infected, zombified, vaccinated, and recovered. an update rule determines how a node changes its state from one compartment to another. one can formulate models with as many compartments as desired [ ] , but investigations of how network structure affects dynamics typically have employed examples with only two or three compartments [ , ] . researchers have studied various extensions of compartmental models, contagions on multilayer and temporal networks [ , , ] , metapopulation models on networks [ ] for simultaneously studying network connectivity and subpopulations with different characteristics, non-markovian contagions on networks for exploring memory effects [ ] , and explicit incorporation of individuals with essential societal roles (e.g., health-care workers) [ ] . as i discuss in section . , one can also examine coupling between biological contagions and the spread of information (e.g., "awareness") [ , ] . one can also use compartmental models to study phenomena, such as dissemination of ideas on social media [ ] and forecasting of political elections [ ] , that are much different from the spread of diseases. one of the most prominent examples of a compartmental model is a susceptibleinfected-recovered (sir) model, which has three compartments. susceptible nodes are healthy and can become infected, and infected nodes can eventually recover. the steady state of the basic sir model on a network is related to a type of bond percolation [ , , , ] . there are many variants of sir models and other compartmental models on networks [ ] . see [ ] for an illustration using susceptible-infectedsusceptible (sis) models. suppose that an infection is transmitted from an infected node to a susceptible neighbor at a rate of λ. the probability of a transmission event on one edge between an infected node and a susceptible node in an infinitesimal time interval dt is λ dt. assuming that all infection events are independent, the probability that a susceptible node with s infected neighbors becomes infected (i.e., for a node to transition from the s compartment to the i compartment, which represents both being infected and being infective) during dt is if an infected node recovers at a constant rate of µ, the probability that it switches from state i to state r in an infinitesimal time interval dt is µ dt. when there is no source of stochasticity, a dynamical process on a network is "deterministic". a deterministic dynamical system can take the form of a system of coupled maps, odes, pdes, or something else. as with stochastic systems, the network structure encodes which entities of a system interact with each other and which do not. there are numerous interesting deterministic dynamical systems on networksjust incorporate nontrivial connectivity between entities into your favorite deterministic model -although it is worth noting that some stochastic features (e.g., choosing parameter values from a probability distribution or sampling choices of initial conditions) can arise in these models. for concreteness, let's consider the popular setting of coupled oscillators. each node in a network is associated with an oscillator, and we want to examine how network structure affects the collective behavior of the coupled oscillators. it is common to investigate various forms of synchronization (a type of coherent behavior), such that the rhythms of the oscillators adjust to match each other (or to match a subset of the oscillators) because of their interactions [ ] . a variety of methods, such as "master stability functions" [ ] , have been developed to study the local stability of synchronized states and their generalizations [ , ] , such as cluster synchrony [ ] . cluster synchrony, which is related to work on "coupled-cell networks" [ ] , uses ideas from computational group theory to find synchronized sets of oscillators that are not synchronized with other sets of synchronized oscillators. many studies have also examined other types of states, such as "chimera states" [ ] , in which some oscillators behave coherently but others behave incoherently. (analogous phenomena sometimes occur in mathematics departments.) a ubiquitous example is coupled kuramoto oscillators on a network [ , , ] , which is perhaps the most common setting for exploring and developing new methods for studying coupled oscillators. (in principle, one can then build on these insights in studies of other oscillatory systems, such as in applications in neuroscience [ ] .) coupled kuramoto oscillators have been used for modeling numerous phenomena, including jetlag [ ] and singing in frogs [ ] . indeed, a "snowbird" (siam) conference on applied dynamical systems would not be complete without at least several dozen talks on the kuramoto model. in the kuramoto model, each node i has an associated phase θ i (t) ∈ [ , π). in the case of "diffusive" coupling between the nodes , the dynamics of the ith node is governed by the equation where one typically draws the natural frequency ω i of node i from some distribution g(ω), the scalar a i j is an adjacency-matrix entry of an unweighted network, b i j is the coupling strength on oscillator i from oscillator j (so b i j a i j is an element of an adjacency matrix w of a weighted network), and f i j (y) = sin(y) is the coupling function, which depends only on the phase difference between oscillators i and j because of the diffusive nature of the coupling. once one knows the natural frequencies ω i , the model ( ) is a deterministic dynamical system, although there have been studies of coupled kuramoto oscillators with additional stochastic terms [ ] . traditional studies of ( ) and its generalizations draw the natural frequencies from some distribution (e.g., a gaussian or a compactly supported distribution), but some studies of so-called "explosive synchronization" (in which there is an abrupt phase transition from incoherent oscillators to synchronized oscillators) have employed deterministic natural frequencies [ , ] . the properties of the frequency distribution g(ω) have a significant effect on the dynamics of ( ). important features of g(ω) include whether it has compact support or not, whether it is symmetric or asymmetric, and whether it is unimodal or not [ , ] . the model ( ) has been generalized in numerous ways. for example, researchers have considered a large variety of coupling functions f i j (including ones that are not diffusive) and have incorporated an inertia term θ i to yield a second-order kuramoto oscillator at each node [ ] . the latter generalization is important for studies of coupled oscillators and synchronized dynamics in electric power grids [ ] . another noteworthy direction is the analysis of kuramoto model on "graphons" (see, e.g., [ ] ), an important type of structure that arises in a suitable limit of large networks. an increasingly prominent topic in network analysis is the examination of how multilayer network structures -multiple system components, multiple types of edges, co-occurrence and coupling of multiple dynamical processes, and so onaffect qualitative and quantitative dynamics [ , , ] . for example, perhaps certain types of multilayer structures can induce unexpected instabilities or phase transitions in certain types of dynamical processes? there are two categories of dynamical processes on multilayer networks: ( ) a single process can occur on a multilayer network; or ( ) processes on different layers of a multilayer network can interact with each other [ ] . an important example of the first category is a random walk, where the relative speeds and probabilities of steps within layers versus steps between layers affect the qualitative nature of the dynamics. this, in turn, affects methods (such as community detection [ , ] ) that are based on random walks, as well as anything else in which the diffusion is relevant [ , ] . two other examples of the first category are the spread of information on social media (for which there are multiple communication channels, such as facebook and twitter) and multimodal transportation systems [ ] . for instance, a multilayer network structure can induce congestion even when a system without coupling between layers is decongested in each layer independently [ ] . examples of the second category of dynamical process are interactions between multiple strains of a disease and interactions between the spread of disease and the spread of information [ , , ] . many other examples have been studied [ ] , including coupling between oscillator dynamics on one layer and a biased random walk on another layer (as a model for neuronal oscillations coupled to blood flow) [ ] . numerous interesting phenomena can occur when dynamical systems, such as spreading processes, are coupled to each other [ ] . for example, the spreading of one disease can facilitate infection by another [ ] , and the spread of awareness about a disease can inhibit spread of the disease itself (e.g., if people stay home when they are sick) [ ] . interacting spreading processes can also exhibit other fascinating dynamics, such as oscillations that are induced by multilayer network structures in a biological contagion with multiple modes of transmission [ ] and novel types of phase transitions [ ] . a major simplification in most work thus far on dynamical processes on multilayer networks is a tendency to focus on toy models. for example, a typical study of coupled spreading processes may consider a standard (e.g., sir) model on each layer, and it may draw the connectivity pattern of each layer from the same standard random-graph model (e.g., an erdős-rényi model or a configuration model). however, when studying dynamics on multilayer networks, it is particular important in future work to incorporate heterogeneity in network structure and/or dynamical processes. for instance, diseases spread offline but information spreads both offline and online, so investigations of coupled information and disease spread ought to consider fundamentally different types of network structures for the two processes. network structures also affect the dynamics of pdes on networks [ , , , , ] . interesting examples include a study of a burgers equation on graphs to investigate how network structure affects the propagation of shocks [ ] and investigations of reaction-diffusion equations and turing patterns on networks [ , ] . the latter studies exploit the rich theory of laplacian dynamics on graphs (and concomitant ideas from spectral graph theory) [ , ] and examine the addition of nonlinear terms to laplacians on various types of networks (including multilayer ones). a mathematically oriented thread of research on pdes on networks has built on ideas from so-called "quantum graphs" [ , ] to study wave propagation on networks through the analysis of "metric graphs". metric graphs differ from the usual "combinatorial graphs", which in other contexts are usually called simply "graphs". in metric graphs, in addition to nodes and edges, each edge e has a positive length l e ∈ ( , ∞]. for many experimentally relevant scenarios (e.g., in models of circuits of quantum wires [ ] ), there is a natural embedding into space, but metric graphs that are not embedded in space are also appropriate for some applications. as the nomenclature suggests, one can equip a metric graph with a natural metric. if a sequence {e j } m j= of edges forms a path, the length of the path is j l j . the distance ρ(v , v ) between two nodes, v and v , is the minimum path length between them. we place coordinates along each edge, so we can compute a distance between points x and x on a metric graph even when those points are not located at nodes. traditionally, one assumes that the infinite ends (which one can construe as "leads" at infinity, as in scattering theory) of infinite edges have degree . it is also traditional to assume that there is always a positive distance between distinct nodes and that there are no finite-length paths with infinitely many edges. see [ ] for further discussion. to study waves on metric graphs, one needs to define operators, such as the negative second derivative or more general schrödinger operators. this exploits the fact that there are coordinates for all points on the edges -not only at the nodes themselves, as in combinatorial graphs. when studying waves on metric graphs, it is also necessary to impose boundary conditions at the nodes [ ] . many studies of wave propagation on metric graphs have considered generalizations of nonlinear wave equations, such as the cubic nonlinear schrödinger (nls) equation [ ] and a nonlinear dirac equation [ ] . the overwhelming majority of studies in metric graphs (with both linear and nonlinear waves) have focused on networks with a very small number of nodes, as even small networks yield very interesting dynamics. for example, marzuola and pelinovsky [ ] analyzed symmetry-breaking and symmetry-preserving bifurcations of standing waves of the cubic nls on a dumbbell graph (with two rings attached to a central line segment and kirchhoff boundary conditions at the nodes). kairzhan et al. [ ] studied the spectral stability of half-soliton standing waves of the cubic nls equation on balanced star graphs. sobirov et al. [ ] studied scattering and transmission at nodes of sine-gordon solitons on networks (e.g., on a star graph and a small tree). a particularly interesting direction for future work is to study wave dynamics on large metric graphs. this will help extend investigations, as in odes and maps, of how network structures affect dynamics on networks to the realm of linear and nonlinear waves. one can readily formulate wave equations on large metric graphs by specifying relevant boundary conditions and rules at each junction. for example, joly et al. [ ] recently examined wave propagation of the standard linear wave equation on fractal trees. because many natural real-life settings are spatially embedded (e.g., wave propagation in granular materials [ , ] and traffic-flow patterns in cities), it will be particularly valuable to examine wave dynamics on (both synthetic and empirical) spatially-embedded networks [ ] . therefore, i anticipate that it will be very insightful to undertake studies of wave dynamics on networks such as random geometric graphs, random neighborhood graphs, and other spatial structures. a key question in network analysis is how different types of network structure affect different types of dynamical processes [ ] , and the ability to take a limit as model synthetic networks become infinitely large (i.e., a thermodynamic limit) is crucial for obtaining many key theoretical insights. dynamics of networks and dynamics on networks do not occur in isolation; instead, they are coupled to each other. researchers have studied the coevolution of network structure and the states of nodes and/or edges in the context of "adaptive networks" (which are also known as "coevolving networks") [ , ] . whether it is sensible to study a dynamical process on a time-independent network, a temporal network with frozen (or no) node or edge states, or an adaptive network depends on the relative time scales of the dynamics of network structure and the states of nodes and/or edges of a network. see [ ] for a brief discussion. models in the form of adaptive networks provide a promising mechanistic approach to simultaneously explain both structural features (e.g., degree distributions and temporal features (e.g., burstiness) of empirical data [ ] . incorporating adaptation into conventional models can produce extremely interesting and rich dynamics, such as the spontaneous development of extreme states in opinion models [ ] . most studies of adaptive networks that include some analysis (i.e., that go beyond numerical computations) have employed rather artificial adaption rules for adding, removing, and rewiring edges. this is relevant for mathematical tractability, but it is important to go beyond these limitations by considering more realistic types of adaptation and coupling between network structure (including multilayer structures, as in [ ] ) and the states of nodes and edges. when people are sick, they stay home from work or school. people also form and remove social connections (both online and offline) based on observed opinions and behaviors. to study these ideas using adaptive networks, researchers have coupled models of biological and social contagions with time-dependent networks [ , ] . an early example of an adaptive network of disease spreading is the susceptibleinfected (si) model in gross et al. [ ] . in this model, susceptible nodes sometimes rewire their incident edges to "protect themselves". suppose that we have an n-node network with a constant number of undirected edges. each node is either susceptible (i.e., of type s) or infected (i.e., of type i). at each time step, and for each edge -so-called "discordant edges" -between nodes of different types, the susceptible node becomes infected with probability λ. for each discordant edge, with some probability κ, the incident susceptible node breaks the edge and rewires to some other susceptible node. this is a "rewire-to-same" mechanism, to use the language from some adaptive opinion models [ , ] . (in this model, multi-edges and selfedges are not allowed.) during each time step, infected nodes can also recover to become susceptible again. gross et al. [ ] studied how the rewiring probability affects the "basic reproductive number", which measures how many secondary infections on average occur for each primary infection [ , , ] . this scalar quantity determines the size of a critical infection probability λ * to maintain a stable epidemic (as determined traditionally using linear stability analysis of an endemic state). a high rewiring rate can significantly increase λ * and thereby significantly reduce the prevalence of a contagion. although results like these are perhaps intuitively clear, other studies of contagions on adaptive networks have yielded potentially actionable (and arguably nonintuitive) insights. for example, scarpino et al. [ ] demonstrated using an adaptive compartmental model (along with some empirical evidence) that the spread of a disease can accelerate when individuals with essential societal roles (e.g., health-care workers) become ill and are replaced with healthy individuals. another type of model with many interesting adaptive variants are opinion models [ , ] , especially in the form of generalizations of classical voter models [ ] . voter dynamics were first considered in the s by clifford and sudbury [ ] as a model for species competition, and the dynamical process that they introduced was dubbed "the voter model" by holley and liggett shortly thereafter [ ] . voter dynamics are fun and are popular to study [ ] , although it is questionable whether it is ever possible to genuinely construe voter models as models of voters [ ] . holme and newman [ ] undertook an early study of a rewire-to-same adaptive voter model. inspired by their research, durrett et al. [ ] compared the dynamics from two different types of rewiring in an adaptive voter model. in each variant of their model, one considers an n-node network and supposes that each node is in one of two states. the network structure and the node states coevolve. pick an edge uniformly at random. if this edge is discordant, then with probability − κ, one of its incident nodes adopts the opinion state of the other. otherwise, with complementary probability κ, a rewiring action occurs: one removes the discordant edge, and one of the associated nodes attaches to a new node either through a rewire-to-same mechanism (choosing uniformly at random among the nodes with the same opinion state) or through a "rewire-to-random" mechanism (choosing uniformly at random among all nodes). as with the adaptive si model in [ ] , self-edges and multi-edges are not allowed. the models in [ ] evolve until there are no discordant edges. there are several key questions. does the system reach a consensus (in which all nodes are in the same state)? if so, how long does it take to converge to consensus? if not, how many opinion clusters (each of which is a connected component, perhaps interpretable as an "echo chamber", of the final network) are there at steady state? how long does it take to reach this state? the answers and analysis are subtle; they depend on the initial network topology, the initial conditions, and the specific choice of rewiring rule. as with other adaptive network models, researchers have developed some nonrigorous theory (e.g., using mean-field approximations and their generalizations) on adaptive voter models with simplistic rewiring schemes, but they have struggled to extend these ideas to models with more realistic rewiring schemes. there are very few mathematically rigorous results on adaptive voter models, although there do exist some, under various assumptions on initial network structure and edge density [ ] . researchers have generalized adaptive voter models to consider more than two opinion states [ ] and more general types of rewiring schemes [ ] . as with other adaptive networks, analyzing adaptive opinion models with increasingly diverse types of rewiring schemes (ideally with a move towards increasing realism) is particularly important. in [ ] , yacoub kureh and i studied a variant of a voter model with nonlinear rewiring (where the probability that a node rewires or adopts is a function of how well it "fits in" within its neighborhood), including a "rewire-tonone" scheme to model unfriending and unfollowing in online social networks. it is also important to study adaptive opinion models with more realistic types of opinion dynamics. a promising example is adaptive generalizations of bounded-confidence models (see the introduction of [ ] for a brief review of bounded-confidence models), which have continuous opinion states, with nodes interacting either with nodes or with other entities (such as media [ ] ) whose opinion is sufficiently close to theirs. a recent numerical study examined an adaptive bounded-confidence model [ ] ; this is an important direction for future investigations. it is also interesting to examine how the adaptation of oscillators -including their intrinsic frequencies and/or the network structure that couples them to each other -affects the collective behavior (e.g., synchronization) of a network of oscillators [ ] . such ideas are useful for exploring mechanistic models of learning in the brain (e.g., through adaptation of coupling between oscillators to produce a desired limit cycle [ ] ). one nice example is by skardal et al. [ ] , who examined an adaptive model of coupled kuramoto oscillators as a toy model of learning. first, we write the kuramoto system as where f i j is a π-periodic function of the phase difference between oscillators i and j. one way to incorporate adaptation is to define an "order parameter" r i (which, in its traditional form, quantifies the amount of coherence of the coupled kuramoto oscillators [ ] ) for the ith oscillator by and to consider the following dynamical system: where re(ζ) denotes the real part of a quantity ζ and im(ζ) denotes its imaginary part. in the model ( ), λ d denotes the largest positive eigenvalue of the adjacency matrix a, the variable z i (t) is a time-delayed version of r i with time parameter τ (with τ → implying that z i → r i ), and z * i denotes the complex conjugate of z i . one draws the frequencies ω i from some distribution (e.g., a lorentz distribution, as in [ ] ), and we recall that b i j is the coupling strength on oscillator i from oscillator j. the parameter t gives an adaptation time scale, and α ∈ r and β ∈ r are parameters (which one can adjust to study bifurcations). skardal et al. [ ] interpreted scenarios with β > as "hebbian" adaptation (see [ ] ) and scenarios with β < as anti-hebbian adaptation, as they observed that oscillator synchrony is promoted when β > and inhibited when β < . most studies of networks have focused on networks with pairwise connections, in which each edge (unless it is a self-edge, which connects a node to itself) connects exactly two nodes to each other. however, many interactions -such as playing games, coauthoring papers and other forms of collaboration, and horse racesoften occur between three or more entities of a network. to examine such situations, researchers have increasingly studied "higher-order" structures in networks, as they can exert a major influence on dynamical processes. perhaps the simplest way to account for higher-order structures in networks is to generalize from graphs to "hypergraphs" [ ] . hypergraphs possess "hyperedges" that encode a connection between on arbitrary number of nodes, such as between all coauthors of a paper. this allows one to make important distinctions, such as between a k-clique (in which there are pairwise connections between each pair of nodes in a set of k nodes) and a hyperedge that connects all k of those nodes to each other, without the need for any pairwise connections. one way to study a hypergraph is as a "bipartite network", in which nodes of a given type can be adjacent only to nodes of another type. for example, a scientist can be adjacent to a paper that they have written [ ] , and a legislator can be adjacent to a committee on which they sit [ ] . it is important to generalize ideas from graph theory to hypergraphs, such as by developing models of random hypergraphs [ , , ]. another way to study higher-order structures in networks is to use "simplicial complexes" [ , , ] . a simplicial complex is a space that is built from a union of points, edges, triangles, tetrahedra, and higher-dimensional polytopes (see fig. d ). simplicial complexes approximate topological spaces and thereby capture some of their properties. a p-dimensional simplex (i.e., a p-simplex) is a p-dimensional polytope that is the convex hull of its p + vertices (i.e., nodes). a simplicial complex k is a set of simplices such that ( ) every face of a simplex from s is also in s and ( ) the intersection of any two simplices σ , σ ∈ s is a face of both σ and σ . an increasing sequence k ⊂ k ⊂ · · · ⊂ k l of simplicial complexes forms a filtered simplicial complex; each k i is a subcomplex. as discussed in [ ] and references therein, one can examine the homology of each subcomplex. in studying the homology of a topological space, one computes topological invariants that quantify features of different dimensions [ ] . one studies "persistent homology" (ph) of a filtered simplicial complex to quantify the topological structure of a data set (e.g., a point cloud) across multiple scales of such data. the goal of such "topological data analysis" (tda) is to measure the "shape" of data in the form of connected components, "holes" of various dimensionality, and so on [ ] . from the perspective of network analysis, this yields insight into types of large-scale structure that complement traditional ones (such as community structure). see [ ] for a friendly, nontechnical introduction to tda. a natural goal is to generalize ideas from network analysis to simplicial complexes. important efforts include generalizing configuration models of random graphs [ ] to random simplicial complexes [ , ] ; generalizing well-known network growth mechanisms, such as preferential attachment [ ] ; and developing geometric notions, like curvature, for networks [ ] . an important modeling issue when studying higher-order network data is the question of when it is more appropriate (or convenient) to use the formalisms of hypergraphs or simplicial complexes. the computation of ph has yielded insights on a diverse set of models and applications in network science and complex systems. examples include granular materials [ , ] , functional brain networks [ , ] , quantification of "political islands" in voting data [ ] , percolation theory [ ] , contagion dynamics [ ] , swarming and collective behavior [ ] , chaotic flows in odes and pdes [ ] , diurnal cycles in tropical cyclones [ ] , and mathematics education [ ] . see the introduction to [ ] for pointers to numerous other applications. most uses of simplicial complexes in network science and complex systems have focused on tda (especially the computation of ph) and its applications [ , , ] . in this chapter, however, i focus instead on a somewhat different (and increasingly popular) topic: the generalization of dynamical processes on and of networks to simplicial complexes to study the effects of higher-order interactions on network dynamics. simplicial structures influence the collective behavior of the dynamics of coupled entities on networks (e.g., they can lead to novel bifurcations and phase transitions), and they provide a natural approach to analyze p-entity interaction terms, including for p ≥ , in dynamical systems. existing work includes research on linear diffusion dynamics (in the form of hodge laplacians, such as in [ ] ) and generalizations of a variety of other popular types of dynamical processes on networks. given the ubiquitous study of coupled kuramoto oscillators [ ] , a sensible starting point for exploring the impact of simultaneous coupling of three or more oscillators on a system's qualitative dynamics is to study a generalized kuramoto model. for example, to include both two-entity ("two-body") and three-entity interactions in a model of coupled oscillators on networks, we write [ ] x where f i describes the dynamics of oscillator i and the three-oscillator interaction term w i jk includes two-oscillator interaction terms w i j (x i , x j ) as a special case. an example of n coupled kuramoto oscillators with three-term interactions is [ ] where we draw the coefficients a i j , b i j , c i jk , α i j , α i j , α i jk , α i jk from various probability distributions. including three-body interactions leads to a large variety of intricate dynamics, and i anticipate that incorporating the formalism of simplicial complexes will be very helpful for categorizing the possible dynamics. in the last few years, several other researchers have also studied kuramoto models with three-body interactions [ , , ] . a recent study [ ] , for example, discovered a continuum of abrupt desynchronization transitions with no counterpart in abrupt synchronization transitions. there have been mathematical studies of coupled oscillators with interactions of three or more entities using methods such as normal-form theory [ ] and coupled-cell networks [ ] . an important point, as one can see in the above discussion (which does not employ the mathematical formalism of simplicial complexes), is that one does not necessarily need to explicitly use the language of simplicial complexes to study interactions between three or more entities in dynamical systems. nevertheless, i anticipate that explicitly incorporating the formalism of simplicial complexes will be useful both for studying coupled oscillators on networks and for other dynamical systems. in upcoming studies, it will be important to determine when this formalism helps illuminate the dynamics of multi-entity interactions in dynamical systems and when simpler approaches suffice. several recent papers have generalized models of social dynamics by incorporating higher-order interactions [ , , , ] . for example, perhaps somebody's opinion is influenced by a group discussion of three or more people, so it is relevant to consider opinion updates that are based on higher-order interactions. some of these papers use some of the terminology of simplicial complexes, but it is mostly unclear (except perhaps for [ ] ) how the models in them take advantage of the associated mathematical formalism, so arguably it often may be unnecessary to use such language. nevertheless, these models are very interesting and provide promising avenues for further research. petri and barrat [ ] generalized activity-driven models to simplicial complexes. such a simplicial activity-driven (sad) model generates time-dependent simplicial complexes, on which it is desirable to study dynamical processes (see section ), such as opinion dynamics, social contagions, and biological contagions. the simplest version of the sad model is defined as follows. • each node i has an activity rate a i that we draw independently from a distribution f(x). • at each discrete time step (of length ∆t), we start with n isolated nodes. each node i is active with a probability of a i ∆t, independently of all other nodes. if it is active, it creates a (p − )-simplex (forming, in network terms, a clique of p nodes) with p − other nodes that we choose uniformly and independently at random (without replacement). one can either use a fixed value of p or draw p from some probability distribution. • at the next time step, we delete all edges, so all interactions have a constant duration. we then generate new interactions from scratch. this version of the sad model is markovian, and it is desirable to generalize it in various ways (e.g., by incorporating memory or community structure). iacopini et al. [ ] recently developed a simplicial contagion model that generalizes an si process on graphs. consider a simplicial complex k with n nodes, and associate each node i with a state x i (t) ∈ { , } at time t. if x i (t) = , node i is part of the susceptible class s; if x i (t) = , it is part of the infected class i. the density of infected nodes at time t is ρ(t) = n n i= x i (t). suppose that there are d parameters , . . . , d (with d ∈ { , . . . , n − }), where d represents the probability per unit time that a susceptible node i that participates in a d-dimensional simplex σ is infected from each of the faces of σ, under the condition that all of the other nodes of the face are infected. that is, is the probability per unit time that node i is infected by an adjacent node j via the edge (i, j). similarly, is the probability per unit time that node i is infected via the -simplex (i, j, k) in which both j and k are infected, and so on. the recovery dynamics, in which an infected node i becomes susceptible again, proceeds as in the sir model that i discussed in section . . one can envision numerous interesting generalizations of this model (e.g., ones that are inspired by ideas that have been investigated in contagion models on graphs). the study of networks is one of the most exciting and rapidly expanding areas of mathematics, and it touches on myriad other disciplines in both its methodology and its applications. network analysis is increasingly prominent in numerous fields of scholarship (both theoretical and applied), it interacts very closely with data science, and it is important for a wealth of applications. my focus in this chapter has been a forward-looking presentation of ideas in network analysis. my choices of which ideas to discuss reflect their connections to dynamics and nonlinearity, although i have also mentioned a few other burgeoning areas of network analysis in passing. through its exciting combination of graph theory, dynamical systems, statistical mechanics, probability, linear algebra, scientific computation, data analysis, and many other subjects -and through a comparable diversity of applications across the sciences, engineering, and the humanities -the mathematics and science of networks has plenty to offer researchers for many years. congestion induced by the structure of multiplex networks tie-decay temporal networks in continuous time and eigenvector-based centralities multilayer networks in a nutshell multilayer networks in a nutshell temporal and structural heterogeneities emerging in adaptive temporal networks synchronization in complex networks mathematical frameworks for oscillatory network dynamics in neuroscience turing patterns in multiplex networks morphogenesis of spatial networks evolving voter model on dense random graphs generative benchmark models for mesoscale structure in multilayer networks birth and stabilization of phase clusters by multiplexing of adaptive networks network geometry with flavor: from complexity to quantum geometry chaos in generically coupled phase oscillator networks with nonpairwise interactions topology of random geometric complexes: a survey explosive transitions in complex networksÕ structure and dynamics: percolation and synchronization factoring and weighting approaches to clique identification mathematical models in population biology and epidemiology how does active participation effect consensus: adaptive network model of opinion dynamics and influence maximizing rewiring anatomy of a large-scale hypertextual web search engine a model for the influence of media on the ideology of content in online social networks frequency-based brain networks: from a multiplex network to a full multilayer description statistical physics of social dynamics bootstrap percolation on a bethe lattice configuration models of random hypergraphs annotated hypergraphs: models and applications hebbian learning architecture and evolution of semantic networks in 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fractal trees synergistic effects in threshold models on networks hipsters on networks: how a minority group of individuals can lead to an antiestablishment majority drift of spectrally stable shifted states on star graphs maximizing the spread of influence through a social network second look at the spread of epidemics on networks centrality prediction in dynamic human contact networks mathematics of epidemics on networks multilayer networks authoritative sources in a hyperlinked environment dynamics of multifrequency oscillator communities finite-size-induced transitions to synchrony in oscillator ensembles with nonlinear global coupling pattern formation in multiplex networks quantifying force networks in particulate systems quantum graphs: i. some basic structures fitting in and breaking up: a nonlinear version of coevolving voter models from networks to optimal higher-order models of complex systems hawkes processes complex spreading phenomena in social systems: influence and contagion in real-world social networks wave mitigation in ordered networks of granular chains centrality metric for dynamic networks control principles of complex networks resynchronization of circadian oscillators and the east-west asymmetry of jet-lag transitivity reinforcement in the coevolving voter model ground state on the dumbbell graph random walks and diffusion on networks the nonlinear heat equation on dense graphs and graph limits multi-stage complex contagions opinion formation and distribution in a bounded-confidence model on various networks network motifs: simple building blocks of complex networks portrait of political polarization six susceptible-infected-susceptible models on scale-free networks a network-based dynamical ranking system for competitive sports community structure in time-dependent, multiscale, and multiplex networks multi-body interactions and non-linear consensus dynamics on networked systems scientific collaboration networks. i. network construction 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avjsqu authors: davies, jennifer l title: using transcranial magnetic stimulation to map the cortical representation of lower-limb muscles date: - - journal: biorxiv doi: . / sha: doc_id: cord_uid: avjsqu the aim of this study was to evaluate the extent to which transcranial magnetic stimulation (tms) can identify discrete cortical representation of lower-limb muscles in healthy individuals. data were obtained from young healthy adults ( women, four men; mean [sd] age . [ . ] years). motor evoked potentials were recorded from the resting vastus medialis, rectus femoris, vastus lateralis, medial and lateral hamstring, and medial and lateral gastrocnemius muscles on the right side of the body using bipolar surface electrodes. tms was delivered through a -mm double-cone coil at sites over the left hemisphere. location and size of the cortical representation and the number of discrete peaks were quantified for each muscle. within the quadriceps muscle group there was a main effect of muscle on anterior-posterior centre of gravity (p = . ), but the magnitude of the difference was very small. within the quadriceps there was a main effect of muscle on medial-lateral hotspot (p = . ) and map volume (p = . ), but no post-hoc tests were significant. the topography of each lower-limb muscle was complex, displaying multiple peaks that were present across the stimulation grid, and variable across individuals. the results of this study indicate that tms delivered with a -mm double-cone coil could not reliably identify discrete cortical representations of resting lower-limb muscles when responses were measured using bipolar surface electromyography. the characteristics of the cortical representation of lower-limb muscles reported here provide a basis against which to evaluate cortical reorganisation in clinical populations. transcranial magnetic stimulation (tms) can be used to study the representation of muscles within the primary motor cortex. although the extent of somatotopy within the primary motor cortex is debated (donoghue et al. ; schieber ; schellekens et al. ) , tms has revealed alterations in the cortical representation of muscles in several clinical conditions (liepert et al. ; schwenkreis et al. schwenkreis et al. , tsao et al. tsao et al. , b schabrun et al. schabrun et al. , te et al. ) . this suggests that tms can identify clinically meaningful differences in cortical representation between groups of individuals. the majority of work on the representation of muscles within the primary motor cortex has been conducted for muscles of the hand and upper limb. for the lower limb, tms has been used to quantify the size and the amplitude-weighted centre (centre of gravity [cog] ) of the cortical representation of the resting tibialis anterior muscle (liepert et al. ; lotze et al. ) , the resting quadriceps femoris muscle (schwenkreis et al. ) , the resting vastus lateralis muscle (al sawah et al. ) , and the active rectus femoris (ward et al. b (ward et al. , a te et al. ) and vastii (te et al. ) muscles. no studies have reported the representation of the hamstring or gastrocnemius muscles. although tms has revealed discrete cortical representation of the deep and superficial fascicles of the paraspinal muscles (tsao et al. a) , the extent to which tms can be used to identify discrete cortical representation of lower-limb muscles is unclear. only one study has quantified the representation of multiple lower-limb muscles in the same individuals. in statistical analysis, there was no main effect of muscle, suggesting similar cortical representation of the active rectus femoris and vastii muscles (te et al. ) . however, the separation between the representation of the three muscles was smaller in individuals with patellofemoral pain than in healthy controls, suggesting that separation between muscles might be a measure of interest (te et al. ) . this supports findings in other chronic musculoskeletal pain conditions, where there was reduced distinction in the cortical representation of two back muscles in individuals with low-back pain (tsao et al. b) , and of two forearm extensor muscles in individuals with elbow pain (schabrun et al. ) . the extent to which tms can be used to identify discrete cortical representation of lower-limb muscles therefore warrants further investigation. in addition, recent studies have identified multiple discrete peaks in the cortical representation of a muscle (schabrun et al. ; te et al. ), but no normative data exists on this measure for the representation of lower-limb muscles. the aim of this study was to evaluate the extent to which tms can identify discrete cortical representation of lower-limb muscles in healthy individuals. cortical representation was mapped for seven resting lower-limb muscles involved in control of the knee joint (rectus femoris, vastus lateralis, vastus medialis, medial hamstring, lateral hamstring, medial gastrocnemius, and lateral gastrocnemius) and was quantified using size, cog, hotspot and number of discrete peaks. these measures were compared between muscles from the same group (quadricep, hamstring, plantar flexor) to evaluate the extent to which tms can identify discrete cortical representation of lower-limb muscles. these data describe the characteristics of the cortical representation of lower-limb muscles in healthy individuals and provide a basis against which to evaluate reorganisation in clinical populations. this study was carried out at the cardiff university brain research imaging centre and was approved by the cardiff university school of psychology ethics committee. a convenience sample of young healthy adults ( women, five men; mean [sd] age . [ . ] years) was recruited from an existing participant database and advertisements placed around cardiff university. all participants were screened for contraindications to tms (including history of seizures, neurological injury or head injury) and to ensure that they met the following inclusion criteria: no recent, recurring or chronic pain in any part of the body, no history of surgery in the lower limbs, and not taking any psychiatric or neuroactive medications. the full screening questionnaire is available on the open science framework (https://osf.io/npvwu/). all participants reported that they were right-leg dominant, defined by the leg they would use to kick a ball. all participants attended a single testing session between january and march , and provided written informed consent prior to the start of the experiment. participants were instructed to have a good night's sleep the night prior to the experiment, not to consume recreational drugs or more than three units of alcohol on the day of or night prior to the experiment, and not to consume more than two caffeinated drinks in the two hours prior to the experiment. surface electrodes (kendall series; covidien, ma) were placed on the following muscles of the right leg according to the seniam project guidelines (http://seniam.org/): rectus femoris, vastus lateralis, vastus medialis, medial hamstring (semitendinosus), lateral hamstring (biceps femoris), medial gastrocnemius, and lateral gastrocnemius. prior to electrode placement the skin was prepared with exfoliant and alcohol swabs. data were passed through a humbug noise eliminator (digitimer, hertfordshire, uk) and a d / amplifier (digitimer) where they were amplified x and bandpass filtered ( - hz; (groppa et al. ) ) before being sampled at samples per second in signal software (version ; cambridge electronic designs, cambridge, uk). electromyography (emg) data were stored for offline analysis and viewed in real time using signal software. tms was delivered through a -mm double-cone coil (magstim, whitland, uk) using a single-pulse monophasic stimulator ( , magstim). throughout the experiment, a neuronavigation system (brainsight tms navigation, rogue resolutions, cardiff, uk) was used to track the position of the coil relative to the participant's head. the vertex was identified as the intersection of the interaural line and the line connecting the nasion and inion. participants were seated and chair height and arm rests were adjusted to optimise comfort. the coil was placed slightly lateral to the vertex, over the left hemisphere. stimuli were delivered and stimulus intensity was gradually increased until motor evoked potentials (meps) were observed in the emg data. participants were instructed to stay relaxed, and this was confirmed by visual inspection of the emg data in real time. for each participant, a stimulus intensity was selected that elicited consistent meps in all muscles, but that would be tolerable for the remainder of the experiment. in two participants (one woman, one man) it was not possible to elicit meps on resting muscles with a tolerable stimulus intensity, and these participants did not participate further. in the remaining participants, the mean (sd) selected stimulation intensity was ( . )% maximum stimulator output (range, - % maximum stimulator output). the neuronavigation software was used to project a x cm grid with -cm spacings over the left hemisphere of a representation of the skull that was visible to the experimenter on a monitor. the front-rightmost corner of the grid was positioned cm to the right and cm anterior to the vertex, and the back-leftmost corner was cm to the left and cm posterior to the vertex (figure ). this resulted in a total of grid sites. the target grid was centred slightly anterior to the vertex based on previous reports that the cog of lower-limb muscles is anterior to the vertex (schwenkreis et al. ; al sawah et al. ; ward et al. b ward et al. , a te et al. ). the target grid was designed to cover a large area to capture the boundaries of the cortical representations. the order of the targets was randomised and each target was stimulated once at the predetermined stimulus intensity. the inter-stimulus interval was at least s. a break of ~ min was then taken, before this was repeated. the purpose of this break was to avoid long blocks of stimuli during which the participant's attention or level of arousal might decline. five sets of stimuli were performed in total. at each target, the experimenter viewed real-time information on the position of the coil and the error from the target position and did not stimulate until there was < mm error in coil position. the position of the coil was recorded for each stimulation. the same experimenter (jd) performed tms for all participants. all processing and analyses were performing using custom-written code in matlab (versions a and a, mathworks, natick, ma, usa). all code used to process and analyse the data is available on the open science framework (https://osf.io/qrsp /). background emg was quantified as mean absolute emg in the ms prior to stimulus onset. trials were automatically discarded if if there was background muscle activity, defined as background emg greater than three median absolute deviations above the median background emg from all trials for that muscle. trials were also excluded if the stimulus was delivered > mm from the target. emg data from the remaining trials were visualised and trials were manually excluded if there were visible artefacts. emg traces for all muscles of all participants (https://osf.io/ k p/), and the raw data on which all processing and analyses were performed (https://osf.io/y uj/) are available on the open science framework (doi . /osf.io/e nmk). emg data from the remaining trials were averaged across all stimuli at each scalp site. planned analysis was that the amplitude of the mep be quantified as the peak-to-peak amplitude of the emg signal between and ms after stimulus onset. in three participants, this window included the beginning of a second mep and was, a posteriori, shortened to finish at ms after stimulus onset. visual inspection of emg data confirmed that the windows captured the mep for all participants and muscles. within each participant and each muscle, mep amplitude was scaled to peak mep amplitude (tsao et al. (tsao et al. , b . map volume was calculated as the sum of scaled mep amplitudes across all sites (te et al. ) . discrete peaks were identified if the scaled mep amplitude at a grid site was greater than %, was at least % greater than the scaled mep amplitude at all but one of the surrounding grid sites, and was not adjacent to another peak (schabrun et al. ; te et al. ). the location of each grid site was expressed relative to the vertex. the scaled mep amplitude, and the medial-lateral (ml) and anterior-posterior (ap) coordinates of the grid site were spline interpolated in two dimensions to obtain a resolution of one millimetre for each axis (borghetti et al. ; ward et al. a ward et al. , b . the interpolated data were used to create a topographical map and calculate cog. cog is the amplitude-weighted indication of map position, and was calculated using the following formulae: is the medial-lateral location of the grid site, is the anterior-posterior location of the grid site, and ‫ݖ‬ is the scaled amplitude of the mep at that grid site. for each outcome and each muscle, the distribution of the data was evaluated using visual the following analyses were conceived and performed after the data had been viewed. cog can be influenced by the presence of multiple discrete peaks in the topography. the stimulation location that elicited the largest mep (hotspot) was quantified as an additional measure of the cortical representation. for each participant, the onset latency of the mep was determined for each stimulation site. latency was initially defined as the first point after stimulation at which the full-wave rectified emg signal was more than five median absolute deviations above the median fullwave rectified emg signal in the ms prior to stimulation and stayed above this threshold for at least ms. the full-wave rectified emg from each simulation site was then visually inspected to ensure that latency was accurately identified. latency was averaged across four stimulation sites (from midline to -cm lateral to midline) at the vertex (central), cm anterior to the vertex (anterior), and cm posterior to the vertex (posterior; see figure ). in some muscles of some participants, a second mep was present with a latency of ~ ms. for each participant and muscle, the presence or absence of a late mep was determined by visual inspection of the emg data. the onset latency of this late mep was determined manually for each muscle by clicking a cursor on a graph where the first deviation from ongoing emg was visible. for several muscles, hotspot ml and hotspot ap were different from the normal distribution. within each muscle group, hotspot ml and hotspot ap were compared across muscles using a friedman test (quadriceps) or a wilcoxon signed rank test (hamstrings, plantar flexors). for several muscles, mep latency at central, posterior, and/or anterior stimulation locations were different from the normal distribution. for each muscle, onset latency was compared across stimulation locations using a friedman test. effect sizes were calculated as described above. data were collected from participants ( women, four men; mean [sd] age . [ . ] years). all participants completed the testing session and did not report any adverse effects. in six participants, stimulation at the most anterior row of grid sites was uncomfortable due to large twitches of the facial muscles. in one of these participants and one additional participant, stimulation at the most lateral column of grid sites was also uncomfortable due to large twitches in hand muscles. these grid sites were not stimulated for these participants. in the remaining nine participants all grid sites were stimulated. for one participant (# ), emg data from the medial gastrocnemius were of poor quality. for another participant (# ), emg data from the medial gastrocnemius and the lateral hamstring were of poor quality. for a third participant (# ) emg data from the medial gastrocnemius and medial hamstring were of poor quality. these five muscles (from three participants) were excluded from further analysis. meps were present in all muscles from all participants. cog and hotspot are shown in figure . mep latency, cog, hotspot, map volume and the number of discrete peaks for each muscle are shown in table . within the quadriceps muscle group there was a significant main effect of muscle on cog ap (analysis of variance p = . ), but the effect size was very small (η = . ). post-hoc tests showed that cog ap was more negative (posterior) for vastus medialis than for vastus lateralis (bonferroni corrected p = . ) and rectus femoris (bonferroni corrected p = . ), but the magnitude of this difference was very small (table and figure a ). cog ap was similar for rectus femoris and vastus lateralis (bonferroni corrected p > ). within the quadriceps muscle group there was also a significant main effect of muscle on hotspot ml (friedman test p = . ; w = . ). no post-hoc tests were significant, and there was no clear trend in the data ( figure j ). within the quadriceps muscle group there was a significant main effect of muscle on map volume (analysis of variance p = . , η = . ). no post-hoc tests were significant, but there was a trend for greater map volume in rectus femoris than in vastus medialis (bonferroni corrected p = . ; table ). there was no significant effect of muscle for any other outcome measure in any other muscle group (table ) . topographical maps for all muscles from one participant are shown in figure . topographical maps for the rectus femoris muscle from several participants are shown in figure ). in all muscles, the tendency was for latency to be longer for stimuli delivered at the anterior location than for stimuli delivered at the central and posterior locations. the detailed results of post-hoc tests are provided in table . emg data from the medial and lateral hamstring muscles from one participant are shown in figure . in these muscles, a second mep was present after the primary mep. this late mep was present in the lateral hamstring of five participants, the medial hamstring of four participants, the vastus medialis and rectus femoris of two participants and the vastus lateralis of one participant. the late meps observed in the quadriceps muscles were all very small, whereas those observed in the hamstring muscles could be sizeable (see figure ). the mean (sd) estimated onset latency for the late mep was ( ) ms for the lateral hamstring (n = ), ( ) ms for the medial hamstring (n = ), ( ) ms for the vastus medialis (n = ), ( ) ms for the rectus femoris (n = ) and ms for the vastus lateralis (n = ). in this study i used tms to map the cortical representation of seven resting lower-limb muscles in healthy individuals. the data indicate that the size, cog, hotspot and number of discrete peaks were largely similar across muscles within each group (quadriceps, hamstrings, plantar flexors). there was a statistically significant different in cog ap and hotspot ml across the quadriceps muscles but the effect size and magnitude of differences was very small. the magnitude of the difference means that it would not be practically possible to differentially target one of the three quadriceps muscles with navigated tms. these results indicate that there was considerable overlap in the cortical representations of the lower limb muscles identified by tms and surface emg, and that tms delivered with a double-cone coil could not identify discrete cortical representations of lower-limb muscles when meps were measured with bipolar surface emg. the topography of the seven lower-limb muscle studied here was often complex, displaying multiple peaks that were present across the stimulation grid, and variable across individuals. this may reflect a large and complex anatomical representation of these muscles within the cortex, with considerable inter-individual variability. however, the impact of the techniques used to quantify the cortical representation must also be considered, particularly the volume of cortical tissue excited by the tms and the potential for peripheral volume conduction (crosstalk) in the surface emg recordings. for all muscles, the average cog was located at, or slightly posterior to, the vertex. in addition, despite the large area covered by the target grid, large meps were often observed in response to stimuli delivered at the edge of the grid. this is in contrast to previous mapping studies of the quadriceps muscles, which have reported an anterior cog ( studied active muscles, in contrast to resting muscles studied here. the double-cone coil used in this experiment consists of two circular coils arranged in a figure-of-eight shape with a fixed angle of about degrees between the two wings. the double-cone coil can stimulate deeper regions of the brain than a circular or figure-of-eight coil, which is advantageous for activating corticospinal projections to the lower-limb muscles. it elicits meps at a lower stimulation intensity than a circular coil and a figure-of-eight coil (dharmadasa et al. ). however, this improved stimulation depth is obtained at the expense of focality (lu and ueno ) . the large area of cortical tissue activated by tms delivered through a double-cone coil means that even when the coil is not centred over the motor cortex, excited tissue will likely include the motor cortex. this could explain the apparent expansion of the cortical representation beyond the borders of the target grid, and the larger area of the cortical representation than previously reported (schwenkreis et al. ; al sawah et al. ; ward et al. b ward et al. , a te et al. ) . nonetheless, if current spread was the only relevant factor, the largest mep may still be expected when the stimulating coil was centred over the motor cortex. the discrete peaks in the topographical maps that occurred at or close to the boundaries of the target grid in some participants suggest a complexity that is difficult to describe solely by current spread. it is possible that the double-cone coil excited cortical tissue beyond the motor cortex, and this resulted in meps. the latency of meps observed in response to stimulation at the anterior of the target grid was often slightly longer than that of meps observed in response to stimulation at the centre or posterior of the target grid. this may suggest that a different pathway was involved in the generation of these meps. corticospinal neurones innervating the lower limb spinal motoneurones are present in the premotor cortex (he et al. ) and supplementary motor area, caudal cingulate motor area on the dorsal bank and the rostral cingulate motor area (he et al. ) , as well as the primary motor cortex. however, it is also possible that the longer latency at anterior stimulation sites is an artefact of a smaller mep size at the grid boundary, and this requires further investigation. the figure-of-eight coil provides a very focal stimulation in superficial cortical regions, but minimal stimulation at increasing depths (lu and ueno ) . if much of the cortical representation of lower-limb muscles is inaccessible to the figure-of-eight coil, the resulting topography will appear less complex than that obtained with a double-cone coil. although the double-cone coil will excite deeper cortical tissue, the larger current spread means that it also excites a large volume of cortical tissue at shallow depths. it is possible that the double-cone coil accessed corticospinal neurones that could not be accessed with the figure-of-eight coil, and thus the complexity of the cortical representation reflects the true anatomical complexity that cannot be uncovered with a figure-of-eight coil. high-density surface emg recordings suggest that meps recorded in forearm muscles using conventional surface emg may contain crosstalk from neighbouring muscles (van elswijk et al. ; gallina et al. ; neva et al. (liepert et al. ; lotze et al. ; schwenkreis et al. ; al sawah et al. ; ward et al. a; te et al. ) and clinical (ward et al. b; te et al. ) populations. the current results indicate bipolar surface emg used with tms delivered through a doublecone coil cannot reliably identify discrete cortical representation of lower-limb muscles in young, healthy individuals. if, as has been suggested, the cortical representation of muscles is functional, rather than anatomical (schieber ; ejaz et al. ; leo et al. ) , then requiring the participant to perform a motor task will engage the specific subset of cortical neurones relevant for that task. the cortical representation revealed by tms may then be biased towards the representation for that specific task, at the expense of the representations for other functions the topography incorporated multiple discrete peaks, is not clear. however, the available data suggest that the cortical representations uncovered were smaller and less complex than those revealed in the present study. this suggests that the difference between the current and previous studies cannot be explained by the state of the muscle, and is more likely a function of the stimulating coil. in some muscles in some participants, there was a second mep that occurred with a latency of - ms. this late mep has previously been reported in the resting hamstrings, quadriceps, tibialis anterior and triceps surae muscles, with a latency of ms, ms, ms and ms, respectively (dimitrijević et al. ) , and in resting and active tibialis anterior and triceps surae muscles with a latency of ~ ms (holmgren et al. ). dimitrijevic et al. reported that this late mep was most prevalent in the hamstrings, where it was of higher amplitude than the primary mep. this is in line with the current findings, where the late mep was observed most frequently and with the largest amplitude in the hamstring muscles. the late mep is not exclusive to the lower limbs, and has been observed in resting and active forearm muscles (holmgren et al. ) and a resting, but not active, intrinsic hand muscle (wilson et al. ). the source of the late mep is not known, and could be central or peripheral. indirect corticospinal or cortico-brainstem-spinal pathways, which originate either from the targeted areas of the motor cortex or from wider cortical areas excited by the stimulation, could play a role. proprioceptive information arising from the primary mep could also play a role. based on the latency of responses from several lower-limb muscles, dimitrijevic et al. argued against a segmental or transcortical stretch reflex origin of the late mep, and against the involvement of gamma motor neurones (dimitrijević et al. ) . the difference in the latency of the late mep between upper-and lower-limb muscles is ~ ms greater than the difference in latency of the early, primary mep (holmgren et al. ), lending the possibility that a slow central pathway is involved. recent evidence indicates the primary motor cortex includes slow pyramidal tract neurones (innocenti et al. ) , which comprise the majority of the pyramidal tract but are not well studied (firmin et al. ; kraskov et al. ) . this may provide one such candidate pathway, but this remains to be studied. responses to tms were evaluated using bipolar surface emg, and the results may have been influenced by peripheral volume conduction (crosstalk) from other muscles. studies using high-density surface emg recordings, similar to those conducted in the forearm muscles (van elswijk et al. ; gallina et al. ; neva et al. ) , are required to elucidate the contribution of cross-talk to meps recorded from lower-limb muscles. however, the finding that tms could not identify discrete cortical representations of lower-limb muscles measured with bipolar surface emg is highly relevant, and all tms studies of the lower-limb to date have been performed using bipolar surface emg. tms was delivered through a double-cone coil. the ability of other coil designs, such as the figure-of-eight coil, to identify discrete cortical representations of lower-limb muscles remains to be determined. when using any coil, the volume of cortical tissue excited by the stimulus, and whether this is likely to encompass the full cortical representation of the target muscle, should be considered. this is particularly relevant for flat coils, where the depth of electric field penetration is lower than for the double-cone coil (lu and ueno ) . the current results are particularly relevant in light of a recent study recommending the standard use of the double-cone coil for lower-limb studies, in preference to a figure-of-eight or circular coil (dharmadasa et al. ) . the results of this study indicate that tms delivered with a -mm double-cone coil cannot reliably identify discrete cortical representations of resting lower-limb muscles when meps are measured using bipolar surface emg. the characteristics of the cortical representation of lower-limb muscles reported here provide a basis against which to evaluate cortical reorganisation in clinical populations. target stimulation sites. target stimulation sites were arranged in a x cm grid with -cm spacings. the front-rightmost corner of the grid was positioned cm to the right and cm anterior to the vertex, and the back-leftmost corner was cm to the left and cm posterior to the vertex. grey shading indicates the four stimulation sites (from midline to -cm lateral to midline) at the vertex, cm anterior to the vertex, and cm posterior to the vertex over which motor evoked potential latency was averaged for the exploratory analysis median across all participants. asterisks indicate significant difference (p < . ). late motor evoked potential. surface electromyography data from the medial (a) and lateral (b) hamstring of one participant. a late motor evoked potential is clearly evident with a latency of approximately ms. effect size is kendall's coefficient of concordance. mep, motor 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investigation with high-density surface electromyography symmetric corticospinal excitability and representation of vastus lateralis muscle in right-handed healthy subjects novel adaptations in motor cortical maps: the relation to persistent elbow pain normalizing motor cortex representations in focal hand dystonia detailed somatotopy in primary motor and somatosensory cortex revealed by gaussian population receptive fields constraints on somatotopic organization in the primary motor cortex reorganization in the ipsilateral motor cortex of patients with lower limb amputation assessment of reorganization in the sensorimotor cortex after upper limb amputation ml hotspot (mm)* - the author declares that they have no conflict of interest. key: cord- - lyw gh authors: guzman, efrain; montoya, maria title: contributions of farm animals to immunology date: - - journal: front vet sci doi: . /fvets. . sha: doc_id: cord_uid: lyw gh by their very nature, great advances in immunology are usually underpinned by experiments carried out in animal models and inbred lines of mice. also, their corresponding knock-out or knock-in derivatives have been the most commonly used animal systems in immunological studies. with much credit to their usefulness, laboratory mice will never provide all the answers to fully understand immunological processes. large animal models offer unique biological and experimental advantages that have been and continue to be of great value to the understanding of biological and immunological processes. from the identification of b cells to the realization that γδ t cells can function as professional antigen presenting cells, farm animals have contributed significantly to a better understanding of immunity. great advances in immunology are usually supported by experiments carried out in animal models and by far, inbred lines of mice and their corresponding knock-out or knock-in derivatives, are the most commonly used animal systems in immunological studies. though with much credit to their usefulness, laboratory mice will never provide all the answers to fully understand immunological processes. also, some answers provided in mouse models are not applicable to other species of animals or humans. large animal models offer unique biological and experimental advantages that have been and continue to be of great value to the understanding of biological and immunological processes. the humble cow, the underestimated pig and the unassuming chicken have greatly influenced our current understanding of human immunology. for most immunologists dedicated to fundamental and applied research, it is easy to forget that b cells were first identified in chickens and vaccination first occurred because of a cow. although there are far too many important events to discuss in this paper, we have chosen to highlight a few of the most important contributions of farm animals to the current understanding of immunology ( table ) . edward jenner published in a booklet entitled "an inquiry into the causes and effects of the variolae vaccinae, a disease discovered in some of the western counties of england, particularly gloucestershire and known by the name of cow pox" ( , ) and although strictly speaking jenner did not discover vaccination, he was the first person to use scientific rigor to prove protection from disease through targeted intervention. the english dairy farmer benjamin jesty ( - ) was the first person known to vaccinate against smallpox ( ) protecting his family against the virus even after numerous exposures ( ) . however, the idea and indeed the term vaccination, only came into the light spot years later thanks to louis pasteur. this time the chicken takes a privileged position and the story was beautifully explained by pasteur himself ( , ) and has been romanticized in paul de kruif 's book "microbe hunters" ( ) . in pasteur inoculated chickens with "stale" cultures of pasteurella multocida. the chickens became sick but recovered so he decided to re-inoculate them with a fresh culture. the chickens that had received the "stale" culture recovered whereas chickens that had not been pre-exposed to the stale cultures died. pasteur recognized the similarities between his studies in chickens and what jenner had published with smallpox. he coined the term "vaccine" ( , , ) in honor of jenner. by the early s, william smith greenfield in the uk ( , ) and pasteur working with henri thullier, charles chamberland and pierre paul Émile roux in france ( , ) had begun developing and testing vaccines against anthrax in sheep and cattle. a decade later, the german scientists friedrich loffler and paul frosch identified the first ever filterable infectious agent in mammals: foot and mouth disease virus (fmdv) and developed a fully protective heat-inactivated vaccine against it ( , ) ; however an effective long-lasting and broadly protective vaccine against fmdv remains elusive. pigs also played an important role in early vaccinology studies. by the late s swine plague or hog cholera (later discovered to be caused by a virus now called classical swine fever virus, csfv ( ) was killing hundreds of thousands of pigs across the word and was particularly of concern to the us pig producing industry, causing an impressive us$ million a year in losses in ( ) and us$ million by . once again, pasteur and thullier developed a vaccine to what is now thought to be the first ever vaccine against a viral infectious disease ( ) and the first mass-vaccination campaign in history. in addition, it is rarely recognized that csfv was the first animal virus ever to be cultured in vitro ( ) and the techniques developed by carl tenbroek continue to be used today. horses have also contributed to the understanding of fundamental immunological mechanisms. in a series of experiments, emile roux working with alexandre yersin and followed by emil von behring and shibasaburo kitasato immunized horses to produce an "antidote" or immune sera against the diphtheria toxin that was eventually used to treat humans, an important step in understanding antibodies and humoral immunity ( ) . behring won the nobel prize for medicine in for this work. another milestone in vaccine development was the generation in the 's of vaccines to control marek's disease (md), a naturally occurring neoplastic disease in chickens caused by an oncogenic herpesvirus ( ) . md vaccines are the first examples of the use of vaccination to protect against cancer ( , ) . with the discovery of molecular biology techniques in the 's and 's, the race was on to develop recombinant vaccines against numerous infectious diseases. the first report of a biosynthesized polypeptide vaccine was published in ( ) . the structural protein vp of fmdv was cloned ( ). swine plague. science , and expressed in e. coli and the purified protein used to vaccinate six cattle and two swine, which developed neutralizing antibodies and were protected against challenge with fmdv ( ) . and new technologies have only helped to highlight the importance of farm animals in vaccine development: using a computational approach to assess protein-protein stability, kotecha and colleagues ( ) used molecular dynamic ranking to predict fmdv capsid stabilities and produced stabilized fmdv capsids based on these predictions, assessed their stability using x-ray crystallography and demonstrated their improved immunogenicity in vivo by vaccinating cattle. this demonstrates the potential value of structure-based design of vaccines to develop stabilized vaccine antigens for animals and humans alike. although the innate immune system of animals is largely conserved, there are significant variations in the pattern-recognition-receptor (prr) structures of various species ( ) . it has been suggested that laboratory mice have not been subjected to the selective pressures that other animals have and so innate immune studies carried out in laboratory animals do not accurately inform human biology ( ) . it has been demonstrated that human and farm animal prr responses to their ligands ( , ) are more similar to each other than human-mouse prr responses ( ) ( ) ( ) . because prr recognition is associated with adaptive immunity, a better understanding of these molecules in farm animals is likely to better inform on their effect in these animals as well as humans. a major contribution of the chicken to fundamental innate immunity was the description in of the first interferon. chicken embryos were exposed to influenza virus by alick issacs and jean lindenmann ( ) and they identified an immune soluble element responsible for regulating virus infection. this discovery was certainly one of the scientific landmarks in cell biology in the twentieth century and one which opened the doors of what we now know as innate immunity. perhaps the most recognizable contribution of the chicken to science, and immunology in particular, was in the definition of the two elements of adaptive immunity: the b-dependent and the t-dependent immunity. the avian bursa of fabricius, named after hieronymus fabricius of aquapendente ( ) is a sac-like structure located in the cloacal passage of the bird and its function remained elusive until well into the twentieth century. bruce glick and timothy chang working at the poultry science department at ohio state university ( , ) described how following the surgical removal of the bursa, chickens injected with salmonella typhimurium "o" antigen failed to develop bacteria-specific antibodies. glick and chang wrote a paper entitled: "the role of the bursa of fabricius in antibody production" and was rejected by leading scientific journals ( ) and eventually published in poultry science ( ) . several years later, the bone marrow in mammals was shown to be the equivalent of the bursa in birds ( ) , and so the term "b-lymphocyte" originated from "bursa-derived lymphocyte". several years later, cooper et al. published a fundamental paper on the demarcation of the thymic and bursal and systems in birds and proposed the existence of equivalent systems in mammals ( ) . the cannulation of lymphatic vessels was developed in the early twentieth century in rats to study the lymphatic system but due to the complexity of the surgical procedure, sheep and then cattle were used extensively in the s and s in lymphatic cannulation studies ( ) . in a series of adoptive transfers of lymph-migrating cells and fluid, hall and colleagues first identified in sheep that antibody-secreting cells (acs) encounter antigens and are activated in the lymph nodes ( ), then migrate via the efferent lymphatics to the circulatory system, and that the immune response depends on an intact lymphatic system. cattle have also contributed to fundamental b cell immunology and the generation of a highly diverse antibody repertoire. most vertebrates encode a large number of variable (v), diversity (d), and joining (j) gene segments and antibody diversity is achieved by recombination of these segments. in contrast, cattle only express a limited number of v genes and so it is thought that antibody diversity is achieved recombination events and endogenous mutation mechanisms in the cdr region ( ) . another unusual feature of bovine antibodies is their exceptionally long cdr regions ( ) . these long cdr and unusual mutation mechanisms result in "microfolds" within the cdr region that allow bovine antibodies to bind antigens that would normally be inaccessible ( ) . a recent report demonstrated that cows can be immunized with a single hiv env trimer and this results in potent hivspecific nabs which are dependent on the length of the cdr loops of bovine ig ( ) . it has been suggested that this could be an efficient way of producing super-antibodies against other human pathogens. transchromosomal cows have been engineered to produce large amounts of full human igg molecules with pathogen specificity: mers-cov ( ) , hanta virus ( ), veev ( ) , and ebola virus ( ) . this technology has the potential to generate prophylactic antibodies against emerging viral diseases. on the other hand, chickens have serum igm and iga both of which are homologs of their mammalian counterparts; in addition, they express igy, not found in mammals but thought to be an evolutionary ancestor for mammalian igg and ige. chickens however do not have either ige nor igd but instead use a distinct process for generating antibody diversity that is distinctly different to mammals ( ) . engineers frequently look to nature for inspiration. antibody engineers are no exception, modeling new therapeutics on molecules found in animals such as camels and cows. indeed, % of bovine antibodies have unusually long heavy-chain cdr s as part of their antigen-recognition sites. stanfield et al. ( ) have solved crystal structures of three new bovine fab fragments and analyzed the five known structures to show that their ultra-long cdr h s all adopt similar architectures composed of a knob domain containing a small conserved β-sheet connected by diverse disulfide-bonded loops that is separated from the antibody surface by a long conserved stalk. they propose that varying the length of the stalk and the positions and number of disulphide links in the knob may help drive antibody diversity. these structural insights could be leveraged to tailor antibody-based therapeutics. in contrast to all other mammals, camelids (dromedaries, camels, llamas, etc) also have an unique antibody type similar igg but with identical heavy chains lacking the ch domain and which do not pair with their corresponding light chains. these "heavy-chain antibodies" (hcabs) display antigenspecific variable domains or "vhh" which are structurally and functionally similar to an igg fv but have only three cdr loops defining the antigen biding sites. camel vhh domains, also called "nanobodies, " have been of great interest because of their stability and small size and strong affinity to their corresponding antigens. in fact, several camel vhh domain antibodies are in early preclinical development in oncology, infectious, inflammatory, and neurodegenerative diseases ( ), the most recent example being the generation of broadly neutralizing antibodies to influenza in llamas ( ) . cytotoxic and helper t cells are generally considered to be phenotypically different due to the mutually exclusive expression of the co-receptors cd αβ and cd and differences in mhcrestriction (class i vs. class ii). however, between (in normal individuals) and % (in certain pathologies) of human peripheral blood lymphocytes have been shown to be cd /cd double positive (dp) t cells ( ) . thymic and extra-thymic development of t cells has been studied mainly in mice and because the expression of cd and cd in mouse t cells for the most part mutually exclusive, cd /cd dp lymphocytes have generally been ignored. nevertheless, the presence of cd /cd dp t cells in many animals makes it impossible to ignore these cells. studies in pigs have shown that cd /cd dp are a distinct subset of activated and/or memory t helper cells ( ) and in humans the increase in circulating cd /cd dp t cell frequency has been identified in autoimmune and chronic inflammatory diseases ( ) ( ) ( ) ( ) ( ) suggesting the importance of this particular t cell population in human health. most circulating t cells in humans and mice are conventional t cells expressing the αβ t cell receptor (tcr) and either cd or cd . unlike mice, other species like cattle, pigs and chickens possess a substantial proportion of t cells expressing the γδ tcr cells in the circulation suggesting that circulating γδ tcr t cells have a more important role immunity than previously thought ( ) . the fact that the phenotype and frequency of circulating and tissue resident t cells is so vastly inconsistent in different species suggests that immune responses to (vaccine) antigens are also distinct. it is assumed that that all animal species have a similar immune response to a particular antigen, but this is a statement to be reviewed in light of each host particularities. in addition, the th /th polarization of t cells observed in response to particular antigens is a phenomenon of certain strains of laboratory mice and not of outbred mammals including farm animals and humans ( , ) . in fact, it has been shown that cytokine profiles defining th /th responses to antigens in cattle are more similar to human responses than those observed in mice ( ). dendritic cells (dc) as such, and their role in immunity were first described in the s and in ralph steinman published a series of papers describing that a cellular receptor called "dec- " (now cd ) was expressed on mouse dc, was involved in antigen processing ( , ) and was detected by the monoclonal antibody nldc- . in fact, it was years earlier in , that chris howard, a bovine immunologist working at the then called "institute for animal heath" in the uk published a series of papers identifying an important and until then uncharacterized marker expressed on pseudoafferent lymph veiled cells (also called aldc) detected by the monoclonal antibody wc (now cc ) ( - ). although steinman's identification of mouse cd helped characterize the binding of cc to bovine cd ( ), the importance of cd in identifying dc was first evident in cattle. as mentioned above, steinman's seminal work in characterizing dc using the mouse system has been one of the most important developments in cellular immunology of the twentieth century, and one which lead to his nobel laureate. however, the idea that a component of the immune system was involved in antigen processing and presentation had been proposed many times before. as mentioned above, cannulation of the lymphatic vessels is more practical in large than small animals, and this technique has been used to investigate dc biology. afferent or peripheral lymph dc were first described in sheep in ( ) as "very phagocytic dendritic macrophages that are involved in long term immunological reactions" that are very potent antigen presenting non-lymphoid cells ( ) and that their phagocytic and antigen presentation capacities differed from "classical" peritoneal macrophages ( ) , therefore indicating that dc and macrophages were different cell types ( ) several years before steinman's observations ( ) . in addition, lymphatic cannulation of sheep has revealed important ontologic, phenotypic and functional characteristics of dc subsets that are relevant in other mammals, particularly humans ( , ) . similitudes and differences between swine and human dc/macrophage populations have recently been described ( ) . in one striking example and in contrast to studies performed in mice, swine and human cdc , which are associated with th responses, both express fcεriα and are localized in or next to the tracheal and bronchial epithelia. these observations have been proposed to imply that swine and humans have similar allergen responses as opposed to mice. this theory is supported by the fact that localization of cdc helps them access antigens such as airborne allergens, and fcεriα expression on these cells might help proliferation observed in allergic responses. as mentioned before and unlike mice, horses and humans, most other animals have a large γδ t cell compartment. for example, up to % of all blood lymphocytes in young calves are t cell expressing the γδ t cell receptor (tcr). although the reasons for the enlarged t cell compartment in cattle, pigs and chickens is still unknown, their large numbers and ease of collection has resulted in great advances in γδ t cell biology knowledge not only for farm animals, but also for humans. for example, apcs were shown to influence γδ t cell proliferation ( , ) . cynthia baldwin's lab has defined antigen-specific bovine γδ t cell responses in various systems ( ) ( ) ( ) and adrian smith's lab has done similar observations in chickens ( , ) . it has also been shown that bovine γδ t are potent regulatory t cells ( ) , an observation that is also true for a subset of human γδ t cells ( , ) . these results in farm animals have and continue to enhance our understanding of human γδ t biology ( ) . perhaps the most important one was the realization that a subset of bovine γδ t cells expressed mhc class ii and co-stimulatory molecules on their surface, a characteristic normally attributed to macrophages, b cells and dc but not t cells ( ) . bovine γδ t cells were also shown to phagocyte antigens and of mhc iirestricted presentation to cd + t cells ( ) . this function of bovine γδ t cells was subsequently reported in pigs ( ) and much later in mouse ( ) and human ( ) ( ) ( ) γδ t cells. perhaps the best known contribution of any farm animal to scientific progress was the somatic cell nuclear transfer that gave origin to dolly, the sheep ( ) . although nuclear transfer itself is not a direct contribution to immunology, nuclear transfer technology has directly influenced many immunological concepts underpinned by technologies such as induced pluripotent stem (ips) cells and crispr-cas systems. clustered regularly interspaced short palindromic repeats (crispr) is a rna-guided endonuclease used both in vivo and in vitro. genetically modified animals becoming more common and their availability can be exploited in many applications such as comparative immunology, physiology and disease, to generate in vivo bioreactors to produce complex proteins, or to produce genetically modified organs for transplantation in humans ( ) . although the majority of pharmaceutical research is performed in laboratory mice models, it is clear that humans are not "large mice." by a large extent, studies in laboratory mice have been the victim of over interpretation; for example, by extrapolating successful pre-treatment in mice to therapeutic treatment in men. the weakness of the mouse model in pharmaceutical research was recently highlighted in a study showing that inflammatory responses in mouse models do not correlate with human inflammatory disease ( ) . an additional study showed a close similarity in expression profiles of immune-related genes between humans and pigs ( ) . cattle, pigs, and chickens, are useful, valid, and valuable models to study human infectious diseases and important clinical targets in their own right. both humans and cattle are the natural hosts for tuberculosis (being infected with the geneticallyrelated mycobacterium bovis and mycobacterium tuberculosis, respectively) and the bovine and human diseases share many similarities in terms of immunity and pathology ( ) , whereas the mouse model of tuberculosis does not provide a faithful representation of the disease in humans ( ) . similarly, bovine respiratory syncytial virus (brsv) is closely related to human (h) rsv and the pulmonary pathology, immune responses and epidemiology seen in young calves and children are very similar ( ) . swine have been shown to be a more faithful model for human influenza infection and immunity studies and the same strains of influenza infect both humans and pigs because the distribution of influenza virus receptors and physiopathology are similar in both species. the transfer of maternal-derived antibodies (mda) to new born pigs enables fancy vaccine study design to elucidate the role of mda in immunity ( , ) , vaccine efficacy and in enhancement of respiratory disease ( ) . gnotobiotic piglets have been used to study various human gastrointestinal pathogens. for example, human noroviruses are antigenically and genetically related to swine noroviruses and unlike mice, humans and pigs show genetic susceptibilities to noroviruses depending on their histoblood group antigen phenotypes and the virus strain. similarly, gnotobiotic pigs have been used in rotavirus research to study disease pathogenesis and identify virus-specific iga and asc as correlates for protection and vaccine efficacy in children ( ) . pigs have also been proposed to be better models than mice for many other infectious diseases including female genital infection with chlamydia trachomatis, helycobacter pylori, neisseria meningitides, and nipah virus among others because of the natural susceptibility of pigs to these pathogens ( ) . endogenous retroviruses were first discovered in pig kidney cell lines in ( ) and are now known to be present in most, if not all, mammalians. the presence and potential reactivation of endogenous retroviruses has very important consequences in both allo-and xeno-transplantation. immunotherapy is becoming more popular in clinical trials and vaccine efficacy studies. the success of immune cell therapies partially depends on the effective delivery of cells to target organs, a process that invariably involves the lymphatic system. dc migration in mice has not proven to be very informative, however, dc migration in pigs may be able to answer several question on dc migration that cannot be addressed otherwise. these studies demonstrate that using large animals to investigate immune cell trafficking will help improve immunotherapies in humans ( ) . in the french surgeon mathieu jaboulay ( - ) implanted a pig's kidney into one woman and a goat's liver into another thus starting the idea of xenotransplantation; unsurprisingly, both women died ( ) . the acceptance or rejection of a donor's organ or cells is fundamentally an immunological event. cellular rejection involves nk and t cells that recognize foreign antigens on the grafted tissue. using xenotransplantation models (pig-to-rat, pig-to-primate, and pigto-human), the main mechanism for organ and tissue rejection has been proposed to involve arteriosclerosis, or thickening of the arterial walls. this process if thought to be caused by activated and allo-reactive lymphocytes that migrate over time to the transplanted organ ( ) . arteriosclerosis is a major cause of chronic organ rejection ( ) . studies in laboratory mice have underpinned many concepts of immune tolerance and the generation of immune responses in the neonate. however, the peripheral immune system in mice remains unpopulated during pregnancy and it is only after birth that b and t cells begin to emmigrate to the periphery. in contrast, lymphoid cells circulate through the fetus in humans and large animals well-before birth and specialized lymphoid tissues are also well-developed and populated by the time of birth and are able to respond to a number of antigens ( , ) . certainly the immune system in neonate humans and large animals is not matured, but calves, lambs and piglets can be more useful than mice in understanding immune responses during pregnancy and in new borns and these studies can be used to better inform human developmental immunology. this advantage over mice has recently been used to develop extracorporeal support technologies using neonatal lambs with the ultimate objective to use these technologies in premature children ( ) . perhaps one of the most common uses of large animal models is in the development of vaccines with several advantages over mice. the serial collection of peripheral blood from animals such as pigs, cattle, chickens, and horses allows for immunokinetic studies to be possible in response to vaccination or infection at the level of the individual. these immunokinetic studies can be used to correlate immune responses generated with protection after challenge with the relevant pathogen. in vaccinology studies using mice, the typical approach would be to sacrifice groups of mice sequentially and harvest spleen and blood, so the immune response to vaccination at the individual level is not normally achieved. large animals also provide several advantages over mice when investigating mucosal immunity. when mice are vaccinated or inoculated intranasally, it is common for the inoculum to be digested because anesthetized mice can both swallow and inhale the material placed on their nose. in addition, the structure of the mucosal associated lymphoid tissue (malt) differs significantly in mice from that of large animals and humans; for example mice do not have tonsils but instead have undefined networks of malt, whereas cattle, pigs and sheep have well-defined tonsils ( ) ( ) ( ) . in the case of vaccine delivery through the skin, cattle, and pigs appear to be better suited than mice for these studies. skin thickness, structure of the epidermis and the presence and distribution of langerhan's cells are among many characteristics that humans and cattle and pigs have in common and which are practically relevant in transcutaneous immunizations ( ) . the process of selecting a relevant and appropriate animal model is a balanced and complicated exercise due to the diversity in vertebrate physiology, adaptive and innate immunity. studies in mice, for example, have shown the efficacy of vaccines against fmdv, however these efficacy studies have failed to be translated to the target species (cattle and pigs), presumably due to fundamental differences in the immune systems of model organisms and target species and the ability of the virus to mutate in these animals ( ) . it has recently been shown that because immunoglobulin subclass diversification occurred after speciation ( , ) a particular immunoglobulin subclass in one species bears no functional homology to one of the same name of another species ( ) . thus, our knowledge of the functions of igg in mice cannot be extrapolated to other mammals. characterizing generating reagents for each animal model hinders the development and usefulness of any of these models and therefore limiting the usefulness of cows, cattle or chickens as models for human immunology. mice and rats are and will probably continue to be the chosen model organisms over farm animals. mice can be readily mutated (knock in or knock out) to study immunological pathways; so far this has been proven to be very difficult-and expensive-in large animals. as mentioned above, the availability of reagents to study immune cells and processes in mice far out competes the availability of these reagents for large animals. pharmacokinetic and toxicology studies would be prohibitively expensive in pigs, horses or cattle, so small rodents and rabbits are the best organisms to use in these studies. in addition, studies in mice have been fundamental in the discovery of antibiotics, chemotherapy agents and more recently car-t cell therapies that can be directly applied to humans. genetic homogeneity, low cost, the availability of biologically-relevant mutants and reagents make the mouse the optimal animal model for many academic and industrial researchers. farm animals have historically contributed and continue to contribute to fundamental and applied immunology. the use of these animals in research is not difficult as long as the appropriate facilities and reagents are available. dedicated housing, cost, biosecurity, and genetic variability are some of the many disadvantages confronted when using farm animals in research. however, selecting an appropriate animal model should be more than just a matter of accessibility and common practice ( ) but should be based on the scientific question to be addressed and its relevance. not just a country doctor: edward jenner, scientist edward jenner and the eradication of smallpox new light in the dawn of vaccination on the germ theory on chicken cholera: study of the conditions of non-recidivation and of some other characteristics of this disease microbe hunters the life and works of louis pasteur lectures on some recent investigations into the pathology of infective and contagious diseases professor superintendent, the brown animal sanatory institution 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implications therapeutic administration of broadly neutralizing fi antibody reveals lack of interaction between human igg and pig fc receptors model organisms: there's more to life than rats and flies all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. the authors were funded by the uk's bbsrc grants bbs/e/i/ and bbs/e/i/ . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © guzman and montoya. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - pok authors: nan title: a smartphone magnetometer-based diagnostic test for automatic contact tracing in infectious disease epidemics date: - - journal: ieee access doi: . /access. . sha: doc_id: cord_uid: pok smartphone magnetometer readings exhibit high linear correlation when two phones coexist within a short distance. thus, the detected coexistence can serve as a proxy for close human contact events, and one can conceive using it as a possible automatic tool to modernize the contact tracing in infectious disease epidemics. this paper investigates how good a diagnostic test it would be, by evaluating the discriminative and predictive power of the smartphone magnetometer-based contact detection in multiple measures. based on the sensitivity, specificity, likelihood ratios, and diagnostic odds ratios, we find that the decision made by the smartphone magnetometer-based test can be accurate in telling contacts from no contacts. furthermore, through the evaluation process, we determine the appropriate range of compared trace segment sizes and the correlation cutoff values that we should use in such diagnostic tests. witnessing an alarmingly large number of novel pandemics in this century such as sars, swine flue, mers, ebola, and zika, there has been growing concerns on the ''next big one'' [ ] . many worry that we deal with them using strategies established over a century ago and technology that has been around for decades, with little innovation generated [ ] . consequently, there are calls for technology-based preparedness [ ] , especially in the areas of infection prevention, case finding, case investigation, and contact tracing [ ] . among others, the information technology (it) sector should respond to the calls, and continuously expand and finesse the arsenal of technologies in each of these areas. in this paper, we tackle one of the areas that need the technological revamping: contact tracing. on the brink of an infectious disease epidemic, the most urgent task is to trace those who possibly made contacts with the infected person(s), in order to cut the chain of infection and prevent it from growing into a wider epidemic. but the traditional contact tracing technique has been predominantly analog. namely, contact graphs are constructed through interviews with confirmed cases, by asking who they met and where they visited. this is a hugely costly and time consuming task. worse yet, there is the issue of recall [ ] . meanwhile, recent outbreaks have been fundamentally different from those of the past -highly mobile populations [ ] and the spread into densely populated cities [ ] -which exacerbate the problem with the traditional contact tracing approach. when there are many potential contacts that an infected person cannot identify or recollect as in our typical urban life, a potent tool we can marshal is the mobile devices such as smartphones. the mobile-based epidemic monitoring is nothing but a logical next step because only the mobile devices that move with people can keep up with the contacts they make. indeed, there have been increasing number of proposals for smartphone-based contact tracing. the employed technologies range from similar global positioning system (gps) positions [ ] , similar wi-fi fingerprints [ ] , bluetooth peer discovery [ ] , and identical cells in mobile communication [ ] . unfortunately, they either provide position information too coarse to be used for infectious contact detection [ ] (gps, cellular/wi-fi fingerprinting), require the infrastructure nearby (cellular/wi-fi), cannot be used indoors (gps), consumes too much power for extended monitoring use (gps) [ ] , or could compromise privacy by exposing the identity of the device and eventually its owner (bluetooth beacons). however, some recent works including our earlier pilot study [ ] , [ ] present a new possibility by demonstrating that a magnetometer traces-based approach can detect close contacts. they exploit the fact that the magnetic field strength is rich in spatial features (e.g. m − to . m − ) [ ] due to various distortions by ferromagnetic materials used in buildings such as reinforced concrete and metal doors. the magnetometer-based approach overcomes most of the aforementioned issues. first, thanks to the omnipresent geomagnetic field, the similarity comparison of the two magnetometer traces works both indoors and outdoors, and does not need any infrastructure support. second, it offers better privacy protection by not revealing any identity of the device or the location of the trace generation. third, it detects the coexistence only in close proximity. the lowpower smartphone magnetometers can only be affected by ferromagnetic structures within a few meters [ ] , [ ] . only the co-existing smartphones within this distance can bear sufficient similarity in their magnetometer readings [ ] , [ ] . this last characteristics is especially important as many infectious disease transmissions occur in close distances. public health policies for tracing close contacts or infection control guidance often use a distance of up to meters or feet [ ] . when the disease control authority performs an epidemiological investigation, they can use the smartphone magnetometer traces of the person confirmed infected and of the one suspected of a contact with the infected, in a system depicted in fig. . when people make contacts, they are recorded in their individual phones in the form of similar magnetometer readings. when they want to check if they could have met an infected person, they can ask the system to compare their traces with that of the infected person. since it is a pairwise comparison, it works for the case many people gather at a location. each individual pair from the gathering can be checked using the pairwise comparison method depicted in fig. . in this paper, we assume that it is an emergency situation, and people are cooperating with the disease control authority by downloading an application that records the magnetometer readings and submits it through the phone's cellular connection if necessary. indeed, there are recent efforts that seek such public participation to prepare for the next pandemic outbreak [ ] . in this effort by british broadcasting corporation (bbc), people are encouraged to download an app and activate it for helping model the spreading dynamics in future pandemics. the app then meticulously record the trajectory of the smartphone holder, before it reports the trajectory information to a central server. considering this precedent, our own system model in fig. is not excessively unrealistic. the rationale behind such cooperation from the public could be the fear from the lack of information [ ] . under the depicted scenario, not only the disease control authority but also each individual user can check herself whether or not there has been close contact with an infected person. indeed, world health organization (who) strongly recommends that disease control authorities ensure at-risk populations have the information they need, thereby minimizing social and economic disruption [ ] . although the existing magnetometer-based works have confirmed the feasibility of the idea, they are a far cry from a serious alternative to the traditional contact tracing method. first, many of the operating parameters are still to be determined. in particular, the exiting works [ ] , [ ] , [ ] consider only two extreme and impractical cases: either continuous contact or no contact during the whole duration of comparison. however, when two people, possibly strangers, make a contact, its duration can be very small compared to the entire span of comparison (which can match the most active transmission period of the disease). in fig. , if the infected person a was confirmed infected at time t and the disease transmissible duration is l tx , the similarity measure between the traces r a and r b can be computed low if the duration of contact l l tx . but this will be generally the condition that we will face in reality. therefore, we need to define the window of comparison t w that we slide over the entire trace pair to find any contact (i.e., the similarity measure over a threshold) to make it a valid test method. second, when new diagnostic tests are introduced, it is necessary to evaluate the comparative diagnostic accuracy and feasibility of this new test in comparison to the existing tests or the gold standard [ ] . this ability and diagnostic accuracy can be quantified by calculating various measures such as sensitivity and specificity, positive and negative likelihood ratios, diagnostic odds ratio,etc. in this paper, we address these issues by defining the desirable length of t w , the decision threshold θ c , and by evaluating the quality of the contact diagnosis under these parameter values. as to the nature of the technology we propose in this paper, one can argue that it is only supplementary. in that we believe that the final confirmation about the infection event should be always made by human experts, it is true to a certain degree. it can be used to quickly identify possible contacts with relatively high accuracy, so that the human experts can focus on the most likely ones that have been identified by technology. however, at the same time, the technology covers areas that the traditional method could not. without the technical support, it may be not only costly and time-consuming but impossible in many contact events. first, the authority may not be able to catch up with the speed of spreading when the epidemic is full-blown. second, in many urbanized societies of today, we do not even know or remember those who happen to sit next to us in the bus or train or in a restaurant. in largescale epidemics, the technology can quickly pan out even such contacts that cannot be recovered from the memory of the infected person. in these second sense, the technology will be indispensible. the rest of this paper is organized as follows. in section ii, we briefly summarize the related work that exploits the geomagnetic field strength to detect location and coexistence. in section iii, we first discuss how we measure the similarity of two traces that signals a possible contact. then we identify the parameters that determine the performance of the magnetometer-based contact test, and discuss how we will measure it. in section iv, we evaluate the performance of the test using a set of real-life smartphone magnetometer traces. finally, we conclude the paper in section v. before delving into the discussion, we list the acronyms used throughout the paper in table . there is rich literature on co-presence detection or its use on epidemiology and social studies. in terms of the employed technology, existing works range from sensors to communications to social media. we summarize them below, with brief remarks on their relevance to our problem or the relation to our approach. although the disease transmissibility check in contact tracing needs not necessarily absolute but relative coordinates (i.e., relative to the infected person), one may well consider using gps trajectories to determine the distance of contact. for instance, qi et al. use gps to track and visualize space-time activities for a flu transmission study [ ] . unfortunately, gps is a power-inefficient sensor. as we need to amplify the signal and achieve a high processing gain due to the small received power, a significant reduction in battery time is inevitable. for instance, it can drain a smartphone battery in much less than a typical charge interval even with minimal activity [ ] , [ ] . in attempts to mitigate the problem, we could activate gps only when user movement exceeds the accuracy bound, or turn it off indoors by detecting the condition through other means such as the received signal strength (rss) fingerprints of cell towers. even if the power issue is resolved, however, problems remain. first, the distance estimate between two gps sensors may include a large error because each can have an average error over meters, when a few meters matter in disease transmissibility check. second, and more importantly, gps is incapable of checking for possible infection events indoors. many studies have used radio frequency identification (rfid) or sensor network technologies to understand infection and to prevent it in hospitals [ ] and in schools [ ] . isella et al. use active tags to track contacts that take place in a pediatric ward for analyzing the structure of the contact data, it identifies the central groups that need close attention to prevent nosocomial infection prevention. salathe et al. use telosb motes carried by students in a school to obtain close proximity interactions data and develop a more effective vaccination strategy. it finds the small world phenomenon, and suggests a vaccination strategy based on the structure that is more effective than random vaccination. it is also used in social studies [ ] and for security based on proximity [ ] . shafagh and hithnawi [ ] use ambient radio signals to detect other nodes in close proximity, for authentication between iot devices before they connect. bolić et al. use an enhanced rfid tags to mutually detect proximity to track. when attached to people, it can be applied for tracking interactions at social events [ ] . but the biggest drawback of these approaches is that today's smartphones hardly support rfid or personal area network (pan) technologies other than bluetooth. due to the lack of deployment base among general public, they do not serve our purpose of massive mutual contact monitoring between strangers. recently, there have been efforts to introduce social media such as twitter to epidemic monitoring, for early detection, management, and control of epidemic outbreaks [ ] - [ ] . in particular, participatory surveillance using social networks to collect symptom reports to detect infectious disease outbreaks has been tried. however, most studies limit their scope to common and seasonally recurring health events such as influenza due to the noisy nature of twitter [ ] . moreover, this post-symptomatic reporting can take long time because some diseases go through long incubation period (e.g. weeks in middle east respiratory syndrome (mers) [ ] ). moreover, subjective symptom reports do not provide information specific enough for disease control authorities to construct contact traces and obtain contact contexts. also, it gives us only collective statistics at coarse granularities, while contact tracing requires information on person-to-person interactions. in the same vein, search-based global disease trend tracking services [ ] are not directly helpful to contact tracing in emergency response. for its prevalence, wi-fi is extremely popular for indoor localization. for example, there is a recent work that leverages on participatory sensing [ ] . again, as in gps, we could consider using wi-fi assisted location information to determine the distance of contact, although the disease transmissibility check in contact tracing needs not necessarily absolute but relative coordinates. however, there are not many works in co-locating two devices using the technology. existing works based on wi-fi are mostly centered around proximity detection and its applications. but mutual proximity detection is not in the design of wi-fi, so it requires significant manipulation such as exploiting portable hot spot (phs) mode [ ] . carreras et al. [ ] use wi-fi to mutually discover smartphones in proximity and determine the distance using received signal strength indication (rssi). in line-of-sight condition, they argue that . m resolution is achievable using the rssi of the discovered smartphone and machine learning algorithms. as to the closeness estimation, most previous works rely on rssi [ ] - [ ] . the applications include authentication [ ] , [ ] and epidemic prediction [ ] . in particular, nguyen et al. [ ] show that the co-presence in disease transmissible distance can be determined through rssi signatures from public wi-fi access points. a drawback of using wi-fi is that access points may be unavailable or prove insufficient to fix positions with a consistent precision. also, the technology is not stellar in energy efficiency, especially for long and continual monitoring. for proximity detection, bluetooth is a popular technology for its relatively high precision in short distances [ ] . it has been mostly used for studies on social behavior and interaction such as duration and proximity [ ] , and those in mass gathering situations [ ] . liu et al. [ ] show that bluetooth can be used to detect face-to-face interaction within . m by mapping bluetooth rssi to distance. in this work, smartphones attempt to detect other bluetooth smartphones every seconds. compared with wi-fi and cellular location, they show that bluetooth can provide an order of magnitude more precise proximity detection. montanari [ ] proposes to use bluetooth low energy (ble) to measure the duration and the proximity of social contact using ble-enabled wearables. it has been also used to measure, understand, and predict how individuals change their social behavior in response to infectious diseases [ ] . yoneki [ ] uses bluetooth to collect proximity devices data to measure, understand, and predict how individuals change their social behavior in response to infectious disease. jamil et al. [ ] use ble tags and smartphones to track group dynamics in a massive religious gathering. it investigates the best configurations for the ble tags and the scan durations for smartphones. compared with the infection study, the group dynamics study requires detection in farther distances at more than m. also, the tags unilaterally advertise, and the smartphones unilaterally scan. it also does % duty cycling, with minutes of hibernation between seconds scans. therefore, this does not fit with the continuous monitoring need for infectious contacts that could happen any time. a recent study also points out the inefficiency of the bluetooth (le) protocol in connectionbased interactions when there are hundreds or even thousands of ble devices in the communication range of each other [ ] . harris et al. [ ] consider the dense ble deployment scenario where hundreds or even thousands of tags interact with a large number of scanning devices such as smartphones. it raises the message collision and consequent energy waste issues of the ble active scanning mode, and proposes an optimization scheme to solve them. although bluetooth technology has many desirable properties, it relies on the beacon exchange to detect each other. the beacons can reveal the identity of the transmitting device, threatening the privacy of the user. communication traces obtained by mobile phones are known to be good proxies for the physical interaction network, and they may provide a valuable tool for contact tracing. for example, calls and messaging activities were used to construct human contact networks [ ] . mobile network data or call detail records (cdrs) have also been used to model population flows, major mobility hubs, and movement typologies, and how they change as the ebola outbreak unfolds [ ] . we could even use two phones attaching to an identical cell as a signal for a possible physical contact. however, the coverage of a single cell tower is at least a few hundred meters in radius, so it would be too coarse to identify infectious physical contact events within a few meters [ ] . one important instance of the mix encounters with strangers is public transport such as train or bus, which people can share for long enough time to enable several modes of disease transmission. for example, an infected person openly coughing in the bus can infect fellow riders in case of aerosol or droplet transmission diseases. when there are many potential contacts that a confirmed case cannot identify or recollect as in public transports, a potent tool we can exploit is the mobile devices such as smartphones. these devices can be leveraged to detect co-location, which can be a good proxy for the physical contacts. for instance, two smartphones located in adjacent cars in a train, both close to the doors dividing the cars, will probably exhibit high similarity in all their measures. but on a multi-car vehicle, a more relevant question in the context of epidemic infection is whether two passengers are on the same car or on different cars. so, in this letter, we explore how we can differentiate locations in the same train at car-level granularity. our study reveals that accelerometer readings during train stop and start events tend to be characteristic of different car positions, so they can be used to generate a strong co-location signature on the car level. thanks to the movements of the train, it does not require a complex communication infrastructure on the train for classification [ ] , but an accelerometer. common ambient sound detection using the microphone sensor [ ] - [ ] can be a technology of choice. but using the microphone sensor has its own issues. first, the number of samples at its typically high sampling frequencies (e.g. . khz) is too large for continuous and indefinitely long monitoring required for detecting contacts that can happen at any time. second, privacy can be violated because any conversations are also recorded. finally, there is the possibility of false detection. for example, two people watching the same tv channel or listening to other broadcast sounds in different places can be classified as coexistent. the smartphone magnetometer has been extensively used for indoor localization and tracking (but not much for coexistence detection). researchers found that the indoor magnetic field is rich in spatial features [ ] , and easy to sense [ ] . moreover, the field is stable over long periods of time [ ] - [ ] . the richness and the stability of the magnetic field enables mapping (a.k.a. fingerprinting) and magnetic map-based applications. the first application is indoor location. chung et al. [ ] showed that the geomagnetic anomaly can provide signatures for indoor locations that can be leveraged for sub-meter-level location accuracy. frassl et al. [ ] used magnetic maps with centimeterlevel accuracy to localize a human or robot. li et al. [ ] discussed possible issues that can affect the precision and the feasibility of the fingerprinting approach for indoor location. angermann et al. [ ] found that the use of all three field components provides good resolution of ambiguities in a small indoor area. carrillo et al. [ ] used the three components of the measured magnetic field by smartphone magnetometers instead of just the intensity to improve accuracy. the second application is navigation. brzozowski and kazmierczak [ ] discussed ways of recording, visualizing, and mapping local magnetic field changes in d that can be used as a support for indoor navigation systems for unmanned aerial vehicles (uavs). riehle et al. [ ] considered a leader-follower style navigation application for visually impaired people where there is time gap between traversals, without relying on expensive indoor magnetic fingerprinting. a follower could compare its own magnetometer trace and the leader's to determine if the follower reached a waypoint and if the follower went off-route. the third application also does not require fingerprinting, and it is of our interest in this paper -coexistence detection. nguyen et al. [ ] used only smartphone magnetometers to detect co-location of passengers in public transport. they exploited the fact that the passengers share the trajectory between at least two consecutive stations, and the magnetometer traces exhibit high similarity, which was measured by the distance in derivative dynamic time warping (ddtw). kuk et al. [ ] showed that even in outdoors the magnetometer traces can be compared to detect contacts within a few meters where the current gps can have an order-ofmagnitude larger errors. it showed that two closely located smartphones generate highly correlated magnetometer traces, which can be exploited to detect coexistence. kuk et al. showed that they could lower the frequency to hz without significantly harming the detection performance, but increasing the battery life significantly. the smartphone magnetometer overcomes undesirable properties of other technological alternatives. it can detect contacts within very short distances that fit infectious disease transmissions monitoring, and it can work indoors. it is supported by all smartphones, and works without any infrastructure support. it consumes relatively small power compared with other sensors, and has little privacy concerns. in this paper, therefore, we focus on the contact detection on smartphone magnetometers and explore their potential to provide a diagnostic tool for potentially infectious contacts made between smartphone holders. as to the privacy concern of some of the technologies above, it may not an issue in the event of an epidemic. authorities may legally have purpose-based access to the phone data of the infected or so suspected person, or rather, volume , users may voluntarily give consent to the authority to use their trajectory data. indeed, we assume such model in subsequent discussions. finally, it is worthwhile to mention that any combination of the magnetometer-based method proposed in this paper with other technologies is possible. for instance, the cost of comparing two traces for checking close contacts could be avoided if their gps coordinates or cellular attachments show totally different values. many valuable combinations could be conceived, but in this paper, we focus on the smartphone magnetometer-based method first so that it can be used in such combination in a more intelligent way. in this section, we discuss how we measure the similarity of two magnetometer traces. among many similarity measures we can use, we pick the pearson correlation coefficient. it is a good measure of linear correlation, which fits the linear correlation that two magnetometers in close proximity show in their ambient magnetic field strength readings. fig. shows real traces generated by two phones held by the people walking side-by-side through a corridor in a university campus building. here, we let the phones measure the ambient magnetic field strength in µt at the rate of hz. in (a), the horizontal axis is the sample number of measured magnetometer values, and the vertical axis is the strength of the magnetic field vector perpendicular to the ground. we observe that these two time series do exhibit similar fluctuations. the fluctuations are the results of the magnetic distortions to the geomagnetic field by ferromagnetic materials such as steel doors, pillars, and rebars among others in the building the smartphone users are passing by. the synchronized fluctuations of the two magnetometer readings have a linear correlation, as shown by (b). therefore, when each phone records such trace while the user moves around in daily life, we can let the users or the disease control authority later check for possible contacts with an infected person using the strength of the linear correlation. for example, as in fig. , a susceptible user can check if her smartphone has a trace segment that computes a high correlation with an infected person's trace that can be provided by the disease control authority. in order to compute the similarity of two smartphone magnetometer traces, we need to use a similarity measure. there are numerous similarity measures, but some popular ones in the literature are cosine similarity, dynamic time warping (dtw) distance, euclidean distance, kullback-liebler distance, jaccard similarity, pearson correlation, among others [ ] . in the areas of epidemiology and psychology, however, the measure of association is frequently analyzed by correlation analysis and regression analysis [ ] . in this paper, we use the correlation analysis. as for the correlation measures, there are pearson, kendall, and spearman correlation coefficients [ ] . among these, we pick the pearson correlation coefficient, as it is a good measure for a linear correlation. to start with, table lists the notations we use in the subsequent discussion on how we compute the pearson's correlation coefficient of two magnetometer traces. two strangers are compared, we do not know whether or when the contact was made. as we discussed in section i, we need to inspect the traces in a window of time t w , as we slide the inspection window (blue box in fig. ) over the entire span of the traces that we are interested in (l tx ). given t w and the magnetometer sampling rate f s , the pearson correlation coefficient for the samples in the window n w = t w · f s starting from the k th sample is defined to be: where a k+i and b k+i are (k + i) th individual magnetometer readings from phones a and b, respectively. µ and σ are the mean and the standard deviation of the measured values in two phone's compared traces in the inspection window. recollect that the existing works [ ] , [ ] compute the similarity over the entire span of samples l = l tx · f s , essentially making n w = l. unfortunately, at an arbitrary length l, we cannot control the false detection possibilities at all, whether positive or negative. therefore, we will use a window n w l to slide over the compared traces to find any interval for which ρ k (a, b) > θ c , ≤ k < l − n w + , where θ c is the cutoff threshold for the contact decision. when l tx significantly increases, there are two aspects we need to consider: memory to store a trace (at smartphones) and correlation computation (at contact tracing check server). first, in terms of memory, the smartphones should keep the samples collected during the long transmissible duration. in our implementation, each magnetometer measurement sample is a vector, whose size is bytes. at hz sampling, we generate data at bytes/second. for an hour of continuous recording, it is approximately . mb. for one week, it is approximately mb. modern smartphones typically have a few tens of gigabytes of memory, so it will not be an excessive burden, especially in the emergency situation (i.e., infectious epidemic). in terms of computation, the trace comparison is performed not on the smartphones but on a server to which the traces are submitted by users who want to check if they were in a close distance with the infected person. the correlation computation will take proportionally long to the length of the compared traces. but the computation itself is not extremely heavy. we tested the correlation computation with the sliding inspection window of seconds over two continuous traces of , samples collected at hz (or l tx = , seconds or minutes). it takes approximately . seconds on a server that has an i - k processor with the clock speed of . ghz, using only a single core. for week-long traces, it will be slightly over minutes. note that the type of contact we aim to detect in this paper is coexistence [ ] that will enable the 'same-placesame-time' (spst) disease transmission. this contact type is more common in infectious disease transmissions than the 'same-place-different-time' (spdt) type [ ] . since the smartphone users are assumed to stay/move together in this type of contact, we do not need to align the traces for the time gap and the moving speed differences by using such schemes as dynamic time warping (dtw) [ ] . finally, we focus on the contacts made in the indoor contexts, because urban life is % indoors [ ] , and indoors is where most infection events take place. as the length of the traces l over which the search is performed should be defined by the given disease of concern, e.g. by its incubation period [ ] or the duration of active transmission, we do not consider this parameter further in this paper. as for the window size n w , it should be long enough to find the contacts of the critical duration that can enable the transmission. however, it is hard to definitely characterize the duration as it will be disease-specific. so, in this paper, we focus on the technical side. namely, we investigate the minimum window size that we can effectively use for the comparison, which will be equivalent to defining the granularity of inspection that smartphone magnetometers can offer. longer contacts than the window size will manifest as a series of consecutive or densely grouped positive decisions, as we slide the window over the entire trace. finally, we will show that the decision cutoff threshold θ c is related with the window size n w for a given target detection accuracy. if the magnetic field strength had a stationary distribution, we could easily draw earlier works on the sample size planning for clinical research [ ] . specifically, the required sample size n w over which the correlation is computed can be estimated as a function of the targeted cutoff θ c . in particular, n w decreases as θ c or the confidence interval increases. unfortunately, the distribution of the magnetic field strength measured by a moving smartphone is not stationary [ ] . without the stationarity of the magnetometer values in our environment, we cannot analytically derive the window size but turn to the measurement-based approach to estimate n w to meet the given θ c . in order to see whether we can use the similarity check of the smartphone magnetometer traces as a diagnostic test, we evaluate its discriminative and predictive power. in particular, we need to evaluate it under different choices of n w and θ c . in clinical studies, numerous metrics are used to evaluate the quality of a diagnostic test. some of them are: sensitivity, specificity, accuracy, positive and negative likelihood ratio, positive and negative predictive value, odds ratio, relative risk, risk difference, number needed to treat, etc. among these, we will use the ones that are not affected by the prevalence, which can only be artificial in our setting. given the ground truth (contact vs. no contact) and the decision using the smartphone magnetometer traces, there can be four cases among which true positive (tp) and true negative (tn) are desirable, and false positive (fp) and false negative (fn) should be minimized ( table ) . as in any other accuracy assessment of diagnostic tests, we use the × table. as to how the false detections (fp and fn) arise in our setting, we can consider two possibilities. suppose the length of the contact duration represented in the traces is t c , and the number of samples generated during the duration l c = t c · f s . then, let us consider fig. , where two people move indoors with the smartphone magnetometers measuring the ambient field strength at hz. the two people come from different places (a vs. b ), meet in the middle, and move together in the region labeled ''a + b '' for t c = seconds, and then part and return to their initial locations (a vs. b ). fig. shows the correlation coefficients obtained as we slide the inspection window over the entire trace under two different n w values. the x-axis is the sample number k in ( ) at which the coefficient is computed, and the y-axis is ρ k . the shaded region represents the duration of contact. it is approximately from samples , through , in both graphs. there are two subcases in this case. first, if n w is very small, it can cause many spurious contact detections since coincidental high correlations may not be sufficiently averaged out. for example, with n w = and , , fig. (a) and (b) show their pearson correlation coefficients, respectively. the circles in fig. (a) show that two spurious detection events are possible for n w = and θ c = . . second, even if n w is large there are still chances for false detections, but only negative. it is because increasing n w decreases ρ k (a, b) as a consequence of the non-stationarity of the magnetic field strength distribution [ ] , when the human smartphone holder moves through space. using our coexistent trace pairs, we indeed confirm that the larger window sizes significantly reduce the correlation coefficient (fig. ) . in this case, a possible consequence is that the adjacent measurement samples outside the coexistence duration that happen to be included in the window decreases the cross correlation, possibly leading to a false negative decision depending on θ c . observe that for high cutoff thresholds such as θ c > . , fig. (b) will falsely determine that there was no contact, whereas the former will correctly detect the contact. either way, these problems can lead to false decisions about the contact, so it is clear that we need to determine figure . pearson correlation coefficient ρ k with % confidence interval, for a large number coexistent trace pairs. the appropriate window size n w as well as the cutoff threshold θ c . for our measurement-based study, we use indoor magnetometer traces collected in the korea university campus in seoul, korea. below, we first discuss how we collect the traces. then we evaluate the smartphone magnetometerbased contact detection using the measures mentioned in section iii-c. to collect the magnetometer traces, we developed and installed a magnetometer sensing app for android smartphones, samsung galaxy s , s , s and lg g and g . we confirmed that our app works correctly on all these platforms. among the phones, we used two galaxy s 's to collect the traces used in this section. we synchronized their sensing activity through the network time protocol (ntp) [ ] for later comparison of their magnetometer traces. we collect the magnetometer traces in five different buildings in the campus. we picked three places in each building. at each place, we repeated the trace collection six times along the same walking path. so, in total, there are traces, and each trace is seconds long. there are c( , ) = pairs of traces per place to be judged co-existent. since there are different places from which the traces were collected, we have · c( , ) = co-existent trace pairs in total. on the other hand, there are c( , ) · c( , ) · c( , ) = · · · = , non-coexistent pairs. we measured the magnetic field strengths at the default sampling frequency of hz, a popular magnetometer sensing rate in the literature [ ] . the magnetometer readings are obtained in three phone-specific axes: x, y, and z. in order to simulate typical indoor walking dynamics, we let the smartphone holders walk approximately at the 'preferred' walking speed [ ] . it is known that people prefer to walk at approximately . m/s (or . km/h) irrespective of cultures, as they find slower or faster speed uncomfortable. each trace was produced in narrow corridors, and we saw to it that the traces do not deviate from each other more than an 'arm's length' to simulate the typical personal gap [ ] . as the smartphones can have arbitrary attitudes when and while the contact is made, the measured magnetic strengths in their x, y, and z axes will generally be misaligned. for comparison, therefore, they should be translated to a common coordinate system. for this, we use android getrotationmatrix() method to translate the phonespecific coordinates to the absolute coordinate (i.e., north, east, etc). a desirable property of the geomagnetism is that it has absolute reference directions such as the east and the north. smartphones will change attitudes freely, but the translation method lets us readily compare the traces from different phones regardless of their attitudes. as to the robustness of the method against the accumulation of errors over a long duration of continuous operation, it is a research issue of its own [ ] . in this paper, we assume that such calibration is being done to maintain the precision of the magnetometers. fig. illustrates the alignment operation in our measurement system. under the misalignment (a), it is not straightforward to choose the axis for the comparison (b). the traces in (b) shows that the x-axis of phone is aligned with the y-axis of phone , which is the ground truth as shown in (a). but after the translation, the readings from the two coexistent but misaligned phones are cleanly separated along the three absolute axes (c). we notice that the z-axis traces from (b) are identical to up-axis traces in (c), because the phones were held parallel to the ground (a) in the generation of the traces in (b). finally, the east is simply the cross product of the two vectors north and up, so it is redundant. thus in our implementation, we choose whichever axis between north and up that shows the highest correlation in the decision. here, we compute the evaluation measures for the combinations of the window size and the decision threshold. in particular, we will compute them for the first n w samples from each trace pair, i.e., k = in ( ). but first, there is a caveat. in total, there are , and non-coexistent and co-existent trace pairs in our data set, totaling at , . the prevalence in our data set is thus / , = . %. however, this is artificial -we could have made it higher or lower by producing more of coexistent or noncoexistent traces, respectively. naturally, it is meaningless to calculate the measures affected by the prevalence, where the prevalence of disease is artificially controlled [ ] . sensitivity and specificity are not generally related to the prevalence of the disease in the population considered, since these are properties of the diagnostic tool. unlike sensitivity and specificity, measures such as predictive values, accuracy, relative risk and risk difference are affected by the prevalence. therefore, we exclude them, and use the measures that are not affected by the prevalence to evaluate the magnetometerbased contact test. sensitivity is expressed as the proportion of correctly classified as true positives among the total contacts tp/(tp + fn ). in other words, it is the ability of the magnetometer-based test to correctly identify the trace pairs with a real contact. a highly sensitive test is useful, when we do not want to miss a contact (with an infected person) in screening the population. the specificity is the ability to identify the no contacts, expressed as tn /(tn + fp). a specific test will rarely misclassify the trace pairs without a contact as having made a contact. the sensitivity and specificity show the discriminative powers of a diagnostic test. fig. shows the sensitivity and the specificity of our smartphone magnetometer-based test, as functions of n w and θ c . we first find that larger n w does not necessarily mean the higher sensitivity. although happening at different values of n w ( | θ c = . ∼ | θ c = . ), the sensitivity begins to decrease beyond a certain n w at each cutoff threshold. it implies that the correlation decreases when computed for an excessively long trace segment used as the inspection window. this is due to the non-stationary property of the magnetometer measurement value distribution [ ] . the specificity, on the other hand, steadily increases as we use larger n w . the lesson here is that when we use the magnetometer-based diagnostic test, we need to examine the similarity of the two traces using the time window of n w = ∼ to achieve the highest sensitivity. then, the choice of the exact cutoff threshold will depend on the target specificity. also, we find in fig. that the sensitivity is higher with lower cutoff values, whereas the specificity is higher with higher cutoff values. this tension is natural, and can be summarized in the receiver operating characteristic (roc) curve. fig. shows the roc curves for different parameter combinations. although we cannot show the area under curve (auc) itself due to the absence of very low specificity data points, it is clear that the auc's for various n w are very high. namely, the magnetometer-based test is of high diagnostic quality. among the inspection sample window sizes, very small n w ( , ) and very large n w ( , ) lead to poorer auc than those in the middle (n w = ∼ ) as shown in fig. (b) . n w = achieves the best overall auc. in order to obtain the cutoff value θ c that achieves the highest auc for a given n w , we can compute the shortest distance volume , likelihood ratio (lr) is the mostly widely applied measure of diagnostic accuracy. also, it can serve as a predictive measure. in our context, lr tells us how many times more likely a decision is in the trace pairs with the contact than in those without contact. when both probabilities are equal (i.e., lr = ), such test is of no value. the lr for positive test results (lr+) is defined as tp tp+fn / fp tn +fp . the higher the lr+, the more indicative the test is of the contact. good diagnostic tests have lr+ > and their positive result has a significant contribution to the diagnosis [ ] . on the other hand, the lr for negative test result (lr−) is defined as fn tp+fn / tn tn +fp , and it represents the ratio of the probability that a negative result will occur in trace pairs with the contact to the probability that the same result will occur in trace pairs without the contact. good diagnostic tests have lr− < . [ ] . the lower the lr−, the more significant contribution of the test is in ruling-out. lr's do not depend on prevalence of disease of population, as only sensitivity and specificity values are used to calculate them. as a result the lr's of one study could be used in another setting with the condition that the definition of contact is not changed. the likelihood ratios of the smartphone magnetometerbased contact test are shown in fig. . in (a), we observe that it is highly useful for positive identification of contacts. the criterion lr+ > tells us that the positive likelihoods can be a significant contribution to the diagnosis. we also note that we do not need large n w to have lr+ > , especially when we use higher cutoff thresholds of θ c ≥ . . less than measurement samples at hz, or equivalently seconds, is enough to qualify for a good test for positive identification of contacts. on the other hand, fig. (b) shows that the higher cutoff thresholds cannot achieve lr− < . regardless of n w . it implies that using the higher cutoffs can produce a high fraction of false negatives. however, this issue may be mitigated if we require that a contact duration be composed of a series of positive decisions as we slide the inspection window. for example, in fig. (a) , hundreds of adjacent positive decisions will occur as we slide up k in ( ). interspersed false negatives will less affect the final decision then. diagnostic odds ratio (dor) is a relative measure for diagnostic accuracy, used for the estimation of discriminative power of diagnostic procedures [ ] . dor of a test is the ratio of the odds of positivity in traces with the contact relative to the odds in traces without contact. it is calculated according to the formula: dor = (tp/fn )/(fp/tn ). dor depends significantly on the sensitivity and specificity of a test. a test with high specificity and sensitivity with low rate of false positives and false negatives has high dor. with the same sensitivity of the test, dor increases with the increase of the test specificity. for example, a test with sensitivity > % and specificity of % has a dor greater than . the diagnostic odds ratio ranges from zero to infinity, although for useful tests it is greater than one, and higher diagnostic odds ratios are indicative of better test performance. fig. shows that the dor of the smartphone magnetometer-based contact test is much larger than one for most n w values. so, this measure also confirms that the magnetometer-based test is useful. if we use or = as the example criterion, the figure tells us that higher cutoff thresholds qualify with less measurement samples n w to look at (θ c = . has fn = at n w = , so it should qualify although we cannot plot it). for these higher cutoffs, less than samples (or equivalently seconds) or less is enough to achieve the high dor. above, we evaluated the quality of the smartphone magnetometer traces comparison as a clinical test for potential (infectious) contact. all evaluation metrics that we used for the evaluation, namely sensitivity, specificity, likelihood ratio, and diagnostic odds ratio, point to the fact that the number of magnetometer readings to be compared between two traces (n w ) can be small. these metrics produce slightly different optimal numbers for the required readings, but if we need one good number to apply in real-life cases, it is samples (or equivalently seconds at the hz sampling frequency). it leads to the best or close-to-thebest performance in all the evaluation measures. our recommendation is that when two magnetometer traces from two smartphones are compared, one needs to use a window of samples for the pearson correlation computation to achieve the most precise decision as to whether the contact was really made between the smartphone holders. one further recommendation is that the correlation coefficient value used as the decision threshold can be high. specifically, θ c = . is a good match for the -sample inspection window. note that these two numbers n w and θ c to produce the most precise decision are closely related, and other combinations than ( , . ) can be inferred from the results in the previous section. when a large-scale epidemic crisis unfolds in the highly urbanized society today, the traditional contact tracing method of medical personnel interviewing the infected persons will become highly costly, slow, and ineffective. in this paper, we discuss how smartphones carried by most people can be harnessed to automatize the contact tracing in such situation. we exploit the fact that smartphone magnetometers show high linear correlation when two phones coexist within a short, disease-contractible distances, such as less than two meters. then, we use a battery of metrics to evaluate the value of such smartphone magnetometer traces comparison as a clinical test that medical personnel can use in reallife with a high trust level. our evaluation reveals that the magnetometer-based method qualifies for a valid clinical test, if used with certain parameter values in the correlation computation. specifically, our finding and recommendations are as follows. first, the size of the sliding window of trace section to be compared is best to be what corresponds to seconds of samples. second, the decision threshold that matches the comparison window size is . , for the most precise contact decision. these two parameters are inversely related with respect to the precision of the contact detection, and other combinations around the recommended values are also possible. in future, we will further test the reliability of the proposed method with the recommended and other parameter settings in more extensive real-life environments, for instance with different smartphone movement speeds, with obstacles, people or objects between or around the smartphone holders, and with interferences such as power lines close to the smartphones. the artificial traces that we generated in a controlled environment could have biased our experiment results and our conclusion. therefore, we will need to optimize the proposed method further against the real-life traces in a building or in public places to make it more reliable and actually usable in the real-life epidemic 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room-level proximity detection based on rss of dual-band wi-fi signals multimodal indoor social interaction sensing and realtime feedback for behavioural intervention hybrid participatory sensing for analyzing group dynamics in the largest annual religious gathering bluetooth low energy in dense iot environments epidemic contact tracing via communication traces commentary: containing the ebola outbreak-the potential and challenge of mobile network data ambient sound-based proximity detection with smartphone sound-proof: usable two-factor authentication based on ambient sound a wearable, ambient sound-based approach for infrastructureless fuzzy proximity estimation characterization of the indoor magnetic field for applications in localization and mapping magnetic maps for indoor navigation how feasible is the use of magnetic field alone for indoor positioning? magicfinger: d magnetic fingerprints for indoor location magnetic field mapping as a support for uav indoor navigation system indoor waypoint navigation via magnetic anomalies clustering of time series data-a survey applied multiple regression/correlation analysis for the behavioral sciences sample size requirements for estimating pearson, kendall and spearman correlations airborne disease propagation on large scale social contact networks using dynamic time warping to find patterns in time series the national human activity pattern survey (nhaps): a resource for assessing exposure to environmental pollutants quantifying the risk and cost of active monitoring for infectious diseases internet time synchronization: the network time protocol the pace of life in countries dynamic stride length adaptation according to utility and personal space magnetometer calibration for portable navigation devices in vehicles using a fast and autonomous technique measures of diagnostic accuracy: basic definitions her research interests include digital disease detection and mobile computing seungho kuk received the b.e. degree in computer science and engineering from korea university from to , he was a research scientist with bell communications research. he is currently a professor with korea university key: cord- - n iaogb authors: tenckhoff, bernhard; siegmann, silvester title: krisenmanagement date: - - journal: vernetztes betriebssicherheitsmanagement doi: . / - - - - _ sha: doc_id: cord_uid: n iaogb erfolgreiche unternehmen und organisationen entwickeln ein umfassendes bewusstsein für mögliche krisen, sie betreiben professionelle krisenprävention. das rezept für eine dauerhafte und erfolgreiche krisenprävention lautet: jeden tag nach vorne schauen, risiken wahrnehmen, richtig abschätzen und möglichst neutralisieren. systematische verfahren zur früherkennung von warnsignalen spielen dabei eine wichtige rolle. krisenpotenziale erkennen und antizipieren, infrastrukturen schaffen, abläufe einüben und mitarbeiter schulen sind gute voraussetzungen, um in krisenhaften situationen souverän zu agieren, statt in einen schockzustand zu verfallen. leider sind noch zu viele unternehmen und organisationen von diesem ideal weit entfernt. krisen gefährden außerdem immer das image und die reputation eines unternehmens. in diesem zusammenhang darf das mediale interesse nicht unterschätzt werden. die mediengesellschaft, in der wir leben, liebt und produziert deswegen unentwegt große und kleine krisen, indem sie schlicht jede gelegenheit zur berichterstattung nutzt. schließlich ist die krise – mehr noch als die bloß schlechte nachricht – ein hervorragend verkäufliches gut (mörle ). leider verfügen noch zu wenig unternehmen über professionelle konzepte, die auch den aspekt der krisenkommunikation hinreichend berücksichtigen. auch ist einzelnen studien zu entnehmen, dass das bewusstsein in deutschland für das thema krisenprävention und ‑bewältigung in einigen branchen noch nicht hinreichend ausgeprägt ist. veränderungskrise: hierbei handelt es sich um krisen, die durch schlecht gemanagte veränderungen (z. b. fusionen) hervorgerufen werden können. ein gutes "change-management" gehört für jedes unternehmen zum pflichtprogramm in der vergangenheit bzw. gegenwart haben sich folgende branchen als besonders krisenanfällig gezeigt: gesundheit/pharma chemie lebensmittel energie fleischerzeugung versicherungen/banken luftfahrt automobil aufgrund der globalisierung und schnelllebigkeit unserer industriegesellschaft kann eine unternehmenskrise aber auch jede andere branche jederzeit treffen. die ursachen und wechselwirkungen, die zu einer unternehmenskrise führen können, sind komplex und lassen sich in der regel auf die summe verschiedener interner und externer ereignisse und entwicklungen zurückführen. unternehmenskrisen können entstehen durch: zahlreiche beispiele aus der vergangenheit haben aufgezeigt, dass ereignisse mit krisenpotenzial erst durch die medien zu krisen gemacht wurden. nur wenige unternehmen verfügen jedoch über professionelle pläne, die auch den aspekt der krisenkommunikation berücksichtigen. fehler bei der kommunikation können ein kritisches ereignis drastisch verschlimmern. primäres ziel der krisenkommunikation ist die aufklärung der Öffentlichkeit und der medien und der direkt betroffenen gruppen bezüglich der krise. des weiteren versucht das unternehmen, der Öffentlichkeit und den medien zu vermitteln, dass es alles mögliche unternimmt, um größere schäden abzuwenden und die situation wieder unter kontrolle zu bekommen (abb. . ). Öffentlichkeitsarbeit während der krise: information wird sofort und ohne verzögerung weitergeben innerhalb der ersten zwei stunden eine pressekonferenz einberufen und das sofort ankündigen informationsstrom nicht abbrechen lassen ( gerade die erste information der medien und somit der Öffentlichkeit innerhalb der ersten stunde benötigt eine möglichst hohe effizienz und effektivität. kommunikationsmaßnahmen bzw. -instrument müssen perfekt aufeinander abgestimmt eingesetzt werden. alleine daraus ergibt sich, dass die krisenkommunikation im vorfeld proaktiv vorbereitet werden muss. oft ist es sinnvoll, sich dafür mit profis in verbindung zu setzen. eine möglichkeit der optimalen abstimmung der einzelnen instrumente ist die orientierung an den sechs krisen-w's: was ist passiert und was wird unternommen? wer wird informiert bzw. einbezogen? wie kann das vertrauen erhalten bzw. wiedererlangt werden? wann wird die Öffentlichkeit informiert? warum kam es zur krise? wo wird stellung genommen? i tipp eine effektive und effiziente krisenkommunikation ist unbedingt erforderlich und sollte im vorfeld von profis vorbereitet werden! leider reicht es nicht mehr aus, nur finanztechnische kennzahlen zur früherkennung von unternehmenskrisen heranzuziehen. immer mehr unternehmen realisieren, dass die traditionelle krisenprävention den gestiegenen ansprüchen nicht mehr genügt, wie auch zahlreiche studien belegen. angesichts der härter werdenden wettbewerbsbedingungen macht es sinn, auch mehr aktivitäten in die "technische" krisenprävention zu investieren. hierzu gehören insbesondere das durchspielen von "worst-case"-szenarien, das aufzeigen von bottle necks in der produktion, die kontinuierliche durchführung angemessener instandhaltungsmaßnahmen sowie die einführung eines issue profiling für arbeitsmittel. in diesem zusammenhang muss das zentrale präventionsinstrument "gefährdungsbeurteilung" gemäß arbeitsschutzgesetz (arbschg), betriebssicherheitsverordnung (betrsichv) und weiterer verordnungen besser von den unternehmen angenommen und genutzt werden. hier geht es noch um potenzielle krisen. in dieser phase befindet sich das unternehmen noch im normalzustand und es gibt noch keine in den unternehmensintern zu bestimmenden kennzahlen wahrnehmbaren krisensymptome. die zentrale aufgabe des antizipativen krisenmanagements ist die gedankliche vorbereitung auf mögliche krisen. man muss hier den mut haben das undenkbare zu denken. sehr häufig ist hier im vorfeld ein geeignetes "issues management" hilfreich. das präventive krisenmanagement bezieht sich auf die zweite phase des krisenprozesses, der latenten unternehmenskrise. die krisensymptome sind teilweise noch verdeckt, die kennzahlen liefern aber bereits den erhärteten verdacht, dass bereits eine gefährdung der erfolgspotenziale vorliegt. die krise ist bereits eingetreten und das unternehmen ist sich dessen auch bewusst. sie ist aber noch beherrschbar. die zentrale aufgabe ist die erfolgreiche zurückschlagung und die daraus folgende bewältigung der unternehmenskrise. dies ist die letzte phase, die krise nicht mehr beherrschbar und führt zur katastrophe. es kann zu einer möglichen liquidation des unternehmens kommen. tipp alle organisationseinheiten eines unternehmens haben die erforderlichen maßnahmen zu ergreifen, um den eintritt einer krise zu verhindern (krisenprävention) bzw. beim eintreten einer krise negative auswirkungen für das unternehmen so weit wie möglich einzugrenzen! eines der instrumente des antizipatives krisenmanagements ist das "issues management" dies ist bereits seit den er jahren des vorherigen jahrhunderts in den usa ein wichtiges management-thema und findet seit ende der er jahre im europäischen raum ebenfalls anwendung. diese bezeichnung "issue" entstammt dem angelsächsischen sprachraum. eine wörtliche Übersetzung ins deutsche wäre "thema, angelegenheit etc.", darunter versteht man in diesem zusammenhang insbesondere ein thema öffentlichen interesses mit hohem konfliktpotenzial. issues management dient als früherkennungssystem für schwache signale aus dem unternehmensumfelds. der "vater" des konzepts, der amerikanische pr-berater william howard chase, definiert issues-management um herum wie folgt: issues management is the capacity to understand, mobilize, coordinate and direct all strategic and policy planning functions and all public affairs/public relations skills toward achievement of one objective: meaningful participation in creation of public policy that affects personal and institutional destiny. durch das "issues management" hat ein unternehmen oder organisation also die möglichkeit, schnell, flexibel und vor allem sensibel auf jede nachricht und entwicklung zu reagieren, die für die identität und wahrnehmung der unternehmensmarke wichtig ist und bei nichtbeachtung krisenpotenzial entwickeln kann. das issue management lässt sich dabei in fünf phasen unterteilen: "scanning": die identifikation von issues als grundlegende und somit fast schon wichtigste phase "issues monitoring": analyse und beobachtung der öffentlichen meinung und medien "strategic issue diagnosis": die strategische prüfung und einstufung "message formating": wahl der grundlegenden reaktion/antwort auf strategische issues. hierbei kann noch mal zwischen einem proaktivem und reaktivem vorgehen unterschieden werden "incorporation into strategic plan": integration in die strategische planung issues durchlaufen im öffentliche fokus einen lebenszyklus: je weiter das issue in seinem lebenszyklus voranschreitet, desto geringer wird gleichzeitig die einflussmöglichkeit des betroffenen unternehmens. in den usa gilt das issues management mittlerweile als selbstverständlichkeit, in deutschland hingegen hinkt man dieser entwicklung noch hinterher. Über geeignete und im notfall auch sofort zur verfügung stehende dokumentationen (verfahrenanweisungen, notfallpläne, gefahren-abwehr-pläne (gap)), einer entsprechend ausgelegten infrastruktur, spezifischer instrumente und auch mit hilfe von personelle ressourcen muss ein notfallmanagementsystem in den das krisenmanagement implementiert und manifestiert werden. elektronische, softwaregesteuerte notfallmanagementsysteme können den mitarbeitern helfen schnell zu reagieren. es muss aber unbedingt beachtet werden, das die it von der energieversorgung abhängig und diese im notfall ausfallen kann! das notfallmanagement muss den mitarbeitern also auch in ausgedruckter version zur verfügung stehen. um auf notfälle vorbereitet zu sein, empfehlen sich regelmäßige notfallübungen und räumungsübungen. diese können neben dem sicherstellen des prinzipiellen funktionierens des notfallmanagements auch erkenntnisse liefern, welche optimierungspotenziale noch ungenutzt sind. dabei sollten die Übungsszenarien so realitätsnah wie möglich geplant und durchgeführt werden, um alle parameter des notfallplans zu überprüfen -die funktionalität und effektivität von organisatorischen, personellen und materiellen vorkehrungen aber auch die qualifikationen des einsatz-und hilfspersonals. ein typisches notfallszenario ist ein großbrand im betrieb. anlagen und gebäude können nach einem brandschaden mit mehr oder weniger großem aufwand instand gesetzt, zerstörte betriebs-und arbeitsmittel neu beschafft werden. die beeinträchtigung der gesundheit oder gar der verlust von menschlichem leben durch den brand und seine nebenwirkungen wiegen dagegen ungleich schwerer als der sachschaden. nach angaben des gesamtverbandes der deutschen versicherungswirtschaft e. v. ergibt sich für großschäden in der industriellen sachversicherung folgende statistik der brandschäden für die jahre bis (tab. . ). in der obigen aufstellung wurden nur schäden mit einem schadenaufwand von mindestens . c (bis mio. dm) berücksichtigt. zwar ist die anzahl der großschadensereignisse tendenziell rückläufig, jedoch stieg im gleichen zeitraum die durchschnittliche schadenssumme deutlich an. sachschäden durch brände wurden in der vergangenheit zwar seltener, dafür aber auch deutlich teurer. aus praktischen erwägungen sollten alle oben aufgeführten eventuell auftretenden gefährdungen separat aufgelistet und bewertet werden. die anforderungen an die struktur der flucht-und rettungswege sowie der dazugehörigen pläne ergeben sich aus der technischen regel für arbeitsstätten (asr) a . "fluchtwege und notausgänge, flucht-und rettungsplan" vom . august und sind meistens (sollten) bereits in der planungsphase von architekten und genehmigungsbehörden berücksichtigt worden. grundsätzlich ist immer dann eine sicherheitsbeleuchtung vorzusehen, wenn das arbeitsstättenrecht oder das baurecht diese vorschreiben. darüber hinaus kann die gefährdungsbeurteilung weitere anwendungsfälle sowohl für die sicherheitsbeleuchtung als auch bodennahe sicherheitsleitsysteme ergeben. bei möglicher verrauchung ist im allgemeinen ein bodennahes sicherheitsleitsystem erforderlich. dieses kann grundsätzlich entweder elektrisch oder auch lang nachleuchtend ausgeführt werden. die gefährdungsermittlung kann auch ergeben, dass kombinationen unterschiedlicher sicherheitsleitsysteme erforderlich sind. im allgemeinen kann davon ausgegangen werden, dass bei hoher personenbelegungsdichte der einsatz eines elektrisch betriebenen systems vorteilhaft ist. bei geringer personenbelegungsdichte kann ein lang nachleuchtendes system ausreichen. die anforderungen an sicherheitsleitsysteme ergeben sich für den landbereich aus der normenreihe din ( - ) "deutsche industrie norm für langnachleuchtende pigmente und produkte", bgv a "sicherheits-und gesundheitsschutzkennzeichnung am arbeitsplatz" und bgr "optische sicherheitsleitsysteme". folgende anforderungen müssen von einem sicherheitsleitsystem grundsätzlich erfüllt werden: zuverlässig -"funktioniert" auch bei stromausfall sichtbar -auch bei starker verqualmung deutlich erkennbar und auch nach vielen stunden noch zu erkennen lückenlos -durchgehende markierung bis zum nächstgelegenen notausgang insbesondere in sonderbauten mit einer vielzahl von personen ist in der regel ein zweiter baulicher rettungsweg erforderlich. die rettung einer vielzahl von personen über das rettungsgerät der feuerwehr ist nicht in angemessener zeit möglich. um die sich im gebäude befindlichen personen in die lage zu versetzen, sich schnell und sicher orientieren zu können, müssen flucht-und rettungspläne erstellt werden, die eine möglichst einfache darstellung der baulichen gegebenheiten sowie eine unproblematische lesbarkeit aufweisen. grundlage der flucht-und rettungspläne ist die arbeitsstättenverordnung (arbstättv). auch wenn es derzeit nicht direkt gesetzlich vorgeschrieben ist, hat sich in der praxis die bestellung von brandschutzbeauftragten bewährt. der brandschutzbeauftragte sollte eine mehrjährige praxis im vorbeugenden brandschutz besitzen und/oder eine ausreichende ausbildung im vorbeugenden brandschutz haben. vergleichbar den fachkräften für arbeitssicherheit sollte der brandschutzbeauftragte unmittelbar der leitung des werkes oder betriebes unterstellt sein, für dessen brandschutz er zuständig ist. zu allen den brandschutz betreffenden fragen des unternehmens -auch bei der planung -sollte er gehört werden. zu seinen aufgaben und pflichten gehört das erkennen von gefahren sowie ihre beurteilung. er hat dafür zu sorgen, dass sie beseitigt und schäden möglichst gering gehalten werden. unter den bedingungen, die bei einem brand herrschen, kann aus unbedenklichen stoffen, gelagerten gegenständen, arbeitsmitteln (z. b. kopierer) oder bauteilen eine vielfalt an verbrennungsprodukten und rückständen entstehen, deren gefahrenpotenzial nur sehr schwierig einzuschätzen ist. mit den daraus folgenden anforderungen an den umweltschutz, die sicherheit und den gesundheitsschutz der personen, die die sanierungsarbeiten an den brandstellen durchführen, befasst sich die gdv-richtlinien zur brandschadensanierung -vds ( ). die vds schadenverhütung ist ein unternehmen des gesamtverbandes der deutschen versicherungswirtschaft (gdv). die richtlinien vds wurde im oktober neu strukturiert und grundlegend überarbeitet. sie berücksichtigen die vorgehensweisen und schutzkonzepte der gefahrstoff-und biostoffverordnung sowie der zugeordneten technischen regeln zur gefährdungsbeurteilung (trgs "gefährdungsbeurteilung für tätigkeiten mit gefahrstoffen", trba "handlungsanleitung zur gefährdungsbeurteilung und für die unterrichtung der beschäftigten bei tätigkeiten mit biologischen arbeitsstoffen", etc.) und die festlegung von schutzmaßnahmen. zusätzlich berücksichtigen die richtlinien die aufgaben und verantwortlichkeiten, die sich aus der baustellen-verordnung und der bgr "arbeiten in kontaminierten bereichen" ergeben. demnach entspricht die richtlinien vds ( ) einer der ersten schritte ist die unverzügliche meldung des schadenfalles an den zuständigen versicherer. insbesondere bei bränden mit höherem gefahrenpotenzial kann dies entscheidenden einfluss auf die gesamte schadenabwicklung haben. diese frühzeitige meldung des schadenfalles versetzt den versicherer in die lage, rechtzeitig zu reagieren und dadurch u. a. sicherzustellen, dass eine rasche und qualifizierte beurteilung der schadensituation vor ort durch den versicherer veranlasst werden kann (abb. . ). vor beginn der sanierungsarbeiten sind unterschiedliche bewertungen vorzunehmen und entscheidungen zu treffen. in der regel hat der betroffene hierzu professionelle hilfe wie etwa durch den versicherer, durch sachverständige oder sanierungsunternehmen nötig. nur mit dieser hilfe kann das unternehmen schnellstmöglich wieder die produktion aufnehmen und den verlust von vertrauen und einen imageschaden verhindern. letztendlich könnte dies auch zum verlust der kunden und somit der wirtschaftlichen grundlage führen. auf basis der erstbegehung durch die o. a. experten erfolgt die einteilung der brandstelle in die gefahrenbereiche. in allen betrieben und auf baustellen muss erste-hilfe-material bereitgehalten werden. geeignetes erste-hilfe-material ist z. b. im kleinen verbandkasten nach din sowie im großen verbandkasten nach din enthalten. in abhängigkeit von der betriebsart und zahl der versicherten gelten für die ausstattung mit verbandkästen die in der folgenden tabelle aufgeführten richtwerte. die verbandkästen sollen auf die arbeitsstätten so verteilt sein, dass sie von ständigen arbeitsplätzen höchstens m wegstrecke oder höchstens eine geschosshöhe entfernt sind. sie sollen überall dort aufbewahrt werden, wo die arbeitsbedingungen dies erfordern. im zuge der globalisierung verstärken immer mehr unternehmen ihr auslandsgeschäft. die zahl der auslandseinsätze der mitarbeiter nimmt ständig zu. jedoch werden die risiken, die damit verbunden sind, häufig einfach ausgeblendet. es wird zu oft leichtfertig mit dem höchsten gut eines unternehmens, den mitarbeitern, umgegangen, obwohl die risiken ständig zunehmen. unternehmen, die mitarbeiter ins ausland entsenden, müssen regionsabhängig stets mit naturereignissen wie erdbeben, seuchen wie sars, vogelgrippe oder dengue-fieber, terror und kriminellen akten wie bombenanschlägen oder entführungen rechnen. erstaunlich sei auch, dass unternehmen sich häufig nur um das visum und die schutzimpfungen des entsendungspersonals kümmern, nicht aber um eine umfassende sicherheitsvorsorge. dabei umfasst ein gutes basis-schutzpaket weitaus mehr aspekte. die richtige personalauswahl vor der entsendung, die arbeitsmedizinische vorsorgeuntersuchung incl. reisemedizinischer beratung, die einweisung in das jeweilige land und dessen landesspezifischen gefahren, die unterstützung bei der unterkunftssuche, die auswahl geeigneter kontaktpersonen vor ort und eine gesicherte ansprechbarkeit der unternehmens rund um die uhr müssen unbedingt berücksichtigt werden. für den ernstfall sind jedoch noch zu wenig unternehmen professionell vorbereitet. sie verlassen sich offensichtlich nur auf die geschicke des außenministeriums. die pandemie-planung hat inzwischen auch viele betriebe erreicht. für die weltgesundheitsorganisation ist der ausbruch einer grippe-pandemie nur eine frage der zeit. experten rechnen damit, dass in diesem fall rund % der arbeitnehmer ausfallen. eine pandemie ist eine weltumspannende epidemie. erste pandemien sind bereits seit der antoninischen pest (ca. - n. chr.) tatsächlich belegt. neben einer durch influenzaviren verursachten pandemie zählt z. b. auch aids zu dieser kategorie, das (seit ) bisher etwa mio. todesopfer forderte. im . jahrhundert ereigneten sich drei influenzapandemien: die "spanische grippe" - (bis zu mio. tote), die "asiatische grippe" (ca. mio. tote) und die "hong kong grippe" mit etwa . toten. basis einer rationalen planung ist ein gutes verständnis der saisonalen, aviären und pandemischen influenza. die trennung dieser entitäten ist wichtig, gerade weil sie in der Öffentlichkeit nicht immer klar vollzogen wird. seit zirkulieren viren zweier influenza a subtypen (h und h ) sowie des typs b in der menschlichen bevölkerung und rufen die saisonale grippe hervor. der anteil der virustypen bzw. subtypen an den influenza-erkrankungen schwankt von jahr zu jahr, die saison / war z. b. von influenza b-viren dominiert und relativ schwach, während in der saison / influenza a/h n -viren vorherrschten und eine viel stärkere (und für das gesundheitssystem folgenreichere) grippewelle verursachten. typischerweise baut sich eine saisonale welle, häufig im süden beginnend, in wenigen wochen auf, bevor ganz deutschland erfasst ist. die hierbei handele es sich um optimistische schätzungen, da man hierbei von einer pathogenität der "honk kong-grippe" ausgegangen ist und nicht von der höheren pathogenität der "spanischen grippe". welcher betrieb kann einen so hohen ausfall im bereich human ressources verkraften? wie viele betriebe haben sich darauf vorbereitet? wer denkt auch an die angehörigen seiner mitarbeiter? kein familienvater wird in dieser situation mit seinen gedanken im betrieb sein wenn er überhaupt zur arbeit kommt (siehe anlage ). abb. . chancenpotenziale bei unternehmenskrisen. (pachurka und siegmann ) (bcp)) ist als "geschäftsaufrechterhaltungs-und -fortsetzungsplanung" bestandteil des risikomanagements und indirekt aus dem kontrag abzuleiten (keitsch ) . hiefür sind präventiv im rahmen des bcm betriebsaufrechterhaltungs-und -fortsetzungsprozeduren zu entwickeln und zu trainieren um die unternehmenswerte proaktiv zu schützen. im bereich des bcm gibt es einen internationalen standard, den iso/pas : - "societal security -guideline for incident preparedness and operational continuity management" (sicherheit und schutz des gemeinwesens -leitfaden für planung, vorbereitung und operationelle kontinuität), der einige hilfestellungen bietet (abb. . ). viele der betriebsprozesse erstrecken sich entlang der logistischen wertschöpfungskette (supply chain): die besonderen eigenschaften des (gesamt-)systems "supply chain" ergeben sich dabei aus dem spezifischen dynamischen zusammenwirken der lieferkettenglieder. der begriff des "supply chain management (scm)" wurde anfang der er jahre des vergangenen jahrhunderts in den usa geprägt. hier entstand der gedanke der integration von verschiedenen unternehmensaktivitäten. in den er jahren rückte der begriff vermehrt in den bereich der logistik. aus der perspektive des scm folgt die logistik einem prozessorientierten ansatz. Übersetzen lässt sich scm mit dem management von versorgungsketten, lieferketten bzw. wertschöpfungsketten. daran wird sofort erkennbar, welches krisenpotenzial hier zu finden ist. durch das "issues management" hat ein unternehmen oder organisation also die möglichkeit, schnell, flexibel und vor allem sensibel auf jede nachricht und entwicklung zu reagieren, die für die identität und wahrnehmung der unternehmensmarke wichtig ist und bei nichtbeachtung krisenpotenzial entwickeln kann. das issue management lässt sich dabei in fünf phasen unterteilen: "scanning": die identifikation von issues als grundlegende und somit fast schon wichtigste phase "issues monitoring": analyse und beobachtung der öffentlichen meinung und medien (dazu zählen mittlerweile natürlich auch die "social media") "strategic issue diagnosis": die strategische prüfung und einstufung "message formating": wahl der grundlegenden reaktion/antwort auf strategische issues. hierbei kann noch mal zwischen einem proaktivem und reaktivem vorgehen unterschieden werden "incorporation into strategic plan": integration in die strategische planung issues durchlaufen im öffentliche fokus einen lebenszyklus: je weiter das issue in seinem lebenszyklus voranschreitet, desto geringer wird gleichzeitig die einflussmöglichkeit des betroffenen unternehmens. in den usa gilt das issues management mittlerweile als selbstverständlichkeit, in deutschland hingegen hinkt man dieser entwicklung noch hinterher. im zeitalter des internets bieten spezialisierte firmen z. t. bereits ein professionelles "online-monitoring" an. sie durchsuchen dabei regelmäßig im auftrag ihres kunden das internet nach bestimmten begriffen und begriffskombinationen, die in den interessenbereich des kunden fallen und können so frühzeitig auf entwicklungen mit krisenpotenzial hinweisen. nach angaben des pr-trendmonitor september (faktenkontor gmbh) führten bis % der pressestellen bzw. agenturen bereits ein regelmäßiges webmonitoring für ihre unternehmen bzw. kunden durch. die gleiche studie ergab, dass die pressestellen und agenturen die "social media allgemein" als aktuelle top-no.- -herausforderung auch für sich selbst ansahen. auch kleine und mittlere unternehmen sollten zumindest die regionalen medien und fachzeitschriften ihrer branche regelmäßig beobachten. es ist kein großer aufwand sich von einem mitarbeiter des unternehmens einen wöchentlichen pressespiegel zusammenzustellen zu lassen zu allen relevanten themen, die das unternehmen betreffen. heutzutage hat fast jedes unternehmen eine eigene internetpräsenz (abb. . ). viele nutzen diese präsenzen auch, um in krisensituationen schnell reagieren zu können. krisen laufen fast immer unter einem enormen zeitdruck ab. unternehmen sollten daher schon im "normalzustand" im rahmen ihrer krisenprävention z. b. so genannte "dark-sites" vorbereiten (abb. . ). hierbei handelt es sich um webseiten mit hintergrundinformationen über das unternehmen und seine produkte, die im krisenfall freigeschaltet werden. der wert solcher "dark sites" ergibt sich aus unterschiedlichen aspekten. einerseits können sich journalisten, anwohner und die Öffentlichkeit unmittelbar über die ereignisse informieren, andererseits dokumentiert das unternehmen durch die schnelle reaktion im internet, dass es die ereignisse ernst nimmt. der verdacht, etwas würde verharmlost und vertuscht, kommt somit gar nicht erst auf. auch bekommen die journalisten "futter", sie brauchen sich keine informationen ausdenken, sondern können auf fertige texte und bilder zurückgreifen. "klassiker" wie die suchmaschine "google" beschreiten in der krisenkommunikation neue interaktive wege: nach dem verheerenden erdbeben in japan wurden etliche menschen vermisst und mit dem "person finder" konnte vielen geholfen werden, informationen über den aufenthaltsort oder gesundheitszustand der vermissten in erfahrung zu bringen (http://google.org/personfinder/global/home.html). dieses beispiel zeigt, welche innovativen möglichkeiten sich auch im katastrophenschutz durch die neuen anwendungen bieten. zwischenfälle, die betriebsabläufe stören oder unternehmen schädigen, treten häufig auf. notfälle ereignen sich seltener, katastrophen sind die ausnahme. außergewöhnliche ereignisse können ein unternehmen jederzeit treffen. sie können plötzlich eintreten, oder sich langsam anbahnen. man kann sich von ihnen überraschen lassen, oder man kann glauben, darauf vorbereitet zu sein. die auswirkungen können in beiden fällen verheerend sein. viele unternehmen sind auf unerwartete ereignisse nicht ausreichend vorbereitet. beim eintritt eines ernstfalls sind nicht nur technische anlagen und die funktionsfähigkeit des unternehmens bedroht sondern auch das leben von menschen. zwischenfälle, notfälle und katastrophen wie zum beispiel naturereignisse, sabotageakte oder terroranschläge treten in unterschiedlicher ausprägung, zu jeder tages-und nachtzeit sowie meist vollkommen unerwartet auf. nicht immer kann man alle ursachen vorhersehen, wohl aber deren folgen für das unternehmen. die auswirkungen von notfällen und katastrophen können für unternehmen erheblich sein und im schlimmsten fall zu einer existenzbedrohenden krise führen. ein krisenmanagement ist optimal, um sich und seine mitarbeiter bereits im vorfeld auf solch außergewöhnliche situationen vorzubereiten. ziel ist es, ausfallwahrscheinlichkeiten zu reduzieren und handlungsfähigkeiten zu gewährleisten. technische krisenprävention" verlag technik und information krisen erkennen, bewältigen und vorbeugen -unternehmenswerte proaktiv schützen lexikon risiko-management weiterführende literatur krisenmanagement -strategien im krisen-und notfallmanagement belfor international journal information security management gefährdungs-/belastungsanalysen, in: arbeitsmedizin und arbeitsschutz aktuell systematik zur durchführung von gefährdungsanalysen, verlag für neue wissenschaft gmbh schutz von unternehmenswerten durch krisenmanagement günter schulz, prof. bernd tenckhoff -vernetztes betriebssicherheitsmanagement als teil der unternehmensstrategie für die zukunft -sicherheitsingenieur ganzheitliche anforderungen erfordern ganzheitliche systeme -dguv forum gefährdungsbeurteilung und risikomanagement. ecomed sicherheit key: cord- -axyx eg authors: brocal, francisco; sebastián, miguel a.; gonzález, cristina title: advanced manufacturing processes and technologies date: - - journal: management of emerging public health issues and risks doi: . /b - - - - . - sha: doc_id: cord_uid: axyx eg a general framework of the emerging risks linked with advanced manufacturing processes and technologies is showed. for this, the systemic and occupational nature of said risks is considered. to achieve this general objective, the chapter is organized in two parts. in the first part, a theoretical basis is developed. this theoretical basis is configured by an explanation of the emerging risk concept, as well as by the development of an overview of advanced manufacturing processes and technologies. in the second part, contents and tools of practical application are exposed. to do this, the main emerging risks are shown first. among the fields of application of these risks, some of the most important cross-cutting manufacturing technologies have been selected. one of the main risk governance frameworks is shown. subsequently, this framework is deployed with two of its main applications on emerging risks: the management and characterization of the risk. the first three industrial revolutions came about as a result of mechanization, electricity, and information technology (it). germany has proposed the concept of smart manufacturing to realize the fourth industrial revolution, that is, industry . (qian et al., ) . industry . is a "strategic pcast ( ) focuses in its report on advanced manufacturing, a family of activities that ( ) depend on the use and coordination of information, automation, computation, software, sensing, and networking, and/or ( ) make use of cutting-edge materials and emerging capabilities enabled by the physical and biological sciences, for example, nanotechnology, chemistry, and biology. according to that report, this involves new ways to manufacture existing products, and especially the manufacture of new products emerging from new advanced technologies technological change can reduce some risks while aggravating others or even creating new ones (oecd, ) . ilo ( ) indicates that in the occupational safety health (osh) field, recent decades have seen significant technological advances in the workplace, which, together with rapid globalization, have transformed work for many throughout the world. the effects of such changes on osh have also been significant, so that in some cases, more traditional hazards and risks have been reduced or eliminated, for example, through plant automation, but new technologies have also created new risks (ilo, ) . advanced manufacturing processes are characterized by innovative variables of a technological and organizational nature that tend to change with workplaces, processes and conventional work practices, and can generate, as well as traditional occupational risks, other so-called new and emerging risks (ners) (brocal and sebastián, a) . this raises new challenges for workers and companies and, in turn, creates political demands, administrative and technical approaches that ensure high levels of safety and health at work (adapted from eu-osha, ). the general objective of this chapter is to show a general framework of the emerging risks linked with advanced manufacturing processes and technologies. for this, the systemic and occupational nature of said risks is considered. to achieve this general objective, the chapter is organized in two parts. in the first part, a theoretical basis is developed. this theoretical basis is configured by an explanation of the emerging risk concept, as well as by the development of an overview of advanced manufacturing processes and technologies. among these manufacturing technologies, three of the most important have been selected in order to provide a broader overview. these three manufacturing technologies are: intelligent manufacturing, iot-enabled manufacturing, and cloud manufacturing. in the second part, contents and tools of practical application are exposed. to do this, the main emerging risks are shown first. among the fields of application of these risks, some of the most important cross-cutting manufacturing technologies have been selected in order to develop them in greater detail. these manufacturing technologies are: automation; human machine interfaces (hmis), and information and communication technologies (icts) . second, one of the main risk governance frameworks is shown. subsequently, this framework is deployed with two of its main applications on emerging risks: the management and characterization of the risk. finally, a brief summary of the chapter is presented, accompanied by the main conclusions. risk is defined in many ways (aven, ) , and many perspectives on risk are developed and used for specific disciplines and areas (aven, ) . however, considering the most common definitions of risk found in scientific standards and literature, aven ( ) states that the common feature of all these definitions is that the concept of risk comprises events (initiating events, scenarios), consequences (outcomes), and probabilities. as aven ( ) believes, though it is impossible to present and discuss all definitions of the risk concept suggested and used in scientific risk fields, the author proposes the categories of definitions/ perspectives on risk as used in professional/scientific contexts (table . ). the iso : standard (iso, a defines risk as the effect of uncertainty on objectives. likewise (continuing with the definitions of this standard), objectives can have different aspects (such as financial, health and safety, and environmental goals) and can apply at different levels (such as strategic, organization-wide, project, product and process). risk is often expressed in terms of a combination of the consequences of an event (including changes in circumstances) and the associated likelihood of occurrence (iso, a) . in the specific field of occupational risk, ohsas standard defines risk as the combination of the likelihood of an occurrence of a hazardous event or the exposure and the severity of injury or ill health that can be caused by the event or exposure (aenor, ) . and the iso standard (the current version is the iso/dis . : ) defines an occupational health and safety risk as the combination of the likelihood of an occurrence of a work-related hazardous event or exposure, and the severity of injury or ill health that can be caused by the event or exposure (iso, a) . the standard is currently being developed by a committee of occupational health and safety experts, and will follow other generic management system approaches such as iso (iso, a) and iso (iso, b) . it will take into account other international standards in this area such as ohsas , the international labor organization's ilo-osh guidelines, various national standards, and the ilo's international labor standards and conventions (iso, b) . the revision of this new standard, iso , is in its final stage (aenor, ) . the final publication of the standards is expected in march (iso, b). risk ¼ probability of an (undesirable) event (r ¼ p) risk¼objective risk ¼ probability and scenarios/consequences/ severity of consequences (r ¼ p&c) risk ¼ event or consequence (r ¼ c) risk risk is the effect of uncertainty on objectives (r ¼ iso) irgc ( a) considers that there are emerging risks of a systemic nature, which typically span more than one country, more than one economic sector, and may have effects across natural, technological, and social systems. these risks may be relatively low in frequency, but they have broad ramifications of human health, safety and security, the environment, economic well-being, and fabric of societies. irgc ( b) aims to shed light on emerging risks with a perspective that has three elements: sources of risk, drivers of risk, and governance issues. irgc ( a) defines as emerging a risk that is new, or a familiar risk that becomes apparent in new or unfamiliar conditions. the concept of familiarity assumes the existence of recognizable patterns and management regimes that are relatively stable and have proven to be effective if implemented according to certain rules. by contrast, emerging risks are characterized mainly by uncertainty regarding their potential consequences and/or probabilities of occurrence (irgc, a) . irgc ( ) eu-osha defines ner through the reports cited as listed following by ner definition. in this definition, unlike the original sources cited (flaspöler et al., ; brun et al., a brun et al., , b brun et al., , , it is the codification (ci), which in this chapter is called "conditions," that defines an ner (brocal et al., ) . ner definition: any occupational risk that is both new and increasing: • by "new," we mean that: c . the risk did not previously exist and is caused by new processes, new technologies, new types of workplace, or social, or organizational change; or, c . a long-standing issue is considered as a new risk due to a change in social or public perceptions; or, c . new scientific knowledge allows a long-standing issue to be identified as a risk. • the risk is "increasing" if the: c . number of hazards leading to the risk is growing; or c . the exposure to the hazard leading to the risk is increasing (exposure level and/or the number of people exposed); or c . the effect of the hazard on workers' health is getting worse (seriousness of health effects and/or the number of people affected). the ner definition adopted by the eu-osha has been integrated into cwa : (cen, ). however, as brocal et al. ( ) consider, such integration implies two modifications, a terminological modification and another of interpretation. so, instead of using the term "new and emerging risk," the term "emerging risk" is employed, which has been defined as "any risk that is both new and/or increasing." in this regard, one can deduce that the term "new and emerging risk" has been simplified from "emerging risk" (brocal et al., ) . such simplification can be seen, for example, in the work developed by . similarly, houtman et al. ( ) state that an "emerging osh risk" is often defined as "any occupational risk that is both new and increasing." in any case, as brocal ( ) indicates, these quotes reveal a clear problem with consensus about terminology and interpretation of the ner concept. similarly, flage and aven ( ) feel that the term "emerging risk" has an intuitive appeal and meaning, although a consistent and consensual definition is lacking. in addition, the ner definition has been developed without following any risk model reference, thus hindering its use in the ner identification and analysis (brocal and sebastián, a,b) . to solve this problem, brocal and sebastián ( a) have developed a new risk model whose objective is to characterize the ner. nevertheless, this model has two limitations (brocal et al., ) . first, the model does not contemplate the possibility of considering independently the new risks (nr) and increasing risks (ir). second, this model does not allow monitoring the evolution of ner over time, therefore it is not currently possible to determine, in general, the temporary validity of such risks. in order to solve these problems, brocal et al. ( ) have developed a theoretical framework for the modeling of the ner that allows its monitoring through the technology lifecycle (tlc), especially in industrial processes. zhu et al. ( ) considered two major classifications of manufacturing processes: the first by swift and booker ( ) classifies processes into casting, cutting, forming, and fabrication. the second by kalpakjian and schmid ( ) is more comprehensive; they classify processes into casting, machining and finishing, joining, sheet metal, polymer processing, and bulk deformation processes. as the traditional classifications have some difficulties in the identification of newly developed manufacturing technologies (zhu et al., ) , nassehi et al. ( ) proposed a technology-based classification method consisting of five categories, namely joining, dividing, subtractive, transformative, and additive technologies. zhu et al. ( ) , briefly describe these five categories: • joining technology: consists of processes by which two or more workpieces are joined to form a new workpiece; • dividing technology: dividing processes are the opposite of joining processes; • subtractive technology: subtractive/negative operations are material removal processes, by which material is removed from a single workpiece, resulting in a new workpiece; • transformative technology: a single workpiece is used to create another workpiece and the mass does not change; • additive technology: material is added to an existing workpiece to build a new workpiece, where the mass of the finished workpiece is greater than before. advanced manufacturing relies on new technologies that enable flexibility, agility, and the ability to reconfigure, as for example the following applicable areas: biomanufacturing, semiconductors, advanced materials, additive manufacturing, and nanomanufacturing (esmaeilian et al., ) . emerging technologies can have game-changing impacts on manufacturing models, approaches, concepts, and even businesses (zhong et al., a) . as an and ahn ( ) indicate, several organizations have forecasted emerging technologies and reported on their forecasts, so that some well-known forecasts are "disruptive technologies" (mckinsey global institute), " breakthrough technologies" (mit), "next in " (ibm), and "top strategic technologies" (gartner group). the main results of these forecasts are discussed next. as mckinsey global institute indicates, economically disruptive technologies transform the way we live and work, enable new business models, and provide an opening for new players to upset the established order (manyika et al., ) . mckinsey global institute considers that the potentially economically disruptive technologies are: mobile internet; automation of knowledge work; iot; cloud technology; advanced robotics; autonomous and near-autonomous vehicles; next-generation genomics; energy storage; d printing; advanced materials; advanced oil and gas exploration and recovery; and renewable energy (manyika et al., ) . every year mit technology review publishes a list containing breakthrough technologies. the list for the year is as follows (mit, ): reversing paralysis; self-driving trucks; paying with your face; practical quantum computers; the -degree selfie; hot solar cells; gene therapy . ; the cell atlas; botnets of things; and reinforcement learning. ibm ( ) considers five innovations that will change our lives in the next years: with artificial intelligence (ai), our words will be a window into our mental health; hyperimaging and ai will give us superhero vision; macroscopes will help us understand earth's complexity in infinite detail; medical labs "on a chip" will serve as health detectives for tracing disease at the nanoscale; and smart sensors will detect environmental pollution at the speed of light. every year gartner publishes a list containing the top strategic technology trends. the list for the year is as follows(gartner group, ) : ai and advanced machine learning; intelligent apps; intelligent things; virtual and augmented reality; digital twin; blockchain and distributed ledgers; conversational system; mesh app and service architecture; digital technology platforms; and adaptive security architecture. the evolution of integrated and intelligent manufacturing (i m) technology is driven not only by the market demand but also by technological advances. there are major technologies ( fig. . ) that can be identified as the key elements of the new manufacturing paradigm (chen, ) . zhong et al. ( a) consider that the three major advanced manufacturing technologies are the following: intelligent manufacturing, iot-enabled manufacturing, and cloud manufacturing. these manufacturing technologies are briefly described in the following sections. intelligent manufacturing systems represent one of the latest developments in both manufacturing technologies and organization (dumitrache and caramihai, ) . the basic idea involves flexibility in systems, monitoring, and adaptation to changing needs (kumar, ) . the targeted objective is to gain the most benefit for product quality, cost reduction, and increase of efficiency in general (zillner et al., ) . qian et al. ( ) consider four objectives in relation to the layer of engineering technology: digitization, intellectualization, cyberization (to develop cyber-physical systems), and automation. at the same time, these authors consider four objectives in relation to enterprise production and operation: agility, high efficiency, environmental sustainability, and safety. intelligent manufacturing makes use of advanced computational technologies and the advancements in digital systems and machine learning processes to support decision-making, run self- ten major technologies for i m (chen, ). sufficiently via networks of distributed control, and to self-adjust and self-correct should problems arise (oliff and liu, ) . systems' theory, communications, and nanotechnologies, all characterized by solid formal supports, have contributed decisively to a transition from the c paradigm (computer-control) to the c paradigm (computer, communication, cognition for control) (dumitrache, ) . intelligent manufacturing systems represent a reality in the framework of c paradigm and should be analyzed and designed as cyber-physical systems (cpss) (dumitrache and caramihai, ) . currently, there is an ongoing transformation of classical products and machinery toward cpss (becker and stern, ) . cpss link the physical world seamlessly with the virtual world of information technology and software (sztipanovits and ying, ) . main features of these systems are the real-time data exchange between various technical and computational elements enabled by communication technologies and data processing ability provided by embedded systems (becker and stern, ) . the introduction of both cps and the iot, where things are supposed to initiate both a process of preparation, design, planning, optimization, tasks for tools, and humans if necessary, is leading in a fourth industrial revolution referring to the future (mrugalska and wyrwicka, ) . this idea is represented schematically through table . . qian et al. ( ) consider that the intrinsic safety of the manufacturing process as well as enterprise information security must be ensured, and to realize the safe running of manufacturing processes by means of fault diagnoses and self-healing control techniques. for example, with advancements in networking and internet technologies, cyberattacks on physical systems are becoming a growing phenomenon (desmit et al., ). tarkoma and ailisto ( ) indicate that recent advances in radio, network, mobile, and cloud technologies have supported the development of the first-generation iot services and products. these authors consider that iot as a concept is not new as it was proposed over years ago in the mit auto-id center, so that the concept has since gained momentum and importance, and now it is considered in europe, the united states, and asia, not only on the implementation and deployment level but also on the societal level. early iot applications are based on radio frequency identification (rfid) and wireless sensor network (wsn) technologies, and deliver tangible benefits in several areas, including manufacturing, logistics, trade, retail, and green/sustainable applications, as well as in other sectors (kefalakis et al., ) . the adoption of iot in manufacturing enables the transition of traditional manufacturing virtualization and integration systems into modern digitalized ones, generating significant economic opportunities through industries' reshaping (mourtzis et al., ) . for example, iot technologies such as rfid and wireless communications are used for capturing real-time machine status (zhong et al., b) . industrial iot devices for manufacturing include sensors/actuators, computers with wireless networks, etc., which have contributed significantly to different aspects of manufacturing such as automation and tracking (he et al., ) . trappey el al. ( ) divides the iot architecture into four layers, as briefly described in table . . iot-enabled manufacturing is an advanced principle where typical production resources are converted into smart objects that are able to sense, interconnect, and interact with each other to automatically carry out the manufacturing activities (zhong, ) . considering the work developed by tao et al. ( ) , it can achieve the complete connection between terminal devices and the enterprise information management system for the automatic control of the iot-enabled manufacturing execution in workshops. therefore, three functions should be addressed: the access, identification, and control of the physical manufacturing execution process from materials and semifinished products to the final products (tao et al., ) . computers play an essential role in increasing efficiency, capability, and adaptability of manufacturing systems, as for example: computer integrated manufacturing, distributed manufacturing, agile manufacturing, cps, and cloud manufacturing (yu et al., ) . cloud computing is emerging as one of the major enablers for the manufacturing industry; it can transform the traditional manufacturing business model, help it to align product innovation with business strategy, and create intelligent factory networks that encourage effective collaboration (xu, ) . in general, cloud-related manufacturing research can be categorized into two types: cloud computing in manufacturing industry, and cloud manufacturing systems . perception deals with the types of sensors (e.g., sensors are used to detect temperature, weight, motion, vibration, etc.) and actuators that help the physical object to perceive. perception is used in upper layers to achieve the final goal of micro intelligence at the application layer. transmission transmission is the second layer in the iot ecosystem. the next step after perception (collection of sensor information) is to transmit the information to the upper layers. computation the computation layer describes means of receiving data, processing data, making decisions, and delivering the decisions to the application layer. the computation layer consists of hardware, software, algorithms, cloud computing, big data analysis, and security. application the application layer provides tactical understanding by using information collected and transmitted from lower layers. in , bohu et al. ( ) put forward the concept of cloud manufacturing (xin-yu and wei-jia, ). cloud manufacturing is a new, networked, and intelligent manufacturing model that is service oriented, knowledge based, high performance, and energy efficient. in this model, state-of-the-art technologies such as informatized manufacturing, cloud computing, iot, semantic web, and highperformance computing are integrated in order to provide secure, reliable, and high-quality ondemand services at low prices for those involved in the whole manufacturing lifecycle (bohu et al., ) . ooi et al. ( ) note that cloud technology enables on-demand service and resource access, which revolutionized the computing service market. these authors consider that the flexibility and borderless resources provided by cloud technology motivated manufacturers to take advantage of the cloud application service in an effort to improve and innovate their manufacturing process. the cloud application service provided manufacturers with cloud-based software application, web-based management dashboard, and cloud-based collaboration, which bring the manufacturing process, management, and monitoring to the cloud, hence the birth of cloud manufacturing (ooi et al., ) . compared with cloud computing systems, one of the biggest challenges cloud manufacturing faces involves numerous types of physical resources, e.g., machine tools and robots . according to yu et al. ( ) , cloud manufacturing may either apply or not apply the cps s cyber architecture. in other words, cyber systems in cloud manufacturing may choose to be networked or not, and more significantly, cloud manufacturing tries to imitate the paradigm of cloud computing in which design, manufacturing, and other resources are encapsulated as services (yu et al., ) . mobile cps has emerged with advances in cloud computing and wireless sensing technologies (chen, ) . liu et al. ( ) consider that a cps for manufacturing is not a manufacturing cloud if it does not use virtualization technique in cloud computing and service-oriented architecture in service computing. on the other hand, these authors note that a manufacturing cloud is not a cps if it does not have components for direct interactions with machine tools and other physical devices. liu et al. ( ) point out that it is necessary to integrate cloud manufacturing and cps for providing manufacturing services, which can directly operate and monitor machine tools in a manufacturing cloud. as a result, said authors indicate that a new paradigm of a cyber physical manufacturing cloud (cpmc) is created, so that a cpmc is a type of manufacturing cloud where machining tools can be directly monitored and operated over the internet from clouds . this section discusses the main emerging risks linked with advanced manufacturing processes and technologies. first, a general framework on emerging systemic risks related to new technologies is shown. next, this framework is expanded specifically to the osh field. third, some of the most important cross-cutting manufacturing technologies have been selected in order to develop them in greater detail. these manufacturing technologies are: automation, hmis, and icts. these manufacturing technologies are related to both occupational accidents and major accidents. therefore, the related emerging risks have a broad nature. in the report on emerging systemic risks developed by the oecd ( ), five categories of such risks are addressed: natural disasters, industrial accidents, infectious diseases, terrorism, and food safety. according to this report, three aspects of emerging technologies will influence risk: connectedness, the speed and pervasiveness of technological change, and the fundamental changes in the landscape they might induce. the analysis and main conclusions from oecd ( ) eu-osha took the first steps in the field of ners with the publication of four specific reports on emerging physical risks (flaspöler et al., ) , emerging biological risks (brun et al., a) , emerging psychosocial risks (brun et al., b) , and emerging chemical risks (brun et al., ). the methodology used in these reports to identify ners was based on the delphi method, using the likert -point scale to classify such risks. for each risk identified, the mean values (mvs) and the standard deviations were calculated. in this way, each risk was classified as "risk strongly agreed as emerging," "risk agreed as emerging," "undecided," "there is agreement that the risk is not emerging," or "there is strong agreement that the risk is not emerging." the top ners of each of these reports are shown in tables . e . . for this, the mvs have been taken into account. these ners are classified as "risks strongly agreed as emerging" and "risks agreed as emerging." brocal and sebastián ( b) analyzed the ners included in the four specific reports on emerging physical risks (flaspöler et al., ) , emerging biological risks (brun et al., a) , emerging psychosocial risks (brun et al., b) , and emerging chemical risks (brun et al., ) . these authors considered only the ners classified as "risk strongly agreed as emerging" and "risk agreed as emerging." each risk classified as "undecided," "there is agreement that the risk is not emerging," and "there is strong agreement that the risk is not emerging," cannot be considered as ner. among the results obtained, brocal and sebastián ( b) identified a set of technological aspects (table . ) and organizational aspects (table . ) linked to advanced manufacturing processes. in the prospective study on ner associated with new technologies in (ellwood et al., ) , the key technologies that may be introduced in green jobs by , which may lead to ners in the workplace, are the following: wind energy (industrial scale); green construction technologies (buildings); bioenergy and the energy applications of biotechnology; waste processing; green transport; green manufacturing technologies and processes, including robotics and automation; electricity transmission, distribution, and storage, and domestic and small-scale renewable energy; and nanotechnologies and nanomaterials. it is expected that by approximately % of all goods manufactured around the world will be based to some extent on the use of nanotechnology (ilo, ). remote operation in environmentally sensitive areas. emerging risks related to interaction between natural hazards and technologies at the community level. adapted from cwa : . poor awareness of thermal risks among low-status worker groups exposed to unfavorable thermal conditions (e.g., migrant workers in agriculture and construction areas working overtime in hot/cold areas such as greenhouses/cold stores). · high density of animals in confined spaces in contact with humans leading to increasing zoonosis cases (diseases jumping the species barrier from animals to humans). · high population density and increase in business trips, tourism, and immigration helping zoonoses and other infectious diseases to spread quickly worldwide. groups particularly at risk of contamination: staff involved in producing, processing and transporting livestock; airport staff and air crews; staff involved in border controls and policing; staff in health care sector; public transport; and staff in public services. the risk is often underestimated, which leads to a lack of preventive measures. poor or difficult assessment of biological risks. . general increased use of antibiotics for human health care and for animal breeding in the food industry leading to the appearance of drug-resistant pathogens (e.g., methicillin-resistant staphylococcus aureus (mrsa), tubercule bacillius). health effects observed: increase in staff infected with mrsa in western hospitals; increasing antibiotic resistance of livestock farmers and in the population in general. lack of information on biological risks in different workplaces (e.g., office workplaces, agriculture). poor maintenance of air-conditioning (whose use is increasing) and water systems (e.g., legionella, aspergillosis in hospitals). new knowledge about the presence of legionella will help the correct diagnosis of symptoms so far wrongly attributed to other diseases like flu. . inadequate training, poor knowledge of osh, or even poor basic awareness of risks of local authorities' staff (e.g., sewage, excavations, waste collection, etc.). biohazards in waste treatment plants (e.g., selective sorting, manufacture of compost) leading to allergies, infectious diseases (bacteria, viruses), toxinic diseases (endotoxins, mycotoxins), and cancers (oncogenes). especially in composting facilities where there is a wide variety of microorganisms present at the different stages of the composting process, the risks are not completely identified yet. bioaerosols and chemicals, the combined effects of which have been very little studied but lead to allergies. more knowledge will help identify the real multifactorial causes of symptoms for which monocausal explanations have been given so far. in the manufacturing sector, the following osh issues in particular need to be considered (this list is not comprehensive) (ellwood et al., ) : • new processes and materials leading to potential exposure to new (green) substances, including nanomaterials, or substances used or emitted (including dust) from new (green) manufacturing processes; • the extent of chemical use and the potential for exposure as manufacturing is distributed to smaller units as a result of rapid manufacturing techniques ( d printing); • the difficulty in monitoring osh in distributed manufacturing taking place in smaller businesses; • the increasing use of lasers in techniques such as rapid manufacturing; the potential physical risks from human-robot interaction as robots gain increasing autonomy and become freeroaming; • potential psychosocial risks: (a) the high cognitive load of the hmi, "lean" production and justin-time principles all have the potential to contribute to job intensity and pressure and to result in psychosocial problems; (b) the potential effect of renewable energy, with its intermittent nature currently, on shift work in those companies that take interruptible supplies, resulting in more unpredictable working hours; • workers possibly resorting to human performance-enhancing technologies as they feel the need to keep up with coworkers, and maybe even with robots (this could also be a feature of other sectors); and • a focus on safety as opposed to health in competitive scenarios owing to the greater impact of accidents on productivity (this could also be a feature of other sectors). cross-cutting issues are inevitably more general than those concerning specific technologies, but they are important, often more so. in the field of manufacturing processes, the following cross-cutting technologies can be specially considered (based on the study by ellwood et al., ( ) ): • rapid innovation: it may lead to a variety of osh risks, with new materials and new processes, and little time to learn how to use them safely; endotoxins: high concentrations in various industrial settings (e.g., in workplaces exposed to organic materials (straw, wood, cotton dust), waste treatment, poultry houses, swine confinement buildings) leading to asthma, loss of lung function, etc. . molds in indoor workplaces due to new construction methods and materials, due to the aim of saving energy and due to the lack of maintenance: exposure to fungal spores for office workers and especially workers involved in building restoration, leading to sensitization and allergies. . • new materials: they have the potential for major unexpected impacts on health and environment: nanomaterials, new insulating materials, new composites, smart materials, new organisms, biofuels, and by-products; • automation: it is most likely to be very positive for safety in the long term, but absolute reliability is essential; • human-machine and human-ict interfaces: they can give rise to complex risks (e.g., ergonomics and high cognitive load) and overreliance on ict. isocyanates leading to allergic reactions: exposure occurs not only at the production stage but also during further processing (e.g., thermal or chemical degradation of polyurethane, grinding and welding of products containing polyurethane, for example, in car repair shops). man-made mineral fibers (e.g., refractory ceramic fibers, carbon/graphite fibers, or composites): lack of knowledge on health effects of (new) fiber substitutes for asbestos, the use of which is increasing and for which exposure levels seem high enough for concern in certain areas. potential health effects: respiratory diseases, cancer. construction industry (civil and industrial sector, including demolition, rebuilding, and renovation activities): exposure to dangerous substances (crystalline silica dust, asbestos, wood dust, diesel engine exhaust, welding fumes) leading to occupational cancers. the automation of manufacturing processes can produce emerging risks, linked both to the process and a whole (system), and to specific components, such as robots, hmis, and icts (brocal et al., ) . in this way, among the different manufacturing technologies that have been studied throughout this chapter, automation, hmis, and icts can be selected in order to expand them as possible sources of emerging risks. ellwood et al., ( ) indicate that in the field of manufacturing processes, such technologies can be considered as cross-cutting technologies. therefore, studying them can expand the overview of emerging risks. automation is the growing phenomenon of human labor being replaced by machinery and robotics (euromonitor international, ) . industrial automation is a vast and diverse discipline that encompasses processes, machinery, electronics, software, and information systems working together toward a common set of goals: increased production, improved quality, lower costs, and maximum flexibility (mehta and jaganmohan, ) . the reasons to automate manufacturing processes include increased quality and efficiency demands, as well as the presence of hazardous working conditions and the high cost of specialized manual laborers (botti et al., ) . granell et al. ( ) conducted a survey with the aim of discovering the perception of production managers and manufacturing engineers working in industry about concepts such as levels of automation and automation strategy, and to determine the current status of manufacturing and automation issues. the sample was derived from the association of swedish engineering industries. the factors and results when making decisions about automation are shown in table . . in the expert forecast on emerging physical risks related to osh published by eu-osha (flaspöler et al., ) , these emerging risks related to automation were identified: "automation leading to an increase in occupational accidents in maintenance and production tasks" and "automation leading to poor job content (repetitive and monotonous work) and consequently to musculoskeletal disorders (msds) and stress." thus, the two ners above can be classified into two groups, in light of their consequences (brocal et al., ) : a. accident risks: flaspöler et al. ( ) state that increased automation involves an increased risk of accidents from human errors, and they usually affect users, butdespecially in the case of high-risk industriesdhave the potential for serious consequences beyond the operator to include fellow workers, the community at large, and the environment. with this increased automation, operator tasks have been reduced from directly controlling the process to monitoring the control systems and controlling them (chidambaram, ) . this drop in hands-on knowledge and responsibility can lead to a lack of sufficient understanding of the underlying process, which can in turn lead to an accident due to someone doing something inappropriate or not knowing what to do when something goes wrong (stacey et al., ) . new technologies, when introduced poorly, entail the risk of increasing operators' mental workload and also of reducing their degree of control over the task, so that if the measures taken are not appropriate increases the risk of negative health effects such as stress and is likely to result in overreliance on automated safety systems (flaspöler et al., ) . b. psychosocial and musculoskeletal risks: these risks are intimately related, and can be studied together or independently, depending on prevention targets. from the joint perspective on new and emerging risks, flaspöler et al. ( ) states that in automated processes, psychosocial and musculoskeletal problems are caused by reduced physical activity, more static postures, and higher mental workload (e.g., when monitoring and controlling); less privacy at work (as technology allows closer and more intrusive supervision); and more decision-making problems. from the latest eu-osha european survey of enterprises on new and emerging risks (esener ) conducted in (eu-osha, ), and from the analysis of the survey performed by irastorza et al. ( ) , one can see that the risk of accidents with machines or hand tools is the most frequently reported risk factor in mining and quarrying ( % of establishments in the sector in the eu- ); water supply, sewerage, waste management and remediation activities ( %); construction ( %); electricity, gas, steam, and air conditioning supply ( %); agriculture, forestry and fishing ( %); and manufacturing ( %). in the esener analysis conducted by irastorza et al. ( ) , the measures to prevent psychosocial risks are generally less likely to be implemented in construction and manufacturing, as well as in agriculture, forestry, and fishing. the two most frequently reported psychosocial risk factors across all sectors are: "having to deal with difficult customers, patients, pupils, etc." and "time pressure." regarding musculoskeletal problems, irastorza et al. ( ) synthesize that in the manufacturing sector, after risk of "accidents with machines or hand tools %," the most frequent risk factor was "repetitive hand or arm movements ( %)." stacey et al. ( ) indicate that use of computers and automated systems at work leads to immobile body postures and physical inactivity at work. physical inactivity is associated with increased health risks such as coronary heart disease, being overweight and obesity, certain types of cancers, and psychological disorders such as depression and anxiety (stacey et al., ) . the basic requirements for system control, including networks and hmis, are introduced together with basic principles of safety and guarding for automation systems (wilson, ) . the hmi governs the flow of information from the machine to the user (in terms of displays, warning sounds, etc.) and from the user to the machine (in terms of input or control devices such as keyboards, switches, levers, etc.) (flaspöler et al., ) . the use of ict is radically changing the collective experience of work, from a group of people who interact physically to a dispersed community of contacts, whose interaction may be more ad hoc (eurofound, ) . as messenger and lutz ( ) note, forms of ict, like smartphones and tablets, promise historic changes in the way we work, as they provide new possibilities for working anytime and anywhere. this new spatial independence changes the role of technology in the work environment, bringing both new opportunities and new challenges (messenger and lutz, ) . increasing complexity and increasing use of icts in automated manufacturing has led to hmi issues (ellwood et al., ) . ict, including ict-enabled technologies (ict-ets) such as robotics and ai, are likely to have major impacts on the nature and location of work over the next years (stacey et al., ) . how humans will react to the continuous high cognitive load of increasingly complex hmis is still unknown (ellwood et al., ) . workers are more likely to make mistakes due to a poorly designed hmi (stacey et al., ) . there is also evidence that over the next decade there are likely to be significant and accelerating changes in relation to ict-ets, which will considerably change the nature of work across europe and affect most people in some way (stacey et al., ) . the expert forecast on emerging physical risks related to osh published by the eu-osha (flaspöler et al., ) states that a poor design of the interface may result in increased mental and emotional demands on the operator. hence a potential increase in the incidence of stress, human errors, and accidents. flaspöler et al. ( ) published a report that aimed to follow up the results obtained in this expert forecast. this report further researched hmi as an emerging risk, exploring whether complexity of hmis leads to safety and health risks such as increased mental and emotional strain for users. thus, the conclusions drawn in the report that merit mention include: • the design of hmi has important consequences for health and safety at work because of the growing potential for accidents and ill poor health for machine operators; • deficiencies in the hmi present two different risks: (a) psychosocial risk (stress can result from cognitive overload or underload); and (b) accident risk (occupational accidents and major accidents can result from operating errors); • the majority of machine users work in smes, mostly in manufacturing. according to koshti and joshi ( ) , many will recognize the most typical hmi "issues," as shown in table . . irgc ( ) puts forward an integrated analytic framework for risk governance that provides guidance for the development of comprehensive assessment and management strategies to cope with risks, in particular at the global level. the council of the society for risk analysis (sra) has developed a glossary about key generic concepts within the field of risk analysis (sra, ) . the glossary terms are divided into three categories: ( ) terminology on basic concepts; ( ) terminology on related concepts, methods, procedures; and ( ) terminology on risk management actions. the definition of risk governance is one of these key terms. its definition is the following: risk governance as the application of governance principles to the identification, assessment, management, and communication of risk. governance refers to the actions, processes, traditions, and institutions by which authority is exercised and decisions are taken and implemented. risk governance includes the totality of actors, rules, conventions, processes, and mechanisms concerned with how relevant risk information is collected, analyzed, and communicated and management decisions are taken. irgc risk governance framework (irgc, ) is illustrated in fig. . . this framework is divided into three main phases: preevaluation, appraisal, and management. a fourth phase that includes the characterization and evaluation of risk is added. irgc ( ) places this phase between the appraisal and management phases and, depending on whether those charged with the assessment or those responsible for management are better equipped to perform the associated tasks, it can be assigned to either of themdthus concluding the appraisal phase or marking the start of the management phase. the risk process has communication as a companion to all phases of addressing and handling risk and is itself of a cyclical nature (irgc, ) . the user must constantly switch between screens to perform the steps required by a single procedure. display confusion this is the reverse problem, when too much information is presented on the same screen, with no obvious purpose or structure. inconsistent controls similar controllable objects that react differently depending on which display they are located, or conversely, many dissimilar controls all related to the same task. additional procedural steps unrelated to the equipment being controlled, but required by internal functions of the control system. this happens frequently when users are forced through several levels of submenus in order to reach the desired action. although computer performance keeps improving, software complexity increases at the same rate. the problem of slow response to user actions will usually be inferior during a plant upset, precisely when fast response is the most important. this happens when operators are deluged with system messages and alarms. the determination of the cause of equipment "trip" becomes a painstaking investigation through interminable alarm logs. this is perhaps the area where recent systems have improved the most. however, application programmers still require significant time investment to modify or expand existing systems. the risk science and safety literature and leaders in the study of human behavior agree that in many organizations, there is not enough effective communication (irgc, ) . however, the clear sequence of phases and steps offered by this process is primarily a logical and functional one and will not always correspond to reality (irgc, ) . irgc risk governance framework (irgc, ) is a worthwhile basis for diagnosing governance deficits, and is broad and flexible enough to be adapted to diverse governance issues and contexts. thereby, various organizations use this framework to structure their thinking and inspire guidelines, roadmaps, or models, for example (irgc, ) since the publication of the irgc framework, other guidance documents or frameworks have been published, such as iso : , risk management e principles and guidelines (irgc, ) . iso : standard provides principles and generic guidelines on risk management, and it can be used by any public, private, or community enterprise, association, group, or individual. therefore, iso : is not specific to any industry or sector. it can be applied throughout the life of an organization, and to a wide range of activities, including strategies and decisions, operations, processes, functions, projects, products, services, and assets. likewise, iso : can be applied to any type of risk, whatever its nature, whether having positive or negative consequences (iso, a) . iso : standard defines risk management as coordinated activities to direct and control an organization with regard to risk. this process includes the activities shown in fig. . . sra ( ) defines risk management as those activities to handle risk such as prevention, mitigation, adaptation, or sharing. it often includes trade-offs between costs and benefits of risk reduction and choice of a level of tolerable risk. among these activities ( fig. . ), the risk assessment process can be considered key to successful risk management. iso : standard defines risk assessment as the overall process of risk identification, risk analysis, and risk evaluation. sra ( ) defines risk assessment as systematic process to comprehend the nature of risk, express and evaluate risk, with the available knowledge. the perspectives of the glossaries included in iso : and sra ( ) should be considered different. in this regard, sra ( ) indicates that its glossary is unique in its approach compared to existing risk analysiserelated glossaries (including the iso on risk management terminology), with its incorporation of different perspectives and its between overall qualitative concepts and their measurements. in the case of emerging risks, irgc ( ) focuses on these risks in technology and industry. such focus is on how to overcome the major obstacles that commonly prevent organizations from improving their emerging risk management. for this, irgc ( ) presents themes for the three categories of risk a, b, and c described in table . . each theme derives from one obstacle and is described in such a way as to provide clarity and operational significance for managers who have the task of identifying, assessing, evaluating, prioritizing, and managing the early phases of development of an emerging issue. these themes interact considerably, but for conceptual clarity, irgc ( ) has grouped them according to the relevant dimensions of risk governance within which they fit best. these four dimensions of risk governance (from the broad, foundational level to the more specific) and the themes are shown in fig. . and are summarized in table . . the irgc guidelines for emerging risk governance (irgc, a) describe key steps and associated methodologies for early identification and management of emerging risks. the process is described schematically in fig. . , and covers an overarching, flexible, and adaptable set of guidelines designed to deal with complex, evolving, and uncertain environments (irgc, b). irgc ( b) reviews other existing emerging risk governance frameworks. these emerging risk governance frameworks are the following: european union agency for network and information security, the european food safety authority, the swiss reinsurance company (sonar system), the cwa : , and the dutch framework for identifying and managing emerging risks involved in the use of chemicals. the cwa : is one of the main results of integ-risk. this project has summarized its results in a set of major deliverables, labeled, at the end of the project by the developers, as the "big " of integ-risk. they are (jovanovi c and balo s, ): ( ) integ-risk catalogue of emerging risks e riskears system; ( ) integ-risk framework for emerging risk management; ( ) the library of cwa : gives guidance on steps for applying/implementing the proposed ermf in industrial organizations. this ermf is heavily influenced by the irgc ( ) risk governance framework and the iso : risk management process. the main difference is that some issues that are included in other steps in irgc ( ) and/or iso : have been explicitly addressed as a separate step in the ermf, and a particular focus on capturing the earliest signs of an emerging risk (cwa : ) . the steps of the ermf are illustrated in fig. . . risk characterization allows decision makers to distinguish scientific facts from policy orientations when analyzing risk assessment results (cwa : ) . the most controversial phase of handling risk, risk characterization and evaluation, aims at judging a risk's acceptability and/or tolerability (irgc, ) . in the report on emerging systemic risks developed by the irgc ( b), it is indicated that some emerging risks do not prove to be as significant as originally feared, but others may prove to be worse than expected, with a high potential for major losses. in this report it is added that typically, the future consequences of emerging risks cannot be defined in monetary terms, at least not to any satisfactory degree of precision, so the conventional approaches to projecting loss size, relative frequencies, or probability distributions over time or severity of consequences are ineffective. indeed, it is often difficult to establish causality between the source of the emerging risk and its consequences using conventional technical or scientific data (irgc, b). brocal and sebastián ( a) have developed a new risk model whose objective is to characterize the ner. this characterization is based on the so-called risk model. this model describes a risk as a structure consisting of five components, being: the source of risk, causes, events, consequences, and the likelihood. this risk model is compatible with the risk characterization defined by sra ( ) . this definition is the following: risk characterization as a qualitative and/or quantitative picture of the risk; i.e., a structured statement of risk usually containing the elements: risk sources, causes, events, consequences, uncertainty representations/measurements (for example, probability distributions for different categories of consequencesdcasualties, environmental damage, economic loss, etc.) and the knowledge that the judgments are based on. irgc ( ) concludes that managers need to be open to the possibility of adverse future events and to plan for them, using the best information they can obtain. investments in increased vigilance, and the skills needed to identify and characterize emerging risks, could be highly beneficial in avoiding disasters (irgc, ) . from the work done by brocal and sebastián ( a) , brocal et al. ( ) have developed a theoretical framework for the modeling of the ner that allows its monitoring through the tlc, especially in industrial processes. this new framework allows characterizing and differentiating the new qualities from the increasing qualities associated to risk. with this theoretical framework, brocal et al. ( ) have designed a technique that is compatible with standards iso : : (iso, a and iso/iec : (iso, b and permits the ners generated by a system and its components to be identified and characterized. this technique is called the technique to identify and characterize ners (tichner) and is one of the main results of the spanish research project a nersys ("analysis and assessment of technological requirements for the design of a new and emerging risks standardized management system"). tichner can be applied at any stage of the lifecycle of a manufacturing process and it may also be used in combination with other risk identification techniques (brocal et al., ) . as scientific understanding of emerging risks may be changingdsometimes quite rapidlydthe challenge for risk managers in government and industry can be serious, and it is easy to criticize managers after a risk has emerged, regardless of how competent their ex ante decisions may have been, given the information available (irgc, b). with this chapter, a general framework of the emerging risks linked with advanced manufacturing processes and technologies has been shown. these processes and technologies are part of the fourth industrial revolution, called industry . , whose scientific interest is clearly increasing during the last years. the main aspects of this general framework are summarized herein along with their most relevant conclusions. from a global perspective, several organizations have forecasted emerging technologies and reported on their forecasts, such as "disruptive technologies" (mckinsey global institute), " breakthrough technologies" (mit), "next in " (ibm), and "top strategic technologies" (gartner group) . from a specific perspective, the advanced manufacturing processes can be classified into five categories, namely (nassehi et al., ) : joining, dividing, subtractive, transformative, and additive technologies. zhong et al. ( a) consider that the three major advanced manufacturing technologies are the following: intelligent manufacturing, iot-enabled manufacturing, and cloud manufacturing. the general framework of the emerging risks linked with advanced manufacturing processes and technologies has been shown using the cwa : as main reference. next, this framework is expanded specifically to occupational field. in this field, the main results of reports by eu-osha have been shown. among these results, three emerging manufacturing technologies linked to emerging risks have been developed: automation, hmis, and icts. irgc ( ) puts forward an integrated analytic framework for risk governance, in particular at the global level. complementarily, the irgc ( ) focuses on these risks in technology and industry. since the publication of the irgc framework, other guidance documents or frameworks have been published, such as iso : standard. this standard provides principles and generic guidelines on risk management. cwa : gives guidance on steps for applying/implementing the proposed emerging risk management framework (ermf) in industrial organizations. this ermf is heavily influenced by the irgc ( ) and iso : frameworks. the irgc framework is divided into four main phases: preevaluation, appraisal, management and characterization, and evaluation of risk. risk characterization allows decision makers to distinguish scientific facts from policy orientations when analyzing risk assessment results (cwa : ) . from the work done by brocal and sebastián ( a) , brocal et al. ( ) have developed a theoretical framework that allows characterizing and differentiating the new qualities from the increasing qualities associated to risk. with this theoretical framework, brocal et al. ( ) have designed a technique called tichner that permits the ners generated by a manufacturing process to be identified and characterized. the study of the emerging risks linked with advanced manufacturing processes and technologies requires risk governance approaches that consider the systemic and occupational nature of the risk. in this regard, the relationship between the prevention of the occupational accident and the major accident is especially important. for this, these approaches need to continue the path of improvement and development considering the scientific and technological aspects of the emerging risk. sistemas de gestión de la seguridad y salud en el trabajo (management systems the safety and health at work). ohsas nueva norma iso emerging technologiesdbeyond the chasm: assessing technological forecasting and its implication for innovation management in korea on how to define, understand and describe risk the risk conceptdhistorical and recent development trends. reliability engineering and system safety future trends in human work area design for cyber-physical production systems introduction to cloud manufacturing integrating ergonomics and lean manufacturing principles in a hybrid assembly line incertidumbres y retos ante los riesgos laborales nuevos y emergentes [uncertainties and challenges when facing new and emerging occupational risks analysis and modeling of new and emerging occupational risks in the context of advanced manufacturing processes identification and analysis of advanced manufacturing processes susceptible of generating new and emerging occupational risks theoretical framework for the new and emerging occupational risk modeling and its monitoring through technology lifecycle of industrial processes technique to identify and characterize new and emerging risks: a new tool for application in manufacturing processes expert forecast on emerging biological risks related to occupational safety and health expert forecast on emerging psychosocial risks related to occupational safety and health expert forecast on emerging chemical risks related to occupational safety and health. euosha (european agency for safety and health at work) integrated and intelligent manufacturing: perspectives and enablers perspectives on human factors in a shifting operational environment an approach to cyber-physical vulnerability assessment for intelligent manufacturing systems deutsches institut für normung (din), . industry . : new iso strategic advisory group from model-based strategies to intelligent control systems intelligent manufacturing: a new paradigm green jobs and occupational safety and health: foresight on new and emerging risks associated with new technologies by . eu-osha (european agency for safety and health at work) the evolution and future of manufacturing: a review european survey of enterprises on new and emerging risks (esener) [interactive survey european risk observatory a review on the future of work: robotics. discussion paper. eu-osha. bilbao: eu-osha (european agency for safety and health at work) second european survey of enterprises on new and emerging risks (esener) [interactive survey sixth european working conditions survey (ewcs) e overview report. publications office of the european union euromonitor international, . the impact of automation and robotics on the global labour market industrial revolution brings industry back to europe. press release, brussels managing emerging technology-related risks emerging risk e conceptual definition and a relation to black swan type of events. reliability engineering and system safety expert forecast on emerging physical risks related to occupational safety and health. eu-osha (european agency for safety and health at work) the human machine interface as an emerging risk gartner identifies the top strategic technology trends for controlling levels of automation e a model for identifying manufacturing parameters smart manufacturing for the future statistical process monitoring for iot-enabled cybermanufacturing: opportunities and challenges monitoring new and emerging risks. osh. wiki. eu-osha (european agency for safety and health at work) risk management d risk assessment techniques. iso/iec : , geneva. international labour organization (ilo), . emerging risks and new patterns of prevention in a changing world of work white paper on risk governance. towards an integrative approach. irgc, geneva. available at: www.irgc.org. international risk governance council (irgc), a. the emergence of risks. contributing factors. irgc, geneva. available at: www.irgc.org. international risk governance council (irgc), b. emerging risks sources, drivers and governance issues. irgc, geneva. available at: www.irgc.org. international risk governance council (irgc), . improving the management of emerging risks. irgc, geneva. available at: www.irgc.org. international risk governance council (irgc), a. guidelines for emerging risk governance. international risk governance council (irgc), lausanne. available at: www.irgc.org. international risk governance council (irgc), b. guidelines for emerging risk governance. appendix. international risk governance council (irgc) european agency for safety and health at work (eu-osha) esener- ) overview report: managing safety and health at work. european agency for safety and health at work integ-risk project: concept and first results recommendations for implementing the strategic iniciative industrie . . acatech e national academy of science and engineering manufacturing engineering and technology chapter -open source semantic web infrastructure for managing iot resources in the cloud design of human machine interface for plc based automation system methods and materials for smart manufacturing: additive manufacturing, internet of things, flexible sensors and soft robotics. manufacturing letters. available online cyber-physical manufacturing cloud: architecture, virtualization, communication, and testbed mckinsey global institute chapter : industrial automation. industrial process automation systems three generations of telework industrial big data as a result of iot adoption in manufacturing towards lean production in industry . . procedia engineering , e using formal methods to model hybrid manufacturing processes towards industry . utilizing data-mining techniques: a case study on quality improvement. procedia cirp , e cloud computing in manufacturing: the next industrial revolution in malaysia? expert systems with applications oecd reviews of risk management policies report to the president on ensuring american leadership in advanced manufacturing fundamental theories and key technologies for smart and optimal manufacturing in the process industry. engineering , e society for risk analysis (sra), . sra glossary. sra key trends and drivers of change in information and communication technologies and work location. foresight on new and emerging risks in osh. working report. european agency for safety and health at work (eu-osha) manufacturing process selection handbook: from design to manufacture strategic r&d opportunities for st century, cyber-physical systems, connecting computer and information systems with the physical world cciot-cmfg: cloud computing and internet of thingsbased cloud manufacturing service system the internet of things program: the finnish perspective a review of essential standards and patent landscapes for the internet of things: a key enabler for industry . . advanced engineering informatics ubiquitous manufacturing system based on cloud: a robotics application. robotics and computer-integrated manufacturing , e chapter -automation system components research and application of the management and control platform oriented the cloud manufacturing services from cloud computing to cloud manufacturing computer-integrated manufacturing, cyber-physical systems and cloud manufacturing e concepts and relationships. manufacturing letters , e internet of things (iot) enabled smart manufacturing for smes an iot-enabled real-time machine status monitoring approach for cloud manufacturing a review of hybrid manufacturing processes e state of the art and future perspectives towards intelligent manufacturing, semantic modelling for the steel industry. ifac-papers online ( ), e key: cord- -ytlnhyxr authors: zhao, kuan; li, li-wei; jiang, yi-feng; gao, fei; zhang, yu-jiao; zhao, wen-ying; li, guo-xin; yu, ling-xue; zhou, yan-jun; tong, guang-zhi title: nucleocapsid protein of porcine reproductive and respiratory syndrome virus antagonizes the antiviral activity of trim by interfering with trim -mediated rig-i ubiquitination date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: ytlnhyxr porcine reproductive and respiratory syndrome (prrs) is caused by prrs virus (prrsv), and is characterized by respiratory diseases in piglet and reproductive disorders in sow. identification of sustainable and effective measures to mitigate prrsv transmission is a pressing problem. the nucleocapsid (n) protein of prrsv plays a crucial role in inhibiting host innate immunity during prrsv infection. in the current study, a new host-restricted factor, tripartite motif protein (trim ), was identified as an inhibitor of prrsv replication. co-immunoprecipitation assay indicated that the prrsv n protein interferes with trim –rig-i interactions by competitively interacting with trim . furthermore, n protein inhibits the expression of trim and trim -mediated rig-i ubiquitination to suppress interferon β production. furthermore, with increasing trim expression, the inhibitory effect of n protein on the ubiquitination of rig-i diminished. these results indicate for the first time that trim inhibits prrsv replication and that the n protein antagonizes the antiviral activity by interfering with trim -mediated rig-i ubiquitination. this not only provides a theoretical basis for the development of drugs to control prrsv replication, but also better explains the mechanism through which the prrsv n protein inhibits innate immune responses of the host. the tripartite motif protein (trim ) is an e ubiquitin ligase involved in various cellular processes, including regulating the innate immune response against viruses (heikel et al., ) . immediately after a viral infection, pathogen-associated molecular patterns are sensed by host pattern recognition receptors (prrs), thereby allowing cells to distinguish self from non-self and activate an immune response (sparrer and gack, ) . for rna viruses, the main cytoplasmic prr is retinoic acid-inducible gene i (rig-i), which can directly recognize and bind viral ′-ppp rna and short double-stranded rna (chan and gack, ) . after recognition, the n-terminal caspase recruitment domains (cards) of rig-i are modified by ubiquitin which is mediated by trim . such modification is essential for activating a signaling cascade, ultimately resulting in the transcriptional activation of type i and iii interferons (ifns), mediating viral clearance, and inhibiting viral replication and spread (gack et al., ; martin-vicente et al., ) . various viruses are inhibited by trim , e.g., coxsackie b virus and poliovirus (schoggins et al., ) . porcine reproductive and respiratory syndrome (prrs) is endemic in most pig-producing countries (han and yoo, ) , and is caused by prrs virus (prrsv). in , a highly pathogenic prrsv (hp-prrsv) emerged in china, causing high fever, high morbidity, and high mortality . prrsv is an enveloped, single-stranded positive-sense rna virus. its genome is approximately . kb and encodes at least open reading frames (orfs) (han and yoo, ) . orf a and orf b encode two large nonstructural polyproteins, ppla and pplb, which are processed into at least nonstructural proteins (snijder and meulenberg, ; ziebuhr et al., ) . orf a-orf encode structural proteins, including four membrane-associated glycoproteins (gp a, gp , gp , and gp ), three unglycosylated membrane proteins (e, orf a, and m), and a nucleocapsid protein (n) (dea et al., ; snijder and meulenberg, ) . among these, nsp , nsp , nsp , nsp , and been identified and characterized as ifn antagonists (lunney et al., ) . further, the n protein is the most abundant protein during infection, accounting for approximately % of virion proteins (snijder and meulenberg, ) . it plays essential roles in the virus life cycle, including encapsidation of viral rna (spilman et al., ) . currently, it is only known that n protein suppresses ifn-β induction by antagonizing irf activation (sagong and lee, ) . however, other mechanisms through which n protein inhibits ifn-β production are not clear. the nucleocapsid protein of severe acute respiratory syndrome (sars) inhibits type i ifn production by interfering with trim mediated rig-i ubiquitination (hu et al., ) . whereas prrsv and sars both belong to the nidovirales order, whether prrsv n protein inhibits the host innate immune response by interfering with trim mediated rig-i ubiquitination is not clear. herein, we show that trim plays an important role in inhibiting prrsv replication. the n protein interacts with trim and suppresses the ubiquitination of rig-i. further, trim expression is reduced upon co-transfection with the n protein or prrsv infection. together, these findings suggest a novel mechanism through which prrsv n protein inhibits host innate immunity, and provide improved understanding of the mutual regulatory mechanism between prrsv and the host. human embryonic kidney (hek t) cells and african green monkey kidney (marc- ) cells were cultured in dulbecco's modified eagle's medium containing % fetal bovine serum (gibco, thermo fisher scientific, waltham, ma). cells were maintained at °c with % co . the highly pathogenic prrsv strain hun (genbank no. ef ) was used for all the experiments (tong et al., ) . total rna was extracted from porcine alveolar macrophages (pams) using rneasy mini kit (qiagen, hilden, germany), following the manufacturer's instructions. reverse transcription reactions were performed at °c for min and °c for h using the m-mlv reverse transcription polymerase system (takara, dalian, china). full-length trim -and rig-i-encoding sequences were amplified with specific indicated primers. the sequences were then cloned into pcaggs vector to generate pcaggs-trim -ha, pcaggs-trim -flag, pcaggs-trim -myc, pcaggs-rig-i-ha, pcaggs-rig-i-flag, or pcaggs - card-flag. orf of prrsv hun was cloned into pcaggs to generate pcaggs-n-ha, pcaggs-n-flag, or pcaggs-n-myc. all plasmids were constructed by homologous recombination using the nebuilder® hifi dna assembly master mix (new england biolabs; ipswich, ma) according to the manufacturer's instructions. sequences of all primers used for gene amplification will be made available upon request. to investigate the effect of trim on prrsv replication, marc- cells cultured in -well plates were transfected with μg of pcaggs-trim -flag using x-treme gene hp dna reagent (roche applied science, penzberg, germany). next, h post-transfection (hpt), the cells were infected with prrsv hun (multiplicity of infection, moi, of . ). after inoculation for h at °c, the supernatants were discarded, and cells were washed three times with phosphate-buffered saline (pbs). the supernatant was harvested , , , and h post-infection (hpi), and the cells were lysed using ripa lysis buffer (thermo fisher scientific). viral titers in the supernatants were determined using a microtitration assay, according to the method of reed and muench (reed and muench, ) . the amount of the n protein was then detected in cell lysates by western blotting (wb) using a mouse anti-n polyclonal antibody ( : ) produced by the authors. small interfering rnas (sirnas) against macaque trim (genbank no. xm_ . ) were synthesized by genepharma (shanghai, china). the sirna molecules used were ccu gga gua uua cgu uaa att (sirna-trim - ) and gca ucu acc aua gca ccu utt (sirna-trim - ). the control sirna (nc) sequence was uuc ucc gaa cgu guc acg utt. marc- cells were seeded in well plates and transfected with pm sitrim or nc using lipofectamine™ rnaimax transfection reagent (cat. no. , , ; thermo fisher scientific) . the cells were lysed in ripa lysis buffer after h of transfection and the effects of sirnas were analyzed by wb using an anti-trim monoclonal antibody (cat. no. , ; cell signaling technology; danvers, ma; : ). the knockdown efficiency of sirna was analyzed by grayscale scanning with image j software. next, efficient sirna and nc were selected for transfection; hpt, the cells were infected with prrsv hun at an moi of . . the supernatant and cells were harvested , , , and hpi, and analyzed based on virus titers and by wb. cell lysates were prepared by harvesting virus-infected or plasmidtransfected cells in ripa or ip-lysate buffer( mm tris•hcl ph . , mm nacl, % np- , mm edta, % glycerol) at °c for min containing mm phenylmethylsulfonyl fluoride and mg/ml of protease inhibitor cocktail (roche). after centrifuging at , × g for min, the supernatants of cell lysates were mixed with × sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) sample loading buffer (beyotime), and placed in boiling water for min. the proteins were then separated by sds-page and transfected onto a nitrocellulose membrane. the membrane was blocked in % skim milk at room temperature for h and then incubated with the indicated primary antibody at °c overnight. after washing three times with trisbuffered saline with . % tween , the membrane was incubated with horseradish peroxidase-conjugated goat anti-mouse igg(h + l) and/or goat anti-rabbit igg(h + l) secondary antibody ( : ) for h at room temperature. the target proteins were visualized by treating the membrane with pierce ecl wb substrate (thermo fisher scientific). for the quantification of target proteins, their levels were normalized to the levels of β-actin. to detect the interaction between trim and prrsv n protein, hek t cells grown in -well plates were co-transfected with pcaggs-trim -flag and pcaggs-n-ha ( μg each) using x-tremegene dna transfection reagent. at hpt, the cells were lysed with ice-cold ip lysis buffer containing mm phenylmethylsulfonyl fluoride and mg/ml of protease inhibitor cocktail (roche) at °c for min. approximately % of the lysate supernatant was used as an input control and the remaining lysates were incubated with anti-ha agarose beads (cat. no. a ; sigma-aldrich; st. louis, mo) or ezview™ red anti-flag ® m affinity gel (cat.no. m ; sigma-aldrich) for h at °c. the beads were washed five times with the ip lysis buffer and then analyzed by wb using rabbit anti-flag monoclonal antibody (cat. no. f ; sigma-aldrich; : ) or mouse anti-ha monoclonal antibody (cat. no. h ; sigma-aldrich; : ). the interaction between trim and rig-i was tested using the same methods with anti-ha agarose beads and wb using mouse anti-ha monoclonal antibody (cat. no. h ; sigma-aldrich; : ) and mouse anti-myc monoclonal antibody (cat. no. ; cell signaling technology; : ). in order to investigate whether the n protein k. zhao, et al. veterinary microbiology ( ) - competitively affects the interaction between rig-i and trim , hek t cells grown in -well palate were co-transfected with pcaggs-rig-i-ha ( μg) and pcaggs-trim -myc ( μg) with or without pcaggs-n-flag ( μg) for immunoprecipitation. further, to detect the interaction between endogenous (host) trim and n protein in prrsv-infected cells, pam cells were grown in -cm-diameter dishes and infected with prrsv at an moi of . for h. the cells were lysed in μl of ip lysis buffer and the supernatants were precleared with protein g-agarose beads (cat. no. ; roche). the supernatant was incubated with μg of anti-n polyclonal antibody for h. next, μl of protein g-agarose beads was added, followed by an additional h incubation. ip pellets were washed five times with μl of ip lysis buffer, boiled in sds-page sample loading buffer, and analyzed by wb with rabbit anti-trim monoclonal antibody (cat. no. ab ; abcam; : ) and anti-n polyclonal antibody. hek t cells were co-transfected with pcaggs-n-ha and pcaggs-trim -flag, or pcaggs-rig-i-ha and pcaggs-trim -flag. at hpt, the cells were fixed in % paraformaldehyde for min, blocked with % bovine serum albumin for h, and permeabilized with . % triton x- for min. the transfected cells were incubated with mouse anti-ha monoclonal antibody (cat. no. h ; sigma-aldrich; : ) and rabbit anti-flag monoclonal antibody (cat. no. f ; sigma-aldrich; : ) for h at °c, and then washed three times with pbs. the cells were then incubated at °c for h with donkey anti-mouse igg(h + l) antibody conjugated with alexa fluor (cat. no. ; life technologies; : ) and goat anti-rabbit igg(h + l) antibody labeled with alexa fluor (cat. no. ; life technologies; : ). samples were then stained with μg/ml of ′, ′-diamidino- -phenylindole (dapi) for min and examined using a zeiss confocal system. besides, in order to investigate the colocalization of endogenous (host) trim and n protein in prrsv-infected cells, the pam cells infected with prrsv were send to colocalization detection with anti-trim and anti-n protein antibody. hek t cells seeded in a -well plate were co-transfected with control plasmids or the indicated expression plasmids ( . μg per well) together with ng of a luciferase reporter plasmid; prl-tk plasmid ( ng) was used as a control of transfection efficiency (huang et al., ) . next, hpt, cells lysates were harvested and luciferase activity in the lysates was analyzed using a dual-luciferase reporter assay kit (promega, madison, wi), following the manufacturer's instructions. the results are expressed as the means ± standard deviation (sd) from three independent experiments. statistical analysis was performed using graphpad prism (graphpad, la jolla, ca). the measured values are expressed as the mean with sd. the statistical significance was assessed using student's t-test, with p < . considered statistically significant. to examine the effects of trim on prrsv replication, specific sirna molecules were synthesized to knockdown trim expression in marc- cells and efficiently reduce trim expression. using sirna- , the knockdown efficiency was approximately % (fig. a) . this sirna molecule was used in the subsequent interference experiments. as shown in fig. b , n protein levels increased upon transfection with sirna- , especially and hpi, compared with those in nctransfected cells. virus titers in the culture supernatants of cells transfected with sirna- were also increased, which was consistent with the expression levels of the n protein, with a significant difference hpi (p < . ; fig. c ). by contrast, when trim was overexpressed, the n protein levels of hun were lower than those in the control cells (fig. d) . furthermore, there was a significant difference in virus titers between cells transfected with pcaggs-trim -flag or pcaggs, with an approximate . log decrease in virus titers from to hpi (p < . ; fig. e ). these observations suggested that trim is a cellular antiviral factor that represses prrsv infection. as shown in fig. d , the expression of trim -flag decreased gradually upon prrsv infection. therefore, we speculated that the virus might inhibit the expression of trim . to verify this hypothesis, marc- cells infected with prrsv or uninfected cells were lysed , , , and hpi, and analyzed by wb. the analysis revealed that the expression of trim decreased in prrsv-infected cells at every time point, as compared with the uninfected cells ( fig. a) . furthermore, trim expression was significantly suppressed when pcaggs-trim -myc and pcaggs-n-ha were co-transfected into hek t cells. these findings indicated that the n protein inhibits the expression of trim . furthermore, the n protein inhibited trim expression in a dose-dependent manner (fig. b) . since we demonstrated that prrsv infection and n protein accumulation suppress the expression of trim , we next investigated whether the n protein of prrsv exerts an inhibitory effect on trim expression by interacting with it. to test this, the interaction between trim and n protein was investigated by co-ip. based on precipitation with anti-flag agarose, flag-tagged trim interacted with hatagged n protein (fig. a) . furthermore, n protein was efficiently coimmunoprecipitated with trim using anti-ha agarose (fig. b) . we also investigated whether trim co-localizes with the n protein in hek t cells co-transfected with pcaggs-trim -flag and pcaggs-n-ha. trim was mainly located in the cytoplasm ( fig. c-a) , whereas the n protein localized in the cytoplasm and nucleus ( fig. c-b) . as shown in fig. c -d, trim and n protein colocalized in the cytoplasm. to examine the interaction of n protein and endogenous trim in the context of prrsv infection, virus-infected pam cells were stained with anti-n polyclonal antibody and anti-trim monoclonal antibody or immunoprecipitated using anti-n polyclonal antibody and the pellets were detected with anti-trim and n antibody. as shown in fig. d and e, endogenous trim was co-localized with n protein and coimmunoprecipitated with the n protein. these observations confirmed that the endogenous trim indeed interacts with the n protein of prrsv in prrsv-infected cells. in the current study, we demonstrated that the n protein of prrsv could interact with trim . further, it has been reported that trim interacts with rig-i n-terminal cards and e ubiquitin-conjugating enzymes (gack et al., ) . whether the n protein can competitively regulate the interaction between trim and rig-i was unknown. we first detected the interaction between trim and rig-i by co-ip, confirming that trim could be immunoprecipitated with rig-i (fig. a ). in addition, co-localization analysis demonstrated that rig-i and trim co-localize in the cytoplasm (fig. b) . further, we k. zhao, et al. veterinary microbiology ( ) - validated the effect of n protein on the interaction between rig-i and trim . when rig-i-ha and trim -myc were co-transfected with n-flag, the interaction between trim and rig-i was markedly diminished. meanwhile, when trim -myc and rig-i-ha were co-transfected without n-flag, the interaction between trim and rig-i was not affected (fig. c) . these results suggested that the n protein inhibits the interaction between trim and rig-i via competitive binding to trim . to investigate whether trim -mediated rig-i ubiquitination is regulated by the prrsv n protein, hek t cells grown in -well plates were co-transfected with pcaggs-flag-rig-i ( . μg per well) and ha-ubiquitin ( . μg per well), and the indicated amounts of the myc-n expression plasmids. cells lysates were immunoprecipitated using mouse anti-ha monoclonal antibody or rabbit anti-flag monoclonal antibody. the experiment revealed that trim -mediated rig-i ubiquitination was potentiated by sendai virus (sev) infection but was substantially suppressed by increasing the prrsv n protein expression, in a dose-dependent manner (fig. ) . when viral rna is recognized by rig-i, this protein is modified by ubiquitin, which is mediated by the e ligase trim . hence, this enzyme is essential for activating a signaling cascade that ultimately results in the transcriptional activation of type i and iii ifns. to examine regulation of rig-i activity by the prrsv n protein, a luciferase reporter under the control of the ifn-β promoter (ifn-β-luc) was used to quantify promoter activation. consistent with the inhibition of rig-i ubiquitination by the n protein, ifn-β promoter activation induced by rig-i or rig-i card domain overexpression was significantly inhibited by prrsv n expression, in a dose-dependent manner (fig. a, b) . however, co-expression of trim with prrsv n significantly counteracted this inhibitory effect mediated by the n protein (p < . ) (fig. c ). in addition, suppression of rig-i ubiquitination by the prrsv n protein was partially rescued by trim overexpression (fig. d) . these observations indicated that the n protein inhibits rig-i ubiquitination and that ifn production can be restored by trim overexpression. prrsv has been a major threat to global industrial pig farming ever since its emergence in the late s (shi et al., ) , and especially or pcaggs ( μg) were inoculated with prrsv at an moi of . . the cells and supernatant were harvested at the indicated times, and the replication of prrsv was evaluated by wb with an anti-n polyclonal antibody (d) and tcid assay (e). the data are presented as the mean ± sd from three experiments. the statistical significance of differences was determined using student's t-test (*p < . ). since the outbreak of hp-prrsv in . to date, although there is some understanding of the mechanisms through which prrsv escapes innate immunity, very little is known about how viral proteins of prrsv antagonize the host innate immune response. therefore, understanding the interactions between prrsv and host proteins will be beneficial for the development of effective therapies to control outbreaks of this disease in the future. in the current study, we demonstrated that trim functions by restricting prrsv replication, and that it could act as a host-derived inhibitor of the virus. previously, many host factors that result in cellular resistance to prrsv via different mechanisms were identified song et al., ; wang et al., ; zhao et al., zhao et al., , . for example, cholesterol -hydroxylase protects against prrsv infection by converting cholesterol to -hydroxycholesterol, which can be used as a natural antiviral agent to combat prrsv infection (song et al., ) . further, galectin- inhibits replication prrsv by interacting with viral nsp in vitro . in the current study, using marc- cells, we demonstrated that the overexpression of trim restricts the replication of prrsv, whereas knocking down this protein promotes the replication of prrsv (fig. ) . therefore, trim acts as a novel host factor that inhibits the replication of prrsv. trim is an e ubiquitin ligase enzyme that can regulate the innate immune response against viruses (martin-vicente et al., ) . trim -mediated ubiquitination of the cytosolic prr rig-i is an essential step for the initiation of an intracellular antiviral response (gack et al., ) . data presented herein revealed that upon trim overexpression, the n protein-mediated inhibition of rig-i ubiquitination and ifn-β promoter activity were diminished (fig. ) . the host innate immunity was hence activated, leading to a series of signaling cascades and thereby inhibiting prrsv replication. trim can activate the host innate immune system and simultaneously induce a series of antiviral responses by promoting the ubiquitination of rig-i and activation of ifn-β promoter activity. however, in the course of natural infection, prrsv can complete the replication cycle and efficiently spread. hence, prrsv has evolved several general strategies to evade the innate immune response. it has been reported that some viral proteins interact with trim and inhibit rig-i activation. for example, the non-structural protein (ns ) of influenza a virus interacts with the cc domain of trim preventing its dimerization and the k -linked ubiquitination of rig-i cards, thereby suppressing rig-i signal transduction (gack et al., ) . further, trim interacts with the n protein of sars-cov, thereby inhibiting the activation of rig-i (hu et al., ) . in the current study, we found that the n protein of prrsv inhibits the ubiquitination of rig-i by competitively interfering with the interaction between rig-i and trim . this might be the mechanism through which prrsv inhibits the antiviral effect of trim . furthermore, trim levels decreased when the cells were infected with prrsv. in addition, when plasmids expressing trim and the n protein of prrsv were cotransfected into cells, the expression of trim was significantly suppressed. based on this, it would be difficult for trim to exert an antiviral effect upon prrsv infection. this might represent another mechanism through which prrsv antagonizes the antiviral response of trim . besides, the n protein of pedv, another coronavirus, is also able to antagonize ifn-β production (ding et al., ) . since prrsv, sars, and pedv all belong to nidovirales, we speculate that the respective n proteins may exert a similar effect of inhibiting trim mediated ubiquitination of rig-i. however, the effect of pedv n protein on the inhibition of rig-i ubiquitination requires further research. in the present study, we confirmed that trim inhibits prrsv replication. further, prrsv can antagonize the antiviral activity of this protein by decreasing its expression and modulating the trim mediated ubiquitination of rig-i. in addition, the n protein of prrsv inhibits ifn-β production. all these mechanisms improve the understanding of the effect of trim on prrsv replication and will further co-ip experiment was performed using agarose beads conjugated with anti-flag monoclonal antibody or (b) agarose beads conjugated with anti-ha monoclonal antibody. the precipitated proteins were analyzed by wb using antibodies against the flag and ha tags. (c) co-localization of the n protein with trim . hek cells were transfected with plasmids expressing ha-n protein and flag-trim . after h, the cells were fixed and analyzed by ifa to detect tagged n protein and trim , using mouse anti-ha and rabbit anti-flag monoclonal antibodies, respectively. the nucleus is stained with dapi (blue) in the merged images. (d and e) colocalization and interaction of prrsv n and endogenous trim . pam cells infected with prrsv were fixed h after infection, and then subjected to indirect immunofluorescence to detect n protein and trim . in addition, cell lysates from prrsv-infected or mock-infected pam cells were co-ip with mouse anti-n polyclonal antibody, and the precipitates were immunoblotted with the indicated antibodies (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). k. zhao, et al. veterinary microbiology ( ) - help to understand how prrsv evades the trim -mediated innate immune response via the n protein. hence, the current study not only offers a new target for the development of drugs to control prrsv spread but also provides an explanation of the mechanism through which prrsv n protein modulates host innate immune responses. in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. . the prrsv n protein suppresses the trim -mediated rig-i ubiquitination. hek t cells were transfected with the indicated plasmids for h, and were infected (with or without sev) for h. anti-flag immunoprecipitates prepared from the cell extracts were analyzed by wb using the indicated antibodies. k. zhao, et al. veterinary microbiology ( ) - viral evasion of intracellular dna and rna sensing current knowledge on the structural proteins of porcine reproductive and respiratory syndrome (prrs) virus: comparison of the north american and european isolates porcine epidemic diarrhea virus nucleocapsid protein antagonizes beta interferon production by sequestering the interaction between irf and tbk trim ring-finger e ubiquitin ligase is essential for rig-i-mediated antiviral activity influenza a virus ns targets the ubiquitin ligase trim to evade recognition by the host viral rna sensor rig-i engineering the prrs virus genome: updates and perspectives the role of trim in development, disease and rna metabolism the severe acute respiratory syndrome coronavirus nucleocapsid inhibits type i interferon production by interfering with trim -mediated rig-i ubiquitination porcine reproductive and respiratory syndrome virus nonstructural protein antagonizes beta interferon expression by targeting the nf-κb essential modulator galectin- inhibits replication of porcine reproductive and respiratory syndrome virus by interacting with viral nsp in vitro porcine reproductive and respiratory syndrome virus (prrsv): pathogenesis and interaction with the immune system trim in the regulation of the antiviral innate immunity. front. immunol. , . reed porcine reproductive and respiratory syndrome virus nucleocapsid protein modulates interferon-beta production by inhibiting irf activation in immortalized porcine alveolar macrophages pan-viral specificity of ifn-induced genes reveals new roles for cgas in innate immunity molecular epidemiology of prrsv: a phylogenetic perspective the molecular biology of arteriviruses cholesterol -hydroxylase is an interferon-inducible factor that protects against porcine reproductive and respiratory syndrome virus infection intracellular detection of viral nucleic acids cryo-electron tomography of porcine reproductive and respiratory syndrome virus: organization of the nucleocapsid emergence of fatal prrsv variants: unparalleled outbreaks of atypical prrs in china and molecular dissection of the unique hallmark highly pathogenic porcine reproductive and respiratory syndrome the interferon-induced mx inhibits porcine reproductive and respiratory syndrome virus replication porcine ', '-oligoadenylate synthetase inhibits porcine reproductive and respiratory syndrome virus replication in vitro mov inhibits replication of porcine reproductive and respiratory syndrome virus by retaining viral nucleocapsid protein in the cytoplasm of marc- cells virus-encoded proteinases and proteolytic processing in the nidovirales a and b) hek t cells seeded in -well plates were co-transfected using the firefly luciferase reporter plasmid ifn-β-luc and the renilla luciferase control reporter plasmid prl-tk. for the experiment, pcaggs-rig-i-flag ( . μg), or pcaggs - card ( . μg), pcaggs-n-ha were co-transfected. (c) pcaggs - card-flag ( . μg), pcaggs-n-falg ( . μg) and pcaggs-trim -myc ( . μg) plasmids were cotransfected for the experiment, hpt, the cells were infected with sev, and hpi, whole-cell lysates were analyzed by immunoprecipitation using the indicated antibodies to detect the ubiquitination of rig-i-card. the data are presented as the mean ± sd from three experiments. the statistical significance of differences was determined using key: cord- - svaq at authors: ogrodzinski, martin p.; lunt, sophia y. title: metabolomic profiling of mouse mammary tumor derived cell lines reveals targeted therapy options for cancer subtypes date: - - journal: biorxiv doi: . / sha: doc_id: cord_uid: svaq at breast cancer is a heterogeneous disease with several subtypes that currently do not have targeted therapy options. metabolomics has the potential to uncover novel targeted treatment strategies by identifying metabolic pathways required for cancer cells to survive and proliferate. here, we used tumor-derived cell lines derived from the mmtv-myc mouse model to investigate metabolic pathways that are differentially utilized between two subtypes of breast cancer. using mass spectrometry-based metabolomics techniques, we identified differences in glycolysis, the tricarboxylic acid cycle, glutathione metabolism, and nucleotide metabolism between subtypes. we further show the feasibility of targeting these pathways in a subtype-specific manner using metabolism-targeting compounds. breast cancer is a heterogeneous disease with subtypes that vary by morphology, in . % of human breast cancers and is more common in high grade tumors and the basal-like subtype. as a transcription factor, myc affects numerous biological processes including metabolism. , notably, myc expression regulates several genes in glucose, amino acid, and nucleotide metabolism. - therefore, investigating metabolism of the mmtv-myc model system may reveal metabolic features common to human cancer and could present new targeted therapeutic options. here, we present a study investigating the metabolic profiles of two histologically distinct to determine metabolic profiles of histologically distinct mouse mammary tumor subtypes, polar metabolites were extracted from tumor-derived cell lines and quantitated using lc-ms/ms. we found metabolites involved in several central carbon metabolic pathways to be differentially abundant between emt and papillary tumor-derived cell lines (figure ). in the emt subtype, both oxidized and reduced forms of glutathione, a key metabolite in redox homeostasis, are elevated ( figure b ). increased levels of both reduced and oxidized glutathione imply that the emt subtype has elevated glutathione biosynthetic activity. this could reflect a greater dependency on glutathione biosynthesis in the emt cells and targeting glutathione biosynthesis would therefore be more effective against the emt subtype. metabolites increased in the papillary subtype include fructose bisphosphate (fbp; glycolysis); acetyl-coa indicating relative metabolite differences between emt and papillary tumor derived cell lines. metabolites are sorted by relationship to metabolic pathways. yellow and blue boxes indicate increased or decreased metabolite levels relative to the average of the papillary subtype, respectively. data for each sample is normalized to the average signal for all metabolites in the analysis. metabolites with statistically significant differences (p-value < . ) are bolded and marked with asterisks (*). (b) representative bar graphs of metabolites with statistically significant differences between emt and papillary subtypes. data are displayed as means ± s.d., n = . a b * * * * metabolic pool size measurements to reveal more complete metabolic profiles. we find the papillary subtype has proportionally lower abundance of and atp, as well as ribulose- -phosphate and ribose- -phosphate, two intermediates in the ppp production or decreased nucleotide consumption, we applied the same isotope labeling molecular portraits of human breast tumours non-redox-active lipoate derivates disrupt cancer cell mitochondrial open source clustering software java treeview-extensible visualization of microarray data isocor: correcting ms data in isotope supplementary figure : c-isotope labeling from glutamine into the tca cycle is similar between subtypes. cells were incubated in c-glutamine containing media for four hours and extracted for metabolites. grey boxes represent the unlabeled proportion for each metabolite at four hours. green boxes represent the sum of all potential isotopologues for each metabolite supplementary figure : c-isotope labeling from glucose into ribose -phospahte, serine, and glycine and from glutamine into aspartate is similar between subtypes. cells were grey boxes represent the unlabeled proportion for each metabolite at four hours. colored boxes represent isotopologues for each metabolite and are sorted based on carbon source key: cord- -szkiilb authors: gautret, philippe; angelo, kristina m.; asgeirsson, hilmir; duvignaud, alexandre; van genderen, perry j.j.; bottieau, emmanuel; chen, lin h.; parker, salim; connor, bradley a.; barnett, elizabeth d.; libman, michael; hamer, davidson h. title: international mass gatherings and travel-associated illness: a geosentinel cross-sectional, observational study date: - - journal: travel med infect dis doi: . /j.tmaid. . sha: doc_id: cord_uid: szkiilb background: travelers to international mass gatherings may be exposed to conditions which increase their risk of acquiring infectious diseases. most existing data come from single clinical sites seeing returning travelers, or relate to single events. methods: investigators evaluated ill travelers returning from a mass gathering, and presenting to a geosentinel site between august and april , and collected data on the nature of the event and the relation between final diagnoses and the mass gathering. results: of ill travelers, % were female and the median age was years (range: – ). over % returned from a religious mass gathering, most frequently umrah or hajj. only % returned from the olympics in brazil or south korea. other mass gatherings included other sporting events, cultural or entertainment events, and conferences. respiratory diseases accounted for almost % of all diagnoses, with vaccine preventable illnesses such as influenza and pneumonia accounting for % and % of all diagnoses respectively. this was followed by gastrointestinal illnesses, accounting for . %. sixty-three percent of travelers reported having a pre-travel encounter with a healthcare provider. conclusions: despite this surveillance being limited to patients presenting to geosentinel sites, our findings highlight the importance of respiratory diseases at mass gatherings, the need for pre-travel consultations before mass gatherings, and consideration of vaccination against influenza and pneumococcal disease. attendance at an international mass gathering (mg) may expose travelers to health risks related to crowded conditions, population movement, and inadequate sanitation [ , ] . according to the world health organization, an event can be classified as a mg if the number of people attending is sufficient to strain the planning and response resources of the community, state, or nation hosting the event [ ] . however, much of the available literature describes mass gatherings as those exceeding , persons. we describe demographic characteristics and diagnoses among travelers who attended a mg and presented with a travel-associated illness to a geosentinel site. august , , and april , , were collected by geosentinel, a global clinicianbased surveillance network that monitors travel-related illnesses among international travelers and migrants [ ] . geosentinel was established in as a collaboration between the centers for disease control and prevention and the international society of travel medicine. it consists of clinical sites in countries. geosentinel records ill persons' visits to network sites; well travelers are not captured. attendance at a mg is routinely recorded. during the study period, investigators were directed to enter supplemental details on the nature and location of the mg and to evaluate whether the diagnosis was likely associated with mg attendance. records were excluded if the mass gathering was likely to have < , attendees or if data were missing regarding the type of mg. geosentinel's data collection protocol has been reviewed by a human subjects advisor at cdc's national center for emerging and zoonotic infectious diseases and is classified as public health surveillance and not human subjects research. additional ethics clearance was obtained by participating sites as required by their respective institutions or national regulations. a total of records of ill travelers attending a mg during international travel had surveys completed by the investigator providing their care. thirty-one records were excluded. of ill travelers included, ( . %) were female and the median age was years (range: - ; iqr: - ). purposes of mgs were religious ( cases; . %), cultural (e.g., music, dance, carnival) ( cases; . %), the world scout jamboree ( cases; . %), major sport events ( cases; . %), or a large conference ( cases; . %). the top three specific mgs were umrah or hajj in saudi arabia with cases ( . %), including at umrah and at hajj, followed by the world scout jamboree in japan with cases ( . %), and the olympics in brazil and south korea with cases ( . %) ( [ ] [ ] [ ] [ ] ). sixty-four percent of ill travelers who attended umrah or hajj were hospitalized because of their illness; one traveler who attended the world scout jamboree and no travelers who attended the olympics were hospitalized. overall, of ( %) ill travelers with information available reported having a pre-travel encounter with a healthcare provider. the majority ( of , . %) of acquired illnesses were directly associated with mg attendance, while the relation between the illness and the mg was not ascertainable for travelers ( . %), and the illness was travel-related but not linked to mg attendance for travelers ( . %). only three of nine ( . %) diagnoses among travelers who attended the olympics were associated with attending the mg. a total of diagnoses were reported among the ill travelers whose illness was associated with mg attendance (table ) . respiratory diseases were the most frequently reported disease category with diagnoses ( . %), followed by gastrointestinal diseases ( diagnoses; . %). diagnoses related to attendance at the three most common mgs -umrah or hajj, world scout jamboree, and olympics -are presented in table . geosentinel sites evaluated pilgrims with umrah-or hajj-related illnesses among an estimated million foreign mg attendees over the study period [ ] , scouts among , attendees [ ] and olympic spectators among million attendees [ , ] . it should be noted, however, that geosentinel collects data only on ill travelers presenting to a network site and some geosentinel sites may care for more mg attendees than others, which may not be representative of all travelers attending a mg. in particular, the scouts reported to geosentinel were from a single site in sweden and were identified because of an international alert following a meningococcal outbreak (w st serotype) among six scottish and swedish nationals who attended this event [ ] . ill mg attendees seen at a geosentinel site most frequently attended umrah or hajj, likely due to the large number of travelers to these pilgrimages. these findings are consistent with previously published literature demonstrating that outbreaks are not frequently reported during or after mgs other than umrah and hajj pilgrimages, although they sometimes occur at muslim, christian, and hindu religious events, sports events, and large-scale open-air festivals [ , ] . its size, international recognition, unique multinational component, and yearly recurrence, likely account for the preponderance of umrah and hajj among international mgs responsible for outbreaks. our data are also consistent with previous reports regarding the older age of umrah and hajj travelers [ ] , and the younger age of scout jamboree travelers. the finding that almost three-quarters of ill travelers who attended umrah or hajj were hospitalized is likely due to a recruitment bias, given that ill travelers seen at geosentinel sites may be sicker because of the specialized infectious disease or tertiary care nature of these sites; or to initial clinical suspicion for middle east respiratory syndrome, resulting in hospitalization and isolation pending testing. the paucity of reported trauma is also likely a reflection of the infectious disease specialization of most geosentinel network sites, and of the fact that trauma usually requires immediate attention and is most likely to occur during travel [ ] . the predominance of respiratory tract infections reported in this analysis, including cases of pneumococcal disease with one death, corroborates results obtained in saudi hospitals of pneumonia diagnoses among umrah and hajj attendees [ ] as well as the high number of influenza virus infections observed among both patients in saudi hospitals and patients hospitalized on returning to their home countries from umrah and hajj [ ] . crowded conditions with close proximity of large numbers of attendees at umrah and hajj are a likely explanation for the frequency of respiratory infections among pilgrims, given the transmissibility of these infections. our results confirm the importance of influenza vaccination for umrah and hajj travelers [ ] . although the availability of influenza vaccine may be limited depending on the time of year hajj occurs [ ] , immunization with expired influenza vaccine from the recently ended influenza season may have few associated adverse events [ ] . by contrast, most illnesses among travelers attending the olympics were linked to travel, but not to attending the olympics, and these illnesses' were mild, which may be due to the physical separation of various sporting events at the olympics, the propensity to hold the olympics in high income countries, the relatively short travel duration of attendees and participants, and the relatively young age of participants. this is supported by the rarity of documented outbreaks during olympic games between and [ , ] . our identification of respiratory tract infections, especially pneumonia, among umrah and hajj attendees suggests the need for additional research to document responsible pathogens. such data may have important consequences regarding vaccine recommendations before travel. in particular, it may be possible to validate that the influenza serotypes found are representative of strains circulating during the prior northern winter. these data also may provide justification for recommending pneumococcal vaccination for some high-risk travelers [ ] [ ] [ ] . however, one-third of all travelers in this report did not attend a pre-travel consultation, and this will likely be a barrier to implementation of such recommendations. many countries have a staging area where umrah and hajj attendees gather before departure, or have pre-hajj classes at local mosques. leaders there could raise awareness of recommended (but not required) vaccines and personal protective measures such as carrying and routinely using hand sanitizer may lead to higher level of protective behaviors of traveling pilgrims [ ] . also, primary care physicians should inquire about planned travel to mass gatherings and vaccinate travelers against meningococcus, influenza, and pneumococcus, as appropriate. given its broad international catchment, geosentinel plays a role in identifying emerging infectious diseases with epidemic potential, thus contributing to efforts to create enhanced international multidisciplinary surveillance of mg-associated illnesses, as recently recommended by experts [ ] . since our surveillance was limited to patients presenting to geosentinel sites, to better understand travelassociated illnesses acquired at mgs improved global surveillance mechanisms are needed. geosentinel, the global surveillance network of the international society of travel medicine (istm), is supported by a cooperative agreement (u ck ) from the centers for disease control and prevention (cdc), as well as funding from the istm and the public health agency of canada. the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of cdc. global perspectives for prevention of infectious diseases associated with mass gatherings public health for mass gatherings surveillance for travel-related disease -geosentinel surveillance system japan . rd world scout jamboree available at available at: https:// stillmed.olympic.org/media/document% library/olympicorg/games/summer-games/games-rio- -olympic-games/media-guide-for-rio- /ioc-marketing-report-rio- .pdf, accessed date olympic organizers say tickets are sold, but where are the people? meningococcal disease outbreak related to the world scout jamboree in japan communicable diseases as health risks at mass gatherings other than hajj: what is the evidence? infectious diseases and mass gatherings hajj: infectious disease surveillance and control morbidity and mortality amongst indian hajj pilgrims: a -year experience of indian hajj medical mission in mass-gathering medicine clinical respiratory infections and pneumonia during the hajj pilgrimage: a systematic review hajj-associated viral respiratory infections: a systematic review expected immunizations and health protection for hajj and umrah -an overview mandating influenza vaccine for hajj pilgrims notes from the field: administration of expired injectable influenza vaccines reported to the vaccine adverse event reporting system -united states enhanced surveillance at mass gatherings mismatching between circulating strains and vaccine strains of influenza: effect on hajj pilgrims from both hemispheres. hum vaccines immunother pneumococcal disease during hajj and umrah: research agenda for evidence-based vaccination policy for these events travellers and influenza: risks and prevention preparing australian pilgrims for the hajj mass gathering medicine: public health issues arisinf from mass gathering religious and sporting events key: cord- -n ms m h authors: wu, jian-lin; ji, fenfen; zhang, hongna; hu, chuanqin; wong, ming hung; hu, di; cai, zongwei title: formation of dioxins from triclosan with active chlorine: a potential risk assessment date: - - journal: j hazard mater doi: . /j.jhazmat. . . sha: doc_id: cord_uid: n ms m h triclosan, a widely used antimicrobial agent, can increase colitis-associated colon tumorigenesis, and induce liver fibrosis and cancer in mice through mechanisms which may be relevant in humans. in this study, an analytical method using gas chromatography-mass spectrometry (gc–ms) and high resolution gas chromatography-high resolution mass spectrometry (hrgc-hrms) was developed to measure dioxins and chlorinated derivatives from triclosan in the presence of active chlorine in seawater matrix. formation yields of dioxins and chlorinated triclosans were assessed at different initial precursor concentrations under dark and uv light irradiation conditions. results showed that triclosan was rapidly transformed to its chlorinated derivatives, i.e. tetraclosans and pentaclosans, of which the formation yields peaked after h of reaction. uv light was the key factor to promote the formation of dioxins. with the same initial triclosan/active chlorine ratio, the highest yield of dioxins was observed with lower initial concentrations of triclosan under uv irradiation. five dioxins, including , -dcdd, , , -trcdd, , , -trcdd, , , , -tecdd, and , , , -tecdd, were identified and quantified. , , , -tecdd, the most toxic dioxin, was firstly reported as the photo-transformation product of triclosan in aquatic solution. results presented here are useful for a comprehensive understanding of the fate and toxicity of triclosan in contaminated waters. triclosan ( -chloro- -( , -dichlorophenoxy) phenol) is a widely used broad-spectrum antimicrobial and antibacterial agent. it is suspected that a large amount of household disinfectants and antiseptics containing triclosan were used after the outbreak of severe acute respiratory syndrome (sars) in due to the need to against sars coronavirus [ ] . being an anti-bacterial and anti-fungal agent, triclosan has also been included in a wide range of pharmaceutical and personal care products since then [ ] . this leads to the discharge of a large amount of triclosan in densely populated cities such as hong kong, via domestic sewage effluent. studies have revealed that triclosan would degrade to dioxins when exposed to sunlight in wastewater and seawater [ ] [ ] [ ] [ ] [ ] . it has been detected not only in rivers and lakes in hong kong [ ] [ ] [ ] , but also in the human serum of hong kong residents [ ] , indicating a widespread triclosan exposure in the region. recent animal toxicological study showed that triclosan could increase colonic inflammation and associated colon tumorigenesis [ ] . long term exposure of triclosan could also promote liver fibrogenesis and tumorigenesis in mice, and the mechanism of triclosan-induced mouse liver toxicity may be relevant to human [ ] . one of the main concerns about the environmental fate of triclosan is the formation of polychlorinated dibenzo-p-dioxins through photo-degradation [ , ] . it is commonly known that dioxins are potent multisite carcinogens, and of all the dioxin congeners, , , , -tetrachlorinated dibenzo-p-dioxin ( , , , is the most toxic [ , ] . therefore, it is reasonable to speculate that the potential adverse human health effects resulting from the long-term exposure to triclosan and its photo-transformation products, e.g. dioxins, could be even more severe than that expected from the triclosan-only exposure. some laboratory studies have been carried out to investigate the formation of dioxins from the direct photolysis of triclosan and its chlorinated derivatives under uv and/or sunlight. , , , -tecdd was found as the photo-degradation product of , , -trichloro- -( , -dichlorophenoxy)phenol under uv radiation of - nm [ , ] . kanetoshi et al. [ ] detected , -dichlorinated dibenzo-p-dioxin ( , -dcdd), , , -trichlorinated dibenzo-p-dioxin ( , , -trcdd), and , , -trcdd from the -h uv irradiation of triclosan, , -dichloro- -( , dichlorophenoxy) phenol, and , -dichloro- -( , -dichlorophenoxy) phenol, respectively. however, , , , -tecdd was not detected from the photolysis of , , -trichloro- -( , -dichlorophenoxy)phenol [ ] . in another study conducted by this group, solid state triclosan and its three above-mentioned chlorinated derivatives were exposed to sunlight on a glass plate for h, and all these above-mentioned dioxins including , , , -tecdd were determined [ ] . in the subsequent photo-transformation studies of triclosan, , -dcdd was identified as one of the major transformation products in aqueous environments through a photochemical cyclization reaction [ ] [ ] [ ] [ ] [ ] [ ] . however, none of these studies reported , , , -tecdd as the transformation product of triclosan in the environment. among the various oxidants used for water disinfection, chlorine is by far the most widely used. previous investigations have reported that dioxins and other toxic byproducts, such as chlorinated phenols, chlorinated phenoxy-phenols, and trihalomethanes, were formed from the photo-degradation of triclosan in the presence of free chlorine or through the free-chlorine-mediated oxidation of triclosan [ ] [ ] [ ] [ ] [ ] . these byproducts are , , -trichlorophenol, , -dichlorophenol, , dichloro- -( , -dichlorophenoxy) phenol, , -dichloro- -( , -dichlorophenoxy) phenol, chloroform, and so on. some of these compounds, e.g. chloroform and , , -trichlorophenol, are classified as potential human carcinogens by the u.s. epa [ ] . in addition, polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans are also reported as the products from the electrochemical oxidation of triclosan [ ] . given the well-recognized toxicity and carcinogenicity of dioxins, it is of great environmental and toxicological importance to study the fate of triclosan and its chlorinated derivatives in the environment. obviously, more laboratory studies on their aqueous photo-transformation and chlorination chemistry are needed to comprehensively investigate their roles in the formation of dioxins and dioxin-like compounds in aquatic environments, especially in the presence of chlorine. in this study, a sample preparation method using acidic silica gel/acidic alumina/florisil columns was developed to extract the trace amount of dioxin derivatives from samples solutions containing triclosan and its chlorinated derivatives. the enriched dioxin derivatives were then quantitatively analyzed using gas chromatography-mass spectrometry (gc-ms) and high resolution gc-high resolution ms (hrgc-hrms). using this developed analytical technique, we are not only able to characterize dioxins and chlorinated triclosans from the transformation of triclosan in the presence of active chlorine in seawater mixture, but also investigated their formation yields and possible formation mechanisms and pathways under both dark and uv irradiation conditions. to the best of our knowledge, it is the first time to identify , , , -tecdd as the aquatic photo-transformation product of triclosan in the presence of active chlorine. this indicates potential adverse human health effects could be caused by the long-term exposure to triclosan and its transformation products in the aquatic environment. dichloromethane (dcm) and n-hexane used in various experiments were of absolv grade (tedia company, inc., fairfield, ohio, usa). silica gel, acidic alumina (sigma-aldrich, inc., steinheim, germany), nonane (≥ %, fluka chemie gmbh, buchs, switzerland) were of gc grade. anhydrous sodium sulphate (granular) was of ar grade. triclosan was bought from nippon kasei chemical co., ltd. (tokyo, japan). other standards including the photo-transformation of triclosan was carried out in a uv reactor, which was made by the workshop of hong kong baptist university. sixteen w nm uv lamps (philips, netherlands) were mounted on the walls of the reactor as the radiation source. the experiment was performed at room temperature (about ℃) and one electric fan was used to cool down the temperature inside the reactor. seawater collected from tai po harbor in hong kong was filtered and used as the matrix solution, because our previous study showed that it contains very low level of triclosan and no photo-transformation products were detected in it [ ] . given the wide use of sodium hypochlorite (naocl) as a disinfectant/bleaching agent in daily life and its presence in environmental waters, naocl was chosen as the free chlorine precursor in this experiment. free chlorine stock solutions were prepared using purified grade naocl (acros organics, new jersey, usa). the concentration of total active chlorine in the sample solution is the sum of [hocl] and [ocl − ], and it was measured by the dpd/fas titrimetric method. total triclosan includes triclosan and phenolate-triclosan. given the pka values of hclo and triclosan are . and . respectively, and the ph of seawater matrix was between . and . , the predominant active chlorine and triclosan species got involved in the experimental system were hclo and phenolate-triclosan. naocl solution and triclosan (dissolved in methanol) were added into the ml seawater matrix and homogenized using a stirring magnetic bar. initial concentrations of triclosan and active chlorine in the samples were set at . and . μg ml − and . and μg ml − , respectively. the concentrations of triclosan were within those detected in the aquatic environment [ ] . the percentage of methanol was kept below . % of the sample volume in all experiments. after triclosan reacted for min, min, min, min, and min under uv ( nm) radiation and min, min, min, min, min, days, days, days, and days in dark, the excess of chlorine was removed by the addition of sodium thiosulphate. under dark condition, the glass bottles were covered by aluminum foil and securely stopped to avoid sample contamination. after the reaction was quenched by adding an excessive amount of sodium thiosulphate, sample solutions were adjusted to ph . and pretreated by liquid-liquid extraction and acidic silica/acidic alumina/florisil column clean up procedure described below. the detailed experimental conditions under both dark and uv irradiation conditions are listed in tables and . the sample was first extracted with ml of dcm by vigorously shaking the reaction bottles for min, and the organic portion was collected. the extraction was repeated for three times. the combined organic phase was dried using anhydrous sodium sulphate and then evaporated to dryness under a gentle pure n flow. the residue was redissolved in ml of hexane, and the hexane solution was then concentrated to ml. an aliquot of μl was taken out and diluted to ml with nonane. c -labeled triclosan with the final concentration of ng·ml − was added as the internal standard prior to gc-ms analysis. for the hrgc-hrms analysis of dioxins, samples were further cleaned up by consecutive acidic silica gel/acidic alumina/florisil columns to remove triclosan, its chlorinated derivatives and other impurities. an acidic silica gel column (upper column) and an acidic alumina column (lower column) were prepared by packing ml of acidic silica gel and ml of acidic alumina into two glass chromatographic columns ( . -cm inside diameter, -cm length), respectively. the two columns were connected in series and rinsed with ml of hexane before usage. samples re-dissolved in ml of hexane were loaded onto the spe columns. the sample vial was rinsed with ml of hexane, which was also loaded onto the columns. after adding three portions of . ml of hexane directly onto the columns, the upper acidic silica gel column was run dry and taken away. the lower acidic alumina column was further washed with four portions of . ml of dcm/ hexane ( %, v/v) mixture. the reaction products were then eluted with ml of dcm/hexane ( %, v/v) mixture and dried by the pure n flow. the dried residue was re-dissolved in three portions of . ml of hexane, loaded onto an ml florisil column, and then eluted with . ml of hexane and . ml of dcm sequentially. the analytes were in the dcm fraction. after blown dry, the sample was reconstituted in μl of nonane. c -labeled , , , -tecdd with the final concentration of ng·ml − was added as the internal standard prior to hrgc-hrms analysis. a gc-ms method was developed for the analysis of triclosan and its chlorinated products. samples were analyzed in both selective ion monitoring (sim) mode and full scan mode. in this project, sim mode was used for quantification and "aquire scan and sim data mode" was used for qualitative analysis. an agilent n gas chromatography equipped with a split/splitless injector and a hp- ms capillary column ( m × . mm, . μm film thicknesses) coupled to agilent mass spectrometer was used. one microliter of the sample extract was injected in splitless mode with injector temperature of °c, with helium as the carrier gas. the gc column was held at °c for min, followed by an increase of . °c/min to °c and held for min. the gc-msd transfer line temperature, source temperature, and electron ionization (ei) voltage were set at °c, °c, and ev, respectively. triclosan and its chlorinated products were identified and quantified by comparing the retention time and mass spectrum to those of the authentic standards. a hrgc-hrms method was developed to determine dioxins as the transformation products of triclosan. an agilent series gc equipped with a db- ms (j&w scientific, ca) fused-silica capillary column ( m × . mm, . μm film thickness), and coupled to an autospec ultima mass spectrometer (micromass, manchester, u.k.) was used. the gc conditions were optimized to achieve the best separation capacity. the initial oven temperature was held at °c for . min, followed by an increase of °c/min to °c and held for min. it was then increased to °c at . °c/min, held at °c for min, and finally to °c at °c /min and held for min. ei was used and operated in the sim mode at , resolving power ( % valley definition). the two most abundant ions in the [m] + cluster were monitored at a ms dwell time and a delay time of ms. dioxins were identified and quantified by comparing the retention time and mass spectrum to those of the authentic standards. six-point calibration curves for triclosan, , -dcdd, , , -trcdd, , , , -tecdd and , , , -tecdd were constructed with concentrations ranging from ng·ml − to ng·ml − . good linearity was achieved over the whole concentration range with r > . . quantifications of , -dcdd, , , -trcdd, and , , , -tecdd were performed using external standard method, and the isotope dilution method was used for triclosan and , , , -tecdd. the c -labeled triclosan and c -labeled , , , -tecdd were added into the standards with the final concentration of ng·ml − . recovery tests were carried out at three concentrations, which were pg·ml − , pg·ml − and pg·ml − , respectively (table s ). triclosan and dioxin standards were spiked into ml of filtered seawater, and pretreated and analyzed following the procedures described above. triplicate spiked samples were analyzed for each concentration level. recoveries of all the analyzed compounds were within the range of . - . %. the intra-day and inter-day precisions were higher than . % and . %, respectively. the limit of detection (lod) was defined as the concentration of the analyte required to produce a signal with amplitude of at least times of the baseline noise (s/n ≥ ). the lods of tricolsan and dioxins were found to be within the range of . - . pg·ml − for gc-ms analysis and . to . pg·ml − for hrgc-hrms (table s ), which were well below the levels of these compounds observed in the water samples. the mixture of standards, including , -dcdd, , , -trcdd, , , , -tecdd, , , , -tecdd, , , , -tecdd, , , , -tecdd, , , , -tecdd, , , , -tecdd, , , , -tecdd, , , , -tecdd and , , , -tecdd, were well separated under the gc-ms condition described in section . (table s ). the photo-degradation products of triclosan in seawater were first treated by liquid-liquid extraction and analyzed by gc-ms in full scan mode. as shown in fig. , many products were detected in the triclosan photo-transformation experiment, including several dioxins and chlorinated triclosan derivatives. three tetraclosans, i.e. -chloro- -( , , -trichlorophenoxy)phenol (tetraclosan ), , -dichloro- -( , -dichlorophenoxy)phenol (tetraclosan ), and , -dichloro- -( , -dichlorophenoxy)phenol (tetraclosan ), and two pentaclosans, i.e. , -dichloro- -( , , -trichlorophenoxy)phenol (pentaclosan ) and , , -trichloro- -( , -dichlorophenoxy)phenol (pentaclosan ) were identified by comparing their mass spectra with the previously reported data [ , , , ] and their retention times relative to other known compounds in the gc chromatograms. several dioxins, such as , -dcdd, , , -trcdd, , , -trcdd, were also detected by gc-ms. however, for , , , -tecdd ( . min) and , , , -tecdd ( . min), their retention times were close to that of tetraclosan ( . min). since the concentration of tetraclosan was much higher than these two tecdds, they were buried underneath the huge peak of tetraclosan and could not be seen in fig. . therefore, further cleanup of the sample using the tandem acid silica gel/acidic alumina/ florisil spe columns was performed to remove triclosan and its chlorinated derivatives. the dioxin products were then identified and quantified using both gc-ms and hrgc-hrms. . the former was determined as , , -trcdd by comparing to the authentic standard and the latter (figs. s b and s b), was identified as , , -trcdd, which was also reported by kanetoshi et al. [ , ] . the two tetracholorodibenzo-p-dioxins, , , , -and , , , -tecdds, were clearly observed in the cleaned sample, with their retention times and mass spectra well matched those of the standards (figs. s c and s c) . the eic of dioxin products in spiked seawater sample using hrgc-hrms is shown in fig. table s . as shown in fig. d , molecular ions at m/z . were also observed at . and . min, which were from the cl -subsitituted isomers of tecdd. this doubly confirmed the presence of , , , -tecdd and , , , -tecdd in the sample. our finding further proved that dioxins could be photo-chemically transformed from triclosan in the presence of free chlorine in seawater matrix. most importantly, the most toxic dioxin, , , , -tecdd was also determined as the photo-transformation product of triclosan. in the presence of . μg ml − of active chlorine in dark, triclosan rapidly decayed and its half lifetime was about min ( table ). the chlorinated triclosans were rapidly formed, and the concentrations of tetraclosan , tetraclosan and pentaclosan reached their maxima within min (table ). this indicated that triclosan could be easily chlorinated to form tetraclosans and pentaclosans, due to the high oxidabillity of active chlorine. under dark condition, a trace amount of dcdd was detected at the beginning of the reaction. its concentration was only . ± . pg·ml − after min, and kept increasing to . ± . pg·ml − after the system (table ) . as mentioned earlier, dioxins were often considered as the photo-transformation products of triclosan. our finding suggested that under weak base (naocl) condition, triclosan could be transformed to dioxins in the presence of excessive active chlorine even in dark. however, their formation rates were rather slow and the formation yields of dioxins decreased with increasing number of chlorine substitutes. as shown in fig. , , -dcdd could be formed through a slow cyclization reaction of triclosan under this condition. contrary to dioxins, much higher levels of chlorinated triclosan derivatives were observed in the system. almost all of them reached their highest concentrations at min, and gradually decayed as reaction continued for days (table ) . several studies have reported the production of tetraclosan and and pentaclosan from aquatic triclosan chlorination. these three chlorinated triclosan derivatives were proposed to be formed via bimolecular electrophilic substitution of triclosan and their formation was kinetically favorable [ , ] . results shown in table confirmed that these three chlorinated triclosans were the major degradation products of triclosan in dark in the presence of free chlorines. besides, small amounts of tetraclosan and pentaclosan were also detected and the formation pathways of all measured chlorinated triclosans were proposed in fig. [ , ] . similar to triclosan, these chlorinated derivatives may also undergo a slow cyclization reaction in dark, which led to the formation of trcdd and tecdd in the system (fig. ) . in the simulated photolysis experiments of tricolsan, uv light at nm was chosen as the radiation source given that triclosan has a high light absorption in the near uv region of solar spectrum. triclosan decayed much faster under uv radiation than in the dark, and˜ % of it reacted after min of radiation (table ). with the same initial concentrations of triclosan and free chlorines, much more dioxins were formed under uv radiation and all of them, including , , , -tecdd were detected as the photo-transformation products of triclosan, given enough reaction time. among the measured dioxin products, trcdds were the most abundant, followed by dcdd and tecdds. concentrations of almost all dioxins increased with increasing reaction time, except for , -dcdd, which peaked after min of radiation and then gradually decreased as reaction continued for h. this demonstrated that , -dcdd was the first generation product from the photolysis of triclosan and it would further degrade to form the higher generation products. one of the possibility was , -dcdd could generate a biradical intermediate upon photochemical excitation, which led to the production of dihydroxy dichlorinated biphenyls and lower chlorinated phenoxyphenols [ ] . however, they were not measured in the experiments due to the lack of authentic standards. in this paper, the formation yield of the transformation products from triclosan was defined as: [ ] is the concentration of the transformation product a (ng/ml), and triclosan is the concentration of reacted triclosan (ng/ml). as shown in fig. a , the formation yield of total dioxins under uv radiation was substantially higher than that under dark condition. this confirmed that light was the dominant factor to promote the production of dioxins from triclosan. it is interesting to note that with the same initial triclosan/active chlorine ratio, a higher yield of dioxins was observed in the experiment with lower concentrations of triclosan and free chlorines. given that the chlorination reactions of triclosan were kinetically favorable, this phenomenon was probably due to the higher possibility for triclosan to undergo chlorination, instead of photolysis when a higher level of active chlorines was present in the system (fig. ) [ ] . as discussed above, , -dcdd would further degrade through photolysis. we also proposed that , , , -tecdd and , , , -tecdd were the rd generation products from the transformation of triclosan through the direct photolysis of pentaclosan and , respectively ( fig. ) [ ] . therefore, it was not surprising to observe that trcdd had the highest formation yield of all three types of dioxins and tecdd had the lowest. it was worth noting that the formation yield of pentaclosan was about a thousand times higher than that of pentaclosan under the same experimental conditions (table ) . similarly, a much lower yield of tetraclosan was observed than those of tetraclosan and . this indicated that the pathway to form , , , -tecdd was minor, compared with those pathways to form trcdd and , , , -tecdd. however, no significant difference was observed between the yields of , , , -tecdd and , , , -tecdd (table ) . this indicated that another photo-transformation pathway might exist for , , , -tecdd. overall, the formation yields of total dioxins were rather low, which was below . % under the tested conditions. as a result, a logarithmic function between the yield of dioxins and reaction time was observed (figs. a, c and d) . the formation yields of all chlorinated triclosans reached their maximum values after min of reaction and then gradually decayed as reaction continued (fig. ). as shown in fig. b , the formation yields of tetraclosans were about the same under dark and light conditions, which indicated that light was not the key factor to promote the formation nor transformation of these compounds (fig. ) . however, for pentaclosans, their total formation yield was much lower under uv irradiation than that in dark. this indicated that photolysis was an important pathway for the decay of pentaclosans, which might be more significant than the further chlorination of these compounds. this is because pentaclosans possess higher pka values than that of triclosan (pka = . - . ) [ , ] . in the weak basic solution, a smaller fraction of them was in the phenolate forms, and therefore less reacted with active chlorines to get further chlorinated. besides tecdds, other photo-transformation products of pentaclosans could be dihydroxy polychlorinated biphenyls and lower chlorinated phenoxyphenols [ ] , although they were not monitored in our study. it was interesting to note that the formation yields of tetrachlosans and pentaclosans were almost the same under the two photolysis experiments. obviously, it was the initial triclosan/active chlorine ratio but not the initial concentrations of these two precursors that really influenced the production of chlorinated triclosans from triclosan under uv irradiation. chlorinated triclosan derivatives and dioxins, including the most toxic , , , -tecdd, were measured as the photo-transformation products of triclosan in the presence of active chlorine in seawater matrix under both uv irradiation and dark conditions. both uv light and the initial concentrations of triclosan and active chlorine played important roles in the photo-transformation of triclosan to dioxins. higher yield of dioxins was observed in the experiment with lower concentrations of triclosan and free chlorines and trcdds were found to be the most abundant dioxin products. on the other hand, the formation yields of chlorinated triclosans from triclosan was mainly influenced by the initial triclosan/active chlorine ratio, and uv light promotes the transformation of these compounds to dioxins, including the most toxic , , , -tecdd. possible formation mechanisms of dioxins and chlorinated triclosan derivatives from the transformation of triclosan under both dark and uv irradiation conditions were proposed. results from this study suggest that the transformation products of triclosan could be even more toxic and persistent in the environment than itself. more public awareness should be paid to the potential adverse human health effect resulting from the long-term exposure to triclosan and its transformation products in environmental waters. the antiviral action of common household disinfectants and antiseptics against murine hepatitis virus, a potential surrogate for sars coronavirus a weight-of-evidence approach for the bioaccumulation assessment of triclosan in aquatic species quantification of triclosan, chlorinated triclosan derivatives, and their dioxin photoproducts in lacustrine sediment cores new insights into the environmental photochemistry of -chloro- -( , -dichlorophenoxy)phenol (triclosan): reconsidering the importance of indirect photoreactions photolytic degradation of triclosan in freshwater and seawater photodegradation of micropollutants using v-uv/uv-c processes; triclosan as a model compound faster photodegradation rate and higher dioxin yield of triclosan induced by cationic surfactant ctab investigation of levels and fate of triclosan in environmental waters from the analysis of gas chromatography coupled with ion trap mass spectrometry triclosan determination in water related to wastewater treatment determination of triclosan metabolites by using in-source fragmentation from high-performance liquid chromatography/negative atmospheric pressure chemical ionization ion trap mass spectrometry organochlorine isotopic pattern-enhanced detection and quantification of triclosan and its metabolites in human serum by ultra-high-performance liquid chromatography/quadrupole time-offlight/mass spectrometry a common antimicrobial additive increases colonic inflammation and colitis-associated colon tumorigenesis in mice the commonly used antimicrobial additive triclosan is a liver tumor promoter the florence statement on triclosan and triclocarban on the need and speed of regulating triclosan and triclocarban in the united states toxic equivalency factors (tefs) for pcbs, pcdds, pcdfs for humans and wildlife the world health organization reevaluation of human and mammalian toxic equivalency factors for dioxins and dioxin-like compounds chromatographic evidence for the formation of chlorodioxins from chloro- -phenoxyphenols chlorination of irgasan dp and formation of dioxins from its chlorinated derivatives formation of polychlorinated dibenzo-p-dioxins upon combustion of commercial textile products containing , , ′-trichloro- ′-hydroxydiphenyl ether (irgasan® dp ) dibenzodichloro-p-dioxin as a photodegradation product of triclosan in water and wastewater samples photochemical conversion of triclosan to , -dichlorodibenzo-p-dioxin in aqueous solution fate and mass balance of triclosan and its degradation products: comparison of three different types of wastewater treatments and aerobic/ anaerobic sludge digestion aquatic degradation of triclosan and formation of toxic chlorophenols in presence of low concentrations of free chlorine formation of chloroform and chlorinated organics by free-chlorine-mediated oxidation of triclosan chemical changes of organic compounds in chlorinated water: xiii. gas chromatographic-mass spectrometric studies of the reactions of irgasan dp [ -chloro- -( , -dichlorophenoxy)phenol] with chlorine in dilute aqueous solution degradation of triclosan by chlorine dioxide: reaction mechanism, , -dichlorophenol accumulation and toxicity evaluation degradation behavior of triclosan by co-exposure to chlorine dioxide and uv irradiation: influencing factors and toxicity changes hydroxyl radical generation and oxidative stress in carassius auratus liver as affected by , , -trichlorophenol fate and hazard of the electrochemical oxidation of triclosan. evaluation of polychlorodibenzo-p-dioxins and polychlorodibenzofurans (pcdd/fs) formation triclosan: occurrence and fate of a widely used biocide in the aquatic environment: field measurements in wastewater treatment plants, surface waters, and lake sediments aquatic photochemistry of chlorinated triclosan derivatives: potential source of polychlorodibenzo-p-dioxins experimental and theoretical insights into the involvement of radicals in triclosan phototransformation dioxin photoproducts of triclosan and its chlorinated derivatives in sediment cores phototransformation of triclosan in surface waters: a relevant elimination process for this widely used biocide laboratory studies, field measurements, and modeling ab initio and in situ comparison of caffeine, triclosan, and triclocarban as indicators of sewage-derived microbes in surface waters the authors would like to thank for the financial support from national natural science foundation of china ( ), general research fund ( ) and collaborative research fund (c - e) of hong kong research grants council. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.jhazmat. . . . key: cord- -j wj ah authors: paul, michael title: preiskommunikation in krisenunternehmen – eine betrachtung aus praxis-sicht date: - - journal: preiskommunikation doi: . / - - - - _ sha: doc_id: cord_uid: j wj ah unternehmenskrisen werden meist von preiskrisen begleitet. der preis als „fieberthermometer“ offenbart probleme, wie etwa mangelnde wettbewerbsstärke oder nicht marktgerechte angebote, häufig schon bevor die krise insgesamt sichtbar wird. in der krise selbst führt ein erheblicher liquiditätsdruck nicht selten zu weiteren preissenkungen. dass sich die „macht“ der kunden und des wettbewerbs gegenüber einem geschwächten gegner vergrößert, verschärft die herausforderung für das management. dieser beitrag stellt ein systematisches vorgehen zur preiskommunikation in krisenunternehmen vor, das mit der definition der preisziele und preispolitischen maßnahmen beginnt und daraus den stil und die kanäle der preiskommunikation ableitet. dabei wird insbesondere auf die liquiditätsgetriebene kurz- und die am unternehmenswert und der nachhaltigen rentabilität orientierte mittelfristperspektive bezug genommen. • die stakeholderkrise, die durch uneinigkeit bzw. handlungsunfähigkeit der eigentümer gekennzeichnet ist. • die strategiekrise, die davon geprägt ist, dass das unternehmen über kein klares geschäftsmodell mehr verfügt bzw. dieses nicht mehr funktioniert (z. b. aufgrund heftiger disruptionen im markt). • die produkt-und absatzkrise, in deren verlauf das unternehmen gegenüber dem wettbewerb im markt an boden verliert und zumindest stagnierende marktanteile realisieren muss. • die erfolgskrise, in der es dem unternehmen nicht mehr gelingt, im wettbewerbsumfeld übliche renditen zu erzielen bzw. sogar verluste anfallen. • die liquiditätskrise, in der die zahlungsfähigkeit des unternehmens nicht mehr gesichert ist. • die insolvenzlage, in der das unternehmen zahlungsunfähig ist und ein gesetzlich normiertes verfahren der sanierung durchlaufen muss. parallel zu den bekannten krisenphasen verläuft in der weit überwiegenden zahl von unternehmen, die später möglicherweise in existenzbedrohende situationen kommen, in den meisten fällen eine sich verschärfende "preiskrise". aus sicht der preispolitik nämlich ergeben sich häufig schon in den frühen krisenphasen probleme: geringere preisspielräume werden nicht selten schon in der strategiekrise sichtbar. eine unklare positionierung, geringe fokussierung im sortiment oder den bearbeiteten märkten etc. führen zum abschmelzen eines preispremiums oder verhindern, dass ein solches überhaupt erreicht werden kann. in den phasen bis verschärft sich diese problematik. häufig werden in einer gewissen panik unkontrolliert preise gesenkt, um damit strategische probleme sowie einhergehende misserfolge zu überdecken. in diesem beitrag stehen insbesondere die beiden letzten phasen - und -im mittelpunkt. die situation des unternehmens ist in diesen beiden phasen anders als in den ersten in der regel öffentlich bekannt. das management steht unter einem besonderen druck: "mit zu-nehmender insolvenznähe steigt die notwendigkeit, schnell greifende sofortmaßnahmen umzusetzen. wird eine akute illiquidität festgestellt, müssen unverzüglich, d. h. innerhalb von längstens drei wochen, maßnahmen zu deren beseitigung konkretisiert und umgesetzt werden" (idw , s. tz ) . in dieser situation des handlungsdrucks müssen zahlreiche aktionen gesetzt werden, von der sicherung der lieferantenbasis bis hin zu einer engen Überwachung der liquidität. dies erfordert eine klare prioritätensetzung und fokussierung auf überlebensnotwendige aktionen von den beteiligten. tendenziell wird dabei eher auf die ausgaben-als auf die einnahmenseite geachtet, erwartet man sich doch hier schnellere effekte. das ist insbesondere deshalb problematisch, als die umsatzseite und hier nicht zuletzt das thema "preis" für eine bewältigung der krise von enormer bedeutung sind, sowohl für die Überwindung der akuten situation als auch für den weiterbestand des unternehmens. dabei sieht sich das management besonderen rahmenbedingungen gegenüber. mögliche ziele der sanierung als wesentliche rahmenbedingung wesentliche rahmenbedingung sind die ziele, die von eigentümern/gläubigern, dem insolvenzverwalter oder dem management teilweise sogar parallel verfolgt werden (müssen). geht es darum, den erhalt des unternehmens in bisheriger form durch schnelle wiedererlangung der zahlungsfähigkeit zu erhalten? "sanierungsfähig ist ein erwerbswirtschaftliches unternehmen nur dann, wenn […] durch geeignete maßnahmen auch nachhaltig die wettbewerbsfähigkeit wiedererlangt werden kann (nachhaltige fortführungsfähigkeit i. s. einer sanierungsfähigkeit)" (idw , s. , tz ) . nach einer sanierung muss also auf jeden fall wieder wirtschaftliches arbeiten möglich sein. dies erfordert auch die erzielung von preisen, die eine nachhaltige fortführung ermöglichen. nur über kostensenkungen und mengensteigerungen werden dauerhaft keine ausreichenden cash-flows oder gar eine im marktvergleich übliche rendite erzielbar sein. schon im zeitpunkt der erstellung einer fortbestehensprognose muss es deshalb einen klaren plan geben, wie auch die preispositionierung im markt "saniert" werden kann. opportunistische preisvariationen wirken dem eher entgegen, selbst wenn sie kurzfristig helfen würden, die liquidität zu verbessern. das ziel einer fortführung erfordert also einen besonders sensiblen umgang mit dem thema "preis". im falle eines herstellers von haushaltsgeräten mit starker marke war der hohe lagerbestand mit entsprechender kapitalbindung wesentlicher teil der betriebswirtschaftlichen krisenursachen. im sanierungsteam wurde deshalb intensiv darüber diskutiert, mittels eines abverkaufs mit massiven preissenkungen überhöhte lagerbestände abzubauen und notwendige liquidität zu generieren (vgl. klein , s. sowie helbing gruppe . starke bedenken gingen in die richtung, dass durch diese aktion die preispositionierung massiv geschwächt werden könnte. bislang war es immer gelungen, sich aus den preiskämpfen der branche heraus zu halten. es bestand die befürchtung, dass nun durch diese "sanierung" eine neue krisenursache geschaffen werden könnte. zweite wesentliche rahmenbedingung preispolitischer aktionen in der krise ist also die asymmetrie der handlungsspielräume und erwartungen der akteure. die bewältigung der preiskrise erfordert eine eigene, klare strategie, die im einklang mit den zielen und rahmenbedingungen sowie vor allem dem gewählten lösungsansatz zur bewältigung der unternehmenskrise stehen muss. sie ist die grundlage des vorgehens in der preiskommunikation. in anlehnung an diller (diller , s. ) lassen sich die preispolitischen optionen allgemein folgendermaßen darstellen (darunter immer die jeweiligen besonderheiten in der krise, vgl. abb. ). die neubewertung von zielsegmenten wurde bereits mehrfach angesprochen. wesentlich dabei ist die beurteilung der reaktion der segmente auf preispolitische aktionen und eine einschätzung der aktuellen und zukünftigen bedeutung für das unternehmen. verkürzt gesprochen, rücken segmente in den mittelpunkt der betrachtung, mit denen kurzfristig liquidität erzielbar ist, ohne für die zukunft preispolitische spielräume zu sehr einzuengen. dass dafür unter umständen höhere "zäune" zwischen den segmenten aufgebaut werden müssen, etwa durch nutzung anderer vertriebswege, wurde bereits dargestellt. einer der "zäune" zwischen marktsegmenten, die mit mehr oder weniger aggressiver preispolitik angesprochen werden, könnte die räumliche distanz sein. ein international tätiger porzellanwarenhersteller wollte sein hohes preisniveau in europa, nordamerika und asien nicht gefährden. im arabischen markt hingegen verfügte man aufgrund geringer distribution über eine nur schwache marktstellung. eine wesentliche idee der sanierung bestand darin, gemeinsam mit einem arabischen großhändler in dortigen shoppingmalls die bekannte marke zu einem deutlich günstigeren preisniveau anzubieten. dies erfordert wiederum -letztes element der preisstrategie -eine bewusste, krisenbezogene strategische preiskoordination. wesentlich war so auch im fall des porzellanwarenhersteller die koordination. so galt es zum beispiel zu vermeiden, dass zwischenhändler im arabischen raum größere mengen aufkaufen, um diese doch wieder in den europäischen markt zu bringen. dies erfolgte insbesondere über eine bewusste steuerung der preisabstände zwischen den verschiedenen regionalen märkten ("preiskorridore"). die preiskommunikation in der krise muss im gesamtrahmen der krisenkommunikation gesehen werden. behringer , s. ; thießen , s. - ) . trotzdem kann in bestimmten konstellationen auch ein passiver kommunikationsstil der krise angemessen sein. insbesondere dann, wenn negative meldungen noch beherrschbar erscheinen und das unternehmen nach außen noch handlungsfähig auftreten kann. geht es um besonders vertrauenssensible leistungen (zuvor wurden ja bereits bespielhaft finanzdienstleistungen, reiseangebote oder serviceunternehmen erwähnt), so entwickelt sich unter umständen bei bekanntwerden negativer entwicklungen eine eigendynamik, die nicht mehr beherrschbar sein könnte. in solchen konstellationen kann auch ein eher passiver kommunikationsstil angezeigt sein. grundsätzlich sind auch kombinationen beider kommunikationsstile eine mögliche option in der preiskommunikation. unabhängig davon, ob wegen der krise veränderungen in der preispolitik vorgenommen werden oder nicht, ist dies eine information mit signalwirkung. entweder wird kommuniziert, dass an der bisherigen linie festgehalten wird, was z. b. als signal der stärke aufgenommen werden kann, oder dass sich das preisniveau ändert, was als signal zum handeln (mehr kaufen, anders kaufen) in den markt wirken kann. dieses signal kann in verschiedener weise verbreitet, bzw. bewusst thematisiert werden (vgl. simon und fassnacht , s. ) . dabei bieten sich dem unternehmen dieselben optionen der preiskommunikation wie bei einem unternehmen im "normalen modus" an, wobei sich diese in zwei große gruppen unterteilen lassen: • die preiswerbung. diese umfasst sämtliche maßnahmen, in denen der markt durch den einsatz verschiedener medien über den preis informiert wird. diller spricht hier auch von der "preisdeklaration", sie "umfasst alle schriftlichen ausweise des jeweils gültigen preises am produkt oder am verkaufsregal ("preisauszeichnung") sowie in preislisten, prospekten, katalogen und anderen werbemitteln des anbieters" (vgl. diller , s. kombiniert man den aktiven bzw. den passiven kommunikationsstil und die beiden optionen der umsetzung der preiskommunikation als preiswerbung oder persönliche preisvereinbarung, so ergibt sich das in abb. gezeigte bild. eine passive preiswerbung könnte in der praktischen umsetzung etwa in einer anpassung von preislisten und preisauszeichnungen bestehen, sollte dies auf druck des marktes notwendig sein. von einer passiven "werbung" zu sprechen, mag paradox klingen. doch selbst, wenn anpassungen beispielsweise der preislisten oder preisauszeichnungen "aktiv" vorgenommen werden, so erfolgt dies doch in einem rahmen, der nicht zwangsläufig zu einer größeren aufmerksamkeit in einer breiteren marktöffentlichkeit führt. in der praktischen umsetzung würde das unternehmen z. b. die entwicklung des umsatzes in der krise weiter beobachten und nur dann anpassungen vornehmen, wenn es hier zu erheblichen negativen reaktionen kommt. ein solches vorgehen hat den vorteil, dass zunächst einmal "business as usual" in den markt ausgestrahlt und die lage als "unter kontrolle" dargestellt wird. dieser vorteil ist zugleich auch der größte nachteil eines solchen vorgehens. insbesondere wenn es darum geht, durch preisseitige aktionen zusätzliche liquidität durch höhere umsätze zu schaffen, oder größere warenbestände abzuverkaufen, ist dieses vorgehen ungeeignet. es eignet sich insbesondere dann, wenn man zumindest in einzelnen marktsegmenten keinerlei veränderung an der preispolitik in der krise vornehmen möchte. selektiver lässt sich dieser ansatz im e-commerce verfolgen, je besser die daten des unternehmens über seine kunden sind, desto selektiver können preise angepasst werden. je nach informationsbedürfnissen und preisbereitschaften kann dann sehr viel gezielter gesteuert werden. eine aktive preiswerbung hingegen setzt unterschiedliche medien ein, um wahrnehmung für veränderte preise zu schaffen. im gegensatz zur passiven vorgehensweise wird sie eine veränderte preisgünstigkeit besonders hervorheben. bei allen strategien, die darauf setzen, zusätzliche umsätze zu generieren bzw. warenbestände abzubauen, wird sie das mittel der wahl sein (vgl. simon und fassnacht , s. ) . sie muss allerdings nicht nur bei preisänderungen zum einsatz kommen. auch die breite kommunikation, dass ein Überleben des unternehmens für den kunden von nutzen ist bzw. dass sich aus der aktuellen situation keine risiken ergeben, kann den einsatz aktiver preiswerbung erfordern, zum beispiel auch im sinne einer solidarisierung zwischen kunden und unternehmen. aufgrund ihrer breitenwirkung darf die wirkung aktiver preiswerbung im sinne eines "signaling" in richtung des wettbewerbs nicht unterschätzt werden kap. " signaling gegenüber wettbewerbern -erkennen und verhindern von preiswettbewerb und "preiskrieg"". durch die breite streuung und einfache auffindbarkeit sendet die aktive preiswerbung aufmerksamkeitsstarke signale. reaktionen bis hin zur angesprochenen gefahr des preiskrieges werden wahrscheinlicher. die persönliche preiskommunikation, sowohl aktiv als auch passiv, funktioniert nur in solchen branchen, in denen es direkte, persönliche (vertriebliche) kontakte zu kunden gibt. sie spielt deshalb insbesondere bei hochwertigeren konsumgütern, immer seltener im klassischen einzelhandel, eher im personalisierten online-handel und in vielen bereichen des b b eine zentrale rolle. die aktive variante der persönlichen preiskommunikation spielt vor allem dort eine rolle, wo es um die vereinbarung von sanierungsbeiträgen oder der modifikation von konditionen geht. wesentliche voraussetzung solcher vereinbarungen ist das vertrauen der kunden. sie werden nur dann bereit sein, einem unternehmen trotz krise zugeständnisse zu machen, wenn sie eine gewähr dafür haben, dass sich diese auch auszahlen. dies setzen die verlässlichkeit des managements und eine hohe wahrscheinlichkeit des Überlebens voraus. insofern ist das aktive zugehen auf die kundenunternehmen essenziell für den erfolg dieses vorgehens. die kunden müssen das gefühl haben, dass sie vom management des krisenunternehmens immer rechtzeitig, möglichst mit als erste verlässliche informationen erhalten. dazu gehören eine transparente darstellung der unternehmenssituation und konkrete vorschläge zum beitrag der entsprechenden kundenunternehmen. deshalb setzt ein einsatz aktiver persönlicher preiskommunikation voraus, dass die im vorhergehenden abschnitt dargestellten strategischen optionen bereits durchdacht sind und dem kunden konkrete vorschläge unterbreitet werden können. selbst wenn es nicht um sanierungsbeiträge geht, sondern lediglich um ein halten des preises oder ein maßvolles absinken, spielt die persönliche kommunikation als wesentliche maßnahme der reduktion des empfundenen risikos von kunden eine entscheidende rolle. dies erfordert entweder vertriebliche kontakte auf der arbeits-, oder sogar gespräche auf managementebene. insofern verschiebt sich an dieser stelle nicht selten die durchführung der preiskommunikation in der hierarchie. sie wird zur "chefsache". diese einschaltung der top-ebene des krisenunternehmens ist insbesondere wegen der bedeutung des vertrauens wichtig. zugeständnisse oder vereinbarungen mit einer gewissen bindungswirkung werden nur akzeptiert werden, wenn die kompetenz der verhandelnden außer frage steht. befindet sich das unternehmen bereits in der insolvenz, wird sogar die kompetenz des managements in vielen fällen nicht ausreichen. es wird dann häufig von kunden die mitwirkung der insolvenzverwaltung in der verhandlung erwartet. die passive persönliche preiskommunikation schließlich bietet sich dann an, wenn in den erwähnten branchen und märkten noch keine breiteren reaktionen auf die krise erkennbar sind. in diesen fällen kann es möglicherweise sinn machen, nur dann in persönlichen gesprächen gezielt zu reagieren, wenn entsprechende anfragen von seiten der kunden kommen. in der praktischen umsetzung zeigt sich allerdings häufig, dass ein solches vorgehen nur in ausnahmefällen und dann auch nur für einen begrenzten zeitraum sinnvoll ist. je besser der informationsstand der kunden ist, desto mehr anfragen werden das unternehmen erreichen. dies kann so massiv werden, dass es zu vertrieblichen kapazitätsengpässen kommen kann. ein sonderfall der krise (z.b. durch die covid- -pandemie) sind situationen, in denen kurzfristig zumindest komplette branchen (z.b. einzelhandel, tourismus etc.) wenn nicht sogar das gesamte volkswirtschaftliche umfeld in eine rezession geraten. alle relevanten akteure befinden sich im krisenmodus und nicht nur einzelne unternehmen. die gefahr besteht hier insbesondere in einem kommunikativen "herdenverhalten" mit z.t. dauerhaften nachwirkungen. in der branchenkrise der luftfahrt, die schon vor co-vid- begann und zu teilweise ruinösen preiskriegen führte, kam es z.b. zu einer anhaltenden preislichen umpositionierung des fliegens bei potentiellen fluggästen. "signaling" ist deshalb gerade in solchen umfassenden krisen sehr wesentlich: eine begründete und offensiv kommunizierte eigene zurückhaltung bei preissenkungen kann ein wesentliches signal auch in die eigene branche hinein sein, auf preisliche amokläufe zu verzichten. begründungen können etwa die sicherung der qualität oder die (system-)relevanz der eigenen branche sein und die daraus resultierende notwendigkeit wirtschaftlicher stabilität durch auskömmliche preise (als eine art beitrag aller zur aufrechterhaltung bestimmter angebote). das vorgehen in der konzeptionierung der preiskommunikation unterscheidet sich generell nicht von dem zuvor geschilderten. wesentlich sind die schonungslose analyse und zielsetzung. hat das unternehmen eine realistische perspektive, die über die krise hinausreicht, ist mit der preiskommunikation wesentlich vorsichtiger umzugehen, als wenn die krise wahrscheinlich die liquidation des unternehmens bedeuten wird. im falle eines geplanten fortbetriebs sind bei der konzeptionierung des vorgehens die relative liquiditätsstärke gegenüber anderen anbietern und die flexibilität im abbau von kapazitäten sowie der ausgabenwirksamen fixkosten kritische größen: je mehr spielraum ein unternehmen hier aufweist, desto vielfältiger sind auch die handlungsoptionen und desto eher ist es möglich, auch ohne aggressive preiskommunikation zu überleben -was im sinne des langfristigen werterhalts des unternehmens auf jeden fall zu bevorzugen ist. krisen stellen eine besondere herausforderung der preiskommunikation dar: durch sich schlagartige verändernde ziele und rahmenbedingungen der unternehmensführung -höhere bedeutung der (kurzfristigen) liquidität, möglicherweise suche nach investoren etc. einerseits und sich verändernde stärkeverhältnisse im dreieck unternehmen, kunden, wettbewerb andererseits. war der preis nicht ohnehin schon teil der krisenursachen, so kann er jetzt doch schnell zum problem werden: das preisniveau zu halten, wird schwierig dadurch, dass kunden preiszugeständnisse erwarten. sie wollen die kompensation eines als höher eingeschätzten risikos und einen preis, der ihrer wahrgenommenen zusätzlichen stärke entspricht. dieser preisdruck verschärft sich, wenn wettbewerber neue chancen durch die abwerbung von kunden des krisenunternehmens erkennten und diese preisaggressiv umwerben. dem krisenunternehmen selbst liegen preissenkungen aber häufig auch gar nicht fern: es besteht die verlockung, durch aggressive preispolitik massiv den absatz zu steigern. sie wird allerdings durch eine wenig flexible nachfrage und die mittelfristig starke, negative nachwirkung einer solchen vorgehensweise relativiert. in diesem beitrag wurde dargelegt, dass es einer konsistenten preispolitik in der krise bedarf, die auf die situativen besonderheiten eingeht. zu dieser krisenstrategie ist das passende kommunikative vorgehen zu wählen. zur entwicklung von strategie und kommunikation bedarf es eines stringenten aktionsplans, der auf einer kurz-und mittelfristigen analyse der preispolitischen effekte an sich und im zusammenhang beruht. unternehmenssanierung: ursachen -krisenfrüherkennung -management unternehmen erfolgreich restrukturieren und sanieren: herausforderungen und lösungsansätze für den turnaround preiswettbewerb in krisen: auswirkungen der terror-attentate des gutachterliche kommentierung idw-s , bgh-rechtsprechung und restrukturierungsplanung management letter -wege aus der unternehmenskrise. zürich, helbing corporate finance ag idw-standard: anforderungen an sanierungskonzepte strategie und taktische maßnahmen in krisenzeiten: eine analyse über das verhalten von führungskräften in der wirtschaftskrise train wrecks: asset pricing and the valuation of severely distressed assets. ucla anderson school and nber marketing arbeitsbuch: aufgaben -fallstudien -lösungen. gabler, wiesbaden plinke w ( ) die geschäftsbeziehung als investition bewertung sanierungsbedürftiger unternehmen: wertbausteine, risikoverteilung und kapitalkosten preismanagement: strategie -analyse -entscheidung -umsetzung bewertung von unternehmen im financial distress handbuch krisenmanagement er widmet sich in der beratung schwerpunktmäßig der sanierung und restrukturierung von unternehmen sowie distressed m&a. beginnend mit seiner promotion hat er sich immer wieder mit themen der optimierung von pricing und vertrieb beschäftigt. dass eine sanierung auch diese aktionsfelder im auge behalten muss und nicht rein kostenorientiert erfolgen kann, ist eine wesentliche erfahrung, die er in zahlreichen projekten machen konnte. nach den standards der eacva ist er "certified valuation analyst" (cva) für unternehmensund markenbewertungen und seit key: cord- -yzb yo authors: popovich, michael l.; watkins, todd; kudia, ousswa title: the power of consumer activism and the value of public health immunization registries in a pandemic: preparedness for emerging diseases and today’s outbreaks date: - - journal: online j public health inform doi: . /ojphi.v i . sha: doc_id: cord_uid: yzb yo public health immunization registries and the immunization ecosystem have evolved over the past two decades to become significant population health data assets. clinical providers and pharmacists are reporting the immunizations given to their patients to public health registries in states and all territories, creating consolidated immunization event patient records. most of these immunization events are reported through the provider’s electronic health record system (ehr), pharmacy management system (pms), online, or through data uploads. meaningful use and health data standards (hl ) became the drivers that accelerated reporting to immunization registries and significantly improved the quantity and quality of the data. the infrastructure supporting the immunization ecosystem (ie) has enabled real-time compliance reporting and, more importantly, real-time patient queries. the provider community now has online access to a patient’s immunization history in over three quarters of the states, and growing. this access includes a forecast of the patient’s immunization gaps provided by public health decision support tools based upon the most recent acip recommendations. this is creating an opportunity for the provider and the patient to work together to reduce their risk of suffering a vaccine-preventable disease. this ie and the data in an immunization information system (iis) are especially useful as pharmacies expand their immunization practices and create opportunities to reduce the adolescent and adult immunization gaps. in a few states, this provider-public health ecosystem has begun to extend to individuals by allowing them to access the iis online through the use of myir. myir provides them with the electronic version of their immunization "yellow cards," recommendations for immunizations due, and the ability to print official certificates. this emerging consumer engagement creates opportunities to empower individuals to be more proactive in their family’s health care. this paper builds upon early experiments to empower individuals in this ecosystem by leveraging the value of these public health data assets and trusted communications, illustrating the possibilities for engaging consumers to support reducing the impact of emerging diseases, outbreaks and the next pandemic. this paper will suggest the value of the ie and the role individuals can play within their own social networks to advance public health efforts to manage disease events. in turn, this social mission would encourage consumers to be more proactive in managing their own healthcare. most of these immunization events are reported through the provider's electronic health record system (ehr), pharmacy management system (pms), online, or through data uploads. meaningful use and health data standards (hl ) became the drivers that accelerated reporting to immunization registries and significantly improved the quantity and quality of the data. the infrastructure supporting the immunization ecosystem (ie) has enabled real-time compliance reporting and, more importantly, realtime patient queries. the provider community now has online access to a patient's immunization history in over three quarters of the states, and growing. this access includes a forecast of the patient's immunization gaps provided by public health decision support tools based upon the most recent acip recommendations. this is creating an opportunity for the provider and the patient to work together to reduce their risk of suffering a vaccine-preventable disease. this ie and the data in an immunization information system (iis) are especially useful as pharmacies expand their immunization practices and create opportunities to reduce the adolescent and adult immunization gaps. in a few states, this provider-public health ecosystem has begun to extend to individuals by allowing them to access the iis online through the use of myir. myir provides them with the electronic version of their immunization "yellow cards," recommendations for immunizations due, and the ability to print official certificates. this emerging consumer engagement creates opportunities to empower individuals to be more proactive in their family's health care. when asked what scared him the most and kept him up at night, tom frieden, the former director of the cdc, replied: "the biggest concern is always for an influenza pandemic." [ ] a recent cnn article [ ] outlined the world health organization's (who) review of potential public health emergency diseases that included the top global concerns: crimean-congo haemorrhagic fever (cchf), ebola virus disease and marburg virus disease, lassa fever, middle east respiratory syndrome coronavirus (mers-cov) and severe acute respiratory syndrome (sars), nipah and henipaviral diseases, rift valley fever (rvf), zika, and finally disease x. currently there are no vaccines to prevent these. the who's disease x was included in this "list of blueprint priority diseases" [ ] because the world does not know what pathogen can cause the next epidemic. globally, as well as in the u.s., epidemiologists and public health professionals work endlessly to ensure the risk and impact of existing and emerging diseases are minimized, and that neither turns into a pandemic. as the world increasingly becomes interconnected through travel and technology, timely information and accurate data become more imperative. early warning of disease occurrence and assessing the resulting impact on the public are paramount. early warning systems referred to as "syndromic surveillance," along with mandated notifiable disease reporting, capture data and electronically process the information to present public health with a view into future potential impacts. over the past two decades, new information systems have played a key role in improving public health's early warning and case management for disease outbreaks. as improved analytics are used to predict risk in populations, researchers and epidemiologists open new doors to disease cures, clinical research develops new medicines, and providers develop new care models. the role of technology and public health to support these efforts therefore becomes more valuable. health information systems are traditionally considered to be used for electronic medical record or payer billing systems. they are not paired with technology advancements that exist in the ojphi hands of consumers which could encourage patients to be more proactive with their healthcare. efforts today to link information and technology to engage consumers are championed by health plans and healthcare providers. engaging and empowering individuals to be proactive when presented with their medical records is not a simple problem to solve. it is not just a matter of making data available but making it actionable. actionable information may not achieve the desired success. but what if… this was augmented with another social mission? what if… the health community engaged consumers to help them fight disease outbreaks; what if… consumers become frontline activists to report occurrences and outcomes, and become "intelligent connections" to extend the right information in their social networks? this would encourage today's consumer technology to be better integrated with the clinical health information technology (hit). it would encourage continued investment in evolving and sustaining critical public health ecosystems. it would create opportunity to engage consumers, empowering each to be more proactive in supporting population health and their own healthcare. another large part of the health information system are immunization information systems (iis) where individuals who have received vaccines are documented in a confidential computer based system in a specific geographic area [ ] . the iis can be used for disease surveillance purposes and provide valuable information to public health authorities [ ] . as an extension of one of the existing iiss, myir was created where any state iis, pharmacy or provider can provide patients direct access to family state immunization records -regardless of the type of immunization information system used. providers can communicate to patients using myir to increase patient engagement. automated vaccine reminders can be sent using this system as well. during a pandemic, the need for accurate and timely information is vital. we propose that if there were direct public health agency communication channels to individuals -by building on existing immunization networks, the public would receive correct information quicker. furthermore, there is value that can be leveraged from social networks to advance public health efforts to manage disease events and in turn encourage consumers being more proactive in managing their own health care. one could envision a public-health-engagement-approach to empower consumers begins by offering individuals a challenge and a mission they care about. a mission that allows them to contribute to the social good with the added benefit of making them more attentive to their own healthcare. placing this mission in the palms of their hands through every cell phone is the first objective. the second is to create value for this phone's owner to become active in supporting the mission. the third objective is to provide a commonly understood health event that is a cornerstone component to this phone owner's health and welfare that helps launch them toward utilizing their complete health information profiles. universally the most significant public health event in the th century was the power of vaccines and applied immunizations to individuals and large populations [ ] . online journal of public health informatics * issn - * http://ojphi.org * ( ):e , the most significant action an individual can take to reduce their risk of a vaccine-preventable disease is to become vaccinated and stay up-to-date on their immunizations. however, there is a significant gap between believing in the value of an immunization and in ensuring one's own (and one's family's) immunizations are current. how many individuals really understand what the immunization schedule recommends or are proactive in ensuring they have no immunization gaps? how many of these same individuals in an outbreak or with news of a new disease ask the question "is there a vaccine and what are my risks?" the challenge is how to engage individuals directly, empower them to be advocates for their own health and in an outbreak become sources of trusted public health messages as they communicate within their social network, effectively supporting the higher-level mission. the lesson learned from every outbreak? that the public demands accurate, timely, and transparent communication from the government. if this public health information is communicated directly to this new evangelistic network, there is the potential to expand to larger consumer networks. a few separate experiments were conducted using myir. the first experiment aimed to increase sustainment. the engagement project was to reach out in an effort to increase sustainment by contacting users who had not used myir and accessed their data from the iis in over days. the baseline looked at number of users that were logging into myir more than once a month. the target was non-engaged users. in this category there were nearly , accounts. the second experiment aimed to engage the consumer for the flu shot. for this campaign, on november st, , an email was sent to , users that asked them a simple question, "did you get your flu shot?" if they selected yes, they saw a funny meme and received positive affirmation. if they clicked no, the message was an encouragement to get their flu shot before thanksgiving . in the third experiment, consumer engagement effort was initiated january , . capitalizing on the popularity of new year's resolutions around wellness this engagement experiment created a healthy lifestyle page within myir. it featured an arizona food blogger, simple, sassy and scrumptious, who offers readers nutritious easy meal ideas. the fourth experiment focused on outreach efforts where louisiana targeted users who had failed to complete the two step process to fully enroll for access to their immunization histories. forty-nine states have established immunization information systems (iis) to capture and consolidate patient immunization events as reported by their clinicians or pharmacists [ ] . hl is a set of standards for the transfer of clinical and administrative data between applications among the healthcare system's stakeholders. in nearly all these states, and soon all, secure hl data exchanges exist, allowing electronic health records (ehrs) and pharmacy systems to report a patient immunization event to the state iis in real time. furthermore, this infrastructure allows a clinician or pharmacist to review a patient's vaccine history and actionable immunization intelligence provided by public health decision support tools that using the latest advisory committee on immunization practices (acip) recommendations to identify immunization gaps and establish opportunities for closing these through communication at the patient point-of-care. the immunization ecosystem is based on what public health has spent the past quarter century developing. this is based on statewide population health environments consisting of key data assets and technical infrastructure for reporting, accessing and assessing immunization activity. additionally, these systems layer decision support, analytics, and communications across the entire population health environment. the value that the immunization ecosystem creates is the ability for all key "players" to integrate or connect. thus, a national immunization ecosystem exists today and, as it continues to evolve, the framework and platform are primed to impact the cost of health care and improve patient outcomes. the key is to fully extend the framework to the consumer, creating end-to-end communication channels between trusted health authorities and individuals. pandemic preparedness planning that takes advantage of iiss, their infrastructure and their players while engaging individuals to be consumer activists in an emergency response network, is potentially a new approach to accelerating individual health accountability while offering public health the ability to communicate valuable outbreak or pandemic information. by doing so, the outcome should influence day-to-day consumer behavior to mitigate the outbreak's impact on the population. the sars outbreak (with a -billion-dollar economic impact) [ ] , the h n pandemic, and the zika outbreak confirmed that the questions asked by the public in each of these events were: • is there a vaccine for this [ the ability for continued government and health authority responses to these questions is an important step toward managing the welfare of concerned populations while outbreaks are studied, and public health mitigation plans are put into place. the velocity of information, both accurate and inaccurate, cannot be controllable through current communication channels available to disseminate information to individuals, especially those most at risk. public health has an expectation and relies on their disease specific health education reaches at risk individuals, and is clearly understood and accepted. this reliability can be impacted by the social media and online consumer world today. much of the public's information and behavior during a pandemic will be influenced through these social media and online networks [ ] . individuals will search for data they choose to believe [ ] . once found, they will push this information to their family and friends and their social media sphere of influence [ ] . information from social media can pose as a challenge to stability for the trusted information that public health wishes to deliver in order to inform and encourage those at risk to take action [ ] . when a pandemic occurs, the need for accurate and timely information accelerates. if the odds of receiving accurate information during a pandemic are against you in the social media world, consider the opportunity if there were direct public health agency communication channels to individuals -by building on existing immunization networks. accurate communications would be the goal during a public health event. in washington and louisiana currently, over , consumers access their immunization records through real-time connections to the state iis through myir. the "players" in these states encourage individuals to enroll for access. once enrolled, an individual is connected to one of their health data attributes through the immunization platforms, which also establishes information access to trusted health agencies. it creates the opportunity to accelerate consumer engagement and activism in a pandemic or outbreak by layering digital communication to individuals to include those most at risk, and supporting the extension of this trusted communication through the individual's social network. . using existing infrastructure for immunization data exchange, establish communication links to trusted public health authorities supporting the following actions: a. inform and engage consumers by proactively alerting and notifying each individual of their immunization coverage gaps. b. identify the nearest locations capable of providing specific immunizations and antivirals to high risk populations. c. capture feedback and monitor outcomes or concerns of individual immunization events at their time of occurrence as well as over an extended period of time. d. capture surveillance of personal (family) health events, such as influenza or influenza-like illness. . integrate provider, pharmacy and laboratory patient influenza tests, public health influenza reporting, and overall tracking of the most recent outbreak. . integrate outbreak occurrences through alerts and visual displays (risk and outbreak maps). . provide social media exchanges to link immunization ambassadors and general social networks that can support the concept of "layering" to reduce the impact of an outbreak and to ensure a controlled, consistent and accurate dissemination of information. since , a number of immunization consumer engagement campaigns have been undertaken by stc to explore the power of direct links to individuals who have enrolled and have access to their immunization records. in a -day period from mid-august to mid-november, the stc consumer tool myir had , total user visits to the state immunization registry. of these % were returning users. in that same period , users visited myir more than once a month. they spent an average of minutes on the site each time and viewed seven different pages. typically, this included the initial login, requests for their immunization records, and reviewing the forecast or immunizations due or past due for each family member. in reviewing the individuals that enroll for user access to the immunization registry, they currently are from a specific demographic. the majority are within the ages of - , with % being female. this suggests the people who see the most value in myir are likely to be women with a family. % of people opened the initial email and . % of these individuals used myir within days to access their immunization records. % answered the question with % of these saying "yes" they got their flu shot. as a result, new immunizations were administered to these individuals within days. one nine-month-old family member received a full series of age appropriate immunizations. a . % increase in returning users were tracked and a . % increase in engaged users, again defined as logging in more than once a month. there was also a . % increase in average session duration. five hundred fifty-six emails were sent and a % open rate was achieved. the email contained a single step to finalize the enrollment and thus access to their immunization medical records. fifty of those who opened the email followed through and completed the process. respondents were asked: "did you get a flu shot this year? do you feel like you got the flu this year?" % responded they did receive the flu shot this year. of these . % felt they got the flu this year which equates in this group to a . % efficacy rate for this year's influenza vaccine. in february, cdc had determined the interim estimates for the effectiveness of the influenza were % [ ]. these consumer engagements were early experiments. they initially targeted to increase consumer access and utilization of the information contained in a public health immunization registry. they have moved toward soliciting input from active users to test the concept of empowering advocates to support a larger public health mission and engage with trusted communications from government. they created the thought processes that lead to the concept of extending the ie to proactively engage individuals to support public health missions, notably outbreaks and the next pandemic. the data, technical frameworks and infrastructure of the iiss form the environment to engage and empower consumers to be field assets to support outbreak mitigation efforts and be instrumental within the social networks if a pandemic were to occur. our aim was to illustrate examples where public health agencies using direct communication channels to individuals -by building on existing immunization networks, could increase the efficacy of reaching the public with correct information. the illustrations used to demonstrate the ojphi consumer engagement potential were not designed to test the hypothesis that consumer activism and the value of public health immunization registries in a pandemic would prove effective. they do demonstrate the potential of engaging individuals that have enrolled to access their immunization records from public health registries. they demonstrate that a subset of these individuals will provide information requested from public health authorities. it was through these early experiments and the growing data assets in state immunization systems that create a framework and technical platform to accelerate the potential value of engaging individuals in response plans for pandemic preparedness planning and support of today's outbreak. the next step is to begin to engage individuals to establish those that would be willing to provide ongoing information to public health specific to immunizations and disease occurrences. this would include developing experiments that test a social campaign to engage these individuals over the course of a year to ensure they remain activists in this network and then initiate efforts to encourage them to be proactive in their health care. the impact would be monitored in order to begin to establish a model with key parameters that would allow scaling across states and different demographics of users. the experiments illustrated were not statistically tested or compared to other sources.they were not designed to collect specific and more detailed information in support of an outbreak to determine if it is possible to enroll advocates and ensure consumer activism. however, sufficient assets and a growing community of individuals with access to their online immunization histories suggest a specific demonstration project to test this hypothesis is warranted. the paper presents a concept of empowerment and a few example consumer engagement tests that indicate the possible opportunity for public health. the results of the consumer engagements were not measured against a specific research goal to determine their effectiveness. there were no comparisons to other public outreach and education methods that would established the potential. the consumer outreach efforts were not regional, culturally, or demographic specific and as such there was no intent to determine the true public health education to populations approach. these are all recommendations for next steps although there is some justification to simply begin to enhance and expand the current process and develop a stronger strategy to be tested. this is the start. the immunization ecosystem is a robust environment to build upon. consolidated immunization information systems and technology supported by public-private partnerships have reached the dynamic where data and information is available across wide networks of users, stakeholders, and consumers. while health plans, providers and pharmacists struggle to engage their networks, by encouraging patients to be proactive in their healthcare, public health immunization assets may be the tipping point to accelerate this movement since the single most common health event is an immunization, required from birth to death. furthermore, pandemic preparedness planning is primed to step beyond just response actions and traditional communication plans to reach directly into areas where outbreaks are occurring, ojphi soliciting consumer activism through this ecosystem to report disease occurrences and disseminate accurate information as public health works to mitigate the outbreak. the hypothesis can extend one step further: if you are active in supporting a higher cause and technology is the facilitator, the belief is this would not only reduce the risk of outbreaks and help mitigate the economic impact and costs, but also create momentum for more people to become proactive with all their healthcare. continued investment through government immunization programs, centers of medicare and medicaid services (cms) / match programs, office of national coordinator (onc) efforts supporting innovation, and consumer empowerment are essential to continue to evolve and sustain the immunization ecosystem and data assets. as these assets create added value to each stakeholder, the government investment begins to create a positive return. the private sector benefits also grow. their investment in this same ecosystem is necessary. today, opportunities to use this ecosystem to drive down healthcare costs and improve patient outcomes are unlimited. the value of this virtual ecosystem to the nation is untapped. being ready for the next pandemic through everyday practice is unique. we are in unique times. players in this sense represent all key stakeholders that utilize immunization information and include public health, providers, pharmacists, payers, employers, school nurses, foster care, consumers, and more. for example, feedback such as: did you receive this year's influenza immunization? do you feel like you have the flu? (the dollar cost of the flu on the u.s. economy, according to cdc health affairs - , was $ . billion.) in a pandemic with a new vaccine, the question might be: do you feel you had a reaction to the vaccine? federal funding provided by onc has partially supported the expansion and testing of stc's myir since . sustainment of public health immunization registries and technical infrastructure is the subject of a separate stc paper, "sustaining the public health immunization ecosystem through public private partnerships." the content and concepts of this paper are based upon stc's years of working in the immunization registry sector, which includes implementing state public health iis, implementing electronic hl connections to every state iis from over , pharmacies and , providers, and using data and analytics to tell the story of the power of the ecosystem. chief talks about agency's successes-and his greatest fear. the washington post world health organization gets ready for 'disease x'. cnn [internet] list of blueprint priority diseases. world health organization about immunization information systems immunisation information systems -useful tools for monitoring vaccination programmes in eu/eea countries centers for disease control and prevention (cdc). . ten great public health achievements--united states immunization information systems (iis) fundamentals:overview and development. centers of disease control and prevention estimating the global economic costs of sars learning from sars: preparing for the next disease outbreak: workshop summary pandemic (h n) : frequently asked questions centers of disease control and prevention (cdc). questions about zika. cdc frequently asked questions on severe acute respiratory syndrome (sars). who [internet the effects of media reports on disease spread and important public health measurements why facts don't change our mind. the new yorker news sharing in social media: the effect of gratifications and prior experience a new dimension of health care: systematic review of the uses, benefits, and limitations of social media for health communication. eysenbach g we would like to thank theresa munanga for her editorial assistance. we would also like to thank all stc employees, especially those who assisted with the myir studies. key: cord- -y htkvux authors: yang, jun; jing, ming; yang, xiaoge title: association between genetic polymorphisms and osteonecrosis in steroid treatment populations: a detailed stratified and dose-response meta-analysis date: - - journal: biosci rep doi: . /bsr sha: doc_id: cord_uid: y htkvux steroid treatment has become recognized as an important risk factor for avascular osteonecrosis of the femoral head. however, not all patients who receive long-term, high-dose steroids develop osteonecrosis, indicating that there are individual differences in occurrence. we explored the relationship between polymorphisms and steroid-induced osteonecrosis of the femoral head (sonfh) incidence with variables. we used a multilevel mixed-effects logistic regression model, which is an expansion of logistic regression, for each type of steroid, primary disease, drug dose, applied duration, and single-nucleotide polymorphism (snp). we also conducted a dose-response meta-analysis to analyze the cumulative dosage and sonfh risk in mutation carriers. there were significant correlations between the abcb rs mutant and sonfh in the prednisone-use and methylprednisolone/prednisone-use populations. the abcb rs mutant homozygote had a protective effect in the methylprednisolone/prednisolone renal transplant population. for apob rs , mutation increased the incidence of sonfh in prednisone-use and methylprednisolone/prednisolone-use populations and renal transplant patients. for apob rs , mutation increased the risk of sonfh in the prednisone-use population. the pai- rs mutation had a protective effect on the sonfh risk prednisone-use and renal transplant populations. abcb rs mutations have a protective effect against sonfh, and apob rs and rs increase the sonfh risk. cumulative dosage and treatment duration had little effect on the results. in addition, there was a dose-effect correlation in abcb rs and rs mutation carriers. steroid-induced osteonecrosis of the femoral head (sonfh), which leads to collapse of the femoral head and articular dysfunction, has an incidence of - % amongst patients receiving steroid treatment [ ] . the exact pathology of sonfh is still unclear and might be related to lipid metabolism disorders, abnormal microcirculation, insufficient blood supply, inflammation, and bone marrow mesenchymal stem cell osteogenesis differentiation dysfunction. the abnormal blood supply leads to the apoptosis of osteocytes and osteoblasts, followed by bone loss and reduced bone mineral density [ ] . lipid metabolism dysfunction is also an important pathology, and steroid application may lead to an increase in blood lipid levels and then to the development of intravascular lipid embolism in the microvasculature [ ] . embolism accumulation might affect microcirculation and increase the pressure of the intramedullary cavity, eventually leading to the death of bone cells. long-term or mass steroid application is the critical pathogenesis of sonfh [ ] . however, not all patients who receive long-term, high-dose steroids will develop sonfh, indicating that there are individual differences in the occurrence of sonfh. to date, several meta-analyses have been published and shown that pai- g/ g (rs ) [ ] , abcb c t (rs ) [ ] [ ] [ ] and cyp a activity [ ] are associated with sonfh incidence. however, the results of the abcb g t/a polymorphism are still disputed [ ] [ ] [ ] . our previous research reported only the single-nucleotide polymorphisms (snps) that appeared in more than three studies and indicated that abcb rs has a protective effect on sonfh in an allelic model and that the apob rs and rs mutations promote the pathogenesis of sonfh. abcb rs , mthfr rs , and pai- rs were not correlated with sonfh incidence. however, heterogeneity still exists in the previous results, and further analysis of the characteristics of the included studies is needed. therefore, this research further analyzes the effects of primary disease, type of steroids, cumulative steroid dosage, and treatment duration on the results. we used the meta-analysis of observational studies in epidemiology guidelines in the present study [ ] . two authors independently performed a literature search of the pubmed, embase, cochrane library, and chinese public databases, including the china national knowledge infrastructure, the china biology medicine database, the china science periodical database (wanfang database), and the vip journal integration platform. the search included studies published through july . the following terms were used in the search strategy: hormone, glucocorticoid, steroid, corticosteroid, osteonecrosis, femoral, femur, femoris, whirlbone, polymorphism, snp, genetic, mutation, genotype, allele, allelic, and variation. the search strategy is shown in supplementary table s . divergence in the search results was resolved by discussion. the studies were included in our meta-analysis if they met the following criteria: ( ) case-control or cohort studies comparing a population that suffered sonfh with a population that did not suffer after steroid treatment, ( ) studies assessing the associations between genetic polymorphisms and sonfh, and ( ) studies reporting the frequencies of specific alleles or the effect sizes of individual genotypes between cases and controls. studies were excluded from the analysis for the following reasons: ( ) noncase-control or noncohort studies, ( ) the case group included sonfh patients with other etiologies or sonfh patients who were not reported separately, ( ) the control group included an onfh population without steroid application or a healthy population, ( ) non-snp-related studies, ( ) studies about family heredity, and ( ) studies that did not report data pertaining to allelic frequencies or effect size. in addition, conference reports, editor comments, reviews, case reports, and academic dissertations were excluded from the analysis. two authors independently extracted the following data from each eligible study: first author's name, publication year, research location, sample size, average subject age, primary disease, steroid type, cumulative steroid dose, treatment duration, and genes of interest. if the average or median value of the cumulative steroid dosage in each group was not reported, it was estimated. the cumulative dose was generally calculated by multiplying the daily average dose and the treatment duration. for studies with a fixed treatment strategy, the cumulative dosage was calculated by multiplying the dosage of each treatment cycle by the total number of cycles. in the present study, we defined the average body surface area as . m and the average body weight as kg. for closed-interval categories, the value assigned to each dosage or duration, such as the average or median, was the midpoint. for open-interval categories, the value was estimated as the limit value multiplied by . [ ] . because of the different types of steroids used in the included studies, it was necessary to convert the doses of different types of steroids to the equivalent dose of prednisone. the conversion standard is mg prednisone = mg prednisolone = mg methylprednisolone = . mg dexamethasone [ ] . the newcastle-ottawa scale (nos), a validated tool for evaluating the quality of observation studies, was used to evaluate the methodological quality of the included studies; the scale includes the following three subscales: selection, comparability, and exposure [ ] . association analysis was performed using five genetic models: allelic (w vs m), dominant (ww+wm vs mm), recessive (ww vs wm+mm), heterozygous (wm vs mm), and homozygous (ww vs mm) models [ ] . w is the wild allele and m is the mutation allele. the odds ratios (ors) and their % cis were used to assess the strength of the associations between polymorphisms and sonfh incidence. the i statistic was used to estimate the degree of heterogeneity amongst the studies. cochran's q statistics were also taken as measures of between-study heterogeneity. generally, a significance level of p< . suggests the existence of significant heterogeneity to expand a test's sensitivity [ ] . if the i ≥ % (q test, p< . ), the random-effect model was used; otherwise, the fixed-effect model was used. then, we attempted to use a multilevel mixed-effects logistic regression model, which is an expansion of logistic regression, for each type of steroid, primary disease, drug dose, applied duration, and snp. the factors of different genotypes and other variables were included as fixed effects, and different studies were considered random effects. finally, we conducted a dose-response meta-analysis across the cumulative steroid dosage [ ] . to derive the dose-response curve, we modeled the dose using restricted cubic splines with three knots at fixed percentiles of , , and % of the distribution [ ] . all tests were two-tailed, and a p-value less than . was considered statistically significant except in cochran's q test. our research returned english articles and chinese articles after removing duplicates. after screening the titles and abstracts, of these articles were excluded. the full texts of articles were assessed, amongst which studies were excluded for the following reasons: the control group did not receive steroid therapy ( ); reviews ( ); non-snp-related studies ( ); basic studies ( ); studies without frequencies or effect size results ( ); non-steroid-related onfh studies ( ); case reports ( ); microrna-related studies ( ); duplicated reports ( ); and heredity sonfh study ( ) . ultimately, we included studies that assessed a total of patients in our meta-analysis (figure , table ). all studies were case-control designs, except one study, which was a cohort design [ ] . the primary diseases included organ transplantation, hematological disease, systemic lupus erythematosus (sle), rheumatoid arthritis (ra), nephrotic syndrome, and ophthalmic disease. in addition, two studies by wei he [ , ] , two studies by weibao fang [ , ] , and studies by masaaki and kyoko [ , ] included the same patient groups but did not assess the same snps. the nos scores ranged from six to nine points, and the overall quality of the observational studies was ideal ( table ). the details of nos scores for each study are shown in supplementary table s . amongst the preliminary results of snps that were included in more than three studies, an allelic model showed that the abcb rs mutation had a protective effect against sonfh (or: . ; % ci: . figure ). other snps were not included in the analysis because they did not appear in more than three studies. a further analysis was performed in the present study using a multilevel mixed-effects logistic regression model. the details of the included studies for each group analysis are listed in supplementary table s . in the results of the abcb rs multilevel mixed-effects regression model, the mutant homozygote and the mutant carrier could significantly protect against sonfh after steroid application (tt vs cc genotype: or: . ; % ci: . primary disease showed no significant results. it should be noted that the studies that did not report primary disease type or that included several types of primary disease were not included in the categorical analysis of primary disease ( figure ) . for abcb rs , the negative results did not change after considering cumulative dose and treatment duration. in the categorical analysis, the protective effect of the mutant was only found in the methylprednisolone/prednisolone-use renal transplant population (or: . ; % ci: . - . ; p= . ). however, this result was based on a single study and had a large standard error (se = . ); thus, this result needs to be confirmed ( figure ) . for apob rs , mutation increased the incidence of sonfh (ct figure ). due to a large number of zero events, the regression results did not converge after considering the cumulative dose. the results were not changed after considering treatment duration. in addition, in prednisone-use and methylprednisolone/prednisolone-use populations and renal transplant patients, mutation increased the risk of sonfh. for apob rs , mutation increased the risk of sonfh (ga vs gg: or: . ; % ci: . - . ; p= . ) ( figure ). the results were not obviously changed after considering cumulative dose and treatment duration. in the categorical analysis, mutation increased the risk of sonfh in the prednisone-use population (or: (figure ). these results differed from previous results because in previous research a random-effect model was used due to large heterogeneity, and a mixed-effect model was adopted in this research. the results did not change after considering cumulative dose and treatment duration. in the categorical analysis, the protective effect existed in the prednisone-use and renal transplant population. however, there was no significant correlation in the methylprednisolone/prednisolone-use population ( g g vs g g: or: . ; % ci: . furthermore, we sought to analyze the cumulative dosage and sonfh risk in mutation carriers by a dose-response meta-analysis. sonfn/onfh incidence in mutation carriers cannot be obtained when steroids are not used. therefore, the minimum dose must be used as a reference, and whether increasing the dose affects sonfh incidence must be assessed. two dose assessment methods were used: an absolute dose and multiple doses. for abcb rs , there was a correlation between the cumulative dose and the occurrence of sonfh in the mutation carriers. as the dose increased, the risk of sonfh decreased (absolute dose: p= . ; multiple dose: p= . ) ( figure a,b) . this negative correction also persists in homozygous mutation (tt genotype, absolute dose: p= . ; multiple dose: p= . ) and heterozygous mutation patients (ct genotype, absolute dose: p< . ; multiple dose: p< . ) (supplementary figure s a-d) . in wild-type populations, there was no correlation between the dose and the risk of sonfh (absolute dose: p= . ; multiple dose: p= . ). although there was no significant correlation between the abcb rs mutation and the risk of sonfh, the cumulative dose in the mutant population was correlated with the occurrence of sonfh. as the dose increased, the risk of sonfh decreased (absolute dose: p= . ; multiple dose: p= . ) ( figure c these findings may suggest that mutation carriers still have a risk of sonfh when using steroids, similar to the wild-type population. if sonfh does not occur after small-dose steroid application, increasing the steroid dose in mutation carriers will not increase but rather decrease the risk of sonfh. for apob rs , although sonfh was correlated with the dose in the mutation carriers (absolute dose: p= . ; multiple dose: p= . ), a correlation was also found in the wild-type population (absolute dose: p= . ; multiple dose: p= . ). for apob rs , there was no correlation between the cumulative dose and sonfh risk in the mutation carriers (absolute dose: p= . ; multiple dose: p= . ) or in the wild-type population (absolute dose: p= . ; multiple dose: p= . ). apob rs and rs have too few homozygous mutation patients to analyze separately. we did not perform a dose-response analysis for mthfr rs and pai- rs due to the small number of included studies. abcb rs mutations have a protective effect against sonfh, and mutations in apob rs and rs increase the sonfh risk. the present study further analyzed the effects of each specific characteristic, such as the primary disease of the included patients, the types of steroids, the cumulative dosage, and the treatment duration, on outcomes. however, with the increase in variable factors, the results will be more dependent on the results of single studies, which will reduce the stability of the results. however, the analyses performed here can provide additional clues and research directions for further basic and clinical research studies. in previous research, we analyzed the associations between snps and sonfh by traditional meta-analysis, and more than three snps were included. ultimately, abcb rs , rs , apob rs , rs , mthfr rs , and pai- rs were included. in previous meta-analyses, one study analyzed the association between abcb gene polymorphism and sonfh and the t (rs ) and t/a (rs ) alleles of abcb were identified, which may reduce the risk of gc-induced onfh [ ] . however, our study included more reports and found that rs had no correlation with sonfh. our result is the same as that from another meta-analysis that analyzed the correlation between gene polymorphism and sonfh occurrence with the grade method to assess the level of evidence. the results showed that abcb rs could reduce the occurrence of sonfh but had a very low level of evidence. apob rs and rs mutations increased the risk of sonfh and had a moderate level of evidence [ ] . the results of our study are essentially the same as those of the above two studies, except for rs . however, our work further analyzed the associations between snp and sonfh based on steroid type, primary disease, drug dose, and applied duration using a mixed-effects logistic regression model. we also conducted a dose-response meta-analysis to analyze the cumulative dosage and sonfh risk in mutation carriers. our work expanded the research results and found that cumulative steroid dosage and treatment duration had little effect on the results. there was a dose-effect correlation in abcb rs and rs mutation carriers. another study analyzed the associations between onfh and genetic polymorphisms of vascular endothelial growth factor rs , endothelial nitric oxide synthase rs , and abcb rs . however, the included studies contained sonfh-and onfh-related studies. the results showed that the mutations of rs and rs increased the risk of onfh, and the mutation of rs increased the risk of onfh in an allelic model. there were errors in the present study, as the authors proposed that the mutation of abcb rs increased the risk of onfh, but the data showed that it reduced the risk [ ] . our study analyzed sonfh risk, but not onfh, which is different from the above study. in the present study, there were significant correlations between the abcb rs mutant and sonfh in the prednisone-use and methylprednisolone/prednisone-use populations. the abcb rs mutant homozygote had a protective effect in the methylprednisolone/prednisolone renal transplant population. for apob rs , mutation increased the incidence of sonfh in the prednisone-use and methylprednisolone/prednisolone-use populations and renal transplant patients. for apob rs , mutation increased the risk of sonfh in the prednisone-use population. the pai- rs mutation had a protective effect in the sonfh risk prednisone-use and renal transplant population. there were different outcomes for the different types of steroid-use populations, possibly because the results were based on even fewer studies. these differences may also indicate that different types of steroids have subtle differences in affecting the risk of sonfh. it is generally believed that prednisone must be transformed to an active form through the liver, while prednisolone does not require this change, which may be the reason for the different results of the two drugs in abcb -related mutation states. in addition, more evidence is needed to determine whether the different results for the types of steroids in apob and pai- gene polymorphisms were due to different effects on adipocytes. currently, a registered study is analyzing the pharmacokinetics of different steroids in children, but the results have not yet been reported (nct ). an earlier study also reported that the cumulative dosage of methylprednisolone, but not prednisone, is correlated with avascular necrosis incidence [ ] . therefore, defined steroid types should be evaluated in further genetic polymorphism-and sonfh-related studies. although there are reports about the effect of primary disease on sonfh risk, most have focussed on the influence of steroids used in disease treatment. it was also reported that in renal transplant patients, cyclosporine versus tacrolimus as well as gender factors may be independent risk factors for avascular necrosis [ ] . a meta-analysis of the avascular necrosis risk in sle patients also showed, that in addition to hormonotherapy, arthritic, cushingoid, hypertension, cytotoxic drugs, etc., also affect the occurrence of sonfh [ ] . therefore, the primary disease and the treatment strategy are also possible risk factors for sonfh incidence. it is generally believed that the occurrence of sonfh is due to long-term and/or high-dosage steroid application. a dose-response meta-analysis showed that the incidence of osteonecrosis is correlated with both the cumulative dose and treatment duration [ ] . however, there is still no related research on specific mutant carriers. first, the present study found that the abcb rs mutation has a protective effect on sonfh, and the risk will be further reduced with increasing cumulative steroid dosage. it can also be concluded that some mutant carriers will have sonfh when the cumulative dosage is low; however, if sonfh does not occur, the risk of sonfh will not increase when the cumulative dose of steroids is increased. therefore, for mutant carriers, the critical time period for prevention and diagnosis of sonfh is during the early stage of steroid application, not the late stage. a negative correlation was also found in abcb rs mutation carriers, even though the mutations were not significantly associated with sonfh in general. the present study still has several limitations. first, our study was performed at the study level instead of the individual level. second, in stratified analyses, with the increase in variable factors, the results become more dependent on the results of single studies, which will reduce the stability of the results. third, in dose-response analyses, sonfn/onfh incidence in mutation carriers without steroid application cannot be calculated; therefore, the minimum dose must be used as a reference. fourth, our study only analyzed snps that were examined in more than three studies, but with emerging research and evidence, more snps may be studied in the future. therefore, the present study was limited by the research available at the time. the role of autophagy in steroid necrosis of the femoral head: a comprehensive research review vascular anatomy and microcirculation of skeletal zones vulnerable to osteonecrosis: vascularization of the femoral head incidence of genetic polymorphisms involved in lipid metabolism amongst chinese patients with osteonecrosis of the femoral head the pathogenesis of steroid-induced osteonecrosis of the femoral head: a systematic review of the literature genetic risk factors for glucocorticoid-induced osteonecrosis: a 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and elderly adults: a dose-response meta-analysis generalized least squares for trend estimation of summarized dose-response data polymorphisms in the glucocorticoid receptor gene and associations with glucocorticoid-induced avascular osteonecrosis of the femoral head influence of bcl l polymorphism on osteonecrosis during treatment of childhood acute lymphoblastic leukemia genetics of glucocorticoid-associated osteonecrosis in children with acute lymphoblastic leukemia the correlation between apolipoprotein gene polymorphisms and steroid-induced osteonecrosis of the femoral head genetic association of the p-glycoprotein gene abcb polymorphisms with the risk for steroid-induced osteonecrosis of the femoral head in chinese population mdr gene polymorphisms are associated with glucocorticoid-induced avascular necrosis of the femoral head in a chinese population association of apolipoprotein a genetic polymorphisms with steroid-induced osteonecrosis of femoral head in a chinese han population relationship between apolipoprotein b gene polymorphism and tendon-vessel stagnation syndrome of steroid induced osteonecrosis of the femoral head genetic polymorphisms in plasminogen activator inhibitor- predict susceptibility to steroid-induced osteonecrosis of the femoral head in chinese population association of a polymorphism in pon- gene with steroid-induced osteonecrosis of femoral head in chinese han population association between polymorphisms of steroid induced osteonecrosis of the femoral head and traditional chinese medicine syndrome types relationship between gene polymorphisms and syndrome types in steroid-induced femoral head osteonecrosis relationship between gene polymorphisms and hereditary susceptibility of steroid-induced femoral head osteonecrosis in linyi city of shandong province polymorphism in the pai- (serpine ) gene and the risk of osteonecrosis in children with acute lymphoblastic leukemia incidence of genetic polymorphisms involved in lipid metabolism amongst chinese patients with osteonecrosis of the femoral head genetic susceptibility of corticosteroid-induced osteonecrosis of femoral head combination analysis of three polymorphisms for predicting the risk for steroid-induced osteonecrosis of the femoral head a pai- (serpine ) polymorphism predicts osteonecrosis in children with acute lymphoblastic leukemia: a report from the children's oncology group roles of tnf-alpha gene polymorphisms in the occurrence and progress of sars-cov infection: a case-control study genetic association of a polymorphism of the camp-responsive element binding protein-binding protein with steroid-induced osteonecrosis after kidney transplantation mdr (abcb ) gene polymorphisms associated with steroid-induced osteonecrosis of femoral head in systemic lupus erythematosus low molecular weight phenotype of apo(a) is a risk factor of corticosteroid-induced osteonecrosis of the femoral head after renal transplant apob c t polymorphism predicts risk for steroid-induced osteonecrosis of the femoral head after renal transplantation thrombophilia and avascular necrosis of femoral head in kidney allograft recipients association of corticosteroids and factor v, prothrombin, and mthfr gene mutations with avascular osteonecrosis in renal allograft recipients pharmacogenetic risk factors for osteonecrosis of the hip amongst children with leukemia relationship between postrenal transplant osteonecrosis of the femoral head and gene polymorphisms related to the coagulation and fibrinolytic systems in japanese subjects genetic analysis of steroid-induced osteonecrosis of the femoral head abcb c t and g t/a polymorphism decreased the risk for steroid-induced osteonecrosis of the femoral head after kidney transplantation association of plasminogen activator inhibitor- genotype with avascular osteonecrosis in steroid-treated renal allograft recipients lipid transporter activity-related genetic polymorphisms are associated with steroid-induced osteonecrosis of the femoral head: an updated meta-analysis based on the grade guidelines vegf, enos, and abcb genetic polymorphisms may increase the risk of osteonecrosis of the femoral head avascular necrosis of bone after cardiac transplantation. prevalence and relationship to administration and dosage of steroids cyclosporine use and male gender are independent determinants of avascular necrosis after kidney transplantation: a cohort study the risk factors of avascular necrosis in patients with systemic lupus erythematosus: a meta-analysis the authors declare that there are no sources of funding to be acknowledged. nos, newcastle-ottawa scale; or, odds ratio; ra, rheumatoid arthritis; sle, systemic lupus erythematosus; snp, single-nucleotide polymorphism; sonfh, steroid-induced osteonecrosis of the femoral head. y.j. designed the study; performed statistical analysis; edited and reviewed the manuscript; guarantor of integrity of the entire study. y.x.g. performed literature research and data acquisition and analysis. j.m. performed literature research and data acquisition and analysis; prepared and reviewed the manuscript. all authors approved the final manuscript. the authors declare that there are no competing interests associated with the manuscript. key: cord- -ecmayzr authors: savarin, carine; dutta, ranjan; bergmann, cornelia c. title: distinct gene profiles of bone marrow-derived macrophages and microglia during neurotropic coronavirus-induced demyelination date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: ecmayzr multiple sclerosis (ms) is a chronic inflammatory disease of the central nervous system (cns) characterized by demyelination and axonal loss. demyelinating lesions are associated with infiltrating t lymphocytes, bone marrow-derived macrophages (bmdm), and activated resident microglia. tissue damage is thought to be mediated by t cell produced cytokines and chemokines, which activate microglia and/or bmdm to both strip myelin and produce toxic factors, ultimately damaging axons and promoting disability. however, the relative contributions of bmdm and microglia to demyelinating pathology are unclear, as their identification in ms tissue is difficult due to similar morphology and indistinguishable surface markers when activated. the cd t cell-induced autoimmune murine model of ms, experimental autoimmune encephalitis (eae), in which bmdm are essential for demyelination, has revealed pathogenic and repair-promoting phenotypes associated with bmdm and microglia, respectively. using a murine model of demyelination induced by a gliatropic coronavirus, in which bmdm are redundant for demyelination, we herein characterize gene expression profiles of bmdm versus microglia associated with demyelination. while gene expression in cns infiltrating bmdm was upregulated early following infection and subsequently sustained, microglia expressed a more dynamic gene profile with extensive mrna upregulation coinciding with peak demyelination after viral control. this delayed microglia response comprised a highly pro-inflammatory and phagocytic profile. furthermore, while bmdm exhibited a mixed phenotype of m and m markers, microglia repressed the vast majority of m -markers. overall, these data support a pro-inflammatory and pathogenic role of microglia temporally remote from viral control, whereas bmdm retained their gene expression profile independent of the changing environment. as demyelination is caused by multifactorial insults, our results highlight the plasticity of microglia in responding to distinct inflammatory settings, which may be relevant for ms pathogenesis. introduction multiple sclerosis (ms) is a chronic inflammatory disease of the central nervous system (cns), characterized by demyelination and axonal damage. active demyelinating lesions are characterized by cd t cells, cd t cells expressing both th and th cytokines, bone marrow-derived macrophages (bmdm) and activated cns resident microglia ( , ) . myeloid cells activated by t cell effector functions are thought to participate in tissue damage by removing or "stripping" myelin ( ) , and secreting toxic factors, such as reactive oxygen species, nitric oxide and the pro-inflammatory cytokines, tumor necrosis factor (tnf), and il- β ( , ) . activated microglia also secrete chemokines, which recruit innate and adaptive immune cells into the parenchyma, further amplifying the destructive inflammatory response ( ) . however, both bmdm and microglia effector functions are highly heterogeneous depending on the environment and may not only contribute to disease progression but also to resolution ( , ) . for example, by removing apoptotic cells and debris, their phagocytic activity favors tissue repair and is essential for disease resolution ( ) . in addition, both cell populations secrete anti-inflammatory cytokines, such as il- and tgf-β, as well as trophic factors, which provide an environment that promotes tissue repair and neuronal protection ( ) . the heterogeneity of the inflammatory response associated with ms lesions at the cellular and functional levels, thus makes it difficult to establish detrimental versus disease resolving functions of bmdm and microglia in ms pathogenesis. in addition to the inherent limitations associated with sampling cns tissues for longitudinal studies, the individual role of bmdm versus microglia as pathological mediators remains ambiguous due to morphological similarities and lack of reagents uniquely identifying each population. however, increasing evidence from animal models supports the concept that microglia and bmdm comprise two effector populations with distinct origins (derived from progenitors in the embryonic yolk sac and circulating monocytes respectively) and functions during ms and other neuroinflammatory disorders ( ) . a variety of murine models, including autoimmune-and viral-induced demyelination, have been developed to study pathogenic features of ms ( ) . the most common is the experimental autoimmune encephalomyelitis (eae), an autoreactive cd t cell-induced autoimmune demyelination characterized by infiltration of myelin-specific th and th cells, bmdm and microglial activation ( , ) . pathogenesis during eae is associated with temporally distinct microglial activation and bmdm cns infiltration. early microglia activation is insufficient to trigger clinical disease, whereas delayed cns recruitment of bmdm directly correlates with disease progression. importantly, depletion of bmdm but not microglia inhibits eae ( , ) . similarly, mice deficient in ccl (ccl −/− ), a chemokine essential for inflammatory monocyte recruitment into the cns ( ) , are resistant to eae ( ) . in support of detrimental bmdm functions, a combined histological and gene profiling study showed that demyelination is mediated by bmdm associated with nodes of ranvier, whereas debris clearance is achieved by microglia ( ) . altogether, studies in the eae model demonstrate that bmdm recruitment into the cns is essential for the process of myelin loss and clinical manifestation. inflammatory demyelination is also induced following infection with two natural viral mouse pathogens, theiler's murine encephalomyelitis virus (tmev) and members of the neurotropic mouse hepatitis viruses (mhv). tmev infection induces an autoimmune disease in which bmdm are essential for both viral persistence and demyelination ( , ) . however, the function of bmdm as a main reservoir of active viral replication during chronic tmev infection, limits efforts to assess their role in demyelination independent of virus load ( ) . in contrast, infection with the non-lethal glia tropic mhv strain designated jhmv predominantly targets oligodendrocytes (olg) and to a lesser extent microglia and astrocytes. viral replication peaks at day post infection (p.i.), but infectious virus is reduced below detection by day p.i. acute infection initiates rapid cns recruitment of predominantly bmdm, but also neutrophils and nk cells, followed by infiltration of both cd and cd t cells, as observed in active ms lesions. the t cell response, which is essential to reduce viral replication, is highly th polarized with no evidence of il- or gm-csf production ( ) ( ) ( ) . importantly, t cell-mediated virus control coincides with initiation of demyelination, which peaks between days - p.i. after infectious virus is cleared ( , ) . although olg tropism is a requirement for demyelination, immunodeficient mice demonstrated that infection of olg in the absence of adaptive immunity is insufficient to cause demyelination. however, transfer of either virus-specific cd or cd t cells into virus infected immunodeficient mice leads to demyelination ( , ) . furthermore, ifn-γ dependent control of infectious virus within olg and no evidence for olg apoptosis, suggested that direct t cell-mediated cytolysis of olg does not play a major role in myelin loss ( ) . this implicates t cell activated bmdm and microglia as the most probable mediators of myelin destruction. moreover, both myeloid populations are abundant in lesions and occasionally associated with damaged axons ( ) . however, in contrast to eae, genetic or chemical depletion of monocytes during jhmv infection does not alter disease severity, virus replication or myelin loss ( , ) , suggesting that bmdm are dispensable for jhmv-induced demyelination. this study takes advantage of the distinct tissue environments established during eae and jhmv infection to characterize temporal alterations in gene expression profiles of bmdm versus microglia in a th biased demyelination model. to date, we are not aware of any reports evaluating the signature profile of microglia associated with pathogenic functions during demyelination. the results reveal that cns infiltrating bmdm rapidly establish a characteristic profile including m and m markers, which prevails throughout infection as the population declines. by contrast, gene expression in microglia is only prominently altered remote from viral control concomitant with demyelination; distinct from bmdm, the gene expression pattern is skewed to a highly pro-inflammatory and phagocytic profile. the results overall highlight the plasticity of microglia responses in distinct inflammatory settings, which may be relevant for ms pathogenesis at distinct stages of disease. wild-type (wt) c bl/ mice were purchased from the national cancer institute (frederick, md, usa). homozygous ccl deficient (ccl −/− ) mice were originally obtained from b. j. rollins (dana-farber cancer institute, boston, ma, usa). cx cr gfp/gfp (b . p-cx cr tm litt /j) and ccr rfp/rfp (b . (cg)-ccr tm . ifc /j) mice were purchased from the jackson laboratory (bar harbor, me, usa) and crossed to generate cx cr gfp/+ ccr rfp/+ mice. transgenic mice were bred and maintained at the biological research institute under sterile conditions. all procedures were preformed in compliance with the cleveland clinic institutional animal care and use committee approved protocols. the glia tropic jhmv neutralizing monoclonal antibody (mab)derived . v- variant was used for all infections ( ) . mice of both sexes between and weeks of age were infected in the left hemisphere with , pfu of jhmv diluted in endotoxin-free dulbecco's phosphate-buffered saline (pbs) in a final volume of µl. mice were monitored daily for clinical disease severity according to the following scale: , healthy; , hunched back and ruffled fur; , partial hind limb paralysis or inability to maintain the upright position; , complete hind limb paralysis; , moribund or dead. for analytical flow cytometry, anesthetized mice were perfused with ice-cold pbs, and resected brains and spinal cords homogenized using a ten-broeck tissue grinder as described ( ) . tissue homogenates were adjusted to % percoll (pharmacia, uppsala, sweden) and underlaid with ml % percoll prior to centrifugation at g for min at °c. cns mononuclear cell were recovered from the / % interface, washed and resuspended in facs buffer (pbs + % bovine serum albumin). cells were blocked with anti-mouse cd /cd (clone . g ) mab for min on ice prior to staining. staining was performed for min on ice using fluorescein isothiocyanate, phycoerythrin, peridin chlorophyll protein complex (percp), or allophycocyanin (apc) conjugated mab (all from bd biosciences except where indicated) specific for cd (clone ly- ), cd b (clone m / ), f / (serotec, raleigh, nc, usa) and major histocompatibility complex (mhc) class ii (clone g ). cells were then washed twice in facs buffer prior to analysis using a bd accuri flow cytometer (bd biosciences) and flowjo software (tree star inc., ashland, or, usa). for cell purification, spinal cords from pbs-perfused cx cr gfp/+ ccr rfp/+ mice were finely minced with a razor blade. minced tissues were enzymatically digested in rpmi medium containing % fetal calf serum, . % collagenase d ( mg/ml) roche, basel, switzerland and % dnase i ( mg/ml) (sigma aldrich, st. louis, mo, usa) for min at °c. collagenase was then inactivated by addition of % . m edta for min at °c prior centrifugation at g for min at °c. spinal cord-derived cells from seven mice were pooled and isolated using percoll gradients as described above and then stained with cd b-percp and cd -apc for min on ice. spinal cord-derived bmdm (cd hi cd b + ccr rfp+ ) and microglia (cd low cd b + cx cr gfp+ ) were purified using a facsaria cell sorter (bd biosciences) and resuspended in trizol. yields from -pooled mice ranged between . - × cells for bmdm and . - . × cells for microglia depending on the time p.i. microglia from naïve mice were used to assess baseline expression, whereas circulating monocytes were used as controls for cns infiltrated bmdm after infection. monocytes were isolated from blood treated with gey's solution to lyse red blood cells prior to staining and cell sorting. gene expression profiling using ncounter analysis rna was prepared by extraction with trizol reagent (invitrogen, carlsbad, ca, usa) and direct-zol rna mini prep (zymo research, irvine, ca, usa) according to the manufacturer's instructions. gene expression profiles were analyzed using the ncounter mouse myeloid innate immune panel comprising targets representing all major myeloid cell types and generated according to the manufacturer's protocol (nanostring technologies, seattle, wa, usa). the nanostring ncounter system directly captures and counts individual mrna transcripts using a multiplexed measurement system thereby omitting cdna based amplification ( ) . analysis was performed using nsolver analysis software v . and ingenuity pathway analysis (qiagen, hilden, germany). venn diagrams from individual gene lists and protein-protein interaction networks were constructed using genespring (agilent, inc.) and string software (http://www. string-db.org). to confirm validity of nanostring ncounter analysis, a small set of selected genes were analyzed by real-time pcr ( figure s in supplementary material). following rna extraction as described above, first-strand cdna was synthesized using reverse transcriptase (invitrogen) with oligo-dt and random primers (promega, madison, wi, usa) as described ( ) . gene expression analysis was performed using a fast real-time pcr system (applied biosystems, foster city, ca, usa), sybr green master mix (applied biosystems) and the following primers: gapdh, ′-catggccttccgtgttccta- ′ (forward) and ′-atgcctgcttcaccaccttct- ′ (reverse); il , ′-tg aggctggcattcatgtctt- ′ (forward) and ′-tccagtt ggcctctgttttagg- ′ (reverse); il rn ′-agatagacatg gtgcctattgacctt- ′ (forward) and ′-catctccagac ttggcacaaga- ′ (reverse) and arg ′-tgggtggatgct cacactga- ′ (forward) and ′-caggttgcccatgcaga tt- ′ (reverse). transcripts levels were normalized to the housekeeping gene gapdh and converted to a linearized value using the following formula: (c t gapdh-c t gene) × , , where ct represents the threshold cycle value. following pbs perfusion, spinal cords were fixed in % neutral buffered formalin, embedded in paraffin and sections stained with luxol fast blue as described to visualize demyelination ( ) . for analysis of iba + cells spinal cords from ice-cold pbs-perfused mice were quickly embedded in oct and kept at − °c until µm sections were prepared. sections were fixed with paraformaldehyde for min, treated with blocking solution for min and then stained with rabbit anti-iba mab (wako, osaka, japan) overnight at °c. goat anti-rabbit secondary ab (invitrogen) was added for h at room temperature and sections mounted with vectashield mounting medium (vector laboratories, burlingame, ca, usa). sections were analyzed using a leica tcs confocal microscope. to better characterize reactivity of microglia and infiltrating bmdm following jhmv infection, we initially monitored cns infiltration of bmdm, as well as upregulation of mhc class ii as an activation marker on both cns bmdm and microglia by flow cytometry. bmdm with a typical cd hi cd b + f / + phenotype comprised the majority of inflammatory leukocytes as early as day p.i. and then progressively decreased as virus replication is controlled by t cells. at the onset of demyelination at day p.i., the bmdm population stabilized at ~ % of the infiltrating leukocytes ( figure a ). bmdm initially infiltrated as mhc class ii lo expressing cells, but the vast majority upregulated mhc class ii by day p.i. mhc class ii expression on microglia was sparse at days and p.i., but rapidly increased by day p.i. and then gradually declined by day p.i. (figure a) . these kinetics supported that microglia and bmdm activation peaks delayed relative to peak bmdm accumulation and coincides with peak t cell ifn-γ production ( , ) . enhanced activation of microglia at day p.i., compared to earlier times p.i., was also supported by progression of morphological changes, evidenced by enlarged cell bodies and retracted and thickened processes ( figure b) . the decline of bmdm, but an ongoing activation phenotype of microglia at the time of evident demyelination implicated microglia as mediators of tissue damage during jhmv encephalomyelitis. biochemical depletion of peripheral monocytes indeed supported that bmdm are not essential to tissue destruction in jhmv-infected mice ( ) . data from our own laboratory further demonstrated that the chemokine ccl is essential for bmdm accumulation within the cns ( ) . the absence of ccl resulted in an ~ % reduction of bmdm at all time points, including day p.i. ( ) and ( figure c ) when demyelination is prominently evident in wt mice. nevertheless, microglia activation, as monitored by mhc class ii expression, was independent of ccl ( figure d ). most importantly, the absence of ccl -dependent bmdm within the cns did not alter demyelination ( figure e ). similar myelin loss at day p.i. comparing wt and ccl −/− infected mice supported the concept that microglia mediate demyelination during jhmv infection. we next evaluated effector functions of bmdm versus microglia associated with jhmv-induced demyelination by comparing gene expression profiles using ncounter analysis of mrna isolated from purified bmdm and microglia of infected cx cr gfp/+ ccr rfp/+ mice. characteristic expression of cx cr gfp and ccr rfp on cd high cd b + bmdm (population # ) and cd low cd b + microglia (population # ) is shown in figure throughout days - p.i. microglia were characterized by high expression of cx cr and undetectable ccr expression (figures b,c) similar to other inflammatory models ( , ) . in contrast, cns infiltrating bmdm expressed ccr and low levels of cx cr compared to microglia ( figure b ). co-expression of ccr and cx cr was maintained on bmdm at all time points p.i. and no cx cr − cells were detectable ( figure c ). as both microglia and infiltrating bmdm retained their phenotype throughout infection, cd low cd b + cx cr gfphi ccr − and cd hi cd b + cx cr gfplow ccr + populations were isolated by facs from spinal cords at days , , , and p.i. for subsequent mrna expression analysis. age-matched naïve animals were used to isolate microglia and blood circulating monocytes as precursors of cns-infiltrating bmdm. gene expression profiles for all purified populations were obtained using ncounter analysis and the innate myeloid immune panel. the respective naïve populations were used to assess signature gene expression profiles under homeostatic conditions (figure ) . figure a shows the top highly expressed genes within each population relative to three ncounter platform housekeeping genes, namely g pdx, polr b, and tbp, selected for three high, medium, and low expression, respectively, in this part of analysis platform. figure b lists the top enriched genes specific for microglia compared to monocytes, or monocytes versus microglia, respectively. among the top genes highly expressed in microglia, were also specific and included genes of the complement cascade (c qa, c qb, c qc, and c ar ) and trem , encoding a cell surface receptor involved in phagocytic functions and known to be expressed by microglia ( ) . other genes, such as adamts , f r or hpgds, found within the top enriched genes expressed by microglia ( figure b ) were also previously described as microglia specific ( , ) . cx cr mrna encoding the fractalkine receptor and used as a marker for microglia ( ) , was also among the top highly expressed genes ( figure a) , but not unique, consistent with the cx cr lo phenotype on circulating monocytes. similarly, ccr expression characteristic of monocytes was confirmed by ccr mrna as the second in place of the top expressed genes specific for circulating monocytes (figure b) . other specific signature genes of monocytes are related to motility and migration/tissue invasion, e.g., s a , s a , s a , fn , sema , mmp , and to a lesser extent mhc class ii antigen presentation, e.g., h -ab , cd , and fas. microglia and circulating monocytes also shared highly expressed genes, including genes characteristic of the myeloid lineage such as csf r, a gene coding for a cell surface receptor essential for hematopoietic figure a ). interestingly however, three genes among the common and highly expressed genes were regulated differently. ctss mrna, encoding for cathepsin s, a lysosomal cysteine proteinase participating in the mhc class ii molecule antigen presentation pathway as well as nociception ( , ) , was specifically upregulated within bmdm, with highest levels observed at days and p.i. (figure a) , when demyelination increases. by contrast, microglia transiently downregulated ctss mrna at day p.i. opposite regulation was also observed for dusp mrna (figure a ). dusp mrna encodes the dual specificity protein phosphatase , an enzyme involved in the cellular stress response and a negative regulator of cell proliferation ( ) . while dusp mrna levels were vastly upregulated early following bmdm accumulation, but declined by day p.i. and thereafter, levels were rapidly downregulated within microglia ( figure b) . finally, tyrobp mrna encoding for the trem adaptor dap and known to regulate microglia phagocytic functions ( ) , was downregulated in bmdm throughout infection, but specifically upregulated within microglia at days and p.i. (figure b ). this expression pattern on microglia correlated with the onset of myelin loss and supported trem signaling specifically by microglia in response to tissue damage. to determine whether apparently differential functions of bmdm and microglia associated with jhmv-induced demyelination are reflected in distinct gene profiles, we monitored overall up-and downregulation of gene expression relative to basal levels in each population. analysis times were chosen to correlate with innate responses (d p.i.), peak t cell effector function (d p.i.), resolution of infection and initiation of demyelination (d p.i.), and finally viral clearance and overt demyelination (d p.i.). at day p.i. a higher number of genes were differentially regulated within infiltrating bmdm compared to microglia ( versus ; figure a ). moreover, almost % of the genes showing altered expression in early infiltrated bmdm were increased compared to basal levels, while only % were increased in microglia; the remaining differentially expressed mrnas were decreased ( figure b) . the overall number of differentially expressed genes slightly declined in bmdm by day p.i., when t cells exert maximal effector function ( ) , and remained fairly constant throughout day p.i. (figure a) . furthermore, the relative decline in the proportion of upregulated mrnas coincided with an increased proportion of downregulated genes, reaching a roughly equal distribution at days - p.i., when virus is largely controlled (figure b ). in contrast, microglia altered their gene expression pattern extensively at day p.i. ( genes, figure a ) with % of differentially regulated genes showing increases ( figure b ). by day p.i., overall altered gene expression remained stable relative to day p.i., with equal proportions showing increases and decreases. however, as myelin loss progresses by day p.i., differentially regulated genes increased again in numbers, with the proportion of upregulated mrnas reaching % (figure a ). altogether these data show unique regulation of gene profiles in bmdm compared to microglia throughout the course of infection. while most changes were evident in bmdm following initial cns accumulation, microglia revealed most pronounced changes at the time of myelin loss. we further analyzed differential gene expression across time points focusing on upregulated genes using venn diagrams to reveal the relative proportion of genes that were commonly increased at all time points (figure c ). of the upregulated genes in bmdm at day p.i., were unique to day . on the other hand, genes (representing % of all upregulated genes) remained highly expressed at all other time points (figure c ). of note, not a single gene transcript was specifically upregulated at day p.i., and only three overlapped with sustained upregulation at days and p.i. similarly, only gene transcripts were specifically elevated at day p.i., were unique to both days and , and only one was unique to day p.i. these results indicate that the gene expression profile characterizing bmdm is established early following infection, with sparse unique alterations as bmdm decline during infection. in stark contrast, only of gene transcripts upregulated in microglia at day p.i. were unique to day , and no gene transcript was commonly upregulated across all time points analyzed. furthermore, distinct from bmdm, gene transcripts were figure | gene expression characterizing microglia and circulating monocytes under homeostatic conditions. spinal cord-derived microglia (cd low cd b + cx cr gfp+ ) and circulating blood monocytes (cd b + ccr rfp+ ) were purified from naïve cx cr gfp/+ ccr rfp/+ mice by facs and rna subjected to ncounter analysis using the myeloid cell probe panel. panel (a) depicts the top highly expressed genes and (b) the enriched genes uniquely characterizing each population. in (a) * highlights genes that are both highly expressed and enriched in each population, while # highlights genes highly expressed and common to both microglia and circulating monocytes. uniquely upregulated by day p.i., with none common to day p.i., and only six overlapping with those upregulated at day p.i. although no gene transcripts were upregulated uniquely at day p.i., overlapped with those upregulated at day p.i. a further genes, comprising % of all upregulated genes at day p.i., were specifically expressed at elevated levels at day p.i. coinciding with overt myelin loss ( figure c) . these profiles reveal a dynamic range of responses and extensive plasticity of gene expression profiles in microglia throughout jhmv infection ( figure c ). we next used a protein-protein network connection constructed based on differential gene expression to specifically examine upregulation of gene transcripts within bmdm and microglia correlating with demyelination at day p.i. for comparison, we also analyzed the network connection at day p.i., when expression profiles were most prominently altered in bmdm, but more modestly in microglia. this comparative analysis was chosen to provide clues about specific functions and involvement of microglia relative to bmdm in tissue destruction (figures and ) . our initial focus was on temporally altered networks in bmdm (figure ) . at day p.i., early infiltrated bmdm expressed a wide array of chemokines regulating cns infiltration of both innate (cxcl , ccl , ccl , ccl , ccl , and ccl ) and adaptive (cxcl , cxcl ) immune cells (figure a) . a large cluster of molecules regulating the innate immune response, essential to limit early viral replication ( , ) , was also expressed by bmdm (figure a) . these include pathogen recognition receptors such as tlrs (tlr - and tlr ) and molecules linked to the tnf pathway (tnf, traf , etc.). finally, bmdm expressed molecules involved in antigen presentation, including tap and tap , as well as t cell activating co-stimulatory molecules (cd and cd ) or il- , which induce th differentiation (figure a) . these results indicate that early infiltrating bmdm orchestrate the acute innate immune response crucial for limiting cns viral spread, as well as initiating the adaptive immune response by recruiting and activating t cells. at day p.i., correlating with peak demyelination, a more restrained number of mrna transcripts were upregulated in bmdm (figure b) . the cluster of chemokines mobilizing immune cells was sustained ( figure b) . in contrast, the molecular network extended from tnf was more limited at d p.i. compared to d p.i. (figures a,b) . molecules regulating primarily the cd t cell response were expressed at day p.i. and comprised gene transcripts involved in antigen presentation including mhc class ii (h -ab ) and co-stimulatory molecules such as cd and cd (figure b) . interestingly, among the more restricted number of gene transcripts upregulated at day p.i. in bmdm, several were transcripts encoding m molecules, which included arg , il rn and tgm (figure b) . in contrast to the vast number of genes upregulated early following infection in bmdm, a significantly lower number of upregulated genes characterized microglia at day p.i. (figure a) . transcripts for chemokines regulating migration of both innate and adaptive immune cells, such as ccl , ccl , ccl , ccl , cxcl , and cxcl , were expressed by microglia at day p.i. (figure a) . transcripts for tnf and other inflammatory cytokines generally associated with the innate responses, e.g., il a, il b, and il were also upregulated early in microglia ( figure a) . another extended network of tnf comprised psmb and psmb , subunits of the immunoproteasome, essential for antigen presentation by mhc class i molecules. by day p.i., the number of upregulated transcripts extensively increased in microglia (figure b) . the most clustered network comprised proteins like ccl , cxcl , cxcl , cxcl , cxcl , and cxcr , all chemokines and chemokine receptors regulating migration and arrest of adaptive immune cells within the cns during inflammation ( ) . this chemokine cluster was linked to tnf and inflammatory cytokines previously detected at d p.i., e.g., il a, il b, and il . another extended network of tnf comprised psmb and psm , also present at d p.i., ctnnb encoding b-catenin, a cellular adhesion molecule, and cdkn a, a cyclin inhibitor. other upregulated gene transcripts associated with class i antigen presentation, e.g., tap and tap , were also linked through a network associated with complement component genes (c , c ar , c b, c qa, c qb, c qc), which are highly expressed within microglia under homeostatic conditions (figure ) . similarly, tyrobp and trem , which formed phagocytic synapses ( ), are both highly expressed in microglia during myelin loss. finally, a wide variety of upregulated gene transcripts are associated with mhc class ii antigen presentation and modulation of t cell function. this includes h -ab , encoding for the mhc class ii molecules and h -dm, encoding for a second accessory protein, which facilitates peptide loading. similarly, genes associated with the invariant chain of mhc class ii were increasingly expressed within microglia, such as cd and ctss (cathepsin s, which cleaves invariant chain thereby promoting loading on mhc class ii). in addition, genes encoding for modulators of the cd t cell response, such as itgax (cd c), cd and cd were also expressed by microglia ( figure b) . overall, the upregulated networks are related to complement activation, enhanced class i and class antigen processing and presentation (potentially related to ifn-γ responses) as well as migration and phagocytic activity. however, there does not appear to be a bias toward phagocytic receptors over other components activated by pro-inflammatory mediators. pathogenic versus protective functions of myeloid cells following activation have also been correlated to expression of key molecules defined as m versus m markers. while the strict classification of myeloid cells into the m or m category has been tempered based on a more dynamic and mixed phenotype during inflammatory responses ( , ) , the m and m markers remain helpful to gage overall effector functions. among the analyzed gene transcripts in the nanostring myeloid panel related to m /m polarization ( m and m genes), between and % were upregulated across the course of jhmv infection with no difference comparing m versus m genes ( figure a) . among the total upregulated m markers, about % were commonly increased within both infiltrating bmdm and microglia; representative genes were il b and il ra (figures a,b) . however, while high levels of il b mrna were observed in both bmdm and microglia at d p.i., expression was only sustained in microglia at day p.i. and thereafter (figure b ). by contrast, il ra was increased in both bmdm and to a lesser extent in microglia at d p.i., but was decreased in both populations at later time points p.i. (figure b ). in addition, between and % of m markers were specifically expressed by infiltrating bmdm during the course of jhmv infection (figure a) , including cd , atf , ifng, ptgs , ccr , cxcl , and cxcl transcripts ( figure b and data not shown). however, only m related gene transcripts were specifically upregulated in microglia at the time of demyelination, including ccl , fas, cxcl , tnfsf , and psmb . (figures a,b and data not shown) . importantly, the most prominent difference between microglia and bmdm was noted in m marker regulation. among the - % m gene transcripts upregulated following jhmv infection, only a small proportion ( - %) was expressed by microglia ( figure a) . fn was the only m marker specifically expressed by microglia at the time of demyelination ( figure a) . although il rn transcript expression was elevated in both bmdm and microglia, the increase was at best modest in microglia ( figure c) . the majority ( - %) of m markers upregulated during jhmv infection were rather expressed by infiltrating bmdm, including arg , erg , il- , and ccl (figures a,c and data not shown). increased transcript levels were most pronounces at days and p.i., but dropped off thereafter. altogether, these data showed that while infiltrating bmdm express a mixed phenotype of m and m markers during jhmv infection, microglia expressed primarily pro-inflammatory genes while not expressing m markers. microglia and infiltrating macrophages are major components of ms active lesions ( ) . their effector functions are highly heterogeneous as evidenced by both pathogenic and protective functions during the course of ms ( ) . they can promote tissue damage by releasing toxic and pro-inflammatory molecules, mediate demyelinated axons through phagocytosis as well as propagate inflammation by recruiting and activating adaptive immune cells. on the other hand, both populations also display protective functions by clearing myelin debris, which facilitates remyelination, as well as releasing trophic and anti-inflammatory factors, which promote tissue repair. while it remains a challenge to distinguish infiltrating macrophages from microglia in ms lesions due to morphological and phenotypic similarities, they are disparate effector cells based on animal ms models ( , ) . questions relating to the pathogenicity of infiltrating macrophages and/or microglia in ms remain unanswered. can both populations display protective functions? do they display dynamic functions throughout the evolution of ms lesions? deciphering the respective roles of macrophages versus microglia in facilitating tissue damage and/or repair is essential to our understanding of ms pathogenesis and development of effective therapeutic strategies. in the murine eae model of ms, infiltrating bmdm are essential in mediating demyelination ( ) . gene expression profiles demonstrated that bmdm are indeed highly phagocytic and inflammatory at disease onset, while microglia display a repressed phenotype ( ) . by contrast, during jhmv-induced demyelination, recruited bmdm are dispensable for the demyelinating process ( ) . distinct from eae, where microglia activation precedes cns infiltration of bmdm ( ) , jhmv infection elicits early bmdm infiltration, prior to microglia activation. these distinct kinetics of bmdm recruitment relative to microglia activation thus appear to correlate with the apparently opposing roles of microglia as demyelinating populations. these data further suggest that early responses set the stage or imprint subsequent effector functions of bmdm and microglia. using a similar approach with nanostring analysis as in eae, the present study used gene expression profiling to characterize both bmdm and microglia myeloid functions at various times post jhmv infection. analysis of overall gene expression patterns revealed that the most extensive changes in bmdm were evident early after infection, while microglia showed a more dynamic profile throughout the course of viral encephalomyelitis. importantly, the most drastic gene upregulation in microglia was observed coincident with demyelination, at which time peak viral load and t cell effector function have substantially subsided ( ) . our data contrast with eae ( ) , where bmdm upregulated far more genes compared to microglia at disease onset, supporting opposing functions of bmdm and microglia in mediating demyelination in these two models. further, while bmdm exhibited a mixed expression profile of both pro-and anti-inflammatory markers, microglia expressed a highly pro-inflammatory profile and repressed most of the m markers across the entire time course of jhmv encephalomyelitis. analysis of protein-interacting networks within genes upregulated in microglia at the time of myelin loss revealed several key functions linked to promoting tissue damage. genes associated with complement activation were notably increased, although they were already highly expressed by microglia under homeostatic conditions. complement activation as a pathogenic component in ms has been reported following detection of deposits of the activated products of the complement component c in ms lesions ( ) . the classical complement pathway has also been shown to mediate olg death thus promoting demyelination ( ) . microglia phagocytic activity may also initiate tissue damage by directly removing myelin from axons, especially at the node of ranvier ( ) . genes associated with trem /dap signaling were also highly expressed by microglia at time of demyelination. trem modulates phagocytic capacity of myeloid cells via dap signaling ( ) and is expressed on myelin-loaded myeloid cells in ms lesions ( ) , supporting a role in ms pathogenesis. similar to jhmv infection, trem is predominantly expressed by microglia during eae and cuprizone-induced demyelination ( ) ( ) ( ) . however, trem -modulated phagocytic functions are essential for removal of myelin debris and remyelination implicating repair-promoting functions of microglia in the specific tissue environments defining these two demyelination models ( , ) . preferential trem expression within microglia compared to bmdm following jhmv infection support a more pathogenic role of trem- in jhmv-induced demyelination, potentially by promoting myelin stripping after recognition of glycolipids and phospholipids exposed on damaged myelin. in this context, it is critical to note that jhmv infection is associated with extensive transient production of ifn-γ and its inducible genes, i.e., inos, and cxcr ligands, which may drive a more phagocytic pathway in microglia in efforts to remove damaged proteins and lipids ( ) . further investigation is required to define inflammatory conditions under which trem- modulated or other phagocytic pathways promote tissue damage or repair and whether these are transient and reversible. microglia may also induce demyelination by secreting toxic factors including inflammatory cytokines that are highly expressed by microglia at time of demyelination, including tnf. tnf can induce olg death ( , ) and is expressed in active ms lesions, as well as elevated tnf in serum and cerebral spinal fluid correlates with enhanced ms pathology ( , ) . finally, microglia functions during jhmv infection were also associated with promoting adaptive immune response. an extensive network of chemokines and chemokines receptors relating to the recruitment and arrest of t and b cells within the inflamed cns were highly expressed by microglia. similarly, several genes associated with antigen processing and presentation by mhc class i and ii molecules were upregulated within microglia, suggesting that microglia promote t cell reactivation upon cns entry. however, microglia explanted during eae, tmev as well as jhmv infections failed to support myelin-specific cd t cell responses ex vivo, despite detection of internalized myelin ( ) ( ) ( ) . a potential deficit in antigen processing was supported by the ability of exogenous peptide to overcome the inability of microglia to prime myelinspecific cd t cells ( ) . nevertheless, this notion is opposed by our microglia profiling showing upregulation of genes involved in protein degradation and class ii peptide loading, e.g. cd (invariant chain), h -dma (peptide loading), ctss (cathepsin s), which cleaves invariant chain. the apparent inability of microglia to elicit cd t cell effector function ex vivo thus remains intriguing. our present study reveals new insights into the plasticity of microglia in adapting to inflammation and expressing pathogenic functions associated with demyelination, characteristics which have previously been ascribed to bmdm ( ) . moreover, altered bmdm expression profiles coincided with their early infiltration into the cns and remained largely similar throughout infection. while altered microglia gene expression coincided with the time of early, yet robust demyelination, it remains to be determined whether these changes are sustained at later time points during jhmv persistence, when clinical disease improves and remyelination occurs. it will be of specific interest to assess whether the microglia pro-inflammatory phenotype evolves to an anti-inflammatory, repair-promoting phenotype, as evidenced by the plasticity of myeloid cells in cns autoimmunity ( ) . furthermore, our study emphasizes that the distinct tissue environments during eae and jhmv infection drive opposite effector functions of microglia versus infiltrating macrophages. the interplay between t cells, infiltrating macrophages and microglia, as well as astrocytes drives ms pathogenesis, yet mechanisms ultimately leading to loss of repair remain unclear. taking advantage of demyelinating models characterized by distinct inflammatory factors such as both th and th cells in eae ( ) , strong th polarized responses during jhmv infection, distinct kinetics of bmdm recruitment versus glia activation promises to reveal essential new insights into the interplay of microglia and bmdm functions in debris clearance versus active myelin stripping and ongoing axonal damage. longitudinal studies will aid in developing efficient future therapies to combat ms pathogenesis. all procedures were performed in compliance with protocols approved by the cleveland clinic institutional animal care and use committee. author contributions cs designed and performed experiments, collected and interpreted the data, and wrote the manuscript. rd analyzed data and edited manuscript. cb designed the research, provided materials, interpreted the data, and wrote the manuscript. all authors approved the final manuscript. the authors would like to thank natasha towne for her exceptional technical assistance, as well as jennifer powers for performing facs purification. this work was supported by the national institutes of health grant ns and national ms society research grant nmss rg . rd is supported by the national institutes of health grant ns . the supplementary material for this article can be found online at https://www.frontiersin.org/articles/ . /fimmu. . / full#supplementary-material. figure s | validation of nanostring ncounter gene expression analysis by real-time pcr. to validate nanostring ncounter data, expression of il- , il rn, and arg was analyzed by q-pcr in naïve circulating monocytes and microglia, as well as in bone 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plasticity in the evolution of central nervous system autoimmunity the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -vovas d authors: yokobayashi, yohei title: aptamer-based and aptazyme-based riboswitches in mammalian cells date: - - journal: curr opin chem biol doi: . /j.cbpa. . . sha: doc_id: cord_uid: vovas d molecular recognition by rna aptamers has been exploited to control gene expression in response to small molecules in mammalian cells. these mammalian synthetic riboswitches offer attractive features such as small genetic size and lower risk of immunological complications compared to protein-based transcriptional gene switches. the diversity of gene regulatory mechanisms that involve rna has also inspired the development of mammalian riboswitches that harness various regulatory mechanisms. in this report, recent advances in synthetic riboswitches that function in mammalian cells are reviewed focusing on the regulatory mechanisms they exploit such as mrna degradation, microrna processing, and programmed ribosomal frameshifting. molecular recognition by rna aptamers has been exploited to control gene expression in response to small molecules in mammalian cells. these mammalian synthetic riboswitches offer attractive features such as small genetic size and lower risk of immunological complications compared to proteinbased transcriptional gene switches. the diversity of gene regulatory mechanisms that involve rna has also inspired the development of mammalian riboswitches that harness various regulatory mechanisms. in this report, recent advances in synthetic riboswitches that function in mammalian cells are reviewed focusing on the regulatory mechanisms they exploit such as mrna degradation, microrna processing, and programmed ribosomal frameshifting. envisioned practical applications of mammalian synthetic biology frequently require gene switches that recognize endogenous or exogenous chemical signals and turn on or turn off expression of proteins, which in turn regulate synthetic genetic circuits inside the cell. these chemical gene switches need to be flexible enough to be tailored to diverse chemical species, engineered to function as on-switch or off-switch, fine-tuned to adjust the sensitivity, and have a minimal genetic and metabolic footprint. protein-based engineered transcription factors (tfs), such as tet-on and tet-off systems derived from a bacterial tf, are among the most widely used tools to control mammalian gene expression in response to small molecule triggers [ ] . however, there are a number of drawbacks of tf-based gene switches as generally applicable switches for mammalian and biomedical applications [ ] [ ] [ ] : (i) adapting an engineered tf to respond to a new compound is challenging, (ii) expression of engineered tfs can trigger immunogenicity, (iii) genetic size and expression of an engineered tf can burden the host cell or the vector, and (iv) switch performance can be influenced by the expression level of the tf; therefore, optimization may be required. rna aptamers for desired ligands can be obtained relatively easily by in vitro selection (selex) [ , ] ; therefore, riboswitches can potentially be engineered to respond to a variety of compounds more readily than engineered tfs. moreover, aptamers are typically small ( to nt). even with the additional nucleotides necessary to regulate gene expression, genetic footprints of riboswitches (few hundred nt) are small enough to satisfy vectors that have limited capacity (e.g. adeno associated virus). furthermore, the lack of any translated proteins is expected to result in lower immunogenicity and metabolic burden on the host cell. in , werstuck and green [ ] demonstrated that insertion of an rna aptamer selected to bind the hoechst dye h in the untranslated region of mrna allows repression of gene expression in response to the ligand in cho cells, presumably by blocking ribosome binding or scanning. it should be noted that this work predated the discovery of natural bacterial riboswitches [ , ] , demonstrating that small molecules can directly and specifically affect gene expression in the absence of mediator proteins. importantly, rna aptamer-based regulation of gene expression allows bioengineers to harness the rich diversity of gene regulatory mechanisms that involve rna, for example, translation initiation, rna interference and micrornas, and rna splicing. for each rna mediated gene regulatory mechanism, there are multiple ways to couple its outcome with aptamer-ligand interaction, further enriching the potential diversity of synthetic riboregulators. an excellent review on rna-based gene switches in mammalian cells was recently published by ausländer and fussenegger [ ] . therefore, this article reviews more recent developments in synthetic mammalian riboswitches with a focus on the diversity of the regulatory mechanisms harnessed by the riboswitches. in , yen et al. demonstrated chemical regulation of gene expression in mammalian cells by embedding a hammerhead ribozyme in the untranslated regions (utrs) of an mrna [ ] . as the and utrs are indispensable for translation of eukaryotic mrnas, self-cleavage within the mrna was expected to suppress gene expression. addition of toyocamycin to the cell culture medium resulted in nonspecific incorporation of the antiviral nucleotide analog into the mrna and statistical inactivation of the ribozyme activity [ ] . while the ribozyme was not specifically regulated by a small molecule via an aptamer, this work paved the way for the subsequent riboswitches that employ allosterically regulated ribozymes (aptazymes) embedded in the and/or utr to chemically regulate gene expression in mammalian cells (figure a ) [ ] [ ] [ ] [ ] . this strategy continues to be popular, and the recent advances highlight new approaches to design and optimize aptazymes. earlier aptazyme-based riboswitches in mammalian cells were designed via trial-and-error, or based on mediumthroughput or high-throughput screening in bacteria or yeast systems. however, it has been reported that ribozyme activity in living cells are not highly correlated among different cell types (bacteria, yeast, or mammalian cells) which is understandable considering the differences in translational mechanism, intracellular environment (rna binding proteins, ribonucleases, etc.), and mode of gene regulation by ribozymes in different cell types [ ] . because high-throughput screening of riboswitches directly in mammalian cells is technically challenging, alternative design strategies focusing on aptazymes that function in mammalian cells are desirable. the farzan group demonstrated an intriguing strategy that combines empirical experimental data with quantitative modeling [ ] . they first synthesized a panel of aptazymes and measured their riboswitch performance in cultured mammalian cells, and attempted to correlate the experimental results with various design parameters such as calculated annealing energy and the number of hydrogen bonds in the communication module. the researchers concluded that the proximity of base pairing (or lack thereof) within the communication module to the ribozyme affects the ribozyme activity, and devised 'weighted hydrogen bond score (whbs)' as a calculable parameter that correlates with the ribozyme performance. they used whbs as a guide to design aptazyme-based riboswitches using three aptamers with good switching characteristics [ ] . it remains to be seen if the strategy can be extrapolated to different aptamers, ribozymes, and aptazyme architectures. dohno et al. addressed the aptazyme design problem with a unique approach [ ] . instead of starting from an existing aptamer, they started with a rationally designed molecule targeted to mismatched dna/rna sequences. the naphthyridine carbamate tetramer with z-stilbene mammalian riboswitches yokobayashi linker (z-ncts) designed by the group is known to bind xgg/xgg mismatches in dna and rna through watson-crick type hydrogen bonding between the naphthyridine moieties with the unpaired guanines [ ] . they used this ligand as a 'molecular glue' to induce a tertiary contact between loops i and ii of a hammerhead ribozyme that is critical for ribozyme activity (figure b) . the tertiary contact between the two loops was disrupted by introducing an agg/ugg mismatch that is recognized by z-ncts. the engineered ribozyme was inserted into the utr of an mrna encoding firefly luciferase whose expression was reduced by -fold upon addition of z-ncts ( . mm) in hela cells. while it is notable that the switching was observed with a low ligand concentration, z-ncts exhibited cellular toxicity (lc = . mm) probably due to nonspecific interactions with cellular rnas. in contrast to these rational or semi-rational design efforts, high-throughput screening of aptazymes has mostly been executed in escherichia coli or saccharomyces cerevisiae. however, screening is generally labor and cost intensive while yielding sequence information for a handful of 'hits'. screening also yields limited sequence--function information that can be exploited for further optimization or library design. to address the lack of comprehensive sequence-function relationship data for ribozymes, we developed an in vitro high-throughput ribozyme assay method using deep sequencing (figure ) [ , , ] . a library of ribozyme or aptazyme mutants are transcribed in vitro in a single tube as a mixture, and the resulting ribozymes (cleaved or uncleaved) are converted to dna templates for deep sequencing. the number of cleaved and uncleaved reads for each ribozyme variant are then counted to calculate cleavage efficiency under the reaction condition. this strategy yields a complete sequence-function relationship for all members of the ribozyme library up to variants or more [ ] , depending on library preparation and sequencing output. this method can unambiguously identify functional aptazymes if they exist, and provide a broader view of the sequence-function relationship that can be used to synthetic biology schematic illustration of in vitro aptazyme assay strategy based on deep sequencing. an aptazyme library is prepared as a dna mixture which is transcribed into rna in vitro by t rna polymerase. the aptazyme pool is then converted to dna sequencing templates by reverse transcription and pcr. deep sequencing yields read counts of the cleaved and uncleaved fragments for every mutant in the library from which cleavage efficiency (% cleaved) is calculated. plotting cleavage efficiency of the mutants in the presence and absence of the ligand reveals aptazyme candidates which are subsequently evaluated as riboswitches. build a mechanistic model and/or to further optimize the aptazyme design. we used deep sequencing to identify guanine-activated ribozymes based on an hdv-like ribozyme which were subsequently used to control gene expression in hek cells [ ] . use of a low mg + concentration during in vitro transcription may have contributed to the positive correlation of the ribozyme activity in vitro and in mammalian cells. similar methods were also used to fine tune mammalian gene expression levels [ ] and to screen for active ribozymes (but not aptazymes) directly in mammalian cells [ ] . programmed - ribosomal frameshifting (- prf) [ ] can result in translation of an alternative polypeptide from an mrna by shifting the translation reading frame by - nucleotide. a canonical - prf element consists of a -nt slippery sequence x xxy yyz (trinucleotides xxy and yyz representing the original frame and xxx yyy representing the - frameshift) followed by a stable secondary structure such as a pseudoknot or a stem-loop. the chang group first controlled - prf in mammalian cells (figure a ) by incorporating theophylline and s-adenosyl-l-homocysteine (sah) aptamers [ ] . an on/off ratio of was reported but the frameshifting efficiency (fe) remained low. the same group used another - prf stimulating pseudoknot from sars coronavirus to engineer theophylline inducible - prf switches with up to fivefold activation of gene expression [ ] . more recently, matsumoto et al. used the analogs of naphthyridine carbamate tetramers described above to control the formation of a pseudoknot, thereby chemically inducing - prf (figure a , top) [ ] . although up to -fold activation was observed in hela cells, the maximum fe was low ( . %) in the presence of the pseudoknot inducing ligand. it is possible that the toxicity of the ligand prevented observation of higher fes in hela cells, as an fe as high as % was observed in rabbit reticulocyte lysate. besides controlling translation via an aptamer embedded in the targeted mrna, another strategy aims to control the microrna (mirna) processing pathway by an rna aptamer. the mirna product subsequently targets one or more mrnas in trans. an et al. first demonstrated that an aptamer incorporated in a mirna precursor allows chemical regulation of rna interference (rnai), in this case, by modulating the dicer mediated cleavage of a short-hairpin rna (shrna) [ ] . the smolke group has developed an alternative strategy that controls the drosha mediated processing of primary mirna substrates by embedding an rna aptamer in the vicinity of the drosha cleavage site [ ] . binding of the ligand to the aptamer inhibits drosha cleavage and upregulates the expression of the gene targeted by the mirna. recently, the group adapted an aptamer that they selected for ( r)-folinic acid (fa) to control a synthetic mirna precursor targeting il- receptors (figure b ) [ ] . although the modulation of the targeted gene expression by the ligand was rather modest (up to % activation), it was sufficient to observe a robust regulation of cell proliferation. the suess group reported a new strategy to control the dicer processing by inserting an aptamer that binds to tetr protein [ ] near the cleavage site blocking the reaction in the presence of tetr [ ] . they showed that addition of the tetr ligand doxycycline dissociates tetr from the mirna precursor and increases the formation of the mature mirna (figure c ). doxycycline was able to tightly control the mature mirna level ( %- % relative to unmodified mirna precursor), although the on/off ratio of the targeted reporter gene was more moderate ( ). although this system requires an exogenous protein factor, it allows the use of doxycycline which has been used extensively in mammalian cells and animals. the pei group adapted another drosha modulation strategy originally reported by our group [ , ] to control endogenous gene expression in cancer cell lines to induce apoptosis. a theophylline activated aptazyme was inserted between an inhibitory strand that blocks the single-stranded region of the pri-mirna, thereby preventing the rna from being processed by drosha (figure d ). the researchers targeted map k in hepg cells [ ] and bcl- in mcf- cells [ ] , and observed fourfold to fivefold increase in apoptosis. vogel et al. showed that exon skipping can be controlled by a tetracycline rna aptamer inserted near the splice site along with a suicide exon (figure e ) [ ] . tetracycline induces exon skipping resulting in -fold activation of gene expression. in combination with an aptazyme device, the dynamic range increased to -fold in hek cells. an mrna can be targeted by an endogenous mirna by inserting one or more mirna target sequences in the utr, a strategy sometimes used to achieve cell type-dependent gene expression. mou et al. controlled the accessibility of the mirna target sequence by an aptamer embedded near the target site in the mrna [ ] . addition of tetracycline occluded the mirna target site resulting in upregulation of gene expression (figure f ). the performance of the switch, as expected, was dependent on the mirnas and cell types used, but they observed tight regulation of gene expression with up to -fold activation in hela cells with a mir- target site. the diversity of gene regulatory mechanisms that involve rna has and will continue to inspire synthetic rnabased gene switches in mammalian cells. it can be anticipated that riboswitches based on different regulatory mechanisms have advantages for different applications. for example, mirna-based riboswitches are more convenient for controlling endogenous gene expression because they operate in trans as opposed to the utr embedded aptazymes that function in cis. however, the existing mammalian riboswitches still exhibit poor and variable dynamic range and ligand sensitivity compared to the conventional tf-based gene switches. with further optimization and increasing availability of new aptamers and ligands for cellular applications, synthetic riboswitches should emerge as useful tools in synthetic biology of mammalian cells. controlling mammalian gene expression with small molecules generation of cells expressing improved doxycycline-regulated reverse transcriptional transactivator rtta s-m selecting the optimal tet-on system for doxycycline-inducible gene expression in transiently transfected and stably transduced mammalian cells tet-on systems for doxycycline-inducible gene expression systematic evolution of ligands by exponential enrichment: rna ligands to bacteriophage t dna polymerase in vitro selection of rna molecules that bind specific ligands controlling gene expression in living cells through small molecule-rna interactions sensing small molecules by nascent rna: a mechanism to control transcription in bacteria thiamine derivatives bind messenger rnas directly to regulate bacterial gene expression synthetic rna-based switches for mammalian gene expression control exogenous control of mammalian gene expression through modulation of rna self-cleavage identification of inhibitors of ribozyme self-cleavage in mammalian cells via high-throughput screening of chemical libraries controlling mammalian gene expression by allosteric hepatitis delta virus ribozymes genetic control of mammalian t-cell proliferation with synthetic rna regulatory systems conditional control of mammalian gene expression by tetracycline-dependent hammerhead ribozymes a ligand-dependent hammerhead ribozyme switch for controlling mammalian gene expression highly motif-and organism-dependent effects of naturally occurring hammerhead ribozyme sequences on gene expression this work surveyed a panel of ribozymes in various cellular and genetic contexts and showed that ribozyme activity is highly context dependent. the results imply that care must be taken when translating screening results obtained in nonmammalian systems to mammalian cells rational design of aptazyme riboswitches for efficient control of gene expression in mammalian cells restoration of ribozyme tertiary contact and function by using a molecular glue for rna the authors used a 'molecular glue' to restore a tertiary contact in a hammerhead ribozyme, presenting an alternative to canonical aptamers for controlling gene expression in mammalian cells naphthyridine tetramer with a pre-organized structure for : binding to a cgg/cgg sequence high-throughput assay and engineering of self-cleaving ribozymes by sequencing deep sequencing was used to exhaustively assay aptazyme mutants without screening or selection in vitro. the aptazymes were then shown to function as riboswitches in mammalian cells high-throughput mutational analysis of a twister ribozyme applications of high-throughput sequencing to analyze and engineer ribozymes deep sequencing analysis of aptazyme variants based on a pistol ribozyme analyzing and tuning ribozyme activity by deep sequencing to modulate gene expression level in mammalian cells direct screening for ribozyme activity in mammalian cells changed in translation: mrna recoding by - programmed ribosomal frameshifting synergetic regulation of translational reading-frame switch by ligand-responsive rnas in mammalian cells rational design of a synthetic mammalian riboswitch as a ligand-responsive - ribosomal frame-shifting stimulator small synthetic molecule-stabilized rna pseudoknot as an activator for - ribosomal frameshifting artificial control of gene expression in mammalian cells by modulating rna interference through aptamer-small molecule interaction design of small molecule-responsive micrornas based on structural requirements for drosha processing regulation of t cell proliferation with drug-responsive microrna switches an rna aptamer that induces transcription although this system includes a protein component (tetr), tight control of natural microrna maturation was demonstrated using a widely used small molecule (doxycycline) in mammalian cells modulating endogenous gene expression of mammalian cells via rna-small molecule interaction conditional rna interference mediated by allosteric ribozyme regulation of map k gene expression by rna interference through an engineered theophyllinedependent hepatitis delta virus ribozyme switch inducible bcl- gene rna interference mediated by aptamer-integrated hdv ribozyme switch a small, portable rna device for the control of exon skipping in mammalian cells conditional regulation of gene expression by ligand-induced occlusion of a microrna target sequence a new mode of engineered rna-based gene regulation in mammalian cells was demonstrated by controlling the accessibility of a mirna target site by aptamer-ligand interaction nothing declared. work in the author's laboratory mentioned in the article was financially supported by okinawa institute of science and technology graduate university. papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest key: cord- -ah jclgw authors: feng, qilin; cai, hao; chen, zhilong; yang, yibin; lu, jingyu; li, fei; xu, jiheng; li, xianting title: experimental study on a comprehensive particle swarm optimization method for locating contaminant sources in dynamic indoor environments with mechanical ventilation date: - - journal: energy build doi: . /j.enbuild. . . sha: doc_id: cord_uid: ah jclgw source localization is critical to ensuring indoor air quality and environmental safety. although considerable research has been conducted on source localization in steady-state indoor environments, very few studies have dealt with the more challenging source localization problems in dynamic indoor environments. this paper presents a comprehensive particle swarm optimization (cpso) method to locate a contaminant source in dynamic indoor environments with mechanical ventilation and develops a multi-robot source localization system to experimentally validate the method. three robots were used to test the presented method in a typical dynamic indoor environment with periodic swinging of the air supply louvers of a cabinet air conditioner. the presented method was validated with two typical source locations, ds (in the downwind zone) and rs (in the recirculation zone). for ds and rs, and experiments out of experiments were successful, with success rates of % and . %, and each robot moved an average of . and . steps, respectively. the presented method was also compared with the standard particle swarm optimization (spso) and wind utilization ii (wuii) methods for locating the source at ds. for the spso and wuii methods, only and experiments out of experiments were successful, with success rates of % and % and averages of . and . steps, respectively. the experimental results show that the presented method not only has a much higher success rate than the spso and wuii methods but also has higher source localization efficiency. in modern society, people spend as much as % of their time in indoor environments [ ] . with the improvement of living standards, people are increasingly aware of the importance and urgency of indoor air pollution control. improving indoor air quality has become a trending topic worldwide [ , ] . in recent years, the continuous growth of the electronic information, aerospace, precision instrument, medical and health industries has greatly promoted the development of the clean room industry. in industrial and biological clean rooms, it is often necessary to consume a large amount of energy to strictly control airborne particles and gaseous contaminants to meet specific process requirements [ ] . to achieve an ideal steady state. the main reasons are as follows: first, the air supply velocity of mechanical ventilation systems always fluctuates slightly; second, the air supply direction sometimes periodically swings up and down or left and right according to the needs of indoor personnel; and finally, various interference factors, such as the switching of ventilation modes, movement of personnel or equipment, and changes in heat source intensity, can disturb the indoor airflow field. compared with steady-state indoor environments, dynamic indoor environments not only have more complex air distribution characteristics but also have more complex spatial and temporal contaminant distribution characteristics and therefore pose new challenges in source localization [ ] . the available methods for source localization can be roughly categorized into two types: stationary sensor network methods and robot active olfaction methods. stationary sensor network methods require the installation of one or more sensors in the indoor space in advance, after which the source location must be identified from sensor readings by using forward or backward models. most of the available studies on stationary sensor network methods have focused on steady-state indoor environments [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (a detailed review is provided in [ , ] ), and only very few attempts have been made to locate sources in dynamic indoor environments. wang et al. [ ] proposed an adjoint probability-based method for identifying the contaminant source location in dynamic indoor environments. the method was validated by computational fluid dynamics (cfd) simulations and assumed that the simulated airflow data were consistent with those obtained in real-world conditions. however, most real-time dynamic airflows are difficult to accurately simulate by cfd models due to the uncertainty and complexity of dynamic airflows, which poses a great challenge to stationary sensor network methods. robot active olfaction methods have been inspired by many animal behaviors, such as foraging, finding mates and evading predators [ , ] . this type of method normally uses mobile robots equipped with gas or airflow sensors to initiatively track and locate release sources and decomposes the task of locating a source into three phases [ ] : plume finding (contacting the gas), plume traversal (approaching the source), and source declaration (confirming the source). compared to stationary sensor network methods, active olfaction methods have better adaptability in unknown environments because they can be executed by directly using sensor readings independent of numerical simulations; furthermore, active olfaction methods do not require environmental model construction and environmental sensor installation in advance. according to the number of robots used, this type of method can be divided into two categories: single-robot and multi-robot active olfaction methods. in recent years, multi-robot active olfaction methods have attracted increasing attention due to their high efficiency, good scalability, and strong robustness [ ] . to date, most research on robot active olfaction methods [ , [ ] [ ] [ ] [ ] [ ] has been conducted in steady-state airflow environments, and only a few attempts have been made in recent years to locate sources in dynamic indoor environments. compared to steady-state indoor environments, dynamic indoor environments pose two major challenges to robot active olfaction methods. first, the airflow velocity and direction at any position in the indoor space can fluctuate greatly with time, which not only makes it more difficult to accurately detect the airflow velocity and direction but also reduces the timeliness of the detected information. therefore, the airflow velocity and direction are difficult to use in active olfaction methods. second, the contaminant concentration distribution in indoor spaces can change greatly with time, which not only leads to the generation of more local extremum areas (where the concentration is higher than that in surrounding area) but also changes the locations of these local extremum areas. therefore, variations in the contaminant concentration distribution will make it more difficult for the robots to continuously track the plume and easier for the robots to become trapped in local extremum areas. considering the difficulties of locating sources in dynamic environments, most available works have used multi-robot active olfaction methods. typical methods include the adapted ant colony optimization (aco) method [ , ] , the particle swarm optimization (pso) method [ ] , and modified pso methods [ ] [ ] [ ] [ ] [ ] . although there have been some preliminary explorations of the source localization method for dynamic indoor environments, most of these methods were validated by numerical simulations, and only very few experimental studies have been reported [ , ] . simulation studies based on time-averaged equations cannot easily reflect the turbulence characteristics of indoor airflows, intermittent contaminant concentrations with large fluctuations, or disturbances to the airflow and concentration fields caused by robot movement. therefore, from the perspective of practical applications, experimental research in real environments is more valuable and meaningful than numerical simulation research. among the available multi-robot active olfaction methods, the most commonly used algorithm is the pso algorithm because this method is characterized by fast convergence, high cooperation efficiency, few parameters to be adjusted and ease of use [ ] . however, this algorithm can easily cause the robots to become trapped in local extremum areas. to overcome this drawback, we previously developed an improved particle swarm optimization (ipso) algorithm by combining concentrations and airflows to enable robots to continuously track the plume [ ] . however, this algorithm was only validated in steady-state indoor environments that were simulated by cfd. in this study, considering the complexity of airflow and concentration distributions in dynamic indoor environments, we present a comprehensive particle swarm optimization (cpso) method that integrates the ipso algorithm with a source confirming algorithm and a strategy for escaping from local extremum areas to improve the success rate and efficiency of source localization. we further experimentally validated the cpso method and compared it with a standard pso method (spso) and a modified pso method with wind utilization ii (wuii) [ ] . the main contributions of this paper are as follows: ( ) a cpso method was developed for source localization in dynamic indoor environments with mechanical ventilation. this method integrates our previously presented ipso algorithm with a source confirming algorithm and a strategy for escaping from local extremum areas to improve the success rate and efficiency of source localization. ( ) a multi-robot source localization system was developed, and three robots were used to validate the presented method at two typical source locations (ds, in the downwind zone, and rs, in the recirculation zone) in a dynamic indoor environment with the air supply louvers of a cabinet air conditioner periodically swinging from left to right. the presented method was tested by performing independent experiments at each source location. ( ) in the same experimental environment, the cpso method was compared with the spso and wuii methods with respect to success rate and efficiency; the cpso method not only has a much higher success rate than the spso and wuii methods but also has a higher efficiency. according to the cpso method presented in this paper, the robots have three basic motion modes in the source localization process: ( ) plume finding mode. after departing from the starting positions, the robots use a plume finding algorithm to continuously search for unknown areas until one of the robots detects a specific contaminant in the air (this process is called plume finding). ( ) plume tracking mode. with a plume tracking algorithm, the robots continuously track the plume of the contaminant and move toward higher concentrations until a local extremum area is found. "local extremum area" refers to an area in which the concentration is higher than that in the surrounding area. ( ) source confirming mode. the robots use a source confirming algorithm to determine whether the source has been found. according to the three motion modes, the presented method integrates three core algorithms: a plume finding algorithm, a plume tracking algorithm, and a source confirming algorithm. in addition, the method integrates an obstacle avoidance algorithm to prevent collisions among robots and avoid obstacles. fig. shows the basic procedure of source localization by using the presented cpso method. first, after departing from the starting positions, the robots start in the plume finding mode. when one of the robots finds the plume, all the robots switch to the plume tracking mode. in the plume tracking process, if the robots find a local extremum area, they switch to the source confirming mode. if the robots determine that the source has been found, they terminate the source localization process and report the source location; otherwise, they restart the plume finding mode to escape from this local extremum area. after escaping from the local extremum area, the robots switch back to the plume tracking mode to find the source again. the presented method uses a divergence search strategy in the plume finding stage [ ] ( fig. ) . after the contaminant is released, all the robots depart from the starting positions and move toward different directions at the same speed to quickly find the plume and search the largest possible area. the moving trajectory of each robot is a straight line, and the angle between the lines is the same ( fig. ) . during the plume finding process, if a robot hits the building boundaries, it will change its direction of movement according to the law of reflection. if a robot detects a contaminant concentration that exceeds the preset threshold c min , all the robots will switch to the plume tracking mode. in practical applications, c min can usually be set to the detection threshold of the specific sensor carried by the robot [ ] . in our previous papers [ , ] , we introduced the basic principles of the standard pso algorithm and the ipso algorithm in detail, as shown in fig. . in the following, we only briefly describe the principle of the ipso algorithm. in this paper, the contaminant concentration detected by the i -th robot r i ( i = , , ..., n) at time step t ( t = , , …) is represented by c i ( t ), which is used to evaluate the fitness value of r i . according to the ipso algorithm, the location and velocity vector of r i from time step t to t + are updated as: where p i ( t ) (m) and v i ( t ) (m) are the location and velocity vector of r i at time step t , respectively; w is a dimensionless inertia weight; and l , l and l are dimensionless learning factors (for a detailed explanation, refer to [ ] ). according to previous research [ ] , w, l and l are normally set to . , . and . , respectively. in this study, l is set to . , because a greater value of l allows the robot to follow the airflow more quickly; r , r schematic of the plume tracking principle using the (a) standard particle swarm optimization (spso) algorithm [ ] and (b) improved particle swarm optimization (ipso) algorithm [ ] . r i at time step t (m), expressed as: where v max is the maximum step length (m), namely the maximum moving distance of a robot at a time step; v s i is the airflow velocity of r i obtained at time step t (m/s); v s min is the threshold of the airflow sensor (m/s); and v r is a dimensionless random direction vector uniformly distributed within a range of [ − , ] that is added to the upwind velocity of r i when the air velocity is lower than the sensor threshold. if the variation in the global maximum location p * g (t) within a time step t is not greater than the maximum step length of the believe that they have found a local extremum area and cannot easily find a higher concentration by using the ipso algorithm. at this time, the robots switch to the source confirming mode and use the maximum concentration method to determine whether the global maximum location is in the vicinity of the source or in a local extremum area away from the source. the maximum concentration method is used to confirm the source and terminate the source localization process. after the robots find a local extremum area, if the concentration detected at the global maximum location is not less than the preset concentration threshold c max for source confirming, they will determine that the global maximum location is in the vicinity of the source and terminate the source localization process. otherwise, the robots will determine that the global maximum location is in a local extremum area away from the source and will diverge to find higher concentration values and a better global maximum location. if the robots confirm that they have been trapped in a local extremum area away from the source, they will use the divergence search algorithm ( fig. ) to escape from the local extremum area and find a higher concentration. in the divergence search process, if the robots find a concentration higher than c * ( t ) within the time , they conclude that they have escaped from the local extremum area and then adopt the ipso algorithm ( section . ) to track the plume. if the robots have not yet detected a concentration higher than c * ( t ) after the time step t , they conclude that they have escaped from the local extremum area (because after the divergence search in the time step t , the robots are far from the local extremum area and will not easily be trapped in this area again) and cannot find a concentration higher than c * ( t ); then, the robots turn to find a concentration higher than c min to rediscover the plume, namely, in real-world applications, robots can rely on video vision, infrared rays, laser radar and other techniques to avoid obstacles, but this process sometimes consumes too much time. to shorten the time for avoiding obstacles and speed movement away from obstacles, we developed a simple obstacle avoidance algorithm for each robot. the algorithm uses obstacle locations and building boundaries to avoid obstacles according to the reflection law, as described in [ ] . in addition, to reduce collisions among robots during their moving processes, we also specify in the obstacle avoidance algorithm that if the next position calculated by a robot is at the current position or next position of other robots, this robot will move one step in the opposite direction with a step length r × v max ( r is a random number evenly distributed between [ , ]). the experiments were conducted in a laboratory with a length of . m and a width of . m ( fig. ) . the laboratory contained a test area ( . m × . m) where the robots were able to move and an operation area ( . m × . m) where personnel stayed during experiments. an armchair, a table, a cabinet air conditioner and a source release device were placed in the operation area. during each experiment, the air supply louvers of the air conditioner periodically swung from left to right (the swing range was about - °) to create a typical time-varying mechanical ventilation environment. in addition, the doors and windows with rubber seals of the laboratory were closed during each experiment to prevent the influence of the outdoor environment. ethanol vapor was used as a tracer gas because it is minimally toxic, volatile and easy to detect and has been widely used in source localization research [ ] . during each experiment, a water bath ( °c) was used to heat ethanol liquid in a flask to control its evaporation rate at approximately . mg/s, and an air pump was used to transport the ethanol vapor through a rubber pipe to the release location ( fig. (b) ). to validate the effectiveness of the presented method, two typical source locations were set: ds and rs (see fig. (c) and table ). the height of the source was set to . m, which is roughly the height of the nostrils when an average adult is sitting [ ] . ds was a typical source location in the downwind zone of the laboratory because it was close to the air supply outlet of the air conditioner and was strongly affected by the air supply. in contrast, rs was a typical source location in the recirculation zone of the laboratory because it was far from the air supply outlet and was less affected by the air supply. to test the dynamic airflow characteristics in the laboratory, two d ultrasonic anemometers (sampling frequency of hz) were used to measure the airflow directions and velocities at the two typical source locations (ds and rs) ( fig. (c) ). the ultrasonic anemometer can accurately measure the air velocity as low as . m/s [ ] . three turtlebot mobile robots developed by willow garage were used; the robots have the characteristics of flexible movement (two side-by-side driving wheels and two fore and aft driven wheels for moving and rotating), high positioning accuracy (line error: ± cm, angle error: ± °), and autonomous navigation (autonomous obstacle avoidance and path planning). each robot car- ried an embedded single-board computer (nvidia jetson tk ), a laser ranging radar system (rplidar a ), an ultrasonic anemometer (windsonic, gill) and a gas sensor (mics ) ( fig. ) . the singleboard computer controlled the robots through the robotics middleware robot operating system (ros). the laser ranging radar system combined with the robot's own odometer for navigation and to determine the robot's location. ultrasonic anemometers were used to collect air velocity and direction information, and gas sensors were used to measure the ethanol concentration. the detailed parameters are shown in table . before each experiment began, the windows and doors were opened to remove the remaining ethanol vapor until the indoor ethanol concentration was less than ppm. then, the windows and doors were closed, the air conditioner and the air pump were turned on, and the robots were in standby at their starting positions ( fig. (c) and table ). after ethanol vapor had been released for s, the robots departed to locate the source. the robots adopted the "move-stop-move" strategy; that is, after each robot moved forward one step, it stayed for a while to collect airflow and concentration information and then continued to move [ ] . a central computer was used to communicate with the robots and coordinate their movements. after each robot collected the concentration, air velocity and direction information, the information and the current position of each robot were sent to the central computer. the computer first calculated the time-averaged values of the concentration, air velocity and direction and then calculated the target position of each robot in the next step according to the source localization algorithm. after each robot received the target position from the computer, it moved to that position and collected information again. the use of the central computer facilitated recording the robot's positions, processing the collected information, and analyzing the source localization process in a real-time manner. each robot moved forward one step for approximately s and then stopped and collected information at a sampling frequency of hz for s. in addition, each robot moved with a maximum linear speed of . m/s and a maximum step length of . m to reduce the influence of movement of the robots on the airflow and contaminant concentration. in each experiment, the concentration threshold for each robot to find the plume was set to ppm. during the plume tracking, if the global maximum location of the three robots changed by less than or equal to . m (equal to one step) in consecutive steps, the robots concluded that they had entered a local extremum area and then used the concentration maximization method to confirm whether they had reached the source. the concentration threshold for source confirmation was set to ppm. in addition, to avoid spending too much time on a single trial, if the robots had moved steps (approximately min), the robots terminated the source localization algorithm. after the source localization algorithm was terminated, if the distance between the source location determined by the robot (global maximum location) and the actual source location did not exceed . m, it was considered that the robots had successfully found the source; otherwise, the source localization trial was considered to have failed. the reason for this designation is first that this localization error ( . m) can generally meet the requirements of practical applications [ ] ; second, when the source is close enough, the robots can use a camera or other device to determine the source location [ , ] . fig. shows the airflow information measured for s at the two source locations, ds and rs. as shown in the figure, the airflow direction fluctuated greatly at both ds and rs; the fluctuation at ds exhibited obvious periodicity, while the fluctuation at rs was chaotic. moreover, the airflow velocity also fluctuated at both ds and rs. the magnitude of the airflow velocity fluctuation at ds was significantly larger than that at rs, and the airflow velocity at ds fluctuated periodically, while the airflow velocity at ds fluctuated chaotically. in addition, the time-averaged airflow velocity at ds was significantly higher than that at rs. the above results show that the airflow directions at both ds and rs fluctuated greatly; the airflow velocities at ds fluctuated more greatly than that at rs; the fluctuation in the airflow directions and velocities at ds exhibited obvious periodicity because this location was more affected by the air supply of the cabinet air conditioner. the presented method was tested by using three robots to locate the source in two release scenarios with different source locations: ds and rs ( fig. ) . in each scenario, independent experiments were conducted. as shown in table , for source location ds, the source was successfully located in all experiments, and the success rate (the number of successful experiments divided by the number of total experiments) was %; for source location rs, experiments were successful (only experiment failed), and the success rate was . %. these results indicated that the presented method is robust in a time-varying mechanical ventilation environment. table also lists the averages and standard deviations of the number of steps required to locate the source at ds and rs. note that each robot moved by the same number of steps in each experiment. therefore, the average is the average number of steps moved by any one of the robots in all successful experiments. for ds and rs, the average steps were very similar, and the difference between the points was less than one step. however, the standard deviation in steps for ds was slightly larger than that for rs. the larger the standard deviation is, the greater the dispersion of the data. the reason for the observed difference may be that the periodic swinging of the cabinet air conditioner louver from left to right causes periodic changes in the air supply; furthermore, the effect of air supply at ds is significantly greater than that at rs. in other words, the variation in the airflow and concentration field near ds over time is much larger than that near rs. fig. shows the source localization process of the robots (r -r ) and the maximum time-averaged concentration collected by the robots at each step in a typical successful experiment (the video of the experiment is provided in the supplemental material). in this experiment, each robot moved steps. after the robots departed, they first found the plume, then tracked the plume and continuously confirmed whether they had found the source during the tracking process. from finding the plume to finally locating the source, the main stages experienced by the robots are becoming trapped in a local extremum area, escaping from the local extremum area, rediscovering the plume, and then tracking the plume. after departing from the starting positions, the robots (r -r ) used a divergence search strategy to move straight in different directions to quickly find the plume. at the th step, r found the plume ( fig. (a) ). after the th step, the robots switched to the plume tracking mode. at the th step, r detected a higher time-averaged concentration ( fig. (b) and (c)), which was less than the threshold c max set in section . ; thereby, the robots determined that a local extremum area had been found according to the maximum concentration method. subsequently, the robots used the divergence search strategy to quickly escape this area and try to find a higher concentration. at the th step, r detected a higher concentration, and then r -r restarted plume tracking. further analysis of fig. reveals that although robot r was closer to the source from the th step to the th step, it failed to detect a higher concentration, which caused it to move away from the source in the next step. the above results indicate that due to the periodic variation in the air supply direction of the cabinet air conditioner, the airflow direction and contaminant concentration near the source fluctuated greatly with time, which misled the robots, causing them to move away from the source, and made it more difficult for the robots to continuously track the plume. after the th step, the robots escaped from the local extremum area and tracked the plume again. at the nd step, r detected a higher time-averaged concentration and reached the vicinity of the source ( fig. (b) and (c) ). subsequently, r started searching near the source, trying to find a higher concentration; at the same time, r and r continued to approach r . all the robots failed to find a higher concentration until the th step. because the maximum time-averaged concentration detected by the robots at the nd step was greater than c max , the robots determined that the source had been successfully located according to the maximum concentration method and ended the source localization process. the distance between the source location determined by the robots and the actual source location was . m (less than . m); therefore, we consider that this experiment was successful. further analysis of fig. also reveals that after the th step, r could continuously track the plume and quickly move to the source, indicating that the presented method has a good ability to continuously track plumes in a time-varying indoor airflow field and thus can find the source with fewer moving steps (or shorter time). fig. shows the source localization process and the maximum time-averaged concentration collected by the robots at each step in a typical successful experiment (the video of the experiment is provided in the supplemental material). in this experiment, each robot moved steps, and the main stages of source localization were similar to those illustrated in fig. . the detailed analysis of the source localization process is as follows. after the robots set off, they used a divergence search strategy to move straight in different directions, and r found the plume at the th step ( fig. (a) ). trapping in and escaping from a local extremum area. after the th step, the robots switched to the plume tracking mode. at the th step, r detected a higher time-averaged concentration ( fig. (a) and (c) ), and r -r considered that they had found an optimum concentration. subsequently, r -r used the maximum concentration method to confirm whether the location was close to the source. after the robots determined that they were trapped in a local extremum area rather than near the source, they used the divergence search strategy to quickly escape this area and try to find a higher concentration. at the th step, r detected a higher concentration, and then r -r restarted the plume tracking mode. after escaping from the local extremum area and restarting the plume tracking mode, r detected a higher time-averaged concentration and reached the vicinity of the source at the th step ( fig. (b) and (c) ). subsequently, r began to search near this location in an attempt to find a higher concentration, while r and r continued to approach r . at the th step, r detected a higher concentration and returned to the vicinity of the source. subsequently, the robots determined that the source had been successfully located according to the maximum concentration method and ended the source localization process. the distance between the source location determined by the robots and the actual source location was . m (less than . m), indicating that this experiment was successful. the results of the only failed experiment for locating rs are shown in fig. (the video of the experiment is provided in the supplemental material). each robot moved steps in this experiment, and the main stages of source localization were similar to those illustrated in fig. . in the following, we will briefly describe the source localization process and highlight the differences between figs. and . after departure, the robots used the divergence search algorithm to find the plume and switched to the plume tracking mode due to the discovery of the plume by r at the th step. at the th step, r detected a higher time-averaged concentration and further determined that the robots were trapped in a local extremum area, according to the concentration maximization method. subsequently, the robots escaped from this area using the divergence search algorithm. at the th step, r detected a higher timeaveraged concentration and then tracked the plume again. at the st step, the robots found an optimum location again, determined that the source had been found according to the concentration maximization method and ended the source localization process. the distance between the source location determined by the robots and the actual source location was . m (slightly greater than . m), indicating that this experiment failed. further analysis of fig. also reveals that the distance between r and the source was less than . m at the th step and that the distance between r and the source at the th step was also less than . m, indicating that the two robots met the standard for source finding in sequence. however, at these two time steps, neither of the robots detected a higher concentration, so they subsequently moved away from the source. in the end, the robots mistakenly believed that r had found the source at the th step and ended the source localization process according to the maximum concentration method. the above results indicate that due to the periodic variation in the air supply direction of the cabinet air conditioner, the airflow direction and the contaminant concentration near the source fluctuated greatly with time, which not only made it more difficult for the robots to continuously track the plume but also may have caused the source localization process to fail according to the maximum concentration method. the presented cpso method was experimentally compared with two other pso-based active olfaction methods: spso and wuii [ ] . considering the limitations of the experimental workload, we only chose source location ds to compare and analyze the performances of the three methods. one of the reasons is that compared with the airflow field at other locations, the airflow field at this location was more affected by the periodic variation in the air supply direction ( fig. ) and the time-dependent characteristics of the airflow field at this location were more significant. another reason is that when the source is in the downwind zone, the spread of contaminants or hazardous substances is usually faster; therefore, ds is also a more noteworthy location. to ensure that the benchmarks for comparison were consistent, the three experimental methods used the same plume finding strategy, obstacle avoidance algorithm and other settings, such as the number of robots used and the maximum step length of each robot. table summarizes the experimental results of the three methods. as shown in table , the cpso method achieved the highest success rate, reaching %, which was significantly higher than those of the spso and wuii methods ( % and %, respectively). the success rates of the spso and wuii methods were less than %, indicating that neither method can meet the requirements of practical applications in time-varying airflow fields. from the perspective of source localization efficiency, the average number of steps of the cpso method was the lowest and was significantly lower than those of the spso and wuii methods. the average number of steps of the cpso method ( . ) was . % and . % lower than that of the spso method ( . ) and wuii method ( . ), respectively. these results reveal that the presented method not only has a much higher success rate but also has a higher source localization efficiency than the spso and wuii methods in time-varying indoor airflow environments with mechanical ventilation. the experimental results in table also show that the success rate of the wuii method was significantly higher than that of the spso method; however, the average number of steps of the wuii method was higher than that of the spso method, indicating that the introduction of the upwind effect in the wuii method helps improve the success rate of source localization but may not improve the efficiency of source localization in time-varying indoor airflow environments with mechanical ventilation. to quickly locate a contaminant source in a time-varying indoor environment with mechanical ventilation, we presented and experimentally validated a cpso method that was developed based on the ipso algorithm presented in our previous study [ ] . this study extended the applications of the multi-robot active olfaction method from steady-state indoor environments to time-varying indoor environments with mechanical ventilation. the presented ipso method used a maximum concentration method for source confirming. the accuracy of this method is closely related to the choice of concentration threshold. in this study, this threshold was the average of concentrations measured before the experiments at points equally spaced on a circle centered on the source and having a radius of . m. in experiments at two different source locations (ds and rs), only one experiment failed ( fig. ) , and the success rate was close to %, indicating that the maximum concentration method was effective and the concentration thresholds had been appropriately selected. in practical applications, we recommend that before the robots are put into use, users should first analyze the release characteristics of the source according to the specific application scenario and then determine an appropriate concentration threshold by robot experiments or simulations. in future research, we will incorporate other auxiliary information (such as airflow [ ] and vision information [ ] ) into the concentration maximum method to further improve the success rate of source confirming. the presented method considers the free movement of sensors in the three directions of length, width and height, and all experiments were conducted in a three-dimensional indoor environment. however, to reduce the complexity of the robot mechanism, the sensors carried by the robots only moved in a two-dimensional plane in the experiments. in future research, we will modify the robots to realize the movement of sensors in the height direction and further analyze the effects of the sensors' movement in the height direction and the height of the contaminant source on the success rate and efficiency of source localization. the performance of the presented method can be affected by the environmental factors (e.g., the characteristic of the dynamic airflow, ventilation mode, source location and source release rate profile), the parameters of the method (e.g., learning factors, concentration threshold for finding plume and confirming source), the robots (e.g., the number, step length and starting position) and the performance of sensors (e.g., sensor threshold and measurement error). in practice, we recommend that the users should fine tune the source localization system through real robot experiments or numerical simulations to ensure that the system can achieve the expected success rate in the specific environment. in future study, we will combine real robot experiments with numerical simulations to study the influencing factors and influence laws of the presented method, and further study how to optimize the method. this study presented a comprehensive particle swarm optimization method (cpso) for locating a contaminant source in timevarying indoor airflow environments with mechanical ventilation. this method uses an ipso algorithm that combines concentration and airflow information to enable the robots to continuously track the plume. in addition, this method integrates a source confirming algorithm and divergence search strategy to guide the robots to escape from local extremum areas and finally find the source. the effectiveness of the method was validated by three robots in a typical time-varying airflow environment with the air supply louvers of a cabinet air conditioner periodically swinging from left to right. moreover, the performance of the method was compared with the standard particle swarm optimization (spso) and wind utilization ii (wuii) methods in the experimental environment. the experimental results led to the following conclusions: ( ) for the two typical source locations, ds (in the downwind zone) and rs (in the recirculation zone), and experiments were successful, respectively, out of experiments using the presented method (the corresponding success rates were % and . %), indicating that the method has a high success rate and strong robustness in a timevarying airflow environment with mechanical ventilation. for ds and rs, the average numbers of steps moved by each robot were very close, . steps and . steps, respectively, and the standard deviations were . steps and . steps, respectively. the standard deviation in steps for ds was slightly higher than that for rs, indicating that the airflow and concentration fields near ds were more affected by the periodic swinging of the cabinet air conditioner louver than those near rs, which caused the larger difference in steps obtained when locating the source at ds. ( ) analysis of the source localization processes in the experiments shows that the presented method confers the robots a good ability to continuously track the plume and the ability to successfully escape from a local extremum area and continue to approach the source location when they are trapped in such an area. in the experiment, due to the periodic change in the air supply direction of the cabinet air conditioner, the airflow direction and contaminant concentration near the source fluctuated greatly with time, which not only increased the difficulty of continuously tracking the plume but also may lead to misjudgment of the source confirming algorithm based on the maximum concentration method. ( ) for the source location at ds, experiments were conducted by using the cpso, spso and wuii methods, with , and successful experiments; success rates of %, % and %; averages of . , . , and . steps for source localization; and standard deviations of . , . and . steps, respectively. the experimental results show that the presented method not only has a much higher success rate than the spso and wuii methods but also has higher source localization efficiency. we declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. automated mobile sensing: towards highgranularity agile indoor environmental quality monitoring smart ventilation energy and indoor air quality performance in residential buildings: a review profile of occupant activity impact on indoor air -method of its determination validation and application of the personnel factor for the garment used in cleanrooms distribution characteristics, 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source a method for single odor source declaration in three-dimensional airflow environments isocs/ieee international symposium on olfaction and electronic nose this study was supported by the national natural science supplementary material associated with this article can be found, in the online version, at doi: . /j.enbuild. . . . key: cord- -rzy mejb authors: duricki, denise a.; drndarski, svetlana; bernanos, michel; wood, tobias; bosch, karen; chen, qin; shine, h. david; simmons, camilla; williams, steven c.r.; mcmahon, stephen b.; begley, david j.; cash, diana; moon, lawrence d.f. title: corticospinal neuroplasticity and sensorimotor recovery in rats treated by infusion of neurotrophin- into disabled forelimb muscles started h after stroke date: - - journal: biorxiv doi: . / sha: doc_id: cord_uid: rzy mejb stroke often leads to arm disability and reduced responsiveness to stimuli on the other side of the body. neurotrophin- (nt ) is made by skeletal muscle during infancy but levels drop postnatally and into adulthood. it is essential for the survival and wiring-up of sensory afferents from muscle. we have previously shown that gene therapy delivery of human nt into the affected triceps brachii forelimb muscle improves sensorimotor recovery after ischemic stroke in adult and elderly rats. here, to move this therapy one step nearer to the clinic, we set out to test the hypothesis that intramuscular infusion of nt protein could improve sensorimotor recovery after ischemic cortical stroke in adult rats. to simulate a clinically-feasible time-to-treat, twenty-four hours later rats were randomized to receive nt or vehicle by infusion into triceps brachii for four weeks using implanted minipumps. nt increased the accuracy of forelimb placement during walking on a horizontal ladder and increased use of the affected arm for lateral support during rearing. nt also reversed sensory deficits on the affected forearm. there was no evidence of forepaw sensitivity to cold stimuli after stroke or nt treatment. mri confirmed that treatment did not induce neuroprotection. functional mri during low threshold electrical stimulation of the affected forearm showed an increase in peri-infarct bold signal with time in both stroke groups and indicated that neurotrophin- did not further increase peri-infarct bold signal. rather, nt induced spinal neuroplasticity including sprouting of the spared corticospinal and serotonergic pathways. neurophysiology showed that nt treatment increased functional connectivity between the corticospinal tracts and spinal circuits controlling muscles on the treated side. after intravenous injection, radiolabelled nt crossed from bloodstream into the brain and spinal cord in adult mice with or without strokes. our results show that delayed, peripheral infusion of neurotrophin- can improve sensorimotor function after ischemic stroke. phase i and ii clinical trials of nt (for constipation and neuropathy) have shown that peripheral, high doses are safe and well tolerated, which paves the way for nt as a therapy for stroke. ischemic stroke occurs in the brain when blood flow is restricted, causing brain cells to die rapidly. movements on the opposite side of the body are frequently affected . stroke victims also often exhibit lack of responsiveness to stimuli on their affected side. the w.h.o. estimates that, worldwide, there are million stroke survivors, with another million new strokes annually. the vast majority of stroke victims are not eligible for the few therapies that improve outcome because they arrive in hospital too late for reperfusion to be effective . treatment six hours or more after ischemic stroke is usually limited to rehabilitation: therapies that reverse sensory impairments and locomotor disability are urgently needed, and these must work when initiated many hours after stroke. neurotrophin- is a growth factor which plays a key role in the development, and function of locomotor circuits that express nt receptors, including descending serotonergic and corticospinal tract (cst) axons and afferents from muscle and skin that mediate proprioception and tactile sensation [ ] [ ] [ ] . however, peripheral levels of nt drop in the postnatal period . we and others had shown that delivery of nt into the cns promotes recovery in rodent models of spinal cord injury [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] but this involved invasive routes of delivery (e.g., intraspinal injection or intrathecal infusion) or gene therapy. we also recently showed that injection of an adeno-associated viral vector (aav) encoding full-length human nt (prepront , kda) into forelimb muscles hours after stroke in adult or elderly rats improved sensorimotor recovery . we had originally expected that aav would be trafficked from muscle to the spinal cord retrogradely in axons and that this would enhance secretion of nt by motor neurons, leading to sprouting of the spared cst , , and sensorimotor recovery. although nt protein was overexpressed in injected muscles, to our surprise we found little evidence for expression of the human nt transgene in the spinal cord or cervical drgs , using this dose and preparation of aav. this serendipitous result led us to reject our original assumption that sensorimotor recovery required expression of the human nt transgene in the spinal cord and to wonder whether peripheral infusion of nt protein would suffice. accordingly, here, we test the hypothesis that infusion of the mature form of the nt protein ( kda) into disabled forelimb muscles improves sensorimotor recovery. this is consistent with work by others including a study showing that a signal from muscle spindles can improve neuroplasticity of descending pathways and can enhance recovery after cns injury . notably, nt protein is synthesised by muscle spindles and can be transported from muscle to sensory ganglia and spinal motor neurons in nerves , , and from the bloodstream to the cns [ ] [ ] [ ] . this route of administration and time frame is clinically feasible so to take this potential therapy one step nearer the clinic, we next set out to determine whether intramuscular infusion of human nt protein (mature form, kda) would improve outcome after stroke (i.e., bypassing the use of gene therapy and spinal surgery). importantly, the mature form of the nt protein has excellent translational potential: phase i and ii clinical trials have shown that repeated, systemic, high doses of nt protein are well-tolerated, safe and effective in more than humans with sensory and motor neuropathy (charcot-marie-tooth type a) or constipation including in people with spinal cord injury [ ] [ ] [ ] [ ] [ ] . in contrast to other neurotrophins, nt does not cause any serious adverse effects such as pain probably because its principal high affinity receptor trkc is not expressed on adult nociceptors , . these studies pave the way for nt as a therapy for stroke in humans. we now show in a blinded, randomized preclinical trial that treatment of disabled upper arm muscles with human nt protein reverses sensory and motor disability in rats when treatment is initiated in a clinically-feasible timeframe ( hours after stroke). rats received unilateral focal cortical stroke or underwent sham surgery , (fig. a,b) . twentyfour hours after stroke, rats were allocated to treatment using nt or vehicle, infused into affected triceps brachii muscles for one month via implanted catheters and subcutaneous osmotic minipumps. experiments were performed in accordance with guidelines from the stroke therapy academic industry roundtable (stair) and others and our findings were reported in accordance with the arrive (animals in research: reporting in vivo experiments) guidelines. all surgical procedures, behavioural testing and analysis were performed using a randomised block design. all surgeries, behavioural testing and analysis were performed with investigators blinded to treatment groups. rats were randomised to surgery by drawing a rat identity number from an envelope and then a stroke/sham allocation from an envelope. allocation concealment was performed by having nt and vehicle stocks coded by an independent person prior to loading pumps. behavioural testing was conducted blind and codes were only broken after behavioural analyses were complete. lister hooded (~ months; - g) outbred female rats (charles river, uk) and adult c bl/ mice ( - weeks) were used. all procedures were in accordance with the uk home office guidelines and animals (scientific procedures) act of . rats were maintained (specific pathogen free) in groups of to in plexiglas housing with tunnels and bedding on a : hour light/dark cycle with food and water ad libitum. focal ischemic stroke was induced in the hemisphere representing the dominant forelimb ( supplementary fig. ), as determined by the cylinder behavioural test. stroke lesions (n = ) were performed as previously described . briefly, animals were transferred to a stereotaxic frame (david kopf instruments, usa) where a midline incision was made, the cortex was then exposed via craniotomy using the following co-ordinates [defined as anterioposterior (ap), mediolateral (ml)]: ap mm to − mm, ml mm to mm, relative to bregma. endothelin- (et- , pmol/µl in sterile saline; calbiochem) was applied using a glass micropipette attached to a hamilton syringe. µl of et- to was applied to the overlying dura to reduce bleeding, and immediately thereafter, the dura mater was incised and reflected. four μl volumes of et- were administered topically and four µl volumes were microinjected intracortically (at a depth of mm from the brain surface) at the following co-ordinates (from bregma and midline, respectively): ap + . mm, ml . mm ap + . mm, ml . mm ap + . mm, ml . mm ap - . mm, ml . mm temperature was maintained using a rectal probe connected to a homeothermic blanket (harvard apparatus, usa) placed under the animal which maintained rectal temperature at ± °c. prior to suturing, the animal was left undisturbed for minutes. modifying previous work , the skull fragment was then replaced and sealed using bone wax (covidien, uk). % ( / ) of rats survived this stroke surgery. sham-operated (n = ) rats received all procedures up to, but not including, craniotomy or endothelin- injection. animals were given buprenorphine ( . mg/kg, subcutaneously) for postoperative pain relief. our method of inducing stroke with et- is advantageous for evaluating regenerative stroke therapies for four reasons: ) our model produces ischemic lesions that model small focal human strokes rather than larger "malignant" strokes that tend to be fatal in humans ; ) our model targets sensorimotor cortex; ) our stroke model involves only low mortality rates and has reasonable reproducibility with a proven ability to detect therapies that induce neuroplasticity and functional recovery , ; ) our model causes sustained sensorimotor deficits (e.g., impaired use of limbs) which are common neurological symptoms of human stroke. structural images were obtained prior to stroke, hours after stroke and at one and eight weeks after stroke. mr imaging was conducted on a tesla (t) horizontal bore vmris scanner (varian, palo alto, ca, usa). animals were anaesthetised using . % isoflurane, in . l/min medical air and . l/min medical oxygen in an induction chamber. once anaesthetised they were secured in a stereotaxic head frame inside the quadrature birdcage mr coil ( mm internal diameter, id) and placed into the scanner. each animal's physiology was supervised throughout the procedure using a respiration monitor (biopac, usa) and a pulse oximetry sensor (nonin, usa) that interfaced with a pc running biopac. additionally, an mri compatible homeothermic blanket (harvard apparatus, usa) placed over the animal responded to any alterations in the body temperature identified by the rectal probe, and maintained temperature at ± °c. the t weighted mr images were acquired using a fast spin-echo sequence: effective echo time (te) ms, repetition time (tr) ms, field of view (fov) mm x mm and an acquisition matrix x , acquiring x mm thick slices in approximately minutes. at the end of the study (to avoid affecting blinding or randomization), lesion volumes at the hour time point were measured using a semi-automatic contour method in jim software under blinded conditions (xinapse systems ltd). functional magnetic resonance imaging (fmri) was performed in a subset of rats that did not receive intracortical injections of bda tracer (n = /group). images were acquired prior to stroke and at one and eight weeks after stroke during non-noxious somatosensory stimulation of the affected or less-affected wrist . this involves delivery of small electrical currents to a wrist whilst the subjects were kept anaesthetised using medium dose alpha-chloralose suitable for recovery and longitudinal (repeated) imaging . alpha chloralose anesthesia was prepared by mixing equal amounts of borax decahydrate (sigma) and alpha chloralose-pestanal (analytical standard, < % beta isoform, , sigma, uk) in physiological saline each at a concentration of mg/ml in a glass beaker at °c prior to filtering using a . µm filter. rats were first anaesthetised using % isoflurane, . l/min medical air and . l/min oxygen. a tail cannulation was performed and the animal was transferred to the mri machine. a bolus of mg/kg alpha chloralose-pestanal was injected intravenously and then the isoflurane was switched off after minutes. an infusion line for continuous application of alpha chloralose was then attached to the cannula at mg/kg/h over the experimental time. medical air ( . l/min) and oxygen ( . l/min) was continuously delivered throughout the scanning period . mr images were obtained using a tesla scanner (varian, agilent). initially, a t -weighted structural scan was acquired, using a fast spin echo (fse) sequence with repetition time (tr) = ms, echo time (te) = ms and field of view (fov) of x mm, yielding slices with voxel size of . x . x mm in approximately min. fmri scans were acquired using a multi-gradient-multi-echo sequence (tr = ms, tes = , , ms, voxel size . mm x . mm x mm, resolution x x , scan time s). volumes were acquired with a pseudo-random onoff stimulation of the forepaw at hz ( μs, ma pulse) using a platinum subdermal needle electrode (f-e , grass technologies, usa) and a tens (transcutaneous electrical nerve stimulation) pad. it has previously been shown that the use of a tens pad results in this intensity of stimulation being innocuous rather than noxious: whereas blood pressure is not altered at ma stimulation, it is increased with . ma stimulation (see references in ). the order of paw stimulation was also randomised. animals were closely monitored following the end of the scanning, given ml of saline (subcutaneous, room temperature) and were kept in a warmed incubator ( deg c) until fully conscious: this takes to hours due to slow pharmacokinetics of alpha chloralose. two animals died following alpha chloralose anaesthesia due to breathing difficulties in recovery. scans with obvious imaging artefacts were discarded, leaving final group numbers of n= , , and n= , , at weeks , and for nt and vehicle treated groups respectively. the resulting images were analyzed with spm- (statistical parametric mapping, fil, ucl). in order to make sure all lesions were in the same side of the brain, images with righthand side strokes were rotated about the sagittal mid-plane, so that the lesioned hemisphere always appeared on the left. functional scans were initially realigned to the first image in the timeseries in order to correct for movements of the head. the first volume of the functional scan was then spatially registered to the structural image, which was, in turn, linearly warped to a template brain. linear warping was used in this step in order to avoid deforming the lesion region. warping parameters obtained during registration of structural image to template were applied to the realigned functional time-series, resulting in structural and functional images that are all in a standard space. finally, functional images were smoothed using a gaussian kernel with full width half maximum of . x . x mm (twice the voxel size). because of the relatively long effective tr of the functional images, a pet basic model (one-sample t-test) was used for firstlevel analyses with covariates consisting of the pseudo-random stimulation pattern (paradigm), and the estimated movement parameters of each individual rat. volumes signal intensity was globally scaled and individual masks, generated from the fast spin echo (fse) structural scan for each rat brain at each time-point using a d pulse-coupled neural network (also registered to the template), were used as explicit masks for the first-level statistical analysis. contrast images from the first-level analysis were then carried onto a second-level (random effects) group analysis. effects of group (i.e. nt or vehicle treated) and stimulated paw (i.e. affected or lessaffected) were used to create statistical comparisons. a flexible factorial analysis was used to compare the difference between the nt and vehicle treated groups in the change from baseline to weeks . statistical parametric maps were generated using an uncorrected threshold of p < . ; images show group mean activations and t values are given. hours after stroke (immediately after mri), rats were allocated to treatment using a randomized block design. allocation concealment was performed by having nt and vehicle stocks coded by a third party. rats were anaesthetised as above and a small incision was made between elbow and axilla, and a small subcutaneous space was formed to the lower back. the osmotic pump with the catheter attached was positioned in this subcutaneous space and then an ultrafine, flexible catheter was implanted approximately millimetres into the proximal end of the long head of the triceps brachii muscle on the disabled side and this was sutured in place (prolene / , ethicon, uk). triceps brachii was selected as the site for infusion because this large muscle is involved in forelimb extension during walking and for postural support during rearing) rearing. note that in the triceps brachii, the end plates are located in the belly of the muscle . the catheter was made from stretchable and flexible silicone tubing (id = . inches, wall thickness = . inches, trelleberg, sf medical, uk, sfm - ) attached to the osmotic pump via larger gluedon tubing (id . mm, vwr international). a second section of this stiff tubing ( millimetres long) was inserted to guide the flexible catheter into the triceps; the guide was slid back after the silicone tube was implanted. catheters were connected to subcutaneous osmotic minipumps ( ml , alzet) containing either vehicle ( . % saline containing . % bovine serum albumin; sigma; a ; - - ) or vehicle containing recombinant human nt (kind gift of genentech inc., usa). pump flow moderators were mr-compatible (peek micro medical tubing, durect, # ). the original vials contained . mg/ml recombinant human ("rhu") nt in mm acetate, mm nacl, ph . . sds page and proteomic analysis indicates that this is the amino acid mature nt protein obtained after proteolytic cleavage of the amino acid proneurotrophin- (uniprot p ) ( supplementary figures and ). the nt dose ( µg/ hours) was selected based on previous experiments . pumps were replaced after two weeks and removed after four weeks. skin was sutured and analgesic administered as above. all rats survived this surgery. sham rats did not undergo this surgery. pilot experiments showed that the pump flow rate ( µl/hour) was sufficient to deliver substances ( . % saline containing % fast green, sigma) to the entire volume of the triceps muscles. rats (n = /group) were terminally anesthetised ( weeks following stroke, before pumps were removed) and triceps brachii and c spinal cord were rapidly dissected and snap frozen in liquid nitrogen prior to storage at - °c. tissue was homogenised in ice cold lysis buffer containing mm nacl, mm tris-hcl (ph . ), % np , % glycerol, mm phenylmethanesulfonylfluoride, μg/ml aprotinin, μg/ml leupeptin and . mm sodium vanadate, using approximately times the volume of buffer to the wet weight of tissue ( µl/mg tissue). protein content was measured and nt elisa was carried out according to manufacturer's instructions (emax, promega). n.b., promega nt elisa kits are no longer available. however, it was recently discovered that when performing elisa using other nt kits (r&d using "reagent diluent" and abcam using diluent a), measurements of nt from skeletal muscle lysates do not provide reliable quantitative data. this is due to so-called "matrix effects" as shown by poor recovery of spiked-in nt (< % or > %) and non-linear relationship between concentration of input material and estimated nt concentration based on a dilution series of muscle homogenate. to overcome the effect of interfering substances, samples should be diluted and appropriate diluents to prepare standards and diluted samples must be used. the abcam kit used with diluent b (not a), however, provided reliable quantitative results (dr. aline barroso spejo, unpublished results). we assessed sensory and motor deficits after stroke using the cylinder test (to assess postural support by forelimbs during rearing), adhesive patch test (to assess responsiveness to tactile stimuli), horizontal ladder (to assess forelimb and hindlimb skilled locomotion), a grip strength test and a test used to monitor unusual responses to cold stimuli (cold allodynia) , . all behavioural testing was carried out by an experimenter blinded to surgery and treatment groups. rats were handled and trained for three weeks on the horizontal ladder before the study began. preoperative baseline scores for the horizontal ladder, the vertical cylinder and the grip strength test were collected one week before surgery. the "adhesive patch" test was used to measure ) the time taken to contact stimuli on the wrists, ) the time taken to remove stimuli from the wrists, and ) the magnitude of lack of responsiveness to stimuli on the affected wrist , , , . for each trial, a round adhesive patch ( mm diameter, ryman) was applied to each wrist on the dorsal side and the animal was returned to its home cage. two times were recorded for both forepaws: ( ) contact and ( ) remove; where "contact" represents the time taken for the animal to contact an adhesive patch with its mouth, and "remove" represents the time taken for the animal to remove the first adhesive patch from its wrist. to determine whether the rats preferentially removed a sticker from their less-affected wrist before their more-affected wrist, the order and side of label removal was recorded. this was repeated four times per session until a > % preference had been found; if this was not the case a fifth trial was conducted. the magnitude of asymmetry was established using the seven levels of stimulus pairs on both wrists as previously described (figure a) . from trial to trial, the size of the stimulus was progressively increased on the affected wrist and decreased on the less affected wrist by an equal amount ( . mm ), until the rat removed the stimulus on the affected wrist first (reversal of original bias). the higher the score, the greater the degree of somatosensory impairment. walking: to assess impairments in forelimb and hindlimb function after stroke, rats were videotaped as they walked along a horizontal ladder. rats were videotaped crossing a horizontal ladder ( m) with irregularly spaced rungs ( to cm spacing changed weekly) weekly, times per session. any slight paw slips, deep paw slips and complete misses were scored as errors. the mean number of errors per step was calculated for each limb for each week (foot faults are routinely normalized "per step" after stroke although analysis of foot fault data with or without normalization led to the same conclusions being drawn). the cylinder test was used to assess asymmetries in forelimb use for postural support during rearing within a transparent cm diameter and cm high cylinder . an angled mirror was placed behind the cylinder to allow movements to be recorded when the animal turned away from the camera. during exploration, rats rear against the vertical surface of the cylinder. the first forelimb to touch the wall was scored as an independent placement for that forelimb. subsequent placement of the other forelimb against the wall to maintain balance was scored as "both." if both forelimbs were simultaneously placed against the wall during rearing this was scored as "both." a lateral movement along the wall using both forelimbs alternately was also scored as "both." scores were obtained from a total number of full rears to control for differences in rearing between animals. once scores had been acquired, forelimb asymmetry was calculated using the formula: × (ipsilateral forelimb use + / bilateral forelimb use)/total forelimb use observations (hsu and jones ). figure a ; mjs technology, uk). both the affected and less affected forelimb strength were each measured (simultaneously) at baseline, week , week and week following stroke. a pair of force transducers were used in parallel to measure the peak force achieved by a rat's forelimbs as its bilateral grip was broken by the experimenter gently pulling the rat by the base of the tail horizontally away from the transducer. the average of three strength readings was noted down per session and an average taken for both arms. the difference in grip strength was taken by subtracting the affected forepaw grip strength from the less affected grip strength. the presence or absence of cold allodynia was assessed using standard methods . rats were placed in a transparent cylinder ( cm diameter, cm height) atop a mesh wire floor. a drop ( µl) of acetone was placed against the centre of the forepaw. in the following s after acetone application the rat's response was monitored. if the rat did not withdraw, flick or lick its paw within this s period then no response was recorded for that trial ( , see below). however, if within this s period the animal responded to the cooling effect of the acetone, then the animal's response was assessed for an additional s, a total of s from initial application. the reasons for taking a longer period of time to assess the evoked behaviour were to measure only pain-related behaviour evoked by cooling and not startle responses that can occur following the initial application of acetone . moreover, the behaviour evoked by acetone is often an interrupted series of behaviours, thus it is important to give enough time to see all pain-related behaviours. responses to acetone were graded to the following -point scale: , no response; , quick withdrawal, flick or stamp of the paw; , prolonged withdrawal or repeated flicking of the paw; , repeated flicking of the paw with licking directed at the ventral side of the paw. acetone was applied alternately three times to each paw and the responses scored categorically. cumulative scores were then generated by adding the scores for each rat together, the minimum score being (no response to any of the six trials) and the maximum possible score being per forepaw. to visualize uninjured cst axons, six weeks after stroke, % biotinylated dextran amine (bda; , mw, invitrogen) in pbs (ph . ) was microinjected unilaterally into the uninjured sensorimotor cortex. animals were placed in a stereotaxic frame and six burr holes were made into the skull at the following coordinates (defined as anterioposterior (ap), mediolateral (ml): ) ap: + mm, ml: . mm; ) ap: + . mm, ml: . mm; ) ap: + . mm, ml: . mm; ) ap: + . mm, ml: . mm; ) ap: + . mm, ml: . mm; ) ap: - . mm, ml: . mm, relative to bregma. at each site, . μl injections of bda ( % in pbs) were delivered using a glass micropipette attached to a hamilton syringe inserted mm from the skull surface and delivered at a rate of . μl/min. animals were subsequently left for weeks before being perfused. tract tracing was not performed in rats that were to undergo functional mri or neurophysiology. as described below, we recorded from the ulnar nerve on the affected side and stimulated the ipsilateral median nerve or, in the pyramids, the corticospinal tract corresponding to the affected or less-affected hemisphere. at the end of the study, rats ( rats per group) were anaesthetised with an intraperitoneal injection of . g/kg urethane (sigma-aldrich). the rat was kept at °c with a homeothermic blanket system and rectal thermometer probe. tracheotomy was performed and a tracheal cannula inserted. the pyramids were then exposed ventrally by blunt dissection and removal of a small area of bone. the brachial plexus of the affected forelimb was exposed from a ventral approach by dissecting the pectoralis major. the ulnar and median nerves were dissected free from surrounding connective tissue and cut distally (to prevent twitches of target muscles). skin flaps from the incision formed a pool, which was filled with paraffin oil. the median and ulnar nerves supply flexor muscles in the forearm, wrist and hand. stimulation of afferents in the median nerve can generate responses in the ulnar nerve motor neurons. the proximal segment of each nerve was mounted on a pair of silver wire hook electrodes (with > cm separation). electrical stimuli of increasing amplitude from µa to µa, in μa steps, (single µs square wave pulse at . hz) were delivered from a constant current stimulator (nl a neurolog, digitimer) to the proximal segment of the cut median nerve. ulnar nerve responses to each stimulus were recorded from the pair of silver wire hook electrodes connected to a differential pre-amplifier and amplifier (digitimer) coupled via a powerlab (ad instruments) interface to a personal computer running labchart and scope software (ad instruments). an average of sweeps at µa was calculated online for each nerve and used to find the difference in amplitudes of monosynaptic reflexes evoked by median nerve stimulation. this was achieved using software to calculate the absolute integral of any response between . ms and ms, regardless of whether a response was observed qualitatively. cst stimulation experiment with ulnar recordings: ulnar nerve recordings were obtained during stimulation of each pyramid in turn. the concentric bipolar stimulation electrode (fhc cbbpc ) was located mm lateral to the midline and gently lowered through the pyramid up to a maximum depth of . mm while stimulating at μa ( pulses, pulse width µs; frequency hz). at the electrode location providing maximal ulnar nerve response, stimuli of increasing intensity were applied in the range of µa to µa, in µa increments. five sweeps were captured at each stimulus intensity. the number of spikes % greater than the noise, and falling between and ms, was calculated for each sweep. the average number of spikes for sweeps at each amplitude was calculated and the difference in the number of spikes elicited by stimulation of the pyramids from the lesioned or contralesional hemisphere. furthermore, the signal was rectified and the area under the curve was measured between and ms for each sweep and averaged for the sweeps at each intensity. each parameter was analysed using twoway repeated measures anova. graphs show mean and standard error of the mean for the area under the curve for stimuli given at µa. eight weeks after stroke surgery and two weeks after injection of bda, rats were terminally anesthetized with sodium pentobarbital ( mg/kg; i.p.) and perfused transcardially with pbs for minutes, followed by ml of % paraformaldehyde in pbs for minutes. the brain, c -c spinal cord, c and c drgs and both arms were carefully dissected and stored in % paraformaldehyde in pbs for hours and then transferred to % sucrose in pbs and stored at - °c. spinal cord segments c and c was embedded in oct and μm transverse slices were cut using a freezing stage microtome (kryomat; leitz, germany). ten series of sections were collected and stored in tbs/ . % azide ( mm tris, mm nacl, . mm nan , ph . ) at °c. cst axons were counted that crossed the midline, at two more lateral planes and at an oblique plane (figure a ) at c and c . for each rat, we estimated the number of cst axons per cord segment by calculating the average number of cst axons per section and then multiplying by a scaling factor (number of sections cut per segment). the total length of serotonergic processes was measured using a standard method designed specifically to measure serotonergic sprouting after neurotrophin treatment (see refs in ) and which is well suited for quantification of dense terminal arbors (e.g., in the dorsal horn of the spinal cord). processes were identified using the "adjust threshold" function in imagej and fiber lengths were measured in three areas: the dorsal horn, intermediate grey and ventral horn (fig. a ) in sections per rat. we calculated the ratio of the sides ipsilateral and contralateral to nt treatment for the three areas separately. immunofluorescence was visualized under a zeiss imager.z microscope or a confocal zeiss lsm laser scanning microscope. photographs were taken using the axio cam and axiovision le rel. . or the lsm image browser software for image analysis. nt protein was radiolabelled with µci ( . mbq) n-succinimidyl [ , - h]propionate ( h-nsp) and separated from unbound h-nsp using an Äktaprime purification system using a modification of a previous method the same treatment was repeated for mice h after stroke, with an incubation period of mins (n = ). in this set of experiments, . mbq of c sucrose (vascular marker) was injected towards the end of the incubation and the brain tissue samples also taken for capillary depletion analysis to distinguish nt or albumin in vascular endothelial cells from that in brain parenchyma. in brief, brain tissue was homogenized in physiological buffer ( µl per mg of tissue) and % dextran ( µl per mg of tissue) as described previously . the homogenate was subjected to density gradient centrifugation ( , × g for min at °c) to give an endothelial cell-enriched pellet and a supernatant containing the brain parenchyma and interstitial fluid (isf). the homogenate, pellet and supernatant samples were solubilized and counted as described above. distribution volume, vd, was calculated for all samples, including the endothelial pellet and brain parenchyma (isf). the values were corrected for c sucrose. data was analysed for capillary fraction, parenchyma and whole brain using one way anova and post hoc (bonferroni) t-tests. ug of protein was subjected to denaturing or non-denaturing sds page and visualised using colloidal coomassie brilliant blue staining. each band was excised separately, digested enzymatically (with trypsin) and subjected to lc/ms/ms analysis (dr. steve lynham, proteomics facility, kcl). in-gel reduction, alkylation and digestion with trypsin were performed prior to subsequent analysis by mass spectrometry. cysteine residues were reduced with dithiothreitol and derivatised by treatment with iodoacetamide to form stable carbamidomethyl derivatives. trypsin digestion was carried out overnight at room temperature after initial incubation at o c for hours. lc/ms/ms: peptides were extracted from the gel pieces by a series of acetonitrile and aqueous washes. the extract was pooled with the initial supernatant and lyophilised. each sample was then resuspended in l of mm ammonium bicarbonate and analysed by lc/ms/ms. chromatographic separations were performed using an ultimate lc system (dionex, uk). peptides were resolved by reversed phase chromatography on a m c pepmap column using a three step linear gradient of acetonitrile in . % formic acid. the gradient was delivered to elute the peptides at a flow rate of nl/min over min. the eluate was ionised by electrospray ionisation using a z-spray source fitted to a qtof-micro (waters corp.) operating under masslynx v . . the instrument was run in automated data-dependent switching mode, selecting precursor ions based on their intensity for sequencing by collision-induced fragmentation. the ms/ms analyses were conducted using collision energy profiles that were chosen based on the mass-to-charge ratio (m/z) and the charge state of the peptide. database searching: the mass spectral data was processed into peak lists using proteinlynx global server v . . with the following parameters: (ms survey -no background subtraction, sg smoothing iterations channels, peaks centroided (top %) no de-isotoping; ms/ms -no background subtraction, sg smoothing iterations channels, peak centroiding (top %) no de-isotoping). the peak list was searched against the uniprot database using mascot software v . using the following parameter specifications (precursor ion mass tolerance . da; fragment ion mass tolerance . da; tryptic digest with up to three missed cleavages; variable modifications: acetyl (protein n-term), carbamidomethylation (c), gln->pyro-glu (n-term q) and oxidation (m). lc/ms/ms analysis and interrogation of the data against the uniprot database identified nt from the excised and digested d gel bands. the results of the analysis and database searches are given in supplementary figure . database generated files were uploaded into scaffold (v . ) software (www.proteomesoftware.com) to create the .sfd file (pr lm d gel ). all samples were aligned in this software for easier interpretation and used to validate ms/ms based peptide assignments and protein identifications. peptide assignments were accepted if they contained at least two unique peptide assignments and were established at % identification probability by the protein prophet algorithm . the result table includes probability scores (mowse) for each peptide identified from the protein sequence. the threshold identity score corresponds to a % chance of incorrect assignment. peptides identified below these probabilities were accepted following manual inspection of the raw data to ensure that fragment ions correctly match the assigned sequence. the sequence coverage for each identified protein is represented in supplementary figure in yellow highlights. statistical analyses were conducted using spss (version . ). graphs show means ± sems (except where otherwise stated) and 'n' denotes number of rats. asterisks (*,**,***) indicate p≤ . , p≤ . and p≤ . , respectively. threshold for significance was . . histology and molecular biology data were assessed using kruskal-wallis and mann-whitney tests (due to small sample sizes). serotonergic fibre lengths was analysed by region using one way anova and post hoc (bonferroni) t-tests. pkcγ data was analysed using kruskal wallis and mann whitney tests. behavioural and mri data were analysed using linear models and restricted maximum likelihood estimation to accommodate data from rats with occasional missing values . akaike's information criterion showed that the model with best fit for the horizontal ladder data had a compound symmetric covariance matrix, whereas for the sensory test and mri data an unstructured covariance matrix was used. the model with best fit for the vertical cylinder had a compound symmetric covariance matrix, according to the - restricted log likelihood information criterion. baseline scores were used as covariates. degrees of freedom are reported to nearest integer. normality was assessed using histograms. t-tests were two-tailed unless otherwise specified. sample size calculations were presented previously . magnetic resonance imaging (mri) confirmed that infarcts included the forelimb and hindlimb areas in sensorimotor cortex (fig c) . there was no difference in the mean infarct volume between stroke groups at h, one or eight weeks after stroke (fig. d) . loss of cst axons was assessed at weeks in the upper cervical spinal cord using protein kinase c gamma (pkcγ) immunofluorescence , (fig. e) . stroke caused a % loss of cst axons in the dorsal columns relative to shams (fig. f) with no difference between vehicle and nt treated rats. together, the mri and pkcγ histology data indicate that there were no confounding pre-treatment differences in mean infarct volumes and that nt did not act as a neuroprotective agent, as expected, based on our previous results and given that treatment was initiated after the majority of cell death will have occurred. we used the "adhesive patch" test to assess forepaw somatosensory function. a sensory score was obtained by attaching pairs of adhesive patches to each rat's wrist on the dorsal side (fig. a) : a high score (e.g. ) denotes that a rat preferentially removed the smaller stimulus from their less-affected wrist (i.e., did not first remove the larger stimulus on their affected wrist). the two stroke groups exhibited a similar lack of responsiveness to stimuli on their affected wrists after one week (fig. b) . delayed treatment with nt caused recovery compared to vehicle: whereas vehicle-treated stroke rats showed a deficit relative to sham rats which persisted for eight weeks. importantly, there were no confounding differences in the time taken to contact or to remove a patch from either their less-affected or affected paw: after stroke, nt -treated rats and vehicletreated rats took longer to contact an adhesive patch relative to shams, but there was no difference between nt and vehicle treated rats (supplementary fig. a) . moreover, neither stroke nor nt treatment caused any deficit in the additional time taken after contact to remove the patch (supplementary figure b) . thus, delayed treatment of disabled forelimb muscles with nt improved responsiveness to tactile stimuli after ischemic stroke. walking was assessed using a horizontal ladder with irregularly spaced rungs (fig. c) . accurate paw placement during crossing requires proprioceptive feedback from muscle spindles . after one week, the two stroke groups made a similar number of errors with their affected forelimb when crossing a horizontal ladder (fig. d) . delayed nt treatment caused a progressive recovery after stroke whereas vehicle treated animals remained persistently impaired until the end of the study. this is consistent with previous work from our lab , . stroke also caused a modest unilateral hindlimb impairment on the ladder; infusion of nt into the forelimb triceps brachii did not improve this (supplementary figure ) . neurotrophin- also restored the use of the affected forelimb for lateral support while rats reared in a vertical cylinder (fig. e) . after stroke and vehicle treatment, rats used their affected forelimb less often than shams. nt -treated rats showed more frequent use of the affected forelimb relative to vehicle-treated rats (fig. f) . we used force transducers to measure grip strength of each forelimb (supplementary fig. ) . stroke caused transient weakness in both groups but infusion of nt into triceps brachii did not modify grip strength. we also found no evidence for pain (cold allodynia) on the affected or treated forelimbs, assessed by application of ice-cold acetone to the centre of the forepaw. cold allodynia was induced neither by stroke nor nt treatment (supplementary fig. ). in summary, infusion of nt protein into the triceps brachii induced recovery on both sensory and motor tasks that require control of muscles by pathways including corticospinal pathways, serotonergic raphespinal pathways and proprioceptive circuits. accordingly, we hypothesised that nt would induce neuroplasticity in multiple pathways. we examined anatomical neuroplasticity in the c cervical spinal cord because we knew from experiments using adult and elderly rats that the less-affected corticospinal tract sprouts at this level (as well as other levels) after injection of aav-nt into muscles including triceps brachii . indeed, anterograde tracing from the less-affected hemisphere (fig. b) revealed that infusion of nt protein increased sprouting of the cst in the c spinal cord (fig. a,b) across the midline and into the affected side at two more lateral planes, and also from the ventral cst. we assessed neural output in the ulnar nerve on the affected side, whose motor neurons are also found in c (range: c to c ) that supply muscles in the forearm including the hand , . to do this, we recorded responses during electrical stimulation of either the spared less-affected corticospinal tract (fig. c) or the partially-ablated corticospinal tract (fig. f) in the medullary pyramids. nt treatment led to enhanced responses in the ulnar nerve during stimulation of the less-affected (fig. d, e) and more-affected (fig. g, h) pathways. this result is consistent with the sprouting of traced cst axons (fig. a,b) and indicates that cst axons from both the stroke hemisphere and the contralesional hemisphere formed new synapses and/or strengthened preexisting connections in the cord on the treated side, most likely on pre-motor interneurons that lie between cst axons and motoneurons , . however, we did not find any evidence that nt strengthened the short-latency reflex from afferents in the median nerve to motor neurons in the ulnar nerve (fig. i-l) . we also found that nt treatment caused serotonergic axons to sprout in the ventral c spinal cord (fig. a-d) . anatomical and functional plasticity of corticospinal and raphespinal pathways is consistent with their expression of receptors for nt , , - . we conclude that nt caused neuroplasticity in multiple descending locomotor pathways including the raphespinal and the spared corticospinal tracts. these data are consistent with previous findings from our lab , that peripherally-administered nt can, directly or indirectly, enhance supraspinal plasticity after stroke. accordingly, next, we assessed the biodistribution of nt after peripheral administration. we measured the amount of total (rat and human) nt in the triceps brachii and c spinal cord. elisa was performed using a subset of five rats per group withdrawn at random from the study at the four-week time point: this revealed an increase in total nt protein levels in the triceps brachii on the treated side (fig. a ) and, surprisingly, on the untreated side (perhaps due to nt in endothelial cells; see below). we were not able to detect any increase in total nt in the c spinal cords (supplementary fig. ). however, elisa cannot distinguish exogenous human nt from endogenous rat nt because the amino acid sequences for mature human and rat nt are identical , . because elisa did not allow us to detect any small increases in exogenous human nt against the background of endogenous rat nt in the cns, we next used a more sensitive method for measuring trafficking of nt across the blood-cns barrier. recombinant nt protein was radiolabelled and purified. [ h]nt was injected intravenously into adult mice. radiolabelled albumin was used as a control because it does not enter the cns efficiently from the bloodstream. after , , , , , or minutes, brain, spinal cord and serum were taken for scintillation counting. nt progressively accumulated in the intact brain (fig. b) and cervical spinal cord (fig. c) . in plasma, the half-life of nt was short (fig. d) . our data is consistent with that from others who have shown that radiolabelled nt rapidly crosses the barriers between the blood and an intact cns [ ] [ ] [ ] and that a small amount of intact nt accumulates in the brain and cervical spinal cord (although the majority of nt is cleared rapidly from the bloodstream) [ ] [ ] [ ] . for example, after injection of nt into the brachial vein (which provides drainage from the triceps brachii), nt accumulates in the cortex, striatum, brainstem, cerebellum, sciatic nerve (and other regions of the nervous system involved with locomotion) . ischemia. minutes later, tissues were taken for scintillation counting. in contrast to [ h]albumin, [ h]nt accumulated in the brain (fig e) . to confirm entry of [ h]nt into brain parenchyma beyond endothelial cells, capillaries were depleted by gradient centrifugation to yield a supernatant containing brain parenchyma and an endothelial cell-enriched pellet . [ h]nt entered parenchyma (depleted of endothelial cells) at a level above that seen for [ h]albumin (fig. f ). transport of nt into the cns is apparently a receptor-mediated process as shown by ) the expression of nt receptors in rodent and human cns capillaries , and ) the ability of non-radiolabelled nt to compete for uptake of radiolabelled nt into the cns , , . in addition, we and others have shown that nt enters the pns after peripheral administration: after intramuscular overexpression of aav encoding nt , nt levels are elevated in the blood stream and nt accumulates in the ipsilateral drg , . we also found some evidence that nt is retrogradely transported from muscle to ipsilateral motor neurons . this is consistent with data showing that ) the blood-nerve barrier in drgs is permeable to proteins like nt , ) that after intravenous injection, radiolabelled nt accumulates in the sciatic nerve and ) that nt is retrogradely transported from muscle to the spinal cord or drg in nerves , , , . we conclude that neuroplasticity occurred in multiple locomotor pathways because peripherally-administered nt bound to receptors in the pns and cns. to explore the mechanism whereby nt improved responsiveness to stimuli attached to the affected wrist (fig. b) , we performed functional brain imaging (bold-fmri) during low threshold (non-noxious) intensity electrical stimulation of the affected wrist ( supplementary fig. ). as expected, prior to stroke, stimulation of the wrist resulted in a higher probability of activation of the opposite somatosensory cortex (supplementary fig. a ). fmri performed one week after stroke confirmed that somatosensory cortex was not active when the affected paw was stimulated in either vehicle or nt treated rats (p> . , supplementary fig. b ). this supports our claim above that there were no early differences between groups that could be explained by neuroprotection. fmri performed eight weeks after stroke revealed a trend towards perilesional re-activation of somatosensory cortex in both vehicle and nt treated groups (p< . , supplementary fig. c ). this is in line with human brain imaging studies showing that spontaneous sensory recovery is increased after stroke when more-normal activity patterns are observed on the affected side of the brain . however, these probabilities of re-activation were not big enough to survive correction for testing of multiple voxels (p-values> . ) although clearly they are in a location that might mediate recovery of somatosensation. a longitudinal analysis showed that at weeks (relative to pre-stroke baseline), there was some evidence that rats treated with neurotrophin- showed increased probability of activation of perilesional cortex (supplementary fig. d, p< . ) and showed decreased probability of activation of somatosensory cortex on the less-affected hemisphere (p< . ) relative to vehicletreated stroke rats. however, these apparent differences did not survive correction for testing of multiple voxels (p< . ). we conclude that both groups showed partial, spontaneous restoration of more-normal patterns of somatosensory cortex activation , but, conservatively, that nt did not further increase probability of activation of any supraspinal areas. these conclusions are consistent with previous fmri data from our laboratory ; we propose that the additional recovery of somatosensory function after nt treatment (fig. b) is due to changes in the spinal cord rather than in supraspinal areas. the batch of neurotrophin- protein that we used was produced more than a decade ago by genentech. we sought to determine whether any degradation had occurred and to confirm its amino acid sequence so that identical preparations of neurotrophin- could be made for future experiments. supplementary figure depicts results from a non-denaturing gel showing a higher molecular weight band ( kda) and a lower molecular weight band ( kda) consistent, respectively, with dimeric mature nt and monomeric mature nt . there was no evidence of degradation or aggregation. each band was excised separately, digested enzymatically (with trypsin) and subjected to lc/ms/ms analysis. proteomic analysis was consistent with both bands being mature nt with no evidence of residual prepro sequences (supplementary figure ) . we conclude that the higher molecular weight band is not prepront (~ kda) but rather corresponds to dimeric mature nt . this facilitated our ongoing experiments to evaluate nt as a therapy for stroke because most commercial preparations of nt consist of mature nt rather than prepront . treatment of disabled arm muscles with nt protein, initiated hours after stroke, caused changes in multiple locomotor circuits, and promoted a progressive recovery of sensory and motor function in rats. the fact that nt can reverse disability when treatment is initiated hours after stroke is exciting because the vast majority of stroke victims are diagnosed within this time frame . in contrast, the gold-standard drug for ischemic stroke, tpa, needs to be given within a few hours and is only administered to a minority. thus, nt could potentially be used to treat an enormous number of victims. nt has good clinical potential. firstly, phase ii clinical trials show that doses up to µg/kg/day are well tolerated and safe in healthy humans and in humans with other conditions , - . we used a threefold lower dose ( µg/kg/day) in this study: in future experiments we will optimize the dose and duration of treatment because it is possible that a higher dose of nt would promote additional recovery after stroke. secondly, there is good conservation from rodents to primates including humans in the expression of receptors for nt in the locomotor system , , [ ] [ ] [ ] . thirdly, in none of our rodent experiments has nt treatment caused any detectable pain, spasticity or muscle weakness (in line with the human trials); rather, after bilateral corticospinal tract injury in rats, intramuscular delivery of aav-nt reduced spasticity, slightly improved grip strength and showed a trend towards reducing mechanical hyperalgesia . in this study and in a previous study we used functional mri combined with electrical stimulation of the wrist in an effort to discover what neuroplasticity underlies recovery of somatosensory responsiveness to adhesive patches attached to the wrist. we confirmed work by others that recovery correlated well with more-normal patterns of increased bold signal surrounding the infarct (potentially in spared somatosensory cortex) , , , but we did not find strong evidence that nt further increased peri-lesional (or other) activation (either in this study or in our previous study ). instead, we now propose that nt increased somatosensory recovery by inducing neuroplasticity in spinal circuits involving cutaneous afferents. this is plausible because cutaneous afferents which mediate tactile sensitivity express trkc receptors . moreover, others have shown that dl spinal interneurons can gate cutaneous transmission . we have previously shown that nt normalises post-activation depression of output from spinal circuits evoked by stimulation of low threshold afferents from the treated wrist (which might include cutaneous afferents as well as proprioceptive afferents) although in those experiments we measured motor output rather than sensory transmission. in the future one might examine whether nt modulates gating of somatosensory inputs from the wrist to spinal interneurons . however, in the present work, the deficits in somatosensory responses were modest and might be difficult to dissect. with regard to corticospinal neuroplasticity, we have shown twice previously (in adult and elderly rats) that the less-affected corticospinal tract sprouts across the cervical midline after injection of aav-nt into affected forelimb muscles . others have shown that intrathecal infusion of nt induces sprouting of the corticospinal tracts and that injection of vectors encoding nt into muscles or nerve can induce corticospinal tract sprouting. here, our anatomical tracing confirmed that the less-affected corticospinal tract sprouted after infusion of nt protein into triceps and in future we will trace both tracts. this is because, in the present study, neurophysiology revealed that both corticospinal tracts underwent plasticity after unilateral infusion of nt protein. we propose that spared cst axons sprouted after nt entered the cns from the systemic circulation. this is consistent with data from us and others showing that radiolabelled nt entered the brain and spinal cord after intravenous injection [ ] [ ] [ ] . moreover, it has been shown that endogenous muscle spindle-derived cues induce sprouting of descending pathways after spinal cord injury in adult mice ; given that muscle spindles make nt endogenously , it is plausible that infusion of supplementary nt to muscle might enhance corticospinal sprouting after stroke. it is also notable that infusion of nt into a proximal forelimb extensor improved the accuracy of use of the affected forelimb when walking on a horizontal ladder but did not improve the accuracy of use of the affected hindlimb; this implies that circulating nt is not sufficient to improve hindlimb movements. moreover, we did not find any evidence that nt strengthened the short-latency reflex between afferents in the median nerve and motor neurons in the ulnar nerve; this may be because we infused nt into the triceps brachii whose afferents do not run in the median or ulnar nerve. this is consistent with previous work of ours showing that a reflex may be strengthened when its afferent comes from a muscle expressing higher levels of nt but not when its afferent comes from a muscle lacking transgenic expression of nt . finally, infusion of nt protein into triceps brachii did not improve forelimb grip strength: however, the grip strength task probably depends more on strength in hand and digit flexor muscles (into which nt was not infused) than on triceps brachii (elbow extensors). indeed, in previous work, injection of aav-nt into proximal and distal flexor muscles did modestly improve grip strength . taken together, these results indicate that it may be important to target nt to multiple muscles. however, it is not straightforward to reconcile all our findings with a single mechanistic explanation. it is possible that, additionally, nt was trafficked from triceps brachii in axons to motor neurons and/or by drg neurons where it induced expression of a molecule that was secreted and induced cst sprouting (e.g., bdnf or igf , ). nt is certainly trafficked to ipsilateral motor neurons and drg after intramuscular delivery , , , , and in this study we also showed, unexpectedly, a small increase in contralateral triceps (perhaps from nt in endothelial cells). diffusion of nt within neuropil is inefficient but spinal motor neuron dendritic arbors can be very large; some even extend across the midline and these might provide a widespread source of cues for supraspinal axonal plasticity (e.g., across the midline). to seek drg-secreted factors, we have performed rnaseq of cervical drg after injection of aav-nt into forelimb flexors , . in the future we will also seek motor neuron-derived cues. finally, it is interesting that the recovery continues even after infusion of nt is discontinued at four weeks. this is encouraging, from a translational perspective. we propose that the four-week long nt treatment induces changes in target neurons that persist (e.g., due to sustained modifications in gene expression). indeed, longer treatment with nt induces different intracellular signalling events in sensory axons than does brief treatment, thereby enhancing terminal branching . in the future, we will seek factors that are persistently increased in target neurons after nt treatment is discontinued. additionally, it may be that nt induces sprouting of cst axons that (after cessation of treatment) is followed by selection of synapses (e.g., strengthening or pruning) by a mechanism that is independent of nt . for example, it is known that corticomotoneuronal axon synapses are pruned by repulsive plexina -sema d interactions . to begin to dissect the mechanisms whereby nt promotes neuroplasticity and recovery after peripheral delivery, we are setting up a mouse model of stroke. in summary, treatment of disabled arm muscles with nt (initiated in a clinically-feasible timeframe) induces multilevel spinal and supraspinal neuroplasticity, improves walking and reverses a tactile sensory impairment. and hours later infusion of nt or vehicle into the disabled triceps brachii was initiated for one month. six weeks after stroke, anterograde tracer was injected into the contralesional hemisphere (blue). rats underwent weeks of behavioural testing. structural mri was conducted on all rats at hours, week and weeks after stroke and fmri was conducted in a subset of rats at baseline, week and week . electrophysiology was performed in the subset of rats which did not receive bda tracing. all surgeries, treatments and behavioural testing were performed using a randomized block design and the study was completed blinded to treatment allocation. c) t mri scans hours after stroke, immediately prior to treatment, showing infarct in coronal sections rostral (mm) to bregma. d) there were no differences between stroke groups in mean lesion volume at hours (mann whitney p values = . ). e) photomicrographs showing loss of figure : delayed nt treatment improved responsiveness to somatosensory stimulation, improved walking and partially restored use of the affected forelimb for lateral support during rearing. a-c) somatosensory deficits were assessed using pairs of adhesive patches attached to the rat's wrists. b) treatment with nt caused improvement compared to vehicle (linear model; f , = . , p< . ; post hoc p= . ): whereas vehicle-treated stroke rats showed a deficit relative to sham rats which persisted for eight weeks (linear model; f , = . , corticospinal axons were anterogradely traced from the less-affected cortex and were counted at the midline (m), at two more lateral planes (d and d ) and crossing into grey matter from the ipsilateral, ventral tract (ipsi). b) nt treatment caused an increase in the number of axons crossing at the midline (f , = . , p= . ; post hoc p value= . ), at two lateral planes denoted as d (f , = . , p< . , post hoc p value< . ) and d (f , = . ,p< . , post hoc p value< . ) and from the ventral cst on the treated side (f , = . , p= . , post hoc p value= . ). although stroke by itself caused sprouting at the midline at c (planned comparison p= . ), nt did not promote additional sprouting at c . n= /group were used for tract tracing. c-e) the cst from the less-affected hemisphere or f-h) lesioned hemisphere was stimulated in the medullary pyramids (before the decussation) and the motor output was recorded from the ulnar nerve on the treated side. d, g) the majority of spikes were detected between ms and ms, latencies consistent with polysynaptic transmission, in both vehicle-treated rats (grey) and nt treated rats (blue) when the less-affected or affected cst was stimulated. e, h) stimulus intensity was increased incrementally from µa to µa and the area under the curve were measured (between and ms) after stimulation of the affected or less-affected hemisphere. nt treatment caused increased output in the ulnar nerve during stimulation of either the affected cst (two-way rm anova intensity* group interaction f , = . , p= . , n= vehicle, nt ) or less affected cst (f , = . , p= . , n= vehicle, n= nt ). i, j) the heteronymous reflex from median afferents to ulnar motor neurons was recorded in the axilla. k) the monosynaptic component was measured. l) nt did not increase the strength of the monosynaptic component. the rats held bilaterally on to the pair of force transducers (top left) and the rat was pulled away horizontally and perpendicularly (towards the right) until the bilateral grip was broken. the force transducers provide a measure of strength (grams) for each upper limb. an average of three trials was taken per rat per week. grip strength (grams) are presented as group means ± sems. b) grip strength of the affected limb was subtracted from the unaffected limb strength, as an internal control (e.g., to control for differences in motivation, etc.). c) grip strength for the affected limb. d) grip strength for the less-affected limb. stroke caused a weak trend towards a transient decrease in strength on the limb affected by stroke (relative to shams; time f , = . , p= . ; p-values p= . and . , respectively) but multiple pairwise comparisons did not show significant differences at any timepoint (all p> . ). there was no difference between nt and vehicle treated rats overall (group f , = . restoring brain function after stroke -bridging the gap between animals and humans a comprehensive review of prehospital and in-hospital delay times in acute stroke care trkc-like immunoreactivity in the primate descending serotonergic system local and remote growth factor effects after primate spinal cord injury influences of neurotrophins on mammalian motoneurons in vivo expression and coexpression of trk receptors in subpopulations of adult primary sensory neurons projecting to identified peripheral targets the neurotrophins bdnf, nt- , and ngf display distinct patterns of retrograde axonal transport in peripheral and central neurons expression of neurotrophins in skeletal muscle: quantitative comparison and significance for motoneuron survival and maintenance of function nt- , but not bdnf, prevents atrophy and death of axotomized spinal cord projection neurons muscle injection of aav-nt promotes anatomical reorganization of cst axons and improves behavioral outcome following sci neurotrophin- expressed in situ induces axonal plasticity in the adult injured spinal cord adeno-associated viral vector-mediated neurotrophin gene transfer in the injured adult rat spinal cord improves hind-limb function differential effects of brain-derived neurotrophic factor and neurotrophin- on hindlimb function in paraplegic rats intramuscular aav delivery of nt- alters synaptic transmission to motoneurons in adult rats either brain-derived neurotrophic factor or neurotrophin- only neurotrophin-producing grafts promote locomotor recovery in untrained spinalized cats neurotrophin- enhances sprouting of corticospinal tract during development and after adult spinal cord lesions intramuscular neurotrophin- normalizes low threshold spinal reflexes, reduces spasms and improves mobility after bilateral corticospinal tract injury in rats spinal electromagnetic stimulation combined with transgene delivery of neurotrophin nt- and exercise: novel combination therapy for spinal contusion injury delayed intramuscular human neurotrophin- improves recovery in adult and elderly rats after stroke retrograde viral delivery of igf- prolongs survival in a mouse als model immune activation is required for nt- -induced axonal plasticity in chronic spinal cord injury expression of neurotrophin- promotes axonal plasticity in the acute but not chronic injured spinal cord activity-dependent increase in neurotrophic factors is associated with an enhanced modulation of spinal reflexes after spinal cord injury muscle spindle feedback directs locomotor recovery and circuit reorganization after spinal cord injury selective expression of neurotrophin- messenger rna in muscle spindles of the rat permeability of the blood-brain barrier to neurotrophins permeability at the blood-brain and blood-nerve barriers of the neurotrophic factors: ngf, cntf, nt- , bdnf penetration of neurotrophins and cytokines across the bloodbrain/blood-spinal cord barrier nt- promotes nerve regeneration and sensory improvement in cmt a mouse models and in patients neurotrophin- improves functional constipation tolerability of recombinant-methionyl human neurotrophin- (r-methunt ) in healthy subjects recombinant human neurotrophic factors accelerate colonic transit and relieve constipation in humans nerve growth factor-and neurotrophin- -induced changes in nociceptive threshold and the release of substance p from the rat isolated spinal cord unbiased classification of sensory neuron types by large-scale singlecell rna sequencing sustained sensorimotor impairments after endothelin- induced focal cerebral ischemia (stroke) in aged rats rodent models of focal stroke: size, mechanism, and purpose delayed treatment with chondroitinase abc promotes sensorimotor recovery and plasticity after stroke in aged rats on the use of alpha-chloralose for repeated bold fmri measurements in rats robust automatic rodent brain extraction using -d pulse-coupled neural networks (pcnn) contrast weights in flexible factorial design with multiple groups of subjects spatial characterization of the motor neuron columns supplying the rat forelimb ethosuximide reverses paclitaxel-and vincristine-induced painful peripheral neuropathy chondroitinase abc promotes plasticity of spinal reflexes following peripheral nerve injury the labelling of proteins to high specific radioactivities by conjugation to a i-containing acylating agent heat shock protein-based therapy as a potential candidate for treating the sphingolipidoses graphical evaluation of blood-tobrain transfer constants from multiple-time uptake data the distribution of the anti-hiv drug, ' '-dideoxycytidine (ddc), across the blood-brain and blood-cerebrospinal fluid barriers and the influence of organic anion transport inhibitors a statistical model for identifying proteins by tandem mass spectrometry analysis of longitudinal data from animals with missing values using spss chondroitinase abc promotes functional recovery after spinal cord injury cervical motoneuron topography reflects the proximodistal organization of muscles and movements of the rat forelimb: a retrograde carbocyanine dye analysis lack of monosynaptic corticomotoneuronal epsps in rats: disynaptic epsps mediated via reticulospinal neurons and polysynaptic epsps via segmental interneurons electrophysiological actions of the rubrospinal tract in the anaesthetised rat trka, trkb, and trkc messenger rna expression by bulbospinal cells of the rat motoneuron-derived neurotrophin- is a survival factor for pax -expressing spinal interneurons bdnf and nt- , but not ngf, prevent axotomy-induced death of rat corticospinal neurons in vivo human and rat brain-derived neurotrophic factor and neurotrophin- : gene structures, distributions, and chromosomal localizations neurotrophin- : a neurotrophic factor related to ngf and bdnf a revised role for p-glycoprotein in the brain distribution of dexamethasone, cortisol, and corticosterone in wild-type and abcb a/bdeficient mice the cell biology of the blood-brain barrier nerve growth factor-induced protection of brain capillary endothelial cells exposed to oxygen-glucose deprivation involves attenuation of erk phosphorylation expression of cannabinoid receptors and neurotrophins in human gliomas vascularization of the dorsal root ganglia and peripheral nerve of the mouse: implications for chemical-induced peripheral sensory neuropathies neurotrophin- administration attenuates deficits of pyridoxineinduced large-fiber sensory neuropathy neurotrophin- is a target-derived neurotrophic factor for penile erection-inducing neurons reemergence of activation with poststroke somatosensory recovery: a serial fmri case study correlation between brain reorganization, ischemic damage, and neurologic status after transient focal cerebral ischemia in rats: a functional magnetic resonance imaging study early prediction of functional recovery after experimental stroke: functional magnetic resonance imaging, electrophysiology, and behavioral testing in rats expression of mrnas for neurotrophic factors (ngf, bdnf, nt- , and gdnf) and their receptors (p ngfr, trka, trkb, and trkc) in the adult human peripheral nervous system and nonneural tissues trka and trkc expression is increased in human diabetic skin neurotrophin- -like immunoreactivity and trk c expression in human spinal motoneurones in amyotrophic lateral sclerosis changes in cortical activation patterns accompanying somatosensory recovery in a stroke patient: a functional magnetic resonance imaging study longitudinal changes in cerebral response to proprioceptive input in individual patients after stroke: an fmri study circuits for grasping: spinal di interneurons mediate cutaneous control of motor behavior intraspinal rewiring of the corticospinal tract requires target-derived brain-derived neurotrophic factor and compensates lost function after brain injury igf-i specifically enhances axon outgrowth of corticospinal motor neurons differential distribution of exogenous bdnf, ngf, and nt- in the brain corresponds to the relative abundance and distribution of high-affinity and low-affinity neurotrophin receptors distinct limb and trunk premotor circuits establish laterality in the spinal cord rnaseq dataset describing transcriptional changes in cervical sensory ganglia after bilateral pyramidotomy and forelimb intramuscular gene therapy with aav encoding human neurotrophin- . data in brief sad kinases sculpt axonal arbors of sensory neurons through long-and short-term responses to neurotrophin signals control of species-dependent cortico-motoneuronal connections underlying manual dexterity cst axons in the upper cervical dorsal columns weeks after stroke (right) relative to sham surgery (left), visualised using pkcγ immunofluorescence. f) stroke caused a significant loss of cst axons relative to shams in the dorsal columns there were no differences between nt and vehicle treated rats at one week (t-test p= . ). c) accuracy of paw placement by the affected forelimb during walking was assessed using a horizontal ladder with irregularly spaced runs. d) one week after stroke, nt and vehicle treated rats made a similar number of misplaced steps (t-test p= . ), expressed as a percentage of total steps. importantly, the nt group progressively recovered compared to the vehicle group (group f , = . , p< . ; post hoc p= . ) and differed from the vehicle group from weeks to (group x time f , = . , p< . ; post hoc p values< . ) and whereas the vehicle group remained impaired relative to shams from weeks to (p values< . ), from weeks to the nt group made no more errors than shams (post hoc p-values> . ). e) the vertical cylinder test assessed use of the affected forelimb for lateral support during rearing. f) stroke caused a reduction in the use of the affected forelimb during rearing in a vertical cylinder in both nt and vehicle treated rats relative to shams (group f , = . , p= . ; post hoc p values= . and . , respectively) with no differences between stroke groups at one week (p= . ). nt treatment caused a progressive recovery in the use of the affected forelimb grey circles) against time after iv injection (n= - mice/time) in adult mice. t-tests for nt vs albumin all significant for incubation times of , , , and (p values from < . to < . ***). the volume of distribution of [ h] nt or [ h] albumin in brain (vd =am/cp) is calculated as a ratio of counts per minute (cpm) in µg of brain and cpm in µl of serum for each time point and plotted against exposure time given by the term ∫ t cp(τ)dτ/cp. the rate of influx (ki) was calculated from the patlak plot of vd for  - μl/mg/s). c) [ h]nt entered the cervical spinal cord more abundantly than [ h]albumin. d) plasma half-life of nt for the normal adult mouse is ~ min (estimated from /normalised serum values). e) twenty-four hours after cortical ischemia, [ h]nt entered the brain more abundantly than [ h]albumin, measured minutes after iv injection. f) twenty-four hours after cortical ischemia there are no conflicts of interests of the authors. correspondence and requests for materials should be addressed to l.m (lawrence.moon@kcl.ac.uk) figure : focal cortical stroke caused impairment of the affected forelimb but modest or no impairment of the three other limbs. a) after stroke, nt treated rats recovered function of their affected forelimb on the ladder test relative to stroke vehicle controls and sham rats. nt treated rats recovered fully relative to shams (linear model and t-tests, p≤ . ). *** denotes group difference, p < . ; † denotes interaction of group with time, p< . . this subpanel is reproduced from figure to allow comparison with other subpanels). b) shows photograph of the horizontal ladder set up and insert shows a rat traversing the ladder. c) there was no difference in the number of foot faults made in any of the groups using the less affected supplementary figure : cold allodynia was caused neither by focal cortical stroke nor by treatment with neurotrophin- . the acetone test was used to see whether stroke and/or nt treatment caused any change in cold allodynia pain responses. the test involves applying a drop of acetone to the a) affected or b) less affected forelimb, and then allocating a score between and : higher numbers denote a heightened pain response. there is no evidence of painful behaviour based on this test in either forelimb. rm ancova with bonferroni post hoc tests. figure : elisa revealed that infusion of nt into triceps brachii did not cause detectable elevation of nt in homogenates of cervical spinal cord hemicords on the infused or non-infused side of the body (mann whitney p-values= . , . , respectively). figure : functional brain imaging during stimulation of the affected wrist revealed no enhanced probability of perilesional activation by neurotrophin- . the same rats were imaged prior to stroke and then one week and eight weeks after stroke and intramuscular treatment with either nt or vehicle. scans with obvious imaging artefacts were discarded, leaving final group numbers of n= , , and n= , , at weeks , and for nt and vehicle treated groups respectively. red voxels denote greater probability of activation during stimulation (versus stimulation off) whereas blue voxels denote lesser probability of activation during stimulation (versus stimulation off). a) prior to stroke, stimulation of the dominant paw led to a strong probability of activation in the opposite somatosensory cortex. b) one week after stroke, this activation was abolished by infarction. c) eight weeks after stroke, there was a slight trend towards a small perilesional area of reactivation in both groups. d) there was a slight trend towards greater perilesional reactivation in the nt group versus the vehicle group at weeks (relative to their baselines). however, all these heat maps of groups of rats show t-values obtained by statistical parametric map analysis without correction for multiple testing (p< . ) and there were no differences between the two groups for any voxels when the threshold for significance was corrected for multiple testing (p< . ; this data is not shown as the heat map was black). red voxels denote greater probability of activation during stimulation for the nt group than for the vehicle group whereas blue voxels denote lesser probability of activation for the nt group than for the vehicle group. when stimulating the less-affected wrist, there were no differences between the two groups for any voxels when the threshold for significance was corrected for multiple testing (p< . ; this data is not shown as the heat map was black). figure : different amounts of recombinant nt were run in four lanes of an sds page gel. two sizes of band of interest (lm _ and lm _ ) were detected following staining with colloidal coomassie brilliant blue. these protein bands were excised prior to separate enzymatic digestion and lc/ms/ms analysis. the apparent molecular weight of the upper band (~ kda) is consistent with either the pro-neurotrophin- precursor form or a dimer of the mature nt protein, whilst the lower band (~ kda) is consistent with the mature nt protein. sequencing revealed that both bands represent the mature nt protein. key: cord- -r rg iqq authors: scaggs huang, felicia a.; schlaudecker, elizabeth title: fever in the returning traveler date: - - journal: infectious disease clinics of north america doi: . /j.idc. . . sha: doc_id: cord_uid: r rg iqq millions of children travel annually, whether they are refugees, international adoptees, visitors, or vacationers. although most young travelers do well, many develop a febrile illness during or shortly after their trips. approaching a fever in the returning traveler requires an appropriate index of suspicion to diagnose and treat in a timely manner. as many as % of patients with recent travel history are diagnosed with routine infections, but serious infections such as malaria, enteric fever, and dengue fever should be on the differential diagnosis due the high morbidity and mortality in children. millions of children travel annually, whether they are refugees, international adoptees, visitors, or vacationers. [ ] [ ] [ ] [ ] in , the international tourism organization reported . billion overseas trips. , although most young travelers do well, many develop febrile illnesses during or shortly after their journeys. in a study of european children, % of all pediatric patients with travel-related infections were visiting friends and relatives (vfrs), . % were tourists, and . % were immigrants. most illnesses are selflimited childhood infections that do not require subspecialist consultation. however, % of , ill american travelers sought care at travel clinics after returning home. additionally, young children with fevers can present a diagnostic dilemma because they may not report symptoms and can be at risk for severe disease, such as malaria. as awareness of tropical illnesses rise in parents, such as the increase in multidrug-resistant bacteria worldwide or the emergence of epidemics with zika virus in south america, families may be more anxious about serious infections as an etiologic factor of fevers. approaching fevers in the returning traveler requires an appropriate index of suspicion to diagnose and treat the child in a timely manner. this article offers a framework on how to address these issues by discussing diseases based on geography, incubation period, and affected organ systems, as well as risk factors, diagnostic techniques, and resources. a thorough history is an important initial step when evaluating a pediatric traveler with a fever ( table ) . discussing a detailed travel itinerary develops a timeline of exposures that can be unique to an urban or rural setting ( table ) . many children receive vaccinations and/or antimicrobial prophylaxis, but reported adherence does not preclude an illness with a particular pathogen. up to % of travelers do not adhere to the recommended malaria prophylaxis. many travel vaccines, including typhoid vaccine, provide only partial protection despite proper administration of these immunizations. a medically complex individual may have sought care outside of the united states due to necessity or medical tourism, which can increase the risk of infection through body fluid exposures. multidrug-resistant pathogens can also be associated with health care exposure. up to half of hospitalized children in zimbabwe are colonized with extended spectrum beta lactamase producing enterobacteriaceae on admission to the hospital, a problem that is increasingly seen worldwide. underlying medical conditions, such as asplenia or immunosuppression from chemotherapy, may predispose children to overwhelming infections and sepsis. refugee children from countries such as syria are susceptible to vaccine-preventable diseases such as polio due to infrastructure breakdown. fever is a common and anxiety-provoking sign for parents that can be exacerbated by overseas travel. up to % of patients with recent travel history are diagnosed with routine infections. of the , cases of infection in travelers from to reported to geosentinel, a worldwide data collection network on travel-related diseases, % of cases were considered to be life-threatening. a study in swiss children showed that . % of emergency room visits were due to travel-related morbidities with fever and gastrointestinal symptoms being the most common complaints in % and % of patients, respectively. the temporality of travel to the onset of fever can offer important clues to the etiologic factors of fevers ( table ) . because the causes and clinical outcomes associated with fevers in pediatric travelers vary from self-limited to deadly, a systems-based approach can lead to prompt diagnosis and treatment that evaluates for the most likely and serious diseases early in the illness course. according to geosentinel, % of patients with an acute, life-threatening illness will present with fever. there are a broad range of potential tropical infections, including malaria, dengue fever, and enteric fever. the incidence of emerging infections such as zika virus and chikungunya are not yet known. in both adults and children, pneumonia, sepsis, meningococcemia, and urinary tract infections that were acquired at home or overseas should be on the differential diagnosis. the initial workup of a febrile child without a clear source will be based on the history, physical examination, and risk factors but commonly includes a complete blood count, liver function tests, creatinine, urinalysis, and blood cultures. , malaria smears are also frequently helpful. other tests to consider include serologies for dengue fever scaggs huang & schlaudecker or other potential etiologic agents, polymerase chain reaction for zika virus or other pathogens, chest radiographs, and cultures of the urine and stool. patients with altered mental status may require head imaging and lumbar puncture. the most common and concerning causes of fever in a returning pediatric traveler are highlighted next. [ ] [ ] [ ] fever in the returning traveler plasmodium falciparum malaria is one of the most common tropical infections. approximately % to % of all imported malaria cases are diagnosed in the pediatric population in industrialized countries each year. malaria is transmitted via the nocturnal-feeding anopheles genus of mosquito. children who are vfrs are more likely to become infected with malaria than traditional tourists. nonimmune children are also susceptible to severe malaria from other malaria strains such as plasmodium vivax and many young patients can present with atypical symptoms such as abdominal pain and vomiting. older children may present with paroxysmal fever, fatigue, myalgias, headache, abdominal pain, back pain, hepatosplenomegaly, and hemolytic anemia. additionally, severe malaria is more common in children after the first month of travel due to the incubation period of p falciparum ( - days), especially in those who visited sub-saharan africa. , overall, sub-saharan africa is one of the most common geographic regions for acquisition, comprising . % of cases according to a geosentinel study of travelers migrating or returning to canada from to . malaria should remain on the differential diagnosis for up to a year in an acutely ill, febrile child after travel to an endemic area where p vivax and p ovale strains are present. interestingly, % of malaria cases can be acquired during trips as short as weeks with less utilization of pretravel services being a contributing factor. a minimum of thick and thin blood smears must be performed before malaria can be excluded, preferably collected during febrile episodes. the specificity of blood smears is high but the sensitivity can be low depending on the experience of the individual interpreting the slides. rapid diagnostic tests that detect specific proteins or lactate dehydrogenase are alternatives for diagnosis at medical centers with limited experience in microbiologic evaluation for malaria. the result should be confirmed, however, through the state public health department. in general, a febrile child without a localizing source or splenomegaly, thrombocytopenia, or indirect hyperbilirubinemia, in addition to exposure to an endemic area, should be presumptively approached as having malaria until an alternative diagnosis can be made. treatment of malaria is well-established by the centers for disease control and prevention (cdc) guidelines. children with acidosis, hypoglycemia, hyperparasitemia, end-organ dysfunction, and severe anemia meet the criteria for severe malaria and require prompt administration of parenteral medication. there is a growing body of evidence that artesunate may reduce mortality compared with quinidine and is becoming more common as first-line therapy in pediatric patients. , artesunate must be obtained through the cdc malaria hotline ( - - - ) because it is not routinely available in the united states. quinidine may be initiated until the medication arrives. completion of therapy with an oral regimen for uncomplicated chloroquine-resistant p falciparum, such as atovaquone-proguanil, can be offered when the child is able to tolerate the medications and the parasite burden has decreased to less than %. severe disease is less common in p vivax and p ovale and infection can be treated with chloroquine or hydroxychloroquine in most areas outside of indonesia and papua new guinea. enteric fever accounts for % of the cases with life-threatening tropical diseases reported to geosentinel. most recorded cases were from the indian subcontinent and in vfrs. infection with salmonella typhi and salmonella paratyphi are clinically indistinguishable with fever, abdominal pain, nausea, vomiting, myalgias, and arthralgias. diarrhea is greater than . times more common in infants than older children or adults, although constipation can also be seen. patients can exhibit a fever in the returning traveler typhoid mask with dull features and confusion, as well as a stepladder fever progression with rising temperatures over time in untreated individuals. relative bradycardia and rose spots are also classic signs. complications such as gastrointestinal bleeding are more common in young children who have been ill for weeks or more. transmission is fecal-oral, and humans, especially adults, may be chronic carriers. diagnosis of enteric fever is confirmed through cultures. the most sensitive sterile site is bone marrow ( %- %). blood culture has the highest yield during the first week of illness ( %), and stool cultures are more sensitive as the duration of illness increases. stool studies should be performed on all fellow travelers, and they must be monitored for signs of illness. other abnormal laboratory findings include transaminitis and a normal or decreased white blood cell count. the antimicrobial of choice for treatment varies based on the area in which the infection was acquired because multidrug resistance is increasing. empiric treatment with ceftriaxone or fluoroquinolones is typically recommended. strains in latin america and the caribbean can be susceptible to ampicillin and trimethoprimsulfamethoxazole. south and southeast asian serovars more frequently require azithromycin or cefixime. , children with multidrug-resistant strains have more complications such as myocarditis and shock than children infected with susceptible strains but case fatality is similar ( . % vs . %, respectively). relapse of infection can occur despite appropriate therapy, with the highest mortality in young children ( %). dengue remains an important cause of fever in travelers returning from all tropical regions except africa. the prevalence is rising, even in the united states, with to million global cases reported yearly and , deaths, primarily in children. risk factors are dissimilar from those for malaria because transmission occurs in urban areas during the daytime due to the vector aedes aegypti, whereas malaria transmission is more common in rural areas from dusk to dawn with the anopheles species mosquito. some patients may be asymptomatic, whereas others have hemorrhagic fever and shock. the illness presents as distinct phases: ( ) febrile phase over to days characterized by myalgias, headache, retroorbital pain, and rash; ( ) critical phase of to days with plasma leakage; and ( ) convalescent phase. a rising hemoglobin and gallbladder wall thickening due to increased vascular permeability suggests the development of severe dengue in children. repeat infections with a different strain may lead to more severe disease. serologies are most commonly used for diagnosis, although some rapid diagnostic tests are available. in cases in which infection is unclear, it may be helpful to repeat serologies weeks after initial testing to monitor for an increase in titers. other common laboratory findings include leukopenia and thrombocytopenia. treatment consists of hydration and avoidance of salicylate-containing products to decrease the risk for bleeding. children who develop severe dengue with hemorrhage and shock may require blood products. no antivirals or vaccines are currently available. in recent years, arboviral illnesses transmitted via infected aedes aegypti mosquitos have caused epidemics of zika virus and chikungunya in south america. a european study of travelers returning from brazil in to reported that of the % of patients with travel-related complaints, % had dengue fever, % had chikungunya, and % had zika virus infection. the prevalence of yellow fever, which is seen throughout low-resource settings and shares the same vector, has remained stable. these infections are difficult to distinguish clinically with fever, retroorbital pain, conjunctivitis, and myalgias. knowledge on perinatal infection with zika and the neurodevelopmental sequelae of affected infants is rapidly evolving. a canadian study found that % of travelers developed neurologic complications such as guillain-barre syndrome with zika, suggesting there is much to learn with this disease in nonperinatally acquired infections. at this time, treatment is primarily supportive. additional tropical diseases associated with fevers are outlined in table . vomiting and diarrhea are common complaints in returning travelers. up to % of children less than years of age may develop diarrhea, with % requiring medical services. fevers, nausea, and vomiting can be seen with norovirus that occurs worldwide and is frequently associated with contaminated food and water on cruise ships. rotavirus, however, is one of the most frequent causes of diarrheal illnesses worldwide and is a common cause of infant mortality in low-resource settings. the hepatitides present with a broad range of disease from mild abdominal pain and vomiting to fulminant liver failure, although serious complications are uncommon in pediatric travelers. community-acquired clostridium difficile is uncommon in children but infection should be considered if the patient received recent antimicrobials. geosentinel data reported that % of patients diagnosed with clostridium difficile after travel were to years of age. there are many other causes of both febrile and nonfebrile gastrointestinal illness in children ( table ) . in the pediatric population, common respiratory infections may be seen on return from international trips including pharyngitis, sinusitis, otitis, and pneumonia from pathogens commonly seen in the united states, such as streptococcus pneumoniae and rhinovirus. , local epidemiology of infections can be helpful in diagnosis and management and is available through the cdc. in some tropical regions, influenza may occur throughout the year and should hence remain on the differential for patients who warrant treatment with oseltamivir. mycobacterium tuberculosis is an important etiologic factor of lower respiratory tract disease worldwide and should be considered in children with risk factors or who do not recover with antimicrobials for bacterial pneumonia. of note, children younger than years of age are more likely to present with miliary tuberculosis or neurologic involvement than adult patients. there are also many other less common causes of febrile respiratory tract infections ( table ) . children who present with dysuria, hematuria, and fevers may require urinalysis and culture to evaluate for urinary tract infection and/or pyelonephritis. gross hematuria with the passage of clots in an afebrile child with exposure to freshwater in africa, the middle east, china, and southeast asia should be tested for the helminth parasite from the genus schistosoma via serologies or microscopic identification of eggs in stool. praziquantel is the treatment of choice and may improve anemia and nutrition in some children. patients who may have early disease or a high parasite burden may require a repeat treatment. children who are at risk for sexual abuse and adolescents should undergo testing for sexually transmitted infections such as chlamydia trachomatis and neisseria gonorrheae. rashes are a source of concern for parents without the context of travel and may be even more worrisome after going abroad. the differential diagnosis includes typical childhood illnesses, such as roseola or staphylococcal cellulitis, in addition to tropical infections. a study of canadian travelers from to found that cutaneous larva migrans ( %) and skin and soft tissue infections ( . %) were some of the most common infectious dermatologic complaints among tourists. in countries where vaccination rates are low, varicella zoster virus or rubella may cause disease, especially in young children who have not completed their immunization series. measles remains an important risk, with tourists comprising % of the cases reported to geosentinel from to , and % of patients being younger than years of age, although this may represent underreporting due to the surveillance system's primarily adult focus. petechiae on the extremities in an illappearing child may indicate a serious systemic process such as meningococcal or rickettsial infection. there are many other infections with primarily dermatologic manifestations that may not cause fevers ( table ) . as the numbers of children who travel abroad continues to increase, clinicians need to remain up-to-date on potential etiologic factors for febrile illnesses on families' return home. after ruling out life-threatening disorders that can be acquired locally or internationally, physicians are able to develop a focused diagnosis and management plan best suited to the patient's clinical picture. there is a growing body of resources to assist clinicians, such as the cdc (www.cdc.gov/travel/) and geosentinel (www.istm.org/geosentinel) for data on epidemiology, geography, and other risk factors. in the future, physicians will need to be prepared to deal with the global epidemic of antimicrobial drug resistance, evolving epidemics and pandemics caused by emerging pathogens, reemerging infections due to vaccine hesitancy or international conflicts, and medical tourism in both healthy and medically complex children. fever after international travel etiology and outcome of fever after a stay in the tropics imported malaria in children: a review of clinical studies evaluation of the sick child following travel to the tropics estimate of worldwide rotavirusassociated mortality in children younger than years before the introduction of universal rotavirus vaccination programmes: a systematic review and meta-analysis international tourist arrivals up % reach a record . billion in spectrum of disease and relation to place of exposure among ill returned travelers management of children with travel-related illness evaluated in a pediatric emergency room fever in returned travelers: results from the geosentinel surveillance network the practice of travel medicine: guidelines by the infectious diseases society of america vaccines for preventing typhoid fever. cochrane carriage of antibioticresistant enterobacteriaceae in hospitalised children in tertiary hospitals in harare syrian crisis: health experts say more can be done multicenter geosentinel analysis of rickettsial diseases in international travelers plasmodium vivax and mixed infections are associated with severe malaria in children: a prospective cohort study from papua new guinea diagnosis and treatment of malaria in children treatment of malaria in the united states: a systematic review malaria in travellers returning or migrating to canada: surveillance report from cantravnet surveillance data rapid diagnostic tests for malaria parasites does this patient have malaria? south east asian quinine artesunate malaria trial (seaquamat) group. artesunate versus quinine for treatment of severe falciparum malaria: a randomised trial artesunate versus quinine in the treatment of severe falciparum malaria in african children (aquamat): an open-label, randomised trial artesunate is available to treat severe malaria in the united states an appraisal of the clinical features of pediatric enteric fever: systematic review and meta-analysis of the age-stratified disease occurrence evaluation and management of illness in a child after international travel enteric (typhoid) fever in travelers azithromycin versus ceftriaxone for the treatment of uncomplicated typhoid fever in children systematic review of the global epidemiology, clinical and laboratory profile of enteric fever seasonality, annual trends, and characteristics of dengue among ill returned travelers dengue in travelers arboviral and other illnesses in travellers returning from brazil yellow fever vaccines and international travelers clinical impact of non-congenital zika virus infection in infants and children surveillance report of zika virus among canadian travellers returning from the americas incidence and clinical features of traveler's diarrhea in infants and children traveler's diarrhea: updates for pediatricians epidemiology of travel-associated and autochthonous hepatitis a in austrian children analysis of clostridium difficile associated diarrhea in pediatric patients with antibiotic-associated diarrhea clostridium difficile infection in returning travellers bordetella pertussis infections in travelers: data from the geosentinel global network update: influenza activity-united states and worldwide schistosomiasis in travellers and migrants single dose metrifonate or praziquantel treatment in kenyan children. ii. effects on growth in relation to schistosoma haematobium and hookworm egg counts dermatoses among returned canadian travellers and immigrants: surveillance report based on cantravnet data measles in the st century, a continuing preventable risk to travelers: data from the geosentinel global network dermatologic conditions of the ill returned traveler: an analysis from the geosentinel surveillance network b-virus and free-ranging macaques fever on return from abroad. in: acute medicine -a practical guide to the management of medical emergencies approach to fever in the returning traveler the yellow book: health information for international travel key: cord- -yfjme q authors: magtoto, ronaldo; poonsuk, korakrit; baum, david; zhang, jianqiang; chen, qi; ji, ju; piñeyro, pablo; zimmerman, jeffrey; giménez-lirola, luis g. title: evaluation of the serologic cross-reactivity between transmissible gastroenteritis coronavirus and porcine respiratory coronavirus using commercial blocking enzyme-linked immunosorbent assay kits date: - - journal: msphere doi: . /msphere. - sha: doc_id: cord_uid: yfjme q this study compared the performances of three commercial transmissible gastroenteritis virus/porcine respiratory coronavirus (tgev/prcv) blocking enzyme-linked immunosorbent assays (elisas) using serum samples (n = ) collected over a -day observation period from pigs inoculated with tgev strain purdue (n = ), tgev strain miller (n = ), prcv (n = ), or with virus-free culture medium (n = ). elisa results were evaluated both with “suspect” results interpreted as positive and then as negative. all commercial kits showed excellent diagnostic specificity ( to %) when testing samples from pigs inoculated with virus-free culture medium. however, analyses revealed differences between the kits in diagnostic sensitivity (percent tgev- or prcv-seropositive pigs), and all kits showed significant (p < . ) cross-reactivity between tgev and prcv serum antibodies, particularly during early stages of the infections. serologic cross-reactivity between tgev and prcv seemed to be tgev strain dependent, with a higher percentage of prcv-false-positive results for pigs inoculated with tgev purdue than for tgev miller. moreover, the overall proportion of false positives was higher when suspect results were interpreted as positive, regardless of the elisa kit evaluated. importance current measures to prevent tgev from entering a naive herd include quarantine and testing for tgev-seronegative animals. however, tgev serology is complicated due to the cross-reactivity with prcv, which circulates subclinically in most swine herds worldwide. conventional serological tests cannot distinguish between tgev and prcv antibodies; however, blocking elisas using antigen containing a large deletion in the amino terminus of the prcv s protein permit differentiation of prcv and tgev antibodies. several commercial tgev/prcv blocking elisas are available, but performance comparisons have not been reported in recent research. this study demonstrates that the serologic cross-reactivity between tgev and prcv affects the accuracy of commercial blocking elisas. individual test results must be interpreted with caution, particularly in the event of suspect results. therefore, commercial tgev/prcv blocking elisas should only be applied on a herd basis. tality in piglets in tgev/prcv-naive herds. the virus was first described by doyle and hutchings in in the united states and subsequently reported worldwide ( ) ( ) ( ) . porcine respiratory coronavirus is a naturally occurring spike gene deletion ( to kda) mutant of tgev first isolated in belgium in ( ) . it infects the upper respiratory tract, tonsils, or lungs, with limited intestinal replication ( , ) . porcine respiratory coronavirus itself does not appear to be an important primary pathogen, with the exception of its contribution to the porcine respiratory disease complex ( ) . tgev and prcv share biological and molecular features but differ in their epidemiology, clinical presentation, and pathogenesis. real-time reverse transcription-pcr (rrt-pcr) and multiplex microarray hybridization using primers targeting the = region of the s gene spanning the deletion region in pcrv strain are commonly used for the diagnosis of tgev and differentiation of tgev and prcv ( ) ( ) ( ) ( ) . serum antibodies provide serological evidence of tgev or prcv infection, but prcv-infected pigs produce antibodies that cross-react and cross-neutralize tgev, i.e., conventional serological tests cannot differentiate between tgev-and prcv-infected animals. this presents a complication for tgev seroprevalence studies and serological surveys of sows or slaughterhouse swine tested for international trade ( ) . to address the issue of cross-reactivity, monoclonal antibodies targeting antigenic regions of tgev that have been deleted from the prcv s protein ( ) ( ) ( ) ( ) ( ) ( ) ( ) have been used to develop blocking enzyme-linked immunosorbent assays (elisas) for tgev/ prcv differential serodiagnosis ( ) ( ) ( ) ( ) . several commercial tgev/prcv blocking elisas are available, but comparative test performances have not been reported in recent publications. in this study, the diagnostic test performances of three commercial tgev/prcv blocking elisa kits were evaluated using serum samples of precisely known porcine coronavirus immune status. clinical observations and virus shedding. mild watery diarrhea was observed in pigs inoculated with tgev miller strain between to days postinoculation (dpi). no clinical signs were observed in pigs from the negative-control, tgev purdue, and prcv-inoculated groups throughout the experiment. all animals survived until the end of the study ( dpi). all fecal samples collected from pigs in the tgev strain purdue, tgev strain miller, prcv-inoculated, and negative-control groups were tested by rrt-pcr and found to be tgev and prcv negative prior to the inoculations. likewise, all oral fluid samples, with the exception of one false-positive (threshold cycle [c t ], . ) result in the tgev purdue group, were negative before inoculation. the detection of tgev (s and n genes) and prcv (n gene) in pen-based feces and oral fluids by rrt-pcr is shown in fig. . transmissible gastroenteritis virus was detected in feces between and dpi by rrt-pcr in pigs inoculated with tgev strain miller ( fig. a and c) . no significant fecal shedding was detected in pigs inoculated with tgev strain purdue or prcv throughout the study ( fig. a and c). viral shedding was specifically detected by rrt-pcr in oral fluid samples collected from pigs inoculated with tgev strain purdue ( to dpi), tgev strain miller ( to dpi), and prcv ( to dpi) ( fig. b and d) . tgev antibody response over the course of the experimental inoculation. serum samples collected from all groups of pigs prior to inoculation were antibody negative for porcine coronaviruses (tgev, prcv, porcine epidemic diarrhea virus [pedv] , and porcine delta coronavirus [pdcov]). all pigs in the negative-control group remained tgev and prcv seronegative throughout the monitoring period when tested with any of the three tgev/prcv differential blocking elisa kits evaluated in this study ( the percentages of tgev antibody-positive serum samples reported by the three commercial elisa kits evaluated over the -day study period for pigs inoculated with tgev strains purdue and miller are presented in fig. a to f, respectively. suspect results are presented as positives or negatives. the first tgev-specific antibody detection was reported between and dpi in both tgev-inoculated groups (strains purdue and miller). the number of tgev antibody-positive pigs detected increased through the study, regardless of the elisa kit used. for the tgev purdue inoculation group, no significant differences (p Ͼ . ) were found in the percentages of tgev-seropositive pigs reported by the three elisas regardless of the time postinoculation and interpretation of suspect results. in contrast, for the tgev miller inoculation group, we found that a significantly higher (p Ͻ . ) percentage of tgev-seropositive animals was detected by the swinecheck tgev/prcv recombinant elisa than by both the ingezim corona diferencial elisa (dpi ) and svanovir tgev/prcv-ab elisa ( , , and dpi) only when suspect results were interpreted as negative. a nonspecific tgev antibody response to prcv-inoculated pigs was reported by the three elisa kits between and dpi ( fig. g to i). no significant differences in the percentages of tgev false positives were found between elisas over the monitoring period when tgev suspect results were interpreted as negative (p Ͼ . ). however, the overall proportion of false positives was higher when suspect results were interpreted as positive, regardless of the elisa kit evaluated. moreover, the percentage of tgev-false-positive results reported by the svanovir tgev/prcv-ab elisa was significantly greater (p Ͻ . ) than those obtained with the swinecheck tgev/prcv recombinant elisa ( , , and dpi) and ingezim corona diferencial elisa ( to dpi). prcv antibody response over the course of the experimental inoculation. with the exception of one false-positive result reported ( dpi) by the ingezim corona diferencial elisa kit, the negative-control group remained seronegative for prcv by the three commercial elisas throughout the study. on prcv-inoculated animals, an early prcv-specific antibody response was first detected at to dpi by the three commercial elisas and lasted through dpi ( fig. g to i ). an analysis of the proportion of prcv-seropositive animals showed significant differences between elisa kits over time following prcv inoculation, regardless of the interpretation of suspect results (p Ͻ . ). the percentage of prcvseropositive animals detected by the svanovir tgev/prcv-ab elisa was significantly lower than those with both the swinecheck tgev/prcv recombinant elisa (dpi to ) and ingezim corona diferencial elisa (dpi to and ) (p Ͻ . ). no differences were found between the swinecheck tgev/prcv recombinant and ingezim corona diferencial elisas. on tgev-inoculated animals, a nonspecific prcv serum antibody response was detected by the three commercial elisas. this serologic cross-reactivity seemed to be tgev strain dependent, with a higher percentage of prcv-false-positive results in the tgev strain purdue-inoculated group ( fig. a to c) than in the tgev strain miller group ( fig. d to f). the cross-reactivity appeared more marked at early stages postexposure ( to dpi). no differences were found between elisa kits over the course of the study, except at dpi when the proportion of prcv-false-positive animals detected by the svanovir tgev/prcv-ab elisa was significantly greater (p Ͻ . ) than that by the ingezim corona diferencial elisa regardless of the interpretation of suspect results. comparative diagnostic performance of the tgev/prcv differential elisas. the analytical specificity and diagnostic sensitivity and specificity of the three commercial differential tgev/prcv elisa kits evaluated in this study are presented in table . diagnostic parameters are presented relative to the interpretation of suspect results as positive or negative. overall, the diagnostic sensitivity for the tgev miller inoculation group was higher than for the tgev purdue group, independent of the commercial elisa kit evaluated. the swinecheck tgev/prcv recombinant elisa kit showed the highest diagnostic sensitivity for antibody detection in pigs inoculated with tgev strain miller ( %), regardless of the interpretation of suspect results. the diagnostic sensitivity for the tgev strain purdue-inoculated group was higher with the svanovir tgev/prcv-ab elisa kit ( %) when suspect results were considered positive, while it was slightly higher with the ingezim corona diferencial elisa kit ( %) when suspect results were interpreted as negative. the diagnostic sensitivities for prcv antibody detection obtained with both swinecheck tgev/prcv recombinant elisa ( %) and ingezim corona diferencial elisa ( to %) were greater than that of the svanovir tgev/prcv-ab elisa kit ( %). with the exception of the ingezim corona diferencial elisa, the diagnostic sensitivity calculated for each elisa kit was not affected by the interpretation of suspect results (positive versus negative). the three commercial elisas showed % diagnostic specificity for both tgev and prcv detection, with the exception for one prcv-false-positive result reported by the ingezim corona diferencial elisa, which resulted in a diagnostic specificity of . % for prcv detection. the diagnostic specificity of any of the evaluated elisa kits was unaffected by the interpretation of suspect results. the ingezim corona diferencial elisa kit showed the highest analytical specificity (less cross-reactivity) for tgev-specific antibody detection ( . %). for all three commercial elisas, we found higher cross-reactivity to prcv within the tgev purdue inoculation group than in the tgev miller group. the highest analytical specificity for prcv was obtained with the ingezim corona diferencial elisa ( %) when suspect results were interpreted as positive or with the svanovir tgev/prcv-ab elisa kit ( %) when suspect results were interpreted as negative; however, the svanovir tgev/ prcv-ab elisa kit showed the lowest analytical specificity ( %) of the kits when suspect results were interpreted as positive. the greatest risk of introducing tgev is through the importation of pigs from endemically infected countries. seronegative pigs of all ages are susceptible to infection with tgev ( , , ) , with the route of infection typically being oral or oral-nasal ( , ) . to prevent tgev from entering a naive herd, it is important to introduce only animals from tgev-free and serologically negative herds, along with disciplined biosecurity. therefore, quarantine and testing for tgev-seronegative animals are requirements for export/import into a herd. moreover, the detection of tgev antibodies, particularly in reproductive animals, can assist in diagnosis and control of the disease. however, tgev serology is complicated by cross-reactivity with prcv ( ), an s protein deletion mutant of tgev with altered tissue tropism (nonenteropathogenic but respiratory) that circulates subclinically in most swine herds worldwide ( , , ) . this cross-reactivity may explain why a region endemic for prcv has less tgev-associated disease ( ) . porcine respiratory coronavirus does not cause significant losses in swine except for its contribution to the porcine respiratory disease complex. historically, prcv diagnosis was based on recognition of respiratory disease in growing pigs that have high antibody titers to tgev but have had no clinical enteric disease or lesions of atrophic enteritis. conventional serological tests, such as virus neutralization, indirect fluorescent antibody, agar-gel precipitation, indirect immunoperoxidase, radioimmunoprecipitation, and some elisas based on polyclonal tgev antibodies, cannot be used for differentiation between tgev and prcv, because the two viruses share antigenic determinants located on the spike (s), membrane (m), nucleoprotein (n), and envelope (e) structural proteins ( , ) . the exception is a large deletion ( amino acids in length) in the amino terminus of the prcv s protein that is responsible for the loss of hemagglutination activity ( ) . this deletion provided the opportunity to develop blocking enzyme-linked immunosorbent assays for differentiation of prcv and tgev infections and laid the basis for the commercial tgev/prcv differential elisas that are currently available ( ) ( ) ( ) ( ) . few field reports have described the use of tgev/prcv blocking elisas to differentiate antibodies to tgev and prcv and their utility at the herd level ( , , ( , , ) . specimens primarily used for tgev virus detection include feces and intestinal contents, and those for prcv detection include nasal swabs or lung homogenates. in this study, the verification and monitoring of the tgev or prcv viral shedding status were performed using rrt-pcr testing of pen-based feces and oral fluids. this process provided further information on the dynamics of viral shedding in feces versus oral fluids. viral shedding was detected in both feces and oral fluids. however, for pigs inoculated with prcv and, interestingly, for tgev purdue, virus shedding was only detected in oral fluids. this could be related to differences in pathogenicity and tissue tropism, which were beyond the scope of this study. these results indicated that oral fluids may replace feces as a more suitable specimen for direct detection and surveillance of tgev/prcv by rrt-pcr. overall, we observed differences in the test performances of the three commercial elisa kits evaluated in this study. however, the differences were more marked for the svanovir tgev/prcv-ab elisa kit, compared to either the swinecheck tgev/prcv recombinant elisa or the ingezim corona diferencial elisa, for which test performance was comparable. this could be due to differences/similarities in assay design. for instance, both the swinecheck tgev/prcv recombinant elisa and the ingezim corona diferencial elisa use a tgev recombinant s protein antigen on a plate, while the svanovir tgev/prcv-ab elisa claims to use noninfectious tgev antigen of an unknown source, which could detect antibodies to a variety of viral proteins. the way the antigen is presented may also affect assay performance. the antigen can either be bound directly to the plate wells (as in the swinecheck tgev/prcv recombinant elisa and svanovir tgev/prcv-ab elisa) or may be captured by antigen-specific antibodies immobilized on the plate (as in the ingezim corona diferencial elisa). in addition, the svanovir tgev/prcv-ab elisa is the only kit using unlabeled tgev and tgev/prcv mouse monoclonal antibodies (mabs), which requires an additional incubation step with anti-mouse horseradish peroxidase (hrp)-conjugated secondary antibody. undisclosed differences in buffer composition could also be involved in the differences in assay performance among the kits. all three elisa kits showed higher diagnostic sensitivity in the detection of anti-tgev antibodies in pigs inoculated with tgev strain miller ( . to . %) than in pigs inoculated with tgev strain purdue ( . to . %), regardless of the kit used. the differences in diagnostic sensitivity among tgev-inoculated groups could be due to strain-related differences in virulence, which may have impacted the magnitude of the immune response. indeed, in this study, pigs inoculated with tgev strain miller showed mild watery diarrhea between and dpi, while no clinical signs or viral shedding in feces were observed in the pigs inoculated with tgev purdue. the diagnostic specificity of the three commercial elisa kits was evaluated on pigs of precisely known negative porcine coronavirus immune status (i.e., the porcine coronavirus negative-control group) to rule out potential cross-reactivity with other porcine coronaviruses (i.e., pigs virologically and serologically negative for pedv, pdcov, porcine hemagglutinating encephalomyelitis virus [phev], tgev, and prcv). all three kits evaluated in this study showed excellent diagnostic specificity, ranging from % (svanovir tgev/prcv-ab elisa) to % (swinecheck tgev/prcv recombinant elisa and ingezim corona diferencial elisa). these results were consistent with previous reports in the field where a tgev/prcv blocking elisa (i.e., swinecheck and biovet) showed a diagnostic specificity of % in wild boar populations historically free from coronavirus disease ( ) . with the final goal of maximizing both diagnostic sensitivity and specificity (but understanding that there is a balance between the two parameters), diagnostic specificity is of paramount importance during tgev/prcv screening due to the impact of false-positive results on global pig trade operations. the false-positive rate of any diagnostic test is a function of the specificity of the test and the prevalence of the disease. the assessment of the selectivity of the antigen-antibody response (analytical specificity) was conducted on each of the three commercial blocking elisas using a panel of heterologous monotypic known-status-positive sera generated under experimental conditions. specifically, in this study, the analysis of the analytical specificity was circumscribed to the cross-reactivity between tgev (strains purdue and miller) and prcv. however, the potential cross-reactivity between tgev/prcv against other swine coronaviruses has previously been reported ( ) . a test was considered analytically specific for tgev or prcv when it did not react against heterologous positive sera. as with the diagnostic sensitivity, the overall analytical specificity varied among and across kits and groups of inoculation, even with intrakit variations depending upon the interpretation of suspect results. serological cross-reactivity between tgev and prcv was previously reported by use of an immunoblotting assay based on tgev/prcv structural proteins (s, m, and n) and polyclonal antisera ( ) . this study further confirms that the serological cross-reactivity between tgev and prcv is significant even when using tgev/prcv differential blocking elisas. overall, in this study, we observed a poor analytical specificity (i.e., high cross-reactivity) for prcv antibody detection in pigs exposed to tgev strain purdue ( to %), with no significant differences among kits. however, with the exception of the svanovir tgev/prcv-ab elisa ( to %), we found an acceptable analytical specificity for prcv antibody detection in pigs exposed to tgev miller using the swinecheck tgev/prcv recombinant elisa ( to %) and the ingezim corona diferencial elisa ( %). current tgev/prcv differential immunoassays are based on a blocking elisa format, which is inherently more specific than indirect elisas. in the blocking elisa format, the degree to which specific antibodies in the test serum sample prevent binding of an agent-specific mab is measured. therefore, the higher the levels of specific antibodies are in a serum sample, the lower the likelihood for cross-reactivity. in fact, the overall higher tgev antibody detection rate observed within the group inoculated with tgev strain miller correlates with the lower cross-reactivity against prcv. that would also explain the overall higher rate of serologic cross-reactivity (regardless of the elisa kit used) reported during the first few weeks postinoculation, when specific antibody levels are still low. previous reports indicated that the accuracy of the commercial tgev/prcv blocking elisas for differentiating between u.s. tgev and prcv strains was low ( , , ) . individual test results must be interpreted with caution, particularly in the event of "suspect" results. we have demonstrated that interpretation of suspect results can impact significantly the diagnostic performance of any of the commercial kits evaluated in this study, with differences in their robustness in response to changes in the interpretation of suspect results. while analytical specificity improved overall when suspect results were interpreted as negative, this often comes at the cost of diagnostic sensitivity. in the event of suspect, undetermined, or unexpected positive results, it is recommended that serology testing on paired serum samples be conducted, in which a second sample should be drawn to days after the first sample collection. paired serum samples should be tested together to maximize the diagnostic value of test results. moreover, false-positive results reported at early stages postexposure (when animals are actively shedding virus) could be confirmed by rrt-pcr on oral fluid specimens, as evidenced in this study. the presence of tgev remains a barrier to international livestock trade ( ) . the blocking elisa format alone was determined to be useful in large swine populations for the detection of tgev-infected herds. moreover, the ability to specifically detect prcv antibodies minimized the probability of false-positive tgev results and subsequent exclusion of those herds for trading. however, this study demonstrates that the serologic cross-reactivity between tgev and prcv at the individual pig level affects the accuracy of commercial blocking elisas. therefore, it is important to remember that the tgev/prcv blocking elisas should only be applied on a herd basis ( , , , ) . experimental design. "known-status" serum samples (n ϭ ) collected from pigs experimentally inoculated with tgev purdue (n ϭ ), tgev miller (n ϭ ), prcv (n ϭ ), or with culture medium (minimum essential medium [mem] life technologies, carlsbad, ca) (negative control; n ϭ ) were used to evaluate the diagnostic performance (diagnostic sensitivity and specificity) and antibody crossreactivity (analytical specificity) of the following three commercial tgev/prcv blocking elisas: . at % confluence of the cell monolayer, the maintenance medium was decanted, and the monolayer was washed twice with maintenance medium. thereafter, each flask of cells was inoculated with l of each virus mixed with postinoculation medium, i.e., mem supplemented with . % tryptose phosphate broth (sigma-aldrich) and . % yeast extract (sigma-aldrich). the cells were incubated at °c with % co for h to allow virus adsorption. after incubation, ml of postinoculation medium was added to each flask without removing viral inoculum. the flasks were incubated at °c with % co and subjected to one freeze-thaw cycle at Ϫ °c when % cytopathic effect (cpe) was observed. cell debris was removed by centrifugation at , ϫ g for min at °c, and the virus content was harvested by collecting the supernatant. virus titration was performed on confluent st cell monolayers grown in -well plates (costar; corning) using a method described elsewhere ( ) . animals. the animal study was conducted at the iowa state university (isu) livestock infection disease isolation facility (lidif) with the approval of the iowa state university office for responsible research. forty-eight -week-old conventional pigs were acquired from a commercial wean-to-finish farm with no history of porcine coronavirus infection. during the herd prescreening process and prior to the beginning of the study, fecal and nasal swabs from all animals were tested for tgev, prcv, porcine hemagglutinating encephalomyelitis virus (phev), porcine epidemic diarrhea virus (pedv), and porcine delta coronavirus (pdcov) by real-time reverse transcription-pcr (rrt-pcr) assays. serum samples were tested for all of the pathogens listed above using antibody-based methods, as described elsewhere ( ) ( ) ( ) . upon arrival, all animals were ear-tagged and randomly assigned to one of four inoculation groups (n ϭ per group in the tgev miller, tgev purdue, prcv, and negative-control groups). pigs within each group were housed in pens of pigs in the same room. each pen was equipped with nipple drinkers, and pigs were fed twice daily with an antibiotic-free commercial diet (heartland co-op, west des moines, ia, usa). details regarding viral dose and route of inoculation for each group are presented in table . the infectious dose used for each virus was previously determined in a pilot study (data not shown) with the objective of stimulating a humoral response effectively. pigs were evaluated clinically twice daily throughout the study. the tgev and prcv infectious status of every pig was established and monitored throughout the study by rrt-pcr tests on fecal and oral fluid samples and elisa-based tests on serum samples. sample collection. blood samples were collected on Ϫ , , , , , , , , , , and days postinoculation (dpi) from the jugular vein or cranial vena cava using a single-use blood collection system (becton dickinson, franklin lakes, nj) and serum separation tubes (kendall, mansfield, ma). serum was separated by centrifugation at , ϫ g for min, aliquoted into -ml cryogenic tubes (greiner bio-one gmbh, frickenhausen, germany), and stored at Ϫ °c until use. floor fecal samples were collected daily from each pen ( pigs per pen) within each inoculation group ( pens per group) from Ϫ to dpi. approximately ml of feces was placed in a -ml cryogenic tube (bd falcon). fecal samples ( l) from each group on the same day postinoculation were pooled into a -ml cryogenic tube (bd falcon) at the end of the study for rrt-pcr testing. oral fluid samples were collected from each pen within each inoculation group twice a day ( : a.m. and : p.m.) from Ϫ to dpi. in brief, -strand . -cm % cotton rope (web rigging supply, inc., carrollton, ga) was hung from a bracket fixed to one side of each pen for min, during which time the pigs chewed on and interacted with the rope. after min, the wet end of the rope was severed, sealed in a plastic bag, and then passed through a clothes wringer (dyna-jet, overland park, ks). the oral fluid accumulated in the bottom of the bag was decanted into -ml conical tubes (corning), aliquoted into -ml cryogenic tubes (greiner bio-one gmbh), and stored at Ϫ °c. real-time reverse transcription-pcr. feces and oral fluid samples were submitted to the isu veterinary diagnostic library (vdl) for porcine coronavirus screening by rrt-pcr. transmissible gastroenteritis virus-specific spike (s) and tgev/prcv-specific nucleocapsid (n) genes were targeted and amplified by dually labeled probe-based rrt-pcr. in brief, sample rna was extracted and eluted using the ambion magmax viral rna isolation kit (life technologies) and a kingfisher magnetic particle processor (thermo fisher scientific) following the procedures provided by the manufacturers. the tgev s gene and the tgev/prcv n gene-based rrt-pcr were modified from previous procedures ( ) and performed routinely at the isu-vdl using the taqman fast -step mastermix (thermo fisher scientific). the rt-pcrs were conducted on an abi fast instrument (life technologies) as follows: °c for min, °c for s, °c for s ( cycles), and °c for s. the rrt-pcr results were analyzed using an automatic baseline, with a threshold value of . . a quantification cycle (c t ) value of Ͻ was considered positive for both s and n gene-based rrt-pcr. samples were considered positive for tgev when both s and n genes were rrt-pcr positive. for prcv, samples were considered positive when the s gene rrt-pcr was negative and the n gene rrt-pcr was positive. transmissible gastroenteritis virus and prcv c t data were reported as "adjusted c t ," calculated as shown in the following equation: adjusted c t ϭ ͑ cutoff c t value Ϫ sample c t value ͒ tgev/prcv differential blocking elisas. serum samples were tested for tgev-and prcv-specific antibodies using the following three commercially available tgev/prcv differential blocking elisa kits: (i) swinecheck tgev/prcv recombinant (biovet, canada), (ii) ingezim corona diferencial (ingenasa, spain), and (iii) svanovir tgev/prcv-ab (svanova, sweden). all assays were performed according to the manufacturers' instructions. the pig serum samples and controls were first added to the tgev antigencoated wells for the first incubation step. in the ingezim corona diferencial elisa, plate wells are coated with a recombinant tgev s protein which is captured by a specific monoclonal antibody (mab). the swinecheck tgev/prcv recombinant elisa is also based on a recombinant tgev s protein but directly coated on plate wells, while the svanovir tgev/prcv-ab elisa uses noninfectious tgev antigen (source unknown) as a coating antigen. if anti-tgev or anti-prcv antibodies are present in the test sample, they will bind to the viral (tgev) antigen in the wells and block the antigenic sites. in contrast, if anti-tgev or anti-prcv antibodies are absent in the test sample, these sites will remain free. then, after a washing step to eliminate unbound antibodies, a horseradish peroxidase (hrp)-conjugated mouse anti-tgev mab targeting the n-terminal region of the s glycoprotein that is deleted in prcv, or an anti-tgev/prcv mab that binds to conserved regions of both tgev and prcv, is added into the first well (odd column) or second well (even column), respectively, and attaches to specific free sites of the virus. thus, when antibodies in the test sample occupy the binding sites on the antigen, the binding of the conjugated mabs is blocked. if anti-tgev or anti-prcv antibodies are absent in the test sample, these sites will remain free. the amount of antibody in the test sample that is bound to the tgev antigen is inversely proportional to the intensity of the color. antibody elisa results were expressed as the percentage of inhibition (swinecheck tgev/prcv and svanovir tgev/prcv-ab) or optical density (ingezim corona diferencial) and interpreted (positive, negative, or suspect/inconclusive) for tgev and prcv according to the manufacturers' instructions. the diagnostic sensitivity, diagnostic specificity, and analytical specificity of the three commercial elisas were calculated based on the true status of the samples compared to the specific detection of tgev (miller and/or purdue) and/or prcv antibodies. specifically, porcine coronavirus-negative serum samples (n ϭ ) were used to estimate diagnostic specificity for the three elisa kits. serum samples positive for tgev (purdue and miller strains) (n ϭ ) collected between and dpi and prcv-positive samples (n ϭ ) collected between and dpi were used to calculate the time of detection, antibody detection over time, and the overall diagnostic sensitivity of the three elisa kits for tgev and prcv ab detection, respectively. a transmissible gastroenteritis in pigs molecular basis of transmissible gastroenteritis virus epidemiology porcine coronaviruses isolation of a porcine respiratory, non-enteric coronavirus related to transmissible gastroenteritis porcine respiratory coronavirus related to transmissible gastroenteritis virus polymicrobial respiratory disease in pigs respiratory and fecal shedding of porcine respiratory coronavirus (prcv) in sentinel weaned pigs and sequence of the partial s-gene of the prcv isolates development of a reverse transcription-nested polymerase chain reaction assay for differential diagnosis of transmissible gastroenteritis virus and porcine respiratory coronavirus from feces and nasal swabs of infected pigs molecular characterization and pathogenesis of transmissible gastroenteritis coronavirus (tgev) and 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between tgev and prcv challenge with transmissible gastroenteritis virus of pigs immune after infection with the porcine respiratory coronavirus diseases of swine sero-surveillance of transmissible gastroenteritis virus (tgev) and porcine respiratory coronavirus (prcv) in south korea genetic evolution and tropism of transmissible gastroenteritis coronaviruses tgev corona virus orf encodes a membrane protein that is incorporated into virions transmissible gastroenteritis coronavirus, but not the related porcine respiratory coronavirus, has a sialic acid (nglycolylneuraminic acid) binding activity seroprevalence of porcine respiratory coronavirus in selected korean pigs field validation of a commercial blocking elisa to differentiate antibody to transmissible gastroenteritis virus (tgev) and porcine respiratory coronavirus and to identify tgev-infected swine herds detection of transmissible gastroenteritis virus by rt-pcr and differentiation from porcine respiratory coronavirus in situ hybridization technique for the detection of swine enteric and respiratory coronaviruses, transmissible gastroenteritis virus (tgev) and porcine respiratory coronavirus (prcv), in formalin-fixed paraffin-embedded tissues prevalence of and risk factors associated with viral and bacterial pathogens in farmed european wild boar antigenic relationships among porcine epidemic diarrhea virus and transmissible gastroenteritis virus strains simulation of the economic impact of transmissible gastroenteritis on commercial pig production in australia a simple method of estimating fifty per cent endpoints pathogenesis of porcine epidemic diarrhea virus isolate (us/iowa/ / ) in -week-old weaned pigs reactivity of porcine epidemic diarrhea virus structural proteins to antibodies against porcine enteric coronaviruses: diagnostic implications multiplex real-time rt-pcr for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus this work was funded in part through the iowa pork producers association through the national pork board (pork checkoff funds, grant - ; des moines, ia) and the iowa state university veterinary diagnostic laboratory (ames, ia). data analysis. analyses were conducted using commercial statistical software (sas version . . ; microsoft corporation, usa). the percentage of results for tgev/prcv elisa were interpreted (positive, negative, or suspect) according to the manufacturer's recommendations. the percentage of seropositive animals per day postinoculation detected by each kit were analyzed in two ways, by considering suspect results to be positive or by considering suspect results to be negative. significant differences (p Ͻ . ) in elisa-positive rates relative to tgev (purdue or miller strains) and prcv were calculated using pearson's chi-square test or fisher's exact test. the diagnostic sensitivity and specificity of the three commercial elisas were evaluated based on experimental serum samples of precisely known porcine coronavirus immune status (i.e., samples collected on Ͻ dpi were negative, and serum samples collected on Ͼ dpi were positive). analytical specificity (bidirectional cross-reactivity) between tgev versus prcv was evaluated using samples (n ϭ ) collected between and dpi from animals inoculated with tgev strain purdue (n ϭ ), tgev strain miller (n ϭ ), and prcv (n ϭ ) in each given case. figures were created using commercial graphics software (sigmaplot version . ; systat software, inc.). the funders had no role in the study design, data collection and interpretation, or the decision to submit the work for publication.we declare no conflicting interests with respect to authorship or the publication of this article. key: cord- -bvh o r authors: galasiti kankanamalage, anushka c.; kim, yunjeong; damalanka, vishnu c.; rathnayake, athri d.; fehr, anthony r.; mehzabeen, nurjahan; battaile, kevin p.; lovell, scott; lushington, gerald h.; perlman, stanley; chang, kyeong-ok; groutas, william c. title: structure-guided design of potent and permeable inhibitors of mers coronavirus cl protease that utilize a piperidine moiety as a novel design element date: - - journal: european journal of medicinal chemistry doi: . /j.ejmech. . . sha: doc_id: cord_uid: bvh o r abstract there are currently no approved vaccines or small molecule therapeutics available for the prophylaxis or treatment of middle east respiratory syndrome coronavirus (mers-cov) infections. mers-cov cl protease is essential for viral replication; consequently, it is an attractive target that provides a potentially effective means of developing small molecule therapeutics for combatting mers-cov. we describe herein the structure-guided design and evaluation of a novel class of inhibitors of mers-cov cl protease that embody a piperidine moiety as a design element that is well-suited to exploiting favorable subsite binding interactions to attain optimal pharmacological activity and pk properties. the mechanism of action of the compounds and the structural determinants associated with binding were illuminated using x-ray crystallography. proteins, s (spike glycoprotein), e (envelope protein), m (membrane glycoprotein), and n (nucleocapsid protein), which play a critical role in virion-cell receptor binding, replication and virion assembly, are located at the end of the genome [ , ] . coronavirus entry is initiated by the binding of the spike protein (s) to cell receptors, specifically, dipeptidyl peptidase (ddp ) and angiotensin converting enzyme (ace ) for mers-cov and sars-cov, respectively [ e ] . entry into cells requires host proteases for cleavage at two sites in the s protein, in the case of most cov [ , ] translation of the genomic mrna of orf a yields polyprotein pp a, while a second polyprotein (pp b) is the product of a ribosomal frame shift that joins orf a together with orf b. orf a encodes a papain-like cysteine protease (plpro) and a c-like cysteine protease ( clpro). polyproteins pp a and pp b are processed by clpro ( cleavage sites) and plpro ( cleavage sites) resulting in sixteen mature nonstructural proteins, including rna-dependent rna polymerase (rdrp) and helicase, which play important roles in the transcription and replication of coronaviruses (fig. ). both proteases are essential for viral replication, making them attractive targets for drug development [ , , e ] . mers-cov clpro is a chymotrypsin-like cysteine protease having a catalytic cys -his dyad and an extended binding site [ e ] . the protease displays a stringent primary substrate specificity for a p gln residue [ ] and has a strong preference for a p leu residue. the p residue side chain is oriented toward the solvent while the s subsite is shallow, preferring a small hydrophobic p residue (ala). functional and structural studies have delineated the similarities between the clpro of coronaviruses that can be exploited in the design of broad-spectrum inhibitors [ ] . we have recently reported the first demonstration of clinical efficacy of a coronavirus protease inhibitor (a dipeptidyl aldehyde bisulfite adduct inhibitor designated gc ) [ , ] . specifically, administration of gc to cats infected with fipv, a coronavirus that is % fatal in cats, reversed the progression of fatal fip and resulted in clinical remission in a majority of animals (> %). since fip disease progression is quite rapid and its pathogenesis primarily immune-mediated, features shared by mers-cov, we hypothesized that a viral protease inhibitor could reverse the pathogenesis of mers-cov in affected hosts. interrogation of this hypothesis entailed, as a first step, the design of a new and versatile class of peptidomimetic inhibitors of mers-cov cl protease. we describe herein the structure-guided design of inhibitors of mers-cov clpro that embody a piperidine moiety as a novel design element, as well as pertinent structural and biochemical studies. these inhibitors were also examined against other coronaviruses, including sars-cov, fipv and mhv to evaluate the spectrum of activity against multiple coronaviruses. the structure-guided design of inhibitor (i) encompassed the following steps: (a) we first determined a high resolution x-ray crystal structure of mers-cov clpro in complex with gc ( fig. /panel a) . examination of the active site of the complex revealed that the aldehyde bisulfite adduct had reverted to the precursor peptidyl aldehyde, which subsequently formed a tetrahedral hemi-thioacetal upon reaction with the active site cys . notably, the electron density at this stereocenter was consistent with the formation of both r and s enantiomers at the covalent binding site (also observed for the other structures described in the following sections). the structure reveals a network of backbone hydrogen bonds which ensure correct positioning of the inhibitor to the active site, as well as two critical hydrogen bonds with the p gln surrogate [ ] side chain. the inhibitor p leu side chain is ensconced in the hydrophobic s subsite of the enzyme. importantly, the structure shows a hydrophobic-driven interaction between the benzyl group of the inhibitor and the g-lactam ring of the gln surrogate side chain; (b) based on the forgoing, we reasoned that extending the "cap" would allow the inhibitor to assume an extended conformation and orient the phenyl ring toward the hydrophobic s pocket of the enzyme. validation of this idea was obtained by synthesizing extended inhibitor gc and determining a high resolution x-ray crystal structure of the mers-cov clpro:gc complex ( fig. /panel b) . the m-cl phenethyl side chain is clearly shown to occupy the hydrophobic s subsite. in addition to an array of h-bonds with gln , glu , and gln and the backbone of the inhibitor, which serve to correctly position the inhibitor at the active site, the inhibitor interacts with the s , s and s subsites, but not the s subsite; (c) we hypothesized that the attachment of a piperidine ring to the peptidyl component would yield a structurally novel peptidomimetic (i) capable of ( ) orienting recognition elements r and r in a correct vector relationship for optimal interactions with the s and s subsites, ( ) rendering a dipeptidyl inhibitor equivalent to a tetrapeptidyl inhibitor with potentially diminished pk liabilities and, ( ) providing a flexible means for the structure-guided parallel optimization of admet/pk and physicochemical properties using diversity sites r and r in inhibitor (i) (fig. ) . in summary, the piperidine-based design strategy is a hitherto unrecognized effective means of rendering a dipeptidyl inhibitor equivalent to a tetrapeptidyl inhibitor capable of engaging in optimal binding interactions with all four s -s subsites but which, however, is anticipated to display diminished pk liabilities due to its reduced peptidyl character. furthermore, the aforementioned piperidine-based design strategy has wide applicability and can be extended to any protease with an extended binding site. preliminary evidence in support of this approach is provided by the results of enzyme and cell-based screening of derivatives of (i) (tables and ) , as well as the results of structural studies (vide infra) (see table ). the synthesis of final compounds (a-f) and (a-f) is outlined in scheme . -boc- -piperidinone was reacted with different grignard reagents to yield the corresponding -boc- -piperidinol derivatives ( c and e). refluxing (l) leucine methyl ester hydrochloride with trichloromethyl chloroformate yielded the isocyanate which was reacted with ( c and e, or commercially-available nsubstituted -piperidinol a) to form the corresponding carbamate adducts ( a, c and e) that were hydrolyzed to the corresponding acids ( a, c and e) with lithium hydroxide in aqueous thf. subsequent coupling with glutamine surrogate methyl ester hydrochloride afforded the desired dipeptidyl esters ( a, c and e) which were either treated with lithium borohydride directly or were first treated with dry hcl in dioxane followed by reaction with an alkyl sulfonyl chloride or alkyl chloroformate, to yield esters ( b, d and f) prior to reduction with lithium borohydride, to yield alcohols (a-f). dess-martin oxidation, followed by flash chromatography, yielded pure aldehydes (a-f). the enantiomeric purity of the aldehydes was consistently high, with the amount of epimerized aldehyde ranging between and %. the corresponding bisulfite adducts (a-f) were readily obtained as white solids by stirring the aldehydes with sodium bisulfite in an ethyl acetate/ water mixture. the synthesized compounds are listed in table . the inhibitory activity of the synthesized compounds against clpro of mers-cov, sars-cov or fipv, and the antiviral activity of two representative compounds (compounds a and c) in a cellbased system including mers-cov, fipv and mhv were evaluated as described in the experimental section. the ic , ec , and cc values, are listed in tables and these are the average of at least two determinations. it is evident that derivatives of (i) function as highly potent inhibitors of all tested coronaviruses in enzyme (table ) and cell based assays (table and fig. ). more importantly, representative aldehyde bisulfite adduct compounds a and c display potent inhibition toward mers-cov in both enzyme and cell-based systems, with low cytotoxicity (cc > mm) ( table and fig. ). for example, compound a has a selectivity index (si ¼ cc /ec ) of > . with the exception of compounds e- e, the aldehyde and aldehyde bisulfite adducts were found to have comparable in vitro potency toward mers-cov clpro. furthermore, pharmacological activity was found to be dependent on the nature of the r group (compounds e- e are -fold less active toward mers-cov clpro than compounds a-d, a-d and f- f). in order to establish the mechanism of action of (i), as well as obtain structural information that can be used to guide the optimization of pharmacological activity, the high resolution x-ray crystal structures of several derivatives of (i) bound to mers-cov clpro were determined, including the cocrystal structure of the mers-cov clpro:inhibitor c complex (fig. a) . the formation of a tetrahedral adduct via the reaction of the aldehyde, generated from aldehyde bisulfite adduct c under the crystallization conditions used [ , ] , with the active site cysteine (cys ) is clearly evident, confirming the mechanism of action of (i). inspection of the structure reveals the presence of prominent electron density consistent with the structure of inhibitor c; however, the n-bocpiperidinyl moiety was disordered. the position and orientation of the benzyl group suggest that the piperidine ring is likely projecting toward the s subsite. inhibitor c is bound to the active site of the enzyme via a network of backbone h-bonds with gln , gln , and glu (fig. b ). additionally, a h-bond with his serves to stabilize the hemi-thioacetal tetrahedral adduct. also clearly evident are three critical h-bonds involving the p gln surrogate ring oxygen and nitrogen with glu , his and phe . the h-bonding interactions are near identical to those of inhibitor gc (fig. /panel b) . the structural complementarity of inhibitors c and gc is also evident in the electrostatic surface representation of the enzyme with the two inhibitors nestled in the active site (fig. ) . the cocrystal structure of the mers-cov clpro:aldehyde bisulfite adduct e complex also showed that, under the crystallization conditions used, the aldehyde bisulfite adduct reverted to the precursor aldehyde, which subsequently formed a tetrahedral adduct with the active site cysteine (cys ) (fig. a ). the piperidinyl moiety was disordered and consequently its precise location could not be discerned. however, inhibitor e is engaged in the same h-bonding interactions as inhibitor c (fig. b ). mers-cov constitutes a global public health concern. there are currently no licensed vaccines or antiviral drugs for the prevention and treatment of coronavirus infections. we disclose herein for the first time the design and utilization of a general class of piperidinebased peptidomimetic inhibitors of coronavirus cl proteases. attachment of the piperidine moiety to a dipeptidyl component permits the resultant hybrid inhibitor to engage in favorable binding interactions with the s and s subsites of the enzyme. more importantly, the approach disclosed herein can be extended to other proteases of medical relevance. finally, the disclosed compounds potently inhibit mers-cov, and their mechanism of action and mode of binding to mers-cov cl protease have been illuminated using x-ray crystallography. reagents and dry solvents were purchased from various chemical suppliers (aldrich, acros organics, chem-impex, tci america, and bachem) and were used as obtained. silica gel ( e mesh) used for flash chromatography was purchased from sorbent technologies (atlanta, ga). thin layer chromatography was performed using analtech silica gel plates. visualization was accomplished dropwise under a n atmosphere to a solution of -boc- piperidinone ( mmol) in dry thf ( ml) in an ice bath kept at c. the reaction mixture was stirred for h at room temperature under a n atmosphere while monitoring completion of the reaction by tlc. the reaction mixture was diluted with water ( ml) and the solution was acidified to ph~ using % hydrochloric acid. the solvent was removed on the rotary evaporator and the residue was extracted with ethyl acetate ( ml) and the layers separated. the organic layer then washed with brine ( ml) and dried over anhydrous sodium sulfate, filtered and concentrated to yield a colorless oily product which was purified by flash chromatography to yield c and e. . . . synthesis of (l) leucine methyl ester isocyanate (l) leucine methyl ester hydrochloride ( mmol) was placed in a dry -ml rb flask and then dried overnight on the vacuum pump. the flask was flushed with nitrogen and dry dioxane ( ml) was added followed by trichloromethyl chloroformate ( . g, mmol), and the reaction mixture was refluxed for h. the solvent was removed on the rotary evaporator and the residue was vacuum distilled to yield pure isocyanate as a colorless oil [ , ] . table crystallographic data for mers-cov clpro in complex with compounds gc , gc , c and e. mers-cov clpro: gc mers-cov clpro: compound c mers-cov clpro: compound e hkl. c r factor ¼ Ʃ hkl jjf obs (hkl) j -jf calc (hkl) jj/Ʃ hkl jf obs (hkl)j; rfree is calculated in an identical manner using % of randomly selected reflections that were not included in the refinement. d r meas ¼ redundancy-independent (multiplicity-weighted) r merge. [ , ] r pim ¼ precision-indicating (multiplicity-weighted) r merge. [ , ] . e cc / is the correlation coefficient of the mean intensities between two random half-sets of data [ , ] . . . . synthesis of substituted piperidine-derived carbamates a, c and e. general procedure a solution of substituted or unsubstituted -boc- -piperidinol ( c, e or a) ( mmol) in dry acetonitrile ( ml) was treated with triethylamine ( . g, mmol) followed by the amino acid methyl ester isocyanate ( mmol). the resulting solution was refluxed for h and then allowed to cool to room temperature. the solution was concentrated and the residue was taken up in ethyl acetate ( ml). the organic layer was washed with % hcl (  ml) and brine ( ml). the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, leaving compounds a, c and e as colorless oils. . . . synthesis of acids a, c and e. general procedure a solution of ester ( a, c or e) ( mmol) in tetrahydrofuran ( ml) was treated with m lioh ( ml). the reaction mixture was stirred for h at room temperature and the disappearance of the ester was monitored by tlc. most of the solvent was evaporated off and the residue was diluted with water ( ml). the solution was acidified to ph~ using % hydrochloride acid ( ml) and the aqueous layer was extracted with ethyl acetate (  ml). the combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated to yield the corresponding compounds a, c and e as colorless oils. . . . synthesis of compounds a, c and e. general procedure edci ( . g, . mmol, . eq) and hobt ( . g, . mmol, . eq) were added to a solution of compound ( a, c or e) ( mmol) in dry dmf ( ml) and the mixture was stirred for min at room temperature. in a separate flask, a solution of deprotected glutamine surrogate ( . g, mmol) in dmf ( ml) cooled to e c was treated with diisopropylethylamine (diea) ( . g, mmol, eq), stirred for min, and then added to the reaction mixture containing the acid. the reaction mixture was stirred for h while monitoring the reaction by tlc. the solvent was removed and the residue was partitioned between ethyl acetate ( ml) and % citric acid (  ml). the layers were separated and the ethyl acetate layer was further washed with saturated aqueous nahco ( ml), followed by saturated nacl ( ml). the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to yield a yellow-colored oily product. purification by flash chromatography yielded esters a, c and e as white solids. . . . synthesis of compounds b, d and f. general procedure m hcl in dioxane ( ml) was added to a solution of compound ( a, c and e) ( mmol) in dry dcm ( ml) and the mixture was stirred for h at room temperature. the solvent was removed and the residue was dried under high vacuum for h before the product was dissolved in dry thf ( ml). an appropriate alkyl sulfonyl chloride or alkyl chloroformate derivative ( mmol/ . eq) was added to the solution with stirring. the reaction mixture was stirred for h at room temperature and the residue was dissolved in ethyl acetate ( ml) and washed with % hcl (  ml). the ethyl acetate layer was further washed with saturated nacl ( ml). the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to yield a crude product. purification by flash chromatography yielded the corresponding esters b, d and f as white solids. lithium borohydride ( m in thf, . ml, mmol) was added dropwise to a solution of ester ( or ) ( mmol) in anhydrous thf ( ml), followed by absolute ethyl alcohol ( ml) and the reaction mixture was stirred at room temperature overnight. the reaction mixture was then acidified by adding % hcl and the ph adjusted tõ . removal of the solvent left a residue which was taken up in ethyl acetate ( ml). the organic layer was washed with brine ( ml), dried over anhydrous sodium sulfate, filtered, and concentrated to yield compounds (a-f) as white solids. compound (a-f) ( mmol) was dissolved in anhydrous dichloromethane ( ml) under a nitrogen atmosphere and cooled to c. dess-martin periodinane reagent ( . g, . mmol, . eq) was added to the reaction mixture with stirring. the ice bath was removed and the reaction mixture was stirred at room temperature for h (monitoring by tlc indicated complete disappearance of the starting material). a solution of % aqueous sodium thiosulfate ( ml) was added and the solution was stirred for another min. the aqueous layer was removed and the organic layer was washed with % aqueous sodium thiosulfate ( ml), followed by saturated aqueous sodium bicarbonate (  ml), water (  ml) and brine ( ml). the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. the yellow residue was purified by flash chromatography (silica gel/methylene chloride/ ethyl acetate/methanol) to yield a white solid (a-f). absolute ethanol ( ml) was added to a solution of aldehyde (a-f) ( mmol) in dry ethyl acetate ( ml) with stirring, followed by a solution of sodium bisulfite ( mg; mmol) in water ( ml) and the reaction mixture was stirred for h at c. the reaction mixture was allowed to cool to room temperature and then vacuum filtered. the solid was thoroughly washed with absolute ethanol and the filtrate was dried over anhydrous sodium sulfate, filtered, and concentrated to yield a yellowish oil. the oily product was treated with ethyl ether (  ml) to form a white solid. the white solid was stirred with ethyl ether ( ml) and ethyl acetate ( ml) for min. careful removal of the solvent using a pipette left the corresponding aldehyde bisulfite adducts (a-f) as white solids. the fret protease assay was performed by preparing stock solutions of the substrate (dabcyl-ktsavlq/sgfrkme-edans derived from the cleavage sites on the viral polyproteins of sars-cov) and inhibitor in dmso and diluting into assay buffer which was comprised of mm hepes buffer, ph , containing nacl ( mm), edta ( . mm), glycerol ( %), and mm dithiothreitol (dtt). the expression and purification of the clpro of mers-cov, sars-cov or fipv was performed by a standard method described previously by our lab [ , ] . the protease ( clpro of mers-cov, sars-cov or fipv) was mixed with serial dilutions of each compound or with dmso in ml of assay buffer and incubated at c for min, followed by the addition of ml of assay buffer containing substrate. fluorescence readings were obtained using an excitation wavelength of nm and an emission wavelength of nm on a fluorescence microplate reader (flx ; biotec, winoosk, vt) h following the addition of substrate. relative fluorescence units (rfu) were determined by subtracting background values (substrate-containing well without protease) from the raw fluorescence values, as described previously [ ] . the dosedependent fret inhibition curves were fitted with a variable slope by using graphpad prism software (graphpad, la jolla, ca) in order to determine the ic values of the inhibitors. the effects of compounds a and c on the replication of mers-cov, fipv or mhv-a were examined in vero , crfk or ccl . cells, respectively [ ] . briefly, confluent and semi-confluent cells were infected at an moi of . pfu/cell. following adsorption, cells were incubated with medium containing dmso (< . %) or each compound (up to mm) for h. after incubation, viral titers were determined with a tcid (fipv or mhv) or plaque assay (mers-cov). ec values were determined using graphpadprism software [ ]. the cytotoxic dose for % cell death (cc ) for compounds a and c was determined in vero , crfk or ccl . cells. confluent cells grown in -well plates were treated with various concentrations ( e mm) of each compound for h. cell cytotoxicity was measured by a cytotox nonradioactive cytotoxicity assay kit (promega, madison, wi). the in vitro therapeutic index was calculated by dividing the cc by the ec . purified mers-cov clpro, in mm nacl, mm tris ph . , was concentrated to mg/ml ( . mm). stock solutions of mm gc , gc , compound c or compound e were prepared in dmso and the complex with mers clpro was prepared by mixing the concentrated protein supplemented with mm compound and incubating overnight at c. all crystallization experiments were conducted using compact (rigaku reagents) sitting drop vapor diffusion plates at c using equal volumes of protein and crystallization solution equilibrated against ml of the latter. crystals of mers clpro in complex with gc , compound c and compound e that displayed a prismatic morphology were obtained from the index ht screen (hampton research) condition g ( % (w/v) peg , mm bis-tris ph . , mm mgcl ) in e days. crystals of the gc complex were obtained from the index ht screen (hampton research) condition e ( % (v/v) peg mme, mm bis-tris ph . , mm cacl ). samples were transferred to a fresh drop containing % crystallant and % (v/v) peg before storing in liquid nitrogen. x-ray diffraction data were collected at the advanced photon source beamline -id using a dectris pilatus m pixel array detector. intensities were integrated using xds [ , ] using autoproc [ ] and the laue class analysis and data scaling were performed with aimless [ ] , which suggested that the highest probability laue class was /m and space group c . structure solution was conducted by molecular replacement with phaser [ ] using a previously determined isomorphous structure of mers clpro (pdb: rsp [ ] ) as the search model. structure refinement and manual model building were conducted with phenix [ ] and coot [ ] , respectively. disordered side chains were truncated to the point for which electron density could be observed. structure validation was conducted with molprobity [ ] and figures were prepared using the ccp mg package [ ] . coordinates and structure factors for the mers clpro inhibitor complexes were deposited to the worldwide protein data bank (wwpdb) with the accession codes: wkj (gc ), wkk (gc ), wkl (inhibitor c) and wkm (inhibitor e). coronaviridae in field's virology coronaviruses: important emerging human pathogens sars and mers: recent insights into emerging coronaviruses update on human rhinovirus and coronavirus infections middle east respiratory syndrome and severe acute respiratory syndrome pathogenesis of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease middle east respiratory syndrome coronaviruses: drug discovery and therapeutic options antiviral drugs specific for coronaviruses in preclinical development mers-cov vaccine candidates in development: the current landscape role of the spike glycoprotein of human middle east syndrome coronavirus (mers-cov) in virus and syncytia formation middle east respiratory syndrome infection mediated by the transmembrane serine protease tmprss host cell entry of middle east respiratory distress syndrome coronavirus after two-step, furin-mediated activation of the spike protein an overview of severe acute respiratory distress syndrome-coronavirus 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data processing and analysis with the autoproc toolbox an introduction to data reduction: space-group determination, scaling and intensity statistics phaser crystallographic software ligand-induced dimerization of middle east respiratory syndrome (mers) coronavirus nsp protease ( clpro): implications for nsp regulation and the development of antivirals phenix: a comprehensive python-based system for macromolecular structure solution features and development of coot molprobity: all-atom structure validation for macromolecular crystallography developments in the ccp molecular graphics project scaling and assessment of data quality improved r-factors for diffraction data analysis in macromolecular crystallography global indicators of x-ray data quality linking crystallographic model and data quality resolving some old problems in protein crystallography towards automated crystallographic structure refinement with phenix.refine supplementary data related to this article can be found at https://doi.org/ . /j.ejmech. . . . key: cord- -nvwnbp j authors: gaspard, philippe; mosnier, anne; simon, loic; ali-brandmeyer, olivia; rabaud, christian; larocca, sabrina; heck, béatrice; aho-glélé, serge; pothier, pierre; ambert-balay, katia title: gastroenteritis and respiratory infection outbreaks in french nursing homes from to : morbidity and all-cause lethality according to the individual characteristics of residents date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: nvwnbp j background: gastroenteritis (ge) and respiratory tract infection (rti) outbreaks are a significant issue in nursing homes. this study aimed to describe ge and rti outbreaks with infection and all-cause lethality rates according to the individual characteristics of nursing home residents. methods: clinical and virological surveillance were conducted ( to ). virus stratifications for the analysis were: outbreaks with positive norovirus or influenza identifications (respectively nov+ or flu+), episodes with no nov or influenza identification or testing (respectively nov- or flu-). associations between individual variables (sex, age, length of stay (los), autonomy status) and infection and lethality rates were tested with univariate and mantel-haenszel (mh) methods. results: ge outbreaks and rti oubreaks (total outbreaks) were recorded involving respectively and residents. in univariate analysis, higher infection rates and age were associated in nov+, nov-, and flu+ contexts, and lower infection rates were associated with longer stays (nov+ and nov-). in mh stratified analysis (virus, sex (female/male)) adjusted for los (< or ≥ years), the odds of being infected remained significant among older residents (≥ years): nov+/male (odds ratio (or(mh)): . , % confidence interval (ci): . – . ) and flu+/female and male (respectively or(mh): . , ci: . – . and . , ci: . – . ). in univariate analysis, lower autonomy status (nov+, flu+ and flu-) and increased age (flu+) were associated with higher lethality. in mh adjusted analysis, significant or(age) adjusted for autonomy was: flu+/ ≥ years compared with < years, . ( . – . ) and or(autonomy) adjusted for age for the more autonomous group (compared with the less autonomous group) was: flu+, . ( . – . ); flu-, . ( . , . ). conclusion: the residents of nursing homes are increasingly elderly and dependent. the specific infection and lethality risks according to these two factors indicate that surveillance and infection control measures are essential and of high priority. introduction gastroenteritis (ge) and respiratory tract infection (rti) outbreaks represent a significant burden of illness in nursing homes. viruses cause the majority of these outbreaks, and noroviruses and influenza viruses are the most common pathogens [ , ] . previous studies have suggested that viral respiratory infections and norovirus outbreaks are a common cause of hospitalization or death, particularly among elderly individuals [ ] [ ] [ ] . the impact of outbreaks has been described in terms of both frequency and epidemiology, but little is known about infection rates and all-cause lethality in ge and rti nursing home outbreaks in relation to the individual characteristics of the residents [ ] . the residents of these institutions are increasingly elderly and dependent, and the impact of this trend on the seasonal outbreak burden requires in-depth investigation. the results of studies focused on this issue could yield valuable information for nursing homes, allowing them to adapt their infection control strategies, in particular for improved assessment of infection risk. our objective was to describe ge and rti infection and all-cause lethality rates according to the individual characteristics of nursing home residents (sex, age, length of stay, autonomy status), and to identify specific susceptibility patterns related to these types of viral outbreaks in these facilities. the present study explored outbreaks in sites ( units with geriatric nursing home activities for a total of beds) caring for dependent people in southern alsace (an area in northeastern france). data were collected between september and august [ , ] . each site was geographically independent and autonomous for social and care management. units were located within the larger sites and were defined as a place having a dedicated team at one location. during outbreaks at one site, only the residents in the units with confirmed cases were included. outbreak inclusion depended on institutional alert to the hygiene team. surveillance was done in each unit independently, and the members of staff had to inform a physician or charge nurse when two or more potential related cases of pneumonia or ge were observed within four days and when three or more cases were observed for other rti. units also had to inform the hygiene team when these threshold values were exceeded. for influenza, the first suspected case led to a local alert and the hygiene team was contacted. a practitioner from the hygiene team collected the information and evaluated the clinical signs, the virology information and the epidemiological context with the physician in the affected unit. the detected cluster was only put under surveillance if the hygiene team considered that there was a potential outbreak phenomenon. the duration of days was in relation with the national protocol with alert to the authorities when cases occurred within days [ , ] . on a local level and in addition to the clusters reported to the authorities, clusters with at least cases within a period of seven days in one unit could be recorded if they were reported to the hygiene team. because several outbreaks could potentially occur in the same unit during the surveillance period, a resident could be included repeatedly in different clusters. as a result, the observed patterns reflected the characteristics of an institutional population with longitudinal and pluriannual exposures. personal information and clinical information was collected by a practitioner from the hygiene team directly from the residents' health care records. personal information was collected for all those present the first day of the outbreak. the collected information included: month and year of birth, sex, date of arrival at the nursing home and autonomy status. the autonomy status of residents in french nursing homes is assessed using the aggir scale (autonomy gerontology groups iso-resources), which is the legal instrument for evaluating dependency in the elderly and whose primary purpose is the allocation of means and resources [ ] . with the aggir scale, autonomy is classified into iso-resource groups (gir): gir (bedridden or armchair-bound persons, mental functions seriously altered and requiring continuous presence), gir (bedridden or armchair-bound persons, mental functions not totally altered and requiring assistance in most activities of daily living, or mental functions altered with preserved ability to get around), gir (preserved mental autonomy with partially preserved motor autonomy and assistance several times a day for physical autonomy), gir (moves around the home and sometimes assistance for washing, dressing, physical activities or eating), gir (only occasional assistance for washing, meal preparation, and housework), gir (autonomy for essential tasks of daily living). in outbreaks where the influenza virus was identified, influenza vaccination status and oseltamivir prescriptions were recorded as well. a file is transmitted in the supporting information with all previous data (s data). ge was defined as the sudden onset of vomiting and/or diarrhea over a h period: (i) diarrhea � episodes, (ii) and/or vomiting � episodes, (iii) or diarrhea or vomiting < episodes with two or more other symptoms (diarrhea, vomiting, stomach ache, abdominal cramps, nausea, fever, mucus in stools) [ ] . rti presentation in older adults may be atypical, like for other acute illnesses in this age group [ ] . we used the recommended definitions for rti surveillance in geriatric units, divided in subcategories: (i) common cold syndromes or pharyngitis (at least two of the following criteria: runny nose or sneezing, stuffy nose (i.e. congestion), sore throat or hoarseness or difficulty swallowing, dry cough, swollen or tender glands in the neck (cervical lymphadenopathy)), (ii) influenza-like illness (both the following criteria must be met: fever and at least three other symptoms (chills, new headache or eye pain, myalgia or body aches, malaise or loss of appetite, sore throat, new or increased dry cough)) and (iii) lower respiratory tract infection (both of the following criteria must be met: at least two respiratory signs or symptoms (new or increased cough, new/increased sputum production, o saturation < % or reduced > % from baseline, abnormal lung examination (new or changed), pleuritic chest pain, respiratory rate � breaths/min and one or more constitutional signs/symptoms (fever, leukocytosis, confusion, acute functional decline)) [ ] [ ] [ ] [ ] . infection corresponding to one of these three subcategories was included in this study and classified as rti. for both infection types, the practitioner from the hygiene team obtained clinical information from the patient's health care records, and members of the health care team were consulted if necessary to complete any missing information. at the end of the episode (within seven days after the last identified case), case inclusion as exposed and not infected (eni) or exposed and infected (ei) was determined with a resident physician. in order to study the lethality, the presence of each infected resident was evaluated once at least days after the last case of each outbreak. each resident was followed up retrospectively during eighth -day interval (i n, n = to , total days, between the date of onset of symptoms and the fifty-sixth day of the studied period) with three different possibilities: present (alive and officially residing in the institution), lost to follow-up (alive at the date of departure but no longer residing in the institution (return home, transfer to another institution)) or death (death recorded in the health care record). the dates of death and lost to follow-up were recorded. testing for the virus was not systematic and was decided by the physicians in each institution in the presence of clinical signs. for ge, stool samples were sent to the national reference centre for gastroenteritis viruses in dijon for laboratory testing, as previously described [ ] . for rti surveillance, rapid tests were used to identify the influenza virus. the rapid immunoassay diagnosis tests used for influenza detection were: clearview exact influenza a and b (inverness medical, cologne, germany) from to and influenzatop (alldiag, strasbourg, france) from to . given the low sensitivity of influenza rapid tests, they were no longer used once the control measures had been implemented and the influenza outbreak was under control. samples were also occasionally sent to hospital laboratories or to the national reference centre for influenza viruses to detect viruses with real-time rt-pcr [ ] . most testing targeted the norovirus (nov) and influenza virus, but other tests were occasionally performed by the national reference centre for influenza viruses (rhinovirus, respiratory syncytial virus, human metapneumovirus, parainfluenza , , and , and coronavirus) and the national reference centre for enteric viruses (rotavirus, astrovirus, and adenovirus). because testing for the viruses was variable (from one institution/physician to another, not used in some outbreaks, types of virus sought) and considering the poor sensitivity of the rapid influenza tests, these two sources of data were used to define the epidemiological context of confirmed outbreaks. consequently, individual cases were included consistently in all episodes according to clinical signs and medical evaluation. when virus testing was negative, the clinical signs were recorded and medical evaluation was used as previously to classify the included residents as infected or not infected. the epidemiological context of each outbreak was defined according to whether the virus had been identified or not. one or more positive samples led to the qualification of a nov (nov+) or flu (flu+) context. the other episodes were qualified as flu or nov outbreaks with no specific identification or testing (nov-and flu-). flu and nov contexts did not eliminate other potential enteric or respiratory pathogens. sex, age, length of stay (los, in years) and autonomy status were described for all exposed residents. influenza vaccination and oseltamivir administration rates were calculated for confirmed influenza outbreaks. dichotomous or categorical variables were expressed as percentages. in univariate analysis, the categories were specific in order to obtain a precise description of the age and los variables. class intervals were years for age and one year for los. the residents classified as gir to (sometimes, occasional and no assistance) were grouped together because they were few. for the multi-level analysis with x tables, a median value was used to define the two-level age categories. for los, assessing the longest stays was necessary to identify the effect of longer exposure in a nursing home. consequently, a four-year cutoff was chosen to create the two categories. for autonomy, the two most dependent categories (gir � ) were grouped together and compared with the more autonomous categories (gir � ). the outbreak epidemiological contexts were used with the four categories: nov+, flu+, nov-and flu-. other ge or rti viruses were occasionally identified, but the number of results was too limited to develop separate analyses. however, all the results are available in the tables about the virus investigations along with nov and influenza identifications. for the different categories, infection rate (ei/exposed residents (er), in percentage) was calculated according to sex, age group, los and autonomy status. to investigate all-cause lethality and define the appropriate period for the -day monitoring (d to ), the all-cause lethality rate per -day interval (lr n /i n, n = to ) was calculated: (number of deaths during interval i n /(ei alive the first day of n th studied interval minus lost to follow-up ei during the interval i n ) � ). seeing as successive clusters could occur within the same site, potentially influencing allcause lethality, the serial interval in days (si d ) was calculated. the si d was the time period between the onset of symptoms of the last case in initial outbreak (n) and the onset of symptoms of the first case in the following outbreak (n+ ). investigations were performed when si d was shorter or equal to the length of the previous d to and the following parameters were evaluated for these specific situations: number of episodes, residents infected in both outbreaks, and death among the identified individuals. finally, according to the death rate and the impact of successive outbreaks, the number of -day intervals (n.i n ) to take into account was defined, and the all-cause lethality rate was analyzed during these periods (i to n th .i n or d to � n ). all-cause lethality rates were calculated with the following formula: (number of deaths from d to � n /(ei number at d minus lost to follow-up among ei during the period d to � n ) � ). estimation of the turnover rate per -day interval among the infected residents was calculated on the base of the los (median in years) with the following formula: (proportion of discharged residents: . % in the case of the median)/[(median los � )/ )]. the average rate of residents discharged per -day period was calculated: [(number of lost to follow up during the period d to � n /number of exposed and infected residents at d )/n -day interval] � . as some residents were included in several outbreaks during the surveillance, the observations were not completely independent; non-parametric tests were used as a result. in univariate analysis, chi-square or fisher exact tests (expected number of frequencies fewer than ) were used to compare infection and lethality rates according to the studied parameters and the odds ratio was calculated by median-unbiased estimation. the kruskal-wallis test was used to compare median values. confidence intervals for medians were calculated with bootstrap methods. covariate adjusted analyses were performed with two tables ( x ). the respective impact of each individual factor was tested with mantel-haenszel chi-squared tests. the equality of the stratum odds ratios was tested with the woolf test of homogeneity. finally, for each virus context, multiple tables ( x ) were generated and tested with confounding variables, effect modifiers or covariables. statistical analyses were done using r for mas os x version r . . software with rstudio version . . . a file is transmitted in the supporting information with all r codes and the packages used (s r codes). differences were considered significant at p � . . the french data protection authority approved data collection and analysis (de- - ) and the local ethics committee (espace local de réflexion ethique, centre hospitalier de rouffach) approved the study protocol (erle- ). according to the french law for biomedical research and human experimentation, individual written consent was not required from the patients or their relatives for data collection. each year, the referring local practitioner of the study coordinated with the doctors working in the nursing home. at the beginning of the surveillance period, information regarding participation in the study was displayed in the family vising area, including a document about their right to access and rectify personal data. after collection, data were rendered anonymous. no specific authorization was needed to retrospectively analyze anonymous data collected during routine care in the context of routine surveillance. a total of outbreaks were recorded in the sites. rti outbreaks were more frequent than ge outbreaks ( outbreaks and exposed residents vs. outbreaks and exposed residents, respectively). overall, of the exposed residents were women and were men. the median age was . years old (interquartile range: . - . years). virus investigations (respectively samples for rti and for ge with all the detailed results in s -s tables) confirmed a considerable number of norovirus-related ge outbreaks ( / ) and influenza-related rti outbreaks ( / ). for ge outbreaks, residents were in a nov+ context versus in a nov-context, and for rti outbreaks, residents were in a flu+ context versus in a flu-context. for ge surveillance in the nov+ context, there were ei residents versus eni residents, whereas in the nov-context, there were ei residents versus eni residents. therefore, the infection rate was higher in the nov+ context ( . %) than in the nov-context ( . %, (odds ratio (or): . , % confidence interval (ci): . - . ), p < . ). for rti surveillance, the rates of infection were similar with and without confirmed influenza: . % (n = ei residents / exposed residents) vs. . % (n = ei residents/ exposed residents, or: . , ci: . - . , p = . ). moreover, infection rate in the nov + context was higher than the three other contexts: nov-(or: . , ci: . - . ), flu+ (or: . , ci: . - . ) and flu-(or: . , ci: . - . ). in univariate analysis, certain individual characteristics were associated with significant variations in the infection rate (s table) . the infection rate increased with age (except in the flucontext) and, decreased with los during ge outbreaks. the covariate adjusted analysis revealed specific significant effect modification according to sex (nov+) and los (nov-) (s table) . in analyses stratified according to virus and sex, age adjusted for los remained significant for flu+ and nov+ outbreaks (males). in nov-context, the effect modification of los remained significant (table ) . finally, when autonomy was included and adjusted for age (virus, sex, los stratification), the less autonomous residents (female/los< years/age< / gir - ) were affected more severely by flu+ outbreaks with specific effect modification according to age (s table) . the study of lethality rates in infected residents over the days after onset indicated that there were significant variations for rti but no change for ge (table ). significant differences appeared after days in the context of flu+ outbreaks and other rti outbreaks. the analysis of successive or simultaneous clusters in the same institutions was performed when the time period between the onset of symptoms of the last case in outbreak n and the onset of symptoms of the first case in outbreak n+ was � days (s table) . of the outbreaks ( . %) were identified, and of the exposed and infected residents contracted multiple infections. the percentage of exposed and infected residents implicated in more than one virus stratification was . % (( � )/ ). moreover, two deceased residents were included in the nov-na and flu lethality analyses because death occurred within days for both infections. the analysis of virus stratification of the outbreaks showed the absence of successive clusters for the same category. the same analysis for the first four -day intervals (days to ) showed the respective values: outbreaks ( . %), residents (( . % (( � )/ )), one dead resident. finally, according to the higher lethality impact during the first four -day intervals and to limit the impact of successive clusters in the same site, all cause lethality rates were studied according to individual parameters for the four days intervals with the respective number of according to the surveillance type (ge or rti), the lethality rates differed significantly: . % versus . % (respectively nov+ and nov-contexts, or: . , ci: . - . , p = . ) and . % versus . % (respectively flu+ and flu-, or: . , ci: . - . , p = . ). in univariate analysis (s table) , low autonomy status in the nov+, flu+ and flu-contexts was most significantly associated with increased all-cause lethality, and age was associated with higher lethality in the flu+ context. in the adjusted analysis, no significant statistical differences were identified in ge outbreaks. for rti episodes, the adjusted analysis showed that autonomy had a significant impact when adjusted for sex, age or los (flu+ and flu-na) and that age had a significant impact when adjusted for sex, autonomy or los (flu+) (s table) . in table , the specific effects of age or autonomy were tested. significant or age adjusted for autonomy were: flu+/age � years (compared with the < group), . ( . - . ). or autonomy adjusted for age were for gir - (compared with gir - ): flu+, . ( . - . ); flu-, . ( . , . ). finally, despite the low number of residents and deaths per category, and consequently the limited robustness of the results, autonomy adjusted for age with stratification according to virus, sex and los showed that the effects were higher among subgroups of less autonomous residents (female or male/los< years/gir - ) in flu+ outbreaks, and there was also higher mortality in the small subgroup of autonomous men with los � years (higher mortality) (s table) . in the flu+ context, data regarding vaccination status and oseltamivir prescriptions were available but not used in this study. in the present study, surveillance data obtained during ge and rti outbreaks in nursing homes were used to construct stratified analyses and to identify specific infection and all-cause lethality rates according to the residents' individual characteristics. the infection rates observed here were similar to those found in previous studies of nov and influenza outbreaks (odds of being infected during a flu+ outbreak were around % less than during a nov+ outbreak). reported infection rates were close to . % in influenza outbreaks and . % in nov outbreaks [ ] [ ] [ ] . older age appeared to increase the likelihood of ge and influenza infection, with increasing rates among older residents. age is a well-known factor for influenza and norovirus severity in the elderly and in nursing homes [ , ] . for nov, the highest incidence estimates ( -year age strata) was found in the � year-category (approximately men and for , women per , inhabitants). in our study, univariate analysis (nov+) showed that the odds of being infected were . to . times higher if a resident was older than . moreover, an adjusted analysis of ge outbreaks highlighted different effects among subgroups of residents according to sex and los. indeed, multiple and/or repeated exposure to ge viruses while institutionalized may lead to susceptibility or possible increased immunity in some residents [ ] . for the sex variable, two factors could explain the effect: a possible selection bias with men reporting mild infections less than women (particularly in the < years subgroup) or that male susceptibility was different (age, los, immunity,. . .). a german study from also reported a greater impact in women [ ] . moreover, when age analysis was stratified by sex and los, no epidemic impacts in nursing homes significant impact was observed in women; the only significant differences were fewer infections in men in the < -subgroup (except in the nov-with los < years). for the rti outbreaks, sex and los variables did not have a significant effect. in residents older than , the odds of being infected in flu+ context were . times higher for women and . for men. in univariate analysis, contrary to the other virus contexts where odds ratios were rarely above , residents over years old had increased odds of infection of � . compared with the -year-old category, and for the year-old group the odds were approximately . . in the flu+ context, autonomy adjusted for age (virus, sex and los stratification) revealed a possible increase in infection rates among less autonomous residents. a previous study found that when elderly residents were exposed to the a(h n ) virus, there were higher rates of infection and reinfection, and more significant effects on the institution than with other influenza types/subtypes. in the community, the relative illness ratio (rir) in the [ , ] . the incidence of influenza infection and the associated risks were well described by age group, but the specific impact according to age was not studied. the results of this work highlighted the specific age distribution of influenza illnesses among the nursing home residents and the more significant impact among the older residents. this specific susceptibility could be a critical factor in the institutional exposure and dissemination of influenza and could partly explain the high infection impact in the elderly institutional population. in this work, autonomy status was not the main factor associated with infection (no significant impact in ge and in flu-contexts). however, in flu+ outbreaks, a high level of dependency was associated with a higher risk of falling ill. this observation implies that staff could play a role in the spread of infection (highly dependent and less mobile residents are less likely to contaminate themselves) or that the more active residents may be less fragile and/or have a greater involvement in the recommended infection control measures. finally, improving compliance with personal hygiene measures both for nursing staff and residents might be expected to have a beneficial effect on infection rates. previous studies identified higher nov infection rates in highly dependent individuals, but the results were not adjusted for age and los to take into account the potential correlation with the autonomy status [ ] . lethality is difficult to assess in nursing homes because death is frequent. our ge and rti episodes occurred during the winter seasons, and there are possible interactions between outbreaks and increased mortality at this time of the year [ ] . the all-cause lethality rate of the infected residents in our study reflected global mortality including ge and rti outbreaks and the global epidemiological context. not surprisingly, a higher all-cause lethality rate was observed in the influenza contexts, as reported in previous studies [ ] [ ] [ ] . age and autonomy had similar effects in the different contexts, but in ge and to a lesser degree in flu-outbreaks, the relatively small number of deaths could have limited the power of the statistical tests. in nursing homes, residents are generally discharged due to death. the number of residents lost to follow up was low ( . % in the first days), so the -day interval turnover rate calculated on the base of the median los provides a good indication of the average case fatality rate. the lethality rate for nov+ outbreaks was similar to the estimated -day interval turnover rate ( . %) indicating that this context had a limited impact on the death rate. the all-cause lethality rate was most affected by age and autonomy. both individual characteristics were significant in the flu+ outbreaks, and autonomy adjusted for age was significant in the flu-episodes. the influence of age on mortality in a context of influenza has already been described: a very high mortality rate ( / , inhabitants) was reported in persons years of age and older compared with those aged - years ( / , inhabitants) [ ] . in our univariate analysis, the higher risk was observed in the � group whose risk of death was at least . higher than the < group. when adjusted for autonomy, the impact of age was not significant in more autonomous residents in the flu+ context and not at all in the flucontext. the opposite analysis (autonomy adjusted for age) showed higher global impact in the less autonomous group (flu-) or only in the � age group (flu+). age and autonomy are a reflection of resident's level of frailty. clinical frailty scores were not used in this study, but in a previous study of patients with critical illness, they were associated with greater mortality, regardless of age [ ] . this suggests that in addition to age, autonomy can be a valuable indicator for the assessment of outbreak impact in outbreak surveillance. other studies have suggested that age and certain comorbidities are independent risk factors for the influenza mortality rate or that mortality increase according to the number of risk factors [ , ] . comorbidities and underlying diseases of various severities could reflect overall frailty and consequently the risk of death. in nursing homes, information about autonomy and age are easier to collect and interpret than data on comorbidities. these various approaches should be evaluated and compared in the goal of optimizing risk assessment among nursing home residents. the present work has two main limitations. first, the virus information was incomplete (limited identification, mainly influenza rapid tests for the rti). consequently, some episodes in the levels with no available identification may also have been associated with influenza or norovirus, and multiple contaminations could have been underestimated or not taken into account. moreover, vaccination and oseltamivir prescriptions were recorded but not included because the influenza genotype was not determined and identification was limited. secondly, the deaths of uninfected residents were not recorded in this protocol even though such data would have provided valuable information about the global epidemiological context. in conclusion, specific susceptibility patterns were observed among exposed residents. in this cohort of nursing homes, infection rates varied according to virus, sex, length of stay and age, and there were major differences in lethality depending on virus, age and autonomy score. the collected data were easy to record and could be used to improve the characterization of seasonal outbreaks in nursing homes, whose residents are particularly vulnerable. finally, as the average age and dependency level of residents continues to increase, subsequently increasing the risk of infection and death, health care staff will have to be increasingly vigilant during seasonal outbreaks and targeted interventions should be implemented. supporting information s data. (csv) s r codes. (r) s table. (xlsx) s table. (xlsx) s table. 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with critical illness pneumonia and influenza deaths during epidemics: implications for prevention all the medical, nursing, management teams in the institutions for their commitment to this key: cord- -jld ygxt authors: neidermyer, william j.; whelan, sean p. j. title: global analysis of polysome-associated mrna in vesicular stomatitis virus infected cells date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: jld ygxt infection of mammalian cells with vesicular stomatitis virus (vsv) results in the inhibition of cellular translation while viral translation proceeds efficiently. vsv rna synthesis occurs entirely within the cytoplasm, where during transcription the viral polymerase produces mrnas that are structurally indistinct to cellular mrnas with respect to their ′ cap-structure and ′-polyadenylate tail. using the global approach of massively parallel sequencing of total cytoplasmic, monosome- and polysome-associated mrna, we interrogate the impact of vsv infection of hela cells on translation. analysis of sequence reads in the different fractions shows > % of total cytoplasmic and polysome-associated reads map to the viral genes by hours post-infection, a time point at which robust host cell translational shut-off is observed. consistent with an overwhelming abundance of viral mrna in the polysome fraction, the reads mapping to cellular genes were reduced. the cellular mrnas that remain most polysome-associated following infection had longer half-lives, were typically larger, and were more au rich, features that are shared with the viral mrnas. several of those mrnas encode proteins known to positively affect viral replication, and using chemical inhibition and sirna depletion we confirm that the host chaperone heat shock protein (hsp ) and eukaryotic translation initiation factor a (eif a)—encoded by such mrnas—support viral replication. correspondingly, regulated in development and dna damage (redd ) encoded by a host mrna with reduced polysome association inhibits viral infection. these data underscore the importance of viral mrna abundance in the shut-off of host translation in vsv infected cells and link the differential translatability of some cellular mrnas with pro- or antiviral function. introduction infection of mammalian cells by vesicular stomatitis virus (vsv) results in a profound shut-off of host cell gene expression. this host cell shut-off occurs at the level of mrna transcription through inhibition of rna polymerase ii by the viral-encoded matrix protein (m) [ ] [ ] [ ] . the m protein also forms a complex with ribonucleic acid export (rae ) and nucleoporin (nup ) [ ] thus suppressing host cell mrnp export from the nucleus, including that of mature cellular mrnas [ ] [ ] [ ] [ ] . vsv infection also inhibits protein synthesis by manipulation of the host-cell translation machinery, particularly at the level of translation initiation [ , ] . eukaryotic initiation factor e (eif e)-the rate limiting factor for translation initiation-recognizes the m gpppn mrna cap structure as part of the eif f complex, and in concert with other translation initiation factors facilitates the recruitment of the small s ribosomal subunit to the mrna prior to scanning to the initiating methionine where the s subunit joins [ , ] . vsv infection results in the rapid dephosphorylation of eif e itself, for which the functional consequences are unclear, and of its binding protein (eif e-bp ) leading to eif e sequestration and the suppression of translation initiation [ , ] . viral gene expression evades the shut-off mechanisms employed to suppress host gene expression. as vsv rna synthesis occurs entirely within the cytoplasm, viral rna synthesis is not subject to the inhibitory effects of m on rna polymerase ii and mrna export from the nucleus. the vsv rna synthesis machinery comprises a ribonucleoprotein complex of the negative-sense genomic rna completely encased by a nucleocapsid protein (n) sheath and associated with the viral polymerase complex [ ] . the viral transcriptase copies the n-rna template into monocistronic mrnas that are structurally indistinct to those of the host-cell with respect to their cap and polyadenylate tail [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the enzymes necessary for mrna synthesis, namely an rna dependent rna polymerase (rdrp) and a set of capping enzymes, reside within the viral large protein (l) [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . vsv l protein cannot engage the n-rna template directly, but instead depends on the viral phoshoprotein (p) to facilitate the interaction [ ] [ ] [ ] [ ] [ ] [ ] . messenger rna polyadenylation is also catalyzed by l through reiterative transcription by the rdrp of a u tract that resides at the end of each gene [ ] [ ] [ ] [ ] [ ] . this program of viral transcription results in the cytoplasmic synthesis of mrnas that depend upon the host machinery for their translation, and must therefore avoid the shut-down mechanisms that effectively suppress host mrna translation. metabolic labeling studies demonstrate that hours post vsv infection of baby hamster kidney cells in culture, total translation is suppressed to about % the level of uninfected controls [ ] . extraction of mrna from infected cells coupled with its in vitro translation confirmed that the cellular mrnas remain intact and are competent for translation [ ] . the vsv mrnas are present in an approximately - fold excess of the total cellular mrna, leading to the model that competition between viral and cellular mrnas for ribosomes results in the dominance of viral translation [ , ] . polysome analysis also demonstrates that the cellular mrnas are associated with significantly fewer ribosomes in infected cells [ ] . for example, infection results in the movement of actin mrna from polysomes containing or more ribosomes to those containing [ ] . this movement reflects the competition between viral and cellular mrna for ribosomes and the limited pool of eif e. the competition model predicts that the kinetics of viral mrna synthesis and the levels of viral mrna should correlate closely with host shut-off. tests of this prediction yielded conflicting results. the kinetics of host shut-off and viral mrna accumulation correlate well for many strains of vsv, consistent with the competition model [ , ] . inhibition of host protein synthesis is, however, largely unaffected following coinfection of cells with increasing quantities of defective interfering (di) particles that suppress viral mrna levels up to -fold [ ] . a similar result was obtained for a vsv mutant that is restricted for genome replication at ˚c, and yields only % of the wild type levels of viral mrna [ ] . collectively, these studies suggest additional mechanisms may contribute to the shut-off of host cell protein synthesis. specific features of the viral mrnas that contribute to their efficient translation have not been defined. the untranslated regions of vsv mrnas are short, being - nucleotides for the viral n, p and l mrnas that encode the proteins required for rna replication [ , ] . how such short utrs serve as effective initiators of translation is unclear. evidence for differential translation of viral mrna comes from small interfering rna suppression of eif e, which inhibits host gene expression but has no impact on viral gene expression [ ] . viral translation is also hypersensitive to the loss of ribosomal protein l , suggesting different mrna features facilitate translation of viral versus host mrna [ ] . flanking cellular or reporter genes by the conserved viral -nt gene-start and -nt gene-end sequences, and inserting them into the viral genome is sufficient to mediate their efficient translation [ ] . by contrast, expression of the same genes following transfection of plasmid dna into cells and subsequent vsv infection does not offer this translational advantage [ ] . thus, transcription of the mrnas from the viral genome appears to contribute to their efficient translation. in the present study, we interrogate global mrna translation in vsv infected cells using rnaseq analysis of the cytoplasmic mrna transcriptome, and parallel sequencing of polysome-associated mrnas. we obtain support for the model that an overabundance of viral mrna contributes to host shut-off by leading to a re-distribution of cellular ribosomes onto viral mrna. by combining this rnaseq analysis with examining the distribution of specific viral and cellular mrnas within polysomes, we also demonstrate that mrnas shift to smaller polysomes. analysis of cellular mrnas less-sensitive to this global shut-down of translation identifies several host proteins that promote viral replication. similar analyses revealed the abundance of viral mrna contributes to the host-cell shut-off for other viruses including coronaviruses, influenza and vaccinia [ ] [ ] [ ] . to interrogate the impact of vsv infection on global translation we isolated total cytoplasmic, monosome-and polysome-associated mrna from hela cells at and hpi and compared the relative sequence reads obtained by deep-sequencing ( fig a) . statistical analysis of sequencing reads between biological replicates from each fraction yields a pearson correlation of > . for cytoplasmic, monosome-and polysome-associated mrna pools validating reproducibility between the replicates. as visible in the polysome profiles (fig b) , vsv infection results in a small but reproducible increase in the pool of monosomes and large polysomes at hpi, and a collapse of large polysomes and an increase in monosomes by hpi. mapping of the sequence reads to the viral and host genome highlights that by hpi > % of the total reads in the cytoplasmic and polysome fractions are viral (fig c) . this increase from the < % observed at hpi (fig c) emphasizes the impact of the exponential phase of viral rna replication and secondary transcription of the viral genome on mrna production. the viral sequence reads map to all genes, with clear dips in coverage at gene-junctions (s fig) . consistent with the order of transcription of the viral genome and the localized transcriptional attenuation at gene-junctions [ ] [ ] [ ] [ ] , the relative reads that map to each viral gene generally diminish with distance from the single promoter ( fig d and s fig) . analysis of the sequencing reads that map to cellular genes supports that like the viral mrnas, the level of reads in the polysome fraction mirrors that in the total cytoplasmic fraction at and hpi ( fig c) . this result demonstrates that the majority of mrnas are polysome-associated in proportion to their abundance. the abundance of the viral mrnas at hpi supports the model that viral mrnas outcompete cellular mrnas for ribosomes [ ] . we note that viral mrnas are, however, underrepresented ( %) and cellular mrnas overrepresented ( %) in the monosome fraction at hpi, compared to their cytoplasmic abundance ( fig c) . this finding is consistent with a differential effect on viral versus host mrna translation. to determine how vsv infection affects the distribution of the population of mrnas between total cytoplasmic, monosome or polysome fractions, we plotted the transcript per million (tpm) for each individual mrna mapped to the human and viral genome in all fractions (fig ) . at hpi, reads that map to the viral genes in each fraction are similar in abundance to those reads that map to highly expressed cellular genes (fig a) . the reads that map to any given cellular gene alter within a relatively narrow range, with few genes showing a greater than -fold change in the relative number of sequence reads (fig b) . for the population of mrnas, the relative reads obtained from the polysome fraction mirrored the relative reads in the total cytoplasmic fraction, consistent with the abundance of an mrna being a determinant of its translatability. by hpi, reads that map to each of the viral genes-with the exception of l-exceed the reads that map to any individual cellular gene (fig c, red triangles) . this is concurrent with a decrease in reads that map to the majority of cellular genes in cytoplasmic, monosome and polysome-associated fractions (fig c) . there were, however, some distinctions between the monosome and polysome fractions. for the majority of the population of cellular mrnas, hela cells were infected with vsv at a moi of and cytoplasmic extracts were prepared at and hpi for mrna isolation and polysome profiling. messenger rna was isolated from fractions corresponding to s monosomes, or polysomes containing or more ribosomes, and used for deep sequencing. (b) polysome analysis of uninfected (black) or vsv (red) infected hela cells. cytoplasmic extracts were sedimented through a - % sucrose gradient and . ml fractions were collected while continuously monitoring absorbance at λ = nm. (c) distribution of fragments mapping to the concatenated hg (human) and vsv genomes for cytoplasmic, monosome, and polysome samples at and hpi. trimming and mapping was performed in clc genomics workbench. (d) distribution of reads among the viral genes at and hpi. expression level is presented as transcripts per kilobase million (tpm) to normalize for gene length and library size, error bars denote standard deviation from two biological replicates. reads were most reduced in the polysome fraction compared to the total cytoplasmic fraction ( fig d) . a smaller reduction in reads was observed in the monosome fraction, and some cellular mrnas even showed an increase in reads compared to the total cytoplasmic fraction ( fig c) . this may reflect differences in the movement of cellular mrnas from large polysomes to monosomes or out of the pool of translating ribosomes. we next mined our sequence data for evidence of differential translation of cellular mrnas following vsv infection. for this analysis we divided the polysome tpm by the total cytoplasmic tpm as an indicator of the efficiency with which any given mrna is translated. we also performed a similar analysis for the monosome pool. we are cognizant of the fact that such ratios ignore the movement of any given mrna from larger to smaller polysomes, and will likely represent an underestimate of the extent of any translational regulation. to identify the subset of the population of cellular mrnas with the highest probability for translational regulation in infected cells, we plotted the fold change in tpm at and hpi (fig a- d ). at hpi the monosome or polysome-associated reads changed within a narrow range for the majority of cellular genes ( fig a) . the marked shut-off of host protein synthesis observed by metabolic labeling suggests that at hpi the association of cellular mrna with polyribosomes would alter significantly at the population level. although we observe a global reduction in polysome-associated reads for the bulk of the population of cellular mrnas the reduction is less than - fold. accompanying this global reduction in polysome-associated reads, we also observe an increase in monosome-associated reads with more than half the mrnas within the population exhibiting a > -fold increase (fig b and d ). from the above ratios we selected the subset of cellular mrnas that exhibit the largest changes in relative polysome-associated reads at hpi to determine whether those mrnas shared any common features. for this purpose, we selected those mrnas that change > standard deviations of the mean and thus exceed the % confidence interval. this analysis identified cellular mrnas as candidates for translational upregulation and cellular mrnas as candidates for translational downregulation following vsv infection (fig b and d ). consistent with monosome and polysomeassociated reads at hpi changing within a narrow range, only genes with increased and with decreased polysome association, overlap between and hpi (fig c and d ). within the monosome fraction genes with increased and with decreased monosome association overlap from to hpi. we next determined whether shared functional or sequence elements are present within the specific subsets of mrnas with increased polysome association, or the mrnas with decreased polysome association (fig b and d , blue and green dots). for the genes with significantly increased polysome-associated reads, gene ontology analysis identifies functions in rna binding, helicase and ntpase activities, among others ( fig e and s dataset) . the genes with decreased polysome-associated reads are associated with cellular responses to stimuli and signaling activities ( fig f and s dataset) . this gene ontology analysis reveals that the up and down regulated transcripts comprise distinct functional groups. at hpi the cytoplasmic abundance of cellular mrnas correlates with their polysome association at hpi ( fig a and b ), consistent with mrna abundance being a determinant of translatability. as described above, we use as an indicator of translation efficiency (te) of an mrna the ratio of polysome to total cytoplasmic associated reads. to determine whether there are shared features between the mrnas with evidence of enhanced polysome association or the with reduced polysome association, we extracted mrna sequences and annotations from the ucsc genome browser. assisted by published datasets we examined whether the half-life, size, gc content or poly(a) tail length correlate with increased or decreased polysome association (fig c- f and s fig) [ , ] . cellular mrnas with increased polysome-associated reads tended to have a longer half-life, were typically larger, and were more au-rich ( fig c- e and s fig) . correspondingly, those with decreased polysome-associated reads tended to have shorter half-lives, higher gc content, and were typically smaller. the correlation between higher au content and increased polysome-associated reads was most evident for the coding region and utr (s fig). the effect of length was predominantly a determinant of the orf and not the or utr (s fig). there was no correlation between poly(a) tail length and polysome-associated reads at hpi ( fig f) . this analysis highlights that the cellular mrnas that exceed the % confidence interval for increased polysome-associated reads in response to vsv infection are most similar to the viral mrnas in that they are typically longer and more au rich. we next examined whether cellular mrnas that exhibit increased polysome association encode proteins that are pro-or antiviral. among the cellular mrnas with increased polysome association several encode known proviral factors including the heat shock proteins (hsp) , , and . previous work demonstrated that inhibition of hsp inhibits viral replication, and linked inhibition of those chaperones to defects in l protein folding [ ] [ ] [ ] . we independently verified the proviral function of hsp using the inhibitor -[ -(dimethylamino)ethyl]amino- -desmethoxygeldanamycin ( -dmag) [ , ] . infection of hela cells with vsv that expresses egfp as a marker of infection demonstrates that -dmag has no effect on the fraction of cells infected, but slows the rate of egfp expression ( fig a and s fig) . this was not simply due to defects in egfp folding, as metabolic labeling of viral rna substantiates the defect in gene expression (s fig) . we also found that polysome association of the mrna encoding eukaryotic initiation factor subunit a (eif a), increases after infection. to test whether this reflects a specific proviral function of eif a, we used sirna depletion to reduce eif a and measured viral gene expression using reporter viruses expressing egfp or luciferase. both reporter viruses displayed a sensitivity to the loss of eif a (figs b and s ). as expected, depletion of eif a also reduced cellular translation in uninfected cells, but that reduction was modest as evidenced by expression of a cmv promoter driven renilla luciferase reporter ( fig b) . translation of the cmv driven reporter, however, reflects the accumulated steady-state levels of luciferase mrna. we therefore measured the effect of eif a depletion on viral vs host translation by metabolic incorporation of [ s]-methionine in infected and uninfected cells (fig b) . following eif a depletion we observed a % reduction in viral m protein synthesis over a minute time period, which is similar to the % reduction in host protein synthesis measured in mock infected cells. this analysis supports a proviral role for cellular mrnas that encode proteins with important house-keeping functions that remain polysome-associated following vsv infection. among the cellular mrnas that exhibit reduced polysome association following infection was dna-damage inducible transcript (ddit ) which encodes regulated in development and dna-damage response (redd ) [ ] [ ] [ ] . existing studies demonstrate that ddit /redd restricts the replication of negative-strand rna viruses, including vsv. depletion of ddit /redd by sirna increased viral gene expression as evidenced from infection of cells with vsv-egfp, and by metabolic labeling of viral protein synthesis (fig c, s fig) . consistent with the enhancement of viral gene expression following ddit depletion, we obtained an approximately -fold increase in viral titers ( fig c) . depletion of ddit /redd also increases cellular protein synthesis likely reflecting its role as a negative regulator of mtor ( fig c and s fig) . the above analysis supports that the polysome association of some host mrnas following vsv infection correlates with their pro-or antiviral functions, but does not directly demonstrate that the level of polysome association is associated with a change in synthesis of the corresponding protein. to independently examine whether changes in polysome association of host mrnas affect synthesis of the corresponding protein we selected the heat shock protein (hsp ) and y-box binding protein (ybx ) as representative mrnas with increased and decreased polysome association, respectively. we selected those mrnas based on their highlevels of expression, stability, and availability of antibodies suitable for the selective immunoprecipitation of the corresponding protein. we compared the effect of vsv infection on protein synthesis by selective immunoprecipitation of proteins following metabolic incorporation of [ s]-methionine from - hours post infection (fig d) . synthesis of hsp - hpi is indistinguishable to that synthesized during a h period from mock infected cells (fig d) . by contrast, ybx synthesis decreases more than two-fold ( fig d) . this result confirms for cellular mrnas that the extent of polysome association observed by our rnaseq analysis is reflected in synthesis of the corresponding host proteins. analysis of cellular mrnas with high cytoplasmic abundance (purple) or low cytoplasmic abundance (orange) as compared to mrnas with cytoplasmic abundance within standard deviations of the mean abundance (gray) in uninfected cells. cytoplasmic abundance by tpm is from the data set published with this paper. ��� p< . x − ; �� p< . x − ; � p< . ; all others p> . as determined by the wilcoxon rank sum test compared to mrnas with relative abundance levels within the % confidence interval of the mean. hinges correspond to the th- th percentiles, and whiskers denote . times the inter-quartile range. (b) analysis as in a for cytoplasmic abundance in infected cells. (c-f) mrna characteristics for mrnas with increased polysome association (blue) or decreased polysome association (green) at hpi, as defined in fig . data for rna half-life and poly(a) tail length were from [ , ] . analysis was performed as in a. for our experiments we pooled fractions that contained or more ribosomes prior to sequencing of the polysome-associated mrna. as a result, we do not assess the impact of the redistribution of mrnas toward smaller polysomes. we therefore selected a subset of cellular mrnas (fig a) , and interrogated their distributions across polysomes using reverse transcription and quantitative pcr. as controls, we analyzed the distribution of n and g mrnas as representative viral transcripts translated by soluble and endoplasmic reticulum-associated ribosomes, respectively [ ] . consistent with the robust production of viral proteins at hpi, the vsv n and g mrnas were localized in fractions corresponding to or more ribosomes (fig b) . for two cellular mrnas with increased polysome tpm-collagen type iv alpha (col a ) and alpha-actinin- mrna (actn )-the mrnas remained associated with larger polysomes in infected cells (fig c) . two cellular transcripts that were largely unaltered in their polysome associated tpm-β-actin (actb) and glyceraldehyde -phosphate dehydrogenase (gapdh)-remained polysome-associated, although there was a shift toward smaller polysomes and some gapdh transcripts exited polysomes (fig d) . for two representative cellular mrnas with decreased polysome tpm-transforming growth factor b induced factors (tgif ) and ubiquitin conjugating enzyme e b (ube b)-the mrnas largely exited the polysome fractions, and those that remained were predominantly present on smaller polysomes ( fig e) . in all cases examined, dissociation of polysomes with edta shifted the mrna distribution toward the fractions corresponding to free ribosomal subunits (s fig). these qpcr data highlight the shift towards smaller polysome fractions for some cellular mrnas, which also likely contributes to suppression of host protein synthesis. this shift might also explain our finding that hsp protein synthesis is relatively unaffected by viral infection (fig d) , although the mrna exhibits increased polysome association. the abundance of viral mrna and the suppression of translation initiation through reducing the pool of eif e will both contribute to the movement of mrnas toward smaller polysomes. recognition of the mrna cap-structure by eif e requires that the guanine-n- position of the mgpppnmn cap is methylated [ ] . we previously reported a panel of recombinant vsvs containing amino acid substitutions within the l-encoded mrna cap methylase domain that are defective in viral mrna cap methylation [ ] . mutants vsv-l g a and vsv-l g a contain substitutions in the binding site for the methyl donor s-adenosyl methionine (sam) and ablate all viral mrna cap methylation (vsv-l g a ) or guanine-n but not ribose- -o methylation (vsv-l g a ) [ ] . as vsv mrna is relatively insensitive to the loss of eif e [ ] , we would anticipate that the methylation status of the mrna cap-structure would have little impact on polysome association. analysis of the distribution of vsv n and g mrna within polysomes at hpi revealed a similar distribution in cells infected with wild-cells infected with wild-type vsv. the position of viral proteins is noted to the right. presented is a representative gel from two independent replicates. (c) vsv gene expression and replication in ddit depleted cells. a representative histogram of egfp expression is shown along with the mfi of cells normalized to a non-targeting sirna control. error bars denote the standard deviation from the mean of three independent replicates. for metabolic labeling a representative gel of two independent replicates is presented. kinetics of viral replication were measured by titration of yields at various times post infection of sirna treated hela cells. error bars denote the standard deviation from the mean of independent replicates. (d) immunoprecipitation of cellular proteins synthesized post-infection with vsv wt . shown is a representative gel from two independent replicates. a quantitative analysis of the hsp and ybx bands is shown in the bottom two panels, error bars denote the standard deviation from two independent replicates. https://doi.org/ . /journal.ppat. .g control of vsv protein synthesis type virus as well as those infected with vsv-l g a and vsv-l g a (fig a- c) . correspondingly, the rate of viral protein synthesis in cells infected with vsv-l wt and vsv-l g a measured by a -minute pulse of [ s]-methionine is similar (s fig). these results demonstrate that defects in viral mrna cap methylation do not significantly alter the rate of viral protein synthesis, consistent with a reduced dependence on eif e [ ] . to directly test whether manipulating eif e levels affects viral translation we depleted eif e levels approximately -fold using a peptide-conjugated morpholino (ppmo) and measured the rates of vsv-l wt and vsv-l g a viral protein synthesis by a -minute pulse of [ s]-methionine at various times post-infection (fig d- f) . depletion of eif e decreased the rate of viral protein synthesis in vsv-l a infected cells, but not l wt infected cells ( fig e and f) . this was not due to sequestration of eif e by differential activation of eif e-bp between vsv-l wt and vsv-l g a infected cells, as the kinetics of eif e-bp dephosphorylation are the same during both infections (s fig). we previously reported that although vsv-l a is defective in mrna cap methylation, up to % of the mrna cap-structures are guanine-n methylated. we interpret this finding as indicative of an eif e dependent mechanism of translation early in infection. we obtained two snapshots into the complex battle for control of protein synthesis in cells infected with vesicular stomatitis virus by sequencing of polysome-associated mrnas at and hpi. those snapshots provide further evidence that the abundance of vesicular stomatitis virus mrnas is a determinant of the dominance of viral protein synthesis in infected cells, but highlight several additional attributes of this complex relationship. those include the demonstration that some host mrnas that remain polysome associated encode proteins that support viral replication, and some of those that exhibit reduced polysome association encode proteins that are antiviral. we also obtained further insight into the seemingly paradoxical observations that viral infection results in a reduction of the available pool of eif e -the rate limiting factor for translation initiation-yet viral mrnas contain a cap structure that is indistinct to that of host mrnas. through the use of a viral mutant defective in mrna cap methylation, and suppression of eif e levels we provide evidence consistent with a transition from an eif e dependent phase of viral translation to one less-dependent on eif e. the sequence data reported here provides some support for the model that the vsv mrnas overwhelm the pool of cellular mrna leading to a redistribution of ribosomes onto viral messages [ ] . evidence in support of this model is based on massively parallel sequencing of mrnas associated with polysomes, compared with those present in the cytoplasm. as a fraction of the total cytoplasmic mrna, the vsv mrnas represent~ % by hpi, but more than % by hpi, illustrating the power of exponential amplification of the viral genome. as a result, the viral n, p, m and g mrnas far exceed the abundance of any given cellular mrna, and even the least abundant viral mrna-that encoding the l polymerase-is present at similar levels to the most abundant cellular mrna. thus, one contributor to host cell shut-off in vsv infected cells appears to relate to the synthetic capabilities of the viral polymerase in transcription of viral mrna. similar conclusions have recently been reached for other viruses. infection of cells with mouse hepatitis virus (mhv) a positive-strand rna coronavirus that replicates within the cytoplasm results in - % of the cytoplasmic mrna being viral by hpi [ ] . for influenza a virus, a segmented negative-strand rna virus that replicates in the nucleus, > % of the total mrna in the cytoplasm is viral [ ] . in this case however, the viral endonuclease pa-x degrades cellular mrna which further contributes to the dominance of viral mrna [ , ] . for vaccinia virus, a dna virus that replicates entirely within the cytoplasm, degradation of host mrna through the viral encoded decapping enzymes d and d also helps the viral mrnas overwhelm those of the host cell [ , , ] . collectively these studies indicate that one shared mechanism for host cell shut-off in virus-infected cells is competition for host cell ribosomes through tipping the balance between viral and host mrna. earlier work concludes that viral mrna abundance is not the determinant of host cell shut-off [ ] . when vsv mrna levels were suppressed up to -fold by using defective interfering particles of vsv or a viral mutant defective in transcription, host shut-off was still observed. we did not directly test how suppressing viral mrna levels impacts host shut-off in this study, but rather conclude that abundance is only part of the mechanism by which the virus induces host cell shut-off-as discussed below. we also obtained evidence in support of additional mechanisms that contribute to host cell shut-off in vsv infected cells. we confirmed earlier work that demonstrated a suppression of the pool of the rate limiting factor for initiation, eif e, by altering the phosphorylation status of its negative regulator, eif e-bp , which results in eif e sequestration [ ] . differences in sensitivity to reductions in eif e may contribute to the overrepresentation of cellular mrna we observe in monosome fractions during infection. we also provide new evidence in support of a phase of vsv gene expression that is dependent on eif e through the use of a viral mutant partially defective in guanine-n methylation [ ] and by the suppression of cellular pools of eif e. when eif e levels are suppressed -fold, we unmask a defect in viral protein synthesis in cells infected with vsv-vsv-l g a a mutant with a -fold defect in methylation at the guanine-n -position of the cap-structure. we suspect that this significantly underestimates the eif e dependent phase of viral replication since transformed cell lines, like the hela cells used here, have higher constitutive levels of eif e [ ] . our findings are consistent with a model where viral mrnas initially compete with cellular mrnas and translate in an eif e dependent manner. as infection progresses and the shut-down of host transcription, mrna export and eif e sequestration continue, the process of initiation is increasingly less dependent on eif e. the mechanism by which the viral mrnas become less dependent on eif e remains uncertain, but earlier studies demonstrate that neither the or utr of viral mrnas facilitate this efficient translation. ongoing transcription of viral mrna from the viral genome has also been linked to efficient protein synthesis [ , ] . whether this reflects the fact that the virus is an efficient producer of mrna that supports the competition model, or whether there is a temporal requirement for continued viral mrna synthesis is uncertain. as obligate intracellular parasites, viruses depend upon host cell functions for their replication. our sequence analysis provides evidence that vsv infection differentially impacts the polysome association of cellular mrnas. several host mrnas increased in polysome association include genes with known "proviral" functions for entry and replication including heparan sulfate, clathrin, and hsp [ , [ ] [ ] [ ] . similarly, host mrnas with decreased polysome association included genes with published roles in restricting vsv replication such as interferon regulatory factor (irf ), ddit , and txnip [ , , ] . it is difficult to definitively determine whether this reflects evolution of the virus to contend with the environment in which it finds itself or a bona-fide pro and antiviral effect of a given host protein. our efforts to address this are confounded by the essential house-keeping nature of many of the proteins encoded by host mrnas that remain polysome associated. an example of this is provided by enhanced polysome association of eif a on vsv infection-a protein that is required for assembly of the multisubunit eif complex. that complex also includes eif d which has demonstrated cap-binding ability, and directs eif e-independent translation of select mrnas [ ] [ ] [ ] [ ] . depletion of eif a, however, resulted in an equivalent reduction in the rates of viral and host translation-inconsistent with a specialized need for eif components in vsv protein synthesis. in this study, we validated that the effect of vsv infection on the polysome association of host mrnas-hsp and ybx -had a concordant impact on protein synthesis. although synthesis of hsp did not increase per se, this is likely explained by the shifting of many cellular mrnas towards smaller polysomes. this finding highlights the fact that the designation of rnas as having "increased" or "decreased" polysome association is imprecise, and reflects the complexities of how any given host gene is regulated. nevertheless, the general finding that mrnas with "increased" polysome association on vsv infection are typically larger, have longer half-lives and higher au content-features that are shared with the viral mrna-highlights commonalities among mrnas that remain polysome-associated and thus are more efficient in competing for ribosomes during host shut-off. the cellular mrnas that exhibit reduced translation efficiency were shorter, have shorter half-lives and higher gc content. although we validated changes in translatability and differential impacts on viral gene expression for a few cellular genes, it would be of significant interest to perform stable isotope labelling by amino acids in cell culture (silac) to non-radioactively label newly synthesized cellular proteins, quantify them on a genome-wide scale and correlate those data with the rnaseq results presented here. in addition to suppression of host translation through mrna synthesis and eif e manipulation, the vsv matrix protein inhibits host rna polymerase ii transcription [ , ] , and blocks nuclear export of mature mrnas through complex formation with the nuclear pore components rae and nup [ ] [ ] [ ] [ ] [ ] . a well characterized viral mutant (vsv-m m r ) fails to interact with the nuclear pore complex and exhibits a delayed kinetics of host shut-off [ , , ] . a similar analysis to that described here of cells infected with such a mutant may help delineate the extent to which ongoing synthesis and export of cellular mrna impacts host cell shut-off. we anticipate that over the time course of vsv infection, the contribution of ongoing synthesis and export of host mrna from the nucleus will result in a relatively modest increase in the fraction of the cytoplasmic mrna that is cellular. this study highlights a strategy shared among distinct viruses to commandeer the host translational machinery by outcompeting cellular mrnas. precisely where the tipping point between viral and host mrna levels with respect to this shut-off occurs is uncertain. for vsv, a viral mutant that makes less mrna than the wild type yet still exhibits host cell shut-off suggests that shut-off can be achieved with less than the % of total cytoplasmic mrna observed here [ ] . additional work will be required to define whether a specific tipping point exists and how this is influenced by other viral strategies such as eif e suppression or blocking host gene transcription. hela cells (a gift from james hogle) were maintained in dulbecco's modified eagle medium (dmem; invitrogen, carlsbad, ca) supplemented with % fetal bovine serum (fbs; tissue culture biologicals, tulare, ca). viral stocks were grown on syrian golden hamster kidney bsrt cells (a gift from k. conzelmann), and purified on linear - % sucrose gradients prepared in nte ( mm tris ph . , mm nacl, mm edta). viral titers were determined by plaque assay on african green monkey kidney vero cells (atcc, ccl- ). for infections, cells were first washed with hanks' balanced salt solution (hbss) and incubated with virus for hour at ˚c in serum free medium, washed with hbss and subsequently incubated with medium supplemented with % fbs. for polysome profiling, hela cells were treated with dmem containing μg ml - cycloheximide at ˚c for minutes. cells were washed twice with x ice-cold phosphate buffered saline (pbs) containing μg ml - cycloheximide, and kept on ice or at ˚c. cells were scraped into a . ml microcentrifuge tube in x pbs with μg ml - cycloheximide, and pelleted at × g for minutes. cells were resuspended in μl of a hypotonic buffer of mm tris (ph . ), . mm mgcl , . mm kcl, and rnasin (promega, madison, wi), supplemented with cycloheximide to μg ml - and dl-dithiothreitol (dtt) to μm. the detergents triton x- . % (vol/vol) and sodium deoxycholate . % (wt/vol) were then added sequentially, cells were briefly vortexed, and incubated for minutes on ice and clarified by centrifugation at , × g for min. polysomes were separated on sucrose gradients prepared on a gradient master station (biocomp, fredericton, canada) using % and % (wt/ vol) sucrose dissolved in mm tris (ph . ), mm mgcl , and mm nacl supplemented with rnasin and μg ml - cycloheximide. following centrifugation for hours at , × g in a beckman coulter ultracentrifuge using an sw ti rotor, μl fractions were collected from the top of the gradient while monitoring absorbance at λ = nm on a gradient master station. rna was extracted from total cytoplasmic, polysome, or monosome fractions using ls trizol (invitrogen) according to the manufacturer's protocol. equal amounts of rna as determined by spectrophotometry using absorbance at nm on a nanodrop (thermo fisher, waltham, ma) were subject to library preparation using the illumina truseq vii rna library preparation kit (illumina, san diego, ca), and sequenced at the whitehead institute (cambridge, ma) on an illumina hiseq . reads were trimmed and mapped to the concatenated hg and vsv genomes using clc genomics workbench (qiagen, redwood city, ca). mapping parameters were as follows; mismatch cost , insertion cost , deletion cost , length fraction . , similarity fraction . , and a maximum of hits per read. raw sequence data is available from the ncbi sequence read archive (sra) under the primary accession code srp . transcripts per kilobase million (tpm) was calculated for genes with or more mapped reads in the cytoplasmic fraction of both uninfected and infected cells using the total number of mapped exon reads. to identify cellular mrnas that were potential targets for translational regulation in infected cells, we determined the tpm in the polysome fraction/tpm in the total cytoplasmic fraction for each individual mrna. this ratio was determined for uninfected and infected cells, and presented as the log fold change. gene ontology analysis was performed in r using goseq. utrs and cds sequences were downloaded from the ucsc table browser using "knowncanonical" mrna identifiers. non-protein coding rnas were excluded from the analysis. poly (a) tail length and mrna half-lives were from published data sets [ , ] . graphs and statistical analyses were performed in r using the "wilcox_test" statistical test, the "density" kernel density estimation, and "geom_boxplot" or "geom_density" functions in ggplot and cowplot. total rna was recovered from polysome fractions using ls trizol according to the manufacturer's protocol. rna ( ng) was annealed with oligo d(t) and reverse-transcribed using superscript iii reverse transcriptase (thermo fisher) at ˚c for hour. following digestion of the rna strand with rnasea and rnaseh for min at ˚c, reactions were diluted : for cellular gene-specific qpcr or : for viral gene-specific qpcr. quantitative pcr was performed using power sybr green (thermo fisher) and relative amounts determined by images of rvsv-egfp infected cells were acquired using a × objective on a nikon eclipse te microscope (nikon instruments, melville, ny) equipped with a spot rt se monochrome camera (diagnostic instruments, sterling heights, mi). for cytometry, cells were washed in hbss, trypsinized, fixed in % paraformaldehyde at ˚c for minutes and measured using a facscalibur (cytek development, freemont, ca). cytometry data was analyzed using flowjo (flowjo inc, ashland, or). for mean fluorescence intensity (mfi) we gated on live cells identified by forward and side-scatter. to measure the % infected cells we subtracted those cells that fell within the gate established from uninfected control cells. for luciferase assays, where indicated hela cells were transfected with sirna, and the medium replenished at h. cells were transfected h later with ng prl-cmv (promega), and activity measured h later. for viral driven luciferase reporters sirna transfected cells were infected at h and monitored h later. luciferase expression was measured in a spec-tramax l microplate reader using the appropriate reagents according to the manufacturer's instructions (promega, e and e ). for depletion of host factors by peptide-conjugated morpholinos (ppmos) approximately . x hela cells per well of a well plate were treated h later with μm of the indicated ppmo. at h post treatment, the media was replaced with fresh medium containing μm ppmo, and used for testing h later. cells were washed twice with ice-cold x pbs and lysed in rose lysis buffer consisting of mm tris-hcl (ph . ), mm edta, . % w/v sodium deoxycholate, and % v/v np- on ice for minutes. rose lysis buffer was supplemented with phosphatase inhibitor cocktail (sigma-aldrich, st. louis, mo) and halt protease and phosphatase inhibitor cocktail (thermo fisher) for detection of phospho-eif e-bp . lysates were clarified, protein input was normalized by bradford assay and proteins resolved on polyacrylamide gels- % for eif a and eif e or %, eif e-bp . proteins were transferred to nitrocellulose membranes for minutes, eif e and eif e-bp , or minutes, eif a, at v. membranes were blocked with odyssey blocking buffer in pbs (li-cor, lincoln, ne) for h at room temperature, and incubated overnight at ˚c with the following primary antibodies: rabbit anti-eif a (cell signaling, # ), rabbit anti-eif e (cell signaling, # ), rabbit anti- e-bp (cell signaling, # ), rabbit anti-phospho- e-bp ser (cell signaling, # ), rabbit anti-phospho- e-bp thr / (cell signaling, # ), mouse anti-actin (millipore, #mab ), mouse anti-actin (sigma, #a ). membranes were washed x with x pbs-t for minutes at room temperature, and incubated with the relevant secondary antibodies: goat anti-mouse irdye rd (li-cor, # - ) or goat anti-rabbit irdye cw (li-cor, # - ), for hour at room temperature. membranes were washed again and kept in x pbs, and scanned on an odyssey clx (li-cor). hela cells were starved in dmem lacking l-methionine (corning, # - -cl) for minutes, prior to addition of [ s] express protein labeling mix (perkin elmer, waltham, ma) at . mci ml - . cell lysates were prepared as described above and separated on a low-bis % polyacrylamide gel. the gel was dried for . h at ˚c in a vacuum gel drier and detected using a phosphoimager. quantitative analyses of band intensities was performed in image-quant tl v . (ge healthcare, marlborough, ma). for radioimmunoprecipitations, . x hela cells plated in cm dishes (corning) were starved of methionine for h at h post-plating, and labeled with [ s]-express for h. cells were washed twice with ice-cold x pbs, collected by scraping and subsequent centrifugation for minutes at ˚c and , × g and lysed in ml of mm tris (ph . ), mm nacl , mm edta, % v/v np , mm dtt, supplemented with pierce protease inhibitor (thermo fisher) on ice for minutes. protein input was normalized by bradford assay, sds was added to . %, and μl lysate was pre-cleared for h at ˚c on a nutator with μl prewashed pierce protein a agarose beads. protein a beads were pelleted by centrifugation for minutes at ˚c and , × g and the labeled supernatant incubated with primary antibody at ˚c overnight. the antibodies used for immunoprecipitation were μg anti-yb (abcam, #ab ) and μg anti-hsp (enzo, #adi-spa- ). immune complexes were isolated using μl pre-washed pierce protein a agarose beads, by incubating for hours at ˚c. bead complexes were collected by centrifugation for minutes at ˚c and , × g, washed x with μl ice-cold ip lysis buffer with . % sds, on an orbital shaker for minutes at room temperature. protein-antibody complexes were eluted by boiling in x sds loading buffer, the beads pelleted for minutes at ˚c in a microcentrifuge and the supernatant loaded on a % polyacrylamide gel. after electrophoresis the gel was dried and imaged using a phosphoimager. approximately . x hela cells were plated per well of a -well dish and hours later incubated with phosphate free media (gibco, - ) for h. thirty minutes before infection, actinomycin d-mannitol (sigma, #a ) was added to a final concentration of μg ml - . infections were carried out in phosphate free media supplemented with μg ml - actinomycin d and . μm -dmag. at hpi cells were washed, fresh medium added, and supplemented h later with [ p]-orthophosphoric acid (perkin elmer, #nex h) . μci μl - . cells were harvested at hpi in rose lysis buffer, and total rna was extracted using ls trizol. rna was separated on a m urea-agarose gel as previously described, and detected using a phosphoimager [ ] . vesicular stomatitis virus matrix protein inhibits host cell-directed transcription of target genes in vivo the role of vesicular stomatitis virus matrix protein in inhibition of host-directed gene expression is genetically separable from its function in virus assembly effect of vesicular stomatitis virus matrix protein on transcription directed by host rna polymerases i, ii, and iii vesiculoviral matrix (m) protein occupies nucleic acid binding site at nucleoporin pair (rae * nup ) inhibition of ran guanosine triphosphatase-dependent nuclear transport by the matrix protein of vesicular stomatitis virus the matrix protein of vesicular stomatitis virus inhibits nucleocytoplasmic transport when it is in the nucleus and associated with nuclear pore complexes vesicular stomatitis virus matrix protein inhibits host cell gene expression by targeting the nucleoporin nup vsv disrupts the rae / mrnp mrna nuclear export pathway vesicular stomatitis virus infection alters the eif f translation initiation complex and causes dephosphorylation of the eif e binding protein e-bp inhibition of host and viral translation during vesicular stomatitis virus infection. eif is responsible for the inhibition of viral but not host translation the mechanism of eukaryotic translation initiation: new insights and challenges quantitative studies of mrna recruitment to the eukaryotic ribosome dissociation and reconstitution of the transcriptase and template activities of vesicular stomatitis b and t virions ribonucleic acid synthesis of vesicular stomatitis virus. . multiple complementary messenger rna molecules polysomal ribonucleic acid of vesicular stomatitis virus-infected hela cells translation of vesicular stomatitis messenger rna by extracts from mammalian and plant cells in vitro synthesis of methylated messenger rna by the virionassociated rna polymerase of vesicular stomatitis virus translation and identification of the mrna species synthesized in vitro by the virion-associated rna polymerase of vesicular stomatitis virus methylated and blocked ' termini in vesicular stomatitis virus in vivo mrnas ribonucleic acid synthesis of vesicular stomatitis virus, ii. an rna polymerase in the virion l protein requirement for in vitro rna synthesis by vesicular stomatitis virus transcriptional activity and mutational analysis of recombinant vesicular stomatitis virus rna polymerase amino acid residues within conserved domain vi of the vesicular stomatitis virus large polymerase protein essential for mrna cap methyltransferase activity a single amino acid change in the l-polymerase protein of vesicular stomatitis virus completely abolishes viral mrna cap methylation a unique strategy for mrna cap methylation used by vesicular stomatitis virus unconventional mechanism of mrna capping by the rna-dependent rna polymerase of vesicular stomatitis virus a conserved motif in region v of the large polymerase proteins of nonsegmented negative-sense rna viruses that is essential for mrna capping rebinding of transcriptase components (l and ns proteins) to the nucleocapsid template of vesicular stomatitis virus location of the binding domains for the rna polymerase l and the ribonucleocapsid template within different halves of the ns phosphoprotein of vesicular stomatitis virus structure of the vesicular stomatitis virus nucleoprotein-rna complex structure of the vesicular stomatitis virus nucleocapsid in complex with the nucleocapsid-binding domain of the small polymerase cofactor molecular architecture of the vesicular stomatitis virus rna polymerase critical phosphoprotein elements that regulate polymerase architecture and function in vesicular stomatitis virus synthesis of poly(a) in vitro by purified virions of vesicular stomatitis virus in vitro synthesis of rna that contains polyadenylate by virion-associated rna polymerase of vesicular stomatitis virus site on the vesicular stomatitis virus genome specifying polyadenylation and the end of the l gene mrna aberrant polyadenylation by a vesicular stomatitis virus mutant is due to an altered l protein cis-acting signals involved in termination of vesicular stomatitis virus mrna synthesis include the conserved auac and the u signal for polyadenylation translational control of protein synthesis after infection by vesicular stomatitis virus vesicular stomatitis virus mrna and inhibition of translation of cellular mrna-is there a p function in vesicular stomatitis virus? effect of intracellular vesicular stomatitis virus mrna concentration on the inhibition of host cell protein synthesis complete intergenic and flanking gene sequences from the genome of vesicular stomatitis virus complete sequences of the ribosome recognition sites in vesicular stomatitis virus mrnas: recognition by the s and s complexes translation of mrnas from vesicular stomatitis virus and vaccinia virus is differentially blocked in cells with depletion of eif gi and/or eif gii a ribosome-specialized translation initiation pathway is required for cap-dependent translation of vesicular stomatitis virus mrnas preferential translation of vesicular stomatitis virus mrnas is conferred by transcription from the viral genome high-resolution analysis of coronavirus gene expression by rna sequencing and ribosome profiling a systematic view on influenza induced host shutoff ribosome profiling reveals translational upregulation of cellular oxidative phosphorylation mrnas during vaccinia virus-induced host shutoff localized attenuation and discontinuous synthesis during vesicular stomatitis virus transcription transcriptional mapping of vesicular stomatitis virus in vivo order of transcription of genes of vesicular stomatitis virus determination of molar ratios of vesicular stomatitis virus induced rna species in bhk cells genome-wide determination of rna stability reveals hundreds of short-lived noncoding transcripts in mammals tail-seq: genome-wide determination of poly(a) tail length and ' end modifications antiviral activity and rna polymerase degradation following hsp inhibition in a range of negative strand viruses hsp inhibitors exhibit resistance-free antiviral activity against respiratory syncytial virus hsp chaperoning in addition to phosphoprotein required for folding but not for supporting enzymatic activities of measles and nipah virus l polymerases inhibition of heat shock protein hsp -pp v-src heteroprotein complex formation by benzoquinone ansamycins: essential role for stress proteins in oncogenic transformation crystal structure and molecular modeling of -dmag in complex with human hsp txnip potentiates redd -induced mtor suppression through stabilization of redd chemical inhibition of rna viruses reveals redd as a host defense factor apobec g-regulated host factors interfere with measles virus replication: role of redd and mammalian torc inhibition separate pathways of maturation of the major structural proteins of vesicular stomatitis virus biophysical studies of eif e cap-binding protein: recognition of mrna ' cap structure and synthetic fragments of eif g and e-bp proteins an overlapping protein-coding region in influenza a virus segment modulates the host response selective degradation of host rna polymerase ii transcripts by influenza a virus pa-x host shutoff protein characterization of a second vaccinia virus mrna-decapping enzyme conserved in poxviruses vaccinia virus d protein has mrna decapping activity, providing a mechanism for control of host and viral gene expression malignant transformation by a eukaryotic initiation factor subunit that binds to mrna ' cap new mrnas are preferentially translated during vesicular stomatitis virus infection pathway of vesicular stomatitis virus entry leading to infection entry pathway of vesicular stomatitis virus into different host cells vesicular stomatitis virus enters cells through vesicles incompletely coated with clathrin that depend upon actin for internalization systematic identification of type i and type ii interferoninduced antiviral factors eif targets cell-proliferation messenger rnas for translational activation or repression ' utr m( )a promotes cap-independent translation eif d is an mrna cap-binding protein that is required for specialized translation initiation dynamic m( )a mrna methylation directs translational control of heat shock response ability of the matrix protein of vesicular stomatitis virus to suppress beta interferon gene expression is genetically correlated with the inhibition of host rna and protein synthesis rna molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination we thank members of the whelan lab for scientific discussions and suggestions; we further thank carl novina and cailin joyce for help with polysome profiling, keith ketterer for assistance with clc genomics workbench, and alos diallo for help with r and statistical analyses. we thank david stein and hong moulton, of oregon state university, for design and production of the ppmos used in this study. key: cord- -v eh authors: chughtai, abrar ahmad; khan, wasiq title: use of personal protective equipment to protect against respiratory infections in pakistan: a systematic review date: - - journal: j infect public health doi: . /j.jiph. . . sha: doc_id: cord_uid: v eh like other low-income countries, limited data are available on the use of personal protective equipment (ppe) in pakistan. we conducted a systematic review of studies on ppe use for respiratory infections in healthcare settings in pakistan. medline, embase and goggle scholar were searched for clinical, epidemiological and laboratory-based studies in english, and studies were included; all were observational/cross-sectional studies. the studies examined ppe use in hospital (n = ), dental (n = ) or laboratory (n = ) settings. policies and practices on ppe use were inconsistent. face masks and gloves were the most commonly used ppe to protect from respiratory and other infections. ppe was not available in many facilities and its use was limited to high-risk situations. compliance with ppe use was low among healthcare workers, and reuse of ppe was reported. clear policies on the use of ppe and available ppe are needed to avoid inappropriate practices that could result in the spread of infection. large, multimethod studies are recommended on ppe use to inform national infection-control guidelines. healthcare workers are at the frontline when treating infectious disease cases and at high risk of acquiring influenza and other respiratory infections [ ] [ ] [ ] . several outbreaks of new infectious diseases have occurred in recent decades, such as the outbreak of severe acute respiratory syndrome coronavirus (sars-cov) in - [ ] , influenza pandemic (h n ) in [ ] , middle east respiratory syndrome coronavirus (mers-cov) in [ ] and ebola virus diseases in - [ ] . many healthcare workers were infected and died during these outbreaks because of a lack of infection control [ , , , ] various infection control strategies are used to protect healthcare workers from respiratory and other infections in healthcare settings [ , ] . these strategies can be broadly classified as administrative control measures, environmental control measures and the use of personal protective equipment (ppe). administrative control measures include developing policies and procedures, implementing triage protocols and providing health education and trainings. environmental control measures includes ensuring proper ventilation, establishing airborne infection isolation and negative pressure rooms, developing systems for cleaning and waste disposal. [ , ] . ppe is commonly used in healthcare settings as standard or transmission based precaution to protect healthcare workers from infections and to prevent further spread to patients around them [ , ] . ppe is generally ranked lowest in the infection control hierarchy due to less effectiveness compared to other control measures and high expenditure in the long run. therefore, most infection control guidelines recommend using ppe together with other administrative and environmental control measures. however, ppe is important during the early stage of an outbreak or a pandemic when drugs, a vaccine and other control measures are not available, or access is limited. commonly used ppe to protect from respiratory infectionsare; face masks, respirators, gloves, and goggles or face shields [ ] . face masks (or medical masks) and respirators are the most commonly used ppe to protect from influenza and other respiratory infection in healthcare settings. however, these two products are not the same. face masks are not designed for respiratory protection and are used to avoid respiratory droplet and spray of body fluids on the face. they are also used by sick patients to prevent spread of pathogens to others (referred to as "source control"), or by surgeons in the operating theatre to maintain a sterile operating field. face masks are not fit to the face and have varying filtration capacities [ ] . respirators ratory protection and are used to protect from respiratory aerosols [ ] . a properly fitted respirator provides better protection again respiratory infections than a face mask. gloves are used to protect hands from blood and body fluids, including respiratory secretions. goggles and face shields are used to prevent transfer of respiratory pathogens into the eyes from contaminated hands and other sources. gowns, coveralls, surgical hoods and shoe covers can also be used where procedures on infectious patients generate aerosols or when a new respiratory virus has emerged [ ] . there is an ongoing debate about the selection and use of various types of ppe in healthcare settings. this is mainly because of a lack of high quality studies on the use of ppe. most studies are observational and on the use of masks and/or respirators [ ] . to date, only five randomized clinical trials have been conducted on use of ppe in hospital settings and all were on face masks/respirators [ ] . moreover, most studies on ppe use were conducted in high/middle income countries and currently there are limited data from lowincome countries where the burden of infectious diseases is high. it is therefore important to examine the use of ppe in low resource countries to inform infection control policies. pakistan has a population of about million. as a low-income country, its gross domestic product is low, as is its expenditure on health [ ] . the country has one of highest rates of infant and maternal mortality in the south asia region. infectious diseases are still among the main causes of death, particularly in young children. health and surveillance systems are generally weak and limited data are available on infection prevention and control strategies. the aim of this study was to examine the use of ppe for respiratory infections in healthcare settings in pakistan. a systematic review was conducted using the preferred reporting items for systematic reviews and meta-analyses (prisma) guidelines. we searched for studies on the electronic databases medline and embase using selected key words. a combination of keywords were used including: 'face mask' or 'mask' or 'medical mask' or 'surgical mask' or 'cloth mask' or 'respirator' or 'gloves' or 'gowns' or 'coverall' or 'surgical cap/hood' or 'shoe/boot covers' or 'goggles' or 'face shield' or 'eye protection' and ' respiratory infection' or 'respiratory tract infection' or 'respiratory diseases', 'outbreaks' or 'infectious disease' or 'influenza' or 'pandemic influenza' or 'flu' or 'tuberculosis' or 'pneumonia' and 'pakistan' or 'punjab' or 'sindh' or 'balochistan' or 'khyber pakhtunkhwa'. we used an open date strategy up to december . we anticipated that studies published in local journals might not be indexed on the medline or embase, therefore, an additional search was made on google scholar using the same keywords. we set a limit of results per page on google scholar and first three pages were reviewed for each keyword search. after the initial search; we reviewed titles and abstracts and selected studies for full text review (fig. ) . clinical, epidemiological and laboratory-based studies conducted in any part of pakistan and published in english were included in the review. the focus of this systematic review was on the use of ppe for prevention of respiratory infections. therefore, we only included those studies which examined the use of facemask and/or respirator in healthcare settings, with or without other ppe. we only included those studies where ppe was discussed for respiratory infections. studies where ppe was examined for general infection control were also included, given respiratory protective equipment (face masks and/or respirators) was mentioned. we excluded studies on the use of ppe only for bloodborne infections. conference abstracts and poster presentations were also excluded. a total of studies were found in the initial search. after reviewing titles and abstracts, studies were selected for full text review. finally, articles were included in this review (table ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . we only found observational/cross-sectional studies on the use of ppe for infectious diseases in healthcare settings in pakistan. in all studies data were collected through questionnaires or interviews. no clinical trials or laboratory-based studies on the use of ppe in such settings were found. seven studies examined the use of ppe in hospital [ ] [ ] [ ] [ ] ] and among those, two examined the ppe perceptions among medical students [ ] or pharmacy students [ ] . two studies were conducted in the laboratory settings [ , ] while, four in dental settings [ ] [ ] [ ] [ ] two studies focused on the use of ppe for influenza [ , ] , two were for tuberculosis [ , ] and nine studies were on multiple respiratory diseases, including influenza [ , ] or general infections [ , [ ] [ ] [ ] [ ] [ ] [ ] . only two studies examined the use of ppe alone [ , ] , while other studies examined other infection control practices as well [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . guidelines and standard operating procedures on ppe do not exist in most of the hospitals [ ] or laboratories [ , ] in pakistan. two studies examined the guidelines and current practices on the use of face masks/respirators for influenza, tuberculosis and sars in pakistan [ , ] . recommendations on the use of masks were reported to be inconsistent and different types of product were recommended and used in various healthcare settings [ , ] . face masks were the most commonly used ppe to protect from respiratory infections in most hospitals in pakistan. medical masks were generally used to protect from influenza, tuberculosis and other respiratory infections, while the use of respirators was limited to high-risk situations [ , ] . in a cross-sectional survey among final-year pharmacy students in seven universities of karachi, about % of participants highlighted the need to cover the nose or mouth to protect from influenza and about % highlighted the use of face masks, gloves and other ppe [ ] . laboratory coat and gloves were the most commonly used ppe in the laboratories in pakistan while face masks and eye covers were rarely used [ , ] . a survey of dentists working in various settings (dental colleges, hospitals and private clinics) showed that face masks and gloves were also commonly used ppe [ ] . the use of ppe was also reported to be low among health workers. according to a hospital-based survey, face masks are not provided to patients with tuberculosis and respirators are not provided to the healthcare workers [ ] . another survey showed that % of participants used ppe for patients with suspected tuberculosis and % used ppe for patients with confirmed tuberculosis [ ] . a study in a ward for patients with multidrug-resistant tuberculosis reported that % of the healthcare workers used n respirators and % were provided with a mask [ ] . a study on biosafety level (bsl) laboratory workers showed that ppe was not used by about half of the staff ( . %) [ ] . a countrywide survey showed that almost one third ( . %) of bsl- laboratory workers did not routinely use ppe [ ] . both gloves and laboratory coats were used by only . % of the personnel, while a laboratory coat or gloves alone were used by . % and . %, respectively. less than % of all the respondents across pakistan reported using eye covers [ ] . in a survey of medical students during pandemic (h n ) , % said that they would use a face mask to protect from infection. students with less risk perception were more hesitant to use face masks [ ] . the use of face masks was common in dental practice and according to various surveys, - % of dentists wear masks during dental procedures [ ] [ ] [ ] [ ] . across all the studies in dental settings, more than % also used gloves. among the ppe, face masks were considered the most bothersome to use by wearers. reuse of ppe was also reported in many studies, mainly because of unavailability of ppe and lack of training. gowns are shared among the healthcare workers in hospital many times [ ] . two surveys in dental clinics showed that more than half of the dentist reuse masks during routine work [ , ] . the availability of ppe was generally low in all healthcare settings [ , , ] and varied according to the type [ ] ; gloves and masks were available while gowns and n respirators were not available in several wards [ ] . a shortage of ppe was also reported during sars and pandemic (h n ) [ , ] . a lack of training was a common issue reported and most healthcare workers were not trained in the use of ppe. most of the studies ( / ) discussed other infection control practices as well, in addition to the use of ppe. other non-standard infection control practices included reuse of syringes, improper waste disposal, a lack of hand hygiene practices, non-isolation of infectious cases and low influenza vaccination among healthcare workers. we reviewed the use of ppe in various healthcare settings in pakistan. a lack of guidelines and standard operating procedures, inconsistent policies and practices, low compliance, and non-availability and reuse of ppe were the main issues highlighted in this study. evidence is lacking on the use of ppe in hospitals and other healthcare settings in pakistan and most studies are of low quality. clinical studies should be conducted to examine the effectiveness of ppe and improve the compliance. reuse of ppe may increase the risk of self-contamination to the wearer and this practice should be discontinued. there is a need to improve the availability of ppe and healthcare workers should be trained. ppe is generally considered lowest in the infection control hierarchy and is generally recommended in combination with other control measures. other infection control practices in such settings should also be examined. different types of ppe are used by healthcare workers in pakistan, which reflects a lack of standard policies and guidelines. the different policies and practices may be because of the different recommendations by the world health organization (who) and the united states (us) centers for disease control and prevention (cdc) [ , ] . debate continues about the selection and use ppe for different infections, for example, face masks versus respirators, gowns versus coveralls, face shields versus goggles [ , , , ] . selection of ppe mainly depends on mode of transmission, however, several individual and organizational factors also contribute the selection and use of ppe, such as risk perception, presence of adverse events, pre-existing medical illness, availability and cost [ ] . respiratory infections are generally transmitted through contact, droplet and/or airborne routes. gloves should be used to protect from infections transmitted through contact (e.g. respiratory syncytial virus and adenovirus), face masks should be used for droplet infections (e.g. influenza and coronavirus) and a respirator should be used to protect form airborne infection (e.g. tuberculosis and measles). however, infection transmission is rarely by only one route and most infections are transmitted by more than one route [ ] . for example, influenza and sars primarily transmit through droplet and contact routes, but airborne transmission has also been reported [ , ] . similarly, ebola primarily transmits through direct contact with blood and body fluids [ ] , but animal studies have shown that airborne transmission is also possible [ ] . the risk of transmission further increases during aerosol-generating and other high-risk procedures [ , ] . moreover, uncertainty exists about how pathogens transmit during outbreaks and pandemics [ , , , ] . therefore, superior ppe should be used where the mode of transmission is uncertain, the case-fatality rate is high and pharmaceutical interventions are not available [ ] . infection control guidelines in pakistan need to be updated urgently to reflect these recommendations. given that mers cov is circulating in the eastern mediterranean region (emr), policies and practices on the use of ppe in other countries of the region should also be examined. our study also reported low availability of ppe in hospital, dental and laboratory settings in pakistan. the availability of ppe is a challenge, not only in low-resource counties, but also in highincome countries, particularly during outbreaks and pandemics when the use of ppe greatly increases [ , ] . this may result in non-standard practices such as reuse and extended use of ppe. shortages of ppe were even reported in many high-income countries during the influenza h n pandemic and staff had to use various alternatives [ ] [ ] [ ] . the availability of ppe is important to ensure proper use and compliance. low use of ppe among laboratory workers in pakistan may be due to non-availability and a lack of resources. for example, ppe use was relatively higher in laboratory workers in punjab, which is an affluent province, than other provinces [ ] . moreover ppe use was reported more in the private sector in pakistan than the public sector which has fewer resources [ ] . proper use of ppe depends on several factors such as availability, knowledge, training, risk perception and comfort [ , , ] . this study showed the compliance with the use of ppe was generally low among healthcare workers and was mainly due to unavailability of ppe, discomfort and a lack of training. while the use ppe depends on many factors, a greater perception of risk was positively associated with compliance [ ] . continuous use of face masks and respirators may have psychological and physiological effects on the wearer and result in more adverse events [ ] [ ] [ ] . compliance with the use of face masks has been shown to be based on the nature of the disease, infectiousness of patients and the performance of high-risk procedures [ ] . previous studies have tested the precede (predisposing, reinforcing and enabling) framework to examine healthcare workers' compliance with universal precautions [ ] . the results showed that reinforcing factors, such as availability of ppe and less job hindrance, and enabling factors, such as safety climate and regular feedback, were significant predictors of compliance with ppe [ ] . in addition, the health belief model [ ] was also used to examine the compliance and use of face mask during the sars outbreak [ , ] . perceived susceptibility (vulnerability to acquiring sars and close contact with case), perceived benefits (that face masks can prevent infection) and cues to action (someone asked them to use face masks) were significant predictors of protective behaviour and use of face masks [ ] . our study showed that most healthcare workers were not trained on the use of ppe in pakistan. the risk of infection can be reduced with proper training and availability of policies and standard operating procedures [ ] . however, regular monitoring is also required to make sure that healthcare workers are using ppe according to the protocols. a study in the us reported many deviations from the protocols even though all healthcare workers were trained [ ] . this may result in self-contamination to the wearers and the spread of infection to others [ ] . training programmes should be arranged for newly recruited staff and then annual refresher courses should be provided. our study had some limitations. the initial search was made on medline and embase but very few studies were retrieved because many papers are not indexed on these databases. therefore, we also searched google scholar but we only reviewed the first pages after each search so some studies could have been missed. however, we checked the references lists of the relevant studies and could not find any other studies. our search was up to and studies in were not included. we only considered ppe in this study and did not examine other infection control practices. the use of ppe is generally recommended with other administrative and environmental control measures. the selection and use of ppe vary according to the type of healthcare worker and working environment. face masks and gloves were the most commonly used ppe to protect from respiratory and other infections. overall, compliance with the use of ppe was low, and non-availability and reuse of ppe were reported. most studies were observational and large-scale prospective studies are needed to collect more evidence about the use of ppe in healthcare settings in pakistan. no funding sources. aac tested the filtration of mask samples by min in another study; m products were not used in this study. wk declares none. as this was a systematic review of published data, ethics approval was not required. aac devised the structure and topic areas for this review and made the initial search. wk and aac reviewed titles and abstracts and selected studies for full text review. aac prepared the first draft of manuscript and both authors contributed equally to the final manuscript. influenza and rhinovirus infections among health-care workers nosocomial transmission of measles among healthcare workers tuberculosis among health care workers emergencies preparedness, response. summary of probable sars cases with onset of illness from world health organization. emergencies preparedness, response. pandemic (h n ) -update comparative epidemiology of middle east respiratory syndrome coronavirus (mers-cov) in saudi arabia 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protection in a health care setting the cookie monster muffler: perceptions and behaviours of hospital healthcare workers around the use of masks and respirators in the hospital setting behavioral-diagnostic analysis of compliance with universal precautions among nurses social learning theory and the health belief model factors influencing the wearing of face masks to prevent the severe acute respiratory syndrome among adult chinese in hong kong practice of habitual and volitional health behaviors to prevent severe acute respiratory syndrome among chinese adolescents in hong kong personal protective equipment for the ebola virus disease: a comparison of training programs risk of self-contamination during doffing of personal protective equipment key: cord- -m ox z authors: rogers, jonathan p; pollak, thomas a; blackman, graham; david, anthony s title: catatonia and the immune system: a review date: - - journal: lancet psychiatry doi: . /s - ( ) - sha: doc_id: cord_uid: m ox z catatonia is a psychomotor disorder featuring stupor, posturing, and echophenomena. this series paper examines the evidence for immune dysregulation in catatonia. activation of the innate immune system is associated with mutism, withdrawal, and psychomotor retardation, which constitute the neurovegetative features of catatonia. evidence is sparse and conflicting for acute-phase activation in catatonia, and whether this feature is secondary to immobility is unclear. various viral, bacterial, and parasitic infections have been associated with catatonia, but it is primarily linked to cns infections. the most common cause of autoimmune catatonia is n-methyl-d-aspartate receptor (nmdar) encephalitis, which can account for the full spectrum of catatonic features. autoimmunity appears to cause catatonia less by systemic inflammation than by the downstream effects of specific actions on extracellular antigens. the specific association with nmdar encephalitis supports a hypothesis of glutamatergic hypofunction in catatonia. catatonia is a psychomotor disorder characterised by diverse clinical signs, including mutism, negativism, ambitendency, stereotypy, posturing, waxy flexibility, and echophenomena. the structure and neural mechanisms of the disorder are reviewed elsewhere in this issue of the lancet psychiatry by walther and colleagues. understanding the pathophysiology of this severe disorder is crucial given its high rate of medical complications, including pressure ulcers, infections, and venous thromboembolism. moreover, such under standing might aid the comprehension of other neuro psychiatric disorders. although catatonia has numerous possible symptom combinations, compelling reasons to study it as a single entity exist. clinical and demographic factors can dis tinguish catatonia from other psychotic and affective disorders. different forms of catatonia (retarded catatonia, malignant catatonia, and neuroleptic malignant syndrome) are highly comorbid. in terms of treatment, response rates to benzodiazepines and electroconvulsive therapy (ect) are high, regardless of the cause of the catatonia. moreover, catatonia is not a common disorder, so pragmatically, to study it in depth, considering it as a whole is useful. immune dysregulation is gaining interest as a patho physiological mechanism underlying neuro psychi atric disorders as diverse as narcolepsy, some dementias, depression, and psychosis-with converging evidence from biochemical, neuroimaging, genetic, and post mortem studies. , roles for both the innate immune system, which concerns the rapid, undirected response to pathogenassociated or injuryrelated signals, and the adaptive immune system, which functions over a longer timescale and involves the selection and maturation of antigenspecific tcell and bcell mediated responses, have been identified. in this series paper, we discuss the evidence for the involvement of the immune system in catatonia. this line of enquiry appears to be valuable, given the wide range of infective and inflammatory conditions that can cause catatonia (tables , ). we address whether the immune system has a role in catatonia, using some direct and some more circumstantial evidence, and endeavour to establish specific models. we consider immunity in terms of innate and adaptive systems for the purposes of clarity, while acknowledging that strictly demarcating the two is not always possible. a systematic review reported that % of catatonia has a general medical cause, of which cns inflammation (comprising both infective and immune causes) accounts for %. numerous infectious diseases have been reported to cause catatonia. here, we present the results of a new systematic search of the literature (table ; appendix). we identified cases, the majority of which were published as case reports, with the remaining reported as case series. laboratory evidence of infection (such as isolation of the organism in the serum, or viral dna in the cerebrospinal fluid [csf] ) was reported in of the cases ( %). a robust temporal association between the infection and catatonia was reported in of the cases ( %). a previous psychiatric disorder was recorded in cases ( %) and a previous medical disorder in cases ( %), although the absence of a preexisting condition was often not stated. only of the cases ( %) recorded the presence of at least two features from the bushfrancis catatonia screening instrument (bfcsi); the remainder had insufficient description or only one feature. in some cases, the catatonia resolved with antimicrobial therapy, but in others, it required treatment with benzodiazepines or ect. how infection might result in catatonia is unclear from the literature. possibilities include a direct neurotoxic effect, a psychological reaction to the infection, or mediation by an acutephase response. out of the cases where a specific virus was implicated, of these involved known neurotropic viruses, suggesting a direct neuro toxic series effect. some bacterial agents, such as borrelia burgdorferi and treponema pallidum are also known to infect the cns. the immunological response might also be important, given that in some neurological disorders, such as meningoencephalitis, damage is caused primarily by an immune reaction. in several cases, an explicit immune response was suggested by the authors to explain the catatonia, such as in paediatric autoimmune neuropsy chiatric disorders associated with streptococcal infection (pandas), or in nmethyldaspartate receptor (nmdar) encephalitis purportedly triggered by yellow fever vaccination, herpes simplex virus infection, or epsteinbarr virus infection. in cases of pyrexia of unknown origin in which an infective cause was often assumed, a yet uncharacterised disorder might have been responsible. , although cases of overt catatonia in the context of infections are dramatic, the more common neuro psychiatric presentation of infection is a broader pheno type of illness behaviour that resembles depression. this presentation includes reductions in motor activity, oral intake, and social interaction, all of which are seen in catatonia. psychomotor activity is also slowed in mild experimentally induced infection. this might be due to impaired spatial memory performance and aberrant activity in parts of the brain involved in interoception. hence, the brain's response to inflammation, if severe, could result in a complex movement disorder such as catatonia. , in response to an acute stressor, immune cell trafficking occurs, with the movement of leukocytes to, and within, a target organ. however, in chronic stress, increased monocyte production and microglial activation result in neuroinflammation and are associated with depressive behaviour. depression is often associated with raised levels of proinflammatory cytokines, granulocytes, and monocytes. regarding subtypes of depression, atypical depression (characterised by mood reactivity, hyperphagia, hypersomnia, and leaden paralysis) is most associated with raised inflammatory markers. conversely, psycho motor retardation is more commonly seen in melancholic depression, which is less associated with a peripheral pro inflammatory state. seasonal affective disorder has also been associated with a proinflammatory state, but there has been little research to date on the motor phenotype of this disorder. neuroleptic malignant syndrome is a neurological emergency precipitated by antipsychotic use and charac terised by muscular rigidity, autonomic dysfunction, and altered consciousness. patients treated with anti psychotics who have preexisting catatonia are at an increased risk of developing neuroleptic malignant syndrome compared with those who do not have catatonia ( · % vs given that no clinical features exist that can reliably distinguish neuroleptic malignant syndrome from malignant catatonia, some authors consider neuroleptic malignant syndrome to be a specific form of antipsychoticinduced malignant catatonia. residual cata tonia frequently remains after the resolution of the full syndrome of neuroleptic malignant syndrome. some suggest that inflammation is important to the pathophysiology of neuroleptic malignant syndrome, with acutephase responses such as leukocytosis, thrombo cytosis, and low serum iron frequently reported. , low serum iron has emerged as a promising biomarker. , it has been hypothesised that in neuroleptic malignant syndrome, proinflammatory cytokines might reduce the levels of the neuroprotective kynurenic acid, impairing the activity of dopaminergic neurons in the midbrain, causing exquisite sensitivity to a further antipsychotic induced reduction in dopamin ergic signalling. however, an inflam matory profile in the blood might be the consequence of rhabdomyolysis, rather than the primary pathology. serotonin syndrome, a rare adverse effect of anti depressant medication, has also been described as a form of druginduced catatonia, but to the authors' knowledge no research has been published linking it to the immune system. the acutephase response is a core part of the innate immune system. the response is initiated by the activation of monocytes and macrophages by a stimulus, such as muscle breakdown, infection, physical injury, or psychological stress. in response to these stimuli, cells release proinflammatory cytokines such as interleukin (il ), il , and tumor necrosis factoralpha (tnfα), which in turn act on receptors throughout the body to promote fever, anorexia, muscle catabolism, and activation of the hypothalamicpituitaryadrenal axis. importantly, these cytokines also alter protein synthesis in the liver, causing increased production of acutephase proteins such as creactive protein (crp), procalcitonin, ferritin, and fibrinogen. , some features of malignant catatonia bear notable similarities to the acutephase response, including fever, motor hypoactivity, and autonomic disturbance. here, we include a summary of the evidence for the presence of systemic inflammation, as measured by acutephase reactants and related proteins (table ) . creatine kinase (ck) is not an acutephase marker, but as it is a marker of muscle breakdown, the enzyme is sometimes raised as a downstream consequence of the acutephase response. the evidence for ck elevation in catatonia is equivocal and could be argued to be the result of muscular rigidity and excessive series immobilisation rather than indicating a primary muscular pathology. in one study, a raised ck predicted a good response to treatment with lorazepam. one study found the acutephase marker, and fibrin degradation product, ddimer to be raised in all catatonic patients tested, with a mean value times higher than in noncatatonic psychiatric patients. this finding is consistent with the increased risk of venous thromboembolism in catatonia, but has not yet been replicated. highsensitivity crp concentration was measured in one study and found to be raised in catatonic patients, but the absolute concentration of crp was not very elevated ( · mg/dl). low serum iron was originally hypothesised to be present in catatonia given the similarities to neuroleptic malignant syndrome. low serum iron is an established feature of the acutephase response and arises because of the upregulated production of ferritin and hepcidin by the liver. two uncontrolled studies have shown that between % and % of catatonic episodes were accompanied by serum iron concentrations below the reference range. , when catatonic patients have been compared with psychiatric controls however, the results have been ambiguous. , , the authors of one of the negative studies that used unmedicated patients speculated that iron might have been reduced in other reports because of the effect of antipsychotic medi cations. in several studies, low serum iron in catatonia has been associated with the subsequent development of neuroleptic malignant syndrome. , , this association might exist because iron is a cofactor for dopamine synthesis, so a combination of low iron impairing dopamine production and antipsychotic medications blocking dopamine receptors could result in the patho logical hypodopaminergic signalling charac teristic of neuroleptic malignant syndrome. abnormalities of cerebral white matter, which is composed of glial cells, have been associated with schizophrenia, depression, and autism. ʹ, ʹcyclic nucleotide ʹphosphodiesterase (cnp) is a myelin protein that is specific to oligodendrocytes. in a mouse model, hetero zygotes for a cnp lossoffunction genotype showed axonal degeneration and lowgrade inflammation, along with a depressive and catatonic phenotype. furthermore, this behaviour was alleviated by ablation of microglia, suggesting that microgliamediated neuroinflammation was underlying the phenotype. when this polymorphism was examined in individuals with schizophrenia, a striking association existed with catatonicdepressive behaviour, a finding that was replicated in an independent cohort. mutations of mouse genes encoding two other myelin proteins (myelin basic protein [mbp] and myelin proteolipid protein [plp]) also result in a catatonic phenotype. how glial dysfunction-because of relevant polymorphisms or other factors-might contribute to the psychomotor features of catatonia clearly represents an important focus of future research. the mainstay of current treatment for catatonia is benzodiazepines and ect, neither of which is classically understood as an immunomodulatory therapy. benzo diazepines are positive allosteric modulators at the γaminobutyricacid (gaba)a receptor. although research into the function of gaba in the immune system is at an early stage, evidence suggests that gabaergic signalling has a role in suppression of immune responses. lymphocytes express gabaa receptors, and activation of these receptors reduces production of proinflammatory cytokines. however, one study specifically on catatonia found higher monocyte counts predicted benzodiazepine nonresponse. data distinguishing different benzodiazepines are sparse, but some benzodiazepines, such as diazepam and lorazepam (both recognised treatments for catatonia) but not clonazepam, also bind to translocator protein (tspo), a mitochondrial protein associated with phagocyte activity, immune cell migration, and cytokine function. , in rats, diazepam reduces tspo in the brain and decreases the number of cns inflammatory cells, giving it a protective function against experimental autoimmune encephalo myelitis. reports on other gabaa receptor modulators are scarce, but epidemiological studies exist that indicate that zolpidem use is associated with higher rates of infections (including of pyelonephritis, which would be unlikely to be related to respiratory depression), suggesting the drug might also have an immuno suppressant role. , regarding ect, a single session appears to activate the immune system, increasing concentrations of the cytokines il β, il , il , and tnfα. however, a course of several sessions appears to downregulate immune system activity, at least in animal studies. minocycline is an antimicrobial drug that also has anti inflammatory properties. the drug has been shown to prevent stressinduced microglial changes in rodents and has been proposed as an adjunctive treatment for schizophrenia. some evidence suggests that minocycline might reduce negative symptoms in schizophrenia, some of which (such as poverty of speech, affective blunting, and avolition) overlap with catatonia. however, a double blinded, randomised study which specifically aimed to examine the effect of this drug on negative symptoms did not find any benefit. no studies of which we are aware have investigated minocycline specifically for catatonia, but reports exist of two patients with schizophrenia and prominent catatonic features who responded well to minocycline in the absence of infection. , the evidence for innate immunity we have argued that psychological stress and infection both cause a release of proinflammatory cytokines, series which result in a state of motor hypoactivity. in a normal psychomotor response, this event might be adaptive, allowing conservation of energy for eliminating a pathogen or avoiding a stressor, and resolving when the stressor ends. however, in depression, a prolonged pro inflammatory state might be maladaptive and cause further dysfunction. immobilisation itself can also result in activation of the innate immune system. studies specifically in catatonia have been sparse and conflicting. an argument could be made that catatonia is an exaggerated version of inflammatory depression, in which extreme psychomotor retardation culminates in stupor and mutism-neurovegetative features of catatonia hypothesised to be due to disordered topdown cortico subcortical signalling. however, this hypothesis would not explain the perseverativecompulsive behaviours exhibited in catatonia (posturing, stereotypy, mannerism, echophenomena, and perseveration), which have been proposed to arise due to disrupted corticocortical signalling. the infective causes of catatonia are largely pathogens that infect the cns (table ), which suggests that the causality is mediated by neurotoxic mechanisms, rather than by a systemic inflammatory responsealthough a maladaptive immune response to the pathogen might contribute. a plethora of autoimmune neurological diseases exist, many of which, such as multiple sclerosis, neuromyotonia, and sydenham's chorea, feature prominent movement disorders. we have chosen the examples of stiff person syndrome and narcolepsy to show some particular points of similarity to catatonia. stiff person syndrome is a rare neurological disorder characterised by gradually progressive increased muscle tone with the preservation of muscle power, sensation, and cognitive function. most patients have autoantibodies against the enzyme glutamic acid decarboxylase (gad ). gad is an enzyme that converts glutamate to gaba, although the pathogenicity of gad auto antibodies in stiff person syndrome is not fully established. given that the disorder bears several similarities to catatonia, one author has suggested testing for gad autoantibodies to distinguish between the two disorders. the syndromes share immobility, an emotionless facial expression, and marked anxiety. moreover, hypertonic episodes in stiff person syndrome can have psychological triggers. as with catatonia, the mainstay of treatment for stiff person syndrome is benzodiazepines; however, immuno therapy in the form of intravenous immuno globulin, cortico steroids and the antibcell monoclonal antibody rituximab are increasingly used. a stiff person syndrome variant, progressive encephalomyelitis with rigidity and myoclonus, responds dramatically to immuno suppression. , narcolepsy type is a sleep disorder that arises due to depletion of the orexinproducing neurons in the hypothalamus. evidence that this event is immune mediated comes from linkage to hla-dqb * : and outbreaks coinciding with epidemics of, and vaccination to, the h n influenza virus, suggesting a possible role for molecular mimicry. a small study published in suggested that some patients with narcolepsy have autoantibodies to the nmdar, without the seizures or autonomic disturbance characteristic of nmdar auto immune encephalitis. narcolepsy type also features cataplexy, a sudden loss of motor tone usually triggered by positive emotions. cataplexy usually lasts for up to min, but occasionally status cataplecticus lasting for hours to days can occur. this occurrence has been hypothesised to be due to either a prolonged emotional response to the original stimulus, or an emotional response to the cataplexy per se. here, we show a comparison between cataplexy and catatonia (table ). we did a systematic literature search for autoimmune disorders causing catatonia (table ; appendix). most are presented in case reports and case series, with some larger case series for nmdar encephalitis (table ) . of the cases ( %) recorded at least two features from the bfcsi. patients ( %) had previously had a psychiatric disorder and ( %) had previously had a medical disorder, although an absence of a preexisting condition was often not stated. in some cases, the autoimmune disorder appeared to be the proximal cause of the catatonia. in other cases, the autoimmune disorder was a more distal cause, as in one patient with autoimmune polyendocrine syndrome who developed autoimmune destruction of the adrenal gland (addison's disease), resulting in hyponatraemia and subsequent in addition, q . deletion syndrome, which features thymic aplasia and a resultant absence of peripheral t cells, has also been linked to catatonia. whether this association is due to immunodeficiency, the high rates of various autoimmune disorders present in the syndrome, or to another cause remains unclear. the most noteworthy result from our systematic review is that % ( / ) of all cases of autoimmune catatonia reported were due to nmdar encephalitis, despite the disorder only being described in (table ) . before discussing this finding of autoimmunity directed against the cns in depth, we will illustrate the complexity of autoimmune catatonia with three examples of peripheral autoimmunity. two cases of catatonia in pernicious anaemia have been reported, both of whom responded to vitamin b supplementation. , dietary vitamin b deficiency might also cause catatonia. , in thyroid disease, catatonia has been reported in patients with thyroid autoantibodies with hyper thyroid, [ ] [ ] [ ] hypothyroid, , and euthyroid , states. however, catatonia has also occurred in hypothyroidism due to thyroidectomy; whether thyroid status or the presence of the autoantibodies is the causally relevant factor therefore remains unclear. in systematic lupus erythematosus, cases of catatonia have been reported, generally with high titres of antinuclear antibody and antidoublestranded dna; however, making further comparisons is difficult because testing panels have varied across studies (appendix). one group reported cases of paediatric catatonia of which they suspected had an autoimmune origin, including two patients with evidence of inflammation who were responsive to immunosuppression but who could not be diagnosed with any known disorder. pandas and the broader concept of paediatric acute onset neuropsychiatric syndrome (pans) are characterised by an abrupt onset of obsessive behaviours or motor tics. this behaviour might be due to molecular mimicry, whereby antigens on the infective agent bear a similarity to and provoke a host immune response to selfcns antigens. antistreptococcal antibodies are often positive, although results of immunotherapy have been equivocal. one case has been reported of a boy who developed catatonic symptoms in addition to obsessionality following infection with group a streptococcus; he responded well to lorazepam and plasmapheresis. autoimmune encephalopathies, as examples of auto immune disorders directed at cns targets, merit special consideration. tcell mediated disorders, such as acute demyelinating encephalomyelitis can occasionally present with catatonia. however, catatonia is more commonly a feature of autoimmune encephalitides associated with antineuronal antibodies. these antibodies can cause internalisation of the antigen, inhibiting its function. that catatonia has been reported in two patients with gabaa receptor antibodies is unsurprising, given the centrality of benzodiazepines in treatment for catatonia. , catatonia might be more common than these case reports would suggest, as careful psychiatric phenotyping has not been done among this population. in one of the patients reported with catatonia, gabaa receptor antibodies were present in the serum on the original presentation, but not in the context of relapse, highlighting that testing serum only on a single occasion might increase the risk of missing a clinically significant syndrome. nmdar encephalitis is increasingly considered as an organic cause of psychosis, although controversy exists as to whether this idea is only relevant in the context of the classical encephalitis or also in isolated psychiatric presentations. , the association nmdar encephalitis with catatonia seems to be even stronger than the association with psychosis. where catatonia is reported, it is often malignant catatonia and tends to cooccur with psychosis and mania. nmdar encephalitis is strongly linked to neuroleptic malignant syndrome, with one study suggesting as many as out of ( %) patients with nmdar antibody encephalitis who were administered antipsychotics developed suspected neuro leptic malignant syndrome. of the total cases of nmdar encephalitis documented across seven studies where rates of catatonia were reported, ( %) cases of catatonia were identified (table ). the range of catatonic features reported is wide and includes echolalia, grimacing, posturing, and alternating hypermotor and hypomotor activity. a few studies have examined comparative rates of nmdar autoantibody positivity among different diagnostic groups. of patients with psychiatric disorders, two had igg antibodies against the nmdar subunit nr a in serum and csf; both had catatonia and another study found higher nmdar positivity among patients with catatonia than in a control group of healthy volunteers (although controls were younger than the patients and the investigators used an unusual continuous measure of nmdar immuno fluorescence). one study examined bushfrancis catatonia rating scale scores in patients with firstepisode psychosis and found that catatonic features were actually less common in patients with antineuronal antibodies. a study published in by our group with individuals at ultra high risk of psychosis suggests more severe catatonic features in individuals with nmdar antibodies. nmdar encephalitis has only been described in the last decade but has led to a reevaluation of encephalitis lethargica, first recognised in , due to notable similarities. encephalitis lethargica is characterised by profound sleep impairment (insomnia, hypersomnia, or sleep inversion), extrapyramidal movement deficits, and neuropsychiatric symptoms. although historically linked to the influenza pandemic, the evidence for a causal association is sparse. more recently, investi gations have found a high prevalence of antibodies to the nmdar and the dopamine d receptor in the serum of children with encephalitis lethargica, raising the intriguing prospect that many patients exhibiting cata tonia previously diagnosed with the disorder, might have instead had antibodymediated encephalitis. when we considered the role of the innate immune system, we considered the possibility that inflammation itself was responsible for the stuporous aspects of catatonia. as far as adaptive immunity is concerned, the specificity of the antigen might be the most important determinant of the resulting neuropsychiatric phenotype, including catatonia. the downstream effect of immune activation is dependent on the antigen targeted. autoimmune neurological disorders present differently depending on the target for autoantibodies or t cells; frequently these targets are neurotransmitter receptors with ensuing downstream effects on receptor dysfunction. in autoimmune encephalitis, the presen tation depends on the specific antibodies present. in the specific case of nmdar encephalitis, often little evidence exists of complement activation and neuronal degeneration. the fact that ketamine and phencyclidine-both nmdar antagonists-cause catatonia suggests that nmdar antagonism is responsible, the implication being that nmdar antibody encephalitis is more usefully understood as a synaptopathy. genetic hypofunction of the nmdar due to grin mutation also appears to predispose to psychosis. similarly, benzodiazepine withdrawal presents similarly to gabaa receptor encephalitis. , autoimmunity, therefore, appears to cause catatonia primarily by specific action against central or peripheral antigens; however, secondary inflammation could perpetuate a phenotyperelevant immune response. the close association between nmdar encephalitis and catatonia might provide valuable insight into the patho physiology of catatonia. nmdar encephalitis causes internalisation of the nmdar, resulting in a reversible reduction in the number of receptors and impaired αamino hydroxy methyl isoxazolepropionic acid (ampa) receptormediated longterm potentiation. , this idea is consistent with catatonia also resulting from use of the recreational noncompetitive nmdar antagonists, ketamine, and phencyclidine. , to integrate findings of effective treatment with gabaa receptor agonists and nmdar antagonists, northoff has proposed a model of catatonia in which the normal inhibition of excitatory glutamatergic cortico cortical association fibres by gabaergic neurons in the orbitofrontal region is impaired. in mice, the nmdar antagonist dizocilpine (also known as mk ) shows a bimodal effect on grooming and rearing behaviour: at low doses, this behaviour is suppressed, but as the dose increases, behaviour normalises, before being suppressed again at higher doses. , this effect might explain why catatonia is characterised not only by immobility, but also occasionally by catatonic excitement. an explanation for this finding might rely on the knowledge that the nmdar is expressed by excitatory glutamatergic neurons and by inhibitory gabaergic neurons. moreover, both reduced and excessive nmdar activity can result in neuronal apoptosis, dalmau and colleagues have proposed a model for antinmdar encephalitis, in which increasing nmdar blockade results initially in behavioural and psychotic symptoms, and at higher antibody titres, neurological and autonomic dysfunction. one hypothesis would be that catatonia occupies the ground between these two states (figure). pharmacological or antibody mediated nmdar hypofunction could both cause catatonia and result in progression to malignant catatonia. catatonia is heterogeneous in presentation and cause. however, the generally favourable response to treatment with benzodiazepines or ect suggests a common pathophysiology. activation of the innate immune system can lead to the neurovegetative features of catatonia, but the evidence for the acute phase response in catatonia is preliminary and sometimes conflicting. moreover, whether any peripheral inflam mation in catatonia arises secondary to immobility and muscle breakdown is unknown. examining the asso ciation of catatonia with the adaptive immune system reveals a strong and specific association with nmdar encephalitis, which can cause the full range of catatonic features. this finding suggests that adaptive immunity can cause catatonia through action at specific extra cellular antigens, rather than immune activation per se. additionally, this illustrates the importance of gluta matergic function in catatonia. as more autoimmune disorders are characterised, more cases of catatonia might be explained in this way. although we have considered the innate and adaptive immune systems separately, in reality, they are deeply interconnected. for instance, nmdar encephalitis (a disorder of the adaptive immune system) entails a very high risk of neuroleptic malignant syndrome (a disorder with prominent activation of the innate immune system). malignant catatonia remains an enigmatic entity that could possibly be accounted for by autoimmune disorders such as nmdar encephalitis. finally, is it possible to conclude whether catatonia is due to activation of the immune system? in many cases, little compelling evidence exists for this. however, where infection or autoimmunity are directed at specific targets in the cns or periphery, a high risk of catatonia is apparent. further investigations based on this concept (panel) might assist in elucidating pathophysiological mechanisms and improving the treatment of catatonia. • is catatonia overrepresented in individuals with autoimmune disorders? • what are the rates of catatonia in gaba-ar encephalitis when systematic detection methods are used? • in studies of depression, is there evidence that raised inflammatory markers are associated with catatonic features? • do relapsing and remitting inflammatory markers exist in periodic catatonia? • are patients with catatonia more likely to have antibodies to n-methyl-d-aspartate (nmda) receptor, γ-aminobutyric-acid (gaba)-a receptor, glutamic acid decarboxylase (gad ) and other encephalitis-associated antibodies? • will large genetic studies of catatonia show linkage to hla or other immune-specific regions? • do inflammatory markers predictors response to electroconvulsive therapy? • does a reduced density of nmda receptors exist in catatonia in single-photon-emission ct (spect) or pet studies? • will the use of specific ligands reveal cns immune activation (eg, mediated by microglia or astrocytes) in catatonia? • is immunomodulatory therapy (such as with corticosteroids, plasmapheresis, or rituximab) an appropriate treatment option in catatonia? • is minocycline an effective treatment for catatonia? • is immunosuppressant therapy effective for neuroleptic malignant syndrome? we searched pubmed for articles published up to sept , , with the terms "catatoni*" in association with any of the following terms: "immune*", "autoimmun*", "inflame*", "t-cell", "b-cell", "glia*", "microglia*", "acute phase", "innate", "adaptive", "encephalitis", "antibod*", "infect*", "interleukin", "cytokine", "monocyte", "macrophage", "leukocyte", "lymphocyte", "granulocyte", "phagocyte", "tnf", "c-reactive protein", "dendritic cell", and "immunoglobulin". this primary search along with the references of selected review articles revealed three areas that were suitable for systematic summaries of the literature: infective causes of catatonia, autoimmune causes of catatonia, and inflammatory markers in catatonia. to investigate these areas , we searched six databases (amed, bni, cninahl, embase, medline, psycinfo, and pubmed) for catatonia and mesh terms (or equivalent) in conjunction with relevant specific search terms (eg, "infect*", "virus", "bacteria"). after deduplication, articles were screened via their titles and abstracts before relevant full-text articles were reviewed by the first author and systematically included in tables in the manuscript. only articles with full texts or sufficiently detailed abstracts written in english were included. the manuscript was planned by jpr, tap, gb, and asd. jr did the literature search and drafted the manuscript. tap, gb, and asd reviewed the manuscripts, made amendments, and added further references. all authors approved the final manuscript. we declare no competing interests. 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effects of (s)ketamine and n,ndimethyltryptamine (dmt): a doubleblind, crossover study in healthy volunteers paradoxical effects of very low dose mk bimodal effects of mk on locomotion and stereotypy in c bl/ mice nmda receptor blockade and catatonia: a complex relationship glutamatenmethyld aspartate receptor modulation and minocycline for the treatment of patients with schizophrenia: an update the dichotomy of nmda receptor signaling key: cord- -xo ruswo authors: new, r.r.c.; moore, b.d.; butcher, w.; mahood, r.; lever, m.s.; smither, s.; o'brien, l.; weller, s.a.; bayliss, m.; gibson, l.c.d.; macleod, c.; bogus, m.; harvey, r.; almond, n.; williamson, e.d. title: antibody-mediated protection against mers-cov in the murine model() date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: xo ruswo murine antisera with neutralising activity for the coronavirus causative of middle east respiratory syndrome (mers) were induced by immunisation of balb/c mice with the receptor binding domain (rbd) of the viral spike protein. the murine antisera induced were fully-neutralising in vitro for two separate clinical strains of the mers coronavirus (mers-cov). to test the neutralising capacity of these antisera in vivo, susceptibility to mers-cov was induced in naive recipient balb/c mice by the administration of an adenovirus vector expressing the human dpp receptor (ad -hdpp ) for mers-cov, prior to the passive transfer of the rbd-specific murine antisera to the transduced mice. subsequent challenge of the recipient transduced mice by the intra-nasal route with a clinical isolate of the mers-cov resulted in a significantly reduced viral load in their lungs, compared with transduced mice receiving a negative control antibody. the murine antisera used were derived from mice which had been primed sub-cutaneously with a recombinant fusion of rbd with a human igg fc tag (rbd-fc), adsorbed to calcium phosphate microcrystals and then boosted by the oral route with the same fusion protein in reverse micelles. the data gained indicate that this dual-route vaccination with novel formulations of the rbd-fc, induced systemic and mucosal anti-viral immunity with demonstrated in vitro and in vivo neutralisation capacity for clinical strains of mers-cov. the middle east respiratory disease syndrome (mers) first emerged in in saudi arabia [ , ] . since then, there have been an estimated laboratory-confirmed cases with deaths, reported from a total of countries in the eastern mediterranean region and from countries elsewhere [ ] . saudi arabia, however, remains the main focus of infection and a disease outbreak in south korea involving cases was traced back to an index case who had travelled from saudi arabia. whilst the incidence of mers cases in saudi arabia peaked in , there are still a significant number of cases reported from the country and in the period september -may , there were cases including deaths with a case fatality rate of . % [ ] . mers coronavirus (mers-cov) is a member of the betacoronavirus genus [ ] and as for other betacoronaviruses, bats may provide a natural reservoir for the virus [ , ] , but high levels of antibodies to mers-cov in dromedary camels [ ] suggest that the dromedary camel is the principal source for animal-to-human transmission of mers-cov [ ] . however, evidence of human-to-human transmission comes from the reporting of outbreaks in countries remote from saudi arabia such as the uk, europe, usa, and china where small outbreaks have also occurred [ ] . mers-cov is an enveloped, positive-sense, single-stranded rna virus [ ] . the virus possesses an envelope-anchored trimeric spike protein which binds to the human receptor dipeptidyl peptidase (dpp or cd ) and gains host cell entry by the fusion of viral and host membranes [ ] . the spike protein comprises an s sub-unit and a membrane fusion s sub-unit. in the coronaviruses, the s sub-units are further divided into n-terminal and c-terminal subdomains and for mers-cov, it is the c-terminal sub-domain that comprises the receptor-binding domain (rbd) [ ] . the rbd also incorporates a receptor-binding motif at its c-terminal and the crystal structures of mers-cov rbd [ ] and of the rbd bound to the extracellular domain of human dpp have been reported [ ] . the rbds of the coronaviruses represent vaccine and therapeutic targets and the rbd of mers-cov as a vaccine antigen has been demonstrated to induce neutralising antibody [ ] and to protect mice transduced with a viral vector expressing hdpp , or nonhuman primates from viral challenge [ ] [ ] [ ] [ ] [ ] . there are significant ongoing efforts to develop vaccines for mers-cov infection, predominantly involving live attenuated viral vectors such as adenovirus, modified vaccinia ankara or measles [ ] to induce anti-viral immunity and some of these vaccines are already in clinical trials. here, we were interested to determine the relative importance of inducing systemic and/or mucosal immunity in vaccination to protect against mers-cov, an infection predominantly of the respiratory tract and lungs. to this end, we have used a dual route immunisation regimen in balb/c mice to induce both systemic and mucosal immunity, to generate rbd-specific murine antisera. initially, we immunised balb/c mice sub-cutaneously (s.c.) with rbd-fc in the mf adjuvant to induce rbd-specific igg. subsequently, we have immunised further groups of balb/c mice by s.c. priming and per oral (p.o.) boosting with the rbd-fc, to induce both systemic igg and mucosal iga responses. to do this, we have used novel formulations of rbd-fc coated onto microcrystals formed from histidine or glutamine and also incorporating calcium phosphate for sub-cutaneous priming [ ] , whilst the formulation for oral boosting comprised rbd-fc in reverse micelles dispersed in a self-emulsifying oil phase, which has been optimised from previous formulations [ , ] . the advantages of these formulations are that they are very stable under extremes of temperature [ ] . furthermore, on translation to the clinic, only one injected priming dose would be required, followed by a p.o. booster dose; the latter could be self-administered in capsule form. we have compared the relative abilities of the two sources of antiserum to neutralise clinical isolates of mers-cov in vitro. to do this, we have used two clinical strains of mers-cov (erasmus medical center or emc and london - :), each of which were derived from severely-ill individuals who had contracted the virus in the middle east in . subsequent sequencing of the polymerase gene from these isolates indicated them to be newly-emerged members of the betacoronavirus genus with a close sequence homology and phylogenetic relationship to the bat coronaviruses hku and hku- [ , ] . mice are not naturally susceptible to mers-cov infection, but susceptibility can be induced by the administration of an adenovirus vector which induces expression of the human receptor (hdpp /cd ) for the virus in vivo for a limited time, providing a non-lethal murine model of the disease [ ] . we have used this transduced mouse model to test the capacity of the antiserum derived from the dual route immunisation to neutralise mers-cov in vivo, by passive transfer prior to challenge with the emc strain and we have demonstrated a significant reduction in viral load in lung tissue in transduced mice. the rbd was synthesised and expressed according to methods adapted from du et al. [ ] . in brief, a single dna fragment containing an in-frame fusion of the coding sequences for the human il signal peptide, the rbd and human igg -fc was synthesised. this was transferred into the plasmid pef-dest (invitrogen) so that the target sequence was expressed as a secreted protein with a c-terminal human igg fc tag. this construct was transfected by cationic transfection into human embryo kidney (hek) cells in suspension (fshek) or adherent hek cells stably expressing the sv large t antigen ( ft), using serum-free media and incubated for - days. small scale purifications of rbd-fc were performed using protein a chromatography. for this, medium from the transfected cells was treated with ammonium sulphate to precipitate the protein, prior to dialysis and resuspension in buffers for binding to protein a beads. the latter were washed and eluted with buffer containing m urea. protein concentration was determined by uv absorbance spectroscopy and purity was estimated by sds-page with coomassie staining and subsequent optical densitometry using a syngene g:box imaging system. the rbd-fc was incorporated on glutamine calcium phosphate (cap) microcrystals for s.c. immunisation, using methodology adapted from [ ] . briefly, aqueous mixtures of rbd-fc with sodium orthophosphate and glutamine were precipitated as cap protein-coated microcrystals (cap-pcmc), by addition to of a fold excess of isopropanol containing dissolved calcium chloride. the resultant suspension contained self-assembled microcrystals comprising a glutamine core with the rbd-fc protein embedded in a thin surface layer of cap (now termed rbd-fc-pcmc). the pcmc were isolated by vacuum filtration and dried to a powder. protein content and integrity was determined by elisa and sds-page. for oral dosing, the rbd-fc was incorporated in mineral oil with added excipients using methodology adapted from [ , ] . the oral formulation comprised rbd-fc with the mucosal adjuvant cholera toxin b sub-unit (ctb), retinoic acid (ra), vitamin d, e and trehalose debehenate (tdb), a synthetic analog of the mycobacterial trehalose dimycolate [ ] and imiquimod, a tlr / agonist [ ] . specific pathogen-free female balb/c mice ( - weeks of age) were obtained from a commercial breeder and used throughout this study. on receipt, mice were randomised for allocation to cages and given free access to food, water and environmental enrichment. mice were fully acclimatised to the animal housing facility for at least five days prior to any procedure. all animal procedures were performed in accordance with uk legislation as stated in the uk animal (scientific procedures) act . the institutional animal care and use committee approved the relevant project licence. naïve mice were randomised for allocation to a treatment group (typically per group) and immunised in one of two regimens: either with a s.c. priming dose followed by two s.c. doses, given at and days after the prime; alternatively, mice received a s.c. priming dose followed by an oral or s.c. booster dose days after the prime (table ) . for s.c. immunisation, mice received . lg of rbd-fc-pcmc in . ml pbs injection volume, whereas for all per oral (p.o.) dosing, mice received lg of rbd-fc in a total volume of . ml mineral oil (mo), by oral gavage. where rbd-fc was administered s.c. in the conventional adjuvants mf or alhydrogel, mf (novartis, us) was used in a : ratio by volume with rbd-fc in pbs, whilst alhydrogel (brenntag biosector, denmark) was used in a : ratio with rbd-fc by volume in pbs. at selected intervals after dosing, mice were blood-sampled from the tail vein for assay of specific antibody titre. at the end of the immunisation schedule, individual mice were terminally anaesthetised for collection of blood by cardiac puncture, then culled prior to removal of small and large intestines for collection of faecal pellets for extraction of iga. titres of rbd-fc-specific antibody in serum samples were determined by elisa. in brief, test sera were bound to microtitre plates pre-coated with rbd-fc and antibody binding was detected with an hrpo-labelled secondary antibody to mouse igg, igg , igg a or iga (bio-rad). a standard curve for calibration comprising the relevant murine ig isotype (sigma) captured with an anti-fab reagent, was included on each plate. plates were developed by the addition of , -azino-bis( -ethylbenzothiazoline- -sulfonic acid) diammonium salt (abts) substrate (sigma) and optical density (od) was read at nm (multiskan plate reader). for assay of antibody in faecal samples, faecal pellets were extracted in supplemented pbs as described previously [ ] . in brief, ml of cold pbs was prepared, supplemented with tablet of complete mini protease inhibitor cocktail (sigma) and ll tween were added. to . g faecal pellets, ml of supplemented pbs was added and left at room temperature for min. samples were vortexed for approximately s, incubated on ice for a further min. and then centrifuged ( , g, min.). supernatants were retained and stored at À °c pending assay. the faecal extracts were assayed for specific igg and iga content, by elisa, as for serum samples. antibody concentrations in all samples were determined from the relevant standard curves using ascent software with fourparameter logistic curve-fitting and reported in ng/ml or lg/ml serum or faecal extract, as appropriate. to determine if the antibody induced by immunisation with to rbd-fc was neutralising for mers-cov in vitro, plaque assays were performed. for this, two strains of mers-cov were used: london - (genbank accession number kc . ) [ ] and erasmus medical center (emc genbank accession number jx ) ( ) . the london - strain was obtained from the national collection of pathogen viruses, phe porton, salisbury, uk and the emc strain was kindly provided by the erasmus university medical center rotterdam, the netherlands. both strains were prepared in serum-free media (gibco) at a multiplicity of infection (moi) of . , equivalent to plaque-forming units (pfu). the murine antiserum for testing was prepared at a dilution range from undiluted to : in pbs. virus was incubated overnight ( °c) with murine antiserum, negative control antibody (nibsc, uk) or media, prior to infection of a confluent monolayer of vero e cells (ecacc, salisbury uk) with ll of the mixture. the neutralising ability of the murine antiserum was tested in duplicate or triplicate at each dilution. after incubation ( h, °c), an overlay comprising a : dilution of carboxymethyl cellulose with serum-free media was added to the cells and incubation continued for a further days ( °c) prior to fixing ( . % formaldehyde) and staining ( . % crystal violet) with enumeration of the number of plaques per ml. mice are not naturally susceptible to infection with mers-cov, since they lack the human dpp receptor. to induce transient susceptibility in balb/c mice, we used an ad construct (oxford genetics) to express the human dpp receptor (ad hdpp ), as previously described ( ) . mice were administered the ad hdpp construct ( .  pfu in ll) by the intra-nasal (i.n.) route under light sedation with inhalational isofluorane and then monitored by serial blood sampling for serum levels of hdpp /cd by elisa (thermoscientific). at peak levels of expression of hdpp (days - ), mice were lightly sedated as before and challenged by the i.n. route with mers-cov (emc strain) at pfu in ll per mouse. mice were weighed prior to challenge on each subsequent day to monitor changes in body weight during infection. to test the in vivo neutralising capacity of murine antiserum raised to the rbd-fc construct, naïve mice (n = per treatment group) were passively immunised by the i.p. route at h. prior to i.n. challenge with the mers co-v (emc strain), as described above. the murine antiserum, pooled from mice who had been primed with rbd-fc pcmc and boosted orally (regimen , treatment group ), was delivered at a dilution of : in pbs and delivered in a total volume of ll per mouse. a further group of mice received a purified polyclonal human igg at a single dose level ( lg/mouse in ll i.p.), which had been raised to inactivated mers-cov. control mice received a non-specific human igg at a single dose-level ( lg/mouse in ll, i.p.). both sets of human igg (specific and non-specific) were raised in a bovine transchromosomal model and purified prior to use. a further group of negative control mice were included, which received pbs in place of either the ad dpp construct or the mers-cov-specific antibody, and were also challenged i.n. with mers-cov (emc strain) at pfu/mouse. to determine the protection afforded by the passive immunisation, pairs of mice from each treatment group were culled on days - after challenge and their lungs were removed and weighed and then rapidly frozen (À °c) prior to the determination of viral load. pairs of lungs from each of mice per treatment group were individually thawed and homogenised in serum-free media ( ml). rna was extracted from ll of each homogenate using the qiaamp viral rna kit (qiagen), following the manufacturers' instructions. real-time pcr was conducted on duplicate ll the amount of virus in tested samples was determined in duplicate using the standard curve and reported as pfu/g lung tissue. all data were analysed using graph pad prism software v. and expressed as mean ± s.e.m. statistical comparisons were made using one-way anova or unpaired t-test. the rbd-fc protein was expressed in both adherent ft and suspension human embryo kidney (hek) cells, but with greater expression in adherent cells (fig. ) . purification of protein from adherent cells with protein a was very effective, yielding protein which was > % pure, with molecular weight of approximately kda (fig. a) . the use of m urea for elution was optimum, as it was sufficient to solubilise the protein without denaturing it, yielding rbd-fc in optimum yield ( . mg/ml) and predominantly in a dimeric form (fig. b) . this method of protein purification was therefore selected for forward use. rbd-fc, formulated for either sub-cutaneous (s.c.) or per oral (p.o.) immunisation, was tested for immunogenicity and the formulations optimised in an iterative approach. initially, a s.c. dosing regimen was used in which rbd-fc was formulated in either alhydrogel or mf to deliver . lg of protein on each of three occasions at , and days. mice were monitored for days after the final boost and igg titre determined ( fig. a) . at day , the total igg titres achieved with rbd-fc in alhydrogel or mf did not differ significantly. to determine if the presentation of rbd-fc in either alhydrogel or mf influenced the ability to develop virus-neutralising antibody, antisera were selected from mice in each immunisation group and tested in a plaque assay for neutralisation of both the emc and london - strains of mers-cov ( fig. b and c) . all four sera gave some neutralisation of viral activity, although at a : dilution, sera and were most potent, against both viral strains. sera and were derived from the treatment group immunised with mf adjuvanted rbd-fc, whereas sera and were derived from alhydrogel-adjuvanted rbd-fc ( fig. a) . based on this pilot data, we subsequently used mf as the conventional adjuvant for rbd-fc, to compare with some novel formulations. having demonstrated to proof-of-principle that the rbd-fc, when delivered in mf can induce a high titre of antibody with neutralising activity, we next investigated how to tailor an rbd-fc vaccine optimally to induce both systemic and mucosal immunity, with the aim also of reducing to a -dose immunisation regimen and increasing functional antibody. for this, we selected novel formulations in which rbd-fc protein was presented as rbd-fc-pcmc for s.c. priming and incorporated into mineral oil (mo) for p.o. boosting. we compared the serum igg response achieved from this -dose dual route immunisation with that induced to rbd-fc delivered in mf in a -dose s.c. regimen (fig. ) . at month after the booster dose, at day , there was no significant difference in the serum igg titres achieved, so that the -dose dual-route immunisation with rbd-fc-pcmc for s.c. priming and incorporated in mo with excipients for p.o. boosting, was just as immunogenic as the -dose s.c. immunisation with rbd-fc in mf (fig. a) . at day , the serum response to rbd-fc in the dual-route regimen was predominantly igg biased, whereas s.c. dosing with rbd-fc in the presence of mf induced both igg and igg a (fig. b) . since dual route immunisation effectively induced serum igg to rbd-fc, it was of interest to determine whether it could also effectively induce mucosal immunity. in this study, the rbd-fc-specific iga response was determined in serum and in faecal pellet extracts from individual animals on day . in this case, the rbd-specific iga responses of mice immunised in the -dose dual route regimen were compared with that of mice immunised by the oral route twice, and with mice immunised by the s.c. route in mf twice, on exactly the same days ( , ) (fig. ) . this comparison showed that s.c. immunisation in mf did not induce serum iga. however s.c. priming with rbd-fc-pcmc with p.o. boosting effectively induced rbd-fc-specific iga and was not inferior to oral priming and boosting in this effect in either serum (fig. a ) or faecal extracts (fig. b ). however mice primed and boosted orally did not develop rbd-specific systemic igg (data not shown). additionally, day sera from mice in all treatment groups were tested for their ability to neutralise either strain of mers-cov in vitro (table ) . from this it can be seen that sera from out of mice in the dual route regimen were fully neutralising neutralisation of mers-cov in vitro. in in vitro for both strains (emc and london - ), when tested at : dilution ( fig. a and b) . in order to test whether the in vitro neutralising activity translated into viral neutralisation in vivo, sera from these mice (highlighted in table ) were pooled in equal aliquots at : dilution to enable a subsequent passive transfer study. in order to design the passive transfer study, it was necessary to define the duration of expression of cd in murine lungs in vivo, following induction with the ad hdpp construct. mice dosed with ad hdpp i.n. at t were culled in pairs and lung homogenates prepared and assayed for cd expression. cd in lung tissue was expressed in a time-dependent manner, with levels peaking at day and declining to day (fig. a) , setting a sufficient window to use the model for the determination of the protection against viral challenge afforded by the passive transfer of mers-specific antibody. to determine the protection afforded by the passive transfer of murine antiserum raised in the dual route immunisation regimen, against infection, susceptibility to mers-cov was induced at t with i.n. administration of ad hdpp to groups of mice. passive transfer by the i.p. route of the pooled serum sample derived from the mice highlighted in table , which had previously been shown to be neutralising in vitro (table ) was conducted days later and mice were challenged after a further h with mers-cov emc . additional groups of mice, which had been transduced with ad hdpp , were passively immunised with a mers-cov specific human igg and a non-specific human igg. at - days after challenge, pairs of mice were culled for the determination of viral load in lungs, which was determined to peak at days p.i. (data not shown). at days p.i., the pooled murine antiserum significantly reduced viral titres in lungs, to the same extent as the specific human igg, and contrasting with the negative control human igg, demonstrating significant in vivo neutralising activity (fig. b ). fig. . a. expression of cd was induced in lung tissue by the administration of ad hdpp ( .  pfu) to mice by the i.n. route at t . subsequently, mice were culled in pairs on the days shown and their lungs assayed for the expression of cd . the plot shows the time-course of cd expression from to days post-induction. all data points were normalised for background values from control mice. b: content of mers-cov (emc strain) in murine lungs (pfu/g tissue) determined by rt-pcr at day post-infection, (equivalent to day after passive transfer with murine antisera to rbd-fc which had previously been shown to neutralise the emc strain in vitro). mice received either a mers-cov-specific human igg ( lg) or non-specific human igg ( lg) in ll /mouse i.p.; or murine antisera to rbd-fc, which had been pooled from murine donors and which was delivered at : dilution ( ll/mouse i.p.). negative control mice received pbs in place of ad hdpp or antiserum all mice were challenged with mers-cov emc i.n. at pfu/mouse. statistical significance was determined at the p < . level by one way anova and unpaired t-test. no significant differences in body weight were detected between treatment groups challenged with mers-cov, which was attributed to the short time period of the study. mers is a serious endemic respiratory infectious disease for which there is no licensed vaccine, although there are several vaccines in clinical trial currently including adenovirus-vectored delivery of the spike protein and sub-units [ ] , dna vaccines and nanoparticle-delivered sub-unit approaches [ ] . we were interested in determining the advantage of r a vaccine which could induce mucosal as well as specific systemic immunity to the key target, the rbd protein, in order to achieve optimum protective efficacy. vaccination to induce effective immunity at mucosal surfaces, should prime the immune system to respond rapidly to invading pathogens such as mers-cov. previous studies have used adenovirus delivery of the mers spike protein with intra-muscular (i.m.) or intra-gastric (i.g.) delivery to induce neutralising systemic igg, but not iga; further, whilst i.m. delivery also induced specific t-cell immunity, i.g. delivery did not [ ] . others have shown that intra-nasal delivery of a live attenuated adenovirus-vectored subunit vaccines does induce specific mucosal as well as systemic immunity, although translation of this approach to the clinic may raise safety issues [ ] . here, we have relied on novel formulations of a sub-unit protein to enable dual route vaccination (parenteral and oral) to induce mucosal as well as systemic immunity. in this study, we have achieved the expression and purification of a recombinant rbd-fc protein in milligram quantities. we have also demonstrated that when formulated as a sub-unit vaccine, the construct induced murine antibody which effectively neutralised two different clinical strains of mers-cov in vitro. additionally, we have shown that these murine antisera, when passively transferred into naïve mice transduced to express the hdpp /cd receptor, conferred protection against viral challenge in the recipients, with significantly reduced viral loads in the lung tissue of the recipient mice. whilst the use of conventional adjuvants such as mf or alhydrogel to formulate the rbd-fc protein resulted in high titres of specific igg in serum, the mf formulation did not induce specific iga in serum. in order to promote both systemic and mucosal immunity to rbd-fc, we have formulated this protein for injected priming and p.o. boosting, entailing the optimisation of the cap pcmc and of the reverse micelles in oil emulsion, respectively. this has enabled the achievement of a vaccination regimen comprising only two doses and rapidly inducing rbd-fc-specific systemic and mucosal immunity. whilst the pcmc formulation of rbd-fc was as effective as rbd in mf in inducing a primary igg response, we have shown that an oral formulation of rbd-fc in mineral oil with selected immunostimulants was as effective as mf when used as a booster immunisation. additionally, we have shown that non-invasive oral priming and boosting is as effective at inducing a specific mucosal response, measured as specific iga in serum and faeces, as is injected priming with rbd-fc in the pcmc formulation together with oral boosting, leading to the exciting concept of a potential orally-dosed sub-unit vaccine for mers. whilst both alhydrogel and the combination of pcmc and oral formulations are th -polarising, as evidenced by the predominantly igg titres raised to rbd-fc, the influence of mf on the response to rbd-fc was a mixed th- /th effect, with a significant induction of specific igg and igg a. to counter a viral infection, it would be expected that a th response would be most appropriate. however, the fact that neutralising antibody to rbd-fc was raised under either th or th -polarising influences, suggests that either isotype can be protective and primes the immune system sufficiently and would allow for cross-presentation to occur on subsequent exposure to the virus [ ] . in this study, we have not examined the induction of a cell-mediated memory response to the rbd-fc protein, although this will play a significant role in protection against the virus. currently, we are presenting the rbd protein in our formulations with an fc tag, derived from human igg and useful in purifying the protein. the fc tag may contribute additional adjuvantising activity by engaging antigen-presenting cells in the vaccinee [ ] and it may aid mucosal immunity since the fc receptor, an mhc transmembrane protein, is also expressed at mucosal surfaces e.g. in the respiratory tract [ ] . vaccination of the zoonotic host, the dromedary camel, may also effectively curb outbreaks of mers in endemic regions and limit the risk of viral recombination [ ] and significant progress with an orthopox-vectored vaccine for mers has recently been reported [ ] . the potential use of a sub-unit vaccine for mers in camel vaccination could be aided by varying the sequence of the rbd protein [ ] and substituting the human fc tag with an alternative tag recognised by the camel, to design approaches tailored for animal vaccination, bearing in mind that a single dose vaccine would be ideal in this context. however, future work in our laboratory will also address the value of retaining or removing the fc tag from the rbd protein for clinical or veterinary iterations of the vaccine. in this study we have determined vaccine efficacy by demonstrating in vitro and in vivo neutralising ability of murine antisera raised in the dual route two-dose regimen against two virulent clinical strains of mers-cov which have greater than % genome sequence homologyy. in future work, it will be worthwhile to test the efficacy of this approach against other clinical isolates of mers-cov. this is the first report of a dual route dosing regimen applied to a sub-unit vaccine for mers-cov. future development of this approach would require the direct testing of efficacy in the immunised transduced mouse model. as well as giving a direct readout of vaccine efficacy, this will enable the identification of the immune correlates of protection, ready for transitioning this candidate vaccine into more extensive pre-clinical testing and clinical development. the authors declare that all the data supporting the findings of this study are available within the paper. ksa mers-cov investigation team. hospital outbreak of middle east respiratory syndrome coronavirus genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans mers-cov origin and 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fc-fusion proteins: new developments and future perspectives fc-fusion proteins and fcrn: structural insights for longer-lasting and more effective therapeutics co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels recombinant receptor-binding domains of multiple middle east respiratory syndrome coronaviruses (mers-covs) induce cross-neutralizing antibodies against divergent human and camel mers-covs and antibody escape mutants the authors acknowledge with thanks the expert technical assistance of lin eastaugh, louise thompsett and vicky roberts. this work was supported by sbri awards and from innovate uk to rrcn and na, respectively and on independent research commissioned from na and funded by the nihr policy research programme, [ ]. the views expressed in the publication are those of the author(s) and not necessarily those of the nhs, the nihr, the department of health, 'arms' length bodies or other government departments. the authors declare no conflict of interests. supplementary data to this article can be found online at https://doi.org/ . /j.vaccine. . . . key: cord- -r p helx authors: aggarwal, sadhna; verma, sumit singh; aggarwal, sumit; gupta, subash chandra title: drug repurposing for breast cancer therapy: old weapon for new battle date: - - journal: semin cancer biol doi: . /j.semcancer. . . sha: doc_id: cord_uid: r p helx abstract despite tremendous resources being invested in prevention and treatment, breast cancer remains a leading cause of cancer deaths in women globally. the available treatment modalities are very costly and produces severe side effects. drug repurposing that relate to new uses for old drugs has emerged as a novel approach for drug development. repositioning of old, clinically approved, off patent non-cancer drugs with known targets, into newer indication is like using old weapons for new battle. the advances in genomics, proteomics and information computational biology has facilitated the process of drug repurposing. repositioning approach not only fastens the process of drug development but also offers more effective, cheaper, safer drugs with lesser/known side effects. during the last decade, drugs such as alkylating agents, anthracyclins, antimetabolite, cdk / inhibitor, aromatase inhibitor, mtor inhibitor and mitotic inhibitors has been repositioned for breast cancer treatment. the repositioned drugs have been successfully used for the treatment of most aggressive triple negative breast cancer. the literature review suggest that serendipity plays a major role in the drug development. this article describes the comprehensive overview of the current scenario of drug repurposing for the breast cancer treatment. the strategies as well as several examples of repurposed drugs are provided. the challenges associated with drug repurposing are discussed. breast cancer, one of the most common cancer in women, is globally diagnosed by physical examination, breast scan and tissue biopsies. a total of . million new cases of breast cancer were identified in , of which ∼ , cases died. this accounted for approximately % of all cancer deaths among women globally [ ] . in , the highest numbers of breast cancer cases were reported from asia ( , , ) followed by europe ( , , ) , north america ( , , ) , latin america and caribbean ( , ), africa ( , , ) and oceania ( , ) [ ] . although very common in women, breast cancer rarely occurs in men [ ] . a vast heterogeneity in breast cancer molecular profiling has been reported [ ] . based on the er/pr and her expression, breast cancer can be of types: (i) er/pr+, her +; (ii) er/ pr−, her +; (iii) er/pr+, her −; and (iv) er/pr−, her − [ ] . the most aggressive breast cancer is er/pr−, her − (also called triple negative breast cancer, tnbc) because no receptor is present on these tumors. it is estimated that % of the breast cancers are hormone receptor positive ( %) and can be treated with corresponding therapies [ , ] . in addition to hormone receptors, other biomarkers used for clinical, pathological and molecular characterization of breast cancer include ki , p , ca , cea, brca /brca etc. [ ] . these molecular biomarkers in breast cancer are used for diagnosis, staging and grading, therapeutic intervention, prognosis as well as clinical management of recurrent and metastatic cases [ ] . are given as neoadjuvant therapy and only trastuzumab is continued post-surgery [ , ] . surgery is mostly followed by chemotherapy or radiotherapy to further destroy the remnant micrometastatic cancer cells that have escaped the breast or lymph nodes, to decrease the chances of recurrence and to increase the overall patient survival [ , ] . the adjuvant (additional) therapies i.e. hormone or targeted therapy are provided to breast cancer patients depending on the expression of hormone receptor or target protein, respectively [ ] . the er/pr + breast cancer patients are given tamoxifen and aromatase inhibitor together or alternatively after the surgical resection [ , ] . likewise, trastuzumab (herceptin) is mostly recommended to the her + breast cancer patients [ ] . tnbc patients are difficult to treat, with unfavorable prognosis and are generally administered with the standard chemotherapy along with the parp inhibitors or the dnatargeting platinum drug (carboplatin) [ , ] . another strategy is based on the holistic approach of patient recovery i.e. complementary and alternative medicine. in the holistic approach, the patients are advised for several life style changes like exercise, yoga, meditation, acupuncture and/or ayurvedic treatments in addition to the standard therapies [ ] . the united states fda has approved a number of drugs for the breast cancer therapy (table ) . however, these drugs are very costly and produce numerous side effects [ , ] . the common reported side effects in patients include fatigue, headaches, pain, numbness, dental issues, lymphedema, musculoskeletal symptoms, heart problems, blood clots, menstrual and menopausal symptoms, infertility, bone loss, and memory loss. these side effects are hard to be tolerated by the patients who are already weakened by the disease. eventually these also affect the decision for further choice of treatment and impair the quality of life. this necessitates the development of novel drugs for breast cancer therapy. we have previously reviewed the overall process involved in cancer drug development [ , ] . the drug development for breast cancer like other cancer types is a multi-step process involving the designing, synthesis, characterization, testing for efficacy and toxicities, and approval. overall, the process is lengthy, involving huge investment of money. it is estimated that the development of a new drug usually requires - years of time and approximately - billion dollars. on the top of all this, nearly % of the drugs fail during the clinical trial stage due to unexpected lack of appropriate efficacy, unacceptable side effects or regulatory norms [ , ] . therefore, the approaches to expedite the drug development process are required. one approach might be finding new uses for old clinically approved drugs (drug repositioning). the drug repositioning (aka drug repurposing, drug reprofiling, drug re-tasking, drug redesigning, drug resorting, drug reindication, indication switching, therapeutic switching) can be defined as exploring the new uses for an old clinically approved drug, with reduced risk, time and cost. drug repositioning circumvents the usual route of substantially higher rates, slow pace and side effects etc. [ ] [ ] [ ] . it is becoming an attractive concept for pharmaceutical industries. it is estimated that the repositioning for drug development requires much lesser time ( - years) and money ($ . billion) with at least known and approved side effects; and higher probability of success [ , [ ] [ ] [ ] [ ] . repositioning of old drugs offer rapid transition from bench to bedside as already approved by fda and other regulatory norms, passed through the clinical trials and are approved for human use. these drugs have well defined pharmacodynamics, pharmacokinetics, dose, side effects, metabolic profiles and targeted molecular pathway etc [ , ] . here, the clinical development can directly start from the phase ii trial to assess the efficacy for new indication/disease target. the successful example of drug repositioning includes sildenafil (viagra) and thalidomide. viagra was originally launched for hypertension and angina pectoris patients by pfizer ( ) . however, the patients developed penile erections as side effects in phase i clinical trial. the drug was later developed for erectile dysfunctions. in , the total sale of viagra was . billion dollars [ ] . similarly, the sedative thalidomide was serendipitously repurposed for erythema nodosum leprosum (enl) in and for multiple myeloma and other cancers in [ ] [ ] [ ] [ ] . recent advances in genomics, proteomics, transcriptomics and metabolomics have provided vast and deep knowledge about the molecular and metabolic alterations that occurs in cancers. the fundamentals of drug repurposing through the integration of system biology and bioinformatics depends on basic concepts i.e. activity based and in silico drug discovery [ ] . activity based drug repurposing is the experimental approach where the drug candidate is evaluated for the anticancer activity directly. the drug that is structurally similar would tend to have similar target, shared biological activity and indications [ ] . in the two diseases where same metabolic pathway or signaling is affected, then the drugs that target the specific pathway can be used in both of the diseases irrespective of their structural dissimilarity [ , ] . the drug that shows strong efficiency for any off-target (side effect) in one disease can be explored further i.e. the off-target binding and effect can be used as the novel indication of that drug in some other disease [ ] [ ] [ ] . in silico drug repurposing approach is based on the association and/or overlaps in the disease-gene-drug paradigm. the large data about drug-disease, gene expression (microarrays) or protein-protein interactions or gene-protein interactions or signaling pathway mapping, signature matching, genome-wide association studies (gwas) can be used for providing drug-disease gene association networking by the systematic integration and coordination of computation and bioinformatics, modeling (such as docking for structural modeling), experimentation, statistics, machine learning etc. [ ] [ ] [ ] [ ] . the various data resources are available that can be used for bio-informatics based exploration followed by experimental validation for any drug repurposing possibilities in any disease. the similar gene expression profile for drug phenotype in patients with different diseases as mapped by connectivity map (cmap), library integrated network based cellular signatures (lincs), expression signatures may have similar therapeutic applications [ ] [ ] [ ] . other important public data resources that are used for network-based drug repositioning includes gene set enrichment analysis (gsea) for drug-drug similarity network [ ] ; drugbank [ ] , online mendelian inheritance in men (omim) [ ] and geo [ ] for predicting drug-disease network; kegg [ ] , string [ ] , biogrid [ ] , happi [ ] and reactome [ ] for pathway and/or proteinprotein interactions; stitch drug-gene/protein database [ , ] , ttd therapeutic target database [ ] , sfinx for drug-drug interactions [ ] ; and sider for drug side effects [ ] . also, the recently reported 'drug repurposing from control system theory (decost)' is a comprehensive platform for drug repurposing that encompasses various limitations in the previous databases like variation in number of copies of gene of interest, mutations, lack of reference for normal range of gene expression etc. in different diseases [ ] . vigorously exploited 'network-based approach' by integrating the mentioned resources for identifying potential target, pathway and drug has been reported as the most potential way for drug repositioning [ ] . in the following section, we discuss the clinically approved drugs that were originally used for diseases other than breast cancer. however, these drugs are now being used or explored for breast cancer therapy. the clinically approved repositioned drugs for breast cancer are grouped based on their mode of action (table ). these drugs are diverse in terms of their chemical structures (fig. ) . abbreviations: cdk: cyclin-dependent kinase; erd: er down-regulator; lhrh: luteinizing hormone-releasing hormone; serd: selective estrogen receptor degrader; serm: selective estrogen receptor modulator; er: estrogen receptor; pr: progesterone receptor, her: human epidermal growth factor receptor. these are dna damaging agent that alkylate the guanine base thereby rendering them unable to bind to complementary strand. this leads to inhibition of dna replication and cancer cell growth. the alkylating agents affect all phases of cell cycle. cyclophosphamide is a known immuno-modulator that inhibits the suppressive regulatory t cells and enhances the effector t cells in tumor microenvironment. it shows the biphasic effect i.e. at low doses it imparts the immuno-suppressive function and at higher doses it functions as an alkylating agent leading to the death of tumour as well as the lymphoid cells [ , ] . usually mg/m² cyclophosphamide is intravenously (iv) administered to breast cancer patients in combination with other chemotherapeutics (docetaxel, paclitaxel, doxorubicin). the recommended regimens for robustly healthy breast cancer patients include cycles of cyclophosphamide, methotrexate and -fluorouracil (cmf) or cycles of adriamycin and cyclophosphamide (ac) [ , ] . thiotepa is a derivative of n,n',n"-triethylenephosphoramide (tepa) that was launched ( ) as immunosuppressive drug for transplantation in hematological diseases [ ] . subsequently, the drug was recommended for solid tumors in [ ] and for breast cancer ( . to . mg/kg iv repeated every - weeks) in [ ] . thiotepa in combination with vinblastine, adriamycin, and halotestin (vath) was effective in patients with relapse after adjuvant therapy and in metastatic breast cancer patients [ ] . anthracyclins are antibiotics that intercalates between the adjacent base pairs of dna in such a manner that it forms anthracyclin-dna-topoisomeraseii ternary complex. the ternary complex disrupts the resealing activity of enzyme leading to inhibition of dna and rna synthesis in highly replicating cancer cells [ , ] . some of the common anthracyclins are doxorubicin, daunorubicin, epirubicin and idarubicin. these are generally extracted from streptomyces bacterium. approved for medical use in , doxorubicin ( -hydroxylated version of daunorubicin) [ ] was originally extracted from streptomyces peucetius [ ] . there was a long lapse of years between first clinical usages of anthracyclins to their proven relevance in breast cancer treatment. however, once established the chemotherapeutic regimens that contained doxorubicin [usual dose: - mg/m² intravenously (iv) every days] were used regularly with dose-intense and dosedense schedules. some of the regimens containing doxorubicin are ac (adriamycin, cyclophosphamide), tac (taxotere, ac) and fac ( fluorouracil, ac) [ ] . unlike doxil (the pegylated form of doxorubucin), myocet is the non-pegylated liposomal version of doxorubicin that is approved for treatment of metastatic breast cancer in combination with cyclophosphamide in europe and canada [ ] . antimetabolites are the chemo-drugs that interfere with the metabolic pathways in the cancer cells primarily by acting as structural analogues to important cellular metabolites. -flourouracil ( -fu) is a pyrimidine antimetabolite, an uracil analogue that inhibits the activity of thymidylate synthetase by converting -fu to fdump and futp, instead of uracil to thymidine conversion. therefore, -fu not only inhibits the dna synthesis in the cell but also inhibits rna synthesis due to fdutp incorporation in rna; leading to highly toxic effects on the growth of rapidly multiplying cells like cancer cells. klein and colleagues introduced -fu for medical use in for the treatment of self-healing, squamous cell tumors of skin i.e. keratoacanthomas (kas) [ ] . -fu was subsequently used for other cancer types including breast cancer [ ] . general drug dosage of -fu is mg/m or mg/m iv on day and day of every -day cycle (six cycles in total). lokich et al ( ) first introduced the infusion of -fu in combination with methotrexate (m) for breast cancer [ ] . since then various regimes of combination therapy containing -fu (f) with other drugs like doxorubicin (a), cisplatin (c), cyclophosphamide (cx) and epirubicin (e) (ecf, cmf, caf) have been continuously used for treating metastatic breast cancer [ ] . the other combinations such as -fu with paclitaxel in [ ] and high dose -fu/paclitaxel with doxorubicin [ ] were found successful for breast cancer treatment. methotrexate is a folate/folic acid antagonist that binds to the dihydrofolate reductase (dhfr) enzyme and inhibits the synthesis of building blocks of dna and rna itself. folic acid is converted to dihydrofolate by dhfr, which is then reduced to tetrahydrofolate (thdf) for further action by thymidine synthetase. methotrexate binding to dhfr decreases the synthesis of purines and pyrimidines in the cells that inhibits the cell cycle progression in s-phase. methotrexate was discovered in s as a substitute to folic acid, because the cases with decreased leukemic cell count (acute lymphoblastic leukemia) due to dietary deficiency of folic acid were being presented in clinics [ , ] . methotrexate was first shown to remit breast cancer in by jane c wright [ ] . in s and s, methotrexate was being used as single cytotoxic agent for advanced breast cancer treatment [ ] . however, gianni and bonadonna group ( ) showed the first treatment with cycles of cmf combination in mastectomised women with advanced cancer and positive lymph nodes and reported only . % treatment failure with acceptable toxicity [ , ] . in fact, cmf regimen was the first ever schedule for breast cancer treatment. further, cmf was found to be equally effective even at cycles. immunosuppressive activity of methotrexate has been explored for its clinical use in autoimmune diseases like rheumatoid arthritis [ ] . general dosage of cmf regimen is defined as - cycles of cyclophosphamide ( mg/m ); methotrexate ( mg/m ), and fluorouracil ( mg/m ) at -day intervals. capecitabine, a pro-drug for -fu, is another pyrimidine antimetabolite. the conversion of capecitabine to -fu requires the three systematic enzymatic reaction cascades through intestine, liver and tumour cells. the enzyme that catalyzes the last step of conversion is highly expressed in tumour cells as compared to the normal cells. therefore, the tumour selective conversion of capecitabine to -fu prevents the systemic exposure of body to -fu [ ] . also, capecitabine is easier to administer and safer than -fu with better efficacy. the capecitabine was first used against colon cancer in [ ] . capecitabine ( mg/m given orally twice daily for days followed by a -day rest period in a -day cycle for cycles) is generally used to treat paclitaxel or docetaxel resistant advanced and metastatic breast cancers [ ] . it is used either alone or in combination with cabazitaxel [ ] , vinorelbine [ ] or ixabepilone [ ] . gemcitabine is a pro-drug that gets tri-phosphorylated inside the cell (dfdctp) by sequential enzyme catalyzed reactions. this dfdctp masquerades as an analogue of cytidine and gets incorporated in the newly synthesized dna generating irreparable error that inhibits dna replication leading to cell death [ , ] . gemcitabine was primarily manufactured in by larry hertel's group at eli lilly and company to be used as an anti-viral drug against enteroviruses [coxsackievirus b (cvb )]. the drug was also used against cvb , ev , human rhinoviruses (hrvs), human immunodeficiency virus (hiv), hepatitis c virus (hcv), poliovirus, influenza virus, zikv and mers-cov [ , ] . it was then pre-clinically tested for its anti-tumour attribute. the drug was approved by fda for pancreatic cancer in [ ] and for non-small lung cancer in [ ] . finally, gemcitabine was approved for metastatic breast cancer in in combination with paclitaxel [ , ] . after anthracycline-containing adjuvant chemotherapy failure, gemcitabine ( mg/m² iv infusion over min on days and of each -day cycle) and paclitaxel ( mg/m² on day as a h infusion before gemcitabine) combination is used as the first-line of treatment against metastatic cancer. other combinations such as gemcitabine/vinorelbine (gemvin), gemcitabine/cisplatin (gemcis), gemcitabine/capecitabine (gemcap) have also shown increased response rate and overall survival in pretreated metastatic breast cancer patients [ ] . palbociclib is a cdk / inhibitor that halts the progression of cells from g -to s-phase. palbociclib ( mg, -day cycle with aromatase inhibitor) is used as the targeted therapy against er + /her − advanced and metastatic breast cancer in conjunction with hormone therapy. the open label paloma (palbociclib: ongoing trials in the management of breast cancer) clinical trials were designed recently with either aromatase inhibitor (letrozole) (paloma- or paloma- trials) [ , ] or fulvestrant hormone therapies (paloma- trails) [ ] . an improvement in the overall survival of metastatic breast cancer patients was reported in paloma- phase iii clinical trials. inhibiting cdk / activity delays the resistant to hormone therapy and significantly improves the progression free survival (pfs) of patients [ , ] . the hormonal therapy or endocrine therapy is usually given for - years. this therapy either directly targets the hormone (estrogen and/or progesterone) production or negatively regulates the functional effects in hormone sensitive breast cancer patients (er + and/or pr + ). selective estrogen receptor modules (serms) serve as anti-estrogens by binding to the hormone receptors as antagonists. the widely used serms that have been repositioned as breast cancer drugs are tamoxifen ( ), toremifene ( ) and raloxifene ( ) . tamoxifen is the oldest serm that has been in use for more than years for early stage breast cancer treatments in pre-and post-menopausal women. toremifene and raloxifene are equally effective but safer alternatives of tamoxifen that are used in only post-menopausal women with advanced breast cancer [ , ] . multiple outcomes raloxifene evaluation (more) clinical trial was an osteoporosis treatment trial in postmenopausal women, with secondary objective of evaluating the effects on breast cancer risk reduction. more lead to the designing of further clinical trials such as continuing outcomes relevant to evista® (core), raloxifene use for the heart (ruth), and study of tamoxifen and raloxifene (star) [ ] . aromatase inhibitor hormone therapy is administered only in postmenopausal women to treat er + early and/or late stage breast cancer [ ] . it acts on the aromatase enzyme that still produces estrogen hormone in fat tissue of post-menopausal women or women without active ovaries. thus, the aromatase inhibitor reduces the amount of estrogen in post-menopausal women with breast cancer that would otherwise feed the breast cancer cells for further growth. the aromatase inhibitors (anastrazole, exemestane and letrozole) were initially used for ovary stimulation and induction of ovulation in infertile females or polycystic ovary syndrome. aromatase inhibitors can be used as neoadjuvant or adjuvant therapy, mostly alone or in combination, which were introduced as an alternate to tamoxifen in postmenopausal patients [ ] . selective estrogen receptor degrader (serd) such as fulvestrant, are pure anti-estrogens that blocks estrogen receptor and degrades the receptor without any agonist effect [ ] . fulvestrant was first used in as a 'serd hormone therapy' against hr + her − advanced and metastatic breast cancer in post-menopausal women that were resistant to other hormone therapy [ ] . it is used in combination with cdk / inhibitors like palbociclib (paloma- ) and ribociclib (monaleesa- ) and anti-pi k/akt/mtor pathway drugs such as pictilisib (fergi) and buparlisib (belle- and belle- ) [ ] . luteinizing hormone releasing hormone (lhrh) analogs interfere with the signaling mechanism that activates the estrogen synthesis in ovaries causing temporary menopause. in , goserelin was used for the assisted reproduction and prostate cancer treatment. goserelin was then approved for the treatment of pre-menopausal women with hormone sensitive breast cancers in . it is used alone or in combination with other hormone therapies [ ] . goserelin is currently under phase ii clinical trial as an additional drug into the standard neoadjuvant therapy for tnbc patients. the goserelin phase ii trial is expected to complete by [ ] . everolimus is an mtor kinase inhibitor that inhibits the pi k/akt/ mtor signaling pathway. everolimus was originally approved for renal cancer in , as immunosuppressant during renal transplants in and for pancreatic cancer in . a phase iii clinical trial 'breast cancer trial of oral everolimus- (bolero- )' that included everolimus in combination with exemestane was successfully completed in leading to the approval of everolimus by us fda for the treatment of hr + , her − advanced metastatic cancers that are resistant to letrozole or anastrazole [ , ] . mitotic inhibitors terminate the cell division or mitosis by disrupting the microtubule dynamics. this leads to g /m phase cell cycle arrest or inhibition of spindle formation. some of the common examples of mitotic inhibitor include docetaxel, paclitaxel and vinblastine. while docetaxel and paclitaxel induces g /m cell cycle arrest, vinblastine is known to inhibit spindle formation. docetaxel and paclitaxel are used as neoadjuvant or adjuvant therapy as single agent or in combination with other chemotherapeutic agents for the treatment of early, advanced and metastatic breast cancer in pre-and postmenopausal women. paclitaxel was isolated from pacific yew in . it was used as drug for arterial restenosis. docetaxel and paclitaxel were initially used as therapeutics in prostate and ovarian cancer ( ) respectively. thereafter, docetaxel ( mg/m² iv h after doxorubicin and cyclophosphamide weeks x cycles) and paclitaxel ( mg/m² iv over h weeks times with doxorubicincontaining regimen) were repositioned as chemotherapy adjunct in breast cancer treatment regimen [ ] [ ] [ ] . taxanes are now used as the part of standard chemotherapy in metastatic breast cancer. numerous combinatorial chemotherapy containing taxanes are used in routine practice for treating breast cancer by clinicians around the globe [ ] . vinblastine is a naturally occurring vinca alkaloid found in white flowered periwinkle, vinca rosea. this was discovered by robert noble and charles t beer in . the discovery of vinblastine is a beautiful example of serendipity in drug development. the group rather aimed to evaluate the anti-diabetic effect of extract in rats and observed pseudomonas mediated septicemia that was accompanied with the rapid wbc fall and granulocytopenia. further study in this direction showed peripheral granulocytopenia and leukopenia in vinca rosea extract treated rats. finally, they reported carcinostatic activity of vinca rosea extract/vinblastine in rats with transplantable mammary adenocarcinoma and sarcoma [ , ] . vinblastine was approved for lymphoma in . also, since s, vinblastine ( - mg/mm iv once weekly or every other week) in combination with mitomycin or mvp (mitomycin c, vinblastine and cisplatin) has been used as chemotherapy against advanced and metastatic breast cancer [ , ] . with a drastic increase in the number of new cases, the cost of the cancer treatment is rising even at a higher speed. this is because the disease demands molecular dissection at gene and protein level to find newer strategies, appropriate targets and corresponding drugs. this requires a lot of time and the use of high-end techniques by pharmaceutical companies thereby increasing the overall financial investments and eventually the cost of the drug [ ] . the antibody-based immunotherapies or cell-based cart therapy, dc vaccine, act types of drug treatment might prove promising. however, these therapies are unaffordable by most patients in developed as well as developing countries. this necessitates the development of drugs with higher efficacy, lower side effects and practically lower cost. one of the smart ways is to repurpose an old, existing and approved drug for a newer indication. the advantage of this approach is that the approved drugs have existing information about molecular targets, off-targets, modes of action, safety level and side effects. this would not only save a lot of time that is otherwise required for discovery, designing, clinical trials and approvals of a new drug but also reduce the overall cost of anticancer drug. this will cut the cost involved in preclinical development and phase i trial. further, the old drugs would be off-patent and hence cheaper than the initial costs. in this article, we discussed in detail the advantages of drug repurposing for breast cancer treatment. we provided several drugs that have been successfully repurposed for breast cancer treatment. the triple negative breast cancer (tnbc) is highly heterogeneous, aggressive and complex form of breast cancer without expression of er, pr, or her receptors. tnbc is untreatable with regular hormone therapy. however, the combination of chemotherapies and the drug repositioning approach has offered promising outcomes by preclinical studies. flunarizine, a n-ras inhibitor, has been approved for migraine or vertigo. flunarizine has also shown promise in tnbc mouse models by inducing autophagy [ ] . a recent publication showed that the combination of metformin and hemin, used for type diabetes or porphyria respectively, could inhibit the growth of breast cancer cells. bioinformatics tools revealed that the bach expression is significantly elevated in tnbc and the hemin sensitizes the tnbc to metforminmediated degradation of bach [ ] . the completion of repurposing drug in oncology (redo) project has provided evidence for the new uses of drugs for breast cancer. originally these were discovered for indications other than breast cancer [ ] . these drugs include mebendazole (anti-helminthic), cimitedine (anti-acid), nitroglycerins (heart attack preventing), itraconazole (anti-fungal), and diclofenac (anti-inflammatory). other drugs such as l-nmma (tilarginine acetate, a nitric oxide synthase inhibitor) [ ] , pro-viral integration moloney virus kinase (pim)- inhibitors (olaparib) [ ] , l-asparginase [ ] , and fenofibrate [ ] are being repurposed for breast cancer. these drugs were originally used in patients with cardiogenic shock, against viral infection, leukemia, helminthic infection, and in patients with high serum cholesterol and triglycerides, respectively. implementation of network pharmacology in examining the potential of natural herbs i.e. ayurvedic formulations and traditional chinese medicines (tcm) are also clinching the attention of researchers for anticancer drug repositioning. these are based on multi-targeted synergistic drug approach instead of one target-one drug approach and thus appear to be promising for breast cancer therapy. the integration of super-computation, simulations, network pharmacology and bioinformatics can detect the target and efficacy of herbs. triphala is the mixture of at least ayurvedic formulations meant for the treatment of many diseases. the bioactive-target-pathwaycancer type networking revealed the link of triphala with cancer types including breast cancer through bioactive and targets. this information can be explored further for either bioactive-or target-based drug repositioning and/or new drug development. however, some challenges and concerns associated with drug repurposing for breast cancer therapy require thorough consideration. the breast tumor heterogeneity, poorly defined molecular signatures, and poorly identified drug dosage provides a roadblock to the drug repurposing. moreover, the current strategies often ignore tumor grade. it is crucial to search the new therapeutic strategies for certain very stringent and hard to treat molecular subgroups of breast cancers such as basal subtype, tnbc, mbc and tumors resistant to standard treatment therapy. the molecular alterations in tnbc, mbc, and resistant tumor types should also be thoroughly investigated. aforementioned bach inhibition by targeting the energy generating mechanism of rapidly growing cells is indeed the novel approach against otherwise resistant-breast cancer. understanding and targeting the tumour microenvironment instead of tumor itself appears more promising in detailing the heterogeneity in breast cancer patients. the overlapping area that consists of tumour and normal cells in tumor microenvironment should be thoroughly characterized at the molecular level [ , ] . identifying the molecular basis of super-responders and non-responders also holds valuable insight for deeper exploration [ ] . another area that needs attention while strategizing drug repositioning for breast cancer is personalized medicine i.e. to extend the repositioning strategy to treat upto single patient design, as the patients under same molecular subgroup often presents further variations leading to unexpected response. structurally similar drugs may, at times, target functionally dissimilar protein hence pathway driven repurposing strategy is better for multi-targeted diseases like cancer. the orphan drugs generally have short or no patent and thus repurposing them is associated with lower cost. repurposing the off-patent drugs eventually blocks the patenting rights on repurposed drug and hence future investors [ ] . inclusion and exclusion criteria for the selection of trial group for repurposed drug is very crucial as different physiological responses are anticipated in comparison to the original group. for example, the pregnant women (first trimester) with cancer are excluded for thalidomide treatment due to risk of amelia and phocomelia [ ] . drug companies that expand the repositioning in similar therapeutic areas, such as reusing ovarian cancer drug as breast cancer drug, had success rate of % in comparison to % success rate when explored in different area [ ] . a comprehensive, large-scale data mining and research is required before jumping on to actual repurposing procedure in order to save the time and finances because smallest of errors in computation or simulations can mislead the entire study. faultless selection of most relevant pharmacological target using most appropriate database (tcga and metabric) for data mining and accurate repurposing strategy is indispensable for the successful drug repositioning for breast cancer. repurposed drugs mainly include the phase iii clinical trial, which still is the most challenging phase due to the longest duration, huge investments and inclusion of largest number of patients as compared to the phase i and phase ii clinical trials. the repurposed drug for breast cancers usually does not work in monotherapy but as poly-pharmacology/combinations. the parp inhibitors are synthetic lethal with brac mutation [ ] . hence, initially proving the repurposed drug efficacy as single drug agent becomes difficult. the toxicity of repurposed drug in pretreated patients or in combination therapy is unknown. for example, valproate-doxorubicin treatment caused toxicityinduced death of two patients in a group of cancer patients [ ] . the overall success rate of development of new drugs ( %) and repositioning of drugs does not vary much as the ultimate efficacy remains same [ ] . also, the repositioned drug anyhow requires clinical re-assessment for optimizing its efficacy and cytotoxicity if the route of administration or drug dosage is different from the older indication; thereby increasing the overall cost of repositioning scheme. nonetheless, the animal model used for drug testing does not represent the exact patient phenotype and hence is less predictive of efficacy in real [ ] . despite many benefits of drug repositioning, much attention is required to lower the drug dosage and toxicity without mitigating efficacy and resolve above discussed limitations for cost effective and more efficient drug development. in this article, we have comprehensively explained the current scenario of repurposed drugs for breast cancer. we have discussed in detail the need and strategies of drug repositioning in breast cancer and several classical examples of drugs that have been repositioned as breast cancer chemotherapy. the futuristic potential of non-cancer drugs that are under investigations for breast cancer as well as the challenges and bottlenecks of drug repositioning were also discussed. we thus conclude that comprehensive approach of selecting the most appropriate gene-protein-pathway-target-drug modeling via integration of system biology and bioinformatics holds the high potential of providing more efficient, safer and cost-effective chemotherapeutics for treatment of even the most stringent forms of breast cancer (metastatic and triple negative). the scientists, researchers and clinicians must continue to work together for extension of the present pharmacological databases, multi-omics and 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chen, yuehong; guo, yan; li, jiangfan; zhao, guangyu; zhou, yusen; sun, shihui title: complement receptor c ar inhibition reduces pyroptosis in hdpp -transgenic mice infected with mers-cov date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: vdeffy l middle east respiratory syndrome coronavirus (mers-cov) is a highly pathogenic virus with a crude mortality rate of ~ %. previously, we established a human dpp transgenic (hdpp -tg) mouse model in which we studied complement overactivation-induced immunopathogenesis. here, to better understand the pathogenesis of mers-cov, we studied the role of pyroptosis in thp- cells and hdpp tg mice with mers-cov infection. we found that mers-cov infection induced pyroptosis and over-activation of complement in human macrophages. the hdpp -tg mice infected with mers-cov overexpressed caspase- in the spleen and showed high il- β levels in serum, suggesting that pyroptosis occurred after infection. however, when the c a-c ar axis was blocked by an anti-c ar antibody (ab), expression of caspase- and il- β fell. these data indicate that mers-cov infection induces overactivation of complement, which may contribute to pyroptosis and inflammation. pyroptosis and inflammation were suppressed by inhibiting c ar . these results will further our understanding of the pathogenesis of mers-cov infection. middle east respiratory syndrome coronavirus (mers-cov), the second highly pathogenic coronavirus to emerge after severe acute respiratory syndrome coronavirus (sars-cov), causes severe acute respiratory failure and extra-pulmonary multi-organ damage accompanied by severe systemic inflammation [ ] [ ] [ ] . however, the pathogenesis of mers-cov still needs to be explored. complement activation and pyroptosis are two proteolytic cascades that defend the host against dangerous pathogens. they are important parts of the innate immune system and have some similar characteristics, including pore-formation and proinflammatory characteristics. pyroptosis is a lytic and inflammatory mode of regulated cell death catalyzed by the caspase family [ ] . activation of caspase- relies on assembly of inflammasome complexes, which contain nlrp b, nlrc , nlrp , and aim . different inflammasomes are activated by different pathogen-associated molecular patterns (pamps) or danger-associated molecular patterns (damps) via particular pattern recognition receptors (prrs). the best-characterized inflammasome is the nlrp inflammasome, which responds to a variety of bacterial, viral, and fungal agents [ , ] , damps (e.g., atp, monosodium urate crystals, and amyloid-β aggregates) [ , ] , and even environmental and industrial particles such as silica and asbestos [ ] . the nlrp inflammasome comprises the nlrp scaffold, the asc (pycard) adaptor, and pro-caspase- . the activated nlrp inflammasome promotes transformation of pro-caspase- to its active form, which proteolytically cleaves gasdermin d, pro-il- β, and pro-il- to yield their bioactive forms. the n-terminal domain of cleaved gasdermin d perforates the cell membrane, resulting in osmotic lysis [ ] , whereas mature il- β and il- act as proinflammatory cytokines [ ] . the complement system is an ancient molecular cascade; indeed, homologs have been found in sea urchin [ ] and mosquitoes [ ] . complement is activated via three pathways: the classical, lectin, and alternative pathways. during the process of activation, an enzyme named c convertase cleaves c to c a and c b, which are recruited to the c convertase to form the c convertase. c convertase catalyzes cleavage of c to c a and c b to initiate the terminal complement pathway, resulting in formation of the membrane attack complex, which has pore-forming properties. during this process, two split products, c a and c a (known as anaphylatoxins), promote inflammation or serve as chemoattractants by engaging their cognate receptors [ , ] . in a previous study we demonstrated that aberrant complement activation contributes to severe outcomes in hdpp transgenic mice infected with mers-cov, and that preventing over-activation of the complement system may be an effective clinical therapy for mers [ ] . here, we examined the role of pyroptosis in the pathogenesis of mers, along with the relationship between pyroptosis and complement. the results may help us to better understand the mechanism underlying severe outcomes after mers-cov infection. all animal experiments were approved by the institutional animal care and use committee (iacuc) of the beijing institute of microbiology and epidemiology (iacuc permit no: bime - ; permit date: march ). animal studies were carried out in strict accordance with the recommendations set out in the guide for the care and use of laboratory animals. human monocytic cells (thp- ) were purchased from the american type culture collection (manassas, va, usa, atcc number: tib- ) and cultured in rpmi medium supplemented with % heat-inactivated fetal bovine serum (fbs), u/ml penicillin, µg/ml streptomycin sulfate, × glutamax-i (l-glutamine alternate), and . mm -mercaptoethanol. thp- (differentiated) macrophages were obtained by exposing the cells to nm phorbol- -myristate- -acetate (pma) for h, followed by culture for a further h in complete growth medium without pma. mers-cov (hcov-emc/ strain) was propagated and titrated on vero cells in an approved biosafety level laboratory. thp- differentiated macrophages cultured in -cm flasks were infected with mers-cov at a multiplicity of infection of . . the virus was adsorbed at • c for h and unbound virus was washed away. hdpp -tg mice ( weeks old, female) [ ] were maintained in a pathogen-free facility and housed in cages containing sterilized feed and drinking water. following intraperitoneal anesthetization with sodium pentobarbital ( mg/kg body weight), mice were inoculated intranasally with mers-cov ( . % tissue culture infectious dose (tcid )) in µl dulbecco's modified eagle's medium (dmem). mice in the sham group received the same volume of dmem. for the experiments of c ar inhibition, mice were received an intravenous (i.v.) injection ( µg/kg) of a monoclonal ab (mab) specific for mouse c ar (hycult biotech, uden, the netherlands) to block the interaction of c a to c ar or an injection of phosphate-buffered saline (pbs) (sham treatment control) at the same time as mers-cov inoculation. all infectious experiments related to mers-cov were performed in an approved biosafety level facility. thp- differentiated macrophages were lysed in trizol™ reagent (life technologies, carlsbad, ca, usa) at h post-infection with mers-cov. total rna and proteins were isolated according to the reagent user guide. mice were euthanized by overdose inhalation of carbon dioxide at different time points after infection with mers-cov. lungs were harvested and total rna was extracted and purified using an rneasy extraction kit (qiagen, hilden, germany). to detect expression of inflammasomes and complement components in mers-cov-infected thp- differentiated macrophages and hdpp -tg mice, µg of total rna from cells or the lung of mice we used as template for first-strand cdna synthesis. the resulting cdna was subjected to quantitative pcr using power sybr ® green pcr master mix (life technologies, carlsbad, ca, usa) to determine the relative abundance of inflammasome and complement components. the forward and reverse primers used for each component are listed in the table . the relative amount of each gene was obtained by normalization against an endogenous control gene (gapdh) and calculated using the comparative −∆∆ct method. sham-infected monocytes and sham-infected hdpp -tg mice were used as respective calibrators. an identical amplification reaction comprising (i) polymerase activation and dna denaturation at • c for min, (ii) cycles each of the denaturation at • c for s, and (iii) an annealing/extension step at • c for s, was used for each gene analyzed. proteins isolated from thp- monocytic cells and thp- differentiated macrophages were electrophoresed in a % sds-page gel and transferred to a pvdf membrane (ge healthcare, dassel, germany). pvdf membranes were then blocked for h at room temperature in % non-fat cytokines in mouse serum were measured using a milliplex mouse cytokine/chemokine magnetic panel kit (merck millipore, burlington, ma, usa). a panel of inflammatory cytokines (il- β, il- , tnf-α, and ifn-γ) was detected according to the manufacturer's protocol. sections of paraffin-embedded spleen and lung tissues ( µm thick) were prepared and stained to detect antigen expression. briefly, retrieved sections were incubated overnight at • c with the following antibodies: mouse anti-caspase- mab (adipogen, san diego, ca, usa), polyclonal rabbit anti-cd (abcam, cambridge, ma, usa), and polyclonal anti-ifn-γrα (santa cruz biotechnology). biotinylated immunoglobulin g was then added, followed by an avidin-biotin-peroxidase conjugate (beijing zhongshan biotechnology co., ltd., beijing, china). immunoreactivity was detected using , diamino benzidine (dab). slides were counter-stained with hematoxylin. statistical analyses were performed using graphpad prism software, version . (graphpad software, san diego, ca, usa). student's t test was used to compare two groups with respect to relative expression of mrna and cytokine levels in serum. p values < . were considered significant. unlike abortive infection of sars-cov in human macrophages, mers-cov can establish a productive infection in macrophages and induce production of proinflammatory cytokines and chemokines [ ] . many rna viruses, such as ev , h n , h n influenza a virus, and zika virus, can infect macrophages and trigger il- β secretion via the nlrp inflammasome [ ] [ ] [ ] [ ] . to evaluate the response of macrophages to mers-cov infection, we inoculated thp- monocytic cells and thp- differentiated macrophages with mers-cov or rpmi medium (sham-infection). we then examined expression of nlrp , pro-caspase- , and pro-il- β h later by rt-qpcr. as shown in figure , mers-cov infection induced relatively higher expression of pro-caspase- ( figure a ) and pro-il- β ( figure b ), but not nlrp ( figure c ), in both thp- monocytes and macrophages. expression of pro-il- β in monocytes increased by -fold, whereas that in macrophages increased by -fold (on average). we verified expression of caspase- , il- β, and mers nucleocapsid protein (np) by western blotting ( figure d ). mers-cov-infected thp- macrophages expressed higher levels of pro-caspase- , pro-il- β, and activated il- β (p ) than sham-infected thp- macrophages or mers-cov-infected thp- monocytes. mers np was detected in both mers-cov-infected thp- monocytes and macrophages. these results indicate that mers-cov infection induces high levels of proinflammatory il- β secretion and thp- macrophage pyroptosis. to determine whether mers-cov infection induces pyroptosis in mice, we used rt-qpcr to detect mrna encoding nlrp , pro-caspase- , and pro-il- β in lung tissue from hdpp transgenic mice at day post-mers-cov infection. although there was no significant difference in expression of nlrp and pro-caspase- between the sham-infected and mers-cov-infected groups ( figure a ,b), expression of pro-il- β mrna was significantly higher after mers-cov infection ( figure c ). in addition, we measured the concentration of il- β in serum. the results showed that mers-cov infection induced production of il- β ( figure d ). furthermore, we examined expression of caspase- in the lung and spleen at day post-mers-cov infection by ihc. in line with the mrna results, there was no significant difference in expression of caspase- in the lung of sham-infected and mers-cov-infected mice. however, the spleens of mice infected with mers-cov showed higher expression of caspase- than those of mice in the sham group ( figure e ). the results indicated that mers-cov infection could induce pyroptosis in mice. (d) samples of total protein were subjected to western blotting to detect pro-caspase- , pro-il- β, activated il- β, and mers np. to determine whether mers-cov infection induces pyroptosis in mice, we used rt-qpcr to detect mrna encoding nlrp , pro-caspase- , and pro-il- β in lung tissue from hdpp transgenic mice at day post-mers-cov infection. although there was no significant difference in expression of nlrp and pro-caspase- between the sham-infected and mers-cov-infected groups (figure a,b) , expression of pro-il- β mrna was significantly higher after mers-cov infection ( figure c ). in addition, we measured the concentration of il- β in serum. the results showed that mers-cov infection induced production of il- β ( figure d ). furthermore, we examined expression of caspase- in the lung and spleen at day post-mers-cov infection by ihc. in line with the mrna results, there was no significant difference in expression of caspase- in the lung of sham-infected and mers-cov-infected mice. however, the spleens of mice infected with mers-cov showed higher expression of caspase- than those of mice in the sham group ( figure e ). the results indicated that mers-cov infection could induce pyroptosis in mice. il- β plays an important role in mediating autoinflammatory diseases and in generating inflammatory responses to infection [ ] . therefore, to assess the inflammatory responses in mice, we measured tnf-α, ifn-γ, and il- in serum at day post-mers-cov infection. as shown in figure a -c, serum from mice in the mers-cov-infected group contained more tnf-α, ifn-γ, and il- than that from sham-infected mice. ihc examination of cd and ifn-γ receptor expression also suggested greater macrophage infiltration and activation in the lung and spleen of mice at days post-mers-cov infection ( figure d ). these results indicate that mers-cov infection causes systemic inflammation, as reported in clinical mers patients and mers-cov infected animal models [ , ] . il- β plays an important role in mediating autoinflammatory diseases and in generating inflammatory responses to infection [ ] . therefore, to assess the inflammatory responses in mice, we measured tnf-α, ifn-γ, and il- in serum at day post-mers-cov infection. as shown in figure a -c, serum from mice in the mers-cov-infected group contained more tnf-α, ifn-γ, and il- than that from sham-infected mice. ihc examination of cd and ifn-γ receptor expression also suggested greater macrophage infiltration and activation in the lung and spleen of mice at days post-mers-cov infection ( figure d ). these results indicate that mers-cov infection causes systemic inflammation, as reported in clinical mers patients and mers-cov infected animal models [ , ] . the complement system links activation of toll-like receptors to transcription of il- β mrna [ , ] . it has been studied that intracellular c is converted to biologically active c a and c b by the protease cathepsin l [ ] , and c a activates nlrp and triggers il- β production in human monocytes by regulating efflux of atp [ ] . in addition, c a is believed to induce a proinflammatory or anti-inflammatory response when ligated to c ar or c ar respectively [ ] [ ] [ ] . thus, we used rt-qpcr to examine expression of complement components and their receptors c ar, c ar , and c ar . as shown in figure , c and c ar expression by both thp- monocytes and macrophages was highly upregulated (by - -fold) after mers-cov infection. c ar was upregulated, whereas c ar was downregulated, after mers-cov infection. the complement system links activation of toll-like receptors to transcription of il- β mrna [ , ] . it has been studied that intracellular c is converted to biologically active c a and c b by the protease cathepsin l [ ] , and c a activates nlrp and triggers il- β production in human monocytes by regulating efflux of atp [ ] . in addition, c a is believed to induce a proinflammatory or anti-inflammatory response when ligated to c ar or c ar respectively [ ] [ ] [ ] . thus, we used rt-qpcr to examine expression of complement components and their receptors c ar, c ar , and c ar . as shown in figure , c and c ar expression by both thp- monocytes and macrophages was highly upregulated (by - -fold) after mers-cov infection. c ar was upregulated, whereas c ar was downregulated, after mers-cov infection. our previous study demonstrated that mers-cov infection results in dysregulated host immune responses and severe tissue damage [ ] and inhibiting c ar alleviates mers-cov infection-induced tissue damage by regulating host immune responses [ ] . here, we used an anti-c ar ab to block the c a-c ar axis. the antibody was administered at the same time as mers-cov infection. we then measured expression of caspase- in the spleen and il- β in the lung and serum. compared with the pbs-treated group, mice receiving the anti-c ar ab expressed less caspase- in the spleen at day post-mers-cov infection ( figure a ). although there was no significant difference between the two groups with respect to pro-il- β mrna expression ( figure b ), serum levels of il- β were lower in the anti-c ar ab-treated group than in the pbs-treated group at day ( figure c ) and day [ ] post-mers-cov infection. these results suggest that complement inhibition decreased the expression of pyroptosis indicators, il- β and caspase- , in mice infected with mers-cov. our previous study demonstrated that mers-cov infection results in dysregulated host immune responses and severe tissue damage [ ] and inhibiting c ar alleviates mers-cov infection-induced tissue damage by regulating host immune responses [ ] . here, we used an anti-c ar ab to block the c a-c ar axis. the antibody was administered at the same time as mers-cov infection. we then measured expression of caspase- in the spleen and il- β in the lung and serum. compared with the pbs-treated group, mice receiving the anti-c ar ab expressed less caspase- in the spleen at day post-mers-cov infection ( figure a ). although there was no significant difference between the two groups with respect to pro-il- β mrna expression ( figure b ), serum levels of il- β were lower in the anti-c ar ab-treated group than in the pbs-treated group at day ( figure c ) and day [ ] post-mers-cov infection. these results suggest that complement inhibition decreased the expression of pyroptosis indicators, il- β and caspase- , in mice infected with mers-cov. our previous study demonstrated that mers-cov infection results in dysregulated host immune responses and severe tissue damage [ ] and inhibiting c ar alleviates mers-cov infection-induced tissue damage by regulating host immune responses [ ] . here, we used an anti-c ar ab to block the c a-c ar axis. the antibody was administered at the same time as mers-cov infection. we then measured expression of caspase- in the spleen and il- β in the lung and serum. compared with the pbs-treated group, mice receiving the anti-c ar ab expressed less caspase- in the spleen at day post-mers-cov infection ( figure a ). although there was no significant difference between the two groups with respect to pro-il- β mrna expression ( figure b ), serum levels of il- β were lower in the anti-c ar ab-treated group than in the pbs-treated group at day ( figure c ) and day [ ] post-mers-cov infection. these results suggest that complement inhibition decreased the expression of pyroptosis indicators, il- β and caspase- , in mice infected with mers-cov. at day after mers-cov infection, we measured proinflammatory cytokines (ifn-γ, tnf-α, and il- ) in serum. ifn-γ levels in mice treated with the anti-c ar ab were much lower than those in the pbs-treated group ( figure a ). to further evaluate the effect of complement inhibition on the local inflammation at later time after mers-cov infection, we examined expression of cd and ifn-γ receptor in lung and spleen at days post-mers-cov infection. ihc revealed that macrophage infiltration and activation were lower in the anti-c ar ab-treated group ( figure d ). taken together, these results suggest that inhibiting complement dampens the over-activated inflammatory response in mice infected with mers-cov. at day after mers-cov infection, we measured proinflammatory cytokines (ifn-γ, tnf-α, and il- ) in serum. ifn-γ levels in mice treated with the anti-c ar ab were much lower than those in the pbs-treated group ( figure a ). to further evaluate the effect of complement inhibition on the local inflammation at later time after mers-cov infection, we examined expression of cd and ifn-γ receptor in lung and spleen at days post-mers-cov infection. ihc revealed that macrophage infiltration and activation were lower in the anti-c ar ab-treated group ( figure d ). taken together, these results suggest that inhibiting complement dampens the over-activated inflammatory response in mice infected with mers-cov. macrophages play important roles in host defense by clearing dead cells, ingesting and destroying microbes, and presenting antigens to t lymphocytes. in addition, macrophages produce the full array of complement components [ ] and prrs, which are closely associated with inflammasome activation and pyroptosis. the accumulated studies indicate that macrophages play an important role in the pathogenesis of sars and mers [ , ] . macrophages infected with mers-cov secrete proinflammatory cytokines and chemokines [ ] . widespread distribution of these macrophages throughout many organs is one of the reasons underlying multi-organ damage and systemic inflammation after virus infection. pyroptosis or inflammasome activation plays an important role in virus-mediated pathogenesis. for example, abortive hiv- infection in quiescent macrophages play important roles in host defense by clearing dead cells, ingesting and destroying microbes, and presenting antigens to t lymphocytes. in addition, macrophages produce the full array of complement components [ ] and prrs, which are closely associated with inflammasome activation and pyroptosis. the accumulated studies indicate that macrophages play an important role in the pathogenesis of sars and mers [ , ] . macrophages infected with mers-cov secrete proinflammatory cytokines and chemokines [ ] . widespread distribution of these macrophages throughout many organs is one of the reasons underlying multi-organ damage and systemic inflammation after virus infection. pyroptosis or inflammasome activation plays an important role in virus-mediated pathogenesis. for example, abortive hiv- infection in quiescent lymphoid cd t cells leads to cd t cell pyroptosis independent of the nlrp inflammasome [ ] . ev d and zikv ns activate the nlrp inflammasome by interacting with nacht and the lrr domain of nlrp [ , ] . the pb -f protein of avian influenza a virus h n or h n induces inflammation by activating the nlrp inflammasome [ , ] , and deficiency of nlrp or caspase- protects mice against h n infection-associated morbidity and mortality [ ] . here, we demonstrate that the dysregulated macrophage-mediated immune responses after mers-cov infection may contribute to a severe outcome. several studies demonstrate relationships between complement and inflammasomes. for example, c -/-mice display reduced inflammasome activation in an intracerebral hemorrhage (ich) model [ ] . engagement of c a and c ar on cd + t cells generates reactive oxygen species, which are a classical damp, thereby triggering inflammasome assembly [ ] . in monocytes, c a alters metabolic programming and increases atp efflux, leading to nlrp activation and il- β generation via the receptor p × [ ] . thus, the complement system is considered to be an essential regulatory component of the cellular alarm system that controls inflammasome activation [ ] . here, we inhibit mers-cov infection-induced inflammation and pyroptosis by blocking the c a-c ar axis with an anti-c ar antibody. in our study, we first demonstrated that virus infection leads to pyroptosis by measuring activation of caspase- and il- β in human macrophages infected with mers-cov ( figure ). next, we found that mers-cov infection induced transcription of pro-il- β in the lung and expression of caspase- in the spleen. activated caspase- could be released into the extracellular space and delivered to the lung via exosomes [ ] or the systemic circulation; it then cleaves pro-il- β into its bio-activated form, which is secreted into the serum ( figure d ). we cannot exclude the possibility that pyroptosis occurs in the spleen because we did not detect expression of pro-il- β. meanwhile, examination of cd and ifn-γ receptor expression revealed macrophage infiltration and activation in the lungs and spleen ( figure d ). thus, pyroptosis could occur in both organs. in our previous study, we showed that inhibiting c ar increased splenic cell regeneration and decreased splenic cell apoptosis, thereby alleviating mers-cov infection-induced tissue damage [ ] . the spleen happens to be a large reservoir for myeloid lineage cells such as macrophages, dendritic cells and cd + t cells in which pyroptosis mainly occurred [ , , ] . here, we studied and demonstrated that inhibiting c ar suppressed caspase- activation ( figure a ) and macrophage infiltration and activation in the spleen ( figure d ), and thereby reduced the secretion of il- β into the serum ( figure c ) and the systemic inflammation ( figure ). however, in the lung tissue, there was no significant difference of pro-il- β transcription between the ab and sham-treated groups ( figure b ), which may due to the limited macrophages, the main cell type in which pyroptosis occurred, when compared to that in spleen. although many studies have focused on the link between complement and pyroptosis, the pathways that link them remain unclear. here, our results showed that mers-cov infection induces pro-il- β transcription, and complement activation, which leads to pyroptosis in macrophages. induction of pyroptosis was related with complement activation and may be promoted by ligation of c a and c ar , which was confirmed by the blockade of anti-c ar antibody (figure ) . in summary, these data indicate that mers-cov infection induces overactivation of complement, which may contribute to pyroptosis and inflammation. pyroptosis and inflammation were suppressed by inhibiting c ar . these research will further our understanding of the pathogenesis of mers-cov infection. occurred, when compared to that in spleen. although many studies have focused on the link between complement and pyroptosis, the pathways that link them remain unclear. here, our results showed that mers-cov infection induces pro-il- β transcription, and complement activation, which leads to pyroptosis in macrophages. induction of pyroptosis was related with complement activation and may be promoted by ligation of c a and c ar , which was confirmed by the blockade of anti-c ar antibody (figure ) . clinical course and 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pneumonia (cap), hospital-acquired pneumonia (hap) and ventilator associated pneumonia (vap) are all associated with significant mortality and cause huge expense to health care services around the world. early, appropriate antimicrobial therapy is crucial for effective treatment. syndromic diagnostic testing using novel, rapid multiplexed molecular platforms represents a new opportunity for rapidly targeted antimicrobial therapy to improve patient outcomes and facilitate antibiotic stewardship. in this article we review the currently available testing platforms and discuss the potential benefits and pitfalls of rapid testing in pneumonia. lower respiratory tract infections were accountable for an estimated . million deaths in , making them the third most common cause of death worldwide . community acquired pneumonia (cap) caused nearly , deaths in england and wales in and costs europe around € million annually . it is estimated that per , adults are hospitalised with pneumonia each year . hospital acquired pneumonia (hap) is defined as occurring > h after admission to a healthcare facility. it is caused by a different spectrum of more antibiotic resistant bacterial pathogens than those occurring in the community. ventilator associated pneumonia (vap) is defined as occurring > h after intubation for invasive artificial ventilation. the two entities combined (hap and vap) are the most common nosocomial infection in the developed world with hap complicating around % of hospital admissions . the incidence of vap in intubated patients is around % and is associated with mortality of around % . a retrospective matched cohort study by kollef et al. found patients who developed vap were intubated for longer, spent longer on icu, and were in hospital for a greater period of time. they estimated the additional cost of vap from to be $ , per patient. large amounts of empirical 'broad spectrum' antibiotics are used to treat pneumonia which inadvertently promote antimicrobial resistance (amr): a problem identified by the who as one of the leading threats to global health today. the o'neill report, commissioned by the uk government in , has highlighted the need for developed nations to take a lead in tackling amr. as part of this there is a specific recommendation that all antibiotic prescriptions should be supported by diagnostic tests where available by . the uk government recently published a five-year action plan for tackling amr, which emphasised the need for improved diagnostics to support antibiotic prescribing. this included a target to be able to report the percentage of antimicrobial prescriptions which are supported by a diagnostic test or decision making tool by . timely administration of appropriate antibiotics is a central tenant of care for patients with pneumonia , and yet the goldstandard for microbiological diagnosis remains traditional, slow, culture based methods. these take greater than h to identify an organism and often greater than h to provide phenotypic antibiotic sensitivity data. culture is insensitive, only detecting a pathogen in - % of patients with clinically diagnosed pneumonia , - and an even smaller proportion after the administration of antibiotics. in recent years several rapid syndromic molecular tests for pneumonia have been developed. these offer the potential to revolutionise treatment by providing information to clinicians in 'real-time' on the pathogens present and their likely antibiotic sensitivity by also detecting genotypic markers of resistance. multiple studies have demonstrated the superior diagnostic accuracy of pcr based platforms for detecting bacterial pathogens in the sputum compared with standard culture [ ] [ ] [ ] [ ] . this review will discuss the commercially available syndromic molecular panels for pneumonia, their potential clinical impact and the challenges to implementing them as a 'front line' diagnostic test. the greatest potential clinical benefit of a rapid syndromic test for pneumonia is being able to better utilise antibiotics. the superior diagnostic yield of multiplex pcr means that a pathogen is detected rapidly in a much greater proportion of patients, so therapy can be quickly tailored to the responsible organism. in some situations, this will allow narrowing of antimicrobial therapy: for example, identification of streptococcus pneumoniae facilitating a change of antibiotics to penicillin, in geographical areas with a low prevalence of penicillin resistant s. pneumoniae . in other cases, it may facilitate a change or escalation of antimicrobial therapy: for example, the identification of methicillin resistant staphylococcus aureus (mrsa) which would not be covered by empirical regimens in many areas. the absence of detection is also helpful: the sensitivity when compared to culture of molecular assays is very high so can reassure clinicians that organisms are not present and so support decisions to stop unnecessary antibiotics or to deescalate antibiotics that were used empirically to cover organisms subsequently not detected. the impact of this improved use of antibiotics are twofold: firstly, earlier appropriate antibiotics should improve clinical outcomes including mortality and length of stay. secondly, it prevents unnecessary broad-spectrum antibiotic use, which facilitates antibiotic stewardship and reduces antibiotic related adverse events. the aetiology of cap and hap/vap are highly variable between different regions and times, and this is reflected in studies of causative microbial agents as identified by culture. patients with underlying lung diseases, for example chronic obstructive pulmonary disease, can be colonised with microbial flora which are more typical pathogens of hap. as a result, they may develop community acquired infections caused by these agents. s. pneumoniae, haemophilius influenzae, s. aureus, moraxella catarrhalis and 'atypical' organisms including mycoplasma pneumoniae and legionella pneumophila are all cultured from the sputum of patients with cap. many of these organisms have predictable resistance patterns when interpreted with local epidemiological data. gadsby et al. developed and internally validated their own syndromic molecular assay for pneumonia. they used this to test sputum samples of adults admitted to hospital with cap . their assay detected a pathogen in % of patients (as opposed to % of patients using only routine culture). as a result, they proposed that % of antibiotic prescriptions in cap could have been deescalated based on results from multiplex pcr testing. the majority of these potential interventions involved stopping clarithromycin when atypical organisms were not detected or 'narrowing' antibiotics when a likely sensitive pathogen had been detected. in hap and vap, frequently cultured bacterial pathogens include s. aureus, pseudomonas aeruginosa, klebsiella species, escherichia coli, acinetobacter species and enterobacter species . empirical regimens are therefore broad spectrum and large numbers of antibiotics are consumed. the absence of certain organisms (for example p. aeruginosa ) could facilitate a narrowing of the antimicrobial spectrum with a knock-on effect of reducing antibiotic related adverse effects and improving stewardship. furthermore, common gram negative isolates are increasingly resistant in pneumonia surveillance studies . rapid molecular detection of these resistance genes should facilitate earlier initiation of effective antibiotics and this should lead to better outcomes. in adults, respiratory viruses are found in approximately one third of community acquired pneumonia cases , . one study found that % of patients admitted to intensive care with pneumonia were positive for a respiratory virus, with a broad range of viruses detected . detection of certain viruses such as influenza and adenovirus which are known to cause pneumonia, coupled with the absence of detection of bacteria and low levels of serum biomarkers such as procalcitonin (which is elevated in patients with bacterial infection), could support decisions to stop or use an abbreviated course of antibiotics. the respoc trial was a pragmatic randomised controlled trial that tested patients with community acquired acute respiratory illness using the biofire respiratory panel (which tests comprehensively for respiratory viruses and atypical bacteria) at the point-of-care. it found that patients who were tested with the filmarray were significantly more likely to receive a single dose or shorter course of antibiotics than those who were not. it also found a significant reduction in length of hospital stay in the intervention group along with improved use of neuraminidase inhibitors (nai) in patients with influenza. currently there are no licenced antiviral agents for respiratory viruses other than influenza. the benefit from nai treatment is greatest when they are started within h of symptom onset but there is evidence in adults to suggest ongoing benefit when started beyond this time and a recent study suggests that treatment earlier in admission to hospital improves outcome irrespective of overall duration of illness . as such, timely identification and treatment is critical. antiviral treatments for other respiratory viruses, including respiratory syncytial virus (rsv) are in development. since the s infection control methods including patient source isolation and deep cleaning with targeted decolonisation have been highly successful at reducing the spread of mrsa. enhanced infection control practices are recommended for a number of pathogens that may be present in patients with pneumonia. early identification of these should reduce the spread of these organisms, especially in hospitalised patients. some examples of these which are found on commercially available molecular tests are extended spectrum beta lactamases (esbls), carbapenemase producing enterobacteriaceae (cpes), mrsa, influenza and rsv. in the uk there is a mandatory requirement to report certain infectious diseases to public health england, so they can be investigated. l. pneumophilia is associated with outbreaks from devices that aerosolize water. there were cases in the uk in , earlier sensitive detection of these would allow outbreak investigation to occur sooner and potentially stop further cases occurring. at the current time there are fda approved, ce marked syndromic molecular panels for pneumonia which are commercially available: the filmarray (biofire diagnostics llc, salt lake city, utah, us) pneumonia panel and the unyvero (curetis gmbh, holzgerlingen, germany) hospitalised pneumonia (hpn) panel. fast track diagnostics respiratory panel (fast track diagnostics sarl, luxembourg) is another available platform with a large number of targets, but insufficient bacterial targets for it to be considered a true pneumonia panel so this will only be considered in brief. the commercially available platforms are summarised in table . the authors are aware of further panels in development from mobidiag, bruker, accelerate and axo science but published data is only available for the latter. there are also several research groups who have developed their own syndromic molecular pneumonia tests, most notably gadsby et al . there are a multitude of other 'respiratory pathogen' multiplex panels which have targets only for respiratory viruses, atypical bacterial targets or a very small range of typical bacteria. these are beyond the scope of this review article. we have only included assays with targets for a wide range of typical pathogens for pneumonia. this is an fda approved and ce marked platform that uses nested real-time pcr to detect clinically important respiratory targets ( semi-quantitative bacterial targets, qualitative atypical bacterial targets, [ ] [ ] [ ] furthermore, the pneumonia panel detects pathogens in a much higher proportion of samples than culture. buchan et al reported that the filmarray detected a bacterial target in % more specimens than routine culture, equating to over % increase in total bacterial detections. the relative abundance of organism for the bacterial targets is estimated based on real-time pcr relative to a material of known quantity and is grouped for reporting into bins. these represent approximately , , and > genomic copies of bacterial nucleic acid per millilitre of specimen respectively. concordance with reference molecular testing is very high but as expected the overall concordance between bin and reference sputum culture (cfu/ml) concentration was lower at around % and was highly variable between organisms. as such the manufacturer advises clinical correlation in interpretation of semi-quantitative results. to date there have been no published prospective interventional studies evaluating the clinical impacts of using the pneumonia panel in patients with pneumonia. observational data based on lower respiratory tract assays which preceded the final, fda approved pneumonia panel suggested change of antibiotics could be supported in > % of cases , . the hpn panel is ce marked and runs on the unyvero platform which includes the unyvero lysator, the unyvero cockpit and the unyvero analyzer. amplicons generated by parallel multiplex pcr reactions are qualitatively detected by hybridisation on arrays in a single use cartridge. it has a wide range of bacterial and resistance gene targets including the assay is validated for use on sputum (including expectorated sputum, bal and et aspirate). like the filmarray, the unyvero is a platform designed as a 'sample-to-answer' solution taking min of low skill hands-on time with a total turnaround time of - h. an equivalent test, the lower respiratory tract panel (lrt) has fda approval in the us but is only validated for use on tracheal aspirates. manufacturer reported diagnostic sensitivity for bacterial detection (when compared to reference culture and molecular detection in cases of discrepancy) is between and % with the majority of targets in the diagnostic performance data presented by the manufacturer, resistance marker detection aligned poorly with organism antibiogram: for example, matching in only / meca detections or / quinolone resistance markers in e. coli . this issue was noted by gadbsy et al for the p assay where the sensitivity for antibiotic resistance detection was %. two predecessors to the hpn cartridge have been developed and ce marked: the earlier p , and the later p . the former of these was evaluated most extensively by personne et al. who found the test to be sensitive for bacterial detection but with a run failure rate of . % and extensive discrepancies with regards to sensitivity testing . furthermore, the test was unable to differentiate s. pneumoniae from the s. mitis group. papan et al. reported that the p had a low sensitivity for gram positive organisms (when evaluated on paediatric samples) . the resistance panel on the p was broad but lacked several key emerging carbapenemase gene targets. the p panel rebalanced this by removing less clinically relevant resistance genes. it added targets for s. pneumoniae and m. pneumoniae . again, the sensitivity for bacterial detection remained high when assessed by ozongwu et al. albeit with a high overall run failure rate of % . the targets on the panel for the hpn are the same as the p , but the manufacturer claims it has a higher sensitivity and specificity. to date there are no published randomised controlled trials evaluating the clinical impact of the unyvero hpn system in patients with pneumonia. jamal et al performed a non-randomised interventional study using the p assay where antibiotics were adjusted based on the results and pathogens detected were compared to culture. the turnaround time for result was very quick ( ∼ h) compared to culture ( - h) and a large proportion of patients had antibiotics changed based on the p results, however the small number of patients studied and the lack of a comparator group make definitive conclusions impossible. gadsby et al. retrospectively tested bal samples with the p and reviewed patient notes. they reported that . % of patients who had positive standard of care microbiology could potentially have had a change in antibiotics earlier based on p results . conversely, they reported a false negative p result in ∼ % of those with a positive culture which could have caused harm if acted upon. the respiratory pathogens panel differs from the first two tests discussed in that it is exclusively a laboratory centred assay. the ce marked respiratory pathogens kit can be used on several standard laboratory cyclers. as such there is no reported standard turnaround time although it is greater than h. positive signals are detected from eight multiplex real-time pcr reactions. it is not an automated process so will have a considerably longer hands-on time requiring skilled extraction and setup. the there is very little published data comparing different syndromic molecular pneumonia tests. enne et al. and the inhale group presented data at eccmid where they compared the unyvero and the filmarray on surplus intensive care respiratory tract samples . the filmarray had slightly greater sensitivity for common pathogens, fewer major discordances (defined as routine culture finding or more undetected organisms) and fewer machine failures. the unyvero had slightly higher specificity and overall concordance with reference culture. whilst the data presented for syndromic molecular test for pneumonia clearly demonstrates high accuracy and the detection of many more pathogens than culture, no data has yet been published showing that this translates into improved antibiotic use or clinical benefit. other molecular diagnostics studies for blood stream infection have shown improved diagnostic performance, but negligible impact on clinical outcomes when results were not provided to clinicians along with infection specialist advice. it seems likely that such a wealth of information generated will require careful interpretation by an infection specialist in consultation with the clinicians directly caring for the patient, for these benefits to be maximised. rapid syndromic molecular platforms have the potential to significantly improve the use of antibiotics and clinical outcomes in patient with pneumonia, but high quality randomised controlled trials are urgently required to evaluate their clinical impact. we are aware of trials that are currently underway or in set up that may address this evidence gap: the saripoc study is a single centre randomised controlled trial (rct) recruiting critically unwell patients with pneumonia in southampton, uk. the inhale study is a uk multicentre, rct recruiting critically unwell patients with hap and vap. pibcap is a uk multicentre rct recruiting patients with cap. the norcap trial, in norway is a single centre rct in set up, also aiming to recruit patients with cap. a further single centre rct in edinburgh is using molecular testing for broader community acquired lrti microbial diagnosis. the first of these two studies are testing patients at the point-of-care, whereas the others use rapid laboratory-based testing. antibiotic de-escalation based on results is a key component of antibiotic stewardship and is widely accepted as good practice. trials looking at the efficacy and safety of antimicrobial de-isolation based on culture results are sparse. the vast majority of published studies are observational and comparison between studies for so many variables (hap, cap, vap, icu/ non-icu, severe sepsis etc.) are fraught with difficulties. furthermore, due to the geographic variability in causative organisms and prescribing practices, they are often poorly transferrable between regions. to our knowledge no interventional studies have looked at the safety or efficacy of antimicrobial de-escalation based on multiplexed pcr for pathogens of pneumonia. studies to date have made their de-escalation intervention after at least h when the patient has stabilised, and culture results are available. both the idsa and the national institute for clinical excellence (nice) cite an urgent need for well-run rcts on the impact of de-escalating antimicrobial therapy , . the idsa and the american thoracic society advise antibiotic de-escalation in hap/vap according to culture results on the basis of expert opinion, citing a high level of confidence that it 'reduces costs, burdens, and side effects, and that it is very likely that deescalation also reduces antimicrobial resistance' . there a small number of interventional studies looking at antibiotic de-escalation based upon microbiological culture results in hap/vap which have suggested this practice is safe , . high quality data for outcomes, including length of intensive care stay and antibiotic savings, are lacking and conflicting. a meta-analysis by khan et al of observational studies reviewing antibiotic de-escalation in pneumonia in icu (hap and vap only) found no difference in mortality between those who were de-escalated according to culture result and those that weren't. in the context of cap, both the idsa and nice/bts guidelines recommend organism directed therapy when a pathogen has been identified by culture. high quality data is lacking but observational data and limited interventional data suggests this is safe [ ] [ ] [ ] . a systematic review by paul et al included studies with cap, hap, vap and blood stream infection. the reviewers found no association between de-escalation and survival with pneumonia (or . , % ci . - . ). concern has been raised that the high sensitivity of molecular tests will lead to excessive detection of colonising flora which may paradoxically increase unnecessary antibiotic use. this is particularly pertinent in expectorated sputa where small numbers of potentially pathogenic bacteria can be present in the absence of disease. a potential solution to this is the development of semi-quantitative molecular methods such as with the biofire r filmarray r pneumonia panel. this provides a representation of the amount of bacterial dna present which is highly concordant with reference molecular techniques. as highlighted by studies using the unyvero , , molecular detection of resistance genes may correlate poorly with phenotypic sensitivity in its current form. detection of genes from 'off panel' organisms, for example mec a genes in colonising coagulase negative staphylococci, may be incorrectly attributed to those organisms which are on the panel. as such, clinicians will need to be cautious in interpreting these results. as well as having relatively quicker run times, syndromic multiplex molecular tests could potentially be deployed at the pointof-care. the resppoc trial by brendish et al., demonstrated with a respiratory viral panel that this was logistically feasible and associated with a number of clinical benefits compared to routine clinical care . a post hoc analysis of patients who tested positive for respiratory viruses in the trial highlighted an association between rapid turn-around time (defined as < . h), shorter hospital admission and shorter durations of antibiotic therapy. it is our belief that point-of-care testing represents the ideal strategy for new, rapid diagnostic test platforms allowing clinicians to maximise the benefit from such accurate tests early in the decision-making process. clearly, rigorous quality assurance is essential for any diagnostic test irrespective of the site of testing. it should also be noted that the tests described in this article are not currently clia waivereda requirement for use at the point-of-care in the us. rapid syndromic molecular tests for pneumonia have improved diagnostic accuracy compared to the current gold standard of culture and can provide results in real time. in the era of widespread amr their use has the potential to dramatically improve the rational use of antibiotics and to improve clinical outcomes in patient with pneumonia. high quality data from well conducted randomised controlled trials are now urgently needed to assess the impact of these platforms on antibiotic use and patient outcome. poole, s. -declarations of interest: none. clark, t.w. has received speaker fees, reimbursement for travel and honoraria from biofire llc and biomerieux and has also received equipment and consumables from these companies for the purposes of independent research. no commercial entities had any input into this manuscript. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. global, regional, and national life expectancy, all-cause mortality, and causespecific mortality for causes of death, - : a systematic analysis for the global burden of disease study - ): directly standardised rate, all ages, annual trend: nhs digital risk 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kwon, sungjun; lee, donghyun title: predicting infectious disease using deep learning and big data date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: c v jz infectious disease occurs when a person is infected by a pathogen from another person or an animal. it is a problem that causes harm at both individual and macro scales. the korea center for disease control (kcdc) operates a surveillance system to minimize infectious disease contagions. however, in this system, it is difficult to immediately act against infectious disease because of missing and delayed reports. moreover, infectious disease trends are not known, which means prediction is not easy. this study predicts infectious diseases by optimizing the parameters of deep learning algorithms while considering big data including social media data. the performance of the deep neural network (dnn) and long-short term memory (lstm) learning models were compared with the autoregressive integrated moving average (arima) when predicting three infectious diseases one week into the future. the results show that the dnn and lstm models perform better than arima. when predicting chickenpox, the top- dnn and lstm models improved average performance by % and %, respectively. the dnn model performed stably and the lstm model was more accurate when infectious disease was spreading. we believe that this study’s models can help eliminate reporting delays in existing surveillance systems and, therefore, minimize costs to society. infectious disease occurs when a person is infected by a pathogen from another person or an animal. it not only harms individuals, but also causes harm on a macro scale and, therefore, is regarded as a social problem [ ] . at the korea center for disease control (kcdc), infectious disease surveillance is a comprehensive process in which information on infectious disease outbreaks and vectors are continuously and systematically collected, analyzed, and interpreted. moreover, the results are distributed quickly to people who need them to prevent and control infectious disease. the kcdc operates a mandatory surveillance system in which mandatory reports are made without delay to the relevant health center when an infectious disease occurs and it operates a sentinel surveillance system in which the medical organization that has been designated as the sentinel reports to the relevant health center within seven days. the targets of mandatory surveillance consist of a total of infectious diseases from groups to by the kcdc. the targets of sentinel surveillance include influenza from group along with infectious diseases from group . overall, a total of infectious diseases in six groups are monitored. in the current korean infectious disease reporting system, if there is a legally defined infectious disease patient at a medical organization, a report is made to the managing health center through the infectious disease web reporting system. the managing health center reports to the city and province health offices through another system and the city and province health offices report to the kcdc. in the conventional reporting system, some medical organizations' infectious disease reports are incomplete and delays can occur in the reporting system. for instance, in the traditional influenza surveillance system, around two weeks elapses between when a report is made and when it is disseminated [ ] . the kcdc has been running an automated infectious disease reporting system as a pilot project since . however, by , only . % of all medical organizations were participating in the pilot project. in medical organizations using the conventional infectious disease reporting system, a large number of missing and delayed reports can occur, which hinders a prompt response to infectious disease. as such, it is necessary to create a data-based infectious disease prediction model to handle situations in real time. furthermore, if this model can understand the extent of infectious disease trends, the costs to society from infectious disease can be minimized. an increasing number of researchers recognize these facts and are performing data-based infectious disease surveillance studies to supplement existing systems and design new models [ ] [ ] [ ] [ ] [ ] [ ] [ ] . among these, studies are currently being performed on detecting infectious disease using big data such as internet search queries [ ] [ ] [ ] [ ] [ ] [ ] . the internet search data can be gathered and processed at a speed that is close to real time. according to towers et al., internet search data can create surveillance data faster than conventional surveillance systems [ ] . for example, when huang et al. predicted hand, foot, and mouth disease using the generalized additive model (gam), the model that included search query data obtained the best results. as such, it has been reported that new big data surveillance tools have the advantage of being easy to access and can identify infectious disease trends before official organizations [ ] . in addition to internet search data, social media big data is also being considered. tenkanen et al. report that social media big data is relatively easy to collect and can be used freely, which means accessibility is satisfactory and the data is created continuously in real time with rich content [ ] . as such, studies have used twitter data to predict the occurrences of mental illness [ ] and infectious disease [ ] [ ] [ ] [ ] in addition to predictions in a variety of other scientific fields [ ] [ ] [ ] [ ] . in particular, a study by shin et al. reported that infectious diseases and twitter data are highly correlated. there is the possibility of using digital surveillance systems to monitor infectious disease in the future [ ] . when these points are considered, using search query data and social media big data should have a positive effect on infectious disease predictions. in addition to these studies, there are also studies that have used techniques from the field of deep learning to predict infectious disease [ , , , ] . deep learning is an analysis method and, like big data, it is being actively used in a variety of fields [ ] . deep learning yields satisfactory results when it is used to perform tasks that are difficult for conventional analysis methods [ ] [ ] [ ] . in a study by xu et al., a model that used deep learning yielded better prediction performance than the generalized linear model (glm), the least absolute shrinkage and selection operator (lasso) model, and the autoregressive integrated moving average (arima) model [ ] . as such, methods of predicting infectious disease that use deep learning are helpful for designing effective models. there are also examples of infectious disease prediction based on environmental factors such as weather [ ] [ ] [ ] [ ] . previous studies have confirmed that weather data comprises a factor that has a great influence on the occurrence of infectious diseases [ ] [ ] [ ] . liang et al. showed that rainfall and humidity are risk factors for a hemorrhagic fever with a renal syndrome [ ] . in addition, a study by huang et al. reported that trends in dengue fever show a strong correlation with temperature and humidity [ ] . previous studies indicate that infectious disease can be predicted more effectively if weather variables, internet big data, and deep learning are used. most previous research has attempted to predict infectious disease using internet search query data alone. however, as discussed above, it is necessary to also consider various big data and environmental factors such as weather when predicting infectious disease. in addition, in the case of models that use deep learning, it is possible to improve prediction performance by optimizing the deep learning model by optimizing its parameters. therefore, the aim of this study is to design a model that uses the infectious disease occurrence data provided by the kcdc, search query data from search engines that are specialized for south korea, twitter social media big data, and weather data such as temperature and humidity. according to a study by kwon et al., a model that considers the time difference between clinical and non-clinical data can detect infectious disease outbreaks one to two weeks before current surveillance systems [ ] . therefore, this study adds lag to the collected dataset to take temporal characteristics into account. in addition, in the design process, a thorough testing of all the input variable combinations is performed to examine the effects of each resulting dataset on infectious disease outbreaks and select the optimal model with the most explanatory power. the model's prediction performance is verified by comparing it with an infectious disease prediction model that uses a deep learning method and an infectious disease prediction model that uses time series analysis. ultimately, using the results obtained by this study, it should be possible to create a model that can predict trends about the occurrence of infectious disease in real time. such a model can not only eliminate the reporting time differences in conventional surveillance systems but also minimize the societal costs and economic losses caused by infectious disease. the remainder of this paper is organized as follows. section describes the data sources and standards used in this study and introduces the analysis methodology used to design the prediction model. in section , the analysis results are described and their implications are discussed. section discusses the results. section concludes the paper. as mentioned above, this study uses four kinds of data to predict infectious disease, which includes search query data, social media big data, temperature, and humidity. the standards for the non-clinical data are as follows. data from days between january, and july, was used. the infectious diseases selected for this study are subject to mandatory reporting. unlike those diseases subject to mandatory reporting, diseases subject to sentinel reporting aggregate data on a weekly basis. since prediction is also performed on a weekly basis, it is difficult to cope with infectious diseases in real time. therefore, diseases that are subject to sentinel reporting were excluded from the study. moreover, the study excluded infectious diseases with an annual occurrence rate of less than as well as infectious diseases that have a statistically insignificant model with an adjusted r-squared value of less than . when regression analysis is performed using all variables. three infectious diseases satisfied all conditions, which include malaria, chickenpox, and scarlet fever. the search data was collected from the naver data lab (https://datalab.naver.com/keyword/trendsearch.naver). the usage share data provided by internettrend (http://internettrend.co.kr/trendforward.tsp) on search engines in the health/medicine field in the first half of shows that the naver search engine had the highest usage share ( . %) in south korea. therefore, it was chosen as the search engine for extracting search data. note that the collected search data consists of only korean terms because the search engine is specific to south korea. the search queries used in this study consisted of the infectious disease's proper name and symptoms (e.g., "chickenpox" and "chickenpox symptoms" in korea). the frequency of inquiries using these search queries were used as the search data. the number of searches were normalized with respect to the largest number of searches within the study period. weather data (temperature and humidity) were collected from the korea meteorological administration's weather information open portal (https://data.kma.go.kr). hourly data collected from weather stations nationwide was converted into daily average data for each station. in gyeonggi-do province, where around half of south korea's population lives, there are many weather stations crowded together. there was a concern that simply finding the averages of the daily data for each station would cause errors to occur, so the following process was performed. first, the averages of the data from each station were collected for the eight provinces in south korea (gyeonggi-do, gangwon-do, chungcheongnam-do, chungcheongbuk-do, jeollanam-do, jeollabuk-do, gyeongsangbuk-do, and gyeongsangnam-do). next, the averages of the data for each of the eight provinces were found to obtain south korea's national average weather data. average temperature (degrees celsius) and average humidity (percentage) were recorded. social media big data was collected for each infectious disease from twitter through a web crawler that used the python selenium library. for the twitter data, the daily number of tweets mentioning infectious disease was recorded. lastly, infectious disease data was collected from the infectious disease web statistics system (https://is.cdc.go.kr/dstat/index.jsp). this data consists of the daily number of people who were infected throughout south korea. table shows the sources and descriptions of the data. table shows the statistics for each of the infectious disease variables used in this study. in the case of temperature and humidity, the same conditions were used, which means they were put in a shared category. the data in table shows that an average of . people are infected with chickenpox daily with a standard deviation of . and the daily naver frequency average is . with a standard deviation of . . we observed that all the statistics for chickenpox are higher than those for other infectious diseases. figure shows the overall framework of the model used in this study including the data collection process and the comparison of models designed using the deep neural network (dnn) method, the long-short term memory (lstm) method, the autoregressive integrated moving average (arima) method, and the ordinary least squares (ols) method. this study constructed an infectious disease surveillance model that uses non-clinical search data, twitter data, and weather data. to design the optimal prediction model, the ols models that use all possible combinations of variables in the dataset were created. the adjusted r-squared values of each model were compared. in addition, lags of - days were added to each infectious disease and their adjusted r-squared values were compared in a preliminary analysis. a lag of seven days, which had high explanatory power for all infectious diseases, was selected as the optimal lag parameter. the optimal parameters were used to create the ols, arima, dnn, and lstm models. before analysis, this study applied a lag of seven days between the input variables (optimal variable combination) and their associated output variable (disease occurrence). the ols dataset was divided into a training data subset and a test data subset using a ratio of : . this means all rows of collected data were divided such that there were rows for the training data subset and rows for the test data subset. the training data subset was only used for model training. the test data subset was only used for prediction and performance evaluation in the model after training. the arima dataset was also divided into a training data subset and test data subset using a ratio of : , but only the disease occurrences were required for arima. similarly to the data above, the rows of disease occurrence data were divided into rows for the training data subset and rows for the test data subset. in the dnn and lstm models, the whole dataset was divided into training, validation, and test data subsets at a ratio of : : and training was performed. this means all rows of collected data were divided into rows for the training data subset, rows for the validation data subset, and rows for the test data subset. the training data subset was used for model training. the validation data subset was only used for performance evaluation during training. the final model after training was the model that yielded the best performance when the validation data subset was used in training. the test data subset was only used for the prediction and performance evaluation. to compare the models, the root mean squared error (rmse) was used to evaluate the prediction rates. rmse is a common measurement for the difference between predicted and actual values. it is usually used in the other fields as well as in the prediction of infectious diseases [ , , ] . rmse is calculated using the equation below. ( ) this study constructed an infectious disease surveillance model that uses non-clinical search data, twitter data, and weather data. to design the optimal prediction model, the ols models that use all possible combinations of variables in the dataset were created. the adjusted r-squared values of each model were compared. in addition, lags of - days were added to each infectious disease and their adjusted r-squared values were compared in a preliminary analysis. a lag of seven days, which had high explanatory power for all infectious diseases, was selected as the optimal lag parameter. the optimal parameters were used to create the ols, arima, dnn, and lstm models. before analysis, this study applied a lag of seven days between the input variables (optimal variable combination) and their associated output variable (disease occurrence). the ols dataset was divided into a training data subset and a test data subset using a ratio of : . this means all rows of collected data were divided such that there were rows for the training data subset and rows for the test data subset. the training data subset was only used for model training. the test data subset was only used for prediction and performance evaluation in the model after training. the arima dataset was also divided into a training data subset and test data subset using a ratio of : , but only the disease occurrences were required for arima. similarly to the data above, the rows of disease occurrence data were divided into rows for the training data subset and rows for the test data subset. in the dnn and lstm models, the whole dataset was divided into training, validation, and test data subsets at a ratio of : : and training was performed. this means all rows of collected data were divided into rows for the training data subset, rows for the validation data subset, and rows for the test data subset. the training data subset was used for model training. the validation data subset was only used for performance evaluation during training. the final model after training was the model that yielded the best performance when the validation data subset was used in training. the test data subset was only used for the prediction and performance evaluation. to compare the models, the root mean squared error (rmse) was used to evaluate the prediction rates. rmse is a common measurement for the difference between predicted and actual values. it is usually used in the other fields as well as in the prediction of infectious diseases [ , , ] . rmse is calculated using the equation below. the optimal variable combinations for the model were selected by considering all possible models in the regression analysis. the models are combinations of the four types of data in the dataset (naver searches (n), twitter searches (tw), temperature (t), and humidity (h)). figure shows the adjusted r-squared values of regression models for each infectious disease. among the observed regression models, the models that are combinations of all variables had the best explanatory power. therefore, this combination was chosen as the optimal variable combination. the optimal variable combinations for the model were selected by considering all possible models in the regression analysis. the models are combinations of the four types of data in the dataset (naver searches (n), twitter searches (tw), temperature (t), and humidity (h)). figure shows the adjusted r-squared values of regression models for each infectious disease. among the observed regression models, the models that are combinations of all variables had the best explanatory power. therefore, this combination was chosen as the optimal variable combination. previous results [ ] have shown that it is possible to predict infectious disease at an early stage if a model is designed to consider the time difference between clinical data and non-clinical data. based on this observation, our model was designed to consider the time difference in each data set. in this situation, "lag" refers to the time delay between the date the data is collected and the date at which the effects actually occur. this means analysis was performed by establishing the time difference between the four input variables used in this study and the output variable that is actually affected. for example, a lag of means that the output variable of january is calculated using the input variables of january . figure shows the adjusted r-squared values of regression models when - days of lag were tested for each of the infectious diseases in order to select the optimal lag. in the case of chickenpox, it was found that lags of , , and days yielded the highest explanatory power. for scarlet fever, it was found that lags of , , and days yielded the highest explanatory power. in the case of malaria, it was found that lags of , , and days yielded the highest explanatory power. for chickenpox and malaria, the lag with the highest explanatory power was one day. however, it was decided that this lag was not suitable for the ultimate goal of reducing the length of delay from reporting to dissemination. in the observed regression models, the explanatory power of a lag of seven days was high for all infectious diseases. therefore, it was decided that this lag was the most suitable and was used for later predictions. previous results [ ] have shown that it is possible to predict infectious disease at an early stage if a model is designed to consider the time difference between clinical data and non-clinical data. based on this observation, our model was designed to consider the time difference in each data set. in this situation, "lag" refers to the time delay between the date the data is collected and the date at which the effects actually occur. this means analysis was performed by establishing the time difference between the four input variables used in this study and the output variable that is actually affected. for example, a lag of means that the output variable of january is calculated using the input variables of january . figure shows the adjusted r-squared values of regression models when - days of lag were tested for each of the infectious diseases in order to select the optimal lag. in the case of chickenpox, it was found that lags of , , and days yielded the highest explanatory power. for scarlet fever, it was found that lags of , , and days yielded the highest explanatory power. in the case of malaria, it was found that lags of , , and days yielded the highest explanatory power. for chickenpox and malaria, the lag with the highest explanatory power was one day. however, it was decided that this lag was not suitable for the ultimate goal of reducing the length of delay from reporting to dissemination. in the observed regression models, the explanatory power of a lag of seven days was high for all infectious diseases. therefore, it was decided that this lag was the most suitable and was used for later predictions. in this study, the ols model was used to select the optimal parameter values. it was also used as a comparison model to evaluate the prediction performance of the deep learning models. linear regression is a regression analysis technique that models the linear correlation between the output variable y and one or more input variables x in the collected data. the model has the following form. ols is the most simple and commonly used form of linear regression. it is a technique that minimizes the sum of squared errors and can solve the mathematical expression for ß, which is the parameter to be predicted, by using the equation below. ols analyses were performed by r version . . (https://www.r-project.org/). because ols is the simplest form of linear regression analysis, it is not sufficient for comparison with deep learning models. therefore, we also compare the arima model, which is often used for the prediction of infectious diseases [ ] [ ] [ ] . this will more clearly compare traditional analysis methods (ols and arima) with deep learning (dnn and lstm). the arima model is a method for analyzing non-stationary time series data. one characteristic of arima analysis is that it can be applied to any time series. in particular, it shows the detailed changes when the data fluctuates rapidly over time. in this study, we used seasonal arima because the collected data is seasonal. the seasonal arima model is denoted as arima(p, d, q)(p, d, q)s. where p is the order of the autoregressive part, d is the order of the differencing, q is the order of the moving-average process, and s is the length of the seasonal cycle. (p, d, q) is the seasonal part of the model. the seasonal arima model is written below. in this study, the ols model was used to select the optimal parameter values. it was also used as a comparison model to evaluate the prediction performance of the deep learning models. linear regression is a regression analysis technique that models the linear correlation between the output variable y and one or more input variables x in the collected data. the model has the following form. ols is the most simple and commonly used form of linear regression. it is a technique that minimizes the sum of squared errors and can solve the mathematical expression for ß, which is the parameter to be predicted, by using the equation below. ols analyses were performed by r version . . (https://www.r-project.org/). because ols is the simplest form of linear regression analysis, it is not sufficient for comparison with deep learning models. therefore, we also compare the arima model, which is often used for the prediction of infectious diseases [ ] [ ] [ ] . this will more clearly compare traditional analysis methods (ols and arima) with deep learning (dnn and lstm). the arima model is a method for analyzing non-stationary time series data. one characteristic of arima analysis is that it can be applied to any time series. in particular, it shows the detailed changes when the data fluctuates rapidly over time. in this study, we used seasonal arima because the collected data is seasonal. the seasonal arima model is denoted as arima(p, d, q)(p, d, q) s . where p is the order of the autoregressive part, d is the order of the differencing, q is the order of the moving-average process, and s is the length of the seasonal cycle. (p, d, q) is the seasonal part of the model. the seasonal arima model is written below. where y t refers to the value of the time series at time t, µ is the mean term, a t is the independent disturbance, b is the backshift operator, φ(b) is the autoregressive operator, and θ(b) is the moving average operator. φ s b s and θ s b s are the seasonal operators of the model. the arima analyses were carried out using the r version . . . the dnn model is a feedforward analysis method that is a basic model for deep learning. dnn is composed of a minimum of three node layers and, with the exception of the input node, each node uses a nonlinear activation function. dnn uses a supervised learning technique called backpropagation. in this study, an infectious disease prediction model that uses dnn was designed and the basic dnn model was compared with this more advanced deep learning model. the variables used in dnn are bias b, input x, output y, weight w, calculation function σ and activation function f (σ). each neuron in dnn uses the following equation. figure shows the structure of a neuron in the dnn model. the dnn analyses were carried out using the "dense layer" option of the keras package in the python version . . (https://keras.io/). there are parameters available in the dense layer. we only modified the units, activation function, and dropout. the rest of the parameters used the default values (e.g., use_bias = true and kernel_regularizer = none). where refers to the value of the time series at time , is the mean term, is the independent disturbance, is the backshift operator, ( ) is the autoregressive operator, and ( ) is the moving average operator. ( ) and ( ) are the seasonal operators of the model. the arima analyses were carried out using the r version . . . the dnn model is a feedforward analysis method that is a basic model for deep learning. dnn is composed of a minimum of three node layers and, with the exception of the input node, each node uses a nonlinear activation function. dnn uses a supervised learning technique called backpropagation. in this study, an infectious disease prediction model that uses dnn was designed and the basic dnn model was compared with this more advanced deep learning model. the variables used in dnn are bias , input , output , weight , calculation function and activation function ( ). each neuron in dnn uses the following equation. figure shows the structure of a neuron in the dnn model. the dnn analyses were carried out using the "dense layer" option of the keras package in the python version . . (https://keras.io/). there are parameters available in the dense layer. we only modified the units, activation function, and dropout. the rest of the parameters used the default values (e.g., use_bias = true and kernel_regularizer = none). the lstm model is suitable for predicting time series data when there is a time step with a random size [ ] . it was thought that prediction performance could be improved by creating an infectious disease prediction model using lstm and the time series data collected in this study. an important advantage of recurrent neural networks (rnns) is that contextual information is available when mapping io sequences. however, there is a gradient problem in that the effect of a given input on the hidden layer can be increased or decreased significantly during the circular connection. as new inputs are overwritten, the sensitivity of the first input decreases over time. therefore, the network is "forgotten". the input gate, output gate, and forget gate are non-linear summation units that control the activation of the cell. the forget gate multiplies the previous state of the cell while the input and output gates multiply the io of the cell. the activation function f of the gate is a logistic sigmoid. the io activation functions g and h of the cell usually use hyperbolic tangents or logistic sigmoids. however, in some cases, h uses the identity function. as long as the forget gate is open and the input gate is closed, the memory cell continues to remember the first input. in this way, lstm is an algorithm that resolves a problem in traditional rnns [ ]. the lstm model is suitable for predicting time series data when there is a time step with a random size [ ] . it was thought that prediction performance could be improved by creating an infectious disease prediction model using lstm and the time series data collected in this study. an important advantage of recurrent neural networks (rnns) is that contextual information is available when mapping io sequences. however, there is a gradient problem in that the effect of a given input on the hidden layer can be increased or decreased significantly during the circular connection. as new inputs are overwritten, the sensitivity of the first input decreases over time. therefore, the network is "forgotten". the input gate, output gate, and forget gate are non-linear summation units that control the activation of the cell. the forget gate multiplies the previous state of the cell while the input and output gates multiply the io of the cell. the activation function f of the gate is a logistic sigmoid. the io activation functions g and h of the cell usually use hyperbolic tangents or logistic sigmoids. however, in some cases, h uses the identity function. as long as the forget gate is open and the input gate is closed, the memory cell continues to remember the first input. in this way, lstm is an algorithm that resolves a problem in traditional rnns [ ] . the equations for forgetting, storing, renewing, and outputting information in the cell are shown below, respectively. when data (x t ) is input to the lstm cell in equation ( ), function f t determines the information to be forgotten in the cell layer. in equations ( ) and ( ), information that will be newly saved in the cell layer is created in i t and c t in equation ( ), the cell layer c t is renewed using f t , i t , and c t in equation ( ), the cell layer's information is used and h t is the output. in equation ( ), the cell state gets a value between − and through the tanh function. the values of c t and h t are kept for the next iteration of lstm. lstm analyses were carried out using the "lstm layer" of the keras package in the python version . . . there are parameters available in the lstm layer. we only set the units, activation function, return sequence, and dropout. the rest of the parameters used the default values (e.g., use_bias = true, recurrent regularizer = none, recurrent_constraint = none, and unit_forget_bias = none). figure shows the parameter selection method for the deep learning approach used in this study. the adadelta, adagrad, adam, adamax, nadam, rmsprop, and stochastic gradient descent (sgd) optimizers were compared. all parameters of each optimizer used the default values of the keras package. for instance, in sgd, the learning late is . , the momentum is , the decay is , and the nesterov momentum is false. in addition, the following activation functions were evaluated: exponential linear unit (elu), rectified linear unit (relu), scaled elu (selu), and softplus. lastly, various numbers of epochs ( , , , and ) were evaluated. the other parameters were fixed as follows: number of hidden layers = , number of units in each hidden layer = , batch size = , and drop out = . prediction models with variable and fixed parameters were trained on the data and the resulting models were compared to determine the optimal prediction model. to ensure the amount of dnn model data was the same as that of the lstm model, previous data from the same time period as the lstm was inserted. all deep learning models were implemented using the keras package in the python version . . . and drop out = . prediction models with variable and fixed parameters were trained on the data and the resulting models were compared to determine the optimal prediction model. to ensure the amount of dnn model data was the same as that of the lstm model, previous data from the same time period as the lstm was inserted. all deep learning models were implemented using the keras package in the python version . . . the regression model was formed based on days of data in which a lag of seven days was applied to each infectious disease dataset. the dataset was divided up in an : ratio and each part was used for constructing the regression model and prediction. table presents the ols results. each regression model had results that were below the level of significance (p < . ). the adjusted r-squared value was greater than . for all three infectious diseases, which means the models can be said to have significant explanatory power. of the infectious disease regression models, the chickenpox model yielded significant results for the naver search queries, temperature, and humidity. the scarlet fever model yielded significant results for the naver search queries and humidity. additionally, the malaria model yielded significant results for the naver search queries and temperature (p < . ). looking at these results together, the naver search query data was significant for all three infectious diseases and the twitter data was not significant for any of the three. it can be seen that the internet search query data can be used to design an infectious disease prediction model, which was reported by previous studies. however, the results for the twitter data differ from the results of previous studies. this is believed to be because naver accounted for the largest share ( . %) of korean search engine use in the health/medicine field for the first half of while twitter accounted for the smallest share ( . %) of social media use in the health/medicine field for the same time period (http://internettrend.co.kr/trendforward.tsp). however, the twitter data had an effect on the process of finding the model with the highest adjusted r-squared value. therefore, it is expected to have an effect on future analysis as well. the temperature had a significant relationship with all infectious diseases except for scarlet fever and humidity had a significant relationship with all infectious diseases except for malaria. the values of the coefficients show that the most significant variables for chickenpox and scarlet fever was the naver search query data ( . and . , respectively) and, for malaria, it was the temperature values ( . ). the effect of naver search query data in particular was significant for all three infectious diseases, which confirms that it can be suitable for predicting infectious disease. the seasonal arima model was evaluated using the same data used for ols. the autocorrelation function and the partial autocorrelation function were checked for the seasonality of infectious diseases and seasonality was observed. it was considered inappropriate to select the parameters (e.g., p, d, q) because cuts off and tails off are unclear. therefore, the optimal model for each infectious disease was selected based on the akaike information criterion (aic) and rmse. the aic and rmse were used to compare the arima models. table shows the aic and rmse of the seasonal arima model for each infectious disease, which allows us to identify the top three arima model. in addition, the choice of parameter values did not substantially affect the aic and rmse of the model for a single infectious disease. to compare the performance of each model, figure shows the models with the lowest rmse of the test data subset. the numbers inside the parentheses in each model's name represent the optimizer, the activation function, and the number of epochs used in the models (e.g., dnn ( , , ) indicates that the optimizer, the activation function, and the number of epoch are adadelta, relu, and , respectively). the metric used to compare the models is rmse, which shows the difference between the actual and predicted values. a smaller rmse value indicates a smaller difference between the actual and predicted values and indicates a higher prediction performance. table s shows the rmse and prediction graphs of the dnn and lstm models with the lowest rmse for chickenpox. it can be seen that the prediction graphs for each analysis method have similar shapes overall. the dnn models for chickenpox had a mean rmse of . and a standard deviation of . , which shows stable model performance. when the prediction performances of each model were compared based on rmse, the top dnn models showed a . % performance improvement compared to the arima model. the mean rmse of the lstm models was . , which is higher than the dnn models. the standard deviation was . , which shows that the difference among lstm models was more marked than the difference among dnn models. despite this, the top lstm models achieved an . % performance improvement over the arima model on average. there was a difference between the dnn and lstm models' average figures and standard deviations. however, in the models with the lowest rmse for each analysis method, there was not a big difference, which indicates that there was not a large difference in performance when the optimal parameters for each analysis method were used. table s shows the rmse and the prediction graphs of the dnn and lstm models with the lowest rmse for scarlet fever. the shapes of the graphs for the dnn models are similar. for the lstm models, the shapes of the graphs are similar except for the model with the lowest rmse. unlike the graphs of the other lstm models, the graph lstm model with the lowest rmse showed a strong tendency to follow the actual trend. this result infers that a prediction model that is better than existing prediction models can be designed by changing the deep learning parameters to achieve optimization. as is the case with chickenpox, the mean rmse of the dnn model ( . ) for scarlet fever was lower than that of the lstm model ( . ) . the standard deviation of the dnn model ( . ) was also lower than that of the lstm model ( . ) . when comparing each model based on rmse, the top dnn models showed a . % performance improvement over the arima model and the lstm models showed a . % performance improvement over the arima model. table s shows the rmse and the prediction graphs of the dnn and lstm models with the lowest rmse for malaria. like other infectious diseases, the dnn model prediction graphs have a similar shape. however, the shapes of the lstm model prediction graphs have a tendency to not follow the trend. the rmses of each prediction model, excluding the arima model, showed little difference. this is believed to be because the number of malaria occurrences is fewer than those of the other infectious diseases. therefore, adequate predictions could not be formed. diseases, the dnn model prediction graphs have a similar shape. however, the shapes of the lstm model prediction graphs have a tendency to not follow the trend. the rmses of each prediction model, excluding the arima model, showed little difference. this is believed to be because the number of malaria occurrences is fewer than those of the other infectious diseases. therefore, adequate predictions could not be formed. it is difficult to understand the special characteristics of each analysis method by simply comparing rmse figures alone. therefore, a detailed comparison was performed on the basic comparison models (ols and arima) and the analysis methods that use deep learning (dnn and lstm) . the deep learning models used for comparison were the models with the optimal performance and lowest rmse so that they could best represent each analysis method. with the best performance figure shows the chickenpox predictions of the model with the lowest rmse out of the models with the lowest rmse for each analysis method. the dnn model with the best performance it is difficult to understand the special characteristics of each analysis method by simply comparing rmse figures alone. therefore, a detailed comparison was performed on the basic comparison models (ols and arima) and the analysis methods that use deep learning (dnn and lstm). the deep learning models used for comparison were the models with the optimal performance and lowest rmse so that they could best represent each analysis method. with the best performance. figure shows the chickenpox predictions of the model with the lowest rmse out of the models with the lowest rmse for each analysis method. the dnn model with the best performance had the following specifications, which include optimizer = adadelta, activation function = relu, and number of epochs = (dnn ( , , ) ). the lstm model with the best performance had the following specifications, which include optimizer = nadam, activation function = softplus, epochs = (lstm ( , , ) ). the ols model's predictions had a smaller range of fluctuation than the deep learning models. from day , it seems to follow the trend, but it does not follow the small changes. after day , it cannot predict the downward shape even within a stable graph model. in short, the ols model is not suitable as a prediction model. the arima model's prediction graph has a very simple shape. this model is cyclic and there is a slight increasing trend in which the predicted value per cycle increases by a factor of about . each time. this model cannot predict the trend at all. it can only predict a stable cyclic behavior. in contrast, the dnn ( , , ) predictions followed the actual occurrence trend well. moreover, it had a large range of fluctuation, which means it made accurate predictions overall. however, when the number of occurrences rose rapidly in days - , it was unable to follow these values. the lstm ( , , ) predictions had a smaller range of fluctuation than the dnn ( , , ) model. its range of variance was small, which means it had a stable shape and it performed better than dnn ( , , ) when the number of occurrences rose rapidly. predictions overall. however, when the number of occurrences rose rapidly in days - , it was unable to follow these values. the lstm ( , , ) predictions had a smaller range of fluctuation than the dnn ( , , ) model. its range of variance was small, which means it had a stable shape and it performed better than dnn ( , , ) when the number of occurrences rose rapidly. figure shows the scarlet fever predictions of the models with the best performance for each analysis method. the dnn model with the best performance had the following specifications: optimizer = adadelta, activation function = elu, and number of epochs = (dnn ( , , ) ). the lstm model with the best performance had the following specifications: optimizer = adamax, activation function = elu, number of epochs = (lstm ( , , ) ). the ols model's predictions were completely unable to follow the trend, which was similar with the chickenpox case. the arima model's prediction has no particular merit because it also predicts a simple cycle. much like its figure shows the scarlet fever predictions of the models with the best performance for each analysis method. the dnn model with the best performance had the following specifications: optimizer = adadelta, activation function = elu, and number of epochs = (dnn ( , , ) ). the lstm model with the best performance had the following specifications: optimizer = adamax, activation function = elu, number of epochs = (lstm ( , , ) ). the ols model's predictions were completely unable to follow the trend, which was similar with the chickenpox case. the arima model's prediction has no particular merit because it also predicts a simple cycle. much like its chickenpox prediction, it can only predict a stable cyclic behavior. the dnn ( , , ) predictions were relatively close when the number of occurrences was low, but they were too low when the number of occurrences was high. lstm ( , , ) had a larger range of variance than dnn ( , , ) and its predictions were close when the number of occurrences was high. in the prediction graphs for all of the top performing scarlet fever models, none of the models were able to follow the trend on days - when there was a severe variance in the number of actual occurrences. looking at the mean of each model's predicted value for the number of occurrences, the lstm model ( . ) was larger than the mean of the dnn model ( . ). these same results were also seen in the case of chickenpox (dnn model mean = . , lstm model mean = . ). this showed that more suitable results can be obtained if the lstm model is used to predict the maximum value for the number of occurrences and the dnn model is used to predict the minimum value. figure shows the malaria predictions of the models with the best performance for each analysis method. the dnn model with the best performance had the following specifications: optimizer = adamax, activation function = softplus, number of epochs = (dnn ( , , ) ). the lstm model with the best performance had the following specifications: optimizer = adadelta, activation function = softplus, number of epochs = (lstm ( , , ) ). the predictions of the analysis methods were not satisfactory, but the dnn ( , , ) model's predictions seemed to follow the trend relatively well. the arima model predicts values close to . it is believed that the occurrences in the malaria data are less than those of other diseases and, therefore, not suited to time series analysis because occurrences are concentrated in the summer seasons. as seen in section . , the lowest rmses of each prediction model excluding the arima model showed little difference. lstm ( , , ) had a lower range of variance than ols and it seemed completely unable to make predictions. as mentioned before, the reason that predictions were inadequate for all of the models in addition to lstm ( , , ) was that figure shows the malaria predictions of the models with the best performance for each analysis method. the dnn model with the best performance had the following specifications: optimizer = adamax, activation function = softplus, number of epochs = (dnn ( , , ) ). the lstm model with the best performance had the following specifications: optimizer = adadelta, activation function = softplus, number of epochs = (lstm ( , , ) ). the predictions of the analysis methods were not satisfactory, but the dnn ( , , ) model's predictions seemed to follow the trend relatively well. the arima model predicts values close to . it is believed that the occurrences in the malaria data are less than those of other diseases and, therefore, not suited to time series analysis because occurrences are concentrated in the summer seasons. as seen in section . , the lowest rmses of each prediction model excluding the arima model showed little difference. lstm ( , , ) had a lower range of variance than ols and it seemed completely unable to make predictions. as mentioned before, the reason that predictions were inadequate for all of the models in addition to lstm ( , , ) was that the number of malaria occurrences was small and proper results could not be produced. the deep learning model showed outstanding performance compared to the traditional arima method. of all the dnn and lstm prediction models for chickenpox, the optimal models with the lowest rmse yielded . % and . % better performance than the arima model, respectively. the top dnn models for chickenpox improved performance by an average of . % and the lstm models improved performance by an average of . %. the lowest rmses of the dnn and lstm prediction models for scarlet fever showed . % and . % improved performances compared to arima models. the top dnn models for scarlet fever improved performance by an average of . %. the lstm models improved performance by an average of . %. as noted in the previous sections, it was difficult to predict infectious diseases when the number of infections was small and concentrated in one season. in effect, we observed that the incidence of malaria was high over days - and after day . this period corresponds to the summer season in korea. predicting infectious diseases with this particular data set was difficult and it was not suitable for the arima analysis. even using this particular data set, when dnn was used, the trend of infectious diseases was followed comparatively (figure ). moreover, there is a possibility that the performance would be improved in the dnn model if more diverse parameters were adjusted. this means using deep learning has the advantage of scalability and this can be further investigated in future studies. the arima model that was used in this study was observed to be effective if the number of incidences of infectious diseases was regular and had no increasing or decreasing trends. however, when the predictions were compared according to each analysis method, the dnn and lstm deep learning models performed better than the ols and arima models by assuming that there was a sufficiently large number of occurrences. when comparing the dnn and lstm models, the best models had similar performance, but the dnn models were better in terms of average performance. however, when the number of occurrences was large, the lstm model made close predictions. it seems to be an analysis method that is suitable for circumstances when the number of occurrences is rapidly increasing and infectious disease is believed to be spreading. the deep learning model showed outstanding performance compared to the traditional arima method. of all the dnn and lstm prediction models for chickenpox, the optimal models with the lowest rmse yielded . % and . % better performance than the arima model, respectively. the top dnn models for chickenpox improved performance by an average of . % and the lstm models improved performance by an average of . %. the lowest rmses of the dnn and lstm prediction models for scarlet fever showed . % and . % improved performances compared to arima models. the top dnn models for scarlet fever improved performance by an average of . %. the lstm models improved performance by an average of . %. as noted in the previous sections, it was difficult to predict infectious diseases when the number of infections was small and concentrated in one season. in effect, we observed that the incidence of malaria was high over days - and after day . this period corresponds to the summer season in korea. predicting infectious diseases with this particular data set was difficult and it was not suitable for the arima analysis. even using this particular data set, when dnn was used, the trend of infectious diseases was followed comparatively (figure ). moreover, there is a possibility that the performance would be improved in the dnn model if more diverse parameters were adjusted. this means using deep learning has the advantage of scalability and this can be further investigated in future studies. the arima model that was used in this study was observed to be effective if the number of incidences of infectious diseases was regular and had no increasing or decreasing trends. however, actual data can have trends and be irregular. therefore, deep learning can be an excellent analytical method when analyzing such data and predicting future situations. according to the results of the previous analyses, the deep learning model follows increasing and decreasing trends sufficiently well. moreover, the dnn and lstm models were observed to be sensitive to decreasing trends and increasing trends, respectively. infectious disease is a social problem in that it can cause not only personal damage but also widespread harm. for this reason, research is being conducted to minimize social losses by predicting the spread of infectious diseases. the aim of this study was to design an infectious disease prediction model that is more suitable than existing models by using various input variables and deep learning techniques. therefore, in this study, the optimal parameters were set using a variable selection method based on ols. the relationship between actual instances of disease occurrence and the internet search query data tends to have a time lag, which means a lag was added to each infectious disease's dataset to find the future trend. next, an analysis of arima, dnn, and lstm was performed with optimal parameters. the results of ols analysis using optimal parameters showed that the regression models for each infectious disease had significant results. of the four input variables, the naver search frequency had a significant relationship with all three infectious diseases. the performance of the ols and arima analysis was used to evaluate the deep learning models. looking at the results for dnn and lstm, both the deep learning models made much better predictions than the ols and arima models for all infectious diseases. moreover, the dnn models had the best performance on average, but the lstm models made more accurate predictions when infectious diseases were spreading. however, in the case of malaria, there were few occurrences of the disease compared to other infectious diseases, which means the predictions were not comparatively accurate. this study was also able to reveal special characteristics of the dnn and lstm models. the dnn model produced smaller values than the lstm model on average when predicting infectious diseases. suitable predictions can be made using the dnn model when predicting the minimum value for disease occurrence and using the lstm model when predicting the maximum value. in previous studies, deep learning algorithms were not used [ ] [ ] [ ] [ ] [ ] [ ] ] or the amount of data considered was small [ , , , ] . this study used social media big data and weather data, which have not been sufficiently considered in existing studies. it also used deep learning analysis, which yields high prediction performance to increase the performance of infectious disease predictions. the results showed that, when selecting the optimal parameters, adding all input variables had the highest explanatory power. this means that, by adding various data, it was possible to design a model with higher explanatory power. moreover, the lstm model results for scarlet fever indicate that it is possible to optimize a deep learning model by changing its parameters in various ways and, therefore, design a prediction model that is better than existing prediction models. this study has reviewed the factors involved with infectious disease occurrence using search query data and social media big data, which exist because of the development of the internet as well as temperature and humidity weather data. it also constructed traditional prediction models such as ols, arima, and deep learning prediction models such as dnn and lstm and compared their prediction performance to confirm that the models that use deep learning are the most suitable for infectious disease prediction. it is believed that infectious disease prediction models that employ deep learning can be used to supplement current infectious disease surveillance systems and, at the same time, predict trends in infectious disease. if this can reduce the time differences in reporting systems so that infectious disease trends can be known immediately, it is expected that immediate responses to infectious disease will become possible and costs to society can be minimized. according to a study by shin et al., an emerging infectious disease known as the middle east respiratory syndrome (mers) has a deep correlation with internet search data [ ] and it will become possible to expand these methods to the real-time surveillance and prediction of emerging infectious diseases as well. however, this study has three limitations, which include a relatively short data collection period, regionally combined predictions, and a consideration of a narrow range of parameters in the deep learning model. the search query data collection time period used in this research was relatively short extending from january to july . the particular spatial ranges of data were averaged across the whole of south korea. it is believed that, if the data is expanded and the spatial ranges are subdivided, the model's performance will improve. in addition, an effort was made to change the dnn and lstm model parameters and create a variety of prediction models, but the deep learning prediction models used in this study did not cover all the prediction models that could be implemented. parameters such as hidden layers and batch size were not considered. therefore, it is difficult to conclude that the most effective model was created. if more parameters are considered and more prediction models are made in future research, it is believed that prediction performance can be increased somewhat. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , table s : the root mean squared error (rmse) and prediction graphs of top deep neural network (dnn) and long-short term memory (lstm) models for chickenpox. the seasonal autoregressive integrated moving average (arima) model is denoted as arima(p, d, q)(p, d, q) s . where p is the order of the autoregressive part, d is the order of the differencing, q is the order of the moving-average process, and s is the length of the seasonal cycle. (p, d, q) is the seasonal part of the model. the numbers in parentheses indicate each deep learning model's optimizer, activation, and number of epochs, respectively. (optimizer) : adadelta, : adagrad, : adam, : adamax, : nadam, : rmsprop, and : sgd, (activation function) : elu, : relu, : selu, and : softplus, (number of epochs) : , : , : , and : , table s : the rmse and prediction graphs of the top dnn and lstm models for scarlet fever, table s : the rmse and prediction graphs of top dnn and lstm models for malaria. infectious disease, safety, state: history of infectious disease prevention and mers situation a profile of the online dissemination of national influenza surveillance data multiscale mobility networks and the spatial spreading of infectious diseases modeling the worldwide spread of pandemic influenza: baseline case and containment interventions seasonal transmission potential and activity peaks of the new influenza a(h n ): a monte carlo likelihood analysis based on human mobility modelling disease outbreaks in realistic urban social networks strategies for mitigating an influenza pandemic controlling pandemic flu: the value of international air travel restrictions mitigation measures for pandemic influenza in italy: an individual based model considering different scenarios monitoring pertussis infections using internet search queries disease surveillance based on internet-based linear models: an australian case study of previously unmodeled infection diseases advances in nowcasting influenza-like illness rates using search query logs correlation between national influenza surveillance data and google trends in south korea dynamic forecasting of zika epidemics using google trends influenza forecasting with google flu trends mass media and the contagion of fear: the case of ebola in america monitoring hand, foot and mouth disease by combining search engine query data and meteorological factors assessing the usability of social media data for visitor monitoring in protected areas forecasting the onset and course of mental illness with twitter data high correlation of middle east respiratory syndrome spread with google search and twitter trends in korea defender: detecting and forecasting epidemics using novel data-analytics for enhanced response applying gis and machine learning methods to twitter data for multiscale surveillance of influenza forecasting influenza-like illness dynamics for military populations using neural networks and social media twitter in the cross fire-the use of social media in the westgate mall terror attack in kenya real-time diffusion of information on twitter and the financial markets bibliographic analysis of nature based on twitter and facebook altmetrics data frequent discussion of insomnia and weight gain with glucocorticoid therapy: an analysis of twitter posts forecasting influenza in hong kong with google search queries and statistical model fusion construction and evaluation of two computational models for predicting the incidence of influenza in nagasaki prefecture deep learning applications and challenges in big data analytics deep learning for digital pathology image analysis: a comprehensive tutorial with selected use cases dermatologist-level classification of skin cancer with deep neural networks deep learning based tissue analysis predicts outcome in colorectal cancer time series analyses of hand, foot and mouth disease integrating weather variables short term effects of weather on hand, foot and mouth disease weather and virological factors drive norovirus epidemiology: time-series analysis of laboratory surveillance data in england and wales imported dengue cases, weather variation and autochthonous dengue incidence in cairns a large temperature fluctuation may trigger an epidemic erythromelalgia outbreak in china implications of temperature variation for malaria parasite development across the impact of variations in temperature on early plasmodium falciparum development in anopheles stephensi mapping the epidemic changes and risks of hemorrhagic fever with renal syndrome in shaanxi province a threshold analysis of dengue transmission in terms of weather variables and imported dengue cases in australia monitoring seasonal influenza epidemics in korea through query search forecast model analysis for the morbidity of tuberculosis in xinjiang, china time series analysis of dengue incidence in guadeloupe, french west indies: forecasting models using climate variables as predictors the authors declare no conflict of interest. key: cord- - kuh njb authors: elkholy, amgad a.; grant, rebecca; assiri, abdullah; elhakim, mohamed; malik, mamunur r.; van kerkhove, maria d. title: mers-cov infection among healthcare workers and risk factors for death: retrospective analysis of all laboratory-confirmed cases reported to who from to june date: - - journal: j infect public health doi: . /j.jiph. . . sha: doc_id: cord_uid: kuh njb background: approximately half of the reported laboratory-confirmed infections of middle east respiratory syndrome coronavirus (mers-cov) have occurred in healthcare settings, and healthcare workers constitute over one third of all secondary infections. this study aimed to describe secondary cases of mers-cov infection among healthcare workers and to identify risk factors for death. methods: a retrospective analysis was conducted on epidemiological data of laboratory-confirmed mers-cov cases reported to the world health organization from september to june . we compared all secondary cases among healthcare workers with secondary cases among non-healthcare workers. multivariable logistic regression identified risk factors for death. results: of the laboratory-confirmed mers-cov cases reported to who, were healthcare workers and were non-healthcare workers. compared with non-healthcare workers cases, healthcare workers cases were younger (p < . ), more likely to be female (p < . ), non-nationals (p < . ) and asymptomatic (p < . ), and have fewer comorbidities (p < . ) and higher rates of survival (p < . ). year of infection ( – ) and having no comorbidities were independent protective factors against death among secondary healthcare workers cases. conclusion: being able to protect healthcare workers from high threat respiratory pathogens, such as mers-cov is important for being able to reduce secondary transmission of mers-cov in healthcare-associated outbreaks. by extension, reducing infection in healthcare workers improves continuity of care for all patients within healthcare facilities. middle east respiratory syndrome coronavirus (mers-cov) was first detected in a patient living in saudi arabia in september of [ ] . subsequent cases have included human infections across the arabian peninsula, occasional importation of cases outside the arabian peninsula and associated clusters in other regions of the world. outbreaks of non-sustained, human-to-human transmission have occurred primarily in healthcare settings [ ] . while mers-cov appears to be inefficient at transmitting between humans in the general community, about half of the reported mers-cov infections have occurred in healthcare settings [ ] . healthcare-associated transmission of mers-cov has been reported in france, jordan, saudi arabia, united arab emirates, republic of korea and the united kingdom and has on occasion resulted in large outbreaks [ ] [ ] [ ] [ ] [ ] [ ] [ ] . secondary transmission has occurred between patients, from patients to healthcare workers, and from patients to visitors of the hospital. to date, there has been limited evidence of transmission documented between healthcare workers in saudi arabia [ ] and anecdotal evidence from healthcare workers to patients in saudi arabia [ ] . because hcw have not been consistently tested for mers-cov infection, nor has detailed outbreak investigations occurred within all hospital outbreaks, the role of healthcare workers in onward transmission remains unclear. given the large number of hcws infected to date, it is possible that hcw can propagate an outbreak within a healthcare facility. among reported secondary mers cases in such outbreaks, a substantial proportion have been healthcare workers [ , , [ ] [ ] [ ] [ ] [ ] . the clinical spectrum of mers-cov infection ranges from asymptomatic infection to severe pneumonia with acute respiratory distress syndrome and other life-threatening complications [ ] [ ] [ ] [ ] . mild symptoms are non-specific and can include headache, tiredness, fever, mild cough, sore throat and runny nose. some patients may present with gastrointestinal symptoms, including diarrhoea. the non-specificity of mers signs and symptoms poses several challenges, not only for the timely identification and isolation of infected patients, but also for reducing secondary transmission within the healthcare facility, particularly to healthcare workers and between patients. preventing mers-cov infection in healthcare workers is critical because of their role in the clinical management of patients and in ensuring adequate infection prevention and control measures are implemented in healthcare facilities. unnecessarily exposing healthcare workers to mers-cov affects the safety of both the healthcare workers and other patients in the healthcare facility, potentially propagating secondary transmission in healthcare-associated outbreaks. understanding mers-cov infection in healthcare workers to date and the risk factors for adverse outcomes is important for preventing future infection of healthcare workers, for informing and updating infection prevention and control measures in healthcare facilities and for reducing secondary mers-cov transmission within healthcare settings. the international health regulations [ ] require all laboratoryconfirmed cases of mers-cov to be reported to the world health organization (who) within h of laboratory confirmation [ ] . in this study, we use the epidemiological data of all mers cases reported to date to who to describe secondary cases of mers-cov infection among healthcare workers and to identify the risk factors for death among healthcare workers with secondary infection. a retrospective analysis was conducted on epidemiological data of laboratory-confirmed mers-cov cases reported to who [ ] from september to june . we looked specifically at healthcare workers involved in the delivery of health care to patients within the same healthcare facility as a confirmed or suspected mers-cov case. primary cases were defined as cases with laboratory confirmation of mers-cov infection with no epidemiological link to a suspected or confirmed human mers case. secondary cases were defined as those with laboratory confirmation of mers-cov infection and with a direct epidemiological link with a confirmed or probable mers-cov case. prior to , data from individual cases including occupation, signs/symptoms, exposures and risk factors for infection, etc. was not collected systematically. following the large mers outbreak in jeddah and riyadh in , case report forms and policies related to the investigation of cases and contacts became more systematic from onwards. given the inconsistencies in the collection and reporting of epidemiologic data from mers-cov cases to who prior to , we performed a secondary analysis with data reported only from january to june . descriptive analysis was performed for all mers-cov cases reported to who from to . for all statistical analyses, p < . was considered statistically significant and all analyses were performed using the epidemiological data display package in r, version . . . (https://cran.r-project.org/ package=epidisplay). we compared all secondary cases in healthcare workers with all secondary cases among non-healthcare workers, using the student t-tests for continuous variables and chi-squared tests for categorical variables. we also aggregated survival outcomes of healthcare workers with mers-cov infection by year of infection. to identify risk factors for adverse outcomes in healthcare workers with secondary infection, we performed multivariable logistic regression analysis on all healthcare worker secondary cases. variables considered were dichotomous (sex, residency, symptomatic clinical presentation, presence of any comorbidities), categorical (year of infection) or continuous (age). the final multivariable model, constructed using the backward, stepwise elimination method, included all variables with an adjusted p < . . our analysis considered all cases of mers-cov reported to who up to june . among these cases, were primary cases, secondary cases, cases for which the information reported was insufficient to be able to determine whether it was a primary or secondary case, and cases with missing case information. among all cases, ( . %) deaths occurred. of the cases, were reported as healthcare workers. the mean age of healthcare workers was . (interquartile range . - . ) years and . % were women. five were primary cases, were secondary cases, cases had insufficient information to be able to determine whether it was a primary or secondary case and cases had missing case information. fig. shows the epidemic curve of cases of mers-cov among healthcare workers and non-healthcare workers reported from to june . table provides further description of the healthcare worker cases, as well as the healthcare worker cases reported to who from january to . table compares all secondary healthcare worker cases and all secondary non-healthcare worker cases. compared with secondary non-healthcare worker cases, secondary healthcare worker cases were younger (p < . ), had a higher proportion of women (p < . ), were non-national residents (p < . ), and had asymptomatic infection (p < . ), fewer comorbidities (p < . ) and higher survival (p < . ). further comparison between laboratory-confirmed mers-cov cases in healthcare workers and non-healthcare workers are shown in the supplementary material. tables a and b shows the survival outcomes of all infections by year of infection. tables a shows outcomes of all infections by year of infection for healthcare workers, while table b shows outcomes of all infections by year of infection for non-healthcare workers. there have been no fatal mers infections among healthcare workers since . table shows the regression coefficients and adjusted odds ratios ( % confidence interval) for the two variables retained in the final multivariable risk model for death in secondary cases among healthcare worker. year of infection and having no comorbid conditions were found to be independent protective factors against death in healthcare workers with secondary mers-cov infection. healthcare workers include, but are not restricted to: doctors, nurses, pharmacists, physiotherapists, radiologists, rehabilitation staff, infection prevention and control staff, intensive care staff, ambulance staff, respiratory therapists, auxiliary healthcare workers, attendants, laboratory, x-ray and ultrasound technicians, and healthcare administrators. a lack of consistency in reporting specific job titles prohibited subgroup analysis by different roles of healthcare worker. healthcare workers continue to constitute a substantial proportion of secondary mers-cov infections. that is, among all cases of mers-cov reported to who as of june , healthcare workers accounted for . % of all mers-cov cases and . % of secondary mers-cov cases. this is similar to other respiratory pathogens: in the outbreak of severe acute respiratory syndrome, % of cases in hong kong, % in canada and % in singapore were healthcare workers [ ] . protecting health care workers from infectious hazards is paramount to ensuring their safety in delivering health care. in addition, being able to protect healthcare workers, constituting the front line response against high-threat respiratory pathogens, such as mers-cov, is important for reducing secondary transmission in healthcare-associated outbreaks. reducing infection in healthcare workers, even if their infection does not cause morbidity or mortality, will improve the continuity of care for all patients in the same healthcare facility. we also found that the demographic and clinical profile of secondary infections in healthcare workers was different from secondary infections among non-healthcare workers. healthcare workers infected with mers-cov were younger, more were female and non-national residents, and had fewer comorbidities, more asymptomatic infections and higher survival. importantly, no deaths have occurred among healthcare workers with secondary mers infection since the end of . these results are similar to the findings in individual outbreaks reports [ , , [ ] [ ] [ ] [ ] [ ] [ ] ] . the highest burden of mers cases to date is in saudi arabia where healthcare workers are more likely to be female, a large proportion of whom are expatriates. the younger age and fewer comorbidities may partly explain the higher survival observed among healthcare workers, as well as the possibility of earlier identification or suspicion of mers among healthcare workers. indeed, the case fatality rate among all healthcare workers was . % compared with . % among all non-healthcare workers. this is the first study to perform multivariable logistic regression to identify risk factors for death among healthcare workers with secondary infections reported to date. the analysis showed that year of infection ( - ) and having no comorbidities were independent protective factors against death. the decline in risk of death since may reflect the substantial improvements in surveillance and the infection prevention and control measures that have been introduced in affected countries in recent years. in saudi arabia, for example, the ministry of health regularly review and update national infection prevention and control guidelines, according to which, healthcare workers who had unprotected "high-risk exposure" (within . m of the patient) or have suggestive symptoms regardless of exposure type are required to stop performing their duties immediately; to have a nasopharyngeal swab tested for mers-cov; to not resume their duties until cleared by the infection control team and to delay travel until cleared by infection control team [ ] . any healthcare worker who tests positive for mers, any healthcare worker who develops mers suggestive symptoms and any healthcare worker who had unprotected high-risk exposure are considered clear and able to resume work if they meet all of the following criteria: asymptomatic for at least h, and the -day observation period is over, and they have at least one negative rt-pcr [ ] . the enhanced infection prevention and control (ipc) efforts which have been introduced since include regular training of healthcare workers on ipc, auditing of ipc in healthcare facilities, improved case notification and isolation within emergency departments, and comprehensive contact-tracing and testing of all contacts, including healthcare workers, regardless of the development of symptoms. guidelines on contact tracing were revised after to include the testing of all contacts of confirmed mers cases (including healthcare workers) for mers-cov. prior to , contacts were only tested if they developed symptoms. this has increased the detection of asymptomatic or mildly symptomatic cases, which in turn, may have decreased the case fatality rate. specific ipc measures which may have contributed to the decreased case fatality rate include more systematic use of appropriate personal protective equipment and increased testing of asymptomatic personnel which effectively increases the detection of asymptomatic healthcare workers who are less likely to die, therefore increasing the denominator of healthcare worker cases and decreasing the case fatality rate. in addition, earlier detection of cases, and more efficient contact tracing have likely identified mers-cov infections in healthcare workers at an earlier stage of infection, enabling more timely treatment and clinical management, and, as a result, a reduction in the case fatality rate. further efforts to prevent and manage emerging respiratory disease infections, including mers among healthcare workers, still need to be made. these include improving identification and rapid diagnosis of mers, further understanding of the mecha- nisms of transmission in healthcare settings, optimizing the layout of emergency departments for better triage of patients with respiratory symptoms, standardization of infection prevention and control practices and (re)training at facilities with high hospital staff turnover, and auditing of healthcare facilities for adherence to infection prevention and control measures. these will be critical to minimizing transmission in healthcare facilities, particularly until interventions are introduced to stop the virus entering the human population from the dromedary camel reservoir. the role of environmental contamination was evaluated in a number of hospitals following the mers outbreak in the republic of korea and collaborative, experimental studies were conducted to evaluate the viability and persistence of mers-cov on surfaces and in the air [ ] [ ] [ ] . the role of mild or asymptomatic cases in transmission chains, however, remains unclear [ ] [ ] [ ] [ ] . further epidemiologi- outcomes cal and environmental studies to determine route(s) of secondary transmission will also be critical. the results of our study are strengthened by the size of the study, which included all cases reported to who since the first case was notified in . our database has cases from all countries reporting cases globally. however, since , some inconsistencies have occurred in the way data have been collected and reported to who, for example in the use of a systematic data collection tool and in reporting the outcome of all cases. challenges also exist in classifying cases based on the available information at the time of reporting. for example, thorough outbreak investigations, which include full genome sequencing to clarify the transmission chains within the outbreak or separate introductions from outside, may find that cases that were initially classified as secondary case are in fact primary cases [ , ] , but this information is not systematically relayed to who. efforts are currently being made to retrospectively review and update the epidemiological data for all cases reported to who to date, particularly before . healthcare workers still make up a significant proportion of secondary mers-cov infections. understanding transmission to healthcare workers, preventing infection and improving clinical management of infected healthcare workers will all be critical to further reducing the incidence of secondary infections in healthcare settings. isolation of a novel coronavirus from a 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middle east respiratory syndrome coronavirus: a report of nosocomial transmission state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans critically ill patients with the middle east respiratory syndrome: a multicenter retrospective cohort study middle east respiratory syndrome a cohort study of patients suspected for mers-cov in a referral hospital in saudi arabia geneva: world health organization middle east respiratory syndrome case definition for reporting to who: interim case definition severe acute respiratory syndrome (sars) and healthcare workers middle east respiratory syndrome coronavirus: guidelines for healthcare professionals -version . environmental contamination and viral shedding in mers patients during mers-cov outbreak in south korea extensive viable middle east respiratory syndrome (mers) coronavirus contamination in air and surrounding environment in mers outbreak units stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case middle east respiratory syndrome coronavirus (mers-cov) viral shedding in the respiratory tract: an observational analysis with infection control implications a case of long-term excretion and subclinical infection with middle east respiratory syndrome coronavirus in a healthcare worker infectivity of an asymptomatic patient with middle east respiratory syndrome coronavirus infection the authors would like to thank the many individuals involved in the collection of individual case data and in the care of mers-cov infected patients in the affected countries. the opinions expressed in this article are those of the authors and do not necessarily reflect those of the institutions or organizations with which they are affiliated. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.jiph. . . . all authors contributed to the study design, and reviewed and edited the manuscript. aae, rg and me performed the statistical analysis. all authors were involved in the interpretation of the findings. no funding sources. none declared. the case-patient data reported to who is anonymized, thus neither informed consent, nor approval from an institutional review board were required by who or countries providing this data to who under the international health regulations ( ). key: cord- -o wpcxdp authors: schmidt-küntzel, anne; dalton, desiré l.; menotti-raymond, marilyn; fabiano, ezequiel; charruau, pauline; johnson, warren e.; sommer, simone; marker, laurie; kotzé, antoinette; o’brien, stephen j. title: conservation genetics of the cheetah: genetic history and implications for conservation date: - - journal: cheetahs: biology and conservation doi: . /b - - - - . -x sha: doc_id: cord_uid: o wpcxdp from allozymes in to whole genomes in , genetic studies of the cheetah have been extensive. in this chapter we provide an overview of the available literature. overall, patterns of genetic variation provided evidence of low variability and suggest this loss occurred thousands of years ago. differences between published subspecies were supported genetically. at a local scale, populations were generally considered panmictic with minor genetic structure. although cheetahs have persisted despite low genetic variability, important questions arise from these findings: does the cheetah have the ability to adapt to and evolve with future changes in environmental and infectious pressure? how would cheetahs cope with further loss of genetic diversity? connectivity in the wild should be maintained via prevention of habitat loss, while management of small isolated populations may require reestablishing gene flow. genetics could assist captive-breeding decisions and provide forensic evidence as to the geographical origin of illegally traded animals. the cheetah (acinonyx jubatus) is one of the most recognized examples of the important links between evolutionary history, genetic variation, and conservation. its value to the biodiversity of the world is not only warranted by its unique physical characteristics, such as being the fastest land mammal (chapter ), but also its unique evolutionary lineage as the only extant representative of its genus, acinonyx. concerns over levels of genetic variation among cheetahs were first raised as captive programs grappled with difficulties in breeding cheetahs (chapter ). these observations led to research investigating the biological basis of the low rates of captive breeding success ( %- %) and the concurrent high rate of infant mortality ( %) . this research led to the discovery of low levels of genetic diversity in the cheetah, which were attributed to one or several severe population bottlenecks. as a consequence, debates arose regarding the impact of low genetic diversity on the survival of the species, and the cheetah has been featured in genetic textbooks since the s. early research on cheetah represented one of the first studies in the new field of conservation genetics. in the last century, cheetah numbers have declined drastically due to loss of habitat and prey, persecution due to real or perceived livestock depredation, and removal from the wild to supply captive facilities and private individuals (chapters , , , and ) . the reduction in numbers and fragmented distribution add to the urgency to preserve the genetic diversity left today. in this chapter, we review the current status of cheetah genetics and its impact on the species' conservation. while publications on cheetah genetics may appear contradictory at times, the fundamental conclusions have been consistent for over years, with various measures confirming low genetic diversity (section "genetic diversity"), which was shown to have originated thousands of years ago (section "historic demography"), and with a relatively recent divergence of extant published subspecies (section "subspecies definition and divergence"). the differences debated among geneticists only affect the interpretation of genetic results regarding precise timing of events and extent of reduced genetic diversity. this chapter also covers the cheetah's phylogenetic (evolutionary relation based on genetic data) position among other felids (section "species-level taxonomy"), the genetic structure of the subspecies and within geographical regions (section "phylogeography"), an overview of additional genetic studies including kinship (section "additional insights into cheetah genetics"), and implications of genetic findings for cheetah conservation (section "discussion"). the cheetah is a member of the family felidae ( fig. . ), which comprises living species that are distributed throughout the world, with the exception of australasia and the polar regions (kitchener et al., ) . one of the most striking aspects of molecular genetic studies in the felidae was how rapidly felids evolved into eight different lineages (over a -million-year period), each with unique biogeographical histories. earlier groupings of felid lineages were largely based on morphological features and life-history patterns. the cheetah was generally considered to be an early divergence from the felid radiation due to some of its unique adaptations, including its incompletely retractile claws (chapter ). however, the advent of genetic approaches has provided clarity to more confidently reconstruct felid evolutionary history, and today the cheetah is included in the puma lineage, which was the sixth of eight lineages to branch off during felid evolution [ million years ago (mya); johnson et al., ; li et al., ; werdelin et al., ; fig. . ] . the cheetah's closest living relatives are known to be the puma (puma concolor) and the jaguarundi (herpailurus yagouaroundi) . the cheetah (johnson et al., ; li et al., ; werdelin et al., ; fig. . ) , with whom the cheetah likely shared a common ancestor and evolutionary history prior to their divergence. today the cheetah is found in the old world, and the puma and jaguarundi in the new world (chapter ). the cheetah has chromosomal pairs (i.e., chromosomes) like most felid species . the cheetah is the only extant representative of its genus. genetic variation (polymorphism) forms the raw material of evolution. novel variation rises from dna mutations. dna mutation rates that alter the amino acid sequence and may be detectable in allozyme migration rates (section "allozymes") are several orders of magnitude slower than those observed in the mitochondrial dna (section "mitochondrial dna") or in repetitive elements in nuclear dna, such as microsatellites (section "microsatellites"). while significant gain of genetic variation is limited by mutation rates and takes numerous generations, variation can be lost within a single generation if only a subset of the population reproduces due to either high death rates or a high number of nonreproducing individuals. the first study to indicate reduced genetic diversity of the cheetah documented low variation at protein-based markers compared to other felids and mammals (o'brien et al., ; o'brien et al., ; et al., ) . thus, it was surprising that analyses of southern african cheetahs failed to identify genetic polymorphisms across a total of allozyme loci (o'brien et al., ; o'brien et al., ; however, insights from these studies were limited, because protein-based markers only assess amino acid changes that alter the electrophoretic mobility of the protein. dna changes, such as silent/synonymous substitutions are masked in proteins, resulting in an underestimation of the amount of genetic variation in all species. in addition, protein-based markers are more likely to be linked to functional differences and as such allele frequency differences may be susceptible to selective pressure (environmental conditions affecting survival of organisms with a particular characteristic). the subsequent development of dna-based markers provided more detailed information about the degree of genetic diversity in cheetahs. concurrently with allozyme studies, functional studies demonstrated that reciprocal skin allografts between unrelated cheetahs and siblings showed no signs of acute graft rejection, whereas xenografts (from domestic pokorny et al. ( ) ; in gir; sachdev et al. ( ) . et al., ) . these results were attributed to reduced functional allelic variation at the cheetah's major histocompatibility complex (mhc), an important immune gene family, which encodes cell surface proteins responsible for distinguishing foreign from self molecules. the inferred reduced functional variation was ultimately supported by molecular studies (section "major histocompatibility complex"). as sequencing techniques became available, low levels of genetic variation were also observed in mitochondrial dna (mtdna). mtdna is independent from the nuclear genome, and represents the maternal demographic history. the mitochondrial genome evolves faster than the nuclear coding genome, with the control region (cr) being the most rapidly evolving region of the mitochondrial genome. mtdna, in particular the cr, has been informative for investigations of diversity patterns, population structure, and phylogeography (phylogenetic structure in relation to location). the complete cheetah mtd-na genome has , bp (burger et al., ) and has % similarity with the mtdna genome of the domestic cat (lopez et al., ) . in the s, a study based on restriction fragment length polymorphism (rflp) inferred low levels of nucleotide variation ( . % diversity) in cheetah mtdna relative to comparable studies in other species (menotti-raymond and o'brien, ; table . ). nucleotide variation was even lower when a short sequence of the mtdna coding region was included with the mtdna-cr (charruau et al., ) , or when the entire mtdna genome was evaluated (dobrynin et al., ; table . ). dobrynin et al. ( ) found a % reduction in nucleotide variation across cheetahs ( namibian, tanzanian) relative to other mammals (dobrynin et al., ; microsatellites (nuclear dna markers consisting of variable numbers of tandem repetitions of - nucleotides; also called str or short tandem repeats) accumulate new variation quickly as they have high mutation rates (several orders of magnitude higher than dna coding for proteins), and are usually not associated with any function (i.e., they do not code for a protein) and thus are not subjected to selection pressure. these markers have been used widely in conservation genetics, population genetics, and wildlife forensics. most non-domestic felid studies have selected a subset from polymorphic microsatellite loci, which were mapped and characterized in the domestic cat (menotti-raymond et al., ) . initial studies, with microsatellite markers in cheetahs, demonstrated low observed heterozygosity ( . ; menotti-raymond and o'brien, ; table . ). expected heterozygosity was slightly higher ( . - . ) when measured in randomly selected microsatellite markers in cheetahs (driscoll et al., ; table . ). these estimates were comparable to the heterozygosity levels of other felids in small isolated populations (e.g., pumas from idaho, lions from serengeti and ngorongoro crater), but lower than in domestic cats (driscoll et al., ; table . ) . subsequent studies of cheetahs only included a subset of these microsatellite markers, which were not selected randomly, but instead for their known relatively high level of polymorphism, leading to higher heterozygosity estimates. hence, these increased heterozygosity estimates do not reflect the genetic diversity of the cheetah species per se, but are summarized in table . for informational purposes. terrell et al. ( ) suggested a reduction in genetic diversity in the wild population from a dataset spanning years. the study was based . the cheetah on individuals born between and from south africa and namibia, which were genotyped (characterized on a genetic level) with microsatellite markers. while microsatellite markers demonstrated levels of heterozygosity in the cheetah that were not always significantly lower than for other species (table . ), this does not contradict findings of low genetic diversity in cheetahs at other, more slowly evolving markers. it merely indicates that the variation at microsatellite loci is more recently evolved in origin. the length of time needed to accumulate this new microsatellite variation can inform estimates of the timing of events that led to the loss of genetic variation (section "historic demography"). the mhc is one of the most polymorphic loci known in vertebrates and has important immune functions. an increasing number of studies have documented an association between the diversity of mhc genotypes or individual alleles with disease susceptibility in wildlife, thus confirming the importance of the selection pressure from pathogens on the mhc (reviewed in sommer, ) . most comprehensive studies of the mhc in felids (also known as the feline leucocyte antigen), have been conducted in the domestic cat (winkler et al., ) . the cat is a good model as the general architecture of the mhc appears relatively conserved within each class of vertebrates; the number of mhc class i or ii genes, however, can vary substantially among species. the domestic cat mhc region is located on chromosome b and includes mhc i and mhc ii genes (yuhki et al., ) . the cheetah mhc sequence resolved genes with complete homology to the domestic cat for all mhc ii and most mhc i genes. its structural organization was also found to be highly similar to that of the domestic cat (dobrynin et al., ) . an early study of cheetah mhc i based on rflp markers showed reduced genetic diversity in cheetah (observed heterozygosity = . - . ) compared to other species, which was only comparable to that of lions from isolated populations (gir forest and ngorongoro crater; yuhki and o'brien, ; table . ). only mhc i alleles were identified in individual cheetahs through sequencing (yuhki and o'brien, ; table . ) and mhc ii-drb alleles were identified in individuals through reference strand-mediated conformational analysis (drake et al., ; (table . ). while a -fold increase in the number of mhc i alleles identified in may appear like an increase in genetic diversity, the identification of only additional alleles despite a -fold increase in the number of study animals is in fact further confirmation of low levels of allelic diversity in the cheetah. the low level of allelic diversity was further confirmed by comparison to other species, which harbor more alleles in fewer individuals (table . ). more recently, a %- % reduction in single nucleotide variants (snvs) was observed when the complete mhc sequence of cheetahs ( namibian, tanzanian) was compared to that of human (homo sapiens), dog (canis familiaris), and an outbred domestic cat; only variants affecting the amino acid sequence were detected in the mhc i coding region of cheetahs (dobrynin et al., ; dobrynin et al. ( ) described patterns of diversity across the entire genome of the cheetah. five commonly employed metrics confirmed the genic and genomic lack of diversity of the species: snv incidence was % less than that observed in a feral domestic cat; snv density was - × less than in the domestic cat, . the cheetah european wildcat (felis silvestris silvestris), or human (however it was higher than in lions from the gir forest); regions of continuous homozygosity (identical alleles at a given locus/set of loci) were - × longer than in the domestic cat; heterozygosity levels were %- % of the levels observed in the domestic cat, tiger (panthera tigris), and human; snvs in coding genes were % reduced compared to the domestic cat or european wildcat (dobrynin et al., ; table . ; fig. . ). since the initial discovery of reduced genetic diversity in the cheetah in the s, conservation, and scientific interest have turned toward identifying its cause (section "historic demography") and assessing the impact of low genetic diversity on the cheetah's chances of long-term survival (section "importance of low genetic diversity on cheetah survival"). the cumulative results, indicative of reduced genetic diversity in the cheetah, were consistent with a genetic bottleneck or a series of demographic reductions over time and space. menotti-raymond and o'brien ( ) proposed a scenario of distant past, rather than recent, reduction in the global population size. the demographic event causing this drastic loss of diversity was estimated to have occurred during the end of the pleistocene ( , - , years ago; table . ). this proposal was based on the time estimated for the near-reconstitution of genetic variation at rapidly evolving minisatellite (nuclear dna marker consisting of variable numbers of tandem repetitions of - nucleotides) loci. the authors obtained similar estimates with mtd-na rflp data calibrated on estimates of divergence of the species from the panthera genus. this estimated timeframe was later corroborated with microsatellite markers (driscoll et al., ) , with the time needed for nuclear alleles to reach fixation in the mhc (castro-prieto et al., ) , and with whole genome data (dobrynin et al., ) (table . ). in the asiatic cheetah population a more recent, independent, bottleneck was inferred based on a significant heterozygosity excess observed in the microsatellite loci tested (charruau et al., ) . several alternative hypotheses to the single bottleneck scenario have been proposed to explain the severe loss of genetic variation. first, that the uniformity resulted from a persistent low effective population size (ne; theoretical number which roughly reflects the number of animals genetically contributing to the population), possibly resulting from the high reproductive variance linked with the cheetah's presumed polygynous mating system (pimm et al., ) . second, that low effective population sizes were maintained by a continuous cycle of extinction of subpopulations followed by recolonization, that is, metapopulation dynamics (gilpin, ; hedrick, ; pimm et al., ) . the availability of snvs derived from whole genome data of individuals from both eastern and southern africa permitted more robust analyses of historical demographic patterns (dobrynin et al., ) . these analyses support the premise that cheetah populations expanded uniformly following a founder event , years ago. this was followed by a more recent split into an eastern and southern population, which were subjected to a bottleneck around , - , years ago. an alternative scenario of a gradual decline in the effective population size was supported by analyses of diploid whole genome sequence data to estimate past population sizes (table . ). fabiano et al. (in preparation) suggested a gradual decline in population numbers, commencing at least , years ago, based on different coalescent-based approaches applied to published microsatellite profiles in the namibian cheetah. while there was evidence . the cheetah of a continuous decline during this time period, some methods suggest an accelerated decline around , and , years ago (table . ). prior to the availability of genetic data, the species' taxonomy was based on morphological and geographical information. according to these taxonomic criteria, the extant cheetah populations were classified into four african and one asiatic subspecies (smithers, ) a. j. jubatus and a. j. raineyi were the first two subspecies to be assessed with molecular tools. initial allozyme analyses in detected some minor differences (o'brien et al., ; table . ). the separation of the two sub-saharan populations was further supported with mtdna-cr (freeman et al., ) , microsatellites (driscoll et al., ) , and whole genome variation (dobrynin et al., ; . the cheetah single subspecies (kitchener et al., ) . additionally, kitchener et al. ( ) put forward that as additional data become available the four subspecies that the iucn cat specialist group currently recognizes may be further merged in the future. however, this does not affect the amount of diversity found between the populations identified to date, which we will present here. a. j. soemmeringii was first compared to a. j. jubatus in based on mtdna (freeman et al., ) (kelly et al., ) ; years (modified from kelly et al., ) ; years (marker and o'brien, ) . c cal: reference time point used as calibration; µ: mutation rate given per generation; mya, million years ago. d /µ × g (nei, ) ; coal, coalescent methods, msvar . (storz and beaumont, ) , vareff (nikolic and chevalet, ) , diyabc-fda (cornuet et al., ) ; dadi, diffusion approximation to the allele frequency spectrum (gutenkunst et al., ) ; fixation n e , fixation of a neutral nuclear marker is expected after n e generations (nichols, ) ; psmc, pairwise sequential markovian coalescent (li and durbin, ) ; smm, stepwise mutation model (valdes et al., ) . e an accelerated decline may be present , and , years ago. a. j. hecki was not specifically assessed, as none of the studies was able to include confirmed a. j. hecki samples from west africa. historically, the distributions of each subspecies were defined based on morphological differences and presumed connectivity between populations. once the subspecies were confirmed genetically, the distribution of each subspecies and their genetic structure could be verified (fig. . a) . charruau et al. ( ) obtained a sample collection of cheetahs from countries from the extant and historical cheetah range. these samples were expected to represent four of the five cheetah subspecies recognized at that time. a. j. jubatus was confined to individuals from southern african countries, which included botswana, south africa, and namibia. these samples consistently clustered (grouped) together, with both nuclear (microsatellite) and mtdna data. depending on the type of analyses, a single cheetah sample from the democratic republic of congo grouped with a. j. jubatus, or slightly outside. the mtdna haplotype (linked group of variants that is inherited together) group of a. j. jubatus was the most diverse ( haplotypes) of the investigated sample collection and was centrally positioned in the mtdna haplotype networks, with the haplotypes of the other subspecies radiating from it. haplotypes assigned to a. j. raineyi were confined to east african countries, which included kenya and tanzania. however, the maternal lineages (mtdna) fell into separate haplotype groups, one of which clustered with a. j. jubatus, separately from other a. j. raineyi haplotypes. as a consequence, a. j. raineyi has been included in a. j. jubatus by kitchener et al. ( ) a. j. venaticus was confined to extant samples from iran. historical samples of asiatic cheetahs (oman, jordan, india, iraq, medieval iran) clustered with the extant iranian cheetah samples, as observed with both nuclear and mtdna data. additionally, one sample from the extinct population in northeastern egypt also clustered with the asiatic cheetah samples of a. j. venaticus. historical samples from libya appeared distinct based on mtdna data. with microsatellite data the libyan sample clustered more closely to, but separately from, the asiatic cheetah (bootstrap support of % for the divergence between the branch of the asiatic samples and the libyan sample). cheetahs from southern egypt, western sahara, and algeria shared the mtdna haplotype with the libyan samples, and were separate from the a. j. venaticus haplotypes. thus the mtdna data supported the asiatic subspecies . the cheetah (marker and o'brien, ) ; cal, reference time point used as calibration; clo, reference time point used for molecular clock; µ, mutation rate given per generation; mya, million years ago. d migr, isolation with migration rate = , conservation method (wakeley and hey, ) ; (δµ) , microsatellite genetic distance (goldstein and pollock, ; zhivotovsky and feldman, ) ; apd, average percent difference; coal, coalescent method (gaggiotti and excoffier, ) ; d, nei's raw number of nucleotide differences between populations (nei and li, ) ; d a , net number of nucleotide differences between populations (nei and li, ) ; d sw , stepwise weighted genetic distance (shriver et al., ) ; ima, isolation with migration, demographic method (hey and nielsen, ) ; k p, kimura's -parameter model (kimura, ) ; prop unique, proportion of unique alleles. . the cheetah boundaries suggested by nowell and jackson ( ) for a. j. venaticus, with cheetahs from the northern sahara being of a differtent subspecies (belbachir, ; krausman and morales, ) . the cheetahs from the northern sahara may be of the same subspecies as the west african cheetah (a. j. hecki), but this could not be confirmed, as no west african samples were available. a. j. soemmeringii was confined to northeast african countries, which included sudan, djibouti, ethiopia, and somalia. although these samples clustered together, some substructuring was identified. in freeman et al. ( ) , of the a. j. soemmeringii mtdna haplotypes was more similar to the a. j. raineyi haplotype ( substitution apart) than to the other a. j. soemmeringii mtdna haplotypes ( substitutions apart). this likely reflects a more complex migration/colonization history, imperfect lineage sorting, or weakly defined boundaries of subspecies. all the a. j. soemmeringii mtdna haplotypes shared a -amino acid deletion in the mtdna-nd protein, which, if confirmed, could be used as diagnostic site to trace illegally traded specimens. globally, there was no evidence of gene flow between the subspecies in recent generations, as no admixture was detected with bayesian analysis of population structure. genetic differentiation was further supported by the neighbor-joining (nj) phylogenetic tree based on microsatellite markers. patterns of genetic variation identified broad regional populations in southern africa: namibia, the kalahari, and south africa, when analyzing microsatellite loci in cheetahs from botswana, namibia, and the northwestern parts of south africa (fig. . b ; kotze et al., ) . f st values between namibia and south africa ( . ) and namibia and the kalahari ( . ) were higher than those between south africa and the kalahari ( . ). however, these values remain below the f st values recommended for high statistical certainty of population assignment ( . - . ; manel et al., ) . also, while the significance of the differentiation between populations (p < . ) suggests that accurate population assignment may be possible, attempts to assign cheetahs of unknown origin to a specific region (from a bayesian exclusion test method and a frequency-based method) were inconclusive. the efficacy of assignment testing in cheetahs will most likely be improved through more comprehensive sampling (more subpopulations and more individuals per region; manel et al., ) . mtdna data may also help to resolve questions of origin within southern africa, given that haplotypes were identified in a bp sequence of mtdna in botswana, namibia, and south africa (charruau et al., ) . the namibian cheetah population was the first national population to be assessed genetically. marker et al. ( ) analyzed microsatellite markers in unrelated individuals whose distribution covered most (over %) of the cheetah's natural distribution in the country (fig. . b ). no regional structure was detected when analyzing samples individually. when individuals were grouped by region, very modest support was given to a tentative grouping of the north-western regions (outjo, grootfontein; nj tree, bootstrap support of %) and of the southern regions (windhoek and gobabis; bootstrap support of %). multiple component analysis distributed the regions somewhat according to their geographical location, with the most northern region (outjo) appearing to be most distinct, but f st values for okahanja and outjo (f st = . ) were below the recommended threshold for separate population assignment ( . - . ; manel et al., ) . overall it was concluded that the namibian cheetah population is panmictic (without barrier to breeding and without population structure), with gene . the cheetah flow maintained between the studied regions and precluding the emergence of any major structure. connectivity was further supported by identification of two migrants to and from the outjo region. more recently, castro-prieto et al. ( ) reached similar conclusions through analyses of exon of mhc class i, and class ii-drb genes that were genotyped with the single strand conformational polymorphism method in individuals from the north-central region and individuals from the east-central region (fig. . b ). all identified alleles and resulting haplotypes were present in both regions. no regional differentiation was detected for mhc ii-drb, although haplotype frequency for mhc class i varied between north-central and eastcentral namibia with a moderate f st support (f st = . ; p < . ). the difference in allele frequency at mhc class i (which targets intracellular pathogens) was attributed to different viral selective pressures in the two regions. botswana dalton et al. ( ) conducted an analysis of the botswanan population ( fig. . b) in unrelated animals with microsatellite loci. although this study included a small sampling number, it still provided essential insights into the lack of population structure in botswana. all animals were assigned to one unique group. absence of substructure was further supported from the analysis of molecular variance ( % of variation shared among localities) and low genetic distance between the two largest sampled populations (ghanzi vs. jwaneng f st = . ; p < . ). weak subdivision among the geographical populations suggests that gene flow occurs, which can be attributed to natural cheetah movements. the moremi population appeared slightly "distinct" from the neighboring ghanzi population (f st = . ), which was tentatively attributed to the presence of the okavango delta as potential natural barrier between these two populations. genetic analyses of fecal samples in the serengeti identified multiple paternities for subsequent litters ( females), as well as within litters ( / litters); observed adoption events could also be confirmed genetically (gottelli et al., ) . in namibia of females with cubs were confirmed as biological mothers, and of presumed sibling groups without a dam were confirmed to be related (marker et al., ) ; in all exceptions ( mothers and sibling groups) individuals had been sampled from captive facilities with suspected humaninduced animal grouping. coalition males appeared to be related in namibia based on of male coalitions (marker et al., ) ; while in botswana, of wild-caught coalitions appeared to include at least one unrelated individual (dalton et al., ) . all three unrelated namibian groups and one of the unrelated botswanan groups dispersed at the time of release, suggesting that the grouping may have been an artifact of capture. it was also shown that females with higher levels of genetic relatedness had greater homerange overlap (marker et al., ) , suggesting a matriarchal society. the identification of the infectious agent responsible for one of the better documented viral outbreaks in the captive cheetah population (chapter ) was made possible due to samples properly stored since the outbreak in the early s (pearks wilkerson et al., ) . using a phylogenetic approach, the virus was identified as a coronavirus, very similar to domestic cat coronavirus responsible for feline infectious peritonitis. however, in cheetahs, morbidity . the cheetah was % (symptoms included diarrhea, jaundice, and seizures) and overall mortality was % within years ( % in cubs). this was significantly higher compared to the %- % mortality in domestic cat or the corresponding coronavirus (sars) outbreak in humans in , making this the deadliest documented death toll of a coronavirus. the authors attributed the high death toll to the lack of genetic diversity and the naïveté of the cheetah population to this virus, which appeared to have jumped from the domestic cat into the cheetah species. a new species of infectious blood-borne parasite, babesia lengau, was characterized genetically in cheetah (bosman et al., ) . while, as opposed to its effect in domestic cats, babesia is not currently known to cause any pathology in the cheetah, additional research is continuing to investigate the effect of babesia on specific health parameters (schmidt-küntzel et al., in preparation) . a single nucleotide polymorphism (snp), described in the serum amyloid a (saa) gene, was genotyped in captive cheetahs to assess whether there was a correlation between amyloidosis disease status (chapter ) and the snp genotype (franklin et al., ) . it was found that the snp had a semidominant effect on the associated protein level within each study population (n = ), but that the institution at which the animals were housed had an even larger effect. in addition, there was no significant association between genotype and disease status (n = ). thus, the genetic impact of saa on amyloid levels in cheetahs is minimal and outcompeted by other factors. no correlation could be detected between variants in the mtdna genome and myelopathic pathology (burger et al., ) . and to date no correlation could be identified between oxalate nephrosis and the coding regions of published candidate genes (cheetah conservation fund and national zoological gardens of south africa, unpublished data). the cheetah has long been known for its poor sperm quality, with less than % viable sperm observed in reproductive studies (chapter ). in a whole genome study comparing the cheetah sequence to that of other species, several mutations affecting gene function were found in a-kinase anchor protein (akap ), a gene involved in spermatogenesis, and were shown to be likely fixed (only allele present in the population) in the cheetah (dobrynin et al., ) . those mutations may be in part responsible for the documented poor sperm quality. other phenotypes (heritable traits that can be seen/measured) of interest observed in the cheetah are kinked tails, crowded incisors, palatal depression, and coat variations (chapter ). while no molecular work has been performed on the morphological traits to date, insight was gained on several coat related phenotypes. genes from the keratin-associated protein family were found to be expressed at higher levels in the yellow background of the cheetah fur (kaelin et al., ) , which is consistent with observations that fur of the black spots is softer relative to the coarser textured yellow background. conversely, genes responsible for pigmentation were expressed at higher levels in the black spots relative to the less pigmented yellow background of cheetah fur (hong et al., ) . another gene whose expression was increased in the black spots was a paracrine hormone, which was hypothesized to be involved in coordination of the spot pattern (kaelin et al., ) . the genetic basis for the king cheetah coat variant was determined to be a mutation in the transmembrane aminopeptidase q gene (kaelin et al., ) . rare cases of gross morphological deformities have occurred . the cheetah in the past, but no literature is available to substantiate whether they were based on low genetic diversity, inbreeding in a captive setting, or teratogenic influences. in a recent genome-wide analysis of the cheetah (dobrynin et al., ) signatures of positive selection (by comparison with lion, tiger, cat, human, and mouse) were identified in close to a thousand genes. ten of the genes were involved in muscle contraction (both cardiac and striated muscles), specifically the mitogen-activated protein kinase pathway (which is linked to stress), and in the regulation of catabolic processes, indicating that the cheetah underwent some degree of specialization in these pathways. in the same study over million nucleotides of segmental duplications were identified, and affect genes that are believed to be involved in energy balance, nutrition, and sensory adaptation. in addition, gene expansion was observed in the mhc extended class i region, which includes vomeronasal receptors, as well as olfactory and g-coupled receptor genes; these gene expansions were tentatively linked to behavior (pheromones) and physiology (e.g., ldh-a and ldh-b are linked to a carnivorous diet). both segmental duplications and gene expansions lead to temporary redundancy, allowing new gene functions to arise. evidence of historical positive selection on antigen binding sites that interact directly with pathogen-derived proteins was detected for both mhc classes, particularly mhc i (castro-prieto et al., ) . signatures of selection in the mhc were also identified in the whole genome study (dobrynin et al., ) . a study of the cytochrome p gene (cyp d ), involved in drug metabolism, showed considerable genetic diversity and signs of relaxed selection pressure in felids, including cheetahs (schenekar et al., ) . the most notable and still poorly understood feature of cheetah evolutionary history is how the cheetah has persisted in spite of remarkably low levels of genetic variation. genomic variation is generally considered to be crucial for long-term survival of species as it provides potential for adaptive responses (natural selection of advantageous genetic variation) to environmental changes, such as climate change (chapter ), and adaptability of immunity to disease outbreaks (o'brien and evermann, ) . therefore, the initial discovery of genetic uniformity of the cheetah was quickly followed by concerns about the species' chances of long-term survival. however, the discovery that the event or events leading to the loss of diversity could be placed over , years ago and that cheetah numbers had recovered by the th century, indicate the cheetah's ability to survive and thrive, despite reduced levels of genetic diversity, over extended periods of time. however, this does not guarantee the cheetah's survival in the future, as lack of genetic diversity limits the ability to adapt and evolve, in particular in the light of major changes in environmental conditions or pathogenic pressure. reduced genetic variation, particularly at adaptively important mhc loci, has been associated with high susceptibility to infectious diseases in captive cheetahs o'brien et al., ; o'brien and evermann, ) . a prime example is the high death toll caused by a coronavirus outbreak in a north american zoo (section "genetic investigations of infectious diseases affecting cheetahs," chapter ). despite this, free-ranging cheetahs from eastern and southern africa show robust health (caro ; munson et al., ; munson et al., ; thalwitzer et al., ) and do not seem to have compromised immunocompetence (castro-prieto et al., ) . in addition, in the wild the cheetah's . the cheetah large home ranges (chapter ) reduce the risk of infectious disease transmission. however, this may not be sufficient to protect the species in the event of an emerging disease, especially given the low levels of mhc diversity. at an individual level manifestation of deleterious traits caused by excessive levels of homozygosity (inbreeding depression) appear to be limited compared with the puma population in florida (florida panther), which suffered from atrial defects, poor sperm quality, cryptorchidism, and high disease load (roelke et al., ) . the cheetah is only known to suffer from poor sperm quality; none of the other traits observed in the cheetah (e.g., kinked tails, crowded incisors; section "investigations of the molecular basis for heritable traits") are detrimental to individual health, or the capacity to survive and reproduce. this, and evidence of positive signatures of selection on genes involved in muscle contraction and stress metabolism (section "signatures of selection, copy number variation, and changes in gene families"), suggests that the low levels of genetic diversity were caused, or followed by, strong selective pressures, which perhaps purged deleterious alleles from the species (e.g., hedrick and garcia-dorado, ). despite limited sperm quality, cheetah matings produce sufficient viable cubs (up to cubs per litter every years; chapter ) to maintain and even increase the population. however, further loss of genetic diversity could impair reproductive success, which is the ultimate requirement for species survival. indeed, increased infant mortality was observed in captive inbred individuals . with the reduction of its natural range, cheetah numbers are declining (durant et al., ; chapter ) , and most cheetah populations today are fragmented with loss of connectivity between them (chapter ). small populations, such as the critically endangered iranian cheetah (a. j. venaticus; chapter ), which has the lowest amount of genetic diversity of all the currently recognized cheetah subspecies (charruau et al., ) , are particularly at risk of losing further genetic diversity. therefore, it is crucial to maintain or regain sufficiently large population sizes and connectivity, while preserving existing variation through viable long-term storage of sperm and oocytes (chapter ). whenever possible, animals should remain in, or if captured, be returned to the wild (chapter ). captured animals that are not suitable for release back into the wild should be considered for breeding programs (section "genetic diversity and ex situ cheetah conservation"). wild cheetah populations of the same subspecies and geographical region were generally panmictic; minor population structure was only observed in allele frequencies of the rapidly evolving immune response genes. as such, translocations of wild caught individuals performed as part of conservation actions within these populations, only mimic natural connectivity (which may be restricted by anthropogenic barriers to gene flow). an additional level of complexity arises when populations are from different subspecies. while in principle exchange between populations of different subspecies should be avoided as they may be considered evolutionary significant units (moritz, ) , a compromise between preserving the existing structure and the urgency to rescue a small population at risk of disappearing, may have to be reached. relatively recent times of subspecies divergence and the merging of two subspecies in , are additional considerations during such decision making processes (section "subspecies definition and divergence"). this dilemma may have to be addressed for the iranian population at some point if the numbers remain below individuals (chapter ). additional information on the genetic health of the existing population is critically and urgently needed to determine if this population is likely to survive without management actions. captive cheetah populations have been established in part to serve as a reservoir for the wild population (chapter ). breeding decisions are guided by the genealogical data managed through the regional and international cheetah studbooks (chapter ). reputable captive programs (chapter ) aim to retain % of genetic diversity over years (lacy, ) . however, this may not be sufficient in the long term as the small number of founders only represents a subset of the genetic diversity found in the wild (founder effect) and the % genetic diversity lost is irreversible. this loss of diversity can only be compensated for by the recovery of lost breeding lines through "reinjection" of viably preserved reproductive material (e.g., gametes) of founders into the captive population, addition of new founder individuals (or gametes) obtained from the wild, or inclusion of unrelated captive individuals to the breeding pool. as with wild populations, interconnectivity of captive populations should be maintained as much as possible, through regional and interregional exchange of animals or reproductive material. as the cost of genetic research continues to decrease, it will provide the possibility to assess the genetic makeup of all captive cheetahs, and thus the integration of animals of unknown origin into the cheetah breeding pool, if genetic evaluation determines that they represent a new breeding lineage. of note, inbred individuals (high homozygosity levels) can also be used for further breeding if they represent a unique breeding lineage, as homozygosity is not heritable. the field of conservation genetics will see more advanced analyses emerging, including the evaluation of heritable traits, landscape genetics, and increased precision in assessing the extent and timing of the events that cause the loss of genetic diversity. non invasive samples are increasingly employed to provide answers regarding populations that have not yet been intensely studied (chapter ). additional research involving contemporary and museum samples is currently under way to fill existing knowledge gaps regarding the published subspecies (léna godsall bottriell, personal communication). however, it is crucial to remember that data obtained from genetic studies published to date agree sufficiently in confirming the low genetic diversity of the species at nonrepetitive loci (section "genetic diversity"), dating the origin of the low diversity to more than , years ago (section "historic demography"), providing support for genetic differences, although short divergence times, between the populations corresponding to most published subspecies (section "subspecies definition and divergence"), and showing only minimal population structure within geographical regions (section "phylogeography"). this in turn enables a joint message in terms of recommendations for cheetah conservation, as well as in situ and ex situ management (sections "genetic diversity and in situ cheetah conservation" and "genetic diversity and ex situ cheetah conservation"). we hope that by presenting all available data on cheetah genetics, this chapter provides clarity to the results and conclusions arising from the 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nucleotide polymorphism of the major histocompatibility complex in the domestic cat dna variation of the mammalian major histocompatibility complex reflects genomic diversity and population history exchanges of short polymorphic dna segments predating speciation in feline major histocompatibility complex class i genes microsatellite variability and genetic distances key: cord- -ax i d f authors: karampourian, arezou; ghomian, zohreh; khorasani-zavareh, davoud title: exploring challenges of health system preparedness for communicable diseases in arbaeen mass gathering: a qualitative study date: - - journal: f res doi: . /f research. . sha: doc_id: cord_uid: ax i d f background: infectious diseases are common problems in mass gatherings, especially when there is a lack of health system preparedness. since iran is one of the most important countries on the walking path of arbaeen and has a vital role in providing health services to pilgrims, the experiences of health challenges by participants is of key importance. the aim of this study is to explore stakeholders’ experiences on the health system's preparedness and challenges, and to provide suggestions for preventing infectious diseases during the arbaeen mass gathering. methods: a qualitative research method was used with a conventional content analysis approach. the number of participants was , including executive managers and health policymakers who entered the study among participants. semi-structured interviews were used to generate the data. interviews were analyzed by means of content analysis after face-to-face interviews. results: data analysis resulted in the extraction of four main themes and sub-themes. health infrastructure defects in iraq has three sub-themes (health abandonment in iraq, the weaknesses in health culture and problems related to the health system); poor control of the causative factors of infectious diseases has three sub-themes (the underlying factors of the prevalence of contagious diseases, health system response to communicable diseases and ignoring the risks of the arbaeen ceremony); the low perception of risk in pilgrims has three sub-themes (lack of awareness in pilgrims, fatalism in pilgrims and unhygienic belief in pilgrims); and the ineffectiveness of health education has two sub-themes (training shortage in the targeted group and educational content problems) that shows participant’s experiences of the health system's challenges for coping with infectious diseases during the arbaeen ceremony. conclusion: pilgrim-based training, planning and controlling other challenges may change these threats to opportunities and improve the health of participants of the mass gathering of arbaeen in the region. according to the definition of the world health organization (who), any structured or spontaneous event leading to a certain number of people gathering in a particular site, for a specific aim in a determined period, putting pressure on the response resources and social programs, is called a mass gathering. mass gatherings are divided into different types based on their purpose. the expansion of interconnectivity between societies and increases in the number of national and international events in communities has led to an increase in the number of mass gatherings, which, despite the benefits like cultural exchange, have health challenges such as infectious disease transmission and, should therefore be considered by health planners . one of the health challenges of mass gatherings is the prevalence of infectious diseases and the outbreak of diseases, which, along with, complicated health needs of participants increases the health burden on the host country. the public health system can be under severe pressure, even with advanced equipment and the proper resources for prevention and control of infectious diseases - . various factors, such as the type and location of gathering, the number of participants and the lack of access to health facilities, can affect the incidence of infectious diseases in mass gatherings. planners must therefore pay attention to these factors in preparation , [ ] [ ] [ ] . since a mass gathering is a collection of many people together in one particular site, the possibility of infectious disease transmission due to the high population density always exists. studies on mass gatherings such as hajj, ashura day in karbala, and kumbh mela and sabarimala in india show the prevalence of infectious diseases in these ceremonies [ ] [ ] [ ] [ ] [ ] [ ] . the dates of religious ceremonies like hajj and arbaeen are decided using the lunar calendar (the islamic or hijri calendar) which is not only shorter than the gregorian calendar, meaning that these events occur days earlier each year and can synchronize with different seasons and season-associated diseases. accordingly, planners and policy makers of public health are faced with changing goals, requiring health system preparedness , . one of the world's largest religious gatherings is the arbaeen ceremony, which happens on the th day after the anniversary of imam hussein's martyrdom, the third shiite imam. in the ashura event, pilgrims walk to karbala, south of baghdad. based on the statistics of , the number of iranian pilgrims taking part in the arbaeen ceremony was , , . iran is a neighboring country of iraq and shares a common land border with it. pilgrims from other neighboring countries of iran, such as afghanistan and pakistan, cross iran to reach iraq and the arbaeen ceremony. on the basis of the mutual memorandum of cooperation between the two countries of iran and iraq, iran is committed to providing health services to other pilgrims in addition to iranian pilgrims . therefore, it is necessary to have a plan for preparedness in dealing with infectious diseases during arbaeen ceremony. if a mass gathering is not carefully managed, it can lead to the distribution of infectious diseases. in mass gatherings, infectious diseases are threats to global health security and even the political security of countries. therefore, planning, communication and public health supervision are important in these religious ceremonies , . mass gatherings are different from structured disasters so that in case of occurrence, many people will be affected . since the arbaeen ceremony is held with the presence of many pilgrims from many different countries and, like the hajj pilgrimage, is based on the lunar calendar, and it is held in iraq, there is the possibility of the occurrence and transmission of infectious diseases. it is therefore essential to be prepared to control and prevent these diseases. dealing with infectious diseases in arbaeen is considered a challenge for policy makers . according to arbaeen's social and cultural context, it seems essential to take a deeper look in this field. on the other hand, there is relatively little knowledge about the arbaeen ceremony; therefore, a qualitative method for clarifying the concept and challenges of health system preparedness in arbaeen ceremony is necessary. the aim of this study was to explore challenges of health system preparedness for communicable diseases in arbaeen ceremony. we collected data from june to march . since the study attempted to explore preparedness challenges of health systems, a qualitative research method with the approach of conventional content analysis was used . the health system's challenge in arbaeen is multidimensional, owing to the cultural difference between iran and iraq, the challenges faced by the health system during the arbaeen pilgrimage should be investigated in both countries. indeed, the cultural practices of the participants, especially those surrounding health, differs during the arbaeen ceremony. therefore, a qualitative research method, with the aim of describing phenomena, providing new knowledge, insight and a practical health guide, is the method used in this study , . the study was conducted using in-depth interviews, based on stakeholder's experiences in iraq-iran land terminals (mehran, shalamche, and chazaba) and also in health care posts in iraq. the interviews were conducted with health care providers and policy makers, as well as pilgrims in the arbaeen ceremony. participants were chosen among executive managers and policymakers of the ministry of health and medical education, medical training and treatment and other related organizations, including the red crescent organization, mobilizing the medical society, the hajj and pilgrimage organization, medical universities in the border cities and the social security organization. in total, participants, consisting of executive managers and health policymakers, were selected in this study through purposeful sampling with the aim of exploring challenges of health system preparedness for communicable diseases in the arbaeen ceremony. the existence of practical experience in planning or participate in arbaeen ceremony and the ability to communicate and willingness to participate were inclusion criteria in the research. we recruited participants either by phone call or by approaching them in person. "maximum variety sampling" was used to explore the experiences of the participants in those selected so that they were chosen from the ministry of health and medical education, medical training and treatment and other related organizations, including the red crescent organization, mobilizing the medical society, the hajj and pilgrimage organization, medical universities in the border cities and the social security organization, with different experiences of work, education and gender. inclusion criteria included the existence of practical experience in planning or participate in arbaeen ceremony, the ability to communicate and willingness to participate in the research (table ). the study was done through face-to-face interviews followed by telephone interviews for concept saturation. the data was collected using audio recorders with permission from the participants. ak conducted the interviews, ak and dkz transcribed the data. ak and dkz and zgh coded the data. dkz and zgh performed rigor. initially the first two interviews were conducted in a non-structured format, with the following interviews being semi-structured. open questions used to generate the data were developed by experienced and/or knowledgeable policy makers and health care providers. the individual's experiences and beliefs were used without considering their specialty . interviews were continued until data and concept saturation were reached . the interview duration was between and minutes, based on the tolerance, amount of information and desire of the participants. interviews were performed individually and based on participants' willingness in terms of time and site. firstly, interview questions began with the following general questions based on the participants' level and the main questions of the research: "how was your organization preparedness plan to deal with infectious diseases in arbaeen ceremony?"; "please express your experiences of related challenges in infectious diseases in arbaeen ceremony"; "what problems were in your preparedness plan?"; "what problems did you face in the vaccination program of the health team and pilgrims in arbaeen ceremony?"; and "what is your offer to pilgrims for a safe pilgrimage?" following this, exploratory questions were gradually used to clarify the concept and deepen the interview process: e.g. "please explain further what you mean?" and "why?" at first, the interviews were transcribed verbatim and then typed up using microsoft word office. the data were analyzed through a conventional content analysis method . first, the main researcher converted the interviews to written texts. the digital files were listened to several times and the texts were read repeatedly. next, the meaning units were determined based on the aim and the question of the research. meaning units were a collection of words and sentences that were related to each other in terms of content and were grouped together. meaning units reached the level of abstraction and conceptualization and were coded considering the research question. the key points and subjects were extracted as open codes. these codes have been put under the broader headings based on the present similarities and differences; in other words, the data were reduced in order to describe the phenomenon and gain a better understanding, and this abstraction process continued until concept extraction . data first emerged as meaning units, then condensed meaning units, codes, sub-themes and finally themes. the study was approved by ethics committee of shahid beheshti university of medical sciences on / / , no. ir.sbmu. retech.rec. . . the interviews were conducted and recorded with participants' consent. written or verbal consent was taken from participants to participate in the study. verbal consent was only taken for telephone interviews; this was due to the distance between interviewer and interviewee. anonymity, confidentiality and the right of resignation were informed to participants and considered during the study. the interview time was set according to participants' willingness. the researchers used the trustworthiness criteria recommended by guba and lincoln to establish rigor . all authors were engaged in the environment and field of research. in addition, the principle investigator always had suitable involvement with the participants for in-depth interviews. credibility was established by the prolonged engagement of researchers with participants. researcher triangulation was also used to verify the accuracy of the coding process. the research team also retained raw data, codes, and themes for control of the reliability. at the same time, sampling was carried out with maximum diversity in order to provide triangulation by means of credibility and confirmability. a detailed description of the method was used to establish transferability. engaging participants in the research increases the interaction between researchers and participants and then the credibility . the research supervisor monitored the data collection and data analysis process. it is necessary to mention that the research team participated in the arbaeen ceremony as pilgrim and conducted field notes. also, memberand peer-checking were used to ensure credibility. therefore, many interviews with related topics were sent to some external expert reviewers and participants (policymakers of the ministry of health and medical education, medical training and treatment and the red crescent organization) to be checked, and they were requested to assess the degree of relevance between the findings and raw data. moreover, transferability was established by sampling with maximum variation from various centers, including the ministry of health and medical education and other peer organizations including the red cross organization, the medical community mobilization, the hajj and pilgrimage organization, the country's medical universities in the border cities and the social security organization with different experiences of work, education and gender. a completed srqr checklist is available in supplementary file . themes and sub-themes the mean age of participants was and the mean time of work experience was years (table ) . overall, original codes were extracted and after integration using conventional concept analysis, four original themes consisting of sub-themes were identified. the theme of "health infrastructure defection in iraq" had three sub-themes: "health abandonment in iraq", "the weakness of health culture" and "problems related to health system". the theme of "poor control of factors effective in infectious diseases" had three sub-themes: the "underlying factors in prevalence of contagious diseases", "health system response to communicable diseases" and "ignoring the risks of the arbaeen ceremony". the theme of "low perception of risk in pilgrims" had three sub-themes: "lack of awareness in pilgrims", "fatalism in pilgrims" and "unhygienic belief in pilgrims". the theme of "ineffectiveness of health education" had two sub-themes: "training shortage in the targeted group" and "educational content problems" (table ) . according to the findings of this study, the main theme is pilgrim-based education. it seems that the biggest issue with the prevention of communicable diseases in the arbaeen ceremony is pilgrim-based education. educating pilgrims can directly help people to create or reform health infrastructures in iraq. education can also help to identify health risks and respond to them by identifying effective factors in combating infectious diseases. the training of health instructors and guidelines from people who are trusted and accepted by pilgrims, such as missionaries and religious leaders, can have a positive impact on their beliefs. the main point of training is to determine the targeted group both at the level of pilgrims and health directors and consider the training needs of each group in preparing training guidelines. health abandonment in iraq. most of the participants believed that the iraqi health system has been abandoned because foodstuff distribution is not under the supervision of a special organization and does not have a special trustee in the execution and monitoring of health rules or, if there is one, he is inconspicuous. the existence of a trustee or supervisor in the health system can prevent the delivery and distribution of unsafe food and reduce the prevalence of infectious diseases. unhealthy foodstuff preparation, production and distribution, and a lack of food evaluation and supervision system can lead to gastroenteric disease. based on the participants' experiences, the lack of a health system trustee means system weakness. the following quotation is an example of the above: "sometimes donations are prepared in an unhealthy manner and so lead to acute digestive problems… the health system is weak in iraq because health rules are not enforced and there is no supervision for these centers…" (executive manager, male, - years, - years job experience). the weakness of health culture. according to the participants' point of views, observation of unhealthy behaviors, such as neglect of the individual and public health standards, and the existence of cultural differences between iran and iraq, are considered by pilgrims to bring about an unsafe culture. policy makers and executives should be familiar with the kind of health culture in iraq so that they can develop a program to prevent contagious diseases. in iranian culture, the non-use of spoons and forks is considered as lack of health belief and neglect of health, whereas in iraq, eating food with the hands is part of the food culture. iran's health system can help train the iraqi people in sanitary practices alongside iranian pilgrims. the following quotation is an example of the above: "some food providers don't meet health … the culture of using spoons and forks for food serving is different in iran and iraq… one of the cultural works which we can do in iraq is health education…" (executive manager, male, - years, - years job experience). problems related to health system. most participants acknowledged that despite annual health care improvements in iraq, there are also some shortages in this field due to the lack of a long history of a health service system. health system weakness, incomplete health service implementation and insufficient supervision of environment health are indicative of the weakness of the health infrastructure; this has made it impossible to provide environmental health, sanitation and waste disposal. the following quotation is an example of the above: "health background in iraq was poor… there were no waste bins and waste sanitation there… failure to implement health services has led to failure in meeting health conditions … there was no health the probability of an outbreak of endemic and non-native diseases the ineffectiveness of the health system in screening the impossibility of requirement flu vaccination the inability of the system to provide services in an epidemic the underlying factors in prevalence of contagious diseases. most participants have identified underlying factors of infectious diseases as one of the challenges affecting the preparedness of the health system in dealing with infectious diseases. various factors, such as population density and diversity, not paying attention to the principles of personal and general health, for example through a lack of health facilities, weather conditions during the trip and changes in nutrition can cause the spread of infectious diseases. identifying these factors helps pilgrims and planners to prevent infectious diseases. the following quotation is an example of the above: "there were few toilets or no healthy facilities. somehow, pilgrims would have to sleep in the desert or in a limited space with a lot of people… congestion of pilgrims from different countries increases the risk of spreading infectious diseases…" (executive manager, male, - years, over year job experience). health system response to communicable diseases. on the basis of the view of participants, mass population movements from different countries and their gathering with different population diversity can transfer and spread endemic diseases and also emerging diseases such as plague and anthrax. there is also the possibility of bioterrorism events occurring during the arbaeen ceremony. the health system needs facilities such as equipped laboratories to diagnose and treat infectious diseases in a timely manner. the inaccessibility or lack of experimental equipment for disease identification leads to the failure of timely diagnosis of diseases, a lack of disease control and finally the incidence of epidemics. syndromic surveillance system can be used to diagnose infectious diseases. there is a need to correctly pass the treatment period of infectious diseases in order to prevent epidemics, although drug shortages can affect treatment completion and is one of the causes of epidemics. vaccination also helps to prevent infectious diseases, but the requirement of pilgrims to vaccinate is always up to the host country and since vaccination is not one of iraq's priorities, the ministry of health and medical education can only advise pilgrims to vaccinate. the following quotation is an example of the above: "there is the probability of spread of local and new-appeared diseases and even bioterrorism due to the gathering of pilgrims from different countries… some diseases can't be diagnosed due to lack of facilities but syndromic surveillance system can be used. …the large number of pilgrims and lack of medication have led to not having complete course of antibiotic treatment… the need for vaccination is one of the requirements of the host country". (policy makers, male, - years, - years job experience). the arbaeen pilgrimage has special features that distinguish it from other mass gatherings. participation of different peoples with a diverse range of socio-demographic statuses, cultures and nationalities makes this distinction. the arbaeen pilgrimage has some specific hazards like other trips that are sometimes neglected. the population of arbaeen pilgrims and its time of year are changeable. financial management of travel expenses at the ceremony is carried out by volunteers. considering these features is essential for the readiness program. in the opinion of the majority of participants, one of the challenges of the health system is the lack of attention to the risks of arbaeen ceremony and the lack of planning based on these features. the following quotation is an example of the above: "arbaeen is a new phenomenon that children and adults, men and women with different cultures and ethnicities take part in the ceremony … even with the knowledge of the dangers of the route, people attend arbaeen ceremony… arbaeen is a spontaneous and popular event and doesn't cost much … the population is moving and the time of the ceremony changes every year …" (executive manager, male, - years, - years job experience). lack of awareness in pilgrims. one of the challenges in the view of the participants, especially executives, is the pilgrims' low awareness of health hazards, such as not using personal hygiene products and non-compliance with health standards. unhealthy and dangerous practices, such as unsanitary food consumption, are not understood by pilgrims, and this low awareness and inadequate knowledge of the risks are the causes of infectious diseases in the pilgrims. respect for personal and public health, such as hand washing, providing food from health food centers and avoiding overeating, is effective in preventing digestive diseases. the following quotation is an example of the above: "some pilgrims do not wash their hands or do not use personal hygiene products …or they do not get foods from healthy centers … overeating and then digestive problems are one of the pilgrims' problems … the other problem is not being familiar with arabic language …" (executive manager, female, - years, less than years job experience). participants believed that one of the health challenges is belief in destiny and fatalism; so that most pilgrims who participate in the arbaeen walking ceremony, based on their belief in destiny, have a relatively low understanding of the dangers and diseases, and begin their trip merely confident in allah and without any plan for dealing with infectious diseases, such as vaccination, and continue the way using food and drinks that are often unhealthy. the following quotation is an example of the above: "it's enough to decide to travel and you do not need to have a special plan … if you think openly, you won't get sick, and nothing bad will happen…" (policy makers, male, - years, - year job experience). based on the participants' experiences, low perception of danger in pilgrims is sometimes seen as insanitary beliefs in preventing medication consumption and disregarding hygiene recommendations. believing in the use of medication during illness, preventing self-curing and trying to abide by hygiene recommendations prevents infectious diseases. the following quotation is an example of the above: "sometimes we see pilgrims using traditional treatments instead of antibiotics consumption… they don't pay attention to hygiene recommendations such as masking and not using suspicious food…" (executive manager, male, - years, - year job experience). training shortage in the targeted group. based on stakeholders' views, one of the present challenges is the quantitative and qualitative shortage in the stakeholder's training level. this means that providing a personal and public health plan should cover all stakeholders, including executives, policymakers and volunteers in the arbaeen ceremony, and be considered with respect to each participant. furthermore, the timing of training is also important and should be before the days of arbaeen ceremony in order to have a greater effect on the individuals' knowledge. indeed, training courses should be held separately for each of the groups, pilgrims, executive managers, and policymakers, and at a proper time before arbaeen. the following quotation is an example of the above: "training should not exactly be in the days of arbaeen. personal and public health training must be held several months before arbaeen… the training is not only for pilgrims, but also anyone involved in arbaeen ceremony. everyone should be trained from pilgrims to policymakers…" (policy maker, male, - years, - year job experience). another problem was the provision of educational content. most of the participants believed that training should be fitted with pilgrims' needs and respond to the problems of pilgrims. pilgrims should be divided into different groups based on the level of education, their problems and illnesses, and individual and general education should be planned accordingly. participants also believed that training in the area of personal, general and nutritional health should be provided more comprehensively. the following quotation is an example of the above: "we should divide pilgrims to diverse groups and send targeted training messages to each group, not the same training for everyone… the benefactors should be trained…pilgrims should have more training…" (policy maker, male, - years, - year job experience). the aim of this study was to explore the challenges of health system preparedness for infectious diseases in the arbaeen ceremony as the first qualitative study in iran. the most important findings of the study are the ineffectiveness of health training, the low perception of risk in pilgrims, poor control of the causative factors of infectious diseases, and deficient and defective health infrastructure in iraq. based on the views of the majority of participants, pilgrim-based training is the most effective factor in health system readiness in dealing with infectious diseases in the arbaeen ceremony. ineffectiveness of health training is one of the challenges of health system preparedness. one of the plans which should be considered to ensure arbaeen ceremony preparedness is health training. educational planning must be done before holding the arbaeen ceremony, with consideration of the training content and targeted groups. indeed, according to the needs of the targeted group, training must be given to pilgrims, executives and volunteer treatment teams, and the training content for pilgrims should be different to that of executives and policy makers. past conducted studies of religious gatherings such as the hajj in saudi arabia and ashura day in iraq, as well as other mass gatherings, such as the tamworth country music festival, australia, indicate the reality that a crowd of people from diverse nations and cultures is a source of infectious disease; disease transmission is one of the most important challenges of public health in these kinds of events, so using training strategies in relation to hand washing, masking and vaccination is an important factor in preventing the diseases and health improvement , , , - . since religious ceremonies are rooted in people's beliefs and have great popularity among people, people-centered education therefore has great potential to reduce the gap between knowledge and practice in pilgrims, empowering individuals and enhancing their ability to deal with health threats. this goal is achieved by identifying accurate and targeted needs, developing relevant content and through educational planning . in this study, like other studies on mass gatherings, a personal health training plan, such as the importance of hand washing, using healthy food and masking, should be included in the pilgrims' training plan, and public health training such as vaccination, environmental health, monitoring donations, cooking and distribution, controlling bioterrorism and establishing mobile toilets should be considered in executives and policymakers' preparedness plan. regarding the aim of holding the arbaeen ceremony, which is a popular religious-ideological ceremony, training can be performed in mosques before the ceremony by clergy. a low understanding of the risks of infectious diseases in pilgrims is one of the other challenges mentioned by the majority of participants, especially policymakers. a lack of risk understanding refers to the inability to identify and respond to dangerous situations. top documents such as sendai framework have identified that understanding risks is the first priority to decrease the incidence of disasters, and that it needs people-based and vast preventive approaches. besides this, the hyogo framework and sustainable development goals emphasize the role of training in increasing risk understanding and decreasing the vulnerability of individuals to hazards - . in this study, one of the health issues was fatalism and a low understanding of risk. a study concerning the beliefs and methods of infection control in hajj pilgrims residing in australia also showed that the majority of participants had low understanding of the occurrence of respiratory infections and the need for an influenza vaccine in hajj, and refused the vaccine, using trust in allah as an excuse and belief in destiny when dealing with the risk of disease . another problem is also the prevalence of self-treatment among pilgrims. in a study aimed at assessing the knowledge, attitude and the performance of australian pilgrims on using antibiotics in hajj showed that they did not have proper understanding of using these drugs and used medications arbitrarily, meaning that more training on proper use is needed . a lack of understanding of health instructions and disregard for public and personal health in any situation can endanger human health. it seems that the fatalism of pilgrims with regards to diseases increases their vulnerability if the understanding of danger is reduced. islamic instructions state that the person is obliged to preserve his health and life in any location and position, even in holy lands, and to avoid risks . given that arbaeen is a religious gathering, religious leaders have an important position in ceremony implementation and can affect the pilgrims' beliefs and understanding of risks during pilgrimage. the influence of religious leaders on the people's beliefs can provide the opportunity to promote health. we should consider that religious scholars must be trained first and then transfer health instructions and methods of infectious disease control to the people in cultural and religious gatherings such as mosques. another problem in this field is the poor control of the effective factors on infectious diseases. one of the most common causes of infectious disease is the epidemiology triangle, which has three components-pathogen, host and environment. the interaction of these agents causes infectious diseases, and considering these factors can help control infectious diseases . considering the three factors in arbaeen is very important. the 'host' factor includes different pilgrims with diverse cultures and nationalities. the 'environment' factor includes holding the ceremony in iraq, which, due to the many years of internal and external conflicts, has had little attention paid to its health infrastructure, and also the overall environmental factors effective in the occurrence of infectious diseases. different studies , , have shown that factors such as crowd size, equipment, climate, the event duration and location, the type of ceremony, and features and behavior of participants affect the occurrence of diseases in mass gatherings, and planners must consider it during preparations. to prevent the occurrence of contagious diseases and their consequences, comprehensive planning, rapid diagnosis and effective management is required , , . one of the factors that affects the occurrence of disease is the time of year that the arbaeen ceremony is held. arbaeen ceremony is held based on the lunar calendar and so its needs and challenges differ and are dependent on the season that the ceremony is held, so that if the ceremony is held in cold seasons, respiratory diseases are more common and if is held in hot seasons, the majority of infectious diseases are of the digestive system. additionally, population movement among different countries leads to the transfer of local diseases, so health considerations and cooperation between states are needed.. policymakers should consider three components-pathogen, host and environment-before the beginning of arbaeen mass gathering. it is also necessary that the health system is aware of all possible scenarios and the methods for dealing with them. the preparedness plan at the local level includes assessing the risk, resources capacity, equipment, surveillance system and an expert team for providing services to pilgrims. health infrastructure defects in iraq was another challenge to the health system from the participants' perspective. the term 'health infrastructures' refers to health facilities and their related factors. the infrastructure includes staff instructions, processes and the development of systematic approaches related to personnel resources and medical support plans . various studies indicate that readiness for structured mass gatherings depends on investing in health infrastructures and the size of gatherings, and strengthening infrastructures and post-event coordination of mass gatherings must be continued. the inappropriate location of gatherings, the weakness of facilities and the lack of infrastructure increase the vulnerability of communities , . the remoteness of health facilities and a lack of needed road infrastructure can make medical services and emergency assistance ineffective. limitation of infrastructure and medical care system, increase the incidence of injuries , . arbaeen is held in a country that has long been involved with interior and exterior wars; therefore, it seems that due to economic difficulties, it does not have the capacity to support the necessary health infrastructure required by pilgrims. although according to the participants, the health system in iraq seems somewhat weak, given that arbaeen ceremony is of particular popularity among shia muslims and is held annually, therefore, some of the activities serving pilgrims and its management during the arbaeen ceremony is voluntarily conducted. in recent years, numerous health facilities have been constructed on religious places, as well as along the path of pilgrimage using pilgrims' donations. management of pilgrims' donations can help to build and maintain health infrastructure in iraq. with this policy, the iranian pilgrims will benefit from the arbaeen ceremony, and the level of health in the region will be improved. this study is the first qualitative study on the experiences of arbaeen, so it provides rich information in this regard; however, since the results have been collected from semi-structured interviews, it is considered subjective. it is recommended that in future studies, by creating a quantitative instrument for examining the challenges and measuring, the subjective concepts can be objectively transformed and analyzed. the present study can be used as a basis for this purpose. the ineffectiveness of health training, low perception of risk in pilgrims, poor control of the effective factors on infectious diseases and deficient health infrastructure in iraq are important challenges of the health system in dealing with contagious diseases in the arbaeen ceremony from the stakeholder's perspective. therefore, pilgrim-based educational planning, along with the control of other challenges, represents an opportunity to improve the health of pilgrims taking part in the arbaeen ceremony. the full data for this study are not provided because the transcripts of the interviews contain identifiable and sensitive information. researchers can apply to access limited deidentified transcripts of interviews from the first author, arezou karampourian (a.karampourian@sbmu.ac.ir) under no strict conditions. please note that transcripts are only available in persian. the study was supported by the shahid beheshti university of medical sciences, tehran, iran. . . the paper is interesting and adds to the medical literature of arbaeen ceremony which has been poorly addressed from a public health research perspective. we hope this paper will serve as a key reference on health aspect of arbaeen ceremony in near future. a few minor issues should be addressed before the manuscript is published. the reference list needs to be revised, we note some misplacement error. for example, in the third paragraph of the introduction (p ), "based on the statistics of , the number of iranian pilgrims taking part in the arbaeen ceremony was , , ", the reference given for this is number . ineffectiveness of health education. emerging of all four themes has been verified by the participants' comments which made results acceptable. the method of analysis is appropriately explained which can facilitate the reproducibility. the authors conclude that the main factor in preventing communicable disease is pilgrim-based training long before the ceremony. this conclusion is adequately supported by the results. is the study design appropriate and is the work technically sound? yes are all the source data underlying the results available to ensure full reproducibility? yes no competing interests were disclosed. i have read this submission. i believe that i have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. the benefits of publishing with f research: your article is published within days, with no editorial bias you can publish traditional articles, null/negative results, case reports, data notes and more the peer review process is transparent and collaborative your article is indexed in pubmed after passing peer review dedicated customer support at every stage for pre-submission enquiries, contact research@f .com establishment of public health security in saudi arabia for the hajj in response to pandemic influenza a h n knowledge, attitude and practice (kap) survey concerning antimicrobial use among australian hajj pilgrims fatalism at the arbaeen ceremony methodology of applied research in medical sciences letter to editor: mortality trends of pilgrims in hajj: an implication for establishment of surveillance system. health in emergencies and disasters qurterly should cities hosting mass gatherings invest in public health surveillance and planning? reflections from a decade of mass gatherings in pubmed abstract | publisher full text | free full text human stampedes during religious festivals: a comparative review of mass gathering emergencies in india pattern of morbidity and mortality in karbala hospitals during ashura mass this study is part of a phd thesis. the authors thank shahid beheshti university of medical sciences for approving the study, as well as all participants in this study who participated in the study, despite being busy. click here to access the data. competing interests: we have read this submission. we believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. it is better to discuss the results respectively as the results mentioned. so, the reader can insure . it is better to discuss the results respectively as the results mentioned. so, the reader can insure that all results have considered in the discussion part. it is better to explain what pilgrim-based education is and how it controls cd in discussion part and conclusion as well. are all the source data underlying the results available to ensure full reproducibility?no source data required no competing interests were disclosed. this is an interesting and well-written piece of work. infectious disease prevention strategies in mass gatherings, especially religious or cultural gathering, need to start with such the qualitative study. having a predetermined program, implementation, evaluation, and upgrade of the strategies in preventing and responding to communicable diseases requires the proper recognition of the contextual factors and challenges, experiences and perception of healthcare providers and decision-makers of the key agencies involved. the authors carried out a qualitative study with conventional content analysis approach about the arbaeen ceremony, a religious mass gathering in iran and iraq and found four main themes: -health infrastructure defects in iraq, poor control of the causative factors of infectious diseases, -the low perception of risk in pilgrims, and ineffectiveness of health education. emerging of all four themes has been verified by the participants' key: cord- -tl qtz x authors: jost, ferdinand; peter, pascal; weickert, joachim title: compressing flow fields with edge-aware homogeneous diffusion inpainting date: - - journal: nan doi: nan sha: doc_id: cord_uid: tl qtz x in spite of the fact that efficient compression methods for dense two-dimensional flow fields would be very useful for modern video codecs, hardly any research has been performed in this area so far. our paper addresses this problem by proposing the first lossy diffusion-based codec for this purpose. it keeps only a few flow vectors on a coarse grid. additionally stored edge locations ensure the accurate representation of discontinuities. in the decoding step, the missing information is recovered by homogeneous diffusion inpainting that incorporates the stored edges as reflecting boundary conditions. in spite of the simple nature of this codec, our experiments show that it achieves remarkable quality for compression ratios up to : . motion estimation has many practical applications such as traffic surveillance, object tracking in driver assistance systems and robotics, or prediction in video compression. especially in video compression many scientific contributions to this problem have been made. this is due to an important trade-off: on one hand the stored motion fields should be as accurate as possible for a good prediction, on the other hand they have to be represented compactly. therefore, it is crucial for this application to encode motion fields efficiently and accurately. most modern video codecs like hevc [ ] use block matching algorithms to compute motion vectors for coarse blocks of pixels. the resulting piecewise constant motion fields can be encoded very efficiently, but introduce block artefacts. alternatively, optical flow methods have been used to compute dense flow fields [ ] , [ ] . these describe the motion between frames more accurately, but are harder to encode efficiently. accurate flow fields of natural image sequences are usually piecewise smooth. compression methods with diffusion-based interpolation work well for this kind of data. in contrast to classical transform-based codecs such as jpeg [ ] or bpg (better portable graphics) [ ] they exploit sparsity in the spatial domain instead of a transform domain. they only store a few selected pixels and reconstruct missing data by interpolation, also called inpainting. especially specialized approaches that use additional edge information excel for piecewise smooth images [ ] , [ ] . we present a framework for the compression of flow fields based on edge-aware homogeneous diffusion inpainting. our method benefits from the piecewise smooth structure of motion fields by storing additional edge information for the inpainting process. we propose an example method with established, easy to implement components to show that our framework can achieve favourable quality in this setting. our codec relies on three major components for encoding: edge detection, selection of mask pixels, and quantisation. we use the marr-hildreth edge detector [ ] together with hysteresis thresholding to detect boundaries between smooth regions of the flow field. this gives us connected edges that we can store efficiently with chain codes. as our framework uses inpainting for the reconstruction, we additionally have to select mask pixels with known flow vectors. we choose a regular grid that allows us to encode the position of mask pixels very efficiently. additionally, our codec quantises the flow values of stored mask pixels with a simple uniform quantisation. the decoder reconstructs the missing data using homogeneous diffusion inpainting that incorporates edge structures. compared to classical homogeneous diffusion, our newly proposed edge-aware operator ensures the preservation of discontinuities in the motion field. compression methods with diffusion-based inpainting were introduced by galić et al. [ ] in . their method stores only a few selected pixels of an image and reconstructs missing data with edge-enhancing anisotropic diffusion [ ] . the r-eed algorithm of schmaltz et al. [ ] improves this idea with an efficient tree structure to adaptively encode mask pixels and can beat the quality of jpeg . the concept of diffusion-based inpainting has also been extended to video compression. peter et al. [ ] developed a method based on r-eed that allows decoding in real time. however, this approach compresses each frame individually and does not exploit temporal redundancies. andris et al. [ ] proposed a proof-of-concept video codec that additionally uses optical flow methods for inter frame prediction. the motion fields are compressed with a simple subsampling, resulting again in block artefacts. ottaviano and kohli [ ] developed a motion estimation algorithm that incorporates coding costs for a wavelet-based compression of the resulting flow field. our method is related to codecs for the compression of depth maps, as they also have a piecewise smooth structure. the approach of gautier et al. [ ] stores mask pixels on both sides of edges and uses homogeneous diffusion inpainting to reconstruct smooth regions in-between. a similar approach by mainberger et al. [ ] for cartoon-like images also selects mask pixels along edges. hoffmann et al. [ ] extended this idea by explicitly storing segment boundaries with chain codes and selecting mask pixels on a hexagonal grid. in section ii we introduce an edge-aware inpainting operator based on homogeneous diffusion. using this concept we propose the encoding step of our method in section iii and the corresponding decoding in section iv. we compare our approach with jpeg and bpg in section v and present our conclusions in section vi. the centrepiece of our codec is the edge-aware homogeneous diffusion that is used to reconstruct a flow field with only a small amount of known data. this method relies on additional edge information to preserve discontinuities of the original flow field. let us consider a flow field f (x) : Ω → r where x := (x, y) denotes the position in a rectangular domain Ω ⊂ r . we assume that flow vectors are only known at mask points k ⊂ Ω. furthermore, we assume that a set of edges e of f is known. the reconstructed flow field u(x) can then be computed using homogeneous diffusion [ ] by solving the laplace equation with reflecting boundary conditions where n denotes the normal vector to the boundary ∂Ω. the values of known mask points are not altered: as an extension to classical homogeneous diffusion we prevent any diffusion across the edge set e by introducing additional reflecting boundary conditions across the boundaries of e: this problem can be discretised with finite differences where edges are given at between-pixel locations. we solve the resulting linear system of equations with a conjugate gradient solver [ ] . the result of this edge-aware homogeneous diffusion inpainting gives us a flow field with discontinuities along edges e and smooth transitions in between. in contrast to the segment-based homogeneous diffusion of hoffmann et al. [ ] , our method allows arbitrary edge structures and does not require a segmentation with closed contours. in this section we describe the encoder of our framework. it gathers and stores all information for the proposed edge-aware homogeneous diffusion inpainting. as a first step our framework collects edge information at between-pixel locations that act as boundaries for the homogeneous diffusion process. then it selects the pixel mask needed for inpainting and quantises the selected pixel values for a more compact representation. our approach uses the marr-hildreth operator combined with hysteresis thresholding similar to canny [ ] as an edge detector. this method aims to detect zero-crossings of the laplacian of a gaussian-smoothed image with a standard deviation of σ. as the laplacian is a second order derivative operator, zero-crossings indicate extrema in the gradient. we apply an additional hysteresis thresholding on the gradient magnitude of all edges detected by the marr-hildreth operator to keep only important structures. edges where the gradient magnitude exceeds a threshold t become seed points. we then recursively add all candidates that are adjacent to seed pixels and exceed a threshold t < t to the set of edges. for high quality flow fields with sharp discontinuities this method gives well-localised and connected edges. the resulting relevant edges in xand y-direction lie at between-pixel locations on two different grids. this gives us two binary images to encode (fig. ). an alternative to storing edges as binary images are chain codes as used by hoffmann et al. [ ] . these are very efficient in our setting as there are only three possible directions to follow. we first extract different kinds of t-junctions as starting points for our chains (fig. ) . as there can also be isolated edges without such t-junctions, we add two other types of starting elements to cover remaining edges (types and ). for each starting element we have to store a reference point and a type. we then obtain the chain code by following the contours from starting elements until there is no more edge to encode. it is a sequence of four different symbols: three symbols for each possible direction and a terminating symbol indicating the end of an edge. our edge-aware homogeneous inpainting algorithm also requires a set of mask points to be able to reconstruct a flow field. to reduce the coding cost of our method we choose mask pixels on a regular grid instead of arbitrary positions. mask positions are uniquely determined by the image dimensions and a density parameter d. adaptive approaches like the rectangular subdivision of schmaltz et al. [ ] usually give better results for diffusion processes. they accumulate mask pixels around edges to compensate the inability of homogeneous diffusion to preserve edges. our codec, however, already stores edge information explicitly. this way adaptive schemes do not provide a large benefit over a regular mask. in this case the small improvement in quality that these methods provide cannot compensate for the overhead of storing a more complex mask. as edges can form isolated segments that do not contain a point of the regular grid, we additionally store the average flow value of such segments. this is equivalent to storing a single mask pixel at an arbitrary position in this segment. we further reduce the amount of stored data by quantising the values of mask pixels. flow vectors are usually given as two -bit floating-point numbers. this representation is not very convenient for compression purposes, as optical flow fields do not use the entire range of possible values. we want to represent the range of actually occurring flow values as integers q ∈ { , ..., k − }. one of the simplest methods to achieve this is uniform quantisation. this allows us to quantise each channel of the flow field individually. let min be the minimal and max the maximal value occurring in one channel of the flow field, and let x ∈ [min, max] be a flow value. we also define the length of a quantisation interval as a := max−min k− . the quantised value q can then be computed as in order to reconstruct a flow value x q we can simply compute x q = min+a·q. note that the first and last interval only have a width of a . this is necessary to ensure that the values of min and max are preserved. as we have to store the minimal and maximal value of each channel explicitly, this ensures that the range of values does not shrink after multiple quantisations. after the quantisation step we have all data necessary to represent a flow field. our file structure consists of a header and a data part: the header contains the image dimensions, the density d needed to reconstruct the inpainting mask, the quantisation parameter k, and the minimum and maximum flow values for each channel. we also have to store the size of all starting elements and the chain code to be able to split the different types of data. the data part of our encoded file contains the edge information in form of starting elements and chain codes, and the quantised flow values. as we have a high spacial correlation for this type of image, we store flow values channel wise. the entire file is then entropy encoded using lpaq [ ] . the decoding step of our codec is a straightforward process. we first read the header information and split the data into t-junctions, chain codes, and flow values. the header information then allows us to reconstruct the regular inpainting mask. next we follow the chain codes starting at t-junctions in each direction until we encounter a terminating symbol to reconstruct edges. finally, we restore flow vectors from the stored quantised values and place them on their corresponding mask positions. the flow field is then reconstructed by solving the edge-aware homogeneous diffusion inpainting proposed in section ii. we use a conjugate gradient solver with a relative residual norm decay of − as a stopping criterion. in this section we show the potential of our method by comparing it to jpeg and bpg, an adaption of hevc's intra-coding mode for the compression of still images [ ] . as both methods are not designed to handle float-valued pixel data, we quantise the input flow fields to integer values in the range { , ..., } with the uniform quantisation described in section iii. we also use these quantised flow fields as input to our method. as an implementation of jpeg we choose kakadu [ ] and use the original implementation of fabrice bellard [ ] for bpg. we select two ground truth flow fields of the mpi sintel flow dataset [ ] as test images. this provides us with high quality flow fields with realistic complexity. we choose a simple flow field of the scene alley and a more complex one of the scene market shown in fig. . to optimise the parameters of our approach we perform a simple grid search. we fix the standard deviation σ in the edge detection step to . as this gives good results for sharp edges. fig. shows the results for jpeg , bpg, and our edgeaware approach for a compression ratio of : . note that the compression ratio is computed relative to the quantised input flow fields. transform-based codecs like jpeg have problems preserving sharp edges. this results in unpleasant wave-like artefacts around object boundaries. bpg performs much better than jpeg in preserving edges, but still blurs boundaries slightly. in contrast, our edge-aware homogeneous diffusion inpainting reconstructs all important edges very well. in a quantitative comparison we measure the peak-signal-tonoise ratio (psnr) of the different approaches. fig. shows that our method outperforms both jpeg and bpg for almost all compression ratios. vi. conclusions and outlook our new framework for flow field compression combines homogeneous diffusion with explicitly stored edge data. a concrete implementation with straightforward edge detection achieves a remarkable quality and consistently outperforms state-of-the-art competitors such as jpeg and bpg. this shows the significant potential of inpainting-based compression for motion data. the modular nature of our framework allows to adapt and improve our method by simply changing the different components. for example, more advanced edge detection or even segmentation might improve the quality of our method for flow fields with blurry edges. future work should evaluate such methods for actual video compression. to this end the residual signal of hybrid video coding should be further analysed. in this context it is also interesting to investigate optical flow methods that give suitable results for edge-aware approaches 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comparison of jpeg , bpg, and our edge-aware approach for two flow fields and a compression ratio of : . both jpeg and bpg show artefacts around edges compression ratio (x : ) psnr (db) compression ratio (x : ) psnr (db) comparison of our method with jpeg and bpg in terms of psnr (higher is better). our codec outperforms jpeg and bpg consistently on both alley (left) and market a proof-of-concept framework for pde-based video compression compressible motion fields efficient depth map compression based on lossless edge coding and diffusion picture coding symposium basic theory on normalization of pattern (in case of typical one-dimensional pattern) iterative methods for sparse linear systems a computational approach to edge detection adaptive weighing of context models for lossless data compression performance analysis of hevc-based intra coding for still image compression kakadu jpeg a naturalistic open source movie for optical flow evaluation key: cord- -ent vu z authors: tan, joshua; sack, brandon k; oyen, david; zenklusen, isabelle; piccoli, luca; barbieri, sonia; foglierini, mathilde; fregni, chiara silacci; marcandalli, jessica; jongo, said; abdulla, salim; perez, laurent; corradin, giampietro; varani, luca; sallusto, federica; sim, b kim lee; hoffman, stephen l; kappe, stefan h i; daubenberger, claudia; wilson, ian a; lanzavecchia, antonio title: a public antibody lineage that potently inhibits malaria infection by dual binding to the circumsporozoite protein date: - - journal: nat med doi: . /nm. sha: doc_id: cord_uid: ent vu z immunization with attenuated plasmodium falciparum sporozoites (pfspz) has been shown to be protective, but the features of the antibody response induced by this treatment remain unclear. to investigate this response at high resolution, we isolated igm and igg monoclonal antibodies from tanzanian volunteers who were immunized by repeated injection of irradiated pfspz and who were found to be protected from controlled human malaria infection (chmi) with infectious homologous pfspz. all igg monoclonals isolated bound to p. falciparum circumsporozoite protein (pfcsp) and recognized distinct epitopes in the n-terminus, nanp repeat region, and c-terminus. strikingly, the most effective antibodies, as assessed in a humanized mouse model, bound not only to the repeat region, but also to a minimal peptide at the pfcsp n-terminal junction that is not in the rts,s vaccine. these dual-specific antibodies were isolated from different donors and used vh - or vh - alleles carrying tryptophan or arginine at position . using structural and mutational data, we describe the elements required for germline recognition and affinity maturation. our study provides potent neutralizing antibodies and relevant information for lineage-targeted vaccine design and immunization strategies. investigate this response at high resolution, we isolated igm and igg monoclonal antibodies from tanzanian volunteers who were immunized by repeated injection of irradiated pfspz and who were found to be protected from controlled human malaria infection (chmi) with infectious homologous pfspz. all igg monoclonals isolated bound to p. falciparum circumsporozoite protein (pfcsp) and recognized distinct epitopes in the n-terminus, nanp repeat region, and cterminus. strikingly, the most effective antibodies, as assessed in a humanized mouse model, bound not only to the repeat region, but also to a minimal peptide at the pfcsp n-terminal junction that is not in the rts,s vaccine. these dual-specific antibodies were isolated from different donors and used vh - or vh - alleles carrying tryptophan or arginine at position . using structural and mutational data, we describe the elements required for germline recognition and affinity maturation. our study provides potent neutralizing antibodies and relevant information for lineage-targeted vaccine design and immunization strategies. malaria is a serious global health threat, causing , deaths and million clinical cases in . much of the effort to develop a vaccine against the disease has focused on plasmodium falciparum sporozoites (pfspz), the asymptomatic parasite stage that is injected by mosquitoes into the host skin to initiate a malaria infection. after entering the skin, pfspz migrate to the liver, multiply in hepatocytes and then emerge in the blood, where the parasites cause malaria symptoms and differentiate into sexual stages for transmission. while natural infection by pfspz elicits little or no protective immunity to this stage of the life cycle , , subunit or whole organism vaccines based on pfspz can induce robust immune responses [ ] [ ] [ ] . the most advanced malaria vaccine candidate, rts,s, incorporates part of the p. falciparum circumsporozoite protein (pfcsp), which coats the pfspz surface and plays a key role in parasite migration out of the skin, entry into the liver parenchyma and invasion of hepatocytes [ ] [ ] [ ] [ ] [ ] [ ] . multi-site clinical trials in sub-saharan africa have shown that rts,s confers significant but modest and short-lived protection against clinical illness , , . an alternative approach that has shown promise is the use of whole attenuated pfspz as immunogens. this line of research is based on key early discoveries that immunization with irradiated p. berghei sporozoites protected mice against subsequent challenge and that immunization of humans with > irradiated mosquitoes carrying pfspz conferred sterilizing protection against controlled human malaria infection (chmi) [ ] [ ] [ ] . these studies have led to efforts to develop whole attenuated pfspz as a vaccine , and recent trials have shown that immunization with attenuated pfspz was highly protective in malaria-naïve volunteers and gave significant protection in malian adults , [ ] [ ] [ ] . while these results are promising, the specific mediators of this protective immune response have yet to be fully elucidated. studies of the antibody response have mainly investigated polyclonal serum responses to pfspz and pfcsp , [ ] [ ] [ ] , and highresolution analysis of the monoclonal antibodies generated by vaccination and their target antigens on the pfspz surface remains to be performed. these experiments could provide useful information for the improvement of whole sporozoite-based vaccines and for the identification of new antigens as subunit vaccine candidates, as well as to generate tools for prophylaxis of p. falciparum infection. we characterized the antibody response of tanzanian volunteers living in malaria-endemic regions who were immunized by repeated intravenous injection of irradiated pfspz (pfspz vaccine) and then underwent chmi with live homologous parasites (fig. a) . serum igm and igg antibodies, as measured by flow cytometry on live pfspz, increased following immunization, but did not show a clear association with protection from chmi (fig. b,c and supplementary fig. a,b) . memory b cells from five protected individuals were immortalized and screened by staining of intact pfspz to isolate human monoclonal antibodies against any surface antigen on pfspz ( supplementary fig. c) . most of the igg monoclonal antibodies bound to pfspz with high affinity (fig. d,e) . interestingly, in the two donors from whom both pfspz-specific igm and igg monoclonal antibodies were isolated, the igm antibodies were recovered at much higher numbers (fig. f ). the igm antibodies had fewer, but still substantial, mutations compared to the igg antibodies, consistent with an origin from igm memory b cells (fig. g ). in particular, the finding of an antibody lineage containing both igm and igg members suggests an incomplete switch in this response despite repeated immunization ( supplementary fig. d ). these results demonstrate that immunizations with pfspz vaccine induce a robust antibody response that retains a significant igm component. to identify the features of the most effective neutralizing monoclonal antibodies produced by the protected individuals, we tested a panel of igg antibodies in vitro for their capacity to inhibit pfspz traversal and invasion of a human hepatocyte cell line (fig. a) . the invasioninhibitory activity varied among antibodies and was significantly correlated with binding affinity to pfspz (fig. b) . a subset of antibodies was further tested in an in vivo mouse humanized liver model for their capacity to protect against natural, mosquito bitetransmitted infection by pfspz. some antibodies, such as mgg , mgg , mgh , mgu and mgu , were very potent in reducing liver burden by up to . %, while others, such as mgg , mgh , mgh and mgu , were less effective (fig. c) . these findings suggest that in vivo neutralizing activity may be related to the fine specificity of the antibodies. next, we set out to identify the target antigens of the monoclonal antibodies. strikingly, although we had used an antigen-agnostic approach to identify antibodies that bound to the pfspz surface regardless of specificity, we found that all of the antibodies bound to recombinant pfcsp (fig. d,e) , confirming that this is the most immunogenic protein on the pfspz surface [ ] [ ] [ ] . to understand the basis for effective neutralization, we mapped the specificity of the monoclonal antibodies using synthetic peptides that cover the n-terminus, the nanp repeat region, the n-terminal junction (connecting region between the n-terminal domain and nanp repeats), and the c-terminus of pfcsp. binding to the classical nanp repeats (nanp peptide), the pfcsp c-terminus, recombinant pfcsp or pfspz did not correlate with efficacy ( supplementary fig. a-d) . interestingly, however, binding to a minimal -mer peptide (npdp ) that covers the junction between the n-terminal domain and the nanp repeats was a shared characteristic of the most potent in vivo neutralizing antibodies (fig. f) . these potent antibodies also recognized the nanp peptide, suggesting that the capacity to bind both to the nanp repeats and to the n-terminal junction of pfcsp is the main feature of efficient neutralization. another distinctive characteristic of the most potent antibodies was the common usage of vh - family alleles carrying tryptophan or arginine at position (vh - f , here defined as including vh - , vh - - , vh - - and vh - alleles sharing > % identity) (fig. e , supplementary fig. , , supplementary table ). strikingly, vh - f was the most common vh gene used by igm antibodies isolated from donors g and u, with almost % of such antibodies carrying w or r ( supplementary fig. e ). collectively, these data indicate that the most potent antibodies have a dual specificity and share common vh gene usage. importantly, such antibodies were isolated from four out of five donors, suggesting that these antibodies belong to a public lineage and therefore have the potential to be readily induced by vaccination. to investigate the influence of somatic mutations on binding of vh - f antibodies to pfcsp, we focused on two clonally related and highly mutated antibodies, mgu and mgu (fig. a) . the unmutated common ancestor (uca) of these antibodies, which carries tryptophan at position , was able to bind to pfcsp and pfspz with low affinity, while substitution to serine , which is commonly found in vh - alleles, resulted in loss of binding (fig. b,c) . these findings, in conjunction with the high frequency of w in the igm sequences and the identification of putative vh - f alleles carrying w in the germline sequences obtained from non-b cells of donors g, u and w ( supplementary fig. , ) , identify vh - f alleles carrying w as a preferred feature for the initiation of this lineage-specific antibody response to pfcsp. the branch point of this clone achieved, through several mutations, high affinity binding to pfspz, pfcsp and nanp , while further mutations in mgu , but not mgu , increased breadth by conferring the unique ability to bind to npdp (fig. b-e, supplementary fig. ). despite their similarities in binding to pfspz, pfcsp and nanp , mgu was substantially more potent than mgu in the in vivo assay (fig. c) , suggesting that acquisition of binding to npdp is the key factor for potent neutralization. interestingly, mutagenesis studies of mgu suggest that w remains a critical residue for binding to npdp , but becomes dispensable for high affinity binding to nanp and full-length pfcsp in the fully mutated antibody (fig. f ). in contrast, in a second clonal family consisting of mgu and mgu , full binding to pfcsp, nanp , npdp and pfspz was already achieved by the branch point while the remaining mutations appeared redundant ( supplementary fig. a -e). to investigate the original specificities of germline vh - f antibodies, we analysed binding of the ucas of various vh - f antibodies to pfcsp peptides using a more sensitive beadbased assay. most ucas bound to nanp but not to npdp , suggesting that the antibodies generally started as nanp-specific and gained affinity for npdp through somatic mutations ( supplementary fig. f-m) . these data delineate a pathway of antibody development that is dependent on specific vh alleles and leads to antibodies with dual specificity. to identify the minimal residues recognized by antibodies at the pfcsp nterminal junction, we performed a mutational analysis on npdp , a -mer version of npdp that was used to provide a longer scaffold for binding (fig. g) . the loss of binding to certain peptides identified a specific motif (dpnanp) that was recognized by most antibodies regardless of vh gene usage. these findings, combined with data from peptide array experiments ( supplementary fig. ) , identify the n-terminal junction binding site of the most potent neutralizing antibodies as including the first unit of the nanp repeat region and flanking non-repeat sequences, providing a molecular basis for the dual specificity of these antibodies. to gain structural insights into the recognition of the n-terminal junction, we attempted to crystallize several vh - f antibodies and successfully crystallized mgg in complex with the ac- kqpadgnpdpnanp -nh peptide ( fig. a and supplementary table ) . only the c-terminal half (npdpnan, residues - ) of the peptide was visible, with most of the contacts being made by heavy-chain residues as shown by the fab buried surface area (fig. b) . specifically, the heavy-chain cdr loops form a groove in which the peptide resides ( fig. a and supplementary fig. a ). in addition, three interfacial waters are involved in an extensive hydrogen-bonding network, connecting the side chain of n to the base of cdr h and cdr h ( supplementary fig. b ). the ch/π interaction between p and w ( fig. a and supplementary fig. b ) confirms that the latter residue is critical for binding as indicated by the mutagenesis experiments. while most peptide residues, except for n , display a relatively large buried surface area (fig. c) , weak electron density for the residues visible at the peptide's termini (n , p , a and n ) ( fig. e and supplementary fig. c ) indicates structural flexibility and highlights the sequence dpn as the principal binding motif. interestingly, the dpn sequence displays a pseudo turn, which is stabilized by hydrogen bonding of the aspartate side chain to the asparagine backbone amide. such a conformation is very similar to turns observed for unbound nanp peptides in solution and in crystal structures of free and antibody-bound peptides (fig. d) [ ] [ ] [ ] [ ] [ ] . an isolated dpn motif is also present in the - c-terminal peptide, which may explain its binding to mgg (fig. e) , notwithstanding the significant differences between the dpn flanking residues in the - c-terminal and the n-terminal junctional peptides (ndpnr versus pdpna, respectively). binding to both npdp and nanp repeats suggests that the aspartate residue in the dpn motif is interchangeable with an asparagine. binding studies of mgg to npdp peptide mutants validate the specificity of mgg toward dpn and npn motifs (fig. g) . only when the central dpn and npn motifs within the npdp peptide are mutated to dpa and aan is mgg binding completely abrogated. in contrast, a third dpn motif present at the c-terminus of the npdp peptide does not contribute to binding, possibly indicating the importance of flanking residues for optimal binding. overall, these data imply that potential peptide-binding promiscuity allows mgg to bind to diverse epitopes on pfcsp. since other vh - f antibodies cannot bind to the - peptide, we expect that they will be less promiscuous and bind slightly larger or more specific sequences. the finding that the most potent monoclonal antibodies recognize a defined n-terminal junctional peptide suggests that this region could be a component of an effective subunit vaccine. in an initial attempt to investigate whether the npdp peptide might be sufficient to induce a protective response, we immunized balb/c mice with npdp conjugated to klh. all mice produced igg antibodies that were specific for the npdp peptide, but were at best weakly reactive for the nanp peptide ( supplementary fig. a,b) . strikingly, in spite of their ability to bind to pfspz, the mouse sera were unable to inhibit pfspz invasion of a hepatocyte cell line in vitro, suggesting that dual specificity for nanp and the n-terminal junction may be required for potent neutralizing function ( supplementary fig. c,d) . the reliance of the dual-specific antibodies that we isolated on specific human vh - f alleles suggests that mice, which do not have the counterparts of human vh - f genes, may not be the most suitable model organism to test a novel csp-based vaccine. rather, an organism such as the aotus monkey, which has a more similar vh gene repertoire to humans and contains vh - f -like genes carrying the equivalent of w , may be a more suitable choice. this study shows that the antibodies produced by vaccinated and protected african individuals contain a highly mutated igg component, as well as an important igm component with fewer but substantial mutations, as also seen in the response to blood-stage plasmodium antigens . the large igm component would be consistent with stimulation of marginal zone b cells in the spleen following intravenous immunization with a particulate antigen . strikingly, all of the igg antibodies that we identified recognized pfcsp, consistent with previous studies describing the immunodominance of this protein and its abundance on the pfspz surface [ ] [ ] [ ] , . our findings are in agreement with previous work that separately describes the importance of the pfcsp nanp repeat region and of the n-terminus , - , but, importantly, highlight the fact that antibodies that target epitopes in both regions simultaneously are more potent than antibodies that exclusively recognize each individual site. interestingly, the structural analysis and peptide mutational data indicate that these antibodies, which are originally specific for nanp motifs, do not acquire a completely unrelated specificity, but rather gain promiscuity for sequences centred on a dpn motif. this dual specificity is to a large extent encoded by vh - f alleles, but also requires extensive somatic mutations. the importance of dual specificity is highlighted by the fact that immunization with a single npdp peptide is not sufficient to confer protection despite eliciting pfspz-binding antibodies. the increased potency of the dual-specific antibodies could be due to the proximity of the n-terminal junction to region i (klkqp) of pfcsp, which is involved in the cleavage of the n-terminus to allow pfspz invasion of hepatocytes , . this n-terminal junction region is not included in the most advanced malaria vaccine candidate rts,s, which may explain its limited efficacy in malaria-endemic regions . these findings support continued work to develop whole pfspz vaccines, which contain the entire pfcsp, and provide a rationale for further attempts to develop refined vaccination approaches to elicit dual-specific antibodies using prime-boost strategies with improved carriers , . the finding that the most potent antibodies share common vh gene usage in multiple donors is consistent with a public antibody response that can be readily induced by vaccination. these results are reminiscent of previous work on the use of particular vh genes and their allelic forms in the response to the stem of influenza haemagglutinin , and justify further efforts to investigate the role of vh gene polymorphisms in protective antibody responses. nevertheless, whether these antibodies are sufficient to protect humans and why some individuals were not protected remain to be established. possible reasons for the latter include the lack of a vh - f allele, the incomplete maturation of the vh - f antibodies, or insufficient production of the potent antibodies. the antibodies described could be used to obtain proof of concept that antibodies alone can be protective in vivo in humans, as previously shown in mice and non-human primates [ ] [ ] [ ] , and pave the way for the use of antibodies in the prophylaxis of p. falciparum infection and for the development of improved antibody-based subunit malaria vaccines. aseptic, purified cryopreserved pfspz of the nf strain provided by sanaria ® were used in serum and monoclonal antibody binding experiments. pfspz produced by the center for infectious disease research, seattle was used in all in vitro and in vivo functional assays. following informed consent, blood samples used in this study were collected from malaria pre-exposed volunteers during a clinical phase clinical trial of the safety, immunogenicity and protective efficacy of the sanaria ® pfspz vaccine in bagamoyo, tanzania for serum preparation, whole blood was collected in vacutainer tubes (bd) containing clot activators and kept at room temperature until a clot was formed. the tube was centrifuged at , × g for min at °c and the serum fraction was stored at − °c. peripheral blood mononuclear cells (pbmcs) were isolated from whole blood by ficoll density gradient centrifugation and resuspended in freezing medium for long-term storage in liquid nitrogen. cryopreserved pfspz (sanaria ® ) were thawed and stained with different concentrations of tanzanian sera or monoclonal antibodies in . × sybr green i (thermofisher scientific) for min at °c. the pfspz were washed twice by centrifugation at × g for min. human serum antibody binding was detected using . μg ml − alexa fluor -conjugated goat anti-human igg (jackson immunoresearch, - - ) or alexa fluor conjugated goat anti-human igm (jackson immunoresearch, - - ). mouse serum antibody binding was detected using μg ml − pe-cy -conjugated goat anti-mouse igg (biolegend, ) or pe-cy -conjugated rat anti-mouse igm (bd biosciences, ). facs diva (version . ) was used for acquisition of samples and flow-jo (version . ) was used for facs analysis. the pfspz were gated based on high fluorescence in the fitc channel. median fluorescence intensity (mfi) of the pfspz in the alexa fluor or pe-cy channel was calculated to quantify igg or igm binding. the concentration of antibody needed to achieve mfi , (conc ) was calculated by interpolation of binding curves fitted to a sigmoidal curve model (graphpad prism ) as a measure of affinity. the gating strategy can be found in supplementary figure . igm or igg memory b cells were isolated from frozen peripheral blood mononuclear cells (pbmcs) by magnetic cell sorting with . μg ml − anti-cd -pecy antibodies (bd, ) and mouse anti-pe microbeads (miltenyi biotec, - - ), followed by facs sorting using . μg ml − alexa fluor -conjugated goat anti-human igg (jackson immunoresearch, - - ), μg ml − alexa fluor -conjugated goat anti-human igm (invitrogen, a ) and / pe-labeled anti-human igd (bd, ). as previously described , sorted b cells were immortalized with epstein-barr virus (ebv) and plated in single cell cultures in the presence of cpg-dna ( . μg ml − ) and irradiated pbmc-feeder cells. two weeks post-immortalization, the culture supernatants were tested (at a / dilution) for binding to pfspz by flow cytometry using a no-wash protocol. briefly, cryopreserved pfspz were thawed, stained with the supernatants in . × sybr green i for min at room temperature, and incubated with . μg ml − alexa fluor -conjugated goat anti-human igg or anti-human igm for hour at °c. only supernatants that did not bind to control beads were selected to exclude polyreactive antibodies. the ability of monoclonal antibodies and serum to prevent pfspz invasion and traversal in vitro was tested as previously described , . briefly, monoclonal antibodies at μg ml − were mixed with freshly dissected pfgfp_luc sporozoites in dmem media containing fitc-dextran, % fbs, pen/strep, fungizone and l-glutamine and incubated at °c for min. these pfspz were then added to hc hepatoma cells plated one day prior at , cells/well in a -well plate for a final moi of . ( , pfspz: , hc ). plates were then spun at × g for min and the pfspz were left to infect for min at °c. cells were then fixed, stained with μg ml − of the monoclonal antibody a conjugated to alexafluor- and analyzed by flow cytometry for invaded cells ( a /alexafluor- positive) or traversed cells (fitc-dextran positive). frg huhep mice were purchased from yecuris, inc. and infected by bite of pfgfp_luc mosquitos - hours following intraperitoneal injection of μg/mouse of each monoclonal antibody or human igg control as described previously . parasite liver burden was determined by bioluminescent imaging using an ivis imager at day at the peak of liver burden. reductions in liver burden were calculated by normalization to the mean of control mice injected with an equivalent dose of human igg within each bite experiment. all animal procedures were conducted in accordance with and approved by the center for infectious disease research institutional animal care and use committee (iacuc) under protocol sk- . the seattle biomed iacuc adheres to the nih office of laboratory animal welfare standards (olaw welfare assurance # a - ). cdna was synthesized from selected b-cell cultures and both heavy chain and light chain variable regions (vh and vl) were sequenced as previously described . the usage of vh and vl genes and the number of somatic mutations were determined by analyzing the homology of vh and vl sequences of monoclonal antibodies to known human v, d and j genes in the imgt database (version . . ) . antibody-encoding sequences were amplified and sequenced with primers specific for the v and j regions of the given antibody. sequences were aligned with clustal omega (version . . ) . unmutated common ancestor (uca) sequences of the vh and vl were inferred with antigen receptor probabilistic parser (arpp) ua inference software, as previously described , or constructed using imgt/v-quest . phylogenetic trees were generated with the dna maximum likelihood program (dnaml) of the phylip package, version . , . antibody heavy and light chains were cloned into human igg , igκ and igλ expression vectors and expressed by transient transfection of expi f cells (thermofisher scientific) using polyethylenimine. cell lines were routinely tested for mycoplasma contamination. the antibodies were affinity purified by protein a chromatography (ge healthcare). the kqpadgnpdpnanp peptide was ordered from innopep inc. with a purity of > % and containing chlorine counter ions. the peptides have n-terminal acetylation and cterminal amidation to eliminate charges at the peptide termini. the mgg -peptide complex was crystallized from a solution containing mgg at . mg ml − in tbs buffer ( mm tris-hcl, mm nacl, . mm kcl, ph . ) with a : molar ratio of ac-kqpadgnpdpnanp-nh peptide to fab. crystals were grown using sitting drop vapor diffusion with a well solution containing . m kh po , % glycerol, % peg at k and typically appeared within days. crystals were cryo-cooled without additional cryoprotection. x-ray diffraction data were collected at the advanced light source (als) . . . data collection and processing statistics are outlined in supplementary table . data sets were indexed, integrated, and scaled using the hkl- package (version ) . the structures were solved by molecular replacement using phaser (version . . ) with a homology model (swiss-model - and pigspro ) for mgg as a search model. after refinement of the fab using phenix.refine (version . - ) combined with additional manual building cycles in coot (version . . ) , positive fo-fc density was observed in the fab combining site for the peptide. the peptide was manually built into the difference density fo-fc map, followed by additional rounds of refinement of the complex in phenix.refine and manual building cycles in coot . buried surface areas (bsa) were calculated with the program ms (version . ) , using a . -Å probe radius and standard van der waals radii . total iggs were quantified using half-area, high-binding -well plates (corning) with μg ml − goat anti-human igg (southernbiotech, - ) using certified reference material (erms-da , sigma-aldrich) as a standard. to test specific antibody binding, elisa plates were either directly coated with μg ml − of recombinant pfcsp (sanaria ® , sequence previously shown ), μg ml − of peptide - or μg ml − of peptide - , or first with μg ml − of avidin (sigma-aldrich), followed by μg ml − of nanp (nanpnanpnanpnanpna), npdp (kqpadgnpdpnanpnvdpn), npdp (kqpadgnpdpnanpn) or various npdp peptide mutants. non-specific binding to plates coated with an irrelevant control peptide was tested to exclude polyreactivity of the antibodies. all peptides and mutants were synthesized with biotin attached to the c-terminus (a&a labs). plates were blocked with % bovine serum albumin (bsa) and incubated with titrated antibodies, followed by / ap-conjugated goat anti-human igg (southern biotech, - ). plates were then washed, substrate (p-npp, sigma) was added and plates were read at nm. streptavidin beads with different levels of fitc labelling (svfb- - k, spherotech) were coated with μg ml − of biotinylated nanp , npdp , npdp or a negative control peptide for min at room temperature. the beads were washed and incubated with titrations of monoclonal antibodies for min at room temperature. antibody binding was detected with . μg ml − alexa fluor -conjugated goat anti-human igg or anti-human igm. the ucas were compared for binding to nanp and npdp at a concentration of μg ml − (supplementary fig. f ). biotinylated npdp and nanp peptides were diluted ( nm) in hepes buffered saline (hbs) ( mm hepes, ph . , mm nacl, mm edta, . % surfactant . hbs was also used as running buffer. an irrelevant biotinylated -mer peptide was used as a control for non-specific interactions. a neutravidin-immobilized nlc proteon sensor chip (biorad) was pre-conditioned with an nacl solution ( m) and the biotinylated peptides were injected onto the chip. the monoclonal antibodies were diluted and titrated in hbs ( - . - . - . - . nm) and injected onto chip; one channel of the chip was injected with hbs and used as reference for the analysis. all injections were made at a flow rate of μl/min. injection time and dissociation time were s and s, respectively. each binding interaction of the monoclonal antibodies with the biotinylated peptides was assessed using a proteon xpr instrument (biorad) and data were processed with proteon manager software (version . . . ). k a , k d and k d were calculated by applying the langmuir fit model. peptides of -amino acid lengths spanning the entire pfcsp (with a shift of a single amino acid between peptides) were synthesized and coated onto a microarray chip (peppermap® linear epitope mapping, pepperprint gmbh). the peptides were incubated with μg ml − of monoclonal antibodies for h at °c, followed by incubation with dylight conjugated goat anti-human igg to detect antibody binding. female balb/c mice ( - weeks of age) were obtained from envigo laboratories. all procedures were performed in accordance with guidelines by the swiss federal veterinary office and after obtaining ethical approval from the ufficio veterinario cantonale, bellinzona, switzerland (approval number: ). keyhole limpet haemocyanin (klh)conjugated npdp (genscript) was reconstituted in water and formulated with % mf (addavax, invivogen) according to the manufacturer's instructions. mice were immunized subcutaneously with μg of peptide on day and . mice were bled on day . recovered sera were used for staining of pfspz by flow cytometry and for binding to pfcsp and pfcsp peptides by elisa. the number of mutations in the heavy chains of igg (n= antibodies) and igm (n= antibodies) isolated from the tanzanian volunteers were compared by a two-sided t-test. results are shown as mean ± s.d.. in this test, n refers to the number of antibodies, p = . , t = . , df = . a two-tailed spearman's correlation was performed to correlate invasion with binding affinity to pfspz (from n= representative experiment out of ), p = . . in the in vivo test of the monoclonal antibodies, error bars show s.d. and were calculated from n = or mice for each antibody. a one-sided anova with kruskal-wallis post test was used to compare the percentages of liver burden with that of control mice injected with irrelevant human igg; for the anova, p = . , f = . , df = , df = . for the kruskal-wallis post test, the results for each individual antibody are presented as *p≤ . , **p≤ . , ****p< . . a two-tailed spearman's correlation was performed to correlate affinity for npdp , pfcsp, nanp , - or pfspz with in vivo antibody efficacy (from n= representative experiment out of ). p = . , . , . , . and . , respectively. the confidence intervals were not determined by prism as n< for each correlation. in all other cases, n refers to the number of independent experiments. sequence data of the monoclonal antibodies isolated in this study will be deposited in genbank (https://www.ncbi.nlm.nih.gov/genbank/). the x-ray structure factors and coordinates have been deposited in the protein data bank (pdb id bqb). refer to web version on pubmed central for supplementary material. a, binding interface of mgg in complex with the kqpadgnpdpnanp peptide; only residues to of the peptide have interpretable electron density (indicated in bold). the peptide is shown in the cartoon representation with sidechains as sticks, while the heavy and light chains of mgg are shown as dark and light grey surfaces respectively. the cdr loops are in the cartoon representation: cdrh (green), cdrh (blue), cdrh (magenta), cdrl (light green), cdrl (light blue) and cdrl (pink). the w sidechain is shown as blue sticks and the interfacial waters are highlighted as red spheres. b, buried surface area (bsa) for the heavy chain (hc) and light chain (lc) with the peptide. c, bsa for individual peptide residues with the fab. d, pseudo turn for the dpn motif of the bound peptide (yellow carbons) and type i β-turn for the previously published crystal structure of the unbound anpna peptide (green carbons) . stabilizing hydrogen bonds between the sidechain of d /n and the amide backbone of n /n in the two structures are highlighted by the dashed line. e, fo-fc electron density map for the n-terminal peptide contoured at . σ (dark blue) and . σ (light blue). naturally acquired antibodies to sporozoites do not prevent malaria: vaccine development implications an intensive longitudinal cohort study of malian children and adults reveals no evidence of acquired immunity to plasmodium falciparum infection the rts,s malaria vaccine antibody and b cell responses to plasmodium sporozoites protection against malaria at year and immune correlates following pfspz vaccination the basolateral domain of the hepatocyte plasma membrane bears receptors for the circumsporozoite protein of plasmodium falciparum sporozoites malaria circumsporozoite protein binds to heparan sulfate proteoglycans associated with the surface membrane of hepatocytes the plasmodium circumsporozoite protein is proteolytically processed during cell invasion efficacy and safety of rts s/as malaria vaccine with or without a booster dose in infants and children in africa: final results of a phase individually randomised controlled trial heparan sulfate proteoglycans provide a signal to plasmodium sporozoites to stop migrating and productively invade host cells the malaria circumsporozoite protein has two functional domains, each with distinct roles as sporozoites journey from mosquito to mammalian host four-year efficacy of rts,s/as e and its interaction with malaria exposure seven-year efficacy of rts,s/as malaria vaccine among young african children protective immunity produced by the injection of x-irradiated sporozoites of plasmodium berghei immunization of man against sporozoiteinduced falciparum malaria sporozoite induced immunity in man against an ethiopian strain of plasmodium falciparum protection of humans against malaria by immunization with radiationattenuated plasmodium falciparum sporozoites development of a metabolically active, non-replicating sporozoite vaccine to prevent plasmodium falciparum malaria protection against malaria by intravenous immunization with a nonreplicating sporozoite vaccine sterile protection against human malaria by chemoattenuated pfspz vaccine safety and efficacy of pfspz vaccine against plasmodium falciparum via direct venous inoculation in healthy malaria-exposed adults in mali: a randomised, double-blind phase trial rationale for development of a synthetic vaccine against plasmodium falciparum malaria the circumsporozoite protein is an immunodominant protective antigen in irradiated sporozoites natural parasite exposure induces protective human anti-malarial antibodies conformational preferences of synthetic peptides derived from the immunodominant site of the circumsporozoite protein of plasmodium falciparum by h nmr crystal structure of an npna-repeat motif from the circumsporozoite protein of the malaria parasite plasmodium falciparum structural basis for antibody recognition of the nanp repeats in plasmodium falciparum circumsporozoite protein t-dependent b cell responses to plasmodium induce antibodies that form a highavidity multivalent complex with the circumsporozoite protein identification of five different ighv gene families in owl monkeys (aotus nancymaae) somatically hypermutated plasmodium-specific igm + memory b cells are rapid, plastic, early responders upon malaria rechallenge human marginal zone b cells total and putative surface proteomics of malaria parasite salivary gland sporozoites interrogating the plasmodium sporozoite surface: identification of surfaceexposed proteins and demonstration of glycosylation on csp and trap by mass spectrometrybased proteomics an immunologically cryptic epitope of plasmodium falciparum circumsporozoite protein facilitates liver cell recognition and induces protective antibodies that block liver cell invasion the n-terminal domain of plasmodium falciparum circumsporozoite protein represents a target of protective immunity proteolytic cleavage of the plasmodium falciparum circumsporozoite protein is a target of protective antibodies versatile virus-like particle carrier for epitope based vaccines design of a hyperstable -subunit protein icosahedron rapid development of broadly influenza neutralizing antibodies through redundant mutations vaccine-induced antibodies that neutralize group and group influenza a viruses monoclonal, but not polyclonal, antibodies protect against plasmodium yoelii sporozoites inability of malaria vaccine to induce antibodies to a protective epitope within its sequence humoral protection against mosquito bite-transmitted plasmodium falciparum infection in humanized mice synthesis and immunological characterization of -mer and -mer peptides corresponding to the n-and c-terminal regions of the plasmodium falciparum cs protein molprobity: all-atom structure validation for macromolecular crystallography an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus development of a quantitative flow cytometrybased assay to assess infection by plasmodium falciparum sporozoites efficient generation of monoclonal antibodies from single human b cells by single cell rt-pcr and expression vector cloning imgt, the international immunogenetics information system a new bioinformatics analysis tools framework at embl-ebi reconstructing a b-cell clonal lineage. i. statistical inference of unobserved ancestors co-evolution of a broadly neutralizing hiv- antibody and founder virus processing of x-ray diffraction data collected in oscillation mode phaser crystallographic software swiss-model: modelling protein tertiary and quaternary structure using evolutionary information protein structure homology modeling using swiss-model workspace the swiss-model workspace: a web-based environment for protein structure homology modelling pigspro: prediction of immunoglobulin structures v phenix: a comprehensive python-based system for macromolecular structure solution features and development of coot the molecular surface package side-chain torsional potentials: effect of dipeptide, protein, and solvent environment we would like to thank m. nussenzweig (rockefeller university) and h. wardemann (german cancer research center) for providing reagents for antibody cloning and expression. this work was supported in part by the swiss the authors would like to thank first and foremost the study volunteers for their participation in the study. we also thank the entire study team at the bagamoyo branch of the ifakara health institute and the manufacturing, quality control, regulatory and clinical teams at sanaria, inc. for their contributions to the conduct of the trial. we would like to thank prof. marcel tanner (former director of the swiss tropical and public health institute, basel) for his vision and support of the development of the clinical trial platform enabling whole sporozoite-based malaria vaccine trials in bagamoyo, tanzania. key: cord- -vox xsgd authors: deng, xufang; baker, susan c title: an “old” protein with a new story: coronavirus endoribonuclease is important for evading host antiviral defenses date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: vox xsgd here we review the evolving story of the coronavirus endoribonuclease (endou). coronavirus endou is encoded within the sequence of nonstructural protein (nsp) , which was initially identified as a component of the viral replication complex. biochemical and structural studies revealed the enzymatic nature of nsp /endou, which was postulated to be essential for the unique replication cycle of viruses in the order nidovirales. however, the role of nsp in coronavirus replication was enigmatic as endou-deficient coronaviruses were viable and replicated to near wild-type virus levels in fibroblast cells. a breakthrough in our understanding of the role of endou was revealed in recent studies, which showed that endou mediates the evasion of viral double-stranded rna recognition by host sensors in macrophages. this new discovery of nsp /endou function leads to new opportunities for investigating how a viral endou contributes to pathogenesis and exploiting this enzyme for therapeutics and vaccine design against pathogenic coronaviruses. we provide a brief outline of the major research findings related to coronavirus (cov) endoribonucleases (endou) in table . in the text below, we describe the experimental approaches that led to these findings and compare the activity of cov endou with reports of other viral and host ribonucleases. initial studies focused on identifying the products of the cov replicase polyprotein, pp ab (depicted in fig. a ). heusipp et al. used a murine monoclonal antibody ( g ) that recognizes amino acid residues - of human cov e pp ab (heusipp et al., ) . they identified a -kda polypeptide in virus-infected cells and found that this polypeptide was a product of the viral c-like proteasemediated cleavage of pp ab. this protein localizes to the perinuclear region, as detected by immunofluorescence assay, similar to other pp ab-derived polypeptides (heusipp et al., ) . shi and coworkers obtained similar findings while studying mouse hepatitis virus (mhv) (shi et al., ) . they generated a rabbit antiserum against amino acids - of mhv pp ab and found that this antiserum detected a -kda product in infected cells. they also found that this protein colocalized with de novo synthesized viral rna, and therefore postulated that this viral protein associated with the viral rna replication/ transcription machinery (shi et al., ) . later, the corresponding polypeptides that these antibodies recognized were defined as the th cleavage product of pp ab (called nsp ), counting from the aminoterminus to the carboxyl terminus of pp ab (ziebuhr et al., ; snijder et al., ) . after the outbreak of severe acute respiratory syndrome (sars) in [ ] [ ] , and once a cov had been confirmed as the etiological agent of sars, researchers intensively scrutinized cov genomic sequences to better understand this novel human pathogen. by comparative genomic characterization of cov replicases, snijder et al. reported that the c-terminus of nsp has high sequence similarity to the xenopus laevis poly(u)-specific endoribonuclease and therefore predicted that nsp possesses endou activity (snijder et al., ) . based on the available sequence information of viruses from the order nidovirales at that time, the endou was considered a nidovirus-specific marker (called nendou) (fig. b) (snijder et al., ) . the members of the family arteriviridae, including equine arteritis virus (eav) and porcine respiratory and reproductive syndrome virus (prrsv), also have endou domains within nsp . however, it was later discovered that the presence of the endou domain is not universal in all nidoviruses. nam dinh virus, the first insect nidovirus belonging to the family mesoniviridae, and roniviruses that infect invertebrates, do not encode an endou domain (nga et al., ; lauber et al., ) . these findings suggest that the endou domain may only serve as a signature for vertebrate nidoviruses, including covs and arteriviruses (fig. ). previous reviews have comprehensively summarized the biochemical and structural features of nidovirus ribonucleases (ulferts and ziebuhr, ; snijder et al., ) . here, we highlight the key experiments with respect to nidovirus endou and provide recent updates. studies performed by ivanov et al. ( ) and bhardwaj et al. ( ) first demonstrated the endou activity of sars-cov nsp in vitro (ivanov et al., ; bhardwaj et al., ) . the wild-type (wt) nsp and its mutants with alanine (ala) substitutions of the putative catalytic residues were expressed in e. coli. the recombinant nsp -wt, but not the mutants, could efficiently cleave single-stranded (ss) and doublestranded (ds) rnas. in contrast, neither ssdna nor dsdna molecules could be processed by nsp , demonstrating its predicted ribonuclease-as opposed to deoxyribonuclease-activity. blocking either the ′ or the ′ terminus of the rna substrates by covalent modifications with fluorescein or puromycin, respectively, had no effect on the rna cleavage (bhardwaj et al., ) , confirming that nsp is an endorather than an exoribonuclease. similar endou activity was detected in other cov nsp s, including human cov e, mhv, infectious bronchitis virus (ibv), and turkey cov (ivanov et al., ; bhardwaj et al., ; cao et al., ) , and in the nsp s of arteriviruses eav and prrsv (nedialkova et al., ) . hexamerization of nsp is critical for its endou activity. guarino and coworkers found that cov nsp could be present in solution as either monomers or hexamers in a protein concentration-dependent manner. the hexamer is the fully active form of endou that binds rna and executes optimal endou activity (guarino et al., ) . the residues in the n-terminal domain of nsp are critical for hexamer formation (guarino et al., ) . in addition, nsp requires divalent metal ions as a co-factor for rna cleavage and prefers mn + over mg + or other divalent cations (ivanov et al., ; bhardwaj et al., ) . addition of mn + significantly affects the protein conformation, enhances rna binding, and increases endou activity (ivanov et al., ; bhardwaj et al., ) . in contrast to cov nsp , arterivirus nsp forms dimers in solution and does not require divalent cations as a cofactor for activity in vitro (nedialkova et al., ; shi et al., ) . instead, a concentration of mn + that greatly stimulated the activity of cov nsp inhibited the endou activity of eav and prrsv nsp s. cov endou was revealed biochemically to hydrolyze the ′ end of pyrimidines, with a preference for uridylates, and release products with ′, ′-cyclic phosphate and ′-hydroxyl ends (ivanov et al., ; bhardwaj et al., ) . this finding seems to implicate a broad range of targets; however, the endou activity of cov nsp can be affected by secondary structure and modification of the rna substrate. bhardwaj et al. found that nsp preferentially cleaved unpaired uridylates in hairpin-structured rnas and that the neighboring nucleotides of targeted sites also influenced hydrolysis (bhardwaj et al., ) . on the other hand, ivanov et al. found that ′-o-ribose methyl groups present on the substrate rna blocked endou-mediated cleavage (ivanov et al., ) . these data suggest that multiple factors might limit the range of endou targets. this is reasonable because the endou activity of nsp is likely to be tightly regulated during infection in cells to avoid unwanted cleavage on viral and/or cellular targets. for example, cov nsp is a ′-o-ribose methyltransferase, whose function could theoretically block the endou-mediated cleavage of viral rnas. similar to cov nsp , arterivirus nsp also prefers ′ of uridylates for cleavage and yields products with ′, ′-cyclic phosphate ends (nedialkova et al., ) . further studies are needed to address whether the endou activity of arterivirus nsp is restricted by rna modifications or secondary structures. guarino and coworkers visualized single particles of sars-cov nsp using electron microscopy (guarino et al., ) and the same group later reported an . Å structure of nsp by cryoelectron microscopy (bhardwaj et al., ) . they reported that the nsp hexamer comprises a dimer of trimers and proposed that the rna substrate binds to the inter-trimer interface. x-ray crystal structures of sars-cov and mhv nsp s were first solved by ricagno et al. and xu et al., respectively (ricagno et al., ; xu et al., ) . these high-resolution structures define cov nsp as a separate endou family with unique folds that differ from cellular endoribonucleases (fig. a) . the monomer of sars-cov nsp consists of three domains: a small nterminal domain (ntd) (residues - , cyan), a middle domain (residues - , magenta), and a large c-terminal domain (ctd) (re-sidues - , green). the endou is located in the ctd. the monomeric structure of mhv nsp can be superimposed onto that of sars-cov nsp , except for a flexible loop structure in the middle domain of mhv nsp (fig. b ). this flexible loop is encoded by a viral rna packaging signal sequence, which is present in mhv nsp but not in sars-cov nsp (kuo and masters, ) . these structural studies demonstrated that the presence of the flexible loop did not alter the overall folding of mhv nsp relative to sars-cov nsp . in addition, through these and other structural studies (ricagno et al., ; xu et al., ; joseph et al., ; bhardwaj et al., ) , the nsp hexamer was again confirmed to be a dimer of trimers. as shown in fig. c , three monomers form a trimer and two trimers interact headto-head to form a hexamer. the ntds line up in the center of the hexamer, while the ctds face outward. this architecture allows the nsp hexamer to possess six active sites. the extensive contact between subunits through the ntd and middle domain is critical for hexamerization. the crystal structure of arterivirus nsp was also recently reported (shi et al., ; zhang et al., ) . these structural studies revealed that the monomer of nsp contains only two domains: ntd and ctd (no middle domain). the monomeric structures of cov nsp and arterivirus nsp could not be superimposed except in the ctd (catalytic domain). distinct from the hexameric structure of cov nsp , nsp monomers assembled into an asymmetric dimer (shi et al., ) . virus-encoded endoribonuclease is a genetic signature of nidoviruses that infect vertebrates. a phylogenic tree of representative nidoviruses was generated based on a conserved region of rdrp (sequnces and genbank assession numbers are available upon request). multiple sequence alignment and phylogeny analyses were conducted with the programs muscle and phyml, respectively (available at http:// www.phylogeny.fr/). the phylogenic tree was generated using dendroscope software version with default parameters. virology ( ) - since the crystallographic studies of cov nsp revealed that the active site of endou is structurally similar to that of rnase a, researchers evaluated small molecule inhibitors of rnase a for their ability to inhibit nsp activity. ortiz-alcantara et al. tested several commercially available rnase a inhibitors (benzopurpurin b, congo red, and others) and reported that their % inhibitory concentration (ic ) values for inhibiting the endou activity of sars-cov nsp ranged from . μm to μm. benzopurpurin b was shown to have a broad-spectrum activity and could inhibit nsp orthologs from mhv and ibv with ic values of . μm and . μm, respectively (ortiz-alcantara et al., ). these rnase a inhibitors had variable effects on cov replication in cell cultures. in plaque formation assays, treatment with congo red resulted in -fold and -fold reductions in mhv and sars-cov titers, respectively, while benzopurpurin b led to marginal inhibition of both covs (ortiz-alcantara et al., ) . although the impact of these rnase a inhibitors on cov replication requires more comprehensive investigation, these early results suggest that the similarity between nsp /endou and rnase a may provide a basis for exploiting small molecule inhibitors to modulate viral endou activity. due to its unique enzymatic activity and co-localization with the replicating viral rna, nsp /endou was initially thought to play an important role in virus replication. however, mhv encoding catalyticdefective endou exhibited only a subtle defect in rna synthesis and a slight reduction in viral titers (~ log) compared to wt virus when evaluated in fibroblasts (kang et al., ) . similar results were obtained for sars-cov and hcov- e nsp mutants generated using either reverse genetics or replicon systems (ulferts and ziebuhr, ) . these data indicate that the endou activity of nsp is not essential for cov replication, as was initially proposed (snijder et al., ; ivanov et al., ; bhardwaj et al., ) . intriguingly, nsp may indirectly affect virus replication through other mechanisms. ivanov et al. demonstrated that when the conserved aspartic acid (asp)- of hcov- e nsp was replaced with an ala, its endou activity was eliminated, viral rna synthesis was completely abolished, and no viable virus was recovered (ivanov et al., ) . similar phenotypes were observed for an mhv nsp mutant with an ala substitution of asp- (equivalent to asp- of hcov- e) (kang et al., ) . it is unclear how the asp residues affect viral rna synthesis. kang et al. predicted that asp- is critical for an ionic-bond network and observed that the ala substitution resulted in an insoluble protein when mhv nsp was expressed in e. coli (kang et al., ) . these data suggest that the ala substitution may prevent the nsp protein from folding correctly. since the proteins adjacent to nsp are critical replicative components, any protein-folding issue with nsp may have an effect on the proteolytic processing of the neighboring components, thereby leading to a nonviable phenotype. overall, current evidence indicates that the endou activity of cov nsp is dispensable for viral rna synthesis and virus replication in cell culture. further work is required to address any non-endou-mediated role of nsp protein in virus replication. mutagenesis of arterivirus nsp revealed pleiotropic effects of endou on the viral life cycle (posthuma et al., ; sun et al., ) . similar to cov nsp , mutations in the asp- and asp- residues (corresponding to mhv nsp asp- and asp- , respectively) in eav nsp resulted in a nonviable phenotype. compared with the mild effect of mutating the catalytic histidine (his) residues (hi-s ala and his ala) of mhv nsp , eav infectious clones with catalytic residue mutations (his ala/gln and his ala/gln) exhibited smaller plaque sizes, reduced rna synthesis, and dramatic titer reductions up to log units. other substitutions at conserved, noncatalytic residues of eav nsp resulted in an intermediate phenotype: intermediate plaque sizes and~ - log reduction in titers. similar results were obtained with the prrsv nsp mutant viruses (sun et al., ) . these data indicate that arterivirus nsp may be involved in virology ( ) - viral rna synthesis. however, similar to the asp ala mutation of mhv nsp , both asp ala and asp ala mutations in eav nsp rendered the protein insoluble, again raising the question of whether or not these mutations influence viral rna synthesis through interfering with proteolytic processing of the orf b polyprotein. the absence of an endou domain in insect nidoviruses and invertebrate roniviruses further indicates that endou activity is not required for the unique rna synthesis strategy of nidoviruses (nga et al., ; lauber et al., ) . the aforementioned studies highlight the extensive efforts of the field to investigate the characteristics of nsp /endou and its role in virus life cycle. the endou activity of cov nsp was found to play a non-essential role in viral rna synthesis and replication in immortalized fibroblast cells. recent work with different cell types and in vivo experiments revealed a novel function of nsp /endou in virus replication and pathogenesis and provided a new direction of study with respect to this "old" protein. cov nsp was first suggested to possess interferon (ifn) antagonism capabilities through ectopic expression experiments. frieman et al. used an alphavirus replication-defective vector (vrp) to screen sars-cov proteins that suppress vrp-induced ifn responses. one of the identified ifn antagonists of sars-cov was nsp (frieman et al., ) . later, arterivirus nsp was also identified as an ifn antagonist and its endou activity was found to mediate inhibition of ifn-beta induction (beura et al., ; shi et al., ) . several other studies also reported that cov nsp and arterivirus nsp inhibit cellular innate responses in ectopic expression experiments (lei et al., ; wang et al., ) . these data seem to indicate that these two proteins function as ifn antagonists; however, the endou activity of overexpressed nsp /nsp may unexpectedly affect the activities of the reporters used in these assays. we found that transfected mhv nsp reduced the signal of both ifn-reporter firefly luciferase and the internal control renilla luciferase (hackbart m, deng x, and baker s, unpublished data). a similar result was obtained with the overexpressed prrsv nsp (shi et al., ) . these observations imply that nsp /nsp may execute non-specific cleavage when ectopically expressed. since cov nsp is part of the viral replicase/transcriptase complex (rtc), it is reasonable to predict that its endou activity is tightly regulated during viral infection to avoid unwanted cleavage. in line with this prediction, we and others reported a specific, punctate, perinuclear localization of cov nsp during viral infection (heusipp et al., ; shi et al., ; deng et al., ; athmer et al., ) , while ectopically expressed nsp was distributed throughout the cytoplasm (cao and zhang, ) . hence, we advise caution when interpreting the results of overexpression studies, as the nature of the endou activity was only revealed after studying nsp /nsp in the context of viral infection. it was first discovered that the endou activity of nsp mediates the evasion of host recognition of viral dsrna by infecting primary macrophages with endou-deficient covs (deng et al., ; kindler et al., ) . infection with mhv endou-deficient mutants stimulated mouse bone-marrow derived macrophages (bmdms) to produce a remarkably high level of type i ifns during the early phase of infection compared to wt infection. this ifn response is mda -dependent, as both the ifn mrna and protein levels were not elevated in mda -deficient bmdms. moreover, the replication of endou-deficient covs was severely impaired in primary macrophages. interestingly, the ifn-induced antiviral response is not the only player responsible for this replication defect, as the titers of the endou-deficient covs were not completely restored in bmdms that lack critical genes (e.g. mda , mavs, and irf / / ) involved in the ifn response (deng et al., ; kindler et al., ) . these data suggest that other antiviral pathways may also contribute to the observed replication defect. in support of this, it was found that the infection of endou-deficient covs also activate the pkr and the oas-rnase l pathways (deng et al., ; kindler et al., ) , which both execute potent antiviral functions, discussed further below. indeed, the replication of endou-deficient covs could be partially restored in pkr/ rnase l-double knockout cells (kindler et al., ) . replication was only fully restored in type i ifn receptor-knockout macrophages, as these cells not only have a defect in ifn signaling but also express very low basal levels of pkr and oas relative to wt cells (deng et al., ; birdwell et al., ) . taken together, these studies using live viruses in primary cells effectively illustrated the ifn antagonistic properties of cov endou. as mentioned above, infection with endou-deficient covs also activates the pkr and oas-rnase l pathways in macrophages (deng et al., ; kindler et al., ) . pkr is a dsrna-activated protein kinase and serves as a dsrna sensor. activated pkr phosphorylates eukaryotic initiation factor α (eif α), resulting in inhibition of host and viral mrna translation. thus, pkr-mediated translation shutoff plays an important role in the host antiviral defense (barber, ) . the endou-deficient cov-infected macrophages exhibited increased levels of phosphorylated eif α and decreased levels of translation (deng et al., ; kindler et al., ) , indicating that pkr was activated during infection. another piece of evidence of pkr activation was that endou-deficient covs induced rapid apoptotic cell death in infectedmacrophages (deng et al., ) . it has been shown that pkr-mediated translation shutoff leads to apoptosis in macrophages (hsu et al., ) . when the endou-deficient cov-infected macrophages were treated with a pkr-specific inhibitor (c ), the level of apoptosis was significantly reduced (deng et al., ) . this result further supports the hypothesis that endou-deficient covs activate pkr. interestingly, loss of pkr expression or inhibition of its activity only partially restored the replication of endou-deficient covs in macrophages (kindler et al., ) . moreover, treatment with the pkr inhibitor did not affect ifn induction or rnase l-mediated ribosomal rna degradation in the endou-deficient cov infected-macrophages (deng et al., unpublished data) . these results imply that the infection of endou-deficient cov activates multiple host dsrna sensors independently, including mda , pkr, and oas. oas is a protein family of ′, ′-oligoadenylate ( - a) synthetases. upon activation, oas can synthesize - a, which binds to and activates rnase l. rnase l is a host ribonuclease that executes global degradation of host and viral rnas. thus, oas and rnase l constitute a potent host antiviral system. macrophages infected by the endou-deficient covs exhibited an early, rnase l-mediated degradation of ribosomal rna, demonstrating that the oas-rnase l system was activated (deng et al., ; kindler et al., ) . lack of mda expression or treatment with the pkr inhibitor did not affect virus-induced rna degradation (deng et al., ; kindler et al., ) , suggesting that the nsp -mediated blockage of oas-rnase l activation is independent of the mda -ifn and pkr pathways. loss of rnase l expression does not restore the replication of endou-deficient covs in macrophages. taken together, these results suggest again that multiple antiviral pathways, including mda -ifn, pkr, and the oas-rnase l system, were activated during infection with endou-deficient covs. the antiviral defense executed by these pathways contribute together to the replication defect of endou-deficient covs in macrophages. interestingly, some covs encode a ′, ′-phosphodiesterase (pde) (e.g. mers-cov orf b and mhv ns ) (thornbrough et al., ; zhao et al., ) . this cov pde also prevents rnase l-mediated rna degradation through digesting the - a produced by oas. thus, the presence of two antagonists of the oas-rnase l system in some cov genomes represents a functional redundancy or tissue-specific roles for these antagonists. in fact, not all covs encode a pde, and it has been reported that loss of the pde activity mitigated mhv pathogenicity in the mouse liver but not in the brain, suggesting a liver-specific effect of cov pde activity in vivo (roth-cross et al., ; zhao et al., ) . several endou-deficient covs have been evaluated in fibroblast cell lines and no marked phenotypes were obtained (mild reduction of viral rna synthesis and replication) (discussed above). we speculate that the activation of an ifn response and apoptosis in macrophages is due to high levels of basal expression of host sensors, such as mda , pkr, and oas (deng et al., ; birdwell et al., ) . indeed, when the expression of oas was induced by pre-treatment with ifn, the immortalized fibroblast cells also exhibited rna degradation upon infection with endou-deficient cov (kindler et al., ) . in line with this, without the stimulation of ifn, constitutively expressed oas and pkr in the mda -deficient macrophages is capable of sensing the endou-deficient cov infection and implementing antiviral processes (deng et al., ) . although the production of ifn is dispensable for the activation of the oas-rnase l system and pkr, type i ifn receptors or a direct signal is required for maintaining the high basal expression of these ifn-inducible genes (deng et al., ; birdwell et al., ) . this is biologically relevant because macrophages and other myeloid cells are quick responders to early virus infection, responding even before ifn is highly induced. it is unclear whether other cell types, such as epithelial cells, behave similar to macrophages, but at least mouse embryonic fibroblasts have been shown to display the nsp /endoumediated effects (kindler et al., ) . due to robust activation of antiviral responses, mhv endou-deficient mutants also exhibited a marked attenuation in vivo relative to mhv-wt. depending on the inoculation route, mhv infection can result in hepatitis or encephalitis in c /bl mice. strikingly, regardless of which infection route (intraperitoneal or intracranial injection) was used, we found that mhv endou-deficient mutants were highly attenuated (deng et al., ) . when mice were inoculated intraperitoneally using a high dose of the mutant virus, there was no detectable viral titer in the liver or spleen at day post-infection and only a minimal detection of viral mrna in mesenteric lymph nodes at day post-infection. when a sensitive encephalitis model was used, mice infected with mhv endou-deficient mutants exhibited only a transient loss of body weight and recovered completely. this significant attenuation is attributed to the loss of endou-mediated evasion of host antiviral defenses. we found that endou-deficient mutants maintained wt-level virulence in type i ifn receptor knockout (ifnar -/-) mice (deng et al., ) . similar results obtained by kindler et al. showed that the mutant virus was only detected in the organs from ifnar -/mice but not wild-type or other gene-deficient (mda -/-, tlr -/-, and mda -/-/tlr -/-) mice at day post-infection (kindler et al., ) . interestingly, even though mhv endou-deficient mutants were highly attenuated and exhibited limited replication in vivo, the pre-infected mice were protected from subsequent lethal challenges of wild-type mhv in both disease models (deng et al., ) . these results demonstrate that nsp plays an important role in virus pathogenesis and illustrate the potential use of endou-deficient covs as vaccine candidates. . . how does nsp mediate the evasion of host sensors? as an endou, it is plausible that nsp may degrade viral dsrna to prevent host recognition. previous studies of pestivirus envelope glycoprotein (e rns ) and lassa virus nucleoprotein linked viral ribonuclease activity to type i ifn antagonism through degrading viral dsrna (python et al., ; qi et al., ; hastie et al., ) , although no direct evidence for this linkage has been obtained. for cov nsp , kindler et al. detected an increased level of dsrna in endou mutantinfected cells by flow cytometry using a dsrna-specific monoclonal antibody (mab) (kindler et al., ) . this mab recognizes dsrna molecules longer than bp in length, regardless of the sequence, meaning that a long dsrna could potentially bind multiple mab molecules. to saturate the mab-dsrna binding, we tested serial concentrations of antibody but did not detect any significant change of dsrna levels by either flow cytometry or immunofluorescence analysis (deng et al., ) . the discrepancy between these two studies may be ascribed to the experimental settings in respective experiments. however, it is also possible that the methods used in these studies may not be sensitive enough to detect changes in dsrna levels if the wt-nsp produces dsrna cleavage products that are > bp, such that the uncleaved and cleaved dsrnas are indistinguishable for binding by the anti-dsrna antibody. importantly, it has been demonstrated that the endou activity of cov nsp is influenced by secondary structures and modifications of dsrna (ivanov et al., ; bhardwaj et al., ) . because these factors may limit the number of cleavage events and/or the targets of endou, nsp -mediated cleavage may produce few overall cleavage products that are < bp. consequently, nsp mediated cleavage may not reduce the level of total dsrna in the cell, but rather may hydrolyze long dsrnas into shorter cleavage products that are sufficiently short to evade recognition by host sensors (e.g. mda ) but not by the anti-dsrna antibody. it has been documented that cov dsrnas form cytoplasmic aggregated foci during replicating in cells (becares et al., ; hagemeijer et al., ) . these dsrna foci co-localize with the viral rtcs early during infection. interestingly, we noted that during the early stages of infection, dsrna foci were not co-localized with the rtcs in the endou-deficient cov-infected cells. this decrease in colocalization relative to wt infection resulted in a dispersed pattern of dsrna foci, such that some dsrnas did not appear to associate with the rtcs. it is not known whether these "free" dsrnas trigger host sensors, but this dispersed distribution of dsrna does notably coincide with early activation of the ifn response and other dsrna sensors (e.g. pkr and oas). overall, additional studies with new methods are needed to characterize the intracellular localizations and the fates of dsrna in cov-infected cells. the detailed strategy used by cov endou to evade host recognition remains enigmatic. more studies are needed to address several key questions: (i) what is the natural target of endou? (ii) how does the endou activity of nsp alter the fate of dsrna? (iii) how is this endou activity regulated to avoid unwanted cleavage events? (iv) do any interaction partners (viral or cellular) of nsp participate in regulating its endou activity (bhardwaj et al., ; athmer et al., ) ? answers to these questions will be essential for understanding the mechanism of the endou-mediated evasion of host dsrna sensors. additionally, it is possible that endou serves as a conserved antagonist in vertebrate nidoviruses. a similar phenotype has been observed in human blood-derived macrophages infected with the hcov- e nsp mutant virus (kindler et al., ) , which is a representative alphacoronavirus. whether arterivirus nsp also functions as an ifn antagonist during infection is still unknown. one obstacle to studying arterivirus nsp mutants in cell culture is that mutations of catalytic residues have been reported to severely affect viral replication even in ifn-deficient cells (posthuma et al., ; sun et al., ) . nonetheless, given the striking roles of cov endou in macrophages and in vivo, it will be important to understand the underlying mechanism of endou-mediated evasion of host antiviral defenses in order to advance an ultimate goal of exploiting this enzyme as a target for therapeutics and vaccine design against existing and emerging pathogenic covs. in situ tagged nsp reveals interactions with coronavirus replication/transcription complex-associated proteins the dsrna-dependent protein kinase, pkr and cell death mutagenesis of coronavirus nsp reveals its potential role in modulation of the innate immune response porcine reproductive and respiratory syndrome virus nonstructural protein beta modulates host innate immune response by antagonizing irf activation structural and functional analyses of the severe acute respiratory syndrome coronavirus endoribonuclease nsp the severe acute respiratory syndrome coronavirus nsp protein is an endoribonuclease that prefers manganese as a cofactor rna recognition and cleavage by the sars coronavirus endoribonuclease the coronavirus endoribonuclease nsp interacts with retinoblastoma tumor suppressor protein activation of rnase l by murine coronavirus in myeloid cells is dependent on basal oas gene expression and independent of virus-induced interferon comparative in vivo analysis of the nsp endoribonuclease of murine, porcine and severe acute respiratory syndrome coronaviruses turkey coronavirus non-structure protein nsp -an endoribonuclease coronavirus nonstructural protein mediates evasion of dsrna sensors and limits apoptosis in macrophages severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of irf and nf-kappab signaling mutational analysis of the sars virus nsp endoribonuclease: identification of residues affecting hexamer formation visualizing coronavirus rna synthesis in time by using click chemistry crystal structure of the lassa virus nucleoprotein-rna complex reveals a gating mechanism for rna binding identification and subcellular localization of a kda, polyprotein ab processing product in human coronavirus e-infected cells the protein kinase pkr is required for macrophage apoptosis after activation of toll-like receptor major genetic marker of nidoviruses encodes a replicative endoribonuclease crystal structure of a monomeric form of severe acute respiratory syndrome coronavirus endonuclease nsp suggests a role for hexamerization as an allosteric switch biochemical and genetic analyses of murine hepatitis virus nsp endoribonuclease early endonuclease-mediated evasion of rna sensing ensures efficient coronavirus replication functional analysis of the murine coronavirus genomic rna packaging signal mesoniviridae: a proposed new family in the order nidovirales formed by a single species of mosquito-borne viruses mavs-mediated apoptosis and its inhibition by viral proteins biochemical characterization of arterivirus nonstructural protein reveals the nidovirus-wide conservation of a replicative endoribonuclease discovery of the first insect nidovirus, a missing evolutionary link in the emergence of the largest rna virus genomes small molecule inhibitors of the sars-cov nsp endoribonuclease site-directed mutagenesis of the nidovirus replicative endoribonuclease nendou exerts pleiotropic effects on the arterivirus life cycle efficient sensing of infected cells in absence of virus particles by blasmacytoid dendritic cells is blocked by the viral ribonuclease erns cap binding and immune evasion revealed by lassa nucleoprotein structure crystal structure and mechanistic determinants of sars coronavirus nonstructural protein define an endoribonuclease family organ-specific attenuation of murine hepatitis virus strain a by replacement of catalytic residues in the putative viral cyclic phosphodiesterase ns colocalization and membrane association of murine hepatitis virus gene products and de novo-synthesized viral rna in infected cells endoribonuclease activities of porcine reproductive and respiratory syndrome virus nsp was essential for nsp to inhibit ifn-β induction a dimerization-dependent mechanism drives the endoribonuclease function of porcine reproductive and respiratory syndrome virus nsp unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group lineage the nonstructural proteins directing coronavirus rna synthesis and processing nonstructural protein of porcine reproductive and respiratory syndrome virus suppresses both mavs and rig-i expression as one of the mechanisms to antagonize type i interferon production nidovirus ribonucleases: structures and functions in viral replication the nonstructural protein of porcine reproductive and respiratory syndrome virus inhibits nf-κb signaling by means of its deubiquitinating activity new antiviral target revealed by the hexameric structure of mouse hepatitis virus nonstructural protein nsp structural biology of the arterivirus nsp endoribonucleases antagonism of the interferon-induced oas-rnase l pathway by murine coronavirus ns protein is required for virus replication and liver pathology cell-type-specific type i interferon antagonism influences organ tropism of murine coronavirus virus-encoded proteinases and proteolytic processing in the nidovirales we thank robert c. mettelman and aaron volk for assistance with editing the manuscript. our studies are supported by national institutes of health r ai (to scb). key: cord- -oudj q authors: al-tayib, omar a. title: an overview of the most significant zoonotic viral pathogens transmitted from animal to human in saudi arabia date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: oudj q currently, there has been an increasing socioeconomic impact of zoonotic pathogens transmitted from animals to humans worldwide. recently, in the arabian peninsula, including in saudi arabia, epidemiological data indicated an actual increase in the number of emerging and/or reemerging cases of several viral zoonotic diseases. data presented in this review are very relevant because saudi arabia is considered the largest country in the peninsula. we believe that zoonotic pathogens in saudi arabia remain an important public health problem; however, more than million muslim pilgrims from around islamic countries arrive yearly at makkah for the hajj season and/or for the umrah. therefore, for health reasons, several countries recommend vaccinations for various zoonotic diseases among preventive protocols that should be complied with before traveling to saudi arabia. however, there is a shortage of epidemiological data focusing on the emerging and reemerging of zoonotic pathogens transmitted from animal to humans in different densely populated cities and/or localities in saudi arabia. therefore, further efforts might be needed to control the increasing impacts of zoonotic viral disease. also, there is a need for a high collaboration to enhance the detection and determination of the prevalence, diagnosis, control, and prevention as well as intervention and reduction in outbreaks of these diseases in saudi arabia, particularly those from other countries. persons in the health field including physicians and veterinarians, pet owners, pet store owners, exporters, border guards, and people involved in businesses related to animal products have adopted various preventive strategies. some of these measures might pave the way to highly successful prevention and control results on the different transmission routes of these viral zoonotic diseases from or to saudi arabia. moreover, the prevention of these viral pathogens depends on socioeconomic impacts, available data, improved diagnosis, and highly effective therapeutics or prophylaxis. rudolf virchow , one of the foremost th century german leaders in medicine and pathology [ ] , noted a relationship between human diseases and animals and then introduced the term "zoonosis" (plural: zoonoses) in [ ] . later, the world health organization (who) in specified that "zoonoses are those diseases and infections which are naturally transmitted between vertebrate animals and man" [ ] . venkatesan and co-authors reported that the term zoonosis is derived from the greek word "zoon" = animal and "noso" = disease [ ] . zoonotic pathogens causing different kinds of diseases are of major public health issues worldwide [ ] . these zoonotic diseases include frequent mixing of different animal species in the markets in densely populated areas, and the human intrusions into the natural habitats of animals, have facilitated the emergence of novel viruses. the most important zoonotic viral diseases of which eight were diagnosed (in dead or diseased animals or through antibody detection) on the arabian peninsula over the last years include rabies, middle east respiratory syndrome (mers-cov), influenza virus (ifv), alkhurma hemorrhagic fever, crimean-congo hemorrhagic fever (cchf), rift valley fever (rvf), west nile fever (wnv), and dengue fever virus. among these eight zoonotic viral diseases, two (alkhurma and mers-cov) were first reported in a patient in and , respectively in saudi arabia [ , ] . these two were transmitted later to several other countries, not only in the middle east but also to africa, asia, and europe. rabies is an almost invariably fatal zoonotic disease, which belongs to the genus lyssavirus of the rna family rhabdoviridae. rabies virus is considered an endemic viral infectious disease in animals in saudi arabia. recent scientific data on rabies cases reported in camels at al-qassim region (one of the thirteen administrative regions of saudi arabia) showed that there is an increasing number of this fatal virus disease [ ] . however, the most significant animal bites which have been recorded in saudi arabia were caused by different species of animals including dogs, cats, rodents, and foxes [ ] . later, al-dubaib reported rabies in dromedaries in saudi arabia in and suggested an incidence of about . % for rabies that was reported among camel herdsmen looking after more than animals [ ] . interestingly, another survey was conducted between and in the al-qassim region of central saudi arabia among camels and showed that about . % of clinical rabies incidence is caused by dogs (may be cause it highly used as a perfect guard for camels), followed by foxes; furthermore, the diagnosis of viral rabies in that region was confirmed among dogs, foxes, camels, and cats [ ] . lately, the relevant government authorities (the moh and ministry of agriculture in saudi arabia) in an updated report between and showed that there were a total of , animal bites to humans in saudi arabia [ ] . furthermore, most cases of animal bites were caused by dogs ( . %) and cats ( . %), followed by mice and rats, camels, foxes, monkeys, and wolves [ ] . moreover, dogs, particularly feral dogs and foxes, are considered the most important host for rabies virus; however, bats are also considered as reservoirs of this disease. humans can become rabid by direct contact with animal mucosal surfaces via bites. according to the moh and ministry of agriculture data in saudi arabia, pets are responsible for most animal bites in humans [ ] , and it is well-known that insufficient vaccination coverage of pets are among the most common hallmarks of the endemic status of rabies worldwide [ ] . more recently, many saudi and expatriate families are keeping pets; however, there are limited number of specialized veterinary clinics (~ ) within the kingdom of saudi arabia that have fully licensed veterinary laboratories with state of the art technologies and veterinary staff. globally, almost % of all human deaths caused by rabies occur in africa and asia [ ] . however, saudi arabia, as one of the asian countries, has scarce publications and epidemic data on rabies status [ , ] . moreover, memish et al., between and in saudi arabia, reported the histologic detection of the virus by identifying negri bodies in the brain samples of animal rabies cases. the study showed that among the suspected rabies cases, (~ . % of all cases) were found to be positive; thus confirming rabies cases among dogs, foxes, sheep, camels, goats, wolves, and cows [ ] . furthermore, more recent data confirmed the transmission of rabies virus in saudi arabia by feral dogs [ ] . in spite of these facts, there are very few studies available, and no case of human rabies has been reported in recent decades from saudi arabia [ ] . however, in march , a scientific work was reported as the first confirmed case of human rabies in saudi arabia from makkah city, which has now been published [ ] . indeed, several previous global epidemiological data confirmed that rabies accounted for , to , human deaths per year [ ] , and more than % of these cases occur in children < years of age [ ] . in september , a -year-old saudi man, presented with different clinical features-such as nausea, vomiting, and epigastric pain, with significant features suggestive of gastritis-at makkah hospital. his past medical history was significant for hypertension and diabetes type . during the clinical diagnostic procedure of this case, he developed respiratory distress and tachycardia, for which he was transferred to the intensive care unit [ ] . because, his case worsened with chest pain and ventricular tachycardia he was referred to the king abdullah medical city in makkah for further management. the written diagnostic report indicated that he had acute anteroseptal myocardial infarction, had coronary angiogram which suggested that two-vessels were diseased with left main involvement, and surgical intervention was planned. after the decision for surgery, he was found to have leukocytosis and severe retching while attempting to drink water (hydrophobic behavior), which necessitated further review by the infectious disease consultants based on the patient's clinical symptoms. the consultant team discovered the history of an unprovoked scratch on the patient's face by a dog in morocco a month prior to the admission at the hospital. also, the patient stated that he only received tetanus vaccine. all diagnostic tests including neurologic examination were unremarkable and his saliva polymerase chain reaction (pcr) test confirmed rabies virus. he was administered verorab rabies vaccine and human hyperimmune rabies immunoglobulin ( iu/kg) intramuscularly (im) [ ] . in addition, he had troponin i ( . ng/ml), creatine kinase isoenzyme mb (ckmb) was found ( . ng/ml), and serum glucose ( mg/dl). on the fifth day of hospital, he had recurrent episodes of ventricular tachycardia, progressively worsening of hemodynamic parameters, and he succumbed to his infection on that day. there is no vaccine against rabies recommended for travelers from/to saudi arabia, and no rabies treatment is offered to pet dogs. however, vaccination is given to dogs before they are infected; otherwise they are euthanized if infected. according to a previous study, most patient injuries from animal bites in saudi arabia showed some variations due to the monthly incidence and/or, according to the animal species [ ] . bites by dogs and cats were reported frequently throughout the year, with a decrease in april and between august and october. however, bites by foxes increase between august and september while camel bites were more frequent between december and march of the subsequent year. the same previous study suggest that these seasonal variations of injuries might be due to the saudi population habits, with people going to the desert for leisure activities during good weather periods. laboratory diagnosis of rabies viral disease occur with the use of the rabies virus direct fluorescent antibody test (dfat) on brain samples and hippocampal tissue [ ] . while rabies is considered nearly % fatal, it is also % preventable, and thus vaccination to pets is the key element to prevent the risk of rabies zoonotic infection [ ] . reports of the epidemiology of rabies virus worldwide, and particularly in saudi arabia, suggest that it is on the increase, thus the implication of this virus' potential to spread across borders from high to low prevalence countries was highlighted [ ] . the mers-cov infection is considered to be a new respiratory disease with a dire global concern [ ] . mers-cov infections are caused by a newly emerging coronavirus (cov), belonging to the designated lineage c of betacoronavirus of the rna family coronaviridae. with respect to viral origin and transmission, bats are thought to be the reservoir host of betacoronaviruses, and the african neoromicia bats in particular are the natural reservoir of mers-cov [ , ] . since its emergence in in saudi arabia, when an elderly patient ( years old) with respiratory illness died after admission to a hospital in jeddah [ ] , the disease was subsequently reported to have been transmitted to several countries worldwide, and has affected more than patients with over % fatality [ , [ ] [ ] [ ] . moreover, a -year-old saudi man was admitted to a private hospital in jeddah, saudi arabia in june with a history of fever, severe acute respiratory syndrome with cough, expectoration, and shortness of breath. he did not smoke; and for the disease, which was suggested to be due to an animal transmission of coronaviruses, he was treated with oseltamivir, levofloxacin, and piperacillin-tazobactam. on day , he died [ ] . after this, a -year-old saudi male with hypertension and diabetes with no history of smoking, reported for surgery. at the time of admission, he was asymptomatic. he was initially screened using nasopharyngeal swab, endotracheal aspirate, and serum sample for mers-cov per protocol with the mers rrt-pcr assay. the results confirmed mers-cov infection. he died three days after admission. it was discovered that the patient owned a dromedary camel barn in saudi arabia, and had a history of close contact with camels, as well as a habit of raw milk consumption of an unknown duration [ ] . two studies have suggested a relationship between the infection and contact with dromedary camels [ , ] . in addition to this, serological diagnostic methods have been used to confirm mers-cov infections in dromedary camels for at least - decades and has thus confirmed camels as an intermediate host for this virus [ , ] . thus, in , a novel coronavirus (mers-cov) was isolated from two fatal human cases in saudi arabia and qatar; and since then, more than clinical cases of mers-cov have been identified, and the great majority of the cases were from saudi arabia [ ] . this previous report author raised a thoughtful comment related to the emerging viral diseases "why we need to worry about bats, camels, and airplanes" [ ] . moreover, another study suggested that mers-cov infection is usually transmitted from human's direct contact with dromedary camels, especially when people drink the milk or use camel's urine for medicinal purposes [ ] . more recently, a metagenomics sequencing analysis of nasopharyngeal swab samples from mers-cov-positive live dromedary camels marketed in abu dhabi, united arab emirates, showed at least two recently identified camel coronaviruses, which were detected in . % of the camels in that study [ ] . however, limited human-to-human infections have been reported. the prevalence of mers-cov infections worldwide still remains unclear. in addition to this, the who reported about cases of these infections since june , with about deaths in different countries, worldwide. recently, a study was conducted from june to july , during which samples were collected from mers-cov infected individuals, from the national guard hospital in riyadh (the saudi arabian capital city), the moh in saudi arabia, and other gulf corporation council countries, to determine the prevalence of mers-cov [ ] . the epidemiologic data that were collected, showed that the highest number of cases (about of patients) were reported from saudi arabia (~ %). among the mers-cov cases from saudi arabia, riyadh was the worst-hit area with infected cases ( . %), followed by the western region of makkah where cases ( . %) were reported [ ] . furthermore, this study also showed that the incidence of mers-cov infections was highest among elderly people aged ≥ years [ , ] ; with speculation that there might be certain conditions or factors involved. it is considered that mers-cov infection might have a peculiar gender predisposition [ ] . recent data examined the mortality in patients with mers-cov and the gender relationships, looking at the survival of cases among females and males. it was suggested that males have a higher risk of death [ , ] ; however, this was contradicted by the findings from two other studies which suggested that males have a low risk of death [ ] ; while another survey which examined the influence of gender on -day and -day survival, found a low risk of death especially in the older age group [ ] . on the other hand, badawi et al., suggested that mers-cov infections could be mild and may only result in death among patients suffering from any kind of immune system disorder and/or any chronic disease [ ] . more recently, data regarding the mortality in patients with mers-cov have been published. according to saudi arabia's moh daily statements, dated from february through march , laboratory-confirmed new cases of mers-cov and deaths occurred [ ] . recently, on february , patients infected while hospitalized at riyadh included two men ( and years old) in stable condition, who were not healthcare workers. according to a february update, a new case involved a -year-old man from the city of buraydah who later died. meanwhile, on march , another mers-cov infection in a riyadh hospital patient, a -year-old man who was listed in critical condition and who likewise had contact with camels, as the other two patients, was reported. thus, the moh stated that the spillover from camels is thought to be the main source of mers-cov in saudi arabia, since all these patients were exposed to the animals before reporting ill [ ] . furthermore, an -year-old patient from riyadh, and other two patients who had camel contacts from hail city in the north central part of saudi arabia were listed in critical condition. the illness in these patients was reported on march . according to a march statement, another patient, a -year-old man from najran located in southern saudi arabia, was reported. the man was listed in a stable condition. of these new cases, only one death, involving the -year-old man from riyadh, according to the march moh statement, was reported [ ] . still, much work is needed to detect the mers-cov infection risk in saudi arabia, because data showed increasing number of cases exist among the eight countries including saudi arabia. thus, the emergence of mers-cov in the region and its continuing transmission from - , currently poses one of the biggest threats to global health security [ ] . most cases (over %) reported to date have been from countries in the region (e.g., egypt) notably from saudi arabia, with cases including deaths [ ] . influenza viruses are considered to be important infectious viral diseases, which is caused by three virus types (a, b, and c) [ ] . due to their zoonotic spread, influenza type a infects both humans and animals, and causes moderate to severe illness, with more likelihood of fatalities in young children and the elderly [ , ] . other types of influenza, including type b and c, infect only humans [ ] . furthermore, influenza a viruses, members of the rna family orthomyxoviridae, are further classified into human, swine, and avian influenza viruses. however, during the influenza pandemic, swine influenza virus infected one-third of the world's population (an estimated million people) and caused approximately million deaths [ ] . since , several infections with this virus have been recorded from various areas worldwide, including saudi arabia [ ] . at the end of april , an outbreak of a new type of influenza, a/h n , started in mexico and the usa [ ] . the who declared the pandemic influenza a (h n ) as a "public health emergency of international concern" following the first few initial cases in mexico, and subsequently in the usa [ , ] . in saudi arabia, the epidemiological data for influenza virus were collected using a predesigned questionnaire with the first confirmed pandemics influenza a (h n ) cases identified by the infectious diseases department from the moh, and the database during the period covered from june to july [ ] . however, according to the saudi moh data, the number of laboratory-confirmed cases of the virus in saudi arabia as at december was , , with deaths [ ] . the virus later spread worldwide, causing a pandemic, and the most recorded cases then, as reported by the who in the middle east, were in saudi arabia with , cases, followed by kuwait [ ] , egypt, and oman; with less number of infected patients [ , ] . nevertheless, between and , a serosurveillance outcome of swine influenza virus from egypt provided evidence of laboratory diagnosis and very early confirmation of the virus in human patients [ ] . in saudi arabia, the influenza surveillance system has been established since . moreover, among people with certain chronic medical diseases or conditions, a trivalent influenza vaccine (tiv), which contains inactivated antigens for two different subtypes of influenza viruses (types a and b), became available in saudi arabia [ ] . indeed, h n is now in the post-pandemic period and has become a seasonal influenza virus that continues to circulate with localized outbreaks of varying magnitude in saudi arabia [ ] . a previous data was collected using a predesigned questionnaire for the first cases of pandemic influenza a (h n ) from different hospitals in saudi arabia. the age of patients enlisted in the data ranged from to years. the age groups with the highest percentage of cases were between: and years ( %), and and years ( %). there were males and females, and % patients had some contacts with infected persons within saudi arabia while about % had history of travels into saudi arabia and/or the philippines [ ] . these facts are similar to the previous relationship noted between the occurrence of zoonotic viral diseases and the gender of patients and/or their ages, as reported for another viral (e.g., mers-cov) infection; provided certain conditions are met [ ] [ ] [ ] [ ] . interestingly, among elderly patients, influenza cases were higher in females than males. this relationship with viral infection occurring particularly with respiratory viral diseases, might pave the way and play a big role of more significant importance in the detection of these diseases, taking into account the influence of climate change and the different environmental factors [ , , ] . nevertheless, between september and october , about samples were taken from several patients presenting with respiratory symptoms to king abdulaziz university hospital, jeddah, saudi arabia. however, during this study conducted to detect the susceptibility to the influenza viruses circulating in the western part of saudi arabia, out of all the tested samples, ( . %) respiratory samples were positive for influenza h n virus, ( . %) was positive for influenza h n virus, and ( . %) were positive for influenza b virus [ ] . furthermore, h n , now in the post-pandemic period, has become a seasonal influenza virus that continues to circulate with localized outbreaks of varying magnitude [ ] . interestingly, the presentation of influenza virus infections in humans usually vary from mild, self-limiting respiratory-like illness, to severe cases that may result in death [ , ] . nevertheless, a recent study has shown that subclinical infection in human exists, as revealed by the serological surveillance [ , ] . therefore, the epidemiological surveillance of influenza in saudi arabia is highly important especially with the fact that influenza cases have also been highly reported can spread globally [ ] . thus, geographic influences on influenza virus infection in saudi arabia must be of concern [ ] . this is relevant because a remarkably high number of egyptian muslims visit saudi arabia yearly to participate in the umrah and/or the hajj pilgrimage; in addition, the impact of the poultry industry in egypt is also worth considering, with an estimated billion birds and several millions of engaged laborers with/without surveillance [ ] . it is well-known that the influenza drugs, antiviral agents, and the current seasonal influenza vaccines are effective in reducing the incidence and severity of the disease, sickness, and/or complications. however, the important strategy for influenza management includes the provision of prophylaxis and treatment [ ] . however, it is possible for widespread drug resistance against antiviral agents or vaccines to emerge in patients who extensively abused the drugs, in addition to those who have never received such treatment, globally [ ] [ ] [ ] . furthermore, influenza viruses pose a challenge to vaccine developers and manufacturers due to the fact that these viruses are continually changing in nature, including hemagglutinin and neuraminidase [ , ] . moreover, while resistance to neuraminidase inhibitors (e.g., oseltamivir and zanamivir) have been reported to sporadically occur, the resistance to oseltamivir has been widely reported since , with a worldwide spread [ ] . this highlights why there is an urgent need for the public health system to monitor continuously via globally active influenza surveillance programs. furthermore, there is need to monitor the circulating influenza viruses strains, as well as the occurrence of any resistance, using appropriate diagnostic methods. this is considered highly essential in saudi arabia. interestingly, survey data has shown an increasing report of the viral infection from egypt, since hajj egyptians has ranked in the top list of countries with the highest number of mecca pilgrims in the last years [ ] . influenza is highly susceptible to antiviral drugs such as oseltamivir, according to a more recent epidemiological study [ ] . although millions of muslims, globally, travel annually to saudi arabia to perform hajj and/or umrah in the holy places including both makkah and al-madinah for very limited period (~ days), this gathering could play a major role in the introduction of new influenza viruses, not only to saudi arabia but also to the rest of the world [ , ] . unfortunately, there is no such influenza surveillance program in saudi arabia, thus this pose a serious public health concern. recently, in a study of pilgrims screened on arrival at the hajj season in saudi arabia, ( . %) had influenza a virus ( out of the had h n virus) [ ] . additionally, the epidemiological data showed that the pilgrims had the potential not only to introduce these viruses to saudi arabia, but also to export the influenza virus back to their home countries [ , ] . this can occur in saudi arabia, despite the availability of a tiv containing inactivated antigens for influenza virus types a and b, which can protect against the influenza virus infection [ ] . importation of resistant and highly pathogenic viruses including influenza viruses can occur worldwide. despite this, there is lack of studies and data on drug susceptibility, and a very limited number of studies and reports on viral isolates, except for one study conducted in jeddah, according to the best of our knowledge [ ] . most importantly, this highlights the importance of circulating influenza viruses in saudi arabia, hence there is need to ensure effective use of antivirals for prophylaxis and treatment of influenza. furthermore, the rate of vaccination against influenza is very low among pilgrims and healthcare workers [ , ] . moreover, studies are needed to provide a clear picture on the impact of drug resistance on saudi arabia's endemic pathogens, including the influenza viruses. in a recent communique, the ministry of environment, water, and agriculture for saudi arabia reported two cases of h n avian influenza in the kharj governorate [ ] . the latest update by the ministry revealed that the number of samples collected from saudi regions since the start of the influenza outbreak had reached , . positive results from samples and laboratory tests indicate positive cases, and the saudi authorities have taken action by culling as many as , birds within a -h period [ ] . in contrast, several epidemic zoonotic cases of influenza h n have been reported in domestic cats in several countries in asia, europe, the usa, and italy [ ] . moreover, the epidemic of influenza in dogs might be related to a serious public health issue and could be shown to have resulted from zoonotic diseases from pets, similar to the avian influenza h n outbreak reported in pet dogs in south korea in [ ] . nevertheless, a recent study has shown that the role of pets, particularly cats and dogs in the epidemic of influenza as a source of human infection seems limited. however, cats were shown to be fully susceptible to experimental infection, and infected cats were able to infect naive cats [ ] . in , pandemic h n infection in a domestic cat in the usa from iowa was diagnosed by a novel pcr assay; thus, human-to-cat transmission was presumed [ ] . despite this prior evidence, the role of pets including cats and dogs seem even more limited in the dispersal of avian influenza to humans. rather, humans may be the source of pet infection, as suggested for influenza h n and/or h n virus infections [ , [ ] [ ] [ ] . most importantly, epidemic zoonotic cases of influenza among pets has highlighted the importance of circulating influenza viruses globally; especially, to ensure the effective use of antivirals for the prophylaxis and treatment of influenza, in particular, with the increase in the number of pets stores in saudi arabia, especially in riyadh [ , ] . surprisingly, previous data focused on the occurrence of zoonotic infection of different influenza virus types, and particularly, the transmission of avian influenza virus h n to domestic dogs [ ] . several studies have examined and confirmed the occurrence of zoonotic infection of the influenza a virus h n pandemic, especially in domestic cats [ , ] . nevertheless, epidemiological studies on different zoonotic infections among the pets in saudi arabia including cats, dogs, and/or baboons are very rare. however, a previous case report confirmed a relationship between some zoonotic diseases causing respiratory symptoms, such as influenza, among pets [ ] . this study suggests that severe lung infection with dry cough and severe anemia should lead to the suspicion of a secondary infection with zoonotic balantidiasis, which infected a hamadryas baboon from saudi arabia in a research center for pets in riyadh [ ] . furthermore, two other epidemiological zoonotic study on balantidium coli protozoan zoonotic infection in camel was reported from riyadh [ ] . in addition, another previous data confirmed the occurrence of toxocara canis zoonotic infection based on respiratory symptoms reported at the pet clinics in saudi arabia (and also in riyadh where the symptoms occurred in dogs) [ ] . still, more such studies are needed to highlight the important issues and/or provide clearer pictures of the zoonotic pathogens among pets in saudi arabia; however, pet ownership has been growing rapidly as well as the number of pet stores among the saudi population. alkhurma hemorrhagic fever virus (ahfv) in humans was discovered in [ ] . the first case reported in a butcher from the city of alkhurma, a district south of jeddah in saudi arabia, died of hemorrhagic fever after slaughtering a sheep. the viral infection has a reported fatality rate of up to % [ ] . interestingly, one of the previous reports regarding this disease showed a misunderstanding of the real name of this infection, called alkhurma, not alkhumra [ , ] . because subsequent cases were diagnosed in patients from the small town known as alkhurma in jeddah from where the virus got its scientific name; the name was accepted by the international committee on taxonomy of viruses [ ] . thus, based on evidence, the first case was confirmed to be the butcher, following the slaughtered sheep [ ] . therefore, a study was conducted among affected patients to address this disease as a public health issue. blood samples were collected from household contacts of patients with laboratory-confirmed virus for follow-up testing by enzyme-linked immunosorbent serologic assay (elisa) for ahfv-specific immunoglobulin (ig) g. samples from persons seeking medical care were tested by elisa for ahfv-specific igm and igg using ahfv antigen. viral-specific sequence was performed by reverse transcription pcr (tibmolbiol, lightmix kit; roche applied science, basel, switzerland). a total of cases were identified through persons seeking medical care, whose illnesses met the case definition for ahfv, and another cases were identified through follow-up testing of household contacts [ ] . subsequently, the virus was isolated from six other butchers of different ages (between and years) from the city of jeddah, with two deaths. the diagnosis was established from their blood sample tests. the serological tests later confirmed four other patients with the disease [ ] . from to , the study on the virus initial identification in the city of alkhurma again identified other suspected cases; with laboratory confirmation of the disease in (~ %) of them. among the , ( %) had hemorrhagic manifestations and ( %) died [ ] . the virus was later identified in three other locations: from the western province of saudi arabia (ornithodoros savignyi and hyalomma dromedarii were found by reverse transcription in ticks) and from samples collected from camels in najran [ , ] . ahfv virus was considered as one of the zoonotic diseases; however, the mode of transmission is not yet clear. recently, it was suggested that the disease reservoir hosts may include both camels and sheep. the virus might also be transmitted as a result of skin wounds contaminated with the blood or body fluids of an infected sheep; through the bite of an infected tick, and through drinking of unpasteurized or contaminated milk from camels [ , ] . in humans, this zoonotic disease may present with clinical features ranging from subclinical or asymptomatic features to severe complications. it is related to kyasanur forest disease virus, which is localized in karnataka, india [ , ] . however, epidemiologic findings suggest another wider geographic location for the disease in western (including jeddah and makkah) and southern (najran) parts of saudi arabia, and the virus infections mostly occur in humans [ , , ] . a study was conducted by alzahrani et al. in the southern part of saudi arabia particularly in the city of najran (with populations of~ , ), an agricultural city in saudi arabia, where domestic animals are reared at the backyard of owners. after the initial virus identification, from january through april , persons with positive serologic test results were identified. infections were suspected if a patient had an acute febrile illness for at least two days; when all other causes of fever have been ruled out [ ] . additionally, data analysis indicated that patients infected with the virus were either in contact with their domestic animals, involved in slaughtering of the animals, handling of meat products, drinking of unpasteurized milk, and/or were bitten by ticks or mosquitoes. symptoms consistent with ahfv infection-including fever, bleeding, rash, urine, color change of the feces, gum bleeding, or neurologic signs-then develop [ ] . fortunately, infected patients responded to supportive care (including intravenous fluid administration and antimicrobial drugs when indicated), with no fatal cases. in summary, ahfv is a zoonotic disease with clinical features ranging from subclinical or asymptomatic features to severe complications. another study highlighted different characteristics of the exposure to the blood or tissue of infected animals in the transmission of ahfv to humans. of the patients confirmed with infections, % were butchers, shepherds, and abattoir workers, or were involved in the livestock industry [ ] . more recently, a study on infection using c bl/ j mice cells showed that the clinical symptoms of the disease were similar to the presentations in humans [ ] . however, alkhurma disease resulted in meningoencephalitis and death in wistar rats, when high titers to the infection occurred [ ] . in addition, exposures to mosquito bites are regarded as potential sources of transmissions of the infection; however, very few available data support this [ ] . although, available data shows that alkhurma virus has been isolated following mosquito bites [ ] . however, another study suggested that mosquitoes may play a role only as a vector in the transmission of the disease [ ]. cchf is a zoonotic viral disease from the bunyaviridae family, and the principal vector for the disease is ticks of the genus hyalomma. it is most commonly endemic in africa, middle east, asia, and eastern europe [ , ] . it is an acute, highly-contagious, and life-threatening vector-borne disease responsible for severe hemorrhagic fever during outbreaks, and a fatality rate of up to % [ , ] . the infectious disease was recognized first in the crimean peninsula in , and it was named crimean hemorrhagic fever virus because the virus was isolated for the first time from a febrile child in from stanleyville (now kisangani), democratic republic of congo [ ] . currently, the virus infects both humans and animals following tick bites [ ] . however, a human can be infected by the animal through contact with the blood or tissues of the infected animal, in particular, exposures at the abattoirs are common. therefore, workers in contact with animals (e.g., veterinarians, farmers' and workers in slaughterhouses) form a high percentage of those affected [ ] . also, different species of infected animals-such as camels, cattle, sheep, goats, and ostriches-might be infected with no clinical signs [ ] . in addition, human-to-human transmission is also documented, mostly through a form of nosocomial or in-house infection [ , [ ] [ ] [ ] . lately, antibodies to the virus have been detected in different animal species, as reported in , in egyptians animals' sera [ ] . the preliminary seroepidemiological survey detected antibodies to the virus in . % of camels' sera and . % of sheep sera, but no antibody was detected against the virus in the sera of other animals such as donkeys, horses and mules, pigs, cows, and buffaloes [ ] . the epidemiology and distribution of cchf in saudi arabia are unclear, but there are reports of cchf as a result of the trading and importing of infected livestock from neighboring countries to saudi arabia [ ] . in , the cchf virus (cchfv) caused an outbreak involving seven individuals in makkah, although the virus had not been reported previously in saudi arabia. therefore, a study on the epidemiology of this virus was carried out in makkah, jeddah, and taif from - . about out of different species of ticks that were capable of transmitting the disease were collected from camels, cattle, sheep, and goats, but camels had the highest rate of tick infestation ( %), and h. dromedarii was the commonest tick ( %). an investigation in makkah between and , which included a serological survey of abattoir workers in contact with sheep blood or tissue, identified human cases of confirmed or suspected cchf with fatalities [ ] . the report from the investigation stated that the virus might have been introduced to saudi arabia through the jeddah seaport via infected ticks on imported sheep; since then, it has been endemic in the western province of saudi [ , ] . in addition, another previous study confirmed that the highest seropositivity rate of the virus in saudi arabia localities was associated with animals imported from sudan [ ] . furthermore, the who reported countries with cchf including saudi arabia; however, all the remaining countries are either close to saudi arabia or are islamic countries with high numbers of muslims who travel annually to saudi arabia for hajj pilgrimage. the same who epidemiological data suggest that in these countries including saudi arabia, in recent years, there has been report of steadily increasing number of sporadic human cases, incidence, and outbreaks of the virus [ ] . furthermore, another study by who investigating cchfv in the eastern mediterranean region (emr) stated that cchf is a clear and growing health threat in the who emr. cases are being reported in new areas, showing a geographical extension of the disease that is probably linked to the livestock trade and the spread of infected ticks by migratory birds. according to ecological models, the increase in temperature and decreased rainfall in the who emr could have resulted in the sharp increase in distribution of suitable habitats for hyalomma ticks and the subsequent drive of cchfv infection northwards [ ] . jazan province, the red sea port city on saudi arabia's southern border with yemen, serves as the east-west portal from sub-saharan africa at djibouti and the south-north route across the yemeni frontier. it is a heavily traveled corridor for humans and animals entering saudi arabia, particularly during the annual hajj pilgrimage. in november , a total of ( %) enrolled soldiers reported symptomatic illness during deployment, ( %) of whom were hospitalized. reported signs and symptoms included fever (n = ), rash (n = ), and musculoskeletal complaints (n = ). a surveillance study was conducted to detect the causes of the several outbreaks through that area, which was reported as endemic over a wide geographic range. from the surveillance, serologic testing for cchfv, ahfv, denv, and rvf was completed for saudi military units from several saudi arabian provinces. these units were previously stationed in other parts of the country, and were deployed to jazan province; the initial screening for igg of each of these viruses was conducted by igm testing for all igg-reactive samples. among the samples from all military forces, the study identified reactive serum samples with a combined seroprevalence of . cases/ soldiers tested. a confirmed serologic status of soldiers who were evaluated for igg and igm elisa reactivity against cchfv, rvf, ahfv, and denv infections were positive for , , , and sample, respectively [ ] . rvf is a common arbovirus zoonotic disease caused by the rvf virus. the virus belongs to the genus phlebovirus and family bunyaviridae. it is most common in domestic animals, and causes mild to life-threatening infections in humans. the name of the disease was derived from the great rift valley of kenya, when the disease was described for the first time in [ ] . epidemiological tests have since been described after a highly fatal epizootic occurred there in [ ] . rvf is a viral zoonosis with evidence of widespread occurrence in humans and animals in africa and the arabian peninsula. the epidemiology of this virus in saudi arabia might be closely related to the ecological factors that are prevalent, as shown from another area, along the great rift valley, which traverses ethiopia and kenya to northern tanzania with the drainage ecosystems [ ] . saudi arabia has many of the world's mosquito vectors of parasitic and arboviral diseases. however, few studies have addressed their geographic distribution and larval habitat characteristics [ ] . there are complex interactions between these factors that significantly impact mosquitoes ecological fitness and vectorial capacity for disease transmission, with important implications for vector management and control at the local and regional levels [ , ] . therefore, studying these factors for different mosquito fauna will help in monitoring potential modifications of larval habitats due to rains, global climate change, or man-made activities. previous studies on the ecology, distribution, and abundance of mosquito species in kingdom of saudi arabia are generally few and sporadic; and most of these studies were conducted in the western and southern regions. these studies were conducted in the asir province in - and - [ , ] [ , ] . these studies reported the presence of many species from many genera, the most important of which are anopheles, aedes, and culex. among these studies, only a few provided the description of habitats of the larvae of these vectors. even fewer studies provided evidence on the active role of some species on disease transmission; the existing ones were mainly for anopheles vectors of malaria [ , , ] , as well as aedes and culex vectors of arboviruses such as sindbis and dengue fever [ , , ] . rvf is not considered a major type in the arboviruses family, which mostly are adapted to a narrow range of vectors; however, among this family, the rvf infection has a very wide range of vector including mosquitoes such as aedes and culex, flies, and often, ticks [ ] . interestingly, for different rvf species, rvf vectors have special roles about how they sustain the transmission of the disease ecologically to humans [ ] . in some cases, the impact of rainfall, soil type, water, the persistence of breeding, and often wind, have significant effect on vector distribution [ ] . epizootics studies indicate that rvf disease follows unusually severe rainy seasons, a situation that may likely favor the breeding of a very large insect population, needed as a vector prerequisite. globally, rvf epidemiology was first reported in africa with the rvf epizootics in kenya when laboratory test reports confirmed virus isolation [ ] [ ] [ ] . in , the disease, for the first time, affected humans and livestock outside africa, with the larger rvf disease incidence following outbreaks, reported in saudi arabia [ ] and yemen. lately, rvf infections have been associated with minimal genetic diversity, epidemiologically; which has lately been considered to be a newly introduced single lineage of rvf viral disease [ ] . epidemiological reports from both saudi arabia and yemen showed that the outbreak, which occurred in , resulted in about human infections, and deaths [ ] . furthermore, the fatality rate reported in southern saudi arabia then, reached %, and was considered the most severe epidemic in that area ever since [ ] . moreover, the disease outbreak was thought to have been transmitted in countries such as saudi arabia by infected imported ruminants from east africa via the port of djibouti and probably from kenya and/or sudan [ ] . however, the fact remains that the rvf epidemic has been around for more than years, with infections occurring at prolonged intervals in eastern and southern africa [ , ] . consistent with this, another report showed that the same virus strain was implicated in the - rvf outbreaks in kenya and the outbreaks in saudi arabia and yemen [ ] . the outbreaks in kenya later resulted in about , human infected with about patients deaths [ , ] . surprisingly, in , jup et al. found the mosquito species that was identified as a potential vector, which led to the assumption that the zoonotic viral disease in saudi arabia was transmitted by culex tritaeniorhynchus [ ] . other species of mosquitoes were implicated in the transmission of this viral disease in other countries closer to saudi arabia [ ] [ ] [ ] . furthermore, another study reported the unexplained rvf virus infection among people from saudi arabia, with isolation and genetic virus characterization associated with illness in livestock, along the southwestern border of saudi arabia in september [ ] . the study reported that vertical transmission of the virus in the epidemic mosquito vector occurred in saudi arabia. in addition, the study stated that the most abundant culicine mosquitoes collected were aedes vexans arabiensis, culex pipiens complex, and culex tritaeniorhynchus, which were considered to be the most important epidemic and epizootic vectors of rvf virus in saudi arabia [ , ] . however, the same study, focusing on a very important issue which occurred during the rainy seasons; suggested that aedes vexans arabiensis has the potential to be an important epidemic and epizootic vector because of the tremendous numbers of individual mosquitoes produced after a flood [ ] . characteristically, once the virus is introduced into permissive ecologies, it becomes zoonotic; thus, they are able to enhance vulnerability of the area to periodic outbreaks, with the potential to spread further into non-endemic environments with favorable conditions [ , ] . saudi arabia is considered a region where rvf virus has circulated actively. noticeable data regarding zoonotic infection from animal to human from the arabian peninsula including saudi arabia has recently showed that it may be due to the consumption of unpasteurized camel milk [ , , ] . wernery reported camelus dromedarius as the animal host and/or reservoir of rvf zoonotic infection, which was diagnosed in the arabian peninsula [ ] . due to the scientific data regarding rvf disease, it is quite clear that globalization of trade and altered weather patterns are a concern for the future spread of more infections, since the causative agent of this viral disease is capable of utilizing a wide range of vectors for its transmission. thus, this poses a significant challenge to outbreak prediction, with inherently complex methods of infection control; therefore, mitigation and management of the virus will require concerted efforts [ , , ] . dengue hemorrhagic fever (dhf) viral disease is a serious global mosquito-borne infection. the clinical manifestation ranges from mild febrile illness to severe sickness which may include dengue shock syndrome [ ] . the dhf virus belongs to the genus flavivirus in the flaviviridae family, which can usually be spread by mosquitoes of the genus aedes aegypti, but less often through the genus aedes albopictus [ , ] . also, this virus is a single-stranded positive-sense rna virus that exists as four different serotypes (den- , den- , den- , and den- ) [ ] . in saudi arabia, the disease is limited to the western and southwestern regions, such as jeddah and makkah where aedes aegypti exists. however, all dhf cases in saudi arabia presented as a mild disease [ , ] . in fact, the first experience of dhf virus isolation from saudi arabia was recorded during an outbreak of the virus in [ ] , where the confirmed cases reported in jeddah were caused by denv- [ ] [ ] [ ] . however, during this first outbreak, in both summer and rainy season, at the end of the year, both denv- and denv- were isolated. in , during the rainy season in jeddah, there was an emergence of the denv- virus [ ] . in subsequent years, from - ; the emergence of dhf occurred with the three identified serotypes (denv- , denv- , and denv- ) isolated in jeddah [ ] . khan [ , ] . however, egger suggested that the reemergence of the disease in saudi arabia might be explained by the growing levels of urbanization, international trade, and travels [ ] . in keeping with the findings of most previous studies, the epidemiological occurrence of dhf infection using the saudi's national data indicated that the majority ( %) of patients with dengue virus infection were saudi nationals [ ] . on the contrary, from the epidemiological report based on saudi's national data in previous publications, an estimated % of patients with dhf presented in jeddah [ ] . kholedi [ ] . in yet another recent study, the virus was reported as % in saudi patients [ ] . all of these saudi studies were conducted in jeddah. from makkah city, the reported epidemiological study identified . % of dhf infection cases among saudi nationals [ ] . similarly, a later study puts the estimate at more than % of saudi nationals [ ] . these previously published studies suggest that differences in proportions may exist between saudi nationals infected with dhf virus in jeddah and makkah city. contrary to previous data from jeddah, in makkah, it was clear that the majority of patients presenting with clinically significant dhf were saudi nationals. therefore, these results emphasized the fact that saudi nationals are at greater risk of dhf infection. the awareness of these results is considered a cornerstone to enhancing the ability of healthcare professionals' identification of the disease; and this might play an important role in the development of effective eradication strategies for the disease in saudi arabia localities. furthermore, the first cases of the virus, confirmed in al-madinah in , showed that the isolated virus serotypes were denv- and denv- [ ] . in , the moh in saudi arabia reported a total of cases of the dhf infection, with an estimated case fatality rate of about . per thousand in saudi arabia [ ] . in august , several countries in asia, including malaysia, singapore, and pakistan reported about , , , and dengue cases including deaths, respectively. in the same period ( ), saudi arabia reported confirmed dengue cases in makkah, of which occurred in august , suspected cases, and cases pending laboratory confirmations. from these epidemic data indicating the reemergence of dhf infection in saudi arabia; jeddah, makkah, and al-madinah were shown to be the more susceptible areas, for this infectious disease, and this could be due to the fact that these cities are the sites of both the annual hajj pilgrimage and/or the minor umrah pilgrimage, which draw millions of muslims to saudi arabia [ , ] . currently, there are few epidemiological studies on dhf virus infection in saudi arabia. a study by al-azraqi et al. was conducted in hospitals and primary healthcare centers in two cities in the southern province of saudi arabia, particularly in jizan, and aseer. the study, which was limited to the seroprevalence among clinically suspected hospital-based patients, detected about . % positive cases of dengue virus igg among randomly selected patients attending the outpatient clinics for any reason. the associated risk factors were male gender, younger age ( - years) , lack of electricity, and having water basins in the house [ ] . the authors suggested that the virus may occur in sporadic cases in jizan, due to the nature of the city. jizan is relatively flat and located at sea level [ ] ; thus the likelihood of the formation of small stagnant water following the rainfall in the city is high [ ] . interestingly, a retrospective cross-sectional study, which compared the clinical findings and/or the diagnostic laboratory results in uncomplicated patients, and patients who developed dhf, was conducted at dr. soliman fakeeh hospital in jeddah, between january and june . about patients with a discharge diagnosis of dhf or dengue shock syndrome were identified [ ] . of these, ( %) were adult patients within the age range - years, and % were children with age ranging from months to years. however, among all these patients, % of the adults and % of the pediatric cases were males. the clinical data from the hospital showed that in the adult patients, about % made a full recovery without complications while two patients died [ ] . more recently in january , the moh began an intensive campaign to eradicate the dhf virus from saudi arabian cities, to enhance public health awareness, and facilitate a change in hygiene behavior of citizens and residents. this resulted in a . % reduction in the number of dhf infection among inpatient cases in jeddah when compared to the same period in the previous year. however, the overall drop in dhf cases reached % in , compared to the previous year [ ] . furthermore, recently, it is well-known that in saudi arabia, the dhf infection has been limited to the western and southwestern regions such as jeddah and makkah where aedes aegypti exists. however, all dhf cases in jeddah, saudi arabia, were mostly mild cases [ , , ] and the prospect of dengue virus control lies with vector control, health education, and possibly vaccine use. west nile fever is one of the emerging zoonotic infections, which is caused by an arthropod-borne virus belonging to the genus flavivirus, of the rna family flaviviridae. the virus' main reservoir, which is responsible for the transmission of the disease, is the genus culex mosquitoes [ , ] . the west nile virus (wnv) derived the name from the site where the first case was isolated in , from the blood of a woman with mild febrile illness living in the west nile district of uganda [ ] . the first outbreak, in - , was reported in israel [ ] . this constituted a turning point in the epidemiology of the virus, because it was thought to have originated from israel following the introduction from africa, and later introduced to the usa in [ , ] . subsequently, the infection was documented across the globe [ ] , with the exception of antarctica [ ] , in various species of vertebrates, including humans, mammals, non-human primates, birds, rodents, reptiles, and amphibians [ ] . however, birds are considered as one of the main reservoirs of the virus [ ] . saudi arabia is geographically close to several of the countries where wnv had circulated actively or had been reported; thus, there is a high risk of the disease being introduced into saudi arabia. wnv is known to cause neurological disease in both humans and horses. however, the clinical manifestations of the disease in horses include ataxia, paralysis of the limbs, recumbency, hyperexcitability, and hyperesthesia. in al-ahsa, saudi arabia, a study was performed on horses to test the incidence of the virus using the clinical examination and serologic elisa test. however, from this previous study, while clinical examination for neurologic signs detected no significant findings, wnv antibodies were positively identified at serology among . % of the tested population [ ] . in , lanciotti et al. found this virus to be responsible for an outbreak of encephalitis in two fatal human cases from northeastern usa in late summer; and suggest a closely relation between this outbreak in the usa to a wnv infection which was isolated from a dead goose in israel in [ ] . the first cases of wnv in horses was identified in egypt and france in the s [ ] ; ever since, wnv has had significant public health impact worldwide due to its resurgence and dynamic epidemiologic features in humans and animals. between and , a study in iran identified wnv antibodies in horses, and the results confirmed the highest activity of the virus reported in the western and southern provinces with seroprevalences of up to % in some areas of iran [ ] . although human cases and/or animal infections with wnv including horses have also been reported in jordan and lebanon (direct and close neighbors of saudi arabia) between and [ ] [ ] [ ] ; however, the reported wnv in patients or horses in these areas might have circulated in natural transmission cycles with close relationship to the wnv isolated from human and horses in jordan, lebanon, and iran in , , and , respectively. humans and horses (incidental hosts), are unable to develop sufficient viremia to infect mosquitoes, hence, they are not included in the wnv lifecycle [ ] . more recently, in , using standard procedures, the central veterinary research laboratory in dubai, the united arab emirates, described the first wnv isolation in a dromedary calf; and this supports the conclusion that wnv is present in the country [ ] . the wnv zoonotic infection was probably transmitted through the human-animal interface; that is through the well-known contact with infected arabian camels in saudi arabia. interestingly, dromedary are exported from the united arab emirates to saudi arabia and vice versa; due to the closely related wnvs genes and their circulation through the natural transmission cycles worldwide, a complete genome sequencing for more wnvs strains, as well as comparative genomic and phylogenetic studies in saudi arabia, are needed to ascertain whether the dromedary infection with wnv exists in the country or not. however, the same facts have been suggested recently ( ), when it was suggested that wnv infection was introduced into turkey at the time of the outbreaks in saudi arabia and yemen. it was further suggested that the virus may have been introduced via unlawful entrance of viremic domestic or wild animals through the borders or through vectors that carry the virus into turkey [ ] . camels play an important role in public health issues regarding zoonosis and they have been involved in most of the zoonotic infections which occurred in saudi arabia in the last three decades. they are reported as sources of infections-including rabies, mers-cov, alkhurma virus, cchfv, and rvf virus [ , , , , , , ] -via direct physical contacts with camels and/or indirectly by having camels within or near the household in saudi arabia. however, some zoonotic infections among camels are sometimes asymptomatic; thus, they play a vital role in the mechanism of transmission of various diseases [ ] . furthermore, wernery et al. reported that wnv can be transmitted by mosquito bites in different species including to humans, horses, camelids, and many other mammalian species as well as reptiles and birds [ , , ] . to the best of our knowledge, there is still no extensive surveillance data regarding this disease among wildlife animals in saudi arabia. strikingly, several of the human zoonotic cases that involve camels-which included different viral, bacterial, and parasitic infections on the arabian peninsula-have recently been highlighted as being caused by the consumption of unpasteurized camel milk [ ] . currently, in this review, some aspects of the most common viral diseases of zoonotic importance in saudi arabia were summarized; these are presented in table . however, data regarding emerging and reemerging zoonotic viral diseases are reported as they occur from time to time from the same, new, and/or different localities from saudi arabia. while other viral zoonotic infections occur in other countries, which are considered to be close to saudi arabia, some infections spread to some localities within saudi arabia because of the geographical proximity as shown in figure . interestingly, some of these zoonotic viral pathogens were first exotic to saudi arabia (e.g., mers-cov and ahfv) and should be of more concern when reported in prevalence studies, and whenever they are detected by saudi authorities. epidemiological data should be focused more on both the trade routes and wildlife migration across the region, since these are potential risks for saudi arabia (e.g., from yemen, egypt, gulf areas, and sudan). fortunately, there are many ways and/or approaches to improve the control of such different zoonotic pathogens in animals and humans in saudi arabia. however, the control measures of these viral zoonotic pathogens will not only benefit saudi arabia or arabian peninsula but will also be of high benefit to other countries, especially those with low prevalence, by stopping or controlling the spread of the epidemic worldwide. prevention, control, and management of several zoonotic diseases usually require several important measures including the following. having vaccination protocols for all suspected animal species by the use of up to date vaccines and compliance with the standards needed for all animals. taking into account the highly needed and important investigation for these zoonotic viral diseases vectors, including vector breeding control (including vectors, hosts, and arthropods), and control of the animals (livestock) movements, with respect to trade and export [ , ] . because an intensive livestock trade exists between saudi arabia and its neighboring countries, there may be increased risk of reemerging viral diseases of all kinds [ , ] . this is supported by several previous studies concerned with the route of livestock trade between saudi arabia and the neighboring countries (e.g., rabies through yemen and/or oman [ , ] interestingly, some of these zoonotic viral pathogens were first exotic to saudi arabia (e.g., mers-cov and ahfv) and should be of more concern when reported in prevalence studies, and whenever they are detected by saudi authorities. epidemiological data should be focused more on both the trade routes and wildlife migration across the region, since these are potential risks for saudi arabia (e.g., from yemen, egypt, gulf areas, and sudan). fortunately, there are many ways and/or approaches to improve the control of such different zoonotic pathogens in animals and humans in saudi arabia. however, the control measures of these viral zoonotic pathogens will not only benefit saudi arabia or arabian peninsula but will also be of high benefit to other countries, especially those with low prevalence, by stopping or controlling the spread of the epidemic worldwide. prevention, control, and management of several zoonotic diseases usually require several important measures including the following. having vaccination protocols for all suspected animal species by the use of up to date vaccines and compliance with the standards needed for all animals. taking into account the highly needed and important investigation for these zoonotic viral diseases vectors, including vector breeding control (including vectors, hosts, and arthropods), and control of the animals (livestock) movements, with respect to trade and export [ , ] . because an intensive livestock trade exists between saudi arabia and its neighboring countries, there may be increased risk of reemerging viral diseases of all kinds [ , ] . this is supported by several previous studies concerned with the route of livestock trade between saudi arabia and the neighboring countries (e.g., rabies through yemen and/or oman [ , ] ; rvf through kenya, djibouti, and/or egypt [ , , ] ; cchf through sudan [ ] ; influenza through oman and egypt [ , , , [ ] [ ] [ ] ; wnv through emirates, egypt, jordan, and israel [ , , , , ] ; and dhfv through egypt [ ] ; as well as mers-cov and ahfv viral infections, which originated and are transmitted globally from saudi arabia) [ , , , , ] . therefore, it is clear that a huge gap still exists in the sharing of published data about the acknowledged epidemiology of zoonotic diseases in saudi arabia, which rigorously prohibits speculations about the health burden of people. currently, there are surveillance activities for some viral diseases-such as rabies, mers-cov, and influenza-but these are still being weakly addressed or neglected, especially at the human-animal interface. the important role of vaccination both in the prevention and control of animal diseases and the need to check the human sources in food or water must not be neglected. also, management of animals, both outdoors and indoors must be taken seriously. however, owners of pets clinics and pets stores should be held responsible in ensuring that they keep their pets' vaccination protocols up to date, and prevent any kind of animal behavior that might result in zoonotic risks to humans through bites or scratches by pets. therefore, pet clinics and/or pets stores should be always considered a serious public health issue and vaccination should be obligatory. therefore, the importance of the annual vaccination routine programs for all stray dogs against rabies, and regular investigation of other animals, should be considered. in addition to this, pet clinics and stores should monitor pets' health records, and their owners should be held fully responsible in ensuring that their animals remain healthy and fully vaccinated. this will guarantee for them and their neighbors a zoonotic disease-free environment (e.g., against rabies virus particularly in dogs). this is particularly important in view of the case of human rabies reported in march from a makkah hospital. this involved a -year-old saudi man who was admitted to the hospital with a history of an unprovoked scratch on his face by a dog. a month after his admission, his saliva pcr test confirmed rabies virus [ ] . nevertheless, rabies is endemic in animals in the arabian peninsula, with increasing numbers of reported cases form certain countries in the area including saudi arabia, yemen, and oman [ , ] . kuwait, qatar, and the united arab emirates are considered to be rabies-free, whereas there is no available information about bahrain [ , ] . furthermore, animal rabies cycle and cases reported in these endemic countries including saudi arabia are characterized by different animal species such as camels, cattle, goats, and sheep; however, the majority of cases are reported in feral dogs [ , ] . fortunately, studies about pets with different zoonotic infections from pet clinics and/or pet stores in saudi arabia have been rarely detected among cats, dogs, and baboons. however, there was a previous study, which reported the occurrence of toxocara canis infection in pets (dogs) in riyadh, saudi arabia [ ] . there were also two previous reports regarding a protozoan zoonotic infection of some pets with clinical manifestation, particularly in papio hamadryas baboon in riyadh [ , ] . in addition to this, another report highlighted the protozoan zoonotic infection in camels, in riyadh [ ] ; however, more of these kind of studies are needed, because, they provide important opportunities to present a clear picture about indoor and outdoor animals and zoonotic pathogens such as viral, bacterial, fungal, etc. which involved, in saudi arabia. by enhancing biosecurity and management in animal farms, the risk of reemerging pathogens particularly responsible for zoonotic diseases caused by viruses, can be reduced. this is a matter of economic importance; in view of the large livestock trade existing or that existed between countries in the indian ocean and eastern africa countries where several zoonotic diseases are endemic. however, a phylogenetic study strongly suggests that some zoonotic infections have been introduced into saudi arabia through ruminant trade [ , ] . furthermore, following the adoption of the recommended guidelines of the world organization for animal health through its office international des epizooties (oie) code, if such policies regarding the exportation and/or importation of animals are exactly followed, these would greatly limit the extent of this risk [ ] . furthermore, an emphasis should be made on surveillance to detect any sign of zoonotic disease that might occur in any animal kept directly in a quarantine station in any country of origin for days prior to shipment to another country to ensure no clinical sign develops during that period. in addition, the longer quarantine periods or restriction of imported animals-particularly pets (e.g., dogs, cats, rodents, and monkeys) or goats, sheep, and camels-from endemic countries may be effective in reducing the introduction of zoonotic viruses. of such measures, the control of vectors (e.g., ticks and mosquitoes), particularly the intermediate hosts and animal reservoirs, should be key components in the intervention strategy for zoonoses in saudi arabia. while the improving, enhancing, providing, and upgrading of laboratory techniques and/or testing in both veterinary and human medicines are fundamental to early detection and containing of any zoonotic disease or transmitted infection. indeed, epidemiologic evidence should be linked with the seasonal time during the year for different zoonoses, and/or with any symptoms related to zoonotic infections that occur on the mainland a few years earlier. up to date ecological factors on evolutionary issues, social movements, economic, and epidemiological mechanisms affecting zoonotic pathogens' or their persistence and emergence, are not yet well understood. however, studies on the ecological, socioeconomic, and health issues are needed to assess the sustainability and acceptability of measures by breeders, as well as information that ensures appropriate slaughtering or consumption practices, which will decrease the risk of infection to humans [ ] . due to these facts about the ecological cascade and evolutionary perspectives, authorities can provide valuable insights into pathogen ecology and can inform zoonotic disease control programs; and thus evaluate their global effect in terms of actual disease and its socioeconomic correlations. enhancing biosecurity and management in the treatments of various zoonotic infections may result in appropriate use of vaccinations, drugs, and antibiotics, however, the overuse of these agents result in various types of resistance. furthermore, regardless of the influenza virus resistance level to treatment, according to a serosurveillance, the enzootic influenza virus h n in egypt is endemic [ ] . the same result to oseltamivir-resistant influenza viruses are reported globally, with a high susceptibility to these antiviral drugs among all reported cases of the virus from egypt. resistance was also found in most infected viral cases that are usually acquired in humans through intensive contacts, particularly with backyard birds, among women and children [ , , , ] . therefore, drug regimens in saudi must include vaccines against this virus during hajj and umrah seasons, for egyptians. most importantly, epidemic zoonotic cases of influenza among pets has highlighted the importance of circulating influenza viruses globally, and the importance of ensuring the effective use of antivirals for the prophylaxis and treatment of influenza, especially because of the increased number of new pets stores in saudi arabia, particularly in riyadh [ , ] . thus, studies on drug resistance are considered to be of a high public health importance, although, this might demonstrate the best scenario of how drug resistance in saudi arabia can pave its own way and/or role into the reemerging of different zoonotic pathogens. on the other hand, few studies have been done in this area to identify the relationship between different gatherings and the occurrence of signs and clinical symptoms of viral infections, especially among humans of different ages and gender. however, there are several suggestions and information regarding zoonoses (e.g., influenza and mers-cov infections) in saudi arabia among the elderly, based on age and gender [ , ] . more recently, increased availability of limited public health data on the prevalence of some zoonotic diseases and associated risk factors or data that identifies the relationship between different zoonotic pathogen antibodies in pregnant women, are of importance [ , ] . central to the profound worldwide changes in religious beliefs and activities is the birth of a new era of both emerging and reemerging diseases that could be arranged under the umbrella of social movements, along with its own role in the spread of zoonotic diseases. thus, any prevention and/or control strategies against any zoonotic pathogen have to take this point of view into account. furthermore, annually, saudi arabia hosts the largest international gathering of hajj where many millions gather in a small geographical area. this puts saudi arabia in the front line of threats of pandemic diseases [ ] . thus, saudi arabia must keep a high level of alertness in monitoring the situation of these pathogens, particularly in view of the potential for global spread of pandemic viruses especially during winter and around the hajj season (e.g., mers-cov infections, ahfv, and influenza viruses). therefore, there is need to prevent further spread of the virus locally, regionally, and internationally. interestingly, with wnv outbreaks, the israeli-like wnv that was isolated in white storks in egypt in - suggests that migrating birds do play a crucial role in the geographical spread of the virus [ ] . recently, the same fact was again suggested in , when the same infection by this virus was introduced into turkey at the time of the outbreaks in saudi arabia and yemen; it was stated that the wnv virus might have been introduced via unlawful entry of the viremic domestic or wild animals through the borders, or by vectors carrying the virus to turkey [ ] . more recently, epidemiological data of zoonotic viral pathogens from saudi arabia and/or from other neighboring countries after it was confirmed through laboratory test isolation from dromedaries (e.g., rabies, mers-cov, rvfv, and wnv) may enhance a high interest in the search for other novel zoonotic viruses in dromedaries [ , , , , , ] . furthermore, the habits of ingestion off unpasteurized milk from camels as a rare delicacy by saudi people need to be checked. moreover, viral pathogens such as rvfv are acquired through the importation of camels, while the remaining pathogens (e.g., rabies and influenza viruses) are endemic worldwide. of these (e.g., influenza virus), there is need for a highly preventive zoonotic control in saudi, due to fact that the isolation and genetic characterization of h n was reported in among vaccinated meat-turkeys flock in egypt, a neighboring country, that was previously reported to have more than , travelers to saudi arabia during hajj pilgrimage seasons, annually [ ] . this might be considered as one of such important risk factor of possible introduction or spread of influenza pathogen in saudi arabia [ , ] . lastly, increased zoonotic pathogens surveillance, particularly influenza, during the hajj season, increased infection control interventions, screening, and quarantine of suspected cases, provision of adequate medical treatment, sustainable awareness, increased education and training of target groups at high risk (e.g., doctors, nurses, veterinarians, and animal workers such as farmers and abattoir workers, etc.) are of great importance to reduce the burden of zoonoses among saudi arabian localities. fortunately, in collaboration with three organizations-including the moh in saudi arabia, the usa centers for disease control and prevention, and the who-a successful preparedness plan during the hajj season was put in place to vaccinate all pilgrims before leaving their home countries [ ] . altogether, there is an urgent need for collaborative surveillance and intervention plans for the control of zoonotic pathogens in saudi arabia. with saudi arabia, the focal point of the ongoing zoonotic pathogens outbreak could be due to the large number of religious pilgrims congregating annually particularly in makkah, jeddah, and al-madinah, the main three cities for hajj and umrah, which drastically increased the potential for uncontrolled global spread of zoonotic infections [ ] . a zoonotic pathogen outbreak could be dramatically decreased among the annual saudi pilgrims if we take into account the fact that: jeddah governorate, the main seaport in saudi arabia is considered to be the main entry point for over million pilgrims coming for hajj or umrah annually. all these numbers of pilgrims arrive through the jeddah islamic port before going on to makkah, for the start of their umrah and/or hajj. surprisingly, the current review showed that during an outbreak, each of these eight most zoonotic viruses (rabies, mers-cov, influenza, ahfv, cchfv, rvfv, dhfv, and wnv) which occurred and/or cases confirmed in saudi arabia particularly from (jeddah and/or makkah) areas with at least one or all of these eight zoonotic viral pathogenic diseases [ , , , , [ ] [ ] [ ] [ ] , , , ] . the spread could also have been due to the fact that jeddah is the main port for animal importation to saudi arabia. at the same time, it is the closest area to several countries where some zoonotic outbreaks were reported. to enhance this spread, the role of the active circulation of zoonotic viruses, during their natural transmission cycle, has been reported, however, an importation might increase risk of disease introduction to saudi arabia. • almost annually, from the more than million pilgrims who come to makkah and madinah from different countries worldwide during hajj and umrah, the kingdom's revenue in was put at more than billion saudi riyals (~about . billion us dollars), % up from the figures. this hajj revenue accounted for % of the gross domestic product for the kingdom of saudi arabia. to avert all that number of health hazards from zoonotic diseases in view of economic facts, the global community and particularly the pilgrims need more gift items made in saudi arabia to control and prevent the spread of zoonotic diseases which could be transmitted among hajj and umrah pilgrims. therefore, the following recommendations are suggested in order to improve public awareness and/or health education of zoonotic viral diseases in saudi arabia: based on findings of previous studies, health education strategies could enhance the awareness of the saudi population regarding viral zoonotic diseases through health education program experiences of other countries, particularly during hajj and umrah seasons. this response can draw on the availability of several studies on how to improve, control, and prevent the spread of several zoonoses in both animals and humans, worldwide [ , [ ] [ ] [ ] [ ] . public health authorities must highlight the importance of promoting health education and facilitate the outcomes of studies for reducing patient cases in saudi arabia. the saudi authorities and government bodies such as the moh should also launch different programs and workshops to increase public awareness about these zoonotic infections. this should involve the cooperation of the saudi regime, and the private and public sectors. different activities may be needed in saudi arabia-such as the practice of self-protection against these diseases, adult control strategies, control activities, and regular workshops-to achieve control and prevention. enhancing of self-awareness among people through health education programs or other strategies for the prevention of viral zoonotic diseases, which require vectors (such as mosquitoes, ticks, and fleas) for their transmission; are important issues on which the saudi population should be educated. they should also be educated about the adverse effects of arbitrary application of insecticides without prior knowledge on dose, resistance, and side effects. increasing the knowledge about the biology and ecology of the animal vectors in society is also crucial. furthermore, the saudi ministry of culture and information should establish intensive health education programs on television channels, radio, and newspapers to increase public awareness and to maintain hygiene conditions within the kingdom and in saudi houses. the saudi ministry of agriculture could play a big role by regularly controlling the application of vaccinations and/or antibiotics on animals which used in the veterinary sector, and also accounting the misuse of such agents following other developed and developing countries on controlling and/or accounting drug strategies [ , , ] . thus, veterinary regulations of animal antibiotics-including overuse of drugs and their application-must be enforced to alleviate the serious public health problems. funding: this research received no external funding. the authors thank the dental oral rehabilitation (dor) research center at king saud university, college of dentistry, kingdom of saudi arabia, riyadh for 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countries: causes and control strategies key: cord- - qri n authors: shi, mang; zhang, yong-zhen; holmes, edward c. title: meta-transcriptomics and the evolutionary biology of rna viruses date: - - journal: virus res doi: . /j.virusres. . . sha: doc_id: cord_uid: qri n metagenomics is transforming the study of virus evolution, allowing the full assemblage of virus genomes within a host sample to be determined rapidly and cheaply. the genomic analysis of complete transcriptomes, so-called meta-transcriptomics, is providing a particularly rich source of data on the global diversity of rna viruses and their evolutionary history. herein we review some of the insights that meta-transcriptomics has provided on the fundamental patterns and processes of virus evolution, with a focus on the recent discovery of a multitude of novel invertebrate viruses. in particular, meta-transcriptomics shows that the rna virus world is more fluid than previously realized, with relatively frequent changes in genome length and structure. as well as having a transformative impact on studies of virus evolution, meta-transcriptomics presents major new challenges for virus classification, with the greater sampling of host taxa now filling many of the gaps on virus phylogenies that were previously used to define taxonomic groups. given that most viruses in the future will likely be characterized using metagenomics approaches, and that we have evidently only sampled a tiny fraction of the total virosphere, we suggest that proposals for virus classification pay careful attention to the wonders unearthed in this new age of virus discovery. metagenomics is transforming the study of virus evolution, allowing the full assemblage of virus genomes within a host sample to be determined rapidly and cheaply. the genomic analysis of complete transcriptomes, so-called meta-transcriptomics, is providing a particularly rich source of data on the global diversity of rna viruses and their evolutionary history. herein we review some of the insights that meta-transcriptomics has provided on the fundamental patterns and processes of virus evolution, with a focus on the recent discovery of a multitude of novel invertebrate viruses. in particular, meta-transcriptomics shows that the rna virus world is more fluid than previously realized, with relatively frequent changes in genome length and structure. as well as having a transformative impact on studies of virus evolution, meta-transcriptomics presents major new challenges for virus classification, with the greater sampling of host taxa now filling many of the gaps on virus phylogenies that were previously used to define taxonomic groups. given that most viruses in the future will likely be characterized using metagenomics approaches, and that we have evidently only sampled a tiny fraction of the total virosphere, we suggest that proposals for virus classification pay careful attention to the wonders unearthed in this new age of virus discovery. our knowledge of the virosphere is scant. although viruses are the most abundant source of nucleic acid on earth, with every species of cellular life likely harboring multiple viruses, until recently most studies of virus biodiversity and evolution were of limited scope, with a strong focus on aquatic environments and prokaryotic dna viruses (angly et al., ; culley et al., ; desnues et al., ; paez-espino et al., ; philosof et al., ) . far less is known about the diversity of rna viruses in terrestrial organisms. this has begun to change following advances in bulk genome sequencing that have initiated a new age of virus discovery, in which it is now possible to rapidly document the entire virome of groups of host organisms (li et al., ; shi et al., a) . as well as greatly expanding our knowledge of virus diversity, including the 'dark matter' of highly divergent viruses that often elude characterization, these new data will enable us to determine the fundamental evolutionary and ecological processes that shape the virosphere, and better understand the virus-host interactions that lead to disease emergence. it is also clear that the virus diversity generated from these genomic studies will radically shake-up attempts to classify the virus world (simmonds et al., a) . one genomic technique that is already having a major impact on studies of virus diversity and evolution is rna-seqa whole transcriptome shotgun sequencing approach that enables enormous amounts of rna sequence to be generated rapidly (palacios et al., ; we describe the technique in more detail below). as the transcriptome data generated by rna-seq is able to provide an unbiased and likely comprehensive view of all the viruses present within a host samplethat is, their complete viromeit can also be thought of as 'metatranscriptomics'. the data generated by meta-transcriptomics is a rich source of evolutionary and ecological information. as a case in point, meta-transcriptomic studies of invertebrates have unearthed remarkable levels of untapped virus genetic diversity, such that the virosphere is evidently far broader and more complex than previously anticipated (li et al., ; shi et al., a; webster et al., ) . for example, an analysis of species from nine invertebrate phyla identified a remarkable novel rna viruses, as well as potentially novel genera and families (or orders) (shi et al., a) . aside from its evolutionary utility which we will discuss in more detail below, meta-transcriptomics allows the identification of novel microbial pathogensthat is, those associated with overt disease in their hostson clinically actionable time-scales (wilson et al., ) . indeed, it is possible that with a continually declining cost meta-transcriptomics may eventually be used for routine microbiological diagnostics. a key advantage of this over other diagnostic techniques is that it has the potential to detect, in an unbiased fashion, any pathogen that produces an rna molecule (dna viruses, bacteria, fungi, eukaryotes) , as well as the obvious case of rna viruses. hence, if appropriate tissues are analyzed meta-transcriptomics may provide a one stop diagnostic shop. as much as metagenomics is transforming studies of virus evolution, it is also the case that it has shone a bright light on fundamental gaps in our understanding of the virus world. most obviously, it is evident that we have only just begun to scratch the surface of the true diversity of viruses that make up the virosphere, and the factors that shape this diversity and evolution within ecosystems and over long-term evolutionary scales are largely unknown. herein, we will review what, in our opinion, meta-transcriptomics has told us about virus diversity, evolution and taxonomy, and provide some suggestions for future work in this area. before the advent of dna sequencing, new viruses were discovered using a variety of approaches, including filtration, cell culture, electron microscopy, and serology. many of these techniques remain important in virology (leland and ginocchio, ) . indeed, the propagation of viruses in cells, accompanied by the visualization of virus particles by electron microscopy and the successful replication of infection in animal models, can still be considered the gold standard for virus discovery. however, the substantial time and effort required for work of this kind means that it is often impossible. in addition, most viruses are not culturable and there are not enough cell lines to meet the diversity of viruses. more modern approaches of virus discovery involve the determination and comparison of viral nucleic acids. this combination of pcr and sequencing can be used to screen for infectious agents using degenerative primers targeting conserved genomic regions, thereby identifying novel, but related, viruses with great sensitivity. this approach has been very successful in virus discovery, with notable examples including bat influenza a virus (tong et al., ) and rodent hepaciviruses (drexler et al., ) . however, the drawback of consensus pcr is that it is heavily dependent on currently available sequences and hence has limited capability to detect more divergent viruses. it can also be tedious to design and run consensus pcr for a large number of different virus families. the most robust, although costly, method of virus discovery is through a coupling of metagenomics and high-throughput sequencing technology. indeed, metagenomics provides an unbiased survey of the genetic material within a sample, and has revolutionized virus discovery in terms of speed, accuracy, sensitivity, and the amount of information generated (firth and lipkin, ) . among the various metagenomics approaches are available, meta-transcriptomics has recently come to the fore. this approach involves gathering total transcriptome information from a host sample after depletion of ribosomal (r) rna, as this is the dominant component of the host transcriptome. compared to metagenomics protocols that involve viral particle enrichment (reviewed in kumar et al., ) , this method is far simpler yet still achieves a high level of sensitivity, generality, and efficiency for virus discovery (fig. ) . previous methodologies were often based on removing as much nucleic acid outside viral particles as possible by filtering, centrifugation, lysis, and nuclease treatment, although this seldom results in a complete depletion of host rna (firth and lipkin, ; mokili et al., ) . in contrast, in meta-transcriptomics total rna (i.e. the transcriptome) is directly extracted from untreated homogenates and used for library preparation without filtering and nuclease digestion steps. another benefit of meta-transcriptomics is that it provides a ready way to quantify each virus present in a sample. specifically, the percentage of reads that map to a particular virus genome is a good indication of how abundant any virus is, especially in the context of conserved host genes (shi et al., a; shi et al., ) . in turn, abundance level can provide important pointers to disease associations, whether viruses are segmented (such that genomic components have similar or different expression levels), and help identify those viruses that are in fact derived from other eukaryotic organisms present in the host sampled, such as in undigested food or prey, gut micro flora, and parasites, or simply contamination (and the greater the virus abundance, the more likely that active viral infection has occurred in the host under consideration). in addition, compared to genomic nucleic acid, the transcriptome comprises compact information that is more balanced across domains of life, thereby preventing the over-dominance of genetic information from large cellular organisms. those meta-transcriptomic studies undertaken to date have transformed our understanding of the extent and nature of viral biodiversity, making it abundantly clear that we have only sampled a tiny fraction of rna virus biodiversity (as will also be true of dna viruses). indeed, it is likely that the diversity of uncharacterized viruses far exceeds that of those that have been classified to date (fig. ) . these studies also highlight the inherent bias toward studying viruses that can be cultured, or associated with overt disease, which in turn reflects a longerstanding historical preference to studying viral infections in humans and economically important plants and animals. as is discussed in more detail below, it is possible that such highly biased sampling has distorted our view of virus evolution. what is perhaps more daunting is that these studies have only been conducted in a small number of sampling locations, often in china. it is therefore simple to predict that we will identify a legion of new viruses in the near future, especially given that only a minuscule fraction of the perhaps eight million eukaryotic species (many of which are marine) have ever been sampled for viruses. indeed, it was recently estimated that approximately . % of the eukaryotic virosphere remains undiscovered or unclassified . the reality, therefore, is that our study of virus diversity and evolution, and hence taxonomy, has only just begun. a powerful example of how meta-transcriptomics is changing our understanding of virus diversity was the discovery of chuviruses in (li et al., ) , that have recently and rapidly been accepted as a new family of negative-sense rna viruses by the international committee on taxonomy of viruses (ictv). although the chuviruses form a monophyletic group in phylogenetic trees of the rna-dependent rna polymerase (rdrp), they contain a diverse array of genome structures, including both segmented and unsegmented representatives, as well as a potentially circular form that would be unique among rna viruses. it is highly likely that similarly diverse new families will be identified in the future. there are also huge differences between the diversity revealed by previous culturing and pcr-base methods and by metagenomics, again highlighting the biases that detection method may have introduced into our understanding of natural viromes. for example, considerable effort has been directed toward isolating and culturing mosquito viruses that are relevant to humans, such as flaviviruses, alphaviruses and orthobunyaviruses. in reality, however, these disease agents represent a tiny fraction of the mosquito virome (hall et al., ; junglen and drosten, ; vasilakis and tesh, ) , which in fact comprises representatives from every major virus group, that are more prevalent in the mosquito population, have much higher abundance, and are often transmitted vertically (cook et al., ; vasilakis and tesh, ; shi et al., ) . the new wealth of diversity revealed by meta-transcriptomics also shows that the virus world is far more connected than we previously thought. new broad-scale rdrp phylogenies have shown that virus families, orders, floating genera, and undefined lineages can often be amalgamated into larger groups, such that they exhibit an evolutionary continuity (shi et al., a) , in turn providing compelling evidence for their common origin (koonin et al., ) . it is obvious that the increasing number of newly described viruses from diverse hosts will continue to fill 'gaps' in phylogenetic diversity (i.e. the long branches present in inter-virus phylogenies) resulting in a more robust and stable depiction of virus evolutionary history. it is now clear that invertebrates carry a huge diversity of rna viruses, including the potential ancestors of many those viruses found in vertebrates (junglen and drosten, ; li et al., ; marklewitz et al., ; nga et al., ; shi et al., a; webster et al., ) . given their vast diversity, abundance and often huge population sizes, it is no surprise that invertebrates harbor such a high number and diversity of rna viruses. although they are the most sampled group, arthropods may be especially important in this evolutionary arena because of their strong ecological relationship with both plants and vertebrates, and a phylogenetic mix between these taxa is becoming increasingly apparent (li et al., ; shi et al., a) . what is far less clear is how frequently this huge array of invertebrate viruses is associated with overt disease in their hosts and, if invertebrates are largely refractory to disease, how this is mediated. the orthomyxo-like viruses provide an informative example of how the sampling of invertebrate viruses has changed our perspective on virus evolution. prior to the orthomyxoviruses comprised a small group of vertebrate (mammal and bird) and tick-associated rna viruses that were best known through influenza virus and classified into five genera (allison et al., ; presti et al., ) . however, subsequent studies have revealed a remarkable diversity of orthomyxo-like viruses in invertebrates, including mosquitoes, cockroaches and earthworms, that fell both basal to, and interleaved among, the previously known genera on phylogenetic trees (li et al., ) . hence, the gaps on the tree have been dramatically filled and the previous genera no long appear as phylogenetically distinct groups. in addition, that all orthomyxo-like viruses currently sampled are segmented shows that this form of genome organization is an ancient innovation in this group. despite the recent dramatic expansion in the number of invertebrate viruses, it is striking that some families rna viruses remain vertebratespecific and contain no invertebrate viruses, with the arenaviridae, paramyxoviridae and picornaviridae providing important examples. clearly, the monophyletic nature of vertebrate-specific viruses implies that have had a long-term evolutionary association with vertebrate hosts. also, although some invertebrate viruses appear basal to vertebrate viruses, the distance between them are often substantial and phylogenetic relationships are not always stable. therefore, while it is tempting to conclude that most, if not all, families of vertebrate viruses will have their ultimate ancestry with invertebrates, particularly as so very few of the latter have been sampled, it would be wrong to think that this a forgone conclusion. determining the host range of viruses is essential to understanding the process of cross-species transmission that underpins disease emergence. meta-transcriptomic data provide a ready means to determine what viruses are present in which hosts and allows a simple measure of virus abundance. equally important is that the meta-transcriptomic sampling of an increasing number and diverse set of hosts has fundamentally changed the view of the host structure of major virus groups. fig. . comparisons of virus enrichment and meta-transcriptomics approaches for rna virus discovery. the workflow of a typical virus enrichment approach is marked in blue, whereas that of a metatranscriptomics approach is marked in red. before the metagenomics revolution the virus diversity within a specific family was often dominated by particular host groups; so, for example, vertebrate, insect, and plant viruses often fell into distinct taxonomic groups. this has changed dramatically with meta-transcriptomics. for example, the family totiviridae, previously thought to be largely associated with fungi, are now commonly found in metazoa. similarly, some previously defined families of plant viruses, such as the tombusviridae and luteoviridae, have expanded to include viruses from arthropods, nematodes, molluscs, and protists (shi et al., a) . given such a complexity of host structure, combined with still very sparse sampling, it is dangerous to construct detailed ancestor-descendant relationships on the currently available data. for example, arthropods were initially proposed to be the ancestral hosts of bunyaviruses (marklewitz et al., ) , although more divergent viruses in this group have now been discovered in other invertebrates, fungi, and protists (akopyants et al., ; shi et al., a) . the combination of meta-transcriptomics and phylogenetics has also told us that virus evolution is a complex interaction between cross-species transmission and virus-host co-divergence, with the evolutionary history of many virus groups reflecting an interweaving of both processes . however, given their complexity and the often great genetic distances between virus genomes, determining the precise sequence of cross-species transmission and codivergence events that have shaped the evolutionary history of a particular group will undoubtedly be challenging and require a denser sampling of host taxa. indeed, the greater diversity of hosts sampled, the more cases of species jumping we are likely to document . although the occurrence of virus-host co-divergence has long been suggested, meta-transcriptomic-based studies indicate that this may extend even further back in time than previously suspected. for example, one interpretation of the evolutionary relationships within the narna-levi clade of rna viruses is that there has been virus-host co-divergence since the α-proteobacteria became endosymbionts (shi et al., a) . at the same time, however, it is clear that cross-species transmission has occurred frequently, even among phylogenetically divergent taxa, and is likely the dominant mode of fig. . current taxonomy of rna viruses in the context of the genetic diversity revealed by meta-transcriptomics. the phylogenies are based on rdrp amino acid sequences from a broader analysis as performed by shi et al. ( a,b) (and see this paper for a description of branch lengths and rooting schemes). the taxonomic groups (i.e. genus, family, and order) established by ictv are shown to the left of each phylogeny. finally, although meta-transcriptomics has profound implications for our understanding of virus evolution, it likely undermines biodiversity-based attempts to predict the virus source of the next major disease pandemic (olival et al., ) . although the bulk sequencing of potential animal reservoir species as been proposed as a way to better predict of what types of virus may emerge in human populations in the future, and where this may occur, in reality disease emergence is a nuanced process that entails a complex interaction of ecological and genetic factors (parrish et al., ; plowright et al., ) . metatranscriptomics tells us that there are so many viruses in nature that trying to establish which will ultimately appear in a new host from diversity sampling alone is almost certainly a futile exercise. this is apparent in the current vogue to study bat viruses. since the emergence of sars coronavirus in humansa pathogen that has its ultimate ancestry in batssampling bat viruses as a means to determine which next might emerge in humans has received considerable attention (smith and wang, ) . while these studies have made it clear that bats indeed harbor an enormous number of viruses (anthony et al., ; luis et al., ; olival et al., ) , at the same time they clearly show that the vast majority of these viruses have not jumped to humans. the true goal of studies of disease emergence should therefore be to reveal that combination of genetic and ecological factors that underpins successful cross-species transmission and emergence. one of the most important impacts of metagenomic data has been to change our understanding of the structure of virus genomes and the evolutionary processes that have given rise to them. suffice to say, rna virus genomes are more diverse, have more complex structures, and a wider range of lengths than previously anticipated. although the reasons for this diversity and the birth of individual genes are uncertain, one process of undoubted importance is inter-specific recombination, including lateral gene transfer (krupovic et al., ) . this evidently occurs more frequently than previously anticipated, and can involve both structural and non-structural genes, with even evidence that cellular genes can be integrated into viral genomes (shi et al., a) . indeed, an emerging view is that rna viruses experience as complex processes of genome evolution as in dna organisms. to better determine the evolutionary processes that shape viral genome structures, and hence how new viruses are created, it is important to use the new wealth of meta-transcriptomic data to carefully determine the frequency, pattern and history of gene duplications and losses, lateral gene transfers, and genomic rearrangements; combined, these will provide a more complete picture of genome-scale evolutionary processes obtained. another component of rna virus genome organization that has proven more fluid than previously envisioned is segmentation. families of rna viruses were generally thought to be characterized by a specific segmentation type, such as the presence/absence of segmented genomes or certain number of segments. however, segmentation no longer appears to be a strong taxon defining trait, and a combination of segmented and unsegmented genomes has now been observed within families of rna viruses. an informative example is presented by the flaviviridae and their relativesthe so-called 'flavi-like' viruses. traditionally, flaviviruses were considered to be small (∼ kb) unsegmented positive-sense rna viruses that infected vertebrates; if invertebrates were involved then it was as vectors of these viruses among vertebrates, particularly mosquitoes and ticks (simmonds et al., b) . meta-transcriptomic studies have radically changed this view, including the identification of a large number of 'insect-specific' flaviviruses (bolling et al., ; may et al., ; qin et al., ; shi et al., b) . indeed, flavi-like viruses now appear to be a group of predominantly invertebrate rna viruses with the potential to have very large genomes (∼ kb) and which can be arranged in four or five segments (ladner et al., ; qin et al., ) . even more dramatic is that some of these flavi-like viruses appear to comprise distinct virus particles such that they are multipartite, a form of genome organization that was previously thought to be the exclusive domain of plant rna viruses (ladner et al., ) . despite such a data revolution, one key feature of rna virus genomes that has held firm in the metagenomics revolution is an upperlimit on genome length of < kb, with ball python nidovirus exhibiting the largest rna virus genome reported to dateat . kb (stenglein et al., ) . although there is still debate as to the cause of this size limit, it is tempting to think that it reflects the high rate of rna virus evolution and the mutational burden this entails, particularly since single-stranded dna viruses, that also mutate rapidly, similarly possess small genomes (holmes, ) . of course, it is possible that the length profiles of viruses will radically change with increased sampling, and an rna virus with the length and complexity of a large doublestrand dna virus stands represents something of a virological holy grail. the lessons learned from evolutionary studies of meta-transcriptomic data clearly have important implications for rna virus taxonomy and classification, and we will consider some of these here. most obviously, that the virosphere is vast and we have only searched a tiny fraction of it leads us to believe that the 'traditional' way to perform virus taxonomy is dead. given the huge number of viruses that exist in nature , it is both practically impossible and inherently pointless to isolate of all these, determine their structure, and measure their ability of replicate in cells of different types. indeed, there is now a growing recognition that the primary way in which viruses will be characterized in the future will be through metagenomic surveys (simmonds et al., a) , with complete 'classical' virological investigations only being performed on that subset of viruses that may be of special interest or that can be considered as markers of specific groups. metagenomics has already revealed the challenges facing current virus classification, with increased sampling challenging the criteria proposed to define many groups (simmonds et al., a) . a key issue is that the genome structures that have been used as criteria for classification, such as segmentation and orf arrangement, are no longer 'conservative' enough over broad evolutionary timescales. an informative example is provided by the mononegaviralesan order of viruses originally characterized by unsegmented negative-sense rna genomes and which has recently been the subject of considerable attention from the ictv. although use of the taxonomic term 'mononegavirales' is growing in popularity, it now makes little sense in its strict literal definition as rdrp-based phylogenies show that this group contains segmented viruses, so that they no longer fulfil the criterion of possess a single ('mono') negative-sense rna molecule (li et al., ) , with genome segmentation evolving a number of times independently. similar stories can be told for the flaviviridae and the totiviridae that were originally defined based on single segment but are now found to be closely related to viruses with multiple segments (li et al., ; qin et al., ; sasaya et al., ) , and the partitiviridae and picobirnaviridae that were thought to be bisegmented yet now include viruses containing one to six segments. these growing number of these 'exceptions' have often been classified as separate families or floating genera, in doing so ignoring their evolutionary relationships. another important limitation of the current classification system is that equivalent taxonomic groups can vary enormously in their component genetic diversity. although this is a common problem in classification, and in large part reflects the fact that some families have a much longer evolutionary history than others, it is especially prominent in rna viruses. the reason for such imbalance again points to the sometimes shaky criteria used for viral classification. for example, the 'hepe-virga' clade (also known as the alpha-like supergroup) are relatively closely related in rdrp phylogenies yet the ictv divides them into one order (tymovirales), eight families (the virgaviridae, togaviridae, bromoviridae, closteroviridae, endoranviridae, alphatetraviridae, hepeviridae, and benyviridae), and three floating genera (negevirus, idaeovirus, and cilevirus) . although this clade does possess some divergent genome structures, with differences in segmentation, orf arrangement, genome length, and even the genome sense, its rdrp diversity is no larger than that of reoviruses that are still classified as a single family thanks to a stable genome plan. in other cases these taxonomic differences appear to be largely arbitrary. for example, in the newly established order bunyavirales (https://talk. ictvonline.org/taxonomy/), the jonviridae, feraviridae and phasmaviridae are defined as separate families, although they form a single rdrp cluster whose diversity is significantly smaller than those of some individual families, such as the phenuiviridae and the peribunyaviridae. although there have been clear improvements in making virus classifications more compatible with underlying phylogenetic relationships, there are notable exceptions. for example, the togaviridae comprise two genera, alphavirus and rubivirus, that do not share common ancestry in phylogenies of either their replicase or structural proteins. at the very least proposals for individual taxonomic groups should be monophyletic, which is not always the case (kuhn et al., ) . we also contend that it is naive to think that the structure of virus diversity in nature, and the phylogenetic analysis of this data, will necessarily produce a simple and stable classification scheme. first, the boundaries we draw to mark higher virus taxa are inherently arbitrary, rather than reflecting a hard evolutionary 'rule', and we should not expect nature to provide neat boundaries for classification. as noted above, the gaps apparent in many phylogenetic trees will likely be filled by newly discovered lineages as our sampling becomes more extensive. hence, phylogenetic gaps do not necessary reflect a fundamental evolutionary process, but are likely an artefact of sparse and inadequate sampling. indeed, from a metagenomic perspective virus species will simply be points in phylogenetic space, and viruses 'species' differ fundamentally from those in diploid outcrossing animals in which the term has a real biological meaning. at a lower taxonomic level, using genetic distance cut-offs to determine taxonomic differences within virus species, particularly genotypes, is also fraught with difficulties as different schemes are used in different viruses and all such rules of distance may break down if there is extensive rate variation among taxa and if our sampling is biased toward specific geographic locations. it is also important to recall that virus gene trees are not the same as species trees, such that phylogeny-based classifications will often be only genic in nature. because of high levels of sequence divergence it is necessarily the case that most deep (particularly inter-family) virus phylogenies are based on the analysis of rdrp alone. however, given the dynamic nature of virus genome organization, particularly the occurrence of lateral gene transfer, it is certain that in many cases the phylogeny of the rdrp will not match that of the virus genome as a whole. for example, the luteoviridae are currently defined based on the relatedness of the structural proteins, although the replicase sequences of these viruses do not form a monophyletic group. unfortunately, phylogenetic analyses of other genes, particularly those that encode structural proteins, often present an unsurmountable challenge for sequence-based analytical methods because of the huge sequence distances involved (holmes, ; zanotto et al., ) . it is therefore an inconvenient truth that while phylogenies based on the rdrp can sometimes accurately depict the evolutionary history of that gene, they do not necessary reflect that of the virus as a whole. although there are pros and cons to using either replicase or structural genes to determine phylogenetic relationships, the fact that they often give contrasting views of evolutionary history clearly complicates virus classification. most importantly, phylogenetic trees are only ever able to depict the relationship among those viruses that are present in the sample of viruses under study; as our sample is likely negligible, so our classification is necessarily incomplete. a more fundamental question is whether the current classification scheme can withstand the onslaught of metagenomic data? the proliferation of 'family-like' viruses revealed from meta-transcriptomic surveys amply highlights the scale of the challenge facing taxonomists. as emphasized throughout this paper it is clear that we are still only scratching the surface of the virosphere and that we evidently have a great deal to learn about virus diversity and evolution. as well as revealing an abundance of new virus taxa, and determining the evolutionary processes that have shaped this diversity, it is undoubtedly the case that viruses exist in hosts that have not been screened for rna viruses or that are so divergent in sequence that they cannot readily be detected by standard homology-based methods (such as blast) or included in phylogenetic analyses. if the nature of this dark matter can be resolved it will surely shed new light on the ultimate origins of viruses as well as their deep phylogenetic relationships. the situation is particularly acute in the case of the archaea in which only a single putative rna virus has been described to date (bolduc et al., ) , and which in large part may reflect our current inability to identify viruses that possess highly divergent genome sequences. it is therefore of critical importance to perform unbiased metagenomics surveys of prokaryotic taxa that have not been examined to date, followed by novel bioinformatics analyses that are able to accurately identify viruses and reveal their phylogenetic relationships. this will entail the characterization of the unknown biodiversity of rna viruses in prokaryotes and basal eukaryotes and, in parallel, developing and utilizing new computational tools to robustly extract sequence information from highly divergent genome sequences. similarly, the increasingly frequent detection of recombination and lateral gene transfer also poses a major challenge to current phylogenetic protocols and may require a new computational tool-kit (iranzo et al., ; koonin and dolja, ) . our knowledge of the evolutionary processes that have generated the diversity of the virosphere has been strongly skewed by a focus on those viruses that act as agents of disease in economically important animals and plants and those that can be easily cultured. importantly, recent work has shown that animals harbor enormous uncharacterized viral diversity, only some of which has been associated with disease. however, these viruses still only reflect a tiny proportion of those in nature and therefore provide an incomplete picture of the major processes of virus ecology and evolution. key questions for future research that can be addressed with the new wealth of meta-transcriptomic data include (i) determining the flow of viruses between host taxa and the processes that shape virus ecosystems; (ii) revealing the mechanisms of long-term virus macroevolution, particularly lineage birth and death, and (iii) revealing the mechanisms and evolutionary processes that structure viral genomes. rather than simply surveying biodiversity and classifying, the goal for the future should be to perform more ecologyfocused studies to reveal fundamental patterns and processes. it is critical that studies of virus diversity evolution shape our attempts to classify these infectious agents, rather than classification schemes guiding how we think that viruses have evolved. finally, we contend that is perhaps premature to construct inflexible and overly hierarchical classification schemes for rna viruses when we have clearly sampled so little of what is there in nature. the new age of virus discovery will undoubtedly provide many new challenges for the science of virus classification. the authors declare that no competing interests exist. a novel bunyavirus-like virus of trypanosomatid 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reevaluation of the higher taxonomy of viruses based on rna polymerases this study was supported by the special national project on research and development of key biosafety technologies ( yfc ), the th five-year major national science and technology projects of china ( zx - ), and national natural science foundation of china (grants , ). ech is funded by an nhmrc australia fellowship (gnt ). key: cord- -lmkfxme authors: schafrum macedo, aline; cezaretti feitosa, caroline; yoiti kitamura kawamoto, fernando; vinicius tertuliano marinho, paulo; dos santos dal‐bó, Ísis; fiuza monteiro, bianca; prado, leonardo; bregadioli, thales; antonio covino diamante, gabriel; ricardo auada ferrigno, cassio title: animal modeling in bone research—should we follow the white rabbit? date: - - journal: animal model exp med doi: . /ame . sha: doc_id: cord_uid: lmkfxme animal models are live subjects applied to translational research. they provide insights into human diseases and enhance biomedical knowledge. livestock production has favored the pace of human social development over millennia. today's society is more aware of animal welfare than past generations. the general public has marked objections to animal research and many species are falling into disuse. the search for an ideal methodology to replace animal use is on, but animal modeling still holds great importance to human health. bone research, in particular, has unmet requirements that in vitro technologies cannot yet fully address. in that sense, standardizing novel models remains necessary and rabbits are gaining in popularity as potential bone models. our aim here is to provide a broad overview of animal modeling and its ethical implications, followed by a narrower focus on bone research and the role rabbits are playing in the current scenario. rabbits have been used for decades by researchers in diverse scientific fields. however, only recently have they been targeted as potential bone models. with great importance in age-related bone loss research. , here, we first present a broad historical review and some key ethical points in animal modeling. we then take a closer look at bone research and the role rabbits play in this field. animal domestication was a significant turning point for mankind. human society developed into what it is today due to livestock production, and animals still provide us with food, clothing, transportation, protection, and companionship. , nowadays they contribute to human well-being in additional ways: by helping people with visual impairment or diabetes, by taking part in police enforcement, or even by entertaining people in animal shows, zoos, and social media. animals have also been pivotal to our medical knowledge and health status since ancient greece. , the first animal studies provided understanding of biological pathways and disease mechanisms. animal dissection proved to be a valuable substitute for human dissection -an illegal practice in ancient times. several philosophers and physicians, from aristotle to diocles and erasistratus, experimented on animals. alcmaeon of croton ( - bc) was the first physician to document and publish anatomical observations of canine dissections. , he established brain control over intelligence and sensory perceptions. centuries later, aelius galenus (also known as galen of pergamon, - ad) would make pivotal discoveries based on animal experimentation. galen served as a doctor to different roman emperors. his public demonstrations of cutting laryngeal nerves in squealing pigs made him famous. he also made important anatomic observations on cranial and spinal nerves. he included findings from more than animal species. , in the late nineteenth century, claude bernard set the foundations of experimental medicine by developing rigorous guidelines for controlled studies. , , animal-based research has been the cornerstone of health sciences ever since. it accounts for more than % ( / ) of all physiology or medicine nobel laureates' studies. research on the diphtheria vaccine-developed in guinea pigs (cavia porcellus)-received the very first prize in . other fundamental discoveries, like the insulin mechanism and pasteur's and koch's studies, are also credited to animal research. , , animal welfare has not always been a concern. proper acknowledgment of an animal's moral status as a sentient being is a recent development. , for most part of the history, animals were considered senseless to pain and were treated with little or no respect in research, teaching, and demonstrations. , for centuries, animals were perceived mainly as useful tools. , most greek philosophers excluded animals from moral judgments, especially those derived from stoic and epicurean beliefs. other philosophical strands, such as cynicism, were more empathetic to the well-being of animals. nevertheless, assumptions that animals are entitled to ethical consideration and can indeed perceive pain and negative feelings only emerged during the renaissance. , , french philosopher rené descartes ( - ) acknowledged that animals could perceive sensations, but in a purely mechanical way. based on this cartesian perspective, scientists justified the use of animals without concern for their feelings for centuries afterwards. , , when william harvey demonstrated blood circulation on conscious dogs, the attending public believed the painful screams were part of a "beast machinery," like an automatic sound. , only by the second half of the nineteenth century, in victorian europe, animal rights would be debated among the mainstream philosophers. , jeremy bentham's introduction to principles of morals and legislation ( ) was a turning point. emphatic attitudes displayed by influential thinkers like rousseau and schopenhauer helped shaping a new approach towards animal welfare. , darwin's evolutionary insights (published in ), emphasized our moral duty towards animals. [ ] [ ] [ ] the cruelty to animals act-passed in -was the first official legal document to set boundaries on animal experimentation. however, the dominant approach to animal research remained utilitarian. , in the late s, russell and burch developed the "three rs concept" to rationalize animal use by replacing, reducing, and refining resources. these guidelines aim to minimize animal distress and emphasize our duty to search for alternative technologies. bioethical principles are now mandatory for any animal experimentation. , today, the internet reflects public opinion on animal welfare. the attitude of young people towards animals is much more empathetic now than in previous generations. consequently, bioresearch elicits heated debates. some groups with radical views advocate banning animal research altogether. nevertheless, the unlimited potential and importance of animal-based discoveries cannot be denied. five key bioethical points are considered when assessing the moral status of animal subjects in research: the presence of life, the ability to feel and perceive stimuli, the level of cognitive behavior, the degree of sociability, and the ability to proliferate. scientific proof of animal consciousness and sentience is a recent achievement. however, there is no global consensus on the value people attach to particular animals. in some cultures, the western household dog is no more than a food source. the same is true for research. using animals like monkeys, dogs or cats as models will likely evoke adverse reactions nowadays. the social perception of the animal's "worthiness" is called "speciesism". at this point in time, animal research cannot be entirely replaced by in vitro testing. developing alternative methods is essential. scientists can now create and cultivate microfluid organ-on-a-chip models. but these new technologies are still under development. hopefully future studies will provide the means to replace animal experiments. until then, ethical treatment and rational use of all living forms are still necessary. , in that context, characterizing alternative models remains a goal. rabbits, for instance, may be potentially useful bone models. they are already used as laboratory subjects in several medical fields. even though they are also praised as household pets, particularly in europe, their use in laboratory is well accepted. , many species can be suitable models for different diseases. the research question will dictate what type of model should be considered. undoubtably, rodents are the most popular laboratory subjects worldwide. rats (rattus norvegicus) have been part of medical studies since the nineteenth century ( ). they reached peak importance with the development of the wistar strain, in . although mendel started studying the laws of inheritance on mice (mus musculus), he shifted his methods to peas after facing religious restrictions on his animal model. , rodents became the standard choice for genetic experimentation after watson and crick published their dna study. during the s, the first "gene knockout" mouse was developed. this study won a nobel prize. , using models is very attractive because one can easily ensure homogeneity between subjects-unachievable otherwise. then future studies can reproduce similar conditions. , for obvious reasons, the greater the model's similarity to humans, the greater are the moral implications. the planning phase is the moment to define the best model to answer the research question, avoiding unnecessary enrollments. the "ideal model" does not exist. no single animal-aside from humans-can perfectly exhibit human responses. researchers must choose the most suitable option, considering the objectives of the study. careful planning is mandatory. it should be kept in mind that sometimes more than one type of model might be necessary to answer the research question. multi-level assessment is required to identify the possible advantages and challenges of any given model and table provides a template guide. animal models have taught us much about bone disorders and have been central to developing many treatments throughout history. their contribution remains paramount for assessing bone physiology and immunology, since in vitro alternatives cannot fully reproduce whole-organism physiological behavior. they remain beneficial to the whole orthopedic field. either by mimicking diseases in arthrology and oncology studies or by allowing surgical training, animals are still essential to medicine. nonhuman primates are our best biological representation. for that reason, using them nowadays for scientific purposes elicits public. aside from moral implications, their size and ease of handling in experiments are difficulties, besides being financially demanding. working with primates also requires very well-trained staff (owing ta b l e schematic compilation of traits and possible challenges to consider when planning to use an animal model , , purpose/approach to their unpredictable aggressive behavior and zoonotic potential), which limits their research potential. our second closest model on the structure of bone is dogs. despite individual variations on macrostructure, their bone remodeling is somewhat similar, and they exhibit similar haversian structure. dogs used to be popular research subjects due to their medium size, ease of handling, and docile behavior. , today, these classical models are no longer feasible. , over recent decades, a paradigm shift regarding animal use in research has occurred. the fields of laboratory sciences, animal welfare and alternative methods for replacing animal use have expanded considerably to overcome the lack of public acceptance of the classical models. one of the most studied-and prevalent-disorders nowadays is osteoporosis. age-related osteopenia is a public health concern of growing importance. demographic aging and the urban lifestyle of western societies have led to this modern disease. the world health organization considers osteoporosis a significant age-related disease and has developed global strategies for its prevention, management, and surveillance. osteoporosis causes unbalanced bone formation/resorption and decreases bone mass. the weakened bones are more prone to suffer a fracture, even with low-impact injuries. pathological fractures occur mainly at the hip joint and vertebrae. they may even go unnoticed in elder patients. the domestic rabbit (oryctolagus cuniculus) is a small digging lagomorph of the family leporidae. in the modern age, there are only two living families: leporidae (rabbits and hares) and ochotonidae (pikas), with genera currently recognized. more than rabbit breeds exist worldwide. rabbits exhibit desirable traits for bone research. these calm and easily handled creatures have a short lifespan and breed readily in captivity. the new zealand white rabbit is the most popular research breed. furthermore, rabbits are phylogenetically closer to primates than rodents. they reach skeletal maturity between and weeks of age (females earlier). adults display some haversian remodeling and their bone metabolism is somewhat similar to humans. however, surgical castration is not enough to mimic satisfactory bone loss and other techniques must be associated. , rabbits display less cancellous bone than humans. , they have more fragile cortices. , cortical thickness and the diameter of drilled holes contribute to the high complication rate of fracture repair in this species. their functional anatomy allows their peculiar high-speed hopping to evade predators. cage confinement and exercise restriction might be harmful to their bone development and researchers should consider alternative in-house systems, as opposed to small cage confinement. in their natural habitat, rabbits are a prey species, which explains their curious but easily scared behavior and also explains some anatomic features that enable them to escape at high speed when in danger. their peculiar appendicular skeleton (figure ) must be light weight but also resistant to allow their burrowing and food-seeking behaviors. their hindlimbs have high power hip extensor muscles concentrated at the proximal part. muscle mass in the front limbs is distributed more distally and accounts for approximately % of the total body mass. their fibula fuses to the middle shaft of the tibia. their four long webbed toes on each hindlimb allow accelerated digitigrade hopping. their small clavicles resemble those of domestic cats and make them more agile. , a survey of the terms "rabbit" and "experimental model" in pubmed resulted in articles of indexed journals published between and , with almost from the past decade. rabbits were pivotal to the discovery of the atropine esterase enzyme, in the nineteenth century. since then, they have been used in several studies by nobel laureates. they helped characterizing the mechanisms involved in insulin production and diabetes. , , rabbits are appealing models for bone research. studies involving rabbits are now commonplace in orthopedics, and multi-species assessments of model suitability have rated rabbits as potential bone models after primates and dogs. biomechanical forces act during stance and walking in any living animal. measuring these forces is important to determine a model's bone strength, but biomechanical data on rabbit bones are still scarce. a study published the effects of in vivo loading of rabbit tibiae. biomechanical data on axial compression and bending moments in the rabbit tibia were given. the authors concluded that rabbit tibia can endure higher strain levels than goats can, therefore rabbits were better models. in another study describing the qualitative differences between mice, rats, dogs, nonhuman primates and rabbits, the authors concluded that the skeletal characteristics of rabbits were the least suitable for extrapolating to humans, but highlighted their lack of biomechanical data. rabbits are a standard model in periodontal research. they are part of diverse studies such as measurement of parathyroid hormone effects on osseointegration in osteoporosis, some recent studies have explored the potential of rabbits as models for cartilage and meniscal tears repair. they have also been used in other studies on arthrology and tendon healing. one study focused on intra-articular injections of chondroitin sulfate carried by hydrogel. others assessed tendon healing by reproducing biceps tenosynovitis, and anterior cruciate ligament and rotator cuff tears. rabbits have also increased in importance as pets. they are the third most popular companion animal in the uk, after dogs and cats. more than two million pet rabbits are estimated to have existed in the past decade. they are the most popular exotic animal in us private veterinary practices. in view of these trends, the demand for higher standards of rabbit medicine is increasing and thus the need to enhance veterinary knowledge also exists. more recent studies focus on clinical and surgical aspects of the pet rabbit. , , [ ] [ ] [ ] [ ] [ ] [ ] in a recent paper, the authors evaluated the effect of three different screw-hole diameters and torsional properties of rabbit femora. however, more in-depth biomechanical studies are lacking. there are scarce data on the torsional properties, [ ] [ ] [ ] but the main focus of these studies was bone healing and bone grafting. fracture repair in the pet rabbit remains a major challenge. rabbit bones are very thin and brittle, an important complicating factor that results in frequent implant failure. , another study has defined vertebral safe corridors for implant insertion using computer tomography. but rabbit research still has unexplored gaps to be addressed. the human-animal bond has sculpted medical knowledge. animal models play a significant role in enhancing our understanding of emerging pathologies. current in vitro technologies are 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of long bone fracture repair in rabbits survey of the husbandry, health and welfare of pet rabbits welfare assessment in pet rabbits anesthesia and analgesia in rabbits and rodents case report surgical correction of patellar luxation in a rabbit analysis of mechanical symmetry in rabbit long bones comparison of cyclic loading versus constant compression in the treatment of long-bone fractures in rabbits physical properties of autoclaved bone: torsion test of rabbit diaphyseal bone bsava manual of rabbit surgery, dentistry and imaging computed tomographic study of safe implantation corridors in rabbit lumbar vertebrae key: cord- -oecpqf j authors: nan title: aspho abstracts date: - - journal: pediatr blood cancer doi: . /pbc. sha: doc_id: cord_uid: oecpqf j nan myelodysplastic syndrome (mds) and frequently arise in the context of inherited bone marrow failure (bmf) syndromes, such as shwachman diamond syndrome (sds). monosomy /del( q) is associated with high grade mds and propensity to progress to acute myelogenous leukemia, a major cause of morbidity and mortality for patients with inherited bmf. development of non-transplant strategies to treat bone marrow failure without simultaneously stimulating outgrowth of malignant clones remains a major challenge. objectives: the aim of this study is to investigate the molecular consequences of del( q) in the context of bmf with the goal of developing more effective treatments. design/method: to study the biological and molecular consequences of monosomy/del( q) in bmf, induced pluripotent stem cells were generated from sds patients (sds-ipsc) . a deletion of the mds-associated region of the long arm of chromosome was then introduced using a previously published modified cre-lox approach. results: the sds ipsc phenocopied bone marrow failure with slow proliferation and impaired hematopoietic differentiation. we next explored whether deletion of q conferred a relative fitness advantage within the context of bone marrow failure. proliferation of the sds-del( q) ipscs was reduced below that of both the isogenic sds ipscs and normal controls without an increase in cell death. sds-del( q) demonstrated reduced hematopoietic differentiation compared with isogenic sds cells. these data demonstrate that deletion of q fails to confer a relative growth advantage relative to isogenic sds ipscs and results in further impairment of hematopoiesis. to gain insight into the mechanisms of del q-associated clonal evolution in sds, we performed rna sequencing (rnaseq) of sds+/-del( q) ipsc. expression of tgf pathways and their downstream targets were reduced in sds-del( q) ipscs compared to isogenic sds ipsc. single cell rnaseq analysis of primary sds bone marrow cells confirmed that the tgf pathway is hyperactivated in sds. western blot analysis showed increased phospho-smad levels in sds ipscs compared to sds-del( q) and normal controls, while total levels of smad were unchanged. pharmacological targeting of tfg with small molecule inhibitors resulted in selective improvement of sds hematopoietic colony formation and myeloid differentiation without stimulating outgrowth of the isogenic sds-del( q) cells or normal controls. these results demonstrate that del( q) reverses the tgf pathway hyperactivation of sds. furthermore, inhibition of tgf selectively rescues hematopoiesis in sds but not in isogenic del q cells, suggesting a potential strategy to treat bone marrow failure without stimulating del q clonal outgrowth. background: standard therapy of medulloblastoma consists of treatment with alkylating agents and radiation after surgical resection. although a statistically significant increase in survival is reported with this regimen, / rd recur and become resistant this class of agents ultimately leading to mortality. large numbers of somatic mutations were observed in recurrent medulloblastoma (rm) after alkylating agent and radiation treatment. high mutation rates in tumors can have twofold effect; ) a large number of non-synonymous mutations that have no role as drivers can still cause functional tumor antigens increasing the neoantigen burden and immunogenicity. moreover, ) such tumors can gain mutations in canonical or non-canonical dna repair pathways leading to a gain in the number of mutations as seen in case of glioblastoma, this can lead to even higher accelerated mutational rate. evidences suggest that high mutational load can cause higher neoantigen burden thereby making the tumor more susceptible to immune checkpoint inhibition. we propose that post therapy recurrent medulloblastoma gain mutational signature and immunophenotype of malignancies demonstrating clinical response to immune checkpoint therapy. objectives: ) rm has molecular signatures identical to tumors with high immunogenicity and clinical response to immune check point inhibition. ) rm has the immune inflammatory phenotype; harboring high percentage of tumor infiltrating lymphocytes (tils), macrophages and monocytes. design/method: to test our hypothesis, we downloaded the raw bam files of previously published data from international cancer genome consortium (icgc) . this set of about matched primaries and recurrent medulloblastoma cases forms our discovery cohort. we have called somatic variants using the gatk pipeline by the broad institute. to validate our key findings, we have procured human medulloblastoma specimens and are conducting whole exome sequencing. the primary assays utilized to assess immunogenicity are immunohistochemical (ihc) staining of formalin fixed and embedded recurrent medulloblastoma tissue to identify tils, tumor associated macrophages and other markers. mg/m had dlts of dyspnea (grade )/hypoxia (grade ) but no dlts were observed in any other cohort. adverse events were generally mild to moderate, consistent with the safety profile observed in adults. across the desc cohorts, plasma concentrations were dose-proportional and steady state concentrations were lower on day vs. day . mean systemic exposure in the mg/m cohort was ∼ -fold greater compared with the adult rp d of mg bid. a pk:pd relationship between tazemetostat exposure and h k me levels in peripheral blood monocytes and granulocytes was observed in the desc phase. consistent and significant post-dose reductions in h k me occurred at doses ≥ mg/m . further analysis of twelve patients treated at the rp d confirmed that h k me inhibition was maximally inhibited. doses - mg/m showed confirmed objective responses (cr/pr) per recist/rano in patients with es (n = ), chordoma (n = ), and atrt (n = ). background: previous studies established that the platelet/ fibrin(ogen) axis promotes metastatic potential by impeding the clearance of newly formed micrometastases by natural killer (nk) cells. however, multiple important questions remain, including the potential of fibrin(ogen) to promote metastasis through interactions with cells other than platelets (e.g., inflammatory cells), and the fundamental question of whether fibrin polymerization is required for metastasis. objectives: determine the role of fibrin polymerization and fibrin(ogen) engagement of integrins iib and m in metastasis. design/method: we performed experimental and spontaneous metastasis assays in immunocompetent mice carrying specific fibrinogen structure/function alterations. results: expression of a mutant fibrinogen lacking the binding motif for the leukocyte integrin m (fib - a) significantly decreased metastatic potential relative to wildtype fibrinogen, suggesting a role for fibrin(ogen)inflammatory cell interactions mediated by m in metastasis. to directly determine the importance of thrombinmediated fibrin polymerization in metastasis, we analyzed metastatic potential in fibaek mice, which carry a form of fibrinogen essentially "locked" in the soluble state due to a mutation in the a chain thrombin cleavage site. metastatic potential in fibaek mice was diminished relative to control mice, speaking to the importance of thrombin-mediated fibrin polymerization in the metastatic process. however, the fibaek mice retained significant metastatic potential relative to complete fibrinogen deficiency, indicating that fibrinogen monomer retains significant prometastatic properties. in order to better define the role of fibrin(ogen)-platelet interactions in metastasis, we compared metastatic potential in control and fib Δ mice, carrying a form of fibrinogen lacking the chain binding motif for the platelet integrin iib . surprisingly, this mutation had no impact on metastatic potential. together, these studies suggest fibrinogen plays a multifaceted role in metastasis. fibrin(ogen)-leukocyte interactions mediated by m appear to have a role in metastasis. previous studies showed that macrophages promote the metastatic potential of circulating tumor cells, which may represent at least one important m expressing cell type whose prometastatic behavior is influenced by fibrin(ogen) interactions. these studies show that thrombin-mediated fibrin polymerization promotes metastasis, but soluble fibrinogen retains some significant prometastatic capacity. surprisingly, loss of the fibrinogen chain iib binding motif had no impact on metastasis. given the established importance of platelets in metastasis, these findings suggest that fibrin (ogen) is capable of platelet stabilization through mechanism(s) independent of this iib binding motif. platelets may bind polymerized fibrin at other sites, and/or fibrin interactions with other matrix proteins capable of binding iib are sufficient to support platelet functions required for metastasis. the role of platelets in hemostasis and thrombosis is well defined, but it is becoming increasingly evident that platelets also assist in host defense and inflammation. platelets participate in the innate immune system through direct antimicrobial activity and interactions with effector cells (chapman , garlanda , kapur ). in the adaptive immune system, platelets recruit and costimulate t-cells, and promote b-cell differentiation and antibody class switching (kapur , morrell ). the question remains: which mechanisms influence platelet immune function and are they developmentally regulated? preliminary studies in the palis lab have revealed significant dif-ferences in embryonic versus adult platelet gene expression, including regulators of immune and inflammatory responses such as beta -microglobulin (b m) and major histocompatibility complex class i (mhc ). mhc is expressed on all cell surfaces except red blood cells and its molecular chaperone b m is a marker of inflammation highly expressed in platelet alpha granules (zufferey ). preliminary data from the morrell lab reveals a mass release of b m during platelet activation, which drives monocyte differentiation to an inflammatory phenotype through tgfb receptor signaling. we therefore sought to determine whether developmental changes in platelet b m expression mediate differences in platelet-mediated monocyte activation. with trilineage hematopoiesis with a predominance of early myeloid precursors, with full maturation. microarray, elane and sbds sequencing and deletion/duplication analyses were negative. immunologic evaluation was significant for agammaglobulinemia and an absence of memory (cd +cd +) b cells. a gene primary immunodeficiency panel revealed two variants of unknown significance-c. g>a and c. g>t in dnmt b; one previously reported in association with icf . parental testing demonstrated parental heterozygosity. centromeric instability was confirmed in mitogen stimulated lymphocytes showing characteristic, multibranched chromosomes containing at least arms of chromosome and joined near the centromere. decondensation of the qh and qh regions and triradial configuration of chromosome was noted, and a diagnosis of icf syndrome was made. the patient was started on monthly intravenous immunoglobulin (ivig). prophylaxis for pneumocystis jiroveci pneumonia and respiratory syncytial virus was initiated. a / matched sibling hsct is being planned. demonstrated the diagnosis of high grade osteosarcoma. the patient was started on multi-agent chemotherapy with planned a whole femur prosthesis at time of local control. cases of osteosarcoma have been described in the literature in patients with nf (median age; years, range - years) with slightly male predominance ( cases). the femur was the most common site of involvement ( cases). four patients died of metastatic disease despite surgery and multi-agent chemotherapy. conclusion: nf represents a major risk factor for development of malignancy and uncommonly osteosarcoma in adolescents and adults. we report a rare case of an extensive involvement of osteosarcoma of the left femur in a child with known diagnosis nf . this presentation should alert the pediatric oncologists to monitor for bone tumors in patients with nf by physical exam and detailed medical history. hasbro children's hospital, providence, rhode island, united states background: dysautonomia is a paraneoplastic syndrome most commonly described in adult malignancies. despite current therapies aimed at symptoms management, it is often debilitating. we present a case of a -year-old girl who initially presented with autonomic dysfunction and was subsequently found to have hodgkin lymphoma. objectives: describe hodgkin lymphoma presenting with dysautonomia and discuss symptom management with rituximab design/method: case report a year-old-girl presented with severe symptoms of orthostatic hypotension necessitating prone positioning to prevent syncopal episodes. additionally, she reported anhidrosis, xerostomia, urinary retention, and constipation. she had unmanageable peripheral neuropathic pain despite multiple analgesia medications. initially, it was suspected that her symptoms were caused by an atypical presentation of guillain-barre syndrome. she was treated with intravenous immunoglobulin g, without response. due to a suspicion of a paraneoplastic syndrome a positron emission test/cat scan (pet/ct) was performed and revealed widespread fdg-avid nodal and splenic disease. pathology from a thoracoscopic biopsy of a mediastinal lymph node demonstrated classical hodgkin lymphoma. she was classified as stage ivb. a paraneoplastic panel obtained during the first cycle of chemotherapy revealed elevated anti-amphiphysin antibodies and glutamic acid decarboxylase (gad) antibodies. therapy was initiated with abe-pc (doxorubicin, bleomycin, etoposide, prednisone, cyclophosphamide) ; vincristine was held given her significant neuropathy. due to persistence of autonomic symptoms following her first cycle and presence of antiamphiphysin and gad antibodies, rituximab was incorporated into her treatment. following two cycles abe-pc, she had a rapid early response by fdg-pet/ct. she completed an additional three cycles of abd-pc. end of therapy imaging demonstrated complete response with a single persistent mildly fdg-pet avid lymph node (deauville ) and her antibodies were negative. she continues treatment of maintenance rituximab with significant improvement, but not resolution, of her orthostatic hypotension. at this time, the patient can ambulate with assistance. constipation and urinary retention have fully resolved and, her peripheral neuropathy, xerostomia, anhidrosis have improved. conclusion: this is rare case of a pediatric hodgkin lymphoma patient developing dysautonomia associated with antiamphiphysin and glutamic acid decarboxylase antibodies and subsequently managed with chemotherapy and rituximab. clinicians should be suspicious of a paraneoplastic syndrome when a neurologic disorder fails to improve with standard treatment. results: labs obtained at an outside hospital one month prior to presentation showed absolute neutrophil count (anc) and hemoglobin . g/dl. she presented to our institution with days of fever, hepatomegaly cm below costal margin, a white plaque on her tongue, and circumferential perianal ulceration. labs were significant for anc and hemoglobin . g/dl. anti-granulocyte antibody testing was positive. bone marrow biopsy showed arrest of neutrophil maturation. after initiation of filgrastim ( . mcg/kg/day), her anc increased to > and repeat bone marrow biopsy demonstrated left shifted myelopoiesis. biopsy of her oral lesion demonstrated invasive actinomyces prompting a prolonged course of antibiotics. biopsies of her oral and anal lesions were reported as myeloid sarcoma without mll rearrangement. chemotherapy was not initiated due to complete resolution of both lesions within weeks of initiating filgrastim and appropriate antibiotic coverage. she has not developed any further lesions concerning for malignancy. testing for common genes associated with severe congenital neutropenia and autoimmune lymphoproliferative syndrome was negative. her immunoglobulin levels and the measurement of age-appropriate vaccine responses were normal. after her lymphocyte subpopulation analysis indicated a selective deficiency in cd positive t-lymphocytes (absolute cd cell count ), the severe combined immunodeficiency panel from genedx showed compound heterozygous mutations in results: a male infant was born with a large thigh mass. the child was clinically well aside from restricted movement of affected leg. mri showed mass expanding into pelvis without other lesions. an interventional-radiology guided core biopsy of the mass was reported as high-grade spindle cell sarcoma without etv rearrangement. surgery was deferred because of concern that it would result in excessive morbidity. the mass was treated with vincristine and dactinomycin per infantile fibrosarcoma protocols. after months of therapy, no significant change in size of the mass was noted on physical exam or imaging. repeat biopsy was obtained to confirm diagnosis and allow for expanded tumor testing. this biopsy showed triphasic distribution of adipose, fibrous and mesenchymal tissue consistent with fhi with rare sarcomatous foci. additional chemotherapy was deferred and the child was followed clinically. his tumor has remained approximately the same size and still unresectable. next generation sequencing of tumor utilizing panel based technology revealed braf-erc fusion consistent with braf activating mutation. this mutation was confirmed by fluorescent in situ hybridization (fish) probe for braf. braf and mek inhibitors have been pursued as treatments to decrease size of tumor and allow for resection. conclusion: braf mutations have been characterized in a variety of malignancies. inhibition of braf and downstream signaling components has produced promising results in a variety of patients. this is the first case report of a braf mutation in a fhi. although management of fhi is typically surgical, this does suggest a potential therapeutic target and may allow for improved surgical outcomes especially in cases where up-front surgery would result in unacceptable morbidity. genetic sequencing of fhi and other rare tumors is an important tool and has the potential to identify mutations amenable to targeted therapies. background: icf is a rare autosomal recessive disorder characterized by hypo-or agammaglobulinemia and often opportunistic infections suggesting t-cell dysfunction. it is further categorized into subtypes - based on mutations in dna methylation. mutations in the helicase-lymphoid specific (hells) gene, which is required for t-cell proliferation and participates in de novo dna methylation, are characteristic of icf type (icf ). of approximately reported cases of icf, less than percent are characterized as icf . while malignancy has been reported in icf (angiosarcoma, acute lymphoblastic leukemia), and icf (hodgkin lymphoma), here we describe the diagnosis and management of an icf patient with neuroblastoma and neutropenia, which has not been previously described. objectives: describe a novel phenotype and mutation of icf and its management to further expand our understanding of this disease. results: a month ex- week premature male with bronchopulmonary disease and failure to thrive presented with acute respiratory failure in the setting of recent viral bronchiolitis with associated chronic diarrhea. he was subsequently diagnosed with multiple infections including pjp pneumonia, norovirus, parainfluenza, rhinovirus, and pseudemonal cellulitis. he presented with profound neutropenia and agammaglobulinemia with presence of b and t cells on lymphocyte phenotyping. ct revealed a paraspinal mass that was mibg-avid on further study, strongly suggesting neuroblastoma. bone marrow was normocellular and negative for malignancy, however revealed marked granulocytic hypoplasia and maturation arrest concerning for severe congenital or, less likely, immune-mediated neutropenia. metastatic workup was negative. whole exome sequencing revealed a homozygous variant of unknown significance (c. t>c) in the hells gene, portending a working diagnosis of icf syndrome. immunoglobulin supplementation, pentamidine prophylaxis, and g-csf were initiated. he was able discontinue g-csf after months of treatment. his neuroblastoma, initially categorized as l , met criteria for observation. however, followup mri revealed interval growth nearing the spinal canal. he underwent tumor resection, confirming mycn non-amplified, favorable histology neuroblastoma. after infectious prophylaxis and immunologic support were initiated, he incurred two other hospitalizations, the first for g-tube cellulitis and the second for parainfluenza respiratory illness. he now has stable neutrophil counts off g-csf and remains in remission from neuroblastoma. current plan is to proceed with bone marrow transplantation for immunodeficiency. conclusion: icf has not previously been described with neutropenia or neuroblastoma. this report not only describes a novel mutation and phenotype of icf and the management thereof, but also reveals the potential curative role of bone marrow transplantation in such disease. staten island university hospital -northwell health, staten island, new york, united states background: desmoid tumors are rare tumors that arise from highly differentiated fibroblasts. they occur in isolation or as part of the disease spectrum of familial adenomatous polyposis (fap) . fap mutations between codons - typically correlate with increased extraintestinal disease such as desmoid tumors and upper gastrointestinal polyps. we describe a patient with a large intra-abdominal desmoid tumor who is heterozygous for a c. c>t (p.arg cys) apc gene mutation. we are not aware of any other patients reported with this germline apc mutation presenting with a desmoid tumor. objectives: to discuss a novel apc mutation and the presentation of a rare case. design/method: review of clinical presentation, genetic analysis and management of a rare tumor. a -year-old female with no significant medical history presented with abdominal asymmetry and intermittent pain. she reported urinary urgency, shortness of breath, early satiety, decreased appetite and a -pound weight loss over the course of months. ct scan of the abdomen demonstrated a × cm abdominal tumor abutting the local organs but no presence of bowel obstruction. a biopsy revealed a spindle cell neoplasm favoring fibromatosis. there was no known family history of fap, colon cancer, or desmoid tumors. apc gene mutation analysis demonstrated a c. c>t (p.arg cys) heterozygous gene variant. due to size and location of the tumor, it was initially deemed unresectable. the patient was started on a course of monthly liposomal doxorubicin. she tolerated the initial cycles well and interval ct after cycles of chemotherapy revealed a % decrease in tumor volume. variability exists in phenotypic presentation with regards to the location of the afp mutation locus. while fap mutations associated with desmoid tumors typically have changes in the - codon region, our patient presented with a heterozygous mutation resulting in a missense mutation at codon . due to the change in polarity and size, the mutation is not considered to be of conservative nature. we are only aware of one other report of this mutation, which occurred in an individual with a personal and family history of colon cancer. we are not aware of any patients with desmoid tumors who also have this germline apc gene mutation. our case report highlights an apc gene mutation that is not well-described; we are not aware of any other cases of this mutation reported in patients with desmoid tumors. future evaluation and tracking of this mutation may lead to the determination of further clinical significance. background: over time, advanced care planning for location of death has been associated with increased deaths at home rather than in the hospital. in some cases, however, complex management and symptom control can prevent families from achieving their goal of keeping their child out of the hospital and at home at the end of life. ascites is a sequelae of many conditions including malignancy that might lead to significant morbidity. increasingly, interventional procedures are being utilized. peritoneovenous "denver" shunts are placed internally with one end in the peritoneal space and the other buried within a major vessel such as the svc. a one-way valve and pump buried under the skin allows the patient to pump fluid from the peritoneal to the vascular space. the shunt is used frequently in adults, but has not seen much use in pediatric oncology patients. objectives: to describe a case of a terminally ill patient with refractory wilms tumor with ivc involvement who received symptomatic relief with denver shunt placement. results: an -year-old female was diagnosed with relapsed, refractory, metastatic wilms tumor with pulmonary and hepatic involvement, with tumor extension to the hepatic veins and ivc. multiple chemotherapeutic regimens and palliative radiation to the ivc were administered, but her disease continued to progress, leading to pressure on the portal vein and portal hypertension. the resulting ascites was causing the patient significant pain and was difficult to manage. the patient's code status was changed to dnr/dni after discussion with her mother, who identified a desire to have the child die at home as comfortably as possible. a peritoneovenous shunt was placed in order to control the patient's pain and avoid frequent medical procedures and therapies. despite initial anxiety, the patient was able to utilize the pump and achieve significant improvement in her ascites and pain. she was able to spend the remaining six weeks of her life at home. ascites is a common phenomenon of end stage disease. peritoneovenous shunts are a treatment modality that may be considered to allow for pain control at the end of life for pediatric oncology patients with ascites. the procedure is relatively low risk, allows for self-control of the pump to maintain comfort, and is easy enough to use by the patient or family. background: extraneural metastases (enm) from pediatric glioblastoma multiforme (gbm) are rare, with an estimated frequency of . %. etiologic factors include multiple neurosurgical procedures and sarcomatous dedifferentiation. their occurrence can seriously affect the patient's quality of life and survival. while enms have been well documented in adults, pediatric cases have not been previously summarized. a year old male with a cerebral gbm developed extension of disease outside of the neuraxis approximately months post initial presentation and at the time of disease progression. metastases included exracranial temporal lesions, cervical and mediastinal lymph nodes and s of s bilateral lung nodules. a large pleural-based soft tissue metastatic focus was identified on imaging when the patient presented with respiratory distress secondary to a right tension pneumothorax, which was recognized and managed promptly. we summarize the main reported cases in literature to better define risk factors for and evaluate the proposed mechanisms underlying these systemic metastases. design/method: we performed a literature review on the pubmed database using the terms gbm and enm. patients under years of age who met the weiss criteria for the diagnosis of enm from primary cns tumors were included. results: our patient fulfilled two of the three weiss criteria with confirmed gbm at the primary site with all enm in the temporal soft tissue and cervical lymph nodes displaying histopathologic features similar to the primary cns tumor. the intrathoracic adenopathy and lung nodules detected upon chest imaging during workup for respiratory distress were assumed to represent additional metastatic foci. our literature review identified pediatric patients with enm from gbm with a median age of years (range . - years) and a slight female predominance ( % females vs. % males). the most common sites of metastases reported were pleura/lungs, bones, lymph nodes and liver. in of patients, metastases were associated with csf shunting. conclusion: pediatric oncologists should have an increased index of suspicion when caring for patients with gbm, particularly those who have undergone shunting procedures and present with systemic symptoms including bony pain, respiratory changes, transaminitis or cytopenias which should prompt timely investigation for enm. although enm of cns tumors carry very poor prognosis, their diagnosis has potential therapeutic importance because treatment of metastatic lesions may alleviate symptoms and improve the quality of life. additional studies may be warranted to evaluate the incidence of enm that can provide valuable insight into the pathogenesis and biology of high-grade gliomas. nicklaus children's hospital, miami, florida, united states background: sinusoidal obstruction syndrome (sos) has been reported in patients undergoing intensive chemotherapy and as a complication post-hematopoietic stem cell transplan-tation. sos may be complicated by portal hypertension, hepatorenal disease or multi-organ failure. however, despite treatment, there may be further potential complications that can be anticipated in patients with history sos. we report two patients with history of sos presented with post-procedural bleeding after gastric tube placement. we believe that their presentations may be associated to their previous diagnosis of sos. design/method: pubmed search was done with search for terminology including "sinusoidal obstruction syndrome" "defibrotide", and "bleeding". papers relevant to our cases were selected for literature review. results: case : a year-old female with history of desmosplastic medulloblastoma status-post resection and intensive chemotherapy was diagnosed with sos one month after her second part of planned tandem transplant. she was managed with paracentesis and defibrotide. due to malnourishment, patient had a gastric tube placement months after she completed therapy and had an episode of upper gastrointestinal bleeding postoperatively from the g tube site. case : similarly, a year-old male diagnosed with anaplastic medulloblastoma status post resection and adjuvant multiagent chemotherapy. his treatment course was complicated with sos after the second cycle of induction chemotherapy which responded to -day course of defibrotide. likewise, the patient had a major bleeding event from the g-tube site approximately two months after sos diagnosis. defibrotide was discontinued in both cases before g-tube placement. both patients had no previous history of bleeding disorders or relevant family history. in addition, comprehensive laboratory evaluations were within normal limits before both procedures. in sos, there is blockage of fluid out of the liver that leads to congestion, ascites, ischemia of the liver, and post-sinusoidal portal hypertension. two related causes of sos should be considered as an explanation for g-tube bleeding. similar patients should have close monitoring postoperatively or if possible surgical intervention should be delayed until the sos process has been evolved. nicklaus children's hospital, miami, florida, united states background: the development of treatment related acute myeloid leukemia (t-aml) and myelodysplastic syndromes (t-mds) is a potential complication after cytotoxic chemotherapy or radiation therapy. the incidence of development of t-aml/t-mds varies from - % depending on the treatment regimen used. cutaneous myeloid sarcoma (ms) is a common presentation of extramedullary leukemia and usually occurs in the setting of aml. we report a rare case of cutaneous ms in an adolescent female after successful treatment for ovarian yolk sac tumor (yst) stage i with bep (bleomycin, etoposide and cisplatin) therapy. the ms was managed only with biopsy and close observation. design/method: a pubmed search was conducted for queries including t-aml/t-mds, cytotoxic agents, cutaneous myeloid sarcoma, regression. relevant papers were selected for literature review. a year-old female was diagnosed with a left ovarian yolk sac tumor, for which she underwent left salpingooophorectomy and successfully completed cycles of bep over months. during routine follow-up months after initiation of treatment for ovarian yst, she was noted to have a small, non-tender, indurated nodule on the left side of her upper back approximately cm in diameter. punch biopsy of the skin nodule was performed and pathology was positive for cutaneous myeloid sarcoma. at the time of next follow-up less than one month later, the skin lesion had resolved. two subsequent bone marrow aspirates were performed one month apart and were negative for leukemic involvement or mds. examinations and work-up including whole body pet with ct scan were negative for evidence of disease. although cutaneous ms can be regarded as the herald of systemic myeloid disease rather than a localized process, our patient was monitored periodically with physical exam and laboratory evaluations. she remains free of disease more than four years after the presentation of cutaneous ms without any further treatment. spontaneous regression ms has been previously reported. the authors would like to stress that a conservative approach with close observation could be an option in cutaneous ms even with history of chemotherapy exposure. nesreen ali, iman sidhom, sonia soliman, sherine salem national cancer institute, cairouniversity, egypt children cancer hospital egypt, egypt background: acute leukemia is the commonest malignancy in childhood. the coincidental occurrence of leukemia with hemophilia is extremely rare. hemophilia is a congenital rare x linked bleeding disorder. the main complication of the two diseases is bleeding diathesis which may be lifethreatening due to many factors, deficiency of coagulation factors in hemophilic patients, thrombocytopenia from disease and chemotherapy in leukemic patients, certain cytotoxic drugs such as asparaginase which may result in coagulation disorders and infection which may lead to disseminated intravascular coagulation. objectives: reporting such a case is imperative to set up treatment guidelines for prevention of bleeding and to optimize the therapeutic approach for these patients. design/method: seventeen years old boy, presented to children cancer hospital egypt in june with pallor and multiple ecchymoses.he was diagnosed with precursor b acute lymphoblastic leukemia, cerebrospinal fluid (csf) was free, the chromosomal analysis revealed hypodiploidy , xy. he had moderate type of hemophilia a since birth, factor viii level was . % at time of diagnosis, coagulation profile revealed prolonged partial thromboplastin time (normal - ), factor viii was low %, prothrombin concentration and prothrombin time were normal % and seconds, virology screening for hepatitis b core igg/igm, hbs ag, hiv and hc igg /igm were negative.the patient started induction total xv sjcrh protocol, factor viii unit/kg was given at presentation before doing bone marrow aspiration(bma), csf and as a prophylactic before intramuscular asparaginase injection, intrathecal and bma. it was given immediately within hours before the procedures and platelets transfusion was given regularly to maintain platelets count about , . the minimal residual disease by flow cytometry was . % and . % at d and d induction. results: our patient received his induction and reintensification chemotherapy without any major bleeding event which reveals the success of our guidelines for the prevention of bleeding. he developed very early relapse at w maintenance by the same clone. he received salvage chemotherapy but didn't achieve remission and died out of disease and resistant clone. the development of leukemia on top of hemophilia is a major problem. bleeding complication during chemotherapy can be prevented by regular prophylactic factor viii and platelets concentrate transfusion with good supportive care. life threating bleeding complication may be correlated with the severity of hemophilia. we need to collect data about the biology of leukemic cells, complications, and cause of death to optimize care for these patients. background: mucoepidermoid carcinoma (mec) is a rare malignancy that arises from exocrine glands in the upper aerodigestive tract and tracheobronchial tree. conventionally, mec diagnosis is based on histology, with prognosis based on the extent of resection and detection of metastases. mec is characterized by a translocation of chromosomes q and p resulting in a fusion between the mect and maml genes, that occurs in - % of cases. this fusion transcript has been recognized to have a favorable impact on disease features and prognosis of mec. however, recent studies indicate that high grade mec can have mect -maml fusion positivity and multiple other genomic imbalances that have not been studied in much detail. owing to the rarity of mec tumors, more definitive data related to the clinical and prognostic significance of these molecular markers are limited. objectives: . identify the presence or absence of mect -maml fusion in the tissue of our patient. . analyze the incidence of the fusion in mec cases in children and young adults retrieved from the iowa cancer registry. . determine if fusion status correlates with clinical, pathological and outcome data in our cohort. design/method: we describe the case of a year-old caucasian male who presented with recurrent pneumonia, persistent cough and radiographic evidence of right lobar collapse. bronchoscopy revealed an endobronchial lesion and the patient underwent right upper lobe sleeve resection. pathology report was consistent with low grade muco-epidermoid carcinoma. we retrieved archived formalin-fixed paraffinembedded (ffpe) specimens of pediatric and young adult mec cases (ages - ) reported in iowa from - using the iowa cancer registry. testing for the mect -maml fusion in the index case and ffpe specimens will be done using a custom-designed laboratory validated next generation sequencing (ngs) assay with the ability to detect novel fusion partners. clinical, pathological and outcome data (age, sex, tumor site, tumor size, nodal metastases, clinical stage, histologic grade, treatment and follow up) will be analyzed to correlate with fusion status. the mect -maml fusion tested positive in our index patient. we will obtain irb approval to test for the fusion in the archived ffpe specimens and correlate clinical, pathological and outcome data. conclusion: mect -maml fusion is a frequent event in mec that has prognostic and potential therapeutic applications in adults. the results of this study may enlighten the clinical management of mec in children and young adults. children 's mercy hospital, kansas city, missouri, united states background: mutations in the samd gene are associated with a rare syndrome comprising of myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes and enteropathy (mirage syndrome). diagnosis is made through exome sequencing. in the largest reported case series, of eleven patients diagnosed with mirage syndrome, two developed loss of chromosome . given the potent growth restricting activity of samd mutants, the loss of chromosome is considered the first documentation of adaptation by aneuploidy mechanisms in humans and led to myelodysplastic syndrome (mds), with deaths occurring from related complications at and years of age. objectives: to report a case of mirage syndrome with congenital thrombocytopenia progressing to bone marrow failure, managed uniquely with bone marrow transplantation. results: male born at weeks gestation with prenatal diagnosis of iugr, two vessel cord, oligohydramnios was found to have ambiguous genitalia, adrenal insufficiency, partial panhypopituitarism and congenital thrombocytopenia with bone marrow showing absence of megakaryocytic precursors. severe thrombocytopenia was present from birth. bone marrow evaluation demonstrated a hypocellular marrow with markedly reduced megakaryocytic and myeloid precursors and no evidence of myelodysplasia. he required gastric tube placement for failure to thrive, had a laryngeal cleft repaired and developed focal segmental glomerulosclerosis. mpl gene testing for congenital amegakaryocytic thrombocytopenia was negative. testing for fanconi anemia, shwachman-diamond syndrome and dyskeratosis congenita was also negative. approximately % of cells had loss of heterozygosity on chromosome q. exome sequencing showed that he is heterozygous for a de novo gain of function variant, c. g>a (p.arg gln), identified in the samd gene, confirmed by sanger sequencing and consistent with a diagnosis of mirage syndrome. at years of age, he developed pancytopenia requiring frequent transfusions with platelets and packed red blood cells. he underwent a successful bone marrow transplant at years of age without significant complications, and remains transfusion independent without cytopenias greater than months from bone marrow transplantation. conclusion: it is imperative to pursue work up for persistent congenital thrombocytopenia in a stepwise multidisciplinary manner. to the best of our knowledge, this is the first case of mirage syndrome associated bone marrow failure treated with bone marrow transplant. due to the individual rarity of mirage syndrome and pediatric myelodysplastic syndrome, it is important to maintain an index of suspicion given their association and explore bone marrow transplant as a therapeutic option. results: the patient demonstrated disease regression, initially, and continued without disease progression for months. the regimen has been well tolerated with only minimal side effects of dry skin (ctcae grade ) and a transient episode of brief erythrodysesthesia (ctacae grade ) that resolved spontaneously. the combination of sorafenib and capecitabine was effective and well tolerated in this adolescent patient with fl-hcc. our observations, although in a single patient, lend support for further testing of this novel oral chemotherapy regimen in patients with fl-hcc, a disease for which there is no effective standard chemotherapy approach. background: epstein-barr virus (ebv) is a ubiquitous virus associated with a broad range of malignancies due to its oncogenic potential. history of organ or bone marrow transplantation, immunosuppressive therapy, and primary or acquired immunodeficiency syndromes increases the risk of ebvassociated tumors. epstein-barr virus associated smooth muscle tumors (ebv-smt) are unique and rare neoplasms typically discovered in immunocompromised patients. most information related to pathogenesis and therapeutic options is limited to case reports and case series of adult patients. there are several gene expression pathways that ebv utilizes, the most notable of which is the mammalian target of rapamycin (mtor) pathway. the mtor pathway performs a key role through integrating various cell growth signals and factors to regulate protein synthesis and metabolism related to smooth muscle proliferation. sirolimus is an immune modulating therapy that targets the mtor pathway to block activation of lymphocytes. objectives: several case reports have demonstrated shortterm clinical remission of ebv-smt in adult patients with the use of sirolimus. we report the first case of long-term background: bilateral neuroblastoma is characterized as neuroblastoma arising in both adrenal glands, a rare presentation with little data on its genetic make-up. a two-monthold patient was diagnosed with bilateral neuroblastoma in our clinic. her risk assignment was based on biopsy of the left adrenal lesion, which showed mycn amplification, an unfavorable genetic marker. treatment regimen was intensified accordingly and after courses of chemotherapy tumors were excised. patient went on to receive a stem cell transplant and immunotherapy. with no knowledge of genetic similarity between the two tumors it is unclear whether biopsy of the right lesion would have yielded similar results or whether bilateral biopsies are needed for risk assessment of bilateral neuroblastoma. objectives: utilize whole exome sequencing (wes) to characterize the genomic signature of bilateral adrenal neuroblastomas excised following chemotherapy treatment. design/method: paraffin-embedded samples from left (l) and right (r) tumors underwent wes at the broad institute. we analyzed resulting data including somatic variant calls, indel mutations, and copy number variants (cnvs) using ingenuity software to evaluate and compare differences between the two tumor samples. preliminary analysis of the data shows important descriptive information on the two tumor samples. out of somatic mutations in the r tumor cells and mutations in the l tumor cells, only two common somatic mutations were present. out of cnv calls in the r tumor and in the l tumor, cnvs were common between the two tumors, or % of each tumor's cnv calls. there was a fold higher frequency in gains versus losses. the median size of the common cnvs was , (range to , , bp). cancerrelated genes with increased copy numbers included transcription factors, receptors for signal transduction pathways, and histone methylation proteins. conclusion: preliminary analysis of the wes results of the two adrenal tumors show some genomic divergence. because the tumor tissue was exposed to chemotherapy prior to excision it is difficult to determine whether genomic divergence is a result of independently originated tumors or subsequent adaptation to chemotherapy of a clonal cell population. the high number of common cnvs in the two tumors points to a common cell of origin, however the low number of common somatic mutations does not fit that picture. a future study to help elucidate the question will be wes of the original biopsy tissue to provide information on tumor mutations prior to the effects of chemotherapy. baylor college of medicine, houston, texas, united states background: although there has been significant improvement in the overall survival rates of children with cancer many children will still die from their illness or complications secondary to treatment. research surrounding the deaths of children who succumb to their disease is warranted to ensure we are providing the best care possible for these patients. objectives: this case series aims to explore pediatric cancer deaths by focusing on perhaps the most extreme cases of high intensity end of life care. we explore those patients whom we know are dying or our very likely to die as evidence by their do not resuscitate (dnr) orders. in all of these cases despite the patients very grim prognosis, their great likelihood of death and limitations placed of resuscitation methods all patients continued end of life care in the pediatric intensive care unit (picu). the primary medical records of all children with a cancer diagnosis who died between february , and january , in the picu with a dnr order seven days or earlier prior to death. each medical history included disease-directed treatment history and response with particular attention to the events surrounding the terminal admission. results: eight patients met criteria for this study representing . % of all cancer patients who died during this time period and . % of those who died in the icu. the average time between dnr and death is . days ( days - days). the average length of terminal admission was . days ( day - days). the average time between diagnosis and dnr is . months ( months - months). the average time between diagnosis and death is . months ( months - months). conclusion: these cases highlight the journey that patients, families and providers endure leading up to death. medical care is complex, there are very few absolutes that are encountered when caring for patients and decisions around limiting or withdrawing medical care are made in a context of the prior journey. . these cases help to understand the complexity of death and how two seemingly opposite ideals can be congruent in the event of an anticipated death. most of these cases show the need for improved anticipatory guidance surrounding death and greater consideration for de-escalation of care when death is expected. the hospital for sick children, toronto, ontario, canada background: rhabdomyosarcoma (rms) is the most common soft tissue sarcoma in children, with embryonal (erms) and alveolar (arms) representing the most common subtypes. arms tumors are associated with inferior outcome when compared to erms, and they are characterized in about % of the cases by a t ( ; ) or t( ; ) chromosomal translocation with creation of a pax -foxo or pax -foxo fusion gene, respectively. it is increasingly clear that the pax-foxo fusion status is an important poor prognostic factor, thus the histological classification tends to be replaced by the fusion status, particularly in terms of risk stratifica-tion in contrast to arms, there are no recurrent chromosome alterations in erms; however, there are multiple numerical chromosome changes that are frequent in these tumours: gain of chromosome , , and have been found in to % of emrs karyotypes. moreover, erms tumors show frequently allelic loss, the .p . chromosomal region being the most frequently involved. recently, novel gene fusions have been described also in erms tumours. these fusions involved mainly the ncoa and or the vggl genes. the rearrangement partners are variable, and include, i.e. pax ( q ), srf ( q ) and tead ( p ). objectives: to present a patient who died as a consequence of brain metastases while on therapy in the setting of an foxo negative rms and the identification of a new translocation t( ; )(q ;q ). design/method: case report and retrospective review of the literature. we report a case of pelvic embryonal rhabdomyosarcoma in a -month old boy. he was treated as per cog arst intermediate risk group, but unfortunately was found to have a large cerebellar tumour during the course of his chemotherapy treatment and he subsequently passed away. a novel translocation between chromosomes and was observed in of metaphase cells by g-band analysis in the autopsy sample of the brain lesion. breakpoints of the translocation were estimated to be at q and q . there were no additional clonal chromosome abnormalities in the tumour cells. conclusion: erms tumors with fusion genes involved have been exclusively described in patients less than months of age; they seem to be associated with spindle cell histology and, a favorable outcome. in our patient, a novel ( ; ) translocation was found and clinically, the patient had a dismal outcome. further studies are indicated to inquire whether this finding is of significance in term of prognosis for these patients. children 's national medical center, washington, district of columbia, united states background: iatrogenic immunodeficiency-associated lymphoproliferative disorders (lpds) are a group of lymphoid s of s proliferations or lymphomas that are well known to be associated with an immunosuppressed state. these disorders most commonly occur following hematopoietic or solid organ transplantation (called post-transplant lymphoproliferative disorders or ptld), but cases have also been described during the treatment of autoimmune and rheumatologic disorders by immunosuppressive and immunomodulatory medications. these disorders are strongly associated with infection by the epstein-barr virus (ebv) as a result of impaired immune function in the immunosuppressed state. while this phenomenon has been well documented in autoimmune conditions, cases affecting pediatric patients while on antileukemia chemotherapy are lacking. background: atypical teratoid/rhabdoid tumor (at/rt) of the central nervous system (cns) in children younger than years old has a prevalence of % to % and accounts for . % of all pediatric cns tumors. only - % of patients have leptomeningeal dissemination. rhabdomyosarcoma is the most common soft tissue tumor in childhood, but represent only - % of all pediatric cancers. rarely, it can metastasize or even directly extend into the cns, but typically, cases of cns involvement arise either from parameningeal areas or other primary sites. primary spinal or meningeal rhabdomyosarcoma is extremely rare. objectives: our objective is to describe two unique cns malignancies presenting as rare, primary leptomeningeal disease. design/method: case a -month-old female presented with vomiting, fatigue and listlessness, despite a normal head ct and brain mri. csf showed hypoglycorrhachia and mild pleocytosis. ceftriaxone was started, but she developed nuchal rigidity and cranial nerve vii palsy. repeat brain mri showed evolving leptomeningeal enhancement concerning for meningitis. she gradually developed worsening opisthotonus and ultimately a brain biopsy of the temporal lobe was consistent with at/rt. case a -year-old male presented with new generalized tonic-clonic seizure activity and intermittent headaches with photophobia, phonophobia, and vomiting. brain mri was significant for enhancement of interpenducular and suprasellar cisterns extending to the optic nerves and chiasm most consistent with meningitis. neurosurgery ultimately placed a lumbar drain for hydrocephalus, and a tissue biopsy demonstrated primary meningeal rhabdomyosacroma. results: in case , our patient's temporal lobe biopsy demonstrated grade iv malignant tumor cells consistent with atypical teratoid/rhabdoid tumor. fish demonstrated a homozygous deletion of smarcb ( q . ). she was started on chemotherapy per the dana farber at/rt protocol but ultimately was discharged home on hospice. in case , our patient's lumbar arachnoid biopsy demonstrated cellular tumor consistent with group iiia embryonal rhabdomyosarcoma. immunostaining was positive for cd , desmin, myogenin, and myo-d with neural markers ema and gfap highlighting the meninges but without a neural component to the tumor. he completed craniospinal radiation to gy total with lumbar boost to . gy total. he is currently receiving chemotherapy per arst protocol. conclusion: these two cases are particularly instructive because of their similar initial presentations and neuroimaging, but with very different and unique diagnoses. university of iowa, iowa city, iowa, united states background: ebf -pdgfrb fusion causes ph-like b-cell acute lymphoblastic leukemia (b-all), which has a philadelphia positive phenotype without the bcr-abl translocation. this is one of several mutations associated with ph-like b-all and leads to downstream overexpression of tyrosine kinase. ebf -pdgfrb fusion accounts for about % of children with ph-like b-all. patients with ph-like b-all previously had poorer outcomes with conventional chemotherapy. the addition of tyrosine kinase inhibitors (tki), like imatinib, has improved the outcome for many patients predicted to have tki sensitive mutations. objectives: to review clinical characteristics and outcomes of two cases of ph-like b-all at the university of iowa stead family children's hospital and to compare these outcomes to similar cases reported in the literature. design/method: a retrospective chart review was performed for two cases of ph-like b-all diagnosed and treated at the university of iowa stead family children's hospital. results: both patients were males diagnosed at years of age with high wbc count ( , and , ) and positive for ebf -pdgfrb gene fusion. patient (pt ) was cns b at presentation while patient (pt ) was cns negative; neither had testicular involvement. both started treatment according to cog protocol aall . peripheral blasts cleared by induction day for pt and induction day for pt . at end of induction, pt had m bone marrow and pt had m bone marrow but mrd %. dasatinib was started induction day for pt and induction day for pt . pt was still not in remission at end of consolidation; bone marrow cell culture for tki resistance showed best response to dasatinib. pt proceeded to anti-cd car t-cell therapy followed by tbi-based matched unrelated donor bone marrow transplant. pt had negative mrd at the end of consolidation and continues chemotherapy according to aall , dasatinib arm. both patients are currently clinically well. our patients had the same tyrosine kinase gene fusion and similar initial clinical courses. while both patients had persistent disease at end of induction, pt had almost % blasts while pt had significant reduction of disease burden before starting tki. pt showed good response with the addition of dasatinib while pt did not. these findings suggest that response to conventional chemotherapy may potentiate the effect of tki and may predict overall outcome. there are likely additional factors which must be taken into account when determining response to tki for patients with ph-like b-all which have not yet been identified. background: medulloblastoma is the most common malignant brain tumor of childhood. classically, medulloblastoma presents as a well-defined mass lesion in the cerebellum, with a high rate of metastatic dissemination. primary leptomeningeal medulloblastoma (plmb) is an exceedingly rare type of medulloblastoma presentation with a dismal prognosis in which patients present with isolated leptomeningeal disease without an associated mass. to our knowledge, only three pediatric and three adult cases of plmb (ages - years) have been reported, all of which died within months of diagnosis. this is the first case of plmb to report a molecular classification. objectives: to report the case of a pediatric patient with plmb in which histopathologic and molecular characterization was performed and to describe the patient's treatment and clinical course. design/method: retrospective review of the patient's electronic medical record and review of the literature. a -year-old boy presented with headache, vomiting, diplopia, and fatigue. physical examination revealed upward gaze palsy, left-sided extremity and facial weakness, and ataxia. magnetic resonance imaging (mri) of the brain revealed diffuse cerebellar leptomeningeal enhancement and edema without an identifiable mass and moderate hydrocephalus. mri of the spine and cerebral spinal fluid analysis were normal. a diagnosis of cerebellitis was rendered, and the patient underwent placement of a ventriculoperitoneal shunt. an extensive infectious, neurologic, rheumatologic, and oncologic workup did not identify an etiology. empiric antibiotics, high-dose steroids, and intravenous immunoglobulin therapy yielded minimal improvement. two months later, repeat mri of the brain performed for declining mental status demonstrated progressive thickening of cerebellar leptomeningeal disease. a suboccipital craniectomy with decompression and cerebellar biopsy were performed. pathologic examination revealed a diagnosis of plmb, classic histology, non-wnt/non-shh, without gain/amplification of myc/mycn, and p wild type pattern. craniospinal radiation to cgy with a cgy boost to the posterior fossa was delivered with concurrent carboplatin/vincristine over six weeks. two months following chemoradiation, mri of s of s the brain demonstrates significantly reduced pathological leptomeningeal enhancement of the cerebellum, and the patient is awaiting initiation of systemic chemotherapy while recovering from a surgical wound infection. conclusion: plmb is extremely rare but should be considered in patients with cerebellitis and diffuse leptomeningeal involvement who are refractory to medical management or in whom an etiology has not been identified. cerebellar biopsy is recommended early to enable timely treatment and improved outcomes. molecular classification should be performed in cases of plmb to further characterize this disease, inform treatment decisions, and improve clinical outcomes. background: primary intracerebral osteosarcoma is extremely rare and limited to case reports. ptpn gain of function is associated with noonan syndrome, which has increased risk of multiple cancer types including brain tumors, but osteosarcoma has never been described. ptpn mutations have been reported in many cancers as both oncogenes and tumor suppressors, however no ptpn mutations have been described in osteosarcoma. pdgfr-a is a growth factor receptor whose activation is implicated in several malignancies. pdgfr-a and ptpn concurrent mutations are described in glioblastoma. there is no known link between holoprosencephaly, noonan syndrome, and osteosarcoma. we report a case of multifocal intracerebral osteosarcoma in a child with lobar holoprosencephaly and chronic subdural hemorrhage and discuss the genetic changes found in the tumor. design/method: a seven-year-old caucasian female, with a known diagnosis of lobar holoprosencephaly, chronic subdural hemorrhage and well controlled seizure disorder presented with status epilepticus shortly after completing antibiotic therapy for infection of subdural hematoma. mri showed diffuse dural thickening with mass lesions in the frontal lobe, temporal lobe, and the parasagittal region, the largest of which was contiguous with the subdural space but none of the lesions were associated with bone on mri or by direct neurosurgical visualization. tissue obtained for concern for recurrent infec-tion resulted in a diagnosis of high grade osteosarcoma. dna analysis was performed to help guide treatment choice. results: standard metastatic work-up was negative for skeletal primary tumor or metastatic lesions outside of the brain. she was treated with high dose methotrexate for two cycles per modified aost . despite maximal supportive care, she quickly developed rapid tumor growth as well as intratumoral hemorrhage with resultant herniation and death from respiratory failure just three months after diagnosis. tumor gene sequencing discovered three mutations with described roles in cancer: pdgfra d >vr, kdm a loss of exons - , and ptpn a v. conclusion: to our knowledge, primary multifocal extraosseus intracerebral osteosarcoma has not been previously described. despite known cns penetration of high dose methotrexate, this tumor proved resistant and aggressive. holoprosencephaly is associated with a multitude of known genetic drivers, but none are found in this case. furthermore, the genetic changes in this tumor are not typical for osteosarcoma. pdgfr-a over-expression is described in osteosarcoma, but is not clearly correlated with worse overall survival. further research is required to determine the role of ptpn in osteosarcoma. background: anaplastic lymphoma kinase (alk) encodes a receptor tyrosine kinase whose activation induces pathways associated with cell proliferation, angiogenesis, and cell survival. alk rearrangements are rare in neuroblastoma, while alk mutations and gene amplification occur more frequently. alk mutations have been found to be associated with increased alk protein expression that is associated with a worse prognosis. alk is commonly mutated in neuroblastoma at three hotspots (f , r , and f ). the eml -alk rearrangement has mostly been associated with lung adenocarcinomas, with only a few cases of non-lung cancers found. it has never been reported in neuroblastoma. multimodal therapy and to report the successful management of treatment related iron overload. results: a -year old male presented with abdominal swelling and ct showed a right kidney mass and bilateral lung nodules. he underwent right radical nephrectomy with lymph node sampling. pathology was reviewed centrally and revealed wilms tumor with diffuse anaplasia with rhabdomyosarcoma arising within the stromal component and of nodes positive. he received adjuvant intensive chemotherapy and radiation to the hemiabdomen and whole lungs. the -week chemotherapy regimen was vincristine, doxorubicin, cyclophosphamide (per cog arst ) alternating with carboplatin and etoposide (per cog aren revised uh- ). treatment was complicated by multiple episodes of fever and neutropenia and anorexia requiring g-tube placement. post-therapy, he had persistent neutropenia and thrombocytopenia without related complications. every months for evaluations he underwent a bone marrow which revealed normocellular marrow with maturing trilineage hematopoiesis. evaluation for a bone marrow failure syndrome was unrevealing. starting at months into therapy and all posttherapy imaging showed splenomegaly. he received units of packed red blood cells through the duration of therapy. he was diagnosed with iron overload based on serum ferritin and imaging, including t *mri. he received therapeutic phlebotomy for years with normalization of serum iron studies, t * of the heart, and liver iron concentration. he is more than years from completing therapy with no evidence of recurrent disease. asymptomatic cytopenias persist and he has no evidence of iron overload. conclusion: though a rare development, clonal sarcomatous transformation can occur in wilms tumor. our patient's tumor was successfully treated with intensive multimodal therapy targeting the diffusely anaplastic wilms and the rhabdomyosarcomatous component. treatment-related iron overload in a pediatric patient with a solid tumor was successfully treated with phlebotomy. consideration should be given to screen patients with solid tumors who receive multiple packed red cell transfusions for iron overload at the completion of cancer therapy. primary children's hospital, university of utah, salt lake city, utah, united states background: malignant solid tumors are less frequently encountered in infants. primitive myxoid mesenchymal tumors of infancy (pmmti) are a myofibroblastic malignancy and cases are rarely reported in the literature. cure is achieved in the majority of cases with surgical resection, however treatment for unresectable cases remains an enigma. recently published literature postulates that the newly discovered bcor duplication found in pmmti is tumorigenic via an epigenetic pathway. this molecular signature resembles that of clear cell sarcoma of the kidney (ccsk) and the growing number of bcor mutated sarcomas. a similar chemotherapeutic backbone and local control used for ccsk, has been proposed for the unresectable subset of pmmti. utilizing this approach a month-old with relapsed disease has remained disease free for months. however, given the rarity of this disease and the lack of published literature, there is no known standard of care treatment for unresectable and/or recurrent ppmti. we report a case of unresectable recurrent pmmti, a rare infant tumor, with less than cases reported. design/method: medical record, radiological studies, pathology and literature was reviewed. results: our patient is a now month-old female who presented with constipation and lower extremity weakness in the first weeks of life. an mri demonstrated a large lumbar epidural mass with spinal cord impingement. given prolonged (> days) neurological symptoms and location, emergent chemotherapy was initiated. biopsy showed a bcor positive, primitive myxoid mesenchymal tumor of infancy (pmmti). she was treated with ifosfamide, carboplatin and etoposide, and demonstrated clinical and radiographic response. we gave two additional cycles of cyclophosphamide, carboplatin and etoposide until surgical resection was feasible followed by two post-surgical cycles of chemotherapy. unfortunately, four month post-therapy mri demonstrated two new lesions; an unresectable paraspinal soft tissue mass and a left iliopsoas groove mass. given bcor association and reported successful therapy with vincristine, doxorubicin, cyclophosphamide alternating with ifosfamide and etoposide, we elected to incorporate vinca-alkaloid and anthracycline into her regimen. she is being treated with vdc/ie with plan for radiation consolidation. conclusion: pmmti is a locally aggressive tumor, for which surgical resection is curative. for those not amendable to resection, best care practices are still being determined. we report a case of pmmti initially responsive to chemotherapy, but not curative. this is the second case to conclusively demonstrate chemo-responsiveness. bcor mutation seems to be a common feature of this cancer; its role in the pathogenesis and as a target is an area of investigation. medical college of wisconsin, milwaukee, wisconsin, united states background: atypical teratoid/rhabdoid tumors (atrt) are central nervous system (cns) tumors that most commonly occur in very young children. there is no widely accepted standard of care for atrt patients, and while survival rates are improving they are historically poor. patients with metastatic disease to the spine at diagnosis have a worse prognosis, and for patients > years old, the presence of metastatic disease often results in the use of craniospinal radiation. the importance of correctly identifying metastatic disease at diagnosis aids in decision making and can have both prognostic and therapeutic implications. mr imaging at diagnosis is used to identify metastatic disease; however, here we present a case of diffuse leptomeningeal enhancement that spontaneously resolved after resection of a primary supratentorial atrt. objectives: to describe the resolution of diffuse leptomeningeal enhancement after resection of a primary atrt tumor in a -month-old prior to any adjuvant therapy. results: a -month-old male presented with a month history of vomiting and weight loss, regression of gross motor developmental milestones, and left hemiparesis. a brain mri demonstrated a × . × . cm solid and cystic right atrial mass with diffusion restriction and post-contrast enhancement. smooth diffuse enhancement was noted along the surface of the brainstem and within the interpeduncular fossa. a spine mri demonstrated diffuse circumferential post-contrast enhancement along the surface of the entire spinal cord. the patient underwent a successful near total surgical resection of the primary mass. pathology confirmed the loss of ini- staining in tumor cells, consistent with a diagnosis of atrt. no immediate adjuvant radiation or chemotherapy was given. repeat imaging was completed days after resection. brain mr demonstrated expected post-operative changes within the surgical cavity without definitive residual mass or leptomeningeal enhancement. spine mr demonstrated complete resolution of the previously seen circumferential enhance-ment along the entire spinal cord. csf evaluation at that time was negative for tumor cells. after recovery from surgery, chemotherapy treatment was initiated. conclusion: leptomeningeal enhancement at the time of diagnosis of atrt has historically been considered clear evidence of metastatic disease. this case raises questions about the previously accepted etiology of these imaging changes and suggests that widespread leptomeningeal enhancement should be carefully interpreted in future patients with similar imaging findings. in this setting, clinicians should consider repeat imaging following primary surgical resection in order to provide appropriate prognostic information and inform therapeutic decisions. poster # primary ewings sarcoma of cervical cord mimicking cauda equina syndrome sucharita bhaumik, joshua chan nyu winthrop hospital, mineola, new york, united states background: ewing's sarcoma (es) is a malignant primary bone tumor usually involving long bones. primary es of spine is quite uncommon ( . %) and its location in the cervical spine is even more rare. cauda equina syndrome (ces) is symptoms due to damage to the bundle of nerves below the end of the spinal cord known as the cauda equina (low back pain, radiating shooting pain down the legs, paraplegia, and loss of bowel or bladder control). it often occurs with lesions of lumbosacral spine. treatment with high-dose steroids may provide pain relief and improved neurologic function (by reducing edema) while awaiting diagnostic studies objectives: to demonstrate an unusual clinical presentation and emergent management of cervical es presenting with ces like symptoms. : year old male presented with a left sided posterior neck mass. soon after, he developed weakness of left arm, urinary and stool retention and inability to walk or bear weight in both legs. on physical exam a left tempero-occipital × cm fixed, non-tender, non-fluctuant mass was noted as well as motor and sensory impairment of left upper extremity, bilateral spastic paraplegia and loss of sphincter control. mri cervical spine showed a left cervical tumor with moth eaten appearance involving the vertebral bodies of c -c , adjacent muscles, displacing vital structures of the neck and compressing the cervical spinal cord. the thoracic and lumbosacral spine had no disease involvement. due to rapidly worsening spinal cord compression he was emergently treated with high dose steroids. he gained back all function in his extremities and regained bowel and bladder control. this eliminated need for urgent neurosurgical intervention. results: biopsy of the neck mass showed small blue round cells consistent with es with ewsr gene rearrangement. staging work up revealed no additional metastatic involvement. he then initiated treatment for localized es with systemic chemotherapy and radiotherapy and has had excellent response to treatment so far. conclusion: this is the first known case of non metastatic primary cervical es mimicking ces where an acutely enlarging mass presented with rapidly progressive neurologic deficits due to compression of anterior spinothalamic tract. in these unusual presentations of ces without lumbodorsal involvement it is important to consider cervical lesions. early rapid steroid initiation should be considered while awaiting biopsy results to prevent worsening cord compression followed by es focused treatments. this increases the chance of a successful outcome. the initial improvement with steroids may confuse the tumor with being a lymphoma children 's mercy hospital, kansas city, missouri, united states background: von willebrand disease (vwd) is a relatively common bleeding disorder with a high degree of genotypic and phenotypic variation. bleeding is usually mucocutaneous but can be severe and include muscle and joint bleeds especially in type vwd patients. most common bleeding management consists of desmopressin, anti-fibrinolytics, and/or plasma-derived antihemophilic factor/von willebrand factor (ahf/vwf) complex. a recombinant vwf has become available in the last few years. anaphylaxis and inhibitor development in vwd are rare. objectives: to describe the rare clinical manifestation of anaphylaxis to factor concentrate in a patient with severe type vwd. results: a -year-old female with severe type vwd [baseline vwag %, activity < %, factor viii (fviii) %] originally presented with heavy menstrual bleeding (hmb) leading to anemia requiring blood transfusion. she underwent placement of a levonorgestrel-releasing intrauterine device (lngiud) and began norethindrone. her hmb continued despite the lngiud and an increase in norethindrone dosing. plasma derived ahf/vwf complex was administered, which she had previously received. following the infusion, the patient developed anaphylaxis with hives, wheezing, tachycardia, and itching requiring doses of diphenhydramine and dose of hydrocortisone with resolution of symptoms. subsequently, she received recombinant vwf without incident. however, due to her low fviii level, she also required treatment with a full length recombinant fviii product. she again developed hives and itching after this infusion. she has since received recombinant vwf with recombinant fviii/fc fusion protein without further allergic reaction. there was no evidence of an inhibitor with her most recent post-infusion vwf level was %, factor viii %. conclusion: anaphylaxis to plasma derived factor products has been documented far less frequently within the vwd population compared to those with hemophilia and is typically seen in those with large gene deletions, usually with type disease. therefore, similar type vwd patients with severe disease may benefit from gene sequencing. it is unclear in this patient's case to which aspect of her treatment she is allergic, as she reacted to plasma-derived ahf/vwf and full length recombinant fviii, but not recombinant vwf or recombinant fviii/fc fusion protein. we hypothesize that she may be allergic to an epitope in the fviii b domain, or that the presence of fc fusion may have had a protective effect. further investigation including genetic analysis is planned. nodules. biopsies were consistent with neuroendocrine carcinoma, large cell type (g ). next generation sequencing revealed a khdrbs -braf fusion. he received conventional cytotoxic chemotherapy regimens both with cisplatin/doxorubicin, capecitabine/temozolomide, and doxorubicin/etoposide, but achieved a minimal response followed by rapid disease progression, massive ascites, and renal failure secondary to bilateral ureteral obstruction. results: based on his prior genomic testing, therapy with single agent mek inhibitor (trametinib) was initiated. this produced a rapid, dramatic response with greatly reduced disease burden at all sites, resolution of ascites and return to completely normal activity within months. this response lasted for approximately months before the tumor again progressed. further therapy with an erk inhibitor was ineffective, and the patient expired from progressive disease. located on the chromosome q , the braf oncogene, as part of the ras/mapk pathway, is involved in cellular proliferation, differentiation, migration, and apoptosis. braf mutations are recognized in a wide range of adult malignancies: thyroid cancers, non-small cell lung cancer, cholangiocarcinoma, ovarian cancers, and multiple myeloma. braf mutations have also been described in adult neuroendocrine carcinoma of the colon. trametinib is a highly specific inhibitor of mek /mek , a downstream mediator in the braf pathway. it has demonstrated activity in a number of tumors including advanced melanoma and gliomas. trametinib was chosen for this patient based on his atypical braf fusion. we believe this is the first documented case of its successful use in neuroendocrine carcinoma in the pediatric population. conclusion: this case demonstrates the presence of braf fusion in a case of pediatric neuroendocrine carcinoma and significant response to single agent mek inhibition in this context. this cases raises the question as to whether the combination of a targeted inhibitor, in addition to either conventional chemotherapy or other braf inhibitors, might offer a better approach to therapy than current treatment options. albany medical center, albany, new york, united states background: warm autoimmune hemolytic anemia (waiha) is characterized by autoantibody, and occasional complement binding of protein antigens, on the surface of red blood cells at temperatures ≥ oc resulting in targeted destruction. we describe the case of a year old male with a history of evan's syndrome, poor immune response to vaccines and lymphoid hyperplasia, presenting with altered mental status and severe anemia, found to have a warm igg pan agglutinin with evidence of both intra and extravascular hemolysis. his course was complicated by respiratory failure requiring intubation, pulmonary emboli, enterococcus bacteremia and hypertension. he received multiple transfusions with only transient increases in hemoglobin. the aiha was refractory to multiple rounds of treatment with high dose steroids, ivig, rituximab, cyclophosphamide, bortezomib, plasma exchange and mycophenolate mofetil (mmf). objectives: given the refractory nature of our patient's aiha the decision was made to trial eculizumab, a monoclonal antibody targeting c complement, preventing its cleavage and activation, and shown to be effective in treatment of atypical hemolytic uremic syndrome and hemolysis due to an igm cold agglutinin. prior to eculizumab infusion, ch and sc b- assays were significantly elevated. design/method: the patient was given two doses of eculizimab days apart. results: his hemoglobin steadily rose independent of red cell transfusions with a corresponding decrease in reticulocyte count, ldh and ch levels. the patient has remained stable with a normal hemoglobin ( - g/dl) on maintenance steroids and mmf. although we cannot definitively conclude that eculizumab directly caused his recovery, the clinical course post-eculizumab suggests this may be an efficacious treatment for aiha. genetic testing showed monoallelic frameshift mutation of the nfkb gene and monoallelic missense mutation of the dock gene. given the role of nfkb in both immunodeficiency and autoimmunity, it is thought that the patient's phenotype is due to nfkb haploinsufficiency and he is currently considering hematopoietic stem cell transplant. st. joseph's regional medical center, paterson, new jersey, united states background: heterozygous -thalassemia typically manifests as thalassemia minor, characterized by mild microcytic hypochromic anemia with minimal clinical ramifications. coinheritance of -globin gene triplication has been reported to exacerbate the clinical and hematological phenotype ofthalassemia trait, due to increase in the alpha/non-alpha-chain imbalance. reported phenotypes range from asymptomatic thalassemia minor to moderate thalassemia intermedia, usually diagnosed in adulthood without transfusion dependence. this combination has been described in mediterranean, european and asian populations, but rarely reported in hispanics. objectives: to report two cases of unusually severethalassemia intermedia in hispanic patients with heterozygosity for triplicated -globin gene and a ( )-thalassemia allele. results: case : sixteen-month-old male of mexican descent presented with persistent microcytic anemia and jaundice. peripheral smear showed nucleated rbcs with basophilic stippling and target cells. hemoglobin electrophoresis revealed: hba- %, hbf- %, hba - . %. -globin gene testing revealed heterozygosity for ( ) mutation (ivsi-i, g→a). given the unusually severe anemia, -gene testing was performed which showed -globin gene(anti . ) triplication ( / ). at four years, he had splenomegaly and bilateral maxillary prominence. head ct showed irregular contour of the parieto-occipital region due to medullary expansion. due to significant persistent anemia ( - g/dl) and progressive bony deformities of the skull, patient began chronic transfusions at age eight after family declined splenectomy.case : fifteen-year-old female, of peruvian and honduran descent, presented for evaluation prior to cholecystectomy for gallstones and recurrent ruq pain. father had known thalassemia trait. her hb was . g/dl with hypochromia, microcytosis, and target cells. electrophoresis indicated -thalassemia trait (hba- %, hba - . %, hbf- . %), confirmed by gene testing (heterozygous for a ( ) mutation in codon c>t). given jaundice and gallstones, -globin gene analysis was ordered showing triplication ( / ). ruq pain resolved post-cholecystectomy, but she developed persistent painful splenomegaly. she began hydroxyurea to increase gamma-globin production and decrease excess alpha chains, but it was discontinued due to hematological toxicity. due to recurrent luq pain and progressive splenomegaly, she underwent laparoscopic splenectomy at age with resolution of symptoms and improved hemoglobin. conclusion: -globin gene testing should be considered in -thalassemia carriers with an atypical clinical presentation including hispanic patients. the wide variability in the phenotypic expression of (anti . ) mutation andthalassemia trait suggest interplay of other genetic factors which remain undefined. the clinically significant presentation amongst certain subjects, as in our two cases, makes it imperative to identify these factors to aid in phenotype prediction and genetic counseling. ashley bonheur, shivakumar subramaniyam, jogarao vedula, sucharita bhaumik nyu winthrop hospital, mineola, new york, united states background: wilms tumor (wt) is one of the most common solid malignant neoplasms in children. a diverse range of genes and mechanisms are implicated in wt pathogenesis. predisposing syndromes result from a disruption of wt gene, crucial for renal and gonadal embryogenesis. another gene is wt gene locus at p , an area of imprinting. the p tumor suppressor gene on chromosome p . is seen in patients with anaplastic histology. in addition to these genes, whole and partial chromosome gains of q, , q, , , & and losses of p, p, q, q, as well as loss of heterozygosity (loh) are commonly seen. some genetic markers appear to be predictive of outcome and are now incorporated into the assigning of risk-directed therapy. patients with loh at chromosome p and q are treated with more intensive chemotherapy, as they have been associated with increased risk of relapse and mortality. objectives: to describe a new complex translocation involving chromosome , , and in a case of pediatric wt. design/method: a four-year old female presented with abdominal pain and emesis. on exam, patient had a firm and large abdominal mass. radiologic studies revealed a complex lobulated right renal mass. right radical nephrectomy was performed. histopathologic studies showed wt with triphasic histologic features with blastema predominance, invasion of the lymphovascular and perinephric adipose tissues, perinephric lymph node involvement and no anaplasia. chest ct scan showed bilateral lung metastases. tumor cytogenetics showed an abnormal karyotype, a complex translocation of , , and . the rearrangement occurred due to translocation between chromosomal bands q and q , with an insertion of q - on the q region. pcr based genotyping using microsatellite markers additionally identified loh for chromosome p and q . the patient was treated for high risk stage iv wilms tumor with favorable histology and received intensive chemotherapy and radiation therapy to the flank and the lungs. she is now in remission months after, with no evidence of recurrence on surveillance scans. complex translocations associated with wt have not been rigorously studied. a question for further study is whether there is any relationship between recurrence potential with a complex translocation compared to common chromosomal abnormalities. further knowledge of the molecular pathology and genetic changes in wt will help the development of new targeted therapies, as well as new biomarkers to aid diagnosis, risk stratification, and monitoring of treatment and relapse. results: a week-old girl was referred for evaluation of an abnormal newborn screen. mother was a known carrier of hb khartoum trait while father was a known carrier of thalassemia trait. patient's hemoglobin quantification performed by capillary zone electrophoresis showed hbf %, hb variant %, and no detectable hba. the hb variant ran in the d zone, a pattern consistent with mother's hb. alkaline agarose gel electrophoresis banding pattern showed f/s. acid agarose gel electrophoresis pattern showed v/f. later testing revealed abnormal isopropanol stability with + precipitation at minutes. this electrophoresis pattern is consistent with the pattern previously reported of hb khartoum. clinically, the patient is a healthy, active child whom we have followed for two years. she has not had any significant anemia outside of her physiologic nadir. she has not had any hemolytic episodes, and her bilirubin levels have always been within the normal range conclusion: to the best of our knowledge, this is the only reported case of hb khartoum/ thalassemia. the proline to arginine substitution of hb khartoum introduces a charged group on the chain at the site of contact. the resulting unstable chains can dissociate into monomers and favor the formation of methemoglobin, leading to hemoglobin instability. we had wondered if this unstable hemoglobin might result in clinical hemolysis when challenged with oxidative stress, such as in periods of infection. however, in the two years we have followed this patient, she has never had a hemolytic episode. at two years of age, she has hbf . %, hb khartoum . %, and hba . %. whether hbf elevation is protective from oxidative stress remains to be determined as we continue to follow this child. university of puerto rico -medical science campus, san juan, puerto rico, united states background: gm gangliosidosis is a lysosomal disorder caused by -galactosidase deficiency due to mutations in the glb gene. it is a rare autosomal recessive neurodegenerative disorder with an incidence of about : , - : , live births worldwide. this neurological disorder has three clinical forms. gm type , or infantile form is characterized by psychomotor regression by the age of months, visceromegaly (hepatosplenomegaly), macular cherry red spot, facial and skeletal abnormalities, seizures, and profound intellectual disability. we present a -year-old female with gm type and acute lymphocytic leukemia (all). design/method: she was diagnosed with gm type at the st months of age and family history was remarkable for an older sister with gm type . diagnostic studies reveal homozygous exon of the glb gene for a sequence variant defined as c. c>t, predicted to an amino acid substitution p.aarg cs. results: patient presented to our hospital with petechiae in lower extremities, pallor and intermittent tracheal bleeding. physical examination shows a hemodynamically stable girl that is chronically ill dependent of mechanical ventilation, severe mental retardation and scatter petechiae at upper and lower extremities. laboratory workup revealed severe normocytic anemia (hgb: . g/dl) with immature peripheral cells and thrombocytopenia ( × /l). serum chemistry revealed increase ldh ( u/l), increase hepatic enzymes (ast: u/l), normal uric acid level. there was no evidence coagulopathy. chest x ray was unremarkable except for evidence of chronic pulmonary illness. abdominal sonogram hepatosplenomegaly. during hospitalization, bone marrow aspirate and biopsy was performed which was diagnostic of b cell acute lymphoblastic leukemia (all) with . % lymphoblast and orderly myeloid/erythroid maturation. flow cytometry: % b lymphoblast with aberrant phenotype c/w b-acute lymphoblastic leukemia. karyotype revealed hyperdiploid female of favorable prognosis. cytogenetic by fish: hyperdiploid all with extra copies of runx and igh (no bcr-abl translocation). family was oriented about the new diagnosis and the dismal prognosis in conjunction to her primary condition. parents agree on no chemotherapy treatment for all with only supportive treatment. to this date, there is no evidence in literature that has previously described association of gm and leukemia. life expectancy of patient's primary condition is null therefore, correlation with leukemia might not be a coincidental finding. this patient opens the possibility of malignancy as part of gm type thus, malignancy diagnosis should be considered as part of their medical lifetime course. university of south florida, tampa, florida, united states background: hematological manifestations related to hiv infection are not uncommon, with thrombocytopenia having an estimated prevalence of - %. the pathophysiology is likely multifactorial. studies suggest that the primary mechanism may be immunologic resulting in accelerated platelet destruction. additional theories suggest that infection of megakaryocytes may also play a role causing inadequate platelet production. treatment of hiv-related thrombocytopenia is challenging. first-line treatments include initiation and optimization of antiretroviral therapies, immunoglobulin (ivig), and glucocorticoids. however, this approach is not effective in all patients and second line treatment options are less well studied, particularly in the pediatric population. objectives: we aim to present and discuss the case of a year old patient with perinatally acquired hiv- infection and persistent thrombocytopenia who, after failing first line therapies, showed normalization of platelet count on the novel thrombopoietin receptor agonist, eltrombopag. design/method: a retrospective chart review of the case patient's medical record was conducted. additionally, a thorough literature review was performed on this topic including the pathophysiology of hiv related thrombocytopenia and its treatment modalities. the patient required monthly ivig infusions for about year, but did not show a sustained response, often with platelet count dropping to less than , in between infusions. after initiation of mg eltrombopag daily the patient showed a sustained increase in platelet count (range , - , ). during a brief week lapse in eltrombopag treatment his platelet count dropped to , . upon re-initiation of therapy his count increased to , . the patient has remained asymptomatic, off of ivig for over one year, with undetectable hiv viral load and greater than cd t cell counts. no side effects or grade laboratory abnormalities were reported. conclusion: treatment of hiv-related thrombocytopenia can be challenging. first line therapies, including ivig and glucocorticoids, are not effective in all patients. several other treatment modalities have been utilized, including anti-d immunoglobulin, dapsone, danazol, interferon alfa, vincristine, thrombopoetic growth factors including romiplostim and eltrombopag, or splenectomy, but these are less well studied. this represents the first reported case of a pediatric patient with hiv who showed a positive response to eltrombopag with a sustained improvement in platelet count and no adverse effects from treatment. eltrombopag may be a safe alternative to first line therapies in those patients with hiv and refractory thrombocytopenia, however additional studies are needed. university of illinois college of medicine at peoria, peoria, illinois, united states background: achromobacter xylosoxidans is a gram negative rod with peritrichous flagella which causes rare opportunistic infections most commonly encountered by immunocompromised patients. it is primarily associated with uncomplicated bacteremia, cather-associated infections, and pneumonia. most reports of bacteremia associated with a. xylosoxidans are nosocomial, associated with neoplasm, and occurring mainly in adults. most reported infections with a. xylosoxidans in children are associated with cystic fibrosis. there are very few reported cases of septic shock from a. xylosoxidans bacteremia and pneumonia in the pediatric oncology population. objectives: to describe a rare case of a. xylosoxidans septic shock in a pediatric patient with relapsed neuroblastoma results: a -year old boy with history of stage iv highrisk neuroblastoma underwent standard frontline therapy with chemotherapy, hematopoietic stem cell transplant, radiation therapy, and immunotherapy, followed by a dfmo trial for maintenance. his -month follow-up scans demonstrated relapse and he was subsequently treated with additional chemotherapy, surgical resection, and mibg therapy, crizotinib for an eml -alk fusion and finally ifosfamide, carboplatin and etoposide (ice). he developed neutropenic fevers and was started on cefepime, vancomycin and fluconazole. blood cultures were initially negative. on the th day of fever, his previously scheduled pet scan was performed during hospitalization and showed new pulmonary opacities. he did not have respiratory symptoms, but therapy was escalated to meropenem, vancomycin and amphotericin. emergent bronchoscopy was performed the same day, with all bacterial and fungal cultures remaining negative. overnight, he developed tachypnea and saturations in the upper s, requiring nasal cannula. ir-guided lung biopsy was performed the next day, a flexible bronchoscopy was done to remove blood clots in the airway, the patient was placed on a ventilator, femoral lines were placed, granulocytes ordered and pressors were started for deterioration to presumed septic shock. arterial and femoral lines were placed but patient continued to have hemodynamic instability on multiple pressors. the following day, blood and respiratory cultures returned positive for results: at days after the start of iti, the inhibitor was < . bu and continued undetectable months after initiation of iti therapy. in this patient, iti with high-dose plasma-derived factor viii and von willebrand factor (vwf) complex was well tolerated and effective. genetic analysis confirmed a large factor viii gene duplication of exons to . we believe our patient developed inhibitor so quickly ( exposure days) due to the possibility of this mutation causing a frameshift that introduces a premature termination codon. this might be functionally similar to a deletion in the factor viii gene which poses the highest risk for inhibitor development in patients with severe hemophilia a. this variant has only been identified previously in two unrelated patients diagnosed with severe hemophilia a. this duplication is not listed in dbsnp variant database, nor observed in the general population database. our case proves the effectiveness of this method for patients with severe hemophilia a and an inhibitor. it also shows that more research is needed to identify patients at risk for inhibitor development. background: mercaptopurine ( -mp) is a prodrug that is a core component of maintenance chemotherapy for patients with a diagnosis of acute lymphoblastic leukemia (all). suppression of the neutrophil count is used to demonstrate adequate dosing of -mp during this phase of therapy. bone marrow suppression is mediated by the active metabolite -thioguanine ( -tgn), whereas the metabolite -methylmercaptopurine nucleotides ( -mmpn) has been shown to cause hepatotoxicity. allopurinol has been used infrequently in all maintenance therapy in the setting of skewed metabolism when adequate myelosuppression is difficult to achieve due to excessive hepatic toxicity. when given in combination with allopurinol a reduced dose of -mp may result in both increased -tgn levels and decreased -mmpn levels. objectives: describe the characteristics and clinical course of patients treated with allopurinol and reduced dose -mp during maintenance chemotherapy for all. we performed a retrospective chart review of patients at aflac cancer and blood disorders center of children's healthcare of atlanta with new diagnoses of b or t-cell all who received allopurinol during maintenance chemotherapy. we identified eleven patients with b-cell or tcell all who received allopurinol adjunctive therapy during maintenance chemotherapy at a single institution between - . these patients received adjunctive allopurinol for - weeks (median weeks) with reduced -mp ( - % of full dose). all ten patients with genetic testing for thiopurine s-methyltransferase (tpmt) had wildtype genotype associated with normal enzyme levels. indications for allopurinol use were most commonly unfavorable -mp metabolite levels, transaminitis (n = ), pancreatitis (n = ) and hyperbilirubinemia (n = ). favorable metabolite shift was achieved in all patients. liver enzymes improved in of patients with transaminitis after initiation of allopurinol/reduced -mp. three patients who experienced pancreatitis during maintenance did not have recurrence after initiation of allopurinol ( of these patients previously reported). six patients developed pancytopenia while on allopurinol, and two of those patients developed pancytopenia severe enough to require allopurinol cessation. four patients developed isolated anemia (hgb < . g/dl) without thrombocytopenia or severe neutropenia. no patient has experienced a recurrence of leukemia. overall, treatment with allopurinol and reduced dose -mp was successful in producing a favorable -mp metabolite distribution and reducing toxicity. therapy was generally tolerated; however a major and notable side effect was pancytopenia, in two cases severe enough to stop allopurinol treatment. anemia may be more prominent with allopurinol usage. allopurinol effect is variable among individual patients despite normal tpmt genotypes. baylor college of medicine, houston, texas, united states background: congenital sideroblastic anemia, b-cell immunodeficiency, periodic fevers and developmental delay syndrome (sifd) is a rare inherited sideroblastic anemia syndrome, first described in with clinically similar cases. genetic variations of trnt were identified as causative. objectives: to present an unusual presentation of a patient with sifd complicated by diagnosis of concomitant alpha thalassemia trait. design/method: retrospective chart review. a five month old male infant was referred to our hematology center for evaluation of elevated hemoglobin barts identified on newborn screen. despite numerous attempts, blood work was unable to be collected. at seven months of age he had microcytic anemia (hemoglobin . g/dl, mean corpuscular volume fl) more severe than what would be expected with alpha thalassemia trait. no variant hemoglobin was identified with isoelectric focusing or high performance liquid chromatography. by nine months of age he developed growth failure, intermittent emesis with fevers, developmental delays (predominantly gross motor), hearing loss, a disproportionally large head and coarse, thinning hair. over the next ten months, he was seen by numerous specialists for seemingly unconnected problems including sensorineural hearing loss, elevated liver enzymes and growth hormone deficiency. alpha globin analysis revealed deletion of two alpha globin genes. at months of age, he was admitted with one week of fevers, jaundice, and emesis. peripheral blood smear showed microcytic hypochromic anemia with marked anisopoikilocytosis including target cells, elliptocytes, tear drops, spherocytes, poikilocytes, marked polychromasia, and coarse basophilic stippling. given the inconsistency of his laboratory findings with the diagnosis of alpha thalassemia trait and clinical syndromic findings, bone marrow biopsy was performed which revealed rare ringed sideroblasts. one month later whole exome sequencing revealed trnt splicing variant c. - c>g and novel missense variant c. a>t consistent with sifd. hemoglobin barts on newborn screen with moderate to severe microcytic anemia directed initial diagnostic work-up towards variant alpha thalassemia. as additional medical conditions developed the focus shifted to a unifying syndrome. compared to previously described cases, our patient was diagnosed at an older age, presented with anemia rather than episodes of febrile illnesses, and had rare sideroblasts on bone marrow examination. diagnosis in this case led to identification of the novel c. a>t variant in his sister who had similar, but milder, features. sifd is a rare disease with variable phenotypic severity making diagnosis challenging without high index of suspicion which is crucial for appropriate management. wiseman, blood, . chakraborty, blood, background: cholelithiasis is uncommon in childhood. cholelithiasis is known to occur more frequently in children with predispositions, including female sex, obesity, parenteral nutrition, previous abdominal surgery, use of oral contraceptives, family history of gallstones, chronic hemolytic anemias, hepatobiliary disease, or exposure to specific drugs. although there have been occasional case reports linking cholelithiasis to childhood leukemia or leukemia therapy, the prevalence and risk factors of cholelithiasis in patients with childhood leukemia remain unclear. objectives: to estimate the prevalence of cholelithiasis in patients diagnosed with childhood acute lymphoblastic leukemia (all), and to evaluate possible risk factors for the development of cholelithiasis in patients with childhood all. we performed a computer-assisted review of the electronic medical records of patients diagnosed for b or t-cell all at children's healthcare of atlanta in the period from to . patients with diagnoses of cholelithiasis, cholecystitis or who had a cholecystectomy were identified. possible risk factors of age, sex, bmi, history of abdominal surgery and parenteral nutrition use were abstracted. patients with underlying chronic hemolytic anemia or pre-existing gallbladder disease were excluded. results: seventeen cases of cholelithiasis and cases of cholecystitis without documented cholelithiasis were identified. among patients with cholelithiasis, were female. median age at diagnosis of cholelithiasis was . (range . - . ) years. seven patients had no symptoms referable to cholelithiasis at the time of diagnosis. the median age of leukemia diagnosis among these patients was . (range . - . ) years. the median interval from diagnosis of leukemia to gallbladder disease was . years. four patients had bmi over the th percentile for age. two patients had a prior history of intraabdominal surgery. no patient received oral contraceptive pills. six patients received parenteral nutrition for more than days. there was no documented family history of cholelithiasis. seven patients did not receive any cholelithiasis directed therapy. two patients were managed with medical management only, with endoscopic retrograde cholangiopancreatogram with stone extraction, and with cholecystectomy. our study estimates the prevalence of cholelithiasis in childhood lymphoblastic leukemia to be . %, higher than the reported prevalence in the general pediatric population of . - . %. although our cohort size is small, it appears that all therapy and supportive care modalities associated with all are likely to play a larger role in the development of cholelithiasis than known predisposing factors in the general population. further studies are warranted. background: an uncommon side effect of intravenous immunoglobulin (ivig) administration is clinically apparent, sometimes severe hemolysis. we describe a severe case of coombs-positive hemolytic anemia secondary to ivig administration. ivig is a blood derivative manufactured from pools of , to , individual plasma donations. ivig is not abo-type restricted, so anti-a, anti-b and anti-a,b isoagglutinins are detectable. objectives: to describe a rare but serious type of transfusion reaction leading to gross hemolysis after ivig administration. results: a -year-old male with a past medical history of obstructive sleep apnea and obesity was admitted to the pediatric intensive care unit for adenoviral pneumonia and subsequent respiratory failure requiring mechanical ventilation. he had a complex hospital course with many complications including acute respiratory distress syndrome (ards), septic-shock, and coombs-positive hemolytic anemia. the patient was treated with commercial ivig (baxter/baxalta) -mg/kg daily for five days. he had two isolated episodes of severe hemolysis in relation to ivig administration requiring multiple transfusions of packed red blood cells (prbc). examination of pre-transfusion peripheral blood smear showed spherocytosis with rouleaux formation and large clumped rbc aggregates. the patient's blood type was classified as blood group a, rh-negative and his initial prbc transfusions were of this type. subsequently, the patient's coombs test was found to be positive using polyspecific and anti-igg typing sera. the patient's antibody screen against reagent group o screening cells was negative ruling out autoimmune hemolytic anemia. however, type specific anti-a antibodies were detected in his plasma as well as the acid eludate prepared from the coombs-positive red blood cells. it was concluded that the patient's hemolysis was due to anti-a antibodies presumed to arise from ivig. the patient's rbc transfusions were changed to o-negative blood and the hemolytic process resolved. the patient ultimately died due to complications of ards. although hemolysis is a known side effect of ivig, it is rarely considered when deciding to administer ivig. in addition, it has rarely been described in the pediatric population. ivig is used in the treatment of a growing number of medical conditions. due to the critical nature of many of these patients, hemolysis secondary to ivig may not be considered and continued blood transfusions with the patient's specific blood type may be used. it is crucial to remember that severe hemolysis can occur from ivig, and the importance of transfusing with blood group o, rh-negative blood when applicable. university of maryland medical center, children's hospital, baltimore, maryland, united states background: coagulopathy is a well-described complication of acute promyelocytic leukemia (apml), and remains a leading cause in induction failure. with treatment, coagulopathy associated with apml has been shown to rapidly improve. multiple organ dysfunction syndrome (mods) in apml, including acute respiratory distress syndrome (ards), has been associated with infection, traumatic injury, malignant infiltration, and cytokine release syndrome. when mechanical ventilation is no longer sufficient, extracorporeal membrane oxygenation (ecmo) can be considered; however, coagulopathy, severe end-organ damage, and malignancy are all relative contraindications to initiation of treatment. we report the case of a -year-old female presenting in respiratory failure, disseminated intravascular coagulopathy (dic), with intracranial hemorrhage, and mods, diagnosed with apml, successfully treated with ecmo therapy. design/method: retrospective case analysis and literature review. our patient, a -year-old female was admitted in respiratory failure and altered mental status, following a fall shortly prior to presentation. initial laboratory values were notable for pancytopenia, dic, and acute renal failure. a non-contrast head ct showed left temporal lobe intraparenchymal hemorrhage. she was diagnosed with apml by peripheral smear, later confirmed by fish for t( : ), and was started immediately on high-risk induction chemotherapy as per cog protocol aaml , including all-trans retinoic acid, arsenic trioxide, idarubicin, and dexamethasone. cvvhd was required for acute renal failure. despite maximal respiratory support, she remained hypoxemic, with oxygenation index of , pao /fio ratio of . ecmo was initiated hours after start of induction, hours after admission. coagulopathy resolved on day of induction, ecmo was discontinued after days, mechanical ventilation and cvvhd were stopped after days and she continued to improve, eventually achieving remission with few neurologic side effects. despite relative contraindications to ecmo, this patient was successfully treated with ecmo without significant neurologic side effects. the correction of her coagulopathy was multifactorial: ) restoration of adequate oxygen delivery via ecmo improving endothelial function; ) successful organ support to allow sufficient response to induction chemotherapy with atra leading to the terminal differentiation of leukemic blasts; ) complement and contact system activation through contact with ecmo circuitry. this case illustrates that ecmo can still be considered in patients despite coagulopathy and end organ damage. sinai hospital of baltimore, baltimore, maryland, united states background: primary polycythemia vera is an extremely rare diagnosis in the pediatric patient and is defined by a marked elevation of red blood cells due to erythropoietin-independent mechanisms. presentations of this disorder range from the asymptomatic person to severe thrombotic events, such as budd-chiari syndrome or cerebrovascular stroke. mutations in the jak gene are found in adult and pediatric patients with polycythemia vera; however, the jak v f mutation is less commonly identified in pediatric patients. we describe an otherwise healthy -year-old female who presented with a significantly elevated total erythrocyte count, hemoglobin, and platelets, incidentally discovered upon routine annual blood work obtained by her pediatrician. design/method: this is a report and discussion of a rare case. demonstrated cellular marrow with trilineage hematopoiesis and no dysplasia. cytogenetics were not assessed. his hemoglobin and platelet count recovered but leukopenia and neutropenia persisted. follow-up evaluation at three months revealed fevers, ongoing cytopenias, a one-month of a nodular skin rash on the trunk and extremities resembling erythema nodosum, and hepatitis (peak alt and ast of , and , , respectively). following clinical evaluation, a skin biopsy was performed and was remarkable for atypical lymphocytes within the subcutis with t-cell markers, a high ki- , and positive tia- , perforin, and -f immunoperoxidase stains. negative stains for cd , cd , and ebv were noted. these results are consistent with sptcl. additional evaluation did not support a diagnosis of hlh. a staging evaluation was performed. pet-ct showed widespread hypermetabolic subcutaneous activity in the legs, trunk and skull and diffuse marrow hyperplasia. bone marrow demonstrated involvement with precursor b-cell acute lymphoblastic leukemia, with a mll gene rearranagement. his skin biopsy was retrospectively stained with tdt, cd , pax- , cd a, and cd with negative results, and a blood smear taken at the time of the skin biopsy did not demonstrate leukemic cells. conclusion: this is the first report of a patient with sptcl having a synchronous malignancy. the patient is doing well, currently in the maintenance phase of treatment for his all, and his skin disease has resolved on pet-ct. while it is possible that his presentation was a function of chance, the possibility of an underlying immune dysfunction or cancer predisposition warrants further investigation. cincinnati children's hospital medical center, cincinnati, ohio, united states background: hereditary xerocytosis (hx) is a rare red blood cell (rbc) dehydration disorder, characterized by variable hemolysis and propensity to iron overload. hx is often misdiagnosed as hereditary spherocytosis (hs). while splenectomy is curative for hs, it is relatively contraindicated in hx due to a substantial thromboembolism risk, signifying the importance of delineating these diseases. blood smear abnormalities are variable and often insufficient to make an accurate diagnosis. osmotic-gradient ektacytometry and genetic confirmation are critical in distinguishing these overlapping disorders. objectives: describe a family with hx, initially misdiagnosed as hs. discuss the importance of distinguishing these disorders and the utility of ektacytometry in making this distinction. design/method: a -year-old caucasian male was diagnosed with hs after presenting with prolonged neonatal jaundice starting on the first day of life. he described mild scleral icterus and history of intermittent jaundice and dark urine, without need for transfusions. his father, paternal uncle and paternal grandmother were all diagnosed with hs during childhood and underwent cholecystectomy. additionally, his father underwent splenectomy for abdominal pain. the child's blood counts revealed compensated anemia (hb . gm/dl) and reticulocytosis (arc × /mcl) with increased mcv ( . fl) and mchc ( . gm/dl). blood smear showed increased polychromasia and poikilocytosis with rare spherocytes and few stomatocytes. while the child had normal ferritin, his father had iron overload (ferritin ng/ml) despite no prior transfusions. osmotic-gradient ektacytometry profile of the child and father's rbcs showed a characteristic left-shifted, bell-shaped curve with decreased omin and ohyp, diagnostic of hx. the family is currently undergoing genetic studies. despite clinical similarities between hs and hx, distinguishing these diseases has significant management implications. hx is a disorder of rbc permeability, causing shortened rbc survival. stomatocytes on blood smear can raise suspicion for hx, but are insufficient to make an accurate diagnosis. identifying characteristic biomechanical membrane properties using osmotic-gradient ektacytometry is the gold standard for clinical diagnosis, which can then be confirmed by molecular studies. hs and hx can be easily and reliably distinguished using ektacytometry, as both disorders have very distinctive curves representing different rbc deformability patterns. after hx diagnosis was made, we counseled the family against splenectomy, as the risk of thromboembolism is significantly increased in hx compared to hs, and the father was diagnosed with iron overload. conclusion: hx is commonly misdiagnosed as hs. this case highlights the importance of making this distinction, and the utility of osmotic-gradient ektacytometry in reliably distinguishing these conditions. penn state health children's hospital, hershey, pennsylvania, united states background: relapsed acute myeloid leukemia (aml) presenting as an isolated central nervous system myeloid sarcoma (cns ms) is very rare and its treatment is not well-defined. thiotepa, vinorelbine, topotecan and clofarabine (tvtc) has been successful for re-induction therapy to induce remission prior to hematopoietic stem cell transplant (hsct). objectives: to describe our experience in utilizing tvtc therapy in two children with no extramedullary disease at initial diagnosis who presented with relapsed aml as intracranial myeloid sarcomas. results: case : month-old female was diagnosed with flt negative aml and completed treatment per the children's oncology group (cog) aaml study on the low risk arm without bortezomib. cerebral spinal fluid (csf) negative at diagnosis. fish testing positive for tcf gene deletion of unknown significance. mrd was undetectable after induction i and remained undetectable after each cycle. nine months off therapy, recurrent headaches prompted mri imaging which revealed two posterior fossa masses. csf and bone marrow testing were negative. stereotactic biopsy of the larger mass confirmed recurrence of aml. patient underwent two cycles of tvtc with a total of seven doses of intrathecal cytarabine with almost near resolution of the cns ms. completed cranial radiation and proceeded to allogeneic stem cell transplant with unrelated cord marrow donor and is disease free at approximately day + .case : year-old female diagnosed with flt and mll negative aml and completed treatment per cog aaml study on the low risk arm without bortezomib. csf negative at diagnosis. mrd was undetectable after induction i and completed therapy without complications. two months off therapy, a retrospective analysis of her diagnostic bone marrow by the cytogenetic laboratory to test a new panel identifying novel q partners revealed a cryptic insertional : (mllt /mll(kmt a) translocation. at four months off therapy, acute mental status changes prompted mri imaging which revealed two intracranial ms and lumbar spine involvement. resection of the larger lesion for symptomatic relief confirmed the mllt /mll(kmt a) fusion. csf positive for blasts and marrow negative for relapsed disease. patient completed two cycles of tvtc with a total of seven doses of it cytarabine with near resolution of cns disease (only mm contrast enhancement in the medulla). she received craniospinal radiation and is awaiting improvement in her cardiac function before proceeding to hsct. conclusion: tvtc is a successful reinduction regimen for relapsed aml with cns ms prior to hsct. background: acute severe anemia can be a life-threatening medical condition. the differential is quite broad for possible etiologies of acute severe anemia, including autoimmune hemolytic anemia (aiha) and atypical hemolytic uremic syndrome (ahus). autoimmune hemolytic anemia is an antibody-mediated process that targets the protein antigens located on the surface of red blood cells. treatment options for aiha include corticosteroids, with up to % of patients being responsive, with some requiring splenectomy. atypical hemolytic uremic syndrome is a medical urgency, defined as the triad of microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury. the etiology is usually due to genetic causes, or less commonly, due to autoantibodies or idiopathic reasons. prognosis is very poor. objectives: differentiating between autoimmune hemolytic anemia and atypical hemolytic uremic syndrome can be a time-sensitive diagnostic dilemma while the patient is in critical condition, but this important delineation can vastly alter therapeutic options. design/method: here we discuss two cases highlighting the diagnostic workup involved in differentiating between atypical hemolytic uremic syndrome and autoimmune hemolytic anemia. patient a is a -year-old male who presented in extremis with severe anemia, uremic encephalopathy, and severe acute renal injury requiring hemodialysis and multiple blood transfusions. patient b is a -month-old male, who also presented in extremis with respiratory failure secondary to adenovirus/rhinovirus/enterovirus, with acute progressive renal failure and microangiopathic hemolytic anemia, requiring hemodialysis and cardiorespiratory support. : patient a underwent a full hematologic and infectious disease workup. subsequent laboratory studies confirmed enteropathogenic e.coli (epec) in the patient's stool; blood cultures remained negative. renal biopsy results were consistent pigment nephropathy. bloodwork indicated positive direct coombs. patient a was ultimately treated with steroids mg/kg/day, with significant improvement. patient b also included a full hematologic work-up, including adamts activity and ahus genetic panel, as well as full infectious disease work-up. subsequent laboratory test-ing revealed blood cultures growing streptococcus pneumoniae, with adamts activity at % (adult ref range: >/ = %), and normal complement levels. imaging findings also supported diagnosis of ahus. the management of a critically ill patient with acute severe anemia requires a thorough hematologic and infectious disease work-up. while molecular and genetic are helpful in definitive diagnosis of ahus, the utility of such results is limited by time. overlapping clinical presentation of a patient in extremis due to acute severe hemolytic anemia with progressive renal failure presents a rather broad differential, with time-sensitive treatment and prognostic implications. the favorable response to steroids delineates aiha from hus. background: d- -hydroxyglutaric aciduria (d- -hga) is a rare metabolic disorder characterized by developmental delay, hypotonia, and bi-allelic mutations in d- hydroxyglutarate dehydrogenase (d hgdh) or isocitrate dehydrogenase (idh ). metaphyseal chondromatosis with d- -hydroxyglutaric aciduria (mc-hga) is a type of d- -hga that has been previously reported in seven patients (omim ; pmid ), three of whom had somatic mosaicism for r variants in isocitrate dehydrogenase (idh ). we describe a -year-old boy with mc-hga who subsequently developed acute myeloid leukemia (aml) and was found to have a r variant in idh in a leukemic bone marrow sample. we report the first case of aml with this metabolic disorder. design/method: a -year-old hispanic boy presented with short stature, developmental delay, abnormal skin pigmentation, and unilateral congenital cataract. workup revealed multiple skeletal enchondromatosis and elevated urine d- -hydroxyglutaric acid levels. he was diagnosed with mc-hga. no pathogenic variants in d hgdh, idh and idh were identified in peripheral blood. germline testing with biopsies of skin lesions was declined by the family. two years later, he presented with streptococcal sepsis and pancytopenia. blasts were noted on peripheral smear. bone marrow morphology was consistent with acute myelomonocytic leukemia (∼ % blasts). chromosome analysis showed normal xy, and molecular testing by pyrosequencing idh and idh revealed a r c variant in idh ( % mosaicism). the patient is being treated as per the cog study aaml . end of induction i bone marrow aspirate was hemodiluted, but there was no obvious residual disease by flow cytometry ( . - . % sensitivity) or morphology. the previously identified idh variant was no longer detectable (limit of detection < %). although targeted therapy for aml with idh mutation is currently in phase i clinical trials in adults, there is no safety or efficacy data for using idh inhibitors in children. treatment with ivosidenib is therefore not currently an option for our patient. conclusion: this is the first case of aml reported with this rare metabolic disorder. somatic r variants in idh have been identified in three other mc-hga cases. this same mutation leads to the accumulation of d- -hydroxyglutarate in gliomas and aml. without any confirmed germline mutation or somatic mosaicism testing of multiple specimen sources, we can only speculate that the patient has an underlying somatic idh mutation associated with mc-hga which subsequently led to leukemogenesis. we present the first case of this association, to increase index of suspicion for development of aml in children with metabolic disorders associated with variants in idh . background: congenital combined deficiency of the vitamin k-dependent coagulating factors (vkcfd) is a rare heterogeneous autosomal recessive bleeding disorder. vkcfd is caused by mutations in the genes of either gamma-glutamyl carboxylase (ggcx) or vitamin k epoxide reductase complex (vkorc), which are responsible for the gammacarboxylation of vitamin k dependent proteins (vkdps) allowing for their activation. the clinical presentation ranges from no bleeding to intracranial hemorrhage. to date, vkcfd has been reported in few patients worldwide. objectives: we report a case of a girl with novel homozygous mutation of the ggcx gene, highlighting her clinical and biochemical characteristics with a review of the literature. a -month-old girl of consanguineous emirati parents, presented to our hospital with a history of bleeding from puncture site after receiving her second-month vaccine. that was associated with episodes of mild mucosal bleeding. review of systems was negative for jaundice, steatorrhea and failure to thrive and physical exam was unremarkable. investigations revealed markedly prolonged pt and aptt with high inr. fibrinogen, hemoglobin and platelets were always normal. activities of vitamin k-dependent factors including fii, fvii, fix, fx, protein c and s were all low. a measurement of proteins induced by vitamin k absence (pivka-ii) was done and came very high. this was associated with a mild elevation in liver enzymes but normal liver function test. the picture was supporting vitamin k deficiency, and as a result, she was started on oral vitamin k supplements of mg/day. she responded partially to vitamin k and required higher doses to stabilize her inr. after excluding acquired causes and due to her requirement of high doses of vitamin k, a mutation in either ggcx or vkorc genes was suspected. genetic analysis was conducted for her which revealed a novel missense homozygous mutation in the ggcx gene (c. a>t) confirming the diagnosis of combined deficiency of vitamin k-dependent clotting factors type . the asymptomatic parents were both heterozygous for the same mutation. results: she is currently stable on mg/day of vitamin k supplements. conclusion: vkcfd is a rare bleeding disorder with an overall good prognosis due to the availability of several effective therapeutic options. the function of the mutated gene is unknown. our patient demonstrated a partial response to vitamin k supplements suggesting presence of a residual carboxylation capacity and a possible role of this gene in the enzymesubstrate interactions. university of alabama at birmingham, birmingham, alabama, united states s of s background: gata is a zinc finger transcription factor that plays a critical role in the regulation of hematopoiesis and lymphatic angiogenesis. mutations leading to gata deficiency (gd) have been linked to a variety of clinical conditions. patients with gd have a striking predisposition to develop myelodysplastic syndrome (mds), acute myeloid leukemia (aml), or chronic myelomonocytic leukemia (cmml). acute lymphoblastic leukemia (all) has not been associated with gd, although the association of bcell all and gd has been previously reported. objectives: to describe a unique association of gata deficiency and t-cell all in a young child. results: an -year-old female presented with a one-week history of fever and malaise. she had a significant past medical history of verruca plantaris and self-resolving leukopenia associated with febrile illnesses. significant family history included sister with neutropenia and human papilloma virus (hpv) infection, and mother with neutropenia, monocytopenia, atypical mycobacterial infections, and hpv infection. peripheral blood revealed hemoglobin . g/dl, hematocrit . %, platelets , /ul, and white blood cell , /ul (neutrophils /ul, lymphocytes /ul, monocytes /ul). patient underwent a bone marrow biopsy demonstrating lymphoblast infiltration. flow cytometry analysis demonstrated monoclonal lymphoid blast population that co-expressed cd , cd , cd , nuclear tdt, cd , however, lacked expression of cd , cd , cd , cd , hla-dr, or myeloperoxidase. findings were consistent with tcell all with aberrant myeloid markers. cytogenetics analysis revealed ,xx,dic( ; )(p . ;p . ). patient began treatment as per children's oncology group aall and achieved remission at the end of induction. course of therapy was complicated by episodes of fever, reciprocating junctional tachycardia, asparaginase-associated thrombosis, viral meningitis, recurrent episodes of verruca plantaris, and resistant streptococcus pneumoniae or haemophilus parainfluenza infections causing chronic cough. later, she was also found to have low igm levels; after completion of therapy, she developed monocytopenia. lymphocyte subset panel revealed absent b cells, decreased number of natural killer (nk) cells, and cd /cd inversion. further work-up included gata sequence analysis that showed heterozygous nonsense mutation (c. c > t/c; reference nm_ ) likely resulting in gata haploinsufficiency. patient continues to be in remission, is receiving monthly immunoglobulin replacement and is on azithromycin for atypical mycobacterial prophylaxis. surveillance bone marrow biopsies have shown no evidence of mds or leukemia, however, have demonstrated persistent hypocellularity. the possibility of undergoing an allogeneic bone marrow transplant is actively being discussed given its curative potential. clinicians should be aware that t-cell all may be associated with gata deficiency. cincinnati children's hospital medical center, cincinnati, ohio, united states background: treatment for severe hemophilia a is centered on factor viii (fviii) replacement therapy. development of an alloantibody (inhibitor) against fviii is a significant treatment complication occurring in as many as - % of patients. high titer inhibitors render treatment with factor viii ineffective, necessitating the use of bypass agents that may not achieve hemostasis with the same efficacy. considering the substantial ramifications of inhibitor development on treatment, eradication of inhibitors is of great importance to achieve adequate hemostasis in this patient population. desensitization by immune tolerance induction (iti) is the primary method of inhibitor elimination. however, not all patients respond to iti. immunomodulation may be considered as the next line of therapy, although controversy remains in regards to agent selection and use. objectives: there is incomplete data on the use of immunomodulation therapy for inhibitor eradication in severe hemophilia a. we present a case of a pediatric patient with severe hemophilia a and high titer inhibitor who failed initial iti therapy to better illustrate potential treatment options for the future. design/method: a retrospective chart review was performed on a patient with severe hemophilia a at cincinnati children's hospital medical center. results: an -year-old caucasian male with severe hemophilia a secondary to intron inversion, was initially diagnosed following extensive bleeding after circumcision at birth. he was identified as having an inhibitor ( bethesda units (bu)) at months of age after exposure days of treatment. he failed multiple attempts of iti, with recombinant and plasma-derived (pd) fviii. he was advanced to immunomodulation therapy in combination with pdfviii, however demonstrated anaphylaxis to rituximab and ofatumumab. he underwent tolerization to rituximab, and received a six month course with a partial response (nadir of . bu). months following last dose of rituximab, a rising inhibitor titer ( . bu) was found. mycophenolate mofetil (mmf) was initiated with subsequent inhibitor stabilization and a decreasing titer ( . bu) over the course of the following year. mmf has been well tolerated without major side effects or infection throughout therapy. conclusion: development of an inhibitor against fviii is a considerable complication in patients with severe hemophilia a. use of immunomodulatory therapies following iti failure remains controversial. mmf has not been well studied in this patient population. we report a case of a patient who is being successfully treated with mmf with minimal side effects. further prospective studies should be considered to further define the role of mmf immunomodulation therapy. background: down syndrome (ds) children with aml (ds aml) have higher cure rates than their non-ds counterparts. outcomes for refractory/relapsed cases, however, remain dismal. somatic mutations of the gene encoding the transcription factor gata in ds aml patients are responsible for the observed hypersensitivity of ds aml blasts to cytosine arabinoside (ara-c). in view of excellent survival rates (approaching %) of ds aml patients, the ongoing children's oncology group (cog) aaml study seeks to determine the feasibility of treating standard risk (minimal residual disease/mrd negative) ds aml patients using a reduced dose ( -fold decrease) ara-c backbone. although results from japanese trials with this approach are promising, north american and european data are conflicting. although chromosome rearrangements in ds aml do not appear to carry the same adverse prognostic significance as in non-ds aml, monosomy in ds aml patients has been associated with a moderately worse outcome. isochromosome q, however, is rare and has only been reported in previous cases of ds aml. objectives: to report our institutional experience of very early relapse involving cases of ds aml patients treated per the reduced dose ara-c arm ( . g/m ) of the aaml study. design/method: we hereby report the disease course and cytogenetics of the above ds aml patients. : patient is a month old caucasian female who had gata mutation negative aml. patient is a -year old caucasian male whose chromosomal analysis revealed isochromosome q ( copies of the long arm of chromosome ). both patients achieved negative mrd (< . %) after induction i chemotherapy with thioguanine, low-dose ara-c and daunorubicin and proceeded per the reduced dose ara-c arm of aaml . patient relapsed immediately after completion of chemotherapy. salvage chemotherapy with mitoxantrone/high dose ara-c (hidac) failed to induce a second remission and the patient subsequently died of disease. patient relapsed within months from end of therapy. the patient underwent salvage chemotherapy utilizing a hidac backbone and remains in disease remission. the noted very early relapse following a reduced dose ara-c regimen in our above ds aml children suggests that testing for gata mutation and chromosome rearrangements may play a useful role in the development of future risk-stratified treatment strategies for ds aml. university of rochester, rochester, new york, united states background: in developed countries in the st century, severe nutritional deficiency is not an often considered differential diagnosis of unexplained childhood anemia. aside from iron deficiency anemia, vitamin deficiency severe enough to impact hematopoiesis is uncommon in the general pediatric population. here we present the unique case of a -monthold infant who presented with intermittent emesis, failure to thrive (ftt), developmental delay, macrocytic anemia, and neutropenia which was initially concerning for a congenital bone marrow failure syndrome. instead, she was discovered to have an underlying, potentially familial deficiency of b . objectives: . to describe the unique case of an infant with b deficiency. . to outline the importance of including b deficiency in the differential diagnosis of unexplained megaloblastic anemia in children. a -month-old exclusively breastfed infant presented for gastroenterology evaluation due to persistent emesis and poor weight gain over the course of months. her history was notable for delayed developmental s of s milestones and hypoactivity. marked pallor prompted hematologic evaluation, which revealed concern for macrocytic anemia (hemoglobin . g/dl, mcv ), reticulocytopenia ( . × ^ / l), and neutropenia (anc . × ^ /l). an otherwise reassuring physical examination and laboratory evaluation was notable only for the discovery of an undetectable b level and marked hyperhomocysteinemia ( mol/l). her hemoglobin (hgb) continued to decline (to . g/dl) over the first few days after presentation, and she required red blood cell (rbc) transfusion. within only a few days of initiation, daily cyanocobalamin injections resulted in a robust reticulocytosis response, improved hgb, immediate normalization in the neutrophil count, and resolution of hyperhomocysteinemia. additional history and laboratory evaluation from the patient's mother revealed a concurrent, asymptomatic maternal b deficiency as well as a history of a need for b supplementation in the maternal grandfather, raising concern for an inherited etiology. despite the rarity of vitamin-deficient hematologic abnormalities in the general pediatric population, b deficiency should be considered as a potential cause of an otherwise unexplained megaloblastic anemia, especially in the setting of concurrent ftt and neurodevelopmental delay. a detailed family history should be obtained in such cases and may have helped to prevent this patient's clinical sequelae had the deficiency been discovered sooner. our patient has experienced a favorable clinical response to b supplementation, attesting to the importance of vitamin b in early childhood growth and development. background: peg-asparaginase is universally utilized in the treatment of pediatric acute lymphoblastic leukemia (all). despite its high efficacy in this disease, it is associated with hypersensitivity and allergy in - % of patients. protracted anaphylaxis has been described in circumstances such as severe food allergy with ongoing allergen exposure; however, it has not yet been described in relation to peg-asparaginase. we describe the first reported case of protracted anaphylaxis after peg-asparaginase administration, provide guidance as to time course and management of protracted anaphylaxis, as well as evidence that erwinia asparaginase may be safely administered even in this high risk population. objectives: to provide guidance regarding the duration, course and management of protracted, severe anaphylaxis after peg-asparaginase therapy. a year old male with very high risk all presented for consolidation therapy with peg-asparaginase (intramuscular) and vincristine. one hour after administration, he developed generalized hives and angioedema, for which he was given diphenhydramine. he then quickly developed progressive hives, angioedema, subjective throat and chest tightness, and wheezing. he was treated with diphenhydramine, epinephrine, albuterol, and methylprednisolone with resolution of symptoms. one hour later, symptoms recurred and the patient became hypotensive; he was retreated with methylprednisolone and epinephrine, and was transferred to the pediatric intensive care unit (picu). in the picu, he was placed on an epinephrine drip, and continued on methylprednisolone, diphenhydramine, cetirizine, albuterol, and ranitidine. the epinephrine drip was successfully discontinued after hours, and his other medications were gradually weaned over the course of two weeks. of note, the patient did have st segment changes in his electrocardiogram during the first hours of anaphylaxis. these were associated with normal ventricular function as per echocardiogram, and resolved within one week. this patient has subsequently tolerated multiple doses of erwinia asparaginase (intramuscular) without premedication. this patient was acutely managed in the pediatric intensive care unit with steroids, anti-histamines, and continuous infusion epinephrine. symptoms consistent with severe anaphylaxis including hives, angioedema, throat and chest tightness, wheezing, and hypotension persisted for a total of four days before finally resolving. he has thus far tolerated multiple doses of erwinia asparaginase without any symptoms of allergy, hypersensitivity, or anaphylaxis. protracted severe anaphylaxis after peg-asparaginase therapy can be successfully managed with multi-agent therapy, including antihistamines, steroids, and continuous infusion epinephrine. re-challenge with an alternate form of asparaginase may be tolerated, even in a patient with protracted anaphylaxis to peg-asparaginase. ucsf benioff children's hospital oakland, oakland, california, united states background: vincristine (vcr) is widely used in pediatric cancers. unlike most cytotoxic agents, hematopoietic toxicity is uncommon. vcr-induced anemia has been observed but its mechanism has not been well studied. vinca alkaloid-induced membrane changes were seen in early studies of hereditary spherocytosis (hs) and anecdotal cases suggest vcr may increase hemolysis in such patients. here we describe a case involving severe vcr-induced anemia in a patient with hs and an explanation as to the mechanism. objectives: to describe the mechanism of vcr-induced anemia in hs. design/method: case report. a year-old female with hs was diagnosed with t-lymphoblastic lymphoma. she had required packed red blood cell (prbc) transfusions as a neonate and thereafter had done well without episodes of acute hemolysis or aplasia. complete blood counts (cbc's) demonstrated a compensated hemolysis, and she did not require further transfusions until she commenced chemotherapy. by the start of maintenance she had received many more prbc transfusions than the average patient. intermittent drops in hemoglobin (hb) did not correlate with any particular agent, and she had stable, mild splenomegaly. a clear pattern emerged during maintenance. her hb was - g/dl at monthly clinic visits, when she received vcr, intermittent intrathecal methotrexate, and corticosteroids. within - days, her hb dropped to . ± . g/dl, and reticulocyte count decreased from . to . ± . %. transfusion at day corrected hb, and the reticulocytes and hb returned to baseline. white blood cell and platelet counts did not change after vcr. blood samples from pre, immediately post, and days post vcr were analyzed and rbc characteristics and markers of hemolysis were not significantly different. ektacytometry showed identical curves, indicating no change in rbc deformability. in vitro incubation of patient blood samples with vcr also did not affect the osmotic deformability, confirming that a change in rbc rigidity was unlikely the reason for the drop in hb. these data indicate that a dysregulation of erythropoiesis was responsible for the anemia after vcr, rather than damage of peripheral rbc's. in most patients, maintenance therapy for lymphoblastic lymphoma does not cause severe anemia, likely because a temporary reduction in erythropoiesis in patients with a normal rbc survival and low reticulocyte count is not noticed. however, in a patient with decreased rbc survival and a brisk reticulocytosis, a disruption in rbc generation is more apparent. in conclusion, vcr administration to patients with an rbc disorder warrants close observation for potentially severe vcr-induced anemia. background: the addition of tyrosine kinase inhibitors (tki) to conventional chemotherapy has improved outcomes for pediatric patients with philadelphia chromosome-positive (ph+) acute lymphoblastic leukemia (all), however there remains an increased risk of relapse compared to other types of childhood all. typically, in relapsed disease the philadelphia chromosome persists and several mechanisms of resistance involving acquired mutations of the bcr-abl chimeric oncoprotein have been reported. objectives: describe a unique case of a pediatric patient with ph+ b-precursor all relapsing with b-precursor all without the philadelphia chromosome. results: an -year-old boy was diagnosed with ph+ bprecursor all with the presence of the t( ; )/bcr-abl translocation by cytogenetics and fluorescence in situ hybridization (fish), respectively. additional abnormalities included gains of runx and loss of one copy of etv . a remission bone marrow with negative minimal residual disease (mrd) was achieved at the end of induction with dasatinib and the esphall chemotherapy backbone. duration of tki therapy was two years post diagnosis. nearly one year after the completion of therapy, cytopenias prompted a bone marrow investigation. relapsed b-precursor all was established by immunophenotyping, however fish analysis did not identify the bcr-abl rearrangement. moreover, quantitative reverse transcriptase pcr was negative for the bcr-abl fusion transcript. again fish analysis of the bone marrow revealed multiple additional copies of runx and mono-allelic loss of etv , similar to the initial diagnostic sample. the patient was re-induced per aall anticipating a ph+ all relapse. however, with confirmation of the loss of the ph+ clone, tki therapy was not re-initiated. due to positive mrd of . % at the end of re-induction therapy, the patient was salvaged with blinatumomab therapy and subsequently underwent an allogenic stem cell transplant with a sibling donor. conclusion: this is the first known report of a pediatric patient with ph+ b-precursor all who developed recurrent b-precursor all without the philadelphia chromosome. the persistent findings of gain of runx and loss of etv makes it unlikely that a second unrelated b-precursor all developed following successful treatment of the original disease. this case highlights the possibility of a genetically distinct subclone present at the onset of disease that shared abnormalities of runx and etv but did not contain the philadelphia chromosome. nevertheless, the subclone harbored leukemogenic potential in the absence bcr-abl expression. it is plausible that the predominant clone present at diagnosis was effectively treated with dasatinib and extinguished, but the bcr-abl -negative clone persisted in the face of tki therapy. background: ligneous conjunctivitis is a rare form of pseudomembranous conjunctivitis that develops specifically in patients with type plasminogen deficiency. lack of plasmin activity in those patients result in defective fibrinolysis and formation of fibrin-rich membranous material/ masses that develops on the palpebral conjunctiva as well as other sites in the body.current management involve surgical excision of the masses that is usually complicated by multiple recurrences. recently, use of topical plasminogen concentrates helped delaying recurrence, but currently, those concentrates are not commercially available. we report on a -year-old omani girl, with hypoplasminogenemia who required optimization of plasminogen level at the time of surgery to delay/ prevent recurrence. objectives: case report on the peri-operative use of ffp versus cryopricipitate transfusion as an alternative replacement of plasminogen during surgical excision of ligneous conjunctivitis. design/method: pharmacokinetic study was performed to assess plasminogen recovery after ffp ( ml/kg) and precipitate ( bag/ kg) transfusion results: plasminogen levels remained subnormal after either ffp or cryoprecipitate administration. with ffp, the maximum concentration reached was almost % of normal. although half-life of plasminogen is known to be - . days, the patient seemed to have a high catabolic rate after receiv-ing cryoprecipitate, with plasminogen levels reaching basal levels within hours. because of the better recovery profile with ffp, we opted to give ffp before and after surgery. peri-operative management included ffp transfusion at ml/kg/ hours one day before and for days post operatively, followed by ml/kg once daily from day - , then ml/kg on th post-operative day. topical treatment was initiated using antibiotic and steroids ed on the day of surgery, followed by heparin ed on the second day. on follow up, she used topical heparin, cyclosporine, prednisolone, and topical lubricant eye drops for variable duration. clinical picture remained stable for almost year post operatively, when she started to develop recurrence of ligneous lesions again. background: ponatinib (inclusig®, ariad pharmaceutical) is a rd generation multi-targeted tyrosine kinase inhibitor (tki) approved for treatment of adults with chronic myeloid leukemia (cml) and philadelphia chromosomepositive acute lymphoblastic leukemia (ph+ all) resistant to or intolerant of other tkis. ponatinib has numerous drug-drug interactions and a black box warning for associated serious adverse vascular events and hepatotoxicity. for this reason, ponatinib use has been confined to specific high-risk populations. however, in patients who prove refractory to other therapies, the potential benefits of ponatinib may outweigh risks. to date, ponatinib has not been studied in the pediatric/adolescent and young adult (aya) population. furthermore, literature describing the use of ponatinib alone or in combination with other agents in pediatric oncology patients is scarce. objectives: to describe a single institutional experience using ponatinib in the pediatric patients with ph+ all. design/method: two cases of ponatinib use in pediatric ph+ patients resistant to other tkis were identified at our institution and are described. peripheral blood samples obtained from both patients identified bcr-abl p fusion transcripts and sanger sequencing was used to identify resistant mutations. results: our first case is a -year-old female who received upfront multi-agent chemotherapy plus dasatinib for ph+ all. relapse was confirmed on end-of-therapy bone marrow evaluation, thus bcr-abl mutation testing was performed and revealed a t i mutation. ponatinib was initiated then discontinued after one week due to clinically significant fluid retention with peripheral edema and bilateral pleural/pericardial effusions. the second case is a lateadolescent female with ph+ all who relapsed -years after stem cell transplant (sct). following relapse, tki therapy included both imatinib and dasatinib. due to persistence of bcr-abl fusion transcript despite tki therapy she was switched to ponatinib. shortly following initiation of ponatinib she developed a diffuse, maculopapular rash, which persisted despite dose reduction, resulting in ultimate discontinuation of the drug. bcr-abl mutation testing identified f l and f v resistance-conferring mutations. to date, there is scant existing literature detailing the use of ponatinib in pediatric patients. appropriate dosing is undefined and side effect profile not well described, particularly when used concurrently with other chemotherapeutic agents. thus, this case series reporting the response to and toxicity of ponatinib in pediatric ph+ all patients has important clinical implications. additionally, this is the first report of a pediatric ph+ all patient with documented t i mutation underscoring the importance of bcr-abl mutational testing, particularly at the time of relapse. cooper university hospital, camden, new jersey, united states background: myh -related disorder is a rare autosomal dominant disease, encompassing several subtypes: may hegglin anomaly, epstein syndrome, fechtner's syndrome, and sebastian syndrome. heterozygous mutations are seen in the gene encoding non-muscle myosin heavy chain iia (nmmhc-iia) which is involved in cell motility as well as functions to maintain cellular shape and integrity. the presentation of myh -rd is mainly characterized by macrothrombocytopenia, but various related expressions exist: nephritis often leading to renal failure, cataracts and sensorineural deafness ( ). a -year-old girl with history of extensive dental caries, hyperactivity, and speech delay due to suspected hearing loss was incidentally found to have thrombocytopenia at the time of genetic evaluation. she did not have any bruising or excessive bleeding. she did not respond to observation, immunoglobulins, or steroid therapy. her platelet count remained persistently low ( - k/ul). she underwent extensive evaluation to rule out platelet disorder vs. coagulation defect. her peripheral smear showed enlarged platelets by giemsa stain but no inclusion bodies were noted in granulocytes. her platelet aggregation and platelet surface glycoprotein by flow cytometry were negative. her coagulation profile was also normal. objectives: this case report summarizes the complexity in diagnosing myh -rd in a pediatric patient. design/method: since a unifying diagnosis for her clinical presentation was not apparent, whole exome sequencing (wes) was undertaken. results: wes revealed the r c heterozygous pathogenic variant, located in exon in the myh gene. myh gene alteration explained the patient's clinical features of macrothrombocytopenia and hearing loss. this mutation was paternally inherited, and her father demonstrates mosaicism. he was asymptomatic with normal platelet count but his morphology showed enlarged platelets with no inclusion bodies in granulocytes. when dealing with patients who have mild or no symptoms of bleeding diathesis but evidence of persistent macrothrombocytopenia, considering a platelet disorder belonging to myh -rd can help delineate certain predisposing syndromes and guide clinical management. patients are likely to benefit from early genetic testing while receiving supportive therapy. wes can highlight syndromes and provide information on recurrence risk for families. the renal and hearing abnormalities are indistinguishable between epstein and fechtner's syndromes, but the pathogenic variants differ ( ). the genotype-phenotype correlation implies that our patient may have either syndrome, although clinical features compatible with nephritis have yet to manifest. patients should be monitored closely for long-term progression of myh disease, and treatments should be initiated accordingly. we present an -year old female evaluated by genetics at birth due to prenatal microcephaly. chromosomes and microarray were normal. at age she developed standard risk pre-b-cell acute lymphoblastic leukemia (all). she completed treatment in and has been doing well in the interim, remaining in complete clinical remission. during and after treatment she exhibited developmental delay and neurocognitive deficits. at age her height and weight were at or below the th centile and head circumference was below the nd centile (approximately standard deviations below the mean and corresponding to the th centile for a -month-old girl). bone age was appropriate. she had a distinctive triangular face with micrognathia and a pointed nose resembling a seckel-like syndrome. the patient also had clinodactyly of the th toes, zygodactylous triradius involving the nd and rd left toes, tendency to sydney line in the right palm and a radial loop in the left middle finger. the patient's unique clinical presentation prompted a more thorough genetic evaluation, which led to a novel finding we feel is clinically significant with regard to the development of malignancy. design/method: whole exome sequencing (wes) was performed on the patient as well as her biological parents (trio). a de novo heterozygous mutation in the gene pcdh with potential relation to the phenotype was discovered. this c. dupa variant causes a frameshift starting with codon asparagine , changing this amino acid to a lysine residue and creating a premature stop codon at position of the new reading frame denoted p.asn lysfsx . this variant is predicted to cause loss of normal protein function via protein truncation or nonsense-mediated mrna decay. conclusion: pcdh is a member of the protocadherins family which is important in cell-to-cell adhesion and synaptic function in the central nervous system and is highly expressed in areas of the brain involved in higher cortical function and speech. aberrant expression of protocadherins has been associated with the development of malignancies in many organ systems. with regards to leukemia, the methylation status of this gene at diagnosis has been implicated in the prognosis of all and could be used as a biomarker to predict relapse. this patient's de novo mutation and clinical presentation are unique to what has been previously presented in the literature. we feel that this mutation is a clinically significant finding that may shed light on the role of this gene in the development of hematopoeitic malignancies. background: acquired hemophilia a (aha) is an uncommon and potentially life-threatening hemorrhagic disease characterized by sudden onset of bleeding in patients with neither personal nor family history of bleeding dyscrasia. it is usually seen in adults with autoimmune diseases, solid tumors, lymphoproliferative diseases, pregnancy or during the postpartum period; occurrence in the pediatric population has rarely been reported. we report a case of an otherwise healthy teenager who was found to have aha when he presented with acute onset of atraumatic soft tissue hematoma. results: a -year old male of middle eastern descent with history of congenital absence of the right external ear, but otherwise in good general health, presented to our emergency department with a three day history of progressive worsening of right lower leg pain, swelling, and paresthesia, without preceding history of trauma. evaluation by the pediatric orthopedics service documented significantly elevated compartment pressures, necessitating immediate four-compartment fasciotomy. pre-operative labs were significant for prolonged activated partial thromboplastin time (aptt) of . ( . - . ) seconds with normal prothrombin time (pt) and international normalized ratio (inr). ptt did not correct on mixing studies, suggesting the presence of a circulating anticoagulant. factors xii and xi were in the normal range; factor ix was elevated, ( - ). factor viii level was % and fviii inhibitor level was . bethesda units (< . ), confirming the diagnosis of aha. work up for autoimmune disease was negative. his bleeding and surgical hemostasis were managed with recombinant factor vii (novoseven) mcg/kg every hours for hours post operatively, with gradual interval prolongation. factor viii antibody eradication was managed with prednisone mg/kg/day. factor viii and inhibitor levels normalized by day of hospitalization. recombinant factor vii was discontinued; steroids were gradually tapered and discontinued at discharge (hospital day ). conclusion: acquired hemophilia is likely an underdiagnosed condition in pediatrics. while it is typically seen in adults with underlying autoimmune disease, solid tumors, lymphoproliferative disease, or during pregnancy or the postpartum period, pediatric cases may have no identifiable etiology. this case highlights the importance of considering this diagnosis in any patient with unexplained bleeding regardless of their age, so as to intervene early and prevent adverse consequences. university of oklahoma, oklahoma city, oklahoma, united states background: myeloid neoplasms associated with eosinophilia is a rare subtype of chronic leukemia characterized by clonal eosinophilia. the true incidence is unknown due to its rarity and possible classification as idiopathic hypereosinophilia syndrome. the most common chromosomal aberrations involve platelet-derived growth factor receptors (pdgfrs). we report one such rare case in a pediatric patient. most of the pediatric management of this entity is derived from adult case reports and case series. objectives: to describe a case of chronic leukemia presenting as eosinophilia results: a previously healthy year old caucasian male presented with a several week history of migrating joint pain, splenomegaly, and abnormal blood counts with leukocytosis, thrombocytopenia and absolute eosinophilia. white blood cell differential showed myeloid precursors suggestive of chronic myeloid leukemia. bone marrow evaluation showed % blasts and % eosinophils. bcr-abl testing was negative, ruling out cml. fish analysis for eosinophilic clonality revealed deletion of chic gene, resulting in fip l /pdgfra fusion gene, diagnostic for myeloid neoplasm with eosinophilia associated with pdgfr abnormalities. treatment was started with tyrosine kinase inhibitor (tki), imatinib mg daily. within months, fish analysis for fusion gene was negative. after approximately months of daily imatinib, he was switched to maintenance dose of mg weekly. he is approximately months since diagnosis and doing well on maintenance imatinib. in , the who revised its classification of some chronic eosinophilic leukemias to myeloid and lymphoid neoplasms associated with eosinophilia and rearrangement of pdgfra, pdgfrb, fgfr . the most common abnormality is the fip l /pdgfra fusion gene. other less common abnormalities include fusion genes kif b-pdgfra and etv -pdgfrb and point mutations in pdgfra . some features of chronic eosinophilic leukemia include absolute eosinophilia, splenomegaly, elevated vitamin b and tryptase levels, and organ damage from eosinophil infiltrates and cytokine release. patients with rearrangements or mutations involving pdgfra are usually very responsive to imatinib. starting doses have not been well studied or established. experts recommend co-administration of corticosteroids during the first few days of imatinib therapy in patients with a history of cardiac involvement and/or elevated serum troponin levels to prevent myocardial necrosis, a rare complication of imatinib therapy in eosinophilic patients. fortunately our patient did not have cardiac involvement and to date has not exhibited signs of chronic tki toxicity. conclusion: myeloid neoplasms with eosinophilia constitute a rare form of chronic leukemias. they are often associated with pdgfr abnormalities and are usually very responsive to tyrosine kinase inhibitor therapy. walter reed national military medical center, bethesda, maryland, united states background: germline samd l mutation is a rare cause of constitutional bone marrow failure with a unique propensity for clonal evolution to monosomy and mds. objectives: previous case series have demonstrated diverse clinical outcomes in patients with a germline samd l mutation. our case presents a novel samd l mutation (p.val leu). additionally, the case highlights the challenges in clinical decision making for a patient with a gene mutation that is known for clonal evolution towards monosomy with risk of progression to myeloid malignancy, but also known for self-correction through uniparental disomy or inactivating mutations which results in disease remission. design/method: a retrospective chart review and review of the literature was performed. dna was isolated from peripheral blood and used for whole exome sequencing. a peripheral blood sample from the patient's mother and father showed no samd l mutation. skin biopsies of the patient and parents were evaluated for uniparental disomy or new mutations. to determine the pathogenicity of this novel mutation, the specific samd l mutant dna was transfected into the human embryonic kidney cell line to assess its role in inhibiting cell proliferation. our patient presented at months of age with pancytopenia and hypocellular bone marrow in the setting of s of s sepsis. he had evidence of dysfunctional immune activation with hemophagocytosis and elevated soluble il with simultaneous severe hypogammaglobulinemia. analysis of the peripheral blood showed no increase in chromosomal breakage, normal telomere length, and normal flow cytometry. gene testing for primary hemophagocytic lymphohistiocytosis and inherited bone marrow failure were negative. after the patient recovered from his presenting illness, a repeat bone marrow biopsy demonstrated improved cellularity with myelodysplasia and cytogenetics significant for monsomy .whole exome testing demonstrated a novel samd l mutation. the patient continued to require intermittent ivig and failed to demonstrate appropriate leukocytosis with intermittent infections. on repeat bone marrow evaluation over the course of months, the patient demonstrated no evidence of evolution towards self-correction and had a persistent monosomy clone. the patient is scheduled to undergo a matched unrelated donor bone marrow transplant. our case highlights the unique clinical picture associated with constitutional marrow failure and clonal evolution secondary to a novel samd l mutation which is thought to cause pancytopenia by inhibiting cellular proliferation and often results in the development of monosomy which rescues hematopoiesis but with a risk for malignancy. background: notable labs developed a flow cytometricbased assay with a custom robotic platform to test fdaapproved drugs for anti-cancer activity against individual patient's tumor cells. this personalized assay is a potential method for identifying novel agents and drug combinations to treat aml patients who have failed standard therapies. objectives: to present the case of a teen who underwent successful treatment of relapsed aml post-sct with bortezomib, panobinostat, and dexamethasone-a regimen selected based upon results of notable lab testing. results: a -year-old male with m -aml had an isolated bone marrow relapse months after completion of scheduled therapy. at relapse, his aml was flt -itd positive. he achieved a second remission with negative mrd and underwent matched sibling donor bmt after busulfan/cyclophosphamide conditioning. bma performed on day + was mrd positive ( . %). repeat bma done on day + showed . % mrd. he started sorafenib on day + . he received donor lymphocyte infusion (dli) on day + , then received cycles of azacitadine (aza) followed by dli. marrow mrd by flow after sorafenib alone, sorafenib with dli, and sorafenib with aza/dli were %, . %, and . %, respectively. treatment was complicated by varicella meningitis, grade i skin agvhd, febrile neutropenia and c. difficile colitis, and metapneumovirus pneumonia. despite extremely low levels of leukemia (marrow mrd . %), notable lab testing performed on the patient's leukemia cells from marrow collected after aza/dli/sorafenib revealed sensitivity of his leukemic blasts to a combination of bortezomib, panobinostat, and dexamethasone. because of prolonged cytopenias, multiple infectious complications, and persistently positive mrd, he discontinued aza/dli/sorafenib and on day + started bortezomib . mg/m iv on days , , , and ; panobinostat mg po on days , , , , , ; and dexamethasone mg po on days , , , , , , , and . chemotherapy cycle started days later. he tolerated treatment without side effects and with resolution of rash and cytopenias. he achieved full donor chimerism, negative flt -itd, and complete remission by morphology and flow after two cycles. notable lab testing is a powerful tool for evaluating the sensitivity of small populations of leukemic blasts to novel drug therapy. results from notable lab testing may serve as a useful guide for treatment selection after failure of standard aml therapy. this patient achieved morphologic and mrd remission post-sct with bortezomib, panobinostat, and dexamethasone-a regimen predicted to be efficacious based upon notable lab results. maria ahmad-nabi, christine knoll, sanjay shah, esteban gomez, lori wagner phoenix children's hospital, phoenix, arizona, united states background: development of inhibitors in patients with factor ix deficiency (fixd) is a well-recognized complication occurring in - % of patients. within this subset a small percentage can develop anaphylaxis to factor. desensitization with cyclophosphamide, an alkylating agent used in the management of various oncologic malignancies, and reported for use in factor viii desensitization has been previously unreported for use in desensitization in patients with fixd. rituximab, an anti-cd antibody, however has been used. objectives: to induce immune tolerance (it) in patients with inhibitors to factor ix with either novel or under reported methods using cyclophosphamide and/or rituximab. we report a case series of patients at phoenix children's hospital with fixd who achieved it with cyclophosphamide and/or rituximab. results: patient one was a year old male with severe fixd, who at the time of desensitization had inhibitor levels of bu. he was desensitized with cyclophosphamide, then admitted for infusion of recombinant factor ix. he experienced a few minor symptoms of intolerance including an urticarial rash which was self-limited, and hemarthrosis of the right elbow on day which responded to novo . he tolerated the remainder of his infusion without issues. he continued recombinant factor ix daily, and returned to clinic for monthly cyclophosphamide for months. he did develop urticaria with hemarthrosis and spontaneous muscle bleeds which were tempered with zantac, zyrtec, solumedrol, and benadryl. he remained without a recurrence of inhibitors, however did have intermittent hemarthrosis of his ankles thereafter requiring prophylactic twice daily dosing recombinant factor ix. patient two was a year old male with severe fixd and a family history of anaphylaxis to factor causing early death in all male relatives with the disease. he had never received factor ix and did not have a detectable inhibitor prior to desensitization. he successfully underwent desensitization to recombinant factor ix with rituximab in the icu, and returned to clinic for weekly infusions x . he experienced no adverse reactions concerning for anaphylaxis. he continued to tolerate factor ix products without evidence of intolerance, development of inhibitors, and continues on as prophylactic dosing of recombinant factor ix every other day. our experience at a single institution proves cyclophosphamide as a novel agent for inducing it in those with fixd and anaphylaxis. it also provides further evidence that rituximab can desensitize patients with severe fixd. differences include longer duration for cyclophosphamide therapy ( months vs month). background: cartilage-hair hypoplasia (chh) is an autosomal recessive chondrodysplasia associated with defective cell-mediated immunity caused by mutations in the ribonuclease mitochondrial rna processing (rmrp) gene. cancer incidence is -fold higher in patients with chh than in the general population, especially non-hodgkin lymphoma. the use of rituximab, an anti cd antibody, results in decreased host b-cell number and impaired humoral function for - months. the safety of rituximab in pediatric patients with cancer and immunodeficiency is not well documented. a diagnosis of underlying immunodeficiency may discourage physicians from using rituximab due to the risk of severe bacterial infection or viral re-activation. objectives: to report a case of burkitt lymphoma in a young adult female with chh and defective cellular immunity successfully treated with rituximab. results: an -year old amish female with disproportionate short stature presented to our center for management of stage iv biopsy proven burkitt lymphoma with myc rearrangement. she had presented a week earlier with cervical, occipital, and submandibular lymphadenopathy, splenomegaly; fevers, night sweats, and weight loss for - weeks. on exam, her height was three feet associated with brachydactyly, mild bowing of the legs, normal size head without frontal bossing, fine and sparse hair. she had normal intelligence. her pattern of dysmorphisms was suggestive of chh (genetic testing not performed at time of diagnosis). pet-ct scan showed stage iv disease with involvement of cervical lymph nodes, spleen, iliac bone and bone marrow. treatment with standardintensity fab/lmb therapy (group c) with the addition of rituximab was initiated. she had an incomplete response to cop (∼ % reduction of tumoral masses) but achieved complete remission after copadam . her course was complicated with severe varicella zoster but she completed therapy and remains in complete disease remission for months after treatment completion. genetic testing subsequently performed proved homozygosity for chh with a n. a>g variant. she had no other opportunistic infections during or after therapy. conclusion: the use of rituximab was both safe and beneficial in our patient despite defective cell mediated immunity secondary to chh suggesting that rituximab may be safe to use in patients with cellular immune deficiencies. background: hemophilia a and b are bleeding disorders characterized by deficiency in factor viii or ix, respectively. spontaneous or provoked hemarthrosis is a known complication of hemophilia. repetitive episodes of hemarthrosis can lead to debilitating hemophilic arthropathy. lyme disease is a tick-born infection which is endemic to increasing parts of the united states. chronic lyme disease, the phase in which lyme arthritis typically develops, occurs months to years after initial infection and is characterized by swelling of one or more large joints generally in the absence of systemic symptoms. objectives: review cases of hemophilia a and b patients with episodes of provoked hemarthrosis refractory to intensive recombinant factor replacement therapy found to have concurrent lyme arthritis. design/method: we report two clinical cases and review relevant literature. results: first, we report a year-old male with moderate hemophilia a with a provoked knee hemarthrosis which failed to improve despite months of intense factor replacement therapy requiring multiple hospitalizations. factor replacement regimens included twice daily standard half-life recombinant factor viii products or daily to every other day extended half-life recombinant factor viii products with trough levels aimed as high as - %. factor viii pk studies were obtained for dosing, to confirm adherence, and to evaluate for subclinical inhibitors (inhibitor testing was negative). given protracted symptoms additional workup for hemarthrosis was pursed. lyme titers were positive for ( )igg, though negative for igm. he was treated with days of doxycycline during which time hemarthrosis greatly improved on examination and imaging, and he was able to recover function through physical therapy. second, we report a year-old male with moderate hemophilia b who required multiple hospital admissions for a provoked knee hemarthro-sis with no improvement in symptoms despite weeks of daily or twice daily factor replacement with standard halflife recombinant factor ix products aiming for % correction. we performed inhibitor testing (which was negative) and pk studies to assess for non-detectable inhibitors, dosing and adherence. lyme testing was positive for ( )igg, though negative for igm. he was treated with amoxicillin for days during which time hemarthrosis significantly improved on examination and imaging. diagnosis and follow-up imaging studies for both patients included mri and serial bedside ultrasounds performed as per uc san diego school of medicine mskus guidelines. background: relapse/refractory aml following allogeneic hematopoietic stem cell transplant (hsct) holds a high mortality rate. current relapse/refractory therapy modalities for younger patients may include re-induction with a clofarabinebased regimen followed by second allogeneic hsct. even for patients who undergo second hsct, the five-year survival rate is dismal. new therapies, including small molecule inhibitors, are being studied in the post-hsct relapse setting or those unfit for hsct with promising results. venetoclax is a small molecule inhibitor that has received breakthrough designation for aml treatment in elderly patients objectives: to report a young adult aml patient with relapse post hsct who was successfully re-induced with topotecan, vinorelbine, thiotepa, clofarabine (tvtc) and has sustained remission with venetoclax maintenance therapy. this approach appears to be unique in terms of reported literature. results: our patient is now a -year-old female noted to have mll rearranged aml at initial diagnosis when she was years old. she underwent chemotherapy consisting of cytarabine/daunorubicin according to standard + . due to persistent disease, she was re-induced with g-csf, clofarabine, and high-dose cytarabine (gclac) which put her in cr. her course was complicated by sepsis, colitis, gastrointestinal bleed, deep venous thrombosis, and transfusionassociated circulatory overload. given her co-morbidities, she received another cycle of clofarabine/cytarabine, and then proceeded to reduced intensity allogeneic hsct, according to bmt ctn . the patient tolerated hsct well and experienced no transplant-related complications, including no acute or chronic gvhd. unfortunately, she relapsed about month's post-hsct. initial salvage therapy consisted of another course of g-clac, but due to persistent disease the decision was made to re-induce her with topotecan, vinorelbine, thiotepa, and clofarabine (tvtc). during this time however, she was found to have extensive infection with a fusarium species requiring a course of anti-fungal therapy. bone marrow evaluation showed no residual disease with an mrd of < . %. once the absolute neutrophil count recovered, the patient was started on single-agent venetoclax for maintenance therapy, which has been well-tolerated. she remains in morphologic remission for over months. we describe herein a young adult with multiply relapsed aml wherein tvtc re-induction, followed by maintenance with venetoclax were safely used in the post-hsct setting. venetoclax therapy in the relapsed aml setting warrants further study. background: vitamin b deficiency is uncommon in children in developed countries, especially in the absence of risk factors like malabsorption or inadequate dietary intake. it often presents with non-specific symptoms and signs and can elude diagnosis. the recognition and treatment of vitamin b deficiency is critical as it can lead to bone marrow failure as well as severe neurological and developmental problems in children. to increase index of suspicion of vitamin b deficiency anemia in children. we report a rare case of vita-min b deficiency anemia in a child who presented with a severe macrocytic anemia, with signs of hemolysis and concern of malignancy. design/method: an almost three-year-old previously healthy girl presented with a few day history of fever, emesis, fatigue and pallor. she had no dysmorphic features, hepatosplenomegaly or lymphadenopathy on exam, growth and development were normal. laboratory findings showed severe macrocytic anemia (hemoglobin . grams/dl; mcv . fl) with reticulocytopenia. signs of intravascular hemolysis were present with elevated lactate dehydrogenase ( , units/l) and haptoglobin below assay limit. immune-mediated hemolysis was ruled out. initial picture of a hemolytic anemia was compounded by other findings of moderate neutropenia, mild thrombocytopenia and peripheral smear showing occasional blasts. further workup was done with a broad differential diagnosis that included leukemias, hemolytic anemias, bone marrow failure syndromes, and specific deficiencies. results: workup revealed abnormally low vitamin b levels along with significantly elevated homocysteine and methylmalonic acid levels indicating functional vitamin b deficiency. bone marrow evaluation showed megaloblastic anemia and dyserythropoiesis consistent with vitamin b deficiency, and ruled out leukemia. vitamin b deficiency can cause a hemolytic anemia like picture secondary to intramedullary hemolysis due to ineffective erythropoiesis. myeloid precursors are also affected which can lead to neutropenia, thrombocytopenia, and abnormal peripheral blood cells. in our patient, initial symptomatic anemia was treated with blood transfusion, followed by intramuscular vitamin b injections with normalizing lab values. so far, workup for an etiology for vitamin b deficiency is negative except for an equivocal range of anti-parietal cell antibodies raising concerns for pernicious anemia; however it is rare in this age group. another rare condition is an inborn error of the cobalamin transporter. she is currently on oral vitamin b supplementation and further workup will be planned based on response. conclusion: this case highlights the importance of early consideration and thorough evaluation of vitamin b deficiency in children with unclear etiology of anemia, so that prompt treatment can be initiated. memorial hospital/ university of miami, miami, florida, united states background: despite great success in the treatment of acute lymphoblastic leukemia (all), the outcomes for patients with relapsed all remain poor. prognostic indicators include timing and site of relapse. blinatumomab, is the first agent in its class that simultaneously binds cd -positive cytotoxic t cells to cd -positive b cells resulting in lysis of malignant cells. however, mechanisms of leukemia resistance to blinatumomab are unclear. objectives: to describe a case with multiple sites of extramedullary (em) relapse during blinatumomab therapy. results: a -year-old hispanic male with philadelphia positive, cd -positive b-precursor cell all refractory to chemotherapy, had failed a bone marrow (bm) and was placed on blinatumomab and imatinib. he achieved minimal residual disease (mrd)-negative systemic remission, but during his fifth cycle developed bilateral periorbital masses. biopsies confirmed cd -negative isolated em relapsed disease, which was treated with radiation therapy (rt). there was notable resolution of em disease and he continued systemic therapy. subsequently, he presented with a painful left scapular swelling. imaging showed muscle and lung parenchymal em relapse with cd -positivity confirmed on histology. he continued on blinatumomab with localized rt while awaiting car-t cell therapy. his bm mrd remained negative until he developed systemic mrd-positivity with cd -positive blasts following the sixth cycle. primary resistance to blinatumomab is poorly understood. it is proposed that expansion of cd -negative clones or downregulation of cd following blinatumomab may play a role. this was observed in our patient's periorbital relapse; but subsequent em and systemic relapses were cd -positive, consistent with the co-existence of multiple clones in relapsed all. it has also been postulated that em relapse could be linked to the failure of blinatumomab or t cells to migrate to em sites of disease or drug inactivation by the microenvironment. the second em relapse in our patient, with cd -positive disease suggests this as a possible mechanism of relapse. this was reported in patients with cd positive non-hodgkin lymphoma (nhl), and higher doses of blinatumomab however, have shown promising results in this population. despite blinatumomab's effectiveness in inducing remissions in patients with refractory/relapsed all, it appears to have limitations in patients with em disease. these may arise either from the multiclonality associated with relapsed all or due to the emergence of resistance to blinatumomab, including failure to migrate to em sites. background: cyclic neutropenia is a rare hereditary disorder, characterized by recurrent neutropenia, cycling at about week intervals, with variable associated symptoms including oral ulcers and fever. there are reported cases of cyclic neutropenia associated with chronic inflammation leading to development of reactive aa amyloidosis. one patient also presented with amyloid goiter. we report a new case of cyclic neutropenia with associated renal and thyroid amyloid. design/method: a -year-old female presented with a month history of thyromegaly, and recurrent aphthous ulcers associated with fevers. laboratory workup showed severe neutropenia, anemia, azotemia, and abnormal thyroid function, with an absolute neutrophil count - / l, hemoglobin - . g/dl, serum creatinine - . mg/dl, and uric acid - . mg/dl. thyroid stimulating hormone was elevated - . iu/ml, and normal free t . urinalysis showed + protein, + blood, and - urine red blood cells/hpf. chest radiograph showed mild narrowing of the trachea from thyroid compression. bone marrow biopsy showed a hypocellular marrow, with tri-lineage hematopoiesis, left shifted myeloid maturation with very rare mature neutrophils. both renal biopsy and thyroid fine needle aspiration revealed abundant amyloid. of note, her father had aa amyloidosis, resulting in end-stage renal disease (esrd) requiring hemodialysis, and recurrent aphthous ulcers. the family history suggested a familial predisposition. genetic testing revealed a pathogenic elane c. a>t gene mutation with autosomal dominant inheritance confirming the diagnosis of cyclic neutropenia. we treated our patient with daily granulocyte colony stimulating factor to reduce the burden of chronic inflammation induced by cyclic neutropenia, and to preserve renal and other end organ function affected by further amyloid deposition. results: proband with elane gene mutation positive cyclic neutropenia, amyloidosis of thyroid and kidney, with a positive paternal history of aa amyloidosis resulting in esrd. cyclic neutropenia may result in chronic inflammatory states leading to secondary amyloidosis. university of kentucky, lexington, kentucky, united states background: overall survival of burkitt lymphoma (bl), regardless of stage, is greater than % in the pediatric population when treated with multi-agent chemotherapy. adenovirus is a common, usually self-limited infection within the pediatric population; however, findings can vary within an immunocompromised host. hepatitis is a rare complication, with very few reports of radiologic findings in this patient population. we discuss a three year old male with history of bl who presented with clinical and radiographic evidence of relapse but was found to have adenovirus hepatitis. design/method: a case report of a patient with bl in complete remission after completion of standard of care chemotherapy, who presented with return of high fever, elevated ldh, transaminitis and hepatic lesions. we describe the hepatic imaging and pathology consistent with adenovirus hepatitis in this immunocompromised host. our patient presented at three years old with a six week history of worsening abdominal pain and fevers. he was found to have a right sided pleural effusion, multiple lesions of the liver, and diffuse abdominal lymphadenopathy; biopsy of lymph tissue was consistent with bl. he completed therapy per anhl arm b and was in a complete remission at the end of planned therapy. one month after completion of therapy, he returned with high fever, abdominal pain and transaminitis, similar to his initial presentation. ct scan showed multiple hypodense discrete lesions throughout the liver and re-accumulation of right sided pleural effusion. ldh peaked at u/l (uln u/l). uric acid remained within normal limits. bilirubin peaked at . mg/dl, conjugated . mg/dl. liver biopsy was performed, showing smudgy nuclei with immunohistochemical staining positive for adenovirus. there was no evidence of lymphomatous involvement. resolution of hepatic lesions and transaminitis, with normalization of ldh and fever, occurred with symptomatic treatment alone. adenovirus is known to cause systemic disease in immunocompromised patients and rarely hepatitis. no pediatric patients with discrete hepatic lesions secondary to adenovirus have been reported in the literature. three cases of discrete hepatic lesions have been reported in adult immunocompromised patients, two with fatal fulminant liver failure and one who required cidofovir. this case demonstrates that a common pediatric viral infection can present with lesions concerning for metastatic disease in a pediatric lymphoma patient. prompt diagnosis is vital in the management of these patients when recurrent lymphoma is in the differential. background: heparin induced thrombocytopenia (hit) is an immunologic process in which antibodies bind a heparin complex and cause a paradoxical hypercoagulable state. ramifications of this process may include a multitude of thrombotic events and bleeding complications secondary to platelet consumption. in our patient, hit manifested as increased bruising, an acute decrease in platelet count, and continual clotting of her crrt circuit. hit, although rare in pediatrics, should be included in the differential for children with thrombocytopenia who have received heparin products. to present a unique case report of a critically ill pediatric patient who developed hit in the presence of multiorgan system failure and to discuss the challenges encountered with identification of an alternative anti-coagulant. results: a yo obese, caucasian female child presented to our facility with bilateral pulmonary emboli (of unclear etiology). initially, she was started on a continuous heparin infusion, but was transitioned to enoxaparin within days without issue. five days after enoxaparin was initiated, the patient developed acute kidney injury (evidenced by increasing creatinine) attributable to her biventricular heart failure. due to her need for continuous renal replacement therapy (crrt), she was transitioned back to a continuous heparin infusion. whereas her initial platelet count on transition was normal, she developed severe thrombocytopenia ( , ul) within hours. due to intermediate risk but low suspicion for hit, pf antibodies were sent which were positive. after much discussion, she was transitioned to an argatroban infusion which was titrated according to ptt levels. within hours, her platelet count normalized. at discharge, she was prescribed apixaban for anti-coagulant management. conclusion: hit is an uncommon presentation in the pediatric population. given its rarity, there is often a delay in diagnosis which increases risk of complications such as bleeding, stroke, and limb ischemia. even if the diagnosis is suspected or proven, there may be challenges in initiating alternative agents as limited data exists on pediatric options. as argatroban remains the treatment of choice for patients with hit, experience in pediatric patients is limited, and dosing recommendations have been extrapolated from adult studies. anecdotal data exists for use of bivalirudin in children, although studies, primarily, focus on use in specific cardiac cases. in our patient's case, choice was further complicated by renal failure. this case study highlights the need for further research regarding the identification of a secondary anti-coagulant agent for use in pediatric patients with hit. background: subcutaneous panniculitis-like t-cell lymphoma (sptl) is a rare form of non-hodgkin's lymphoma characterized by infiltration of cytotoxic t-cells into subcutaneous tissue. sptl occurs in both adults and children and can present in both patient populations as either alpha/beta or gamma/delta subtypes. patients with the gamma-delta phenotype have an overall poorer survival, although the exact etiology is unclear. interestingly, both subtypes of sptl can present with secondary hemophagocytic lymphohistiocytosis (hlh), and this is associated with a worse prognosis. currently, there are no standardized treatment protocols for sptl, and clinical management includes watchful waiting, corticosteroids/immunosuppression, chemotherapy, and stem cell transplant. the primary objective was to compare how two patients with the same diagnosis responded acutely to therapy. we performed a retrospective chart review of two pediatric patients at our institution who were diagnosed with alpha/beta sptl and secondary hlh. we examined each presentation, treatment course, and outcome. we then completed a brief review of the current literature describing treatment of and outcomes for sptl with secondary hlh. results: these two patients presented in a similar manner with signs and symptoms of hlh. each was then subse-quently diagnosed with alpha/beta sptl after biopsy of cutaneous nodules and each had diffuse disease, as measured by pet. however, they demonstrated vastly different acute responses to therapy. one patient was pre-treated with systemic glucocorticoids before receiving definitive chemotherapy and tolerated therapy well as an outpatient. the other patient started systemic chemotherapy without steroid pretreatment and developed severe cytokine storm characterized by hypotension, cardiac dysfunction, multi-organ failure and cytokine elevation. both patients achieved complete remission (cr) after treatment with chop chemotherapy and remain disease-free - months off therapy. in patients presenting with sptl and secondary hlh, we propose that initial treatment with antiinflammatory or anti-cytokine therapy can decrease, or even prevent, the possibility of life threatening cytokine release as a result of cytotoxic chemotherapy. background: congenital dyserythropoietic anemia type ii (cda ii) is a rare autosomal recessive disorder, rarely presenting in the neonatal period. iron overload often occurs as a late sequela of ineffective erythropoiesis and intramedullary hemolysis. objectives: to report the novel use of iron chelation in an infant with cda ii associated with severe iron overload. the patient is a -month-old, former -week infant with prenatal non-immune hydrops and transfusion-dependent fetal anemia who presented with persistent anemia, reticulocytopenia, hyperbilirubinemia, liver dysfunction, and hyperferritinemia. his initial ferritin was . ng/ml, tibc ug/dl, and transferrin mg/dl. his bone marrow biopsy showed trilineage hematopoiesis and erythroid dyspoiesis characterized by binucleation of late-stage precursors. genetic testing revealed a compound heterozygous missense mutation and splice site mutation in the sec b gene, confirming the diagnosis of cda ii. initial liver biopsy revealed mild portal fibrous expansion, and abundant hepatic iron deposition. his ferritin continued to increase, peaking at , ng/ml, along with liver enzymes peaking at an alanine aminotransferase (alt) of u/l and aspartate aminotransferase (ast) of u/l. ferriscan showed an elevated estimated liver concentration of . mg/g dry tissue. repeat liver biopsy months later showed giant cell hepatitis with worsening mild portal fibrosis and hemosiderosis. additionally, tissue liver iron concentration was mcg/g dry weight. cardiac t * mri revealed mild cardiac iron deposition. given his significant degree of iron overload, deferoxamine was used to reduce hemosiderosis and liver morbidity in preparation for bone marrow transplantation. the patient received deferoxamine mg/kg/day iv x days/week for three months, without any clinically significant adverse events. blood counts and hepatic and renal function were monitored weekly without any abnormalities. growth parameters and liver enzymes significantly improved while receiving chelation therapy. as a noninvasive, cost-effective method, serum ferritin levels were monitored monthly to gauge response to treatment. despite receiving blood transfusions every - weeks, serum ferritin decreased to ng/ml and liver enzymes decreased to alt u/l and ast u/l prior to bone marrow transplantation. we report the use of deferoxamine in a patient with cda ii less than years of age, for treatment of iron overload. our patient tolerated deferoxamine well without significant adverse events or organ toxicity. deferoxamine may be a well-tolerated method of reducing iron burden in young patients with iron-loading pathologies. background: low grade gliomas with kiaa- -braf fusions typically have a favorable prognosis with infrequent rates of high grade transformation, low rates of metastasis and even lower rates of extra cns metastasis. while highgrade transformation has been reported for tumors with braf v e mutations and cdkn a deletions, it has not been pre-viously reported in gliomas with kiaa- -braf fusions. while there are case reports of high-grade cns malignancies metastasizing through a ventriculo-peritoneal (vp) shunt, low-grade gliomas metastasizing in this manner are extremely rare. objectives: to describe a unique case of peritoneal tumor dissemination of a braf fusion positive high grade neuroepithelial tumor in a child with a vp shunt placed for multifocal braf fusion positive low grade astrocytomas results: an eight-year-old male was initially diagnosed with multifocal low-grade astrocytomas of the hypothalamus and c -c spinal cord. initial testing revealed the kiaa- -braf fusion, but no cdkn a or braf v e mutation. initial surgical management included a vp shunt and resection of the cervical spinal lesion. he received vincristine and carboplatin, followed by transition to vinblastine given new thoracic metastatic lesions after months of therapy. at months after diagnosis, scans were concerning for diffuse leptomeningeal progressive disease and new intracranial lesions, necessitating craniospinal radiation. following a near cr, he presented months later with acute onset of abdominal pain. a ct scan revealed peri-renal and perirectal soft tissue masses, confirmed by exploratory laparotomy to be peritoneal tumor dissemination of high grade neuroepithelial tumor. a kiaa -braf fusion was noted and confirmed by rt-pcr, identical to that seen in the original cns tumors. additional findings included deletion of chromosome p (without q loss) and heterozygous and homozygous deletion of cdkn a found by fish. brisk mitotic activity justified a high-grade designation. salvage chemotherapy consisted of cycles of ice with subsequent resolution of pet-avid disease and only minimal peri-nephric tissue remaining. given the favorable response, surgical resection and multiple tissue biopsies were performed which documented no residual active disease. the shunt was revised and he started trametinib for maintenance. we present a unique case of peritoneal dissemination of high grade neuroepitheial tumors with the same kiaa- -braf fusion as multifocal low grade astrocytomas in a child with a vp shunt. this raises suspicion for tumor metastasis and transformation to a higher grade malignancy versus two distinct diseases, which may be indicative of an underlying cancer predisposition. texas children's hospital, houston, texas, united states background: polycythemia is a common referral to hematology. it is important to evaluate for a high oxygen affinity hemoglobinopathy, ensuring appropriate testing is performed for early diagnosis and avoidance of additional tests and procedures. a year old mexican female presented with an elevated hemoglobin and hematocrit, symptoms of plethora of her hands and feet, chest pain, palpitations, and fatigue. further confounding the picture, she also had significant menorrhagia and iron deficiency. she was diagnosed with the rare high oxygen affinity hemoglobin new mexico variant, only previously described once in the literature in a year old black boy. objectives: the patient initially presented at age with a hemoglobin of . g/dl and a hematocrit of . %. initial work up consisted of a hemoglobin electrophoresis which diagnosed sickle cell trait, a co-oximetry panel which was normal, and erythropoietin level of mu/ml, also normal. she was then lost to follow up and re-referred at age . she is a competitive basketball athlete, and at that time, she presented with a hemoglobin of . g/dl, and hematocrit of %. erythropoietin level continued to be normal at mu/ml. design/method: cardiology was consulted regarding chest pain and palpitations with a normal evaluation. chest x-ray was also normal. a bone marrow aspirate and biopsy was performed with results significant for mild erythroid hyperplasia and mild reticulin fibrosis. jak mutation, von hippel lindau, bpgm, and hereditary erythrocytosis mutations including phd , hif a, and epor mutation analysis were sent, all of which were normal. testing to mayo clinic for p rbc oxygen dissociation returned low at mmhg ( - mmhg normal range) and subsequently a hemoglobin electrophoresis identified a hemoglobin variant leading to beta globin gene sequencing. results: patient found to be heterozygous for hemoglobin new mexico, with . % hb new mexico and . % hba, and . % hba . there was no evidence of hbs. when evaluating patients with polycythemia, maintaining a high index of suspicion for high affinity hemoglobinopathies may eliminate further unnecessary and invasive testing for patients. caution should be used when using hemoglobin electrophoresis testing since hb new mexico is known to migrate similarly to hbs on hplc with minimal change that may not be detected in regular laboratories. most high affinity hemoglobinopathies are reported to not have significant symptoms. in this case, our patient complains of fatigue, occasional palpitations and plethora of hands and feet. we will need to further follow this patient for possible attributable symptomatology. divya keerthy, simone chang, warren alperstein, patricia delgado, claudia rojas, ofelia alvarez, matteo trucco university of miami jackson memorial hospital, miami, florida, united states background: improved technology is enabling detection of previously unidentified translocations and mutations in otherwise unclassified sarcomas. one such mutation is the bcl- co-repressor -internal tandem duplication (bcor-itd) allowing for the new classification of bcor positive undifferentiated round cell sarcomas (urcs). this sarcoma has a similar appearance to clear cell sarcoma of the kidney (ccsk), potentially representing an extra-renal manifestation of this tumor, but their clinical pathologic features are not identical. objectives: this case highlights how recombinant polymerase chain reaction (rt-pcr) and bcor immunohistochemical staining can ease the diagnosis of this rare sarcoma. results: a month-old female presented for right sided pre-septal cellulitis and a temporal subcutaneous mass. the detection of multiple other subcutaneous nodules on exam raised the concern for malignancy and she was admitted for evaluation. she had two subcutaneous masses on her abdomen, with more cutaneous masses on her legs, back, shoulder, cheek and submandibular areas. she lacked spontaneous lower limb movement and had bilateral clonus. imaging confirmed multiple masses throughout the body including paravertebral area from t to l , bilateral adrenal glands, left kidney and muscles of upper and lower extremities. initial differential included neuroblastoma, infantile myofibromatosis, rhabdomyosarcoma or atypical presentation of a renal tumor. however, synaptophysin and chromogranin stains were negative. with standard immunohistochemistry, the tumor could be only broadly classified as "undifferentiated sarcoma" maintaining the diagnostic challenge. using rt-pcr in the setting of a morphologically primitive round cell neoplasm with strong bcor expression, two external institutes simultaneously diagnosed the tumor as bcor-urcs. the primary lesion is unknown but potentially may have arose from the kidney. bcor-urcs has a heterogeneous histology with tumor cells appearing monomorphic in nests of - cells separated by septa with uniform nuclei. there is frequently an "orphan annie eye" appearance and sparse cytoplasm to the cells. diagnosis cannot be made solely on evaluation of this nonspecific histology. rt-pcr uses the genetic abnormality in undifferentiated sarcomas to narrow the differential and bcor immunohistochemical staining provides further context. bcor has significant diagnostic value given its sensitivity and specificity in urcs. another potential marker includes ywhae-nutm b fusions, which occur in smaller subset of cases, but requires further study. rt-pcr has helped further classify tumors leading to the diagnosis of a rare undifferentiated sarcoma with bcor overexpression. while this technology is beneficial, its availability is limited. if accessibility improves, earlier identification and treatment may be possible maximizing the chance for a positive outcome. background: hematohidrosis is a rare condition that mimics bleeding disorders. cases present with oozing blood tinged fluid from various sites like eyes, ears, nose, skin, etc. reported causes of this condition were stress or fear, physical activity, psychological disorders. the condition is self-limited and don't affect the general condition of the patients, but it may contributes to psychosocial problems and may increases their stress and anxiety. so this condition needs to be promptly treated. to test the response of this disease and the associated headache to propranolol treatment. design/method: our case female patient years old st offspring of non consanguineous marriage, was admitted with recurrent episodes of oozing blood tinged fluid from eyes, ears and nose months before admission, about . - ml from each orifice, lasted - minutes and subsided spontaneously. it could involve the sites simultaneously or - sites. the number of attacks was - times per day then gradually increased to - times per day. later on the patient developed a bleeding attack from umbilicus. these attacks were aggravated by stress and physical activity and decreased with rest and sleep. the condition was associated with severe headache involving the whole head, throbbing in nature of gradual onset, increased by physical activity and relieved by analgesics. the condition was not associated with vomiting, blurring or diminution of vision, ocular pain, eye discoloration. no earache, tinnitus or diminution of hearing. there was no other form of discharge from eyes, ears or nose. no history of ecchymotic patches, bleeding from other orifices or blood product transfusion. no history of trauma, drug intake, fever or rash. no symptoms of other system affection. past history of recurrent attacks of epistaxis and two operations were done that passed without remarkable bleeding. no similar condition in the family physical examination was free, no evidence of psychological problems. complete blood count, coagulation profile, platelets function, factor and c.t brain were normal. oozing fluid from the patient was analyzed showed the same components as blood. results: our case started oral propranolol . mg/kg/day based on its use in similar cases in literature. the frequency of attacks and headache reduced then stopped after months of treatment and didn't recur after stoppage of propranolol. propranolol can treat this condition successfully. further investigations are needed to determine the link between this condition and severe headache our case was suffering from. background: wilms tumor is the most common renal solid tumors of childhood and is derived from primitive metanephric cells located in the kidney. primary extra-renal wilms tumors (erwt) are extremely rare, estimated to comprise . - % of all wilms tumors. despite similar histologic appearance intrarenal and erwts differ in embryologic tissues of origin. erwts arise from the more primitive mesonephric or pronephric origin and, therefore, can develop anywhere along the craniocaudal migration pathway of these primitive tissues, most often retroperitoneal, inguinal/genital, lumbosacral/pelvic and mediastinal. these tumors are typically staged and treated per national wilms tumor study (nwts) guidelines, and, by definition, are stage ii or greater due to location beyond the kidney borders. based on the cases reported in the literature, outcomes for erwt are comparable to renal wilms tumors with an % local recurrence rate and an % two-year event-free survival. we report the first case of a stage iii testicular extrarenal wilms tumor in an -month-old male with an intrabdominal undescended testis who underwent complete surgical excision followed by chemotherapy and inguinal radiation. results: a full term -month old male underwent orchipexy for an undescended left testicle. the testicle was noted to be grossly abnormal with a pea-sized thickened tissue adherent to the upper pole and a separate mass outside of the scrotum on the superior epididymis. both masses were removed, and s of s pathology demonstrated wilms tumor with favorable histology and negative margins. ct imaging of the chest, abdomen and pelvis were negative for a primary renal tumor, local residual disease, pathologic lymph node enlargement or distant metastases. the tumor was classified per nwts as stage iii due to tumor removal in multiple pieces. the patient completed dd- a treatment with vincristine, doxorubicin and dactinomycin per aren with cgy left inguinal radiation. he is currently months off therapy without clinical or radiographic evidence of recurrent disease. primary erwt is an extremely rare malignant neoplasm associated with challenges in diagnosis, staging and treatment. based on the cases reported in the literature, outcomes are similar to that of intrarenal wilms tumor. there are four pediatric paratesticular wilms tumors reported in the literature and, to the best of our knowledge, this is the first case of stage iii testicular wilms tumor successfully treated with dd- a chemotherapy and radiation. in erwt, nwts guidelines for staging and treatment should be applied with evaluation of both kidneys to exclude an intrarenal primary tumor. background: patient is a yo f, with esrd secondary to atypical hus versus ttp, who presented with thrombotic microangiopathy, aki, thrombocytopenia and anemia after a living unrelated donor kidney transplant. patient initially had downtrending creatinine. on post-op day , hematology was consulted for an increasing ldh and drop in platelets. peripheral smear was notable for an absence of schistocytes. yet, biopsy of the kidney revealed microthrombi. the patient was diagnosed with a thrombotic microangiopathy. plasmapharesis was initiated on day # , at which time ms r was noted to have significantly elevated creatinine. plasmapharesis did not yield any correction in labs and significant bruising developed. patient was started on eculizimab; plasmapharesis was stopped. shortly after, creatinine, anemia and thrombocytopenia corrected to levels at which she was discharged. overall, patient was found to have progressive anemia, thrombocytopenia, an increasing creatinine and ldh ( s) concerning for atypical hus, despite absence of schistocytes on peripheral smear. she responded well to eculizimab, with correction of hematologic changes during induction. she was discharged on eculizimab and continued to respond with normalizing platelet counts and hemoglobin. the differential in light of patient's thrombotic microangiopathy and thrombocytope-nia also included ttp. yet, adamts remained normal. dic was unlikely given normal fibrinogen level and d-dimer. objectives: presentations of atypical hus vs ttp. discuss eculizumab as a treatment of atypical hus. highlight atypical presentations of illness in transplant patients. results: despite absence of schistocytes by smear, pt was diagnosed with atypical hus based on presentation and after failing plasmapharesis, she responded well to eculizumab. though her presentation was abnormal, her response to this antibody that blocks the complement cascade suggests that she was experiencing a complement-mediated process. there are rare documented cases in the literature of atypical hus without schistocytes. hemolytic uremic syndrome (hus) is characterized by hemolytic anemia, thrombocytopenia and acute kidney injury. atypical hus is a diagnosis of exclusion, not due common etiologies such as shiga toxin. among atypical causes are complement-mediated forms, caused by an antibody to complement factor. in addition to plasmapharesis, renal transplant and supportive care, the mainstay of treatment for atypical hus is eculizumab (an antibody that blocks the complement terminal cascade). this case describes a patient unique in that, she was diagnosed with atypical hus without any schistocytes by smear. secondly, she responded to eculizumab, with unremarkable gene studies. finally, this case highlights that transplant patients often have unique presentations. nicklaus children's hospital, miami, florida, united states background: synovial sarcoma is a spindle cell tumor categorized as a soft tissue sarcoma. the chromosomal translocation t(x; ) leading to the ss -ssx fusion protein is unique to this sarcoma. it is a slow growing tumor with common recurrences and often, at presentation, with evidence of metastatic disease. if resection is not feasible, then neoadjuvant with adjuvant chemotherapy is recommended. metastasis carries an unfavorable prognosis given synovial sarcoma historically does not respond well to chemotherapy. trabectedin is a well-tolerated alkylating agent currently indicated for the treatment of liposarcoma and leiomyosarcoma. we present a -year-old male with metastatic synovial sarcoma to the lungs that progressed and was refractory to chemotherapy. he was administered trabectedin as a form of palliative chemotherapy, with significant clinical and radiographic response. design/method: pubmed search was done with search for terminology including "synovial sarcoma" and "trabectedin". papers relevant to our case were selected for literature review. a -year-old male patient presented with a large right axillary mass. initial imaging showed a heterogeneous multiseptated mass invading the subscapularis and teres major muscles along with innumerable lung nodules. biopsy confirmed diagnosis of monophasic synovial sarcoma. the patient was started on protocol arst with ifosfomide, mesna, doxorubicin. he completed cycles followed by radical resection and sessions of radiation. due to progression of disease multiple chemotherapy regimens were tried including topotecan and cyclophosphamide, protocol advl with lorvotuzumab, and pazopanib. imaging of the chest continued to show significant progression of metastasis. the patient's clinical status deteriorated with worsening respiratory status, requiring l of oxygen therapy, and inability to ambulate. he was started on trabectedin . mg/m for palliative care. after cycles of treatment patient was no longer requiring oxygen and was ambulating without assistance. radiological imaging showed significant reduction in number and size of lung nodules. trabectedin is a recently approved alkylating agent for the management of sarcomas resistant to first line treatment. response in synovial sarcoma is scarcely documented in the pediatric population. epidemiology places the most common age group in the young adults and children. our case opens the doors to further consideration of the use of trabectedin in the pediatric patient with metastatic synovial sarcoma. background: gata is an x-linked gene that plays critical role in hematopoiesis. mutations of gata gene can be associated to various blood disorders including diamond blackfan anemia, cytopenia, congenital dyserythropoietc anemia and acute megakaryoblastic leukemia. we report a patient with macrocytic anemia and platelet dysfunction who carries a novel gata mutation that has not been reported. results: a now -month-old male with complex medical history including prematurity at weeks, dysmorphic features, global developmental delay, hyperinsulinism, hypogonadotropic hypogonadism, growth hormone deficiency, micropenis, failure to thrive, patent ductus arteriosus status post ligation, and severe hypotonia, was referred to hematology at months old for resolved, transient thrombocytopenia and macrocytic anemia since month of age. chromosomal microarray showed chromosome deletion of q . , which is the rps gene. he doesn't have a family history of diamond blackfan anemia (dba), despite mom having the same rps mutation. he was then diagnosed with dba. his lab workup showed mild macrocytic anemia (hgb . g/dl, mcv fl), normal to inappropriately low reticulocyte count, normal white blood cell and platelet counts, hgf %, erythroid ada . eu/gm hgb (elevated). he has abnormal pfa- , with prolonged closure time of both adp and epinephrine. he had low von willebrand antigen and ristocetin cofactor activity. he has severe pancreatic insufficiency. bone marrow biopsy showed normocellular marrow with trilineage hematopoietic maturation, without ringed sideroblasts. since mother has the same rps gene mutation, maternal labs were done and showed no evidence of macroytosis or anemia. the diagnosis of dba was questioned. whole exome sequencing did not identify any pathogenic sequence changes in the coding regions of rps gene, but detected a gata mutation r w, which was reported variant of uncertain significance. his mother shares the same mutation and is asymptomatic, but she may not be affected since gata iis xlinked. his father doesn't harbor the gata mutation. conclusion: gata gene encodes zinc finger dna binding hematopoietic transcription factor, which is important during erythroid differentiation. gata mutation r w has not been reported in literature and is a novel variant of gata mutation, which might be contributing to this patient's clinical picture. further studies are warranted to confirm gata mutation r w to be a pathogenic sequence change. alexander boucher, tomoyuki mizuno, alexander vinks, greg tiao, stuart goldstein, james geller cincinnati children's hospital medical center, cincinnati, ohio, united states background: hepatoblastoma (hb), the most common pediatric primary hepatic malignancy, can be associated with specific congenital syndromes. recently, chronic kidney disease and genitourinary anomalies have been linked to hb. cisplatin is a key chemotherapeutic agent in treating hb but its renal clearance and toxicity profile can limit its use for those with end-stage renal disease (esrd). objectives: using an institutional case series, we present data using cisplatin for hb in dialysis-dependent esrd and define recommended dosing for future use. design/method: a chart review of patients with concurrent hb and esrd on dialysis treated with cisplatin at our institution was undertaken. demographic data, diagnostic history, tumor pathology, alpha fetoprotein (afp), hearing assessments, dosing schema, treatment outcomes, and therapyrelated toxicities were reviewed. total cisplatin levels were collected at time points within days after each infusion. free cisplatin levels were also collected for infusions, as were dialysate cisplatin levels. pk parameters were generated using bayesian estimation with a published population pk model as a priori information. results: three patients meeting these criteria were identified. each had "low risk" (non-metastatic resectable) disease at presentation and underwent upfront resections. all had congenital renal anomalies with esrd prior to their hb diagnosis. all cisplatin infusions were given over hours, followed hours later by hemodialysis. patients and received cisplatin at % of children's oncology group's ahep weight-based dosing ( . mg/kg). patient received % of ahep body surface area-based dosing ( mg/m ) during cycle but required a second dose reduction ( mg/m ) for cycle due to prolonged cisplatin exposure (total area under the curve mg⋅h/l; average for all seven evaluable cycles mg⋅h/l) and early sensorineural hearing loss at - hz. no other hearing loss in any patient was identified; mild toxicities also included grade - emesis and grade neutropenia and thrombocytopenia. the median (range) of clearance, volume of distribution at steady-state, and elimination half-life at terminal phase for total platinum were . ( . - . ) l/hour/ kg, . ( . - . ) l/ kg and ( - ) hours, respectively. patients and received cycles with rapid afp normalization. patient required an additional cycles, for a likely second primary hb year after initial therapy. cisplatin can be used successfully in pediatric patients with esrd on hemodialysis to treat hb with minimal morbidity using % standard mg/kg-based dosing ( . mg/kg), achieving pharmacologically appropriate cisplatin exposures. background: treatment for immune thrombocytopenia (itp) has been grouped into rescue and maintenance therapy and often is reserved for patients with bleeding, severe thrombocytopenia, or for improvement in quality of life. splenectomy is considered one of the more invasive but definitive treatments with success rates of - %. treatment of itp can be more difficult in the setting of previous treatment with immune modulation or when the patient is immunocompromised and not a candidate for splenectomy. objectives: present an interesting case of a patient with an autoimmune disease that presented with severe thrombocytopenia, un-responsive to rescue therapy, and requiring emergent splenectomy in the setting of acute intracranial hemorrhage (ich). a year old female with a history of juvenile dermatomyositis presented with a fine purpuric rash on her extremities, wet purpura, and a platelet count of k/ l. bone marrow evaluation at that time was consistent with itp. she was on cyclosporine and plaquenil for dermatomyositis. platelets failed to increase after three doses of intravenous immunoglobulin and high dose steroids. following a two week course of oral prednisone and eltrombopag, she presented with persistent severe thrombocytopenia of k/ l, anemia of . g/dl, and a lower gi bleed. she was started on amicar, novo-seven, rituximab, and given platelet transfusions with no improvement in bleeding. subsequently, she developed a subdural hematoma with midline shift. surgery performed an emergent open splenectomy with concurrent continuous platelet transfusion. results: she was monitored closely post operatively and, due to ich, transfused to maintain platelets greater than k/ l. by week post-op she had normal platelet counts off transfusions. all medications were stopped within three days of discharge. she represented eight days later with abdominal pain and thrombocytosis and was found to have a portal vein, splenic vein and mesenteric vein thrombosis. she was started on lovenox therapy and admitted for monitoring due to her history of ich. it is unknown whether our patient's underlying immune dysregulation and history of treatment with immunosuppressive medications may have contributed to her unresponsiveness to multiple therapeutic agents. in addition, her significant bleeding did not allow us to fully evaluate her response to second tier therapy. this adds to the scarcity of literature of itp response in pediatric patients with autoimmune disease, and may support more aggressive therapy upfront in these patients. background: multivisceral organ transplantation involves concurrent transplantation of the stomach, pancreas, liver, and intestine with splenectomy, and has been classically used in the pediatric population for infants with intestinal failure from disorders affecting foregut integrity. while there is some data demonstrating its efficacy in adults with low-grade abdominal malignancies, it has not been traditionally used for hepatocellular carcinoma treatment. to describe a unique pediatric case of multivisceral organ transplantation as definitive therapy for refractory fibrolamellar hepatocellular carcinoma in an adolescent male. a year old male presents with a history of fibrolamellar hepatocellular carcinoma, tumor invasion of the portal vein, severe portal hypertension complicated by bleeding esophageal varices and hypersplenism. he had two treatments with yttrium- radioembolization, without significant response. he completed six cycles of traditional chemotherapy in combination with sorafenib with resolution of petavidity, but minimal decrease in tumor size and continued portal hypertension. since his disease remained relatively stable for over years, he was evaluated and listed for multivisceral organ transplantation. at approximately years and months after diagnosis, he underwent en bloc liver, pancreas, stomach, small bowel, and colon transplant with splenectomy. a single lymph node was positive for malignancy at the time of resection. in addition to expected post-transplant complications, he also developed skin only acute graft versus host disease at weeks after transplant, treated successfully with a thymoglobulin course. he clinically improved and was back to his baseline activity level, on full oral feedings within months post-transplantation. at three and six month post-transplantation, there is no concern for relapsed hepatocellular carcinoma on comprehensive imaging and evaluation. he is maintained on protocol immunosuppression and posttransplant support. we present the first known case of successful multivisceral organ transplantation in the treatment of refractory pediatric fibrolamellar hepatocellular carcinoma. background: hematohidrosis is a rare disorder that presents with spontaneous excretion of whole blood from intact skin or mucosa. diagnosis is based on clinical observation of the occurrence with the proven presence of erythrocytes and other blood components, without other abnormalities to account for the phenomenon. the existing literature is scarce and consists of primarily case studies. most reports describe bleeding from facial sites around the eyes, ears, and nose. the available literature suggests anxiety and physical or emotional stress reactions as the most common inciting events. little evidence exists regarding the ideal therapeutic approach, however propranolol has been used successfully to reduce bleeding frequency and severity in multiple case reports. a specific genetic etiology has not been elucidated, and no familial cases have previously been reported. we present a pair of half-siblings, both of whom presented with spontaneous cutaneous and mucosal bleeding before two years of age, and report on preliminary results of propranolol therapy. tanzania. at months of age, he became ill and developed spontaneous bleeding from his ears, nose, and scalp. he continued to have frequent bleeding episodes, usually related to illness or physical distress. a bleeding diathesis work-up was unremarkable, however some episodes were severe enough to require transfusions. the patient was subsequently diagnosed with hiv and hepatitis b, presumably acquired via unscreened blood product transfusions. patient b is an infant female born to the same mother as patient a, with a different father. she was healthy until two months of age when she developed spontaneous bleeding from the hairline, eyelids, ears and genital/rectal area. bleeding episodes were nearly always associated with irritability and crying. extensive coagulation workup was unremarkable. results: propranolol therapy was started in both patients, titrated to a goal of mg/kg/day. in both patients, the frequency and duration of bleeding episodes significantly improved. patient b continues to have milder occasional bleeding episodes from her eyes, ears and scalp but has significantly less discomfort and irritability during the episodes. conclusion: to our knowledge, there are no prior reports involving two related patients with hematohidrosis. this case series suggests that there may be a genetic predisposition which has yet to be identified. propranolol has shown effectiveness in reducing symptom frequency and severity. background: gliomas are the most common central nervous system tumors in children. they are classified into different grades based on genotype (idh, braf, tsc, etc.). lowgrade gliomas such as oligodendrogliomas, astrocytomas, and mixed oligoastrocytomas are classified as grades i and ii. of the molecular level alterations this case report focuses on the braf v e mutation. braf is a member of the raf family of serine/threonine protein kinases and it plays an important role in cell survival, proliferation and terminal differentiation. objectives: here we discuss two cases where dabrafenib, a braf kinase inhibitor, was utilized in the management of gliomas. the cases focus on the use of dabrafenib late versus early in disease course. design/method: patient jl is a year old female who was diagnosed with a low-grade glioneuronal tumor (c -t with a metastatic lesion to the brain) in . jl was treated with chemotherapy, radiation, and surgical resection. despite treatment, the patient's disease progressed. she developed lower extremity dysfunction, urinary incontinence, poor truncal control, and hydrocephalus. dabrafenib was started after the braf v e mutation was confirmed. patient lg is a year old female who presented in november with left facial and upper extremity weakness. ct and mri scans demonstrated a mixed solid and cystic lesion extending from the optic chiasm and hypothalamus to the right thalamus and posterior basal ganglia with additional involvement of the right cerebral peduncle. neurosurgical intervention was undertaken and dabrafenib was started after the braf v e mutation was confirmed. results: patient jl's mri scans have demonstrated improvement of the spine with diminished areas of enhancement along thecal margins, decreased volume and enhancement within the trigeminal plate cistern and resolution of ependymal enhancement within the right ventricle. the patient's most recent mri exhibits no disease progression in head or spine. jl has shown improvement clinically since starting dabrafenib. patient lg has shown improvement in strength and recent mri of the brain has shown resolution of enhancement along surgical resection margins, decreased hyperintensity along the inferomedial aspect of the right basal ganglia and no new enhancements. conclusion: low grade gliomas can alter a person's quality of life and even lead to life threatening complications. often the standard chemotherapy, radiation and surgery don't prevent these complications. genetic analysis can help clinicians target therapy towards certain mutations such as braf v e. dabrafenib has shown to decrease tumor burden, early utilization as therapy can help prevent morbidity and mortality. children's hospital of pittsburgh of upmc, pittsburgh, pennsylvania, united states background: copper is an essential cofactor in enzymatic reactions essential to proper hematologic, skeletal, neurologic and vascular function. copper requirements in children over the age of are mg/day, which is readily acquired in a typical diet. copper deficiency is known to occur in patients with the rare x-linked mutation and in older individuals with gastrointestinal bypass surgery; however, it is rarely reported in other conditions. objectives: to highlight individuals with autism spectrum disorders or developmental delay with a limited dietary repertoire are at risk for copper deficiency, thus a high index of suspicion must exist in order to diagnose the disorder. design/method: a y/o boy with a prior diagnosis of global developmental delay and oral aversion presented with slowly progressive fatigue, weakness, gait instability, and weight loss. his longstanding feeding difficulties were refractory to intensive feeding programs. his daily diet consisted of - oz of milk and - individual servings of butterscotch pudding ( - calories/day, . mg iron/day). initial complete blood count demonstrated white blood cell count of . , absolute neutrophil count of , hemoglobin of . , mean corpuscular volume of < , reticulocyte count of . , platelet count of . review of his peripheral blood smear revealed microcytic, hypochromic red cells without marked fragmentation, anisopoikilocytosis and ringed sideroblasts; there were no morphologic abnormalities of his leukocytes or platelets. iron studies demonstrated ferritin of , total iron binding capacity of , and % iron saturation. he had no evidence of b , folate deficiency or blood loss. additional evaluation revealed a serum copper level of (range - ), and cerulosplasmin of . (range - ). results: once a diagnosis of copper deficiency was made, the patient promptly began a course of parenteral copper repletion. he received iv copper mcg/kg/day x days then weekly intravenous infusions. given his malnutrition, a gtube was placed to begin oral copper repletion and enteral nutrition. within weeks his copper level improved as well as his blood counts. unfortunately, although his blood counts and copper levels normalized, his neurologic status remains below his old baseline although, he has made gains in his gross and fine motor abilities. conclusion: acquired copper deficiency in the pediatric population is a rare event but given the hematologic and neurologic consequences, prompt recognition and treatment is important. this patient's clinical course demonstrates the need to have a high index of suspicion of concomitant nutritional deficiencies other than those routinely evaluated such as iron, b and folate. background: lymphoepithelioma-like thymic carcinoma (lelc) is a rare, aggressive neoplasm with a high rate of invasion, metastasis and recurrence. there are no known curative therapies for metastatic lelc. we report the case of a -year-old male who presented with metastatic ebv positive lelc. sites of disease included a large primary anterior mediastinal mass and metastases to hilar lymph nodes, lungs and liver. he was initially treated with cisplatin and fluorouracil followed by mediastinal radiation. he had a partial response to therapy but his end of therapy scans showed disease progression in lungs, liver, and hilar, supraclavicular and axillary lymph nodes. objectives: molecularly targeted therapies tailored to the patient's genetic profile offer a novel approach to obtain improved survival outcomes. design/method: the patient enrolled on a precision medicine trial, nmtrc : molecular-guided therapy for the treatment of patients with relapsed and refractory childhood cancer (nct ). in this study, tumor/normal whole exome sequencing and tumor rna sequencing were performed and a molecular report detailing the results of genomic and gene expression analysis was generated. a treatment plan was designed within a molecular tumor board comprising oncologists, pharmacists, genomicists, and molecular biologists with domain expertise. results: exome sequencing revealed somatic coding point mutations and no structural mutations (focal copy number changes or translocations). candidate somatic driver mutations included tp s x and r w as well as kit n k. both genes have been previously implicated in thymic carcinoma. rna expression analysis demonstrated aberrant activation of biological pathways, including overexpression of kit, hdac , and , tyms, and dhfr. the molecular tumor board selected the combination of pemetrexed ( mg/m ) on day of a day cycle, imatinib ( mg daily), and vorinostat ( mg days - , - , and - ) . on day of cycle , he was admitted with a herpes zoster infection and imatinib was discontinued in order to reduce risk of herpes zoster recurrence. imaging after cycles showed a complete metabolic response on f- fdg pet and a partial response by ct size criteria. as of december , the patient had received cycles of pemetrexed and vorinostat. scans in december showed an increase in the size and metabolic activity of two right lower lobe pulmonary nodules. there were no new sites of disease and imatinib was re-started. background: systemic lupus erythematosus (sle) is a chronic autoimmune disease that affects multiple organ systems and is associated with many different autoantibodies. patients can present with vague constitutional symptoms including fever, rash, fatigue, and weight loss. some of the various hematologic manifestations of sle include anemia of chronic disease, leukopenia, autoimmune hemolytic anemia (aiha), and idiopathic thrombocytopenic purpura (itp). these can be the presenting signs of sle. evans syndrome (es), a disease characterized by itp and aiha, is a rare hematologic manifestation of sle. neurofibromatosis (nf ) is a relatively common neurocutaneous disorder. these patients are at risk of developing benign and malignant tumors. its association with autoimmune disorders, including sle, remains rare. objectives: there are few cases in the literature that have patients with the combination of sle and nf . this is the only case that has a patient with sle, nf , and es. results: a -year-old caucasian female presents with two months of vaginal bleeding, weight loss and petechiae. her exam is remarkable for petechiae and café au lait macules. laboratory findings show severe anemia and thrombocytopenia. she receives blood and platelet transfusions during stabilization, and a bone marrow aspirate is performed to rule out a malignancy which is negative. based on the presence of thrombocytopenia and a positive coombs test, an autoimmune process such as es is considered. screening tests for sle reveal positive antinuclear and anti-double stranded dna antibodies as well as low complement. she receives intravenous immunoglobulin and methylprednisolone and eventually her vaginal bleeding slows and her counts recover. she begins sle therapy with hydroxychloroquine and azathioprine. due to the presence of café au lait macules on her exam, a genetics evaluation is performed and the patient is also diagnosed with nf . to date, there are seven cases of sle with nf reported in the literature, only two of which are pediatric cases. there are no reports of the combination of sle, nf , and es. conclusion: es is a rare hematologic manifestation of sle but can be the initial presentation of this disease. one large study estimates % of childhood-onset sle cases are observed to have es. screening for sle should be considered in all es patients even in the absence of typical clinical findings. association of nf and sle has been rarely described. whether this association reflects a causal relationship or is coincidental needs more investigation. (lube, ped blood & cancer, ) . university of california san diego, la jolla, california, united states background: high grade glioma (hgg) has poor outcomes in adults and children. extraneural metastases are very rare in hgg, and poorly characterized with only a few small case series in adults and only isolated case reports in pediatrics. no genomic data has previously been published for any children with hgg who develop extraneural metastases. objectives: our objective is to describe the natural history of two children with hgg and bony extraneural metastases, comparing their clinical characteristics as well as whole exome sequencing data for both tumors. this information would suggest similar patients should be monitored closely for extraneural metastasis and may benefit from more systemic therapy. design/method: we present a case series of two patients who presented with hgg and had development of bony metastases less than six months after initial diagnosis. both patients had molecular profiling with whole exome sequencing (wes). the first patient was an -year-old male with a tumor found in the left lateral ventricle invading into the fornices, hypothalamus, and left midbrain, who had subtotal resection. bony metastasis were found at . months after diagnosis, and he died months after diagnosis. he initially received radiation, followed by nivolimuab. the second was a -year-old female with a tectal/pineal tumor and multiple spinal cord metastases, who had subtotal resection. she developed bony metastasis at . months after diagnosis and died months after diagnosis. her histologic diagnosis was pineoblastoma, revised to hgg after whole exome sequencing. she received craniospinal radiation followed by chemotherapy per acns (cisplatin, vincristine, and cyclophosphamide) for cycles. when she failed to respond satisfactorily to this therapy, wes of tumor was performed and the findings were consistent with hgg. treatment was transitioned to temodar and lomustine after hgg diagnosis was given. she had ongoing progressive disease despite this therapy as well as trials of nivolumab, everolimus, and vorinostat. neither patient had extraneural metastasis at presentation. in both tumors, whole exome sequencing identified the h f a k m mutation. both tumors also had additional known mutations associated with hgg but no other overlapping mutations. this case series represents the first description of the genetic alterations of pediatric hgg patients who developed extraneural metastases. while h f a k m is a common mutation in pediatric midline hgg, especially dipg, and is associated with more aggressive disease, there has not been an association with extraneural metastasis prior to this series. background: deferiprone-induced agranulocytosis is a well -known albeit rare side effect of the drug. incidence of agranulocytosis varies from . - . %, while milder neutropenia is reported in . % of patients treated with deferiprone. deferasirox is unknown to cause such a complication. clinical trials and post marketing side effect monitoring studied possible correlations between different risk factors and development of agranulocytosis. unfortunately, no studies directly addressed a special risk in a community with background of ethnic neutropenia, like oman. objectives: to report on the incidence of neutropenia among omani children with b thalassemia using different iron chelators design/method: a retrospective study conducted on patients < year-old with b thalassemia treated with different iron chelators. electronic patients records were reviewed to detect episodes of neutropenia either mild (anc . -< . /cmm), moderate (anc . -< ), severe < . , or agranulocytosis anc = ). data were collected including sex, age, personal or family history of ethnic neutropenia, iron chelating agent, infective complications, management and outcome. detailed clinical, laboratory ± radiological information were reported for patients who developed life-threatening agranulocytosis. among young patients with b thalassemia, treated between - in squh, neutropenia, was reported in patients ( . %).severe neutropenia was encountered on occasions in patients ( / : . %) ( on deferiprone including episodes of agranulocytosis, on defersirox, on combined chelation, and off chelation). moderate neutropenia was encountered in patients ( / : . %), on occasions: deferiprone ( ), deferasirox ( ), combined chelation ( ), and episodes off chelation. mild neutropenia was more prevalent, encountered in patients ( . %) on occasions ( on deferiprone, on defersirox, on combined chelation, and off chelation) of patients exposed to deferiprone, patients had neutropenia ( %), higher than previously reported. deferiproneinduced agranulocytosis was encountered in patients ( / = . %). three of them had life threatening complications. one patient developed pneumonia complicated by rupture of pulmonary artery aneurysm-massive hemoptysis, who recovered fully after catheter embolization. the second had facial cellulitis and treatment with gcsf was complicated by frequent ventricular extrasystoles. the third had sepsis, disseminated herpes simplex and required admission to icu for inotropic support. in a community with background ethnic neutropenia, neutropenia is more common to be encountered among thalassemic patients, both on and off chelation therapy. careful monitoring of anc and rational choice/modification of chelating agents is required for optimal management of iron overload and to avoid life threatening complications. objectives: this case control study aimed to evaluate the systolic and diastolic cardiac function in groups of children with ti: non transfused group and a group that received early regular blood transfusion comparing them to healthy controls. design/method: thirteen regularly transfused patients with ti with a mean age of . + . years were compared with eight patients who are non-transfused or minimally transfused (< rbcs transfusion/year); mean age . + . years and healthy controls with a mean age of . ± . years. clinical parameters and standard echocardiographic and tissue doppler imaging (tdi) were compared. results: young non-transfused ti patients had a statistically significant higher peak late diastolic velocity of the left ventricular inflow doppler, a mitral valve a wave duration over the pulmonary vein a wave duration ratio and the pulmonary s of s vein s/d velocities ratio compared to the transfused group with p values of . , . , . respectively. in addition, they have a lower e/a ratio of the mitral valve inflow and a larger left atrial to aortic diameter ratio compared to the control group with p values of . and . respectively. the diameters of the right and left outflow tract were significantly larger in the non transfused group with a trend to have a higher cardiac index compare to the transfused group. systolic function was similar in the studied groups and none of the patients had evidence of pulmonary hypertension. young patients with ti who are receiving early regular blood transfusion have normal systolic function. diastolic function assessment revealed indicators of an abnormal relaxation of the left ventricle in the non transfused group which indicate diastolic dysfunction. the abnormalities affected multiple diastolic function parameters which give an indication that the changes are clinically significant. a statistically significant increase in the diameters of the outflow tracts are likely attributed to high cardiac output status in nontransfused ti patients as they had a trend to have a higher cardiac index. these findings support the early commencing of regular blood transfusion therapy for ti patients to prevent serious cardiac complications in adult life. background: in the -week sustain study, crizanlizumab . mg/kg significantly reduced the frequency of scpcs versus placebo ( . vs . , p = . ) and increased the time to first on-treatment scpc ( . vs . months, p = . ) in patients with sickle cell disease (scd). to evaluate time to first scpc in sustain study subgroups and the likelihood of not experiencing scpc for the duration of the trial using post hoc analyses. design/method: sustain was a randomized, double-blind, placebo-controlled, phase study (nct ). inclusion criteria were: scd patients aged - years; - scpcs in previous months; concomitant hydroxyurea use permitted if ≥ months and stable dose for ≥ months. patients were randomized : : to receive intravenous crizanlizumab . mg/kg, . mg/kg, or placebo. study treatments were administered on days and , then every weeks to week , with the final assessment at week . median time to first scpc after first dose was summarized for crizanlizumab . mg/kg or placebo in these subgroups: - or - scpcs in previous months; scd genotype; and hydroxyurea use at baseline. hazard ratios (hrs) for crizanlizumab . mg/kg versus placebo were calculated based on cox regression analysis, with treatment as a covariate. descriptive statistics were used to summarize the frequency of patients who were scpc event-free for the duration of the study by prior scpc events, scd genotype, and hydroxyurea use at baseline. : patients received crizanlizumab . mg/kg and received placebo. there was a meaningful delay in time to first scpc with crizanlizumab . mg/kg versus placebo observed in the entire study population. the effect was present in both scpc subgroups, and the largest treatment difference was observed in hbss scd versus other genotypes ( . vs . months; hr: . ). in patients taking hydroxyurea who experienced - scpcs in the previous year, time to first onstudy scpc was longer with crizanlizumab . mg/kg versus placebo ( . vs . months; hr: . ). a greater proportion of patients treated with crizanlizumab . mg/kg were scpc event-free versus placebo in each of the analyzed subgroups. one third of patients who were taking hydroxyurea and treated with crizanlizumab . mg/kg were scpc event-free during the study versus . % with placebo, possibly suggesting an additive effect. with crizanlizumab . mg/kg, there was a clinically meaningful delay in time to first scpc and an increased likelihood of being scpc-free versus placebo in all subgroups investigated. cincinnati children's hospital medical center, cincinnati, ohio, united states background: shwachman-diamond syndrome (sds) is an inherited marrow failure syndrome associated with increased risk of myelodysplasia (mds) and acute myeloid leukemia (aml). objectives: this multi-institutional retrospective study investigated clinical features, treatment, and outcomes of sds patients who developed mds or aml by central pathology review. design/method: nine individuals presented with aml ( male, female), mds-eb / ( males, females, with mds ( male and female), and one male with isolated persistent somatic tp mutation. one mds-eb and mds patient progressed to aml. median age (years) at diagnosis of mds was (range . - ), mds-eb / was (range . - ) and aml was . (range . - ). complex cytogenetics were noted in / aml cases, with one having normal cytogenetics. complex clonal cytogenetic abnormalities were noted in of mds-eb /eb patients and clonal abnormalities in of mds patients. follow up was available for aml patients; are deceased. received chemotherapy with intent to proceed to hematopoietic stem cell transplant (hsct). four failed to achieve remission and died with disease without proceeding to transplant. one patient proceeded to hsct without prior chemotherapy. four of six transplanted subjects died with relapsed disease. treatment related mortality was largely infectious or gvhd. the sole surviving aml patient had normal cytogenetics, achieved remission with chemotherapy and underwent hscts with separate stem cell infusions due to two primary graft failures. he remains alive in remission more than years after diagnosis. of the mds-eb / patients, underwent ric hsct, three of whom are alive, one died of infection. the fifth patient has stable disease on continued decitabine monotherapy for . years. of mds patients with treatment data, had upfront hsct therapy, upfront chemotherapy and had no therapy. three patients required ≥ hscts all due to graft failure. follow up is available for , of whom are deceased, with relapsed disease. treatment related mortality was largely infectious or graft failure. one individual died of hepatic failure unrelated to mds. seven mds patients are alive in remission. in summary, prognosis is poor for patients with sds who develop aml due to resistant disease and treatment-related complications. better markers for risk stratification are needed to identify patients who would benefit from early transplant. novel therapeutic strategies are urgently needed to improve outcomes of sds patients with mds or aml. background: unlike primary myelofibrosis (pmf) in adults, which is associated with somatic mutations in jak , mpl, or calr, myelofibrosis in children is rare and the underlying genetic mechanisms remain elusive. here we describe families with autosomal recessive congenital macrothrombocytopenia with focal myelofibrosis (cmtfm) due to germline mutations in the megakaryocyte-specific immune receptor tyrosine-based inhibitory motif (itim) receptor g b-b. objectives: to characterize the clinical phenotype, histological features and identify the causative gene for cmtfm. we performed affymetrix snp . genotyping on the index family to identify shared regions of homozygosity by descent. whole exome sequencing (ws) was performed on all three pedigrees to identify potentially causative mutations. we studied affected children from families, with macrothrombocytopenia, anemia, mild leukocytosis and a distinctive pattern of bone marrow (bm) fibrosis centered around clusters of atypical megakaryocytes. affected children had mild to moderate bleeding symptoms and required platelet and red cell transfusions. none showed evidence of extramedullary hematopoiesis, and all were negative for mutations in jak , mpl, and calr. snp genotyping identified multiple statistically non-significant genomic loci, including the region of the major histocompatibility locus (mhc) on chromosome p (lod = . ). we focused on this region because affected individuals in two families shared a common homozygous human leukocyte antigen (hla) type and had congenital adrenal hyperplasia (cah) due to -hydroxylase (cyp a ) mutation; the cyp a and hla loci are located at p . and p . - p . . wes revealed homozygous frameshift mutations in the megakaryocyte and platelet inhibitory receptor g b-b, encoded within the candidate linkage region. we identified two distinct g b-b frameshift mutations (c. _ + dup; p. fs and c. inst; p. fs) in individuals within these three families. no other mutations that segregated with the phenotype were identified. to validate g b-b as a potential disease-causing gene, we evaluated g b-b expression in bm biopsy specimens from affected patient and control samples by immunohistochemical staining using a monoclonal antibody. g b-b was strongly s of s and selectively expressed in megakaryocytes of control samples, but completely absent in clinically affected individuals. a murine knockout that lacks g b-b has a strikingly similar phenotype with macrothrombocytopenia, myelofibrosis and aberrant platelet production and function, further affirming the causality of g b-b mutations. we showed that autosomal recessive loss-offunction mutations in g b-b cause cmtfm, uncovering the molecular basis of this rare disease. loss of g b-b-dependent inhibition of megakaryocyte activation likely underlies the distinctive focal myelofibrotic phenotype and might be important in other forms of marrow fibrosis. cardinal glennon, saint louis, missouri, united states background: intrauterine transfusion is the method of choice for management of fetal anemia due to red blood cell alloimmunization. despite the decrease in prevalence of anemia due to rhesus d alloimmunization with prophylactic administration of anti-rhd immunoglobulin in rh d negative patients, maternal red red blood cell alloimmunization with other type of red blood cell antigens remains an important cause of fetal anemia. newborn who received intrauterine transfusion for hemolytic disease may have prolonged postnatal transfusion requirement. objectives: -to evaluate clinical outcome of fetuses and newborns who received intrauterine transfusions. -to determine the need of packed red blood cell transfusions until months of age. we conducted a retrospective case series study of all intrauterine transfusions due to anemia secondary to red blood cell alloimmunization performed in our regional center ssm in st louis missouri, between april and january . we evaluated the indications, diagnosis, gestational age, and frequency of intrauterine transfusions, along with the infant's gestational age at birth, duration of admission, timing of blood transfusion and monitoring of hemoglobin. results: intrauterine transfusions were performed in patients. the most common causes of alloimmunization were due to d antibodies (n = , %) and kell antibodies (n = , . %). the median gestational age of the first intrauterine transfusion was . weeks, and the median pre-transfusion hemoglobin was . g/dl. the gestational age at the first intrauterine transfusions was found to be significantly correlated with the number of postnatal transfusions (r = . . p = . ). the median gestational age at birth was found to be weeks ( . - . weeks), with a hemoglobin of . ( . - . ). in our population, patients ( %) received postnatal transfusions, of which were during the first weeks of life, and close monitoring follow up with a hematologist was established in patients at their discharge from the nursery/nicu. one neonatal death occurred and severe morbidity due to severe anemia occurred in one infant. despite the continuing risk factor for persistent anemia, only patients had follow up hemoglobin monitored by their primary care provider. conclusion: infants with anemia due to red blood cell alloimmunization treated with intrauterine transfusion should be monitored closely via regular complete blood count for persistent anemia due to suppression of fetal erythropoiesis. sebastian hesse, piotr grabowski, juri rappsilber, christoph klein dr. von hauner childen's hospital, lmu university hospital, munich, munich, germany background: neutrophil granulocytes are the most abundant leukocytes in the peripheral blood. validated diagnostic options for these cells are limited, leaving many patients with functional neutrophil defects without a defined diagnosis. objectives: here we evaluate proteomics as a new diagnostic tool to investigate defects of neutrophil granulocytes. we analyzed neutrophil granulocytes from children with severe congenital neutropenia (scn) associated with elane mutations, children with chronic granulomatous disease (cgd) with cyba ( ) or cybb ( ) mutations and children with leukocyte adhesion deficiency (lad) due to itgb mutations. in addition we collected samples of children with genetically undetermined neutrophil defects. neutrophils from healthy individuals served as controls. cells were isolated from fresh venous blood using negative selection (purity > %). whole cell proteome analysis was done by data-independent acquisition. showed a correlation coefficient of ∼ . . principal component analysis demonstrated unequivocal separation of the proteome of healthy and diseased cells. differential expression analysis showed minimal proteome aberrations in lad with deficiency in cell surface receptors and upregulation of alpl (total downregulated proteins: / total upregulated proteins: ). analysis of neutrophils from cgd patients also showed limited proteome aberration. cyba and cybb were both diminished independent of genotype, whereas protein clusters around a stat / centered network were increased (total down: / up: ). neutrophils with elane mutations showed the gravest proteome disturbance (total down: / up: ) with an upregulated translational apparatus (srpdependent ribosomes and protein folding complexes) and increased mitochondrial proteins. proteins of each granule subset were dysregulated and metabolic pathways upregulated. a detailed analysis of the proteome from patients with genetically undefined diseases is currently ongoing. one patient with clinical phenotype of cgd was found to have no mutations of nadph oxidase members in whole exome sequencing but critically low levels of ncf on protein level. heterozygosity mapping showed autozytocity in the ncf region warranting current efforts to sequence promoters and intronic regions of the gene. mass spectrometry based proteomics promises exciting new insights into monogenic disease of neutrophil granulocytes and may offer new diagnostic options, in particular in synergy with genome sequencing. by virtue of our international care-for-rare alliance, open to new partners, we hope that our proteome focus may lead to better delineation of as yet unknown disease of neutrophil granulocytes. background: warm autoimmune hemolytic anemia (aiha) is an igg mediated disease. although it can be post-viral, it is often idiopathic and can also be a forme fruste for malignancy or an autoimmune disease. initial management includes steroids. it often relapses on steroid wean and can be refractory to the use of second line treatment such as rituximab. objectives: abatacept (ctla- -ig fusion protein, ctla- mimetic) has been used to ameliorate autoimmune manifestation associated with ctla- haploinsufficiency. we used abatacept as a novel therapeutic agent to manage patients with refractory aiha. design/method: a retrospective case series of two patients at phoenix children's hospital with severe refractory aiha. results: patient , a previously healthy year old female, presented with weeks of icterus, fatigue, and hemoglobinuria. spleen was enlarged cm below the costal margin. laboratory evaluation demonstrated: hemoglobin . g/dl, mild leukopenia /microliter, platelets , /microliter, reticulocytosis . %, positive direct coombs' test, mycoplasma igm and igg positive. bone marrow evaluation showed a hypercellular marrow. she continued to need packed red blood cell (prbc) transfusions despite receiving high dose steroids, ivig and rituximab from may-july . in august, she started sirolimus decreasing her transfusion requirement. after starting abatacept ( mg/kg/dose bi-monthly for three doses and then monthly) in october, she maintained hemoglobin of - g/dl without transfusion. patient , a previously healthy month old male, presented with one week of progressive fatigue, jaundice, and poor feeding. splenomegaly was absent. laboratory evaluation revealed hemoglobin . g/dl, leukocytosis , /microliter, platelets , /microliter, reticulocytosis . %, negative direct coombs' test, and non-specific reactivity on antibody screen. evaluation for inherited hemolytic anemia including a next generation sequencing panel was negative. further evaluation by blood bank showed + positive coombs' for c d due to a warm antibody. cold agglutinin disease was ruled out. bone marrow evaluation was normal. he received high dose ivig as a steroid sparing agent but continued to require prbc transfusions weekly. when prednisone did not seem to slow down hemolysis, treatment with abatacept was initiated and he has not required transfusions for two months. steroids are being weaned. we present successful treatment of two refractory aiha cases with abatacept. patient is steroid and transfusion free and continues on monthly abatacept and sirolimus. patient is also transfusion free and continues on a steroid taper. ctla- is crucial for suppressive function of treg cells. abatacept by binding to cd / seems to enhance treg activity ameliorating autoimmune hemolysis. children's minnesota, minneapolis, minnesota, united states s of s background: transfusional iron overload is common in patients receiving chronic red cell transfusions. as a result, iron chelation is required to minimize toxicity from iron overload. chelation with a single agent can be inadequate at controlling or reducing iron burden. when combination therapy is required deferoxamine may be added to oral chelation. deferoxamine is generally given subcutaneous over - hours for - days a week at - mg/kg/day. many patients struggle to remain compliant with this schedule which has prompted trials of intravenous high-dose (hd) deferoxamine. prior reports of short-term hd deferoxamine have shown minimal side effects however, prolonged use of hd deferoxamine has known toxicity. when compliance is a concern, our center has used hd deferoxamine infusions at mg/kg/hr x hours every to weeks. objectives: evaluate the safety and efficacy of hd deferoxamine at our institution to help guide future therapy. design/method: a retrospective review was completed of patients previously treated with hd deferoxamine between april and september at children's minnesota. final sample included patients ages to years with underlying diagnosis of thalassemia ( ) and diamond-blackfan anemia ( ). deferoxamine infusions were given for hours every - days with a mean length of treatment of days. results: all patients were on combination therapy with deferasirox, however deferasirox was held during deferoxamine infusion. mean pre-deferoxamine liver iron concentration (lic) was . mg/g and mean post lic was . mg/g (p = . ). ferritin mean pre-deferoxamine was ng/ml compared with mean post ng/ml (p = . ). two patients had possible allergy, leading to deferoxamine discontinuation. one patient developed hives, eye swelling and cough while the other had emesis and cough. another patient experienced facial nerve palsy of unclear etiology, which did not recur with resumption of deferoxamine. no respiratory complications were seen. results showed significant decrease in iron burden following combination therapy with high dose deferoxamine and deferasirox. no significant pulmonary, liver, renal, vision, or hearing toxicities were observed. three patients reported reactions to deferoxamine infusions. however, one of these was able to successfully continue deferoxamine without further incident. short-term, hd deferoxamine was effective at reducing lic in combination with oral chelation but requires further evaluation to assess for potential increased risk of toxicity. short-term hd deferoxamine may be considered in the setting of poor compliance of subcutaneous administration or inadequate chelation with single agent therapy. further studies are needed to clarify ideal dosing, timing and risk of toxicity. background: immune thrombocytopenia (itp) is the most common cause of symptomatic thrombocytopenia in childhood but remains a diagnosis of exclusion warranting further evaluation if atypical findings are present. two male children ( months and years old) with newly diagnosed immune thrombocytopenia (itp) were found on initial evaluation to have persistent elevations of lactate dehydrogenase (ldh), alanine aminotransferase (alt), and aspartate aminotransferase (ast). these serum enzyme abnormalities cannot be attributed to itp. in the setting of thrombocytopenia, elevated transaminases and ldh create diagnostic complexity for the hematology/oncology provider as their elevation raises concern for malignancy, hemolytic disease, and other systemic diseases. to raise awareness about an unexpected pattern of duchenne muscular dystrophy in patients undergoing evaluation for itp. to expand the differential of a hematologist/oncologist when abnormal labs support a nonhematologic diagnosis design/method: this case-series of two patients with their clinical and laboratory findings were discovered with retrospective chart review. results: after a thorough evaluation for hemolytic anemias, liver disease and infectious etiologies was negative, bone marrow and liver biopsies were considered. eventually, both children were found to have severely elevated serum creatine kinase (ck). skeletal muscle has the highest concentration of ck of any tissue. thus, significant ck elevation is almost exclusively attributable to muscle injury and is the most sensitive and specific enzyme for diagnosis of muscle disease. referral to a neuromuscular specialist and further genetic testing confirmed the diagnosis of duchenne muscular dystrophy in both children allowing initiation of appropriate interventions. to date, there is no clear genetic predisposition to itp in patients with muscular dystrophy although further investigation may be needed. hematology/oncology providers should consider obtaining a serum ck to rule out muscle disease in any male child with unexplained elevations of serum ldh and/or aminotransferases, as it provides an easy and inexpensive, non-invasive approach to screening. additionally, clinical history and physical examination can aid in the diagnosis of muscular dystrophy, with gross motor delay, abnormal muscle bulk, gower's sign, and proximal muscle weakness all possible findings. objectives: to identify the range of cbcs in patients with ds without infections, hematologic or immune disorders and to create more accurate reference ranges for total white blood count; hemoglobin; hematocrit; mcv; platelet count and absolute neutrophils (anc), lymphocytes, monocytes, eosinophils, and basophils. design/method: a retrospective investigation of healthy pediatric patients with ds who received a cbc between and as part of their medical care at a single, large, pediatric teaching hospital. the study group consisted of children with ds (male = , . %; mean age = . years, sd = . ) at time of blood draw. initially children were reviewed for possible participation in the study; however, patients were excluded due to not meeting the study's inclusion criteria. descriptive statistics were performed on demographic and clinical characteristics. kruskal-wallis h tests, anova, and t-tests were run to determine the significant associations between independent means. results: a significant difference in absolute neutrophils between racial groups, f( , . ) = . , p = . , was observed. there was an increase in anc from . +/- . with african americans to . +/- . in the other racial groups and to . +/- . with caucasians. differences were also found in anc in hispanics/latinos versus non-hispanic/latinos. the results were higher in non-hispanics and latinos, a significant difference of -. ( % ci, -. to -. ), t( ) = . , p = . . preliminary kruskal-wallis h tests run determined that there were significant differences between age groups for total white blood cell, hemoglobin, hematocrit, platelets, lymphocytes and anc. further studies are being run to evaluate in which age groups these differences lie and create reference ranges by age, race and sex. conclusion: among patients with ds, there are differences between racial groups and age groups. this data has been compared to previously established reference ranges for cbcs, but we are currently establishing healthy cbc controls which we will use to validate the reference ranges. these ranges will be published to help guide providers in workup and management of patients with ds. background: transfusion is a critical part of the care provided in the neonatal intensive care unit, but it is not without risks. low birth weight and premature infants can become anaemic from an immature haematopoietic system and frequent phlebotomy. these infants often receive multiple red blood cell transfusions. identifying infants more likely to require such intervention is important in ensuring the appropriate usage of this scarce resource. to determine whether birth weight, gestational age, gender, length of stay and mode of delivery can predict red cell concentrate (rcc) transfusion, units required, donor exposure and time to exposure. design/method: a retrospective chart review of all infants born below weeks gestation and/or birth weight less than , g who received a red blood cell transfusion between july and july in the cork university maternity hospital neonatal unit. results: infants met the inclusion criteria, ( . %) received a rcc transfusion. our study showed lower gestational age (p< . ) and lower birth weight (p< . ) infants are more likely to be transfused. donor exposure increases with a lower birth weight (p = . ). multivariate analysis showed infants with a lower gestational age (or - . per day; p< . ); lower birth weight (or - . per g; p< . ) and a longer length of stay (or . per day; p< . ) are more likely to receive a higher number of rcc transfusions. the time to first rcc transfusion is shorter in those with lower birth weight (or . per g; p< . ) and lower gestational age (or . per day; p< . ). gender and mode of delivery were not found to be predictors of red blood cell transfusion in this study. conclusion: low birth weight and premature infants are more likely to receive a rcc transfusion during admission to the neonatal unit. our study highlights predictors of rcc transfusion, donor exposure and time to transfusion. these can be used in identifying at risk infants, counselling parents and in anticipating transfusion requirements. emily southard, r. grant rowe, david williams, akiko shimamura, taizo nakano children's hospital colorado, aurora, colorado, united states background: the mecom locus encodes transcription factors that regulate hematopoietic stem cell self-renewal and maintenance. overexpression of mecom has been noted in - % of acute myeloid leukemia, several solid tumors, and denotes a poor prognosis. mutations that reduce mecom expression or that disrupt protein function, however, have been implicated in the development of bone marrow failure (bmf) through undefined pathways. an association between mecom mutations and radioulnar synostosis with amegakaryocytic thrombocytopenia (rusat) syndrome has been reported, however further characterization of this phenotype has yet to be explored. to characterize the phenotypic spectrum of a cohort of pediatric patients with novel mecom mutations. we performed a retrospective review of five patients with mecom mutations who were referred to hematology at children's hospital colorado or boston children's hospital. clinical, laboratory, and genetic data was collected on subjects and available family members. results: four of subjects were identified in infancy presenting with congenital cytopenias or physical dysmorphisms that prompted broad genetic screening. platforms for genetic detection included microarray, targeted genetic panels, and whole exome sequencing. three of subjects with cytopenias presented with congenital thrombocytopenia, of whom rapidly progressed to severe aplastic anemia. four of subjects presented with congenital anomalies, of whom demonstrated radioulnar synostosis. additional dysmorphic features identified include craniofacial (low set ears x ), cardiac (pda x , vsd x , aortic root dilation x ), pulmonary (pulmonary hypertension x , arteriovenous malformations x ), and developmental delay. one subject presented at age years with acute pancytopenia, hypocellular marrow, no dysmorphisms, and a mecom variant of unknown signif-icance. the identified mecom mutations include one . mb deletion involving several genes including mecom, one variant affecting a splice acceptor consensus sequence predicted to disrupt splicing, and three novel missense mutations, tyr cys, arg thr, and tyr cys, all of which were absent from public databases and were predicted in silico to be deleterious. we describe the phenotypic spectrum of patients with novel mecom variants. a subset of patients lacked radio-ulnar synostosis and had presence of additional systemic anomalies, demonstrating a varied clinical phenotype that is not isolated to rusat syndrome. a centralized publically accessible database to share clinically annotated mecom variants, together with analysis by experts in mecom function would advance our understanding of the clinical interpretation of mecom variants. mecom should be considered in the differential diagnosis of bone marrow failure and we advocate for the inclusion of mecom in targeted sequencing panels. cairo university, cairo, egypt background: beta thalassemia is regarded as a serious public health problem in the mediterranean region, southeast asia, and the middle east. however, very few studies have been conducted to assess the quality of life (qol) among thalassemia major patients. objectives: to assess the quality of life among b-thalassemia major patients using short form (sf)- questionnaire and to determine the factors associated with their quality of life. design/method: a cross-sectional study was conducted among thalassemia major patients who were attending the hematology outpatient clinic at cairo university hospital, during the study period. data were collected between october and march . the quality of life was assessed for patients aged ≥ years. the mean age of the studied group was . ± . years. the majority ( . %) had one monthly blood transfusion. the mean total score of sf- was . ± . . general health perception domain was the most affected domain with mean score, while vitality was the least affected one. there was no statistically significant difference between males and females regarding different quality domains except for vitality where the mean score was significantly higher in males than females (p = . ). age at onset of disease, and at first blood transfusion were the most documented factors positively correlated with the quality of life among the enrolled thalassemia patients. conclusion: the quality of life in thalassemia major patient was found to be compromised. all thalassemia patients should undergo assessment of the quality of life so that interventions focusing on the affected domains can be implemented. background: international adoption of children with special needs has become more prevalent in recent years leading to tremendous growth in the number of u.s. thalassemia patients adopted from foreign countries. currently % of the , thalassemia patients registered in the cooley's anemia foundation (caf) patient database have been adopted from foreign countries, primarily china. as this population continues to grow, further information is needed in order to provide these families with best supportive care. the primary goal of this study is to characterize the socio-demographics and health statuses of adopted children with thalassemia and their families. a secondary goal is to describe adoptive families' motivations, experiences, challenges, and support resources. design/method: a redcap survey was accessed by families of adopted children with thalassemia through the caf website and caf social media from january to august . following a four-question screen, eligible subjects were directed to complete an adoption questionnaire. families who had at least one adopted child with thalassemia receiving care at a participating thalassemia treatment center or hematology office in the u.s. were considered eligible. descriptive statistics were analyzed using sas . . respondents who were ineligible or who provided incomplete data were removed from the dataset prior to analysis. of survey respondents, qualified and completed the survey. these households had adopted a total of children with thalassemia ( . % male), most from china ( . %), where they had been living in orphanages ( . %). legal guardians identified primarily as christian ( . %). the majority had completed post-secondary education ( . %) with reported household incomes greater than $ , ( . %). most adoptive families were connected to an adoption group or community including online groups, local support groups, and adoption networks ( . %). commonly cited challenges were: ) volume of frequent medical appointments, ) insufficient support from their local care centers, and ) financial burdens. the reality of care for the population of adopted patients with thalassemia in the u.s does not seem to match the expectations set by their providers. we are hopeful this data will be used to assist adoptive families navigating the complexities of thalassemia care. the findings suggest that this population would benefit from additional outreach, education, guidance, and advocacy resources -especially in the early stages of adoption and during initiation of post-adoption medical care. background: in many higher-income countries, thalassemia major has become a chronic disorder; many outcomes are different in emerging countries with more limited resources. most analyzes of health-related quality of life (qofl) in thalassemia have been conducted in high-income settings. objectives: to assess the impact of health status on qofl in thalassemia patients in an emerging country. we assessed qofl in randomly-selected patients ( thalassemia major; with hemoglobin e thalassemia; five thalassemia intermedia) at the national thalassemia center in kurunegala, sri lanka where approximately patients are managed. treatment is free, but compared to north america/europe, access to tertiary staff and other resources are limited. overall, control of body iron as estimated by serum ferritin concentration (mean± sem, ± g/l) was not optimal in many patients. to understand the impact of health status on qofl, we used the sf v health survey, analyzing scores of physical function, pain, general health, social functioning, emotional and mental health, to generate overall physical and mental component scores. results: compared to reports from higher-income countries (american journal of hematology ; : - ), physical function scores (mean±sd, . ± . ) were similar in sri lankan patients; indeed, in three categories (physical role, social function, emotional role), sri lankan scores were slightly higher. by contrast, compared to scores from higherincome settings, those estimating bodily pain, general health, and mental health were significantly lower, resulting overall in a significantly lower physical component score in sri lankan patients. male sri lankan patients reported higher scores than females, and somewhat surprisingly, in four categories (physical function, physical role, social function and emotional role) reported higher scores than those obtained in higher-income settings. lower scores in physical functioning, leading to an overall lower physical component score, were recorded by females. patients with hemoglobin e thalassemia reported generally poorer qofl than those with thalassemia major. the lack of differences in qofl in patients with "high" and "low" hemoglobins was likely related to low pre-transfusion hbs (mean±sem, . ± . g/dl) in nearly all patients. these early data in a small cohort of thalassemia patients in an emerging setting suggest that in many patients bodily pain, reduced mental health, and poorer views of general health affect overall qofl. prospective studies in larger cohorts including evaluation of adequacy of transfusions and chelation therapy, complications, and overall accessibility of care may guide approaches to improve qofl in lower-income settings of thalassemia care. geetanjali bora, anand prakash dubey, tarun sekhri, mammen puliyel, aparna roy maulana azad medical college, new delhi, delhi, india background: in the last two decades, the presence of osteopenia has been described in optimally treated patients with transfusion dependent thalassemia, the pathogenesis of which seems to differ from osteopenia in non-transfused patients. the prevalence rate of low bone mineral density (bmd) in pediatric population is highly variable amongst studies done worldwide. furthermore, the role of metabolic and endocrine factors in determining bone mass in this population is not well understood. objectives: to assess bmd in subjects with transfusion dependent beta thalassemia by dual-energy-x rayabsorptiometry and find its co-relation with clinical, biochemical and hematological parameters. design/method: this is a comparative cross-sectional study and includes patients with transfusion dependent beta thalassemia between ages to years enrolled from a thalassemia day care center in the year - . at the time of enrollment age, sex, bmi z scores, pubertal staging, duration and type of chelation therapy were noted. enrolled subjects were scanned for bmd at lumbar spine l - and left femoral neck using dexa scan. the bmd was expressed in mean values and z scores. age, bmi, ethnicity and gender matched historic controls were used to generate z scores. ml of pre transfusion fasting venous blood samples were obtained to test for serum calcium, phosphate, alkaline phosphatase, pth, thyroid function panel, serum ferritin and serum igf- levels. mean values for pretransfusion hemoglobin and serum ferritin over last months were calculated. results: total no of subjects , median age . years, male ( %), female ( %), ethnicity % asian, bmi < rd centile ( %), pre pubertal %, all receiving transfusion and chelation therapy. prevalence of low (z score < - sd) and very low (< - . sd) bmd was %, % at l -l respectively and %, % at left femoral neck respectively. there was trend of lower bmd z scores with advancing age. statistically significant co-relation (p value < . ) was found between low bmd and low mean pretransfusion hemoglobin, serum phosphate, igf - and vitamin d levels conclusion: a sizable proportion of children and adolescents with transfusion dependent thalassemia have suboptimal bone mineral density and this decline may start as early as - years of age despite being on transfusion regimen highlighting the importance of yearly dexa screening and optimization of pre-transfusion hemoglobin, vitamin d and igf levels. vanderbilt university medical center, nashville, tennessee, united states background: it is well described that iron deficiency anemia (ida) can co-present with thrombocytosis or thrombocytopenia, though cases of thrombocytopenia are less frequent than thrombocytosis. prior reports of thrombocytopenia have included adult and pediatric patients with menorrhagia ( - ), menorrhagia due to uterine fibroids ( ), or other gynecologic abnormalities ( ). our cases highlight the pattern of ida, thrombocytopenia, and menorrhagia in the setting of significant menstrual clotting without observed gynecologic abnormalities in african-american adolescents. objectives: to describe the clinical course of three adolescent females with severe ida, menorrhagia, and thrombocytopenia. results: our cases included three female african-american patients ages - who presented with severe anemia and concurrent thrombocytopenia in the setting of menorrhagia. all three patients reported heavy and prolonged menstrual cycle bleeding with significant clots. two of the three were admitted for transfusions at presentation and noted to have significant menstrual bleeding with continued blood loss requiring additional transfusions until bleeding was controlled with estrogen therapy. these two patients were evaluated with pelvic ultrasounds revealing a prominent endometrium in both patients and hyperechoic material consistent with a clot in one patient. average hemoglobin on presentation was . gm/dl ( . - . ), average platelet count was , /mcl ( , - , ), and average mcv was ( - ). all had severe iron deficiency with an average ferritin of ng/ml ( - ) subsequently treated with oral iron. one patient had a prior history of ida that required transfusion and had subsequent normalization of her complete blood count. two patients had subsequent thrombocytosis before normalization of their platelet counts. two patients received platelet transfusions: one due to recent neurosurgical intervention with a higher goal platelet count and the other to help control menstrual bleeding after a nadir platelet count of , . a review of the clinical history and red cell indices pointed to ida and ongoing blood loss from menorrhagia as the reason for the bicytopenias. the thrombocytopenia in these cases may have been exacerbated by consumption of platelets in the significant clots all three patients reported. it is reasonable to treat with iron supplementation and supportive care which may include transfusions or management of menorrhagia with oral contraceptives or other hormonal methods. background: sickle cell disease is one of the most common inherited red blood cell disorders, yet many are not aware of their carrier status. the american college of obstetricians and gynecologists' guidelines recommend that pregnant women of african, mediterranean and southeast asian descent be screened for hemoglobinopathies with a cbc and hemoglobin electrophoresis . however, adherence to this practice and frequency of improper screening with sickledex is unknown. proper screening and counseling can impact families' knowledge, allowing for establishing relationships with pediatric hematology providers earlier. objectives: we sought to assess prenatal hemoglobinopathy screening practice patterns and methods of obstetrics & gynecology (obgyn) and family medicine providers in the nyc regional area. design/method: a cross-sectional electronic survey was administered to obgyn and family medicine practitioners from four nyc institutions. questions focused on prenatal hemoglobinopathy screening practices using case scenarios with variations on parental trait status and ethnicities. chisquare analyses were used to compare the two provider groups on categorical variables. there were total responses; surveys were complete, of which were obgyn and family medicine providers. respondents were mainly from academic medical centers, with the majority being faculty ( % of the obgyns and % of family medicine). no significant difference was found in frequencies of screening patients with a positive family history of a hemoglobinopathy. when asked about screening practices for patients without a personal/family history of a hemoglobinopathy, % of obgyns versus % of family medicine providers "always" screened for hemoglobinpathies (p = . ). when analyzed by ethnic background, there were significant differences by group in screening patients of white ( % vs %), black ( % vs %), mediterranean ( % vs %), and asian descent ( % vs %) (p≤ . for all). however, in cases where the hemoglobinopathy carrier status of both parents was known, there was no difference in screening with a hemoglobin electrophoresis. furthermore, > % of all respondents use sickledex for screening in the case scenarios. conclusion: this pilot survey highlights a difference in the methods and likelihood of prenatal hemoglobinopathy screening based on the type of prenatal care provider. screening differences can lead to variations in prenatal guidance, diagnostic procedures, informed decision-making and knowledge of families referred to pediatric hematology clinics. this is the first study analyzing prenatal screening for hemoglobinopathies in obgyn and family medicine. improving prenatal screening practices by collaborating with hematologists may decrease health care disparities and allow for earlier relationship building with pediatric hematology. . acog, opinion# , poster # hermansky-pudlak syndrome: spectrum in oman background: hermansky-pudlak syndrome (hps) is a rare autosomal recessive disorder, characterized by the triad of oculocutaneous albinism, a hemorrhagic diathesis resulting from storage pool-deficient platelets, and accumulation of ceroid/lipofuscin-like material in various tissues. before , nine different types of hermansky-pudlak syndrome were identified, which can be distinguished by their signs and symptoms and underlying genetic cause. in , a tenth type was defined based on mutations in the ap d gene. hps type is characterized in addition by severe neutropenia and recurrent sinopulmonary infection. the disease is more common in puerto rico, and this is the first report from oman. to describe the clinical, laboratory and genetic characteristics of hps sub-types in oman, including the first cases of hps type . design/method: this is a retrospective study, including cases with hps that had been suspected clinically and confirmed through genetic mutation analysis. clinical data included sex, age at presentation, initial clinical presentation (skin, eyes, development, neurological involvement, bleeding tendency, recurrent infections) and course of disease. laboratory data (complete blood counts, platelet and absolute neutrophil counts, coagulation screening, platelet function tests by platelet function analyzer, and platelet aggregation studies using different agonist had been recorded. pcr and next generation sequencing for genetic confirmation by testing mutations in hps , ap b , hps , hps , hps , hps , dtnbp ,, bloc s , bloc s genes had been done. results: seven omani cases with hps have been identified ( males and females). their age ranged between (at birth) to years. two patients had hps type , patient had type , while the other cases had hps type . no other sub-types were encountered in oman. all patients were products of consanguineous marriage. one patient had adrenal hge, while the others had mild hemorrhagic phenotype, characterized by recurrent bruising and mild epistaxis. laboratory testing confirmed variable platelet aggregation defects with different platelet agonists. all patients had characteristic hypopigmentation, iris transillumination, nystagmus, and foveal hypoplasia. both patients with hps type had the same homozygous mutation in the ap b gene (c. _ delta), and presented with severe neutropenia. early diagnosis and initiation of gcsf on one of them improved outcome and prevented the development of complications. late diagnosis in the other patient resulted in the development of bronchiectasis as a result of recurrent sinopulmonary infections. background: sickle cell disease (scd), a genetic disorder characterized by defective sickle hemoglobin (hbs), triggers red blood cell sickling, hemolysis, vaso-occlusion, and inflammation. ischemic injury from scd starts in infancy and accumulates over a lifetime, causing pain, fatigue, and progressive end-organ damage that culminates in early mortality. voxelotor (gbt ) is an oral, once-daily therapy that modulates hemoglobin's oxygen affinity, thereby inhibiting hemoglobin polymerization. objectives: to assess the safety, pharmacokinetics, and efficacy of voxelotor in pediatric patients with scd. design/method: this ongoing study is being conducted in parts: part a: a single dose of voxelotor mg in pediatric and adolescent patients; part b: multiple doses of voxelotor mg/d or mg/d for weeks in adolescents. part b's primary objective is to assess the effect of voxelotor on modifying anemia. secondary objectives include measuring other markers of disease modification, such as hemolysis; daily scd symptoms, using a patient-reported outcome (pro) measure; and safety. results: as of november , , patients ( females) had received voxelotor mg and patients ( females) had received voxelotor for ≥ weeks. the median age for the patients was years, % were receiving hydroxyurea (hu), and % had ≥ painful crises in the past year. data for hemolysis measures are available for patients who received voxelotor for weeks. six of the patients achieved a hemoglobin (hb) response of > g/dl increase. laboratory markers of hemolysis improved concordantly; the median reductions in reticulocytes and indirect bilirubin were % and %, respectively. ten of patients showed reduction in total symptom scores (tss) at week , with a % median reduction in tss from baseline. there were no treatmentrelated serious adverse events (aes) or drug discontinuations due to aes. voxelotor mg for weeks in adolescents with scd, the majority receiving hu, demonstrated consistent, sustained efficacy on hb levels and measures of hemolysis; > % of patients showed a > g/dl improvement in hb. improvement in tss in mildly symptomatic patients suggests that the pro is sensitive to treatment effect and supports use in the ongoing hope phase study. voxelotor's reassuring safety profile is consistent with results in adults. these interim results support ongoing clinical evaluation of voxelotor as a potential disease-modifying therapy for adults and children with scd. supported by global blood therapeutics. background: acute kidney injury (aki) is a common complication in sickle cell disease (scd), and a potential risk factor for sickle nephropathy. aki is associated with acute decline in hemoglobin (hb) during vaso-occlusive pain crisis and acute chest syndrome (acs). it is unclear which pathologic factor plays a stronger role in aki development during hb drop: increase in free heme during vaso-occlusive events secondary to hemolysis or hb decline itself. objectives: to investigate if hb decline alone is associated with aki, we tested if the renal function of patients with scd worsened during parvovirus b -induced transient aplastic crisis (tac), in the absence of accentuated hemolysis. design/method: with irb approval, a retrospective study of patients who had laboratory confirmed parvovirus-b was conducted. serum creatinine (scr), both during and within months from the tac event, was collected. comparisons of the clinical and laboratory characteristics were analyzed using the wilcoxon test for continuous variables. aki was defined as an increase in scr by ≥ . mg/dl or a % increase in scr from baseline. to evaluate differences in change in hb on aki risk, changes in scr during tac were compared to those during pain crisis or acs admissions by fitting a generalized linear mixed model for binary outcome. a comparative sample of acs events and vaso-occlusive pain crisis were used to estimate rates of aki according to hb levels. results: three ( %) of the patients with scd developed aki during tac. no association was identified between change in hb from baseline to tac event (p = . ). no cases of aki were identified until hb decreased < . g/dl or the change in hb was ≥ . g/dl from baseline. next, we developed a model to evaluate the impact of change in hb from baseline for patients admitted with tac, pain crisis or acs on aki. with a g/dl decrease in admission hb from baseline, patients with tac had a % probability of developing aki, while acute chest syndrome and pain crisis would have a % and % probability, respectively. our data suggest that aki is still prevalent during parvovirus b -induced tac. however, the risk of aki during a tac event is and times lower than that from severe anemia induced by acute chest syndrome and vasoocclusive pain events, respectively. hemolysis-induced anemia during scd crisis appears to have a more significant role in the development of aki as compared to agenerative anemia. background: the natural history of hemoglobin e beta thalassemia (hbethal), the commonest form of severe beta thalassemia worldwide, has been examined in very few longterm studies. previously, we reported findings in hbethal patients in sri lanka. objectives: to evaluate longterm requirements for transfusion and splenectomy, complications and death in hbethal patients. design/method: all available patients were reviewed - times annually over years. results: patients ( %) died, aged (mean ± sem) . ± . years; the (known) causes commonly included iron overload ( ) and infection ( ); patients surviving patients are aged . ± . years. of patients originally classified by severity (group the mildest, and group the most severe, phenotypes), ( %) were assessed as mild (groups and ), of whom transfusions had been discontinued in . ultimately, / ( %) resumed transfusions, often following shifts to increasingly severe phenotypes including increasing intolerance to anemia. age at resumption of transfusions (following a transfusion-free interval of . ± . years) was . ± . years; in the more severe groups and , regular transfusions were stopped in / patients and resumed in / ( %), at younger ages ( . ± . years) and after shorter transfusion-free periods ( . ± . years) than in "milder" patients. mid-parental height (mph) was ultimately achieved in %. patients ( %) were splenectomized; updated analysis of responses to splenectomy (originally "group " patients), showed that splenectomy (at . ± . years) was followed by an extended, but impermanent, transfusion-free interval ( . ± . years); % patients resumed transfusions, usually related to exercise intolerance or poor growth. in groups and , complications of anemia and ineffective erythropoiesis, including leg ulcers (in % and %) and gallstones ( % and %), were more frequent than in groups and ; fractures were observed ( - %) across all groups, except for regularly-transfused group patients ( %). pulmonary artery pressures > mm were recorded in % patients. evaluation of patients with hbethal requires observations over years, without which definition of patients as "mild" or "severe" may be misleading. while in many patients transfusions may be withheld or reduced in frequency, troublesome complications may surface with advancing age even in "milder" patients. although individual consideration of transfusion requirements is critical, the availability of effective chelation, where this can be provided without prohibitive cost, may alter the balance of risks and benefits of regular transfusions in hbethal. (premawardhena a. lancet ). background: social determinants of health (sdh) are environmental and socioeconomic factors, such as access to food and housing that affect health outcomes. pediatricians are increasingly screening for sdh as part of primary care visits, however less is known about screening for sdh in pediatric hematology. evidence suggests that sdh play a role in disease severity for children with scd, who face significant socio-economic and racial disparities. the goal of our quality improvement (qi) project was to increase the percentage of patients with scd who were connected to community resources for unmet social needs. design/method: we based our intervention on the successful implementation of wecare in our institution's pediatric primary care clinic. eligible patients were identified at the start of each clinic session. on arrival the parent was given a self-reported screening tool for six sdh (childcare, education, employment, food, utilities and housing). results were entered in the electronic health record by the physician or social worker who then printed a pre-existing resource list for patients with a positive screen. we used a series of plan-do-study-act (pdsa) cycles to study tests of change. we tracked process measures (percentage of patients screened, percentage of patients with an unmet social need who received a resource sheet), outcome measures (percentage of patients with an unmet social need who connected with a community resource) and balancing measures (staff, patient and provider satisfaction). run charts were reviewed weekly and then monthly to inform further tests of change. examples of pdsa cycles include who gave the paper survey to patients (social worker or physician versus medical assistant) and length of time between surveys ( to months). results: between august and december screening rates improved from % to %. of the patients screened, % report at least one unmet social need; of those % received a targeted list of community resources in the first month of the project, and % in the fifth month. finally, % of patients reached by phone had connected with a community resource within weeks of the clinic visit. we have successfully implemented universal screening for sdh for patients with scd in our urban pediatric hematology clinic without requiring extra staff. next steps include further pdsa cycles to connect more patients to appropriate resources, and tracking improvement in health care utilization outcomes from addressing sdh in this vulnerable patient population. background: the clinical manifestations of sickle cell disease (scd), chronic hemolytic anemia, and vaso-occlusion occur as a direct result of sickle hemoglobin (hbs) polymerization. voxelotor (gbt ) is a first-in-class, oral, oncedaily investigational agent designed to modulate hemoglobin's oxygen affinity in a targeted approach to inhibit hbs polymerization. objectives: to examine the pharmacokinetics (pk), safety, and dosing of voxelotor in children (aged - years) and adolescents (aged - years) with scd from part a of the gbt - study. design/method: gbt - is an ongoing, open-label, phase a study in patients aged - years with scd (sickle cell anemia or sickle beta zero thalassemia). part a of this study (the focus of this abstract) is examining pk of singledose ( mg) voxelotor. pk samples to measure whole blood and plasma voxelotor concentrations were collected up to days following single-dose administration. separate population pk (ppk) models were developed to describe the concentration versus time profiles of voxelotor in whole blood and plasma using nonlinear mixed effects modeling (non-mem, version . ). ppk modeling and physiologically based pk (pbpk) modeling were used to simulate voxelotor pk parameters and support dose selection for future evaluation in younger children. : part a included adolescents ( females; median age years [range - ]) and children ( females; median age . years [range - ]). mean weight was . kg (range - kg) and . kg (range - kg) in adolescents and children, respectively. voxelotor was well tolerated with no drugrelated grade ≥ adverse events (ae) or serious aes. a compartment model with first-order absorption best described the pk of voxelotor (and was the same model structure used for adults with scd). voxelotor pk exposures in adolescents were comparable to those observed in adults, but higher exposures were observed in children. ppk and pbpk modeling support the use of a weight-based dosing strategy in younger children (aged < years) in future trials. adult voxelotor doses can be used in adolescents. however, based on higher pk exposures, a lower weight-based dosing strategy is recommended in children. ppk and pbpk modeling provides an innovative approach to minimize experimental dosing in children and accelerate dose selection of voxelotor in ongoing and future clinical studies. this abstract is supported by global blood therapeutics. background: hydroxyurea (hu) reduces rates of acute complications, and improves long term outcomes in patients with sickle cell disease (scd) and is now fda approved for children. through previous work we have increased the number of eligible patients on hu in our clinic, however accessing a compounding pharmacy remained a significant barrier to hu adherence for infants and children who cannot swallow capsules. objectives: the objective of our quality improvement project was to improve adherence to hu among pediatric patients with scd at our urban safety net hospital by addressing barriers to obtaining liquid hu. design/method: to begin we met with the leadership of our outpatient pharmacy which offers mail order delivery. however, like most retail pharmacies, they do not have the necessary protective equipment to compound liquid hu. through a series of discussions, we began a unique partnership with our institution's inpatient chemotherapy pharmacy who compounds the liquid hu and delivers it to the outpatient s of s pharmacy, who then dispenses liquid hu to families. using a series of plan-do-study-act (pdsa) cycles we tracked adherence by calculating the medication possession ratio (mpr), defined as the percentage of days in a given period of time that each patient had their medication on hand. the mpr for liquid hu mpr among enrolled patients was tracked by pharmacy staff and reviewed monthly. additional pdsa cycles included adding automatic refills and reminder calls by pharmacy staff and improving communication about delivery. we also tracked patient satisfaction. results: between march and december , a total of thirty pediatric patients were enrolled in our program for on-site compounding and free mail order delivery of liquid hu. mpr for liquid hu is currently . % among enrolled patients, significantly higher than the mpr of % reported in the literature, and has risen steadily since the beginning of the project. families are highly satisfied with the program, specifically appreciating the convenience of mail order delivery, saving on delivery fees, and reminder calls when refills were due. by compounding and dispensing liquid hu directly from our institution's outpatient pharmacy we have significantly improved adherence to this hu therapy in our high-risk population. next steps include analysis of change in clinical outcomes for patients enrolled in this program. as adherence to hydroxyurea is associated with decreased acute care utilization and cost, programs such as ours could play a crucial role in reducing the excessive costs and ed utilization among this patient population. background: experience with the iron-chelator deferasirox is reported widely in higher-income settings. by contrast, real-life experiences in emerging countries are infrequently reported. objectives: to evaluate, in a non-trial setting, the real-life response to deferasirox in an emerging country. design/method: in sri lanka's national thalassemia center which manages patients without tertiary staff, quantitative evaluations of body iron or estimates of extra-hepatic iron, the records of patients who began deferasirox in / were retrospectively reviewed. results: baseline assessments (mean±sem) indicated substantial iron loading [serum ferritin (sf) , ± ug/l; serum alt ± . u/l (normal ≤ u/l)]. deferasirox was introduced at low doses ( . ± . mg/kg/day); many patients started at < mg/kg and, after months, doses remained ≤ mg/kg/day in % patients. after months, sf in % patients remained > , ug/l; only by months had (mean) sf declined to < , ug/l ( ± ; p< . ). similarly, mean alt normalized (to ± u/l) only by months. death and complications were not systematically recorded by staff who had been charged, without provision of additional resources, with the introduction of this new drug in hundreds of patients. these results contrast to those in sri lanka's tertiary thalassemia center where, in patients following the introduction of deferasirox ± . mg/kg/day, sf declined rapidly, even in relatively less ironloaded patients (from , ± to , ± g/l after months; p = . ). these findings underscore the importance, during the implementation of new drug regimens in lowerincome centers with marginal resources, for investments in methods to quantitate body iron burden, hands-on educational initiatives to guide day-to-day management by competent but non-expert staff, and data systems to record efficacy, effectiveness, toxicity and compliance. such investment is critical to optimising therapy and improving complications in thalassemia patients worldwide: even in sri lanka, where resources directed to thalassemia management are greater than in most of asia, results in the oldest living cohort (born - ) indicate under-treatment [elevated iron burdens (sf , ± ug/l) and high prevalences of diabetes ( %) and hypothyroidism ( %)]. even in a younger cohort (born - ) which has benefitted from improved treatments, the prevalence of many complications exceeds those reported from high-income settings. over the next decade, and two decades after the who declaration that the impact of thalassemia on global mortality and morbidity is underrecognized, increased investments by governmental and nongovernmental sources will be necessary to improve outcomes for asian patients with thalassemia. background: a major barrier to success in hydroxyurea (hu) treatment of patients with sickle cell disease (scd) is non-adherence. objectives: to optimize hu adherence in patients with scd. design/method: a care model was designed by the sickle cell (qi) team at children's hospital to improve hu adherence among scd patients. the original model included bimonthly family phone contact, monthly dispensing pharmacy phone contact and lab monitoring. adherence measures included obtaining hu from pharmacy monthly, completion of monthly labs, hb f percentage and mcv, and mtd achievement. from / - / , several pdsa cycles refined our care model. a one-year follow-up survey gathered feedback on the care model. the first-year data involved ∼ patients. the biggest improvements resulted from making pharmacy calls before patient/family calls, shipping liquid hu to outlying patients, and tracking call time/content. the qi goal was % hu adherence by / . the % baseline adherence rate increased to % by / , and has remained in that range. the completion rate of patient/parent phone calls increased from % the first month to % at six months. pharmacy prescription pick-up has increased from % to % per month. lack of liquid hu availability was overcome by shipping the medication to the patient's home. parental hesitance to share information by phone, especially with qi team members with whom they had no established relationship, was overcome by having the longtime sickle cell nurse do many of the early calls. however, survey feedback showed families became comfortable with several clinic personnel calling. the calls gave families the opportunity to ask questions about their child and/or get additional information about scd. the calls also provided an opportunity for seasonal flu shot or tcd testing reminders. the surveys gave information on the optimal time of day to reach each family, providing individualization and further increasing the percentage of completed calls. two families surveyed said they no longer needed two calls a month because they were now able to remember to pick up hu, administer it, and get labs on their own. this qi project has not only improved hu adherence, but also fostered health education/counseling, increased patient/parent satisfaction, and enhanced service utilization. medical team member and patient/family comments demonstrate that it has helped build relationships and trust between families and the medical care system. based on survey feedback, we will further individualize care to increase adherence rate and sustain improvements. cincinnati children's hospital medical center, cincinnati, ohio, united states background: the thalassemias are a heterogeneous group of genetic blood disorders caused by mutations that decrease or eliminate the synthesis of the -and/or -globin subunits of hemoglobin. the phenotype of thalassemia depends on the interaction of the -and -globin gene clusters, because both loci determine the -/ -chain balance. for example, a -thalassemia phenotype can be more severe than expected when coinherited with -globin gene triplication (copy number gain), which exacerbates the -/ -globin imbalance. objectives: describe four individuals with an incorrect diagnosis of -thalassemia trait who were later properly diagnosed by comprehensive genetic testing to have -thalassemia intermedia caused by heterozygous -thalassemia mutations coinherited with triplicated -globin loci. design/method: sequence analysis of the -globin (hba /hba ) and -globin (hbb) genes, and copy number variation analysis of the -and -globin gene clusters by multiplex ligand-dependent probe amplification. results: four unrelated individuals of northern european ancestry were evaluated for signs and symptoms not explained by a diagnosis of -thalassemia trait (previously made by a pediatric hematologist), including growth delay, splenomegaly, moderate anemia, marked elevation of hemoglobin f, thalassemic facies, reticulocytosis, and/or indirect hyperbilirubinemia. genetic testing revealed that all were heterozygous ( / ) for the same, single -globin mutation [hbb.c. c>t (p.q *)] and also heterozygous for an -globin triplication ( / anti- . ). their previous diagnoses of thalassemia trait had been made by complete blood counts, hemoglobin electrophoresis, and/or sequence analysis of the -globin genes only. these individuals' phenotypes ranged from moderate anemia only to multiple stigmata of thalassemia, demonstrating the phenotypic variation of a thalassemia genotype. correct diagnosis was made at an average age of . years. a trial of chronic transfusions was initiated for one patient for growth failure. all were educated about the potential for exacerbations of anemia, gallstones, osteoporosis, and iron overload (even without transfusions). parental genetic testing was recommended to assess reproductive risk, because inheritance of this complex genotype can be apparently autosomal dominant. conclusion: heterozygosity for a -thalassemia mutation does not necessarily indicate -thalassemia minor or "trait". when coinherited with -globin gene triplication, a symptomatic form of -thalassemia can occur. correct and timely diagnosis of thalassemia requires careful consideration of the degree of anemia and examination for organomegaly, bony changes, and jaundice. sequence analysis and copy number variation analysis of both the -and -globin gene clusters is key. hematologists need to be aware of this diagnostic possibility and how to test for it to prevent inaccurate or delayed diagnosis. background: the burden of healthcare costs for sickle cell disease (scd) is nationally estimated at over $ billion. the major components of these costs are inpatient and emergency center (ec) visits, many of which are potentially avoidable. in several chronic conditions, a subset of patients account for most of the avoidable encounters. identifying these patients is the first step in targeted care delivery. objectives: to measure and analyze scd patient utilization patterns in the ec and inpatient at texas children's hospital (tch). we identified all individuals under years old with any encounter at tch associated with an international classification of disease (icd)- or code for scd, including hgb ss, hgb sc, and hgb s/beta thalassemia. for each patient, we identified all inpatient and ec encounters in the days prior to their most recent encounter. finally, each encounter was classified as associated with pain, acute chest syndrome (acs), or "other" using an algorithm of discharge diagnosis codes and pharmaceutical delivery. the total number of scd-associated ec and inpatient encounters over the prior year was calculated for each patient. we stratified each patient according to their utilization patterns: low ( - encounters), intermediate ( - encounters), and high (≥ encounters). we identified unique patients with scd that had at least one encounter from july until june . there were , scd-related encounters in the days prior to their most recent encounter. most ( %, n = ) patients exhibited low-utilization patterns and % (n = ) were intermediate. finally, a small subset ( %, n = ) demonstrated high-utilization patterns and accounted for % of all encounters. high-utilization was associated with older age and public payment mechanisms. pain encounters were predominantly in pre-adolescents and teenagers with high-and intermediate-utilization patterns. acs was most frequent in pre-teens and younger teens in the intermediate-utilization group. finally, the youngest-aged high and intermediate users presented for other reasons such as febrile episodes and splenic sequestration. our findings reflect national trends in that a significant portion of encounters are attributed to a small subset of patients exhibiting a high-or "super-" utilization pattern. at our institution, scd super-utilization is associated with older age and pain. we also identified a group of infants and toddlers with frequent encounters for fever. to comprehensively address this burden, it will be important to design interventions targeted toward age and specific medical needs. background: background: the rarity of diamond blackfan anemia (dba) has hindered describing the spectrum of disease, identifying predictive correlations, and guiding datadriven recommendations. long-term toxicities from steroid or transfusion therapy that start in childhood remain the major clinical problems in patients with dba who do not receive stem cell transplant. objectives: objective: to define the dba patient population at st. jude children's research hospital including treatment responses and toxicities to help inform recommendations on treatment and monitoring. design/method: method: medical records were reviewed for all patients with dba treated at st. jude between and for diagnostic testing, treatment types and regimens, and outcomes. two-sample t-test or wilcoxon rank sum test was used to compare continuous variables in two groups depending on the normality of the data tested by shapiro-wilk test. results: a total of patients with dba were identified with a median age of . years (range months - years) at last follow up. a ribosomal protein gene mutation was identified in / patients ( %) with an rps mutation / ( %). thirteen different congenital malformations were described in / patients ( %). fourteen of twenty ( %) patients treated with corticosteroids had an initial response and of those achieved full remission. three patients became steroid-refractory and were unable to wean to an acceptable dose. five of twenty patients continue on lower-dose steroids. five patients currently require no therapy. univariate analysis revealed no statistically significant genetic predictors of response or remission, however, / rpl patients responded to steroids with / ( %) in long-term remission. ten patients are maintained on chronic transfusions and have undergone successful hematopoietic stem cell transplant. nineteen of treated patients ( %) had a treatment-related toxicity. patients on steroids were more likely to have short stature than patients on transfusions or in remission (p = . ). severe bone mineral density deficit occurred in / ( %) patients, in before age years. eight patients had hepatic iron overload, in one documented by age years. other severe toxicities included restrictive cardiomyopathy from iron overload, pathologic fracture, diabetes mellitus, and premature ovarian failure in one patient each. this genotypically and phenotypically heterogeneous dba cohort had a high rate of treatment-related toxicities, notably growth retardation, bone density loss, and hepatic iron overload even in very young children. these findings underscore the need for early standardized monitoring. background: patients with sickle cell disease (scd) face worsening morbidity and mortality between ages and , when they must transition from pediatric to adult healthcare.( ) an effective curriculum addressing disease knowledge, educational and vocational skills, self-efficacy, and social supports is critical to a successful transition. traditional didactic approaches have not led to durable knowledge retention. ( ) technology-based methods have been attempted, but the best educational approach remains unknown. objectives: . to understand how adolescent and young adult (aya) patients with scd view existing transition education. . to include patient preferences in improving our transition curriculum. we developed a qualitative survey to assess patient views of existing approaches for learning about scd and their opinions about preferred transition topics. thirty patients with scd aged to years old were recruited between january and december . responses were managed using redcap electronic data tools hosted at the university of rochester.( , ) qualitative and quantitative data analyses were performed, including independent t-testing to compare responses between age groups. results: approximately % of subjects were under years of age, while % were or older. seventy-one percent had a computer, and . % had a cell phone, with most reporting daily use. subjects reported greatest satisfaction with learning from their doctor during clinic visits ( . % agree or strongly agree) and websites on a cell phone ( . % agree or strongly agree); the least popular methods were online chat rooms and microsoft® powerpoint presentations. satisfaction was similar across age groups. recommended transition topics were viewed positively, with subjects ranking highest understanding their bloodwork ( . % agree or strongly agree) and understanding laws protecting students with chronic disease ( . % agree or strongly agree). older subjects ( - years old) agreed more strongly with learning about opioid addiction and understanding differences between adult and pediatric doctors than did younger subjects ( - years old) (p < . ). this pilot study was successful in helping us to understand the educational needs of aya patients with scd. preliminary data underscore the importance of education provided by the pediatric hematologist. our results also suggest that the optimal use of technology-based methods requires further investigation and that tailoring transition education by age group may be useful. background: similar to patients with transfusion-dependent beta-thalassemias (tdt-beta), survivors of hemoglobin barts hydrops fetalis (homozygous alpha- -thalassemia, tdtalpha) will require lifelong transfusions of erythrocytes. we have previously shown that a transfusion strategy that is based on the guidelines developed for tdt-beta (conventional transfusion) is suboptimal for these patients owing to the differences in the pathophysiology of anemia in the two conditions: in tdt-alpha, conventional transfusion strategy will lead to a gradual increase in non-functional hbh with subsequent tissue hypoxia and hemolysis. an aggressive transfusion strategy that was based on reduction of hbh and increase in "functional" hemoglobin level resulted in improvement of tissue oxygenation and reduction of hemolysis but was associated with significant increase in transfusional iron burden [amid et al, blood ] . objectives: to define the optimal chronic blood transfusion targets for hbh% and functional hemoglobin in patients with tdt-alpha. design/method: following research ethics board approval, longitudinal data of patients with tdt-alpha ( males, median age . ( . - . ) were retrospectively collected. variables of interest included total pre-transfusion hemoglobin, hbh%, and "functional" hemoglobin [measured as total hemoglobin x ( -hbh/ )]. outcome variables were lactate dehydrogenase (ldh, marker of hemolysis), and soluble transferrin receptor (str, marker of erythropoiesis). hemoglobin analysis was done using high-performance liquid chromatography and capillary zone electrophoresis. we examined the association of "functional" hemoglobin with str, and hbh% with ldh, using repeated-measures anova to adjust for the effect of multiple testing. we constructed receiver operating characteristic curve and calculated the area under the curve to define the best cut-off values for variables of interests. there was a strong association between functional hb and str, as well as hbh and ldh. the optimal cut-off for "functional" hemoglobin that was associated with str < . mg/l was g/l (auc = . , sensitivity and specificity of . % and % respectively). the optimal cut-off for hbh to supress ldh to < u/l was % (auc = . , sensitivity and specificity of . % and % respectively). the optimal pre-transfusion hbh% for reduction of hemolysis was % and the optimal "functional" hemoglobin to adequately supress erythropoiesis was g/l. to meet these hbh% and functional hb targets by simple blood transfusions, patients with tdt-alpha would require a hypertransfusion regimen with a minimum pre-transfusion total hb of g/l and consequently high transfusional iron burden. an alternative approach using exchange transfusion to reduce hbh% and improve functional hemoglobin would be associated with less volume of transfusion and potentially better long-term outcome. hospital sacre coeur, milot, haiti background: initial results of work developing a pediatric sickle cell disease (scd) clinic at the hôpital sacré coeur (hsc) in milot, northern haiti were presented at aspho . the purpose of this clinic is for a pediatrician with a special interest in scd to provide scd care, advising on trait and managing disease with penicillin prophylaxis (pcn) and hydroxyurea therapy (hu) for select patients. this clinic was started in collaboration with a us based hematologist and support from yale-new haven hospital. objectives: to describe the success and challenges of providing pcn and hu in the scd clinic at hsc through a review of patient records. design/method: since this clinic's inception, a database of patients, with basic clinical information has been kept and made accessible, through 'drop-box', to the us hematologist. the records of those that presented to the clinic were reviewed. the hemoglobin diagnosis was made either by clinical history and sickle cell prep or by hemoglobin electrophoresis through alpha laboratory, port-au-prince, haiti. results: ninety-nine individuals were seen in the first years of the program. fifty-six underwent a hemoglobin electrophoresis. of these , are ≤ years old. thirty-two were started on pcn vk, of which / ( %) were ≤ years old. eleven patients were started hu therapy. all patients on hu have shown progressive increases in hemoglobin. there have been no clinical complications of hu therapy. none of the patients taking hu have required hospitalization or transfusion in . three patients (not on hu) were hospitalized in for complications of scd (osteomyelitis, pain). in , with less than half the numbers in the program, there were admissions for severe anemia, pain, stroke and splenic sequestration. with ongoing external support and a local reputation for excellence in sickle cell care, the clinic at hsc has been able to expand services and improve the health of a growing number of patients with scd. early data suggests that pcn and hu therapies are helping to reduce complications and improve quality of life. challenges to date have included lack of funding for transportation to clinics, for hospitalizations and to cover the cost of electrophoreses. at the same time as continuing providing excellent care and gathering data, it is crucial to explore opportunities for collaboration and cooperation in ways that will assure that the clinic can become independently sustainable while continuing to improve the quality of life for the individuals it serves. background: ykl- is an inflammatory glycoprotein expressed by infiltrating macrophages in various inflammatory conditions. it has been found to be elevated in patients with different pathological conditions like acute and chronic inflammations, increased remodeling of the extracellular matrix (ecm), development of fibrosis and cancer. several studies have found elevated ykl- concentrations in sera of patients with liver diseases such as hepatic fibrosis by hepatitis c virus. it has been suggested that ykl- concentrations reflect the degree of liver fibrosis. to evaluate serum ykl- levels in patients with -thalassemia and its relation to viral hepatitis, liver stiffness as assessed by transient elastography (fibroscan, fs) and hepatic iron concentration. design/method: a prospective study included patients with -tm ( males and females) with mean age . ± . years (range: - years). serum ferritin level, liver enzymes (alt and ast), hbs ag, anti hcv ab and serum ykl- using elisa kit were evaluated. all patients were subjected to liver mri t * to detect liver iron content by the sequence and transient elastography (fibroscan, fs) to assess degree of liver stiffness. results: mean fibroscan value was ( . ± . ) kpa with a median . (range . to ) kpa. ( %) patients were categorized as f - and ( %) were stage f - , ( %) patients had severe fibrosis. their median serum ferritin was ng∖ml, with ( %) patients had values exceeding g/l. median cardiac t * was . with patients had values below ms, and the median lic was . mg/g dw with patients showed readings above mg/g dw. nyl- was evaluated as a marker of inflammation and liver fibrosis and showed mean value . (± . ) pg/ml, and range from to pg/ml. mean ykl- was significantly higher among males (p = . ), patients on chelation therapy (p = . ), patients on dfs (p≤ . ), in those with abnormal liver enzymes, splenectomised patients, patients with hbv sero-positivity, those with moderate elevation of t * and patients with high grades of liver fibrosis (p< . ). ykl- showed positive correlation with the rate of transfusion, lic, ferritin, alt and ast but negative correlation with weight, height and t *. roc curve analysis revealed that the cutoff value of ykl- at pg/ml could differentiate -tm patients with and without viral hepatitis with . % sensitivity and specificity of . %, area under the curve (auc) . , positive predictive value . and negative predictive value . (p< . ). roc curve analysis revealed that the cutoff value of ykl- at pg/ml could detect -tm patients with liver cirrhosis with . % sensitivity and specificity of . %, area under the curve (auc) . , positive predictive value . and negative predictive value . (p< . ). conclusion: serum ykl- levels are elevated in patients with -thalassemia and can detect patients with active viral hepatitis and liver stiffness. background: the most common splenic complication in pediatric patients with sickle cell disease (scd) is acute splenic sequestration (ass), which has often been managed with splenectomy. although splenectomy has been a treatment of choice for years, long-term vascular complications have not been thoroughly evaluated. pulmonary hypertension (phtn) is a severe complication of scd. in adults with scd, phtn has been associated with a -month mortality rate of approximately %. it has been reported that splenectomized patients with hemolytic disorders are at even greater risk of phtn. several medications exist to treat phtn, but with few studies of their efficacy or toxicities in patients with scd. additionally, these patients are often treated with either chronic prbc transfusions or hydroxyurea (hu) to raise hemoglobin, reduce hemolysis, and prevent vaso-occlusive events. objectives: to evaluate effect of chronic prbc or hu vs. no intervention, on tricuspid regurgitant jet velocities (trv) in pediatric patients with scd and history of splenectomy. design/method: retrospective chart review of splenectomized patients with hbss followed at marian anderson center at st. christopher's hospital for children, philadelphia, between and . we analyzed trvs ( hu, prbc, and from control group receiving neither treatment) from patients ( hu, prbc, neither). mean age at echo was . +/- . . data was analyzed with linear correlations and analysis of variance (anova), including the post hoc test of least significant difference (lsd) for all pairs of treatment groups. results: trv was not significantly correlated with age at time of assessment or with time between splenectomy and trv. univariate anova among groups yielded trv means of: . +/- . cm/s (hu), . +/- . (prbc), . +/- . (neither). we found a notable difference as the mean of the hu group was almost cm/s lower than the others, but no overall statistically significant association for any of the groups exists. however, when we performed post hoc tests to adjust for multiple comparisons and looked at all pairings within the anova, we found that the lsd between the hu and the prbc groups was statistically significant (p = . ), and that a trend exists between the hu group and the neither treatment group (p = . ). our data suggests that treatment with hu is correlated with a reduction in trv in pediatric patients with scd who underwent splenectomy. given these promising results, we believe our data warrants further study with larger treatment groups. nancy olivieri, gaurav sharma, susmita nath, rajib de, tuphan kanti dolai, prakas kumar mandal, abhijit phukan, amir sabouhanian, robert yamashita, angela allen, david weatherall, prantar chakrabarti background: hemoglobin e thalassemia (hbethal), which accounts for % of all severe beta thalassemia worldwide, has an estimated prevalence of . / , in west bengal, from which little information about clinical findings has been reported. objectives: to document clinical and laboratory findings in patients with hbethal, ultimately to improve resources for clinical management. design/method: we reviewed records from: a database recording patient names; clinic charts; "special" charts containing additional details; and, in transfused patients, transfusion day-care records. additionally, because in india's public hospitals original lab/imaging reports are commonly retained at home, % of families were interviewed to provide additional information. we excluded records of patients aged < years and patients aged < years who had not been reviewed since . results: while at least one visit had been recorded in , hbethal patients at nrs hospital, most patients are not regularly reviewed there. we examined charts [ ( %) aged ≥ years; ( %) aged - years; % male], representing approximately % of regularly-reviewed patients. most families ( . %) reported monthly incomes (< , indian rupees), below the monthly cost of living ( , rupees) in kolkata. mean (±sem) hemoglobin was . ± . g/dl. % patients were receiving eight or more transfusions per year; from , % had been treated with deferasirox, . ± . mg/kg/day. iron control estimated by serum ferritin concentration ( . ± g/l) was highly variable. a total of % patients were splenectomized. a substantial obstacle to documenting complications was the lack of recording, in any of the five sources, of many relevant parameters: for example, the status of sexual maturation (normal, delayed, or absent) was documented in less than %, and measurements of fasting blood glucose in less than %, of records. where recorded, complication rates were high: delayed/abnormal sexual maturation was recorded in % patients aged > years; in the patients aged > years and those aged - years, respectively, hypothyroidism was recorded in % and %, and elevated serum alt in % and %. in most evaluable patients > years, height was measured between the rd- th percentiles. cardiac findings, rarely documented, included pulmonary hypertension and reduced left ventricular ejection fractions in a few patients. despite dedicated attention to many aspects of thalassemia care, insufficient documentation limited a clear understanding of the current morbidity in hbethal patients. investment in personnel and technology will be critical to record relevant information, ultimately to improve clinical management, over the next decade. children's hospital of richmond at vcu health, richmond, virginia, united states background: sepsis is a common cause of death in children with sickle cell disease (scd). recommendations for care of fever in children with scd include immediate medical evaluation including blood culture and initiation of broad-spectrum antibiotic therapy. the increasing availability of pcr-based respiratory pathogen panels (rpp) provide the opportunity to rapidly identify viral causes of fever. the role for rpps in identifying the source of fever in children with scd and how it affects provider practice is not well studied. ( ) to determine the epidemiology of respiratory virus-associated fever in children with scd and ( ) to determine whether a positive rpp is associated with reduced risk of bacteremia in this population. this was a single-center, retrospective cohort study. we identified and reviewed the medical records of all children with scd seen in our emergency department (ed) with temperature ≥ . oc at home or in the ed from january , , through september , , as well as, all febrile children for whom rpps were sent since the introduction of rpps april . we reviewed the results of blood cultures, rpps, chest radiographs, and ed notes and discharge summaries to identify sources of infections. independent t test and chi-square analysis were used as appropriate to compare results using spss©. overall, the rate of bacteremia was %. there were no cases of bacteremia among children with positive rpps. % of children with negative rpps had true bacteremia. a positive rpp did not reduce the likelihood of bacteremia (p . ). patients with bacteremia had higher presenting temperatures than those without bacteremia ( . oc vs . oc, p . ). the most common rpp findings were rhinovirus/enterovirus ( %), human metapneumovirus ( %), and influenza a ( %). sending an rpp did not affect admission rate ( % and % respectively, p . ); however, likelihood of admission was lower in patients with positive rpps ( % vs %, or . [ . - . ], p . ). length of stay (los) was shorter in patients for whom an rpp was not sent ( . vs . days, p . ). as previously reported, bacteremia in febrile children with scd is very low, but remains a serious concern, particularly in the setting of high fever (> oc). a positive rpp did not reduce the odds of bacteremia, but did have a sta-tistically significant impact on both admission rate and los. more work is needed to understand how rpp results impact provider decision-making and care for children with scd. cincinnati children's hospital medical center, cincinnati, ohio, united states background: diffuse myocardial fibrosis is a common, if not defining, feature of the heart in sickle cell anemia (sca) that is strongly associated with diastolic dysfunction. we found diffuse myocardial fibrosis in every patient in a sca cohort (n = ) ranging in age from to years (niss ). the treatment and prevention of this complication of sca has not been studied before. objectives: because diffuse myocardial fibrosis must begin in early childhood, we hypothesized that early initiation and uninterrupted use of disease-modifying therapy for sca can prevent it. design/method: we use cardiac magnetic resonance imaging (cmr) to measure the myocardial extracellular volume fraction (ecv) to quantify diffuse myocardial fibrosis in individuals with sca who have been treated, uninterrupted, with hydroxyurea or chronic transfusion therapy since ≤ years of age. two comparison groups were used: individuals with sca who have not been treated with disease-modifying therapy since ≤ years of age (n = ) and controls without sca (n = ). results: we studied individuals ( m/ f) with a mean age of . years (range - ). mean age at the start of diseasemodifying therapy was . ± . years (range - ). only had evidence of mild diffuse myocardial fibrosis (ecv . ); the other had no detectable diffuse fibrosis (all had ecv < . , the upper limit of normal). mean ecv was . ± . , which was significantly lower than the ecv of individuals with sca who have not received early uninterrupted therapy ( . ± . ; p = . ) and not statistically different from normal controls ( . ± . ; p = . ). none had macroscopic fibrosis by late gadolinium enhancement or evidence of myocardial hemosiderosis by t * imaging. no patient had diastolic dysfunction by echocardiographic classification, right heart catheterization, or both. disease-modifying therapy for sca can prevent diffuse myocardial fibrosis, and possibly diastolic dysfunction, if started in early childhood. prospective trials of disease-modifying and anti-fibrotic therapy are planned to prevent diffuse myocardial fibrosis, which can be monitored noninvasively by cmr, and improve outcomes in sca. (niss, blood, ) . background: a statewide sickle cell surveillance system (sscss) was developed with the goal of determining the prevalence of sickle cell disease (scd) in indiana and the level of care that patients receive throughout the state. persons with scd are at high risk of infection, especially with encapsulated organisms, as well as at increased complications from influenza. utilizing sscss data, the relationship between vaccination status and mortality was explored. to determine if vaccination status is associated with mortality in persons with scd. the project was granted a waiver of consent by the st. vincent irb. death certificates were obtained to identify cause of death. deceased patients (cases) were matched by age, gender, and sickle genotype to living patients (controls). vaccination data were collected from the medical record and the children and hoosier immunization registry program (chirp) through the date of death for each case. cases and controls were assigned a point for completion of the pneumococcus, meningococcus and haemophilus influenza type b (hib) vaccine series and one point if the influenza vaccine was given within a year prior to death of the cases [max vaccine status score (vss): ]. total points were compared between the cases and controls. two tailed t-tests to compare means of continuous data and wilcoxon signed-rank test to compare ordinal data. one thousand forty-eight individuals were included in the sscss. six hundred and seven ( . %) were seen at one institution and included in this analysis (mean age = years). thirty-three of the ( . %) were deceased at the time of analysis. six point one ( . )% of controls and . % of cases received a vss of . the mean vss for cases was . ± . and . ± . for controls. thirty point three ( . ) % of controls had a vss of one or more, compared to % of cases (p = . ). patients who died of infection [streptococ-cus (n = ), pseudomonas (n = ) and unidentified organisms (n = )] were not up to date on vaccination against encapsulated organisms, but two had received the influenza vaccine in the year prior to death. in this sample, mortality occurred exclusively among adult patients, which is consistent with current patterns in developed countries. among these adults, vss and mortality rates were not related. limitations to the study include small sample size and potential incompleteness of vaccine records. vaccination rates and other standard of care indicators should be explored in a larger cohort of patients to determine associations with mortality. background: sickle cell disease (scd) is a genetic disorder resulting in acute and chronic complications, including delayed puberty. delayed puberty can have adverse physical and psychosocial effects on affected children and families. there are no published reports from ghana on pubertal timing in children with scd. the aim of this cross-sectional study was to describe pubertal changes in children with scd at korle bu teaching hospital (kbth), accra, and compare these findings to those in a control group without scd. design/method: children with scd and children with hb aa, ages - years, were consecutively recruited and matched for age, sex and socioeconomic status. investigator-administered questionnaires were used to obtain demographic data for all participants and information on menarche (girls only). pubertal status was assessed by physical examination using tanner staging. testicular volumes were determined in boys using a prader orchidometer. body mass index (bmi) and socioeconomic status (ses) of participants were analyzed to determine if there were any associations with tanner stage. of the with scd, ( . %) were hb ss and ( . %) hb sc. females comprised . % (cases and controls). mean age at onset of breast development was significantly delayed in girls with scd ( . ± . years) compared to controls ( . ± . years) but there was no significant age difference at onset of pubic hair development. mean age at menarche was significantly delayed in girls with hb ss ( . ± . years) and hb sc ( . ± . years), compared to those with hb aa ( . ± . years). in boys, the mean ages at onset of puberty were significantly delayed in those with scd ( . ± . years, for genital development and . ± . years, for pubic hair development), compared to those without scd ( . ± . years and . ± . years, respectively). mean testicular volumes were significantly lower in cases compared to controls, across all age ranges (p< . ). mean bmi in both cases and controls were similar at onset of breast development in girls. however, in boys with and without scd, mean bmi values were significantly different at pubertal onset. in univariate analysis, ses was not associated with tanner stage for both genital and breast development. mean ages at pubertal onset were significantly delayed in children with scd. longitudinal studies are needed to further characterize any associations with bmi and determine potentially modifiable risk factors affecting pubertal onset in scd. background: sickle-cell disease (scd) is a life-threatening genetic disorder associated with multiple chronic and acute complications. specific monitoring and treatment for children is a major part of the medical focus, but there remains a lack of real-world evidence of the disease burden and practice patterns among the pediatric scd population. objectives: to examine the clinical burden and management of scd among pediatric patients. design/method: a retrospective claims study was conducted using the medicaid analytic extracts database from jan - dec . pediatric patients (aged < years) with scd were identified using icd- -cm diagnosis codes ( . - . , . - . ). the first observed scd diagnosis during the identification period was designated as the index date. patients were required to have continuous medical and pharmacy benefits for at least months pre-and months post-index period. patient data were assessed until the earliest occurrence of the following events: disenrollment, death, or the end of the study period. patient demographic and baseline clinical characteristics, clinical outcomes (mortality, incidence of pain crisis, complications), scd management, and healthcare utilization were examined. all variables were analyzed descriptively. results: a total of , patients met the study inclusion criteria, with a mean age of . years. most patients were black ( . %) and had a charlson comorbidity index score of ( . %). mortality during follow-up was . in personyears, and the event rate of pain crisis in the inpatient setting was . in person-years. the three most common complications after pain crisis (highest rates in person-years) were fever ( . ), infectious and parasitic diseases ( . ), and asthma ( . ). rates of life-threatening complications were also examined in person-years, including acute chest syndrome ( . ), stroke ( . ), splenic sequestration ( . ), pulmonary hypertension ( . ), and pulmonary embolism ( . ). . % of patients were prescribed antibiotics during the one-year post-index period. other frequent medications utilized among children were folic acid ( . %), nonsteroidal anti-inflammatory drugs ( . %), opioids ( . %), and hydroxyurea ( . %). . % of patients had a blood transfusion within one year post-index date. patients had frequent health care utilizations in the inpatient ( visit), emergency room ( visits), office ( visits), and pharmacy ( visits) settings during the one-year follow-up period. pediatric scd patients are burdened with a high rate of complications including pain crisis. in addition, patients utilized a substantial amount of health care resources including outpatient office care and acute care visits. background: novel use of hydroxyurea in an african region with malaria (noharm, nct ) is a randomized controlled trial of hydroxyurea for very young children with sickle cell anemia living in uganda. during year , study participants received blinded study treatment of hydroxyurea or placebo; those receiving hydroxyurea had no increased risk of malaria, but had both laboratory and clinical benefits. during year , all study participants received openlabel hydroxyurea treatment. to assess the effects of open-label hydroxyurea treatment in a very young population of children with sickle s of s cell anemia living in uganda. study endpoints included the rates and severity of malaria infections, clinical sickle-related events, and laboratory effects. design/method: all children in the noharm trial were enrolled at mulago hospital sickle cell clinic in kampala uganda. during year , all children received open-label fixeddose hydroxyurea ( mg/kg/day) for months, after previously receiving either hydroxyurea or placebo for months. results: a total of children entered year of the noharm trial and received fixed-dose hydroxyurea, including males and females, at an average age of . ± . years. among children previously on placebo, there were malaria events in children, including with severity grade ≥ , and three deaths (two acute chest syndrome, one sepsis). clinical adverse event rates dropped from . to . per patient year, and hospitalizations were reduced from to . expected hematological benefits of increased hemoglobin, mcv, and fetal hemoglobin, along with decreased neutrophils and reticulocytes, were rapidly achieved. laboratory adverse events were infrequent at . events per patient-year, and only half of those were dose-limiting hematological toxicities. among children previously on hydroxyurea, there were malaria events in children, including with severity grade ≥ , and two deaths (one acute chest syndrome, one sepsis). clinical adverse event rates and hospitalizations were maintained at low rates, the hematological benefits of hydroxyurea continued throughout the extended treatment period, and dose-limiting toxicities remained infrequent. fixed-dose hydroxyurea treatment of young children with sickle cell anemia living in uganda is associated with no increased risk for malaria. clinical and laboratory benefits occur, including children previously on placebo who crossed-over to hydroxyurea treatment. future studies should focus on the optimal dosing and monitoring strategies, in an effort to determine the overall feasibility and safety of introducing hydroxyurea therapy across sub-saharan africa. background: acute chest syndrome (acs) is the second most common cause of hospitalization in patients with sickle cell disease and is a leading cause of morbidity and mortality. in mid- , an algorithm was implemented at cohen children's medical center to initiate transfusions within four hours of diagnosis of acs in order to improve patient outcomes. objectives: the aim of this project was to analyze the effect of early blood transfusion on the outcomes of patients with acs. we focused on the number of total transfusions, need for exchange transfusion, need for intensive care unit (icu) stay, and length of hospitalization. design/method: a retrospective chart review was completed on patients admitted to ccmc with a primary diagnosis of sickle cell disease and a secondary diagnosis of either acs or pneumonia during the years of - . data from the three years directly prior to implementation of the algorithm was compared to data from the three years directly after implementation of the algorithm. a total of patients were analyzed, of which belonged to the pre-algorithm group and to the postalgorithm group. patients from the post-algorithm group had a higher incidence of transfusions ( % with a mean transfusion number of . pre versus % with a mean of . post) as well as exchange transfusion ( % pre versus % post). the post-algorithm group had a shorter overall length of stay (mean of . days pre versus . days post). while the overall percentage of patients requiring an icu admission was similar in each group ( % pre versus % post), the post-protocol group had a lower likelihood of requiring an icu admission for reasons outside of line placement for exchange transfusion, most commonly for icu-level respiratory support ( % pre versus % post). despite a higher total number of transfusions, early recognition and transfusion for acs can lead to decreased lengths of hospitalization as well as decreased need for icu-level respiratory support. further studies comparing different center's clinical practice guidelines are necessary to improve the standard of care. background: novel use of hydroxyurea in an african region with malaria (noharm) was the first placebocontrolled randomized clinical trial of hydroxyurea in sub-saharan africa. in noharm, young children with sca received either hydroxyurea or placebo during year , followed by open-label hydroxyurea for all study participants during year . an ancillary noharm project was designed to determine if hydroxyurea treatment lowers transcranial doppler (tcd) velocities and possibly reduces stroke risk in this very young cohort. objectives: to perform tcd screening on the noharm cohort, measuring the time-averaged mean velocity (tamv) at the end of both year and year . we hypothesized that the maximum tamv would be lower for noharm study participants receiving hydroxyurea compared to those receiving placebo, and that key clinical and laboratory parameters would also influence tcd velocities. design/method: all children enrolled in noharm were eligible to undergo tcd examination at two study time points: month - when they were completing the blinded treatment phase, and again at month - at the end of the open-label treatment phase. tcd measurements included tamv readings from the main intracranial arteries: middle cerebral artery, distal internal carotid artery, and bifurcation on tcd. all tcd examinations were scored and classified as normal (less than cm/sec), conditional ( - cm/sec) or abnormal (greater than or equal to cm/sec), with higher scores correlating to greater risk of stroke. results: at the end of year , tcd exams were conducted of which were suitable for analysis ( hydroxyurea, placebo). based on the maximum tamv, the median velocity was cm/sec (iqr - ) for children on hydroxyurea and cm/sec (iqr - ) on placebo, p = . . maximum tamv values had negative correlations with hemoglobin concentration (- . ), fetal hemoglobin (- . ), and oxygen saturation (- . ); positive correlations were noted with age ( . ) and absolute neutrophil count ( . ). at the end of year , tcd exams were conducted and all were suitable for analysis; the median velocity was cm/sec on open-label hydroxyurea treatment, regardless of previous blinded treatment. all correlations with tamv were maintained except for age. conclusion: compared to placebo, hydroxyurea treatment for young children with sca living in uganda was associated with lower tcd velocities, which have been correlated in other studies with lower risk of primary stroke. tcd velocities were correlated with hematological and clinical parameters that can be improved by hydroxyurea therapy. children's hospital of richmond at virginia commonwealth university, richmond, virginia, united states background: acute chest syndrome (acs), defined by respiratory symptoms and a new pulmonary infiltrate, is a serious complication of sickle cell disease (scd). acs can occur during hospitalization for non-pulmonary conditions, such as a vaso-occlusive crisis or after surgery. nih clinical practice guidelines encourage incentive spirometry (is) which decreases the incidence of acs. it is additionally widely accepted that early, frequent ambulation in post-operative and pneumonia patients decreases the length of stay (los). to decrease acs events in children with scd at our children's hospital, we aimed for is use in % of ageappropriate pediatric sickle cell admissions. design/method: a multidisciplinary team examined inpatient acs prevention practices, including is, at children's hospital of richmond. key drivers were identified, including educational awareness of patients and healthcare staff, order placement, and documentation. we aimed for all scd patients ≥ months of age hospitalized with any admission diagnosis to participate in is with the use of a traditional incentive spirometer or similar age-and ability-appropriate devices (e.g. positive expiratory pressure devices, bubbles, and pinwheels). we secondarily aimed to increase activity events, specifically ambulation and out of bed time. educational and outreach tools included patient informational brochure and incentive program, and staff informational sessions and reference materials at workstations. a disease-specific order set was implemented including desired is and activity orders. data were collected prospectively may through november , during which pdsa cycles were conducted. admissions during the corresponding months of the previous year were reviewed for comparison. independent t-test analysis was performed using graftpad prism statistical analysis software. results: improvements reaching statistical significance included increase in is order placement from % to % of admissions (p < . ), and admissions with documented is use increased from % to % (p < . ). los decreased from a mean of . days to . days (p . ). post-admission development of acs also decreased from % to % of admissions, but did not reach statistical significance (p . ). there was an additional increase in appropriate activity order placement and documentation of activity events. conclusion: improving education and outreach to patients and staff, including implementation of a disease-specific order set, can improve is use and activity events. the decline seen in incidence of acs development during hospitalization, though not statistically significant, and the decreased los are encouraging, and efforts continue to improve on these trends. background: painful vaso-occlusive crises (voc) are a frequent and debilitating complication of sickle cell disease (scd) and are thought to occur due to progressive blockage of the microvasculature with rigid sickle shaped red blood cells. any trigger that decreases the microvascular blood flow (mbf) can promote entrapment of sickled cells in the microvasculature and progression to voc. exposure to cold wind and changes in weather are common triggers of voc and are associated with increased frequency of hospitalizations for pain in patients with scd. there is limited experimental data on the physiologic effects of these factors on peripheral perfusion in scd. to study the effect of graded thermal stimuli on the peripheral mbf in scd. design/method: scd and control (healthy or sickle trait) subjects aging to years were exposed to their individual threshold temperatures for heat and cold detection, heat and cold pain via tsa-ii thermode that was placed on the thenar eminence. mbf was measured on the contralateral thumb using photo-plethysmography (ppg). the vasoconstriction response within the complex ppg signal was detected using cross-correlation technique. mean mbf was derived from the ppg amplitude during each of these stimuli and compared to baseline mbf. cross correlation analysis showed that cold pain caused significant vasoconstriction response in % of the subjects, followed by heat pain ( %), cold detection ( %) and heat detection ( %).there was a significant drop in the mbf during cold pain (p < . ), heat pain (p < . ), heat detection (p = . ) and cold detection (p = . ) when compared to baseline mbf, with cold pain causing the greatest drop in mbf. thermal sensitivity and mbf responses were comparable between scd and controls. conclusion: exposure to graded thermal stimuli causes a progressive drop in mbf with exposure to cold pain eliciting the strongest vasoconstriction response. vasoconstriction occurred in the contralateral hand at an average of seconds after the stimuli, suggesting a neurally mediated mechanism. although there was no significant difference in vasoconstriction responses between scd and controls, the drop in mbf in patients with sickle cell disease can increase the likelihood of entrapment of the sickled red blood cells, leading to vaso-occlusion. these findings are consistent with extensive reports in literature that exposure to cold weather is associated with a higher frequency of voc. this suggests that neurally mediated vasoconstriction is likely an important factor in the pathophysiology behind cold exposure leading to voc in scd. background: vaso-occlusive crisis (voc) is a major cause of hospital admissions in children with sickle cell disease (scd). although the use of clinical biomarkers in voc has been studied, especially with regards to acute chest syndrome (acs), there is less data regarding overall voc severity prediction. in addition new biomarkers such as platelet to lymphocyte ratio (plr), neutrophil to lymphocyte ratio (nlr), and lymphocyte to monocyte ratio (lmr) have been little studied with regards to scd. objectives: to identify whether admission laboratory values, changes from well baseline laboratory values, and new biomarkers such as plr, nlr, and lmr could predict severity of vaso-occlusive crisis in children with sickle cell disease admitted with voc. design/method: this was a retrospective single center observational study of admissions of voc in children aged - years with hbss or hbs-b thal from september to november excluding those on hyper-transfusion protocol or having an admission diagnosis of acs. univariate analysis was done using student's t-test, mann-whitney non parametric test, or fischer's exact test as appropriate depending on the distribution between admission laboratory data of complete blood count (cbc), reticulocyte count, comprehensive metabolic panel, lactate dehydrogenase (ldh), change from well baseline cbc values within months previously, plr, nlr, lmr, and the development of complicated voc. complicated voc was defined as the development of secondary acute chest syndrome, prolonged admission duration > days ( hours), requirement of blood transfusion, and readmission within days. results: a total of admissions were studied. fifty-nine ( . %) were female. of the , ( . %) were complicated with no significant differences in sex (p . ) or age (p . ). univariate analysis revealed significant elevations in total bilirubin (p . ), ldh (p . ), and platelet count (p . ) in those with complicated voc. there is also significant difference in the percentage change of platelet count from baseline with greater decline in uncomplicated voc (p . ). there were no significant differences in plr (p . ), nlr (p . ), or lmr (p . ). conclusion: elevations in total bilirubin, ldh, and platelet count in admission laboratory values are associated with developing complicated voc. in addition, those with complicated voc present with significantly less decline in platelet count from baseline well cbc. plr, nlr, and lmr do not seem to be useful predictive biomarkers for severity of voc. background: sickle cell disease (scd) causes health problems of varying frequency and severity. the only validated biomarker for children with scd is transcranial doppler. if reliable predictors existed for scd severity, children with scd could be treated according to risk category. many patients with scd face psychosocial or economic hardships, but these factors have not been evaluated as risk markers for medical or functional severity of scd. objectives: the goal of this project was to develop and stratify a preliminary list of psychosocial risk factors for health outcomes that could be used as scd severity predictors. st. vincent institutional review board. a list of potential psychosocial risk factors for adverse health outcomes was compiled based on assessment materials utilized by the sickle safe program (indiana's hemoglobinopathy newborn screening follow-up program). this list of items was distributed to child abuse prevention ( ) and scd ( ) experts, who ranked each item on a likert scale of (least important) to (most important). mean scores were calculated using spss version ; assessments were retrospectively analyzed to determine psychosocial risk factor frequency. risk factors occurring in ≥ % of homes were considered high frequency events. overall, there was high agreement among experts on the risk factors that were considered the most important predictors of severe scd outcomes. the risk factor with the highest frequency ( %) was eligibility for public assistance programs. fifteen risk factors were rated ≥ by the experts. four ( . %) were high frequency events occurring in ≥ % of homes: a child with hbss or hbs thalassemia not taking hydroxyurea ( %); parent report that they had treated a fever (> ®f) at home in the past months ( %); tobacco use by someone in the household ( %); and the family reporting significant psychosocial stressors in the past year ( %). tobacco use in the home was significantly correlated with several other risk factors (smoking during pregnancy [r = . ], other health concerns in the child [r = . ], and child having health insurance [r = - . ]), suggesting that it is part of a constellation of health risk. in general, the risk factors that were rated as most important for health outcomes occurred less frequently in the sample. this study represents important progress toward identifying a group of psychosocial risk factors for scd severity, which is a necessary first step for future investigation of empirical relationships between candidate risk factors and scd outcomes. unitversity of cartagena, cartagena, bolivar, colombia s of s background: sickle cell disease is an autosomal recessive disorder characterized by a mutation in the -globin chain, which produces hbs. acute and chronic complications as aplastic crisis, acute chest syndrome, priapism, stroke, leg ulcers and primary/secondary prevention of stroke can be treated with simple transfusion or exchange transfusion. the latter offers advantages as lower iron overload, post-treatment hbs goal control, lower viscosity and improved microvascular circulation. but it is not a widely-used option because is associated with technical difficulties. objectives: standardization of a new partial exchange transfusion protocol in a group of patients with sickle cell disease, within the framework of a chronic transfusion program. design/method: this is a prospective descriptive study, which included patients under years with sickle cell disease ( hbss, hbs-tal), with indication of partial exchange transfusion in a chronic transfusion program, according to the institutional protocol; patients who fulfilled the inclusion criteria were enrolled in the study between february and december . a registry of the medical and technical complications was made in each of the procedures. a database was constructed in excel, and the graph-pad prism® version oc software was used for statistical analysis. the sequence is as follows: isovolemic phlebotomy and transfusion of packed red cells. depending of the recent hemoglobin level ( hrs), we do the phlebotomy there: hb: - . : cc/kg, hb: - . : cc/kg, hb> : cc/kg; isovolemic solution (ns , %) there: hb: - . : cc/kg, hb: - . : cc/kg, hb> : cc/kg and packed red cell transfusion there: hb: - . : cc/kg, hb: - . : cc/kg, hb> : cc/kg. the safety of this exchange transfusion protocol was analyzed in patients with sickle cell disease ( procedures). there were no differences in the sex distribution, and the median age was years. % of the population was homozygous. the indication of transfusion was . %( / ) primary stroke prevention, . %( / ) secondary stroke prevention and . %( / ) was other reason. a low percentage of complications was found ( . %); of which, those of medical origin (hypotension and nausea/vomiting) were only presented in . % of the total procedures. the standardization of this protocol was safe and its use could be extended to other low-income centers that treat patients with sickle cell disease that need chronic transfusion program including patient with hemoglobin level until gr/dl. we suggest do studies for measure the security and efficacy of this protocol in patients with acute complications. background: clinical trials that aim to achieve pain reduction have challenges achieving clinical endpoints as pain has no quantifiable biomarkers and may be unrelated to scd. furthermore, the threshold of seeking medical care differs between patients and vocs that occur at home are missed. we present a non-interventional, longitudinal study to identify vocs in patients with scd. objectives: to examine the longitudinal relationship between pros and biomarkers in subjects with scd before, during, and after a self-reported voc event, in order to build a model of in-home and clinical voc and to collect longitudinal pros and biomarker data from subjects that span voc events in the home, clinic and the hospital. design/method: longitudinal measures of pain, fatigue, function, activity, and biomarkers from scd patients in steady state and voc were studied over a six month period. patients self-reported pain, fatigue, function, and medication use using a novel epro tool. voc was reported in real-time, triggering a mobile phlebotomy team. blood was collected sequentially after self-reported voc (at home or hospital). blood samples were drawn two days after resolution of voc, as reported by the patient. during non-voc periods, blood was drawn every weeks to establish a baseline. biomarkers included leukocyte-platelet aggregates and circulating microparticles, cell and soluble adhesion molecules, cytokines, inflammatory mediators and coagulation factors. patients wore an actigraphy device to track sleep and activity and rest. results: twenty-seven of thirty-five patients experienced a total of days with voc > hr, of which only days resulted in healthcare utilization. voc days had significantly higher pain and fatigue scores. voc days were associated with significantly decreased functional scores, with significantly greater decreases during vocs requiring medical contact compared to at-home vocs. different activity profiles were identified for non-voc, at-home voc and medical contact voc days by actigraphy monitoring. at-home voc days exhibited increased daytime resting compared to non-voc days. medical contact vocs had decreased average and peak activity, and increased daytime resting compared to non-voc days. a sleep fragmentation index trended up for both at-home ( %) and medical contact voc days ( %). significant changes during voc days were observed in: c-reactive protein ( % increase), nucleated rbc ( % increase), monocyte-platelet aggregates ( % increase) and neutrophil-platelet aggregates ( % increase), interleukin- ( % increase), interleukin- ( % increase) and tnfalpha ( % increase). the identification and assessment of at-home vocs through use of epros, actigraphy and biomarkers is feasible as demonstrated by this innovative at-home study design. background: risk-stratifying sickle cell disease (scd) patients and demonstrating response to disease-modifying therapies is challenging due to the phenotypical heterogeneity of scd. a pathogenic role for procoagulant von willebrand factor (vwf) via excess vwf high molecular weight multimers (hmwm) has been proposed, with variable reports of increased vwf and hmwm in crisis vs. steady-state in adults, but less so for vwf in children with scd. moreover, vwf and multimers have not been studied in sickle trait. objectives: our pilot study evaluated the potential for vwf antigen (vwf:ag) and hmwm on densitometric tracings to serve as biomarkers for disease severity or treatment response in children and young adults with scd compared to sickle trait (hbas) siblings. design/method: we evaluated vwf:ag, vwf multimers and retrospective clinical data from hbss, hbsc and hbas subjects at steady state. one hbsc subject also had a crisis sample. median scd age was years ( . - . years). % were female. scd severity was judged by annual vasoocclusive and acute chest events, or stroke/elevated tcd. eight of ( hbss and hbsc) took hydroxyurea. four hbss subjects had severe scd, all of whom were chronically transfused. results: mean vwf:ag (normal - iu/dl) was higher for hbss ( +/- . ) and severe hbss ( +/- . ) compared to hbsc ( +/- . , p = . and . , respectively); however, lacked statistical significance when compared to hbas ( +/- . , p = . and . , respectively). vwf:ag was elevated in / ( %) steady-state, including / ( %) with "severe" disease on chronic transfusion and / ( %) taking hydroxyurea, in hbsc crisis but no hbsc / ( %) at baseline. vwf:ag was high in / ( %) hbas siblings. four ( %) had increased hmwm at baseline: hbss/severe disease/chronic transfusion, hbss/hydroxyurea and hbsc untreated. hmwm were increased only during vaso-occlusive crisis in hydroxyureatreated hbsc subject. no ultra-large hmwm were observed. in this preliminary study, in young scd subjects, vwf:ag trended higher in hbss vs. hbsc and in severe hbss participants at a single time-point, but serial evaluations at baseline, in crisis and with optimized diseasemodifying therapy are needed to determine the potential of vwf:ag and hmwm as biomarkers for severity or treatment response. surprisingly, vwf:ag was high in some sickle trait subjects. since hbas is associated with some health challenges such as increased thrombosis risk, further examination of vwf and endothelial dysfunction in sickle trait may provide novel insights into its role as a biomarker. background: the national heart lung & blood institute(nhlbi) guidelines for acute management of voe recommends rapid evaluation and treatment of pain, including administration of a parenteral opioid within -minutes of triage or -minutes from registration, pain reassessment & repeat opioid delivery within - -minutes. inf use has been increasing in peds due to its rapid onset and ease of administration. objectives: to evaluate ped utilization of inf & its effect on intravenous (iv) opioid administration and pain control for the treatment of voe. design/method: a retrospective review of emr was performed on children with scd± years presenting to a ped with voe (pain scores on a - scale) from jan-june . variables studied were median time (iqr, %ci) from ped arrival to first-parenteral-opioid-administration, time-to-first-iv-opioid, first & final pain score, disposition and readmission rate. time-to-first-iv-opioid was also compared to historical data (jan-dec ,n = ) prior to inf protocol initiation. . additionally, % patients received iv opioids within minutes of ed arrival in the inf+iv opioid vs. % in the iv opioids alone group (p< . ). no differences in -hour-returnrates were found in any of the groups, including inf alone group. conclusion: use of inf in the ped for voe is an excellent strategy to shorten time-to-first-parenteral-opioidadministration, improve pain scores & improve adherence to the nhlbi guidelines. however we had distinct unexpected findings: ( ) delays in iv opioid delivery after inf use & ( ) inf alone appeared to provide sufficient pain control without iv opioids for disposition home in % of voe patients. whether the latter reflects insufficient pain management or that there is a milder subgroup for whom inf alone is sufficient, requires further investigation. this study illustrates our experience with a ped-based inf protocol in terms of unanticipated delays in iv opioids and also discharges after inf alone. efforts are underway to further improve use of inf in voe management. st. christopher's hospital for children, philadelphia, pennsylvania, united states background: folate supplementation is commonly included as standard management in patients with sickle cell disease. however, clear evidence supporting the clinical benefits of this practice is lacking. a single study demonstrated improvement on the occurrence of repeat dactylitis at a higher dose of folic acid. to compare clinical outcomes in pediatric patients with sickle cell disease treated with folate supplementation versus those who were not. design/method: this study was a retrospective chart review that included patients to years old with sickle cell disease type ss and s followed at st. christopher's hospital for children. data collected included information about folate supplementation, red cell indices and the presence or absence of clinical outcomes including vaso-occlusive crisis requiring hospitalization in the last six months, acute chest syndrome, infections, asthma, sleep apnea, nephropathy, cerebral vascular disease, stroke and avascular necrosis. analysis of variance (anova) was used to evaluate mean differences between age, number of infections, number of voc events, hemoglobin, reticulocyte count, and mean corpuscular volumes. additionally, chi square analysis was implemented to evaluate differences in folate and non-folate groups for left ventricular remodeling (lvr), sickle cell nephropathy, asthma, obstructive sleep apnea (osa), nocturnal hypoxia, and avascular necrosis (avn). mean differences between the folate and non-folate groups were compared for patients on and off hydroxyurea therapy. one hundred and seven patients met inclusion criteria following review of clinical data. of the patients included in the study, patients were found to be taking folate ( %), while patients were not ( %). statistical analysis showed that there were no significant differences in the incidence of clinical outcomes between patients on folate versus those who were not on folate. of the patients who were not on hydroxyurea, hemoglobin levels were significantly higher in patients on folate versus those who were not (p = . ), but not significantly different for the patients on hydroxyurea. this study suggests that folate supplementation makes no significant impact on the red blood cell indices of anemia nor on the incidence of adverse clinical outcomes in children with sickle cell disease. however, a larger prospective study is needed to guide future considerations for folate supplementation in sickle cell patients in the clinical setting. background: tanzania ranks rd globally for the number of infants born annually with sickle cell disease (scd) but lacks a national newborn screening program. the prevalence of sickle cell trait (sct) and scd is highest in the northwestern regions around lake victoria served by bugando medical centre (bmc) a teaching and consultancy hospital in mwanza. bmc also houses the hiv early infant diagnosis (eid) laboratory that tests dried blood spots (dbs) from hivexposed infants. dbs can be tested for hiv and then retested for sickle cell trait and disease. to determine the prevalence of sickle trait and disease by region and district in northwestern tanzania using existing public health infrastructure. secondary objectives explored associations between sct, scd, malaria and hiv. design/method: the tanzania sickle surveillance study (ts ) is a prospective year-long cross-sectional study of hivexposed infants born in northwestern tanzania, whose dbs collected by the eid program are tested at bmc and available for further testing of sct and scd. samples from children ≤ months of age were tested by isoelectric focusing (ief) and scored independently by two tanzanian staff as normal, sct, scd, variant, or uninterpretable. dbs samples scored as disease or variant were repeated. over the course of months, ief gels have been run. a total of , dbs samples have been scored, including , from children less than -months old. the overall prevalence of sct is . % and the prevalence of scd is . %, along with . % hemoglobin variants. quality of the laboratory results is extremely high, with only . % dbs samples yielding an uninterpretable result. geospatial mapping of the first , samples revealed a regional scd prevalence ranging from . % up to . % among the regions served by bmc. the prevalence of sct and scd is very high in northwestern tanzania. geospatial mapping will identify high prevalence areas where targeted newborn screening can be started using existing public health infrastructure with minimal start-up cost and training. further data will enhance the accuracy of the map to the district level. background: pediatric patients with sickle cell disease (scd) could develop obstructive, restrictive or mixed abnormalities of pulmonary function (pf). several publications report progressive worsening of pf over time, which could lead to severe morbidity in adult patients with sickle cell disease. in adults with sickle cell anemia up to - % of mortality is related to lung disease. early intervention aimed at improvement of lung function could significantly decrease morbidity and possibly improve life expectancy. among disease modifying approaches commonly used in scd are hydroxyurea (hu) and chronic prbc transfusions. both interventions lead to increase of hemoglobin, decrease of hbs fraction, leading to decreased hemolysis. reports of effect of hu on pulmonary function are conflicting with some suggesting no effect and others proposing a slower decline of pulmonary function. the goal of our study is to evaluate effect of disease modifying therapies, like hu and chronic prbc on change of pulmonary function in pediatric patients with sickle cell disease. design/method: this study utilized a retrospective chart review of children with scd who had multiple pfts. we analyzed pfts from patients done during clinic visits. scd patients were divided into three treatment groups: hydroxyurea, chronic transfusions or neither. data was analyzed with linear correlations and analysis of variance (anova). comparison were made between the three groups specifically observing the changes in absolute numbers on pfts over time using the first and last pft the patient had. results: there were a total of patients with multiple pfts (ranging from - ); control ( ), hydroxyurea ( ) and chronic transfusion ( ). the mean changes of the control, and hydroxyurea for the pft parameters fev (- . the chronic transfusion group demonstrated a small improvement in pfts over time for fev ( . ), fvc ( . ), fef - ( . ), however there was a decline in fev /fvc (- . ). however, there was no statistically significant (p-value < . ) in the difference in any pfts parameters between any of the groups. in children with scd there is a decline of pf parameters over time. although no significant differences were seen between the three groups it appears chronic transfusion may improve or limit the decline in pfts. larger studies need to be done to evaluate difference in pf decline in patients with scd patients. background: the use of mobile technology in health care has been a growing trend. patients with chronic diseases such as sickle cell disease (scd) require close monitoring to provide appropriate treatment recommendations and avoid complications. we conducted a feasibility study for patients with scd hospitalized for pain using our self-developed mobile application (tru-pain: technology resources to better understand pain) and a wearable activity tracker. subjective symptoms such as pain and objective data such as heart rate (hr) were measured. we aimed to ) correlate nursing recordings with mobile technology recordings; ) get feedback from patients about usability. design/method: we enrolled patients with scd > years old and < hours from admission for uncomplicated vasoocclusive crisis, excluding patients admitted to icu. patients were given an ipad and a wearable device. they were instructed to record in the application at least once per day and to keep the wearable on, removing only to charge. prior to discharge, patients completed a feasibility questionnaire. we enrolled patients, % females, median age . (range to ) who were admitted for a median days (range to ) for uncomplicated pain crisis. patients used the application throughout hospitalization and made one entry/day (range to ). pain scores recorded via tru-pain correlated well (r = . , p< . ) with pain scores recorded in emr. there was an average of , data points recorded per day, by the wearable, with a maximum of , data points/day. the median amount of hours of wearable data per day was . (maximum of . ). the hr recorded via the wearable correlated significantly with the hr recorded in emr (r = . , p-value < . ). as for usability, % of patients indicated never having a problem with the technology, % found tru-pain 'very easy' or 'somewhat easy' to use, and % were 'very satisfied' with their participation in the study, indicating that it helped them track their pain. our pilot study during hospitalization shows strong potential for using tru-pain for patients with scd. pain data from application and hr from wearable correlated well to the emr data. according to the feedback received, our application was easy to use and helped patients track their pain. despite limitations of battery life, the use of wearable technology is feasible, providing additional data such as activity. we are optimistic that we can continue to improve our tru-pain system to help improve care in patients with scd. background: hydroxyurea, chronic blood transfusion, and bone marrow transplantation can reduce complications, and improve survival in sickle cell disease (scd), but are associated with a significant decisional dilemma because of the inherent risk-benefit tradeoffs, and the lack of comparative studies. these treatments are underutilized leading to avoidable morbidity and premature mortality. there is a need for tools to provide patients high-quality information about their treatment options, the associated risks, and benefits, help them clarify their values, and allow them to share in the process of informed medical decision making. objectives: to develop a health literacy sensitive, web-based, decision aid (ptda) to help patients with scd make informed choices about treatments, and to estimate in a randomized clinical trial the acceptability and effectiveness of the ptda in improving patient knowledge, involvement in decisionmaking and decision-making quality. design/method: we conducted qualitative interviews of scd patients, caregivers, stakeholders, and healthcare providers for a decisional needs assessment to identify decisional conflict, knowledge, expectations, values, support, resources, decision types, timing, stages, and learning, and personal clinical characteristics, and to guide the development of a ptda. transcripts were coded using qsr nvivo . stakeholders completed alpha and beta testing of ptda. we conducted a randomized clinical trial of adults, and of caregivers of pediatric patients to evaluate the comparative efficacy of the ptda, vs. standard of care. results: ptda (www.sickleoptions.org) was developed per decisional needs described by stakeholders and finalized following alpha testing, and beta testing by and stakeholders respectively. in a randomized trial of subjects considering various treatment options, qualitative interviews revealed a high level of usability, acceptability, and utility in education, values clarification, and preparedness for decision making of the ptda. a median % rated the acceptability of ptda as good or excellent and provided narrative comments endorsing the acceptability, ease of use, and utility in preparation for decision making. the ptda met international standards for content, development process, and efficacy with the exception of having a full range of positive and negative experiences in patient stories. compared to baseline ptda group had statistically significant improvement in preparedness for decision making (p = . ) and informed subscale of decisional conflict (p = . ) but not for decisional self-efficacy, knowledge, choice predisposition, or stages of decision-making. a ptda for patients with scd developed following extensive engagement of key stakeholders was found to be acceptable, useful, easy to use, to improve preparedness for decision making, and decrease decisional conflict. background: painful vaso-occlusive crisis (voc) accounts for the majority of emergency department (ed) visits and hos-pitalizations in sickle cell disease (scd). we are interested in studying mental stress and associated autonomic nervous system (ans) imbalance that cause vaso-constriction as possible triggers of scd pain. to this end, we developed a mobile phone application (app) to record daily pain frequency and intensity as clinical endpoints that might be predicted by ans parameters measured in the laboratory. in particular, we think that the aura may represent ans instability that precedes or even triggers change in blood flow and voc. objectives: to assess the feasibility of using an app to evaluate frequency and severity of voc and its potential association with mental stress and presence of aura. design/method: an app was developed for both ios and android systems to allow patients to track pain, stress, and aura. the idea was to create an app that was easy to use with the intent to only capture pain episodes, rather than detailed description of the pain. all scd patients were eligible and a parent version was available for younger children. de-identified data was automatically transferred to a hipaa compliant database via a cloud-based server interfaced to the main research project database. a feedback questionnaire was implemented after at least a month of utilization to assess usability. of the scd patients enrolled, participants utilized the app and of the participants that provided feedback indicated the app was easy to navigate. the mean pain scale was out of (standard deviation . ) for those that entered they had pain that day. although the mean stress level was out of , there was a statistically significant correlation between increasing stress levels and increasing pain scores (p < . ). aura was reported by patients, with patients reporting more than episodes. moreover, on days aura was present there was greater incidence that pain was present as well (p < . ). however, there was no statistically significant association between pain intensity and presence of an aura (p = . ). conclusion: consistent with prior research, reported pain intensity is significantly associated with reported stress intensity. although there was an association between presence of aura and pain, it did not seem to correlate with pain intensity. this uniquely designed app can monitor scd pain clinically and help understand the role of sickle dysautonomia in the genesis of scd pain. university of florida college of medicine, gainesville, florida, united states background: evidenced-based guidelines recommend the emergent evaluation of fever in children with sickle cell disease (scd). as the prevalence of bacteremia has decreased, outpatient management has become more common. however, fever can sometimes herald other complications of scd, such as acute chest syndrome, vaso-occlusive pain crisis, splenic sequestration, or aplastic crisis. institutional practices regarding fever management in scd remain variable, and little is known about the clinical outcomes of children hospitalized for uncomplicated fever. objectives: the primary objective was to determine the rate of bacteremia or scd-related complications per febrile episode in children with scd admitted to a single institution between january and june for uncomplicated fever. this was a retrospective cohort study of febrile patients up to years of age with scd, any genotype, admitted to the university of florida during the defined study period. eligible patients were identified by a database search using admitting diagnosis codes for scd and fever based on the international classification of diseases th and th revisions. encounters were manually reviewed to confirm eligibility. patients were excluded if they had other indications for hospitalization apparent at the time of admission, such as an acute vaso-occlusive episode requiring parental narcotics, asthma exacerbation, or additional complications of scd. the database search identified encounters, of which were excluded based on confounding indications for hospitalization. sixty-three eligible patients accounted for hospitalizations. the median age was years (range weeks- years); . % were male. mean duration of hospitalization was . days (range - days). eight positive blood cultures were identified; six of these were classified as contaminants. bacteremia or the development of a scd-related complication was identified in ( . %) admissions. these included acute chest syndrome (n = ), bacteremia (n = ), splenic sequestration (n = ), and red cell transfusion (n = ). exploratory analyses of potential predictors of bacteremia or scd-related complications showed no association with the presenting white blood cell count or degree of fever (p = . ). of the patients classified as having a scd-related complication, % had hemoglobin ss disease and % had at least one prior documented complication. % of the patients transfused had at least one prior transfusion. conclusion: while improvements in preventative care have substantially lowered rates of bacteremia in children with scd, fever warrants careful evaluation for other acute scdrelated complications. providers should consider inpatient observation in select cases. additional studies are warranted to define subsets of patients suitable for outpatient fever management. background: children with sickle cell disease (scd) exhibit lower neurocognitive functioning than healthy peers, even in the absence of stroke. among the domains commonly affected, working memory (wm) seems particularly affected by disease processes and wm deficits have significant implications for academic achievement and disease selfmanagement. few interventions to improve working memory in pediatric scd have been evaluated. to determine the effects of cogmed, a homebased computerized wm training intervention, in children with scd using a randomized controlled trial design. design/method: participants (ages - ) with scd completed a baseline neuropsychological assessment and those with wm deficits were randomized to either begin cogmed immediately or enter an -week waitlist. cogmed is a homebased intervention completed on an ipad that consists of increasingly challenging exercises targeting visual-spatial and verbal wm, practiced over sessions. at the end of training, participants completed a post-intervention neuropsychological assessment, including tests of visual-spatial and verbal wm from the wechsler intelligence scale for children-fifth edition (wisc-v). results: ninety-one participants (m age = . , sd = . ; % female; % hbss) enrolled in the study; % (n = ) exhibited wm deficits and were randomized to either begin cogmed immediately or wait - weeks before starting cogmed. among those that have received the intervention and reached the end of their training period (n = ), participants ( %) completed at least cogmed sessions, ( %) finished at least sessions, and finished at least sessions ( %). the mean number of completed cogmed sessions was . (sd = . ). paired samples t-tests revealed significant improvements on the working memory index (t[ ] = - . , p = . ) and on the digit span (t[ ] = - . , p = . ), and spatial span-backward (t[ ] = - . , p = . ) subtests. improvements were especially pronounced for participants completing at least sessions. partial correlations controlling for respective baseline scores indicated that the number of cogmed sessions completed was positively correlated with post-test scores on digit span (r = . , p = . ) and spatial span-backward (r = . , p = . ) subtests. among participants who completed at least cogmed sessions, % scored in the average range or higher on the working memory index at the post-intervention assessment, compared to % at baseline. results support the efficacy of cogmed in producing significant improvements in wm. a dose-effect was observed such that participants who completed more cogmed sessions had greater improvements in wm. home-based cognitive training programs may ameliorate scd-related wm deficits but methods for motivating and supporting patients as they complete home-based interventions are needed to enhance adherence and effectiveness. background: sickle cell disease is associated with myriad complications that lead to significant morbidity and early mortality. hydroxyurea has been used successfully to reduce the incidence of these complications and has led to significant improvements in quality and duration of life. at children's minnesota we recommend hydroxyurea in all patients with hb ss/s thalassemia as early as months of age with a goal of starting all patients before months of age. objectives: the purpose of this study was to evaluate the use of hydroxyurea therapy in young patients with sickle cell disease, with particular attention to those children less than one year of age. design/method: a retrospective chart review was conducted on patients less than years of age with sickle cell disease who began hydroxyurea therapy between january , and december , . the study population was divided into three cohorts based upon age at hydroxyurea initiation: cohort ( - year), cohort ( - years), and cohort ( - years). outcomes included laboratory data, clinical events (hospitalization, dactylitis, pain crisis, transfusion, splenic sequestration, acute chest syndrome), and toxicity occurring in the first years of life. results: a total of patients were included in cohorts (n = , mean age . months), (n = , mean age . months), and (n = , mean age . months). patients in cohort had higher hemoglobin (p = . ) and mcv (p = . ) and lower absolute reticulocyte count (p = . ) when compared to cohort . the wbc (p = . , < . ) and anc (p = . , . ) were significantly lower compared to both older cohorts. however, no patient had therapy held because of neutropenia. the mean baseline hemoglobin f in cohort was . % compared to . % and . % in cohorts and respectively (p = . , p< . ). the mean duration of therapy in cohort was . months, compared to . months in cohort (p = . ) and . months in cohort (p = . ). during this time, hb f levels remained higher in cohort (mean . %) compared to cohorts and (mean . %, p = . and mean . %, p = . ). patients in cohort experienced fewer hospitalizations (p = . ), pain crises (p = . ), and transfusions (p = . ). there was no difference in toxicity between groups. hydroxyurea was used safely in infants to months of age and resulted in more robust hematologic responses and a decrease in sickle-related complications when compared with patients starting hydroxyurea later in life. children's national health system, washington, district of columbia, united states background: children with sickle cell disease (scd) have a significantly greater risk of silent or overt cerebral infarction than the general population. infarcts are associated with declines in cognitive functioning and academic achievement. while infarcts are reliably identified using mri, scans are expensive and occasionally necessitate sedation. moreover, mri's are not recommended for routine monitoring of cerebral infarcts. additional tools are needed for discriminating the presence of a cerebral infarct that are brief, noninvasive, inexpensive, and repeatable. objectives: to evaluate differences in performance on cogstate, a computerized neurocognitive assessment, in patients with scd with and without history of cerebral infarct. design/method: participants included children with scd ages - (m = . , sd = . ; % female; % s of s hbss) enrolled in a cognitive intervention trial. participants completed the cogstate pediatric battery, which measures processing speed, sustained attention, verbal learning, working memory, and executive functioning. history of silent or overt infarct was determined via health record review. participants also completed measures of intelligence (iq) and math fluency. results: participants' standard scores across most neurocognitive measures were lower than expected compared to the standardization sample (mean iq = . , sd = . ). thirty percent of participants (n = ) had a documented history of cerebral infarct. participants with a history of cerebral infarct scored lower on cogstate tasks measuring sustained attention (t[ ] = . , p = . ) and executive functioning (t[ ] = . , p = . ), as well as on a measure of math fluency (t[ ] = . , p = . ). receiver operating characteristic (roc) analyses demonstrated that the cogstate task measuring sustained attention was a fair discriminant of patients with and without a history of infarct (auc = . , ci = . - . , p = . ), whereas iq score was not (auc = . , ci = . - . , p = . ). cogstate processing speed and sustained attention tasks fairly discriminated between patients with at least average or below average intelligence (auc = . , ci = . - . , p = . and auc = . , ci = . - . , p = . , respectively). finally, the cogstate processing speed task was good at discriminating between at least average or below average math fluency (auc = . , ci = . - . , p< . ). multiple tasks in the cogstate pediatric battery appear to adequately identify patients with a history of cerebral infarcts. in addition, cogstate tasks appear to be fair predictors of impairments in iq and academic achievement outcomes. cogstate is inexpensive and can be easily administered in a medical setting with minimal training in approximately minutes. results support the potential for cogstate to be used as a screening tool for medical and neuropsychological abnormalities in children with scd. st. christopher's hospital for children, philadelphia, pennsylvania, united states background: cardiovascular disease contributes to the morbidity and mortality of patients with sickle cell disease (scd). hydroxyurea therapy in scd has known clinical efficacy including improving anemia, decreasing episodes of vasoocclusive crisis and acute chest syndrome, and decreasing mortality. effect of hydroxyurea on cardiac function in children with scd is not well studied. an earlier study suggested the protective effect of hydroxyurea on left ventricular (lv) hypertrophy in scd. we hypothesized that hydroxyurea use would be associated with decreased lv remodeling and improved cardiac function. we aimed to evaluate the association between hydroxyurea use and lv remodeling and cardiac dysfunction in children with scd. design/method: we completed a retrospective study of patients with scd who were to years old, followed at st. christopher's hospital for children and had an echocardiogram completed in the past months. data collected included gender, bmi, scd genotype, hydroxyurea use, chronic transfusion use, and d and doppler echocardiographic parameters. cardiac structure, geometry, systolic function, and diastolic function echocardiogram parameters were included. analysis of variance (anova) tests were performed to assess for statistical significance of differences in cardiac parameters between patients with and without hydroxyurea use. analysis of covariance (ancova) tests were performed to control for age. results: demographic and echocardiogram data was collected on all patients who met inclusion criteria. of the patients included, ( %) were on hydroxyurea therapy. patients on hydroxyurea had significantly lower mean relative wall thickness (p = . ) and significantly higher mean peak early lv filling velocities (p = . ) and peak early lv filling/septal annuli early peak (e/ea) velocities (p = . ); however, only the e/ea velocities remained significant when controlling for age (p = . ). mean peak early lv filling velocities approached significance when controlling for age (p = . ). hydroxyurea therapy resulted in a significantly higher e/ea velocity, suggesting that these patients had worse diastolic function. it is possible that the patients initiated on hydroxyurea already had worse disease manifestations than those not on hydroxyurea, possibly accounting for the decreased diastolic function. when controlling for age, hydroxyurea use did not result in significant differences in cardiac structure parameters, systolic function parameters or cardiac geometry. prospective studies and larger sample size are needed to validate our findings, examine for additional statistically significant differences, and develop preventive strategies for cardiovascular disease in children with scd. background: acute chest syndrome (acs) is now the leading cause of death in children with sickle cell disease; mortality in the u.s. is reported to be - % and is mostly due to respiratory failure. early transfusion improves clinical outcomes. although patients with concurrent asthma are considered at increased risk for poor outcomes, risk factors for respiratory failure in pediatric acs have not been well-defined. to determine whether specific epidemiological and clinical features of children hospitalized with acs are predictive of the need for mechanical ventilation. design/method: data from the kids' inpatient database were reviewed to identify patients age < years with a discharge diagnosis of acs for the years , , , and . outcomes were defined by the international classification of diseases, ninth revision, clinical modification code. data were weighted to estimate total annual hospitalizations according to hospital characteristics in the united states. trends in healthcare costs, length of hospital stay, transfusion, and mechanical ventilation use were analyzed using multivariable linear regression. in addition, multivariable logistic regression was used to ascertain specific clinical or epidemiologic factors associated with mechanical ventilation use after adjusting for patient and hospital characteristics. the total hospitalizations for acs were , in ; , in ; , in ; and , in . reported use of mechanical ventilation ranged from . % to . % and was associated with non-black compared to black children (or, . ; %ci, . to . ) and the fall season (or, . ; %ci, . to . ), but not with age, preexisting asthma or hb-genotype. comorbidities of obesity (or, . ; %ci, . to . ), obstructive sleep apnea (or, . ; %ci, . to . ) and heart disease (or, . ; %ci, . to . ) were associated with mechanical ventilation use. the use of simple and exchange transfusion during all acs admissions ranged from . % to . % and . % to . %, respectively. among pediatric acs patients, those with obesity, obstructive sleep apnea or heart disease were at increased risk for respiratory failure and might benefit from early intervention (e.g., transfusion). surprisingly, asthma in children with acs does not appear to be a distinct risk factor for respiratory failure, and further studies are needed to clarify whether differences in treatment approach (e.g., addition of corticosteroids, bronchodilators) might impact on acs progression and/or severity even in high risk patients without asthma. objectives: to compare pulmonary functions between aa and k children with scd and to assess if a high hb f level contributes to better function. design/method: a cross sectional study was done on children with scd (hb ss disease) followed in comprehensive sickle cell programs. aa patients were followed at brookdale hospital, ny and k patients were followed in mubarak hospital, kuwait. children between the ages of and years who had pulmonary function tests (pft) done as a routine screening were enrolled. pft was done using spirometer and plethysmography. patients with congenital or anatomical lung abnormality, heart disease, pulmonary disease such as acute chest syndrome, acute asthma or pneumonia within weeks were excluded. results: there were children ( in each group) with scd,. restrictive pattern on pft was seen in / ( %) of aa vs. / ( %) of k (p> . ). obstructive pattern was seen in / ( %) of aa vs. / ( %) of the k group (p> . ). in both groups, children ( %) had normal pft. three/ ( %) in the aa group had a hb f> % as compared to / ( %) in the k group (p< . ). abnormal pft was noted in / children ( %) in each group. hbf was > % in / ( %) in the aa group vs. / ( %) in the s of s k group (p< . ). in patients with abnormal pft, mean hbf was . ± . in aa group, compared to . ± in k group (p< . ). conclusion: abnormal pft is highly prevalent among children with scd in both groups. aa children are more likely to have restrictive disease and k to have an obstructive pattern. level of hbf did not seem to protect k patients from abnormalities on pft. this finding should emphasize the importance of performing pft as part of the initial evaluation of all children with scd. background: sickle cell disease (scd) is a life-threatening disease with varied clinical spectrum and severity leading to premature death. there is a lack of validated prognostic marker in scd. recent evidence suggests that inflammation and platelet adhesion plays a critical role in the pathophysiology of vaso-occlusion in scd. elevated mean platelet volume(mpv) values are associated with a higher degree of inflammation in many disease states but it's effect on sickle cell disease or it's severity is unknown. objectives: to analyze the role of mpv in predicting disease severity/mortality in pediatric patients with scd. design/method: this is a single center retrospective study and included patients with sickle cell disease between months and years of age during a -year period ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . demographic information, lab data and clinical information including acute chest syndrome (acs), priapism, transfusions, sepsis, pain crisis, avascular necrosis were collected. all laboratory data were collected in steady state with no crisis in the recent past months. the disease severity score/probability of death was calculated using a validated model to predict risk of death in sickle cell disease (sebastiani et al. blood ) . pearson test was used to analyze correlation between mpv and probability of death. results: total no. of patients = ; male ( . %); female ( . %). median age is . years. all patients were of african-american origin. disease severity, hb ss - ( %); hb sc - ( . %) and sickle-beta thalassemia ( . %). patients on hydroxyurea has significantly lower mpv, p = . and this is independent of hb f levels. mpv has a significant positive correlation with the probability of death, p = . and correlation coefficient, r = . . on subgroup analysis, the correlation is even more significant in the age group between and years, p = . , r = . . using linear regression model, with probability of death as a dependent variable and hydroxyurea, mpv as independent variables, mpv maintains a significant association with probability of death (p = . ). conclusion: mpv is an independent biomarker predicting disease severity and probability of death in pediatric patients with sickle cell disease. hydroxyurea a known disease ameliorating agent is associated with lower mpv values. this effect is independent of the levels of fetal hemoglobin and may be due to anti-inflammatory effect of hydroxyurea or effect on the platelets. background: major success with initial qi projects by the sickle cell care team at children's hospital has precipitated ongoing inclusion of the qi approach to many other aspects of patient care. objectives: to optimize scd patient care utilizing qi processes. design/method: success of the scd qi team's initial project on transcranial doppler studies (tcds) and a second more complex project on hydroxyurea (hu) adherence, led to additional projects on completion of key immunizations, rbc phenotyping, and vitamin d level testing. using similar processes and principles from the hu adherence project, plan-do-study-act (pdsa) cycles were used to conduct smallscale tests of change. patient chart prep sheets, created for bi-monthly pre-appointment chart prep meetings, were significantly modified to include these focused care qi objectives. because of difficulty with emr database capability, data collected from the emr was tracked in excel spreadsheets or other unique tracking vehicles for the various parameters. for example, due to the clinic's diffuse, geographically scattered population, many separate non-shared primary care emrs, and lack of a mandatory state immunization registry; immunization records needed to be retrieved from pcps, outlying hospitals, public health departments, and fqhcs, and added to the emr and excel database. starting in / , all such data was collected and updated monthly. in one year's time ( - ) , the average immunization completion rate for seven key immunizations (pcv , pcv , hepatitis a, hepatitis b, meningococcal a, meningococcal b, and hpv) has increased by %. the biggest improvements were a % and % increase in completion for meningococcal a and meningococcal b, respectively. completion rate for rbc phenotyping rose from . % to . %. patients with at least one vitamin d lab test increased from . % to . %. since starting the tcd project in , the percent of patients who have completed their annual tcd has gone from a baseline of % to a sustained value of > %. conclusion: these qi projects have not only increased adherence to national recommendations for care of scd patients, they have helped establish a scd clinic methodology to create and implement sustainable processes. having the focused care initiatives prominently displayed on the patients' chart prep sheet serve as a reminder to medical team members to check the status of that item. this methodology is currently being used to formulate additional qi projects on annual renal function parameters and specialty visits, such as annual eye and dental exams. background: dominican republic has a high burden of sickle cell disease, and - % of children with homozygous hbss (sickle cell anemia, sca) will develop primary stroke. transcranial doppler (tcd) ultrasonography is an effective screening tool for primary stroke risk, but is not routinely available in dominican republic. hydroxyurea and blood transfusions are available, but no prospective screening and treatment program for stroke prevention has been implemented to date. ( ) to screen a large cohort of children with sca living in dominican republic, using tcd to identify elevated stroke risk; ( ) to determine the effects of treatments for stroke prevention (hydroxyurea for conditional velocities and transfusions for abnormal velocities). we hypothesized that both hydroxyurea and blood transfusions will decrease elevated tcd velocities and help prevent primary stroke. design/method: stroke avoidance for children with república dominicana (sacred, nct ) features a research partnership between cincinnati children's hospital and robert reid cabral children's hospital in dominican republic. the protocol, consent forms, and redcap database were prepared collaboratively and translated into spanish, and then irb approval was obtained at both institutions. in the initial prospective phase, children receive tcd screening over a -month period; those with conditional tcd velocities (maximum time-averaged velocity - cm/sec) receive fixed-dose hydroxyurea at mg/kg/day, followed by dose escalation to maximum tolerated dose, while those with abnormal tcd velocities (≥ cm/sec) receive monthly transfusions for stroke prevention. results: a total of children were enrolled in sacred, with an average age of . ± . years. initial tcd screening revealed ( . %) normal, ( . %) conditional, ( . %) abnormal, and ( . %) inadequate velocities. among children ( males, females, average age . ± . years) who initiated hydroxyurea at mg/kg/day for conditional tcd velocities, completed six months of treatment with expected hematological benefits including significant increases in hemoglobin concentration ( . to . g/dl) and fetal hemoglobin ( . to . %). no clinical strokes have occurred in the treatment group. repeat tcd examination after -months of hydroxyurea treatment revealed % ( / ) with previous conditional velocities had normal tcd velocities. the prevalence of conditional tcd velocities in the dominican republic is high, indicating an elevated stroke risk among children with sca. hydroxyurea treatment is associated with improved hematological parameters, lower tcd velocities, and probable decreased stroke risk. sacred is an important prospective and collaborative research trial providing epidemiological data regarding tcd screening, stroke risk, and hydroxyurea effects among children with sca. background: red blood cell aggregation is a rheologic property that explains the shear-thinning behavior of blood. at lower shear rate blood flow, red cells tend to aggregate, s of s whereas in higher shear rate blood flow, these aggregates are dispersed. this property is especially important in the venous system, where low shear rate blood flow predominates. there is inconsistent data in the literature concerning aggregation and aggregability in sickle cell disease (scd). objectives: because the lorrca and myrenne instruments have been shown to be similarly effective methodologies in red cell aggregation measurements, we aimed to determine whether the measurement of aggregation indices in scd, by myrenne and by lorrca, is consistent in our lab. design/method: we measured aggregation in blood samples corrected to % hematocrit. aggregability was measured using kda dextran in the myrenne but not the lor-rca. aggregation index using lorrca was measured in patients with scd and healthy subjects enrolled in a study of blood flow between and . aggregation and aggregability using the myrenne was measured in patients with scd and healthy subjects enrolled in a separate study of blood flow between and . results: using lorrca, we found that aggregation index in patients with scd was less than that of healthy subjects (p< . ). in the myrenne, aggregation at stasis was slightly higher in patients with scd compared to healthy subjects (p = . ) but aggregation at low shear rotation was not different. aggregability was higher in the patients with scd compared to healthy subjects at both stasis and low shear rotation (p< . ). red cell aggregation is an important determinant of low shear blood flow. deoxygenated venous blood is particularly important to low shear blood flow in patients with sickle cell disease. we found that two different aggregometers predict different aggregation results for scd. it is unclear why there is a systematic difference between the two methods, but there are some possibilities. first, the syllectogram in the lorrca is generated by the backscatter of light from the laser, while the myrenne measures transmitted light. second, the distance between the bob and cup in the lorrca is microns, while the gap between plates in the myrenne is microns, which might affect the disaggregation of red cells. further work is needed to understand the differences in red cell aggregation and aggregability when using these instruments, particularly when using aggregation as a predictor of blood flow and tissue perfusion. background: children with sickle cell disease (scd) are at risk of acute splenic sequestration crisis (assc). assc is a life-threatening complication characterized by splenomegaly, pain and severe anemia. assc most often occurs in young children with the most severe forms of scd and one-third of patients will have more than one episode. treatment is based primarily on expert opinion and includes blood transfusion and surgical splenectomy. objectives: we plan to assess the clinical practice patterns of physicians treating children with assc. design/method: a survey study was performed. the survey included six scenarios of severe scd with variation in age, hydroxyurea-use, and episode number of assc; questions focused on the acute and chronic management of assc. the survey was disseminated on three occasions over a six-month period, using an online survey tool, surveymonkey, to pediatric hematologist-oncologists participating in the american society of pediatric hematology-oncology hemoglobinopathy special interest group. the survey had a response rate of % ( / ). most respondents were recent graduates ( %; / ) practicing in academic urban centers with greater than sickle-cell patients. seventy-nine percent ( / ) recommended hydroxyurea initiation in - m/o with severe scd. prophylactic penicillin after surgical splenectomy was continued by % ( / ) after years. for the acute management of assc results did not vary despite patient age, hydroxyurea use, and the number of previous assc episodes. simple transfusion was preferred by % ( / ), with % ( / ) recommending slow transfusion and % ( / ) recommending routine simple transfusion. for the chronic management of assc, results varied based on patient age and the number of previous assc episodes. for a m/o after the first episode, % ( / ) recommended observation and % ( / ) hydroxyurea initiation. for a m/o with any prior episode of assc, % ( / ) recommended chronic transfusion therapy and % ( / ) surgical referral for splenectomy. for a y/o after the first episode, % ( / ) recommended surgical splenectomy and % ( / ) increasing hydroxyurea dose. for a y/o with any prior assc episode, % ( / ) recommended referral for surgical splenectomy. in this survey, we found most providers continue to recommend simple transfusions for assc and surgical splenectomy after two episodes. the majority of providers continue to delay referral for surgical splenectomy until age two, but earlier referral in children under two and use of chronic transfusion therapy were also reported. variability in chronic management highlights the need for further research of splenic sequestration. background: developing therapies for sickle cell disease (scd) is challenging in part because the accepted endpoint, vaso-occlusive crisis (voc), occurs infrequently, does not measure full disease burden, and is a measure of healthcare utilization. in phase / studies of patients with scd, voxelotor (gbt ) has demonstrated increased hemoglobin (hb) levels and reduced hemolysis and has been safe and welltolerated. voxelotor is being evaluated in the ongoing hope phase trial. objectives: to report the innovative phase / hope trial design with novel primary and secondary outcomes to accelerate drug development. design/method: hope (nct ) is a phase , randomized, placebo-controlled, multicenter study of oral voxelotor in patients with scd (aged - years) with baseline hb . - . g/dl and - episodes of voc in the prior year. to accelerate clinical trials to support drug development, the study combines a phase exploratory, dose-selection phase (group ) with a pivotal phase (groups / ). patients in group will be randomized : : to voxelotor or mg/day or placebo. analysis for dose selection will occur when the final patient has received weeks of treatment. group will continue enrollment with randomization : : until dose selection based on analysis of the group cohort. group will allow for a seamless transition into group , which will randomize patients : to the selected dose or placebo. the final data analysis set will include group patients who received placebo or the selected dose and all group patients. the primary endpoint is an objective laboratory measure and surrogate of clinical benefit, increase in hb > g/dl, from baseline to weeks based on voxelotor mechanism of action (inhibition of hb polymerization). this trial is the first to use a patient-reported outcome (pro), the -item sickle cell disease severity measure, as a secondary endpoint. this novel electronic pro, developed specifically for the hope study following fda guidance, will evaluate changes in scd symptom exacerbation and total symptom score from baseline to weeks. additional secondary endpoints include measures of hemolysis, rates of voc, transfusions, and opioid use. the study was designed to enable selection of pro-defined symptom exacerbations or traditionally defined voc as the key secondary endpoint after the group analysis. results: this study is ongoing. the hope trial, expected to complete enrollment by late , will evaluate the efficacy and safety of voxelotor compared with placebo in patients with scd. supported by global blood therapeutics. background: inflammation, coagulation activation, oxidative stress and blood cell adhesion are elements of sickle cell disease (scd) pathophysiology. patients with scd have low levels of the omega- fatty docosahexaenoic acid (dha) and eicosatetraenoic acid (epa) in plasma and blood cell membranes. dha is a bioactive fatty acid with anti-inflammatory, anti-blood cell adhesion and anti-oxidant properties. altemi-atm is a novel dha ethyl ester formulation with a proprietary delivery platform (advanced lipid technology® (alt®)) that enhances oral dha bioavailability. the scot trial investigated the effects of altemiatm in children with scd. objectives: to demonstrate the effects of altemiatm on blood cell membrane omega- index and selected biomarkers of inflammation, coagulation, adhesion and haemolysis associated with scd. s of s design/method: children with scd, aged - years (n = ), were enrolled. subjects were randomized to receive either placebo or one of three daily oral doses of altemiatm ( - , - or - mg/kg/day dha) for two months. the effects of altemiatm on red blood cell (rbc), white blood cell and platelet membrane omega- fatty acids index (total dha + epa levels) were assessed after four weeks of treatment. the effects of altemiatm on markers of inflammation, adhesion, coagulation, and hemolysis were assessed after eight weeks of treatment. cell membrane dha and epa concentration was determined by using lc-ms/ms method. the percent changes from baseline on blood cell membrane omega- index and select scd biomarkers were compared between the three dose groups and placebo using a mixed-model repeatedmeasures (mmrm) analysis with baseline blood cell membrane omega- index, hydroxyurea use, and treatment as fixed effects and patient as a random effect. after four weeks of treatment, blood cell membrane dha and epa levels were significantly increased in all altemiatm doses (p< . ). after eight weeks of treatment, significant reductions were observed in se-selectin (p = . ), and d-dimer (p = . ) in patients exposed to altemiatm dose level vs. placebo. hemoglobin was significantly increased at altemiatm dose level versus placebo. plasma high-sensitivity c-reactive protein, lactate dehydrogenase, soluble vascular cell adhesion molecule- and white blood cell count showed improvement after weeks of treatment in all three altemiatm doses levels but did not reach significance. conclusion: treatment with altemiatm enriches dha and epa in blood cell membranes of patients with scd and improves select sickle cell disease biomarkers of blood cell adhesion and thrombin generation. these findings provide insight into the mechanisms of action of altemiatm in sickle cell disease. brown university -hasbro children's hospital, providence, rhode island, united states background: despite clinical advances in the treatment of sickle cell disease (scd) in pediatric and young adult patients, pain remains a significant source of disease-related morbidity. physical therapy has been shown to be useful for the treatment of pain in children and young adults with various chronic illnesses of which pain is a significant component, however no data exists regarding potential benefits of physical therapy in pediatric and young adult patients with scd. objectives: to query healthcare providers and others involved in the care of pediatric and young adult scd patients regarding possible benefits of and barriers to physical therapy as a potential treatment modality. design/method: we conducted a web-based survey of healthcare providers within the new england pediatric sickle cell consortium (nepscc) in an attempt to identify potential benefits of and barriers to outpatient physical therapy in this patient population. results: nearly % of survey participants felt that physical therapy had the potential to be "somewhat beneficial" or "very beneficial" in pediatric and young adult patients with scd. a majority of physicians reported having referred patients with scd for physical therapy in the past. the most frequently identified perceived potential benefits included improved functional mobility, improvement of chronic pain symptoms, decreased use of opiates, improved mood symptoms, improved acute pain symptoms, and improved adherence with medications and clinic visits. significant perceived barriers identified included lack of transportation, time constraints, patient lack of understanding, and difficulty with insurance coverage. our study indicates that healthcare providers have an overwhelmingly positive view of the use of physical therapy in the management of pediatric and young adult patients with scd. significant barriers exist which need to be addressed. future research should focus on patient and parent perspectives regarding physical therapy, as well as a randomized controlled trial of a physical therapy intervention in this patient population. background: vitamin-d deficiency is fast becoming increasingly recognized in patients with sickle cell disease (scd). while it is estimated that these patients are five times more likely to develop vitamin-d deficiency, the exact clinical significance of this is largely unknown. given that this deficiency can be inexpensively and easily treated, our study sought to establish the prevalence of vitamin-d deficiency in our patient population and its relationship with disease severity. objectives: to estimate the prevalence of vitamin-d deficiency in patients with scd in our institution and to analyze their disease severity in relation to their vitamin-d level. design/method: through retrospective chart review we analyzed subjects that represent a cohort of patients followed at the adult and pediatric hematology services at university of miami with known diagnosis of scd that had a vitamin-d level drawn between january st, and august st, . we conducted a cross-sectional study and recorded the first vitamin-d level during this period. patient demographics, medical and social history information were collected along with laboratory data. the number of admissions for vaso-occlusive crisis (voc) and acute chest syndrome within one year preceding the collection the vitamin-d level was also recorded. results: a total of charts were reviewed, adult charts and pediatric charts. after exclusion, patients were enrolled. subclinical vitamin-d deficiency is only evident on laboratory blood testing of vitamin-d ( -hydroxy) and according to this laboratory result patients were classified as sufficient (≥ ng/ml), insufficient (< to ng/ml) and deficient (< ng/ml). out of the cases, . % ( / ) were deficient, . % ( / ) were insufficient and . % ( / ) were optimal. after statistical analysis two negative correlations were identified, increasing vitamin-d levels with decreasing white blood cell count (ci %- . (- . , - . )and decreasing incidence voc (ci %- . (- . , - . ). conclusion: this study confirms that there is a significant prevalence of vitamin-d deficiency in patients with scd. furthermore, the results of this investigation proved that vitamin-d deficiency is associated with acute pain and leukocytosis in patients with scd. given the multitude of confounding factors that affect vitamin-d absorption and intake, multivariate analyses are required to truly further investigate this relationship. texas children's hospital, houston, texas, united states background: hemophagocytic lymphohistiocytosis (hlh) is a rare but life-threatening condition of hyper-inflammation that is characterized by splenomegaly, cytopenias, hyperferritinemia, hypertriglyceridemia, hemophagocytosis and coagulopathy. although timely diagnosis is imperative, it is often challenging as these individual signs and symptoms may occur in a variety of clinical conditions. to report a case of undiagnosed sickle cell anemia presenting with severe ebv viremia and associated hemophagocytic lymphohistiocytosis results: a -month-old previously healthy male presented with respiratory distress, increased fatigue, and a focal seizure following a two-week history of cough and lowgrade fevers. physical exam was consistent with hypovolemic shock and revealed significant splenomegaly. laboratory testing revealed severe hypoglycemia, acidosis and electrolyte disturbances including hyperkalemia, hyperphosphatemia, and hyperuricemia. labs showed a leukocytosis (wbc , ), severely low hemoglobin ( . ), and platelets of , . coagulation testing revealed prolonged pt/inr and ptt, hypofibrinogenemia and a highly elevated d-dimer. additional workup was completed to determine etiology of acute presentation, given broad differential diagnosis. infectious studies were consistent with an acute ebv infection (plasma ebv pcr > , ). elevated levels of soluble il- and ferritin completed / criteria for the diagnosis of hlh. bone marrow evaluation showed trilineage hematopoiesis with no abnormal blast population or hemophagocytosis. results from hemoglobin electrophoresis sent from the initial cbc sample were notable for hbs . %, hbf . %, and hba of %, confirming the diagnosis of sickle cell disease. the patient was started on hydroxyurea and penicillin and splenomegaly resolved. with supportive care, he demonstrated gradual improvement in symptoms and laboratory abnormalities, including normalization of soluble il- , ferritin, cd , il- levels, immunoglobulins, and declining ebv titers. nk cell function has remained abnormally low, not eliminating the possibility of acquired hlh despite spontaneous improvement. conclusion: splenic sequestration associated with sickle cell disease in combination with acute infectious mononucleosis could have explained many of the presenting symptoms including anemia, thrombocytopenia, and splenomegaly. however, it does not explain the unusually high ebv titer and degree of inflammation meeting diagnostic criteria for hlh, which raises concern for an underlying immunologic abnormality such as x-linked lymphoproliferative disorder (xlp). although testing for xlp was negative, he will require s of s continued monitoring in the future for signs of relapse. this case illustrates the complexity of diagnosing lymphohistiocytic disorders and the significant overlap in presentation between these disorders and other medical conditions. background: vaso-occlusive crisis (voc) is one of the most distressing occurrences in patients with sickle cell disease (scd). patient controlled analgesia (pca) is recommended by nih and expert opinions favor its early use. we aim to review the use of pca in patients with voc and to evaluate if its early use is associated with faster pain control and reduced length of stay (los). design/method: this retrospective single center study included all pediatric patients admitted and treated with pca for a severe voc from to . "early" use was defined as start of pca within hours of arrival in the emergency department (ed) and "late" use after hours. time to reach adequate analgesia was defined as oucher, verbal scale or faces pain scale < / obtained twice consecutively in a -hours interval. time to reach adequate analgesia and los were compared between early-pca and late-pca groups. results: a total of patients presented episodes of voc treated with pca during the study. sixty-one episodes ( %) were treated with early-pca and ( %) with late-pca. both groups were comparable in terms of age ( . vs . years old), gender ( . % female vs . %), hemoglobin phenotype ( . % hbss vs . %), but median pain score at admission was higher in early-pca than in late-pca ( / vs / , median difference ( % ci , ). early-pca was associated with a median reduction in los of . days ( % ci . , . ) (median early-pca los . vs late-pca . days). time to reach analgesia could be evaluated only in a subset of patients ( in early-pca and in late-pca group). although time to reach adequate analgesia tended to be shorter in the early-pca group, it was not statistically different: median . hours vs . hours, difference of . ( % ci - . , . ). side effects were observed during ( . %) pca treatments ( / ( . %) episodes in early-pca, / ( . %) in late-pca group) among which ( . %) were significant adverse events. these were observed in patients who required interventions: desaturations requiring oxygen without intubation, neurologic abnormalities (hallucinations, visual abnormalities, no stroke), urinary retentions. conclusion: early use of pca for severe voc was associated with a reduced length of hospital stay despite that these patients had higher pain score on admission. prospective studies are needed to support these positive outcomes. background: acute chest syndrome is one of the leading causes of death in children with sickle cell disease - . while the cause of acute chest syndrome most commonly is not identified, fat embolism and infectious causes are believed to be most common. with an extremely high mortality rate, rapid identification and initiation of therapy is essential for survival. case presentation: we describe the case of an -year-old female with sickle cell sc disease who was admitted for vasoocclusive pain crisis and quickly progressed to multi-system organ failure due to fat embolism syndrome and parvovirus b infection objectives: the case highlights the presentation and diagnosis so other providers can optimize outcomes for those with this under-recognized syndrome design/method: her parvovirus studies returned after days which showed: parvovirus b dna pcr detected; parvo igg . (positive > . ); and igm . (positive > . ). the patient experienced an approximately . g/dl drop in hemoglobin( . to . g/dl/ hrs) with progressive thrombocytopenia (from , to , /ul) and a peripheral smear showed microcytic,normochromic red cells with nucleated rbcs and occasional nuclear budding, slight polychromasia, schistocytes, and polymorphic cells with toxic granules that suggested leukoerythroblastosis. she was emergently transferred to the regional quaternary care hospital for ongoing ecmo therapy where she experienced a change in her pupillary exam prompting a stat ct scan that showed severe, diffuse cerebral edema with transtentorial herniation. the decision was made to withdraw life-sustaining therapies and her family refused a post-mortem autopsy examination. fat embolism syndrome is a severe and uncommonly recognized complication of sickle cell disease, seen most commonly in those with a non-ss phenotype and previous mild disease course who present with severe, unrelenting vaso-occlusive pain episode and/or acute chest syndrome that progresses to respiratory distress with altered mental status and cutaneous changes. rapid identification and initiation of exchange transfusion therapy should be initiated with clinical suspicion because of the extremely high mortality rate. although previously considered rare, it needs to be considered in the differential diagnosis of more commonly encountered complications of sickle cell disease. background: patients with sickle cell disease (scd) experience vaso-occlusive crisis (voc), which results in extreme pain, often requiring opioids and admission. genetic and environmental factors affect the frequency and severity of these episodes. previous research has born conflicting evidence on whether environmental temperature is contributory. edmonton, alberta is the northern most city with a population over a million in north america. there is an increasing sickle cell population which is exposed to extreme winter conditions. this provides a suitable population and atmosphere to study the influence on cold external temperatures in scd. this study sought to identify if pediatric patients with scd, experience greater morbidity in cold external temperatures. board approved retrospective case control series. patients were identified through a clinical database, and emergency visit, phone call and admission data was collected over a fiveyear period. the average, minimum and change in temperature on day of presentation, and hours prior, was collected from the government of alberta, and was statistically analyzed using descriptive statistics, to determine the relation to vaso-occlusive events. results: one-hundred and eighteen patients were identified, and voc events reviewed. the mean patient age was . years of age with a range from . - years old. the female to male ratio was equivalent with female ( . %) and male ( . %) voc events. eight records ( %) had docu-mented cold exposures. the analysis between the temperature and the frequency of events did not yield significant correlation. average and minimum temperature on day of admission had the largest percentage of voc events occur at mild temperatures, from - . to • c and - . to respectively. change in temperature on day of admission, and hours had the largest percentage of voc events at a mild to moderate change in temperature of - degrees. data at & hours prior to admission showed similar results. secondary data analysis accounting for the lower proportion of extreme weather days in comparison to moderate temperate days showed no significant impact. there was no correlation of average, minimum or change in temperature on day of admission, or hours prior. multiple cofounding factors likely contribute to these results. as it was a retrospective study many confounding and precipitant factors may not be recorded or identified. a prospective study to better record specific cold exposure is warranted. children's national health system, washington, district of columbia, united states background: achieving optimal anticoagulation with unfractionated heparin (ufh) in pediatric patients receiving extracorporeal membrane oxygenation (ecmo) is often challenging due to antithrombin (at)-mediated heparin resistance (hr). intermittent at dosing during pediatric ecmo support does not maintain adequate at levels. continuous at infusion (cati) presents an alternative strategy to achieving consistent goal at levels and optimizing heparinization. however, cati during pediatric ecmo has not been adequately studied. objectives: to describe our center's experience with an ecmo cati protocol. design/method: in , we modified our ecmo anticoagulation protocols to include ufh titration according to anti-factor xa (anti-fxa) levels and cati in patients with at-mediated hr. the cati rate was calculated using baseline and goal at levels while accounting for the circuit volume. cati was administered with ufh into the circuit via a s of s y-infusion set. at and anti-fxa levels were monitored every hours. recombinant at (r-at) concentrate was used at our center until with subsequent transition to a plasmaderived at (pd-at) concentrate. due to the longer half-life of pd-at concentrate, the protocol was modified so cati is stopped once target at and anti-fxa levels are achieved. we conducted a retrospective study of all patients who received cati during ecmo support at our center. data are reported as median and interquartile range and compared using the mann-whitney u test. two-tailed p-value < . was considered statistically significant. since , patients [ males, age month ( . - )] on ecmo support received catis ( rat, pd-at) per our protocol ( patients received pd-at infusions during one ecmo run). the duration of cati was hours ( - ). cati administration led to significant increases in at and anti-fxa levels from baseline of % ( - ) and . units/ml ( . - . ) to the first level within goal of % ( - ) and . units/ml ( . - . ), respectively (p< . ). the respective times to achieve goal at and anti-fxa levels were hours ( - ) and hours ( - ). the respective peak at and anti-fxa levels were % ( - ) and . units/ml ( . - . ). during cati, no patient required circuit change, patient developed cannula thrombosis and patients experienced non-fatal major bleeding. conclusion: cati in pediatric patients receiving ecmo support with close monitoring of at and anti-fxa levels was associated with significant rapid increase in at, optimization of heparin effect, and reduction in thrombotic complications without increase in major bleeding compared to prior reports. a prospective study of this at dosing strategy is warranted. children's hospital of orange county, orange, california, united states background: inherited factor xiii (f ) deficiency is a rare bleeding disorder with wide heterogeneity in clinical manifestations ranging from mild bruising, and mucosal and umbilical stump bleeding to spontaneous, severe intracranial bleeding. the bleeding phenotype is influenced not just by zygosity of the fxiii mutation alone, but also by co-inheritance of variants in other clotting protein genes that also play a major role in clot formation and stability. we present a series of three siblings found with f a gene variant and platelet dysfunction linked to bleeding phenotype. design/method: retrospective chart review of the index case, coagulation studies and whole gene sequencing. the index patient presented at two years of age with a subdural hematoma after a fall, requiring emergent craniotomy. a week after initial evacuation, she re-bled, prompting an extensive work-up for potential bleeding disorders, including f activity, von willebrand profile, comprehensive fibrinolysis panel, pai- antigen level, platelet mapping thromboelastogram (plt-teg), and f genetic analysis. the patient's identical twin and older sibling, who had symptoms of bruising, underwent a similar evaluation. the index patient demonstrated consistently low f activity ( - %), and platelet function testing revealed decreased response to adp agonists. the twin and older sibling had normal f levels, and only slightly decreased response to adp in platelet studies. whole gene analysis of f and other genes on our next generation panel, revealed several intronic deletions in the index patient that were not shared by her siblings, which likely account for her decrease in circulating f levels. her symptoms have responded well to monthly treatment with factor concentrate. all three children shared the f variant, pro leu, previously described as a risk factor for intracranial hemorrhage. the f mutation, pro leu, has been associated with intracranial hemorrhage in young women, but the presence of the variant alone may not be enough to cause a severe bleeding phenotype. family studies identified novel deletions in the index patient which may account for her decreased f levels, which would have been overlooked with standard sequencing. future studies, including evaluation of 'platelet' f levels, should be performed when platelet dysfunction is detected. further laboratory and clinical evaluation is required to delineate the long term implications of the interaction of even mild f deficiency if present with additional clotting disorders such as the platelet function defect in these siblings. background: acquired hemolytic anemia can occur due to mechanical shearing of red blood cells and is classically seen in patients with prosthetic heart valves. there are reports of this same traumatic effect with other repairs, including annuloplasty. following valvular procedures flow disturbances can exist across the valve that lead to shear stress and hemolysis. although von willebrand disease (vwd) is typically seen due to an inherited disorder in the pediatric population, flow disturbances in the setting of valve abnormalities can lead to acquired von willebrand syndrome (avws). von willebrand factor multimers become unfolded and elongated in the setting of shear stress resulting in increased susceptibility to cleavage by adamsts- . specifically, loss of high molecular weight multimers (hmwms) can lead to a syndrome akin to type a vwd. objectives: to describe a case of mechanical hemolysis with acquired type a vwd design/method: a -month-old girl with history of hypoplastic left heart syndrome and severe tricuspid valve insufficiency underwent norwood procedure, blalok-taussig shunt placement and subsequently a bidirectional glenn and tricuspid valve annuloplasty. during the following month she requires weekly red blood cell (rbc) transfusions due to intermittent anemia. she also experienced bloody stools and dark urine. laboratory evaluation was notable for normocytic anemia, reticulocytosis, elevated lactate dehydrogenase, and low haptoglobin consistent with hemolytic process. immune-mediated hemolysis from transfusion reaction or presence of autoimmune or alloimmune antibodies testing was negative. to investigate gi bleeding, work up for vwd revealed normal vw activity and antigen but with loss of high molecular weight multimers consistent with acquired type a vwd. in consultation with cardiology, it was felt her tricuspid valve insufficiency jet could be leading to mechanical hemolysis and avws. a repeat echo showed persistent moderate tricuspid insufficiency but no other significant changes. due to the patient's continued need for weekly rbc transfusions she was subsequently trialed on pentoxifylline which is used in adult patients to decrease blood viscosity and increase erythrocyte flexibility in patients with mechanical hemolysis. her transfusion needs remained the same and the medication was discontinued after two weeks. she required one transfusion a week later but no transfusions since that time. although not commonly seen in pediatric patients, the diagnosis of mechanical hemolysis accompanied by avws should be pursued in a patient with congenital heart disease with significant anemia and/or bleeding. the work up in these patients is difficult as echocardiograms can be inconclusive thus an extensive hematologic evaluation is usually necessary. objectives: our aim was to assess incidence of and potential risk factors for central line-related dvt at our institution between - . additionally, our goal was to analyze if that incidence differed between the three central line types and identification of line-specific risks. design/method: a retrospective chart review of central line placements in pediatric patients at cleveland clinic between - was conducted. data included demographics, potential risk factors, line characteristics and any related thrombotic events. the study cohort consisted of lines in pediatric patients aged - years of age. there were . thrombi ( % ci . - . ) per , line days. statistically significant risk factors for thrombus include diagnosis group (liquid tumor highest rate of %, solid tumor lowest at %), type of line (picc %, broviac %, and mediport %), location of line, greater number of lines per patient, peg asparaginase ( % vs %), sepsis, and history of procoagulant state. line characteristics such as lumen size and number of lumens were not identified as a significant risk. there was a significantly higher rate of thrombus in than in the previous years when pooled ( % in vs . % from - , p = . ). the incidence of dvt in pediatric patients at our institution was highest with broviac lines, and significant risk factors in our patient population included liquid tumor, femoral vein location, peg asparaginase, sepsis, and history of a procoagulant state. the incidence of thrombi was highest in , and therefore highlights the urgent need for improvement in nationwide hospital practices to minimize risk of thrombi formation and early detection in the higher-risk s of s populations. there is still much to be learned regarding the characteristics specific to different central lines, which would influence thrombi formation. nyu winthrop hospital, mineola, new york, united states background: pediatric immune thrombocytopenic purpura (itp) is an autoimmune disorder with platelet counts < causing increased risk for significant hemorrhage. there is increased immunologic platelet destruction due to production of specific autoantibodies along with inhibition of platelet production. few randomized trials exist to guide management and ultimately each patient requires an individualized treatment plan. itp may be acute (diagnosis to m) or chronic (> months). one of the treatments of chronic itp is laparoscopic splenectomy (ls), which is very well tolerated. a rare complication of ls is splenosis, an autotransplantation or implantation of ectopic splenic tissue within the abdominal cavity or in any other unusual body compartment. splenosis is sometimes associated with relapsed itp due to preserved immune activity. the usual management of symptomatic splenosis is surgical resection. objectives: to describe medical management in a young patient with itp relapsed due to extensive unresectable splenosis following ls design/method: our patient was originally diagnosed at years with itp and was treated with ls at years of age for chronic severe thrombocytopenia and persistent bleeding not responding to first line therapies. she tolerated it well and had a complete response (cr) defined as a platelet count of > measured on occasions > days apart and absence of bleeding. she maintained a normal platelet count for twelve years after which she relapsed (loss of response after cr) with severe thrombocytopenia and hematuria necessitating high dose steroids. ct scans showed multiple wellcircumscribed soft tissue masses in the left lower quadrant adjacent to uterus and left ovary, involving left omentum and the anterior abdominal wall partly. findings were confirmed by damaged rbc nuclear scan to be splenosis. during laparoscopy the splenosis lesions were deemed too extensive and were not resected completely to avoid postoperative morbidity. she was started on sirolimus around the same time for treatment of her relapsed itp and steroids were weaned off. results: eight months since beginning sirolimus with therapeutic levels she remains in cr with no bleeding and has not required any steroids, immunoglobulins or anti d immunoglobulin. conclusion: sirolimus is a safe and effective steroid-sparing agent in treatment of chronic itp. this is the first instance of a patient with poorly resectable splenosis responding well to medications for itp. more data is needed regarding the longterm efficacy of such an intervention and whether it will eliminate the need for a second surgery in relapsed itp patients with extensive splenosis. background: storage pool disorders affecting platelets result in bleeding symptoms related to a deficiency or defect in alpha granules or delta granules. in delta-storage pool disorders (dspd,) there is a deficiency of the delta granules and their constituents, which results in the inability of platelets to properly activate as well as lack of proper constriction of blood vessels during bleeding episodes. amongst patients with dspd, females most commonly present with menorrhagia, while males tend to present with epistaxis and easy bruising. the international society on thrombosis and hemostasis (isth) developed a screening bleeding assessment tool (bat) for mild bleeding disorders, shown to be a validated tool in children. diagnosis of dspd is classically made with a platelet electron microscopy (pem) value < . delta granules per platelet (dg/pl), but recently lower diagnostic thresholds of dg/pl or even . dg/pl have been suggested. objectives: evaluate the correlation between pem and bleeding scores, and also examine various cut-off values used to diagnose and risk stratify patients with dspd. design/method: retrospective chart review of pediatric patients followed by hematology with a diagnosis of dspd was performed. clinicians obtained bleeding scores for each patient as standard of care in the hemostasis clinic. quartile ranges were established to appropriate three stages of severity based upon bleeding scores. statistical analysis was performed using software r and exploratory data analysis to evaluate for a correlation. results: amongst all patients, the average bat score was . and pem was . dg/pl. the average bleeding score for pem between . dg/pl and dg/pl was . , while the average bleeding score for pem below dg/pl was . . the correlation coefficient between pem and bleeding scores is . . using a threshold of dg/pl, % of patients would have met diagnostic criteria. quartile ranges for the bleeding scores are as follows: st quartile was - , nd quartile was - , and rd quartile was > . conclusion: patients with a more marked granule deficiency do not exhibit a more severe bleeding phenotype, suggesting proper platelet function is not solely determined by granule quantity in these patients. bleeding severity may be more appropriately assessed with bleeding scores rather than pem values, and using quartile ranges may aide in risk stratification and therapeutic interventions for dspd patients. further work remains to determine the optimal diagnostic threshold of pem dspd in pediatric populations. texas children's hospital, houston, texas, united states background: warfarin management has many challenging aspects including pharmacogenomics, food and drug interactions, lack of standardized dosing, patient compliance, tracking lab results from multiple lab locations, and the potential for significant bleeding or thrombotic complications. a literature review revealed limited data highlighting anticoagulation monitoring workflow and emr documentation and specifically, no data in the pediatric population. historically, the texas children's hospital cardiology and hematology centers were each documenting anticoagulation data within the epic tm system differently. epic's tm original design for anticoagulation documenting resulted in the necessity to duplicate documentation in order to see at-a-glance critical anticoagulation monitoring information. objectives: the objective of this project was to standardize inr documentation across departments to reduce the risk of patient safety events and improve workflow. design/method: a workgroup assembled consisting of nurses from the cardiology and hematology departments, along with staff members from the epic tm is support group. the workgroup identified current documentation practices, available epic tm tools, and brainstormed ideas to streamline and improve both documentation with the current epic tm tools. physician partners were identified in cardiology, hematology and coagulation laboratory to gain their input. a new anti-coag (ac) encounter was developed and first made available in an epic tm practice environment, then once approved, epic tm written education and training session were completed by both departments' staff. results: surveys were sent to health care providers in the cardiology and hematology centers prior to the new ac encounter, and also to health care providers six months after implementing the ac encounter. six responses were received for each survey. the pre-implementation survey showed the most problematic part of the documentation system for anticoagulation was no single place in the emr to find a complete anticoagulation picture. post ac encounter implementation survey results revealed more health care providers using the epic tm inr reminder pool, less time needed to compile a report of three months of anticoagulation information, less time needed to document individual encounters, less locations needed to document ac information and decreased amount of types of documentation used. standardized ac encounters improves workflow with less time needed to document and compile information, less types of documentation utilized and easier access to patients ac information. next steps include retrospective review of patients' inr time in therapeutic range to determine if there was an impact on patient compliance and continue to evaluate and modify the ac encounter to enhance user friendliness. caitlin tydings, jennifer meldau, christine guelcher, carole hennessey, eena kapoor, michael guerrera, yaser diab s of s children's national health system, washington, district of columbia, united states background: venous anatomic abnormalities (vaas) are considered a risk factor for developing deep vein thromboses (dvts) that occur as a result of significant alterations in venous blood flow. identification of predisposing vaas can be challenging. hence, diagnosis can be delayed or overlooked especially in pediatric patients. dvts in children or adolescents with predisposing vaas have been only described in sporadic case reports and small case series. objectives: to describe characteristics and outcomes of dvts in pediatric patients with underlying vaa treated at our center. design/method: we conducted a retrospective chart review of all pediatric patients with objectively confirmed extremity dvt treated at our institution over a -year period from to and identified all patients with underlying vaas. patients were managed according to standardized institutional protocols based on published guidelines. post-thrombotic syndrome (pts) was assessed at our center using the manco-johnson instrument. relevant data were collected and summarized using descriptive statistics. during the study period, of pediatric patients ( %) [ females, median age years (range - )] diagnosed with extremity dvt at our center were found to have an underlying vaa. vaas included may-thurner anomaly ( patients), venous thoracic outlet obstruction ( patients) and inferior vena cava (ivc) atresia ( patients). additional provoking factors were identified in patients at time of presentation. dvt locations included upper extremity veins ( patients), lower extremity veins ( patients) and lower extremity veins and ivc ( patients). the majority of dvts [ patients, ( %)] were completely occlusive. high risk thrombophilia (defined as inherited deficiency of antithrombin, protein c, or protein s, or antiphospholipid antibody syndrome) was present in patients ( %). all patients were treated with therapeutic anticoagulation with patients continuing indefinite anticoagulation. endovascular interventions were performed in patients and included percutaneous pharmacomechanical thrombectomy and/or catheter-directed thrombolysis ( patients), balloon angioplasty ( patients) and stent angioplasty ( patients). surgical interventions included thoracic decompressive surgery ( patients) and surgical thrombectomy ( patient vvas represent an important risk factor for developing extensive extremity dvt in adolescents. this special population is at risk for short-term and long-term com-plications. early identification and correction of vaas may improve outcomes. however, multicenter, prospective studies are needed for developing optimal evidence-based treatment approaches. alexander glaros, roland chu, sureyya savasan, meera chitlur, madhvi rajpurkar, yaddanapudi ravindranath children's hospital of michigan, detroit, michigan, united states background: acute budd-chiari syndrome (bcs) is a rare thrombotic emergency in children, and etiologies/treatment are less well-defined than in adults. in adults, a systematic approach including anticoagulation, relief of venous obstruction, and treatment of the underlying cause has proven successful. more recently treatment has tilted towards aggressive surgical interventions, which carry significant risk and are often not feasible. objectives: review our experience with three different patients with bcs and suggest a mechanistic based approach to treatment. the records of three patients with bcs were reviewed and their presentations, etiologies, treatment, and outcomes were reported. results: patient a was a -year-old female with paroxysmal nocturnal hemoglobinuria who presented with recurrent worsening abdominal pain over several months. narrowing of inferior vena cava (ivc) and hepatic veins was noted on imaging. liver transplant was not considered surgically feasible. she was treated with eculizumab, steroids, and anticoagulation with restoration of hepatic venous flow in weeks. patient b was a -year-old male with several weeks of right upper quadrant pain, fatigue, and pre-syncopal episodes, with a history of blunt abdominal trauma from football scrimmage weeks earlier. he was found to have near complete occlusion of the ivc and hepatic veins. liver transplant was not considered feasible. he was successfully treated with anticoagulation alone. patient c was a -yearold male with acute myeloid leukemia in induction cycle who developed severe pancytopenia; typhlitis was diagnosed and managed medically. days later he acutely decompensated, arrested, and was placed on extra corporeal membrane oxygenation, and imaging showed complete occlusion of the portal vein, hepatic veins, and ivc to the level of the atrium, with bilateral pulmonary emboli. emergency liver transplant or catheter based interventions was deemed not feasible. treatment with eculizumab was considered for presumed inflammation induced complement activation (c mg/dl [normal - ]; ch was u/ml [normal - ]) as a trigger for thrombosis, but the patient progressed quickly and died before it could be initiated. our experience with bcs shows that invasive interventional options and liver transplant may not be feasible in most patients for multiple reasons. rapid diagnosis and aggressive etiology-based medical management are paramount to successful treatment of this rare complication. eculizumab may be considered in treating bcs with complement activation not only due to innate disorders, but also secondary to acute inflammation when proper laboratory evidence is present. background: platelet aggregation studies are the gold standard for the diagnosis of platelet function defects during the evaluation of a patient with bleeding problems. the platelet aggregation test measures how well platelets clot in response to different concentrations of epinephrine, adenosine diphosphate (adp), collagen, arachidonic acid and ristocetin. because platelet function defects are often under-recognized and under-diagnosed in the pediatric patient, the true incidence is unknown. we report our experience in the diagnosis of platelet defects at our institution over a -year period in order to add some clarity to the limited pediatric data available. objectives: our primary objective is to document correlations/trends between less well-known platelet function abnormalities and clinically significant bleeding at our institution over a -year period. design/method: after appropriate irb approval obtained, we performed a retrospective chart review of all children who had platelet aggregation testing done from to . data collected included demographics (age, sex, race), personal and family history of bleeding, screening for coagulation defects and platelet aggregation test results. symptoms examined in our data were limited to epistaxis and heavy menstrual periods. for each of these symptoms, results were further analyzed to those with abnormal responses to adp and epinephrine. patients with existing bleeding diagnoses and those with incomplete medical records were excluded. we identified patients. of the patients with epistaxis, % had abnormal platelet aggregation testing while only % of those with heavy menstrual periods had abnormal results. within our population, abnormal platelet function assay (pfa- ) results or race did not appear to correlate with abnormal platelet aggregation testing. in the cases of epistaxis, sex was also noncontributory. our preliminary results suggest that platelet aggregation testing was more useful in predicting platelet defects in those with a clinical bleeding history of epistaxis as opposed to heavy menstrual periods. for other presenting symptoms, platelet aggregation testing did not offer diagnostic benefit. abnormal response to adp in the platelet aggregation test was the most common finding in our population; the clinical significance of which is not well understood. going forward, we plan to document whether abnormal results correlated significantly with the subsequent final diagnoses of our patients. background: decision making for severe hemophilia a in previously untreated patients (pups) has recently become a significant ethical debate. recombinant factor viii (rfviii) products previously were recommended to avoid transmission of blood borne pathogens associated with plasma-derived fviii (pdfviii) products. however, the increased incidence of fviii alloantibody inhibitors with rfviii products compared to pdfviii products has challenged this former standard of care. despite the support of the medical and scientific advisory council, recommendations considering pdfviii products for a pup remains controversial. design/method: we used a modified utilitarian approach involving clinical, public health, and research ethics. shared decision making permeates the framework to maximize understanding, minimize bias, respect informed consent or dissent, and provide care that aligns with patient and family values when medically and practically feasible. the framework has three tiers. first, it evaluates whether resources are scarce or abundant for equitable resource allocation. if fviii products are scarce, we s of s recommend developing a central supply for emergency use and then evaluating the needs of the severe hemophilia a patients. prioritization of who receives the factor products would be decided by a designated team based on the availability of the factor products and clinical scenarios, with no preference given to those on research trials. however, if resources are abundant, treatment for acute bleeding and standard of care prophylaxis measures, including primary prophylaxis, could continue. the second tier accounts for whether there is a new infectious epidemic or concern where a pathogen cannot be eliminated. if there is, healthcare and public health workers may limit the use of pdfviii products. if not, pdfviii and rfviii products are to be equally considered. the third tier evaluates whether the clinical scenario is emergent or not. if there is acute, emergent bleeding, the immediately available resource should be used, along with bypassing and/or adjuvant resources as needed until the bleeding has resolved or improved. to align with patient and family preferences, attempts to have both pdfviii and rfviii products available at similar costs in institutions would be ideal. this ethical framework endeavors to balance autonomy, beneficence, nonmaleficence and justice in helping guide discussions among providers, pups with severe hemophilia a, and their families. disclaimer: findings and conclusions are those of the author(s) and do not necessarily represent the official position of the centers for disease control and prevention, emory university, or children's healthcare of atlanta. background: von willebrand disease (vwd) is a common bleeding disorder which affects up to % of the population without gender predilection. bleeding associated with this condition results from a deficiency or abnormality in von willebrand factor interfering with formation of primary hemostasis. ehlers-danlos syndrome (eds) is a group of rare inherited connective tissue disorders which may have an associated bleeding manifestation without abnormalities in coagulation testing. bleeding symptoms reported in eds result from capillary and tissue fragility. joint hypermobility syndrome (jhs) is an inherited condition which is nearly indistinguishable from eds iii. reports of coinheritance of vwd and eds or jhs are infrequent. the objective of this retrospective study was to review patients with coexisting vwd and eds or jhs at the indiana hemophilia and thrombosis center in order to describe the type and severity of bleeding symptoms, physical examination findings, and pertinent laboratory data. design/method: the electronic medical record database of the indiana hemophilia and thrombosis center was queried for patients with a diagnosis of vwd and one of the following descriptors: hypermobility syndrome, hypermobility, hypermobile joints, or ehlers-danlos syndrome. the records of identified patients were reviewed for demographics, type and severity of bleeding symptoms, beighton scores (bs), vwd antigen, ristocetin cofactor, factor viii levels, vwd multimer pattern, vwd subtype, genetic testing for eds, and family history of eds. results: a total of patients with dual diagnoses of vwd and eds and patients with vwd and hypermobility were identified with this query. two patients had completed genetic testing for eds, and one had a col a gene mutation identified. significant bleeding symptoms in the vwd and eds group included hematuria and postoperative hemorrhage. two of these patients had delayed wound healing postoperatively. seven of the patients identified to have type i vwd and jhs had moderately severe and somewhat unusual bleeding episodes reported including hematuria, hematemesis, and hemoptysis; of these patients had significant perioperative bleeding. females composed % of the vwd and eds group and % of the vwd and jhs group. conclusion: coinheritance of vwd and eds is an uncommon phenomenon. patients with vwd and eds or jhs may have atypical and moderately severe bleeding, especially with procedural intervention. incorporation of bs into the assessment of patients with bleeding disorders is useful to identify potential inherited collagen disorders, as diagnosis of these conditions may impact clinical management. in the year-long phase ii study (ro fd ), / khe patients responded. patients were followed for years after study completion, collecting data on growth and development, complications of therapy, unexpected toxicities, and need for continuing sirolimus. objectives: after study therapy treatment of one year, objectives include: . assess long term toxicity over the - year period after study therapy completion . assess unexpected toxicity . assess overall condition of the patient . assess need for restart or continuation of sirolimus therapy design/method: prospective follow-up of patients with a diagnosis of khe from institutions. inclusion criteria: follow-up for - years post-study. results: follow-up included data at year (n = ) and - . year (n = ) time points. average age at the start of treatment was months. of patients were available for follow up. four patients are no longer on sirolimus: one patient completed study therapy and remains off treatment (ot) ( years), required years of treatment and is now . years ot and required an additional treatment course prior to successful discontinuation now and months ot. of the patients still on sirolimus, all restarted medication for symptoms of pain, swelling and/or edema interfering with quality of life and have made an average of . attempts to discontinue sirolimus. no patient had reoccurrence of kmp. all patients had improvement in clinical and radiologic appearance of khe but all have residual lesions noted on imaging and/or clinical exam. no unexpected toxicity, growth delay, developmental issues or other long term toxicity of sirolimus was noted. conclusion: this is the first prospective data on long-term follow up of khe patients treated with sirolimus. although numbers are small, sirolimus is well tolerated; however, over half the patients were still on medication at - year follow up. this stresses the need for continued long term follow up in these young patients and investigation of the mechanism of sirolimus effect. nationwide children's hospital, columbus, ohio, united states background: recent studies have identified that adult persons with hemophilia (pwh) have a higher prevalence of hypertension and renal disease than the general population. while hematuria is a known complication of hemophilia a and b (ha, hb), its long-term impact on pwh is not currently known. by annually screening our patients with urinalysis, our pediatric center identified that just under half of our patients demonstrated hematuria over a four-year period. motivated by a desire to identify early markers of hypertension and renal disease, we sought to determine if this finding is reflected in the pediatric hemophilia population as a whole. objectives: establish the population-wide prevalence of hematuria in pediatric pwh. design/method: we used the pediatric health information system (phis) database, which contains clinical and resource utilization data for inpatients from hospitals nationwide, to analyze the prevalence of hematuria, hypertension, renal disease and related diagnosis codes in pediatric pwh who were admitted from january to september . results: during the five-year period, , unique pediatric pwh accounted for , admissions. while the majority of admissions were for bleeding or infectious concerns, ( . %) patients had an affiliated admission code for hematuria. for admissions as a whole, the median age was years with % of those admitted being infants, % toddlers, % children, % adolescents, % older than . we identified % of admissions were for ha with the remaining % were for hb. there were ( %) admits in which a bypassing agent was administered. the median length of stay for persons with hematuria was days compared to days for nonhematuria/other bleeding. there were ( . %) admissions with hypertension reported; though, only patients received an antihypertensive medication during that admission. additionally, only ( . %) admissions reported a diagnosis code of renal disease. our study demonstrated that pediatric pwh are experiencing hematuria. in general, only patients with persistent hematuria require hospital admission so we suspect this data underrepresents the numbers of pwh experiencing hematuria that is managed in the outpatient setting. we also suspect that hypertension is grossly underreported and undertreated in pediatric pwh. additionally, there are a low number of patients experiencing renal disease requiring hospital admission among this cohort. given that there is little research into the long-term impact of hematuria in hemophilia, we feel these findings support the need for further vigilance of our pediatric pwh. background: gla and gsd can aggressively destroy bone, with significant impact on morbidity and mortality. the mtor inhibitor, sirolimus has been shown to be effective in the treatment of these diseases. based on the addition of mtor inhibition to bisphosphonate therapy in metastatic cancer therapy, regimens have been used for refractory or high risk gla and gsd but there is heterogeneity of diagnosis, and variability of drug regimens and assessment of effectiveness. objectives: . assess the variability of clinical features of gla and gsd . assess the heterogeneity of diagnosis . assess drug regimens and response assessment across multiple institutions design/method: we conducted a retrospective review from institutions of cases of gla and gsd treated with sirolimus and a bisphosphonate for at least months with assessment of clinical features, treatment protocols, response regimens and side effects. results: patients included gla (n = ) and gsd (n = ). the average age at diagnosis was years. clinical features included effusions: gla (n = ), soft tissue lymphatic malformations: gla (n = ), gsd (n = ), multiple splenic lesions: gla (n = ), and soft tissue swelling at the site of bony lesion: gsd (n = ). the presenting symptom in patients was pain with patients (gla) presenting with shortness of breath. fracture was noted in patients: gla ( ), gsd ( ). diagnostic and/or response imaging included mri, ct, bone scan, skeletal survey and dexa scan. treatment consisted of: initial sirolimus use with the addition of bisphosphonate secondary to worsening disease (n = ), initial therapy with other agents (interferon, chemotherapeutic agents, radiation) and change to sirolimus and bisphosphonate secondary to toxicity (n = ), sirolimus and bisphosphonates (n = ) and sirolimus, bisphosphonates and interferon (n = ). seventeen patients had stable disease and patients had improvement of disease. sirolimus protocol was standard; however, bisphosphonate protocol varied in dosing and frequency. side effects were tolerable and expected with no grade iii or iv toxicity. sirolimus and bisphosphonates are a safe and effective therapy for gsd and gla. a consistent medication regimen, redefined response and an improved radiologic classification will be important for the development of a prospective clinical trial. background: hemophilia a is a bleeding disorder from the deficiency of clotting factor viii. the most significant sequelae of hemophilia a is the tendency to develop hemarthrosis that incites joint destruction. the prevalence of overweight and obesity has been increasing in the general and hemophilia population and leads to several morbidities including arthropathy. this is a particular concern for hemophilia a as arthropathy is a consequence of joint bleeding. objectives: the purpose of this study was to detect the relation between body mass index (bmi) and joint health endpoints in a pediatric hemophilia population. design/method: participants in this study included patients from the hemostasis and thrombosis center at children's hospital los angeles. participants were pre-screened and approached for this study during routine follow-up appointments. patients aged - years old who have been diagnosed with hemophilia a, including mild, moderate, and severe, qualified for the study. informed consent was obtained from the patients or parents before enrollment. joint health was objectively measured by physical therapists from children's hospital los angeles using the hemophilia joint health score (hjhs). an hjhs total score is calculated by assessing: swelling, duration of swelling, muscle atrophy, crepitus on motion, flexion loss, extension loss, joint pain, and muscle strength in major joints. subjective data was also obtained by patients recording their annual bleed rate within the past year. of the patients, ( %) were normal weight, ( %) overweight, and ( %) obese. we used chi-square analysis to compare joint scores across bmi classifications (chi square = . , df = , p-value = . ). although, this did not approach statistical significance, the average hjhs score in patients who had a hjhs > shows an increasing trend among bmi classifications: . in normal bmi patients, . in overweight bmi patients, and . in obese bmi patients. the average number of annual bleeds in those with positive values show: in normal bmi patients, in overweight bmi patients, and in obese bmi patients. although a positive effect of adiposity was found in the joints of hemophilia a pediatric patients, the effect shows there was not enough evidence to conclude a difference. future studies are needed to address whether obesity has an effect on hemophilia and to determine whether overweight/obesity can lead to further complications in hemophilic joints. background: stagnant blood flow in slow-flow vascular malformations (vm), particularly in their venous components, can lead to localized intravascular coagulation (lic) that is characterized by elevated d-dimer levels, low fibrinogen and decreased platelet count this coagulation derangement can lead to localized thrombosis or bleeding which can result in pain, functional limitations, and possible progression to disseminated intravascular coagulopathy (dic). the treatment of vm and their associated coagulopathy has proven difficult. patients with complex vm are frequently managed with sirolimus, an mtor inhibitor, and have clinical benefits, including reduction of pain and improvement in functional impairment. it is possible that some of these improvements from sirolimus could be secondary to improvement in the coexisting lic. objectives: this study assessed the use of sirolimus to manage the coagulopathy seen in slow-flow vm. design/method: we reviewed charts of patients with vm who are followed in the vascular anomalies center at arkansas children's hospital and were started on sirolimus. efficacy was objectively assessed through improvement of ddimer, fibrinogen and platelet count. three sets of lab values (pre-sirolimus, - months post-sirolimus, and most recent) were obtained for each patient when available. we identified a total of patients who had been prescribed sirolimus. eighteen were excluded based on underlying condition other than slow-flow vascular malformation and for inadequate medical records. a total of patients ( combined vascular, venous) were included in the study. all had elevated d-dimer levels (mean . mcg/ml feu, median . mcg/ml feu, range ( . - . )) prior to treatment. two patients had an associated low fibrinogen (below mg/dl), indicating severe lic. with treatment, ( . %) patients showed an overall decrease in d-dimer levels with an average decrease of . mcg/ml feu between pre-and post-sirolimus labs, and an average decrease of . mcg/ml feu between pre-sirolimus and most recent values. the two patients with low fibrinogen prior to treatment showed a decrease in d-dimer levels (mean decrease of . mcg/ml feu) and an increase and normalization in fibrinogen (mean increase . mg/dl) after beginning sirolimus. no patient had thrombocytopenia. we report that treatment with sirolimus was effective in improving coagulopathy associated with slowflow vm as evidenced by decreased d-dimer levels and increased fibrinogen and/or platelets. long-term use of this medication in this population may decrease the bleeding and thrombotic complications that these patients experience, especially following invasive vascular procedures. background: safety and efficacy of bay - , a sitespecifically pegylated b-domain-deleted recombinant factor viii, in previously treated adolescents and adults aged - years with severe hemophilia a was demonstrated in the phase / protect viii study and ongoing extension. objectives: this subanalysis examines the efficacy and safety of bay - in adolescents in protect viii and the ongoing extension study (data cutoff, january ). design/method: in protect viii, patients (including adolescents) received bay - on demand or as prophylaxis for weeks. prophylaxis regimens for weeks - were twice-weekly ( - iu/kg), every- -days ( - iu/kg), or once-weekly ( iu/kg) infusions based on bleeding during a -week run-in period of iu/kg twice-weekly prophylaxis. patients continued their prophylaxis regimens in the extension or changed regimens at any time. results: twelve patients aged - years were included in the protect viii intent-to-treat population; s of s additional patient discontinued after dose (included in safety population). for patients receiving prophylaxis before study enrollment, median (range) number of total and joint bleeds in the months before study entry was . ( - ) and . ( - ), respectively. ten patients ( . %) had target joints at baseline (median [range], [ - ] per patient). during weeks - of protect viii for the entire time patients remained on their designated prophylaxis dosing frequency, the median (quartile [q] ; q ) annualized bleeding rate (abr) for patients receiving twice-weekly (n = ), every- -days (n = ), and once-weekly prophylaxis (n = ) was ( ; . ), . ( ; . ), and . ( ; . ), respectively (overall prophylaxis [n = ], . [ . ; . ]). two patients switched from once-weekly to twice-weekly (n = ) or every- -days prophylaxis (n = ), and number of bleeds decreased from to in one patient and to in the other. all patients from the main study continued in the extension; mean abr in the extension was . and varied by dosing regimen (twice weekly [n = ], . ; every days [n = ], . ; once weekly [n = ], . ). two patients changed from every- -days to once-weekly prophylaxis during extension (mean abr, . ). one patient had a nonneutralizing antibody to bay - at baseline; end-of-study titers were negative. no patient developed anti-peg antibodies or factor viii inhibitors or experienced a serious adverse event related to bay - during the main study or extension. in previously treated adolescents with severe hemophilia a, bay - prophylaxis was effective in prevention of bleeds, with less bleeding overall versus prestudy, and was generally well tolerated. funded by bayer. cincinnati children's hospital medical center, cincinnati, ohio, united states background: vascular malformations (vms) consist of a heterogeneous group of congenital disorders characterized by the abnormal development of blood and/or lymphatic vessels, which cause a broad spectrum of clinical manifestations. although considered benign, vms are frequently associated with cutaneous complications that can cause significant morbidity such as nodular overgrowth, skin thickening, pruritus, oozing or bleeding of lymphatic blebs and secondary infection. oral sirolimus has shown to be effective in the treatment of complicated vascular malformations but has known side effects and need for frequent laboratory monitoring. currently, there are limited studies on the use of topical sirolimus for the treatment of cutaneous manifestations of vascular malformations. objectives: to evaluate the efficacy and safety of topical sirolimus in vms with cutaneous complications and propose indications for use. design/method: this is a retrospective review of medical records of patients with vascular malformations treated with topical sirolimus from january to december . response was determined by subjective and objective improvement. results: twenty-four patients, ( %) females and ( %) males, with vascular malformations and cutaneous manifestations were treated with topical sirolimus. age ranged from - years. indications for treatment were: blebs ( %, n = ) causing either leaking, bleeding, pain, pruritus, swelling or recurrent infection; nodular overgrowth % (n = ); pyogenic granuloma % (n = ); bleeding % (n = ) and cosmetic % (n = ). treatment course ranged from - months. no major side effects were reported. one patient reported burning and itching sensation. regarding clinical response: % (n = ) patients had improvement in cutaneous lesions; % (n = ) had a stable lesions; and % (n = ) stopped treatment due to side effects. for prior/concomitant treatment: % (n = ) had prior surgery, laser or sclerotherapy; % (n = ) had concomitant oral sirolimus. of the patients not receiving concomitant systemic sirolimus, only % (n = / ) had been on oral sirolimus. of these patients, % (n = / ) had a very good response to topical treatment. : topical sirolimus appears to be beneficial and well-tolerated with a minimal side effect profile for the treatment of cutaneous manifestations of vascular malformations as a single agent or as adjuvant therapy with systemic sirolimus when symptoms are not adequately controlled. further studies are needed to prospectively analyze efficacy and safety of topical sirolimus in this patient population. objectives: to evaluate the safety and efficacy of long-term romiplostim in children with itp. design/method: all patients received weekly sc romiplostim from - g/kg to target platelet counts of - × ( )/l. median (min-max) treatment for the patients was ( - ) weeks for a total of patient-years, or . years per patient. at baseline, median (min-max) age was ( - ) years; % were female; . % had prior splenectomy. median (min-max) average weekly dose was . ( . - . ) g/kg, including escalation to a stable dose; patients started on g/kg. reasons for discontinuing romiplostim (n = , %) included consent withdrawn (n = ), required other therapy (n = ), and ae (n = ) (asthenia, headache, dehydration, and vomiting in one patient and anxiety in the other; none treatment related). fifty four serious aes occurred in patients but were treatment related in one (concurrent grade thrombocytopenia, grade epistaxis, and grade anemia). anti-romiplostim neutralizing antibodies were detected in one patient who discontinued to receive other therapy; antibodies were absent on retesting. from week on, median platelet counts remained > × ( )/l; median platelet counts were > × ( )/l from weeks - . nearly all ( %, / ) patients had ≥ platelet response (platelet counts ≥ × ( )/l, excluding ≤ weeks after rescue medication). most ( %, / ) patients had a platelet response ≥ % of the time and % ( / ) did ≥ % of the time. sixty ( %) patients (or caregivers) self-administered romiplostim. fifteen ( %) patients had treatment-free periods of platelet counts ≥ × ( )/l for ≥ weeks (ie, remission); these patients ( girls, boys) had had itp for a median (min-max) of . ( . - ) years, none had prior splenectomy, and had received romiplostim for . ( . - ) years. all had platelet counts > × ( )/l for ≥ months and / for ≥ months; the median (min-max) duration of being ≥ × ( )/l was ( - ) weeks. of baseline characteristics such as sex, platelet counts, itp duration, and number of past itp treatments ( , , , > ), only age < years was predictive of developing treatment-free periods ≥ weeks (p = . ). in this seven-year open-label extension, > % of children with itp achieved a platelet response and romiplostim was well tolerated. importantly, % of patients were able to discontinue all itp medications for ≥ months. funded by amgen inc. background: sirolimus is an immunosuppressive drug that is widely used in solid organ and bone marrow transplantation, and more recently for the treatment of vascular and lymphatic anomalies. sirolimus has been associated with decreased immunity in the transplant setting in patients that have received other immunosuppressive drugs or were immunosuppressed from previous chemotherapy. the effects of sirolimus on the immune system in chemotherapy naïve children who have not received other immunosuppressive agents are not well understood, and there is variability in the approach to fever and pcp prophylaxis. to understand the effects of sirolimus on the immune system of patients with non-complicated vascular or lymphatic anomalies by evaluating anc, alc prior to and after sirolimus therapy. design/method: multi-institutional retrospective review was done to include patients with non-complicated vascular or lymphatic anomalies. those with effusions/ascites, multiorgan involvement, or history of vascular-anomaly-related infections prior to treatment were excluded. results: twenty patients with kaposiform hemangioendothelioma (n = ), generalized lymphatic anomaly (n = ), cloves syndrome ( ), and simple vascular malformation (n = ) were included. age at initiation of sirolimus treatment ranged from . - years. male to female ratio was : . sirolimus was initiated due to extensive disease, lack of response to steroids or bisphosphonates, pain, dment, lymphatic drainage, and prevention of ongoing overgrowth. prior to the start of sirolimus (sir- ) the mean anc was and alc was . the target level of sirolimus varied by indication and patient, and ranged from to . after the st steady state level, month after sirolimus (sir- ) the mean anc decreased to and alc was . at months after sirolimus (sir- ) the mean anc was and alc was . the first sirolimus levels (sir- ) mean was . ; and sir- level was . . nine patients were placed on pcp prophylaxis at the start of sirolimus. none of these patients had an infectious complication while on sirolimus at a median f/u of months. one patient had mild neutropenia (anc > ) which normalized after discontinuation of pjp prophylaxis. conclusion: in this small cohort of patients we found that the anc and alc level in patients with non-complicated vascular or lymphatic anomalies at sir- was not different from the sir- or sir- . prospective studies that specifically track anc, alc, igg, and lymphocyte function should be conducted to better understand the effects of sirolimus in the immune system. this data will allow for uniform recommendations regarding prophylaxis and management of febrile episodes. background: acute infections and the associated systemic inflammation can increase the risk of venous thromboembolism (vte) and in certain well-defined clinical scenarios may be the primary trigger of vte in pediatric patients. pediatric data on vte in the setting of acute infection are sparse. objectives: to describe characteristics and outcomes of vte in pediatric patients with acute infections. we conducted a retrospective chart review of all pediatric patients with objectively confirmed vte treated at our institution since and identified all patients in whom an acute infection was identified as a vte trigger. patients were managed according to standardized institutional protocols based on published guidelines. relevant demographic, clinical and laboratory data were collected and summarized using descriptive statistics. since , acute infection was identified as a trigger in of vtes ( %) diagnosed at our center. the median age at time of vte diagnosis in this group was . years (interquartile range . - ). males were more commonly affected than females, representing % of cases. neonatal vte events accounted for % of cases. sepsis was the most common acute infection to be identified as a vte trigger [ / cases ( %)]. most vte events ( %) associated with acute infections were considered hospital-associated vtes. at time of vte diagnosis, % of patients were critically ill. extensive vte (defined as completely occlusive thrombosis involving > venous segment) occurred in % of patients. acute infection was deemed to be the primary trigger for vte in / patients ( %). infection-associated vtes in this cohort included cerebral sinus venous thrombosis due to sinus or cns infection ( patients, %), septic throm-bophlebitis ( patients, %), lemierre's or lemierre's-like syndrome ( patients, %) and osteomyelitis-associated deep vein thrombosis ( patients, %). systemic anticoagulation was prescribed in / patients ( %). anticoagulationrelated major bleeding occurred in / patients ( %). vte complications included vte recurrence ( patients, %), vte progression ( patient), acute pulmonary embolism ( patients) and arterial ischemic stroke ( patients). our study indicates that acute infection is a common risk factor for pediatric vte, especially in critically ill children, and can be the primary trigger in a significant proportion of vte cases associated with acute infections. anticoagulation appeared to be overall safe in this population and was associated with low rates of serious vte-related acute complications. however, our study also suggests that this population may be at increased risk for vte recurrence and anticoagulation-related major bleeding. background: epithelioid hemangiomas (eh) are rare benign vascular tumors that occur in soft tissues and bone and present between the third and sixth decades of life. a subset ( %) of eh harbor fos rearrangement. eh has been described in children, but little is known about the long-term outcomes of pediatric eh. the main objective is to obtain data to be used for improved understanding of this rare disease in order to provide standardization of care and development of future research studies. board-approved retrospective review of clinical, pathologic, and radiographic characteristics, and treatment outcomes in patients diagnosed with eh between and . results: eight patients were male; mean age at diagnosis was . years (range: - ). lesions involved the lower extremities (n = ), cranium (n = ), pelvis (n = ), and spine (n = ). multifocal disease was identified in five patients. the most common presentations involved significant localized pain and neurologic symptoms: headache, cranial nerve injury, loss of consciousness. radiographic studies identified variable features, such as multifocal lytic bony lesions with sclerotic margins, enhancing soft tissue component, and surrounding inflammatory edema. histologically, all specimens were composed of vascular channels lined by epithelioid endothelial cells without significant cytologic atypia; solid cellular areas (n = ). endothelial cells were positive for cd and egr, and negative for camta . fos rearrangement was assessed in only one specimen and detected. mean follow-up time was days (range: - ). patients were treated with surgical resection, intravascular embolization, bisphosphonates, propranolol, interferon, and sirolimus. one patient treated with interferon and one with sirolimus exhibited partial response for mean follow-up of . days. although eh is a benign neoplasm, it is difficult to manage without standard protocols and portends considerable morbidity. our findings suggest medical management, particularly sirolimus, may benefit these patients; however, long-term follow-up is needed in treated children. novel fos inhibitors are in development and may benefit patients with fos rearrangement. penn state health children's hospital, hershey, pennsylvania, united states background: central venous catheters (cvc) are often required in critical care settings in order to provide a secure point of access for life sustaining care. clinical studies identify cvc presence as the single most important risk factor for deep vein thrombosis (dvt) in children. venous thromboembolic event (vte) incidence rates in critically ill children with a cvc range from . - % and . - . per catheter days depending on the population studied. per institutional protocol, the penn state health children's hospital picu (hershey, pa) utilizes a low dose continuous infusion of unfractionated heparin (ldufh) at units/kg/hr as prophylaxis against cvc-related vte and to maintain line patency. the efficacy of this approach has never been evaluated. to determine if ldufh for prophylaxis results in lower incidence of cvc-related vte, catheter dysfunction and central line associated blood stream infection (clabsi) without increasing morbidities. to determine if the incidence of catheter related vte is lower than historical published data, a retrospective chart review was conducted utilizing the institutional electronic medical record for all patients in , aged - . years, who had a cvc during a picu admission. secondary objectives such as the incidence of catheter dysfunction, clabsi, and any associated bleeding complications are also being analyzed. results: interim data analysis revealed cvcs ( nontunneled cvc, totally implantable devices, tunneled lines, peripherally inserted central catheters [picc] ) in total patients with a median age of . years. overall vte incidence was . % ( / ) with vtes associated with non-tunneled cvc and with piccs. sixty one percent of non-tunneled cvcs received ldufh and % ( / ) of the patients with vtes associated with non-tunneled cvcs did receive ldufh prophylaxis. vte incidence rate of nontunneled cvcs with ldufh was . % ( / ) and . per picu catheter days. the only other vte events identified within our study cohort were in the picc group where two patients experienced vte, one of which was receiving ldufh. clabsi incidence was . % ( non-tunneled cvc, tunnel cvc, picc). no major bleeding complications were associated with ldufh. preliminary data demonstrates ldufh is efficacious in preventing cvc-related vte in comparison to published reports. further analysis will compare another similar sized and acuity level picu which does not practice the same method. background: fibroadipose vascular anomaly (fava) is a rare, challenging disorder associated with pik ca mutations. fava often causes painful replacement of muscle and soft tissues with fibrotic and adipose tissue and is associated with ectatic draining veins. treatments for focal lesions are surgical excision, cryoablation or sclerotherapy and the role of medical therapy is unclear. some fava lesions are too extensive or directly involve neurovascular structure, resulting in refractory pain. objectives: to retrospectively evaluate the efficacy of sirolimus in patient with residual symptoms after procedural therapies for fava design/method: retrospective review of individual cases from institutions of fava refractory to other therapies treated with sirolimus for at least months. cases were s of s identified by polling member of the aspho vascular anomalies special interest group. results: all seven patients report improvement on sirolimus therapy. all patients had received prior procedures, including sclerotherapy ( patients), cryoablation ( patients) and/or resection ( patients). mean age at sirolimus initiation was y (range - y). mean length of therapy is . months (range - months). six patients were treated with bid dosing and one adult received daily dosing. goals of sirolimus were improvement in pain or musculoskeletal dysfunction. pain and function improved in all patients, including discontinuation of narcotic use and resumption of participation in sports. time to symptom improvement ranged from - weeks. in four patients for whom dose was lowered, pain recurred in all four and responded to restarting or increasing sirolimus dose. while all patients do not have pre-and postsirolimus imaging, decrease in fava lesion size is seen in cases with available imaging. sirolimus side effects are similar to prior reports, most commonly mouth sores, elevated lipids and acne. we report the first known data supporting a role of sirolimus in refractory fava cases. sirolimus is welltolerated and initial improvement is rapid, within weeks of initiation. whether sirolimus has a role in upfront therapy to reduce lesion size prior to procedures deserves further study. objectives: to assess platelet responses in children with itp receiving romiplostim. design/method: eligible children had itp for ≥ months, ≥ prior therapy, and screening platelet counts ≤ × ( )/l or uncontrolled bleeding. weekly dosing was from - g/kg to target platelet counts of - × ( )/l. bone marrow biopsies were evaluated in europe at baseline and after or years (cohorts and ). as of mar , patients received ≥ dose. at baseline, median (min-max) age was ( - ) years, itp duration was . ( . - . ) years, and platelet count was ( - ) × ( )/l; patients ( %) had had prior splenectomy. the median (q , q ) % time with a platelet response (platelet count ≥ × ( )/l, no rescue medications past weeks) in months - was % ( %, %) (primary endpoint). over the course of the study, % ( / ) of patients had a platelet response. four patients maintained platelet counts ≥ × ( )/l with no itp medications for ≥ weeks. median (min-max) treatment duration was ( - ) weeks for patient-years in total. median (min-max) average weekly romiplostim dose over the course of the study was . ( . - . ) g/kg; the median dose was g/kg at year (n = ) and g/kg at years (n = ). most ( %) patients initiated self-administration. sixty-four patients ( %) discontinued treatment, most frequently for lack of efficacy (n = ), patient request (n = ), and adverse event (ae) (n = ). fortyone ( %) patients had serious aes (saes) including epistaxis ( %) and decreased platelet count ( %). five patients had treatment-related saes: headaches, abdominal pain, and each of presyncope and neutralizing antibodies (ab). there were cases of neutralizing ab to romiplostim (of patients tested), but none to tpo; / had continued elevated platelet counts and in / cases ab were not found on retesting. for cohort , of patients with baseline bone marrow biopsies, had evaluable on-study biopsies scheduled for year; patient had an increase from grade to . there were no findings of collagen or abnormalities. in this interim datacut of a romiplostim openlabel study in children with itp, % of children had a platelet response. overall, the median dose was . g/kg; the median romiplostim dose over time reached g/kg. no new safety signals were observed over patient-years. funded by amgen inc. background: hepatic hemangiomas are benign vascular tumors without a medical home, managed by multiple specialties. the diagnosis has been assigned historically to various vascular lesions affecting the liver with completely different clinical presentations, resulting in difficult standardized management. objectives: the consensus steering committee identified an acute need of clear definitions and evaluation guidelines using the updated international society for the study of vascular anomalies (issva) classification. the goal was to formulate recommendations that will be adopted by all specialties involved in the care of children with hepatic hemangiomas. design/method: we used a rigorous, transparent consensus protocol, with input from multiple pediatric experts in vascular anomalies from hematology-oncology, surgery, pathology, radiology and gastroenterology. in the first section, we precisely define the subtypes of hepatic hemangiomas seen in children (congenital and infantile) using clinical course, histology and radiologic characteristics. inclusion and exclusion limits to the diagnosis are noted. the following two sections describe these subtypes in further detail, including complications to be considered during monitoring and respectively recommended screening evaluations. conclusion: while institutional variations may exist for specific clinical details, a clear understanding of the diagnosis of hepatic hemangiomas affecting the pediatric population and the possible complications that require screening during the monitoring period should be standard. as patients with hepatic hemangiomas are managed by different medical and surgical specialties, a multidisciplinary consensus based on current literature, on the data extracted from the liver hemangioma registry and on expert opinion was required and was accomplished by this manuscript. objectives: to investigate the association between routine prophylaxis with bay - and bleeding outcomes after adjusting for key patient and pharmacokinetic (pk) characteristics. design/method: the leopold kids study evaluated safety and efficacy of bay - prophylaxis in previously treated boys aged ≤ years with severe hemophilia a. patients received bay - - iu/kg x/wk (n = ) or > x/wk (n = ) and were followed up for - months. prophylaxis dose and frequency were assigned by investigators. pk parameters, including area under the curve (auc), half-life, and clearance, were derived from a population pk model and reflect predicted pk values with a -iu/kg dose. patient characteristics were compared between the x/wk and > x/wk groups using wilcoxon rank sum or chi-square tests. negative binomial regression was used to model the association between prophylaxis frequency and annualized bleeding rate (abr) for total bleeds, first without adjustment and then adjusting for age, pk parameters, and bleed history. results: mean ± sd age for patients in this analysis was . ± . years. patients receiving prophylaxis x/wk had more bleeding episodes in the months before study entry (mean ± sd, . ± . [median, . ] for x/wk vs . ± . [ . ] for > x/wk; p = . ) and were more likely to have been treated on demand ( % vs %; p = . ). pk parameters were similar between the x/wk and > x/wk groups. without adjustments, abr during the study was % higher in the x/wk group compared with the > x/wk group (rate ratio [rr], . ; % ci, . - . ; p = . ). abr was % lower in the x/wk group (rr, . ; % ci, . - . ; p = . ) after adjusting for age, auc, and number of bleeds in the prior months. conclusion: abr was numerically lower but not significantly different between the x/wk and > x/wk groups after adjusting for age and pk parameters. these findings suggest that even among patient groups that are homogeneous with respect to age, pk, and bleed history, further individualization of bay - prophylaxis based on other characteristics may help reduce bleeding episodes even at a lower treatment frequency. larger real-world studies are needed to verify these findings. funded by bayer. stanford, palo alto, california, united states s of s background: vascular malformations may be of lymphatic, arterial, venous or capillary endothelial origin. they may be simple or complex, with complex malformations being a combination soft tissue and skeletal overgrowth. although likely present at birth, these malformations often become symptomatic with puberty or infection, and range from little or no clinical impact to life threatening symptoms. in malformations primarily of venous origin, pain may be significant and hypothesized to be caused by phlebolith development (intra-malformation thrombi), inflammation, consumptive coagulopathy, vascular engorgement, and endothelial proliferation. anti-angiogenic and anti-platelet therapies have been reported to relieve pain. however, the use of anticoagulation for pain is not well described. objectives: to report clinical features and outcomes of patients with vascular malformations of venous origin treated with anticoagulation for pain. we performed a retrospective review of patients with vascular malformations followed by the hematology service between january and december who were treated for pain with anticoagulation. pain relief was determined both by wong-baker pain scales and patient report. clinical data were extracted from electronic medical records. we identified five patients with venous malformations (vm) who had received anticoagulation for pain. four patients were female and median age was years old (range to years old) at time of initiation of anticoagulation. all five patients had vm of the extremity, two with vm of the lower extremity, and three patients had vm of the upper extremity. two patients had concomitant coagulopathy and demonstrated decreased d-dimer after initiation of anticoagulation. four patients received enoxaparin, and one adult patient received rivaroxaban. all patients reported improvement in pain after administration of anticoagulation. one patient exhibited mild epistaxis and bruising at the injection site. there was no significant bleeding or other complications. pain is a significant complication in patients with venous malformations. our case series suggests that anticoagulation is a safe and effective therapy for pain relief in this population. further investigation is indicated to compare the effect of anticoagulation to other therapeutic interventions such sclerotherapy, surgery, and sirolimus in the treatment of pain associated with venous malformation. maria ahmad-nabi, christine knoll, sanjay shah, lucia mirea phoenix children's hospital, phoenix, arizona, united states background: estimates of the incidence of dvt in patients with osteomyelitis range widely from %- %, however risk factors and outcomes of dvt in this cohort have not been thoroughly established. objectives: this study aims to estimate the incidence of dvt in patients with osteomyelitis, and to assess risk factors and outcomes of dvt in this cohort. design/method: after irb approval, a retrospective chart review was conducted for patients aged - years seen at phoenix children's hospital between - with icd / codes for osteomyelitis. exclusion criteria included chronic recurrent multifocal osteomyelitis, and chronic dvt. demographics, clinical factors and outcomes were compared between osteomyelitis patients with and without dvt using the fisher-exact and wilcoxon-rank sum tests, as appropriate for the data distribution. results: a total of study subjects with osteomyelitis had a mean (standard deviation) age of . ( . ) years. dvt was present in ( % of ) patients, and ( %), ( %) and ( %) patients received anticoagulation for < , - and ≥ weeks, respectively. patients with vs without dvt were more likely to be male ( % vs %; p-value = . ), and had significantly higher rates of bacteremia ( % vs %; p-value = . ). rates of central lines were comparable between dvt and non-dvt patients ( % vs %; p-value = . ); however patients with dvt vs without dvt had significantly longer mean length of stay ( vs days; p-value < . ) and higher rates of icu admission ( % vs %; p-value < . ). the incidence of dvt among osteomyelitis pediatric patients was estimated at %, with risk increased by male sex and bacteremia. patients with dvt had significantly higher rates of icu admission and longer length of hospital stay. many of these patients had standard practice management of their dvt with - weeks of anticoagulation. our data highlights the need for recognition of high risk patients, and the need for future efforts targeting dvt prophylaxis. baylor college of medicine, houston, texas, united states background: lymphatic malformations (lm) frequently occur in the head and neck and can often be disfiguring and even life-threatening. management options include observation, surgery, sclerotherapy, and sirolimus. the optimal sequence of therapeutic interventions has not been determined due to the lack of comparative clinical trials or established guidelines. thus, prenatal planning with a multidisciplinary team is beneficial. we present a case series of ten children with head and neck lms evaluated in at our multidisciplinary vascular anomalies center. a chart review was performed to assess treatment modalities and recent trends. results: seven of patients ( %) with head and neck lms were diagnosed prenatally. six patients required an ex utero intrapartum treatment procedure. all patients were started on sirolimus at a median age of . months (range days - years). four patients most recently started on sirolimus were less than months of age at the time of initiation. six patients underwent partial excision of lm during the first year of life; none of whom received sirolimus prior to surgery. sirolimus was discontinued in one patient given chronic clostridium difficile infections, and non-compliance in another patient. five patients received sclerotherapy. tracheostomy was necessary in six patients; one patient was de-cannulated after months on sirolimus. all patients have had radiographic and clinical improvement of lm with varying treatment modalities. current clinical observations show improved response with sirolimus and demonstrate tolerability of sirolimus at a young age. conclusion: treatment of pediatric head and neck lms is challenging and a multidisciplinary approach is necessary. as the majority of patients are diagnosed prenatally, prenatal planning and discussion of potential use of sirolimus is beneficial. availability of vascular anomalies experts in the prenatal/neonatal period offers the best management results, and early initiation of sirolimus should be considered for complex lesions. long-term follow up is warranted to investigate the efficacy and timing of treatment options. yale school of medicine, new haven, connecticut, united states background: to mitigate transfusion of pathogencontaminated platelets, amotosalen, a synthetic psoralen compound, is added to sdp components. exposure to uv-a light activates amotosalen and crosslinks dna/rna base pairs, preventing replication of a broad spectrum of viral, bacterial, and other pathogens that may contaminate platelets. pr-sdps were fda approved for clinical use with no age restrictions in . we initiated use of pr-sdps in november of for all patients. we retrospectively analyzed usage of pr-sdp vs conventional (non-pr) platelets (cp) in neonatal and pediatric patients with thrombocytopenia to compare hemostatic efficacy and the incidence of transfusion reactions (tr) for these products, after one year of a dual platelet inventory. design/method: since pr-sdp were fda-licensed, no irb approval was required; pr-sdp and cp were both considered standard of care. we evaluated transfusions for all pediatric patients age - years who received any platelet transfusion between november and november . we determined the volume (mean ml ± sd) of each type of platelet component transfused, the number of platelet transfusion episodes, and reported trs based on cdc hemovigilance guidelines. a subgroup analysis was performed for thrombocytopenic neonates ( - months). results: patients - years who received only cps (n = ) received a total of , ml of platelets ( ± ml/patient) over transfusions ( . ± . episodes/patient). for comparison, in patients who received only pr-sdp, a total of , ml of platelets ( ± ml/patient, p = . ) were infused over transfusions ( . ± . episodes/patient, p = . ). for neonates ( - months, n = ) who received only cps, , ml of cps ( ± ml/ patient) were transfused over episodes ( . ± . episodes/patient). for comparison, those who received only pr-sdp (n = ), received , ml of pr-sdp ( ± ml/patient, p = . ), transfused over episodes ( . ± . episodes/patient, p = . ). for all recipients - years (n = ), including additional patients who received both cp and pr-sdp, there were three reported allergic trs over transfusion episodes, while no allergic reactions were reported with pr-sdp transfusions. one febrile tr was reported to cp transfusion, while three were reported for pr-sdp. in conclusion, pr-sdps, in our pediatric population age - years, were comparable to cp products in regards to volume and episodes of platelet transfusions, and incidence/type of transfusion reactions. pr-sdp were safe and effective for use in this pediatric patient population. background: vascular anomalies are classified as either vascular tumors or vascular malformations. fibro-adipose vascular anomaly (fava) is a newly described entity which presents with distinct clinical, radiographic and histopathologic findings. we present a case in which the diagnosis of fava was complicated by a persistent low platelet count secondary to immune thrombocytopenia (itp). to describe a challenging diagnosis of a novel vascular anomaly (fava) complicated by severe thrombocytopenia. a year old male presented to hospital with bruising and left thigh pain related to a remote sports injury. blood work revealed a platelet count of × /l, but with an otherwise normal complete blood count. the following were also normal: aptt and fibrinogen; d dimer levels were slightly increased. he was treated with one dose of ivig ( . mg/kg) for presumed itp and responded well with his platelet count increasing to × /l. he returned to hospital weeks later with recurrent thrombocytopenia and worsening leg pain. an ultrasound of the left thigh revealed a . cm x . cm x . cm lesion within the vastus medialis. the diagnosis of an intramuscular hematoma secondary to persistent thrombocytopenia was made. the patient presented with multiple episodes of thrombocytopenia over the next several months. his itp did not respond to oral prednisone ( mg/day for days). he continued to have short-lived responses to ivig requiring infusions every other week as his platelet count would fall below × /l. his leg pain progressed, restricting him to a wheelchair. further imaging by mri brought into question the diagnosis of a hematoma and a biopsy of the thigh lesion was performed. the results were consistent with a diagnosis of fava; this was subsequently excised. conclusion: this is a unique case where a vascular anomaly was misdiagnosed as a hematoma due to a patient's persistent thrombocytopenia and history of an injury. fava is a newer entity which, unlike other vascular anomalies, has not been linked to thrombocytopenia or a localized consumptive coagulopathy. after excision of the fava, the patient's chronic pain, and mobility resolved, though his itp persisted. objectives: this preliminary, exploratory analysis of realworld administrative data was conducted to determine units dispensed and factor replacement product-related direct expenditures associated with a currently marketed shl or ehl rfix product. design/method: de-identified claims data from the commercially available truven health marketscan® research u.s. claims database were used to identify direct expenditures and number of international units (ius) dispensed for all patients aged - years with a diagnosis code of icd- . /icd- d who used nonacog alfa or eftrenonacog alfa during the study period (june , to july , ). reference weight measurements from the centers for disease control and prevention national center for health statistics' (cdc nchs) anthropometric data were used to estimate product dispensation on an iu per kg basis. the nonacog alfa and eftrenonacog groups comprised and patients, respectively. the median [iqr] age in the two groups was . [ . ] and . [ . ] years, respectively. while of the patients in the eftrenonacog alfa group had > calendar quarter of available data, only of the patients in the nonacog alfa group had > available quarter. the median rfix product dispensation per quarter was , ius (iqr, , ius) in the nonacog alfa group and , ius (iqr, , ius) in the eftrenonacog alfa group. incorporating attributed weight values, the median rfix product iu dispensation per kg per week was . iu/kg/wk (iqr, . iu/kg/wk- . iu/kg/wk) in the nonacog alfa group, and . iu/kg/wk (iqr, . - . iu/kg/wk) in the eftrenonacog alfa group. applying wac prices (eftrenonacog alfa = $ . /iu; nonacog alfa = $ . /iu), the calculated estimates of $/kg/week were $ and $ in the nonacog alfa and eftranonacog alfa groups, respectively. conclusion: preliminary real-world data derived from a large u.s. claims database revealed differences in product dispensation and factor product-related expenditures among pediatric patients with any severity of hemophilia b to whom an shl or ehl rfix product was prescribed. refinements of these data, potentially to exclude instances of sporadic usage, may shed light on real-world dispensation of rfix products among pediatric hemophilia b patients. background: vascular malformations can be classified as simple (including capillary, venous, lymphatic, arteriovenous), combined, malformations of major named vessels or associated with other anomalies. multiple modalities including laser treatments, sclerotherapy, embolization, surgery and pharmacological intervention (with mtor inhibitors like sirolimus) have been used for treatment of vascular malformations. these interventions have been used alone or in combination with varied outcomes. we present our institution's experience with a multimodal approach to simple and combined vascular malformations. design/method: we performed a retrospective chart review of patients with vascular malformations who were referred to our center for an interventional radiology evaluation from june -july . we included patients (age at presentation: months - years), referred initially for interventional radiology procedures (irp) for vascular malformations. all patients had symptoms of pain and/or swelling/deformity. diagnosis of was based on vascular imaging (doppler ultrasound, mri/a/v). nine patients had venous malformations (vm), five had macrocystic lymphatic malformations (lm), six had lymphatic-venous malformations (lvm), and two arteriovenous malformations (avm). patients initially underwent interventional radiology procedures. all the vm patients responded to sclerotherapy alone. three patients with lm responded to sclerotherapy alone, remainder required surgical intervention. one avm patient responded well to embolization, the other needed surgical resection after embolization. four lvm patients underwent irp with minimal improvement in symptoms ( - procedures attempted), surgical resection was attempted in patients with poor response and patients were started on sirolimus ( . mg/m /dose twice a day). all lvm patients started on sirolimus have responded well (decreased pain and swelling); time to initial symptom response ranged from weeks - month from starting medication. in this case series, patients with simple vm responded well to sclerotherapy alone, avm and lm patients needed irp and/or surgery for complete response. complex lvm did not respond well to surgery or irp; . % had improvement in clinical symptoms with addition of sirolimus to the treatment regimen. response to various modalities of treatment varied based on the type of vascular malformation. a multidisciplinary approach to management of vascular malformations is essential to provide multimodal therapeutic options for rapid symptom relief and improve the quality of life of these fragile patients, especially those with complex malformations. background: von willebrand disease (vwd) is the most common bleeding disorder in humans, affecting ∼ % of the united states' population. desmopressin (ddavp) is a longacting vasopressin analog that induces vasoconstriction and release of vwf. ddavp is used in patients with vwd and as a surgical prophylaxis, but carries anti-diuretic properties. to avoid electrolyte imbalance and hyponatremia, fluid restrictions are recommended in the hours post-ddavp administration. objectives: this study sought to examine perioperative practices and outcomes following ddavp administration and a fluid restriction protocol in a population of pediatric patients with von willebrand disease. design/method: a retrospective chart review was conducted for patients with von willebrand disease who underwent surgical procedures at children's hospital of pittsburgh of upmc between january , and december , . patient age, sex, weight, diagnosis, surgical procedure, total fluids administered, and post-operative sodium level were recorded. the primary outcomes noted were the proportion of patients exceeding % of the recommended fluid consumption for the -and -hour periods post-ddavp s of s administration, as defined by local guidelines. secondary outcomes were the presence of any bleeding requiring an er visit or readmission or hyponatremic seizures within hours of ddavp administration. results: data was compiled for patients ( females, males). the mean age was . years (sd . years), median age was years (range to years). procedures included dental ( ), otolaryngology ( ), orthopedics ( ), gastrointestinal ( ), plastics ( ), neurosurgery ( ), ophthalmology ( ), dermatology ( ), general surgery ( ) and gynecology ( ). % of patients exceeded % of the fluid volume recommended for the first -hour period post-ddavp administration while still in the surgical setting. no patients exceeded % of the fluid volume recommended for the total -hour period post-ddavp administration. post-operative sodium levels were obtained in only of patients. no patients returned to the er or were admitted for bleeding in the hours post-ddavp administration. no patients returned to the er or were admitted for hyponatremia or seizures in the hours post-ddavp administration. maintenance of a fluid restriction protocol effectively deterred negative outcomes in this cohort. however, a significant fluid volume was administered in nearly a third of patients despite the restrictions. given the risk of hyponatremia, and limited compliance with fluid restrictions, postoperative sodium levels should be recorded in following ddavp administration to assess the possibility of a hyponatremia and to reinforce the importance of fluid restrictions and their communication. results: a male fetus required in utero insertion of a pleuroamniotic shunt for bilateral pleural effusions diagnosed antenatally by ultrasound. shortly after delivery at term, he developed respiratory distress and was found to have reaccumulation of the pleural effusions. blood work on day of life showed a platelet count of , / l, which then decreased precipitously. he demonstrated schistocytes on blood-smear, signs of consumptive coagulopathy with hypofibrinogenemia and high d-dimers, and compensatory reticulocytosis. he required multiple transfusions and admissions to the intensive care unit for respiratory support. investigations ruled out congenital ttp, neonatal alloimmune thrombocytopenia, and noonan syndrome. given high clinical suspicion for an underlying vascular lesion causing kmp, a full body mri without contrast was undertaken. this showed a focal area of suspicious signal intensity in the upper paraspinal musculature. an ultrasound and mri with contrast demonstrated an extensive infiltrative vascular lesion involving the paraspinal musculature, prevertebral space, posterior extrapleural space, mediastinum, and neck. the child was commenced on prednisone ( mg/kg/day) and rapamycin ( . mg/m twice/day). there was no clinical or laboratory improvement after one month. a biopsy was performed which confirmed khe. in the second month of rapamycin therapy, the platelet count gradually normalized and the patient was discharged from hospital at . -months of life. prednisone was weaned off at . months of life. a repeat mri at months showed significant reduction in the khe. he is now almost years into therapy and doing well. conclusion: this is a unique case of khe with kmp that initially presented with extensive and recurrent pleural and pericardial effusions. this case demonstrates the importance of suspecting an underlying vascular malformation in the presence of kmp. our patient had a delayed but overall good response to rapamycin. further studies investigating duration of rapamycin therapy is key for the optimal management of these patients. rosa diaz, donald mahoney, lakshmi srivaths, donald yee texas children's hospital, houston, texas, united states background: since von willebrand disease (vwd) is the most common inherited bleeding disorder, it must co-exist with other less common bleeding disorders in some dually affected patients. however, reports of combined deficiencies in factor viii (fviii) and von willebrand factor (vwf) are rare. objectives: to study the prevalence and bleeding phenotype of combined deficiencies of fviii and vwf in males with hemophilia a in a hemophilia treatment center. design/method: we retrospectively reviewed the electronic medical records of males with hemophilia a followed at our institution during the past years. the primary and secondary outcomes for the study were ( ) the prevalence of combined fviii and vwf deficiencies and ( ) the bleeding phenotype of these patients. we identified vwf deficiencies in % (n = ) of the patients with hemophilia a. most (n = , %) patients were tested for vwf deficiency as part of the initial hemostatic evaluation, but one-third were tested due to clinical concern for inadequate response to fviii concentrate. the median duration of follow up was . years (range . to . ). patients were referred to our clinic at a median age of months (range to years) for evaluation of easy bruising (n = , %), mucosal (n = , %) and surgical bleeding (n = , %). primary diagnoses included with severe, moderate and mild discrepant hemophilia a. secondary diagnoses included with low vwf activity, type vwd and with type unclassified. patients experienced episodes of musculoskeletal (n = , %), mucocutaneous (n = , %) and cns bleeding (n = , %). a total of patients received factor prophylaxis. half of the patients were initially treated with fviii concentrates but subsequently changed to combined fviii/vwf products due to the frequency of breakthrough bleeding despite good compliance. all patients are on combined fviii/vwf products at the time of this review. a total of ( %) of this cohort developed chronic joint disease manifest as decreased range of motion and/or abnormal mri findings. combined deficiencies of fviii and vwf were present in % of our center's hemophilia patients. these patients exhibited a severe bleeding phenotype as evidenced by the high frequency of hemarthrosis, need for prophylaxis and high prevalence of chronic joint disease. while the optimal treatment strategy remains to be elucidated, early recognition of a combined deficiency may have important clinical implications, particularly in patients who demonstrate a suboptimal response to fviii concentrate alone. background: childhood neutropenia is heterogeneous and may be congenital or acquired. cerebral cavernous malformation (ccm ) is a neurovascular malformation disorder where lesions consist of low flow, dilated capillary endothelial channels with increased permeability, predisposing to hemorrhage and thrombosis. programmed cell death protein (pdcd ) activity has been implicated in glia and neuron migration, and recently linked to the dysregulation of the actin and microtubule cytoskeleton, thereby affecting cellular morphology and migration. variants of pdcd encoding pdcd have been associated with ccm . ccm causes a greater and earlier disease burden than other ccms, with % presenting younger than years. some patients have associated extra-neuronal manifestations, suggesting that pdcd plays a role in other tissues. we describe a patient with significant blood cytopenias associated with ccm . design/method: retrospective chart review to obtain patient data. results: an -month old female presented with seizure and was found to have multiple intracranial cystic lesions and abscesses due to s. pneumonia serotype f. during her treatment, she developed anemia (hemoglobin . - . g/dl), thrombocytopenia (platelets , - , cells/l), and profound neutropenia (absolute neutrophil counts of zero). initial bone marrow evaluation revealed a normocellular marrow but with marked granulocytic hypoplasia and % hematogones on flow cytometry. florescent in situ hybridization excluded cytogenetic changes characteristic of myelodysplastic syndrome. further evaluation included testing for neutrophil antibodies, chromosome breakage, and telomere length and results were normal. whole exome sequencing excluded mutations affecting congenital neutropenia genes, but detected a de novo pdcd variant (c. + g>a), thereby diagnosing ccm . the neutropenia has responded well to granulocyte colony stimulation factor (gcsf), which is still needed at months of age. moreover, the thrombocytopenia has progressed, requiring periodic platelet transfusions. over time, the bone marrow hematogone population has decreased to % at months of age, though the granulocytic hypoplasia persists. conclusion: our case describes the first patient with neutropenia and thrombocytopenia associated with ccm . we hypothesize the pdcd variant is the etiology of bone marrow dysfunction due to its role in actin and microtubule cytoskeleton formation, akin to the pathophysiology of xlinked neutropenia. supportive features of an underlying genetic cause of marrow dysfunction include the persistence of cytopenias beyond infection resolution as well as presence of hematogones. hematogones were previously reported to occur in patients with other congenital neutropenia disorders, indicating they could be a feature of congenital neutropenia and may be reactive to surrounding cell apoptosis. further testing of pdcd role in hematopoiesis should be explored. background: - % of adult women will suffer from heavy menstrual bleeding (hmb) during their lifetime. % of women with inherited bleeding disorders suffer from hmb. there is a paucity of data about hmb among adolescents and young adults (aya), a population in which hmb may have large social and educational effects. objectives: to study the social and academic implications of hmb in an aya population. design/method: this is a questionnaire based survey conducted in a medium-sized city in california. we recruited females - years of age from one high school and from local university. the questionnaire was set up in research electronic data capture (redcap) at our institute which allowed us to obtain objective data about the respondents' menstrual cycles. a link was sent to the high school students via their online portal schoolloop and to the university students via social media and word of mouth. data was collected over weeks from may to august . we received replies, some were not complete. using regression analysis, data was analyzed from respondents in the age group of - (with a mean age of ) years. we developed a composite score for hmb based on factors including saturation levels, number of pads, duration of bleeding, soaking of a pad within two hours, passage of clots, size and number of clots, and gushing sensation. we conducted statistical analysis of the drivers and implications of hmb based on the composite score. results indicate that having a relative with hmb, having other bleeding problems, and having anemia are drivers of higher hmb score. the results also indicate that hmb adversely affects quality of life as measured by participation in sports, social activities, after-school activities, tiredness, absenteeism, and gpa. hmb is also associated with increased rates of anemia and use of anti-depressants. hmb-driven anemia further adversely affects gpa. under-represented minorities are more likely to have a higher hmb score, as well as an increased adverse effect of hmb on gpa. the results suggest that the social costs of hmb are pervasive in the aya population, and especially pronounced among minorities. a relative with hmb is a significant driver of heavy menstrual bleeding. a hemostatic screen should be included when assessing the aya population with hmb. johns hopkins all children 's hospital, st. petersburg, florida, united states background: propranolol is a non-cardioselective beta blocker medication frequently prescribed for hemangiomas and hyperthyroidism. propranolol inhibits types i and ii iodothyronine deiodinases, enzymes that convert bioinactive thyroxine (t ) into bioactive triiodothyronine (t ). hypothyroidism is a well-recognized complication of diffuse hepatic hemangiomas that produce type iii deiodinase, an enzyme that converts t into bioinactive reverse t and t into diiodothyronine. thyroxine is typically selected for replacement in this population, even though doses up to % above physiologic may be necessary. we hypothesized that low dose, nearly physiologic t would be safer and equally effective because it bypasses propranolol's impact on the pituitarythyroid axis. we report an infant with diffuse hepatic hemangiomatosis and acquired hypothyroidism successfully treated with propranolol, prednisone, and triiodothyronine. design/method: a mo healthy female presented with abdominal distension, poor oral intake, and hepatomegaly. mri confirmed diffuse hepatic hemangiomatosis, the largest lesion measuring . cm by . cm. thyrotropin (tsh) was elevated at . (reference range* . - mcgiu/ml), total t # (rr - ng/dl), and total t ^ . (rr - mcg/dl). treatment was started with prednisone ( mg/kg/day) for three weeks, propranolol ( mg/kg/day) and t ( . mcg/kg/day). the t dose was slowly titrated to a maximum of . mcg/kg/day. thyroid hormone levels rapidly improved on t replacement. after two weeks, the tsh was . , tt , and tt . . after eight months, the tsh was . , tt , and tt . . at twelve months, the tsh dropped to . , tt , and tt . , suggesting decreased tumor production of type iii iodothyronine deiodinase. liver mri confirmed fewer hemangiomas, largest being . cm by . cm. the patient's t dose was reduced. both propranolol and t were discontinued after twenty-four months of treatment. one year off all therapy, this child has normal growth and development, only two < . cm hepatic hemangiomas and no evidence of hypothyroidism (tsh . ; tt ; tt . ). conclusion: t at near physiologic doses corrects the consumptive hypothyroidism associated with diffuse hepatic hemangiomas. t replacement is preferable to thyroxine due to its lower risk of rebound hyperthyroidism as the hemangiomas involute and type iii deiodinase production declines. there are two prior case reports describing t use without t , one employing propranolol and the other utilizing steroids for hemangioma management. this is the first case report with long term follow-up of a child treated with multimodal therapy including propranolol, prednisone, and triiodothyronine. *rr = reference range; #tt = total t ;^tt = total t background: multifocal lymphangioendotheliomatosis with thrombocytopenia (mlt) is a rare congenital disorder first described in that is characterized by multiple vascular abnormalities commonly involving the skin and gastrointestinal tract as well as consumptive coagulopathy often resulting in gi bleeding in infancy( ). to describe an unusual presentation and successful management of mlt in a neonate. design/method: baby h was born at full term after a pregnancy complicated by maternal sinus venous thrombosis requiring anticoagulation beginning at weeks. at birth, she was diagnosed with multiple hemangiomas based on clinical exam. at two weeks of age, she developed melena and hematemesis. cbc revealed platelet count of and she was referred to the ed. abdominal ultrasound was concerning for abnormal hepatic waveform; cxr showed multiple pulmonary nodules. workup revealed no other lesions and no further hematologic abnormalities. biopsy of presumed hemangioma ultimately revealed a smooth muscle-lined vascular proliferation without glut- immunoreactivity, consistent with mlt. her early course was complicated by an acute hemodynamically significant gi bleed; esophagogastroduodenoscopy identified six bleeding vascular malformations within the stomach that were injected with epinephrine and sclerosed with successful hemostasis. she received multiple prbc and platelet transfusions. central access was obtained and she was started on oral sirolimus based on previous reports of successful use in management of vascular malformations given its antiangiogenic and immunosuppressive effects ( ). she has tolerated it well with no evidence of toxicity and has achieved a partial response with stable of hemoglobin > and platelet count > . cutaneous lesions have diminished in intensity and she has had no further signs of gi bleeding. she receives pentamidine for pcp prophylaxis. she continues to have appropriate growth and development. we describe here an unusual presentation of an already rare disease. while cutaneous and gi lesions are typical of mlt, pulmonary involvement is not well-described in the literature. early identification of tissue-based diagnosis enabled timely stabilization and treatment of the patient. five months later, she continues to tolerate sirolimus and has shown significant response with diminished coloration of cutaneous lesions, stable blood counts, and no further bleeding. mlt is a relatively newly-recognized disorder with significant phenotypic variability. given that bleeding secondary to a kasabach-merritt-type consumptive thrombocytopenia is the major cause of morbidity and mortality in the first year of life in children with mlt, it is essential to recognize the diagnosis and initiate appropriate treatment as early as possible. north, arch background: patients with generalized joint hypermobility (jhm) may experience easy bruising or bleeding given the association between these symptoms and abnormalities in collagen, a required component of primary hemostasis. heavy menstrual bleeding (hmb) is a common initial presentation for females with underlying hemostatic defects and may be the sole manifestation of a bleeding disorder. however, limited reports describe jhm as a cause of hmb, leading to under recognition. objectives: to describe the clinical characteristics and management of young women presenting with hmb in the setting of jhm. design/method: this study utilized our hmb research registry. we included subjects - years, seen in the nationwide children's young women's hematology clinic between february and november with both hmb and jhm. medical records were retrospectively reviewed for history of presentation, menorrhagia impact questionnaire (miq): a validated quality-of-life tool for females with hmb, medication profiles and relevant laboratory studies. results: twenty-five patients met inclusion criteria (median age years, range - ) with an average beighton score of . (range to ). participants presented an average of . years (range months to years) after menarche despite % of patients reporting heavy to very heavy menses since menarche. according to the miq responses, most participants expressed hmb-associated limitations in physical activities ( %), social activities ( %), and work or school activities ( %). of the participants, % reported bleeding symptoms in addition to hmb, most commonly easy bruising ( %), epistaxis ( %) and cutaneous bleeding ( %). forty percent of young women presented with anemia due to chronic blood loss. results of hemostatic testing were unremarkable, with the exception of one patient who was also found to have type von willebrand disease. additionally, % of females reported arthralgia, with knees and ankles the most commonly affected joints. at time of presentation, % of participants reported failure of initial therapies and most patients ( %) were managed long-term with oral hormone therapy. in a small population of young women found to have jhm who initially presented with hmb, patients were likely to have prior bleeding symptoms as well as substantial delays from menarche to timing of presentation at our young women's hematology clinic despite limitations in activities of daily life. greater awareness of the associations between bleeding symptoms and jhm, despite typically normal hemostatic laboratory results, is necessary so that patients can more easily be identified and receive appropriate therapy. the objective is to determine the impact of cl care practices involving the home environment on ambulatory clabsi rates. design/method: information for the pi was collected through a comprehensive survey that was completed annually by the ccbdn member hospitals. responses to the questions about cl care practices involving the home environment were selected from the pi for . ambulatory clabsi rates and ambulatory total bloodstream infection (bsi) rates were obtained from another ccbdn database. the proportion of hospitals that did or did not employ a particular cl care practice was tallied. the mean ambulatory clabsi rate and mean ambulatory total bsi rate of the hospitals that did or did not employ a particular cl care practice were compared using generalized linear model techniques assuming an underlying negative binomial distribution. results: twenty-five hospitals submitted responses to the questions about cl care practices involving the home environment. one hospital was excluded for lack of bsi data. sixty-three percent of the hospitals programmatically educated parents about all aspects of the cl care bundle. the mean ambulatory clabsi rate for the hospitals that educated parents was significantly lower than that of the hospitals that did not ( . infections/ cl days vs. . infections/ cl days; p = . ). the mean ambulatory total bsi rate was also significantly lower ( . infections/ cl days vs. . infections/ cl days; p = . ). the mean ambulatory clabsi rates and mean ambulatory total bsi rates were not significantly different for the other cl care practices. conclusion: an analysis of cl care practices involving the home environment reveals that parental education of all aspects of the cl care bundle is associated with a lower ambulatory clabsi rate and lower ambulatory total bsi rate. this finding highlights the importance of systematically teaching family members the proper method of handling cl. background: children undergoing chemotherapy are at a high risk for developing nausea. dr. amy baxter in collaboration with pediatric oncology patients and nurses, developed and validated a pictorial nausea rating scale for children aged - years, called the baxter retching faces (barf) nausea scale. staff nurses at a large, academic, pediatric hospital located within washington, d.c., have identified variability in nursing assessment and documentation of chemotherapy induced nausea and vomiting (cinv) in pediatric oncology patients. the purpose of this quality improvement project was to utilize the barf scale to standardize assessment and documentation of nausea in pediatric oncology patients receiving chemotherapy. the primary aims of this project were to: assess feasibility of the barf scale in clinical practice; increase nursing knowledge about cinv through education sessions; increase documentation of nausea assessments through the use of the scale. the secondary aim of this project was to: increase the recognition of nausea through the use of a standardized assessment tool. design/method: the pdsa model was used to guide the design and implementation plan. in the first phase of the project data was collected to identify the prevalence of nausea in patients admitted for chemotherapy in the prior three months. education sessions discussing cinv and the utilization of the barf scale were conducted. pre and post assessment of nurses' knowledge of cinv and documentation were assessed. in the second phase the barf scale was implemented into practice. nurses were asked to utilize the barf scale to assess and document nausea scores in patients, aged to years, receiving chemotherapy. at the end of the implementation period nurses were surveyed about the feasibility of the scale. post data was collected to identify the prevalence of nausea documented in the electronic health record. this project was undertaken as a quality improvement initiative at children's national and it does not constitute as human subjects research. as such it was not under the oversight of the institutional review board. results: all data has been collected; however complete data analysis will be conducted in the upcoming weeks. background: sickle cell disease (scd) is the most common inherited blood disorder in the united states (us); however, there are few quality measurements to evaluate scd practice. in , the nhlbi published guidelines that include two key interventions for children with sickle cell anemia (sca): the use of transcranial doppler (tcd) screening for stroke prevention and hydroxyurea (hu) to prevent scd pain crisis. we conducted a national survey of scd management sent to providers in over institutions in the us to better assess knowledge of the guidelines and barriers to hu counseling and tcd screening guideline implementation. it was hypothesized that the barriers to tcd screening are different than barriers to hu counseling and prescribing. a -question anonymous survey was sent to providers by mail (follow-up by email). survey themes included nhlbi guidelines knowledge and comfort with understanding and implementing both tcd screening and hu use. the response rate was % ( / ) however one survey was incomplete. thus, were analyzed in the final data set. all of the respondents are in active practice, % s of s in academics and all care for children with scd. the majority of providers ( %) felt "very" or "extremely" confident in their knowledge of tcd screening and interpretation. similarly, % of providers felt "very" or "extremely" familiar with hu dosing and management. for tcd screening, % of providers estimated their screening rates were > % and % providers felt their annual screening rates were - %. the two biggest barriers to tcd screening noted by providers (of moderate to extreme significance) included: lack of support staff ( %) and lack of time during a patient visit ( %). regarding hu prescribing practices, % of providers offered hu to at least % of children with sca over nine months of age. the biggest barrier to hu prescribing noted by % of providers was concerns about patient adherence or access to the medication. only % providers felt that lack of support staff was a moderately significant barrier to hu prescribing. the pediatric scd providers surveyed all have access to the nhlbi guidelines. despite widespread guideline knowledge, there are different barriers for tcd screening versus hu prescribing, which prevent optimal implementation. as a result, although both recommendations are from the same nhlbi guideline, they likely will require different implementation strategies (systems-based interventions for tcd screening; interventions to improve patient adherence for hu counseling) to improve outcomes. background: invasive fungal disease (ifd) is a major cause of mortality and morbidity among pediatric immunocompromised patients such as those who receive chemotherapy or hematopoietic stem cell transplantation. the current diagnostic 'gold standard' of ifd remains culture of infected tissue obtained by biopsy. noninvasive biomarker testing for galactomannan or , -beta-d-glucan (bg) can have low sensitivity and does not provide species-level identification. nextgeneration sequencing (ngs) of cell-free plasma is a promis-ing noninvasive approach to providing species-level identification of ifd via a blood test and can further guide specific treatment. objectives: describe the incidence of positivity for fungal specific pathogens on ngs analysis in a high-risk immunocompromised pediatric population and correlate results with other 'standard' infectious studies if performed. design/method: immunocompromised pediatric patients with suspected ifd were enrolled and plasma was collected at time of enrollment. ngs was performed on extracted dna in cell-free plasma (karius, redwood city, ca). after removing human reads, remaining sequences were aligned to a curated database including pathogens. organisms present at a significance-level above a predefined threshold were reported. results: twenty-seven samples from enrolled patients have been processed thus far. of these subjects, were enrolled for prolonged febrile neutropenia (≥ hours) despite broad-spectrum antibiotics, for recrudescent febrile neutropenia, for abnormal imaging, and with other findings. after evaluation of routine studies performed, patients met criteria for proven ifd, for probable ifd, and for possible ifd using eortc/msg guidelines. the ngs plasma test identified the same pathogen as cultured from infected tissue or blood in % ( / ) of the proven cases. in the probable cases, pneumocystis jirovecii was identified in a patient with a positive bg ( pg/ml) and pneumonia. among the possible cases, toxoplasma gondii was detected in a patient with prolonged febrile neutropenia and lung imaging suggestive of ifd. additionally, candida glabrata was isolated in a patient with prolonged febrile neutropenia but no other criteria for ifd. numerous pathogens were also identified that could explain the above clinical parameters, including hsv , cmv, vzv, hhv , ebv, bk polyoma virus, and ureaplasma parvum. the cell-free plasma ngs test can detect invasive fungal infections from blood. the test identified fungi from proven ifd, detected pathogens in both probable and possible ifd cases, and is a useful diagnostic tool in the evaluation of ifd. supplies and sample shipment and processing supported by karius, inc. baylor college of medicine, texas children's hospital, houston, texas, united states background: practicing medicine is a lifelong learning process. as noted in the institute of medicine's seminal report, 'to err is human,' adverse outcomes do not typically result from individual recklessness; rather, they result from faulty systems, processes, or conditions that provide an environment conducive to making a mistake, or failing to prevent one. learning to systematically review errors and translate lessons learned into quality improvement (qi) initiatives is a critical component of practice-based learning and improvement for practitioners at all career levels. objectives: to develop a methodical, self-reflective and nonthreatening approach to incident analysis and translation of lessons learned into qi initiatives. design/method: we used a validated, structured case audit approach, modified from szostek et al: ) review all documentation relating to the case and identify all health care providers involved; ) interview stakeholders, including those who directly provided and supported care; ) use a qi tool to conduct a root-cause analysis; ) identify a systems issue that contributed to the outcome; and ) propose systems-level interventions and prioritize initiatives based on effort-yield projections. results: pdsa cycle : plan: establish a committee to ) identify potential cases, ) triage cases for conference presentation, ) determine timing and frequency of conferences, ) develop a training manual, ) record identified qi initiatives. do: we established a quarterly section-wide meeting to which all members of the pediatric hematology/oncology service are invited, including administrative and nursing leadership. we developed a training manual and structured presentation template. prioritized cases were discussed in advance during multidisciplinary case review sessions, and presented by senior fellows who were instructed to focus discussion on potential opportunities for qi. study: we identified cases, meeting criteria for mmi presentation. qi initiatives identified from this conference resulted in a number of systemic practice changes; however, we encountered challenges to sustaining these changes over time. act: objectives for the next pdsa cycle are to ) establish a method for tracking the adherence to recommended changes in practice, ) maximize sustainability by integrating qi initiatives into institutional qi leadership and practice standardization committees. we have successfully implemented an mmi conference that meets out of institute of medicine quality domains: safety, effectiveness, patient-centeredness, timeliness, and efficiency. a standardized, consistent approach to mmi presentations that includes identification of contributing factors and specific qi implications has the potential for improving both provider education and patient care/safety. johns hopkins university, baltimore, maryland, united states background: receiving a cancer diagnosis is a life-changing event for patients and caregivers, although little is known about the experience. while some oncologists receive dedicated training in delivering this bad news, the initial conversation is often with a primary pediatrician, and these providers often feel they do not receive adequate training in the communication of a cancer diagnosis. objectives: our objectives were two-fold: first, to better define the experiences of caregivers/patients when told of a cancer diagnosis, and to query how caregivers/patients believe providers can improve the disclosure of this bad news. secondly, to assess what, if any, training primary pediatricians received in this skill, and to assess how comfortable providers in various settings and stages of training are with communicating cancer diagnoses. design/method: from november - , semistructured, in-depth interviews were conducted with pediatric oncology patients and caregivers of patients (n = ) diagnosed in the past year regarding their experiences receiving the diagnosis at our institution. in addition, pediatric residents (n = ), outpatient pediatric primary care physicians and pediatric emergency medicine physicians (n = ) were interviewed regarding their experiences delivering cancer diagnoses. interviews were analyzed following principles of thematic analysis. interviewers with patients and caregivers had two common themes: ) all emphasized their wish for direct and thorough information; ) both patients and caregivers emphasized the gratitude they felt for physicians who gave them hope by emphasizing the good prognosis of their child's cancer. lack of training in this area, as well as lack of comfort delivering this news was common will all providers. additionally, providers report variable approaches to giving bad news, including ) whether to tell caregivers separately or tell the child and parents together, and ) whether to give favorable prognostic information. additionally, attending physicians also differed significantly in their approaches to teaching residents. while some believed residents should give the news to gain experience, others felt that this is not appropriate if residents are inexperienced. only one resident reported ever receiving feedback on his communication skills in this type of discussion. conclusion: we plan to build on these interviews to develop a national survey of patients, caregivers, and providers to better understand the issues surrounding this discussion. we will use the findings to develop a communication curriculum for pediatric residents, focusing on the discussions that occur in the outpatient setting by primary pediatricians. background: human papilloma virus (hpv), common in both females and males, is responsible for pathologies ranging from benign genital warts to cervical and penile cancer. hpv strains and are responsible for , malignancies each year in the united states, and one third of them arise in men. pharmaceutical companies have now developed a vaccine that will help prevent the virus-associated malignancies. the cdc initially recommended that females ages - years receive the vaccine series, then starting in they expanded the eligibility to males ages - years. despite being widely available and highly publicized, only % of eligible females receive the full vaccine series. objectives: this study aims to assess the knowledge of hpv, the attitudes towards the hpv vaccine, and identify barriers preventing its full utilization. once identified, we aim to overcome the barrier(s) in order to improve vaccination rates in eligible adolescents. we distributed a standardized questionnaire to the parents of eligible female and male patients in our pediatric hematology-oncology clinic. it assessed the parents' knowledge of hpv and the vaccine, their views of the vaccine, and reasons why they may oppose it. results: approximately % of parents claim they have been educated about hpv, mostly by their primary care physician. however, % did not know what disorders hpv caused; % felt the vaccine should not be added to the typical vaccine schedule; % of parents do not intend to vaccinate their child. of those that opposed the vaccine, one-third were concerned about potential side effects and nearly % feel they do not have enough information. additionally, % of parents are not aware that the vaccine is available at their child's doctor and only % of parents have discussed the hpv vaccine with their child's doctor. the largest barrier to the utilization of the hpv vaccine that we have identified appears to be lack of educa-tion. as a result, we have begun distributing the cdc's hpv and vaccine patient guide to our patients' families as an intervention. we are currently in the process of re-administering our survey to these families after implementing the intervention to assess its success in increasing both knowledge and utilization of the hpv vaccine. cancer institute, chennai, chennai, tamilnadu, india background: rasburicase is a recombinant urate oxidase enzyme approved for use in tumor lysis syndrome (tls) and it acts by reducing serum uric acid levels. using rasburicase at the recommended dose of . mg/kg/day for days is expensive and it is not known whether this extended schedule is clinically beneficial compared to a single fixed dose of . mg. the aim of the present study was to evaluate the efficacy of single dose rasburicase . mg in prevention and management of tls. design/method: rasburicase is available as single use . mg vial. at our institution a single dose of rasburicase . mg irrespective of bodyweight has been used in adults and in children a dose of . mg/kg (maximum . mg) has been used since for prevention and management of tls and subsequent doses are given based on biochemical response and clinical condition. we retrospectively analysed the case records of patients who had received rasburicase from january to january . the study included patients with hematological malignancies who received rasburicase. children accounted for . % (n = ) patients and males comprised % (n = ). rasburicase was used prophylactically in ( . %) patients, for laboratory tls in patients ( . %) and for clinical tls in ( . %) patients. single fixed dose rasburicase prevented laboratory/clinical tls in % of the prophylactic group and prevented clinical tls in % of the laboratory tls group. none of the patients in prophylactic and laboratory tls group developed clinical tls. however, majority of the patients with clinical tls required more than one dose rasburicase. single dose of . mg ( vial) rasburicase is efficient in preventing and managing laboratory tls and is economically viable in resource constrained settings. nicole wood, lauren amos, nicholas clark, chris klockau, karen lewing, alan gamis children's mercy kansas city, kansas city, missouri, united states background: medication reconciliation for newly diagnosed oncology patients is complicated and cumbersome. these patients are often admitted on no medications, and leave on multiple. chemotherapy and supportive medications are crucial. despite numerous individuals overseeing this process, prescribing errors or omissions still occur. when reviewing the literature, improvement occurs when there is an interprofessional and standardized process to medication reconciliation. objectives: this project's aim was to improve the accuracy of the discharge medication reconciliation process from % to % from february -august . the process measure was the percentage of patients discharged with an accurate checklist. additional time for staff spent in completing the checklist and avoiding an increased error rate by changing the prescribing process were followed as balancing measures. we created a discharge medication checklist which included a list of required home medications prescribed by the resident, ideally hours prior to discharge. it required fellow or attending review and pharmacy to review the list and educate the family. checklists were collected monthly and reviewed against the electronic medical record (emr) for accuracy. results: six pdsa cycles were completed. there were errors during the data collection time frame. in pdsa cycle , a patient received acetaminophen for pain control which is avoided at home. in addition, this patient received diphenhydramine instead of ondansetron, which is preferred as an antiemetic. in pdsa cycle , a patient with a pending diagnosis was sent home with acetaminophen. of note, this patient did not have a checklist completed upon discharge. this project provides a novel and important method to standardize the discharge medication reconciliation process in a complex patient population. it clarifies which types of medications these patients need, provides pharmacy teaching to families which was not done previously, and prescribes discharge medications to families sooner. after the first medication reconciliation error, the checklist was revised. no further errors were made following revision, with the exception of one patient without a completed checklist at dis-charge. our accuracy rate increased from % at baseline to % following implementation. we are in the process of making the checklist electronic and accessible in the emr. in the interim between the end of data collection and implementation into the emr, a leukemia patient was sent home without an epinephrine pen, further demonstrating the importance of this standardized discharge process. for this reason, we have re-instituted the checklist until the electronic version is available. background: survivors of pediatric cancer are at risk of losing pre-existing protective antibodies to vaccine preventable diseases. in a prior study, % of children < years lost humoral immunity to measles as a result of chemotherapy induced alterations in immune system. measles in recipients of immunosuppressive chemotherapy has mortality rates up to %. because of volitional vaccine refusal, there has been a dramatic increase in measles infection from cases in to in , including several statewide outbreaks. small pediatric oncology practices frequently share floor/clinic space with the general pediatric patients putting them at risk for measles since virulence starts hours prior to symptoms. there is no standard protocol for revaccinating post-chemotherapy patients. to assess measles risk based on serial humoral immune status in a cohort of pediatric oncology patients receiving intensive chemotherapy design/method: patients < years age with known vaccination status receiving intensive chemotherapy between july -june at our institution's pediatric oncology practice were included in this prospective study. serial measles igg antibodies were measured at diagnosis, months and months after initiation of chemotherapy using elisa. measles immunity was defined per lab standards. a comparison of pre-chemotherapy and serial post-chemotherapy immunization titers was made for all patients by diagnosis. the study population consisted of children ( male); patients had all, non-hodgkin lymphoma, sarcoma and other solid tumors. two patients ( . %), both unvaccinated had non-protective measles antibody levels at s of s baseline. of the remaining patients, . % patients ( leukemia, lymphoma and sarcoma) lost protective antibody titers at months after initiation of chemotherapy and . % ( leukemia, lymphoma and sarcoma) at months after initiation of therapy. % of the remaining patients who retained measles antibody titers within protective range at months also demonstrated a steady decline in antibody titers at and months from therapy initiation. the loss of protective measles humoral immunity occurred significantly more often in patients with leukemia compared to other malignancies. oncology patients in our practice undergoing intensive chemotherapy demonstrated progressive waning of protective measles igg titers. our data suggests that it should be standard practice to check all patients for measles humoral immunity prior to starting chemotherapy and at completion. larger studies need to be performed to establish guidelines for revaccinating post-chemotherapy pediatric patients, an intervention that is easily applicable and of low cost. background: the accurate determination of glomerular filtration rate (gfr) is important to screen for acute kidney injury, to dose chemo-therapy, and to identify risk for chronic kidney disease.being correlated with inulin clearance, measured gfr by iohexol plasma disappearance (igfr) is a new gold standard for measurement of gfr in pediatric cohort studies. igfr is based on the clearance of an exogenous marker and is unaffected by endogenous compounds or a patient's muscle mass. we compared igfr with -hour urine creatinine clearance ( crcl) and gfr estimating equations using serum creatinine (scr) and serum cystatin c (cystc) in pediatric patients with cancer. we recruited participants who were ages to yrs, continent of urine, and diagnosed with a malignancy in the past years. eligible subjects had stable kidney function for at least two weeks prior to the assessment of igfr. consented subjects had baseline assessments including height, weight and vital signs. blood samples were obtained for serum chemistry, and time zero iohexol. igfr determined by ml iohexol solution infused over - minutes followed by ml of sterile saline. blood was drawn at , , and minutes.at the same time of igfr, the crcl was collected. igfr was calculated using a two-compartment model and area under the curve. we compared igfr to published gfr equations (schwartz et al, kidney int ). results: ten subjects ( female/ male) agreed to participate. the distribution of diagnoses for the subjects: all = , lymphoma = , brain tumors = and hepatocellular carcinoma = . six patients were off therapy. the lower gfrs are noted in patients who had malignancies other than leukemia, likely due to the use of cisplatin based therapy. the average igfr was ml/min/ . m^ whereas crcl was . ml/min/ . m^ ; demonstrating the crcl overestimates gfr compared to igfr. comparing igfr to univariate equations using scr, cystc, and the multivariate equation with both, the univariate cystc equation correlated well with igfr; the others overestimated igfr. we found that crcl overestimated igfr. the univariate cystc equation better correlated to igfr than equations with scr. the poor performance of scr based methods to assess gfr might be due to decreased muscle mass and inadequate nutritional status. creatinine-based determinations of gfr alone, may not be accurate in this population. further study is needed to determine if igfr should be a standard of care to assess gfr in children with cancer particularly who are receiving nephrotoxic medications and incontinent of urine. background: pediatric oncology patients undergoing chemotherapy through indwelling venous catheters are at increased risk for severe sepsis especially when neutropenic due to chemotherapy. rapid triage and early recognition are essential because delayed initiation of antibiotics and fluids in these patients or delayed transfer to higher level of care after initial stabilization is associated with poor clinical outcome. our pediatric oncology out-patient clinic is designated as an article unit whereby the providers can initiate and give treatment such as intravenous fluid, antibiotics, chemotherapy and blood products. objectives: global aim-optimize management of early sepsis and decreased morbidity, mortality and hospital length of stay in the high risk pediatric oncology patients. smart aim-improve timely management with initiation of fluids and antibiotics and transfer of septic patients to higher levels of care by % in months in above patients design/method: multidisciplinary team with physicians and nurses was created. retropective chart review of sepsis patients treated at the clinic from april to october was done using an audit sheet to identify the barriers in the delivery of care. three patients were identified and data analyzed prior to intervention; two were analyzed post interventions. a key driver diagram was created by the group to drive intervention. a process map was designed to identify the different steps in the care of these patients to pinpoint areas needing improvement. different timed data points were used starting from time of arrival to clinic, time to antibiotics and fluids and time to transfer to higher level of care. rapid pdsa cycles were done to improve the processes and delivery of care. run charts were created. there was an improvement close to the goal of % for all data points used. pdsa cycles for improvement included conducting frequent mock codes with appropriate feedback real time coaching and process planning with nursing staff. we partnered with pharmacy for close loop communication with clinic staff and we improved communication between physicans at different levels. conclusion: sepsis in neutropenic pediatric oncology patients is deadly and can be reversed with timely management at different levels. given the promising results of the above project, we want re-inforcement of the processes to be a part of the daily practice of first line clinical staff. eventually we will extend the principles learnt in management and triage of sepsis to other outpatient emergencies chemotherapy related anaphylaxis background: chemotherapy-induced nausea and vomiting (cinv) is a common side effect in children receiving antineoplastic chemotherapy. recommended prophylactic antiemetic medications are based on the classification of chemotherapy emetogenicity. however, despite appropriate use of these antiemetic agents, some patients will still experience nausea and/or vomiting. children's oncology group clinical practice guidelines recommend the addition of olanzapine to prophylactic regimens for management of breakthrough cinv. objectives: our pediatric hematology oncology center implemented a quality improvement (qi) project aimed to increase the use of olanzapine in pediatric cancer patients years of age and older receiving moderately or highly emetogenic chemotherapy and experiencing breakthrough cinv over a month period. design/method: this qi project was conducted utilizing plan-do-study-act (pdsa) cycles. for the first pdsa cycle, baseline data was collected through chart review to determine the rate of olanzapine use for breakthrough cinv over a month period from july to december . breakthrough cinv was defined as use of or more doses of antiemetic agents other than those given for cinv prophylaxis. guidelines for treatment of breakthrough cinv were reviewed with pediatric hematology/oncology attending physicians and fellows. flyers were created that listed chemotherapy regimens considered moderately and highly emetogenic. if a patient experienced breakthrough cinv, a flyer was to be placed in the patient's roadmap binder to signal olanzapine should be added to the next chemotherapy block. data was collected over a month period in september following this first intervention. the second pdsa cycle consisted of didactic education and training of pediatric oncology nurses as well as pediatric residents regarding the addition of olanzapine for breakthrough cinv. rates of olanzapine use were then collected from october through november . results: olanzapine use increased from . % at baseline to . % after the first pdsa cycle ( = . , p = . ). after the second pdsa cycle, olanzapine use increased another . % to . % ( = . , p = . ). the administration of olanzapine was successfully increased by modifying patients' roadmaps after patients experienced breakthrough cinv as well as with education and training of pediatric oncology staff, fellows, residents, and nurses. background: venous thromboembolism (vte) is increasingly affecting children. according to an administrative database study, there was a % increase in the incidence of vte among children admitted to free-standing children's hospitals in the united states from to . risk factors for hospital-acquired vte are well-known and well-studied in adults, with evidence-based preventative measures available. similar guidelines are lacking for children. objectives: there is an ongoing national-initiative to develop and institute methods for screening and preventing hospitalacquired vte in children. in / , nationwide children's hospital instituted an electronic screening form required for all patients admitted ≥ hours. patients were scored and riskstratified based on eight risk-categories. a summated score was used to determine the vte risk level, and used to make prophylaxis recommendations for patients ≥ years; as well as patients ≥ years who were admitted to an intensive care (icu), surgical, or trauma unit. the purpose of this irb exempt, quality improvement initiative was to retrospectively review our experience with this risk-stratification tool. results: hospital-acquired vte events occurred in unique subjects. median age at vte diagnosis was years. only ( %) vte occurred in children ≥ years of age. ( %) vte were deep vein thrombosis (dvt), and ( . %) involved pulmonary embolism. vte was most common in subspecialty units including the pediatric and cardiac icus ( . %); neonatal icu, ( . %); and hematologyoncology, ( . %). ( %) vte were associated with central venous catheters (cvc) and events ( %) were associated with altered mobility. congenital heart disease/heart failure was the most common chronic medical condition associated with vte ( ( . %) events); whereas infection and trauma/surgery were the most common acute medical conditions associated with vte ( ( . %) and ( %) events, respectively). during ( %) events, subjects scored a summated score ≥ . in summary, in this single institution, prospectively maintained database, cvc remains the most common risk factor for vte, followed by cardiac disease, infection and trauma/surgery. most subjects who developed vte scored high (score ≥ ) on our screening tool. only a small proportion of vte occurred in patients older than years and thus eligible for thromboprophylaxis. our results indicate that future vte prevention endeavors should include these age groups in addition to exploring more aggressive prophylactic modalities including pharmacological prophylaxis. background: pediatric fellows are required to have active engagement in quality improvement (qi) activities, and yet a national acgme review found most trainees had "limited knowledge of qi methods" and "limited participation in interprofessional qi teams". the twenty fellows in our pediatric hematology/oncology training program identified blood culture utilization as their qi priority. our institution recently introduced a hospital-wide decision algorithm to guide providers regarding when to obtain blood cultures. there is often a low threshold to obtain blood cultures in immunocompromised pediatric oncology patients, but these are often low-yield or result in falsepositives. our fellows spearheaded a project to implement the algorithm in the inpatient pediatric oncology population and improve the proportion of appropriately drawn blood cultures. we investigated how appropriately the algorithm was being utilized on the inpatient pediatric oncology floor prior to and after several educational steps aimed at disseminating the algorithm to members of the care team. our primary endpoint was to quantify the proportion of culture episodes drawn "inappropriately", with a goal of reducing inappropriate episodes to ≤ %. the algorithm was initially introduced to the nursing staff and residents covering the twenty-bed inpatient unit in september . qi project planning took place with upper level fellows in january . fellows and faculty received intensive training on the algorithm in july-august . we then conducted a retrospective chart review of blood culture episodes drawn between august and november . upper level fellows scored ∼ culture episodes as to whether the decision to culture and number of cultures drawn were "appropriate" or "inappropriate", and catalogued the indications for culture episodes and if applicable, why the episode was found to be inappropriate. additionally, fellows discussed inappropriate culture episodes with the team onservice, to provide direct feedback on where the algorithm failed. results: between august -december on average cultures/ patient-days were drawn. forty-nine percent of culture episodes were inappropriate. from january -october , following targeted education on the algorithm, the rate of blood cultures drawn decreased to cultures/ patient-days. the average proportion of inappropriate culture episodes fell to . %, representing a % decrease in inappropriate culture utilization. correct application of a decision algorithm for blood culture utilization can reduce total cultures drawn on an inpatient pediatric oncology unit. fellow-led education of the multi-disciplinary team decreases the rate of inappropriate culture episodes as well as provides active engagement in qi. background: inadequate understanding of sickle cell disease (scd) is common and can affect patients' compliance and therefore their morbidity and mortality, especially after transition to adult care. optimal clinical care for scd includes disease education, which can be difficult given the breadth of possible topics and limited time in clinic. it is unclear how best to provide personalized, efficient education for adolescents with scd. this quality improvement (qi) study aimed to implement a questionnaire-based system to improve patients' knowledge of their scd and documentation of education by the nurse or physician. the study objective was to improve provider documentation and patient knowledge about their scd by identifying patients' gaps in comprehension. by january , the study aimed to increase education documentation from % to %. by april , the study aimed to increase use of a smart phrase for education documentation from % to %. by june , the study aimed to increase patients' knowledge about their disease by %. design/method: twenty-one scd patients enrolled on an irb approved qi study, with twenty active patients. our comprehensive team generated a questionnaire with knowledgebased questions for two age groups: - and - years old. at each comprehensive visit, a questionnaire was distributed, with at least -month intervals. the provider scored questionnaires and reviewed two educational topics, with wrong answers taking priority. plan-do-study-act (pdsa) cycles included pdsa# : patients completed questionnaire. pdsa# : a smart phrase addressing questionnaire topics was created and shared with providers. pdsa# : patients received education handouts during clinic education. documentation in clinic notes was the process measure and questionnaire scores was the outcome measure. results: pdsa# is complete, pdsa# has four patients remaining, and pdsa# is ongoing. due to variable visit frequency, there are multiple concurrent cycles. after pdsa# , free text documentation was completed an average of % over the course of months. after pdsa # documentation increased to % within months and questionnaire scores increased from an average of % to %. of the questions that patients got wrong on their first visit, they were significantly more likely to improve on retesting if the topic was taught to them than if it was not addressed ( % vs. %, p = . ). we are currently completing pdsa# and collection of post pdsa# data. questionnaire-based scd education coupled with standardized smart phrases improves patients' scd knowledge and documentation by providers. further improvement in knowledge is expected with the addition of handouts. background: exposure to suffering can have a profound impact on the wellness of caregivers, often referred to as the "cost of caring". this cost is especially high in pediatric hematology/oncology. repeated exposure to suffering has the potential to negatively impact resilience and increases the risk of burnout, thus impacting quality of care and patient satisfaction. we have developed a peer support team utilizing the critical incident stress management (cism) model. this model has been successfully used in other professions that frequently face traumatic events such as fire fighters, police and emergency medical technicians. the h.o.p.e.s. team (helping our peers endure stress) consists of volunteer multidisciplinary staff members who have received training to provide both group and peer support following any 'critical incident' that may impact one or more staff members. we hypothesize that implementation of the h.o.p.e.s. team will improve staff resilience, decrease overall rates of burnout and improve compassion satisfaction. s of s design/method: we are using both empiric metrics and anecdotal reports to assess the impact of the h.o.p.e.s. team. prior to the activation of the team, all pediatric hematology/oncology clinical staff members were surveyed using validated tools to assess their levels of resilience, burnout, secondary trauma and compassion satisfaction (proqolv and brief resilience scale). they were also asked to rate the number of times they had experienced critical incidents, as well as their perceived level of distress after dealing with traumatic events. after the h.o.p.e.s. team has been functional for months, we will send the same survey to staff members to measure changes, paying special attention to resilience and rates of burnout and compassion satisfaction. results: enthusiasm for development of the team has been high. of people approached to volunteer their time to participate in the multidisciplinary team agreed, including attending physicians, fellows, nurses, nurse practitioners, child life specialists, social workers, clergy and psychologists. all volunteers participated in a -day training conducted by an instructor from the international critical incident stress foundation. engagement in the first staff survey has been high, with of responding to date. data collection is ongoing. clinical staff in pediatric hematology/oncology may be particularly vulnerable to burnout and decreased resilience by repeatedly witnessing suffering and trauma. peer support interventions following critical incidents may lead to increased resilience and compassion satisfaction while decreasing rates of burnout. enthusiasm for the development of a peer support team has been high. background: monthly blood transfusions are an indicated therapy for pediatric patients with sickle cell disease with certain complications. maximizing transfusion efficiency in a busy infusion clinic requires: ensuring that appropriate blood units are available in the hospital blood bank; laboratory specimens are obtained from patients in advance; and coordination of clinic appointment and nursing availability. we sought to improve clinic efficiency through identifying ways to better communicate with patients/families regarding upcoming laboratory and transfusion appointments, and to assess the efficacy of implementing a web-based personalized text reminder (pinger.com). we measured the baseline frequency with which transfusion appointments were missed by families, moved to later within the week, or delayed due to late labs. a convenience sample of patients receiving monthly transfusions received a questionnaire about patient/parent preferences for appointment reminders and barriers to keeping appointments. those patients/parents who did not opt-out of an additional text reminder received personalized texts from their care team reminding them of lab and transfusion appointments. rates of missed/moved/delayed appointments were compared between the group receiving the additional text messages and the group only receiving standard, hospitalgenerated appointment reminders (telephone call). results: forty-one families ( patients) responded to the survey, capturing information on % of patients receiving chronic transfusion therapy. thirteen families ( %) declined the additional text reminders. families reported a preference for text reminders ( %), more often than email ( %) or telephone ( %), and % of families wanted to receive reminders for both transfusion and laboratory appointments. the majority ( %) of families reported competing work/life priorities as the reason for missed/late appointments. other families noted transportation/travel ( %), fear/illness/pain ( %), and lack of reminders ( %) as the reason for missed appointments. at baseline (twelve weeks), . % of appointments were missed on a weekly basis (range - of available per week), . % were moved, and % of appointments were delayed. during our intervention period (twelve weeks), % were missed, . % were moved, and . % were delayed (combined, both groups). there was no difference in missed ( . % texted vs . % standard), moved ( . % texted vs . % standard) or delayed ( . % text vs . % standard) appointments. though families at our center reported a preference for a text-based reminder, personalized text reminders for appointments did not improve clinic efficiency as measured by missed, moved or delayed transfusion appointments. there was no improvement in appointment adherence in the group receiving personalized texts in addition to standard hospital reminders. university of utah, salt lake city, utah, united states background: childhood cancer outcomes have improved significantly, in large part due to multi-institution collaborative clinical trials run by the children's oncology group (cog). approximately half of eligible children with cancer will enroll on a therapeutic trial, but little is known about the factors affecting caregiver decision-making regarding enrollment or how well the required elements of informed consent are conveyed during the consent process. objectives: . assess coverage of ten of the required elements of informed consent for cog therapeutic trials. . describe factors affecting caregiver decision-making regarding therapeutic trial enrollment. we surveyed families of children who were offered enrollment onto a phase cog therapeutic study for an initial cancer diagnosis in the previous months. fisher's exact or wilcoxon rank-sum tests were utilized to compare demographic and other motivating factors related to enrollment decision-making. results: seventy participants were surveyed. regarding of the basic required elements of informed consent, % knew the trial involved research, % knew consent was required, % knew the enrollment length for the trial, % knew they could continue care independent of enrollment, % knew who to contact with questions, % knew there were options besides enrollment, % knew they could withdraw at any time, % knew the information was confidential, % knew there were risks associated with the trial, and % knew there were benefits. of all participants, % (n = / ) enrolled onto a therapeutic study. among enrollees, % (n = / ) of the primary caregivers had completed college compared to % (n = / ) of those not enrolled (p = . ). when asked about factors impacting their decision, % (n = / ) of those enrolled said they felt there were no risks or did not know if there were risks associated with the study compared to % (n = / ) of those choosing not to enroll (p = . ). of those enrolled, % (n = / ) reported the physician recommendation "somewhat" or "strongly" affected their decision to enroll compared to % (n = / ) of those not enrolling (p = . ). of those who enrolled, % (n = / ) reported feeling pressured to enroll while % (n = / ) of those not enrolled reported pressure (p = . ). of enrollees, % (n = / ) reported they did not have enough time to decide compared to % (n = / ) of those not enrolled (p = . ). failure to convey all required elements of informed consent highlights possible deficiencies in the consent process for cog therapeutic trials. caregivers' perception of being pressured and lack of time to make an informed decision may impact clinical trial enrollment. background: abnormal uterine bleeding (aub) is a frequent adolescent gynecologic complaint. however, limited research exists to guide management, and acute care varies. we sought to improve emergency care for adolescents with aub by developing a clinical effectiveness guideline (ceg) and assessing its impact on quality of care. design/method: a stakeholder engagement group consisting of members from the departments of hematology/oncology, adolescent medicine, general pediatrics, and emergency medicine designed a ceg algorithm for emergency aub management. pediatric residents received ceg training and their knowledge and attitudes were assessed using pre and post intervention surveys. icd- and codes identified electronic health record data for patients presenting to the pediatric emergency department (ed) for aub months before and after ceg implementation. pre-pubertal patients and those with vaginal bleeding from trauma were excluded. a weighted, -point scoring system consisting of prioritized aspects of history, laboratory studies and management was developed to quantify the quality of care provided. t-test, chi square test, wilcoxon rank sum test, and a run chart were used for analysis. of the patients identified, met inclusion criteria. there were % of patients currently using some form of contraception, while . % had bleeding related to a current or recent pregnancy. median aub quality care scores were pre-and post-intervention (p = . ). run chart data showed no shifts or trends (overall median score, -points). both pre and post-implementation, points were deducted most frequently for not assessing personal/family clotting disorder history and inappropriate use/dosing of oral contraceptives. we successfully designed and implemented a ceg and educational intervention for aub management in a pediatric ed. these data suggest our ceg may be an effective tool to improve emergency aub care for adolescents, though additional cycles are needed. background: high-dose methotrexate (hd-mtx) is a common chemotherapy administered inpatient at most centers. its administration is particularly susceptible to error due to the need for frequent drug levels with resulting changes in supportive care. errors can prolong patient stay and cause patient harm. objectives: global aim-to reduce the length of stay (los) of hd-mtx admissions. smart aims-to increase the percentage of patients whose pre-hydration fluids are started by am from % to % by / / , and to increase the percentage of patients who receive hd-mtx by pm from % to % by / / . we used rapid process improvement methods to target earlier methotrexate administration. a key driver of prolonged los was hypothesized to be drug levels returning overnight rather than in the day time due to delayed hd-mtx start. changes implemented have included scheduling hd-mtx patients as the first patients of the day for their exam in clinic and scheduling labs to pass for hd-mtx on the day prior to admission. there are ongoing pdsa cycles to change the location of pre-hydration start from the inpatient room to the clinic exam room in order to meet hd-mtx administration time goals. we are piloting two different education materials to improve patient experience. one explains hd-mtx levels in a red/yellow/green stoplight format and the other reminds patients how to prepare for the admission. other interventions regarding how we test urine ph and safety checks in the ordering process for history of delayed clearance are in the planning stage. the project is ongoing, but as of / / , we start methotrexate by pm % of the time which is improved from a baseline of %. when the project was started, pre-hydration was never started before am. now, fluids are started by am % of the time. pdsa cycles are ongoing and we have yet to sustain reductions in los, but some months have shown decreased los by as much as hours from baseline measurements. rapid cycle improvement can be utilized to decrease los hd-mtx admissions. this has important financial implications as well as the potential to reduce secondary harm from unnecessary time in the hospital. pediatric cancer centers should schedule hd-mtx admissions first thing in the morning so that data regarding kidney injury and drug clearance can be interpreted by the day team and children are not cleared for discharge in the middle of the night. background: education and training for interdisciplinary pediatric oncology providers requires training in principles of palliative and end-of-life (eol) care. the experiences of bereaved parents can inform and enhance palliative care educational curricula in uniquely powerful and valuable ways. the objective of this study is to present an innovative palliative care educational program for oncology providers facilitated by trained bereaved parents who serve as volunteer educators in local and national palliative care educational forums and to describe how incorporation of bereaved parents in these educational forums affects participant comfort with communication and management of children at the eol. design/method: survey tools were adapted to determine how bereaved parent educators affected participant experiences in different educational forums: institutional seminars on pediatric palliative and eol care, role-play based communication training sessions, and an international symposium on pediatric palliative oncology. pre-and post-session surveys with incorporation of retrospective pre-program assessment item to control for response shift were used in the evaluation of institutional seminars and communication training sessions. results from feedback surveys sent to all attendees were used to appraise the participants experience in the international oncology symposium. results: involvement of trained parent educators across diverse, interdisciplinary educational forums improved attendee comfort in communicating with, and caring for, patients and families with serious illness. importantly, parent educators also derive benefit from educational with interdisciplinary clinicians. integration of bereaved parents into palliative and eol care education is an innovative and effective model that benefits both interdisciplinary clinicians and bereaved parents. background: poorly controlled chemotherapy-induced nausea and vomiting (cinv) significantly impairs patients' quality of life and contributes to ongoing medical costs through increased length of stay in the hospital or readmissions and outpatient visits for control of nausea, vomiting or dehydration. lack of adherence to national evidenced-based guidelines that dictate antiemetic prescribing for variably emetogenic chemotherapy leaves patients vulnerable to increased cinv and its ensuing complications. objectives: to review our institution's antiemetic prescribing practices and their consistency with the antiemesis guidelines from the national comprehensive cancer network (nccn) and children's oncology group (cog)-endorsed supportive care guidelines and to further develop tools to increase adherence to these national-based guidelines to improve control of cinv. we performed a retrospective chart review of inpatient chemotherapy encounters. we evaluated emetogenicty of chemotherapy (high, medium, low), initial antiemetic regimen ordered, number of as needed medications required and adherence to national evidenced based guidelines tailored to each level of emetogenicity in the prescription of antiemetics. results: fifty-five total inpatient chemotherapy encounters were reviewed over months. eighteen of these encounters were considered to have been highly emetogenic chemotherapy (hec) with the remaining of these considered to be moderately emetogenic. only out of hec encounters completely included all guideline-recommended agents. there was a demonstrable lack of consistency across providers with dosing of aprepitant and most as needed medications. there was significant variation in order of first, second and third line anti-emetics ordered -with lorazepam and promethazine being used most frequently. with an aim of improving antiemetic prescribing practices for our patients, we are currently rebuilding chemotherapy treatment plans in our electronic medical record to incorporate antiemetic drug order sets that follow evidenced-based guidelines for variably emetogenic chemotherapy. this will be used in conjunction with an education initiative about best practices in supportive care for all prescribers of antiemetics. review of our department's recent inpatient chemotherapy encounters show we are falling short in following nationally recommended standards for appropriate antiemetic coverage during chemotherapy. identification of these deficiencies allows for implementation of quality initiatives to improve prescriber adherence to evidenced-based guidelines for better control of cinv. background: there are currently no consensus guidelines for the management of pediatric oncology patients presenting with fever without neutropenia. historically, these patients had been treated similarly to neutropenic patients with empiric antibiotics. while there has been a shift towards reducing unnecessary empiric treatment, there has been limited research into the outcomes associated with withholding empiric iv antibiotics in this patient population. we assessed the safety and efficacy of our institution's current protocol of observing well-appearing patients who present with fever without neutropenia and compared the outcomes of the patients who did and did not receive empiric iv antibiotics. design/method: this was a prospective, single-institution cohort study. patients were included if they were currently undergoing chemotherapy for an oncologic diagnosis and presented initially as an outpatient with fever and nonneutropenia (defined as anc ≥ cells/mm ). for each episode we recorded lab and blood culture results, signs and symptoms of initial presentation, and clinical outcomes, including antibiotic administration and hospital admission. results: a total of episodes of well-appearing patients with fever without neutropenia were identified. compliance with the institutional protocol was high; . % of patients were observed without receiving empiric iv antibiotics. the majority of patients were discharged home and there were no serious complications or infectious deaths. the incidence of positive blood cultures was low ( . % including several likely contaminants), despite the presence of central venous catheters in the majority ( . %) of patients. there were no significant differences in age, oncologic diagnosis, central s of s line access, anc value, or incidence of bacteremia between patients who did and did not receive empiric iv antibiotics. patients who were admitted to the hospital were significantly more likely to have received iv antibiotics (p < . ) despite documentation of a reassuring exam. however, admitted patients who initially received iv antibiotics were just as likely to discharge within hours compared to patients who were observed. we propose that empiric iv antibiotic administration in febrile, non-neutropenic, otherwise well-appearing patients is unnecessary. our study demonstrated no adverse consequences of observation and no significant differences in clinical outcomes between patients who did and did not receive iv antibiotics aside from rate of hospitalization. this supports the practice of observation without empiric antibiotics for such patients. background: children with hepatoblastoma (hb) undergo repetitive computed tomography (ct) scans to determine response to treatment and assess for relapse. this imaging exposes children to radiation, anesthesia, and imposes financial and emotional burden. objectives: review our institutional experience to determine if afp measurements are sufficient to assess response to treatment and detect relapse. we conducted a retrospective chart review of all patients diagnosed with hb at our institution between - . data collected included serum afp, total number and type of imaging studies during and post treatment, and how relapse or progressive disease was detected. results: thirty-one patients were diagnosed with afp positive hb. during therapy, ct scans were performed: to assess for response to therapy or surgical planning (average scans/patient) and due to concern for progression with rising afp. off therapy, surveillance ct scans were performed (average of . scans/patient) and ( %) included the chest in patients with no lung metastasis at diagnosis. relapsed patients averaged . surveillance scans, . of which were done before relapse was noted on imaging. there were no cases of radiographic evidence of relapse without a prior increase in afp. during treatment, response to therapy based on imaging correlated with a decline in afp in all patients, arguing that repetitive scans are not needed in this setting unless required for surgical planning. only of scans performed during off therapy surveillance displayed evidence of relapse, all of which were preceded by rise in afp. our study represents the largest cohort of hb patients. prior studies suggest similar results, but included fewer patients, lower stage of disease and less than years of surveillance monitoring. at our institution, the cost of a ct c/a/p is $ , with reimbursement varying from - %. in comparison, the cost of an afp measurement is $ . . many scans also require anesthesia and result in emotional toil for families concerned about this procedure as well as the results. thus, afp demonstrates greater sensitivity, with significant cost savings and decreased emotional burden, and should be used for monitoring both during and off therapy, replacing routine serial imaging. background: we observed that our practice of drawing daily blood cultures in hospitalized patients with fever and neutropenia was wasteful; it resulted in excessive negative cultures that did not add to patient care. the smart aim of this quality improvement project was to reduce the number of negative blood cultures drawn on hospitalized patients with fever and neutropenia by % in months. design/method: after reviewing published evidence suggesting drawing daily blood cultures in febrile neutropenic patients was unnecessary, a new blood culture guideline was implemented: cultures were drawn at presentation for fever with neutropenia and, if negative at hours, repeat cultures were not drawn except for clinical change, new fever after being afebrile > hours, or antimicrobials were being changed/broadened. to impact key drivers, we educated staff and changed blood culture order sets to require providers to select a reason for ordering the culture and to eliminate a nursing order to draw daily cultures with fever. we compared the number of blood cultures drawn per central linedays (/ -cld) and the proportion of positive versus negative cultures pre-guideline (july -may ) and postguideline (june -december ). we calculated the cost savings from reducing cultures. to assess patient safety, potential septic events without a corresponding positive blood culture were reviewed. data were analyzed by service (oncology and stem cell transplant). a chi-square test was used to compare rates. in stem cell transplant patients, pre vs. postguideline, there were vs. total cultures drawn/ -cld; vs. positive ( % decrease, p = . ) and vs. negative cultures/ -cld ( % decrease, p< . ). in oncology patients, pre vs. post-guideline, there were vs. total cultures drawn/ -cld; vs. positive ( % decrease, p = . ) and vs. negative cultures/ -cld ( % decrease, p< . ). the decreased positive culture rate among oncology patients may be due to decreased culture contaminants and/or the effect of a concurrent initiative to decrease clabsi in that group. there were safety concerns; however, chart review concluded that the guideline did not lead to missed infections in these patients. for the first months of the guideline, the total cost savings in blood cultures was $ , . . the implementation of our new blood culture guideline successfully led to a substantial reduction in the collection of negative cultures and a cost savings without compromising the detection of bacteremia in hospitalized pediatric patients with fever and neutropenia. background: there are various evidence-based guidelines for treatment of adult cancers, such as the nccn guidelines. previously, care was standardized for most new diagnosis pediatric cancer patients through enrollment on a clinical trial. with decreasing clinical trial availability and enrollment and few, if any, evidence-based guidelines for pediatric cancer, care standardization is challenging for pediatric cancers. objectives: to assess consistency of care, as determined by plan of treatment by diagnosis, for pediatric patients receiving chemotherapy for newly diagnosed cancer at a single center. design/method: patients with a new cancer diagnosis at a large, tertiary care pediatric oncology center in calendar year were identified through reports from the chemotherapy order entry (coe) system. reports included diagnosis (recorded through standardized options) and the plan of treatment. chart review was used to exclude patients who started treatment elsewhere and patients being treated for relapse, to clarify diagnosis if the standardized options in coe were unclear, and to clarify treatment plan if needed. data was entered and analyzed in a redcap database. specific diagnoses were clustered into higher level disease groups and the distribution of treatment plans for patients within each was determined. this project was deemed exempt from irb approval for human subject research as a qualifying quality improvement project. of the patients with a first chemotherapy order in , were excluded due to one or more reasons: stem cell transplant ( ), transfer of care ( ), relapse ( ), and other ( ). an additional patients were excluded because < patients/year/diagnosis. there was no cns tumor disease group with > patients. thus, patients with hematologic malignancies or non-cns solid tumors are the focus of this analysis. for patients with intermediate risk rhabdomyosarcoma, the plan of treatment was the standard arm of a cog protocol, arst for patients and arst for subsequent patient after protocol activation. for all other diseases including lymphoblastic leukemia/lymphoma (excluding infants), classical hodgkin lymphoma, aml (excluding trisomy and apml), stage iii/iv burkitt lymphoma/diffuse large b-cell lymphoma, posttransplant lymphoproliferative disease, wilms tumor, rhabdomyosarcoma, ewings sarcoma, osteosarcoma, neuroblastoma, and retinoblastoma, only one treatment plan per risk category was used. conclusion: this analysis demonstrates highly consistent chemotherapy treatment at a single center for patients with hematologic malignancies and non-cns solid tumors. next steps include exploring strategies to group diagnoses for cns tumors and assessing the quality of evidence supporting the treatments given. background: rapid initiation of empiric antibiotics in patients with fever and neutropenia has been shown to reduce morbidity and mortality. current practice guidelines call for the initiation of antibiotics in these patients within sixty minutes and time-to-antibiotic (tta) has been suggested as a quality-of-care measure. many institutions, including our own, face barriers to meeting this time limit. objectives: utilizing a quality improvement model, determine barriers and implement an intervention to reduce the time-to-antibiotics for pediatric febrile patients with suspected neutropenia who present to the emergency department (ed) at our institution. we have identified and implemented an intervention utilizing the plan-do-study-act model for quality improvement. a twelve-month retrospective review was conducted to evaluate the efficacy of the current practice algorithm at our large, academic tertiary-care hospital. subjects identified were pediatric oncology patients undergoing active chemotherapy who presented to the ed with febrile neutropenia. we identified two specific barriers, triage level assignments and delay in ordering antibiotics. to address these barriers, we have created a wallet sized "fever card" that patients were instructed to show upon arrive to the ed. in collaboration with the ed staff, efforts were also made to educate all pediatric staff on the use of the fever card. post-intervention data collection is currently underway and pre-and post-intervention antibiotic delivery times will be compared. the pre-intervention cohort consisted of thirty-three encounters with a mean time-to-antibiotic delivery of minutes, or seventy-five minutes greater than the accepted standard of care. only one patient received antibiotics within sixty minutes of arrival. post-intervention data collection is currently underway. since identifying two barriers to meeting the standard of care at our institution, we have implemented a quality improvement measure that empowers patient families to direct appropriate triage in the ed as well as simplifying the treatment protocol for ed providers. we expect to identify an improvement in time-to-antibiotics from the pre-intervention to the post-intervention period. background: sickle cell disease (scd) is a genetic disorder in which sickle hemoglobin (hbs) triggers multiple downstream effects, including red cell sickling, hemolysis, vaso-occlusion, and inflammation. scd, a lifelong disease initiated at birth with injury that accumulates over time, causes significant end-organ damage and clinical complications that are undertreated and associated with early death. homozygous mutation (hbss) causes the severe form of scd. individuals with scd are at increased risk of infection, stroke, and retinopathy. clinical guidelines for pediatric patients with scd recommend prophylactic penicillin use (ages - ), annual screening for stroke with transcranial doppler (tcd) imaging (ages - ), and annual ophthalmology exams to assess for retinopathy (ages ≥ ). there are limited real-world data on implementation of these nhlbi-based recommendations. objectives: to describe utilization of penicillin, tcd screening, and ophthalmology care in children with hbss disease. medicaid administrative claims databases were used to identify us patients aged - years at first indication of hbss recorded in each calendar year from to . patients were required to have medical and pharmacy benefits for the calendar year in which they were identified and for months prior to their first recorded hbss indication. prior year utilization of penicillin, tcds, and ophthalmologist visits was measured for each annual cohort. annual cohorts included - commercial (mean age . years, % female) and - medicaid (mean age . years, % female) patients with hbss disease. fewer than half of all patients had received a tcd scan in the previous year, with similar rates seen across all age groups for both payers. ophthalmologist visits increased as patients aged, and while patients aged - years had the highest proportion with an ophthalmologist visit in both payer populations, the overall implementation remained low. in contrast to the low use of tcd and ophthalmology visits, penicillin use was highest in the - year age group: > % use in any given year for both payers. conclusion: although our data demonstrated high penicillin use in the - year age group, consistent with guidelines there is an opportunity to improve implementation of other guidelines-based recommended screening. for example, tcd screening can identify children at risk of scd-related stroke in order to initiate preventive therapies. further research to understand potential barriers to proper screening and to evaluate strategies to improve awareness, adherence, and implementation of recommended screenings in children with scd is warranted. supported by global blood therapeutics. background: childhood cancer therapy has improved where there are many long-term survivors. while psychosocial difficulties in pediatric cancer survivors are recognized, the prevalence of these problems at initial survivorship presentation is unclear. objectives: to examine the prevalence of overall internalizing symptoms (e.g., depression/anxiety) in pediatric cancer survivors presenting to a survivorship clinic and to examine how this is mitigated by receiving psychological services and by evidence of parental depression/anxiety. design/method: pediatric cancer survivors attending their first visit at the reach for survivorship clinic at vanderbilt (ages - ) were included. survivors' parents ( % female) completed the child behavior checklist (cbcl), beck depression inventory-ii, and beck anxiety inventory. survivors > years completed a self-report. the wilcoxon rank-sum and pearson's test were used for univariate analyses. the effect size and % confidence intervals (ci) estimated from the multivariable linear regressions were reported. results: childhood cancer survivors a median of years old and . years off therapy were included. thirty one survivors ( %) showed at least borderline clinical internalizing problems (t score > ) on the cbcl, but only of these patients ( %) reported receiving psychological services. nine other survivors with normal t score ≤ also reported receiving psychological services. parental depressive and anxiety symptoms were correlated to the parental report of survivor overall internalizing symptoms (spearman = . , p = < . and = . , p = < . respectively), however they were not correlated to survivor selfreports. furthermore, parents with mild to severe depressive symptoms or mild to severe anxiety symptoms were more likely to rate their child as having higher overall internalizing symptoms (p = . ; p = . , respectively). multivariable linear regression showed that when adjusted for age, gender, cancer diagnosis and time off treatment, reported utilization of psychological services ( = . , % ci [ . , . ],p = . ), and parent depressive symptoms ( = . , [. , . ],p< . ) were significantly associated with child overall internalizing symptoms. in an otherwise identical alternate model substituting parental anxiety for parental depression, parental anxiety was also a significant risk factor ( = . , [. , . ], p< . ). alternatively, parent anxiety/depressive symptoms were not significantly associated with child self-report of internalizing symptoms. childhood cancer survivors have an elevated prevalence of experiencing internalizing symptoms but seldom report receiving psychological services. childhood cancer survivors' parents with anxious/depressed symptoms are more likely to rate their children as having more internalizing problems, compared to patient self-reports. ongoing longitudinal analyses will help clarify the best timing for potential interventions. background: life expectancy for adults with sickle cell disease (scd) has remained unchanged over the past years despite improvements in pediatric scd survival. at greatest risk are the adolescents and young adults (ayas) transitioning from pediatric to adult care. allen county ranks rd in scd incidence among the counties in indiana, and has board certified pediatric hematologist-oncologists. when children "age out" of the pediatric system, there are few providers knowledgeable about managing adults with scd in the region. a novel partnership between hematologists and the family medicine residency program in allen county was initiated to educate family medicine residents (fps) about scd, hydroxyurea (hu), and management of scd-related complications with the goal to increase the number of knowledgeable providers to care for adults with scd. to determine the effectiveness of online learning modules in educating fps about hu, best practices for aya scd care and transition. three online learning modules about scd (comprehensive care of ayas with scd, hu, best practices in aya transition) were developed and cme-accredited. electronic pre-and post-tests were distributed to fps with five questions for each module covering: contraception; screening tests; hu indications, dosing and monitoring; developmental milestones and scd knowledge assessments. the st vincent irb reviewed the protocol and granted a waiver of consent. results: twenty-six fps ( %) completed the pre-and posttests. over two-thirds correctly identified the clinical benefits of hu on both assessments. knowledge about the rationale for hu therapy increased after the completion of the hu module ( % correct on pre-test vs. % on post-test, p = . ). the proportion of correct responses increased for all comprehensive aya scd care post-test questions, but only the leading cause of death and the priapism-related questions reached statistical significance ( % vs. %, p = . ; % vs. %, p = . , respectively). the proportion of correct responses for of the transition-focused questions was unchanged ( % for both), while the proportion of correct post-test responses on the self-care assessment question significantly increased ( % vs. %, p = . ). after module completion, fps were able to correctly identify common scd complications and why hu is an effective treatment for individuals with scd. the best practices of transition clinic module may need modification to improve physician understanding of the intricacies in establishing and maintaining a scd transition clinic. overall, online training is effective at educating fps and could be used to increase the number of providers knowledgeable about scd care. background: survival rates for pediatric hodgkin lymphoma (hl) exceed % with contemporary therapy. studies of pediatric hl survivors treated in the s- s have shown increased risk for treatment-related chronic health conditions. risk-adapted therapy, including tailored radiotherapy, has been developed to reduce long-term morbidity while maintaining excellent survival. little is known about chronic conditions associated with contemporary therapy presenting during the first years from therapy completion (early outcomes). objectives: to analyze survival and early outcomes of pediatric hl patients treated with contemporary therapy. we conducted a retrospective review of hl patients diagnosed < years of age at our institution from - . three-year overall (os) and event-free (efs) survival were calculated with kaplan meier statistics using sas . . results of standardized screening for targeted toxicities that developed between - years from therapy completion were identified and graded per ctcae criteria. censoring occurred at date of death, years from therapy completion, or december , . data from the last collection point were used for prevalence calculations in cases with multiple evaluations. we identified patients ( % male; % non-hispanic white; mean age at diagnosis . ± . years) with a median time since therapy completion of . years (range . - . ). initial treatment included: ( %) chemotherapy only and ( %) multimodality treatment. all patients received anthracyclines (median dose mg/m ) and % received alkylating agents (median cyclophosphamide equivalent dose [ced] mg/m ). the -year os was % with an efs of % ( % chemotherapy only, % multimodality treatment; p = . ). patients with relapsed/refractory disease received salvage treatment including chemotherapy only (n = ), multimodality therapy (n = ), or multimodality treatment including stem cell transplant (autologous n = ; autologous+allogeneic n = ). no patients developed thyroid dysfunction, cardiac dysfunction, subsequent neoplasm, or male gonadal dysfunction during the study period. pulmonary dysfunction was limited to ctcae grade . anti-mullerian hormone (amh) below the normal range was found in / pubertal females who received ced ≥ mg/m compared to / females who received ced < mg/m . two of the females with low amh also had follicle stimulating hormone > iu/ml. this study is the first to evaluate early outcomes in pediatric hl survivors. the results indicate contemporary chemotherapy and a lower rate of radiotherapy utilization lead to excellent -year survival rates with minimal early toxicities. females exposed to ced ≥ mg/m are at increased risk for gonadal dysfunction and should be prioritized for fertility preservation approaches prior to initiation of cancer therapy. background: cancer is one of the leading disease-related causes of death among individuals aged < years in the united states. recent evaluations of national trends of pediatric cancer used data from before , or covered ≤ % of the us population. objectives: this study describes pediatric cancer incidence rates and trends by using the most recent and comprehensive cancer registry data available in the us. design/method: data from us cancer statistics were used to evaluate cancer incidence rates and trends among individuals aged < years during - . data were from states and covered % of the us population. we assessed trends by calculating average annual percent change (aapc) in rates using joinpoint regression. rates and trends were stratified by sex, age, race/ethnicity, us census region, county-based economic status, and county-based rural/urban classification, and cancer type, as grouped by the international classification of childhood cancer (iccc). we identified , cases of pediatric cancer during - . the overall cancer incidence rate was . per million; incidence rates were highest for leukemia ( . ), brain tumors ( . ), and lymphoma ( . ). rates were highest among males, aged - years, non-hispanic whites, the northeast us census region, the top % of counties by economic status, and metropolitan counties. the overall pediatric cancer incidence rate increased (aapc = . , % ci, . - . ) during - and contained no joinpoints. rates increased in each stratum of sex, age, race/ethnicity (except non-hispanic american indian/alaska native), region, economic status, and rural/urban classification. rates were stable for most individual cancer types, but increased for non-hodgkin lymphomas except burkitt lymphoma (iccc group ii(b), aapc = . , % ci, . - . ), central nervous system neoplasms (group iii, aapc = . , % ci, . - . ), renal tumors (group vi, aapc = . , % ci, . - . ), hepatic tumors (group vii, aapc = . , % ci, . - . ), and thyroid carcinomas (group xi(b), aapc = . , % ci, . - . ). rates of malignant melanoma decreased (group xi(d), aapc = - . , % ci, - . -- . ). this study documents increased rates of pediatric cancer during - , in each of the demographic variables examined. increased overall rates of hepatic cancer and decreased rates of melanoma are novel findings using data since . next steps in addressing changing rates could include investigation of diagnostic and reporting standards, host biologic factors, environmental exposures, or potential interventions for reducing cancer risk. increasing pediatric cancer incidence rates may necessitate changes related to treatment and survivorship care capacity. background: while childhood cancer treatment modalities have improved, the delayed effects of cancer treatment continue to compromise the quality of life in survivors. metabolic syndrome (ms) is diagnosed based on the presence of three of the following findings -obesity, dyslipidemia, hypertension and insulin resistance per the world health organization (who) criteria. the increased risk of ms among childhood cancer survivors was first reported in the 's and is known to increase the incidence of cardiovascular disease in these individuals. objectives: assess the frequency of ms in childhood cancer survivors at our institution. . we conducted a retrospective chart review on pediatric cancer survivors, - years of age, who had been treated at sri ramachandra medical institute and research foundation between august and august . patients who received at least one year of treatment with s of s chemotherapy and/or radiation and surgery were included. medical history, family history of diabetes, cardiovascular diseases, and hypercholesterolemia, tanner staging, weight for height (< y per who criteria), bmi (> y per indian academy of pediatrics iap), blood pressure (nhlbi criteria), fasting blood sugar levels and lipid profile were obtained from the charts. statistical analysis of the data was done using ibm spss statistical software (version ). results: patients were studied, . % were male. . % were under years of age, . % between - years and . % above years. leukemia survivors comprised . % of the sample and non-leukemic's were . %. . % were treated with chemotherapy alone, . % with radiotherapy and chemotherapy, and . % underwent surgery with radiotherapy and chemotherapy. hypertension was found in . % of the study group, dyslipidemia in %, impaired fasting blood glucose in . % and . % were found to be obese. % of the study group was diagnosed with ms based on who criteria. conclusion: % of our study population was found to have ms per who criteria. individual metabolic complications were detected in % of the population. acute lymphoblastic leukemia (all) survivors appeared to be at high risk in our population. ms has been known to increase cardiovascular complications in cancer survivors. a multidisciplinary team approach to management of these patients is important to closely monitor and manage the long-term complications related to ms such as type diabetes and atherosclerosis. such an approach is essential to decrease long term morbidity and mortality from ms in this vulnerable population. background: the -year survival rate for childhood cancer exceeds %. however, up to % of these children require admission to the pediatric intensive care unit (picu) within three years of diagnosis. these children account for approximately % of all picu deaths, with mortality being higher for those post-hematopoietic stem cell transplant (hsct). national guidelines recommend that providers share informa-tion regarding prognosis and treatment options within the first hours of icu admission. these prognostic goals of care conversations (pgocc) are critical to the care of children with malignancies, a subpopulation at risk for increased mortality. to determine the frequency of pgocc as well as describe differences in patient characteristics and critical care therapies by pgocc status. design/method: a retrospective cohort study was conducted using the university of michigan virtual picu system database. picu admissions lasting longer than hours for patients ages to years between july , and june , with an oncologic diagnosis and/or hsct were identified. data on pgocc, patient demographics, diagnoses, picu interventions, and outcomes were recorded and compared between children with pgocc and those without using chi square test for categorical variables and kruskal-wallis test for continuous data. of picu admissions, % were male; the mean age was . years. the leading diagnoses were acute lymphoblastic leukemia ( %), acute myeloid leukemia ( %), lymphoma ( %), neuroblastoma ( %), and brain tumors ( %), and % of patients were post-hsct. pgocc was documented in ( %) patients. in comparison with patients who did not have a pgocc, children with a pgocc were more likely to be readmitted to the picu ( % vs. %, p < . ) and more likely to have had relapse of disease ( % vs. %, p< . ). patients with a pgocc had higher severity of illness scores (p = . ), higher use of non-invasive ( . % vs. . %, p = . ) and invasive conventional ventilation ( . % vs. . %, p< . ), and high frequency ventilation ( . % vs. . %, p < . ). also, patients with pgocc were more likely to receive continuous renal replacement therapy ( . % vs. . %, p< . ), arterial catheterization ( . % vs. . %, p< . ), and cardiopulmonary resuscitation ( . % vs. . %, p< . ). in only in critically ill children with hematologic-oncologic disease is pgocc held. children with pgocc were sicker and received more critical care interventions. future research is needed to evaluate the content of pgocc. background: central nervous system (cns) tumors and autism spectrum disorder (asd) represent significant disease cohorts in the pediatric population. asd diagnoses in children have a prevalence of %, in every children in the united states. additionally, more than , cns tumors are reported in children age to years in the united states with brain tumors being the most common solid tumor and the leading cause of death among all childhood cancers. the genetic etiology of autism and cns tumors is complex. specific gene alterations present in certain cancers have similarly been described and suspected to play a role in asd subtypes. targeted therapy panels, like foundation one (fo), have been beneficial in guiding treatment for some cancers based on distinct gene alterations. given the genetic overlap, the potential for therapeutic benefit and crossover from such actionable gene target panels merit further exploration in asd and cns tumors. we aim to identify and describe genetic alterations with known actionable targets in cancer therapy from fo as potential diagnostic, therapeutic and research targets for neurodevelopmental diseases. we plan to discuss the common genetic alterations between our cancers and neurodevelopmental diseases described in the literature. fo data was extracted and compared to the literature. each reported gene alteration from fo plus the keywords "autism", "psych" were used on pubmed to search for a suspected association if any with a neurodevelopmental disorder. results: twenty-one patients representing a cohort of six unique (astrocytoma-five, ependymoma-six, gbm-four, glioma-three, nerve sheath tumor-one, etmr-two) cns tumors were investigated. fo produced eighty total with sixty unique gene alterations. thirty-one ( %) of these yielded at least one published, suspected association to a neurodevelopmental disorder. the most common gene alterations were tp -four, cdkn a/b-five and braf-four. the main functional categories were cellular: proliferation, structure, differentiation and degradation; chromatin modeling; histone transcriptional modification; dna methylation and repair; strna; and neural signaling. sixty unique gene alterations were found in our cns tumor set using foundation one. thirty-one ( %) of these discrete alterations paired with at least one description in the literature as having been similarly altered in an asd subtype. many of these alterations have actionable targeted therapies presented through foundation one for our cns tumors and may be a relevant guide in the future of targeted therapy and research in asd subtypes. monoclonal antibody therapy usage is associated with significantly improved survival in b-cell nhl aya patients. although the usage has increased in the aya population from to , the magnitude of the increase is low. factors that affect the use of mab include race and insurance s of s type. further research is warranted to identify why privately insured patients are less likely to receive these drugs. background: prevention of chemotherapy-induced nausea and vomiting (cinv) remains a challenge despite advances in pharmacotherapy and the development of cinv clinical practice guidelines by the pediatric oncology group of ontario (pogo) that have been endorsed by the children's oncology group. achieving control of cinv in pediatrics further is complicated by the difficulty young children have vocalizing their symptoms. use of a validated nausea-assessment tool in conjunction with improved adherence to evidence-based guidelines may result in better quantification of symptoms and reduction of both nausea severity and vomiting frequency for pediatric patients undergoing chemotherapy. the pediatric nausea assessment tool (penat) has been validated for children ages - , and its integration into clinical practice may help optimize cinv control. objectives: this single-institution study sought to improve control of cinv in patients admitted for chemotherapy by standardizing the antiemetic regimens prescribed by all providers according to an institutional cinv algorithm developed from the pogo guidelines. we hypothesized that treatment using a standardized guideline would improve cinv control in patients admitted for chemotherapy. a baseline cohort of admissions for chemotherapy completed penat assessments and cinv diaries prior to receiving chemotherapy, four times daily during each admission, and daily for days following completion of chemotherapy from may , to january , . providers then were provided an institutional cinv treatment algorithm based on the pogo guidelines and received education at departmental meetings on appropriate implementation of this algorithm. a second cohort of admissions completed penat assessments and cinv diaries in a similar fashion from july , to december , . results: complete control of vomiting markedly improved following cinv guideline implementation ( % vs %, p <. ) with treatment failure also significantly reduced ( % vs %, p <. ). after controlling for the degree of emetogenicity of chemotherapy received, a patient was . times more likely to vomit prior to guideline implementation (or . , ci . - . ). there was no difference in nausea control, even after adjusting for the emetogenicity of chemotherapy. conclusion: control of chemotherapy-induced vomiting (civ) improved following widespread implementation of an institutional cinv treatment algorithm at a single institution. the severity of nausea reported remained unchanged which may reflect the difficulty of assessing nausea or an inadequate sample size. future research may focus on cinv treatment management through the use of guidelines specifically for breakthrough cinv and delayed cinv. background: aspho's professional development committee (pdc) recognized pediatric hematologists-oncologists (phos) serving in the united states (us) military have unique professional development needs that may not be addressed by aspho or a similar professional society. these individuals may also encounter challenges when transitioning to a civilian career. however, barriers to professional development have not been systematically characterized. the objectives were to characterize the number of phos with current or prior military service (mphos) and to identify any unmet professional development needs. design/method: a working group consisting of pdc members and both senior and early career mphos was formed. initial comments were solicited by email from known mphos regarding potential gaps in professional development and interest in working with aspho to improve support of mphos. a survey was developed and piloted with four members of the advisory group, questions were revised based on their feedback, and a final version was distributed via the aspho website and online community forum. targeted emails were sent to mphos identified through aspho and military databases. eligibility to complete the survey included ) completion of a fellowship in pediatric hematologyoncology, and ) current or prior service as an active duty military provider. quantitative and qualitative information were collected, including demographic data and perceived barriers to professional development. responses were summarized using descriptive statistics. results: sixty-five mphos were identified and surveys were completed for a % response rate. respondents were engaged in a variety of professional activities; % were male, % were serving active duty commitments, and % felt there were professional development gaps. areas of concern were categorized into nine themes with the most concerning being ) limited civilian knowledge of mpho practices ( % of participants), ) inability to attend professional society meetings ( %), and possibility of deployment ( %). participants expressed a desire for educational products to meet their specific needs and for networking opportunities with civilian colleagues. qualitative analyses identified concerns about low patient numbers and practice size. a subset of mphos perceive significant gaps in professional development. additional research is needed to better define areas for intervention, but many of the concerns align with those of similarly sized civilian programs and may be addressed through professional society networking opportunities, such as an aspho special interest group. background: infertility is an established cause of distress and has a negative impact on quality of life among childhood cancer survivors. the american society of clinical oncology has established guidelines on fertility counseling for individuals of reproductive age diagnosed with cancer, with the goal of improving reproductive and psychosocial outcomes. studies have shown that instituting a fertility team that can provide counseling and discuss fertility preservation (fp) options results in improved patient satisfaction in patients with cancer. objectives: the goal of this study was to examine predictors of referrals to the multidisciplinary fertility team, and documented fp interventions among these patients. design/method: an irb-approved retrospective medical record review was performed at a large pediatric academic center. all patients with new cancer diagnoses receiving chemotherapy were included from january (when the fertility team was established) to present. a standardized abstraction form was used to collect information about: age at diagnosis, gender, cancer type, whether a fertility consult was placed, and documented fp interventions. data were summarized descriptively and comparisons were made using nonparametric statistical methods. results: patients met inclusion criteria, of which ( %) were male. cancer types were as follows: leukemia/lymphoma, cns tumors, sarcomas, embryonal tumors, and langerhan's cell histiocytosis (lch). the mean age was . years, (range < - years). overall, % of all patients had a consultation with the fertility team. patients were significantly less likely to have a fertility consult if they were younger (p< . ). further, there were differences in the consultation rate between diagnoses, with % of sarcoma patients completing a consult, compared to % of those with cns tumors, % of those with embryonal tumor, % of those with leukemia/lymphoma and none of the patients with lch. our findings show that many children, adolescents, and young adults newly diagnosed with cancer are still not receiving fertility counseling despite: ) an expanding body of literature supporting the need to provide this counseling, ) guidelines published by several organizations recommending discussions about infertility risk and fp options, and ) presence of a multidisciplinary fertility team. specific strategies need to be developed to improve access for younger children, and for disease groups in whom fertility consults are underutilized, such as youth with cns tumors, embryonal tumors, and leukemia/lymphoma. background: socioeconomic status (ses) has on impact on overall survival in the pediatric oncology population. unfortunately, data are insufficiently detailed to explain the mechanism behind this phenomenon. how parents handle the health management demands placed on them at the time of a child's cancer diagnosis may represent a point of differentiation in health outcomes. objectives: determine the association between socioeconomic factors, cancer literacy, and parents' understanding of home emergency management and their responses to instances of pain, nausea, and fever. in a prospective observational study of parents whose children were newly diagnosed with cancer, we obtained demographic information and, using a validated instrument, (dumenci, ) we evaluated cancer literacy. we tested understanding of the education parents received about home emergency management with a -item multiple-choice vignette-based questionnaire focused on actions needed in home scenarios. we then followed parents' actual behavior through periodic phone calls assessing instances of nausea, pain, and fever and their responses to these episodes. results: preliminary analysis of participants showed an average score of on the -item parental understanding questionnaire (range - ). variables associated with increased score were college-level education by . points ( % ci [. to . ]), private insurance by . points [. to . ] and adequate cancer literacy by . points [. to . ]. actual behavior reported by families indicated that married parents and those with income above $ , were less likely to treat instances of pain by % ( % ci [ to ]) and % [ . to ], respectively. white parents, those with college-level education, and those with adequate cancer literacy were less likely to treat instances of nausea by % [ to ], % [ to ] and % [ to ], respectively. no associations were found between socioeconomic markers and parental responses to instances of fever. our findings suggest an association between demographic and socioeconomic markers and improved parental understanding of home emergency management. paradoxically, the same markers show a decrease in treatment response to pain and nausea. larger prospective studies are needed to link this behavior pattern to health outcomes, and help inform the extent of ses impact on home emergency management. emory university/children's heathcare of atlanta, atlanta, georgia, united states background: cardiovascular disease is a leading cause of morbidity and mortality in childhood cancer survivors (ccs). previous research showed wide practice variation in referral patterns to cardiology from the survivor clinic and in recommendations from cardiologists about the need for further testing or exercise restrictions. to develop a cardio-oncology algorithm in order to standardize referrals to cardiology and provide guidelines for cardiologists evaluating pediatric ccs. design/method: survivorship and cardiology experts developed a weighted scoring system for pediatric ccs who received cardiotoxic therapy based on time since treatment and risk factors identified by the children's oncology group (cog) and american heart association (aha). the cardiooncology algorithm assigned a score of - . the score range was categorized to guide cardiology referral: screening echo only ( - ), consider cardiology referral ( - ), recommend cardiology referral ( - ), and regular cardiology follow-up (≥ ). the algorithm also provides recommendations to cardiologists for screening and exercise modifications based on the score. after establishment of the algorithm, a convenience sample of institutional survivor clinic patient charts were retrospectively reviewed from the first month of each quarter from april -march to validate the algorithm, evaluate referral patterns to cardiology, and assess cardiology recommendations. the retrospective chart review evaluated patients ( % male; % non-hispanic white; % leukemia survivors; median age at diagnosis . years [range - . ]; median time off-therapy . years [range . - . ]). patients ( %) received anthracyclines (median dose mg/m , range - ) and ( %) received cardiac radiation. assigned cardio-oncology scores resulted in: % echo only, % consider cardiology referral, % recommend cardiology referral, and % regular cardiology followup. when evaluating detection rates of late effects by cardiooncology score, survivors ( %) had an abnormal echo: / echo only, / consider referral, / recommend referral, and / regular cardiology follow-up. assessing referral patterns prior to initiation of the algorithm revealed forty-two survivors ( %) referred to cardiology: / echo only, / consider referral, / recommend referral, and / regular cardiology follow-up. of the patients seen by a cardiologist at our institution, had further diagnostic testing ordered (i.e., stress test) and received exercise restrictions. a cardio-oncology algorithm and guidelines will standardize cardiac care for survivors by assigning a score to guide referral and cardiology practice after referral. prospective clinical use has begun and review will occur in one year to determine changes in detection rates of cardiac late effects, referrals, and recommendations from cardiologists. oregon health and science university, portland, oregon, united states background: delirium affects - % of patients (pts) in pediatric intensive care units (picu) and is associated with increased length of stay, decreased attention in school, and post-traumatic stress disorder. the diagnostic and statistical manual of mental disorders (dsm v) defines delirium as a "disturbance of consciousness […] with reduced ability to focus, sustain or shift attention" due to an underlying medical condition. despite the medical complexity of the hospitalized pho population, there are no published prospective studies looking at delirium in these pts. hypothesizing that delirium is under recognized in the pho population, we designed a year-long prospective study using a validated screening tool to determine the frequency of delirium in hospitalized pho pts and to identify associated clinical factors. design/method: baseline frequency of pts with symptoms suggestive of delirium was determined through retrospective chart review using a data mining program of electronic medical records (emr). for the prospective study, pho and picu nurses were trained to use the cornell assessment for pediatric delirium and to record scores within the emr on all pho pts once every -hour shift. predetermined demographic and clinical variables were entered daily into a red-cap database on all hospitalized pho pts. results: baseline frequency of delirium, without active screening, was determined to be . % of hospitalized pho pts. in the first months of the prospective study, consecutive admissions occurred among unique pho pts: oncology, hematology, and stem cell transplant pts. pts had at least positive delirium screen, for a prevalence per admission of . %. statistically significant variables associated with delirium, at p < . by univariate logistical regression, included prolonged length of stay, pt location (picu vs pho unit), and fever. adjusting for length of stay, administration of benzodiazepines and opiates were also significantly associated with delirium, p = . and . , respectively. on average, nurses completed delirium screening in % of each pts' -hour shifts. study accrual ends in jan and final data analyses will be reported in the abstract presentation. conclusion: delirium does occur in the pho hospitalized population and screening by trained nursing staff is feasible. pts at highest risk appear to be pts with prolonged hospital stays, picu admissions, or frequent use of benzodiazepines/opioids. routine screening should improve our recognition of delirium and allow us to promptly intervene, or prevent delirium in an effort to avoid potential acute and long term consequences. background: with high survival rates for children and adolescents with hodgkin lymphoma (hl), treatment regimens are now designed to maximize cure while decreasing risk of long-term health outcomes associated with chemotherapy and radiation therapy. within contemporary treatment regimens, the comparison of toxicities experienced by patients receiving chemotherapy plus radiotherapy (crt) versus only chemotherapy (co) has not been studied extensively. objectives: this study examines select self-reported adverse health outcomes in survivors of contemporarily-treated pediatric hl to better understand the balance between efficacy and toxicity associated with chemotherapy and radiation therapy. (cog) ahod that evaluated a response-based treatment paradigm in pediatric hl. patient who received initial chemotherapy were randomized based on early response to continued chemotherapy, chemotherapy plus radiotherapy or augmented chemotherapy plus radiotherapy. patients completed self-report questionnaires on health problems at , , , and years following therapy. we examined selected patient-reported pulmonary, gastrointestinal (gi), cardiac and endocrine outcomes. kaplan-meier survival curves were used to determine probability of survival without the selected adverse health outcome. log-rank tests were used to compare the co versus the crt group. results: a total of , enrolled patients, patients in the co group and patients in the crt group, completed , questionnaires at a median of . years after s of s completion of therapy (q , q : . , . ) which were analyzed. the cumulative -year incidence of endocrine dysfunction was significantly greater in the crt group versus those in the co group ( % versus %; p< . ), driven by the incidence of hypothyroidism ( % versus %; p< . ). there were no significant differences in cardiac ( % versus %; p = . ), pulmonary ( % versus % p = . ), and gastrointestinal dysfunction ( % versus %; p = . ) between the co and crt patients. conclusion: this study demonstrates low cumulative incidence overall of organ dysfunction early post completion of contemporary therapy for hl. the addition of radiation therapy significantly increased risk for hypothyroidism, but with no higher risk noted for cardiac, pulmonary or gi dysfunction. limitations include self-report status, potential selection bias, and relatively short latency period following end of therapy. longer follow-up is needed to determine more delayed risks for organ dysfunction in order to best define the balance between therapeutic efficacy and long-term adverse health outcomes related to chemotherapy and/or radiation therapy. background: identification of an organism via bronchoalveolar lavage (bal) or respiratory tract biopsy (rtb) has historically been considered the gold standard for diagnosis of invasive fungal infection (ifi); however, data previously published by our group showed that these procedures infrequently lead to a change in management in children with an oncological diagnosis or undergoing hematopoietic stem cell transplant (hsct). there is also a paucity of data on the cost of ifi in this population. to compare the costs of work-up and management of pulmonary ifi diagnosed based on ct scan alone versus ct scan or chest x-ray prompting a bal or rtb. design/method: we collected cost data on patients at ann & robert h. lurie children's hospital of chicago undergoing chemotherapy or within months of hsct who were suspected of having an ifi between and . in order to include sufficient time to account for post-procedure compli-cations but avoid including costs unrelated to ifi, data were included for days from the day of their diagnostic scan or procedure. cost data was available for of the patients previously studied. thirty-six of these patients were diagnosed with suspected ifi based on ct only and patients underwent bal or rtb. when evaluating specific costs, inpatient beds costs were higher in the bal and rtb group (median $ , versus $ , , p = . ), yet there was only a trend towards higher costs for antifungal agents (median $ , versus $ , , p = . ) and respiratory support (median $ versus $ , p = . ). many of the initial ct scans were not captured in the -day evaluation period for the bal or rtb group based on the study design; however, even when accounting for ct scans up to a week prior these procedures, the total cost of ct scans was higher in the ct only group (median $ versus $ , p = . ), as they had more scans. despite this, total costs were significantly higher for patients who underwent bal or rtb versus ct scan only (median $ , versus $ , , p < . ). combined with our previous data that bal and rtb infrequently leads to a change in management in children with an oncological diagnosis or undergoing hsct suspected to have an ifi, the significantly higher costs associated with these procedures makes these invasive diagnostic techniques even less desirable. batra, pediatr blood cancer, . background: while infants > months of age with acute lymphoblastic leukemia (all) have a poor prognosis, infants with acute myeloid leukemia (aml) fare better despite more intensive therapy. there are limited data on this difference, particularly differences in supportive care requirements during induction therapy for infants. objectives: to compare induction mortality and resource utilization in infants relative to non-infants aged < years, separately for all and aml. design/method: we used previously established cohorts of children treated for new onset all or aml at children's hospitals in the us contributing to the pediatric health information system. patients with down syndrome were excluded. follow-up started on the first day of induction chemotherapy and continued until the earliest of: days after commencement of chemotherapy, start of the subsequent course, or death. high acuity of presentation, defined as icu requirements involving or more organ systems within the first hours following initial admission were compared using log binomial regression. -day inpatient mortality was compared using cox regression. resource utilization rates (days of use per inpatient days) were compared using poisson regression. results: a total of all ( infants, non-infants) and aml ( infants, non-infants) were included in the analyses. infants were more likely to present with high acuity compared to non-infants for both all ( % and %, rr = . , % ci: . , . ; p< . ) and aml ( % vs %; rr = . , % ci: . , . ; p = . ). infants with all had higher inpatient mortality compared to non-infants even after accounting for differences in acuity of presentation ( . % vs . %, adjusted hr = . % ci: . , . ; p = . ). in contrast, inpatient mortality was more similar for infants and noninfants with aml ( . % vs . %, adjusted hr = . % ci: . , . ; p = . ) and comparable to rates among infants with all. infants with all and aml had higher rates of utilization of fresh frozen plasma, cryoprecipitate, diuretics, supplemental oxygen, and ventilation relative to non-infants. infants with all also had higher rates of total parenteral nutrition, ecmo, and patient controlled analgesics compared to noninfants. infants with all experienced significantly higher induction mortality compared to noninfants, a difference not entirely explained by acuity at presentation. differences in ru among infants may reflect higher presentation acuity and greater treatment related toxicity. further work is needed to elucidate the contribution of treatment related toxicity to early mortality in infants with all. background: fever in a child with cancer is a medical emergency due to the significant risk of a serious bacterial infection. many attempts have been made to risk stratify these patients. the respiratory pathogen panel (rpp) is a panel of polymerase chain reaction tests that identify seventeen common respiratory viruses and three bacterial infections. samples are taken via nasopharyngeal swab. rpps are frequently sent, but we do not have data to determine whether a positive result can lead to stratification to a lower risk of bacterial infection. ( ) to determine the epidemiology of respiratory virus-associated fever in pediatric oncology patients ( ) to determine whether a positive rpp is associated with reduced risk of bacteremia in this population. this was a single-center, retrospective cohort study. we identified and reviewed the medical records of all pediatric oncology patients seen in our emergency department (ed) with fever from the introduction of the rpp in april to september , . we reviewed the results of blood cultures, rpp, chest radiographs, and discharge summaries to identify sources of infection. we also identified the patients' cancer diagnosis, age, absolute neutrophil count (anc), and absolute lymphocyte count (alc). results: positive rpps were found among pediatric oncology patients who presented to the ed with fever. the most common positive rpp findings were rhinovirus/enterovirus (rev) ( %), parainfluenza ( %), influenza ( %), coronavirus ( %), and polyviral ( %). among patients with a positive rpp, % had bacteremia compared to % bacteremia among all pediatric oncology patients with fever (or . [ . - . ], p . ). all cases of bacteremia were associated with rev. there was no bacteremia identified in patients with rpps positive for other viruses (or . [ . - . ], p . ). rev positivity did not confer a lower risk of bacteremia than rpp negative patients ], p . ). anc (p = . ) and alc (p = . ) less than , and number of patients with severe neutropenia (p = . ) were not statistically different between the rev and non-rev positive rpp groups. rpps positive for viruses other than rev reduced the likelihood of bacteremia in febrile pediatric oncology patients in the ed setting. patients with bacteremia may have concurrent infection with rev. a larger study is warranted to determine if positive rpp results can inform clinical management of a child with febrile neutropenia. emily mueller, anneli cochrane, seethal jacob, aaron carroll s of s background: the usage of mobile health (mhealth), which refers to the application of mobile or wireless communication technologies to health and healthcare, has grown exponentially in recent years. mhealth tools have been used by caregivers of other vulnerable populations, but little has been focused on caregivers of children with cancer. objectives: to conduct a survey to understand the mobile technology usage, barriers, and desired mhealth tools by caregivers of children with cancer. we conducted a mailed cross-sectional paper survey of caregivers of all children who were diagnosed with cancer at riley hospital for children between june, and june, . the survey contained questions, both fixed and open-ended, in both english and spanish. up to three rounds of surveys were sent to those who did not respond. of the respondents, they were primarily parents ( . %), median age was . years (range - ), and most were white ( . %) and non-hispanic/latino ( . %). the top three annual household income brackets included $ , to $ , ( . %), $ , to $ , ( . %) and under $ , ( . %). the majority had an education: . % college graduates, % graduate degree, and . % high school education or ged. nearly all respondents owned a smart phone ( . %) and . % owned a tablet. the majority used an ios operating system ( . %), while . % reported use of a device with an android operating system. all caregivers reported use of at least one mobile website/app regularly for their personal use. while . % of respondents reported no barriers to mobile technology use, the top barrier selected was "data limitations" ( . %). overall, . % wanted at least one medical managementrelated website/app: medical knowledge ( . %), healthcare symptom tracking/management ( . %), and medication reminders ( %). healthcare system-related desires were high, as . % wanted access to their child's medical record and . % wanted a website/app to facilitate better communication with medical providers. there were no significant associations between socioeconomic status (income or education) with barriers or types of websites/apps desired by caregivers. since the vast majority of caregivers use mobile technology with minimal barriers, future research should focus on designing an mhealth tool to address the medical management needs by caregivers of children with cancer. by supporting caregivers through this type of mhealth tool, it could positively impact patient clinical outcomes through greater adherence to medications and treatment protocols. background: in children with fever and neutropenia, early initiation of targeted antibiotic therapy improves outcomes, yet there are no standards for choice of empiric antibiotics. in our institution implemented an early empiric ceftriaxone (eec) protocol to reduce time to antibiotic administration in febrile hematology-oncology patients who are potentially neutropenic when the absolute neutrophil count is not yet know. ceftriaxone is given immediately after obtaining blood for culture and lab studies. in patients found to be neutropenic, ceftriaxone is discontinued and cefepime is initiated. the purpose of this retrospective study was to evaluate our eec protocol in neutropenic patients by assessing ceftriaxone sensitivity of positive blood cultures and comparing rates of adverse outcomes with a cohort of patients treated prior to implementation of the protocol. we are now conducting a prospective study to more thoroughly investigate antibiotic sensitivities of organisms isolated from blood cultures of neutropenic patients. design/method: hematology-oncology patients with at least one positive blood culture between january and december were identified. patient demographics, neutrophil count, antibiotic treatment, isolated organisms and sensitivities, and adverse outcomes (increased respiratory support, hypotension requiring intervention, and icu admission) were obtained by retrospective chart review. fisher exact test was used to compare dichotomous variables between patient groups. we are now prospectively identifying febrile neutropenic patients with positive blood cultures and performing antibiotic sensitivity testing to several antibiotics commonly used as empiric therapy for febrile neutropenia. results: retrospectively, we identified neutropenic patients with a total of bacterial isolates from blood cultures. of organisms isolated, were tested for sensitivity to ceftriaxone and ( %) were not sensitive, / ( %) of gram-positive cultures and / ( %) of gram-negative cultures. ten of ( %) eec patients had an adverse outcome versus / ( %) of non-eec patients (p = . ). notably, % of eec patients required icu admission versus % of non-eec patients (p = . ). thus far our data obtained prospectively is revealing similar rates of ceftriaxone resistance with / cultures not sensitive to ceftriaxone ( %, ci . %- . %). in our retrospective study, no statistically significant difference was seen in overall adverse outcome rate between the two cohorts, though icu admission rates were significantly higher in eec patients. ceftriaxone resistance rates were high in tested isolates, which is further supported by preliminary data from our ongoing prospective study. given these data, eec may not be effective at improving outcomes in febrile neutropenic pediatric hematology-oncology patients. background: approximately in children diagnosed with cancer will die of their disease, despite advances in treatment. results: two focus groups of six parents each met in june . the parents were predominantly female ( female, male) and had lost their children an average of . years prior (range - . years). two parents were in the same family. nearly all patients were offered palliative care ( / ), all were offered hospice and most died at home ( at home, in the icu). parent discussion uncovered six broad themes: beneficial provider qualities, optimal communication, helpful systematic supports, struggles to feel like a good parent, struggles with a loss of control and unmet needs. parents appreciated providers who were consistent, reliable and honest. parents desired communication that was sensitive to the needs of the patient and family with a balance of hope and realism. parents appreciated the tangible supports pro-vided by social work and the emotional support of child life both for the patient and their siblings. some parents struggled to define and advocate for their child's quality of life, especially when it led to disagreeing with the medical team. several parents expressed frustration with unfamiliar caregivers in the hospital, especially trainees. they expressed a strong desire for more anticipatory guidance about the end of life including how to discuss it with their children. they also wished for a cancer-specific support group for bereaved parents. conclusion: bereaved parents of pediatric oncology patients in our focus groups appreciated consistent, reliable providers who communicated with a balance of realism and hope. they appreciated the tangible and emotional support they received and wanted more anticipatory guidance at the end of their child's life. these results can help guide clinical care, especially in communities without strong palliative care support. further research is needed to develop interventions to improve end of life care. background: clinical trials involving human subjects depend on informed consent (ic) to ensure ethical protections for participants. parents of children with cancer often lack full understanding of the basic elements of ic for clinical trials. additionally, the stress of their child's cancer diagnosis may affect their decision-making capabilities. this is especially problematic as these children rely on parents to fully comprehend clinical trials and weigh their benefits and risks. physician communication is critical for effective family-centered care. the acgme mandates that training programs teach and assess trainees' communication skills. however, there are currently no published curricula aimed at training pediatric hematology/oncology fellows to deliver ic effectively for cancer clinical trials. to develop and pilot-test a simulation-based curriculum to enhance communication skills of pediatric s of s hematology/oncology fellows in the delivery of ic for cancer clinical trials. we developed, tested, and implemented the curriculum from to in two phases. in phase- , we reviewed literature on simulation-based curricula and completed a needs assessment to create a clinical scenario and full curriculum using standardized patients. using miller's pyramid model, fellows' assessments included: immediate de-brief, surveys to assess pre/post confidence and knowledge of the basic ic elements ("knows" and "knows how"), and -degree summative assessments compiled from fellow self-assessments, faculty, and standardized patients ("shows how"). after initial testing and refinements done with fellow, in phase- , we implemented the curriculum with our fellows. likert scale ( strongly disagree- strongly agree) and basic p values are reported. results: fellows gave high mean ratings for training relevance ( . ) and standardized patients' preparedness ( ). almost all ( . ) reported they have used the knowledge gained in their clinical practice. increase in self-reported confidence (pre/post) was noted in all domains: general -describing possible benefits of the clinical trial . / vs. . / (p = . ), risks and potential side effects . / vs. . / (p = . ), and explaining alternatives . / vs. . / (p = . ); research -discussing purpose of the clinical trial . / vs. . / (p = . ), and randomization . / vs. . / (p = . ); and family-centered -addressing emotions during ic . / vs. . / (p = . ), and delivering bad news . / vs. . / (p = . ). summative evaluation mean ratings for all fellows were . (range . - . ). our novel simulated-based ic curriculum, significantly increased fellows' self-reported confidence and skills during ic delivery. importantly, our ic curriculum addressed not just research-related content but also management of parental emotional needs during the ic discussion. next phase includes kirkpatrick model program evaluation and dissemination across other training programs in our institution. national kaohsiung normal university, kaohsiung, taiwan, province of china background: taiwan's childhood cancer foundation reported in that the -year survival rate of childhood cancer was %. as a result, many childhood cancer survivors were back in school after treatment. however, childhood cancer survivors' educational outcomes suffered because of their long-term absence from school and late effects of cancer and cancer treatment. a few school reentry protocols have been developed by the nursing professionals in taiwan to facilitate students' return to school but remained experimental in nature and hardly accessible. parents, students, and teachers were left to their own devices to make individual school reentry plans. objectives: this study aimed to examine and uncover the commonalities among three middle school students' successful school reentry experiences from their teachers' perspectives and to analyze the factors contributing to their success. design/method: this is a qualitative interview study. indepth semi-structured interviews were conducted with three middle school teachers in december about their perceptions, observations, and experiences working with adolescent childhood cancer survivors. the students were two boys with leukemia and one girl with bone cancer. they were diagnosed in the first year of middle school when they were - years old and returned to school for the third and the final year. these students met the following criteria for successful school reentry: regular school attendance, average/above average academic performance, friendship maintenance, and high school diploma. the theme -bring the class to the hospital was found to be the key to the adolescents' successful return to school. without a prescribed school reentry protocol and in the face of limited bedside education services, the homeroom teachers, as links between school, home, and hospital, brought the class to their hospitalized students. they doubled as bedside teachers conducting lessons at the hospital or students' homes, became friends with the parents, witnessed firsthand the students' pain and triumph during treatment, brought the students back to school for visits and celebrations, delivered the classmates' wishes and news to the students, encouraged and welcomed classmates' visits to the hospital, and, together with parents and other teachers, developed flexible school reentry schedules for the students. this on-going study demonstrated the critical roles and functions of homeroom teachers in successfully bringing the students back to school during and/or after cancer treatment. further analysis will be focused on how and why these three homeroom teachers were able to carry out this unexpected task on top of their already full workload. jennifer kesselheim, shicheng weng, victoria allen, collaborative group fellowship program directors dana-farber/boston children's cancer and blood disorders center, boston, massachusetts, united states background: a novel, -module, case-based curriculum entitled "humanism and professionalism for pediatric hematology-oncology" (hp-pho) aims to foster pho fellows' reflection on grief and loss, competing demands of fellowship, difficult relationships with patients and families, and physician well-being and burnout. in small group facilitated sessions, fellows work to identify coping strategies and explore how the challenges of fellowship influence both their own doctoring and the patient experience. objectives: to administer the hp-pho curriculum in a prospective, cluster-randomized trial, measuring whether exposure to this educational intervention, compared to standard conditions, fosters humanism and professionalism and improves satisfaction with training. design/method: pho fellowship programs (n = ) were cluster-randomized to deliver usual training in humanism and professionalism (control) or the novel curriculum (intervention) during the - academic year. the primary outcome measure was the pediatric hematology-oncology self-assessment in humanism (phosah). secondary measures included a -point satisfaction scale, the maslach burnout inventory (mbi), the patient-provider orientation scale, and the empowerment at work scale. participating fellows were pre-tested in summer and post-tested in spring . a change score was calculated for each study instrument. we compared each outcome between arms using mixed effect models adjusted for pre-test score as a fixed effect and site as a random effect. results: randomization yielded intervention and control fellows. the two arms did not significantly differ in distribution of fellow age, gender, or post-graduate year. the intervention sites successfully administered of ( %) modules. change scores on the phosah were not significantly different between the control and intervention arms (adjusted mean difference = . ; % confidence interval [ci] - . , . ; p = . ). compared to the control arm, fellows' exposed to the curriculum gave significantly higher ratings on several items within the satisfaction scale including satisfaction with their training on "physician burnout" (adjusted mean difference = . ; % ci . , . ; p< . ), "physician depression" (adjusted mean difference = . ; % ci . , . ; p< . ), "balancing professional duties and personal life" (adjusted mean difference = . ; % ci . , . ; p = . ), and "humanism overall" (adjusted mean difference = . ; % ci . , . ; p = . ). change scores on other secondary measures were not significantly different between study arms. conclusion: exposure to the hp-pho curriculum did not alter fellows' self-assessed humanism and professionalism. however, the curriculum proved feasible to administer and intervention fellows expressed higher levels of satisfaction in their humanism training, indicating the curriculum's positive impact both for fellows and their learning environment. background: recent work has documented significant levels of unmet needs among adolescents and young adults with cancer, particularly psychosocial challenges during the transition to adulthood, (e.g., abrupt disruption to school and social life, and social isolation). given that adolescents and young adults drive mobile app use, a mobile-phone may be an ideal way to deliver a psychosocial intervention to adolescents and young adults with cancer. to use a patient-centered approach to inform a mobile-based mindfulness and social support intervention for adolescent and young adult patients with cancer. design/method: participants were ten aya with sarcoma ( % female; % adolescents); parents of the five adolescents, and six healthcare providers (n = ). formative research involved three steps: ( ) in-depth interviews were conducted with ten aya with sarcoma; parents of the five adolescents, and six healthcare providers (n = ). ( ) adaptations were made to an existing mindfulness app which offers a program for youth. modifications included creating a -week "mindfulness for resilience in illness" program, with relaxation exercises, and the addition of videos featuring two sarcoma survivors as program hosts. content was informed by the mindfulness curriculum for adolescents, learning to breathe. ( ) a private facebook usability group was organized to (i) elicit beliefs about the mindfulness app and potential future enhancements, and (ii) promote social support. results of the in-depth interviews revealed themes around adolescents' functioning and coping, including body image concerns; recurrence-related anxiety; anger over loss; and being overwhelmed by medical information. themes from the interviews were incorporated into a demonstration version of the mobile app. a patient-centered approach is widely recommended in the development of mobile-based health behavior change interventions and may be a useful way to inform development of a mobile-based mindfulness and social support intervention for adolescents and young adults with cancer. background: medical trainees consistently report suboptimal instruction and poor self-confidence in communication skills. despite these deficits, few training programs provide comprehensive pediatric-specific communication education, particularly in the provision of "bad news." an in-depth survey to examine the historical experience and communication needs of pediatric fellows was conducted at a large academic pediatric center as the first step towards the development of a comprehensive communication curriculum. to determine the previous educational and clinical experiences of pediatric subspecialty fellows, assess their levels of comfort in the context of various communication topics, and query potential modalities and topics for future communication training. design/method: the needs assessment survey was developed using previously developed and validated questions and review of the literature. the survey was reviewed by internal and external pediatric oncology and palliative experts and pre-tested with a subset of trainees to enhance content validity. results: thirty-two out of a total of fellows completed the survey ( % completion rate), of which % were pediatric hematology-oncology or subspecialty fellows. most fellows had participated in previous teaching sessions ( %), including those involving role play or simulation ( %). however, few fellows had received feedback from senior clinicians on their communication skills ( % of fellows had received feedback ≤ times). on a scale of -x, with indicating "not well prepared," the mean score for of communication items was < . fellows felt least prepared to lead discussions around informed consent for experimental therapies, end of life care, and autopsy. fellows indicated that didactic educational sessions and additional coursework were less useful strategies for improving their communication skills, whereas small group role play sessions with faculty and/or bereaved parent educators were most useful. fellows' overall communication preparedness score was not correlated with post-graduate year but was positively associated with the number of times they previously had delivered bad news to patients and families. fellows requested additional training on many topics, with greatest interest in learning skills to optimize communication with an angry patient or family. additional topic requests included placing limitations on resuscitation, withdrawing/withholding further therapy, and ageappropriate inclusion of patients in difficult discussions. despite self-report of prior communication skills training, pediatric subspecialty fellows felt underprepared to participate in difficult discussions with patients and families. learners identified role-playing and coaching with real-time feedback from other physicians and bereaved parents as more useful training strategies as compared to didactic sessions. background: when children die of cancer, parents must adjust to their child's absence amidst the lingering turmoil of what preceded their death: witnessing their child undergo painful treatments, making difficult decisions, and anticipating a devastating loss, all the while hoping for a recovery. adjustment to a child's death, as depicted by current bereavement literature, necessitates making meaning of one's loss. professional care staff can help parents make sense of their child's illness, and in turn, of their own parental experience during treatment. however, the extent to which relationships with professional care team members influence parents' ability to make sense of, and successfully cope with, their loss has not been examined. objectives: to examine how bereaved parents' interactions with their deceased child's pediatric oncology professional care team have impacted their grief symptoms design/method: to better understand how interactions with professional care staff relate to parents' grief outcomes, we conducted a mixed-methods study examining staff impact on parental grief. thirty participants whose children died of cancer one to three years ago completed an in-depth interview and psychometrically validated surveys measuring meaningmaking, depression, and grief symptoms. results: correlational analyses of the measures found that an increase in meaning making was associated with lower depressive and grief symptoms. a content analysis of the interviews found that many participants regarded staff "like family," had on-going relationships with staff after their child died, and described various ways staff interactions during treatment and after the child's death helped them make sense of their loss. in particular, participants described how interactions with staff have helped them find benefits in their loss and learn to create a new relationship with their child despite their physical absence. quantifying the interview data and statistically analyzing it along with the measures found that participants' increased frequency of describing staff's positive impact on their grief correlated with higher meaning-making scores and lower grief symptom scores. our study found that bereaved parents who lost their children to cancer were articulate in sharing their experiences of staff engagement and communication during treatment, offering numerous examples of how staff aided them in making meaning of their loss that were reliably associated with their subsequent grief. we hope the results of this mixed methods research encourage further study of the importance of staff interaction with families during the critical period of their children's care, and the lasting impact this can have regardless of the treatment outcome. memorial sloan kettering cancer center, new york, new york, united states background: although resiliency has been recognized as necessary for healthcare professionals, trainees feel unprepared for the emotional challenges inherent in caring for sick and dying patients. compounded by long hours, challenging work environments, and lack of formal training on handling emotionally difficult situations, many institutions are recognizing the need for interventions to reduce trainee distress. the goals of this fellow-led quality improvement initiative were: ) to determine whether there is a need for emotional support amongst pediatric hematology and oncology fellows, ) to provide formal resiliency and debriefing sessions, and ) to measure feasibility, acceptability and effectiveness of implemented curriculum. design/method: an anonymous survey to determine need for resiliency and debriefing sessions following a traumatic event was distributed to active pediatric hematology & oncology fellows at memorial sloan kettering cancer center in january . once need was established, an intervention consisting of a formal curriculum was developed and initiated in june , involving: ) scheduled and ad hoc debriefing sessions in response to traumatic events (including patient death, codes, interpersonal conflicts, end-of-life care); led by a psychiatrist and social worker with fellows and a pediatric oncologist mentor in attendance, and ) a resiliency didactic curriculum, led by a palliative medicine specialist, focused on skills such as contesting cognitive distortions and mindfulness. the effectiveness of these sessions will be measured using follow-up anonymous surveys at months (currently underway) and months post-initiation of intervention. the initial survey demonstrated most trainees ( / ) were present at or more deaths during their training, while less than half of respondents had attended a post-event debriefing session. % of respondents felt there was not sufficient emotional support from the institution for physicians caring for dying patients. a separate pre-intervention survey found all respondents ( / ) expressed a need for regular debriefings, and nearly all anticipated that they would benefit from such debriefings. concerns identified by trainees that would preclude participation in the curriculum included preference to deal with emotional situations privately and time constraints. trainees identified a need for formal debriefings and resiliency skill development. the program was easily implemented, and is both feasible and acceptable with good attendance. feedback received at the -month mark will determine deficits and possible improvements to the curriculum. the -month survey will measure effectiveness of the program and whether it should be continued. background: acute kidney injury (aki) is a common but under-recognized complication among patients with leukemia. it is associated with prolonged hospital stays, increased mortality, progression to chronic kidney disease, and delays or changes in cancer therapy which may affect a patient's prognosis. however, data on aki in pediatric patients with cancer is still lacking overall. we investigated the incidence of aki in patients who were newly diagnosed with all at our center from january to september . we performed a retrospective chart review of all patients who were newly diagnosed with all from neonate to years in our facility. we determined the incidence of aki in our population using the kidney disease: improving global outcomes (kdigo) diagnostic criteria. we also assessed for nephrotoxic exposures, nci all risk stratification and risk of aki, and tumor lysis syndrome (tls). we identified patients diagnosed during the study period who met inclusion criteria. median follow-up time was . months (range . - . ). the cohort was predominantly male ( . %) and hispanic ( . %). our analysis showed . % had aki by kdigo criteria ( % grade , . % grade , and % grade ), . % had aki on presentation, and % had multiple aki episodes during the study period. older age and longer length of hospitalization were associated with aki (p = . and p = . , respectively). there was no association between aki and nci all risk classification, contrast exposure, hyponatremia, elevated white blood cell count, uric acid levels, antimicrobial therapy, or diuretic use in this study. conclusion: aki was a common finding in our study population. the majority had grade aki by kdigo criteria. however, aki was associated with older age and a longer length of stay. further study is needed to determine the short-and long-term impact of aki on pediatric patients with all. st. jude children's research hospital, memphis, tennessee, united states background: in some regions, the availability of trained pediatric oncologists is a limiting barrier for the care of children with cancer. in , the unidad nacional de oncología pediátrica (unop) and the universidad francisco marroquín school of medicine in guatemala established a pediatric hematology/oncology fellowship program sponsored by st jude children's research hospital to provide central america and the caribbean with well-trained specialists. a systematic analysis of the impact of fellowship programs in pediatric oncology has never been done, especially in the context of a regional education program. objectives: this study sought to analyze the impact of the unop fellowship program based on the regional number of providers, pediatric cancer centers and patient volume. in addition, it sought to characterize the jobs and scientific output of the graduates. the impact will be evaluated in the context of a cost analysis. to define the volume of providers, pediatric cancer centers and patients, the directors of pediatric cancer centers in central america were sent an online survey to obtain these data. all the centers contacted maintain an updated hospital-based patient registry. in addition, the graduates of the fellowship program were also sent an online survey, asking about their job at graduation, current role and scientific productivity. the cost analysis will include assessment of direct costs including salaries and stipends for away rotations, as well as the indirect costs of faculty time spent teaching. since the establishment of the unop fellowship program, the region has more providers for pediatric cancer (p< . ) and centers treat a larger volume of patients (p< . ). two new centers have opened with graduates of the program. all but one graduate practice pediatric oncology ( / ) and the majority do it in their country of origin ( / ). no graduate practices outside of this region. almost half of the graduates ( %) hold a leadership role at their institution. the majority of their time is spent in the public sector (> %). the majority of graduates participate in clinical research ( %) and have participated in the creation or implementation of therapeutic protocols ( %). on average, the graduates have published peer-reviewed articles since completion of training. the unop fellowship program has had a favorable impact on pediatric cancer care in the region, contributing to the capacity to treat a larger volume of patients. graduates practice pediatric oncology in the region in the public sector, frequently hold leadership roles and are scientifically productive. background: abandonment of treatment is a major cause of treatment failure and poor survival in children with cancer in low-and middle-income countries. the incidence of abandonment in peru has not been reported. objectives: the aim of this study was to examine the prevalence and associated factors of treatment abandonment in pediatric patients with cancer of peru. we retrospectively reviewed the sociodemographic and clinical data of children referred between january and december to the two main tertiary centers for childhood cancer, located in lima, peru. definition of treatment abandonment was used from the siop (international society of paediatric oncology) podc (paediatric oncology in developing countries) abandonment of treatment working group recommendation. results: data of children diagnosed with malignant solid tumors and lymphomas were analyzed, of which ( . %) abandoned treatment. univariate logistic regression analysis showed significant higher abandonment rates in children living outside the capital city, lima (p< . ); prolonged travel time to a tertiary center (> hours; or . , p = . ); living in a rural setting (or . ; p< . ) and lack of parental formal job (or . ; p = . ). according to cancer diagnosis, children with retinoblastoma were more likely to abandon compared with other solid tumors. in multivariate regression analyses, rural origin and lack of formal parental employment were independently predictive of abandonment. conclusion: treatment abandonment prevalence in our country is high and closely related to socio-demographical factors. treatment outcomes could be substantially improved by strategies that help prevent abandonment of therapy based on these results. st. jude children's research hospital, memphis, tennessee, united states background: to improve the quality of a pediatric hematology/oncology fellowship program, a systematic assessment must be performed that can evaluate its current state and identify areas of opportunity, as well as modifications over time. unfortunately, widely agreed-upon metrics of quality for pediatric hematology/oncology fellowship programs currently do not exist. this is particularly important in this field due to the global shortage of specialists. for this reason, an assessment instrument that is applicable throughout the world must be created. objectives: the st. jude global education program assessment tool (epat) is a novel instrument that seeks to evaluate pediatric hematology/oncology fellowship programs around the world in systematic and objective way. epat will help determine key performance indexes that are relevant for quality education in pediatric hematology/oncology fellowship programs and establish the framework for improvement. design/method: firstly, key domains to be evaluated for program assessment were identified a priori based on the continuum of pediatric hematology/oncology fellowship programs in the context of geography and educational structure. subsequently, questions were formulated to evaluate these key domains, seeking to assess elements involved in ensuring competence in clinical practice, academic productivity and regional impact. due to the novelty of this tool and the lack of defined metrics of quality, epat relies on expert opinion in a two-step process: internally in the department of global pediatric medicine at st. jude children's research hospital and, subsequently, from a panel of experts in global pediatric oncology and medical education from around the world. ten key domains were identified to evaluate all aspects relevant to training programs around the world, regardless of educational and geographic context. questions have been created to assess these domains and, to make epat quantitative, these have assigned weights with a value reflective of their relative importance. this grading system allows for a score in each key domain, permitting monitoring of changes over time. epat is currently at the stage of external expert review, and subsequently will be piloted in five fellowship programs around the world to provide different geographical and patient care contexts for its validation. once epat is finalized, it will be distributed to pediatric hematology/oncology fellowship programs around the world to be applied. epat proposes a novel strategy to assess training programs in a systematic way that includes all aspects relevant for a training program in a global context. this tool will help guide improvements in pediatric hematology/oncology fellowship programs and assure a well-trained workforce. background: with the improvement in pediatric oncology patient survival and outcomes in the past several decades, monitoring for recurrence and long-term effects of therapy has become even more important. the utilization of personalized treatment summaries and survivorship care plans (scps) is one way to communicate this information with patients and families. the american college of surgeons commission on cancer (coc) created a standard regarding provision of scps to % of eligible patients by december , as a metric for accreditation of all cancer centers. the standard applies to all patients with stage i, ii, and iii cancer diagnoses and requires creation of the scp within one year of diagnosis or six months of completing treatment. during implementation at our pediatric cancer center, we identified barriers to use of the guidelines in the childhood cancer setting. objectives: define eligibility for an scp for pediatric oncology patients to include all patients with curative intent and to deliver scps within six months of finishing therapy. design/method: using chart review and a cancer center registry query, we identified childhood cancer patients potentially eligible for an scp by collecting stage, goal of therapy, and dates of treatment. all patients with curative intent were deemed eligible for an scp regardless of stage i-iv. patients being followed in the oncology clinic for posttreatment surveillance and care were included even if they had received an scp in the survivorship program or were greater than six months off therapy at time of implementation. as expected in the pediatric oncology population, acute lymphoblastic leukemia (all) was the most common diagnosis comprising . % of patients. all is stratified into risk groups instead of surgical staging categories, and treatment duration is greater than one year, unlike many adult-onset malignancies. these differences required interpretation of the guidelines to apply to our pediatric population for all and other pediatric diagnoses with non-surgically based staging. our pediatric oncology clinic has to date provided scps to of eligible patients by adapting the guidelines to focus on patients with curative intent to receive an scp by six months off therapy. cancer staging guidelines and goals for curative intent as well as lengths of treatment vary between the pediatric and adult populations. the coc guidelines require adaptation for optimal applicability to the pediatric oncology population. background: education in communication for fellows in fields that require difficult discussions with families are few in nature. adult learning pedagogies such as role play are under-utilized in medical education, and have been shown to be as effective as traditional teaching methods such as lecture. an -module course for fellows in hematology/oncology, hospice and palliative medicine, radiation oncology, and pediatric hematology/oncology was implemented in january/february . fellows participated in the program. topics covered including fundamentals of communication, coping and spirituality, delivery of bad news, communicating with families, sexual dysfunction during treatment, palliative care/death and dying, and burnout. objectives: overall goal of this course is to foster holistic physicians who views their patients as people with cancer, not cancer patients, and physicians that can communicate effectively with their patients throughout the disease continuum. by the end of the course, learners should be able to practice the fundamental principles of good communication. design/method: fellows initially participated in a pre-course osce to establish baseline skills. osce was facilitated by the center for learning and innovation at northwell, and included actors portraying a pediatric patient and family member to whom the fellow had to break bad news. two months later, the course was carried out over the span of eight weeks and included didactic sessions followed by minutes of role play scenarios. five of the eight modules included role play, with faculty members serving as simulated patients. after the course, a second breaking bad news osce was held. both osces were filmed, and feedback was given by the on-site actors. additionally, faculty members were given access to the videos in an on-line format and were given an evaluation tool to assess the fellows' performance pre-and post-intervention. fellows were given subjective surveys pre-and post-course as well. results: subjective data from participants showed a noticeable increase in comfort level in all areas on the pre-and post-course survey. data obtained from osce videos showed improvement in communication skills as assessed by sps and faculty members using a new evaluation tool developed by faculty. initial first-run data shows that this course is successful in improving communication skills as well as increasing fellows' comfort level across several domains of communication. future directions for our course include improving and validating our assessment tool, expanding our topic base to include more aya and pediatric scenarios, faculty development for improved role play, and investigating impact on practice after course completion. background: acute lymphoblastic leukemia (all) is the most common form of childhood cancer with approximately children diagnosed each year. survival rates have improved significantly over the past several years. children with all are at risk for developing musculoskeletal complications during and after completion of treatment, which can contribute to impaired activity, elevated body mass index (bmi), and risk for complications. interventions involving physical activity could improve musculoskeletal strength as well as overall health in these children. the aims of this study are to examine the feasibility of a directed physical activity program for children with newly diagnosed all during the initial intensive phase of therapy and to evaluate the overall health and quality of life of children participating in the directed physical activity program. design/method: all subjects will receive education materials about the importance and safety of physical activity and a nutrition handout. all subjects will also participate in the directed physical activity program under the supervision of a trained physical therapist for at least minutes every week for weeks. the program will entail four stations including a cardiovascular, balance/proprioception, strength and flexibility, and coordination and cardio. feasibility will be assessed by tracking the participation rate throughout the study period. other assessments will be made at study entry, at the end of weeks of physical activity initiative and months after completion of the intervention. assessments include overall strength and flexibility, weight, height, bmi, blood pressure and performance scores. descriptive statistics will be used for this study. results: a total of patients, male and female, enrolled in the study over a . month period. patient ages ranged from - years. half of the patients enrolled have completed the week program and all patients had stability or improvement of their physical functioning scores. further data collection and analysis is ongoing. patients in the early intensive phase of all therapy are at risk for complications that can affect their physical functioning. a directed physical activity protocol may improve their overall physical functioning. patients may not need specific physical therapy; however a directed physical activity program appears to be beneficial for these patients. the main roadblocks to successful completion of the program were difficulty with scheduling, strain on the parents and patient from treatment, unplanned admissions for fever, as well as nausea and fatigue at time of visit. albany medical center, albany, new york, united states background: communication skills are a core competency highlighted by the acgme. increasing resident confidence in delivering difficult news has been shown to lead to more s of s effective communication. currently, the majority of residency programs lack formal training in communication skills. our objective was to demonstrate feasibility and efficacy of integrating a standardized-patient based training program for communication skills into the curriculum of pediatric residents design/method: to date, pediatric and medicine/pediatric residents have participated in the program during the intern year. the program consists of three, two-hour long sessions, in which each resident is given several opportunities to act out case scenarios with a standardized patient. scenarios included informing a parent of their child's new cancer diagnosis and disclosure of a positive hiv test to a teenager. residents received post hoc peer to peer, and preceptor to learner feedback. pre and post-program surveys were completed by residents. results: following course completion residents reported an increase in confidence in multiple areas of communication including giving a difficult diagnosis (p< . ), discussing a poor prognosis (p< . ), responding to different patient/family member emotional responses i.e. crying or anger (p< . ), and organizing vital information to be relayed (p< . ). in conclusion, communication skills training of pediatric residents is feasible and provides a platform for developing valuable skills not taught elsewhere within the curriculum. background: for children with cancer, transitioning back to school during or after treatment can be challenging. literature supports the need for school re-entry programs to ease this transition. however, these programs vary widely among pediatric cancer institutions with little data addressing their program components. data from this study provides information on current school re-entry programs across these institutions. objectives: one objective of this study was to assess for correlation between the presence of a school re-entry program and other factors, such as geographic location and institution size. a second objective was to establish a list of differences between institutions' school re-entry program components. finally, we aimed to describe current school reentry practices, as well as program benefits and perceived areas for improvement. states with membership in the children's oncology group were offered enrollment in this study. a member of each institution was invited to participate in a survey established by the research team. this person was closely associated with the institution's school re-entry practices. each interview queried institution demographics, as well as program components (e.g., participants, target audience, resources). comment was also collected on program benefits and potential for improvements. analysis of transcripts was performed using pearson's correlation to assess for relationships between institution size, geographic location, and program presence. grounded theory was used for analysis of benefits and improvements. results: thirty-nine of forty-one pediatric institutions who were offered enrollment participated in this study. twentynine institutions ( %) indicated the presence of a school reentry program, and ten ( %) stated they had none. no correlation was found between institution size and the presence of a school re-entry program (p = . , ns). there was also no correlation found between institution location and the presence of a school re-entry program (p = . , ns). a major theme surrounding the benefits of having a program included education for the returning student's peers. for those with programs, perceived improvements included increasing staffing and the ability to offer more services. the results do not support the hypothesis that the presence of a school re-entry program is influenced by the size and geographic location of the treating institution. however, data seem to suggest that available staffing may influence the presence of a program. future studies are needed to address other potential influences, as well as to take an evidence-based approach to determine the effectiveness of the interventions present in these programs. cohen children's medical center/ zucker school of medicine at hofstra-northwell, new hyde park, new york, united states background: genetics/genomics is evolving at an extremely rapid pace. current advances lead to individual algorithms toward disease treatment for each disease with multiple branch points. fellows learn only a fraction of the knowledge and there is no formal approach to teaching critical analysis of information and application algorithms toward disease. additionally, as knowledge evolves extremely rapidly, any approach must teach self-acquisition and application of evolving discoveries. objectives: to create, implement and evaluate a novel curriculum for genetics/genomics targeted toward pediatric hematology/oncology fellows design/method: the curriculum includes four components: ) genetic and genomic medical knowledge, with one initial team-based learning session and weekly online multiple choice questions; ) essential pathways, which will teach molecular pathways common in oncogenesis and relevant to targeted therapy in microteaching sessions with using auditory, visual and tactile learning; ) knowledge acquisition and clinical judgment, to allow learners to gain experience into researching data available, then developing and prioritizing potential treatment plans using problem-based learning sessions in which they will stage a patient, research treatment options, prioritize and present findings; and ) synthesis to demonstrate independent ability to research and recommend therapy through an independent project in which the learner, given a case, will present the case and research findings, genetics/genomics, molecular pathways and make recommendations for therapy in molecular tumor board for faculty and fellows. to evaluate, we plan to recruit to institutions, match for size of programs and implement in half and evaluate nd and rd year fellows in both groups by mcq exam and satisfaction surveys. the creation of a multi-module, adult-learning based curriculum for genetics and genomics in pediatric oncology is feasible. implementation and evaluation are necessary to demonstrate efficacy. background: neuroblastoma is the most common extracranial solid tumor in children. chimeric anti-gd antibody ch . (dinutuximab) therapy has improved the survival of children with newly diagnosed high-risk, neuroblastoma patients as well at the time of first relapse/progression. acute neuropathic pain is a well-documented side effect of dinutuximab administration. however, additional adverse effects including sensorimotor neuropathy, ocular symptoms, and behavioral changes have been described. the incidence and severity of these effects are currently not well-documented in pediatric patients. with improved long term survival of patients receiving this modality, it is important to look for the potential late effects of dinutuximab. objectives: to determine the incidence and severity of neurologic, ophthalmologic, or behavioral changes after dinutuximab administration at our institution. we performed a retrospective chart review using our electronic medical record. we included all patients with high-risk neuroblastoma between the ages of and years at our institution diagnosed between and who received dinutuximab. patients with history of opsoclonus-myoclonus syndrome or gross sensorimotor neuropathy prior to receiving dinutuximab were excluded. we examined clinical documentation for subjective reports and objective exam findings of neurologic, ophthalmologic, or behavioral changes. we also looked for referrals made to neurology, ophthalmology, physical medicine & rehabilitation (pm&r), and psychology. : twenty-two patients met inclusion criteria. at the time of chart review, patients were alive and were deceased. eighteen patients received dinutuximab per anbl ; patients received dinutuximab per anbl . of these patients, patients reported symptoms of interest and reported multiple symptoms. six patients reported symptoms that began at least months after completing dinutuximab. nine patients had objective findings on exam, including decreased deep tendon reflexes, abnormal pupils, and nearsightedness. for patients, referrals were made to ophthalmology, pm&r for neuropsychologic testing, or neurology. two patients who reported symptoms of interest were not referred to a specialist. conclusion: neurologic, ophthalmologic, and behavioral symptoms were commonly reported and demonstrated on exam among pediatric patients with high-risk neuroblastoma who received dinutuximab. it is important to identify these effects so that appropriate specialist referrals can be placed for adequate management of these changes. we recognize that these symptoms may not be solely due to dinutuximab as these patients receive other agents including opioids, so a prospective trial is needed to further evaluate the long-term effects of dinutuximab and to determine how best to screen for these effects. akron children's hospital, akron, ohio, united states background: pediatric cancer is the leading cause of diseaserelated death in children in the united states (u.s.). in , over fifteen thousand children were diagnosed with cancer in the u.s. this population is at high risk for malnutrition due to the multimodal therapies they receive: surgery, chemotherapy, radiation therapy, antibody therapy, and/or bone marrow transplant. adverse effects of these therapies include taste changes, loss of appetite, diarrhea, vomiting, and/or mucositis, making it difficult for the children to be able to consume adequate amounts of nutrition during therapy. there is no "gold standard" measurement tool for identifying patients at risk for malnutrition. nutritional status is not frequently evaluated as a component of clinical trials. assessment of anthropometric measurements (weight, height, z-scores) at diagnosis, as well as over the duration of treatment, can assist in the early identification of malnutrition. the incidence and prevalence of malnutrition in this population is unknown at akron children's hospital. the purpose of this study is to describe the nutritional status and provision of nutritional support therapies in pediatric patients during their first year post new oncologic diagnosis. objectives: identify the incidence and prevalence of malnutrition across oncologic diagnostic categories over the first twelve months post diagnosis. we performed a retrospective records review of all patients newly diagnosed with cancer in at akron children's hospital. demographic and anthropometric data was collected at time of diagnosis and nutritional status categorized by z score. anthropometric and nutrition support data was then collected every two months for the first year after diagnosis along with incidence of unplanned inpatient admissions. results: a total of patients were included in the analysis, with . % malnourished at time of diagnosis; . % developed malnutrition the first year. patients with solid tumors represented % of patients with pre-existing or acquired malnutrition. overall, % of patients received at least one nutritional support modality. patients with pre-existing or acquired malnutrition had a non-significant increase in unplanned admissions (p = . ). our study demonstrated that patients with solid tumors were found to be at increased risk of pre-existing and acquired malnutrition, followed by leukemias, and experienced higher incidence of unplanned admissions in the time period observed. prospective, multi-center replication of this study, including detailed collection of nutrition therapies is recommended to guide development of diagnosis specific nutrition support guidelines. background: pediatric and young adult oncology patients treated with intense chemotherapy have a high incidence of transfusional iron overload. iron deposition can lead to heart failure/arrhythmias, liver abnormalities, endocrine dysfunction, ineffective erythropoiesis, and increased cancer and mortality risk. however, there is a paucity of data regarding recommendations for management of transfusional iron overload in these cancer survivors. consequently, long-term complications of transfusional iron overload specific to these patients have not been assessed. objectives: to assess screening and phlebotomy-based treatment algorithms for this population. design/method: a retrospective chart review of pediatric and young adults who completed oncology management, had iron overload, and initiated phlebotomy treatment was conducted. tiered screening occurred in patients that received at least packed red blood cell (prbc) transfusions. patients were recommended for evaluation and possible phlebotomy if: ( ) liver iron concentration (lic) > mg of iron/gram dry weight liver tissue by ferriscan and/or ( ) cardiac mri t * < ms. during phlebotomy, iron status was assessed quarterly and phlebotomy discontinued with lic < or normalization of ferritin/imaging lic verification. descriptive statistics were employed to report the characteristics of the study population. spearman correlations were utilized to describe associations between transfusions, lic, ferritin, iron saturation and number of phlebotomy sessions. results: twenty five survivors underwent phlebotomy. the mean age was . years (sd . ) and ( %) were female. oncologic diagnoses: all ( %), aml ( %), nhl ( %), ewing sarcoma ( %), osteosarcoma ( %), neuroblastoma ( %) and cns ( %). patients received a median of . (iqr - ) transfusions. median number of phlebotomy sessions was (iqr - ) over . years (iqr . - . ). prior to phlebotomy, median lic was . mg/g (iqr . - . ) and ferritin was . ng/ml (iqr - ) . no patients demonstrated abnormal cardiac t * mri (n = ). ( %) patients completed phlebotomy. one discontinued due to poor vascular access. no patients developed iron deficiency. lic was reduced by a median of . mg/g (iqr . - . ) and ferritin by ng/ml . correlation between number of transfusions and phlebotomy sessions was poor (r = . ). conclusion: management guidelines are lacking for transfusional iron overload in pediatric and young adult survivors of cancer. we demonstrate a phlebotomy algorithm that is effective and tolerated. correlation between number of transfusions received and phlebotomy treatments was poor, necessitating serial assessments. using this management algorithm, prospective studies can evaluate the effect of iron removal on iron overload complications in this patient population. penn state children's hospital, hershey, pennsylvania, united states background: cancer therapy leads to an impaired immune system that takes time to recover. it is important to ensure that these survivors have adequate immunity to prevent common yet potentially severe childhood illnesses. no validated guidelines currently exist for surveillance testing or re-immunization in this population. retrospective analysis involving a small cohort of pediatric cancer patients treated at penn state children's hospital showed % of patients screened for varicella immunity after therapy completion did not have adequate disease titers. to determine the proportion of pediatric cancer survivors who have lost humoral immunity to previously received vaccines; to determine the rate of response to single dose boosters or full vaccine series in seronegative subjects after one booster. design/method: pediatric cancer survivors treated at the children's hospital who are at least months from completion of cancer therapy are prospectively tested for antibody levels to hepatitis b, tetanus, varicella, measles, and strains of pneumococcus ( , b, v, c, f, and f). samples are analyzed by the cdc for measles and varicella avidity. seronegative subjects by commercial studies, are eligible to receive booster vaccines. titers are rechecked at least weeks after boosters to re-evaluate immunity; if still seronegative, subjects will receive the entire vaccine series. titers are finally tested at least weeks after the final dose of the vaccine series. immunity analyzed after therapy, after boosters, and after vaccine series. results: of pediatric cancers survivors who completed therapy, % were non-immune to hepatitis b, % nonimmune to > % of pneumococcal strains tested, % nonimmune to measles, % non-immune to varicella, and % non-immune to tetanus. of subjects who received mmr vaccine after therapy and prior to study enrollment did not have protective antibodies to measles. of the subjects who received varicella vaccine after end of therapy and prior to study enrollment, did not maintain protective antibody levels. cdc results for measles and varicella are pending, as well as repeat studies after vaccine boosters and series. conclusion: a significant percentage of pediatric cancer survivors do not retain immunity to hepatitis b, pneumococcus, measles, and varicella. after one booster, a high percentage of subjects did not develop protective immunity to varicella. only subject did not have immunity to tetanus, which is consistent with the high immunogenicity of tetanus toxoid. formal guidelines are needed to protect this population from vaccine-preventable illness post-therapy. children's hospital of richmond at virginia commonwealth university health system, richmond, virginia, united states background: childhood cancer survivors are at risk for being overweight. diet and physical exercise are important in maintaining a healthy lifestyle and weight; however, it has been reported that cancer survivors are less active than their peers. one reason for this may be that there are no clearly established risk-based exercise recommendations for cancer survivors. another reason may be that providers tend to focus s of s recommendations for exercise more towards patients who are overweight. objectives: to describe changes in physical fitness of childhood cancer survivors who exercise. design/method: 'moving forward' is a wellness and physical fitness program that the center for care beyond the cure at chor offers in partnership with the ask childhood cancer foundation and the ymca. the program is available for any childhood cancer survivor between y and y age, being seen at our center. survivors define their fitness or wellness goals and then work with a trainer once a week (at least) for min sessions throughout the year to achieve these goals. baseline and ongoing measurements for core strength, endurance, overall strength and balance were collected. the average of each of the parameters of all participants were compared from the beginning to the end of the program. over the year, there was a % increase in endurance as measured by the average of the miles walked in minutes, % increase in core strength as measured by the average number of sit-ups in secs, an % and % increase in overall strength as measured by the average weight lifted by leg press and the average weight lifted by chest press, and a % increase in balance as measured by the average number of seconds balancing on a single leg. in addition, each child had actually gained weight in the process with an approximately % increase in the average of the weights of all children. there are benefits to regular exercise beyond weight control, and improvements in physical fitness can be seen even without weight loss. regular physical exercise results in improved physical fitness and should be universally advocated to all patients. determining insulin resistance, measuring changes in fatigue and wellness perception following exercise are future directions that we intend to explore. dana-farber cancer institute, boston, massachusetts, united states background: improvements in adolescent and young adult cancer patient (aya) survival rates and quality of life outcomes have lagged behind those of children and older adults, highlighting a need for research targeting this unique population. current literature supports the value of strong ayaclinician communication, notably in facilitating therapeutic alliance, however little is known about aya communication priorities during cancer care and barriers to optimal ayaclinician communication. objectives: to explore aya and oncology clinician communication priorities and to identify barriers and facilitators to aya-oncology clinician communication. design/method: semi-structured interviews were held with aya cancer patients and survivors (ages - years) from a single large academic institution and oncology clinicians (physicians and nurse practitioners) from academic institutions in the northeastern united states. interviews were conducted in english by phone or in person. all interviews were audio-recorded and transcribed verbatim. analyses were aided by nvivo software. ayas identified a wide range of topics as important to discuss with clinicians. the most frequently identified topics were ) side effects of treatment (with an emphasis on physical appearance and function, n = ), ) social issues (including friendship, family, and school, n = ), ) looking ahead to the future (n = ), and ) sexual & reproductive health (including future fertility, contraception, and romantic relationships, n = ). clinicians prioritized ) cancer treatment and side effects (n = ), ) emotional and psychological health (n = ), and ) sexual and reproductive health with a focus on fertility risk and fertility preservation (n = ). aya reported facilitators to good communication including an open and long-established relationship with the clinician (n = ) and clinician engagement in age-appropriate and patient-directed conversations (n = ). barriers included parental presence during visits (n = ). clinicians reported barriers including ) clinician discomfort (not feeling wellequipped to discuss psychosocial topics such as sexual health, spirituality, and relationships with peers, n = ), ) presence of parents/family (n = ), and ) perceived patient discomfort discussing specific topics (such as sexual health, n = ). clinicians acknowledged the need for collaborative efforts with additional team members (i.e. nurses, psychosocial providers) to assist in meeting aya communication needs. conclusion: aya and clinician-reported communication priorities are largely aligned. however, ayas emphasize some topics, such as social function, appearance, and sexual health that are not highly prioritized by clinicians, which may result in gaps in care for ayas in treatment and in survivorship. these data identify opportunities for intervention, including clinician education, patient and family education, clinic-based intervention, and systems-based changes that can be developed and tested. background: primary care physicians (pcps) cite lack of knowledge and inadequate communication with the oncology team as major barriers to providing recommended surveillance for late effects of treatment to childhood cancer survivors. a standardized telephone handoff to pcps posttherapy is a potential strategy to increase survivorship care by pcps through interactive communication. to determine the feasibility of a structured telephone communication using the situation, background, assessment, and recommendation (sbar) communication tool delivered by a trained oncology nurse to increase pcp knowledge and willingness to provide survivorship care. design/method: from / / to / / , a registered nurse expert in childhood cancer survivorship attempted to contact by telephone the pcps of the most recent patients attending yale's childhood cancer survivorship clinic that were < years old, english-speaking, and ≥ years posttreatment. all pcps had been previously sent an individualized survivorship care plan (scp) that listed the patient's previous treatment history and recommended surveillance tests. upon successful contact and after confirming receipt of the scp, the nurse explained the definition of late effects, description of patient's diagnosis and treatment history, and associated potential late complications and schedule of recommended surveillance tests. the pcp was also asked about his/her ability and willingness to provide needed surveillance for late effects in the future. overall, of pcps were successfully contacted with a median of phone call (range: - ) that lasted a median of minutes (range: - ) after a median of business day (range: - ). no pcps ended the call mid-conversation. all pcps were receptive and expressed appreciation for the call. twenty-five of ( %) pcps expressed an understand-ing of the material discussed and endorsed belief in their ability and willingness to provide late effects surveillance for their patients. no pcps questioned discussing their patient's care with a nurse versus a physician. interactive, structured communications between nurses and pcps by telephone are feasible and are associated with high-levels of pcp confidence in providing survivorship care. background: childhood cancer (cc) admissions account for % of non-newborn pediatric hospitalizations. these hospitalizations are longer and more expensive than other hospitalizations. admission payer (medicaid or commercial) reflects both health policy and sociodemographic status. the objective of this study was to determine if length of stay (los) or cost of cc admissions differed by payer. we used the kids inpatient database, a sampling of all pediatric hospital discharges in the united states. analysis for this study was limited to admissions containing a cancer diagnosis in any discharge icd- codes. admissions were further subcategorized by discharge codes according to diagnosis (leukemia, lymphoma, solid tumor and brain tumor) and reason for admission (chemotherapy, procedure, infection, non-infectious toxicity or "other"). charges were converted to costs using cost-to-charge ratios. multivariable linear regression models were performed to control for age, gender, race, reason for admission, and diagnosis. results: there were , weighted admissions for children with a cancer diagnosis in . of these admissions, . % had medicaid, . % had commercial insurance, and less than % had other payers. the mean los for medicaid admissions was . days ( % ci . - . ), compared with . days ( % ci . - . ) for commercial insurance. surgical admissions accounted for the largest difference in length of stay with medicaid admissions being . days longer than those covered by commercial insurance ( . days vs . days), however, the difference was significantly different for all reasons for admission. in multivariable analysis admissions associated with commercial insurance were % shorter s of s (p< . ), accounting for approximately one hospital day, than admissions associated with medicaid after controlling for other variables including race. the mean overall cost for medicaid admissions was $ , ( % ci - ), compared with $ , ( % ci - ) for commercial insurance. in the multivariable model, cost was collinear with race. conclusion: los and cost of admissions associated with medicaid differed from those associate with commercial payers. medicaid admissions were % longer on average than commercial insurance, accounting for a difference in length of stay of approximately one day although the difference varied with the reason for hospitalization (chemotherapy, surgical procedure, infection, other toxicity, other). costs of admissions were not independent of race. further investigation into potential explanations for this difference including differential access to home care needs, outpatient reimbursement differences, social indications for prolonged hospitalization, and provider biases, is warranted. background: pediatric cancer is a major cause of morbidity and mortality among children surpassed only by accidents. despite improved outcomes in high income countries (hic) survival rates remain poor in the developing word. there are various diagnostic and therapeutic limitations contributing significantly for the survival gap. the main objective of the study is to to evaluate the outcomes of pediatric cancer in armenia and identify diagnostic and therapeutic limitations in the country. we conducted a retrospective study among (≤ years old) children with cancer (solid tumors and hematological malignancies), who were diagnosed and treated at the clinic of chemotherapy of muratsan hospital complex of yerevan state medical university between and . those patients, who didn't receive chemotherapy for any reason were not included in the study cohort. epidemiological, social, medical information was collected through the patient charts review. this included patient age at diagnosis, sex, place of residence (city vs village), the educational level and employment status of parents, type of cancer, stage, presentation of symptoms, first medical specialty consulted and the time consulted, initial work-up, the type of treatment received, information on the diagnosis/treatment received abroad. results: at our clinic during the mentioned period of time the majority of patients presented with hematologic malignancies- %. ( . %) patients had information on diagnosis delay. average delay in diagnosis was about days. in % of cases the first contact with "healthcare system" was through pediatrician, and in % with surgeon. out of relapsed patients received salvage treatment in armenia and abroad. from those who stayed for treatment in armenia patients survived. majority of relapsed patients had acute lymphoblastic leukemia. from leukemia patients immunophenotyping and cytogenetics were available for ( . %) patients; the majority of missing cases were between and , when these diagnostic modalities were not available or affordable in the country. ( %) patients received part of diagnosis and/or treatment abroad. the most frequent reason for going abroad was bone marrow transplantation, otherwise none available in armenia. out of patients were lost to follow-up, patients had a fatal outcome. patients were in remission at a median follow up of . years. conclusion: unavailability of cancer registry and several essential diagnostic/treatment modalities, luck of multidisciplinary care and palliative support, high rate of out-of-pocket expenses were among the main challenges of pediatric cancer care in armenia. background: adverse drug reactions (adrs) are increasingly recognized as important and sometimes irreversible complications of cancer treatment. anthracyclines and cisplatin are effective chemotherapeutic agents, but their use can be limited by cardiotoxicity (anthracyclines) and ototoxicity (cisplatin) in up to % of patients. genetic variants that can be used to predict who is most at risk of developing these adrs have been discovered and replicated. objectives: to create pharmacogenetic risk prediction models for anthracycline and cisplatin toxicities and discuss results with oncologists to facilitate incorporation into treatment decision-making when appropriate. design/method: risk prediction models were developed from the linear regression of strongly-predictive genomic variants (odds ratios ≥ ) discovered and replicated in at least three patient populations. these models were used to assess an individual patient's genomic risk of developing cardiotoxicity from anthracyclines or hearing loss from cisplatin. risk results were returned to oncologists showing where the specific patient's genetic risk of toxicity lies on a continuum between the lowest and highest risk groups across all studied patients using a multi-gene model. interviews were conducted with patients, families, and oncologists to determine how results were valued and utilized. results: patients have been genotyped and had their genetic risk results returned to their oncologists. the first patients have been characterized to determine the impact these test results have had on their clinical care. results were described as being useful in decision-making by patients and/or oncologists in % of cases. additionally, for patients in the most extreme risk groups (highest and lowest risk), a change in treatment plan was ordered % of the time for cisplatin patients and % of the time for anthracycline patients. this included increased cardiac and audiological monitoring, the addition of a protective agent, or choosing an alternative treatment protocol if the risk outweighed the benefits of remaining on the current treatment plan. in interviews, patients indicated that they felt more involved in decision making, and felt reassured by understanding their genetic risk of toxicities. genetic risk prediction models for anthracycline cardiotoxicity and cisplatin ototoxicity were highly utilized by patients and oncologists in decision-making. results were found to be an important tool for informing patients of the risk of adrs during cancer treatment, and resulted in patients and their families feeling more involved in decision-making. background: childhood cancer survivors are at increased risk of developing executive dysfunction, and low socioe-conomic status (ses) has been identified as one of the mediators of executive functioning. previous studies have used traditional measures of ses, such as parents' education level, family annual income and occupation. but more recently, area based socioeconomic measures like block group poverty status are deemed to be more useful in monitoring of social inequalities in health in the united states. block groups are statistical divisions of census tracts and generally contain between and , people. the current study aims to understand the association of block group poverty status (percentage of households in family's block group of residence living below the federal poverty level) with executive functioning among cancer survivor children. design/method: we used a retrospective cohort of childhood cancer survivors. relevant information was collected from the medical record, administrative data sets and parent-filled surveys. address information was geocoded using arcgis . to obtain data on the block group poverty status. a priori cut-points were set to represent block groups with families living below poverty level at %, . % to . %, and ≥ . %. executive functioning were assessed through a parent-rated instrument, the behavior rating inventory of executive functions (brief). multiple linear regressions were used to determine the relationship between block group poverty status and the brief scores. results: data was examined from families of childhood cancer survivors, ranging in age from to years. in this sample, . % families reported an annual income <$ , , . % reported income between $ , and $ , while . % reported annual income ≥$ , . primary care giver of . % of cancer survivors had more than more high school education, and . %, . % and . %, of families were living in a block groups with %, . - . % and ≥ % poor households respectively. block group poverty level was not significantly associated with annual income levels (spearman's rho = . , p = . ), or parental education level (spearman's rho = - . , p = . ). in a step-wise multiple linear regression, there was no statistically significant association seen between block group poverty status and executive functioning after adjusting for co-variables in the final model. future prospective study with a bigger sample size, longer follow up period and more robust measures of the executive functioning like a clinician administered test are needed to understand the effect of block group poverty status on executive functioning. to d completion was . days (range - ). all parents strongly agreed/agreed that d was helpful and would recommend d participation to another family. ten parents ( %) reported time spent on d was "just right." no parent felt more worried due to the intervention, though parent found d participation stressful. this interim analysis suggests that parents have a favorable d experience and recommend the intervention. to date, < % of enrolled parents fail to participate. d shows promise as an acceptable interdisciplinary communication intervention targeted to the early treatment period for childhood cancer. children 's hospital and research center oakland, oakland, california, united states background: screening echocardiograms are recommended by children's oncology group (cog) guidelines to assess for anthracycline-induced left ventricular (lv) systolic dysfunction. the yield of screening echocardiograms during chemotherapy and in the immediate post-therapy period is uncertain. objectives: to assess the incidence of lv dysfunction detected by screening echocardiograms during chemotherapy and in the immediate post-therapy period, defined as - months off-therapy. design/method: children diagnosed with cancer between january -march who received anthracycline chemotherapy were identified. echocardiograms were performed as per protocol, institutional and cog guidelines, and were reviewed retrospectively. lv dysfunction was defined as fractional shortening (fs) < % or ejection fraction (ef) < % ( ) results: in this cohort (n = , median age years), the most common diagnosis was all ( . %), followed by aml ( . %). of echocardiograms, ( . %) were performed during treatment and in the immediate posttreatment period. thirty-eight ( . %) patients had a > % decrease in fs compared to their pre-treatment echocardiograms. none of these patients required any treatment modification or cardiac medications. only patient ( . %) had echocardiogram-proven lv dysfunction discovered on a screening echocardiogram during her treatment course. she eventually died due to multi-organ failure following septic shock. this patient was receiving treatment for aml and had received mg/m of doxorubicin-equivalent anthracyclines at the time of the abnormal echocardiogram. one patient with metastatic ewing sarcoma had borderline lv dysfunction with a fs of % detected a month before completion of therapy. she had received mg/m of doxorubicin equivalent anthracyclines at the time of the abnormal echocardiogram. she did not require any therapy modification or additional cardiac medications. serial echocardiograms done on this patient have shown stable ventricular function. no off-therapy screening echocardiograms identified lv dysfunction. in our experience, the yield of echocardiograms to detect anthracycline-related cardiac dysfunction during treatment and in the immediate post-therapy period is very low. one patient developed lv dysfunction during treatment and one had borderline fs, while no lv dysfunction was identified within months of completing chemotherapy. though fs decreased in % of patients, none required intervention. further study is needed to optimize the use of echocardiography screening in children treated with anthracyclines. references: . landier w et al. jco . background: platinum-based chemotherapy increases the risk of sensorineural hearing loss in children with cancer. little is known about the impact of hearing loss on cognitive and emotional functioning in survivors. to determine the association of severe/profound hearing loss after platinum-based chemotherapy with ) cognitive impairment and ) emotional distress (i.e. anxiety and/or depression). cross-sectional study of all patients attending yale's childhood cancer survivorship clinic ≥ years off therapy for cancer diagnosed at < years and treated with cisplatin and/or carboplatin, but with no history of cns tumor, cranial radiation, congenital hearing loss, or developmental delay. hearing loss severity and hearing aid data were abstracted from audiograms and detailed clinical history. cognitive impairment was defined as behavior rating inventory of executive function t score ≥ , assessment by neuropsychologist, and/or history of special education. emotional distress was determined by brief symptom inventory t score ≥ (global or two subscales) or behavioral and emotional screening system t score ≥ , psychologist interview, and/or history of psychotropic medication/psychotherapy. the most recent available patient data were used. logistic regression with sas software, version . was performed. results: overall, patients ( % female, % white) met eligibility criteria with a median age of . years (iqr = . ) at diagnosis and . years at evaluation (iqr = . ) after a diagnosis of sarcoma ( %), neuroblastoma ( %), or other ( %) for which % received cisplatin and % received carboplatin. fifteen patients ( %) had severe/profound hearing loss in at least one ear. patients with severe/profound hearing loss had a significantly increased risk of cognitive impairment (or = . ; % ci = . - . ), but not emotional distress, compared to patients without severe/profound hearing loss. there was no significant association between age at diagnosis, current age, time since diagnosis, sex, race, ethnicity, or diagnosis with either cognitive impairment or emotional distress. similarly, there was no significant interaction between ) age at diagnosis and hearing loss or ) sex and hearing loss with either cognitive impairment or emotional distress. ten of the ( %) patients with severe/profound hearing loss in at least one ear were recommended hearing aids, of which ( %) reported compliance most of the time. we conclude that severe/profound hearing loss is significantly associated with cognitive impairment, but not emotional distress, in childhood cancer survivors. our data supports the need for interventions to improve hearing in these patients, including compliance with hearing aids. background: who grade anaplastic astrocytoma is a high grade glioma dependent on vascular endothelial s of s growth factor (vegf) mediated angiogenesis for its growth and infiltration. bevacizumab is a recombinant humanized monoclonal antibody which binds vegf-a and inhibits angiogenesis. common adverse effects of bevacizumab are hypertension, proteinuria, thrombosis and bleeding. while animal model based studies have shown that bevacizumab may impair ovarian function the effects of bevacizumab therapy on human fertility are not clear. since the physiology of pregnancy involves neovascularization/angiogenesis it is recommended that conception be avoided for at least months following exposure to bevacizumab. to describe the course of a young adult who became pregnant after receiving bevacizumab and radiation therapy for treatment of an anaplastic astrocytoma. a year old woman diagnosed with a localized hemispheric who anaplastic astrocytoma was treated with chemotherapy and radiation (temozolomide/ . gy) followed by cycles of bi-weekly bevacizumab/temozolomide. patient opted not to pursue fertility preservation prior to initiation treatment. she experienced bevacizumab-associated proteinuria and hypertension during treatment but received all protocol mandated doses (cumulative doses: bevacizumab = mg/kg; temozolomide = . gm/m ). she had a spontaneous unassisted pregnancy months after completing treatment. her pregnancy was uneventful and she was normotensive throughout. fetal ultrasonography at , , , weeks revealed no abnormality of the brain, heart, great vessels, kidney, extremities, placenta and umbilical cord. at weeks she delivered a female infant via cesarean section (birth weight: grams, apgars: and ) excessive post-partum hemorrhage was not reported. placenta was bi-lobed and weighed g. histological analysis revealed normal placental villous development and maturation and two small infarcts. conclusion: exposure to bevacizumab in our patient had no detrimental effect on fertility and on placental/fetal vascular development. we hope this report will add to the existing data on the effects of bevacizumab therapy on fertility. children's healthcare of atlanta, emory university school of medicine, atlanta, georgia, united states background: reports of malnutrition incidence and prevalence in young cancer patients are variable and not well established. previous research suggests children, especially less than years old, treated with intensive cancer-directed therapy are at higher risk for malnutrition. however, no standardized assessment has been used to evaluate risk in this population. objectives: we aim to assess the trends of weight-for-age for patients following cancer diagnosis. this study will be the first to use a standardized measure of treatment intensity (intensity treatment rating scale, itr- ) and will assist in targeting interventions for identification and treatment of malnutrition. design/method: this observational, retrospective study obtained data through the center's pediatric cancer registry and electronic medical record. patients were classified by tumor type (brain or non-brain tumor) and treatment intensity (itr- ). itr- incorporates diagnosis, chemotherapy, radiation, and surgery, beginning with lowest intensity ( ) to highest intensity ( ). inclusion criteria included new cancer diagnosis - at less than years old, with weight obtained and available within days of therapy start date. incomplete data, alternate growth charts, or treatment intensity of , were excluded. weight was obtained at start of therapy and through years after treatment initiation (approximately days) and converted to z-scores adjusted for age and sex. weight trajectories were modeled using generalized linear mixed models with subject-specific random intercepts and spline functions. separate functions were constructed for subgroups of interest (tumor type and itr). results: there were patients included: patients with brain tumors ( . %) and with non-brain tumors ( . %). of included patients, had treatment intensity of ( . %), of ( . %) and of ( . %). over the observation period, , valid weights were recorded. at initiation of treatment, no difference existed between z-score by tumor type (p = . ) or by intensity ( vs. , p = . ; vs. , p = . ; vs. , p = . ). tumor type did not affect z-score through the follow up period. z-scores were higher for intensity rating vs. and vs. (p = < . and p = . respectively) at days after the start of treatment and persisted through days (p = . and p< . respectively). higher treatment intensity is associated with decline in z-score and failure to return to baseline. future directions include further analysis on specific risk factors and timing of weight loss, longer-term follow-up of weight trends, and targeted interventions for identification, prevention, and treatment of malnutrition. objectives: asses the pt requirements for bleeding episodes in a prospective cohort of pcp using a < × e threshold compared to a < × e /l threshold in a historical cohort. we collected pt data in all pcps treated at our center between january/ through december/ . diagnosis, prescription for pt (prophylaxis vs bleeding disorder), plt count and transfused units were assessed for each pt. pcps treated from january/ through june received prophylactic pt with a < × e threshold (cohort a), and pts treated from july/ through december/ received prophylactic pt with a < × e threshold. pts done for procedures and pts with concomitant hemorrhagic pathology were excluded. we compared the number of pts prescribed as prophylaxis vs bleeding episode between cohorts. data analyzed: graphpad prims . ®. statistical analysis: percentages with confidence interval (ci); t-student test (parametric variables) and mann-whitney test (nonparametric variables). statistical significance: p< . . we reviewed pts ( in cohort a, cohort b) in patients. % had acute leukemia, % received and auto or allo hsct. diagnoses and the proportion of patients undergoing hsct was comparable in both cohorts. the average number of pts per patient was , in cohort a and , in cohort b (p = ns), but a significant difference was found when hsct patients were excluded from this comparison ( , pt per patient in cohort a vs , in cohort b, p = , ), which resulted in an estimated , % reduction in pts prescription. furthermore ( , %) pts were prescribed for bleeding episodes in cohort a versus ( , %) in cohort b (p = ns). patients receiving hsct in the entire group ver-sus those not receiving hsct had similar pt requirements for bleeding episodes ( % vs , % p = ns) conclusion: a < × e plt count threshold for prophylactic pts is safe in pcp in chemotherapy and hsct. it can result in a significant reduction in pt usage. key words: platelets, transfusions, prophylaxis, cancer, childhood. ucsf benioff children's hospital oakland, oakland, california, united states background: transition of care for adolescent and young adult (aya) survivors of childhood cancer from pediatric to adult-oriented long-term follow-up (ltfu) is complex. loss to follow-up is common, and little is known about the success rates among different models. the survivors of childhood cancer program (sccp) at ucsf benioff children's hospital oakland employs a community-based model for transitional care. our multidisciplinary team provides aya survivors a comprehensive treatment summary and recommendations, then facilitates transition to primary care or adult oncology ltfu programs. evaluate the success rate for transition of care among aya survivors of childhood cancer in our ltfu program, and identify barriers to successful transition. design/method: aya patients seen from november to august in the sccp with intent to transition were asked by email or telephone if they had followed up with their designated provider. the primary outcome was successful transition, defined as establishing care within months of their visit. patients were also asked about barriers to transition and to rate the new provider's familiarity with their cancer history and ltfu needs. results: transition was intended for patients. eightyseven were contacted and responded. of these, ( %) successfully transitioned, while ( %) were lost to followup. ages ranged from to years, at to years since completion of therapy. ten ( %) transitioned to a primary care provider, ( %) to an adult oncology ltfu program, and ( %) to a pediatrician. patients rated their new provider's knowledge above average ( . ) on a -point scale from poor ( ) to excellent ( ). survivors lost to follow up indicated the following barriers to transition: loss/change of insurance ( ), inability to find a provider ( ), too busy/forgot ( ), problems with transportation ( ), concerns about cost/copay ( ), and s of s other ( ). twelve patients requested further assistance with transition. conclusion: two-thirds of responding patients successfully transitioned. more work is needed to overcome various barriers to transition for one third of aya survivors. albany medical center, albany, new york, united states background: the transition from active treatment, to offtherapy follow-up, is a stressful event for parents of children with cancer. the psychosocial needs of parents after therapy have received limited attention in the united states with only published quantitative studies, the largest with parents. we have secured funding for and recruited a transition care coordinator (tcc) to investigate this further. objectives: our objective is to assess and screen parents at the end of their child's treatment, and to develop interventions to support parents during this time and thereafter. design/method: after informed consent, a standardized questionnaire, the psychosocial assessment tool (pat . ), was administered to parents at end of therapy (t ), months later (t ) and year later (t ). the tcc provided "universal" intervention to all families with an end of therapy binder containing a treatment summary, follow-up roadmaps, information on late effects, and survivor scholarships. based on their pat . scores, some parents were provided intervention specific to symptoms (targeted intervention for scores - . ) or referred to a behavioral health specialist through the clinic social worker for counseling (for scores > ). results: analysis of pat data showed that % of parents (n = ) scored in the targeted or clinical ranges; % of parents scored in those ranges at pat . significant gender differences were revealed with the mean score for men of . and for women of . . this was confirmed by showing statistical significance (p = . ) when analysis was conducted for only a subgroup of data composed of couples (n = ). analysis of pat data by couples (n = ) showed the mean score for men was . and for women was . (p = . ). gender differences were most apparent in caregiver stress reaction questions that focused on ptsd symptoms. when the subgroup of couples' scores (n = ) for caregiver stress reaction at pat was analyzed, there was a significant difference (p = . ) in caregiver stress reaction with a mean of . for men versus . for women. [note: subcategory scores range from to ]. this study was initiated in october using a tcc and the pat . screening tool. the results suggest greater stress on mothers after therapy, with a substantial proportion of parents having symptoms of ptsd after therapy. background: hodgkin lymphoma (hl) is a common childhood cancer characterized by an inflammatory microenvironment. chemotherapy and radiation may exacerbate this inflammation and contribute to the development of late effects (pneumonitis or pulmonary fibrosis). in a heterogeneous cohort of childhood cancer survivors exposed to pulmonarytoxic therapy, no association between pro-inflammatory cytokines and late pulmonary dysfunction was observed. our objective was to test this association in a relatively uniform cohort of survivors of hl, given the well-recognized proinflammatory background of this disease. objectives: to characterize off-therapy pulmonary function in survivors of hl treated with contemporary therapy, and to investigate its association with persistent systemic inflammation. design/method: blood samples, clinical data, and pulmonary function tests were obtained from survivors of hl ≥ months off therapy. lung function score (lfs), a validated method for assessing degree of pulmonary dysfunction on a scale of i to iv, was determined from diffusion capacity and forced expiratory volume in one second (fev ). for a control group, blood samples from patients with benign, noninflammatory hematologic conditions were used. plasma concentrations of inflammatory cytokines were measured on a luminex platform (emd millipore). associations between clinical features or cytokine levels and lfs i (normal) vs. ii-iv were evaluated using logistic regression or wilcoxon rank sum tests, respectively. results: of survivors (mean age at diagnosis: years, range: - ; mean time off therapy: . years, range: . - ), % were categorized as lfs ii (mild dysfunction), % as lfs iii (moderate dysfunction), and no survivors as lfs iv (severe dysfunction). higher lfs was associated with female sex (p = . ) but not other demographic, disease, or treatment factors. forty-eight survivors had blood samples collected at a mean age of . years (range: - ) with a mean time since treatment completion of . years (range: . - . ). of controls, the mean age at time of blood collection was years (range: - ). survivors did not have significantly elevated cytokine levels compared to controls. female survivors of hl ≥ months off therapy are at increased risk of pulmonary dysfunction. neither evidence for pulmonary dysfunction, as measured by lfs, nor duration of time off therapy were related to systemic inflammation in this study. pulmonary function deterioration and clinical pulmonary symptoms are rarely observed immediately following therapy but increase over time. future studies may consider exploring the contribution of systemic inflammation to pulmonary late effects in survivors farther off therapy, when risk for this late effect is greater. background: thyroid carcinoma is a very rare tumor in pediatrics, accounting for . - % of childhood carcinomas in the united states and europe. we aim to detect the risk of second malignancies among pediatric thyroid cancer survivors. the cohort analysis consisted of pediatric cancer patients aged less than years diagnosed with a primary thyroid cancer and identified by site code icd- - : c , reported to a seer database between and . they were followed up by death or the end of the study period (december , ) . out of patients diagnosed primarily with thyroid carcinoma, there were patients who had incidences of subsequent malignancies. the mean age of patients at initial diagnosis of thyroid cancer was years. females ( . %) had significantly higher incidence of second malignancies (sm) than males ( . %). the overall standardized incidence ratio (sir) of sm in thyroid pediatric patients was higher than expected (sir = . ). some specific sites showed significantly higher incidences: salivary gland (sir = . ), gum and other mouth (sir = . ) and kidney (sir = . ). the overall risk of sm in patients received radioactive iodine was higher than expected (sir = . ). the cumulative inci-dence of sms from the initial diagnosis of thyroid cancer was calculated with the survival methodology of competing risk, death treated as a competing event. cumulative incidence of sm was . % [ % ci ( . , . %)] at years and substantially expanded after years, reaching . % [ % ci ( . , . %)] at years. the cumulative incidence of each tumor type at years was . % [ % ci ( . , . %)] for breast cancer, . % [ % ci ( . , . %)] for salivary gland, . % [ % ci ( . , . %)] for each one of kidney and cervix uteri and . % [ % ci ( , . %)] for each one of ovary and melanoma of the skin. cumulative incidence of sm was stratified based on race, gender and radiotherapy exposure, but there was no statistical difference in each of them. conclusion: race, gender, histological subtypes, and radioactive iodine may play an important role as prognostic factors for developing sm among pediatric thyroid cancer survivors. identification of underlying mechanisms that raise the risk of sm is important for both treatment and follow-up strategy. background: the ethical practice of informed consent requires it be both voluntary and understood by the research participant. in pediatric oncology, parents must undergo informed consent to enroll their child with cancer into clinical trials, but often it can be difficult to understand especially for parents with low english proficiency. previous research has shown that parents of children with cancer have difficulty understanding voluntariness, and that parental satisfaction with informed consent does not always correlate with adequate comprehension. objectives: to examine socio-demographic and contextual correlates of comprehension of informed consent, voluntariness, and satisfaction in parents who consented to participation of their child in a cancer clinical trial. we focused on characterizing differences between non-hispanics and hispanics, the fastest growing ethnic group in the u.s. design/method: parents/guardians (n = ) of children aged - years with newly diagnosed cancer, who had consented to participation of their child in a clinical trial for cancer treatment at rady children's hospital-san diego were s of s prospectively recruited. parents completed questionnaires assessing comprehension, voluntariness, satisfaction, health literacy, socio-demographics, and acculturation level, if hispanic. comprehension was surveyed at baseline and longitudinally at months. comprehension, voluntariness and satisfaction outcomes were analyzed by socio-demographics, health literacy, and acculturation level using logistic regression. results: of the participants surveyed, ( . %) were hispanic and ( . %) were non-hispanic. we found that higher health literacy was associated with greater objective comprehension (p< . ), voluntariness (p< . ), socioeconomic status (p< . ), and acculturation (p< . ). hispanics reported lower objective comprehension (p = . ), voluntariness (p = . ), health literacy (p< . ) and ses (p = . ) compared to non-hispanics. spanish-speakers reported lower voluntariness (p = . ), health literacy (p< . ), and acculturation (p< . ) compared to englishspeakers. at the -month follow-up, comprehension in hispanics significantly improved (p = . ) compared to their baseline comprehension. satisfaction was moderately high across all subgroups and was not significantly impacted by socio-demographics, health literacy, or acculturation. in this study, with equivalent numbers of hispanic and non-hispanic participants, we found that hispanic and spanish-speaking parents of children with newly diagnosed cancer had inadequate informed consent comprehension, voluntariness and health literacy despite high satisfaction. our study suggests that hispanics and individuals with limited english proficiency are not making truly informed decisions for their child with cancer. to ensure the ethical practice of research in pediatric oncology, the informed consent and decision-making process must be improved with culturally and linguistically interventions for these underserved populations. memorial sloan kettering cancer center, new york, new york, united states background: pediatric oncology patients undergo repeated bone marrow aspirations and biopsies (bma/bx). these potentially painful procedures can exacerbate anxiety and distress. standard practice at memorial sloan kettering (msk) department of pediatrics is to use propofol, which has amnestic but no analgesic properties. we sought to evaluate whether the addition of local anesthetic would improve patient experience with bma/bx. the purpose of reppair: reducing procedural pain and improving recovery of quality of life (qol) (nct ) is to evaluate the efficacy of local anesthesia with ropivacaine in reducing procedural pain and improving post-procedure qol in pediatric neuroblastoma patients undergoing bma/bx with general anesthesia. reppair is a prospective, randomized, crossover clinical trial that opened for enrollment october . eligible patients were - years old with neuroblastoma. participants were observed on trial for two sequential bm procedures; one procedure with intervention a: propofol alone (pa), and the other with intervention b: propofol plus ropivacaine (p+r). participants were randomized to intervention sequence ab or ba and were blinded to the order of interventions. participants and recovery room (rr) nurses, who were also blinded, followed a standardized postprocedure pain management algorithm. the primary endpoint was percentage of participants requiring opioid analgesia in the hours post-procedure. secondary endpoints included total opioid in hours, non-opioid analgesia use, pain scores, time to first opioid, and short-term qol. qol was assessed by a parent-proxy metric that evaluated pain interference with sleep, physical, emotional, and social recovery. as of january , patients were assessed for eligibility and patients were randomized ( have completed both procedures). for the primary endpoint, a slightly higher proportion of participants required opioid for pa than p+r ( % versus %, p = . ). pain scores in the rr were significantly higher for pa than p+r (median [ th, th percentile]: [ , ] versus [ , ], p = . ). there were no statistically significant differences in total opioid or non-opioid analgesia, -and -hour pain scores, median time to first opioid, or pain interference scores. there were no adverse events. conclusion: preliminary findings of the reppair trial suggest that local anesthesia does not reduce the need for opioid analgesia or improve short-term qol in pediatric patients undergoing bma/bx with general anesthesia. local anesthesia did improve pain scores in the immediate recovery period. final results of this study will help establish evidence-based guidelines and optimize the experience of pediatric patients with bone marrow procedures at our center. background: children with advanced cancer experience a range of symptoms throughout treatment or at end of life, some of which are poorly controlled. minimizing suffering, including effective symptom management, in children with advanced cancer is a central value for pediatric oncology clinicians. patient-reported outcomes have been used in symptomrelated research in pediatric oncology patients; however the majority of literature specific to symptoms during palliative care and end of life for children and adolescents with advanced cancer is based primarily upon medical record reviews and to a lesser extent, patient self-report. the purpose of this study was to prospectively describe symptom frequency, severity, and level of distress in children/adolescents with advanced cancer using patient selfreport and parent proxy. design/method: a prospective cohort design was used for this study. five pediatric oncology institutions from across the united states participated. children and adolescents were eligible to participate if they were - years of age, englishspeaking, and had a diagnosis of advanced cancer, defined as a -week history of progressive, recurrent, or non-responsive disease or a decision not to pursue curative-focused therapy. a modified version of the memorial symptom assessment scale (msas) was used to measure symptom frequency, severity, and level of distress and was administered to child/parent dyads electronically via smartphones every two weeks. information regarding disease status and cancer treatment was collected concurrently. data was analyzed using descriptive statistics and univariate logistic regression analysis. results: a total of children and adolescents and parents participated in the study. the median age of child participants was years, with half being male. the median age of parents was years. the child participants had a variety of primary diagnosis, including: leukemia/lymphoma (n = , %), solid tumor (n = , %), and brain tumor (n = , %). the most frequently reported symptoms by children with advanced cancer and parents were pain (n = / , . %), lack of energy (n = / , . %), and nausea (n = / , . %). presence of disease (p = < . ), recent disease progression (p = . ), and receiving cancer therapy (p = . ) were significant factors on the presence of pain. high intensity cancer therapy was a significant factor on pain frequency (p = . ) and level of distress (p = . ). it is feasible to collect data prospectively in children with advanced cancer regarding symptom frequency, severity, and level distress. clinicians' increased understanding of the symptom experience may promote communication with children and adolescents and timely intervention. more research is needed to understand symptom clusters in children with advanced cancer. vanderbilt children's hospital, nashville, tennessee, united states background: febrile neutropenia (fn) is a frequent occurrence in children undergoing chemotherapy. though guidelines recommend adding a second antibiotic to broad-spectrum antipseudomonal coverage in specific scenarios, augmenting empiric therapy with a second antibiotic is common practice. additional empiric antibiotic (aea) use increases the risk of antibiotic toxicity and future antimicrobial resistance. data clarifying the indications for aea are limited in pediatric patients. objectives: to identify risk factors for gram-positive (gp) and gram-negative (gn) bacteremia in patients presenting with fn to determine situations in which aea use is warranted. design/method: a retrospective chart review was conducted of pediatric severe fn with absolute neutrophil count < / l occurring at a single institution between and . potential a priori risk factors based on clinical reasons for antibiotic expansion were chills, hypotension, mucositis, skin or soft tissue infections (sstis), recent administration of highdose cytarabine (hdac), and a diagnosis of acute myeloid leukemia (aml). potential factors for gn bacteremia were chills, hypotension, mucositis, and abdominal pain. the association between each potential risk factor and gp or gn s of s bacteremia was identified. logistic regression was used for multi-variable analysis. the review yielded episodes. gp bacteremia was isolated in cases ( . %) and gn bacteremia in episodes ( . %). in multivariable analysis, hypotension (or . ( % ci . , . ), p = . ) and sstis (or . ( . , . ) , p = . ) were independently associated with increased risk of gp bacteremia, while mucositis (p = . ), recent administration of hdac (p = . ) and chills (p = . ) were not. ten patients with aml didn't receive hdac, thus the association between aml and gp bacteremia could not be reliably estimated. hypotension (or . ( . , . ), p< . ) and chills (or . ( . , . ), p< . ) were independently associated with a higher risk of gn bacteremia, while mucositis (p = . ) and abdominal pain (p = . ) were not. of the gn infections, ( %) were resistant to cefepime, the empiric agent of choice at our institution. patients with fn with sstis, hypotension, or recent hdac had increased risk of gp bacteremia indicating potential benefit of empiric vancomycin in these settings, while mucositis and chills were not associated with gp bacteremia. hypotension and chills were associated with gn bacteremia, potentially warranting empiric antibiotic expansion, while mucositis and abdominal pain were not. identifying specific indications for aea use in pediatric severe fn use may improve antimicrobial utilization, decrease unnecessary antibiotic use, and improve patient outcomes. background: for children/young adults with incurable high grade gliomas (hggs), like diffuse intrinsic pontine glioma (dipg) or glioblastoma multiforme (gbm), oncologists endeavor to align therapy with patient/family goals of care, but may be influenced by providers' preferences or limited resources. ethical challenges can arise around the perceived purpose, risks and benefits of therapy options, provider conflicts of interest, access to care, deciding decisional priority between patients and families, and conflicts around end-oflife care. objectives: evaluate factors that play into longitudinal decision making for children and young adults with hggs, their families and oncologists using a qualitative approach with ethnographic elements. design/method: eligible patients were aged - with dipg, gbm, or secondary hgg. patient exclusions included: non-english speaking, in state custody, death prior to diagnosis, seen by oncology once, or an oncologist declined participation. key decision making visits (e.g. mri reviews) were serially audio-recorded, along with subsequent : semistructured interviews with patients and/or parents about the decision making process. field notes from clinician meetings, chart notes, and oncologist questionnaires were obtained. discussions and interviews were transcribed and independently coded by three investigators. inter-rater reliability was assessed during code book development. discrepancies were discussed until consensus met. constant comparison analysis with maxqda software continued until thematic saturation. results: twenty-two of eligible patients were approached; agreed to participate. one withdrew upon transferring care. mean age was . years (sd . ); % male, % caucasian, % african american, % hispanic, and % asian. four encounters, ( . hours), were recorded on average per patient. parent/patient interview themes included: ) hope (for a cure, prolonged life, and quality of life), ) importance of physician recommendations, ) importance of support systems (family, community, social media), ) food (as cancer etiology, intervention) ) finances (personal, research funding), ) communication (with medical providers, family, community), ) death, and ) god (beliefs, prayer, existential questions). oncologists desired prolonged quality of life, while patients/families transitioned to that hope from hope for a cure. decisions made in the setting of hggs are multi-factorial, ultimately reflecting the competing values of decision makers. optimism about treatment efficacy is held in tension with poor prognosis, allowing for functional hope. acknowledging patients' and families' shifting hopes allows for changes in goals of care and shared decision making. future work is needed to ) develop preference tools for pediatric patients and families to inform medical providers and ) provide training in communication and shared decision making with oncologists. emory university, atlanta, georgia, united states background: bone marrow transplantation (bmt) is a potentially curative but underutilized treatment for scd. our previous work has shown that there is variation in physician philosophy and practice in considering bmt as a treatment option for patients with scd, and physicians may not discuss this with patients and families as a potential treatment option. in a randomized clinical trial to test the effectiveness of a decision aid for disease modifying therapies for sickle cell disease, adult patients with scd as well as caregivers of adult/pediatric patients were interviewed about how they seek or have sought information related to scd, made decisions about treatments for scd, and identified a treatment option they were interested in learning more about using the decision aid tool. we performed a secondary analysis of these baseline data to understand patient information needs and attitudes regarding bmt as a treatment option for scd. the goals of this analyses was to understand patient and caregivers' attitudes and perceived information needs regarding bmt as a treatment option for scd. we performed an analysis of baseline interviews from caregivers of patients with scd or adult patients from a randomized control trial for a decision aid tool for scd. of the interviews belonged to caregivers of patients with scd. in addition to reviewing interviews for discussion of bmt, we interrogated for mention of terms such as 'bone marrow transplant' or 'cure' or 'stem cell transplant'. interviews were coded using nvivo and analyzed for emerging themes. results: of the baseline interviews, interviews met selection criteria. thirteen of the interviews were with caregivers of pediatric patients, and the remainder were with adult patients, including young adult patients with scd. the majority of participants want to learn about bmt or curative options. in many participants, this was expressed despite knowledge that they were not a likely candidate for transplant. desired information about bmt included eligibility, benefits, risks, long-term effects, quality of life and financial aspects related to bmt. of the patients who discussed how they learnt about bmt, approximately half mentioned that their healthcare provider had not previously mentioned this to them. we then examined knowledge of bmt and attitudes with demographic and clinical variables. patients and caregivers of pediatric patients with scd want to learn about bmt as a treatment option. healthcare providers should consider discussing bmt with their patients with scd. natasha frederick, anna revette, alexis michaud, jennifer mack, sharon bober dana-farber cancer institute, boston, massachusetts, united states background: adolescents and young adults (ayas) consistently identify the need for improved patient-clinician communication on sexual and reproductive health (srh) issues. however, oncology clinicians do not routinely integrate srh conversations with ayas through disease treatment and survivorship. little is known about why these conversations do not take place. objectives: explore aya perceptions of and receptiveness to srh communication with oncology clinicians and to identify barriers and facilitators to these conversations. design/method: semi-structured interviews were held with aya cancer patients and survivors (ages - years, men, women). twelve participants were on active treatment and were within years of treatment completion. interviews were conducted in english by phone or in person. the interview transcript underwent pre-testing with ayas. all interviews were audio-recorded and transcribed verbatim. transcripts were analyzed and summarized by two trained qualitative researchers according to standard comprehensive thematic qualitative analysis methods. analyses were aided by nvivo software. results: ayas perceived existing srh communication between ayas and oncology providers as inadequate. all ayas reported a need for improved srh communication with oncology providers, and three key areas of need emerged: ) general education; ) addressing specific srh issues experienced during treatment and survivorship; and ) understanding the long-term impact of cancer and treatment on srh. ayas felt that current srh discussions are limited and too narrow in scope and scale. ayas reported that most srh conversations focus exclusively on fertility (n = ), usually taking place at the start of treatment. other additional yet limited communication reported was about sexual activity (n = ), contraception (n = ), sexual function (n = ). no ayas reported conversations about potential treatment complications related to sexuality other than infertility. key barriers to srh conversations include patient discomfort initiating conversation (n = ) and presence of family members (n = ), with additional reported barriers including perceived provider discomfort (n = ), lack of rapport with provider (n = ), and age/gender differences (n = ). ayas felt that s of s communication tools such as handouts, brochures, and websites would be helpful facilitators to direct communication from the oncology clinician, and wanted conversations to start before treatment initiation and to continue through treatment and survivorship conclusion: ayas identify a key role for pediatric oncology providers in srh care from diagnosis through survivorship, however multiple barriers interfere with discussions about srh on a regular basis. identified barriers suggest that future efforts should focus on provider education and training in srh and srh-related communication in order to optimize care provided to this unique patient population. background: peripherally inserted central venous catheters (picc) provide secure vascular access in pediatric patients for the delivery of necessary therapies. the ease of placement in the inpatient and outpatient settings has expanded their utilization. however, recent data analyses show a significant increase in venous thromboembolism (vte) risk with the use of picc lines. with its rising use, modifiable risk factors need to be understood for preventative measures. objectives: in this study we aim to understand patient and catheter specific characteristics in relation to the development of vte. design/method: with irb approval, a retrospective interrogation of the electronic medical record and a picc database, at rainbow babies and children's hospital, was completed. the study cohort contained patients < years of age who had a picc line placed between january of and december of . data collected included indication for line placement, line dwell time, location of insertion including blood vessel and extremity, number of attempts at line placement, lumen size and indwelling line length. in addition, we collected number of days to vte formation, associated symptoms and location of vte. chi-squared analyses and fischer's exact test were used where appropriate for statistical analysis. we analyzed ( neonatal) newly placed picc lines. fifty line-associated vte events were found, for an incidence of . %. all vte occurred with the placement of the first picc line. intravenous therapies were the most common reason for line placement. no statistical significance was found between various indications for placement. the most common symptom of vte manifestation was extremity swelling, follow by extremity pain. right extremity picc was found to have a higher incidence of vte. larger catheter lumen sizes (> french) had a higher incidence of vte. we found a mean time of . days to vte detection. we were unable to find any clinical, patient or line specific factors leading to increased vte formation after statistical analysis. special consideration should be given to the duration of picc line use as this may reduce the incidence and comorbities associated with vte. there is still much to be understood about catheter associated vte formation as our analyses indicates the need for prospective data collection on a larger scale in hopes to create guidelines related to catheter use in pediatrics. background: the decision to transfuse a patient is a complex one and is never based solely on a number; however, certain hemoglobin or platelet count thresholds have been proposed in aiding physicians make transfusion decisions. in our hospital, the thresholds for packed red blood cell (prbc) and platelet transfusion in pediatric oncology patients are hemoglobin levels below . g/dl and platelet counts below , /mm (< , for brain tumors), respectively. recently, these thresholds have been questioned and we were asked whether we could safely lower the thresholds to < . g/dl of hemoglobin and < , /mm platelet count objectives: to investigate platelet and hemoglobin transfusion thresholds for oncology patients at children hospital of michigan design/method: retrospective chart review over a -month period, examining platelet and hemoglobin pretransfusion levels for each prbc and platelet transfusion given to oncology patients results: over the course of months, eligible oncology patients (median age years) received transfusions ( prbc transfusions and platelet transfusions). the mean pretransfusion hemoglobin level was . ± . g/dl (range . - . ) (n = ) for total prbc transfusions and this was not different among disease categories (p = . ). patients who had anemia symptoms and signs (n = ) had a slightly lower hemoglobin level compared to those who did not (n = ): . ± . vs . ± . g/dl (p = . ). the mean pretransfusion platelet count was , ± , /mm (range , - , ) for total platelet transfusions (n = ); , ± , /mm in patients with brain tumors (n = ); , ± , in patients with leukemia (n = ); and , ± , in patients with solid tumors (n = ). the mean pretransfusion platelet count was significantly higher in transfusions for brain tumors compared to that in the other disease groups (p< . for both). the mean pretransfusion platelet count was not different among those patients who had bleeding/bruising symptoms ( , ± , , n = ) versus those who did not ( , ± , , n = ) (p = . ). the bleeding/bruising rate was slightly but insignificantly higher in those who had platelet counts < , vs those who had ≥ , ( . % vs . %, p = . ). since most patients develop symptoms of anemia at hemoglobin above g/dl and about / of patients develop bleeding/bruising symptoms at platelet counts above , /mm , our current policy so far reflects a safe threshold for transfusion, and further lowering of the thresholds should be investigated in prospective studies. background: renal impairment is an important complication of childhood cancer and its treatment. serum creatinine level is frequently used as a screening test to monitor renal function; however, patients can have significantly decreased glomerular filtration rate (gfr) with normal serum creatinine. to determine the prevalence of chronic kidney disease (ckd) among children with cancer diagnosis, based on calculated gfr. to compare the difference between using serum creatinine value alone versus gfr in detecting ckd. design/method: retrospective review of medical records of patients, age - years, diagnosed between / - / with solid tumors were analyzed. serum creatinine and calculated gfr using schwartz formula were recorded. ckd as classified by the foundation of kidney disease and outcome quality initiative was used: ckd stage : gfr ( to ml/min per . m ) ckd stage : gfr ( to ml/min per . m ) statistical analysis using spss software v. . chi-squared test for proportions within group, and pearson chi-squared and fisher exact tests for statistical differences between groups. p-value < . was considered to indicate significance results: out of the records reviewed, ( %) were males and ( %) females, with mean age of . ± . years. ( . %) patients received one or more of nephrotoxic chemotherapy drugs; cisplatinum, carboplatinum, or ifosphamide mainly in the non-wilms solid tumors group ( . %) compared to ( . %) in the wilms tumor (wt) group. based on calculated gfr (by schwartz formula) ckd stage /or was diagnosed in ( %) patients with overwhelming majority ( %) were in the mild stage ckd, only ( . %) of those patients had abnormally high serum creatinine levels (p = . ). . % of patients who received nephrotoxic chemotherapy developed ckd, compared to . % in those who did not receive it, (p = . ). despite that only / ( %) of wt group patients received nephrotoxic chemotherapy, yet this group had higher percentage of ckd ( . %) compared to non-wt group ( . %) p = . . significantly lower mean gfr . ± was noticed in the wt group compared to . ± in non-wt group (p = . ) conclusion: high prevalence of mild ckd was found among solid tumor patients. using serum creatinine alone as measure of renal function significantly under estimates renal impairment in those patients. early identification of ckd is easily achieved by using calculated gfr, which can helps providers and care givers to avoid potential nephrotoxic antibiotics, contrast media, nsaids and dehydration that may further deteriorate renal function the university of texas southwestern medical center, dallas, texas, united states background: children with down syndrome (ds) have increased risk of developing leukemia. pediatric patients with ds-associated acute lymphoblastic leukemia (ds-all) are known to have significant toxicities with reinduction chemotherapy and historically poor outcomes with stem cell transplant (sct). anti-cd chimeric antigen receptor (car) t-cell therapy, tisagenlecleucel, demonstrated high rates of durable complete remission (cr) and a manageable safety profile in children with r/r b-cell acute lymphoblastic leukemia (b-all). objectives: characterize the efficacy and safety of tisagenlecleucel in pediatric/young adults with ds-all. design/method: pooled data from single-arm, multicenter, phase trials of tisagenlecleucel in pediatric/young-adult patients with r/r b-all (eliana, nct ; ensign, nct ) were analyzed. eight patients with ds-all were enrolled (data cutoff: eliana, november ; ensign, february ). seven were infused with tisagenlecleucel; patient died from all progression and intracranial hemorrhage before infusion. no manufacturing issues occurred during production. / infused patients were male, / had prior sct (age range, - years). / patients achieved cr or cr with incomplete blood count recovery (cri) by day (d) (cr+cri, %); died before d and was not evaluable. analysis of minimal residual disease was negative in bone marrow in responding patients. two patients had cd negative relapses at and months. ongoing remissions in patients without relapse ranged from to months. the safety profile (n = ) appears similar to that in patients without ds in the same trials (n = ). grade (g) / cytokine release syndrome occurred in % ( / ) of patients with ds and in % without ds. rates of other g / adverse events of special interest did not appear to favor a consistent trend between patients with/without ds (febrile neutropenia: % vs %; neurological events: % vs %; tumor lysis syndrome: % vs %). g / infections were not observed in patients with ds ( % vs %). one patient died after infusion due to intracranial parenchymal hemorrhage on d associated with ongoing coagulopathy. time and extent of tisagenlecleucel expansion and long-term persistence were similar between groups. conclusion: this is the first analysis of car t-cell therapy in pediatric patients with r/r b-all and ds. these data suggest that toxicities appear similar to those in patients with b-all without ds, remission rates in ds-all are high, and longterm outcomes with sustained persistence appear promising. further exploration of tisagenlecleucel as an alternative to sct in children with r/r ds-all is warranted. sponsored by novartis. background: hispanic adolescence and young adults are twice as likely to develop acute lymphoblastic leukemia (all) with high risk features as non-hispanic whites. they also have poor prognosis and % higher death rate. b-all with crlf overexpression caused by genetic alteration of the cytokine receptor, crlf is five times more common in this subgroup. approximately % of crlf b-all cases also have ikzf genetic alterations. ikaros is involved in transcriptional regulation of several important genes involved in leukemogenesis. overexpressed casein kinase ii (ck ) impairs functions of ikaros. objectives: understand the molecular mechanisms that regulate crlf expression in crlf b-all. here we present evidence that ikaros-mediated repression of crlf transcription in b-all in hispanic children is regulated by ck . design/method: primary b-all patient samples from hispanic children were used. ikaros retroviral transduction, ikaros shrna transfection, real time-pcr, luciferace assay, quantitative chromatin immunoprecipitation (qchip) coupled with the next-generation sequencing (chip-seq), cytotoxicity assay and western blot. results: ikaros binding to promoter of crlf was confirmed using quantitative chip. functional experiments such as overexpression of ikaros in b-all primary cells results in transcriptional repression of crlf whereas ikaros silencing using shrna resulted in increased transcription. these results suggest that ikaros negatively regulates crlf expression. molecular inhibition of ck with shrna targeting the ck catalytic subunit, as well as pharmacological targeting of ck with cx resulted in transcriptional repression of crlf . ck inhibition was associated with increased ikaros dnabinding to the promoter of crlf . however, the ability of cx to repress crlf is lost or severely reduced, in cells with shrna silencing of ikaros, as compared to cells with intact ikaros. moreover, similar results were noted following treatment with cx in leukemia cells obtained from high risk b-all patients with deletion of one ikzf allele. ikaros binds poorly to promoters of crlf gene in these cells. treatment with cx restores ikaros dnabinding to the promoters of crlf , which is associated with its strong repression. serial qchip analysis of the epigenetic signature at the crlf promoter showed that increased ikaros binding to the crlf promoter, following ck inhibition, is associated with enrichment for the h k me histone modification, which is a marker of repressive chromatin. results demonstrate that crlf expression is epigenetically regulated by the ck -ikaros axis .cx show antileukemic effect via restoration of ikaros tumor suppressor function, resulting in crlf repression suggesting advantage of using ck inhibitors as potential therapeutic approach in crlf altered b-all. results: hypodiploid all (modal chromosome number < and/or di < . ) was identified in patients ( . % of all patients; . % of nci standard risk (sr) and . % of nci high risk (hr)), who were removed from frontline protocol therapy post-induction. overall -year efs and os were . %± . % and . %± . %. transplant status was retrospectively available for / ( %), of whom underwent hsct in cr . five-year efs with hsct was . %± . % vs. . %± . % without (p = . ). -year os with and without hsct was . %± . % vs. . %± . % (p = . ). when corrected for the median time to hsct ( days), there were no significant differences in -year efs or os rates with and without hsct: . %± . % and . %± . % vs. . %± . % and . %± . %. no nci risk group or mrd subset benefitted significantly from cr hsct. sr patients (n = ) had -year efs and os of . ± . % and . %± . % with hsct (n = ) vs. . %± . % and . %± . % without. hr patients (n = ) had -year efs and os of . %± . % and . %± . % with hsct (n = ) vs. . %± . % and . %± . % without. for those with end-induction mrd < . % (n = ), -year efs and os were . %± . % and . %± . % with hsct (n = ) vs. . %± . % and . %± . % without. end-induction mrd-positive patients (n = ) fared poorly with both year efs and os of . %± . % with hsct (n = ) vs. . %± . % and . %± . % without. multivariate regression analysis including nci risk group, mrd, and cr hsct, showed only mrd negativity was significantly associated with efs (hr . , p< . ) and os (hr . , p< . ). patients with hypodiploid all fare poorly, particularly those with end-induction mrd ≥ . %. while cr hsct is a standard treatment approach, it does not confer significant benefit. we were unable to assess bridging therapy prior to hsct, and comparator groups are small. taken together, however, new strategies are urgently needed for these patients. background: ras-pathway mutations are known to play a pivotal role in a significant proportion of myeloid malignancies, including upwards of % of pediatric aml cases. ras-pathway mutations in myeloid malignancy commonly co-occur with mutations of epigenetic regulators, suggesting cooperative leukemogenesis. among the epigenetic modifiers most frequently mutated in myeloid malignancy are regulators of dna methylation. this indicates that the alteration of dna methylation contributes to leukemogenesis. the ten-eleven translocation (tet ) is an epigenetic regulator that plays an important role in regulation of dna methylation through its action of hydroxylation of -methylcytosine, which ultimately leads to passive de-methylation of dna cytosines. in myeloid malignancy, loss of function tet mutation is one of the most frequently co-occurring lesions in ras mutated malignancy. how specifically the altered methylation patterns in ras-pathway driven diseases promotes leukemogenesis is unclear. objectives: we hypothesize in mice with a ras-pathway mutation, that when an epigenetic modifier co-occurs, such as loss of function of tet , this primes stem cells and/or early differentiating progenitors for transformation by preventing the repression of stem cell self-renewal genes, inhibiting differentiation, enhancing ras signaling and leading to leukemogenesis. we have generated a novel murine model with constitutive deletion of tet (tet -/-) combined with an inducible activating krasg d mutation (krasg d/wt). mice have been tracked for evidence of hematologic malignancies and compared to mice with corresponding single genetic lesions. cooperative leukemogenesis will be demonstrated by decreased latency to disease onset, impact on malignancy lineage, in addition to investigating mechanistically through which pathways leukemogenesis may be promoted. results: krasg d/wt/ tet -/-mice demonstrate statistically significant differences in peripheral white blood cell count, hemoglobin, and platelet levels as early as -weeks post ras-pathway activation. peripheral cell lineage analysis demonstrates early skewing toward myeloid differentiation and marked splenomegaly in mice harboring both genetic lesions compared to wild type or mice with single genetic lesions. phospho-flow cytometric analysis reveals increased perk and ps activation in krasg d/wt/ tet -/-sca- enriched bone marrow cells compared to either genetic lesion alone. our study utilizing a murine model to examine how in ras-pathway mutations the addition of a co-occurring epigenetic lesion demonstrates that these lesions appear to cooperate to promote early myeloid differentiation with attendant changes in signaling pathways. this exploration to elucidate the mechanics of ras-pathway mediated disease lay the foundation for identification of patients who may benefit from existing therapies, such as dmtis, or identify new signaling targets for therapeutic exploration. background: the humoral immunogenicity of car , a chimeric antigen receptor (car) with a murine scfv domain developed for treatment with tisagenlecleucel in relapsed/refractory (r/r) pediatric/young-adult acute lymphoblastic leukemia (all), was evaluated in studies. little is known about the presence/impact of preexisting/treatmentinduced anti-murine car (mcar ) antibodies in patients treated with car therapy. objectives: patients from eliana (nct ; n = ) and ensign (nct ; n = ) were evaluated before and after tisagenlecleucel infusion to determine the impact of anti-mcar antibodies on cellular kinetics, efficacy, and safety. design/method: anti-mcar antibodies were determined by flow cytometry and reported as median fluorescence intensity. assay validation included evaluation of the interferences of intravenous immunoglobulin (ivig) treatment with the anti-mcar antibody assay. impact of preexisting and treatment-induced immunogenicity on cellular kinetics, efficacy, and safety was determined. treatment-induced immunogenicity was defined by a positive increase in anti-mcar antibody levels over baseline and was assessed by calculating the fold-change between preexisting (ie, baseline) and postinfusion levels. results: % of patients displayed preexisting anti-mcar antibodies; a similar incidence was detected in healthy volunteer samples during method validation. % of patients developed treatment-induced anti-mcar antibodies. no relationship was identified between tisagenlecleucel expansion (auc - d) and preexisting/treatment-induced anti-mcar antibodies (r < . and r = . , respectively); similar results were seen for cmax. presence of treatment-induced anti-mcar antibodies did not appear to impact transgene persistence or response. kaplan-meier estimates showed that preexisting/treatment-induced anti-mcar antibodies did not appear to impact duration of response or event-free survival. strip plots showed consistent levels of preexisting/treatment-induced anti-mcar antibodies across patients with safety events, including cytokine release syndrome, neutropenia, thrombocytopenia, and neurological events. there was no apparent relationship between treatment-induced anti-mcar antibodies and b-cell recovery categories (≤ months, > and ≤ months, > months, and ongoing sustained aplasia). no association existed between time of b-cell recovery and presence of treatment-induced anti-mcar antibodies. b-cell aplasia requiring ivig occurred following tisagenlecleucel in the majority of patients. the tisagenlecleucel concentration-time profiles in patients with treatment-induced anti-mcar antibodies were categorized by time following ivig administration. time of ivig administration had no impact on in vivo transgene expansion and persistence. we report the first comprehensive assessment of the impact of anti-mcar antibodies on clinical endpoints with car therapy. pediatric/young-adult patients with r/r all had a high frequency of baseline anti-mcar antibodies, and preexisting/treatment-induced anti-mcar antibodies did not impact the cellular kinetics, safety, and efficacy of tisagenlecleucel. cell-mediated immunity studies are ongoing. sponsored by novartis. background: adoptive immunotherapy, using cd engager (cd -eng) t-cells, has shown success in preclinical studies, recognizing and killing acute myeloid leukemia (aml) blasts in vitro and in vivo. cd -eng t-cells secrete bispecific molecules that recognize cd (t-cells) and cd (aml blasts), and are able to direct transduced t-cells and recruit bystander t-cells to kill cd -positive blasts. however, cd -engs do not provide costimulation and have not shown the capability for sequential killing of targets in vitro. we are seeking to improve the expansion, persistence and sequential killing capabilities of cd -engs by genetically modifying these cells with an inducible costimulatory molecule, which can be activated by a chemical inducer of dimerization (cid). we generated a retroviral vector encoding cd -eng and the inducible costimulatory molecule myd .cd linked by a a sequence (cd -eng. a.imc). cd -eng and cd -eng.imc t-cells were generated by retroviral transduction, and their effector function was compared with and without cid. we used flow cytometric analysis to assess transduction efficiency, chromium release assays to evaluate cytolytic activity, and elisa to determine cytokine production. we successfully generated cd -eng.imc tcells and achieved a mean initial transduction efficiency of % that was maintained above % throughout our study period. cd -eng.imc t-cells +/-cid and cd -eng t-cells readily killed cd -positive aml blasts (molm and kg a) in cytotoxicity assays when compared to the cd -negative control (k ). in co-culture assays, cd -eng.imc t-cells secreted increased il- and ifn-gamma in the presence of cid and cd -positive targets (kg a and molm ) when compared to co-culture with cd -positive targets in the absence of cid. in addition, cd -eng.imc t-cells displayed enhanced sequential killing capabilities and ifn-gamma secretion when stimulated weekly with cid and tumor cells at a : ratio when compared to cd -eng t-cells. conclusion: cd -eng.imc t-cells are able to recognize and kill cd -positive aml blasts in an antigen dependent manner. cd -eng.imc t-cells have improved effector function in the presence of cid as judged by cytokine production and their ability to sequentially kill cd -positive target cells. thus, inducible myd and cd costimulation is a promising strategy to improve the effector function of cd -eng t-cells, and warrants further active exploration in preclinical studies. background: eliana (nct ; n = ) is a pivotal multicenter study testing the efficacy of tisagenlecleucel, anti-cd car-t, in children/young adults with r/r b-all. tocilizumab (toci) has been used for management of moderate/severe (grade / ) crs in ≈ % of patients treated with tisagenlecleucel at equivalent doses used in approved nononcological pediatric indications (< kg received mg/kg; ≥ kg received mg/kg [ mg max dose]).( ) crs onset, as graded by the penn grading scale, generally occurred at a median of days (range, - ) after infusion, requiring administration of - toci doses in some patients via a protocol-specific treatment algorithm. toci is a humanized monoclonal antibody that inhibits il- receptor (il- r) signaling. the pharmacokinetics (pk) and pharmacodynamics (pd) of toci in pediatric patients with b-all with carassociated crs have not previously been described. objectives: characterize toci pk/pd for crs management following tisagenlecleucel infusion and describe its impact on cellular kinetics. design/method: toci pk and levels of soluble il- r (sil- r) were determined from serum and quantified using validated assays. maximum toci concentration (cmax) was derived using noncompartmental methods. sil- r, proinflammatory cytokines, and crs resolution time were characterized to describe toci pd. summary statistics and graphical analyses of tisagenlecleucel exposure by number of doses were performed to describe the impact of toci on tisagenlecleucel kinetics in patients responding to tisagenlecleucel infusion. : / patients with crs received the first toci dose at a median of days (range, - ) after crs onset. seventeen patients received dose (range, . - mg/kg); received doses ( - mg/kg); received doses ( - mg/kg), per the crs treatment algorithm. first-dose mean cmax (sd) was ≈ ( . ) g/ml; second dose, ≈ ( ) g/ml. individual patient pd concentration-time profiles showed increased sil- r levels after the first toci dose which remained elevated following the second dose. following toci administration, median time to crs resolution (including fever resolution) was days (range, - ). crs onset coincided with tisagenlecleucel expansion, followed by a peak in serum cytokines, including il- . the geometric mean auc - day and cmax of tisagenlecleucel transgene (by pcr) were % and % higher in tisagenlecleucel-responding toci-treated patients. conclusion: crs symptoms resolved within a median of days after toci administration. toci levels achieved in patients with b-all were similar to reported pediatric nononcological indications (tocilizumab label) and resulted in concentration/time-dependent sil- r increases. transgene continued to expand and persist following toci administration. these data support treatment with toci for crs management. ( ) buechner, eha, . sponsored by novartis. background: in acute myeloid leukemia (aml), mesenchymal stem and stromal cells (mscs) in the bone marrow microenvironment contribute to extrinsically mediated chemo-resistance and are therefore important potential therapeutic targets. the study of patient-derived mscs is at a competitive disadvantage, however, because traditional means of isolating mscs from a bone marrow aspirate interferes with isolating the more highly prioritized leukemic cells. many opportunities to study mscs are therefore missed. objectives: to develop a novel method of isolating mscs using the otherwise discarded portion of a bone marrow aspirate, thereby de-coupling the isolation of primary mscs from the isolation of leukemia cells. design/method: aml patient bone marrow aspirates were obtained prospectively from the children's oncology group. healthy patient marrow was purchased. experimental mscs were isolated from the bottom-most layer (rbc-layer) produced by density-gradient separation of a bone marrow aspirate, which is typically discarded. control mscs were isolated from the buffy coat (mnc layer). non-adherent cells were removed after hours, and adherent cells were cultured at % co with mem-alpha containing % fbs. growth curves were obtained by seeding -well plates with , cells per well. cells were stained using oil red o to observe adipocyte differentiation. results: rbc-layer mscs grow successfully following overnight shipment of the aspirate. identical to mnc-layer mscs, rbc-layer mscs exhibit a fibroblastic morphology and are adherent to plastic. rbc-layer mscs persist in culture up to passages before senescence. they exhibit a slower growth curve relative to mnc-layer mscs, but their overall doubling time is similar at approximately hours. surprisingly, mscs from the rbc-layer exhibit adipocyte differentiation on stimulation, revealing their stem-cell like qualities. we present a method of isolating mscs from the discarded portion of a bone marrow aspirate that does not interfere with the isolation of leukemia cells from the same patient. this portion of the aspirate can be shipped, or can sit for at least hours, without sacrificing its mscs. rbclayer mscs are nearly identical to mscs obtained conventionally. perhaps most importantly, rbc-layer mscs retain a stem-cell like capacity, showing them to be a highly valuable cell population in aml research. future plans include investigating potential selective enrichment of stem-cell mscs in the rbc-layer, which could explain the unexpected difference in growth kinetics. aml researchers now have the opportunity to study this exciting component of the bone marrow microenvironment without sacrificing valuable leukemic cells in the process. background: neutropenia is one of the most frequent side effect of chemotherapy associated with an increase in the risk of infection, especially in the cases when the depth and duration of neutropenia are extended. some genes, as variations of darc, gsdma and cxcl are known to influence white blood cell and neutrophil counts. our previous study conducted in children with acute lymphoblastic leukemia (all), showed that polymorphisms in these genes might play a role in the onset of chemotherapy complications during consolidation and maintenance treatment. objectives: in order to support our previous finding, we have expanded the study to the induction period in a cohort of all children treated at the sainte-justine university health center between july and july . design/method: previous associated single nucleotide polymorphisms (snps) in darc, gsdma and cxcl genes were analyzed for an association with the complications occurring during induction including the duration of low neutrophil count (pnn) and low absolute phagocyte count (apc), proven infections and delay between induction and consolidation phases. results: significant effect was found for all studied polymorphims. minor alleles of darc rs , cxcl rs and gsdma rs were all associated with higher risk of complications during induction treatment, whereas that of darc rs (particularly gg genotype) had a protective effect. the gg genotype of rs was associated with a lower risk of post-induction delay (p = . or = . , %ci . - . ), less frequent febrile episodes (p = . ) and lower number of days with apc/pnn count reduction (p = . for apc< . and p = . for pnn< . ). in contrast, the minor t allele of another darc polymorphism (rs ), was associated with longer apc/pnn count reduction (p = . for apc< . and p = . for pnn < . ), as it was the tt genotype of gsdma rs (p = . for apc< . and p = . for pnn< . ). the patients with the gsdma rs had also a higher risk of documented febrile episodes (p = . or = . %ci - . ). the aa genotype of rs cxcl was associated with a higher risk of post-induction delay due to infection (p = . , or = . , % ci . - . ). conclusion: this complementary study confirmed our previous results, showing overall that variations in darc, gsdma and cxcl genes influence the onset of chemotherapy complications in pediatric all, regardless of treatment phases. these polymorphisms might be useful pharmacogenetics markers possibly guiding an adjustment of chemotherapy intensity. background: pediatric acute myeloid leukemia (aml) has a poor survival rate of about % and there is an urgent need for newer targeted therapies. car t-cell based therapies are effective against all but similar therapies against aml are still under development. recent clinical trials have highlighted the concerns about toxicity and therapy related deaths from car t-cells. antigen selection is the key factor determining the specificity, efficacy and toxicity of car t-cells. while contemporary adoptive t-cell therapies use monoclonal antibodies against tumor associated antigens we employed the naturally occurring flt ligand (fl) to target aml cells expressing flt receptors. flt receptor is expressed on multipotent and myelomonocytic progenitors as well as myeloid leukemia cells. to generate fl containing chimeric tlymphocytes designated flcar t-cells and to evaluate their efficacy against aml cells. design/method: flcar was constructed by fusing the coding sequences of the human fl, cd costimulatory domain, and cd -zeta chain (intracellular region) in series. it was then cloned into the phiv-egfp lentiviral vector for expression in cell lines and primary t cells obtained from healthy donors. the empty phiv-egfp vector was used as a negative control. flcar was expressed on both cd + and cd + t-lymphocytes, confirmed by western blot. cell cytoxicity was evaluated by co-culturing flcar t-cells and aml cells followed by flow cytometric analyses. cytokine production was assessed by analyzing expression of interleukin- using quantitative rt-pcr. results: flcar t-cells were generated from cd + jurkat and cd + tk- cell lines with up to % lentiviral transduction efficiency. the efficiency for primary t cells was lower ( - %). flcar was expressed as a ∼ kda protein in cells and was partially phosphorylated on tyrosine. the expression of flcar on lymphocytes lead to increased basal il- expression in the cells. this was further augmented (by > folds) upon co-incubating flcar t-cells with flt expressing target cells. jurkat cells, tk- cells and primary human t cells expressing flcar suppressed the growth of flt -expressing aml cell lines and primary aml cells in vitro. notably, flcar t-cells generated from healthy donors caused strong inhibition of aml cells even at a lower transduction efficiency. in vivo experiments using nsg-sgm mice xenografted with human aml cells are underway. our data demonstrate that flcar can be effectively expressed on t-lymphocytes and mediate potent cytotoxicity against flt -expressing aml cells in vitro. being a completely human derived chimeric protein, it represents a promising candidate for further therapeutic development. holly pacenta, kelly sullivan, ahwan pandey, kelly maloney, joaquin espinosa children's hospital colorado, denver, colorado, united states background: individuals with down syndrome (ds) have a -fold higher risk of developing acute lymphoblastic leukemia (all) than the typical population. there are several important differences between all in individuals with ds (ds-all) and all in individuals without ds (nds-all): first, patients with ds-all have a lower percentage of favorable cytogenetic features compared to nds-all. second, patients with ds-all are more likely to have activating mutations in jak , crlf overexpression, and ikzf deletions. despite these clear genotypic differences, this knowledge has not yet been exploited for therapeutic purposes in ds-all. when outcomes for ds-all are compared to nds-all with similar cytogenetic features, the survival rates are similar. however, individuals with ds-all have an increased risk of treatment-related mortality (trm). current therapy for ds-all is similar to that for nds-all, with the exception of small changes to decrease toxicities that are more prevalent in ds-all. it was recently identified that interferon signaling is constitutively activated in healthy individuals with t . we hypothesize that aberrant interferon signaling could play a role in the unique leukemias observed in ds patients. objectives: to identify differences in gene expression and intracellular signaling cascades that are unique to individuals with ds-all, relative to both nds-all and healthy individuals with ds that can be exploited for therapeutic use. design/method: bone marrow samples were obtained from ds-all patients and matched nds-all patients based on clinical characteristics and genetic features. rna sequencing of these samples was performed and a total of samples were used for the transcriptome analysis ( ds-all vs. nds-all). the differential expression data was generated by deseq and analyzed using ingenuity pathway analysis. the analysis revealed that the chromosome genes that have been implicated in leukemogenesis are not differentially expressed in the ds-all samples, relative to nds-all. an inflammatory signature was identified, which included interferon gamma as an upstream regulator with predicted activation in ds-all. this finding is consistent with prior observations from healthy individuals with ds. other examples of results with potentially actionable targets include the upregulation of several genes in the ras pathway and genes involved in histone methylation. the increased interferon signaling seen in healthy individuals with ds was also identified in ds-all. this may contribute to the development of mutations in inflammatory pathways such as jak and crlf in ds-all. targeting these common pathways with small molecule inhibitors may have a therapeutic benefit in ds-all. cincinnati children's hospital medical center, cincinnati, ohio, united states background: next-generation sequencing (ngs) guides precision medicine approaches in oncology using therapies targeting molecular alterations found within an individual cancer. increased availability of ngs coupled with a proliferation of targeted drugs in development heightens the need for reliable pre-clinical animal models. here we report a patientderived xenograft (pdx) system with integrated molecular profiling for pre-clinical testing of conventional cytotoxic and novel targeted agents. objectives: to utilize ngs from patients with pediatric leukemia to guide rational pre-clinical trials in pdx leukemia avatars, and to determine pdx mice tolerance of and response to cytotoxic and targeted therapies. pediatric acute lymphoblastic leukemia (all) samples were obtained in adherence to an irb-approved protocol and xenografted into nod/rag/interleukin- (il- )rg (nrg) mice. ngs was performed clinically using the foundationone® heme panel. a de novo all sample bearing mutations involving jak , crlf , ntrk , cdkn a/b, ptpn and wt was used for pre-clinical testing. thirty-seven nrg mice were transplanted with million patient cells/mouse via iv injection. standard -drug induction chemotherapy was administered consisting of vincristine, dexamethasone, pegaspargase, and daunorubicin [vxpd, n = mice], in comparison to vehicle control [n = ]. parallel pdx cohorts were treated with single agent targeted therapies based on ngs findings, including ruxolitinib [n = ], crizotinib [n = ] and loxo- [n = ]. the four-week treatment period began on day + from transplant after confirmation of engraftment. following completion of therapy, residual disease burden was analyzed by flow cytometry (hcd +, mcd -cells) in the bone marrow [bm] . to date, pdx models have been established using over thirty ngs-profiled pediatric all samples, including six samples bearing philadelphia (ph) chromosome or phlike mutations. pre-clinical testing was performed in a repre- conclusion: ngs reveals concomitant mutations in ph-like all that may represent additional targets for therapy, or predict tyrosine kinase inhibitor (tki) resistance. we show that all xenograft nrg mice can tolerate a -week multi-agent cytotoxic chemotherapy induction regimen, as well as rational targeted agents, and serve as a robust pre-clinical model for precision medicine trials. background: osteonecrosis is a well-characterized all therapeutic toxicity attributed to glucocorticoids, asparaginase, and methotrexate that disproportionately affects adolescents. in ccg- , alternate-week dexamethasone during double delayed intensification (di) reduced osteonecrosis vs continuous dexamethasone with single di in rapid early responders (rer) ≥ y. to compare efs and os between hr-all patients with vs without osteonecrosis. design/method: hr-all patients - y on aall ( - ) received cog augmented therapy with a × randomization to: ( ) induction dexamethasone ( mg/m d - ) vs prednisone ( mg/m d - ), and ( ) interim maintenance (im) high-dose methotrexate (hdm) vs escalating-dose methotrexate/pegaspargase (ema). rer received single, and slow early responders (ser) double, im/di. initially, all received monthly dexamethasone maintenance pulses, patients ≥ y received di alternate-week dexamethasone, and patients ≤ y received di continuous s of s dexamethasone. there were osteonecrosis-related amendments: after / all patients ≥ y received di alternateweek dexamethasone; after / all patients ≥ y were assigned to induction prednisone, and all patients received di alternate-week dexamethasone and maintenance prednisone pulses. results: osteonecrosis was confirmed in / patients. the y cumulative incidence (ci) was . % overall and increased with age: - y . %, - y . % (alternateweek dexamethasone . % vs continuous dexamethasone . %; p< . ), ≥ y . % (p< . ). among randomized rer patients ≥ y, ci differed by glucocorticoid (dexamethasone . % vs prednisone . %; p = . ) but not methotrexate assignment (hdm . % vs ema . %; p = . ). among randomized ser patients ≥ y, ci was . % with no difference by regimen. results were similar for patients ≥ y. in the entire study population, patients with osteonecrosis had superior y efs ( . % vs . %; p< . ) and os ( . % vs . %; p< . ) than those without osteonecrosis. y efs was significantly higher among randomized patients ≥ y with vs without osteonecrosis ( . % vs . %; p< . ); this finding was present in different age ranges (≥ y, ≥ y, ≥ y) and rer/ser subsets within each, especially in the ≥ y rer ( . % vs . %; p = . ) and ser ( . % vs . %; p< . ) cohorts. across groups, asparaginase allergy was significantly associated with reduced osteonecrosis risk (≥ y: hr . ; p = . ). patients who develop osteonecrosis have significantly increased efs and os, suggesting host differences that increase sensitivity to develop osteonecrosis and render all cells more chemo-responsive. pennsylvania state university, hershey, pennsylvania, united states background: cdc (cell division cycle protein ) belongs to rho family of small gtpases in ras-oncogene superfamily. pro-oncogenic role of overexpressed cdc in ras driven solid tumors are well known. however, role of cdc in leukemia is yet to be established. ikzf encodes ikaros protein which has important role in regulation of lymphoid development and tumor suppression in leukemia. casein kinase ii (ck ) oncogene is overexpressed in leukemia. ck impairs ikaros function which can be restored by using ck inhibitors. objectives: to investigate role of cdc in leukemia and regulation of cdc by ikaros and ck in b-cell acute lymphoblastic leukemia (b-all). shrna transfection, real time-pcr, luciferace assay, quantitative chromatin immunoprecipitation (qchip) coupled with the next-generation sequencing (chip-seq), cytotoxicity assay and western blot. results: cdc is identified as one of the ikaros target genes by analysis of genome-wide dna binding of ikaros using chip-seq and qchip in b-all primary cells. expression of cdc was also noted to be higher in all patient samples compared to normal bone marrow. functional experiments showed that ikaros overexpression via retroviral transduction results in transcriptional repression of cdc . ikaros silencing using shrna resulted in increased expression of cdc . these data suggest that ikaros negatively regulates transcription and expression of cdc . ck directly phosphorylates ikaros and impairs its function as transcription factor. we noted that molecular inhibition of ck via sirna as well as treatment with specific ck inhibitor, cx also decreases expression of cdc . treatment with cx of primary b-all with ikaros haploinsufficiency restores ikaros binding to cdc promoter and represses cdc expression. however, this effect is evident only in presence of ikaros. treatment with cx in ikaros silenced (ikaros shrna) cells showed no change in expression of cdc . these results emphasizes the importance of ikaros in regulating cdc expression. furthermore, we analyzed the changes in epigenetic signature at the cdc promoter following treatment with cx . results show that loss of histone marker of open chromatin (h k ac) and increased histone marker for repressive chromatin (h k me ), at the cdc promoter. these data suggest that ikaros transcriptionally represses cdc via chromatin remodeling. a specific cdc inhibitor, ml showed cytotoxic effects on primary b-all cells. conclusion: cdc may have important role in hematologic malignancies. expression of cdc in b cell all is regulated by ikaros and ck . these results suggest that targeting cdc could be a potential therapeutic strategy in leukemia. caitlyn duffy, laura hall, justin godown, koyama tatsuki, scott borinstein monroe carell jr. children's hospital at vanderbilt, nashville, tennessee, united states background: systemic corticosteroids are widely used as treatment of acute lymphoblastic leukemia (all) and lymphoblastic lymphoma. there are anecdotal reports of bradycardia in pediatric patients receiving corticosteroids, but a more extensive analysis of this effect is needed. objectives: the aim of this study was to describe the incidence, severity, and timing of steroid-induced bradycardia and document any adverse events associated with bradycardia. design/method: we performed a retrospective review of all newly diagnosed patients at our center ( - ) with all/lymphoblastic lymphoma who received corticosteroids (dexamethasone - mg/m /dose or prednisone mg/m /dose) during induction chemotherapy. patients were excluded if they had a pre-existing cardiac abnormality or if they received prior corticosteroids. the average hour heart rate (hr) was assessed for the period prior to initiating steroid therapy and for the hour period surrounding the nadir following steroid administration. the degree and time of steroid induced bradycardia was assessed. adverse patient events and concomitant medication use was documented to identify other contributing factors to bradycardia. a total of children ( females, males, months- years) were included in the analysis with demonstrating a decrease in mean hr following steroid administration. median hr decrease was . beats per minute (quartiles . - ) from prior to initiating steroids to surrounding nadir. sixty one percent developed bradycardia less than or equal to the st percentile for their age range. nadir occurred doses (range - ) into treatment, which corresponded to hours ( - ) after initiation of therapy. of patients who experienced bradycardia, % were associated with dexamethasone rather than prednisone. hr nadir was not associated with other vital sign abnormalities. after completion of induction chemotherapy, % of patients had documented resolution of bradycardia with hr greater than the th percentile for age. it was observed that the children who continued to have relatively low hr were often younger ( months- years old). examination of nadir hr during subsequent hospitalizations in which steroids were not being administered (excluding hr during procedural sedation) did not demonstrate a significant incidence of bradycardia. concomitant opioid, beta-blocker, or other medication exposure did not contribute to the incidence of bradycardia. corticosteroid-induced bradycardia is extremely common in children, teenagers, and young adults with all receiving induction chemotherapy. bradycardia was not associated with clinical adverse events and resolved after completion of corticosteroid treatment. therefore, further cardiac assessment may not be warranted in the presence of bradycardia suspected to be secondary to steroid administration. baylor college of medicine, houston, texas, united states background: survival in newly diagnosed pediatric acute myeloid leukemia (aml) is approximately %; however survival falls dramatically if a patient relapses. currently, approximately one-third of patients with pediatric aml relapse on standard chemotherapy regimens. aml cells are exposed to proteotoxic stress at baseline due to their rapid and inefficient metabolism; proteotoxic stress increases after chemotherapy due to accumulation of reactive oxygen species resulting in misfolded proteins. this leads to activation of cell stress pathways, such as the unfolded protein response (upr) in the endoplasmic reticulum. because an activated upr can make cells more sensitive to proteotoxic stress, we hypothesize that upr activation correlates with response to chemotherapy. objectives: determine the status of upr in pediatric aml and its correlation with chemosensitivity; design/method: peripheral blood samples from pediatric patients with aml were collected at the start of induction chemotherapy, - hours (h) and h post initiation of systemic chemotherapy. tumor cells were sorted from peripheral blood mononuclear cells. expression of upr proteins was determined by chemiluminescence using an automated capillary electrophoresis system. clinical correlations were performed using an annotated database. we measured five upr proteins: grp (glucose regulated protein kda), phospho-eif , inositol-requiring enzyme (ire ) and activating transcription factor (atf ). patients with aml had - times higher expression of upr proteins (except atf ) at baseline than normal controls. grp -the key upr driver-had the highest level of protein expression in myeloid blasts. there was a wide variability in the level of baseline upr expression. eight out of samples expressed > fold increase in grp above those with the lowest grp levels. similarly, and patients respectively, had a > fold increase in peif and ire , compared to patients with low basal expression of these upr proteins. in our limited sample set, there was a trend towards lower overall survival (os) and event-free survival in patients with low baseline grp and ire . conclusion: upr has a variable expression at baseline in pediatric aml, with a trend towards lower os in patients with a low basal grp and low ire expression, suggesting less chemosensitivity in this subgroup. conversely, it is possible that blasts with an upregulated upr prior to chemotherapy manage proteotoxic stress less effectively, having faster apoptosis and hence a better response to chemotherapy in patients with a high basal upr. we are currently expanding our findings in a larger cohort of patients enrolled in the children's oncology group aaml protocol. background: children with newly diagnosed acute lymphoblastic leukemia (all) undergo chest x-ray (cxr) evaluation during initial diagnostic workup to ensure safe airway management. however, to our knowledge, no systematic assessment of cxr findings has been reported. objectives: to evaluate cxr findings at diagnosis of all and their associations with clinical characteristics. we reviewed the cxr findings at diagnosis of all in patients treated on the total xv and xvi protocols at st. jude children's research hospital. findings were evaluated for associations with clinical characteristics at presentation, and the clinical management of mediastinal masses was reviewed. mediastinal masses were seen in ( . %) of patients evaluated and were more common in older patients (mean age, . years) than in younger patients (mean age, . years) (p = . ), in males than in females (p = . ), and in patients with t-all than in those with b-all (p< . ). also associated with mediastinal masses were a higher white blood cell count (wbc) at diagnosis (mean, . × /l) (vs. a lower wbc; mean, . × /l) (p< . ), cns involvement (vs. no involvement) (p = . ), and standard/high-risk disease (vs. low-risk disease) (p< . ). other cxr findings included pulmonary opacity ( patients [ . %]), bronchial/perihilar thickening ( patients [ . %]), cardiomegaly ( patients [ . %]), and osteopenia/fracture/periosteal lesions ( patients [ . %]). pulmonary opacity was more common in younger patients (mean age, . years) than in older patients (mean age, . years) (p = . ) and in those with t-all (vs. b-all) (p = . ). bronchial/perihilar thickening, cardiomegaly, and osteopenia/fracture/periosteal lesions were also more common in younger patients than in older ones (p< . , p = . , and p< . , respectively) and in those with low-risk disease (versus standard/high-risk disease) (p< . , p = . , and p = . , respectively). of the patients with a mediastinal mass on cxr, underwent a confirmatory chest ct scan, and ( . %) were confirmed to have a mediastinal mass. notably, patients ( . %) had airway compression, and compression of venous structures was identified in of patients ( . %) who received iv contrast. the clinical course was evaluated for patients with mediastinal masses detected by cxr. fifty patients ( . %) required icu admission (mean stay, . days). general anesthesia was used for only patients ( . %), and patients ( . %) had a less invasive peripherally inserted central catheter. no deaths occurred in the acute phase. conclusion: cxr at the time of all diagnosis can detect various intrathoracic lesions and is helpful in planning initial diagnostic workup and management. background: mertk is a receptor tyrosine kinase that is aberrantly expressed in % of pediatric primary aml samples. mertk inhibition with the small molecule tyrosine kinase inhibitor (tki) mrx- decreases tumor burden and prolongs survival in aml xenografts. while treatment with mrx- reduces leukemia in the peripheral blood, it is less effective in the bone marrow, suggesting a role for the marrow microenvironment in therapeutic resistance. the jak/stat pathway has been implicated as a mediator of bone marrow derived resistance to tkis and inhibitors of this pathway are in clinical development for the treatment of aml. to determine the role of the bone marrow stromal niche in mediating resistance to mertk inhibition and to evaluate the efficacy of combined mertk and jak/stat inhibition. design/method: aml cell lines were cultured with or without the hs stromal cell line or hs conditioned medium, then treated with mrx- +/-the jak/stat inhibitor ruxolitinib, or control. induction of apoptosis and cell cycle arrest in aml cells was measured by flow cytometry. expression of h ax and total and phosphorylated stat were determined by immunoblot. results: co-culture with stromal cells significantly reduced aml cell death and g /m phase arrest in response to treatment with nm mrx- compared to no co-culture (cell death: . % versus . %, p< . ; g /m arrest: . % versus . %, p< . ). g /m arrest was accompanied by an increase in h ax expression which was similarly abrogated in co-culture. conditioned medium did not provide protection from mrx- induced apoptosis, g /m arrest, or h ax induction. mrx- inhibited stat phosphorylation but direct co-culture and conditioned medium potently increased basal stat phosphorylation which was not inhibited by mrx- . to determine whether the observed induction of stat phosphorylation was functionally relevant, cocultures were treated with both mrx- and ruxolitinib. while ruxolitinib potently inhibited the phosphorylation of stat in the presence of co-culture, combination treatment did not overcome stromal mediated protection from mrx- induced apoptosis. similarly, the addition of exogenous gm-csf induced stat phosphorylation but did not yield protection from mrx- functional effects in the absence of co-culture. together these data support a model whereby direct cell-cell contact with stromal cells in the bone marrow niche protects leukemia cells from mrx- induced apoptosis, cell cycle alterations, and dna damage. while co-culture potently induces phosphorylation of stat in leukemia cells, this is neither necessary nor sufficient for stromal-cell mediated protection from mertk inhibition and combined treatment with jak/stat inhibitors is unlikely to be therapeutically efficacious. background: mercaptopurine ( -mp) is an immunosuppressive thiopurine drug that is a key component of acute lymphoblastic leukemia (all) treatment. -mp is metabolized into -thioguanine ( -tgn), which is responsible for anti-leukemic effects, as well as -methylmercaptopurine nucleotides ( -mmpn/ -mmp), which are associated with hepatotoxicity. some patients preferentially metabolize -mp to -mmpn/ -mmp, increasing their risk for hepatotoxicity and potentially reducing anti-leukemic effects. hepatotoxicity can cause interruptions or delays in therapy that may jeopardize cure rates. allopurinol has been increasingly used in patients with inflammatory bowel disease (ibd) to shunt -mp metabolism toward -tgn and away from -mmpn to minimize hepatotoxicity and preserve therapeutic effects. objectives: this retrospective chart review expands upon our previously published case series of three patients with all in whom allopurinol was successfully used to redirect -mp metabolism. twelve additional patients have subsequently received allopurinol and -mp combination therapy at texas children's hospital. data from this larger patient sample, with longer follow up, is being analyzed to increase knowledge of the effectiveness and longitudinal effects of adding allopurinol to -mp to reduce risk of hepatotoxicity. design/method: data were abstracted from the electronic medical records of patients with all treated at texas children's hospital from to present, who had been found to have evidence of altered -mp metabolism and in whom allopurinol was added to -mp therapy due to concern for risk or recurrence of hepatotoxicity. metabolite levels, -mp dose, and alanine transaminase (alt) prior to initiation of allopurinol and approximately weeks later were compared. wilcoxon signed-rank test was applied for statistical analysis. : after the addition of allopurinol, patients experienced a significant decrease in mean levels of -mmpn (p = . ), correlating with a significant decrease in mean alt (p = . ). with the initiation of allopurinol, the mean -mp dose was decreased from to mg/m /day over an -week period. mean -tgn levels increased (p = . ). in follow up beyond weeks, no patients had further holds in -mp due to hepatotoxicity. addition of allopurinol appears to shift metabolism from -mmpn toward -tgn, with increases in mean -tgn levels despite a decrease in mean -mp dose. this may limit negative side effects, thus resulting in fewer gaps in therapy and possible improved outcomes. further analysis of -mp dose titration and effects on anc over time as well as effects on overall survival is ongoing. prospective background: alterations in epigenetic patterning are a fundamental feature in acute myeloid leukemia (aml). treatment with dna methyltransferase inhibitors (dnmti) yields responses in aml, but the molecular mechanisms underlying this effect are poorly understood. in prior work, we demonstrated induction of genes involved in the pirna rna (piwi) silencing pathway as a common gene feature of aml cell lines treated with decitabine. the piwi pathway is an rna silencing system, distinct from classical small rna transcriptional silencing, responsible for transposon-silencing in gametogenesis; emerging data suggest a role for this system in somatic cells. based on these data, we postulate that piwi induction plays a crucial role in aml recovery following demethylation and that disruption of this pathway would modulate response and/or recovery from decitabine treatment. to assess the effect contribution of the pirna pathway response following dnmti treatment in aml. design/method: to choose target genes in the pirna pathway for disruption, molm cells were first treated with escalating doses of decitabine. using quantitative rt-pcr, the dose-dependent expression of several pirna-associated genes were analyzed. two genes, mael and piwil , were selected for disruption experiments based on preliminary data suggesting decitabine dose-dependent responses. molm cells were transduced with shrna targeting these genes using a lentivirus delivery system with selection in puromycin. knockdown efficiency was assessed by rt-qpcr. to determine how gene disruption affected cell growth, knockdown cells were treated with decitabine nm. proliferation was assessed by celltiter glo assay following decitabine treatment. clonogenic potential was assessed by colony forming assays of transduced cells after treatment with decitabine at nm and nm. results: following decitabine exposure in molm , there was a markedly increased expression of mael and piwil compared to untreated cells ( : and : , respectively) . thus, these were the candidate genes chosen for disruption. of mael shrna constructs, two resulted in a % relative expression of mael compared to controls. of the piwil shrna constructs, the best knockdown showed % relative expression. there were no significant differences in proliferation or clonogenicity of stably selected mael or piwil knock-down molm cells following decitabine treatment. using gene knockdown procedures, mael and piwl do not appear to have a marked effect on growth and response to decitabine treatment in molm . however, these results may be limited by inefficient knockdown using shrna targeting methods. further work using a cas /crispr based inactivation of these genes is ongoing. children cancer hospital cairo, egypt background: hypodiploidy < chromosomes is very uncommon and have particularly poor outcomes in childhood acute lymphoblastic leukemia (all). it is subdivided into: near-haploid ( - chromosomes), lowhypodiploid ( - chromosomes) and high-hypodiploid ( - chromosomes). to determine if minimal residual disease (mrd) can identify a group of patients with better prognosis in the hypodploid population who can be treated with intensive chemotherapy alone. design/method: a retrospective study that included all patients under age of diagnosed as hypodiploid b-precursor all during the period between january -december and treated at children's cancer hospital egypt on sjcrh total study-xv for ir/hr all. sixteen patients had < chromosomes ( nearhaploid and low-hypodiploid), constituting % of all pediatric patients with b-precursor all during the study period. patients with near-haploid all had a median age of years (range - ), initial leukocyte count (wbc) median of . × /l (range . - . ), ( . %) were males and / ( . %) had hr-nci criteria. four patients ( . %) are alive in complete remission(cr) (range - months, median ), one died in induction and ( . %) had hematological relapse (range . - months, median ). patients with low-hypodiploid all had significantly older age (median years, range - ), median wbc . × /l (range . - . ), / ( . %) were males. one patient ( . %) is alive in cr, one died in induction, one failed to achieve cr post-induction and patients( %) had hematological relapse (range . - . months, median . ). mrd< . % by flow-cytometry on day- and end of induction was achieved in / ( . %) and / patients( %) with near-haploid, compared with / ( . %) and / patients( %) with low-hypodiploid; respectively (p = . , p = . ;respectively). allogeneic transplantation was performed during initial remission only in mrd negative patients (one relapsed and one is in cr) and in the patient with induction failure (relapsed post-transplant). five of the total six patients who had negative mrd on day- and end of induction are alive in cr ( / with chemotherapy alone). all patients with negative mrd at end of induction but with mrd levels≥ . % on day- (range . - . %) relapsed as well as all patients with detectable mrd at the end of induction. the difference in relapse was statistically significant in relation to negative-mrd on day- (p = . ), but not at end of induction(p = . ). conclusion: children with hypodiploid all and negative mrd on day- of induction are highly curable with intensive chemotherapy alone, while patients with negative mrd at the end of induction and detectable mrd on day- had dismal outcome. background: overall survival in pediatric acute myeloid leukemia (aml) has plateaued between - %, with death during induction chemotherapy seen in - % of patients. respiratory complications contribute to morbidity and mortality in pediatric aml induction, however the incidence, patterns, and predictors of respiratory adverse events (aes) during this period are unknown. to estimate the incidence of respiratory aes during induction therapy for de novo pediatric aml, to characterize and grade these respiratory aes, and to identify predictors of respiratory ae development. we conducted a retrospective longitudinal study from presentation to day in institutional de novo pediatric aml patients (≤ years) between march and december . outcomes included any nci ctcae grade - respiratory ae or death from another cause. demographic, disease, and treatment-related data were abstracted. the most specific, best-fitting ctcae category and grade for each ae was determined. descriptive statistics, survival analysis, multivariable logistic regression analysis, and time-toevent distributions were performed (sas v . , cary, nc) . among eligible patients, . % (n = ) experienced discrete respiratory aes. incidence of grade - aes was . % (n = ). a bimodal time-to-event distribution demonstrated peaks at treatment days and . induction death occurred in . % (n = ) including deaths from respiratory failure associated with disseminated fungal disease. in univariate analysis, those experiencing aes differed significantly in regards to older age at diagnosis (p< . ), higher initial wbc (p = . ), higher initial peripheral blast percentage (p = . ), coagulopathy at diagnosis (pt (p = . ), d-dimer (p = . )), fluid overload status (p< . ), occurrence of infection (p = . ), and occurrence of tumor lysis syndrome (tls) (p = . ). patients with hyperleukocytosis (p = . ), fluid overload (p< . ), and fab m morphology (p = . ) each had a significantly decreased probability of completing the follow up period without experiencing a respiratory ae. on multivariable analysis, fluid overload (aor . [ % ci: . - . ) and older age (aor . [ % ci: . - . ) were significantly associated with ae occurrence when gender, hyperleukocytosis, tls, and infection status were held constant. we describe a high incidence of respiratory aes during pediatric aml induction. fluid overload and older age at diagnosis are independently associated with ae development when controlling for other proposed risk factors. interventions focused on conservative fluid management and offset of fluid overload should be explored in newly diagnosed pediatric aml in an effort to reduce respiratory complications during induction. overall, all survival rates are outstanding and have continued to improve with risk-adapted therapy. the most striking improvement occurred in t-all where -year os rates now exceed % and parallel b-all. survival improvements, however, have not been observed uniformly across all subgroups. while the gap in outcome differences narrowed among blacks, outcomes for hispanics have remained static. further, no improvements in survival were observed in infants or ayas and new treatment approaches have been implemented for these populations. background: acute myeloid leukemia (aml) accounts for approximately % of new childhood leukemia cases. chest x-ray (cxr) is performed in all newly diagnosed aml cases to evaluate the safety of airway management for anesthesia during diagnostic procedures; however, cxr results in pediatric patients with aml have not been described. objectives: the primary objective was to evaluate cxr findings at diagnosis in patients with aml. the secondary objectives included assessing associations between cxr findings and clinical characteristics, with the overall goal of aiding in the evaluation of the use of cxrs as an initial diagnostic study in pediatric patients with aml. design/method: cxr findings and clinical characteristics were evaluated in patients with newly diagnosed aml who were enrolled in one of three protocols at st. jude children's research hospital (aml , aml , and aml ). the findings were categorized based on radiologic reports. further, the associations of these findings and clinical characteristics were evaluated. we evaluated cxr findings in a total of patients: from aml ; from aml ; and from aml . common cxr findings were pulmonary opacity (n = , . %), bronchial/perihilar thickening (n = , . %), splenomegaly (n = , . %), mediastinal mass and lymph nodes (n = , . %), pleural effusion/thickening (n = , . %), demineralization/fracture/periosteal lesions (n = , . %), scoliosis (n = , . %), and granulomatous disease (n = , . %). three cxr findings were associated with younger age at diagnosis: pulmonary opacity (median age, . years in patients with positive findings vs. . years in those with negative findings, p< . ), bronchial/perihilar thickening (median age, . years vs. . years, p< . ), and demineralization/fracture/periosteal lesions (median age; . years vs. . years, p = . ). two cxr findings were associated with older age at diagnosis: scoliosis (median age, . years vs. . years, p< . ) and granulomatous disease (median age, . years vs. . years, p = . ). higher white blood cell counts (wbcs) at diagnosis were associated with cxrs showing pulmonary opacity (median wbc; . × ^ /l vs. . × ^ /l, p = . ) or splenomegaly (median wbc; . × ^ /l vs. . × ^ /l, p = . ). french-american-british (fab) m /m subtypes were more frequently associated with pulmonary opacity compared with others (p< . ). we did not find significant differences between female and male patients. conclusion: cxr in patients with newly diagnosed aml showed a variety of thoracic, abdominal, and bony lesions that are important for the initial evaluation and management. pulmonary opacity was the most common finding and was frequently seen in patients who were younger or had higher wbcs at diagnosis or fab m /m . background: children diagnosed with acute lymphoblastic leukemia (all) require a central venous catheter (cvc) to administer chemotherapy safely. both external and internal cvcs carry risks of complications including thrombosis, infection, and possible replacement. internal catheters, such as a port, are generally used for the majority of patients for the duration of treatment since therapy lasts for several years. many institutions place a port at the time of diagnosis. other institutions prefer to start induction therapy via placement of a peripherally inserted central catheter (picc) and defer port placement until the completion of induction therapy due to concerns of increased risk of infectious complications with port placement. objectives: to compare rates of common cvc associated complications by type of cvc placed at start of induction therapy in children treated for newly diagnosed all at the jimmy everest center (jec) at the university of oklahoma health sciences center. design/method: a retrospective chart review analyzed data from newly diagnosed all patients treated at the jec between - . data was collected on complications including thrombosis, bacteremia, insertion site infection, cvc malfunction and need for removal. data collection began at the start of induction and was completed at the end of induction therapy. statistical analysis used a univariate and multivariate logistic regression model to compare complication rates between those who had a port versus those who had a picc placed at start of induction. results: data was collected on patients. fifty-six patients had a port placed at start of therapy while had a picc placed. fourteen percent of patients had a cvc associated complication. univariate analysis showed no statistically significant difference in rates of cvc associated complications between the groups (port %, picc . % p = . ). the rates of hospitalization for cvc associated complications were similar between both groups (port %, picc % p = . ). rates of cvc removal were also similar between both groups (port %, picc % p = . ). multivariate model that included baseline patient characteristics including type of all, patient body surface area, gender, ethnicity and age continued to demonstrate no significant difference in cvc associated complications between both groups. conclusion: this single institution study showed that there was no significant difference in cvc associated complications between port and picc line placement at the start of childhood all induction therapy. port placement can be considered as a safe option at the start of induction therapy. complete remission [cr] or cr with incomplete blood count recovery [cri]) within treatment cycles - . interim data are reported (nct ). results: seventeen patients were enrolled and received ≥ dose of lenalidomide; median age was years (range - ); patients were female. patients received median prior regimens (range - ). nine patients had previously undergone bone marrow transplantation (bmt). four patients had relapsed aml and were refractory to immediate prior treatment. median duration of study treatment was weeks (range - ); patients completed a median of treatment cycle (range - ). all patients were evaluable for primary outcome; achieved morphologic cri after cycles (no patients achieved cr). the responder was a -year-old male with history of r/r aml after first-and second-line treatment, bmt, and salvage chemotherapy. at baseline, he had a complex cytogenetic karyotype (monoallelic − q . , − q, − q . , − p ) with no identifiable molecular mutation; he was also positive for del( q) (− q , − q ). his post-treatment karyotype showed no abnormalities. sixteen patients experienced treatment failure; due to resistant disease, of indeterminate cause, and had treatment failure before a post-baseline assessment was performed. all patients experienced ≥ grade - treatment-emergent adverse event (teae). the most commonly reported were thrombocytopenia (n = ), anemia (n = ), febrile neutropenia (n = ), and hypokalemia (n = ). fifteen patients experienced ≥ teae related to lenalidomide. all patients discontinued treatment; remain in follow-up. the study is now closed to enrollment. ten patients died on study: during treatment, during follow-up. all deaths were attributed to aml or complications due to aml. conclusion: third-line lenalidomide monotherapy was associated with clinical response in of pediatric patients with r/r aml; however, treatment exposure was limited. safety data are consistent with the known profile of lenalidomide. lenalidomide was not an efficacious treatment for r/r pediatric aml. funding: celgene corporation, summit, nj, usa. cook children's medical center, fort worth, texas, united states background: it is well documented that pediatric patients with acute lymphoblastic leukemia (all) often experience significant weight gain during induction therapy and later struggle with obesity. however, some patients experience unintended weight loss during induction therapy; since this issue is not well reported, it often goes undertreated. although malnutrition is reported to be associated with decreased survival, increased risk of infection, and loss of lean body mass, there remains a scarcity of in-depth analysis of prevalence and risk factors that contribute to this problem. our study attempts to address this critical yet unmet need. objectives: our aim was to identify the clinical risk factors and outcomes associated with weight loss during induction therapy for pediatric all. design/method: this was a retrospective chart review of patients between and years of age diagnosed with all at cook children's medical center from / / to / / . for each patient, we collected height, weight, age, body mass index (bmi) z-scores at diagnosis and end of induction therapy, risk stratification, and whether consolidation was delayed. patients with a bmi > th percentile at diagnosis were categorized as being overweight or obese. using logistic regression analyses, we examined which variables predicted whether the patient had an increase or decrease in bmi z-score throughout induction. a critical alpha level of . indicated statistical significance. results: ninety-six patients met our inclusion criteria. of these, % experienced a decrease in bmi during induction therapy. compared to patients whose bmi increased during induction, patients with a decrease in bmi were more likely to be overweight or obese at diagnosis ( % vs. %; p< . ), to be ≥ years of age ( % vs. %; p< . ), to have a high-or very-high-risk stratification ( % vs. %; p< . ), and to experience a delay in the start of consolidation therapy ( % vs. %; p< . ). conclusion: this research highlights a risk not previously identified in the literature that may impact outcomes. patients treated on high-or very-high-risk protocols, who are overweight or obese at diagnosis, and who are ≥ years of age at diagnosis should be monitored closely for weight loss during induction therapy. patients who experience weight loss should receive prompt intervention. it is our hope that this information can be used for future prospective studies and to help develop evidence-based guidelines. background: p abnormalities have been observed in some patients with hematologic malignancies. loss of p function as a tumor suppressor gene in the chromosome plays an important role for development of leukemia. these patients usually have poor outcome due to the chemotherapy and are associated with poor prognosis. objectives: this study aimed to identify frequency of p abnormalities between iranian children and adult patients with aml (acute myeloid leukemia) malignancy. design/method: the p abnormalities were analyzed via bone marrow karyotyping and fish method in acute myeloid leukemia patients. in this study, p abnormalities were observed in ( %) patients out of diagnosed cases. a significant strong correlation between p abnormalities and other high risk factors (poor risk cytogenetic) were observed. from patients with aml malignancy ( p abnormalities), ( %) patients have complex karyotype, ( %) patients monosomal karyotype and ( %) patients have monosomal karyotype accompanied with a complex karyotype. overall, p abnormalities are independent risk factor in acute myeloid leukemia and evaluation of these abnormalities by fish or other complementary techniques prior to treatment, might help for better risk stratification of high risk aml patients. background: hepatotoxicity in treatment of acute lymphoblastic leukemia (all) is well studied and transiently affects most patients receiving antimetabolite therapy. rarely, patients develop liver injury severe or prolonged enough to undergo a liver biopsy. little is known about how these patients differ from patients that develop transient hepatotoxicity. we sought to describe disease and treatment characteristics for all patients that developed hepatotoxicity severe enough to undergo liver biopsy. we also looked for pre-dictive factors for liver biopsy, including signs of early hepatic injury from the initial treatment protocol. design/method: pathology reports of all patients from the liver biopsy database at children's healthcare of atlanta were collected. controls were matched : for age, all subtype, and treatment protocol. demographics, treatment protocols, and overall outcomes were collected through the electronic health record. hepatic lab results for transaminases, coagulation, and albumin were collected for induction, consolidation, interim maintenance, delayed intensification, and maintenance. results: sixteen patients diagnosed between - (median age at diagnosis years, range - ; % male; % pre-b all) were included in the case series. the median time from diagnosis to liver biopsy was . years (range - ). eight patients ( %) were in maintenance at the time of biopsy; none had active disease. eight ( %) were postbone marrow transplant. biopsy results included: steatosis ( ), acute inflammatory/infectious ( ), liver infiltration ( ), fibrosis ( ) and graft-vs-host disease (gvhd) ( ). six patients were deceased; -year all-cause mortality from diagnosis was %. thiopurine methyltransferase (tpmt) status was known in % cases and % controls. all cases had intermediate or wildtype status, which did not differ from controls (p > . ). patients requiring liver biopsy did not have evidence of acute hepatotoxicity (ast/alt > × normal values) during their initial treatment protocol. hepatotoxicity requiring liver biopsy is a rare outcome of all treatment. these patients had elevated rates of relapse, bmt, and -year all-cause mortality, suggestive of a more severe disease process. however, it is difficult to sort out the temporality of relapse, bmt, and hepatoxicity requiring biopsy in this limited sample. additionally, patients with bmt preceding liver biopsy have other confounding factors that makes them difficult to include in the analysis. finally, our limited descriptive data show no notable correlation between early hepatotoxicity and later indication for liver biopsy. future cohort or case-control studies with larger sample sizes are required to further explore early predictive factors for severe hepatotoxicity requiring liver biopsy. nathan gossai, joanna perkins, michael richards, yoav messinger, bruce bostrom background: the majority of chemotherapeutic agents used to treat hodgkin lymphoma are teratogenic. pregnancy screening prior to the start of chemotherapy is supported by clinical guidelines and baseline testing is a standard component in therapeutic trials. there is limited data available on the incidence of pregnancy screening prior to the start of hodgkin therapy but previous studies suggest that pregnancy screening, especially at pediatric institutions, is not consistently completed. objectives: the objective of this study is to evaluate the incidence of pregnancy screening and contraceptive counseling prior to the start of therapy in females diagnosed with hodgkin lymphoma. design/method: a retrospective chart review was performed for all female patients newly diagnosed with hodgkin lymphoma from to at the hospital for sick children in toronto, ontario. all patients who were intended to receive multi-agent chemotherapy were included, regardless of age. data collected included demographic and disease information, chemotherapy regimen and enrollment on clinical trial. all pregnancy testing within two weeks prior to the start of therapy was captured, as well as type of pregnancy test performed, documentation of menstrual status, contraceptive counseling and contraceptive provision. univariate and multivariate analyses were used to describe factors influencing the incidence of pregnancy testing. results: a total of female patients with newly diagnosed hodgkin lymphoma between the ages of and years were identified. sixty patients ( %) had pregnancy testing done prior to the start of therapy. testing modalities included serum and urine screens as well as quantitative beta-hcg measures. older age (p = . ), documentation of menstrual status at diagnosis (p = . ) and diagnosis between and (p = . ) were associated with higher incidence of screening. enrollment on a therapeutic trial was not associated with a higher incidence of screening (p = . ). contraceptive counseling was documented for patients ( %) and patients ( %) were prescribed contraceptive medications during therapy. pre-chemotherapy pregnancy testing was completed on % of females with newly diagnosed hodgkin lymphoma. improvement is required and interventions, including clarification of institutional standards, modification of chemotherapy order sets and staff education, are planned. (rao et al., cancer, ) . university of louisville, louisville, kentucky, united states background: granulocytic sarcomas (also known as chloromas or leukemia cutis) were first described by a. burns in . they are solid tumors comprised of immature granulocytic cells and represent extramedullary manifestations of underlying leukemia. chloromas are most commonly associated with acute myeloid leukemia. they may arise in other myeloproliferative disorders but are rarely seen in b or t cell acute lymphoblastic leukemia (all). objectives: although patients with all rarely have chloromas, it should remain on the differential for patients with unusual swelling or masses. design/method: we present a case series of two patients from our institution diagnosed with b cell all who had a chloroma as the presenting symptom. the first patient is a yo who presented to his primary provider with nasal congestion and a one-week history of bilateral eye swelling and was referred to an allergist when the symptoms did not resolve with anti-histamines. his review of systems was otherwise negative. he was referred urgently to ent two months later for a × cm mass palpated along the medial border of the left eye. an mri showed a left facial mass surrounding the zygoma and extending into the anterior inferior left orbit. biopsy revealed b cell acute lymphoblastic lymphoma, and bone marrow aspirate and biopsy confirmed the diagnosis as b cell all. the second patient is a yo who presented to his primary doctor for rapid growth of a scalp nodule that had been present for about months. he was referred to dermatology and treated for a supposed kerion from tinea capitis. the lesion continued to grow and became more irritated with this treatment. punch biopsy revealed a complicated phenotype of lymphoblastic lymphoma. however, after a lymph node biopsy and bone marrow aspirate and biopsy, the diagnosis was confirmed as b all. his only other positive point on review of systems was a questionably pathologic -pound weight loss and an area of matted cervical lymph nodes. for both of our patients, the chloromas completely disappeared during induction therapy. it is worth noting that both of these patients presented with the chloroma as the only symptom of the underlying leukemia. this led to initial misdiagnosis and delay in identifying their leukemia. therefore, while it is very rare for a patient with b all to present with a chloroma, our experience shows that all should be on the differential for patients presenting with unusual swelling or masses. background: hodgkin lymphoma (hl) is a lymphoproliferative neoplasm that commonly presents with history of adenopathy and a predictable pattern of disease involvement with or without systemic symptoms of fever and/or weight loss. in the hands of an experienced oncologist the diagnosis of hl is usually not a challenge. occasionally a diagnostic challenge is presented by a patient who has an atypical presentation which is suggestive of an alternative diagnosis. we describe a case series of patients diagnosed with hl whose initial clinical presentations lead to a diagnosis different form hl. honduras, nicaragua and the united states. results: six pediatric oncology centers from the american continent conducted a retrospective review of patients diagnosed with hl since . patients that had an initial presentation not suggestive of hl or who were initially diagnosed with a disease other that hl were included for a total of patients. argentina n = , guatemala n = , honduras n = , nicaragua n = , united states n = . five patients were female and male. patient's ages ranged from to years. most patients (n = ) were older than years. three patients ( %) presented with non-immune cytopenias without overt lymphadenopathy, of those one had active hemophagocytic syndrome. five patients ( %) were suspected to have localized solid tumors: ewing sarcoma n = , rhabdomyosarcoma n = , hepatocellular carcinoma n = , and soft tissue tumor of the cheek n = . two ( %) metastatic solid malignancy as they presented with disseminated pulmonary nodules. five ( %) with autoimmune disorders: hashimoto thyroiditis n = , autoimmune hemolytic anemia n = , nephrotic syndrome n = . ten ( %) with chronic infectious processes: brucella n = , tonsillar abscess n = , splenic abscess n = , and tuberculosis (tb) n = . patients with suspected tuberculosis were diagnosed outside of the united states. six of patients were ultimately diagnosed as having both tb and hl. seventeen patients had ann-arbor stage iii or iv, seven patient had stage ii with either b symptoms or bulky disease. patients were treated with various chemotherapy regimens according to the treating center: abvd, abve-pc oepa-copdac, avpc, beacopp. two patients had recurrent disease, one died of disease progression and one died from causes not related to hl conclusion: a small proportion of hl patients have atypical or unusual presentations. hl should be included in the differential diagnosis of solid tumors, autoimmune disorders, infections or cytopenias. the most common atypical presentation is an infectious process. background: acute lymphoblastic leukemia (all) represents the largest group of pediatric malignancies. the high cure rate of childhood all represents one of the most remarkable success stories in the war on cancer. in a lower middle income country (lmic) like the philippines, we reviewed the five year survival in a tertiary referral center. objectives: this retrospective cohort study aims to determine the survival of children - years old with all treated at a tertiary referral center for childhood cancer in the philippines from january to december . design/method: this is a retrospective cohort study that reviewed medical charts of newly diagnosed all ages to years old from january to december . a total of subjects were included in the study. the year overall survival (os) and event free survival (efs) were . % and . %, respectively. the year os for standard risk all was . % and for high risk patients was %. the year os for the patients on remission was . % and for those who relapsed was . %. univariate and multivariate by cox proportional hazards regression revealed wbc count at diagnosis, risk classification, immunophenotyping, and development of relapse showed significant prognostic impact for mortality. age and gender were reported with no prognostic significance. the -year os and efs were lower compared to developed countries but are comparable with other lmics. the prognostic factors for relapse and mortality were compatible with the literature. overall, the adopted treatment protocols for childhood all in this institution showed acceptable results. relapse has a significant prognostic impact for mortality. development of accessibility to care, increase awareness, early detection and resources at hand should be achieved. improvement in the follow up protocol to prevent delays in the treatment, patient education to prevent non-compliance and psychosocial support, to developed better supportive care, and expand facilities should be given emphasis to further improve survival and prevent relapse. objectives: here, we seek to further characterize this entity by describing the pathologic and clinical features of pediatric cases of burkitt-like lymphoma with q aberration. we collected pathologic and clinical data from the medical record on all pediatric high grade b-cell lymphoma (hgbcl) cases diagnosed at our institution over a -year period ( - ) . for those cases classified as neither burkitt lymphoma nor diffuse large b-cell lymphoma (dlbcl), fish for myc, bcl- and bcl- , as well as array comparative genomic hybridization (acgh), were performed. we identified cases of hgbcl, including cases of burkitt lymphoma presenting as purely leukemic phase. of the hgbcl cases, had burkitt lymphoma as defined by myc rearrangements, and had dlbcl. collectively, the majority of these patients had primary disease outside of the head/neck, and most patients presented with advanced stage (iii-iv) disease. of the remaining cases, q aberration was identified in cases using acgh. all cases histologically and immunophenotypically resembled burkitt lymphoma but lacked myc rearrangement, instead showing proximal gains in q -q and telomeric losses in q . qter. all cases involved primary disease in the cervical lymph node and/or tonsil. three of these cases were localized (stage ii), and the fourth case involved a few metabolically active but non-enlarged lymph nodes in the chest and abdomen (stage iii). all patients achieved complete remission with standard therapy for mature b-cell lymphoma, and were alive with no clinical evidence of disease at a median follow-up of months. although the number is small, our results suggest that the majority of non-burkitt, non-dlbcl cases of pediatric hgbcl carry q aberrations. in addition, patients with q aberrations appear to be more likely to present with lower stage disease, thus requiring less intensive therapy, and also tend to have primary disease in the head/neck. these findings further support the classification of burkitt-like lymphoma with q aberration as a distinct pathologic and clinical entity, and we propose that all pediatric non-burkitt, non-dlbcl cases of hgbcl regularly undergo further workup for possible q aberrations. marie claire milady auguste, joseph bernard st damien hospital, port-au-prince, port-au-prince, haiti background: hodgkin lymphoma (hl) and non-hodgkin lymphoma (nhl) account for % of cancers in the united states pediatric population ( , ). in central america and the caribbean, they are in second position among all types of pediatric cancers ( ). a previous study on pediatric cancers in haiti showed that the lymphomas were in fifth place after the leukemias, wilms tumor, retinoblastoma and the sarcomas ( ). the main objective of this study is to present the epidemiological profile of lymphomas managed at a haitian pediatric hospital. design/method: this is a retrospective study conducted on the cases of lymphoma diagnosed and managed at st damien hospital from january to december . key variables such as age, gender, stage at diagnosis, histopathological types and outcome were collected to present the characteristics of this retrospective cohort. of the cases of cancer diagnosed during the study period, ( . %) had the diagnosis of lymphoma. the sex ratio was . ( males for females) and the average age was . years [ - years]. there were cases of hl ( . %) and cases of nhl ( . %). . % of the patients were diagnosed at stages iii and iv. among the hl cases, ( . %) were nodular sclerosis lymphoma, ( . %) with mixed cellularity and ( . %) with lymphocytic predominance. for the nhl cases, ( . %) were burkitt's lymphoma and ( . %) lymphoblastic t-cell lymphoma. among the patients for who immunohistochemistry was found, the cases of hl were cd -positive and out of cases of nhl were cd -negative. only patient was hiv-positive, and patients had a confirmed exposure to epstein-barr virus. patients ( . %) were lost to follow-up, ( . %) were in remission, ( %) relapsed, ( . %) were still in treatment and ( %) were deceased. university of chicago, chicago, illinois, united states background: due to the adoption of risk-adapted therapy, pediatric and adolescent acute lymphoblastic leukemia (all) is associated with high cure rates. despite excellent outcomes in most children, patients with certain blast cytogenetic features do not fare as well. furthermore, african american, native american, and hispanic patients have worse outcomes than caucasian patients. while the outcome discrepancies are certainly multifactorial, and blast cytogenetics are related to age, it remains unclear whether ethnicity and blast cytogenetics correlate. the diverse patient population at the university of chicago provides an opportunity to evaluate for such a correlation. objectives: to describe cytogenetic findings in a racially and ethnically diverse population of patients of all age groups diagnosed with all at university of chicago from to and determine if there is a correlation between race/ethnicity and blast cytogenetics. results: a total of newly diagnosed patients with all between the ages of - from - were included in this study. of those, patients ( . %) had b-all, had t-all ( . %), one had early t-cell precursor all and one had mixed phenotype all (b/t). caucasians accounted for % of patients, african americans (aa) %, hispanics . %, asians . %, and % were of other races. age distribution had a bimodal pattern, with a peak in incidence at and another at years of age, consistent with published data. cytogenetic categories included: t( ; )(p ;q ), q rearrangements (kmt a), iamp , t( ; )(q ;p . ), t( ; ) (q ;q ), hypodiploidy, hyperdiploidy and double trisomy of chromosomes and . aa and hispanic patients with b-all presented more frequently between the ages of - years compared to caucasians (p = . and . , respectively). in aa patients, t( ; ) (q ;p . ) was overrepresented (p = . when compared to caucasians), and was mainly observed in patients between - years. caucasian patients were more likely than non-caucasians to have hyperdiploidy (p = . ), especially in patients aged - years. the rate of t( ; )(q ;p . ) was significantly higher in aa patients in our cohort, in particular in patients between the ages of - years. hyperdiploidy was more likely in caucasians aged - years. these findings may suggest that varying blast cytogenetics could contribute to outcome differences between races. ahmed elgammal, yasser elborai, mohamed fawzy, asmaa salama, eman d el-desouky, lobna shalaby national cancer institute, cairo, cairo, egypt background: hodgkin lymphoma (hl) in children is one of the malignancies that have a high chance of cure. stage iv hl remains a challenge for getting good clinical outcome as in other stages. many treatment protocols used to give combination chemotherapy while combined modality treatment is the mainstay in other treatment protocols. objectives: we aimed in to assess the outcome using consolidation radiotherapy to chemotherapy (combined modality treatment) versus combination chemotherapy alone in treatment of stage iv hl. design/method: we included patients with stage iv hl and whose data were retrieved from the medical records of the pediatric oncology department, national cancer institute, cairo university, egypt from till june and were followed till august . treatment was either to give cycles of abvd (adriamycin, bleomycin, vinblastine, dacarbazine) only or to give cycles of abvd followed by consolidation radiotherapy. the study included cases; were males and were females. mean age was . years ranging from to years. the histopathology subtype was nodular sclerosis in the majority of cases ( cases) followed by mixed cellularity ( cases) then only one case of lymphocyte rich. nine cases were initially bulky while cases were not. constitutional manifestations were present in cases while it was absent in cases. bone marrow was involved in only cases. radiotherapy was given after completion of chemotherapy to cases while cases received chemotherapy only. the -year overall survival for patients who received radiotherapy was superior to those who received chemotherapy alone; % versus . % respectively with statistical significance (p = . ). the -year progression free survival was also higher with radiotherapy than others; % versus . % (p = . ). patients with stage iv hl who received consolidation radiotherapy apparently had a better outcome than those who received chemotherapy only. this suggests that radiotherapy contributes significantly with chemotherapy to the cure rate for those patients. the feinstein institute for medical research, manhasset, new york, united states background: microrna (mirnas) are short non-coding rnas that play a decisive role in cancer biology, including leukemia. exosomes are microvesicles ( - nm) produced by most cells in biological fluids. exosomes represent the fingerprint of the parental tumor and are loaded with bioactive markers such as mirnas, which may regulate tumor growth. exosomal cargo can be transferred into target cells changing their biological properties. our study investigates a functional role for exosomal mir- a in pediatric acute lymphoid leukemia (p-all). objectives: / to demonstrate that p-all exosomes induce cell proliferation / to confirm that exosome-induced cell proliferation is disease-stage specific / to analyze exosomal mir- a expression profiles in p-all / to authenticate that inhibition of exosomal mir- a reduces leukemia proliferation design/method: exosomes were isolated by ultracentrifugation from healthy donors (hd) & p-all serum and conditioned medium (cm) of sup-b , jm , and cl- (control) human cell lines. cell lines were exposed to different sources of leukemia-derived exosomes in a paracrine or autocrine fashion for hrs in triplicates. proliferation was assessed by microscopic cell counting and confirmed by gene expression for proliferation, pro-survival and pro-apoptotic genes. mirna profiling was performed with the human cancer pathway finder microarray (qiagen). silencing of exosomal mir a was carried out by a mir- a inhibitor (qiagen), utilizing exo-fecttm exosome transfection reagent (sbi, system biosciences). further, exosomal mir- a silencing was confirmed by q-pcr. cellular uptake of texred-sirna (sbi, system biosciences) was confirmed by flow cytometry. transfer of exosomal mir a to the target cells was evaluated by q-pcr. we elucidated that cm-derived exosomes from sup-b and jm cell lines induce cell proliferation in sup-b , jm (autocrine and paracrine) and cl- cells (paracrine) (p< . ). serum p-all exosomes promote paracrine cell proliferation in all cell lines compared to hdderived exosomes (p< . ). heatmap analysis of mirna profiles of leukemia exosomes (all cell lines and p-all) identified mir- a significantly upregulated in leukemia exosomes compared to controls. mir- a was also upregulated in all cell lines after exposure to leukemia exosomes that induced proliferation. moreover, exosomal mir- a inhibition reduces leukemic proliferation in pediatric all. our data suggest that all exosomes induce cell proliferation of leukemic cell lines in both paracrine and autocrine fashion. exosomes regulate these phenomena in a highly orchestrated way, by transfer of functional exosomal mirnas such as mir- a. the results of this study suggest s of s that exosomal mir- a inhibition can act as a novel way for growth-suppression of pediatric leukemia. results: a total of disease sites were detected at pet/ct, while sites were detected at contrast-enhanced ct and bone marrow biopsy (bmb). pet/ct showed improved detection of nodal lesions (p < . ) (kappa value = . ), extranodal lesions (p < . ) (kappa value = . ) and bone marrow (p < . ) (kappa value = . ) compared to contrast enhanced ct and bmb. pet/ct had upstaged cases ( %) and down-staged cases ( . %) (p < . ) (kappa value = . ). among the upstaged cases, patients ( . %) were upstaged from stage ii to iii, based on residual in pet/ct not seen in contrast enhanced ct after abdominal mass excision. four patients ( . %) were upstaged from stage iii to iv based on bone marrow uptake in fdg-pet without positivity in bma or bmb.regarding response assessment, sensitivity was % for pet and % for contrastenhanced ct (p = . ). specificity was % for pet and % for ct (p< . ). positive predictive value for pet was %, while was % for ct scan (p< . ). negative predictive value for both pet and ct was % (p = . ). five patients had nd biopsy to confirm viability of the residual lesions, lesions were negative in pathological examination (all of them were metabolic negative in pet/ct; deauville score below ). one lesion was positive in pathological examination (was positive in pet/ct; deauville score of ). conclusion: pet/ct detected additional sites compared with contrast-enhanced ct and resulted in changing stage of disease. pet scan is significantly more specific than ct in the management of children with burkitt lymphoma. background: deep sequencing of the immunoglobulin heavy chain (igh) locus indicates that each b all is composed of innumerable subclones. in many cases, subclones exhibit differing phenotypic qualities. however, it remains unclear whether subclones demonstrate distinct tissue distribution within a patient. objectives: . to quantify the extent of clonal heterogeneity in diagnostic b all specimens; . to identify variability in clonal composition between bone marrow (bm) and peripheral blood (pb) disease sites. design/method: igh sequencing was performed on purified dna from pairs of matched bm and pb patient specimens. multiplex pcr was used to globally amplify the igh locus; next generation sequencing (ngs) was performed using illu-mina® miseq. index clones (defined as ≥ % of all sequence reads in a specimen) and their subclone progeny (defined by shared nucleotide bases immediately upstream of a common jh, or n_jx) were identified using igblast-determined vh and jh alignments (http://www.ncbi.nlm.nih.gov/igblast/) and an established in-house computational pipeline. results: up to index clones per specimen were discovered in of the samples. in the remaining ( bm/pb pairs), pair did not reveal a clonal igh and was eliminated from analysis; in the other, clone frequency did not reach the % index threshold, but predominant clonal precursors were inferred by the prevalence of their subclone progeny. subclone counts ranged from to , per index clone. a combined , subclones derived from pb index clones were observed; in contrast, bm index clones gave rise to only subclones. subclone heterogeneity was observed between all paired specimens. in bm/pb pairs, index clones existed in equivalent proportions between disease sites. in contrast, bm/pb pair demonstrated high-frequency index clones in the bm ( . % & . %) with limited representation of these clones in the pb ( . % & . %, respectively); in this case, the most prevalent clone in the pb ( . %) matched the least frequent index clone in the bm ( . %). similarly, another pair showed a predominant index clone in the pb ( . %) which was below index threshold ( . %) in the bm. in paired patient specimens, index clone predominance was discovered to be overtly distinct between bm and pb. among all pairs, the extent of subclone progeny derived from each index clone showed marked variability, with far higher subclone frequency in the pb than in the bm. our data indicate that b all clonal composition differs between disease sites. valley children's healthcare, madera, california, united states background: tuberculosis (tb) presenting with hodgkin lymphoma (hl) is rare. their coexistence could lead to delay in diagnosis of both tb and hodgkin lymphoma due to the similarities in signs and symptoms of presentation. most cases have been reported in the adult literature. we describe a case series of children that were suspected to have tb and were found to have coexisting tb and hl. results: a retrospective review of hl patients in guatemala and argentina over six years, uncovered patients with simultaneous diagnosis of tb and hl. eight patients were from guatemala (incidence of . %) and from argentina (incidence of . %). there were females and males. age ranged from - years (mean . years, media years). nine patients were suspected to have tb at presentation by the referring physician. two patients were found to have tb at the time of relapse through routine tissue culture. initial systemic symptoms included fever (n = ), weight loss (n = ), and night sweats (n = ). six patients had a second systemic symptom in addition to fever. time for referral to oncology center ranged from weeks to months. nine patients were diagnosed with tb and hl through a tissue cultures and with serum quantiferon. one patient was found to have hl without tb. two patients had no systemic symptoms and the diagnosis of tb came to light through routine tissue culture. five patients had stage iiib and ivb, two stage iia and one iib at diagnosis. hl treatment was given according to the insti-tutional standards depending on stage and risk with abvd, oepa/copdac +/-radiation therapy, and ice for relapse. five patients started anti tb treatment (isoniazid, rifampin, pyrazinamide +/-ethambutol for months followed by isoniazid and rifampin for - weeks) simultaneously with chemotherapy, and three others after completing cycles. the two relapsed patients started tb treatment after cycles of chemotherapy. seven patients are alive and have been followed for months - years. one patient died during therapy, another died for causes not related to tb or hl and one is currently receiving treatment. conclusion: tuberculosis can coexist with hl. in areas were the prevalence of tb is high, microbiology investigations of biopsy specimen should be strongly considered. therapy for tb can be given simultaneously with chemotherapy. coexistence of tb and hl does not appear to affect outcomes. the children's hospital affiliated to the capital institute of pediatrics, united states background: the pi k/akt signaling pathway plays a central role in cell growth, proliferation and survival in physiological conditions. this signal pathway is considered to be an innovative targeted therapy of cancer, and its abnormal activation has been proved to be related to t-cell acute lymphoblastic leukemia (t-all). despite improved treatment strategies, such as multi-drug combination, high-dose chemotherapy and all kinds of application and popularization of hematopoietic stem cell transplantation, children with drug resistance or relapse t-all are still rather worse and its overall outcome and prognosis are much poorer than the more common b-lineage all. objectives: to explore the relationship between the pi k/akt pathway and the pediatric t-all, so as to probe the exact molecular mechanisms of t-all and provide more directions for its treatment. design/method: cases of new or recurrent acute t lymphocyte leukemia children with clinical information were collected in the children's hospital affiliated to the capital institute of pediatrics from dec. to oct. , with age and gender matched healty children as control (all was informed consent). the expressions of key genes in pi k pathway were s of s analyzed by western blot rt-pcr analysis, the pi k enzyme activities were detected by elisa,and the ccrf -cem's proliferation and its apoptosis were tested by mtt and flow cytometry technology on t-all cell lines ccrf-cem in different treatment group. the results of t-all children in clinical showed that pi k protein and gene expression level were higher apparent than the control group (p< . ), and pi k enzyme activity increased as well (p< . ); pi k inhibitor ly made a significant inhibition of cell proliferation and promoted cell apoptosis. ly also enhanced the effectiveness of clinical commonly used chemotherapeutic drug dnr. in combination ly and dnr treatment group cell viability dramatically declined, apoptosis and the apoptosis relation protein casepase expression in t-all patients was obviously higher than the control and the single drug group; pi k/akt signaling pathway related proteins and gene expression level, pi k, akt, gsk transcription in ccrf-cem were significantly higher than the control (p< . ), while pten transcription was significantly lower than the control (p< . ). the abnormal activation of pi k/akt signaling pathway might play an important role in pediatric t-all patients, especially in the cell proliferation or apoptosis. the results might provide new train of thought and direction in targeted suppress this signal pathway or in combination with other chemotherapy drugs therapy in looking for the more effective and less cytotoxic treatment of pediatric t-all. cleveland clinic children's hospital, cleveland, ohio, united states background: non-hodgkin lymphomas (nhls) are a heterogeneous group of lymphoproliferative diseases which comprise % of all childhood malignancies. nhls can be divided in to b cell lymphomas and t cell/natural killer (nk) cell lymphomas depending on immunophenotype, molecular biology, and clinical response to treatment. although nk/t cell lymphomas occurring in childhood and adolescence comprise a small portion of all lymphomas, they present many diagnostic and therapeutic challenges. the role of angiogenesis in lymphoma pathogenesis is becoming more evident. high molecular weight kininogen (hk) is a central compo-nent of the kallikrein-kinin system. it has been previously reported that cleaved hk (hka) induces apoptosis of proliferating endothelial cells and inhibits angiogenesis in matrigel plug and corneal angiogenesis models. however, the role of endogenous kininogen in regulation of angiogenesis is in tumor microenvironment is unknown. objectives: to elaborate the role of hk in lymphoma angiogenesis, we used a murine t-cell lymphoma model and compared angiogenesis and tumor growth between wild-type and kininogen deficient (mkng -/-) mice. we also evaluated the effect of hka on lymphoma cell proliferation. design/method: el- murine t-cell lymphoma cells ( × ^ ) were implanted into wild-type and mkng -/-mice. tumor size was measured using calipers and tumor volume was calculated using the formula volume = length × width^ × . . seventeen days after cell implantation, tumors were harvested and processed by immunoblotting and immunofluorescent staining. cell proliferation assays (mts) were performed to investigate any possible inhibitory effect of hka on el- cell growth, with human umbilical vein endothelial cells (huvec) were used as a positive control. results: el- lymphomas grew more rapidly and to larger sizes in mkng -/-mice compared to wild-type mice, with significant differences apparent by day after tumor implantation (p< . ). by day , the volume of tumors in mkng -/-mice was approximately . -fold larger than in wild-type mice (mean volume ± standard deviation; ± vs. ± mm , respectively, p< . ). mts assays showed that hka does not directly inhibit the proliferation of el- cells in vitro, though it does significantly impair the viability of ecs studied simultaneously. conclusion: these findings suggest that hk is an important endogenous regulator of angiogenesis and tumor growth in this t-cell lymphoma model, and suggests that hka specifically modulates endothelial proliferation in tumor microenvironment. further work is needed to understand the mechanisms underlying these findings and provide future anti-angiogenic approaches to increase the therapeutic options for patients with nhl. bruce bostrom, jack knudson, nathan gossai, joanna perkins, michael richards, jawhar rawwas, susan sencer, julie chu, nancy mcallister, yoav messinger children's minnesota, minneapolis, minnesota, united states background: osteonecrosis causes significant pain and morbidity in older patients treated for acute lymphoblastic leukemia. besides altering the schedule of dexamethasone in delayed intensification there is no other intervention known to reduce the incidence of symptomatic osteonecrosis. pamidronate has been shown to reduce bone pain from osteonecrosis but not to prevent joint collapse when advanced. objectives: to compare the incidence of symptomatic osteonecrosis in patients who received prophylactic pamidronate compared with concurrent controls. to describe any increase in side effects from the use of pamidronate. design/method: patients age to years at time of all diagnosis were given intravenous pamidronate monthly for one year at the discretion of the primary oncologist starting in the first year of therapy. concurrent controls were patients age to who did not receive pamidronate. all patients were treated according to the concurrent cog protocols and received intermittent dexamethasone during delayed intensification. patients with bcr-abl all were excluded as the use of imatinib may increase the risk of osteonecrosis. imaging was only done if osteonecrosis was suspect based on clinical symptoms. patients were censored at the time of relapse. data were analyzed as of / / . this retrospective study was approved by the children's minnesota irb. of the patients evaluated % were male and % female, % had b-cell and % t-cell. the median followup is . years with a range of . to years. pamidronate was given to patients with developing symptomatic osteonecrosis. there were concurrent controls with developing osteonecrosis. there was no significant difference in the leukemia lineage, gender distribution or body mass index (bmi) at diagnosis between groups. for all patients the median bmi was with a range of to . the age at diagnosis was significantly higher in the pamidronate group with a median of . years vs. . in the controls (p = . ). by kaplan-meier analyses the incidence of symptomatic osteonecrosis was significantly lower in the pamidronate group at % vs. % in controls. the log-rank p-value was . and the breslow p-value, which is more sensitive to early events, was . . there were no untoward side-effects from pamidronate. pamidronate infusions significantly reduced the incidence of symptomatic osteonecrosis in patients over the age of compared to concurrent controls who did not receive pamidronate. arahana awasthi, dina edani, janet ayello, christian klein, mitchell cairo new york medical college, valhalla, new york, united states background: mature b-nhl, including bl and pmbl express cd +/cd b+ and have an excellent prognosis, however, subset of patients relapse secondary to chemoimmunotherapy resistant disease and have a dismal prognosis (≤ % yr. efs, cairo et al. blood. ; gerrard/cairo et al., blood, , goldman/cairo et al. leukemia, . pv has been demonstrated to possess significant preclinical activity against indolent cd b+nhl (polson et al. can. res. ). we previously observed that obinutuzumab (anti-cd mab) significantly enhanced cell death and increased overall survival against bl (awasthi/cairo et al., bjh ) in xenografted nsg mice. however, additive/synergistic effects of pv with obinutuzumab against mature pmbl/bl are unknown. to determine the efficacy of the pv or obinutuzumab/rtx alone or in combination against pmbl and rituximab (rtx) sensitive/resistant bl cell lines. design/method: raji rh (provided by m. barth, md, roswell park cancer institute) and raji/ karpas p (atcc, usa) were cultured in rpmi. tumor cells were incubated with pv, and/or anti-cd b, mmae (generously supplied by genentech inc.) with obinutuzumab /rituximab ( ug/ml) for hr with nk cells at : e: t ratio and cytotoxicity was determined by delfia cytotoxicity assay. six to week old female nsg (nod.cg-prkdcscid il rgtm wjl/szj), were divided into groups: pbs, isotype control, pv, anticd b mab and mmae ( mg/kg). mice were xenografted with intravenous injections of luc+ bl and pmbl cells and tumor burden was monitored by ivis spectrum system. results: os of mice receiving pv alone was significantly increased compared to anticd b ab or isotype control in raji ( . vs. vs. . our preliminary data indicates that pv significantly increased survival in bl and pmbl nsg xenografts compared to anti-cd b ab alone. furthermore, pv in combination with obinutuzumab significantly enhances in-vitro cytotoxicity in bl and pmbl compared to obinutuzumab or pv alone. results: maximal grades (g) / , , and crs occurred in , , and patients, respectively. median lowest fibrinogen levels were . , . , and . g/l in patients with maximal g - , , and crs, respectively. %, %, and % of patients with maximal g - , , and crs had lowest reported fibrinogen levels of ≥ to < . g/l. eight patients (all with g crs) had very low fibrinogen levels (< g/l), which occurred before (n = ) or during (n = ) maximal crs grade or at time of improvement (n = ). no patients with maximal g - crs had < g/l fibrinogen levels. at the onset of < g/l fibrinogen levels, patient had concurrent g , and had g - increased international normalized ratio and activated partial thromboplastin. cryoprecipitate was the primary treatment in the us, and fibrinogen concentrate (fc) guidelines for tisagenlecleucel-associated coagulopathy were developed for other countries because administration of fresh frozen plasma can be problematic. fc was available at / sites for infused patients: / (g crs) and / (g - crs). cryoprecipitate was available at / sites for infused patients: / (g crs), / (g crs), and / (g - crs). risk of bleeding increases in pediatric patients with comorbid thrombocytopenia and anticoagulant treatments. / patients had g / decreased platelets within day of < g/l fibrinogen levels. fatal case of intraparenchymal cranial hemorrhage occurred during resolving crs with g hypofibrinogenemia, ongoing thrombocytopenia, and continuous veno-venous hemofiltration with citrate. hypofibrinogenemia was observed more frequently in patients with higher crs grades during/when crs was improving or resolving. fc and cryoprecipitate treatment guidelines were developed. frequent monitoring and fibrinogen replacement are needed in patients with g / crs. sponsored by novartis. its prolonged cns half-life, may allow a reduction in the number of intrathecal injections. objectives: to safely reduce the burden of therapy by reducing the number of it injections and reducing the total dose of doxorubicin with the addition of liposomal cytarabine and rituximab. design/method: patients ( - years) with cd + b-nhl with fab group b good risk (=stage i/ii and stage iii with ldh < xuln), fab group b intermediate risk (=stage iii ldh ≥ xuln and stage iv {bm blasts < %}) and fab group c high risk were eligible. patients received fab backbone therapy with the addition of six rituximab ( mg/m ) doses; two doses prior to each of two induction courses and one dose prior to each of two consolidation courses. cumulative doxorubicin was reduced from to mg/m in gr patients. after systemic methotrexate clearance, patients received age based dosing of it liposomal cytarabine. it injections were reduced from nine to five. the primary outcome is safety and toxic deaths among evaluable patients with an estimated -year survival above %, monitored by an independent dsmb. results: to date, evaluable patients, fab group b and group c ( cns positive), median age years (range - ), males, burkitt/ dlbcl with gr, ir and hr have enrolled. there has been one grade anaphylactic reaction to rituximab and one grade facial nerve palsy. no other serious adverse events were attributable to protocol therapy. there has been death from progressive disease and relapse at a median follow up of months. efs and os are % and %, respectively. our initial results show excellent efs and os, consistent with published standard of care outcomes, with the addition of rituximab and intrathecal liposomal cytarabine despite the reductions in therapy. further enrollment is ongoing and continued long term outcomes are needed to confirm early results. future randomized studies are needed to examine both short term (mucositis, infections, hospitalization days) and long term (late cardiac toxicity) endpoints. . goldman etal, leukemia, . cairo etal, jco st. jude children's research hospital, memphis, tennessee, united states background: bereaved parents identify significant spiritual needs around time of death and throughout their bereavement journeys. spirituality has been identified as a primary means by which bereaved parents can find meaning in their losses, and this ability to find meaning is associated with lower maladaptive grief symptoms. the use of spiritual coping strategies has been associated with improved coping and mental health outcomes among bereaved parents. objectives: to better understand how bereaved parents' experiences with spirituality throughout bereavement effects objective measures of grief, depression, and meaning-making. design/method: thirty participants whose children died of progressive cancer or related complications one to three years prior to participation completed an in-depth semi-structured telephone interview about their experiences with grief. participants were prompted to describe the impact of their spirituality on their bereavement processes. additionally, participants completed surveys related to grief (prolonged grief disorder questionnaire, pg- ), depression (beck depression inventory, bdi), and meaning-making (integration of stressful life experiences scale, isles). results were analyzed using a mixed methods approach including semantic content analysis of qualitative content and kruskal-wallis h test and post-hoc analyses of quantitative data. results: correlation analyses demonstrated significant differences between participants with positive and negative spiritual experiences of bereavement. participants with negative experiences of bereavement had a statistically significant increase in scores on the pg- compared to those with positive spiritual experiences signifying greater symptoms of prolonged grief. participants with negative spiritual experiences with grief had significantly lower scores on the isles, suggesting a lesser degree of adaptive integration of their losses. there were no significant differences in depression scores between groups. conclusion: bereaved parents that have a negative spiritual experience of bereavement are at increased risk for prolonged grief symptoms and are less likely to find meaning in their children's deaths than bereaved parents that describe a positive spiritual experience of bereavement. providers should consider exploration of spiritual beliefs and provision of spiritual care for parents of children facing life-limiting illnesses during treatment and bereavement. background: langerhans cell histiocytosis (lch) is an inflammatory myeloid neoplasia characterized by frequent relapse, with treatment failure associated with higher risk of death and neurodegenerative disease (lch-nd). activating somatic mutations in mapk pathway genes have been identified in almost all cases, with braf-v e in approximately % of lesions. targeted therapies have been successful in treating other refractory cancers with braf v e mutations (such as melanoma). given the central role of mapk pathway activation in lch, mapk pathway inhibition may be an effective therapeutic strategy for children with lch. objectives: the purpose of this study was to report the efficacy and toxicity profile of a retrospective cohort of patients with lch treated with mapk pathway inhibitors. design/method: medical records from pediatric patients with lch (systemic and/or lch-associated neurodegeneration) who were treated with a mapk pathway inhibitor were retrospectively reviewed from five institutions. all patients had failed at least one prior systemic therapy and had a proven mapk pathway mutation. results: all patients in this series were less than years old (median = . years; range: - years) with a median of three prior treatments (range: - ). at the time of initial mapk inhibitor use, nine of the patients had lch-nd diagnosed clinically and/or by radiographic imaging; the remaining three patients had systemic disease. patients were treated for a median of months (range: - months) with various reasons for discontinuation. three patients received combination mapk inhibitor therapies and three patients received other concurrent lch-directed therapies. four of the twelve patients had a grade or toxicity reported and three of these patients required dose reduction in order to be able to successfully resume therapy. overall survival was % with median month follow-up (range: - months) with only one patient achieving transient complete response. the remaining ten patients had partial response or stable disease and four of these patients developed progressive disease while on therapy. conclusion: mapk pathway inhibitors may be a relatively safe salvage therapy for refractory systemic lch and lch-nd but the efficacy and durability of this strategy remains to be defined. combination with cytotoxic chemotherapies may be required in order to eradicate the disease-causing cell. future prospective trials of mapk pathway inhibitors for patients with refractory lch are needed in order to directly compare their efficacy and toxicity relative to other current salvage strategies. cincinnati children's hospital medical center, cincinnati, ohio, united states background: medication adherence during maintenance therapy has been shown to have a direct relationship with disease relapse in pediatric leukemia. previous research determined that patients who are ≤ % adherent to mercaptopurine ( mp) have a greater risk for relapse. the primary aim of the present study is to examine the relationship between metabolite profiles of mp with behavioral adherence rates obtained via electronic monitoring at , , and days. it is hypothesized that patients demonstrating low levels of thioguanine (tgn) and methylated mercaptopurine (mmp) will have lower behavioral adherence rates prior to the blood draw. design/method: in a multisite, prospective study of patients ages - years diagnosed with acute lymphoblastic leukemia (all) or lymphoblastic lymphoma (lbl), mp adherence was measured across months of maintenance therapy using behavioral adherence (electronic monitoring) and pharmacological (metabolites) measures of mp. mp is metabolized into mmp and tgn. cluster analysis was used to generate three mutually-exclusive profiles of mp adherence. behavioral adherence rates were calculated for , , and days prior to the blood draw. results: this study identified three metabolite profiles of mp across months. previous research indicated that low levels of both metabolites suggest nonadherence to medication. low levels of one metabolite with high levels of another metabolite indicate adherence to mp. in this study, . % of the low tgn-low mmp group had -day behavioral adherence rates ≥ % (mean = %); . % had adherence rates < % (mean = . %). in the high tgn-low mmp group, . % had a mean -day adherence of %; . % had adherence rates < % (mean = . %). the low tgn-high mmp group had % of patients with a mean -day adherence level of %; % had adherence rates < % (m = . %). at and -days, to % of patients in the low tgn-low mmp group had adherence rates < %. conclusion: these findings suggest that electronic monitoring and metabolite concentrations can be used to monitor mp medication adherence during maintenance therapy. it is notable that there is a sub-sample of pediatric patients who are identified as being nonadherent to mp based on electronic monitoring, however, metabolite levels indicate adherence to mp. similarly, a sub-sample of patients were identified as being adherent based on electronic monitoring, but metabolite profiles indicated sub-therapeutic levels of mp. our findings underscore the clinical significance of using both objective measures of medication adherence to inform clinical decision making. cincinnati children's hospital medical center, cincinnati, ohio, united states background: hemophagocytic lymphohistiocytosis (hlh) is a life-threatening hyperinflammatory syndrome characterized by non-remitting fevers, rash, hepatosplenomegaly, cytopenias, liver dysfunction and coagulopathy, and can include central nervous system involvement. several genetic diseases cause hlh by impairing normal lymphocyte or macrophage function. the hlh panel at the cincinnati children's genetics laboratories includes genes associated with hlh and other lymphoproliferative diseases, including the genes that cause primary hlh (prf , unc d, stxbp , stx , rab a), x-linked lymphoproliferative diseases (sh d a, xiap), itk deficiency (itk), hermansky-pudlak syndrome types and (ap b and bloc s ), chediak-higashi syndrome (lyst), cd deficiency (cd ), xmen syndrome (magt ) and lysinuric protein intolerance (slc a ). deletion/duplication analysis is available as a reflex test for all genes, as copy number variations (cnvs) are not directly assessed by sequencing. objectives: the prevalence of cnvs among large groups of patients with hlh in north america is unknown. we assessed the frequency of cnvs in the genes on the hlh panel through a retrospective review of orders for deletion/duplication analysis performed after next-generation or sanger sequencing: orders for all genes on the panel, and orders of - genes from the panel. deletion/duplication analysis was performed on a custom × k microarray annotated against ncbi build (ucsc hg , march ). deletion/duplication analysis resulted in a confirmatory diagnosis in of cases ( . %). pathogenic or likely pathogenic cnvs were most common in the three x-linked genes: sh d a ( deletions), xiap ( deletions, duplication), and magt ( deletions). hemizygous deletions in xlinked genes in male patients were typically suspected after amplification failure during previous sequencing. of the autosomal recessive genes, pathogenic cnvs were observed once in each of three genes: rab a (heterozygous), lyst (heterozygous), and stxbp (homozygous). in the two heterozygous cases, a second change was not identified by sequencing, so deletion/duplication analysis did not offer a confirmatory diagnosis. in patients, deletion/duplication analysis was performed after a pathogenic or likely pathogenic variant was identified in an autosomal recessive gene during sequencing; however, in no case was a second mutation uncovered by cnv analysis. we recommend that deletion/duplication analysis be routinely performed in all male patients with hlh who lack a genetic diagnosis after sequencing of hlh-associated genes, especially if any regions failed to amplify. deletion/duplication analysis may be performed in female patients after sequencing if a genetic form of hlh is highly suspected, but the yield is expected to be low. cleveland clinic children's hospital, cleveland, ohio, united states background: the development of post-transplant neoplasia, typically from lymphoproliferative disease (ptld), is a severe complication in transplant recipients and affects approximately % of pediatric solid organ recipients. rates of lymphoma in adult heart transplantation patients are comparatively low, at less two percent at ten years. there are few published reports of the long-term outcomes of neoplasia after pediatric heart transplantation. we aimed to identify the subsequent malignancies that occurred in pediatric heart transplantation patients in a large single institution, and describe their treatment and subsequent clinical course. we performed a retrospective chart review of all pediatric heart transplant recipients followed at the cleveland clinic children's hospital from january to october . we excluded patients who died within days of heart transplantation. we reviewed in depth the history and clinical course of subjects who developed neoplasms. results: between and , patients underwent heart transplantation and survived at least days post transplantation. nine patients ( . %) developed a subsequent malignancy. in this case series, the median age at heart transplant was years old and the median time to develop neoplasia was . months. primary neoplasia included monomorphic ptld ( ), polymorphic ptld ( ), burkitt lymphoma ( ), hodgkin's lymphoma ( ), plasmacytoma-like lymphoma ( ) and epstein-barr virus-associated smooth muscle tumor (ebv-smt) ( ). one patient with hodgkin lymphoma subsequently developed monomorphic ptld, one patient with polymorphic ptld subsequently developed ebv-smt and later, an undifferentiated gastric cancer. one patient with monomorphic ptld developed an ebv-smt. evidence of epstein-barr virus was present in six of nine patients at diagnosis of first malignancy. four of nine patients received reduction in immunosuppression as a primary intervention for the initial malignancy, with two complete responses (cr), one partial response, and one with progressive disease. five patients were treated with chemotherapy, with four cr and one with progressive disease. three patients died of malignancy (recurrent ebv-smt, undifferentiated gastric cancer, and monomorphic ptld post-hodgkin disease) and two patients died of other transplant related complications. conclusion: secondary malignancies represent a significant disease burden to survivors of cardiac transplantation. as expected, much of the malignancy burden is driven by ebv. despite aggressive histology, many malignancies can be successfully cured in this setting with a multidisciplinary approach. stanford university school of medicine, palo alto, california, united states background: current treatment of langerhans cell histiocytosis (lch) is based on extent of organ system involvement and if high risk systems are affected. gastrointestinal (gi) involvement is diagnosed in about % of lch patients, and classically presents in children under years of age with malabsorption, failure to thrive, bloody diarrhea and anemia. although the gi system is considered standard risk, a mortality rate over % occurring within years of diagnosis has been reported. this study was performed due to this discrepancy and the limited number of published cases. objectives: to review the clinical course and outcomes of patients diagnosed with gi lch. design/method: a retrospective chart review of patients with histologically confirmed gi lch diagnosed in the last years identified from the bass center histiocytosis clinical database was performed. two other pediatric hematology/oncology centers (ucsf benioff children's hospital oakland and san francisco) were queried for additional cases. results: four patients with biopsy proven gi lch [ subjects ( . %) from database records and l from center queries] were identified. failure to thrive, hypoalbuminemia, bloody diarrhea and rash were the most common presenting symptoms. lch of the skin was found in all patients. risk organ systems were involved in patients. of note, subjects were of african racial background. the median age at diagnosis was . months ( . months to years), mean albumin . g/dl ( . - . g/dl), mean esr of mm/hr ( - mm/hr). all patients initially received combination therapy per lchiii protocol (vinblastine, prednisone, and mercaptopurine). two patients had recurrent disease and received second line therapy (cytarabine, cda, and local radiation therapy). all patients are alive without active disease at last follow-up ( to months after completion of therapy). a systematic approach to evaluate gi involvement should be performed in children diagnosed with lch. from our experience, combination chemotherapy for patients with lch involving the gi tract is an effective intervention for active disease. cincinnati children's hospital medical center, cincinnati, ohio, united states background: bhatia indicated that rates of mp adherence ≥ % have better clinical outcomes. those with adherence rates ≤ % have an increased risk for disease relapse. the present study investigated patterns of mp medication adherence using group-based trajectory modeling in a large sample of pediatric patients. to describe patterns of behavioral adherence during the maintenance phase of therapy for a cohort of pediatric patients ages - years who were diagnosed with acute lymphoblastic leukemia or lymphoblastic lymphoma (n = ). previous research has documented the relationship between optimal levels of medication adherence with positive health outcomes. it was hypothesized that three groups would be identified: optimal adherence, deteriorating adherence, and chronic nonadherence. it was hypothesized that patients in the optimal adherence group would have adherence rates ≥ %. those with poor adherence would have adherence rates ≤ %. design/method: the present study was a longitudinal, multisite study investigating adherence to -mercaptopurine in a pediatric cohort of patients using electronic monitoring devices. daily adherence rates (electronic monitoring of mp) were examined across -months. health outcomes were measured at quarterly intervals through medical chart reviews. results: unconditional growth curve modeling indicated that the mean percentage of behavioral adherence was . % at baseline and declined to . % at -months. three trajectories of mp behavioral adherence were identified: ) optimal adherence ( % of patients): averaging % behavioral adherence across months; ) moderate adherence ( %): relatively stable nonadherence with rates of % across months; and, ) chronically nonadherent ( %): adherence decreased from % to %. with respect to patterns of medication adherence and relationship to clinically-relevant health outcomes, there were no significant differences in health outcomes between patients in the adherent versus nonadherent trajectories, including mean absolute neutrophil counts (anc), risk for infection as measured by anc, healthcare utilization, or risk for disease relapse. although longitudinal patterns of mp behavioral adherence were not related to health outcomes, it is notable that only % of the current sample had adherence rates ≥ %. in fact, % of the current sample demonstrated adherence rates ≤ %. our findings are important for development of future adherence promotion studies in pediatric cancer. our findings underscore the relative significance of tailoring adherence promotion interventions to subgroups of patients, including those with problematic patterns of adherence. patients who demonstrate adequate levels of adherence could still benefit from less intensive, preventative interventions to sustain and improve adherence. sophie gatineau-sailliant, pascale grimard, marie-claude miron, guy grimard, anne-sophie carret, jean-marie leclerc chu sainte-justine, montreal, quebec, canada background: vertebral involvement in langerhans cell histiocytosis (lch) is still a subject of interest, due to its low frequency and the absence of management's guidelines. objectives: to provide additional information on presentation, treatment and morbidity of pediatric lch vertebral lesions, we report cases of children with vertebral lesion of biopsy-proven lch, between january st and december st , at sainte-justine university health center (montreal, quebec, canada). we conducted a retrospective study by reviewing charts and imaging of vertebral lch in a population of children (median age of . years at lch diagnosis), followed for a median duration of months. symptoms at presentation, treatment modalities and morbidities were collected. results: vertebral lesions were present at lch diagnosis in of cases. they were usually diagnosed secondary to back pain in of cases and were asymptomatic in only one case. despite an epidural extension in of cases, no child developed neurological symptoms. lesions frequently involved vertebral body ( of cases) and were rarely unstable ( of cases). out of vertebral lesions, most of them had a dorsal localization ( of lesions) and of patients had lch in multiple vertebrae. at diagnosis, median vertebral height loss was . % compared to % at last imaging control. most used imaging modalities were pet-scan and plain x-rays. treatments were diverse and consisted in chemotherapy in all children but three and bisphosphonates in only cases. radiation therapy was not used in any patient. six out patients did benefit of an orthosis. a lch recurrence was observed in patients and involved vertebrae in cases. one patient with treatment-resistant lch disease had relapses, and required multiple lines of treatment. all children were alive and disease-free at their last follow-up, patients having radiological vertebral sequelae and only had clinical sequelae. our study is consistent with the epidemiological data described in larger cohorts of children with vertebral lesions of lch and the favorable prognosis associated with such lesions. nevertheless, aggressive treatment and long term follow-up seemed to be essential as recurrences are s of s not rare and spontaneous bone regeneration often incomplete. plain x-rays appears to be a good follow-up tool for vertebral lesions as it allows reliable measures, less exposure to radiation at lower cost. national cancer institue, giza, giza, egypt background: acute lymphoblastic leukemia (all) is the most common type of childhood cancer and also the most complicated in the treatment, so it requires many interventions for both treatment and to alleviate suffer form side effects. pancreatitis is one of the toxicities, which is more common in all as it appears in about % of the patients. it occurs in many drug combinations which induce pre-pancreatitis and even direct destruction of pancreatic tissues. pancreatitis can be induced by many drugs used in the treatment such as chemotherapeutic agents or supportive treatment. lasparaginase is the backbone drug of the treatment of all in which to doses are required to achieve complete remission status in the induction phase of treatment and to doses in the maintenance phase.it is an enzyme that destructs the l-asparagine amino acid into aspartic acid and ammonia thus deplete the asparagine from the extracellular matrix . many drugs are investigated for their effect on treatment of induced pancreatitis such as interleukin- , nsaid as antiinflammatory, glycerin tri nitrates as improvement of microcirculation, tnf-alpha antibody, paf inhibitor as specific anti-inflammatory and low molecular weight heparin .none of the drugs was investigated for their ability to prevent the occurrence of pancreatitis. objectives: this study was designed to evaluate the protective effect of enoxaparin and diclofenac against l-asparaginase induced pancreatitis design/method: acute pancreatitis was induced in rats by intra-muscular injection of l-asparaginase ( i.u/kg) given daily for five days. enoxaparin was given subcutaneous ( i.u/kg) and diclofenac was given intra-peritoneal ( mg/kg) daily for five days. then, markers of pancreatic injury, lipids, immune cell infiltration and oxidative stress were analyzed with histo-pathological examination of the pancreatic tissue results: during acute pancreatitis, oxidative stress markers were significantly changed as indicated by reduced tis-sue glutathione and increased malondialdehyde levels. this was accompanied with significant increase in immune cells infiltration as indicated by high levels of myeloperoxidase and pro-inflammatory cytokine tnf-alpha. triglyceride only showed increase level. treatment with enoxaparin and/or diclofenac restored levels of biochemical markers including serum alpha-amylase, reduced glutathione, malondialdehyde, pro-inflammatory cytokine tnf-alpha, myeloperoxidase and triglyceride. histological injuries of pancreatic tissues as vacuolation and necrosis of epithelial lining pancreatic acini, inflammatory cells infiltration and focal pancreatic hemorrhage were also reduced by treatment with enoxaparin and/or diclofenac. the present study emphasizes the potential protective effect of enoxaparin and diclofenac against l-asparaginase induced pancreatitis background: rosai dorfman disease (rdd), or sinus histiocytosis with massive lymphadenopathy (shml), is a rare condition of immune dysregulation of unknown etiology arising from the massive accumulation of non-langerhans type histiocytic cells inside lymph nodes. the disease classically presents as bulky, painless lymphadenopathy often associated with infection showing distension of lymph node sinuses by abundant histiocytic cells (cd a(-), s- (+)/cd (+)). in some cases, the disease can be self-limiting, but in cases with a prolonged chronic course of exacerbations and remissions, those with extranodal involvement, or disease that threatens vitals structures, treatment may be necessary. there is no treatment consensus. to describe a case of life-threatening, unresectable, recurrent rdd successfully treated with langerhans cell histiocytosis (lch) -inspired therapy. design/method: we compared this case to the current literature on chemotherapeutic treatments for rdd. we searched pubmed, ovid, and google scholar for similar cases. we believe this to be the first reported case of using lch therapy to successfully treat rdd. an -year-old male presented to an outside hospital with two years of massive neck swelling causing torticollis. biopsy confirmed rdd. he was intermittently treated with courses of antibiotics with partial response. surgical removal of the affected lymph nodes was unsuccessful due to proximity to the spinal cord. two years later, the patient presented to our institution. he was initially treated with prednisone with a fast tapering dose, but after a second relapse the decision was made to try chemotherapy following the lch- protocol of weekly vinblastine ( mg/m ), -mp ( mg/m ), and high dose steroid bursts. he experienced two additional relapses off therapy at ages and years old, including cmv(+) associated septic shock and cytokine storm requiring rapid response, picu admission, and ionotropic support. this last episode was treated with a more prolonged induction and maintenance therapy. an extended and slowly tapered maintenance therapy regimen of . years of daily -mp, monthly vinblastine and steroids with a slowly tapered dose during his fourth remission has resulted in -months of continuous complete remission-the longest stretch of his life. no similar cases were found. literature search demonstrated no consensus regarding the most effective treatment of rdd, with no previous cases being successfully treated following lch chemotherapy protocols. we hypothesize that the multi-agent relatively mild lch- therapy mitigates the immune dysregulation of rdd. this case suggests that lch- therapy can be used to treat cases of rdd that is not amendable to surgery or observation. nicklaus children's hospital, miami, florida, united states background: central venous catheters (cvc) are necessary in the management of patients with malignancies, especially children. patients with acute leukemia (al) have higher rates of central line associated complications such as bloodstream infections compared with other malignancies. objectives: to examine the choice of placement of cvc and the differences in outcome between peripherally inserted central catheters (picc) and ports in patients with leukemia during induction. design/method: retrospective chart review of patients with newly diagnosed leukemia at nicklaus children's hospital between and . results: ninety four patients with a new diagnosis of leukemia undergoing induction chemotherapy were identified. the average age was . years. overall, ( . %) patients had a port placed and ( . %) had a picc placed. the decision for picc or port was subjective and physician based. the main outcome measures were local inflammation/infection, bacteremia, thrombophlebitis, blocked catheter and premature removal. the most common complication was bacteremia ( . %). in a multiple logistic regression analysis for predicting whether patients had at least one complication, results showed that having at least one complication is . times the odds in patients with aml compared to patients with all (p = . ). when comparing picc vs. ports, patients with picc had more frequent episodes of blocked catheters ( . %) and premature removal ( . %) compared to the patients with ports ( . % and . %) (p = . and p = . respectively) during induction. local inflammation, bacteremia and thrombophlebitis were not statistically different (p = . , p = . and p = . respectively). the most common place for port placement was the right subclavian vein ( %). there was no significant association between port location and having at least one complication (p = . ). acute lymphocytic leukemia subgroup analysis: fourteen patients ( %) in the picc group had at least one complication and ( %) in the port group but that was not statistically significant (p = . ). our series showed a higher incidence of blocked catheters and premature removals with picc compared to ports in patients with leukemia during induction. the choice of placement of picc vs port was subjective and physician based. patients with all, despite receiving steroids and asparaginase during induction, did not show a statistically significant increase risk in thrombosis or infection but larger numbers may be needed in future studies. university of california, san francisco, san francisco, california, united states background: hemophagocytic lymphohistiocytosis (hlh) is classically a disorder of young children meeting systemic hyperinflammation criteria. presentation in late adolescence is uncommon. furthermore, though cns signs occur in - % of cases, initial isolated neurologic presentation is rare, frequently resembling encephalitis or demyelinating disorders. these cns signs can be isolated or precede systemic disease, delaying hlh diagnosis. hlh declaring in adolescence with predominant psychiatric features has not been well documented. objectives: to describe a case of cns hlh presenting with neuropsychiatric features in absence of classic hlh criteria. design/method: retrospective review of clinical, radiologic, histologic, immunophenotypic, and molecular features of a patient with cns hlh. a -year-old female presented with acute-onset headaches following nine months of progressive anxiety, short-term memory loss, emotional lability, perceptual disturbances, and hypomania. brain mri demonstrated numerous enhancing t hyperintense supratentorial and infratentorial white matter lesions in the left thalamus and caudate head. brain biopsy showed histiocyte-rich inflammation and associated demyelination. extensive evaluation including universal microbial pcr failed to reveal underlying infection or malignancy. past medical history was notable for presumptive pulmonary sarcoidosis diagnosed months prior with progressive respiratory failure with associated granulomatous pulmonary nodules which responded to systemic immunosuppression. at presentation of her neuropsychiatric symptoms, she had normal sil- r, ferritin, fibrinogen, and triglycerides. there was no pancytopenia, coagulopathy, bone marrow hemophagocytosis, fevers, or splenomegaly. given the possibility of partial immune suppression of systemic symptoms and the prominent neurologic symptoms, hlh screening labs were sent and notable for decreased natural killer and cytotoxic t lymphocyte function, normal granzyme expression and cd a mobilization, and absent perforin expression. genetic testing confirmed compound heterozygous mutations in prf (c. g>a, c. a>c) and familial hlh type . she was treated with low-dose dexamethasone and intrathecal chemotherapy per hlh- . due to lack of evidence of systemic inflammation, vp- and high-dose steroids were held. within one week of initiating therapy, she had decreased anxiety and improved cognition, with sustained, incremental neuropsychiatric improvement with additional intrathecal treatments. she tolerated dexamethasone tapering without symptom flare. mri also demonstrated parenchymal lesion improvement. for definitive treatment, she underwent unrelated allogeneic hematopoietic cell transplantation and remains at neurologic baseline as of eight months post-transplant with ongoing imaging improvement. conclusion: this case of familial hlh with compound heterozygous perforin mutations in an adolescent with isolated neuropsychiatric symptoms illustrates that cns hlh may be an underrecognized phenomenon in absence of systemic signs. standard hlh therapy may effectively reverse these symptoms with associated radiologic responses. rush university children's hospital, chicago, illinois, united states background: posterior reversible encephalopathy syndrome (pres), a recognized complication of pediatric leukemia treatment has been reported in up to % patients in various series. hypertension, chemotherapy and cortical spreading depression have been implicated in the pathophysiology. due to the combinations used, it is difficult to identify the offending drug, several have been implicated. since delay of chemotherapeutic treatment in children with high risk leukemia is unfavorable, it is important to recognize the characteristic radiologic findings, manage appropriately and reintroduce the treatment as soon as possible. pharmacoethnicity is now recognized as an important factor for variation in neurotoxicity in children with all. ethnic differences in reported pres events in pediatric patients with all has not been well described in literature. to describe the factors associated with pres in a cohort of high risk pediatric all patients at a single institution. design/method: a total of children with an average age of years ( - years) diagnosed with all between - were retrospectively reviewed for the occurrence of pres. various demographic factors, therapy received, clinical features, radiology related findings and management were reviewed. a search for all published articles on pres in leukemia was conducted using pubmed databases. results: five ( %) children (average age . years) developed pres during days - of induction. % of the patients that developed and % of those that did not develop pres were hispanic. all the patients that developed pres and % of those that did not were diagnosed with high risk all. all patients received vincristine, % received daunomycin and intrathecal methotrexate and % received asparaginase in the week prior to the event. mri findings confirmed pres in all patients with no evidence of methotrexate related leukoencephalopathy or leukemia. at the time of pres all patients were in remission based on mrd and spinal fluid cytology. two-thirds of the patients had seizures and hypertension at the time of the event with no prior history of either. all patients had complete recovery of normal mental status after resolution of pres. a higher incidence of pres than previously reported was noted in our series. hispanic ethnicity, high-risk all and exposure to vincristine, daunomycin and intrathecal methotrexate in induction were associated with pres in our cohort. a new association that emerged was that of hispanic ethnicity with pres .larger studies to understand the importance of pharmacoethnicity in pres may help in individualization of chemotherapy based on ethnic differences. children's hospital of illinois, peoria, illinois, united states background: hyper ige syndrome is a primary immunodeficiency characterized by susceptibility to skin and lung infections as well as increased propensity for malignancy. hemophagocytic lymphohistiocytosis (hlh) is a syndrome characterized by overwhelming activation of t lymphocytes and macrophages occurring as either primary hlh caused by genetic abnormalities or secondary hlh associated with infectious, malignant, metabolic, or immunodeficiency causes. we describe the first case to our knowledge of hlh in a patient with hyper ige syndrome. to describe a case of hlh in a pediatric patient with hyper ige syndrome. results: a -year old caucasian male with known autosomal dominant hyper ige syndrome (stat mutation) was transferred to the pediatric intensive care unit secondary to concern for septic shock. the patient had persistent slow bleeding from oral lesions and central catheter sites despite the addition of aminocaproic acid and recombinant factor viia. he also required numerous blood product transfusions sec-ondary to anemia and thrombocytopenia. clinical suspicion was high for hlh and the patient met criteria for diagnosis of hlh with the following: ferritin > , ng/ml, triglycerides mg/dl, decreased nk cell function with the sample only containing % nk cells, elevated soluble il- receptor at u/ml, splenomegaly, and fever. infectious workup was remarkable for a positive ebv qpcr with , copies/ml suggestive of ebv driven secondary hlh. familial hlh testing was unable to be completed. therapy was initiated based upon the hlh- study. the addition of ruxolitinib and anakinra were considered but the patient declined rapidly prior to treatment. ct of the head was concerning for a stroke with signs of edema and increased intracranial pressure likely leading to the development of symptoms consistent with brain stem herniation. the decision was then made to withdraw care. conclusion: to our knowledge, this is the first report of hlh in a patient with hyper ige syndrome. diagnosing hlh requires a high index of suspicion in critically ill patients, and prompt initiation of therapy is essential. this challenging case of hlh in a patient with hyper ige syndrome highlights the diagnostic challenge, variable presentation, and need for effective therapy in this vulnerable patient population. background: adolescents and young adults (ayas) with cancer are at risk for psycho-social as well as physical symptom burden during cancer therapy. the purpose of this study is to explore psychological and physical symptoms endorsed by aya while receiving therapy for cancer design/method: surveys were given in both inpatient and outpatient settings during cancer therapy. symptom screening in pediatrics tool (sspedi) and memorial symptom assessment scale (msas). symptoms severity was rated by teens on a point likert scale. spss , used for statistical analysis. results: : a total of aya on cancer therapy (age range - . years) % female, % male, . % acute leukemia, . % solid tumors, and . % diagnosis was not reported. % of aya on cancer therapy reported at least or more symptoms, % reported > symptoms cluster. of the physical symptoms that were reported as most distressing to the teens, mouth sores and headaches were the top causes. of the physical symptoms that were most frequently endorsed; fatigue was on the top ( %), followed by change in appetite %, vomiting %, and pain %., the least was bowel habit changes. aya rated sadness as the most frequent psychological symptom %, followed by feeling angry %, and scared %. statistically significant difference was noticed based on gender difference with more females reported symptoms (p = . ), while type of cancer (acute leukemia versus solid tumors) was not statistically different. conclusion: aya with cancer reported multiple physical and psychological symptoms with significant distress. females seem to report more symptoms compared to males. screening aya for cancer therapy related symptoms is feasible during routine visits and adds important information about the aya well-being. background: sinus histiocytosis with massive lymphadenopathy (shml), also known as rosai-dorfman disease, is a rare histiocytic proliferative disorder of unknown etiology. many treatment modalities have been employed; however, no uniform guidelines exist. objectives: literature review of treatment options for shml. design/method: chart review was performed on pediatric patients diagnosed with shml at the children's hospital at montefiore between and after irb approval. inclusion criteria included children between the ages of and years with shml. exclusion criteria included children with cutaneous shml. four cases of shml seen at montefiore are described. a comprehensive review of the literature identified additional cases published between and . manuscripts that did not include the treatment modality or outcome were excluded. results: many of the patients with shml responded to observation alone. of patients, patients were observed, with ( %) having resolution of disease, five having stable disease, and five being lost to follow-up. one patient received subsequent systemic therapy. surgical management was con-ducted upfront in patients. of those, ( %) had resolution of disease, one had stable disease, and one had recurrence with no further therapy noted. of the remaining nine patients, % were successfully treated with systemic therapy, consisting of either steroids ( ) or steroids and chemotherapy ( ). systemic therapy was used as first-line therapy in patients. steroids alone or in conjunction with chemotherapy resulted in resolution of disease in / and / patients ( / , %), respectively, with four patients having stable and three with progressive disease. chemotherapy without steroids resulted in resolution of or stable disease in / patients. radiation was ineffective. conclusion: shml is a rare disease with no published guidelines for treatment. from the results of the cases and a detailed review of the literature, it can be suggested that observation may be considered as first line management in patients providing there are no significant symptoms. for patients who are symptomatic or have significant progression, surgery may be considered. in patients with recurrence or refractory disease, steroids and/or chemotherapy may be used. the presence of nodal or extra-nodal disease did not seem to have a significant impact on the course of treatment. given the rarity of the disease, it is difficult to conduct a randomized control trial. further work, involving collaboration between centers and cooperation with the international rare histiocytic disorders registry would be helpful. boston children's hospital, boston, massechusettes, united states background: increasing census and intensified work compression on the inpatient oncology service at our institution was identified as leading to resident dissatisfaction, impaired resident learning and decreased perceived quality of patient care. objectives: to evaluate the impact of a redesign of a pediatric inpatient hematologic malignancy (ihm) service on resident perceptions of the educational value of the rotation and safety of patient care. design/method: during the - academic year, we initiated a bundled intervention on the ihm service. modifications included ) decreased patient volume: the ihm service was divided into two teams, utilizing an extra attending -a teaching service consisting of residents and fellows and a team comprised of nurse practitioners. ) intentional patient team assignment: patients were deliberately assigned to a care team based on educational opportunities and provider skill sets. ) intentional attending faculty selection: attending faculty with deeper clinical and teaching experience were selected to supervise on the teaching team. ) increased weekend staffing. after completing the service, junior residents completed an electronic survey to evaluate their perceptions of the educational value of the rotation, as well as their ability to deliver safe care while on the rotation. fisher's exact tests were used to compare responses from residents in who experienced the redesign to residents in , whose experience results: survey completion rates were % ( / ) in and % ( / ) in . intervention residents were significantly more likely than comparison group residents to choose the answers "very good" or "excellent" to describe both the overall quality of the rotation ( % intervention vs. % comparison, p< . ) and the educational experience on rounds ( % intervention vs. % comparison, p< . ). intervention residents also reported caring for fewer average primary patients daily on weekdays as compared to comparison residents ( . vs . patients, p< . , % ci - . to - . ). furthermore, intervention residents were more likely than comparison residents to "agree" or "strongly agree" that they could provide safe patient care on weekend days ( % intervention vs. % comparison, p< . ) and on nights ( % intervention vs. % comparison, p< . ) while on the oncology service. a redesign initiative of an oncology service with the development of a new teaching service led to improved resident perceptions of the educational value of the rotation and ability to provide safe care to patients. this approach could be useful to other services and institutions to promote similar outcomes in resident education and patient care. background: alk-positive histiocytosis is a rare histiocytic proliferative disorder that has been reported in three infants presenting primarily with hepatosplenomegaly, anemia, and thrombocytopenia. given the rarity of this disease, there are no standard treatment algorithms for this diagnosis and the disease course and outcomes remain largely unknown. the published series describes treatment ranging from monitoring alone to multi-drug chemotherapy regimens. there was ulti-mately resolution of presenting symptoms in all three cases despite varying treatment strategies. objectives: to report a newly diagnosed case of alkpositive histiocytosis that was treated with a novel approach using cytarabine monotherapy. results: a full term male infant presented at birth with difficulty feeding and hyperbilirubinemia. over the first few weeks of his life, he subsequently developed thrombocytopenia, transaminitis, and profound hypoalbuminemia. by six weeks of life, he was experiencing significant abdominal ascites requiring repeat paracenteses, massive hepatosplenomegaly, respiratory distress secondary to abdominal distension, anemia, and coagulopathy. he underwent numerous diagnostic tests, including a liver biopsy followed by a bone marrow biopsy that showed alk-positive histiocytic infiltrates in both sites. treatment was initiated with cytarabine mg/kg/day x days, repeating every weeks. throughout his course of five cycles of treatment, he experienced intermittent fevers and mild nausea with no other adverse events. by the end of five cycles, his hepatosplenomegaly resolved, his blood counts normalized, he demonstrated weight gain on oral feeds, and his liver enzymes normalized. he is currently months post completion of therapy and remains well with a normal physical exam and laboratory values. conclusion: treatment of alk-positive histiocytosis with lose dose cytarabine resulted in complete resolution of our patient's symptoms with minimal treatment related adverse effects, and few long-term treatment related risks. given the rarity of the diagnosis, the reporting of effective novel treatment options is important for future patient care. background: adult patients with melanoma or lung cancer harboring braf v e have benefitted from the development and subsequent approval of specific braf inhibitors. as such, delineating the subset of similarly targetable pediatric oncology patients may spur development and rational use of these inhibitors in children. importantly, other point mutations and fusions of braf may also be targetable in s of s children analogous to recent emerging data in adult cancer patients. objectives: to define the genomic landscape of known and novel braf alterations and raf fusions in pediatric malignancies and report index cases with clinical response to braf or mek inhibitors. design/method: dna was extracted from microns of ffpe sections of , tumors from pediatric (< years of age) oncology patients, and cgp was performed on hybridization-captured, adaptor ligation based libraries to a mean coverage depth of x for up to cancer-related genes plus introns from genes frequently rearranged in cancer. genomic alterations (ga) included base substitutions, indels, copy number alterations and fusions/rearrangements. a total of ( . %) braf-altered pediatric malignancies were identified. ( . %) harbored a single kinaseactivating braf short variant, indel, or fusion. an alteration resulting in reduced braf kinase activity was identified in ( . %) tumors while ( . %) tumors harbored multiple braf alterations, of which contained at least a single activating short variant. the remaining tumors ( . %) contained functionally uncharacterized variants. kinaseactivating braf alterations were identified in diverse tumor spectra comprised of brain tumors ( . %; subtypes), carcinomas ( . %; subtypes, with melanoma constituting % of cases), hematological malignancies ( . %; subtypes), sarcomas ( . %; subtypes), and extracranial embryonal tumors ( . %; subtypes). seventy-two ( . % of braf-altered cases) braf fusions were identified, ( . %) of which were kiaa -braf; involved the novel fusion partners: stard nl and khdrbs . seven ( . %) raf fusionpositive cases, predominantly brain tumors ( ), were identified; involved the novel fusion partners: tmf and sox . index cases of response to therapy of intracranial tumors will be presented. we describe a population of pediatric patients with targetable braf alterations predominantly enriched in primary intracranial tumors, but spanning diverse solid tumor types and hematologic malignancies. we additionally report a cohort of raf fusion-positive patients. an index case and multiple previous reports suggest raf or mek inhibitors may benefit pediatric patients with either intracranial or extracranial disease, and development of such drugs in pediatric indications is strongly warranted. background: diffuse midline gliomas (dmg) with h k m mutation, including diffuse intrinsic pontine glioma (dipg), are the leading cause of brain tumor-related deaths in children. there are no effective therapeutic strategies and the median survival remains dismal. genomic studies have identified a recurrent mutation in the majority of dmgs involving a lysine to methionine substitution (k m) in histones . and . , resulting in changes in the epigenetic landscape that dysregulate gene expression and promote gliomagenesis. panobinostat, a multiple histone deacetylase (hdac) inhibitor, was found to be one of the most effective agents against dipg patient-derived cell cultures and xenograft models in previous studies and is presently in clinical trial for dipg. hdac inhibition with panobinostat may also exhibit activity against h k m+ diffuse midline gliomas of the thalamus and spinal cord. to evaluate the effect of panobinostat as a single agent against patient-derived thalamic and spinal cord h k m+ diffuse midline glioma cell cultures and in an orthotopic xenograft murine model of h k m+ spinal cord glioma. design/method: patient-derived thalamic and spinal cord h k m+ diffuse midline glioma cell cultures were treated with single agent panobinostat at a range of concentrations. cell viability was evaluated using the celltiter-glo assay. panobinostat was systemically administered to orthotopic xenograft murine models of luciferase-expressing spinal cord h k m+ diffuse midline glioma. response to panobinostat was evaluated with ivis in vivo imaging. results: hdac inhibition with panobinostat significantly decreases cell proliferation with an ic of nm and nm in the spinal cord and thalamic glioma patient-derived cell cultures respectively. panobinostat slowed tumor growth in murine models of spinal cord glioma by . -fold in the brain (p = . , n = ) and -fold in the spinal cord (p = . , n = ) when compared to vehicle controls after week of administration. panobinostat is in clinical trials for dipg. this study suggests that hdac inhibition with panobinostat may also be beneficial for patients with thalamic and spinal cord diffuse midline glioma h k m mutants. background: brain tumors are the most common solid tumor of childhood and the leading cause of childhood cancer deaths. while medulloblastoma is the most common malignant brain tumor of childhood with a -year survival - %, children with high-grade gliomas (hggs) such as glioblastoma multiforme (gbm) fare much worse with a -year survival of - %. implicated in this poor outcome is the presence of treatment resistant brain tumor stem-like cells. gbm stem-like cells (gscs) have been implicated in tumor growth, treatment resistance and patient relapse, making them a key therapeutic priority. antipsychotic drugs (apds) have been used for decades in various psychiatric clinical settings and are associated with a lower incidence of cancer, including malignant brain tumors. currently, atypical apds are being evaluated for their potential to alleviate cancer and treatment induced side effects. furthermore these drugs may have direct anti-tumor effects, potentially via inhibition of dopamine d receptors (drd ). objectives: determine the anti-cancer effects of atypical apds on gbm stem-like cells design/method: the anti-cancer effects of apds (quetiapine and risperidone) were evaluated on gbm stem-like cell lines developed in our laboratory (glio and ) and the group medulloblastoma cell line hdmbo . cell proliferation/viability was determined using trypan blue exclusion and mts assays. the effect of apds on cancer stem cell self-renewal was determined by neurosphere assay. receptor expression and apds effect on cell cycle proteins were examined by western blot analysis. results: western blot analysis of gscs and hdmbo demonstrated robust drd expression indicating a viable therapeutic target. both apds induced dose dependent cell death of all cell lines tested. treatment with only um of either apd for days significantly reduced cell proliferation by % (hdmbo ) and - % (gscs). consistent with these findings, we observed an increase in cell cycle inhibitors p and p . furthermore at day both apds induced a robust increase in gsc death, approximately % compared to only % in non-treated controls. lastly, um apds significantly reduced gsc neurosphere formation compared to untreated controls by up to % suggesting inhibition of gbm stem cell self-renewal. our data indicates that clinically relevant concentrations (low micromolar) of these apds induce anticancer effects in both gscs, which are enriched with tumor initiation/propagation properties, and in the group (myc amplified) medulloblastoma cell line. these apds represent strong candidates as potential adjuvant therapies for the treatment of these brain tumors. background: while the poor prognosis for high risk neuroblastoma (hrnb) underscores the need for new treatment strategies, the elucidation of specific biologic subsets of neuroblastoma suggests a way to improve disease management. the identification of agents that target specific molecular pathways associated with the development or progression of diseases holds promise. dfmo, an inhibitor of odc, has been shown to decrease lin and mycn and target cancer stem cells in preclinical studies. currently % of patients undergoing immunotherapy relapse. dfmo is in studies to prevent relapse after immunotherapy and may be helpful during immunotherapy as well. the hypotheses for this study were that: ) the incorporation of a targeted therapy, selected based upon upfront tumor genomic interrogation, into standard induction chemotherapy for hrnb is safe, feasible and may increase the pr/cr/vgpr response rate at the end of induction therapy; and ) the addition of dfmo as maintenance during immunotherapy is safe and feasible and may decrease the relapse rate for hrnb. a multicenter feasibility pilot trial in subjects with newly diagnosed hrnb within the beat childhood cancer consortium. at diagnosis, patients' tumors underwent dna exome and rna sequencing which were analyzed within a molecular tumor board to identify the single best drug of targeted agents to be added to cycles - of induction chemotherapy. after consolidation with asct and radiation, the patients received dfmo along with standard dinutuximab and retinoic acid and dfmo for years after immunotherapy. patients were evaluated for additional toxicities with the addition of targeted agents and dfmo in addition to induction response. results: the pilot study of eligible patients has shown this process to be feasible. all patients have completed induction portions of the study. the combination of targeted agent with chemotherapy was shown to be safe without any unexpected toxicities. delays between induction cycles were < weeks and related to surgery, infection, or thrombocytopenia. the induction response demonstrated % cr/vgpr/pr rate, which suggests improvement over historical %. in addition, patients were eligible for the combination of dfmo with dinutuximab and retinoic acid was well tolerated and safe without additional toxicities due to dfmo. the pilot study of patients has shown the process of genomic sequencing and addition of a targeted agent to upfront chemotherapy and addition of dfmo to dinutuximab and retinoic acid maintenance therapy in newly diagnosed hrnb patients and is feasible and safe without any unexpected toxicities. background: identifying sub-populations of medulloblastoma tumors with stem cell-like properties holds promise for reducing disease recurrence, but there is no known unifying marker of medulloblastoma cancer stem cells. the granulocyte stimulating factor receptor (gcsf-r or cd ) is well understood in the context of hematopoiesis, but its role in solid tumor pathogenesis is less clear. neuroblastoma and melanoma subpopulations expressing gcsf-r have cancer stem cell properties of chemoresistance and increased tumorigenicity, and are enriched in tumors after chemotherapy. gcsf-r activation leads to signaling through the jak-stat pathway, suggesting a potential therapeutic target. we hypothesized that a subpopulation of medulloblastoma cells would express the gcsf-r and that this subpopulation would demonstrate chemoresistance and response to inhibitors of the jak/stat pathway. objectives: our objective was to identify a subpopulation of medulloblastoma cells expressing the gcsf-r and determine their relative growth rates, tumorigenicity, and responses to chemotherapy and jak/stat inhibition. design/method: medulloblastoma cell lines were sorted via flow cytometry for gcsf-r surface expression. subpopulations of gcsf-r-positive and -negative medulloblastoma cells were then monitored for growth by continuous live cell imaging. responses to chemotherapy were measured in subpopulations of gcsf-r-positive and -negative medulloblastoma cells using continuous live cell imaging to measure percent cell confluence and cell viability assays. ic values were calculated for each cell line and each agent. parental medulloblastoma cell lines and isolated gcsf-r-positive and -negative subpopulations were also treated with the jak / inhibitor ruxolitinib and growth rates, viability, and ic values were calculated. results: gcsf-r surface expression was identified on . - . % of medulloblastoma cell lines. isolated gcsf-r positive cells demonstrate a slower growth rate compared to gcsf-rnegative or parental unsorted medulloblastoma cells. gcsf-r positive cells are more resistant in vitro to vincristine, etoposide, and carboplatin, when compared to the gcsf-r negative population and an unsorted population of the same cell line. ruxolitinib is cytotoxic to medulloblastoma cells in vitro, with higher ic values noted in gcsf-r positive cells compared to unsorted and gcsf-r negative cells. we show that a subpopulation of gcsf-r positive cells are present in multiple medulloblastoma cell lines via flow cytometry, and that isolated gcsf-r-positive cells have a slower growth rate than gcsf-r-negative or unsorted populations. we also show that ruxolitinib has in vitro activity against medulloblastoma cell lines. we propose that jak inhibition may represent an adjunct therapy targeting overall tumor burden and specifically targeting the gcsf-r-positive subpopulation of medulloblastoma cells that may drive tumor recurrence. we investigated the efficacy of intensified adjuvant chemotherapy in osteosarcoma patients. design/method: we retrospectively analyzed the medical records of children with osteosarcoma treated at asan medical center between and . all patients received a -drug induction consisting of cycles of cisplatin and doxorubicin along with cycles of methotrexate (map), and proceeded to surgical resection. adjuvant ct was map or map with the additional ifosfamide and etoposide (mapie), and mapie was mainly considered for poor responders (tumor necrosis below %) or patients with metastases. results: among patients, patients had metastases at diagnosis. surgery was conducted in patients who responded to induction ct, and showed over % tumor necrosis. among patients who proceeded to adjuvant ct, and patients received to map and mapie protocols. with a median follow-up of months, the -year overall survival (os) and event-free survival (efs) rates of all patients were % and . %. of those patients, patients recurred, and of them died of disease progression. relapsed patients received salvage ct and/or surgery, and were rescued after autologous stem cell transplantation (sct). three patients developed treatment-related acute myeloid leukemia, and they are alive after allogeneic sct. according to the response to neoadjuvant ct, the os rates of good responders (n = ) and poor responders (n = ) were % and . % (p = . ), and efs rates were . % and . % (p = . ). of the poor responders, patients received map as adjuvant ct, and the other received mapie. the os rates of map and mapie group were . % and . % (p = . ), and efs rates were . % and . % (p = . ), respectively. when patients were classified into three groups: . localized disease & necrosis ≥ % (n = ), . localized disease & necrosis < % (n = ), . metastatic disease (n = ), survival rates were in the order of group > > (os = %: . %: . %, efs = . %: . %: %). in each group, intensified adjuvant ct by mapie did not improve survival outcomes. conclusion: initial metastatic disease and poor histological response to neoadjuvant ct were major risk factors for poor survival in osteosarcoma patients. we found that adding ifosfamide and etoposide to map did not improve survival outcomes of patients with adverse risk factors. more effective adjuvant therapy for these patients is needed. background: circulating cell-free dna (cfdna) that shed from tumors into circulation have been used for noninvasive molecular profiling in adult cancers but little is known about its utility in pediatric cancers. pediatric patients with metastatic and refractory solid tumors are known to have poor survival rates, and a key challenge in their management is obtaining biopsy samples especially at times when disease is widely spread or the patient is physically unfit for sampling. the development of a noninvasive profiling strategy is critical for optimizing molecularly guided therapy and assessing response to treatment. in this study, we want to determine the utility of cfdna to noninvasively analyze the molecular profiles of pediatric solid tumors such as neuroblastoma (nb), osteosarcoma (os), and wilms tumor (wt). design/method: tumor, plasma, and matched controls were collected from patients with nb, wt, and os, at diagnosis or time of disease progression. cfdna was extracted from the plasma and analyzed through multiple methodologies including a targeted next generation sequencing panels and shallow whole genome sequencing (swgs). results: fifteen nb patients, os patients, and wt patients had tumor molecular profiles known from different targeted next-generation sequencing platforms. in the cfdna of / nb patients, somatic mutations and copy number alterations previously reported in the tumors were detected, including recurrent nb drivers such as mycn amplification, alk, and atrx mutations. mutations not detected in the original tumor were also found in / nb patients including nras, mll , arid b, some of which are potentially actionable. in os, mutations known from the tumor were found in the cfdna of of patients, including atrx and notch mutations, as well as copy number alterations such as cdk amplification, which has targetable therapeutics available. of the two wt patients analyzed, cfdna revealed the same mutations as tumor in one patient, however in a cohort of patients where tumor was not available, cfdna revealed recurrent driver mutations such as amer , dicer . it is feasible to noninvasively identify somatic mutations and copy number alterations in cfdna of patients with pediatric solid tumors. establishing a platform using cfdna to identify molecular profiles of these tumors can serve as a powerful tool for guiding treatment and monitoring response to treatment. background: despite multi-modality therapy, the prognosis for patients with metastatic osteosarcoma remains poor necessitating development of novel targeted therapies. immunotherapy can be exploited to target osteosarcoma with exquisite specificity but remains limited by insufficient tumor specific targets. objectives: to overcome the dearth in tumor specific antigens, we have explored the use of tumor derived mrna (representing a tumor specific transcriptome) for development of personalized nanoparticle vaccines. design/method: rna-nanoparticles (rna-nps) can be amplified from limited amounts of biopsied tissue for induction of tumor specific t cells against osteosarcoma. since local vaccination strategies are mired by poor overall immunogenicity, we assessed the feasibility, immunogenicity and antitumor activity of intravenously administered rna-nps (tumor mrna complexed to dotap nanoliposomes) in pre-clinical murine and canine tumor models. we identified a clinically translatable np formulation for the delivery of rna to antigen presenting cells (apcs) that induces in vivo gene expression and preserves rna stability over time. tumor derived rna-nps induced antigen specific t cell immunity and mediated anti-tumor efficacy in several pre-clinical solid tumor models (i.e. b f , kr b). when administered intravenously, rna-nps increased expression of co-stimulatory molecules (i.e. cd , cd , cd , ccr ) and pd-l on cd c+ cells throughout reticuloendothelial organs (i.e. spleen, liver, bone marrow) and within the tumor microenvironment; this phenotype was strictly dependent on type i interferon. targeted inhibition of type i interferon signaling (via infar mabs) abrogated anti-tumor efficacy mediated by rna-nps. we enhanced the immunogenicity of this platform by simply combining mrnas encoding for immunomodulatory molecules (i.e. hcv-pamps, gm-csf) or by combining rna-nps with immune checkpoint inhibitors. addition of checkpoint inhibitors (pd-l mabs) to rna-nps increased tumor infiltrating lymphocytes, and intratumoral mhc class i/ii expression, and mediated synergistic anti-tumor activity in settings where pd- or pd-l inhibition alone did not confer therapeutic benefit. we then explored the feasibility of rna-nps in a large animal osteosarcoma model. in ongoing studies for canines with osteosarcomas, we have shown that sufficient amounts of rna can be extracted, amplified, and manufactured into personalized rna-np vaccines. conclusion: rna-nps reprogram systemic immunity and mediate anti-tumor activity providing near immediate immune induction without the complexity of cellular immunotherapy. the immune correlate of preclinical response to rna-nps is hallmarked by interferon dependent pd-l expression on activated apcs (cd c+ mhcii+ cd + cells). based on these findings, we are exploring the preclinical safety, efficacy and immunologic effects of rna-nps targeting canine osteosarcoma before first in-human evaluation. background: ewing sarcoma is an aggressive bone tumor affecting mainly adolescent and young adults. treatments are based on compressed schedule chemotherapy combined with local control (surgery and/or radiation). prognosis is poorer for patients with metastatic disease, older age and central primaries. survival when disease recurs within two years of diagnosis is < %. the ews-fli fusion gene t( ; ) (q ; q ) has been well characterized as a dominant ews driver-gene. the most common variation is ews exon with fli exon ( % of fusion positive patients). we designed a novel pbi-shrna tm ews/fli type lpx which has demonstrated, safety and efficacy in animal model (rao et all). the pbi-shrna strategy silences target gene expression by concurrently inducing translational repression and p-body sequestration as well as post-transcriptional mrna cleavage. to determine the safety and maximum tolerated dose of intravenous administration of pbi-shrna tm ews/fli type lipoplex in patients advanced ews. design/method: phase i study × escalation cohort. testing pbi-shrna tm ews/fli type lpx (starting iv dose of . mg/kg) on patients (≥ age ) with advanced ewing's sarcoma, all with a type translocation. intravenous infusion was given twice a week for weeks with the following escalation schema: % → % → % → % → %. required kps > % and adequate organ function. cytokines induction pre and post-infusion was analyzed (il- , il- , tnf-alpha, il ra). first cohort of patients has been enrolled (ages between - years). three relapsed patients had > lines of therapy and patient had refractory disease, patients received a complete cycle of pbi-shrna tm ews/fli type lpx with twice a week infusions. a total of doses were given. the most prominent related toxicity has been hematological, patient developed transient g neutropenia, another patient developed g anemia that required prbc transfusion, and of note this patient had significant bone and bone marrow involvement. one patient only received two lpx infusions; she developed a fatal rsv pneumonia. other reported grade toxicity includes fatigue and headache. evaluable patients (n ) had stable disease between and months before progression. one patient had sustained response for month before progression, two patients are still alive. our preliminary experience supports the safety and potential efficacy of pbi-shrna tm ews/fli type lpx as novel treatment for advanced ews with limited toxicity. il- increase correlates with higher bi-shrnai ews/fli lpx infusion rate and clinical symptoms. further clinical testing is indicated. background: as more children with cns malignancies (bt) are surviving, the late effects of the therapies they receive are better described. studies show that radiation therapy is particularly harmful to neurocognitive functioning, specifically processing speed, working memory, and attention span. these deficits have negative effects on quality of life, especially in academic and professional settings. a large proportion of s of s adult survivors of bt are unable to reach adult milestones such as living on their own, holding a steady job, and getting married. proton beam radiation therapy (pbrt), is touted for the potential to have fewer and less severe side effects than traditional photon radiation therapy (xrt). because of the properties of protons, the amount of damaging energy released in non-target healthy tissue is reduced when compared to xrt. although a study comparing iq testing between pbrt and xrt found no difference between the two therapies, no studies have compared the specific neurocognitive domains. it would be valuable to evaluate full neurocognitive testing scores (nct) since the specific domains, particularly processing speed (psi), appear to be most vulnerable to radiation therapy. objectives: our primary aim was to assess differences in psi for patients with bt who underwent pbrt versus xrt. a secondary aim was to assess differences in iq (fsiq) and working memory (wmi). we retrospectively evaluated all patients treated for bt at the jimmy everest cancer center within the past years who received rt and had nct post radiation. we examined the full nct results for both subsets of participants to evaluate differences in the specific domains of processing speed, working memory, and iq by measuring percentiles scored in these domains. objectives: we report our experience on imaging children with mm treated uniformly on an institutional melanoma trial. we retrospectively reviewed the clinical and imaging findings of patients with ajcc stage iic-iv cutaneous mm treated on our institutional mel protocol. brain mri/ct, pet/ct, ct chest, abdomen, and pelvis (ctcap) were performed at diagnosis in all patients. on treatment, stratum a patients (peg-interferon; ajcc iic, iiia, iiib) (n = ) had the same imaging repeated every months; stratum b (peg-interferon and temozolomide; unresectable measurable disease metastatic, or recurrent) (n = ) had pet scans every months and brain imaging every months; those in stratum b (peg-interferon and temozolomide; unresectable non-measurable, metastatic, or recurrent) (n = ) had the same imaging performed every months. off therapy all patients continued same imaging every months for years. results: there were patients ( female; median age years). eleven had spitzoid and conventional melanoma. primary sites included head/neck (n = ), trunk (n = ), and extremities (n = ). patients with spitzoid melanoma had imaging studies ( pet, ctcap, ct chest, ct brain, and mri brain) with a median of , , , and studies/patient respectively. median cost per patient was $ , . thirteen studies ( . %) showed suspicious lesions with additional scans and diagnostic biopsies of which one only was positive stratum a with tert promoter mutation and died from disease). for conventional mm, studies ( pet, ctcap, ct chest, ct brain, and mri brain) were performed with a median of , . , , , studies/patient respectively. median cost per patient was $ , . twenty ( %) showed suspicious lesions with additional scans and diagnostic biopsies; four were positive (two at diagnosis); both died of disease; the other two recurred locoregionally and were detected clinically; both are alive and disease free; one patient had diffuse metastases and died shortly after enrollment. after a median follow up of . years (range . - . ) patients are alive and disease free. children with spitzoid melanoma should have minimal imaging at diagnosis and follow-up given the low risk of recurrence and low yield and high cost of aggressive imaging protocols. patients with conventional mm should be imaged according to the adult guidelines. nationwide children's hospital, columbus, ohio, united states background: the role of infections in the long term outcome of patients with bone tumors is controversial. two retrospective studies have shown increased survival in osteosarcoma patients who had a post-operative wound infection, while another showed no changes in overall survival. to determine the relationship between wound infections and/or bloodstream infection (bsi) on survival in pediatric and young adult patients with osteosarcoma and ewing sarcoma treated at a tertiary children's hospital. design/method: a retrospective chart review was performed for patients with diagnosis of osteosarcoma or ewing sarcoma from - . patients received standard chemotherapy regimens for their disease type and stage. local control included surgical resection and/or radiation therapy. presence of infection was determined by bsi or wound cultures while receiving treatment for primary tumor. the median age of patients was (range - years) at diagnosis. % had a diagnosis of osteosarcoma and % had ewing sarcoma. of these, % of patients developed an infection during treatment; % had bsi, % had wound infections, and % had both. patients with bsi had a year os of . %, compared to % in those without bsi (p = . ). those with both bsi and wound infections had the poorest overall survival of %, compared to . % for patients without any infection. patients with wound infections alone had a year os of . %, compared to % of patients without a wound infection. our analysis revealed decreased os in patients with bsi; however, this could be due to other confounding factors in the presence of bsi. those with bsi or bsi and wound infections had the poorest survival. wound infections without bsi were associated with a slight increase in survival; however, this study was limited by the number of patients that had local wound infections. with the use of newer surgical techniques, availability of antimicrobials and routine use of prophylactic antibiotics, the incidence of infections while undergoing treatment is low. however, the importance of this clinical observation indicates a likely enhanced immune system associated with infection, supporting the role of immunotherapy for treatment of these aggressive tumors. background: hypoalbuminemia is a well-recognized effect of cancer and other chronic illnesses and is often regarded as a marker of malnutrition. in adults, hypoalbuminemia has been associated with adverse outcomes in patients with cancers of the lung, pelvis, head and neck, gastrointestinal tract, and bone marrow, as well as in some pediatric patients with ewing sarcoma and hodgkin lymphoma. hypoalbuminemia has not been well studied in children with cancer. to determine the incidence of hypoalbuminemia (using age-specific references) in children with cancer receiving chemotherapy at baseline (prior to starting chemotherapy) and to determine whether hypoalbuminemia is associated with inferior -year overall survival. design/method: we performed a single institution, irbapproved, retrospective review of pediatric oncology patients diagnosed between and . five-year survival was estimated using the kaplan-meier method; groups were compared using cox regression. we identified pediatric patients with a first diagnosis of cancer, brain tumor, or other condition possibly requiring chemotherapy. of these patients, were excluded for reasons including not receiving chemotherapy and missing data, leaving patients who had a serum albumin level within days prior to starting chemotherapy. the mean age was . years (sd . years); % were male; % were non-hispanic. the most common diagnosis was acute lymphoblastic leukemia ( of ; %). one hundred thirty nine of ( %) had hypoalbuminemia prior to starting chemotherapy. there was no statistically significant difference in -year overall survival between those with and without hypoalbuminemia ( % vs. %, respectively; hazard ratio . , % c.i. . - . ). conclusion: hypoalbuminemia at baseline in pediatric oncology patients requiring chemotherapy is common (one in five), and was not associated with inferior -year overall survival in this cohort. leptomeningeal metastases at diagnosis. standard treatment for completely resected, non-anaplastic supratentorial ependymomas is close observation. treatment for anaplastic or incompletely resected non-anaplastic ependymomas is maximal safe surgical resection followed by focal radiation. however, up to % of localized ependymomas recur. the role of chemotherapy in treating ependymomas is under investigation. extraneural metastases of anaplastic ependymomas have rarely been reported and the outcome is dismal. objectives: to report extraneural cervical node metastases of a non-anaplastic ependymoma and successful treatment with surgical resection, radiation, and systemic chemotherapy. design/method: retrospective review of patient medical records, including radiographic imaging and tumor tissue pathology, and comprehensive literature review. results: a previously healthy -year-old girl underwent gross total resection (gtr) of an isolated right parietal lobe ependymoma (who grade ii). at age years, magnetic resonance imaging (mri) revealed an isolated localized recurrence. she underwent gtr followed by observation. at age years, she again experienced isolated localized recurrence and underwent gtr followed by . gy focal conformal photon radiation. at each recurrence, pathology revealed a non-anaplastic ependymoma, and cerebral spinal fluid (csf) cytopathology and spine mri were negative. at age years, she developed an enlarged right posterior cervical chain lymph node. subsequent mri revealed a large rim-enhancing, t hyperintense lymph node and multiple abnormally enhancing regional nodes consistent with metastases. biopsy revealed a non-anaplastic ependymoma. mri of the brain and spine, computed tomography of the chest, abdomen, and pelvis, and csf and marrow evaluations were unremarkable. chemotherapy according to acns was initiated. mri after course demonstrated significant node size reduction. she underwent right neck node dissection. only one right level ii lymph node showed metastases. she was treated with . gy irradiation to the neck and additional courses of chemotherapy. she remains in remission months and months after diagnosis of metastatic disease and end of therapy, respectively. literature review reveals rare reports of extraneural metastatic disease of anaplastic ependymomas to bone, lung, or liver, and only involving lymph nodes, all associated with a poor outcome despite multimodal therapy. to our knowledge, this is the first report of extraneural metastases of a non-anaplastic ependymoma. extraneural metastases should be considered in children previously treated for non-anaplastic ependymomas who experience systemic symptoms, even in absence of cns relapse. multimodal treatment offers potential long-term disease control with acceptable toxicity. arun gurunathan, joel sorger, andrew trout, joseph pressey, rajaram nagarajan, brian turpin cincinnati children's hospital medical center, cincinnati, ohio, united states background: pigmented villonodular synovitis (pvns) is a benign neoplasm of the synovium. standard treatment is surgery, but post-operative recurrence rate is as high as %. radiation therapy can be used for local control, but is associated with late effects. while pvns is rarely fatal, aggressive disease and/or extensive surgery can result in substantial functional impairment. colony stimulating factor- (csf ) overexpression, often due to chromosomal translocation involving csf , drives pvns through recruitment of synovial-like mononuclear cells expressing the csf -receptor. tyrosine kinase inhibitors such as imatinib are active against the csf -receptor, and have shown benefit in the post-surgical relapse setting. however, questions remain regarding the broader application of imatinib and regarding optimal response assessment. to present three patients with pvns, each with different clinical scenarios, who demonstrate clinical response to imatinib monitored by changes in metabolic activity (maximum suv) on pet/ct. results: three patients with pvns demonstrate pet/ct response to imatinib, guiding management of their challenging clinical scenarios. patient is a year-old female with left hip pvns and high grade articular cartilage loss, with decrease in metabolic activity (suvmax . to . in months) on neoadjuvant imatinib, enabling total hip replacement surgery planning. patient is a year-old female with left knee pvns with recurrences after synovectomies, spared subsequent surgical control attempts after clinical improvement correlating with pet/ct response to imatinib (suvmax . to . in months). patient is a year-old male with right knee pvns that recurred after total knee replacement, now with clinical improvement correlating with pet/ct response to imatinib (suvmax . to . in months). all patients would have been characterized as stable disease by response evaluation criteria in solid tumors (recist). in each of these patients, imatinib has been tolerated well, with no therapy interruptions and absent or easily managed side effects (one patient takes dronabinol for decreased appetite, one patient takes prn immodium for diarrhea). all patients are currently still taking imatinib, with therapy length ranging from five to eleven months. in our series of three patients with pvns, imatinib shows promise for disease management in neoadjuvant and adjuvant settings with a tolerable side effect profile. imatinib should be considered in the treatment of pvns to spare surgical and radiotherapy related morbidity, and treatment effect can be monitored by pet/ct. background: metastatic rhabdomyosarcoma (rms) carries a poor prognosis with three-year event free survival rates ranging between %- % (depending on oberlin risk factors) due to the lack of significantly effective breakthroughs in the recent past. there is an urgent and unmet need for new treatment strategies against this disease. metastatic rms cell lines exhibit increased expression of the erm family membrane-cytoskeleton linker protein ezrin. knockdown of ezrin expression using sirnas decreases the metastatic potential of these cells, whereas forced expression of ezrin results in increased degree of metastasis. the activity of ezrin is controlled by its phosphorylation at the threonine (thr ) residue at the c-terminus of the protein, suggesting that alteration of ezrin phosphorylation may control rms growth and metastasis. our goal was to determine if pharmacological inhibition of thr phosphorylation in ezrin affects the growth, survival and metastasis in rms in vitro as well as in vivo. design/method: rms cell lines representative of the alveolar and embryonal histological subtypes were used. rms cells were treated with a small molecule inhibitor of ezrin, nsc , which specifically dephosphorylates ezrin at the thr residue. baseline expression of ezrin and perm levels as well as the effect of nsc on perm levels in the rms cell lines was determined by western blotting of cell lysates. viability of cells was assessed by trypan blue exclusion, and morphology visualized by bright field microscopy. the extent of apoptosis was detected by imaging caspase / activation using fluorescent microscopy. motility of rms cells was examined by performing a wound-healing assay. subcutaneous and orthotopic xenografts were established in nsg mice using rd cells (embryonal rms). mice harbor-ing xenografts were treated with intraperitoneal injections of nsc or dmso. results: ezrin is constitutively phosphorylated at the thr residue in a majority of the rms cell lines examined. nsc dephosphorylates ezrin at the thr residue in these cell lines. treatment with nsc inhibits growth, induces apoptosis and inhibits the migration of rms cell lines in vitro. further, treatment of nsg mice bearing subcutaneous or orthotopic embryonal rhabdomyosarcoma xenografts with nsc significantly impedes tumor progression without any obvious adverse effects. our findings suggest that dephosphorylation of ezrin at the threonine residue may have the potential to be a novel therapeutic strategy for rms patients. all india institute of medical sciences, new delhi, new delhi, delhi, india background: the role of laparoscopy in the management of pediatric intra-abdominal solid tumors is yet to be established. the safety of laparoscopic management of pediatric intra-abdominal tumors is still questionable. we study the results of the initial case series of pediatric intraabdominal tumors managed laparoscopically at our institute from july onwards. design/method: total children ( -males, females) who presented to us with pediatric intra-abdominal tumors were included. the tumors included wilms tumor (n = ), neuroblastoma(n = ), adrenal cortical tumor(n = ), ovarian teratoma(n = ).children were between months - years and received neo-adjuvant chemotherapy. a -port laparoscopic nephrectomy and lymph node sampling for wilms tumor and adrenalectomy for adrenal tumors was performed. the tumors were removed in-toto with no rupture (except in one). specimens were retrieved through a lumbar incision (n = ) or an inguinal incision(n = ). all the children are under regular follow up. two children with wilms tumor had recurrence. the neuroblastoma child underwent open surgery for recurrence later. conclusion: laparoscopy/laparoscopic assisted removal of pediatric intra abdominal tumor is a feasible and safe option. it has the advantage of less postoperative pain, shorter hospital stay and a better cosmetic result. proper patient selection, port placement and laparoscopic experience are contributory. background: targeting of proteins and cell surface antigens specific to cancer cells with monoclonal antibodies has proven to be an effective form of treatment in many forms of cancer. gd is a cell surface disialoganglioside that is expressed on the cell surface of some normal tissues including nerve cells, melanocytes, and mesenchymal stromal cells and is overexpressed in some pediatric cancers like neuroblastoma and osteosarcoma. dinutuiximab is a chimeric monoclonal antibody that is fda approved for the treatment of patients with high risk neuroblastoma and under investigation for the treatment of relapsed osteosarcoma. little is known about the patterns of gd expression in other pediatric malignancies. objectives: we sought to describe the patterns of gd expression in the following pediatric sarcomas: synovial sarcoma, rhabdomyosarcoma and ewing sarcoma. design/method: synovial sarcoma (n = ), rhabdomyosarcoma (n = ) and ewing's sarcomas (n = ) formalin fixed, paraffin embedded cores were obtained from the seattle children's research institute tissue microarray (tma) biorepository. tma blocks consisting of melanoma cores stained with and without gd antibody were used as positive and negative controls, respectively. slides were incubated with anti-ganglioside gd antibody clone q (ab from abcam) diluted : in % normal goat serum and % bsa in tbs overnight at ˚c. the negative control of human melanoma section was incubated in % normal goat serum and % bsa in tbs without primary antibody. the expression of gd was indicated by characteristic brown diaminobenzidine staining. the intensity and location of tissue staining were assessed and compared to positive and negative controls. staining was considered positive (+++) if the intensity of the staining was consistent with that of the positive control with - % of cells staining positive. classification of intermediate gd expression (++) was assigned to slides in which - % of cells stained positive. slides were classified as sporadic staining (+) if - % of cells stained positive. tissue was considered (-) if there was complete absence of staining, similar to the negative control. objectives: to evaluate the clinical presentation, management and treatment outcomes of children with malignant germ cell tumor at our institute design/method: a prospective study was conducted from june to dec in the department of pediatric surgery in a tertiary care institute in a developing country. all patients were evaluated for local disease and metastatic disease by imaging and tumor markers. risk stratified chemotherapy was used with low risk tumor receiving no chemotherapy, intermediate risk: courses of peb chemotherapy and high risk: courses of peb + courses of pe. upfront resection of the primary or the residual disease after neoadjuvant chemotherapy if feasible was performed. follow up was done with monthly tumor markers for months and imaging studies every - months for initial years. five year overall survival and disease free survival was calculated. results: during the study we treated children who formed the study group. of these ( %) were gonadal ( ; % testicular and ; % ovarian) and the remaining ( %) were extragonadal with sacrococcygeal (sct) being the most common site ( %). one hundred and thirteen children ( %) presented to us primarily while the remaining had received treatment elsewhere. stage or stage disease at presentation was present in ( %) children. recurrence was noted in ( %) patients. respectively. patients with testicular mgct and children with age - years and males had significantly poor rfs rates. conclusion: patients with mgct should be staged correctly and adjuvant chemotherapy is advisable to all patients except stage i endermal sinus tumor of testis. awareness regarding the same is still lacking in our country. meticulous follow up is needed as more than % of will recur. cure rates are dismal in children with recurrent mgct especially those who are not chemotherapy naïve. nemours children's specialty care, jacksonville, florida, united states background: radiotherapy for pediatric head and neck tumors often results in mucositis, limiting oral intake and compromising patients' nutritional status. this may be reduced through the improved conformality offered by proton therapy. despite widespread use of enteral tube feeding through a percutaneous gastrostomy (peg) or nasogastric tube (ngt), there is little data available regarding overall incidence of ngt/peg placement and perspectives of pediatric patients and caregivers. objectives: to (a) estimate the need for ngt/peg support and (b) characterize patient and caregiver perceptions surrounding enteral feeding in children with head and neck tumors undergoing proton therapy. design/method: dependent on development stage, patient (n = ) or parents (n = ) filled out a series of customized surveys according to a prospective irb approved study. seventythree percent of patients also received concurrent chemotherapy. questions addressed their current feeding route and perception, for example, "what aspect(s) of tube feedings are beneficial to you?" and "what aspect(s) of tube feeding worry or scare you?" fifty-five surveys were distributed before and after radiation, and with any change in feeding route. results: at the start of proton therapy, patient had a ngt and patients had peg. of these, patients ( %) had a ngt/peg in place exclusively for the administration of medication; only patient ( %) needed a ngt/peg for nutrition. in those patients without ngt/peg, % would "consider" enteral feeds. in patients without ngt/peg, the most commonly cited benefit was "maximizing my nutrition" ( %) and the most common negative aspect was "fear" of tube placement ( % of patients). all sub-populations ( % of patients) cited change in appearance as a negative aspect. in patients without ngt/peg at the start of proton therapy, % of patients/caregivers felt enteral feeding to be "unnecessary," and % of these patients would not "consider" ngt/peg even if their "physician advised it." over the course of proton therapy, the patients/caregivers who deemed enteral feeding "unnecessary" decreased from % to %. at completion of treatment, patients ( %) were using a ngt/peg tube for nutritional support but only one ( %) patient relied exclusively on their enteral feeds. two patients (without ngt/peg) ( %) required parenteral support. our data does not support prophylactic placement of ngt/peg in of children with head and neck tumors undergoing proton therapy. ongoing research is needed to identify which patients will need ngt or peg to supplement their diet. in this cohort, anticipatory counseling should focus on pain, cosmesis, and utility. children's national medical center, washington, district of columbia, united states background: ovarian sex cord-stromal tumors (osct) are rare neoplasms that typically present with signs/symptoms of an adnexal mass and signs of hormonal production approximately % of ovarian sex cord-stromal tumors in children are sertoli-leydig cell tumors (slct) with median age of presentation years overall. to our knowledge the youngest reported case in the literature describes a -month old female in china with a slct that was treated with oophorectomy alone. some studies have found an association in families between pleuopulmonary blastoma and osct with a germline mutation leading to dicer syndrome, which has been associated with a younger age at diagnosis. , objectives: to describe an unusual case presentation of slct in an infant results: -month old, twin female, ex- week premature infant presented to the emergency department on multiple occasions for abdominal distention and feeding intolerance initially thought to be related to previous omphalocele repair and umbilical hernia. an ultrasound demonstrated an × cm mass arising from the right ovary with large volume ascites. she required admission to the intensive care unit due to s of s respiratory distress from her significant ascites. serum tumor marker including hcg, afp and ldh were negative. patient underwent right oophorectomy with tumor capsule noted to be open at time of surgery. further imaging post operatively demonstrated no other sites of disease. the patient was classified as figo stage ic due to the presence of her significant abdominal ascites that was presumed to be malignant pre-operative tumor rupture. the pathological diagnosis was challenging and eventually resulted as a mixed germ cell sex cord stromal tumor with pattern of sertoli cell tumor with neuroendocrine differentiation. based on the staging of figo ic with pre-operative rupture, the decision was made to treat with a standard platinum based regimen as there is a higher incidence of relapse in stage ic patients when compared to ia treated with observation alone. our patient tolerated four cycles of chemotherapy well and end of therapy scans showed no evidence of disease. interestingly, her dicer mutation genetics performed by ion torrent tm next generation sequencing was negative in germline and tumor studies. to our knowledge, our patient is the youngest described with slct. she will continue to be followed with serial imaging alone as she had no evidence of elevated tumor markers at diagnosis. , due to young age and unusual diagnosis, she was referred to cancer genetics team. background: approximately % of patients with wilms tumor (wt) have metastatic disease at diagnosis and often have a grave prognosis. limited cell lines are available for the study of metastatic wt and long-term passaged cell lines do not always recapitulate the human condition. focal adhesion kinase (fak) is a non-receptor tyrosine kinase that controls cellular pathways involved in the tumorigenesis of pediatric renal tumors. using a novel patient-derived xenograft (pdx) model from a patient's primary wt (coa ) and matched isogenic metastatic wt (coa ), we previously demonstrated that fak is expressed and its inhibition led to decreased tumorigenicity of both the primary and metastatic pdxs. kinomic profiling is an innovative, high-throughput method used to investigate kinase signaling to identify potential therapeutic targets. to date, the kinomic profile of primary and metastatic wt has not been examined. objectives: investigate baseline kinomic differences between primary and metastatic wt and evaluate kinases upstream and downstream of fak as potential targetable therapies. design/method: cells from coa and coa were treated with pf- , (pf), a small molecule fak inhibitor. protein from cell lysates of treated and untreated coa and coa were combined with kinase buffer, atp, and fluorescently labeled antibodies and loaded into a phosphotyrosine kinase or serine-threonine kinase pam-chip® per the uab kinome core protocol. phosphopeptide substrate analysis with the pamstation® kinomics workstation (pamgene® international), pamchip® protocol using evolve software, and bionavigator v. . were used to analyze kinases upstream and downstream of fak. the primary wt had increased epha , ror sgk and decreased pdgfrb relative to the paired metastatic wt at baseline. treatment with pf increased ron, pdgfrb, p s kb, mak, camk g, vacamkl, camk d, ck a and pskh in the primary wt. treatment with pf decreased tnk , lmr , cck , epha , pdk , sgk , lkb and increased pskh in the paired metastatic wt. primary wt displayed a different kinomic profile compared to metastatic wt in a matched isogenic pdx model. these data reveal that alternative therapies to specifically target metastases are needed. furthermore, fak inhibition resulted in diverse kinomic alterations between primary and metastatic wt. inhibitors targeting many of these pathways, such as pdgfrb inhibitors, are currently available and potentially could be combined with fak inhibitors in the treatment of wt. the results of the current study indicate that kinases upstream and downstream of fak in primary and metastatic wt warrant further investigation. background: use of high-dose methotrexate (hd-mtx, g/m^ ) is a mainstay of standard therapy for pediatric osteosarcoma (os) in north america. in pediatric os, there is a narrow therapeutic window for hd-mtx, with decreased tumor response rate with mtx concentrations < m and decreased survival due to severe toxicity with concentrations > m. risk factors for hd-mtx toxicity have been defined in adults, including body mass index (bmi) and male gender, but such studies have not been conducted in children. we sought to examine the relationship between mtx levels and toxicities during hd-mtx infusion for pedi-atric os, thereby identifying risk factors for increased toxicity and providing a framework for therapeutic drug monitoring. design/method: this retrospective chart review included patients treated at texas children's hospital with hd-mtx as first-line therapy for os from - . data abstracted from electronic records included patient characteristics, bmi and body surface area (bsa), baseline and post-treatment laboratory values, mtx levels and hours after dose given ( h, h), hour mtx cleared (mtx < . um), grade / mucositis, myleosuppression, persistent lft elevation (ctace v . ), and % tumor necrosis. correlation between h mtx level and other covariates was summarized using descriptive statistics. we reviewed hd-mtx infusions corresponding to patients. bmi was found to significantly impact h mtx level (p< . ). female gender was also significantly associated with higher h mtx level (p< . ). percent necrosis (available in patients) was associated with h mtx levels at near-statistical significance (p = . ). h mtx level was not found to contribute to toxicities or associate significantly with mtx clearance. analysis in a larger cohort is ongoing. we have identified at least one patient factor (bmi) that significantly impacts h mtx levels and is of potential use for future modeling, as current models incorporate bsa only. our findings concord with studies in adult os in that bmi significantly impacts h mtx level but diverge in that female gender is associated with higher h levels. importantly, these data support targeting h mtx levels to ensure that minimum concentration for adequate tumor necrosis is reached. these results do not suggest that monitoring h levels would prevent toxicities, thus necessitating further characterization of any intrinsic patient factors that associate with toxicity. overall, our definition of the clinical factors that associate with h mtx levels contributes to a framework for therapeutic drug monitoring in pediatric os. children 's mercy hospital kansas city, kansas city, missouri, united states background: post consolidation immunotherapy with dinutuximab, aldesleukin (il- ), granulocyte macrophage colony stimulating factor (gmcsf) and isotretinoin is standard of care for children with high risk neuroblastoma. dinutuximab is combined in alternating cycles with s of s gmcsf or il , followed by a th cycle with isotretinoin alone. il- is administered as a hour continuous infusion on days - at miu/m /day followed by a higher infusion dose, . miu/m /day, in combination with dinutuximab on days - of cycles and . the miu/m /day dose may be administered inpatient or in the ambulatory setting. objectives: to retrospectively compare the incidence of inpatient and outpatient side effects and complications associated with low dose ( miu) il to provide the tolerability data necessary to evaluate these venues for future administration options. design/method: this study was a descriptive, singlecentered definitive study utilizing a retrospective convenience sample population of children with high risk neuroblastoma who received low dose il either as an inpatient or an outpatient without exclusion from may to june . subjects were identified by a tumor registry query post irb approval. electronic and paper medical records were reviewed for the dates and location of the infusions, the home health company used if applicable and all documentation regarding clinical status, side effects and toxicity. demographics was limited to age and gender. results: infusion venue was chosen by provider preference. twenty-six infusions, inpatient and outpatient via separate home health companies were all administered in entirety and without interruption. there were males and females ranging from - years of age. two children received a single outpatient infusion due to intolerance of il when combined with dinutuximab and received therapy in both settings. fever, inpatient and outpatient was the only common side effect. no source of infection was ever identified. there was one incidence of diarrhea and one patient with pruritus in both the outpatient and inpatient settings respectively. no planned outpatient infusions required subsequent admission however the outpatient fever did necessitate an er evaluation. conclusion: low dose il can successfully be administered outpatient. the medication has minimal side effects with fever occurring in %, none of which were associated with infection. no outpatient infusion required a subsequent admission. no patients who received cycle infusions outpatient opted to receive the next cycle inpatient. baylor college of medicine, houston, texas, united states background: metastatic ewing sarcoma (es) has an extremely poor overall survival, necessitating investigations into molecular mechanisms to identify novel targets and develop new therapies. we previously performed an in vivo study, using our mouse model, designed to provide insights into transcriptomic and proteomic signatures for metastatic es to identify potential therapeutic targets. comparing profiles of primary tumors to corresponding metastatic lesions, we identified aberrant expression of integrin ß (itgb ) and downstream activation of integrin-linked kinase (ilk) in metastatic lesions compared to primary tumors, implicating this pathway as a key regulator in the ability of es to establish and enhance metastasis. our hypothesis is that upregulation of itgb and its downstream signaling events play a key role in es metastasis and are viable therapeutic targets. objectives: to investigate the role of itgb and its downstream signaling pathways in driving the establishment and enhancement of metastasis in es and to investigate this pathway as a potential therapeutic target. to investigate the role of itgb and ilk in es metastasis, we used sirna to knock down itgb and ilk expression in established es cell lines and then performed functional assays in vitro, including cell proliferation and invasion/migration assays. we also tested inhibition of this itgb signaling pathway using available small molecule inhibitors targeting itgb , ilk and the downstream target ap- , using cilengitide, compound and sr , respectively. we are currently using these small molecule inhibitors as treatment in vivo and assessing rates of metastatic tumor formation. we generated stable itgb and ilk overexpression and knockdown cell lines, which we are using for similar in vitro and in vivo investigations. knockdown of itgb and ilk in our sirna cell lines resulted in decreased cell proliferation and decreased invasion and migration compared to controls. we also found significantly decreased cell proliferation using each of the small molecule inhibitors in vitro. our preliminary studies using compound in vivo established a safety profile and dose escalation is underway to assess the effectiveness of inhibiting es metastasis. these results support our hypothesis that itgb and its downstream signaling events play a key role in the ability of es to establish metastatic foci and may serve as a potential therapeutic target. we continue to investigate this pathway in vitro. we are also using our small molecule inhibitors and itgb and ilk overexpression and knockdown approaches to study these effects on metastatic tumor development in vivo using our mouse model. background: neuroblastoma (nbl) is characterized by phenotypic heterogeneity. outcome is excellent for patients with low-(lr) and intermediate-risk (ir) disease, whereas only % of high-risk (hr) patients will survive. -hydroxymethylcytosine ( hmc) is an epigenetic marker of active gene transcription, and hmc profiles are prognostic in many types of adult cancers. we hypothesized that hmc profiles will serve as robust biomarkers in children with nbl tumors, refining current risk stratification. objectives: analyze genome-wide hmc in nbl tumors and correlate hmc deposition with chromosomal copy number and gene expression. design/method: hmc was quantified by nano-hmc-seal-seq from the dna extracted from hr, ir and lr nbl tumors. read counts and clinical data were analyzed with deseq to identify genes with differential hmc patterns between risk groups. chromosomal copy number was assessed by chromosomal microarray analysis (cma) in a subset of samples ( lr and hr). expression of genes located on chromosome p was evaluated using publically available microarrays (e-mtab- ) of hr nbl tumors with known p loh status. results: globally, lr tumors had more hmc peaks ( , ) than ir ( , , p = . ) tumors, or hr tumors ( , , p = . ). , genes had different patterns of hmc deposition in hr versus lr tumors. ( %) of these genes mapped to chromosome p and had decreased hmc in hr versus lr tumors (padj < . ). in the cma analysis p deletion was detected in of the tumors tested. in the tumors with p loss, genes that map to p showed decreased hmc deposition compared to the hr tumors without p loss (p< . ). further, compared to the tumors without p loss, the expression of of the p genes was decreased (p< × - ), including chd , camta , and arid a, known and proposed tumor suppressor genes in nbl. conclusion: different patterns of hmc accumulation are associated with neuroblastoma risk classification. nano-hmc-seal-seq is sensitive to copy number variations and has the potential to identify these changes in patient tumors. our results suggest that hmc deposition contributes to the silencing of tumor suppressor genes in p and may also regulate the transcription of other genes that drive tumor phenotype. background: metastatic osteosarcoma has a -year survival rate of - %. pulmonary metastases remain a major treatment challenge in osteosarcoma. current treatment with conventional chemotherapy shows inadequate activity towards metastases and has toxic systemic side effects. chloroquine is a widely used anti-malarial drug and has been shown to have promising anti-cancer and anti-metastatic activity. polymeric drugs have been shown to have multiple advantages over their small molecular parent drugs, including enhancing the therapeutic efficacy, an improved pharmacokinetics profile and decreased systemic toxicity. we hypothesized that by developing chloroquine into a polymeric drug and combining it with conventional chemotherapy it will improve the treatment of metastatic osteosarcoma. objectives: to identify the optimal combination of polymeric chloroquine (pcq) with conventional chemotherapy active in osteosarcoma as a new means of treating metastatic disease in a murine osteosarcoma model. we synthesized and developed pcq and evaluated its anti-invasive activity using an osteosarcoma cell migration and invasion assay. we evaluated the efficacy of cell killing using combination drug therapies with pcq and a panel of conventional chemotherapy agents (doxorubicin, docetaxel, cisplatin and paclitaxel) using celltiter blue cell viability assay. to develop the murine osteosarcoma model, we intravenously injected luciferase-expressing human osteosarcoma cells b into nsg mice. we administered the drug combination that showed the strongest in vitro synergy to the mice and evaluated their anti-cancer and anti-metastatic effects in vivo. tumor growth and suppression were evaluated using whole body bioluminescence imaging. results: we successfully synthesized pcq that contains . % chloroquine with a molecular weight of . kd. pcq was also found to decrease the toxicity of the parent chloroquine. pcq showed strong inhibition of osteosarcoma cell migration with % inhibition compared to % by chloroquine. we screened the combination drug therapies and found the combination of pcq and doxorubicin to show the strongest synergism. the pcq/doxorubicin combination is currently being evaluated in the murine model. combination drug therapy using pcq and doxorubicin showed synergistic cell killing and inhibition of cell migration in vitro. the combination represents a promising treatment strategy for pulmonary metastatic osteosarcoma. emory university/children's healthcare of atlanta, atlanta, georgia, united states background: survival for relapsed high-risk neuroblastoma (rnb) is < %, underscoring the critical need for novel therapies. rnbs have increased ras/raf/mapk mutations and increased yes-associated protein (yap) transcriptional activity. yap is a transcriptional co-activator that binds with tea-domain (tead) transcription factors to regulate cellular proliferation, self-renewal, and survival. we found that shrna inhibition of yap decreases nb cell proliferation and sensitizes ras-mutated nbs to mek inhibitors, supporting yap as a tractable therapeutic target. verteporfin (vp), a photodynamic drug used for macular degeneration, is the only drug found to inhibit yap expression or yap:tead binding to kill tumor-derived cells. peptide is a mer yap peptidomimetic that also disrupts yap:tead interactions. we sought to determine whether these compounds are potent in nb via yap direct effects. design/method: yap expressing (nlf, sk-n-as) or yap null (ngp, lan , sk-n-as-shyap) human-derived nbs were incubated with vp, with and without direct light exposure, or with peptide . celltiter-glo and immunoblots were used to assess for cell death and yap-downstream protein expression, respectively. results: without direct light exposure, vp inhibits yap expression at nm dosing, yet no nb cell death was observed at equal or higher concentrations. egfr and erk / were inhibited along with yap, confirming yap/ras pathway coregulation. when vp was exposed to direct incandescent light for minutes, > % nb cell death occurred in all nbs tested, even those lacking yap. peptide caused no cell death or yap inhibition up to um. neuroblastomas are resistant to vp at doses sufficient to inhibit yap expression. in macular degeneration, light-activated vp produces reactive oxygen species, which we hypothesize is the off target mechanism killing nbs independent of yap. given the off target effects and the need for light activation, vp is not an ideal preclinical or clinical yap inhibitor. accordingly, peptide has poor cell permeability and low tead affinity, leading to its lack of efficacy. given the relevance of yap in rnb and other cancers, we are chemically optimizing a yap peptidomimetic with enhanced permeability, nuclear localization, and tead affinity to create a bonafide yap inhibitor for preclinical and clinical application. kayeleigh higgerson, aaron sugalski, rajiv rajani, josefine heim-hall, jaclyn hung, anne-marie langevin ut health san antonio, san antonio, texas, united states background: osteosarcoma is the most common bone malignancy in children, adolescents, and young adults. most study cohorts have to % hispanic patients that encompass many different hispanic backgrounds. the university of texas health science center at san antonio (uthscsa) sarcoma team serves a latino population that is predominantly mexican american, thus providing a unique opportunity for evaluation this population. this study expands on previous data collected from january to december from the same institution, providing increased insight into outcomes of mexican american children, adolescents, and young adults with osteosarcoma. objectives: to further understanding of osteosarcoma in latino children, adolescents and young adults. design/method: a retrospective analysis of demographics, tumor characteristics, response to treatment, and survival outcome of all localized osteosarcoma of the extremity patients below years of age diagnosed and treated by the uthscsa sarcoma team between january and june was performed. results: in our original cohort from january to december , we observed a significantly decreased -year eventfree survival (efs) in patients diagnosed before age (preadolescent) relative to patients diagnosed between ages and ( % vs. %, p< . ). patients had a -year overall survival (os) and event-free survival of % and % respectively. in our expanded cohort from january to june we evaluated sixty-six patients with a median age of (range, to y) with localized high-grade osteosarcoma of the extremity. the expanded cohort was % mexican american, with a median follow-up of months (range, to ). the analysis of our expanded cohort is ongoing and we postulate that the findings will hold true, as we increase the cohort size and length of follow-up. conclusion: analysis of our previous cohort, predominantly of mexican american ethnicity, showed that preadolescent patients had an increased rate of relapse when compared with previous large studies. we also showed a trend towards decreased efs for the entire cohort. we hypothesize that we will further validate these findings with this expanded cohort and this will support further investigation into potential causes of poor outcome in this vulnerable latino population. background: neuroblastoma in infants has the potential to regress or mature spontaneously. growing literature showed that some cases subjected to initial observation didn't show inferior outcome compared to actively treated similar categories. objectives: we investigated whether early active treatment can be safely avoided/deferred in selected favorable cases at the children's cancer hospital-egypt (cche). design/method: patients enrolled on the watch and see strategy (w&s) at cche had small primary tumor; inss stage - , uncomplicated stage s or stage infants (< days). tissue biopsy was not mandatory for infants below months of age with localized adrenal mass (stage - ). on progression, immediate intervention took place according to stage and risk of disease after biological characterization. results: thirty four nbl patients were enrolled on w&s strategy; m/f: . / . eighteen patients had stage s disease, patients had stage - and were stage . primary adrenal site was reported in patients ( . %), patients ( . %) had small mass measuring ≤ cm in its largest diameter. the -year os & efs were . ± . % and . ± %, respectively, with months median follow-up (range: - months). spontaneous total/near total resolution of mass occurred in / patients ( %). median time to eliciting regression was . months (range: . - . months), and . months (range: - months) till complete resolution. only / patients ( . %) witnessed progression ( local, distant and combined local and distant progression); median time to progression was months (range: - months) with / deaths after starting chemotherapy. watch and see strategy is a safe approach in localized and uncomplicated stage s neuroblastoma. progressive cases could be rescued. baylor college of medicine, houston, texas, united states background: ga- dotatate binds to somatostatin receptor expressed in neuroendocrine tumors (nets). it was approved by fda in for use with pet/ct scan for localization of somatostatin receptor positive nets in adult and pediatric patients. pediatric approval was based mainly on extrapolation of data from adults. objectives: to describe the use of ga- dotatate pet/ct scan in children with neuroendocrine tumors and compare with other imaging modalities. design/method: patients with nets enrolled in texas children's rare tumor registry between february and october were reviewed and those patients who underwent ga- scan were included. results: four patients with nets underwent ga- scans without any adverse reactions. first patient was a -yearold female with small bowel net with multiple liver metastases. mri abdomen and fdg pet at diagnosis showed s of s multiple liver metastases but could not identify the primary lesion. ga- scan was able to accurately identify the enlarged lymph nodes in the small bowel and was better than fdg pet in delineating the liver metastases. second patient was a -year-old female with recurrent small bowel net with liver, lung and paraspinal metastases. the lesions were initially detected by ct scan. octreotide scan failed to show any uptake in the identified lesions while ga- was taken up by the liver lesions, lung lesions > cm in size and the paraspinal lesion. third patient is an year-old male with pancreatic net with peripancreatic lymphadenopathy, multiple liver metastases and cardiophrenic lymph node involvement. the primary lesion in the pancreas could not be identified by ct scan, ct angiogram, mibg scan, or octreotide scan. in addition, there was uncertainty about involvement of the enlarged cardiophrenic lymph node. in addition to clearly identifying the primary lesion, ga- scan was able to detect multiple peripancreatic lymph nodes not detected by other scans and revealed uptake in the cardiophrenic lymph node confirming its involvement by the tumor. fourth patient is a -year-old female with malignant abdominal paraganglioma with solitary lung metastasis. both mibg scan and ga- scan were able to identify the primary lesion. ga- scan was performed after the lung metastasis was removed and thus its ability to detect it could not be confirmed. background: neuroblastoma is the most common extracranial solid tumor of childhood, with overall survival for high-risk patients (hrnbl) near %. the outcomes of hrnbl have improved with high dose chemotherapy followed by autologous stem cell rescue (abmt). data about factors influencing the rate of hematopoietic recovery following abmt in hrnbl is lacking in the literature. our objective was to identify factors influencing the rate of hematopoietic recovery following abmt in hrnbl. design/method: this was a retrospective chart review of patients with hrnbl treated at texas children's hospital from to . neutrophil engraftment was considered the first of three consecutive days with post-transplant neutrophil count greater than cells/ul. red blood cell and platelet engraftment were considered at a hemoglobin greater than g/dl and platelets greater than , /ul three days after the last transfusion. race and conditioning regimen were analyzed using one-way anova; amount of infused cells was analyzed using pearson correlation coefficients; chemotherapy delay and bone marrow (bm) involvement after cycle of induction chemotherapy were analyzed using independent sample t-tests. the study included males and females with a median age at diagnosis of . years. thirtyeight patients were caucasian, african-american, hispanic, asian, and did not have race documented. the mean dose of infused cd + cells was . × ^ cells/kg. forty-five patients received conditioning therapy with carboplatin/etoposide/melphalan (cem), received busulfan/melphalan (bu/mel), and received thiotepa/cyclophosphamide (thiotepa/cpm). the conditioning regimen administered was significant (p = . ) for time to engraftment of neutrophils, with bu/mel at . days, cem at . days, and thiotepa/cpm at days. a delay of chemotherapy during induction (n = ) was significant (p = . ) for time to platelet engraftment of greater than , /ul and trended towards significance (p = . ) for time to neutrophil engraftment. bm involvement at diagnosis and after cycle of induction was not significant for time to engraftment. dose of stem cells infused was the only variable significant for hemoglobin engraftment. background: osteosarcoma (os) is the most prevalent aggressive primary malignancy of the bone affecting children and young adults. approximately % to % of patients have metastatic disease at initial presentation, and % of those patients have isolated pulmonary metastases. although overall survival in patients with os has improved with advances in therapy, there have been no significant improvements in survival outcome in patients with metastatic disease. recent studies suggest that tumor-associated vascular cell adhesion molecule (tvcam- or cd ) plays a critical role in the metastatic progression of various tumors. indirect evidence from these studies suggest that vcam- / integrin signaling promotes tumor survival and metastatic progression by changing the tumor niche and associated immune response. to determine if interfering vcam- / signaling between pulmonary metastatic osteosarcoma (pos) and macrophages (macs) by down-regulating vcam- , depleting macs or blocking vcam- / signaling will reduce pos and improve overall disease-free survival. design/method: we used a pair of spontaneous, high-grade murine os cell lines from balb/c mouse (h- d), k and k m (derived from in vivo k metastasis). we used lentiviral shrnas to knockdown vcam- mrna and protein expression in k m (vcam- kd). we introduced luciferase into k , k m and various k m shrna cell lines to follow lung metastasis by bioluminescence (bli). we depleted macs by intranasal administration of liposomal clodronate formulation. we tested the ability of k and k m supernatants to polarize m macs into m or m phenotype in vitro. we also administered anti- monoclonal antibody (anti- mab) intranasally to assess the outcome of functional blockade of vcam- / signaling. results: k m over-expressed vcam- compared to k . mac depletion in k m -bearing animals exhibited reduced pos. weekly administration of anti- mab resulted in % tumor-free rescue among mice with established k m pos. interestingly, supernatant from k m but not k preferentially induced m -like macs, suggesting a novel integrin-mediated mechanism of m differentiation. validation data with additional os cell lines will be presented. despite aggressive multimodal therapy, overall outcome for patients with pos remains dismal at - %. for this reason, novel and directed therapy approaches are desperately needed. molecular targeted approaches for therapy are challenging, due to the complex genetic heterogeneity of os. immune-modifying therapy is a promising new alternative approach for pos. university of chicago, chicago, illinois, united states background: only half of all patients diagnosed with high-risk neuroblastoma achieve long-term survival. imetaiodobenzylguanidine (mibg) scans are routinely used to evaluate disease at diagnosis and following treatment, and the extent of disease is quantified using the curie scoring system. a previous study by yanik et al., has shown that for high-risk patients with mycn non-ampliified tumors, scores less than versus greater than following cycles chemotherapy are associated of superior survival, whereas scores less than versus greater than were prognostic in patients with mycn-amplified tumors. however, the prognostic significance of specific sites of metastatic disease at diagnosis is not known. to determine if site of metastatic disease determined by i-metaiodobenzylguanidine (mibg) imaging in high-risk patients at the time of diagnosis was associated with outcome design/method: we performed a retrospective chart review of high-risk neuroblastoma patients treated at comer children's hospital and lurie children's hospital in chicago between and with positive mibg scans at the time of diagnosis. we collected imaging data as well as other clinical data including bone marrow status. sites of disease were defined as curie regions with any positive value. kaplan-meier analysis was performed to evaluate the association with disease sites and survival. pearson correlation coefficients were calculated to compare bone marrow disease to sites of positivity on mibg scan. the cohort consisted of high-risk patients. had skull disease, and had pelvic disease. the presence of mibg positive disease in the skull and in the pelvis trended toward worse efs. efs at years for patients with disease in the skull at diagnosis was ± % and for patients without skull disease was ± % (p = . ). efs at years for patients with and without pelvic disease was ± % and ± % (p = . ). consistent with prior data, we found that the presence of bone marrow disease was associated with worse survival with year efs of ± % and ± % with and without marrow disease at diagnosis (p = . ). there is the highest correlation between pelvic disease on mibg scan and bone marrow disease with pearson coefficient . . pelvic disease noted on mibg scan likely reflects underlying bone marrow disease. in patients with high-risk neuroblastoma, skull disease and pelvic disease on mibg scan at diagnosis may predict worse event free survival. background: osteosarcoma is one of the deadliest cancers in the pediatric population with little progress in morbidity and recurrence rates since the 's. oncolytic herpes simplex- virus (ohsv) is an attenuated virus that has shown encouraging results against certain solid tumors. programmed cell death protein (pd)- -mediated t cell suppression via engagement of its ligand, pd-l , is also of particular interest due to recent successes in selected cancers, especially those with high genetic mutational loads. most pediatric cancers do not have a wide variety of mutations; however, osteosarcoma has a chaotic genome, prone to genetic mutations. it has been shown through numerous other studies that pd- inhibition alone is not sufficient to result in statistically significant tumor growth delays in osteosarcoma models and patients. we hypothesize the addition of ohsv therapy as an immunologic stimulus to pd- inhibition is efficacious for osteosarcoma. ( ) to determine whether ohsv therapy enhances response to pd- inhibition in immunocompetent murine models of osteosarcoma and ( ) to quantify and characterize the anti-tumor t-cells infiltration after treatment with ohsv and pd- inhibition individually and in combination. we utilized an immunocompetent transplantable murine model using a cell line derived from a spontaneous metastatic osteosarcoma (k m , balb/c background). we transplanted established tumor wedges subcutaneously and monitored tumor volume by caliper measurement. once tumors reached - mm , we administered intratumoral injections of hsv ( × plaque-forming units) every other day for a total of injections. we then gave intraperitoneal injections of ug anti-pd- or control antibody twice weekly, up to weeks, starting from the last dose of virus treatment. we monitored tumor growth via calipers twice weekly until tumors reached mm or cm diameter. we quantified and characterized innate and adaptive immune cell infiltrates in tumors using flow analysis. we found significantly prolonged survival with our combination therapy group compared to all other groups. we found that anti-pd- by itself had little impact on t cell recruitment while the combination group had higher influx of cd + cells with a reduced amount of t-regulatory cells (cd +foxp +cd +). we also found an increase in cd + effector memory cells. osteosarcoma is a deadly cancer with therapeutics remaining unchanged for the last years. here, we describe prolonged murine survival after treatment with combination of pd- inhibition and ohsv injection. the combination treatment changed the microenvironment to be more inflammatory. our data support further preclinical and clinical studies. background: neuroblastoma is the second most common cause of cancer related death in children. treatment for high-risk neuroblastoma has improved significantly over the past twenty years, however cure rates remain below %. immunotherapy has emerged as an effective therapy for neuroblastoma, however new modalities and targets are needed to improve outcomes. objectives: our lab has developed a chimeric antigen receptor (car) that targets b -h (cd ), an immune checkpoint molecule overexpressed on many cancers, including neuroblastoma. we hypothesized that b -h would be a good target for car based immunotherapy for neuroblastoma. design/method: neuroblastoma tissue microarrays of primary patient samples were screened for b -h expression by immunohistochemistry and cell lines were screened using flow cytometry. b -h car t cells were tested in vitro by measuring tumor cell killing and cytokine production after coculture with tumor cell lines and in vivo in an orthotopic model of neuroblastoma. results: b -h expression was detected by ihc on % of the screened neuroblastoma patient samples. b -h was expressed at high levels ( + or +) in more than half of these samples ( %). almost all cell lines screened were homogeneously positive for b -h by flow cytometry. retrovirally transduced b -h . - bb. car t cells were cocultured with three b -h positive neuroblastoma cell lines (sk-n-be , kcnr, and chla ) and robust tumor cell killing was demonstrated using an incucyte assay. supernatant from the co-cultures was harvested after hours and both interferon gamma and il- production were detected by elisa.in an orthotopic subrenal capsule xenograft model of neuroblastoma, mice treated with b -h car t cells show significant reductions in tumor growth and prolonged survival compared to those treated with untransduced control t cells. however, the treatment is not always curative.b -h car t cells express high levels of exhaustion markers (pd , tim , and lag ) when compared to cd car controls. in order to overcome inhibition from exhaustion, b -h car t cells were co-cultured with neuroblastoma cell lines and pd- blocking antibody. nivolumab significantly increased the production of il- and interferon-gamma by b -h car t cells. further studies are underway to determine if b -h car t cell activity is enhanced in vivo by treating animals with pd- blockade along with car t cells. conclusion: b -h is expressed on a majority of neuroblastoma samples and appears to be a promising candidate for car t cell therapy. b -h car t cells demonstrate activity against neuroblastoma xenografts that may be enhanced by the addition of pd inhibitors. helen devos children's hospital, michigan state university, grand rapids, michigan, united states background: osteosarcoma is the most common bone tumor in children. it is often metastatic at diagnosis and in this scenario less than % of children survive. polyamines, small molecules found in all cells, are involved in many cell processes including cell cycle regulation, immune modulation, cell signaling and apoptosis. they are also involved in tumor development, invasion and metastasis. in neuroblastoma, inhibition of the polyamine biosynthesis pathway with odc inhibitor alpha-difluoromethylornithine (dfmo) results in decreased cell proliferation and differentiation. these finding have led to multiple phase i and phase ii multicenter clinical trials in pediatric neuroblastoma patients. dfmo is an attractive drug as it is oral, well-tolerated, can be given for prolonged periods and is already used in pediatric patients. the polyamine pathway has not been evaluated in osteosarcoma. objectives: evaluate effect of inhibition of polyamine biosynthesis with dfmo on osteosarcoma proliferation and cell differentiation. design/method: up to three osteosarcoma cell lines were used: mg- , u- os and saos- . cells were exposed to mm dfmo for days with replacement of media and dfmo on day . intracellular polyamine levels were measured by high performance liquid chromatography (hplc). cell numbers were obtained with a hemocytometer using trypan blue. flow cytometry cell cycle distribution (facs) and propidium iodide were used to evaluate for cell cycle arrest. the protein expression of several osteosarcoma differentiation markers was measured by sds-page and western blot using differentiation specific antibodies. a bioluminescent cell viability assay was used to measure cell recovery over several days after dfmo was removed and replaced with standard media. results: dfmo exposure resulted in significantly decreased cell proliferation in all cell lines. after treatment, intracellular spermidine levels were nearly eliminated in all cells. cell cycle arrest at g was observed in u- os. cell differentiation was most pronounced in mg- and u- os cells as determined by increased osteopontin levels. remarkably, cell proliferation continued to be suppressed for several days after removal of dfmo. conclusion: based on our findings dfmo is a promising new adjunct to the current osteosarcoma therapy for high risk patients. it is a well-tolerated oral drug that is currently in phase ii clinical trials in pediatric neuroblastoma patients as a maintenance therapy. the same type of regimen may also improve outcomes in metastatic or recurrent osteosarcoma patients for whom there have been essentially no medical advances in the last years. background: recent studies demonstrate that lower levels of the ews-fli fusion oncoprotein are associated with enhanced metastatic capability in ewing sarcoma. the nf-kb transcription factor is a critical mediator of cxcr and cxcr -driven metastasis in multiple cancers, and increased cxcr and cxcr expression have each been associated with increased metastasis and poor prognosis in ewing sarcoma. we thus sought to investigate the impact of ews-fli on cxcr /cxcr -dependent nf-kb signaling in ewing sarcoma. objectives: the goals of this study are ) to determine the impact of cxcr /cxcr signaling on metastasis-associated nf-kb target gene expression in ewing sarcoma and then ) to investigate how the ews-fli fusion oncoprotein modulates this response . design/method: we utilized multiple ewing sarcoma cells lines including a , chla , chla , tc and tc . cxcr /cxcr cell surface expression was determined by flow cytometry. ews-fli level was modulated using sirna and expression levels were confirmed by western blot and rt-pcr. p dna binding was measured via elisa. nf-kb target gene expression was assessed via rt-pcr. results: consistent with ihc analysis of primary and metastatic patient tumor samples, the paired primary and metastatic ewing sarcoma cell lines chla and chla showed dramatic differences in cxcr and cxcr expression, with the metastatic chla line demonstrating much higher expression of both receptors. other cell lines (nonpaired) showed variable cxcr /cxcr expression. genetic knock-out of cxcr lead to significant decrease in expression of both cxcl /sdf- and il- , two nf-kb transcriptional targets known to play a key role in tumor metastasis. knock-out of cxcr did not alter endogenous ews-fli mrna levels. conversely, lowering the level of ews-fli using sirna lead to enhanced nf-kb signaling, indicated by an increase in p dna binding. consistent with this observation, treating ewing cell lines with ews-fli sirna also resulted in significantly increased nf-kb target gene expression compared to control cells and target gene expression was then further enhanced upon cxcr /cxcr receptor stimulation with the receptor ligand cxcl /sdf- . our findings indicate that the ews-fli oncoprotein negatively modulates cxcr /cxcr -dependent nf-kb signaling. this suggests that ews-fli low, cxcr /cxcr high cells, which are associated with enhanced metastasis and poor prognosis, would be anticipated to exhibit enhanced expression of key nf-kb target genes. importantly, the nf-kb pathway is a druggable target that could potentially serve as an "achilles heel" in this subset of high risk tumors. current work is evaluating nf-kb inhibition as an approach to treating metastatic and refractory ewing sarcoma. background: acute graft versus host disease (agvhd) is a major cause of morbidity and mortality following allogeneic bone marrow transplant (bmt) in pediatric patients. gastrointestinal (gi) agvhd is the most serious manifestation. recently, decreased paneth cell (pc) in a predominantly adult cohort was shown to correlate with agvhd clinical grading and response to treatment. we aim to demonstrate the relationship between pc counts and gi agvhd stage and response to therapy. design/method: charts of patients who underwent endoscopy following bmt between - were reviewed. for repeated biopsies during the course of agvhd, only the first was included for analysis. one pathologist retrospectively reviewed the biopsies and counted pcs in high powered fields; the average pc count was analyzed. twenty-six percent of biopsies were reviewed by a second blinded pathologist. statistical associations between pc counts and day (d ) response, agvhd stage, and other study covariates of interest were gauged using general linear regression. agreement in pathologist pc counts was quantified by intraclass correlation (icc). the research was approved by the children's healthcare of atlanta irb. results: seventy-eight biopsies were included in the analysis. mean age at transplant was . years ± . (range: months - years). most patients underwent transplant for hematologic malignancies ( , %). the majority of transplants used a matched unrelated donor graft -including cords ( , %) and myeloablative conditioning regimens ( , %) - % received total body irradiation. of these, % were diagnosed clinically with gi agvhd (stage , %; stage , %; stage , %; stage , %). icc showed good agreement ( . ) between the pathologists. mean pc was . for patients with no gut agvhd, . for stage , . for stage , . for stage and . for stage (p = . ). on multivariate analysis pc was strongly associated with gi agvhd stage (p< . ) after controlling for age, preparative regimen intensity, and diagnosis (malignant vs. non-malignant). mean pc counts were significantly lower in patients with no response to steroid therapy at d (complete response (mean . ) vs. persistent disease ( . ) vs. partial response ( . ) (p = . )). patients diagnosed with gi agvhd with pc counts less than had a higher risk of mortality (hr . , % ci: . , . ; p = . ). lower pc count correlated with stage gi agvhd, refractory disease at d , and mortality. incorporating pc count in pathology review during gi agvhd work-up may help in agvhd risk stratification. background: there have been increasing discussions pressuring health care teams and institutions for potentially bearing the cost of clostridium difficile infections (cdi) as a health care-associated infection in the recent years. the pediatric oncology patient population, though small, accounts for significant portion of all cdi with - -fold increased risk. hematopoietic stem cell transplant (hsct) recipients constitute a unique subset with distinct risk factors, such as severe immune deficiency state and graft versus host disease (gvhd). although there is ample data on cdi in adult hsct recipients, reports on pediatric experience are limited. objectives: to evaluate the incidence and patterns of cdi among pediatric hematology, oncology and hsct inpatients at our institution. a retrospective review of all clostridium difficile (cd) stool tests performed using toxin enzyme immunoassay and later, polymerase chain reaction targeting toxin genes between and in a large, urban academic children's hospital was performed. the data were analyzed for hematology, oncology, hsct inpatient population and all the other cases separately and statistical comparisons were performed. results: a total of samples were submitted to the microbiology laboratory for cd testing during the study period. while hematology patients constituted . %, oncology . %, hsct . % and others . % of the cases on whom cd testing was done; per patient average test number was . , . , . , and . , respectively. of all the cd tests per-formed, . % were positive. test positivity was higher in hsct ( . %) and oncology ( . %) cases tested compared with hematology ( . %) and other cases ( . %) with statistical significance (p< . ). overall recurrence rate was . %; hsct patients had the highest recurrence with a rate of % followed by oncology ( . %), hematology ( . %) and other ( . %) cases, again reaching statistical significance (p< . ). again, hsct patients had the highest average number of recurrences at . ( - ) followed by oncology . ( - ), general . ( - ) and hematology . ( - ) groups. there was no seasonal variability in the incidence of cdi among populations analyzed. prolonged hospital stay/antibiotic use and persistent diarrhea due to gvhd are the likely reasons for higher rate of cd testing in hsct as a result of increased monitoring and thus might have even caused underrepresentation of positive cd test frequency. higher incidence and frequencies of recurrence underscores the inevitable nature of cdi in hsct population as a consequence of the current therapies and may lead to future radical treatment approaches like fecal implantation. background: viral infections remain a challenge to treat post hct in children, and significantly contribute to morbidity and mortality. virus specific t cells (vsts) have shown tremendous clinical efficacy in treating viral infections post-hct, with minimal toxicity and long term efficacy. we have used donor-derived vsts in individual patients, however not all donors are agreeable to the process, and numerous patients may benefit from vsts who do not have an identified donor/have other disease indications objectives: we sought to actively build a third-party vst bank, for "off the shelf" use in eligible patients. design/method: vsts targeting cmv, adenovirus and ebv were manufactured using one of techniques. initially ebv transformed b cells were genetically modified with an ad f pp vector and used as antigen presenting cells (apc) to stimulate and expand ebv, ad and cmvpp specific t cells. more recently, vsts were expanded using s of s apc pulsed with commercially available peptide pools (pep-mixes) to expand ebv/cmv/ad specific t cells. products were entered into the "bank" via two mechanisms: a) left over products from our "donor-derived" protocol when patients no longer required vsts or were not at risk of developing viral infections, or b) by targeting regular blood donors based on their hla typing to ensure an appropriate mix of high frequency hla types for optimal patient matching and antigen presentation based on current knowledge of antigen presentation. results: a total of products are currently in the thirdparty vst bank ready for use. twenty seven of these are from our donor derived protocol, and three from targeted donors. all vst products met safety and in vitro efficacy testing. thirteen vst infusions have been given to patients. eleven infusions have been given for cmv and two for adenovirus. five out of seven patients responded to thirdparty vst infusions, with a median of vst infusions per patient (range - ). the median hla matching was out of per patient (range to ) no patients experienced adverse reactions, gvhd or other toxicity related to the vst infusion. a third-party vst bank is feasible and produces clinically appropriate vsts for use in patients with viral infections. hla typing and matching of vst products is essential to reduce toxicity and promote appropriate antigen presentation and expansion of vsts in vivo. further work is underway to further characterize the vsts using epitope mapping to better define the hla restriction and immunogenicity of each vst product. akron children's hospital, akron, ohio, united states background: acute graft-versus-host disease (agvhd) is a well-known complication of hematopoietic stem cell transplant (hsct) and a major cause of post-transplant related morbidity and mortality. first line therapy of agvhd involves corticosteroids and calcineurin inhibition. in patients with severe refractory gvhd, mortality can reach up to %. currently, there is no standard of care for the treatment of steroid refractory agvhd. many centers have looked at the use of antibody mediated control of agvhd to competitively inhibit the inflammatory cascade. basiliximab, a chimeric monoclonal antibody against the t-cell il- receptor, has been used in adults with steroid refractory agvhd. patients receiving this medication have demonstrated complete and partial responses to therapy with minimal toxicities. objectives: report the successful use of basiliximab in the treatment of agvhd in a -year-old following matched unrelated (mud) hsct. design/method: a -year-old male underwent mud transplant for high risk aml with monosomy . conditioning regimen included busulfan, fludarabine and equine atg. his clinical course was complicated by fever, mucositis and agvhd (stage skin; stage gi-biopsy proven). gvhd prophylaxis included tacrolimus and methotrexate, however with progressive skin rash, diarrhea, and early satiety, gvhd treatment with corticosteroids was initiated. as the patient continued to have worsening symptoms, basiliximab therapy was started. the patient received doses ( mg) iv basiliximab on two consecutive days and then received weekly therapy for a total of doses leading to initial improvement. the patient further developed acute on chronic gvhd on day + , and subsequently received a second course of basiliximab. after initial administration of basiliximab, the patient had near complete resolution of symptoms. however, with a small wean in his tacrolimus dose, the patient experienced another skin gvhd flare prompting the second basiliximab course. the patient was subsequently weaned off all immunosuppression by day + . the only acute complication the patient experienced while receiving basiliximab was right toe paronychia and asymptomatic low ebv titer. the patient is currently off all immunosuppression at the time of report without evidence of cgvhd. conclusion: this single case report, in a young pediatric patient, demonstrates the use of basiliximab may be a safe and efficacious treatment for pediatric patients with agvhd. university of california, san diego, la jolla, california, united states background: clinical outcomes after allogeneic hematopoietic stem cell transplantation (hsct) depend on restoration of t lymphocyte populations. association between recovery of cd +foxp + regulatory t cells (tregs) and protection from chronic graft versus host disease (cgvhd) has been described in adult hsct. in adults, t cell recovery is driven by expansion of donor t cells and treg reconstitution is hypothesized to result from peripheral conversion. restoration of t cells in pediatric patients has a larger contribution from thymopoiesis, however, the relationship between thymopoiesis and treg recovery is undefined. objectives: we hypothesized that effective thymopoiesis is important for restoration of treg populations and protection from cgvhd in pediatric hsct patients. design/method: we performed longitudinal flow cytometry of peripheral blood t cells from pediatric hsct patients and age-matched healthy donors. laboratory data were correlated with clinical outcomes to evaluate impact. recovery of tregs occurred in / ( . %) patients by post-transplant day . day treg frequency in patients that developed cgvhd ( . ± . % of cd + t cells) was reduced compared to cgvhd-free patients ( . ± . %). failure to restore tregs to > . % of cd + cells by day was associated with increased risk of cgvhd in the first year post-hsct (rr = . , p = . ). a majority ( . ± . %) of tregs from patients recovering the peripheral treg compartment expressed helios, a marker of thymic-derived tregs; only . ± . % of tregs expressed helios in patients failing to restore adequate tregs. this prompted examining the relationship between defects in thymopoiesis and inability to restore tregs. we evaluated thymic function by flow cytometry quantification of cd ra+cd +ptk + recent thymic emigrant (rte) cd + cells (confirmed by qpcr for trec content). most ( / , . %) hsct patients had detectable rtes by day post-hsct. thymic production of rtes was persistently absent in patients that developed cgvhd (< / ^ cd + cells in / patients), compared to cgvhd-free patients ( / patients > rte/ ^ cd + cells by day , average . ± . / ^ cd + cells). post-hsct thymic activity as measured by rte enumeration correlated with treg restoration; / ( %) rte+ patients restored tregs, compared to / ( %) of rte-patients. conclusion: failure to restore tregs after allogeneic hsct results in increased risk for cgvhd. in pediatric patients thymic generation of new t cells is an important contributor to restoration of the treg compartment. this data supports further investigation into mechanisms impairing post-hsct thymopoiesis and suggests peripheral blood tregs may be a prognostic biomarker for cgvhd. background: haploidentical stem cell transplantation (haplo sct) is riddled with unique challenges. objectives: we present our experience in the use of haplo sct with post-transplant cyclophosphamide (ptcy) and the adaptations required for each disorder for optimal outcome. design/method: we performed a retrospective study at the pediatric blood and marrow transplant unit, apollo cancer institutes, chennai, india. children up to years of age, diagnosed to have benign disorders and underwent haplo sct with ptcy from to july were included. results: ptcy was used in i.e. % haplo transplants for children with benign disorders. the underlying conditions included fanconi anemia , severe aplastic anemia , mds , jmml , hemoglobinopathy , prca , xld and primary immunedeficiency disorders (pid) . source of stem cells was peripheral blood in %, bone marrow in %. conditioning included fludarabine with treosulphan or cyclophosphamide for pids and aplastic anemia respectively. neutrophil engraftment by day+ - with a durable graft was noted in % transplants with graft versus host disease in %, cmv reactivation in %. mortality rate was % with infants less than months of age developing severe fatal cytokine release syndrome. the median follow up is year with years being the longest. no significant late effects have been noted with chronic skin gvhd in children. survival rate was superior among children with pids with survival of % in this group. haplo sct with ptcy is a feasible and costeffective option for cure in children with life-threatening benign disorders with no compatible family or matched unrelated donor. careful patient selection, reducing cyclophosphamide related free radical toxicity with the use of n acetylcysteine, limiting t cell numbers by capping cd at × /kg, post-transplant viral monitoring protocols are required to reduce morbidity and mortality. we have been working on universal access to care for children from s of s all socioeconomic background and incorporating innovations to reduce the cost of hsct without compromising outcomes. haploidentical hsct using tcr / depletion costs usd as compared to ptcy priced at usd. children with severe aplastic anemia and pids can be transplanted using reduced intensity conditioning and ptcy. in hemoglobinopathies, pretransplant immunosuppression is required to prevent graft rejection. graft versus host disease remains the main cause of mortality in children with fanconi anemia. mortality in infants less than months after ptcy has been high, tcr / depletion would be superior in this cohort. cincinnati children's hospital medical center, cincinnati, ohio, united states background: fanconi anemia (fa) is a congenital bone marrow failure syndrome with hsct the only curative option for associated bone marrow failure. patients with fa undergoing hsct may experience increased toxicity related to either their underlying disease, or the effects of medications, resulting in the inability to tolerate prophylactic medications or sideeffects from anti-microbial therapy. objectives: we postulated that increased cd cell dose would be associated with a rapid immune reconstitution and therefore early withdrawal of anti-infective prophylactic medications. design/method: patients with fa transplanted at cchmc from an unrelated donor had peripheral blood stem cell grafts collected and cd selection performed. where possible, patients had serial measurements of their immune system performed at varying intervals post hsct. we defined immune reconstitution as normalization of lymphocyte subsets-cd , cd , cd and cd cells, as well as a normal response to mitogen stimulation including phytohemagglutinin, concanavalin a and pokeweed. the first measurement of either normal cell number or mitogen response was recorded for each patient. results: a total of patients underwent hsct for fa at cchmc between and . patient demographics included a median age of years at hsct, the vast majority of patients having a fully matched or one anti-gen mismatched donor, and the majority of patients transplanted for bone marrow failure. there was a statistically significantly decreased time post-transplant to immune cell recovery in patients receiving > × /kg cd cells (median . ) compared to those receiving < × /kg cd cells (median . ). the median time to normalization of cd count was days (cd count > /kg) versus days (cd count < /kg), cd count days (cd count > /kg) versus days (cd count < /kg), cd count days (cd count > /kg) versus days (cd count < /kg) and cd count days (cd count > /kg) versus days (cd count < /kg). time to normalization of mitogen response was decreased posttransplant in those patients receiving increased cd cell dose at time of transplant, though this was not significant, reflecting low number of patients with evaluable responses. no patients in either group experienced gvhd or graft failure. patients with fa who are transplanted with higher cd cell doses have quicker immune reconstitution than those who receive lower cell doses. along with benefit to patients including less risk of infection and early termination of immune-prophylaxis medications, this supports the use of high dose cd selected grafts in this vulnerable population. background: parvovirus b (pvb ) infection after transplantation was first reported in . since then, numerous cases of pvb infections after hematopoietic stem cell transplantation (hsct) and solid organ transplantation (sot) have been reported. most report anemia as the predominant clinical manifestation. however, pvb has been associated with pancytopenia, hepatitis, myocarditis, and allograft rejection. we present a patient with acute lymphoblastic leukemia who developed bone pain and pancytopenia following hsct in the setting of pvb infection. to describe an unusual presentation of pvb in a patient with acute lymphoblastic leukemia following hsct. design/method: a search of the english-language medical literature was performed using pubmed and medline databases. a review of the patient's medical history was performed. a year old male with relapsed b-cell all and history of "fifth disease" in infancy presented four months after hsct with focal left arm pain and difficulties fully extending the arm. bone mri showed enhancement of the medullary space centered within incomplete transverse cortical fracture interpreted as pathologic fracture due to neoplastic involvement of the ulna with no history of inciting injury. subsequently, peripheral blood counts decreased from low normal values to wbc . k/microl, anc /microl, plt k/microl, and hemoglobin . g/dl. the patient's chimerism remained % donor. a bone marrow biopsy and aspirate were performed to assess for recurrent leukemia given persistence of bone pain and developing pancytopenia. marrow findings included morphologic cytopathic effects with erythroid precursors and strong parvovirus staining with no signs of red cell aplasia or recurrent b-cell disease by morphology or flow cytometry. pvb was detected in blood by pcr and immunoglobulins with resolution of cytopenia and bone pain. this case highlights an unusual constellation of symptoms following hsct in a child with all. unexplained bone pain and medullary infiltrates with pancytopenia suggestive of recurrent leukemia were likely triggered by pvb infection. the question remains if he had reactivation of pvb , a primary infection by a new strain, or the virus was aquired through stem cells. bone biopsy could not be justified in light of clinical improvement. so far, bone lesions have only been described with congenital pvb infection. pvb appears to be uncommon after hsct, with a review of literature yielding pediatric cases. however, it may be underestimated due to lack of routine screening. our patient's presentation supports that evaluating for pvb may be warranted in hsct patients presenting with symptoms suggestive of relapsed leukemia. background: cardiac injury may occur during hematopoietic stem cell transplant (hsct) in pediatric patients and can be asymptomatic for many years. recommendations for screening are available for patients who received anthracyclines or chest irradiation, but no guidelines exist for unexposed longterm survivors. we sought to define the prevalence of echocardiographic abnormalities in long-term survivors of pediatric hsct and determine the need for screening in asymptomatic patients. design/method: we analyzed echocardiograms performed on long-term survivors (≥ five years) who underwent hsct at cincinnati children's hospital between and . we analyzed echocardiograms for left ventricular ejection fraction (ef), end-diastolic dimension (lvedd), septal thickness, posterior wall thickness, and global longitudinal strain (gls). we normalized linear measurements for age and patient body surface area. we included for further analysis patients who had echocardiogram obtained for routine surveillance. results: a total of patients underwent hsct and were alive more than years after transplant in , with having an echocardiogram obtained ≥ five years postinfusion. those with an echocardiogram were transplanted more recently (median vs. ). however, no difference between screened and unscreened individuals was noted for age at transplant, sex, transplant indication, anthracycline exposure, chest irradiation, or cyclophosphamide based preparative regimen. indications for echocardiograms included: cardiac symptoms ( . %), congenital cardiac anomalies ( . %), hypertension ( . %), known cardiac or pulmonary disease ( . %), routine post-hsct surveillance ( . %), and unknown ( . %). the mean time post-hsct was . years. among routine surveillance echocardiograms, the mean ef z-score was - . . mean lvedd zscore was - . , mean septal thickness z-score - . , mean posterior wall thickness z-score - . , and mean gls - . %. for patients that had echocardiogram performed for routine surveillance, / patients ( . %) had ef measured, and / ( . %) had ef z-scores ≤ - . (abnormally low). patients exposed to anthracyclines had a mean z-score ef of - . vs. unexposed patients - . (p = . ). among individuals who received neither anthracyclines nor tbi only / ( . %) was found to have an abnormal ef, . % (z-score - . ) or gls (- . %). only one patient who had a normal ejection fraction (z-score - . , ef . %) had an abnormal gls, - . % (normal ≤ - . ). long-term survivors of pediatric hsct who are asymptomatic and did not receive radiation or anthracyclines likely do not require surveillance echocardiograms, unless indicated by clinical symptoms. patients exposed to anthracyclines or tbi require close echocardiographic s of s screening and clinical monitoring for the development of cardiac complications. duke children's hospital, durham, north carolina, united states background: children undergoing pediatric blood and marrow transplants (pbmt) experience significant symptom distress. mobile health (mhealth) technologies can be leveraged to collect and monitor patient generated health data, and subsequently enhance our understanding of pbmt symptom clusters, patterns, and trajectories. better understanding of symptom complexity can foster development of precision health strategies to improve patient outcomes. however, limited research exists in integrating mhealth technology into pbmt management. we aimed to explore the feasibility, acceptability, and usability of using a pbmt specific mobile application to collect and monitor symptoms and wearable technology (apple watch) to measure objective data such as heart rate (hr) and activity. design/method: an exploratory mixed method design began in october to monitor pbmt symptoms for patients using real-time data from: ) a self-developed mhealth application (app) to collect subjective symptom data; and ) apple watch to collect physiologic measures such as heart rate and number of daily steps. data is collected pre-transplant through days. acceptability will be assessed through satisfaction surveys at study completion. we have enrolled patients to date who are all currently using the app and watch. patients' average frequency of daily charting in the app %. the wearable average daily recorded measurements are for hr and for step count. most common symptoms recorded within the app include fatigue and pain. we have noted trends in data including a decrease in activity following transplant and gvhd and an increase following engraftment. patients have stated "the app is helpful to keep track of how my pain is doing day to day" and "i try to take more steps each day than the day before". patients often remove the watch for charging, then forget to put it back on, but consistently put it on upon reminder. finally, parents often were required to make app entries with patients too sick to record. we continue to enroll patients with enthusiasm from both patients and parents to use mhealth during pbmt. preliminary findings suggest feasibility of using the mhealth devices is strongly correlated to the patient's post-transplant stage and is facilitated by caregiver participation with device management (charging devices, reminders to wear watch and record in app). patients reported satisfaction and ease of use with devices, but found it difficult to keep up with charging and charting. these findings indicate using mobile devices may be useful methods to collect patient generated health data. cincinnati children's hospital medical center, cincinnati, ohio, united states background: bacterial bloodstream infections (bsi) are a common complication following hematopoietic stem cell transplantation (hsct) in both pediatric and adult populations, and are associated with poor outcomes. there is limited data describing the outcomes and characteristics of patients who develop three or more bsi after hsct. objectives: to describe the characteristics and outcomes of pediatric patients who develop three or more blood stream infections in the first-year post hsct. design/method: we performed a retrospective chart review of consecutive patients who underwent hsct at our institution from through to compile this case series. data were collected through the first year post-hsct including: patient demographics, underlying disease and therapy characteristics; and transplant complications such as thrombotic microangiopathy (tma), graft versus host disease (gvhd) and overall survival. bsis were classified according to current center of disease control guidelines. results: of patients, ( %) developed or more bsi in the first-year post transplant (total bsi cases = including all patients). of the cases, the majority underwent allogeneic hsct (n = / ; %). most cases were from unrelated donor (n = / , %). more than half of patients had grade - gvhd (n = / , %). sixteen ( %) had tma. of these cases, tma preceded the first bsi in n = / ( %). the majority of bsis were classified as central line-associated bloodstream infections (clabsis, n = / , %), followed by mucosal barrier injury laboratory-confirmed bloodstream infections (n = / , %) and secondary bsi (n = / , %). the majority of isolated organisms ( %) were associated with mucosal barrier injury pathogens. one-year overall survival in the cohort was % (n = / ). pediatric patients undergoing hsct who develop or more bsis in the first-year post transplant demonstrated an increased rate of tma compared to the overall institutional incidence of roughly %. tma diagnosis preceded the first bsi in over half of patients, suggesting that tma may predispose to recurrent bsi. improved strategies for early detection and treatment of tma as well as prevention of clabsis may help reduce the number of bsis ultimately leading to decreased morbidity and mortality in this patient population. background: in neutropenic pediatric patients, infection remains a significant cause of morbidity and mortality. while granulocyte transfusions have been utilized for decades to treat infections, including in the pediatric population, the efficacy of this intervention remains poorly described. previous guidelines have primarily utilized information from adult populations. furthermore, recruitment of donors typically involves friends or relatives of the patient with periodic involvement of community donors. the use of a readily available local donor population to improve availability has yet to be well described. as the immunocompromised population is particularly susceptible to worsening infection and clinical deterioration, the ability to rapidly harvest and deliver granulocytes warrants further investigation. to investigate the efficacy, safety, and outcomes of severely immunocompromised patients receiving granulocyte transfusions from a local altruistic granulocyte program in a pediatric tertiary care center. design/method: a retrospective review was performed to evaluate the context for receiving a transfusion as well as primary outcomes including infection clearance, survival to discharge, and overall mortality. the indiana blood bank assisted with timing the interval from initial order placement to onset of first granulocyte infusion. results: among the patient population reviewed, patients received separate granulocyte regimens. ages ranged from - years with a mean neutrophil count of at time of first transfusion. indications for transfusions included bacteremia (n = ), fungal pneumonia (n = ), and fungemia (n = ). primary outcomes included clearing infection ( %) and surviving to discharge ( %). the median time from initial order placement to infusion was hours, although there was no significant difference between responders who cleared the infection and non-responders who did not. however, additional investigation found that ward patients had a % chance of surviving to discharge while patients in the icu at time of initial transfusion had a % chance of survival to discharge. the readily available granulocyte transfusion program allows patients to quickly receive therapy in neutropenic settings. this is beneficial for patients as transfusion prior to clinical decompensation correlates with increased likelihood of infection clearance, and subsequently improved mortality. further investigation is needed, likely as a prospective study, to better explore circumstances that are beneficial for granulocyte transfusions. background: donor lymphocyte infusions (dli) are composed of immune cells to treat relapse after hematopoietic cell transplantation (hct). to date, data regarding its efficacy is limited in pediatric populations. furthermore, while outcomes related to cd content have been characterized, to our knowledge, the relationship between outcomes and other cellular content in dli has never been reported. objectives: determine whether the primary hematological malignancy, presence/absence of graft-versus-host disease s of s (gvhd), and unique phenotypic content of each dli impact overall survival (os) in pediatric patients with hematological malignancies. design/method: irb-approved, retrospective study investigating all consecutive dlis given to patients at the children's hospital of wisconsin. analyses were conducted using mann-whitney, fisher's exact, and chi-square. from from - patients ≤ years old with hematologic malignancies [myeloid (aml/ mds/cml/jmml),n = ; lymphoid (all),n = ] underwent dlis ( %% ≥ dlis). the median time between hct and dli was . (range, . - . ) years. there were significant differences between the lymphoid and myeloid groups, respectively, in regard to median age at hct ( . vs . yrs, p = . ) and at first dli ( vs years, p = . ). ultimately, there were no statistically significant differences in gvhd or os in products with either higher or lower cd , cd , cd , cd , or cd cellular content. however, the median cd /kg content was more than double in the patients who developed gvhd as compared to patients who exhibited no gvhd after dli ( . × vs . × , p = . ). patients receiving one dli had a -year os of ± % vs those receiving + dli of ± % (p = . ). with a median follow-up of . (range, . - . ) years, the year estimated os of patients in the lymphoid group was higher at ± % vs ± % in the myeloid group, although not significant (p = . ). our results indicate a survival benefit when using dli in a subset of patients who relapse after hct. unlike adult studies demonstrating little effect of dli in lymphoid diseases, many children with all achieved durable remission. while our analysis did not demonstrate that dli cellular content had a statistically significant effect on gvhd or os, it is possible that differences could be found if a larger population and more targeted cell doses were studied. more data will be needed to further define these relationships and identify patients who stand to benefit most. cincinnati children's hospital medical center, cincinnati, ohio, united states background: many arabic speaking muslim parents of children requiring bone marrow transplantation (bmt) receive medical care in the united states. providers may not understand the impact of islamic parents' religious beliefs and practices on their health care experience. objectives: to explore how islamic parents used religion in decision making and to understand the impact of their religious beliefs and practices on their overall health care experience. design/method: we used grounded theory, an inductive method gathering data from interviews and analyzing text, to identify core themes. ten caregivers of bmt children from middle eastern countries were interviewed by an arabicspeaking provider; interviews were coded by an interdisciplinary team. we identified key themes: . patience is a core belief in islam. patience results from the acceptance of allah's will. behaviors showing patience include praying rather than questioning and crying. . al qur'an provides comfort, healing, and protection. families listen to recitations of al qur'an in the patient's room because they feel that this practice not only comforts them but promotes healing as well. for some, certain portions of the qur'an were especially meaningful such as surat al-baqara, which explains that while we may think something is bad for us, allah will know it is good for us. . religious care in the medical center helped families feel respected. religious care in the medical center included interactions with chaplains, who were understood to be "religion experts," and provision of space for prayer and religious resources. . seeking religious consultation. religious consultation from imams or religious scholars (muftis or sheikhs) provides interpretations of the qur'an applied to the family's specific situation helps families make difficult decisions and follow allah's plan. . muslim beliefs guided decision making; muslim practices brought comfort, strength, and peace. drawn from the parents' understanding of islam. parents who addressed this topic said they would only do what islam allowed. they did indicate that most aspects of healthcare were understood to be allowed within islam. additionally, muslim practices of prayer, reading/listening to qur'an, and giving alms all provided comfort, strength and peace. we identified several recurring themes through our interviews that allowed us to understand how families use their muslim faith to deal with their children's illnesses and how it influences their decision making. we believe this better understanding will allow for more informed conversations about patients' health care and decision making, and shows respect for religious beliefs and practices. nemours/dupont hospital for children, wilmington, delaware, united states background: virtually all children will be infected with human herpesvirus (hhv- ) by the age of two. hhv- reactivation after stem cell transplantation causes multiorgan toxicities, including encephalitis, with inflammation and destruction of the temporal lobes and hippocampi, memory loss, and seizures. catatonia is characterized by posturing, immobility, mutism, and autonomic instability, and it's associated with various psychiatric and medical conditions. we describe a patient with hhv- encephalitis and unusual neurologic sequelae, including cognitive and neurobehavioral dysfunction and catatonia, which may impact our understanding of the pathophysiology of hhv- reactivation encephalitis. objectives: describe a case of hhv encephalitis with practice implications for stem cell transplantation. results: our patient was diagnosed with acute myeloid leukemia at age . within years, he relapsed and received two stem cell transplants. on the th day after his second transplant, he developed hyponatremia and refractory seizures. brain mri showed edema in the medial right temporal lobe with linear ischemic change. eeg showed diffuse encephalopathy. cerebrospinal fluid (csf) demonstrated white blood cells, red blood cells, and hhv- by pcr. his prophylactic antiviral was switched to foscarnet and ganciclovir. repeat mri showed abnormal signals in bilateral medial temporal lobes and the right insula. three months later he developed episodes of diaphoresis, hypothermia, agitation, mutism, and unusual posturing, recurring almost daily, recognized as catatonia. mri showed improvement of the abnormalities in the bilateral medial temporal lobes and hippocampi. eegs showed diffuse slowing. after months of antiviral therapy, csf was negative for hhv- . over the ensuing years, he had numerous episodes of diaphoresis, hypertension, hypothermia, pruritis, confusion, agitation, cogwheel rigidity, and bizarre posturing. dopamine blocking agents did not help. clonazepam helped reduce their frequency, and hot showers helped break acute episodes. further mris showed generalized cortical volume loss. he suffered from depression and severely impaired sleep and cognitive function. we describe a novel, debilitating outcome of hhv encephalitis which may provide diagnostic considerations as we continue to improve our understanding of the breadth of possible neurologic sequelae in transplant patients. hhv- is understood to infect and destroy the temporal lobes and hippocampi, but our patient's autonomic dysfunction indicate involvement of the hypothalamus and basal ganglia. antidopaminergic agents may worsen catatonia, and they were not effective for our patient. treatment of catatonia includes benzodiazepines; electroconvulsive therapy was not attempted in this case but may also be useful. background: epstein-barr virus (ebv)-related posttransplant lymphoproliferative disorder (ptld) is a lifethreatening complication in patients following hematopoietic stem cell transplantation, with a frequency estimated at . % and a cumulative incidence of mortality estimated as high as %. studies of ebv have hypothesized that the tonsils are critical for propagating this infection, as tonsillar epithelial cells have been shown to be the site of primary viral infection and continued viral shedding; however, to date no studies have been performed assessing the role of tonsillectomy in patients with ebv ptld. objectives: identify patients with localized ebv ptld treated with tonsillectomy to identify prognostic factors that may be able to help guide future treatment decisions. design/method: patients treated at memorial sloan kettering cancer center who had received hematopoietic stem cell transplantation and had billing codes for both ebv and tonsillectomy were eligible for inclusion in this study. a retrospective chart review was performed, assessing patient demographics, transplant characteristics, laboratory values, tonsillar pathology, and clinical course. any patient who did not have unilateral or bilateral tonsillectomy performed or who had non-localized disease (defined as disease involvement outside of the oropharynx and neck) was subsequently immunodeficiency; % (n = / ) fanconi anemia (fa); % (n = / ) hemoglobinopathy; % (n = / ) non-fa marrow failure and % (n = / ) a metabolic disorder. seventy one percent (n = / ) had normal amh for age pre-transplant, % (n = / ) had low amh for age pre-transplant; of these, % (n = / ) had an oncologic diagnosis; % (n = / ) had fa; % (n = / ) had previously treated hlh; % (n = / ) had non-fa marrow failure; one had a metabolic disorder and one a hemoglobinopathy. of the patients with post-transplant amh measurement % (n = / ) had low levels. of the patients with previously normal pre-transplant amh % (n = / ) underwent myeloablative conditioning (mac) regimen with a % (n = / ) having low amh levels post-transplant compared to %(n = / ) who underwent reduced intensity conditioning (ric) regimen with % (n = / ) having low amh levels post-transplant (p . ). fifteen percent (n = / ) had low levels pre-transplant and underwent mac regimen with % (n = / ) remaining low; % of these patients (n = / ) had fa. nine percent (n = / ) had low levels and underwent a ric regimen with % (n = / ) of amh levels remaining low; % (n = / ) of these patients had hlh treated prior to transplant. conclusion: amh levels can be used for detection of premature ovarian failure and fertility counseling. there is a higher risk of premature ovarian failure with mac regimens and prior chemotherapy vs ric regimens. follow up of this cohort will provide more information to understand the effects of hsct in ovarian function and the usefulness of amh as a predictor of fertility potential. background: there are no proven strategies to prevent blood stream infections (bsi) secondary to oral mucosal barrier injury after hematopoietic stem cell transplant (hsct). additionally, we recently reported progressive gingivitis and dental plaque accumulation in hsct recipients despite our current oral standard of care (three times daily oral rinse). xylitol is a non-fermentable sugar alcohol that reduces dental caries, plaque accumulation, and oral disease progression by inhibiting bacterial growth. we hypothesized that the addition of xylitol to standard oral care will decrease dental plaque accumulation, gingivitis and bacteremia from oral flora. objectives: identify a clinically effective strategy to improve oral health and prevent bsi secondary to bacterial translocation through the oral mucosa in patients undergoing hsct. we are conducting a prospective randomized control study to test our hypothesis. those in the intervention arm receive our current standard of care (three times daily oral rinse) in addition to daily xylitol wipes; controls receive oral standard of care alone. oral exams are performed at baseline and weekly for the first days post hsct. metagenomic shotgun sequencing (mss) of gingival samples is performed at all time points to evaluate microbiome diversity and pathogenic bacterial load. finally, we performed whole genome sequencing of pathogenic bacterial isolates causing bacteremia to assess for genetic relatedness to corresponding strains present within the patient's oral microbiome preceding the infection. : preliminary interim analysis of patients demonstrates improved oral health in patients receiving xylitol (n = ) over those receiving standard of care (n = ), measured by the oral hygiene index (p = . ) and gingivitis index (p = . ). in the nine patients having complete oral mss analysis, xylitol appeared to be associated with decreased streptococcus mitis/oralis domination in the oral microbiome. finally, patients receiving xylitol had no incidence of streptococcus mitis/oralis bacteremia through the first days compared to three patients ( %) in standard of care arm. interestingly, streptococcus mitis/oralis comprised % of the oral microbiome in one child who subsequently developed a streptococcus mitis/oralis bsi. we expect to complete this study in the next months (n = ). the addition of xylitol to oral standard care appears to decrease dental plaque and gingivitis in patients undergoing hsct. xylitol may also impede streptococcus mitis/oralis dominance in the oral microbiome with potential reduction in blood stream infections. (range: - days). twenty-one mdli ( %) were administered because of lymphopenia, fourteen of them ( %) in patients with concomitant viral/opportunistic infections. mixed chimerism/graft failure was the motive of % of the mdli (n = ) and six ( %) were administered to accelerate immune reconstitution. all infusions were well tolerated without appearance or worsening of gvhd. an increase in t-cell counts was observed following six mdli ( . %), although it was a transitory response ( - weeks) in five cases. viral/opportunistic infections were controlled in five cases ( . %), requiring a median of mdli to achieve this response. none of the mdli administered in cases of mixed chimerism/graft failure were effective in reverting this situation. our preliminary data suggests that mdli, is a safe adoptive immunotherapy strategy even with high dose of t-cells without infusion side effects or gvhd complications. some efficacy has been observed in patients with lymphopenia and opportunistic infections, with no positive results in patients with mixed chimerism/graft failure, up to date. however, to determine the real efficacy of this strategy, prospective studies are required. jun zhao, kristen beebe, lucia mirea, alexandra walsh, shane lipskind, alexander, ngwube phoenix children's hospital, phoenix, arizona, united states background: male adolescents undergoing myeloablative hematopoietic stem cell transplantation (hsct) develop infertility with impaired spermatogenesis with reported rates ranging from % to %. in nonmalignant diseases, myeloablative regimens have been replaced with reduced intensity conditioning (ric) with the hopes of better survival rate, less organ toxicity and improved quality of life. despite the increased use of ric regimens for hsct, the effects of ric on fertility remain unknown. objectives: to assess fertility following ric hsct in young adult males. we assessed gonadal function and semen characteristics in adolescent males (> years) who received a single ric hsct at phoenix children's hospital for nonmalignant diseases during - . male patients who were a minimum of year from ric hsct and had postpubertal development at tanner stage iii or above were eligible for this study. gonadal status was assessed by measuring fsh, lh, testosterone, and inhibin b levels, and semen anal-yses assessed fertility indicators (semen volume, sperm concentration, motility, viability, forward progression, morphology, and total count). results: hormone levels and semen analysis have been obtained for patients thus far. the median time between transplant and semen analysis was years. post hsct, ( %) patients showed abnormally elevated lh levels, but fsh, testosterone (total and free), and inhibin b levels were within normal range for all patients. sperm morphology and viability testing were not able to be performed due to low concentrations and volumes. as a result, the total motile sperm count, the most useful estimate for fertile potential, is essentially for all patients. conclusion: recruitment is ongoing, but so far our limited results suggest that ric hsct may have detrimental longterm effects on male fertility. a multi-institutional trial may be appropriate due to small patient numbers at each institution. we are currently exploring options to expand to other centers. further consideration is warranted regarding decisions made by providers, ways to improve anticipatory counseling provided to patients and their families prior to transplant, and how to augment the preventive care of these patients in longterm follow-up. currently all male patients being considered for ric transplant should be counseled to sperm bank prior to transplant. background: a previous systematic literature review identified all published studies of defibrotide treatment for patients of all ages with vod/sos. to assess day+ survival for defibrotidetreated pediatric patients (≤ or ≤ years, per study) all patients exhibited infectious complications with at least viral infection. four patients also had bacterial infections. of note, no patient developed evidence of fungal infections. conclusion: early institution of ecp in patients with high risk acute gvhd (grade - ) was very effective at treating agvhd, allowed for an aggressive steroid taper and contributed to excellent overall survival rates ( %). infectious complications were primarily viral and bacterial, with no fungal infections in this very high risk population. background: vod/sos is a life-threatening complication of hsct conditioning. vod/sos with multi-organ dysfunction (mod) may be associated with > % mortality. defibrotide is approved to treat hepatic vod/sos with renal/pulmonary dysfunction post-hsct in the us and severe hepatic vod/sos post-hsct patients aged > month in the eu. there are few published data on survival of neuroblastoma patients with vod/sos post-hsct. objectives: to report day+ survival and safety post hoc for patients with neuroblastoma and vod/sos post-hsct in the defibrotide t-ind trial. design/method: vod/sos was diagnosed by baltimore or modified seattle criteria or biopsy, with/without mod, after hsct or chemotherapy. defibrotide treatment ( mg/kg/day) was recommended for ≥ days. this post hoc analysis is based on adult and pediatric patients receiving ≥ dose of defibrotide, including with mod. results: among patients with neuroblastoma, developed vod/sos after hsct. for these post-hsct patients, . % were male and . % were female, median age was years (range - years): . % aged - months, . % - years, . % - years, and patient > years. day+ survival data were available for / of these neuroblastoma patients ( with mod and without mod); had autologous and had allogeneic transplants. kaplan-meier estimated day+ survival for the neuroblastoma group was . % ( % confidence interval [ci] , . %- . %). for the mod and no mod subgroups, kaplan-meier estimated day+ survival was . % ( % ci, . %- . %) and . % ( % ci, . %- . %), respectively. in the overall t-ind hsct population aged ≤ years (n = ) and pediatric autologous hsct subgroup (n = ), kaplan-meier estimated day+ survival was . % and . %, respectively. treatment emergent adverse events (teaes) occurred in . % (n = / ), with serious teaes in . % ( / ; most common: multi-organ failure, . % [ / ]). teaes lead to treatment discontinuation in . % (n = ; most common: pulmonary hemorrhage, n = ); death occurred in . % (n = ; > %: multi-organ failure, . %; vod/sos, . %). treatment-related adverse events, as assessed by investigators, occurred in . % (n = ; most common: pulmonary hemorrhage, . %). this post hoc analysis found kaplan-meier estimated day+ survival of . % in patients with neuroblastoma and vod/sos post-hsct, which was consistent with outcomes in pediatric patients after autologous hsct. the safety profile of defibrotide in neuroblastoma patients was consistent with the overall hsct population in this study and other defibrotide studies in pediatric patients. cincinnati children's hospital medical center, cincinnati, ohio, united states background: blood stream infections occur in nearly % of patients undergoing hematopoietic stem cell transplant (hsct) and fever is often the first symptom. timely administration of antibiotics is associated with improved outcomes, thus, early recognition of fever is paramount. current standard of care (soc) includes episodic monitoring of temperature in hospitalized patients, which may delay fever detection. therefore, continuous real-time body temperature measurement may detect fever prior to the current soc. temptraq is a food and drug administration cleared class ii medical device and consists of a soft, comfortable, disposable patch that results: of patients, were started on a pca in the days post hct. % were male with median age of y. % had all, and % aml. matched related donors were used in % and % received tbi. pca was initiated median d+ . oral mucositis alone was the most common indication ( %). a majority of patients were started on hydromorphone ( %); % started on morphine and % started on fentanyl. % started on continuous infusion. pca was used for a median of days (range - days). median pain score was highest d+ of pca use, however, there was inconsistency in charting of numerical pain scores. on d+ , patients had insufficient data to determine efficacy of pain control; of the remaining patients, % had good pain control while % had moderate and % had poor pain control using our devised scale. the most common toxicity observed was respiratory depression (∼ %), however, etiology was often multifactorial and not due to opiates alone. analysis is ongoing to assess variables predicting pca use as well as efficacy of pain control and correlation between current reporting scales and patient perception. conclusion: pca use is common in pediatric hct yet pain control remains inadequate. there's a need for better evaluation of pca management, especially uniform assessment of pain, thereby improving quality of life post hct. children's national health system, washington, district of columbia, united states background: actinomycosis is a rare invasive anaerobic gram-positive bacterial disease caused by actinomyces spp. that may colonize the oropathynx, gastrointestinal tract and urogenitial tract and can lead to abscesses. respiratory tract actinomycosis is characterized by pulmonary cavities, nodules, consolidations and pleural effusions. although actinomyces are nearly always sensitive to penicillin they are frequently resistant to cephalosporins and variable sensitives to fluoroquinolones. although rare in children, immunosuppressed patients are at increased risk for actinomycosis. to describe a case of next-generation sequencing identification of actinomycosis. a -year-old male with a history of very high risk b-cell acute lymphoblastic leukemia who was months status post a / matched unrelated donor bone marrow transplant complicated by prolonged fevers, persistent weight loss, and splenic lesions, treated with posaconazole and levofloxacin developed fever and cough in the setting of neutropenia. blood cultures demonstrated staphylococcus epidermidis. ct showed micronodules and effusion not consistent with s. epi, prompting bronchoscopy. all bacterial cultures were negative. patient was prescribed a three-week course of vancomycin with rapid improvement. design/method: s next generation sequencing (ngs) from bronchoalveolar levage sample was performed at the university of washington laboratory results: ngs assay from bronchoalveolar lavage showed major abundance of actinomyces most closely related to meyeri or oodontolyticus. demonstrated actinomyces. the patient was started on a six month course of amoxicillin with continued clinical improvement. in retrospect, the splenic nodules that were presumed fungal disease were likely actinomycosis, partially treated with levofloxacin. this case highlights the potential utility of ngs in the diagnosis of rare diseases in immunocompromised patients. actinomycosis was only demonstrated through ngs and led to a change in treatment regimen and durable clinical improvement. because actinomyces often mimics malignancy, tuberculosis or nocardiosis, the use of this novel test both targeted appropriate therapy and reduced the exposure to unnecessary medications to treat the differential diagnosis. finally, we highlight that actinomyces should be considered in patients who present with unexplained fevers, weight loss, and night sweats. haneen shalabi, cynthia delbrook, maryalice stetler-stevenson, constance yuan, bonnie yates, terry j. fry, nirali n. shah center for cancer research, national cancer institute, national institute of health, bethesda, maryland, united states background: car-t therapy, while effective, may not be durable for all, and antigen negative escape is a growing problem. hct, in relapsed/refractory all, can be curative, particularly for those in an mrd negative remission. we demonstrated that cd directed car-t therapy effectively rendered patients into mrd negative remissions (by flow cytometry) and the leukemia free survival post-hct was high . in pastorek, jesssica bruce, michael a. pulsipher, chloe anthias, peter bader, andre willasch, jennifer sees, jennifer hoag, wendy pelletier, brent logan, pintip chitphakdithai, lori wiener university of pittsburgh, pittsburgh, pennsylvania, united states background: more than , pediatric hscts are performed in north american and europe each year. the ethics of exposing a healthy child to donation procedures which have some risks and no direct medical benefits continue to be a topic of debate. pediatric donors may experience psychological distress and poorer quality-of-life during and after donation compared to healthy controls. although there are fact/jacie requirements related to the management of pediatric donors, it is unclear what standardized practices exist for psychosocial assessment/management of this group. objectives: to describe transplant center practices for psychosocial evaluation/ management of pediatric donors (< years) and to examine differences in practices by location (cibmtr/ebmt) and number of harvests (volume). design/method: data were collected via a single crosssectional survey distributed electronically to cibmtr and ebmt centers between / / and / / . : / ( %) of cibmtr and / ( %) of ebmt centers completed the survey. most centers had written eligibility guidelines for pediatric donors ( %). most also had a process for ensuring that donors were freely assenting to donate ( %), managed by a transplant physician ( %). a single physician often jointly managed donor/recipient care ( %). half of centers had a pediatric donor advocate ( %), who was most often a physician ( %) or social worker ( %). cost was the largest barrier to having a donor advocate ( %). most centers performed psychosocial screening of donors ( %) but rarely declined donors based on psychosocial concerns ( %). less than half of centers provided post-donation psychosocial follow-up ( %). comparisons by center location indicated that ebmt centers were more likely to have a physician doing joint donor/recipient care ( % vs. %; p = . ), less likely to have a psychosocial assessment policy ( % vs. %; p = . ), less likely to have a donor advocate ( % vs. %; p = . ), but marginally more likely to do post-donation psychosocial follow-up ( % vs. %; p = . ). large volume centers were more likely to have a psychosocial assessment policy than their medium/smaller counterparts ( % vs. %, %; p = . ) â€"there were no other differences on key psychosocial management variables by volume. although most centers have written guidelines for pediatric donor eligibility and mechanisms for ensuring assent, substantial numbers of donors do not undergo psychosocial assessment, are jointly managed with the recipient by a single physician without an assigned donor advocate, and do not receive psychosocial follow-up. the field would benefit from guideline development for the psychosocial management of pediatric donors. background: germline mutations in samd and samd l genes cause mirage (myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes and enteropathy) and ataxia-pancytopenia syndromes, respectively, and are associated with chromosome deletions, mds and bone marrow failure (bmf). there are limited data on outcomes of hct in these patients. to describe outcomes of allogeneic hct in patients with hematologic disorders associated with samd /samd l mutations. results: seven patients underwent allogeneic hct for primary mds (n = ), congenital amegakaryocytic thrombocytopenia (camt)(n = ), and dyskeratosis congenita (n = ). retrospective exome sequencing revealed gain-of-function mutations in samd (n = ) or samd l (n = ) genes. constitutional mosaic monosomy was present in cases. two samd patients had features of mirage syndrome. unusual findings of panhypopituitarism, laryngeal cleft, and glomerulosclerosis were noted in one case. in another case with a samd mutation hypospadias & bifid scrotum were the only findings. the remaining patients had no phenotypic abnormalities. median age at hct was y (range: . - . ). patients received transplants from bone marrow (matched unrelated (n = ) & hla identical sibling (n = )), or unrelated cord blood (ucb) (n = ). five mds patients received myeloablative s of s conditioning (busulfan-based (n = ) or tbi-based (n = )); patients (mds (n = ); camt (n = )) received reducedintensity conditioning (ric) (fludarabine, cyclophosphamide, with ratg or alemtuzumab). syndrome-related comorbidities (diarrhea, infections, malnutrition, electrolyte imbalance, lung disease and hypoxia) were present in both patients with mirage syndrome. one patient with a familial samd l mutation, mds and morbid obesity failed to engraft following ric double ucbt. she died one year later from refractory aml. all other patients achieved neutrophil and platelet engraftment, at a median (range) of ( - ) and ( - ) days, respectively. posttransplant complications included severe hypertension (n = ), pericardial effusions (n = ), veno-occlusive disease of liver (n = ), and recurrent aspiration pneumonias (n = ). one patient developed grade iii agvhd which resolved with treatment. one patient developed mild skin cgvhd and suffers from chronic lung disease. all surviving patients had resolution of hematological disorder and sustained peripheral blood donor chimerism ( - %). overall survival was % with a median follow-up of years (range: . - . y). patients with hematological disorders associated with germline samd /samd l mutations tolerated transplant conditioning without unusual, or unexpectedly severe toxicities. allogeneic hct led to successful resolution of mds or bmf, with excellent overall survival. more data is needed to refine transplant approaches in samd /samd l patients with significant comorbidities, and develop guidelines for their long-term follow-up. shyamli singla, tiffany simms-waldrip, andrew y. koh, victor m. aquino background: steroid-refractory acute graft versus host disease (agvhd) is a potentially fatal complication of allogeneic hematopoietic stem cell transplantation (hsct). basiliximab (anti-il -r monoclonal antibody) as a single agent or in combination infliximab (anti-tnf-monoclonal antibody) has demonstrated efficacy in adult cohorts with steroid-refractory agvhd, but has not been well studied in the pediatric population. we adopted the use of basiliximab and infliximab as our institutional standard of care for steroid-refractory agvhd in pediatric hsct patients. to determine the response and survival of hsct children who received basiliximab and infliximab for the treatment of steroid-refractory agvhd. design/method: we retrospectively reviewed children who received basiliximab and infliximab for steroid-refractory agvhd refractory between september and december . complete response (cr) was defined as resolution of all clinical signs of agvhd. partial response (pr) was defined as at least one grade reduction in one target organ (e.g. skin, gut or liver) without increased grade in another target organ. no response was defined as either no improvement or progressive worsening of agvhd in at least one organ. baseline demographics, transplant details, laboratory findings, and treatment outcomes were also evaluated. results: of the evaluable hsct patients, children (median age yrs, range mo- yrs) with steroid-refractory agvhd received combination monoclonal antibody (mab) therapy. the median time from the start of steroid therapy to initiation of mab was days. the overall glucksberg grade of agvhd at the time of initiating mab therapy was grade i (n = , . %) ii (n = ; %), iii (n = ; %) or iv (n = ; %). the overall response rate was %, with ( %) patients achieving cr, ( . %) patients achieving pr, and ( . %) patients with no response at days following the start of mab therapy. the median overall survival was , , and days for patients who exhibited cr, pr, and no response, respectively. the overall survival at year following start of mab therapy was %. background: the role of high dose chemotherapy (hdc) and autologous stem cell rescue (ascr) in patients with high risk (advanced metastatic or relapsed) soft tissue sarcomas is controversial. despite multimodal chemotherapy, radiotherapy, and local control measure advancements, prognosis of patients with advanced metastatic or unresectable and relapsed sarcomas remains poor, with less than % years disease free survival. objectives: to determine if consolidation with myeloablative hdc and ascr improves relapse free (rfs) and overall survival (os) outcomes in a high risk patient subgroup. we performed retrospective review of all high risk soft tissue sarcoma patients who underwent hdc and ascr at the children's hospital at montefiore, bronx, ny between october and january . the protocol was approved by albert einstein college of medicine institutional review board. results: patients ( primary metastatic high risk disease, relapsed or recurrent disease) received hdc with ascr. primary diagnoses were rhabdomyosarcoma (rms) (n = , alveolar histology), primary site nasopharynx (n = ) and lower extremity (n = ). ewing's sarcoma (ews) (n = ), axial site (pelvic) in patients ( %). median age years (range - years), ( %) were male. all patients were in complete metabolic remission before transplant. median pre transplant comorbidity index was (range - ). patients ( rms and ews) received conditioning with carboplatin, etoposide and melphalan. remaining patients with ews received conditioning with busulfan, melphalan and topotecan. all patients received peripheral blood mobilized hematopoietic stem cell transplantation. stem cell mobilization achieved with high dose filgrastim in all patients except one who required addition of plerixafor. median cd +/kg s of s recipient body weight cell dose infused was . × ^ (range . - . × ^ ). median times to neutrophil and platelet (> , / l) engraftment were (range - ) and ( - ) days respectively. patients ( %) developed bk viuria (one with grade iii hemorrhagic cystitis); ( %) developed cmv viremia; and one patient ( %) had asymptomatic ebv viremia. there was no graft failure, sinusoidal obstruction syndrome or transplant related mortality. median follow up post-transplant was days (range - days). year probability of os and rfs were % and % respectively. hdc with ascr is a promising therapeutic strategy to consolidate remission and improve survival in select high risk soft tissue sarcoma patient subgroups. prospective clinical trials will inform the impact of disease status prior to hdc and ascr on outcome, optimal conditioning and long term relapse free and overall survival. background: absence of minimal residual disease is paramount for cure of pediatric acute lymphoblastic leukemia (all). the testis may harbor occult leukemia and this disease may result in treatment failure. objectives: the purpose of this study was to assess the longterm outcomes of boys with or without testicular leukemia pre-hematopoietic stem cell transplantation (hsct). design/method: retrospective analysis of boys with high-risk de novo ( with hypodiploidy all) or recurrent/refractory all was conducted. flow cytometry of bone marrow mononuclear cells was used to determine remission status. testicular evaluations were performed by physical examination and wedge biopsy pre-hsct. the median age at time of transplant was . years. all patients were in remission by flow cytometry of bone marrow mononuclear cells at the time of transplant and none had evidence of clinically apparent testicular disease. testicular leukemia was detected in patient and he underwent bilateral orchiectomy. he developed acute graft versus host disease (gvhd) of the duodenum and sigmoid colon which resolved, and the leukemia remains in second complete remission and he is free of hsct-related morbidity . months post-hsct. of the patients without testicular leukemia died a median of . months (range, . to . ) post-hsct ( with adenovirus infection and each with thrombotic microangiopathy and aspergillus pneumonia); experienced infection (staphylococcus species, corynebacterium, enterococcus, klebsiella, citrobacter, e. coli, epstein barr virus, adenovirus, bk virus, human herpesvirus- , candida albicans, fusarium, aspergillus, yeast, and other fungus); experienced gvhd ( of the gi tract, of the skin, of the liver, of the eyes, of the mouth, and of the lungs); and developed a second neoplasia (right lower leg leiomyosarcoma). one patient developed bone marrow minimal residual disease ( . % phenotypically abnormal cells detected months after / matched sibling hsct). reinduction therapy comprised weekly doses of rituxan, courses of blinatumomab and donor lymphocyte infusions with il- . two subsequent bone marrow evaluations were minimal residual disease negative. thirteen months post-hsct residual disease recurred ( . %) and he will receive inotumumab. overall median survival post-transplant of the boys is . months (range, . to . ) and of the surviving boys is . months (range, to . ). conclusion: testicular biopsy can detect occult leukemia pre-hsct. testicular leukemia pre-hsct does not appear to increase the risk of subsequent relapse or other hsct-related adverse events compared to those without it. yaya chu, nang kham su, sarah alter, emily k. jeng, peter r. rhode, mathew barth, dean a. lee, hing c. wong, mitchell s. cairo new york medical college, valhalla, new york, united states background: rituximab has been widely used in frontline treatment of b-nhl including burkitt lymphoma (bl), however, some patients retreated with rituximab relapse, which limit patient treatment options. novel therapies are desperately needed for relapsed/refractory b-nhl patients. several strategies for overcoming rituximab-resistance are currently being evaluated, including engineering immune cells with chimeric antigen receptors (car), as well as second-generation anti-cd antibodies. nature killer (nk) cells play important roles in the rejection of tumors. however, nk therapy is limited by small numbers of active nk cells in unmodified peripheral blood, lack of tumor targeting specificity, and multiple mechanisms of tumor escape of nk cell immunosurveillance. our group has successfully expanded functional and active peripheral blood nk cells (expbnk). b t m was generated by fusing alt- , an il- superagonist, to four single-chains of rituximab. b t m displayed tri-specific binding activity through its recognition of the cd molecule on tumor cells, activated nk cells to enhance adcc, and induced apoptosis of b-lymphoma cells. objectives: to examine if b t m significantly enhances the cytotoxicity of expbnk against rituximab-sensitive and -resistant bl cells. design/method: expbnks were expanded with lethally irradiated k -mbil - bbl and isolated using miltenyi nk cell isolation kit. alt- and b t m were generously provided by altor bioscience. nk receptors expression and cytotoxicity were examined as we previous described. ifng and granzyme b levels were examined by elisa assays. equal doses of rituximab, alt- , rituximab+alt- , obinutuzumab (obinu) were used for comparison. igg was used as controls. anti-cd car expbnk cells were generated as we previously described by mrna electroporation. rituximab-sensitive raji andresistant bl cells raji- r and raji- rh, were used as target cells. results: b t m significantly enhanced expbnk cytotoxicity against rituximab-sensitive raji cells, rituximab-resistant raji- r cells and resistant raji- rh cells compared to the controls igg, rituximab, alt- , rituximab+alt- , obinu (p< . , e:t = : ). furthermore, we confirmed the enhanced cytotoxicity by measuring ifn-g and granzyme b production. b t m significantly enhanced ifn-g and granzyme b production from expbnk against raji, raji- r and raji- rh compared to igg (p< . ), rituximab (p< . ), alt- (p< . ), rituximab+alt- (p< . ), and obinutuzumab (p< . ). when compared to anti-cd car expbnk cells, b t m + expbnk had the similar cytotoxicity against raji, raji- r and raji- rh as anti-cd car expbnk cells did (p> . ). conclusion: b t m significantly enhanced expbnk activating receptor expression and in vitro cytotoxicity against rituximab-sensitive and -resistant bl cells. the in vivo functions of b t m with expbnk against rituximab-sensitive and -resistant bl cells using humanized nsg models are under investigation. background: cardiac dysfunction, including left ventricular systolic dysfunction (lvsd), is a known complication in stem cell transplant (sct) survivors. while detection of lvsd by echocardiography is important in this population, there has been minimal research to determine if subclinical cardiac dysfunction exists in sct patients. cardiopulmonary exercise testing (cpet) is a valuable tool to assess cardiac function, and to determine how the heart responds to the stress of exercise. no studies have been performed to determine if sct patients with normal lvsd on standard echocardiography may have abnormal cpet. to determine the feasibility of cpet, as well as additional echocardiographic parameters, to detect dysfunction in sct patients with a normal ejection fraction on echocardiogram. design/method: we performed a cross-sectional analysis of sct survivors who were at least years post sct, years of age or older and with an ejection fraction > % (low end of normal range) on echocardiogram. we assessed the exercise capacity of all patients with cpet, and sub-clinical cardiac dysfunction through tissue doppler and strain analysis from the echocardiogram. results: seven patients ( male) have qualified and completed this study so far with an average age of . ± . years. the median time from transplant is . ± . years. all seven patients had a normal ejection fraction, however four patients had abnormalities on their cpet. these abnormalities included abnormal predicted peak oxygen consumption (vo ) ( %± . , normal > %) (the best predictor of functional capacity), predicted oxygen pulse ( %± . , normal > %) (measure of cardiac stroke volume) and ventilatory efficiency (ve/vco slope) ( ± . , normal < ). submaximal exercise data, used when patients are unable to complete a maximal effort test, demonstrated low-normal predicted vo at anaerobic threshold ( . %± . %, normal > % of was . days while patients who received autologous infusions had a mean number of days to engraftment of . . engraftment after hsct needs to be prompt to minimize duration of neutropenia and maximize survival rates . our data demonstrates that the infusion of hematopoietic stem cell products with a syringe or iv pump is an effective method of delivery for stem cell products and does not delay the time to engraftment. the median days to neutrophil engraftment was . days. this is comparable to data from the nmdp, which reports engraftment occurs within - days. the main limitation to this study was its small sample size due to the number of transplants done at our center. however, it does provide evidence to support that infusion of stem cell products via pump mechanism is a safe alternative to the infusion by gravity method in the process of the hematopoietic stem cell administration. johns hopkins all children 's hospital, st. petersburg, florida, united states background: leukemic relapse remains the most common cause of treatment failure after allogeneic hematopoietic cell transplant (allohct) for myeloid malignancies. most children who relapse post-allohct will die of their disease, making interventions to minimize this risk a high priority. objectives: to evaluate the safety and efficacy of posttransplant azacitidine for relapse prevention in children undergoing allohct for myeloid malignancy. design/method: we retrospectively reviewed the charts of children undergoing allohct for myeloid malignancies between february and november at johns hopkins all children's hospital. results: during the study period, children (ages to years, median ) underwent allohct for myeloid malignancies: de novo acute myeloid leukemia (aml), ; mixed phenotype acute leukemia, ; treatment-related aml, ; juvenile myelomonocytic leukemia with aml transformation, ; and myelodysplasia/aml, . thirteen were in first complete remission, were in cr or greater. most patients ( / ) received fludarabine/melphalan/thiotepa conditioning; received hla-identical related or unrelated donors, and received haploidentical bone marrow grafts with post-transplant cyclophosphamide. three patients never received planned azacitidine ( early relapse; early trm), leaving evaluable patients. azacitidine ( mg/m /dose for days, in -day cycles for up to cycles) was started at a median of days post-transplant (range - ). two-thirds ( / ) of patients received eight or more cycles. of five patients who stopped therapy early, only one was due to toxicity; other reasons included severe gvhd ( ), parental preference ( ), and relapse ( ). cycle delays occurred in patients, with a median cycles delayed per patient, mostly for mild myelosuppression with early cycles. no patient required blood product transfusion during therapy, but g-csf was used in three patients to maintain anc> / l. dose-modifications were made in patients (renal tubular acidosis, acute kidney injury, and myelosuppression). there were relapses ( %), two of which occurred in patients in cr , for a relapse incidence of % in patients in cr , with a median follow-up of months (range . to ). no patients who received azacitidine died of transplant-related mortality. conclusion: administration of azacitidine in children undergoing allohct for myeloid malignancies is safe and feasible, with most patients successfully receiving all planned cycles. toxicity was acceptable and there was no trm or secondary graft failure. despite the limitations of a small cohort, relapse incidence-particularly in patients transplanted in cr suggests a potential benefit in disease control that warrants investigation in follow-up studies. background: despite significant improvements in the success rate of hematopoietic cell transplantation (hct), graft failure remains an important complication in patients transplanted for severe aplastic anemia (saa). second allogeneic hct can salvage patients, but -year overall survival (os) rates have been reported as low as % . objectives: identify patients who developed dropping donor chimerism, graft rejection, and/or graft failure after first hct for saa, necessitating additional hcts or cellular boosts (defined as stem cell products infused without preceding chemotherapy), and evaluate treatment-related complications and os. with vod/sos with and without multi-organ dysfunction (mod) pubmed and embase databases were searched for "defibrotide and retrospective chart reviews; excluded publication types were: case reports (< cases); meta-analyses; reviews; animal, modeling, pharmacokinetic, chromatography, and adult-only studies; guidelines; articles; and letters. resulting reports were screened for exclusion criteria. full-text articles were then reviewed for eligibility. study characteristics of selected publications were summarized, and publications were categorized by patients' mod status. when necessary, additional data tables were requested. a random effects model was used for pooling data for efficacy. interstudy heterogeneity was assessed with cochran's q-test. percentage of total variation across studies due to heterogeneity (i ) was evaluated we quantified ∼ proteins in each sample. reproducibility for one donor at different time points children 's minnesota, minneapolis, minnesota, united states background: pediatric and young adult hodgkin lymphoma (hl) has five-year survival rates > %. chemotherapy required to achieve this rate is associated with a lifetime risk of cardiac deaths, second malignancies, pulmonary disease and infertility. as effective salvage therapy exists, outcomes may be improved by de-intensifying initial therapy to lessen toxicity.objectives: we piloted a regimen in low and intermediate risk hl patients using agents without known association to significant late effects. this retrospective chart review was approved by children's minnesota irb.design/method: the bvg(p) regimen incorporated bortezomib ( . mg/m day , , , ); vinorelbine ( mg/m day , ); gemcitabine ( mg/m day , ) every days and prednisone ( mg/m /dose bid x days). we treated newly diagnosed patients, ages - years, with non-bulk stage iia (n = ) or iib (n = ) hl. two patients received bvg and received bvgp with the addition of prednisone.results: newly diagnosed patients were all pet negative after the first or second cycle and remained pet negative at end of therapy, cycles. nausea was well controlled with -ht antagonists and scopolamine. pegfilgrastim was not necessary due to the high absolute neutrophil count nadir [median . and minimum . × /l]. there were no episodes of febrile neutropenia, infection or transfusion need. no patients experienced alopecia. one patient developed sensory neuropathy after the eighth dose of bortezomib that was controlled with gabapentin and a switch to subcutaneous bortezomib administration. of the five newly diagnosed patients, four remain in remission at , , , days; relapsed at previous disease sites at days and subsequently achieved remission with bvgp with the addition of brentuximab. this series provides early evidence to stimulate expansion of this pilot experience and subsequent multiinstitutional study leading to a randomized trial of bvgp and current chemotherapy for low and intermediate hl. st jude affiliate clinic at st francis hospital, tulsa, oklahoma, united states background: symptoms suggestive of morning hypoglycemia has been noticed in children receiving all chemotherapy. only few small studies looked at this therapy related complication. factors increase risk of hypoglycemia in all patients include accelerated starvation, steroid induced adrenal suppression, mercaptopurine therapy and prolonged fasting for procedures.objectives: to study the prevalence and risk factors for hypoglycemia during all therapy design/method: medical records of of children (up to years old) treated for all between - ( patients) were studied for evidence of morning hypoglycemia defined as blood sugar (bs) < mg/dl. statistical mean differences between the subgroups were analyzed with spss using a nonparametric mann-whitney u test.results: fifty two percent ( %) of patients developed hypoglycemia during all treatment, with an average of . episodes/patient. % were males and % females. almost / ( %) of patients with hypoglycemia were in maintenance phase of therapy. % of hypoglycemic episodes occurred in % of patients. majority of hypoglycemic episodes ( . %) occurred on the day of procedure when patients were fasting overnight. . % of hypoglycemic episodes occurred in children ≤ years, with . % in ≤ years. patients who developed hypoglycemia were significantly younger (mean age at time of diagnosis of all was . ± . at the hypoglycemia group versus the non-hypoglycemia ( . ± . ) p< . . no statistically significant difference was found regarding sex, or tpmt genotype. % of hypoglycemic children-all < years of age-presented with life threatening hypoglycemia symptoms including seizure and loss of consciousness. this study showed high prevalence of hypoglycemia during childhood all therapy. younger age, especially ≤ years, is associated with higher risk of hypoglycemia as well as life-threatening episodes. to decrease fasting hypoglycemia during therapy for childhood all, we recommend that children under the age of years receive bed time snack high in proteins and complex carbohydrates, and to get them up early the day of procedure to take clear sugary drink. hospital for sick children, toronto, ontario, canada ann & robert h. lurie children's hospital of chicago, chicago, illinois, united states background: childhood brain tumors are the most common solid malignancy and the leading cause of cancer-related mortality in children. the most aggressive type of pediatric central nervous system (cns) tumors is diffuse intrinsic pontine glioma (dipg). despite decades of clinical trials, there has been no substantial improvement with respect to therapeutic outcomes with most children eventually succumbing to the disease. research on adult high-grade gliomas has shown a targetable pathway through the inflammationinduced expression of indoleamine , dioxygenase (ido ) and its recognized ability to suppress the anti-tumor immune response. a limited understanding into the role of ido in pediatric central nervous system tumors serves as the foundation of this research project. furthermore, the integration of nanotechnology is a fundamental step for the investigation and targeting of ido . spherical nucleic acids (snas) composed of nanoparticles have been shown to transverse cellular membranes, exhibit stability in physiological environments, escape from degradation, and create precise targeting in brain tumors.objectives: the purpose of our project is to delineate the role of ido in pediatric dipg, and develop small inhibitory (si)rna oligonucleotides and snas aimed at therapeutically inhibiting the gene expression of immunosuppressive ido . our specific aims are to: ( ) confirm the gene expression ido in different human dipg cell lines; ( ) generate and characterize sirna oligonucleotides targeting human ido in vitro; and ( ) generate and characterize gold nanoparticles for targeted inhibition of ido .design/method: unique patient-derived dipg cell lines were grown in culture, stimulated with increasing concentrations of the proinflammatory cytokine, ifn , and analyzed for mrna levels. sirna specific to ido was transfected into cells. sna generation is in progress.results: ido is expressed in multiple human pediatric dipg cell lines. sirna targeting ido among exons and results in a significant decrease in overall ido expression by dipg cells. sna generation for targeting ido with improved penetration & stability is ongoing, with preliminary results demonstrating a robust ability to inhibit ido expression. the grim prognosis of children with dipg, the lack of effective therapies, and the expression of ido by human dipg cells emphasize the importance of developing the treatment capability to inhibit ido gene expression, as a excluded from this study. the remaining patients were analyzed using descriptive statistics.results: a total of patients meeting inclusion criteria were identified. of these, patients ( . %) received tonsillectomy alone, ( . %) underwent tonsillectomy and decreased immunosuppression, ( . %) received tonsillectomy and rituximab, and another ( . %) received tonsillectomy with additional therapy (including ebv-specific cytotoxic tlymphocytes, donor leukocyte infusion, and chemotherapy). of the patients who received tonsillectomy with or without a decrease in immunosuppression, all were diagnosed with high-grade lymphoma and achieved clinical remission following tonsillectomy with no evidence of relapse to date. on further analysis looking at ptld risk factors, all patients were under years of age, all received t-cell depleted grafts, and none had significant graft-versus-host disease (gvhd) at the time of ptld diagnosis. we have identified a population of patients with localized ebv ptld that achieved clinical remission with no evidence of recurrence following tonsillectomy, suggesting that tonsillectomy alone may be an adequate treatment for localized ebv ptld in a specific subgroup of patients. further analysis is needed to identify characteristics of this subgroup to determine which patients would be most likely to respond to this treatment. university of rochester, rochester, new york, united states background: malignant central nervous system (cns) tumors in young children have a poor prognosis and pose a significant therapeutic challenge. consolidation therapy with carboplatin and thiotepa was piloted in ccg- , cog acns , and cog acns with the goals of intensifying therapy and omitting or delaying radiation.objectives: to document outcomes for patients undergoing carboplatin/thiotepa consolidation with autologous stem cell rescue (ascr) and to demonstrate the feasibility and toxicity of this regimen.design/method: patients up to years old (median age: months) with malignant cns tumors treated at the university of rochester from - with at least one cycle of carboplatin ( mg/kg/day x days) and thiotepa ( mg/kg x days) followed by peripheral blood ascr were included in retrospective analysis. data were recorded on time to engraftment (defined by absolute neutrophil count (anc) recovery to > . × ^ /l), length of hospitalization, toxicity with each consolidation cycle, progression free survival (pfs) and overall survival (os). stem cell harvest data were also collected.results: eleven patients with malignant cns tumors ( atypical teratoid/rhabdoid tumor, primitive neuroectodermal tumor, glioblastoma multiforme, and pineoblastoma) received a total of cycles of carboplatin/thiotepa. of these, underwent stem cell harvest at our institution, with complications limited to procedure-related hypotension for patient with known autonomic instability, and catheter-associated deep vein thrombosis (dvt) for patient. four patients were in complete remission (cr) /status-post gross total resection, was in cr , and had residual tumor at the time of consolidation. nine patients received planned consolidation cycles, patient (of ) planned cycles, and patient of an anticipated cycles thus far. average time to engraftment for these cycles was . (+/- . ) days, with a mean hospital length of stay of (+/- . ) days. fever occurred in of cycles ( %); infectious toxicity included documented bacterial infection in cases (enterococcus faecalis bacteremia in , klebsiella pneumoniae in ). there were no regimenrelated deaths. with a mean follow-up of months, survivors have not yet completed all therapies, and patients have relapsed ( have died of disease). of the survivors, have been disease-free for > months. background: autologous hematopoietic stem cell transplantation (auto-hsct) has resulted in improved survival for patients with high-risk neuroblastoma. treatment intensification is however associated with greater complications. data on early infectious complications in low-and-middle income countries are limited.objectives: to review the early infectious complications following auto-hsct in patients with high-risk neuroblastoma.design/method: a retrospective chart review of pediatric patients with high-risk neuroblastoma who underwent auto-hsct at the american university of beirut medical center between and was conducted. infectious complications during the first days post-transplant were reviewed.results: forty-three patients ( males and females) with a median age at diagnosis of . years [range: . - . ] years underwent auto-hsct during the above-mentioned period. conditioning regimen consisted of melphalan, etoposide and carboplatin. all patients received antiviral and antifungal prophylaxis. median time for neutrophil engraftment was days [range: - ]. bacteremia and clostridium difficile infections occurred in ( %) and ( %) patients respectively. seven ( %) patients developed enterocolitis diagnosed by imaging, were adenovirus induced. cmv viremia was diagnosed in ( %) patients, of whom required treatment. varicella zoster reactivation, parvovirus viremia, toxoplasmosis encephalitis, bk virus cystitis ( patients) and central nervous system ebv related post-transplant lymphoproliferative disorder were diagnosed in different patients. there was no invasive fungal infection. sixteen ( %) patients have died, of whom died in the early post-transplant period, due to disease progression and ( . %) due to infectious complications. among the patients who died due to infection, developed toxoplasmosis encephalitis, developed severe enterocolitis, of which were adenovirus related. the mean igg level within one week post-transplant was lower in patients with clinically significant viral infection compared to others ( vs . mg/dl, p: . ). the mean igg level at the time of clinically significant bacterial infection was lower in infected patients compared to others ( . vs . mg/dl, p: . ). neither absolute lymphocyte count nor absolute neutrophil count at day post-transplant affected the incidence of clinically significant infections. our results show that the rate of infections during the early post auto-hsct period is higher than what has been described in developed countries and has a significant impact on mortality. prevention, early detection and improvement in the treatment is required to improve outcome. university of miami, miami, florida, united states background: allogeneic hematopoietic stem cell transplantation (allo-hsct) is a curative treatment for many malignant and non-malignant (bone marrow failure, immunodeficiency, or metabolic diseases) in pediatrics. despite advances in medicine, graft-versus-host-disease (gvhd) remains a significant cause of non-relapsed morbidity and mortality, specifically in those with malignant diseases.objectives: to highlight the complexity to acute gvhd management and seldom-described treatment approach. a year male with a history of high risk acute myeloid leukemia (aml) due to failed induction therapy. he received a matched ( / ) unrelated donor hsctmarrow product-conditioned with busulfan, fludarabine, and anti-thymoglobulin (atg). his post-transplant course was complicated by hhv- viremia, pres (prompting a change from prograf to cyclosporine), mucositis, and grade iii acute gvhd (skin s , gut s , liver s ) around post transplant day , which later morphed to ocular involvement by d+ . he was started on mg/kg steroids with good response but flared up with each attempt to taper steroid dose. a course of rituximab and later atg were tried without success in weaning off steroids. switching cyclosporine to sirolimus did not provide any additional benefit either. extracorporeal photopheresis (ecp) was started times a week. he initially responded well, yet was not able to wean off steroids. in addition, he developed a flare when ecp session was reduced to days per week. ecp was therefore increased to days per week, which appeared to stabilize skin lesions. a trial of weekly methotrexate was attempted to wean off steroids and photopheresis, which provided no response. finally, a trial of bortezomib on days , , , and of a day cycle as published in a case series of multiple myeloma patients who developed post hsct gvhd. skin lesions improved remarkably however dose had to be reduced due to related pancytopenia. given the response to therapy, he was continued on a weekly dose of bortezomib, receiving a total doses, which has permitted the slow taper of prednisone that has since been discontinued without a major flare. he however is currently maintained on ecp times per week, which is now been slowly withdrawn.conclusion: management of acute gvhd in pediatric patients after hsct can be challenging with no definite options for those who fail steroids or become steroid dependent after initial response. in these situations, bortezomib could be a valid therapeutic option. background: neuroblastoma (nbl) is the second most common solid tumor in children and despite recent treatment advances, overall survival for high risk nbl remains < %. the addition of immunotherapy has improved survival and includes anti-gd antibody therapy. the success of antibody therapy in neuroblastoma is primarily due to natural killer (nk) cell mediated antibody dependent cellular cytotoxicity. we previously demonstrated that nk cells from patients with high risk nbl can be successfully isolated and expanded to large numbers and exhibit potent anti-tumor effects against nbl ( ). thus, infusions of autologous expanded nk cells in high risk nbl in combination with anti-gd antibody are being studied in clinical trials. toll-like receptors (tlr) present on the surface of leukocytes are responsible for pathogen recognition, and activation of these receptors stimulate the production of cytokines that critically link innate and adaptive immune responses. the tlr agonist, poly(ic) is a synthetic analog of dsrna that has previously been shown to directly stimulate cytokine production and improve cytotoxicity in primary nk cells through activation of genes regulated by interferon-response elements (ire) ( ). we hypothesized that ex vivo activation of tlr pathways in nk cells during our normal -day expansion using k feeder cells expressing membrane bound il- would enhance their function.design/method: nk cells were isolated from peripheral blood mononuclear cells and expanded with our previously described expansion protocol in media containing il- and ug/ml poly(ic) ( ). at the end of the -day expansion, nk cells expanded with poly(ic) were compared to controls using a calcein cytotoxicity assay to measure cytotoxicity against high risk neuroblastoma and cytometric bead array to measure cytokine production. : surprisingly, the addition of poly(ic) during nk cell expansion did not improve proliferation, cytokine production or cytotoxicity compared to our standard expansion method. rnaseq demonstrated that our standard expansion method results in a modest decrease in tlr expression at the transcriptional level, but significant upregulation of several ireregulated genes. we conclude that either our standard approach interferes with tlr signaling or saturates the innate immune response pathway such that co-stimulation with poly ic does not produce an additive effect. we are performing expression analysis on nk cells receiving poly(ic) during expansion to further explore this hypothesis. background: gonadal dysfunction leading to infertility is a complication after hematopoietic stem cell transplant (hsct). anti-müllerian hormone (amh) is a marker of ovarian reserve; it is not controlled by gonadotropins and has minimal inter-cycle variations, therefore, it can be used as a marker of ovarian reserve and aid in fertility counseling.objectives: assess ovarian reserve in hsct patients utilizing amh levels. background: tgf beta is an immune suppressive cytokine frequently elevated in the tumor microenvironment causing tumor immune evasion. acute tgf beta treatment potently inhibits nk cell cytotoxicity, cytokine secretion, and proliferation. however, tumor infiltrating nk cells receive chronic inhibitory tgf beta signals in conjunction with activating signals from tumor cells. objectives: to this end, we hypothesized that long-term tgf beta-cultured nk cells would induce functional and phenotypical changes on nk cells that differ from short-term tgf beta treatment.design/method: to explore this, primary human nk cells were cultured with the leukemia cell line, k , alone or with exogenous tgf beta for weeks. : surprisingly, nk cells cultured in tgf beta proliferated faster, and upon challenge with a variety of cell line targets they secreted much greater quantities of ifnÎ ( -to -fold increase against / cell lines) and tnf ( -to -fold increase against / cell lines). further, the high cytokine secretion induced in these nk cells was no longer inhibited by adding additional tgf beta. degranulation was also increased ( / cell lines), however cytotoxicity was not enhanced in a -hour cytotoxicity assay. after resting in il- , the cytokine hypersecretion of tgf betacultured nk cells was maintained for several weeks suggesting this functional change might involve cellular reprogramming. we investigated the mechanism behind these functional changes and profiled genes involved in tgf beta signaling. we found significant reduction of smad transcription which corresponded to a striking decrease in smad chromatin accessibility. we also found significantly increased smad and decreased tgfbr expression. phenotypic analysis revealed that tgf beta also induced remodeling of the nk receptor repertoire with decreased nkp , cd , and klrg and upregulation of trail. the functional consequences of these tgf beta-induced changes on in vitro and in vivo nk cell function are currently under investigation. background: the use of t-cell depleted grafts in haploidentical stem cell transplantation (hsct) has been associated with a delay in early t-cell recovery which increases the risk of viral infections, relapse or graft rejection. conventional donor lymphocyte infusion (dli) after hsct transplantation is effective but conditioned because of a high prevalence of gvhd. the infusion of selected lymphocyte subpopulations with low aloreactivity is emerging as an effective strategy to rectify this issue. the depletion of cd ra+ naive lymphocytes, preserving cd ro+ memory t-cells, could provide a safe source of functional lymphocytes with anti-infection, antileukemic and anti-rejection properties, and lower rates of adverse effects. our objective is to present data of patients that have received cd ro+ memory t-cells dli (mdli) and assess its safety and outcome. we present data of mdli performed after hsct in cases of mixed chimerism, persistent lymphopenia, viral/opportunistic infections or as a strategy to accelerate immune reconstitution.results: fifteen patients with diagnosis of all (n = ), aml (n = ), mds (n = ), saa (n = ), sideroblastic anemia (n = ) and cgd (n = ), received mdli after hsct. a total of forty-three mdli were infused. the median dose of cd ro+ memory t-cells infused was . × /kg (range: . × - . × /kg), with a median dose of cd ra+ naive t-cells of . × /kg (range: - . × /kg). the mdli were infused at a median of seventy-seven days after hsct (range: - days), with a median interval between mdli of thirty-four days results: eight published studies reported survival outcomes for pediatric vod/sos patients (n = ), across all defibrotide doses. estimated day+ survival ( % confidence interval) was % ( %- %). for vod/sos with mod, studies were identified (n = ) with pooled estimated day+ survival of % ( %- %). only one openlabel expanded-access study, the treatment-ind, reported outcomes separately for pediatric vod/sos patients without mod (n = patients aged ≤ years). the day+ kaplan-meier estimated survival for those patients was % ( %- %). safety results were not pooled due to differences in reporting methodology; however, study results were consistent with the safety profile of the phase historicallycontrolled trial in vod/sos patients with mod ( % pediatric), in which / defibrotide-treated patients and all controls experienced ≥ ae. hypotension was the most frequent ae ( %, defibrotide; %, controls); common hemorrhagic aes (ie, pulmonary alveolar and gastrointestinal hemorrhage) occurred in % of defibrotide-treated patients and % of controls. in this pooled analysis of studies with defibrotide-treated pediatric patients with vod/sos, estimated day + survival was % (without mod, %; with mod, %). safety results in individual studies were generally consistent with the known safety profile of defibrotide. taken together, these results show a largely consistent defibrotide treatment effect in pediatric patients treated with defibrotide for vod/sos, with or without mod. results: six patients met inclusion/exclusion criteria. all patients were started on ecp while concurrently receiving . to mg/kg steroid therapy for agvhd plus a calcineurin inhibitor. patients had initiation of ecp within a maximum of weeks from initial diagnosis of agvhd (range - days). patients had grade - agvhd ( / patients with grade ) with skin, liver, and gi gvhd represented. patients received ei-ecp - times per week for the first weeks and then had ei-ecp frequency tapered based on initial response.after weeks of therapy patient had a decrease in overall gvhd grade by grade. all patients were able to have steroids tapered, with doses decreased by an average of % ( % - % decrease).at weeks of therapy, one patient with grade agvhd died of mof associated with infections. three patients had complete resolution of agvhd and patients decreased by grade. steroid doses were decreased by an average of % ( % - % decrease). continuously measures axillary temperature and wirelessly transmits real time-time data. the primary aim of the study was to evaluate the feasibility, safety and tolerability of continuous temperature monitoring in hsct patients using temptraq. we are performing a prospective observational study of pediatric patients ( - years of age) undergoing hsct at cincinnati children's hospital in cincinnati, ohio. enrolled patients wore a temptraq patch for days. a - rating scale survey was completed by the parent/guardian at the end of the study to determine tolerability, ease of use, satisfaction and desire for future use in the inpatient and outpatient setting. temperature data from the temptraq patch was compared to the standard episodic temperature monitoring to determine detection of febrile episodes. seven of ten patients have completed screening. we anticipate completion of the study in early february. the temptraq patch was well tolerated by study subjects (mean tolerability rating of . / ). one patient developed skin breakdown at the site of the temptraq patch attributed to recent thiotepa. the patch was easy to apply with an easy of application rating of . / . parents were overall satisfied (rating . / ) and would like to use the temptraq patches in future hospitalizations (rating . / ) and at home (rating . / ). temptraq patch identified fever (≥ . • f) in patients. the fever was never detected by episodic monitoring (soc) in patients and significantly delayed in the other patients (> hours). temptraq was well tolerated in pediatric hsct patients. timely fever detection was improved in temptraq over the current soc. background: serotherapy is commonly used in patients undergoing hematopoietic stem cell transplant (hsct) to reduce the incidences of engraftment failure and graft versus host disease. however, one well-known side effect is fever. as children undergoing hsct have compromised immune defenses, fever may also be an early indicator of bloodstream infection, which would warrant prompt use of broad-spectrum antibiotics. in a subset of patients with serotherapy-associated fever, antibiotics, which may induce antibiotic resistance and increase costs, may be unnecessary. we aimed to determine the incidence and characteristics of serotherapy-related fever, as well as the likelihood of concomitant bacteremia, in our institutional experience. a -year retrospective chart review was conducted of pediatric patients who received serotherapy as part of hsct conditioning at the university of minnesota. one-hundred sixty eight consecutive hsct patients who received serotherapy -either atg (n = ) or alentuzumab (n = ) -were identified. the median age at hsct was -years (range, . - years). a total of patients ( %) developed fever while on serotherapy (atg = , alentuzumab = ). one-hundred sixteen patients presented fever following the first infusion, and the median onset of fever was hours after commencing infusion (range, . - hours). fever resolved at a median hours (range, - hours). one hundred and fourteen patients ( %) underwent blood cultures. only seven patient were not started on ( %) empiric antibiotics, while % (n = ) were on antibiotic treatment prior to serotherapy for previously known or suspected infections. nine patients ( % of febrile patients, % of all patients) had positive blood cultures (atg = ; alentuzumab = ). no infection-associated deaths were observed.conclusion: while fever is common during serotherapy conditioning in children undergoing sct, episodes of concomitant bloodstream infection are rare. ongoing analysis identified potential risk factors for bacteremia as recent history of infection, first episode of fever following second or subsequent infusions, and previous central line placement. further analysis is being conducted to identify subgroups of patients for whom close monitoring alone may be safe. background: hsct is potentially curative for caya with high-risk leukemias; however, most lack an hla-matched aspho abstracts related donor. the risk of gvhd is increased with unrelated (urd) or partially matched related (pmrd) donors. selective t-cell depletion based on the elimination of t cells carrying and chains of the t-cell receptor may greatly reduce the gvhd risks, while allowing the maintenance of mature donor-derived alloreactive nk cells and / (+) t cells, which may augment the anti-leukemia effect.objectives: this is a prospective study of caya with acute leukemia who underwent hsct with mmrd or urds and tcr / /cd depletion. outcomes included engraftment, toxicities, viral reactivation, and relapse.design/method: this study included caya with acute leukemia transplanted between october and may . all received a myeloablative preparative regimen with targeted busulfan (n = ) or tbi ( cgy/ fractions) (n = ), with thiotepa ( mg/kg) and cyclophosphamide ( mg/kg). atg ( mg/kg x ) was given to those receiving haploidentical grafts and to the first who received urd grafts. immune suppression was not given post-hsct. the stem cell source was mobilized peripheral blood stem cells (pscs), which then underwent tcr / /cd depletion utilizing the clinimacs device under gmp conditions in the chop cellular immunotherapy lab.results: median age was (range . - . ). diagnoses included all ( -b-cell, -t-cell) and aml ( ; -secondary aml). urd were used for ; were / allele matched and were / matched. haploidentical donors were used for . median cd (+) dose - . × , / (+) cd (+) cells - . × , and b cells - . × . all patients achieved an anc at a median of d+ ( - ), and % had platelet engraftment at median d+ ( - ). nine patients ( %) developed acute gvhd (all skin, grades i-iv). five developed chronic gvhd (skin, gut, lung): limited in , extensive in . viral reactivations included: adenovirus ( , %), bk virus ( , %), cmv ( , %), and hhv ( , %). nine ( %) patients relapsed at a median of days (range - ) post-hsct, including aml patients ( . %) and all patients ( . %). transplant-related mortality was %; causes included sepsis ( ) and ards ( ). os was %; efs was % (gvhd-free efs %, lfs %). hsct with tcr / /cd depletion demonstrates excellent engraftment kinetics with limited gvhd without immune suppression. elimination of post-hsct immunosuppression may offer an excellent platform to augment anti-leukemic immune therapy or to enhance immune reconstitution. background: hematopoietic cell transplantation (hct) is the only curative treatment available for patients with sickle cell disease (scd). low bone mineral density (bmd) has been described in scd, but little is known about the impact of curative hct on this outcome. to determine the prevalence of low bmd and variables associated with low bmd in scd patients after hct. we conducted a retrospective chart review of scd patients who underwent hct at children's healthcare of atlanta (choa) between / and / and survived ≥ year post-hct. transplant characteristics, post-hct dual-energy x-ray absorptiometry (dexa) scan results, vitamin d levels, graft-versus-host-disease (gvhd) status, and fsh levels were reviewed. for patients - years of age, height corrected z-scores were calculated using a nihvalidated calculator, with t-scores used for older patients. bmd was categorized as low if between - and - sd below the mean and clinically significantly low if >- sd, in accordance with the children's oncology group long-term follow-up guidelines. vitamin d levels < ng/mol were considered deficient, and fsh levels > miu/ml suggestive of premature ovarian failure. fisher's exact test was used to compare variables in those with normal versus abnormal dexa scan results, with p< . considered significant.results: hct was performed on patients with scd, with surviving ≥ year post-hct. dexa scans were obtained in patients ( % female), with mean time from hct to dexa scan being years ( . - . years) and mean age at time of dexa . years ( . - . years). patients with and without dexa scans did not differ by sex, donor source, age at transplant, or vitamin d status. low bmd was noted in patients ( . %), with these patients more likely to be > years (pubertal; . versus . %, p = . ). acute gvhd was more common in patients with low bmd ( . versus . %), but not statistically significant (p = . ). clinically significant low bmd was noted in patients ( . % of those with dexa scans). these patients were older ( . years at testing), were more likely to be male ( . %), and all had acute and chronic gvhd, while none had evidence of gonadal failure.conclusion: clinically significant low bmd is uncommon after hsct for scd. patients at risk for low bmd include older patients and likely those with gvhd. this preliminary data suggests routine dexas may not be indicated for all patients who undergo hct for scd, but further data is needed. background: causes of renal dysfunction after hematopoietic cell transplantation (hct) include damage from radiation, nephrotoxic medications, graft vs. host disease (gvhd), hepatorenal syndrome, viral infections, or transplant associated microangiopathy. we sought to investigate the incidence of, and risk factors for, acute kidney injury in pediatric hct patients and associated risk with mortality.design/method: data from patients who underwent hct between and at a single institution were sequentially retrospectively captured on irb approved protocol. acute kidney injury (aki) was defined at multiple time points post-hct using the standardized criteria: kidney disease: improving global outcomes (kdigo). interval differences between values were analyzed using wilcoxon rank sum testing and categorical variables were analyzed using chi-square analysis.results: ninety-eight patients were included in the study: allogeneic (n = ) and autologous (n = ), mean age . years, of whom % were african american, % asian, % caucasian, % latino, and % mixed race. forty-seven percent of patients developed aki within the first years of hct. increased risk for aki was associated with a lower pre-transplant creatinine level (p = . ), abnormal pretransplant bun (p = . ) and an unrelated donor (p = . ) while preparative regimen intensity, race, or primary disease were not. twenty-six percent of patients developed aki within days of hct. of those with aki, % were exposed to either cidofovir, aminoglycosides, and/or ambisome for at least days versus % without aki and % were exposed to vancomycin compared to % without aki. evaluating outcomes at year after hct, of those with stage aki: % had reduced gfr and % died, while % had reduced gfr and % had died for patients with aki stage or . the absence of aki by day was associated with % reduced gfr and % death at -year after hct. overall, those with aki at any time in the first year post-hct had a . fold increased risk of death compared to those without. for patients who required renal replacement therapy (rrt, n = ), the risk of death was . fold greater compared to those who did not. in the % of patients who survived rrt, both recovered renal function within years.conclusion: acute kidney injury is common after pediatric hct, and may be associated with low creatinine, abnormal bun, unrelated donor pre-hct, and renal toxic medications. early-onset aki post hct is associated with an increased risk of mortality. these data should be validated in a larger prospective study but may offer opportunities to intervene and enhance outcomes. background: myeloablative hematopoietic stem cell transplant (hct) for pediatric malignant disease is associated with significant morbidity with % patients experiencing mucositis. patient controlled analgesia (pca) utilizing opioids is an effective strategy for pain management. we sought to describe and analyze pca use in d+ days post myeloablative hct for malignancies at lurie children's hospital of chicago from - .design/method: utilizing retrospective chart review, pca details were collected: indication, initiation day, pca duration, team managing pca (anesthesia or palliative), medication and dose in morphine equivalents, and pca toxicities. efficacy of pca was evaluated on pca day + , + , + , + using demands %, maximum pain score (rflacc, faces, vas) and subjective patient, parent and/or pain team perception of pain control. we devised a scale based on the above to designate pain control as good, moderate or poor. variables being analyzed include recipient age, sex, donor type, source, diagnosis, tbi use, gvhd/trm. this analysis, we analyze the depth of remission, car-t persistence, and post-transplant gvhd on our phase i anti-cd car-t protocol (nct ) to better understand the role of car-t in the peri-hct setting.design/method: children and young adults with relapsed/refractory cd + all treated on our phase i anti-cd car-t protocol were analyzed. mrd was assessed by flow cytometry (fc) in all, with pcr-based mrd analysis using igh or tcr testing assessed in select patients. hcts were performed at each patient's local institution based on standard of care and included varying conditioning regimens, donor types, stem cell source, and gvhd prophylaxis.results: on our cd car trial, patients were treated, the majority of patients (n = ) having relapsed following a prior hct. / patients ( %) attained a cr, of whom were mrd negative by fc. concurrent pcr based mrd analysis available in patients demonstrated that all patients achieved pcr based negativity. in , this was simultaneous with the month mrd negative fc, and in , pcr negativity was achieved over time (fc remained negative). patients proceeded to hct at a median time of days (range: - days) post-car-t, which was a first hct in . these two patients remain in an mrd negative cr, year post-car-t. no patients developed acute or chronic gvhd. car persistence was seen in patients who had detectable car-t cells on the pre-hct marrow suggesting the possibility of ongoing anti-leukemia surveillance prior to initiation of the conditioning regimen.conclusion: by inducing pcr negativity, car-t therapy may have a synergistic role with hct to improve leukemia free survival, prior to emergence of antigen negative leukemia, without an increased risk of gvhd. while the sample size is small, car-t therapy may offer an effective bridge to hct, particularly for those who are pcr negative, and those who have not had a previous transplant. given the underlying risk of hct related trm, pre-hct car may potentially allow for hct conditioning de-intensification as it may not be needed to eradicate residual disease. lee dw, ash abstract , background: post-transplant lymphoproliferative disease (ptld) is a complication after solid organ transplantation (sot) that is frequently due to epstein -barr virus (ebv) as a decrease in ebv-specific t cell immunity due to immune suppression allows for uncontrolled proliferation of ebv-infected b cells. outcomes for ptld are suboptimal with relapse rates approaching %. however, ebv-infected b cells in ptld express the ebv antigens lmp and lmp that can be targeted with immune therapy.objectives: we hypothesize that third party "off the shelf" lmp-specific t cell products may improve outcomes and decrease associated co-morbidities for patients with ptld by not only target the lymphoproliferating ebv-infected b cells but also restoring ebv-specific immunity.design/method: lmp-specific t cells (lmp-tcs) are manufactured from eligible donors with a broad range of hla types in our gmp facility to be used in a children's oncology group (cog) trial (anhl ) for patients with ptld after sot. lmp-tc products are manufactured from healthy donors using autologous monocytes and lymphoblastoid cell lines (lcl) transduced with an adenoviral vector expressing Δlmp and lmp as antigen presenting cells. lmp-tc products undergo comprehensive characterization by ifn-elispot assay to determine lmpspecific epitopes, class i and/or ii response, and hla restriction to guide selection of lmp-tc product for each patient.results: thus far, lmp-tc products have been manufactured. lmp-tcs were active against lmp (mean: sfu/ × ^ cells; range: - ), lmp ( ; - ), and lcl ( ; - ) as determined by ifn-elispot assay. at the time of cryopreservation, the lmp-tc products comprised a mean of % cd + t-cells, % cd + t-cells, and % nk cells. no b cells or monocytes were detected in the final products. thus far, we have identified novel lmp epitopes (lmp specific: n = ; lmp specific: n = ). approximately % of the lmp-tc products have lmp-specific activity through multiple hla alleles, and % have a mixed class i and class ii response. conclusion: thus, lmp-specific t cell products can be expanded from healthy donors to creat a third party bank, and identifying epitopes and hla alleles with lmp activity will facilitate selecting the most appropriate product for patients. while lmp-specific t cells have previously demonstrated safety and efficacy in phase i studies, anhl is the first trial using cellular therapy within a cooperative group setting. children's cancer hospital at the university of texas md anderson cancer center, houston, texas, united states background: in , the united states food and drug administration (fda) approved the first chimeric antigen receptor t cell (car-t) therapy; tisagenlecleucel. this cd -directed genetically modified autologous t cell immunotherapy has shown response rates of almost % among children and young adults with b-cell precursor acute lymphoblastic leukemia (all) that are refractory or in second or later relapse. cytokine release syndrome (crs) and car-t cell related encephalopathy syndrome (cres) are well described toxicities associated with car-t therapy. crs is a systemic inflammatory response and is typically characterized by fever, hypoxia, tachycardia, hypotension and multi-organ toxicity. cres may occur concurrently or following crs, or without any associated crs symptoms and is characterized by encephalopathy, delirium, seizures and rarely cerebral edema. almost half of patients who receive tisagenlecleucel may require pediatric intensive care unit (picu) support. crs and cres are generally reversible but may be associated with fatal outcomes. pediatric specific management guidelines, comprehensive training of multidisciplinary staff, effective communication and phased infrastructure ensure that adequate resources are available to facilitate early diagnosis and appropriate management of pediatric patients with crs and cres and allow for optimal patient outcomes and accreditation by the foundation for accreditation of cellular therapy (fact).objectives: develop a comprehensive program to ensure safe administration of immune effector cell (iec) therapy to pediatric patients.design/method: an inter-disciplinary pediatric cartox (car t cell therapy associated toxicity) committee consisting of cell therapy and picu physicians, neurologists, fellows, nursing leadership, advanced practice practitioners, pharmacists, registered nurses and social workers was created to monitor patient toxicity and establish specific clinical guidelines and diagnostic and treatments algorithms for pediatric patients receiving iec therapy. educational modules were developed as (i) live in-services and (ii) an online module with a competency based assessment. electronic medical record (emr) order sets and documentation and warning systems were also developed by the committee. the pediatric cartox committee developed a diagnostic and treatment algorithm for patients receiving iec therapy. emr orders and flowsheets were developed to support adherence to the algorithm. inter-disciplinary staff training and competency assessments were closely tracked. almost % of identified staff have completed training and achieved competency including, pediatric cell therapy staff, emergency center, picu, outpatient clinic/triage, neurology and sub-specialty staff and nocturnalists.conclusion: an inter-disciplinary approach can assist in institutional readiness for an iec program, promote quality assurance and perhaps fact iec accreditation. future directions include a program for ongoing staff competency assessments. predicted peak vo ) and abnormal oxygen uptake efficiency slope at the anaerobic threshold ( . ± . . , normal ± ). additionally, on echocardiogram three patients had evidence of diastolic dysfunction as evidenced by an elevated e/a ratio ( . ± . ) on tissue doppler. three patients demonstrated depressed longitudinal peak systolic strain (- . ± . ), indicating dysfunction not captured by ejection fraction. in this feasibility study, sct patients without evidence of lvsd on standard measures by resting echocardiogram can demonstrate abnormal exercise capacity. additionally, they can demonstrate systolic and diastolic dysfunction by measures not always included in standard echocardiography. these data suggest the need for a more thorough screening of survivors, and will be further validated as additional patients are recruited for this study. background: in hematopoietic transplantation, the t lymphocytes of the inoculum play a determining role in promoting hematopoiesis, transferring immunity to pathogens and acting as mediators of the graft-versus-leukemia effect (gvl). however, they are also responsible for graft-versus-host disease (gvhd), the main cause of post-transplant morbidity and mortality. the depletion of cd ra lymphocytes, by eliminating naive t lymphocytes from the inoculum, aims to conserve the gvl without producing gvhd.design/method: since april , patients ( boys and girls), with a median age of years, have undergone an allogeneic hematopoietic transplant from an hla donor identical with cd ra/cd depletion. the indication for transplant was: acute lymphoblastic leukemia ( ), acute myeloblastic leukemia ( ), myelodysplasia ( ) and medullary aplasia ( ). the donor was familiar in cases and unrelated in . the conditioning regimen was with fludarabine, busulfan and thiotepa. the median of cd + cells infused was . × / kg. on the day , + and + a programmed infusion of × / kg lymphocytes cd ra-was performed.results: all the patients grafted with a median leukocyte (> . × / l) and platelet (> × / l) engraftment time of and days, respectively. only one patient has developed acute gvhd grade i and no patient has developed chronic gvhd. immune reconstitution was early and rapid in all t cell subsets no patient has relapsed so far and only patient with myelodysplasia has developed an aml. she has received a nd transplant and has died of relapse. there was no case of toxic mortality. the event-free survival (sle) was ± % with a median follow-up of months. at present, patients are alive, out of immunosuppressive treatment and doing well. allogeneic transplantation with cd lymphocytes ra depletion resulted on very encouraging results, with a very low incidence of acute and chronic gvhd, but preserving the gvl effect by infusing cd ra-donor lymphocytes. miami children's health system, miami, florida, united states background: hematopoietic stem cell transplantation (hsct) using autologous or allogeneic progenitor cells is a potentially curative treatment for patients with high-risk malignancies and nonmalignant conditions. the american society for blood and marrow transplantation developed a task force to establish consensus guidelines for defining patient care in hsct and advocated for further studies to delineate safe procedural steps as an increasing amount of hsct are being offered to patients. there is limited evidence to support engraftment in recipients who receive their infusions via iv or syringe pump. we present novel data from patients who achieved neutrophil engraftment following hsct by a pump mechanism.objectives: to provide evidence supporting the use of pump (intravenous or syringe) infusion method in hematopoietic stem cell transplantations.design/method: a retrospective review was completed for patients who underwent hsct between and . inclusion criteria included patients who had received hematopoietic stem cell transplants between and and who were ages months to years old. the main outcome measure was days to neutrophil engraftment (defined as the first of three consecutive days with an anc > × /l).results: among patients who received infusion of hematopoietic stem cell products via pump mechanism, patients ( . %) received autologous products and ( . %) received stem cells from allogeneic donors. neutrophil engraftment (anc > × /l) occurred in a median of . days after stem cell infusion. the mean number of days to engraftment for patients who received allogeneic infusions s of s design/method: a retrospective chart review was performed at the children's hospital of wisconsin. statistical analyses included kaplan-meier estimate for os, mann-whitney test for comparing outcomes between subjects, and descriptive analyses.results: from - , patients with a median age of . ( . - . ) years at st hct were identified. patients were conditioned with cy/atg (n = ), cy/flu/atg (n = ), or cy/flu/atg/tbi (n = ) and received marrow (n = ) or cord blood (n = ) with median cd /kg dose of . ( . - . ) × . two patients developed grade i acute graftversus-host disease (gvhd); none developed chronic gvhd. due to dropping chimerism, graft rejection, or graft failure, nd hct (n = ) or boost (n = ) was offered. the median cd chimerism prior to hct/boost was ( - )%. median time between st hct and nd hct or boost was days ( days- . years). in patients receiving nd hct, used the same donor, of which used the same stem cell source (marrow) and switched to peripheral blood stem cells (pbsc). in patients who switched donors, used pbsc and used cord blood. most patients receiving nd hct underwent a uniform conditioning regimen of cy /flu /equine atg/ gy tli (n = ) or cy /flu /rabbit atg/ gy tbi (n = ); one received cy/atg. acute and chronic gvhd (limited seen in %) developed in % and % of patients, respectively. four patients required additional boosts and additional hct. after final intervention, cd and whole-blood chimerism at last follow-up was between - % (n = ) and - % (n = ), respectively. with a median follow-up of . ( . - . ) years, of patients are alive with an estimated -year os of . ± . %, having performance status ≥ % (n = ) or % (n = ). one patient developed chronic extensive gvhd and died of fungal infection . years after nd hct. our single-center experience demonstrates excellent ability to salvage patients who develop graft failure after initial hct. transplant-related complications such as gvhd and infections remain significant concerns. key: cord- -qfim nu authors: oem, jae-ku; lee, soo-young; kim, young-sik; na, eun-jee; choi, kyoung-seong title: genetic characteristics and analysis of a novel rotavirus g p[ ] identified in diarrheic feces of korean rabbit date: - - journal: infect genet evol doi: . /j.meegid. . . sha: doc_id: cord_uid: qfim nu group a rotaviruses (rvas) are important gastroenteric pathogens that infect humans and animals. this study aimed to analyze the complete genome sequence, i.e., genome segments of the lapine rotavirus (lrv) identified in the intestine of a dead rabbit in the republic of korea (rok) and to describe the genetic relationships between this lapine isolate [rva/rabbit-wt/kor/rab / /g p[ ] (rab )] and other lapine isolates/strains. rab possessed the following genotype constellation: g -p[ ]-i -r -c -m -a -n -t -e -h . the p[ ] genotype was found to originate from rabbits and was for the first time identified in the rok. phylogenetic analysis showed that rab possessed vp - and vp genes, which were closely related to those of the bat strain lzhp ; nsp - genes, which were closely related to those of the simian strain rrv; and vp , vp , and nsp genes, which were closely related to the genes obtained from other rabbits. interestingly, a close relationship between rab and simian rva strain rva/simian-tc/usa/rrv/ /g p[ ] for gene segments was observed. rrv is believed to be a reassortant between bovine-like rva strain and canine/feline rva strains. rab and canine/feline rvas shared the genes encoding vp , vp , vp , nsp , and nsp . additionally, the genome segments vp (i ), nsp (n ), and nsp (h ) of rab were closely related to those of bovine rvas. this is the first report describing the complete genome sequence of an lrv detected in the rok. these results indicate that rab could be a result of interspecies transmission, possibly through multiple reassortment events in the strains of various animal species and the subsequent transmission of the virus to a rabbit. additional studies are required to determine the evolutionary source and to identify possible reservoirs of rvas in nature. group a rotaviruses (rvas) are major pathogens associated with acute gastroenteritis in various host species, including birds and mammals, throughout the world (bresee et al., ) . rvas belong to the family reoviridae and have genome segments composed of double-stranded rna encoding six structural viral proteins (vp -vp , vp , and vp ) and five or six non-structural proteins (nsp -nsp ) (estes and cohen, ; pesavento et al., ) . the infectious rotavirus particle is composed of three concentric layers (the inner, middle, and outer layers). the outer layer is formed by the two capsid proteins vp and vp , which are most frequently used to classify rvas into g (for glycoprotein) and p (for protease-sensitive) genotypes, respectively (abe et al., ; matthijnssens et al., ) . this dual classification system has accelerated the comparison according to species-specific patterns among various animal species. to date, based on genetic characterization, g and p genotypes have been identified in humans and animals (li et al., ) . lapine rotavirus (lrv) strains have been isolated in canada, china, japan, italy, hungary, and the united states (banyai et al., ; bonica et al., ; ciarlet et al., ; guo et al., ; hoshino et al., ; martella et al., ) , and those that have been characterized belong to the vp g genotype. the g genotype has been described in various different host species, such as humans, rabbits, pigs, birds, bats, cats, dogs, monkeys, horses, mice, cows, and lambs (bonica et al., ) . the p [ ] and p [ ] genotypes of vp have mainly been reported in lrvs (banyai et al., ; martella et al., ; martella et al., ) . additionally, the p[ ]/[ ] genotype has been identified in porcine (collins et al., ; tonietti et al., ) . lrv is recognized as a potential source of human infection (bonica et al., ) ; however, to date, little is known about the molecular characteristics of rotavirus infection in rabbits in the republic of korea (rok (matthijnssens et al., ; parreno et al., ; schoondermark-van de ven et al., ) . the objective of this study was to analyze an lrv isolated from the intestine of a dead rabbit in in the rok by performing a complete genomic sequence analysis of the genome segments and to characterize the phylogenetic relationships between our isolate and other lapine isolates/strains. in , young rabbits died of acute enteritis in a flock of approximately rabbits. a domesticated rabbit that died suddenly was sent to the animal disease diagnostic division, animal and plant quarantine agency, rok, for post-mortem examination, where routine bacterial examination of the intestinal content was also performed. rabbit enteritis-related virological examination was performed, and rotavirus was detected using rt-pcr. the rabbit rotavirus rva/rabbitwt/kor/rab / /g p[ ] (rab ) was isolated from the intestinal content of the rabbit. viral rna was extracted from fecal suspensions using a rneasy mini kit (qiagen, hilden, germany) according to the manufacturer's instructions. the total rna was eluted in μl of rnase-free water and stored at − °c until use. rt-pcr was performed using × one-step rt-pcr smart mix (solgent, daejeon, korea). the primers used for amplifying vp -vp , vp , vp , and nsp -nsp were as described previously (de leener et al., ; matthijnssens et al., ; zeller et al., ) . briefly, rna was denatured at °c for min and quenched on ice. reverse transcription was performed at °c for min, followed by °c for min, and then cycles of amplification [at °c for s, °c for s (vp , vp , vp , and nsp -nsp ), °c for s (vp -vp and nsp ), °c for min (vp -vp and nsp ) and °c for min (vp , vp , and nsp -nsp )], and a final extension step at °c for min (vp -vp and nsp ) and °c for min (vp , vp , and nsp -nsp ). each pcr product was purified using the accupower pcr purification kit (bioneer, daejeon, korea). pcr amplicons were directly cloned into pgem®-t easy vector (promega, madison, wi, usa), which was then used for direct sequencing (macrogen inc., daejeon, korea). the obtained sequence data were analyzed using the basic local alignment search tool of the national center for biotechnology information database. homologous sequences were analyzed using the chromas software (version . , http://www.technelysium.com.au/ chromas.html) and aligned using clustalx (version . ). phylogenetic trees were constructed based on the genome segments of lrv using the maximum-likelihood method and a kimura -parameter (kimura, ) substitution model by employing mega software (kumar et al., ) . to construct each phylogenetic tree, additional sequences were obtained from genbank (http://www.ncbi.nlm.nih.gov). the complete nucleotide sequences of the genome segments of rab obtained in this study were assigned the accession numbers mk and mk -mk . the intestinal content of the rabbit was tested and found to be positive for rotavirus alone. no other enteric pathogens, such as e. coli and coronavirus, were detected. the nucleotide sequences of the genome segments of rab were completely identified, whereas the vp gene ( bp) of rab was partially sequenced. based on the nucleotide sequence identities, the vp , vp , vp , vp - , and nsp - genome segments of rab were observed to possess g -p[ ]-i -r -c -m -a -n -t -e -h , which is different from the constellation observed in previously characterized lrv strains (table ) . on comparison of the genotype of rab with that of the italian lapine strain - , chinese lapine strain n , and the dutch lapine strain k , differences were found in five (vp - , vp , nsp , and nsp ), five (vp , vp , nsp , nsp , and nsp ), and eight genes (vp - , vp , nsp , nsp , and nsp ), respectively (table ) . interestingly, rab shared eight identical genes with rch (vp - , vp - , nsp - , and nsp ) and rrv (vp - , vp - , and nsp - ). furthermore, rab shared seven genotypes with the canine strain cu- , canine-like human strain hcr a, bat strain lzhp isolated in china, and equine strain e (table ) . the sequences of genome segments of rab were analyzed and the genetic relationships between rab and other known lrvs as well as rvas were compared. the vp genome segment of rab was most closely related to the chinese bat strains, myas ( . %) and lzhp ( . %), and the argentine horse strain e ( . %) but not to the lrvs ( fig. and table ). the vp sequence was compared with the currently known representative rabbit strains. the vp gene of rab exhibited a maximum nucleotide similarity of . % with the lapine strain - from hungary followed by . %, . %, and . % similarity with the - , - , and - italian rabbit strains; phylogenetic analysis revealed that the vp gene from rab clustered in the p[ ] genotype with lrvs ( fig. and table ). the vp gene of rab clustered most closely with the lapine strain - ( . %) as well as with the human lapine-like strains b ( . %) and be ( . %), which were previously demonstrated to have a lapine origin (matthijnssens et al., ) ; this vp gene of either simian, bovine, or human origin belonged to the i genotype (fig. ) . the vp gene of rab was closely related to lzhp ( . %) belonging to the r genotype. the vp gene of rab belonged to the c genotype and included bat, simian, and equine rva strains. rab clustered most closely with the bat strain myas ( . %). the vp gene segment of rab was closely related to rrv ( . %, table ). the nsp , nsp , and nsp genes of rab were closely related to rrv ( . %, . %, and . %) and e ( . %, . %, and . %) and belonged to the a , t , and e genotypes, respectively. the nsp gene segment of rab clustered in the a genotype with rrv ( . %). the nsp and nsp genes of rab showed genetic relatedness to rrv ( . % and . %) and lzhp ( . % and . %), whereas the nsp and nsp genes of the lapine strain - and the human lapine-like strains b and be clustered in the t and e genotypes, respectively. the nsp and nsp genes of rab clustered in the n and h genotypes and were closely related to the strains - ( . % and . %), k (both genes . %), b ( . % and . %), and be ( . % and . %), respectively ( fig. and table ). although k originated in a rabbit, it was completely divergent from the n strain and shared three genotypes with rab and six with the - strain (fig. ) . to date, g and p genotypes of rotavirus have been identified (li et al., ) . among them, g has been detected in a broad spectrum of host species, including humans, indicating that g is more likely to undergo interspecies transmission than g , g and g . additionally, g is the only genotype identified in rabbits having vp specificity (banyai et al., ; guo et al., ; martella et al., ) . p [ ] for vp has been found in humans, goats, and rabbits (martella et al., ; matthijnssens et al., ; parreno et al., ) . previous studies have also identified p [ ] in the lapine-like human strains b and be and in the human bovine-like strain rch (bonica et al., ; donato et al., ) . consequently, p[ ] specificity in humans may be attributed to interspecies transmission. however, recently, p[ ] rotaviruses have rarely been reported in humans. moreover, p[ ] has mainly been found in rabbits in hungary and italy (banyai et al., ; martella et al., ) and also identified in porcine (collins et al., ; tonietti et al., ) . this seems to be related to virus evolution. our results revealed that the p[ ] genotype was detected in a rabbit for the first time in the rok, suggesting that p[ ] has greater species specificity than p [ ] . because little lrv sequencing data are available, further studies are necessary to investigate the presence of the p[ ] genotype in other animal species in the rok. rab shared three genes with two lrvs ( - and n ), namely vp (g genotype), vp (m genotype), and nsp (a genotype), whereas the remaining eight genes (vp , vp , vp , vp , nsp , nsp , nsp , and nsp ) were different from each other (table ) . considering the lapine origin, the reason underlying differences in genotypes remains unclear. it is therefore presumed that its ancestor/origin is different. interestingly, of the genotypes, rab shares eight identical genotypes with rrv and rch . however, rch showed ≤ % sequence identify for a majority of rab gene segments. rrv, which was isolated from a juvenile rhesus macaque with diarrhea, shared gene segments (vp , vp , nsp - ) and showed ≥ % sequence identity with rab . rrv was most closely related to rab with regard to the vp , nsp , and nsp genes ( fig. and table ). additionally, rrv shared eight genotypes with the canine strain cu- , feline strain cat , and canine-like human strain hcr a (mino et al., ; tsugawa and hoshino, ) and three genotypes with the bovine-like rva strain (vp , vp , and nsp ) (matthijnssens et al., ) (table ) . rrv may present strong evidence for reassortment among different animals rva strains via interspecies transmission. rrv appears to have undergone reassortment a long time ago. so far, the infection source and host origin of rab remains unknown. these results indicate that rab is most closely related to the canine/feline ancestor of rrv. our genomic analysis showed that rab was more related to other animal strains, such as simian, bat, and equine, than rabbit. this implies that rvas can easily cross between different host species and are able to spread successfully and cause diseases in a new host. several studies have reported that human and animal rvas originate from complex animal-human reassortment or interspecies transmission events, presumably because of the close proximity of humans to livestock and companion animals (banyai et al., a; banyai et al., b; ghosh et al., ; guo et al., ; matthijnssens et al., ; matthijnssens et al., ) . rab shared the following genes with a previous phylogenetic analysis revealed that rab showed a high sequence identity for vp ( . %), nsp ( . %), and nsp ( . %) genes with the human strain be , representing a rabbit to human interspecies transmission which can cause disease development in humans; rab was closely related to the bovine strain (bonica et al., ) . another lrv strain, k , has a genotype constellation identical to that of a bovine strain, which was isolated from a -month-old boy with gastroenteritis in slovenia (steyer et al., ) . interestingly, lapine and lapine-like strains have been derived from a population of previously characterized bovine strains and bovine-like ancestral strains, suggesting a past reassortment event between a bovine and lapine. rab was closely related to the bat strain lzhp with regard to vp ( . %), vp ( . %), vp ( . %), and nsp oem, et al. infection, genetics and evolution ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ( . %) genes. however, numerous genes of bat strains possessed canine/feline characteristics. bats may be important zoonotic reservoirs that readily transmit animal rvas, facilitating reassortment events. the present results indicate that rab possesses genes derived from various host species; however, it was impossible to accurately determine the host species that transmitted rab . therefore, rab identified in the rok may have originated from other animal strains, instead of rabbits, as a result of multiple reassortment events. in conclusion, the present findings suggest that rab is closely related to the simian strain rrv and not lrvs. g p[ ] identified in this study has newly emerged in rabbits in the rok and may have more species specificity. the results revealed that rab is an interspeciestransmitted virus, which may have developed owing to multiple reassortments among canine, feline, and bovine hosts and subsequent transmission to a rabbit. the results of this study reinforce the important role of animals in the ecology and evolution of rvas as well as highlight the potential of this virus to cross between species. additional studies should emphasize on the importance and the need for continued surveillance of rvas in animals. oem, et al. infection, genetics and evolution ( ) - rva/cat-tc/aus/cat / /g p rva/rabbit-tc/ita/ - / /g p[ ] rva/rabbit-tc/ita/ - / /g p[ ] rva/cat-tc/aus/cat / /g p[ ] rva/rabbit-tc/ita/ - / /g p[ ] rva/rabbit-tc/ita/ - / /g p[ ] rva/cat-tc/aus/cat / /g p[ ] rva/rabbit-tc/ita/ - / /g p[ ] rva/rabbit-tc/ - / /g p[ ] whole genome characterization of new bovine rotavirus g p[ ] and g p[ ] strains provides evidence for interspecies transmission identification of the novel lapine rotavirus genotype p [ ] from an outbreak of enteritis in a hungarian rabbitry genetic diversity and zoonotic potential of human rotavirus strains genetic heterogeneity in human g p[ ] rotavirus strains detected in hungary suggests independent zoonotic origin complete genome analysis of a rabbit rotavirus causing gastroenteritis in a human infant update on rotavirus vaccines comparative amino acid sequence analysis of the outer capsid protein vp from four lapine rotavirus strains reveals identity with genotype p[ ] human rotaviruses detection and characterisation of group a rotavirus in asymptomatic piglets in southern ireland human infection with a p[ ], g lapine rotavirus genetic characterization of a novel g p[ ] rotavirus strain causing gastroenteritis in year old australian child rotavirus gene structure and function full genomic analysis and possible origin of a porcine g rotavirus strain ru full genomic analysis of rabbit rotavirus g p[ ] strain n in china: identification of a novel vp genotype characterization of neutralization specificities of outer capsid spike protein vp of selected murine, lapine, and human rotavirus strains a simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences mega : molecular evolutionary genetics analysis version . for bigger datasets rva/rabbit-tc/ita/ - /g p[ ] identification of novel and diverse rotaviruses in rodents and insectivores, and evidence of cross-species transmission into humans molecular characterization of the vp , vp , vp , and nsp genes of lapine rotaviruses identified in italy: emergence of a novel vp genotype lapine rotaviruses of the genotype p[ ] are widespread in italian rabbitries full genomic analysis of human rotavirus strain b and lapine rotavirus strain / provides evidence for interspecies transmission are human p[ ] rotavirus strains the result of interspecies transmissions from sheep or other ungulates that belong to the mammalian order artiodactyla? simian rotaviruses possess divergent gene constellations that originated from interspecies transmission and reassortment equine g p[ ] rotavirus strain e related to simian rrv and feline/canine-like rotaviruses based on complete genome analyses molecular characterization of the first isolation of rotavirus in guanacos (lama guanicoe) rotavirus proteins: structure and assembly rabbit colony infected with a bovine-like g p [ ] rotavirus strain whole genome sequence analysis of bovine g p[ ] rotavirus strain found in a child with gastroenteritis phylogenetic analyses of the vp and vp genes of porcine group a rotaviruses in sao paulo state, brazil: first identification of g p[ ] in piglets whole genome sequence and phylogenetic analyses reveal human rotavirus g p[ ] strains ro and hcr a are examples of direct virion transmission of canine/feline rotaviruses to humans full genome characterization of a porcine-like human g p[ ] rotavirus strain isolated from an infant in belgium the authors declare no conflicts of interest. supplementary data to this article can be found online at https:// doi.org/ . /j.meegid. . . . key: cord- - z fp u authors: magdi, mohamed; rahil, ali title: severe immune thrombocytopenia complicated by intracerebral haemorrhage associated with coronavirus infection: a case report and literature review date: - - journal: eur j case rep intern med doi: . / _ sha: doc_id: cord_uid: z fp u immune thrombocytopenic purpura (itp) is an autoimmune disorder that causes isolated thrombocytopenia. many viruses have been identified as triggering the autoimmune process, including hiv, mcv, ebv, parvovirus, rubella and measles. however, itp in association with coronavirus infection has not previously been reported. we describe the case of a healthy man who presented with severe itp complicated by intracranial haemorrhage following upper respiratory tract infection. an infection screen revealed coronavirus infection. learning points: coronavirus can cause severe immune thrombocytopenic purpura (itp). intracerebral haemorrhage is an uncommon presentation of itp. intravenous immunoglobulin and steroids are very effective treatments for severe itp. immune thrombocytopenic purpura (itp) is a rare haematological disorder formerly known as idiopathic thrombocytopenic purpura before the autoimmune mechanism was identified and found to be triggered by many, mostly viral, infectious agents. most cases of itp are benign with only minor mucosal bleeding as the main presentation. we present a case of severe thrombocytopenia complicated by intracranial haemorrhage following infection with a coronavirus, which has not previously been reported. a -year-old healthy man presented to the emergency department with prolonged gum bleeding and a -day history of a diffuse skin rash on his extremities and abdomen. he was in his usual state of health until week before presentation when he developed fever and a runny nose. he reported no joint pain, oral ulcers or raynaud's phenomenon. he denied intake of any medications. his family history was not contributory. upon arrival at the emergency department, the patient was awake and oriented, with blood pressure / mmhg, heart rate bpm, temperature . °c and respiratory rate breaths/min. physical examination revealed signs of gum bleeding. examination of the skin was remarkable for petechiae over the chest and limbs. the remainder of the systemic examination was unremarkable. laboratory findings were as follows: haemoglobin: g/dl; wbc: . × /mm ; platelets: , /µl; ptt: . sec; pt: . sec; inr: . . a peripheral blood smear was normal. respiratory viral panel pcr was positive for coronavirus hku . autoimmune work-up including ena and anca serology was negative. frequent platelet transfusions, intravenous immunoglobulin g/kg for days and intravenous dexamethasone mg for days were initiated. the next day, the patient developed a headache and vomiting, and his level of consciousness deteriorated to a glasgow coma scale score of / . urgent ct of the head showed a large left frontal intracerebral haemorrhage extending into the ventricles with a midline shift of mm. after days, the platelet count rose to , /µl and then , /µl. the patient`s level of conscious and muscle power gradually recovered. after month of rehabilitation, he was discharged in a good state of health. itp is characterized by isolated low levels of circulating platelets (plt < , /µl) secondary to autoimmune destruction of platelets or inhibition of synthesis. it varies from mild to severe disease with lethal sequelae. the severe form includes bleeding requiring treatment which usually occurs with a platelet count < , /µl. itp may present acutely or chronically; the chronic form is defined as thrombocytopenia of more than months' duration since initial clinical presentation. acute itp is common in children (< years) in contrast to the chronic form which is more common in adults. the exact mechanism of itp is poorly understood, with many hypotheses claiming that viral infection triggers the disease after which preformed antibodies cross-react with platelet antigens [ , ] . clinical presentation varies from the more common petechiae, purpura and mucous membrane bleeding (epistaxis or gum bleeding) to the rare severe gastrointestinal or intracranial bleeding, which has been reported in . % of patients [ ] . viruses thought to cause itp include hiv, hcv, cmv, ebv, herpes viruses and vzv [ ] [ ] [ ] . in , wright [ ] reported a case of severe thrombocytopenia secondary to asymptomatic cmv infection. in , hamada et al. [ ] described a patient with severe thrombocytopenia associated with varicella zoster infection whose platelet count returned to normal after antiviral treatment. interestingly, zea-vera and parra [ ] reported a case of itp exacerbation that was secondary to zika virus infection. an association between itp and some bacterial infections such as tuberculosis and helicobacter pylori has been documented [ ] . however, a connection between itp and infection with coronavirus, even though the virus is common, has not previously been reported in the literature. infection with coronavirus (cov) has been associated with severe acute respiratory syndrome (sars). haematological changes in patients with sars are common and notably include lymphopenia and thrombocytopenia. the development of thrombocytopenia may involve a number of mechanisms. although the development of autoimmune antibodies or immune complexes triggered by viral infection may play a significant role in inducing thrombocytopenia, sars-cov may also directly infect haematopoietic stem/progenitor cells, megakaryocytes and platelets, inducing their growth inhibition and apoptosis [ ] . in contrast, we report severe thrombocytopenia following mild coronavirus upper respiratory tract infection. coronavirus infections were considered benign until the sars outbreak of when the their virulence attracted increased attention and new group members like cov.hku were identified. cov.hku was discovered in in hong kong in an adult with chronic pulmonary disease [ ] and is now considered to be associated with acute respiratory infections. coronavirus is not a well-known cause of immune thrombocytopenia even though it may possibly cause severe itp. therefore, a respiratory viral panel test should be used in the initial assessment of a patient with immune thrombocytopenia. characterization of autoantibodies against the platelet glycoprotein antigens iib/iiia in childhood idiopathic thrombocytopenia purpura varicella-associated thrombocytopenia: autoantibodies against platelet surface glycoprotein v severe bleeding events in adults and children with primary immune thrombocytopenia: a systematic review cytomegalovirus can make immune thrombocytopenic purpura refractory viral infection of megakaryocytes in varicella with purpura idiopathic thrombocytopenic purpura after human herpesvirus infection severe thrombocytopenia secondary to asymptomatic cytomegalovirus infection in an immunocompetent host a case of varicella-associated idiopathic thrombocytopenic purpura in adulthood zika virus (zikv) infection related with immune thrombocytopenic purpura (itp) exacerbation and antinuclear antibody positivity effects of eradication of helicobacter pylori infection in patients with immune thrombocytopenic purpura: a systematic review thrombocytopenia in patients with severe acute respiratory syndrome characterization and complete genome sequence of a novel coronavirus, coronavirus hku , from patients with pneumonia key: cord- -ktet wl authors: lim, jong-min; do, eunju; park, dong-chan; jung, go-woon; cho, hyung-rae; lee, seo-young; shin, jae wook; baek, kyung min; choi, jae-suk title: ingestion of exopolymers from aureobasidium pullulans reduces the duration of cold and flu symptoms: a randomized, placebo-controlled intervention study date: - - journal: evid based complement alternat med doi: . / / sha: doc_id: cord_uid: ktet wl aim: the objective of the study was to assess the efficacy of exopolymers from aureobasidium pullulans (eap) on the incidence of colds and flu in healthy adults. methods: we conducted a randomized, double-blind, placebo-controlled study at the onset of the influenza season. a total of subjects ( – years of age) were recruited from the general population. the subjects were instructed to take one capsule per day of either eap or a placebo for a period of weeks. the duration of cold and flu symptoms, a primary variable in assessing effectiveness, and serum cytokine levels as well as wbc counts as secondary variables were also evaluated. results: eap was associated with a statistically significant decrease in the duration of cold and flu symptoms, a primary variable in assessing effectiveness. although cold and flu symptom levels were not significantly different at a significance level of %, the cold and flu symptom levels of the eap group were less severe compared to the placebo group. no statistically significant changes of serum cytokine levels as well as wbc counts were observed. conclusion: the results showed that eap is a useful pharmaceutical and functional food material for preventing and treating colds and flu. the immune system has evolved to protect multicellular organisms, including humans, from pathogens. immunostimulatory effects are regulatory actions that counteract reduced immune responses caused by immunodeficiency diseases, viral infections, malnutrition, tumors, and aging. two strategies have been developed to stimulate the immune system. one method is to increase antigen-specific or nonspecific immune responses, and the other method involves the use of appropriate immunosupplements for antigen administration [ ] . respiratory viruses are among the most infectious pathogens in humans, and many differences occur in the kinds of pathogens, their antigenicities, and their worldwide infection patterns. recently, respiratory diseases such as middle east respiratory syndrome, severe acute respiratory syndrome, and the flu caused by novel swine-origin influenza a strains have emerged and begun to spread rapidly [ ] [ ] [ ] . as the associated respiratory viruses show very strong tendencies to spread, there is a growing interest in foods that promote immune functions as well as a global monitoring system for influenza [ ] . the results of several researches have revealed a positive correlation between food nutrition and the control of human immune functions, and various foods have been recognized as functional foods that can improve immune functions. these food materials may be derived from vegetables [ ] , evidence-based complementary and alternative medicine seafood [ ] , mushrooms [ ] , microbes such as lactic acid bacteria [ ] , chitosan [ ] , and peptides [ ] . - , / , -glucan is derived from yeast cell walls and modulates many in vivo and in vitro activities [ , ] . its main immunopharmacological activities [ ] [ ] [ ] are associated with antitumor effects [ ] , increased host resistance to viral, bacterial, and parasitic infections [ ] , and adjuvant effects [ ] . proximate composition of extracellular polysaccharides of aureobasidium pullulans is carbohydrate . %, sugar . %, crude protein . %, and moisture . %. namely, above % of the proximate composition is polysaccharides and there is a small amount of protein and fat (personal comm. dr. dong-chan park). it has been reported that the extracellular polysaccharides isolated from aureobasidium pullulans are pullulan (poly-- , -maltotriose-based exopolysaccharide, eps) [ ] , aubasidan ( - , -d-glucan with - , -branches to - , side chains) [ ] , and -glucan ( - , / , -d-glucan) [ ] . however, the physiological activity of polysaccharides produced by aureobasidium pullulans has been mainly studied for - , / , -d-glucan [ ] . exopolymers purified from aureobasidium pullulans sm- (eap; its solubility is about % in water), the test food material used in this study, containing % - , / , -glucan [ ] , are used in foods and have shown potent immunomodulatory activities in a mouse model [ ] . in addition, eap showed no adverse events in clinical studies designed to evaluate its effectiveness on bone metabolism improvement [ , ] and on atopic dermatitis [ ] . therefore, -glucan is considered safe for human ingestion. many reports have described the immunomodulatory activities of -glucan against immune-related diseases [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , but only a few reports have described its effects against colds and flu. for this reason, a clinical study is now being performed to evaluate the efficacy and safety of -glucan from aureobasidium pullulans against colds and flu in patients. in this study, we selected adult subjects who provided written, informed consent to participate, who satisfied our selection criteria, and who did not meet exclusion criteria. we randomly administered eap or a placebo, where subjects took a daily dosage of mg for weeks. we evaluated immunological improvements of the test food by observing functional changes in the immune system before and after administering the test food materials in the eap and placebo groups. we also observed the duration and total levels of cold and flu symptoms during the test period. the eap and placebo products used in this clinical research study were packaged inside boxes provided by glucan co., ltd. (busan, korea) and were supplied and distributed by aribio co., ltd. (seongnam-si, gyeonggi-do, korea). some eap samples were deposited in the herbarium of silla university (busan, korea; encoded as eap choi ). sixty-five capsules were provided to each subject, taking into consideration an -week dosage ( days, capsules), a visitation window of days ( capsules), and storage products ( capsules). the test food was manufactured as maroon-colored hard capsules, and each capsule of test food contained mg of eap and . mg of microcrystalline cellulose, whereas the placebo food contained . mg of microcrystalline cellulose. the test food was supplied to a testing agency in sealed white plastic bottles, each containing capsules intended for person. the test foods used in this study were identical in appearance and description and would be difficult to distinguish with the naked eye. the experiment was conducted in doubleblind format for the subjects and researchers by attaching identical labels to the test foods. the main ingredients, generic names, and lot number were not recorded on the test food labels to avoid exposing the differences between the test groups. subjects took eap hard capsule once daily for weeks on an empty stomach, with a sufficient amount of water. the subjects were healthy adults who did not have any disease that required proactive treatment. the number of subjects assigned to each group was ( overall). in consideration of a potential % dropout rate, the number of subjects in each group would decrease to , the minimum number of subjects for an effectiveness analysis ( figure ). subjects included in this study had to meet the following criteria: adults between and years of age; individuals with a white blood cell (wbc) count of - × / l; individuals who could be monitored throughout the test period; individuals who listened to a thorough explanation of the study's purpose and contents and voluntarily signed an informed consent form to participate in the experiment. subjects for whom the following criteria were applicable were excluded from the experiment: individuals with a body mass index (bmi) under or over ; individuals who exceeded the normal maximum alanine transaminase and aspartate transaminase levels by -fold; females who were pregnant or were breast-feeding; females of childbearing age who did not agree to use contraceptives via medically proven methods (e.g., condoms, lubricant, and femidom) during the test period; individuals with a fasting plasma dextrose concentration over mg/dl; individuals with high blood pressure (systolic blood pressure of mm hg or diastolic blood pressure of mm hg); individuals continuously using medicine that could affect the effectiveness assessment (hyperlipidemia medicine, steroid medicines, hormone medicines, immunosuppressants, and antibiotics); individuals who require continuous treatment for psychiatric disorders such as anorexia, depression, and manic depression; individuals with systemic diseases such as immunity-related diseases, serious hepatic and renal insufficiencies, malignant tumors, pulmonary disease, collagenosis, multiple sclerosis, allergic skin conditions, and other autoimmune diseases; individuals with a medical history of drugs and clinically significant allergic reactions; individuals with a history of gastrointestinal disorders that could affect the absorption of the test foods or a history of gastrointestinal surgery (excluding a simple appendectomy or hernia operation); individuals who consumed medicine or herbal medicines within a month of participation in the experiment which could affect immunity; individuals who participated in a different human study or clinical test and took experimental products within months of participation in this experiment (excluding human studies with cosmetics); and individuals whom the researchers otherwise determined might have difficulty completing the experiment. dosages of the experimental products were temporarily suspended at instances where it was appropriate to stop the experiment for the welfare of the subjects. events that may have occurred. in the following cases, administration of the test foods was temporarily suspended: (a) a temporary interruption on the occurrence of an adverse reaction or for the treatment of an adverse reaction; (b) when showing adverse events related to the safety of the test foods; and (c) in case of showing acute reactions (allergies and hypersensitivity) to the test foods. however, in the case of a temporary interruption for the occurrence of an adverse reaction or for the treatment of an adverse reaction, if there was no problem in its compliance, the study was continued. cases that were processed as dropouts included those in which test foods were allocated and the subject or a legal representative withdrew consent from participation in the experiment, cases where results for a final inspection could not be obtained, cases where subjects did not adhere to the test protocol resulting in a significant effect on the test results, cases where there was an interruption in contact with the subject (i.e., when the subject could not be traced), cases where the subject failed to make visits, subjects for whom a disease was discovered which went unnoticed during the screening inspection, cases where compliance with usage of the test food fell below %, as assessed with the usage log, cases in which, without the direction of the researcher, the subject used a medicine or product while they were taking the test food which could affect the research results or interpretation of data, cases in which the subject had an average weekly drinking quantity as ethanol of g/week throughout the test period, or cases where the lead researcher determined that the research proceedings were inappropriate or that there could be a significant effect on the safety of the subjects or the experiment results. subjects stopped taking test foods and were processed as dropouts for the following reasons, in which it was not possible to continue the human study: cases in which a serious adverse event occurred with the subject, cases in which an adverse event made participation in the experiment impossible, cases in which the researcher determined that the usage and observation of the test foods would be impertinent, or cases in which clinical tests were not possible due to the death of a subject or emergence of a disease. subjects who took the test foods and later dropped out of the study, where a portion or the entire estimate value of the final assessment (visit ) was omitted, were included in the itt group's analysis data. missing values were processed after a dropout occurred using missing value processing method for effective assessments. the results of all subjects who dropped out were excluded from assessment of the per protocol (pp) group ( figure and table ). . . . drinking, diet, and exercise among the subjects. during the experiment, subjects maintained regular levels of drinking, diet, and exercise which were similar to levels prior to participation in the experiment. to verify this, on the day of base evaluations, we examined the dietary contents evidence-based complementary and alternative medicine for contiguous days prior to the visitation day and the weekly exercise types and times prior to the visitation day. the number of alcoholic drinks and the amount of ethanol consumed during the week prior to the visitation day were also examined. subjects kept a daily record of whether they consumed the test foods and recorded contiguous days of their diets, including saturday or sunday. exercise, alcoholic drinks, and test food consumption were recorded in a chart provided by the testing agency, a comparison was made against these quantities prior to participation in the experiment, and observations were made as to whether there were any changes that could have significantyl affected the experiment results. subjects were assigned to the eap group (the group taking eap) and the placebo group (the group taking the placebo) based on a randomized list produced with a random sampling table generated using microsoft excel on microsoft windows ( figure ). the exclusion criteria used to select subjects during the screening visit (visit ) were verified, and individuals who met selection criteria and had met no exclusion criteria were selected as subjects for this study. food materials were allocated to the subjects during visit . the subjects selected for the experiment were issued a subject number in the order of visits made during visit and were provided with the test foods, which were randomly allocated. the foods used in the human test were provided only to the participating subjects, based on the process described in the human test protocol ( table ) the visitation dates allowed for the set base visitation days + additional days. those who were selected as test subjects received the test foods within days from the base evaluation date (selection inspection date) and began taking the test foods by the next morning. demographic data including the initials, age, gender, birthdate, weight, and height of the subjects and basic information such as medical history, medicinal intake history, combined treatment, concomitant drugs, smoking, drinking, and exercise habits were recorded in detail. the contents during the last years were recorded in the medical histories, and contents during the last weeks were recorded in the medicinal intake history. the types of exercise and the exercise times were examined for week prior to the time of the visitation day. diet and exercise were compared during the base evaluation period and experimental period and observed to determine whether any important changes occurred which could affect the test results. the number and quantity of alcoholic drinks consumed during the week preceding the visitation day were examined, and that information was converted into the number of grams of ethanol consumed and recorded. levels. blood pressure, body temperature, and pulse were measured and recorded as vital signs. obesity levels were measured using a body fat analyzer (inbody . , biospace, korea), and the bmi (kg/m ), percent evidence-based complementary and alternative medicine my daily life was very uncomfortable due to symptoms, and proactive treatment was needed. in the case of severe discomfort, i may stop working or studying or be admitted to the hospital body fat (%), waist-hip ratio, and visceral fat area (cm ) were recorded. hematological examination of wbcs, red blood cells, hemoglobin levels, hematocrits, and platelets was performed using an automated analyzer (sysmex xp- ; sysmex, kobe, japan). the levels of blood urea nitrogen, creatinine, uric acid, total bilirubin, albumin, total protein, alkaline phosphatase, glucose, alanine transaminase, aspartate transaminase, gamma-glutamyl transferase, triglyceride, total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol were measured using a clinical chemistry analyzer (dimension xpand plus; siemens, munich, germany). all urine samples were stored in the dark at ∘ c and were analyzed within two hours of collection. the ph, specific gravity, and protein and glucose levels were measured using a semiautomated analyzer (uriscan pro ii; yd diagnostics, seoul, korea). pregnancy testing was performed using a kit detecting human chorionic gonadotropin. serum concentration of cytokines was analyzed by using hmaglxsa ( plex) multiplex elisa kit (r&d systems, minneapolis, mn). cytokine levels were determined using luminex (luminex, austin, tx, usa) and the data were reported as median fluorescent intensities. comparisons. if immune functions improve, the disease duration and symptom levels should decrease due to increased resistance to viral diseases such as colds and flu. accordingly, we examined the duration and levels of cold and flu symptoms after consuming the test food products as a functional index of immunological improvements. for the survey following the administration of test foods, a form was provided in which subjects made a daily record of their health conditions, with a daily record of their test food dosages. nine symptoms were monitored to inspect the health conditions of the subjects in the survey: fever, chills, runny nose, nasal congestion, coughing, sneezing, sore throat, headache, and gastrointestinal symptoms (nausea, vomiting, diarrhea, and abdominal pain). subjects marked i for "symptoms exist" and × for "no symptoms." to record the symptom levels, subjects used a -step scoring point system, where they marked ( ) for no symptoms, ( ) for minor symptoms, ( ) for moderate symptoms, and ( ) for serious symptoms [ ] and marked whether they took early leave or were absent from work or school due to symptoms. table shows a detailed explanation of the criteria used for reporting symptom levels. to assess the effectiveness of the experimental food products, subjects kept daily records in their dosage logs of cold and flu symptom durations and symptom levels during the testing period. compliance with ingesting test foods was evaluated based on the food-usage logs. during the final visit, the number of remaining foods collected was compared with the usage log records. in cases where there were differences between the recorded and actual amounts of food taken, compliance was verified when the differences were small. primary effectiveness assessment variables included the duration and total score of symptom levels for cold and influenza. secondary effectiveness assessment variables included serum il- , il- , il- , il- , tnf-, and inf-levels, as well as wbc counts and differential blood counts. evidence-based complementary and alternative medicine on significance comparisons between the eap and placebo groups. the methodologies described below were followed for detailed analysis. analysis sets obtained from the test subjects were composed largely of an itt analysis group and a pp analysis group. the itt group comprised the primary data set used for effectiveness assessments, and the pp group was additionally analyzed to assess the effectiveness. subjects in the itt group included those who had taken the test food materials and whose assessment variables were measured at least once during visit . subjects in the pp group included those in the itt group who completed the test according to the study protocol. it has been predicted that fewer subjects will contract viral diseases such as cold and flu if their immune functions are enhanced and that the duration and levels of symptoms will decrease if one of the diseases is contracted. the activation state of principal cells related to immunity can be determined by measuring changes in the serum contents of cytokine il- , il- , il- , il- , tnf-, and inf-. to assess the effectiveness of the test food materials, the significance of the mean difference (visits - ) was tested for each variable included in the effectiveness test. in other words, a statistical comparison was made between the average variation before and after food ingestions in the eap and placebo groups. to this end, normality was tested for the evaluation data. the parametric method of a twosample t-test was chosen if normality was satisfied, whereas the nonparametric method of a wilcoxon's rank-sum test was chosen if normality was not satisfied. in addition, the above significance tests were performed on mean differences between the eap and placebo groups between each visit after test food ingestions. to analyze the prevalence of cold symptoms, subjects recorded the duration and levels of symptoms when they contracted a cold or the flu prior to ingesting the test food. during visit , subjects recorded the duration and total symptom scores of cold and influenza symptoms during the test period. accordingly, it was not meaningful to compare the mean difference between visits, considering that the rating scales used in visits and were different. in other words, we confirmed homogeneity between the test groups in visit and evaluated the effectiveness by comparing the results between test groups in visit . forward (locf) method (a single-imputation method) for data missing in the final effectiveness assessment. typically, the locf method is easy to use in clinical trials, despite the restricting assumption where missing data are treated as a constant. the locf method is commonly used because it provides conservative results. however, there are no commonly recommended methodologies for processing missing values [ ] . in this study, the statistical analytic data collected was comprised of only the baseline assessment prior to the ingestion of test food materials and the final assessment after ingestion of test food materials. the missing values during the final assessment were not replaced by the base assessment values but were substituted by the average of each group and assessed. in this study, we screened individuals who voluntarily agreed to participate in this human study, and subjects who met the selection criteria and did not meet any exclusionary criteria were enrolled. among the subjects, were assigned to the eap group and were assigned to the placebo group ( figure ). four instances were verified in which subjects violated the study protocol while consuming the test foods. these subjects were processed as dropouts. among the subjects who were selected, individuals from the eap group dropped out due to violations in the visitation schedule and shortfalls in compliance, and individuals from the placebo group dropped out due to withdrawal of informed consent and shortfalls in compliance. the remaining subjects adhered to the test protocol and completed the test. the current states of dropouts are separated according to the test group ( figure ) . compliance with ingesting test food materials was assessed in visit . the contents recorded in the daily record filled out by the subjects as well as the quantity of remaining products returned during the final visit were confirmed and assessed. compliance was calculated using the following formula: compliance (%) = (number of food materials ingested/number of food materials that should have been ingested) × , where the number of food materials ingested = number of food materials provided − number of food materials returned. subjects whose compliance was below % were set as dropouts, and there were dropouts in this study due to compliance shortfalls. the compliance of test food material ingestion was . ± . % in the eap group and . ± . % in the placebo group. a significant difference in compliance between the test groups was not observed (table ) . a homogeneity test was conducted between test groups for the following: measurement values prior to food material ingestion regarding demographic information of the subjects including age, gender, medical history, treatment history, medication history, and concomitant medication and measurement values before and after ingestion of food materials regarding items that could affect the test results based on changes in lifestyle habits during the test period including regular exercise, smoking, and drinking. the results of a preliminary investigation on the age, gender, medical history, treatment history, medication history, concomitant medication, exercise, smoking, and drinking among the subjects are summarized in tables and . no significant differences were found between the test groups regarding any of the items examined. missing values were replaced with averages and used in the itt group data analysis. (a) (b) figure : foundational statistics and comparisons of cold symptom durations (days; (a)) and levels (b) between test groups: eap group (black columns) and placebo group (gray columns). variables in the itt group. the results of a -test performed to evaluate significant differences between groups before and after ingesting the test food materials verified that the duration of cold symptoms ( value = . ) and cold symptom levels ( value = . ) at visit were not significant at a significance level of %. statistical analysis also showed that no difference occurred between test groups prior to the ingestion of food materials. during visit , the duration of cold symptoms in the experiment group ( . ± . days) was lower than that of the placebo group ( . ± . days). the difference between the test groups was statistically significant ( value = . ). the eap group also had a lower score for cold symptom levels than the placebo group ( . ± . versus . ± . , resp.). this difference was significant at a level of % ( value = . ) but not % (figure ). were verified with respect to the mean difference of each measured item between the visitation times to assess the effectiveness of the test food materials on the blood test results. the results were not significantly different between the eap and placebo groups (table ) at a significance level of %. for wbc, basophil, and lymphocyte counts that wilcoxon's rank-sum test was used to analyze variables that did not follow normality. missing values were replaced with averages and used in the itt group data analysis. did not follow normality, a nonparametric wilcoxon's ranksum test was conducted. the results were not significantly different between the eap and placebo groups (table ) at a significance level of %. to assess the effectiveness of the test food materials, a -test was conducted to evaluate significance differences in the mean cytokine concentrations between visits - . the results were not significantly different between the eap and placebo groups (table ) at a significance level of %. il- , il- , il- , and tnf-values did not satisfy normality; a nonparametric statistics wilcoxon's ranksum test was conducted, revealing no significant differences in any of the items at a significance level of % (table ) . adverse events were observed in individuals from the placebo group and in individual from the eap group. in the placebo group, nausea and pruritus were confirmed in subject e , asthenopia and rash in subject e , and a sore throat in subject e . no other adverse events were observed aside from these. in the eap group, headache was confirmed in subject e , but no other adverse events were confirmed aside from this. all these adverse events subsided within a few days. in the case of subject e , who ingested the test food materials, the headache was completely alleviated after days, and the subject was judged as having no cause-and-effect relationship with ingesting the test food materials (data not shown). while the sgot or sgpt exceeds normal upper limit by fold, the finding is not considered to represent an abnormal phenomenon. however, the -gtp value was found to be abnormally high during visit for subject e , but examination of the results showed that the abnormal values were not related to the ingestion of test food materials. instead, the results were due to excessive drinking, where the subject consumed alcoholic drinks frequently ( days/week) and a high quantity of ethanol (average = g/week) (data not shown). with the occurrence of adverse events and abnormal changes in clinical test results, no items were found to correlate with the use of test food materials or were suspected to have a correlation. thus, the test foods were found to be safe in the context of this study. the various biological activities of -glucan were verified through in vitro testing, animal testing, and clinical trials [ ] . recently, the effectiveness of -glucan on respiratory diseases has been studied in rodent [ ] and human [ ] [ ] [ ] because their prevalence has increased rapidly. eap, exopolymers purified from aureobasidium pullulans sm- , containing % - , / , -glucan [ ] , are used in test foods and have shown potent immunomodulatory activities in a mouse model [ ] . however, this study is the first to evaluate the use of eap against respiratory diseases. to assess the ability of eap to improve human immunological responses and to alleviate cold or flu symptoms, adults aged - years with a white blood cell count of - x / l were screened. individuals were enrolled as subjects who satisfied the selection criteria and did not fall under any exclusion criteria (data not shown). in an aspect of subject's respiratory morbidity, none of the subjects had a respiratory disease at the time of enrollment. within weeks prior to the screening date of the subjects, there were subjects with light respiratory disease. after confirming no symptoms at the enrolment, the subjects were proceeded to obtain informed consent. the subjects (administrated with the test foods for a period of weeks) were randomly assigned to either eap or a placebo group. blood tests and surveys of cold or flu symptoms were conducted before and after ingesting test food materials, and the duration and symptom level of colds or flu, as well as the results of the blood test and cytokine analysis, were compared. the eap and placebo groups were composed of individuals that exhibited homogeneity and showed no statistically significant differences in their compliance of ingesting test foods or in gender, age, compliance, regular exercise, smoking, drinking, medical history, medicinal history, or concomitant drug administration. clinical data have shown that immunity-promoting functions can prevent acute respiratory infections from cold or influenza viruses and reduce the frequency of contracting colds or flu, symptom levels, and symptom durations based on effectiveness criteria [ ] . in this study, statistical analysis of the duration and level of cold symptoms, a primary variable for assessing effectiveness, showed that there were no differences in assessment values between the eap and placebo groups before ingesting test food materials. however, the duration of cold and flu symptoms after ingesting test food materials was significantly lower in the eap group than in the placebo group at a % significance level. the eap group also showed lower levels of cold and flu symptoms, which were statistically different at the % (but not %) significance level than placebo group. it is predicted that individuals whose immune functions are enhanced will contract fewer viral diseases such as colds and flu, with reduced durations and levels of symptoms. the activation state of primary cells related to immunity can be determined by measuring the serum levels of the cytokines il- , il- , il- , il- , tnf-, and inf- [ ] . according to the "guideline for safety and efficacy assessments of functional food on immune function improvement" from the ministry of food and drug safety of korea, changes in cytokine levels, wbc counts, t cell counts, and nk cell activity in the peripheral blood should be measured when determining the functionality of immunity reinforcement in humans [ ] . in this study, secondary variables including serum cytokines (il- , il- , il- , il- , tnf-, and inf-) and wbcs (neutrophil, eosinophil, basophil, lymphocyte, and monocyte) for assessing effectiveness did not exhibit statistically significant differences before and after ingestion of test food materials at a significance level of %. these results are consistent with those of choi et al. ( ) [ ] , where the daily intake level of eap (polycan tm ) or placebo food materials was set at mg, and changes in serum cytokines were studied (with the intention of observing effectiveness in immunity improvements of eap), but no statistically significant changes were found. although eap has shown potent immunomodulatory activities in a mouse model [ ] , no immunity improvement of eap on the human was observed in this study. the cytokine is a factor that needs to change suddenly according to the individual health condition and then return to the normal range [ ] . on the normal range of blood cytokine concentration, its individual variation is very large. thus, a large number of subjects are required to measure the normal range of cytokine concentrations [ ] . indeed, in a study of normal korean cytokine levels, subjects were studied [ ] , and in a study of normal american cytokine levels, subjects were examined [ ] to estimate the normal range of blood cytokine concentration. serval studies have shown that no changes in blood cytokine levels have been observed following ingestion of multivitamin and mineral supplement [ ] , probiotic supplement [ ] , fish oil [ ] , and cordyceps militaris [ ] , which are known to help improve immunity. this may be because the cytokine level increased during the test period and then returned to normal, or the number of subjects was small, indicating that the individual deviation range was not statistically significant. in this study, the total number of subjects was : in the test group and in the control group, respectively. therefore, the results of this study may not statistically overcome the individual variation range of the subject's cytokine level due to the small number of subjects. further research is needed on extensive clinical studies with clear statistical numberings to confirm the effects of eap on plasma cytokines. no significant adverse events occurred during the period of test food material intake, and adverse events (excluding colds) were observed in individuals from the placebo group (nausea, pruritus, asthenopia, rash, and sore throat) and in individual from the experiment group (headache). all these adverse events subsided within just a few days and were determined to have no cause-and-effect relationship with the ingestion of test food materials. changes in blood test values were also observed in the eap group with an increase in sgpt, tg, and -gtp values, but this was difficult to determine as being clinically abnormal. in addition, in an aspect of the respiratory morbidity, the subjects who suffered from cold during the test period appeared and then fully cured after the treatment, and no respiratory morbidity was shown in all subjects. therefore, eap was verified to be harmless to the human body. collectively, our data revealed that eap was associated with a statistically significant decrease in the duration of cold and flu symptoms, a primary variable in assessing effectiveness. although cold symptom levels were not different at a significance level of %, the cold and flu symptom levels of the eap group were less severe compared to the placebo group. no statistically significant changes were observed in the serum cytokine concentrations, wbc, and differential blood counts (secondary variables for assessing effectiveness). therefore, our data showed that eap is a useful medicine and functional food material for preventing and treating colds and flu. the data used to support the findings of this study are available from the corresponding author upon request. the authors declare that there are no conflicts of interest. human infection with mers coronavirus after exposure to infected camels from sars to avian influenza preparedness in hong kong whole-genome characterization of a novel human influenza a(h n ) virus variant, brazil epidemiological characterization of respiratory viruses detected from acute respiratory patients in seoul a review on immunostimulatory plants bioactive compounds from marine processing byproducts-a review mushroom immunomodulators: unique molecules with unlimited applications probiotic immunomodulation in health and disease application of chitin and chitosan derivatives in the pharmaceutical field bioactive peptides: production and functionality clinical and physiological perspectives of -glucans: the past, present and future functional food for immune regulation-beta-glucan a week randomized, double-blind human trial to compare the efficacy and safety of aureobasidium pullulans cultured solution and placebo on improvement of immune in subjects immunomodulatory activities of oat -glucan in vitro and in vivo immunomodulatory effects of aureobasidium pullulans sm- exopolymers on the cyclophosphamide-treated mice effect of glucan on natural killer (nk) cells: further comparison between nk cell and bone marrow effector cell activities purification of solubleglucan with immune-enhancing activity from the cell wall of yeast glucan as an adjuvant for a murine babesia microti immunization trial characterization and enzymatic hydrolysis of hydrothermally treated - , - , -glucan from aureobasidium pullulans an aubasidan-like -glucan produced by aureobasidium pullulans in thailand the structure of the carbohydrate moiety of an acidic polysaccharide produced by aureobasidium sp. k- production of ß , / , glucan by aureobasidium pullulanssm safety and efficacy of polycalcium for improving biomarkers of bone metabolism: a -week open-label clinical study randomized, doubleblind, placebo-controlled trial of the effects of polycan, ß-glucan originating from aureobasidium pullulans, on bone biomarkers in healthy women a clinical study for the efficacy and safety of ß-glucan from aureobasidium pullulans in mild to moderate atopic dermatitis patients a placebo-controlled trial of korean red ginseng extract for preventing influenza-like illness in healthy adults korea food and drug administration (kfda, guidelines for clinical trial statistics sample size calculations with dropouts in clinical trials Β-glucan derived from aureobasidium pullulans is effective for the prevention of influenza in mice immunomodulatory effect of pleuran ( -glucan from pleurotus ostreatus) in children with recurrent respiratory tract infections preventive effect of pleuran ( -glucan from pleurotus ostreatus) in children with recurrent respiratory tract infections -open-label prospective study placebo-driven clinical trials of yeast-derived ß-( , ) glucan in children with chronic respiratory problems efficacy of cold-fx in the prevention of respiratory symptoms in community-dwelling adults: a randomized, doubleblinded, placebo controlled trial guidelines for functional evaluation of health functional foods, 'can help improve immune function conceptual and methodological issues relevant to cytokine and inflammatory marker measurements in clinical research serum cytokine profiles in healthy young and elderly population assessed using multiplexed bead-based immunoassays baseline levels and temporal stability of multiplexed serum cytokine concentrations in healthy subjects the effects of a multivitamin/mineral supplement on micronutrient status, antioxidant capacity and cytokine production in healthy older adults consuming a fortified diet probiotic supplement consumption alters cytokine production from peripheral blood mononuclear cells: a preliminary study using healthy individuals proand anti-inflammatory cytokines in healthy volunteers fed various doses of fish oil for year regulation of human cytokines by cordyceps militaris this research was performed with the support of the industry core technology development project (no. ), ministry of trade, industry, and energy, republic of korea. jong-min lim and eunju do contributed equally to this work. key: cord- - tre rn authors: premanand, balraj; zhong wee, poh; prabakaran, mookkan title: baculovirus surface display of immunogenic proteins for vaccine development date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: tre rn vaccination is an efficient way to prevent the occurrence of many infectious diseases in humans. to date, several viral vectors have been utilized for the generation of vaccines. among them, baculovirus—categorized as a nonhuman viral vector—has been used in wider applications. its versatile features, like large cloning capacity, nonreplicative nature in mammalian cells, and broad tissue tropism, hold it at an excellent position among vaccine vectors. in addition to ease and safety during swift production, recent key improvements to existing baculovirus vectors (such as inclusion of hybrid promoters, immunostimulatory elements, etc.) have led to significant improvements in immunogenicity and efficacy of surface-displayed antigens. furthermore, some promising preclinical results have been reported that mirror the scope and practicality of baculovirus as a vaccine vector for human applications in the near future. herein, this review provides an overview of the induced immune responses by baculovirus surface-displayed vaccines against influenza and other infectious diseases in animal models, and highlights the strategies applied to enhance the protective immune responses against the displayed antigens. the existing battle between the human immune system and pathogens has a long-lasting history and is destined to continue. due to the advancement in the field of infectious diseases and the discovery of effective vaccines, therapies in recent history have aided in controlling the spread of diseases. however, the looming danger of re-emerging pathogens poses a serious issue and threatens to tilt the newfound equilibrium. among the types of vaccines available, inactivated vaccines are generally considered safe and stable. given their inactive nature, these vaccines can be given to people with weakened immune systems without the serious complication of opportunistic infection. however, the immune responses induced by inactivated vaccines are mainly humoral, and repeated immunization is required to improve immunity to the targeted disease. moreover, an inactivated vaccine may not retain the correct antigenic conformation and thus induces poor immune response against the targeted antigen [ ] . in contrast, live attenuated vaccines are more effective in inducing protective immune responses in vaccinated individuals. the major risks of live attenuated vaccines are the possibility of reversion and recombination with circulating strains, as well as the incompatibility of the vaccine with the immunocompromised, the elderly, the chronically ill, and the pregnant [ ] . recombinant subunit vaccines, on the other hand, can generally be used regardless of health status but possess other disadvantages. some of their disadvantages include the need for supporting adjuvants to improve immunogenicity, and purification issues due to the hydrophobic nature of antigens, which complicates the purification process, reducing the cost-effectiveness of vaccine production [ ] . dna vaccine utilizes genetically engineered dna capable of inducing humoral and cell-mediated immune responses against parasites, bacteria, and viruses [ , ] . modification of elements in the dna, such as promoters or enhancers, can enhance the expression of the encoded protein in vaccine recipients. moreover, a mixture of plasmids encoding a plethora of immunogenic genes can potentially be used to generate broad-spectrum vaccines. however, dna vaccine may induce antibodies against dna, resulting in autoimmune responses and the development of immunologic tolerance in recipient host [ ] . since , viral vaccine vectors have emerged, and such vectors have a more favorable safety profile than live attenuated virus vaccines, while having better immunogenicity than inactivated vaccines. viral vaccine vectors present the desired antigens in their native conformation, resulting in a stronger immunogenic response, while maintaining a higher level of foreign gene expression in vivo compared to dna vaccines [ , ] . however, some considerations exist for the use of viral vectored vaccines in humans: ( ) pre-existing vector immunity may have a serious impact on vaccine vector efficacy and ( ) increased transgene size could cause genetic instability and decrease viral yield. to overcome the issues of pre-existing vector immunity, vaccine vectors utilizing recombinant viruses of nonhuman origin have been developed to avoid neutralization of viral vector by pre-existing antibodies. a class of virus infecting insect cells, known as baculoviruses, has been developed for use as nonhuman viral vectors. among the various baculoviruses, autographa californica multicapsid nucleopolyhedrovirus (acmnpv) is the most widely studied. acmnpv is a large, double-stranded dna virus with a genome size of . kbp and contains open reading frames (orfs) [ ] . acmnpv has a biphasic life cycle resulting in the production of two forms of virus, budded virus (bv) and occlusion-derived virus (odv). the odv is responsible for the primary infection of the host insect, while bv is released from the host cells for cell-to-cell infection. moreover, the bv is produced during early stages of infection, whereas the odv is produced during late stages of viral infection, becoming concentrated in the nucleus and occluded within polyhedra [ ] . generally, acmnpv expresses each gene at specific time phases during its replication cycle. the genes encoding proteins that are responsible for viral replication and/or late gene expression (e.g., dnapol, lef - ) are expressed at an early time; structural protein genes (e.g., vp , p . , and e ) are expressed at late times; some genes are expressed at both early and late phases (e.g., ie , pp , and gp ); and p and polyhedrin (polh) are highly expressed at late and very late times of infection [ ] . based on published reports, the acmnpv promoters polyhedrin (polh) or p have been extensively used for expression of foreign proteins. in addition to the conventional acmnpv and other viral promoters, polh and p showed high levels of recombinant protein expression [ ] . interestingly, the combination of some of these promoters exhibited higher levels of protein expression compared to standard late promoters alone [ ] [ ] [ ] [ ] . moreover, it is well known that the polyhedrin promoter is active in the late stage of infection, and budding baculoviruses in the early stage of infection are unable to efficiently incorporate the target protein on the baculovirus envelope. hence, the use of immediate-early promoter may improve incorporation of target protein into the viral envelope. its salient features, including the ease of high-titer virus production, capability for simultaneous delivery of multiple genes, potential transduction ability, as well as its nonpathogenic and nonreplicative nature in humans, have led to the use of acmnpv in the production of complex eukaryotic protein in vitro, ex vivo, or in vivo [ ] [ ] [ ] and also for the vaccine development using surface display technology [ ] . in this review, we describe the immune responses elicited by recombinant baculovirus displayed vaccines against various infectious diseases and explore the unique strategies used to enhance the protective immunity against baculovirus displayed antigens (table ) . nt-not tested. since , influenza virus has been considered as an ever-present threat to humans, causing annual epidemics and infrequent pandemics, resulting in emergence of new virulent strains that pose a sustained alarm as a public health emergency [ ] . currently, the best method for the prevention and control of influenza virus infections is via vaccinations. however, a traditional influenza virus vaccine, while effective at managing infections, faces severe challenges. limited vaccine production capacity, long production time, poor growth properties of selected vaccine strains, and necessity of high-level biocontainment facilities are some major issues limiting vaccination production. particularly, uncertainty in prediction of emerging pandemic viral subtypes poses an obstacle in the prompt production of vaccines during an influenza pandemic. thus, a novel platform for the rapid development of vaccines, which overcomes limitations of currently available traditional vaccines, is required for swift response to influenza outbreak. hemagglutinin (ha) is a major surface glycoprotein of the influenza virus and is the main target for generating protective immunity against influenza virus [ ] . known to be a key determinant of host specificity, introduction of new ha subtypes was found to be associated with influenza strains responsible for pandemic outbreaks [ ] [ ] [ ] [ ] . hence, ha could serve as an important ideal target for development of influenza vaccines. recombinant subunit vaccines present one such method for targeting ha. recombinant subunit vaccines targeting ha were developed using an insect cell expression system and have been extensively evaluated in humans and animals as influenza vaccines. previously, a trivalent recombinant ha influenza vaccine-flublok ® , produced in insect cells using a baculovirus expression vector-was approved by the us fda. however, the need for a high dose with adjuvants and vaccine purification issues due to the hydrophobic nature of ha pose major setbacks in the development of recombinant subunit vaccines. an alternative strategy in vaccine development is the use of the baculovirus surface display technique. with baculoviral vaccines, targeted protein can be expressed on the surface of the virus without affecting the replication of the virus. moreover, insect cells possess the necessary cellular machinery to perform extensive post-translational modifications, such as glycosylation [ ] , phosphorylation [ ] , and disulfide bond formation [ ] , essential for the expression of many complex eukaryotic proteins, which require such modifications to maintain structural integrity and biological activities. furthermore, it has been reported that baculoviruses have strong adjuvant properties and are capable of inducing humoral and cellular immune responses against vaccine antigens [ ] . glycoprotein (gp ) is a major envelope protein of acmnpv which is essential for viral infection in insect cells. through the addition of gp domains (signal sequences, transmembrane and cytoplasmic regions), foreign proteins such as glutathione-s-transferase, human immunodeficiency virus (hiv) gp protein, rubella virus envelope protein, and synthetic igg binding domains can be displayed on the baculoviral envelope [ ] [ ] [ ] . alternatively, certain membrane proteins can be displayed on the budded virus itself without the need for gp domains. for example, vesicular stomatitis virus (vsvg) protein and measles virus receptor can be independently displayed on the viral envelope [ , ] . similarly, ha protein can be incorporated into the viral envelope without gp domains. the different regions of gp , such as the signal sequences, transmembrane domain, and cytoplasmic domain, have been widely used for the expression of secretory proteins in baculovirus/insect cell expression systems, and have been exploited to display foreign proteins or peptides on viral envelopes [ , ] . in a case study, the efficiency of h ha incorporation into the baculoviral envelope was shown to differ between recombinant baculoviruses carrying ha with different cytoplasmic domains (ctds), despite equal expression of recombinant ha. bac-ha , which expresses histidine-tagged ha with gp -ctd (cytoplasmic domain of gp ), incorporates ha to the viral envelope more efficiently than bac-ha, which expresses ha with ha-ctd (cytoplasmic domain of ha). moreover, bac-ha elicited higher hemagglutination inhibition titer (hi) compared to bac-ha. the result indicates the importance of the cytoplasmic domain and the degree of palmitoylation on the domain in the incorporation of ha on the viral envelope and on vaccine potential [ ] . in a second study, a modified bac-ha vaccine was developed by employing baculovirus p and cytomegalovirus (cmv) promoters with ha, which demonstrated surface display and endogenous expression of ha after transduction. an immunization study on the vaccine vector system revealed superior or equivalent immune responses against ha compared to other vaccine forms. the immune response is mediated by interaction with antigen-presenting cells (apcs) via the major histocompatibility ii (mhc-ii)-mediated antigen presentation pathway [ ] . in general, vaccines targeting respiratory viral infections, like influenza, act by inducing neutralizing antibodies that neutralize virions or block viral attachment and entry into host cells. however, currently available conventional vaccines induced humoral responses against only homologous strains and provided inadequate protection against heterologous strains. in contrast, t-cell-mediated cellular immune responses against conserved internal regions of antigens mediate protective immunity against heterologous strains [ , , ] . in previous studies, stimulation of helper t-lymphocytes (th cells) and cytotoxic t-lymphocytes (ctls) by nucleoprotein amino acids np - and np - provided complete cross-protection in challenge studies [ , ] . sridhar et al. [ ] observed that individuals having a higher number of pre-existing cd + t-cells against conserved cd epitopes showed milder illness after infection with pandemic h n influenza virus [ ] . furthermore, wilkinson et al. [ ] showed that, in the absence of antibody responses, pre-existing cd + t-cells respond to influenza internal proteins, reduce the severity of illness, and demonstrate lower shedding of virus [ ] . recently, zhang et al. [ ] developed a recombinant baculovirus-based vaccine containing three tandem copies of the highly conserved extracellular domain of influenza m protein (m e) (bv-dual- m e); a second vaccine had an additional mucosal adjuvant heat-liable enterotoxin b (bv-dual- m e-ltb). mice inoculated with bv-dual- m e-ltb vaccine induced higher levels of mucosal antibodies and more efficient cellular immunity against different h n clades (clade , . . . , . . , and ) compared to non-adjuvanted bv-dual- m e vaccine. interestingly, it was discovered that mucosal immunity alone was insufficient for protection from lethal h n challenge, whereas adjuvanted vaccine provided enhanced protection against similar challenge through cd + t-cell response. to validate the role of cd + t-cells in enhancing protective immunity, mice with depleted cd + or cd + t-cells were inoculated with bv-dual- m e-ltb. mice with depleted cd + t-cells displayed reduced lung viral titers, but no significant effect on survival rate was observed. comparatively, depletion of cd + t-cells or both cd + t-cells and cd + t-cells markedly increased lung viral titers and decreased survival rate. further research on enhancing ha-specific immune responses utilized a baculovirus vaccine vector with additional elements to improve immunogenicity. these elements included cag promoter, mhc class i signal sequence (mhciss), mhc class i trafficking domain (mitd), and woodchuck hepatitis virus posttranscriptional regulatory element (wpre). cag promoter enables robust expression of ha, while mhciss and mitd enhance mhc class i and ii antigen presentation, which stimulates the epitope-specific cd + and cd + t-cells. wpre, on the other hand, elevates the ha gene expression and increases immunogenicity. as shown in the study, mice immunized with baculovirus possessing the additional elements induced higher levels of ha-specific igg and igg a, higher hemagglutination inhibition titers, and better th and ifn-γ + /cd + t-cell responses compared to baculoviruses without the additional elements [ ] . furthermore, the role of wpre and itrs (inverted terminal repeats) in enhancement of ha expression and their effect on induction of humoral and cellular immune responses was studied. two series of baculovirus that use different promoters to drive the expression of ha were generated: bv-s series (bv-s-ha, bv-s-itrs-ha, bv-s-con-ha), which utilizes the cmv promoter for the expression of ha, and bv-a series (bv-a-ha, bv-a-itrs-ha, bv-a-con-ha), which utilizes white spot syndrome virus (wssv) immediate-early promoter one (ie ) for the expression of ha. results from immune studies indicate that baculoviral vaccine with wssv ie promoter induces a higher level of humoral and cellular immunity compared to similar baculoviral vaccine utilizing the cmv promoter [ ] . in another study, recombinant baculovirus vectors with an egfp expression cassette under p and cmv promoters were examined. the baculoviral vectors were modified to express ha using baculovirus polyhedrin (polh) promoter (vac-ha) or using dual-promoter with polh and cmv promoter (vac-ha-dual). while ha was expressed efficiently in sf insect cells for both constructs, mice vaccinated with vac-ha induced higher levels of igg and iga compared to mice vaccinated with vac-ha-dual. in addition, stimulation of splenocytes in immunized mice with ha-specific peptide triggered th immune response for vac-ha-immunized mice, whereas vac-ha-dual-immunized mice showed th -biased immune response. moreover, vac-ha-dual vaccine was more effective in inducing ha-specific cd + and cd + t-cell response in vitro [ ] . it has been reported that the signal peptide of membrane proteins is important in directing the protein to the endoplasmic reticulum membrane and in protein trafficking [ ] . the transmembrane (tm) domain plays a role in protein trafficking, membrane anchoring, membrane fusion, and viral budding [ , ] , whereas the ct domain is crucial for envelope incorporation, virus budding, interaction with the components of viral core [ , ] , and incorporation of influenza ha into the baculovirus envelope [ ] . supporting these observations was the research by tang et al. [ ] on the functional role of signal peptide (sp), ctd regions of gp , and tm of ha in the incorporation of ha into the baculovirus envelope. the study demonstrated enhanced ha incorporation and increased hi titers for baculovirus with gp sp, ctd, and ha tm. in stark contrast, recombinant baculoviruses containing different combinations of gp or ha domains (gp sp or ha-sp with ha-tm and ha-ctd; gp -tm or ha-tm with ha-sp and gp ctd; gp sp or ha-sp with gp -tm and gp -ctd) showed the opposite effect on ha incorporation and hi titers. based on the results, the second generation of tetravalent baculoviral vaccine was developed using gp -sp, ha-tm, and gp -ctd. the vaccine displays has from four subclades of h n influenza viruses and induces strong humoral and cellular immunity against homologous and heterologous h n strains [ ] . in another study, efficiency of wssv ie promoter-mediated h ha expression by recombinant baculovirus was examined [ ] . also, they confirmed that ha expressed by the baculovirus was able to successfully translocate to the plasma membrane of the infected cells and observed that surface-displayed ha was able to sustain its antigenic conformation and authentic cleavage [ ] . it has been reported that baculovirus pseudotyped with vesicular stomatitis virus glycoprotein (vsvg) from vesicular stomatitis virus (vsv) effectively increased transduction levels in mammalian cells [ ] and has the capability to increase the amount of expressed protein displayed on the surface of the entire viral envelope [ , ] . in addition, recombinant baculovirus which displays ha and co-expresses vsvg under wssv ie showed ha displayed on the baculovirus surface has high ha activity. intranasal or intramuscular immunization of chickens with baculovirus containing wssv ie induced strong ha-specific antibodies and hi titer compared to baculovirus with cmv promoter. nevertheless, co-expression of vsvg by both viruses contributed to increased anti-ha antibody level and hi activity [ ] . similarly, recombinant baculovirus pseudotyped with vsvg (bv-g-ha) was shown to successfully transduce into mammalian cell, mediate gene delivery, and enable efficient expression of h ha. an immunization study revealed that the vaccine candidate induced higher levels of h ha-specific antibodies and cellular immunity compared to the mice vaccinated with µg of dna vaccine. furthermore, immunized chickens, administered with varying doses, demonstrated complete protection from a lethal dose of highly pathogenic h n avian influenza virus [ ] . however, the vaccine did not efficiently protect mice against an evolutionarily distant strain [ ] , possibly due to a high degree of genetic divergence of the influenza virus [ ] . for example, current monovalent inactivated whole virus vaccine can induce neutralizing antibodies against the homologous strain but shows lower magnitude of response against heterologous strains [ ] . hence, it is necessary to develop a vaccine that expresses antigens covering the major circulating viruses [ ] . therefore, to broaden the protective efficacy of monovalent bv-g-ha vaccine, a pseudotyped baculovirus-based bivalent vaccine that encodes ha from clade . . and clade influenza viruses was developed. mice immunization with pseudotyped baculovirus-based bivalent vaccine induced significantly higher humoral and cellular immune responses and provided complete protection against h n viruses from clade . . and clade compared to other candidates used in the study [ ] . similarly, the cross-protective efficacy of wssv ie promoter-controlled bivalent baculovirus surface-displayed ha of a/indonesia/ / and a/anhui/ / (bivalent-bacha) was tested in another study. mice orally vaccinated using live bivalent-bacha vaccine showed both systemic and mucosal immunity, unlike mice immunized subcutaneously with live or inactivated bivalent vaccine, which induced only robust systemic immunity. the level of systemic immunity induced by oral bivalent vaccine in immunized mice was lower compared to subcutaneous immunization. however, mice orally immunized with bivalent-bac-ha vaccine demonstrated strong ha-specific mucosal iga response. in addition, oral vaccination induced potent cross-clade neutralizing antibodies against distinct clades ( , . . , . . . , . . , . . , , , and ) of h n viruses which are absent in monovalent inactive whole h n vaccination, whereas mice subcutaneously vaccinated with live or inactivated bivalent-bacha vaccine showed low neutralization titer against clade h n compared to other clade of viruses. enzyme-linked immunospot assay revealed that mice immunized orally or subcutaneously with live bivalent vaccine triggered both ifn-γ-secreting th cells and il- -secreting th cells, while mice immunized subcutaneously with inactive adjuvanted bivalent vaccine stimulated only the il- response. moreover, mice vaccinated orally or subcutaneously with live bivalent bac-ha vaccine showed complete cross-protection against clade and clade . . . h n viruses, suggesting that this bivalent vaccine can, potentially, protect against cross-clade h viruses during pandemic situations [ ] . since baculovirus has been proved to efficiently express foreign genes and is capable of transduction in a variety of cell lines, researchers utilized this virus as a potential vaccine vector against various infectious agents. furthermore, additional elements, like vsvg protein, were included by researchers to increase the efficiency of transduction, gene delivery, and expression in the recipient host [ ] . while recombinant baculoviral vector expressing both vsv-g and influenza ha was shown to evoke both humoral and cellular immune responses and provided effective protection against lethal virus challenge in mouse and chicken hosts [ ] , the high cytotoxicity of vsv-g protein [ ] and its immediate inactivation by serum complement systems impedes the use of the element in a vaccine delivery vehicle [ ] . recent reports have shown that human endogenous retrovirus (herv) envelope-coated recombinant baculovirus-based human papillomavirus l vaccine significantly improved the expression of l in mammalian cells and induced humoral immune responses which are comparable with the level obtained using commercial cervical cancer virus-like particles vaccine [ ] . further, it has been reported that herv envelope-coated baculovirus-based ha of the h n pandemic influenza vaccine induced a strong cellular immune response compared to the commercial vaccine [ ] . in another study, prabakaran et al. [ ] developed a recombinant baculovirus displayed ha vaccine under the control of wssv ie promoter (bacha) against the pandemic h n virus. the vaccine showed successful ha expression on its envelope, and mice vaccination studies showed that both the live and adjuvanted with inactive form of recombinant baculovirus induced ha-specific antibody responses and offered complete protection against lethal viral infection [ ] . studies have shown that high neutralizing antibodies generated against influenza virus target specific regions in the globular head of influenza ha. moreover, the ha stalk domain is well conserved and antibodies targeting this region are capable of neutralizing the heterologous influenza viral subtypes [ ] [ ] [ ] [ ] [ ] [ ] . for example, chimeric ha constructs expressing the globular head and stalk regions from different subtypes induced antibodies that showed broad protection [ , , ] . similarly, mice immunized with recombinant baculovirus displayed ha of the pandemic h n influenza virus induced ha-stalk-specific antibodies and demonstrated protective immunity against homologous and heterologous h n subtype viruses [ ] . in recent decades, avian h influenza virus was responsible for numerous influenza outbreaks among poultry industries in europe and north america. since , none of these poultry-adapted viruses has evolved to widely infect humans or cause a pandemic. however, there are cases where the virus acquired the ability to infect mammals, as evidenced by the largest outbreak of highly pathogenic h n (nl/ . ) in the poultry industry of the netherlands, where human infections were reported [ ] . h n viruses isolated from the patients were shown to possess replicative potential in human conjunctiva and ability for human-to-human transmission. interestingly, the virulence of the virus in chickens and turkeys increased with acquisition of additional amino acids at the hemagglutinin (ha) cleavage site [ ] . in , human infections with avian influenza a h n subtype were first reported in china and caused several waves of human infection. it was speculated that the virus possesses high mutagenicity, which contributed to its ability to infect humans. higher mutagenicity may be the cause of enhanced pathogenicity, as supported by recent human cases, where the isolated virus was found to have mutations associated with reduced susceptibility to neuraminidase inhibitors used in antiviral treatment [ ] . since h subtype viruses pose a major challenge for public health and the poultry industry, numerous vaccines have been generated against these subtype viruses using various platforms. recently, baculovirus displayed ha vaccine against h n (nl ) was developed and its efficacy was tested via intranasal (i.n.) or subcutaneous (s.c.) administration in mice. it was observed that intranasally immunized mice with live baculovirus displayed ha vaccine induced higher or comparable humoral, mucosal, and cellular immune responses compared to other vaccine candidates, irrespective of immunization route. additionally, the immunization provided % complete protection, whereas no protection was observed in mice vaccinated with other vaccine candidates [ ] . in another study, recombinant baculovirus-based vaccine (bacha) against avian h n virus with high pandemic potential was generated, and its immunogenicity and cross-protective efficacy was evaluated in mice. they found that live bacha i.n. immunized mice elicited high levels of ha-specific iga mucosal immune response and humoral immune response, comparable to mice administered subcutaneously with live bacha. on the other hand, subcutaneous immunization with adjuvanted inactive bacha stimulated robust humoral immune response and induced higher neutralizing antibodies against h n viruses and other h subtype (h n and h n ) viruses compared to other vaccine candidates. in addition, subcutaneous immunization with inactive whole h n vaccine protected % against rg-h n challenge but demonstrated insufficient protection against rg-h n (nl ) subtype, whereas mice vaccinated subcutaneously with live or inactive recombinant baculovirus against h n (nl ) protected only - % of mice against h n challenge. furthermore, mice immunized i.n. with live bacha of h n or h n showed complete protection against both h n and h n infection in a challenge study [ ] . it has been reported that the differences between the immunogenicity of ha of h n (nl ) and ha of h n might be due to the amino acid changes at a t, which introduced a potential glycosylation site at position n, masking the epitopes of h ha (nl ) [ , ] . to account for this difference in immunogenicity, kumar et al. [ ] compared the ha of h n and h n viruses and selected three positions in their study: (a) , (b) , and (c) , which are located within or near the receptor-binding site of h n ha protein. by site-directed mutagenesis, they generated six mutant constructs by single or double amino acid substitution at these three positions (t a, t a, i v, t a- a, t a-i v, and i v-t a) . among the generated mutant constructs (bac-ha m ), mice vaccinated with constructs t a, t a-i v, and i v-t a induced higher hemagglutination inhibition and neutralization titer that neutralizes both h n and h n viruses compared to mice immunized with bacha or other mutant vaccines. the result shows that the substitution of amino acids responsible for forming potential glycan motif-epitope masks would improve immunogenicity of the vaccine candidates. in another study, mice i.n. immunized with baculovirus-displaying ha of low pathogenic influenza h subtype triggered higher ha-specific humoral, mucosal, and cellular immunity compared to mice immunized with inactivated influenza virus. moreover, regardless of immunization route, it conferred % protection against lethal homologous mouse-adapted h n virus [ ] . moreover, recombinant baculovirus with cmv-polyhedrin dual promoter for expressing chimeric ha of h n was shown to efficiently express ha in both mammalian and insect cells, induce strong immune response, and provide % protection against lethal h n viral challenge in mice, unlike other vaccine candidates observed [ ] . shrimp immunity relies on an innate immune mechanism, and their lack of adaptive immunity hinders vaccine development against shrimp pathogens. though limitations exist in developing shrimp vaccines, the presence of quasi-immune responses after natural or experimental infection with white spot syndrome virus (wssv) in p. japonicas and the resistance against experimental rechallenge with wssv due to neutralizing factors in the survivors' plasma [ , ] encouraged the researchers to continue the development of vaccines against the wssv virus. several attempts have been made to develop vaccines by targeting the immunogenic vp protein of wssv virus [ ] [ ] [ ] [ ] syed musthaq et al. [ ] , developed a recombinant-based vaccine (bac-vp ) that displays recombinant vp (rvp ) on its envelope. confocal and transmission electron microscopy analysis confirmed that rvp was able to localize on the plasma membrane of infected insect cells, and the virus also successfully acquired rvp on its envelope from the insect cell membrane during the budding process. approximately . µg/ml of vp protein was anchored on the bac-vp envelope ( viral particles). furthermore, its protective efficacy against wssv infection was tested via injection, oral, or immersion methods in shrimp. the bac-vp -injected shrimp successfully transcribed and translated vp and showed survival rates of . % and . % against wssv virus challenge at and days postvaccination, respectively [ ] . on the other hand, the transcriptional levels of lipopolysaccharide and β- , -glucan binding protein (lgbp) and signal transducer and activator of transcription (stat) genes (which are crucial for triggering innate immune responses) [ ] were altered at different days postinfection (dpi) in shrimp vaccinated with bac-vp by oral or immersion routes. the expression level of stat gene in the control group was downregulated at various dpi as wssv infection progressed, but in the vaccinated group, stat gene level decreased until dpi and started to increase at dpi and dpi. moreover, the levels of lgbp gene in the control group was upregulated as infection progressed, whereas the vaccinated group showed an increasing level at early infections, which later decreased as wssv infection cleared. these results confirm that oral vaccination of bac-vp triggers the innate immune responses and, possibly, was responsible for higher survival rates of . % and . % upon wssv challenge at and days postvaccination. also, shrimp vaccinated with bac-vp immersion vaccine showed % and . % survival rates compared to % mortality obtained with the control group in a challenge study [ ] . porcine reproductive and respiratory syndrome virus (prrsv), classical swine fever virus (csfv), and porcine circovirus type (pcv ) are the major pathogens continuously affecting the pig industry and cause significant economic losses worldwide. to date, researchers all over the world have contributed various forms of vaccines utilizing different strategies targeting these pathogens. virus-based vectored vaccines were developed using modified vaccinia virus ankara, canine adenovirus- , pseudorabies virus, and semliki forest virus [ ] [ ] [ ] [ ] . however, these vectors are of mammalian origin and pose a biohazard threat. in contrast, recombinant baculoviruses possess good safety profiles and have been utilized to develop vaccines against prrsv, csfv, and pcv viruses. using gp sp, tm, and ct domains, xu et al. [ ] developed recombinant baculovirus-based bivalent vaccine vector (bacsc-dual-gp -cap) that surface-displayed gp (major immunogenic inducer of neutralizing antibodies against prrsv infection) and pcv (major immunogenic capsid (cap) protein). a swine vaccination study demonstrated that this vaccine candidate stimulated potent gp and cap-specific neutralizing antibodies and induced higher lymphocyte proliferation responses compared to control groups, which indicates that the bivalent vaccine can potentially be used as a vaccine against a mixed prrsv and pcv infections. in another study, the orfs of prrsv were successfully displayed on the baculovirus envelope using the gp sp, h n tm, and gp ctd regions. the antibody responses elicited by the vectored vaccines were sustained for up to days after the booster, whereas the titers in the inactivated prrsv-vaccinated group were reduced dramatically at this time point. the enhanced immune responses could be due to the in vivo transduction of the prrsv genes by baculovirus vaccine, which activates the endogenous antigen-processing and -presenting pathways. amongst the vaccines developed in this study, baculoviruses that displayed orf a, orf , and orf induced higher antibody responses in mice compared to other vaccine candidates [ ] . a recombinant baculovirus-based vaccine that contains the pcv cap and vsvg protein (bv-gd-orf ) was developed. the expression of pcv was placed under the control of cmv and polh promoters, whereas expression of vsvg was placed under the p promoter. due to vsvg-mediated transduction and the successful expression of the pcv cap protein in transduced cells, bv-gd-orf behaved as a subunit/dna vaccine and induced robust cap protein-specific humoral and cellular immune responses [ ] . studies reported that e or e rns glycoprotein are the main immunogenic proteins of csfv, which can induce neutralizing antibodies and provide protective immunity against csfv. xu et al. generated recombinant baculoviruses that surface-displayed either e (bacsc-e ) [ ] or e rns (bacsc-e rns ) [ ] as fusion forms with gp domains. the presence of the gp domains did not alter the viral titers of the recombinant viruses. moreover, a mice vaccination study revealed that bacsc-e or bacsc-e rns induced stronger e -or e rns -specific antibodies, respectively, compared to other vaccine candidates, suggesting that the vaccine candidates could potentially be applied in the treatment of csfv infection. respiratory syncytial virus (rsv) is a common contagious virus that causes acute lower respiratory tract infection in infants, young children, elderlies, and immunocompromised individuals [ ] . currently, there is no approved vaccine available to treat this infection in humans. studies have reported that immunization with recombinant viral vector expressing rsvg protein showed immune pathological issues by enhancing th skewing of the cd + t-cell response [ ] [ ] [ ] . countering the issue, zhang et al. [ ] developed recombinant baculovirus vaccines that showed reduced immune pathological conditions in mice. four vaccine constructs were developed: bac-tf (displays the f ectodomain (tf) on the envelope), bac-cf (expresses full-length f protein in transduced mammalian cells), bac-cf/tf (displays tf on the envelope and expresses f in cells), and bac-cf/tf -visa (contains additional virus-induced signaling adaptor (visa gene). bac-cf and bac-cf/tf showed an increased mixed th /th cytokine response, higher levels of igg a/igg antibody responses, and reduced immunopathology upon rsv challenge compared to bac-tf . moreover, co-expression of visa reduced the th immune responses induced by bac-cf/tf and protected lungs from inflammatory immune response upon a subsequent rsv challenge. therefore, the inclusion of visa elements could be an effective alternate vaccine strategy against rsv infection [ ] . rabies virus (rabv) is highly infectious and poses a constant threat to humans and animals. traditionally, vaccine development against rabv mainly focused on inducing neutralizing antibodies [ ] [ ] [ ] . however, studies have shown that, in addition to humoral immunity, cellular immune responses are important for providing protective immunity against rabv [ , , ] . previously, a recombinant baculovirus viral vector vaccine against rabv glycoprotein (rvg) was constructed (bv-rvg/rvg). the vector expresses two types of rvg antigen (baculovirus-expressed rvg and in vivo expressed rvg) to enhance specific immune responses against the rvg antigen. based on immunization study, bv-rvg/rvg-immunized mice displayed higher humoral and cellular immune responses and offered complete protection against rabv compared to mice immunized with other vaccine candidates. the enhanced immunogenicity could be due to immediate recognition of rvg incorporated into bv-rvg/rvg by immunocompetent cells, resulting in presentation to dendritic cells (dcs) and, subsequently, activation of b cells to secrete viral-specific neutralizing antibodies. furthermore, cmv-mediated in vivo rvg expression by bv-rvg/rvg activated the endogenous antigen presentation pathway, inducing persistent and specific immune responses [ ] . in another study, feng et al. [ ] displayed the entire ectodomain of sars-cov spike protein (s) on the baculovirus envelope using gp signal peptide and vsv-g membrane anchor. in mice, the vaccine induced specific neutralizing antibodies against s protein in the absence of adjuvants [ ] . in a subsequent study, researchers developed recombinant baculovirus vaccines against avian infectious bronchitis virus (ibv), japanese encephalitis virus (jev), and bovine herpesvirus- (bhv- ) with the gp domains (sp, tm, and ctd). chickens immunized with baculovirus displayed s protein of ibv vaccine induced more humoral and cellular immune responses and showed better protection ( %) compared to other vaccines studied [ ] . on the other hand, glycoprotein e of jev displayed on the baculovirus envelope induced strong neutralizing antibodies in mice and swine and offered % protection in vaccinated mice against a lethal jev challenge [ ] . similarly, bhv- envelope glycoprotein (gd) displayed on the baculovirus envelope elicited strong neutralizing antibody against bhv- in mice [ ] . enterovirus (ev ) immunogenic capsid protein (vp ) is a non-membrane protein which requires additional regions, like sp, tm, and ctd of gp , ha, or other domains for successful incorporation into envelope, while membrane proteins like ha or na (neuraminidase) can be translocated to the plasma membrane and incorporated onto the envelope during viral budding. meng et al. [ ] generated recombinant baculoviruses, with wssv ie (bac-pie -gp -vp ) or polyhedrin promoter (bac-pph-gp -vp ), which surface-displayed chimeric vp (vp was fused in between gp sp and mature domain). transmission electron microscopy analysis confirmed that vp was successfully anchored on the recombinant baculoviral envelope. bac-pie -gp -vp -immunized mice were found to induce slightly higher vp -specific antibodies with potent neutralizing activity (can neutralize c subgenogroups and heterologous subgenogroups) than bac-pph-gp -vp and to show neutralizing activity comparable with inactivated ev vaccine. subsequently, premanand et al. [ ] generated the recombinant baculovirus (bac-vp ) that surface-displayed the chimeric vp using minimal fusion partners, like gp sp ( aa), h n -ha transmembrane domain ( aa), and gp ctd ( aa) to facilitate better incorporation of vp and higher baculovirus titers. expectedly, bac-vp contains -fold higher vp ( ng/µg) on its envelope compared to vp on bac-pie -gp -vp envelope ( ng/µg). bac-vp -immunized mice elicited stronger vp -specific antibody responses and cross-neutralization activity against homologous or heterologous strains compared to bac-pie -gp -vp and bac-pph-gp -vp vaccines. moreover, bac-vp provided % protection against a mouse-adapted lethal strain of ev [ ] . in both studies mentioned above, vp was anchored on the viral envelope similar to type transmembrane protein with unrestricted n-terminus exposure. mimicking other types of transmembrane protein, kolpe et al. generated recombinant baculovirus (bac-na-vp ) which surface-displayed vp using influenza a neuraminidase (na), resulting in a type ii transmembrane-anchored pattern with a distal c-terminus. mice immunization studies revealed that bac-na-vp induced both humoral and cellular immune responses and provided % protection of suckling balb/c mice against mouse-adapted ev -b strain challenge [ ] . infectious bursal disease virus (ibdv) is the causative agent of infectious bursal disease, which affects chickens and causes considerable economic loss for the poultry industry [ , ] . it has been reported that live attenuated vaccines elicited lower level of antibodies and failed to protect the chickens against ibdv [ ] . to target this issue, xu et al. [ ] generated a recombinant baculovirus that surface-displayed his -tagged vp protein using the gp tm and ct domains (bacsc-vp ). bacsc-vp -immunized chickens demonstrated higher levels of neutralizing antibodies and better protective immunity against a very virulent ibdv strain than control groups. furthermore, ibdv-specific lymphocytes were observed in bacsc-vp -vaccinated chickens. also, the major immunogenic capsid protein vp of canine parvovirus type (cpv- ) and immunogenic proteins σc and σb of avian reovirus (arv) have been utilized to develop a baculovirus-based vaccine against cpv- and arv, respectively. fusion of gp domains (sp, tm, and ctd) with the capsid proteins allows their incorporation into the baculoviral envelope. an animal vaccination study revealed that baculovirus that surface displayed vp of cpv- induced higher neutralizing antibodies in mice compared to other vaccines tested [ ] . moreover, recombinant baculoviruses that displayed immunogenic proteins of either σc or σb of arv elicited strong antibody titers with higher neutralizing activities compared to antibody responses by other vaccine candidates [ ] . malaria, a global disease caused by plasmodium parasites, is endemic in tropical and subtropical regions and results in up to million deaths annually. a recombinant protein-based subunit vaccine containing a part of a major surface protein from plasmodium falciparum circumsporozoite protein (pfcsp) provided short-lived protective immunity, and the effective period was affected by age group and malaria transmission intensity [ , ] . another vaccine plasmodium falciparum sporozoite (pfspz) was reported to provide protection against homologous and heterologous human malaria infection [ ] [ ] [ ] . aiming to curb the spread of malaria, the who has set to a goal of % vaccine efficiency by the year . novel and alternate strategies will be required to ensure the development of more effective vaccines against malaria infection. to develop a vaccine with dual functions (such as a malaria vaccine that can act as a liver-directed gene delivery vehicle), yoshida et al. [ ] developed the recombinant baculovirus-based vaccine that displayed rodent malaria parasite plasmodium berghei circumsporozoite protein (pbcsp). in the immunization study, the vaccine induced both humoral and cellular immune responsea and provided % protection against sporozoite challenge [ ] . subsequently, a series of baculovirus-based vaccines expressing pfcsp (with deleted glycosylphosphatidylinositol gpi anchor region) under the control of polh or cmv promoter was developed. acnpv.cs vector expresses the cs protein in apcs under the control of the cmv promoter to enable (preferentially) induction of cd + t-cells upon mhc class i presentation. acnpv.cs vector expresses a cs protein with gp as a fusion form using the polyhedrin promoter in insect cells, which allows the incorporation of cs on the baculovirus envelope during the budding process (mimicking the p. falciparum sporozoite). uptake of acnpv.cs into apcs triggers cd + cells via mhc class ii presentation and induces a potent humoral immune response. acnpv.cs-cs combines expression and presentation of recombinant cs antigens in mammalian cells to induce both cs-specific cd + and cd + t-cells. a mouse vaccination study showed that the acnpv.cs-cs vaccine induced efficient humoral and cellular immune responses with intramuscular immunization compared to other vaccine candidates [ ] . recombinant baculovirus dual expression-based malaria vaccine, which drives pbcsp expression by polh and cmv promoters, was developed. mice immunized with the vaccine induced both th and th responses after intramuscular inoculation and provided complete protection against sporozoite challenge [ ] . another baculovirus-based vaccine candidate which displays the major surface antigen of p. falciparum, ppfs , was developed as a transmission-blocking vaccine against human malaria. both i.m. and i.n. immunized mice showed high levels of pfs -specific antibodies and effective transmission-blocking response, with an % (intranasal) and % (intramuscular) reduction in oocyst intensity [ ] . in another case study, a recombinant baculovirus-based plasmodium vivax transmission-blocking vaccine using autographa california nuclear polyhedrosis virus (acnpv-dual-pvs ) was developed. acnpv-dual-pvs not only displayed pvs on the acnpv envelope in native conformation, but also expressed pvs upon transduction of mammalian cells. acnpv-dual-pvs induced pvs -specific antibodies in intranasally or intramuscularly immunized mice. moreover, in a rabbit immunization study, the vaccine induced significant transmission-blocking response, regardless of immunization route (subcutaneous, intramuscular, and intranasal) [ ] . yoshida et al. [ ] developed a baculovirus-based nasal drop vaccine (acnpv-pymsp surf ) that surface-displayed the carboxyl terminus of merozoite surface protein (pymsp ) from plasmodium yoelii. intranasal and intramuscular immunization of mice with acnpv-pymsp surf induced mixed th /th immune responses and provided % protective efficacy. however, intranasal immunization elicited higher pymsp -specific antibody titers and stronger natural boosting after a challenge study [ ] . in another study, a baculovirus dual-expression system-based pfcsp protein (bdes-pfcsp) vaccine was generated. based on an immunization study, the vaccine was found to induce mixed th /th immune responses in mice, generate a higher level of pfcsp-specific antibodies, and confer significant protection against malaria. in addition, sera from immunized monkeys prevented sporozoite invasion in hepg cells [ ] . a multistage malaria vaccine was developed using the baculovirus platform to target and inhibit the plasmodium parasite in its pre-erythrocytic and sexual stages. the generated baculovirus displayed correct folding of both p. vivax circumsporozoite protein (pvcsp) and p (pvs ) protein in fusion form (bdes-pvs -pvcsp) and acted as a pre-erythrocytic and a transmission-blocking activity vaccine. the bdes-pvs -pvcsp vaccine induced higher antibody levels against pvs and pvcsp while providing % protection against malaria and % transmission-blocking activity [ ] . in a recent study, human decay-accelerating factor (hdaf) was incorporated into a baculovirus vector, which effectively displayed hdaf and pfcsp on the surface of the baculoviral envelope to prevent serum complement inactivation. following intramuscular immunization, the modified vaccine conferred higher protective efficacy ( %) compared to control group ( %) in a challenge study [ ] . mucosal surfaces (respiratory, gastrointestinal, and urogenital tracts) provide the major route of entry for various microorganisms, and the mucosal immune system acts as first line of defense against microbial infection [ ] . therefore, triggering of the mucosal immune system is important in preventing infection, and this can be achieved by mucosal vaccinations via intranasal, oral, pulmonary, rectal, or vaginal routes of administration. among these routes, oral or intranasal immunizations are the two most viable options for vaccine delivery. the nasal route, in particular, offers several advantages, like the possibility of self-administration, the induction of both mucosal and systemic immunity in the gastric mucosa, respiratory tracts and genital tracts, as well as the lower doses of antigen and adjuvant required compared with oral vaccination. several nonhuman viral vectors have been developed in recent years, among which is the baculoviral vector. baculovirus has been reported to possess strong adjuvant properties in mammals, promoting enhanced humoral, mucosal, and cellular immune responses against co-administered antigens. supporting this are previous reports which indicated that mice immunized with baculovirus genomic dna and ovalbumin showed humoral and cytotoxic t-lymphocyte responses against co-administered antigens. the abundance of cpg motifs in the baculovirus improves its ability to induce innate immune responses through the tlr /myd -dependent endosomal recognition pathway and dna-dependent activator of interferon regulatory factor-mediated pathway [ ] . other studies have shown that intranasal inoculation of mice with wild-type baculovirus provided protection against h n , h n , and other influenza b strains [ ] , which highlighted the potential of the baculoviral vector in the development of vaccines against mucosal acquired pathogens. intranasal immunization of mice with live recombinant baculovirus (bac-ha) vaccine induced significantly higher mucosal iga immune response against h n -ha compared to other intranasally delivered vaccines. the higher immune response could be attributed to the efficient transduction of baculoviral vector or the strong adjuvant property of baculovirus due to the presence of unmethylated cpg motifs. in addition, i.n delivery of bac-ha stimulated higher amounts of ifn-γ and il- compared to s.c immunization of live bac-ha, which is similarly reported in other studies [ , ] . the enhancement of cytokine levels could be due to recall response in splenocytes or due to the presence of nasopharynx-associated lymphoid follicles in the nasal mucosa that trigger antigen-specific th lymphocytes, ctls, and iga immune responses. a challenge study in mice revealed that i.n immunized mice with live bac-ha showed % protection against lethal viral challenge compared to other vaccines, irrespective of route of administration. even though s.c immunized mice achieved a higher level of igg antibodies, the incapability of bac-ha in protecting against lethal viral challenge could be due to lack of mucosal iga, which is more crucial in clearing upper respiratory tract infection than igg [ ] . in another study, a recombinant baculovirus-based vaccine against another pandemic potential avian h n virus was generated (bac-ha). they found that mice intranasally immunized with live bac-ha elicited higher levels of ha-specific mucosal and humoral immune responses. mice vaccinated subcutaneously with live or inactive baculovirus displayed ha of h n (nl ) only protected - % of mice against h n challenge, but mice immunized intranasally with live bac-ha of h n or h n showed complete protection against both h n and h n infection in a challenge study. the result indicates the importance of additional mucosal immunity in recovery and protection during h subtype infection [ ] . the inclusion of adjuvant improves the immune response induced by the vaccine, and the cause of the better immune response in mucosal adjuvants, in particular, is due to the induction of protective local and systemic immune responses against various infectious diseases [ ] . also, intranasal immunization of recombinant baculovirus-displaying ha of h n virus induced high levels of mucosal iga and serum igg immune responses in mice. combination of recombinant mucosal adjuvant ctb further enhanced serum igg and iga responses compared to unadjuvanted log or log ha titer bac-ha alone. log ha titer live bacha with µg of rctb conferred % protection against homologous and heterologous h n strains [ ] , suggesting that the combination of safe adjuvants with vaccines would be ideal for optimal efficacy of intranasal vaccines. oral administration is considered as another efficient route for delivering the mucosal vaccines. it is non-invasive, safe, and shows good patient compliance. for example, oral polio vaccine against highly contagious poliovirus helped to eradicate poliovirus infection in most parts of the world [ ] . moreover, studies have shown that oral vaccination can prevent infection of lungs and is capable of inducing mucosal immune responses (iga) in the respiratory tract, providing protection against influenza viruses. prabakaran et al. [ ] reported that live recombinant baculovirus displaying ha of h n virus (bac-ha) was able to express ha in epithelial cells of intestines of orally vaccinated mice and induced strong systemic and mucosal immune responses. mice immunized with live bac-ha of h n without any inclusion of adjuvants conferred % protection against homologous and heterologous h n strains, while immunization with inactivated bac-ha resulted in a lower survival rate of . %. the reduced immune responses of the inactivated bac-ha could be due to loss of transduction ability, whereas the live bac-ha was able to bind to the receptors that are expressed in the intestinal cells, resulting in gene delivery and stimulation of cell-mediated immune responses [ ] . moreover, the formulation of inactivated vaccine can have a huge impact on its immunogenicity. in one study, the immunogenicity lost by inactivated baculovirus displayed ha (bac-ha) vaccine was recovered by encapsulation using reverse micelle of phosphatidylcholine adjuvant. mice orally immunized with encapsulated inactive bac-ha induced systemic and mucosal immune response comparable to live bac-ha [ ] . however, there are limitations in the use of mucosal adjuvants in humans. aside from effectiveness of the adjuvant, the major concern in using mucosal adjuvants is its possible detrimental impact on humans. therefore, adjuvant used in mucosal immunization has to be proven safe for use. premanand et al. [ ] utilized naturally occurring lipid-based vesicles (bilosomes) as a mucosal adjuvant to coat the recombinant baculovirus (bac-vp ). an oral immunization study revealed that bac-vp , associated with bilosomes, induced higher vp -specific immune responses and conferred complete protection in a challenge study [ ] . to overcome the hurdles of selecting safe mucosal adjuvants, data analysis can be applied to search for suitable mucosal adjuvants and to understand their mechanism of action. in addition to new technologies, novel concepts are welcomed in the development of suitable mucosal adjuvants. finally, considerations on the delivery route and the appropriate combination of vaccine and adjuvant are essential in ensuring optimal effectiveness of vaccination in humans. baculovirus surface-displayed vaccines have been proven effective in inducing protective immune responses in animal models. however, further advancement in the context of immunogenicity will be made by utilizing hybrid promoters for the enhanced expression and anchoring of complex protein on recombinant baculoviral envelopes. in addition, inclusion of novel regulatory elements and molecular adjuvant property-carrying motifs in the vector will certainly boost the immunogenicity of antigen displayed on the baculovirus envelope. the main obstacle in implementing baculovirus surface-displayed vaccine as a human vaccine candidate is the lack of safety guidelines for human immunization. however, international organizations, like organization of economic co-operation and development in [ ] directorate-general in [ ] , concluded that no adverse effects of baculovirus on human health have been observed in safety tests, and they are not pathogenic, carcinogenic, or genotoxic in mammalian cells. however, environmental and ecological impacts of the baculovirus in invertebrates will be reduced by modification of baculovirus essential genes required for replication and budding, allowing the development of a 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subcutaneous vaccination against pulmonary tuberculosis comparative evaluation of intranasal and subcutaneous route of immunization for development of mucosal vaccine against experimental tuberculosis mucosal adjuvants: opportunities and challenges oral vaccines: directed safe passage to the front line of defense reverse micelle-encapsulated recombinant baculovirus as an oral vaccine against h n infection in mice consensus document on information used in the assessment of environmental applications involving baculovirus; series on harmonization of regulatory oversight in biotechnology number european commission's health & consumer protection directorate-general we are grateful for the financial support received from temasek life sciences laboratory, singapore. the authors declare no conflict of interest. key: cord- -z qga v authors: ji, chao; kriaucionis, skirmantas; kessler, benedikt m.; jiang, chengyu title: from herbal small rnas to one medicine date: - - journal: sci china life sci doi: . /s - - -y sha: doc_id: cord_uid: z qga v nan in this special topic "herbal small rnas, novel precision medicine from mother nature", we present four original articles that study the entry of small rnas derived from herbs used in traditional chinese medicine into mammalian cells and their function within human and mouse cells, and one review paper focusing on current trends in herb genomics (du et al., ; huang et al., ; zhang et al., ; li et al., ; xin et al., ) . controversy over whether plant-derived micrornas could enter and function in mammalian cells and organisms began following publications by zhang chenyu in (zhang et al., ) and dickinson et al. in (dickinson et al., . although the current knowledge may not be able to explain how plant derived small nucleic acids enter mammalian cells and survive the nuclease degradation within, plant-derived small rnas have been successfully detected in mammalian cells and tissues (chin et al., ; zhu et al., ) . before we believe that regulation by small rna occurs across phylogenetic kingdoms, three critical questions need to be answered: ( ) what is the quantity of plant-derived small rnas entering mammalian cells and tissues? ( ) by which routes does entry occur? ( ) what protects small rnas from degradation after entry? in the manuscript "large-scale analysis of small rnas derived from traditional chinese herbs in human tissues", huang et al. discovered thousands of unique small rna sequences derived from traditional chinese medicine herbs in human blood samples and mice lung tissues (huang et al., ) . over % of the small rnas (srnas) sequences from the herbs chuan xin lian and hong jing tian identified in the study could be mapped to herb genomes, confirming their plant origins. these data suggest that large amounts of plant-derived small rnas can enter mammalian cells and tissues, and the cross-kingdom regulation by mi-crornas proposed by zhang chenyu group may be a common phenomenon (zhang et al., ). an online publication by du et al. in provided an answer to the question of how plant-derived small rnas enter mammalian cells and tissues: "plant-derived phosphocholine facilitates cellular uptake of anti-pulmonary fibrotic hjt-srna-m " (du et al., ) described herbderived phosphocholines able to facilitate the entrance of small rnas into mammalian cells. many traditional chinese medicines are administered by ingestion of a decoction, a concentrated plant essence produced by boiling herbs with water for - min, and consumed orally by patients. li et al. discovered that a heat-stable decoctosome-an exosomelike nanoparticle isolated from a decoction and consisting of lipids, proteins, chemicals, and small rnas-could enter mammalian cells and tissues by oral administration. the results are published in this issue: "herbal decoctosome is a novel form of medicine". a simple bencaosome, composed of synthetic lipids and srnas, is a simulation of a decoctosome constructed for medical applications. li et al. found that for optimal bencaosome preparation, lipids and small rnas should be mixed at degrees for min . using a critical micelle concentration assay, small rnas in the bencaosome were found to co-assemble with the lipid layers, suggesting that these small rnas may be protected from degradation. a previous study reported that ′-o-methyl groups on small interfering rnas (sirnas) can stabilize the sirnas in serum without affecting their activity in mammalian rna interference (yu et al., ) . plant micrornas have a natural occurring methyl group on the ribose of the last nucleotide (khvorova and watts, ) , indicating that herbal micrornas are naturally stable in mammalian cells. other phosphodiester or ribose modifications may impact both stability and affinity of micrornas to their targets. the main instability of rna comes from ′oh group in the ribose. it acts as a nucleophile, reacting with the adjacent phosphate and resulting in cleavage of the rna backbone. thus, the ribose or phosphodiester modifications are likely to impact the stability of rna. moreover, rna backbone modifications can impact base-pairing affinity. for example, ′-o-methylnucleotides have been demonstrated to have elevated melting temperature of resulting duplex molecules (majlessi et al., ) . future studies are necessary to explore additional modifications that might protect srnas from nuclease degradation in cells. in this special topic, the molecular mechanisms of some herbal-derived small rnas activities were elucidated. pgy-srna- was identified in a screen of the anti-inflammatory herb pu gong ying and shown to target the rela gene, a component of transcription factor nf-κb . bencaosomes with pgy-srna- inhibited poly(i:c) induced inflammation both in vitro and in vivo . hjt-srna-m was selected from anti-fibrotic herb hjt and targets three fibrotic genes simultaneously: alpha-sma, fibronectin, and collagen type iii alpha . the design from mother nature is fantastic: the bencaosome with hjt-srna-m improved lung fibrosis both in vitro and in vivo . furthermore, rnas resembling mi-crornas were identified in ophiocordyceps sinensis, a renowned traditional chinese medicine used to stimulate the immune system, decrease blood pressure and inhibit tumor growth . the target predictions of these microrna-like rnas and go analysis have confirmed their functions . the large amounts of small rnas we have consumed from plants over time may precisely regulate human gene expression, suggesting that the phrase "you are what you eat" may have scientific foundation. further studies are necessary to discover srnas from herbs that target all human genes, so that a healthy balance of gene expression can be achieved. it appears that mother nature may have provided precise remedies for many human diseases. useful nucleic acid therapeutics, including antisense oligonucleotides, aptamers, and small interfering rnas, are understood to inhibit specific functions of particular genes involved in disease. while hundreds of clinical trials are underway around the world, few are approved by the fda (vitravene , macugen , knamro , exondys , spinraza , defitelio . although quite a few compounds have been shown to provide effective treatment, delivery remains an important hurdle since oligonucleotides do not enter cells by diffusion. in this issue, the bencaosome has been used as a novel therapeutic to deliver oligonucleotides to mice by oral administration, and showed marked treatment effects . by mimicking a decoctosome, the bencaosome could consist of synthetic lipids, srnas, chemicals, and proteins targeting different genes in combination to achieve an optimal therapeutic effect. if the problem of oligonucleotide delivery is resolved, the paradigm of the drug market will be shifted: mother nature may also provide the drug delivery tools for precision medicine. globally, many medicines including chinese, ayurveda, unani, siddha, persian, islamic, muti, and mayan, as well as traditional european medicine, were based on herbal treatment with functional components extracted from herbs as the therapeutic agents. sources of modern pharmaceuticals include chemicals, proteins, and nucleic acids. the variety and richness of srnas may provide far more treatment options than chemicals and proteins. different medicines-be they western or eastern, traditional or modern-may share common modes of action to relieve symptoms and cure disease. future studies may elucidate the molecular mechanisms of these treatments and allow traditional and alternative therapies to be seen together as one medicine, regardless of origin. the author(s) declare that they have no conflict of interest. cross-kingdom inhibition of breast cancer growth by plant mir lack of detectable oral bioavailability of plant micrornas after feeding in mice plant-derived phosphocholine facilitates cellular uptake of anti-pulmonary fibrotic hjt-srna-m large-scale analysis of small rnas derived from traditional chinese herbs in human tissues the chemical evolution of oligonucleotide therapies of clinical utility herbal decoctosome is a novel form of medicine advantages of '-omethyl oligoribonucleotide probes for detecting rna targets methylation as a crucial step in plant microrna biogenesis exogenous plant mir a specifically targets mammalian ldlrap : evidence of cross-kingdom regulation by microrna identification of microrna-like rnas in ophiocordyceps sinensis plant micrornas in larval food regulate honeybee caste development followed by postdoctoral training at massachusetts general hospital, affiliated to harvard medical school before joining pumc/cams in . her research is to elucidate molecular pathogenesis of acute respiratory distress syndrome induced by rna viruses such as sars-cov, avian and swine influenza, and ebola. her group has also studied molecular mechanism of lung fibrosis, psychiatry and cancer. she has proposed a number of repurposing drugs after elucidation of disease molecular targets. her recent studies are focused on herbal small rnas as functional components of medicine. she published extensively in peer-reviewed journals including nature key: cord- -ajnlbno authors: domokos, g'abor; jerolmack, douglas j.; kun, ferenc; torok, j'anos title: plato's cube and the natural geometry of fragmentation date: - - journal: nan doi: nan sha: doc_id: cord_uid: ajnlbno plato envisioned earth's building blocks as cubes, a shape rarely found in nature. the solar system is littered, however, with distorted polyhedra -- shards of rock and ice produced by ubiquitous fragmentation. we apply the theory of convex mosaics to show that the average geometry of natural d fragments, from mud cracks to earth's tectonic plates, has two attractors:"platonic"quadrangles and"voronoi"hexagons. in d the platonic attractor is dominant: remarkably, the average shape of natural rock fragments is cuboid. when viewed through the lens of convex mosaics, natural fragments are indeed geometric shadows of plato's forms. simulations show that generic binary breakup drives all mosaics toward the platonic attractor, explaining the ubiquity of cuboid averages. deviations from binary fracture produce more exotic patterns that are genetically linked to the formative stress field. we compute the universal pattern generator establishing this link, for d and d fragmentation. solids are stressed to their breaking point when growing crack networks percolate through the material , . failure by fragmentation may be catastrophic , (fig. ), but this process is also exploited in industrial applications . moreover, fragmentation of rock and ice is pervasive within planetary shells , , , and creates granular materials that are literally building blocks for planetary surfaces and rings throughout the solar system [ ] [ ] [ ] [ ] [ ] (fig. ). plato postulated that the idealised form of earth's building blocks is a cube, the only space-filling platonic solid , . we now know that there is a zoo of geometrically permissible polyhedra associated with fragmentation (fig. ) . nevertheless, observed distributions of fragment mass [ ] [ ] [ ] [ ] and shape [ ] [ ] [ ] [ ] are self-similar, and models indicate that geometry (size and dimensionality) matters more than energy input or material composition , , in producing these distributions. fragmentation tiles the earth's surface with telltale mosaics. jointing in rock masses forms three-dimensional ( d) mosaics of polyhedra, often revealed to the observer by d planes at outcrops (fig. ) . the shape and size of these polyhedra may be highly regular, even approaching plato's cube, or resemble a set of random intersecting planes . alternatively, quasi- d patterns such as columnar joints sometimes form in solidification of volcanic rocks . these patterns have been reproduced in experiments of mud and corn starch cracks, model d fragmentation systems, where the following have been observed: fast drying produces strong tension that drives the formation of primary (global) cracks that criss-cross the sample and make "x" junctions [ ] [ ] [ ] (fig. ) ; slow drying allows the formation of secondary cracks that terminate at "t" junctions ; and "t" junctions rearrange into "y" junctions , to either maximise energy release as cracks penetrate the bulk [ ] [ ] [ ] , or during reopening-healing cycles from wetting/drying (fig. ) . whether in rock, ice or soil, the fracture mosaics cut into stressed landscapes (fig. ) , form pathways for focused fluid flow, dissolution and erosion that further disintegrate these materials . experiments and simulations provide anecdotal evidence that the geometry of fracture mosaics is genetically related to the formative stress field . it is difficult to determine, however, if similarities in fracture patterns among different systems are more than skin deep. first, different communities use different metrics to describe fracture mosaics and fragments, inhibiting comparison among systems and scales. second, we do not know whether different fracture patterns represent distinct universality classes, or are merely descriptive categories applied to a pattern continuum. third, it is unclear if and how d systems map to d. here we introduce the mathematical framework of convex mosaics to the fragmentation problem. this approach relies on two key principles: that fragment shape can be well approximated by convex polytopes ( d polygons, d polyhedra; fig. a ); and that these shapes must fill space without gaps, since fragments form by the disintegration of solids. without loss of generality (si section . ), we choose a model that ignores the local texture of fracture interfaces , . fragments can then be regarded as the cells of a convex mosaic , which table s . may be statistically characterised by three parameters. cell degree (v) is the average number of vertices of the polytopes, and nodal degree (n) is the average number of polytopes meeting at one vertex : we call [n,v] the symbolic plane. we define the third parameter ≤ p ≡ n r /(n r + n i ) ≤ as the regularity of the mosaic. n r is the number of regular nodes in which cell vertices only coincide with other vertices, corresponding in d to "x" and "y" junctions with n = and , respectively. n i is the number of irregular nodes where vertices lie along edges ( d) or faces ( d) of other cells, corresponding in d to "t" junctions of n = (fig. b) . we define a regular (irregular) mosaic as having p = (p = ). for d mosaics we also introducef as the average number of faces. in contrast to other descriptions of fracture networks , our framework does not delineate stochastic from deterministic mosaics; networks made from random or periodic fractures may have identical parameter values (fig. ) . this theory provides a global chart of geometrically admissible d and d mosaics in the symbolic plane. in this paper we measure the geometry of a wide variety of natural d fracture mosaics and d rock fragments, and find that they form clusters within the global chart. remarkably, the most significant cluster corresponds to the "platonic attractor": fragments with cuboid averages. discrete element method (dem) simulations of fracture mechanics show that cuboid averages emerge from primary fracture under the most generic stress field. geometric simulations show how secondary fragmentation by binary breakup drives any initial mosaic toward cuboid averages. the geometric theory of d convex mosaics is essentially complete and is given by the formula : which delineates the admissible domain for convex mosaics within the [n,v] symbolic plane (fig. ) -i.e., the global chart. boundaries on the global chart are given by: (i) the p = and p = lines; and (ii) the overall constraints that the minimal degree of regular nodes and cells is , while the minimal degree of irregular nodes is . we constructed geometric simulations of a range of stochastic and deterministic mosaics (see si section ) to illustrate the continuum of patterns contained within the global chart ( fig. ) we describe two important types of mosaics, which help to organise natural d patterns. first are primitive mosaics, patterns formed by binary dissection of domains. if the dissection is global we have regular primitive mosaics (p = ) composed entirely of straight lines which, by definition, bisect the entire sample. these mosaics occupy the point [n,v] = [ , ] in the symbolic plane . in nature, the straight lines appear as primary, global fractures. next, we consider the situation where the cells of a regular primary mosaic are sequentially bisected locally. irregular (t-type) nodes are created resulting in a progressive decrease p → and concomitant decreasē n → toward an irregular primitive mosaic. the valuev = , however, is unchanged by this process (fig. ) so in the limit we arrive at [n,v] = [ , ] . in nature these local bisections correspond to secondary fracturing , . fragments produced from primary vs. secondary fracture are indistinguishable. further, any initial mosaic subject to secondary splitting of cells will, in the limit, produce fragments withv = (si section ). thus, we expect primitive mosaics associated with the linev = in the global chart to be an attractor in d fragmentation, as noted by , and we expect the average angle to be a rectangle (fig. ) . we call this the platonic attractor. as a useful aside, a planar section of a d primitive mosaic (e.g., a rock outcrop) is itself a d primitive mosaic (fig. ) . the second important pattern is voronoi mosaics which are, in the averaged sense, hexagonal tilings [n,v] = [ , ] . they occupy the peak of the d global chart (fig. ). we measured a variety of natural d mosaics (si section ) and found, encouragingly, that they all lie within the global chart permitted by eq. . mosaics close to the platonic (v = ) line include patterns known to arise under primary and/or secondary fracture: jointed rock outcrops, mud cracks, and polygonal frozen ground. mosaics close to voronoi include mud cracks and, most intriguingly, earth's tectonic plates. hexagonal mosaics are known to arise in the limit for systems subject to repeated cycles of fracturing and healing (fig. ) . we thus consider voronoi mosaics to be a second important attractor in d. horizontal sections of columnar joints also belong to this geometric class; however, their evolution is inherently d, as we discuss in section . it is known that earth's tectonic plates meet almost exclusively at "y" junctions; there is debate, however, about whether this "tectonic mosaic" formed entirely from surface fragmentation, or contains a signature of the structure of mantle dynamics underneath , , . we examine the tectonic plate configuration as a d convex mosaic, treating the earth's crust as a thin shell. we find [n,v] = [ . , . ], numbers that are remarkably close to a voronoi mosaic. indeed, the slight deviation from [n,v] = [ , ] is because the earth's surface is a spherical manifold, rather than planar (si section . ). while this analysis doesn't solve the surface/mantle question, it suggests that the tectonic mosaic has evolved from episodes of brittle fracture and healing. the rest of our observed natural d mosaics plot between the platonic and voronoi attractors (fig. ). we suspect that these fractured landscapes, which include mud cracks and permafrost, either: initially formed as regular primitive mosaics and are in various stages of evolution toward the voronoi attractor; or were voronoi mosaics that are evolving via secondary fracture towards the platonic attractor. there is no formula for d convex mosaics analogous to the p = line of eq. that defines the global chart. there exists a conjecture, however, with a strong mathematical basis ; at present this conjecture extends only to regular mosaics. we define the harmonic degree ash =nv/(n +v). the conjecture is that d <h ≤ d− , where d is system dimension. for d mosaics we obtain the known result , h = , consistent with the p = substitution in eq. . in d the conjecture is equivalent to <h ≤ , predicting that all regular d convex mosaics live within a narrow band in the symbolic [n,v] plane (fig. ) . plotting a variety of well-studied periodic and random d mosaics (si section ), we confirm that all of them are indeed confined to the predicted d global chart (fig. ) . unlike the d case, we cannot directly measurē n in most natural d systems. we can, however, measure the polyhedral cells: the average numbersf ,v of faces and vertices, respectively. values for [f ,v] may be plotted in what we call the euler plane, where the lines bounding the permissible domain correspond to simple polyhedra (upper) where vertices are adjacent to three edges and three faces, and their duals which have triangular faces (lower; fig. ). simple polyhedra arise as cells of mosaics in which the intersections are generic -i.e., at most three planes intersect at one point -and this does not allow for odd values of v. as in d, d regular primitive mosaics are created by intersecting global planes. these mosaics occupy the point [n,v] = [ , ] on the d global chart (fig. ) . cells of regular primitive mosaics have cuboid averages [f ,v] = [ , ] , . this is the platonic attractor, marked by thev = line in the global chart. d voronoi mosaics, similar to their d counterparts, are associated with the voronoi tessellation defined by some random process. if the latter is a poisson process then we obtain (fig. ) . prismatic mosaics are created by regarding the d pattern as a base, that is extended in the normal direction. the prismatic mosaic constructed from a d primitive mosaic has cuboid averages, and is therefore statistically equivalent to a d primitive mosaic. the prismatic mosaic created from a d voronoi base is what we call a columnar mosaic, and it has distinct statistical properties: [n,v] = [ , ] , [f ,v] = [ , ] . thus, the three main natural extensions of the two dominant d patterns are d primitive, d voronoi, and columnar mosaics. regular primitive mosaics appear to be the dominant d pattern resulting from primary fracture of brittle materials . moreover, dynamic brittle fracture produces binary breakup in secondary fragmentation , , driving the d averages [f ,v] towards the platonic attractor. the most common example in nature is fractured rock (figs. , ) . the other two d mosaics are more exotic, and seem to require more specialised conditions to form in nature. columnar joints like the celebrated giants causeway, formed by the cooling of large basaltic rock masses , , (fig. ) , appear to correspond to columnar mosaics. in these systems, the hexagonal arrangement and downward (normal) penetration of cracks arise as a consequence of maximising energy release [ ] [ ] [ ] . the only potential example of d voronoi mosaics that we know of is septarian nodules, such as the famous moeraki boulders (fig. ) . these enigmatic concretions have complex growth and compaction histories, and contain internal cracks that intersect the surface . similar to primitive mosaics, the intersection of d voronoi mosaics with a surface is a d voronoi mosaic. we hypothesise that primary fracture patterns are genetically linked to distinct stress fields, in order of most generic to most rare. in a d homogeneous stress field we may describe the stress tensor with eigenvalues |σ | ≥ |σ | and characterise the stress state by the dimensionless parameter µ = σ /σ , whose admissible domain is µ ∈ [− , ]. in d this corresponds to eigenvalues |σ | ≥ |σ | ≥ |σ |, stress state µ = σ /σ , µ = σ /σ , i = sgn(σ ), and domain µ , µ ∈ [− , ], |µ | ≥ |µ | (and it is double covered due to i = ± ) (fig. ). there is a unique map from these / s ). the d pattern generator is described by the single scalar functionv(µ) (andn can be computed from [ ] ), while the d pattern generator is characterised by scalar functions computing the full pattern generator is beyond the scope of the current paper, even in d. instead, we perform generic dem simulations , of a range of scenarios to interpret the important primary mosaic patterns described above (see methods). in d, we find that pure shear produces regular primitive mosaics (fig. ) , implyingv(− ) ≈ . this corresponds to the platonic attractor. in contrast, hydrostatic tension creates regular voronoi mosaics ( fig. ; si section ) such that v( ) ≈ -the voronoi attractor. both are in agreement with our expectations. in d, we first conducted dem simulations of hard materials at [µ , µ ] locations corresponding to shear, uniform d tension and uniform d tension ([− . , − . ], [ , − . ] and [ , ]), respectively (methods, si section ). the resulting mosaics displayed the expected fracture patterns for brittle materials: primitive, columnar and voronoi, respectively (fig. ) . to obtain a global, albeit approximate, picture of the d pattern generator we ran additional dem simulations that uniformly sampled the stress space on a × (∆µ= . ) grid, for both i= + and i= − (si section ). the constructed pattern generator demarcates the boundaries in stress-state space that separate the three primary fracture patterns (fig. ) . the vast proportion of this space is occupied by primitive mosaics, which are also the only pattern generated under negative volumetric stress. such compressive stress conditions are pervasive in natural rocks. columnar mosaics are a distant second in terms of frequency of occurrence; they occupy a narrow stripe in the stress space. most rare are voronoi mo- ( ) table s . patterns ( ) ( ) are generated by generic dem simulation (see methods): ( ) general stress state with eigenvalues σ > σ ; and ( ) isotropic stress state with saics which only occur in a single corner of the stress space (fig. ) . boundaries separating the three patterns shifted somewhat for simulations that used softer materials (fig. s ), but the ranking did not. these primary fracture mosaics serve as initial conditions for secondary fracture. while our dem simulations do not model secondary fracture, we remind the reader that binary breakup drives any initial mosaic toward an irregular primitive mosaic with cuboid averages (si section ) -emphasising the strength of the platonic attractor. based on the pattern generator (fig. ) we expect that natural d fragments should have cuboid properties on average, [f ,v] = [ , ] . to test this we collected particles from the foot of a weathering dolomite rock outcrop (fig. ) and measured their values of f and v, plus mass and additional shape descriptors (see methods; si section ). we find striking agreement: the measured averages [f ,v] = [ . , . ] are within % of the theoretical prediction, and distributions for f and v are centred around the theoretical values. moreover, odd values for v are much less frequent than even values, illustrating that natural fragments are well approximated by simple polyhedra (fig. ) . we regard these results as direct confirmation of the hypothesis, while also recognising significant to better understand the full distributions of fragment shapes, we used geometric simulations of regular and irregular primitive mosaics. the cut model simulates regular primitive mosaics as primary fracture patterns by intersecting an initial cube with global planes (fig. ) while the break model simulates irregular primitive mosaics resulting from secondary fragmentation processes. we fit both of these models to the shape descriptor data using three parameters: one for the cutoff in the mass distribution, and two accounting for uncertainty in experimental protocols (see methods; si section ). the best fit model, which corresponds to a moderately irregular primitive mosaic, produced topological shape distributions that are very close to those of natural fragments (mean values [f ,v] = [ . , . ]). we also analysed a much larger, previously collected data set ( particles) containing a diversity of materials and formative conditions . although values for v and f were not reported, measured values for classical shape descriptors , could be used to fit to the cut and break models (si section ). we find very good agreement (r > . ), providing further evidence that natural d fragments are predominantly formed by binary breakup (si, fig. ). finally, we use the cut model to demonstrate how d primitive fracture mosaics converge asymptotically toward the platonic attractor as more fragments are produced (fig. ). table s . the application and extension of the theory of convex mosaics provides a new lens to organise all fracture mosaicsand the fragments they produce -into a geometric global chart. there are attractors in this global chart, arising from the mechanics of fragmentation. the platonic attractor prevails in nature because binary breakup is the most generic fragmentation mechanism, producing averages corresponding to quadrangle cells in d and cuboid cells in d. remarkably, a geometric model of random intersecting planes can accurately reproduce the full shape distribution of natural rock fragments. our findings illustrate the remarkable prescience of plato's cubic earth model. one cannot, however, directly 'see' plato's cubes; rather, their shadows are seen in the statistical averages of many fragments. the relative rarity of other mosaic patterns in nature make them exceptions that prove the rule. voronoi mosaics are a second important attractor in d systems such as mud cracks, where healing of fractures reorganises junctions to form hexagonal cells. such healing is rare in natural d systems. accordingly, columnar mosaics arise only under specific stress fields, that are consistent with iconic basalt columns experiencing contraction under directional cooling. d voronoi mosaics require very special stress conditions, hydrostatic tension, and may describe rare and poorly understood concretions known as septarian nodules. we have shown that earth's tectonic mosaic has a geometry that likely arose from brittle fracture and healing, consistent with what is known about plate tectonics (fig. ) . this opens the possibility of inferring stress history from observed fracture mosaics. space missions are accumulating an evergrowing catalogue of d and d fracture mosaics from diverse planetary bodies, that challenge understanding (fig. ) . geometric analysis of surface mosaics may inform debates on planetery dynamics, such as whether pluto's polygonal surface (fig. c) is a result of brittle fracture or vigorous convection . another potential application is using d outcrop exposures to estimate the d statistics of joint networks in rock masses, which may enhance prediction of rock fall hazards and fluid flow . initial samples were randomised cubic assemblies of spheres glued together, with periodic boundary conditions in all directions. the glued contact was realised by a flat elastic cylinder connecting the two particles which was subject to deformation from the relative motion of the glued particles. forces and torques on the particles were calculated based on the deformation of the gluing cylinder. the connecting cylinder broke permanently if the stress acting upon it exceeded the tresca criterion . stress field was implemented by slowly deforming the underlying space. in order to avoid that there is only one percolating crack, we have set a strong viscous friction between the particles and the underlying space. this acts as a homogeneous drag to the particles which ensures a homogeneous stress field in the system. for any given shear rate the fragment size is controlled by the particle space viscosity and the tresca criterion limit. we set values that produce reasonable-sized fragments relative to our computational domain, allowing us to characterise the mosaics. another advantage of the periodic system was that we could avoid any wall effect that would distort the stress field. we note here that it is possible to slowly add a shear component to the isotropic tensile shear test and obtain a structure which has average values ofn andv that are between the primitive mosaics and the voronoi case. details of mechanical simulations are discussed in si section . field data in the simulation we first computed a regular primitive mosaic by dissecting the unit cube with randomly chosen planes, resulting in × fragments. we refer to this simulation as the cut model. subsequently we further evolved the mosaic by breaking individual fragments. we implemented a standard model of binary breakup , to evolve the cube by secondary fragmentation: at each step of the sequence, fragments either break with a probability p b into two pieces, or keep their current size until the end of the process with a probability − p b . the cutting plane is placed in a stochastic manner by taking into account that it is easier to break a fragment in the middle perpendicular to its largest linear extent. inspired by similar computational models , we used p b dependent on axis ratios (see si section ). this computation, which we call the break model, provides an approximation to an irregular primitive mosaic; this secondary fragmentation process influenced the nodal degreen, but not [v,f ]. in order to compare numerical results with the experimental data obtained by manual measurements, we have to take into account several sampling biases. first, there is always a lower cutoff in size for the experimental samples. we implemented this in simulations by selecting only fragments with m > m , m being the cutoff threshold. second, there is experimental uncertainty when determining shape descriptors -especially marginally stable or unstable equilibria for the larger dataset (see si section ). we implemented this in the computations by letting the location of the centre of mass be a random variable with variation σ chosen to be small with respect to the smallest diameter of the fragment. we kept only those equilibria which were found in % of the cases. third, there is experimental uncertainty in finding very small faces. we implemented this into the computations by assuming that faces smaller than a p will not be found by experimenters, where p denotes the smallest projected area of the fragment. using the above three parameters we fitted the seven computational histograms to the seven experimental ones by minimising the largest deviation, and we achieved matches with r max ≥ . from all histograms (see si section for details). results for the small data set are shown in fig. . / fractals and chaos in geology and geophysics statistical physics of fracture, friction, and earthquakes fractures and fracture networks crushing and grinding process handbook subduction controls the distribution and fragmentation of earth's tectonic plates tidal reorientation and the fracturing of jupiter's moon europa vigorous convection as the explanation for pluto's polygonal terrain an evolving view of saturn's dynamic rings clastic polygonal networks around lyot crater, mars: possible formation mechanisms from morphometric analysis size distribution of particles in saturn's rings from aggregation and fragmentation plato's error and a mean field formula for convex mosaics uniform tilings of -space fragmentation of long thin glass rods self-organized criticality in fragmenting fragmentation of shells new universality class for the fragmentation of plastic materials scaling behaviour of fragment shapes universality of fragment shapes fractal behavior and shape characteristics of fragments produced by the impact of quasi-brittle spheres reconstructing the transport history of pebbles on mars an automaton for fractal patterns of fragmentation universal dynamic fragmentation in d dimensions characterizing rock joint geometry with joint system models. rock mechanics rock engineering evolving fracture patterns: columnar joints, mud cracks and polygonal terrain universal scaling of polygonal desiccation crack patterns desiccation cracks and their patterns: formation and modelling in science and nature evolution of mud-crack patterns during repeated drying cycles evoluton of polygonal fracture patterns in lava flows sequential fragmentation: the origin of columnar quasihexagonal patterns why hexagonal basalt columns? crack formation and self-closing in shrinkable, granular packings breaking rocks made easy. blending stress control concepts to advance geomorphology tensile fracturing in rocks stochastic and integral geometry tensile fracturing in rocks on some average properties of convex mosaics hierarchical crack pattern as formed by successive domain divisions how many plates? an updated digital model of plate boundaries discrete models for two-and three-dimensional fragmentation solution for the fragment-size distribution in a crack-branching model of fragmentation disclosing the temperature of columnar jointing in lavas the moeraki boulders; anatomy of some septarian concretions septarian crack formation in carbonate concretions from shales and mudstones on folding during three dimensional progressive deformation fast parallel algorithms for short-range molecular dynamics a contact model for the yielding of caked granular materials rock fractures and fluid flow: contemporary understanding and applications this research was supported by nkfih grants k (gd) k (jt) and k (fk) and emmi fikp grant viz (gd, jt) the authors thank krisztián halmos for his invaluable help with field data measurements. gd originated the concept of the paper, led mathematical and philosophical components, and contributed to field data. djj contributed to geophysical interpretation, and led formulation of the manuscript. fk led interpretation of fragmentation mechanics, and contributed to field data. jt performed geometric and dem computations, and led data analysis. all authors contributed to writing the manuscript. key: cord- - mrzaxi authors: kim, byunghyun; kim, kyuseok; lee, jieun; kim, joonghee; jo, yoo hwan; lee, jae hyuk; hwang, ji eun title: impact of bacteremia prediction rule in cap: before and after study date: - - journal: the american journal of emergency medicine doi: . /j.ajem. . . sha: doc_id: cord_uid: mrzaxi abstract objective in cases of community acquired pneumonia (cap), it has been known that blood cultures have low yields and rarely affect clinical outcomes. despite many studies predicting the likelihood of bacteremia in cap patients, those results have been rarely implemented in clinical practice, and use of blood culture in cap is still increasing. this study evaluated impact of implementing a previously derived and validated bacteremia prediction rule. methods in this registry-based before and after study, we used piecewise regression analysis to compare the blood culture rate before and after implementation of the prediction rule. we also compared -day mortality, emergency department (ed) length of stay, time-interval to initial antibiotics after ed arrival, and any changes to the antibiotics regimen as results of the blood cultures. in subgroup analysis, we compared two groups (with or without the use of the prediction rule) after implementation period, using propensity score matching. results following the implementation, the blood culture rate declined from . % to . % (p = . ) without significant changes in -day mortality and antibiotics regimen. the interval to initial antibiotics ( min vs. min, p = . ) and length of stay ( min vs. min, p = . ) were not significantly changed. in subgroup analysis, the group that use the prediction rule showed min faster antibiotics initiation (p = . ) and min shorter length of stay (p = . ) than the group that did not use the rule. conclusion implementation of the bacteremia prediction rule in cap patients reduced the blood culture rate without affecting the -day mortality and antibiotics regimen. blood culture before antibiotics treatment has been a routine diagnostic test in patients presumed to be infectious [ ] . however, in patients with community acquired pneumonia (cap), it has been known that the rate of true positive blood cultures is b % [ , ] . in emergency departments (ed), the true positive results from blood cultures rarely affected patient management [ , ] . additionally, ed overcrowding may increase the risk of blood culture contamination [ , ] . as part of an effort to reduce unnecessary blood culture and improve the yield of blood culture in cap, there have been many studies describing bacteremia prediction rules [ ] [ ] [ ] . despite those studies, in a recent study, it has been reported that the blood culture rate in cap patients is still increasing [ ] . therefore, whether those decision rules clinically impacted the reduction of blood cultures is questionable. for clinical decision rules to have an impact on standard treatment, the clinical decision rules should pass three stages: derivation, validation, and implementation [ , ] . in our previous studies, we derived the bacteremia prediction model in cap patients and tested the generalizability of the rule by a multicenter external validation [ , ] . here, we evaluated the impact of implementing the bacteremia prediction model on the rate of blood cultures. we hypothesized that implementation may reduce the blood culture rate without changing the mortality rate. also without blood culture, the time interval from patient presentation in the ed to the first intra-venous antibiotics administration could be shortened as well as the length of ed stay. this study is an uncontrolled before and after study based upon a retrospective review of a patient registry database. before implementation of the bacteremia prediction rule, a prospective registry of patients diagnosed with pneumonia in the ed had been in place since . the registry includes the patient's baseline characteristics and co-morbidities. time variables such as ed arrival time and ed discharge time were included in the registry as well as the initial antibiotics administration time. the registry also provided the patient's initial vital signs and laboratory findings including the blood culture results if a blood culture had been performed. finally, the patient's dispositions from the ed including admission to the ward or icu as well as the -day mortality were included in the registry. this study was performed at a -bed tertiary academic hospital with an annual ed census of , patients and was approved by the institutional review board of the hospital. the implementation of the bacteremia prediction rule was initiated in january of . we retrospectively analyzed the patient registry data from january to january . exclusion criteria were the same as in previous studies [ , ] . in short, we excluded patients who were diagnosed with hospital-acquired pneumonia (hap), health care-associated pneumonia (hcap), and ventilator-associated pneumonia (vap). we also excluded patients who were transferred from our hospital because of difficulty in follow-up and limited information on the changes of antibiotics. for ease of use of the bacteremia prediction rule, we created an excel file in which the recommendations for blood culture appeared with risk-stratified scores, calculated automatically by entry of variables ( supplementary fig. ). because the resident physicians in the ed changed monthly according to a rotation system with other hospitals, the instructions for the excel file and the explanations of the prediction rule were introduced on the first wednesday of every month. all emergency physicians were encouraged to record the score calculated with the excel file on electronic medical record (emr) of patients suspected of pneumonia regardless of the performance of blood cultures. the prediction rule use records for each emergency physician were reported as a feedback three times a week. the primary outcome was the blood culture rate before and after implementation of the bacteremia prediction rule. the secondary outcomes included the -day mortality rate, the ed length of stay, the time-interval to initial antibiotics after ed arrival, and any changes to the antibiotics regimen as a result of the blood culture findings. for the cost analysis of reduced blood culture usage due to this intervention, we compared the mean cost for blood cultures per patient before and after implementation of the prediction rule. for any positive blood culture results, we thoroughly reviewed the results, including preliminary reporting and final reporting times. in preliminary results, only gram staining results of the species growing in culture bottle were reported. in the final report, the exact names of the identified pathogens were reported with the antibiotics susceptibility test results. for changes to the antibiotics regimen following the blood culture results, we reviewed the daily antibiotics administration with the exact times until h after the final result report time. for adverse events due to contaminations, we defined the unnecessary administration of vancomycin as the start of administration after the preliminary reports and its discontinuation following the identification of contaminants in the final reports. additionally, unnecessary followup blood cultures were defined as blood cultures performed after preliminary reports that were confirmed as contaminants in the final reports and the follow-up results were negative. the following isolates were identified as contaminants: coagulase-negative staphylococcus, bacillus species, micrococcus species, corynebacterium species, and propionibacterium species. continuous variables were presented as the means and standard deviations. binomial variables were presented as the frequency of occurrence. wilcoxon's rank-sum test, the chi-square test, or fisher's exact test was performed, as appropriate, for comparisons before and after intervention. all p values were -tailed, and p values b . were considered statistically significant. we adopted a piecewise regression discontinuity approach using generalized linear models (using odds for binary results) to evaluate the impact of the intervention. for adjusted intervention effects, we constructed multivariable linear regression models using variables with statistical significance (p value b . ) from comparisons before and after the intervention. additionally, we performed box-pierce tests to identify statistically significant autocorrelations in time series models we used (p value b . ). because irrespective of prediction rule, the blood culture rates could be reduced only by education introducing the low yield of blood culture in cap, we performed subgroup analysis only using the patients after intervention. for the subgroup analysis, we used propensity score matched analysis to compare the groups that use the prediction rule and did not. we compared the blood culture rate, time-interval to initial antibiotics, and ed length of stay of both groups. all analyses were performed using stata (version ; statacorp, college station, tx) and any patients with missing variables were not included in analyses. of the registered patients during study period, a total of patients were included in the study after patients were excluded (fig. ). among the eligible patients, and patients were assigned to the before and after intervention groups, respectively. among the patients after intervention, patients ( . %) had the bacteremia score record on their emr. table summarizes the baseline characteristics of the two groups. after implementation of the bacteremia prediction rule, the blood culture rate decreased from . % to . %, representing . % reduction ( % confidence interval [ci] = . to . , p = . ) ( table ) . additionally, the monthly trend of the blood culture rate changed from increasing to decreasing (fig. ) . after model adjustment, the blood culture rate decreased from . % to . %, which represents . % reduction ( % ci = . to . , p = . ) reduction. the monthly trend of the blood culture rate still appeared to decrease after implementation of the intervention ( table ). after the implementation, the -day mortality rate decreased from . % ( % ci = . to . ) to . % ( % ci = . to . , p b . ). however, after model adjustment, this difference was not statistically significant (p = . ) ( table ) . the time-interval to initial antibiotics after ed arrival decreased from min to min but did not reach statistical significance (p = . ). after model adjustment, no statistically significant difference was found between the two groups (table ) . after implementation, the ed length of stay also decreased from min to min but the difference between the two groups was not statistically significant (table ) . table shows the blood culture results before and after intervention. the overall blood culture contamination rate was . % ( / ). after the implementation, the blood culture contamination rate was not changed (p = . ). antibiotics regimen changes based on positive blood culture results were not significantly different between the two groups ( . %; / before implementation vs. . %; / after implementation, p = . ). among the patients with contaminations, . % ( / ) had to undergo unnecessary phlebotomy for follow-up blood cultures and . % ( / ) were administered unnecessary vancomycin treatment. after score implementation, the total adverse event rates decreased from . % ( / ) to . % ( / ). however, this difference did not reach statistical significance (p = . ). in the propensity score matched subgroup analysis, we compared two groups that used the prediction rule and did not, during intervention period (supplementary table ). the baseline characteristics of the two groups were similar but, the group with the bacteremia score had the lower blood culture rate than the group that did not use the bacteremia score ( . % vs. . %, p b . ). additionally, the group that use the prediction rule had shorter time-interval to initial antibiotics ( min vs. min, p = . ) and reduced ed length of stay ( min vs. min, p = . ). in our time series analysis study, we reported that the implementation of the bacteremia prediction rule successfully reduced the blood culture rate without any significant -day mortality and antibiotics regimen changes. blood culture technique is time-consuming and resource-consuming considering the need for an aseptic technique and venous access to more than two sites [ , ] . especially in the ed, it is well known that blood culture contamination is common, and many eds have higher contamination rates than clinical and laboratory standards institute (clsi) recommendation of % [ ] [ ] [ ] . false positive results from blood cultures are associated with increased medical cost and hospital length of stay [ , ] . clinically, blood culture contaminations result in unnecessary antibiotics administration [ ] . in one previous study, a % of additional unnecessary glycopeptide administration in pseudobacteremia cases was observed, which is consistent with our results (table ) [ ] . as multidrug resistance pathogens in pneumonia have been increasing, concerns exist about treatment failure without initial blood cultures [ ] . however, in a recent study, multidrug resistance pathogens were uncommon in cap patients, which accounted for . % [ ] . according to our bacteremia score system, blood cultures could be omitted in patients in the low-risk group, of which the true bacteremia rate was b % [ ] . in this study, the true bacteremia in the low-risk group was . % ( / ) and antibiotics step up due to multidrug resistance organisms isolated from blood cultures was . % ( / ). concerning the adverse event rate due to false positive blood cultures of . % ( / ), it seems reasonable to omit blood culture in the low-risk group (table ) . contrary to our hypothesis, the time to initial antibiotics and ed length of stay tended to decrease, but no statistically significant differences were measured between the before and after intervention groups (table ). according to the sepsis guideline, initial antibiotics should be administered within h after recognition of sepsis [ ] . for patients with severe initial presentation, ed physicians may urge the initiation of iv antibiotics and blood cultures without adherence to the bacteremia score. calculation of the bacteremia score takes n . h because the scoring system includes albumin and c-reactive protein results. this fact may be why the bacteremia score adherence group had mild presentations and lower pneumonia severity index (psi) scores (supplementary table ). for patients with mild symptoms and stable initial vital signs, ed physicians may wait for the laboratory tests before the initiation of iv antibiotics. those patients may be stratified into the low-risk group for bacteremia and could have more chances to avoid unnecessary blood cultures of low yield. therefore, by the time a decision had to be made regarding initiating iv antibiotics without blood culture, n min had already passed, and the time-saving effect of omitting blood cultures may have been diminished. this study has several limitations. first because this study was a time series before and after study based on a retrospective review of a registry. missing not at random (mnar) which occupied a small fraction ( / ; . %) but not included in analysis, may have created a bias. also there could be many confounders influencing the results besides the intervention. second, the baseline characteristics of the two groups before and after intervention were significantly different. the middle east respiratory syndrome coronavirus (mers-cov) outbreak in may have increased the sensitivity of the ed visit population to respiratory symptoms, which may be one of the reasons accounting for the difference [ ] . third, the adherence rate for the bacteremia score was . % which may be too low to expect clinical and statistical significance. generally, evidence-based changes in the clinical practice of physicians take a substantial amount of time and effort [ , ] . in addition to those periods of adaptation, the time needed for calculation of the score make a decrease of adherence inevitable in severe patients for whom blood cultures and iv antibiotics were urgent. additionally, the calculation of the score with multiplication and addition is difficult and the use of excel file could be another cumbersome step to emergency physicians in a crowded ed. finally the target population only included patients with cap and this would limit the broad application of the table blood culture results with contamination and antibiotics regimen changes before and after implementation of bacteremia prediction model. ⁎ adjusted by variables with statistical significance (p value b . ): age, sex, history of diabetes mellitus, hypertension, heart failure, cerebrovascular disease, renal disease, liver disease, and known neoplasm, systolic blood pressure, respiratory rate, pulse rate, body temperature, white blood cell count, hematocrit, platelet count, glucose, albumin, bun, psi, admission rate. a the numbers of patients with any missing variables used in analysis was and those patients were not included in analyses. rule in the clinical field. despite those limitations, we could observed a statistically significant reduction in blood cultures after intervention without affecting -day mortality as well as decreasing trends in the initial antibiotics administration time and ed length of stay. considering the low adherence rate to the score system, a more thorough education and feedback is needed to achieve statistically significant clinical impacts on the time of initial antibiotics administration and ed length of stay. in conclusion, the implementation of the bacteremia prediction rule in cap patients reduced the blood culture rate without affecting the day mortality and antibiotics regimen. and bacteremia prediction score was independently associated with a reduction in initial antibiotics administration time and ed length of stay. further prospective multicenter studies should be performed to evaluate the impact of wide 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potential contaminants in blood cultures among patients in a medical center risk factors for drug-resistant pathogens in community-acquired and healthcare-associated pneumonia epidemiology and predictors of multidrug-resistant community-acquired and health care-associated pneumonia the surviving sepsis campaign sepsis guideline preventive behaviors by the level of perceived infection sensitivity during the korea outbreak of middle east respiratory syndrome in from best evidence to best practice: effective implementation of change in patients' care what drives change? barriers to and incentives for achieving evidence-based practice none. supplementary data to this article can be found online at https://doi. org/ . /j.ajem. . . . key: cord- -s pnq j authors: li, maiquan; huang, weisu; jie, fan; wang, mengmeng; zhong, yongheng; chen, qi; lu, baiyi title: discovery of keap −nrf small−molecule inhibitors from phytochemicals based on molecular docking date: - - journal: food chem toxicol doi: . /j.fct. . sha: doc_id: cord_uid: s pnq j various phytochemicals have been reported to protect against oxidative stress. however, the mechanism underlying has not been systematically evaluated, which limited their application in disease treatment. nuclear factor erythroid −related factor (nrf ), a central transcription factor in oxidative stress response related to numerous diseases, is activated after dissociating from the cytoskeleton−anchored kelch−like ech−associated protein (keap ). the keap –nrf protein–protein interaction has become an important drug target. this study was designed to clarify whether antioxidantive phytochemicals inhibit the keap –nrf protein–protein interaction and activate the nrf -are signaling pathway efficiently. molecular docking and d−qsar were applied to evaluate the interaction effects between antioxidant phytochemicals and the nrf binding site in keap . the nrf activation effect was tested on a h( )o( )−induced oxidative−injured cell model. results showed that the phytochemicals could be divided into high−, medium−, and low−total−score groups depending on their binding affinity with keap , and the high−total−score group consisted of compounds with abundant oxygen or glycosides. meanwhile, these compounds could bind with key amino acids in the structure of the keap −nrf interface. compounds from high−total−score group show effective activation effects on nrf . in conclusion, phytochemicals showed high binding affinity with keap are promising new nrf activators. oxidative stress, which is caused by an imbalance between reactive species and antioxidative stress defense systems in cells, plays a key role in many diseases, including cancers (reuter et al., ) , cardiovascular diseases (anatoliotakis et al., ) , neurodegenerative diseases (emerit et al., ) et al. the nuclear factor erythroid −related factor (nrf ) pathway is the major pathway that responds to reactive species and redox potentials, and nrf activation is a key defense mechanism against oxidative stress (h et al., ) . however, under physiological conditions, repressor kelch−like ech−associated protein (keap ) holds nrf in the cytoplasm and promotes its ubiquitination (mcmahon et al., ) . by contrast, under pathological conditions, nrf is released from keap , translocates to the nucleus, and triggers the transcriptional activation of are−dependent genes and antioxidative enzymes (dhakshinamoorthy and jaiswal, ) . thus, targeting the nrf -are signaling pathway is a logical strategy to discover therapeutic agents for diseases and conditions induced by oxidative stress. the direct inhibition of the keap -nrf protein-protein interaction is an alternative for the discovery of small−molecule nrf activators (magesh et al., ) . compounds that interact with keap and occupy the nrf binding site in the protein can cause the dissociation of keap from nrf and finally induce the transcriptional activation of nrf (pang et al., ) . phytochemicals have significantly contributed to drug discovery. many studies have shown that phytochemicals in edible flowers, vegetables, and fruits prevent or mitigate chronic diseases in humans (kumar et al., ; steinmetz and potter, ) . studies in the last few decades have demonstrated the benefits of phytochemicals counteracting oxidative stress. phenolic compounds as primary antioxidants can scavenge free radicals, thereby delaying or inhibiting the initiation step and interrupting the propagation step of lipid oxidation (kiokias et al., ) . vitamin e functions as a peroxyl radical−scavenging antioxidant and as an inhibitor of lipid peroxidation in vitro and in vivo (niki, ) . carotenoids can act as chemical quenchers undergoing irreversible oxygenation. epidemiological studies and clinical trials showed that adequate carotenoid supplementation may significantly reduce the risk of several disorders mediated by reactive oxygen species (fiedor and burda, ) . natural alkaloids are effective under oxidative stress in dpph, frap, and teac assays (rehman and khan, ) . some of these natural compounds exhibit antioxidant effects through activating the nrf pathway; however, details on this finding are few. molecular docking is widely used to predict the conformation of small−molecule ligands within the appropriate target binding site accurately (meng et al., ) . quantitative structure-activity relationship (qsar) modeling is a well−used computer−aided drug design method because it can amalgamate statistics and computational chemistry as well as complement the experimental approach. comparative molecular field analysis (comfa) (cramer et al., ) , which is a useful method in ligand−based drug design strategy, holds that appropriate sampling of steric and electrostatic fields around molecules provides information necessary for understanding their biological activities. statistics is computed by partial least squares (pls) regression analysis. in this study, molecular docking and d−qsar were applied to evaluate the interaction effects between phytochemicals and the nrf binding site in keap . phenylethanoid glycosides showed better effects than other compounds. to verify the presumptive model above, compounds from high−, medium−, and low−total−score groups were further studied on a h o −induced oxidative−injured cell model, and compounds from different groups showed significantly different activation effects on nrf . a search for phytochemicals was carried out using pubmed, web of science, and google scholar. keywords used for the search were "antioxidant" and "nrf activators." natural antioxidants were selected artificially according to the classification standard of phytochemicals. then the d conformer of these phytochemicals were searched in pubchem. a compound library of phytochemicals, whose d conformer could be found in pubchem, was created (supplement table ). . rpmi− medium and fetal bovine serum were procured from hyclone (logan, utah, usa), . % trypsin edta, penicillin, and streptomycin were purchased from keyi (hangzhou, china). antibodies to nrf , histone h , and β−actin; anti−mouse−horseradish peroxide (hrp) igg; and anti−rabbit-hrp-igg were acquired from abcam (london, uk). α−tocopherol was used as a positive control. keap d structure (protein data bank (pdb) codes: l b) was obtained from pdb and prepared with sybyl x . (ji et al., ) . a total of phytochemicals that were proven to have good antioxidant properties were selected and prepared in accordance with the procedure in section . . the sdf formats of their d structures were obtained from pubchem chemical library. the semi−flexible docking of natural compounds as ligands for keap structures was evaluated using sybyl x . . surflex-dock module of sybyl is a molecular docking unit that performs flexible alignments. the results are presented as docking accuracy and screening utility (ferreira et al., ) . the docking procedure was started with protomol generation using a ligand−based approach. for each protein-ligand pair, three top−ranked docked solutions were saved. in consideration that the results of comfa studies are sensitive to the alignment of molecules, the alignment of d structures plays a vital role during comfa analysis. the lowest energy conformer of analog was chosen as a template structure for the molecular alignment of the data set. molecules in their respective lowest energy conformations were superimposed on the template using the rigid−body fit option in sybyl−x . (singh et al., ) . following the standard procedure, sybyl−x . was used to create a database of phytochemicals. the pls algorithm was used to obtain the relationship between the structural parameters and the binding affinity. cross−validation analysis was performed using the leave−one−out method, wherein compounds were removed from the dataset and their activity were predicted using the model derived from the rest of the dataset. the cross−validated r that resulted in the optimum number of components and lowest standard error of prediction was selected (zhang and zhong, ) . rat adrenal pheochromocytoma line (pc cells) was obtained from the institute of biochemistry and cell biology, sibs (cas, shanghai, china). the cells were maintained in rpmi− (hyclone) containing % fetal bovine serum (hyclone), u/ml penicillin, and . mg/ml streptomycin at °c with % co . the medium was changed every other day. pc cells were seeded in mm petri dishes at × cells/dish. after attachment, the cells were treated with the selected compounds ( and μm) for h and then incubated with h o for another h with the previous compounds removed. pc cells were seeded in -well plates at × cells/well. after attachment, cells were incubated with tested compounds at and μm for h. after incubation, cells were treated with mg/ml mtt for h at °c. then the media were carefully removed, the formazan crystals were dissolved in μl of dmso, the absorbance was measured at nm on a plate reader. controls utilized the same concentration of medium with dmso alone. cell viability was normalized as the percentage of control. cytosolic and nuclear proteins were isolated with a beyotime kit. the growth medium was removed after cell culture, and the cells were washed twice using ml of pbs, added with ml of pbs, and then scraped into centrifuge tubes on ice. the cells were centrifuged at ×g for min. the cells were added with buffer a containing % pmsf on ice for min, vortexed for s, and then placed on ice for min. then, the cells were added with buffer b, vortexed for s, and then placed on ice for min. the tubes were centrifuged at , ×g for min at °c, added with buffer n containing % pmsf on ice for min, vortexed every - min, and then centrifuged again at , ×g for min at °c. the protein concentration of the samples was detected by a beyotime bca protein assay kit, and all samples were stored at − °c for western blot analysis. western blot was performed using standard methods. in brief, protein samples ( - μg) were separated by sds−page and transferred onto pvdf membranes. the membranes were blocked in % milk−tbst and then incubated overnight at °c in primary antibodies. antibodies used included nrf (abcam), histone h (abcam), and β−actin (abcam). peroxidase−conjugated anti−mouse igg (abcam) or anti−rabbit igg (abcam) was used as the secondary antibody. protein bands were visualized using chemiscope series (clinx science instruments, shanghai, china). the gray value of protein bands was quantified using imagej (national institutes of health, bethesda, maryland, usa). one−way anova with lsd analyses was performed using spss. all results were confirmed from three independent experiments. data were expressed as the means ± standard error of the mean. data were considered significantly different at p < . . keap d structure was prepared with sybyl x . , and the binding pocket, which easily interacted with other compounds, was obtained ( fig. ) . a library of phytochemicals (supplement table ) was prepared as mentioned in section . . the phytochemicals were divided into varieties according to their structural features: alkaloids, anthocyanins, carotenoids, chalcones, coumarins, flavanols, flavanones, flavones, flavonols, isoflavonoids, organosulfurs, phenolic acids, phenylethanoid glycosides, quinones, stibenes, terpenes, tocopherols, and others. according to the rule of sybyl x . , c_score shows hydrogen bonding, metal-ligand interaction, lipophilic contact, and rotational entropy, along with an intercept term. total_score shows the binding affinity between the ligand and protein. when total_score > , the ligand is considered as a candidate for further study, meanwhile the c_score ≥ , the candidate is considered to be more promising. here average total_score is the average value of total_score of all the compounds with similar structural features. this value shows the binding affinity between the different classification of compounds and protein, was used to compare the binding affinity with keap of varieties of compounds. docking calculation results (table ) showed that their binding affinity with keap in descending order was shown and the average total_score were as follows: phenylethanoid glycosides > tocopherols > flavones > flavanols > anthocyanins > flavonols > stibenes > flavanones > chalcones > carotenoids > isoflavonoids > phenolic acids > quinones > others > coumarins > terpenes > alkaloids > organosulfurs. these compounds could be divided into three groups, the high−total−score group (average total_-score > ), the medium−total−score group ( > average total_score > ), and the low−total−score group (average total_-score < ). as shown in table , compounds showed strong binding affinity with keap and most of them have c_score ≥ . the interactive amino acids including y , s , l , n , s , p , d , n , i , v , s , s , l , y , s . the d structure of binding mode between keap and the compounds above were shown in supplement fig. . the varieties of compounds were divided into two groups, the compounds containing glycosides (i.e., anthocyanins, flavanols, flavanones, flavones, flavonols, isoflavonoids, and phenylethanoid glycosides) and the compounds without glycosides (i.e., alkaloids, carotenoids, chalcones, coumarins, organosulfurs, phenolic acids, quinones, stibenes, terpenes, tocopherols, and others). the binding affinity with keap of the compounds containing glycosides was better than the compounds without glycosides. the number of glycosides was positively correlated with the binding affinity with keap . as shown in table , phenylethanoid glycosides followed this typical pattern, and the binding affinity with keap of phenylethanoid trisaccharides (ech), phenylethanoid disaccharides (acteoside, isoacteoside), and phenylethanoid monosaccharides (salidroside) were in descending order. compounds in table also showed better binding affinity with keap when they existed in the form of glycosides. the more glycosides these compounds combine with, the higher score they obtained. meanwhile, the number of oxygen molecules was related to the binding affinity with keap . for example in table , ternstroside a, ternstroside d, ternstroside c, ternstroside b, and ternstroside e were all phenylethanoid monosaccharides, and the only difference in their chemical formula was the number of oxygen molecules. ternstroside a, ternstroside d, and ternstroside c contain oxygens, whereas ternstroside b and ternstroside e contain oxygens, which resulted in a significant difference in their binding affinity with keap : ternstroside a > ternstroside d > ternstroside c > ternstroside b > ternstroside e. the number of oxygen molecules was more sensitive than that of glycosides in predicting the binding affinity with keap . in accordance with the method described in section . , the relationship between the structural parameters and the binding affinity was evaluated by d comfa studies in the qsar module in sybyl−x . . as shown in previous study, the r > . was satisfactory. the r between the experimental and predicted values was . , which indicates that the structure was a key element in binding to keap . the cross−validation analysis, wherein compounds were randomly selected by the system, showed a good relationship between the structural parameters and the binding affinity. the plot of the actual versus predicted total_score values is shown in fig. . in the results above, phytochemicals were divided into three groups depending on their binding affinity with keap . to evaluate their activation effect on nrf , different compounds including c s, l g, rut, api, kaem, αt, cop, mag, pip, ea, and ech from high−, medium−, and low−total−score groups were selected for further studied on a cell model. the cytotoxicity assays was conducted at , , μm, l g, αt, and kaem significantly reduced cell viability at μm (fig. ) . so the compounds were tested at or μm in further study. as shown in fig. , the nrf expression level in the nucleus increased after treatment with c s, l g, rut, api, kaem, αt, cop, mag, pip, ea, and ech at or μm. results of western blot and gray density analyses showed that all of these tested compounds exerted no significant influence on nrf expression in the cytosol (fig. a and b) (p > . ). c s, l g, rut, αt, and ech whose total_score were > increased nrf expression in the nucleus (fig. c and d) (p < . ), and their effect on nrf activation in descending order is as follows: c s ( . %, . %) > αt ( . %, . %) > l g ( . %, . %) > ech ( . %, . %) > rut ( . %, . %). this order was consistent with their total_score value (except ech). meanwhile, api and kaem from the medium−total−score group and cop, mag, and pip from the low−total−score group showed no effect on nrf activation (except pip at μm). the results above indicated that the compounds showing potential binding affinity (total_score > , c_score ≥ ) with the nrf binding site in keap l can activate nrf under oxidative stress. in the present study, phytochemicals were investigated to make sure whether they can effectively activate the transcription factor nrf using molecular docking methods. the main findings of this study were as follows: ( ) structure of the compounds determined their inhibitory effect on the keap -nrf protein-protein interaction. compounds with abundant oxygen or glycosides effectively inhibited the keap -nrf interaction. ( ) the steric/acceptor/hydrophobic contribution instead of the electrostatic/donor contribution was the predominant element affecting the binding affinity. ( ) compounds with higher total_score (total_score > ) in binding affinity with keap showed significantly positive effects on the nuclear translocation of nrf in the h o −induced oxidative−injured cell model. molecular docking is a very powerful technique for screening large numbers of new compounds as active drug candidates, and this technique has been successfully used in the discovery of antimicrobial (shen et al., ) , alzheimer's disease related cyclophilin d inhibitors (valasani et al., ) , human coronavirus nucleocapsid protein inhibitors (chang et al., ) . previous studies paid great attention to antioxidants and nrf activators; however, few of these studies systematically screened nrf activators from known antioxidants. wu et al. ( ) utilized arec cells that contain a luciferase gene under the control of antioxidant response element promoters to screen nrf activators. in this study, phytochemicals were selected from the literature and directly explored whether they can activate nrf by inhibiting the keap -nrf protein-protein interaction using the molecular docking method. with the help of the software, candidates were obtained, and the results showed that the number of glycosides and oxygen positively correlated with the binding affinity of compounds with keap . the procedure above was easy to operate and time saving. further study showed that the c s, l g, rut, αt, and ech from the candidates can significantly increase the nrf level in the nucleus. several studies have reported about the antioxidant effects of αt (niki, ) and ech (and and kitts, ) . masoudi et al. ( ) reported that αt activates the transcription factor nrf in neuronal cells and that ech is a representative antioxidant compound in herba cistanches (tu et al., ) . c s (cheng et al., ) was subjected to dpph scavenging activities, aop, and reducing power assessments; a dose−dependent antioxidant activity was observed. l g (jung et al., ) in korean milk thistle strongly protects t−bhp−treated hepg cells from oxidative damage, and these protective effects might be attributed to nrf activation. rut (tian et al., ) significantly attenuates oxidative stress and upregulates nrf expression in a rat model. previous studies are consistent with our results. the candidates bind with the special amino acids on keap , such as ser , ser , ser and ser , which have been reported to be the key amino acids in the structure of the keap −nrf interface (lo et al., ) . the results indicated that the compounds with high binding affinity with keap could directly inhibit keap −nrf protein−protein interaction as nrf activators. antioxidants have the potential to activate nrf by inhibiting the keap −nrf protein−protein interaction, and the effect is largely determined by their structures. compounds with abundant oxygen or glycosides can inhibit the keap −nrf interaction more effectively and phenylethanoid glycosides showed the best effect among the varieties. high−total−score group (total_score > ) show significant effects on the nuclear translocation of nrf in h o −induced oxidative−injured cell model. these results may provide a new method for discovering nrf activators that interfere with keap −nrf protein−protein interaction. fig. . graph between actual total_score and predicted. total_scorethe x axe: total_score is the true value from molecular docking. the y axe: predicted total_score is the virtual value from d comfa studies the blue spots stand for tested compounds, the red circle stand for phenylethanoid glycosides. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) fig. . cytotoxicity study of tested compounds on pc cells. cell viability was detected by mtt assay. ck: control group, c s: cyanidin -sambubioside treated group, l g: luteolin- -o-glucoside treated group, rut: rutin treated group, api: apigenin treated group, kaem: kaempferol treated group, αt: α-tocopherol treated group, cop: coptisine treated group, mag: magnoflorine treated group, pip: piperine treated group, ea: ellagic acid treated group, ech: echinacoside treated group. #p < . , versus control group, ##p < . , versus control group. pc cells were incubated with tested compounds ( μm μm) for h, and then incubated with μm h o for another h after the previous compounds removed. (a) the expression of nrf protein in cytoplasm was detected by immunoblotting using specific antibody. β-actin was used as loading control. (b) the quantitative densitometric analyses of nrf protein in cytoplasm. (c) the expression of nrf protein in nucleus was detected by immunoblotting using specific antibody. histones h was used as loading control. (d) the quantitative densitometric analyses of nrf protein in nucleus. all data are expressed as means ± sd and represent three independent experiments. ck: control group, model: h o treated group, c s: cyanidin -sambubioside treated group, l g: luteolin- -o-glucoside treated group, rut: rutin treated group, api: apigenin treated group, kaem: kaempferol treated group, αt: α-tocopherol treated group, cop: coptisine treated group, mag: magnoflorine treated group, pip: piperine treated group, ea: ellagic acid treated group, ech: echinacoside treated group. α−tocopherol was used as a positive control. #p < . , versus h o treated group, ##p < . , versus h o treated group. myeloperoxidase: expressing inflammation and oxidative stress in cardiovascular disease nuclear factor erythroid -related factor signaling in parkinson disease: a promising multi therapeutic target against oxidative stress, neuroinflammation and cell death studies on the antioxidant activity of echinacea root extract 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hepg cells in vitro activity of vitamins, flavonoids, and natural phenolic antioxidants against the oxidative deterioration of oil-based systems promising therapeutics with natural bioactive compounds for improving learning and memory-a review of randomized trials structure of the keap :nrf interface provides mechanistic insight into nrf signaling small molecule modulators of keap -nrf -are pathway as potential preventive and therapeutic agents the adjuvant component α-tocopherol triggers via modulation of nrf the expression and turnover of hypocretin in vitro and its implication to the development of narcolepsy keap -dependent proteasomal degradation of transcription factor nrf contributes to the negative regulation of antioxidant response element-driven gene expression molecular docking: a powerful approach for structure-based drug discovery role of vitamin e as a lipid-soluble peroxyl radical scavenger: in vitro and in vivo evidence caffeic acid prevents acetaminophen-induced liver injury by activating the keap -nrf antioxidative defense system advances in antioxidant potential of natural alkaloids oxidative stress, inflammation, and cancer: how are they linked? free radical blueprint for antimicrobial hit discovery targeting metabolic networks d-qsar comfa study on oxindole derivatives as cyclin dependent kinase (cdk ) and cyclin dependent kinase (cdk ) inhibitors vegetables, fruit, and cancer prevention: a review rutin ameliorates diabetic neuropathy by lowering plasma glucose and decreasing oxidative stress via nrf signaling pathway in rats analysis of phenylethanoid glycosides of herba cistanchis by rp-hplc. yao xue xue bao structure based design, synthesis, pharmacophore modeling,virtual screening, and molecular docking studies for identificationof novel cyclophilin d inhibitors screening of natural compounds as activators of the keap -nrf pathway docking and d-qsar studies of -hydroxycoumarin derivatives as ck inhibitors the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. baiyi lu and maiquan li designed the study. maiquan li performed the laboratory work, data analysis and wrote the paper. all authors revised the manuscript. the authors hereby declare no conflict of interest. supplementary data to this article can be found online at https:// doi.org/ . /j.fct. . . key: cord- -v n ff authors: ahn, inkyung; heo, seongman; ji, seunghyun; kim, kyung hyun; kim, taehwan; lee, eun joo; park, jooyoung; sung, keehoon title: investigation of nonlinear epidemiological models for analyzing and controlling the mers outbreak in korea date: - - journal: journal of theoretical biology doi: . /j.jtbi. . . sha: doc_id: cord_uid: v n ff abstract much concern has arisen regarding serious epidemics due to the middle east respiratory syndrome (mers) coronavirus. the first mers case of korea was reported on may , and since then, the mers outbreak in korea has resulted in hundreds of confirmed cases and tens of deaths. deadly infectious diseases such as mers have significant direct and indirect social impacts, which include disease-induced mortality and economic losses. also, a delayed response to the outbreak and underestimating its danger can further aggravate the situation. hence, an analysis and establishing efficient strategies for preventing the propagation of mers is a very important and urgent issue. in this paper, we propose a class of nonlinear susceptible-infectious-quarantined (siq) models for analyzing and controlling the mers outbreak in korea. for the siq based ordinary differential equation (ode) model, we perform the task of parameter estimation, and apply optimal control theory to the controlled siq model, with the goal of minimizing the infectious compartment population and the cost of implementing the quarantine and isolation strategies. simulation results show that the proposed siq model can explain the observed data for the confirmed cases and the quarantined cases in the mers outbreak very well, and the number of the mers cases can be controlled reasonably well via the optimal control approach. first confirmed on may , the latest outbreak of middle east respiratory syndrome coronavirus (mers-cov) infections in korea accounted for laboratory-confirmed cases, including deaths, recovered individuals discharged from hospitals, and remaining in hospitals up to july , a de facto end date of the outbreak ( ministry of health and welfare , moh ). it spread remarkably fast in hospitals, which has caused the largest mers outbreak outside the middle east. the case fatality rate of . % is, however, much lower than the reported rate of . % prior to the outbreak in korea, according to the world health organization (who). the mers-cov is a novel betacoronavirus which was first identified from the sputum of a -year-old man in fall ( zaki et al., ) . clinical features of mers range from mild illness to fatal conditions such as acute respiratory distress syndrome * corresponding authors. e-mail addresses: ahnik@korea.ac.kr (i. ahn), parkj@korea.ac.kr (j. park). and multi-organ failure resulting in death, especially in patients with underlying comorbidities ( zumla et al., ) . although it is initially known as a zoonotic disease, human-to-human transmission occurs in health care settings and now is linked with significant morbidity ( oboho et al., ) . this outbreak of the mers-cov infection, including the index cases roommates, their caregiver, and even the healthcare workers, in what is called as a super-spreading event ( kupferschmidt, ) , raised several important issues for global public health surveillance and raised several issues regarding infection control policies, including the control of nosocomial transmission to avoid a repeated outbreak ( keeling and rohani, ) . in this study, we carry out an epidemiological assessment of the mers-cov outbreak in korea in order to provide a mathematical framework for understanding the complex dynamics of the pathogen spread and establishing efficient guidelines for implementing quarantine and isolation strategies. more specifically, we propose a class of nonlinear susceptible-infectious-quarantined (siq) models for analyzing and controlling the mers outbreak in korea. the proposed siq model is innovative, in that the mers https://doi.org/ . /j.jtbi. . . - /© elsevier ltd. all rights reserved. transmission probability is time-dependent, monotone decreasing, and squashing-type. more specifically, it is initially almost flat, then decreasing rapidly, and finally gradually reaching a saturation point, which is reasonable because this can reflect the change in individuals' hygienic behaviour with time. recently, there has been much interest in investigating some aspects of the time-dependent nature of the disease transmission probability. in particular, several methods have been considered to model the time-dependence due to the impact of media coverage ( cui et al., ; liu et al., ; sun et al., ; xiao et al., ) . however, these studies have mostly focused on only a single factor under consideration. in this paper, we consider all relevant factors that can affect the mers transmission probability ( e.g. , media coverage, increased awareness, etc.) collectively, and try to model the resultant time-dependent transmission probability using a sigmoidal function. note that the sigmoidal functions are quite popular in the field of machine learning when one needs to address monotone and squashing-type phenomena. for the proposed siq model, we perform the task of parameter estimation, and apply optimal control theory to the controlled siq model, with the goal of minimizing the infectious compartment population and the cost of implementing the quarantine and isolation strategies. simulation results show that the proposed siq model can explain the observed data for the confirmed cases and the quarantined cases in the mers outbreak very well, and the number of the mers cases can be controlled reasonably well via the optimal control approach. finally in the last two sections of this paper, discussion and concluding remarks are given along with brief descriptions of data treatment. in this section, we will propose a nonlinear susceptibleinfectious-quarantined (siq) model for analyzing the mers outbreak in korea. the siq model is a generalization of an epidemiological population model involving susceptible, infectious, and quarantined compartments ( hethcote et al., ; keeling and rohani, ; lenhart and workman, ; xiao et al., ) . in the siq model, the four compartment populations are used as the model's state variables: s ( t ), the number of individuals that are susceptible to the mers disease at time t; i ( t ), the number of individuals that are actively infectious at time t; s q ( t ), the number of quarantined susceptible individuals at time t ; and c q ( t ), the number of confirmed cases at time t . note that in the siq model, q stands for the super-compartment comprising s q and c q (see fig. ). also, note that c q ( t ) is a collective sink-type compartment, which includes the number of the mers patients under treatment, the recovered cases, and the dead cases altogether at time t . as shown in fig. , the siq model considers two kinds of non-pharmaceutical interventions: quarantine of the susceptible and infected individuals, and isolation of the infectious individuals following contact tracing. as a result of a contact tracing, a proportion, q , of individuals who are contacted in connection with a mers patient is quarantined. the quarantined individuals can move to compartments c q or s q , depending on whether they are exposed to the mers coronavirus or not. hence, the quarantined individuals, if uninfected, move to the compartment s q at a rate of c( − β(t)) q, where c is the contact rate, i.e. , the average number of contacts of the whole population per unit time, and β( t ) is the probability of the mers transmission per contact at time t . note that in the siq model, the mers transmission probability, β( t ), is time-dependent, monotone decreasing, and squashing-type. obviously, using timedependent transmission probability is more reasonable than using the constant one, because it can reflect the change of individuals' hygienic behavior with time. as shown in fig. , if infected, the quarantined individuals move to the compartment c q at a rate of c β( t ) q . also, the remaining proportion ( i.e. , the proportion missed from the contact tracing), − q, can move to compartment i or stay in compartment s , depending on whether they are exposed to the mers coronavirus or not. the transmission dynamics of the siq model is illustrated in fig. , and its state equations are as follows: the first equation of ( ) describes the rate of change of the susceptible compartment population, with four terms on its right-hand side. its first term concerns the transition from s to s q due to quarantine of susceptible individuals. this term can be explained in terms of a bilinear incidence law having a contact rate c together with β( t ), the probability of the mers transmission per contact at time t , and q , the probability of quarantine per contact. the second and third terms model the transition from s to c q and the transition from s to i , respectively. the fourth term represents the transition from s q to s , and this transition means the release from quarantine into the wider community. in the second equation of ( ) , the rate of change of the infectious compartment population is described by two terms. the first represents the transition from the susceptible state to the infectious state, and the second term denotes the transition from i to c q due to detection and isolation of the infectious patients. the remaining equations of ( ) describe the rates of change of s q and c q in the super-compartment q , respectively, and the exact meaning of their terms can be explained similarly. the natural birth and death rates are not considered in the siq model, and this omission allows us to focus on the core theme of the paper. note that consideration of these additional aspects is relatively straightforward, and would lead us to some further related observations. for example, if the natural birth rate of a susceptible population, , and the natural death rate, d , are consid- ered, the resultant reduced siq system with a constant transmission probability, β , would have the disease-free equilibrium point as previously mentioned, the mers transmission probability, β( t ), of our siq model, is time-dependent, monotone decreasing, and squashing-type. since many factors ( e.g. , media coverage, increased awareness, etc.) can alter individuals' hygienic behavior, we employ the squashing-type function of fig. for β( t ). more specifically, we utilize where σ ( ·) is the logistic sigmoidal function ( bishop, ) defined as σ (x ) = / ( + exp (−x )) ; t β is the inflection point of β( t ); s β is the parameter determining the slope of β( t ) at its inflection point. note that the use of logistic sigmoidal functions is quite popular in the field of machine learning ( bishop, ) when one needs to represent monotone and squashing-type phenomena. also, note that using the logistic sigmoidal function of ( ) lead to the following simplification when we compute its derivative: this property can be utilized effectively in further studies on the qualitative properties of the siq model ( e.g. , study of the global stability of the disease-free and endemic equilibrium points of the siq model). an explanation of the siq parameters is given in table . by fitting the siq model to the reported data for the confirmed cases and the quarantined cases, we obtain the following tant approach that can be addressed along these lines is the event-dependent approach, where the transmission probability could be dependent on the number of the newly-added or accumulated mers cases. parameters: we performed simulations based on the estimated parameters. simulation results ( fig. ) show that the proposed siq model can explain the observed data ( fig. ) for the confirmed cases and the quarantined cases in the mers outbreak very well. our parameter estimation results show that the mers transmission probability, β( t ), is initially almost flat, then decreasing rapidly, and finally gradually reaching a saturation point (see the solid line of fig. ). this has a natural interpretation, in that as information on the mers outbreak becomes more widely known with the passage of time, health authorities' and individuals' effort s against the epidemic intensify accordingly, which results in the mers transmission probability decreasing in the squashing-type fashion, as in fig. . in retrospect, if the initial effort to reduce the mers transmission probability were more effective, the magnitude of β could be decreased further. in order to investigate this aspect, we additionally performed simulations for a scenario with β reduced to % of its estimated value (see the dashed line of fig. ). fig. shows that the infectious population could be reduced significantly if the aforementioned effort were successful. in order to clarify why it is important to consider the incidence rate of the type shown in ( ) , we also considered the constant beta case, and provided the corresponding simulation results ( fig. ) . comparing figs. and shows the superiority of using the time-dependent β. since quarantine and isolation strategies are the most important and effective measures against the outbreaks of disease when one does not have valid medicines and vaccine (see e.g. , castillo-chavez et al., ; day et al., ; yan and zou, ; yan et al., ) , one can view the effort s of implementing quarantine and isolation strategies as the actions that control the entire model. in this paper, we utilize optimal control theory ( lenhart and workman, ; lewis and syrmos, ) for the possibility of improving our control effort s. note that recent applications of optimal control theory are increasingly used in communities of biological systems ( e.g. ( buonomo and messina, ; joshi et al., ; jung et al., ; kirschner et al., ; de pillis et al., ) ), and in particular, they have been widely used and discussed in the control of epidemics ( e.g. caetano and yoneyama, ; feng et al., ; gupta and rink, ; joshi et al., ; lenhart and workman, ) . by incorporating the control inputs, q * ( t ) and θ * ( t ) into our siq model ( ) , one can obtain the following state equations for the controlled model: note that for the optimal control of ( ) , it is enough to consider the first three variables only. hence, we consider the following controlled siq (c-siq) model: from the data fitting based on the siq model, we have already obtained the estimation results for the quarantine probability, q , and the isolation rate, θ . in retrospect, however, control-theoretic investigation is desirable for the purpose of improving our response to the outbreak. in the following, we consider the problem of improving the quarantine probability and isolation rate further with additional effort s ( q * ( t ) and θ * ( t )) by minimizing an objective function in the form of j = t f g(i(t ) , q * (t) , θ * (t)) dt. note that in this problem, the quarantine probability and isolation rate at time t are represented by and the control inputs to be determined via optimal control theory are the additional effort s described by q * ( t ) and θ * ( t ). here g ( · ) should be reasonably chosen to reflect the relative importance of the quarantine and isolation effort s over the infection. more precisely, in order to minimize an objective function comprising the infection cost ( i.e. , the infectious compartment population) and the cost of implementing quarantine and isolation strategies, we consider the following optimal control problem: subject to the c-siq state equations ( ) . in the cost rate of this problem, c q and c θ are the trade-off constants defining the relative importance of the implementation costs over the infection cost. note that the cost rate considers quadratic cost terms for the control inputs, which is a commonly used strategy in related control problems dealing with epidemicmodel-based systems ( e.g. , lenhart and workman, ) . also, note that the existence of an optimal control and corresponding optimal state trajectory comes from the convexity of the integrand of the objective function with respect to the control and the lipschitz property of the state system with respect to the state variables (see, e.g. , fleming and rishel, ) , and based on the existence, we can now rely on the pontryagin maximum principle (pmp) ( pontryagin et al., ) for an optimal solution. as is well known, the necessary conditions for an optimal solution of ( ) can be obtained via the pontryagin maximum principle. for this, the hamiltonian h of the optimal control problem ( ) is defined as and its costate equations can be obtained via - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - t s q (t) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - from the optimality conditions, ∂h ∂ q * = and ∂h ∂ θ * = , we can also obtain the following condition for optimal control: also, if upper bounds for the nonnegative control inputs are forced, then by confining the control input to be nonnegative and subject to a positive upper bound q max and θ max , the optimal control of ( ) can be written in the following form: from the above steps, we can conclude that any solution to the optimal control problem ( ) must satisfy the following: s( ) = s , by solving this boundary value ordinary differential equation (ode) problem numerically, we can obtain optimal control inputs for problem ( ) . in order to illustrate the optimal control policy, we simulated the optimally controlled c-siq system. the parameters considered for the simulations are set to be the same as the estimation results except the control inputs, q * ( t ) and θ * ( t ). for the trade- off constants of ( ) , we used c θ = , , c q = . the initial conditions for the simulations were also taken from the estimation results, i.e. , s = , , i = , and s q = . the simulations considered two scenarios. the first scenario does not have bounds on the control inputs, while the second scenario has the bounds of q max = . and θ max = . . with the goal of keeping the infection level low with reasonable control effort s, we solved the boundary value ode problem ( ) using bvp c , the matlab function to solve boundary value problems for ordinary differen-tial equations). figs. - show the simulation results for the first scenario. figs. and show that under the optimal control of the first scenario, the best method of fighting the infection is to initially enter large amounts of q ( t ) and θ ( t ), and later after days to slowly reduce them to zero. the resultant state trajectory of i ( t ) (the dashed line of fig. ) shows that, with the optimal control strategy, the infectious compartment population can be reduced significantly compared to the original case (the solid line of fig. ) . to consider the robustness of the quarantine and isola- tion control, we also conducted sensitivity analysis for the following cases: ( ) when c is % lower and % higher; ( ) when λ is % lower and % higher. for each case we simulated the resultant controlled system, and the results on the number of infectious individuals are shown in figs. and , respectively, for cases ( ) and ( ). comparing figs. and shows that c is more important for the quarantine and isolation control. simulation results for the second scenario, which deals with the bounded input case, are shown in show that under the optimal control of the second scenario, the best method of fighting the infection is to apply the maximum amounts of the control inputs from the start, and then to slowly reduce them after days to zero. the state trajectory of i ( t ) resulting from the optimal control is shown in fig. . note that the infectious compartment population can be reduced significantly even with the bounds on control inputs. from the simulation results ( figs. - ), we can conclude that the mers disease spread can be properly handled by the optimal control approach, and we can obtain a practical guideline, whereby quarantine and isolation effort s in the early stage are critically important in effectively controlling the mers outbreak. in recent years, global pandemic viral infections, including the severe acute respiratory syndrome, the h n influenza, and the ebola outbreak, have been devastating but provided valuable experience in outbreak responses. for public health control, increased vigilance by health professionals and voluntary compliance by the public are essential in implementing rapid effective response interventions. in this study, we carried out an epidemiological assessment of the mers-cov outbreak in korea in order to provide a mathematical framework for understanding the complex dynamics of the pathogen spread and establishing efficient guidelines for implementing quarantine and isolation strategies. the following have been observed by the assessment: • by fitting the siq model, which employs a squashing-type function of fig. for β( t ) , to the real data on the confirmed cases and the quarantined cases, we obtained reasonable performance, as shown in fig. . also, it turned out that the resultant estimated parameters belonged to plausible ranges. by comparison, the conventional siq model utilizing a constant transmission probability could not explain the observed data well. • our nonlinear epidemiological models showed that the mers transmission probability decreased in the squashing-type fashion and then approached a saturation point in a timedependent manner. as information on the mers outbreak be-came widely known in the nation, effort s against this epidemic, including individuals hygienic behavior, and interventions by health care facilities and by authorities were accordingly strengthened. in our siq-based analysis, the inflection point for transmission probability was found to be t β = . , corresponding to a couple of days after june . interest-ingly, june was the day when the korean government revealed the names of mers-affected hospitals to the public. after releasing the names of affected medical facilities, the rate of increase in new confirmed cases abated. as a practical guideline to avoid another similar unexpected outbreak, we draw the conclusion that combined effort s in the early stage are critically important, and sharing information including the names of affected hospitals or countries, clinical situations, and prevention methods might be important for global public health control. • we applied optimal control theory to the controlled siq model with the goal of minimizing the infectious compartment population and the cost of implementing the quarantine and isolation strategies. simulation results show that the number of the mers cases can be controlled reasonably well via the optimal control approach. in conclusion, this paper proposes a nonlinear epidemiological ode for the mers outbreak in korea, the siq model, in which the state variables are defined as the populations of four compartments ( s ( t ), i ( t ), s q ( t ), and c q ( t )), and the mers transmission probability, β( t ), is modelled by the time-dependent sigmoidal function. we performed the task of parameter estimation for the siq model, and the data fitting results explained the observed data for the confirmed cases and the quarantined cases in the mers outbreak very well. we also applied optimal control theory to the controlled siq model with the goal of minimizing the infectious compartment population and the cost of implementing the quarantine and isolation strategies. our simulation results show that mers propagation was controlled reasonably well via the optimal control approach. in future work, we will conduct further simulation studies, with the aim of revealing the strengths and weaknesses of the proposed method, and investigate stability and control issues for its various extensions, including a stochastic differential equation (sde) approach. we retrieved publicly available data ( ministry of health and welfare , moh ) from the centers for disease control and prevention and the ministry of health and welfare in the republic of korea. the data included information on the cumulative number of reported cases. the first mers case in korea was confirmed on may , and the numbers of newly confirmed cases and suspected patients who had been quarantined to prevent possible spread of the mers have reached cases and , cases, respectively, as of july . the data also included a brief description of each confirmed case with exposure date and onset of symptoms, and they were sufficient to estimate our siq model for the mers outbreak epidemiology. in this model, we assumed mers is unlikely to spread to another region. we implemented the parameter estimation procedure as a matlab program.we used fminsearch , a matlab function for unconstrained nonlinear optimization, along with some changes of variables in order to carry out the data fitting procedure for the siq model ( ) with the time-dependent variable β( t ) specified in ( ) . the performance index (pi) used in the optimization for parameter estimation was defined as follows: p i = w × ( square d error of c q ) + w × ( square d error of s q ) . the objective function of ( ) , pi , was based on the numbers of the confirmed mers cases and the quarantined cases between may and july. the weight values ( w = and w = ) were obtained empirically via a tuning process based on the training data. simulation results show that the above set of weight values yielded reasonably good fitting results. finally, note that a more sophisticated markov chain monte carlo (mcmc) algorithm ( e.g. , rasmussen et al., ) could be used for parameter estimation of the siq model. however, the use of fminsearch , which was simpler and more transparent, was sufficient for the purposes of this paper. pattern recognition and machine learning impact of vaccine arrival on the optimal control of a newly emerging infectious disease: a theoretical study optimal and sub-optimal control in dengue epidemics mathematical models of isolation and quarantine the impact of media on the control of infectious diseases when is quarantine a useful control strategy for emerging infectious diseases? timely identification of optimal control strategies for emerging infectious diseases deterministic and stochastic optimal control optimal control of epidemics effects of quarantine in six endemic models for infectious diseases optimal control methods 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quarantine and isolation strategies in epidemics control isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome this work was supported by a korea university grant. key: cord- - mu yp authors: syomin, b. v.; ilyin, y. v. title: virus-like particles as an instrument of vaccine production date: - - journal: mol biol doi: . /s sha: doc_id: cord_uid: mu yp the paper discusses the techniques which are currently implemented for vaccine production based on virus-like particles (vlps). the factors which determine the characteristics of vlp monomers assembly are provided in detail. analysis of the literature demonstrates that the development of the techniques of vlp production and immobilization of target antigens on their surface have led to the development of universal platforms which make it possible for virtually any known antigen to be exposed on the particle surface in a highly concentrated form. as a result, the focus of attention has shifted from the approaches to vlp production to the development of a precise interface between the organism’s immune system and the peptides inducing a strong immune response to pathogens or the organism’s own pathological cells. immunome-specified methods for vaccine design and the prospects of immunoprophylaxis are discussed. certain examples of vaccines against viral diseases and cancers are considered. immunoprophylaxis has long been used to control infectious diseases exerting an enormous impact not only on the global health but also on the safety of numerous populations of agricultural animals. in the past decades, the focus on vaccine production technologies has changed from handling intact pathogens to producing recombinant subunit vaccines based on isolated target antigens [ ] . intensive development of recombinant vaccines began in the s when it became possible to clone the desired dna sequences into expression plasmids and produce the target proteins. the construction of a recombinant vaccine takes advantage of the knowledge of the nucleotide sequences of genes encoded by a pathogen, and involves identification of the antigenic determinant, synthesis of the nucleotide sequence encoding the antigen, its cloning into an expression vector, and production of the target peptide in a certain expression system. the expression systems for individual heterologous proteins has been developed based on the prokaryotic and eukaryotic cells, which allow us to produce target proteins both on laboratory and industrial scales [ ] . the interest in recombinant vaccines is a consequence of the emergence of new infectious diseases, most often zoonotic ones. among them are the outbreaks of human diseases caused by the ebola virus, zika virus, marburg virus, the middle east respiratory syndrome, and severe acute respiratory syndrome coronaviruses [ ] . oncology also places great hopes in recombinant vaccines, which may help to overcome immune tolerance in the case of cancer treatment [ ] . moreover, there exists a constant threat of the emergence of new highly virulent strains of well-known viruses due to unceasing mutational processes in virus genomes [ ] . against this background, a new scientific concept, vaccinomics [ ] , which consists of identifying the minimum subset of antigens, which are able to induce a competent immune response to a pathogen or tumor by their specific interaction with the b and t immune cells, is being actively developed. using this approach it appears possible to design vaccines based on the minimum subset of antigens which most specifically characterize the pathogen in its interaction with the immunome (the set of all immune receptor sequences, which are present in the individual organism) [ ] . target detection of epitopes, the regions located on the surface of the protein, is possible with the aid of x-ray analysis, which is rather labor-consuming since it requires obtaining protein crystals. presently, the -d protein structure modeling has found broad use. this approach is based on identification of the physicochemical and electrostatic characteristics of the polypeptide chain regions and correlation of these characteristics with antigenic properties. two strategies are currently utilized, namely, modeling by homology and abbreviations: vlp, virus-like particles. udc . + : : - . de novo modeling. in the first case, the protein data bank (http://www.ncbi.nlm.nih.qov/genbank/) database of -d protein structures is used to model the protein. the commonly used modeller [ ] , as well as other software such as i-tasser [ ] , swiss-model [ ] , esypred d [ ] , d-jigsaw [ ] , phyre [ ] , and cphmodels [ ] , can be used as homology modeling tools. the limitation of this approach lies in the requirement that the structures of homologous proteins should demonstrate more than % identicality [ ] . the de novo modeling strategy is based on the computer simulation of the protein folding process (rosetta [ ] and tasser [ ] software). in this case, all possible variants of polypeptide chain folding are considered, and the most energetically favorable conformation, i.e., the one with the lowest potential energy, is chosen. predicted antigenic determinants are synthesized using genetic engineering vector techniques and heterologous protein expression systems, and then are experimentally tested using, for example, the antibody neutralization assay. using protein expression systems it is possible to produce virus-like particles (vlps), which are made up of monomers, which are able to multimerize into vlps, and display the antigenic determinants of target pathogens on their surface. this point will be addressed further in the review, we will just observe here that even complex epitopes represented by trimers may be obtained using heterologous expression systems. this has been demonstrated, for instance, for the trimeres of the human immunodeficiency virus (hiv- ) envelope glycoprotein [ , ] and influenza virus haemagglutinin [ ] . in order to choose the most specific antigens of the pathological agent of a new infectious disease, it appears inevitable to work with the infected material if only to isolate and sequence the nucleic acids containing the genetic information of the pathogen. however, in order to produce a vaccine based on epitopes characteristic for the target pathogen, there is no need to work with the pathogen itself. therefore, apart from all other advantages of the vaccines of this kind, they appear to be improved in terms of biological safety. vaccines based on the presentation of a subset of antigenic determinants of the infectious agent are characterized by a high level of reproducibility when commercially manufactured and are highly effective [ ] , since in this case, the immune response is directed exclusively against the most significant antigenic elements of the pathogenic microorganism or tumor. a number of vaccines specifically interacting with certain immune system receptors have been tested to date. for example, a vaccine containing only a single epstein-barr virus epitope specifically recognized by cd + t-cells is proposed for the prophylaxis of mononucleosis in human [ ] . this vaccine prevents the development of the disease, although it does not protect the organism from the entry of the virus. another example is classical swine fever virus (csfv). it has been demonstrated that the e viral protein produced in the baculovirus expression system induces the synthesis of csfv-neutralizing antibodies in pigs [ ] . vaccines are designed to produce immunity to a disease. although a single epitope is able to induce a strong immune response, it appears usually insufficient to induce protective immunity. the in vitro synthesized peptide properly representing the main antigenic determinant of the pathogen is highly likely not to be itself a strong immunogene primarily because of its small size (< nm) and high risk of proteolytic degradation. this problem may be overcome by the use of nanoparticles. it has been shown in a considerable number of works that in order to induce a strong immune response, antigenic determinants should be exposed on the surface of nanoparticles whose shape and size ( - nm) mimic those of the virus (fig. ) . in this respect also, vlps, the nanoparticles composed by viral proteins capable of self-assembly (multimerization) into structures morphologically resembling viruses, are the choice selection for the role of a strong immunogene. antigenic determinants multiply repeatedly on the vlp surface and promote complement fixation and b-lymphocyte receptor clusterization leading to the activation of the immune response. additionally, the same antigen multiply displayed on the surface of the particle promotes the multiplication of the pool of autoreactive b-cells, which is the primary objective when designing vaccines against autoimmune diseases and tumors [ ] . natural sources for vlp bioengineering and the specific features of their assembly structural proteins of various viruses are capable of autonomous self-assembly into vlps. they interact with the formation of globular, icosahedral, or rodlike structures. so far, the structural proteins of several dozen viruses have been obtained in heterologous expression systems, and almost all of them proved able to form vlps. the vlp size varies from to nm and is similar to the size of the corresponding viruses [ ] . being similar to viruses allows vlps to penetrate into the lymph and to be efficiently entrapped by antigenpresenting cells [ ] . currently, several heterologous expression systems and the corresponding expression vectors which can be used with them are available. these systems are based on both the bacterial cells (the pet system based on escherichia coli cells is often exploited) and different eukaryotic cells. in the case of eukaryotic cells, the most commonly used systems are yeast expression systems and the ones which rely on drosophila [ ] and spodoptera frugiperda insect cell lines [ ] . mammalian cells are also utilized, the most pop-ular among them being cho and hek [ ] . the cost of heterologous protein production in bacterial systems is lower than in eukaryotic cells. however, when studying the functional activity of the protein or vlp associated, for example, with the potential glycosylation sites or interaction with ubiquitin, or other ubiquitin-like peptides [ ] , no alternative to eukaryotic expression systems is available. it should be noted that the choice of the expression system is up to the researcher and is driven by the research task. for example, in different laboratories different eukaryotic systems for viral protein expression, including plant cells, are used to produce vlps which are used for vaccination against the hepatitis c virus (hcv) [ ] . in most cases, vlps assembled from a virus protein are considered as a candidate vaccine against this very virus, since protein monomers in multimeric configuration (vlp) induce a more potent immune response than protein monomers [ , ] . the strong immunogenic properties of vlps are determined by several factors. first of all, the dominant epitope of the structural protein is displayed as a part of the particle and is present in a multimeric form as is the case with a native virion [ ] . second, vlps stimulate b-lymphocytes and induce t-lymphocytes in the same manner as does the virus infecting the host organism [ ] . for example, vlps formed by the main capsid protein l of the human papilloma virus of several serotypes are successfully used as vaccines against cervical cancer [ ] . a vaccine against hepatitis b virus has been produced which contains vlps in the lipid membrane envelope [ ] . the vaccine against coxsackievirus a contains vlps assembled from the virus capsid proteins produced in the baculovirus expression system [ ] . scientists from china [ ] have demonstrated that the capsid protein (vp ) of the rabbit hemorrhagic disease virus (rhdv) efficiently multimerize into vlps, and a single intramuscular injection of the obtained vlp preparation completely protects the immunized rabbits against rhdv infection for at least days. the porcine circovirus capsid protein is also able to efficiently assemble into vlps when synthesized either in the human embryonic kidney cell culture (hek ) [ ] , or yeast [ ] and baculovirus [ ] expression systems, as well as in bacteria [ ] . footand-mouth disease vlps are assembled from three structural polypeptides vp , vp , and vp (naturally produced as a result of processing the p - a precursor polypeptide), simultaneously produced in e. coli [ ] . it has been recently demonstrated that the polyprotein ) liposome-based particles [ ] , ( ) nondegradable spherical nanoparticles (for example, metal nanoparticles) [ ] , ( ) polymer nanoparticles [ ] , ( ) graphene nanosheets [ ] and nanotubes [ ] . antigen of the duck hepatitis a virus produced in the baculovirus expression system assembles into vlps immediately in the cultured spodoptera frugiperda (sf ) cells, while immunization of ducklings with the obtained vlps induces a high level humoral immune response and protects them from developing the disease [ ] . the abilities of vlps resulting from multimerization of the cloned virus protein to play the role of an immunogen are not limited just to the presentation of their proper epitopes but may also be taken advantage of to display heterologous proteins. multimerization of capsid proteins into a particle requires the presence of nucleic acid, while for in vitro assembly, a short oligonucleotide ( - nt) is sufficient [ ] . therefore, vpls formed by capsid proteins are free from the infectious virus rna or dna. moreover, the above-mentioned property, which triggers protein monomer multimerization by nucleic acid, may be taken advantage of to package rna or dna into a particle with two different objectives. hence, antisense rna can be used to suppress virus expression. for example, in the case of the vaccine against foot-and-mouth disease, researchers from china [ ] not only displayed the vp epitope of the foot-and-mouth disease virus on the surface of vlps but also packaged the antisense rna complementary to the fragment of the viral genomic rna into a particle. another goal of nucleic acid packaging into a particle lies in the presentation of viral nucleic acids leading to the activation of specific immune receptors which induce the synthesis of type i interferons (inf) and other cytokines triggering the antivirus response [ ] . it has been demonstrated that long dna and rna molecules may be incorporated into vlps. for example, mrna for the reporter protein, red fluorescent protein, was packaged into particles representing the hepatitis e capsids [ ] . the plasmid containing the green fluorescent protein (gfp) gene was encapsidated into the vlps assembled in vitro from the main capsid protein of the hamster polyoma virus [ ] . a strategy for the packaging of up to kbp doublestranded circular dna into the particles was developed using the structural protein of the sv simian retrovirus expressed in baculovirus system [ ] . there exist two fundamentally different approaches for nucleic acid incorporation into vlps: ( ) in vitro vlp assembly from protein monomers in the presence of rna or dna, which is to be encapsidated into the vlp; ( ) exposure of the assembled vlps to osmotic shock in the presence of nucleic acids. osmotic shock is produced by using a solution with a low ionic strength; nucleic acid enters into a vlp as a result of the shift in the surface structures [ ] . however, the incubation of vlps in the presence of nucleic acids without exposure to osmotic stress results in a certain part of the nucleic acids becoming associated with the particles [ ] . a far more predictable approach is the in vitro assembly of vlps from the mixture of protein monomers and nucleic acids which should be encapsidated in them. in this case, after synthesis in a heterologous system, the obtained protein monomers are purified to completely remove the contaminating nucleic acids from the protein preparation; then the proteins are denatured in - m urea, nucleic acids which should be packaged are added, and the proteins are assembled into the particles by eliminating urea from the solution. the use of this strategy was demonstrated, for instance, in the works [ , , ] . protein association into a particle is a reversible process. vlp dissociation can be easily achieved by the addition of a denaturing agent. further, it may be removed and vlps may be reassembled in vitro [ ] with the encapsidation of the target rna or dna into the particle. this approach was implemented for the p protein of the hepatitis b virus. virus proteins produced in the heterologous expression system were denatured in m urea solution, and their assembly into vlps in the presence of the nucleic acid was further induced [ ] . protective immunity is controlled by specific humoral and cellular mechanisms activated by the antigen. posttranslational protein modifications and covalent bonds between the modifying molecules and the functional groups in the polypeptide chains are of great importance for immune homeostasis during the antiviral response. the balance between phosphorylation, ubiquitination, methylation, acetylation, sumoylation, adp-ribosylation, and glutamilation of a certain antigen performs the fine adjustment of the host's antiviral response [ , ] . the structural proteins for vlp production when synthesized in the eukaryotic expression systems undergo a number of posttranslational modifications. in particular, the gag gypsy monomer proved to be a natural substrate for type casein kinase [ ] . moreover, while produced in the eukaryotic cell, gag gypsy monomers become associated with ubiquitin and sumo, the cellular protein partners of the viral structural protein [ ] . generally, ubiquitin and sumo can bind with any lysine residue within the protein molecule, although there are preferable binding sites; it should also be noted that phosphorylation determines which of these two partners binds with the protein [ , ] . additionally, binding with a limited number of the above-mentioned signal peptides, such as monoubiquitination [ ] , usually plays a regulatory role and controls the transport of the protein itself, or the particle formed by it. in the case of polyubiquitination, the protein is destined for proteasome degradation [ , ] . by constructing recombinant polypeptides based on the viral capsid proteins it is possible to obtain vlps bearing several antigens. for example, dna encoding the vp capsid protein of the porcine parvovirus was fused with the dna fragment encoding amino acid residues of the main antigen of porcine circovirus (pcv ) nucleoprotein. the resulting hybrid dna was used to produce protein in heterologous cells which further formed vlps. these vlps induced a significantly stronger immune response against pcv than the recombinant adenovirus encoding the open reading frame (orf ) of pcv [ ] . another interesting example is represented by the vlps formed by the influenza virus matrix protein and displaying a toxoplasma gondii antigen on their surface [ ] . there exists another approach to designing therapeutic vaccines based on vlps. in this case, the vlp surface displays the variable fragment of an antibody specific to the antigen of the target virus [ ] . moreover, knowing which receptor is bound by the virus proteins renders it possible to produce particles possessing tropism to the cells of certain tissues. for example, the pre-sprotein of the hepatitis b virus exposed as a ligand on the vlp surface provided for their specific binding with hepatocytes [ ] . vlps assembled from the structural proteins of bacteriophages are also considered as carriers for human and animal vaccine production. for example, the dna fragment encoding foreign protein was inserted into the dna region encoding the n-terminal β-hairpin of the coat protein of the e. coli ms phage. it has been demonstrated that the expression of the obtained construct in bacterial cells resulted in the production of the recombinant protein in which a foreign peptide was present in the central part of the hairpin. the obtained chimeric protein monomers were able to self-assemble into particles morphologically similar to the phage capsid both in vivo and in vitro [ ] . using the mentioned property of the ms coat protein, vlps displaying the ep - epitope of the foot-and-mouth disease vp structural protein on their surface were obtained. these chimeric vlps induced string immune response in the animals, which allowed regarding them as a promising base for the development of a prophylactic vaccine [ ] . it is worth noting that the exposure of ep - on the vlp surface resolved a long-standing problem of how to make use of the beneficial properties of this peptide. researchers from several laboratories have demonstrated that this antigenic determinant of the footand-mouth disease not only induces the production of neutralizing antibodies but also stimulates t-lymphocytes [ ] . however, when isolated, ep - is not able to induce the immune response protecting animals against the foot-and-mouth virus infection [ ] . many attempts were made to solve this problem, for example, by combining the antigenic determinant with large molecules, such as for instance, t cell-specific molecules [ ] . however, only integration of ep - into vlps resulted in the possibility of using this antigenic determinant as a strong immunogen for vaccine production [ ] . practically all structural proteins of phages infecting various species of bacteria are able to autonomously form vlps. for example, salmonella typhimurium phages are able to autonomously multimerize into vlps on whose surface the epitopes of eukaryotic viruses, including the epitopes of the human influenza virus may be exposed [ ] . at the same time, however, bacteriophages are apparently incapable of presenting large epitopes, whose length exceeds amino acid residues [ ] . structural proteins of retroviruses and retrotransposons have a higher capacity compared to bacteriophage proteins. this means that protein monomers forming the particle may be fused with a longer peptide and still retain the ability to multimerize. in particular, it has been shown for the gag gypsy capsid protein [ ] , that at least % of its amino acid sequence (more than amino acid residues) is of little importance for multimerization into vlps [ ] . therefore, the truncated form of this protein may be fused with a heterologous peptide with the length similar to the length of the deleted fragments and a recombinant protein may be obtained which will retain its ability to self-assemble into vlps. in such a way, the truncated gag gypsy fused with the heterologous peptide formed particles when it was synthesized in bacterial cells. we were also able to assemble particles from the purified protein monomers in vitro [ , , ] . the obtained particles resembled the native gypsy virus by their morphology [ ] . the substitution of the deleted region in the dna encoding gag gypsy by the nucleotide sequence encoding the main antigenic determinant of the foot-and-mouth virus vp protein (ep - ) and his -tag resulted in the formation of particles which displayed ep - as the target antigen. the advantage of the technique used is that vlps formed by the protein containing his -tag can be readily isolated and purified by affinity chromatography [ ] . chimeric vlps may be obtained through the construction of recombinant dna molecules encoding both the corresponding virus protein and a foreign peptide or protein. another way is vlp pseudotyping. this approach was elaborated using the hamster polyomavirus. the v capsid protein of this virus first forms pentamers which further assemble into vlps comprised of pentamers. when v is expressed together with the minor capsid protein v , the latter binds with the central part of each v pentamer. it has been demonstrated that the n-terminal part of v is not involved in this interaction and therefore may be removed and substituted by the target epitope [ ] . another approach is also known. antigen is immobilized on the vlp via covalent binding between the reactive groups of the amino acid residues of the antigen and vlp. this approach proved to be ineffective due to the disruption of the native conformation of the antigen attached to the particle surface [ , ] . this problem was countered in the following way. glygly-lysglygly sequence was inserted into the monomer subunits for vlp assembly. in this environment, the reactive ε-amino group of lys residue is exposed and ready to interact with the cys-group of any protein in the presence of the cross-linking agent, such as m-maleimidobenzoyl-n-hydroxysulfosuccinimide ester (sulfo-mbs) [ ] , or succinimidyl- -[(β-maleimidopropionamido)hexanoate] (smph) [ ] . although the authors claimed that the described system of molecular assembly may be used to induce strong blymphocyte response against most antigens and prospective vaccine prototypes were suggested, this platform became the prototype only for a single prospective preparation based on vlps, the medication for lowering blood pressure in patients with hypertension [ ] , due to the development of the new improved techniques for antigen immobilization of the vlp surface, which will be discussed below. one of them takes advantage of the ability of his tag to bind with multivalent tris-nitrilotriacetic acid (trisnta) which in turn binds with a broad range of molecules, in particular, with biotin. the possibilities of this elegant technique for the functionalization of noninfectious viral nanoparticles were demonstrated in the case of vlps formed by the norovirus (nov) structural proteins. using the baculovirus expression system, the authors obtained nov vlps, displaying his -tag on their surface, which was first used to purify vlps, and further to bind trisnta molecules conjugated with a fluorescent dye, or biotin which in turn successfully bound streptavidin [ ] . notwithstanding some authors suggesting the described technique to be promising for vlp vaccine production, this approach continues to be only a prototype and has not advanced further than model experiments. the search for techniques providing efficient and stable immobilization of the antigen on the vlp surface led to the development of a versatile platform which allows us to covalently attach large antigens to the vlp surface [ ] . this molecular assembly system uses the tag-catcher conjugation system which was derived from the cnab domain of fibronectin-binding protein fbab from streptococcus pyogenes. as a result a highly reactive spytag peptide ( amino acid residues) was obtained which efficiently interacted with the spycatcher protein with the formation of isopeptide bonds in a broad range of buffer solutions [ ] . the spytag peptide was inserted into the vlp-forming polypeptide of the acinetobacter-infecting phage (these particles were called ap ), so that each monomer forming ap displayed two spytag peptides. a recombinant antigen consisting of the target protein and the spycatcher peptide was produced using bacterial or baculovirus expression systems [ ] . the obtained antigen efficiently bound with the vlp surface and induced a very strong immune response. almost the same effect was observed in the case of the reverse combination of the conjugating peptides, that is when spycatcher was incorporated into vlps, and spytag was fused to the antigen. this system is already utilized to design different types of vaccines, including the vaccines against tuberculosis and malaria [ , ] . a recent report has demonstrated that it was successfully used to develop a vaccine against breast cancer [ ] . this work is remarkable due to the fact that the high density of the her antigen (human epidermal factor receptor ) synthesized in the cultured drosophila melanogaster cells could be obtained on the ap vlp surface [ ] . the obtained particles induced a strong immune response to the antigen in the patients, which rendered it possible to overcome the immune tolerance to her known for patients with the her -dependent breast cancer [ , ] . in summary, the analysis of the discussed published data allows us to conclude that the nature of the vlp-forming protein is not as important for vlp functionalization as the technique which makes it possible to obtain high antigen density on the surface of vlps (table ) . the product range a number of vlp vaccines are available on pharmaceutical markets in many countries. the most wellknown are the cervarix® and gardasil® vaccines against cervical cancer, which have been successfully used for prophylaxis of this disease in girls for more than ten years. both vaccines are based on vlps formed by the main capsid protein l of the human papilloma virus belonging to several serotypes [ ] . engerix and recombivax hb vaccines against the hepatitis b virus representing vlps enveloped in a lipid membrane [ ] , as well as hecolin and xiamen innovax vaccines against hepatitis e [ ] , were developed and commercialized. the anti-malaria vaccine malaria rts has been certified, which represents the c-terminal part of the cs protein of plasmodium falciparum attached to the surface antigen of the hepatitis b virus (hbsag), which is also used in the certified vaccines against hepatitis b. to stabilize recombinant vlps, the fused protein is expressed together with the hbsag protein in saccharomyces cerevisease [ ] . a vaccine against the porcine circovirus representing vlps assembled from the vp pcv capsid protein synthesized in a heterologous system has also been commercialized [ ] . an increasing number of works have appeared that report on the development of vaccines based on nanoparticles, including vlps and/or replicationinactive viruses. among the apparent advantages of vaccines of this kind are their high specificity, efficiency, and good pharmacokinetic characteristics. it has been demonstrated that in an organism vlps reach lymphatic nodes in less than min, while the particle mixture bearing different antigens may be pro-cessed by the same antigen-presenting cells simultaneously [ ] . it should be noted that nanoprticle-based vaccines offer new possibilities in the development of immunoprophylaxis strategies, especially, the development of injection-free formulations, in particular, intranasal vaccines and inhalers. the administration of these vaccines may not involve humans, which is especially important in the case of industrial animal breeding in large agricultural enterprises characteristic of presentday russia [ ] , since it proves to be extremely difficult to administer identical injections to a large number of animals at the same time. this problem may be resolved by nebulizing aerosol vaccines in the area where animals are kept. the potential of this approach was demonstrated for the first time in with mice [ ] ; then, in the s- s, attempts were made to introduce this approach into animal and poultry farms in many countries. starting from the s, attempts were also made in the soviet union [ ] . however, at that time, the method of inhaled administration of vaccines was not introduced in practice since protective immunity could only be obtained when the tolerable dose was exceeded many times. for example, the inhaled administration of the vaccine against the newcastle disease led to - % lethality in birds [ ] . however, due to the introduction of vaccines based on the exposure of the epitopes of a pathogen on the surface of nanoparticles, including recombinant vlps, together with the development of innovative approaches to aerosol production, inhaled vaccines again became a topical issue. for example, biodegradable polymer nanoparticles formed by poly(glyceroladipate-co-ω-pentadecalactone), pga-co-pdl, proved themselves as an effective antigen carrier for inhaled vaccination [ ] , while the vaccine based on the adenovirus with the particles ranging from to µm induced stable protective immunity when administered via inhalation to monkeys [ ] . lastly, a method for vlp-vaccination via inhalation was proposed as protection against the human papilloma virus [ ] . one of the most important properties of vlps is mimicking virus particles and the consequent ability to induce a strong immune response to the antigen which they demonstrate irrespective of the source of the monomers which multimerize into vlps, these being either insect viruses, in particular the gypsy virus [ , ] , or plant viruses [ ] . vlps based on the structural proteins of plant viruses produced in plants [ ] make it possible to obtain vaccines with another nonconventional way of administration, edible vaccines [ ] . vaccines of this type may be synthesized directly in plant forage, with the oral vaccination of this kind inducing an immune response. expression vectors for foreign protein production in plants have been developed based on plant viruses, which allows obtaining plant-producing recombinant viruses or vlps displaying the target antigen on their surface [ , ] . vlps assembled from the structural proteins of plant viruses are also used to design functionalization platforms for antigen binding. these platforms are based on the strategies described above. in particular, they take advantage of the reactivity of the lys ε-group to immobilize the antigen on the surface of the plant's vlps [ ] [ ] [ ] [ ] . using the tobacco mosaic virus [ ] and potato x virus [ ] , it has been shown that a considerably large antigen can be immobilized on the surface of the corresponding vlps via the formation of the streptavidin-biotin complex. finally, based on the structural protein of the plant virus, the platform for antigen immobilization using the tag-catcher conjugation system is being developed [ ] . it should be noted that among the drawbacks of vlp vaccines manufactured using proteins which are not specific for mammals, for example, the plant virus proteins, include the development of the immune response to the structural component of the particles, which is the plant virus protein. undoubtedly, there is no therapeutical benefit in developing such immunity; however, whether there are adverse effects will be demonstrated by further studies. model molecules including biotin, alexa (fluorescent dye), and gfp protein conjugated with trisnta [ , ] isopeptide bond formation between spytag (integrated into vlp monomers) and spy-catcher (fused with antigen) peptides vaccine against breast cancer (her antigen presentation). it is also implemented to develop vaccines against malaria and tuberculosis [ ] [ ] [ ] [ ] [ ] the use of vlps for vaccination in medicine is becoming increasingly widespread. this point is reinforced by the list of clinical trials of vlp vaccines prepared by the united states national institute of health. currently, more than hundred vlp vaccines, which are directed against human and avian influenza viruses, the norwalk virus, norovirus, hiv- , and the chikungunya virus, the foot-and-mouth disease virus, as well as against melanoma, adenocarcinoma, papillomatoses, and cervical cancer, are undergoing clinical trials (http://clinicaltrials.gov/ct /search/index). it may be expected that the variety of nanoparticle vaccines against different cancers and viral infections will grow steadily, and vlps which can be used for immunization against microorganisms belonging to different taxons as well helminthes will also be developed. we thank the designers oleg vasil'ev and galina podzolkova (institute for statistical studies and economics of knowledge (issek), national research university higher school of economics) for their assistance in preparing the illustrations. the article was prepared within the framework of the basic research program at the national research university higher school of economics (hse) and supported within the framework of a subsidy by the russian academic excellence project - . the authors declare that they have no conflict of interest. this article does not contain any studies involving animals or human participants performed by any of the authors. assessing sequence plasticity of a virus-like nanoparticle by evolution toward a versatile scaffold for vaccines and drug delivery identifying and engineering promoters for high level and sustainable therapeutic recombinant protein production in cultured mammalian cells going to bat(s) for studies of disease tolerance mesothelin virus-like particle immunization controls pancreatic cancer growth through cd + t cell induction and reduction in the frequency of cd + foxp + icos-regulatory t cells quasispecies and virus vaccinomics approach 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psoriasis, alzheimer's and cat allergy. npj vaccines. , modified tobacco mosaic virus particles as scaffolds for display of protein antigens for vaccine applications use of plant viruses and virus-like particles for the creation of novel vaccines key: cord- -wea mmt authors: liu, bao hua; zhao, miao miao; liang, zi; gao, li jun; gao, fei; wu, qun hong; hao, yan hua; ning, ning title: factors associated with field epidemiology investigation: a cross-sectional study in china date: - - journal: biomedical and environmental sciences doi: . /bes . sha: doc_id: cord_uid: wea mmt nan doi: . /bes . the public health problem caused by disease outbreak cause increasing global concern. field epidemiology investigation (fei), which involves the timely use of epidemiology to solve urgent public health problems, is a crucial core capacity for the public health workforce [ ] . when an acute public health problem occurs, there is an urgent need to identify the origin, path, cases, and vulnerable population, and to implement timely intervention. fei can be conducted not only for infectious disease epidemics but also for non-communicable events [ ] such as poisoning, vaccination accidents, and environmental pollution. while situations do not always meet the statutory standard of a public health emergency, it remains necessary to conduct an investigation to prevent event from becoming a serious public health problem. in view of the fundamental role of fei in coping with outbreaks, numerous training programs have been conducted worldwide. this concept has been gradually accepted in china, and a -year on-the-job chinese field epidemiology training program (cfetp) was established in by chinese center for disease control and prevention. the training model for the cfetp originated from the epidemiological information service (eis) in the united states established in [ , ] , which was promoted as a global epidemiological training network by the world health organization. fei is viewed as an essential skill that all professionals in a given field should be competent [ ] , suboptimal investigation and control measures based on it could jeopardize the reputations of public health and government. the response to outbreak of severe acute respiratory syndrome in the early phase of the epidemic in reflected the severe shortage of well-trained epidemiological professionals in china [ ] ; therefore, measures were taken to improve the overall competency in the public health system. since then, the task of conducting fei in china has been undertaken mainly by the centers for disease control and prevention (cdcs) at all levels, with multi-sectoral cooperation. however, fei capacity of cdc professionals remains unclear after years of institutions building and health-related human resource construction. the results of feis are often reported, but there is limited information reflecting the factors associated with fei in the literature. this study aimed to gather first-hand information regarding fei capacity and determine factors associated with investigation, via a case study conducted in heilongjiang province, to provide evidence-based options for future capacity building programs of cdc staff. a stratified cluster sampling method was used to select cdcs. to counties in each of the city (prefecture) in heilongjiang province were selected at random to participate in the study. in total, cdc institutions were sampled. eligible target participants were the professionals who had worked at the cdcs for more than year and were members of the health emergency detachment of the cdcs. after excluding unqualified participants and incomplete questionnaires, we obtained , valid responses. in-depth interviews were conducted among cdc emergency managers to understand the factors affecting fei, and obtain their opinions about potential causes of hindering personnel capacity building from the perspectives of individual, institution and society. of the , cdc professionals, . % were men and . % were women. almost half ( . %) had completed a college education or higher, and . % had background of preventive medicine education. in addition, . % had conducted fei more than two times during the preceding year. furthermore, . % and . % had never participated in training and drills related to epidemic response, respectively. only . % of the cdc professionals rated themselves as 'good' in terms of fei capacity (table ) . the dependent variable in the study was measured using the item, 'what do you think of your overall capacity to perform field epidemiology investigation?' the answer was given using a -point likert scale ranging from ('very poor') to ('very good'). for modelling purposes, we created a dichotomous variable, where respondents with , , and on the likert scale were classified as 'not good', and , were classified as 'good'. the independent variables included sex, age, educational level, professional title, work time, efi frequency, training, drills, and the level of cdc institutions. logistic regression was conducted to identify factors influencing individual fei capacity (performed using spss . ). the significance level was set at p < . . participants were asked to rate the degree of factors ( items) hindering efi performance, including ( ) lack of professional investigators, ( ) complexity of methods and processes, ( ) insufficient funding, ( ) lack of operational guidelines, ( ) poor laboratory testing ability, ( ) lack of training and drills, ( ) uncooperative cases, and ( ) lack of a support network. the scores of the items ranged from to . the results of the multivariate analysis suggested that participants who had conducted fei more than two times (or = . , % ci: . - . ), who majored in preventative medicine (or = . , % ci: . - . ), and who had been trained to conduct investigations (or = . , % ci: . - . ) tended to exhibit higher self-ratings compared with other participants. this study showed that only . % of the participants had education background in the field of preventive medicine. field epidemiology is an interdisciplinary methodology, and the knowledge domains of fei include epidemiology, statistics, clinical medicine, social science, management, and communication. professional background in preventive medicine indicated that such participants had completed basic education in most of these necessary knowledge in colleges, which would contribute to the capacity to address the complexity of fei tasks. in addition, fei capacity could also be improved through on-the-job public health practice and formal professional training. fei is an interdisciplinary field, and the technology used in fei is complex and varies according to the situation, requiring both individual and teamwork skills. further, experience in emergency response activities is invaluable to improve the capacity of cdc workforce during complex investigations. indeed, fei practice as a must for training is an important course for both eis and cfetp, in which 'keep learning by doing' is promoted [ , ] . participation in targeted fei training programs was another key factor in self-rated fei capacity for public health workforce. professionals should gain theoretical knowledge, practical skills and abilities in systematic formal training for the response to public health emergencies. however, the schedules of grassroots cdc professionals are often occupied by heavy workloads and daily routines, and training often gives place to organization's other priorities which usually incorporated only a small number of investigative skills. in the current study, participants were asked to score items related with the factors that hinder fei (figure ). lack of a support network was shown to be the most significant barrier to fei, with a score of . . the law endows cdc investigators with a duty to conduct fei in china, but they often encounter resistance due to the lack of concretely stipulated laws and regulations that meet the specific requirement of fei. moreover, the investigations require cooperation with other departments and access to premises or records related to emergency [ ] ; however, it is difficult to obtain substantial support when there is a disagreement between departments, which hinders the availability of resources within a community and limits timely intervention. previous studies have shown an overlap of responsibilities, poor communication between cdcs and other health sectors [ ] . furthermore, rising concern on privacy also plays a role in causing difficulties that hinder fei. another important obstructive factor was the lack of targeted training ( . ); consistent with the findings of the logistic regression analysis. the complexity of the methods and processes of fei ( . ) was the third most important obstacle reported. in addition, previous research has shown that the capabilities of data collection and statistical analysis are always important but weak in public health workforce [ , ] . local cdc professionals are obliged to conduct investigations; however, even those who have performed fei for numerous times may not be valued as epidemiologists. in fact, grass-roots investigators usually have a lower education level and lack of training and investigation experience; therefore, the methods and processes involved in conducting fei are usually complicated and difficult for them. when an investigation is in a jam or does not know how to execute it, grass-roots cdcs have to turn to the higher level for help, and this may miss the best time for intervene. the findings of the quantitative investigation were partly verified by the interviews. investigators often lacked of sufficient and targeted training for fei. moreover, support networks were not conducive to the implementation of fei. investigators are empowered to conduct investigations on site and gain access to data which was private and confidential, and this requires supports from the public, communities, organizations, and other government agencies; however, they are often challenged by those who question their rationality for enquiries, particularly when interests are threatened. other unfavourable factors for the investigation were as follows: heavy work schedules and lower incomes led to increased staff turnover and negative attitudes; new staff members' education background did not match the job requirements when they were recruiteds; and deficiency of laboratory capacity at the district and county level, resulting in difficulties in providing accurate and timely support for investigations. in practice, fei is often conducted by a team of professionals through cooperation [ ] . field epidemiology is a branch of applied epidemiology, but the lack of sufficiently competent staff in this area has been identified as a major challenge in the public health field for many years [ ] . not all professionals are expected to be equally capable in all steps and types of investigation, but they should have the capacity to conduct epidemiological surveys [ ] . so, it is necessary to develop a work manual for fei, which is scarce in china now, to describe the basic skills in each step of fei, address the knowledge and skills deficiency and outline the detailed tasks to be accomplished for each investigation step. in addition, new tools, such as epidemiological analysis and investigation software based on artificial intelligence, should be developed and made easy available for investigators, to reduce the barriers to fei resulting from weaknesses in identifying biological characteristics of diseases, and application of statistical method. in conclusion, almost % of the professionals in the current study were not confident in their fei capacity. in addition, fei experience, formal training, and educational background had influences on fei capacity. moreover, defective support network, the complexity of fei methods and processes, and lack of training and drills were considered as major factors hindering fei. it is necessary to consider the key obstruction of factors in decision making, personnel recruitment systems involving highly relevant educational background should be established, tasks and responsibilities should be clarified in multi-sectoral collaboration and co-operation in fei. the funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the article. the corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. the authors wish to thank those who participated in the questionnaire survey and qualitative interview. a strategic approach to public health workforce development and capacity building outbreak investigations--a perspective building global epidemiology and response capacity with field epidemiology training programs applied epidemiology and public health: are we training the future generations appropriately competencies for master and doctoral students in epidemiology: what is important, what is unimportant, and where is there room for improvement? frontline field epidemiology training programs as a strategy to improve disease surveillance and response field epidemiology: methodological constraints and limitations in the developing world determinants of emergency response responsibility perceptions in the local public health workforce after china's health sector restructuring implementation of a competency-based medical education approach in public health and epidemiology training of medical students the cdc/council of state and territorial epidemiologists applied epidemiology fellowship program key: cord- -x taxwkx authors: singh, amandeep; halgamuge, malka n.; moses, beulah title: an analysis of demographic and behavior trends using social media: facebook, twitter, and instagram date: - - journal: social network analytics doi: . /b - - - - . - sha: doc_id: cord_uid: x taxwkx abstract personality and character have major effects on certain behavioral outcomes. as advancements in technology occur, more people these days are using social media such as facebook, twitter, and instagram. due to the increase in social media's popularity, the types of behaviors are now easier to group and study as this is important to know the behavior of users via social networking in order to analyze similarities of certain behavior types and this can be used to predict what they post as well as what they comment, share, and like on social networking sites. however, very few review studies have undertaken grouping according to similarities and differences to predict the personality and behavior of individuals with the help of social networking sites such as facebook, twitter, and instagram. therefore, the purpose of this research is to collect data from previous researches and to analyze the methods they have used. this chapter reviewed research studies on the topic of behavioral analysis using the social media from to . this research is based on the method of previous publications and analyzed the results, limitations, and number of users to draw conclusions. our results indicated that the percentage of completed research on the facebook, twitter, and instagram show that % of the studies were done on twitter, % on facebook, and % on instagram. twitter seems to be more popular and recent than the other two spheres as there are more studies on it. further, we extracted the studies based on the year and graphs in which indicated that more research has been done on facebook to analyze the behavior of users and the trends are decreasing in the following year. however, more studies have been done on twitter in than any other social media. the results also show the classifications based on different methods to analyze individual behavior. however, most of the studies have been done on twitter, as it is more popular and newer than facebook and instagram particularly from to , and more research needs to be done on other social media spheres in order to analyze the trending behaviors of users. this study should be useful to obtain knowledge about the methods used to analyze user behavior with description, limitations, and results. although some researchers collect demographic information on users’ gender on facebook, others on twitter do not. this lack of demographic data, which is typically available in more traditional sources such as surveys, has created a new focus on developing methods to work out these traits as a means of expanding big data research. criteria, and data analysis [ ] . the next section is the result section which provides the statistical analysis and the percentage of research completed on different social media. the result section includes a table which provides the research paper analysis according to the year along with pie chart figures, data collection, and behavior analysis methods and classifications based on different methods with line graphs [ ] . the next section is a discussion on the given topic and the last section is the conclusion of this research work. data were collected from different conference papers published in the ieee. from these papers, different methods of analyzing the user behavior [ ] was assessed. this report is based on a review of the published articles and analyzes the methods they have used. the data are given in a tabular form. data were collected from various journal papers from the ieee library regarding the analysis of the user behavior using social media from to . the collected data were related to facebook, twitter, and instagram in different countries [ ] . the attributes that were used for data collection were: applications, methods used, description of the method, number of users, limitations, and results. this raw data is presented in table [ ]. the different data attributes used to analyze the papers are given in table . this included the following: author name, applications, methods used, detail of methods, number of users, limitations, and results. data were gathered relating to different social networking sites [ ] . in our analysis, the different methods that have been used by researchers to analyze the user's behavior are explored. in this research, three different social media datasets have been collected, which represents the methods and technologies used to understand the behavior of the users. the raw data presented in table specifies the attributes that were used to conduct this research. we pooled and analyzed studies based on the impact of variables used in their studies. the descriptive details of the study based on the publication year were then analyzed to observe the behavior of the social media user from to . a comparison of the methods they used to investigate the behavior of users was then done. this research included papers from the last years from to . all papers used data from facebook, instagram, and twitter. privacy is major concern algorithm for news feed is not known filtering is not done properly [ ] it has been observed that individuals who are friends with each others have similar interests two evaluation metrics were used to judge the performance of classifier roc and pr used to the aim of this research is to know the methods used by researchers to predict the behavior of social media users. in this research, data were collected based on the use of three different social networking sites such as facebook, instagram, and twitter. a random user list was used to analyze the behavior. in our final analysis, we pooled the data, which showed a statistically significant difference in various parameters (published year, methods, results, and limitations) for different social media sites. the results section includes the percentage of research on the three social networking sites, research papers according to year with bar graph representations, data collection and behavior analysis methods and classification based on the different methods with line graph representations. we performed statistical analysis to organize the data and predict the trends based on the analysis. this showed the different social media sites used based on the data given in table . as shown in fig. and table , % of data was based on facebook users, % of data was based on instagram users, and % of data was based on twitter users. as such, it is clear that twitter is used more than other two social media sites for the analysis of the behavior of users. table data collection techniques and behavior analysis methods used by different studies are shown in table . the behavior of users can be analyzed using different methods as shown in table . fig. is based on the classification of papers based on the different methods used and it is clear that the researchers have used analysis techniques more than others and they have rarely used coding rules. in this analysis, we observed that the amount of studies on facebook and instagram in the period from to was low, so there is a need of more research in these important areas. this review study will help the readers to understand the different methods that the authors have used in their research studies on behavior analysis in social media. an examination of the different methods of behavior analysis carried out with the help of social media is the main aim of this research. thirty research studies were collected and analyzed to understand the personality of individuals who use social media such as facebook, twitter, and instagram. only three types of social network sites were included in this research. this analysis from the reported studies gives an overview of methods used to predict the personality of social media users. as seen from fig. , % of research was done on twitter from to , whereas as the other two social networking sites, facebook and instagram, only had % and %, respectively. moreover, some studies [ , ] proposed more than one method to analyze individuals' behavior. a major issue in this area is the security and privacy of the information that the users put on the social media. however, some of the studies included in this review provided suggestions and methods to help secure the personal information of users. many authors also discussed machine learning technique to observe the personality of social networking site users. the results showed that most of the research completed in were on twitter rather than facebook and instagram. in , most research was done on facebook and the least research was done on instagram. on the other hand, in twitter has the highest numbers of research papers and facebook had the lowest numbers. in , twitter and instagram had the highest number of research paper while facebook had none at all. data collection and behavior analysis methods provided by authors were collected as raw data and analyzed. a classification based on the methods used by the authors for analysis was created. previous review studies did not include the limitations and number of users' attribute in their analysis. we have included these two attributes in table to make the research more specific and easy to understand for the readers [ ] . the analysis of the papers indicated that twitter has been the most used to predict the personality of social media users. considering table , there is a need for more variety in research methods on instagram to understand the behavior of users. a cut-based classification method was used to analyze the behavior of twitter users by bhagat et al. [ ] . from the analysis done by these authors, they have concluded that cut-based classification method can be extended in the future to provide gui for users for polarity classifications and subjectivity classifications. real-time user messaging can also be analyzed in the future [ ] . this review study is based on the analysis of behavior of individuals, who use social network in their daily life. this study benefits readers as it helps to identify the methods used by different researchers and the number of researchers that applied these methods. this review study provides a clear description of the methods, limitations, and results that have been used by previous researches in studies during - . more than % people of the world use social media; however, the way social media users interact with each other vary greatly. there are demographic and behavioral trends from the facebook, twitter, and instagram that are discussed in table . in this review paper, we have reviewed and analyzed data collected from different published articles from to on the topic of behavior analysis using social media. it is found that there were different methods used by the researchers to analyze their table demographic and behaviour trends from the different social media according to age: age group between and use more facebook than twitter and instagram. more than % of this age group use facebook according to current trend use of smart phones: another reason of using social media have been increased in the past year is smart phones. smart phones have more visual interaction and people can access the social media easily. advancement in the mobile phones play very important role in the increased users of social media according to location: more people use the social media while they go out for dinner with family and friends. other locations where people like to use social media is gym, cinema and home specially in lounge room area more than other rooms according to time: more than % people use internet in the evening and % people use as a first thing in the morning. there is minimum use of social media during breakfast, lunch, at work and commuting frequency of using social networking sites: more than % people use social media more than five times a day as compared to % people who never use social networking site in a day. there are only % people who use once a week apps: more than % use apps to access the social media and fewer people use websites to access the social media data. from these methods, the most common technique to analyze the behavior of individuals was analysis techniques. from this study, it is clear that there is need for more research to predict the personality and behavior of individuals on the instagram. this study found that % of research was done on twitter and different analysis techniques were sued. while reviewing the research articles, it was clear that the researchers have used more than one method for data collection and behavior analysis. table has all the data analysis of the paper reviewed in the study. furthermore, unlike past research papers, this chapter included the attributes of the number of users and the limitations of the work done. these studies mostly focused on twitter with some research on facebook and instagram. in this research paper, we have attempted to fill the gap by including the number of users and limitation attributes. there are some challenges to find the solutions to the issues that have been discussed, but these require urgent attention. this study should be useful as a reference for researchers interested in the analysis of the behavior of social media users. facebook user's like behavior can reveal personality social media user personality classification using computational linguistic, international conference on information technology and electrical engineering (icitee) trendminer: large-scale analysis of political attitudes in public facebook messages cut-based classification for user behavioral analysis on social websites, green computing and internet of things (icgciot) analyzing emotions in twitter during a crisis: a case study of the middle east respiratory syndrome outbreak in korea, big data and smart computing (bigcomp) prediction of cyberbullying incidents in a mediabased social network, international conference on advances in social networks analysis and mining (asonam) media use of nursing students in thailand, international symposium on emerging trends and technologies in libraries and information services human behaviour in different social medias: a case study of twitter and disqus, international conference on advances in social network analysis and mining do private and sexual pictures receive more likes on instagram? demographic analysis of twitter users, advances in computing, communications and informatics (icacci) spotting suspicious behaviors in multimodal data: a general metric and algorithms characterizing behavior of topical authorities in twitter, international conference on innovative mechanisms for industry applications back to # d: predicting venezuelan states political election results through twitter, edemocracy & egovernment (icedeg) quad motif-based influence analyse of posts in instagram, advanced information and communication technologies (aict) the analysis of instagram technology adoption as marketing tools by small medium enterprise, information technology investigating link inference in partially observable networks: friendship ties and interaction consumer acceptance and use of instagram analyzing deviant behaviors on social media using cyber forensics-based methodologies analysis of the behavior of customers in the social networks using data mining techniques, international conference on advances in social networks analysis and mining (asonam) hiding in plain sight: characterizing and detecting malicious facebook pages fuzzy sentiment classification in social network facebook' statuses mining, international conference on sciences of electronics, technologies of information and telecommunications (setit) can visualization techniques help journalists to deepen analysis of twitter data? exploring the "germany x brazil" case measuring the controversy level of arabic trending topics on twitter, international conference on information and communication systems (icics) predicting temperament from twitter data, international congress on advanced applied informatics can twitter posts predict stock behavior, cloud computing and big data analysis (icccbda) probabilistic inference on twitter data to discover suspicious users and malicious content correlation of weather and moods of the italy residents through an analysis of their tweets, international conference on future internet of things and cloud workshop predicting personality traits of chinese users based on facebook wall posts, wireless and optical communication conference (wocc) monitoring adolescent alcohol use via multimodal analysis in social multimedia, big data (big data) personal preferences analysis of user interaction based on social networks, computing, communication and security (icccs) empirical analysis of user behavior in social media, international conference on developments of e-systems engineering fbmapping, an automated system for monitoring facebook data age distribution of active social media users worldwide as of rd quarter key: cord- - uxg tu authors: guenette, alexis; husain, shahid title: infectious complications following solid organ transplantation date: - - journal: critical care clinics doi: . /j.ccc. . . sha: doc_id: cord_uid: uxg tu infections in solid organ transplant recipients are complex and heterogeneous. this article reviews the clinical syndromes that will likely be encountered in the intensive care unit and helps to guide in the therapy and management of these patients. immunosuppression plays an integral role in solid organ transplant (sot) recipients because it increases graft survival; however, there are unintended consequences, such as infectious complications. one strategy aimed at assessing the functionality of the immune system consists of non-pathogen-specific immune monitoring, consisting of serum immunoglobulins, serum complement factors, peripheral blood lymphocyte subpopulations, soluble cd , and iatp in cd t cells. ideally, these would help to demonstrate one aspect of the overall "net state of immunosuppression." the "net state of immunosuppression" comprises all factors that may contribute to the risk of infection; this includes preexisting immune deficits, colonization with antimicrobial-resistant pathogens, immunosuppressive agents, acquired immunodeficiency, prior antimicrobial therapies, mucocutaneous barrier integrity, fluid collections, neutropenia, lymphopenia, and viral coinfections. disclosure statement: a. guenette has no relationships with a commercial company that has a direct financial interest in subject matter or materials discussed in article or with a company making a competing product. s. husain has received grants from astellas, pfizer, and merck. a division of infectious disease, university health network, university of toronto, university avenue, vaccinations, surgical prophylaxis, universal prophylaxis, preemptive or presymptomatic therapy, targeted prophylaxis, and education avoidance are preventative strategies that have been implemented in sot recipients. trimethoprim/sulfamethoxazole prophylaxis is given in most institutions for months to a lifetime to prevent pneumocystis pneumonia along with toxoplasma gondii, cyclospora cayetanensis, and many nocardia and listeria species. antiviral prophylaxis along with nucleic acid-based assays to prevent cytomegalovirus (cmv) and other herpesvirus infections has also transformed posttransplant care. infections in sot recipients reflect the net balance between the recipient's epidemiologic exposures and immunosuppression. alterations to the balance can be seen with antimicrobial prophylaxis, immunosuppression, and improved graft survival. this balance is also affected during a period of graft rejection or intensification of immunosuppression (fig. ). in this article, the authors review infectious syndromes encountered in intensive care units among sot recipients. bloodstream infections (bsis) are associated with poor outcomes along with being the leading cause of mortality and morbidity in sot. [ ] [ ] [ ] mortality as high as % , , has been described, and in fact, once septic shock develops, mortality can reach %, , although kalil and colleagues demonstrated that there may be a decrease in mortality of transplant patients compared with nontransplant patients. it is thought that sot recipients do not necessarily clinically behave in the same manner due to underlying immunosuppression, and in fact, tend to present with organ failure and thrombocytopenia during sepsis. universal risk factors for sepsis, regardless of transplanted organ, are cmv serology mismatch, particularly positive donor to negative recipient, cmv disease, which inherently demonstrates an immunomodulatory effect predisposing recipients to higher rates of bacterial and fungal sepsis, prolonged duration of graft cold ischemia, prolonged duration of surgical transplantation procedure, and requirement of large amounts of blood transfusion. , management should consist of rapid initiation of intravenous antibiotics, rapid diagnosis, source control, aggressive search for pathologic condition that mimics severe sepsis, and reduction in immunosuppressive drugs. nosocomial bsis are associated with an even increased risk of septic shock and failure of cure when compared with other bsis in sot patients. [ ] [ ] [ ] , grampositive bacteria are the most frequent source of bsis and are likely to be associated with intravascular catheters, especially in the nosocomial setting. , however, with kidney transplant recipients (ktrs), gram-negative bacteria likely related to urinary tract infections (utis) are the primary source, regardless of the time period. the overall incidence of multidrug-resistant organisms (mdro) is increasing. mdro gram-negative organisms accounted for about % of isolates. fluconazoleresistant candida spp accounted for up to % of cases of candidemia according to moreno and colleagues. , vancomycin-resistant enterococci (vre) have become an emerging pathogen with studies documenting an incidence of up to . % of nosocomial enterococcal spp bsis consistent with vre. these findings along with previous microbiological history and local antibiotic resistance patterns should be considered when determining empiric antimicrobial therapy. overall, there are limited studies on infective endocarditis (ie) in sold organ transplant recipients. the incidence of ie in a single center was %, with an estimated -fold higher incidence as compared with the general population, with an overall mortality up to %. there are limited data on the mode of infection and predisposing factors in sot recipients. underlying structural abnormalities may not appear to be a risk factor for ie as compared with the general population. , according to paterson and colleagues, % of infections were due to aspergillus fumigatus or staphylococcus aureus, and % were due to viridans streptococci, which is in contrast to the general population. a combination of antibiotic therapy as described in infectious diseases society of america (idsa) guidelines, infective endocarditis in adults: diagnosis, antimicrobial therapy, and management of complications and surgical management, if warranted, is the current management. empiric management of suspected sepsis/bsi should include gram-positive coverage, in the presence of intravascular catheters, and broad gram-negative coverage. the choice of the antibiotic is dependent on the local epidemiology and previous microbiological data. empiric candida or antifungal coverage is not required, because the early initiation of antifungal has not been shown to improve the outcome in randomized controlled trials of mostly immunocompetent patients. , bacterial pneumonia is the most common cause of lower respiratory tract infections. [ ] [ ] [ ] [ ] [ ] [ ] [ ] according to giannella and colleagues, community-acquired pneumonia (cap) was found in . % and hospital-acquired pneumonia (hap) was found in . % of sot recipients treated for pneumonia. in lung transplant recipients, bacterial pneumonia or bronchitis accounts for % to % of all infections with the incidence of bacterial pneumonia peaking in the first to postoperative weeks and then declines by the fourth month. perioperative antibiotics, which are focused on preoperative cultures from the recipient and donor, reduce the incidence of early bacterial pneumonia to less than %. , regarding cardiac, hepatic, and renal transplants, the incidence of early bacterial pneumonia is %, %, and % to %, respectively, [ ] [ ] [ ] , with a mortality of % to % in liver and kidney transplant recipients. however, mortality between nosocomial and community-acquired infection was extreme at % compared with % - with mechanical ventilation and nosocomial infections at a higher increased risk for death. , [ ] [ ] [ ] in the initial perioperative infectious complications period, nosocomial pathogens, such as pseudomonas aeruginosa, escherichia coli, klebsiella species, acinetobacter species, and s aureus, including methicillinresistant s aureus (mrsa), should be considered in the immediate perioperative period; however, prolonged mechanical ventilation following transplant increases the risk for nosocomial pneumonia. [ ] [ ] [ ] community-acquired pathogens, such as haemophilus influenzae, streptococcus pneumoniae, and legionella species, may be seen. with the implementation of trimethoprim-sulfamethoxazole prophylaxis, the incidence of nocardia pneumonia has decreased; however, it is still reported. , empiric treatment should take into consideration previous microbiological data, local epidemiology, and recent clinical history with regards to empiric antibiotic coverage. respiratory viral infections are a significant cause of mortality and morbidity among transplant recipients, including influenza, respiratory syncytial virus, parainfluenza virus, rhinovirus, human metapneumovirus, and coronavirus. the seasonal pattern usually follows that of the general public. [ ] [ ] [ ] disease can consist of mild congestion and rhinorrhea to more severe tracheobronchitis, bronchiolitis, and pneumonia. clinical manifestation can range from mild or atypical symptoms, including absence of fever, with lung transplant recipients presenting with a more severe clinical course and complications. , viral shedding is usually prolonged and seen even with the use of antivirals. , transplant recipients are at higher risk of infectious complications, including fungal and bacterial pneumonia. respiratory viral infections appear to be a risk factor for both acute and chronic rejection, especially in lung transplant recipients. , [ ] [ ] [ ] [ ] diagnostic workup should consist of a nasopharyngeal swab, wash, or aspirate. if upper tract samples fail to document the cause or if there is clinical or radiological evidence of lower tract involvement, bronchoalveolar lavage (bal) should be considered. polymerase chain reaction (pcr)-based assays are commercially available with many centers adopting them because they are the most preferred mode of testing given the high sensitivity along with most allowing for simultaneously detecting a broad range of respiratory pathogens from a single sample. treatment, as is outlined in fig. , includes supportive care and reduction in immunosuppression. adenovirus is a nonenveloped, lytic double-stranded dna virus that can be acquired de novo, through reactivation of a latent infection of the recipient, or from transplant organ. transmission occurs by respiratory route, person-to-person contact, or fecal-oral route. the true incidence among sot recipients is unknown, with most infections occurring within the first year after transplantation. clinical manifestations can vary; however, when affecting lung transplant recipients, it can produce a range of clinical manifestations, including acute flulike illness, diffuse alveolar damage, or necrotizing pneumonia along with chronic changes, such as bronchiolitis obliterans, interstitial fibrosis, or bronchiectasis. [ ] [ ] [ ] [ ] viral culture, direct antigen detection, molecular methods, and histopathology are available for diagnosis with histopathologic evaluation, as the gold standard for the diagnosis of invasive disease. rapid antigen detection kits, in particular, immunofluorescence assays when processing respiratory specimens, are commercially available, which yield rapid and specific results. pcr, qualitative and quantitative, has emerged as a widely used tool for detection because it is highly sensitive and rapid. recovery of adenovirus from respiratory samples does not necessarily confirm disease because patients can shed asymptomatically for a prolonged period of time; therefore, it is essential to correlate with clinical findings along with detection of virus from other sites and histopathological findings. cidofovir has the best evidence to support its use in the treatment of adenoviral infections. brincidofovir, the lipid conjugate of cidofovir, has also demonstrated in vitro susceptibility and appears to be promising in vivo with regards to sot recipients; however, further studies are warranted. cmv is a major pathogen in sot recipients, with the ability to cause end-organ disease. the immunomodulatory effects of cmv, impaired t-cell and phagocytic function, and cytokine dysregulation can lead to opportunistic infections, rejection, graft loss, and reduced survival. , the transplant recipients who are at highest risk are seronegative recipients of seropositive organ, d /rÀ, because they have no preexisting immunity, and seropositive recipients, dÀ/r , are at intermediate risk. there is little difference between d /r and dÀ/rÀ groups, with potentially worse outcomes in d /r (fig. ) . clinical presentation consists of dyspnea, fever, and malaise with the identification of characteristic cmv cells in lung tissue. radiographic changes are nonspecific and include diffuse haziness, focal haziness, focal lobar consolidation, and no change. diagnosis is made via cell culture viral isolation; however, this can be time consuming. therefore, the detection of cmv dna by pcr in the peripheral blood leukocytes, providing a sensitivity of % and specificity of % for cmv pneumonitis, along with the bal cmv dna pcr, is an alternative means to diagnosis. , however, with regards to the bal findings, it would be imperative to differentiate between infections versus shedding; hence, the concomitant peripheral blood leukocyte pcr along with the clinical picture is necessary to help determine the diagnosis. treatment consists of ganciclovir or the oral alternative, valganciclovir. foscarnet and cidofovir are alternative options; however, they are primarily reserved when there is a concern for resistance or documented resistance because their side-effect profiles are less desirable. cmv immunoglobulin in conjunction with treatment offers limited efficacy. maribavir, brincidofovir, and letermovir are novel agents that may provide alternative options for treatment; however, further studies are warranted. candida is a frequent colonizer of lung transplant recipients, but less than % of patients colonized develop invasive disease. , bronchial anastomotic infections may occur early in the postoperative setting, which can lead to anastomotic failure, parenchymal lung infection, and mediastinitis. [ ] [ ] [ ] [ ] [ ] [ ] artificial bronchial stents can serve as a potential site for infection. , candida tracheobronchitis is based on visual inspection and histologic confirmation along with positive cultures from an appropriate specimen. aspergillus species, a saprophytic organism, has higher rates of mortality, up to %, and an incidence of % in lung, . % in heart, % in liver, and . % in kidney transplant recipients. infection may be due to reactivation or de novo infection following inhalation of the mold. renal failure, hemodialysis, repeated bacterial infections, leukopenia, cmv disease, high levels of immunosuppression, retransplantation, chronic exposure of the transplanted lung to the environment, and abnormal anatomic and physiologic function of the transplanted and, if still present, the native lung, airway ischemia, hypogammaglobulinemia, cystic fibrosis, and bronchial stent are all risk factors for invasive aspergillosis. , [ ] [ ] [ ] [ ] in lung transplant recipients, % to % are colonized with aspergillus with complicated infections affecting up to % of all patients. , [ ] [ ] [ ] [ ] colonization can also lead to bronchiolitis obliterans syndrome after lung transplantation. , clinical manifestations can range from asymptomatic colonization to tracheobronchitis, invasive pulmonary aspergillosis, empyema, and disseminated disease, , with symptoms including purulent sputum, fever, malaise, respiratory distress, and hemoptysis. , aspergillus tracheobronchitis can cause airway obstruction, ulcerations, and pseudomembrane formation. nonaspergillus mycelial fungi are also increasing with frequency, as high as %, and an overall mortality of % , ; however, zygomycosis (rhizopus and mucor species) and non-aspergillus hyalohyphomycosis (scedosporium apiospermum and fusarium species) have an even higher mortality of up to %. endemic fungi, coccidioides immitis, histoplasma capsulatum, blastomyces dermatitidis, and cryptcoccus are additional pathogens that may need to be considered. radiological findings may demonstrate nodules, cavitary lesions, focal consolidation or patchy densities, wedge-shaped pleural-based lesions, air-filled bronchi with an intraluminal lesion, "air crescent" sign, and halo of decreased density. tissue invasion by fungal organisms is the gold standard for diagnosis of invasive fungal pneumonia. however, this may be difficult to obtain; therefore, international society for heart and lung transplantation developed guidelines, a working formulation for the standardization of definitions of infections in cardiothoracic transplant recipients, to assist in the diagnosis of not only fungal infections but also bacterial and viral infections. these definitions may also be applied to abdominal transplant recipients. treatment is as follows in fig. ; however, be aware of drug-drug interactions with regards to immunosuppressive agents. it is important to note, however, that empiric management of mold infections in sot recipients is seldom necessary. the optimal approach is to pursue the diagnostic workup aggressively and treat accordingly even in lung transplant recipients. pneumocystis jirovecii, t gondii, mycobacterium tuberculosis, and nontuberculosis mycobacteria are other pathogens to also consider as possible causes of respiratory tract infections along with the other pathogens listed in fig. . central nervous system (cns) infections can account for approximately % to % of cns lesions in transplant recipients. , routine prophylaxis aimed at opportunistic infections along with a more conservative approach regarding immunosuppression has led to noticeable trends in infections in transplant recipients. for example, routine administration of trimethoprim-sulfamethoxazole for p jirovecii has likely contributed to the reduction in infections owing to t gondii, nocardia, and listeria monocytogenes along with acyclovir and valganciclovir attributing to the likely decline in herpesviridaerelated infections. , clinical presentation will vary and may include fever, headache, meningismus, kernig and brudzinski signs, new-onset seizure, papilledema, altered sensorium, and/or focal neurologic deficits; however, because of their underlying immunosuppression, these may be subtle or absent. , as listed in fig. , possible causes range from community-acquired organisms, donor-derived infections, reactivation, to opportunistic pathogens. metastatic or direct lesions along with "pulmonary-brain" syndrome are also considerations in such organisms as cryptococcus, nocardia, aspergillus, zygomycetes, strongyloides, and toxoplasma; therefore, further investigations are warranted (eg, computed tomography of sinus and computed tomography of chest). , evaluation should include neuroimaging along with lumbar puncture as soon as possible if no contraindication exists. cerebral spinal fluid examination should always include cell counts and differential, glucose, protein; routine smears; and cultures for bacteria, fungi, and mycobacteria. , additional specialized testing, such as viral pcr, antigen or antibody, and s ribosomal rna, may be required depending on the clinical scenario. , however, brain biopsy with appropriate staining may be the definitive diagnosis if findings are inconclusive. empiric treatment should cover common bacterial and viral pathogens; however, additional agents may be needed depending on the epidemiologic history. cholangitis is a common infection after liver transplant, and in fact, is the most common infection more than year after liver transplant. most cases occurred within years and were associated with primary sclerosing cholangitis and rouxen-y anastomosis. the most frequently identified bacteria are enterococcus spp and e coli , ; however, other gram-negative bacilli and anaerobes should also be considered. , bile leaks can occur in % to % of cases after liver transplantation, especially in living liver donor transplants. clinical presentation varies with extent of the leak; however, symptoms can include abdominal pain, fever, or any sign of peritonitis. however, because of underlying immunosuppression, they can also be asymptomatic. in these cases, elevations in serum bilirubin, fluctuations in cyclosporine, or bilious ascites should raise suspicion for a bile leak. biliary strictures at the site of anastomosis can also present with fever, abdominal pain, but also jaundice and asymptomatic biochemical cholestasis. bilomas can represent an additional source of infection. , hepatic artery thrombosis, although more common in living donor liver transplants (ldlts), is uncommon with deceased donor liver transplant (ddlt) with an overall incidence up to % and can lead to complications, including hepatic abscesses, necrosis, sepsis, and graft loss. vancomycin-resistant enterococcus faecium (vref) is of particular concern in liver transplant. pretransplant colonization increases rates of intra-abdominal and bsis after transplantation. , in fact, hospital and intensive care unit stays are longer for patients with vref versus vancomycin-sensitive e faecium infections. liver, pancreas, and intestinal recipients are at particular risk for fungal infections, most often caused by candida species. , , regarding ddlt versus ldlt, there are variations with infectious complications. the rate of infection appears to be similar to ddlt; however, because of the more complex nature of the surgery, there are observable difference and specific concerns as detailed in box . [ ] [ ] [ ] the clinical syndrome of hepatic dysfunction can range from mild elevated liver enzymes to hepatitis to fulminant hepatic failure. hepatic dysfunction can present in any sot recipient; however, of utmost concern would be liver transplant recipients. causes can range from infectious to noninfectious with noninfectious causes primarily an issue with liver transplant recipients regarding postoperative complications, recurrence of primary disease, drug-induced complications, and rejection. the infectious causes listed in fig. can range from donor-derived infections, postoperative complications, community-acquired organisms, reactivation, to opportunistic pathogens. evaluation should include imaging (eg, ultrasound, computed tomography, or mri) along with the appropriate infectious workup (eg, blood cultures, serum pcr, serology, antigen, and/or antibodies). if the diagnosis remains inconclusive, a liver biopsy may need to be pursued with appropriate staining obtained. diarrhea following transplantation is frequently observed and is estimated to occur in % to % of patients. [ ] [ ] [ ] [ ] [ ] it can be associated with allograft loss and increased mortality. , , , in fact, it results in , hospitalizations and deaths annually. , the severity and cause of diarrhea can lead to hypovolemia and/or septic shock. diarrhea is a recognized side effect of some immunosuppressive agents; however, infectious causes should be considered based on the clinical picture. causes are detailed in fig. . evaluation should include stool culture, ova and parasite and giardia antigen along with appropriate pcr, antigen testing, and/or special staining. imaging may include a computed tomography to evaluate for bowel wall edema along with colonic wall thickening and dilation. esophagogastroduodenoscopy (egd) and/or colonoscopy along with biopsy may also be warranted. cmv colitis is diagnosed via histopathology obtained during a biopsy. serum pcr can be low to undetectable in this setting. if there is a clinical suspicion (eg, elevated serum pcr along with diarrhea, however the patient is unable to undergo colonoscopy), this may warrant empiric treatment. clostridium difficile infection (cdi) is among the most common health careassociated pathogen and is the most common cause of nosocomial infectious diarrhea. the highest incidence of cdi in sot occurs within the first months following the transplant and is likely related to frequent exposure to antimicrobials, health care settings, and immunosuppressants. proton pump inhibitors (ppis) are known to be a risk for cdi and may still be used in the setting of mechanical ventilation, versus h blockers. hospitalized patients who use ppis are twice as likely to develop cdi. , fulminant colitis develops in up to % of sot recipients with cdi. , relapsing disease is common, and protracted courses of therapy are often essential. , , idsa and society for healthcare epidemiology of america recently updated the clinical practice guidelines for clostridium difficile infection in adults and children for , and although this is for immunocompetent patients, these guidelines can be applied to sot recipients because there have been limited studies into the treatment of cdi in sot patients. the most common infectious complication in sot is uti, accounting for % to % of all infections, and % of all hospitalizations for sepsis in ktrs. , [ ] [ ] [ ] [ ] [ ] most uti are seen within the first months after transplant; however, it can occur any time after transplantation. , , empirical treatment is imperative, as it has been demonstrated that inappropriate antibiotic therapy is associated with an increase in mortality. , , to guide empirical therapy, local epidemiologic data, patient's microbiological history, and prior antibiotic use need to be taken into account. , the most frequent organisms causing utis are gram-negative bacilli , ; however, when a urinary catheter is involved, enterococci and s aureus should also be considered. the duration of treatment varies from - days depending on the clinical syndrome. recurrent uti, defined as or more episodes of symptomatic utis over a month period or in the previous months, should prompt further investigation regarding anatomic and functional abnormalities along with behavioral modifications (eg, postcoital voiding) and may need a prolonged course of antibiotics, possibly to weeks. , , asymptomatic bacteriuria should only be treated during the early postoperative period and up to month after transplant in renal transplant patients. the data on uti related to candida spp in sot are limited and mostly include ktr. in ktr, candida spp are the most frequently isolated fungal cause of uti. unfortunately, there are no clinical trials in the management of candida uti in sot. candiduria is frequent, occurring in up to % of ktr; however, it is mostly asymptomatic. asymptomatic candiduria is usually treated as there is concern regarding the allograft and potential for complications regarding the upper urinary tract; however, it should only be treated if the patient is neutropenic, or undergoing a urologic procedure. , candiduria should be classified based on risk factors for disseminated candidiasis. urinary catheters should be removed or exchanged, and candiduria should be confirmed with a second, clean voided urine culture. disseminated candidiasis may be considered if clinical manifestations are consistent (eg, positive blood cultures, a second urine culture after removal or replacement of the urinary catheter, funduscopic examination, cultures from any other significant site, and kidney imaging). persistent candiduria along with infectious complications no indwelling catheters should prompt imaging of the kidneys and collecting system to exclude renal abscess, fungus balls, or other urologic abnormalities. treatment options can range from fluconazole treatment of choice, to echinocandins, amphotericin b, and flucytosine depending on the clinical situation and organism sensitivities with durations from at least days for uti to to weeks for pyelonephritis (box ). sot recipients are a complex group of patients with diverse causes given the underlying immunosuppression. as with all infectious processes, rapid identification of the always consider previous microbiological data and local epidemiology with regards to empiric antibiotics if an intravascular catheter is involved, consider broad gram-positive cocci, including mrsa, coverage in addition to broad gram-negative coverage, including esbls and cres, if warranted empiric antifungal is not needed, unless there is a high index of suspicion always consider previous microbiological data and local epidemiology with regards to empiric antibiotics cap should include empiric coverage for atypicals along with community-associated organisms hap and vap should include broad gram-positive coverage, especially mrsa, along with broad gram-negative coverage, including esbls and cres if warranted influenza is the only virus with approved treatment, oseltamavir; therefore, this should be started empirically if there is a concern antifungals should not be started empirically, even in lung transplant recipients; however, fungal infections should be worked up thoroughly pathogen, source control, and adjustment of immunosuppression is the hallmark of treatment. there is a summary of the management of infections in sot recipients included in box . empiric therapy should consist of ceftriaxone and vancomycin ae acyclovir always consider previous microbiological data and local epidemiology with regards to empiric antibiotics if vre positive, will need to consider coverage using high-dose daptomycin or linezolid along with broad gram-negative coverage, including esbls and cres, if warranted regarding c difficile infections, oral vancomycin should be initiated empirically, if suspected, as first-line therapy always consider previous microbiological data along with local epidemiology with regards to empiric antibiotic decisions asymptomatic bacteriuria should only be treated in renal transplant patients during the first month posttransplantation antimicrobials should be tailored to the causative agent, with durations that generally range from to days depending on the clinical context fluconazole is the treatment of choice for cystitis and pyelonephritis if candida is the causative organism abbreviations: cre, carbapenem-resistant enterobacteriaceae; esbl, extended spectrum betalactamase inhibitors; vap, ventilator-associated pneumonia. clinical immune-monitoring strategies for predicting infection risk in solid organ transplantation from the classic concepts to modern practice epidemiology and risk factors for nosocomial bloodstream infections in solid organ transplants over a -year period bloodstream infections among transplant recipients: results of a nationwide surveillance in spain autopsy-determined causes of death in solid organ transplant recipients nosocomial bloodstream infections in a nationwide study: comparison between solid organ transplant patients and the general population bloodstream infection after kidney transplantation: epidemiology, microbiology, associated risk factors, and outcome bacteremia and septic shock after solidorgan transplantation is bacteremic sepsis associated with higher mortality in transplant recipients than in nontransplant patients? a matched case-control propensity-adjusted study sepsis in the severely immunocompromised patient sepsis and solid organ transplantation statins are associated with improved outcomes of bloodstream infection in solid-organ transplant recipients risk factors and outcomes of bacteremia caused by drug-resistant eskape pathogens in solid-organ transplant recipients severe endocarditis in transplant recipients-an epidemiologic study infective endocarditis in solid organ transplant recipients empirical fluconazole versus placebo for intensive care unit patients: a randomized trial empirical micafungin treatment and survival without invasive fungal infection in adults with icu-acquired sepsis, candida colonization, and multiple organ failure: the empiricus randomized clinical trial pulmonary infections in liver transplant recipients receiving tacrolimus. changing pattern of microbial etiologies infectious complications after kidney transplantation: current epidemiology and associated risk factors pneumonia infection in organ transplant recipients pneumonia in solid organ recipients: spectrum of pathogens in episodes microbiologic features and outcome of pneumonia in transplanted patients infectious pulmonary complications in lung transplant recipients a standardized protocol for the treatment of severe pneumonia in kidney transplant recipients pneumonia in solid organ transplant recipients: a prospective multicenter study the diagnosis of pneumonia in renal transplant recipients using invasive and noninvasive procedures pulmonary complications in cardiac transplant recipients nocardiosis following solid organ transplantation: a single-centre experience rna respiratory viruses in solid organ transplantation respiratory viral infections in immunocompetent and immunocompromised persons clinical implications of respiratory virus infections in solid organ transplant recipients: a prospective study respiratory viral infections in transplant recipients respiratory viruses and chronic rejection in lung transplant recipients respiratory viral infections are a distinct risk for bronchiolitis obliterans syndrome and death clinical impact of community-acquired respiratory viruses on bronchiolitis obliterans after lung transplant a single-season prospective study of respiratory viral infections in lung transplant recipients respiratory viruses in transplant recipients: more than just a cold. clinical syndromes and infection prevention principles adenovirus in solid organ transplantation adenovirus pneumonia in lung transplant recipients adenovirus infection in the lung results in graft failure after lung transplantation assessment of adenovirus infection in adult lung transplant recipients using molecular surveillance ecil- ): guidelines for diagnosis and treatment of human respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, and coronavirus viral infections in solid organ transplant recipients: novel updates and a review of the classics late-onset cytomegalovirus disease as a significant complication in solid organ transplant recipients receiving antiviral prophylaxis: a call to heed the mounting evidence increased human cytomegalovirus (hcmv) dna load in peripheral blood leukocytes after lung transplantation correlates with hcmv pneumonitis infectious complications of lung transplantation. impact of cystic fibrosis invasive fungal infections in solid organ transplant recipients fungal infection in lung transplantation diagnosis and outcome of early pleural space infection following lung transplantation trends in invasive disease due to candida species following heart and lung transplantation infection in the transplanted and native lung after single lung transplantation anastomotic infections in lung transplant recipients risk factors for invasive aspergillosis in solid-organ transplant recipients: a case-control study a risk profile for invasive aspergillosis in liver transplant recipients invasive aspergillosis in the recipients of liver retransplantation risk factors for invasive aspergillosis in liver transplant recipients aspergillus infection in lung transplant patients: incidence and prognosis pulmonary complications of solid organ and hematopoietic stem cell transplantation spectrum of aspergillus infection in lung transplant recipients: case series and review of the literature nebulized amphotericin b prophylaxis for aspergillus infection in lung transplantation: study of risk factors aspergillus colonization of the lung allograft is a risk factor for bronchiolitis obliterans syndrome prevalence and outcome of invasive fungal infections in , thoracic organ transplant recipients: a multicenter retrospective study. italian study group of fungal infections in thoracic organ transplant recipients aspergillus infection in single and double lung transplant recipients opportunistic mycelial fungal infections in organ transplant recipients: emerging importance of non-aspergillus mycelial fungi cns infections in solid organ transplant recipients infections of the central nervous system in transplant recipients central nervous system syndromes in solid organ transplant recipients infectious complications more than year after liver transplantation: a -decade nationwide experience the importance of late infections for the long-term outcome after liver transplantation biliary complications following liver transplantation etiology and management of hepatic artery thrombosis after adult liver transplantation infections in solid-organ transplant recipients hepatitis g virus in patients with cryptogenic liver disease undergoing liver transplantation life-threatening infection in transplant recipients fungal infections in transplant and oncology patients epidemiology and risk factors for infection after living donor liver transplantation infections after living donor liver transplantation in children infectious complications in living-donor liver transplant recipients: a -year single-center experience abnormal liver tests after liver transplantation diagnostic yields in solid organ transplant recipients admitted with diarrhea severe diarrhea in renal transplant patients: results of the didact study incidence and risk factors for diarrhea following kidney transplantation and association with graft loss and mortality gastrointestinal complications in liver transplant recipients: mitos study gastrointestinal complications in renal transplant recipients: mitos study gastrointestinal complications in renal transplant patients: a large, single-center experience diarrhoea following renal transplantation diarrhea in organ transplant recipients clinical practice. acute infectious diarrhea clostridium difficile infection in solid organ transplant patients histamine- receptor antagonists vs proton pump inhibitors on gastrointestinal tract hemorrhage and infectious complications in the intensive care unit strategies to prevent clostridium difficile infections in acute care hospitals fulminant clostridium difficile: an underappreciated and increasing cause of death and complications clostridium difficile infection in solid organ transplant recipients early and late onset clostridium difficileassociated colitis following liver transplantation urinary tract infections in solid organ transplantation urinary tract infections in renal transplant recipients hospitalizations for bacterial septicemia after renal transplantation in the united states infective complications in renal allograft recipients: epidemiology and outcome acute pyelonephritis represents a risk factor impairing long-term kidney graft function management of urinary tract infection in solid organ transplant recipients: consensus statement of the group for the study of infection in transplant recipients (gesitra) of the spanish society of infectious diseases and clinical microbiology (seimc) and the spanish network for research in infectious diseases (reipi) management of urinary tract infections and lymphocele in renal transplant recipients bloodstream infections caused by antibioticresistant gram-negative bacilli: risk factors for mortality and impact of inappropriate initial antimicrobial therapy on outcome mortality and delay in effective therapy associated with extended-spectrum beta-lactamase production in enterobacteriaceae bacteraemia: a systematic review and meta-analysis multidrug-resistant bacterial infection in solid organ transplant recipients key: cord- -nquozaxt authors: sieg, michael; busch, johannes; eschke, maria; böttcher, denny; heenemann, kristin; vahlenkamp, annett; reinert, anja; seeger, johannes; heilmann, romy; scheffler, kira; vahlenkamp, thomas w. title: a new genotype of feline morbillivirus infects primary cells of the lung, kidney, brain and peripheral blood date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: nquozaxt paramyxoviruses comprise a large number of diverse viruses which in part give rise to severe diseases in affected hosts. a new genotype of feline morbillivirus, tentatively named feline morbillivirus genotype (femv-gt ), was isolated from urine of cats with urinary tract diseases. whole genome sequencing showed about % nucleotide homology to known feline morbilliviruses. the virus was isolated in permanent cell lines of feline and simian origin. to investigate the cell tropism of femv-gt feline primary epithelial cells from the kidney, the urinary bladder and the lung, peripheral blood mononuclear cells (pbmc), as well as organotypic brain slice cultures were used for infection experiments. we demonstrate that femv-gt is able to infect renal and pulmonary epithelial cells, primary cells from the cerebrum and cerebellum, as well as immune cells in the blood, especially cd (+) t cells, cd (+) b cells and monocytes. the cats used for virus isolation shed femv-gt continuously for several months despite the presence of neutralizing antibodies in the blood. our results point towards the necessity of increased awareness for this virus when clinical signs of the aforementioned organs are encountered in cats which cannot be explained by other etiologies. paramyxoviruses constitute a large family of single stranded, negative-sense, enveloped rna viruses that can lead to important diseases in humans and animals [ ] . historically, paramyxoviridae have been subdivided into seven genera based on biochemical properties and sds-page patterns of viral structural proteins: rubulavirus, avulavirus, respirovirus, henipavirus, morbillivirus, ferlavirus and aquaparamyxovirus. taking genome sequences and protein data into account many currently described paramyxoviruses are assigned as unclassified, e.g. rodent-borne tailam virus [ ] , nariva virus [ ] and bank vole virus [ ] , as well as paramyxoviruses detected in bats [ ] . in recent years, the genus morbillivirus has received growing attention, due to the discovery of a new feline morbillivirus (femv, formerly abbreviated as fmopv) associated with tubulo-interstitial nephritis in stray cats from hong kong [ ] . subsequently, the prevalence was reported from other countries including japan, usa, turkey, brazil, thailand, italy and germany [ ] [ ] [ ] [ ] [ ] [ ] [ ] . percentage of femv-positive urines ranged from % to % in the us [ ] and japan [ ] , respectively. seroprevalence data of femv available from hong kong and japan showed . % [ ] , . % [ ] , . % [ ] and % [ ] of investigated cats to be femv-positive using nucleo-or phosphoproteins as antigens. while some of these studies established a link between an infection with femv and the presence of kidney diseases in affected cats [ , , , , ] , others could not confirm such an association [ ] [ ] [ ] ] . these discrepancies may be due to the complexity of chronic kidney disease (ckd) pathogenesis in general, making it difficult to link cases of feline ckd to only one specific trigger [ ] . in some cats, feline morbilliviruses may induce a persistent infection of the urinary tract [ ] . so far it is not clear whether an acute or chronic infection can cause or support the development of ckd. during our current studies an unknown feline paramyxovirus was detected in urine samples from domestic cats [ ] . although this virus was initially linked to femv strains from japan, whole genome sequencing revealed a different genotype of femv, tentatively named feline morbillivirus genotype (femv-gt ). here we show that the femv-gt -gordon strain replicates in primary feline epithelial cells from different organs and is able to infect primary feline t and b cells, as well as monocytes in vitro. we demonstrate that femv-gt readily infects feline organotypic brain slice cultures with cells of the cerebrum and cerebellum being comparably susceptible. the molecular and biological characterization of femv-gt shows that the diversity of feline paramyxoviruses extends beyond the formerly known femv isolates, which must be further studied in detail. all cell lines and primary cells used were maintained at • c, % humidity and % co . llc-mk and vero ccl cell lines were purchased from the instituto zooprofilattico sperimentale della lombardia e dell'emilia romagna «bruno ubertini» (izsler), italy, whereas crfk, mdbk, mdck-ii, hek , bhk- , ma- , marc- , a , fmn-r, mgn-r, ran- -r, fln-r and ke-r were kindly provided by the friedrich-loeffler-institute (fli), germany. all cell lines were grown in dulbecco's modified eagle medium (dmem) containing . g/l glucose, % fbs, glutamax™ supplement, × mem non-essential amino acids solution and mm sodium pyruvate. fcwf cells (atcc ® crl ™) were purchased from the american type culture collection (atcc), usa and cultivated in rpmi medium containing mm l-glutamine, . g/l sodium bicarbonate, . g/l glucose, mm hepes, . mm sodium pyruvate, . mm -mercaptoethanol, % fbs or in dmem with % fbs, respectively. the organ material used in this work was provided by the institute of pathology, faculty of veterinary medicine, leipzig university and derived from dead animals euthanized for medical reasons unrelated to this study. primary feline kidney cells were isolated by adapting a previously described protocol [ ] . briefly, kidneys from dead animals were removed aseptically and stored in ice cold hank's buffered salt solution (hbss) without cacl and mgcl until further processing. kidneys were de-capsulated, bisected, and the renal cortex was removed and cut into small pieces. tissue was dissociated by collagenase ii ( mg/ml) treatment at • c for min. this step was repeated three times and the collected cell suspensions were passed through a µm cell strainer to remove cell aggregates. cells were pelleted by centrifugation at × g for min and re-suspended in complete culture medium (dmem:f [ %: %] medium containing ng/ml human epidermal growth factor, nm hydrocortisone, µg/ml insulin, µg/ml transferrin, nm sodium selenite and iu/ml penicillin-streptomycin) and seeded into cm plastic flasks. the cells were cultured at • c under % co in a humidified atmosphere until the monolayer became nearly confluent. similarly, primary feline urinary bladder cells were prepared by collagenase treatment of the urinary bladder mucosa. dissociated cells were than cultured in keratinocyte serum free medium (ksfm) including ng/ml egf, . % (v/v) of bovine pituitary extract (bpe, thermo fisher) and ng/ml choleratoxin (sigma-aldrich, st. louis, mo, usa) and iu/ml penicillin-streptomycin. to isolate primary feline pulmonary epithelial cells fresh lungs were rinsed three times with dmem through the primary bronchus to remove alveolar macrophages. pulmonary epithelial cells were dissociated from the surrounding tissue with . % trypsin and . % edta followed by an incubation for min at • c. finally, cells were collected by rinsing the lungs with dmem and a centrifugation step for min at × g. cells were seeded into a cm plastic flask and cultured in dmem containing . g/l glucose, % fbs, glutamax™ supplement, × mem non-essential amino acids and mm sodium pyruvate, iu/ml penicillin-streptomycin until the monolayer reached approximate confluence. urine samples from cats were collected as part of the routine physical examination at the department of small animals, leipzig university. the material was stored at − • c (for not more than weeks) before rna isolation was conducted. for virus isolation vero, crfk and llc-mk cells were infected with ml urine mixed with ml pure dmem (containing iu/ml penicillin-streptomycin) in cm cell culture flasks. after h incubation at • c, % co and % humidity the inoculum was replaced by ml cultivation medium (dmem, sodium pyruvate, non-essential amino acids, % fbs, iu/ml penicillin-streptomycin) and were cultured for days at the indicated conditions. the cell culture supernatant from this first passage was used to infect fresh cells. this procedure was repeated several times and the resulting cell culture supernatants were continuously tested for the presence of paramyxovirus-rna by rt-pcr. for virus infection assays different cell lines (see section . ) were infected with the llc-mk -adapted (passage no. ) 'gordon' strain of femv-gt in a -well format at an moi~ . for two hours at • c. infection medium was replaced by cultivation medium (see above) and cells were further incubated at • c, % co and % humidity for five days. percentage of virus-positive cells were semi-quantified by immunofluorescence staining targeting the femv-gt -nucleoprotein followed by microscopic analysis. viral particles were concentrated from cell culture supernatants of femv-gt -infected llc-mk via ultracentrifugation. in brief, ml virus-containing supernatant was sublayered viruses , , of with ml of a % sucrose cushion and centrifuged using an sw ti rotor (beckman coulter) at , rpm for . h at • c. the semi-purified virions were re-suspended in pbs prior to loading onto formvar-coated copper grids (plano gmbh, wetzlar, germany) and were negatively stained with % uranyl acetate. grids were viewed using an tem libra (carl zeiss, jena, germany) transmission electron microscope. detection of femv-gt in feline urine samples or in cell culture supernatants was performed as previously described [ , ] using the primer pair hen-res-mor-r and hen-res-mor-f /f . for whole genome sequencing, degenerated primers were designed based on the available femv-sequences (primer sequences are available in supplementary files, table s ). amplification was performed with the "superscript™ iii one-step rt-pcr system containing platinum™ taq high fidelity dna polymerase" (thermo fisher scientific, waltham, ma, usa). pcr products were visualized by agarose gel electrophoresis with × tris-acetate-edta buffer ( mm tris-acetate, mm edta, ph . ) containing . µg/ml of ethidium bromide. for dna-sequencing, specific pcr fragments were cut out of the gel and purified using a gel/pcr dna fragments extraction kit (geneaid, new taipei city, taiwan). sequencing was performed by sanger's dideoxy termination method (microsynth seqlab gmbh, göttingen, germany) and each pcr product was sequenced twice. nucleotide sequences were analyzed using the basic local alignment search tool (blast, www.ncbi.nlm.nih.gov/blast/), and genetic distances of whole genome sequences were calculated applying the general time reversible model with gamma distributed invariant sites (gtr + i) at the nucleotide level using the mega software. a phylogenetic tree was built by the maximum likelihood method with bootstrap replicates [ ] . sequences determined in the course of this work have been deposited in genbank, with the accession numbers mk and mk . cells were fixed with % (w/v) paraformaldehyde for min at • c, permeabilized with . % triton x- in pbs for min, blocked with % bsa-pbs for min at room temperature and probed with a cytokeratin pan type i/ii antibody cocktail (clone ae /ae , thermo fisher scientific, waltham, ma, usa) at a dilution of : . femv-gt was probed using a polyclonal rabbit antibody raised against the nucleoprotein of femv (generated in house) at a dilution of : . after overnight incubation at • c cells were washed three times with pbs and incubated with an alexa fluor -conjugated goat-anti-mouse igg (h+l) or with an alexa fluor -conjugated goat-anti-rabbit igg (h+l) secondary antibody (thermo fisher scientific, waltham, ma, usa) in % bsa-pbs at a dilution of : containing , -diamidino- -phenylindole (dapi, nm) and incubated for one hour at • c in a humidified chamber. after three pbs-washing steps images were acquired using an ax microscope (olympus, tokio, japan). virus titration was performed by the endpoint dilution assay in llc-mk cells followed by visualization of virus positive dilutions using immunofluorescence staining. viral titers were expressed as % tissue culture infective dose (tcid ) calculated after the reed-muench method [ ] . to test for femv-gt -neutralizing antibodies a serum neutralization tests (snt) was established. briefly, cell monolayers of llc-mk -cells were used at approximately % of confluence in a -well microtiter format. feline serum samples were incubated at • c for min to inactivate complement components followed by the preparation of two-fold serial dilutions in pure dmem starting with a four-fold dilution. each sample was then was mixed with~ infectious viral particles (tcid ) of femv-gt -gordon strain in an equal volume of dmem followed by an incubation period of one hour at • c. this suspension was used for infection of llc-mk -cells for two hours at • c. samples were then replaced by dmem including % fbs, sodium pyruvate, non-essential amino acids and iu/ml penicillin-streptomycin. four days later, viral plaques were visualized applying the described immunofluorescence protocol (see section . ). each experiment was performed in duplicates. neutralizing titers were defined as the reciprocal of the serum dilution at which the number of plaques was reduced to % relative to virus-only controls. feline blood samples were obtained from healthy cats by venipuncture into vacutainer tubes containing edta as an anticoagulant (bd vacutainer ® , ml, beckton dickinson, heidelberg, germany). peripheral blood mononuclear cells (pbmc) were isolated as described previously by using histopaque- (sigma-aldrich, st. louis, mo, usa) [ ] . freshly isolated cells were resuspended in rpmi medium (without supplements) and infected with femv-gt-gordon strain, moi = . for one hour at • c. the infection medium was removed by centrifugation at × g for min, cells were re-suspended and further cultured for h at • c in a -well cell culture plate (greiner bio-one, kremsmünster, austria) at a density of million cells per ml. rpmi medium supplemented with % fbs, mm sodium pyruvate, % (v/v) non-essential amino acids and iu/ml recombinant human interleukin- (thermo fisher scientific, waltham, ma, usa) was used for cultivation. mock-infected pbmc from each animal served as negative controls and were treated similarly. after the indicated incubation period cells were centrifuged at × g for min and the cell pellet washed twice with pbs. for discrimination of dead cells in the consecutive flow cytometric analysis, femv-gt -and mock-infected pbmc were stained with fixable viability dye efluor (thermo fisher scientific, waltham, ma, usa) according to the manufacturer's protocol. subsequently, cells were incubated with % (v/v) goat normal serum (thermo fisher scientific, waltham, ma, usa) for min on ice to avoid non-specific fc binding. staining of surface markers was performed using fitc-anti-cat cd (clone vpg , bio-rad, herkules, ca, usa), alexa fluor ® -anti-human cd (clone h , bio-rad, herkules, ca, usa) and pe-anti-human cd (clone b , thermo fisher scientific, waltham, ma, usa). the corresponding isotype controls were all purchased from ebioscience. surface staining was performed by incubation of cells with fluorochrome-conjugated antibodies in facs-buffer (pbs with % fbs) for min on ice. stained cells were fixed for min at • c with % (w/v) paraformaldehyde. for intracellular detection of femv-gt , cells were permeabilized using . % saponin in facs-buffer for min at room temperature (rt). the virus was labeled by incubation with a rabbit polyclonal anti-femv-nucleoprotein antibody (generated in-house) at µg/ml (in facs-saponin-buffer for min at room temperature followed by two washing steps in facs-saponin buffer. finally, cells were incubated with a : dilution of pacific blue™-anti-rabbit antibody (thermo fisher scientific, waltham, ma, usa) in facs-saponin buffer for min at rt followed by two consecutive washing steps with facs buffer. the cells were acquired with a bd lsr fortessa tm flow cytometer (becton dickinson, heidelberg, germany) and viable cells were analyzed using the flowjo . . software (treestar inc., ashland, or, usa). the gating strategy is shown in supplementary files, figure s . to investigate neuronal tropism of femv-gt we established an in vitro model of the feline brain by generating organotypic slice cultures from cerebrum and cerebellum. brains from animals euthanized for medical reasons unrelated to this study, were dissected and the cerebrum was separated from the cerebellum. following preparation the tissue was immediately submerged in ice-cold hbss and slices were generated using a vt s (leica, wetzlar, germany) microtome. due to the difference in tissue rigidity, slices of the cerebrum were cut to µm and of the cerebellum were cut to µm thickness. freshly prepared brain slices were transferred to cell culture inserts ( . µm, merck millipore) pre-equilibrated with hbss and placed into -well cell culture plates (greiner bio-one, austria). for cultivation, minimum essential medium (mem) containing % (v/v) heat-inactivated horse serum, mm l-glutamine, mm hepes, mm hbss and iu/ml penicillin-streptomycin was used and changed daily. cultures were incubated at • c, % humidity and % co . after days of cultivation each slice was infected by direct application of ffu of femv-gt -gordon strain to the tissue. negative controls (mock infections) were generated by applying an equivalent volume of dmem with % fbs to the brain tissue. days after infection slices were washed with pbs and fixed with % (w/v) paraformaldehyde for min. slices were permeabilized with . % triton x- in pbs (tpbs) for min and blocked in tpbs with % horse serum and % bsa for min. the primary antibody raised against the femv-nucleoprotein was diluted ( : ) in blocking buffer and incubated with the samples for h at • c with constant agitation. after washing with pbs a goat anti-rabbit igg (h+l) cross-adsorbed, alexa fluor ® labeled secondary antibody (thermo fisher scientific, waltham, ma, usa) was diluted ( : ) in blocking buffer, applied to the slices and incubated for two hours. nuclei were counter-stained using dapi ( nm). after two subsequent washing steps with pbs the slices were visualized using a leica sp confocal microscope. as part of the german surveillance program for paramyxoviruses in domestic cats, urine samples from animals were analyzed using the described paramyxovirus consensus rt-pcr [ ] . six samples ( . %) were positive for a previously unknown paramyxovirus, tentatively named feline morbillivirus genotype (femv-gt ). based on physical examination and urinalysis findings all six animals were diagnosed with diseases of the urinary tract (i.e. acute or chronic kidney disease; table ). no viral rna was detectable within the available serum and edta-blood samples from these urine-positive animals. two cats ('gordon' and 'tv ') were sampled for several months and were found to be positive for femv-gt -rna by pcr within the urine at all time points tested (table ) . electron microscopy analysis and successful virus isolation from fresh urine samples of these two animals revealed that intact viral particles were released. interestingly, corresponding serum samples from 'gordon' and 'tv ' showed high titers of femv-gt -specific neutralizing antibodies (titer > ). abbreviations: ckd = chronic kidney disease; flutd = feline lower urinary tract disease; iris = international renal interest society; n.a. = not available; = no follow-up urine samples were available due to euthanasia, = no serum sample available, nab = neutralizing antibodies. to characterize this virus further, isolation attempts from fresh urine samples of affected animals (appropriate samples were only available from the animals 'gordon' and 'tv ') were conducted using vero, crfk and llc-mk cells. after the third passage cell culture supernatants were tested for the presence of paramyxoviruses using rt-pcr. we successfully isolated femv-gt strains from both animals. the results revealed that all three cell lines were susceptible to femv-gt with llc-mk cells being more permissive in comparison to vero and crfk cells. these findings were also confirmed by immunofluorescence staining targeting the femv-gt -nucleoprotein ( figure ). no visible cpe was induced on any tested cell line. initial virus titer on llc-mk cells was low at passage no. viruses , , of ( × tcid /ml), but could be enhanced to × tcid /ml after passage no. . virus titer remained low (~ × tcid /ml) on crfk and vero cells independent from the passage number (tested until passage no. ). both virus strains, femv-gt -gordon and femv-gt -tv , exhibit similar growth kinetics. as a consequence, virus stock was produced on llc-mk cells and was used for further infection experiments. mk cells being more permissive in comparison to vero and crfk cells. these findings were also confirmed by immunofluorescence staining targeting the femv-gt -nucleoprotein ( figure ). no visible cpe was induced on any tested cell line. initial virus titer on llc-mk cells was low at passage no. ( × tcid /ml), but could be enhanced to × tcid /ml after passage no. . virus titer remained low (~ × tcid /ml) on crfk and vero cells independent from the passage number (tested until passage no. ). both virus strains, femv-gt -gordon and femv-gt -tv , exhibit similar growth kinetics. as a consequence, virus stock was produced on llc-mk cells and was used for further infection experiments. to exclude contamination with other feline viruses, viral stock was concentrated via ultracentrifugation and tested for feline herpesvirus (fehv), feline coronavirus (fcov), feline calicivirus (fcv) and feline parvovirus (fpv) using specific pcr systems [ ] [ ] [ ] [ ] . none of the aforementioned viruses were found to be present in the femv-gt preparations. to visualize femv-gt virions, cell culture supernatants were semi-purified and examined by transmission electron microscopy. the observed viral particles showed typical paramyxoviral morphology represented by pleomorphic, enveloped virions sized between - nm (figure a ). to exclude contamination with other feline viruses, viral stock was concentrated via ultracentrifugation and tested for feline herpesvirus (fehv), feline coronavirus (fcov), feline calicivirus (fcv) and feline parvovirus (fpv) using specific pcr systems [ ] [ ] [ ] [ ] . none of the aforementioned viruses were found to be present in the femv-gt preparations. to visualize femv-gt virions, cell culture supernatants were semi-purified and examined by transmission electron microscopy. the observed viral particles showed typical paramyxoviral morphology represented by pleomorphic, enveloped virions sized between - nm ( figure a ). both animals. the results revealed that all three cell lines were susceptible to femv-gt with llc-mk cells being more permissive in comparison to vero and crfk cells. these findings were also confirmed by immunofluorescence staining targeting the femv-gt -nucleoprotein ( figure ). no visible cpe was induced on any tested cell line. initial virus titer on llc-mk cells was low at passage no. ( × tcid /ml), but could be enhanced to × tcid /ml after passage no. . virus titer remained low (~ × tcid /ml) on crfk and vero cells independent from the passage number (tested until passage no. ). both virus strains, femv-gt -gordon and femv-gt -tv , exhibit similar growth kinetics. as a consequence, virus stock was produced on llc-mk cells and was used for further infection experiments. to exclude contamination with other feline viruses, viral stock was concentrated via ultracentrifugation and tested for feline herpesvirus (fehv), feline coronavirus (fcov), feline calicivirus (fcv) and feline parvovirus (fpv) using specific pcr systems [ ] [ ] [ ] [ ] . none of the aforementioned viruses were found to be present in the femv-gt preparations. to visualize femv-gt virions, cell culture supernatants were semi-purified and examined by transmission electron microscopy. the observed viral particles showed typical paramyxoviral morphology represented by pleomorphic, enveloped virions sized between - nm ( figure a ). in addition, characteristic free nucleocapsids were found in all virus preparations varying between - nm in diameter ( figure b ). no differences in viral morphology were detected between samples originating from urine or from cell culture supernatants. to investigate the in vitro growth spectrum of femv-gt , several cell lines of different animal origin were tested for their susceptibility and efficiency for virus production. experiments were done with the llc-mk -adapted femv-gt -gordon strain at passage no. . as summarized in table , fcwf- and llc-mk cells were most susceptible to femv-gt , with~ % of the cells being femv-gt positive by ifa five days after infection. crfk, vero, marc- and bhk- were also susceptible, but to a lesser extent ( table ) in order to determine the phylogenetic relationship between already known feline morbilliviruses and the isolated strains from this study, whole genome amplification was performed. primers were designed from conserved regions of available femv sequences. femv-gt fragments were amplified and gaps were filled by applying the primer walking strategy. near-complete genome sequences of two femv-gt strains ('gordon' and 'tv ', accession numbers mk and mk ) from domestic cats were revealed. nucleotide sequences from strains were . % identical, differing in only nucleotides mainly located in the non-coding regions of the genome. the genome organization of femv-gt resembled that of classical femv strains, encoding six ( -n-p/v/c-m-f-h-l- ) open reading frames (orfs), typical for other morbilliviruses. pairwise alignments of the orfs between femv and femv-gt demonstrated nucleotide identities ranging between . % (phosphoprotein) and . % (matrix protein) and an overall whole genome nucleotide identity of about . % (table ) . amino acid conservation between femv and femv-gt was found to be higher than % for all structural proteins, except for the phosphoprotein where the amino acid identity of the femv-gt-gordon strain to femv was only % (table ) . phylogenetic analysis based on whole genome sequences demonstrated that femv-gt is genetically closely related to (figure ) , but yet distinct from, previously described feline morbilliviruses [ ] . therefore, we suggest that femv-gt should be considered as a different genotype of feline morbilliviruses. to elucidate the target tissues of femv-gt we established protocols for the isolation of primary feline cells from various organs of cats (see section . .) experimental in vitro infection was performed using the llc-mk -adapted femv-gt -gordon strain. we initially investigated primary cells from the urinary tract and successfully established a method for the preparation and cultivation of feline to elucidate the target tissues of femv-gt we established protocols for the isolation of primary feline cells from various organs of cats (see section . ). experimental in vitro infection was performed using the llc-mk -adapted femv-gt -gordon strain. we initially investigated primary cells from the urinary tract and successfully established a method for the preparation and cultivation of feline primary kidney cells as described in materials and methods. isolated cells represented a mixture (approximately %: %) of cytokeratin-positive epithelial cells and vimentin-positive non-epithelial cells. an in-depth analysis further showed that cells also expressed the renal collecting duct cell marker aquaporin- [ ] , the distal tubulus cell marker calbindin d k [ ] and the proximal tubulus cell marker alkaline phosphatase [ ] . hence, it was concluded that the isolated primary feline kidney cells express the terminal differentiation markers also present in naïve tissues and are therefore suitable for in vitro infection experiments. the cells were infected at an moi of . for two hours and thereafter cultured for five days prior to femv-gt nucleoprotein staining. mock-infected cells served as negative controls. we observed approx. % of primary feline kidney cells to be femv-gt positive, whereas no signal was detected in the negative control (figure ) , showing that primary feline kidney cells were susceptible to femv-gt infection in vitro. no cytopathic effects (e.g. formation of syncytia) or significant cell death in comparison to the mock-infection was detected. these observations were reproduced from cell preparations of three individual animals. the nature of the femv-gt -susceptible cell types was elucidated by cytokeratin staining (marker for cells of epithelial origin). renal epithelial cells were shown to be the main target of femv-gt (figure ) , although a minority (~ %) of non-epithelial cells were also susceptible to this virus. to elucidate the involvement of adjacent organs in virus shedding, primary feline bladder epithelial cells were isolated and infected with femv-gt as described above. as shown in figure , primary feline bladder cells were successfully isolated from clinical material and stained positive for cytokeratins as expected for cell types of epithelial origin. urinary bladder cells were shown to be viable, due to trypan blue exclusion, and due to the possible cultivation for at least three passages. in contrast to kidney cells, only a few urinary bladder cells were susceptible to femv-gt . this finding was reproduced from isolated urinary bladder epithelial cells of different animals (n = ). to elucidate the involvement of adjacent organs in virus shedding, primary feline bladder epithelial cells were isolated and infected with femv-gt as described above. as shown in figure , primary feline bladder cells were successfully isolated from clinical material and stained positive for cytokeratins as expected for cell types of epithelial origin. urinary bladder cells were shown to be viable, due to trypan blue exclusion, and due to the possible cultivation for at least three passages. in contrast to kidney cells, only a few urinary bladder cells were susceptible to femv-gt . this finding was reproduced from isolated urinary bladder epithelial cells of different animals (n = ). represent µm. to elucidate the involvement of adjacent organs in virus shedding, primary feline bladder epithelial cells were isolated and infected with femv-gt as described above. as shown in figure , primary feline bladder cells were successfully isolated from clinical material and stained positive for cytokeratins as expected for cell types of epithelial origin. urinary bladder cells were shown to be viable, due to trypan blue exclusion, and due to the possible cultivation for at least three passages. in contrast to kidney cells, only a few urinary bladder cells were susceptible to femv-gt . this finding was reproduced from isolated urinary bladder epithelial cells of different animals (n = ). to determine the capability of femv-gt to infect feline immune cells we isolated pbmc from healthy cats and performed in vitro infection experiments. cats were proven to be femv-and femv-gt -negative by immunofluorescence analysis of serum samples and by rt-pcr analysis of corresponding urine samples. intracellular virus in different immune cell populations was quantified h after infection by flow cytometry (figure ). the results of these investigations demonstrated that cd + t cells, cd + b cells and fsc hi monocytes are susceptible to femv-gt infections in vitro. the percentage of virus-positive cells varied from - % for cd + t cells and from - % for both cd + b cells and fsc hi monocytes ( figure a ). in contrast, cd + t-cells were less susceptible to femv-gt ( figure b ) with percentages of virus positive cells varying between - %. in addition, flow-cytometric analysis of femv-gt infected pbmc revealed that about % of non-t and non-b lymphocytes were also positive for femv-gt . these cell types likely represent natural killer (nk) cells, but due to a lack of specific antibodies to phenotype feline nk-cells this could not be confirmed. to determine the capability of femv-gt to infect feline immune cells we isolated pbmc from healthy cats and performed in vitro infection experiments. cats were proven to be femv-and femv-gt -negative by immunofluorescence analysis of serum samples and by rt-pcr analysis of corresponding urine samples. intracellular virus in different immune cell populations was quantified h after infection by flow cytometry (figure ). the results of these investigations demonstrated that cd + t cells, cd + b cells and fsc hi monocytes are susceptible to femv-gt infections in vitro. the percentage of virus-positive cells varied from - % for cd + t cells and from - % for both cd + b cells and fsc hi monocytes ( figure a ). in contrast, cd + t-cells were less susceptible to femv-gt ( figure b ) with percentages of virus positive cells varying between - %. in addition, flow-cytometric analysis of femv-gt infected pbmc revealed that about % of non-t and non-b lymphocytes were also positive for femv-gt . these cell types likely represent natural killer (nk) cells, but due to a lack of specific antibodies to phenotype feline nk-cells this could not be confirmed. due to the fact that other morbilliviruses infect epithelial cells of the lung, feline primary pulmonary epithelial cells were also used for femv-gt infection experiments. cells were probed with a pan-cytokeratin antibody to discriminate cells of epithelial origin, alveolar macrophages and fibrocytes. representative results of distinct experiments are shown in figure . due to the fact that other morbilliviruses infect epithelial cells of the lung, feline primary pulmonary epithelial cells were also used for femv-gt infection experiments. cells were probed with a pan-cytokeratin antibody to discriminate cells of epithelial origin, alveolar macrophages and fibrocytes. representative results of distinct experiments are shown in figure . no contaminating alveolar macrophages or fibrocytes were detected in these cell preparations via ifa analysis. it can be stated that the vast majority of the cultured cells represented pulmonary epithelial cells. these primary epithelial cells were infected with femv-gt -gordon strain at an moi of . for two hours and further cultured for five days before analysis. the experiments revealed that about - % of the cultured pulmonary epithelial cells were susceptible to femv-gt . all viruspositive cells were also cytokeratin-positive. no cytopathic effect (e.g. formation of syncytia) or significant cell death was observed. we were able to cultivate brain slice cultures from adult cats for at least four weeks. when infecting these cultures with femv-gt -gordon strain, it became evident that both the cerebrum and cerebellum were highly susceptible (figure ). no contaminating alveolar macrophages or fibrocytes were detected in these cell preparations via ifa analysis. it can be stated that the vast majority of the cultured cells represented pulmonary epithelial cells. these primary epithelial cells were infected with femv-gt -gordon strain at an moi of . for two hours and further cultured for five days before analysis. the experiments revealed that about - % of the cultured pulmonary epithelial cells were susceptible to femv-gt . all virus-positive cells were also cytokeratin-positive. no cytopathic effect (e.g. formation of syncytia) or significant cell death was observed. we were able to cultivate brain slice cultures from adult cats for at least four weeks. when infecting these cultures with femv-gt -gordon strain, it became evident that both the cerebrum and cerebellum were highly susceptible ( figure ) . interestingly, infected slice cultures of the cerebellum displayed small syncytia ( - nuclei per cell) ( figure b ). this finding was the first evidence that femv-gt can induce cytopathic effects to particular cell types as it is known for other paramyxoviruses. in contrast, cpe was not visible in femv-gt -infected slice cultures of the cerebrum ( figure d ). femv-gt -infected cells from the cerebrum and the cerebellum showed large amounts of nucleoprotein in the entire cell body and cell protrusions. all infected cells retained a typical morphology of brain cells. virus-positive cells grouped together in distinctive patterns so that virus transmission via cell-to-cell contact might have occurred. all described observations were verified on at least three slice cultures from the cerebrum and the cerebellum. the specific nature of the susceptible cell types (e.g. neurons, astroglia and microglia) could not be determined definitely, but staining of the brain slice cultures with a gfap-specific antibody (glial fibrillary acidic protein, marker for glia cells) showed no co-localization interestingly, infected slice cultures of the cerebellum displayed small syncytia ( - nuclei per cell) ( figure b ). this finding was the first evidence that femv-gt can induce cytopathic effects to particular cell types as it is known for other paramyxoviruses. in contrast, cpe was not visible in femv-gt -infected slice cultures of the cerebrum ( figure d ). femv-gt -infected cells from the cerebrum and the cerebellum showed large amounts of nucleoprotein in the entire cell body and cell protrusions. all infected cells retained a typical morphology of brain cells. virus-positive cells grouped together in distinctive patterns so that virus transmission via cell-to-cell contact might have occurred. all described observations were verified on at least three slice cultures from the cerebrum and the cerebellum. the specific nature of the susceptible cell types (e.g. neurons, astroglia and microglia) could not be determined definitely, but staining of the brain slice cultures with a gfapspecific antibody (glial fibrillary acidic protein, marker for glia cells) showed no co-localization of femv-gt -positive cells with gfap-positive structures. neurons could not be stained, due to the lack of specific feline antibodies. the family of paramyxoviridae is currently divided into seven distinct genera. the number of unassigned paramyxoviruses is still increasing because well-established classification schemes, like sequence comparisons based on the viral rna-dependent rna polymerase (vrdrp) or pairwise analysis of sequence complementarity (pasc) of all orfs is often connected to several difficulties among new paramyxoviruses [ ] . feline morbilliviruses (femv) were found to be clearly distinguishable from the classical morbilliviruses, such as cdv or mev based on phylogenetic analyses [ , ] despite the fact that the size and organization of their genome reflects properties as defined for the genus morbilliviruses. here, we describe a second morbillivirus isolated from domestic cats, tentatively named feline morbillivirus genotype (femv-gt ). this virus grouped together with previously described feline morbilliviruses (femv), but could be separated from these isolates based on whole genome sequences (figure ). amino acid identities between the femv-gt and the femv structural proteins ranged between - %. these differences are comparable to the species cetacean morbillivirus (cemv) where currently five strains from different animal species exhibit a similar genetic diversity as identified for femv and femv-gt in our study [ ] . in addition, electron microscopy revealed a similar morphology of the femv-gt particle as described for femv [ ] and other morbilliviruses. we observed a wider ribonucleocapsid protein (rnp) in femv-gt preparations, but this could be due to a preparation or staining artefact, but may also be an intrinsic feature of feline morbilliviruses. therefore, we provisional propose that femv-gt should be considered as a different genotype of femv. to clarify the taxonomic status of femv-gt more information about the genetic diversity of circulating strains is needed and should be addressed to future studies. the prevalence of femv-gt was low in urine ( . %), but this may be influenced by the storage conditions. urine samples were collected as part of the routine clinical work and for practical reasons they were stored for some weeks at - • c before rna extraction could be performed. thus, rna degradation may have occurred influencing the sensitivity of the pcr analysis. two cats were femv-gt -positive for several months till years, with virus shedding in their urine emphasizing that the virus is able to establish persistent infections. this is in accordance with published data about prolonged femv-infections in domestic cats [ , , ] . interestingly, virus secretion within the urine in two persistently infected cats ('gordon' and 'tv ', table ) continued despite high titers of femv-gt -specific neutralizing antibodies in the blood. this is in contrast to other morbillivirus infections were neutralizing antibody titers higher than or prevent virus secretion in cdv [ ] and mev [ ] , respectively. the reason for this phenomenon remains speculative at the moment, but can be the result of femv-gt replication in epithelial cells of the kidney, femv-gt escape mutants or impaired t-cell response in infected animals. all femv-gt -positive cats were affected with diseases of the urinary tract, but the number of positive cases was too small to suggest the virus as the cause of the observed illnesses. an alternative explanation could be that femv-gt can only replicate on already damaged tissues of the urotract. in addition, it is known that ckd is common in geriatric cats with prevalence ranging between - % [ , ] leading to the possibility of a statistical connection between femv-gt and ckd. to further elucidate whether femv-gt infection is a cause or effect of acute and/or chronic urotract diseases, controlled animal infection experiments are required. femv-gt was propagated in various kidney cell lines from primate, rodent and feline origin although no cpe was observed in any of the tested cell lines. this is in contrast to published results from femv as this virus is able to induce syncytia formation in crfk cells [ , ] . these discrepancies could be due to lower viral titers (~ -fold) of femv-gt in comparison to femv [ , ] or may be a result of the viral adaption process to cell line not capable of expressing the wild type entry receptor which might be necessary for inducing cytopathic effects as described for mev [ ] . we showed that feline primary kidney cells are susceptible to femv-gt infection in vitro and that epithelial cells are the primary target of femv-gt in our in vitro model. whether this virus location is also reflected in natural infections needs to be clarified by (immune)-histopathology in diseased or experimentally infected cats. feline urinary bladder epithelial cells were significantly less susceptible to femv-gt . the reproducibility of the results excludes the possibility that this observation was caused by the cat's individual medical histories. data generated from in vitro infection experiments might, however, differ from the in vivo situation. the cultivation medium used for primary kidney cells, for instance, might influence the expression of terminal differentiation markers and serial passaging of these cells can induce dedifferentiation of primary distal renal tubular cells [ ] . equally, primary bladder epithelial cells might not display their physiological surface markers, so-called uroplakins, when cultured in vitro [ ] . therefore, it is possible that the femv-gt tissue tropism observed in our in vitro experiments differs from the in vivo conditions. on the other hand, femv was shown to replicate in renal tubular cells in naturally infected cats [ , , ] , supporting our results for a kidney-associated femv-gt replication. although persistent infections with femv-gt are evident, none of the analyzed blood samples harbored detectable amounts of viral rna. this implicates that viremia is present-if at all-only for a short period of time. this observation is supported by previous investigations in femv-infected cats where viremia was not detectable in the vast majority of blood samples [ , , ] . there is only one report from a shelter in thailand that found femv to be present in the blood of animals [ ] . currently, the mode of transmission of feline morbilliviruses is unknown. related viruses (e.g. canine distemper and measles virus) establish infections in their susceptible hosts by transmission via the oro-nasal route. following an initial replication in resident immune cells (tissue macrophages or dendritic cells) these viruses infect different lymphocyte subtypes resulting in a short-term viremic state [ ] . here we show that femv-gt can infect several cells of the immune system in the peripheral blood including cd + t cells, b cells, monocytes and to a significantly lesser extent cd + t cells. feline pbmc were shown to be susceptible to femv-gt infection without prior stimulation supporting the assumption that the in vitro results reflect the in vivo situation. percentage and identity of femv-gt -positive immune cells resemble findings from in vivo infection experiments with cdv in a ferret model [ ] . similar results were also observed in a macaque model of mev-infections [ ] . recently it was also shown that femv can be detected in the spleen and lymph nodes in naturally infected cats [ ] . the in vitro infection experiments using primary feline pulmonary epithelial cells, as well as cells from the cerebrum and the cerebellum revealed that they are susceptible to femv-gt . whether alveolar macrophages may also contribute to virus replication in the lung could not be investigated in this study, due to the lack of specific antibodies for phenotyping feline alveolar macrophages. alongside infections of the lung, cdv and mev are able to infect and induce severe damage to the brain under certain circumstances [ ] . although the mechanism of how cdv and mev enter the brain is not completely understood, a leukocyte-associated virus transport as a source of hematogenous brain infections is hypothesized [ ] . our in vitro findings show that such a mechanism might also be responsible for the spread of femv-gt in the body of affected animals as brain slice cultures were susceptible to the femv-gt -gordon strain. although the specific nature of the affected cell types could not be determined definitely, it is likely that neurons are a target as gfap-positive cells (representing glia cells) were found to be femv-gt -negative in our in vitro experiments. these data revealed that femv-gt is able to infect feline brain cells integrated into a physiological tissue environment. however, this needs further investigation in controlled animal infection experiments. although the observed in vitro tropism of femv-gt is in line with naturally femv-infections [ , , ] and resembles the pathology of other morbilliviruses as cdv or mev [ ] it cannot be completely excluded that these are in part the result of viral mutations emerged through the cell culture adaption process. it is well known that vero-adapted mev strains acquired mutations leading to the usage of cd as a viral entry receptor which is not a characteristic of mev wild type strains [ ] . the possibility that femv-gt have gained mutations might explain our observation that viral titer increased about -fold on llc-mk cells between consecutive passages. on the other hand, titers remained low in crfk, vero and bhk- cells independent from the number of blind passages. therefore, raising titers in the first passages of llc-mk cells can also be addressed to a low concentration of augmentable virus in urine samples. in depth sequencing approaches of femv-gt -positive urine samples and the respective cell culture adapted viral isolates will help to interpret our in vitro findings in the context of viral pathogenesis and should be conducted in future studies. nevertheless, clinicians should be aware of femv-gt when respiratory or neurological signs are encountered. although the aforementioned organs are well known as infections sites for cdv and mev [ ] , they have not been characterized as target tissues for femv in the past. until recently, the feline kidney was focused on being the target of feline morbilliviruses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . our data clearly demonstrate that femv-gt can infect primary cells obtained from other organs and that the pathologic potential of femv and femv-gt might currently be underestimated. hence, further research is needed to elucidate the impact of feline morbilliviruses on animal health, especially for domestic cats. in summary, we isolated and characterized a new genotype of feline morbillivirus (femv-gt ) and showed the in vitro tropism to primary feline cells from the kidney, lung and the immune system, as well as to organotypic slice cultures from the feline cerebrum and cerebellum. our results indicate that veterinarians should be aware of this virus when symptoms of the aforementioned organs are encountered which cannot be explained by other reasons. part of the work reported in this manuscript resulted in the patent entitled "new paramyxovirus and uses thereof" with the international application no.: pct/ep / . the following are available online at http://www.mdpi.com/ - / / / /s , table s : primer sequences used for the amplification of nearly whole genome sequences of femv-gt strains. figure s : gating strategy used in flow cytometric analyses of pbmc. paramyxoviridae: the viruses and their replication complete genome sequence of a novel paramyxovirus, tailam virus, discovered in sikkim rats complete genome sequence of nariva virus, a rodent paramyxovirus genetic characterization of bank vole virus (bavv), a new paramyxovirus isolated from kidneys of bank voles in russia bats host major mammalian paramyxoviruses feline morbillivirus, a previously undescribed paramyxovirus associated with tubulointerstitial nephritis in domestic cats existence of feline morbillivirus infection in japanese cat 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morbillivirus receptors and tropism: multiple pathways for infection we thank grit müller and ines müller for assistance in sample collection and urinalyses. we thank sven reiche for providing cell lines from rodent and bat origin and uwe gerd liebert for providing access to the leica vt s microtome. the authors declare no conflict of interest. the founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. key: cord- - d yedn authors: chastant, sylvie; mila, hanna title: passive immune transfer in puppies date: - - journal: anim reprod sci doi: . /j.anireprosci. . . sha: doc_id: cord_uid: d yedn the puppy, born without immunoglobulins g (igg), acquires a passive systemic immunity thanks to colostrum intake during the two first days of life. the quality of passive immune transfer (i.e. blood igg concentration at two days of age), highly variable between litters and between puppies within litters, depends mainly on the time elapsed between birth and ingestion of colostrum, with limited influence of colostrum igg concentration. deficit in passive immune transfer, impacting puppy’s health and neonatal mortality rate, can be indirectly diagnosed through blood gammaglutamyltransferases assay and evaluation of growth rate over the two first days of life. in the absence of maternal colostrum, few homo- and heterospecific immune sources are available and canine colostrum banking remains the optimal solution. whereas passive immune transfer is crucial for survival during the neonatal period, it later interferes with response to vaccination. in addition to systemic passive immune transfer, maternal antibodies (mainly iga) would provide local (digestive) immunity, ensuring mid-term protection of the puppies’ gut together with probably long term training of the digestive immune system. neonatal period in the canine species, defined as the first three weeks of life, is a critical period with high risk of mortality: about % of all live-born puppies die between birth and days of age (mugnier et al., ) . survival rate during this period depends on the ability of the newborn to adapt to the extra-uterine life. once cardiorespiratory system has been able to switch from a placental to an aerial oxygen provision, the newborn dog goes through new challenges, both immune and nutritional. from the nutritional point of view, the circulation of nutrients via placenta is interrupted at birth; nutrients supply relies on implementation of new strategies by the newborn, including mammary gland approach, suckling and milk digestion. the immune situation of the newborn dog is critical, as the endotheliochorial structure of the placenta drastically limits transplacental transfer of macromolecules, including immunoglobulin g (igg), to the newborn's bloodstream. whereas exposed to high concentrations of infectious agents immediately after birth when delivered out of the uterus, puppies are born with very low systemic immunity, with mean serum igg concentration at about . g/l versus - g/l in the adult dog (poffenbarger et al., ; bouchard et al., ; chastant-maillard et al., ; mila et al., a) . transfer of passive immunity from dam to the offspring is thus essentially lactogenic in the canine species, colostrum ensuring both nutrients and immunity provision: at two days of age, mean serum igg concentration in the puppy rises up to - g/l, with - % of the immunoglobulins originating from the colostral transfer (pollock and carmichael, ; poffenbarger et al., ; schäfer-somi et al., a; greene and schultz, ; day, ; chastant-maillard et al., ; fig. ) . even when evaluated to its maximum (two days of age), immunoglobulins concentrations or specific antibody titers acquired by the puppy after colostrum intake remain lower than in the adult dog, reaching between and % of the maternal level (mila et al., a; gillespie et al., ) . unlike in the piglet or calf, the passive immune transfer (pit) was investigated in the dog only recently. the aim of this review is to present different factors influencing quality of the pit (defined as the serum igg concentration acquired at two days of age), to describe the impact of pit on puppies' health, and to comment on the alternatives to colostrum. physiology of pit in the dog and the methods of its evaluation will be also addressed. adequate pit is essential for the puppy as its low quality is associated with an increased risk of neonatal mortality: mortality between birth and three weeks of life in puppies with serum igg concentration at or below . g/l was . % versus . % in puppies with higher igg concentration (p = . ; mila et al., a) ; mean blood igg concentration at day of life was . g/l in puppies dying between day and vs . g/l for puppies still alive at days of age). the minimal protective level of igg concentrations are very different depending on species: - g/l in the foal, g/l in the calf and g/l in the piglet (weaver et al., ; cabrera et al., ; liepman et al., ) . in order to determine a real prevalence of the deficit in pit, data on large number of puppies from different kennel conditions still remain to be obtained. this deficit was diagnosed in one out of puppies ( %) according to gooding and robinson ( ) and in out of puppies ( . %) according to mila et al. ( a) . indeed, the prevalence of the deficit in pit varies strongly among kennels, but may vary also among breeds, as only out of labrador puppies ( . %) presented igg concentration below . g/l (unpublished data). in case of free suckling (uncontrolled by the breeder), quality of pit is strongly variable among litters, but also among puppies from the same litter, both evaluated via serum igg concentration (general immunity) and via cpv -specific antibody titer (specific immunity; fig. ) (mila et al., b) . fig. . pattern of immunoglobulins g, m and a concentrations in puppies' blood. n= beagle puppies from one kennel, with free suckling. mean ± standard deviation. elisa assay (method described in chastant-maillard et al., ) . heterogeneity of passive immune transfer (evaluated through blood igg concentration at day of age) inter and intra litter. n = beagle puppies from litters from one kennel, with free suckling. the horizontal line indicates the threshold defining failure of passive immune transfer ( . g/l). in litters , and , all puppies reached a sufficient passive immune transfer; in litter , all were in failure of passive immune transfer; within litter and , passive immune transfer was heterogeneous, some above, others below the threshold. although igg is not the only molecule with immune activity provided to the newborn via colostrum intake (see below), its absorption witnesses colostrum intake, making serum igg concentration assay at days of age the reference method for pit evaluation (poffenbarger et al., ; chastant-maillard et al., ; mila et al., a) . igg assay performed via an immuno-enzymatic method (elisa -enzyme-linked immunosorbent assay) is not an automated process and requires about six hours of laboratory work, limiting its use to scientific purposes. in practice, pit can be evaluated indirectly through blood gamma-glutamyltransferases (ggt) activity at two days of age: ggt activity in canine colostrum is ten times higher than that in the female blood, whereas ggt activity in puppies' serum at birth is almost absent. any increase of ggt activity in the newborn's serum is indicative of an ingestion of colostrum (center et al., ) , with an activity below u/l diagnosing a deficit in pit with a . % sensitivity and a % specificity (mila et al., b) . pit can be also evaluated via specific antibody titer. indeed, at two days of age, serum igg concentration and cpv specific-antibody titer are positively correlated, with concordant diagnosis of adequate pit in % of puppies; adequate pit was defined as igg concentration > . g/l for general immunity and cpv -antibody titer > : for specific immunity evaluation . easy-to-use, but also non-invasive, tests of the evaluation of the quality of pit remain still to be developed for the canine species. namely, all above mentioned methods require blood sampling of the newborn, in newfoundland puppy of mean birth weight of g, as well as in a g chihuahua (mean birth weight in france, a. mugnier, personal communication) . knowing that dog breeders are forbidden to draw blood samples in most countries, and taking into account the cost of veterinary consultation (ideally at the kennel, since speaking of two-day-old puppies) and the cost of the blood test itself, such blood screening cannot be implemented in practice. an easier and costless method of indirect pit evaluation is early growth monitoring, i.e. the percentage of weight gain from birth to two days of age, since colostrum provides together energy and immunoglobulins. a growth rate over the first two days of life below − . % allowed to identify the deficit of pit in - % of cases, both in terms of general (igg) and specific (cpv specific antibodies) immune transfer (table ; mila et al., ) . in any species, final blood igg concentration at two days of age depends on the quantity of igg ingested by the newborn and on the proportion of igg absorbed from the newborn gut into its bloodstream. three factors thus influence the quality of pit: i) the immunological quality of the colostrum (evaluated via igg concentration), ii) the volume of colostrum ingested by the newborn, and iii) the time elapsed between birth and colostrum ingestion. immunoglobulin concentrations in canine mammary secretions are elevated during the first two days post-partum compared with later in lactation, defining this period as the colostral phase in the canine species. three immunoglobulin classes are present in the canine colostrum (iga, igm and igg), ige remaining at undetectable concentrations (chastant-maillard et al., ) . iga represent between and % of total colostral immunoglobulins at the onset of lactation, becoming the principal immunoglobulin class in the mature milk (schäfer-somi et al., b; chastant-maillard et al., ) . as igm, iga are mainly produced locally by the lymphocytes of the mammary tissue (hurley and theil, ) . once ingested into the gut, iga participate locally in the immune defense mechanisms of the digestive epithelium. apart from this local role, a part of iga passes through the intestinal wall, being absorbed into the bloodstream and subsequently redistributed onto the mucous surfaces, digestive or not (salmon et al., ; chastant-maillard et al., ) ; they thereby participate for example in the immune defense of the respiratory tract. iga concentration in the colostrum is between five and ten times higher than in adult serum, whereas that of igm is much lower than in the serum, only about - % of the serum concentration (day, ) . igg, main actor of the systemic immunity, is the major immunoglobulin class of the colostrum ( - % of total immunoglobulins). only a very small proportion of colostral igg are produced by the mammary tissue, with the vast majority originating from the maternal blood. at the end of gestation, blood igg circulating in the maternal bloodstream are trapped into the mammary tissue and stored until lactation onset. the involvement of the fcγr n receptors (fragment, crystallizable receptor, neonatal) in the igg mammary storage remains unclear and has never been studied in the canine species (cervenak and kacskovics, ). the drop in blood progesterone concentration at parturition induces the increase of prolactin secretion and the onset of the lactation: igg are then released into the mammary alveoli lumen and excreted into the colostrum (hurley and theil, ) . mean igg table diagnostic value of growth rate over the first two days of life at or below − . % for the diagnostic of failure of passive immune transfer. n= puppies of various breeds within one kennel. hi: hemagglutination inhibition. i.e. - times higher than in the maternal serum (between . and . times depending on the dam). immunological quality of the colostrum is amazingly variable between bitches: mean concentration of colostrum calculated as the mean of concentrations obtained separately from the five pairs of mammy glands of an individual varies from to g/l, without any influence of the breed or litter size (mila et al., ) . age may have an impact, with colostrum of better immunological quality in females below six years of age than in older bitches. in addition to this huge inter-individual difference, colostrum igg concentration also varies for one given bitch among the different pairs of mammary glands, with a mean intra-individual coefficient of variation of ± %. for one given bitch, the highest and the lowest igg concentrations as assayed by mammary pairs differ in average by a factor of . (chastant-maillard et al., ) . however, when numbered (m anterior thoracic to m posterior inguinal), none of the five pairs of mammary glands secreted systematically colostrum of better (or worse) immunological quality. no practical recommendation can be thus drawn for dog breeders to encourage suckling of a specific pair of mammary gland to optimize pit. in the cow, posterior quarters produce colostrum of higher igg concentration (gross et al., ) , whereas in the sow, reported results are controversial (klobasa and butler, ; wu et al., ) . such a difference in colostral igg concentration could be due to differences in vascularization between the different pairs, or to different densities in fcγr n receptors. the eventual impact of such a variability in colostrum quality between teats on the quality of pit depends on the suckling behavior of the puppy, and more precisely on the choice of the teat. unlike piglets or kittens, puppies' siblings do not develop any competitive behavior for teat appropriation. in average, a puppy suckles ± teats over the first h of life (before the intestinal barrier closure), with m being the most often suckled (fig. ) . such multiple teat shifts contribute to erase the impact of the difference in colostral immune quality per teat. later, data obtained during the second and third day of lactation showed that a puppy suckles an average of . ± . teats per feeding session (arteaga et al., ) . to date, no method is available for igg concentration evaluation in canine colostrum in kennel conditions. refractometry, routinely used in the cow and mare, is not a reliable method of colostrum quality evaluation in the bitch (mila et al., ) . as no correlation between colostral and serum igg concentrations was evidenced in the bitch (chastant-maillard et al., ; mila et al., ) , prediction of the colostrum quality according to maternal blood igg level is neither possible. evaluation of the repeatability of colostrum quality along with lactations of one given bitch would be desirable for breeding selection purposes, but also to decide which neonates would require the administration of a colostrum replacer. nevertheless, no correlation has been demonstrated between mean colostrum igg concentration (mean value from pairs of mammary glands per bitch) and serum igg concentration in the newborn at two days of age in a study on bitches and their puppies from various breeds (aggouni, ) . no effect on the incidence of deficit of pit (see below) was either evidenced. according to a theoretical approach presented in fig. , a puppy receiving an adequate volume of colostrum at an adequate time (see below) would meet the passive immune transfer requirements (serum igg at or below . g/l) only if provided with colostrum with more than . g/l of igg. as only one out of the bitches tested by mila et al. ( ) had colostrum below this threshold, colostrum immunological quality does not seem to be a frequent limiting factor for pit in the canine species. gastric capacity in the newborn dog is estimated at four ml per g of body weight, with a mean gastric emptying time of two hours. however, the actual mean volume of colostrum ingested by the newborn dog remains unknown to date. based on calculation presented in fig. , the quantity of a colostrum of medium quality (igg concentration of g/l) necessary to achieve the minimum blood igg level ( . g/l) is . ml for each g of newborn body weight. the delay elapsed from birth decreases both the colostrum immune quality and the intestinal potential to absorb immunoglobulins. on the maternal side, colostrum igg concentration drops dramatically after whelping with a mean decrease of % ( ± %) between and h post-partum (albaret et al., ) . for the neonate's part, intestinal barrier closure is one of the main factors determining the quality of pit. histological description of this phenomenon, non available in the dog, is known in the bovine and porcine species. at birth, tight junctions between enterocytes as well as brush border of these cells are undeveloped. macromolecules, including immunoglobulins, can pass through digestive mucosa and be absorbed into the lymph and later into the bloodstream. this transport seems to be non specific and most probably independent from fcγr n receptors (cervenak and kacskovics, ). bioavailability of colostral immunoglobulins is highly due to a weak proteolytic activity of the digestive system at birth, immature digestive microbiota but also thanks to a high concentration of trypsin inhibitors in the ingested colostrum ( times higher than in the mature milk in the cow) (levieux and ollier, ) . as early as birth, over the first hours of life intestinal permeability decreases: puppies at birth absorb in average % of ingested igg, whereas absorption rate drops at % h later, and at only % h later. after the first - h of life, igg absorption rate is absent (chastant-maillard et al., ) . thus, in order to optimize pit, the colostrum intake must take place as early as possible, and in any case during the first h of life. spontaneous suckling behavior in early life is largely unknown in the canine species. recently, we observed during the first h of life five labrador litters with free suckling. in the included puppies, the first suckling was observed between five minutes and six hours after birth, with % of puppies suckling for the first time during the first two hours. over the first h of life, each puppy suckled during in average ± min ( % of the total duration of life) in sessions (viaud, ) . suckling behavior seems to naturally promote an adequate pit. after intestinal barrier closure in the newborn, serum igg concentration drops exponentially, with an igg half-life from . to . days. depending on their specificity, maternally derived antibodies (mda) persist until - days of life (gooding and robinson, ; pollock and carmichael, ; greene and schultz, ; mila et al., b) . in the dog, the mda concentration falls at to % of the initial level as early as days after birth (chappuis, ) . however, this drop of circulating mda tends to be accelerated in presence of pathogens in the environment or due to repeated vaccinations, by consumption within the immune response. immune complexes formed are then secreted into the intestinal lumen through fcγrn receptors (rath et al., ; greene and schultz, ) . the postnatal drop in mda seems breed-dependent, with shorter half-time in rapidly growing puppies (chappuis, ) . in parallel to the decrease in mda, the newborn dog is able to produce its own antibodies since birth, with a significant increase of antibody concentration visible as early as - days of age (fig. ) . [ ] with a blood volume equivalent to % body weight and a hematocrit of the newborn puppy at %, the total volume of serum within a puppy is . milliliters for g body weight ( g x % x ( -hematocrit)). [ ] the minimal serum concentration to be reached by the puppy at two days of age is . g/l, representing an absorbed igg amount of . mg ( . ×serum volume). [ ] the igg absorption rate was % in average between birth and h of life (chastant-maillard et al., ). an . mg absorbed amount corresponds to . mg ingested igg. [ ] a puppy ingests ml/ g body weight and performs meals over the period of intestinal permeabilty: circulating igg are thus absorbed from ml colostrum per g body weight. [ ] the minimal colostrum concentration is . g/l (ingested igg× / ). the quality of pit at two days of age correlates with the specific immunity level and thus with health status of the neonate. in a kennel with spontaneous cpv circulation, puppies with cpv -specific antibody titer above : at two days of age were longer protected against parvovirus infection and excreted cpv significantly later in the pre-weaning period than puppies with lower mda levels ( vs days of age, respectively; fig. ). mortality rates between and days of life were also significantly different in the two groups: % ( / ) and % ( / ), respectively (mila et al., b) . the survival rate over the entire neonatal period is thus markedly influenced by the quality of specific pit, with mda probably preventing the infection thanks to virus neutralization (mila et al., b) . although puppies with an adequate pit may not exhibit any clinical signs of infection, they may still actively participate in the virus circulation by shedding virus . they may put at risk some vulnerable puppies (i.e. with failure of pit), if introduced into a kennel naïve to cpv . indeed, mda persistence at the time of vaccination at about weeks of age is problematic to dog breeders. although puppies are able to produce antibodies in response to natural infection or vaccination since birth (and even during the fetal life), seroconversion occurs only in the presence of very low levels of specific mda (gooding and robinson, ; chappuis, ; toman et al., ; day, ) . a so called immunological gap appears, with remaining mda preventing the mount of a correct response to vaccination whereas no more providing puppy with adequate immune protection (decaro et al., ) . among puppies vaccinated against cpv between and weeks of age, eight puppies did not develop a protective response due to the presence of mda (thibault et al., ) . taking into account the late presence of colostral mda, a third vaccination at about weeks of age was implemented in into the international recommended vaccination protocol (wsava, ; day, ) . beside the transfer of mda, colostrum also ensures the acquisition of other immune compounds, suspected in the canine species based on information obtained in large animals. colostrum -and later, milk-contributes to local digestive immunity by iga, participating into the enteropathogens neutralization within the intestinal lumen. except iga, other nonspecific colostral compounds, such as lysozyme and lactoferrin, participate in the newborn immune response, even though they are considered of minor importance (handl et al., ) . in other domestic animals, white blood cells, such as macrophages, neutrophils and lymphocytes are other compounds of the pit: these cells are able to cross the intestinal barrier and enhance neonatal immunity during the first month of life (liebler-tenorio et al., ; langel et al., langel et al., , . they release iga locally when in contact with digestive pathogens (wheeler et al., ) . globally, colostrum increases the digestive immunity thanks to the presence of non-specific antimicrobial factors, controls the development of the digestive microbiota, modulate development of peyer's patches and digestive epithelium, participate in the adequate immune response. stimulation of the intestinal immune system (the most developed one of the entire organism) during the first days of life is essential to adapt the immune response and to limit infections, inflammatory diseases and allergies (kelly and coutts, ; rogier et al., ) . facing the marked unexplained increase in the prevalence of dysimmune diseases and inflammatory processes (including obesity) in the dog (sundburg et al., ; banfield, ) , the impact of systemic, but also digestive immunity established during the first days of life on the long-term health remains to be investigated. in some situations, colostrum is not available to puppies, or in limited quantities. in the absence of the dam, absence of maternal fig. . cpv -specific antibody titers (histogram) and fecal viral loads (squares at day , , of age). puppies (n= from various breeds within one kennel) were classified in two groups depending on their titer at day (hemagglutination inhibition): puppies with hi titer > : (black), puppies with hi ≤ : (grey). the protective titer is : . viral load was evaluated by rt pcr (significant above copies/ml). neocare unpublished data. methods are described by mila et al. ( b) . behavior, insufficient colostrum production according to the litter size and puppy's inability to suckle, an alternative to maternal colostrum should be administrated. an optimal colostrum replacer should provide puppies with an adequate energy but also immune supply, the last one making this nutritional solution a challenge for veterinary industries. numerous colostrum replacers are potentially available, homo-or heterologous, designed from mammary secretions, blood or egg products. only a few of them were scientifically evaluated regarding puppies' pit and/or health. besides the effect on the systemic pit, evaluation of the local digestive immune action, as well as its short and long term consequences on the health status of the supplemented animal would be of interest. whatever the type of colostrum replacer, it should be administrated, similarly as maternal colostrum, during the first eight hours of life, via a baby bottle or a feeding tube. although bottle feeding is a procedure closer to the physiological suckling, feeding tube allows to control the volume and the time of colostrum ingestion. dog breeders, often reluctant to tube feeding, should be encouraged to learn this technique, in order to deal with urgent situations and be able to increase chances of appropriate pit. . . colostrum/milk . . . canine colostrum frozen canine colostrum is to date the best alternative to maternal colostrum, as practiced in bovine and equine species. the optimal time for colostrum collection from the donor bitch is a compromise between the physiological drop in colostral immune quality after parturition ( % of the initial igg concentration as early as h post-partum) and the time required for the donor's own litter to acquire pit (intestinal barrier closure at - h of life). the donor bitch can thus be milked during its second day of lactation. preferably, the colostrum should be obtained from bitches housed in the same kennel, recently boosted with vaccines before whelping, and whose previous litters exhibited low mortality rates and high growth rate over the first two months of age. manual milking of a bitch is rather easy to proceed, but oxytocin injection may be helpful in some females ( - ui sc a couple of minutes before milk collection). after teats are cleaned and dried, colostrum is collected into - ml plastic tubes to be immediately stored at − °c. attention must be taken concerning the hygiene conditions at the collection, as the collected secretions are intended to be later administered to highly vulnerable individuals. in human medicine, the recommended maximum duration for frozen colostrum storage is months; one year in bovine and equine neonatology. thawing using microwave oven has to be avoided, as this process destroys immune potential of antibodies. it is rather recommended to thaw colostrum at °c, preferably using baby-bottle warmer or water-bath. after thawing, colostrum is administrated to the newborn dog at the dose of . ml per g of body weight. to date, colostrum banking is the best solution aiming to replace the maternal colostrum, providing the newborn with energy and immunity, but also with hormones and growth factors, even though white blood cells are destroyed. mean igg concentration in canine mature milk is - g/l, compared with g/l in the colostrum, and far below the minimal threshold avoiding failure of pit in puppies. no data are available in puppies, but no significant increase in serum igg concentration has been evidenced in kittens treated with mature milk of a donor queen (claus et al., ) . igg concentration in pseudopregnancy secretions ( . ± . g/l) was not significantly different from those measured in the colostrum ( . ± . g/l), but significantly higher than in the mature milk ( . ± . g/l). similarly, as for the colostrum, igg concentration may vary by a factor of . to . for one given bitch (abrard et al., ) . immune transfer obtained in puppies after ingestion of pseudopregnancy secretions is to be evaluated to validate their interest as colostral substitutes. bovine colostrum is an alternative source of antibodies, easily available in large quantities. however, its immune interest for the canine newborn, never evaluated, would be probably limited, as bovine colostrum is free from antibodies directed against major canine pathogens, such as antibodies targeting cpv . no canine immunoglobulins are present in industrial milks, modified from bovine milk. no pit can thus be expected from these formulas. despite containing antibodies directed against canine pathogens, canine blood products display igg concentration at about three times lower of that of the canine colostrum. in case of oral administration of canine serum to colostrum-deprived newborn puppies, the increase in their blood igg concentration was insignificant compared with that achieved after ingestion of maternal colostrum and below the threshold defining failure in pit (poffenbarger et al., ; bouchard et al., ; igg threshold at . g/l from mila et al., a) . oral administration of the canine plasma during the first eight hours after birth to puppies with free access to the dam and the colostrum did not allow to reduce the proportion of puppies at deficit of pit (mila et al., a) , but a tendency for a lower morbidity rate in supplemented puppies was observed (mila et al., a) . only parenteral administration, with associated risks, was suggested to allow pit: a subcutaneous injection of ml or ml/ g body weight at birth allowed to reach mean igg concentrations of respectively . and . g/l (bouchard et al., ) . however, some severe lesions have been reported after such a treatment: "impressive subcutaneous pockets of liquid" according to bouchard et al. ( ) until large (> cm) skin necrotic zones in our own experience. no study was conducted in puppies with administration of heterospecific serum for pit acquisition. in kittens, equine serum and purified equine antibodies given orally transferred no significant passive immunity (crawford et al., ) . besides the effects on systemic immunity, early supplementation (before the intestinal barrier closure) with canine plasma was associated to an increased diversity of microbial digestive communities, whose long term consequences remain to be explored (mila et al., a) . hens vaccinated against canine antigens produce antibodies targeting those antigens. these antibodies (called igy, for yolk immunoglobulins) are secreted in the egg yolk at high concentrations and can thus be easily available, in large amounts and in a non invasive way. benefits of cpv -specific and e. coli-specific igy on the canine neonate have been recently demonstrated, as large breed puppies receiving igy orally once before the intestinal barrier closure presented greater growth rate over the neonatal period (d -d ) than controls: ± g in igy supplemented puppies vs ± g in controls, n= puppies (mila et al., a) . such colostrum alternative containing cpv -specific and e. coli-specific igy is available for dog breeders (puppyprotech, royal canin, aimargues, france). igy supplementation is a strategy validated in other species (but after intestinal barrier closure; diraviyam et al., ) and promising for the canine newborns since igy directed against numerous canine pathogenic bacteria (as for example salmonella), viruses (canine coronavirus ccov, canine herpesvirus chv …) and parasites (giardia) could be produced. igy half-life in the puppy's bloodstream is unknown to date, but probably short, as the half-life of igy administrated to piglets at h of life was of . h versus days for homologous igg (yokoyama et al., ) . nevertheless, the effect of igy administration in the newborn probably relies not only on passive immune transfer, and its role on the digestive microbiota and digestive immune competences are to be explored. systemic passive immune transfer in puppies depends mainly on the time elapsed between birth and colostrum ingestion rather than on the immune quality of colostrum. while pit is recognized as essential for neonatal health in other species, such as bovine, equine and porcine, it is often neglected in the canine. scientific knowledge in this area is to be transferred to breeders, encouraging colostrum banking and early colostrum ingestion. controlled suckling in canine newborns could contribute to decrease neonatal mortality rate, improving financial situation in kennels and animal welfare. authors research on passive immune transfer in puppies was partially supported by merial (lyon, france) and royal canin r&d (aimargues, france). authors patented antibodies against various pathogens administered before h of age or between h and up to days of age in the dog to 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type infection in puppies by colostrum-derived antibodies immunoglobulin g concentration in canine colostrum: evaluation and variability natural and artificial hyperimmune solutions: impact on health in puppies indirect detection of passive immune transfer in puppies general and type parvovirus-specific passive immune transfer in puppies -evaluation by early growth low birth weight as a risk factor for early neonatal puppy mortality use of adult dog serum as a substitute for colostrum in the neonatal dog maternally derived immunity to canine parvovirus infection: transfer, decline, and interference with vaccination the immunologic functions of the neonatal fc receptor for igg lessons from mother: long-term impact of antibodies in breast milk on the gut microbiota and intestinal immune system of breastfed offspring humoral and cellular factors of maternal immunity in swine immunoglobulins in nasal secretions of dog puppies from birth to six weeks of age gonadectomy effects on the risk of immune disorders in the dog: a retrospective study evaluation of the impact of residual maternally-derived antibodies against canine parvovirus on the efficacy of a standard primary vaccination protocol postnatal development of leukocyte subset composition and activity in dogs le comportement de tétée du chiot et son implication dans le transfert passif d'immunité passive transfer of colostral immunoglobulins in calves immune components of colostrum and milk-a historical perspective guidelines for the vaccination of dogs and cats differential composition of proteomes in sow colostrum and milk from anterior and posterior mammary glands detection of passage and absorption of chicken egg yolk immunoglobulins in the gastrointestinal tract of pigs by use of enzyme-linked immunosorbent assay and fluorescent antibody testing authors want to thank all breeders and all veterinary students who participated in improvement of knowledge about passive immune transfer in the dog, and particularly charlotte aggouni, amélie albaret, morgane delebarre, leslie garrier, milène gonnier, barbara hanse, morgane mantelli, maelys martin, cynthia olivier, laurène plante, chloé robic, lisa rossig, camille viaud. key: cord- - dk authors: gulla, krishna mohan; balaji, arvind; mukherjee, aparna; jat, kana ram; sankar, jhuma; lodha, rakesh; kabra, sushil k title: course of illness after viral infection in indian children with cystic fibrosis date: - - journal: j trop pediatr doi: . /tropej/fmy sha: doc_id: cord_uid: dk objective: to study the clinical impact of respiratory viral infection in children with cystic fibrosis (cf). design: retrospective cohort study. setting: tertiary care referral centre for cf in india. participants/patients: children with cf attending a pediatric chest clinic. methods: case records of the children with cf who had a pulmonary exacerbation with documented acute respiratory viral infection between october and december (group i) and an equal number of controls (group ii) with pulmonary exacerbation in absence of acute respiratory viral infection were reviewed. outcome measures: the two groups were compared for the following outcomes over a period of – months: bacterial colonization, antibiotics usage, pulmonary exacerbations, numbers of outpatient visits, hospitalization and oxygen therapy and spirometric parameters. results: in total, children [ each with viral infection (group i) and without viral infection (group ii)] of age – months were enrolled; baseline clinical status and pulmonary function tests were comparable. mean (sd) follow-up duration in those who had viral infection and who had no viral infection was . ( . ) and . ( . ) months, respectively. on follow-up, children with viral infection (group i) had adverse outcome in form of greater worsening of shwachman clinical scores, number of pulmonary exacerbations requiring antibiotic usage [ ( . %)] and [ . ( . %)], need for intravenous antibiotics . % vs. . %, hospitalization rates . % vs. . % and mortality . % vs. . %, respectively. conclusion: acute viral infection in children with cf affected course of illness on follow-up, including frequent and severe pulmonary exacerbations requiring hospitalization, intravenous antibiotics, decline in cf scores and increased mortality over next – months. respiratory failure is the major cause of morbidity and mortality in patients with cystic fibrosis (cf); bacterial infections are generally thought to be the major cause of clinical deterioration [ ] . however, literature suggests that respiratory viruses do play an important role in pulmonary exacerbations, disease progression and an increase in bacterial adherence to the cf airways [ ] . even with appropriate use of antibiotic therapy, chronic obstructive airway disease continues to develop in patients with cf and is the major cause of morbidity and mortality. in children with cf, approximately half of the pulmonary exacerbations are associated with viral infections and are associated with worsening of spirometric parameters over time, especially in young children [ ] [ ] [ ] [ ] [ ] . if respiratory virus infections do lead to secondary bacterial infection in cf, there may be implications for the management of cf. hence, we conducted a retrospective cohort study to compare the effects of viral infection on the clinical course of children with cf over a period of months. in this retrospective cohort study, data were collected from the case records of cf patients attending the pediatric chest clinic of an indian tertiary care centre. cf was diagnosed based on typical clinical characteristics and two elevated sweat chloride levels on different occasions. nasopharyngeal aspirates/ nasal swabs of children with cf who presented with pulmonary exacerbations during october to december had been tested for viral pathogen using real-time polymerase chain reaction, pcr (fast-track diagnostics, luxembourg) as a part of another study done at the same institute [b. arvind, unpublished dissertation]. this pcr test could detect rhino, human corona, influenza a and b, parainfluenza, adeno, respiratory syncytial, human metapneumo and echo viruses. children with cf were routinely followed up in the pediatric chest clinic every months or earlier if indicated. the clinical information is recorded using a predesigned performa. children with cf whose nasopharyngeal aspirates/nasal swabs were tested and found positive for viral infection during an exacerbation were included as group i. children with cf and pulmonary exacerbation, whose throat swabs tested negative for viral pathogens were enrolled as controls after matching for age and sex (group ii). the children had been followed up every months or earlier, if needed. details of the follow-up visits during the months after throat swab collection were collected in a predesigned performa, which included the following: age; gender; weight; height at time of throat swab collection and during follow-up; bacterial colonization; intravenous and oral antibiotics usage; episodes of outpatient visits; hospitalization; percentage of predicted fev (forced expiratory volume in s), fvc (forced vital capacity), mmef (maximum mid-expiratory flow) and fev /fvc for the age and height; and clinical cf score. clinical cf score designed by shwachman and kulczycki [ ] was used to assess the clinical status of the children in follow-up. assessment of pancreatic insufficiency was done with history of frequent, foul-smelling and oily stools. written informed consent had been taken from parent/guardian before collecting respiratory specimen for viral detection in the earlier study [b. arvind, unpublished dissertation]. ethical clearance had been obtained from the institutional ethics committee for the current study. as we collected data from case record forms of children with cf who are routinely followed up in the pediatric chest clinic of the institute, consent for this retrospective study was not obtained from parent/guardian. data were analysed using stata ver. (college station, tx, us) and significance of difference of various parameters between groups i and ii were examined using student's t-test and mann-whitney test, according to the distribution of the parameter. weight for age and height for age z-score were calculated using the anthroplus software (who, geneva). the visit where throat swab for viral detection was collected was considered as the baseline visit for this study. multivariate analysis was performed using performing logistic regression model. twenty-three children with cf were included in each group. the baseline characteristics of the study children were comparable in both the groups and are presented in table . the distribution of viruses detected in the respiratory specimen in the enrolled children is shown in intravenous antibiotic usage and episodes of hospitalizations were significantly more in children who had a viral infection as compared with the others. mortality was also significantly higher in children in whom virus had been detected ( ; . %) as compared with those in whom no virus could be detected in respiratory specimen ( ; . %), p ¼ . (table ). in total, children in group and children in group could perform spirometry and parameters were shown in table . of the seven children who died in group i, rhinovirus had been detected in five, adenovirus in one and coronavirus in one during the pulmonary exacerbation evaluated for viral infections. the course of illness of children infected with rhinovirus was compared with those who were infected with other viruses (table ). in group i, three patients were newly colonized with pseudomonas aeruginosa and one patient was colonized with beta-haemolytic streptococcus. whereas in group ii, three patients were colonized with pseudomonas and two patients were colonized with staphylococcus aureus after virus isolation. allergic bronchopulmonary aspergillosis (abpa) was present in three children in group i and five children in group ii. one patient was home oxygen-dependent this retrospective cohort study demonstrated that cf children who had suffered a viral respiratory infection had worse outcome in follow-up in the form of lower cf scores, higher use of intravenous antibiotics, higher rate of hospitalization and higher mortality as compared with matched controls who did not have viral respiratory infection. respiratory viruses associated with the respiratory exacerbations of cf include influenza a and b, respiratory syncytial virus (rsv), parainfluenza virus (piv) types - , rhinovirus, human metapneumovirus, coronavirus and adenovirus. viral infections predispose the respiratory epithelium to secondary bacterial infections by multiple mechanisms resulting in frequent pulmonary exacerbations [ , ] . rhinovirus has been reported as major viral pathogen in cf with detection rates reaching up to % from samples collected both during exacerbations and routine visits [ ] . in our cohort too, rhinovirus was the most commonly detected ( . %) virus. in a prospective multi-birth cohort study by armstrong et al. [ ] on viral infection in infants with cf during infancy, children of ( %) were hospitalized for respiratory distress and of them had viral infection, and they concluded that viral infections are important cause of hospitalization in children with cf. abman et al. [ ] followed infants with cf over a period of . months (range - months); ( %) infants had been hospitalized times for respiratory distress. among these children, rsv was detected in infants. reports from earlier research on cf children have documented that patients with virus-associated infections had higher use of antibiotics at the longterm follow-up, compared with those without virusassociated respiratory tract infections [ , ] . in our cohort, usage of oral antibiotic was similar in both groups. but the requirement of intravenous antibiotics was significantly higher during follow-up of cf patients with viral respiratory infection compared with those who did not have any virus detected in the respiratory specimen ( . % vs. . %, p ¼ . ). in our cf patients with viral infection, of ( %) children required hospitalization for pulmonary exacerbation during their follow-up period, whereas only patient of ( . %) from the other group required hospital admission. hospitalization was higher in cf children in whom rhinovirus was detected as compared with other virus. over few decades, it has been presumed that viruses may be the harbinger of infection with typical bacterial cf pathogens. a temporal link was proposed between viral infections and new pseudomonas infection [ ] [ ] [ ] . there are currently limited data available to determine the effects of viral infection on the cf lung microbiota. in vitro studies have shown that rsv and influenza virus increase the adherence of pseudomonas to human airway [ , ] . it was also shown that rsv promotes growth of pseudomonas biofilms through modulation of the immune response and pathogen iron metabolism [ ] , while rhinovirus promotes the free movement of pseudomonas from biofilms [ ] . in contrast to what was believed, a recent study on dynamic interaction between respiratory viruses and the lung microbiota in children recently reported that viral infection had no impact on bacterial diversity [ ] . in our cohort, three children in each group were found to have new pseudomonas infection in follow-up, which suggests that viral infection may not be the only factor involved in colonization of pseudomonas in airway epithelium of cf children. viral infections in cf patients are associated with decline in pulmonary function. the majority of studies had shown declining trend of fev and fvc in long-term follow-up in those cf patients who had virus-associated lower respiratory tract infection [ , , ] . some studies like that by ramsey et al. [ ] showed no effect on pulmonary function tests (vital capacity (vc) or residual volume (rv)/total lung capacity (tlc)) in the follow-up of children with cf after viral infection. a recent review on clinical impact of respiratory viruses in cf suggests that high burden of viral respiratory infection in children with cf is likely to be linked with progression of cf lung disease [ ] . in our cohort, both the groups had shown decrement of lung function tests over the follow-up period with more decrement in group , though statistically not significant. it suggests that lung disease progresses over the period of time in these patients irrespective of viral infection. disease progression in cf is assessed through clinical data, chest radiography and tomography and pulmonary function tests. shwachman-kulczycki (sk) [ ] score is the most commonly used score to follow the progression of disease activity in cf children. we reviewed clinical data on general activity, physical examination and nutrition in our cohort. at the baseline, both the groups were comparable in above said three domains. however, at the last visit, children in group i had poorer scores in the domains of general activity, physical examination and nutrition as compared with group ii suggesting that viral infection may play a role in worsening of clinical status in children with cf. nevertheless, the subjectivity in the score needs to be kept in mind. apart from morbidity of cf caused by pulmonary exacerbations, the annual number of exacerbations has a considerable impact on survival also. in our cohort, . % of mortality was constituted by group i who had viral infection. this can be attributed to the severe pulmonary exacerbations requiring hospitalization in this group of children. in total, of ( . %) children who required hospitalization in group i (cf with viral respiratory infection) died by the end of follow-up period. in a predictive model, cf patients who required hospitalization more than twice a year were . times more likely to die within years [ ] . in a similar survival prediction model comprising data from cf patients selected from cystic fibrosis foundation patient registry, each acute pulmonary exacerbation within the first year of follow-up increased the risk of death by . times in the following years [ ] . in a prospective cohort study conducted on adult cf patients, more than two exacerbations per year had a greater risk of lung transplant or death during the study than those with less than one exacerbation per year (adjusted hazard ratio, . ) [ ] . all of our patients had at least moderate pancreatic insufficiency as suggested by presence of history of frequent, foul-smelling and oily stools. we also note that our patients were malnourished at baseline in both the groups along with low fev , which might have contributed to the high mortality in part. supportive care apart from antibiotics during pulmonary exacerbation plays an important role in the care of cf patients. in our clinic, we do advise and demonstrate regarding chest physiotherapy, inhalation therapy such as hypertonic saline ( % saline) and daily enzyme replacement therapy. however, we do not routinely use dnase, potentiators or correctors because of cost constraints. we also want to highlight the fact that all of our patients were pancreatic insufficient because patient with milder diseases were not suspected to have cf because of lack of awareness and belief that cf does not occur in indian children. strengths of the study include the use of highly sensitive assay for detection of virus and long-term follow-up of months in indian children with cf. the major limitation of the study is its retrospective design. the determination of viral aetiology of pulmonary exacerbation was done only at enrolment; subsequent exacerbations were not investigated for viral etiology. c o n c l u s i o n following the viral respiratory infections in children with cf who were already nutritionally compromised, there were frequent and severe pulmonary exacerbations requiring hospitalization, intravenous antibiotics, decline in cf scores and increased mortality over next - months. the seattle virus watch. ii. objectives, study population and its observation, data processing and summary of illnesses community study of role of viral infections in exacerbations of asthma in - year old children detection of viral, chlamydia pneumoniae and mycoplasma pneumoniae infections in exacerbations of asthma in children the role of respiratory viruses in cystic fibrosis the art and science of the use of antibiotics in cystic fibrosis severe viral respiratory infections in infantswith cystic fibrosis effects of upper respiratory tract infections in patients with cystic fibrosis the effect of respiratory viral infections on patients with cystic fibrosis respiratory viral infection predisposing for bacterial disease: a concise review rsv mediates pseudomonas aeruginosa binding to cystic fibrosis and normal epithelial cells long-term study of one hundred five patients with cystic fibrosis; studies made over a five-to fourteen-year period viral and atypical bacterial infections in the outpatient pediatric cystic fibrosis clinic role of respiratory syncytial virus in early hospitalizations for respiratory distress of young infants with cystic fibrosis prevalence and impact of respiratory viral infections in young children with cystic fibrosis: prospective cohort study respiratory infections in cystic fibrosis patients caused by virus, chlamydia and mycoplasma-possible synergism with pseudomonas aeruginosa seasonal onset of initial colonisation and chronic infection with pseudomonas aeruginosa in patients with cystic fibrosis in denmark adherence of pseudomonas aeruginosa to tracheal cells injured by influenza infection or by endotracheal intubation respiratory syncytial virus infection enhances pseudomonas aeruginosa biofilm growth through dysregulation of nutritional immunity rhinovirus infection liberates planktonic bacteria from biofilm and increases chemokine responses in cystic fibrosis airway epithelial cells insights into the respiratory tract microbiota of patients with cystic fibrosis during early pseudomonas aeruginosa colonization infective respiratory exacerbations in young adults with cystic fibrosis: role of viruses and atypical microorganisms association of respiratory viral infections with pulmonary deterioration in patients with cystic fibrosis the diagnosis and management of respiratory viral infections in cystic fibrosis developing cystic fibrosis lung transplant referral criteria using predictors of -year mortality predictive -year survivorship model of cystic fibrosis exacerbation frequency and clinical outcomes in adult patients with cystic fibrosis key: cord- -vd ctqro authors: hua, chen; zhu, yun; wu, congquan; si, lulu; wang, qian; sui, long; jiang, shibo title: the underlying mechanism of -hydroxyphthalic anhydride-modified bovine beta-lactoglobulin to block human papillomavirus entry into the host cell date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: vd ctqro we have previously demonstrated that -hydroxyphthalic anhydride ( hp)-modified bovine beta-lactoglobulin ( hp-β-lg) is highly effective in inhibiting entry of pseudovirus (psv) of high- and low-risk human papillomavirus (hpv) into the target cell. intravaginally applied hp-β-lg-containing vaginal gel could significantly inhibit hpv infection and reduce viral load in the cervical region. however, we still do not understand the underlying molecular mechanism by which hp-β-lg is able to inhibit hpv infection. here, though, we showed that hp-β-lg did not inactivate hpv psv, but rather blocked entry of hpv psv into the target cell via its interaction with virus, not cell. it bound to the positively charged region in the hpv l protein, suggesting that hp-β-lg binds to hpv l protein through the interaction between the negatively charged region in hp-β-lg and the positively charged region in hpv l protein, thus competitively blocking the binding of hpv to the receptor on the basement membrane in vaginal mucosa. although hp-modified chicken ovalbumin ( hp-ova) also carries high net negative charges, it exhibited no anti-hpv activity, suggesting that the interaction between hp-modified protein and hpv l protein relies on both electrostatic and matchable conformation of the binding sites in both proteins. when topically applied, hp-β-lg did not enter the host cell or blood circulation. these findings suggest that hp-β-lg targets hpv l protein and blocks hpv entry into the host cell, thus being safe and effective for topical application in the treatment of hpv infection. cervical cancer is the second most common cancer among women in china (chen et al., ) . almost all these cases result from the persistent infection of human papillomavirus (hpv) (bosch et al., ) . hpvs are a large viral family consisting of about different types (bernard et al., ) . among them, types have high oncogenic properties, and these are regarded as the high-risk types, such as hpv , hpv , and hpv . the high-risk types of hpv have relatively low self-clearance rate compared to the low-risk types of hpv (safaeian et al., ) . to prevent the infection of these highrisk hpvs and the occurrence of cervical cancer, several prophylactic vaccines, like cervarix, gardasil, and gardasil (deschuyteneer et al., ; mccormack, ) , have been developed and approved for marketing in many countries. however, no therapeutic vaccines or drugs have been approved for clinical use to treat hpv-infected patients. hpv is a non-enveloped, double-stranded dna virus. its capsid is made up of two proteins, major protein l and minor protein l . they are both involved in receptor binding and viral entry. analysis of the atomic structure of native t = hpv virus-like particle (vlp) reveals that l pentamers form the icosahedral shell of hpv (baker et al., ; li et al., ) . the l pentamer strongly binds to hpv receptors, such as heparan sulfate proteoglycans (hspgs), on the basement membrane to mediate viral entry and infection (kines et al., ) . the highly potent neutralizing antibodies elicited by hpv vaccines were found to bind to the l protein and prevent hpv infection (li et al., ) . thus, the l pentamer is regarded as the most important target for development of antiviral agents against hpv infection. viral entry inhibitors represent a class of antiviral agents that inhibit virus infection by blocking virus entry into the host cells. jiang et al. reported the first peptide-based hiv entry inhibitor, sj- , in (jiang et al., and the first hiv entry inhibitor-based anti-hiv drug, enfuvirtide, was approved by u.s. fda for clinical use in (dando and perry, ) . later, jiang and colleagues have reported that anhydride-modified proteins such as -hydroxyphthalic anhydride ( hp)-modified proteins are potent virus entry inhibitors against a number of enveloped viruses, such as hiv , herpes simplex virus (hsv) , and ebola virus (ebov) (li et al., ) . in , jiang's group reported that hp-modified bovine beta-lactoglobulin ( hpβ-lg) could also inhibit entry into the target cell of the pseudovirus (psv) of non-enveloped virus, hpv (high-risk hpv and hpv , and low-risk hpv ) (lu et al., ) . in the clinical trial, the topical application of vaginal gel containing hp-β-lg was proven to be very safe and highly effective in suppressing hpv infection and reducing viral load in vaginal mucosa (guo et al., a,b) . however, we still do not understand the molecular mechanism by which hp-β-lg inhibits hpv infection. in this study, we found that while hp-β-lg could not inactivate hpv psv, it was effective in inhibiting hpv psv entry into the host cell via its interaction with virus, not the cell. unlike β-lg, hp-β-lg could bind with l protein and the peptides derived from the positively charged region in the hpv l protein, suggesting that hp-β-lg inhibits hpv infection by binding, via its negatively charged region, to l protein, and thus competitively blocking hpv binding to the receptor on the basement membrane in vaginal mucosa, which then inhibits hpv from entering and replicating in the host cell. we have also shown that topically applied hp-β-lg does not enter the host cell and blood circulation. therefore, the hp-β-lg-containing formulations can be safely used in vagina for treatment of hpv infection and prevention of cervical cancer development. reagents -hydroxyphthalic anhydride (hp), , , -trinitrobenzenesulfonic acid (tnbs), human serum albumin (hsa), β-lactoglobulin (β-lg), chicken ovalbumin (ova), and bovine serum albumin (bsa) were purchased from sigma. horseradish peroxidase (hrp)-conjugated goat anti-human iga and ige antibodies were purchased from abcam (uk). hrp-conjugated rabbit anti-mouse igg antibody was purchased from dako (denmark). the hpv -and hpv- -l /l -expressing plasmids and luciferase pclucf plasmid were kindly provided by dr. john schiller at the laboratory of cellular oncology, national cancer institute, nih, md, usa. anhydride-modified proteins were produced as previously described (lu et al., ) . briefly, each kind of protein was dissolved with . m phosphate buffer (ph . ) at a final concentration of mg/ml. afterward, protein solutions were mixed with anhydrides (hp at m in dmso) to a final concentration of mm by the addition of five equal aliquots at -min intervals, while the ph was adjusted to . with m naoh after each mixing. all mixtures were kept at °c for two more hours and then dialyzed against pbs. the hpv pseudovirus was produced as previously described . briefly, t cells, which had been seeded in a -cm culture dish ( × cells) at h before transfection, were transfected with a mixture of hpv -l /l -expressing plasmid (p shell) and pclucf plasmid for hpv psv, or a mixture of hpv -l / l -expressing plasmid (p shell) and pclucf plasmid for hpv psv, using vigofect (vigorous biotechnology corp.). the cells were suspended in . ml of lysis buffer and incubated for h at °c with slow rotation. the lysate was cooled on ice for min, mixed with m nacl solutions to adjust the concentration of nacl to . m, and further kept on ice for min. then, the lysate was centrifuged at , × g for min at °c, and quantified for the concentration of l /l capsid protein levels. to remove aggregates, the pseudovirion stock was filtered before use for the neutralization assay. the frontiers in microbiology | www.frontiersin.org inhibitory activity of hp-β-lg against hpv psv entry into hela or hacat cells was detected as previously described (surviladze et al., ; kwak et al., ) . briefly, hela or heccatt cells were seeded at . × cells in μl of % fbs dmem (dmem- ) per well in a -well plate, followed by a culture at °c overnight. hp-β-lg or β-lg was serially diluted in dmem and incubated with μl of hpv psv (equal to ng l ). mixtures were added to hela or hacat cells and incubated at °c for h. after replacement of the culture supernatants with free medium and culture for additional h, cells were lysed for measurement of luciferase activity, according to the manufacturer's manual (promega, madison, wi, usa). hp-β-lg diluted in pbs was coated onto all wells of a -well polystyrene plate (corning, usa) at °c overnight. after blocking with protein-free blocking buffer (thermo fisher scientific, usa), hpv l (type or ) protein at μg/ml was added into the wells for μl/well, and the plate was incubated at °c for h. after washing with pbst three times, mouse anti-hpv l (type or ) antibody (abcam, uk) was added at a : , dilution. after incubation at °c for h and washing again, hrp-conjugated rabbit antimouse igg antibody (dako, denmark) was added for h. after washing and adding tetramethylbenzidine (sigma, usa), the absorbance was measured at nm. similar protocols were used for detection of hp-β-lg in the serum of rhesus monkeys and hp-β-lg-specific iga and ige in the vaginal swab eluates, which were leftover samples from the clinical trials for evaluating the in vivo safety and efficacy of the intravaginally applied hp-β-lg-containing vaginal gels (registry no.: chictr-trc- ) (guo et al., a,b) . measurement of the binding ability of -hydroxyphthalic anhydride modified bovine beta-lactoglobulin and beta-lactoglobulin to peptides two positively charged peptides of hpv l protein, l -i (residues - : lkakpkftlgkrkat) and l -ii (residues - : ststtakrkkrkl), were synthesized by a standard solid-phase fmoc method as previously reported (he et al., ) . the peptide solution ( mm) was prepared in mm phosphate buffer (ph . ) and added into the titration sample cells. hp-β-lg and β-lg were also dissolved in phosphate buffer to μm and then added to the titration needle. isothermal titration calorimetry (itc) analysis was carried out at constant temperature of °c with stirring speed of , rev/min. each titration volume was μl, and the titration interval was s for the first drop and s for the others. hela cells were plated in a -well plate (for . × /well) overnight. then, cells were incubated with hp-β-lg at a final concentration of μg/ml at . h before or , . , , , , , , , and h after addition of hpv psv (equal to ng l ), followed by an incubation at °c for h. after replacement of culture supernatant with fresh medium and an incubation for additional h, cells were lysed to determine the entry inhibition ratio, according to the manufacturer's manual (promega). hela cells in -well plates were preincubated with μl of hp-β-lg ( μm) or β-lg ( μm) at °c for h and then washed with dmem three times before addition of hpv psv. for the control, hp-β-lg-or β-lg-pretreated hela cells were not washed before hpv psv was added. hpv psv entry into hela cells was detected as described above. the plated hela cells and hp-β-lg mixture were both prechilled at °c for min. then, the cells were incubated with the hp-β-lg mixture at °c for h. after washing with cold pbs buffer twice, the cells were further incubated at °c for h. then the cells were lysed to determine luciferase activity, according to the luciferase assay system manual (promega, usa). the experiment on rhesus macaques was conducted under ethical guidelines and approved by the ethics committee of the institute of laboratory animal science, chinese academy of medical sciences and peking union medical college (approval number: ilas-vl- - ). in brief, the rhesus macaques were randomly divided into treatment group and control group. in the treatment group, vaginal gel containing hp-β-lg ( . mg per dose) was dispersed into carbomer gel and administered intravaginally or rectally. in the control group, only carbomer gel was applied. then, ml of blood was collected before or after injection at − h, h, min, h, h, h, h, h, and h. the serum was separated after condensation. to determine whether hp-β-lg could enter into hela or t cells, cells ( × ) were seeded onto coverslips in a -well plate. then, hp-β-lg ( . μg/ml) was added for h. then, the supernatant was replaced with dmem containing % fbs. after days, the cells were fixed by % paraformaldehyde (pfa; sigma-aldrich, st. louis, mo), perforated by . % triton x- , and blocked with % bsa (amresco, llc, solon, oh). next, cells were incubated overnight with anti- hp-β-lg mab or anti-β-actin mab ( : , ) at °c. after five washes, the cells were incubated with alexa fluor -labeled donkey anti-mouse igg ( , , , thermo fisher scientific, wilmington, de, usa) at room temperature for h. after five washes, the coverslips were sealed with prolong gold antifade reagent with , -diamidino- phenylindole (thermo fisher scientific) and scanned with the leica sp confocal microscope. frontiers in microbiology | www.frontiersin.org our previous studies have shown that -hydroxyphthalic anhydride-modified bovine beta-lactoglobulin ( hp-β-lg) is a promising hpv entry inhibitor against entry of psv of both high-risk and low-risk hpv types (lu et al., ) . this inhibitory activity was attributed to the interaction of the increased net negative charges on β-lg after -hydroxyphthalic anhydride modification (lu et al., ) . one may ask whether all proteins with increased net negative charges can inhibit hpv entry into the target cell. to answer this question, we compared the inhibitory activity of hp-β-lg with that of -hydroxyphthalic anhydride-modified human serum albumin ( hp-hsa) and -hydroxyphthalic anhydride-modified chicken ovalbumin ( hp-ova) against entry of hpv psv into hela cells, using the unmodified β-lg, hsa, and ova as controls. as shown in figure a , hp-β-lg exhibited potent anti-hpv activity in a dose-dependent manner with an ic (the half maximal inhibitory concentration) of . μg/ml, which is more potent than that of hp-hsa (ic : . μg/ml). hp-ova and all three unmodified proteins at the concentration as high as μg/ml showed no significant inhibitory activity against hpv psv entry into the target cell. these results confirm that not all, just some, proteins, such as β-lg and hsa, modified by -hydroxyphthalic anhydride gain the ability to inhibit hpv infection. in our previous studies, we proved the antiviral activity of hp-β-lg against entry of the high-risk hpv types and and low-risk hpv type into the target cell (lu et al., ) . aside from these three representative hpv types worldwide, another important high-risk type, hpv , is found with high incidence in china. to determine whether hp-β-lg is also effective against hpv , we constructed the hpv pseudovirus and tested its sensitivity to hp-β-lg (control: β-lg). as shown in figure b , hp-β-lg could also inhibit hpv psv entry into the target cell in a dose-dependent manner (ic of . μg/ml), confirming that hp-β-lg has broad-spectrum anti-hpv activity. considering that the hacat cells express abundant integrin and heparan sulfate proteoglycan (hspg), the receptors for hpv (aksoy et al., ; kumar et al., ) and act as target cells for hpv infection, we also evaluated the inhibitory activity of hp-β-lg against hpv and hpv psv entry into hacat cells. as shown in figures c,d frontiers in microbiology | www.frontiersin.org against entry of hpv and hpv psv into hacat cells, suggesting that hp-β-lg acts on the virus, rather than the target cell. bovine beta-lactoglobulin binds to the l protein and the peptides derived from the positively charged residue-enriched region in human papillomavirus l protein as we proposed before, hp-β-lg inhibited hpv psv entry into the target cell possibly through the binding of the negatively charged residues in hp-β-lg with the positively charged residues in a protein on the surface of the viral particle, thereby blocking the interaction between viral protein and receptor on the target cell (lu et al., ) . however, it is still unclear which protein on the virus surface may be involved in this interaction. the most abundant protein on the hpv particle is l protein. here we detected the interaction between hp-β-lg and hpv l protein by elisa. as shown in figures aa,ab, hp-β-lg could strongly bind to the l protein of both hpv and hpv , while the unmodified β-lg protein could not. the c-terminal positively charged region of hpv l was proved to regulate the binding of hpv to the receptor (sibbet et al., ; bousarghin et al., ) . this region was exposed on the viral surface, and involved in the binding to cell surface receptors or neutralizing antibodies. therefore, we synthesized two positively charged peptides in this region of l protein, l -i (residues - ) and l -ii (residues - ). the binding ability of hp-β-lg and β-lg to these peptides was measured by isothermal titration calorimetry (itc). as shown in figure b , β-lg had no obvious binding to l -i or l -ii peptide, while hp-β-lg showed high binding ability against both l -i (binding constant k α value equals . × /m) and l -ii (binding constant k α value equals . × /m). these results suggested that hp-β-lg may bind to the positively charged sites in the c-terminal region of l protein on the hpv surface to block the interaction between the viral particle and cell receptor. to determine whether hp-β-lg could inactivate hpv psv, we incubated hpv psv with hp-β-lg at room temperature for h and then removed hp-β-lg by peg- precipitation. as shown in figure a , the treated virus could still infect the target host cell with no reduced activity, compared the control virus (dmem-or β-lg-treated), suggesting that the binding between hp-β-lg and hpv psv may be noncovalent and reversible so that hp-β-lg cannot permanently inactive the virus. hpv entry into the host cell takes more than h, a relatively slow process compared to other viruses (aksoy et al., ) , which provides a long window period for an entry inhibitor to bind with the viral protein responsible for interaction with the receptor on the target cell and block viral entry. here, we performed a time-addition assay to pinpoint the window of functional hp-β-lg blocking of hpv entry into the target cell. as shown in figure b , hp-β-lg ( μg/ml) could fully inhibit hpv psv entry when it was added to the cells . h before and . , , , , and h after addition of hpv psv, respectively, while about , , and % of hpv psv entry were blocked when hp-β-lg was added to the cells , , and h after addition of hpv psv, respectively ( figure b ). this result suggests that during this relatively long period of entry process (aksoy et al., ) , the entry inhibitor hp-β-lg remains sufficiently active to inhibit hpv psv entry into the target cell. to confirm the functional stage of hp-β-lg in blocking viral entry, we used a temperature shift assay to slow down the viral infection process. at °c, hpv psv could bind to cell receptors, but could not fully enter the cytoplasm because the speeds of membrane fusion and cytoskeletal transformation are largely reduced at such low temperature. we incubated hpv psv and hela cells at °c for h so that hpv could bind to the cell surface. then we washed away the unbound hpv before shifting the cells to °c for further h of incubation. when hp-β-lg was added into the cells at the beginning and then removed by washing, no viral entry occurred (figure c ). this proved that hp-β-lg blocked the attachment between psv and cell receptor at the earliest stage of infection. to determine whether hp-β-lg could also bind to the receptor on the target cell surface to block hpv entry, we incubated hela cells with hp-β-lg (control: β-lg) at °c for h. the cells were washed with dmem to remove the unbound proteins, or not washed (as control), before addition of hpv psv. under the washing conditions, hpv psv entry into the hela cells was not blocked, while under the non-washing conditions, hpv psv entry into the hela cells was effectively blocked. no inhibition was observed in the β-lg control groups, no matter whether the β-lg-treated hela cells were lg was unable to inactivate hpv psv. hpv psv was incubated with hp-β-lg (control: β-lg) at room temperature for h and then separated by peg- to analyze its entry activity. the dmem-treated virus was taken as negative control. (b) hp-β-lg blocked hpv psv entry into the target cell. hela cells were incubated with hpv psv, while hp-β-lg ( μg/ml) was added at different time points (− . , , . , , , , , , , and h) to analyze the inhibitory activity. (c) hp-β-lg blocked hpv psv entry by targeting virus. temperature shift assay for anti-hpv activity of hp-β-lg (control: β-lg). diluted hp-β-lg or β-lg at μg/ml was mixed with hpv psv and prechilled at °c for min; then they were added to hela cells for h. after washing twice, the cells were further incubated at °c for h and then analyzed for fluorescent integrated density. (d) hp-β-lg inhibited hpv psv entry by targeting the virus, not the host cell. hela cells were incubated with hp-β-lg (control: β-lg) at °c for h with/without washing by dmem before addition of hpv psv. each sample was tested in triplicate, and the experiment was repeated twice. data from a representative experiment are presented as mean ± sd. asterisks represent significant differences. ***p < . . frontiers in microbiology | www.frontiersin.org washed or not washed ( figure d) . these results suggest that hp-β-lg inhibition of hpv psv entry into hela cells does not result from its binding to hpv's receptor on the host cell. bovine beta-lactoglobulin could not penetrate into the cell and the vaginally applied -hydroxyphthalic anhydride modified bovine beta-lactoglobulin did not enter into the blood circulation to determine whether intravaginally applied hp-β-lg could enter into epithelial cells, we incubated hela cells and t cells with hp-β-lg for h and then washed the cells three times using pbs. the cells were stained with mouse anti- hpβ-lg antibody or anti-β-actin antibody, as a control, and alexa fluor -labeled donkey anti-mouse igg. different from the anti-β-actin antibody-treated cells that showed strong fluorescence signals, anti- hp-β-lg antibody-treated cells displayed no significant fluorescence signals inside or outside the cells (figure ) . these results indicate that hp-β-lg could neither enter into the cell nor bind the cell surface proteins, including the receptor(s) for hpv. to investigate whether the topically applied hp-β-lg enters the blood circulation, a carbomer gel with or without hp-β-lg was topically administered in the vagina or anus of rhesus macaques. their blood samples were collected at different time points before and after gel application. the concentration of hp-β-lg in the blood samples and that in the gel (as positive control) were quantified with elisa using anti- hp-β-lg mab. as shown in figure , no hp-β-lg was detected in the blood samples of rhesus macaques vaginally or rectally administered with carbomer gel with or without hp-β-lg, confirming that the topically applied hp-β-lg does not enter the blood circulation. about - % of acute hpv infections become persistent, leading to various forms of cancer (shanmugasundaram and you, ) , such as the cervical cancer. although several multivalent prophylactic hpv vaccines have been used in clinics to prevent hpv infection and cervical cancer development, they have no effect against pre-existing hpv infection in middle-aged and elderly women, the population at high risk for cervical cancer. therefore, it is essential to develop effective and safe therapeutic agents for treatment of hpv infection in order to reduce the morbidity of cervical cancer. viral entry inhibitors have been proven effective and safe for the treatment of viral infections (jiang et al., ; dando and perry, ; lu et al., ; li et al., ; su et al., ) . we have previously reported that an hpv entry inhibitor, hp-β-lg, is highly effective in blocking the entry of hpv, both high-and low-risk types, into the host cell (lu et al., ) . in a randomized clinical trial, topical application of the vaginal gel containing hp-β-lg has shown excellent efficacy and safety to treat high-risk hpv infections (guo et al., a,b) . however, how hp-β-lg inhibits hpv entry into the target cell is still unclear. figure | hp-β-lg could not enter into the target cell. after incubation with hela and t cells, hp-β-lg in the cells was detected by immunofluorescence assay using anti- hp-β-lg mab (green). β-actin was used as positive control. cell nuclei were stained by , -diamidino- -phenylindole (blue). in our previous study, we demonstrated that the inhibitory activity of hp-β-lg on hpv psv entry into the target cell is closely correlated with the number of the positively charged lysine and arginine residues in hp-β-lg, as modified, i.e., the net negative charges on hp-β-lg (lu et al., ) , suggesting that the negatively charged residues on hp-β-lg play an important role in hp-β-lg-mediated inhibition of hpv infection. as shown in figure a , the unmodified bovine β-lg contains positively charged residues ( lys and arg) and negatively charged residues ( asp and glu) (qin et al., ) , thus having a net charge of − . in hp-β-lg, out of the lys and all arg were modified by hp at mm (lu et al., ) , resulting in the neutralization of of the positive charges. therefore, the net charge of hp-β-lg becomes − , making hp-β-lg be active in binding to a viral protein with high positive net charges and inhibiting virus infection. in this study, we demonstrated that hp-β-lg could strongly bind to the l protein on the hpv psv, while the unmodified β-lg protein could not (figure a) . using itc analysis, we showed that unlike β-lg, hp-β-lg could strongly interact with the positively charged residue-enriched c-terminal regions (residues - and - ) of l protein, which is responsible for the attachment of the hpv particle to the host cell, possibly through its interaction with the cellular receptor(s). one may argue that hp-β-lg may bind to any virus with high net positive charges on its surface protein and inhibit its infection. indeed, hp-β-lg can also inhibit infection of simian immunodeficiency virus (siv) (wyand et al., ) and hsv , but it is not effective against infection of mers-cov and vsv (unpublished data) . in this study, we showed that hp-modified hsa ( hp-hsa) was also effective, while hp-modified ova ( hp-ova) was not effective in inhibiting hpv psv entry (figure ) , even though hp-ova also contains high net negative charges on its surface. thus, aside from having high net negative charges, these results suggest that the active site in hp-β-lg for binding to the viral protein must possess a conformation that fits with that in the corresponding site in the viral protein (figure b) , enabling hp-β-lg to strongly and closely interact with the viral protein to block hpv entry into the host cell. indeed, the molecular docking analysis indicated that hp-β-lg could strongly bind to the positively charged region of hpv l protein, which is the binding site for the negatively charged hspg receptor for hpv ( figure b ). hp-β-lg has many more negative charges on the surface, and the interface between hp-β-lg and hpv-l is much larger than the hspg molecule. therefore, hp-β-lg can competitively bind to hpv l to block the binding of the hspg receptor. based on the results obtained from this study, we proposed a mechanistic model of hp-β-lg for inhibiting hpv infection in the cervical mucosa ( figure c) . the newly invaded hpv accesses the basal cell layer through micro lesions or breaks in the cervical epithelium and binds to the hspg receptor on the basement membrane through interaction between the positively charged region in l protein of hpv and the negatively charged region in hspg. this interaction results in a series of conformational changes in the capsid, which leads to protease digestion of l and exposure of its n terminus, thus initiating entry of hpv into to the epithelial cells (kines et al., ; pyeon et al., ) . after hpv infects the epithelial cell, it gradually matures along with the host cell cycles and moves toward the top layer of epithelia, followed by eventual release from the epidermal cells. the newly released a b figure | hp-β-lg could not enter into blood circulation when applied through the vagina or anus. (a) concentration of hp-β-lg in sera of monkeys through vaginal administration. (b) concentration of hp-β-lg in sera of monkeys through rectal administration. hp-β-lg was formulated in a carbomer gel ( . mg/each) and administered through the vagina or anus of rhesus macaques. their blood samples were collected at different time points before and after the application (− to h). hp-β-lg in the blood samples was analyzed by elisa. each sample was tested in triplicate, and the experiment was repeated twice. data from a representative experiment are presented as mean ± sd. the asterisks represent significant differences: *p < . ; **p < . ; ***p < . . frontiers in microbiology | www.frontiersin.org hpv particles access the basement membrane through micro lesions of the cervical epithelium and start another infection and replication cycle, gradually establishing persistent infection and promoting the development of cervical cancer. hp-β-lg that is topically applied in the vagina can bind to the l protein of the newly invaded or the newly released hpv and block the attachment of hpv to the basement membrane, thereby inhibiting the entry of hpv into the host cells for replication. after treatment with hp-β-lg for a certain period of time (e.g., months), the newly produced hpv-free epithelial cells in the lower part of the mucosal epithelium are expected to move upward and push the hpv-infected cells away from the vaginal mucosa (this movement is accelerated during the menstrual period), making all layers of mucosal epithelium free of hpv infection. considering that the presence of micro lesions or breaks in the cervical epithelium, possibly caused by sexual activity, is the requirement for hpv to access the hspg receptor on basement membrane (bousarghin et al., ) , combinational use of some biocompatible materials for mucosa wound healing, such as human collagen proteins (hua et al., ) , may have synergistic or complementary effects against hpv infection. the results from the time-of-addition assay, cell washout assay, and virus inactivation indicate that hp-β-lg could interact with the virions, rather than the cells, to effectively block the entry of hpv psv into the target cell, but could not inactivate the virions (figure ) . these biological properties of hp-β-lg make it an ideal anti-hpv agent for topical application to treat hpv infection because it can interact with hpv particles on the vaginal mucosa and block the virions binding to the receptor, e.g., hspg, on basement membrane. since hp-β-lg cannot enter into the cell or the blood circulation, it is not expected to cause systemic toxicity to human or induce harmful hp-β-lg-specific antibodies. therefore, the topical formulations containing hp-β-lg can be safely used to treat local hpv infection. the increased net negative charges on β-lactoglobulin after modification of the protein with -hydroxyphthalic anhydride. the structure of hp-β-lg was modeled by coot (emsley et al., ) using the crystal structure of β-lg (pdb entry: beb). the structures were shown as an electrostatic surface by pymol (alexander et al., ) . (b) predicted interactions between hpv l pentamer and hp-β-lg. crystal structure of hspg-bound hpv l pentamer (pdb entry: w o) was shown as electrostatic surface (dasgupta et al., ) , and the docking structure between hpv l and hp-β-lg was generated by autodock (trott and olson, ) . (c) schematic illustration showing how hp-β-lg inhibits hpv entry and infection. the newly invaded or newly released hpv particles access the basal layer through the mucosal lesion, or breaks, and bind to the hspg receptor on the basement membrane, resulting in the entry of hpv into and replication in mucosal epithelial cells. hp-β-lg can bind with hpv l protein, through the electrostatic interaction between the negatively charged region in hp-β-lg and the positively charged region in l protein, to block the attachment of hpv to the hspg receptor, resulting in inhibition of hpv infection in vaginal mucosa. all datasets generated for this study are included in the manuscript/supplementary files. the animal study was reviewed and approved by institute of laboratory animal science, chinese academy of medical sciences and peking union medical college (approval number: ilas-vl- - ). sj, lsu, and yz conceived and designed the experiments. ch, cw, lsi, and qw conducted the experiments. yz performed the computer modeling and analysis of the interaction between hp-β-lg and hpv l protein. ch, yz, lsu, and sj analyzed the data and wrote the manuscript. all authors have read and approved the final manuscript. this work was supported by an intramural fund of fudan-jinbo functional protein joint research center, fudan university. hpv infection of hacats is dependent on beta integrin, and alpha integrin processing hpv entry into cells bcl::cluster: a method for clustering biological molecules coupled with visualization in the pymol molecular graphics system structures of bovine and human papillomaviruses. analysis by cryoelectron microscopy and three-dimensional image reconstruction classification of papillomaviruses (pvs) based on pv types and proposal of taxonomic amendments the causal relation between human papillomavirus and cervical cancer positively charged sequences of human papillomavirus type capsid proteins are sufficient to mediate gene transfer into target cells via the heparan sulfate receptor cancer statistics in china structural basis of oligosaccharide receptor recognition by human papillomavirus molecular and structural characterization of the l virus-like particles that are used as vaccine antigens in cervarix, the as -adjuvanted hpv- and - cervical cancer vaccine features and development of coot safety evaluation of chemically modified beta-lactoglobulin administered intravaginally a randomized open-label clinical trial of an anti-hpv biological dressing (jb -bd) administered intravaginally to treat high-risk hpv infection. microbes infect conserved residue lys in the cavity of hiv- gp coiled-coil domain is critical for six-helix bundle stability and virus entry characterization by high-resolution crystal structure analysis of a triple-helix region of human collagen type iii with potent cell adhesion activity hiv- inhibition by a peptide the initial steps leading to papillomavirus infection occur on the basement membrane prior to cell surface binding intermediate heparan sulfate binding during hpv- infection in hacats impact of inhibitors and l antibodies upon the infectivity of diverse alpha and beta human papillomavirus types rational design of a triple-type human papillomavirus vaccine by compromising viral-type specificity the c-terminal arm of the human papillomavirus major capsid protein is immunogenic and involved in virus-host interaction chemically modified human serum albumin potently blocks entry of ebola pseudoviruses and viruslike particles chemically modified bovine beta-lactoglobulin inhibits human papillomavirus infection development of small-molecule hiv entry inhibitors specifically targeting gp or gp quadrivalent human papillomavirus (types , , , ) recombinant vaccine (gardasil®): a review of its use in the prevention of premalignant anogenital lesions, cervical and anal cancers, and genital warts bovine beta-lactoglobulin modified by -hydroxyphthalic anhydride blocks the cd cell receptor for hiv a herpesvirus inhibitor from bovine whey establishment of human papillomavirus infection requires cell cycle progression functional implications of structural differences between variants a and b of bovine beta-lactoglobulin determinants of incidence and clearance of high-risk human papillomavirus infections in rural rakai targeting persistent human papillomavirus infection alpha integrin is not the obligatory cell receptor for bovine papillomavirus type a peptidebased hiv- fusion inhibitor with two tail-anchors and palmitic acid exhibits substantially improved in vitro and ex vivo anti-hiv- activity and prolonged in vivo half-life essential roles for soluble virion-associated heparan sulfonated proteoglycans and growth factors in human papillomavirus infections autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading effect of -hydroxyphthaloyl-beta-lactoglobulin on vaginal transmission of simian immunodeficiency virus in rhesus monkeys we thank dr. jing xue at the institute of laboratory animal science, chinese academy of medical sciences and peking union medical college for performing the experiments on rhesus macaques; dr. john schiller at the laboratory of cellular oncology, national cancer institute, nih, md, usa for providing plasmids for constructing hpv psv; and yuhong fu for assistance in preparing anhydrate-modified proteins. key: cord- - gzont l authors: guo, nan; zhang, bingzhou; hu, han; ye, shiyi; chen, fangzhou; li, zhonghua; chen, pin; wang, chunmei; he, qigai title: caerin . suppresses the growth of porcine epidemic diarrhea virus in vitro via direct binding to the virus date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: gzont l porcine epidemic diarrhea (ped) has re-emerged in recent years and has already caused huge economic losses to the porcine industry all over the world. therefore, it is urgent for us to find out efficient ways to prevent and control this disease. in this study, the antiviral activity of a cationic amphibian antimicrobial peptide caerin . against porcine epidemic diarrhea virus (pedv) was evaluated by an in vitro system using vero cells. we found that even at a very low concentration, caerin . has the ability to destroy the integrity of the virus particles to block the release of the viruses, resulting in a considerable decrease in pedv infections. in addition, caerin . showed powerful antiviral activity without interfering with the binding progress between pedv and the receptor of the cells, therefore, it could be used as a potential antiviral drug or as a microbicide compound for prevention and control of pedv. alphacoronavirus of the family coronaviridae with single-stranded positive-sense rna [ ] . it is the causative agent of an acute infectious enteric disease known as porcine epidemic diarrhea (ped) that is clinically manifested by severe watery diarrhea, vomiting, and dehydration in the suckling piglets [ ] . with the high mortality in the piglets, ped infection finally caused enormous economic losses to the global swine industry, especially after its recent re-emergence caused by variant pedv strain throughout the world [ , ] . however, the classical ped vaccines could not provide appropriate protection against the variant pedv infection. considering this, relevant studies of new antiviral materials are needed to prevent and control emerging or re-emerging infectious diseases such as ped [ ] . antimicrobial peptides (amps) are important components of the nonspecific immune system of animals to eradicate invaders [ ] , and the skin secretion of anuran amphibians are rich sources for collecting amps [ ] [ ] [ ] . amps have a wide spectrum of antimicrobial activity against microorganisms such as bacteria, viruses, fungi, and parasites [ , ] , but are very friendly to host cells [ ] [ ] [ ] . moreover, amps can work as growth and health promoters to improve the performance of pigs by enhancing the immune status, improving the intestinal health, and alleviating the toxic effects of deoxynivalenol in pigs [ ] . there are also research findings that amps can modulate immune responses like chemokines, cytokine production, and pro-inflammatory responses [ , ] . caerin . is a peptide from the granular glands within the skin of the australian green tree frog with -residues (gllsv lgsva khvlp hvvpv iaehlnh ). nuclear magnetic resonance (nmr) of caerin . in the membrane mimetic environments showed that it has two α-helices and a flexible hinge region composed of two prolines [ ] . both prolines are essential for the antimicrobial activities [ ] . some studies have reported that caerin . exhibits antibacterial and antiviral properties by forming pores on the membrane to destroy the integrity of the particles [ , ] . meanwhile, it has been confirmed that caerin . has a complete inhibitory effect against hiv by preventing viral fusion to target cells and disrupting the hiv envelope, and, remarkably, that caerin . is also highly effective in inhibiting the transfer of hiv from dendritic cells (dcs) to t cells even when dcs are continuously exposed to peptides for h after virus capture, and that caerin . has a bacteriostatic activity against escherichia coli and bacillus subtilis [ , ] . although the mechanism that suppresses the activities of some bacteria and viruses has been illustrated clearly, it remains unknown whether caerin . can inhibit the growth of pedv. so, this study is aimed to investigate the function of caerin . . for this purpose, we investigated the inhibitory ability and antiviral mechanisms of caerin . against different pedv strains in vero cells, which will provide an insight into amps' antiviral mechanisms and its application as antiviral drugs or as drug loading compounds. vero cells (african green monkey kidney cell lines) were propagated at • c in a % co humidified incubator using dulbecco's modified eagle medium ((dmem), gibco, langley, va, usa) containing % fetal bovine serum (invitrogen, carlsbad, ca, usa). cells were infected with three different pedv strains: yn (accession no. kf ), cv (accession no. kt ), and dr (accession no. jq ). the yn strain ch/ynkm- / was isolated from a suckling piglet suffering from acute diarrhea. the cells infected with pedv strains were cultured in dmem supplemented with µg/ml trypsin. caerin . and n-terminus fitc labeled caerin . (purity: both > %) were chemically synthesized by bioyeargene (wuhan, china). caerin . was initially dissolved in . % acetic acid to reach the concentrations of mg/ml and mg/ml as stock solutions, respectively and stored at − • c until further use [ ] . the cytotoxicity of caerin . in vero cells grown in -well culture plates was assessed by mtt ( -( , -dimethyl- -yl)- , -diphenyltetrazolium bromide, mg/ml) assay. the cells were incubated with caerin . at different concentrations for h, then µl/well mtt was added and cultured for additional h. the supernatant was then removed and dimethylsulfoxide (dmso) was added to culture plates ( µl per well), then the plates were shaken at room temperature (rt) for min. finally, the optical density (od) value was measured at the wavelength of nm [ ] . the plaque forming assay was performed on vero cells cultured in -well plates. pedv ( pfu/well) was incubated using different concentrations of caerin . for h at • c before infection and then incubated pedv was added on vero cells covering % monolayer. after h of viral adsorption, the supernatant was removed, and the cells were rinsed with pbs and overlaid with µg/ml trypsin supplemented with sodium carboxymethyl cellulose-containing medium. the plates were fixed with % formaldehyde after h infection, and then stained with crystal violet solution [ ] . vero cells cultured in -well plates were washed with pbs for times and inoculated respectively with single medium, or single pedv, or pedv pre-incubated with different concentrations of caerin . . the cells were rinsed with pbs after h infection, the cells were fixed with % formaldehyde for min at rt. then the cells were incubated with an anti-pedv monoclonal antibody (made in our laboratory) for h and fluorescein isothiocyanate (fitc)-conjugated goat anti-mouse antibody ( : dilution) for h at • c. the immunofluorescence images were taken with a nikon eclipse ti microscope (nikon, tokyo, japan) [ ] . vero cells were cultured in -well plates and infected with pedv, and treated with caerin . at different time points. the cells were rinsed three times with pbs at h post infection (hpi), and treated with µl/well lysis solution containing protease inhibitors (pmsf). then µl of sodium dodecyl sulfate (sds) loading buffer was added to the cell extracts and the samples were boiled for min. the proteins were separated by % sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page) and then transferred into pvdf membranes (millipore, mississauga, on, canada). membranes were blocked with % skimmed milk for h at • c and then incubated with primary antibodies over night at • c. the blots were then incubated with corresponding horseradish peroxidase (hrp)-conjugated secondary antibodies (abclonal, wuhan, china). the membranes were washed times at each step. the protein bands were visualized using the clarity™ western ecl blotting substrate (bio-rad, hercules, ca, usa). the protein blots were quantified by image j software (national institutes of health, bethesda, md, usa). pedv-infected cell culture was prepared and centrifuged at • c for min at × g to get rid of the cell debris. the supernatants were ultracentrifuged at • c for h at , × g. then the purified virus pellets were re-suspended with pbs and treated with caerin . ( µg/ml) for h at • c and the viruses treated in the same manner were used as positive control. afterwards, the cocktail was dripped on the copper grid and negatively stained with phosphotungstic acid (pta). finally, the samples were examined using the electron microscope (hitachi h , tokyo, japan) [ ] . pedv suspensions containing different concentrations of caerin . were pre-incubated for h at • c, and were serially diluted before they were inoculated on the % confluent vero cell monolayers grown in the -well plates, followed by washing times with pbs. about hpi, the inoculates were removed and the cells were washed again with pbs for times and incubated for another days with dmem containing trypsin ( µg/ml). the infectivity was calculated by tcid (tissue culture infectious dose ) following the reed-muench method established by l. j. reed and h. muench [ ] . the vero cell monolayers were infected with pedv ( pfu). at hpi, the cells were washed twice with pbs and then treated with different concentrations of caerin . , and at hpi, the culture supernatants and the cells were collected respectively [ ] . the cell samples were frozen and thawed times to completely release the intracellular virus particles. both the extracellular and intracellular virus yields were determined by tcid . the vero cells in -well plates were inoculated with pedv in the presence or in the absence of caerin . at different time points: a. h before infection (pre); b. incubation of caerin . and pedv for h before infection (co); c. hpi (post). the supernatants and the cells were collected together at different time points and the samples were treated in the same way mentioned above in an infectious virus yield reduction assay. on the other hand, the inhibitory ability of caerin . against pedv was examined using a -fold higher concentration for further understanding antiviral activities in the pre and post treatment situations. finally, the cell lysates were collected in a parallel set for western blot assay. cells were pre-chilled at • c for h before infection and were chilled again after inoculated with pedv for h, then the cells were washed with ice cold pbs times, replenished with dmem containing trypsin, were incubated at • c for h, and the inhibitory effects were determined by tcid . caerin . was separately added at two time points: together with pedv at the time of viral absorption, and after the cells were washed at the time of viral entry to reach a highest final concentration of µg/ml. the cell lysates were processed in the same procedures with western blot analysis [ ] . vero cells were seeded onto some glass coverslips and were separately treated with fitc-caerin . ( µg/ml), pedv ( pfu), and both fitc-caerin . ( µg/ml) and pedv ( pfu), then incubated at • c for h. cells were fixed with % formaldehyde followed by permeabilization for min with . % triton x- , and were blocked with % bsa for h. the cells were washed times with pbs at each step. cell nuclei were counterstained with . % , -diamidino- -phenylindole ((dapi), invitrogen), and the antibodies used here were: pedv-s monoclonal antibody (made in our laboratory) and alexa fluor -conjugated affinity pure donkey anti-mouse igg (h+l) (ant gene, wuhan, china). the samples were examined using a confocal microscope (lsm meta, carl zeiss, munich, germany) [ ] . all experiments were performed with three independent experiments, and the calculated results were presented as mean ± standard deviation (sd). statistical analyses were performed using student's t-test. graph pad prism . was used to analyze the statistics in this study. the statistical significances were defined as p < . (*), and the higher significance was denoted by p < . (**) and p < . (***). the result of cytotoxicity assay indicated that cell viability increased with the decrease of the concentration of caerin . , that the viability of cells remained over % when treated with caerin . at the concentrations not exceeding µg/ml, and that the cell morphology was not affected even at the concentration µg/ml with a cell viability value of %, as seen in figure . thus, µg/ml caerin . was chosen as the highest final concentration in most of the subsequent experiments. we used µg/ml, µg/ml, µg/ml, and . µg/ml of caerin . in the ifa assay and in the addition assay. different incubating conditions of pedv in the presence of caerin . can directly affect the antiviral activity of caerin . . in this experiment, we determined the titer of pedv under different incubating temperatures and time durations. the results showed that the most suitable incubating temperature was °c, shown in figure a , and time length was h, shown in figure b . low temperature and inadequate incubation time would obviously affect the inhibition activity of caerin . against pedv. different incubating conditions of pedv in the presence of caerin . can directly affect the antiviral activity of caerin . . in this experiment, we determined the titer of pedv under different incubating temperatures and time durations. the results showed that the most suitable incubating temperature was °c, shown in figure a the results are presented as the mean ± sd of three independent experiments. the statistical analysis was performed using graph pad prism . . significance was defined as p < . (*), and higher significance was defined as p < . (**). to fully understand the inhibitory effect of caerin . against pedv-yn strain, several experiments were performed including cytopathic effects (cpe) observation, plaque reduction assay, tcid , and ifa. the cpe observation results showed that there were remarkable differences between the cells infected with pedv in the absence or presence of caerin . . caerin . -treated cells were in a relatively normal status, and the cpe appeared in caerin . -treated group about h later than in the pedv control group, as seen in figure a . with the addition of caerin . , the number of plaques declined at a surprising speed and the diameters decreased notably. similar results can be observed even with pfu of pedv treated by caerin . . the cells infected with pfu pedv exactly remained the same as the cells in negative control group, as seen in figure b . caerin . significantly inhibited the multiplication of pedv in a dose-dependent manner, demonstrating that pedv infection to vero cells was blocked to a large extent. the results of tcid , shown in figure c , and ifa, in figure d , indicated that the titer and the fluorescence intensity of pedv decreased significantly with the increase in the concentration of caerin . . to fully understand the inhibitory effect of caerin . against pedv-yn strain, several experiments were performed including cytopathic effects (cpe) observation, plaque reduction assay, tcid , and ifa. the cpe observation results showed that there were remarkable differences between the cells infected with pedv in the absence or presence of caerin . . caerin . -treated cells were in a relatively normal status, and the cpe appeared in caerin . -treated group about h later than in the pedv control group, as seen in figure a . with the addition of caerin . , the number of plaques declined at a surprising speed and the diameters decreased notably. similar results can be observed even with pfu of pedv treated by caerin . . the cells infected with pfu pedv exactly remained the same as the cells in negative control group, as seen in figure b . caerin . significantly inhibited the multiplication of pedv in a dose-dependent manner, demonstrating that pedv infection to vero cells was blocked to a large extent. the results of tcid , shown in figure c , and ifa, in figure d , indicated that the titer and the fluorescence intensity of pedv decreased significantly with the increase in the concentration of caerin . . the inhibitory effects of caerin . against another two different pedv strains were examined to evaluate the anti-pedv potential of caerin . based on ifa. as shown in figure , vero cells were infected with pedv ( pfu) pre-incubated with different concentrations of caerin . . compared with the virus control, treatment group exhibited excellent inhibitory effects in a dose dependent manner even at extremely low concentrations of caerin . . these results revealed that the intracellular viruses evidently reduced in the presence of caerin . in a dose dependent manner. groups (pedv) were not treated by caerin . . the results of three independent experiments are presented as the mean ± sd. the statistical analysis was performed using graph pad prism . . the significance was defined as p < . (*), and the higher significance was defined as p < . (**), p < . (***); (d) fluorescence intensity of pedv in the presence of caerin . at different concentration. the inhibitory effects of caerin . against another two different pedv strains were examined to evaluate the anti-pedv potential of caerin . based on ifa. as shown in figure , vero cells were infected with pedv ( pfu) pre-incubated with different concentrations of caerin . . compared with the virus control, treatment group exhibited excellent inhibitory effects in a dose dependent manner even at extremely low concentrations of caerin . . these results revealed that the intracellular viruses evidently reduced in the presence of caerin . in a dose dependent manner. to better understand the inhibitory effects of caerin . against the progeny virus production of pedv, we added caerin . at hpi to pedv infected cells. and both the intracellular and extracellular virus titers were determined separately by using tcid , as shown in figure a . the progeny virus titer decreased significantly as compared to that of virus control in a dose dependent manner, as seen in figure a . on the other hand, extracellular virus titers were also determined at different time points and different caerin . concentrations. the results showed that the increase of extracellular virus titer slowed down under the treatment of caerin . , as seen in figure b . incubated with caerin . at the concentrations of . (c), (d), (e) and (f) μg/ml for h. scale bar, μm. to better understand the inhibitory effects of caerin . against the progeny virus production of pedv, we added caerin . at hpi to pedv infected cells. and both the intracellular and extracellular virus titers were determined separately by using tcid , as shown in figure a . the progeny virus titer decreased significantly as compared to that of virus control in a dose dependent manner, as seen in figure a . on the other hand, extracellular virus titers were also determined at different time points and different caerin . concentrations. the results showed that the increase of extracellular virus titer slowed down under the treatment of caerin . , as seen in figure b . in the fluorescent confocal experiment, the fluorescence of caerin . and pedv around cell nucleus could be observed when we incubated them with vero cells respectively. additionally, the distribution of caerin . and pedv were almost the same, so the results of confocal laser scanning microscopy verified that caerin . could combine with pedv, and that the antiviral function of caerin . in the cell plasma could be observed especially in the areas showing severe cpe, as seen in figure . in the fluorescent confocal experiment, the fluorescence of caerin . and pedv around cell nucleus could be observed when we incubated them with vero cells respectively. additionally, the distribution of caerin . and pedv were almost the same, so the results of confocal laser scanning microscopy verified that caerin . could combine with pedv, and that the antiviral function of caerin . in the cell plasma could be observed especially in the areas showing severe cpe, as seen in figure . in caerin . -pedv pre-incubation process, the morphological changes of pedv particles were observed clearly. so caerin . could destroy the structure of the virus to block the infection of the host cells, as seen in figure a , resulting in the inhibition of viral release, which in turn could reduce the transmission of the progeny virus among the adjacent cells. as shown in the growth curve, pedv co-incubated with caerin . showed the lowest titer, while pedv was only slightly inhibited in preincubation process of caerin . -cells and the delayed usage of caerin . , as seen in figure b . in a western blot experiment, the protein band could barely be observed in pedv-caerin . co-incubation group. however, no obvious differences in pedv-n protein expression were observed among the cell-caerin . pre-incubation group, the post treatment group, and pedv control group, as seen in figure c . to further explore whether caerin . could bind to some virus receptors in host cells to interfere with the attachment and entry process, we kept the cells at °c to maintain the attaching status. caerin . did not show obvious inhibitory effects during the attachment and entry process. there were no significant differences in virus titers between the virus control groups and caerin . treated groups during both attachment period and entry period, even if the concentration of caerin . was increased tenfold, as seen in figure d . the western blot assay did not exhibit any obvious differences in protein expression among attachment period treated group, entry period treated group, and the pedv control group, shown in figure e , either. in caerin . -pedv pre-incubation process, the morphological changes of pedv particles were observed clearly. so caerin . could destroy the structure of the virus to block the infection of the host cells, as seen in figure a , resulting in the inhibition of viral release, which in turn could reduce the transmission of the progeny virus among the adjacent cells. as shown in the growth curve, pedv co-incubated with caerin . showed the lowest titer, while pedv was only slightly inhibited in pre-incubation process of caerin . -cells and the delayed usage of caerin . , as seen in figure b . in a western blot experiment, the protein band could barely be observed in pedv-caerin . co-incubation group. however, no obvious differences in pedv-n protein expression were observed among the cell-caerin . pre-incubation group, the post treatment group, and pedv control group, as seen in figure c . to further explore whether caerin . could bind to some virus receptors in host cells to interfere with the attachment and entry process, we kept the cells at • c to maintain the attaching status. caerin . did not show obvious inhibitory effects during the attachment and entry process. there were no significant differences in virus titers between the virus control groups and caerin . treated groups during both attachment period and entry period, even if the concentration of caerin . was increased tenfold, as seen in figure d . the western blot assay did not exhibit any obvious differences in protein expression among attachment period treated group, entry period treated group, and the pedv control group, shown in figure e , either. figure b . gapdh (glyceraldehyde- phosphate dehydrogenase) was used as a loading control; (d) tcid showed that caerin . had no obvious inhibitory effects during the attachment and entry process. caerin . was added at pedv attachment and entry periods, then the titers of pedv were detected; (e) western blot analysis of the expression level of pedv-n protein when caerin . was added in different ways as shown in figure d . gapdh was used as a loading control. pedv is one of the most important pathogens leading to diarrhea in pig industry. the aim of this study is to find whether caerin . has the antiviral activity against the three pedv strains and to reveal the antiviral mechanism of caerin . . according to published data, caerin . is a cationic peptide with two α-helices and has proved to be an effective agent against hiv by damaging the virus envelope and stopping the infection of hiv to t cells [ , ] . some α-helical peptides such as melittin and cecropin have been reported to exert antiviral activities by direct disruption of virus membranes or inhibition of the virus replication [ ] . the current study found that caerin . was able to destroy the structure of viral particles and reduce the viruses capable of infecting the cells and decrease their titers almost up to logs. this is the first attempt to reveal that caerin . has strong antiviral activity against pedv infection. figure b . gapdh (glyceraldehyde- -phosphate dehydrogenase) was used as a loading control; (d) tcid showed that caerin . had no obvious inhibitory effects during the attachment and entry process. caerin . was added at pedv attachment and entry periods, then the titers of pedv were detected; (e) western blot analysis of the expression level of pedv-n protein when caerin . was added in different ways as shown in figure d . gapdh was used as a loading control. pedv is one of the most important pathogens leading to diarrhea in pig industry. the aim of this study is to find whether caerin . has the antiviral activity against the three pedv strains and to reveal the antiviral mechanism of caerin . . according to published data, caerin . is a cationic peptide with two α-helices and has proved to be an effective agent against hiv by damaging the virus envelope and stopping the infection of hiv to t cells [ , ] . some α-helical peptides such as melittin and cecropin have been reported to exert antiviral activities by direct disruption of virus membranes or inhibition of the virus replication [ ] . the current study found that caerin . was able to destroy the structure of viral particles and reduce the viruses capable of infecting the cells and decrease their titers almost up to logs. this is the first attempt to reveal that caerin . has strong antiviral activity against pedv infection. some amps including cecropin d (cd-prrsv, salps-aiv) were reported to have blocked viral attachment and invasion by interacting with the receptors of the host cells [ ] . based on these findings, so we tried to keep pedv infection within the periods of the attachment and entry through the temperature control, and the results showed no evident differences in protein expression among attachment period treated group, entry period treated group, and the pedv control group. the number of virus infecting cells did not diminish. this means that caerin . did not interfere with the attachment and entry process. considering the fact that caerin . does not compete with the virus to conjugate with the cells, and the fact that viral attachment-entry processes are very fast and caerin . will not have enough time to act against the viral membrane, it can be concluded that caerin . has less significant antiviral activities in the pre-incubation of caerin . -cells progress. to further understand the mechanism in the post-treatment process, we examined the antiviral activity during the replication period. the results showed that caerin . did not have obvious antiviral effect during viral replication period, but it can control the infection progress by blocking the release of pedv particles to reduce viral transmission among the adjacent cells. antiviral activities of caerin . is expected to be improved through the combination usages with other different amps or other antiviral agents like graphene oxide (go), which could make caerin . a good weapon to deal with the diseases caused by viruses including pedv [ ] . as a matter of fact, we did a combination treatment with piscidin which was also proved to be effective against pedv [ ] , but the results were not as good as expected based on our unpublished work. the mechanisms still remain to be further investigated. although the joint use of piscidin and caerin . did not work as well as expected, other candidates can be explored for the prevention of pedv and other pathogens in drug combination studies. on the other hand, some related research work has been done in our laboratory. our other unpublished work has preliminarily testified the extraordinary ability of caerin . against some other viruses including prv (pseudorabies virus) which is a herpesvirus with double-stranded dna that can infect many mammal species. therefore, many potential functions of caerin . are still to be explored in the future. in this study, we investigated the antiviral activities of caerin . against pedv strains and its antiviral mechanism. the results show that caerin . can maintain the integrity of the host cells since it has very low cytotoxicity, and that it exhibits excellent virucidal activity in a dose-dependent manner even at very low concentrations. caerin . can reduce the viruses infecting the cells by destroying the integrity of the viral membrane, resulting in the decrease in the virus replication and protein expression. caerin . can also interfere with the virus release process essential for the inhibition of intracellular infection. therefore, caerin . is an excellent antiviral material against pedv. in conclusion, our study 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molecules for screening challenging species inhibition of porcine reproductive and respiratory syndrome virus by cecropin d in vitro antiviral activity of graphene oxide: how sharp edged structure and charge matter comparative pharmacokinetics and preliminary pharmacodynamics evaluation of piscidin against prv and pedv in rats antimicrobial peptide ifa indirect immunofluorescent assay nmr nuclear magnetic resonance mtt -( , -dimethyl- -yl)- , -diphenyltetrazolium bromide ped porcine epidemic diarrhea pedv porcine epidemic diarrhea virus tcid tissue culture infectious dose key: cord- -xjbz fw authors: ahmed, qanta a.; memish, ziad a. title: from the “madding crowd” to mass gatherings-religion, sport, culture and public health date: - - journal: travel med infect dis doi: . /j.tmaid. . . sha: doc_id: cord_uid: xjbz fw human behavior has long engaged in collective behavior assembling in crowds. the christian pilgrimage to the holy land has been recorded since the th century, while the hajj, islam's great pilgrimage, has existed for fourteen centuries, of which a body of literature devoted to the travelogues of the hajj has been recorded for over ten centuries. football is a sport played worldwide by more than . million teams and in , clubs. most however play outside of the officially organized sphere: more than percent of the global population plays football, including million amateur players. assembling for specific events is a uniquely human behavior, though the formal study of crowds did not begin until the mid-twentieth century. today mass gathering medicine focuses on the public health challenges to hosting events attended by a large enough number of people, at a specific site, for a defined period of time, likely to strain both the planning and response to the mass gathering of a community, state, or nation. all of us can recall attending a mass gathering, whether it be watching one's favorite rock group in performance or assembling for religious pilgrimage. certainly, the event itself is transporting and transforming and the unison of behaviors and activities can be enormously enriching, uplifting and overwhelming, just as much as they may be at times dangerous and high risk. this review seeks to draw contrasts and comparisons between sporting gatherings and religious gatherings with a chief focus on hajj, among the largest of all mass gatherings today. we will find there are some powerful similarities as well as stark differences. each bequeaths a legacy which can inform the other and, as we make our observations, we join with you and the legions of other investigators who continue to remain fascinated and enthralled by mass gatherings which are among the most beloved and beholden events of modern humanity. human behavior has long engaged in collective behavior assembling in crowds. built years bce, stonehenge in the british isles is thought to be the first monument and evidence of mass gatherings in the pre-historic era [ ] . numerous burial sites have been found at the now designated unesco world heritage site adding to the belief that it was a focus of spiritual energy and ritual. christian pilgrimage to the holy land has been recorded since the th century. the hajj, islam's great pilgrimage, has existed for fourteen centuries of which a body of literature devoted to the travelogues of the hajj has existed for over ten centuries [ ] . in , thomas gray's 'elegy written in a country churchyard' referred to the 'madding crowd's ignoble strife' at once holding forth the sobriety on common ordinary country folk in contrast to the madding crowd which drove men to disgraceful uncontrolled and even violent behavior captured in the timeless phrase [ ] . in charles mckay published his treatise on "extraordinary popular delusions and the madness of crowds" writing that 'men, it has well been said, think in herds; it will be seen that they go mad in herds, while they only recover their senses slowly, and one by one,". in , the great writer thomas hardy wrote a novel which still remains recognized as a masterpiece of english literature, "far from the madding crowd,' focusing on the lives of rural dwellers who were left behind by the madding crowd of industrialization. even today the 'madness of crowds' continues to fascinate and enthrall. assembling for specific events is a uniquely human behavior, though the formal study of crowds would not begin until the mid-twentieth century. the civil rights movements sweeping across the united states in the s and the crowds that cycles of violent lynchings had drawn prior, first captured the attention of behavioral scientists interested in public gatherings. psychologists referred to the 'collective mind' (first observed by thinker gustav le bon) that developed when large numbers of people gathered to be together, and the 'de-individuation' that followed as a result whereby not individuals, but the larger group determines actions accounting for violence destruction and even murder that could result [ ] . two schools of investigation developed, those focusing on disaster reliefthe public health needs of large number of people escaping or evacuating from a threat-and those who studied protest events. while many theories of behavior were proposed, researches remained mostly removed from the gatherings themselves and very little research was performed by direct observation of public gatherings themselves. at the time, modern travel had not yet permitted some of the massive scale of gatherings which have become commonplace since the late twentieth century. today mass gathering medicine focuses on the public health challenges to hosting events attended by a large enough number of people at a specific site for a defined period of time to strain the planning and response of a community, state, or nation. the definition is purposefully not linked to the size of the gathering or the number of people because each community has a varying capacity to manage crowds of people. all of us can recall attending a mass gathering, whether it be watching one's favorite rock group in performance or assembling for religious pilgrimage. certainly, the event itself is transporting and transforming and the unison of behaviors and activities can be enormously enriching uplifting and overwhelming, just as they may be at times dangerous and high risk. this review seeks to draw contrasts and comparisons between sporting gatherings and religious gatherings with a chief focus on hajj, among the largest of all mass gatherings today. we will find there are some powerful similarities as there as stark differences. each bequeaths a legacy which can inform the other and as we make our observations we join with you and the legions of other investigators who continue to remain fascinated and enthralled by the mass gatherings which are among the most beloved and beholden events of modern humanity [ ] . as this article goes to press, one of the most awaited mass gatherings in modern historythe fifa world cup is underway [ ] . the size of football is difficult to comprehend, nothing else compares in scale: football dwarfs the united nations, and even coca-cola, in international reach. across the globe, almost most five million referees, assistant referees and officials are directly involved in football, a sport played worldwide by more than . million teams and in , clubs. most people however play outside of the officially organized sphere. more than percent of the global population plays football, including million amateur players. . monotheisms, long congregating in mass gatherings, are on the rise most people are religiously affiliated. according to the pew research center report examining the global religious landscape published in december , over . billion people-adults and children-are religiously affiliated [ ] . at the time of this survey conducted examining more than sovereign nations and territories, this amounted to % of the world population. while religions diversity is panoramic, three theisms predominate: christianity, islam and hinduism. the pew study examined censuses surveys and other registers of population finding . billion christians ( % of the world's population), . billion muslims ( % of global population) and . billion hindus ( % of global population). a half billion identify as buddhist and million as jews-followers of judaism [ ] . some billion follow folk or traditional religions (including african traditional religions or chinese folk religions or native american religions). and, while well known, a tiny proportion-less than % of the earth's population-follow other religions including the yazidism, the bahai faith, sikhism, zoroastrianism and others. worth noting is that the religiously unaffiliated are themselves a group of over billion- % of the global population as populous as some leading theisms. the unaffiliated include atheists, agnostics and those declining to identify with any religion on surveys [ ] . yet the fastest growing population in the world is the muslim population-it is expected to increase by % in the next twenty years [ ] . by the global muslim population is expected to reach . billionrising more than twice the rate of the non-muslim population at a . % growth rate compared to . % annual growth rate for non-muslim populations. while slower than the . % annual growth rate of muslims in the past thirty years, by these calculations, muslims will account for . % of the . billion global population estimated by . while the muslim world is long familiar with hosting one of the world's largest mass gatherings-the hajj, islam's pinnacle of worship which centers on mecca in saudi arabia, and to where millions of muslims travel from within the muslim world to the saudi kingdom -a sporting event of the scale of the world cup, itself one of the world's larger mass gatherings is an unprecedented mass gathering event. among religious gatherings, it is also the best studied informing the public health risks of mass gatherings across the world. in some ways football, the beautiful game reaches the dimensions of a religion given its vast appeal and global reach and devoted followers. but it is useful to consider the world cup as a sporting event on a par with the summer olympics and the more recently developed para-olympics while the hajj as a mass gathering centering on islamic belief is better understood within the context of other mass gatherings centered on other theist belief systems including the hindu kumbh mehla, world youth day and other pilgrimages [ ] [ ] [ ] . christian mass gatherings include the world youth day, held every two or three years by the catholic church when almost a quarter of a million young catholics attend a mass gathering centered in varying cities for a live event anchored by the pope, first initiated by the late pope john paul ii in [ ] . world youth day will be next held in more well-known and rooted in centuries of observance may be the christian pilgrimage to lourdes in france, a year-round event drawing more than million in worship. lourdes, a small town in the southern pyrenees, itself has a population only of , yet because of the pilgrimage which has existed since , it is now the second most visited city in france, second only to paris. in the catholic world, it is the third most important site of pilgrimage after the holy land and rome [ ] . in asia, on one day, almost million catholics gather in christian procession for the feast of the black nazarene. the black nazarene is life-sized wooden statue of jesus christ crowned with thorns believed to have been brought from mexico to manila on a galleon in by spanish missionaries. the galleon that carried it to the philippines caught fire, but the charred statue survived and was named the black nazarene. devotees gather for the -h procession around the holy icon which followers believe delivers miracles. the world's largest mass gathering however occurs in india. the kumbh mela is held periodically over twelve-year cycles, rotating in four different indian cities-allahabad, nasik, ujjain and hardwar. over million hindus gather in each location at the banks of major rivers, including the ganges and the yumna in which pilgrims bathe as an act of worship on up to six important bathing days [ , [ ] [ ] [ ] . worshippers assemble at each location over a three-month period during which time the festival is held, alternating at the different locations four times in every twelve-year cycle. festivals go on for days in total. the bbc has reported that on a single day up to million worshipers have gathered simultaneously [ ] . these mass gatherings dwarf hajj, islam's pinnacle of worship centered on mecca, though both share the risk of human stampede [ ] . islam's hajj however is the best studied of all mass gatherings and has driven much of the scientific enquiry into the modern mass gathering. as many as three and a half million muslims from countries around the world gather for what is considered the final pillar in islam returning them to islam's birthplace in order to perform hajj. hajj is a series of week-long religious rites retracing the prophet mohammed's final visit to mecca during which he drew the lines of islamic pilgrimage which still stand today and delivered his final sermon revealing the final verses of the quran and sealing the foundations of islamic belief. even today, true to the prophet mohammed's final hajj, the modern hajj incorporates the prophet abraham's footsteps centuries before islam's inception and the legends of hagar, ishmael, adam and eve. with the rise in muslim population the hajj can be expected to grow in attendance and saudi arabia is preparing for these increased capacities investing in massive construction and infrastructure developments accordingly saudi arabia has committed $ billion in both land and infrastructure expansion with much of this work ongoing [ ] . the king abdul aziz international airport and the expansion of the haram sharif-the grand mosque surrounding the ka'aba-is to be completed / . 'haramain' a high speed above ground railway linking mina to medina, the second holiest city in islam (often included in hajj rites) will be completed this year but opened to the public after extensive testing in . the mecca-metro project connecting a four kilometer long station and two metro stations to the tune of $ . billion will be completed by the decade's end, greatly relieving congestion at the focal site of hajj. possibly the most beloved sporting events of all time are the olympics inspired by the athletic games held in ancient greece as far back as the th century bce. the modern olympics were conceived in with the formation of the international olympic committee which has governed every olympics since the games were first revived in [ ] . olympic games draw mass gatherings. the london olympics in drew million in attendance paying high prices for tickets. the more controversial and beleaguered rio olympics for sold only % of the million tickets available even though the tickets were significantly cheaper [ ] . regardless, the olympics, whether summer or winter and its counterpart, the special olympics, draw millions in attendance to watch the pinnacle of elite amateur sports. nation states win an intensely competitive bidding process to secure the event, which (like the fifa world cup) can define a nation and celebrate its culture. hosting the olympics is considered a rare privilege and nations, once winning the bid, work intensely during the seven-year lead time to the event to prepare both an appropriate legacy and also the necessary infrastructure for the events: olympic villages for the athletes, and the necessary response systems needed for health, security and disaster response. these are enormously costly events. perhaps it is the modern olympic games which have most inspired the foundations of studying medicine during sporting events physicians as david s jones noted physicians 'only slowly became interested' 'mostly in marathons' when the winner of the st. louis olympic marathon required four physicians in attendance after the race, due both to heat exposure and the pre-competition strychnine he had taken for performance enhancement [ ] . by , medical examinations for athletes had become mandated and in . the united states sent its first physician with its olympic team to paris. slowly medicine began to interface with sports, igniting the field of sports medicine for the elite athlete. almost a hundred years later, today all attendees-spectators and athletes alike-are known to be exposed to risks which could endanger health during these spectacular events and the science of mass gathering medicine has come into view informed by the experience of managing the public health at these huge events. equally prestigious and just as hotly contested is the fifa world cup held every four years. this year's fifa world cup in is being held in russia, and the subsequent has been awarded to qatar, in . interrupted only by world war two, the world cup has continued since its founding in held in uruguay, which, even then, drew more than half a million attendees. the brazil world cup in drew . million attendees. in all more than million people around the world have attended a world cup event yet today in the televised era it commands audiences of billions [ ] . the revenue generated by the fifa world cup is extraordinary -$ . billion during brazil -because of the cumulative television audiences watching all the matches and lucrative sponsorship dealsmore than . billion watched the world cup [ ] . audiences can only be expected to be bigger in the era of live-streaming, enabling anyone with an internet connection to tune in, sometimes to watch games more than once. because of its scale, the fifa world cup is the most viewed sporting event today. given its unique experience and unparalleled popularity, fifa has contributed important understandings to the management of mass gatherings in both public health and public security aspects. legacy building has been very critical to fifa and is a deal-breaking aspect of each competitive bid, and often a focus of criticism after events. the crowds at mass gatherings are a captive audience and vulnerable to environmental, physical, infectious and non-infectious hazards. they are also increasingly prime targets for terrorism including bioterrorism. whether a sporting or religious gathering these risks are shared though their nature may vary [ ] [ ] [ ] . both religious and sporting events can be at risk of stampedes which can evolve whenever crowds gather. while human stampede is lethal and devastating (forces of up to n/m can be generated by several people pushing in panic) with death resulting from acute venous hypertension, traumatic asphyxia, crush injuries to the torso and other catastrophic trauma, investigators in the field of human stampede note even in this modern era that there is a serious lack of high-level epidemiological data examining human stampedes. as a result, both experts and casual observers are mistakenly influenced by unbalanced media focus which trains its scrutiny on hajj each year and may be mislead in believing it is the only mass gathering at risk of stampede, and the single most frequent site of human stampedes. neither of these assumptions are true. hsu et al. searched both research and unconventional sources including media reports for evidence of human stampede incidents [ ] . [ ] . certainly, of the top ten most lethal human stampedes, seven occurred at religious gatherings, not exclusively the hajj but also other religious events. in india, % of all human stampedes occurred at religious festivals according to a recent exhaustive review [ , ] . non-religious mass gatherings were also the site of human stampedes including political gatherings, sports events, entertainment venues and spontaneous gatherings. in phnom penh in a shopping stampede on black friday (usually implying major sales) resulted in deaths [ ] . but most human stampedes occurred not in the arabian peninsula -home of the hajj-but in developing nations in africa and southern asia. together south asia and africa suffer over half of the world's human stampede. yet each year, media coverage focuses intensely on saudi arabian hajj sites (mecca, medina, muzdalifah and arafat) devoid of this context. when stampedes occur in the developing world, mortality is more than eight-fold greater than when the stampede occurs in advanced societies like saudi arabia. this increased fatality is a result of lack of planning, inexperience with crowd management and crowd control, scant emergency services, limited trauma care and infield emergency services and limited onsite and first responder communication capabilities. saudi arabia each year goes to intense effort to prevent stampede, one of many physical hazards associated with hajj and as a result stampedes have been few and rare. despite a spiritual reluctance to bar any eligible muslim from hajj, saudi arabia does limit hajj attendees through global visa quotas. also, once a local muslim in ksa has been to hajj, hajj visas cannot be issued for the next five years limiting frequent performers of hajj. yet the major improvements in hajj crowd management have been through engineering: the multilevel bridge at jamarat, site of the stoning ritual which has been a previous site of stampedes at hajj; the re-engineering of the pillars at mina into elliptical columns in place of cylindrical columns to dissipate crowd densities and reduce crowd turbulence from developing, and the development of massive pedestrian causeways which are color coded, one way, and temporally controlled to ensure steady throughput of pilgrims by controlling pilgrim ingress into these causeways through optimized schedules. movement of the hajj crowds in these areas of high density and high emotion -both risk factors for stampede-is monitored in realtime through video graphic analysis. intervention can be implemented real-time, which can be both life-and limb-saving. detailed video recordings achieved at hajj examining crowd dynamics are primarily the means of assessing the flow of hajj crowds. in the future, more sophisticated assessment is likely to be achieved in a number of additional ways-through fixed-laser scanning devices, closed circuit television and fixed gps monitors [ , ] . while a much smaller scale, human stampede at a british football match has been more exhaustively examined than any other stampede in history. the hillsborough stadium stampede was among the most disastrous events in football and examining its causes has been instructive if extremely painful for the football community and britain in particular [ , ] . the fa cup semi-final was held in the hillsborough stadium in sheffield on april th, drawing two intense rivals-liverpool and nottingham forest -for a contest place in the final for the fa cupthe biggest sporting event in british football [ ]. , liverpool fans traveled to sheffield for the event. authorities knew of the rivalries between both teams and because of the propensity for vandalism and hooliganism among fans on both teams, their entry to the stadium was deliberately segregated, as were the 'pens' designated for fans of each team. because of the bottleneck at the entrance and the large numbers of attendees, fans initially poured into two central 'standing only' pens-sections of the stadium demarcated by wire barriers, unaware that to either side of them thousands of empty seats were available. no one directed the congregating crowds to these more lateral areas on the right and left. as the time for kick-off was only minutes away, the chief superintendent (the police commander in charge of the event) allowed an additional gate to open as lines of liverpool supporters outside the stadium had built up. unaware that in the central standing pens crowd density was already high, the surge of additional people resulted in deaths-men women and children and more than injuries. all except one were liverpool fans. hillsborough remains the worst disaster in british sporting history. in , british jurors, delebrating for days (the longest inquest in british history)-emerged to convict the chief superintendent "responsible for manslaughter by gross negligence" due to a breach of his duty of care. the tragedy was compounded by the fact that the hillsborough match had been the first mass gathering under his command. this spiritual commitment which is of the utmost priority for them to complete hajj to the best of their abilities adding to a religious intensity and focused commitment despite harsh and difficult physical conditions imposed on the pilgrim whether by climate or congestion often in the face of sleep deprivation and other forms of self-denial required in the spiritual state of the hajjee. pilgrims are therefore vulnerable not only to their surroundings but to a fear of not completing hajj. stampedes are known to be triggered by fear, panic and as many investigators have noted, even by rumor. live surveillance of hajj crowds is vital to help pilgrims achieve their rites safely. hajj authorities can assess mounting crowd densities, blockage of foot traffic, bottlenecks and dangerous nascent crowd turbulence which can precipitate human stampede. supervision of the crowds at hajj is unlike any other mass gathering in the world [ , ] . interagency communication between various authorities overseeing the hajj (security forces, civil defense and special forces experienced in crowd control) and is continuous and announcements through public communication can be made if needed. sms capable networks (instant messaging via cellphones) are also available and have been used for urgent health messaging to communicate to the vast numbers of attendees simultaneously. because the hajj is so trying and muslims, enjoined by the maker, remain so committed to completing it peacefully and without infringement or desecration of anyone else's efforts in pilgrimmage, the hajj crowd works informally together, shifting to accommodate the weak, the vulnerable, the disabled who are in the teeming crowds next to them. in this way hajj, like other religious mass gatherings, is infused with a collective spirituality that can be of enormous public health benefit. further, this spirit of protecting the entire muslim community is clearly deleinated in the teachings of islam to be a metaphor for life beyond hajj for all the world's muslim community-a reminder that we must live together in harmony to peacefully collaborate and support one another through hardship and vulnerability. additionally, these values greatly enhance the pilgrim populations' receptivity to public health planners and on site security in ensuring disaster management and crisis aversion can be achieved with as much cooperation as possible. unlike religious gatherings, mass gatherings connected to sport or music events can be complicated by the availability of alcohol, recreational and illicit drugs, all of which impede the ablity of an individual to remain safe within the mass gathering and impede the ability of the crowds to behave protectively towards the vulnerable [ ] . these events are usually open air, often held in undesignated locations (particularly when considering electronic dance raves and may not benefit from experienced professional organizers). sometimes events are held in underground locations which were never designed to accommodate such capacities. participants are generally younger in age than those attending religious gatherings or the diverse ages seen in attendance of sports gatherings. music festivals, particularly the electronic dance movement event, are increasingly associated with intoxication and injuries [ ] . targeting young people aged to they draw large crowds, sometimes in unregulated venues, but increasingly today in purpose-built locations. alchohol overuse and recreational drug use are commonly associated with these events. both mdma ( , methylenedioxy-n-methylamphetamine) and the notoriously named date rape drug (gamma hydroxybutyric acid) are liberally used at these events. lund et al. report that in a fifteen year period deaths were recorded of drug related overdose at electronic music dance events. violence at such events has also been recorded with participants reporting stabbingssome severe enough to result in tube thoracostomy. matters are worsened by the fact that many participants self report ' preloading'drinking alcohol prior to entry to the event, a common practice at american events which can occur during 'tailgating'when americans often picnic around the trunk of their cars in the parking lots of the venues. while for most american families this means an innocent meal of hamburgers and hot dogs, for youngsters attending rave events this could mean 'preloading' particularly if attendees to the raves are under legal drinking age, adding to their vulnerability. excessive alcohol consumption with or without recreational drug use increases the risk of injury, sexually transmitted disease and extreme behaviors like the recently fire jumping and more traditional risks of sexual assasult. while the focus of this paper has been primarily planned mass gatherings, mass gatherings can erupt spontaneously. one such phenomenon is the celebratory riot which can develop for instance when a sporting team wins a match or tournament. hawkins et al. describe the spontaneous mass gathering of , fans which assembled when the university of north carolina men's basketball team played in the national collegiate athletic association, final four semifinal and national championship games in st. louis, missouri in the united states [ ] . as a result of the matches, back in the team's home state of north carolina, two mass gatherings assembled on two consecutive nights drawing a total of , fans. celebrating their team, they lit bonfires in the downtown area of chapel hill where they were congregating and began fire jumpingjumping and dancing through the flames by way of a victory dance. a total of revelers needed medical care including from the on-the-ground ems responders and a total of who needed emergency room admission. the average age of the injured was . years and they were predominantly male. most - %had medical complaints relating to alcohol and didn't need hospital admission. of those who were admitted to hospital, one third had burns from firejumping. these injuries are a function of the sponetnaeity and unplanned nature of these mass gatherings which unlike planned mass gatherings lack well defined boundaries. revelers have no idea of how ems will reach them should the need arise, nor of the disruption to access, and public health demands by their incohrenent mass activities. further, such spontaneous gatherings are componded by the use of excessive alcohol and illicit drugs. the crowds unlike a catholic crowd at world youth day or a muslim crowd at hajj is widely diverse in their compositionwhile some revelers maybe hardcore hooligans or hoodlums others have never attended a mass gathering. the propensity for deliberate violence and even sexual assault can be created especially when vulnerable inexperrinced attendees find themselves caught in the melee. even the mood of the crowd-the collective mind-as earlier researchers referred to it-can vary from benign to malignant, from celebratory to activitely destructive. sexual assault can manifest in such circumstances. perhaps one of the most extraordinary sponetaneous mass gatherings of the last decade is now dubbed "tahrir square" first at the time of the arab spring reaching egypt in feburary and later, in response to newly elected mohammed morsi being deposed by a military coup in july . while no academic papers exist in the literature at the time of writing concerning sexual assault at tahrir square, the mainstream media reported extensively on sexual assaults impacting women protestors at tahrir sqaure, shocking many in the region, particularly in the muslim majority world. sampsel et al. published the first reports of mass gathering associated sexual assault [ ] . important data reported by sampsel et al. reveals that sexual assault occurs at mass gatherings peaking on specific holiday events-new years eve, canada day, halloween and university freshmans' week. women were more often assaulted if they were of younger age, had consumed alcohol or drugs and unlike most sexual violence which befalls women aged to , the assailant at mass gatherings was not previously known to the victim. more often than not, victims declined to release the findings of their 'rape kit' as evidence to the police in the hope of seeking prosecution. the sexual assaults at these mass gatherings occurred both within a friend's home and also outdoors, as in tahrir square. unlike sexual assaults occurring independent of mass gatherings, the majority of women did not know their assailants suggesting perpetrators may seek out mass gatherings as cover for predicating sexual assault on vulnerable victims. sampsel noted the increase in sexual assault events in conjunction with mass gatherings which fell on canadian holidays and surmised that in this occassions the consumption of excess alcohol was more likely. sexual assault transpires much more often when the victim has consumed excess alcohol often spiked with covert drugs in an effort to render the victim unconscious and unable to resist assault. these patterns support the view that the nature of the revelry, and the young female revelers assembling in this gatherings are additionally vulnerable because of social behaviors at such mass gatherings leading to drug faciliated sexual assault. sixteen years post / , it is impossible to consider mass gatherings independent of terrorism irrespective of their location. because of both the magnitude of citizens gathering at mass gathering events whether for celebratory or spiritual purposes, the prize of disrupting a civilian event often garnering extraordinary mass media attention proves very tempting for nefarious actors [ ] . terrorists seek two goals: one to physically disrupt, kill and maim as many innocents simultaneously, and do so with maximum digitally transmitted reverberations to enhance the impact of their attacks. but they also act to instill fear and immobilization in the target population both at the event targeted, but more importantly in the desire to return to normal life. mass gatherings present perfect targets for both these goals. vulnerable events include political, sporting, entertainment events-political party conventions, sporting tournaments like the superbowl, the olympics or the fifa world cup. us experts in homeland security remark that when events tie mass gatherings of the american public with specific national events of celebration -independence day celebrations for example, or events honoring the military or the nation's patriotism threats are not only perceived to be tangible but escalating in risk. hazards that must be considered are diverse. biological agents, ever since the anthrax attacks immediately after / targeting government officials, remain a major concern. many events-consider a presidential inaugrationare open air. access is very difficult to limit and the dissemination of a biological agent over such massive crowds (exceeding a million people at president obama's first inauguration) pose terrifying consequences, particularly if military grade biological weapons were released. one agent could kill hundreds or hundreds of thousands of people depending on its virulence and the dose released. it is not inconceivable that sophisticated terrorists release a biological pathogen in a mass gathering of a population with no immunity to this agent. in , homeland security notes that participants in an outdoor concert contracted hepatitis a, causing morbidity among a population never vaccinated for this virus [ , ] . american cities hosting mass gathering events take this public health security threat very seriously. as far back as , when miami hosted the superbowl xli bio-surveillance activities were expanded by three county health departments and the florida state department of health. they were prepared to identify a bioterrorism attack which might have been invisible during the mass event but become apparent within a two-week window of the superbowl xli. because of the enhanced bio surveillance, public health officials identified more illnesses, injuries, accidents and absenteeism than usual. most importantly, three different public health departments were able to successful data share and coordinate their responses to real-time findings. the federal bureau of investigation (fbi) is charged with identifying and generating intelligence about potential and actual terrorist attacks throughout the united states including attacks that can endanger mass gathering events. critical to the success of advance warning for these events is a clear line of communication and excellent education of the fbi concerning mass gatherings. a key recommendation the us committee on homeland security made was for the development of a national medical intelligence program which would enable combined knowledge of public health concerning bioterrorism to be located within a knowledge base of domestic intelligence concerning potential actors. critical to all these events is the collaborative approach. sharing resources across sectors and agencies whether public or private entities is critical to mass gatherings being safe-guarded. without such collaboration, agencies may compete with one another and hoard information, jeopardizing not only the mass gathering event but also the host city which may well swell in population to become transiently some of the largest cities in the country for the duration of the mass gathering event. global health security is a relatively novel concept: galvanizing public health responses to threats which could imperil the global community. most recently three pathogens have captured the imagination driving the movement to formalize a global health security agenda-literally a world prioritization of threats to public health security [ ] . the ebola virus epidemic, the influenza pandemic and the mers-corona virus outbreaks in the arabian peninsula and south east asia all demonstrated the need for urgent coordinated international responses to avert devastating international impact. periodically health ministers from around the world meet to set the global health security agenda and streamline a multidisciplinary and coherent response to contain these threats. while a global health security agenda for mass gatherings has not been proposed, saudi arabia, in its experience of managing the hajj recognized at the inaugural meeting on mass gathering medicine in jeddah formalized the discipline of mass gathering medicine. six years on, it is time the mass gathering community call for proposals to set a global health security agenda for planned mass gatherings around the world. certainly, yellow fever while not yet a global threat, is a serious consideration for all mass gatherings receiving international visitors. certainly, each country hosting a mass gathering, whether religious or sporting, must enact surveillance and disease reporting mechanisms during the mass gathering events themselves to be able to identify case clusters or even infectious disease outbreaks. control measures and means to prevent these infections being exported back by the attendees to the attendees' countries of origin must be in place. countries receiving participants in events whether a religious pilgrimage or a mass sporting event must be ready with a public health surge capacity to respond to returning travelers especially at a time of heightened awareness of outbreak potential. surge capacity is especially important if host country's health systems are not to be depleted or placed under undue strain during mass gathering events. both authors declare no conflict of interest. one thousand roads to mecca: ten centuries of travelers writing about the muslim pilgrimage paperback the myth of the madding crowd (social institutions and social change) by clark mcphail (author) the psychology of health and wellbeing in mass gatherings: a review and a research agenda qatar steps up to global health security: a reflection on the joint external evaluation religious mass gatherings: connecting people and infectious agents a comprehensive review of the kumbh mela: identifying risks for spread of infectious diseases an influenza outbreak among pilgrims sleeping at a school without purpose built overnight accommodation facilities the practice of pilgrimage in palliative care: a case study of lourdes world's largest mass bathing event influences the bacterial communities of godavari, a holy river of india using mobile technology to optimize disease surveillance and healthcare delivery at mass gatherings: a case study from india's kumbh mela safeguarding the faithful -saudi arabia takes the long view olympic medicine twenty years of the fifa medical assessment and research centre: from 'medicine for football' to 'football for health the quest for public health security at hajj: q.a. ahmed the who guidelines on communicable disease alert and response during mass gatherings advancing the global health security agenda in light of the annual hajj pilgrimage and other mass gatherings cambodian bon om touk stampede highlights preventable tragedy the impact of crowd control measures on the occurrence of stampedes during mass gatherings: the hajj experience human stampedes during religious festivals: a comparative review of mass gathering emergencies in india crowd and environmental management during mass gatherings the hillsborough tragedy mass-gathering medicine: risks and patient presentations at a -day electronic dance music event fire jumpers: description of burns and traumatic injuries from a spontaneous mass gathering and celebratory riot characteristics associated with sexual assaults at mass gatherings committee on homeland security. majority staff report examining; public health, safety, and security for mass gatherings key: cord- -sfa d ux authors: lei, h.; li, y.; xiao, s.; lin, c.‐h.; norris, s. l.; wei, d.; hu, z.; ji, s. title: routes of transmission of influenza a h n , sars cov, and norovirus in air cabin: comparative analyses date: - - journal: indoor air doi: . /ina. sha: doc_id: cord_uid: sfa d ux identifying the exact transmission route(s) of infectious diseases in indoor environments is a crucial step in developing effective intervention strategies. in this study, we proposed a comparative analysis approach and built a model to simulate outbreaks of different in‐flight infections in a similar cabin environment, that is, influenza a h n , severe acute respiratory syndrome (sars) coronavirus (cov), and norovirus. the simulation results seemed to suggest that the close contact route was probably the most significant route (contributes %, % confidence interval [ci]: %‐ %) in the in‐flight transmission of influenza a h n transmission; as a result, passengers within rows of the index case had a significantly higher infection risk than others in the outbreak (relative risk [rr]: . , % ci: . ‐ . , p = . ). for sars cov, the airborne, close contact, and fomite routes contributed % ( % ci: %‐ %), % ( % ci: %‐ %), and % ( % ci: %‐ %), respectively. for norovirus, the simulation results suggested that the fomite route played the dominant role (contributes %, % ci: %‐ %) in most cases; as a result, passengers in aisle seats had a significantly higher infection risk than others (rr: . , % ci: . ‐ . , p = . ). this work highlighted a method for using observed outbreak data to analyze the roles of different infection transmission routes. knowledge about the relative importance of different transmission route(s) is fundamental to developing effective intervention strategies for infectious respiratory and enteric diseases in indoor environments. epidemiological analysis together with in-depth environmental investigations often provides useful insights, and meta-analysis may also be carried out for a particular disease. in this study, we proposed an alternative comparative analysis approach in which we studied outbreaks of different diseases in the same environment using the same approach, by examining differences in the spatial infection patterns. this approach could partly overcome the limitation of traditional individual outbreak analysis that outbreak cannot be repeatedly observed, because the comparative analysis of different diseases in the same environment is like that one disease happened several times. our hypothesis is that the different transmission routes of infection lead to different spatial patterns of secondary cases. for example, close contact transmission always happens with - m of the source, which means that secondary cases infected via close contact route would be close to the index case(s). the airborne transmission may occur over long distance, and the secondary cases infected via airborne route would distribute uniformly in a space when the air is fully mixed. aircraft cabins were selected as the context for our study. the more or less fixed seating arrangement in aircraft cabins permits a spatial pattern of the secondary cases to be identified in some outbreaks. the norovirus is the leading cause of nonbacterial gastroenteritis in humans. , the major possible routes in aircraft cabins are close contact, airborne, and fomite. in this study, a mathematical model was built to study the in-flight infection transmission process, based on the studies by atkinson and wein and nicas and jones. this technique enables detailed physical and biological processes to be modeled and the impact of environmental parameters to be easily integrated. we compared the simulated relative importance of different transmission routes in in-flight outbreaks with the reported spatial distribution of the secondary cases. we performed a literature search for in-flight outbreaks of influenza a h n , sars cov, and norovirus in appendix s . all chosen outbreaks occurred in boeing aircraft cabins with flight duration . or hours. the main criteria for identifying suitable outbreaks include the availability of detailed seating information for both the infected and noninfected, airplane type and flight duration. figure illustrates the detailed spatial distribution of the secondary cases in the chosen outbreaks. the definitions for the relevant transmission routes ( figure ) in our multiroute model are as follows. the airborne route refers to direct inhalation of an infectious agent through small droplet nuclei, that is, the residue of large droplets containing microorganisms that have evaporated to an aerodynamic diameter of less than microns (termed respirable). these respirable droplets can deposit in the respiratory tract. close contact route includes direct contact and large droplet transmission. direct contact refers to infection through person-to-person contact with the index source passenger, such as handshaking. we assume there is no body-to-body contact between index source passenger(s) and other passengers during the flight. we only consider large droplet transmission in the model, which refers to the inhalation of the virus carried in respirable airborne particles with a diameter between and microns (termed inspirable), and the droplet spray of large droplets (> microns in diameter) onto facial target membranes. the fomite route refers to infection by touching objects or surfaces that have earlier been contaminated by hands or by direct deposition • our identification of the dominated routes, that is the close contact route (large droplet) for influenza, the fomite route for norovirus, and all routes for sars cov, suggested the relative importance of different environment intervention for different infectious diseases in air cabins and probably also in other indoor environments. for minimizing in-flight fomite transmission, the aisle seatbacks and toilets should be cleaned and disinfected effectively. f i g u r e spatial distribution for in-flight infection outbreaks, (a) norovirus, (b) sars cov, and (c) influenza a h n of infectious pathogens from the index source passenger, which is also sometimes termed indirect contact route. for respiratory disease, coughing is used as surrogate to model the virus-containing droplets from all respiratory activities such as breathing, talking, and sneezing, as the size distribution of the droplets from coughing, talking, and sneezing is similar, and the amount of droplets generated due to breathing is negligible. assume that cough frequency for infector is f c per hour and that one cough can produce n c droplets with the size distribution f c r . then, the generation rate (number/h) of droplets with radius r (μm) from individual i is given by: for enteric disease, such as norovirus, virus-containing droplets are emitted from the infector in vomit and/or diarrhea. a study by simulated vomiting device showed that the volume of the aerosolized droplets ranged from . to ml, with a mean value of . ml. to the best of our knowledge, there is no study on the size distribution of the droplets from vomit, and we assume that these droplets have same size distribution as those from coughing. for respirable droplets with aerodynamic diameter of less than microns, they could move a long distance with the airflow and dis- for inspirable droplets with a diameter between and microns, we assumed that the maximum horizontal distance they could move was m because of gravity and the relatively high deposition rate on environmental surfaces. they distributed within m of the source, and the volume of this space was denoted to be a rapid death rate of pathogens atomized into air had been observed, , and evaporation of droplets was believed to play an important role. xie et al found that there was a fast viability decline stage when the droplets completely evaporated, when viability decreased to about % and then slowly declined. here, the survival ratio due to evaporation was defined as e r ,s = l r ,t ∕l r , where l r ,t is the concentration of viable viruses (tcid /ml or genome copies/ml) in droplets with initial radius r (μm) at the time t (s) after being exhaled; t e r is the evaporation time (s) for the drop- illustration of different transmission routes considered in this study. note that all sizes of droplets are involved in the fomite route assume there is no resuspension of droplets from environmental surfaces into the air. in the air cabin, on the one hand, viable virus is generated from index case(s) at rate ( ) was the exposure through each route was modeled separately, and then the dose-response model was used to assess an integrated risk. the dose to individual i via the airborne route in the lower and upper respiratory tracts is denoted as d i al and d i au (tcid or genome copies), and for a flight duration t, they can estimated as follows: where r a is the largest radius for airborne droplets and r a = μm ; p is the pulmonary ventilation rate and p = . m /h ; r is the droplets' initial radius; and r is the final radius after complete evaporation. here, we assume that r = r / ; l (r) and u (r) are the deposition fraction of droplets with radius r in the lower and upper respiratory tracts, respectively. the model from icrp was used in this study. transmission by close contact refers to either inhalation of the virus carried in airborne particles with a diameter between and microns, or the spray of large droplets on the susceptible individuals' mucous membranes. for norovirus transmission, it is difficult for the "large" droplets generated from vomiting to move to the inhale air of the seated susceptible passengers, which is always more than one meter above the ground, because of the high downward velocity, gravity, and the relatively high deposition rate. therefore, the close contact route was not considered in the norovirus transmission. then the dose via inhalation of inspirable droplets in upper respiratory tract (d i cr (tcid or genome copies)) was where r b was radius of the maximum inspirable droplets and for the spray of large droplets on mucous membranes, because of the seating arrangement we assumed that there was no face-to- the negative exponential dose-response model was used to estimate the infection risk, which implies that a single particle can start an infection, all single particles are independent of each other. the infection risk of individual i during the flight can be calculated according to the following equation where η l η l , η u , and η m are the dose-response rates (/tcid or/genome copy) in lower/upper respiratory tracts and on mucous membranes, respectively. here it is also assumed that η u = η m . table ( ) d i cm = t ∫ o ∞ ∫ r a a m a v c i c (r) kr v t πr l r ,t drdt ( ) c j s � k + � = � c j s (k) + ∑n p i= (c i h (k)a hs τ hs −c j s (k)a hs τ sh )ps i,j (k) a s � e −b s ×Δt ( ) c i h � k + � = � c i h (k) − ∑n p i= (c i h (k)a hs τ hs −c j s (k)a hs τ sh )ps i,j (k) a h − pm i (k)a m h τ hm c i h (k) a h � e −b h ×Δt ( ) d i fm = n t ∑ k= pm i (k) a m h τ hm c i h (k) ( ) p i = − e −(η l d i al +η u d i au )−(ηud i cr +η m d i cm )−ηmd i fm in this study, we built a mathematical model to study the inflight transmission of different viruses, using a novel comparative analysis approach. the results suggested that the dominant transmission routes in air cabins are probably the close contact route for influenza, the fomite route for norovirus, and all routes (airborne, close contact, and fomite routes) for sars cov. the dominant transmission routes vary for the viruses, mainly depending on the pathogen-specific t a b l e infection risks of passengers within rows of the index case(s) and others from the simulation results and reported outbreak data, respectively (with the tuned range of the virus shedding magnitudes) outbreak data within rows (others) as far as we are aware there are no data on the dose-response rate of sars cov either on mucous membranes or in the respiratory tracts of humans. we assume that the dose-response rate on human mucous membranes is the same as that on mice mucous membranes, and the dose-response rate in the human respiratory tract is times that of mucous membranes (similar to the influenza a h n data). we also performed the sensitivity analysis of the dose-response rates, with different ratios of median dose-response rate, that is (in lower respiratory tract)/(on mucous membranes) (see detail in appendix s ), and we found that all routes were important in in-flight sars cov transmission for different ratios of median dose-response rate. the fecal-oral spread is known to be the primary mode of transmission of norovirus. the fomite route transmission of norovirus is well supported by the reported widespread environmental contamination with norovirus. , our simulation of a norovirus outbreak confirms that the fomite route is dominant in transmission. it is also suggested that vomiting can produce aerosol droplets containing norovirus particles, and inhaled by exposed susceptible individuals, depositing in the upper respiratory tract and subsequently swallowed along with the respiratory mucus. airborne norovirus was detected from an air sample in one outbreak. a study of a norovirus outbreak in a hotel found an inverse relationship between the infection risk and the distance from the person who vomited when a food source was not implicated. this study simulated both the airborne and fomite route transmission of norovirus. and the results showed that in most cases, the fomite route plays the dominant role. the predicted infection risk from the fomite route for aisle seat passengers is . times higher than that for nonaisle seat passengers. the aisle passenger-tonon-aisle passenger relative risk in the outbreak ( . ) is much higher than . , and may be attributable to a small sample size of secondary cases ( ). in conclusion, a mathematical model was built to simulate the physi- additionally, this study highlights a way to use observation outbreak data 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environmental influenza interventions: how the host, agent, and environment alter dominant routes of transmission likely transmission of norovirus on an airplane transmission of the severe acute respiratory syndrome on aircraft influenza a(h n )pdm during air travel who technical advice for case management of influenza a(h n ) in air transport high infectivity and pathogenicity of influenza a virus via aerosol and droplet transmission transmission of influenza a in human beings an outbreak of influenza aboard a commercial airliner role of air distribution in sars transmission during the largest nosocomial outbreak in hong kong experimental study of dispersion and deposition of expiratory aerosols in aircraft cabins and impact on infectious disease transmission severe acute respiratory syndrome coronavirus on hospital surfaces widespread environmental contamination with norwalk-like viruses (nlv) detected in a prolonged hotel outbreak of gastroenteritis a norovirus outbreak at a longterm-care facility: the role of environmental surface contamination norovirus, gastroenteritis, and indoor environmental quality detection and quantification of airborne norovirus during outbreaks in healthcare facilities evidence for airborne transmission of norwalk-like virus (nlv) in a hotel restaurant additional supporting information may be found online in the supporting information tab for this article. key: cord- -phv xe authors: mukhopadhyay, urbi; chanda, shampa; patra, upayan; mukherjee, anupam; komoto, satoshi; chawla‐sarkar, mamta title: biphasic regulation of rna interference during rotavirus infection by modulation of argonaute date: - - journal: cell microbiol doi: . /cmi. sha: doc_id: cord_uid: phv xe rna interference (rnai) is an evolutionary ancient innate immune response in plants, nematodes, and arthropods providing natural protection against viral infection. viruses have also gained counter‐defensive measures by producing virulence determinants called viral‐suppressors‐of‐rnai (vsrs). interestingly, in spite of dominance of interferon‐based immunity over rnai in somatic cells of higher vertebrates, recent reports are accumulating in favour of retention of the antiviral nature of rnai in mammalian cells. the present study focuses on the modulation of intracellular rnai during infection with rotavirus (rv), an enteric virus with double‐stranded rna genome. intriguingly, a time point‐dependent bimodal regulation of rnai was observed in rv‐infected cells, where short interfering rna (sirna)‐based rnai was rendered non‐functional during early hours of infection only to be reinstated fully beyond that early infection stage. subsequent investigations revealed rv nonstructural protein to serve as a putative vsr by associating with and triggering degradation of argonaute (ago ), the prime effector of sirna‐mediated rnai, via ubiquitin–proteasome pathway. the proviral significance of ago degradation was further confirmed when ectopic overexpression of ago significantly reduced rv infection. cumulatively, the current study presents a unique modulation of host rnai during rv infection, highlighting the importance of antiviral rnai in mammalian cells. to its target. the ultimate fate of the target mrna is determined by the degree of complementarity between the guide strand and the target sequence; perfect complementarity (for sirnas) triggers cleavage (slicing) of the mrna whereas imperfect complementarity (generally for mirnas) sequesters mrna away from polyribosomes into cytoplasmic processing bodies (p bodies) resulting in translational repression. at the catalytic core of risc are the argonaute (ago) family of proteins of which mammals encode four homologs (ago - ). in spite of evidences of functional redundancy among the four agos, slicer activity is exhibited only by the isoform ago in mammalian cells (bartel, ; bartel, ; siomi & siomi, ) . during viral infection, replicative or transcriptive viral dsrna intermediates may succumb to processing by host cellular rnai yielding virus-derived small interfering rnas (virnas), which might further direct viral rna cleavage ultimately resulting in heavily attenuated viral replication. as a coevolutionary counterstrategy, viruses have adopted rnai-evasive virulence measures in the form of producing virus-encoded-suppressors-of-rnai (bivalkar-mehla et al., ; burgyán & havelda, ; ding, han, wang, & li, ; wu, wang, & ding, ) . experimental evidence of virna generation during the course of natural viral infection is abundant in plants, worms, and insects (siu et al., ) . physiological relevance of rnai as an antiviral innate immunity, however, has been greatly debated in differentiated, somatic cells of higher vertebrates because of the evolution of a superior, interferon (ifn)-based innate immune system (cullen & cherry, ; goubau, deddouche, & e sousa, ; maillard et al., ; samuel, ; schneider, chevillotte, & rice, ; ten oever, ) . alternatively, mammalian viruses, at least in some contexts, have been experimentally shown to exert strong rnai evasive mechanisms by encoding highly potent vsrs (li, lu, han, fan, & ding, ; qiu et al., ) , justifying near absence of virnas in mammalian cells during natural course of infection (parameswaran et al., ) . mammalian vsrs characterised till date include ebola virus vp (haasnoot et al., ) , influenza a virus ns (bucher, hemmes, de haan, goldbach, & prins, ) , vaccinia virus e l (li et al., ) , nodamura virus b (johnson, price, eckerle, & ball, ), hepatitis c virus core (wang et al., ) , hiv- tat (bennasser & jeang, ; bennasser, le, benkirane, & jeang, ) , coronavirus nucleocapsid protein n (cui et al., ) , and virus-associated rnai and rnaii of adenoviruses (andersson et al., ; lu & cullen, ) . vsrs are generally dsrna-binding proteins (excepting adenoviral vsr), which inhibit activity of either dicer, ago, or both. interestingly, all vsrs identified from mammalian viruses possess ifn or protein kinase r antagonistic properties and are essential for replication and pathogenesis, suggesting that rnai and other innate antiviral responses are interrelated. the genome of rotavirus (rv), a diarrheagenic virus in infants and young animals, consists of segmented double-stranded rnas, which are usually enclosed within viral double-layered particles and, at proliferative stage of infection, within highly dynamic encagements called viroplasms. single-stranded viral mrnas, produced within double-layered particles, are either translated into six structural (vp to vp , vp , and vp ) and six nonstructural (nsp to nsp ) proteins or used as templates for viral rna replication (desselberger, ; estes & greenberg, ) . however, oozing of rotaviral dsrna intermediates in unmasked cytosolic environment has been hypothesised based on rnase iii-sensitive and dsrna-positive scattered signals in infected host cell cytoplasm (rojas, arias, & lópez, ; zhu et al., ) . accidental exposure of viral genomic dsrnas or even transcriptive dsrna intermediates in the cytoplasm may potentially evoke rnai-based surveillance mechanism. strikingly, previous reports emphasised retention of competent rnai pathway in the context of rv infection enabling functionality of both small interfering rnas (sirnas) and short hairpin rnas (shrnas; déctor, romero, lópez, & arias, ; arias et al., ; lópez, rojas, ayala-bretón, lópez, & arias, ; chen et al., ) . indeed, successfully knocking down expression of viral and host proteins has been proven to be excellent loss-of-function studies to unravel complex host-rv interaction networks (dhillon & durgarao, ; mukherjee, patra, bhowmick, & chawla-sarkar, ; oceguera, peralta, martínez-delgado, arias, & lópez, ; silva-ayala et al., ; trujillo- alonso, maruri-avidal, arias, & lopez, ; yin et al., ) . moreover, rnai-based potential therapeutic approaches targeting rv genes have also been advocated (arias et al., ) . notably, in most of these experiments, rnai functionality was assessed at relatively late hours of infection (beyond -hr postinfection). while studying loss-of-function attributes of selected cellular determinants in context of rv-sa infection, intriguingly, we found differential sensitivity of cellular genes to rnai at different time points of infection. rnai pathway was found to be inhibited during early hours ( - hr) of rv-sa infection only to be restored to full functional competence beyond that early phase. blocking of rnai was confirmed in the form of ablated functioning of sirna and shrna but not mirna. interestingly, hindrance to rnai during the early stage of rv-sa infection coincided with the loss of protein level of ago . further mechanistic studies revealed that rv-nsp interacts with and degrades ago in ubiquitin-proteasome-dependent way. together, the data highlight a fascinating and unique modulation of rnai pathway in response to rv infection. to assess the sensitivity of ectopic gfp expression to synthetic sirna during early and late hours of rv infection, gfp (pegfp-n )-transfected ma cells were cotransfected with either sigfp or scrambled sirna, and -hr posttransfection cells were further mock-infected or infected with rv-sa for and hr. interestingly, analyses of gfp expression by immunoblotting showed sirna-mediated targeted knock-down of gfp to be apparent in mock-infected cells as well as in cells infected with rv-sa for hr but not in rv-sa -infected cells harvested at -hr postinfection ( hpi; figure a ). we ruled out transfection of sigfp to have any off-target effects on overall rv infection as relative expression of rv structural and nonstructural proteins remained unperturbed ( figure a ; figure s a ). visualisation by immunofluorescence microscopy ( × magnification) in ma cells ( figure b ) and hek cells ( figure s b ) also revealed ectopic expression of gfp to be sensitive to rnai only at hpi but not at hpi, suggesting time point-dependent differential regulation of rnai during rv-sa infection. rhodamine-conjugated nsp visualised in rv-infected samples under higher magnification ( × oil immersion) ensured progressive infection (figure b, panel ). next, we investigated whether rnai targeted against any endogenous cellular protein was also regulated similarly during early and late hours of rv-sa infection. ma cells, transfected with scrambled sirna or ringbox protein (rbx )-specific sirna for hr, were subsequently mock-infected or infected with rv-sa . consistent to our previous results, rbx expression was successfully knocked down in response to rbx sirna in rv-sa -infected cells lysed at hpi as well as in mock-infected control but not in rv-sa -infected cells harvested figure host rna interference is blocked during early hours of rv-sa infection. (a) ma cells transfected with gfp (pegfp-n ) were further cotransfected with scrambled sirna or sigfp as indicated in the figure (designated by +, −); -hr posttransfection, cells were mock infected or infected with rv-sa . after and hpi, cells were harvested, and gfp expression was analysed by western blot analysis. (b) gfp (pegfp-n )-transfected ma cells were cotreated with scrambled sirna or sigfp as indicated in the figure (designated by +, −). cells were fixed at indicated time points postinfection (mock/rv-sa ) with paraformaldehyde, permeabilised, blocked, and subsequently stained overnight with anti-nsp antibody (as a marker of virus infection). secondary staining and mounting were performed with rhodamine conjugated anti-rabbit antibody and dapi, respectively. cells were finally visualised by confocal microscopy ( ×); scale bar μm. (lower panel) rhodamineconjugated nsp was visualised in rv-infected samples under higher magnification to ensure progressive infection ( × oil immersion). (c) ma cells were transfected with scrambled sirna or rbx sirna. -hr posttransfection, cells were either mock infected or infected with rv-sa for indicated time points; rbx expression was finally assessed by immunoblot analysis. (d) ma cells, transfected with either empty vector (plko. -trc) or rv-vp -shrna (cloned into plko. -trc vector) for hr, were further infected with rv-sa for and hr. expression of rv-vp in whole cellular extracts was checked by western blot analysis. (e) scrambled mir/mir- b mimic transfected ma cells were mock infected or infected with rv-sa for and hr. cellular lysates prepared at indicated time points were subjected to western blot analysis to check expression of p α subunit of pi k. (f) pmir-report luciferase construct containing the ′-utr of p α-pi k was transfected in hek cells in association with mir- b mimic ( and nm). -hr posttransfection, cells were either kept mock-infected or infected with rv-sa for and hr. percentage of relative luciferase activity was finally represented after normalising firefly luciferase with corresponding renilla luciferase control at hpi (figure c ), further indicating hindrance of rnai mechanism during early hours of rv-sa infection. furthermore, sensitivity of rv protein expression to shrna-mediated rnai in infection scenario was analysed. ma cells expressing shrna against vp for hr were subsequently infected with rv-sa , and expression of vp was checked after and hr of infection. in agreement with previous reports (arias et al., ; déctor et al., ) , expressions of vp rna and protein were found to be silenced at hpi ( figure s c; figure d ), suggesting sensitivity of viral protein expression to rnai at late phase of rv-sa infection. interestingly, shrna-mediated silencing of vp expression was attenuated at hpi ( figure s c; figure d ), affirming our previous observation that functional competence of sirna is lost during early phase of rv-sa infection. we further checked modulation of mirna-mediated rnai by evaluating target repression capacity of ectopically administered mirna mimic during early and late hours of rv-sa infection. ma cells, transfected with either mir- b mimic or scrambled mir for hr, were subsequently infected with rv-sa for and hr. one set of transfected cells was mock infected. interestingly, following mir- b overexpression, reduction of mir- b target p α subunit of pi k (park, lee, ha, nam, & kim, ) was evident in both mockinfected cells and rv-infected cells for (early time point) and hr (late time point; figure e ). consistently, much alike to mock-infected group, sensitivity of luciferase expression from pmir-report luciferase vector containing ′-utr of p α subunit of pi k in hek cells to cotransfected mir- b mimic ( and nm) was also found to be unaffected at both early ( hpi) and late ( hpi) hours of rv-sa infection ( figure f ). collectively, the results suggest that though inhibition of sirna and shrna-mediated rnai occurs during early hours of rv-sa infection, functionality of mirna is retained during the same infection window. figure s a ). however, ago protein level was found to be reduced during late hours of rv-sa infection ( - hpi; figure a ; figure s a ). levels of ago and ago , two other members of ago family proteins in mammals, as well as dicer, remained unchanged with respect to their respective mock-infected controls throughout indicated time period ( - hpi) of rv-sa infection ( figure a ; figure s a ). confocal microscopy and subsequent quantitation of corrected total cell fluorescence (ctcf) also revealed ago expression to get reduced at hpi compared with the mock-infected control only to restore and be distinctly visible again at hpi ( figure b ). intracellular distribution of ago , however, was found to be somewhat different at hpi from that in mockinfected control (figure b) . furthermore, infection with uv-treated rv-sa (smirnov, kapitulets, amitina, ginevskaya, & kaverin, ) had no effect on ago protein levels postinfection ( figure s b ). together, the data suggest that actively replicating rv-sa triggers attenuation in protein levels of ago leading to functional blocking of rnai during early time points ( - hpi) of infection. we further speculated involvement of viral protein(s) in mediating reduction of ago protein levels during - -hr post rv-sa infection. owing to previous reports of rv-nsp being involved in triggering degradation of multitude of host proteins, which are regulators of host cellular innate immunity (arnold, barro, & patton, ; bagchi, bhowmick, nandi, kant nayak, & chawla-sarkar, ; barro & patton, ; bhowmick, halder, chattopadhyay, nayak, & chawla-sarkar, ; bhowmick, mukherjee, patra, & chawla-sarkar, ; graff, ettayebi, & hardy, ; graff, ewen, ettayebi, & hardy, ; graff, mitzel, weisend, flenniken, & hardy, ; qin et al., ) , the involvement of rv-nsp was investigated in the process. gradual reduction of ago protein levels in a dose-dependent manner in response to escalated concentration of transfected rv-nsp further ascertained the role of this viral nsp in modulating ago levels ( figure s b ). furthermore, infection with bovine rv strain a - possessing wild-type nsp (taniguchi, kojima, & urasawa, ) also resulted in reduced ago protein levels ( - hpi) whereas corresponding nsp mutant rv strain a - (taniguchi et al., ) figure s d ). in addition to the putative e ubiquitin ligase activity intrinsic to rv-nsp , co-opted host e ubiquitin ligase complex cullin has recently been implicated to be interacting with (ding et al., ; lutz, pace, & arnold, ) and be involved in mediating proteasomal degrada- figure s f ). notably, ubiquitinated ago levels were found to be low at hpi compared with hpi, justifying their corresponding cellular levels represented in the input lanes ( figure s f ). the observation that rv-nsp can cause proteasomal degradation of ago prompted us to check whether rv-nsp can also function as a suppressor of rna silencing. to evaluate this, rbx protein expression was assessed in ma cells transfected with either rbx sirna alone or pcdnsp in addition to rbx sirna. immunoblotting revealed inefficient knocking down of rbx expression by sirna in the presence of rv-nsp ( figure a ). sensitivity of ectopic gfp (pegfp-n ) expression to sigfp was also reduced in rv-nsp overexpressing cells ( figure s a ), indicating that rv-nsp might figure rotaviral nonstructural protein triggers ubiquitination and proteasomal degradation of ago . (a) whole cellular extracts from pcdnsp -and pcdnsp -transfected ma cells were subjected to western blot analysis for checking expressions of ago , ago , pan , and ser pdrp . (b) ma cells were mock-infected or infected with rv-a - and rv-a - ( - hpi). ago and p expression was checked in cellular extracts of infected cells prepared at indicated time points. nsp protein expression was analysed in the same lysates to confirm rv-a - and rv-a - infection. (c) ma cells were kept untransfected or transfected with pcdnsp ; -hr posttransfection, cells were treated with vehicle (dmso) or -μm lactacystin. cellular lysates prepared after hr of transfection were subjected to sds-page followed by immunoblotting and expression of ago was checked. his protein expression was checked as a marker of pcdnsp transfection. (d) pcdnsp -transfected ma cells were treated with either vehicle (dmso) or -nm mln ( hr after transfection). whole-cell lysates were prepared ( -hr posttransfection), and immunoblotting was done to check expressions of ago , his (nsp ), cullin , and cullin . neddylated form of cullin and cullin is indicated by arrows. (e) ma cells ectopically expressing pcmv-flag-ub were cotransfected with either empty vector (pcdna b) or pcdnsp . (left panel) cellular lysates prepared after hr of transfection were immunoprecipitated with anti-flag antibody, and immunoprecipitates were further subjected to immunoblotting with anti-ago antibody. input lysates were probed with anti-ago and anti-his (nsp ) antibodies. (right panel) reciprocal coimmunoprecipitation was done with anti-ago antibody followed by immunoblotting with anti-flag antibody. expressions of ago , his (nsp ) were checked in the input lysates. (f) ma cells were trasnfected with either empty vector (pcdna b), full-length nsp (cloned in pcdna b vector), or truncated domains of nsp (ring and Δring domain cloned in pcdna b vector). after hr of transfection, cell lysates were subjected to western blot analysis to check levels of ago and p . expression of his tag was used as a marker of pcdnsp , pcdring-nsp , and pcdΔring-nsp transfection. (g) equal amount of lysates from pcmv-flag-ub overexpressed ma cells cotransfected with pcdring nsp or pcdΔringnsp were immunoprecipitated with anti-ago antibody. immunoprecipitates were then probed with anti-flag antibody. simultaneously, his (nsp ) and ago expressions were assessed in the input lysates serve as an rna-silencing suppressor. furthermore, silencing of vp expression by vp shrna at hpi was observed only in rv-a - together, the results suggest that rv-nsp can be regarded as a putative rv-encoded suppressor of rnai, which inhibits functionality of sirna and shrna but not of mirna. we further looked for any possible association between rv-nsp and ago , which might lead to proteasomal degradation of ago in the presence of rv-nsp . pcdnsp -transfected ma cells were lysed -hr posttransfection and subsequently immunoprecipitated with anti-his antibody. the presence of ago in the immunoprecipitate indicated an association between ago and rv-nsp ( figure a ). the presence of p and absence of ago in the immunoprecipitate served as positive and negative control, respectively, for this interaction assay (figure a ). similar observation was also made in hek cell line ( figure s a ). as both ago (oceguera et al., ) and rv-nsp (hua, chen, & patton, ) were previously reported to interact with viral rna, we went on to investigate whether the association between rv-nsp and ago required viral rna intermediates. interestingly, ago -rv-nsp association was found to be unperturbed in the presence of both single-stranded rna-specific rnase i and double-stranded rna-specific rnase iii (figure b ), excluding the possibility of viral rna scaffold to have a mediating role. ago activity has been reported to be regulated by phosphorylation (bridge et al., ; horman et al., ) . phosphorylation of rv-osu-nsp by caesin kinase ii has also recently been shown to be a prerequisite for nsp -βtrcp association (davis et al., ) . to further explore whether association between rv-nsp and ago is influenced by phosphorylation status of either nsp or ago , coimmunoprecipitation analysis was carried out in the presence of lambda protein phosphatase (lambda pp). nsp -ago association was not hampered following dephosphorylation of lysates with anti-his coupled resin was incubated with cellular extracts from mock-transfected or pcdnsp -transfected ma cells pretreated with rnase i and rnase iii. immunoprecipitates were subsequently subjected to western blot analyses with anti-ago antibody. input lysates were probed with anti-his and anti-ago antibodies. (c) ma cells were transfected with empty vector or pcdnsp for hr followed by whole-cell lysate preparartion. cellular lysates were incubated with active lambda pp or heat inactivated lambda pp as described in the experimental procedures section followed by immunoprecipitation with anti-his antibody. expression of ago was analysed in the immunoprecipitates by immunoblot analysis. expression of ago and his (nsp ) was analysed in the input lysates (d) ma cells were transfected with pcdring-nsp or pcdΔring-nsp . after hr, cell lysates were immunoprecipitated with anti-his antibody. immunoprecipitates were then resolved on sds-page, transferred on to pvdf membrane, and finally probed with anti-ago and anti-p antibodies. expressions of ago , p , and his (nsp ) were checked in the input lysates. (e) ma cells were mock infected or infected with rv-sa for and hr. equal amount of lysates were immunoprecipitated with anti-ago antibody followed by immunoblotting with anti-nsp antibody. expressions of ago and nsp were checked in the input lysates. (f) (left panel) schematic representation of ago mutants showing span of deletion (ago -mt - ). (right panel) ma cells transfected with pcdnsp were further cotransfected separately with ago mutants (ago -mt - ). after hr, equal amounts of whole-cell lysates were immunoprecipitated using anti-flag antibody. immunoprecipitates were then subjected to immunoblot analysis with anti-nsp antibody. expressions of his (nsp ) and flag (for ago domain mutants) were analysed in the input lysates input lysates of lambda pp-treated cells confirmed lambda pp enzymatic activity ( figure s b ). ago proteins consist of n-terminal (n), paz (piwi/ago/zwille), mid (middle), and piwi (p-element-induced whimpy testes) domains (faehnle & joshua-tor, ) . we therefore went on to characterise the region of ago needed for association with rv-nsp . coimmunoprecipitation assay was performed using four flag-tagged in agreement with the previous results, nsp -ago association was found in the presence of flag-paz but not of flag-piwi, confirming involvement of paz domain of ago in association with nsp ( figure s c ). whole-cell lysates were subjected to western blot analyses to check expressions of rv structural (vp and vp ) and nonstructural (nsp ) proteins. (b) end point viral titers ( , , and hpi) were determined by plaque assay from rv-sa (moi . )-infected ma cells in the presence and absence of overexpressed hago gfp ( μg) and expressed as virus plaque forming units/ml. (c) ma cells were transfected with empty vector (flag; μg) or ago domain mutants (ago -mt - ) and subsequently infected with rv-sa (moi ; hr). end point viral load was measured by plaque assay and represented as viral plaque forming units/ml. (d) ma cells transfected with full-length hago gfp or catalytic-dead ago mutants (designated as ago -d a or ago -d a) were infected with rv-sa for hr followed by viral titer measurement. empty vector (gfp; μg) and ago -mt -transfected cells served as negative control and positive control, respectively, for this experiment proteins in ma cells at hpi (figure a ) without bystander cytotoxicity ( figure s a ). concurrently, a dose-dependent reduction in the percentage of rv infectivity was observed at hpi in response to increasing concentration of overexpressed hago gfp ( figure s b ). for further experiments, the concentration of μg of hago gfp was chosen, as this concentration showed prominent antiviral effect without stochastic cytotoxicity. attenuation of rv rna upon ago overexpression was also significantly prominent at hpi in conventional rt-pcr-based studies ( figure s c ). moreover, antiviral potency of ago overexpression was found to be evident at as late as and hpi (figure b) . notably, overexpression of ago (gfp ago ) had no effect on either rv protein expression or rv progeny yield ( figure s d loss of antiviral potency of ago , however, is more prominent in the presence of piwi domain deleted ago mutants (ago -mt and ago -mt ) compared with piwi domain containing ago mutants (ago -mt and ago -mt ; figure c ; figure s f ). notably, overexpression of ago -mt showed slightly yet consistently better anti-rotaviral property than that of ago -mt (figure c ; figure s f ). due to lack of paz domain, ago -mt fails to associate with rv-nsp . subsequent increased half-life of ago -mt during infection scenario might be the reason behind better anti-rotaviral property of ago -mt compared with ago -mt . ago catalytic activity is reported to be antiviral against a broad range of viral infections . for this purpose, ago site-directed (catalytic-dead) mutants (ago -d a and ago -d a) were transfected in ma cells followed by rv-sa infection (for hr). end point viral titer measurement showed attenuated rv infection in the presence of slicing-competent wild-type ago to restore in ma cells transfected with catalytic-dead ago mutants (figure d ). piwi domain deleted ago mutant (ago -mt ) was taken as positive control for this experiment ( figure d ). similar results were reflected when expression of rv proteins was checked in the presence of wild type and catalytically impotent ago constructs ( figure s g ). together, the data suggest that the full-length, slicing-competent ago , especially encompassing the piwi domain, is a critical anti-rotaviral host determinant. physiological relevance of rnai as an antiviral innate immune measure in plants and nonchordates follows three fundamental lines of arguments: (a) ample virnas are produced during natural course of viral infection in these species, (b) host mutants defective in mounting rnai-mediated defense response are more susceptible to viral infec- (vsrs); vsr-mutant viruses show less virulence in wild-type hosts but acquire important virulence rescue phenotype in rnai-defective host mutants (gammon & mello, ; sledz & williams, ; younis, siddique, kim, & lim, ) . in spite of their universality and ubiquitousness, vsrs are highly divergent within and across kingdoms with no sequence homology and have been hypothesised to have evolved independently in different viruses (burgess, ) . virusesinfecting mammalian cells, on the other hand, are only sensitive to synthetic sirnas that bypass the requirement of dicer for being processed but produce very little virnas during natural course of infection (cullen & cherry, ) . this is possibly due to decreased dicerprocessing activity, which occurs during cellular differentiation perhaps via modification of its internal auto-inhibitory helicase domain. interestingly, inability to mount rnai-based innate immunity in response to invading dsrnas has well been superseded in higher cumulative evidences have emerged in support of functional rnai-dependent antiviral immunity in mammals (berkhout, ; jeang, ; qiu et al., ) . according to this view, the very establishment of infection over cellular defence requires production of extremely potent and efficient vsrs. this view is (arias et al., ; chen et al., ; déctor et al., ; dhillon & durgarao, ; lópez et al., ; montero, rojas, arias, & lópez, ; mukherjee et al., ; oceguera et al., ; patra et al., ; silva-ayala et al., ; trujillo-alonso et al., ; yin et al., ) . intriguingly, we found infection time point-dependent bimodal regulation of rnai, where rnai showed differential sensitivity at early and late hours of rv infection. sirna-guided rnai targeting ectopically expressed gfp (figure a ,b, figure s a , b), and endogenous gene rbx (figure c) and shrna-mediated rnai targeting rotaviral vp gene (figure d , figure s c ) were all found to be blocked at early hours of rv-sa infection only to regain full functionality at relatively late hours. evidence of abrogated functionality of synthetic sirnas suggests that hindered dicer activity cannot be the exclusive reason for rnai inhibition during early hours of rv-sa infection. in contrast, functionality of mirna is retained, as mirna mimics successfully knocked-down target expression both at early and late hours of rv-sa infection (figure e,f) . this is in agreement with our previous findings, where mirna mimics preserved their target repression potency in context of rv-sa infection (chanda, nandi, & chawla-sarkar, ; mukhopadhyay et al., ) . we also did not find global change of mirna population characteristic of abrogated mirna biogenesis in our mirna microarray data (chanda et al., ) . therefore, in the absence of ago , loading in risc and mirnaguided knock-down can be taken over by other nonslicing reserve agos to sustain cell viability. in agreement with this is the observation that all agos have been shown to bind to mirnas and sirnas indiscriminately of sequence, to interact with a common set of helicases and mirna-binding proteins and to localise to p bodies in mammalian cells (ruda et al., ) . indeed, availability of nonslicing agos during early hours of rv infection (figure a , figure s a figure s a , b, g), to trigger protein level reduction of ago . rv-nsp is a multifunctional protein involved in ifn antagonism, activation of pro-survival pi k/akt pathways, denucleating cytoplasmic p bodies, and also harbouring rna binding as well as cytoskeleton localisation domain (bagchi et al., ; bhowmick et al., ; ding et al., ; lutz et al., ; morelli, ogden, & patton, ) . ifn antagonistic properties of rv-nsp lies in its ability to trigger proteasomal destruction of irf , , , , β-trcp, traf , and rig-i, whereas p body destabilising property of nsp includes degradation of pan by ubiquitin-proteasome system. rv-nsp possesses a highly figure s a , c). this is in contrast to the fact that substrate binding specificity of rv-nsp generally lies in its highly variable c-terminal half (lutz et al., ; morelli et al., ) . the association between ago and rv-nsp (figure e , figure s b ) as well as ubiquitin enrichment of ago ( figure s f) were also observed at early hour of rv-sa infection. interestingly, at relatively late hours, both association with rv-nsp (figure e , figure s b ) and ubiquitination of ago decreased ( figure s f ), partially explaining restoration of ago protein levels at these late time points of infection. rv-nsp has recently been reported to interact with (davis et al., ; ding et al., ; lutz et al., ) and degrade β-trcp by co-opting cullin ring e ubiquitin ligases (crls; ding et al., ) . reversal of ago degradation, however, was observed in presence of neither dominant negative forms of cullin and cullin nor pan-cullin inhibitor mln , suggesting that rv-nsp mediated degradation of ago is not dependent on co-opted crls ( figure d; figure s e ). molecular mechanisms governing time point-dependent bimodal regulation of ago -nsp association and possible involvement of co-opted host e ubiquitin ligase, however, remain to be investigated in future. moreover, on the basis of our previous observation of p -nsp interaction to get regulated in a similar bimodal fashion , we also cannot rule out the possibility of mutual inaccessibility of rv-nsp or its interaction partner to each other during late phase of infection as a result of differential subcellular relocalisation. in agreement with schuster, tholen, overheul, van kuppeveld, and van rij ( ) and schuster, miesen, and van rij ( ) , however, we can make a hypothesis on the physiological relevance of time point-dependent bimodal ago regulation. according to this notion, biphasic ago modulation by rv-nsp relies on the prioritisation preference of the latter to get invested either in crippling antiviral rnai during relatively early phase of infection or antagonising host-triggered antiviral ifn response during the later phase. from the perspective of rv-nsp as a putative vsr, degrading ago to disarm si/shrna-mediated rnai is advantageous during early hours of infection. but as infection progresses and rv dsrnas accumulate above a certain threshold, host pattern recognition receptors (rig and mda ) can start recognising viral replication intermediates as non-self entities and respond by triggering antiviral ifn signalling (broquet, hirata, mcallister, & kagnoff, ) . to counteract this, rv employs nsp to degrade ifn regulatory factors ultimately escaping host immune response. evidence for viral ifn antagonist to also serve as a vsr is strikingly abundant (cárdenas et al., ; hartman, towner, & nichol, ) . vsrs have been reported to interfere at different stages of rna silencing pathways: viral rna sensing and dicing, risc assembly, ago activity/levels, and also by targeting accessory components of risc. most of the vsrs are dsrna-binding proteins that interact with either dicer substrates, sirna duplexes, or both and sequester them away from rna silencing pathway (bronkhorst & van rij, ; schuster et al., ) . in this respect, it is noteworthy that bacterial dsrna-binding proteins can also inhibit rnai pathway nonspecifically by virtue of their binding to dsrnas and thereby withdrawing dsrnas away from rnai machinery. there are a few reports of degradation of ago in case of insect and plant viruses (baumberger, tsai, lie, havecker, & baulcombe, ; nayak et al., ) , but no report is available for mammalian viruses. consistent with the ability of rv-nsp to degrade ago proteasomally, rv-nsp also showed putative vsr property as its ectopic expression prevented sirna-mediated rnai (figure a ; figure s a ). concurrently, shrna-mediated rnai was also found to be functional during early and late hours of a - infection (figure b , figure s b ). only attain high pathogenicity in rnai-defective host cells. nsp mutant rv strain a - has also been reported to have slow growth rate and forms smaller plaques compared with wild-type rv strain a - (taniguchi et al., ; bagchi et al., ) . however, the difference in the virulence phenotype between wild-type a - and nsp mutant a - may arise, at least partially, from activation of premature apoptosis and failure of ifn antagonism. so, it poses an experimental challenge to uncouple attenuated pathogenicity stemming from failure to antagonise ifn and premature apoptosis from that due to failure of rnai inhibition. indeed, as also observed previously during infection, mirna mimics retained their functionality in the presence of rv-nsp (figure c,d) . antiviral potency of ago is widely accepted for plant, insect, and mammalian viruses (alvarado & scholthof, ; carbonell & carrington, ; harvey et al., ) . ago catalytic activity is especially reported to restrict influenza a virus, encephalomyocarditis virus, and vesicular stomatitis virus infection in mammalian cells . in our study too, overexpression of ago but not ago showed potent anti-rotaviral activity (figure a ,b; figure s b-e). this is rationally consistent with recent reports of increased rv infectivity in the absence of ago (dhillon & durgarao, ; oceguera et al., ) . moreover, all the functional domains of ago were found to be important for exerting the best antiviral potency, the catalytic piwi domain being the major determinant scientific) and % (v/v) antibiotic-antimycotic ( -thermo fisher scientific) within humidified °c incubator with % co . cell culture-adapted rv strains sa (simian), a - (bovine), and a - (bovine) were used for this study. unless otherwise mentioned, cells were infected at a multiplicity of infection (moi) as described previously (dutta et al., ) (groene & shaw, ) . for calculating viral infectivity, plaque assay was performed as described previously (dutta et al., ). viral plaque forming units (pfu) were calculated as pfu/ml (of original stock) = /dilution factor × number of plaques × /(ml of inoculum/plate; smith, estes, graham, & gerba, ) . in some experiments, viral infectivity was represented as "percentage of infectivity" of infected test cells compared with infected control cells (infectivity of infected control was considered as %). the ′-utr luciferase reporter construct of p α-pi k was generated by cloning the pcr-amplified human p α mrna ′-utr (mir- b ma and hek cells seeded in four-well chamber slides (bd pharmingen) were treated as mentioned in figure b , s b, b. cells were then fixed with paraformaldehyde ( % [wt/vol] in phosphatebuffered saline) for min at room temperature and further processed as described previously (mukherjee et al., ) . after overnight incubation with primary antibodies (anti-ago , anti-nsp , and anti-vp ) at °c, slides were treated with rhodamine conjugated and lysates from infected/transfected ma /hek cells were subjected to coimmunoprecipitation using pierce co-ip kit ( ) according to the manufacturer's instructions. for resin controls, cell lysates were incubated with antibody-uncoupled resin and further processed according to the protocol. to immunoprecipitate equal amount of lysates, protein concentration was measured by bradford assay (sigma-aldrich) or pierce™ bca protein assaykit (thermo fisher scientific). a % volume of each lysate was kept as input. to analyse rna-independent interaction, antibody coupled resins were incubated with equal amount of cell extracts pretreated with rnase iii ( μg/ml) (brown & gordon, ) . after elution, immunoprecipitates were boiled in sds-page loading buffer for min followed by gel electrophoresis and immunoblot analyses. short to determine cytotoxicity of increasing concentrations of hago gfp overexpression in ma cells, cell viability assay was conducted. all results are presented as mean ± standard deviation from at least three (n ≥ ) independent experiments. statistical significance of data was analysed by mann-whitney test or student's t test using graphpad prism . . statistical significance of data is marked by asterisks ( * p ≤ . ; ** p ≤ . ; *** p ≤ . ). p value of <. was 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genomic constellation rotavirus nsp inhibits expression of type i interferon by antagonizing the function of interferon regulatory factors irf , irf , and irf micrornas: target recognition and regulatory functions metazoan micrornas rna silencing in plants the polerovirus silencing suppressor p targets argonaute proteins for degradation hiv- tat interaction with dicer: requirement for rna evidence that hiv- encodes ansirna and a suppressor of rna silencing rotavirus-encoded nonstructural protein modulates cellular apoptotic machinery by targeting tumor suppressor protein p rotavirus disrupts cytoplasmic p bodies during infection viral rna silencing suppressors (rss): novel strategy of viruses to ablate the host rna interference (rnai) defense system argonaute utilization for mirna silencing is determined by phosphorylation-dependent recruitment of lim-domain-containing proteins the long and short of antiviral defense: small rna-based immunity in insects rig-i/mda /mavs are required to signal a protective ifn response in rotavirus-infected intestinal epithelium the stimulation of pp v-src kinase activity by vanadate in intact cells accompanies a new phosphorylation state of the enzyme the influenza a virus ns protein binds small interfering rnas and suppresses rna silencing in plants small rnas: antiviral rnai in mammals viral suppressors of rna silencing antiviral roles of plant argonautes ebola virus vp protein binds double-stranded rna and inhibits alpha/beta interferon production induced by rig-i signaling rotavirus-induced mir- - p elicits proviral milieu by targeting non-canonical transforming growth factor beta signalling and apoptosis in cells tyrosine phosphorylation modulates mitochondrial chaperonin hsp and delays rotavirus nsp -mediated apoptotic signaling in host cells short hairpin rna-mediated silencing of bovine rotavirus nsp gene prevents diarrhoea in suckling mice the nucleocapsid protein of coronaviruses acts as a viral suppressor of rna silencing 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activation by inducing proteasome-dependent degradation of β-trcp: a novel mechanism of ifn antagonism zinc-binding domain of rotavirus nsp is required for proteasome-dependent degradation of irf and autoregulatory nsp stability interferon regulatory factor is a cellular partner of rotavirus nsp psoralen preparation of antigenically intact noninfectious rotavirus particles theebola virus vp protein is a suppressor of rna silencing rna interference a c-terminal basic amino acid motif of zaire ebolavirus vp is essential for type i interferon antagonism and displays high identity with the rna-binding domain of another interferon antagonist, the ns protein of influenza a virus an antiviral defense role of ago in plants akt-mediated phosphorylation of argonaute downregulates cleavage and upregulates translational repression of microrna targets deletion mapping of the rotavirus metalloprotein ns (nsp ): the conserved cysteine-rich region is essential for virus-specific rna binding rnai in the regulation of mammalian viral infections identification of substrates for f-box proteins nodamura virus nonstructural protein b can enhance viral rna accumulation in both mammalian and insect cells generation of recombinant rotaviruses expressing fluorescent proteins by using an optimized reverse genetics system reverse genetics system demonstrates that rotavirus nonstructural protein nsp is not essential for viral replication in cell culture modification of the trypsin cleavage site of rotavirus vp to a furin-sensitive form does not enhance replication efficiency technical insights into highly sensitive isolation and molecular characterization of fixed and live circulating tumor cells for early detection of tumor invasion quantitative analysis of argonaute protein reveals microrna-dependent localization to stress granules interferon antagonist proteins of influenza and vaccinia viruses are suppressors of rna silencing induction and suppression of antiviral rna interference by influenza a virus in mammalian cells rna interference functions as an antiviral immunity mechanism in mammals the c-terminal half of human ago binds to multiple gw-rich regions of gw and requires gw to mediate silencing argonaute is the catalytic engine of mammalian rnai reduced expression of the rotavirus nsp gene has a pleiotropic effect on virus replication adenovirus va noncoding rna can inhibit small interfering rna and microrna biogenesis rotavirus nsp associates with components of the cullin ring ligase family of e ubiquitin ligases inactivation of the type i interferon pathway reveals long double-stranded rna-mediated rna interference in mammalian cells a lentiviralrnai library for human and mouse genes applied to an arrayed viral high-content screen rotavirus infection induces the phosphorylation of eif α but prevents the formation of stress granules silencing the alarms: innate immune antagonism by rotavirus nsp and vp rotaviral nonstructural protein triggers dynamin-related protein -dependent mitochondrial fragmentation during infection synchronized orchestration of mir- b and let- g positively regulates rotavirus infection by modulating a viral protein restricts drosophila rnai immunity by regulating argonaute activity and stability rotavirus rnas sponge host cell rna binding proteins and interfere with their subcellular localization six rna viruses and forty-one hosts: viral small rnas and modulation of small rna repertoires in vertebrate and invertebrate systems mir- mirnas activate p by targeting p α and cdc ra- , a selective agonist of nrf /are pathway, exerts potent anti-rotaviral efficacy in vitro rotavirus nonstructural protein antagonizes innate immune response by interacting with retinoic acid inducible gene i human virus-derived small rnas can confer antiviral immunity in mammals purified argonaute and ansirna form recombinant human risc protein kinase r is responsible for the phosphorylation of eif α in rotavirus infection the roles of individual mammalian argonautes in rna interference in vivo antiviral actions of interferons interferon-stimulated genes: a complex web of host defenses antiviral rnai in insects and mammals: parallels and differences. viruses deletion of cytoplasmic double-stranded rna sensors does not uncover viral small interfering rna production in human cells egfr modulates microrna maturation in response to hypoxia through phosphorylation of ago genome-wide rnai screen reveals a role for the escrt complex in rotavirus cell entry on the road to reading the rna-interference code antiviral rna interference responses induced by semliki forest virus infection of mosquito cells: characterization, origin, and frequency-dependent functions of virus-derived small interfering rnas rna interference in biology and disease effect of uv-irradiation on rotavirus a plaque assay for the simian rotavirus sa nondefective rotavirus mutants with an nsp gene which has a deletion of nucleotides, including a 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providing catalytic-dead ago mutants (pcdna -ago d a, pcdna -ago d a). dr. asim biswas is duly acknowledged for his technical assistance in confocal microscopy. authors declare no conflict of interest. mamta chawla-sarkar https://orcid.org/ - - - key: cord- -muxrxvyo authors: markotter, wanda; geldenhuys, marike; jansen van vuren, petrus; kemp, alan; mortlock, marinda; mudakikwa, antoine; nel, louis; nziza, julius; paweska, janusz; weyer, jacqueline title: paramyxo- and coronaviruses in rwandan bats date: - - journal: trop med infect dis doi: . /tropicalmed sha: doc_id: cord_uid: muxrxvyo a high diversity of corona- and paramyxoviruses have been detected in different bat species at study sites worldwide, including africa, however no biosurveillance studies from rwanda have been reported. in this study, samples from bats collected from caves in ruhengeri, rwanda, were tested for the presence of corona- and paramyxoviral rna using reverse transcription pcr assays. positive results were further characterized by dna sequencing and phylogenetic analysis. in addition to morphological identification of bat species, we also did molecular confirmation of species identities, contributing to the known genetic database available for african bat species. we detected a novel betacoronavirus in two geoffroy’s horseshoe bats (rhinolophus clivosus) bats. we also detected several different paramyxoviral species from various insectivorous bats. one of these viral species was found to be homologous to the genomes of viruses belonging to the jeilongvirus genus. additionally, a henipavirus-related sequence was detected in an egyptian rousette fruit bat (rousettus aegyptiacus). these results expand on the known diversity of corona- and paramyxoviruses and their geographical distribution in africa. bats (order chiroptera) account for % of all mammalian species and are distributed worldwide. with the advancement in detection techniques and increased surveillance, bats are being increasingly recognized as hosts for many zoonotic viruses [ ] , including filo-, paramyxo-, corona-and lyssaviruses [ ] [ ] [ ] [ ] . regions in africa are considered a hotspot for emerging infectious diseases with more than % of recently emerging diseases originating from wildlife species on this continent [ , ] . although several surveillance studies have been implemented to detect potential zoonotic viruses in bats, including from countries in the congo basin and east africa, limited information is available for rwanda. importantly, in the bordering democratic republic of congo and uganda, marburg and ebola disease outbreaks in humans have occurred [ ] , and corona-and paramyxoviruses have been reported to circulate in bats [ , [ ] [ ] [ ] . coronaviruses are positive-sense rna viruses with the potential to cause respiratory, gastrointestinal, hepatic, and neurological diseases in their hosts [ ] and are divided into four genera namely alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus [ ] . bats host a large diversity of coronaviruses and the expanding research can be largely attributed to the emergence of novel coronaviruses of public health and veterinary importance. three such viruses emerged in the last years, including the severe acute respiratory syndrome (sars) coronavirus in , middle east respiratory syndrome (mers) coronavirus in , and the swine acute diarrhoea syndrome (sads) coronavirus in [ , ] . bat coronaviruses have been shown to be associated with particular bat genera and similar viruses have been identified throughout the geographical distribution of their hosts [ , , ] . diverse bat coronaviruses related to sars coronavirus (now termed the sarbecovirus subgenus) have been identified from the rhinolophus bat genus (horseshoe bats) in asia, europe and africa [ , , ] . continued surveillance within these bats species in china identified lineages of recombinant sars-related coronaviruses nearly identical to human sars coronaviruses, capable of using the same receptor molecules [ ] [ ] [ ] . as a result, these viruses have therefore been postulated to be capable of direct human infection [ , ] . bats from various african countries, including kenya, ghana, nigeria, tanzania, uganda and south africa, have yielded a large diversity of novel coronaviruses [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . some of the bat coronaviruses identified have been shown to be genetically related to known human coronaviruses such as hcov- e, hcov-nl , and mers coronavirus [ , , [ ] [ ] [ ] . paramyxoviruses are negative-sense single-stranded rna viruses capable of infecting a diverse host range including mammals, birds, reptiles and fishes [ ] . the taxonomic classification of viruses in the paramyxoviridae family has recently undergone several changes [ ] . in an attempt to accommodate the rapidly growing number of paramyxoviruses described, the previously known avulavirus and rubulavirus genera were elevated to the sub-family level (avula-and rubulavirinae) each with two new genera. in addition, several unclassified rodent-borne viruses were classified to newly established genera (narmovirus and jeilongvirus) in the sub-family orthoparamyxovirinae to which the henipa-, morbilliand respirovirus genera belong. several zoonotic paramyxoviruses have emerged as important public health threats in the past three decades. the emergence of hendra and nipah viruses (henipavirus genus) during the s in australia and southeast asia respectively, marked the first report of zoonotic paramyxoviruses of considerable public health importance [ , ] . these viruses are characterized by high morbidity and mortality rates and outbreaks have been reported on a near annual basis. in addition, another paramyxovirus, sosuga virus (pararubulavirus genus), emerged as the etiological agent of a single non-fatal human infection contracted in uganda, africa [ ] . the natural wildlife reservoir for these zoonotic viruses was determined to be the fruit bat species occurring in these areas, i.e., flying foxes from the pteropus genus for the henipaviruses [ , ] , and the egyptian rousette bat (rousettus aegyptiacus) for the rubulavirus [ ] . viruses related to the henipaand orthorubulaand pararubulavirus genera as well as a number of unclassified viruses have been described from countries bordering rwanda as well as other african countries [ , , , [ ] [ ] [ ] . r. aegyptiacus, hipposideros spp. and miniopterus inflatus have tested positive for paramyxoviral rna that is closely related to known human pathogens including the henipaviruses, human mumps virus, human parainfluenza virus and human parainfluenza virus [ , ] . in this study, we report the detection of a novel betacoronavirus in rhinolophus clivosus sampled in the ruhengeri cave system in rwanda. in addition, we report on the detection of jeilongvirus and related sequences in hipposideros spp. as well as a henipavirus-related sequence in the fruit bat species r. aegyptiacus. these results expand on the known diversity of these virus groups and their geographical distribution in africa. in december , a team from the university of pretoria, national institute for communicable diseases, and rwanda tourism board and national park authority visited two cave sites in ruhengeri, rwanda (gps coordinates: • . " s • . " e; figure ) where bats were caught at night using mist nets in the surrounding areas, and two bank g forest strainer harp traps (bat conservation and management, inc., usa, australia) at the cave entrances. when collecting samples in the field, personal protective equipment used included tyvek suits (dupont tm , wilmington, de, usa), disposable over gowns (stylianou medisupplies ltd, middle east), m full powered air purifying respirators ( m, maplewood, mn, usa), gumboots (bata industries ® , pinetown, south africa), double layer nitrile gloves (lasec, cape town, south africa) and leather gloves (evrigard, johannesburg, south africa). in december , a team from the university of pretoria, national institute for communicable diseases, and rwanda tourism board and national park authority visited two cave sites in ruhengeri, rwanda (gps coordinates: ° ' . "s ° ' . "e; figure ) where bats were caught at night using mist nets in the surrounding areas, and two bank g forest strainer harp traps (bat conservation and management, inc., usa, australia) at the cave entrances. when collecting samples in the field, personal protective equipment used included tyvek suits (dupont tm , wilmington, de, usa), disposable over gowns (stylianou medisupplies ltd, middle east), m full powered air purifying respirators ( m, maplewood, mn, usa), gumboots (bata industries®, pinetown, south africa), double layer nitrile gloves (lasec, cape town, south africa) and leather gloves (evrigard, johannesburg, south africa). bats were placed in individual cotton bags before processing. bats were morphologically identified [ ] and data including sex, reproductive status, forearm length and weight were also recorded. samples collected from bats included fecal and oral swabs, wing biopsies in % ethanol and blood (serum) for use in viral surveillance studies. oral swabs were collected by gently swabbing the inside of the mouth (cheeks and tongue) with a sterile swab (vwr critical swab, atlanta, ga, usa). fecal material or swabs (vwr critical swab, atlanta, ga, usa) were collected from the bat or the cotton bag, when it was available. sterile foreceps were used to collect fecal pellet(s) and place them in ml microcentrifuge tubes (sarstedt, nümbrecht, germany). urine was collected with a sterile swab (vwr critical swab, atlanta, ga, usa) from individual bats as was available. in instances where bats died during processing, necropsies were performed and various organs and tissues, including kidney, spleen, heart, pectoral muscles, liver, lung, stomach, bladder, tongue, brain and lymph nodes, were collected and placed in ml cryotubes (sarstedt, nümbrecht, germany). all samples were collected in rnalater preservative inactivation solution (qiagen, hilden, germany), stored at °c, then transported to and tested in south africa at the national institute for communicable diseases (nicd) and university of pretoria. permits were obtained from the rwanda development board/tourism & conservation and animal ethics was obtained from the animal ethics committee, university of pretoria. for viral rna detection, rna was extracted from kidney, spleen, urine, fecal, rectal and intestinal samples (table s ). rna from kidney (n = ), spleen (n = ) and urine (n = ), were extracted using the bats were placed in individual cotton bags before processing. bats were morphologically identified [ ] and data including sex, reproductive status, forearm length and weight were also recorded. samples collected from bats included fecal and oral swabs, wing biopsies in % ethanol and blood (serum) for use in viral surveillance studies. oral swabs were collected by gently swabbing the inside of the mouth (cheeks and tongue) with a sterile swab (vwr critical swab, atlanta, ga, usa). fecal material or swabs (vwr critical swab, atlanta, ga, usa) were collected from the bat or the cotton bag, when it was available. sterile foreceps were used to collect fecal pellet(s) and place them in ml microcentrifuge tubes (sarstedt, nümbrecht, germany). urine was collected with a sterile swab (vwr critical swab, atlanta, ga, usa) from individual bats as was available. in instances where bats died during processing, necropsies were performed and various organs and tissues, including kidney, spleen, heart, pectoral muscles, liver, lung, stomach, bladder, tongue, brain and lymph nodes, were collected and placed in ml cryotubes (sarstedt, nümbrecht, germany). all samples were collected in rnalater preservative inactivation solution (qiagen, hilden, germany), stored at • c, then transported to and tested in south africa at the national institute for communicable diseases (nicd) and university of pretoria. permits were obtained from the rwanda development board/tourism & conservation and animal ethics was obtained from the animal ethics committee, university of pretoria. for viral rna detection, rna was extracted from kidney, spleen, urine, fecal, rectal and intestinal samples (table s ). rna from kidney (n = ), spleen (n = ) and urine (n = ), were extracted using the trizol reagent (invitrogen, carlsbad, ca, usa), and fecal material/swabs (n = ), rectal and/or intestinal samples (n = ) (table s ) were extracted using the duet rna/dna extraction kit (zymoresearch, ca, usa) from samples homogenized in µl of phosphate buffered saline (lonza, basel, switzerland). both extraction methods were performed according to the manufacturer's instructions without deviations. dna was extracted using the dneasy blood & tissue kit (qiagen, hilden, germany) from heart tissues. confirmation of species identification of bats, in which viral rna was detected, was performed by amplifying the cytochrome b (cyt b) or cytochrome oxidase one (coi) gene region and determining the dna sequence. selection of cytochrome region for amplification was based on availability of credible comparative sequences in public databases (ncbi genbank and bold). previously reported pcr primers targeting these two regions were used or modified [ ] [ ] [ ] . following the pcr analysis, reactions were subjected to agarose gel electrophoresis on a . % agarose gel (lonza, basel, switzerland) and pcr amplicons were gel-purified using the wizard ® sv gel dna clean-up system (promega, madison, wi, usa) according to the manufacturer's instructions and without deviation. all amplicons were subjected to sanger sequencing for both the forward and reverse reactions on an abi dna sequencer (ae applied biosystems) at the sequencing facility of the university of pretoria. host gene sequences were subsequently compared to bat sequences available in the public domain (on the ncbi genbank and bold databases), results were interpreted and compared with the respective morphological field identifications. fecal, rectal and/or intestinal samples from bats (table s ) were extracted and analysed for coronavirus rna. complementary dna (cdna) was prepared using ng random hexamers (ie hplc purified, integrated dna technologies, coralville, ia, usa) with u superscript iv reverse transcriptase (thermo scientific, waltham, ma, usa). additionally, cdna was treated with u rnase h (thermo fisher scientific) incubation at • c for min and inactivated at • c for min. presence of coronavirus rna was detected with a coronavirus genus-specific hemi-nested rt-pcr assay which targets the rna dependent rna polymerase (rdrp) gene for amplification as described in geldenhuys et al. [ ] . as the hemi-nested rt-pcr assay produced only short amplicons (approximately bp), the rdrp-grouping unit (rgu) assay was used to extend the sequenced region of the identified betacoronaviruses to bp [ ] . a hemi-nested rt-pcr assay was performed using the randomly primed cdna prepared as well as forward primers from drexler et al. [ ] (sp '-ctt ctt ctt tgc tca gga tgg caa tgc tgc- ' and sp '-ata ctt tga ttg tta cga tggt ggc tg- ') in combination with a reverse primer (p beta_rev '-cat crt cas dia rda tca tcat-' ) from the geldenhuys et al. [ ] assay. assay conditions from geldenhuys et al. [ ] were used with modifications to cycling conditions including longer annealing and extension cycles ( cycles of • c for s, • c for s and • c for s). agarose gel electrophoresis and purification of all pcr products were performed as previously described for molecular host species identification. kidney from insectivorous bats, spleen from frugivorous bats and urine from both groups (n = ; table s ) were tested with the use of two broadly-reactive assays targeting the avula-rubulavirinae (ar) sub-families, and the respiro-morbilli-henipvirus (rmh) genera. for both assays, published primers targeting the conserved polymerase (l) gene [ ] were used in combination with adapted two-step hemi-nested rt-pcr protocols. samples were tested with the ar assay as previously described [ ] . for the rmh assay, the samples were tested as previously described [ ] , with minor variations in the protocol. for the first-round pcr, mm mgcl (thermofisher scientific, waltham, ma, usa) was added and the nuclease-free water (ambion, foster city, ca, usa) was adapted for a final reaction volume of µl. all cycling conditions and the protocol for the hemi-nested pcr remained the same as for ar. agarose gel electrophoresis and purification of all pcr products were performed as previously described for molecular host species identification. sequencing was performed as previously described for molecular host species identification. sequences were viewed, edited and a consensus generated using the bioedit sequence alignment editor software version . . [ ] . cipres was used for clustalx alignments, determining the best dna substitution model for nucleotide sequence analysis using the jmodeltest software and for constructing bayesian phylogenies using the beast version . software [ ] [ ] [ ] . bayesian mcmc chains were set to million iterations, sampling every steps for optimal ess scores. output files were visually inspected to check for convergence using the tracer software version . [ ] . the final phylogenies were constructed in treeannotator with a burn-in value of %. for visualization and manipulation of the phylogenetic tree, the figtree version . . software was used. pairwise similarities between sequences were analysed in mega x with complete deletion [ ] . in total, samples from bats constituting five genera were tested for coronaviruses (table and table s ). of these, two samples contained coronavirus rna, originating from two individuals of the rhinolophus genus. barcoding and molecular identification confirmed the host species to be rhinolophus clivosus (table s ). coronavirus sequences were extended to bp with rgu assay primer sets [ ] . the two sequences (rh-btcov/ /rwanda/ and rh-btcov/ /rwanda/ ) share . % nucleotide identity; pairwise similarities and phylogenetic analysis group these sequences with other lineage b betacoronaviruses (figure ). the closest relative to the rwandan betacoronavirus was reported from kenya, btcovky (tao et al. unpublished; genbank accession number ky . ), though the rhinolophus species is not specified. the sequences share very close sequence similarities ( . % nucleotide identity and % amino acid identity), suggesting that similar betacoronaviruses may be harbored by both kenyan and rwandan rhinolophus bats. full genome comparisons will be able to determine if kenyan and rwandan rhinolophus bats are infected by the same betacoronavirus species. within the analyzed conserved rdrp gene segment, this rwandan rhinolophus betacoronavirus also shares pairwise similarities of % nucleotide identity ( . % amino acid identity) to the bulgarian betacoronavirus rh-btcov/bm - /bgr/ [ ] , as well as close similarities ( . - . % nucleic acid similarity and . - . % amino acid identity) to asian rhinolophus sars-related coronaviruses such as sarsr-rh-btcov/rp and sarsr-rh-btcovwiv [ , ] . other coronavirus surveillance in rwanda, and surrounding countries such as uganda and tanzania also report sars-related coronaviruses from the rhinolophus genus [ ] . unfortunately, the sequences cannot be compared as an assay targeting a different conserved peptide of the rdrp gene was used [ ] . boldface indicates positive samples. twenty-four samples from bats were tested for paramyxovirus rna (table s ), none of which tested positive with the avula-rubulavirinae (ar) assay. an overall percentage positivity for paramyxovirus rna, detected using the respiro-morbilli-henipavirus (rmh) assay, was found to be . % (n = ). three of the viral sequences were detected in the insectivorous bat species hipposideros ruber and otomops martiensseni, while one other sequence was detected in the frugivorous bat species rousettus twenty-four samples from bats were tested for paramyxovirus rna (table s ), none of which tested positive with the avula-rubulavirinae (ar) assay. an overall percentage positivity for paramyxovirus rna, detected using the respiro-morbilli-henipavirus (rmh) assay, was found to be . % (n = ). three of the viral sequences were detected in the insectivorous bat species hipposideros ruber and otomops martiensseni, while one other sequence was detected in the frugivorous bat species rousettus aegyptiacus. host identification of positive samples was confirmed using molecular analysis (table s ). phylogenetic analysis of the sequences indicated that the insectivorous bat-borne viral sequences grouped with the jeilongvirus genus as well as in a jeilongvirus-related clade (figure ). one of the h. ruber sequences (batpv/hip_rub/up /rwa/ ) described from this study potentially groups within the jeilongvirus genus. the second h. ruber-derived viral sequence (batpv/hip_rub/up /rwa/ ) grouped with a paramyxoviral sequence detected in a bat from the same genus sampled in cameroon in , however, was not identical. the detection of two diverse viruses from bats of the same species and same population has previously also been reported in insectivorous bats sampled in other african countries [ ] . these observations can in part be explained by the generation of viral quasi-species populations due to the high mutation rate of rna viruses as a consequence of rna proofreading deficiency of the rna dependent rna polymerase [ ] . a larger pool of diverse viruses within a bat population and the co-roosting of several cave-dwelling bat species may facilitate viral sharing between different bat species [ ] . however, ongoing biosurveillance in these cave-dwelling bat species will be required before active viral sharing can be shown. the paramyxoviral sequence detected in the o. martiensseni bat (batpv/oto_mar/up /rwa/ ) was near identical to the viral sequences previously described from several individuals of the same species sampled in kenya in [ ] . these sequences shared a . % similarity on both nucleotide and amino acid level. the r. aegyptiacus-derived viral sequence (batpv/rou_aeg/up /rwa/ ) grouped within a henipavirus-related clade and was near identical to a paramyxoviral sequence detected in the same host species previously reported from kenya [ ] . sequence similarity shared between these two sequences was found to be . % and . % on nucleotide and amino acid level, respectively. to our knowledge, this study reports on the first evidence of paramyxovirus rna in bats from rwanda. two of the four viral sequences detected in h. ruber, were not closely related to the paramyxovirus sequences previously reported (sharing nucleotide and amino acid similarities of less than % and . %, respectively) and might represent novel viral species. however, a more rigorous analysis with variable genes such as the fusion and hemagglutinin gene will be required before putative species can be inferred. as observed in previous studies, viral sequences from frugivorous bats were mostly found to belong to the henipavirus genus or a related clade, while insectivorous bat-associated viral sequences have been linked to other genera including morbilliand jeilongvirus [ ] . this observation was again reflected in the current study. the detection of highly similar viral sequences from bats in rwanda and kenya, which are more than km apart, can be explained by either the phenomena of metapopulations or the hypothesis of co-evolution of paramyxoviruses with their bat hosts [ , , ] . rna viruses have an exceptionally high mutation rate commonly associated with quasi-species populations and with the potential to cross the species barrier, among others. this is evident in the high diversity of paramyxoviruses described to date and the wide host range associated with these viruses [ , , , ] . additionally, due to the emergence of sars, mers and sads, it is widely accepted that coronaviruses are capable of readily adapting to new hosts [ ] . the rwandan caves are considered an ecotourism site and guano is also mined on a small scale, providing an ideal bat-human interface. several insectivorous bat species co-roost with the egyptian fruit bats in these caves and future studies should investigate viral sharing. the egyptian fruit bat also uses these caves as a maternity roost and studies have shown that increased viral shedding is linked to reproductive cycles [ ] . longitudinal biosurveillance studies can therefore identify high risk periods in the future. as such, the detection of a sars-related bat coronavirus potentially circulating within the rhinolophus population and a henipavirus-related paramyxovirus in r. aegyptiacus in the ruhengeri region may merit further investigation to determine exposure, and the potential for spill-over events to occur. to date, emphasis of paramyxovirus surveillance has mostly been placed on fruit bats, the henipavirus genus and related viruses due to the association of other henipavirus species with zoonotic events [ , ] . however, research regarding the zoonotic potential of the insectivorous bat-associated viruses is still lacking. one major aim regarding surveillance of wildlife populations is to identify potential zoonotic agents and to evaluate any threat to the public as well as domestic animal health. though these bat-borne viruses are unlikely to pose a significant threat, it still merits continued monitoring of the chiropteran species within these caves as well as mammalian species that inhabit the surrounding area. , however, was not identical. the detection of two diverse viruses from bats of the same species and same population has previously also been reported in insectivorous bats sampled in other african countries [ ] . these observations can in part be explained by the generation of viral quasi-species populations due to the high mutation rate of rna viruses as a consequence of rna proofreading deficiency of the rna dependent rna polymerase [ ] . a larger pool of diverse viruses within a bat population and the coroosting of several cave-dwelling bat species may facilitate viral sharing between different bat species [ ] . however, ongoing biosurveillance in these cave-dwelling bat species will be required before active viral sharing can be shown. the paramyxoviral sequence detected in the o. martiensseni bat (batpv/oto_mar/up /rwa/ ) was near identical to the viral sequences previously described from several individuals of the same species sampled in kenya in [ ] . these sequences shared a . % similarity on both nucleotide and amino acid level. the r. aegyptiacus-derived viral sequence (batpv/rou_aeg/up /rwa/ ) grouped within a henipavirus-related clade and was near identical to a paramyxoviral sequence detected in the same host species previously reported from kenya [ ] . sequence similarity shared between these two sequences was found to be . % and . % on nucleotide and amino acid level, respectively. for countries where the bat-human interface is more pronounced, as a result of ecotourism, guano mining or bat hunting and consumption, surveillance is key to identify the diversity of viruses present and their potential host species. longitudinal and well-structured surveillance programmes would better characterize the circulation and shedding periods of the paramyxo-and betacoronaviruses within the ruhengeri cave system. such information would be most valuable towards well-considered zoonotic disease risk assessments and mitigation strategies. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , table s : bats collected and tested in this study. host and viral traits predict zoonotic spillover from mammals filoviruses in bats: current knowledge and future directions bats host major mammalian paramyxoviruses bat lyssaviruses global epidemiology of bat coronaviruses global capacity for emerging infectious disease detection emerging infectious diseases of wildlife-threats to biodiversity and human health diagnostics for filovirus detection: impact of recent outbreaks on the diagnostic landscape a recently discovered pathogenic paramyxovirus, sosuga virus, is present in 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of the southwest indian ocean we would also like to thank the staff of the rwanda tourism board and national parks and wendy white from the kwazulu-natal bat interest group for assisting in the logistics and fieldwork. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. key: cord- -kzmch j authors: yang, li; zhang, jia hao; zhang, xiao li; lao, guang jie; su, guan ming; wang, lei; li, yao lan; ye, wen cai; he, jun title: tandem mass tag-based quantitative proteomic analysis of lycorine treatment in highly pathogenic avian influenza h n virus infection date: - - journal: peerj doi: . /peerj. sha: doc_id: cord_uid: kzmch j highly pathogenic h n influenza viruses (hpaiv) cause rapid systemic illness and death in susceptible animals, leading to a disease with high morbidity and mortality rates. although vaccines and drugs are the best solution to prevent this threat, a more effective treatment for h strains of influenza has yet to be developed. therefore, the development of therapeutics/drugs that combat h n influenza virus infection is becoming increasingly important. lycorine, the major component of amaryllidaceae alkaloids, exhibits better protective effects against a/ck/gd/ / (h n ) (gd ) viruses than the commercial neuraminidase (na) inhibitor oseltamivir in our prior study. lycorine demonstrates outstanding antiviral activity because of its inhibitory activity against the export of viral ribonucleoprotein complexes (vrnps) from the nucleus. however, how lycorine affects the proteome of aiv infected cells is unknown. therefore, we performed a comparative proteomic analysis to identify changes in protein expression in aiv-infected madin-darby canine kidney cells treated with lycorine. three groups were designed: mock infection group (m), virus infection group (v), and virus infection and lycorine-treated after virus infection group (l). the multiplexed tandem mass tag (tmt) approach was employed to analyze protein level in this study. in total, , proteins were identified from the three groups of cells by using tmt proteomic analysis. in the v/m group, , proteins were identified, of which differentially expressed proteins (deps) were determined during hpaiv infection; among the , proteins identified from the lycorine-treated group, proteins presented significant change. here, proteins showed significant upregulation or downregulation of expression in the virus-infected/mock and virus-infected/lycorine-treated comparisons, and the proteins in each fraction were functionally classified further. interestingly, lycorine treatment decreased the levels of the nuclear pore complex protein (nup , e rsv ), which is associated with nuclear–cytoplasmic transport. in addition, western blot experiments confirmed that the expression of nup was significantly downregulated in lycorine treatment but induced after viral infection. our results may provide new insights into how lycorine may trap vrnps in the nucleus and suggest new potential therapeutic targets for influenza virus. highly pathogenic influenza a viruses resulting from routine seasonal epidemics and global pandemics continue to circulate in nature. it is an extremely contagious and aggressive disease that causes rapid systemic illness and death in susceptible birds. moreover, certain strains, such as h n subtypes, are capable of cross-species transmission and thus can infect humans with high morbidity and mortality (sun et al., ) . the new subtypes have emerged through accumulation and antigenic shift (zhu, wang & wang, ) , which may result in rare influenza a virus pandemics. resistance and timeliness to clinical antiviral therapy are the major problems in curing these infections (blanton et al., ; hu et al., ) . m inhibitors, such as amantadine and ramantadine, cannot protect the host from virus infection because of its amino acid mutation (wang et al., ) . to date, oseltamivir is the only commercial drug effective in human, where it inhibits na activity. however, drug-resistant strains have emerged because of one single amino acid residue substitution (h y in n ) (yusuf et al., ) . therefore, novel influenza inhibitors must be developed. virus-host interactions involve a complex interplay of host cellular and viral networks. the discovery of host factors that may regulate viral replication is a promising approach. in this respect, genomic and proteomic studies have provided abundant information on host genes and proteins associated with viral infection and pathogenesis (wang et al., ; zhang et al., ) . the mode of rnp exit of influenza viruses from the nucleus complements active crm -dependent export mechanisms via the nuclear pore complex (npc) and ensures the efficient production of infectious virus progeny (muhlbauer et al., ) . some of the host factors involved in viral replication and/or pathogenesis could be targeted for the treatment of aiv infection without obvious side effects. traditional chinese medicines (tcms) contain effective components that deal with different types of diseases, such as malaria and influenza a virus (crunkhorn, ; nonaka et al., ) . lycorine is a tcm that exhibits acetyl-cholinesterase-inhibitory and butyryl-cholinesterase-inhibitory activities (wang et al., ) . in russia, it is clinically used as an expectorant to treat chronic and acute inflammatory processes in lungs and bronchial diseases (henry et al., ) . over the past years, lycorine has attracted great research interest owing to its superlative biological potential and pharmacological actions, including antiangiogenic, antiviral, antibacterial, antimalarial, anti-parasite, antioxidant, hepatoprotective, analgesic, anti-inflammatory activities, and inhibition of ascorbic acid synthesis (kim et al., ; lamoral-theys et al., ; cedron et al., ; giordani et al., ; citoglu et al., ; ye et al., ; wang et al., ; cembrowska-lech & kepczynski, ; bendaif et al., ) . different drugs may affect different molecular pathways that may be affected within disease models. for instance, several studies have shown that lycorine induces apoptosis to suppress the propagation of carcinoma cells (li et al., ; yu et al., ) . other studies have demonstrated that its antitumor effect is through inhibiting inflammatory factors and suppressing p and stat activation (kang et al., ) . although lycorine exhibits a wide range of biological activities, the mechanism underlying its effects remains unclear. in our previous study, the efficacy and inhibitory effects of lycorine (ec = . µm) were determined in madin-darby canine kidney (mdck) cells. results showed that lycorine can completely prevent h n infection as indicated by the absence of any cytopathic effect (he et al., ) . unlike the mechanism of oseltamivir, the vrnps are retained in the nucleus by lycorine treatment instead of directly targeting viral proteins and rna polymerase activity (he et al., ) . these results strongly suggest the potential of lycorine as an antiviral agent for h n strain. however, the molecular mechanism of the antiviral/inhibitor action of lycorine remains unknown. therefore, we performed a comparative proteomic analysis to determine the effects of lycorine at the protein level in gd -infected mdck cells to understand its mode of action. gd (n . ay - ) and mdck cells were obtained from the key laboratory of veterinary vaccine innovation of the ministry of agriculture, p. r. china. the mdck cells were cultured in dulbecco's modified eagle's medium (dmem, invitrogen) containing % (v/v) fetal bovine serum (fbs, gibco). a plaque assay was used to determine viral activity in mdck cells (he et al., ) . three separate biological cell cultures were made for the tandem mass tag (tmt) proteomic experiments. lycorine (fig. a ) was obtained as previously described (wang et al., ) . all experiments involving live h n influenza virus were carried out in biosafety level- facilities. three groups were designed: ( ) m, mock group (control); ( ) v, virus-infected group. they were then inoculated at multiplicity of infection (moi) for h (v), removed, and inoculated with dmem containing % fbs until h post-infection (h.p.i.); and ( ) l, lycorine-treated post-viral infected group. after h adsorption of moi gd , lycorine was added at . µm for h.p.i. for each sample, the proteins were diluted in µl of sdt buffer (containing % sds, mm tris-hcl, and mm dtt, ph . ) following a standardized protocol (wisniewski et al., ) , homogenized, heated at • c for min, and then centrifuged at , × g for min at room temperature. each sample (one µl) was taken and quantified using the bicinchoninic acid method. the remaining lysate was frozen at − • c until use. protein digestion was conducted using the fasp procedure (wisniewski et al., ) . in brief, mg of proteins were loaded onto an ultrafiltration filter ( kda cutoff; sartorius, cells from each group were harvested, underwent trypsin digestion, and then labeled by tandem mass tag (tmt)- plex, and high-ph reverse-phase liquid chromatography was used to fractionate the pooled tmt-labeled peptide mixtures before nano liquid chromatography-mass spectroscopy tandem (lc-ms/ms) analysis. all raw files were searched together using proteome discover . . the quantification data matrix was further used for statistical trend analysis and visualization by trelliscope. finally, western blot experiments were conducted for trend verifications. full-size doi: . /peerj. / fig- goettingen, germany) containing µl of ua buffer ( m urea, mm tris-hcl, ph . ), centrifuged at , × g for min, and then washed with µl of ua buffer. then, µl of mm iodoacetamide in ua buffer was subsequently added to the filter to block reduced cysteine residues. the samples were incubated for min at room temperature in the dark and then centrifuged at , × g for min. the filters were washed three times with µl of ua buffer and then centrifuged at , × g for min after each washing step. next, µl of dissolution buffer (applied biosystems, foster city, ca, usa) was added to each filter and then centrifuged at , × g for min, which was repeated three times. the protein suspensions were then digested with µl of trypsin (promega, madison, wi, usa) buffer ( µg trypsin in µl of dissolution buffer) at • c for h. finally, the filter unit was transferred to a new tube, and µl of dissolution buffer was added followed by centrifugation at , × g for min. the resulting peptides were collected as a filtrate, and the peptide concentration was analyzed at od . nine serial samples from each subject and the common reference sample were included in one tmt labeling experiment set as shown in fig. c . this labeling strategy avoids the potential for missing value issues resulting from data-dependent ms/ms acquisitions (zimmer et al., ) . tmt-labeled peptides were subjected to high-ph reversed-phase fractionation in series hplc value system (agilent) equipped with a gemini-nx (phenomenex, f- -e ) column ( . × mm, µm, Å) (wang et al., ; batth, francavilla & olsen, ) . the peptides were eluted at a flow rate of . ml/min. buffer a consisted of mm ammonium acetate, ph . , and buffer b consisted of mm ammonium acetate, % v/v acetonitrile (acn), ph . . both buffers were filter sterilized. the gradient to perform the separation consisted of several steps: ( ) % buffer a for min, %- % buffer b for min; ( ) %- % buffer b for min; ( ) %- % buffer b for min; ( ) %- % buffer b for min; ( ) %- % buffer b for min; ( ) % buffer b for min; and ( ) % buffer a for min. the elution process was monitored by measuring absorbance at nm, and fractions were collected every . min. the collected fractions (approximately ) were finally combined into pools. each fraction was concentrated via vacuum centrifugation and reconstituted in µl of . % v/v formic acid. all samples were stored at − • c until lc-ms/ms analysis. the labeled samples were analyzed using the easy-nlc nanoflow hplc system connected to the thermo scientific tm orbitrap fusion tm tribrid tm mass spectrometer (thermo fisher scientific, san jose, ca, usa). a total of µg of each sample was loaded onto the thermo scientific easy column (two columns) using an autosampler at a flow rate of nl/min. sequential separation of peptides on the thermo scientific easy trap column ( µm × cm, µm, Å, c ) and analytical column ( µm × cm, µm, Å, c ) was accomplished using a segmented min gradient from % to % solvent b ( . % formic acid in % acn) for min followed by %- % solvent b for min and then % solvent b for min. the column was re-equilibrated to its initial highly aqueous solvent composition before each analysis. the mass spectrometer was operated in a positive ion mode, and ms spectra were acquired over a range of - , m/z. the resolving powers of the ms and ms/ms scans at m/z for the fusion were set as , and , , respectively. the data-dependent mode was top speed, cycle time was s, and ions were fragmented through high energy collisional dissociation. the maximum ion injection times were set at ms for the survey scan and ms for the ms/ms scans, and the automatic gain control target value was set to e for ms and to e for ms/ms. the dynamic exclusion duration was s. all raw files were analyzed using the proteome discoverer . software (thermo fisher scientific). a search for fragmentation spectra was performed using the mascot search engine (matrix science, london, uk; version . ) embedded in proteome discoverer against the uniprot canidae protein sequence database (released in november , , sequences). several search parameters were used: ( ) monoisotopic mass; ( ) trypsin as the cleavage enzyme; ( ) two missed cleavages; ( ) tmt labeling; ( ) cysteine carbamidomethylation as fixed modifications; ( ) peptide charges of +, +, and +; and ( ) methionine oxidation. the mass tolerance was set to ppm for precursor ions and to . da for the fragment ions. peptide spectral matches were filtered to a % false discovery rate. the relative quantitative analysis of the sample proteins was based on tmt reporter ion ratios from all unique peptides representing each protein. this analysis was performed using proteome discoverer (version . ). the relative peak intensities of the tmt reporter ions released in each of the ms/ms spectra were used. the final ratios obtained from the relative protein quantifications were normalized based on the median average protein quantification ratio. only unique peptides obtained with a confidence percentage of > % were included in the ratio ≥ . , or ≤ . -fold cutoff value was used to identify upregulated and downregulated proteins with p < . . the mass spectrometry proteomic data have been deposited to the proteomexchange consortium via the pride partner repository with the dataset identifier pxd lycorine (accession number pxd ). protein-protein interaction (ppi) analysis of dep proteins string (search tool for the retrieval of interacting genes/proteins) version . was employed in this study for the potential ppi analysis of the deps proteins (http://string-db.org/) (szklarczyk et al., ; shannon et al., ) . the parameter for confidence score was set to . , and the yield ppi results were visualized by cytoscape software (shannon et al., ) . the light blue nodes indicated upregulated-proteins and the light green nodes indicated downregulated-proteins. the edge thickness was proportional to the combined score of the proteins. bioinformatics and enrichment analysis was performed as previously described (hui bin huang, ) . cells were homogenized on ice for min in × ripa buffer with cocktail protease inhibitors (roche) and then centrifuged at , × g for min at • c. the supernatant was collected and then stored at − • c. for western blot analysis, equal amounts of proteins were separated using sds-page and subsequently transferred onto a polyvinyl fluoride membrane (bio-rad). the membrane was blocked with % bsa and then incubated with monoclonal antibody nup (ab ) and monoclonal antibody antiβ-actin (cell signaling technologies) at • c overnight. membranes were washed in phosphate-buffered saline with tween and then incubated for h at rt with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody ( : , ; cell signaling technologies). statistical analyses were conducted as previously described (hui bin huang, zhang & li, ) . the concentration and exposure time of lycorine were determined to avoid any cytotoxic effects on the host cells and to ensure that the proteome of the host cells reflected the true response to the presence of lycorine. a prior study found through cck- assay that . µm lycorine is not toxic to cells at h post-infection (h.p.i.) (he et al., ) . in addition, the time of lycorine addition was optimized. results showed that the earlier lycorine was added, the better effect on viral titers. therefore, a single replication cycle of influenza virus ( h.p.i.) within lycorine treatment at . µm was chosen because it reflects a single viral replication and is a non-toxic concentration of lycorine at the time addition assay. dose-effect relationship experiments showed that inhibition of lycorine of aiv becomes invalid within high-dose virus (jun he et al., ; he et al., ) . hence, we ensured that the response of the whole cell proteome is caused by early infection and that the activity of lycorine is effective. thus, moi was selected as the minimum infectious dose for a single cycle of viral infection for studying the single viral replication cycle (peschel et al., ) . therefore, moi of aiv infections, lycorine concentration of . µm, and h of incubation time were selected as the optimal dose and exposure time for further experiments. in this study, we applied a tmt method, which has been widely applied for quantitative proteomic cell biology studies and many model organisms, to explore potent anti-viral agents and different signaling pathways (lombardi et al., ; burton et al., ) . a schematic of the experimental workflow and venn diagram of v/m, l/v, and l/m is shown in fig. c . each experiment group consisted of three biological replicates (n = ). in total, , proteins were identified and relatively quantified at . % fdr in the proteome using maxquant (table s ). among the statistically significant proteins detected by anova (p < . ), protein abundances that changed < . -fold, > . -fold, and p > . were discarded. the proteome profiles of mock control and infected cells were compared (v/m) to determine the effects of aiv infection on mdck cells. among those protein groups, proteins were identified, of which deps were determined in the v/m group. the proteome profiles of the infected cells and lycorine-treated infected cells were compared to determine the effects of lycorine treatment on the infected cells. a total of proteins were identified in the v/l group, of which were deps. these results indicated wide differences of protein expression in the hpaiv infection and lycorine treatment. inoculating with hpaiv and lycorine resulted in identified deps ( + + + + + + ) among the three groups of mdck cells in response to hpaiv infection and lycorine treatment, as shown in the venn diagram (fig. b) . the proteome profiles of the gd -treated mdck cells and mock control were compared (v/m) to determine the effects of gd influenza virus on mdck cells. the v/m group contained upregulated (> . -fold) and downregulated (to < . -fold) proteins in the v/m group (table s ) . some of the proteins in the same network were direct interaction or through intermediate partner at the ppi level, as shown in fig. s . gene ontology analyses of the proteins upregulated and downregulated by gd were performed to map the genes involved in different cellular processes, including biological and functional events ( fig. a) . a large number of biological process go terms were identified. the deps were strongly represented by ''catabolism, metabolism, and physiological process''; the infected cells were also assigned to numerous molecular functions, of which ''binding, catalytic and molecular transducer activities'' were the main ones, and cellular components, of which ''extracellular region part, organelle and membrane'' were dominant. go enrichment analysis was conducted on cell periphery ( proteins), plasma membrane ( proteins), intrinsic component of membrane ( proteins) and molecular transducer activity ( proteins) to further study the impact of deps in cell physiological processes and discover their internal relations (fig. b) . several identified proteins have known interactions with one another and function in the same biochemical pathways. therefore, a kegg pathway-based enrichment analysis was applied to identify the main pathways that were potentially affected by the deps in the v/m group (fig. c) . the enriched pathways showed that the proteins were involved in pathway in cancer (path: ko ), extracellular matrix (ecm)-receptor interaction (path: ko ), focal adhesion (path: ko ), mitogen-activated protein kinase signaling pathway (path: ko ), and pi k-akt signaling pathway (path: ko ). these data indicated that there are many changes in the protein profile in response to influenza virus infection at h.p.i. the proteome profiles of the infected cells and the infected cells within lycorine treatment were compared (v/l) to determine the effects of lycorine treatment on the infected cells. in total, deps were found (table s ). the ppi network was performed to determine whether these deps interact with each other and form protein complexes (fig. s ) . twohundred six proteins with at least . -fold upregulation between the lycorine-treated and virus control group were found (v/l), and proteins were significantly downregulated. the go terms of deps were strongly represented by ''cellular process, single-organism process, and biological regulation'' in the biological process and ''binding, catalytic and molecular transducer activities'' in molecular functions; the lycorine-treated cells were also assigned to numerous cellular components, of which ''membrane-enclosed lumen, organelle and membrane'' were dominant (fig. a) . go enrichment analysis showed that the three main parts were nuclear division ( proteins), chromosome segregation ( proteins), and condensed chromosome (eight proteins) (fig. b) . the functional classification of deps was conducted by kegg enrichment analysis, and each protein was assigned to at least one of the following pathways: human t-lymphotropic virus- infection pathway (path: ko ), cell adhesion molecules (cams) (path: ko ), epidermal growth factor receptor tyrosine kinase inhibitor resistance (path: ko ), janus kinase-stat signaling pathway (path: ko ), and pancreatic cancer (path: ko ) (fig. c) . these results showed differences between v/m and v/l, and further analyses of these genes that were co-changed in the two groups may shed light on the antiviral mechanism of lycorine. the proteins co-regulated in v/m and v/l were selected for further analysis. these deps were investigated and annotated using go terms and subjected to go functional analysis (table s ) . fifty-four proteins were upregulated in the v group but downregulated in the l group; this finding indicated that some of the proteins may participate in viral infections and may be blocked by lycorine to avoid progeny virus budding. seventeen proteins were downregulated in the v group but induced in the l group, implying that these proteins were downregulated after influenza virus infection but increased after lycorine treatment. a total of proteins showed upregulated or downregulated difference in abundance, and a heat map of these proteins was obtained (figs. a and c). all co-upregulated and co-downregulated proteins were further analyzed by go enrichment analysis, and each protein belonged to at least one term. among the upregulated proteins, the three main terms in the bp category were cellular process ( proteins), metabolic process ( proteins), and organic substance metabolic process ( proteins). the main terms in the mf category were transcription factor activity ( proteins) and nucleic acid binding transcription factor activity ( proteins). the main terms in the cc category were membrane part ( proteins) and cell junction ( proteins). in the downregulated proteins, the main terms in the bp category were localization ( proteins), immune system process ( proteins), and reproduction ( proteins). the main terms in the mf category were structural molecule activity ( proteins) and binding ( proteins). the main terms in the cc category were extracellular region ( proteins) and membrane part ( proteins) (figs. c and d ). the analysis suggested that these biological functions may be affected by the treatment of aiv-infected mdck cells with lycorine. in addition, pathway analysis showed that these proteins were involved in viral replication-related pathways. for instance, oligoadenylate cyclase (oasl ), serine/threonine protein kinase (stk ), and other proteins ( / ) were involved in adenosine and guanine triphosphatase (atp and gtp) activity-related pathways; other pathways such as nup , mitochondrial translational initiation factor (mtif ), and other proteins ( / ) participated in host cell responses to lycorine-related transcription or translation processes, such as cellular responses to stimulus and oxoacid-related metabolic processes. this may or may not be related to the antiviral properties of lycorine. according to our prior study, vrnp is retained in the nucleus under conditions of lycorine treatment to stop the cycle of influenza virus, which leads us to search for the proteins involved in the vrnp export pathway (he et al., ) . for several pathways relevant to proteins targeting nuclear export were enriched in the ''biological process'' category. the proteins with the same trend in the v/m and v/l groups were our candidate proteins. fortunately, a gene directly related to nuclear transport, nup , was upregulated by . -fold, in contrast to the low expression level ( . ) found in lycorine treatment over controls (table s ) . from the go terms, nup is related with export of material from the nucleus, such as mrna, trna, viral material, and viral transcription (table s ) . given its possible role in viral inhibition (muhlbauer et al., ) , western blotting assay with specific antibodies was performed to determine if the expression regulation for these proteins is at the protein level. as shown in fig. , the trends in nup level changes in the infected cells and lycorine treatment groups were similar to the changed patterns as was observed. moreover, the protein levels of nup in the uninfected cells but treated with the same concentrations of lycorine were not changed as such as the cells treated with lycorine after influenza virus infection. these results indicate that nup expression was induced after influenza virus infection but was dramatically decreased after lycorine treatment at . µm. this concentration did not exhibit toxicity to the uninfected cells, which was in accordance with the tmt results. as a result, aiv infection may induce nup to complete the viral cycle, and the process of protein targeting into nup after lycorine treatment may partly be explained by the blockage of vrnps in the host cellular nucleus. an effective drug for hpaiv treatment is still a worldwide problem (arai et al., ) . our previous study found that lycorine exhibits stronger anti-influenza activity against gd than oseltamivir and retains vrnp in the nucleus (he et al., ) . hence, a comprehensive description of changes in viral and cellular proteins during aiv infection and lycorine treatment based on multiplex tmt-based quantitation was carried out. the interaction of the host cellular proteins with lycorine and the factor of nucleus transport are two of our main research interests. proteomics is the large-scale study of molecules known as proteins, the critical building blocks of both host and viruses (drayman et al., ; kim et al., ; kleiner et al., ; vasilijevic et al., ) . influenza virus modifies and hijacks numerous processes and cell organelles during its replication cycle (de castro martin et al., ) ; it also regulates the subcellular localization of protein complexes. in the present study, these phenomena were observed in the hpaiv-infected results ( fig. and table s ) in conjunction with the total number of significantly regulated proteins ( proteins). on the basis of the numbers of proteins being significantly modulated and the pathways associated with those proteins, the gd influenza virus induced more profound responses to ecm-receptor interaction, focal adhesion, and pi k-akt signaling pathway through kegg pathwaybased enrichment analysis. yek et al. ( ) showed that the major function modulated by myxovirus (influenza virus) resistance infection is predominantly enriched in the ecm-receptor interaction and focal adhesion pathways. liu et al. ( ) showed that infection with a h n hpaiv strain with an moi of . cannot infect much of the cells in their study. thus, moi or higher infection doses are better for studying one single viral replication cycle. considering the dose effects and efficiency of lycorine and gd infection, we chose moi for the single cycle. therefore, a suitable time point ( h.p.) must be selected to harvest virus-infected cells that have been treated by lycorine and can be used for proteomic analysis. after endocytic cell entry, vrnps are released into the cytoplasm and enter the nucleus where viral mrna synthesis and replication occur. the viral genomes are encapsulated with nucleoproteins (nps), and they are associated with trimeric polymerase complexes (the viral proteins m , nep/ns , and np) and recognized by crm to the cytoplasm via the npc (flatt & greber, ) . npc is a highly conserved protein complex that is localized at the nuclear periphery and required for the import and export of proteins and rna (labade, karmodiya & sengupta, ) . the influenza virus could induce proteins and pathways that it uses on dependently, replicate its genome in the nucleus, and export it to the cytoplasm via npcs. the nuclear pores promote the nuclear export of newly synthesized rnps, which is an important process that regulates cellular functions and facilitates viral npc assembly. muhlbauer et al. ( ) proved that virus-induced cellular caspase activities cause a widening of nuclear pores, thereby facilitating nucleocytoplasmic translocation. however, they focused on another npc protein, nup , which is correlated with the import and export pathway for influenza virus infection. in the present study, the expression of nup was upregulated by . -fold after gd infection at h. however, the different npc proteins and different functions render direct comparison with our results impossible. the mdck cells infected with gd exhibited tremendous changes in the levels of many proteins and pathways. interestingly, the npc protein nup was confirmed to be directly related to the export of mrna/trna from the nucleus. we will pay close attention to the changes in nup expression after lycorine treatment. lycorine, the main constituent from lycoris radiata bulbs, exhibits a wide range of biological activities, including antiviral (masi et al., ), antimalarial (cho et al., , antibacterial (bendaif et al., ) , anti-parasitic and anti-inflammatory (park, ) . the first reported activity of lycorine is the inhibition of the termination of protein synthesis in poliovirus infection (vrijsen et al., ) . subsequent studies found that lycorine exhibits antiviral activity toward herpes simplex virus (renard-nozaki et al., ) , hiv- (lin et al., ) , coronavirus (li et al., ) , poliovirus (hwang et al., ) , west nile virus, dengue and yellow fever viruses (zou et al., ) , enterovirus (liu et al., ) , influenza virus (he et al., ) , hepatitis c virus (guo et al., ) , and adult zika virus (masi et al., ) . although lycorine is a compound with various antiviral activities, the molecular mechanism underlying the effects of lycorine is still unclear. compared with other pharmacological activity mechanisms, studies on anti-cancer activity have gained deep insights (lamoral-theys et al., ) . potential targets for lycorine action include bcl- family proteins bcl- and mcl- , hdac, tnf-α, stat, and hmgb . however, no specific target for lycorine-induced anticancer effect has been identified so far. in the present study, it is most obvious by examination of lycorine treatment after hpaiv-infected results ( fig. and table s ). on the basis of proteins being significantly modulated and the pathways associated with those proteins, the lycorinetreated cells induced more profound responses to cams, egfr-related pathway, and jak-stat signaling pathway through kegg pathway-based enrichment analysis. shen et al. authenticated that lycorine directly interacts with egfr and inhibits egfr activation (shen et al., ) . hu et al. ( ) and jin et al. ( ) showed that lycorine inactivates the jak-stat signaling pathway to inhibit the proliferation of cancer cells. furthermore, our present results agree with these specific signaling pathways. in addition, go enrichment analysis showed that proteins involved in nuclear division were differently expressed upon lycorine administration. notably, nup expression was decreased upon lycorine treatment. the deps that were co-upregulated or co-downregulated in both v/m and v/l groups were selected as candidates ( fig. and table s ). among them, candidate proteins were increased by gd infection but decreased by lycorine treatment. viral infection played a significant down-modulatory role to the candidate proteins that were upregulated by lycorine. however, the deps detected in the current study hardly match those determined by silac analysis conducted in and published in (hui bin huang, ) by our group. this result might be due to the different instruments applied and databases used. the mass spectrometer q-exactive was applied for silac experiment in the previous study, whereas fusion-lumos instrument was used in the present study. at present, we found that nup protein was inhibited by lycorine treatment, which aroused our great interest. the same topic will be the focus of our follow-up work. to explore how lycorine affects nucleus transport, we analyzed the protein levels of nup by western blot assay and found that nup levels were increased after hpaiv infection but lowered with lycorine treatment. additionally, nup had the same levels in both lycorine control group and mock group. lycorine significantly reduced nup expression after influenza virus infection, which may have affected nucleocytoplasmic transport instead of lycorine toxicity in the host cells. npcs are composed of approximately proteins known as nucleoporins. nup is one of the major subcomplexes of the npc and is responsible for the correct assembly of the npc (sachdev et al., ) . given its time-of-addition effect, lycorine is involved in the early steps of influenza virus replication aside from virus binding and viral rnp activity (he et al., ) . nucleocytoplasmic transport is integral to the majority of the influenza virus explicative cycle and critical for the efficient replication of influenza virus. importantly, shuttling of specific proteins out of the nucleus is essential for the regulation of the basic functions of the host cells. the functions of nucleo-cytoplasmic transport in regulating tumor growth, cell cycle, and apoptosis have become the therapeutic target of cancer (gravina et al., ; hill et al., ; rosebeck et al., ) . recent data have indicated that viral genome transport through the npc is not always smooth sailing but can be blocked. this result may be the basis for host defense mechanisms that evoke an intrinsic antiviral response (flatt & greber, ; kirli et al., ) . furusawa, yamada & kawaoka ( ) showed that viral rna accumulates in the nucleus of nup -depleted cells. this observation suggests that nup is involved in the nuclear export of viral rna of the viral life cycle. a deeper understanding of how aiv rna is exported from the nucleus via npcs will thus help in the development of new antiviral drugs. lycorine is an inhibitor of influenza virus that effectively inhibits aiv infection. comparative proteomic analysis revealed that treatment with lycorine alters the expression of a number of proteins in aiv-infected cells. this finding suggests that lycorine affects the protein expression in aiv-infected cells. among the deps that were modulated in both v/c and v/l groups, candidate targets were predicted to collectively inhibit viral infection (pathway analysis). go and kegg pathway analyses revealed that nup , as an important npc component that directs nucleocytoplasmic transport, was induced by virus infection but sharply decreased by lycorine treatment. therefore, nup may serve as a potential antiviral target for genetic manipulation. however, 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with the g /g cell cycle arrest lycorine alkaloids from hymenocallis littoralis identification of human host proteins contributing to h n influenza virus propagation by membrane proteomics lycorine reduces mortality of human enterovirus -infected mice by inhibiting virus replication evaluation of phosphopeptide enrichment strategies for quantitative tmt analysis of complex network dynamics in cancer-associated cell signalling alkaloids with activity against the zika virus vector aedes aegypti (l.)-crinsarnine and sarniensinol, two new crinine and mesembrine type alkaloids isolated from the south african plant nerine sarniensis influenza virus-induced caspase-dependent enlargement of nuclear pores promotes nuclear export of viral ribonucleoprotein complexes screening of a library of traditional chinese medicines to identify anti-malarial compounds and extracts synthesis and characterization of norbelladine, a precursor of amaryllidaceae alkaloid, as an anti-inflammatory/anti-cox compound comparison of influenza virus yields and apoptosis-induction in an adherent and a suspension mdck cell line effect of alkaloids isolated from amaryllidaceae on herpes simplex virus synergistic myeloma cell death via novel intracellular activation of caspase- -dependent apoptosis by carfilzomib and selinexor the c-terminal domain of nup is essential for assembly of the structural backbone of nuclear pore complexes cytoscape: a software environment for integrated models of biomolecular interaction networks we would like to thank prof. ming liao for providing bsl- facilities. we gratefully acknowledge shanghai omicsspace biotechnology co. ltd. for technical assistance with the peptide/protein identification. this work was financially supported the national natural science foundation of china (grant no. ) and the natural science foundation of guangdong province (no. a ). there was no additional external funding received for this study. the the following grant information was disclosed by the authors: national natural science foundation of china: . natural science foundation of guangdong province: a . the authors declare there are no competing interests. • li yang performed the experiments, prepared figures and/or tables, authored or reviewed drafts of the paper.• jia hao zhang performed the experiments, contributed reagents/materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper.• xiao li zhang, guang jie lao and guan ming su performed the experiments.• lei wang and yao lan li contributed reagents/materials/analysis tools.• wen cai ye contributed reagents/materials/analysis tools, approved the final draft.• jun he conceived and designed the experiments, analyzed the data, authored or reviewed drafts of the paper, approved the final draft. the following information was supplied regarding data availability: raw data is available at pxd (iprox): http://proteomecentral.proteomexchange.org/cgi/ getdataset?id=pxd . supplemental information for this article can be found online at http://dx.doi.org/ . / peerj. #supplemental-information. key: cord- -c mv eve authors: christensen, paul a; olsen, randall j; perez, katherine k; cernoch, patricia l; long, s wesley title: real-time communication with health care providers through an online respiratory pathogen laboratory report date: - - journal: open forum infect dis doi: . /ofid/ofy sha: doc_id: cord_uid: c mv eve we implemented a real-time report to distribute respiratory pathogen data for our -hospital system to anyone with an internet connection and a web browser. real-time access to accurate regional laboratory observation data during an epidemic influenza season can guide diagnostic and therapeutic strategies. we implemented a real-time report to distribute respiratory pathogen data for our -hospital system to anyone with an internet connection and a web browser. real-time access to accurate regional laboratory observation data during an epidemic influenza season can guide diagnostic and therapeutic strategies. keywords. analytics; epidemic; epidemiology; influenza; laboratory. the us centers for disease control and prevention (cdc) provides data regarding influenza activity, aggregated from state data sources that generally lag or more weeks behind the date of release [ ] . however, real-time data summarizing regional hospital system observations are more relevant for local clinical decision-making. clinicians frequently request updates from the microbiology laboratory on influenza test positivity, in addition to other common respiratory pathogens, during the respiratory virus season to help inform their daily practice. in addition, clinical laboratories should routinely monitor local influenza data to determine if epidemics are occurring, if continued testing is necessary, or if patients can be treated based on positive symptoms alone [ , ] . to address these local needs in a major us metropolitan area, our clinical microbiology laboratory implemented an online dashboard to distribute respiratory pathogen data for our -hospital system to clinicians, epidemiologists, infection control practitioners, system leadership, and the public. the report provides easy access from any workstation or mobile device with an internet connection. development of this report began in the fall , before the respiratory virus season, during which influenza reached an epidemic status across the united states that resulted in supply shortages, testing difficulties, and a widespread public health crisis [ , ] . respiratory pathogen panel test result data were extracted from our laboratory information system (scc soft computer). the extracts included de-identified laboratory result data, including specimen collection date, facility, and result for all influenza and respiratory pathogen tests. the data were further analyzed and aggregated to produce interactive charts published to a public-facing web server. the accuracy of the report was validated by comparison with data generated natively by our laboratory information system, as well as a manual review of all test results from day. we gathered visitor statistics from the server log files. internet protocol addresses were mapped to internet service provider, country, city, and organization using ipinfo.io [ ] . four distinct data summary analyses were performed. first, for our most commonly detected pathogens (influenza a, influenza b, respiratory syncytial virus, and rhinovirus/enterovirus), we calculated the number of positive tests for each day and week. second, we calculated the number of positive tests at each facility. third, we calculated the daily and weekly positivity rates of our respiratory pathogen molecular test. fourth, we calculated the frequency with which each pathogen was identified by our molecular test. these counts reflected anonymized and aggregated data devoid of protected health information. to present users with interactive and dynamic data, we elected to use hypertext markup language (html) [ ] and javascript [ ] as our visualization modality. we used chart.js [ ] as the framework for producing our interactive charts. the data analyses generated javascript arrays that were stored in chart.js data structures to produce charts ( figure for both chart and chart , we created radio buttons that allowed the user to toggle between a weekly or daily summary. for chart , we built a radio button to switch between basic view and detailed view. in basic view, the influenza a molecular and antigen results are grouped together; in detailed view, the subtypes of influenza a detected by our molecular platform are graphed separately. three distinct time intervals were supported, including the most recent weeks, the past year, and -present. the charts were packaged into an html report and uploaded to a public-facing web server. we unveiled the report and requested informal feedback at the system infection control meeting, the system antimicrobial stewardship meeting, and the hospital infection prevention and control committee meeting. we included a link to the report in an e-mail distributed to all employees from the executive vice president of the hospital system. we updated the graphs daily. over the subsequent weeks, the report was accessed times, and over the next weeks, the report was accessed times. approximately % of the originating ip addresses were from within our hospital system, and % were from locations outside the united states. views on mobile devices accounted for % of the traffic, and % of views were referred from the department of pathology and genomic medicine website. views peaked at per hour right after the link was distributed by our executive vice president. during the first week, % of all hour intervals saw at least page view, with an average of views per hour. daily view counts decreased as the influenza season ended and stabilized at views per week on average. at the height of the influenza epidemic at our hospital system, % ( / on december , ) of all influenza antigen tests and % ( / on december , ) of respiratory pathogen molecular tests were positive for influenza a or influenza b. forty-six percent ( / on december , ) of these molecular tests were positive for any respiratory pathogen. vendor supply stocks were limited nationwide [ ] , and in january , our supply of universal transport media diminished, requiring the creation of : aliquots to preserve material for sample collection. based on these data and following cdc/world health organization (who) guidelines for epidemics [ , ] , our primary care group stopped testing for influenza and treated symptomatic patients as if they were influenza positive. our interactive website provided near real-time data, which allowed this decision to be made a week earlier than otherwise would have been possible using federal and state data. furthermore, our inpatient pharmacy was able to anticipate oseltamivir utilization and stock accordingly while remaining prepared for potential drug shortages. brief report • ofid • we developed a near real-time report that presents statistics regarding respiratory pathogen testing from our microbiology laboratory. the population tested includes all inpatient and outpatient individuals across our -hospital system ( operating beds) and all patients in the associated free-standing emergency and primary care clinics. the report is available to any device with an internet connection and is updated daily to provide critical data to clinicians, epidemiologists, infection prevention and control committees, hospital leadership, and the public. we developed the site with mobile devices in mind, which allows the graphs and fonts to be readable on any platform. the user can switch between daily and weekly data aggregations using radio buttons. the time interval of interest can be modified using preconfigured buttons. data can be filtered by clicking the data labels in the chart legends. these features are possible because of our decision to develop a web-based report, as opposed to a pdf, spreadsheet, or word processing document. anecdotal feedback collected at the time of rollout was universally positive. interest in the report quickly peaked after the initial announcement but continued to be viewed daily. as the influenza season ended, infectious diseases clinicians asked that we add a rhinovirus/enterovirus trend to the website so they could track the summer respiratory virus season as well. at least clinician changed ordering practice in early september after identifying a spike in rhinovirus/enterovirus positivity frequency. in november , the chief physician executive and specialty physician group ceo sent an e-mail to physician leaders across the system regarding a significant uptick in respiratory syncytial virus isolates and rising influenza a pathogens detected by laboratory testing, which was identified through our website report. based on the data provided by our laboratory in this report and cdc/who guidelines for epidemics, our primary care group stopped laboratory testing for influenza and treated symptomatic patients as if they were influenza positive. access to accurate real-time data during an epidemic influenza season can guide diagnostic and therapeutic strategies. during the epidemic, there was a nationwide shortage of testing reagents [ ] . earlier identification of high positivity rates by monitoring real-time data allows for an institution to implement "treat don't test" guidelines earlier, preserving test reagents for critical situations where they are most needed. in summary, our microbiology laboratory implemented a near real-time internet report to distribute respiratory pathogen data for our -hospital system to clinicians, hospital epidemiologists, infection control committees, system leadership, and the public. facile access to accurate real-time data during an epidemic influenza season can guide diagnostic and therapeutic strategies. the report is available at https://flu.houstonmethodist.org/. fluview: a weekly influenza surveillance report prepared by the influenza division guide for considering influenza testing when influenza viruses are circulating in the community world health organization. who recommendations on the use of rapid testing for influenza diagnosis centers for disease control and prevention. summary of the - influenza season labs take stock of surprising flu season ip address api and data solutions -geolocation, company, carrier info, type and more world wide web consortium. w c html ecmascript language specification financial support. this work was supported by the department of pathology and genomic medicine at houston methodist hospital. this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.potential conflicts of interest. all authors: no reported conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord- - iecsho authors: wen, xiaohong; huang, qiuling; tao, hong; zou, weihua; gao, min; guo, huihui; yao, xing; cui, dawei; wang, xiang title: clinical characteristics and viral etiologies of outpatients with acute respiratory infections in huzhou of china: a retrospective study date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: iecsho background: viruses are commonly found in patients with acute respiratory infections (aris). however, the viral etiologies and clinical characteristics of outpatients with aris are poorly understood in china. here, we identified the viral etiologies in outpatients with aris in huzhou, china. results: our results indicated that of outpatients, were positive for viruses. of them, were positive for a single virus, including influenza a, which comprised h n ( . %) and pandemic h n ( ) ( . %), enterovirus ( . %), and influenza b ( . %). other single viruses were detected at less than . %. twenty-five patients were positively coinfected with two viruses. the prevalent viruses in coinfections were rhinovirus and h n virus ( . %). viruses were major pathogens in young children (< years) ( . %). coinfections were prevalent in older adults ( . %) and young children ( . %). virus-positive outpatients presented higher temperatures and more sore throat, fatigue and shortness of breath than virus-negative outpatients. aris and most virus detections peaked during the winter, but enteroviruses emerged between april and september. conclusion: viruses are major agents of aris among outpatients in huzhou, china. there was a variation in the distribution of viruses across different age groups and seasons. these findings are beneficial for planning prevention and treatment services for outpatients with aris. acute respiratory infections (aris) are common and major public health threats, causing high morbidity and mortality worldwide, particularly in developing countries [ , ] . many pathogens can result in aris, and viruses have been identified as major causes in aris among various populations; the most common viruses of aris include influenza a and b virus (flua and flub), rhinovirus (rhv), respiratory syncytial virus (rsv), parainfluenza virus (piv) type - , enterovirus (ev), adenovirus (adv), human metapneumovirus (hmpv), human bocavirus (bov), and coronavirus (cov)- e, nl , oc and hku [ ] [ ] [ ] [ ] [ ] [ ] . currently, there are few available vaccines to prevent respiratory virus infections [ , ] . it is important to investigate the epidemic viral etiologies of aris to efficiently prevent and control viral epidemics in the future. it is well known that the early and rapid molecular detection of respiratory viruses is valuable to prevent and control aris [ , ] . however, only a portion of patients with aris have their viral etiologies detected because of the expensive testing costs in china and other developing countries [ , , , ] . moreover, the spectrum of viral etiologies is closely correlated with various factors, such as age, season, geographical region, medical condition, and immune status [ , , [ ] [ ] [ ] [ ] . in this study, we collected clinical and demographic data from outpatients including children and adults with aris, and their specimens were tested for viruses. this study aims to provide basic data to direct local disease prevention and control measures for aris in huzhou, china. this study was conducted from january to april in two general hospitals in huzhou city located beside tai lake of southeast china. demographic and clinical data from all enrolled aris outpatients of any age were collected, and clinical specimens from the upper respiratory tract of these outpatients were tested for viruses by the multiplex rt-pcr method using a seeplex® rv ace detection kit (seegene, korea). furthermore, a positive sample with flua virus was discriminated for seasonal h n (sh n ), seasonal h n (sh n ) and pandemic h n ( ) viruses by a one-step real-time rt-pcr assay from shanghai zj bio-tech co., ltd. (shanghai, china). outpatients of any age were enrolled from january to april at the department of fever outpatient clinic of the first people's hospital of huzhou and at the huzhou central hospital, huzhou, china. outpatients suffering from fever now or in recent days, such as influenza-like illness (ili) and aris patients, were seen at the fever outpatient clinic. a case definition of ari was described in a previous report [ ] . briefly, patients with aris presenting with at least one of the following symptoms: cough, sore throat, shortness of breath or coryza as an acute onset of symptoms within days were judged by a clinician for an infection. clinical specimens from the upper respiratory tract of patients, including throat swabs, nasal aspirates and washes, or sputum specimens, were collected and kept in ml of viral transport medium stored at − °c until testing for respiratory viruses. clinical data including age, oral body temperature, clinical symptoms and other information were recorded in case report forms during face-to-face interviews. statistical analysis was conducted with spss software (v . , spss, chicago, il, usa). descriptive statistics were used to analyze the seasonal and age distribution and infection rates of different respiratory viruses. chi-squared tests were used to compare different age groups, gender, and clinical characteristics between age virus-positive and virus-negative outpatients with aris. p-values < . were considered to be statistically significant. a total of outpatients with aris were enrolled from january to april in this study. of them, specimens ( . %, / ) were positive for at least one virus, and single infections accounted for . % ( / ) of cases. coinfections were observed in . % ( / ) of cases (fig. a) . of the single virus infections, flua virus was the most frequent virus, identified in . % ( / ) of the cases, comprising ( . %) cases of sh n virus and ( . %) cases of pandemic h n ( ) virus patients, followed by ev ( . %, / ), flub ( . %, / ), adv ( . %, / ), rhv ( . %, / ), hmpv ( . %, / ), and other viruses that were identified under . %, respectively (fig. b) . of the coinfections, rhv + sh n viruses were predominantly identified and accounted for . % ( / ) of cases. rhv + ev, rhv + adv and rhv + piv- viruses equally accounted for . % ( / ) of cases. adv + hmpv, adv + rsv-b, and flub+piv- also equally accounted for . % ( / ) of cases. other coinfections were identified in . % ( / ) of cases (fig. c) . sh n virus was not detected in this study. demographic and clinical characteristics of outpatients with aris are shown in table . of outpatients with aris, ( . %) were males, and ( . %) were females. the distribution of viruses did not significantly differ between males and females (χ = . , p = . ). however, the distribution of viruses notably differed among the different age groups (χ = . , p < . ). in addition, . % ( / ) of aris outpatients suffered from high fever (body temperature ≥ °c), followed by cough ( . %), sore throat ( . %), fatigue ( . %), and other respiratory symptoms. moreover, a significant difference was observed in clinical symptoms including fever (χ = . , p = . ), sore throat (χ = . , p < . ), fatigue (χ = . , p < . ), and shortness of breath (χ = . , p = . ) between virus-positive and virus-negative cases, and the proportion of patients with abdominal pain was greater among virus-negative cases (χ = . , p = . ). additionally, physical examinations showed abnormal lung auscultation ( . %) and x-rays ( . %). the distribution of the viral etiologies of the four age groups is shown in table . the highest proportion was observed in young children ( . %), and lowest proportion was observed in adults - years of age ( . %). similarly, the positive rate of cases with a single virus infection was highest in the young children ( . %) and lowest in adults of - years of age ( . %). moreover, the positive rate of the cases with coinfections was highest in older adults ( . %), followed by young children ( . %). the predominant viruses among four age groups differed. flua virus and subtypes were the most prevalent viruses in the older adults (≥ years), and the converse was true in the young children (< years). ev, rsv and piv viruses predominated in the young children (< years), and flub, rhv and adv viruses were more prevalent in the young adults ( ~ years). the virus detection rates for different seasons are shown in fig. . the proportion of positive viruses exhibited two waves corresponding to winter and spring, including jan to mar , and nov to feb . similarly, the flua virus also occurred more frequently in winter and spring. conversely, ev infections were predominant between april and september. other viruses occurred almost sporadically throughout the year without obvious seasonal trends, and a small number of ari outpatients with virus infection were observed between june and september, excluding ev infections. viruses are major agents contributing to the high morbidity and mortality of patients with aris, particularly in children under years of age [ , , ] . many reports describe the etiology and epidemiology of hospitalized aris patients, including children and/or adults worldwide [ , [ ] [ ] [ ] , although the study of outpatient aris in children and adults is more limited [ , ] . in this study, some clinical characteristics significantly differed between virus-positive and virus-negative outpatient aris, such as fever, cough, sore throat, fatigue, and other respiratory symptoms. these findings implied that viruses were the most common causes of aris, easily eliciting severe clinical symptoms. similar results were found in previous reports [ , [ ] [ ] [ ] [ ] . laboratory diagnosis of viruses is commonly conducted by conventional methods (such as culture or antigen detections), and real-time and multiplex rt-pcr assays have been considered to be important tools for identifying the etiologies of aris [ , , , ] . recently, a commercial multiplex pcr assay with a seeplex® rv ace detection kit was used to simultaneously and precisely identify the viruses of aris in many laboratories [ ] [ ] [ ] [ ] . the methods used in this study expanded and improved the capacity for testing viruses ( viruses and subtypes of flua virus). in this study, . % were positive for at least one virus, which was similar to the morbidity rates reported in previous studies in pittsburgh ( . %) and vitória of southeast brazil ( . %) [ , ] , but was different in china and compared with other reports [ , - , , ] . the single infection ( . %) was predominant in our study, particularly the flua virus ( . %), which was consistent with previous reports in shandong province, beijing, of china and other countries [ , [ ] [ ] [ ] [ ] [ ] . sh n and rhv coinfections were predominant among viral coinfections, which was different from previous reports [ , , , , ] . the discrepancy of the predominant coinfections might be closely associated with principal epidemical viruses in the local region. the proportion of respiratory viruses notably differed across different age groups; the virus positive rate was the highest in young children under years but was lowest in adults ( ~ years) in this study. these findings indicated that the viruses were the predominant pathogen found in young children with aris, and similar morbidity rates have been reported in previous studies [ , ] , although the morbidity rate in this study also differed from those in other studies [ ] . the flu a virus was the predominant virus among the three age groups, excluding young children, and was highest among older adults (≥ years) and lowest among young children; however, ev was the highest in young children among all age groups. some studies show that evs and rhvs can be difficult to discriminate with rt-pcr primers unless accompanied by amplicon sequencing, and flua virus and rhv might fail to detect rhv due to competition between the amplification reactions [ ] [ ] [ ] . therefore, all positive rhv and/or ev specimens and flua virus specimens with random selection were identified table age distribution of viruses from outpatients with aris aris, acute respiratory infections by sequencing assay, respectively, and among them, four rhv positive and ev positive specimens were not sequenced due to low viral load in the specimens. additionally, viral coinfections predominantly occurred in young children (< years) and older adults (≥ years), which was consistent with previous reports in china and other countries [ , , , , , ] . these results indicated that the detection rate of viruses in aris was closely associated with the age of outpatients because age affects immune status, exposure opportunities to viruses, and other lifestyle behaviors of people. generally, young children and older adults have weak immune systems against viruses, which might contribute to their higher susceptibility to viruses than young adults and adults who have strong immune status against viruses [ , ] . moreover, young children have more opportunities for exposure to evs than young adults and adults, which might cause higher incidences of ev infection than in other age groups [ , , ] . many studies have indicated that viral aris are affected by seasonal distributions and occur commonly in spring, autumn, and winter [ , , , , , ] . in this study, viruses from outpatient aris were detected throughout the whole year and commonly occurred in spring and winter, with peaks occurring in february and december . similar results were reported in other studies [ , , ] . moreover, our results demonstrated that ev infections occurred between april and september , with the peak detection rate in august , which was in accordance with previous reports [ ] . many studies have shown that evs are often detected in summer and autumn [ , [ ] [ ] [ ] [ ] . we speculate that climate conditions might be an important factor for ev detection in temperate regions. these findings indicated that the geographical diversity of surveillance areas with warm and wet climate conditions beside tai lake might contribute to the variability in seasonal trends and viral etiologies of aris. our study had some limitations. first, there were only outpatients with aris enrolled in the two local hospitals; because this was a small sample size, it may be difficult to estimate the disease severity of outpatient aris in detail. more large-scale surveillance for aris will performed, and analyses over additional years will provide a more accurate picture of seasonal variation in respiratory virus circulation in this community in the future. second, we detected only viruses, but did not test for bacterial pathogens in the respiratory tracts of outpatients with aris; therefore, our data regarding pathogens causing aris were not comprehensive, which also affected our ability to determine the relationships between pathogens and disease severity in outpatients with aris. taken together, our results were valuable to a certain degree for assessing outpatients and clinical treatments. in summary, this study provides important epidemiologic data regarding the clinical characteristics, viral spectrum, age distribution and seasonality of viruses in outpatients with aris in huzhou, china. these findings contribute to evaluating the burden of virus infections in outpatients, including young children and adults. timely and accurate diagnosis of pathogens in outpatients with aris is required to reduce the burden caused by these diseases. viral etiology of acute respiratory infection in gansu province prevalence of respiratory viruses among children hospitalized from respiratory infections in shenzhen, china viral etiology of acute respiratory infections among children and associated meteorological factors in southern china estimates of worldwide distribution of child deaths from acute respiratory infections viral etiology of acute respiratory tract 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microbiologically unexplained acute respiratory tract infections in hospitalized pediatric patients new epidemiological and clinical signatures of pathogens from respiratory tract infections based on a -year study hand, foot, and mouth disease in china, - : an epidemiological study burden, seasonal pattern and symptomatology of acute respiratory illnesses with different viral aetiologies in children presenting at outpatient clinics in hong kong we thank the outpatients, nurses and clinicians of the participating hospitals for their participation and cooperation in this study. not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -srym kh authors: de rosa, nicoletta; giampaolino, pierluigi; lavitola, giada; morra, ilaria; formisano, carmen; nappi, carmine; bifulco, giuseppe title: effect of immunomodulatory supplements based on echinacea angustifolia and echinacea purpurea on the posttreatment relapse incidence of genital condylomatosis: a prospective randomized study date: - - journal: biomed res int doi: . / / sha: doc_id: cord_uid: srym kh introduction. hpv infection is a highly infectious disease; about % of partners of individuals with genital warts will develop genital condylomatosis. only in - % it regresses spontaneously and relapse rates range deeply ( - %). echinacea extracts possess antiviral and immunomodulator activities. the aim of this study was to evaluate the efficacy of the therapy, using a formulation based on hpvadl ® (on dry extracts of mg echinacea purpurea (ep) roots plus e. angustifolia (ea)), on the posttreatment relapse incidence of genital condylomatosis. materials and methods. it is a prospective single-arm study. patients with a satisfactory and positive vulvoscopy, colposcopy, or peniscopy for genital condylomatosis were divided into two random groups and subjected to destructive therapy with co laser. group a (n= ) immediately after the laser therapy started a -month treatment with oral hpvadl ®; group b (n= ) did not undergo any additional therapy. patients were subjected to a follow-up after , , and months. differences in relapse incidence between the two groups during follow-up controls were evaluated by χ -test; the groups were stratified by age, gender, and condylomatosis extension degree. results and discussion. gender, age, and condyloma lesions' extension degree showed no statistically significant differences between the two trial groups. the relapse incidence differs statistically between the two studied groups and progressively decreases during the months after treatment in both groups. statistically significant reduction of relapse rates has been shown in group a in patients over years old. this difference is significant for both men and women. the relapse incidence is superior in case of extended condylomatosis. conclusions. in conclusion, the presence of a latent infection causes condylomatosis relapse; in order to reduce the relapse risk an induction of a protective immune response seems to be essential to allow rapid viral clearance from genital areas surrounding lesion and treatment zones. echinacea promotes this process. ep and ea dry root extracts seem to be a valid adjuvant therapy in reducing relapse incidence of lesions in patients treated for genital condylomatosis. hpv infection is one of the most common sexually transmitted infections in the world. more than % of sexually active adults contract the infection during their life. in the two years after a sexual debut the sexual risk of infection varies from to % depending on the studied population and the hpv type [ ] . there is a similar incidence of genital condylomatosis in males and females ( - % and - %) [ ] [ ] [ ] . in men, compared to women, infections with multiple genotypes and low-oncogenic risk genotypes are more frequent [ ] . only - % of the genital condylomatosis regresses spontaneously. this is a highly infectious disease; about % of partners of individuals with genital warts will develop genital condylomatosis. the risk of infection and the risk of progression of hpv-associated lesions are related to several factors including number of sexual partners experienced during the life and early age of the first intercourse; tobacco smoking; and eating habits [ ] [ ] [ ] . it has long since known that the above-ground portion and the roots of echinacea angustifolia (ea) and of e. purpurea (ep) possess anti-inflammatory and immunostimulatory properties. numerous in vitro and in vivo studies have been recently conducted in an effort to validate some of the traditional uses of echinacea extracts [ ] . early studies have shown that only a few echinacea extracts possess significant antiviral activity. in particular, above-ground portions and roots of ep show a strong antiviral activity, as they have a virucidal effect against influenza virus, herpes simplex virus, and coronaviruses [ , ] . the ep appeared much less effective against intracellular viruses [ , ] , which could be resistant to the ep inhibitory effect; on the contrary, viral particles located in the extracellular fluids appeared to be vulnerable. therefore, ep can act during an initial contact with virus, that is, at the beginning of infection and also during the transmission of the virus from the infected cells. numerous viral and bacterial infections cause an increase of expression of proinflammatory cytokines, in particular, of il- and il- , which are therefore considered as markers of an inflammatory state [ , ] . any compound or herbal extract that inhibits or inverts the increase of il- / can be considered a potential anti-inflammatory agent. all the portions of the roots, leaves, stems, and flowers of ep show this effect [ ] . these studies make it evident that echinacea not exactly acts as an "immunostimulant" or "immune system booster," but more likely has an immunomodulatory action, rather than a generalized immunostimulatory effect [ ] [ ] [ ] [ ] . the aim of the present study was to evaluate the efficacy of the therapy, using a formulation based on mg of hpvadl (equal to mg polyphenols plus . mg of echinacosides), on the post-treatment relapse incidence of genital condylomatosis. between july and july , all patients with a genital condylomatosis diagnosis received in the colposcopy and cervical-vaginal pathology unit of university federico ii, naples, were invited to participate in a prospective randomized trial. patients were properly informed and provided their written consent to participate in the trial and to undergo ambulatory diagnostic examinations; afterwards, colposcopy or peniscopy was conducted and, if appropriate, biopsy examinations. all procedures performed in the study were in accordance with the ethical standards of the institutional and/or national research committee and with the helsinki declaration and its later amendments or comparable ethical standards. the criteria for participation in the trial were as follows: satisfactory and positive colposcopy / peniscopy for genital condylomatosis (cervix, vagina, perianal vulva, or perineum for females and penis, scrotum, or anal region for males) and / or histological examination for koilocytosis or condylomatosis in case of positive cervical biopsy. patients with h-sil cytological diagnosis, cin - histologic diagnosis, or invasive cervical carcinoma, pregnant women, immunosuppressed patients, and individuals infected with human immunodeficiency virus (hivpositive) were not enrolled in the trial. colposcopy and peniscopy were conducted after an application of % acetic acid. visible acetowhite lesions have been classified in accordance with the criteria of the international federation of cervical pathology and colposcopy [ ] . in case of genital condylomatosis, to standardize extension of the lesions, genitals were divided into genital areas for women, that is, cervix, left/right vaginal wall, left/right major labia, left/right minor labia, clitoris, pubis, perineum, and perianus and into genitals arear for men, that is, pubis, scrotum, glans, preputial balanus grooves, and penis. patients were classified into lesion degrees, according to the number of genital areas affected by condylomas and the number of the condylomas: ( ) from to condylomas on - genital areas (mild and localized condylomatosis) ( ) > condylomas on - genital areas (mild and diffuse condylomatosis) ( ) > condylomas on > genital areas (extended condylomatosis). patients with low grade (ztag ) or high grade (ztag ) cervical lesions were subjected to a targeted biopsy using a biopsy forceps (cfs chimo schumacher pliers) with - mm jaw in order to obtain - mm tissue specimens. two serial micron sections of the formalin-fixed and paraffin-embedded sample were stained with hematoxylin and eosin. the specimens were examined by optical microscope and classified as normal, cin , cin , and cin carcinoma in situ or microinvasive carcinoma according to the criteria of the world health organization. patients with low grade (cin ) or high grade (cin - ) preneoplastic lesions were excluded from the trial and carried on all the therapeutic and diagnostic procedures as recommended by national and international guidelines. patients with genital condylomatosis, diagnosed through colposcopy, vulvoscopy, peniscopy, and/or biopsy examinations, were included in the study. all enrolled individuals were divided into two random groups and subjected to destructive therapy with co laser. group a immediately after the laser therapy started a month treatment with oral immunomodulatory supplements based on hpvadl ; group b did not undergo any additional therapy (control group). the medical device administered to group a was composed of mg of hpvadl (equal to mg polyphenols plus . mg of echinacosides), mg vitamin c, mg of zinc, and . mg of copper. patients were subjected to a follow-up colposcopy after , , and months. in case the infection persisted and relapse condyloma lesions occurred, patients were again subjected to destructive therapy until the full lesion elimination. all colposcopy, peniscopy and biopsy examinations and therapies were performed by our team. statistical analysis of the data was executed by spss software . (spss inc., chicago, il, usa). data with p-values < . were considered statistically significant. demographic and clinical data of the two groups were compared by student's t-test for the data with parametric distribution (age) and by -test for ordinal variables (gender and condylomatosis extension degree). differences in relapse incidence between two groups during follow-up controls were evaluated by -test; the groups were stratified by age, gender, and condylomatosis extension degree. one hundred and forty women appeared to be suitable for destructive therapy with co laser and were divided into group a (n = ) and group b (n = ) at random. of these, patients did not undergo a required operation and patients did not undergo a programmed follow-up or interrupted the therapy before the -month period expired. one hundred and twenty-five patients, ( %) women and ( %) men, completed the diagnostic-therapeutic procedure as scheduled by the protocol and were therefore included in the analysis. of the studied population, women ( . %) underwent echinacea therapy after the treatment (group a) and ( . %) did not undergo any additional therapy (group b, control group). the mean age of female patients in group a is . ± . years, in group b . ± . years (p = n.s.); the mean age of male patients in group a is . ± . years, in group b . ± . years (p = ns). table shows epidemiological data and condyloma lesions' extension degree for groups a and b. there were no statistically significant differences in these data in the two trial groups. no severe side effects were recorded in group a. only ( . %) patients reported some digestive difficulties. the relapse incidence differs statistically between the two studied groups (table , figure ) and progressively decreases during the months after treatment in both groups. therapy does not seem to modify the relapse incidence in very young female patients under the age of . instead, statistically significant reduction of relapse rates has been shown in patients over years old. this difference is significant for both men and women. the relapse incidence is superior in case of extended condylomatosis (extension degree n. ) (table , figure ). clinical trials conducted on patients with genital condylomatosis show quite different relapse rates, depending on the studies and on the treatment and range from % to % [ ] [ ] [ ] [ ] . our data show a global relapse rate of about %. therapy with hpvadl is effective in reducing relapse incidence of lesions in patients treated for genital condylomatosis. our data prove, indeed, that the relapse incidence of lesion is greater in the control group compared to the treatment group at the first, second, and third follow-up controls. spontaneous remission of genital condylomatosis is possible, but not frequent; the percentage of spontaneously recovered patients varies considerably and ranges from % to % [ , ] . most commonly used therapy is cryotherapy or diathermocoagulation ( % and %); drug therapy is much less frequent ( %). approximately % of patients undergo a single treatment procedure; the number of patients that undergo more than one treatment procedures progressively decreases; % of patients undergo or more treatments [ ] . this pattern is similar for both sexes and is according to the anatomical site [ ] . in compliance with these data, the difference in relapse incidence between the two trial groups is statistically significant even when these are stratified by gender and extension degree of the lesion. on the other hand, age appears to be a determinant factor; in fact, in individuals under the age of , the therapy does not seem to influence significantly the relapse incidence of lesion. the small numbers of younger age groups, however, cannot induce us to generalize this data. based on these data, it follows that in very young individuals additional therapy with hpvadl could be superfluous. moreover, individuals under the age of show greater relapse incidence at the first follow-up. the relapse incidence decreases progressively in both groups as the time passes and is related to the extension degree; in fact, the extension degree of condylomatous lesions corresponds to a higher relapse incidence than degrees and . the presence of a latent infection causes lesion relapse; in order to reduce the relapse risk after the treatment of condyloma lesions, an induction of a protective immune response seems to be essential to allow rapid viral clearance from genital areas surrounding lesion and treatment zones. introduction of an immunostimulatory substance such as echinacea seems to promote this process. the hpv-induced immune response is both humoral and cell mediated. a humoral immune response to hpv capsid protein l is weak during natural infection. the humoral immune response to the viral capsid can be detected averagely starting from months after the infection, though - % of patients with persistent infection will never present a seroconversion [ ] . the seropositivity to the infectious genotype persists only in % of the cases, even when the initial lesion transformed to a cervical cancer [ ] . when viral dna has been eliminated, specific antibodies can be detected only in half of cases after years [ ] . hpv infection promotes a cellular immune response, especially in the active phase of the clearance of genital condylomatosis infection, when a cell infiltration of macrophages and t cells develops in correspondence to the lesion [ ] . in the blood, an immune response of cd + t cells against e , e , and e proteins is associated with hpv and hpv infection and occurs in particular in early disease phases and in case of regressing lesions, less when a persistent disease takes place. in individuals with a deficiency of cell-mediated immune response, hpv infection, genital condylomatosis, or precancerous lesions are destined to persist. therefore, this type of response seems to be essential for the viral clearance. the ep immunomodulatory effect has been widely demonstrated. ep extract was used for the preventive care and for the treatment of various viral infections [ ] . in vitro studies have shown that ep acts directly on a number of cell types, including natural killer cells [ ] , polymorphonuclear leukocytes [ ] , and macrophages [ ] . ep induces a proliferation of t cells. this has been conferred to the activation of macrophages that stimulates a production of ifn-and, consequently, a secondary activation of t lymphocytes [ ] . ifn-is one of the fundamental mediators for the latency prevention [ ] ; it has been proven that this mechanism is responsible for reducing the latency incidence of herpes virus simplex infection and, consequently, reducing the relapse risk of hsv lesions [ ] . it is possible that an analogous mechanism induces a cell-mediated response to hpv infection, which allows the reduction of the persistence of infection and, therefore, the lesion relapse. this study has some limitations: this is a single institution study with a small number of participants and it lacks placebo controls. on the other hand, the strengths of this study are as follows: the rigorous inclusions criteria, the evaluation of patients at colposcope (so not only grossly visible genital warts were evaluated and treated but also small lesions), and the treatment modality with laser co for all patients. in conclusion, hpvadl seems to be a valid adjuvant therapy in reducing relapse incidence of lesions in patients treated for genital condylomatosis. the data used to support the findings of this study are included within the article. the authors state that there are no conflicts of interest. meta-analysis of human papillomavirus infection concordance trends in genital warts and genital herpes diagnoses in the united kingdom anogenital warts incidence, medical management and costs in women consulting gynaecologists in france epidemiology and cost of treatment of genital warts in spain genital human papillomavirus (hpv) concordance in heterosexual couples risk of female human papillomavirus acquisition associated with first male sex partner highrisk human papillomavirus is sexually transmitted: evidence from a follow-up study of virgins starting sexual activity (intercourse) the role of high-risk hpv-dna testing in the male sexual partners of women with hpv-induced lesions echinacea plants as antioxidant and antibacterial agents: from traditional medicine to biotechnological applications characterization of antiviral activities in echinacea root preparations echinacea purpurea aerial parts contain multiple antiviral compounds antiviral properties and mode of action of standardized echinacea purpurea extract against highly pathogenic avian influenza virus (h n , h n ) and swine-origin h n (s-oiv) induction of multiple pro-inflammatory cytokines by respiratory viruses and reversal by standardized echinacea, a potent antiviral herbal extract the influence of traditional herbal formulas on cytokine activity anti-inflammatory compounds of plant origin. part ii. modulation of pro-inflammatory cytokines, chemokines and adhesion molecules antiinflammatory activities of echinacea extracts do not correlate with traditional marker components echinacea and anti-inflammatory cytokine responses: results of a gene and protein array analysis alkylamides from echinacea modulate induced immune responses in macrophages pharmacokinetics and immunomodulatory effects of phytotherapeutic lozenges (bonbons) with echinacea purpurea extract echinacea-induced macrophage activation colposcopic terminology of the international federation for cervical pathology and colposcopy the diagnosis and treatment of human papillomavirus-mediated genital lesions an evidence-based review of medical and surgical treatments of genital warts external genital warts: diagnosis, treatment, and prevention management of genital warts a retrospective analysis of the costs and management of genital warts in italy comparison of human papillomavirus types , , and capsid antibody responses following incident infection human papillomavirus and l serology compared across anogenital cancer sites determinants of human papillomavirus serological conversion and persistence in a population-based cohort of women in costa rica characterization of human antibody-reactive epitopes encoded by human papillomavirus types and treatment of the common cold with unrefined echinacea: a randomized, double-blind, placebocontrolled trial echinacea purpurea and melatonin augment natural-killer cells in leukemic mice and prolong life span the effect of aerial parts of echinacea on the circulating white cell levels and selected immune functions of the aging male sprague-dawley rat echinacea stimulates macrophage function in the lung and spleen of normal rats the current trend in genital herpes: progress in prevention cd t cell control of acute and latent murine gamma herpes virus infection requires ifn key: cord- -buc dd y authors: dong, rui; he, lily; he, rong lucy; yau, stephen s.-t. title: a novel approach to clustering genome sequences using inter-nucleotide covariance date: - - journal: front genet doi: . /fgene. . sha: doc_id: cord_uid: buc dd y classification of dna sequences is an important issue in the bioinformatics study, yet most existing methods for phylogenetic analysis including multiple sequence alignment (msa) are time-consuming and computationally expensive. the alignment-free methods are popular nowadays, whereas the manual intervention in those methods usually decreases the accuracy. also, the interactions among nucleotides are neglected in most methods. here we propose a new accumulated natural vector (anv) method which represents each dna sequence by a point in ℝ( ). by calculating the accumulated indicator functions of nucleotides, we can further find an accumulated natural vector for each sequence. this new accumulated natural vector not only can capture the distribution of each nucleotide, but also provide the covariance among nucleotides. thus global comparison of dna sequences or genomes can be done easily in ℝ( ). the tests of anv of datasets of different sizes and types have proved the accuracy and time-efficiency of the new proposed anv method. with the rapid development of next generation sequencing technology, more and more information of the genome sequences is available. studying sequence similarity is a crucial question in research and can explain phylogenetic relationships by constructing trees. one of the most commonly used methods, multiple sequence alignment (msa) uses dynamic programming, a regression technique that finds an optimal alignment by assigning scores to different possible alignments and taking the one with the highest score (yu et al., a) . however, the computational cost of msa is extremely high and msa may not produce accurate phylogeny for diverse systems of different families of rna viruses (yu et al., b) . alignment-free approaches have been developed to overcome those limitations. published alignment-free methods include markov chain models (apostolico and denas, ) , chaos theory (hatje and kollmar, ) , and some other methods based on the statistics of oligomer frequency and associated with a fixed length segment, known as k-mer (sims et al., ) . yau and his team proposed the natural vector method, which takes the position of each nucleotide into consideration. the natural vector method performs well on many datasets (deng et al., ; yu et al., b; hoang et al., ; li et al., ) , however, it only considers the number, average position and dispersion of positions of each nucleotide. relationships between nucleotides are also important, especially when the functions may be related to interactions of nucleotides, such as the folding of a chromosome. in this paper, we propose a new accumulated natural vector (anv) method, which not only considers the basic property of each nucleotide, but also the covariance between them. in the traditional natural vector (nv) method, each sequence is uniquely represented by a single point in r . the traditional natural vector approach is firstly introduced in deng et al. ( ) : for a sequence of length n, n α (αǫ{a, c, t, g}) denotes the number of nucleotide α in the sequence. s [α] [v] is the distance from the first nucleotide (regarded as origin) to the v th nucleotide α in the dna sequence. t α = n α v= s [α] [v] denotes the total distance of each set of a,c,g,t from the origin, αǫ{a, c, t, g}. µ α = t α n α , is the mean value of the distances of nucleotide α from the origin. , is the normalized central moment of order , which can also be seen as the variance of the positions of nucleotide α. therefore, a dna sequence can be represented by a -dim vector: in this paper, we propose an accumulated natural vector approach, which projects each sequence into a point in r , where the additional six dimensions describe the covariance between nucleotides. obviously, anv can provide more information than the traditional nv method, and doesn't include the human intervention, such as choosing the optimal value of k in the k-mer method. therefore, it can distinguish different sequences and classify species into correct clusters with higher accuracy and less time cost. the following six datasets were used to validate the method. the coronaviruses dataset includes viral genomes, in which viruses are from the exact same dataset with (woo et al., ; yu et al., ; hoang et al., ) and the other two viruses are new members in coronavirus. the second dataset consists of the genomes of influenza a viruses, which is a classic dataset to test if a new proposed method performs well. the third dataset includes viruses from zheng et al. ( ) , which focuses on the classification of ebolaviruses. the fourth one is from our colleagues' previous paper (li et al., ) which includes viruses chosen randomly under some criteria. the fifth one is the mitochondrial genomes of mammals, which can be clustered into seven well-known categories. all the sequence materials can be found on ncbi with the reference number provided in the appendices. we also generated different mutations by simulation in a dna sequence and constructed phylogenetic trees of simulated sequences to test our anv method. all computations in this paper are done on a dell laptop equipped with intel i processor under windows home premium with gb ram, together with the matlab (version r a) and mega x. for a given genomic sequence, we first define four indicator functions (u) for adenine, cytosine, guanine and thymine, respectively: if α appears at the i th position of the sequence , if α doesn ′ t appear at the i th position of the sequence ( ) where αǫ{a, c, t, g}, and i = , , . . . , n. heren is the length of the whole sequence. for example, if the genomic sequence is "atctagct, " then the four indicator functions are shown in table . here are some simple properties about the indicator functions: . each column has the sum of . . each row has the sum of the number of corresponding nucleotide. now we define four accumulated indicator functions as the following:ũ the four accumulated indicator functions for the example above ("atctagct"), are shown in table . here are some properties about the accumulated indicator functions: . the i th column has the sum of i. α∈{a,c,g,t}ũ . the last column is the total number of the nucleotideα in the sequence. µ k is the average position in the natural vector in deng et al. ( ) . property and can be easily proved by the definition of indicator function (u α ) and accumulated indicator function (ũ α ), now we prove the property , which builds up the relationship between the accumulated indicator function and the average position of a specific nucleotide. if we assume that the positions of nucleotide α are t , t , . . . , t n α , where n α is the number of nucleotide α in the sequence, then the basic form of accumulated indicator function should be, which satisfies ≤ t < t < . . if we add up those n elements above and denote the sum as α and t = , we have: therefore, we use to describe the average position of nucleotide α, which indicates the distance of the average position to the end of the sequence. for two finite point sets with equal number of elements: a = {a , a , . . . , a n }, b = b , b , . . . , b n in r, which satisfy a < a < . . . < a n and b < b < . . . < b n , the covariance of two sets can be defined as follows: where u a = n i = a i /n and u b = n i = b i /n. now we apply the covariance formula above to the accumulated indicator functions. a set is a collection of definite, distinct objects, known as the elements or members of the set. now for each nucleotide, we have an array of n elements which is the accumulated indicator function for the nucleotide α ∈ {a, c, g, t}: [ , , . . . , , , , . . . , , , , . . . , , . . . (n α − ) , however, those n elements cannot build up a set of n elements since many of them are replicated. hence, we extend the definition of set to a generalized concept, where the elements in a set can be the same. in this generalized definition, each nucleotide has a set of n elements and they can be arranged in the ascending order, i.e., from the smallest to the biggest number. thus, we can use the covariance formula ( ). as the example of sequence "atctagct, " the covariance of nucleotide a and c can be computed in this way: the generalized set of nucleotide a is { , , , , , , , } and of c is { , , , , , , , }. each generalized set has n = elements and the generalized covariance would be similarly, we can get cov (a,g), cov (a, t), cov (c, g), cov (c, t), cov (g, t). for two nucleotides like α and β, the covariance formula is then it is obvious that when α = β, the corresponding formula should be the formula above defines the variance of the positions of nucleotide α. for a given nucleotide sequence, now we can build up its accumulated natural vector. the first four dimensions describe the number of each nucleotide, denoted as n a , n c , n g , n t , which are the last column of the accumulated indicator functions. the second four dimensions describe the average distance of nucleotides to the end of the sequence, denoted as ( ). the third four dimensions describe the divergence of each nucleotide, denoted ( ). please note that this d α is a little different from the d α in the traditional natural vector method since the previous definition of variance cannot be extended to a reliable definition of covariance. the last six dimensions describe the covariances between each two nucleotides, denoted as cov (a, g), cov (a, t), cov (c, g), cov (c, t), cov (g, t) as formula ( ). and the universal form of accumulated natural vector is from section . . to section . . , we introduce how a dna sequence is represented by a vector in r space. therefore, the distance between two sequences can be measured by the euclidean distance between two vectors. suppose that now we have two sequences in r w (in our case, w = ), denoted as x = (x , ..., x w ) and (y , ..., y w ), the euclidean distance between them is for a dataset of m different sequences, we can construct a distance matrix d = (d ij ) m×m , and d ij (≥ ) represents the euclidean distance between sequence i and sequence j. d is a symmetric matrix and the diagonal element is zero. in this research, we use mega x to build up phylogenetic trees. in order to eliminate the influences of different algorithms of constructing trees, we apply the unweighted pair group method with arithmetic mean (upgma) algorithm (sneath and sokal, ) for analysis on the four datasets. for comparison with other common alignment or alignmentfree method, we also perform k-mer and msa (clustalw or muscle) on the same dataset. the feature frequency profile (ffp) (woo et al., ) , which is based on k-mer frequency, calculates the frequency of each k-mer in the sequence and turns a dna sequence into a vector in a k -dimensional space. the euclidean distance between two k-mer vectors can also be computed by formula ( ). we apply msa method, clustalw on several datasets as well, with the default parameters in mega x. clustalw is much slower than another msa algorithm, muscle, while clustalw can give a better result. muscle is applied on the fourth dataset of viruses and after we get the alignment result of the viruses, distance matrix is calculated using hamming distance, to find the nearest neighbor of each virus. hamming distance between two strings of equal length is the number of positions at which the corresponding symbols are different. it measures the minimum number of substitutions required to change one string into the other or the minimum number of errors that could have transformed one string into the other. since alignment approaches are to arrange the sequences to identify regions of similarity between the sequences, the alignment would provide the performance of each sequence on a fixed number of positions. therefore, the hamming distance can be calculated by simply counting the number of pairwise differences in character states. in the simulated dataset, we use the pairwise alignment distance by the "seqpdist" function inside matlab bioinformatics toolbox, which uses the jukes-cantor algorithms as the correct tree, since the sequences are simulated according to a base sequence. then the distance matrices are compared using robinson-foulds distances, which can measure the congruence to the reference topology. we apply the accumulated natural vector method on five datasets, and compare the results with common methods, such as msa, k-mer (ffp) and the traditional natural vector method. from comparison, the results of accumulated natural vector are more accurate and the calculation cost is very small compared to others. a dataset of viruses has also been tested, and laptop cannot bear such a heavy burden of calculation of aligning them but alignment-free can still be done in a reasonable time. we also use a server to align segments of sequences, to compare the results to anv and other methods. anv also gives the best performance on this dataset. besides, we simulate another dataset of sequences from a randomly generated sequence with length of , bp, and test the phylogenetic trees from this and other methods. we have chosen those datasets of different sizes (number of sequences, and lengths of sequences), which to test if anv can be suitable in all cases. most datasets have been analyzed by previous researches, therefore we can compare our results to others to evaluate the performances. four datasets consist of viruses that are closely related to human health, and the mammal's dataset and simulated dataset show that this method can perform on other types of sequences as well. coronavirus belongs to the subfamily coronavirinae in the family coronaviridae, in the order nidovirales. in this paper, we construct a dataset with coronaviruses, in which viruses are from the exact same dataset with (woo et al., ; yu et al., ; hoang et al., ) . the other two viruses are two new members in coronavirus. details of the coronaviruses can be found in table s . the new chinagd (lu et al., ) was identified in guangdong province (china) in and is an imported middle east respiratory syndrome coronavirus. the other one mers-cov/kor is from south korea (kim et al., ) . as of june , the mers-cov was spreading in south korea, and the chinagd case was a south korean national who traveled to guangdong in may . therefore, those two members were considered highly correlated with each other. the genomic size of coronaviruses ranges from about to kbp, with the average of , nucleotides. using our accumulated natural vector and upgma method (sneath and sokal, ) , we can build up a phylogenetic tree as shown in figure . figure shows that the two new members are clustered together with group , which is also well-known as sars (severe acute respiratory syndrome). between november and july , an outbreak of sars in southern china caused an eventual , cases, resulting in deaths reported in countries. both mers-cov and sars viruses are beta-coronaviruses, however, they belong to different lineages, for more details please see (drexler et al., ; hilgenfeld and peiris, ) . the phylogenetic tree indicates that the chinagd and mers-cov/kor forms a monophyletic clade, sister to the sars clade, which may possibly be a variant from some sars viruses. we also performed the same procedure with k-mer method on the coronaviruses dataset. however, how to choose an optimal k-value is an interesting topic that requires manual intervention. sims et al. showed in woo et al. ( ) that the location of the peak in the distribution of k-mers, i.e., the k with the largest vocabulary, is related to the sequence length n. the k with maximum information is empirically determined but may be closely approximated by where is the alphabet size. they have shown in sims et al. ( ) that reliable tree topologies are typically obtained with k-mer resolutions where k > k hmax whereas lengths below k h max yield unreliable trees. the upper limit of resolution can be empirically determined by a criterion that the tree topology for feature length k is equal to that of k+ , i.e., tree topologies converge. according to this principle, we have ≤ k ≤ . we show the result of k = in figure a , and the results of k = and k = are in the figures s , s . the four outgroup viruses cannot be clustered together as another branch from the tree of coronaviruses, meanwhile the group was divided into smaller groups. the traditional clustalw algorithm of multiple sequence alignment (msa) is also applied on the same dataset, and the result is shown in figure b . msa cannot cluster viruses from same groups together either. from this example, we can see that our anv method is better than the k-mer and msa method. influenza a viruses are single-stranded rna viruses, which have been a major health threat to both human society and animals . influenza a viruses' nomenclature is based on the surface glycoproteins: hemagglutinin (ha) and neuraminidase (na) (obenauer et al., ) . ha has subtypes and na has subtypes, which forms different combinations. the ncbi number of the analyzed influenza a viruses can be found in table s . our result agrees with previous work by hoang et al. ( ) . furthermore, we find that all the influenza a viruses are clustered with the same h and n type in figure , with only one exception of a/turkey/minnesota/ / (h n ). there is no specific research about this virus and we infer that it may be the intermediate from h n to h n . h n had an outbreak in july , causing millions of poultry's infection, but there is no report of infection from human to human yet. however, h n was identified in shanghai, china at the end of march . considering that the ha glycoprotein of those two subtypes are the same and the close outbreak date, we indicate that the h n on march might be a variant from h n , and a/turkey/minnesota/ / (h n ) plays a key role in this variation. we get the same conclusion in another work as well (dong et al., ) . more biological research on this virus should be done to deepen our understanding of influenza a viruses to accelerate the invention of an effective vaccine and to prevent more dangerous variants. the k-mer method and msa are also performed on this dataset as shown in figures a,b . the k-value is determined in the same procedure as in the coronaviruses dataset as . in figure a , the viruses from h n and h n are mixed up together with each other, while msa has a worse result in figure b . the results also indicate that k-mer and msa cannot reveal the real relationships among the viruses. to get a direct image of the relationships between influenza a viruses, we draw the natural graph of them. natural graph was first introduced by zheng et al. ( ) . in figure , the blue lines represent the -level connected components and the red ones -level. classes are marked in different colors and it is obvious that after the construction of two levels, the influenza a viruses with the same h and n are clustered together, including the a/turkey/minnesota/ / (h n ) which is number in figure . h n and h n are clustered together in level , indicating that they have a closer relationship, which accords with our previous conjecture. to illustrate that the new proposed anv method is an important improvement of the traditional natural vector method, a ebolaviruses dataset is tested, which is a subset of the viruses used in zheng et al. ( ) . it consists of ebola virus (ebov), sudan virus (sudv), reston virus (restv), taï forest virus (tafv), bundibugyo virus (bdbv), marburg virus (marv) and lloviuvirus (llov). details of this dataset are shown in table s . in figure a , the phylogenetic tree shows that from the novel accumulated natural vector method classifies all viruses into the right groups, however, in figure b , the traditional natural vector method divides ebov class into two clusters and sudv is misclassified with some ebov virus. this is an indication that including covariance between nucleotides helps improve the accuracy of classification. hence this is an important improvement to the traditional natural vector and other alignment-free methods. we also test a large dataset of viruses in li et al. ( ) , and the details of this dataset can be found in table s . the average length of them is , nucleotides and it makes alignment methods on a laptop impossible. only server or cloud computing can finish such a task. here we use -nearest neighbor ( -nn) method (li et al., ) to see the accuracy of the prediction. this evaluation is inspired by the high rate of missing labels in many databases of viruses. for example, if a virus with missing family label has been added to the database, and it should share the same family label with the virus (stored in the database already) that is closest to it, then we can predict the missing family label according to the information of its nearest neighbor. therefore, for a dataset with no missing labels, we can count how many viruses share the same label with its neighbor. "nearest neighbor" of a specific virus can be defined as the virus that has the smallest euclidean distance in the dataset to it for the alignment-free methods. for alignment results, we use the hamming distance to measure the distance between two sequences. if the virus shares its distance with its neighbor, we consider it as a "correct" one, since even if its label is missing we can still predict it from its nearest neighbor. the accuracy can be computed by dividing the number of correct ones by the number of all viruses, in this case, by . we compare the result of anv to the k-mer method since they are all alignment-free methods, and the results are shown in table . the optimal choice of k is made by the same procedure in the other datasets. from table , it is evident that anv has much higher accuracy than the k-mer method, meanwhile using much less time. thus, we have proved that anv can apply to practical use with high time-efficiency and high-accuracy. for the alignment in this part, we tried to align all the sequences with full length on our server, but it fails to give a reliable result. therefore, we extract , bp from the beginning and align pieces of segments all with length of , bp. the results are shown in table as well and the accuracy is still not as good as what anv gives. our accumulated natural vector performs well not only on virus datasets, but also on other common species. we extract mammalian mitochondrial genomes with the average length of , nucleotides, and the ncbi numbers of them can be found in table s . the genomes are from seven known clusters: primates, carnivora, cetartiodactyla, perissodactyla, eulipotyphla, lagomorpha, and rodentia. the accumulated natural vector method can still distinguish the differences among the seven clusters, as shown in figure a . ffp (k-mer) method has also been tested as well (the optimal k-value for this dataset is ), as shown in figure b . since the species that includes in different paper are not all the same, it is hard to compare the whole topology of phylogenetic trees, however, our work still only has a small difference from the previous work in murphy et al. ( ) and tarver et al. ( ) . the difference can be attributed to that mitochondrial genomes in mammals may not always reflect the organismal evolutionary history (morgan et al., ) , however, it still keeps more information than k-mer does in figure b , since the distance within each group is smaller than the distances among groups, we can still distinguish clusters based on current dataset. in ladoukakis and zouros ( ) , point out that most of the information researchers gained about the tree of life through the use of mtdna remains valid, while we should pay more attention to its role in the function of the organism and its value as a tool in the study of major evolutionary novelties in the history of life. therefore, the result implies that our anv method can capture the key information hidden inside the dna sequences and gives us a reliable topology among mammals. to verify is the similarity distance by our method can be used for clustering dna sequences effectively, we also generated different mutations in dna sequences and constructed phylogenetic trees by various methods. we simulated a sequence of length , bp as a base sequence, and generated two new sequences named "a_original" and "b_original" using point mutations. both a and b have nucleotides different from the original sequence. we then similarly evolved a and b into different mutants by four different mutations (substitutions, deletion, insertion, and transposition) as did in yin et al. ( ) . table is the detailed description on the simulated dna sequences with different mutations. since the sequences are mutated slightly based on an exon sequence, we take the aligned result as the "correct" relationships among the sequences, and the alignment is done by the "seqpdist" function in matlab bioinformatics toolbox. this function uses the classical jukes-cantor algorithm and we calculate the pairwise alignment distance. for comparison, we use the anv method, ffp method (we test k = , , in this case, since the lengths of sequences are about , bp). the upgma trees of alignment, anv and ffp (k= ) methods are shown in figures , a ,b separately. among these trees, it is not very obvious which one is more similar to the alignment results, therefore we calculate the robinson-foulds distances between the distance matrix and the "correct" matrix and the results are shown in table . here we apply the program named "robinson-foulds" (robinson and foulds, ) when calculating table . the simulated dataset is in table s . actually, the differences among trees mainly lie in the branch of sequences generated from b, and anv gives a more similar result, since the order is slightly disorganized by b and the transpositional sequences, while in figure b , the whole branch of b is different from the alignment result. in this paper, we propose a novel vector named accumulated natural vector to analyze sequences, genomes and their phylogenetic relationships. results from our analysis largely agree with the earlier studies, which indicates that our approach can detect the similarity and difference among sequences. therefore, constructing phylogenetic trees only by sequence data could be done accurately in a very reasonable time, without using large computing platforms or conducting biological experiments of high cost. our method can be applied in a global comparison of all genomes and provide a new powerful tool by including the correlations of nucleotides. we are working on 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measure of dna sequence similarity by fourier transform with applications on hierarchical clustering protein sequence comparison based on k-string dictionary real time classification of viruses in dimensions a novel construction of genome space with biological geometry ebolavirus classification based on natural vectors ss-ty and rh conceived the idea of covariance. rd implemented the idea and wrote the first draft of the manuscript. lh discussed and revised the first draft. rd, lh, rh, and ss-ty all contributed to the writing of the manuscript and agreed with the manuscript results and conclusions. they jointly developed the structure and arguments for the paper, made critical revisions and approved final version, and reviewed and approved the final manuscript. this study is supported by the national natural science foundation of china ( ) (to ss-ty), tsinghua university start-up fund (to ss-ty). the corresponding author would like to thank national center for theoretical sciences (ncts) for providing excellent research environment while part of this research was done. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fgene. conflict of interest statement: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © dong, he, he and yau. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - fsl authors: wathes, d. claire; oguejiofor, chike f.; thomas, carole; cheng, zhangrui title: importance of viral disease in dairy cow fertility date: - - journal: engineering (beijing) doi: . /j.eng. . . sha: doc_id: cord_uid: fsl many viral diseases are endemic in cattle populations worldwide. the ability of many viruses to cross the placenta and cause abortions and fetal malformations is well understood. there is also significant evidence that viral infections have additional actions in dairy cows, which are reflected in reduced conception rates. these effects are, however, highly dependent on the time at which an individual animal first contracts the disease and are less easy to quantify. this paper reviews the evidence relating to five viruses that can affect fertility, together with their potential mechanisms of action. acute infection with non-cytopathic bovine viral diarrhea virus (bvdv) in mid-gestation increases abortion rates or causes the birth of persistently infected calves. bvdv infections closer to the time of breeding can have direct effects on the ovaries and uterine endometrium, which cause estrous cycle irregularities and early embryo mortality. fertility may also be reduced by bvdv-induced immunosuppression, which increases the susceptibility to bacterial infections. bovine herpesvirus (bhv)- is most common in pre-pubertal heifers, and can slow their growth, delay breeding, and increase the age at first calving. previously infected animals subsequently show reduced fertility. although this may be associated with lung damage, ovarian lesions have also been reported. both bhv- and bhv- remain latent in the host following initial infection and may be reactivated later by stress, for example associated with calving and early lactation. while bhv- infection alone may not reduce fertility, it appears to act as a co-factor with established bacterial pathogens such as escherichia coli and trueperella pyogenes to promote the development of endometritis and delay uterine repair mechanisms after calving. both schmallenberg virus (sbv) and bluetongue virus (btv) are transmitted by insect vectors and lead to increased abortion rates and congenital malformations. btv- also impairs the development of hatched blastocysts; furthermore, infection around the time of breeding with either virus appears to reduce conception rates. although the reductions in conception rates are often difficult to quantify, they are nevertheless sufficient to cause economic losses, which help to justify the benefits of vaccination and eradication schemes. although viral disease remains a major cause of financial loss to the modern cattle industry, its potential impact on fertility is generally underestimated, and the main mechanisms of action are often unclear. factors including trade globalization, increases in herd size, and environmental change have contributed to the spread of existing pathogens and the introduction of disease into regions and animal populations that were previously free of it [ ] . poor fertility and udder health/milk quality remain the two major causes of concern among dairy producers [ ] . in terms of fertility, the ability of viruses to cause abortions and fetal malformations has probably received the most attention [ ] . the outcome is generally dependent on the stage of pregnancy during which the initial infection occurs. the effects of viral diseases on reproductive performance are, however, much more pervasive and can have many subtle effects through reductions in conception rates and increased risk of culling through failure to conceive in a timely fashion. excluding fertilization failure, approximately % of bovine embryos die in the first three weeks after service or insemination, with cows returning to estrus after - d. a further %- % of embryos are lost between days - of gestation [ ] . in comparison, abortion rates on cattle farms are usually quite low ( %- %) and have many potential etiologies that are often difficult to diagnose reliably [ ] . in addition to the loss of the fetus, an abortion does, however, often have adverse effects on the fate of the dam. depending on the stage of gestation when it occurs, the cow either may need to be rebred (thus increasing her calving interval) or may start the next lactation prematurely. in one study, for example, . % of holstein heifers aborted. this increased their risk of leaving the herd without completing a first lactation . times. one third of the animals which did not complete a first lactation either died or had to be culled within d of calving [ ] . the present short review focuses on five different viral infections showing a variety of mechanisms that can have an impact on dairy cow fertility. bovine viral diarrhea virus (bvdv) is discussed first and in the most detail, as its effects on fertility have been the most widely studied; thus, more is understood about its potential underlying mechanisms. bvdv is a flaviviridae pestivirus that is endemic in many countries worldwide, with a prevalence of %- % in individual cattle and %- % in cattle herds [ , ] . it comprises a single-stranded, positive-sense rna genome that is classified by sequence differences as type or (bvdv- or bvdv- ). there is also a third type, bvdv- (a hobi-like, atypical pestivirus). the virus exists as either non-cytopathogenic (ncp) or cytopathogenic (cp) biotypes, with the ncp biotype causing the majority of field losses [ ] . bvdv exhibits vertical transmission from mother to fetus, has a broad tissue tropism, and can infect the host either transiently or persistently [ ] . such rna viruses display significant genetic variation, facilitating the emergence of new species [ ] . mammalian cells normally produce type i interferons (ifns) in response to viral infection, which then trigger a cascade of antiviral pathways. bvdv causes immunosuppression through its ability to inhibit ifn production, thereby delaying the host's responses and enhancing the ability of the virus to complete its replication cycle [ , ] . bvdv infection generally occurs via the oronasal route, but direct transmission to the reproductive tract via semen or embryo transfer is also possible [ , ] . acutely infected animals usually eliminate the virus within - d, but transmissible virus can persist for much longer in some animals that have apparently recovered [ ] . in rare cases, bulls develop a persistent infection of the testes-an immune-privileged site. more commonly, bvdv is detectable by reverse transcription polymerase chain reaction (rt-pcr) in semen for some months after an initial acute infection, although the continued risk of viral transmission appears to be unlikely after nine weeks [ ] . fetal infection with ncp bvdv before the development of immune competence (i.e., prior to gestation day ) results in early embryonic death, later abortion, or the birth of an immunotolerant calf that is persistently infected (pi) [ ] . the pi calf can continuously shed virus from all secretions, and is therefore a major source of infection within a herd. the effects of acute bvdv infection vary extensively depending on both biotype and virulence, and this can lead to either avoidance or initiation of apoptotic and innate immune responses. ncp bvdv can dampen innate immune responses in several ways [ , ] . the virus is first detected by toll-like receptor (tlr)- or tlr- /tlr- located in intracellular compartments or by cytoplasmic pattern-recognition receptors (rig-i, ddx ), which detect single-stranded rna. the downstream signaling pathway from tlr- involves the ifn regulatory factor (irf)- and irf- , which usually upregulate the transcription of type i ifns. the bvdv protein n pro targets irf- toward proteasomal degradation, thus inhibiting downstream signaling and preventing the ifn rise [ ] . guanylate-binding protein (gbp ), an ifn-inducible gtpase, can also inhibit this pathway while leaving nf-jb signaling intact [ ] . in addition, the secreted bvdv structural protein e rns degrades viral rna through its extracellular function as a ribonuclease [ ] . many of the economic losses attributed to bvdv are due to suboptimal fertility, in addition to causing abortion and fetal deformity at later stages of gestation [ , ] . bvdv-induced immunosuppression increases susceptibility to other diseases, which may then also affect fertility. conception rates fell by up to % following experimental infections with bvdv either d before or d after insemination [ ] . the review by fray et al. [ ] cited many similar results that have been reported following ncp bvdv infection in the field, in spite of the occasional report to the contrary. since then, rüfenacht et al. [ ] measured fertility parameters in swiss dairy herds with a high prevalence of bvdv using individual seroconversion measurements to assess the time of likely exposure. infection during the first d of gestation did not influence non-return rates, but infection in mid-gestation was associated with an increased abortion rate from . % to . %. the timing of exposure is clearly critical, as rodning et al. [ ] reported that when pi animals were introduced to naïve heifers d prior to the start of breeding, they developed active immunity and there was no adverse effect on reproductive performance. newcomer et al. [ ] undertook a meta-analysis of studies to determine the potential benefits of vaccination against bvdv on three reproductive outcomes. vaccinated cows experienced a reduction in both abortion and fetal infection rates of nearly % and %, respectively, compared with unvaccinated cohorts, while the risk of becoming pregnant was smaller but nevertheless improved by about %. it is likely that a change of this magnitude would fail to reach significance in smaller studies due to a lack of statistical power. a variety of mechanisms have been suggested to account for such reductions in fertility via effects on the ovary, uterus, and early embryo. bvdv antigen was detectable in ovaries d after acute infection [ ] and in oocytes and follicular cells of pi heifers [ ] . animals infected with bvdv develop oophoritis [ ] and have impaired ovulation and ovarian steroidogenesis [ ] [ ] [ ] . when heifers were infected with acute ncp bvdv, follicular growth patterns were affected through the subsequent two estrous cycles, including reduced growth of dominant follicles [ ] . similarly, when heifers were infected d before a synchronized estrus, luteinizing hormone (lh) pulsatility was decreased, there was a delay from ovulation to the progesterone rise, and subsequent progesterone levels were lower [ , ] . these results align with studies showing that various types of stress can either delay or inhibit ovulation mechanisms [ , ] , while both heat stress and intramammary infection can reduce follicular steroidogenesis, disrupt follicular dominance, and reduce the pre-ovulatory lh surge [ ] . any acute infection occurring at this critical stage of the estrous cycle is likely to have a similar effect. the uterine endometrium is also recognized as a major site for bvdv infection [ , ] . bvdv was found in the uterus - d after infecting heifers with bvdv by either intravenous inoculation or by breeding to a pi bull [ , ] , while ncp bvdv was isolated from uterocervical mucus d after initial infection [ ] . bvdv antigen was also detected in macrophage-like cells of the endometrium in % of cows examined in a slaughterhouse survey [ ] . there is good evidence for two mechanisms by which the uterine presence of bvdv may have detrimental effects on fertility: first, by predisposing cows toward the development of endometritis; and second, by interference with the establishment of pregnancy. the bovine uterus is colonized with many bacterial species following calving in over % of cows [ ] . these bacteria should be cleared rapidly using mucosal defense systems and an innate immune response involving endometrial epithelial and stromal cells in addition to professional immune cells [ , ] . this early innate response is crucial to avoid the development of uterine disease; nevertheless, many dairy cows do develop metritis and/or endometritis (estimated at around % and % of all animals, respectively) [ ] . in cultured bovine endometrial cells, experimental infection with ncp bvdv inhibited a variety of immune pathways normally activated in response to a challenge with bacterial lipopolysaccharide (lps), including downregulation of many interferon-stimulated genes (isgs), which are an important part of uterine defense mechanisms [ , ] . infection with ncp bvdv was also able to switch endometrial prostaglandin production from prostaglandin (pg)f a to pge [ ] . pgf a is recognized as an immune enhancer, while pge acts as an immune suppressor and is luteotrophic [ , ] ; therefore, this switch may also reduce the endometrial immune response to bacteria and increase the likelihood of a cow developing a persistent corpus luteum [ ] , which is often found in association with uterine disease [ ] . maternal recognition of pregnancy in cows is achieved through the production of interferon tau (ifnt) by the trophectoderm of the elongating conceptus [ , ] , which inhibits the development of endometrial oxytocin receptors, thereby preventing luteolysis [ , ] . ifnt is a type i ifn that is structurally related to ifn-a and ifn-b but lacks viral responsive elements in its promoter and is therefore not upregulated by viral infection [ ] . ifnt does, however, bind to the same ifn-a/ifn-b receptor on the uterine endometrium. together with progesterone, ifnt programs the uterine endometrium to develop a receptive environment for implantation, including upregulation of many isgs [ , , ] . these are likely to have crucial roles in the establishment of pregnancy via modulation of uterine immunity, stromal remodeling, hyperplasia of the endometrial glands, and development of the uterine vasculature [ , ] . acute infection with ncp bvdv alone has been shown to have a limited influence on endometrial gene expression in vitro [ ] . however, infection did interfere with the isg regulatory irf-stat and stat pathways to inhibit ifnt-induced isg expression including isg , herc , usp (involved in protein modification via isgylation), ddx , ifih (cytosolic detection of viral rna) and ifit , mx , rsad , and samd (immune regulators with antiviral activity) [ ] . upregulation of the endometrial isgylation pathway is an important process in early pregnancy that is conserved across mammalian species [ ] . therefore, dysregulation of the antiviral ifn response by bvdv can undoubtedly interfere with ifnt signaling in the endometrium, suggesting another mechanism whereby infection in early gestation may reduce conception rates. there has been considerable research on the effects of bvdv on bovine embryos following concern that naïve cows might develop bvdv following embryo-transfer procedures. embryos produced using both in vivo and in vitro techniques have been infected with either ncp or cp virus at all stages from oocyte to hatched blastocyst. the affinity of bvdv for in vivo-derived embryos varied according to the strain of bvdv [ , ] . uterine inoculation with ncp bvdv- in the medium used for embryo transfer on day of a synchronized estrous cycle resulted in / heifers becoming pregnant d later, but these pregnancies had been lost within the following d [ ] . although bvdv replicated efficiently in cumulus cells surrounding bovine oocytes, this did not affect the development of the blastocysts subsequently produced by in vitro fertilization [ ] . similarly, when oocytes, zygotes, -cell embryos, morulae, and hatched blastocysts were infected with either ncp or cp virus, development was only adversely affected with cp bvdv and when the zona pellucida was not present [ ] . in a more recent study, cumulus-oocyte complexes were infected with bvdv- , bvdv- , or bvdv- at different doses [ ] . bvdv- had no effect on the embryos that did develop, and bvdv- infection actually increased cleavage rates but did not affect blastocyst rates. in both cases, however, the degenerate embryos tested positive. overall, the oocytes infected with bvdv- and bvdv- developed normally but carried the virus. bvdv- (hobi-like virus) reduced both cleavage and blastocyst rates, so would be expected to cause preimplantation embryo loss in vivo. bielanski et al. [ ] used semen from a pi bull on superovulated cows, collected day embryos, and transferred washed embryos to clean recipients. although bvdv was detected in the pre-transfer embryos, it did not infect the new host. from this work, it was concluded that the risk of transmission of bvdv to host cows via embryo transfer was minimal providing correct washing procedures were applied, as recommended by international embryo transfer society guidelines [ ] . this resulted in low copy numbers of virus, as measured by a sensitive quantitative polymerase chain reaction (qpcr) technique [ ] . in summary, acute ncp bvdv infection causes intracellular changes to ovarian and endometrial tissues through combined effects on pathways regulating immunity. these effects can reduce cow fertility by causing estrous cycle irregularities, early embryo mortality, and immunosuppression. infections during midgestation increase abortion rates or may give rise to the birth of pi calves. infectious bovine rhinotracheitis (ibr) is a highly contagious respiratory disease caused by bovine herpesvirus (bhv)- that is characterized by acute inflammation of the upper respiratory tract. bhv- is a virus of the family herpesviridae and subfamily alphaherpesvirinae. although some countries have achieved ibr eradication [ ] , the disease remains endemic in dairy herds in many parts of the world, including britain and ireland [ , ] . a recent metaanalysis found a pooled prevalence of bhv- of % in chinese cattle [ ] . it is a major contributing factor in calf pneumonia, which remains the most common cause of mortality and morbidity in dairy calves between and months of age [ ] . bhv- can also cause conjunctivitis, abortions, encephalitis, and generalized systemic infections [ , ] . after the first infection, the virus is never fully eliminated, remaining latent in nerve cells of the brain. from there, it can be reactivated in times of stress, mediated via increased glucocorticoids [ ] [ ] [ ] . bhv- is only one of a diverse range of pathogens that can contribute to bovine respiratory disease (brd) including several other viruses (i.e., bovine respiratory syncytial virus (brsv), parainfluenza iii virus (pi ), bvdv, and corona viruses), bacteria (e.g., mannheimia haemolytica, haemophilus somnus, pasteurella spp., and mycoplasma), and fungal genera (e.g., aspergillus) [ ] . numerous epidemiology studies in various countries around the world have determined that up to % of calves contract brd [ , ] . for the calves that survive, there is mounting evidence of longer term consequences of juvenile disease on adult performance [ , ] . brd-affected animals have reduced growth rates [ , ] , which in turn delay the age at first breeding and first calving. this is often associated with bronchopneumonic lesions and pleural adhesions [ ] . for example, first parity was delayed by a median of six months in heifers that had brd in the first three months of life [ ] . bach [ ] reported that calves experiencing four episodes of brd before first calving had . ± . greater odds of failing to complete their first lactation in comparison with healthy calves. another study found that calving intervals were increased by % in mature cows that had experienced severe brd as calves during their first three months [ , ] . in irish herds with a seasonal calving pattern that were identified as positive by a bulk tank bhv- enzyme linked immunosorbent assay (elisa), the three-week calving rate was significantly lower in multiparous cows in comparison with bhv- negative herds [ ] . two related epidemiology studies in ethiopia found significantly higher rates of uterine infection and retained fetal membranes in cows that were seropositive for bhv- [ , ] . a meta-analysis of over animals showed an overall decrease in abortion risk of % in pregnant cattle vaccinated against bhv- [ ] . a number of studies have investigated the effects of treating cattle with modified live ibr vaccine around the time of breeding. heifers inoculated at estrus [ ] , the day after [ ] , or on days or post-breeding [ ] developed mild oophoritis characterized by foci of necrosis, a few necrotic follicles, and mononuclear cell accumulation in the corpus luteum. heifers inoculated on days or post-breeding did not have lesions in the corpus luteum, but there were numerous necrotic follicles [ ] . such lesions were not found in ovaries from which bhv- was not isolated [ ] . vaccination at estrus was followed by a reduction in circulating progesterone [ , ] ; conception rates were also reduced [ , ] . although this review relates primarily to cows, there is evidence that young bulls exposed to bhv- at about six months of age had reduced sperm quality six months later [ ] . givens [ ] recently reviewed the effects of a number of viral diseases on bulls and the transmission risks of these diseases via semen. in summary, a high proportion of dairy calves experience brd, which is often associated with bhv- infection. this slows growth, leading to an increased age at first calving. fertility, risk of culling, and abortion rates are all subsequently increased. information on the direct effects on the reproductive tract is sparse, but there is some evidence that infection can have a direct effect on ovarian function. bhv- is a double-stranded dna virus that is highly prevalent in some dairy herds and has been associated with reduced fertility [ , ] . in common with other herpes viruses, it can remain latent in the host following an initial infection in several cell types including macrophages. this results in a persistent infection [ ] , which can be reactivated in vitro by glucocorticoids [ , ] . there is evidence from measuring seroconversion that it can also be reactivated in vivo during the periparturient period [ ] and in association with clinical metritis [ ] . like bvdv, bhv- can readily infect the uterus and has been associated with metritis and endometritis; however, its role in fertility is somewhat unclear, as it has often also been found in control cows that did not have uterine infection. in addition, tested cows were usually also positive for recognized bacterial pathogens including escherichia coli, trueperella pyogenes, streptococcus spp., and histophilus somni [ ] [ ] [ ] [ ] . nevertheless, there is evidence that bhv- can be associated with reduced fertility. a comparison between cows requiring one or two inseminations to conceive and those needing more than two inseminations found a higher prevalence of bhv- in the cows requiring more inseminations [ ] . klamminger et al. [ ] also recorded reduced risks of infected animals either being inseminated before d after calving or conceiving within d. unlike ncp bvdv, bhv- is cytopathic, and infection can kill endometrial epithelial and stromal cells [ , ] . accumulating evidence supports the view that bhv- can act as a co-factor with established uterine pathogens to promote the development of endometritis [ , , ] . replication of bhv- depends on immediate early gene (ie ) transactivation, and it has been shown that this promoter is upregulated by pge , tumor necrosis factor-a (tnf-a), escherichia coli, and lps, all of which are associated with bacterial infection of the endometrium [ , ] . bhv- in turn activates the interleukin (il)- gene promoter in endometrial cells [ , ] . this is a key chemokine that attracts granulocytes to the uterus. in a recent study, tebaldi et al. [ ] measured global gene transcription caused by the bhv- infection of cultured bovine endometrial stromal cells. in addition to il- , another main pathway that was activated involved the upregulation of matrix metalloproteinase (mmp)- . mmps are involved in the remodeling of the postpartum endometrium [ ] . they are also important in controlling the balance of immune responses. on the one hand, their proteolytic activity can promote immune cell migration and activate cytokines such as il- , il- , tnf-a, and defensins [ ] . on the other hand, over-activation of mmps has been associated with many immunopathological outcomes (reviewed in ref. [ ] ). in summary, the evidence to date suggests that bhv- infections are quite common in dairy cows. the virus on its own probably does not cause clinical uterine disease, but it can be reactivated from latency in the endometrium following calving and then act together with bacterial pathogens to increase the risk of uterine disease by disrupting innate immunity and impairing uterine repair mechanisms. schmallenberg virus (sbv) first emerged in europe in . phylogenetic analysis showed that it belongs to the simbu serogroup of the genus orthobunyavirus [ ] . sbv is transmitted by culicoides midges and affects both domestic and wild ruminants including sheep, goats, and cattle. the clinical signs of disease in adult cows are quite mild and include fever, a drop in milk yield, and diarrhea with peak viremia - d post-infection [ ] . sbv can both persist in and cross the placenta to replicate in the fetus itself [ ] . depending on the time of exposure, this may result in abortion or severe congenital malformations causing dystocia and the birth of non-viable calves [ , ] . a case control study on swiss dairy farms found that the abortion rate increased to . % in when the sbv infection started, in comparison with a rate of . % the year before [ ] . while these effects on the fetus are the most obvious sign of disease, there is also evidence for adverse effects on the establishment of pregnancy and/or early embryo development. similar to bvdv, it is possible that sbv infection during early pregnancy may disrupt ifnt production, thus compromising the survival of the conceptus. like bvdv, sbv uses a non-structural protein (in this case nss) that degrades cellular rna polymerase ii, resulting in the inhibition of type i ifn production and an increase in virulence [ ] . the impact of the epidemic on the productivity of dairy cattle in the netherlands and parts of germany was assessed at the herd level in a study by veldhuis et al. [ ] , who compared milk production, fertility, and mortality during the epidemic with those from an earlier reference period. in both countries, there was a small but demonstrable decline in fertility parameters during the epidemic, including a significant increase in the number of repeat inseminations required and a decrease of about % in the -day nonreturn rate (from . % to . %). a further analysis was undertaken based on the effects of sbv on swiss dairy cows [ ] . this was analyzed at the individual animal level and similarly found that the number of inseminations per cow was higher during the epidemic for cows showing clinical signs of infection in comparison with non-clinical animals from case and control herds. in this study, the non-return rate was not affected, although this may have been influenced by farms with affected animals stopping their services during the period of active infection. bluetongue virus (btv) is an important orbivirus virus infection of both domestic and wild ruminants. its geographical distribution is primarily dependent on the distribution of culicoides midges, which are the insect vectors [ ] . many serotypes of btv exist, including the btv- strain, which is currently circulating in europe [ ] . in addition to potentially causing high morbidity and mortality and reduced milk production, btv affects reproductive performance in dairy cows [ , ] . the virus can cross the placenta, and bovine fetuses infected before d of gestation develop fatal malformations of the central nervous system [ ] . later studies on btv- also found a higher incidence of congenital malformations in newborn calves [ ] . cows that were seropositive for btv in a californian study were significantly older at first calving [ ] . fetal mortality increased during an outbreak of btv- in belgium in [ ] . an early epidemiological study provided evidence for lower conception rates and longer calvingto-conception intervals in cattle [ ] . more recently, this was confirmed from data obtained after an outbreak of btv- in the netherlands [ ] . this study found that infected cows were five times more likely to return to service within d after their first artificial insemination (ai) and required . times more inseminations. using a different analytical approach, nusinovici et al. [ ] provided evidence that french cows infected with btv- experienced reduced fertility if they had been inseminated from four weeks before until five weeks after the date of disease detection within the herd. together, these studies provide good evidence that btv- infection prevents initial conception and/or has an adverse effect on early embryos. experimental infection of pre-implantation cattle embryos was only cytopathic in embryos with damaged zona pellucidae; there was no evidence of btv transmission to the early embryo in viremic donors [ ] . days - hatched blastocysts were, however, susceptible to btv- infection, showing growth arrest and increased apoptosis [ ] . there is again evidence that btv has the ability to inhibit ifn synthesis. in this case, viral ns protein is able to counteract the host's immune response by downregulating the expression of type i ifn and isgs [ ] . as discussed above for bvdv, this may potentially negate the signals normally associated with the maternal recognition of pregnancy. this literature review confirms that many common viral infections of cattle have adverse effects on dairy cow fertility. abortions and fetal abnormalities are easy to quantify, although in many cases the causal factor remains unknown. in contrast, reductions in conception rates are much more difficult to detect reliably. the effects are dependent on the exact stage of the reproductive cycle when the animal becomes infected, and are influenced by herd and season. some viruses can remain latent, and reactivation around calving is likely in association with the metabolic stress of early lactation. others have synergistic actions with other infectious agents, either directly or indirectly by promoting immunosuppression in the host. this may interrupt reproductive processes such as ovulation and implantation as well as predisposing the animals to bacterial infections of the reproductive tract. determination of significant effects on fertility rates in the field is dependent on having significant power in the study to detect potentially small changes. it is also complicated by our inability to capture reliable data on many other factors that influence fertility, such as the previous disease and vaccination history and the current metabolic status of individual cows. in vitro studies using primarily uterine endometrial cells and embryos have provided useful evidence on mechanisms of action. however, very few studies have made a thorough examination of the effects on the reproductive tract of viral infection in vivo. this is understandable, given the costs involved and the practicalities of maintaining infectious cows in containment facilities over a sufficient period of time. despite these limitations, the available data do strongly suggest that viral disease plays a key but currently under-recognized role in reducing cow fertility. given the importance of viral diseases in global cattle production, attempts to eradicate-or at least 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epidemic: impact on milk production, reproductive performance and mortality in dairy cattle in the netherlands and kleve district association of clinical signs after acute schmallenberg virus infection with milk production and fertility in swiss dairy cows culicoides and the emergence of bluetongue virus in northern europe bluetongue in europe: vectors, epidemiology and climate change bovine infection with bluetongue virus with special emphasis on european serotype quantification and at-risk period of decreased fertility associated with exposure to bluetongue virus serotype in naïve dairy herds the impact of bluetongue virus on reproduction a cross-sectional study of bluetongue virus and mycoplasma bovis infections in dairy cattle: ii. the association between a positive antibody response and reproduction performance bluetongue in belgium: episode ii bluetongue virus serotype (btv- ) infection reduces fertility of dutch dairy cattle and is vertically transmitted to offspring bluetongue virus and embryo transfer in cattle susceptibility of in vitro produced hatched bovine blastocysts to infection with bluetongue virus serotype bluetongue virus ns protein is an interferon antagonist and a determinant of virus virulence unmapped reads from cattle rnaseq data: a source for missing and misassembled sequences in the reference assemblies and for detection of pathogens in the host the work in the authors' laboratory was funded by contributions from the royal veterinary college, the china scholarship commission, and the commonwealth scholarship commission. the authors thank professor joe brownlie and miss olivia anstaett for their generous provision of bvdv for use in in vitro experiments. rvc manuscript number is pps_ .compliance with ethics guidelines d. claire wathes, chike f. oguejiofor, carole thomas, and zhangrui cheng declare that they have no conflict of interest or financial conflicts to disclose. key: cord- -kzaowysv authors: joshi, lok r.; okda, faten a.; singrey, aaron; maggioli, mayara f.; faccin, tatiane c.; fernandes, maureen h. v.; hain, kyle s.; dee, scott; bauermann, fernando v.; nelson, eric a.; diel, diego g. title: passive immunity to porcine epidemic diarrhea virus following immunization of pregnant gilts with a recombinant orf virus vector expressing the spike protein date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: kzaowysv passive immunity is critical for protection of neonatal piglets against porcine epidemic diarrhea virus (pedv). here, we investigated the immunogenicity of an orf virus (orfv) vector expressing the full-length spike (s) protein of pedv (orfv-pedv-s) in pregnant gilts and its ability to confer passive immunity and protection in piglets. three doses of orfv-pedv-s were given to two groups of pedv-negative pregnant gilts, with the last dose being administered two weeks prior to farrowing. one of the two groups immunized with the orfv-pedv-s recombinant virus was also exposed to live pedv orally on day post-immunization (pi). antibody responses were assessed in serum, colostrum and milk of immunized gilts, and passive transfer of antibodies was evaluated in piglet sera. the protective efficacy of orfv-pedv-s was evaluated after challenge of the piglets with pedv. pedv-specific igg, iga and neutralizing antibody (na) responses were detected in orfv-pedv-s-immunized and orfv-pedv-s-immunized/pedv-exposed gilts. pedv na, igg and iga were detected in the serum of piglets born to immunized gilts, demonstrating the transfer of antibodies through colostrum and milk. piglets born to immunized gilts showed reduced morbidity and a marked reduction in mortality after pedv challenge in comparison to control piglets. piglets born to gilts that received orfv-pedv-s and were exposed to live pedv showed stronger na responses and lower clinical scores when compared to piglets born to gilts immunized with orfv-pedv-s alone. these results demonstrate the potential of orfv as a vaccine delivery platform capable of eliciting passive immunity against pedv. porcine epidemic diarrhea virus (pedv), an alphacoronavirus in the family coronaviridae, is a single-stranded, positive-sense rna virus that causes porcine epidemic diarrhea (ped) in pigs [ ] . pedv infects pigs of all age groups resulting in an enteric disease with high morbidity. high mortality rates ( %- %) are observed in neonatal piglets; however, mortality is usually low in older animals [ ] . ped is characterized by vomiting, watery diarrhea and dehydration, which are usually followed by death in suckling piglets [ ] . after its introduction in the usa in , pedv caused the deaths of over million piglets, resulting in significant economic losses to the us swine industry [ ] . since then, significant investments and efforts to develop pedv vaccines have been made, with a few vaccines receiving a conditional license from the usda. the efficacy of these vaccines in protecting newborn piglets, however, is still unknown. piglets are born agammaglobulinemic due to the impermeable nature of the epitheliocorial swine placenta, and their immune system is immature, which makes them highly susceptible to pedv in the first weeks ( - ) of life. therefore, passive transfer of antibodies through colostrum and milk is critical for protection of neonatal piglets against pedv [ , ] . previous studies with transmissible gastroenteritits virus (tgev), a coronavirus that is closely related to pedv, have shown a high correlation between milk antibody levels and protection in piglets [ , ] . hence, a requirement for an effective pedv vaccine is the ability to induce high levels of antibodies in colostrum and milk with their subsequent transfer to piglets born to immunized sows. although several attenuated, killed, or subunit pedv vaccines have been developed, most of them fail to induce sufficient levels of lactogenic immunity and protection in newborn piglets [ , ] . thus, it is critical to develop improved alternatives to currently available pedv vaccines. recently, we demonstrated that a recombinant orf virus (orfv) expressing the full-length spike (s) protein of pedv (orfv-pedv-s) is capable of eliciting protective immune responses in immunized pigs [ ] . three-week-old piglets immunized intramuscularly (im) with the orfv-pedv-s recombinant virus were protected from clinical signs of ped after oral challenge with pedv and showed reduced virus shedding in feces [ ] . in the present study, we investigated the immunogenicity of orfv-pedv-s recombinant virus in pregnant gilts and its ability to induce passive immunity and protection in piglets born to immunized animals. the recombinant orfv-pedv-s was previously generated and characterized in our laboratory [ ] , propagated and titrated in primary ovine fetal turbinate cells (oftu, provided by d.l. rock, university of illinois) [ ] . pedv strain usa/co/ (co ) was obtained from the national veterinary services laboratory (nvsl) and propagated in vero- cells (atcc ® crl- ™) in the presence of . µg of tpck-treated trypsin per ml (sigma aldrich, st. louis, mo). six primiparous gilts were randomly assigned to three experimental groups as follows: group (g ), control (mem; n = ); group (g ), orfv-pedv-s-immunized (n = ); group (g ), orfv-pedv-s-immunized/live pedv-exposed (n = ) ( table ) . animals from g and g were immunized intramuscularly (im) with ml of the recombinant orfv-pedv-s containing . tissue culture infectious dose (tcid )/ml in mem. all animals received the first immunization on day and two booster immunizations on days and post-immunization (pi). the second booster was administered two weeks prior to the anticipated parturition date. in addition to the immunization regimen with orfv-pedv-s described above, gilts in g were exposed to live pedv orally ( × tcid in ml) on day pi. animals from g were sham-immunized with ml mem as described above. animals were housed in bsl- animal rooms, and two weeks prior to farrowing, each gilt was transferred to individual farrowing crates (two crates per room). all gilts received prostaglandin-f α ( mg) on day pi by im injection to induce parturition. animals from g and g farrowed on day pi, while animals from g farrowed on day pi. twelve piglets from each sow were randomly selected, kept with their mothers (n = per group) for days and allowed to suckle colostrum and milk ad libitum. excess piglets were euthanized to keep all sows with an equal number of piglets (n = ). all piglets (g , g and g ) were challenged orally on day post-birth with a virus suspension containing . × tcid of pedv strain co ( ml/piglet). animals were monitored daily for clinical signs and mortality, and the experiment was terminated on day pi or day post-challenge (pc). clinical scores were evaluated based in four criteria, which were modified from a scoring method described by lohse and collaborators [ ] as follows: a) well-being: normal = , slightly depressed = , depressed and lethargic = ; b) defecation: normal feces = , semi-solid and pasty = , watery feces = ; c) vomiting: no = , yes = ; d) body condition: normal = , × . × thin = , emaciated = . mean daily group scores and mortality rates were calculated and compared between different groups. piglets showing severe dehydration and emaciation were euthanized based on the independent evaluation of sdsu's veterinarian. serum was collected from sows on days , , , , , , , , , , and pi. additionally, colostrum and milk were collected from sows on days (day of farrowing) , , , , post-farrowing. serum and rectal swabs were collected from piglets on days (pre-colostrum), (pre-challenge), , , and post-birth. all animal studies were conducted at the sdsu animal resource wing (arw), following the guidelines and protocols approved by the sdsu institutional animal care and use committee (iacuc approval no. - a). indirect elisas using a truncated s protein (aa - ) were used to assess igg and iga antibody responses in animals immunized with the recombinant orfv-pedv-s virus as described previously [ ] . neutralizing antibody responses elicited by immunization with the recombinant orfv-pedv-s were assessed by fluorescent focus neutralization assay (ffn) as described previously [ ] . pedv s-specific igg and iga antibody responses were assessed in colostrum and milk by fmia. optimal assay conditions (amount of antigen, colostrum/milk and secondary antibody dilutions) were determined by a checkerboard titration [ ] . virus shedding was assessed in rectal swabs by rt-qpcr. viral rna was extracted from rectal swabs using a zymo viral rna extraction kit (zymo research, ca, usa) according to the manufacturer's instructions. primers and a probe targeting the pedv nucleocapsid protein were designed using primerquest tool (integrated dna technologies inc., usa). rt-qpcr was performed using a sen-sifast™ probe lo-rox one-step kit (bioline, ma, usa) following the manufacturer's instructions. genome copy numbers per ml were determined using a relative standard curve method. the amount of viral rna detected in feces was expressed as log genome copies/ml. gilts from both immunized groups (g and g ) developed pedv s-specific igg and iga responses ( fig. a and b). igg and iga antibodies were first detected on day pi in animals in g , and the highest level of antibodies was detected on day pi. similarly, in g , igg and iga antibodies were first detected on day pi, and their levels increased after the booster immunization on day . an anamnestic antibody response was observed in g gilts after they were exposed to live pedv on day pi (fig. a and b) . no spike-specific igg or iga antibodies were detected in the serum of control sows (g ). the ability of orfv-pedv-s to induce neutralizing antibody (na) responses against pedv was assessed using a ffn assay. na were first detected in serum a week after the first booster immunization (day pi; fig. c ). an increase in na levels was observed in both immunized groups (g and g ) after day pi (fig. c) . gilts in g , which were immunized only with orfv-pedv-s, had the highest neutralizing antibody titers on day pi, a week after the second booster immunization (day pi; fig. c ), whereas gilts in g , which were immunized with orfv-pedv-s and exposed to live pedv (day pi), had the highest antibody titers on day pi, with titers remaining constant thereafter until the end of the experiment (fig. c) . similar to the igg and iga responses, higher neutralizing antibody responses were observed in gilts in g when compared to g animals. no neutralizing antibodies against pedv were detected in control gilts (g ) in serum samples collected pre-farrowing/pre-challenge. animals in g seroconverted to pedv, presenting detectable levels of igg, iga and na a week after challenge of the piglets (day post-birth; fig. ). notably, a strong correlation between na and igg and iga levels was observed in sow serum (na vs. igg: r = . , p < . ; na vs. iga: r = . , p < . ; fig. d and e). these results confirmed our previous findings demonstrating the immunogenicity of orfv-pedv-s in pigs [ ] . high levels of s-specific igg antibodies were detected on days and post-farrowing, decreasing thereafter in animals from g and g ( fig. a) . colostrum collected from sows in g , exposed to live pedv, had higher levels of igg when compared to g on day post-farrowing ( fig. a) . high levels of s-specific iga antibodies were also detected in colostrum of animals from g and g on day post-farrowing (fig. b) . notably, the levels of s-specific iga antibodies remained elevated in milk up to day post-farrowing (fig. b) . iga antibody levels were higher in milk from animals in g when compared to animals in g after day post-farrowing (fig. b) . no spike-specific igg or iga antibodies were detected in colostrum/milk collected from control sows (g ) ( fig. a and b ). these results demonstrate the ability of orfv-pedv-s to induce s-specific igg and iga antibodies in colostrum and milk. passive transfer of antibodies from immunized sows to their offspring was assessed by elisa and ffn assays performed on serum samples collected from piglets pre-and post-ingestion of colostrum/milk. no s-specific antibodies were detected in serum samples collected on day one prior to ingestion of colostrum (fig. ) . notably, high levels of s-specific igg and iga antibodies were detected in the serum of piglets from g and g on day post-birth. piglets born to immunized sows had igg and iga antibodies in their serum until the end of the experiment on day post-birth (fig. ) , with higher levels of igg antibodies being detected until the end of the experiment. both igg and iga levels were higher in piglets born to sows in g when compared to piglets born to sows in g . no spike-specific igg or iga was detected in serum of piglets born to control sows (g ; fig. ). in addition to igg and iga, nas were detected in piglet serum on day post-birth. high levels of na were detected in piglets born to orfv-pedv-s-immunized sows until the end of the experiment on day pi (fig. c) . no neutralizing antibodies were detected in the piglets born to control sows (g , fig. c ). notably, a significant correlation between na titers in sow serum and piglet serum was observed (r = . , p = . ). additionally, a strong correlation between na and igg and iga antibody levels was observed in piglet serum (na vs. igg: r = . , p < . ; r = . ; fig. e and f). together, these results demonstrate passive transfer of pedv-specific igg, iga and nas from immunized sows to piglets through ingestion of colostrum and/or milk. all piglets born to immunized or control gilts were challenged orally with virulent pedv strain co on day post-birth. piglets were monitored daily for characteristic clinical signs of ped and mortality. daily average clinical scores were calculated for each group, and the mean daily scores are presented in fig. a . all piglets born to sows in g and g showed clinical signs of ped starting on day pc which lasted until day - pc. the highest clinical scores were observed in g piglets between days and pc. piglets in g started showing clinical signs on day pc, when moderate diarrhea was observed and continued until day pc. as shown in fig. a , more-severe clinical signs were observed in piglets from g (average daily scores ranging from to ), followed by g (average daily scores ranging from to ) and then g (average daily scores ranging from to . ). it is important to note that, while piglets in g and g experienced vomiting and diarrhea, none of the piglets in group experienced vomiting during experiment. virus shedding in feces was assessed by rt-qpcr using rectal swabs. piglets in g had a significantly lower level of viral rna in their feces (p < . ) in comparison to piglets in g and g on day pc (fig. b) . however, no significant differences in virus shedding in feces were observed between the three groups thereafter. the daily mortality recorded for piglets in the three groups is presented in fig. c . notably, twelve out of results are presented as group mean s/p ratios or na titers. the error bars represent +/-standard error of mean (sem). statistical significance was determined using two-way anova, and multiple comparisons were ran using tukey's test. a, b, and c represent statistical significance for g vs. g , g vs. g , and g vs. g , respectively. the significance level is < . . s/p, sample-to-positive ratio; the arrowhead represents the day of challenge with pedv. (d) correlation of piglet serum na levels with sow mean na levels. (e) correlation of group mean igg antibodies in piglet serum with piglet serum na levels. (f) correlation of group mean iga antibodies in piglet serum with piglet serum na levels. correlations were calculated using the spearman method with a % confidence interval using graphpad prism rt-qpcr in piglet feces expressed as log genome copy number per milliliter. data are presented as group means. error bars represent +/-sem. statistical significance was determined using two-way anova, and multiple comparisons were ran using tukey's test. a, b, and c represent statistical significance for g vs. g , g vs. g , and g vs. g , respectively. the significance level is < . . (c) survival curve demonstrating piglet mortality. the survival curve was gener-ated using the kaplan-meier method. statistical comparison between groups was performed using the log-rank test in graphpad prism . the p-value was adjusted using the bonferroni method. (d) correlation of piglet mortality with piglet group mean na levels. correlation of piglet mortality with group mean iga antibodies in sow colostrum/ milk. (f) correlation of piglet mortality with group mean igg antibodies in sow colostrum/milk. correlations were calculated using the spearman method with % confidence interval using graphpad prism ( / ; %) piglets in control g died by day pc, while only one piglet ( / ; %) in g died on day pc, and none of the piglets ( / ; %) in g died after the pedv challenge. interestingly, when the correlation between antibody levels in piglets and sows was compared to survival rates in piglets, moderate to strong correlations between na levels in piglet serum (r = . , p = . ) and iga and igg antibody levels in the sow colostrum/milk (r = . , p < . ; r = . , p = . , respectively) were observed (fig. d, e and f) . together, these results demonstrate decreased morbidity and mortality in piglets born to immunized gilts and a strong correlation between survival and antibody levels in colostrum and milk. in the present study, we assessed the ability of orfv-pedv-s to induce passive immunity against pedv following immunization of pregnant gilts. one of the goals of the study was to assess whether immunization with orfv-pedv-s alone would be sufficient or if live exposure to pedv would be required for protection of piglets born to immunized gilts. this underlies our experimental design, in which pregnant gilts were either immunized with orfv-pedv-s (g ) or with orfv-pedv-s followed by oral exposure to live pedv (g ). similar to our findings in -week-old weaned pigs [ ] , im immunization of gilts with orfv-pedv-s elicited pedv-specific igg, iga and nas. as expected, the booster provided by live pedv exposure in gilts from g led to higher antibody responses in serum, colostrum and milk of these animals. notably, passive transfer of antibodies from gilts to piglets was observed in both g and g , as pedv-specific igg, iga and nas were detected in serum of piglets born to immunized gilts following ingestion of colostrum and milk. na antibodies detected in piglet serum showed a strong correlation with igg and iga levels ( fig. e and f). although the antibody responses elicited by immunization with orfv-pedv-s alone were lower in comparison with those induced by immunization with orfv-pedv-s followed by live pedv exposure, the levels of antibodies induced by im immunization with orfv-pedv-s were sufficient to reduce neonatal mortality after oral challenge with pedv. antibodies present in sow colostrum and milk are derived either from serum or produced locally in the mammary tissue [ ] , and transfer of pathogen-specific antibodies from the sow to the piglet via colostrum and milk is critical for protection against the pathogens during the neonatal phase of the piglet's life. in this context, iga plays a key role in protection against enteric pathogens such as pedv, mainly because it is stable to proteolytic degradation in the intestine. by remaining in the gut lumen, pathogen-specific iga can effectively inhibit/ decrease virus infection/replication in the gut epithelium [ , [ ] [ ] [ ] . the results of this study show that increasing levels of iga in milk of immunized animals paralleled lower disease morbidity and mortality in piglets born to immunized gilts after challenge with pedv. while severe ped and high mortality ( %) were observed in piglets born to control gilts in g (no antibody responses detected), piglets born to gilts in g and g (high levels of iga in g and even higher in g ) showed less-severe clinical signs and reduced mortality rates ( and %, respectively). these results suggest that pedv-specific iga detected in piglets in g and g may have contributed to reducing disease severity and mortality in piglets. the possibility that other antibody isotypes or t cell-mediated immunity may have played a role in protection, however, cannot be formally excluded. one of the most important observations of our study was the reduced mortality in piglets from g ( % versus % in the control group, g ), which were born to gilts immunized im with orfv-pedv-s. notably, the lower piglet mortality in this group paralleled s-specific igg, iga and na responses detected in the gilts and passively transferred to piglets. together, these observations show that parenteral immunization with orfv-pedv-s is sufficient to induce protective levels of passive immunity against pedv. these results corroborate the findings of previous studies in which pedv specific-antibodies have been detected in serum and colostrum/milk and passively transferred to piglets after parenteral immunization with live, inactivated and/or subunit vaccine candidates [ ] . although the mechanism(s) underlying this phenomenon remain unknown, it is possible that systemic antibodies could be transferred from the sow's serum to colostrum and milk and then to the piglets. alternatively, the immunomodulatory properties of the orfv vector used here could potentially lead to migration of antibody-secreting plasma cells to the mammary gland, resulting in local antibody production. future studies assessing local immune cells in the mammary gland and antibody isotypes in the intestinal lumen of piglets born to immunized sows will be critical for dissecting the precise mechanism(s) underlying the protective immune responses elicited by im immunization with orfv-pedv-s observed here. in summary, this study demonstrates the ability of orfv-pedv-s to confer passive immunity against pedv following immunization of pregnant gilts. similar to our previous findings in -week-old weaned pigs [ ] , im immunization of gilts with orfv-pedv-s elicited pedv-specific igg, iga and nas responses. additionally, passive transfer of antibodies from gilts to piglets was observed, as pedv-specific igg, iga and nas were detected in serum of piglets born to immunized gilts following ingestion of colostrum and milk. porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines porcine epidemic diarrhoea: new insights into an old 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parapoxvirus expressing the spike protein of porcine epidemic diarrhea virus experimental infection of young pigs with an early european strain of porcine epidemic diarrhoea virus and a recent us strain development of an indirect elisa, blocking elisa, fluorescent microsphere immunoassay and fluorescent focus neutralization assay for serologic evaluation of exposure to north american strains of porcine epidemic diarrhea virus the transfer of immunoglobulins igg, iga and igm from serum to colostrum and milk in the sow mucosal and systemic isotype-specific antibody responses and protection in conventional pigs exposed to virulent or attenuated porcine epidemic diarrhoea virus does circulating antibody play a role in the protection of piglets against porcine epidemic diarrhea virus? strategies for design and application of enteric viral vaccines evaluation of antibody response of killed and live vaccines against porcine epidemic diarrhea virus in a field study we thank the staff of the sdsu animal resource wing (arw) for excellent care and handling of animals and for their invaluable help with the animal experiments. funding this study was funded by the national pork board grant no. - and in part by the national institute of food and agriculture hatch project no. sd - . the authors declare that there is a patent pending related to this work (us patent pending, p us ).ethical approval all applicable international, national, and/or institutional guidelines for the care and use of animals were followed. all animal experiments were reviewed and approved by the sdsu iacuc (approval no. - a). this article does not contain any studies with human participants performed by any of the authors. key: cord- -ru ubss authors: roingeard, philippe; raynal, pierre‐ivan; eymieux, sébastien; blanchard, emmanuelle title: virus detection by transmission electron microscopy: still useful for diagnosis and a plus for biosafety date: - - journal: rev med virol doi: . /rmv. sha: doc_id: cord_uid: ru ubss transmission electron microscopy (tem) is the only imaging technique allowing the direct visualization of viruses, due to its nanometer‐scale resolution. between the s and s, tem contributed to the discovery of many types of viruses and served as a diagnostic tool for identifying viruses directly in biological samples, either in suspension or in sections of tissues or mammalian cells grown in vitro in contact with clinical samples. the diagnosis of viral infections improved considerably during the s, with the advent of highly sensitive techniques, such as enzyme‐linked immunosorbent assay (elisa) and pcr, rendering tem obsolete for this purpose. however, the last years have demonstrated the utility of this technique in particular situations, due to its “catch‐all” nature, making diagnosis possible through visualization of the virus, without the need of prior assumptions about the infectious agent sought. thus, in several major outbreaks in which molecular techniques failed to identify the infectious agent, tem provided the answer. tem is also still occasionally used in routine diagnosis to characterize infections not diagnosed by molecular assays. it is also used to check the microbiological safety of biological products. many biopharmaceuticals are produced in animal cells that might contain little‐known, difficult‐to‐detect viruses. in this context, the “catch‐all” properties of tem make it possible to document the presence of viruses or virus‐like particles in these products. however, the last years have demonstrated the utility of this technique in particular situations, due to its "catch-all" nature, making diagnosis possible through visualization of the virus, without the need of prior assumptions about the infectious agent sought. thus, in several major outbreaks in which molecular techniques failed to identify the infectious agent, tem provided the answer. tem is also still occasionally used in routine diagnosis to characterize infections not diagnosed by molecular assays. it is also used to check the microbiological safety of biological products. many biopharmaceuticals are produced in animal cells that might contain little-known, difficult-to-detect viruses. in this context, the "catch-all" properties of tem make it possible to document the presence of viruses or virus-like particles in these products. microscope had a much higher resolution than any of the light microscopes available at the time and promised to revolutionize many aspects of science, including cell biology and virology. ernst ruska was a physicist ( nobel prize winner in physics), but his younger brother, helmut ruska, who had trained in medicine, rapidly recognized the potential of this microscope for investigating the nature of viruses. in the early s, viruses were classified according to their hosts and the clinical symptoms they caused. despite the lack of established methods of biological sample preparation for transmission electron microscopy (tem) at this time, helmut ruska was able to characterize the morphology of several viruses and he developed a rough viral classification based on the size and shape of the viral particles. tem was rapidly adopted for its first major use in clinical virology: the differential diagnosis of smallpox, caused by the variola virus abbreviations: elisa, enzyme-linked immunosorbent assay; em, electron microscopy; emea, european medicines agency; fda, food and drug administration; fpert, fluorescent product-enhanced reverse transcription; lcmv, lymphocytic choriomeningitis virus; pcr, polymerase chain reaction; sars, severe acute respiratory syndrome; sfts, severe fever with thrombocytopenia syndrome; tem, transmission electron microscopy from the poxvirus family, and chickenpox, caused by the varicellazoster virus of the herpes family, based on investigations of fluid samples from the vesicles on the patients' skin. the chickenpox virus appeared to be spherical and to nm in diameter, with a central body, a structure clearly different from that of the much larger, brick-shaped smallpox virus. "dirty" clinical samples, such as plasma, urine and feces in the s constituted a major breakthrough for studies of these viruses. the etiologic agents of hepatitis b and a were detected in plasma and stool samples, respectively. the bk virus, a polyomavirus, was identified for the first time in the urine of patients undergoing renal transplantation. rotaviruses were also identified by tem as the main cause of epidemic gastroenteritis in humans and animals. , however, many other viruses were also found to cause gastroenteritis. the first of these viruses was the norwalk virus, identified during an outbreak of gastroenteritis in norwalk, ohio, usa. , viruses with a similar morphology were subsequently discovered elsewhere and called "norwalk-like" viruses, to reflect the similarity of their appearance on tem, before being officially renamed "noroviruses." other viruses from the adenovirus, astrovirus, , and calicivirus families were also identified in the stool samples of children with gastroenteritis. tem was, thus, widely used on negatively stained samples for routine diagnosis, as a rapid, "catch-all" method for distinguishing between the diverse viruses potentially implicated in human gastroenteritis, providing a diagnosis within minutes of the arrival of the sample in the laboratory. although more time-consuming, due to the need to embed a sample in resin and cut ultrathin sections with an ultramicrotome, the tem has also proved useful in medical virology, in searches for viruses in tissues. panels e and f in figure illustrate a parapoxvirus (orf virus) visualized in a skin biopsy specimen from a patient with a severe finger ulcer this was the case, in particular, for the diagnosis of viral gastroenteritis, for which molecular techniques capable of identifying most of the virus families involved in human gastroenteritis have been established. [ ] [ ] [ ] [ ] a similar shift in practice occurred in veterinary medicine, with elisas and pcr progressively replacing tem for the routine diagnosis of viral infections. [ ] [ ] [ ] [ ] in human medicine, the use of tem to differentiate between smallpox virus and the other viruses present in the fluids of cutaneous vesicles is no longer required, since the successful eradication of the variola virus in thanks to a worldwide vaccination program. it has been argued that tem remains potentially useful for this application in a context of bioterrorism. , however, the risk of smallpox reappearing is extremely small, and even in the unlikely event of this happening, molecular techniques would undoubtedly outperform tem for this diagnosis. consequently, the number of laboratories making use of tem for diagnostic purposes has decreased considerably. sometimes revealing unexpected infectious agents, while molecular methods require previous knowledge of the virus to be tested. tem has also the advantage of being able to potentially identify double or multiple infections caused by more than one virus, which could be missed by molecular or antigen tests. moreover, the nature of the samples to be analyzed can be diverse, from body fluids or biopsies analyzed directly or after cell culture. in some cases, tem has also been used to confirm a diagnosis previously established with molecular techniques. [ ] [ ] [ ] [ ] figure illustrates most of the viral particles produced by these cells such as intracytoplasmic or intracisternal a-type particles are defective and noninfectious ( figure ) . however, other particles such as c-type particles bud at the cell surface and may infect nonrodent cells ( figure ). some murine retroviruses have been shown to be tumorigenic in primates, and cases of leukemia have been reported in children with severe combined immunodeficiency treated by gene therapy involving the use of murine retroviral vectors. all these elements demonstrate the relevance of tracking the presence of retroviruses in biological products derived from rodent cells. reverse transcriptase assays performed on bulk harvests are often hampered by high background levels due to cell-derived dna polymerases. tem may, therefore, help to document the presence of retrovirus-like particles in these bulk harvests ( figure ). tem can also be used to gauge the concentration of viral particles to validate the clearance of retroviruses or any other virus suspected to be present in the master cell bank. fortunately, the endogenous retroviruses present in the chinese hamster ovary (cho) cell line, the main rodent cell line used to produce biological products in the biotech industry, have been shown to be noninfectious. testing requirements are now lower for this well-characterized cell line than for other cell lines with which experience is more limited. nevertheless, detection of retrovirus-like particles by transmission electron microscopy (tem) with negative staining in bulk harvests of rodent cells used for the production of biological products. scale bars represent nm in both panels novel cell substrates, including insect cell lines in particular, are now being introduced into the biotech industry. their use will carry new concerns about unknown viruses for which there is a potential risk of contamination, and tem will undoubtedly be useful for documenting the presence of viruses or virus-like particles in these cells and the products derived from them. in conclusion, although tem is sometimes seen as a somewhat "oldfashioned" technique, it still has an important role to play in virus detection. it is particularly useful for identifying unknown agents involved in particular outbreaks or transmission clusters. in routine diagnosis, it may be useful to confirm or even, in some cases, to guide the diagnosis of a viral infection. tem can also be used to check the viral safety of biopharmaceutical products. this technique has several disadvantages, such as the cost of electron microscopes and their maintenance, the need for well-trained microscopists, and timeconsuming analysis, particularly if the samples must be embedded in resin for the cutting of ultrathin sections. however, all the techniques available have benefits and disadvantages, and their complementary natures mean that there are advantages to be gained by using them in combination. in this respect, the principal advantage of tem is its ability to provide an image of the virus, providing additional confidence in the result. nobel lecture: the development of the electron microscope and of electron microscopy die bedeutung der ubermikroskopie fur die virusforschung versuch zu einer ordnung der virusarten the use of the electron microscope in diagnosis of variola, vaccinia, and varicella a negative staining method for high resolution electron microscopy of viruses direct electron-microscopy of organ culture for the detection and characterization of viruses viruses in the stools virus-like particles in serum of patients with australia antigen-associated hepatitis hepatitis a: detection by immune electron microscopy of a virus-like antigen associated with acute illness new human papovavirus (b.k.) isolated from urine after renal transplantation virus particles in epithelial cells of duodenal mucosa from children with acute non-bacterial gastroenteritis virus particles in 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virus (bpv) type and unclassified bpvs poxvirus infection in a great tit (parus major) outbreak of cutaneous form of poxvirus on a commercial turkey farm caused by the species fowlpox an outbreak of pseudocowpox in fattening calves in southern brazil fatal outbreak in tonkean macaques caused by possibly novel orthopoxvirus, italy the use of convalescent sera in immuneelectron microscopy to detect non-suspected/new viral agents guidance for industry: a viral safety evaluation of biotechnology products derived from cell lines of human or animal origin adventitious agent issues one-step fluorescent probe productenhanced reverse transcriptase assay retroviruses produced by hybridomas genomic organization and expression of endogenous retrovirus-like elements in cultured rodent cells helper virus induced t cell lymphoma in nonhuman primates after retroviral mediated gene transfer lmo -associated clonal t cell proliferation in two patients after gene therapy for scid-x evaluation of a quantitative productenhanced reverse transcriptase assay to monitor retrovirus in mab cell-culture characterisation of endogenous retrovirus in rodent cell lines used for production of biologicals virus detection by transmission electron microscopy: still useful for diagnosis and a plus for biosafety we wish to thank fabienne arcanger, sonia georgeault, christine the authors have no competing interest.orcid philippe roingeard http://orcid.org/ - - - key: cord- - sldbte authors: nkengasong, john n; onyebujoh, philip title: response to the ebola virus disease outbreak in the democratic republic of the congo date: - - journal: lancet doi: . /s - ( ) - sha: doc_id: cord_uid: sldbte nan the unfolding outbreak of ebola virus disease in the democratic republic of the congo (drc) dominated discussions at last month's world health assembly (wha) in geneva, switzerland. several funding pledges were made and who estimated that us$ million will be required to control the outbreak. on may , , the drc government declared an outbreak of ebola virus disease, initially in a remote area of the equateur province (figure). as of june , , the government of drc reported cases of ebola virus disease and deaths (case fatality rate · %). of the cases, were laboratory confirmed, were probable where it was not possible to collect laboratory specimens for testing, and were suspected. of the confirmed and probable cases, ( %) are from iboko, ( %) from bikoro, and four ( %) from wangata health zones. a total of five health-care workers have been affected, with four confirmed cases and two deaths. although this is the ninth outbreak of ebola virus disease in drc since , certain features of the current epidemic have caused serious concerns in the international community. first, four cases have been identified in wangata, a district of mbandaka, the capital of the equateur province with an estimated population of · million inhabitants. thus, this is the first time the drc government and partners are response to the ebola virus disease outbreak in the democratic republic of the congo aaa screening. routine management includes a ct angiography of the whole aorta for large screeningdetected aaa and an ultrasound of the popliteal arteries for all screening-detected aaas to detect concomitant popliteal artery aneurysms. sometimes, another aneurysm in the thoracic aorta or a more complex aortic aneurysm can be detected, which needs more careful planning and advanced interventional techniques. according to a recent report of screening-detected aaa, % of individuals who had surgery did not have a straightforward and simple evar. instead, they had a complex procedure associated with increased operative morbidity and mortality. an increasing proportion of popliteal artery aneurysms are found and operated because of screening for aaa. these scenarios have not been taken into account in the debate about the benefits and harms of aaa screening. smoking is eight times more common in individuals with aaa than in healthy controls and is implicated in % of aaa cases. the decreasing prevalence of smoking in sweden (from % of the population in , to % of the population in ) should be viewed as the main cause of the decreasing incidence and mortality of aaa. every percentage drop in the prevalence of smoking will have a huge effect on smoking-related diseases such as cancer and aaa. primary prevention programmes to reduce the prevalence of tobacco smoking is a top priority, whereas screening for aaa is not. department of clinical sciences, lund university, malmö , sweden stefan.acosta@med.lu.se i declare no competing interests. tackling an outbreak of ebola virus disease in an urban city. second, the other epicentres of the ebola virus disease outbreak are in rural remote areas: bikoro and iboko are located close to the river congo, which serves the two neighbouring countries of the central africa republic and the republic of the congo, creating an increased risk of the disease spreading to these countries if the outbreak is not rapidly controlled in drc. the - outbreak of ebola virus disease in west africa, which resulted in deaths, showed how rapidly the disease could spread into neighbouring countries. although the outbreak of ebola virus disease in drc is ongoing, two features of the response are noteworthy: the swiftness of the response time and the introduction of ring vaccinations, an innovative, pre-emptive strategy to vaccinate those most at risk of infection. the global health community learned from the - west africa ebola virus disease outbreak that a speedy response was vital to control the outbreak. the response to the current outbreak in drc has been rapid at the national, continental, and international levels. the drc health minister, oly ilunga kalenga, has led his country's response with pragmatism and expediency, both in kinshasa and at the provincial levels, by developing a comprehensive response plan and establishing appropriate technical committees and mobilising the requisite political, financial, and technical support. at the continental level, within days of declaration of the outbreak the africa centres for disease control and prevention (africa cdc), which one of us (jnn) leads, had activated its emergency operation center in addis ababa, ethiopia; deployed an advance team of epidemiologists to kinshasa to assist the ministry of heath; and briefed an extraordinary session of the permanent representative committee of the african union member states. in addition, the director of the africa cdc (jnn) led a delegation to kinshasa, bikoro, and mbandaka for an assessment of the gaps in the responses requiring support from africa cdc. the africa cdc team worked with the staff from who to assist the ministry of health to develop three strategies: surveillance and contact tracing; focal health-care zones to ensure adequate control measures; and laboratory testing. africa cdc is also deploying more than health-care workers, including epidemiologists, infection control and laboratory experts, and anthropologists. globally, who has had a crucial role by rallying appropriate attention to the outbreak and mobilising essential international partners to action. who has augmented the drc health ministry's response plan by deploying many health-care workers in the field and is supporting a ring vaccination programme with a merck-produced ebola vaccine. the vaccination programme is led by drc's ministry of health and supported by who and partners. this is the first time such a vaccine has been used outside of the west africa - epidemic. importantly, the vaccine is being deployed as the outbreak unfolds as part of a new international approach for rapid mitigation of outbreaks through multiple interventions. anthropological and sociological determinants of the uptake of the vaccination programme will be crucial as the intervention is scaled up. moreover, issues of access, equity, and ethical considerations in deploying the vaccine will need to be considered. containment centres at the affected sites to minimise transmission and improve clinical outcomes for detected patients. several other partners are assisting with the response, including the us centers for disease control and prevention, the international federation of red cross and red crescent societies, and others. however, six important gaps in outbreak governance and logistics management must be addressed to ensure a more comprehensive response to the ebola virus disease outbreak in drc. the first is leadership of the response. much has been learned from dealing with past outbreaks of ebola virus disease in west africa but each new outbreak has unique challenges. it is the responsibility of each country to ensure the health security of its citizens. however, effective leadership of the government of drc may be challenged given the weak health system of the country due to its long history of conflict and resulting economic and political difficulties. all efforts should be made to strengthen their leadership. as such, financial, human, and material assistance from the global community to drc's leadership will be central to ensure an effective response. second, coordination-but not controlof contributing partners' effort is essential to create efficiencies to control the outbreak. third, translating global material and financial commitment into countrylevel disbursements must be accelerated. fourth, commitments made to support the response in drc must be fulfilled. there have been huge logistical challenges with airlifting supplies and health-care workers from kinshasa to mbandaka, iboko, and bikoro, because no commercial flights exist from kinshasa to mbandaka and motorable roads from mbandaka to the other affected areas are non-existent. although financial and material support was expressed at the wha, ensuring that these commitments reach drc in a timely way is not yet evident. fifth, there has been inadequate support of the focal health-care zones (bikoro, iboko, mbandaka, and equateur province) to establish appropriate control measures and minimise transmission. finally, laboratory testing has been challenged by insufficient supplies and a shortage of experienced staff. the outbreak of ebola virus disease in drc is far from over and may take several months to bring under control. the steps taken in the next few weeks will be crucial to the trajectory of the outbreak and it is vital that effective coordination of partners' efforts and rapid provision of requisite and well tested interventions are put in place. in this context, fiscal and infrastructural support to drc will be important for rapid containment of the outbreak. as more partners join the fight to control this outbreak, standard operating procedures for engaging with the government, coordinating with other partners, allocating financial resources, and deployment in the field should be established quickly to ensure effective coordination, but not control, of partners' efforts. such standard operating procedures for outbreak governance will ensure that aid is not a burden to drc but an asset in the response. any successful public health response is based on trust between practitioners, patients, government, and communities. trust is essential for effective outbreak control and must be underpinned by active case finding, contact tracing and follow-up, and engagement with communities. in addition, health-care infrastructure needs to be strengthened and supported to provide health care for non-ebola patients. strengthened health-care systems are needed to address existing endemic health challenges and respond to future ebola outbreaks and other emerging infections in drc. alongside the response to the outbreak, post-ebola recovery plans for drc must include supporting the country to develop a functional national public health institute. in future, the response to a potential tenth outbreak of ebola virus disease in drc must be led by the country's national public health institute. in november, , an outbreak of the severe acute respiratory syndrome caught china unprepared. in response to that outbreak, the government of china established the chinese center for disease control and prevention (china cdc). today, if faced with a similar disease threat, china cdc, and not the international community, would lead the response in china. this is what should be done in drc and all african countries. this is a vision africa cdc and the african union are promoting as a new public health order for africa's health and economic security. to ensure that this vision is achieved, the african union commission and africa cdc will be convening an international conference on ebola in drc: response in july, , to raise funding and advocate for sustained support for the drc's response efforts. cardiac troponin assays were introduced into clinical practice in for the diagnosis of acute myocardial infarction. originally, this assay could not help to reliably rule out myocardial infarction until about h after symptom onset. consequently, there has been a drive to develop more sensitive and reliable troponin assays that would facilitate an earlier exclusion of myocardial infarction, ideally in the emergency department. [ ] [ ] [ ] the new high-sensitivity cardiac troponin (hstrop) assays are able to detect troponin at much lower concentrations than those detected previously. this is in keeping with the universal definition of myocardial infarction, which recommends that a troponin assay used to diagnose myocardial infarction should have a coefficient of variation of % or less at the threshold concentration representing the th percentile upper limit of a so-called normal reference population. modern hstrop assays can detect troponin in more than % of the general population, with some assays able to detect some troponin in nearly everyone. however, there are important implications of this increased detection sensitivity on the interpretation of positive hstrop results by front-line clinicians, particularly in the context of the well established subclassification of myocardial infarction, the most clinically relevant being type and type myocardial infarction. type myocardial infarction is a classic heart attack, in which erosion or fissuring of the surface of an atherosclerotic plaque attracts a plateletmediated thrombus, thus reducing the coronary artery flow. by contrast, type myocardial infarction is due to a myocardial oxygen supply-demand mismatch, which occurs in conditions such as tachyarrhythmias, anaemia, and sepsis. evidence unequivocally supports the use of hstrop as a rule-out test for type myocardial infarction, allowing for early discharge of patients with a low subsequent clinical event rate. as a rule-in test for type myocardial infarction, however, the hstrop test is subject to considerable troponin cardiac protein molecule swedish national board of health and welfare. screening for abdominal aortic aneurysm-recommendation and assessment bases benefits and harms of screening men for abdominal aortic aneurysm in sweden: a registry-based cohort study outcome of the swedish nationwide abdominal aortic aneurysm screening program psychosocial consequences in men taking part in a national screening program for abdominal aortic aneurysm cost-effectiveness of screening for abdominal aortic aneurysm in combination with medical intervention in patients with small aneurysms population screening and intervention for vascular disease in danish men (viva): a randomised controlled trial clinical practice guidelines of the european society for vascular surgery on the complexity of screening detected abdominal aortic aneurysms: a retrospective observational multicenter cohort study increasing the elective endovascular to open repair ratio of popliteal artery aneurysm the aneurysm detection and management study screening program: validation cohort and final results tackling the tobacco epidemic in the nordic countries and lower cancer incidence by / in a -year periodthe effect of envisaged scenarios changing smoking prevalence as aid workers move to the heart of congo's ebola outbreak, "everything gets more complicated ebola outbreak in the dr congo: lessons learned la situation épidémiologique de la maladie à virus ebola ebola outbreak in west africa democratic republic of the congo. strategic response plan for the ebola virus disease outbreak democratic republic of efficacy and effectiveness of an rvsv-vectored vaccine in preventing ebola virus disease: final results from the guinea ring vaccination, open-label, cluster-randomised trial (ebola Ça suffit!) infectious disease trends in china since the sars outbreak africa centres for disease control and prevention, african union headquarters, po box , w k addis ababa, ethiopia nkengasongj@africa-union.org jnn is director of africa centres for disease control and prevention and po is senior adviser for policy and strategy in the office of the director at africa centres for disease control and prevention. we declare no other competing interests. key: cord- -fzcuu ma authors: jo, seri; kim, hyojin; kim, suwon; shin, dong hae; kim, mi‐sun title: characteristics of flavonoids as potent mers‐cov c‐like protease inhibitors date: - - journal: chem biol drug des doi: . /cbdd. sha: doc_id: cord_uid: fzcuu ma middle east respiratory syndrome‐coronavirus (mers‐cov) is a zoonotic virus transmitted between animals and human beings. it causes mers with high mortality rate. however, no vaccine or specific treatment is currently available. since antiviral activity of some flavonoids is known, we applied a flavonoid library to probe inhibitory compounds against mers‐cov c‐like protease ( clpro). herbacetin, isobavachalcone, quercetin ‐β‐d‐glucoside and helichrysetin were found to block the enzymatic activity of mers‐cov clpro. the binding of the four flavonoids was also confirmed independently using a tryptophan‐based fluorescence method. the systematic comparison of the binding affinity of flavonoids made it possible to infer their scaffolds and functional groups required to bind with mers‐cov clpro. an induced‐fit docking analysis revealed that s and s sites play a role in interaction with flavonoids. the experimental and computational study showed that flavonol and chalcone are favourite scaffolds to bind with the catalytic site of mers‐cov clpro. it was also deduced that some flavonoid derivatives with hydrophobic or carbohydrate attached to their core structures have a good inhibitory effect. therefore, we suggest that flavonoids with these characteristics can be used as templates to develop potent mers‐cov clpro inhibitors. coronaviruses (covs) are a positive sense, single-stranded rna virus coated with viral particles (fehr & perlman, ) . together with arteriviridae and roniviridae, covs belong to the coronaviridae family in the order nidovirales. these covs can infect a wide variety of hosts, including avian, swine and humans. human coronaviruses (hcovs) represent a major group of covs associated with various respiratory diseases from common cold to serious pneumonia and bronchitis (mesel-lemoine et al., ) . today, hcovs are recognized as one of the most rapidly evolving viruses originated from their characteristic high genomic nucleotide substitution rates and recombination. in human, their infection is known to cause approximately one-third of common cold. severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) covs are highly pathogenic and caused a fatal first major outbreak in and , respectively (de wit, doremalen, falzarano, & munster, . sars-and mers-covs genomes contain two open reading frames orf a and orf b translated to two respective viral polyproteins pp a and pp ab by host ribosomes. orf a encodes two cysteine proteases, a papain-like protease (plpro) and a c-like protease ( clpro). while plpro cuts the first three cleavage sites of its polyprotein, clpro is responsible for cleavage of the remaining eleven locations resulting in release of a total of non-structural proteins (nsp) in both sars-and mers-covs. the homodimeric form of clpro is active in the presence of substrates. the crystal structure of clpro showed that each monomer is composed of three structural domains: domains i and ii form a chymotrypsinlike architecture with catalytic cysteine and are connected to a third c-terminal domain via a long loop (neddle, lountos, & waugh, ) . in the proteolytic site, all clpros prefer glutamine at p position and leucine, basic residues, small hydrophobic residues at p , p and p positions, respectively (chuck, chow, wan, & wong, ) . at p ′ and p ′ positions, small residues are required. since the autocleavage process is essential for viral propagation, clpro is a good drug target for anti-coronaviral infection. in this study, we used a proteolytic method to probe mers-cov clpro inhibitory compounds with a synthetic peptide labelled with the edans-dabcyl fret (fluorescence resonance energy transfer) pair (liu et al., ) . since emission wavelengths of edans are widely overlapped with absorbance wavelengths of dabcyl, the energy emitted from edans will be quenched by dabcyl in a close proximity ( - Å). therefore, an increment of fluorescence can be a sign to judge whether a substrate is cleaved or not in this design. hence from the fluorescence intensity change, the proteolytic activity of protease could be detected. with a synthetic peptide with the fret pair, a flavonoid library was screened to search mers-cov clpro inhibitory compounds. recent studies showed that some flavonoids have antiviral activity in some viruses (frabasile et al., ; jucá et al., ; kiat et al., ; yang, lin, zhou, liu, & zhu, ; zakaryan, arabyan, oo, & zandi, ) . therefore, we assayed flavonoids and tried to induce their structural property crucial to bind with mers-cov clpro. the coding sequence of mers-cov nsp , a c-like protease (ncbi ref. seq. nc_ . ) was synthesized chemically by bioneer and cloned into a bacteriophage t -based expression vector. the plasmid dna was transformed into e. coli bl (de ) for protein expression. e. coli bl (de ) cells were grown on luria-bertani (lb) agar plates containing μg/ml ampicillin. several colonies were picked and grown in capped test-tubes with ml lb broth containing μg/ml ampicillin. a cell stock composed of . ml culture and . ml glycerol was prepared and frozen at k for use in a large culture. the frozen cell stock was grown in ml lb medium and diluted into , ml fresh lb medium. the culture was incubated at k with shaking until an od of . - . was reached. at this point, expression of mers-cov clpro was induced using isopropyl-β-d- -thiogalactopyranoside (iptg) at a final concentration of mm. the culture was further grown at k for hr in a shaking incubator. cells were harvested by centrifugation at , g ( , rev min − ) for min in a high-speed refrigerated centrifuge at k. the cultured cell paste was resuspended in ml of a buffer consisting of mm tris-hcl ph . , mm nacl, mm imidazole, mm phenylmethylsulfonyl fluoride (pmsf) and μg/ml dnase i. the cell suspension was disrupted using an ultrasonic cell disruptor (digital sonifier ; branson). cell debris was pelleted by centrifugation at , g ( , rev min − ) for min in a highspeed refrigerated ultra-centrifuge at k. the protein was purified by affinity chromatography using a ml hi-trap q column (ge healthcare) followed by a ml hi-trap blue column (ge healthcare). the custom-synthesized fluorogenic substrate, dabcyl-ktsavlqsgfrkme-edans (anygen), was used as a substrate for the proteolytic assay using mers-cov clpro (kuo, chi, hsu, & liang, ) . this substrate contains the nsp /nsp cleavage sequence, gvlq↓sg (wua et al., ) , and works as a generic peptide substrate for many coronavirus including mers-cov clpro. the peptide was dissolved in distilled water and incubated with each protease. a spectramax i x multi-mode microplate reader (molecular devices) was used to measure spectral-based fluorescence. the proteolytic activity was determined at k by following the increase in fluorescence (λ excitation = nm, λ emission = nm, bandwidths = , nm, respectively) of edans upon peptide hydrolysis as a function of time. assays were conducted in black, -well plates (nunc) in μl assay buffers containing protease and substrate as follows: for the mers-cov clpro assay, . μl of . mm protease containing mm tris ph . was incubated with . μl of . mm substrate at k for hr before measuring relative fluorescence unit (rfu). before the assay, the emission spectra of flavonoids were surveyed after illuminating at nm to avoid the overlapping with the emission spectrum of edans. every compound was suitable to be tested. the final concentration of the protease, peptide and chemical used at the assay was , . and μm each. at first, mers-cov clpro and chemical were mixed and preincubated at room temperature for hr. the reaction was initiated by the addition of the substrate, and each well was incubated at k for hr. after hr, we measured the fluorescence of the mixture on the black -well plate using the end-point mode of spectramax i x where the excitation wavelength was fixed to nm and the emission wavelength was set to nm using , nm bandwidth, respectively. all reactions were carried out in triplicate. among the first forty flavonoids (table s ), four of them were picked up to further assay at a concentration range of μm- μm. ic value which is the value causing % inhibition of catalytic activity of mers-cov clpro was calculated by non-linear regression analysis using graphpad prism . (graphpad software). the proteolytic assay using mers-cov clpro in the presence of triton x- has been performed to differentiate artificial inhibitory activity of chemicals through non-specific binding with proteases by forming aggregate or complexation. the concentration used in this study was . %. to confirm the feasibility of the assay method independently, the fluorescence spectra from tryptophans of mers-cov clpro with candidate inhibitors were investigated (lin, lan, guan, sheng, & zhang, ). the fluorescence measurements were recorded with a spectramax i x multi-mode microplate reader (molecular devices) at excitation and emission wavelengths of nm and - nm, respectively. the optimal excitation and emission wavelengths were determined by softmax pro. five tryptophans of mers-cov clpro showed a strong fluorescence emission with a peak at nm at the excitation wavelength of nm. in contrast, the flavonoids were almost non-fluorescent under the same experiment condition. each μm chemical was incubated with μm mers-cov clpro for hr, and the fluorescence intensity of mers-cov clpro was measured. all the docking and scoring calculations were performed using the schrödinger suite of software (maestro, version . . ). the compounds were extracted from the pubchem database in sdf format and were combined in one file. the file was then imported into maestro and prepared for docking using ligprep. the atomic co-ordinates of the crystal structure of mers-cov clpro ( wkj) were retrieved from the protein data bank and prepared by removing all solvent and adding hydrogens and minimal minimization in the presence of bound ligand using protein preparation wizard. ionizer was used to generate ionized state of all compounds at target ph . ± . . this prepared low-energy conformers of the ligand were taken as the input for docking analysis. grids for molecular docking were built using receptor grid generation. compounds were docked using ligand docking mode with postdocking minimization including poses per ligand. the docked poses were then refined using an xp (extra precision) option with the threshold value for rejecting minimized pose of . kcal/mol. the energies were calculated using the opls force field. the induced-fit docking protocol (sherman, day, jacobson, friesner, & farid, ) was run from the graphical user interface accessible within maestro . . . receptor sampling and refinement were performed on residues within . Å of each ligand for each of the ligand-protein complexes. with prime (jacobson et al., ) , a side-chain sampling and prediction module, the side-chains, as well as the backbone of the target protein, were energy minimized. a total of induced-fit receptor conformations were generated for each of the eight test ligands. re-docking the test ligands into their respective structures within . kcal/mol of their lowest energy structure. finally, the ligand poses were scored using a combination of prime and glidescore scoring functions . the amount of cell harvested for purification of mers-cov clpro was . g per , ml of e. coli culture. the protein was purified by ion chromatography using a ml hi-trap q column (ge healthcare). the column was equilibrated with a buffer consisting of mm bis-tris ph . , and the pooled fractions were loaded. the column was eluted using a linear nacl gradient to . m nacl, and the protein was eluted at . m nacl. the pooled fractions were loaded on a ml hi-trap blue column (ge healthcare) equilibrated with a buffer consisting of mm sodium phosphate ph . . the target protein was detected in unbound fractions. sds-page showed one band around ( , . da) kda, corresponding to the molecular weight of mers-cov clpro. the protein was concentrated to . mg/ml for protease assays in a buffer consisting of mm sodium phosphate ph . . the purified mers-cov clpro was different from the previous method (kumar et al., ) where the native form was obtained after removing of a his -tag wih factor-xa. in our experiment, the enzyme activity was -fold decreased when the his -tag protein was used (data not shown). that is due to the location of the his -tag connected to the n-terminus of mers-cov clpro. the published crystal structure showed that the active site pocket might be easily hindered by the flexible n-terminal his -tag. a flavonoid library consisting of ten different scaffolds was also built (figure ). it contains three isoflavones, one isoflavane, six flavones, nine flavonols, four flavanols, five flavanones, one flavanonol, one prenylflavonoid, eight chalcones and two unclassified flavonoids (table s ). we applied the library to assay mers-cov clpro. since flavonoids are known to aggregate through a complexation and thus non-specifically inhibit various proteases, the assay in the presence of triton x- was performed. before assay, we tested the influence of triton x- on the catalytic activity of mers-cov clpro. as shown in the figure s , only a slight increment of the catalytic activity was observed even up to . % triton x- . therefore, we performed the experiment at the concentration of . % triton x- where no significant interference was detected. using forty flavonoids, an inhibitory effect of each compound at μm was tested. among them, herbacetin ( , ′, , , -pentahydroxyflavone), isobavachalcone ( ′, , ′-trihydroxy- ′-( -methyl- -butenyl)chalcone), quercetin -β-d-glucoside ( , ′, ′, , -pentahydroxyflavone -β-d-glucoside) and helichrysetin ( , ′, ′-trihydroxy- ′methoxychalcone) were found to have prominent inhibitory activity (figure ). the four compounds showed the severely reduced fluorescent intensity and thus represented their mers-cov clpro inhibitory activity. the experimental data were plotted as log inhibitor concentration versus per cent fluorescence inhibition (figure ) . ic values obtained from the dose-dependent inhibitory curves of herbacetin, isobavachalcone, quercetin -β-d-glucoside and helichrysetin are . , . , . and . μm, respectively. in order to confirm the inhibitory activity of the flavonoids independently, a general tryptophan-based assay method was employed. tryptophan was well-known to emit its own fluorescence. therefore, if tryptophan is positioned adequately in proteins, the change in fluorescence intensity can reflect the binding state of chemicals and be used to judge interaction between proteins and chemicals. mers-cov clpro contains five tryptophan residues and thus displays a high fluorescence peak at nm at the tryptophan excitation wavelength of nm. we monitored the change in the fluorescence intensities of mers-cov clpro depending on the presence or absence of four chemicals. since each compound in the flavonoid library was almost non-fluorescent under the experiment condition, a change in fluorescence intensity reflects interactions between mers-cov clpro and chemicals. only the four inhibitory compounds obviously reduced the fluorescence intensity of mers-cov clpro (figure ) . the decreased emission intensity confirmed the complex formation between mers-cov clpro and inhibitory compounds. in order to deduce the binding modes of the inhibitory flavonoids, an in-depth theoretical investigation through an induced-fit docking study was carried out. the interactions between mers-cov clpro and the inhibitory flavonoids were analysed to predict their binding affinities. top ranked structures (according to the glide scores) from the induced-fit docking results for herbacetin (− . ), isobavachalcone (− . ), quercetin -β-d-glucoside (− . ) and helichrysetin (− . ) were selected and hypothesized to be biological complexes. in order to compare binding affinities to their closest homologues ( figure s figure s . it is very obvious that the s site and hydrophobic s site of mers-cov clpro play a key role in predicted complexes. flavonoids are natural compounds with multiple pharmacological characteristics such as antioxidant, anti-inflammatory, analgesic, anti-carcinogenic, antibacterial infection, antifungal and antiviral properties (frabasile et al., ) . naringenin has therapeutic effects on various neurological, cardiovascular, gastrointestinal, rheumatological, metabolic and malignant disorders (rani et al., ) . it also represents antiviral function (zakaryan et al., ) . fisetin is a commercially available nutraceutical with anti-inflammatory, antioxidant, anti-tumorigenic, anti-invasive, anti-angiogenic, anti-diabetic, neuroprotective and cardioprotective effects (pal, pearlman, & afaq, ) . interestingly, fisetin has an anti-noroviral activity by inducing cytokines (seo & choi, ) . although some of flavonoids show an antiviral effect, a molecular mechanism of their antiviral activity was rarely known. in this study, we assayed the inhibitory activity of various flavonoids against mers-cov clpro. for the trial, a flavonoid library composed of nine different scaffolds plus one undefined group was constructed. they are classified based on a c -c -c skeleton and differ in the overall hydroxylation patterns and in the saturation of the heteroatomic ring c together with the position of the attached aromatic ring b (at the positions c- or c- of ring c) (jucá et al., ) . the flavonoid library was tested, and thus, a systematic analysis was possible. among the ten groups, isoflavones, isoflavane, flavanols and flavanones did not show any inhibitory activity over mers-cov clpro. the other groups contains some flavonoids displaying moderate inhibitory activity. however, four flavonoids, herbacetin, isobavachalcone, quercetin -β-d-glucoside and helichrysetin exhibited prominent inhibitory activity. the immediate inference of the primary scaffold required for binding with mers-cov clpro was as follows: first, the orientation of the aromatic ring b at the position c- of ring c is essential as shown in isoflavones and isoflavane. second, the saturated heteroatomic ring c is not preferred as shown in flavanols and flavanones. third, chalcone and flavonol scaffolds show a promising binding property. the more detailed structural comparison also provided valuable structural information required for the binding affinity of each flavonoid. helichrysetin is a chalcone derivative (van puyvelde et al., ) . therefore, we compared its inhibitory activity with a chalcone, cardamonin ( ′, ′-dihydroxy- ′-methoxychalcone) which is the structurally identical homologue except one hydroxyl group at the -position of the benzyl moiety of chalcone. the inhibitory activity of cardamonin at the concentration of μm was lower than that of helichrysetin ( figure ). it implies that the -hydroxyl group of helichrysetin is functionally important to bind with mers-cov clpro. the structural comparison of cardamonin with isobavachalcone also indicated the modification effect of acetophenone ring of chalcone: the hydrophobic modification at the ′-position of the acetophenone ring moiety of isobavachalcone improves its binding affinity to mers-cov clpro. intriguingly, the docking study shows that the -hydroxyl group of helichrysetin forms a hydrogen bond with the hydroxyl group of tyr of mers-cov clpro. since tyr is located deep inside of the hydrophobic s site, helichrysetin is inserted into deeper than cardamonin ( figure s a ). as a result, helichrysetin seems to have a better affinity and thus represents a better inhibitory activity. in the flavonoid library, there are quite similar homologues of herbacetin, isobavachalcone and quercetin -β-d-glucoside. they are kaempferol ( , ′, , -tetrahydroxyflavone), , ′, ′-trihydroxychalcone and quercitrin ( , ′, ′, , -pentahydroxyflavone- -l-rhamnoside), respectively ( figure s ). their mers-cov clpro inhibitory activity is lower than their corresponding compounds at μm. herbacetin is a kaempferol derivative where one more hydroxide is attached at -position of kaempferol. the docking study indicates kaempferol derivatives occupy the s and s sites of mers-cov clpro ( figure s b ). the hydroxyl group at -position looks important to bind at the s binding site. a f i g u r e comparison of inhibitory activity between homologue flavonoids. the two homologue flavonoids are compared side by side. each of two bars represents the effect of two homologue inhibitory compounds at μm against mers-cov clpro compared to the control. each bar is expressed as the mean ± standard error of the mean (n = ). rfu = relative fluorescence units bit better inhibitory activity of herbacetin indicates the hydroxide at -position is favourable for its binding affinity to mers-cov clpro. in the predicted complex, the hydroxyl group at -position of herbacetin induces a hydrogen bond with his at the s site. the improved activity of isobavachalcone compared with , ′, ′-trihydroxychalcone again points out the importance of its hydrophobic substituent. coincided with the experimental result, the prenyl moiety of isobavachalcone makes hydrophobic interactions with met and leu at the hydrophobic s site ( figure s c) . quercetin -β-d-glucoside is a homologue of quercitrin where rhamnoside is substituted for glucoside. the better inhibitory activity of quercetin -β-d-glucoside means that hydroxymethyl is preferred to hydroxide in this position of the glucoside to interact with mers-cov clpro. the docking study represents that the hydroxymethyl group of the glucoside moiety makes a hydrogen bond with glu and thus contributes its tighter binding to the s site than the rhamnoside moiety of quercitrin ( figure s d ). helichrysetin and isobavachalcone inhibiting the activity of mers-cov clpro are chalcone derivatives. chalcone has immunomodulation, antibacterial, antifungal, antiviral, anti-inflammatory, antioxidant, anticancer and anti-diabetic activities (yadav, prasad, sung, & aggarwal, ) . recently, the non-competitive inhibition of cardamonin against the dengue virus protease has been reported (kiat et al., ) . as discussed above, cardamonin is a strong homologue of helichrysetin. the analysis of the crystal structures of three viral proteases, mers-cov clpro, dengue virus ns b/ns protease and norovirus clpro were performed, and their active sites were compared ( figure s ) (erbel et al., ; nakamura et al., ) . despite their sequential divergence, their overall architecture resembles each other. furthermore, the spatial positions of their catalytic residues were also highly conserved. the observation implies that chalcone and flavonol can be used as templates to develop a potential antiviral agent by targeting these viral proteases. in this study, we showed that the antiviral effects of some flavonoids may be directly from their inhibition of main viral proteases and thus nullify a process of virus peptides. therefore, one of antiviral mechanisms of flavonoids may be originated from their direct binding capability to viral proteases. consequently, flavonoid derivatives can be used as a template to design not only for broad-spectrum but also for virus-specific antiviral agents. a further study is going on to develop better inhibitory lead compounds derived from this study. we built a flavonoid library to systematically search mers-cov clpro inhibitory compounds with a fret method. we found four flavonoids effectively reducing the proteolytic activity of mers-cov clpro. the binding of the flavonoids was independently proven by a tryptophanbased fluorescence method. the assay in the presence of triton x- also eliminated the chance of the false-positive result from the aggregation of flavonoids. the analysis of the four compounds with their homologs using an induced-fit docking study provided an insight of flavonoid scaffolds required to bind with mers-cov clpro. the prominent activity of helichrysetin and isobavachalcone indicates that the flexibility of the chalcone scaffold is good to bind with mers-cov clpro. in contrast, the comparable activity of flavones with a rigid γ-pyrone ring indicates that their spatial rearrangement together with various functional groups may be an alternative strategy to interact with mers-cov clpro. therefore, this study suggests that an antiviral effect of some flavonoids is directly from their structural characteristics binding to viral proteases. profiling of substrate specificities of c-like proteases from group , a, b, and coronaviruses sars and mers: recent insights into emerging coronaviruses structural basis for the activation of flaviviral ns proteases from dengue and 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a norovirus protease structure provides insights into active and substrate binding site integrity structures of the middle east respiratory syndrome coronavirus c-like protease reveal insights into substrate specificity fisetin and its role in chronic diseases pharmacological properties and therapeutic potential of naringenin: a citrus flavonoid of pharmaceutical promise inhibitory mechanism of five natural flavonoids against murine norovirus novel procedure for modeling ligand/receptor induced fit effects isolation of flavonoids and a chalcone from helichrysum odoratissimum and synthesis of helichrysetin prediction and biochemical analysis of putative cleavage sites of the c-like protease of middle east respiratory syndrome coronavirus the role of chalcones in suppression of nf-κb-mediated inflammation and cancer activity of compounds from taxillus sutchuenensis as inhibitors of hcv ns serine protease flavonoids: promising natural compounds against viral infections the authors declare no conflict of interest. the data that support the findings of this study are available from the corresponding author upon reasonable request. https://orcid.org/ - - - mi-sun kim https://orcid.org/ - - - key: cord- -ipa wz authors: poland, g. a.; ovsyannikova, i. g.; kennedy, r. b. title: personalized vaccinology: a review date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: ipa wz abstract at the current time, the field of vaccinology remains empirical in many respects. vaccine development, vaccine immunogenicity, and vaccine efficacy have, for the most part, historically been driven by an empiric “isolate-inactivate-inject” paradigm. in turn, a population-level public health paradigm of “the same dose for everyone for every disease” model has been the normative thinking in regard to prevention of vaccine-preventable infectious diseases. in addition, up until recently, no vaccines had been designed specifically to overcome the immunosenescence of aging, consistent with a post-wwii mentality of developing vaccines and vaccine programs for children. it is now recognized that the current lack of knowledge concerning how immune responses to vaccines are generated is a critical barrier to understanding poor vaccine responses in the elderly and in immunoimmaturity, discovery of new correlates of vaccine immunogenicity (vaccine response biomarkers), and a directed approach to new vaccine development. the new fields of vaccinomics and adversomics provide models that permit global profiling of the innate, humoral, and cellular immune responses integrated at a systems biology level. this has advanced the science beyond that of reductionist scientific approaches by revealing novel interactions between and within the immune system and other biological systems (beyond transcriptional level), which are critical to developing “downstream” adaptive humoral and cellular responses to infectious pathogens and vaccines. others have applied systems level approaches to the study of antibody responses (a.k.a. “systems serology”), [ ] high-dimensional cell subset immunophenotyping through cytof, [ , ] and vaccine induced metabolic changes [ ]. in turn, this knowledge is being utilized to better understand the following: identifying who is at risk for which infections; the level of risk that exists regarding poor immunogenicity and/or serious adverse events; and the type or dose of vaccine needed to fully protect an individual. in toto, such approaches allow for a personalized approach to the practice of vaccinology, analogous to the substantial inroads that individualized medicine is playing in other fields of human health and medicine. herein we briefly review the field of vaccinomics, adversomics, and personalized vaccinology. vaccines have been one of the most effective public health strategies in preventing infectious diseases. a decade ago, we described the idea of vaccinomics and adversomics, based on the immune response network theory [ , ] , which utilizes immunogenetics/imunogenomics and systems biology approaches to understand the basis for inter-individual variations in vaccineinduced immune responses in humans, as well as the basis for adverse side effects from vaccines [ ] . vaccinomics and adversomics explore the influence of genetic and non-genetic regulation on the heterogeneity of vaccine-induced immune responses at both the personal and population levels [ ] . in particular, vaccinomics and adversomics utilize high-throughput, high-dimensional systems biology approaches, which aim to predict variations in protective and maladaptive innate and adaptive immune responses to vaccines [ ] [ ] [ ] [ ] , ] . in this regard, the basis of personalized (and predictive) vaccinology is the assessment of an individual's genetic background, sex, as well as other factors that may impact vaccine immunogenicity, efficacy, and safety [ ] [ ] [ ] [ ] . we and others have widely published on the applicability of the tools and concepts of vaccinomics, including immunogenetics and immunogenomics, to the knowledge-based directed development of new and improved vaccine candidates [ ] [ ] [ ] [ ] . the application of these concepts is likely to allow for explanation, quantification, and prediction of vaccine-induced protective immune responses-including the http://dx.doi.org/ . /j.vaccine. . . - x/Ó elsevier ltd. all rights reserved. development of predictive immune signatures in response to vaccines. indeed, we have previously published what we believe is the first draft of a mathematical model and predictive equation describing the non-random events that lead to a pre-determined immune response [ ] : b i x iþe y = measure of immune response b o = intercept b i = coefficient for the ith variable x i and indicates the amount of change in y for a unit change in x i Ε = random deviations from the model we recognize that such an equation, given the current state of the science, is incomplete and cannot yet predict immune responses. but we present it as an early directional attempt to quantify such an equation. such an approach begins to move us into a st-century model of directed vaccine development and an advanced understanding of how, and by what mechanisms, vaccines and vaccine adjuvants trigger both useful and maladaptive innate and adaptive immune responses. we believe that vaccinomics and adversomics represent approaches counter to the standard methods of vaccine development until recently. historically, vaccine development has been empirical, despite many emerging and re-emerging complex, hyper-variable pathogens-many with elaborate immune escape mechanisms. in addition, vaccine coverage rates continue to suffer as society is risk-averse toward vaccines and demands levels of safety that may not be achievable. finally, the ''one-size-fits-all" approach to the practice of vaccinology ignores the complexity and diversity of the human immune system and host genome. thus, the promise of vaccinomics and related paradigms is to identify specific immune response profiles, immunosignatures, and biomarkers that predict vaccine safety and/or efficacy, and which may lead to new vaccine candidates. vaccinomics provides the opportunity to examine not only immune response genes likely to be involved in vaccine response, but also the possibility of identifying the influence of new (uncharacterized) genes on vaccine-induced immunity. in turn, the identification and directed study of such genetic variants allows recognition, often at the molecular level, of the effects of differential binding, processing, and expression/presentation of antigenic viral peptides used in vaccine development, identification of the differential range of presented peptides (genetic restriction), altered secretion patterns (cytokines) in response to vaccines or vaccine adjuvants, altered transcription of important genes (signaling molecules) and gene products, altered binding of virus/antigens by membrane-based receptors (tlrs, other), differential receptor function, expression, and affinities, and the impact of epigenetics on vaccine-induced immune responses. we have utilized this knowledge in our own laboratory to create a research-oriented paradigm of ''discover-validate-characterize-apply," which may be used in new candidate vaccine development ( fig. ) [ ] . in this paradigm, we have been able to utilize vaccinomics approaches to discover genetic variants that are significantly associated with subsequent downstream immune responses, validate that such variants are indeed associated, then seek to characterize the mechanism whereby such effects occur and, finally, apply this knowledge-often in functional studies that confirm the effect on immunity. such knowledge can be exploited in developing immune strategies to enhance or circumvent genetic restrictions, for example, in triggering vaccine-associated immune responses, by ''reverse engineering" around a given genetic or other obstacle to generating protective immune responses. there are a growing number of studies reporting unbiased genome-wide assessments of genetic variation and its influence on adaptive (humoral and cellular) vaccine-induced immune responses across multiple viral and bacterial vaccines. for example, candidate and gwas immunogenetic and phamacogenetic studies have identified polymorphisms in hla, kir, mica, and btn genes associated with immune responses to pathogens causing disease in humans, such as hepatitis c [ ] , mycobacterium leprae [ , ] , human immunodeficiency virus [ ] , and measles [ ] [ ] [ ] . similar studies have identified novel genes impacting immune responses to vaccines, including hepatitis b, rubella, influenza a, smallpox, anthrax, and mumps [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . our gene association studies of measles-mumps-rubella (mmr) vaccines have demonstrated that inter-individual variations in measles vaccine virus-induced humoral and cellular responses are significantly associated with polymorphisms in immune response genes and, together with hla alleles, explain % of the inter-individual variability in humoral response [ , [ ] [ ] [ ] . these findings, which illustrated the importance of key hla alleles in the adaptive humoral immune response to measles vaccine, led to the identification of naturally processed and presented measles-derived peptides isolated from specific hla polymorphisms associated with vaccine non-and hyper-response [ , ] . these peptides containing specific components (adjuvants and biodegradable nanoparticles) are now being utilized in a reverse-engineering strategy to develop peptide-based candidate measles vaccines. likewise, homan et al. have attributed diminished protection to differential hla presentation of t and b cell epitopes between vaccine and wild type strains of mumps virus [ ] . this diminished efficacy could theoretically be overcome by incorporating defined critical immunogenic peptides into an improved vaccine. tlr genes represent an important link between the innate and the adaptive immune system [ , ] . as an example, we have demonstrated that measles vaccine-induced humoral responses are significantly associated with coding polymorphisms in the tlr (rs ) and tlr (rs ) genes [ ] . for the rubella vaccine and tlr gene, a tlr gene snp rs was associated with rubella-specific gm-csf production [ ] . our recent mumps vaccine study has identified and replicated tlr snps associated with a % decrease in antibody titer, and a tlr snp associated with a % increase in t cell response (unpublished data). these data strongly suggest that robust tlr activation by measles, mumps, and rubella viruses is crucial for optimal vaccine response. supporting these findings is a study demonstrating that an inactivated mumps vaccine containing a protollin-based tlr / adjuvant is highly immunogenic in a mouse model; it led to superior total igg levels, higher neutralizing antibody titers, greater mucosal iga production, and enhanced th /th cytokine secretion [ ] . one potential application of this finding is to identify the specific and critical interactions between tlrs (and other genes) and virus, leading to advances in our knowledge of the precise mechanisms driving immunity to mmr vaccine. significant sex differences in humoral and cellular immune responses to vaccines are apparent [ , ] . additionally, local and systemic adverse rates are generally higher in females versus males. protective antibody responses are significantly higher in females than males after vaccination against influenza, yellow fever, measles, mumps, rubella, hepatitis a and b, herpes simplex (hsv) , rabies, smallpox, and dengue viruses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . sex-based differences in humoral immune responses are observed through various age groups [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , suggesting that sex steroid hormones are not the singular mediators of sex differences in humoral immune responses to vaccines [ , ] . this suggests that genetic, or other, factors may be an important driver of sex-related differences in humoral immune response [ ] . despite significant evidence of immune response differences between the sexes, for the most part, vaccine studies have not examined and analyzed immune response outcomes by sex [ , ] . in fact, little information is known about potential mechanisms for sex-based effects, which should be a priority for vaccine research studies. discovery of specific factors involved in sex-based differences in immune response may allow the identification of new correlates of vaccine immunogenicity. in a cohort of older (ages - ) and younger (ages - ) previously vaccinated individuals, the seasonal trivalent influenza vaccine induced > . -fold higher a/h n -specific hai antibody titers in women than men across both age groups [ ] . similarly, a study of standard seasonal influenza vaccine and high-dose influenza vaccine responses in a sex-balanced cohort of elderly subjects (ages - ) demonstrated significantly higher rates of seroconversion in females than in males [ ] ; however, no significant differences in antibody measures were found between males and females after seasonal influenza vaccination in another cohort of older adults (ages - ) [ ] . a study by furman et al. examining gene expression, serum cytokines/ chemokines, cell subsets, and phosphorylation events found several serum markers (lept, il- ra, crp, gm-csf, and il- ) to be more highly expressed in females than males after influenza vaccine [ ] . this same report used a systems biology approach to identify a gene cluster involved in lipid biosynthesis that is regulated by testosterone and significantly correlated with poor humoral responses following influenza vaccination in men [ ] . these data suggest that this gene cluster (e.g., genes involved in lipid metabolism) could be an important driver of sex-related differences in humoral immune response. this collective knowledge could substantially assist future personalized vaccine development efforts through the generation of new knowledge and the identification of targets and biomarkers that predict vaccine responses in specific populations (e.g., females vs. males; young vs. old; obese vs. lean). further research is needed to clarify the effects of sex on immune response. identification of molecular immune signatures of sex differences in innate and adaptive immune responses to vaccines may provide evidence necessary for additional efforts in designing personalized vaccination and vaccinomics approaches (i.e., in which males and females might be vaccinated differently using different doses or different vaccines) to provide equal protection while reducing side effects [ , , ] . a significant global public health issue is the aging of the population. as individuals age, immunosenescence develops, leading to poorer immune responses to vaccines. immunosenescence is an age-related dysregulation of the immune system due to ageassociated changes in innate and adaptive immune system components, which leads to impaired immunity and protection following immunization or infection [ ] [ ] [ ] . published data reveal that innate and adaptive immunity is decreased with age, but the systems-level mechanisms for these findings are unclear [ , ] , particularly in regard to influenza and other viral vaccine responses where the morbidity, mortality, and associated healthcare costs are greater in older individuals [ ] . major signs of innate immune dysfunction commonly observed in the elderly include, but are not limited to, altered cytokine secretion; decreased nk cell activity; reduced tlr expression; and a chronic inflammatory state (elevated levels of il- b, mcp- , tnf-a, and serum il- ) known as ''inflamm-aging" [ , [ ] [ ] [ ] . age-related humoral immune dysfunction, for example, might be overcome through optimal stimulation of innate and/or th cell-specific genes, which may be different in males and females. for example, adjuvanted zoster subunit vaccine (hz/su) reduced the risks of herpes zoster, and postherpetic neuralgia in immunocompetent persons years of age and older [ ] . this hz/su vaccine contains varicella zoster virus glycoprotein e and a novel as b adjuvant system aimed to improve and preserve with age zoster-specific cd + t cell responses [ ] . a tlr agonist gla-se (glucopyranosyl lipid adjuvant formulated in a stable emulsion) has been shown to enhance th responses to influenza vaccine in older adults [ ] , suggesting a potential mechanism for targeting innate receptor agonists (e.g., tlrs) that enhance innate immune responses against influenza. given the substantially diminished efficacy of influenza and other vaccines with age and the importance of developing improved vaccines [ ] , data from vaccinomics studies could be used to inform directed and rational development of next-generation influenza vaccines-potentially circumventing immunosenescence-related factors. systems biology approaches provide a unique opportunity to identify biomarkers likely to be involved in immune responses to vaccination [ ] [ ] [ ] [ ] , , ] . fourati et al. applied a systems vaccinology approach to examine gene signatures and molecular pathways of age-related hyporesponse to hepatitis b vaccine (hbv) in naïve older adults [ ] . they observed the b cell signaling pathway (and higher memory b cell frequencies) and inflammatory pathway (and increased frequencies of activated pro-inflammatory innate cells) were strongly correlated with higher and low antibody responses to hbv, respectively. this signature, including serum cytokine profiling and flow cytometric correlates of response, predicted the antibody response to hbv with up to % accuracy [ ] . this study demonstrates that a systems biology approach can be used to predict age-related immune response to vaccination. obesity is another major public global health concern. in the us, % of adults and nearly % of children and adolescents are now overweight or obese [ ] . weight gains across all countries have been demonstrated to be associated with increasing socioeconomic status. obesity has been shown to be a predictor of impaired immunogenicity (e.g., decreased antibody response) to hepatitis b, tetanus toxoid, rabies, and influenza vaccines [ ] [ ] [ ] [ ] , and as such can be considered a marker, or state, of immunosuppression at its extremes. these data suggest that obesity is correlated with poorer vaccine-induced immune responses in humans, and further research is required to understand the immune mechanisms that are altered in obesity. as individuals age, their circulating leptin levels rise with a concomitant reduction in leptin signaling; this results in leptin resistance, which is a finding associated with obesity [ ] . leptin resistance has been shown to adversely affect the immune response in obese subjects, including responses to influenza virus [ , ] . for example, obese individuals demonstrate decreased activation of influenza-specific cd + t cells compared to healthyweight persons, including decreased production of ifn-c and granzyme b, suggesting that influenza vaccination may not be as effective in the obese population as in healthy-weight individuals [ ] . given only moderate seroprotection of influenza and other vaccines in obese older adults [ ] , and the importance of developing improved influenza vaccines [ ] , systems biology studies designed to identify the mechanisms for improved immune response are needed. in fact, data from vaccine studies could be used to inform directed and rational development of personalized vaccines that optimally stimulate innate and adaptive immune responses in males and females and overcome immune deficiencies induced by obesity [ ] . careful vaccine studies comparing lean and obese persons could provide foundational data used to improve vaccine-induced protection in the obese, a subpopulation with an elevated risk for serious vaccine-preventable illnesses and suboptimal vaccine-induced protective responses [ ] . adversomics utilizes tools-much like those used in vaccinomics-to identify, characterize, and predict adverse, or maladaptive, immune responses to vaccines [ , , ] . the promise of adversomics would be to develop or identify either predictors or immune signatures of maladaptive immune responses that lead to harm rather than benefit, and to better understand the generation and mechanisms of such maladaptive immune responses. we have asked the question, as have other scientists, ''does it make sense in the st century to give the same vaccine, dose, and at the same frequency to everyone, regardless of age, weight, gender, race, genotype, and medical condition?" for example, we give adult males and females the same dose, and the same number of doses of vaccines, ignoring the findings that females nearly always have superior humoral immune responses to males for all vaccines studied, and yet experience significantly more side effects-more adverse events, of greater duration, and of higher intensity [ , , ] . while the field is young in implementation, research has already revealed associations between specific genes or snps and adverse immune outcomes. for example, associations between cytokine gene expression and fever after smallpox vaccine have been identified [ ] . other studies have demonstrated correlations between smallpox vaccine-induced fevers and il a and il snps [ ] . other smallpox vaccine-induced adverse events such as fever, rash, and enlarged lymph nodes have been significantly associated with mthfr, irf , and il snps haplotypes [ ] . while smallpox vaccine is not used in the general population, such studies stand as examples of the usefulness of vaccinomic approaches. finally, other recent studies have identified generic fever gene networks (tnfa) after vaccine administration [ ] , and relationships between mmr vaccine administration and snps in ifi l, cd , scn a, a, and tmem (ano ) genes [ ] . despite the tremendous success of vaccines, vaccinologists face several current challenges, including difficulty in developing vaccines for hypervariable viruses (hiv, rhinovirus, hepatitis c virus, coronavirus) and complex pathogens (malaria, mycobacterium tuberculosis); newly emerging pathogens, such as zika virus (zikv); complications imposed by aging and immunosenescent populations; inadequate understanding of the neonatal and newborn immune systems; increasingly immune deficient or immunocompromised populations due to hiv, cancer, or medications; sexbased differences in vaccine response and adverse-event rates; enhanced scrutiny of vaccine safety; and as noted global increases in age and weight. in addition, vocal and active anti-vaccine groups whose messages are not easily countered by facts or scientific studies have materially and detrimentally affected vaccine coverage rates [ ] [ ] [ ] . vaccinomic approaches can be utilized to better understand these issues; this information can then be used to inform new approaches, new understandings, and new vaccine candidates. just as new technologies have created exciting new opportunities in personalized medicine, they have brought with them novel challenges in addition to those mentioned above. in order for the full potential of personalized vaccines to be achieved, we must overcome additional challenges, such as the need for the following: larger genotype:phenotype datasets (often in the many thousands to ten thousands) integrating increasingly diverse high-throughput, highdimensional data types biomarkers that can reliably distinguish which product patients receive based on the likelihood of their response or an adverse side effect vaccines with different mechanisms of action may require a move away from humoral correlates of protection for licensure; in this regard, correlates of protection based on cellular immune outcomes are likely to play an important role in future vaccines more sophisticated biostatistical and bioinformatics approaches that can identify patterns and causative networks within terabyte levels of extremely high dimensional data types from the economic side: methods of technology transfer and funding mechanisms to move novel vaccines developed through vaccinomic approaches into low and middle-income countries who often most need specific vaccines (malaria, others) we have seen the shift from ''vaccinology . ," which is the empirical ''isolate-inactivate-inject" paradigm, to ''vaccinology . "-the use of recombinant technology and novel adjuvants. however, even this paradigm is limited by our incomplete mechanistic understanding of adjuvants and innate immunity. as we adopt approaches such as those listed above, we envision a movement of the field into an era of ''vaccinology . ," during which we expect to see the use of vaccinomics and systems-level approaches to develop new vaccines; innovative vaccine-antigen packaging methods; and adjuvant development targeted at the innate response pathways best suited for a given pathogen. a common reaction to this paradigm of personalized vaccinology is questioning cost and economics. at one level, such considerations are simply ''too soon" in the development of the science to effectively answer. however, like progress being made in individualized medicine, it is likely that being able to provide the right vaccine to the right patient-for the right reasons and at the right dose-will lead to improved medical outcomes and reduced costs at the population level. personalized vaccinology is the goal of applying the concept of personalized medicine to vaccines. rapid strides in omics technologies and foundational work applying systems biology, computational immunology and reverse vaccinology have facilitated modern approaches to vaccine design and development enabling us to create vaccine formulations for new and re-emerging pathogens. egg-based influenza vaccines take > months to create. the recent licensure of cell culture-based influenza vaccines demonstrate that rapid, scalable processes can now be implemented in order to create vaccine against emerging influenza strains (e.g., h n , h n , h n , h n , h n ) within weeks [ ] and can be safely administered to individuals with egg allergies [ ] . the ebola outbreak in liberia, sierra leone, and guinea in provides an example of the need to rapidly develop vaccine candidates [ ] . dna vaccines, virus-like particle vaccines, and replicating/nonreplicating viral vector vaccines have all been created and tested. among the most promising are a replication-competent, recombinant vesicular stomatitis virus vector expressing the glycoprotein of ebola zaire (rvsv-zebov), [ ] a variety of adenovirusvectored vaccines expressing ebola glycoprotein, [ , ] a modified vaccinia virus ankara-based vaccine encoding the ebola zaire glycoprotein (mva-bn-filo), [ , ] and dna-based vaccinesone expressing glycoproteins from both zaire and sudan, and the other expressing the marburg glycoprotein [ ] . although the rvsv-based vaccine elicits high titers of neutralizing ab, it is contraindicated in children and those with compromised immune systems. viral vector vaccines present the problem of developing robust immunity to the vector as well as the target immunogen, limiting their usefulness to a single vaccination. the availability of vaccines in multiple vector backbones opens up the possibilities for prime-boost vaccination strategies for ebola, similar to those that have been applied to hiv, malaria, and tuberculosis [ ] [ ] [ ] [ ] . in this regard, a prime-boost regimen using the mva-based vaccine as the booster vaccination has shown considerable promise [ ] . another example of modern vaccine development being applied to a new pathogen can be seen with the response to zika virus. a purified, formalin-inactivated vaccine (zikv piv) has been developed by the walter reed army institute of research (wrair) [ ] and is being evaluated in several clinical trials (nct , nct , nct ), while other inactivated vaccines are in preclinical development [ ] . two variants of a plasmid dna vaccine containing the prm-env proteins have been developed by niaid and one of the formulations is currently in a phase i clinical trial (nct ) [ ] . inovio pharmaceuticals developed their own plasmid dna vaccine (also expressing prm-env), which is currently in two clinical trials (nct , nct ). rna-based vaccines [ ] and a variety of subunit and viral vector-based vaccines are also in development [ , , ] . dna and rna-based vaccines can be rapidly made at minimal costs compared to other formulations and are fairly stable, without the cold-chain requirements of live virus-based vaccines. subunit vaccines are typically safer than whole virus-based products, which represents an active area of investigation not only for pathogens with no existing vaccines, but also for improving on established vaccines. our group and others have identified pathogen-derived epitopes as preliminary steps in the development of safe, stable, and effective peptide-and protein-based vaccines for smallpox, influenza, measles, tuberculosis, staphylococcus, and myriad other viral and bacterial pathogens [ , [ ] [ ] [ ] [ ] [ ] . parallel efforts by different groups to create new vaccines result in a spectrum of potential products that can be uniquely tailored to specific population groups. live viral vaccines rapidly inducing robust immunity can be used in healthy individuals where time is of the essence (e.g., in outbreak scenarios), while inactivated or subunit vaccines can be used in vulnerable populations such as pregnant women or those with immunocompromising conditions, or in young children where the presence of maternal antibody interferes with whole virus vaccines. vaccines based on different viral vector backbones can be combined into effective primeboost regimens. vaccines with specific adjuvants may be most appropriate for the elderly in order to overcome immunosenescence, or in the very young in order to compensate for immune system immaturity. we, along with increasing numbers of other scientists, believe that personalized vaccinology will revolutionize the practice of vaccinology to the benefit of human health. as part of the development of this field of science, vaccinomics and adversomics will allow us to develop molecular immune signatures of adaptive and maladaptive immune responses to vaccines, develop early biomarkers of vaccine response in vaccine trials, identify who should get what vaccine and at what dose, and increase safety and public confidence in vaccines by reducing the likelihood of serious adverse events related to vaccines. in many ways, however, personalized vaccinology is most challenged by the difficulty in moving the field away from the post-wwii population-level paradigm of ''one dose of every vaccine for everyone," toward an individualized or personalized approach based on the unique factors relevant to a given individual. in his book, the structure of scientific revolutions [ ] , thomas kuhn recognized that ''we wrongly believe scientific progress is a process of linear accretion of knowledge, that science is predicated on the belief that the scientific community understands what the world is like, and that we suppress or resist 'fundamental novelties' because they are seen as subversive to our firmly held beliefs of what the world is like." later in his book, he suggests that ''new advances always have and always will reveal that science and medicine includes bodies of belief incompatible with beliefs we hold today, and that advancements come when we reject a time-honored scientific theory in favor of another incompatible with it." these cognitive biases have, in our opinion, been manifest in our discussions with scientific colleagues as we developed this field of science. schopenhaur, the german philosopher, suggested that new discoveries are at first ridiculed, then opposed, and finally accepted as self-evident. vaccinomics and adversomics appear to be moving from the ridiculed and opposed steps, and into the not-yet quite self-evident phase of the continuum. part of the challenge is that often the concept of personalized vaccinology suggests to the reader that a unique vaccine will be developed for each individual. while that is one tactic being used in the cancer-vaccine field, it is neither necessary nor practical for the prevention of infectious diseases. rather, the personalized vaccinology approach would suggest the development of specific vaccines based on factors that relate to overcoming the potential for poor immunogenicity and the potential for adverse events. an excellent example is influenza vaccines. a mere decade or so ago, only a trivalent injectable influenza vaccine was available. quadrivalent vaccines were unavailable. for with one exception, everyone received the same vaccine and dose, regardless of age, weight, immunosuppression state, etc. at the current time in the us, multiple influenza vaccines are available so that the right vaccine, for the right patient, can be given at the right time. for example, laiv (live attenuated influenza vaccine) can be used in younger subjects or the needle-phobic. high-dose or mf -adjuvanted vaccines can be chosen for the elderly. recombinant vaccines can be chosen for those with egg allergy, and so on. this is the approach that should be taken with all vaccines. in some cases it may mean merely adjusting the dose based on weight, gender, or age. in other cases it may mean utilizing an adjuvanted or non-adjuvanted vaccine based on immune status. other examples include the recently licensed mf adjuvanted influenza vaccine (fluad Ò ), which has demonstrably higher immunogenicity and efficacy than its nonadjuvanted counterparts, [ ] [ ] [ ] or the highly effective as adjuvanted zoster glycoprotein e vaccine, which does not contain live virus and may be more broadly suitable for administration to older individuals [ , ] . thus, the movement toward a new paradigm of vaccine practice, based on a personalized approach, is occurring in the st century based on new scientific knowledge, market demand, safety considerations, immunogenicity concerns, public health trends (age, obesity, other), and the simultaneous pull of individualized medicine in other medical arenas. the net result is likely to be higher vaccine coverage rates, increased public confidence in vaccines, improved immunogenicity and adverse event rates, and a reduction or elimination in the morbidity and mortality related to vaccine-preventable diseases. as a result, we anticipate a new era of personalized ''predictive vaccinology," whereby we abandon a ''one size and dose fits all vaccine approach" in order to design and develop new vaccines, and acquire the ability to make the following predictions for each individual: whether to give a vaccine based on likelihood of response (and perhaps need); the likelihood of a significant adverse event to a vaccine; and the number of doses likely to be needed to induce a protective response to a vaccine [ ] . current vaccine development is largely empirical. vaccines are tested by trial and error, are mass produced, and given to the entire population using the same antigen dose, route of administration, number of vaccinations, and at the same age. in contrast, the new vaccine-development paradigm begins with the ''discovery" of new knowledge by integrating unbiased, comprehensive analysis of the genome, transcriptome, proteome, metabolome, microbiome, and immunome-along with the assessment of multiple measures of immune function-in order to under-stand and evaluate perturbations of the immune system. findings are then ''validated" in replication cohorts or additional model systems. the new knowledge is then ''applied" to the creation of new vaccine formulations that can undergo additional testing to start a new round of ''discovery," or can move into clinical trials in order to develop vaccine products engineered to elicit (or avoid) specific effects on the immune system. each product is tailored to specific subgroups such that robust, protective immunity can be elicited in the old and young, lean and obese, or male and female, while avoiding inappropriate immune responses due to genetics, metabolism, race, gender, malnutrition, immunosuppression, and other host factors or underlying conditions. r ai , and contract no. hhsn c (n ai ). the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health. the authors would like to acknowledge the contributions of the centers for disease control and prevention (cdc), which provides financial support to the world health organization initiative for vaccine research (u ck ). dr. poland is the chair of a safety evaluation committee for novel investigational vaccine trials being conducted by merck research laboratories. dr. poland offers consultative advice on vaccine development to merck & co. inc., avianax, dynavax, novartis vaccines and therapeutics, emergent biosolutions, adjuvance, seqirus, and protein sciences. drs. poland and ovsyannikova hold three patents related to vaccinia and measles peptide research. dr. kennedy has received funding from merck research laboratories to study waning immunity to mumps vaccine. these activities have been reviewed by the mayo clinic conflict of 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surfaces and infection control date: - - journal: colloids surf b biointerfaces doi: . /j.colsurfb. . . sha: doc_id: cord_uid: d i ix according to world health organization, every year in the european union, million patients acquire a healthcare associated infection. even though some microorganisms represent no threat to healthy people, hospitals harbor different levels of immunocompetent individuals, namely patients receiving immunosuppressors, with previous infections, or those with extremes of age (young children and elderly), requiring the implementation of effective control measures. public spaces have also been found an important source of infectious disease outbreaks due to poor or none infection control measures applied. in both places, surfaces play a major role on microorganisms’ propagation, yet they are very often neglected, with very few guidelines about efficient cleaning measures and microbiological assessment available. to overcome surface contamination problems, new strategies are being designed to limit the microorganisms’ ability to survive over surfaces and materials. surface modification and/or functionalization to prevent contamination is a hot-topic of research and several different approaches have been developed lately. surfaces with anti-adhesive properties, with incorporated antimicrobial substances or modified with biological active metals are some of the strategies recently proposed. this review intends to summarize the problems associated with contaminated surfaces and their importance on infection spreading, and to present some of the strategies developed to prevent this public health problem, namely some already being commercialized. microorganisms' spreading, and consequent infection propagation is a serious concern worldwide, yet there are still very few guidelines or legislation for infection propagation control in public spaces. the exception is made for healthcare facilities with the existence of few guidelines and orientations for infection prevention and control, nevertheless hospital acquired infection (hai) remains a tremendous problem. one of the main causes for the high number of hai reported by the world health organization (who) is related to the ability of the microorganisms to survive for long periods in hostile environments such as dry surfaces [ , ] . rooms occupied with infected patients often have their surfaces contaminated with pathogens leading to the contamination of hands and gloves of medical staff, and consequent transfer of these microorganisms to patients or onto other surfaces. this cross-contamination mechanism has already been recognized and lead to the implementation of hygiene procedures for hands when contacting with patients. still, it is more likely for a patient to get an infection when staying in a room previously occupied by an infected patient. thus, infected patients are the primary source of surface and material contamination. visitors and/or asymptomatic carriers also contribute to the spread of microorganisms, especially in situations where there is an apparent sense of safety [ ] . as said before, there are guidelines for surface cleaning and disinfection in hospitals, however, those recommendations are not sufficient to solve such a problem and the incorrect following of the disinfection instructions can even cause greater contamination problems [ ] . this review considered english-language articles retrieved from pubmed database literature searches, bibliographies from published articles, and infection-control books and chapters, in a total of references published between and , considering the following criteria: the most recent studies performed on microbiological analysis on different surfaces reporting samplings performed on food contact surfaces, public spaces and hospital surfaces, where microorganisms occur naturally. surfaces studies, the most recent and relevant works developed on surface modification or functionalization were selected and two main types of surfaces were considered for this review -anti-adhesive surfaces and antimicrobial surfaces, along with their subtypes. finally, products already commercially available with enough reliable manufacturers' information about the product and its adequacy to the topics discussed in this review were also included. contamination spreading by contact can occur either by direct contact with an infected patient, or indirectly through a contaminated object or surface [ ] . surfaces represent an important way of transmission of diseases since they can act as a reservoir of microorganisms that may spread to whoever contacts with the surface. especially in crowded spots as public transports or public spaces and places susceptible of the presence of microorganisms like healthcare facilities, surfaces may represent a serious source of infection spreading [ ] . table describes the microorganisms detected in hospital, food handling, and other environmental surfaces. it is estimated that each year, in the united states alone, . million episodes of foodborne illness occur resulting nearly , hospitalizations and over deaths. the microorganisms most commonly associated with this phenomenon are norovirus, salmonella spp., clostridium perfringens and campylobacter [ ] . food contamination can result from several factors and occur at different points of the preparation process. from farming to the moment of consuming, food goes through several steps where contamination by microorganisms can occur. temperature and storage conditions are important factors to prevent food spoilage however, there are other factors that can cause microbiological contamination [ ] . food-contact surfaces' contamination can cause food contamination with pathogens during processing or packaging. bacteria are naturally present in plants and animals and it is easy for them to attach to a food contact surface during handling. due to adhesion mechanisms bacteria can remain on the surfaces and contaminate other foods [ ] . chopping boards, knifes and preparation tables are just some of the food preparation instruments found contaminated with bacteria [ ] . saad et al. [ ] evaluated hygiene conditions on food preparation facilities by analysing food-contact surfaces and the results were quite table examples of microorganisms isolated from different surfaces and some of their associated pathologies. adverse, suggesting fecal contamination. coliform bacteria were found in dining tables ( %), food trays ( %), cooking pots ( %) and kitchen faucets ( %). in addition, e. coli was identified in some cooking pots. in this study, surfaces with a count of total mesophilic aerobes of > cfu/cm or > cfu/cm for e. coli and coliforms were considered to fail hygiene criteria, according to the commission of the european communities guidelines [ ] for hygiene conditions in fresh meat processing facilities. although most countries have guidelines about hygiene measures and good practice on food processing facilities not always the cleaning procedures are sufficient to prevent microorganisms propagation [ ] . most of the times the cleaning cloths used in cleaning can become contaminated if soaked with disinfectants in the wrong dilution (common fault in food handling facilities), contributing to microorganisms' spread to previously uncontaminated surfaces [ ] . in the food industry, the occurrence of biofilm formation is also a common event due to inefficient cleaning and disinfection. the remaining microorganisms can survive on the surfaces, especially if food residues remain. this will promote the development and multiplication of bacteria with consequent biofilm formation that is extremely hard to eliminate, becoming even more difficult to disinfect the surface [ ] . schools and day-care centers are hot spots of infection spreading. lack of hygiene habits, common among very young children, is one of the main causes of contamination between kids, leading to the contamination of other children, staff, parents or people in the community, either by direct contact or by contaminating surfaces. [ ] . ibfelt et al. [ ] evaluated the presence of bacteria and viruses in day-care centers in denmark and different surfaces from toilets, kitchens and playrooms were analyzed. the results proved contamination by several bacteria and virus. despite the main bacteria present were non-pathogenic, several surfaces revealed the presence of coliform and nasopharyngeal bacteria. respiratory viruses were mainly present on toys but also on pillows and playroom tables. gastrointestinal viruses although less prevalent were found in some surfaces, mainly on playroom pillows and toilet surfaces. a study by jerković-mujkić et al. [ ] analyzed potential contamination in public telephones and the results showed high presence of microorganisms on the surfaces. staphylococcus epidermidis ( . %) and bacillus subtilis ( %) were the most common bacteria identified, however more species were isolated. mukherjee et al. [ ] evaluated the presence of microorganisms on different surfaces of a fitness center. the samples were collected from skin-contact surfaces as floor mats and exercise instruments, regularly shared by many people. in total, species were identified with the presence of some pathogenic or potential pathogenic bacteria like salmonella, staphylococcus or klebsiella. staphylococci were present in the majority of the surfaces being one the most prevalent species, namely s. aureus, s. epidermidis and staphyloccocus saprophyticus. kandel et al. [ ] developed a study with stupefying results. they compared microbiological contamination of toilet surfaces with buttons on hospital elevators, and the buttons showed higher colonization ( %) than the toilets ( %). the most common bacteria on both surfaces were staphylococcus and streptococcus, however other bacterial species were founded, as wells as fungi. otter and french [ ] studied the presence of microorganisms in hand-touch surface in public transport system. the samples were collected from several surfaces in trains, stations and buses of london. the presence of bacteria was confirmed, and most sites presented a median bacterial concentration of cfu cm − . even though there are no guidelines for acceptable values for bacteria's presence on surfaces of public spaces this value is higher than the recommended for foodprocessing surfaces [ ] and hospital hand-contact surfaces [ , ] . it has been recognized that hospitals' contaminated surfaces and medical equipment contribute to the contamination of patients and medical staff [ , ] . several factors contribute to this occurrence but the mains reason is associated with the high prevalence of microorganisms in healthcare facilities. pathogens with the ability to cause high rates of infection come from different sources such as patients' endogenous flora ( - %), hands of medical staff and assistants ( - %), antibiotic-driven changes in patients' flora ( - %) and other, including contamination of the environment ( %) [ ] . carling et al. [ , ] have shown that in some cases less than % of hospital surfaces can be considered clean after terminal disinfection procedures. however, for specific surfaces the rate can be even more alarming with less than % of bedpan cleaners, bathroom hand-holds, light switches, and doorknobs being cleaned [ ] . contact between hands and gloves of medical staff with contaminated surfaces leads to pathogens' spreading to other surfaces and people [ ] . particularly if they are immunocompromised patients, the risk of developing an infection is very high [ ] . stiefel et al. [ ] found out that the level of contamination of gloves on medical staff after contact with patients' skin sites was actually quite similar ( % vs %) to the level of contamination after contacting with surfaces on methicillin-resistant staphylocococcus aureus (mrsa) carriers' rooms, suggesting that environmental surfaces may pose serious risks. an identical study from guerrero et al. [ ] found similar results for c. difficile contamination, proving that the risk of hand contamination after contact with patients' skin was the same than when contacting with rooms' highly-touched surfaces ( % vs %). this issue is also related to the compliance of hospital workers on performing disinfection protocols. a recent study from stahmeyer et al. [ ] showed that only % of healthcare workers washed their hands before contact with a patient, and % washed their hands after contact with the patient. also, this study found out that . % of healthcare workers' hand hygiene procedure lasted less than s (average . s), when the recommendations is that it should last around s. another study evaluating disinfection compliance by healthcare workers showed that only % of the enquired radiologists affirmed to disinfect their workstation daily, % admitted never to have disinfected their workstation, and % said to have never received any recommendation on how to perform the disinfection [ ] . several studies have proved that surfaces in rooms of patients infected with important pathogens are frequently contaminated, and that a person admitted to a room previously occupied by an infected patient has an increased likelihood of developing colonization or infection with that pathogen [ , ] . a study performed in portugal by geadas farias et al. [ ] revealed hospital surfaces' contamination with different microorganisms, mostly pseudomonas and staphylococcus. several surfaces were contaminated but some of those with higher microbiological presence were surfaces associated with water distribution system as sinks, taps, showers and drains. viana et al. [ ] performed an analysis on hospital mattresses, and resistant bacteria were found on about % of the evaluated mattresses. a. baumannii was the main species found, being present in . % of the mattresses, but also other pathogens were collected as k. pneumoniae and p. aeruginosa. this study also identified the microorganisms found on mattresses and showed that in % of the cases the microorganisms were not related to the current patient admitted in the room but to the previous one, concluding that even after cleaning microorganisms still remain on the mattresses being a possible source of contamination to other patients. despite most studies evaluating the effectiveness of room disinfection have focused on some specific surfaces considered dirty (sink, toilets, etc.), all spots must be considered since contamination can be present on unsuspected surfaces [ ] . actually, white et al. [ ] found out in an hospital that microbiologically the sinks and floors were cleaner than hand touch sites as chairs, beds and cardiac monitors buttons. shek et al. [ ] developed a study where hospital curtains of burn and plastic surgery units were microbiologically tested. the results proved a considerable presence of bacteria on the curtains, even multi resistant bacteria as mrsa. levin et al. [ ] found that in situations of poor infection control practices it is possible for patients to become a source for unresolved contamination of portable radiographic equipment with pathogenic bacteria, namely resistant bacteria. lestari et al. [ ] performed a similar study but evaluating ecg lead wires, and among the lead wires analyzed only were non-contaminated with some type of microorganism. coagulase negative staphylococci and aerobic spore forming bacteria, two important types of pathogens, were present in % and % of the wires respectively. the ineffective cleaning associated with the ability of microorganisms to survive on surfaces under hostile conditions, for long periods of time, is a serious cause of pathogens spreading to people and to other surfaces [ ] . the presence of biofilms, the substances used in the cleaning process, and the method and frequency of cleaning, all interfere in the grade to which microorganisms resist to the cleaning and disinfection procedures. badly executed cleaning procedures may bring bigger problems than not cleaning at all. the transmission of microorganisms from a contaminated surface to a clean one can occur if the cleaning method is not correct. also, most detergents do not have antimicrobial activity they only act in the microorganisms' removal, leading to the contamination of the cleaning cloths with viable microorganisms, and its consequent spread to non-contaminated surfaces [ ] . the frequency of cleaning is also an important factor, since surfaces such as toilets, sinks, and other known as dirty surfaces tend be cleaned frequently [ ] . however, other surfaces thought safe but still highly touched are often neglected, being poorly cleaned. for example, according to the practical guidelines for infection control in health care facilities [ ] , mattresses without plastic covers must be steam cleaned if contaminated with body fluids. however, if body fluid contamination does not occur the mattresses are not so often cleaned, allowing microorganisms to accumulate with time and use, as proved by viana et al. [ ] . also geadas farias et al. [ ] have found that surfaces usually not considered dirty but highly touched as light switches or nursing trays were contaminated with several microorganisms. a very recent study by johani et al. [ ] revealed the presence of biofilms in many surfaces of intensive care units despite regular disinfection procedures. several highly touched surfaces were analyzed and in %, the presence of biofilm was proved by scanning electron microscopy. in many situations the lack of adequate training of the environmental cleaning services or of the medical staff ultimately promotes the use of inappropriate detergents or germicides promoting the survival of microorganisms on the surfaces, even after cleaning [ ] . among the most used disinfectants are substances based on hypochlorite, alcohols, aldehydes, phenols and quaternary ammonium compounds [ ] . however, not all of these substances are efficient against some specific types of microorganisms, namely spore forming pathogens like c. difficile [ ] . some disinfectants may cause resistance on the microorganisms if not used within specific parameters [ ] . there are several guidelines [ , , ] for infection control that suggest cleaning and disinfection methodologies to be applied in healthcare facilities. these guidelines not only suggest how to disinfect medical equipment but also how to clean environmental surfaces as floors, furniture or toilet seats. however, different guidelines present different opinions on the frequency of cleaning or advisable disinfectants/detergents for each situation since there is no standardization for the methods and cleaning agents to be used. in addition, there is a lack of guidelines suggesting effective standardized methods to assess environmental cleaning. visual evaluation is still the most used method to determine a surface cleanness, however even if a surface is apparently clean it can contain a high microbial load [ ] . according to the commission of the european communities [ ] guidelines for hygiene conditions in fresh meat processing facilities, food-contact surfaces with > cfu/cm for total viable count and > cfu/cm for enterobacteriaceae are considered unacceptable. there are no established limit values for microorganisms on surfaces for public spaces, and there is poor control on cleaning performance. as depicted in section . surfaces in these places are often contaminated showing values higher than those allowed on healthcare facilities ( . - cfus/cm ). more regulation should be made to assess the cleaning and disinfection efficacy and compliance on such places, namely on spaces with more susceptible people as schools, day-care centers or elderly homes. more guidelines about how to clean and how often would be a good option, as well as a higher microbiological assessment on surfaces, that is rarely or never done in public spaces. for healthcare facilities, there is some controversy about the acceptable number of microorganisms on surfaces since there are no established thresholds, only tentative suggestions from researchers. for some authors, clean hand-contact surfaces in hospitals must have less than cfus/cm , with an increased risk of infection for values above that [ ] . other researchers have proposed the maximum value of . cfus/cm as the limit level of contamination in clean hospital surfaces [ , ] . for some specific microorganisms, considered indicator organisms, the limit value must be even lower. the presence of such microorganisms (for example s. aureus, c. difficile, vancomycin-resistant enterococci or acinetobacter spp.) is considered indicative of the need for cleaning even at levels of cfus/cm [ ] . some reference limit-values either from governmental entities or from research studies are listed in table . the limit of < cfus/cm for indicator microorganisms makes sense since it has been shown that an infectious dose of cfus/cm was sufficient to cause c. difficile infection on mice [ ] . also, it was observed that very low doses of norovirus have the capacity to cause infection [ ] . these results show that microbiological contamination on surfaces poses a real risk of infection, since the environmental infectious dose can be very low for some microorganisms. there is still the need for reaching a consensus and creating general guidelines for healthcare facilities. establishing clear limit values and implementing frequent microbiological assessment protocols are some of the steps that should be in the horizon. to avoid contamination and consequent infection propagation on surfaces, several alternative strategies have been developed recently. application of aerosols or uv light on contaminated surfaces are good examples of no-touch strategies, and they seem to be a good option to be applied in hospital environment [ ] . however, these approaches still present some limitations. uv light, in hospital facilities, can only be applied in empty rooms, therefore it cannot be used for a daily cleaning routine. also, it only disinfects areas directly reached by the uv light, so many objects and surfaces remain contaminated after this disinfection approach. aerosols are efficient but take a long time to decontaminate the surfaces compared to conventional cleaning or uv light. besides that, these strategies are also exclusively used in empty rooms. this factor increases the necessary time to do the final disinfection of the room and perform the beds turnover in hospitals [ ] . cold plasma technology is another disinfection strategy that has recently been applied on surfaces. plasma is the fourth state of mater and it can be generated from gases that became ionized with lots of ions, electrons and free radicals that will provide good electric conductivity. plasma is known to have high levels of energy and it can change molecules composition making them highly ionized and with many free radicals moving randomly in all directions. for example, when a molecule of hydrogen peroxide is exposed to this technology the double bond on the molecule is broken, creating reactive oxygen species like hydroxyl radicals that in need to seek equilibrium will bind to surrounding microorganisms destroying them by oxidation [ , ] this technology has already been tested in food industry to decontaminate vegetables [ ] and meat [ ] with good results obtained and also to decontaminate surfaces as packaging materials. puligundla et al. [ ] have tested the elimination of different bacteria such as e. coli, salmonella typhimurium and s. aureus, from several materials often used on food packages such as glass, polypropylene, polyethylene, nylon and paper foil. the results were quite promising with several log reductions after to minutes of treatment. even though the results of this emerging technology are quite encouraging as a possible strategy to disinfect surfaces, more studies are needed to assure its safety and profitability to disinfect larger surfaces. surface modification/functionalization is another common procedure to obtain anti-adhesive or antimicrobial properties on different materials. this approach has several advantages over conventional disinfection techniques. for example, antimicrobial surfaces are in constant process of activity oppositely to no-touch technologies or conventional cleaning. this way the antimicrobial charge on the surfaces is reduced immediately after contact preventing its propagation and consequent contamination of surrounding surfaces or people [ , ] . other advantage is the possibility of these surfaces to be present in populated environments since they cause no harm to people with no need to remove people from the rooms to perform the disinfection. in addition, modified surfaces are not restricted to external surfaces. medical devices such as urinary catheters, central venous catheters, prosthesis or contact lenses are some of the surfaces that have been functionalized to obtain better compatibility properties and lower infection risk when inserted on the human body [ ] [ ] [ ] [ ] . self-disinfecting surfaces are an emerging topic with more and more products coming up. some of the most recent strategies developed to obtain surfaces with anti-adhesive and antimicrobial properties are discussed below. several natural surfaces have suffered diverse evolutionary processes that turned them resistant to microorganisms' colonization. natural and bio-mimicked surfaces of insects' wings, sharks' skin, and lotus' leaves exhibit antibiofouling properties by preventing different cells, particles or microorganisms from attaching to their surface [ ] . considering this, some of these natural anti-adhesive or self-cleaning surfaces have been investigated for their potential application on micro/nanostructured surfaces development. however it is also possible to give anti-adhesive properties to a surface by modifying the material's chemistry, namely using self-assembled monolayers or polymer brushes immobilized on the surface (fig. ) ( [ ] ; [ , ] ). there are several ways to produce materials with anti-fouling properties acting at the level of surface modification with chemical structures. functional groups exhibited by the material's surface interact with those in the microorganism' cells determining the kinetics of microbial adhesion. for instance, surfaces highly negatively charged, i.e. polyanionic surfaces have the ability to repulse the bacteria with polyanionic glycocalice (most of gram-positive bacteria) through electrostatic interactions. however, gram-negative bacteria have policationic glycocalices, so this surface modification mechanism is only effective against some bacterial species and sometimes only against specific strain types [ ] . zwitterionic materials, such as carboxybetaine, sulfobetaine, and phosphobetaine also have anti-fouling properties. zwitterionic polymers have an equal number of anionic and cationic groups present in their repeating unit. this fact gives these polymers ultra-hydrophilicity and consequently great hydration ability [ , ] . studies concerning surface modification with zwitterionic polymers have proved to be a promising strategy to reduce microorganisms' adhesion [ , ] . very recently, a study using a tyramine-conjugated sulfobetaine polymer, grafted on polyurethane showed a great reduction in s. aureus adhesion to the surface [ ] . coating with polymeric brushes can also prevent microorganisms' adhesion. apart from avoiding direct contact between microorganisms and the surfaces, the polymers used for brushes are usually hydrophilic, so water will be attracted into the brush forming a repellent layer in aqueous environment. proteins and microorganisms encountering the brush surface will be repelled by steric hindrance due to the water bound in the brush and the elasticity of the polymer chains [ ] . polyethylene oxide (peo) and polyethylene glycol (peg) are two examples of polymers used to produce hydrophilic brush coatings on biomaterials. in a recent study by. hadjesfandiari et al. [ ] it was proved that peo brushes covalently immobilized reduced % the adhesion of staphylococci and e. coli to a surface. apart from the chemistry, also topography of a surface can be structured to increase anti-adhesive properties. as an attempt to find efficient alternatives over more classic antimicrobials, micro/nanostructured materials present themselves as possible solutions. recently, have been done an attempt to elucidate if alterations on micro or nanotopography of a surface could influence colonization and consequent contamination. an example of structure modification is the application of superficial nanostructures (nanoparticles, nanofibers or nanotubes) reducing the area available for microorganisms to attach [ ] [ ] [ ] . as said previously, mimicking the topography of anti-adhesive surfaces innately present in nature is an innovative way to obtain new self-disinfecting surfaces. many plants have special surface properties namely wettability. for instance, lotus leaves have micropapillae structures covered by nanostructures with fine branch-like shape. these structures along with epicuticular needle-shaped wax tubes that cover them give the lotus leaf a superhydrophobic surface and consequently antifouling properties. water droplets roll down the lotus leaf due to its great hydrophobicity dragging out any possible particles present, and maintaining its surface clean [ ] . other example of nature that inspired anti-adhesive surfaces' coating is sharkskin. sharks have a special scale micropattern, which consist of a rectangular base embedded in the skin with tiny spines on the surface. the ribbed texture of these scales is responsible for the selfcleaning, anti-biofouling, hydrophobic and drag reducing properties of shark skin [ , ] . such properties have made of sharkskin one of the most mimicked surfaces, not only in research environment but in industrial area too. sharklet af (sharklet technologies, alachua, fl, usa) is a company that uses shark skin topography in different surfaces in order to avoid bacterial adhesion and biofilm formation [ , ] . a study comparing sharklet micropattern surfaces with regular surfaces in a clinical simulated scenario proved that shark skin microtopography reduced the number of attached bacteria by about fold [ ] . there are several materials with intrinsic antimicrobial properties, as silver, zinc, cooper, or chitosan. nevertheless, materials can also be modified to acquire bactericidal activity [ ] . chemical or physical changes in the surface can produce antimicrobial effect. chemical substances such as antibiotics or biocides can be incorporated in the materials to give it antimicrobial properties. however, a balance between these properties and biocompatibility must be observed since the materials will be in direct contact with users' organism raising toxicity concerns. photo-activated surfaces are also an example of self-disinfecting materials recently developed. fig. presents some examples of different types of antimicrobial surfaces. some materials have intrinsic antimicrobial properties. they do not need antimicrobial loading to exert its activity since the material by itself has the natural ability to eliminate microorganisms. among the most known natural materials with antimicrobial properties are metals as silver, copper, or zinc and polymers like chitosan [ ] . for many metals with antimicrobial properties, it is the ionic form that presents higher bactericidal activity and not the elemental metal. silver and copper are good examples of that. silver has been used in medical field for many centuries due to its antimicrobial properties. its bactericidal mechanism is based on the binding of silver atoms with thiol (sh) and disulfide (s-s) groups present in the proteins of bacterial cell membranes, leading to its disruption and eventual cell death [ ] . silver has been used for many applications on medical field especially when added in the form of silver sulfadiazine to creams or wound dressings [ ] . several wound dressing containing silver are available on market, however, not always they comply the expectations. cavanagh et al. [ ] analyzed different silver dressings on market and only two of them showed bactericidal activity against s. aureus. apart from that, silver dressing may be related with increased serum silver levels with an associated decrease on white blood cells [ ] . this leads us to conclude that the delivery system of the antimicrobial is very important. a delivery mechanism that acts by contact without leaching silver ions to the surrounding areas or tissues would be good strategy to achieve antimicrobial action without compromise the safety of the product. more recently, silver nanoparticles have been greatly studied and used to produce potential antimicrobial materials such as catheters and surgery sutures [ , ] . however, for biomaterials implanted inside the organism there are some possible risks associated with releasing of silver ions causing local toxicity and possible accumulation in organs [ ] . silver may also have different ranges of efficacy according to the class of bacteria. gram-positive bacteria's cell walls do not have an outer membrane as gram-negative bacteria do, but they have several layers of peptidoglycans, making their cell wall thicker [ ] ; this, along with their negative charge results in the trapping of silver ions (positively charged) preventing their entrance into the bacteria [ ] . agc glass (ags glass uk ltd., rugby, great britain) developed an antibacterial glass by incorporating silver ions inside the glass. the company claims that this glass eliminates % of bacteria and prevents fungi proliferation which they demonstrated in the three bacterial and two fungi strains evaluated [ ] . surfacine development company (tyngsborough, ma, usa) is another company that developed a silver coating (surfacine ® ) that can be applied in several materials, from medical devices to food preparations and packaging industry or water distribution systems. this coating showed good antimicrobial activity against several bacteria, fungi and yeast [ ] . other company, purethread technologies inc. (cary, nc, usa) has developed purethread ® , a system that embeds silver salts into textile fibers, obtaining antimicrobial textiles. according to the company this system does not weaken or disappears during washing and kills . % of microorganisms after four hours of contact with the fabric [ ]. a scientific paper about a study involving hospital curtains incorporating the antimicrobial textile developed by this company proved that those curtains take more time to be contaminated for the first time when compared to regular curtains [ ] . similarly, to silver, copper has been used for many centuries as an antimicrobial agent especially for water treatment and transportation. copper was also very used in the nautical field, to prevent adhesion and growth of organisms in hulls of ships [ ] . the bactericide mechanism of copper is related to the release of copper ions that cause damage in the bacterial envelop and consequent leakage of the cell content and influx of copper ions into the bacteria. this will generate toxic radicals causing oxidative damage to cellular components and dna degradation [ ] . more recently copper came up as an alternative for preventing hospital acquired infections through its application in hospital surfaces and medical equipment [ ] . a study by souli et al. [ ] performed on a hospital intensive care unit, compared two rooms, one with copper coated equipment (beds, side table, intravenous pole stands, etc.) and the other with regular equipment. the copper coated equipment room presented a reduced percentage of colonized surfaces ( . %) compared to the regular compartment ( . %). this study showed both reductions in colonization by gram-negative and gram-positive bacteria and in total bioburden ( . vs . cfu/ cm ). sifri et al. [ ] also developed a study where a section of an acute care unit was equipped with copper impregnated materials, hard surfaces and textiles-patients' gowns, sinks, bed rails, tray tables, sheets and blankets. the results were quite promising with copper equipped section presenting patients' infection reductions of % when caused by c. difficile and % when caused by multi drug resistant organisms, comparing to a non-modified section in the same hospital unit. two companies that collaborate with each other, cupron medical textiles (cupron inc., richmond, va, usa) and cupron enhanced eos surfaces (eos surfaces, norfolk, va, usa) provided the copper impregnated materials used for this study. the product developed is a hard-antimicrobial surface impregnated with copper that continuously kills . % of harmful bacteria in two hours, according to its specifications. according to the company this surface is effective against s. aureus, enterobacter aerogenes, mrsa, e. coli and p. aeruginosa [ ] . there are several substances with antimicrobial activity that can be added to a surface to obtain an antimicrobial surface. antibiotics were one of the first substances to be applied to surfaces such as prosthesis or textiles for wound dressing production, to obtain antimicrobial action. despite the promising results, it is well known that the major disadvantage of antibiotics use is the microorganisms' ability to develop resistance. this drawback has led to an increasing search for alternatives to be used as antimicrobials. quaternary ammonium compounds, chlorhexidine, benzalkonium chloride or nitric oxide are some examples of substances that were already tested as an alternative [ ] [ ] [ ] . quaternary ammonium compounds are disinfectants used in hospitals and healthcare facilities for several clinical purposes as disinfection of surfaces and medical material. however, more recently the quaternary ammonium compounds have been tested as antimicrobial loading agents to different surfaces and materials, namely polymers [ , ] . antimicrobial peptides (amps) are a class of peptides, components of innate immune system, with a broad activity against bacteria, virus, fungus and more, providing a non-specific defense against a broad spectrum of invaders [ ] . recently, numerous studies [ , ] refer its use as antimicrobial loading agents for their excellent characteristics, presenting several advantages over classic antibiotics, namely fast and broad spectrum of action with low susceptibility to induce bacterial resistance [ ] . in contrast with the mechanisms of action of antibiotics, which are based in slow processes of enzymatic inhibition and target specific cellular activities as dna or protein, the amps are effective against different species of microorganisms and also reduce the risk of resistance development [ ] [ ] [ ] . for all these reasons, amps are being applied in the production of antimicrobial surfaces, either by simple adsorption on the surface or covalent immobilization [ ] . nevertheless, studies suggest that covalent immobilization offers many advantages toward physical adsorption, including higher local and long-term stability, and lowering toxicity [ , ] . all those antimicrobial substances are loaded to the surface either by immobilization or by incorporation on the bulk material; recent studies on the application of each type of loading strategy are summarized next. in materials' incorporation process, the antimicrobial substances are added to the ingredients during the phase of production, to obtain a homogeneous mixture of the bulk material with the antimicrobial. this system allows antimicrobial activity throughout the bulk material, even in deeper layers and not only on the surface. this allows the material to retain its antimicrobial activity even when worn out [ , ] . in a very recent study, ferreira et al. [ ] used levofloxacin to load a bone cement, achieving good results against s. aureus not only on its planktonic form but also on biofilm. that was quite positive since this bacterial strain is greatly associated with biofilm formation on bone implants. ciprofloxacin incorporation on textile fibers by electrospinning was tested by li et al. [ ] this strategy aimed to produce a bandage to prevent wound infection and the results in vivo (rats animal model) were quite positive for e. coli and s. aureus. in han et al. [ ] study they incorporated quaternary ammonium methacrylates in dental adhesives to prevent caries by avoiding the accumulation of bacteria and biofilm formation on teeth, testing the presence of three staphyloccocal species (staphylococcus mutans, staphylococcus gordonii, and staphylococcus sanguinis) and the results showed a decreased biofilm formation for the adhesives containing the quaternary ammonium methacrylates. nanoparticles containing quaternary ammonium polyethylenimine were tested in a study about endodontic sealers by barros et al. [ ] . the results were quite positive for enterococcus faecalis, with the endodontic sealers showing good antimicrobial activity. the amp, ll- , was incorporated by cassin et al. [ ] in a membrane of collagen and hyaluronic acid for the production of antiinfective films to cover injured tissues. antimicrobial coatings can be obtained either by adsorption of the antimicrobial substance, by covalently binding it to the material or by immobilizing it using self-assembled monolayers. this strategy can be an advantage for some substances that are too toxic to be used as bulk material but that immobilized in small amounts as surface coatings can exert their antimicrobial activity without the toxicity drawback [ , ] . the coatings can also be divided according to their mechanism of action. they can deliver the antimicrobial substance by leaching, releasing it to the surrounding area or direct contact with the surface where the coating is immobilized. some coatings release substances with antibacterial properties that will interact with microorganisms acting away from the bulk. this leaching mechanism has the advantage of a larger perimeter of action around the surface where it is immobilized. it is important, though, that this release into the surrounding area is controlled to avoid toxic effects by excess of substance [ ] . coatings can also attach certain antimicrobial molecules to the materials' surface. several coatings can be classified as "contact biocides" since they use non-leachable substances that kill by contact with the bacteria and with no need to be released from the surface. this strategy is very interesting for both the self-sterilizing effect and the long-lasting activity, acting just through a direct contact with bacteria without consuming itself releasing from the surface without need [ , ] . alt et al. [ ] realized a study where coating of rifampicin and fosfomycin was applied to prosthesis showing good efficacy against s. aureus, even the methicillin resistant strain, on a rabbit animal model. neut et al. [ ] performed a similar study using a gentamicin-releasing coating that was able to prevent staphylococci growth on a prosthetic hip surface for at least hours, showing promising results. in a work by iyamba et al. [ ] , quaternary ammonium compounds (hexadecyltrimethyl ammonium bromide and hexadecylbetainate chloride) were immobilized on medical catheters reducing s. aureus adhesion to its surface. zanini et al. [ ] also developed a work in which quaternary ammonium silanes were used to coat polyurethane catheters. the results were promising with this coating proving its antimicrobial activity against e. coli bacteria. in a recent study, casciaro et al. [ ] successfully covalently immobilized frog skin derived amp on contact lenses. this strategy not only reduced the adhesion of p. aeruginosa, a species highly associated with contact lenses associated keratitis, but it also proved to be nontoxic to mammalian cells and it did not compromise the lenses physical properties. amps have also been immobilized on nanoparticles as loading strategy. ma et al. [ ] immobilized the amp hhc- on titanium oxide (tio ) nanotubes by physical adsorption with the nanotubes acting as nanocarriers for peptides delivery. they obtained an antimicrobial and anti-fouling surface showing a reduction in s. aureus adhesion and great antimicrobial activity (> % activity). braun et al. [ ] immobilized the human peptide ll- on porous silica nanoparticles, achieving an antimicrobial delivery system. all the studies presented above prove the good potential of modified surfaces to be applied on infection control in different scenarios; still some aspects must be taken into account and more studies are still needed. most studies evaluating the antimicrobial/anti-adhesive activity potential of engineered surfaces do not take into account realistic physiological environments. this can affect the results, since the host's conditioning film may affect the surface in many different ways, for example, covering the surface creating a deterrent layer preventing the antimicrobial substance to leach or to contact directly with the microorganisms, reducing its antimicrobial efficacy [ ] . in addition, the composition of the surface may determine which components of the conditioning film will adhere and that on its turn will influence other cells adhesion, as bacteria. felgueiras et al. [ ] showed that different concentrations of octadecyl acrylate (c ) immobilized on polyurethane surfaces will affect the deposition of albumin and fibrinogen when the surface is in contact with human plasma, that on its turn will affect microorganisms' adhesion to the surface since albumin avoids bacteria adhesion and fibrinogen promotes it. besides, coatings with antimicrobial peptides can be degraded by some components of conditioning films such as proteases [ ] . despite the pointed weaknesses there are already on the market some successful cases of such as biocote limited (coventry, united kingdom) that developed an antimicrobial additive technology, biocote ® , that can be incorporated on textiles, polymers, ceramics and more. these additives are based on different antimicrobial substances such as silver, zinc or phenolic compounds, chosen according to their application and support material [ ] . sanitized ag (burgdorf, switzerland) is another company that developed an antimicrobial additive that can be incorporated in polymers and textiles for different areas of application such as healthcare, public transportation and food industry. this additive technology, sanitized ® , uses different active ingredients as silver, zinc pirithione, silane quat or isothiazolinone that can be added on liquid form, powder or paste according to specific production requirements of the final product [ ] . photo-activated materials have been used in different technological fields namely in antimicrobial surfaces development. the most studied material used in photo-activated surfaces production is tio . tio has high photocatalytic properties and is highly used in cosmetics and skin care products since it is not absorbed by human skin [ ] becoming a very interesting material for development of antimicrobial surfaces. in fact, its antimicrobial efficacy has already been proved against bacteria, fungi, protozoa and virus. the main advantage of this strategy is that tio is not degraded so its activity can be maintained for long periods. tio surfaces are only activated when irradiated by specific photon energy. when this irradiation occurs, reactions of photo-oxidation involving o and h o take place on the surface with consequent formation of oxygen free radicals. these free radicals will attack the cell wall and cytoplasmic membrane of the microorganism by peroxidizing its lipids, leading to leakage of cellular components and lately cell lysis [ , ] . recently, adán et al. [ ] published a study where a water microfiltration system using photocatalytic membranes produced with tio and porous steel showed good inactivation for the retained bacteria. in another study by joost et al. [ ] thin films with tio nanoparticles were illuminated with uv light and after min of exposure no bacteria (e. coli) had survived. the analysis of bacterial cell membrane showed modifications in the chemical structure of unsaturated fatty acids and decomposition of saturated fatty acids faster than normal, confirming the peroxidation of membrane lipids. in the market, there are already some products for microorganism elimination based on tio photocatalytic action. purehealth™ (orion, florence, italy) is a system developed by orion that coats walls and floors and is activated by special lamps of solar spectrum. this system promises to eliminate virus and bacteria [ ] . this review points out the great contribution of surfaces for infection spreading, not only in healthcare facilities but also in other public spaces and food processing facilities. there is an urgent need to pay more attention to surface contamination in different spaces since there is still a lack of guidelines for microbiologic assessment and established safe thresholds for health care facilities. in addition, it is important to establish infection control procedures for public spaces, namely schools since they often present high microbiological charges on surfaces, posing a real risk for children whose immunological system is still developing. both on public spaces and on healthcare facilities, it should be determined how to assess microbiologic presence on surfaces as well as how frequently this assessment should be made, and which methods should be used to obtain a realistic analysis rather than performing visual assessments. the implementation of standard acceptable limits of microorganisms on surfaces is also a key point for a better control of infection spreading. standardized definition of which microorganisms can be considered indicator microorganisms should also be achieved. the existing guidelines for cleaning and disinfection methodologies in healthcare facilities should come to a consensus about the disinfectants to be used in each setting, the frequency of cleaning and the methods to apply. the implementation of self-disinfecting surfaces is a good strategy for infection control, and this review presents some successful research already developed in this area. these surfaces show clear advantages over the regular surfaces with traditional cleaning: the state of continuous disinfection and the antimicrobial activity that permanently eliminates the microorganisms. there are still some issues to improve, like the long-term efficacy of the antimicrobial/anti-adhesive action, or the high cost of implementation of these surfaces in large areas. biomaterials, modified surfaces, or new scientific products in general, have to face some big challenges before they reach the market. problems such as costs of manufacturability, scalability on production, intellectual property issues or regulatory aspects are just some of the main obstacles those products will have to overcome in order to be commercialized [ ] . some products even with great results on laboratory will fail when brought to larger scale due to their high cost of production, making them commercially unattractive. but, looking at the numbers nosocomial bacteremia alone can cost a hospital over million euros per year [ ] , and antibiotic resistance-associated infections may cost more than million euros per year in european union. in fact the societal cost of infections due to the selected antibioticresistant bacteria were estimated at about eur . billion each year, between hospital related costs and losses in productivity due to patients' absence from work [ ] . therefore, maybe it is worthy to invest on new technologies, betting on infection prevention and control not only to avoid higher costs but also to avoid the harm and loss of human lives. self-disinfecting surfaces are a step forward to the future of infection control policies. more studies involving self-disinfecting surfaces 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challenge catheter-related infections in patients with haematological malignancies: novel preventive and therapeutic strategies the authors acknowledge the funding provided by fundação para a ciência e a tecnologia (fct -portugal) through the scholarship sfrh/ bd/ / (micaela machado querido) and the project "b-safecoat -desenvolvimento de novas tintas com propriedades autodesinfetantes" (poci- - -feder- ). key: cord- -inndwa authors: jung, kwonil; miyazaki, ayako; saif, linda j. title: immunohistochemical detection of the vomiting-inducing monoamine neurotransmitter serotonin and enterochromaffin cells in the intestines of conventional or gnotobiotic (gn) pigs infected with porcine epidemic diarrhea virus (pedv) and serum cytokine responses of gn pigs to acute pedv infection date: - - journal: research in veterinary science doi: . /j.rvsc. . . sha: doc_id: cord_uid: inndwa abstract serotonin is a critical monoamine neurotransmitter molecule stored and released from enterochromaffin (ec) cells into the gut submucosa, transmitting the vomiting signal to the brain. we studied one mechanism by which vomiting is induced in pigs infected with porcine epidemic diarrhea virus (pedv) by characterization of swine ec cells by immunohistochemistry. conventional or gnotobiotic (gn) -day-old pigs [pedv-inoculated (n = ); mock (n = )] were inoculated orally ( . – . log genomic equivalents/pig) with pedv pc a strain or mock. this is the first identification of serotonin-positive ec cells in swine by immunohistochemistry and mainly in intestinal crypts, regardless of infection status. they were morphologically triangular-shaped or round cells with or without apical cytoplasmic extensions, respectively. at post-inoculation hour (pih) or , when vomiting was first or frequently observed, respectively, pedv infection resulted in significantly reduced numbers of serotonin-positive ec cells in duodenum, mid-jejunum, ileum, or colon. however, two of three pedv-inoculated gn pigs that did not yet show vomiting at pih had numbers of serotonin-positive ec cells in duodenum, ileum and colon similar to those in the negative controls. these findings suggest that serotonin release from ec cells (increased serotonin levels) into the gut submucosa might occur early pedv post-infection to stimulate the vagal afferent neurons, followed by vomiting. serotonin might be involved in the mechanisms related to vomiting in pedv-infected piglets. we also found that mid-jejunum was the primary site of acute pedv infection, and that systemic innate and pro-inflammatory cytokine responses were induced during the acute stage of pedv infection. vomiting is a defensive reaction of the body to rapidly remove ingested toxins from the gastrointestinal tract. vomiting is generally induced when either of the two medullary centers in the brain, integrative vomiting center and chemoreceptor trigger zone (crtz), is activated via a variety of their surface chemoreceptors, such as -ht (endo et al., ; sikander et al., ; spiller, ) . the integrative vomiting center and crtz are triggered by activation of vagal afferent neurons in the gastrointestinal tract and circulating toxins in blood, respectively. there are vagal or enteric afferent neurons in the gut submucosa. vagal afferent neurons are stimulated by a monoamine neurotransmitter molecule, serotonin ( -hydroxytryptamine, -ht), released from enterochromaffin (ec) cells in the gastrointestinal tract. subsequently, vomiting signals are transmitted to the integrative vomiting center and then to the central nervous system (cns). emetic efferent signals from the cns reach the gastrointestinal and abdominal muscles, leading to their expulsive actions by which vomiting is induced (endo et al., ; sikander et al., ; spiller, ) . in humans, approximately % and % of serotonin is stored in the gut and brain, respectively. in the gut, % of serotonin is deposited in the secretory granules of ec cells mainly located in intestinal crypts, and % is in neurons in the submucosa (endo et al., ; spiller, ) . in humans or rats, by immunohistochemistry (ihc) using antibodies against serotonin, ec cells were serotonin-positive and triangular-shaped with apical cytoplasmic extensions (gustafsson et al., ; spiller, ) . the ec cells likely originate from intestinal crypt stem cells. a mechanical, biological, or chemical stimulus causes ec cells to secret serotonin into the lamina propria or lumen that acts on serotonin receptors ( -ht ) at the terminals of the vagal afferent neurons (endo et al., ; gustafsson et al., ; spiller, ) . serotonin also functions to promote immune activation through the receptors expressed on macrophages, dendritic cells and t and b cells (li et al., ; shajib and khan, ) . porcine epidemic diarrhea virus (pedv) (family coronaviridae, genus alphacoronavirus) causes acute diarrhea, vomiting, decreased or loss of appetite, dehydration and high mortality in neonatal piglets . diarrhea is frequently accompanied by vomiting in pedv-infected nursing piglets during the acute stage of infection, exacerbating dehydration . however, the mechanisms by which vomiting is induced in pedv infection are poorly understood. we hypothesized that: i) serotonin is involved in the mechanisms related to vomiting; ii) serotonin release from ec cells into the intestinal lamina propria or lumen occurs early pedv post-infection to stimulate the vagal afferent neurons, followed by vomiting; and iii) expression levels of serotonin in intestinal ec cells or numbers of serotonin-positive ec cells in the small or large intestine differ between pedv-infected and uninfected pigs. the in situ distribution and characterization of ec cells in swine intestine are also unknown. in our study, therefore, we aimed to develop an ihc to detect and characterize ec cells in swine intestine and to determine whether pedv infection alters the number of serotonin-positive ec cells in the small and large intestines (primary sites of pedv infection) of infected gnotobiotic (gn) or conventional, -day-old piglets during disease progression. we also aimed to detail the pathogenesis of acute pedv infection, including intestinal distribution of pedv antigen and serum innate and pro-inflammatory cytokine profiles in infected gn pigs to understand the relationship of acute pedv infection with the frequency of serotoninpositive ec cells in the small and large intestines. the us virulent (wild-type) pedv strain pc a was obtained from intestinal contents of a diarrheic -day-old piglet in an ohio farm in june (jung et al., ) . the original sample was serially passaged times in gn pigs, and the intestinal contents were negative for other enteric viruses [transmissible gastroenteritis virus, porcine deltacoronavirus (pdcov), porcine rotavirus groups a-c, etc.] by pcr/rt-pcr and electron microscopic examination. the titer of gn pig ndpassaged pc a was . log ge/ml and was used as virus inoculum after dilution in minimal essential medium (mem) (invitrogen, carlsbad, ca, usa), as described previously . six large white × duroc crossbred gn pigs were acquired by hysterectomy from a pedv-seronegative pregnant sow obtained from a pedv-free, specific-pathogen-free (spf) (confirmed by history and seronegative sows; lack of qrt-pcr-positive fecal samples) swine herd of the ohio state university. the spf herd was seronegative for antibodies to porcine reproductive and respiratory syndrome virus, porcine respiratory coronavirus, transmissible gastroenteritis virus and porcine circovirus type . six -day-old gn piglets were randomly assigned to one of two groups: pedv-infected (n = ; pigs - ) and mock (n = ; pigs - ). pigs were inoculated orally with ml of pedv strain pc a [ . log genomic equivalents (ge)/ml] [ . log ge (≈ . log pfu) per pig], a dose similar to that ( . log ge/pig) used in the previous study , or mock inoculated with mem. after pedv inoculation, the pigs were monitored frequently for clinical signs, such as diarrhea, appetite, activity, etc., especially vomiting. inoculated and negative control pigs (n = /group at each time-point) were euthanized for virological and pathological examination at an acute-stage of infection when or shortly after vomiting was first detected, i.e. approximately post-inoculation hour (pih) in this study. diarrhea was assessed by scoring fecal consistency. fecal consistency was scored as follows: = solid; = pasty; = semi-liquid; = liquid, with scores of or more considered diarrheic. the institutional animal care and use committee (iacuc) of the ohio state university approved all protocols related to the animal experiments in this study. the other tissue samples tested were archival formalin-fixed, paraffin-embedded tissues acquired from twenty -day-old [pedv-infected (n = ) and mock (n = )] conventional pigs inoculated orally with . log ge of pedv strain pc a or mock (mem) . the clinical disease, fecal virus shedding, and gross and histopathology were described in a previous paper . however, the previous report included only limited information to understand intestinal distribution of serotonin-positive ec cells at the different locations of the intestine and the relationship of the frequency of serotonin-positive cells with vomiting. therefore, more detailed clinical and histopathological observations relevant to the aims of the current study were described in the results section. pigs (n = - /time-point) were euthanized for pathologic examination at post-inoculation days (pids) , , and . rectal swabs and serum samples were collected from gn pigs - at pih . two rectal swabs were suspended in ml mem. the rna was extracted from μl of centrifuged ( ×g for min at °c) fecal suspensions using the mag-max viral rna isolation kit (applied biosystems, foster city, ca, usa) according to the manufacturer's instructions. pedv rna titers in rectal swabs and serum samples were determined as described previously (jung et al., ) . there were increases in serum innate (ifnα) and pro-inflammatory (tnfα and il- ) cytokine levels in pedv-infected conventional pigs ( -day-old) at pid and increased mrna levels of pro-inflammatory cytokines (il- β, il- , il- and tnfα) in ipec-j cells infected with pedv (lin et al., ) . therefore, serum innate (ifnα and il- ) cytokines, which are known to play a role in antiviral immune responses (gimeno brias et al., ; , and pro-inflammatory (tnfα, il- , and il- ) cytokines were evaluated in gn pigs - at pih to confirm the previous findings as well as to investigate whether acute pedv infection induces systemic innate and pro-inflammatory immune responses. serum ifnα, il- , il- , il- , and tnfα cytokine levels were quantitated by elisa in the serum samples collected from gn pigs - at pih , as described previously azevedo et al., ; chattha et al., ) . briefly, nunc maxisorp -well plates were coated with anti-porcine il- ( . μg/ml, goat polyclonal antibody), anti-porcine il- ( . μg/ml, goat polyclonal antibody), antiporcine ifnα ( . μg/ml, clone k ) (r&d systems, minneapolis, mn), anti-porcine il- ( . μg/ml, rabbit polyclonal antibody), and antiporcine tnfα ( . μg/ml, goat polyclonal antibody) (kingfisher biotech, saint paul, mn) overnight at °c or °c (for ifn-α only). biotinylated anti-porcine il- ( . μg/ml, goat polyclonal antibody), antiporcine il- ( . μg/ml, goat polyclonal antibody), anti-porcine ifnα ( . μg/ml, clone f ) (r&d systems, minneapolis, mn), anti-porcine il- ( . μg/ml, rabbit polyclonal antibody), and anti-porcine tnfα ( . μg/ml, goat polyclonal antibody) (kingfisher biotech, saint paul, mn) were used as detection antibodies. porcine ifn-α detection antibody was biotinylated using a commercial kit, as described previously (chattha et al., ) . plates were developed and cytokine concentrations were calculated, as previously described azevedo et al., ) . the samples were tested in duplicate, and cytokine levels were expressed as the mean values. detection limits of our elisa were pg/ml for ifnα and il- , pg/ml for tnfα and il- , and pg/ml for il- , respectively. small intestinal tissues [duodenum, proximal (distant by - cm from the pylorus), middle (mid-location of the small intestine (duodenum to ileum) dissociated from the mesentery) and distal jejunum (distant by - cm from the ileocecal junction), and ileum] and large (cecum/colon) intestinal tissues were examined grossly and histologically. ihc results from only the mid-jejunum and cecum/colon of the conventional pigs were reported in the previous paper . therefore, duodenal, mid-jejunal, ileal, and colonic tissues of conventional or gn pigs were also conducted or re-tested by ihc, as described previously (jung et al., ; jung et al., ) , for the detection of pedv antigen (using monoclonal antibody c - specific to pedv provided by dr. daesub song, korean research institute of bioscience and biotechnology, korea) (jung et al., ) . for gn pigs - euthanized at pih , proximal and distal jejunum were additionally tested by ihc for the detection of pedv antigen to clarify the tissue tropism of the virus during the early stage of infection. duodenal, midjejunal, ileal, and colonic tissues of conventional or gn pigs were conducted by ihc, as described previously (jung et al., ; jung et al., ) , for the detection of serotonin [using monoclonal antibody ht-h against human serotonin (novus biologicals, littleton, co, usa)]. monoclonal antibodies c - and ht-h were diluted in and in , respectively, in phosphate-buffered saline. mean ratios of jejunal or ileal villous height to crypt depth (vh:cd) were calculated in gn pigs, as described previously . only well-orientated, hematoxylin and eosin-stained jejunal or ileal sections were measured and care was taken to ensure that only transverse sections cut perpendicularly from the villus tip to the muscularis mucosa were included. villous height and crypt depth were estimated by measuring - villi and crypts throughout the section from each gn pig. pedv antigen-positive scores were computed by estimating the number of ihc-positive cells in the intestinal section, as described previously (jung et al., ) , as follows: +/− (few but clearly positive), < % of villous epithelial cells showed staining; + (low), %- % of villous epithelial cells showed staining; ++ (moderate), %- % of villous epithelial cells showed staining; +++ (high), %- % villous epithelial cells showed staining; and -(negative), no cells showed staining. mean numbers of serotonin-positive ec cells were evaluated per microscopic area, at × magnification by a leitz light microscope, where approximately - intestinal crypts and - entire or lower half of villi were included. the variable numbers were dependent on the location of intestinal tissues. in pedv-inoculated pigs at pids - , unlike mock-inoculated pigs, entire jejunal and ileal villi were infrequently observed even at × magnification, because the villi were severely atrophied. only single crypt layer-included areas in well-orientated intestinal tissue sections were evaluated to count the number of serotonin-positive ec cells. mean numbers of serotonin-positive ec cells were estimated by measuring at least different tissue areas for each pid and from pedv-inoculated or mock-inoculated pigs. all values were expressed as the means ± standard deviation of the means (sdm). mean numbers of serotonin-positive ec cells per microscopic area in duodenum, mid-jejunum, ileum, and colon between pedv-and mock-inoculated pigs at the same time-points were analyzed and compared by a student's t-test using graphpad prism software (graphpad prism inc.). a value of p < . was considered statistically significant. . . clinical observations and histologic lesions in conventional -day-old nursing pigs inoculated orally with pedv watery diarrhea ( / pigs) and vomiting ( / pigs) were first detected at pid in pedv-inoculated piglets. at pids - , however, all the inoculated piglets exhibited watery diarrhea, lethargy, and dehydration, but not all showed vomiting, whereas none of the mock-inoculated pigs showed clinical signs. in pedv-inoculated piglets, histologic lesions were limited to the jejunum and ileum, and included diffuse, severe atrophic enteritis at pids - . the duodenum showed only mild villous atrophy at pids - . no histologic lesions were evident in the large intestine and other organs of the inoculated nursing pigs and negative controls. pedv antigens were mostly found in the villous epithelial cells. of pedv-inoculated pigs at pid , pigs showed moderate numbers of pedv antigen-positive cells in the mid-jejunum and ileum only, and pig showed low numbers of pedv antigen-positive cells in all intestinal segments (duodenum to colon). at pid , all pedv-inoculated pigs showed high numbers of pedv antigen-positive cells in the mid-jejunum and ileum and few to low numbers of positive cells in the colon. however, there were no pedv antigen-positive cells in the duodenum. at pid , all pedv-inoculated pigs tested showed high numbers of pedv antigen-positive cells in the mid-jejunum and ileum only. no pedv antigen-positive cells were detected in the intestines of the negative control pigs. . . identification, characterization, and distribution of serotonin-positive enterochromaffin cells in duodenum, mid-jejunum, ileum, and colon of conventional -day-old nursing pigs inoculated orally with pedv or mock in duodenum, mid-jejunum, ileum, and colon of mock-inoculated nursing pigs at pids - ( - days of age), serotonin-positive ec cells were found mainly in the crypts (fig. a -e) and occasionally, in the lamina propria or epithelium of the lower half of the villi (fig. b) (except for colon, where ec cells were detected in the entire colonic epithelium). in intestinal crypts, the majority of serotonin-positive ec cells were triangular-shaped or round cells with or without apical cytoplasmic extensions, respectively, that project into the lumen ( fig. b and c). occasionally, serotonin-positive ec cells were closely associated with the basement membrane of crypt cells. they were spindle cells with extensions surrounding mucus in crypt cells. in the lamina propria or epithelium of villi, serotonin-positive ec cells mostly appeared to be round cells, whereas no serotonin-positive cells were detected in the submucosa. on the other hand, in the duodenum, mid-jejunum, ileum, and colon of pedv-inoculated nursing pigs at pids - ( - days of age), the morphology and intestinal distribution of serotonin-positive ec cells were similar to those found in the mock-inoculated control pigs. however, a few serotonin-positive ec cells were also detected in the entire epithelium of the atrophied jejunal or ileal villi (fig. f) . . . serotonin-positive ec cells in duodenum, mid-jejunum, ileum, and colon of conventional -day-old nursing pigs inoculated orally with pedv or mock during disease progression at pid when vomiting was observed in pedv-inoculated pigs, mean numbers of serotonin-positive ec cells per microscopic area (× ) were significantly (p < . ) reduced in the duodenum, midjejunum, ileum, and colon, compared with the corresponding negative controls (table ; fig. b , d, e, and f). at pid when vomiting had ceased, mean numbers of serotonin-positive ec cells per microscopic area (× ) were significantly (p < . ) increased in duodenum but reduced in ileum of the pedv-inoculated pigs, compared with the corresponding negative controls, but they did not differ in mid-jejunum and colon (table ) . at pid , mean numbers of serotonin-positive ec cells per microscopic area (× ) in duodenum, mid-jejunum, ileum, and colon did not differ between the pedv-and mock-inoculated nursing pigs (table ) . . . clinical observations and pedv rna titers in fecal and serum samples in gnotobiotic -day-old pigs inoculated orally with pedv during the subclinical incubation stage of infection (prior to pid - when conventional -day-old pigs showed clinical signs) the significantly reduced numbers of serotonin-positive ec cells in the small and large intestines of pedv-inoculated conventional piglets at pid , compared with the corresponding negative controls, might be a result of the early release of serotonin from ec cells into the intestinal lamina propria or lumen to stimulate the vagal afferent neurons, followed by vomiting. in an attempt to explore this effect, a further study using six -day-old gn pigs inoculated orally with . log ge/pig (pigs - ) or mock (pigs - ) was conducted to investigate alterations in the number of serotonin-positive ec cells in the small and large intestines at an earlier time-point concurrent with or shortly after vomiting was first detected, as compared with pid when most of the conventional -day-old pigs tested already exhibited vomiting. after virus inoculation, vomiting and other clinical signs such as diarrhea were monitored frequently to determine an earlier time-point appropriate for euthanasia. in our experiments, at pih , pig began to exhibit vomiting (but no diarrhea), whereas pigs and did not show any clinical signs. thus, inoculated pigs - and negative control pigs - were euthanized at pih for virological and pathological examination to determine their infection status and to understand the relationship of acute pedv infection and frequency of serotonin-positive ec cells in the small and large intestines. the results are summarized in table . at pih , by qrt-pcr, no pedv-inoculated gn pigs - or negative control pigs - had detectable viral rna (< . log ge/ml) in the feces. however, pigs - had moderate viral rna titers in serum at pih , ranging from . to . log ge/ml, whereas no negative control pigs - had detectable viral rna (< . log ge/ ml) in the serum. . . trend toward increased innate (ifnα and il- ) and proinflammatory (tnfα, il- , and il- ) cytokine levels in the sera of gnotobiotic -day-old pigs inoculated orally with pedv during the subclinical incubation stage of infection as compared with uninfected gn pigs - at pih , mean serum ifnα, il- , tnfα, il- , and il- levels in the infected gn pigs - at pih were increased by -fold, -fold, -fold, -fold, and -fold, respectively (table ) . since there were large standard deviations of mean values of most of the cytokines in infected pigs as well as too few pigs (n = ) in each group, statistical analysis was not done. interestingly, the infected gn pig with vomiting at pih showed increased or higher serum ifnα, il- , tnfα, il- , and il- cytokine levels compared with the other infected gn pigs and with no vomiting, or uninfected gn pigs - . as compared with uninfected gn pigs - at pih , serum ifnα, il- , tnfα, il- , and il- cytokine levels in the infected gn pig at pih were increased by -fold, -fold, fold, -fold, and -fold, respectively (table ) . the data clearly indicate that infected gn pigs exhibited systemic innate and pro-inflammatory cytokine responses to acute pedv infection concurrent with the detection of pedv rna in serum. . . gross and histologic lesions and intestinal distribution of pedv antigen in gnotobiotic -day-old pigs inoculated orally with pedv during the subclinical incubation stage of infection (prior to pid - when conventional -day-old pigs showed clinical signs) at pih , neither pedv-inoculated gn pigs or exhibited gross lesions in the small and large intestines, whereas pedv-inoculated gn pig had accumulation of moderate amounts of watery liquid in the small intestinal lumen, but not large intestine. no gross lesions were evident in the other organs of the pedv-inoculated gn pigs - and negative controls - . histologic lesions and the distribution based on table mean numbers ( ± sdm) of serotonin-positive enterochromaffin cells by immunohistochemistry in the crypt layers and entire or lower half of villi of duodenum, mid-jejunum, ileum, and colon per microscopic area, at × magnification, of conventional -day-old nursing pigs inoculated with virulent us pedv strain pc a or mock at post-inoculation days (pids) , , and . in pedv-inoculated pigs at pids - , unlike mock-inoculated pigs, entire jejunal and ileal villi were infrequently observed even at × magnification, because their villi were severely atrophied during the period. c bold numbers, p < . (statistically significant differences between the pedv-inoculated and mock-inoculated pigs by student's t-test). intestinal locations are summarized in table . duodenum and proximal jejunum of pedv-inoculated gn pigs and showed normal mucosa, submucosa, and serosa. however, their mid-jejunum and ileum showed acute, diffuse, mild (pig ) to moderate (pig ) swelling and coagulative necrosis of villous epithelial cells lining up to % of the villous epithelium, accompanied by diffuse, mild subepithelial edema and multifocal, mild exfoliation of enterocytes located on the villous tip. also, for pig , multifocal, moderate suppurative inflammation was evident among the affected villi in the lumen. the length of the villi appeared normal. distal jejunum showed diffuse, lipid accumulation- table design and results of a pathology study of the original us pedv strain pc a in gnotobiotic -day-old pigs at post-inoculation hour (pih) when or shortly after vomiting was first detected. pig no. , and oral inoculum, log ge/ml ml, . ( . log ge/pig) ml, . ( . log ge/pig) ml, . ( . log ge/pig) ml, mock mean (sd) c ( ) ( ) ( ) ( ) ( ) a the samples were tested in duplicate, and cytokine levels were expressed as the mean values. detection limits of our elisa were pg/ml for ifnα and il- , pg/ml for tnfα and il- , and pg/ml for il- , respectively. b bold fonts and numbers, pig with vomiting at pih showed higher serum ifnα, tnfα, il- , il- , and il- cytokine levels compared with the other infected gn pigs and with no vomiting, or uninfected gn pigs - . c since there were no or little standard deviations of mean values of some cytokines in mock-inoculated pigs and large standard deviations of mean values of most of the cytokines in infected pigs as well as too few animals in each group, statistical analysis was not conducted. related, cytoplasmic vacuolation of enterocytes lining up to % of villous epithelium, with normal length of the villi. on the other hand, pedv-inoculated gn pig had more progressed lesions compared with pigs and . duodenum and proximal jejunum mostly had normal mucosa, submucosa, and serosa; however, mild, multifocal subepithelial edema was observed in villi of the proximal jejunum. midjejunum showed acute, diffuse, severe coagulative necrosis of villous epithelial cells, and the affected villi was shortened, with villous height: crypt depth (vh:cd) ratios ranging from to (vs. . to . in negative controls). distal jejunum showed diffuse, lipid accumulationrelated, cytoplasmic vacuolation of enterocytes lining up to % of the villous epithelium, with normal length of the villi. ileum showed acute, mild, diffuse swelling and coagulative necrosis of villous epithelial cells, accompanied by diffuse, moderate subepithelial edema and diffuse, mild exfoliation of enterocytes located on villous tip. however, the length of the villi appeared to be within the normal range, as the vh:cd ratios ranged from to (vs. . to . in negative controls). no histologic lesions were evident in the large intestine and other organs of the pedv-inoculated gn pigs - and negative controls - . . . intestinal distribution of pedv antigen in gnotobiotic -day-old pigs inoculated orally with pedv during the subclinical incubation stage of infection (prior to pid - when conventional -day-old pigs showed clinical signs) like pedv-inoculated conventional pigs tested earlier, pedv antigens were mostly found in the villous epithelial cells, not the crypt epithelial cells ( fig. a-f) . at pih , pedv-inoculated gn pig showed moderate to high numbers of pedv antigen-positive cells in up to % of the mid-jejunal and ileal epithelium ( fig. b and c) , low to moderate numbers of pedv antigen-positive cells in the villus-crypt interface of the proximal and distal jejunum ( fig. a) , few positive cells in the villus-crypt interface of the duodenum, and no positive cells in the colon. pedv-inoculated gn pig showed high numbers of pedv antigen-positive cells in up to % of the mid-jejunal epithelium, low numbers of pedv antigen-positive cells in the villus-crypt interface of the proximal and distal jejunum and ileum, and no positive cells in the duodenum and colon. pedv-inoculated gn pig showed high numbers of pedv antigen-positive cells in up to % of the epithelium of atrophied mid-jejunal villi (fig. e ), low to moderate numbers of pedv antigen-positive cells in the villus-crypt interface of the proximal and distal jejunum and ileum (fig. f) , low numbers of positive cells in the villus-crypt interface of the duodenum (fig. d) , and no positive cells in the colon. no pedv antigen-positive cells were detected in the intestines of the negative control pigs. . . serotonin-positive ec cells in duodenum, mid-jejunum, ileum, and colon of gnotobiotic -day-old pigs inoculated orally with pedv or mock during the subclinical incubation stage of infection (prior to pid - when conventional -day-old pigs showed clinical signs) the morphology and intestinal distribution of serotonin-positive ec cells identified in pedv or mock-inoculated gn pigs - were similar to those observed in the pedv-or mock-inoculated conventional pigs. at pih , mean numbers of serotonin-positive ec cells per microscopic area (× ) were significantly (p < . ) reduced in duodenum, midjejunum, and colon, but not ileum, of pedv-inoculated gn pigs - , compared with the corresponding negative controls (table ) . however, further analyses were done based on presence or absence of onset of vomiting in the pedv-inoculated gn pigs tested. in pedv-inoculated gn pigs and with no vomiting, mean numbers of serotonin-positive ec cells per microscopic area (× ) in duodenum, ileum, and colon, but not mid-jejunum, were comparable to those in the corresponding negative controls (table ). like pedv-inoculated conventional pigs tested at pid , however, pedv-inoculated gn pig with vomiting exhibited a trend toward reduced numbers of serotonin-positive ec cells per microscopic area (× ) in the duodenum, mid-jejunum, ileum, and colon, compared with the corresponding negative controls (table ) . our study demonstrates that the monoclonal antibody ht-h is useful for the detection of serotonin in ec cells in swine intestinal tissues. similar to serotonin-positive intestinal ec cells identified by ihc in humans or rats (gustafsson et al., ; spiller, ) , swine ec cells were also mainly localized in intestinal crypts, and they were morphologically triangular-shaped or round cells with or without apical cytoplasmic extensions, respectively. like ec cells in humans, swine intestinal ec cells were the most frequent in duodenum compared with the other intestinal segments (tables and ) (endo et al., ; spiller, ) . at pih or pid , when vomiting was first or frequently observed in pedv-inoculated gn or conventional pigs, respectively, the number of serotonin-positive ec cells was significantly reduced in the small and large intestines, compared with the negative controls. however, two of three pedv-inoculated gn pigs (pigs and ) that did not exhibit vomiting at pih concomitantly showed numbers of serotonin-positive ec cells in the small (but not mid-jejunum) and large intestines similar to those in the negative controls. these findings suggest that serotonin release from ec cells into the intestinal lamina propria or lumen (increased serotonin levels in the gut submucosa) occurred early post-infection to stimulate the vagal afferent neurons, followed by vomiting. the procedure may be essential for inducing vomiting in pedv-infected piglets. at pid , reduced numbers of serotonin-positive ec cells were observed from the duodenum to the colon of conventional pigs, although extensive villous atrophy with a large amount of pedv antigen was confined to the jejunum and ileum. in contrast, the duodenum and colon showed no or mild villous atrophy with little pedv antigen at pid . we speculate that regardless of the tissue tropism of the virus, ec cells in all intestinal segments (duodenum to colon) might be involved in the induction of vomiting in pedv-infected piglets. in our study, the detailed pathogenesis of pedv in infected gn pigs was examined to understand the relationship of acute pedv infection and frequency of serotonin-positive ec cells in the small and large intestines. also, there was little information on the pathogenesis of acute pedv infection in young piglets prior to onset of clinical signs. at pih , pedv rna was not detected in feces but was detected in serum ( . - . log ge/ml) of pedv-inoculated gn pigs. our observations are similar to a previous study, showing that only of ( %) -dayold, caesarean-derived, colostrum-deprived (cdcd) pigs inoculated orally with pedv plaque-forming units/pig were positive for pedv rna in the feces at pih (madson et al., ) . in the same study, however, of pigs ( %) were positive for pedv rna in serum at the same time-point. based on these and our observations, viremia (viral rna) may be detected earlier than fecal virus rna shedding in pedvinfected piglets. as our ihc study also revealed large amounts of pedv antigens in the small intestine of pedv-inoculated gn pigs - at pih , viremia might be a result of diffusion or spread of replicated pedv from the infected intestine to the blood, although the detailed mechanisms need to be studied further. based on our histopathologic and ihc observations in pedv-inoculated gn pigs - at pih , pedv initially and mainly infected the mild-jejunum and to a lesser extent, ileum; however, pedv infection of the duodenum, proximal jejunum, and distal jejunum was less compared with the mid-jejunum and ileum. in addition, our ihc study did not find any pedv antigen in the pylorus of the pedv-inoculated pigs - . our pilot study also included another location of proximal jejunum distant by approximately cm from the pylorus. we found that infectivity of pedv in this intestinal segment was more similar to that found in the mid-jejunum compared with the other proximal jejunum segment (distant by - cm from the pylorus) closer to the pylorus. these observations underscore that the mid-jejunum and ileum may be the primary sites of acute pedv infection , although the exact cause of the distinct tissue tropism remains obscure in terms of virus-host cell interactions at the molecular level. it is debatable whether pedv induces innate immune or pro-inflammatory responses in infected pigs, because most of the research has been conducted in cell cultures in vitro . however, our previous study showed increased serum ifnα and il- at pid in conventional -day-old nursing pigs infected with pedv . our current study also clearly showed that infected gn pigs exhibited systemic innate (ifnα and il- ) and pro-inflammatory (il- , tnfα, and il- ) cytokine responses after acute pedv infection, similar to the reported increased mrna levels of pro-inflammatory cytokines (il- β, il- , il- and tnfα) in ipec-j cells infected with pedv (lin et al., ) . in particular, -to -fold increases in the pro-inflammatory cytokines observed in infected gn pigs - coincided with the decreased or loss of appetite following acute pedv infection in young suckling piglets (langhans, ) . ec cells secrete serotonin in response to mechanical stimulation as well as recognition of pathogens via toll-like receptors (worthington, ) . several studies showed that serotonin functions to promote immune activation through the receptors expressed on macrophages, dendritic cells, and t and b cells (li et al., ; shajib and khan, ) . in our pilot study, increased serum levels of pro-inflammatory and innate cytokines coincided with reduced numbers in serotonin-positive intestinal ec cells, i.e. serotonin release from ec cells in the gut submucosa, during the early stage of infection (pih to pid ) (tables and ). this suggests a possible involvement of ec cells and serotonin, not only in triggering an emetic response but also in development of innate or pro-inflammatory immune responses to acute pedv infection. however, the potentially beneficial or detrimental effects or roles of those innate and pro-inflammatory cytokines and serotonin in infected piglets need to be studied further. as tested in pedv-inoculated gn pigs - at pih , our ihc observations revealed that early localization of pedv antigen is evident in the villous-crypt interface of the duodenum, jejunum, and ileum during the acute or incubation stage of infection ( fig. a-f) , similar to previous observations (madson et al., ) . a similar observation was also detected by ihc in -day-old, cdcd pigs inoculated with a porcine deltacoronaivrus at pih - (jung and saif, unpublished) , implying a similar tissue tropism between these two viruses during the early stages of infection. the pedv antigen-positive regions appeared first in the villus-crypt interface and subsequently expanded to the upper and then entire villous epithelium of the jejunum to ileum. our data indicate that enzymatically immature (young) enterocytes in the villous-crypt interface of the small intestine, relative to the enzymatically mature (older) enterocytes located at the villous tips, might be the major infection site of pedv. during the incubation period, similar to vibrio cholera (cholera toxin) or rotavirus (non-structural viral protein ) (spiller, ) , pedv itself or undefined secondary mediators from the infected host might act to trigger serotonin release from ec cells. because the infection site of pedv is close to the crypt layer where the majority of serotonin-positive ec cells are located, it might be beneficial for pedv itself or secondary mediators to access ec cells in the crypt layers. in our study, although pedv antigen-positive cells were confined to villous epithelium (not crypt epithelium where the majority of serotonin-positive ec cells were localized), whether pedv can also infect ec cells needs to be studied further. at pid when vomiting disappeared completely, the numbers of serotonin-positive ec cells in the small and large intestines of the conventional pigs recovered and were comparable to those in the negative controls. the rapid recovery could imply a rapid turnover of ec cells to compensate for the reduced numbers. however, the turnover time of ec cells ( - days) is much slower than for enterocytes ( - days) in rats (de bruine et al., ) . thus, the rapid recovery of the number of serotonin-positive ec cells might be a result of increased synthesis and deposition of serotonin in ec cells. collectively, serotonin might be involved in the mechanisms related to vomiting in pedv-infected nursing pigs. serotonin receptor ( -ht ) antagonists may be useful targets as a therapeutic intervention to suppress acute vomiting in pedv-infected pigs, especially in cases when the clinical signs are severe. therefore, further studies are needed to test whether -ht antagonists are effective to inhibit or suppress acute vomiting in pedv-infected pigs. to evaluate the disease mechanisms and comparative pathogenesis of pedv strains in young piglets during the acute or incubation stage of pedv infection, the mid-jejunum and ileum are the most critical and useful intestinal locations for histological analysis. our study also revealed that acute pedv infection with evidence of pedv rna in serum, induces systemic innate (ifnα and il- ) and pro-inflammatory (il- , tnfα, and il- ) cytokine responses in young gn pigs. none of the authors of this paper have a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. table mean numbers ( ± sdm) of serotonin-positive enterochromaffin cells by immunohistochemistry in the crypt layers and lower half of villi of duodenum, mid-jejunum, ileum, and colon per microscopic area, at × magnification, of gnotobiotic -day-old pigs inoculated with virulent us pedv strain pc a or mock at postinoculation hour (pih) when or shortly after vomiting was first detected. pedv-inoculated gn pigs at 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etiology, epidemiology, pathogenesis and immunoprophylaxis altered pathogenesis of porcine respiratory coronavirus in pigs due to immunosuppressive effects of dexamethasone: implications for corticosteroid use in treatment of severe acute respiratory syndrome coronavirus pathology of us porcine epidemic diarrhea virus strain pc a in gnotobiotic pigs comparative pathogenesis of us porcine epidemic diarrhea virus (pedv) strain pc a in conventional -day-old nursing piglets vs. -day-old weaned pigs anorexia of infection: current prospects serotonin activates dendritic cell function in the context of gut inflammation differential protein analysis of ipec-j cells infected with porcine epidemic diarrhea virus pandemic and classical strains elucidates the pathogenesis of infection characterization of porcine epidemic diarrhea virus isolate us/ iowa/ / infection in -day-old cesarean-derived colostrum-deprived piglets the role of serotonin and its receptors in activation of immune responses and inflammation role of serotonin in gastrointestinal motility and irritable bowel syndrome serotonin and gi clinical disorders the intestinal immunoendocrine axis: novel cross-talk between enteroendocrine cells and the immune system during infection and inflammatory disease immune evasion of porcine enteric coronaviruses and viral modulation of antiviral innate signaling suppression of type i interferon production by porcine epidemic diarrhea virus and degradation of creb-binding protein by nsp we thank dr. juliette hanson, andrew wright, megan strother, and ronna wood for assistance with animal care; and xiaohong wang, zhongyan lu, john blakenship, and thavamathi annamalai for technical assistance. salaries and research support were provided by state and federal funds appropriated to the ohio agricultural research and development center, the ohio state university. key: cord- - l cqhe authors: gawie-rotman, moran; hazan, guy; fruchtman, yariv; cavari, yuval; ling, eduard; lazar, isaac; leibovitz, eugene title: purpuric rash and fever among hospitalized children aged – years: comparison between clinical, laboratory, therapeutic and outcome features of patients with bacterial versus viral etiology date: - - journal: pediatr neonatol doi: . /j.pedneo. . . sha: doc_id: cord_uid: l cqhe background: the evaluation of children with purpuric rash and fever (prf) is controversial. although many of them have viral infections, on occasion such patients may be infected with neisseria meningitidis. we described all children aged – years with prf in southern israel during the period – and compared their microbiologic, laboratory, clinical and outcome characteristics in relation to various etiologies of this syndrome. methods: data were summarized from electronic patient and microbiology files. viral diagnoses were made by serology and/or pcr. results: sixty-nine children with prf were admitted; ( . %), ( . %) and ( . %) had a syndrome of bacterial, viral or non-established etiology, respectively. n. meningitidis infection was diagnosed in / ( . %) patients and in / ( . %) patients with bacterial etiology; / ( . %) patients suffered from a non-invasive bacterial disease ( with rickettsial disease). adenovirus and influenza b ( and cases, respectively) represented the most frequent etiologic agents among patients with viral etiology. more patients with prf of bacterial etiology were older, of bedouin ethnicity, looked ill on admission, had higher rates of meningitis and were treated more frequently with antibiotics compared with patients with non-bacterial prf. fatality rates among patients with bacterial, viral and non-established etiology were / ( . %), % and / ( . %). conclusions: although pfr was uncommon, high rates of meningococcal infections were recorded in children with prf, which was associated with high fatality rates. rickettsial infections were frequent, emphasizing the need for a high index of suspicion for this disease in endemic geographic areas. the occurrence of a purpuric rash in children raises many diagnostic dilemmas related to need of hospitalization and invasive procedures and administration of antibiotic therapy when a suspicion of sepsis and/or meningitis exists, particularly when the infective agent is neisseria meningitides. in the past, when conventional microbiological tests were used for diagnosis, the possibility of bacteremia/septicemia in children with purpuric rash and fever (prf) was estimated to be around % when the skin lesions were larger than mm in diameter. , , in general, no etiologic agents may be identified in about % of the patients with prf, and a virologic diagnosis could be established in % of the cases. acute meningococcemia may be similar in its initial presentation to viral infections or to non-meningococcal bacterial diseases, and the clinical presentation may be characterized by pharyngitis, fever, weakness, vomiting, diarrhea, and/or headache. an initial maculopapular rash may be present in about % of these cases. in patients with fulminant meningococcemia, the disease advances rapidly and within a few hours develops to septic shock, characterized by diffuse petechial rash, hypotension, disseminated intravascular coagulation, metabolic acidosis, adrenal hemorrhage, renal insufficiency, myocardial insufficiency and coma, with or without meningitis. of patients younger than years with invasive meningococcal disease diagnosed during , % were febrile, % had hypotension or impaired peripheral infusion and % had purpura fulminans. viruses, of which respiratory syncytial virus (rsv), epstein barr virus (ebv), cytomegalovirus (cmv), enterovirus, adenovirus and parvovirus b represent the most common etiologic factors, were also reported in the medical literature as associated with occurrence of purpuric rashes. furthermore, relatively newly identified viruses, like human metapneumovirus, coronavirus and bocavirus have also been associated with upper respiratory infections and may also cause purpuric rashes. e the purpose of the present study is to describe all the cases with prf occurring in children aged years diagnosed and hospitalized at the pediatric departments of the soroka university medical center, beer-sheva, israel, during the period , and to compare their microbiologic laboratory, clinical, therapeutic and outcome characteristics in relation to the various etiologies of this syndrome. we conducted a retrospective study enrolling all the children aged - years hospitalized at the pediatric departments of the soroka university medical center, beer-sheva, israel, during the period / / , with a diagnosis of purpuric rash accompanied by a fever > c. all patients were seen first at the pediatric emergency department (ped); the diagnosis was made at ped or during the hospitalization. in this retrospective study, the following diagnoses were searched from the medical archives of the hospital (icd ): database analysis was completed with spss software version . missing data were < %. descriptive analysis included analyses of distribution of single variable, central tendency and dispersion, graphical or tubular format. we checked normal distribution for all continuous variables. uni-variable analysis was completed with chi-square or ttest. for ordinal variables, we computed several dichotomous variables preliminary to the analysis. statistical significance was considered as p value < . . during the study period, , children aged years visited the pediatric emergency department, of whom , ( . %) had a body temperature > c. two hundred and twenty-three patients with henoch-schonlein purpura and with idiopathic thrombocytopenic purpura hospitalized during the study period were excluded from the study. sixty-nine ( . % of the febrile children examined at the pediatric emergency department) children with prf were subsequently admitted to the pediatric departments and represent the study population (table ) . of the children with prf, ( . %) and ( . %) suffered from a syndrome of bacterial or viral etiology, respectively. the remaining ( . %) had a syndrome where the final microbiological diagnosis could not be established. viral pcr examination (from nasal washes and stool samples) was performed in all patients diagnosed with a syndrome of probable viral etiology, and in / ( . %) and fourteen ( . %) patients suffered from a non-invasive bacterial disease ( , and cases of tonsillopharyngitis, acute bacterial gastroenteritis and rickettsial disease, respectively). one patient had group a streptococcus tonsillopharyngitis and a strong clinical suspicion of rickettsial disease (negative serology). there were cases of prf with appropriate clinical picture and positive serology (igg and igm) for rickettsia spp. adenovirus, influenza b, parainfluenza, ebv, cmv, human metapneumovirus and enterovirus ( , , , , , and cases, respectively) represented the etiologic agents identified in the patients with viral etiology. one patient had a mixed infection caused by ebv together with serologically-proven rickettsia spp. of the patients with prf without a definitive microbiological diagnosis, ( %) were discharged with a diagnosis of viral syndrome, ( %) with acute otitis media, ( . %) with possible rickettsial disease (negative serology) and ( . %) had a discharge diagnosis of "post-vaccination reaction". seven ( . %) of these patients received antibiotics in the community prior to hospitalization and had a discharge diagnosis of culture-negative septicemia without meningitis. two ( . %) of these patients were diagnosed with pneumonia and suspicion of scarlet fever, respectively. one patient was diagnosed during hospitalization with acute leukemia. table describes the clinical and laboratory characteristics of the patients with prf in relation to their bacterial or proven viral etiology. the patients with bacterial etiology were older than those with viral etiology (p z . ). no differences were recorded between the group with bacterial etiology and the group with viral etiology in patient distribution among the three age subgroups ( e , e and > years) and seasonal distribution of cases. no differences were recorded between the patients with bacterial etiology vs. those with viral etiology in terms of gender, ethnicity and diagnoses at discharge. two ( . %) of the patients with proven meningococcal disease received prior antibiotic treatment in the community. fifteen ( %) of the patients in the group with bacterial etiology and ( . %) from the group with viral etiology had a fever > c at admission. twenty ( . %) of the patients with bacterial etiology looked ill at admission, compared with only / ( . %) of those with viral etiology (p z . ). more patients with viral etiology had rhinorrhea compared with those with bacterial etiology. of a total of patients who patients, together with vancomycin in patients and together with doxycycline in patient). in the group of patients with proven rickettsial disease, doxycycline alone was administered in patients and in combination with other antibiotics in additional patients. seven ( . %) of the patients with prf of viral etiology received antibiotic treatment at admission (ceftriaxone and doxycycline in and of them, respectively). table compares the clinical and laboratory characteristics of the patients with bacterial etiology and those seven ( . %) of all patients died on admission or during hospitalization, ( . %) diagnosed with a bacterial etiology ( with meningococcemia and with acute gastroenteritis, hypovolemic shock and growth of c. jejunii from stool culture) and ( . %) with non-established etiology (both with septic shock on admission and prior antibiotic treatment in the community). the fatality rates among patients with proven bacterial etiology, proven viral etiology and non-established etiology were / ( . %), / ( %) and / ( . %). the aim of the present study was to determine the etiology of all cases of prf occurring in children aged years hospitalized in southern israel during the period , and compare their microbiologic, laboratory, clinical, therapeutic and outcome characteristics in relation to the various etiologies of this syndrome. the study was completed at the soroka university medical center, a primary and tertiary referral hospital which is the only hospital in the negev area of southern israel and provides medical services for around , children. the pediatric emergency department of the hospital receives around , visits/year. the study enrolled all the children presenting at the pediatric emergency department and hospitalized due to occurrence of a new purpuric rash accompanied by fever > c, after exclusion of idiopathic thrombocytopenic purpura and henoch-schonlein purpura. the patients with prf were grouped and analyzed according to their inclusion in one of the following etiologic entities: patients with disease of bacterial, viral or non-established (microbiologically) etiology. our present study showed the following: . prf was uncommon. . a bacterial etiology of prf was established in . % of the patients while the rest of . % and . % had a syndrome of non-established or viral etiology, respectively. . n. meningitidis infection was diagnosed in . % patients hospitalized with prf. . rickettsial disease was diagnosed in a considerable number of patients ( . %). . more patients were recorded in the group aged > year among the patients with bacterial etiology than in those with non-bacterial etiology. . higher bands counts and prolonged inr were more common in patients with bacterial etiology compared with those with viral etiology. . the fatality rate in prf of a bacterial etiology patients was high ( . %) and n. meningitidis infection was reported in % of the fatal cases with established etiology. we showed that . % of all enrolled children and . % of all children with an established bacterial etiology who presented with prf had meningococcal infection. the rate of meningococcal infection in our study was higher than previously described, both among all patients with prf enrolled ( . % %) e and also among all patients with proven bacterial etiology ( . %). , the overall mortality rate in our series was . % and reached . %% among the patients with prf associated with a bacterial infection. four of the fatalities recorded in our series were due to proven meningococcal invasive disease and two additional fatalities were recorded in patients without recovery of any pathogen and a high suspicion of meningococcal disease, based on their clinical presentation and possible masking of the etiology by previous antibiotic therapy. in a study from enrolling patients with prf, ( %) had meningococcal disease, but the most common bacterial association was with streptococcus pyogenes ( patients). viral infections were documented in patients. patients with invasive bacterial disease appeared more ill, were more likely to have neck stiffness and were more likely to have petechiae on the lower extremities than those with no bacteremic disease. a higher peripheral white blood count and absolute band form count was found in the patient with invasive bacterial disease. nguyen et al. reported in on patients hospitalized with prf in syracuse, ny, and they found that patients ( . %) had culture-proven bacterial infection ( and had n. meningitidis and haemophilus influenzae type b, respectively). as in our study, the authors could not document any single laboratory test as sensitive enough to detect all patients with life-threatening bacterial infections. mandl et al. reported in on patients with prf examined at the emergency department of the pediatric teaching hospital in boston. eight ( . %) patients had bacteremia or clinical sepsis and of them had serious invasive bacteremia (n. meningitidis recovered in patients). none of the well-appearing patients had serious invasive bacteremia. this study, by reporting invasive bacteremia less frequently than in previous series and being able to identify this condition by clinical criteria, supported the concept of the treatment of selected wellappearing children with prf as outpatients. in a study examining infants and children younger than years presenting with a non-blanching rash during the period ̶ in the uk, % had proven meningococcal infection. the authors showed that most children with meningococcal infection were ill at presentation, had a purpuric rash, were febrile and had a delayed capillary refill. none of the children with a non-blanching rash confined to the distribution of the superior vena cava had meningococcal infection. in israel, ben-shimol et al. described the dynamics of childhood invasive meningococcal disease during a -year period ( ). the authors reported on a total of cases, . % of them with meningitis and . with meningococcemia only. the mean annual incidence was cases/ , children aged years, an incidence similar to those reported from other developed countries. the mean mortality incidence was . ae . cases/ , children and this was higher in children without meningitis ( . %) compared to children with meningitis ( . %). in israel, meningococcal vaccine was not introduced in the national immunization program and it is administered only to high-risk patients. it is understandable that preventive, rather than therapeutic strategies will have a major contribution to the decrease of the serious morbidity and mortality associated with invasive meningococcal disease. in this study, we grouped all cases with proven viral etiology together with those with a non-established etiology and obtained a group of patients with non-bacterial etiology, which was further compared with the group of patients with prf of bacterial etiology. we found that more patients with prf of bacterial etiology were older, of bedouin ethnicity, looked ill on admission, had more meningitis and received more antibiotic treatments compared to the patients with prf of non-bacterial etiology. these findings may be useful, at least in part, in the management of the patients with prf at the emergency room and during their hospitalization. israel is considered endemic for types of spotted fevers, caused by rickettsia conorii and rickettsia typhi. e the diagnosis is based on nonspecific clinical findings (fever, weakness, myalgia) and a specific petechial rash; it has a benign course in the majority of cases but sometimes can be severe and may reach mortality rates of . . %. , , in the present study, we treated with doxycycline ( . %) patients with prf caused by a proven rickettsial infection and ( . %) additional patients with a clinical picture appropriate for rickettsial infection. therefore, taking into consideration the high rates of rickettsial infections reported in israel and particularly in the southern area of the country and into the bedouin population, we consider that rickettsial infection should be highly suspected, in our geographic area, in the initial investigation of patients presenting with prf and empiric treatment has to be administered to the patients with a high index of suspicion for this disease. we found a definitive viral etiology in a minority ( %) of the patients with prf, with adenovirus and influenza b identified as the most commonly isolated viruses in these patients. in a prospective study completed in germany during , schneider et al. described febrile and afebrile children aged - years examined at the emergency room with a purpuric rash < mm in diameter, symptoms of viral disease and exclusion of meningococcal disease. the authors reported that a viral pathogen was found in % of the pcr examinations performed from the nasopharynx. the most frequent pathogens associated with prf were cmv and ebv ( % each of all patients) followed by enteroviruses and rotavirus ( % each) and h n and human bocavirus ( % each); % of the patients were diagnosed with more than one viral pathogen. this study emphasizes that novel viral rapid detection methods (like multiplex q-pcr) are available and should be used in determining the possible viral etiology in a considerable number of patients with prf and thereby avoiding unnecessary treatments and hospitalizations. our study had some limitations, derived mainly from its retrospective nature, which could have caused to some missing or inexact information in the data collection from the medical and laboratory charts. we were not able to determine, after a thorough search of the patient files, valuable data on the number, location and diameter of the lesions of the enrolled patients. another limitation is related to the relatively small number of patients enrolled and of virologic investigations performed, particularly in the group of patients with non-established microbiological diagnosis, making it possible that the number of infectious etiologies among our study population was even higher. in conclusion, we report in this study that prf is an uncommon occurrence in hospitalized febrile children. we showed high rates of meningococcal infections in the etiology of prf in children, associated with higher fatality rates than in the published medical literature. rickettsial infections were also frequently diagnosed in our study population, emphasizing the need for a high index of suspicion for this disease in endemic geographic areas. compliance with ethical standards: the study was conducted in accordance with the ethical standards of the soroka university medical center. incidence of invasive bacterial disease in children with fever and petechiae fever and petechiae in children incidence of bacteremia in infants and children with fever and petechiae the child with a non-blanching rash: how likely is meningococcal disease? diagnostic assessment of haemorrhagic rash and fever neisseria meningitidis (meningococcus) clinical characteristics of children with viral single-and co-infections and a petechial rash generalized petechial eruption induced by parvovirus b infection the management of fever and petechiae: making sense of rash decisions frequency of detection of picornaviruses and seven other respiratory pathogens in infants frequent detection of viral coinfection in children hospitalized with acute respiratory tract infection using a real-time polymerase chain reaction dynamics of childhood invasive meningococcal disease in israel during a -year period murine typhus is a common cause of febrile illness in bedouin children in israel clusters of mediterranean spotted fever in israel mediterranean spotted fever retrospective study of rocky mountain spotted fever in children no potential conflict of interest was reported by the authors. the study was conducted following the approval of the helsinki committee of the soroka university medical center. key: cord- -vcrbzzhu authors: lello, joanne; mcclure, susan j.; tyrrell, kerri; viney, mark e. title: predicting the effects of parasite co-infection across species boundaries date: - - journal: proc biol sci doi: . /rspb. . sha: doc_id: cord_uid: vcrbzzhu it is normal for hosts to be co-infected by parasites. interactions among co-infecting species can have profound consequences, including changing parasite transmission dynamics, altering disease severity and confounding attempts at parasite control. despite the importance of co-infection, there is currently no way to predict how different parasite species may interact with one another, nor the consequences of those interactions. here, we demonstrate a method that enables such prediction by identifying two nematode parasite groups based on taxonomy and characteristics of the parasitological niche. from an understanding of the interactions between the two defined groups in one host system (wild rabbits), we predict how two different nematode species, from the same defined groups, will interact in co-infections in a different host system (sheep), and then we test this experimentally. we show that, as predicted, in co-infections, the blood-feeding nematode haemonchus contortus suppresses aspects of the sheep immune response, thereby facilitating the establishment and/or survival of the nematode trichostrongylus colubriformis; and that the t. colubriformis-induced immune response negatively affects h. contortus. this work is, to our knowledge, the first to use empirical data from one host system to successfully predict the specific outcome of a different co-infection in a second host species. the study therefore takes the first step in defining a practical framework for predicting interspecific parasite interactions in other animal systems. jl, - - - ; mev, - - - it is normal for hosts to be co-infected by parasites. interactions among co-infecting species can have profound consequences, including changing parasite transmission dynamics, altering disease severity and confounding attempts at parasite control. despite the importance of co-infection, there is currently no way to predict how different parasite species may interact with one another, nor the consequences of those interactions. here, we demonstrate a method that enables such prediction by identifying two nematode parasite groups based on taxonomy and characteristics of the parasitological niche. from an understanding of the interactions between the two defined groups in one host system (wild rabbits), we predict how two different nematode species, from the same defined groups, will interact in co-infections in a different host system (sheep), and then we test this experimentally. we show that, as predicted, in co-infections, the blood-feeding nematode haemonchus contortus suppresses aspects of the sheep immune response, thereby facilitating the establishment and/or survival of the nematode trichostrongylus colubriformis; and that the t. colubriformis-induced immune response negatively affects h. contortus. this work is, to our knowledge, the first to use empirical data from one host system to successfully predict the specific outcome of a different co-infection in a second host species. the study therefore takes the first step in defining a practical framework for predicting interspecific parasite interactions in other animal systems. co-infecting parasite species can interact with one another, potentially altering both within-host infection dynamics [ ] [ ] [ ] and between-host transmission (e.g. by increasing or decreasing parasite reproductive output or by altering host susceptibility) [ , [ ] [ ] [ ] [ ] . in turn, changes in infection dynamics within hosts can alter host disease severity and/or duration [ ] [ ] [ ] and may directly or indirectly confound attempts to control parasite infection [ , , ] . in most cases, whether or not particular parasite species interact, and the nature of such interactions are unknown. despite the important consequences of co-infection, the potential interactions among parasites are, therefore, rarely considered in either clinical settings or during the design of infection control programmes. one possible solution to this problem would be to discover and define rules that determine when and how parasites interact. such a concept has been explored at a broad scale for macroparasite-microparasite interactions using a meta-analysis of different infection combinations in mice [ ] . this meta-analysis demonstrated that macroparasite-microparasite co-infection would normally result in increased numbers of microparasites owing to helminth-induced impairment of the anti-microparasite immune response, but that such effects would be moderated where resource competition was also present. this was a seminal contribution to the field of co-infection biology, highlighting the potential to predict co-infection using easily obtained parasite traits. however, because of the necessarily broad categorizations in this analysis, and the focus on a single model host system, application of these findings in a clinical or public health setting is difficult. two key questions therefore follow logically from this meta-analysis: (i) can predictions also be made at a species-specific scale appropriate for use in clinical and public health settings? and (ii) can patterns of parasite interspecific interaction be robustly predicted across different host species? in earlier work, we demonstrated, using previously published data, that if parasites were grouped according to both the immune responses they stimulate and those which affect them [ ] , it was possible to predict the result of a co-infection. this approach was limited, however, by the necessity for detailed immunological data for each of the co-infecting parasites. here, we develop and extend this approach by using taxonomic and parasite niche traits (i.e. resource use, site of infection) to assign parasite species to groups, making the assumption that organisms assigned to these groups will interact with the host immune system in a similar manner to one another. subsequently, we infer what the immune interaction of each parasite group will be with its host, and hence the likely immune relationship between the groups, based on a known example of a co-infection interaction between representative species from those groups. in a previous study of the parasite community of wild rabbits (oryctolagus cuniculus), we described a range of interspecific interactions, including the interaction between two gut nematodes; the blood-feeding stomach worm graphidium strigosum and the intestinal worm trichostrongylus retortaeformis, a mucosal browser [ ] . we showed that an increasing abundance of g. strigosum was associated with increased infection intensity of t. retortaeformis but, conversely, that the presence of t. retortaeformis was associated with a reduced intensity of g. strigosum. we further proposed that these effects occurred because (i) g. strigosum downregulated anti-worm immune response in the host, and t. retortaeformis was given an advantage by this suppression, while (ii) t. retortaeformis induced an immune response which, though reduced in co-infection, acted against g. strigosum [ ] . in sheep, there are parasite species that are taxonomically and functionally equivalent to the parasite groups found in the rabbit; specifically, the nematode haemonchus contortus, which lives in the abomasum (stomach) of the sheep and feeds on host blood, and trichostrongylus colubriformis, which lives downstream in the small intestine and feeds on the host mucosa. we predict that these two parasites of sheep will interact with the same pattern, and by the same process, as the functionally equivalent parasite species in the rabbit. this is, to our knowledge, the first empirical attempt to predict the consequences of a hitherto untested interspecific interaction and to do so using data from different host and parasite species. not all parasitic nematodes are equal in the immune responses that they stimulate, or that affect them [ , ] . while the immune control of the majority of gut nematodes is associated with a t-helper cell (th ) immune response [ , ] , many nematodes are able to subvert this response to varying degrees. such immunomodulation may be particularly important for blood-feeding species. these nematodes are usually very harmful to their host, causing both tissue damage and anaemia, with heavy infections sometimes proving fatal [ ] . in addition, blood-feeding nematodes are frequently found at a high prevalence in their host populations [ , ] . therefore, it would be reasonable to expect hosts to evolve strong immune responses against bloodfeeding nematodes. yet age-prevalence and age-intensity curves for these parasites show that they cause chronic infections and/or repeatedly reinfect the host [ ] , suggesting that immune responses are functionally unsuccessful against them. furthermore, many blood-feeding nematode species have been shown to have wide-ranging immunomodulatory capacities (e.g. ancylostoma duodenale, ancylostoma caninum, necator americanus, angiostrongylus cantonensis, h. contortus [ - ] ). while these species do induce a strong th response [ , ] , many simultaneously subvert that response through a range of mechanisms [ ] . these immunomodulatory effects may have consequences for other co-infecting parasite species. in contrast to blood-feeding nematode species, trichostrongylus spp. browses on intestinal mucosa and bacteria, and shows limited invasion and penetration into host tissues [ ] . these nematodes tend to produce shorter-lived infections than those of blood-feeding species, being more rapidly and effectively controlled by the host [ , ] . while there is evidence that trichostrongylus spp. may have some immunomodulatory capacity, it does not appear to be as immunologically broad ranging as that observed among the blood-feeding species [ , ] . further evidence of the different immune responses to these parasite groups is seen in rabbits, where the temporal pattern of natural and laboratory infections suggests that t. retortaeformis is effectively removed by the host, while g. strigosum is not [ , ] . in summary, we therefore propose that how these two parasite groups interact with their hosts' immune responses will result in predictable interspecific interactions. here, we test our hypothesis in sheep experimentally co-infected with h. contortus and t. colubriformis (comparing them to sheep mono-infected with each species, and with uninfected controls), by measuring nematode intensity and the host immune response. we specifically predict that in co-infections (i) the blood-feeding h. contortus will suppress aspects of the host immune response, thereby facilitating the establishment and/or survival of t. colubriformis, and (ii) the t. colubriformis-induced immune response will negatively affect h. contortus. following approval by the fd mcmaster laboratory, chiswick, animal ethics committee, at weaning, merino wethers (castrated rams) were brought into csiro livestock industries animal house where faecal samples were analysed using a modified mcmaster technique (as in [ ] ) to diagnose any helminth infection. animals were then treated with a mixture of abamectin and praziquantel, levamisole and benzimidazole, using the manufacturers' recommended doses. twelve days later a second faecal screen for helminth infection was performed to confirm that animals were helminth-free. all animals were blood-sampled via jugular venepuncture to provide a pre-infection baseline immune and health status measure. animals were then assigned to one of four treatment groups using a stratified random assignment (where groups were balanced for body mass, body condition and original faecal egg count). the four treatment groups were: an overview of the experimental protocol is shown in figure . animals in the co-infected and mono-infected groups were each infected twice weekly for weeks with larvae of h. contortus and/or larvae of t. colubriformis. for animals in the co-infection groups, doses of both parasite species were given simultaneously as an additive dose. differential dosing was used because of the different size and pathogenicity of the two helminth species, t. colubriformis being considerably smaller and less pathogenic than h. contortus [ ] . animals in the control, uninfected group, were handled in the same manner as other animals. throughout the experiment animals were maintained on raised slatted floors to prevent self-reinfection, provided fresh water ad libitum, and fed daily with a ration of g of standard pellets consisting of lucerne ( g kg ), wheat ( g kg ), pollard ( g kg ), bran ( g kg ), salt ( g kg ) and ammonium chloride ( g kg ), the quantity of which was set for normal growth. at weeks , , and post-initial infection (where initial infection indicates the first day of larval dosing), all animals were blood-sampled, as above, and body mass and body condition (assessed using the industry-standard scale of - , www.lifetimewool.com.au/conditionscore.aspx) were recorded. at each of these four sample points, a subset of animals ( for each infection group, and three for the control, uninfected group) was humanely slaughtered and tissue collected, and processed as described below. from killed animals, the abomasum and small intestine were sampled in sections, placed into separate dissecting trays, the tissue opened and the contents gently washed into collecting jars to remove all adult nematodes. the number of worms in subsamples was then counted to determine the total number of worms of each species infecting each animal. samples of abomasal and jejunal tissues ( cm squares) were fixed in bouin's solution for later histological analysis. haemonchus contortus larvae can developmentally arrest within the host at the l stage, a form of diapause known as hypobiosis. hypobiosis does not occur in the strain of t. colubriformis used in our study. remaining abomasal tissue was, therefore, digested in phosphate-buffered saline containing % v/v hcl to release any arrested h. contortus fourth-stage larvae, which were then counted. we measured the number of immune cells in the fixed abomasal and jejunal tissue, which, following standard sectioning, were stained with haematoxylin and eosin, and toluidine blue [ ] . for both tissue samples, cell counts and scores were estimated per villus -crypt unit (i.e. from the tip of one villus to the next). for the abomasal tissue, we determined the number of globule leucocytes, mast cells and eosinophils, and scores for lymphocyte infiltration ( ¼ no infiltration, to ¼ heavy infiltration). for jejunal tissue the same cell counts and scores were made, but in addition the number of goblet cells and a score of the proportion of goblet cells containing granules ( ¼ no cells contained granules, to ¼ most cells contained granules) were also recorded, together with a score of the thickness of the smooth muscle layer ( ¼ very thin, to ¼ thick). we determined the concentration of igg antibodies against h. contortus and against t. colubriformis l antigens using previously described enzyme-linked immunosorbent assays (elisas) [ , ] . one animal was removed from the control group prior to infection because of ill health, leaving a control group sample size of animals. one animal was also removed from each of the coinfection and h. contortus mono-infection groups prior to the -week sample point, due to ill health unrelated to the helminth infections, leaving a sample size of sheep for each of these two groups. a small number of other sampling losses owing to processing problems are detailed in the electronic supplementary material, s , which provides an overview of sample size by sample point for all analyses. analyses were conducted in r v. . . [ ] . the effect of infection treatment group on the number of adult t. colubriformis worms, the number of adult h. contortus worms and the number of h. contortus arrested larvae were assessed in three general linear models (glms). infection group (mono-or co-infected), days post-initial infection (i.e. cull day; included as a categorical variable) and their interaction were included as independent variables. in addition, the faecal egg count pre-anthelminthic treatment and animals' total gain in mass were also accounted for by inclusion as independent terms. following preliminary model assessments, the number of arrested larvae of h. contortus was square root transformed (sqrt(x þ )) to normalize the residuals of that glm. neither poisson nor negative binomial error distributions provided better model fits for any model (electronic supplementary material, s ). we used two steps to determine how treatment group affected the measures of immune responses in the abomasum and jejunum. first, two principal components (pcs) analyses were conducted separately on the abomasal and jejunal measures of immune responses, using a singular value decomposition of the centred and scaled data matrix [ ] . all scores were treated as numeric data and scaling was applied. the measures of the abomasal immune responses were compared between the h. contortus mono-and co-infection groups; and measures of the jejunal immune responses were compared between t. colubriformis mono-and co-infection groups; in both cases, this separation reflects the location of these species within the animals. the pc explaining the majority of the variation in each analysis was then used as the dependent variable in a glm where treatment group, time of sampling and their interaction were the explanatory variables. models were refined in a stepwise manner by evaluating the f statistics (terms were rejected when p . . ). where the glm analyses showed significant differences in pc values between treatment groups, the second step in the analysis was undertaken. in these second analyses, the bootstrapped mean value was calculated for each individual measure of immune response, to qualitatively explore the effect of treatment group on anti-h. contortus and anti-t. colubriformis igg titres were assessed in two general linear mixed models (glmms using the r package asreml-r v . ) in which each animal's individual identification number was included as a random term to control for pseudo-replication. the titres of igg were transformed to normalize residuals in the model, as ((x þ ) . ) for anti-t. colubriformis and ((x þ ) . ) for anti-h. contortus responses. results shown here are backtransformed. in these models, treatment group, time of sampling (included as a categorical variable) and their interaction were included as fixed effects. this fixed-effect model was refined in a stepwise manner using the wald test and evaluation of the conditional f statistics (terms were rejected when p . . ). where treatment group was found to be a significant effect, differences between treatment groups were assessed by within-model contrasts. haemonchus contortus was also affected by co-infection, but differently compared with t. colubriformis. to assess the h. contortus infection, we analysed both the number of arrested l -stage larvae in the host tissues along with adult worms (see the electronic supplementary material, s for mean and s.d. of raw counts through time). there were fewer h. contortus arrested larvae in co-infections, compared with h. contortus-only infections (f , ¼ . , p ¼ . ; figure ); the number of these larvae was also affected by the time post-initial infection (f , ¼ . , p , . ; electronic supplementary material, s ). by contrast, the number of adult h. contortus was not affected by co-infection, though numbers did vary through time post-initial infection (f , ¼ . , p , . ; electronic supplementary material, s ). as the number of adults show no evidence of being bolstered by larvae leaving the arrested state in the co-infection group, together these data mean that in co-infections there are overall fewer h. contortus worms. trichostrongylus colubriformis infects the jejunum and to measure the immune responses in this site, we used a pc analysis of jejunal immune measures. all immune measures positively loaded onto pc axis (pc ), which explained % of the variance in these components (electronic supplementary material, s ). pc was subsequently used in the glm analysis and transformed (ln (pc þ ) ), resulting in a normal distribution of the model residuals; the results shown in the figures are back-transformed. the pc scores significantly differed between the co-infection and monoinfection groups, through time post-initial infection (glm analysis of pc scores f , ¼ . , p ¼ . ; figure ). the pc scores for the co-infected group did not vary with time post-initial infection, whereas those of the mono-infected group increased through time. the predicted pc values in the co-infected animals were significantly lower than in the jejunum. haemonchus contortus infects the abomasum, and to measure the immune responses in this site we used a pc analysis of the abomasel immune measures. all abomasel immune measures loaded positively onto pc explaining % of the variance (electronic supplementary material, s ). pc was subsequently used in the glm analysis and transformed (ln (pc þ ) ), resulting in a normal distribution of the model residuals; the results shown in the figures are back-transformed. glm analyses of the abomasal pc scores showed that they did not differ significantly between the co-infected and mono-infected animals, nor did they vary through time post-initial infection. the concentration of anti-h. contortus igg was significantly different between co-infected and h. contortus-only infected animals (effect of treatment group excluding the t. colubriformis mono-infection group f , ¼ . , p ¼ . ; figure ). the response was significantly greater in the co-infected animals, compared with the h. contortus-only infected and control animals, which did not differ from one another ( figure ). in the co-infected animals at weeks post-initial infection the igg response was reduced, coinciding with a reduced number of arrested h. contortus larvae (electronic supplementary material, s ). the concentration of anti-t. colubriformis igg was significantly affected by treatment group (effect of treatment group, excluding the h. contortus mono-infection group, f , ¼ . , p ¼ . ; electronic supplementary material, s ). specifically, these responses were significantly higher in the co-infected and t. colubriformis-only infection groups compared with the control, uninfected group. the co-infected and t. colubriformisonly infection groups were not significantly different from one another (electronic supplementary material, s ). we hypothesized that, by defining parasite groups using taxonomy and parasite traits, we could infer the host response to those groups and hence the expected interaction among co-infecting parasites. our hypothesis was supported. specifically, we demonstrate that immune suppression by the blood feeder h. contortus had a positive effect upon the numbers of mucosal browser t. colubriformis, while the immune response promoted by the mucosal browser negatively affected the numbers of the blood feeder. the presence of h. contortus resulted in comparatively more t. colubriformis adult worms in co-infected sheep. the trajectory of adult worm numbers in the t. colubriformis mono-infected sheep shows a classic convex age-intensity curve, indicative of host immune responses removing adult worms [ , , ] . in the co-infection treatment group the number of worms reached an asymptote, suggesting that adult worms were not being removed by the host immune response. there was, however, some evidence of a reduction in the larval establishment in this co-infection group (though less than in the mono-infected group), probably indicating that an anti-t. colubriformis response was beginning to develop. this is consistent with previous studies that have shown the anti-t. colubriformis immune response acts first against incoming larvae [ ] . as we hypothesized, the difference in the number of t. colubriformis adults between co-infection and mono-infection groups appears to be immune-mediated. our data demonstrate that there was a reduced immune response in the jejunum in the co-infected animals, compared to the t. colubriformis mono-infected animals, and most pronounced in the latter time points (weeks and post-initial infection; figures and and electronic supplementary material, s ). this differentiation between the infection groups suggests that the immune suppression we observe is dependent on the adult h. contortus (since by week all larvae would have developed to adulthood or arrested their development). we use the presented immune measures as general indicators of anti-helminth immune responses, rather than implicating individual immune components. nevertheless, all these immune components have been associated with the immune response against helminths in sheep [ ] [ ] [ ] . there was no evidence of an effect of co-infection on the number of h. contortus adults, nor on the abomasal cellular immune response. however, the significantly fewer arrested larvae in the co-infected animals demonstrate that coinfection still has a negative effect on h. contortus (figure ). in natural infections, arrested larvae resume development to adulthood during periods of host stress [ ] . there are significantly less arrested larvae in the co-infection group but no more adults. these missing larvae must, therefore: (i) be lost to the system entirely, or (ii) have replaced adults that have been lost. thus, these larvae either (a) never established in the arrested state in the first place, (b) were destroyed in, or expelled from, the tissues, or (c) following a period in the arrested state, resumed their development and either replaced lost adults, or failed to establish as adults. the difference in the number of larvae found in the arrested state between singly and co-infected groups of sheep is relatively small, approximately larvae, and is thus unlikely to be of clinical significance in these sheep. we highlight, however, that this study is not focused upon clinical significance per se, but upon the ability of our predictive framework to establish the form and direction of the parasite interactions, which we have achieved. nevertheless, even these few larvae, as adults, could contribute substantially to the potential infectious burden on pasture under natural conditions. assuming an average daily fecundity of eggs per female [ ] and a sex ratio of : , adult female worms could be adding more than eggs per day to pasture. as predicted, the loss of h. contortus arrested larvae appears to be immune-mediated. although the abomasal immune components do not differ among infection groups, the concentration of anti-h. contortus igg was significantly higher in co-infected animals (figure ). haemonchus contortus larvae were the source antigen for the igg assay and it is likely that this antibody response reduces larval development, as has been previously been reported [ ] . the host immune response to t. colubriformis and h. contortus in mono-infections is well documented [ ] [ ] [ ] [ ] . a feature of these responses is that they differ in strength depending on host species (sheep or goat), breed [ ] , [ - ] , age [ ] , [ ] and diet [ ] , although the same immune components are implicated in helminth control among these host groups. an important consideration, then, is whether the interactions we have described between the co-infecting parasite groups would be robust to such host differences. as the immune components involved in the host response are the same, we suggest that while there may be quantitative differences in intensity of infection owing to variation in the strength of the immune response, the qualitative result (i.e. positive consequences for a mucosal-browsing nematode and negative for the blood-feeding group) will probably persist. this view is further supported by the identical pattern of interaction seen in the rabbit co-infection system between its blood-feeding and mucosal-browsing nematode parasites. it should be noted that one laboratory study of co-infection with the same rabbit helminths did not find this pattern of interaction during co-infection [ ] . that laboratory study, however, used a single, high-dose infection (rather than the trickle infections we used), which can dramatically alter the form of the elicited immune response [ ] , in turn altering the nature of the interspecific interactions. our hypothesis for the interaction between the sheep nematodes was based on data from a different host and different parasite species, where we defined parasite groups based on their taxonomic and parasitological (i.e. resource use, site of infection) traits. we suggest that this novel approach can be more generally applied to other host and parasite systems. while we have successfully applied this approach here, we acknowledge that this is a single test and that further work is required to confirm that the approach could be applied beyond our defined parasite groupings. however, we note that our predictive ability crossed host species (rabbits and sheep) that are distinct taxonomically, behaviourally and physiologically, suggesting that host similarity does not underlie our successful prediction. regarding the parasites, we also emphasize that our hypothesis of how the sheep parasites would interact came solely from our predictive framework. specifically, despite extensive prior study of these parasites in sheep, the interactions we correctly predicted had never previously been hypothesized. together this suggests that our predictive framework is neither host nor parasite species-specific. future exploration of this topic could include a meta-analysis to determine whether parasite traits can represent patterns of immune function across multiple host types and different forms of parasite (i.e. beyond helminths). notably, the parasite species in our study all belong to the superfamily trichostrongyloidea and it is possible that the interaction observed would be restricted to species within this superfamily -though this would still be an important result. nevertheless, we have described here the common immunomodulatory features of several blood-feeding nematode species, which further supports this parasite grouping and also proposes a mechanism (i.e. suppression of the intestinal cellular immune response) for this groups' potential interaction with other parasite groups. there is less information available to support the grouping of mucosal-browsing nematodes, as the host immunological response to this group has been less well studied. even if we narrow this group to mucosal-browsing trichostrongylus spp., the only immune function studies conducted appear to be on t. colubriformis and t. retortaeformis, the species involved in our studies. it will therefore be interesting to determine whether other members of the group also stimulate, and are controlled by, a classic th response, which underlies the mechanism of their interaction with the blood feeders and, further, whether the group could be expanded to other helminth species displaying similarly low levels of tissue invasion, i.e. browsing nematodes beyond trichostrongylus spp. we propose that the form of acquisition of a given resource is likely to be an important indicator of how the host will respond to any parasite. for example, while nematodes and malaria both use the host blood as a resource, they acquire that resource in a different way. we suggest that taxonomically more related parasites are also more likely to evolve related mechanisms of resource acquisition and therefore that a combined grouping strategy involving location, resource use and parasite taxonomy may be a good indicator of host immune response, the ultimate mechanism of the interspecific parasite interaction in our study. our classification mechanism requires that the resource use of the parasite is known. for some species, this will not be the case. however, using physical location in conjunction with taxonomic similarity to other known species will often be a suitable proxy. haemonchus contortus and t. colubriformis are both economically important parasites, causing substantial production losses in both sheep and goats [ ] . production losses owing to t. colubriformis are likely to be greater in sheep co-infected with h. contortus, because of the higher worm burdens and prolonged infection in such co-infections. notably, the condition and mass of co-infected animals did not significantly differ from the other treatment groups. however, pasture-reared sheep, not provisioned with the high-quality maintenance diet provided in our experiment, would probably experience more severe effects during co-infection. transmission of t. colubriformis in co-infected sheep could be substantially higher owing to the higher worm burdens and prolonged infection during h. contortus co-infection, meaning potentially higher worm burdens at a population level, requiring the use of anthelmintics. however, density-dependent reduction in per capita worm fecundity has been observed for t. colubriformis [ ] , which may ameliorate such effects. nevertheless, host immune response appears to play a role in this densitydependent restriction of fecundity [ ] , and thus such immune effects may be reduced during h. contortus co-infection. a change in h. contortus-induced production losses during coinfection are unlikely, as adult worm burdens of this species were not affected by the co-infection. the economic implications of this co-infection are, therefore, principally a consequence of the altered dynamics of the t. colubriformis infection. this work represents, to our knowledge, a first experimental proof-of-principle that groups of parasites can be identified rspb.royalsocietypublishing.org proc. r. soc. b : and thereafter used to predict the outcome of a previously unexplored interspecific parasite interaction in a different host species. given the ubiquity and multiplicity of co-infection in nature, it is important that we derive such grouping mechanisms. in previous work, we suggested grouping parasites by an immunological profile [ ] . a problem with this idea is that immune profiling is complex and expensive, and reagents may not be available for a novel or lesser-studied hosts. however, the current study offers an alternative mechanism for classification by using taxonomy and more easily identified parasitological traits, to act as a proxy for the immune traits. further, we have demonstrated that we can successfully use these traits to predict the immunologically based interaction of two parasite groups. this work therefore proposes a general framework for predicting the relationships between other parasite groups, and next steps should be to determine how widely applicable such a framework can be. ethics. our work using merino sheep followed australian government guidelines with ethical approval obtained from the fd mcmaster laboratory, chiswick, animal ethics committee. approval number aec . data accessibility. all the data described in this study are available from the dryad digital repository: https://doi.org/ . /dryad. hj [ ] . heligmosomoides polygyrus reduces infestation of ixodes ricinus in free-living yellow-necked mice, apodemus flavicollis generating super-shedders: coinfection increases bacterial load and egg production of a gastrointestinal helminth competition and mutualism among the gut helminths of a mammalian host interactions between macroparasites and microparasites drive infection patterns in freeranging african buffalo 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parasites th cytokine-induced alterations in intestinal smooth muscle function depend on alternatively activated macrophages the mortality and fecundity of haemonchus contortus in parasite-naïve and parasite-exposed sheep following single experimental infections voluntary feed intake and diet selection of merino sheep divergently selected for genetic difference in resistance to haemonchus contortus protection of merino lambs against haemonchus contortus by trickle infection of neonates induction of protective immunity to trichostrongylus colubriformis in neonatal merino lambs haemonchus contortus infection in sheep: parasite fecundity correlates with worm size and host lymphocyte counts a th type of immune response is associated with increased resistance to haemonchus contortus in naturally infected gulf coast native lambs st croix sheep produce a rapid and greater cellular immune response contributing to reduced establishment of haemonchus contortus relative resistance of menz and washera sheep breeds to artificial infection with haemonchus contortus in the highlands of ethiopia influence of breed on susceptibility of sheep and goats to a single experimental-infection with haemonchus contortus population dynamics of trichostrongylus colubriformis in sheep: the effect of host age on the establishment of infective larvae effects of dietary-protein intake on responses of young sheep to infection with trichostrongylus colubriformis effects of single or trickle haemonchus contortus experimental infection on digestibility and host responses of naive creole kids reared indoor chasing helminths and their economic impact on farmed ruminants regulation of teladorsagia circumcincta and trichostrongylus colubriformis worm populations by grazing sheep with differing resistance status data from: predicting the effects of parasite co-infection across species boundaries acknowledgements. we would like to thank the animal house support team at armidale, nsw without whom this work could not have been undertaken, and andrea graham (princeton university) for insightful discussions. authors' contributions. j.l. devised and managed the project, conducted the statistical analyses and in conjunction with m.e.v. wrote the manuscript. m.e.v. was also involved in the initial experimental design and interpretation of the data. s.j.m. advised on and supervised all immunological aspects of the project and undertook the abomasal and jejunal immune component analysis, with the exception of the elisas, which were carried out by k.t. k.t. was also lead technical officer for the project and both supervised and took part in all animal handling and dosing, sampling and tissue preparation. k.t. also undertook all worm enumeration.competing interests. the authors declare that they have no competing interests.funding. this work was funded primarily by a commonwealth scientific and industrial research organisation (csiro), australia, complex systems science grant with match funding from csiro livestock industries. j.l., s.m. and k.t. were employees of livestock industries, but there was no intervention by the wider organization in either analysis or interpretation of data or in the writing of the manuscript. a proportion of the time j.l. spent developing the manuscript was funded by a marie skłodowska-curie fellowship at the fondazione e. mach, italy (h -msca-if- ; grant no. ). key: cord- -lbikoyo authors: beidas, meshal; chehadeh, wassim title: effect of human coronavirus oc structural and accessory proteins on the transcriptional activation of antiviral response elements date: - - journal: intervirology doi: . / sha: doc_id: cord_uid: lbikoyo objectives: the molecular mechanisms underlying the pathogenesis of human coronavirus oc (hcov-oc ) infection are poorly understood. in this study, we investigated the ability of hcov-oc to antagonize the transcriptional activation of antiviral response elements. methods: hcov-oc structural (membrane m and nucleocapsid n) and accessory proteins (ns a and ns a) were expressed individually in human embryonic kidney (hek- ) cells. the transcriptional activation of antiviral response elements was assessed by measuring the levels of firefly luciferase expressed under the control of interferon (ifn)-stimulated response element (isre), ifn-β promoter, or nuclear factor kappa b response element (nf-κb-re). the antiviral gene expression profile in hek- cells was determined by pcr array. results: the transcriptional activity of isre, ifn-β promoter, and nf-κb-re was significantly reduced in the presence of hcov-oc ns a, ns a, m, or n protein, following the challenge of cells with sendai virus, ifn-α or tumor necrosis factor-α. the expression of antiviral genes involved in the type i ifn and nf-κb signaling pathways was also downregulated in the presence of hcov-oc structural or accessory proteins. conclusion: both structural and accessory hcov-oc proteins are able to inhibit antiviral response elements in hek- cells, and to block the activation of different antiviral signaling pathways. human coronavirus oc (hcov-oc ) is an enveloped, positive-sense rna virus classified as a betacoronavirus, the same genus as severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) coronaviruses. hcov-oc infection has been associated mainly with upper respiratory tract symptoms and exacerbation of asthma and pneumonia in some groups and institutional settings [ ] [ ] [ ] [ ] . the virus has also been associated with severe neurological disorders like acute disseminated encephalomyelitis in children [ , ] . while most studies have focused their attention on the immunopathology of sars-cov and mers-cov, there has not been the same interest for hcov-oc . indeed, there are few studies concerned with the molecular mech-intervirology ; : - doi: . / anisms governing hcov-oc infection and its effect on the intracellular host defenses. hcov-oc is over kb in length, and similarly to the other coronaviruses, it is composed of the structural proteins spike (s), envelope (e), membrane (m), and nucleocapsid (n) [ ] . uniquely, there are two accessory proteins interspaced between these structural proteins called ns a and ns a [ ] . these proteins are not essential for replication; however, they might play a role in the pathogenesis of coronavirus infection [ ] . the interferon (ifn) induction and signaling pathway is the major branch in the innate immune response against viruses. the induction of type i ifn is initiated by molecular sensing of viral rna by pattern recognition receptors such as tlr, rig-i, and mda [ ] . the adaptor protein mavs mediates the signals from rig-i and mda to activate the kinases tbk and ikkε, which in turn phosphorylate irf and irf transcription factors [ ] . irf and irf dimerize to undergo nuclear translocation and initiate the transcription of type i ifn. the adaptor proteins myd and trif mediate signals from tlrs and activate irak , traf , and the aforementioned kinases [ ] . the irfs along with nuclear factor kappa b (nf-κb) can then be phosphorylated to translocate into the nucleus and establish the transcription of type i ifn by binding cognate sites on the ifn-β promoter [ ] . nf-κb specifically binds to the nf-κb response element (nf-κb-re) to regulate the expression of pro-inflammatory and cell survival genes [ ] . ifn signaling via the jak/stat pathway is established when type i ifn binds to the ifn receptor. this leads to activation of the kinases jak and tyk . these kinases in turn phosphorylate the transcription factors stat and stat [ , ] . phosphorylated stat and stat then dimerize and undergo nuclear translocation, where they form a complex with irf called the isgf . this complex will bind the ifn-stimulated response element (isre) and initiate the transcription of isgs. these isgs assist the cell in counteracting the viral infection by establishing an antiviral state [ ] . sars-cov and mers-cov were shown to inhibit the transcriptional activity of isre, ifn-β promoter or nf-κb-re [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, the effect of different hcov-oc proteins on the transcriptional activation of antiviral response elements has not yet been investigated. in this study, the ability of hcov-oc structural (m and n) and accessory proteins (ns a and ns a) to antagonize the transcriptional activation of isre, ifn-β promoter, and nf-κb-re was investigated. human embryonic kidney (hek- ) cells (american type culture collection atcc, manassas, va, usa) were cultured in cm tissue culture flasks in dulbecco's minimal essential medium containing glutamax tm (life technologies corporation tm , grand island, ny, usa). the medium was supplemented with % fetal bovine serum, fungizone ® ( µg/ml), penicillin g ( , u/ml), and streptomycin sulfate ( , pg/ ml) (life technologies tm ). monolayers of hek- cell culture flasks were incubated at ° c in the presence of % carbon dioxide (co ) and % humidity. the qiaamp ® viral rna minikit (qiagen tm gmbh, hilden, germany) was used to isolate rna from hcov-oc (atcc vr- ) according to the manufacturer's instructions. the hcov-oc ns a, ns a, m, and n genes were amplified by a twostep reverse transcription-polymerase chain reaction (rt-pcr) using the geneamp ® rna core kit (applied biosystems tm , foster city, ca, usa), and pmol of previously described primers [ ] on geneamp ® pcr system (applied biosystems tm ). the rt reaction conditions were: annealing at ° c for min, denaturation at ° c for min, and cooling at ° c. thereafter, the cdna was amplified by pcr using the following conditions: denaturation at ° c for min, then cycles of denaturation at ° c for s, annealing at ° c for s, and extension at ° c for s, followed by a final extension step at ° c for min, and cooling at ° c. pcr products were run on agarose gel and the bands that were size-specific for the amplified gene of interest, were cut and purified by wizard ® sv gel and pcr clean up system (promega tm , madison, wi, usa) according to the manufacturer's instructions. rna of influenza a virus (h n , a/hong kong/ / strain; atcc vr- ) was also isolated and used to amplify ns gene by rt-pcr using ns -specific primers [ ] . influenza a ns protein was used in the following experiments as inhibitor of the innate immune host response [ , ] . rt-pcr products were cloned using the pacgfp -n in-fusion ® ready vector (clontech tm , takara bio company, mountain view, ca, usa) that encodes a green fluorescent protein (gfp) from aequorea coerulescens allowing the expression of the fusion protein to the n-terminus of acgfp . the ligation reaction was set up by utilizing the in-fusion ® hd cloning kit (clontech tm ). competent top escherichia coli cells (invitrogen tm , carlsbad, ca, usa) were used for transformation. the pureyield tm plasmid miniprep kit (promega tm ) was used to isolate the vector according to the manufacturer's instructions. confirmation of successful cloning was achieved using restriction digestion and direct sequencing (data not shown). hek- cells were seeded at × per well of -well plates, and transfected with ns a-pacgfp , ns a-pacgfp , m-pacgfp , n-pacgfp , or ns -pacgfp vector using lipofectamine ® (promega tm ) according to the manufacturer's instructions. expression of ns a, ns a, m, n and ns proteins was confirmed by indirect immunofluorescence assay using anti-gfp monoclonal antibody ( μg/ml) (clone jl- , clontech tm ) (data not shown). expression of n and ns proteins was further confirmed by immunofluorescence assay using monoclonal antibody against hcov-oc n (emd millipore tm , billerica, ma, usa) and polyclonal antibody against influenza a ns protein (emd millipore tm ), respectively (data not shown). the ifn-β promoter was amplified using previously described primers [ ] , and inserted into the reporter pgl . (luc cp/ne) vector (promega tm ) between the restriction sites bglii and hindiii according to the manufacturer's instructions, creating the pifnβluc plasmid. the reporter vectors coding for firefly luciferase under the control of isre, pisre-luc (pgl . [luc p/isre/hygro]) or nf-κb-re, pnfκb-re-luc (pgl . [luc p/nf-κb-re/hygro]) were purchased from promega tm . following h of transfection with one of the expression vectors cited above, hek- cells were co-transfected with pifnβ-luc, pisre-luc or pnfκb-re-luc vector at . μg/well using lipofectamine ® (promega tm ). renilla luciferase vector (prl-tk) (promega tm ) was used at ng/ well as an internal control for transfection efficiency. hcov-oc ns a, ns a, m, and n protein total rna from transfected and mock-transfected hek- cells was extracted using rneasy ® kit (qiagen tm ) with on-column dnase digestion according to the manufacturer's instructions. the rt first strand kit (qiagen tm ) was used for cdna synthesis. the rt reaction was then added to rt sybr green rox qpcr mastermix (qiagen tm ), and the human antiviral response pcr array (qiagen tm ) was used to profile the antiviral gene expression in hek- cells on abi fast real-time pcr system (applied biosystems tm ). the pcr array monitored the transcriptional activity of different genes involved in the activation of antiviral and pro-inflammatory proteins following amplification of rna transcripts by real-time rt-pcr. however, we only profiled the expression of genes relevant to the antiviral response elements. cycle threshold (c t ) values were exported to an excel file to create a table of c t values. this table was then uploaded on to the qiagen data analysis web portal (http://www.qiagen.com/geneglobe). c t values were normalized to glyceraldehyde -phosphate dehydrogenase. fold regulation comparison was used to profile antiviral gene expression. fold change/regulation was calculated using the delta-delta c t method (∆∆c t ). the fold regulation threshold was ≥ for upregulation and ≤- for downregulation. fold change in firefly luciferase from three different experiments was summarized as mean ± standard deviation. the difference in fold change mean between two groups was determined by independent-samples t test. p values < . were considered sig-nificant. all statistical analyses were performed using spss statistics software version . for windows (ibm corporation, armonk, ny, usa). graphpad prism software (graphpad software inc., la jolla, ca, usa) was used to generate all charts. effect of hcov-oc proteins on the transcriptional activation of antiviral response elements. the transcriptional activity of isre, ifn-β promoter, and nf-κb-re was assessed by measuring the firefly luciferase levels in hek- cells transfected with ns a-pacgfp , ns a-pacgfp , m-pacgfp , n-pacgfp , or ns -pacgfp vector. in control cells nonexpressing viral protein, the firefly luciferase levels were highest following h of stimulation with one of the inducers (data not shown). in the absence of an inducer, the firefly luciferase levels in hek- cells expressing one hcov-oc protein were similar to the background levels ( fig. a, b) . in ifn-α-treated hek- cells, the expression of firefly luciferase under the control of isre or ifn-β promoter was inhibited in the presence of one of the tested hcov-oc proteins or in the presence of influenza a ns protein (fig. a, ) . the transcriptional activity of isre was also inhibited in sev-stimulated hek- cells in the presence of one hcov-oc protein (fig. b) . in mock-transfected hek- cells, sev induced only low levels of firefly luciferase under the control of ifn-β promoter (data not shown), and therefore the effect of hcov-oc proteins on the transcriptional activity of ifn-β promoter in sev-stimulated hek- cells could not be assessed. following the challenge with tnf-α, the transcriptional activity of nf-κb-re was inhibited in the presence of one of the tested hcov-oc proteins or in the presence of influenza a ns protein (fig. ) . to investigate whether the inhibition of the transcriptional activity of antiviral response elements in the presence of hcov-oc proteins can be associated with a downregulation of the expression of antiviral genes, pcr array profiling of antiviral genes was carried out in trans-fected and mock-transfected hek- cells. following sev challenge of hek- cells, the expression of genes involved in the type i ifn and nf-κb signaling pathways was downregulated in the presence of hcov-oc structural or accessory proteins (fig. ) . similar results were obtained in the presence of the influenza a ns protein. the mean negative fold change for tradd gene in the presence of accessory protein ns a was greater than in the presence of other hcov-oc proteins (p < . ). the expression of irf gene was more downregulated in the presence of ns a or n protein than in the presence of other hcov-oc proteins (p < . ), whereas the expression of tlr gene was more downregulated in the presence of ns a protein than in the presence of other hcov-oc proteins (p < . ). remarkably, irf and ifna gene expression was more downregulated in the presence of ns a, ns a or n protein than in the presence of influenza a ns protein. similar to influenza a ns protein, hcov-oc structural (m and n) and accessory (ns a and ns a) proteins were able to inhibit the transcriptional activity of antiviral response elements, isre, ifn-β promoter, and nf-κb-re, and to downregulate the expression of several genes involved in the activation of an antiviral response. the inhibition of the isre, ifn-β promoter, and nf-κb-re activity, and the downregulation of the expression of ifna , irf , tlr , myd , tradd, and mavs in the presence of each hcov-oc accessory protein suggest that hcov-oc ns a and ns a have the potential to block the type i ifn and nf-κb pathways. previous studies have shown that sars-cov and mers-cov accessory proteins have a role in innate immune evasion [ , , [ ] [ ] [ ] . however, hcov-oc accessory proteins are not homologous to sars-cov and mers-cov accessory proteins. also of interest, the murine coronavirus accessory protein a, which is homologous to hcov-oc ns a protein, was shown to antagonize ifn induction [ ] . sars-cov and mers-cov m proteins can suppress the activity of ifn-β promoter and isre [ , , ] . nf-κb activation is also inhibited when sars-cov m protein is expressed in vero or hela cells [ ] . our data showed that the transcriptional activity of isre, ifn-β promoter, and nf-κb-re was inhibited in the presence of hcov-oc m protein. in addition, like the accessory proteins, the hcov-oc m protein was able to downregulate the expression of ifna , irf , tlr , myd , tradd, and mavs. these findings suggest that in addition to its function in the assembly of virions [ ] , hcov-oc m protein is an antagonist of the host antiviral defenses. the coronavirus n protein binds genomic rna to form the helical capsid, and has a critical role in coronavirus replication [ , ] . the sars-cov n protein can activate nf-κb-re in vero e cells [ ] . however, sars-cov n protein inhibits the promoter activity of isre, ifn-β promoter, and nf-κb-re in t cells [ ] . our study showed that the transcriptional activity of isre, ifn-β promoter, and nf-κb-re was inhibited in the presence of hcov-oc n protein. lai et al. [ ] showed that after h of tnf-α treatment, hcov-oc n protein potentiates nf-κb expression in t cells by binding its inhibitor, mirna . mirna expression is induced by stimuli which activate nf-κb, such as tnf-α and lipopolysaccharide [ ] . unfortunately, the authors did not show results for nf-κb-re activation after h of tnf-α treatment, the optimal time for nf-κb-re activation in our experiments. the influenza a virus that is known to inhibit irf , ap- , and nf-κb activation [ , ] was shown to upregulate the mirna expression [ ] . we speculate that the expression of n protein results in transient downregulation of nf-κb expression through downregulation of its mediators (e.g., tlr , myd , tradd), leading to early inhibition of nf-Κb-re activation, and that the accumulation of mirna in cells with time will lead to the formation of n protein/mirna complex and the removal of inhibition on nf-kb and nf-kb-re. in conclusion, hcov-oc has the ability to inhibit the transcriptional activation of antiviral response elements, and the expression of antiviral genes. this would lead to the disruption of several antiviral signaling pathways such as the type i ifn and nf-κb pathways. further studies are needed to confirm the role of hcov-oc proteins in antagonizing the host innate immunity. polymerase chain reaction-based detection of rhinovirus, respiratory syncytial virus, and coronavirus in otitis media with effusion high incidence but low burden of coronaviruses and preferential associations between respiratory viruses human coronavirus-associated influenza-like illness in the community setting in peru the pediatric burden of human coronaviruses evaluated for twenty years detection of coronavirus in the central nervous system of a child with acute disseminated encephalomyelitis human coronavirus oc associated with fatal encephalitis nidovirales: a new order comprising coronaviridae and arteriviridae circulation of genetically distinct contemporary human coronavirus oc strains 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syndrome coronavirus accessory protein a is a type i interferon antagonist middle east respiratory syndrome coronavirus orf b protein inhibits type i interferon production through both cytoplasmic and nuclear targets the structural and accessory proteins m, orf a, orf b, and orf of middle east respiratory syndrome coronavirus (mers-cov) are potent interferon antagonists the membrane protein of sars-cov suppresses nf-kappab activation severe acute respiratory syndrome coronavirus m protein inhibits type i interferon production by impeding the formation of traf .tank.tbk / ikkepsilon complex simplified large-scale sanger genome sequencing for influenza a/h n virus variation in the ability of human influenza a viruses to induce and inhibit the ifn-beta pathway effects of ns variants of h n influenza virus on interferon induction, tnf a response and p activity accessory protein a is a major antagonist of the antiviral action of interferon against murine coronavirus membrane assembly of the 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replication by microrna- through targeting mcpip this work was in part supported by kuwait university research administration grant no. ym / . the authors report no conflicts of interest. key: cord- -ebkwv zo authors: bodmer, bianca s.; fiedler, anna h.; hanauer, jan r.h.; prüfer, steffen; mühlebach, michael d. title: live-attenuated bivalent measles virus-derived vaccines targeting middle east respiratory syndrome coronavirus induce robust and multifunctional t cell responses against both viruses in an appropriate mouse model date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: ebkwv zo cases of middle east respiratory syndrome coronavirus (mers-cov) continue to occur, making it one of the who´s targets for accelerated vaccine development. one vaccine candidate is based on live-attenuated measles virus (mv) vaccine encoding the mers-cov spike glycoprotein (mers-s). mv(vac )-mers-s(h) induces robust humoral and cellular immunity against mers-s mediating protection. here, the induction and nature of immunity after vaccination with mv(vac )-mers-s(h) or novel mv(vac )-mers-n were further characterized. we focused on the necessity for vector replication and the nature of induced t cells, since functional cd (+) t cells contribute importantly to clearance of mers-cov. while no immunity against mers-cov or mv was detected in mv-susceptible mice after immunization with uv-inactivated virus, replication-competent mv(vac )-mers-s(h) triggered robust neutralizing antibody titers also in adult mice. furthermore, a significant fraction of mers cov-specific cd (+) t cells and mv-specific cd (+) t cells simultaneously expressing ifn-γ and tnf-α were induced, revealing that mv(vac )-mers-s(h) induces multifunctional cellular immunity. the middle east respiratory syndrome coronavirus (mers-cov) is a member of the coronaviridae family and emerged in in the kingdom of saudi arabia (zaki et al., ) . coronaviruses typically cause mild infections of the upper respiratory tract, but already in , the severe acute respiratory syndrome cov (sars-cov) with a mortality rate of about % among infected patients was introduced in the human population. sars-cov spread world-wide and caused more than diagnosed infections, but was contained within a year after its emergence (http://www.who.int/csr/sars/country/table _ _ / en/). in contrast, infections with mers-cov are ongoing for more than years, with laboratory-confirmed cases distributed over countries with at least deaths that were reported to the who by november (http://www.who.int/emergencies/mers-cov/en/). this apparent case-fatality rate of % is of grave concern, because epidemic spread as has been observed for sars-cov could result in a disastrous death toll. mers-cov has been introduced zoonotically by transmission from dromedary camels to human patients (alagaili et al., ; haagmans et al., ; reusken et al., a) and serological studies indicate wide-spread and early distribution among this animal host (alagaili et al., ; reusken et al., b) . therefore, a continuous risk of transmission especially to persons in close contact to camels is evident. fortunately, the human to human transmission rate has remained low. aside of individuals with regular contact to camels, only health care workers or relatives of mers-cov patients have a considerable risk of infection (alraddadi et al., ; drosten et al., ) , but still at a modest level. nonetheless, the high case fatality rate, the recurrent outbreaks of mers-cov infections, and especially the risk of virus adaptation potentially resulting in epidemic or even pandemic spread make the development of an effective vaccine against mers-cov an international priority. the efficacy of several vaccine candidates has been demonstrated in different animal models up to even dromedary camels (reviewed in (okba et al., ) ). one of these candidates, mv vac -mers-s(h) (malczyk et al., ) , is based on the measles virus (mv) vaccine platform technology (mühlebach, ) , and encodes the mers-cov spike protein (s) as an additional antigen in the backbone of recombinant mv vac (del valle et al., ) resembling vaccine strain moraten that is authorized and in use in the us since . this candidate induces both robust humoral and functional cellular immuneresponses against mers-cov. moreover, mers-cov viral load and inflammation of the lung were significantly reduced in challenged mice that had been vaccinated with mv vac -mers-s(h), before (malczyk et al., ) . while these experiments provided proof of concept for efficacy of this vaccine candidate, further mechanistic insights into the nature of the induced t cell responses remain to be elucidated. these are of special interest, since it has been shown that t cells are essential for clearance of the infection (coleman et al., ; zhao et al., ) : depletion of cd + t cells increased overall inflammation, bronchiolar inflammation, lymphocyte infiltration, and pleuritis at day post-infection in mice (coleman et al., ) , while mers cov-susceptible mice depleted of all t cells were unable to clear the virus . as an alternative to the spike glycoprotein, conserved (internal) structural proteins such as the nucleocapsid protein n are of special interest as putative target of anti-viral t cell responses to be triggered by future mers vaccines . therefore, we have also generated and characterized mers-cov n protein-encoding vaccine candidates based on the mv vac vaccine platform, in this study. to further characterize the induction of mers cov-specific immune responses, we first analyzed the necessity for viral replication for the induction of mers cov-and mv-specific immune responses using the highly immunogenic mv vac -mers-s(h) vaccine candidate. in addition, we characterized the functionality of cd + and cd + t cell responses in juvenile ( - week old) and adult ( months of age) mice using flow cytometry and functional assays. vero (african green monkey kidney) (atcc# ccl- ) and t (atcc crl- ) cell lines were purchased from atcc (manassas, va, usa) and cultured in dulbecco's modified eagle's medium (dmem, biowest, nuaillé, france) supplemented with % fetal bovine serum (fbs; biochrom, berlin, germany) and mm l-glutamine (l-gln; biochrom). jawsii dendritic cells (atcc crl- ) were purchased from atcc and cultured in mem-α with ribonucleosides and deoxyribonucleosides (gibco brl, eggenstein, germany) supplemented with % fbs, mm l-gln, mm sodium pyruvate (biochrom), and ng/ml murine gm-csf (peprotech, hamburg, germany). dc . murine dendritic cells (shen et al., ) were cultured in rpmi containing % fbs, mm l-gln, % non-essential aminoacids (biochrom), mm hepes (ph , ), and μm -mercaptoethanol (sigma-aldrich, steinheim, germany). cells were cultured at °c in a humidified atmosphere containing % co for a maximum of months of culture after thawing of the original stock. the codon-optimized gene encoding mers-cov-n (genebank accession no. jx ) flanked with aatii/mlui binding sites in plasmid pma-rq-mers-n was obtained by gene synthesis (invitrogen life technology, regensburg, germany). the antigen and the immediate early cytomegalovirus (cmv) promoter (martin et al., ) were inserted into plasmids p(+)br-mv vac -atu(p) (del valle et al., ) or p(+)mv vac -gfp(h) via mlui/aatii and sfii/sacii, respectively, to generate p(+)polii-mv vac -mers-n(p) or p(+)polii-mv vac -mers-n (h). for construction of lentiviral transfervectors encoding mers-cov-n, the orf of mers-n was amplified by pcr with primers encompassing flanking restriction sites nhei/xhoi and template pma-rq-mers-n. details on primers and pcr are available upon request. pcr products were cloned into pcr . -topo (invitrogen life technology) and fully sequenced. intact antigen orf was cloned into pcscw gluc-ires-gfp (hewett et al., ) using nhei/xhoi restriction sites to yield pcscw -mers-n-ires-gfp. lentiviral vectors were produced and used for the generation of antigen-expressing dendritic cell lines as described, before (malczyk et al., ) . in short, hiv- -derived vectors were generated using a standard plasmid system and the transfer vector plasmid pcscw -mers-n-ires-gfp by pei transfection. subsequent purification after harvest of transfected t cells yielded virus stocks used to transduce dc cell lines, which were single cell-sorted by facs and selected for antigen expression. mers-n encoding vaccine candidates mv vac -mers-n(p) and mv vac -mers-n(h) were rescued as described (malczyk et al., ; martin et al., ) . single syncytia were picked and overlaid onto % confluent vero cells cultured in -well plates and harvested as "passage " (p ) by scraping and freeze-thaw cycle of cells at the time of maximal infection. subsequent passages were generated as described for the following viruses. mers-n encoding vaccine viruses in p were used for characterization, viruses in p for vaccination. mers-s encoding vaccine virus mv vac -mers-s(h), and control virus mv vac -atu(p) (malczyk et al., ) were also used in p for vaccination. both as well as mv vac -gfp(p) and mers-cov (isolate emc/ ) (zaki et al., ) used for neutralization assays were propagated and titrated on vero cells by the method of spearman and kaerber (hubert, ; kärber, ) . mv vac -mers-s(h) was inactivated by uv-irradiation using a cl- uv crosslinker (uvp, cambridge, uk). μl of virus suspension in -well-plates on ice were exposed to uv light of nm at cm distance from the uv source of , × μj/cm for min. inactivation of virus was controlled by incubation of vero cells with a control aliquot inactivated, in parallel. all virus stocks were stored in aliquots at − °c. cells were lysed and immunoblotted as previously described (funke et al., ) . a rabbit anti-mers-cov serum ( : ) was used as primary antibody for mers-cov-n and a rabbit anti-mv-n polyclonal antibody ( : , ) (abcam) for mv-n detection. a donkey hrp-coupled anti-rabbit igg (h&l) polyclonal antibody ( : , ) (rockland, gilbertsville, pa) served as secondary antibody for both. peroxidase activity was visualized with an enhanced chemiluminescence detection kit (thermo scientific, bremen, germany) on amersham hyperfilm ecl (ge healthcare, freiburg, germany). all animal experiments were carried out in compliance with the regulations of german animal protection laws and as authorized by the rp darmstadt. six-to -week-old or months old ifnar -/--cd ge mice (mrkic et al., ) deficient for type i ifn receptor and transgenically expressing human cd were inoculated intraperitoneally (i.p.) with × tcid of recombinant viruses or uv-inactivated vaccine preparations on days and either on day or . mice were bled on days , , and post initial infection (p.i.). serum samples were stored at − °c. mice were euthanized on days , , or p.i., and splenocytes were harvested for assessment of cellular immune responses. quantification of vnts was done as described, before (malczyk et al., ) . in brief, mouse sera were serially diluted in -fold dilution steps in dmem in duplicates. a total of pfu of mv vac -gfp(p) or tcid of mers-cov (strain emc/ ) were mixed with diluted sera and incubated at °c for h. virus suspensions were added to × vero cells seeded h prior to assay in -well plates and incubated for days at °c. vnts were calculated as the reciprocal of the highest mean dilution that abolished infection. murine gamma interferon (ifn-γ) enzyme-linked immunosorbent spot (elispot) assays (ebioscience, frankfurt, germany) were performed according to the manufacturer's instructions using multiscreen immunoprecipitation (ip) elispot polyvinylidene difluoride (pvdf) well plates (merck millipore, darmstadt, germany). × isolated medium inoculated mice served as mock control. vnts were calculated as the highest dilution abolishing infectivity. dots represent single animals (n = ); horizontal lines represent mean per group. the y-axis starts at the detection limit; all mice at the detection limit had no detectable vnt. (g) secretion of ifn-γ after antigen-specific re-stimulation of splenocytes harvested days post prime immunization and after co-culture with jawsii (left) or dc . (middle) dendritic cells transgenic for mers-n (black) or untransduced controls (nc, white). (right) to analyze cellular responses directed against mv, splenocytes were stimulated with μg/ml mv bulk antigen (mv bulk) or left unstimulated (sham). the reactivity of splenocytes was confirmed by cona treatment ( μg/ml). tcid , tissue culture infectious dose ; one-way-anova with tukey multiple comparison. *: p < , ; **: p < , ; ***: p < ; ****: p < , . splenocytes were co-cultured with different stimuli in μl rpmi + % fbs, mm l-gln, and % penicillin-streptomycin for h. for re-stimulation of mers n-specific t cells, splenocytes were co-cultivated with × jawsii, dc . dendritic cells, or clones of either cell line encoding mers-n. on the other hand, splenocytes were stimulated with μg/ml mv bulk antigen (serion immunologics, würzburg, germany), μg/ml mers s-derived peptide s (biosynthesis inc., lewisville, tx, usa, (channappanavar et al., ) ), or μg/ml siinfekl control peptide (sin) of ovalbumin (aa - ) (invivogen, san diego, ca, usa), as appropriate. for general t cell stimulation, μg/ml concanavalin a (cona, sigma-aldrich, st. louis, mo, usa) was used. as negative control, splenocytes were left untreated. after h, cells were removed from the plates, and plates were incubated with biotin-conjugated anti-ifn-γ antibodies and avidin-hrp according to the manufacturer's instructions. -amino- ethyl-carbazole (aec; sigma-aldrich) substrate solution for development of spots was prepared according to the manufacturer's instructions using aec dissolved in n,n-dimethylformamide (merck millipore) and used for peroxidase-dependent staining, afterwards. spots were counted using an eli.scan elispot scanner (ae.l.vis, hamburg, germany) and elispot analysis software eli.analyse v . (ae.l.vis). for flow cytometry-based determination of cytokine expression by intracellular cytokine staining (ics), splenocytes of vaccinated mice were isolated, and × splenocytes per mouse were cultivated in μl rpmi + % fbs, mm l-gln, × non-essential amino acids (biochrom), mm hepes, % penicillin-streptomycin, μm βmercaptoethanol, μg/ml brefeldin a (sigma-aldrich), and one of the stimuli also used for elispot analysis. for general t cell stimulation, . μg/ml tetradecanoylphorbol acetate (tpa, sigma aldrich) and . μg/ml ionomycin (iono, sigma-aldrich) were used as positive control, and only medium was used as negative control. splenocytes were stimulated for h at °c. subsequently, cells were stained with fixable viability dye efluor (ebioscience), cd -pe ( : ) (bd, franklin lakes, nj, usa), cd -fitc ( : ) (bd), and, after permeabilization with fixation/permeabilization solution (bd) and perm/ wash buffer (bd), stained with ifn-γ-apc ( : ) (bd) and tnf-α-pe-cy ( : ) (bd). cells were fixed with ice-cold % paraformaldehyde (pfa) in pbs and analyzed via flow cytometry using an lsrii sorp flow cytometer (bd) and fcs express software (de novo software, glendale, ca, usa). since the nucleocapsid protein (n) of cov is quite conserved, it is regarded as an appropriate target to induce anti-viral t cells. therefore, mers-cov n was chosen as an alternative antigen to be expressed by the recombinant mv vaccine platform. full-length mers-n was cloned into two different additional transcription units (atus) either behind p (post p) or h (post h) cassettes of measles vaccine strain mv vac genome, and virus clones were successfully rescued and amplified in vero cells with titers of up to × tcid /ml. the essential verification of antigen expression by western blot analysis of vero cells infected with the mv vac -mers-n vaccines revealed expression of the n antigen with only little impact of the genomic position of the transgene cassette (fig. a) , while growth kinetics showed no impairment of virus replication compared to the respective mv vac -gfp(p) control virus ( fig. b, c) . to test the efficacy of the mv vac -mers-n candidate in vivo, genetically modified ifnar -/--cd ge mice were chosen, since they are the prime small animal model for analysis of mv-derived vaccines (mrkic et al., ) . thus, mice per group were inoculated via the intraperitoneal (i.p.) route on days and with each time × tcid of mv vac -mers-n(p), mv vac -mers-n(h), or empty control virus mv vac -atu(p). medium-inoculated mice served as negative controls. days or four days after boost immunization, sera or splenocytes of immunized mice were sampled, respectively (fig. d) . as expected, all mice immunized with recombinant mv (including the control virus) developed high mv virus neutralizing titers (vnt) ( - vnt, fig. f ). little evidence for induction of neutralizing antibodies against mers-cov was found in all mice, as expected for the intra-particular antigen (fig. e) . no vnts against mv or mers-cov were detected in control mice inoculated with medium alone. to analyze splenocytes of animals vaccinated with mv vac -mers-n (h) or control animals inoculated with medium or mv vac -atu(p) by elispot assay for antigen-specific ifn-γ secretion, the antigen-specific t cells were re-stimulated in vitro by syngeneic murine dc cell lines (jawsii and dc . ), which had been genetically modified by lentiviral vector transduction to stably express mers-n protein and thereby to present the respective t cell epitopes on mhc. single cell clones were derived by flow cytometric sorting of single gfp-positive cells. antigen expression by transduced dcs was verified by western blot analysis (data not shown). elispot assays using splenocytes of vaccinated animals in co-culture with jawsii-mers-n or dc . -mers-n revealed about ifn-γ secreting cells per × splenocytes after immunization with mv vac -mers-n(h) (fig. g) , which was significant over controls. additionally, cellular immune responses targeting mv antigens were detected upon stimulation with mv bulk antigens in vaccinated mice that had received any recombinant virus, as expected. however, mv bulk antigens stimulated about - ifn-γ secreting cells per × splenocytes of mv vaccinated animals, as described, before (malczyk et al., ) . finally, splenocytes of all mice revealed a similar basic reactivity to unspecific t cell stimulation, as confirmed by similar numbers of ifn-γ secreting cells upon cona treatment (fig. g) . thus, the generated mv-based vaccine platform expressing mers-n induces significant mers n-specific cellular immune responses, as desired. in any case, humoral and cellular responses induced by vaccine candidate mv vac -mers-s had been considerably higher in previous analyses under similar conditions (malczyk et al., ) . therefore, further characterization of anti-mers-cov immunity induced by mv vac -derived vaccines proceeded with this mers-s encoding vaccine candidate, which yielded approximately -fold higher numbers of reactive t cells after vaccination. since the mers vaccine candidate mv vac -mers-s(h) induced robust protective humoral and cellular immune responses in ifnar -/--cd ge mice (malczyk et al., ) , we were interested in the necessity of viral replication of this life-attenuated vaccine for the induction of mers cov-specific immunity. for these analyses ifnar -/--cd ge mice were chosen as the animal model, again, since these mice are the standard animal model for analysis of mv-derived vaccines (mühlebach, ) , their genetic composition is compatible with an established mers-cov challenge model , as shown, before (malczyk et al., ) , and their size allows housing under regularly available conditions opposed to dromedary camels, the only know natural host of mers-cov, to date. as all morbilliviruses, the mv-based vaccine virions are highly cellassociated, and transfer of antigenic protein within the vaccine preparation cannot be excluded. therefore, we vaccinated these mv-susceptible mice with either × tcid of live or of the same formulation and quantity uv-inactivated mv vac -mers-s(h) in a primeboost regimen ( fig. a) . mv vac -atu(p), which does not encode any additional antigen, was included as vector control. blood was drawn from naïve mice on day before vaccination, and on days and post-immunization. serum samples were tested for their ability to neutralize mv vac -gfp(p) (fig. b , d, f) or mers-cov (fig. c, e, g) . sera of naïve mice had no neutralizing antibodies against either virus (fig. b, c) . after the first immunization, both live virus preparations induced neutralizing antibodies against mv, with mv vac -atu(p) triggering significantly higher titers ( - vnt) than mv vac -mers-s(h) ( - vnt). after the second immunization, anti-mv vnts increased to titers of - in both cohorts. in contrast, only one out of four animals in the uv-inactivated vaccine group had a borderline neutralizing antibody titer of after the first immunization, and another animal had a titer of after the boost. while mv vac -atu(p) and the uv-inactivated mv vac -mers-s(h) vaccine did not induce neutralizing antibodies against mers-cov above background levels over the course of the experiment, the group vaccinated with live mv vac -mers-s(h) developed titers around after the first immunization and - (mean of ) after the boost. taken together, these data reveal that replication of the vaccine is necessary to induce functional antibody responses against mv and the additional antigen mers-s. to assess the capacity of the different mv vac -mers-s(h) vaccine preparations to induce mers-cov s-specific cellular immune responses, splenocytes of mice, which had already been tested for humoral responses ( fig. a) , were isolated and analyzed days after immunization for antigen(ag)-dependent ifn-γ secretion using elispot assay. the isolated splenocytes were re-stimulated with mers-s immunodominant peptide s (channappanavar et al., ) or mv bulk antigen (mv bulk) to analyze mv-specific cellular immune responses. ovalbumin-derived siinfekl-peptide (sin) served as peptide negative control, or cells were left untreated (mock). stimulation with concanavalin a (cona) was used to confirm general t cell reactivity in splenocyte preparations (fig. h) . while splenocytes of all mice responded to cona with to spots per × splenocytes, only those from animals vaccinated with live mv vac -mers-s(h) could be stimulated with mers s-specific peptide s reaching mean values of spots per × splenocytes. in contrast, splenocytes of the uv-inactivated group or control virus mv vac -atu(p) could not be restimulated to secrete ifn-γ. furthermore, only replication-competent vaccine viruses induced mv-specific cellular immune responses in vaccinated mice. re-stimulation with mv bulk ag induced a mean of and spots per × splenocytes for mv vac -mers-s(h) or mv vac -atu(p) vaccinated mice, respectively. consequently, replication of the vaccine candidate is essential to induce both arms of the immune system with responses against mv as well as the additional mers-s antigen. usually, - weeks old juvenile mice are used for our mers-cov neutralizing antibodies in sera of (b, c) naїve mice, or in sera of mice after (d, e) prime-or (f, g) boost-immunization. one-way anova with tukey multiple comparison. * : p < , ; * *: p < , ; ***: p < ; ****: p < , . (h) secretion of ifn-γ after antigen-specific re-stimulation of splenocytes. ifn-γ elispot analysis using splenocytes of mice vaccinated on days and with indicated vaccines isolated days after boost immunization and after incubation with indicated stimuli (mers-s peptide s , mv bulk antigen (mv bulk), immunodominant ovalbumin-derived siinf-ekl-peptide (sin) as a peptide negative control) or untreated (mock). the reactivity of splenocytes was confirmed by concanavalin a (cona) treatment ( μg/ml). the number of cells per × splenocytes represent the amount of cells expressing ifn-γ upon re-stimulation. dots represent individual animals, horizontal bars mean. one-way anova with tukey multiple comparison. ****: p < , . b.s. bodmer et al. virology ( ) - immunization studies. to evaluate if there is an age-dependent change in vaccination efficacy, approximately months-old mice were vaccinated with mv vac -mers-s(h) in a prime-boost vaccination scheme with weeks between prime and boost vaccination (fig. a ). mice were sacrificed at day post-immunization, and splenocytes were re-stimulated with mv-antigens or mers-s peptide s . we found that reactive ifn-γ-secreting t cells were also specifically induced in mice of this age (fig. b) . a mean of spots per × splenocytes was detected upon re-stimulation with mv bulk antigen, whereas spots per × splenocytes were induced by re-stimulation with the mers s-derived peptide s , illustrating that mv-and mers-cov-specific cellular immune responses are effectively induced in adult mice. to gain more detailed insights in the quality of the observed t cell responses, we further characterized the responsive t cell populations by flow cytometry, determining the expression of cd + and cd + surface markers as well as ifn-γ and tnf-α upon re-stimulation with s or mv bulk antigen. as a positive stimulus for t cell activation tetradecanoylphorbol-acetate and ionomycin (tpa/iono) were used. exocytosis of cytokines was blocked by addition of brefeldin a ( μg/ ml) during stimulation. cells were permeabilized, labelled, and fixed for flow cytometry. the gating strategy excluded duplicates (not shown), selected for living cells (fig. a, upper panel) , and separated cd + and cd + t cells (fig. a, lower panel) . selected cd + t cells were then analyzed for their expression of ifn-γ (fig. b left panel) , tnf-α (fig. b middle panel) , or both ( fig. b right panel) as exemplarily shown for splenocytes re-stimulated with mers-s peptide s . likewise, cd + t cells expressing ifn-γ (fig. c, left panel) , tnf-α (fig. c, middle panel) , or both (fig. c , right panel) are depicted after re-stimulation with mv bulk antigen. vaccination with mv vac -mers-s(h) induced a significant amount of mers s-specific cd + t cells expressing either ifn-γ (fig. d, left panel) or tnf-α (fig. d, middle panel) , with means of . % and . % of total positive cells, respectively. among those, a significant fraction of cells revealed to be multifunctional, with a mean of . % of all cd + cells or % of the tnf-α − responsive cells being positive for both cytokines (fig. d, right panel) . moreover, vaccination induced a significant fraction of vector-specific cd + t cells expressing ifn-γ (fig. e, left panel) , or tnf-α (fig. e , middle panel) upon re-stimulation with mv bulk antigen. among those, multifunctional cd + t cells expressing both cytokines were induced with a mean of about . % (fig. e, right panel) . to conclude, vaccination with mv vac -mers-s(h) induces not only ifn-γ or tnf-α expressing t cells directed against mers-cov and mv, but also a significant fraction of multifunctional cytotoxic t cells specific for mers-s and cd + t cells specific for mv antigens, illustrating that a broad and robust mers-covspecific immune response is induced by vaccination with mv vac -mers-s(h). in this study, we aimed to understand the induction of immunity and the functionality of induced t cell responses after vaccination with mv vac -mers-s(h), a vaccine candidate that induces protective immunity against mers-cov in an appropriate animal model. in parallel, we generated and tested also alternative mv-based vaccine candidates expressing mers-cov n protein as conserved t cell antigen. mv vac -mers-n vaccine candidates indeed induced significant antigen-specific cellular immune responses in vaccinated transgenic mice, revealing that also mers n-expression by recombinant mv may have a useful role to combat mers-cov. since the immune responses induced by mers-s expressing candidates had been nevertheless considerably higher, we proceeded with s-expressing vaccine virus to characterize the induction of anti-mers-cov immunity by mv-based vectors. using mv vac -mers-s(h), robust anti-mers cov immune responses were induced also in older mice, while replication of the vaccine vector was necessary to induce either arm of adaptive immunity against vector or pathogen. furthermore, vaccination with mv vac -mers-s(h) triggered significant numbers of multifunctional mers s-specific cd + t cells and mv-specific cd + t cells, simultaneously producing ifn-γ and tnf-α upon stimulation with respective antigens. since not only numbers, but also the quality of the induced mers cov-specific t cell responses might be relevant for protection against mers-cov, it is quite encouraging to see that approx. % of ifn-γ reactive cd + t cells also expressed tnf-α, whereas in reverse % of tnf-α-reactive cd + t cells co-expressed ifn-γ upon stimulation with the immune-dominant mers-s peptide. this induction of multifunctional t cells is quite in accordance with previous studies, since the potential of mv during natural infection or the recombinant mv to induce multifunctional, antigen-specific t cells has already been demonstrated. infection of macaques with wild-type mv induces polyfunctional t cells specific for mv proteins with increasing numbers of cells secreting il- , tnf-α, as well as ifn-γ over time (nelson et al., ) , and polyfunctional t cells directed against mv-h can be expanded from pbmc of human donors (ndhlovu et al., ) . likewise, hiv-vaccine candidates mv -f , which encode gag, rt, and nef of an hiv- clade b or a clade c strain as foreign antigen, induce antigenspecific multifunctional t cells simultaneously expressing ifn-γ, tnf-α, and il- in mice and also macaques (stebbings et al., (stebbings et al., , . while the combination of ifn-γ and tnf-α indicates functional t cells fig. ) were re-stimulated and subjected to intracellular staining (ics) for ifn-γ and tnf-α and stained for extracellular t-cell markers cd and cd for flow cytometry analysis. (a -c) gating strategy for analysis of cd + or cd + t-cells expressing ifn-γ or tnf-α within splenocytes stimulated with (b) s peptide or (c) mv-bulk ag. duplicates (not shown) and dead cells (a) were excluded from analysis. (b, c) cd + and cd + cells were separately subjected to analysis for ifn-γ-(left panels), tnf-α-(middle panels) or double-positive cells (right panels). quantification of flow cytometry data of (d) cd + -and (e) cd +positive cells after incubation with indicated stimuli (mers s-specific peptide s , mv bulk ag (mv bulk), immunodominant ovalbumin-derived siinf-ekl-peptide (sin) as a peptide negative control, or untreated cells (mock); reactivity of splenocytes was confirmed by tetradecanoylphorbol-acetate and ionomycin (tpa/iono) treatment ( μg/ml). dots represent individual animals, horizontal bars mean. repeated-measures one-way anova with tukey multiple comparison. *: p < , . with higher potency, in general, expression of il- is a sign of the induction of cd + memory t cells (williams et al., ) . in our study, the strong correlation of ifn-γ and tnf-α expression thus indicates a high functionality of induced t cell responses. extension of the antibody panel for il- detection could yield further insight into the durability of these t cell responses induced by the mv vaccine platform in future studies. such multifunctional cd + t cells specific for mers-cov may become especially important, since mouse studies have shown that cd + t cells are crucial for clearance of mers-cov infection (coleman et al., ; zhao et al., ) . noteworthy, for other viral infections such as human immunodeficiency virus (hiv), modified vaccinia virus ankara (mva), or cytomegalovirus (cmv) the amount of just ifn-γ producing t cells does not correlate with ctl killing effectivity, but the multifunctionality of antigen-specific t cells inversely correlated with viral load (betts et al., ; lichterfeld et al., ; precopio et al., ; sandberg et al., ) , further underlining the importance of multifunctionality. besides these cellular immune responses, also considerable humoral immunity was induced in vaccinated animals, here. the mean vnt was somewhat lower than expected (malczyk et al., ) , but still quite high. an alternative vaccine candidate derived from modified vaccinia virus ankara, mva-mers-s, revealed protection in dromedary camels, the natural host for mers-cov (haagmans et al., ) . passive immunotherapy with dromedary immune sera significantly reduced mers-cov titers in lung tissue of challenged mice, starting with a vnt of (zhao et al., ) . neutralizing antibody titers in reconvalescent plasma of human patients diagnosed with mers were determined by microneutralization tests in two previous studies, and were on average at (arabi et al., ) or . (zhao et al., ) . furthermore, a prnt titer of at least just was required to lower virus titers by more than . log in mice challenged after transfer of human reconvalescent plasma (zhao et al., ) . these titers were exceeded in this study. in addition, mv vac -mers-s(h) induced higher anti-mers-s titers in c /bl mice than mva-mers-s in balb/c mice, when comparing studies that used similar virus titers for vaccination (malczyk et al., ; volz et al., ) , thus indicating an at least comparable efficacy. thus, also vnt determined here indicate efficacy and were anyway not statistically different from those published before for mv vac -mers-s(h) (malczyk et al., ) . nevertheless, the exact correlates of protection for mers-cov remain to be determined in future studies, since it will be essential to evaluate the efficacy of the different vaccine candidates against this most important benchmark. in contrast, uv-inactivated mv vac -mers-s(h) did not induce any antibodies able to neutralize mers-cov or mv. while neutralizing antibodies can in principle also be induced by inactivated vaccines or proteins, e.g. full-length or truncated mers-s protein in combination with adjuvant (wang et al., ) . obviously, the amount of mers-s antigen within the mv vac -mers-s(h) vaccine formulation or the adjuvant effect of the inactivate were not sufficient during application. therefore, replication of the mv-derived mers vaccine candidate is necessary for the induction of immune responses both against vector and antigen of interest in vaccinated animals. indeed, the induction of cellular immunity is usually more efficient by de novo expression of antigen after immunization. consequently, the application of a replication competent vaccine platform is justified here to robustly induce potent responses of both arms of the adaptive immune system. these powerful immune responses were not only induced in juvenile mice - weeks of age, but also in adult mice older than half a year of age. this is quite of interest, since adult vaccinees are also the target group for vaccination in response to the mers-cov outbreak, as defined in the target product profile by the who (http://www.who.int/ blueprint/what/research-development/mers_cov_tpp_ .pdf). remarkably, mv vaccine strain virus encoding chikungunya virus (chikv) antigens was already tested in a phase i clinical trial in adult human vaccinees ( - years old) (ramsauer et al., ) . these adult test subjects all developed significant humoral immunity against chikv, despite their adult age and most interestingly also independent from measles pre-immunity. taken together, these study shows that mv vac -mers-s(h) induces surprisingly high numbers of multifunctional t cells specific for mers-s also in adult test subjects, as a result from replication of the recombinant vector. therefore, high quality cellular immune responses are induced in addition to the robust antibody responses by this vaccine candidate, further qualifying mv vac -mers-s(h) for evaluation as vaccine candidate against mers-cov. in parallel, mers-n encoding mv can be a further option to generate protection against mers in future studies and constructs. evaluation of serologic and antigenic relationships between middle eastern respiratory syndrome coronavirus and other coronaviruses to develop vaccine platforms for the rapid response to emerging coronaviruses middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia (e - ) risk factors for middle east respiratory syndrome coronavirus infection among healthcare personnel feasibility of using convalescent plasma immunotherapy for mers-cov infection, saudi arabia hiv nonprogressors preferentially maintain highly functional hiv-specific cd + t cells t cell-mediated immune response to respiratory coronaviruses cd + t cells and macrophages regulate pathogenesis in a mouse model of middle east respiratory syndrome a vectored measles virus induces hepatitis b surface 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effector molecules in human cd + t lymphocytes cloned dendritic cells can present exogenous antigens on both mhc class i and class ii molecules immunogenicity of a recombinant measles-hiv- clade b candidate vaccine immunogenicity of a recombinant measles hiv- subtype c vaccine protective efficacy of recombinant modified vaccinia virus ankara delivering middle east respiratory syndrome coronavirus spike glycoprotein evaluation of candidate vaccine approaches for mers-cov interleukin- signals during priming are required for secondary expansion of cd + memory t cells isolation of a novel coronavirus from a man with pneumonia in saudi arabia recovery from the middle east respiratory syndrome is associated with antibody and t-cell responses rapid generation of a mouse model for middle east respiratory syndrome passive immunotherapy with dromedary immune serum in an experimental animal model for middle east respiratory syndrome coronavirus infection this work was supported by the german center for infection research (dzif; ttu . ). the authors would like to thank vivian scheuplein, jürgen schnotz, and daniela müller for excellent technical assistance. the authors are indebted to ron fouchier for providing mers-cov strain emc/ , kenneth rock for dc . cells, roberto cattaneo for providing the pb(+)mvvac construct, and urs schneider for providing the polii rescue system originally used to generate and to rescue recombinant mv vectors. the authors would further like to thank bakhos tannous for providing pcscw gluc-ires-gfp. moreover, the authors would like to thank veronika von messling for critically reading the manuscript. key: cord- -cdd ngf authors: narkpuk, jaraspim; jongkaewwattana, anan; teeravechyan, samaporn title: the avian influenza virus pa segment mediates strain-specific antagonism of bst- /tetherin date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: cdd ngf bst- is an antiviral protein described as a powerful cross-species transmission barrier for simian immunodeficiency viruses. influenza viruses appear to interact with bst- , raising the possibility that bst- may be a barrier for cross-species transmission. an mdck-based cell line expressing human bst- was generated to study human-derived a/puerto rico/ / (h n ; pr ) as well as two low pathogenic avian influenza viruses (subtypes h n and h n ). the h n and h n viruses were less affected by bst- expression than pr , due to their ability to decrease bst- levels, a function localized to the pa segment of both avian viruses. experiments with pa-mutant and -chimeric viruses confirmed that the avian pa segment conferred bst- downregulation and antagonism. these results indicate a species-specific ability of pa from low pathogenic avian viruses to mitigate human bst- antiviral activity, suggesting that bst- is unlikely to be a general cross-species barrier to transmission of such viruses to humans. bst- (bone marrow stromal cell antigen ), also known as tetherin, cd or hm . , is an interferon (ifn)-induced cellular protein that was initially described in as a restriction factor for human immunodeficiency virus type (hiv- ) (neil et al., ; van damme et al., ) . while bst- has been almost exclusively studied as a mammalian antiviral protein, an in silico study identified a bst- ortholog as far back in vertebrate evolution as the elephant shark, dating the appearance of this gene to over million years ago (heusinger et al., ) . other than in fish, this study also identified orthologs in marsupials, reptiles, and birds, with alligator bst- being tested and found to possess antiviral function against hiv- release. among birds, bst- was found in turkeys and chickens, but appears to have been lost through gene erosion among many bird species. as a type ii membrane protein, bst- possesses a c-terminal gpi (glycophosphatidylinositol) modification and an n-terminal transmembrane domain flanking an extracellular coiled coil central region, and is present on the cell surface as a homodimer (kupzig et al., ) . the resulting four-membrane-anchor conformation is considered central to the ability of bst- to restrict hiv- virion release, where it acts as a "tether" linking the membranes of budded virions to the host cell membrane (perez-caballero et al., ) . as bst- acts directly upon the host cell membrane rather than viral components, bst- does not target a specific virus but rather has been found capable of restricting virion release and spread for a range of enveloped viruses beyond the retroviruses, such as filoviruses (jouvenet et al., ; kaletsky et al., ; sakuma et al., ) , arenaviruses (radoshitzky et al., ; sakuma et al., ) , and various coronaviruses (taylor et al., ; wang et al., ) . many of the viruses described to be sensitive to bst- restriction are zoonotic. most notably, bst- has been described as a cross-species transmission barrier that shaped the evolution of the simian immunodeficiency virus (siv) and hiv (evans et al., ) . a recent publication also demonstrated the possible role of bst- as a crossspecies transmission barrier for various orthobunyaviruses (varela ), with human viruses being restricted by sheep bst- but not the human ortholog, and vice versa. also, equine bst- was observed to restrict the growth of both equine and human influenza viruses more effectively than human bst- (wang et al., ) . early reports examining the interplay between influenza viruses and bst- suggested that virus-like particles (vlps) but not wild-type viruses were susceptible to human bst- restriction (watanabe et al., , bruce et al., . these observations lent credence to the possibility that influenza viruses universally encode an antagonist to bst- . contradictory reports soon emerged, however, of viruses inherently sensitive to bst- restriction (gnirss et al., ; hu et al., ; mangeat et al., ) . differential abilities of various influenza virus neuraminidases (na) in circumventing bst- activity (leyva-grado et al., ; yondola et al., ) made it apparent that influenza virus sensitivity to bst- is likely to be strain-specific. further studies supported the possibility that influenza virus na acts a strainspecific antagonist to bst- (leyva-grado et al., ; mangeat et al., ) . mangeat et al. also reported a reduction in bst- protein expression, which may be associated with hemagglutinin (ha) and na together (gnirss et al., ) or the m protein (hu et al., ) , but their observations of decreased bst- mrna levels during influenza virus infection remained unexplained. given the variety of influenza strains, host cells, and methodologies used to study the bst- -influenza interplay, the conflicting data, at the very least, appear to suggest that a variety of influenza virus strains interact with and counteract bst- in some fashion. given that influenza is and remains an important zoonotic disease, the possibility of bst- being a host restriction factor that acts as a cross-species transmission barrier for avian influenza viruses is intriguing. thus far, however, studies examining the intersection between bst- and influenza viruses have mostly focused on human viruses, whether laboratory-adapted, seasonal, or pandemic. these viruses have been successful in maintaining themselves in the human population, suggesting that they possess an inherent capacity to circumvent or antagonize the antiviral activity of bst- . therefore, we were interested in comparing human and low pathogenic avian influenza virus strains, which are generally not deemed a direct threat to human health, with the goal of identifying differences in their response to bst- . to study the impact of bst- on human and avian influenza viruses, we first generated an mdck cell line stably expressing human bst- cloned from hela cells (narkpuk et al., ) (supplementary fig. ). the mdck-bst- cell line generated by lentiviral transduction was examined by immunofluorescence staining and bst- was observed as punctate clusters on cell surface membranes (fig. a ), in accordance with expected bst- localization. cells were also probed for bst- expression by western blotting and revealed the characteristic pattern of monomeric and dimeric bst- in its various glycosylated forms (fig. b) . to test for strain-specific differences in sensitivity to human bst- , we compared the low pathogenic avian viruses a/duck/hong kong/ / (h n ) and a/duck/suphanburi/ai / (h n ) against a/puerto rico/ / (h n ) (pr for short). these viruses were used to infect mdck and mdck-bst- cells at a low multiplicity of infection (moi), and their supernatants were harvested at h post-infection for plaque titration on mdck cells. as low pathogenic avian viruses have not been reported to cause disease in humans, we expected that the h n and h n viruses would be more susceptible to human bst- antiviral activity than pr . surprisingly, the pr virus exhibited greater sensitivity to bst- , with titers decreasing by around log in mdck-bst- cells ( fig. a) . neither the h n nor h n virus appeared to be negatively affected by the presence of bst- , with h n showing a slight trend towards increased replication in mdck-bst- cells (fig. b, c) . when infected cells were probed to confirm bst- expression, western blotting revealed a dramatic disappearance of bst- protein in mdck-bst- cells infected by h n and h n viruses (fig. d ). similar levels of bst- protein down-regulation were not observed with pr infection prior to complete cell death. analysis of bst- mrna demonstrated that this reduction was accompanied by reduced mrna levels as well (fig. e ). pr infection also resulted in decreased bst- mrna levels, albeit not as drastically as infection with the avian viruses. while this result reflects the ability of influenza a viruses in general to mediate host shut-off (rivas et al., ) , it also suggests a difference in mrna down-regulation that may be associated with decreased levels of bst- . alternatively, it is also possible that mrna and protein down-regulation effects occur through distinct mechanisms. these avian viruses should not have been under selection pressure to develop a specific antagonistic mechanism against human bst- , due to the nature of their host range. these observations therefore pointed to a general host response inhibition factor present in the virus. the influenza virus nonstructural protein (ns ), in particular, is known for its ability to inhibit an ifn-induced antiviral state through a variety of activities such as blocking host sensors of viral rna and signal transduction pathways leading to induction of ifn expression (marc, ) . as bst- expression in our cell line is driven by a constitutive promoter, however, we do not expect that these particular activities will be involved in bst- down-regulation. to determine which viral factors of h n and h n are involved in decreasing bst- levels, we studied the impact of each of their genomic segments on bst- expression in the context of transfection. we amplified each segment from viral rna for cloning into the reverse genetics vector phw . individual genomic segments of pr , h n , and h n were then co-transfected with a bst- expression plasmid into hek t cells for western blot analysis. while none of the pr genomic segments affected bst- expression (fig. a) , the pa of h n and h n notably reduced bst- protein levels ( fig. b, c) . additionally, the h n ns segment also exhibited the capacity to reduce protein expression, which is likely due to ns -associated host shut-off capabilities (marc, ) . as the effect of the pa segment appeared common to both low pathogenic avian viruses, we further tested the effect of pa on bst- among all the pa genes tested, the low pathogenic avian influenza viruses h n and h n resulted in the most significant decrease in bst- protein levels ( fig. d , supplementary fig. a ). the pandemic h n strain (non), which has an avian-derived pa, along with the highly pathogenic h n pa, had a noticeable, albeit very mild, effect on bst- levels. the pr and h n viruses had little observable impact on bst- levels. pa protein levels roughly corresponded to their effect on bst- levels; h n and h n had the greatest amounts of pa protein, followed by the highly pathogenic h n and the pandemic h n . pr and h n pa levels were notably lower, likely due to the presence of bst- itself, which has been previously shown to decrease expression of co-transfected genes (narkpuk et al., ) . indeed, in the absence of bst- , pa of pr and the h n and h n viruses were expressed at higher levels, whereas h n and h n pa were expressed at lower levels (fig. e ). these results suggest that the h n and h n pa-mediated down-regulation of bst- may pre-empt bst- activity, preventing bst- from decreasing pa expression. the main product of the pa segment is the pa protein, which functions as an endonuclease (fodor et al., ; hara et al., ) . interestingly, this segment also encodes an alternative product, pa-x, containing the n-terminal endonuclease domain of pa fused to a frameshifted c-terminal tail (jagger et al., ) . pa-x has been demonstrated to actively target host rna polymerase ii-derived transcripts for cleavage (khaperskyy et al., ) and is thought to play a role in decreasing host competition for resources as well as down-regulating host responses (hayashi et al., ; jagger et al., ) . in addition, it displays host-and strain-specific impact on influenza virus pathogenicity and transmissibility (hu et al., ) . to dissect the roles of both pa segment products in mediating bst- down-regulation, we mutated the pa segment based on work by jagger et al., which had identified the frameshift motif (ucc uuu cgu c; fig. a ) required for pa-x expression as well as narrowed down the essential residues to positions that resulted in under . % frameshifting efficiency when mutated (ucc uuu cgu c to ucc uuc aga c) (jagger et al., ) . they also generated a single c nucleotide deletion mutant (ucc uuu cgu c to ucc uuu -gu c) which shifted the pa-x orf (open reading frame) into a position in-frame with the start codon, resulting in expression of pa-x but not pa. we emulated their mutagenesis approach for pr and the h n virus ( fig. b ), mutating the frameshift motif to either reduce frameshifting into the pa-x orf (fs) or to force expression solely in the pa-x orf (Δc), and assessed which orf is responsible for the observed decreases in bst- levels. wild-type or mutant pa was co-transfected with a bst- expression plasmid into hek t cells, and cell lysates were harvested for western blot analysis (fig. c, supplementary fig. b ). as seen previously, the h n wild-type pa decreased bst- levels to a much greater extent than the pr wild-type pa. the h n Δc pa, which is frameshifted to express only the pa-x orf, resulted in undetectable levels of bst- while the fs pa lost its activity. these results indicate that the effect of the h n pa segment on bst- levels may be attributed to the pa-x orf. similarly, the pr Δc mutant decreased bst- levels while the fs mutant had no effect, further demonstrating that the pr pa-x orf also has the ability to decrease bst- protein levels in transfected cells. however, because pr wild-type and Δc pa both exhibit less down-regulation of bst- protein compared to its h n counterparts, we surmise that pr pa-x expression levels or activity may be lower. since our observations demonstrated impact of the avian pa segment on bst- down-regulation in the context of transfection, we were interested in examining whether or not such impact translated to viral growth in the context of infection. first, we generated chimeric pr and h n viruses where the pa, ns or both pa and ns segments were swapped, as well as pr and h n viruses carrying the fs mutation in their pa segments. mdck-bst- cells were infected with these viruses at an moi of . , and cell lysates were assessed for bst- and viral np (nucleoprotein) expression (fig. a, supplementary fig. c ). the pr -based chimeras carrying h n ns and/or wild-type pa were capable of decreasing bst- levels, with the strongest impact being seen with the wild-type pa segment alone. the pr viruses with fs pa, either its own or that of h n , did not exhibit bst- down-regulation. on the other hand, the h n chimeric viruses carrying either the pr wild-type pa or both pr ns and wild-type pa completely lost their ability to decrease bst- levels. the h n fs mutant lost most of their activity, while the virus carrying pr ns lost some activity but still yielded noticeably decreased bst- levels. these results suggest that h n pa plays the primary role in down-regulating bst- , with some contribution from its ns segment. interestingly, the presence of both h n ns and wild-type pa in pr did not result in an additive effect on bst- levels, but rather resulting in a weaker effect on bst- than h n wild-type pa alone. this suggests that viral context also plays a role in how pa and ns manifest their bst- antagonism. to examine the role of the pa segment further, we infected mdck-bst- cells at an moi of and incubated the cells for h in the absence of tpck-treated trypsin to limit the viruses to a single round of growth. this would allow us to examine the impact of viral infection on bst- levels without being confounded by differences in viral spread due to variable bst- sensitivity. western blotting results reflect our transfection observations, although to a less dramatic extent (fig. b , supplementary fig. d) , likely due to the single-round nature of these infections. the h n virus resulted in the lowest bst- levels, followed by pr (h n pa), the pr virus carrying the h n pa segment. bst- mrna levels were also examined (fig. c) . loss of frameshifting into the pa-x orf resulted in notable losses in the ability to down-regulate bst- mrna, strongly suggesting a role for pa-x in controlling bst- transcription. however, swapping the pa segment as a whole did not affect bst- mrna levels significantly. considering the impact of the pa segment on bst- protein levels ( fig. a and b ), this appears to indicate a distinct role for the pa segment as a whole in bst- protein down-regulation. to assess the role of the pa segment in rescuing viral replication in the presence of bst- , we assessed the growth kinetics of pr (h n pa) and h n (pr pa) alongside the wild-type viruses (fig. d-g) . mdck or mdck-bst- cells were infected and their supernatants were harvested at various time points for plaque titration on mdck cells. the growth kinetics data for the wild-type viruses reflect those observed previously, with pr exhibiting sensitivity to bst- while h n replicated similarly in both mdck and mdck-bst- cells. with the chimeric viruses, the h n pa was able to confer bst- resistance to pr , while h n (pr pa) was rendered sensitive to bst- . all together, our data suggest that the pa segment carries the determinants for bst- resistance in our study system, with the avian h n pa antagonizing bst- by down-regulating its expression at both mrna and protein levels. several studies have examined the impact of bst- on influenza virus replication, and a handful of viral proteins have been proposed as possible antagonists of human bst- . given the zoonotic nature of influenza viruses, we were interested in exploring this question from the perspective of human bst- acting as a cross-species transmission barrier for avian influenza viruses. we report here that, contrary to our initial hypothesis that low pathogenic avian influenza viruses incapable of transmission to human hosts would be strongly restricted by human bst- , h n and h n viruses grew to robust titers in mdck cells constitutively expressing bst- and appeared completely resistant to its antiviral activity. mechanistically, our data suggest that this resistance is likely conferred by the drastic down-regulation of bst- levels by the avian pa segment through the reported ability of pa-x to mediate general host shut-off (jagger et al., ) . before discussing the implications of our pa-associated findings, it is important to note that the h n ns segment also exhibited the ability to decrease bst- protein levels. unlike pr , this h n virus contains the f and m residues in its ns protein that are associated with host cpsf ( -kda subunit of the cleavage and polyadenylation specificity factor) interaction and ns -mediated host-shut-off (das et al., ) . cpsf interaction may not be directly related to bst- down-regulation or the sole contributor to this effect, however, as the h n virus also carries this motif despite having an ns segment that did not decrease bst- protein levels. the h n ns protein is divergent from h n ns at five positions, carrying k , i , i , a , and s instead of r , t , n , v , and f . interestingly, three of these distinct h n ns residues are shared with pr , namely those at positions , , and . residue , in particular, is located in the sh binding motif that is involved in interaction with the pi k (phosphatidylinositol -kinase) p β subunit (shin et al., ) . while we did not extensively examine the role of ns nor these residues in the down-regulation of bst- , ns of a/wsn/ (h n ) and a/texas/ / were previously reported to prevent the induction of bst- upon ifn-α treatment (mangeat et al., ) . this effect is not evident in most influenza virus-bst- studies which have focused on engineered cell lines constitutively expressing bst- (bruce et al., ; winkler et al., ) . even so, our data revealed a strain-specific ability of ns to decrease bst- levels during infection in such a cell line, supporting the diversity of ns functionality in modulating the host innate immune response beyond its role in ifn suppression. the other products of the ns segment, nep (nuclear export protein) and ns are unlikely to be associated with the observed impact of h n ns on bst- , however. the ns splicing motif, an a to g mutation at nucleotide position (selman et al., ) , is not present in any of the three viruses, while the nep protein sequence is perfectly conserved between the h n and h n viruses. additionally, while we focused primarily on the pa segment, our results do not rule out the possibility that other viral proteins encoded on two or more segments may work in concert in order to effect bst- decrease, as previously seen with na and ha (gnirss et al., ; hu et al., ) . neither do these results discount the presence of other anti-bst- mechanisms involved in sequestering or altering bst- localization, which would thereby decrease the impact of bst- on influenza virus replication through means other than direct down-regulation of bst- expression as assessed by mrna and protein levels. early on during this work, we hypothesized that pa-x, first described in by jagger et al. ( ) as a frameshift product of the pa gene with endonuclease and host shut-off activity, might be responsible for bst- down-regulation as both protein and mrna levels were affected. indeed, in the transfection studies, the pa-x orf appears to be strongly involved in mediating the decrease in bst- expression levels. this effect was likely due to endonuclease-mediated cleavage of rna, preventing protein translation. likewise, decreases in both bst- mrna and protein were observed in infected cells. suppression of bst- mrna during infection that has previously been reported (mangeat et al., ) could likely also be explained by the effect of the pa segment as well. however, the interpretation of pa and pa-x orf involvement in bst- down-regulation changes slightly in the context of infection, as bst- is pre-expressed in the mdck-bst- cell line. the ability of h n and h n infection to reduce both bst- mrna and protein levels suggest that there is either post-translational down-regulation or extremely rapid turnover of bst- , or both. if the former effect is indeed the case, it would be interesting to map out the mechanism by which pa-x may have exerted such a dramatic effect on protein stability. nevertheless, our infection studies (figs. and ) show a distinct difference between bst- mrna and protein levels in pr -infected cells, where mrna levels are decreased but protein levels are not. this suggests that the endonuclease and protein down-regulation activities may be distinct. alternatively, the impact of mrna degradation may be amplified at a certain threshold due to high bst- turnover. in our transfection experiments with mutant pa, we found that the h n wild-type pa exhibited a bst- restriction phenotype similar to the Δc pa mutant which expresses only the pa-x orf, whereas the pr wild-type pa phenotype was reflected in the fs pa mutant. these observations suggest that the pa-x orf may be fundamentally different between the two viruses. it is possible that the h n pa-x protein is expressed at very high levels from the wild-type pa gene, similar to that of the Δc mutant. this in turn implies that expression of the full-length pa protein itself may suffer as a result, due to higher rates of ribosomal frameshifting during translation. in other words, h n and pr pa may differ in frameshifting rates as determined by factors outside the identified, highly-conserved frameshift motif. alternatively, activity of their pa-x may be different despite neither having a truncated pa-x associated with changes in protein activity (bavagnoli et al., ; gao et al., ) . the significance of the pa-x orf was further highlighted in our attempts to rescue the h n virus. this virus was not used in the later viral mutagenesis studies because we could not rescue it through reverse genetics in mdck cells. intriguingly, we were able to successfully rescue one virus: the h n fs mutant carrying the frameshift motif mutation in the pa gene, shown previously to decrease pa-x expression (jagger et al., ) . that this mutation was able to render the recombinant h n virus rescuable in cell culture suggests a role for the pa-x orf in modulating viral growth. indeed, pa-x has been described to suppress viral polymerase activity in certain contexts (gong et al., ; hu et al., ) , and its loss was accordingly associated with increased viral replication in both tissue culture and infected animals. it is tempting to speculate that this shifting of balance between polymerase activity and host shut-off may play some role in host tropism and cross-species transmission. a recent publication by hu et al. describes a very similar study to the work we have reported here (hu et al., ) . from the observation that infection by a/wsn/ (h n ) reduced bst- levels, they assessed the major viral proteins individually for their effect on bst- expression and identified m as responsible for bst- down-regulation. while we did not specifically separate m and m expression in our study, the m segments of pr , h n , and h n as a whole did not confer bst- down-regulation. similarly, bruce et al. tested m -deficient pr and found that loss of m did not notably affect alter its sensitivity to bst- (bruce et al., ) . on the other hand, hu et al. did not observe any impact of pa on bst- levels; this indicates that a/ wsn/ pa may be similar to that of pr , whose pa also lacks the activity seen with h n and h n viruses. these disparate, seemingly contradictory results actually point towards a single conclusion-that influenza a resistance to and antagonism of bst- is strain-specific, and that the role of the pa segment may possibly be associated with low pathogenic avian strains. overall, our data further elaborate upon the interaction between the antiviral protein bst- and influenza viruses. we identified the pa segment as a species-specific determinant of sensitivity to bst- , with those of low pathogenic avian influenza viruses exhibiting the ability to down-regulate human bst- in both transfection and infection contexts. that such drastic impact was seen even with high levels of preexpressed protein that cannot be controlled pre-translationally by ns suggests that bst- does not necessarily act as a cross-species barrier for transmission of low pathogenic avian influenza viruses to humans. furthermore, as these effects were surprisingly not observed to the same extent with tested human and human-derived viruses, we hypothesize that the avian pa segment may exhibit a different pa-pa-x balance that would translate into a disproportionately large impact on balancing host-shut off with viral replication compared to human viruses. human embryonic kidney (hek) t and madin-darby canine kidney (mdck) cells were maintained in opti-mem (life technologies) supplemented with % fetal bovine serum (fbs). cells were passaged twice weekly by detaching with . % trypsin, and kept in a °c incubator at % co . influenza viruses, and their proteins, studied in this work comprise a/puerto rico/ / (h n ) (pr ) and a/duck/hong kong/ / (h n ), gifts from dr. robert g. webster, st. jude children's research hospital, tennessee, usa (hoffmann et al., ) the human bst- coding sequence was amplified from phw-bst- (narkpuk et al., ) with primers carrying the bamhi and noti restriction sites for cloning into the lentiviral vector psin-csgw-ubem (a gift from dr. yasuhiro ikeda, mayo clinic, minnesota, usa). hek t cells were transfected with the lentiviral vector along with vsv-g-expressing pmd-g (naldini et al., ) and the packaging plasmid pcmv-Δr . (zufferey et al., ) , and supernatants containing lentiviral particles were harvested h post-transfection. the supernatants were clarified by centrifugation and used to infect mdck cells seeded in a -mm dish. polybrene (sigma-aldrich) was added to the infectious supernatant at a concentration of μg/ml. single clones of transduced mdck cells were prepared and expression of bst- by these clones (mdck-bst- ) was verified by western blotting. mdck-bst- cells were plated at a density of × cells in a lab-tek ii chamber slide (thermo scientific) and incubated overnight. cells were washed with phosphate-buffered saline (pbs) and fixed with % formaldehyde for min. after washing with pbs, cells were blocked with blocking buffer (pbs supplemented with % fbs and % bsa) for one hour at room temperature, probed with a : dilution of a rabbit anti-bst- antibody (santa cruz biotechnology) in blocking buffer for one hour, washed in pbs, and incubated with a : dilution of alexa- -conjugated goat anti-rabbit igg (abcam) for another hour. after a final washing step with pbs, cells were stained with dapi and imaged by fluorescence microscopy. viral rna from the a/duck/hong kong/ / (h n ) and a/ duck/suphanburi/ai / (h n ) viruses were extracted using the viral nucleic acid extraction ii kit (geneaid). the rna was used as a template for rt-pcr using takara's one step rt-pcr kit (clontech) along with primers containing the bsmbi restriction sites and specific for the ′ and ′ untranslated regions of each genomic rna segment (hoffmann et al., ) . amplified cdna products were digested with bsmbi and inserted into the phw vector (hoffmann et al., ) . selected clones were sequenced and compared to the official sequence reports on genbank. any nucleotide position that did not match sequence data on genbank was corrected by site-directed mutagenesis. pa mutants were generated following the mutagenesis approach reported by jagger et al. ( ) . the Δc mutants were generated with primers covering the frameshift motif with a single c nucleotide deletion. the fs (frameshift) mutants were generated with primers covering the frameshift motif with mutations of residues - from ucgu to caga. six-well plates were seeded with co-cultures of . × hek t cells and . × mdck cells overnight prior to transfection with eight phw -based plasmids encoding the eight influenza virus genomic segments ( ng each). twenty-four hours after transfection, the cells were washed and replenished with serum-free opti-mem supplemented with μg/ml tpck-treated trypsin. reverse genetics supernatants were harvested once extensive cell death was observed (typically h post-transfection) and clarified by centrifugation at ×g prior to injection into -day old embryonated chicken eggs. the eggs were incubated at °c for h before the allantoic fluid was collected for virus. allantoic fluid was clarified by centrifugation at ×g and sterilized by passage through a . µm syringe filter before storage at − °c. to verify the sequence of these reverse genetics-derived viruses, rna from the viruses were extracted using the viral nucleic acid extraction ii kit. rt-pcr was performed using the superscript iii one- step rt-pcr system (invitrogen) and the products were then submitted for sequencing ( st base, malaysia). to study growth kinetics, mdck or mdck-bst- cells were plated at a density of × cells per well in -well plates overnight and infected with virus at an moi of . . after an hour of virus adsorption at °c, the cells were washed with pbs and replenished with serumfree opti-mem supplemented with μg/ml tpck-treated trypsin. supernatant samples were collected at various times points for plaque titration. to assess the impact of chimeric and mutant viruses on bst- expression, an moi of was used for infection and cells were incubated in the absence of tcpk-treated trypsin for h prior to harvest. mdck cells were plated at a density of × cells per well in well plates and incubated overnight. influenza viruses from cell culture supernatants were prepared as serial -fold dilutions and appropriate dilutions added to seeded cells. cells were incubated at °c for h and then washed with pbs before being overlaid with agar in minimum essential medium (mem) supplemented with μg/ml tpck-treated trypsin. after another - h at °c, the agar was removed and the cells fixed with . % crystal violet in formaldehyde. viral titers were calculated by counting the number of plaques in the well containing - plaques and then multiplying that number by the dilution factor. log transformation was applied to viral titer readouts prior to assessment of statistical significance with student's two-tailed t-test. infected or transfected cells were lysed in radioimmunoprecipitation (ripa) buffer ( mm tris-cl ph . , mm nacl, % triton x- , . % sodium deoxycholate, % sodium dodecyl sulfate) supplemented with % proteoblock protease inhibitor cocktail (thermo scientific)). lysates were loaded into or % polyacrylamide gels for sds-page, and subsequently transferred onto nitrocellulose membranes. membranes were blocked with % skim milk in . % tween -tris-buffered saline (tbs) for at least one hour before probing with antibody. human bst- was detected with a rabbit anti-human bst- antibody (santa cruz biotechnology), influenza virus pa with a rabbit anti-pa antibody (thermo scientific), and β-actin with a mouse anti-β-actin antibody (santa cruz biotechnology). secondary antibodies (horseradish peroxidase-conjugated goat anti-rabbit igg and goat anti-mouse igg antibodies) were purchased from santa cruz biotechnology. bands were visualized using the clarity western ecl substrate chemiluminescence kit (bio-rad) by film exposure. films were scanned using the molecular imager chemidoc xrs+ imaging system (bio-rad) and analyzed by densitometry using imagej. infected mdck and mdck-bst- cells were harvested in pbs at various time points. half the harvested cells were used for genomic dna extraction using the genejet genomic dna purification kit (thermo scientific), and the remaining half used for rna extraction using the genejet rna purification kit (thermo scientific). extractions were carried out according to manufacturer instructions. quantitative pcr (qpcr) was performed on the genomic dna to assess relative fibronectin copy number using the ssoadvanced universal sybr green supermix (bio-rad) and the two-step qpcr protocol according to manufacturer instructions. rt-qpcr was used to assess mrna levels of the human bst- transgene using the itaq universal sybr green one- step kit (bio-rad) and the associated two-step protocol according to manufacturer instructions. the bio-rad c touch thermal cycler was used to run these qpcr experiments. statistical significance was assessed with student's two-tailed t-test. the novel influenza a virus protein pa-x and its naturally deleted variant show different enzymatic properties in comparison to the viral endonuclease pa release of filamentous and spherical influenza a virus is not restricted by tetherin structural basis for suppression of a host antiviral response by influenza a virus bst- /tetherin: a new component of the innate immune response to enveloped viruses a single amino acid mutation in the pa subunit of the influenza virus rna polymerase inhibits endonucleolytic cleavage of capped rnas twenty amino acids at the c-terminus of pa-x are associated with increased influenza a virus replication and pathogenicity tetherin sensitivity of influenza a viruses is strain specific: role of hemagglutinin and neuraminidase pa-x protein decreases replication and pathogenicity of swine influenza virus in cultured cells and mouse models amino acid residues in the nterminal region of the pa subunit of influenza a virus rna polymerase play a critical role in protein stability, endonuclease activity, cap binding, and virion rna promoter binding influenza a virus protein pa-x contributes to viral growth and suppression of the host antiviral and immune responses early vertebrate evolution of the host restriction factor tetherin a dna transfection system for generation of influenza a virus from eight plasmids universal primer set for the full-length amplification of all influenza a viruses pa-x: a key regulator of influenza a virus pathogenicity and host immune responses pa-x decreases the pathogenicity of highly pathogenic h n influenza a virus in avian species by inhibiting virus replication and host response bst- restricts iav release and is countered by the viral m protein an overlapping protein-coding region in influenza a virus segment modulates the host response broad-spectrum inhibition of retroviral and filoviral particle release by tetherin tetherin-mediated restriction of filovirus budding is antagonized by the ebola glycoprotein selective degradation of host rna polymerase ii transcripts by influenza a virus pa-x host shutoff protein bst- / hm . is a raft-associated apical membrane protein with an unusual topology modulation of an ectodomain motif in the influenza a virus neuraminidase alters tetherin sensitivity and results in virus attenuation in vivo influenza virus partially counteracts restriction imposed by tetherin/bst- influenza virus non-structural protein ns : interferon antagonism and beyond in vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector an unconventional bst- function: down-regulation of transient protein expression tetherin inhibits retrovirus release and is antagonized by hiv- vpu tetherin inhibits hiv- release by directly tethering virions to cells infectious lassa virus, but not filoviruses, is restricted by bst- / tetherin shutoff of host gene expression in influenza a virus and herpesviruses: similar mechanisms and common themes inhibition of lassa and marburg virus production by tetherin adaptive mutation in influenza a virus non-structural gene is linked to host switching and induces a novel protein by alternative splicing sh binding motif in influenza a virus ns protein is essential for pi k/akt signaling pathway activation severe acute respiratory syndrome coronavirus orf a inhibits bone marrow stromal antigen virion tethering through a novel mechanism of glycosylation interference the interferon-induced protein bst- restricts hiv- release and is downregulated from the cell surface by the viral vpu protein strain-specific antagonism of the human h n influenza a virus against equine tetherin bst /cd counteracts human coronavirus e productive infection by tethering virions at the cell surface influenza a virus does not encode a tetherin antagonist with vpu-like activity and induces ifn-dependent tetherin expression in infected cells budding capability of the influenza virus neuraminidase can be modulated by tetherin multiply attenuated lentiviral vector achieves efficient gene delivery in vivo we thank the members of the virology and cell technology laboratory for their insightful comments and critique. this work was funded by the young fellow grant from the national center for genetic engineering and biotechnology (biotec), thailand. the authors declare no conflict of interest. supplementary data associated with this article can be found in the online version at doi: . /j.virol. . . . key: cord- -h sten authors: pérez-arellano, josé luis; górgolas-hernández-mora, miguel; salvador, fernando; carranza-rodríguez, cristina; ramírez-olivencia, germán; martín-echeverría, esteban; rodríguez-guardado, azucena; norman, francesca; velasco-tirado, virginia; zubero-sulibarría, zuriñe; rojo-marcos, gerardo; muñoz-gutierrez, josé; ramos-rincón, josé manuel; sánchez-seco-fariñas, m. paz; velasco-arribas, maría; belhassen-garcía, moncef; lago-nuñez, mar; cañas garcía-otero, elías; lópez-vélez, rogelio title: executive summary of imported infectious diseases after returning from foreign travel: consensus document of the spanish society for infectious diseases and clinical microbiology (seimc) date: - - journal: enfermedades infecciosas y microbiología clínica doi: . /j.eimc. . . sha: doc_id: cord_uid: h sten abstract in a global world, knowledge of imported infectious diseases is essential in daily practice, both for the microbiologist–parasitologist and the clinician who diagnoses and treats infectious diseases in returned travelers. tropical and subtropical countries where there is a greater risk of contracting an infectious disease are among the most frequently visited tourist destinations. the seimc considers it appropriate to produce a consensus document that will be useful to primary care physicians as well as specialists in internal medicine, infectious diseases and tropical medicine who help treat travelers returning from tropical and sub-tropical areas with infections. preventive aspects of infectious diseases and infections imported by immigrants are explicitly excluded here, since they have been dealt with in other seimc documents. various types of professionals (clinicians, microbiologists, and parasitologists) have helped produce this consensus document by evaluating the available evidence-based data in order to propose a series of key facts about individual aspects of the topic. the first section of the document is a summary of some of the general aspects concerning the general assessment of travelers who return home with potential infections. the main second section contains the key facts (causative agents, diagnostic procedures and therapeutic measures) associated with the major infectious syndromes affecting returned travelers [gastrointestinal syndrome (acute or persistent diarrhea); febrile syndrome with no obvious source of infection; localized cutaneous lesions; and respiratory infections]. finally, the characteristics of special traveler subtypes, such as pregnant women and immunocompromised travelers, are described. in a global world, knowledge of imported infectious diseases is essential in daily practice, both for the microbiologist-parasitologist and the clinician who diagnoses and treats infectious diseases in returned travelers. tropical and subtropical countries where there is a greater risk of contracting an infectious disease are among the most frequently visited tourist destinations. the seimc considers it appropriate to produce a consensus document that will be useful to primary care physicians as well as specialists in internal medicine, infectious diseases and tropical medicine who help treat travelers returning from tropical and sub-tropical areas with infections. preventive aspects of infectious diseases and infections imported by immigrants are explicitly excluded here, since they have been dealt with in other seimc documents. various types of professionals (clinicians, microbiologists, and parasitologists) have helped produce this consensus document by evaluating the available evidence-based data in order to propose a series of key facts about individual aspects of the topic. the first section of the document is a summary of some of the general aspects concerning the general assessment of travelers who return home with potential infections. the main second section contains the key facts (causative agents, diagnostic procedures and therapeutic measures) associated with the major infectious syndromes affecting returned travelers [gastrointestinal syndrome (acute or persistent diarrhea); febrile syndrome with no obvious source of according to the world tourism organization, there were around , million international tourist arrivals in , some million more than in (an increase of . %). international tourist arrivals for the year were forecast to increase by %, both worldwide and regionally. regional growth was expected to be highest in asia and the pacific (between % and %) and the americas (between % and %), followed by europe (between . % and . %), and africa and the middle east (between % and %). familitur data showed that international tourist movements made by spaniards were . million in , , , of which were to the african continent, , to america (central caribbean and south) and , to asia. the most frequently visited tourist destinations include tropical and sub-tropical countries where there is a higher risk of contracting an infectious disease. travel to economically less developed countries frequently involves exposure to biological agents that cause infections or to infectious diseases transmitted by different routes (digestive, respiratory, mucocutaneous, vector, and so on). one point that should be highlighted from the outset is the possibility that an infection in the international traveler could also be caused by cosmopolitan agents found within our own country, an example of which would be sexually transmitted diseases (stds), a set of infectious conditions that has been insufficiently studied among travelers. the differential diagnosis therefore should always include diseases that have a restricted geographic distribution as well as those with a global presence. furthermore, the severity of the clinical pictures presented here varies a good deal, so that a special section has been included on managing the seriously ill patient. finally, there are certain situations that are physiological (such as pregnancy) or pathological in nature (for example, the immunocompromised patient, whether or not associated with hiv infection) that have special characteristics that warrant further discussion. from a medical point of view, these guidelines will be useful to primary care physicians as well as specialists in internal medicine, infectious diseases and tropical medicine who treat travelers returning from tropical and sub-tropical areas with infections. the target population in this document is adults with infections imported after returning from international travel. the prevention of imported diseases and infections imported by immigrants are explicitly excluded here, since these have been considered in recent eimc reviews. also left out here in a general sense are other noninfectious illnesses among travelers, although certain aspects will be mentioned in particular sections. various types of professionals (clinicians, microbiologists and parasitologists) have helped produce this consensus document by evaluating the evidence-based information available and making recommendations about the following aspects: • general evaluation of the returned traveler with a potential infection -the need to evaluate the asymptomatic traveler -the main syndromes associated with imported infectious diseases -evaluation of the traveler with severe infectious disease -evaluation of the traveler with potentially transmissible diseases and isolation precautions • main infectious syndromes in the returned traveler -acute or persistent diarrhea -fever of unknown origin -localized cutaneous lesions -respiratory infections -eosinophilia -neurological infections -urinary tract infections • special characteristics of the pregnant traveler • special characteristics of the immunocompromised traveler general methodology of the document a systematic review of the bibliography was performed to evaluate all data concerning the causes, diagnostic methods and therapeutic options for infections imported by travelers. a search of the pubmed database was performed using the following selection criteria: articles published between and march , in english or spanish, and limited to humans only. the search terms used were "travel*" associated with each of the items explored (e.g. "fever", "diarrhea", and so on). this search was complemented with a review of the cochrane database of systematic reviews, using "travel*" as the key term, and also of the international guidelines dealing with each of the separate aspects evaluated. the search was conducted using prisma reporting criteria, and was reviewed by the contributors in the first instance, then by those coordinating the text version. a total of publications were selected, eliminating those that were duplicated or not relevant. the specific set of references selected for each section may be requested from the contributors. the recommendations were based on the international standards used in the consensus guidelines of the infectious disease society of america (idsa) and the appraisal of guidelines research & evaluation (agree) instrument. the coordinators and authors of the document issued a consensus version, which was published on the seimc web site between november and december for external review. the final submitted article was returned with approval for publication. the management board of the seimc will designate coordinators to review this document in the next years. this section indicates the main internationally accepted definitions used by the world tourism organization. with respect to duration, three types may be distinguished: short-term (< weeks), medium-term ( weeks to months), and long-term (> months). depending on the purpose of the trip, there are two main groups: trips for personal reasons and those undertaken for professional reasons. trips for personal reasons can be further sub-divided into: (i) leisure, recreation and holidays; (ii) visiting friends and relatives (vfr); (iii) shopping trips; (iv) trips for educational/training purposes; (v) health tourism; (vi) for religious reasons and pilgrimages, and (vii) travel for cooperation and humanitarian aid. travel for professional reasons includes travel for business/conferences and military purposes. the selected key facts (kf) are indicated in the following sections. the need to evaluate the asymptomatic traveler kf . systematic evaluation is not indicated for all international travelers in the absence of clinical signs and symptoms (a-ii). kf . immigrant travelers visiting friends and relatives may benefit from evaluation, even if they are asymptomatic (c-ii). kf . long-term travelers (> months), those to high-risk areas and/or including high-risk activities may benefit from a directed evaluation (b-ii). kf . travelers who have been in contact with freshwater sources in endemic areas, or who have walked barefoot on contaminated soil may benefit from screening for schistosomiasis and strongyloidiasis respectively (a-ii). kf . any traveler who has engaged in risky sexual practice without protection may benefit from serological testing for the detection of stis, hiv, hepatitis b and c and syphilis (a-ii). kf . health aid workers exposed to patients with active tuberculosis may benefit from the tuberculin skin test or interferongamma release assays (igras) (b-ii). principal syndromes associated with imported diseases by travelers kf . in overall terms, the most common syndromes affecting travelers who return home feeling ill are gastrointestinal (acute or persistent diarrhea), fever of unknown origin, localized skin lesions and respiratory infections (a-ii). kf . the relative frequency of these syndromes varies depending on the geographic area or region visited (b-ii). kf . the severity of these syndromes is variable. fever of unknown origin or associated with other symptoms (such as diarrhea or respiratory problems) accounts for the majority of hospital admissions (b-ii). evaluation of the traveler with serious infectious disease kf . the initial evaluation of the international traveler with severity criteria should be carried out at three levels: assessment of vital functions, syndromic evaluation and diagnostic strategy (b-iii). kf . the immediate evaluation of hemodynamic stability should necessarily include blood pressure, respiratory rate, oxygen saturation, diuresis, heart rate and level of consciousness. other variables to be taken into account are body temperature, the presence of edema, capillary refill and the presence of ileus (a-iii). kf . the syndromic picture should be clearly established, since this will make it possible to select the most appropriate diagnostic tests and prognostic scales (b-iii). kf . the analytical determinations that should be ordered for the seriously ill patient include blood count, biochemical tests including serum transaminase, bilirubin and blood coagulation, renal function, glycemia, arterial blood gas, and an analysis of urine. when possible, c-reactive protein and procalcitonin levels should also be requested (a-iii). kf . the diagnostic strategy requires tests that are able to rule out malaria and dengue fever, and at least two sets of blood cultures, plus serological tests to detect rickettsial diseases, q fever, hiv, hbv or hcv infection (a-iii). kf . it is recommended that a specialist in tropical medicine or infectious diseases assess the patient as quickly as possible (c-iii). kf . in the returned traveler, the clinician should initially evaluate not only the individual disease, but also the possibility that it may involve a current public health alert (a-iii). kf . the recommended methods (in spain) are by phoning the departamento de alertas de salud publica (department of public health alerts) of the appropriate autonomous community, or by consulting the web page of the ministerio de sanidad, servicios sociales e igualdad (ministry of health) (a-iii). kf . different isolation precautions will be applied depending on the clinical syndrome and the traveler's travel itinerary (b-iii). kf . high-level isolation units (hliu) for patient management are indicated for confirmed and suspected cases of specific viral hemorrhagic fevers, highly pathogenic emerging respiratory diseases, multidrug-resistant tuberculosis (mdr-tb) and outbreaks of potentially serious transmissible diseases (pstd) caused by unknown agents (a-iii). kf . a basic pillar of control of pstds involves the selection, education and training of staff. this should be regarded as one more isolation precaution (b-iii). kf . restricting the use of invasive tests is also an isolation precaution. the selection of tests and the staff involved should be agreed by protocols adapted to the center where the patient is being treated (b-iii). kf . all travelers transferred from foreign hospitals should be regarded as potential carriers of multidrug-resistant organisms and should be proactively screened (by rectal smear) (a-iii). diarrhea (acute or persistent) kf . most cases of acute traveler's diarrhea are caused by bacterial pathogens. enterotoxigenic escherichia coli (etec) is the most frequently identified causative agent worldwide (a-iii). kf . in a significant percentage ( - %) of cases of acute traveler's diarrhea, an etiological diagnosis cannot be made (a-iii). kf . there are notable geographical variations with respect to the etiology of acute traveler's diarrhea, independent of the length of the trip (a-iii). kf . for acute traveler's diarrhea, microbiological studies should be restricted to patients who present fever, dysentery, choleriform diarrhea, or who are dehydrated, immunosuppressed or have significant comorbidities (a-iii). kf . the diagnostic method of choice for acute traveler's diarrhea is the conventional stool culture or cultures on selective media (depending on clinical suspicion) together with serial blood cultures if there is fever, although the diagnostic yield is low (a-iii). kf . for any traveler with fever and acute diarrhea arriving from an endemic area, malaria should be ruled out with the appropriate methods (b-iii). kf . before traveling, the patient should be given information about the main self-treatment measures to be taken in case of diarrhea, and told to seek medical care in the presence of high fever, severe abdominal pain, bloody diarrhea, uncontrolled vomiting, or if self-treatment is ineffective (a-iii). kf . for previously healthy adults, rehydration with conventional liquids, especially associated with loperamide, should be enough in cases of mild diarrhea (a-i). kf . rehydration and restoration of electrolyte balance with antidiarrheal drugs and non-absorbable antibiotics (rifaximin) is indicated for moderate diarrhea, and for the old or immunocompromised with no previous history of invasive disease (a-iii). kf . for severe diarrhea with obvious signs of dehydration, intravenous rehydration is recommended to restore the fluid and electrolyte balance (a-iii). kf . the use of antidiarrheal agents is contraindicated in the presence of invasive disease (a-iii). kf . the most useful drugs for the treatment of invasive diarrhea are fluoroquinolones or azithromycin, chiefly in single doses (a-i). kf . the pathogenesis of persistent traveler's diarrhea may fall into one of three major groups: persistent infection or coinfection; post-infectious syndromes (transient lactose intolerance, post-infectious irritable bowel syndrome, small intestinal bacterial overgrowth (sibo) and tropical sprue); or an underlying gastrointestinal disease unmasked during or after the trip (a-iii). kf . the most common infections in persistent traveler's diarrhea are due to protozoan pathogens, for which the diagnostic method of choice is the standard comprehensive parasitology profile, using specific stains based on clinical suspicion, and antigen detection methods and pcr, as available, for increased sensitivity (a-iii). kf . diagnosis of post-infectious irritable bowel syndrome is exclusively clinical (rome criteria iii-iv) and it is especially important to determine the state of digestive health before travel and to consider at an early stage whether there are clinical and analytical signs of alarm/organicity (a-iii). kf . the incidence of tropical sprue may be underestimated. its main differential diagnosis is with celiac disease. upper endoscopy (egd) to examine the jejunum and biopsy are frequently required to differentiate them (a-iii). kf . some authors recommend empirical therapy with nitroimidazoles if giardia intestinalis is highly suspected, even if specific studies are negative (c-iii). causative agents kf . the most common causes of fever of unknown origin in the returned traveler are, in order of frequency: malaria, arbovirus (e.g. dengue, chikungunya, zika) and bacterial infections (typhoid fever, rickettsial infections, q fever and leptospirosis) (a-ii). kf . even though they are rare, serious diseases that are highly contagious, such as viral hemorrhagic fevers (e.g. ebola, lassa, marburg, rift valley fever, crimean-congo hemorrhagic fever) should always be considered (c-iii). kf . the traveler's provenance, period of incubation and specific risk exposures should provide guidance as to the etiology of the febrile process (a-ii). kf . travelers who present with fever of unknown origin after visiting a tropical or sub-tropical area should seek immediate medical attention (a-ii). kf . if there is any possibility of viral hemorrhagic fever (vhf), this should be investigated using appropriate biosafety measures and techniques that require the least handling possible (rapid tests or pcr) (a-ii). a significant proportion of fever episodes post-travel either do not lead to a specific diagnosis (a-ii) or are due to cosmopolitan infections (a-ii). kf . if there is no risk of vhf, malaria should be ruled out in the first instance using microscopy techniques and rapid diagnostic tests (a-ii). kf . if the acute phase of an arbovirus is suspected (< - days of clinical evolution), the patient should be tested for the presence of (ns ) antigens or viral rna in blood and urine (a-ii). kf . if arbovirus in later stages is suspected (> days of clinical evolution), the serologic response (principally igm) should be studied and cases confirmed by neutralization tests (a-ii) . kf . the diagnosis of bacterial infection responsible for fever of unknown origin is based, in the acute phase, on isolating the bacterial organism or using molecular biology techniques, and in later phases, on serologic studies of paired serum samples (a-ii). kf . treatment (etiological or symptomatic) should be based on identifying the causative agent (a-ii). kf . if there is a high probability of malaria and a diagnosis cannot be made, or will be delayed for more than h with no alternative diagnosis, administration of empirical antimalarial therapy is recommended (a-ii). kf . patients with a likely diagnosis of severe acute schistosomiasis or neuroschistosomiasis are treated with corticosteroids in combination with praziquantel (b-ii). kf . in complicated or severe cases of malaria, use of ceftriaxone plus doxycycline is recommended while waiting for confirmation of diagnosis (c-iii). causative agents kf . various cosmopolitan infections, such as superficial mycoses (e.g. pityriasis versicolor, cutaneous candidiasis or dermatophytosis) and some ectoparasitic infections (such as scabies) are more frequent in travelers (a-ii). kf . classic bacterial infections constitute the leading cause of consultation for skin lesions and specifically, for those due to certain strains of methicillin-resistant s. aureus (mrsa) that produce panton-valentine leukocidin (pvl) (a-ii). kf . the primary morphology of the lesion (e.g. papules, pustules, nodules, ulcers, blisters), configuration (e.g. linear) and distribution (exposed versus covered areas, specific parts of the body) are helpful for diagnosis (a-ii). kf . in most cases, diagnosis is clinical, and dermoscopy is useful for some entities (scabies, cutaneous larvae migrans, furuncular myiasis and tungiasis) (b-iii). kf . in an imported dermatosis that develops slowly, cutaneous leishmaniasis, mycobacterial infection or a subcutaneous mycosis should be suspected. histological examination and identification of the causative agent with molecular biology techniques or mass spectrometry is essential (a-ii). kf . for many imported dermatoses, treatment is symptomatic with oral antihistamines (diphenhydramine, hydroxyzine, or loratadine), topical antipruritic therapy (calamine lotion) and topical corticosteroids (low potency for the genitals, medium potency for the trunk or extremities) (a-ii). kf . the treatment of choice for the main imported dermatoses that are linear in pattern (cutaneous larva migrans (ctm), cutaneous larva currens and gnathostomiasis) is ivermectin, with albendazole an alternative option (a-i). kf . surgical removal (associated with antibacterial tetanus chemoprophylaxis where appropriate) is the treatment of choice for furuncular myiasis, tungiasis, and d. repens infections (a-ii). causative agents kf . the most common causes of respiratory infection in the local environment are also the most common among travelers, with exotic imported infections being much less frequent (a-ii). kf . in general, viral and bacterial infections are more frequent than those caused by fungi and parasites (b-ii). kf . most respiratory infections in travelers are mild, affect the upper respiratory tract and are caused by viruses (a-ii). kf . the most serious respiratory infection in travelers is pneumonia, and the most common causes of it in the traveler are similar to those in the autochthonous population, although with a greater incidence of infections due to s. aureus and legionella spp. (a-ii). kf . during short trips, the risk of tuberculosis is low and depends on the incidence of the disease in the countries visited (b-iii). kf . the main causes of respiratory conditions associated with eosinophilia are acute schistosomiasis, löffler's syndrome and paragonimiasis (b-iii). kf . travelers may also have non-infectious problems that have respiratory manifestations such as pulmonary embolism and mefloquine toxicity (b-iii). kf . most respiratory infections during and after travel do not require diagnostic confirmation because they are mild, self-limited upper respiratory tract infections (a-ii). kf . the diagnostic methods for pneumonia are no different from those used on autochthonous patients (b-ii). kf . in patients with pneumonia, the decision as to whether a patient with pneumonia should be admitted to hospital or the icu should be based on severity scales used in the autochthonous population (a-ii). kf . procalcitonin levels can guide the clinical judgment of the attending physician in prescribing antibiotics for respiratory infections (b-ii). kf . if tuberculosis is suspected, or any other infection transmitted via respiratory secretions (such as the mers-cov coronavirus), respiratory isolation and droplet precautions are recommended (a-i). kf . most respiratory infections during and post-travel do not require specific treatment because they are mild and self-limiting. treatment may be given for the symptoms (b-iii). kf . for pneumonia in travelers, antibiotic therapy is initially empiric and should include drugs effective against the most common community-acquired pathogens, including legionella spp. (b-ii). kf . in travelers with influenza and severity criteria or risk factors, treatment with oseltamivir or zanamivir is recommended (b-ii). kf . if the traveler with tuberculosis has come from a country with a high incidence of antimicrobial resistance, it would be reasonable to evaluate treatment with at least drugs and rapid dna-based tests to detect mutations associated with resistance (b-iii). kd . the causes of imported eosinophilia vary a good deal and depend on the characteristics of the patient, particularly whether the person is a traveler or an immigrant, and on the geographical destination, itinerary and length of exposure (b-ii). kd . when a patient presents with imported eosinophilia of non-filarial etiology, it is important to rule out infection due to parasites, principally helminths (a-ii). kd . in patients with imported eosinophilia, the cause is identified as a parasite in - % of cases (b-ii). kd . the three most frequently found parasitic infections are strongyloidiasis, schistosomiasis and soil-transmitted helminth infections, as well as filariasis in the case of equatorial guinea (b-ii). kd . all returning travelers with eosinophilia should request a stool examination to search for parasites using a concentration method and strongyloides stercoralis serology (b-ii). kd . travelers returning from africa should request tests for diagnosing schistosomiasis (examination of the urine sediment and feces), and schistosoma spp. serology (b-ii). kd . in cases where a diagnosis cannot be made with direct techniques, serological methods are a useful diagnostic tool, although their possible drawbacks should not be forgotten (b-iii). kd . when the cause cannot be identified, the empiric treatment for imported eosinophilia should be a combination of oral ivermectin plus albendazole. praziquantel should be added for cases with possible epidemiological exposure to schistosomiasis (b-iii). kd . before starting empiric ivermectin therapy, the possibility of loa loa infection should first be ruled out (a-ii). kd . when empiric anthelmintic treatment is administered, the patient should receive clinical follow-up with monitoring of peripheral blood eosinophil counts, since some parasites, like trichuris spp. and schistosoma spp., have developed resistance leading to anthelmintic treatment failure (a-ii). causative agents kf . the most frequent causes of neurological disease in travelers are malaria, viral infections and bacterial meningitis (a-ii). kf . generally speaking, the main clinical manifestations affect the brain and/or meninges, and more rarely, the spinal cord and peripheral nervous system (b-iii). kf . the main manifestation in the peripheral nervous system is guillain-barré syndrome, which is associated with infection due to campylobacter jejuni, dengue and, more recently, the zika virus (b-ii). kf . any international traveler who returns with fever, severe headaches, sensitivity to light and/or a stiff neck should be evaluated urgently to rule out the presence of meningitis or encephalitis (a-iii). kf . the cerebrospinal fluid (csf) analysis can adopt four different patterns: normal, raised neutrophils, raised lymphocytes, raised eosinophils, which is useful for differential diagnosis (a-ii). kf . direct ophthalmoscopy is recommended for all patients with a diagnosis or suspicion of cerebral malaria, given its high prognostic and diagnostic value for malarial retinopathy (a-ii). kf . the possibility of infection with extended-spectrum betalactamase (esbl)-producing enterobacteriaceae should always be considered in travelers with urinary tract infections, principally those acquired in south and southeast asia (a-iii). kf . in the presence of non-specific complaints and/or hematuria, urinary schistosomiasis (s. haematobium) should be considered in travelers returning from traditionally endemic areas (and other more recently reported ones, such as corsica) (a-ii). the pregnant traveler kf . pregnant women are much more susceptible to certain infectious diseases, such as traveler's diarrhea, listeriosis, typhoid fever and malaria (a-ii). kf . various diseases that are transmitted by eating or drinking contaminated food or water (traveler's diarrhea, hepatitis e, listeriosis and typhoid fever) are more severe in pregnant women (a-ii). kf . mother-to-child transmission of the dengue and chikungunya viruses can occur if the mother has fever in the days close to and during birth, while the main complications of zika appear in the first trimester of the pregnancy (a-ii). kf . azithromycin is the antibiotic of choice for traveler's diarrhea and typhoid fever in pregnancy (b-ii). the immunocompromised traveler kf . the course of malaria in immunocompromised patients (especially those infected with hiv and with low cd + lymphocyte counts) tends to involve more severity criteria than in non-immunocompromised individuals. early diagnosis and treatment is essential, therefore, if there is clinical suspicion (a-iii). kf . immunocompromised individuals who present with traveler's diarrhea should be given the parasitology stool test, including the modified kinyoun stain for detecting coccidian species, such as cryptosporidium spp, cystoisospora belli and cyclospora cayetanensis (b-iii). kf . s. stercoralis hyperinfection syndrome and disseminated strongyloidiasis are more frequently found in immunosuppressed patients (corticotherapy, transplants, htlv- coinfection) and have very high mortality rates. early diagnosis and treatment with ivermectin at a dose of g/kg/per day for at least days is required (a-iii). world tourism organization (unwto) familitur características demográficas, quimio-profilaxis antimalárica e inmunoprofilaxis en . viajeros internacionales atendidos en una unidad monográfica declaración prisma: una propuesta para mejorar la publicación de revisiones sistemáticas y meta-análisis the agree collaboration. appraisal of guidelines for research & evaluation (agree) instrument the authors declare no conflict of interest. supplementary data associated with this article can be found, in the online version, at doi: . /j.eimc. . . . key: cord- -vbuepdm authors: ogbu, kenneth ikejiofor; mira, francesco; purpari, giuseppa; nwosuh, chika; loria, guido ruggero; schirò, giorgia; chiaramonte, gabriele; tion, metthew terzungwe; di bella, santina; ventriglia, gianluca; decaro, nicola; anene, boniface maduka; guercio, annalisa title: nearly full‐length genome characterization of canine parvovirus strains circulating in nigeria date: - - journal: transbound emerg dis doi: . /tbed. sha: doc_id: cord_uid: vbuepdm canine parvovirus type (cpv‐ ) emerged suddenly in the late s as pathogen of dogs, causing a severe and often fatal gastroenteric disease. the original cpv‐ was replaced by three antigenic variants, cpv‐ a, cpv‐ b and cpv‐ c, which to date have gained a worldwide distribution with different relative proportions. all previous studies conducted in africa were based on partial vp gene sequences. the aim of this study was to provide a genome analysis to characterize the cpv strains collected in nigeria, africa. rectal swab samples (n = ) were collected in and tested by means of an immunochromatographic assay. among the positive samples, were selected for further analyses using different molecular assays. the results revealed a high prevalence of cpv‐ c ( . %) compared to the cpv‐ a variant ( . %). the vp gene sequences showed a divergence from the strains analysed in in nigeria and a closer connection with cpv strains of asian origin. the non‐structural gene analysis evidenced amino acid changes never previously reported. the molecular analysis based on genomic sequences evidenced a geographical pattern of distribution of the analysed strains, suggesting a potential common evolutionary origin with cpv of asian origin. this study represents the first cpv molecular characterization including all the encoding gene sequences conducted in the african continent and contributes to define the current geographical spread of the cpv variants worldwide. reading frames (orfs) encoding for two non-structural (ns and ns ) and two structural (vp and vp ) proteins through alternative splicing of the same mrnas (reed at al., ) . soon after its emergence, the original cpv- was replaced by two antigenic variants, cpv- a and cpv- b (parrish et al., ; parrish, o'connell, evermann, & carmichael, ) , and in , a third antigenic variant (cpv- c) was described (decaro & buonavoglia, ) . to date, all three cpv variants are worldwide distributed, with different relative proportions according to the year and country of collection (amrani et al., ; miranda & thompson, ; woolford, crocker, bobrowski, baker, & hemmatzadeh, ) . in the african continent, cpv has been described in south africa and namibia (dogonyaro, bosman, sibeko, venter, & vuuren, ; steinel, venter, van vuuren, parrish, & truyen, ) , zambia (kapiya et al., ) , mozambique (figuiredo et al., ) , ghana (folitse et al., ) , morocco (amrani et al., ) , cape verde (costanheira et al., ) , nigeria (chollom et al., ) and tunisia (touhiri et al., ). in nigeria, only recently the molecular analyses based on the partial vp gene sequence of cpv strains described the circulating cpv variants (apaa, daly, & tarlinton, ; dogonyaro et al., ; fagbohun & omobowale, ) . unfortunately, all previous studies conducted in africa lack a comprehensive sequence analysis including all the viral genome, thus preventing a more in-depth knowledge on the origin and evolution of the circulating cpv strains. the aim of this study was to characterize the cpv strains recently collected in nigeria, analysing the nearly complete cpv genome sequence and comparing the obtained sequences with worldwide related sequences available in genbank. m a.s.l). samples were submitted from eight private veterinary clinics, two from each state, and from kennels/breeders in the same areas. rectal swabs were tested using an in-clinic assay for detection of cpv antigen (senspert ® canine parvovirus test kit, vetall laboratories), according to the manufacturer's instructions. among the positive samples, rectal swabs were selected and submitted for molecular analyses, where they were stored at − °c until use. details are reported in table s . swab homogenates were obtained as previously described . viral dna was extracted from µl of swab homogenate using the dneasy blood & tissue kit (qiagen s.p.a.), according to the manufacturer's instructions. the presence of cpv dna was confirmed using a primer pair (table s ) in a pcr protocol amplifying a -bp fragment of the vp gene (touihri et al., ) , using the commercial kit gotaq ® g dna polymerase (promega italia s.r.l.), as previously described (mira, dowgier, et al., ) . amplicons were checked after electrophoresis on a % agarose gel supplemented with ethidium bromide. ten microlitres of each amplicon was digested with units (u) of restriction endonuclease mboii (new england biolabs ® inc.) in a -µl reaction mix consisting of µl of nebuffer and µl of nuclease-free water, under the following reaction conditions: incubation at °c for hr and inactivation at °c for min. the profile was determined by electrophoresis on a % agarose gel supplemented with ethidium bromide. specimens from each city of collection (n = from makurdi; n = from jos; n = from abuja and n = from lafia), representing dogs with different age, vaccinal and clinical status, were selected to determine the vp gene sequence (table ). the nearly complete vp gene sequence was determined using a primer pair (table s ) , which allows for amplification of a , -bp fragment (battilani et al., ; mokizuki et al., ) , using the commercial kit gotaq ® g dna polymerase (promega italia s.r.l.), as previously described with minor modifications (thermal conditions: min for the annealing step). after electrophoresis on agarose gel supplemented with ethidium bromide, amplicons were purified with illustra™ gfx™ pcr dna and gel band purification kit (ge healthcare life sciences) and submitted to bmr genomics srl for direct sanger sequencing with . pmol of the reverse primer used for amplification and of two additional internal primers (table s ). sequences were assembled and analysed using bioedit software (hall, ) . by excluding the vp gene sequences with complete nt identities, cpv dnas from different cities of collection were further selected for a further sequence analysis by amplifying a long genome sequence encompassing both major orfs, without the ′ and ′ flanking sequences (table ) . sequence analyses were carried out using primer pairs described by pérez et al. ( ) , using the commercial kit gotaq ® g dna polymerase (promega italia s.r.l.), as previously described (mira, dowgier, et al., ) with minor modifications (mira, canuti, et al., ) . after electrophoresis on agarose gel, amplicons were purified and submitted for direct sanger sequencing with a set of primers, as previously described (mira, purpari, lorusso, et al., ) . sequences were assembled, analysed and submitted to nblast program (zhang, schwartz, wagner, & miller, ) to search related sequences into public domain databases. these sequence data were submitted to the ddbj/embl/ genbank databases under accession numbers mk - . phylogenetic analyses based on the full-length ns and vp gene sequences and on the whole genome encompassing the two orfs were conducted using the best-fit model of nt substitution with mega version x software (kumar, stecher, li, knyaz, & tamura, ) , inferred with the maximum-likelihood method based on the tamura -parameter (t ) and hasegawa-kishino-yano (hky) models, with discrete gamma distribution (five rate categories) (g) and invariant sites (i) (bootstrap , replicates), the best-fitting models after the model test analyses (vp : t + g; ns : hky + g; whole genome: hky + g+i). rna was extracted from samples using the qiaamp viral rna mini kit (qiagen s.p.a.), according to the manufacturer's instructions. extracted dna/rna was amplified using a set of gel-based or real-time (rt-)pcr assays useful for the detection of canine distemper virus (cdv) , canine adenovirus (cadv) types and (dowgier et al., ) , canine coronavirus (ccov) (decaro et al., ) and canine rotavirus (crov) (freeman, kerin, hull, mccaustland, & gentsch, ) . among the collected samples, rectal swabs tested positive for cpv by in-clinic assay ( %). the prevalence of the positive samples for each city of collection is reported in table . the presence of cpv dna was confirmed in the selected samples (n = ) using the conventional pcr assay. the same samples tested negative for cdv, cadvs, ccov and crov by gel-based or real-time (rt)-pcr assays. based on the rflp analysis, cpv-positive ta b l e identification code, origin, age, vaccination and clinical status, strain and sequence information of the dogs selected for molecular investigation amino acid change i v in ns also lies at the same residue in the ns -encoding sequence. additional four amino acid changes in the ns -encoding sequences were observed: d e, t a, d n and m v (table ). these changes resulted in silent mutations in the corresponding encoded ns protein. the aa change k r in the vp gene sequence is added to the aa changes of the vp gene sequence lying in the corresponding en- canine parvovirus has still been playing a main role in inducing severe and often fatal gastroenteritis in young or non-immunized dogs. during years, cpv spread and evolution have been well documented in north and south america, europe and asia (miranda & thompson, ; zhou, zeng, zhang, & li, ) . more recently, data about its spread were also obtained from australia and africa (amrani et al., ; castanheira et al., ; chollom et al., ; dogonyaro et al., ; figuiredo et al., ; folitse et al., ; kapiya et al., ; touhiri et al., ; woolford et al., ) . most of these studies were based on the partial or complete vp gene sequence, due to the involvement of the vp capsid protein in host switch and to its fast evolutionary rate nelson, palermo, hafenstein, & parrish, ; shackelton, parrish, truyen, & holmes, ) , with limited information on other cpv encoding fagbohun & omobowale, ) . these analyses conducted in nigeria evidenced firstly the circulation of cpv- a strains (apaa et al., ; dogonyaro et al., ) and only recently of cpv- b/ c types (fagbohun & omobowale, moreover, the vp aa change was due to a nt change in the second base of the codon (vp c g), but this change was common only to cpv strains of asian origin mira, purpari, lorusso, et al., ; wang et al., ; zhuang et al., ) . the potential biological relevance of these changes has not been described yet and needs to be assessed in further studies. as most recent asian cpvs, the nigerian strains displayed other three aa substitutions in the vp sequence (f y, y i and q r). while change at aa residue is predominant in all three cpv variants in asia (geng et al., ; yi, tong, cheng, song, & cheng, ; zhao et al., ; zhou et al., ) , the other changes have been less frequently observed, mainly in china since , and change q r has been detected only in cpv- c strains (geng et al., ; guo et al., ; mira, purpari, lorusso, et al., ; wang et al., ; zhuang et al., ) . these aa substitutions are located in the greatest variable vp gh loop, comprised between aa and , but while residue is not exposed on the capsid surface (chiang, wu, chiou, chang, & lin, ) and may not affect the antigenicity of cpv (xu et al., ) , residues and could have immunological implications or biological relevance. indeed, residue is subject to positive selection (hoelzer, shackelton, parrish, & holmes, ) and is adjacent to residue , which affects binding to the canine transferrin receptor . residue is close to residues associated with the ability of cpv to haemagglutinate, altering the ph dependence of haemagglutination or affecting the canine transferrin receptor (tfr) binding that determines the canine host range (guo et al., ; kaelber et al., ; tsao et al., ) . among the synonymous substitutions observed in the cpv- a vp gene sequences, nt change a g has been previously described in cpv- c strains (amrani et al., ; decaro, desario, amorisco, et al., ; decaro et al., ) . this change, observed in the strains uv and uv , was detected in the binding region of the type- a and type- c specific probes of the minor groove binder (mgb) probe assay (decaro et al., . although this change potentially accounts for the absence of vic fluorescence in the a/ b and b/ c assays, specific additional studies are necessary to evaluate its real implication in the characterization of this cpv- a mutant by the mgb probe assay, as previously done for the same substitution in the cpv- c mutants (decaro, desario, billi, et al., ) . the in-clinic assay used in this study was able to detect the cpv- a showing this substitution, as previously observed for another rapid assay used to test also the cpv- c mutants (decaro, desario, billi, et al., ) . limited studies are available on the cpv non-structural genes (hoelzer et al., ; pérez et al., ) , and, only recently, the analysis of the ns gene sequence was included in the cpv phylogenies from several countries (canuti, rodrigues, whitney, & lang, ; grecco et al., ; mira, canuti, et al., ; zhuang et al., ) . in this study, sequence analysis revealed aa changes previously described mainly in ns /ns gene sequences of cpv- a/ c strains of asian origin. additional changes were also evident, some previously reported in south/north america and others never previously observed. this divergence may suggest the same ancestral origin with the cpv strains of asian origin but a separate evolution, as well as a continuous adaptive process of the virus in separate environments. indeed, some of these changes lay in the potential encoding sequence of functional domains (mira, canuti, et al., ) and, particularly, residues , and are located between the α -and α -helices, between the β -and α -helices and just close to the α -helix of the helicase domain protein sequence, respectively, as illustrated in niskanen, ihalainen, kalliolinna, häkkinen, and vihinen-ranta ( ) . therefore, their role needs to be further evaluated. the molecular analysis based on long genome sequences evidenced the geographical origin of the analysed strains rather than the clustering based only on the cpv antigenic variant. therefore, this study supports further studies aimed to track the viral spread and elucidate the cpv evolution. indeed, the recent evidence of specific aa changes, as well as the divergence from previous circulating strains, does not allow to rule out the possible introduction of these strains from other countries, highlighting the need for further studies on cpv whole genome in different geographical areas. this suggestion is supported by the evidence of genetic signatures typical of cpv or other canine viruses with different origins (decaro, campolo, et al., ; martella et al., ; mira, purpari, lorusso, et al., ) , probably connected with the trading and transport of dogs between countries and continents. in this study, with the aim to investigate the prevalence of the most frequent canine enteric viruses, all collected samples were analysed for selected pathogens. cpv was the only enteric viral pathogen detected in this study and this result confirmed the correlation between cpv infection and development of enteric signs, with a limited role of other viral enteric pathogens (dowgier et al., ) . nevertheless, further studies, based on a wider sampling also including the wild potential susceptible species, are necessary to better elucidate the effective spread of the other viruses in nigeria. this study represents the first cpv molecular characterization including all the encoding gene sequences conducted in the african continent and contributes to define the current geographical spread of the cpv variants worldwide. the evidence of mutations that have not been detected before suggests the need for further investigations in order to determine any biological consequences and underlines the continue evolution of cpv. the authors would like to thank dr. ijeoma chekwube chukwudi, dr. pam dachung luka, dr. emmanuel tumininu obishakin and dr. ukamaka uchenna eze for their skilful technical assistance and also practitioners, private veterinary clinics and dog breeders for sample collection. the authors of this manuscript declare that there are no conflicts of interest. molecular epidemiology of canine parvovirus in morocco. infection canine parvovirus (cpv- ) variants 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sha: doc_id: cord_uid: aimvogs peptidylarginine deiminases (pads) are phylogenetically conserved calcium-dependent enzymes which post-translationally convert arginine into citrulline in target proteins in an irreversible manner, causing functional and structural changes in target proteins. protein deimination causes generation of neo-epitopes, affects gene regulation and also allows for protein moonlighting. furthermore, pads have been found to be a phylogenetically conserved regulator for extracellular vesicle (evs) release. evs are found in most body fluids and participate in cellular communication via transfer of cargo proteins and genetic material. in this study, post-translationally deiminated proteins in serum and serum-evs are described for the first time in camelids, using the llama (lama glama l. ) as a model animal. we report a poly-dispersed population of llama serum evs, positive for phylogenetically conserved ev-specific markers and characterised by tem. in serum, deiminated proteins were overall identified, including key immune and metabolic mediators including complement components, immunoglobulin-based nanobodies, adiponectin and heat shock proteins. in serum, deiminated proteins were identified that were not in evs, and deiminated proteins were found to be unique to evs, with shared deiminated protein hits between both serum and evs. deiminated histone h , a marker of neutrophil extracellular trap formation, was also detected in llama serum. pad homologues were identified in llama serum by western blotting, via cross reaction with human pad antibodies, and detected at an expected kda size. this is the first report of deiminated proteins in serum and evs of a camelid species, highlighting a hitherto unrecognized post-translational modification in key immune and metabolic proteins in camelids, which may be translatable to and inform a range of human metabolic and inflammatory pathologies. lamoids, or llamas, belong to a family of camelids which are economically important livestock and have developed complex features and immunological traits related to their habitat (wu et al., ; saadeldin et al., a) . the llama (lama glama), bactrian camel (camelus bactrianus), dromedary (camelus dromedarius) and alpaca (vicugna pacos) differ in their habitat. the bactrian camel and dromedary are adapted to arid-desert-adapted environments, alpacas to plateaus, and the llama to higher altitudes. llamas are historically found in the andean highlands, specifically the altiplano of southeast perú and western bolivia, as well as in chile and argentina, which has the third highest population of llamas (wu et al., ) . the domesticated llama is closely related to two extant wild south american camelids, the vicuña (vicugna vicugna) and guanaco (lama guanicoe). previous genomic studies have revealed a range of specific adaptions in camelids relating to fat and water metabolism, osmoregulation, blood glucose level regulation, stress responses to heat, aridity, as well as to intense ultraviolet radiation and dust (wu et al., ) . furthermore, a particularly important feature in camelid immunity is the production of small, homodimeric heavy chain-only, antibodies (hcabs) which are of great value for the biomedical industry . peptidylarginine deiminases (pads) are phylogenetically conserved calcium-dependent enzymes which post-translationally convert arginine into citrulline in target proteins in an irreversible manner. this can cause functional and structural changes in target proteins (vossenaar, and intrinsically disordered proteins, while the position of the arginine is also important; arginines sitting next to aspartic acid residues are most prone to citrullination, arginines next to glutamic acid residues are rarely citrullinated and those flanked by proline are poorly citrullinated (nomura, ; tarcsa et al., ; györgy et al., ) . protein deimination can affect gene regulation, cause generation of neoepitopes (witalison et al., ; lange et al., ) and may also allow for protein moonlighting, an evolutionary acquired phenomenon facilitating proteins to exhibit several physiologically relevant functions within one polypeptide chain (henderson and martin, ; jeffrey, ; magnadóttir et al., a) . pads have been identified throughout phylogeny from bacteria to mammals, with tissue specific pad isozymes in mammals, in chicken, in bony fish and arginine deiminase homologues in bacteria (vossenaar et al., ; rebl et al., ; magnadóttir et al., a, a; kosgodage et al., ) . while studies on pads in relation to human pathophysiology, including cancer, autoimmune and neurodegenerative diseases (wang and wang, ; witalison et al., ; lange et al., ; kosgodage et al., kosgodage et al., & and cns regeneration (lange et al., and ) exist, relatively little phylogenetic research has been carried out on pads in relation to normal physiology and evolutionary acquired adaptions of the immune system. recent comparative studies focussing on roles for pads in teleost fish have identified post-translational deimination in key proteins of innate, adaptive and mucosal immunity (magnadóttir et al., a, b; magnadottir et al., a; magnadóttir et al., b) . a recent study in shark also revealed novel insights into this post-translational modification in relation to key immune factors, including shark immunoglobulins (criscitiello et al., ) . as the camelid family has developed unusual small immunoglobulins similar as to shark through convergent evolution, indicating common factors between shark and camelid immunity, we felt that an investigation of post-translationally deiminated proteins in camelids was warranted. as pads have been identified to be a key regulator of extracellular (ev)-release, a mechanism that has been found to be phylogenetically conserved from bacteria to mammals (kholia et al., ; kosgodage et al., kosgodage et al., , gavinho et al., ; kosgodage et al., ) , the characterisation of evs in camelids is of further interest. extracellular vesicles (evs) are found in most body fluids and participate in cellular communication via transfer of cargo proteins and genetic material (inal et al., ; colombo et al., ; lange et al., ; turchinovich et al., ; vagner et al., ) . evs in body fluids, including serum, can also be useful biomarkers to reflect health status (hessvik and llorente, ; ramirez et al., ) . previous work on evs has hitherto mainly been in the context of human pathologies, while recently comparative studies are growing (iliev et al., ; yang et al., ; magnadóttir et al., b; criscitiello et al., ; gavinho et al., ; kosgodage et al., ) . few studies have been performed on evs in camelids but therapeutic effects of evs isolated from camel milk have been identified in halting cancer progression (badawy et al., ) . a recent study in shark identified for the first time deiminated small immunoglobulin proteins as part of ev cargo (criscitiello et al., ) . due to the link between camelid and shark immunity through convergent evolution including the unusual immunoglobulin structure of small heavy chain-only ig's, a comparative study on evs and deiminated ev cargo in a camelid species may provide further insights into shared immunological traits. camelids have an unusual ig repertoire and a large diversity of functional nanobodies has been identified in the llama (harmsen and de haard, ; deschaght et al., ) . this has made camelids an important source for small immunoglobulins that can be used for immunotherapy purposes, including for tumour targeting (van lith et al., ) , as well as for assessing cancer metastasis (ramos-gomes et al., ) . as these nanobodies can also penetrate the blood-brain barrier (Širochmanová et al., ) they are of great value for a range of therapeutic treatment applications, including for brain cancers (iqbal et al., ) . furthermore, as the camelid family has acquired unique metabolic features, they are also of interest as a model species for informing metabolic diseases. in the current study we assessed post-translationally deiminated proteins in llama serum and serum-derived evs, and report for the first time ev-mediated export of deiminated key immune, metabolic and nuclear proteins in serum of a camelid species. llama (lama glama l. ) serum was shared from excess blood collected in routine health checks of a resident male llama at the texas a&m winnie carter wildlife center. blood collected from the jugular vein of this year old llama was allowed to clot at room temperature for h before serum was collected by centrifuging at g for min. serum was aliquoted and immediately frozen at − °c until further use. evs were isolated by step-wise centrifugation according to established protocols using ultracentrifugation and the recommendations of misev (the minimal information for studies of extracellular vesicles ; théry et al., ) . llama serum was diluted : in ultrafiltered (using a . μm filter) dulbecco's pbs (dpbs, μl serum added to μl dpbs) and then centrifuged at g for min at °c for removal of cells and cell debris. the supernatant was collected and centrifuged at , g for h at °c. the pellet was then resuspended in dpbs and washed again at , g for h at °c. the resulting evenriched pellet was resuspended in μl dpbs, diluted / in dpbs and analysed by nta, based on brownian motion of particles in suspension, using the nanosight ns system (malvern, uk). the na-nosight was used in conjunction with a syringe pump to ensure continuous flow of the sample, with approximately - particles per frame and videos were recorded for x s. the replicate histograms generated from the recordings were averaged. evs were isolated from serum as described above, the ev pellets were fixed with . % glutaraldehyde in mm sodium cacodylate buffer (ph . ) for h at °c, resuspended in mm sodium cacodylate buffer (ph . ), placed on to a grid with a glow discharged carbon support film, stained with % aqueous uranyl acetate (sigma-aldrich) and thereafter viewed in tem. imaging was performed using a jeol jem transmission electron microscope (jeol, japan) operated at kv at a magnification of , - , . digital images were recorded using an amt xr ccd camera (deben, uk). llama serum and ev isolates (an ev pellet derived from μl serum, reconstituted in μl dpbs after isolation and purification) were diluted : in x laemmli sample buffer, boiled for min at °c and separated by sds-page on - % gradient tgx gels (biorad uk). approximately μg protein was loaded per lane and transferred to nitrocellulose membranes using semi-dry western blotting. blocking of membranes was performed in % bsa in tbs-t for h at room temperature (rt) and incubation with primary antibodies, diluted in tbs-t, was carried out at °c overnight (f mabn , merck, / ; pad ab , abcam, / ; pad ab , / ; pad ab , / ; cith ab , / ; cd ab , / ; flot- ab , / ). the membranes were washed in tbs-t for × min at rt and thereafter incubated in the corresponding secondary antibody (anti-rabbit igg biorad or anti-mouse igm biorad, diluted / in tbs-t) for h at rt. membranes were washed for × min in tbs-t and visualisation performed using electrochemiluminescence (ecl) and the uvp biodoc-ittm system (thermo fisher scientific, uk). for isolation of total deiminated proteins from llama serum and serum derived evs, the catch and release immunoprecipitation kit (merck, uk) was used together with the f pan-deimination antibody (mabn , merck), which has been developed against a deca-citrullinated peptide and specifically detects proteins modified by citrullination (nicholas and whitaker, ) . for f enrichment, μl serum was used according to the manufacturer's instructions (merck). for evs, total protein was first extracted from ev-enriched pellets derived from μl serum, using μl radioimmunoprecipitation assay (ripa) buffer, containing protease inhibitor cocktail (p , sigma, uk), and shaken gently on ice for h. thereafter proteins were isolated from the evs by centrifugation at , g for min, collecting the supernatant containing the proteins. immunoprecipitation was carried out according to the manufacturer's instructions (merck), using a rotating platform overnight at °c. the f bound proteins were eluted using denaturing elution buffer, according to the manufacturer's instructions (merck). the f enriched eluates were then either analysed by western blotting or by lcems/ms (cambridge proteomics, cambridge, uk). for lcems/ms, the f -enriched eluates were run cm into a sds-page gel and the whole f -enriched eluate was cut out as one band, whereafter it was processed for proteomic analysis (carried out by cambridge proteomics). peak files were submitted to in-house mascot (matrix science; cambridge proteomics). databases used for protein identification (in house, cambridge proteomics) were as follows: camelidae_family_ ( sequences; residues) and also specifically for llama: lama_glama_ ( sequences; residues). evs from llama serum were characterised, following step-wise ultracentrifugation, by size exclusion using nta, by morphological analysis using tem and by western blotting using ev-specific markers ( fig. ) . a poly-dispersed population of evs in the size range of - nm, with main peaks at , , , , and nm was identified by nta analysis (fig. a) . western blotting confirmed that the llama serum evs were positive for the ev-specific markers cd and flotillin- (fig. b) . tem analysis confirmed a poly-dispersed evs population (fig. c) . a cross-reaction with human pad , and isozyme-specific antibodies was observed in llama serum by western blotting, at an approximate - kda size range as expected for mammalian pads ( fig. a) . deiminated histone h was also detected in llama serum by western blotting at the expected approximate kda size ( fig. a) . total deiminated proteins in llama serum-evs were detected by western blotting using the f pan-deimination antibody, revealing a range of proteins between - kda in size (fig. b ). deiminated proteins were also assessed by western blotting after f enrichment, using immunoprecipitation (ip) from llama serum and serum-derived evs (fig. c ). deiminated proteins from the f enriched eluates were further identified by lc-ms/ms analysis. in llama serum, hits for camelid proteins were identified, as listed in table and supplementary table . overall, of these deiminated protein hits were common to whole serum and serum-derived evs, while hits were specific for whole serum (fig. d ). llama serum-derived evs showed positive for deiminated proteins by western blotting, using the pan-deimination f antibody (fig. b ). deiminated proteins were further identified by f enrichment and lc-ms/ms analysis, revealing deiminated protein hits in total for evs, with hits unique to evs (not identified from serum). peptide sequences of hits with camelid proteins and m/z values are listed in table and supplementary table . overlap with deiminated proteins identified in whole llama serum and evs is represented in the venn diagram in fig. d . for the first time deiminated proteins are described in a camelid, using the llama (lama glama) as a model species. post-translational deimination was identified in key immune, nuclear and metabolic proteins. a llama pad homologue was identified at an expected - kda size similar as for human pads by western blotting via cross reaction with anti-human pad , pad and pad antibodies. pad is known to be the phylogenetically most conserved pad form (vossenaar et al., ; magnadóttir et al., a) and has also been seen in shark (criscitiello et al., ) . deiminated histone h , a marker of neutrophil extracellular trap formation (netosis), was also detected in llama serum by western blotting and is described for the first time in a camelid species but was recently described in shark (criscitiello et al., ) . netosis is driven by pads (li et al., ) , is conserved throughout phylogeny and is important in innate immune defences against a range of pathogens including bacteria, viruses and helminths (brinkmann et al., ; branzk et al., ; schönrich and raftery, ) . netosis has also been associated with clearance of apoptotic cells and during tissue remodelling in teleosts (magnadóttir et al., a (magnadóttir et al., , a . furthermore, netosis is linked to a range of autoimmune diseases, due to net activation via neo-epitopes (o'neil and kaplan, ), which can also lead to organ damage (lee et al., ) . netosis is also associated with cancer (gonzalez-aparicio and alfaro, ) and neurodegenerative diseases (pietronigro et al., ) . further deiminated proteins identified in llama serum and serumderived evs by f enrichment and lcems/ms analysis included key proteins of camelid innate and adaptive immunity, nuclear proteins, as well as proteins involved in metabolic function. using string (search tool for the retrieval of interacting genes/proteins) analysis (https:// string-db.org/) for protein-protein interaction, the ppi enrichment value for deiminated proteins in whole llama serum was found to be p < . e- for deiminated proteins out of identified in serum, which indicates that these proteins have more interactions among themselves than what would be expected for a random set of proteins of similar size, drawn from the genome (string analysis, see fig. a ). for deiminated proteins in out of the deiminated ev cargo proteins, the ppi enrichment value was found to be p = . (string analysis, see fig. a ). such enrichment indicates that the proteins are at least partially biologically connected, as a group. as the camelid proteins are not present in the string database, corresponding protein homologues for human were chosen to create the protein-protein interaction networks shown in figs. and . out of the camelid proteins identified as deiminated in serum, protein ids for were present for homo sapiens to perform the assessment of protein-protein interactions and identification of main biological go pathways (response to stress, response to wounding, oxygen transport, small molecule metabolic process, vesicle mediated transport and regulated exocytosis; fig. b ). for llama evs, out of proteins identified as deiminated, corresponding protein homologues were found in the string database for homo sapiens and were analysed highlighting main biological go pathways (response to stress, cytoskeleton organisation and vesicle-mediated transport; fig. b ). deimination protein candidates identified here in llama serum and evs, which are involved in immune, nuclear and metabolic functions, are further discussed below, including where appropriate in a comparative context with relevant human diseases. proteins that have previously been identified as deiminated in other species are listed first. nanobodies are based on immunoglobulin single variable domains, derived from the variable domains of heavy chain-only antibodies, which occur naturally in camelids (deschaght et al., ) . in the llama the heavy chain-only antibodies are comprised of at least two subclasses . these types of homodimeric, light chain-less antibodies have evolved through convergent evolution (brooks et al., ) , in at least two groups (camelids and cartilaginous fish), and their variable binding domains (v h h s ) are of great value for therapeutic and diagnostic applications (muyldermans, ; criscitiello, ; steeland et al., ; könning et al., ; henry et al., ) . nanobodies can act against challenging targets such as small molecules and toxins (wesolowski et al., ; bever et al., ) , viruses (wei et al., ; hassiki et al., ; vanlandschoot et al., ; cohen, ) , enzymes (muyldermans, ) , ion channels (wei et al., ; danquah et al., ) and g protein-coupled receptors gpcrs (cromie et al., ) . llama nanobodies have been shown to tether to early endosomes and to mitochondria (traub, ) , be used for diagnostics (shriver-lake et al., ) , be used for design of cancer immunotherapeutics (hussack et al., ; bannas and koch-nolte, ; rossotti et al., ) and have been approved for passive immunotherapy (sheridan, ) . our current finding, that llama nanobodies can be post-translationally deiminated may shed some light on their observed structural variation which still remains to be fully explained as sequence alignment does not fully elucidate their diversity (mitchell and colwell, ) . as a structurally analogous immunoglobulin in shark, new antigen receptor (nar) (greenberg et al., ; barelle et al., ; flajnik and dooley, ; de silva et al., ) was recently also found to be deiminated (criscitiello et al., ) , our current finding may provide novel insights into function of these immune proteins and be useful for refinement in therapeutic nanobody development. llama single-chain antibodies were here found to be deimination candidates both in llama whole serum and in serum derived evs, highlighting also their ev-mediated export. ig proteins were identified here as being deiminated in llama serum and in serum-derived evs, scoring with ig components from other camelids. immunoglobulins (ig) are key molecules in adaptive immunity but post-translational deimination of ig's has hitherto received little attention. deimination of the igg fc region in patients with bronchiectasis and ra has been identified (hutchinson et al., ) . furthermore, deimination of ig's in teleost fish was recently described (magnadóttir et al., a) , as well as in shark (criscitiello et al., ) . given the increased focus on understanding of ig diversity throughout phylogeny (smith et al., ; zhang et al., ; de los rios et al., ; zhang et al., zhang et al., , stanfield et al., ) and the unique features of camelid immunoglobulins (plasil et al., ) , our current finding of deimination of llama ig's highlights a novel concept that may further understanding of ig diversity throughout evolution. of additional interest is the finding that some deiminated ig proteins were also found to be exported in evs. complement components identified to be deiminated in llama serum included c , c and c q, while only c was identified to be deiminated in serum derived evs. complement component c plays a central role in all pathways of complement activation and can also be directly activated by self-and non-self surfaces via the alternative pathway without a recognition molecule (dodds and law, ; dodds, ) . in camelids, the alternative and classical pathway haemolytic activity of serum has been assessed in the dromedary camel with respect to age and gender (olaho-mukani et al., a; and b) . hitherto little is known about roles for post-translational deimination of complement components throughout phylogeny. in teleost fish, c has been identified in serum in deiminated form (magnadóttir et al., a) and also found to be deiminated in mucosal and serum evs of teleost fish (magnadóttir et al., b (magnadóttir et al., , c , while post-translationally deiminated c was identified in shark total serum but not evs (criscitiello et al., ) . other complement components, including c and c q, which belong to the classical pathway of complement activation were here identified as deiminated in llama whole serum and some of those llama serum evs are positive for the ev-specific markers cd and flotillin- (flot- ). c. transmission electron microscopy (tem) imaging of evs isolated from llama serum shows a polydispersed population; scale bar represents nm. have also recently been reported to be deiminated in teleost fish (magnadóttir et al., a) . the c q subcomponent can bind to the fc region of immunoglobulins that are bound to antigen and activate the classical part of the complement pathway (reid et al., ; reid, ) . interestingly, an essential role for arginine in c q has previously been suggested for c q-igg interaction (kojouharova et al., ) . c q also serves as a potent pattern recognition molecule which recognises self, non-self and altered self-signals (nayak et al., ; reid, ) and may therefore also bind to deiminated neo-epitopes (magnadóttir et al., a ). the complement system has multifaceted roles. it forms part of the first line of immune defence against invading pathogens, acting in clearance of necrotic or apoptotic cells (dodds and law, ; sunyer and lambris, ; fishelson et al., ; carroll and sim, ) . complement also has roles in regeneration (del rio-tsonis et al., ; haynes et al., ) and tissue remodelling (lange et al., a (lange et al., , b lange et al., lange et al., , lange et al., , . furthermore, c is also implicated in multiple non-complement functions including binding of apoptotic cells, cleavage of nuclear antigens and cleavage of mhc class i (lu and kishore, ) . post-translational deimination of complement components may possibly influence their function including cleavage ability, binding, deposition and generation of the convertase. apolipoprotein a-i is primarily involved in lipid metabolism where conformational plasticity and flexibility are regarded as key structural features (arciello et al., ) . apo a-i is associated with regulation of llama pad homologues were identified at the expected size of approximately - kda using the anti-human pad , pad and pad isozyme specific antibodies respectively. deiminated histone h (cith ), representative of neutrophil extracellular traps (nets), was verified in llama serum. b. total deiminated proteins were assessed by western blotting in llama serum evs, using the f pan-deimination specific antibody. c. immunoprecipitated (ip) deiminated proteins after f -enrichment were assessed both in serum-evs and whole serum of llama by western blotting. d. the venn diagram represents deiminated proteins identified in total serum and serum-derived evs by f enrichment and lc-ms/ms analysis. overall, proteins were identified in common with both samples, while proteins were found deiminated in serum only and proteins were identified as deiminated in evs only. (continued on next page) mitochondrial function and bioenergetics (white et al., ) . furthermore, apo a-i has been shown to have a regulatory role in the complement system by affecting membrane attach complex (mac) assembly and thus the final lytic pathway (hamilton et al., ; jenne et al., ; french et al., ) . given the diverse roles of apo a-i, the current finding of deiminated forms in serum may be of quite some relevance and has previously been identified in teleost fish (magnadóttir et al., a) . apo a-i was here found to be deiminated in whole llama serum only. serum albumin is a major acidic plasma protein in vertebrates and serves as a transport molecule for fatty acids, bilirubin, steroids, amino acids and copper, as well as having roles in maintaining the colloid osmotic pressure of blood (peters, ) . in camelids, total albumin levels have been assessed, for example in relation to reproductive efficiency (el-malky et al., ) and as biomarkers of oxidative stress centromere protein j ⱡ ions score is - *log(p), where p is the probability that the observed match is a random event. individual ions scores > indicated identity or extensive homology (p < . ). protein scores were derived from ions scores as a nonprobabilistic basis for ranking protein hits. cut-off was set at ions score . ⱡ ions score is - *log(p), where p is the probability that the observed match is a random event. individual ions scores > indicated identity or extensive homology (p < . ). protein scores were derived from ions scores as a nonprobabilistic basis for ranking protein hits. cut-off was set at ions score . (caption on next page) m.f. criscitiello, et al. molecular immunology ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (el-deeb and buczinski, ). while albumin has been identified as a glycoprotein in some species (metcalf et al., ) investigation of posttranslational deimination has been limited, but was recently identified in teleost fish (magnadóttir et al., a) . serum albumin was identified as deiminated in both whole llama serum and evs. hemopexin is a scavenger protein of haemoglobin and a predominant heme binding protein, which contributes to heme homeostasis (smith and mcculloh, ; immenschuh et al., ) . hemopexin also associates with high density lipoproteins (hdl), influencing their inflammatory properties (mehta and reddy, ) . hemopexin is a plasma glycoprotein that has been previously identified as a deimination candidate in teleost fish (magnadóttir et al., a) and shark (criscitiello et al., ) . here, hemopexin was found deiminated in whole llama serum only. inter-alpha-trypsin inhibitor (heavy chain h ) belongs to the serpin family of proteins, which have protease-inhibitory functions and are involved in diverse physiological and pathological processes including fertilisation, ovulation, coagulation, inflammation, as well as tumorigenesis, metastasis and dementia (zhuo and kimata, ; weidle et al., ) . inter-alpha-trypsin inhibitor is synthesised in the liver, circulates in the blood and has two chains, a light and heavy chain, whereof the heavy-chain (itih) includes a von willebrand domain and can interact with the extracellular matrix (bost et al., ) . itih is downregulated in tumours via methylation and itih is strongly reduced in invasive cancers (hamm et al., ) . while itih was recently identified as a deimination candidate in teleost fish (magnadóttir et al., a) further studies on the regulation of itih via post-translational deimination have not been carried out. itih was here identified as deiminated in whole llama serum only. fibrinogen is a glycoprotein, synthesised in liver (tennent et al., fig. . protein-protein interaction networks of deiminated protein hits identified in whole llama (lama glama) serum. reconstruction of protein-protein interactions based on known and predicted interactions using string analysis. due to annotations for camelids not being present in string, proteins are based on corresponding human protein identifiers. a. coloured nodes represent query proteins and first shell of interactors; white nodes are second shell of interactors. b. biological go processes are highlighted for the same protein network as follows: red = response to stress; blue = response to wounding; green = vesicle mediated transport; yellow = oxygen transport; purple = regulated exocytosis; dark green = small molecule metabolic process. coloured lines indicate whether protein interactions are identified via known interactions (curated databases, experimentally determined), predicted interactions (gene neighbourhood, gene fusion, gene co-occurrence) or via text mining, co-expression or protein homology (see colour key for connective lines in a). protein-protein interaction networks of deiminated protein hits identified in evs of llama (lama glama) serum. reconstruction of protein-protein interactions based on known and predicted interactions using string analysis. due to annotations for camelids not being present in string, proteins are based on corresponding human protein identifiers. a. coloured nodes represent query proteins and first shell of interactors. b. biological go processes are highlighted as follows: red = response to stress; blue = cytoskeleton organisation; green = vesicle mediated transport. coloured lines indicate whether protein interactions are identified via known interactions (curated databases, experimentally determined), predicted interactions (gene neighbourhood, gene fusion, gene co-occurrence) or via text mining, co-expression or protein homology (see colour key for connective lines in a). ) and forms part of the acute phase response as part of the coagulation cascade (tiscia and margaglione, ) . in camelids, fibrinogen is a biomarker for stress and infection (greunz et al., ; el-bahr and el-deeb, ; el-deeb and buczinski, ) . impaired mechanism of fibrinogen formation and fibrin polymerization are implicated with various pathologies including coagulopathies and ischemic stroke (weisel and litvinov, ) , while acquired fibrinogen disorders can be associated with cancer, liver disease or post-translational modifications (besser and macdonald, ) . fibrinogen is indeed a known deimination candidate and this post-translational modification contributes to its antigenicity in autoimmune diseases (hida et al., ; muller and radic, ; blachère et al., ) . fibrinogen was here identified as deiminated both in whole llama serum and evs. tubulin beta-chain participates in cytoskeletal rearrangement and its deimination has previously been linked to ev release (kholia et al., ) . deimination of tubulin may therefore be crucial for facilitating ev-mediated processes in homeostasis, immune responses and in relation to pathologies. here, tubulin was identified as deiminated in both whole llama serum and evs. histone h b was identified as being deiminated in both llama serum and evs and in addition, histone (h ai isoform -like protein) and core histone macro-h a. isoform were identified as deiminated in evs only. histones undergo various posttranslational modifications that affect gene regulation and can also act in concert (latham and dent, ; bird, ) . in addition to acetylation, phosphorylation and ubiquitination, histones are known to undergo deimination, including h b (sohn et al., ) and h a (hagiwara et al., ) , as identified in this study in llama. other histones that are known to undergo deimination include h and h (chen et al., ; kosgodage et al., ) . heat shock protein (hsp ) was here found to be deiminated both in llama whole serum and evs. hsp has been described in camelid (saeed et al., ) . hsp is a phylogenetically highly conserved chaperone protein involved in protein folding, stabilisation of proteins against heat stress, and aids in protein degradation (buchner, ; picard, ) . hsp also stabilizes a number of proteins required for tumour growth and is therefore important in anti-cancer drug investigations (goetz et al., ) . hsp is responsible for most of the atpase activity of the proteasome (imai et al., ) and has an atp binding region, which also is the main binding site of drugs (chiosis et al., ) . in camelids, hsp has been related to adaptive tolerance of camel somatic cells to acute and chronic heat shock (up to h at degrees celsius), which is lethal to many mammalian cells (saadeldin et al., b) . hsp has previously been described to be post-translationally deiminated in rheumatoid arthritis, allowing deiminationinduced shifts in protein structure to generate cryptic epitopes capable of bypassing b cell tolerance (travers et al., ) . it is of some interest to find that hsp is also found deiminated in llama serum, further highlighting protein moonlighting functions of hsp in physiological and pathophysiological context via post-translational deimination. in addition, finding post-translational deimination of the same protein throughout phylogeny also supports translational value between species to further understanding of this post-translational modification in human pathologies. the following deimination protein candidates identified in the current study in llama serum and serum-evs have to our knowledge not been previously reported as deiminated: alpha -macroglobulin is closely related to other thioester containing proteins, such as complement proteins c , c and c (sottrup-jensen et al., ; davies and sim, ) . alpha- -m is phylogenetically conserved from arthropods to mammals and found at high levels in mammalian plasma. alpha- -m forms part of the innate immune system and clears active proteases from tissue fluids (armstrong and quigley, ) . here, alpha- -m was found deiminated in whole llama serum only and has not reported as deiminated before, to our knowledge. adiponectin is the most abundant secreted adipokine with pleiotrophic roles in physiological and pathophysiological processes (fiaschi, ) . it has received considerable interest in the field of metabolic and obesity research (frankenberg et al., ; spracklen et al., ) , as well as in diabetes (yamauchi et al., ) , due to its key function in regulating glucose (yamauchi et al., ) . adiponectin is furthermore linked to longevity (chen et al., a (chen et al., , b , regenerative functions (fiaschi et al., ) and has roles in myopathies, such as duchenne muscular dystrophy and collagen vi-related myopathies (gamberi et al., ) . adiponectin is also implicated in a range of cancers, often in relation to obesity (parida et al., ) . furthermore, adiponectin plays roles in reproduction, embryo pre-implantation and embryonic development (barbe et al., ) . due to the range of functions in relation to key pathophysiologies there is great interest in drug development for modulating adiponectin signalling (fiaschi, ) . given the unique metabolic adaptive features of camelids, the identification of post-translational deimination of this key metabolic protein may be of some interest. recent studies in rheumatoid arthritis made a correlation between inflammation, autoantibodies and adiponectin levels (hughes-austin et al., ; liu et al., ) , while adiponectin itself has not been previously identified to be deiminated to our knowledge. post-translational deimination may be a hitherto unrecognized mechanism for adiponectin, also in humans, to adapt moonlighting functions via changes in protein folding and therefore interaction with other proteins. adiponectin is a small aa protein (np_ . ) in humans and contains unfolded regions and arginine sites, while camel adiponectin has arginine sites. these could be subjected to pad-mediated deimination and therefore modulate adiponectin folding and function, depending on which arginine is deiminated. here, deiminated adiponectin was identified in whole llama serum only. dystonin is a plakin-family adhesion junction plaque protein and was here identified as deiminated in both llama serum and evs. dystonin, along with xvii collagen, form hemidesmosomes and both proteins are autoantigens believed to be responsible for the type ii hypersensitivy pathologic in the pruritic skin disease bullous pemphigoid (bağcı et al., ; basseri et al., ) . dystonin has also been linked to sjögren's syndrome and linked to a hypermethylation status (gonzalez et al., ) , but post-translational deimination of dystonin has not been previously described to our knowledge, although deimination is associated with a range of autoimmune diseases, including sjögren's syndrome (konsta et al., ; selmi et al., ) . xaa-pro dipeptidase, also known as prolidase, is an enzyme that in humans is encoded by the pepd gene. post-translational modifications of prolidase regulate its enzymatic abilities. deficiency in prolidase leads to a rare, severe autosomal recessive disorder (prolidase deficiency) that causes many chronic, debilitating health conditions in humans (viglio et al., ) . these phenotypic symptoms vary and may include skin ulcerations, mental retardation, splenomegaly, recurrent infections, photosensitivity, hyperkeratosis, and unusual facial appearance. furthermore, prolidase activity was found to be abnormal compared to healthy levels in various medical conditions including: bipolar disorder, breast cancer, endometrial cancer, keloid scar formation, erectile dysfunction, liver disease, lung cancer, hypertension, melanoma, and chronic pancreatitis (kitchener and grunden, ) . in some cancers with increased levels of prolidase activity, such as melanoma, the differential expression of prolidase and its substrate specificity for dipeptides with proline at the carboxyl end suggests the potential of prolidase in becoming a viable, selective endogenous enzyme target for proline prodrugs (mittal et al., ) . serum prolidase enzyme activity is also currently being explored as a biomarker for diseases including chronic hepatitis b and liver fibrosis (duygu et al., ; Şen et al., ; stanfliet et al., ) . phosphorylation of prolidase has been shown to increase its activity while dephosphorylation leads to a decrease in enzyme activity. post-translational deimination of prolidase has not been described before to our knowledge and may add to understanding of how this enzyme is regulated. prolidase was here identified as deiminated in both llama serum and evs. dipeptidylpeptidase (dpp , also known as cd ) was here identified to be deiminated in both llama whole serum and evs. dpp controls glucose homeostasis and has complex roles in inflammation and homeostasis, including in liver cytokine expression, while its activity in plasma has been shown to correlate with body weight and fat mass (varin et al., ) . interestingly, in camel milk, ddp inhibitory peptides have been identified and suggested to play roles in the regulation of glycaemia in humans (nongonierma et al., ) . furthermore, roles for ddp in cancer have been found to relate to its posttranslational processing of chemokines, thereby limiting lymphocyte migration to sites of inflammation and tumours (barreira da silva et al., ) . as the success of antitumour immune responses depends on the infiltration of solid tumours by effector t cells, a process which is guided by chemokines, dpp inhibitors have been suggested as a strategy to enhance tumour immunotherapy (barreira da silva et al., ) . furthermore, serum ddp activity levels in primary hiv infection were found to be significantly decreased and to correlate with inflammation and hiv-induced intestinal damage (ploquin et al., ) . middle east respiratory syndrome coronavirus (mers-cov) has been found to utilize dipeptidyl peptidase (dpp ) as an entry receptor, via glycosylated sites (peck et al., ) . therefore the identification of dpp as a deimination candidate may be of some relevance as such post-translational modification can affect dpp structure and function, allowing for moonlighting functions which may vary in pathological compared to pathophysiological milieus. e ubiquitin-protein ligase roquin, belongs to the roquins which are a family of highly conserved rna-binding proteins involved in ubiquination, are crucial for t-cell-dependent b-cell responses (athanasopoulos et al., ) and play important roles in modulating tcell activity (akef and muljo, ) . roquins repress constitutive decay elements containing mrnas and play a critical role in rna homeostasis and immunological self-tolerance essig et al., ; mino and takeuchi, ) . roquin plays multifaceted roles both in the generation of a homeostatic immune response, as well as during chronic inflammation and autoimmunity (schaefer and klein, ; lee et al., ) . while roquin causes post-translational ubiquination of target proteins, post-translational deimination of roquin itself may modulate is function and is described here for the first time. roquin was here identified as deiminated in whole llama serum only. serine-trna ligase, mitochondrial was here identified as a deimination candidate protein in llama evs only. it has been linked to hupra syndrome which is a rare mitochondrial disease characterized by hyperuricemia, pulmonary hypertension, renal failure in infancy and alkalosis (rivera et al., ) . it has furthermore been linked to progressive spastic paresis (linnankivi et al., ) . post-translational deimination of this mitochondrial protein has not been described before to our knowledge. nucleoredoxin (nrx) was here identified to be deiminated in evs only. it is an oxidoreductase of the thioredoxin family of proteins with numerous functions in the redox regulation of metabolic pathways, cellular morphology, and signal transduction (urbainsky et al., ) . nrx has been shown to inhibit wnt-beta-catenin signalling (funato et al., ) and is linked to ca + -mediated mitochondrial reactive oxygen species metabolism (rharass et al., ) . nrx has furthermore been identified as an epigenetic cancer marker related to the oxidative status of human blood (schöttker et al., ) , but hitherto not been described as deiminated. dyslexia-associated protein was here identified to be deiminated in total serum of llama. it is associated with developmental dyslexia (levecque et al., ) and has been shown to have roles in cell-cell interactions and signalling and neuronal migration (velayos-baeza et al., ) , as well as in axon guidance (poon et al., ) . dyslexiaassociated protein has previously been found to follow the classical clathrin-mediated endocytosis pathway and its surface expression seems regulated by endocytosis, indicating that the internalization and recycling of the protein may be involved in fine-tuning its role in neuronal migration (levecque et al., ) . it has been descried to be highly glycosylated in different mammalian cell lines (velayos-baeza et al., ) , while deimination has not been described before. metabotropic glutamate receptor was identified here as deiminated in llama evs only. it has, like other components of the glutamatergic system, a widespread distribution outside the central nervous system (cns) and has been linked to regulation of the brain-gut axis (julio-pieper et al., ) . it has also been linked to pain (acher and goudet, ) and psychotic disorders (matrisciano et al., ) . the group iii mglu receptors have been described in human stomach and colon, revealing a huge potential for these receptors in the treatment of peripheral disorders, including gastrointestinal dysfunction (julio-pieper et al., ) . as post-translational deimination has not been described before in this receptor, but may well affect is tertiary structure, our current finding may be of some relevance in relation to strategies for developing antagonistic probes (wenthur et al., ) . such pharmacological tools originally designed for mglu receptors in the cns may also be directed towards new disease targets in the periphery. ulcerative colitis and crohn's disease are potential targets, as irritable bowel diseases can be co-morbid with anxiety and depression (julio-pieper et al., ) . glutathione synthetase (gss) was here identified to be deiminated in both llama whole serum and evs. gss is the second enzyme in the glutathione (gsh) biosynthesis pathway involved in homeostasis and cellular maintenance and also acts as a potent antioxidant (njålsson and norgren, ) . in camelids, glutathione peroxidase, which also belongs to the gsh biosynthesis pathway and is a potent anti-oxidant, has been identified as a seminal plasma fertility biomarker (waheed et al., ) . furthermore, camel milk has been shown to boost glutathione and total anti-oxidant capacity in sera and exudates in animal studies (arab et al., ) . in a diabetic mouse model, the activities of gsh, alongside glucose insulin and ros levels, were restored after camel whey protein treatment (sayed et al., ) . in humans, gss deficiency is an autosomal recessive disorder with varyingly severe clinical manifestations that include metabolic acidosis, hemolytic anemia, hyperbilirubinemia, neurological disorders and sepsis (guney varal et al., ) . deimination of gss identified here has not been assessed before and may possibly affect the gsh biosynthesis pathway and such posttranslational regulation remains to be further investigated. rootletin, also known as ciliary rootlet coiled-coil protein (crocc), is a protein that is required for centrosome cohesion and is therefore important in mitosis (bahe et al., ; graser et al., ) . it was identified here as deiminated in whole llama serum only. rootletin has been shown to be phosphorylated and to have the ability to form centriole-associated fibers, suggesting a dynamic model for centrosome cohesion based on entangling filaments (bahe et al., ) . deletion of rootletin in mouse models causes photoreceptor degeneration and impaired mucociliary clearance, supporting its key function in rootlet structures (yang et al., ) . post-translational deimination of rootletin, as identified here, may possibly facilitate its dynamic functions. centromere protein j is is a highly conserved and ubiquitiously expressed centrosomal protein, involved in microtubule disassembly and plays a structural role in the maintenance of centrosome integrity, genome stability and normal spindle morphology during cell division (tang et al., ; mcintyre, ) . it was here identified as deiminated in whole llama serum only. knockout mouse models targeting the gene encoding this protein have phenotypes of impaired glucose tolerance, hypoalbuminemia and increased micronuclei, indicative of genomic instability (gerdin, ) . in humans, it is associated with primary autosomal recessive microcephaly (gul et al., ) and the microcephalic primordial dwarfism disorder seckel syndrome (al-dosari et al., ; mcintyre et al., ) . deimination has not bee described before in this protein to our knowledge. telomere-associated protein rif isoform is involved in the repair of double-strand dna breaks in response to dna damage (silverman et al., ; escribano-díaz et al., ; drané et al., ) and protects teleomeres (fontana et al., ) . it was identified here as deiminated in evs only. rif has been associated with cancer via activation of human telomerase reverse transcriptase expression . it is also required for immunoglobulin class-switch recombination during the germinal centre reaction for humoral antibody immunity (di virgilio et al., ) . rif is involved in telomere homeostasis and was recently found to be post-translationally s-acylated, identifying a novel posttranslational modification regulating dna repair (fontana et al., ) . post-translational deimination has hitherto not been described. rabenosyn- -like protein was here identified as a deimination protein candidate in both llama whole serum and evs. it acts in early endocytic membrane fusion and membrane trafficking of recycling endosomes (naslavsky et al., ; rai et al., ) . it plays a role in the lysosomal trafficking of ctsd/cathepsin d from the golgi to lysosomes and also promotes the recycling of transferrin directly from early endosomes to the plasma membrane (navaroli et al., ) . rabenosyn- -like protein also binds phospholipid vesicles and plays roles in regulating protein sorting and recycling to the plasma membrane (nielsen et al., ; de renzis et al., ; naslavsky et al., ) . rabenosyn- was recently identified as an upregulated urinary biomarker associated with malignant upper gastrointestinal cancer (husi et al., ) and found to be increased in diabetic kidney disease (dumont et al., ) . rabenosyn- has been shown to be phosphorylated, regulating its recruitment to membranes (macé et al., ) . post-translational deimination has hitherto not been described.. spermatogenesis-associated protein (spata ) was here identified as deiminated in whole llama serum only. it is expressed in testis and to a lesser extent in spleen, thymus, and prostate (graziotto et al., ) . spata has been identified as a bridging factor that regulates tnf-alpha-induced necroptosis and is instrumental for tnf-induced cell death (elliott et al., ; kupka et al., ; schlicher et al., ; wagner et al., ; schlicher et al., ) . spata ensures normal secretory function of sertoli cells (zhao et al., ) and has recently been identified as a novel predictor in ovarian cancer outcome (wieser et al., ) . while spata has been linked to ubiquination (schlicher et al., ) , post-translational deimination has not been invesigated. vitamin d-binding protein (vdbp) belongs to the albumin gene family, together with human serum albumin and alpha-fetoprotein. it is a multifaceted protein mainly produced in the liver, where its regulation is influenced by estrogen, glucocorticoids and inflammatory cytokines (bikle and schwartz, ) . it is secreted into the blood circulation and is able to bind the various forms of vitamin d including ergocalciferol (vitamin d ) and cholecalciferol (vitamin d ), the hydroxylated forms (calcifediol), and the active hormonal product , dihydroxyvitamin d (calcitriol) (verboven et al., ; norman, ) . it transports vitamin d metabolites between skin, liver and kidney, and then on to various target tissues (norman, ) . vdbp is a macrophage activating factor (maf) and has been tested as an anti-cancer agent via activation of macrophages against cancer cells (yamamoto et al., ) . some association has also been made between polymorphisms of vdbp and the risk of coronary artery disease (tarighi et al., ) . post-translational modifications (which still remain to be identified) of vdbp have been associated with multiple sclerosis (ms) (perga et al., ) , while protein deimination is well known to be associated with ms (moscarello et al., ) . vdbp has previously been identified to be glycosylated (kilpatrick and phinney, ) but was here identified as a deimination candidate in whole llama serum only. post-translational deimination may contribute to various functions of vdbp in physiological as well as pathophysiological processes. pseudopodium-enriched atypical kinase (peak ) was here identified as deiminated in evs only. it is a tyrosine kinase and scaffold protein that transmits integrin-mediated extracellular matrix (ecm) signals to facilitate cell movement and growth. while aberrant expression of peak has been linked to cancer progression, its normal physiological role in vertebrate biology is still relatively unknown. deletion of the peak gene in zebrafish, mice and human endothelial cells (ecs) induced severe defects in new blood vessel formation due to deficiencies in ec proliferation, survival and migration. peak seems therefore to play roles in modulation of cell adhesion and growth factor cues from the extracellular environment necessary for new vessel formation during vertebrate development and cancer (wang et al., ) . peak has not been described as deiminated before. desmoplakin was here identified to be deiminated in llama evs only. desmoplakin is a unique and critical component of desmosomal cell-cell junctions and involved in integrity of the cytoskeletal intermediate filament network (bendrick et al., ) . it has been shown to be required for epidermal integrity and morphogenesis in the xenopus laevis embryo (bharathan and dickinson, ) and novel roles in coordination of cell migration were recently established (bendrick et al., ) . mutations in desmoplakin have been linked to multiple allergies, severe dermatitis and metabolic wasting (sam) syndrome (liang et al., ) . it is also linked to carvajal syndrome, involving altered skin and hair abnormalities, and heart diseases (yermakovich et al., ) , including cardiomyopathies (reichl et al., ; chen et al., a chen et al., , b . desmosomal proteins have been shown to have both tumourpromoting and tumour-suppressive functions, depending of cancer types and can regulate cell proliferation, differentiation, migration, apoptosis, and impact treatment sensitivity in different types of cancers (zhou et al., ) . as the roles of desmosomal proteins in cancer and metastasis are not fully understood, the identification of deiminated desmoplakin in llama evs here, and not described before, may be of some interest and add to understanding of diverse functional ability via such post-translational modification. tsc domain family protein , also called glucocorticoid-induced leucine zipper protein (gilz), was here identified as a deimination candidate in llama evs only. it is a glucocorticoid-responsive molecule involved in immune regulation and glucocorticoid actions. its interactions with signal transduction pathways, many of which are operative in ra and other inflammatory diseases, suggest that it is a key endogenous regulator of the immune response including a key glucocorticoid-induced regulator of inflammation in rheumatoid arthritis (ra) (beaulieu and morand, ) . giltz is a small, -amino acid protein with anti-inflammatory properties and has been shown to inhibit nf-κb and mapk pathways (bereshchenko et al., ; ricci et al., ) . it has also been shown to be induced in response to hypoxia by a hif α-dependent mechanism and in response to cholesterol starvation, leading to downstream shedding of procoagulant evs in ovarian cancer (koizume et al., ) . post-translational deimination of gilz has not been described before but may indeed affect its multifaceted functions, including in inflammation and cancer. in the current study we report deiminated proteins in llama serum and serum-derived evs. due to the fact that the llama genome is not yet fully annotated, the hits identified here may underestimate the amount of deiminated proteins present in llama serum and evs. therefore a wider protein-hit analysis was carried out including other members of the camelid family, using known sequences from camelus ferus, camelus dromedaries, lama guanicoe and vicugna pacos. deimination of key immune factors of innate and adaptive immunity and key metabolic proteins is identified here for the first time in a camelid species, highlighting putative protein moonlighting functions via post-translational deimination. it must be noted that in relation to previously observed increases of deiminated proteins with age (ding et al., ) , the llama used in this study would be considered relatively old at years of age, as typical llama lifespans are - years, with some individuals surviving years or more. our findings presented here furthermore complement expanding research in the comparative ev research field. previous studies on the camel urinary proteome revealed enriched proteins from evs and relevance to stress and immune responses as well as antimicrobial activities (alhaider et al., ) . research on evs is a relatively new field in comparative immunology and to our knowledge; this is the first description of evs in serum of a camelid species. previous studies on evs in camelids focussed on evs in camel milk, which were shown to have anti-cancerous properties (badawy et al., ) . as pads have been identified to play major roles in the regulation of ev release (kholia et al., ; kosgodage et al., and , including in host-pathogen interactions (gavinho et al., ) , such ev-mediated communication may be of great relevance also for addressing diverse zoonotic diseases identified in camels (el-alfy et al., ; zhu et al., ) . in continuation of the current pilot study, the assessment of changes in deiminated proteins in camelid serum, and their lateral transfer via evs, will be of great interest to assess animal health in response to infection and environmental stress. our findings will further current understanding of the roles for evs, pads and posttranslational deimination throughout phylogeny and in relation to adaption to a range of, including extreme, environments. furthermore, novel identification of post-translational deimination in key proteins of metabolism and immunity may reveal hitherto unrecognized moonlighting function of these proteins throughout phylogeny, in relation to physiological and pathological processes, as well as being translational to and informing inflammatory and metabolic diseases. pad-mediated contribution to protein moonlighting and in ev-mediated communication in response to physiological and pathophysiological changes remains therefore a field of further studies. this is the first report of deiminated proteins in serum and serum-evs of a camelid species, using the llama as a model animal. our findings highlight a hitherto unrecognized post-translational modification in key immune and metabolic proteins in camelids, which may be translatable to and inform a range of human 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drugs across the blood-brain barrier for brain cancer therapy in danio rerio novel desmoplakin mutations in familial carvajal syndrome shark igw c region diversification through rna processing and isotype switching new insights into the rna-binding and e ubiquitin ligase activities of roquins preferential combination between the light and heavy chain isotypes of fish immunoglobulins molecular characterization and expression analysis of three subclasses of igt in rainbow trout (oncorhynchus mykiss) deletion of spata by crispr/cas n causes increased inhibin alpha expression and attenuated fertility in male mice the role of desmosomes in carcinogenesis structure and function of inter-alpha-trypsin inhibitor heavy chains a review of zoonotic pathogens of dromedary camels the authors would like to thank yagnesh umrania and michael deery at the cambridge centre for proteomics for the lc-ms/ms analysis, as well as alice blue-mclendon of texas a&m university's winnie carter wildlife center for sharing llama serum. this study was funded in part by a university of westminster start-up grant to sl and u.s. national science foundation grant ios- to mfc. thanks are due to the guy foundation for funding the purchase of equipment utilised in this work. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.molimm. . . . key: cord- -g tf jz authors: huang, ke-yan; yang, gui-lian; jin, yu-bei; liu, jing; chen, hong-liang; wang, peng-bo; jiang, yan-long; shi, chun-wei; huang, hai-bin; wang, jian-zhong; wang, guan; kang, yuan-huan; yang, wen-tao; wang, chun-feng title: construction and immunogenicity analysis of lactobacillus plantarum expressing a porcine epidemic diarrhea virus s gene fused to a dc-targeting peptide date: - - journal: virus res doi: . /j.virusres. . . sha: doc_id: cord_uid: g tf jz porcine epidemic diarrhea virus (pedv) is one of the most important causative pathogens of swine diarrhea, which is widely prevalent throughout the world and is responsible for significant economic losses in the commercial pig industry, both domestic and abroad. the spike (s) protein in the pedv capsid structure can carry the major b lymphocyte epitope, which induces production of neutralizing antibodies and provides immunoprotective effects. moreover, the conserved region encoded by the s gene can be considered a target for establishing a new diagnostic method and is a new candidate for vaccine design. in this study, use of anchorin pgsa' allowed the fusion protein of s-dcpep to express on the surface of recombinant lactobacillus plantarum (nc -psip -pgsa'-s-dcpep) nc strain. mice were immunized by lavage administration of the recombinant nc -psip -pgsa'-s-dcpep, which was observed to induce dc activation and high production of siga and igg antibodies in experimental animals, while also eliciting production of significantly more iga(+)b (+) b cells. more importantly, secretion of cytokines ifn-γ, il- and il- in mice that were vaccinated with nc -psip -pgsa'-s-dcpep was remarkably increased. the results of our study suggest that nc -psip -pgsa'-s-dcpep potently triggers cellular and humoral immune responses. the obtained experimental results can provide a theoretical basis that lays the foundation for production of a novel oral vaccine against ped. porcine epidemic diarrhea (ped) is a highly contagious disease with typical symptoms in piglets that include severe diarrhea, vomiting and intestinal dehydration (ko et al., ) . the morbidity and mortality rates of suckling piglets caused by pedv are more than %, and it is the primary cause of death in piglets and weight loss in fattening pigs (choi et al., ) . this viral disease was first reported in britain in and then became an epidemic in other european countries (yu et al., ) . in , pedv was detected for the first time in china, and since october , pedv outbreaks worldwide have become more frequent (song et al., ) . for instance, ped broke out in minnesota and iowa in the united states in , resulting in huge losses to the pig industry (diep et al., ; lee, ) . as a coronavirus, pedv has a typical structure that consists of four structural proteins, including the nucleocapsid protein (n), a membrane protein (m), a glycosylated spike protein (s) and a envelope protein (e) (kocherhans et al., ) . the pedv spike protein-encoding gene is an immune dominant gene that encodes a protein with a very dense hydrophobic region of amino acids that harbors multiple glycosylation sites and has a high antigenic index . several studies have reported that the s protein is a structural surface protein of pedv, containing both the receptor binding domain, which mediates invasion of the virus into the host cell, and the antigenic epitope, which mediates production of the neutralizing virus antibody . thus, the s-glycoprotein plays a crucial role in viral infection, pathogenicity and adsorption. it has been shown that the s-glycoprotein is immunogenic and has the potential to be a good vaccine candidate (fan et al., ) . lactic acid bacteria (lab) are diverse species that are widely distributed in nature and produce large amounts of lactic acid by fermenting carbohydrates (riaz rajoka et al., ) . lab, which are generally recognized as safe bacteria, colonize the intestines of humans or animals and have potentially beneficial effects (landete et al., ) . the study of lab has many advantages, including that they are easy to culture, convenient to manipulate and highly safe, which is why lab can be regarded as a heterologous antigen protein delivery system (wanker et al., ) . l. plantarum has been manipulated to deliver a number of viral antigens in previous studies (li et al., b; yang et al., c) . the l. plantarum strain nc was isolated from ensilages and has a better antireversion characteristic compared with lab isolated from healthy animal intestines (caggianiello et al., ; park et al., ) . thus, l. plantarum nc is more suitable to act as a mucosal delivery shuttle vector to express protective antigens. recently, poly-γ-glutamic acid synthetase a (pgsa) was identified as being a constitutively expressed protein of the polyglutamate acid (pga) synthetase system of bacillus subtilis. pgsa can stably anchor γ-pga synthetase on the cell membrane, acting as a transport carrier of γ-pga and playing a crucial role in the formation of γ-pga (sung et al., ) . the pgsa protein has a transmembrane region near its n-terminus ( - amino acid residues), providing the necessary characteristics for the establishment of a pgsa transmembrane expression system. the pgsa gene was combined with the target gene for fusion expression, after which l. plantarum was used as a mucosal vehicle to deliver an exogenous antigen. this experiment provided a theoretical basis for the construction of a novel, orally administered, genetic engineered vaccine to immobilize exogenous medicinal proteins on the cell wall surface of l. plantarum (cai et al., ; narita et al., ) . dendritic cells (dcs) are the most powerful functional antigen presenting cells (apcs) and are the exclusive apcs that are capable of inducing initial t and b cell activation . dcs can elicit t cell polarization and differentiation in t cells subsets, which also trigger secretion of ifn-γ (subramaniam et al., ) . dcs are involved in regulating the function of b cells and promoting the differentiation of b cells into plasma cells to produce igg . in the germinal center, dcs can boost b cell antibody affinity maturation and help to form and maintain memory b cells. in addition, siga can play an important role in maintaining intestinal integrity and intestinal symbioses (shaw et al., ) . dcs can restrict penetration of microorganisms into the mucosa and promote uptake of antigens in the intestinal mucosal system (owen et al., ) . it has been reported that nc -psip -pgsa'-s-dcpep can specifically conjugate dendritic celltargeting peptide (dcpep) and induce an immune response (apostolico et al., ) . to make dcs recognize the s-antigen more effectively in the intestinal mucosa, recombinant nc -psip -pgsa'-s-dcpep was constructed using standard molecular biology techniques, and its immunogenicity was further investigated. after balb/c mice were immunized by lavage administration of our recombinant l. plantarum, the results indicated that the in vivo antibody levels were greatly improved. the escherichia coli-lactobacillus shuttle vector psip (sorvig et al., ) and l. plantarum nc were kindly provided by professor a. kolandaswamy (madurai kamaraj university, india). the pm -t-s-dcpep (dc peptide, fypsyhstpqrp) was stored at jilin provincial engineering research center of animal probiotics in jilin agricultural university. the pm -t-s-dcpep plasmid was cut at xbai and hindiii sites within the primers using the appropriate restriction enzymes at °c for - h. next, the digested bp s gene fragment (spike protein gene genbank: jq . (source: - )/spike protein genbank: aew . (source: - )) and dcpep (s-dcpep) were ligated into psip -pgsa' using t dna ligase at °c overnight. the recombinant psip -pgsa'-s-dcpep plasmid was detected and purified using a gel extraction kit (axygen). the recombinant plasmid was introduced into the l. plantarum nc strain by electroporation and erythromycin-resistant bacteria were selected for, after which the plasmid was sequenced. in addition, to meet the rigorous nature of the experimental study, a nc -psip -pgsa'-s-ctrlpep (control peptide, epihpettftnn) was constructed similar to nc -psip -pgsa'-s-dcpep and used as the experimental control group. to examine the expression of the s-protein on the surface of recombinant nc -psip -pgsa', nc -psip -pgsa'-s-ctrlpep and nc -psip -pgsa'-s-dcpep was cultured in mrs with μg/ml erythromycin. as described in previous studies, we added sakacin p (sppip) to the culture medium when the (od ) of the medium was between . and . . after induction at °c for h, cells from each strain were washed with pbs containing % bovine serum albumin. next, cells were resuspended in pbs and × cfu of bacteria were incubated with ml of primary antibody ( / anti-s-produced in rat) for h at °c with shaking at rpm. the pellets were washed with pbs containing . % tween- (pbs-t), after which secondary antibodies (fitc, goat anti-rat igg (h + l), cst) were added and the mixture was incubated for one hour at °c with shaking at rpm in the dark. finally, samples were washed with pbs-t and resuspended in pbs. the cells were examined under a laser scanning confocal microscope (lsm ; carl zeiss, germany) following the manufacturer's instructions. to detect the s-protein expression levels, we induced its expression in recombinant lab as described previously. strains were subsequently resuspended in μl of pbs and were lysed by ultrasonic crushing as previously described (shi et al., ) . samples were assessed by % sds-page and transferred to pvdf membranes (millipore, usa). the s-ctrlpep and s-dcpep proteins were detected using an anti-s polyclonal primary antibody (polyclonal antibody was obtained from serum of mice which were immunized with the purified s protein) and a hrpconjugated goat anti-rat igg secondary antibody (a ; abbkine, usa). the bands were visualized using enhanced chemiluminescence reagents in an ecl plus detection kit (thermo scientific) according to the manufacturer's instructions. for experiments involving animals, thirty-six female balb/c mice without specific pathogen (spf) were bought from beijing hfk bioscience co., ltd., china. according to the provisions of the animal care and ethics committees of jilin agriculture university. all animals were strictly raised with commercialized germ-free feed and sterilized water in the jilin provincial engineering research center of animal probiotics, which provided the pathogen-free environment facilities. six-week-old mice with no specific pathogens were randomly divided into four groups, with mice in each group (n = ). briefly, using oral gavage, four groups of mice were immunized with nc -psip -pgsa', nc -psip -pgsa'-s-ctrlpep, nc -psip -pgsa'-s-dcpep or saline. the entire immunization procedure was divided into three phases: prime immunization (days - ), booster immunization (days - ), and the last immunization (days - ), with cfu/mouse administered by oral gavage. generally, experimental mice were sacrificed by cervical dislocation days after being immunized. lamina propria (lp) cells of the small intestine, pps and mlns were acquired via a previously described method (kikuchi et al., ) on a clean sterile clean bench. small intestine samples were washed with pbs and cut into small pieces. next, samples were digested with lymphocyte separation medium, after which lp cell digestive juice was added to obtain single lp cells from the small intestine in an isotonic percoll solution. the cell suspension was filtered through a μm sterile filter-film and the filtrate was centrifuged at g for min at °c. subsequently, the cells were washed with °c pbs and were eventually resuspended in complete rpmi (containing % fbs, % penicillin and streptomycin). the method used for the analysis of s protein expression in recombinant l. plantarum was consistent with the immunofluorescence assay, with cells analyzed by flow cytometry (bd facs lsr fortessa™ flow cytometer). data were analyzed via flowjo . . software. to test the activation of dc surface costimulatory molecules and b cells, flow cytometry was conducted using a previously published protocol . in brief, single lp cells from the small intestine suspensions obtained from immunized mice were diluted to × cells/ μl and incubated with μl anti-cd c (clone hl ), cd (clone - a ), cd (clone gl ), cd (clone / ) and mhcii (clone / ) antibodies to assess the dc cells. to analyze b lymphocyte cells activation, μl of anti-b (clone ra - b ) and iga (clone c - ) antibodies was added to stain b lymphocyte cells for min at room temperature in the dark. afterwards, cell suspensions were washed twice with facs buffer. after staining, samples were examined using flow cytometry (bd facs lsr fortessa™ flow cytometer). data were analyzed using flowjo . . software. blood samples were obtained on days , and after immunization and were centrifuged at rpm for min at °c to obtain the serum, which was stored at − °c. the fecal samples were prepared according to our previously published method, followed by centrifugation at rpm for min at °c. the supernatants were acquired and stored at − °c for further analysis. elisa was performed to determine the levels of specific igg and siga antibodies (yang et al., d) . in brief, μl of purified s protein ( μg/ml) was added to each well of the -well plate and incubated overnight at °c. on the second day, the blocking solution was added and the plate was incubated overnight at °c. on the third day, the fecal samples ( : ) and serum samples ( : ) were diluted and added separately, after incubating at °c for h, the wells were rinsed. subsequently, goat anti-mouese igg (h + l)-biotin and goat anti-mouse iga-biotin (southern biotechnology, birmingham, al) was added separately, and the plate was incubated at °c for one hour, after which streptavidin-hrp (southern biotechnology, birmingham, al) secondary antibody was added and the plate was incubated at °c for min. finally, tmb substrate solution and stop solution was added, after which the color intensity was measured at nm. the end-point titers (log ) were defined as the highest dilution yielding an absorbance that was two and three times higher than the background for the fecal and serum samples, respectively . the mln cells were stimulated by s protein ( μg/ml) and the supernatant was collected days later and examined for specific cytokines, including ifn-γ, il- and il- a using an elisa kit (liuhebio, wuhan, china) according to the manufacturer's instructions. for determining the pedv neutralizing activity of the serum antibodies we detected sample obtained from mice which were orally immunized with recombinant l. plantarum by antibody neutralization assay (microdetermination) as described before (tian et al., ) with minor modifications. under aseptic conditions serum and fecal samples were collected at , and days respectively after immunization, quantitative virus and serial diluted serum were added to vero cells to evaluate serum neutralizing antibody titers, the neutralizing antibody titer pd (median protective dose refers to the dose of serum or vaccine that protects half ( %) of the experimental animals) was determined by karber's method at h. all results were performed by two-tailed t-tests and one-way analyses of variance (anova) (graphpad prism . , graphpad software) were used to analyze the significance of the difference between means. all data were obtained from at least three independent experiments, and the geometric mean titers were expressed as averages. all data were expressed as the means ± sem. the psip -pgsa'-s-dcpep and psip -pgsa' plasmids were introduced separately into l. plantarum nc strain by electroporation (fig. a) . using flow cytometry, s protein expression levels of recombinant l. plantarum nc -psip -pgsa'-s-dcpep and nc -psip -pgsa'-s-ctrlpep were determined (fig. b) . the green fluorescence marked by white arrows indicated that recombinant lactobacillus of nc -psip -pgsa'-s-dcpep and nc -psip -pgsa'-s-ctrlpep expressed s protein on the surface (fig. c ) than nc -psip -pgsa', which was barely observed with green fluorescence under a confocal microscope. western blot analysis showed that a specific band appeared at kda, which demonstrated that the pedv s protein had been expressed. thus, the experimental results suggest that nc -psip -pgsa'-s-dcpep and nc -psip -pgsa'-s-ctrlpep were expressed in l. plantarum nc . to determine the effect of nc -psip -pgsa'-s-dcpep on dcs, flow cytometry was performed to test the level of costimulatory molecules activated by dcs, which were induced by nc -psip -pgsa'-s-dcpep gavage immunization days after the last immunization ( fig. a) . compared with the saline group, the expression level of cd c + cd + of dcs surface molecules in the lp cells of the small intestine was significantly increased in the nc -psip -pgsa'-s-dcpep group (p < . ) and nc -psip -pgsa'-s-ctrlpep group (p < . ) experimental groups (fig. b) . moreover, our experimental results indicated that nc -psip -pgsa'-s-dcpep can promote greater expression of cd + in lp dcs than the nc -psip -pgsa'-s-ctrlpep (p < . ), nc -psip -pgsa' (p < . ) or saline (p < . ) groups (fig. c) . however, no obvious differences were observed in cd c + cd + expression of dcs surface molecules in the lp cells of the small intestine. the number of iga + b + b cells in pp was determined by flow cytometry days after the last immunization (fig. a) . the results showed that there was a remarkable increase in the percentage of iga + b + b cells within the b cell populations of balb/c mice having received lavage administration with nc -psip -pgsa'-s-dcpep compared with the nc -psip -pgsa', nc -psip -pgsa'-s-ctrlpep and saline groups. in addition, nc -psip -pgsa'-s-dcpep in pp significantly increased the production of iga + b + b cells compared to the nc -psip -pgsa' and saline group control (p < . ) (fig. b) , and the nc -psip -pgsa'-s-dcpep was significantly higher than the nc -psip -pgsa'-s-ctrlpep groups in pp (p < . ) (fig. b) . collectively these results demonstrated that vaccination with the nc -psip -pgsa'-s-dcpep can activate the b cell response in mice. the specific siga levels in fecal intestines were measured at days , and after the first immunization using elisa (fig. ) . however, the concentration of siga in the excrement of mice treated with nc -psip -pgsa'-s-dcpep was higher than that of the nc -psip -pgsa' (p < . ) and saline (p < . ) groups at days (fig. ) , and nc -psip -pgsa'-s-dcpep was higher than that of the nc -psip -pgsa' (p < . ) and saline (p < . ) groups at day (fig. ) . in contrast, no obvious difference was observed with respect to slga was observed at days and in the nc -psip -pgsa'-s-dcpep and nc -psip -pgsa'-s-ctrlpep groups (fig. ) . with respect to the day lavage administration, the nc -psip -pgsa'-s-dcpep group was higher than that of the nc -psip -pgsa' (p < . ) and saline (p < . ) groups. higher expression of siga was also observed in nc -psip -pgsa'-s-dcpep group compared with nc -psip -pgsa'-s-ctrlpep group (p < . ) at day (fig. ) . these data suggested that lavage administration of nc -psip -pgsa'-s-dcpep could stimulate the mucosal immune system in mice. the specific igg-antibodies level after lavage administration of nc -psip -pgsa'-s-dcpep in mice was notably increased compared with the nc -psip -pgsa ', nc -psip -pgsa'-s-ctrlpep and saline groups at days , and after the first immunization. the results showed that the concentration of specific igg in the serum of mice treated with nc -psip -pgsa'-s-dcpep was significantly increased compared to the nc -psip -pgsa' (p < . ) and saline (p < . ) groups at days and (fig. ). in addition, only nc -psip -pgsa'-s-dcpep showed higher levels than the nc -psip -pgsa'-s-ctrlpep (p < . ), nc -psip -pgsa' (p < . ) and saline (p < . ) groups at day (fig. ) , with an increasing trend from days to (fig. ) . these data suggested that lavage administration of nc -psip -pgsa'-s-dcpep could increase iga antibody titers and produce humoral immune responses in mice. specific cytokines in the mln cell secretions were measured days after the last immunization using elisa. the results showed that the concentration of il- in the supernatants of mln cells cultured from the nc -psip -pgsa'-s-ctrlpep and nc -psip -pgsa'-s-dcpep-treated mice were significantly increased compared to those from the saline group (p < . ) (fig. a) . higher level of il- was observed in the group of mice orally immunized with recombinant nc -psip -pgsa'-s-dcpep compared to the group of mice orally administered with saline (p < . ), nc -psip -pgsa'-s-ctrlpep and nc -psip -pgsa' groups (p < . ) (fig. c) . unexpectedly, the level of ifn-γ in the supernatant of mln cells cultured with the strains expressing s-dcpep was significantly higher in the group of mice orally immunized with recombinant nc -psip -pgsa'-s-dcpep compare to the group of mice orally administered with saline (p < . ), nc -psip -pgsa'-s-ctrlpep and nc -psip -pgsa' groups (p < . ) (fig. b ). fig. . detection of activated b cells in pp of immunized mice using flow cytometry. the number of total cells for (a) the quadrants and (b) the bar graphs refer to iga + b + cells. the degree of activation of b cells was analyzed by counting the percentage of iga + b + expression. significant differences relative to the saline and nc -psip -pgsa' controls (p < . , p < . ). significant differences are denoted by an asterisk (*) between saline, nc -psip -pgsa', nc -psip -pgsa'-s-ctrlpep and nc -psip -pgsa'-s-dcpep controls (*p < . , **p < . , ***p < . ). k.-y. huang et al. virus research ( ) - . . nc -psip -pgsa'-s-dcpep induced production of serum/fecalneutralizing antibodies specific pedv antibodies titers in serum and faeces were measured by cell neutralization assays at different time after the first immunization. results showed that serum and fecal antibodies of mice orally immunized with recombinant nc -psip -pgsa'-s-dcpep was higher than that from mice orally administered with and saline at days (p < . ) (fig. ) . the serum antibody of mice orally immunized with recombinant nc -psip -pgsa'-s-dcpep was significantly increased compared with nc -psip -pgsa' at days (p < . ) (fig. a) , however, the fecal antibodies of nc -psip -pgsa'-s-dcpep was no significant difference at days compared with saline and nc -psip -pgsa' at days (fig. b ). significant differences were observed in both serum and fecal antibodies of mice orally immunized with recombinant nc -psip -pgsa'-s-dcpep compared with the other three group at days (fig. ) , particularly, difference were also observed in serum and fecal antibodies between the group of mice orally immunized with recombinant nc -psip -pgsa'-s-dcpep and nc -psip -pgsa'-s-ctrlpep (p < . ). the results suggesting that recombinant l. plantarum nc -psip -pgsa'-s-dcpep has good pedv virus-neutralizing activity in humoral and mucosal immunity. porcine epidemic diarrhea (ped) caused by pedv is a contagious disease which seriously threaten pig production industry (huang et al., ) . ped characterized by high morbidity and mortality in piglets was reported in southern china in fall of (huang et al., . furthermore, in april ped has also spread throughout and to the highest degree destroyed the american pork industry, the homology between the genome of popular strain in american and china is as high as %- %, which has caused immeasurable economic losses (huang et al., ) . the continuing outbreak of ped suggests that it is able elude current immunization strategies (curry et al., ) . currently, there is no effective drug for pedv treatment, although inactivated and live vaccines are the two most commonly used vaccines, although live and inactivated vaccines are already in widespread use, development of effective, safe and economical oral vaccines are still the trend of future. and researchs of developing oral vaccine to prevent pedv have achieved effective protection. l. plantarum is an intestinal predominant flora with immunomodulatory effects that enhance the chemotaxis and phagocytosis of neutrophils, prevent viral infection and maintain intestinal microbial homeostasis (li et al., a,b) . therefore, l. plantarum with probiotic properties has become good exogenous protein vector platform. the anchorin expressed by pgsa gene can help exogenous proteins to locate to the surface of bacterium and display their biological function. other and our team have achieved good protection using pgsa to display exogenous antigens, hence, using pgsa to display exogenous antigens during vaccine development is a viable strategy. the feature of localized intestinal infection of pedv determined that the immunization and prevention is difficult, hence, intestinal mucosal immunity plays an important role in pigs except suckling piglets (temeeyasen et al., ) . resistance to pedv infection often relies on iga secreted by mucosa, which requires the development of vaccines that can effectively increase intestinal immunity. contemporary strategies in vaccination primarily focus on using different vectors to express the identified antigenic regions. antigens delivered by different routes might stimulate the intestinal mucosal immune system to achieve immunoprophylaxis effect. the use of probiotics to express antigens and proliferate them in the intestinal of host can promote the digestion of livestock and poultry; meanwhile, the protective antigen expressed can stimulate the intestinal mucosal immune system to produce protective effect. as a favorable mucosal immunization vaccine vector, l. plantarum can also be used as a natural adjuvant in the mucosal microenvironment and attenuate inflammatory response to enhance the body immunity (yu et al., ) . morever, vaccines constructed by dcpep could provide effective protection (yang et al., a,b) . consequently, we constructed fusion protein by the immunogenic s-protein of pedv and the induced dcpep and expressed it on the surface of l. plantarum, which could activate dcs to initiate mucosal and humoral immune responses against microbial infection via secreting antibodies to clear the antigens. results showed that the dcpep fusion group had better immune protection compared with other groups. dcs are the foremost antigen presenting cells (apcs) and play an important role in inherent and adaptive immunity (sato et al., ) . cd c + is a surface marker of dcs, and cd + and cd + are subsets of cd c + . there was a difference in expression of cd c + cd + between the group of mice orally immunized with recombinant nc -psip -pgsa'-s-dcpep and saline group but not nc -psip -pgsa'-s-ctrlpep and nc -psip -pgsa' groups, however, the mei of group nc -psip -pgsa'-s-dcpep was still higher than the above two groups ( fig. a) . the expression of the cd c + cd + in nc -psip -pgsa'-s-dcpep group was significantly higher than the control group and the nc -psip -pgsa' group in the lp of small intestine (fig. b) . in this study, the expression of cd c + cd + in dcs was no significant difference between nc -psip -pgsa'-s-dcpep and saline group, which should also increased according to previous studies. this difference in results might be due to different host strains and different immunization programs. however, cd c + cd + did not affect the biological function of cd c + cd + and cd c + cd + dcs, which could also identify cd + , cd + , cd + and other co-stimulatory molecules, fig. . s-specific siga titers levels in mouse feces examined by elisa se. the s-specific siga ab titers were detected after prime immunization, booster immunization, and the last immunization. end-point titer (log ) were defined as the highest dilution yielding an absorbance that was two and three times higher than the background for the fecal and serum samples, respectively. significant differences are denoted by an asterisk (*) between stroke-physiological saline solution, nc -psip -pgsa', nc -psip -pgsa'-s-ctrlpep and nc -psip -pgsa'-s-dcpep controls (*p < . , **p < . , ***p < . ). s-specific igg titers levels in sera of mice detected by elisa. the s-specific igg ab titers were detected after prime immunization, booster immunization, and the last immunization. end-point titer (log ) were defined as the highest dilution yielding an absorbance that was two and three times higher than the background for the fecal and serum samples, respectively significant differences are denoted by an asterisk (*) between stroke-physiological saline solution, nc -psip -pgsa', nc -psip -pgsa'-s-ctrlpep and nc -psip -pgsa'-s-dcpep controls (*p < . , **p < . , ***p < . ). present antigen to activated t cells or effector t cells. meanwhile, cd + and cd + of activated b cells could combine with cd expressed on the surface of t cells, which involved in co-stimulatory signal transduction. in addition, the results of s-specific igg in serum and s-specific siga in feces showed that l. plantarum strains expressing s protein could improve the secretion of igg and siga and further enhance immune function, particularly for nc -psip -pgsa . several studies showed that iga secreted by mucosal lamina propria could combine with mucus and cover on the surface of epithelial cells which inhibit the adhesion of microorganisms to epithelial cells and neutralization of toxins produced by microorganisms or enzymes. siga agglutinates and incapacitates pathogens by which it inhibits pathogen adhesion to the mucosal surface and easily cleared in the secretions. therefore, siga disenables pathogens interact with epithelial cell receptors and inhibits the assembly of viral particles within the host cell cytoplasm (kurashima and kiyono, ; motegi et al., ) . our study suggesting that the anti-s mucosal siga responses of recombinant lactobacillus nc -psip -pgsa'-s-dcpep group significantly increased. following four indicators are used to evaluate the effectiveness of a vaccine in mucosal system: connecting closely with lymphoid tissue in mucous membrane; the existence of scattered lymphoid tissues and lymphoid organs with a certain structure; dcs use a special mechanism to uptake antigen; and the presence of many activated and memory lymphocytes in the absence of an infection (chung et al., ; faria and reis, ) . currently, many investigators are focused on the development of mucosal vaccines. our study demonstrated that the recombinant l. plantarum nc -psip -pgsa'-s-dcpep could increase the activation of b cells and further improve the level of humoral immunity (fig. ) . orally administration of recombinant nc -psip -pgsa'-s-dcpep in mice boosted the number and frequency of iga + b + b cells compared with the nc -psip -pgsa' and nc -psip -pgsa'-s-ctrlpep groups. similarly, we also detected anti-s systemic igg in the blood, the results showed that s-dcpep could effectively initiate a mucosal immune response and humoral immunity. subsequently, l. plantarum expressed s-dcpep fusion protein could enhance the secretion of mucosal antibodies against pathogens (mohamadzadeh et al., ; shi et al., ; yang et al., a yang et al., ,b, . in the construction of the recombinant strain nc -psip -pgsa'-s-dcpep, pgsa has been used as an anchorin for its stability and safety in clinical use. besides, pgsa has a wide range of applications for surface expression on some types of lactobacillus compared with other anchorins. therefore, we used pgsa as a surface display element for applying to surfaced of l. plantarum. a recombinant vaccine constructed by s-protein expressed on the surface of l. plantarum and dcpep fusion protein can provide protection since pedv is primarily replicated in the small intestine. the results of western blot, facs and confocal microscopy analyses suggested that s protein was expressed on the surface of l. plantarum. compared with traditional intramuscular injection, gavage administration allows antigens to be presented through the gastrointestinal mucosa and obtain better immunological response, which is a simple, convenient and ideal immunization strategy. several genetically engineered bacteria (salmonella, escherichia coli, lactobacillus) could colonize in the intestines after inoculation that were regarded as promising oral vaccine vectors (garcia-fruitos, ). in addition, peptidoglycan expressed on the surface of l. plantarum, could function as a natural immune adjuvant and be utilized to design a novel vaccine (dieye et al., ) . previous studies have demonstrated that l. plantarum could act as a mucosal delivery system to induce immune response and tolerance (tauer et al., ) . the distinctive feature of the immune response triggered by oral mucosal immunization is the production of specific secretory iga. antigens were taken up by m cells after oral inoculation, then dendritic cells and t cell subsets were activated, cytokines and chemokine were released and mhci and mhcii were expressed (kurashima and kiyono, ) . finally the activation of b cells, the expression of specific integrin, phenotype transformation (especially to iga type conversion) are observed (perdigon et al., ) . th and th responses induced by recombinant l. plantarum were evaluated via cell-specific cytokines analysis. th cell response can helpcytotoxic t cell differentiation mediate cellular immune responses, secrete ifn-γ and activate macrophages, which enhance the ability of cells to kill phagocytized pathogens (maldonado-lopez and moser, ). besides, the primary function of th cells is to stimulate b cell proliferation and produce antibodies, which is related to humoral immunity (mosmann and coffman, ) . secretion of il- increased the ability of monocytes differentiate into macrophages . il- can increase the ability of costimulatory molecules, mhc, antigens and t lymphocytes to stimulate antigen peptide delivery and promote dc maturation . therefore, we analyzed cytokine system ifn-γ, il- , and il- of th /th /th cells secreted by the mln of the immunized animals after treatment with recombinant bacteria. our results showed that nc -psip -pgsa'-s-dcpep could significantly increase cytokine secretion and stimulate th and th cells and th cell response, maintain the balance of th , th immune response. the focus of our study was th and th immune responses induced by nc -psip -pgsa'-s-dcpep, which also stimulated the activation of th cells (fig. ) . taken together, recombinant l. plantarum expressed s-dcpep could improve th /th /th immune response in balb/c mice and induce immune responses, such as the differentiation of b cells and dcs maturation. in this study, we detected pedv-specific neutralizing antibody in serum and fecal obtained from the group of mice orally administrated with l. plantarum expressed pedv-s protein on the surface, which could significantly inhibit the formation of plaque on vero cells. the results indicated that systemic immune response induced by recombinant lactobacillus provided an immunoprotective effect against the virus as shown in (fig. ) . the constructed recombinant l. plantarum might contribute to the development of novel vaccines against pedv fig. . nc -psip -pgsa'-s-dcpep induced mice to produce serum(a)/feces(b)-neutralizing antibodies against pedv. pd (median protective dose refers to the dose of serum or vaccine that protects half ( %) of the experimental animals).significant differences are denoted by an asterisk (*) between stroke-physiological saline solution, nc -psip -pgsa', nc -psip -pgsa'-s-ctrlpep and nc -psip -pgsa'-s-dcpep 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lactobacillus plantarum lactobacillus protects the integrity of intestinal epithelial barrier damaged by pathogenic bacteria advances in reverse genetics to treat porcine epidemic diarrhea virus construction of a bivalent dna vaccine co-expressing s genes of transmissible gastroenteritis virus and porcine epidemic diarrhea virus delivered by attenuated salmonella typhimurium key: cord- -yl jdarm authors: le, aurora b.; brooks, erin g.; mcnulty, lily a.; gill, james r.; herstein, jocelyn j.; rios, janelle; patlovich, scott j.; jelden, katelyn c.; schmid, kendra k.; lowe, john j.; gibbs, shawn g. title: u.s. medical examiner/coroner capability to handle highly infectious decedents date: - - journal: forensic sci med pathol doi: . /s - - - sha: doc_id: cord_uid: yl jdarm in the united states of america, medical examiners and coroners (me/cs) investigate approximately % of all deaths. unexpected deaths, such as those occurring due to a deceased person under investigation for a highly infectious disease, are likely to fall under me/c jurisdiction, thereby placing the me/c and other morgue personnel at increased risk of contracting an occupationally acquired infection. this survey of u.s. me/cs′ capabilities to address highly infectious decedents aimed to determine opportunities for improvement at me/c facilities serving a state or metropolitan area. data for this study was gathered via an electronic survey. of the electronic surveys that were distributed, the overall response rate was n = ( %), with of those respondents completing all the questions within the survey. at least one me/c responded from of states, and the district of columbia. select results were: less than half of respondents ( %) stated that their office had been involved in handling a suspected or confirmed highly infectious remains case and responses indicated medical examiners. additionally, me/c altered their personal protective equipment based on suspected versus confirmed highly infectious remains rather than taking an all-hazards approach. standard operating procedures or guidelines should be updated to take an all-hazards approach, best-practices on handling highly infectious remains could be integrated into a standardized education, and evidence-based information on appropriate personal protective equipment selection could be incorporated into a widely disseminated learning module for addressing suspected or confirmed highly infectious remains, as those areas were revealed to be currently lacking. increased international travel and exchange are factors that escalate the risk for rapid transmission of emerging and reemerging infectious diseases, and highly infectious diseases (hids). while neither the centers for disease control and prevention (cdc) nor the world health organization (who) has formally published a current standard list of pathogens deemed to be highly infectious, multiple 'priority' pathogens (e.g. coronaviruses, viral hemorrhagic fever viruses, bacillus anthracis, yersinia pestis) are frequently cited as requiring advanced resources, protocols, and training to minimize risk of disease transmission and mortality [ ] [ ] [ ] . the - west africa ebola virus disease (evd) outbreak, for example, challenged the capabilities, capacities, and efficacy of healthcare facilities in caring for patients with a highly infectious pathogen both abroad and in the united states [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in addition to the direct clinical care provided to patients with confirmed and suspected evd, public health departments and other affiliated sectors including emergency management, clinical and research laboratories, medical waste management, and mortuary services collaborated with medical providers to optimize and support patient care while reducing the risks to environmental and public safety [ ] [ ] [ ] [ ] [ ] . due to the highly infectious nature of evd, comprehensive aspects of infection prevention-including appropriate handling of highly infectious remains-had to be carefully considered and planned to contain disease spread. careful handling of highly infectious remains was particularly imperative given that viral loads were found to be high immediately after an evd-infected individual died, thereby posing hazardous to pathology and mortuary personnel [ , , ] . the risk of infection for death care sector workers posed by hids was exemplified by the infection of a german mortuary worker with lassa fever following the processing of remains previously unknown to be infected with lassa virus [ ] . at the height of the - outbreak, the cdc published guidelines for handling evd-infected human remains informed by evidence-based best practices to address the transmission risk posed to workers [ ] ; however, a recent gap analysis survey of the u.s. death care sector-specifically of funeral homes and crematories-revealed that most were unaware of these guidelines. overall, the study found a lack of up-to-date education, training and resources provided to this industry to safely manage potential or confirmed highly infectious remains scenarios [ ] . as evd is now re-emerging in other regions of africa [ ] and incidence and mortality of other infectious diseases is globally on the rise, the concerns raised in this study remain ever relevant. thus, it is imperative for those working with potentially infected human remains to receive the requisite training and resources to enable effective disease containment and prevent secondary transmission. in the united states of america (u.s.), medical examiners and coroners (me/cs) investigate approximately % of all deaths. typically, mes are physicians, usually pathologists specializing in forensic pathology, who are appointed government officials. they are charged with investigating unexpected, suspicious, and unnatural deaths in order to determine cause and manner of death and perform autopsies as needed. coroners, conversely, could be non-physicians or nonpathologist physicians who are elected or appointed at the county level; they look into deaths similar to those investigated by mes. coroners largely rely upon pathologists to perform the autopsies. unexpected deaths, such as those occurring due to a deceased person under investigation (pui) for a hid, are likely to fall under me/c jurisdiction, thereby placing the me/c and other morgue personnel at increased risk of contracting an occupationally acquired infection [ , ] . historically, me/cs have often been among the first to encounter infectious disease outbreaks. for instance, me/c offices were instrumental in recognizing outbreaks of diseases such as hantavirus pulmonary syndrome, west nile encephalitis, and novel severe acute respiratory syndrome coronavirus (sars-cov) [ ] [ ] [ ] . while the cdc recommends that no autopsy be performed for a confirmed patient with ebola virus disease, it is likely that if a patient were to die from an unidentified hid that an autopsy would be conducted [ ] . additionally, me/c offices play a critical role in discovering the pathogenesis of infectious diseases as well as providing a means of disease surveillance on a global level [ , ] . in puerto rico, me/c autopsy samples have been used to track dengue virus fatalities, while in south asia they have diagnosed deaths due to emergent nipah virus. me/c offices have also helped demonstrate the lethality of pediatric influenza and confirmed deaths due to creutzfeldt-jakob disease (cjd) [ ] [ ] [ ] . despite the essential role me/c offices play in public health, there have been multiple published reports of considerable obstacles to effective infectious disease and mortality surveillance including the following: inadequate morgue biosafety infrastructure, lack of appropriate staff training/ educational updates, and critical shortages in the numbers of forensic pathologists [ ] [ ] [ ] . uncertainties persist on the capabilities of me/c offices to address increasing baseline case volumes, of which the majority are lower risk infectious disease scenarios. disease containment, in this setting, including prevention of secondary transmission, is critical for the benefit of public health, emergency management, medicine, and the general public. given the critical role me/cs play in this endeavor, and the apparent lack of resources dedicated to this sector of the workforce to protect themselves from potential occupational exposures, this study was conducted to evaluate what protocols are in place for suspected or confirmed highly infectious remains, as well as determining levels of training among u.s. me/cs to handle highly infectious remains. total population purposive sampling was utilized for this nonexperimental design, as each state has its own unique death investigation system [ ] . a contact list of me/c offices serving populations of , or greater was compiled for each state by the national association of medical examiners (name) ad hoc committee for bioterrorism and infectious disease. this minimum population limit was selected in an effort to avoid duplication of survey results, as geographic areas with smaller populations often outsource to larger me/ c offices. an electronic survey with questions created by the authors was distributed via qualtrics© (software version . , provo, ut) through a link in an email solicitation (indiana university institutional review board exemption protocol # ). survey questions included: demographic information (e.g. title, population served, state), personal protective equipment (ppe) worn in different infectious scenarios, procedures performed in different infectious scenarios, duration of training received, biosafety level (bsl) capabilities, and jurisdictional handling of highly infectious remains. the name ad hoc committee for bioterrorism and infectious disease sent email solicitations from december , to february , to encourage responses from me/c offices nationwide, ensuring a comprehensive view of u.s. me/c practices. the survey was closed after weeks. data from qualtrics© was exported and data was analyzed utilizing sas version . (copyright (c) - by sas institute inc., cary, nc, us). frequencies and percentages were used to summarize question responses and chi-square tests were performed to investigate associations between variables; only significant findings were reported and individual states were not named to protect the identity of the state me/c. all responses to questions were voluntary so response rates between questions varied. of the electronic surveys that were distributed, the overall response rate was n = ( %), with of those completing all the questions within the survey. at least one me/c responded from of states, and the district of columbia; three states were excluded because their largest me/c office did not serve a population size of , or greater. medical examiners represented the majority of respondents ( %), followed by coroners ( %) and 'other' titles ( %) (e.g. forensic pathologist, deputy coroner, sheriff-coroner). there appeared to be a difference in distribution of professions across the region, with mes being more evenly distributed than other titles. each u.s. region, as delineated by the department of health and human services, had at least medical examiners, while several regions had zero or one respondent who selected 'coroner" or 'other'. for coroners, % were from the midwest (il, in, mi, mn, oh, and wi) and % of those who selected 'other' were from the west coast (az, ca, hi, and nv) [ ] . twenty-five percent of respondents worked in an office that served a population size between , - , ; % served , - , ; % served , up to million and % of respondents came from an office that served a population greater than million people. when asked which entity was responsible for their office's oversight, % stated a government agency, % public safety or law enforcement, % 'other' (academic medical center or university, city or county health department, political subdivision, or self), % state health department, and % a forensic laboratory. respondents were asked to select all ppe worn when performing standard duties, i.e. when no known infectious disease outbreak was occurring locally or regionally or was reported to the me/c (table ) . 'other' optional items of ppe listed were: a plastic apron over the surgical gown (n = ), waterproof sleeve covers (n = ), hair nets/bonnets (n = ), dedicated autopsy socks (n = ), and one respondent noted use of a tyvek suit for standard duties. for comparison, respondents were asked to select what ppe they would wear when performing duties on suspected or confirmed highly infectious remains (table ) . 'other' optional items of ppe listed were: disposable apron (n = ), waterproof sleeves (n = ), hair net/bonnet (n = ), self-contained breathing apparatus (scba) (n = ), hazardous waste and emergency response standard (hazwoper) gear (n = ), tyvek suit (n = ), and two layers of clothes (cloth and plastic) (n = ). four respondents stated their office would not perform autopsies on such cases. slightly more than half of respondents ( %; / ) stated their office staff had received training on donning and doffing ppe in suspected or confirmed cases of highly infectious remains; nearly one-third ( %) ( / ) reported the amount of cumulative training in hours per person, on average per year, was h or less while % ( / ) spent between and h of training. the entity that provided the ppe training varied widely among respondents. common responses included: in-house staff (n = ), state or local health department (n = ), an affiliated university (n = ), occupational health or a safety and compliance coordinator external to the me/c office (n = ), online-based training (n = ), risk or emergency management (n = ), an infectious disease or infection control specialist (n = ), or individuals highly trained in hazardous materials (hazmat) external to the me/c office (n = ). in the event of suspected highly infectious remains, respondents were asked what procedures would be permissible and performed by their office ( table ). the most frequent responses for 'other' were: dependent on a case-by-case basis/contingent upon suspected pathogen (n = ), sending the remains to the appropriate biocontainment facility (n = ), and one noted a written policy for handling highly infectious remains does not exist and would require discussion with multiple stakeholders, including the safety committee to evaluate risk. for comparison, respondents were asked which procedures or tasks would be performed in the event of confirmed highly infectious remains (table ) . 'other' responses echoed those in a suspected case where it was dependent on the circumstances and suspected pathogen, or only as required by the cdc or local health department (n = ). one respondent stated they would decline jurisdiction if no circumstance beyond the confirmed infectious disease made it reportable. less than half of respondents ( %; / ) stated that their office had been involved in handling a suspected or confirmed highly infectious remains case. the most commonly encountered highly infectious pathogens were: cjd and unclassified prions (n = ); forms of tuberculosis (including extremely drug-resistant [xdr-tb] and multiple drug resistant [mdr-tb]) (n = ); forms of meningitis (streptococcus pneumoniae, meningococcal) (n = ); anthrax (bacillus anthracis) (n = ); suspected cases of ebola and other hemorrhagic fevers (n = ); human immunodeficiency virus (hiv) a percentages add up to more than % because this question was multiple-select a percentages add up to more than % because this question was multiple-select (n = ); h n influenza (swine strain) (n = ); and sars (n = ). while just under half of the respondents stated their office had been involved in handling such remains ( %; / ), those cases do not appear equally distributed across regions. no respondents from upper west coast states (al, id, or, wa) reported office involvement, while all respondents from east coast states (ct, me, ma, nh, ri, vt) reported office involvement. other regions had between % to % of respondents reporting office involvement. eighty-one percent ( / ) of respondents did not have a biosafety level (bsl- ) facility within their office to conduct examinations in suspected highly infectious cases; some did have bsl- capabilities ( %; / ) and bsl- capabilities ( %; / ). table provides definitions of the bsl levels [ ] . in regard to the location at which autopsies were performed in a suspected highly infectious remains case, % ( / ) stated there was a separate autopsy area where no other autopsies were being performed at the same time; % ( / ) stated they do not examine suspected highly infectious remains at their facility; and % indicated 'other', which include not having a separate room/in the regular autopsy area (n = ), altering protocol to limit staff and only have that single case autopsied at the time if there was not a separate room available (n = ), performing autopsy in a negative pressure disaster portable morgue unit (n = ), and that autopsy was contingent on the case load and space available (n = ). one open-ended comment emphasized that it was case dependent, and that "tb gets autopsied, ebola straight to the funeral home" . this statement raises concern as the study by le et al. that surveyed the level of education and training received by u.s. funeral home and crematory personnel on highly infectious disease mitigation and management revealed large gaps in knowledge, including incorrectly marking routes of exposure for evd [ ] . more than half ( %; / ) indicated that their staff was not trained to carry out specialized decontamination procedures following autopsy of suspected or confirmed highly infectious remains. of those who had been trained on decontamination procedures, the most frequent response for the average cumulative length of training in hours per year per person was h or less ( %; / ), followed by h ( %; / ). additionally, a little over one-third of respondents ( %; / bsl- builds upon bsl- but includes additional precautions and facility features which are appropriate for work with moderate-risk microorganisms that are associated with human disease of varying severity. laboratory access is restricted when work is conducted. enhanced engineering controls and personal protection is needed. ppe typically includes lab coats and gloves; eye protection and face shields as needed. in addition to the sink for handwashing, there should also be an eyewash station. all aerosol or splash-generating procedures should be performed in a biological safety cabinet (bsc). there must be an autoclave or alternate method of decontamination for proper waste disposal, and the facility must have self-closing, lockable doors. an example of an organism appropriate for use in a bsl- laboratory is human immunodeficiency virus (hiv). biosafety level- (bsl- ) bsl- builds upon the requirements of bsl- but includes additional precautions and facility features which are appropriate for work with microorganisms which cause serious or potentially fatal disease through respiratory transmission. access to the facility is restricted and controlled at all times. in addition to all the aforementioned ppe, respirators may be worn and are required when experimentally infected animals are present. all microorganisms must be handled within a bsc. a hands-free sink and eyewash station must be available near an exit, exhaust air cannot be recirculated and the facility must have sustained directional airflow from clean areas to more contaminated areas. lastly, entrance into the facility is through two sets of self-closing, locking doors. an example of an organism appropriate for use in a bsl- laboratory is severe acute respiratory syndrome (sars) coronavirus. a these definitions are paraphrased from those provided by the centers for disease control and prevention [ ] ) indicated staff were trained to handle and transport (i.e. package and ship) specimens for suspected highly infectious cases. if staff were trained, specimens were sent most frequently to one or two of the following locations: the cdc (n = ), state reference laboratory (n = ), the national prion disease pathology surveillance center (npdpsc) at case western reserve university for prion diseases (n = ), or an academic medical center/hospital laboratory (n = ). of those that had received training on handling and transporting specimens, the most frequent response for the average cumulative length of training in hours per year per person was h ( %; / ), followed by less than h ( %; / ). there was a statistically significant relationship determined between those answering "no" to their office being involved in handling highly infectious remains and those answering "no" to receiving training to safely handle/transport the specimens. for those who answered "no" to involvement, % had no training for transporting specimens and % did; however, for those who answered "yes" to office involvement, only % had training for transporting specimens and % did not. when asked what their jurisdiction permitted for highly infectious remains, % ( / ) stated embalming was permitted, % ( / ) traditional burial practices, % ( / ) cremation and % ( / ) were unsure. survey respondents also had the opportunity to provide open-ended comments at the end of the survey; respondents did. comments included: a desire for formalized or more frequent training in the area of handling highly infectious remains (n = ); a need for more resources or a lack of preparedness or appropriate facilities to address highly infectious remains (n = ); the difficulty of answering questions pertaining to newly emerging and re-remerging highly infectious diseases because policies had not been written or revised (n = ); a need to formalize and update protocols (n = ); and a need for better funding to attract more prospective forensic pathologists to practice and to purchase greater stocks of ppe since what was available was expired or on back-order (n = ). select direct quotations and themes included that the, "national infrastructure for autopsy biosafety is woefully inadequate" and a perception of being overlooked/neglected in infection control training but still an office "they hand bodies off to" without regard for the limited training and resources. as sudden unexpected deaths fall under medical examiner/ coroner jurisdiction, they may play a fundamental role in the response to infectious disease deaths. if communication between various health sectors is unclear or protocols have not been established by the local health department, there is a risk for occupational exposure for all parties involved, and the potential for a me/c to be exposed to a highly infectious death that has yet to be confirmed. the logistical challenges associated with the response to highly infectious pathogens is demanding for public health sectors focused on the treatment and management of living patients. the role of the death care sector in effective disease surveillance and containment of infectious diseases is often overlooked; including the fundamental role of me/cs. me/cs frequently investigate deaths with little clinical information on the circumstances preceding death. hence, it is crucial to for me/cs to have robust, up-todate education and training in potential highly infectious remains handling, ppe donning and doffing, and clear protocols used when handling human remains that stress universal precautions. to determine what training areas are insufficient or need to be supplemented, this survey evaluated current me/c office capability to handle highly infectious remains. this survey provided a national view of the handling of highly infectious remains by capturing a sample of me/cs from nearly every state and washington d.c. medical examiners comprised the majority of the survey respondents and were more evenly geographically distributed than coroners. nearly half of the respondents served large counties or metropolitan areas with populations of greater than one million people, highlighting the large populations that may be covered by a single me/c office. additionally, most me/c offices, including the body storage areas (morgues), are under government oversight. in order to gauge circumstance-dependent ppe use among me/cs, respondents were asked for standard ppe ensembles worn during routine autopsies and those worn for autopsies on suspected or confirmed highly infectious remains (table ) . slightly more than half ( %) reported wearing an n respirator during routine autopsies and this increased to only % for autopsies on suspected or confirmed highly infectious remains. other higher level ppe such as a powered-air purifying respirator (increased by over %), tyvek suit, hazwoper gear and scba also showed an increase. a surgical mask was worn by % in standard autopsies and by % of me/cs for a suspected or confirmed highly infectious case. typically, at minimum an n is recommended for protection against aerosolized particles arising such as tb, monkeypox, sars and others, rather than a surgical mask [ ] . additionally, an autopsy is inherently an aerosol-generating procedure, even organisms that might normally require only large droplet precautions (i.e. surgical mask) can be aerosolized at autopsy due to oscillating saws, aspirating hoses, etc. and thus require added respiratory precautions (i.e. n respirator or papr) [ , , ] . use of a face shield rather than glasses/goggles also has been shown to reduce contamination of respirators by particles but only % of me/c respondents routinely wear them [ ] . the following ppe changes occurred for suspected or highly infectious remains: the use of inner gloves, a face shield, and boot/shoe cover wear increased by %, %, and %, respectively, while donning eye protection decreased by %. usage of a n respirator increased by more than % and the use of a powered-air purifying respirator notably increased by nearly %. higher level ppe, such as a tyvek suit, hazwoper gear and scba also were used when autopsies were performed on suspected or confirmed highly infectious remains. of concern, these results indicate me/c alter their ppe based on suspected versus confirmed highly infectious remains rather than taking an all-hazards approach. despite some improvements in more protective ensembles in the suspected increased risk cases, the amount of training received by respondents was lacking. little more than half ( %) of respondents had received training on donning and doffing ppe in such scenarios, with the % of those who did have ppe training having spent an average of only h or less per person per year on the topic. additionally, the entity that provided ppe training widely varied (e.g. in-house staff, affiliated university, safety and compliance departments), and no information was collected on the survey on the expertise level of those delivering trainings. the lack of reproducible training time and variability of training entity suggest that more standardized training might be of benefit. designating a knowledgeable public organization to offer standardized training modules could lead to the following: ( ) standardization of the organizational source of training; ( ) content of training materials and modules based on reproducible, evidence-based best practices commonly found in the me/c field; and ( ) subscription to online training as it will likely be the most cost-effective and convenient means of training, as was proven successful in healthcare [ ] . moreover, best practices and evidence-based studies have demonstrated that regular training for donning and doffing high level ppe in highly infectious scenarios provide substantially better occupational safety and health outcomes for the employee [ ] . in the event of suspected or confirmed highly infectious remains, most me/c offices stated that the situation was handled on a case-by-case basis, depending on the pathogen that was suspected and required detailed conversations with all stakeholders. as shown in table , procedures did vary between what would be performed with a suspected highly infectious body versus a confirmed infectious body. in a confirmed case, all but one of the listed procedures as decreased compared to a suspected case (e.g. complete autopsy [ % decrease], washing or cleaning of the body [ % decrease], body storage in freezer [ % decrease]). the only increase was, "bypass office and have body directly transported to funeral home/crematorium" by % which, as previously mentioned, may result in funeral home and crematory personnel being placed at risk. fewer than % of me/c offices having been involved with handling a suspected or confirmed case, demonstrating a lack experience in handling highly infectious remains. when asked which suspected or confirmed pathogens were encountered, however, many noted category a or b pathogens (table ) [ , ] that require specific deactivation and decontamination procedures-of which only approximately onethird ( %) of respondents had received training in. it is possible that after such an event that the me/c office would hire an appropriate contractor to conduct the appropriate deactivation and decontamination; however, the possibility remains [ , ] that the task could go to individuals within the me/c offices without proper training. while most offices did not have a bsl- facility, nearly twothirds ( %) of those without a bsl- did have bsl- capabilities. however, if % have only bsl- capability, then these morgues would essentially be considered appropriate for work only with agents not known to consistently cause disease in healthy human adults per cdc guidelines [ ] . in essence, a sizeable percentage of morgues in the u.s. are not equipped to safely perform autopsies on human remains with a large number of infections, especially those highly infectious disease autopsies. in addition to improved training, more investment in morgue infrastructure would be necessary to enhance their capabilities. anecdotally, some larger me/c offices have a computerized tomography (ct) scanner in which triple bagged sealed infectious remains can undergo virtual autopsy. these bags can be constructed with portals to collect needed specimens for microbiologic/virologic studies. the triple bagging prevents leaks and contamination and the remains can safely be sent to funeral home. it would also be beneficial to have list of pathologists and support personnel in each me/c office who could volunteer to take vaccines to handle certain cases with suspected contagious diseases (i.e. smallpox, etc.). approximately % of respondents reported that they did not examine suspected or confirmed highly infectious remains at their facility. given the lack of proper bsl facilities, this would be appropriate. slightly more than % noted the lack of space and/or a lack of staff in their offices as a limitation for being able to perform autopsies of suspected or confirmed highly infectious remains in a separate room or alone. for biosafety, it is recommended that autopsy facilities should have a minimum of air exchanges per hour, be negatively pressurized relative to surrounding office spaces, and exhaust air outside of the facility and away from areas of high pedestrian traffic. morgue laminar air flow should travel from clean to progressively less clean areas with downdraft table ventilation to decrease personnel exposure to aerosolized pathogens [ , ] . it is likely that significant financial investment would be required to retrofit many existing morgues to meet these standards. another option would be to have jurisdictional planning to transport suspected or confirmed cases to known centers that currently have the necessary bsl capability; again, body transportation would incur costs but likely lower costs than that associated with retrofitting many existing morgues. in addition to improved morgue biosafety, it would also benefit me/c facilities to have better publicized, easily accessed, and clearly laid-out protocols for various infectious scenarios in which limited autopsy (e.g. brain-only in suspected cjd cases) or no autopsy (e.g. evd cases) is currently recommended. when asked about level of training to handle and transport specimens for suspected highly infectious cases, only onethird of respondents had received this training with % spending on average less than h per year per person on the topic. nearly half of respondents ( %) were unsure of what their jurisdiction permitted in the case of highly infectious remains for ultimate disposal, and alarmingly, % of respondents stated their jurisdiction permitted embalming and % traditional burial. for evd, for example, the recommended procedure is cremation to ensure complete deactivation of the virus in order to prevent spread to workers and the environment; those who were killed by the disease will have high viral loads present in their body post-mortem [ ] . while needs for funding, resources, supplies and appropriate capabilities may be universal across the death care sector, this survey's results strongly suggest that it would benefit state or regional-specific me/cs to have standardized education and training throughout the u.s [ ] . likewise, open-ended comments from respondents indicated a need for augmented up-to-date formalized trainings, as well as revised written policies and procedures, and enhanced resources (including facilities and funding). there were general perceptions of unpreparedness to address highly infectious remains, budgetary constraints and a weak national structure regarding autopsy biosafety, and a lack of incorporation of me/c offices into infection control planning despite me/c office involvement with highly infectious remains. there were limitations to this study. because of the study's exploratory nature, the survey was not validated beyond subject matter expert vetting. additionally, the survey only included me/c offices that served larger populations; smaller offices may still encounter hid cases if they do not outsource larger nearby offices. therefore, this study may not be generalizable to smaller offices (i.e. those serving populations < , ). also, the survey instrument was designed to allow respondents to check multiple boxes when asked about the use of ppe. the results, therefore, were not clear whether the respondent meant the ppe would be used simultaneously or one instead of the other. for example, a face shield and respirator may be used simultaneously or a face shield may be used instead of a respirator. additionally, a limitation related to potential response bias may exist. although this study was not funded, there could have been sponsor bias on behalf of the respondents, as the survey was distributed by members of name, thereby potentially affecting the candidness of their responses. lastly, non-responses may have arisen because it would not appeal to prospective participants to take a survey about a topic for which they are not trained out of concern their answers may not be "correct." nevertheless, this study addresses a critical gap about what is known and unknown about u.s. me/c capabilities to handle highly infectious remains. in conclusion, this survey of u.s. medical examiners and coroners' capabilities to address highly infectious decedents presents opportunities for improvement at me/c facilities serving their state or metropolitan area. standard operating procedures or guidelines (sops or sogs) should be updated to take an all-hazards approach, best-practices on handling highly infectious remains could be integrated into a standardized education, evidence-based information on appropriate ppe selection could be integrated into a widely disseminated learning module, and existing relationships with the local health department, funeral homes and crematories could be bolstered to develop a multi-sectoral concept of operations for addressing suspected highly infectious remains. while some issues will require greater capital and resources to address-such as retrofitting facilities to meet better biosafety recommendations, or more financial resources to enhance operation-the hope is that this study will draw attention to these more systemic issues and stimulate a call to action from the appropriate entities. . u.s. medical examiners/coroners play a critical role in death investigation, yet their capabilities to address highly infectious remains are unknown. occupationally-acquired infection. . this survey, with respondents from nearly every u.s. state, revealed current levels of medical examiner/ coroner training and education to address suspected or confirmed highly infectious remains. . questions, and thereby results, focus on permissible autopsy procedures, personal protective equipment, and biosafety-level facility capabilities. . medical examiners/coroners could benefit from updates to standard operating procedures and standardized education on handling suspected or highly infectious remains that taken an all-hazards approach. niaid emerging infectious diseases/pathogens list of blueprint priority diseases infection control in the management of highly pathogenic infectious diseases: consensus of the european network of infectious disease clinical care of two patients with ebola virus disease in the united states ebola virus disease cluster in the united states ebola virus disease: preparedness and infection control lessons learned from two biocontainment units caring for patients with ebola: a challenge in any care facility lessons learned: critical care management of patients with ebola in the united states reflections on interprofessional team-based clinical care in the ebola epidemic: the nebraska medicine experience current capabilities and capacity of ebola treatment centers in the united states safe management of patients with serious communicable diseases: recent experience with ebola virus clinical management of ebola virus disease in the united states and europe nebraska biocontainment unit perspective on disposal of ebola medical waste considerations for safe ems transport of patients infected with ebola virus transport and management of patients with confirmed or suspected ebola virus disease us ebola treatment center clinical laboratory support nebraska biocontainment unit patient discharge and environmental decontamination after ebola care guidance for safe handling of human remains of ebola 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infectious disease outbreaks operational research during the ebola emergency role of the medical examiner in zika virus and other emerging infections cdc grand rounds: discovering new diseases via enhanced partnership between public health and pathology experts emerging infectious diseases and the medical examiner medical examiners, coroners, and biologic terrorism death investigation systems department of health & human services. regional offices hospital respiratory protection program toolkit: resources for respirator program administrators aerosol generation during bone-sawing procedures in veterinary autopsies biosafety considerations for autopsy efficacy of face shields against cough aerosol droplets from a cough simulator personal protective equipment in health care: can online infection control courses transfer knowledge and improve proper selection and use? guidelines for safe work practices in human and animal medical diagnostic laboratories age and ebola viral load correlate with mortality and survival time in ebola virus disease patients acknowledgments we would like to extend our gratitude to the members of the national association of medical examiners (name) ad hoc committee for bioterrorism and infectious disease for their support of this research and distribution of the survey; these members include: second author erin brooks (chair), paul chui, karen kelly, john matthew lacy, micheline lubin, lakshmanan sathyavagiswaran, leah schuppener, suzanne utley-bobak, and steven white. additionally, we acknowledge the national institute of environmental health sciences (niehs) worker training program (wtp) ebola biosafety and infectious disease response training uh information, grant number uh es . while the grant funding did not contribute to the development and distribution of this gap analysis survey, the program did highlight the need to explore research in this area. lastly, we also thank from the university of nebraska medical center: elizabeth beam and at harvard t.h. chan school of public health: paul biddinger for their partnership and support. conflict of interest none of the authors have any conflicts of interest to disclose.ethical approval this study was deemed exempt by indiana university institutional review board (protocol # ).informed consent survey participants were informed of potential risks and benefits prior to taking the voluntary survey. this informed consent survey was reviewed by the institutional review board and approved as part of the exemption in the aforementioned protocol number. key: cord- -o x nj authors: wu, yu; li, wei; zhou, qingfeng; li, qunhui; xu, zhichao; shen, hanqin; chen, feng title: characterization and pathogenicity of vero cell-attenuated porcine epidemic diarrhea virus ct strain date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: o x nj background: porcine epidemic diarrhea virus (pedv) has caused enormous economic losses to the global pig industry. currently available pedv vaccine strains have limited protective effects against pedv variant strains. methods: in this study, the highly virulent epidemic virus strain ct was serially passaged in vero cells for up to generations (p ). characterization of the different passages revealed that compared with p and p , p had a higher viral titer and more obvious cytopathic effects, thereby demonstrating better cell adaptability. results: pathogenicity experiments using p in piglets revealed significant reductions in clinical symptoms, histopathological lesions, and intestinal pedv antigen distribution; the piglet survival rate in the p group was %. furthermore, whole-genome sequencing identified amino acid changes in p , which might be responsible for the attenuated virulence of p . conclusions: thus, an attenuated strain was obtained via cell passaging and that this strain could be used in preparing attenuated vaccines. porcine epidemic diarrhea (ped) is an acute and highly infectious intestinal disease in pigs; it is caused by porcine epidemic diarrhea virus (pedv), which has caused enormous economic losses to the global pig industry [ , ] . ped is characterized by diarrhea, vomiting, dehydration, anorexia, weight loss, and high mortality in suckling pigs. although ped also appears in summer, it mainly occurs in winter. pedv infection causes different symptoms according to the pigs' ages; however, in piglets, the symptoms of pedv infection are particularly serious, including a high mortality rate [ ] . pedv belongs to the order nidovirales, family coronaviridae, and genus alphacoronavirus and is an enveloped virus with a single-stranded, positive-sense rna genome [ ] . the whole pedv genome is approximately kb nucleotides (nts) long and has a ′-cap and ′-polyadenosyl tail; the genome also includes ′-and ′-untranslated regions and at least open reading frames (orf a, orf b, and orf - ) [ , ] . orf a and orf b encode the replicase polyproteins a and ab, respectively, which undergo autoproteolysis by viral proteases to form nonstructural proteins (nspl- ) [ ] , which participate in the basic mechanisms of viral rna transcription and replication. orf - encode four structural proteins [fibrin (s), membrane protein (m), envelope protein (e), and nucleocapsid protein (n)] as well as coprotein orf [ , ] ; these proteins are arranged in the genome in the following order: ′-orf (la/lb)-s-orf -e-m-n- ′ [ ] . in , the pedv strain cv was identified as the cause of the ped outbreak in belgium [ ] . in october , a highly pathogenic pedv was discovered in china, which caused the worst outbreak on record and quickly swept across the country [ , ] . the variant then caused a pandemic in the united states in spring and spread to canada and mexico. in addition, severe ped outbreaks occurred almost simultaneously in many asian and european countries, such as korea, japan, belgium, and france [ , ] . vaccination is considered effective in the prevention of pedv infection on farms [ ] . several attenuated activated and inactivated vaccines for classical pedv strains, such as cv [ ] , dr [ ] , and kpedv- [ ] , have been developed and made commercially available in many countries [ ] ; however, the efficacy of these traditional vaccines against emerging pedv strains is questionable because of the antigenic and genetic differences between the vaccine strains and the prevalent strains [ ] . therefore, there is an urgent need for a new pedv vaccine against new variant strains. in the present study, the ct strain was serially passaged in vero cells. the growth kinetics and biological characteristics of the different passages were determined. in addition, -day-old piglets were used to assess the pathogenicity of these strains. finally, the whole-genome sequences of the different passages were determined. a safe attenuated pedv strain was developed in this study, thereby providing an important basis for the preparation of an attenuated vaccine. the pedv ct strain, which belongs to the g b subgroup in china, was previously isolated by and stored at our laboratory [ ] . vero cells were obtained from the american type culture collection (atcc: ccl- ), regularly cultured in dulbecco's modified eagle's medium (dmem) supplemented with % fetal bovine serum (invitrogen, australia) and % antibiotics ( u/ml penicillin, μg/ml streptomycin, and μg/ml fungizone®; gibco™, usa), and maintained at °c in a humidified % co incubator. mouse anti pedv s monoclonal antibody and y -labeled goat anti-mouse igg antibody were prepared and stored at our laboratory. vero cells were grown in a t flask and washed thrice with phosphate-buffered saline (pbs) at % confluency. the cells were then incubated with ml of the pedv ct strain diluted : in virus growth medium {dmem supplemented with antibiotics ( u/ml penicillin, μg/ml streptomycin, and . μg/ml trypsin [gibco])} for h at °c in a humidified % co incubator. then, ml of the virus growth medium was added to the t flask, which was monitored daily for cytopathic effects (cpes). when cpes were observed in > % of the vero cells, the flask was subjected to three cycles of freezethawing. the cells and supernatants were mixed by pipetting, aliquoted, and stored at − °c. these harvested cells were then used as seed stock for the next passage. finally, the cells were further passaged under each condition for up to passages [ , ] . immunofluorescence assay (ifa) the vero cells were inoculated with pedv in -well plates at a multiplicity of infection (moi) of . . at h post infection (hpi), the cells were washed thrice with pbs, fixed at room temperature (rt) with % paraformaldehyde for min, and then permeabilized with . % triton x- (solarbio, china) for min. after washing as described previously, the cells were blocked with % bovine serum albumin (solarbio) for min at rt. the mouse anti pedv s monoclonal antibody and y -labeled goat anti-mouse igg antibody were then used as the primary and secondary antibodies, respectively. finally, the cell nuclei were stained with ′, -diamidino- -phenylindole (vectorlabs, usa) for min at rt in the dark. the cells were then washed thrice with pbs and observed under a fluorescence microscope. vero cell monolayers were separately inoculated with pedv p , p , or p or were mock infected at an moi of . in -well plates. the culture supernatants and cell lysates were collected at , , , , and hpi. after one round of freeze-thawing, following the reed-muench method, the cell culture samples were analyzed in -well plates to determine the titration for % tissue culture infectious dose (tcid /ml) [ ] . total rna was extracted from the cell culture samples using an rneasy kit (magen, china). all primers used to amplify pedv genomic fragments were designed and preserved at our laboratory [ ] . reverse transcription and polymerase chain reaction (pcr) were performed using the primescript™ rt-pcr kit (promega, usa) and primestar® gxl dna polymerase, respectively. the ′-and ′-end sequences were determined using a ′and ′-competition kit (takara). the pcr products were cloned into a pmd -t vector using the topo® ta cloning® kit (invitrogen, usa) and sent to sangon biotech (shanghai, china) for sequencing. for each amplicon, more than three independent clones were sequenced to determine the accurate sequence of a specific genomic region. all sequencing results were then spliced and aligned using lasergene seqman and lasergene megalign, respectively. all reference sequences were obtained from genbank and used for sequence alignment and phylogenetic analysis. all phylogenetic trees were constructed using mega . software. twenty-two -day-old crossbred (duroc × landrace × big white) conventional piglets were obtained from wen's foodstuffs group co., ltd. (guangdong, china). the piglets were determined to be free of antibodies against pedv and confirmed to be negative for the major porcine enteric viruses pdcov, pedv, tgev, and prov [ ] via virus-specific rt-pcr of fecal swab samples [ , ] . the piglets were then placed in separate cages and randomly divided into four experimental groups. each group was placed in a separate room (biosafety level ). the piglets in each group were orally inoculated with ml of the virus culture medium ( × tcid ) or dmem. the piglets were monitored daily for clinical signs of vomiting, diarrhea, and mortality throughout the experiment [ ] . fecal consistency was scored as follows: , solid; , paste; , semi-liquid; , liquid; and , death [ ] . one piglet from each group was euthanized for histopathological examination at days post infection (dpi). the remaining piglets were euthanized at the end of the study ( dpi). fresh fecal swabs were collected daily, diluted with pbs, stored at − °c, and subjected to three freeze-thaw cycles. viral rna was extracted from the supernatant of the fecal swab samples as described previously. the amount of viral rna in the fecal swab samples was determined using taqman real-time rt-qpcr with the following primers: sense, ′-gaattcccaagggc gaaaat- ′; antisense, ′-ttttcgacaaattc cgcatct- ′. a probe targeting the pedv n gene ( ′-fam-cgtagcaggcttgcttcggaccca-bhq- ′) was also employed. the thermal cycling parameters were as follows: °c for s followed by cycles at °c for s and °c for s. the gross intestinal tract of each piglet was photographed at necropsy, and the intestinal tissue was collected and fixed with % paraformaldehyde. the fixed samples were sent to guangzhou saville for histopathology or ihc with pedc s-specific monoclonal antibodies. in each experimental group, statistical significance was measured using one-way analysis of variance. two-sided probability values < . (p < . ) were considered to indicate statistical significance. correlations between the fc scores and fecal pedv rna drop titers were analyzed using spearman's rank correlation [ ] . the animal study protocol was approved by the south china agricultural university committee of animal experiments (approval id: syxk- - ). all experiments were performed in accordance with the recommendations of the guide for the care and use of laboratory animals of the national institutes of health. biological characteristics of pedv p , p , and p our results indicated that syncytial, vacuole, and cell exfoliation were more obvious after infection with p than with p and p (fig. a) . the three different passage strains were confirmed by ifa using a specific monoclonal antibody against the pedv s protein. red signals were observed in the strain-infected vero cells for all the three passage strains but not in the uninfected vero cells at hpi. obviously, the cells infected with p produced more red pedv antigens (fig. b) . in addition, growth kinetics indicated that p had a higher viral titer than the other two strains at all time points, indicating that p had better cell adaptability (fig. c) . fecal swabs were collected from the piglets and scored for diarrhea during the experiment; the clinical signs of the piglets in each group were also recorded. at dpi, severe diarrhea and vomiting were observed in the p group. in contrast, slight diarrhea and vomiting were observed in the p group. interestingly, there were no obvious clinical symptoms in the p and mock groups (fig. a) . regarding the lesion onset times in the groups, lesions appeared at , , and hpi in the p , p , and p groups, respectively. throughout the experiment, a high diarrhea score was maintained in the p group. in contrast, in the p and p groups, the diarrhea scores began to rise at dpi, peaked at dpi, and gradually returned to normal by dpi (fig. b) . differences in pedv rna shedding in the four experimental groups pedv rna shedding was detected in fecal swab samples using rt-qpcr (fig. ) . during the entire experiment, the p group maintained a high level of pedv rna shedding. in the p group, pedv rna shedding appeared at dpi, peaked at dpi, and gradually recovered by dpi. pedv rna shedding was not observed in the p group until the second day after challenge; the viral shedding peaked at dpi and gradually improved after dpi. the entire experiment lasted for days. the health of the piglets in the p and p groups had recovered by dpi. at dpi, one piglet was selected from each group for dissection and sampling. at necropsy, intestinal tissue, particularly the small intestinal tissue, of the the p group piglet exhibited dilatation and transparency (fig. a-d) . in the p group piglet, the intestinal tissue lesions were more obvious and the intestine was filled with yellow fluid and mesenteric hyperemia (fig. d) . in contrast, the p group piglet exhibited only slight changes in the intestinal tract, and the control animal was extremely healthy with no pathological changes (fig. b) . the jejunum and ileum of each piglet were examined via histopathological and ihc analyses. histopathological analysis revealed severe atrophy and shedding of the intestinal villi of the p group piglet (fig. h and l) ; in contrast, in the p group piglet, the intestinal villi were shortened and fused ( fig. f and j) . intestinal villus atrophy was mild in the p group piglet, and the examined control group piglet exhibited normal histopathology ( fig. e and i) . in contrast, ihc revealed that the pedv antigen was dominant in the cytoplasm of some segments of small intestinal villi. in the p and p group piglets, large amounts of the pedv antigen were detected in severe lesions; in contrast, the antigen was hardly detected in the p group piglet (fig. n and r) ; further, no pedv antigen was found in the small intestine of the control group piglet (fig. m and q) . in the p group, one piglet died at dpi and two others died at dpi. in addition, one piglet each died at and dpi, respectively; thus, the survival rate in the p group was %. in the p group, the survival rate was . % because only one piglet died at dpi. no animals died in the p and control groups ( % survival rate for both groups) (fig. ). the genome length of the pedv ct strain was determined to be , nts. previous studies conducted at fig. biological characteristics of porcine epidemic diarrhea virus strains after , , or passages. a cytopathic effects of the three strains hpi at an moi of . . b immunofluorescence detection results for the p , p , and p strains in vero cells infected at an moi of . at hpi. c growth kinetics of the p , p , and p strains in vero cells at an moi of . . cell lysates were sampled at the designated time points and titrated using the titration for % tissue culture infectious dose infectivity assay. the asterisk means significant difference (***p < . , **p < . , and *p < . ). p , pedv after passages; p , pedv after passages; and p , pedv after passages; moi, multiplicity of infection; hpi, hours post infection our laboratory have reported that the pedv ct strain belongs to clade of the g b subgroup (fig. ) . table lists the changes in the amino acids and nts of the different passage strains. p was observed to contain base mutations, resulting in amino acid mutations. the s gene is a determining feature of pedv's virulence and evolution [ ] . amino acid comparison indicated that there were only four amino acid mutations (d a, f r, s r and c g) between p and p . in addition, several nucleotide point mutations resulted in aa substitutions in orf ab (a s, t n, d g, h y, y s and v i), orf (t m), e (p l) and m (g d) proteins. we found that p had base mutations (thus, amino acid mutations). moreover, compared with p , p contained base mutations (thus, amino acid mutations) ( table ). in addition, p had high homology with the cv vaccine strain ( . %) and the attenuated dr strain ( . %). the complete genome sequences of the ct strains described here have been deposited in genbank under accession no.mn . ped outbreaks have caused huge economic losses to the pig industry in not only china but also the united states, japan, south korea, and other countries [ , ] . currently, vaccination is an effective solution to tackling the virus; however, owing to variations in the virus [ ] , the classical attenuated vaccine does not provide effective protection. thus, the epidemic strains of the virus must be urgently studied to prepare a new attenuated vaccine. in the classical method, attenuated vaccines are prepared via cell passaging [ , , , ] . some previous studies have reported that increased viral titers and enhanced cell adaptability are the characteristics of viruses with weakened virulence [ , , ] . in the present study, we passaged the epidemic pedv ct strain continuously in vero cells for up to generations and observed that p had better cell adaptability than the other two strains; thus, our findings illustrated that increased passaging increases the cell adaptability and titers. finding balance between cell and animal adaptability and then determining the best generation is the key to the preparation of attenuated vaccine candidate strains via continuous passaging [ , , ] . moreover, the adaptability of the strain to the host animal is characterized by the virulence of the strain, which is usually evaluated via pathogenicity testing of the animal infected with the virus. to prove that . e-h h&e-stained jejunum tissue sections of the different groups at dpi. i-l h&e-stained ileum tissue sections of the different groups at dpi. m-p immunohistochemically stained jejunum tissue sections of the different groups at dpi pedv antigen is indicated by arrows). q-t immunohistochemically stained ileum tissue sections of the different groups at dpi (pedv antigen is indicated by arrows). p , pedv after passages; p , pedv after passages; and p , pedv after passages; h&e, hematoxylin and eosin; pedv, porcine epidemic diarrhea virus fig. survival rates of piglets infected with different passages of porcine epidemic diarrhea virus ct strain. **p < . , and *p < . the strain virulence has been weakened, animal experiments were designed to confirm the strain [ , , ] . thus, the virulence of the three pedv strains was assessed to confirm whether the p strain was weakened. according to data from animal experiments, the p strain could significantly reduce clinical symptoms, viral shedding, and histopathological changes in piglets. therefore, our results illustrated that the p strain had relatively weak virulence and that compared with the p and p strains, it caused lesser damage to the piglets. thus, it can be concluded that the p strain is safe and potentially useful as a candidate strain for the preparation of an attenuated vaccine. to further assess the attenuation of the p strain, we sequenced the whole genomes of the three passage strains. and the method to prepare attenuated vaccine by cell passage can also get clues to the key virulence factors of the virus [ ] . the pedv s protein is involved in receptor binding, viral entry, neutralizing antibody production induction, and host cell fusion [ ] . the s protein of pedv has always been used as a marker of viral variation. under the pressure of herd immunity, the s gene of pedv mutates frequently, with some of the missense mutations altering viral antigenicity to aid in the virus's escape from preexisting immunity. thus, periodic vaccine updates may be required to ensure sufficient efficacy against emerging virus variants [ ] . in this study, our result revealed that there is little variation in the s gene among these pedv strains. moreover, s protein is implicated in virulence, which was found out in the other studies that s protein can be variant readily while receiving immune pressure [ ] . mutations, including deletions and/or insertions, in the s protein may change the pathogenicity and tissue tropism of coronaviruses [ ] . comparing to the p strain, there was no nucleotides insertion and deletion in the s gene of the p strain. however, we found that there were four amino acid mutations (d a, f r, s r and c g) between the p strain and the p strain. fig. phylogenetic tree analysis of the whole genomes of different pedv strains. phylogenetic analysis conducted for the whole-genome nucleotide sequence of the pedv strains reported in our study and the reference pedv strain. using the mega . software, the adjacency method was used to construct the tree. the numbers at the branch are the guided values (%) after copies. the front ends of the three different sub-strains have been labeled. pedv, porcine epidemic diarrhea virus these mutations have not been reported previously. this finding indicates that these mutations in the s gene may be related to viral pathogenicity. the orf protein was reported to function as an ion channel and can prolongs s-phase, facilitates formation of vesicles and thus to regulate virus production [ ] . it has been speculated that the deletion or mutation of a base in the orf region promotes the adaptation of viral cells and changes in viral titers [ ] . in this study, the t m mutation was detected in orf of p . furthermore, mutations of amino acids in the orf ab, m, and e regions of p indicate that multiple mutation combinations in the genome cause complete decay of the pedv strain via various molecular mechanisms [ ] . these mutations found in orf ab, m, orf and e proteins have not been reported previously. further studies using reverse genetics are required to determine whether a particular or a combination of genetic changes (point mutations, deletions, and insertions) in pedv strains alter viral infectivity, pathogenicity, and replication efficiency [ ] . in conclusion, an attenuated strain of pedv with better cell adaptability and higher titers than the virulent strain was obtained via cell passaging. the results of our animal experiments illustrated that the pathogenicity of the p strain was reduced and that piglets infected with that strain had a % survival rate. moreover, the results of whole-genome analysis revealed amino acid mutations in the p strain, which may contribute to the weakened virulence of the virus. thus, the attenuated strain could be suitable for use in vaccine preparation. porcine epidemic diarrhea virus: an emerging and reemerging epizootic swine virus epidemic strain yc of porcine epidemic diarrhea virus could provide piglets against homologous challenge novel porcine epidemic diarrhea virus variant with large genomic deletion epidemiology and vaccine of porcine epidemic diarrhea virus in china: a mini-review a newly isolated chinese virulent genotype giib porcine epidemic diarrhea virus strain: biological characteristics, pathogenicity and immune protective effects as an inactivated vaccine candidate development of a live attenuated vaccine candidate against 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isolated in korea porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis cell attenuated porcine epidemic diarrhea virus strain zhejiang provides effective immune protection attributed to dendritic cell stimulation cell culture isolation and sequence analysis of genetically diverse us porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene evolution, antigenicity and pathogenicity of global porcine epidemic diarrhea virus strains detection of antibodies against porcine epidemic diarrhea virus in serum and colostrum by indirect elisa comparative genomic analysis of classical and variant virulent parental/attenuated strains of porcine epidemic diarrhea virus pedv orf encodes an ion channel protein and regulates virus production preparation and characterization of an attenuated porcine epidemic diarrhea virus strain by serial passaging publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to thank wen's group academy, wen's foodstuffs group. authors' contributions fc and yw conceptualized the study. yw and wl devised the experimental methods. yw and zcx curated the data. fc, yw, hqs, qhl, and qfz provided resources. yw prepared the original manuscript draft. fc reviewed the manuscript and edited it. all authors read and approved the final manuscript. the datasets analyzed during this study are available from the corresponding author on reasonable request. the animal study protocol was approved by the south china agricultural university committee of animal experiments (approval id: syxk- - ). all experiments were performed in accordance with the recommendations of the guide for the care and use of laboratory animals of the national institutes of health. the authors declare that they have no competing interests. key: cord- -rcxjjxw authors: xu, zhichao; lin, ying; zou, chuangchao; peng, peng; wu, yanan; wei, ying; liu, yuan; gong, lang; cao, yongchang; xue, chunyi title: attenuation and characterization of porcine enteric alphacoronavirus strain gds via serial cell passage date: - - journal: vet microbiol doi: . /j.vetmic. . sha: doc_id: cord_uid: rcxjjxw porcine enteric alphacoronavirus (peav) is a newly identified swine enteropathogenic coronavirus that causes watery diarrhea in newborn piglets. in this study, an original, highly virulent peav strain gds was serially passaged in vero cells. the virus titers and sizes of syncytia increased gradually with the cell passages. newborn piglets were orally inoculated with peav p , p and p . compared with p and p , p resulted in only mild clinical signs and intestinal lesions in piglets. the virus shedding in feces and viral antigens in intestinal tract were markedly reduced in p -inoculated piglets. importantly, all p -inoculated newborn piglets survived, indicating that p was an attenuated variant. sequence analysis revealed that the virulent strain gds had four, one, six and eleven amino acid differences in membrane, nucleocapsid, spike and orf ab proteins, respectively, from p . furthermore, more differences in the predicted three-dimensional structure of s protein between gds and p were observed, indicating that these differences might be associated with the pathogenicity of peav. collectively, our research successfully prepared a peav attenuated variant which might serve as a live attenuated vaccine candidate against peav infection. porcine enteric alphacoronavirus (peav) was first detected by our team via genomic analysis of samples collected from a diarrhea-outbreak swine herds routinely vaccinated with porcine epidemic diarrhea virus (pedv) vaccine in a farm in guangdong, china in february (gong et al., ) . this novel swine enteric coronavirus emerged in china at least since august and it has been widely detected in guangdong, china, with a prevalence of . % by a retrospective detection study (zhou et al., ) . recently a peav-like strain cn/ fjwt/ was recently discovered in fujian, china , indicating a prevailing trend in pig farms. since the first report, the complete genome of the peav strain gds was sequenced (gong et al., ) . peav is an enveloped, single-stranded, positive-sense rna virus with a genome of appropriately kb in length (gong et al., ) . the genome organization of peav is similar to that of bat-like hku strains of coronavirus, with an order of: ′ untranslated region (utr), open reading frame a/ b (orf a/ b), spike (s), nonstructural protein (ns ), envelope (e), membrane (m), nucleocapsid (n), nonstructural protein a (ns a), and ′ utr (lau et al., ) . the s protein of coronaviruses (covs), the pivotal surface glycoprotein, is involved in virus attachment and entry, and induction of neutralizing antibodies in vivo (cruz et al., ; woo et al., ) . gds strain of peav has the smallest s protein among all covs (gong et al., ) . the clinical symptoms caused by peav in newborn piglets are similar to that by other porcine enteric pathogens such as pedv and transmissible gastroenteritis virus (tgev), which include vomiting, diarrhea, dehydration, and mortality rate as high as % (gong et al., ; zhou et al., b) . since peav was detected (gong et al., ) , another two swine enteric hku -related covs named sads-cov and seacov were identified in the same region, causing same diarrheal disease as peav strain gds by experimental infection (pan et al., ; xu et al., a; zhou et al., b) . currently, live-attenuated vaccine has been widely used to prevent and control porcine enteric pathogens, such as pedv, with good clinical effect (song et al., ) . live-attenuated vaccine is prepared from less virulent strain. the virulence of pathogenic microorganisms was reduced by various methods (blanco-lobo et al., ; jie et al., ; o'donnell et al., ) , such as genetic manipulation , and cell passage is the most commonly used (jie et al., ) . usually, the virulent parent strain was continuously inoculated cell lines more than generations in vitro, and in this process, some mutations will happen in the viral genome, which might lead to the strain's virulence reduce. these cell-passaged strains are then tested in animals to determine whether they have less virulence and higher immunogenicity before being used to prepare live-attenuated vaccines. of note, several groups have used cell passage to prepare porcine covs attenuated strains, such as pedv (lin et al., ) and tgev (motovski et al., ) , which laid a good foundation for preparation of live-attenuated vaccines. peav is an important enteropathogen in pigs, but currently no effective treatments or vaccines against peav infection are available. to develop a live-attenuated vaccine for peav, we generated candidates via serial cell passage of the parental peav gds strain and evaluated the pathogenicity of peav p , p and p in -day-old newborn piglets. further, we identified genetic changes related to attenuation by performing comparative, complete genomic sequence analysis of virulent and attenuated peav variants. vero cells were obtained from atcc (atcc number: ccl- ) (usa), and cultured in dulbecco' s modified eagle medium (dmem) (hyclone, usa) supplemented with u/ml penicillin, u/ml streptomycin, and % fetal bovine serum (fbs) (bovogen, australia). the maintenance medium for peav propagation was dmem supplemented with μg/ml trypsin (gibco, usa). the isolation and identification of peav gds strain were reported previously by our laboratory (xu et al., a) . serial virus passaging and plaque-purification in vero cells were performed as previously described with some modifications (lin et al., ; xu et al., a) . briefly, vero cells were cultured in -well plates, and washed three times with sterile ph . × phosphate buffer saline (pbs) when % confluent. ten μl of peav gds strain together with ml maintenance medium were added to plate and cultured continuously at ℃ in % co to observe cytopathic effect (cpe). around day post inoculation (d.p.i.), the plates were frozen at - ℃ and thawed twice after cpe was evident in the inoculated cell monolayers. the cells and supernatants were harvested together and used as seed stocks for the next passage. peav gds strain was passaged regularly to a total of passages in vero cells every - days and the p , p and p were plaque-purified. finally, t flasks were used for the p , p and p propagation in vero cells. four-day-old crossbred conventional newborn piglets produced by duroc × landrace × native pigs of guangdong of china were procured from wen' s foodstuffs group co, ltd (guangdong, china). all pigs were maintained in our animal facility with a mixture of skim milk powder (inner mongolia yi li industrial group co., ltd, china) with warm water ad libitum for day before the experimentation. the animal study was approved by the institutional animal care and use committee of the sun yat-sen university (guangdong, china) and animals were treated in accordance with the regulations and guidelines of this committee. kinetics of peav replication in vero cells were performed as previously described with some modifications (lin et al., ) . briefly, vero cell monolayer was inoculated with p , p and p at a moi = . . the virus inoculum was removed at h post inoculation (hpi), washed with ph . × pbs twice and replaced by the maintenance medium. culture supernatant and cell lysates were collected at , , , and hpi, respectively. after freezing and thawing once, the mixtures of culture supernatants and cell lysates were subjected to titration for % tissue culture infectious dose (tcid ) in -well plates and calculated using reed-muench method (reed and muench, ) . thirty-two -day-old conventional newborn piglets were randomly divided into four groups and piglets in each group. prior to inoculation, piglets were confirmed to be negative for the major porcine enteric viruses including pdcov, pedv, tgev, prov, peav, by testing of rectal swabs using specific rt-pcr according to previously described method (xu et al., ; zhou et al., a) . in addition, serum of each piglet was collected and a commercial kit (allright biotechono-logy, china) was applied in order to detect pedv-specific antibody according to the manufacturer' instruction. pedv-specific antibodies were not detected in all sera samples (data not shown). after -day acclimation, piglets in group were orally inoculated with ml of maintenance medium and served as uninfected controls. piglets in group , and were orally challenged with ml of maintenance medium containing a total of × tcid of p , p and p , respectively. to reduce the risk of cross contamination among piglets and piglet death caused by non-experimental factors, the entire animal study period was ended in nine days after challenge. all piglets were observed daily for clinical signs of diarrhea and lethargy. diarrhea severity was scored with the following criteria (chen et al., ) : = normal, = soft (cowpie), = liquid with some solid content, = watery with no solid content. rectal swabs were collected daily from each piglet from d.p.i. to d.p.i., submerged into ml sterile ph . × pbs immediately after collection and stored at - ℃ for virus shedding analysis. two piglets from each group were necropsied at d.p.i.. at necropsy, the fresh jejunums were collected and then formalin-fixed. the formalin-fixed samples were used for histopathology and immunohischemistry analysis. in addition, the mortality of newborn piglets in each group was recorded daily. viral rna shedding in piglet feces after infection with peav was detected as previously described with some modifications (xu et al., a) . briefly, total rna was prepared from the supernatant of rectal swab from each piglet using a rneasy kit (magen, china) according to the manufacturer' instruction, and was treated with dnase i. two μg of total rna was used for cdna synthesis by reverse transcription using rt-pcr kit (takara, dalian). the specific primers for the n gene of peav (sense: ′-ctgactgttgttgaggttac- ′; antisense: ′-tctgcc aaagcttgtttaac- ′), and probe ( ′-fam-tcacagtctcgttctcgc aatca-tarma- ′) were designed with reference to the previous publication (zhou et al., a) and synthesized by invitrogen company (shanghai, china). the real-time pcr assay was carried out with an applied biosystem instrument (life technologies, usa). the pcr was performed in a -μl volume containing μl of cdna, μl of thunderbird probe qpcr mix, . μl × rox reference dye (toyobo, shanghai), . μmol/l of probe, and a . μmol/l of each gene-specific primer. the thermal cycling parameters were as follows: °c for s; cycles of °c for s, °c for s. and the n gene was amplified to generate the standard curve from peav gds strain using the specific primers (sense: ′−ccgctcgagatggcaactgtta attgg- ′; antisense: ′-cgcggatcccgattaataatctcatccac- ′) z. xu, et al. veterinary microbiology ( ) that were designed with reference to the published sequence (genbank, accession no: mf . ), and the pcr products were cloned into the pegfp-n (clontech, usa). the known plasmid concentration was fold serially diluted for generating a standard curve in each plate. the quantity of peav viral rna in tested samples was calculated based on the cycle threshold (ct) values for the standard curve. histological and immunohistochemical staining were performed as previously described with some modifications (xu et al., a) . briefly, tissue samples of jejunum of the piglets from the challenged and control groups were separated, routinely fixed in % formalin for h at room temperature, dehydrated in graded ethanol, embedded in paraffin, cut in -μm sectioned, and mounted onto glass slides. after the sections were deparaffinized, rehydrated, and stained with hematoxylin and eosin (h&e), the slides were examined and analyzed with conventional light microscopy. five-μm sections of formalin-fixed paraffinembedded tissues were placed onto positively charged glass slides and the slides were air dried for min at °c. the tissue sections were deparaffinized, rinsed and incubated with target retrieval solution (servicebio, china). after being blocked with % bsa (solarbio, china), the sections were incubated with anti-peav m monoclonal antibody (wen's foodstuffs group co., ltd, china) as the primary antibody and the dilution of antibody was : for h at °c. they were then incubated with peroxidase-labeled goat anti-mouse igg secondary antibody (dako, denmark) and the dilution of antibody was : for min at room temperature, and the samples were finally visualized with a , ′-diaminobenzidine (dab) chromogen kit (dako, denmark). hematoxylin was used for counterstaining. tissues of piglets from uninfected control groups were used as negative samples. the immunohistochemistry slides were evaluated according to the evaluation system of histology and immunohistochemistry by jung et al. ( ) . the complete genomes of p , p and p were sequenced by illuminahiseq as previously described with some modifications (malboeuf et al., ) . briefly, total rna was prepared from the peavinfected vero cells using a rneasy kit (magen, china) according to the manufacturer' instruction, and was purified as described above. cdna synthesis was performed by reverse transcription using rt-pcr kit (takara, dalian). the genomic cdna was disrupted and converted to a sticky end by adding the a-base at ′ end of the cdna. the dna containing index sequence was added at either side of sticky end by complementary bases. the target fragment within the scope of a certain length was collected before magnetic beads selection. the sequencing library was built, tested using pcr amplification index sequence, and combined to the chip by bridge pcr. at last, the complete genome was sequenced by iiiumina hiseq. a genome homology analysis and phylogenetic trees were constructed by using the maximum likelihood method with mega software (http://www.megasoftware.net/) based on the whole-genome nucleotide sequences from peav gds , p , p and p together with other covs, like pedv, pdcov and tgev. sequence alignments of n, m, s and orf ab amino acids (aa) of gds , p , p and p were performed using the dnaman. the s protein of cov, the pivotal surface glycoprotein, plays an important role in virus attachment and entry, and induction of neutralizing antibodies in vivo (cruz et al., ; li et al., li et al., , . to further determine the differences of vero-passaged peav strains, the three-dimensional structures of s proteins of gds , p , p and p were predicted in swiss-model using amino acid homology modeling, based on the existing three-dimensional structure of s glycoprotein trimer of human coronavirus nl (pdb code szs) (walls et al., ) in protein data bank. the pymol molecular graphics system (delano scientific; http://www.pymol.org) was used for figures preparation. statistical comparisons were performed using graphpad prism software. the significance of the differences between the growth rate of p and p or p in the tcid was determined by the anova and mann-whitney accordingly. to generate an attenuated peav vaccine candidate, peav gds was passaged regularly to a total of passages in vero cells and the selected passages including p , p and p were characterized by sequencing and analyzing the complete genome (fig. ) . to determine the growth rate of p , p and p in vitro, we inoculated p , p and p at moi = . in vero cells and detected the live virus count at corresponding time point with a tcid assay. interestingly, we observed that the sizes of peav-induced syncytia increased in p and p ( fig. a) . the diameter of p -syncytia was larger than that of p and p ( fig. a ). in addition, the muti-step growth kinetics of peav in vero cells showed similar growth curve trends of p , p and p , but the titers of p were significantly higher than those of p and p at h.p.i. (p < . ) (fig. b) . to evaluate the pathogenicity of vero-passaged peav strains in newborn piglets, we experimentally infected -day-old conventional newborn piglets with p , p and p at a dose of × tcid / head via oral feeding. as shown in fig. a , the newborn piglets inoculated with p , p or p all showed diarrhea from d.p.i. to d.p.i., as compared with the control. but compared to p and p , the degree of diarrhea of the newborn piglets inoculated with p was the lowest, indicating that viral pathogenicity decreased gradually in day-old newborn piglets with cell passage. in addition, we explored the fecal viral shedding in peav-inoculated piglets from d.p.i. to d.p.i. by qrt-pcr. consistent with the clinical signs, the amount of virus rna was lower in p -inoculated newborn piglets, and no peav rna was detected in the negative control piglets during the study (fig. b) . taken together, these results demonstrated that cell passage gradually decreased viral pathogenicity of gds to -day-old newborn piglets. since peav at selected passages caused varying degrees of diarrhea in newborn piglets, we also recorded the mortality of newborn piglets in each group. as shown in fig. , in p -inoculated group, except two piglets that were necropsied at d.p.i., , , , and death (s) were recorded at , , , and d.p.i., respectively. similarly, except two necropsied piglets, piglets in p -inoculated group died from to d.p.i. no piglets died in p -inoculated group and the control group except two piglets that were necropsied at d.p.i.. taken together, these results suggest that peav p strain is low pathogenic to the newborn piglets. to determine the gross pathological and histological changes in piglets infected with peav gds at selected passages, two piglets from each group were necropsied at d.p.i. gross findings were similar in piglets orally inoculated with p and p . the whole intestinal tract, where yellow watery contents accumulated, were transparent, thinwalled, and gas-distended (fig. b&c) . no lesions were observed in the whole intestinal tract of the p -inoculated piglets and the negative control piglets (fig. a&d) . microscopic lesions were also analysed. as shown in fig. f&g , abruption of intestinal villus was observed in p or p -inoculated piglets, whereas the intestines in p -inoculated piglets and the negative control were normal (fig. e&h) . consistent with the histopathological results, peav antigen was detected in the cytoplasm of the villous enterocytes of the p or p -challenged piglets by immunohistochemical analysis, but no antigen observed in p -inoculated and mock piglets (fig. i-l) . taken together, these results indicate that peav p is an attenuated variant. to determine the cause of reduced pathogenicity of vero-passaged peav strains, the complete genomes of p , p and p were sequenced and analyzed. compared to the complete genome of peav gds (mf . ), p possesses point mutations, a -nt deletion and a -nt insertion; p possesses point mutations, a -nt deletion and a -nt insertion; p possesses point mutations, a -nt deletion, a -nt deletion and a -nt insertion, respectively (data not shown), presenting a clue of different pathogenicity of these three strains. in addition, the parental strain gds shares . %, . %, . % nucleotide identity with p , p and p , respectively (data not shown), indicating that differences in viral genome increased gradually with cell passages. in spite of some differences in viral genome of these strains, phylogenetic analysis showed that peav gds , p , p and p were clustered into a large clade (fig. a) . we further analyzed the aa of proteins which might be associated with the pathogenicity of covs, such as m ( results are representative of three independent experiments. data are represented as mean ± sd, n = . *stands for p < . . z. xu, et al. veterinary microbiology ( ) (hou et al., b) . as shown in fig. b , compared to the m protein of peav gds , p possesses aa mutations, p possesses aa mutations, and p possesses aa mutations. compared to the n protein of peav gds , interestingly, only p possesses aa mutation, while no mutations were observed in p and p . compared to the s protein of peav gds , p possesses aa mutation, p possesses aa mutations, while p possesses aa mutations and aa deletions. in addition, compared to the orf ab protein of peav gds , p possesses aa mutations, aa insertions, p possesses aa mutations, aa insertions, p possesses aa mutations, aa deletion and aa insertions. the detailed information of aa changes might help to explain the low pathogenicity of p to the newborn piglets. . . peav p has significant differences in the s protein structure considering the obvious aa mutations and deletions in s proteins of vero-passaged peav strains, we attempted to predict and analyze the three-dimensional structures of s proteins of these peav strains. after analysing the amino acid sequences, the s protein in gds was found to have . % identity with the s protein of human cov nl (walls et al., ) the s protein structures of these strains were predicted by swiss-model, using the s protein of human cov nl as the template (fig. ) . the predicted structure of gds s protein forms a homologous trimer with an expect-value of . , and each monomer is composed of residues in the aa positions - (fig. a&b) . in z. xu, et al. veterinary microbiology ( ) addition, the predicted three-dimensional structures of the s proteins of peav p , p and p have similar overall structures when compared with gds s protein ( fig. c-f) . however the monomer structural overlap of the four s proteins showed that there were three loops and two β strands having significant structural differences in the main chain, especially between p and gds . since the first report of peav in pigs in early february of in guangdong, china (gong et al., ) , this novel swine enteric cov has been widely detected in guangdong, china, with a prevalence of . % and an emergence in china as early as august by a retrospective detection study (zhou et al., ) . although a few studies have demonstrated peav was highly pathogenic to newborn piglets (pan et al., ; xu et al., a; zhou et al., b) , there are no effective treatments or any vaccines against peav infection. in the present study, attenuated peav variants were generated by serial cell passaging and orally inoculated to newborn piglets to evaluate the attenuation in vivo. further, we identified the molecular basis of attenuation by performing comparative, complete genomic sequence analysis of virulent and attenuated peav variants. swine enteropathogenic covs affects all sectors of the pig industry and all cycles of production, which results in significant economic losses. currently, vaccination remains the most effective measure against swine enteropathogenic covs. of note, several groups have used live-attenuated vaccines to prevent and control pedv and tgev in pigs (aynaud et al., ; song et al., ) . as a newly swine enteropathogenic cov, there are no reports about live-attenuated vaccine for peav. pathogenic microorganisms serially passaged in cells (jie et al., ) is one of the most common methods to prepare live-attenuated vaccine. vero cells as a stable african green monkey kidney cell line (rhim et al., ) is commonly used to attenuate covs like pedv (lin et al., ) . in this study, peav gds was passaged regularly to a total of passages in vero cells and the growth rate of p , p and p was determined in vitro. the muti-step growth kinetics of p , p and p in vero cells were similar, but the titers of p were fig. . the survival rate of newborn piglets after infection with peav p , p and p . the mortality of newborn piglets in each group was recorded from to d.p.i.. significantly higher than those of p and p at hpi, similar to observations in attenuated pedv vaccine (lin et al., ) , indicating that p had better adaptation in cells in vitro. usually, adaptation in cell lines coincides with attenuation of the viruses, such as african swine fever virus (asfv) isolate georgioa and pedv isolate pc a, in swine lin et al., ) . the higher fitness of p in vero cells might suggest the changed pathogenicity of peav. for a vaccine candidate, safety is the top concern. it was reported that swine enteric covs like pdcov, pedv and tgev are age-dependent disease (moon et al., ; shibata et al., ; xu et al., b) . similarly, in another study, we found that peav was highly pathogenic to newborn piglets (xu et al., a) , but did not cause vomiting and death in weaned piglets at a medium dose of × tcid within days (data not shown), for which reason we choose newborn piglets to conduct peav pathogenicity study. it was known that peav infection caused typical clinical symptoms characterized by watery diarrhea via oral feeding in newborn piglets (xu et al., a) . in the study, we infected the -day-old newborn piglets with p , p and p via oral feeding. compared with p and p , p caused slight diarrhea in newborn piglets, suggesting that the pathogenicity of peav changed through cell passage. for enteric viruses, viral shedding is often accompanied by diarrhea, so was peav infection (xu et al., a) . thus, fig. . phylogenetic trees constructed on the basis of the complete genome sequences of peav p , p and p or other covs and sequence alignments of peav at selected passages. (a) the dendrogram was constructed using the neighbour-joining method in the mega software package, version (http://www.megasof tware.net). bootstrap resampling with , replicates was performed, and bootstrap values are indicated for each node. reference sequence obtained from genbank is indicated by strain name. the scale bar represents . nucleotide substitutions per site. (b) sequence alignments of the amino acids of m, n s and orf ab proteins were performed using dnaman software. z. xu, et al. veterinary microbiology ( ) we collected fecal swabs from the peav-challenged piglets and detected the fecal shedding by real-time pcr. consistent with the clinical signs, the amount of virus rna was lower in p -inoculated newborn piglets. interestingly and importantly, the presence of peav rna in fecal samples of p inoculated piglets from d.p.i to d.p.i suggested the successful colonization and replication of p in intestinal tract, which is essential to induce mucosal immunity for an enteric pathogen vaccine candidate. it was reported that the lesions were only observed in intestinal tract but not any other organs after peav infection and peav antigen was detected in the villous enterocytes of the peav-challenged piglets (xu et al., a) . compared to p and p , p strain caused slight macroscopic and microscopic lesions in intestinal tract and no peav antigen was detected in the cytoplasm of the villous enterocytes of the p -challenged newborn piglets by immunohistochemical analysis, indicating that the infectiveness of p was reduced. like other swine enteric covs, such as pedv (shibata et al., ) , peav infection caused fatal disease in newborn piglets (xu et al., a) . thus, death after infection is the key indicator to determine the attenuation of the viral strain. compared to p and p , no piglets died in p -inoculated group and the control group during the study, indicating that p is low pathogenic to the newborn piglets. taken together, these results demonstrated that p strain is safe for pigs and effective in colonizing and replicating in intestine, suggesting its future application as a good peav vaccine candidate. compared to p and p strains, the virulence of p was significantly reduced in -day-old newborn piglets. the s protein of covs is the pivotal surface glycoprotein involved in virus attachment and entry, virus attenuation and induction of neutralizing antibodies in vivo (cruz et al., ; lin et al., ; woo et al., ) . in addition, the virulence of a recombinant pedv with inactivation of the endocytosis signal of the s protein was significantly reduced in piglets (hou et al., a) , indicating that the s protein might be associated with the virulence of covs. compared to the s protein of parental strain gds , p possesses aa mutations in aa positions and , which were also found in p and p , indicating that these aa-mutations are insufficient to cause viral attenuation. of note, compared to p and p , p possesses aa deletions and aa mutation in aa positions , , and , respectively, indicating that the absence and mutation of these amino acids might affect the viral attenuation. usually, protein with complete structure has corresponding function, and the changes in the protein's structure might affect its function. overlap of the s protein structures of gds , p , p and p showed that the difference between p and gds was most significant (fig. ) , indicating a potential relationship between these differences of s protein and pathogenicity of peav. it was reported that the s protein is necessary but not sufficient for the virulence of pedv (wang et al., ) . m protein plays an important role in the process of virus assembly by interacting with s and n proteins (de haan et al., ; nguyen and hogue, ) . n protein can encapsulate the viral genome rna to form the nucleocapsid (spaan et al., ; sturman et al., ) . inactivation of the viral ′-o methyltransferase of nsp could help to engineer a live attenuated pedv vaccine (hou et al., a) . in this study, aa changes were also found in m, n and orf ab proteins in p strain as compared to gds (fig. b ). in addition, no additional mutation was observed by sequencing the viruses shed from the challenged piglets (data not shown). therefore, these results suggest that the complete attenuation of a peav strain might be the result of a combination of multiple mutations along the genome, and can occur via multiple molecular mechanisms. whether individual or a combination of genetic changes of peav strains alter viral infectivity, pathogenicity and replication efficiency need further examination by reverse genetics technology. together, all these results confirmed that p generated in this study was low pathogenic to the newborn piglets and might serve as a good peav vaccine candidate. however, there are still several important questions needed to be addressed. for instance, what is the immunogenicity of p strain as a live-attenuated vaccine candidate in pigs? what is the protective effect of p strain in pigs? elucidation of these questions will help us to develop a safe and efficacious live-attenuated vaccine to control peav. in summary, our research successfully generated an attenuated peav variant p . p has the highest titers of virus load, via serial passaging of the parental gds strain. remarkably, inoculation of newborn piglets with p by oral feeding caused the lowest level of clinical symptoms, including slight diarrhea, fecal viral shedding, and pathological changes with a survival rate of % in newborn piglets. the changes in genomic composition and structures found in p by genomic analysis might account for the underlying molecular mechanisms of peav attenuation. cyx, zcx and yl conceived and designed the experiments; zcx, yl, pp, yw, yl performed the experiments; zcx analyzed the data; ycc, cyx, ccz and lg contributed reagents/materials/analysis tools; zcx wrote the paper. cyx checked and finalized the manuscript. all authors read and approved the final manuscript. the animal study was supervised by the institutional animal care and use committee of sun yat-sen university (iacuc dd- - ) and used in accordance with regulation and guidelines of this committee. the authors declare that they have no conflict interest. induction of lactogenic immunity to transmissible gastroenteritis virus of swine using an attenuated 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by nsp retrospective detection and phylogenetic analysis of swine acute diarrhoea syndrome coronavirus in pigs in southern china development of a taqman-based real-time rt-pcr assay for the detection of sads-cov associated with severe diarrhea disease in pigs key: cord- -l wj dsy authors: Özdemir, rabia bilge Özgül; Özdemir, alper tunga; sarıboyacı, ayla eker; uysal, onur; tuğlu, mehmet İbrahim; kırmaz, cengiz title: the investigation of immunomodulatory effects of adipose tissue mesenchymal stem cell educated macrophages on the cd t cells date: - - journal: immunobiology doi: . /j.imbio. . . sha: doc_id: cord_uid: l wj dsy mesenchymal stem cells (mscs) are strong immunomodulatory cells investigated in numerous clinical studies on fatal pathologies, such as graft versus host disease and autoimmune diseases; e.g., systemic lupus erythematosus, crohn's disease, and ulcerative colitis. macrophages are one of the critical cells linking the innate and adaptive immune system, and it has been shown that mscs can differentiate between pro-inflammatory m phenotype and anti-inflammatory m phenotype of macrophages. however, it has not yet been fully clarified whether these differentiated macrophages are functional. in this study, we compared the immunomodulatory effects on the cd t cells of m , m a and m c macrophages with the macrophages that directly and indirectly cultured with mscs. we analyzed the changes in cd , cd , cd , cd and cd r expression to evaluate macrophage phenotypes, and the changes in cd , ifn-g, il- , il- a and foxp expression to evaluate t helper subsets using the facs method. the changes in il- b, il- , il- , il- p , il- a and ifn-g in the media supernatants were analyzed using the luminex method. we also performed wst- and caspase- elisa analyses to observe the proliferation and apoptosis status of the t cells. mscs were found to differentiate macrophages into a distinctive phenotype, which was close to the m c phenotype, but was not considered as an m c cell due to the low expression of cd , a characteristic marker for m c. while mem-d, mem-id and mscs showed similar inhibitory effects on the th and th cells, the most significant increase in treg cell frequencies was seen in mem-d cells. macrophages can alter their phenotypes and functions according to the stimuli from the environment. the fact that macrophages educated with mscs suppressed the production of all the cytokines we evaluated even after the removal of mscs suggests that these cells may be differentiated by mscs into a suppressive macrophage subgroup. however, the treg cell activation caused by direct interactions between mscs and macrophage cells may be the most prominent observation of this study compared to previous work. as a result, according to our data, the interactions between mscs and macrophages may lead to differentiation of macrophage cells into an immunosuppressive phenotype, and these macrophages may suppress the t lymphocyte subgroups at least as effectively as mscs. however, our data obtained from in vitro experiments should be supported by future in vivo studies. mesenchymal stem cells (mscs) are adult stem cells that can be isolated from many tissues, and they have different types, such as chondrocyte, adipocyte or osteocyte (el, ) . mscs also have strong immunomodulation abilities; e.g., they suppress immune cells through the secretion of cytokines, such as prostaglandin e (pge ), indoleamine , dioxygenase (ido), hepatocyte growth factor (hgf), and soluble human leukocyte antigen g (shla-g) and transform growth factor beta (tgf-b) and interleukin (il- ) (budoni et al., ; Özdemir et al., ) . due to these unique properties, mscs are clinically used in the treatment of graft versus host disease (gvhd) caused by uncontrolled activation of immune cells (sangiorgi and panepucci, ) and certain autoimmune diseases, including rheumatoid arthritis (ra), crohn's disease, and systemic lupus erythematosus (sle) (dazzi and krampera, ) . however, the clinical responses of patients to msc treatment are variable, ranging from good responses to only temporary or no effect (mastri et al., ; silva et al., ) . the immunomodulatory effects of mscs occur in a non-specific and nonselective manner. when mscs are activated, they produce certain cytokines, such as pge , ido, hgf, tgf-b and il- , which suppress almost all immune cells (el, ; Özdemir et al., ; cho et al., ) . however, this suppressive effect only lasts for the lifespan of mscs. in both pathogen-related infections and autoimmune pathologies, antigen presentation is a crucial step for t cell activation and lineage specific differentiation. during t cell activation, antigen-specific memory cells are formed, resulting in a life-long response (berard and tough, ; gray et al., ) . to gain a sustainable immune response, t cells should be activated or inhibited by antigen-presenting cells. mscs are not antigen-presenting cells, and under normal conditions, their major histocompatibility antigen expressions (mhc) are either weak or absent (dominici et al., ; shi et al., ) . macrophages are a part of innate immunity, and they play important roles in the antigen presentation and the regulation of immune response. according to the exposed type of cytokine and antigen, macrophages can alter their phenotype and function. it has been shown that ifn-g, lps and tnf-a stimulation leads to the upregulation of mhc-ii, cd and cd expressions of the cell surface and an increase in inos, il- b, il- and il- production of macrophages. this type of macrophages is called m , which activate th immune cells important to the immune response of intracellular pathogens and tumor resistance (ambarus et al., ; martinez and gordon, ) . another macrophage group, called m macrophages, antagonize the effects of m macrophages. m macrophages are further divided into the subgroups of m a, m b and m c according to cytokine and antigen stimulation. il- stimulation is specific to the development of the m a macrophage phenotype. through il- stimulation, the cell surface expressions of cd r and cd increase but cd decreases. furthermore, the production of arginase and il- increases (ambarus et al., ; van dyken and locksley, ) . m a macrophages are important for the activation of th cells responsible for immune responses, including encapsulation of parasites and allergy (martinez and gordon, ; martinez et al., ) . for the emergence of the m b phenotype, immune complexes and tlr stimulation are required. an appropriate stimulation would increase the il- production of m b cells but decrease il- production (anderson and mosser, ; sironi et al., ) . m c is another subset of m macrophages, which are activated by il- stimulation. an increase in the cd and cd expressions and upregulation of il- and tgf-b production caused by il- stimuli, are specific to the m c phenotype (ambarus et al., ; schwarz et al., ) . in the current literature, there are several studies showing that mscs could inhibit pro-inflammatory m macrophages and stimulate anti-inflammatory m macrophages (cho et al., ; vasandan et al., ) . however, it has not been yet fully clarified whether these differentiated macrophages are functional. as a professional antigen-presenting cell, te macrophages can help to transfer the immunomodulation effects of mscs to the adaptive immune cells. in this study, we aimed to investigate the interactions of macrophage and mscs, and the resulting effects of these interactions on cd t cells. for this purpose, we differentiated the macrophage cells into m , m a and m c phenotypes to observe the effects on cd t cells with known macrophage phenotypes. in addition, we generated msc-educated macrophage (mem) cells by direct and indirect co-culture and compared their effects on cd t cells with known macrophage phenotypes. the protocol, sample collection procedures and informed consent form of this study were approved by ethical committee of ege university ( - . / ). to perform cell culture experiments, after informed consent blood samples collected from three female ( , and age) and three male ( , and age) healthy volunteers. peripheral blood mononuclear cells (pbmcs) were isolated by using density centrifugation (ficoll-paque) method. human adipose tissue derived mscs (pcs- - ™) were purchased from american type culture collection (atcc). frozen cells were thawed in °c and cultured by using dmem-f (biosera, usa) medium that including % fetal bovine serum (fbs) (biosera, usa), u/ml penicillin, μg/ml streptomycin (biosera, usa) and % mm l-glutamate (biosera, usa) at °c and % co incubator. cells were controlled daily, and sub cultured when they reached - % confluence. th passage mscs were used to perform co-culture experiments. to obtain macrophage cells from pbmcs, the cells of each subjects were cultured by using rpmi (biosera, usa) medium that including % fetal bovine serum (fbs) (biosera, usa), u/ml penicillin, μg/ml streptomycin (biosera, usa) and % mm l-glutamate (biosera, usa) at °c and % co incubator for h. after the culture period, non-adherent cells were washed with phosphate-buffered saline (pbs) and cultured in different culture plate by using rpmi complete medium and ng/ml human il- (reprokine ltd, israel) till to co-culture experiments. adherent cells were detached by trypsin-edta (biosera, usa) and divided as × cells per well in two well plates. cells were cultured by using rpmi complete medium and human ng/ml macrophage colony-stimulating factor (m-csf) (reprokine ltd, israel) at °c and % co incubator for days. after the culture period, to stimulation of m macrophage phenotype different wells of each plates were cultured with rpmi complete medium and ng/ml ifn-g (reprokine ltd, israel), ng/ml tnf-a (reprokine ltd, israel) and ng/ml gm-csf (reprokine ltd, israel), to m a phenotype ng/ml il- (reprokine ltd, israel) and to m c ng/ml il- (reprokine ltd, israel) and ng/ml tgf-b (reprokine ltd, israel). to generate the msc educated macrophages (mem), cells were direct co-cultured with × (dazzi and krampera, ) mscs (mem-d) and indirect by using . μm pore sized inserts (mem-id) and added ng/ml m-csf and ng/ml gm-csf. one well of each plate were remained as unstimulated control cells. all cells cultured at °c and % co incubator for days. then, the cells of first plate were used to evaluation of phenotype changes of macrophages by facs analyses. the other plate was used to co-culture experiments. to compare of immunomodulatory effects of mem-d and mem-id cells on cd t cells with known macrophage phenotypes, co-culture experiments were performed by using pbmcs. before adding the pbmc, transwell cultured mscs in the mem-id group were removed. pbmcs of each donor were activated by ng/ml il- and . μl dynabeads human t-activator cd /cd (thermo scientific, usa). than × (mastri et al., ) activated pbmcs were added in all different macrophage phenotypes. in different well plate, same amount of activated pbmcs were co-cultured with ad-mscs only and cultured only as activated control. to evaluation unstimulated control group, × (mastri et al., ) unstimulated pbmcs were cultured in different well. all cells cultured at °c and % co incubator for days. ambarus et al. have systematically validated the specific markers that change in response to macrophage polarization (ambarus et al., ) . they have showed that cd and cd expressions of macrophages increased with ifn-g stimulation. they have demonstrated that cd expression of il- -stimulated macrophages decreased, but that cd r expressions were specifically increased. finally, they have reported a specific increase in cd in il- -stimulated macrophages. in our study, we performed facs analyses by using cd fitc, cd pe, cd pe-cy , cd percp.cy . and cd r pe (biolegend, usa) antibody staining to evaluation of phenotypic changes of macrophage cells. to evaluation of changes of cd t cells cd fitc, ifn-g pe, il- pe, il- a pe (diaclone, france) and foxp (biolegend, usa) antibody staining were performed. all measurements were performed with bd accuri c flow cytometry device (bd biosicience, usa) and all analyses were performed by using flowjo v software (becton, dickinson & company, usa). to evaluation of effects of different macrophage phenotypes on inflammatory cytokines il- b, il- , il- , il- p , il- a and ifn-g levels were measured by using luminex kit (thermo fisher scientific, usa). all measurements were performed with luminex ™ device (merck millipore, usa) that calibrated xponent . compatible calibration kit (emd millipore cat. # - ) and verification kit (emd millipore cat. # - ). obtained signal densities were evaluated with xpotent . software (merck millipore, usa). median fluorescence intensity (mfi) data of each sample were calculated by parameter logistic spline curve-fitting method and cytokine concentrations were determined. to observe the effect of different macrophage phenotypes on proliferation, we performed wst- (roche, germany) analysis. × cells of each macrophage phenotype and ad-mscs were seeded a well plate and cultured for h to adhesion of plate surface. than × (dazzi and krampera, ) activated pbmcs were added on these cells. as control, × (dazzi and krampera, ) activated and unstimulated pbmcs only were seeded different wells. all cells cultured at °c and % co incubator for h. after the culture period, μl wst- solution was added to each well and cultured for h. absorbance changes were measured at a wavelength of nm with the multi-plate reader versamax (sunnyvale, ca, usa). the cd surface expressions, which is activation marker of lymphocytes, were analyzed by flow-cytometry to observe activation status of pre-co-culture t cells. for this, pre-activated and activated t cells were stained with cd fitc and cd pe labeled antibodies and analyzed on bd accuri c device. to assess the apoptosis status of the cells, caspase- levels from the media supernatants which collected after co-culture were analyzed using the elisa kit (mybiosource, usa). data obtained from all experiments were evaluated as mean ± sd and were analyzed by using spss v (ibm, usa). to finding statistical differences of all groups, the one-way anova test was used, and according to levene statistic results the tukey hsd or dunnett t posthoc test results were used. the difference between the groups was considered statistically significant when p < . . to performing graphical charts of statistical results, the prism v. software (graphpad, usa) were used. to observe the effects of macrophage and msc interactions on phenotype, we generated unstimulated (us), m , m a and m c macrophages cells via specific cytokine stimulations, and then compared the phenotypes of each cell type. in microscopic examinations, we observed that m and m c macrophages were predominantly spindleshaped, whereas m a, mem-d and mem-id macrophages were round cells with dendritic extensions. the microscopic images of each macrophage phenotype are shown in fig. . we then analyzed the changes in cd , cd , cd , cd and cd r molecules to evaluate the macrophage phenotypes. to minimize the differences due to biological variations and inter-assay biases, we used the fold of geometric median fluorescence intensity (gmfifold) values obtained from the gmfi values of stimulated (s) and unstimulated (us) macrophages [gmfifold=(s-gmfi/us-gmfi)- ]. the histogram plots obtained from facs analyses are presented in fig. , and the comparison of the gmfifold values of the groups is given in fig. . in our experiments, the cd expression of the m and m a cells was lower than that of the usem cells, but this was not statistically significant. even if the cd expressions of the m c, mem-d and mem-id groups were higher compared to the m and m a groups, the difference was only significant between the m a and mem-id groups. the cd expression was increased in all groups except m a cells, and we found that the mem-id group had a significantly higher cd expression than the m a group (fig. ) . the highest and statistically significant cd expression was observed in the mem-d group, and the to observe the effects of the mem cells on cd t cells, we performed co-culture experiments. before the co-culture of pbmcs, we validated the activation status of lymphocytes. for this purpose, we labeled unstimulated and activated pbmcs by cd fitc and cd pe antibodies and analyzed them using facs. for all donors, we detected a significant increase in cd expression in activated pbmcs compared to the unstimulated pbmcs ( supplementary fig- ) . during the co-culture experiments, we observed differences in the interactions between cells. the t cells marked with cd /cd beads were orange under a light microscope due to the deposition of iron on their surface. in the microscopic examinations, we found that t lymphocytes generated clusters similar to antigen presentation, and these clusters were more prominent in m a and mem-d cells (fig. ) . the changes in cd t cell subsets were evaluated by a facs analysis and the dot-plot charts of each t cell subsets are given in fig. . the cell frequencies (%) are summarized in supplementary table- , and the comparison charts between the groups in fig. . the th (cd + ifng + ) cell frequencies of all groups, except the m a and m c groups, were significantly higher compared to the us-pbmc group. in addition, the th frequencies of the m group was significantly higher than that of the msc group. the th (cd + il + ) cell frequencies of the m a and a-pbmc groups were significantly higher compared to the remaining groups. this finding confirmed that m a macrophages were more effective in inducing the th subset, but it also showed that the mem-d and mem-id cells might be as effective as mscs in suppressing the th cells. the frequencies of the th (cd + il a + ) cells of all groups, except the m group, were significantly lower compared with the a-pbmc group. unlike the other groups, the m c group had a significantly higher th frequency than the us-pbmc group. lastly, we detected that the treg (cd + foxp + ) cell frequencies of the mem-d cells were significantly higher than the other groups. this finding suggested that the direct interaction of macrophages and mscs might have led to a more effective increase in treg cell levels. after the evaluation of phenotypic alterations of cd t cells, we evaluated the changes in inflammatory cytokines. the comparative charts of all cytokines are provided in fig. , and the results of all table- . the ifn-g levels of the m groups were significantly higher compared to all co-culture groups, except the m a and a-pbmc groups. the il- levels of the us-m and mem-id groups were significantly lower than those of the other groups (p < . ), except the us-pbmc group. the il- levels of all groups were significantly higher compared to the us-pbmc group. in addition, the il- levels of the mem-d group were significantly lower than all groups, except the msc group. similarly, the mem-id group had significantly lower il- levels than the us-m, m a and m c groups. the il- a levels of the us-pbms group were significantly lower than those of the other groups, except the mem-d group (p < . ), and the m c group had significantly higher il- a levels than the us-m, m a, mem-d and mem-id groups. the il- p levels were similar in all groups, but they were significantly lower in the mem-id groups compared to the msc and a-pbmc groups (p < . ). for il- b, surprisingly, the mem-d and msc groups had significantly higher levels compared with the remaining co-culture groups (p < . ). to compare the effects of all macrophage phenotypes on lymphocyte proliferation and apoptosis, we performed wst- analyses. the comparative charts of all wst- and caspase- results are summarized in fig. and all results are given in supplementary table- . the optical densities (ods) of the activated pbmc groups were significantly higher than those of the co-culture groups, except the m group. in the other hand, the ods of the unstimulated pbmc groups were significantly lower compared to the us-m, m and m a groups. in addition, the ods of the msc group were significantly lower than those of the us-m, m and m a groups. to evaluate apoptosis, we performed caspase- elisa analyses. the caspase- levels of the msc and us-pbmsc groups were significantly lower than those of the m a, m c, mem-d and a-pbmc groups. in this study, we evaluated macrophage polarizations by identifying several distinctive markers for m , m a and m c macrophage phenotypes (fig. ) . one of the markers evaluated was cd , which is a specific marker of monocytes with reduced expression during macrophage maturation (ambarus et al., ) . it is well known that mscs are il- -producing cells (ma et al., ; qu et al., ) , and it has been shown that their maturation can be prevented by il- (schwarz et al., ; allavena et al., ) . we observed that the cd expressions of the mem-d and mem-id groups showed a similar pattern to the m c group, suggesting that mscs can differentiate macrophage cells into the m c-like phenotype through direct or indirect effects. cd is a member of the fcγ family and plays a role in antibodymediated phagocytosis. it has been reported that cd expression in monocytes and macrophages increases in response to ifn-g and lps stimulation but decreases with il- stimulation (hristodorov et al., ) . in another study, ji et al. showed that the cd expression of macrophages increased by m-csf and il- stimulation (ji et al., ) . for cd , our findings were similar to the literature in that the cd expression of macrophages increased in the m and m c group but decreased in the m a group. additionally, the cd expressions of the mem-d and mem-id macrophages were similar to those of the m and m c macrophages. as in the case of cd expressions, this finding supports the idea mscs may alter macrophages into the m c-like phenotype. cd is a molecule involved in the full activation of t cells by interacting with the cd co-stimulatory molecule of t cells during antigen presentation (freeman et al., ) . jiang et al. reported that the cd expressions of lps-stimulated macrophages were suppressed by mscs; however, spaggiari et al. reported no change (jiang et al., ; spaggiari et al., ) . in our experiments, we observed that the cd expressions of the mem-d group were significantly higher compared to the other groups, except m c. as shown in fig. , clusters where t cells interacted, indicating antigen presentation were more common in the mem-d group than in the other groups. these observations suggest that the co-stimulatory properties of macrophages could be increased by the presence of mscs. cd is a scavenger receptor, the expression of which is increased by macrophages in response to il- and tgf-b stimulation, but it is antagonized by ifn-g and il- stimulation (fabriek et al., ) . in this respect, cd is a sensitive marker for m c macrophages, and we found that the cd expressions of the m c macrophages significantly increased compared to the other groups. based on cd and cd expressions observed, we assumed that il- produced by mscs might differentiate macrophages into the m c-like phenotype. however, the absence of a cd increase in the mem-d and mem-id groups did not support this assumption. finally, we found that cd r, a specific mscs (g). the microscopic images of activated pbmcs (h) and unstimulated pbmcs (i). the activated t cells (orange colored) have accumulated on the surface of macrophage cells, however these accumulations were detected as more dense at m a and mem-d co-culture groups compared with the us-m, m and m c co-culture groups. marker for m a cells, decreased in m macrophages and increased in m a macrophages, which is consistent with the literature. we also observed that similar to cd , the cd r expressions of the mem-d and mem-id macrophages were not altered compared to us-m. not only il- , il- and tgf-b, but also various immunosuppressive molecules, such as ido, hgf, and pge are known to be involved in the formation of immunomodulation effects of mscs (chen et al., ) . therefore, macrophages cultured with mscs are likely to be of a different phenotype than known phenotypes, which is also partially supported by our findings. we used limited and specific markers to evaluate the macrophage phenotypes; therefore, based on limited data, we were not able to make a definitive conclusion concerning the phenotype to which the macrophages cultured with mscs were closest. in this study, we focused on the functional comparison of the immunomodulatory effects of mem-d and mem-id cells on cd t cells. th cells, with the main cytokines of ifn-g and il- , orchestrate the elimination of intracellular pathogens (boehm et al., ) , and they are related to certain organ-specific autoimmune pathologies, such as type i diabetes (luckheeram et al., ) . in terms of macrophages, m macrophages stimulate the differentiation of naive t cells into the th phenotype. in contrast, m macrophages stimulate th and/or treg cell differentiation and suppress th differentiation (martinez and gordon, ; charles a janeway et al., ; mills et al., ) . previous research also reported that mscs suppressed the th response and stimulated treg cells (chen et al., ; luz-crawford et al., ) . according to our findings, the cd + ifng + (th ) frequencies of all groups were similar, except the us-pbmc group. however, there were dramatic and significant decreases in the ifn-g levels of the m c, mem-d, mem-id and msc groups. this finding suggests that the cd + ifng + cell frequencies may not be affected by different macrophage phenotypes, but their functions can be suppressed by m c, mem and msc cells. th cells orchestrate the immune response to extracellular parasites, and they are associated with asthma and allergic disorders. the main cytokines of th cells are il- , il- , il- , and il- (deo et al., ; steinke and borish, ) . the presence of a synergistic interaction between the th cells and m macrophages has previously been reported (muraille et al., ) . our data confirmed that il- stimulated macrophages (m a) could significantly stimulate th frequencies and il- production. however, although the th frequencies of the groups except m a were close to each other, the il- levels of the us-m and mem-id groups were significantly lower than those of the other groups. in the current literature, different results have been reported about msc and th interactions. mareschi et al. found that amniotic fluid mscs and placenta mscs could stimulate the il- expressions of activated pbmcs, but not in bone marrow mscs (mareschi et al., ) . Özdemir et al. and genç et al. showed that dental tissue mscs could suppress the il- expressions of t cells (Özdemir et al., ; genç et al., ) . in our experiments, we used adipose tissue mscs and observed that the il- levels significantly increased in the msc and mem-d groups compared with the mem-id and us-pbmc groups. the similarity we observed in us-m and mem-id for il- levels may indicate that mscs may have forced the macrophages to remain in an unstimulated form when there was no direct interaction. th cells have been reported to play a role in the pathogenesis of many autoimmune diseases; e.g., ra, sle and crohn's disease (tabarkiewicz et al., ) . as opposed to th and th cells that are terminally differentiated, th cells can alter their phenotype in response to signals from the environment. for example, bofeng et al. reported that il- induced macrophages contributed to the pathogenesis of colitis by increasing th activity (li et al., a) . haribhai et al. showed that tgf-b-producing m a macrophages differentiated th cells into anti-inflammatory itreg cells (haribhai et al., ) . for mscs and th interactions, qu et al. reported that th cells were suppressed by il- secreted by mscs (qu et al., ) , rozenberg et al. showed that pge secreted from mscs increased the th response but suppressed the th response (rozenberg et al., ) , and ghannam et al. noted that mscs increased treg cells by suppressing th cells (ghannam et al., ) . our findings revealed that the th frequencies of the a-pbmc group was significantly higher than those of the remaining groups, except m and m c. the il- a levels and th frequencies of the m and m c groups were consistent, but il- a remained high in the msc group (fig. ) . according to our data, il- induced macrophages may be important for stimulating both th cell frequency and il- a production. however, even if there is no effect on th frequencies, direct or indirect interaction of macrophages with mscs may have a negative effect on il- a production. treg cells are important since they are responsible for the suppression of the immune system and tolerance to self-antigens. activation of foxp , a specific transcription factor of these cells, leads to the secretion of suppressive cytokines, such as il- and tgf-b (li et al., b) . a co-inhibitor molecule ctla- is highly expressed on treg cells and interacts with cd /cd molecules, leading to the secretion of ido from antigen-presenting cells (oderup et al., ) . . the comparison charts of cd + ifng + , cd + il + , cd + il a + and cd + foxp + t cell frequencies according to experiment groups. data are presented as mean ± sd (standard deviation). there are significant differences (p < . ) between the "a" and the rhombus symbols, "b" and the square symbols, "c" and the star symbols. schmidt et al. reported that m macrophages stabilized the foxp expression of cd t cells, and in this way, led to a decrease in ifn-g and il- production (schmidt et al., ) . similar to m macrophages, mscs have a positive effect on the activation of treg cells. gazdic et al. showed that the interaction between mscs and treg cells played an important role in improving acute liver injury in mouse models (gazdic et al., ) . ozdemir et al. suggested that dental pulp mscs increased the treg cell ratio, probably due to the phenotypic shift of th cells to treg cells (Özdemir et al., ) . in our study, the treg cell frequencies were significantly higher in the mem-d group than in the other groups. however, the il- levels of the mem-d group were significantly lower compared to the other groups, except the msc group. we reviewed the literature to interpret this finding and found studies showing that over-activated th , th and even th cells could produce il- to manage this activation (maynard and weaver, ; o'garra and vieira, ; ye et al., ) . it has been reported that the borrelia burgdorferi and toxoplasma gondii infections lead to an increase in ifng + il- + cells (o'garra and vieira, ) . another interesting study reported that over-activated virus-specific cd + t cells could produce il- in coronavirus infection, and these cells were shown to produce higher levels of pro-inflammatory cytokines and cytotoxic proteins than other cells (maynard and weaver, ) . if we try to interpret our data according to the information in the literature, the direct interaction of mscs and macrophage cells may increase the frequency of treg cells even more than the other macrophage phenotypes, and even mscs themselves. furthermore, overactivation of t cells may be the cause of elevated il- fig. . the comparison charts of ifn-g, il- b, il- , il- , il- p and il- a cytokine levels according to experiment groups. data are presented as mean ± sd (standard deviation). there are significant differences (p < . ) between the "a" and the rhombus symbols, "b" and the square symbols, "c" and the star symbols, "d" and empty square symbols. in the us-m, m , m a and a-pbmc groups. as a result, mem-d, mem-id and mscs may have led to a decrease in il- production due to their potent inhibitory effects on t cell activation. netea et al. and quintin et al. reported that macrophages could develop a memory by being educated against a situation they had previously encountered (netea, ; quintin et al., ) . interestingly, we observed that the mem-id cells had similar effects on the th , th , th , and treg cell frequencies as mem-d cells. besides, these effects were similar for the ifn-g, il- and il- a levels. this suggests that macrophage and msc interactions may cause a kind of memory formation in macrophage cells. in addition, this memory appears to develop in a suppressive direction for all t cell cytokine responses. we performed a wst- analysis to observe the changes in proliferation of t cells as a result of activation. it is known that m macrophages can suppress t cell proliferation via secretion of no and ido or surface molecule pd-l (bingisser et al., ; huber et al., ; munn et al., ) . mscs can also effectively suppress t cell proliferation via cytokines, such as ido, pge , il- , and tgf-b (chen et al., ) . according to our findings, consistent with the literature, the proliferation rates of all co-culture groups, except m macrophages, were significantly lower than those of the a-pbmc group, and the proliferation rates of the m and m a group were significantly higher compared to the msc group. these data suggest that macrophages may not be as effective as mscs in suppressing t cell proliferation. it is also known that macrophages can lead to over-activated t cell apoptosis by secretion of no and by fas/fasl mechanism in acute bacterial infections or ifn-g and lps stimulation (daigneault et al., ; hortelano et al., ) . however, mscs do not cause lymphocyte apoptosis while suppressing proliferation (haddad and saldanha-araujo, ; xu et al., ) . we observed that the caspase- levels were significantly higher in m macrophages than in the mem-id, msc and us-pbmc groups, confirming that mscs can successfully suppress t cell proliferation, but does not lead to apoptosis. overall, our data revealed that msc and macrophage interactions may lead to the formation of a different phenotype than the known macrophage phenotypes. although this phenotype was closer to the m c phenotype, it cannot be considered a m c cell due to the low expression of cd , which is the characteristic marker of m c. while mem-d, mem-id, and mscs had similar inhibitory effects on th and th cells, the most significant increase in treg cell frequencies was seen in mem-d cells. macrophages can alter their phenotypes and functions according to the stimuli from the environment. the fact that macrophages educated with mscs suppressed the production of all the cytokines evaluated even after the removal of mscs suggests that these cells may be differentiated by mscs into a suppressive macrophage subgroup. however, treg cell activation caused by direct interactions between msc and macrophage cells may be the most prominent observation of this study. in conclusion, according to our data, interactions of mscs and macrophages may lead to differentiate macrophage cells into an immunosuppressive phenotype, and these macrophages may suppress t lymphocyte subgroups at least as effectively as mscs. however, considering that we obtained our data from in vitro experiments, our findings should be supported by future in vivo studies. the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. the scientific and technological research council of turkey (tubitak) supported the project for this study, whose reference number is s . il- prevents the differentiation of monocytes to dendritic cells but promotes their maturation to macrophages systematic validation of specific phenotypic markers for in vitro polarized human macrophages a novel phenotype for an activated macrophage: the type activated macrophage qualitative differences between naïve and memory t cells macrophage-derived nitric oxide regulates t cell activation via reversible disruption of the jak /stat signaling pathway cellular responses to interferongamma the immunosuppressive effect of mesenchymal stromal cells on b lymphocytes is mediated by membrane vesicles macrophage activation by armed cd th cells immunomodulatory fig. . the comparison chart of wst- optical densities, and caspase- levels of medium supernatants of each group. data are presented as mean ± sd (standard deviation). there are significant differences mesenchymal stem cells reciprocally regulate the m /m balance in mouse bone marrow-derived macrophages monocytes regulate the mechanism of t-cell death by inducing fasmediated apoptosis during bacterial infection mesenchymal stem cells and autoimmune diseases role played by th type cytokines in ige mediated allergy and asthma minimal criteria for defining multipotent mesenchymal stromal cells. the international society for cellular therapy position statement mesenchymal stem cells: immunology and therapeutic benefits the macrophage scavenger receptor cd the gene for b , a costimulatory signal for t-cell activation, maps to chromosomal region q . - q crosstalk between mesenchymal stem cells and t regulatory cells is crucially important for the attenuation of acute liver injury dental follicle mesenchymal stem cells down-regulate th -mediated immune response in asthmatic patients mononuclear cells mesenchymal stem cells inhibit human th cell differentiation and function and induce a t regulatory cell phenotype novel pathways of antigen presentation for the maintenance of memory mechanisms of t-cell immunosuppression by mesenchymal stromal cells: what do we know so far? tgf-β produced by alternatively activated macrophages synchronizes a tolerogenic itreg-th cell axis contribution of cyclopentenone prostaglandins to the resolution of inflammation through the potentiation of apoptosis in activated macrophages targeting cd mediates elimination of m but not m macrophages in vitro and in cutaneous inflammation in mice and patient biopsies alternatively activated macrophages inhibit t-cell proliferation by stat -dependent expression of pd-l interaction between m-csf and il- on productions of il- and il- and expressions of cd , cd , and cd by human monocytes human mesenchymal stem cells inhibit differentiation and function of monocyte-derived dendritic cells il- engages macrophages to shift th cytokine dependency and pathogenicity during t-cell-mediated colitis foxp + regulatory t cells and their functional regulation cd + t cells: differentiation and functions mesenchymal stem cells generate a cd +cd +foxp + regulatory t cell population during the differentiation process of th and th cells interleukin- contributes to therapeutic effect of mesenchymal stem cells for acute liver failure via signal transducer and activator of transcription signaling pathway immunoregulatory effects on t lymphocytes by human mesenchymal stromal cells isolated from bone marrow, amniotic fluid, and placenta the m and m paradigm of macrophage activation: time for reassessment alternative activation of macrophages: an immunologic functional perspective enhancing the efficacy of mesenchymal stem cell therapy diversity in the contribution of il- to t-cellmediated immune regulation m- /m- macrophages and the th /th paradigm inhibition of t cell proliferation by macrophage tryptophan catabolism th /th paradigm extended: macrophage polarization as an unappreciated pathogen-driven escape mechanism? training innate immunity: the changing concept of immunological memory in innate host defence t h cells control themselves by producing interleukin- cytotoxic t lymphocyte antigen- -dependent down-modulation of costimulatory molecules on dendritic cells in cd + cd + regulatory t-cell-mediated suppression the paracrine immunomodulatory interactions between the human dental pulp derived mesenchymal stem cells and cd t cell subsets mesenchymal stem cells inhibit th cell differentiation by il- secretion innate immune memory: towards a better understanding of host defense mechanisms human mesenchymal stem cells impact th and th responses through a prostaglandin e and myeloid-dependent mechanism modulation of immunoregulatory properties of mesenchymal stromal cells by toll-like receptors: potential applications on gvhd phenotypic and functional characterization of neutrophils and monocytes from patients with myelodysplastic syndrome by flow cytometry impact of interleukin- on phenotype and gene expression during early monocyte differentiation into dendritic cells immunoregulatory mechanisms of mesenchymal stem and stromal cells in inflammatory diseases strategies to improve the therapeutic effects of mesenchymal stromal cells in respiratory diseases differential regulation of chemokine production by fcgamma receptor engagement in human monocytes: association of ccl with a distinct form of m monocyte activation (m b, type ) mscs inhibit monocytederived dc maturation and function by selectively interfering with the generation of immature dcs: central role of msc-derived prostaglandin e th cytokines and asthma -interleukin- : its role in the pathogenesis of asthma, and targeting it for asthma treatment with interleukin- receptor antagonists the role of il- and th lymphocytes in autoimmune diseases interleukin- -and interleukin- -mediated alternatively activated macrophages: roles in homeostasis and disease human mesenchymal stem cells program macrophage plasticity by altering their metabolic status via a pge -dependent mechanism available from the role of il- in inhibition of lymphocyte apoptosis by mesenchymal stem cells il- has a distinct immunoregulatory effect on naive and active t cell subsets the scientific and technological research council of turkey (tubitak) supported the project for this study, whose reference number is s . supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.imbio. . . . key: cord- - qejr o authors: bobade, deepali; khandare, ashwin v.; deval, mangesh; shastry, padma; deshpande, prakash title: hemozoin‐induced activation of human monocytes toward m ‐like phenotype is partially reversed by antimalarial drugs—chloroquine and artemisinin date: - - journal: microbiologyopen doi: . /mbo . sha: doc_id: cord_uid: qejr o plasmodium falciparum malaria is the most severe form of malaria with several complications. the malaria pigment‐hemozoin (hz) is associated with severe anemia, cytokine dysfunction, and immunosuppression, thus making it an interesting target for developing new strategies for antimalarial therapy. monocytes (mo) in circulation actively ingest hz released by plasmodium parasites and secrete pro‐ and anti‐inflammatory cytokines. m and m types represent the two major forms of mo/macrophages (mq) with distinct phenotypes and opposing functions. imbalance in the polarization of these types is reported in many infectious diseases. though the association of hz with immunosuppression is well documented, its role in activation of mo in context of m /m phenotypes remains to be addressed. we report here that natural hz drives human mo toward m ‐like phenotype as evidenced by the expression of m signature markers. hz‐fed mo showed elevated transcript and secreted level of il‐ , ccl , ccl , expression of mannose‐binding lectin receptor (cd ), and arginase activity. hz attenuated hla‐dr expression, nitric oxide, and reactive oxygen species production, which are the features of m phenotype. our data also implicate the involvement of p mapk, pi k/akt, and nf‐κb signaling pathways in skewing of hz‐fed mo toward m ‐like type and suppression of mitogen‐stimulated lymphocyte proliferation. importantly, antimalarial drugs—chloroquine and artemisinin—partially reversed activation of hz‐induced mo toward m ‐like phenotype. considering the limitations in the current therapeutic options for malaria, we propose that these drugs may be re‐examined for their potential as immunomodulators and candidates for adjunctive treatment in malaria. malaria is one of the leading causes of morbidity affecting around million individuals and , , deaths in endemic regions according to who . plasmodium falciparum causes the most severe form of malaria with complications that include cerebral malaria, pulmonary edema, acute renal failure, or severe anemia (buffet et al., ; das, ; medana & turner, ; mohan, sharma, & bollineni, ) . p. falciparum malaria is frequently associated with immunosuppression and increased secondary infections (hu, ; ihekwereme, esimone, & nwanegbo, ; orf & cunnington, ; troye-blomberg & perlmann, ) . immunosuppression enhances parasitemia and reduces immune responses to not only malaria parasite proteins but other pathogens as well. accumulating evidence suggests that a combination of parasite and host factors is involved in the pathogenesis of severe malaria. during the intra-erythrocytic stages, p. falciparum parasites digest hemoglobin in the food vacuole, resulting in the production of potentially toxic heme metabolites (francis, sullivan, & goldberg, ) , which the parasite detoxifies by converting it to an insoluble crystal called malarial pigment or hemozoin (hz) (arese & schwarzer, ) . during schizogeny, hz is released into blood and avidly phagocytosed by human phagocytic cells including monocytes (mo) (shio, kassa, bellemare, & olivier, ) . as a result of hz uptake, several functions of mo such as repeated phagocytosis (schwarzer et al., ) , bactericidal abilities (fiori et al., ) , oxidative burst, mhc class ii expression, antigen presentation (scorza, magez, brys, & de baetselier, ) , and maturation to dendritic cells (urban & todryk, ) are severely affected. mo actively respond to ingested natural hz by secretion of pro-and anti-inflammatory cytokines, tnf-α, il- β, il- ra, il- , il- , and chemokines, ccl , ccl ccl , cxcl , cxcl , cxcl and cxcl (deshpande & shastry, ; giribaldi et al., ) . hz stimulates il- β, tnfα, and ccl production via nf-κb and p mapk kinase in human mo (polimeni et al., ) . purified hz isolated from parasitized rbcs stimulates tlr signaling and induces production of cytokines-tnfα, il- , il- p , ccl in myd -dependent manner in murine spleen and dendritic cells (coban et al., ) . in murine mq cell line b r, nf-κb has been shown to be involved in chemokine production of ccl , ccl , ccl , and cxcl induced by hz (jaramillo, godbout, & olivier, ) . hzinduced activation of nod-like receptor containing pyrin domain inflammasome and il- β production is mediated through src kinase lyn and the tyrosine kinase syk pathways in mice (olivier, van den ham, shio, kassa, & fougeray, ; shio et al., ) . mo are heterogeneous innate immune cells that play a pivotal role in primary response to pathogens, tissue homeostasis, inflammation, resolution, and repair (shi & pamer, ) . mo are highly plastic and polarize toward m phenotype in the presence of inflammatory environment such as lps and ifnγ, while the anti-inflammatory m phenotype is driven by th cytokines il- , il- , il- , and tgfβ. il- -producing m phenotype is associated with antiparasitic and tumor-resistance capabilities, and il- -producing m phenotype is related to wound healing, immune regulation, and resolution of inflammation (mantovani et al., ; martinez & gordon, ; sica & mantovani, ) . th responses, respectively (mantovani et al., ) . a study by murray et al. ( ) described different types of mq phenotypes generated by different activating agents and referred to them as m(il- ), m(ig), m(il- ), m(gc), m(ifnγ), and m(lps); however, a phenotype may not fall into specific categories but may show a spectra related to particular phenotype. the m phenotype displays high expression of mannose-binding receptor cd , high arginase activity, and low nitric oxide (no) production (gordon, ; kobayashi et al., ; mosser, ; tsuchimoto et al., ) . the m type mo are divided into m a, m b, and m c subtypes that produce specific chemokines ccl , ccl , and cxcl , respectively (edwards, zhang, frauwirth, & mosser, ; mantovani et al., ; sironi et al., ) . multiple signaling pathways including jak/stat, pkc/erk, and pi k/akt/mtor are involved in the maintenance of m phenotype. the pathways may act individually or in combination to drive mo toward m polarization (gordon & martinez, ; martinez & gordon, ) .the p mapk and pi k-akt pathway mediated il- -induced m a polarization in mice model (jimenez-garcia, herranz, luque, & hortelano, ) . notch signaling via nf-κb, p mapk, and akt pathways has been demonstrated in m b polarization of murine lupus mouse model (zhang, xu, & xiong, ) . recent studies have enhanced our understanding and importance of phenotypic changes in mo by modulating immune responses in several infectious diseases (buchacher, ohradanova-repic, stockinger, fischer, & weber, ; kobayashi et al., ; nakamura, ito, kobayashi, herndon, & suzuki, ; tsuchimoto et al., ) and highlighted their potential for development of new therapeutics. phenotypic changes in mo have been reported in parasitic diseases such as leishmaniasis, filariasis, and trypanosomiasis (babu et al., ; cabalén et al., ) . while accumulating experimental and clinical data support the significant contribution of hz in immunosuppression, its role in driving the mo toward specific mo phenotypes and the implications therein remains to be unraveled. in the present study, we aimed to (a) investigate the effect of hz in the activation of human mo toward m phenotype, (b) examine the signaling pathways involved in the process, and (c) explore the potential of antimalarial drugschloroquine (chq) and artemisinin (art)-in reversion of hz-driven activation of mo. sterile tissue culture plastics were purchased from corning. rpmi media was purchased from sigma. the parasite and mo cultures were ensured to be mycoplasma free and endotoxin low (< . ). inhibitors sb (sb), parthenolide (par), ly (ly), and drugs chq diphosphate and art were purchased from sigma. natural hz from p. falciparum was isolated as described previously with slight modifications (barrera et al., ; bujila et al., ; schwarzer et al., ; skorokhod, alessio, mordmuller, arese, & schwarzer, ) . briefly, p. falciparum ( d strain; mr /atcc, mycoplasma free) culture was maintained in candle jars at °c (trager & jensen, ) . parasites were cultured in o-positive rbcs at % hematocrit in rpmi medium (sigma) supplemented with mm hepes, . g/l glucose and . g/l nahco , . % albumax ii (thermo fisher scientific), and gentamicin ( mg/ml). the medium was replenished every h. parasite cultures with > % rings were synchronized by % aqueous sorbitol and cultured for h until the rupture of mature schizonts and release of hz. culture supernatants containing hz were carefully aspirated and collected and centrifuged, and the blackish-brown hzcontaining pellet was washed three times with sterile ice cold pbs (ph . ). pigment isolated from different batches of parasite cultures was pooled, aliquoted, and stored in pbs at − °c. the total heme content in isolated hz was determined as previously described (polimeni et al., ) . hz isolated from the supernatants of synchronized parasite cultures after schizont rupture is mostly devoid of parasite material. p. falciparum dna was not detected in natural hz (at concentration used in our experiments) on agarose gel (supporting information figure s ). the study for use of human blood mo was approved by the ethics committee of national centre for cell science, pune, india. buffy coats were obtained from healthy donors recruited for blood collection at government-recognized blood bank, pune. informed consent from donors was not essential as discarded buffy coats obtained after plasmapheresis were used. peripheral blood mononuclear cells (pbmcs) were isolated from buffy coats by histopaque . cd + mo were enriched from pbmcs by positive selection using magnetic beads (macs) (miltenyi biotec, germany), according to the manufacturer's instructions. the purity of mo obtained was %- % cd + and viability > % (supporting information figure s ). adherent mo for phagocytosis was obtained as previously described with some modifications (polimeni et al., ) . briefly, × mo/ ml was seeded per well in six-well tissue culture plates and incubated for h at % co in rpmi medium supplemented with mm hepes, mm glutamine, mg/ml streptomycin, units/ml penicillin g, and % fetal bovine serum (sigma st louis, mo). cells were washed with rpmi medium, and the adherent mo obtained was incubated at °c overnight. the adherence step applied after macs and before phagocytosis further enriched the mo population to contain > % mo. culture medium was replaced with fresh medium before the start of experiments. phagocytosis of hz and latex particles was carried out as previously described (polimeni et al., ) . parasite hz ( μg/ml) and latex beads ( . μm diameter; μl of a -fold dilution of the . % v/v) were exposed to mo. the dose of pigment used was based on hz amounts detected during p. falciparum infection and previous in vitro and in vivo studies (deshpande & shastry, ; jaramillo et al., ) . the latex beads were used as phagocytic control . the plates were incubated in % co incubator at °c for h to allow phagocytosis. un-phagocytized hz and beads were removed by media washes, and the cells were incubated for indicated time intervals. in cell signaling experiments, the mo were pretreated with different pharmacological pathway inhibitors, sb ( μm), parthenolide ( μm), and ly ( μm) h prior to phagocytosis. antimalarial drugs-art ( μm) and chq ( μm)-were added h after exposing the mo to hz pigment. the mo fed with hz and latex beads were collected after h by supplemented with % fbs, and data were acquired on bd facs canto ii (bd biosciences) and analyzed using facs diva software and flowjo software (bd biosciences). all parameters measured were based on ssc versus mo-specific cd + gated cells. the mo fed with hz/latex were collected after h and stored in trizol reagent (life technologies, inc., gaithersburg, md) at − °c until rna isolation. total rna was extracted using the purelink rna kit (ambion, usa) following the manufacturer's protocol of trizol plus purelink columns. the purified total rna was resuspended in rnase-free water and quality was analyzed using nanodrop (nd- spectrophotometer). ng of rna was reverse transcribed with improm-ii™ reverse transcription system (promega) as per manufacturer's instructions. qpcr analysis of mrna expression of m and m phenotype-specific marker genes in hz and latex fed mo was performed using power syber green (thermo fisher scientific) and gene-specific primers. the primers related to m /m phenotype for real-time pcr analysis were designed using ncbi primer software and were custom synthesized from sigma and idt technologies. oligo sequences are listed in supporting information table s . the cycling parameters were as follows: initial denaturation at °c for min to activate the amplitaq gold dna polymerase followed by °c for s, °c for s, and then °c for s for a total of cycles. the comparative threshold cycle (c t ) value for gapdh was used to normalize loading variations in the real-time pcr. the extent of mrna expression for a given condition was represented by the relative value to the lower ddct between those estimated for the different conditions: the control ddct value was fixed at . . the specificity of pcr was confirmed by melting curve analysis. real-time pcr was done on thermocycler applied biosystems (applied biosystems). cytokines tnfα, il- β, il- , il- , and il- p and chemokines, ccl , ccl , and cxcl levels were quantified in the cell freeculture supernatants using elisa kits according to manufacturer's instructions. human elisa kits for ccl and cxcl were purchased from r&d biosystems; ccl , il- , tnfα, and il- were purchased from biolegend; and il- and il- β were purchased from bd biosciences. the mo fed with hz were washed with pbs and lysed with equal volumes of ripa buffer containing × protease inhibitor cocktail on ice and then centrifuged for min at , × g at °c. lysates with equal amounts of protein were separated by % sds-page and then transferred onto pvdf membrane (millipore, bedford, ma, usa), blocked with % milk in tbs containing . % tween , and incubated overnight at °c with primary antibodies. phospho-akt, phospho-p , phospho-p (nf-κb), total p (nf-κb) were purchased from abcam; total p from santa cruz biotechnology; akt from cell signaling technology, iκbα from santa cruz biotechnology, and gapdh from sigma. the membranes were exposed to hrp-labeled anti-rabbit igg secondary antibodies ( : , ) (biorad) for h at rt and detected by enhanced chemiluminescence detection system and visualized using ge image quant chemiluminescence system (ge healthcare lifesciences). adherent mo were prepared and exposed to hz and latex as described above, and autologous lymphocytes previously col- arginase activity was determined by colorimetric assay (sigma) according to the manufacturer's instructions. briefly, mo fed with hz and latex were lysed with mm tris-hcl, ph . containing μm pepstatin a, μm leupeptin, and . % (w/v) triton x- . the samples were centrifuged at , × g for min to remove insoluble material. the samples were incubated with substrate buffer containing arginine buffer and manganese (mn) solution for reaction to occur for h at °c along with appropriate sample blank and urea standard in separate wells. the reaction was terminated by adding urea reagent and incubation for h. the absorbance was recorded at nm and arginase activity was calculated as units/l. the no production in the culture supernatants of hz and latex fed mo was measured as stable end product of no, that is, nitrite using the griess reagent. briefly, μl of cell culture supernatant was added to -well plate in triplicates, followed by μl sulphanilamide and μl n- -napthylethylenediamine dihydrochloride. the plate was incubated in dark for min at °c. absorbance at nm was measured by microplate reader, and nitrite concentration was estimated using a sodium nitrite standard curve. rpmi media without phenol red dye was used for this assay. reactive oxygen species (ros; including superoxide, hydrogen peroxide, and other reactive oxygen intermediates [roi]) was estimated by h o -sensitive probe h dcfda (thermofisher scientific) by flow cytometry. briefly, hz and latex fed mo were incubated with h dcfda ( μg/ml) and pe-anti-human cd at rt for min in the dark. pe-labeled anti-human cd was used to gate mo. fluorescent dcf in the mo emitting green fluorescence and proportional to intracellular ros was acquired on bd facs canto ii, and data were analyzed using facs diva software. all values (expressed as m ± sem) were obtained from three or more independent experiments. comparison between groups was done with the one-way anova (bonferroni correction). statistical comparisons were made as indicated, significance levels: *p < . , **p < . , ***p < . . graphpad prism software was used for statistical analysis. the effect of hz on activation of mo toward the m phenotype was examined in cd + sorted cells and analyzed for a panel of m -and m -associated markers. il- , a well-characterized antiinflammatory cytokine implicated in m polarization, was elevated at transcript level following hz exposure (figure a ). consistent with our previous work (deshpande & shastry, ) , a strong induction of secreted and cellular il- was observed (figure b and supporting information figure s a ). the transcript and secreted level of il- p was assessed in hz-fed mo, and treatment with lps + ifnγ was used as positive control. interestingly, hz phagocytosis did not alter the il- p level. as expected, the production of il- was increased in mo exposed to lps + ifnγ (supporting information figure s b ,c). m phenotype is also induced by microbial products through tlr ligands (murray & wynn, ; murray et al., ; wang, liang, & zen, ) . therefore, additional experiments were conducted to examine tlr engagement in hz-driven mo activation. mo exposed to natural hz were analyzed by flow cytometry and western blotting for expression of tlr . β-hematin (shz) was used as a reference for flow cytometry analysis (coban et al., ) . as shown in supporting information figure s a ,b, natural hz had no effect on tlr expression, sug- results are m ± sem from at least three independent donors. significance levels: *p < . , **p < . , ***p < . in comparison with the control and latex as determined by one-way anova (bonferroni test) addition, hz phagocytosis elevated transcript level and secretion of m b-associated cytokines-tnfα, il- β, and il- (supporting information figure s a ,b). the amount of hz in the range of - μg/ml significantly elevated the secreted levels of il- , ccl , ccl , and surface expression of cd in mo (supporting information figure s c ). interestingly, cxcl and cd (markers related to m c phenotype) were unaltered under similar experimental conditions (supporting information figure s ). mo polarization endows subtypes with specific biochemical and functional properties. m mo are characterized by elevated production of no and roi that are essential for clearance of pathogens. conversely, m mo lack the ability to generate oxidative intermediates, synthesis of ornithine and polyamines through the arginase pathway. we found that hz-fed mo robustly elevated arginase activity ( figure f ) and significantly reduced the no (figure g the role of p mapk, pi k-akt, and nf-κb pathways has been implicated in il- synthesis and m polarization; however, their relevance in hz-induced m -like phenotype is not reported. in this study, exposure to hz led to a significant surge in the phosphoryla- (figure f ). the levels of tnfα, il- β, and il- were markedly reduced with hz in the presence of sb and parthenolide. in contrast, il- β and il- cytokines were further elevated in presence of ly (supporting information figure s a,b) . ly can act on other pathways along with pi k kinases that may be responsible for the regulation of il- and il- β (gharbi et al., ) . to confirm the role of pi k-akt pathway, we used another inhibitor-wortmannin in similar experiments. wortmannin effectively reduced the level of il- β, il- , and il- in supernatants of mo fed with hz, signifying the role of pi k-akt pathway in inhibiting m -like phenotype (supporting information figure s c ). were based on previous studies on mo (jang, choi, byun, & jue, ; wang et al., ) . art in concentrations ranging from to μm did not affect the viability of mo as assessed for cytotoxicity by mtt assay (supporting information figure s a ). in our study, we found that treatment of mo preexposed to hz with art resulted in dramatic reduction of il- levels ( figure a figure s c ). the hz-induced nf-κb activation was inhibited by art as indicated by decreased p phosphorylation and increased iκbα in mo (supporting information figure s d p. falciparum infection manifests itself as varying degrees of severity in malaria, inclusive of uncomplicated, mild, severe, and cerebral malaria. variable susceptibility to the parasite is governed by a constant conflict between immunoprotection and immunopathology. (mantovani et al., ) , it is likely that hz might facilitate recruitment of ccr and ccr -bearing th cells and regulatory t cells of th immunity which contribute to immunosuppression. it is noteworthy in this context that regulatory t cells suppress t-cell responses in malaria (hisaeda et al., ) . furthermore, hz induces production of m b-specific cytokines-tnfα, il- β, and il- in human mo. these data corroborate with reports demonstrating the presence of inflammatory cytokines il- β, tnfα, and il- along with high level of anti-inflammatory il- in plasma of malaria patients (perera et al., ; weinberg et al., ) . functionally, m -type mo are characterized by elevated production of no and roi that are essential for clearance of pathogens (mantovani et al., ) . conversely, m -type mo express high arginase activity which is due to production of arginase- , the enzyme that converts arginine to ornithine and urea causing depletion of arginine and reduction of no (biswas & mantovani, ; galván-peña & o'neill, ; martinez & gordon, ) . in malaria, depletion of no results in low no bioavailability that affects adherence of parasites to endothelium and is associated with severity of the disease. high arginase activity and an inverse relation between disease severity and no production are recently reported in malaria (weinberg et al., ) . in this context, mo fed with hz demonstrate elevated arginase activity compared with untreated and latex-ingested mo. these data along with the findings from studies that plasmodium parasites (olszewski et al., ) and lysed infected erythrocytes (reiter et al., ) release arginase suggest that cumulative elevation of arginase activity from these sources might contribute to m like activity. recent studies have reported hz-induced production of ros (jaramillo et al., ) and no (jaramillo, gowda, radzioch, & olivier, ) in mo. in contrast to these reports, we found that hz significantly reduced production of no and ros levels compared with untreated and latex-ingested mo. the difference in the results may be attributed to the nature of hz used; while the earlier studies were performed with delipidized hz and β-hematin, our experiments were conducted with natural hz. in addition, mitogen-stimulated lymphocyte proliferation was significantly suppressed in the presence of hz-fed mo compared with control and latex-ingested mo. this observation is in agreement with the study that malaria patients have suppressed t-and b-cell functions (zander & butler, ) . the signaling pathways p mapk, pi k-akt, and nf-κb have been implicated in il- synthesis (saraiva & o'garra, ) and m polarization (zhang et al., ) - . in this study, hz f i g u r e proposed model for hemozoin (hz)-induced m -like monocytes (mo) during infection. hz released after schizont rupture is ingested by circulating mo in blood, activates pi k-akt, nf-κb, and p mapk pathways, resulting in increased expression of m markers il- , cd , arginase activity. specifically, hz induces m a (ccl ) and m b (ccl , tnfα, il- β, and il- ) phenotype that regulate th responses. hz attenuates hla-dr expression, nitric oxide production, and reactive oxygen species-features of m phenotype. antimalarial drugs-artemisinin and chloroquine-partially reverse the hz-induced m -like phenotype activated p mapk, pi k-akt, and nf-κb pathways and pharmacological inhibitors for these pathways dramatically down-regulated the expression and secretion of il- , thus, over-riding the induction triggered by hz exposure. in line with this observation, the inhibitors effectively reduced hz-induced m -like phenotypic, biochemical, and functional properties. while these findings implicate the role of p mapk, pi k-akt, and nf-κb pathways, we cannot rule out the possibility of other signaling pathways in hz-driven activation of mo toward m phenotype. since hz does not induce a specific phenotype subset but drives the mo toward m a and m b types, it may be postulated that hz stimulates atypical activation mediated through pi k-akt, p mapk, and nf-κb pathways. we next aimed to identify strategies that could aid in reversing the effect of hz in driving the mo to an m -like phenotype. in this regard, we conducted experiments with chq and art for three reasons; first, they are reported to inhibit pathways crucial for polarization to m type; second, they are widely used for treatment of malaria; and third, they possess immunomodulatory properties. art have been reported to decrease il- , il- β, il- , and tnfα levels (shakir, hussain, javeed, ashraf, & riaz, ; wu et al., ) through mapk (wang et al., ) , nf-κb (prato, gallo, giribaldi, aldieri, & arese, ; wang et al., ) , and pi k-akt (kim et al., ) pathways. chq has also been demonstrated to act through mapk (kono et al., ; weber, chen, & levitz, ) and nf-κb (long, liu, wang, zhou, & zheng, ; yang et al., ) pathways and decrease il- β, il- , and tnfα in mononuclear phagocytes in different diseases (jang et al., ; long et al., ) . in another study, chq has been shown to be an effective anticancer drug in mice by inhibiting tumor resistant mq (m mq) and decreasing tgfβ and il- production. the effect was accompanied by decreased myeloid-derived suppressor cells (mdsc), tregs, and increasing cd + t cells in tumor milieu . the ability of these drugs to inhibit crucial pathways and production of cytokines/chemokines that are signature for m type mo has extended their use for other parasitic diseases, rheumatic diseases, and cancer. in our study, the drugs were found to be more effective in reducing the expression of phenotypic markers-cytokines, chemokines, and surface marker-cd associated with m -like phenotype than reversing altered biochemical and functional properties. art did not reverse arginase activity in hz-fed mo. our finding is supported by an earlier report that art did not significantly alter arginase activity in leishmania donovani-infected mq though it enhanced protective immune responses by reversing no levels (sen, ganguly, saha, & chatterjee, ) . these observations are not surprising as chq and art are known for their antimalarial action through multiple modes on their target, but their mechanism of action as immunomodulators is not clearly elucidated. the m phenotype is previously related to abnormal autophagy and angiogenesis (li et al., ) . we speculate that chq, a known autophagy inhibitor, may attenuate hz-induced m -like mo activation by inhibiting autophagy. previously, falciparum malaria pathogenesis has been related to upregulated angiogenic factors vegf, ang- , and sflt- along with dysregulated and excessive immune responses (muehlenbachs, mutabingwa, edmonds, fried, & duffy, ; silver, higgins, mcdonald, & kain, ) . the antiparasitic action of art is accompanied with disruption of parasite proteins, alteration of mitochondrial functions, and angiogenesis (ak, ; shakir et al., ) . in relation to ability of m phenotype to stimulate angiogenesis, art may attenuate hz-induced m -like phenotype by altering the release of angiogenic factors. the schematic diagram ( figure ) summarizes the hz-induced alterations in m -and m -associated markers, the pathways involved and partial reversal by chq and art in human mo. hz released after schizont rupture is readily ingested by circulating mo in blood; activates pi k-akt, nf-κb, and p mapk pathways resulting in increased expression of m markers; and drives the mo toward m (m a and m b)-like phenotype. antimalarial drugs-art and chq-partially reverse the process of activation of mo toward m -like phenotype. in conclusion, the findings from this study add a new dimension to the understanding of mechanism/s underlying hz-induced immunosuppression by demonstrating its ability to drive the mo toward m -like phenotype. though antimalarial resistance poses a major challenge and discourages the continuation of chq and art, we propose that these drugs may be re-examined for their potential as immunomodulators and candidates for adjunctive treatment in malaria. we thank, dr. s.c. mande, director, national centre for cell sciences research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. http://orcid.org/ - - - ak, d. 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activation and m b polarization key: cord- -g a xf authors: shin, hye jin; kim, chonsaeng; cho, sungchan title: gemcitabine and nucleos(t)ide synthesis inhibitors are broad-spectrum antiviral drugs that activate innate immunity date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: g a xf nucleoside analogs have been frequently identified as antiviral agents. in recent years, gemcitabine, a cytidine analog in clinical use for the treatment of many solid tumors, was also shown to have antiviral activity against a broad range of viruses. nucleoside analogs generally interfere with cellular nucleos(t)ide synthesis pathways, resulting in the depletion or imbalance of (d)ntp pools. intriguingly, a few recent reports have shown that some nucleoside analogs, including gemcitabine, activated innate immunity, inducing the expression of interferon-stimulated genes, through nucleos(t)ide synthesis inhibition. the precise crosstalk between these two independent processes remains to be determined. nonetheless, we summarize the current knowledge of nucleos(t)ide synthesis inhibition-related innate immunity and propose it as a newly emerging antiviral mechanism of nucleoside analogs. nucleoside analogs have been historically used for anti-cancer chemotherapy because they inhibit cellular dna/rna polymerases [ ] . more recently, nucleoside analogs have expanded their therapeutic applications and are being used to develop antiviral drugs against a wide range of serious and life-threatening viruses. some nucleoside analog drugs targeting specific viral polymerases (acyclovir for herpesviruses, zidovudine for human immunodeficiency virus (hiv), and sofosbuvir for hepatitis c virus (hcv)) have been successful in clinical trials [ ] [ ] [ ] [ ] and are currently in use for the treatment of virus-infected patients. another class of nucleoside analog drugs such as ribavirin, more broadly-acting on various viruses, has been used in conjunction with ifn-α [ ] . importantly, extensive studies on the antiviral action of ribavirin have established the underlying molecular framework of nucleoside analogs. the primary mechanism to explain the antiviral effect of nucleoside analogs is based on their direct action on viral polymerization. nucleoside analogs are transported into the cells and phosphorylated by the consecutive action of viral or cellular kinases, eventually generating nucleotide triphosphates. mature nucleotide analogs, which are similar to physiological nucleotides, can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations ( figure ). alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. the other mechanism is based on the modulation of cellular nucleos(t)ide synthesis. there have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos(t)ide synthesis pathways [ ] [ ] [ ] [ ] . by targeting metabolic enzymes(s), nucleoside analogs block the natural flow of nucleos(t)ide synthesis and consequently cause the depletion or imbalance of (d)ntp pools. as viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. a more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferon-stimulated genes (isgs). importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. however, the precise mechanism of this crosstalk remains to be elucidated. there is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. they have been well-summarized in a previous report [ ] . in the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broad-spectrum antiviral activity has been recently reported by many groups including our group. more importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations ( figure ). alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. the other mechanism is based on the modulation of cellular nucleos(t)ide synthesis. there have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos(t)ide synthesis pathways [ ] [ ] [ ] [ ] . by targeting metabolic enzymes(s), nucleoside analogs block the natural flow of nucleos(t)ide synthesis and consequently cause the depletion or imbalance of (d)ntp pools. as viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. a more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferonstimulated genes (isgs). importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. however, the precise mechanism of this crosstalk remains to be elucidated. there is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. they have been well-summarized in a previous report [ ] . in the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broadspectrum antiviral activity has been recently reported by many groups including our group. more importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. figure . the mechanism of antiviral effect of nucleos(t)ide analogs. nucleos(t)ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers [ , ] . however, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of rna viruses, including middle east respiratory syndrome coronavirus (mers-cov), severe acute respiratory syndrome coronavirus (sars-cov), zika virus (zikv), hcv, poliovirus (pv), influenza a virus (iav), hiv, and enteroviruses (ev) [ ] [ ] [ ] [ ] [ ] [ ] . the antiviral activities of gemcitabine against the abovementioned viruses are summarized in table . mers-cov and sars-cov belong to the family of coronaviridae and are causative agents of severe viral respiratory illness in humans. to efficiently select appropriate antiviral drug figure . the mechanism of antiviral effect of nucleos(t)ide analogs. nucleos(t)ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers [ , ] . however, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of rna viruses, including middle east respiratory syndrome coronavirus (mers-cov), severe acute respiratory syndrome coronavirus (sars-cov), zika virus (zikv), hcv, poliovirus (pv), influenza a virus (iav), hiv, and enteroviruses (ev) [ ] [ ] [ ] [ ] [ ] [ ] . the antiviral activities of gemcitabine against the abovementioned viruses are summarized in table . mers-cov and sars-cov belong to the family of coronaviridae and are causative agents of severe viral respiratory illness in humans. to efficiently select appropriate antiviral drug candidates, dyall et al. screened fda-approved drugs in virus-infected vero e cells and identified gemcitabine as one of drugs with antiviral activity against both mers-cov and sars-cov (ec of . µm and . µm, respectively) [ ] . more recently, gemcitabine was shown to effectively suppress zikv infection and replication in human retinal pigment epithelium (rpe) cells, particularly at non-cytotoxic concentrations (ec of . µm vs. cc of > µm) [ ] . zikv, a member of the flaviviridae family, can infect pregnant women and cause congenital abnormalities such as microcephaly in infants, which has attracted increasing public attention as well as extensive research and development into possible treatments. effective antiviral activities of gemcitabine were also found for the replication of hcv in huh- cells and the infection of hiv in u -magi-cxcr cem cells, with estimated ec s of nm and . nm, respectively [ , ] , which were lower concentrations than those used in cancer therapy [ ] . in the case of hiv, the combination of gemcitabine with decitabine, another nucleoside analog in clinical use for cancer therapy, synergistically reduced hiv infectivity by increasing the viral mutation frequency [ ] . in a follow up study, clouser et al. further reported the antiviral effect of gemcitabine against hiv-related retrovirus, murine leukemia virus (mulv), in vitro (ec of . nm) and even in murine aids model [ ] . a significant antiviral effect of gemcitabine on iavs was also reported for rpe cells by denisova et al. (ec of . µm) [ ] . they also tested whether gemcitabine had an antiviral effect on several other viruses of different families and found its strong inhibitory effect on sindbis virus and herpes simplex virus- (hsv- ) (> log reduction in virus titer) but relatively weak effects on semliki forest virus and human echovirus , and minimal effects on bunyamwera virus, measles virus (mev), and vaccinia virus [ ] . the antiviral effect of gemcitabine on evs, initially performed on coxsackievirus b (cvb ), was found from screening fda-approved drugs in cvb replicon-harboring vero cells by our group (ec of . µm) [ ] . its broad-spectrum antiviral activity on evs was further identified by observing a similar inhibitory effect on enterovirus (ev ) and human rhinoviruses (hrvs) (ec s of and - µm, respectively). in the case of hrv, the antiviral effect of gemcitabine was further confirmed in a virus-infected mouse model [ ] . in this study, intranasal administration of gemcitabine significantly lowered the pulmonary viral load and inflammation by decreasing proinflammatory cytokines, including tnf-α and il- β, and the number of lung infiltrating lymphocytes. more recently, zhang et al. also identified gemcitabine as the best anti-pv inhibitor from a screen of fda-approved drugs in pv replicon-harboring hela cells (ec of . µm) [ ] . as previously mentioned, accumulating evidence has definitively demonstrated that gemcitabine is an effective broad-spectrum inhibitor of rna viruses and has a therapeutic potential for the treatment of various virus-associated diseases. moreover, it is possible that gemcitabine is effective for other untested rna viruses. because gemcitabine is a deoxycytidine analog that interferes with dna as well as rna synthesis, dna viruses may not be the exception. consistent with this possibility, there has been a report that the infection of hsv- , which is a representative dna virus classified into the herpesviridae family, was strongly affected by gemcitabine [ ] . most of the abovementioned viruses have, at best, limited prophylactic or therapeutic drugs as possible treatments. this is especially true for newly emerging or re-emerged viruses involving serious illnesses, such as mers-cov, sars-cov, and zikv, which are major threats to public health and which urgently need an effective treatment during their early stages of infection. in this regard, repurposing of gemcitabine for the treatment of patients infected with these deadly viruses is a realistic approach. importantly, it is noteworthy that zikv was the most strongly affected by gemcitabine, with a low nanomolar ec , which was lower than that used in cancer therapy [ , ] . even for other viruses with a relatively high ec , there is an option to treat patients with a combination of gemcitabine with other antiviral agents. in this manner, an effective antiviral treatment may be achieved by the synergistic action of two antivirals with much lower doses for each drug, which minimizes deleterious side effects when used clinically. as an example, the synergistic antiviral effect of gemcitabine in combination with ribavirin, an antiviral drug currently being used against a few rna viruses, was reported against evs such as cvb and ev [ ] . as previously mentioned, the combination of gemcitabine with decitabine synergistically suppressed hiv infectivity both in vitro and in vivo [ , ] . however, the actual use of gemcitabine in virus-infected patients necessitates prior in vivo animal studies and clinical trials. even though most antiviral data have originated from in vitro studies, two recent studies have reported the antiviral effects of gemcitabine in murine models [ , ] . more extensive analyses of gemcitabine in animal models in the near future will accelerate its therapeutic applications in clinical trials. most studies regarding the antiviral activity of gemcitabine lack experimental evidence of the mode of action. however, our group has recently reported that gemcitabine had an anti-ev effect by targeting the salvage pathway of pyrimidine biosynthesis [ ] . moreover, gemcitabine strongly induced the expression of several isgs including cxcl , irf , irf , ifit , and ddx , which were the major effectors in the innate immunity that defended the host against the virus infection. these results were consistent with a previous report that gemcitabine stimulated the production of ifn-β and ifn-γ in iav-infected rpe cells [ ] . importantly, the activation of isgs was well-correlated with the inhibition of pyrimidine biosynthesis, suggesting a link between pyrimidine biosynthesis and innate immunity. similar phenomena in terms of isg activation have been previously reported with a few compounds out of several purine or pyrimidine biosynthesis inhibitors that had antiviral activity, as summarized in table [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . regarding purine biosynthesis inhibitors, ribavirin and mycophenolic acid (mpa) are inhibitors of inosine- -monophosphate (imp) dehydrogenase (impdh), which is a key enzyme of the purine biosynthesis pathway. these inhibitors have been successfully used as clinical antiviral or immunosuppressant agents for decades. both have antiviral activities against viruses such as hcv, hepatitis e virus (hev), mers-cov, dengue virus, yellow fever, hepatitis b virus, west nile virus (wnv), chikungunya virus (chikv), and iav [ ] [ ] [ ] [ ] [ ] [ ] [ ] , majorly through the inhibition of the purine biosynthesis pathway, with the antiviral activity against hcv and hev shown to involve the stimulation of isgs [ , ] . for the antiviral activity of ribavirin against hcv, ribavirin specifically induced the expression of irf , irf , and isg mrnas, which are known to be important for anti-hcv immune responses [ ] . isg activation occurred through an undefined mechanism that was different from the classical ifn signaling, intracellular dsrna sensing pathway, toll-like receptor and nuclear factor b pathways. more importantly, ribavirin-induced isg activation and antiviral activity were suppressed using supplemented guanosine, a natural analog of ribavirin, suggesting impdh inhibition-mediated isg activation as an alternative innate immunity pathway. like ribavirin, mpa remarkably induced the expression of several isgs, including irf , irf , isg , ifi , irf , cxcl , ifit , and ifitm mrnas in naïve or hev-infected huh- cells, and the induction of isgs was at least partially abrogated by the use of supplemented guanosine [ ] . mechanistically, the induction of isgs by mpa was independent of the classical jak/stat system, which is similar to that observed with ribavirin [ ] . similar results were obtained with several impdh or impdh inhibitors, with various affinities, that were custom-designed and synthesized [ ] . as shown in table , most pyrimidine biosynthesis inhibitors target dihydroorotate dehydrogenase (dhodh), an essential enzyme in de novo pyrimidine synthesis. lucas-hourani et al. identified dd as an interferon-sensitive response element (isre)-stimulating compound from high-throughput screening, and further analyses suggested that it was a dhodh inhibitor with a strong antiviral activity against various viruses including mev, chikv, and wnv [ ] . dd enhanced the expression of several isgs, which were almost completely suppressed by the addition of supplemented uridine, indicating dhodh inhibition-mediated isg activation. moreover, the antiviral activity of and isg activation by dd required the interferon regulatory factor (irf ) transcription factor, a master regulator of antiviral gene expression [ ] , which was consistent with the observation that the anti-hcv activity of mpa was partially mediated by irf [ ] . in this study, similar results were shown with brequinar, another well-known dhodh inhibitor. fa- is also an antiviral compound, which inhibits the pyrimidine biosynthesis pathway, probably via targeting dhodh and inducing the expression of isgs such as ifnb , cxcl , isg , and ccl [ ] . however, whether isg activation is mediated by pyrimidine biosynthesis inhibition remains to be determined. the mechanism of nucleotide synthesis inhibitor-induced isg activation is still presently unclear. nevertheless, there has been accumulating evidence showing that nucleotide synthesis inhibitor-induced isg activation is independent of the classical jak/stat-mediated ifn signal [ , , ] . first, wang et al. clearly showed that isg activation and anti-hev activity induced by mpa or brequinar was not mediated by jak [ ] . second, irf induction by ribavirin was not affected by knockdown of stat , while that of ifn-α was strongly affected under the same conditions [ ] . third, our recent study with gemcitabine further confirmed ifn signal-independent isg activation by parallel studies comparing the effects of gemcitabine and ifn-α. in our study, the phosphorylation of stat at tyr , which was dramatically triggered by ifn-α, did not occur when treated with gemcitabine [ ] . moreover, the upregulation of ddx mrnas induced by gemcitabine was not affected by irf knockdown, which was contrary to the result that ifn-α-induced upregulation of ddx mrnas was significantly suppressed under the same conditions. consistent with above observations, there have been some reports that isgs was induced in the absence of jak or stat activation [ , ] . despite limited data, we speculate the scenario of isg activation that is independent of jak/stat-mediated ifn signal. purine or pyrimidine biosynthesis inhibitors could interfere with the metabolic pathway through targeting some key enzymes such as impdh and dhodh, leading to the depletion or imbalance of the (d)ntp pool. inactivation of metabolic enzyme(s) itself or consequently altered nucleos(t)ide pools might trigger a signal, which is ultimately delivered to certain cis-acting elements on the promoter of a subset of isgs, possibly through the relay of kinases and transcription factors. based on the previously mentioned reports, this signal is less likely to be dependent on stat / -irf (ifn-stimulated gene factor ; isgf ), at least for gemcitabine, which is the major transcriptional complex in the ifn-induced jak/stat pathway. it should also be considered that thomas et al. excluded the involvement of an intracellular double-stranded rna sensing pathway, toll-like receptor and nuclear factor κb pathways, as well as a classical ifn signal in the activation of isgs induced by ribavirin [ ] . despite the consensus of isg activation, each purine/pyrimidine biosynthesis inhibitor seems to induce distinct sets of isgs, at least with different patterns [ ] . targeting an enzyme in which pathways (purine or pyrimidine synthesis) or steps (early/late and de novo/salvage) produce different levels of intermediates and nucleos(t)ides will consequently result in diverse outcomes of isg activations. there might be more than one signaling pathway involved. the synergistic antiviral activity of gemcitabine and ribavirin observed in our study might be explained by the possible existence of two separate signaling pathways that mediate each inhibition of nucleotide synthesis toward isg activation. systematic analyses of signaling kinases, irfs, and stats using sirna knockdown and/or pharmacological inhibition and metabolic analyses of corresponding intermediates and nucleos(t)ides should therefore clarify the underlying molecular mechanisms of isg activation by purine/pyrimidine biosynthesis inhibitors. as newly emerging or re-emerged viruses such as sars-cov, mers-cov, and zikv have become a major threat to public health, the need for broad-spectrum antiviral drug has increased. in this regard, nucleoside analogs that directly target viral rna-dependent rna polymerase and present a high barrier to the development of resistant viruses have been considered advantageous. moreover, recent discovery of a new antiviral mode of nucleoside analogs acting through innate immunity strengthens the molecular basis for their therapeutic application as broad-spectrum antiviral drugs. nucleoside analogs probably induce different subsets of isgs, at least with a different pattern, leading to various combinations of isgs and resulting antiviral outcomes. moreover, according to schoggins et al., different viruses are affected by distinct subsets of isgs and some isgs such as irf , mb d , hpse, ddx , mda, and ifitm act broadly on various viruses [ ] . thus, more systematic analyses on the subsets of isgs induced by antiviral nucleoside analogs are required for the identification of better antiviral drugs that can be used broadly or specifically. given the clinical side effects of ifn treatment, nucleotide analogs that differ from ifn in the activation of subsets of isgs need to be considered as alternatives. nevertheless, nucleoside analogs interfering with the host nucleotide synthesis pathway 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and cidofovir on replication of bk virus in an in vitro culture system inhibition of pyrimidine biosynthesis pathway suppresses viral growth through innate immunity broad-spectrum inhibition of common respiratory rna viruses by a pyrimidine synthesis inhibitor with involvement of the host antiviral response broad-spectrum antiviral that interferes with de novo pyrimidine biosynthesis identification of broad-spectrum antiviral compounds and assessment of the druggability of their target for efficacy against respiratory syncytial virus (rsv) inhibition of dengue virus through suppression of host pyrimidine biosynthesis d , a non-nucleoside inhibitor of influenza virus infection that interferes with de novo pyrimidine biosynthesis roles of jaks in activation of stats and stimulation of c-fos gene expression by epidermal growth factor critical roles for both stat -dependent and stat -independent pathways in the control of primary dengue virus infection in mice interferon-stimulated genes: roles in viral pathogenesis author contributions: hye jin shin, chonsaeng kim, and sungchan cho wrote the manuscript. the authors declare no conflict of interest. key: cord- -ds x yym authors: kim, young-seok; son, ahyun; kim, jihoon; kwon, soon bin; kim, myung hee; kim, paul; kim, jieun; byun, young ho; sung, jemin; lee, jinhee; yu, ji eun; park, chan; kim, yeon-sook; cho, nam-hyuk; chang, jun; seong, baik l. title: chaperna-mediated assembly of ferritin-based middle east respiratory syndrome-coronavirus nanoparticles date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: ds x yym the folding of monomeric antigens and their subsequent assembly into higher ordered structures are crucial for robust and effective production of nanoparticle (np) vaccines in a timely and reproducible manner. despite significant advances in in silico design and structure-based assembly, most engineered nps are refractory to soluble expression and fail to assemble as designed, presenting major challenges in the manufacturing process. the failure is due to a lack of understanding of the kinetic pathways and enabling technical platforms to ensure successful folding of the monomer antigens into regular assemblages. capitalizing on a novel function of rna as a molecular chaperone (chaperna: chaperone + rna), we provide a robust protein-folding vehicle that may be implemented to np assembly in bacterial hosts. the receptor-binding domain (rbd) of middle east respiratory syndrome-coronavirus (mers-cov) was fused with the rna-interaction domain (rid) and bacterioferritin, and expressed in escherichia coli in a soluble form. site-specific proteolytic removal of the rid prompted the assemblage of monomers into nps, which was confirmed by electron microscopy and dynamic light scattering. the mutations that affected the rna binding to rbd significantly increased the soluble aggregation into amorphous structures, reducing the overall yield of nps of a defined size. this underscored the rna-antigen interactions during np assembly. the sera after mouse immunization effectively interfered with the binding of mers-cov rbd to the cellular receptor hdpp . the results suggest that rna-binding controls the overall kinetic network of the antigen folding pathway in favor of enhanced assemblage of nps into highly regular and immunologically relevant conformations. the concentration of the ion fe( +), salt, and fusion linker also contributed to the assembly in vitro, and the stability of the nps. the kinetic “pace-keeping” role of chaperna in the super molecular assembly of antigen monomers holds promise for the development and delivery of nps and virus-like particles as recombinant vaccines and for serological detection of viral infections. introduction various types of viral vaccines have been developed over the last century with a wide spectrum of efficacy and safety ( , ) . the manufacturing of most conventional vaccines-live attenuated, inactivated, or subunit vaccines-invariably require the culturing of infectious viruses in cell substrates ( ) . despite dedicated efforts, conventional cell culture often fails to produce sufficient amounts of virus for evaluating the immunogenicity, protective efficacy, and safety of viral vaccines. moreover, some emerging viruses cause high-mortality rates, without options for treatment or prophylaxis, necessitating their manipulation, and manufacture under stringent bio-safety environment ( ) . not surprisingly, alternative technologies that circumvent these limitations are a high priority in the areas of vaccine development and production. nanoparticles (nps), virus-like particles (vlps), and assembly of multimeric peptides each provide attractive platforms for vaccine design ( ) . virus-like particles and nps structurally resemble infectious virions, but are non-infectious due to the lack of viral genomes. recombinant surface antigens from natural virions are assembled into highly ordered conformations as empty particles devoid of genetic material. antigenic epitopes are presented on the multivalent and highly repetitive outer structure of the nps, which leads to the crosslinking of b-cell receptors and the induction of long-lasting immune responses ( ) ( ) ( ) . by mimicking the morphology of the natural infectious virions, the regularly assembled particles are highly immunogenic, and are amenable to diagnostic and prophylactic exploitation. among the simplest targets are the vlps of non-enveloped viruses, such as hepatitis e virus or human papilloma virus, and are composed purely of viral capsid proteins ( ) ( ) ( ) . in contrast to non-enveloped viruses, where virion assembly is exclusive to capsid proteins, enveloped viruses (e.g., coronavirus or flavivirus), require an additional membrane component for assembly into mature virions. consequently, in enveloped vlps, the assembly of matrix proteins provides a molecular scaffold, and viral antigens are embedded into lipid membranes. different types of glycoproteins may be embedded in the lipid membrane as target antigens for generating immunological responses ( ) . however, this process requires multiple proteins (surface antigens and matrix proteins), and the enveloped vlps are not structurally uniform and are difficult to characterize. a promising alternative is to present the target antigens on the surfaces of self-assembled nps, which, in lieu of lipid membranes, serve as the macromolecular scaffold for the presentation of the antigens of interest. in developing np vaccines, consideration should be given regarding the selection of a robust and faithful system for np assembly that enables the cost-effective development and delivery of vaccines in a timely manner. structure-based approaches in silico and their underlying principles are relatively advanced for np assembly ( ) ( ) ( ) . most of the approaches consider the thermodynamic stability of the final assembled nps, without due recognition for the kinetic complexities controlling regular assemblage over random interactions that lead to misfolded aggregations. therefore, it is not surprising that most engineered nps are refractory to soluble expression, which presents practical challenges in production, both at a laboratory-scale and in commercial manufacturing processes. this problem becomes augmented when expressed in bacterial hosts because of a lack of folding assistance in the bacterial cytoplasm for viral antigens. therefore, due to advantages in assisted folding, posttranslational modifications, and the possibility of generating multiple-component nps and vlps, eukaryotic hosts such as yeast, insects, and mammalian cells have been favored over bacterial hosts ( ) ( ) ( ) . however, these systems are significantly more expensive than bacterial systems, are more time-consuming, and the down-stream processes are usually more complex. moreover, the purification of vlps from insect cell systems poses a challenge due to similar physicochemical properties between the vlps and the baculoviruses ( , ) . bacterial systems, if available, would provide a cost-effective means to develop and deliver vaccines, as well as sero-diagnostic antigen kits used to diagnose-specific infection diseases. middle east respiratory syndrome (mers) was first reported in saudi arabia in and has caused multiple cases of infection with high mortality in europe and asia ( , ) . mers is caused by mers-coronavirus (mers-cov), which can be transmitted from camels to humans, and from humans to other humans ( , ) . worldwide transmission is increasing in direct household and community-wide transmission, as well as in nosocomial settings, as exemplified in a outbreak in korea ( , ) . neither effective vaccines nor therapeutic interventions are currently available. because of this, assembly of mers-cov antigens into immunologically relevant conformation as nps would be of interest and may be helpful in developing vaccines, sero-diagnostic tools, and therapeutic monoclonal antibodies. in the current study, we present a novel bacterial np of mers-cov antigen using ferritin as a molecular scaffold for self-assembly. ferritin, which is present in most living organisms, has identical subunits that spontaneously self-assemble and form np complexes with internal and external diameters of and nm, respectively ( , ) . previous studies show that ferritins of helicobacter pylori from a human isolate can be used as scaffold for hiv and influenza np vaccines, using eukaryotic host cells such as human embryonic kidney cells (hek f or hek s) ( , ) . likewise, bacterioferritin (fr), which self-assembles into nanocages with octahedral symmetry, has also been evaluated as a potential drug delivery system ( ) . however, viral antigens of human pathogens are prone to misfolding into aggregates, which necessitates chemical refolding of the insoluble aggregates in order to regain solubility and to allow regular assembly of the antigen ( , ) . in addition, displaying antigens on the surface of multi-molecularly assembled scaffolds in bacterial hosts remains a daunting challenge. we hypothesized that nps displaying the receptor-binding domain (rbd) of the spike protein from mers-cov could be produced in a bacterial system by harnessing the function of a molecular chaperone. conventionally, protein folding and the prevention of non-functional aggregation have been ascribed to molecular chaperones ( ) ( ) ( ) . recently, it has been shown that rna molecules are able to provide novel functions as molecular chaperones ( ) ( ) ( ) . based on novel findings, the concept of chaperna (chaperone + rna) function was established ( ) . in this report, chaperna function was harnessed for the folding and assembly of hybrid ferritin monomers into nps using a bacterial expression system. we also demonstrated that the biophysical properties, including solubility, yield, and stability of mers-cov nps, could be improved by properly controlling the rna-binding affinity, and the concentrations of fe + and salts. the chapernabased np assembly may prove to be a versatile tool for developing and delivering recombinant vaccines and for serological detection of emerging/re-emerging viruses. the expression vector pge-hrid( ) was constructed from the parental vector pge-lysrs ( ) ( ) . the pge-lysrs( ) vector was enzymatically cut with ndei and kpni. the pcr product of hrid, which carries the tev protease cleavage site and a -histidine tag at the c-terminus, was cut using the same restriction enzymes and the digested fragment inserted into the vector to generate pge-hrid( ). fr (genebank accession no. nc_ . ) dna was synthesized by, and purchased from, cosmo genetech (korea). the dna was cleaved with sali and hindiii, and inserted into pge-hrid( ) to generate hrid( )-fr. the receptor binding domain (rbd), n-terminal residues - , of the mers-cov s protein (genbank accession no. afs . ), was generated by gene synthesis, cut with kpni and sali, and inserted into hrid-fr to generate pge-hrid( )-rbd-fr. linker ssg or asg was inserted into the c-terminus of the rbd using overlapping pcr, cleaved with kpni and sai, and ligated into hrid-fr, generating pge-hrid( )-rbd-[ssg]-fr or pge-hrid( )-rbd-[asg]-fr, respectively. the schematic diagrams of each expression vector are illustrated in figure b . the genes of mutant hrid( m) (k a and k a) and hrid( m) (k a, k a, r a, k a, k a, k a, k a, k a, and k a) were generated by gene synthesis, cleaved with ndei and kpni, and inserted into pge-hrid( )-rbd-fr, generating pge-hrid( m)-rbd-fr and pge-hrid( m)-rbd-fr, respectively. the mutation sites and amino acid sequences of the mutants are shown in table s in supplementary material. the resulting expression vectors were transformed into the escherichia coli strain shuffle ® t . the cells were grown in ml of lb medium with ampicillin ( µg/ml) at °c overnight. each type of transformant was inoculated into ml of lb medium with ampicillin, grown at °c until an optical density (od ) of . - . was reached. protein expression was induced with mm iptg for h. each sample was harvested by centrifugation, lysed by sonication in lysis buffer ( mm tris-hcl, ph . ; % glycerol; mm -mercaptoethanol; and . % tween- ). the soluble fraction of each lysate was purified on a ni-affinity histrap™ hp column by atka prime (ge healthcare) and concentrated with centriprep™ (merck millipore ltd.). the purified proteins were treated with tev protease to remove the fusion partner hrid. the assembled nps were purified by gel filtration on / superose™ increase columns (ge healthcare). to examine the size and structure of the purified nps, microscopic evaluations using tem and cryo-em were performed. for tem analysis, a drop of the nps was placed onto a formvar/carboncoated tem grid (spl). the grid was negatively stained with % uranyl acetate, dried, and examined using a jem- electron microscope (jeol) at an accelerating voltage of kv. the particle sizes were calculated using camera-megaview iii (soft imaging system-germany) for measuring the nps in random image fields. for cryo-em, the nps were placed onto plasma-treated formvar/ carbon copper grid (ems) and negatively stained with % uranyl acetate. the grid was accelerated at kv with an fei cryotecnai f cryo-em microscope made available through the korean institute of science and technology. the nps were examined and photographed in high resolution. nanoparticle samples ( ml) were placed into a dispo-h cell, and analyzed using a zeta-potential & particle size analyzer (els- ( , , and °c ) and the cell lysates were separated into total (t), soluble (s), and insoluble (p) fractions by centrifugation (left panel). the solubility of each protein expressed at °c was measured by a gel densitometer and the data were summarized and shown in the right panel (n = ). statistical significance (**p < . , ***p < . ) was indicated for the samples compared with the control using a two-tailed student's t-test. (d) illustration of mers-cov rbd-fr nps using the chaperna-based hrid fusion partner. the hrid facilitated folding of the aggregation-prone rbd-fr through interaction with rna. the monomer of rbd-fr formed a properly folded trimeric structure by cleaving hrid with tev protease. eight trimers assembled and formed into mers-cov-like nps. red triangles indicate the rbd trimer on the fr nps. of the nps was measured twice at °c in water as a solvent with the sample accumulation time at s. effect of salt and fe + concentrations on np assembly and stability cultured cells ( ml) were lysed with lysis buffer in the presence of various concentrations of nacl ( , , , , , , , , and mm) to evaluate the intracellular proteins. all samples were performed in triplicate. the cell lysates were separated into soluble and insoluble fractions by centrifugation, and the protein stabilities analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page). thus, the proteins from cell lysates ( ml culture) were purified using hispur™ ni-nta resin (thermo fisher scientific) in buffer a depending on nacl concentration ( - mm). to evaluate the effects on fe + on np formation, cells were cultured in lb media with various concentrations of fe + ( , , , and , µm). np formation was examined by size exclusion chromatography (sec), sds-page, tem, and dls at the various concentrations of nacl or fe + . the cells were harvested, sonicated with lysis buffer, and separated into soluble and pellet fractions by centrifugation. target proteins in the soluble fraction were purified using hispur™ ni-nta resin (thermo fisher scientific), following the manufacturer's instruction. t (total lysate), s (soluble fraction), p (pellet fraction), w (wash fraction), and e (the elution fraction were analyzed by sds-page. co-purification of the nucleic acids and proteins in the wash and elute were analyzed on a native agarose gel. the nucleic acids were visualized with ethidium bromide (etbr), and the proteins with coomassie staining. cultured cells ( ml) were harvested using the same method described above. the cells were lysed with µl of protein extraction reagent b-per™ ii (thermo scientific) and separated into soluble and pellet fractions by centrifuged , rpm for m. a µl aliquot of each soluble fraction was further treated with µg/ml of rnase a (intron biotechnology) and incubated at °c for min. the nuclease treated samples were clarified by centrifugation at , rpm for min and the soluble supernatants and the pelleted precipitates were analyzed on an sds-page gel followed by western blot analysis. to confirm the proper folding of rbd-fr and its variant (rbd-[ssg]-fr), the binding of the purified proteins with the mers-cov receptor hddp was performed by elisa. fr only and phosphate-buffered saline (pbs) were used as negative controls. nunc -well microtiter immunoplates (thermo fisher scientific) were coated with ng/well of hdpp proteins (abcam) and incubated at °c overnight. the plates were washed and blocked with µl/well of blocking buffer ( % bsa) for h at room temperature. rbd (ssg linker, wt, m, or m)-fr ( ng/ well) were added for h at °c. an anti-penta his antibody ( µl/well; qiagen) was serially diluted ( / to / , ) in tbst [ mm tris-cl (ph . ), . % tween- ], added to the wells, and incubated for h at °c. a secondary goat anti-mouse igg antibody conjugated with hrp in a -µl volume ( : , , sigma-aldrich) was added and incubated for h at °c. the plates were washed three times with tbst at the end of each step. after washing, µl/well of substrate tmb solution (bd biosciences) were added to the well and the plates were incubated at °c for min in the dark. µl of stop solution ( n h so ) was added to the well to stop the colorimetric reaction, and the absorbance at nm was measured using an elisa reader, fluostar optima (bmg labtech). . the coating antigens were removed, and the wells were blocked with pbst ( % skim milk in pbs and tween- ) for h at °c. after h, the blocking solution was removed. twofold serially diluted sera from four patients (cnnh- , , , and ) were added to each well and incubated at °c for h. the antigencoated wells were incubated with peroxidase-conjugated goat anti-human igg antibody (kpl, seracare life sciences, milford, ma, usa) at °c for h. the primary antibody was removed and , ′, , ′-tetramethylbenzidine (tmb; sigma-aldrich) was added to each well as colorimetric substrate. immediately after treatment of the reactions with stopping solution (sigma-aldrich), the od was read at nm. six-week-old female balb/c mice were immunized with µg/ mouse of the rbd-fr, rbd-[ssg]-fr, or rbd protein generated as described above, or with commercially available mers-cov rbd protein (mers-rbd- p; eenzyme) as antigen in bsl- facility in ylarc. antigens were diluted in pbs. for the first group, equal volume of mf adjuvant (addavax, cat. no vacadx- ) ( ) was mixed by pipetting. for the other group, equal volume of antigens and alum adjuvant (thermo fisher scientific) were mixed by pipetting following the manufacturers' protocol. pbs plus adjuvant and fr were used as negative controls. the immunized mice were boosted twice with intramuscular injections on days and . mice were anesthetized on days and for ocular bleeding from the orbital sinus ( figure s in supplementary material). immune sera were processed by centrifugation of the collected blood at , × g for min. the spleen and the balf (bronchoalveolar lavage) were obtained at days after the last immunization from sacrificed mice. balf was taken by washing the airways with ml of pbs. t-cell population from immunized mice were analyzed by flow cytometric analysis ( , ) . the spleens were taken at days after the last immunization from the sacrificed mice. to obtain single-cell suspensions, the tissues were homogenized and passed through µm cell strainers (spl). after centrifugation, erythrocytes were removed by red blood cell lysing buffer (sigma). the cells were washed and resuspended in iscove's modified dulbecco's media containing % fbs. for intracellular cytokine staining, the splenocytes were stimulated with µg/ml rbd protein or phorbol myristate acetate/ionomycin in the presence ng/ml recombinant human il- (biolegend) and brefeldin a ( : , ; ebioscience) at °c for h. after stimulation, the cells were blocked with rat anti-mouse cd /cd (bd biosciences) and surface stained with anti-cd (fitc, clone - . ; biolegend) and anti-cd (pe/cy , clone gk . ; biolegend) at °c for min. the stained cells were fixed in facs lysing solution (bd biosciences) at room temperature for min, and permeabilized with facs buffer ( . % fbs, . % nan in pbs) containing . % saponin (sigma) at room temperature for min. then, the cells were stained with anti-ifn-γ (pe, clone xmg . ; biolegend) and anti-tnf-α (apc, clone mp -xt ; biolegend) at room temperature for min. all data were collected by bd lsr fortessa (bd biosciences) and analyzed with flowjo software (tree star inc., ashland, or, usa). competition elisa was performed to determine whether mers-cov antigen [rbd-[ssg]-fr, rbd-fr, rbd, and fr (negative control)]-immunized mouse serum inhibited binding of rbd protein to hdpp receptor ( , ) . ng/well hdpp protein (abcam) was coated on nunc -well microtiter immunoplates (thermo fisher scientific) and incubated overnight at °c. plates were washed and blocked with µl/well of blocking buffer [ % skim milk in pbs and tween- (pbst)] for h at °c. at the same time, mouse sera immunized with rbd, rbd-[ssg]-fr, rbd-fr, and fr were serially diluted ( / to / ) with ng/well rbd protein (mers-rbd- p; eenzyme) in tbst [ mm tris-cl (ph . ), . % tween- ], added to new wells, and incubated for h at °c. µl solution was added to each well at °c and incubated for h. after that, µl of anti- xhis tag antibody conjugated with horseradish peroxidase ( : , , thermo fisher scientific) was added to each well and incubated for h at °c. plates were washed three times with tbst, and µl/well of substrate tmb solution (bd biosciences) was incubated at °c for min in the dark. µl of stop solution ( n h so ) was added to the well to stop the color reaction and measure the absorbance at nm using an elisa reader fluostar optima (bmg labtech). the hrid facilitated the solubility of mers-cov rbd-fr the spike glycoprotein (s) of mers-cov was used for the generation of mers-cov-like nps. s protein forms trimers, resulting in large spikes on the virus envelope ( ) . it is challenging to express the full-sized s protein (~ kda) in e. coli. thus, the s domain of s protein (~ kda), which includes the receptor-binding ability, was used. our initial attempt to express the s domain, either as s or as an s -fr fusion protein, failed; the expression level and solubility of the protein was below the lower limit of detection by sds-page and western blotting ( figure s in supplementary material). we therefore used the rbd ( - a.a.) of the s protein, which has a pivotal function as illustrated in figure b ( , ) . when expressed alone in e. coli, the rbd is not able to form the trimeric assembly (unpublished observation), due to the lack of the hr domain within the s domain ( ) . to overcome this problem, fr was used as scaffold for the assembly. fr is a spherical np whose subunits form trimers that subsequently result in octahedral structures composed of identical subunits ( ) . we therefore performed computational modeling to evaluate the potential of fr as scaffold for trimer formation of the rbd. possible trimer formation was analyzed by computational modeling using modeler ( , ) and cluspro ( , ) . various linkers, including ssg, asg, and d , were introduced between the rbd and fr with a goal to minimize steric hindrance between the two domains so as to enhance trimer and np formation. in silico analysis showed energy-stable trimeric models of rbd-fr, rbd-[ssg]-fr, and rbd-[asg]-fr, whereas rbd-d -fr failed to form a trimeric structure ( figure a) . the rbd-[ssg]-fr was predicted to be the most stable and well-structured compared with rbd-fr and rbd-[asg]-fr. initial testing of the rbd-fr constructs without hrid fusion showed that none of the constructs were solubly expressed, even under low-temperature culture conditions ( figure c ) ( figure b) . we previously confirmed that by using chaperna, the globular domain of influenza hemagglutinin (ha) is efficiently assembled into a trimeric complex with an immunologically relevant conformation (yang et al., in press) . as shown in figure c , the hrid fusion significantly increased the solubility of both rbd-fr ( . %) and rbd-[ssg]-fr ( . %), indicating that the chaperna platform effectively increased both the solubility and the folding of its fused target proteins. because of the poor expression level and low solubility of the rbd-[asg]-fr construct (figure c) , further experiments were performed using only the rbd-fr and rbd-[ssg]-fr constructs. after purification of the soluble proteins ( figure s in supplementary material), we determined the potential effects of using hrid as a fusion partner for the self-assembly of the nps. as shown in figure s in supplementary material, hrid-rbd-fr failed to form nps. because of this, we performed tev protease cleavage of the hrid. removal of the hrid domain facilitated the self-assembly of the rbd-fr monomers, and also eliminated the immune response against the non-self hrid domain in balb/c mice ( figure s in supplementary material). after hrid cleavage, rbd-fr and rbd-[ssg]-fr were purified using sec (figure a) . as expected, rbd-[ssg]-fr assembled into properly formed nps ( , kda) more efficiently than did rbd-fr nps, which were mainly detected in the void-volume fractions, suggesting they were irregularly assembled soluble aggregates. the size of the rbd-[ssg]-fr nps was further confirmed by tem. tem images of the rbd-[ssg]-fr np structures showed hollow, spherical particles that were more compact than the rbd-fr nps. the average diameter of the rbd-[ssg]-fr nps was - nm (figure b) . in contrast, dls analysis of the rbd-fr np structure without the ssg linker appeared to be smaller with an average intensity diameter of . nm, and this compared with rbd-[ssg]-fr that had an average intensity distribution diameter of . nm (figure c) . consistent with the sec analysis, rbd-fr without a fusion partner was mostly produced in a soluble aggregated form. therefore, we identified that the protein folding did not occur properly without hrid, and the formation of nps was confirmed by both sec and sds-page analyses. as shown in figure s in supplementary material, the purified nps retained their stability over an extended period of time at various temperatures ( , , and − °c). thus, these results indicate that the ssg linker allowed the rbd-fr to generate properly assembled nps. it should also be noted that the efficiency of protein folding and nps formation may be further enhance through appropriate linker selection. it has been reported that ionic strength plays an important role in the stability and self-assembly of ferritins ( , ) . we examined the effect of salt concentration on the formation and stability of the rbd-[ssg]-fr nps at various concentrations ( - mm). consistent with the previous studies, the stability of the protein was highly affected by the concentration of nacl in the lysis buffer by sds-page ( figure a ) (n = ). the solubility of the protein significantly decreased as the concentration of nacl increased from to mm, with the solubility being about . -fold lower at mm compared with mm. unlike previous studies, the solubility of the protein was gradually recovered at higher nacl concentrations (> mm); the solubility at mm was . -fold higher than at mm. furthermore, the yield of soluble of protein per liter of culture increased in a salt concentrationdependent manner (figure b) . to further investigate the effect of salt concentration, the physicochemical and morphological properties of the rbd-[ssg]-fr protein were examined by sec, tem, and dls. in mm nacl, most of the protein was aggregated during the purification process, and the purified protein failed to form spherical structures, but instead, existed predominantly as kda monomers (figures c,d; figure s in supplementary material). in contrast, the protein that was lysed in mm nacl and purified in mm nacl, developed well-structured nps according to tem and dls analyses (figures c,d; figure s in supplementary material). however, based on sec analysis, at high-salt concentrations (> mm), the protein failed to form stable structures with the proteins being eluted predominantly in the void volume, suggesting they were soluble aggregates under the high-salt concentrations ( figure c) . transmission electron microscopy images under the various salt concentrations clearly supported the conclusion, showing that the tendency for aggregation was dependent on the salt concentration ( figure d) . taken together, the results underscored the importance of salt concentration on the solubility of monomers and the quality of multimeric assembly of hybrid nps. ferritin has an intrinsic ability to interact with fe + to form ferritin-iron cores ( ) . thus, it was worth investigating the effect of fe + on the assembly and stability of rbd-[ssg]-fr nps. cells were grown in lb medium with various concentrations of fe + . as shown in figure a , the yield of purified protein was significantly increased from cultures with µm fe + , reflecting a . -fold increase compared with similar cultures µm fe + . the cell growth and purification yield at , µm fe + were slightly decreased, presumably due to the toxicity of ferric acid. np formation under the various concentrations of fe + was analyzed by sec ( figure b) . consistent with the previous results, the proteins were eluted mainly in the fractions expected for the size of assembled nps ( , kda). of note, the ratio between nps and soluble aggregates in the sec analysis showed that np formation was facilitated at high concentrations of fe + (figure b) . the formation of rbd-[ssg]-fr nps at an fe + concentration of , µm was confirmed by tem ( figure c ) and dls ( figure d) . the tem analysis clearly showed that the morphology of the proteins was more compact, and probably highly stable, when assembled at high fe + concentrations ( µm) than at lower concentrations ( µm) ( figure c ). as shown in figure d , the average diameter of nps examined by dls was . nm at high fe + concentration ( - , µm) and . - . nm at lower concentration ( - µm). these results suggest that both fe + and salts concentrations influenced the efficiency and quality of the regular assembly of hybrid ferritin monomers into nps. our previous studies show that an rna-protein interaction is crucial for transducing the chaperone function of rna into the folding of client proteins ( ) . consistent with that, our present study showed that rna facilitated the folding of its interacting proteins. the solubility of hrid(wt)-rbd-fr was . -fold higher than rbd-fr without hrid fusion (figure ) , strongly supporting the previous studies. in addition, the solubility of rbd alone was completely insoluble (figure b ; figure s in supplementary material). it has been shown that the positively charged residues of lysine moieties in hrid contribute to trna binding ( ) . in the current study, the trna binding induced the intrinsically disordered protein (idp) status of hrid to form alpha-helical structures ( figure a) . thus, two rna-binding (table s in supplementary material). the total e. coli lysate (t) was fractionated into the soluble fraction (s) and the pellet fraction (p) by centrifugation. as expected, both rbd and rbd-fr without fusion to hrid domain, were refractory to being produced as soluble proteins ( figure b) . interestingly, the solubility of the rna-binding mutants did not decrease, but actually increased to . % for the m mutant and . % for the m mutant compared with wild-type protein at . % (figure b) . considering that hrid is relatively unstructured in the absence of trna binding, the results are consistent with previous reports that the fusion with idps promotes the solubility of target proteins ( ) ( ) ( ) . following purification of wild-type hrid-rbd-fr (hrid(wt)-rbd-fr), electrophoretic mobility shift assays showed that greater amounts of nucleic acids were co-purified with hrid(wt)-rbd-fr protein than with the mutant hrid-rbd-frs ( and m) under non-denaturing conditions ( figure c ). the relative ratio of nucleic acid based on etbr staining and proteins based on coomassie staining in the eluted fraction confirmed the reduced affinity of mutants to nucleic acids. to test if rna had a role in maintaining the stability of the target proteins, the lysates were treated with rnase a to eliminate rna, and the solubility of each protein was analyzed by sds-page and western blotting. the soluble fractions of the lysates (s) were incubated at °c in the presence and absence of rnase a and the samples were further separated into soluble fraction (ss′) and insoluble fraction (sp′) by centrifugation. as shown in the left panel of figure , rnase a treatment completely abolished the effect of rna on protein solubility as compared with the control (rnase a−) or with samples prior to rnase treatment. parallel experiments with the and m mutants showed much less rna co-purified with the proteins, confirming the reduced affinity to nucleic acids and the complete depletion of rna by rnase a treatment (figure , left panel) . remarkably, the solubility of hrid(wt)-rbd-fr was greatly reduced by depletion of rna as reflected in the ratio of [ss′]/ [sp′] [ . and . for rnase (+) and rnase (−), respectively] by both coomassie staining and western blot analyses (figure , right panel). however, the solubility of the mutants ( and m), was not significantly affected by rnase a treatment, probably due to their lower affinity to rna (figure , right panel) . taken together, the results demonstrate that hrid(wt)-rbd-fr maintained a strong affinity for rna, and that affinity was pivotal for maintaining the solubility of the protein. to further define the rna dependence of solubility of the ferritin hybrids (figure ) , we investigated if the rna binding had a role in the formation of nps. rbd-fr and the various hrid-rbd-fr (wt, , and m) proteins were purified by nickel-affinity chromatography ( figure s in supplementary material) and their physicochemical properties analyzed by sec (figure a) , tem (figure b) , and dls ( figure c) . the soluble yields of rbd-fr (hrid fusion) was approximately . mg/l of culture, representing greater than , -fold higher levels than its hrid (−) counterpart (~ μg/l culture), again confirming the role of hrid as a robust enhancer for solubility and assembly. it was striking to note that the two mutant proteins, despite high solubility (figure b) , were detected at disproportionately higher amounts in the void fractions of sec, indicating that they failed to form nps of a defined size, and existed predominantly as soluble aggregates ( figure a) . however, hrid(wt)-rbd-fr predominantly formed nps of a defined size (~ , kda). it is also interesting to note that there was a slight shift of the rna-binding mutants ( and m) in the elution pattern, suggesting a larger size of nps compared with wild-type nps. overall, the ratio between soluble aggregates in the void volume and the nps of defined size clearly showed that rna binding was crucial for assembly of the monomers into nps. as a control, rbd-fr (without hrid fusion) existed predominantly as soluble aggregates ( figure s in supplementary material). consistent with these results, em analysis confirmed well-structured nps by hrid(wt)-rbd-fr, compared to largely aggregated structures by the mutant proteins ( figure b) . even if multi-molecular structure was formed, the structure becomes unstable, mostly as soluble aggregates. consistently, the intensity distribution diameter of the wild-type protein, as estimated by dls analysis, was nm compared with larger sizes of hrid( m) at . , . nm and hrid( m) at , . nm (figure c ; figure s in supplementary material). it is conceivable that soluble aggregates may shield the exposed -histidine tag, resulting in a decreased binding affinity to nickel resins and elution in earlier fractions compared with wt protein ( figure s in supplementary material). taken together, the data demonstrate that rna binding prevented aggregation into irregular conformations and guided the self-assembly of the hybrid ferritin monomers into nps of a stable structure. the immunological properties of ferritin nps were analyzed by elisa. the hddp (human dpp ) receptor has been previously identified as the receptor for mers-cov human infection ( ) . therefore, using hdpp as a coating antigen, elisa-binding assays between rbd nps and the receptor were performed (figure ) . fr without rbd fusion failed to bind, and was similar to the pbs negative control. strikingly, the binding ability increased in the same order as the rna-binding ability (hrid(wt) > m > m), with highest absorbance observed in the wt with the ssg linker (hrid(wt)-rbd-[ssg]-fr). the results show that the conformation of rbd in the wt nps better resembled the protective antigen of mers-cov rbd from cells, compared with the rna-binding mutants and m. again, judicious choice of linker between the ferritin carrier and the antigen was important for receptor binding and was reflected in its importance for np assembly into a stable conformation (figure ) . finally, the elisa results for np against human patients was investigated using the sera from four mers-cov-infected patients (figure ) . six different proteins, including five recombinant nps (hrid(wt)-rbd-fr, hrid( m)-rbd-fr, hrid( m)-rbd-fr, hrid(wt)-rbd-[ssg]-fr, and fr), and mers-cov rbd protein were compared by elisa using them as capture antigens. strong elisa signals were detected for the four recombinant nps and mers-cov rbd from cells (positive control). the wt form consistently showed a higher response than the rnabinding mutants (hrid(wt) > m > m), with hrid(wt)-rbd-[ssg]-fr being the best binder among constructs tested. these results address to the utility of the e. coli assembled mers-cov rbd-fr nps as useful tools for sero-diagnosis of mers-cov infection. taken together, the results confirmed the immunologically relevant conformation of the mers-cov rbd displayed on the hybrid ferritin particles, and the crucial role of rna in controlling the kinetic pathway for the assembly of viral antigen monomers into stable nps. to evaluate the immunogenicity of ferritin-based nps, balb/c mice (n = ) were immunized with rbd, rbd-fr, and rbd-[ssg]-fr nps antigens. the trnas were found to be removed from the hrid protein during the purification process. before immunization, potential rna contamination in the purified proteins was determined by gel electrophoresis. as shown in figure s in supplementary material, rna was below detection level, if any, after several purification steps, compared with the proteins purified in the first step. previously, mf -adjuvated and alum-adjuvated mers-cov antigen have been reported to increase the antibody and t-cell responses in mice ( , ) . thus, the first group and second group were immunized twice with . µg of antigen containing the equal volume of alum figure s in supplementary material and size exclusion chromatography was used to explore hdpp receptor-binding affinity to the protein. all data are shown as mean ± sd from triplicate samples. fr alone and phosphate-buffered saline were used as negative controls. figure s in supplementary material were used as coating antigens. fr alone and infected cell lysates were used as negative and positive controls, respectively. virus-infected sera from four patients were serially diluted from : (twofold dilution). all data are presented as mean ± sd of duplicate samples. higher than rbd, respectively. the antibody responses by rbd-fr and rbd-[ssg]-fr nps were much stronger than the rbd in all antibody subtypes tested (igg , igg a, and igg b) (figures b-d) . as a test of mucosal immune responses, the rbd-specific iga antibody levels from balf were also analyzed by elisa (figure e) . mf adjuvanted rbd-[ssg]-fr nps presented significantly higher od values than rbd and fr (negative control). these results suggested that rbd-[ssg]-fr nps induces local mucosal immune response stronger than rbd. in addition, it was confirmed that antibody responses of igg, igg (th ), igg a, and igg b (th ) against mf -adjuvated antigens were higher than those from alum-adjuvated antigens. in contrast, pbs and fr control groups failed to, or only weakly induce an antibody response against rbd protein. these results suggest that fr-based nps significantly enhance various antibody responses than monomeric antigens. the cellular immune responses were investigated in mice immunized with protein (rbd, rbd-fr, rbd-[ssg]-fr) and fr (negative control). splenocytes of mice (n = ) were harvested week after the last immunization, stimulated with | nanoparticles-immunized mouse serum inhibited interaction between middle east respiratory syndrome-coronavirus receptor-binding domain (rbd) and hdpp receptor. competition enzyme-linked immunosorbent assay showed that anti-rbd mouse sera ( : , from mice immunized with rbd-[ssg]-fr, rbd-fr, and rbd) blocked binding between rbd ( µg/ml) and hdpp receptor ( µg/ml). fr-immunized mouse serum ( : ) was used as a negative control. all sera were serially diluted from : (twofold dilution). all data are presented as mean ± sd (n = ) and p-values were obtained using student's two-tailed tests (***p < . ). figure | immune responses in receptor-binding domain (rbd) nanoparticles (nps) immunized mice (n = ). endpoint titer of igg (a), igg (b), igg a (c), and igg b (d) antibody binding to middle east respiratory syndrome-coronavirus rbd were detected using mice serum after two immunizations. rbd-specific antibodies were detected after immunizations of rbd nps, rbd, fr with adjuvant (alum and mf ) using enzyme-linked immunosorbent assay. (e). rbd-specific iga antibodies were detected using balf (diluted : ) after immunization of protein with mf . od, optical density. each endpoint titer was shown by individual. all error bars were shown as mean ± sd (n = ) and all p-values were obtained using student's two-tailed tests (**p < . , ***p < . ). rbd protein, and analyzed for cytokines by flow cytometry. in the rbd-immunized group, ifn-γ and tnf-α-producing cd + t-cell responses were detected at low levels. however, ifn-γ and tnf-α-producing cd + t cells were significantly increased in rbd-fr and rbd-[ssg]-fr-immunized groups compared with rbd and fr-immunized group ( figure s in supplementary material) . these results demonstrated that the rbd nps vaccination induced antigen-specific cd + t cells that produced ifn-γ and tnf-α upon antigen stimulation. anti-nps serum effectively blocked rbd protein binding to the hdpp receptor middle east respiratory syndrome-coronavirus infection is mediated by the interaction of rbd and the host receptor hdpp ( , ) . as a correlate of protection, a competition elisa was performed to investigate whether antibodies generated from nps immunization were able to interfere with the binding to hdpp . thus, after incubation of rbd protein with mouse serum ( : ), the binding of serum-mixed samples to hdpp protein was measured. as shown in figure , rbd-[ssg]-fr, rbd-fr, and rbd-immunized sera strongly abolished the binding of rbd to hdpp receptor ( . , . , and . %, respectively). interestingly, the relative efficiency of interference correlates with that of np assemblage (figure ). in contrast, the fr-immunized mouse serum (negative control) failed to inhibit the interaction. taking together, these results demonstrate that immunization of nps greatly stimulates mers-covspecific antibody response that effectively interferes with the cellular receptor binding, suggesting its possibility as a vaccine. however, protection efficacy should ultimately be tested in a live virus challenge model. having key immunologic features, like a highly repetitive nanostructure, provides a designing principle for nps in inducing potent and long-lasting antibody responses. for vlps of non-enveloped viruses, assembly is made purely by capsid proteins. for enveloped viruses, however, additional membrane components and matrix proteins are required to display the target antigens on the surface of assembled vlps. a promising alternative is to present target antigen on the surfaces of selfassembled nps, which, in lieu of lipid membranes and matrix proteins, serve as a macromolecular scaffold for the presentation of antigens of interest ( ) . ferritins, as a substitute for matrix proteins and membranes, have been used as scaffold for the regular assembly of target antigens. however, ferritin-based nps have been produced only in host cells of mammalian or insect origin ( , ) . previously, we showed that influenza ha could be assembled in a soluble, trimeric, and immunologically relevant conformation by exploiting chaperna activity ( ) . the present study is the first report of using rnas as molecular chaperone for supra-molecular structures. here, we present a novel bacterial system for np assembly of hybrid ferritin displayed surface antigens from mers-cov. the nps reacted strongly with sera derived from mers-cov-infected patients (figure ) confirming their utility in sero-diagnosis of infection. moreover, the antisera, generated from immunization of mice, were able to interfere with the binding to the cellular receptor hdpp (figure ), in part of essential protective immune responses. the efficiency of receptor-binding inhibition (figure ) , as well as the ability for inducing the mucosal responses (figure e) , correlated with the regular assembly of nps as examined by dls or em (figure ) , confirming that presentation of antigenic epitopes on a multivalent and highly repetitive structure is indeed important for the quality of immune responses. overall, the quality of nps and consequent immune responses were governed by the rna-mediated assembly of antigens. we hypothesized that chaperna function could be harnessed for presenting target antigens as highly repetitive nanostructures ( figure d) . the hrid is the n-terminal domain of hlysrs and was previously identified as a nucleic acid-binding domain ( figure ) ( ) . in this report, the hrid was exploited as a transducer for chaperna function (tcf) by serving as a docking-tag for cellular rna for the folding/assembly of the hybrid fr containing client antigen proteins [rbd of mers-cov (figure d) ]. the advantage of using hrid as a tcf could be many fold. first, hrid is small ( . kda), monomeric, and was flexible enough to allow the access of site-specific protease for the removal of hrid ( figure s in supplementary material). of note, hrid belongs to idps, which switches into stabile α-helixes upon binding with trnas. second, the bound rna, due to its highly negative charge, may resist uncontrolled intermolecular interactions among monomers into amorphous aggregation. finally, even the naked hrid (in the absence of rna binding), due to its intrinsically flexible nature, may not pose physical hindrance to multiple interactions among monomers, enabling assembly into stable super-structures, upon removal of the hrid. thus, the potential "pace-making" function harnessed with the rna molecule, allows a regular assembly of monomers as highly repetitive nanostructures. consequently, in the current study, hybrid fr was produced in soluble forms, could be purified by one-step affinity chromatography, and most remarkably, assembled into nps of defined sizes upon removal of the hrid ( figure s in supplementary material). consistent with the principles of design, the loss of rna binding by hrid significantly hampered the regular assembly of the ferritin monomers and increased the amount of non-functional misfolded proteins as soluble aggregates (figure ) . thus, the overall yield, as well as the quality of nps, were dependent on the chaperna function transduced by the hrid, which in turn was mediated by interaction with cellular rnas (likely to be trnas). the driving and controlling factors for de novo assembly of biomolecules are poorly understood. historically, host factors like groel/s were initially discovered as molecular chaperones for supporting viral growth in e. coli and supporting the assembly of viral capsid proteins ( , ) . moreover, groel/s also cooperates with rbcx in plant cells for the assembly of multicomponent rubisco, which is the most abundant protein in the biosphere responsible for photosynthesis ( ) . therefore, it is intriguing that rna could provide such a robust folding/ assembly of a supra-molecular structure. we recently confirmed that the present strategy could be successfully applied to the assembly of bacterially synthesized monomers of norovirus into vlps composed of monomers (unpublished observation, seong, b.l.). whether rna can substitute for, or collaborate with pre-existing protein-based molecular chaperones remains an exciting avenue for future investigations. it should be noted that the defined versatile functions are being expanded for rna molecules. as an engineered system for harnessing chaperna function, the present report may prove to be the tip of an iceberg for pivotal function of rna molecules as chaperones for the folding and supra-molecular assembly of proteins in living organisms ( , ) . various factors were identified as important for efficient assembly of mers-cov nps. as an extrinsic factor, the binding affinity of hrid to cellular rnas was crucial for the assembly and the quality of the assembled nps (figure ) . as intrinsic factors, the concentration of salts and fe + also influenced the assembly and stability of nps (figures and ) . the ionic strength played an important role in the stability and self-assembly of ferritins, and aggregation increased with increasing concentrations of nacl ( ) . the assembly of the hybrid mers-cov nps revealed an interesting change in salt dependence, with - mm nacl buffer as optimal condition as confirmed by em and dls analyses (figure ) . the change in salt dependence was probably due to the presence of electrostatic interactions among rbd domains ( , ) . the dependence on fe + was not surprising considering that ferritin has an intrinsic ability to interact with fe + to form ferritin-iron cores ( ) . based on our experience, to enhance the quality of nps, it is advisable to control fe + concentrations, both during the culturing of the bacterial cells and during the purification of the soluble monomer proteins (figure ) . first, the yield of the purified protein was increased in the presence of µm fe + (figure b) , up to . -fold greater compared with the control conditions lacking fe + . second, the ratio between nps and soluble aggregates in sec showed that nps formation was facilitated at high concentrations of fe + , and resulted in a more compact morphology under em (figures b,c) . thus, both the overall yield and the quality of nps were governed by their intrinsic ability to interact with fe + . finally, our data show that the presence and the nature of the linker between the ferritin and the rbd antigen was also important to the assembly of nps. it is possible that a linker with flexibility and sufficient length would accommodate the steric requirements for assembly of multimeric nps. however, it is difficult to precisely predict the effect of the linker, and therefore it is advisable to screen multiple constructs during the early stages of testing the assembly of nps displaying antigens of interest. in conclusion, the chaperna-based antigen assembly platform holds promise for the development and delivery of np-based vaccines to enhance rbd-specific antibody responses, and the serological detection of emerging viruses. various types of designing principles have advanced the structure-based approaches to np assembly ( , ) . however, most of the in silico methods consider the thermodynamic stability of the final assembled nps, but not necessarily the kinetic pathways leading to their successful folding into regular assemblages. consequently, most nps are refractory to soluble expression and fail to assemble as designed, resulting in significant, and practical challenges in the manufacturing process. the chaperna-mediated folding and the "pace-keeping" assembly of monomers into higher ordered structures will enable faithful production of np and vlp-based vaccines against emerging and re-emerging viral infections. this study was carried out in accordance with the recommenda- figure | elucidation of rna-mediated nanoparticle (np) formation of receptor-binding domain (rbd)-fr. (a) size exclusion chromatography analysis of rbd-fr nps purified from the tev protease-cleaved hrid(wt, , or m)-rbd-fr. the fractions ( - ml) estimated as nps were further analyzed by transmission electron microscopy (b) and dynamic light scattering (c) exploiting virus-like particles as innovative vaccines against emerging viral infections traditional and new influenza vaccines vaccine manufacturing: challenges and solutions management of accidental exposure to ebola virus in the biosafety level laboratory vaccine delivery using nanoparticles vaccine delivery: a matter of size, geometry, kinetics and molecular patterns virus-like particles as a highly efficient vaccine platform: diversity of targets and production systems and advances in clinical development the influence of antigen organization on b cell responsiveness structure of the hepatitis e virus-like particle suggests mechanisms for virus assembly and receptor binding self-assembly of human papillomavirus type capsids by 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domain-based subunit vaccines against middle east respiratory syndrome coronavirus protein/peptide-templated biomimetic synthesis of inorganic nanoparticles for biomedical applications self-assembling influenza nanoparticle vaccines elicit broadly neutralizing h n antibodies harnessing an rna-mediated chaperone for the assembly of influenza hemagglutinin in an immunologically relevant conformation host participation in bacteriophage lambda head assembly properties of a mutant of escherichia coli defective in bacteriophage lambda head formation (groe). i. initial characterization coupled chaperone action in folding and assembly of hexadecameric rubisco modeling the influence of salt on the hydrophobic effect and protein fold stability rational design of an epstein-barr virus vaccine targeting the receptor-binding site key: cord- -ywwqnw authors: tomar, jasmine; biel, carin; de haan, cornelis a.m.; rottier, peter j.m.; petrovsky, nikolai; frijlink, henderik w.; huckriede, anke; hinrichs, wouter l.j.; peeters, ben title: passive inhalation of dry powder influenza vaccine formulations completely protects chickens against h n lethal viral challenge date: - - journal: eur j pharm biopharm doi: . /j.ejpb. . . sha: doc_id: cord_uid: ywwqnw bird to human transmission of high pathogenicity avian influenza virus (hpaiv) poses a significant risk of triggering a flu pandemic in the human population. therefore, vaccination of susceptible poultry during an hpaiv outbreak might be the best remedy to prevent such transmissions. to this end, suitable formulations and an effective mass vaccination method that can be translated to field settings needs to be developed. our previous study in chickens has shown that inhalation of a non-adjuvanted dry powder influenza vaccine formulation during normal breathing results in partial protection against lethal influenza challenge. the aim of the present study was to improve the effectiveness of pulmonary vaccination by increasing the vaccine dose deposited in the lungs and by the use of suitable adjuvants. two adjuvants, namely, bacterium-like particles (blp) and advax, were spray freeze dried with influenza vaccine into dry powder formulations. delivery of dry formulations directly at the syrinx revealed that blp and advax had the potential to boost either systemic or mucosal immune responses or both. upon passive inhalation of dry influenza vaccine formulations in an optimized set-up, blp and advax/blp adjuvanted formulations induced significantly higher systemic immune responses than the non-adjuvanted formulation. remarkably, all vaccinated animals not only survived a lethal influenza challenge, but also did not show any shedding of challenge virus except for two out of six animals in the advax group. overall, our results indicate that passive inhalation is feasible, effective and suitable for mass vaccination of chickens if it can be adapted to field settings. outbreaks of avian influenza in poultry are the major source of h n infection in humans [ ] [ ] [ ] . these outbreaks, caused by highly pathogenic strains of avian influenza virus, pose a significant risk of poultry-human transmission. currently, the only remedy to control these outbreaks is to cull the poultry. for safety reasons animals are not only culled on infected farmyards but also on non-infected neighboring farmyards [ , ] . the slaughter of millions of poultry not only leads to severe economic losses but also raises ethical questions and incomprehension within the society. to combat outbreaks of avian influenza virus, it could be useful to rapidly vaccinate the poultry in areas surrounding the outbreak, in a ring fencing strategy. however, this would require that very large numbers of poultry, potentially in the tens or hundreds of millions, should be able to be immunized within a very short time period. this would require a vaccine formulation and route of immunization that are suitable for such mass application. liquid or powder influenza vaccine formulations that can be aerosolized and administered via the respiratory tract could be appropriate formulations for mass vaccination. liquid formulations of inactivated influenza virus have been shown to be inadequate for aerosol vaccination by single administration [ ] . powder formulations, on the other hand, have shown an advantage over liquid formulations because of their long-term storage stability [ ] [ ] [ ] . this long term stability facilitates stockpiling and thus provides ease of administration during mass vaccination [ ] . in a previous study, we have shown that pulmonary immunization by dispersion of a dry powder influenza vaccine directly at the syrinx of chickens (active administration) was able to completely protect these animals against lethal viral challenge [ ] . however, in realistic situations, due to extreme time pressures, active administration of chickens is unsuitable for mass vaccination. a more realistic approach would be to let chickens inhale aerosolized dry powder influenza vaccine formulation during breathing (passive administration). indeed, passive inhalation of dry powder influenza vaccine formulation was feasible in chickens, but the animals were only partially protected indicated by a delay in time to death and reduced virus shedding [ ] . hence, the efficiency of influenza immunization by passive inhalation must be improved for pulmonary vaccination to become feasible. possible approaches could be to increase the concentration of aerosolized vaccine, to expose the animals to aerosolized powder for longer periods of time and to include an adjuvant in the vaccine formulation. an effective adjuvant should not only be cheap, readily available and potent, but also safe. an adjuvant that has been generally recognized as safe is blp. blp are produced by hot acid treatment (ph for min at °c) of lactococcus lactis, a non-pathogenic, food-grade gram-positive bacterium [ , ] . blp act as toll-like receptor- ligand and have shown potent immune boosting properties when administered together with vaccines against influenza, yersinia pestis, malaria, and pneumococcal disease [ ] [ ] [ ] [ ] . besides toll-like receptor ligands, naturally derived polysaccharides, for example advax™ adjuvant comprising an insoluble isoform of inulin, boosts vaccine responses through mechanism that are still not fully characterized [ ] . in (pre) clinical studies advax has been shown to enhance immune responses induced by a wide variety of vaccines including vaccines against hepatitis b, sars coronavirus, listeria and influenza [ ] [ ] [ ] [ ] [ ] [ ] . upon parenteral administration, advax has been shown to have a good safety and tolerability record both in animal studies and clinical trials [ ] [ ] [ ] . the aim of the current study was to investigate whether passive administration with dry non-adjuvanted or adjuvanted influenza formulations has the potential to completely protect chickens against lethal hpaiv challenge. for this, we initially tested whether (a) blp or advax could be co-formulated with influenza vaccine into dry powder formulations that are suitable for pulmonary immunization; (b) the adjuvants have the potential to boost systemic and mucosal immune responses to influenza; (c) passive administration with either non-adjuvanted or adjuvanted influenza formulations would protect chickens against a lethal hpaiv challenge. for immunization, a reassortant virus, nibrg- , prepared by reverse genetics from a/turkey/turkey/ / (h n ) virus and a/pr/ / (h n ) virus was used (nibsc code: / ). the virus was cultured in embryonated chicken eggs by allantoic inoculation of the seed virus. the virus was purified and inactivated as described previously to obtain whole inactivated virus vaccine (wiv) [ , ] . for challenge, a highly pathogenic avian influenza virus strain a/ turkey/turkey/ / (h n ) (clade . . ), obtained from the animal health and veterinary laboratories agency, weybridge, uk was used. for active administration, . , . , and . µg ha of nibrg- wiv was sfd either as such or admixed with adjuvants (blp, mucosis, groningen, the netherlands or advax, vaxine, adelaide, australia) in various ratios. for active administration, the dose of blp and advax in mg of sfd powder was µg and µg respectively. wiv was mixed with blp in weight ratios of ha:blp of : , : and : . for wiv-advax, the weight ratios of ha:advax were : , : and : . likewise, for passive administration, µg ha of nibrg- was sfd either as such or mixed with µg of blp or µg of advax (this dose corresponds to the amount of ha and adjuvants in mg of sfd powder). both non-adjuvanted and adjuvanted formulations were sfd using inulin ( kda, sensus, roosendaal, the netherlands) as the stabilizer. inulin used for stabilization of influenza vaccine is amorphous and water soluble with potentially no adjuvant effect whereas advax (delta inulin) is crystalline and insoluble in water with potent adjuvant activity. in brief, a % (w/v) solution of wiv and inulin with or without adjuvants was pumped at a flowrate of ml/min through a two-fluid nozzle of a büchi mini spraydryer (büchi, flawil, switzerland). an atomizing airflow of l/h was used to spray vaccine preparations in a vessel of liquid nitrogen. then, the frozen vaccine preparations were placed in a christ epsilon - freeze dryer precooled to a shelf temperature of − °c and at a pressure of . mbar. the shelf temperature was slowly increased from − °c to °c within the time period of h. during the next h, the temperature was further increased to °c and pressure was lowered to . mbar. the dry vaccine powder was collected in a climate box with a relative humidity of < % and stored under airtight conditions. a philips cm transmission electron microscope was used to make transmission electron microscopy images. sfd powders containing wiv ( µg ha formulation) with or without adjuvants i.e. blp ( µg in mg of sfd powder), advax ( µg in mg of sfd powder) were reconstituted in sterile filtered water. liquid and reconstituted sfd formulations were placed on a plain carbon grid, rinsed with water and then samples were stained twice with µl of wt-% uranyl acetate. a gatan type ultrascan sp ccd camera at a magnification was used to take images. the receptor binding activity of wiv after sfd with or without adjuvants was assessed by hemagglutination assay. sfd powders were reconstituted in phosphate buffer saline (pbs) to a concentration of µg/ml of ha. then, µl of the preparation was added to -well v bottom plates containing µl of pbs. the entire mixture was two-fold serially diluted, then µl of . % guinea pig red blood cell suspension was added to each well. the plate was allowed to stand undisturbed for two hours at room temperature. hemagglutination titers read after two hours were expressed as log of the highest dilution where red blood cell agglutination could be seen. the capacity of blp to activate nf-κb via toll-like receptor- was evaluated using raw-blue™ cells (invivogen, toulouse, france). raw-blue™ cells have a number of pattern recognition receptors which when bound to agonists leads to activation of nf-κb and thus the production of secreted alkaline phosphatase. a schematic illustration is shown in fig. s . cells were maintained in dmem with high glucose (gibco life technologies bv, bleiswijk, the netherlands), % fbs (lonza, basel, switzerland), µg/ml normocin™ (invivogen, toulouse, france), mm l-glutamine and passaged when - % confluency was reached. approximately × cells were added to -well flat bottom plates and were stimulated with . µg untreated blp or with liquid and reconstituted sfd powder formulations with ( µg ha + µg blp formulation) or without . µg blp. the incubation was maintained for h at °c with % co . to measure alkaline phosphatase levels, µl quanti-blue™ (invivogen, toulouse, france) was added to the cell supernatant and after h, absorbance was measured at nm using a spectrophotometer. sfd-wiv preparations were imaged using a jeol jsm -f microscope (jeol. ltd., tokyo, japan). powders were placed on doublesided sticky carbon tape on a metal disc and the particles were coated with approximately nm of gold using a balzer's b sputtering device (balzer union, liechtenstein). a magnification of × and × was used to capture the images. geometrical particle size distribution of the powders were measured using a helos compact model ka laser diffraction apparatus (sympatec gmbh, clausthal-zellerfeld, germany). to disperse the powders, the highly efficient rodos dispersing system (sympatec gmbh, clausthal-zellerfeld, germany) was used at a pressure of bar. aerodynamic particle size distribution was calculated using the equation described by bhide et al. [ ] . sixty -week old specific-pathogen free chickens (white leghorn) were randomly divided into groups of animals. animals were immunized twice i.e. on day and day . sfd-wiv formulations (either non-adjuvanted or adjuvanted with blp or advax) were administered to the animals using a dp- -c dry powder insufflator (penn-century inc., philadelphia, usa). a custom length delivery tube designed to deliver the powder directly at the syrinx was used. three puffs of ml air were used to aerosolize mg of powder filled in the insufflator. the animals were sacrificed on day . blood samples were taken on day (before the st immunization), day (two weeks after the st immunization) and day (two weeks after the nd immunization). on the day of sacrifice (day ), lung lavages were collected by flushing lungs with ml of pbs as described by holt et al. [ ] . the obtained sera and lung lavages were stored at − °c until further use. an overview of the immunization scheme and groups is shown in fig. a and b. for passive administration of non-adjuvanted and adjuvanted wiv formulations, -week old specific-pathogen free chickens were randomly divided into groups of chickens. one animal died of unknown causes before the start of the experiment. each group was placed in a customized box with a volume of . m . a ml eppendorf tube filled with mg of sfd powder containing . mg ha of nibrg- wiv with or without adjuvants was punctured at the bottom with a g hypodermic needle. the lid of the eppendorf tube was fitted to a pressurized air container while the bottom conical part of the tube was inserted airtight in a hole drilled in the customized box for the dispersion of powders. the sfd powders were aerosolized in the box by using several pulses of pressurized air, resulting in a theoretical concentration of mg ha/m (ha dose for dispersion: . mg, inhalation box volume: . m ). for the first vaccination, the animals were exposed to the aerosolized vaccine for min and after every min short pulses of medicinal oxygen ( s; flow rate . l/min) were supplied through another hole in the box. for the second vaccination ( weeks later i.e. on day ) the exposure time was min with short pulses of oxygen ( s; flow rate . l/min) every minute. based on the respiration rate for chickens of l/h/kg body weight [ , ] these exposure times would result in a maximum theoretical dose of µg ha per animal per application. the eid ( % embryo-infectious dose) titre of the challenge strain stock of a/turkey/turkey/ / ( . log eid /ml) was used to calculate the dilution for the challenge virus. the % chicken lethal dose of a/turkey/turkey/ / virus is . log eid [ ] . on day , animals were challenged with a lethal dose of . log eid of this virus by the combined intranasal/intratracheal route (liquid suspension; . ml each). for back titration, we determined tcid ( % tissue culture infectious dose). the titre ( log tcid /ml) after backtitration of the challenge virus that we received from the stables after the challenge inoculation was . whereas the titre of the original stock was . . thus, a titre of . eid /ml corresponds to . tcid /ml. the titre of . tcid /ml for the back-titration should therefore correspond to at least . eid /ml. since the % chicken lethal dose of the challenge virus is . log eid , a dose of . logeid ( . ml of . eid /ml) is more than sufficient (> × cld ) to kill nonvaccinated animals. in previous studies with the same virus batch and dilution, we found that this dose killed all non-vaccinated control group animals within - days after the challenge [ , ] . in this study, after challenge, animals were observed for clinical signs and on , , , and days post challenge choanal and cloacal swabs were collected for virus quantification. on day , the animals were sacrificed. an overview of the immunization-challenge scheme along with groups is shown in fig. c and d. passive inhalation of dry formulations could be seen in video file (supplementary data). virus titers in choanal and cloacal swabs were determined as per the method described by van der goot et al. [ ] . challenge virus was tenfold serially diluted, followed by rna extraction, and a standard curve consisting of the rna from these serial dilutions was prepared. as per the standard curve, pcr data was converted into equivalent virus titers (eqtcid /ml). serum samples and lung washes were used for the determination of igy and iga antibody titers. for the determination of igy titers, elisa was performed as previously described [ ] , except that the secondary antibody consisted of goat anti chicken igy-hrp (southern biotech, birmingham, usa). igy titers were determined as log of the reciprocal of the sample dilution corresponding to an absorbance of . at the wavelength of nm. iga titers were determined in lung washes using the commercially available chicken iga elisa kit as per manufacturer's instructions (abcam, cambridge, uk). hi assay was performed as described previously [ ] . briefly, hemagglutination units of inactivated virus a/turkey/turkey/ / (h n ) were added to two-fold diluted sera samples. hi titers were recorded as highest serum dilution capable of preventing hemagglutination. hi titers are presented on log scale. micro-neutralization titers were determined as previously described [ ] . briefly, two-fold serial dilutions of serum samples were prepared and tcid of nibrg- virus was added to each well of well plate. after h of incubation at °c, the mixture of serum and virus was transferred to mdck cells (atcc, germany) cultured in -well flat bottom plate. after h of incubation at °c, the supernatant was discarded and replaced with episerf-medium ( u/ml penicillin, mg/ml streptomycin, m hepes and . % sodium bicarbonate, all life technologies™ bv, bleiswijk, the netherlands) supplemented with µg/ml of tpck trypsine (sigma-adrich, the netherlands). after h of incubation, the supernatant was transferred to v bottom plate. micro-neutralization titers were calculated by finding the highest serum dilution capable of preventing hemagglutination. micro-neutralization titers are presented on log scale. mann-whitney one tailed test was used to compare whether the differences between non-adjuvanted and adjuvanted influenza vaccine formulations were significant. levels of significance are denoted as * p ≤ . , ** p ≤ . , *** p ≤ . respectively. in order to investigate the potential of blp and advax as mucosal adjuvants in chickens, it was essential to investigate whether the physical and biological properties of wiv and adjuvants remained unaltered during sfd. to evaluate whether admixing adjuvants with wiv and sfd had an influence on the physical appearance of wiv, blp or advax particles, transmission electron microscopy analysis was performed for these samples before and after sfd. the powder samples were reconstituted prior to use. transmission electron microscopy pictures showed that wiv, blp and advax particles had comparable morphological appearance before and after sfd ( fig. a-c) . thus, neither the stress encountered during sfd nor admixing adjuvants with wiv had any adverse effect on their physical appearance. the biological activity of wiv needs to be preserved both after the addition of adjuvants and after sfd. in order to investigate whether the receptor binding activity of ha was preserved, hemagglutination assay was performed. no differences in hemagglutination titers could be detected either by the addition of adjuvants or by sfd, thus indicating no detrimental effects of adjuvants or sfd on the biological activity of ha (fig. d) . furthermore, the effect of sfd on the biological activity of blp was evaluated. for this purpose, reconstituted blp-adjuvanted sfd wiv formulations were compared to unprocessed dispersion of liquid wiv and blp for their capacity to activate nfκb using raw-blue™ cells. nibrg- derived wiv alone was found to be a poor activator of nfκb. reconstituted blp-adjuvanted wiv formulations activated nfκb to a similar extent as native liquid wiv-blp dispersion. thus, stress encountered during sfd had no effect on the biological activity of blp to activate toll-like receptor (fig. e ). to assess whether the incorporation of blp or advax in the sfd formulation had an effect on the physical characteristics of powder particles, scanning electron microscopy was used to analyze physical appearance of sfd formulations. spherical shaped intact particles with an interconnected porous structure could be seen for both non-adjuvanted and adjuvanted wiv formulations. no major differences could be observed in the morphology of sfd particles by varying the dose of ha and by the addition of adjuvants. representative pictures of nonadjuvanted, blp and advax adjuvanted wiv formulation at an ha dose of . µg per mg of powder are shown in fig. a -c. further, upon determination of geometric particle size, average particle size (x ) was found to be between and µm both for non-adjuvanted and adjuvanted wiv formulations (fig. d) . however, for inhalation, an aerodynamic particle size of - µm is considered to be most suitable [ ] . the average aerodynamic particle size of non-adjuvanted as well as adjuvanted wiv formulations was calculated according to the formula described by bhide et al. [ ] . the average aerodynamic particle size was found to be ≤ . µm and the majority of the particles (x ) had size ≤ µm (fig. e) . particles with a size range between and . µm are considered to be suitable for deposition in the entire respiratory tract of chickens [ ] [ ] [ ] . overall, our results indicated that sfd can produce blp and advaxadjuvanted wiv particles with physical and biological characteristics that make them suitable for pulmonary vaccination. for active administration, the tip of the insufflator was placed directly at the syrinx of chickens and three puffs of ml air were used to disperse the powders. we next evaluated the systemic and mucosal immune responses induced in chickens after active administration. serum igy and hi titers were measured both at day and day whereas serum mn, lung igy and iga titers were measured only at day . on day , non-adjuvanted as well as adjuvanted wiv formulations had induced considerable serum igy titers. advax-adjuvanted wiv formulations induced significantly higher serum igy titers than the corresponding non-adjuvanted wiv formulations (fig. a ). however, no major differences were found among non-adjuvanted and blp-adjuvanted wiv formulations, except between corresponding . µg ha formulations (fig. a) . although, this difference was statistically significant, it was not major. however, on day , significantly higher titers were induced by blp-adjuvanted wiv formulations than by the non-adjuvanted wiv formulation at an ha dose of . and µg; the difference between non-adjuvanted and blp-adjuvanted formulations was not significant at ha dose of µg (fig. b) . for advax-adjuvanted wiv formulation, significantly higher titers than corresponding nonadjuvanted wiv formulation were only induced by the µg ha formulation (fig. b) . systemic immune responses were further assessed by determining serum hi titers. it was found that after the first vaccination i.e. at day , none of the vaccinated chickens had detectable hi titers (fig. c) . however, after the second vaccination, blp-adjuvanted wiv formulations, at a dose of and . µg ha, induced a trend towards higher hi titers (mean hi titers of . log and . log respectively) than corresponding non-adjuvanted wiv formulations (mean hi titers of . log and respectively) (fig. c ). though at an ha dose of µg, the difference was not statistically significant, but at an ha dose of . µg, significantly higher titers could be seen for blp-adjuvanted wiv formulation than corresponding non-adjuvanted formulation. also, compared to non-adjuvanted wiv formulation ( ), a small but non-significant increase in hi titers was seen for the advax-adjuvanted wiv formulation at the lowest ha dose of . µg ( . log ) (fig. c) . at day , micro-neutralization titers were found to be in line with hi titers. in comparison to non-adjuvanted wiv formulation, advax and blp-adjuvanted wiv formulations at a dose of . µg ha induced an increase in the micro-neutralization titers of about five and eight fold, respectively (fig. d) . no major differences were seen at µg and . µg ha dose. mucosal immune responses were assessed by determining iga and igy titers in the lung lavages obtained two weeks after the second immunization. lung igy titers were found to be consistent with serum igy titers after the second immunization. blp-adjuvanted wiv formulations induced significantly higher lung igy titers than respective non-adjuvanted wiv formulations at a dose of . and µg ha (fig. e ). in addition, advax-adjuvanted wiv formulations induced higher lung igy titers than non-adjuvanted wiv formulation at an ha dose of . µg, though the difference was not significant (fig. e) . though non-adjuvanted wiv formulations were found to induce considerable lung iga titers, the inclusion of blp and advax further boosted the immune responses (fig. f) . it was found that at a dose of µg ha, blp-adjuvanted wiv formulation boosted lung iga titers ( . log ) by approximately four fold in comparison to corresponding non-adjuvanted wiv formulation ( . log ) (fig. f) . for advax-adjuvanted wiv formulations, at a dose of . and µg ha, lung iga titers were augmented by three to five fold as compared to corresponding non-adjuvanted wiv formulations (fig. f) . passive administration might be a suitable method for mass vaccination of chickens. for passive administration, chickens placed in a custom made box were allowed to inhale aerosolized vaccine powders during breathing using two applications weeks apart. we next evaluated the systemic immune responses induced in chickens after passive administration of these aerosolized powders. serum igy titers were determined at day and day whereas micro-neutralization titers were determined at day . adjuvanting with blp or advax resulted in significantly higher serum igy titers at day ; an increase of about three to five fold could be seen by the co-administration of blp or advax with wiv (fig. a) . however, after the second vaccination, only blp and advax/blp adjuvanted wiv formulations had significantly higher serum igy titers ( . - . log ) than non-adjuvanted wiv formulation ( . log ) (fig. b) . the augmentation in serum igy titers was about six-fold for blp-adjuvanted wiv and three fold for advax/ blp-adjuvanted wiv (fig. b) . trends in hi titers were in agreement with serum igy titers both after the first and second immunization. after the first immunization, all three formulations i.e. blp, advax and advax/blp adjuvanted wiv formulations showed significantly higher hi titers (mean hi titers between . and . log ) than non-adjuvanted wiv formulation (mean hi titer . log ) (fig. c) . notably, after the second immunization, blp adjuvanted and advax/blp-adjuvanted wiv formulations induced significantly higher hi titers (mean hi titers between . and . log ) than non-adjuvanted wiv or advax adjuvanted wiv (mean hi titers of . log ). in addition, micro-neutralization titers determined after the second immunization were also in line with serum igy and hi titers. blp and advax/blp adjuvanted wiv formulations induced six-fold higher micro-neutralization titers ( . log ) than non-adjuvanted wiv formulation ( . log ) (fig. d ). to assess whether passive administration of non-adjuvanted or adjuvanted influenza vaccine formulations, has the potential to reduce/ diminish the shedding of challenge virus after lethal challenge, virus shedding was determined in choanal and cloacal swabs. in our previous study, we found out that non-vaccinated animals shed virus until they succumbed to infection ( days post challenge) [ ] . in this study, a similar dose of a/turkey/turkey/ / virus was used to challenge chickens by the combined intranasal/intratracheal route. no virus was found either in choanal or cloacal swabs of animals immunized with non-adjuvanted or blp adjuvanted or advax/blp adjuvanted wiv formulations (table ) . only two animals immunized with advax-adjuvanted wiv formulation showed virus in their choanal swabs ( table ) in this study, we demonstrated that blp and advax can be co-formulated with influenza vaccine into a dry formulation that is suitable for pulmonary immunization of chickens. upon active administration of chickens, the adjuvants blp and advax were shown to augment systemic and mucosal immune responses. remarkably, in an optimized setup, passive administration of chickens either with non-adjuvanted or blp or advax adjuvanted wiv formulations induced robust immune responses that were sufficiently high to protect chickens against lethal viral challenge. furthermore, blp and advax/blp adjuvantation significantly raised the level of antigen-specific serum antibodies. in real life situations, where passive inhalation would be performed in large rooms, a portion of the aerosolized vaccine formulation would also end up in food and water. however, previous studies have shown that ha of the influenza vaccine undergoes conformational changes at acidic ph and is thus susceptible to proteolytic cleavage in the acidic environment [ , ] . consequently, little-no neutralizing antibodies are induced by acidic ph treated influenza vaccine [ , ] . therefore, it is speculated that the acidic environment in the stomach of chickens would not really add up to the lung induced mucosal immunity. the crystalline nature of advax and peptidoglycans on the surface of blps might have detrimental effects on influenza vaccine during the production of adjuvanted dry powder formulations. however, the inclusion of blp or advax together with wiv in a formulation led to the formation of dry powder particles of comparable physical and biological characteristics as those of non-adjuvanted wiv formulation. moreover, the physical properties of adjuvants i.e. their size and shape were found to be unaltered after sfd. also, the biological activity of blp was found to be well preserved during sfd. previous studies have shown that administration of blp or advax adjuvanted influenza vaccine formulations via the respiratory tract results in substantial augmentation of systemic and mucosal immune responses in mice [ , [ ] [ ] [ ] [ ] . in this study we showed that after active administration, systemic immune responses (serum igy titers) after one immunization were predominantly enhanced by advax whereas after two immunizations they (serum igy, hi titers, micro-neutralization titers) were mainly enhanced by blp. after two immunizations, both non-adjuvanted and adjuvanted wiv formulations, at an ha dose of µg, induced average hi titers above log which was previously found to be required for protection against lethal dose of influenza virus [ ] . with respect to mucosal immune responses, lung igy titers were mainly enhanced by blp whereas lung iga titers were augmented by both blp and advax. though both blp and advax were found to enhance either systemic or mucosal immune responses or both, the observed discrepancies might be due to the dose of blp ( µg) and advax ( µg) used in this study. although doses of µg of blp and µg of advax were found to be sufficiently high to boost both systemic and mucosal immune responses in mice, however, these doses might be too low for chickens with a x higher body weight. this might have resulted in the adjuvant dose to be a limiting factor in the active administration study. the active administration study revealed a slight but significantly higher effect of both blp and advax adjuvants, in comparison to nonadjuvanted wiv formulations. hence, for the passive administration study, the relative dose of blp was increased to double, i.e µg/mg of sfd powder while keeping the dose of advax similar. since advax particles consisted of wt% of the sfd powder formulation, enhancing the dose of advax further would most likely have compromised the integrity of the powder particles. moreover, a pattern of advax being effective after one and blp after two immunizations could also be determined in the active administration study. therefore, one of the groups of passively administered animals inhaled advax-adjuvanted wiv for the first immunization and blp-adjuvanted wiv for the second immunization. in our previous study, passive inhalation led to in-efficient delivery of influenza vaccine powders to the lungs of chickens and thus provided only partial protection [ ] . hence, for this study, we aimed for complete protection against lethal influenza viral challenge. this was achieved by enhancing vaccine powder delivery to the lungs: by increasing the vaccine concentration (by reducing the size of inhalation box), by increasing the exposure time, and by the use of adjuvants. compared to our previous study, optimization of the vaccine concentration and exposure time resulted in an increment of ∼ fold in the theoretical vaccine dose (this study × µg ha/animal; previous study × µg ha/animal) [ ] . using this optimized set-up, passive inhalation of both non-adjuvanted and adjuvanted wiv formulations not only protected chickens against clinical signs after hpaiv challenge, but also almost completely prevented challenge virus shedding. these results are a significant improvement over our previous study in which chickens showed partial protection and challenge virus shedding even after three immunizations. though the immunological mechanism that governed complete protection in chickens by passive administration of non-adjuvanted and adjuvanted wiv formulations still needs to be investigated, the shedding data indicate that mucosal immune responses were high enough to prevent production of substantial amounts of virus. in addition to mucosal immune responses, an important class of immune responses are those elicited systemically. at both time points, blp and advax/blp adjuvanted wiv formulations, were found to induce higher systemic immune responses than non-adjuvanted or advax-adjuvanted wiv preparation. in addition, we found out that, either of the two adjuvants i.e. advax or blp would be appropriate for first immunization, however, for second immunization, blp was vital. although these adjuvanted wiv formulations elicited significantly higher immune responses than non-adjuvanted wiv formulation, the fact that even the non-adjuvanted wiv formulations provided complete protection, suggests that the use of an adjuvant was not critical for protection via passive inhalation, but it might add to dose-sparing of influenza vaccines for future passive inhalation studies. in conclusion, our results show that, vaccination by passive inhalation of dry influenza vaccine powders is suitable to induce protective immunity in chickens against highly pathogenic avian influenza virus. it not only has the potential to completely protect chickens from morbidity and mortality, and to prevent the virus from spreading, but also seems to be a feasible option for mass vaccination of chickens. the challenge now remains to translate passive inhalation vaccination from the box to real-world field settings. np is affiliated with vaxine pty ltd, which has commercial interests in advax adjuvant. the other authors declare no conflict of interest. this research was funded by the dutch ministry of economic affairs (wot- - - ; kb- - - ). probable limited person-to-person transmission of highly pathogenic avian influenza a (h n ) virus in china human infections with the emerging avian influenza a h n virus from wet market poultry: clinical analysis and characterisation of viral genome limited human-to-human transmission of avian influenza a(h n ) virus avian influenza a virus (h n ) epidemic in the netherlands in : course of the epidemic and effectiveness of control measures influenza vaccines and vaccination strategies in birds a lack of antibody formation against inactivated influenza virus after aerosol vaccination in presence or absence of adjuvantia preservation of the immunogenicity of dry-powder influenza h n whole inactivated virus vaccine at elevated 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delivery of influenza subunit vaccine formulated with gem particles as an adjuvant neonatal mucosal immunization with a non-living, nongenetically modified lactococcus lactis vaccine carrier induces systemic and local th -type immunity and protects against lethal bacterial infection advax™, a novel microcrystalline polysaccharide particle engineered from delta inulin, provides robust adjuvant potency together with tolerability and safety delta inulin: a novel, immunologically active, stable packing structure comprising β-d-[ → ] poly(fructo-furanosyl) αd-glucose polymers a novel hepatitis b vaccine containing advax™, a polysaccharide adjuvant derived from delta inulin, induces robust humoral and cellular immunity with minimal reactogenicity in preclinical testing severe acute respiratory syndrome-associated coronavirus vaccines formulated with delta inulin adjuvants provide enhanced protection while ameliorating lung eosinophilic immunopathology advax, a polysaccharide adjuvant derived from delta inulin, provides improved influenza vaccine protection through broadbased enhancement of adaptive immune responses a gold glyco-nanoparticle carrying a listeriolysin o peptide and formulated with advax™ delta inulin adjuvant induces robust t-cell protection against listeria infection delta inulin polysaccharide adjuvant enhances the ability of split-virion h n vaccine to protect against lethal challenge in ferrets safety and immunogenicity of a delta inulin-adjuvanted inactivated japanese encephalitis virus vaccine in pregnant mares and foals randomized clinical trial of immunogenicity and safety of a recombinant h n / pandemic influenza vaccine containing advax™ polysaccharide adjuvant immunogenicity and safety of advax™, a novel polysaccharide adjuvant based on delta inulin, when formulated with hepatitis b surface antigen; a randomized controlled phase study development of a dried influenza whole inactivated virus vaccine for pulmonary immunization pulmonary delivery of influenza vaccine formulations in cotton rats: site of deposition plays a minor role in the protective efficacy against clinical isolate of h n pdm virus development of a lavage procedure to collect lung secretions from chickens for evaluating respiratory humoral immunity respiratory and heart rates of birds at rest lung volume of meat-type chickens the effect of inoculation dose of a highly pathogenic avian influenza virus strain h n on the infectiousness of chickens genetic versus antigenic differences among highly pathogenic h n avian influenza a viruses: consequences for vaccine strain selection transmission of highly pathogenic avian influenza h n virus in pekin ducks is significantly reduced by a genetically distant h n vaccine enhancement of the immunogenicity and protective efficacy of a mucosal influenza subunit vaccine by the saponin adjuvant gpi- induction of heterosubtypic cross-protection against influenza by a whole inactivated virus vaccine: the role of viral membrane fusion activity an aerosolized fluorescent microsphere technique for evaluating particle deposition in the avian respiratory tract deposition of differently sized airborne microspheres in the respiratory tract of chickens induction of respiratory immune responses in the chicken; implications for development of mucosal avian influenza virus vaccines immunogenicity of low-ph treated whole viral influenza vaccine an improved inactivated influenza vaccine with enhanced cross protection inactivated influenza vaccine adjuvanted with bacterium-like particles induce systemic and mucosal influenza a virus specific tcell and b-cell responses after nasal administration in a tlr dependent fashion enhanced pulmonary immunization with aerosolized inactivated influenza vaccine containing delta inulin adjuvant bacterium-like particles supplemented with inactivated influenza antigen induce cross-protective influenza-specific antibody responses through intranasal administration advax augments b and t cell responses upon influenza vaccination via the respiratory tract and enables complete protection of mice against lethal influenza virus challenge development of advax adjuvant was supported by the national institute of allergy and infectious diseases of the national institutes of health, under contracts hhsn c, hhsn c and u ai . we thank jacqueline de vries-idema for providing the inactivated vaccine, dr. marc stuart for transmission electron microscopy pictures and anko eissens for scanning electron microscopy pictures. also, we would like to thank the animal technicians from wbvr for the execution of the animal experiments, and diana van zoelen and cynthia baars for performing some laboratory tests. the content is solely the responsibility of the authors and the funders played no part in the writing of this paper. supplementary data to this article can be found online at https:// doi.org/ . /j.ejpb. . . . key: cord- -umvi veu authors: subramanian, subbaya; steer, clifford j. title: special issue: microrna regulation in health and disease date: - - journal: genes (basel) doi: . /genes sha: doc_id: cord_uid: umvi veu our understanding of non-coding rna has significantly changed based on recent advances in genomics and molecular biology, and their role is recognized to include far more than a link between the sequence of dna and synthesized proteins [...]. our understanding of non-coding rna has significantly changed based on recent advances in genomics and molecular biology, and their role is recognized to include far more than a link between the sequence of dna and synthesized proteins. micrornas (mirnas) are small regulatory rnas that play a crucial role in posttranscriptional gene regulation. greater than mirnas have been identified and catalogued in humans and many of them are conserved in other species [ ] . mirnas are implicated in almost every facet of fundamental cellular functions including development, senescence and disease. the past decade has experienced a remarkable increase in the understanding of mirna biogenesis, their target genes, mirna biomarkers and potential therapeutics for a growing number of disease conditions. rna-based markers and therapeutics have a potentially significant clinical impact, and many of the mirna-based therapies are at various stages of application and human clinical trial [ ] . as background, non-coding rnas are divided into (i) transcription rnas (including both trna and rrna); (ii) small rnas, which are further subdivided into sirnas, mirnas, snornas, and snrnas; and (iii) most recently, long non-coding rnas, which are now know to transcribe short peptides [ ] . micrornas are single-stranded non-coding rnas that are typically - nucleotides (nts) in length and are best known for their role in the post-transcriptional regulation of gene expression. they are the most abundant class of small endogenous non-protein coding rnas, and make up one of the largest well-conserved gene families found among viruses, plants and animals. the majority of mirna sequences in humans are typically transcribed by introns of non-coding and coding transcripts, with few transcribed by exonic regions. microrna genes are typically transcribed by polymerase ii or iii and generate primary mirnas (pri-mirnas), which can contain sequences for multiple mirnas and be hundreds of nts in length. these structures are then processed and cleaved by drosha-dgcr complex, resulting in the formation of a hairpin-shaped stem-loop structure, which is known as the precursor mirna (pre-mirna) and typically around nts in length. the pre-mirna is exported outside of the nucleus primarily by exportin . further processing takes place in the cytoplasm by dicer -tarbp , which is an rnase iii enzyme, resulting in a two-stranded duplex of mirna-mirna*. typically, it is - nt long, with one strand designated the guide strand and the other as the passenger strand. finally, the guide strand is incorporated into the rna-induced silencing complex (risc), which is a large multiprotein mirna ribonucleoprotein complex that is the effector compound in modulating target gene transcription. alternative pathways have been described that are drosha-dgcr independent as well as dicer-independent, and are likely to greatly advance our understanding of mirna biogenesis and involvement in conditions of health and disease. regulatory interactions between mirna and other noncoding rnas, including long noncoding rnas (lncrna), and circular rna (circrna) are now known to determine the cellular functional status and phenotype. micrornas use seed sequences ( - bases long) to bind microrna response elements (mres) located on their interacting partners, primarily at the 'utr of coding transcripts [ ] . however, it is possible that the frequency of mres in the entire transcriptome of a given cell contributes to the dynamic gene regulatory process by acting as a sponge for mature mirnas, thus regulating their functional availability. thus, the coding and noncoding transcripts sharing common mre sites compete with each other and define the gene expression profile of a given cell. these competing transcripts are collectively called competing endogenous rnas [ ] . thus, gene expression regulation is a complex process involving the dynamic interactions between mirna-mrna-lncrna-circrna. this complexity is increased multifold when these interacting partners are exchanged between cells via extracellular vesicles. recognizing this intricate system will significantly aid in our understanding of the health and disease process. we are beginning to study disease pathogenesis at the cellular, organ, and whole body levels; and the gut microbiota is increasingly recognized as a crucial player [ ] . it is time to acknowledge each of these players as they take center stage in maintaining homeostasis and the normal physiological functioning of an organism. there are still significant gaps in understanding the complex regulatory mechanisms of mirnas; however, the field is advancing rapidly. further, it has been shown that a number of nuclear receptors are involved in the transcriptional regulation of mirna expression, including the small heterodimer partner (shp) and farnesoid x receptor (fxr). in general, mirnas are detected as (i) extracellular circulating mirna bound to different lipoproteins; (ii) part of a non-membrane ribonucleoprotein complex associated with argonaut proteins; and (iii) contained in exosomes as extracellular vesicles, where they act as nano-sized transporters involved in the communication between neighboring cells. the recent finding that mirnas are also transported from one cell to another via tunneling nanotubes underscores their importance in maintaining communication among all cell types, including those associated with cancer. this special issue of genes, entitled "microrna regulation in health and disease" consists of a series of articles spanning the clinical realm from colorectal cancer to pulmonary fibrosis. however, we begin with a research article by liu et al. who reported for the first time the existence of complemented palindromic small rnas (cpsrnas) from sars coronavirus, and propose that cpsrnas and palindromic small rnas (psrnas) constitute a novel class of small rnas [ ] . such a discovery of cpsrnas could pave a way to find novel markers for pathogen detection and to reveal the mechanisms underlying infection or pathogenesis from a different perspective. in a study titled, "a two-cohort rna-seq study reveals changes in endometrial and blood mirnome in fertile and infertile women", rekker et al. compared mid-secretory phase samples between fertile and infertile women [ ] . the study revealed differentially expressed mirnas from the endometrium and one from blood samples. among the novel mirnas, chr _ was validated and showed upregulation in the mid-secretory endometrium. in addition to the novel findings, the authors confirmed the involvement of mir- and mir- family members in mid-secretory endometrial functions. hueso et al. elegantly showed in their article that an exonic switch regulates the differential accession of micrornas to the cd transcript in atherosclerosis progression [ ] . further, they proposed a new mechanism of mirna action, linked to a cryptic splicing site in the target-host gene, that would regulate the differential accession of mirnas to their cognate binding sites. li et al. studied the role of mirna- a- p in inhibiting c c myogenesis via targeting pik r and modulating pi k/akt signaling [ ] . their results showed that mir- a- p was elevated in aged muscles and dexamethasone (dex)-treated myotubes. the up-regulation of mir- a- p significantly reduced the diameters of myotubes accompanied by increased levels of muscular atrophy genes and decreased pi k/akt activities. finally, mir- a- p was demonstrated to directly bind to the '-utr of pik r , thus, repressing pi k/akt signaling. the microbiome appears to interact and perhaps influence an unlimited number of metabolic processes in health and disease. in their article, yuan et al. postulate that the altered nutrient composition and mirna expression in colorectal cancer (crc) microenvironment selectively exerts pressure on the surrounding microbiota, leading to alterations in its composition [ ] . further, the authors present a detailed overview of the current understanding of the role of mirnas in mediating host-microbiota interactions in crc. "single nucleotide polymorphisms in mir contribute to protection against non-hodgkin lymphoma (nhl) in caucasian populations" by bradshaw et al. is first to report a correlation between mirsnps in mir- and a reduced risk of nhl in caucasians [ ] . further, it is supported by significant snps in high linkage disequilibrium (ld) in a large european nhl genome-wide association study (gwas) meta-analysis. axmann et al. compared the mirna profiles in serum and lipoprotein particles of healthy individuals with those of patients with uremia [ ] . they observed a significant increase in levels of cellular mirna level using reconstituted high-density lipoprotein (hdl) particles artificially loaded with mirna, whereas incubation with native hdl particles yielded no measurable effect. based on the results, the authors concluded that there was no relevant effect of lipoprotein-particle-mediated mirna-transfer under in vivo conditions though the mirna profile of lipoprotein particles can be used as a diagnostic marker. mullenbrock et al., carried out an elegant systems analysis transcriptomic and proteomic study on the potential role of mirnas in pulmonary fibrosis [ ] . they specifically targeted fibroblasts and myofibroblasts as the key effector cells responsible for the excessive extracellular matrix (ecm) deposition and fibrosis progression in both idiopathic pulmonary fibrosis (ipf) and systemic sclerosis (ssc) patient lungs. the comprehensive analyses of mrna, mirna, and matrisome proteomic profiles in ipf and ssc lung fibroblasts revealed robust fibrotic signatures at both the gene and protein expression levels and identified novel fibrogenesis-associated mirnas whose aberrant downregulation in disease fibroblasts likely contributes to their fibrotic and ecm gene expression. somatostatin (sst) analogues were used to control the proliferation and symptoms of neuroendocrine tumors (nets) in an article by døssing et al., entitled "somatostatin analogue treatment primarily induce mirna expression changes and up-regulates growth inhibitory mir- and mir- a in neuroendocrine cells" [ ] . two mirnas which were highly induced by sst analogues, mir- and mir- a, were shown to inhibit the proliferation of nci-h and cndt cells. sst analogues also produced a general up-regulation of the let- family members. sst analogues controlled and induced distinct mirna expression patterns among which mir- and mir- a both have growth inhibitory properties. as fda-approved small rna drugs begin to enter the arena of clinical medicine, it is critical to expand both preclinical and clinical research studies for mirnas. a growing number of reports suggest a significant utility of mirnas as biomarkers for pathogenic conditions, modulators of drug resistance, and/or as drugs for medical intervention in almost all human health conditions. the pleiotropic nature of this class of nonprotein-coding rnas makes them particularly attractive drug targets for diseases with a multifactorial origin and few, if any, available treatments. the landscape of both diagnostic and interventional medicine will arguably continue to evolve as candidate mirnas pass successfully through phase and clinical trials. in this special issue of genes, we provide a series of articles that highlight micrornas as diagnostic, predictive and therapeutic agents for human disease. the development of bioinformatics programs to identify mirna-binding sites in target genes and their corresponding biological pathways, along with an expanding platform of in vitro and in vivo preclinical research models, has propelled mirnas into clinical medicine. the first sirna human trial was conducted in and, in , the first sirna drug was approved, paving the way for a class of mirna transcripts whose active investigation began only a little more than years ago. the future of human mirna clinical trials is absolutely guaranteed and that time has arrived. the development of mirna diagnostics and therapeutics is an exciting and potentially new frontier in treating diseases for which few treatment options exist. we believe and hope that this special issue of genes will be an important resource for a wide variety of audiences, including students at all levels, and established investigators who are interested in contributing to the remarkable and ever-expanding field of micrornas in health and disease. the authors declare no conflicts of interest. the potential for microrna therapeutics and clinical research a network of noncoding regulatory rnas acts in the mammalian brain the multilayered complexity of cerna crosstalk and competition cerna cross-talk in cancer: when ce-bling rivalries go awry microrna-mediated tumor-microbiota metabolic interactions in colorectal cancer velthut-meikas, a. a two-cohort rna-seq study reveals changes in endometrial and blood mirnome in fertile and infertile women an exonic switch regulates differential accession of micrornas to the cd transcript in atherosclerosis progression microrna- a- p inhibited c c myogenesis via targeting pik r and modulating the pi k/akt signaling host-microrna-microbiota interactions in colorectal cancer single nucleotide polymorphisms in mir contribute to protection against non-hodgkin lymphoma (nhl) in caucasian populations serum and lipoprotein particle mirna profile in uremia patients systems analysis of transcriptomic and proteomic profiles identifies novel regulation of fibrotic programs by mirnas in pulmonary fibrosis fibroblasts somatostatin analogue treatment primarily induce mirna expression changes and up-regulates growth inhibitory mir- and mir- a in neuroendocrine cells key: cord- - sotgds authors: noll, kelsey e.; ferris, martin t.; heise, mark t. title: the collaborative cross: a systems genetics resource for studying host-pathogen interactions date: - - journal: cell host & microbe doi: . /j.chom. . . sha: doc_id: cord_uid: sotgds host genetic variation has a major impact on infectious disease susceptibility. the study of pathogen resistance genes, largely aided by mouse models, has significantly advanced our understanding of infectious disease pathogenesis. the collaborative cross (cc), a newly developed multi-parental mouse genetic reference population, serves as a tractable model system to study how pathogens interact with genetically diverse populations. in this review, we summarize progress utilizing the cc as a platform to develop improved models of pathogen-induced disease and to map polymorphic host response loci associated with variation in susceptibility to pathogens. pathogens often cause variable disease outcomes across infected individuals, ranging from asymptomatic infection to severe or fatal disease. multiple factors influence an individual's susceptibility to a pathogen, including variation in pathogen dose or virulence as well as host age, prior immune experience, microbiome/coinfection, and genetics. a role for host genetics in pathogen susceptibility is supported both by twin studies (kallmann and reisner, ) and by evidence of pathogen-driven selective pressure on the evolution of the human immune system (cagliani and sironi, ) . human genetic studies have demonstrated a role for host genetics in pathogen susceptibility and identified polymorphic genetic loci and genes associated with variation in susceptibility to specific pathogens (e.g., hiv) (mclaren et al., ) , classes of pathogens (e.g., mycobacteria) (bustamante et al., ) , and more generalized primary immunodeficiencies (pids) that result in severe immune defects (chapman and hill, ) . this information has enhanced our understanding of how host genetic variation affects pathogen susceptibility and how specific genes regulate human immunity and host-pathogen interactions. while advances in human genetic analysis have led to the identification of several host genes that regulate pathogen susceptibility in humans (newport and finan, ) , much of our understanding of how specific genes affect infectious disease pathogenesis in mammals has come from studies using mouse models (masopust et al., ) . this is in large part due to the existence of inbred mouse strains as well as the vast amount of immunological and molecular genetic tools available for the mouse, including the early generation of the mouse reference genome. inbred strains, in which each mouse is essentially genetically identical, allow control of host genetics as well as other factors that can confound human studies, such as pathogen dose, nutrition, and prior immune exposure. additionally, targeted knockout technology has been used in the mouse genome for almost years (bouabe and okkenhaug, ) . while advances in gene editing techniques (e.g., crispr) have recently extended these approaches to other species, genetically modified mice have been an unparalleled resource for studying the role of specific genes in the response to a variety of pathogens (masopust et al., ) . furthermore, the use of classical genetic crossing approaches (e.g., intercrosses), as well as reproducible mouse genetic reference populations (grps), has facilitated the discovery of key regulators of host immunity and pathogen susceptibility such as the innate immune receptor tlr , oligoadenylate-synthetase oas b, and divalent metal transporter nramp (perelygin et al., ; qureshi et al., ; vidal et al., ) . the recent development of multiparental grps, such as the collaborative cross (cc), promises to further extend genetic mapping capabilities, while also leading to the development of mouse models that more accurately reproduce the genetic diversity and phenotypic range seen in human populations (churchill et al., ) . this review will summarize recent advances in the use of mouse grps, with an emphasis on the use of the cc for studying infectious diseases and immunity. the impact of host genetics on pathogen susceptibility can be illustrated by genes exhibiting large effects such as the chemokine receptor ccr and fucosyltransferase (fut ), for which specific variants have a major impact on resistance to viral infection (hiv and norovirus, respectively) (quillent et al., ; thorven et al., ) . likewise, deleterious mutations in immune genes can result in pids, which cause enhanced susceptibility to specific or entire classes of pathogens (carneiro-sampaio and coutinho, ) . to date, over genetically driven pids have been identified in humans (picard et al., ) . the identification of these monogenic resistance and susceptibility genes has enhanced our understanding of human immunity and hostpathogen interactions while contributing to the development of antiviral therapies, such as hiv entry inhibitors, as well as gene therapy and immune replacement therapies (collins and thrasher, ; lenardo et al., ) . though genes of large effect can impact human health, inheritance patterns suggest that susceptibility to infectious disease is largely polygenic (driven by tens to thousands of variants of smaller effect) (hill, ) . this is supported by results from genome-wide association studies (gwas) designed to map loci associated with variation in pathogen susceptibility and vaccine response. for example, gwas of hiv viral load have shown that mutations in the hiv co-receptor ccr and variants in human leukocyte antigen (hla) explain approximately % of the total variance, with an additional % of heritable variance explained by additive effects of other genes (mclaren et al., ) . gwas on malaria susceptibility have identified variants in multiple genes including the calcium atpase atp b , the glucose phosphate dehydrogenase gp d, and hlas, as well as confirming previously identified variants in hemoglobin genes (sickle cell and thalassemia) (hedrick, ; timmann et al., ) . multiple loci have been associated with clearance of hepatitis c virus, including the cytokine il- b and hla genes (duggal et al., ) , and over loci have been associated with leprosy susceptibility (wang et al., ) . while gwas have been applied to and enhanced our understanding of infectious diseases, these studies explain only a fraction of the heritable variation in pathogen susceptibility (hill, ) . this is partly attributable to factors such as small cohort sizes, ethnic striations, and lack of replicates, which limit detection and interpretation of results (du et al., ) . rare genetic variants are unlikely to be detected by gwas, due to both lack of power and lack of tagging on conventional genotyping arrays (auer and lettre, ) . a specific concern for gwas in infectious diseases is that studies can be confounded by pathogen genetics, pathogen dose, and other environmental factors. furthermore, validation and mechanistic analysis can be difficult in humans due to factors such as lack of replicates and inability to routinely access many affected tissue types. for these reasons, the use of appropriate animal models has been a critical resource for identifying and studying pathogen susceptibility genes. while a variety of model genetic systems have been used to analyze host-pathogen interactions (e.g., caenorhabditis elegans, danio rerio, and drosophila melanogaster) (allen and neely, ; dionne and schneider, ; marsh and may, ) , the laboratory mouse (mus musculus) provides the most robust mammalian system for dissecting genetic regulation of immune responses in human-relevant diseases. mice are relatively inexpensive, can be easily controlled in diet and environment, and reproduce quickly. a wide variety of well-characterized mouse immunological reagents are available, as well as genetic tools such as reproducible inbred strains, gene-specific knockouts, genome-wide mutagenesis strategies (e.g., n-ethyl-n-nitrosourea [enu] and crispr/cas ), and transgenic technologies. these systems facilitate the identification and mechanistic characterization of specific host genes that affect disease pathogenesis and/or immunity (pelletier et al., ; takaki et al., ) . though over commercially available inbred mouse strains and outbred stocks are available (beck et al., ) , the majority of research in infectious disease and immunology is conducted in a limited set of strains. one of the major reasons for the focus on specific strains is the need to control for genetic background when analyzing gene-specific knockouts or performing largescale mutagenesis screens. large-scale initiatives such as the international mouse phenotyping consortium (impc) depend upon the ability to control background strain to analyze the impact of specific gene deletions or mutations on a wide range of developmental and immune phenotypes (muñ oz-fuentes et al., ) , and have thus generated these mutants on common backgrounds using largely identical procedures. c bl/ j is the most commonly used inbred mouse strain; it is the source of the mouse reference genome and the genetic background for the majority of knockout mice. other strains, such as the balb/c substrains, also have a rich history of use in immunology and infectious disease research (watanabe et al., ) . while the ongoing use of such strains is driven by the need to compare results across studies, it is important to note that every inbred strain has a unique set of genetic features, and thus no one strain is representative of all mice, let alone of a genetically complex population such as humans. although genetically modified mice have been an essential resource for gene discovery and mechanistic analysis, there has been a growing acknowledgment in the field that knockout approaches have limitations. approximately % of genes cannot be knocked out because they are developmentally essential (nih, ) . similarly, other knockouts have no observable phenotype, due to genetic redundancy and/or lack of robustness in the phenotyping pipeline (barbaric et al., ) . in other cases, knockouts have disparate phenotypes on different genetic backgrounds due to gene-gene interactions (doetschman, ) . furthermore, gene knockouts result in null alleles (complete loss of gene function), whereas naturally occurring genetic variants are most likely to be hypomorphic or hypermorphic alleles (partial loss or gain of gene function; alterations in timing and magnitude of expression). lastly, while single-gene approaches are straightforward, they are isolated systems that do not model the simultaneous contribution of variants in multiple pathways, as would most likely be observed in a natural system. thus, to better model and understand the role of genes in complex phenotypes such as immunity and infectious disease, it is pertinent to consider complex genetic interactions across diverse genetic backgrounds. in contrast to the approaches discussed above, researchers have also studied the role of natural genetic variants by leveraging differential phenotypes across inbred strains, using classical genetic breeding strategies to identify pathogen susceptibility genes such as oas b, immune cell activating receptor ly h, and large interferon-induced gtpase mx (casanova et al., ) . in contrast to mapping crosses between stocks (e.g., f crosses), where each mouse is unique and therefore ephemeral, grps serve as reproducible models to study the role of genetic diversity. grps consist of anywhere from a few to hundreds of inbred lines derived and split from a common ancestral population, and each line has a fixed and known genome that can be replicated indefinitely. the existence of reproducible yet genetically diverse individual mouse strains facilitates study designs that involve case versus control, genotype by environment interactions (gxe), and phenotypic penetrance or threshold traits. these populations further facilitate the integration of phenotypic, genotypic, and molecular data for systems-level data analysis while also allowing retrospective integration of new layers of data. the first recombinant inbred panel, the cxb (balb/cj c bl/ j), was created by donald bailey to facilitate mapping of the mhc locus (bailey, ) . subsequent biparental recombinant inbred panels include the axb/bxa (a/j c bl/ j, c bl/ j a/j), the bxh (c bl/ j c h/hej), and the bxd (c bl/ j dba/ j), which is the largest and most extensively used of these panels (peirce et al., ) . though originally developed to study monogenic traits, use of these panels has been extended to study genetically complex traits such as susceptibility to murine plasmodia (hoffmann et al., ) , murine leukemia virus (panoutsakopoulou et al., ) , group a streptococcus (chella krishnan et al., ) , and influenza a virus (iav) (nedelko et al., ) . as results from these panels accumulated, there was a community-wide recognition that larger and more genetically diverse grps would improve the ability to genetically dissect more complex traits (threadgill et al., ) . based on results from the early grps as well as genetic mapping studies, the complex trait consortium proposed to create a second-generation, multi-parent advanced intercross grp to promote complex trait genetic mapping and systems genetics research (threadgill et al., ) . in contrast to single-gene approaches, systems genetics considers phenotypes in the context of global genetic variation as well as intermediate molecular factors such as gene expression, protein abundance, and environmental interactions. the resource ultimately derived from this proposal was the cc, a large panel of recombinant inbred strains derived from eight genetically diverse founder strains. the cc was designed to facilitate systems genetics approaches with improved resolution and higher statistical power compared to traditional grps, to expand the pool of genetic variation segregating in the population, and to bridge the analysis of genetic and molecular networks across diverse phenotypes (churchill et al., ) . of the eight cc founder strains, five are classical laboratory strains chosen for their rich history of use in mouse genetics (c bl/ j and s /svimj) or their relevance as models of common diseases including cancer, type diabetes, metabolic syndrome, and obesity (a/j, nod/shiltj, and nzo/hiltj). the other three cc founders are wild-derived inbred strains, which represent the three major subspecies of mus musculus: casteneus (cast/eij), musculus (pwk/phj), and domesticus (wsb/eij), and introduce much of the genetic diversity into the cc. these eight founder strains were interbred in a funnel breeding scheme to produce recombinant mice with genomic contributions from each founder, which were then bred to homozygosity to produce recombinant inbred (ri) cc strains (figure ). although the initial goal was to produce , cc strains, only around cc strains survived the inbreeding process due to high rates of infertility and breeding issues, largely caused by genomic incompatibility introduced by the wild-derived strains. extinction of cc strains during the inbreeding process inspired the creation of the diversity outbred (do) panel, an outbred population derived from a set of incipient cc strains (churchill et al., ) . the do now serves as its own important genetic resource that has proven useful for high-resolution genetic mapping as well as exploration of phenotypic diversity ( while the do has only been used in a limited number of infectious disease studies (mchugh et al., ; niazi et al., ) , the do and cc should be viewed as complementary resources for studying how genetic variation affects pathogen susceptibility and immunity. while classical mouse grps remain actively used, and in some ways provide a more straightforward analytical path than the cc due to reduced complexity, several aspects of the cc make it more attractive for discovery-based research. the cc captures approximately % of common genetic variants described within laboratory mice, with up to eight distinct haplotypes at any region of the genome (roberts et al., ) . due to higher genetic diversity with novel allele combinations and epistatic interactions, the cc yields more phenotypic variation and extreme phenotypes than classical grps. the selection of founder strains and the breeding design, as well as higher levels of recombination, result in lower levels of long-range disequilibrium and population structure than classical grps, allowing for higher resolution quantitative trait locus (qtl) mapping (iraqi et al., ; threadgill et al., ) . genetic variation is also more uniformly distributed across the genome, removing several genomic ''blind spots'' that interfere with mapping in classical inbred strains and grps (roberts et al., ) . although the cc has only been available for a relatively short period of time, it has been utilized across a number of disciplines, including response to environmental toxins (venkatratnam et al., ) , nutrition (schoenrock et al., ) , body composition (mathes et al., ) , and immunity and pathogenesis (discussed further here). importantly, these studies have highlighted previously uncharacterized phenotypic diversity, including expansion of the phenotypic range, disassociation of traits previously thought to be connected, and identification of completely novel phenotypes (srivastava et al., ) . the cc is an especially useful resource for studying the role of host genetics in hostpathogen interactions because it allows for control of many of the factors described above that confound infectious disease studies in humans. over the past decade, a variety of studies utilizing the cc, including non-fully inbred incipient cc lines (commonly termed the pre-cc) and derived cc populations (such as cc-f s), have been used to probe the role of host genetics in the response to infectious disease. here we will discuss the current work in this field, including studies focusing on phenotypic characterization (e.g., model development and molecular signatures of disease response) and genetic mapping of disease trait-associated qtl. there is an ongoing debate over the validity of mouse models in recapitulating human disease (seok et al., ; takao and miyakawa, ) . while the utility of the mouse is undeniable in the infectious disease field, it is equally true that individual classical inbred strains do not fully recapitulate human disease and most models recapitulate only a portion of the disease phenotypes observed in humans. in an effort to improve model fidelity to human disease, a number of researchers have turned to the cc to develop new mouse models. genetically diverse cc strains show a range of responses to pathogens, from variation in disease severity to completely novel disease phenotypes. this phenotypic variability better mimics the diversity of disease presentation across the human population, thus providing a more comprehensive platform to develop newer, more representative mouse models. ebola virus ebola virus (ebov) causes severe, and often lethal, hemorrhagic fever in humans. while classical inbred strains are susceptible to mouse-adapted ebov (ma-ebov), they do not display many of the characteristic symptoms observed in severe human cases such as coagulopathy, hemorrhagic manifestations, and rash (st claire et al., ) . rasmussen et al. infected a panel of f crosses between cc strains (cc-f s) following an initial assessment in founder strains (rasmussen et al., ) . they observed significant phenotypic variation following ma-ebov infection, from high resistance to complete lethality, and covering a spectrum of different pathologies similar to the range of clinical disease observed in humans. importantly, some strains presented with severe hemorrhagic disease and liver damage, consistent with human ebov disease. follow-up studies with representative susceptible and resistant strains found differential transcriptional responses, highlighting the central transcriptional regulatory gene tek, for which haplotypes across these cc-f s correlated with weight loss and mortality following ma-ebov infection. west nile virus (wnv), a neuroinvasive flavivirus, induces a diverse spectrum of immune responses and clinical outcomes in humans that classical mouse models do not fully recapitulate (graham et al., ) . graham et al. assessed wnv susceptibility in a panel of cc-f crosses that were heterozygous for the h- b mhc haplotype, which allowed quantification of wnv-specific t cells with the same mhc tetramer. wnv outcome ranged from highly resistant to highly susceptible, including a novel outcome group in which mice were outwardly asymptomatic despite higher viral titers and immunopathology in the brain. graham et al. followed up on one unique cc-f ((cc / geniunc cc /geniunc)-f ), in which half of the mice that survived infection displayed sustained weight loss and persistent viral loads in the cns. they found that these mice exhibited a rapid early innate inflammatory response characterized by increased early expression of ifn-b and the interferonstimulated gene ifn , as well as high early viral titers and the ability to control, but not clear, viral replication in the cns figure . representative cc funnels cc founders were bred in funnel breeding schemes to produce progeny with genetic contributions from each of the eight founders, at which point they were inbred for generations until reaching near homozygosity. many different funnels were set up, each to produce a unique cc ri line. (graham et al., ) . additionally, (cc /geniunc cc / geniunc)-f mice had a unique immunoregulatory signature, with a high number of t regulatory cells in both infected and uninfected animals and a distinctive post-infection gene expression profile that indicated reduced cytolytic ability. the initial graham et al. study (graham et al., ) found that wnv response largely tracked with the haplotype of oas b, a known flavivirus resistance gene, although there was still considerable disease variation within oas b haplotype groups. green et al. continued to dissect the impact of oas b on the innate immune transcriptional landscape following wnv infection (green et al., ) by performing genome-wide micro-array analysis across multiple time points post-infection in seven cc-f s carrying different combinations of functional (wild-derived) or nonfunctional (classical inbred strain-derived) oas b haplotypes. transcriptional correlation analysis of differentially expressed immune genes across cc-f s yielded three gene clusters, and pathway analysis led to the construction of innate immune regulatory networks associated with wnv infection between oas b haplotype groups. mycobacterium tuberculosis (mtb) infection varies broadly in disease presentation and pathology in humans. likewise, the efficacy of the standard tb vaccine, the live-attenuated bcg, is variable across individuals (mangtani et al., ) . smith et al. studied mtb pathogenesis and bcg vaccine efficacy in the cc founders and three ri strains that were selected based on a pilot study assessing mtb susceptibility (smith et al., ) . the authors observed a wide range of susceptibility to mtb and dissociation of previously associated phenotypes such as bacterial burden, immune cell recruitment, and tissue damage. when the bcg vaccine was administered prior to mtb challenge, vaccine efficacy was low overall and not correlated with susceptibility to primary infection. while bcg vaccination reduced mtb burden in four strains, there was an increased bacterial burden in nzo/hlltj mice, a model for obesity and type diabetes. in addition to highlighting the importance of host genetics on mtb susceptibility and bcg-induced vaccine responses, the study illustrates the potential impact of comorbidities, such as type diabetes in nzo/hlltj mice, on vaccine responses in genetically diverse populations. maurizio et al. analyzed the heritability of iav-induced disease using cc-f s as well as f crosses between founder strains (maurizio et al., ) . these studies determined that iavinduced weight loss was % heritable at day post-infection, and that this heritability was mostly composed of additive effects largely attributable to the haplotype of mx , a polymorphic largeeffect anti-iav gene. the genetic dominance of the protective mx haplotype varied depending on subspecies origin, with the m. musculus musculus allele acting dominantly and the cast/eij allele, identified by ferris et al. ( ) , acting additively. consistent with ferris et al. (discussed below) , these authors determined that when controlling for mx , non-mx heritable effects still accounted for % of the phenotypic variation, and this effect was consistent across founder diallel, pre-cc, and cc-f populations. human studies have identified blood transcriptomic and proteomic signatures associated with iav infection that could be used to predict patient outcome. elbahesh and schughart observed that gene signatures from iav-infected humans were reproduced in the peripheral blood of cc founders (elbahesh and schughart, ) . in a follow-up study, kollmus et al. characterized the transcriptional response to iav in the peripheral blood of eleven cc strains (kollmus et al., ) . though both human and mouse datasets were globally heterogeneous, the cc dataset showed that genetic background strongly influenced gene expression, highlighting the importance of genetics in driving the immune response. for the most differentially expressed genes, the transcriptional responses in mice and humans were largely similar, demonstrating the utility of the cc in recapitulating iav-induced host response in humans. xiong et al. characterized transcriptomic variation in response to severe acute respiratory syndrome coronavirus (sars-cov) and iav across the eight cc founder strains (xiong et al., ) . differential gene expression analysis at days and post sars-cov or iav infection showed significant differences driven by mouse strain, infection status, and time point. genes with strain-specific differential expression patterns were largely enriched for immune pathways, while more generic differential expression patterns were enriched for basic biological functions. the study also noted the presence of strain-specific isoforms and novel transcripts not present in the c bl/ j reference annotation. pseudomonas aeruginosa is an opportunistic bacterial pathogen with variable clinical outcomes across susceptible individuals. human studies, including twin studies as well as association studies, have identified multiple genes associated with susceptibility to p. aeruginosa. lorè et al. studied p. aeruginosa infection in pre-cc lines, focusing on survival time and early body weight change, and observed a large amount of variation in these phenotypes (lorè et al., ) . genetic mapping of disease response in the cc as described above, the cc was initially envisioned as a genetic mapping population with approximately , strains (threadgill et al., ) . while the number of available cc ri strains is closer to , this population has proven to be sufficient for successful qtl mapping. mapping studies have identified qtls spanning the genome that are driven by variants from all eight cc founders and show very little overlap across pathogens (figure ; table ; discussed below). overall, infectious disease phenotypes mapped in the cc show a broad range in overall heritability (the proportion of population-wide variation explained by differences between strains) as well as effect sizes (the proportion of population-wide variation explained by specific loci). across the studies described in table , reported estimates for heritability range from % to over %, while reported estimates for effect size range from % (for a qtl of small effect influencing sars-cov viral titer) to over % (for a large effect qtl containing the gene mx on influenza-induced weight loss). authors have narrowed many of these qtls to lists of candidate genes, and a small number of these candidates have been subsequently validated. to date, most publications have highlighted qtls mapped by phenotyping large panels of cc strains; however, this is not the only successful strategy. as mentioned above, some cc strains present with unique disease phenotypes, which may be driven by complex genetic regulatory networks involving multiple loci with epistatic interactions. when one or a few cc strains possess the combination of genetic variants needed to manifest the novel phenotype, population-wide studies may not possess sufficient mapping power. in these cases, researchers have successfully used more traditional f or backcross approaches to identify causal genes (rogala et al., ) . in this section, we will highlight results of both population-wide and intercrossmediated cc studies of relevance to infectious disease research. the first host-pathogen study in the cc examined susceptibility to the fungus aspergillus fumigatus, which is pathogenic in immunocompromised individuals. durrant et al. found that survival time varied between days and over days post-infection in pre-cc lines (durrant et al., ) . the authors mapped multiple genome-wide significant qtls for survival time that were largely driven by wild-derived founder haplotypes. candidate genes were identified using merge analysis, a procedure that compares the pattern of sequence variants (e.g., snps or indels) to the pattern of haplotype effects (impact of each founder strain haplotype on the phenotype) observed under the qtl (yalcin et al., ) . the application of merge analysis to the cc enables more effective gene refinement compared to studies in classic biparental grps, where the presence of only two haplotypes means that every genetic variant in a locus is a putative candidate causal variant. merge analysis supported the candidate gene irf , an interferon regulatory transcriptional factor gene, under the most significant qtl; however, for the remaining qtls there was no strong concordance between a priori and merge-supported candidates. klebsiella pneumoniae vered et al. challenged pre-cc lines with k. pneuomonia, which causes severe pneumonia and sepsis in immunocompromised individuals (vered et al., ) . the pre-cc mice displayed broad and heritable variation in survival time exceeding that observed in classic inbred strains, as well as dissociation of survival time from temperature or body weight changes. the study identified one qtl at day post-infection and two at day post-infection. the authors used merge analysis to refine candidate genes; however, they found that the best merge candidates showed simpler allele effect patterns than the more complex haplotype effects observed under the qtl. the strongest candidate genes identified were ikbkap, a transcriptional elongation factor complex component, and actl a, actl b, and ctnnal , which are involved in cell adhesion and cytoskeletal structure. bottomly et al. analyzed a set of pre-cc lines infected with mouse-adapted influenza a/pr/ / and assessed weight loss, clinical score, and mortality through days post-infection (bottomly et al., ) . a subset of pre-cc lines, classified as high and low responders based on a composite metric of weight loss and histopathological scoring, were selected for transcriptomic profiling. over , transcripts were differentially expressed between susceptible and resistant classes, and mapping identified significant expression qtls (eqtls). twelve of the eqtls were validated in cc founder strains, and structural equation modeling was applied to these candidates to infer reactive expression networks underlying the transcriptional differences. in a companion study, ferris et al. further characterized variation in response to influenza a/pr/ / in pre-cc lines (ferris et al., ) . while disease-associated phenotypes (weight loss, viral titer, and inflammation) were largely correlated, subsets of these pre-cc mice showed breakdowns in these relationships, resulting in novel phenotypic combinations not observed in classical mouse strains (e.g., high weight loss with minimal inflammation). this study identified four significant qtls, including one strong qtl that explained approximately % of iav-induced weight loss, over the iav resistance gene mx . importantly, while mx is well studied, the authors identified a novel allelic variant derived from cast/eij that protects from weight loss but less efficiently inhibits viral replication compared to the functional m. musculus musculus-derived allele carried by nzo/hiltj and pwk/phj. consistent with ferris et al.'s finding of a uniquely functional cast/eij mx variant, leist et al. found that cast/eij mice have a unique response to an h n strain of iav (leist et al., ) . while the mx locus had a dominating effect in the ferris et al. study, large phenotypic variation occurred within mx classes, suggesting the presence of modifier alleles. three additional qtls were mapped when controlling for mx haplotype, corresponding to variation in weight loss, pulmonary edema, and airway neutrophils. notably, aside from the mx locus, none of the qtls identified by ferris et al. matched influenza susceptibility loci found in mapping studies conducted in the bxd population. (boon et al., ; nedelko et al., ) . these differences may reflect the more complex genetics of the cc compared to the bxd and/or differences in the strain of influenza or other experimental variables across studies. salmonella enterica salmonella enterica serovar typhimurium (s. typhimurium) causes typhoid fever, and previous mouse mapping studies have identified multiple loci associated with susceptibility (roy significance threshold levels: *< . , ** . , *** . . genome coordinates marked with refer to genome assembly gscv /mm ; otherwise, coordinates refer to mgscv /mm . et al., ; sebastiani et al., ) . to identify novel loci associated with s. typhimurium susceptibility, zhang et al. infected cc ri strains and measured bacterial load in the liver and spleen at days post-infection (zhang et al., ) . qtl scans for bacterial load mapped multiple significant or suggestive qtl (figure ; table ), which differed from those identified in studies conducted in other mapping populations (roy et al., ; sebastiani et al., ) . zhang et al. used merge analysis in combination with immune cell gene expression, gene ontology, and analysis of known protein functions to narrow the list of candidate genes under the two significant loci. they identified candidate genes cul a (ubiquitin ligase), lamp (lysosomal membrane protein), mcf l (guanine nucleotide exchange factor), pcid (trex- complex component involved in mrna nuclear export), and a high-priority candidate slc f , which has lactate dehydrogenase activity that may be important in the s. typhimurium pyruvate metabolism pathway. the study also noted that one strain, cc /geniunc, had extremely high bacterial loads, suggesting that it may be uniquely susceptible to s. typhimurium. sars-cov causes severe acute respiratory syndrome in humans, but the genes regulating these processes are poorly understood. gralinski et al. infected the cc founder strains and pre-cc lines with mouse-adapted sars-cov and studied disease through day post-infection (gralinski et al., ) . variation in weight loss and viral titer was significant in the cc founders and even broader in the pre-cc mice. similar to iav, phenotypic dissociation was observed in some pre-cc lines, with viral titer not correlated with weight loss. qtl mapping identified a significant main effect qtl, as well as a modifier qtl for vascular cuffing in the lungs, explaining % and % of the variation, respectively. suggestive qtls were identified for eosinophilia ( % of variation) and viral titer ( % of variation) (figure ; table ). the candidate region for the main effect vascular cuffing qtl was narrowed to a small kb region of shared ancestry between the high-response haplotypes. this region contained only one functional gene, trim , a ring zinc-finger-containing protein expressed in smooth muscle, which had not previously been implicated in any immune phenotypes. follow-up validation studies using trim knockout mice, which showed altered chemokine and tight junction gene expression as well as altered inflammatory cell recruitment, confirmed a role for this gene in sars-covinduced vascular cuffing. the authors also noted a high-priority candidate under the modifier qtl, the cadherin family member cdhr , which may be involved in migration of inflammatory cell from the blood into the lung. in a subsequent study, gralinksi et al. used an f cross between two cc-ri strains with highly divergent sars-cov susceptibilities to map five significant loci affecting weight loss, viral titer, and other disease phenotypes, including one main effect qtl that explained between % and % of the variation in every phenotype (gralinski et al., ) . ticam , a tlr adaptor protein, was identified as a high-priority candidate gene under the main effect qtl given the known importance of tlr in sars-cov pathogenesis (totura et al., ) . ticam knockout mice had increased sars-cov-induced weight loss, early viral titers, and pulmonary hemorrhage. in addition to exhibiting high levels of variation in pathogeninduced responses, the cc also exhibits variation in baseline (homeostatic) immunity. variation in immune homeostasis has been associated with variation in vaccine responses in humans (tsang et al., ) , and the cc provides a novel resource to study how variation in pre-existing immunity affects the host response to infection or vaccination. phillippi et al. analyzed subsets of lymphocytes and antigen-presenting cells in the cc founders, cc founder f s, and pre-cc strains (phillippi et al., ) . they observed variation in the pre-cc exceeding that in the founders, as well as novel extreme phenotypes such as lymphopenia and inverted cd /cd ratios. while some immune phenotypes were highly correlated, others showed little or no correlation to other immune populations. mapping identified significant qtls across phenotypes, including b/t cell ratio and mean fluorescence intensity for cd , also known as fc epsilon rii. the cd qtl contains the gene fcer a, which codes for cd itself, and a combination of merge analysis, conditional association, and residue conservation was used to identify specific coding polymorphisms within fcer a driving the qtl. graham et al. also performed an extensive examination of homeostatic immunity in over cc-f crosses, with a focus on t cell populations, expression of activation markers, and production of inflammatory cytokines (graham et al., ) . cc strains exhibited high levels of phenotypic variation that extended well beyond the variation observed between c bl/ j and balb/c, the most common laboratory strains in immunological studies. mapping identified two highly significant qtls driving the frequency of specific t regulatory subsets, and candidates were chosen based on concordance of haplotype effects with founder polymorphisms that were coding or splice variants. similar to the cis-qtl for cd expression identified by phillippi et al., the qtl mapped by graham et al. for cxcr + t regulatory cells contains the cxcr gene itself. kri sti c et al. studied variation in igg glycosylation, which is important for antibody structure and function, in cc strains and observed nearly double the variation observed in humans (kri sti c et al., ) . glycosylation patterns were up to % heritable, and multiple qtls associated with variation in different glycans were identified. variation in different glycans all mapped to the immunoglobulin heavy chain locus. variation in other glycans mapped to a locus containing the glycosyltransferaseencoding gene mgat , which was identified as a strong candidate. commensals and the microbiome in the cc while the focus of this review is on genetic control of immune populations and response to infectious agents, there is a growing appreciation for the role commensals play in shaping health and modulating immune responses. two studies have addressed these responses in the cc (snijders et al., ) and do (carmody et al., ) populations. both found that there were significant contributions of host genetic backgrounds. further, the cc study found strong genomic signals for many bacterial groups (otus) around the genome. given the increasing appreciation and awareness of the microbiome in modulating a variety of biomedically relevant traits, as well as the impact of commensals and the virome (virgin, ) on directly modulating immune responses (masopust et al., ) , future work within the cc will allow for the disentangling of direct roles on host genetic variants on disease responses, as well as indirect roles mediated through effects on commensals or prior immune exposure. the cc is a powerful resource for model development, extreme phenotype discovery, population-based qtl mapping, intercross mapping, validation of candidate genes across genetic backgrounds, and a variety of other approaches. here, we provide an overview of different study designs that can be implemented to study the impact of host genetic variation on pathogen outcomes or immunity using the cc (figure ) . many of the genetic mapping studies described above utilized large panels of cc strains and were able to identify loci of moderate to large effects on pathogen outcomes. however, these types of studies are extremely resource intensive and may not always be the optimal approach. rather, we suggest a tiered approach to study design within the cc, starting with a small-scale analysis in a set of strains (e.g., - ) that sample most of the haplotypes present across the cc genomes, and expanding as necessary. the efficacy of this type of approach is demonstrated by the success of small cc screens that have identified strains with unique phenotypes and proceeded with further targeted studies such as f crosses to reveal complex polygenic networks driving the outlier phenotype (gralinski et al., ; rogala et al., ) . furthermore, even when investigators opt for large mapping studies, they may still identify strains with unique outlier phenotypes that cannot be explained by qtls identified in the initial screen. this missing heritability may be driven by complex genetic interactions such as epistasis, which are difficult to study in complex populations and are more effectively studied in targeted crosses (rogala et al., ) . initially screening small sets of cc strains (e.g., - ) allows the investigator to obtain insight into the distribution of trait variation (e.g., continuous, bimodal), while testing whether any strains show unique or strong outlier phenotypes (gralinski et al., ; rasmussen et al., ; rogala et al., ) . these small screens also facilitate estimation of the proportion of phenotypic variation explained by genetics within the test population (heritability), identification of potential confounding factors (e.g., mouse size and activity levels), and performance of power calculations before embarking on a large-scale screen. in contrast to conducting the initial screen in the cc founders, cc strains exhibit more phenotypic diversity due to re-assortment of allelic variants throughout the genome. furthermore, a specific set of cc strains can be chosen to avoid the presence of large effect resistance genes such as mx or oas b that may dominate the response to some pathogens. likewise, strains can be selected that carry specific gene haplotypes to facilitate specific assays (e.g., mhc haplotypes for antigen-specific t cell analysis) (graham et al., (graham et al., , . following an initial screen, researchers interested in developing new models can pursue the strains with the most relevant phenotypes, while those interested in genetic mapping may either perform a more extensive population-wide study (e.g., across the entire cc) or conduct a focused analysis of one or more strains with extreme or novel phenotypes via classical intercrosses. unlike cc mice, which are fully inbred and genotyped, each mouse in an f or other intercross mapping population will need to be genotyped. in both cases, genetic mapping should identify a subset of the variants impacting the phenotype of interest (e.g., disease). while an in-depth discussion of mapping methods is beyond the scope of this review, several robust mapping strategies have been successfully applied to the cc, including doqtl, bagpipe, and r/qtl (arends et al., ; gatti et al., ; valdar et al., ). once qtls have been mapped, investigators can confirm the underlying haplotype effects by screening additional cc strains with high or low responder haplotypes under the qtls that were not included in the initial screen, or by screening a set of mice from the related do population. while not essential, this step confirms the initial mapping studies and provides confidence in the qtl effects prior to embarking on candidate gene investigation. the identification and validation of candidate genes can be a challenging and intensive process. qtls are often large, containing tens to hundreds of genes. in contrast to mapping in biparental grps or intercross populations, where there are only two haplotypes and every variant is a putative candidate, in the cc, merge analysis (yalcin et al., ) can be used to narrow candidates based on how the pattern of variants (e.g., snps or indels) for each founder matches the distribution of phenotypic responses associated with each founder haplotype. as described above, merge analysis has been a useful tool in the cc; however, a drawback of merge analysis is that it assumes a single snp variant driving the locus. in multi-parental populations, different snps or other mutations within the same gene may phenocopy one another (e.g., different mx null alleles; ferris et al., ) . furthermore, other genetic variants (e.g., copy number variants and transposable elements) are often missed by traditional merge databases. other in silico tools, including baseline tissue and/or haplotype-specific gene expression (e.g., immgen and gecco) (shay and kang, ; http://csbio.unc.edu/gecco) and protein functional consequence prediction (e.g., sift; sim et al., ) , can help narrow candidates to a single high-priority candidate gene or small set of candidates (ferris et al., ; gralinski et al., ) . alternatively, if causal variants are suspected of altering transcript levels, de novo gene expression analysis can be used to further refine candidate genes under the locus. following candidate gene identification, there are several options for testing whether the candidate is actually causal. when the phenotype is driven by an individual cellular factor, such as an antiviral molecule or receptor, gene function can be validated in vitro using techniques such as crispr or ectopic expression of different variants. when the phenotype has a more systemic cause, in vivo validation is generally necessary. this can be facilitated by the wide availability of knockout mouse lines (gralinski et al., (gralinski et al., , . however, in vivo validation studies should take into account factors such as genetic background of the knockout, the effect of the variant, and the distribution of haplotypes (e.g., a knockout on a c bl/ j background may not provide an interpretable phenotype if, in the cc, the c bl/ j haplotype is an extreme response haplotype, or if the c bl/ j allele is hypomorphic or amorphic). more recent advances in crispr technology open up the possibility of knocking out or performing allele swaps in genes directly in cc founders or ri strains, enhancing the utility of the cc as a resource for both identifying and studying polymorphic genes affecting pathogen susceptibility or other phenotypes of interest. in less than two decades since the cc was conceptualized (churchill et al., ) , the cc has proven to be a fruitful resource across many fields, including infectious disease and immunology. expanding use of the cc has highlighted certain challenges and considerations that should be recognized to drive future development in both experimental design and resource expansion. ( ) small screen in a subset of cc strains ( - ), selected based on availability or a genotype of interest (e.g., mhc haplotype). ( ) assess variation across strains, identify outliers, measure heritability, perform power calculations, and identify confounding variables and reassess experimental design if necessary. ( ) perform a larger screen either in the full cc population or a targeted intercross. following mapping, this can be repeated to follow-up on modifier alleles or other unexplained loci. ( ) reassess variation, outliers, and heritability across mapping population. ( ) map qtls driving phenotype of interest; analyze founder haplotype effects if mapping is done in the cc. ( ) rationally select candidate genes using different tools as applicable. ( ) perform validation studies in vitro and/or in vivo to confirm effect of candidate gene on phenotype. genetic mapping studies in the cc have successfully identified loci underlying infectious disease and immune phenotypes; however, relatively few have subsequently validated the initially identified candidate genes. even more challenging than candidate gene validation is the identification of specific causal variants within a gene. while strategies such as merge analysis can help refine candidates and variants, these tools are not always informative. there is a need for more refinement methodologies that can be applied in combination with experimental validation to narrow and identify causal variants. the wide array of genomic, molecular, and immunological reagents available is a valuable asset for mouse research; however, these do not necessarily uniformly work across the cc and should be validated before use. existing cc-specific resources, such as gecco, are not necessarily informative for infectious disease research since lymphoid organs, such as the spleen, thymus, and bone marrow, are not profiled. furthermore, the development of new cc-specific tools, such as cc-derived cell lines (e.g., mefs and ipscs), will expand the utility of the cc across fields, including the infectious diseases and immunology fields. one of the largest challenges across all of model organism research is connecting back to human relevancy. the cc has already proven useful in providing improved models of humanlike disease, as highlighted above. a number of genes that have been highlighted in the cc, such as mx and oas b, have human paralogs relevant in infectious disease susceptibility (lin and brass, ; simon-loriere et al., ) , demonstrating the overlap between important genetically diverse infectious disease pathways in mouse and humans. however, the majority of studies have not yet made direct connections back to human datasets. as cc research develops, it will be increasingly important to continue to extend analysis from the cc to human genetics and vice versa. this will require more crosstalk between mouse and human geneticists to bridge gaps in research and communication. host immunity and susceptibility to infectious disease is a complex response driven by a multitude of factors, from environment and immune experience to the genetics of both the pathogen and the host. studying the role of host genetics in infectious disease directly in humans is challenging because of this complexity, and therefore much of the research in the field leans heavily on the mouse as a model organism. while the role of traditional laboratory strains and genetically modified mice in driving advances in the field cannot be understated, these models do not capture the genetic diversity and phenotypic complexity observed in the natural population. building on the success of classical intercrosses and biparental grps, the cc is a highly diverse and reproducible 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physiology and disease a large-scale genome-wide association and metaanalysis identified four novel susceptibility loci for leprosy innate immune response in th -and th -dominant mouse strains genomic profiling of collaborative cross founder mice infected with respiratory viruses reveals novel transcripts and infectionrelated strain-specific gene and isoform expression using progenitor strain information to identify quantitative trait nucleotides in outbred mice identification of new loci involved in the host susceptibility to salmonella typhimurium in collaborative cross mice we would like to thank sharon taft-benz, sanjay sarkar, emily madden, and brea hampton for critical reading of the manuscript. chromosome ideograms shown in figure were generated using the karyoploter package for r (gel and serra, ) . this work was supported by u ai and p ai . k.e.n. received support from t ai . key: cord- -i loz xb authors: li, tongya; ke, zunlong; liu, weiyong; xiong, ying; zhu, ying; liu, yingle title: human hepatitis b virus core protein inhibits ifnα-induced ifitm expression by interacting with baf date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: i loz xb human hepatitis b virus core protein (hbc) is a structural protein of the hepatitis b virus (hbv) and contributes to hbv regulation of host-cell transcription. however, the mechanisms of transcriptional regulation remain poorly characterized. to dissect the function of hbc, a yeast two-hybrid was performed to identify hbc-binding proteins, and the c-terminal of brg /hbrm-associated factors (baf c) was identified. then, the existence of hbc interactions with baf c and full-length baf was confirmed via co-immunoprecipitation assays in t, hepg and hepg -ntcp cells. furthermore, we show that the binding between hbc and baf was of vital importance to hbc mediated downregulation of interferon-induced transmembrane protein (ifitm ) expression, and the mechanisms for the downregulation were disclosed as follows. basal level of ifitm expression depends on baf , rather than the jak–stat pathway. the interaction of hbc with baf disturbs the stability of the polybromo-associated baf (pbaf) complex and results in the suppression of iftm transcription. finally, the antiviral effects of ifitm on cell proliferation and hbv replication were found to be partially restored when hbc was co-transfected with baf . collectively, our findings indicate that hbc plays a role in hbv resistance against the antiviral activities of ifnα, providing details about hbv evasion of host innate immunity. the human hepatitis b virus (hbv) is a double stranded dna virus in the hepadnaviridae family [ ] . hbv infection could cause acute and chronic hepatitis b (chb), which can progress to cirrhosis and hepatocellular carcinoma, leading to high mortality rates worldwide. antiviral therapy with interferon aims to induce permanent immune control of hbv infection through stimulation of the hosts' innate immune response. nevertheless, experimental data from hbv infected chimpanzees and urokinase-type plasminogen activator/severe combined immunodeficiency (upa-scid) mice have shown that hbv infection does not induce an intrahepatic innate immune response that can be detected [ , ] . this is because early in infection it acts like a stealth virus, remaining undetected and spreading until the onset of the adaptive immune response several weeks later [ ] . besides acting as a stealth pathogen, recent developments have shown that hbv can avoid recognition by the host innate immune system. however, the precise mechanisms are largely unknown. human hepatitis b virus core protein (hbc) is amino acids in length and dimeric in solution [ ] . hbc dimers assemble into t = ( copies) or t = ( copies) capsids of hbv, with t = capsids being the predominant form in vivo [ ] . hbc is composed of an assembly domain (aa - ) and a nucleic acid-binding domain (aa - ) (figure a) , moreover, it not only acts as a structural protein of hbv, but also works as an essential regulator in viral replication [ , ] . the nucleic acid-binding harbors a nuclear localization sequence (nls), which mediates the transport of hbc into the nucleus [ , ] . in vitro studies have shown that hbc binds directly to the covalently closed circular dna (cccdna) upon entering the nucleus, such as the camp response element of hbv, enh i [ ] , and the nuclear factor kappa b binding site of hbv, enh ii [ ] , to regulate hbv transcription. in addition, hbc appears to regulate the activities of host cells by interacting directly with the host genome [ ] . however, the details of hbc function in host transcriptional regulation are not well understood. human swi/snf (mating-type switching (swi) and sucrose non-fermenting (snf)) complexes regulate the expression of numerous interferon (ifn)-inducible genes by mediating atp-dependent chromatin remodeling, exposing the binding sites to the transcriptional machinery. swi/snf complexes are critical for proliferation, differentiation, tumorigenesis, and dna repair [ ] . there are two forms of swi/snf complexes: brg /hbrm-associated factors (baf) and polybromo-associated baf (pbaf). only pbaf can facilitate the ligand-dependent transcriptional activation by interacting with the nuclear receptors [ ] . baf and pbaf complexes share most of their subunits and are distinguished by the presence of two specific subunits-baf and baf -both of which only exist in pbaf [ ] . furthermore, baf , but not baf , is essential for the stability of pbaf, and the depletion of baf leads to the complete inactivation of pbaf [ ] . baf is encoded by arid . besides the n-terminal at-rich interactive domain (arid), baf contains multiple lxxll motifs, which have been shown to participate in the regulation of protein-protein interaction [ ] . additionally, baf has been reported to be a potential tumor suppressor and involved in the ifn signal pathway [ ] . ifns are essential components of the innate immune response and act as the first line of defense against invading microorganisms or pathogens. interestingly, hbv escapes the host innate immune response merely by preventing the induction of ifns [ ] . ifns modulate host defenses against microbial infection through the induction of ifn-stimulated genes (isgs) by the janus kinase (jak)-signal transducer and activator of transcription (stat) signaling pathway. among these isgs, interferon-induced transmembrane protein (ifitm) , , and , which are a cluster of genes encoding membrane proteins, exhibit antiviral capabilities mainly through the inhibition of virus entry [ ] . ifitm restricts the infection of various viruses, including type human immunodeficiency virus [ ] , hepatitis c virus [ ] , severe acute respiratory syndrome (sars) coronavirus [ ] , and influenza a virus [ , ] . however, whether ifitms inhibit hbv infection has not been reported. in this study, we start with the discovery that hbc can interact with baf . then, we focus on functions of the interaction and disclose that overexpressed hbc downregulates the baf -dependent expression of ifitm via disruption of pbaf complex stability. finally, our data demonstrates that the antiviral effects of ifitm on cellular proliferation and hbv replication are partially restored when hbc is co-expressed with baf in hbv-infected cells. these findings enrich details about how hbv counteracts human natural immunity, revealing a potential target for novel therapeutic strategies of hbv infection. baf c ( - nt of arid ) was amplified from the fragment screened by the yeast two-hybrid system using primers-sense, '-gatccatggcaaactcgacggggaa- ' and antisense, '-aattctcactgcagcatttctga- '-and inserted into a pcmv-flag vector (stratagene, san diego, ca, usa). baf (full-length arid ) and hbc cdnas (taxonomy id: ) were synthesized by genscript co. ltd. (nanjing, china). pgc-fu-flag and the phbv . vector were kindly gifted by prof. r xiang (xiangya school of medicine of central south university, china). the pgc-fu-flag vector was ligated and constructed with baf at restriction enzyme site agei. the full-length of hbc was cloned into the pgbkt (clontech, clontech laboratories, inc., mountain view, ca, usa) vector with primers-sense, '-acttccagacttctagggagac- ' and antisense, '-ctgccctgtgacggaattga- '-and cloned into the pcmv-ha vector (clontech) with primers-sense, '-gatccatggacattgaccactataaa- ' and antisense, '-tcgacctaacattgagattcccgaga- '. the matchmaker gal two-hybrid system (clontech) was used for the screening of a human fetal brain cdna library (clontech) with pgbkt -hbc as bait. co-transformants were selected on synthetic dropout (sd), media lacking leucine, and tryptophan (sd/-leu/-trp) and were validated by growth on sd, media lacking leucine, tryptophan, adenine, and histidine (sd/-leu/-trp/-ade/-his) and containing -bromo- -chloro- -indolyl-α-d-galactoside (x-α-gal). then, positive colonies were sequenced (invitrogen, carlsbad, ca, usa). hepg cells and t cells, purchased from cctcc, were cultured in dulbecco's modified eagle medium (dmem, gibco, thermo fisher scientific, waltham, ma, usa), supplemented with % (v/v) fetal bovine serum ((fbs, gibco), % penicillin and . mg/ml streptomycin in a humidified incubator maintained at • c with % co . hepg . . cells, obtained from prof. r xiang, were cultured in medium (gbico) in the presence of g ( µg/ml, sigma-aldrich, st. louis, mo, usa) in a humidified incubator maintained at • c with % co . hepg -ntcp cells and hepaad cells, provided by prof. y zhu (wuhan university, china), were cultured in dmem (gibco), supplemented with % heat-inactivated fetal calf serum (gibco), u/ml penicillin, and µg/ml streptomycin sulfate at • c in % co . all the transfection reactions were performed on indicated cells ( × ) in log phase, using lipofectamine (invitrogen) according to the manufacturer's protocol. co-transfection was performed using a total of µg of plasmids or vectors in a : (w/w) ratio: the pcmv-ha-hbc or pcmv-ha vectors were co-transfected with pcmv-flag-baf c or pgc-fu-flag-baf into indicated cells. the medium was refreshed with serum-free dmem/ h after transfection. the supernatant was harvested h post incubation for co-immunoprecipitation or western blot analyses. the supernatants of hepaad cells were concentrated -fold by ultracentrifugation as hbv inoculums. hbv stock titer (genome equivalents [geq] per milliliter) was measured by using qpcr. for infection, hepg -ntcp cells of a low passage number were seeded onto precooling collagen i-coated plates and incubated in dmem for h, then the medium was replaced by primary hepatocyte maintenance medium (pmm) with % fbs (gibco) for h. after this, cells were infected with geq per cell of hbv in pmm containing % (w/v) polyethylene glycol (peg ) for h. after the virus-containing medium was removed, cells were washed several times and cultured in fresh pmm. the medium was changed every other day [ , ] . pmm is williams' e medium supplemented with an insulin-transferrin-selenium solution (thermo, waltham, ma, usa), ng/ml of human epidermal growth factor (egf, peprotech, rocky hill, nj, usa), mm l-glutamine (thermo), µg/ml of hydrocortisone, % dimethyl sulfoxide (dmso), ng/ml of dexamethasone, µg/ml of streptomycin, and u/ml of penicillin. after transfection, cells were washed with ice-cold pbs after h, harvested by scraping, then lysed using an ripa lysis buffer ( mm tris-hcl [ph . ], mm nacl, % np , . % sds) supplemented with a protease inhibitor cocktail (thermo scientific). after centrifugation at , rpm at • c for min, supernatants were incubated with primary antibody or igg (santa cruz, santa cruz, ca, usa) and protein g agarose beads (ge healthcare, chicago, il, usa) at • c overnight. immunoprecipitates were washed with a washing buffer ( mm tris-hcl [ph . ], mm nacl) three times, then whole cell lysate and immunoprecipitated fractions were used for western blot analysis. the following primary antibodies were used: anti-ha (catalog no. h , sigma-aldrich), anti-flag (catalog no. f , sigma-aldrich), anti-baf (catalog no. a - a, bethyl), anti-hbc (catalog no. b , dako). in order to make sure the loading amounts of the protein were comparable, the protein concentration was quantified by bradford assay [ ] . thirty micrograms of protein was separated by sds polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride (pvdf, thermo scientific) membranes. after blocking with % gelatin in tbst, the membranes were incubated with the indicated primary antibodies at • c overnight. the membranes were washed three times, incubated with secondary antibodies ( : ) conjugated with horseradish peroxidase (hrp) for h at room temperature, and visualized with the ecl system (biorad, hercules, ca, usa) according to manufacturer's instructions. blots were probed with hrp-conjugated secondary antibodies. the following antibodies were used: anti-ifitm , , , (catalog no. , , , cell signaling technology, danvers, ma, usa), anti-baf (catalog no. a - a, bethyl), anti-stat (catalog no. , cell signaling technology), and anti-phospho-stat (catalog no. , cell signaling technology), and β-actin (catalog no. - -ig, proteintech group). the gray density of the western blots was measured by using imagej software (national institutes of health, bethesda, md, usa). rt-pcr assays were performed to determine the relative mrna levels. total rna was extracted by trizol (invitrogen) according to the manufacturer's instructions. the quantity of the rna samples was detected by nanodrop (thermo scientific). one microgram of rna was reverse transcribed using random hexamer primers (fermentas, waltham, ma, usa) and m-mulv reverse transcriptase. the levels of ifitms mrna and intracellular hbv genomic dna were determined by rt-qpcr analysis using sybr green premix (takara, tokyo, japan) on a lightcycler® ii (roche, basel, switzerland) system. the expression of the target genes was normalized to glyceraldehyde -phosphate dehydrogenase (gapdh) by the ∆∆ct method. β-actin was used as control. primers were as follows: ifitm , forward: '-ccccaaagccagaagatgcacaaggag- ', reverse: '-cgtcgccaaccatcttcctgtccctag- '; ifitm , forward: '-catcatcatcccagtgttgg- ', reverse: '-gataaagggctgatgcagga- '; ifitm , forward: '-caaggaggagcacgagg- ', reverse: '-ttgaacagggaccagacg- '; β-actin, forward: '-ctcttccagccttccttcct- ', reverse: '-agcactgtgttggcgtacag- '; gapdh, forward: '-gatggcaagatcttctgcgtg- ', reverse: '-ccgtcgactcacaggaaatagtcggc- '. the sirnas were designed using oligo . software, and synthesized by genscript co. ltd. (nanjing, china) as follows: siifitm : sirna : '-aaaccuucacucaacacuuccuu- ', sirna : '-aaacuuaagagaaauacacacuu- '; sihbc: sirna : '-aaacuuuacugggcuuuauucuu- ', sirna : '-aagagaaacuguccuugaguauu- '; viruses , , of sicontrol: '-guauauaagcaagcauuacuu- '. all the sirnas were transfected using rnaimax (invitrogen) according to the manufacturer's instructions. one hundred microliters of hepg or hepg . . cells of the same passage number were seeded into -well plates at a density of × cells per well. after culturing for h, cells were transfected with pgc-fu-flag vector, sirna, or pgc -fu-flag-baf and incubated for h, then treated with , , , , , or u/ml ifnα for h. afterwards, cell viability was assessed using the mtt assay. the medium was refreshed and mg/ml -( , -dimethlthiazol- -yl)- , -diphenyltetrazolium bromide (mtt, sigma-aldrich) was added at µl per well. after incubating for h, the medium was removed, and cells were lysed in µl dmso. finally, absorbance was measured by a microplate reader (thermo scientific) at nm. to ensure the results of the mtt assays, a trypan blue exclusion assay was performed. after the transfection and treatment of ifnα, cells were harvested. then trypan blue (sigma-aldrich) was added to the cell suspension to a final concentration of . % (w/v), and the mixture incubated at room temperature for min. ten microliters of the suspension was transferred to a hemocytometer and viable cells were counted. the test was repeated at least three times. hepg cells were transfected with phbv . and pgc-fu-flag-baf , or together with pcmv-hbc-ha, in a : ratio. after treatment with ifnα for h, the supernatant was collected to detect the levels of the hepatitis b surface antigen (hbsag) and hepatitis b e antigen (hbeag) using a commercial elisa kit (neobioscience, hangzhou, china) and the hbv dna copy number was quantified by real-time pcr with a commercial pcr-fluorescence quantification kit (bioer, hangzhou, china). for the extraction of the nucleic acid, hepg cells were collected at h post-transfection and lysed in a precooling lysis buffer ( . % np- , mm tris-hcl [ph . ]). after centrifugation at , × g for min, the nuclei were pelleted, and the supernatant was adjusted with mm mgcl and treated with dnase i for h at • c to remove the free dna. the mixture was incubated at • c for min in the presence of mm edta to inactivate the enzymes, then cultured with proteinase k in the presence of % sds to digest proteins. at last, the nucleic acids were purified by phenol-chloroform extraction and ethanol precipitation. for the extraction of extracellular encapsidated hbv dna, free dna and enzymes of µl cell culture supernatant were removed according to the methods mentioned above. then, the mixture was added to µl of a lysis buffer ( mm edta, mm tris-hcl, . % sds, and mm nacl) containing proteinase k and incubated at • c overnight. after this, hbv dna was isolated by phenol-chloroform extraction and ethanol precipitation. hbv dna was subjected to real-time by pcr using primers ( '-agaaacaacacatagcgcctcat- ' and '-tgccccatgctgtagatcttg- ') and probe ( '-tgtgggtcaccatattcttggg- '). data were presented as mean ± standard deviation (sd). all experiments were repeated three times. the statistical analysis was assessed using the student's t-test. differences were considered statistically significant at p < . (**). to dissect the function of hbc (figure a ), we screened a human fetal brain cdna library for novel hbc-interacting proteins using a yeast two-hybrid system. co-transformants were selected on sd/-leu/-trp and were validated by growth on sd/-leu/-trp/-ade/-his/x-α-gal ( figure b) . then, positive colonies were sequenced. finally, the c-terminal of baf (baf c, aa - ) was identified as one of the strongest binding partners of hbc in ah . baf c contains znf domains and can bind to dna, rna, or proteins [ ] (figure a ). because this interaction might play an important role in hbv-infected hepatocytes, our studies focused on the function of hbc interaction with baf . to verify the two-hybrid results, co-ip assays were performed. first, baf c was co-transfected with either empty vectors or hbc into t cells, then the whole cell lysate was immunoprecipitated by an anti-flag antibody and then subjected to western blot by anti-ha antibodies to detect the interacting proteins. the results indicate that the expressed c-terminal of baf co-precipitated with hbc ( figure c ). then, hbc interaction with full-length baf was further assessed in hepg cells. ip was performed against ha-tagged hbc and the co-precipitation was detected with the anti-flag antibody. the data showed that overexpressed baf co-precipitated with hbc ( figure d ). to investigate the endogenous interaction of baf and hbc, hepg -ntcp cells were infected with or without hbv by inoculation with or without the supernatants of hepaad cells. exogenous expression of sodium taurocholate co-transporting polypeptide (ntcp) in human hepg cells (hepg -ntcp) rendered them susceptible to hbv/hdv infection [ ] . hepg -ntcp cells were infected with hbv by inoculation with or without the supernatants of hepaad cells [ , ] . the lysates of hepg -ntcp cells were immunoprecipitated by hbc antibodies or baf antibodies, then subjected to western blot by baf antibodies or hbc antibodies to detect the interacting proteins ( figure e ). the result indicated that endogenous baf also co-precipitated with endogenous hbc, as seen in overexpressed proteins. collectively, the co-ip results confirm the binding between hbc and baf . and can bind to dna, rna, or proteins [ ] (figure a ). because this interaction might play an important role in hbv-infected hepatocytes, our studies focused on the function of hbc interaction with baf . to verify the two-hybrid results, co-ip assays were performed. first, baf c was cotransfected with either empty vectors or hbc into t cells, then the whole cell lysate was immunoprecipitated by an anti-flag antibody and then subjected to western blot by anti-ha antibodies to detect the interacting proteins. the results indicate that the expressed c-terminal of baf co-precipitated with hbc ( figure c) . then, hbc interaction with full-length baf was further assessed in hepg cells. ip was performed against ha-tagged hbc and the co-precipitation was detected with the anti-flag antibody. the data showed that overexpressed baf coprecipitated with hbc ( figure d) . to investigate the endogenous interaction of baf and hbc, hepg -ntcp cells were infected with or without hbv by inoculation with or without the supernatants of hepaad cells. exogenous expression of sodium taurocholate co-transporting polypeptide (ntcp) in human hepg cells (hepg -ntcp) rendered them susceptible to hbv/hdv infection [ ] . hepg -ntcp cells were infected with hbv by inoculation with or without the supernatants of hepaad cells [ , ] . the lysates of hepg -ntcp cells were immunoprecipitated by hbc antibodies or baf antibodies, then subjected to western blot by baf antibodies or hbc antibodies to detect the interacting proteins (figure e ). the result indicated that endogenous baf also co-precipitated with endogenous hbc, as seen in overexpressed proteins. collectively, the co-ip results confirm the binding between hbc and baf . hbc-interacting partners were tested for β-galactosidase activity on an sd/-leu/-trp/-ade/-his/x-α-gal plate. vector: pgadt ; positive control: pgbkt - and pgadt -t co-transformant; negative control: pgbkt -lam and pgadt -t transformant. (c) t cells were co-transfected with pcmv-flag-baf c and pcmv-ha vector or pcmv-ha-hbc, and co-ip assays were performed with anti-flag antibody or control igg. immuno-complexes were detected by western blot assays using the anti-ha antibody or anti-flag antibody (control). (d) hepg cells were co-transfected with the pgc-fu-flag-baf and pcmv-ha vectors or pcmv-ha-hbc, and co-ip assays were carried out with anti-ha antibody or igg. immuno-complexes were detected by western blot assays using the anti-flag antibody or anti-ha antibody (control). (e) hepg -ntcp cells were incubated with supernatants isolated from the supernatants of hepaad cells cultures (containing hbv) at geq for h or not (mock). co-ip assays were performed with hbc antibody or baf antibody, immuno-complexes were detected by western blot assays using baf antibody or hbc antibody. input control assays were performed in whole cell lysates (wcl). because baf was reported to be a critical regulator of ifn signaling [ ] , we first focused on the effect of baf on the expression of ifitms, which are ifnα effectors. the impact of overexpressed baf on ifitm , , and was examined via protein ( figure a ) and mrna (figure b ) levels. the results demonstrated that baf up-regulated ifitm protein expression (figure a) and significantly increased the mrna expression ( figure b ). however, no apparent effect of baf was observed on the expression of ifitm and ifitm (figure b) . furthermore, we observed that the ifitm expression level was suppressed . -fold when endogenous baf was silenced in hepg cells (figure c) . the data suggest that baf specifically mediates basal level expression of ifitm . next, the effect of the hbc interaction with baf on ifitm expression was further investigated. we co-transfected hbc and baf into hepg cells, treated the cells with u/ml ifnα, and detected the expression of ifitm by western blot (figure d ). the results indicate that baf enhanced ifitm expression, while the enhancement was partially reduced when hbc was co-transfected. to further examine the effect of ifnα on hbc function, we treated the transfected cells with ifnα at various concentrations from u/ml to u/ml for h and examined the ifitm mrna levels ( figure e ). as expected, hbc impaired the enhancement of ifitm mrna expression by baf . however, compared to the control, expression was inhibited to a similar degree upon ifnα stimulation with different concentrations. taken together, the data demonstrates that hbc can down-regulate ifitm expression via binding to baf , whereas the binding is irrelevant to the stimulation of ifnα. ifnα at various concentrations from u/ml to u/ml for h and examined the ifitm mrna levels (figure e ). as expected, hbc impaired the enhancement of ifitm mrna expression by baf . however, compared to the control, expression was inhibited to a similar degree upon ifnα stimulation with different concentrations. taken together, the data demonstrates that hbc can down-regulate ifitm expression via binding to baf , whereas the binding is irrelevant to the stimulation of ifnα. furthermore, the mechanism about how hbc regulated ifitm expression was explored. since the jak-stat signaling pathway has been reported to be essential to ifnα-induced antiviral activities [ ] , we examined whether it was involved in baf -mediated ifitm expression. since the phosphorylation of stat is a necessary step for jak-stat signaling, we used western blot assays to detect phosphorylated stat (pstat ) in hepg cells with or without baf (figure a) . the results showed that pstat appeared only upon ifnα stimulation, independent of the presence of baf . it has been shown that baf is essential for the stability of pbaf, and the depletion of baf leads to the complete inactivation of pbaf [ ] . hence, we predicted that the interaction of hbc with baf would interrupt the stability of pbaf complexes, leading to potential baf -dependent ifitm expression. to verify this hypothesis, co-ip assays were carried out to analyze the effect of hb on baf -baf immuno-complexes in hepg cells (figure b) . ip was performed against flag-baf and the co-precipitation of baf was probed. it demonstrated that hbc co-transfection along with baf profoundly reduced the co-precipitation of baf -baf immuno-complexes when compared with the baf transfected alone condition. endogenous baf -baf interaction was further assessed in hbv infected hepg -ntcp cells (figure e) . the co-ip assay showed that the concentration of baf -baf complexes was reduced more than -fold compared to the non-infected mock control. these results imply that hbc de-stabilizes the pbaf complex by preventing the baf -baf interaction, probably by competitive binding to baf . we next determined whether overexpression of hbc regulated ifitm transcription in vitro. flag-baf was transfected with or without ha-hbc into hepg cells. cells were later treated with ifnα ( u/ml) and total mrna was extracted to detect transcription levels of ifitm , , and by rt-qpcr. the data indicates that, upon ifnα stimulation, the suppression of ifitm expression by hbc is specific (figure c ) and statistically significant (figure d ) in vitro. collectively, the data demonstrates that hbc interacts with baf , and the hbc-baf interaction prevents the baf -baf interactions that might abolish pbaf complex stability, which in turn regulates the suppression of ifitm transcription. the pcmv-flag vector and pcmv-flag-baf were transfected into hepg cells, with or without ifnα ( u/ml) treatment for h. phosphorylation of stat was detected via western blot using antibodies against phosphorylated stat (pstat ). meanwhile, stat and baf were detected using anti-flag and anti-stat antibodies as control. (b) hepg cells were co-transfected with pgc-fu-flag-baf and pcmv-ha vector or pcmv-ha-hbc, with or without ifnα ( u/ml) treatment for h. co-ip assays were performed with anti-flag antibody, then baf -baf immuno-complexes were detected by western blot assays using anti-baf antibody or anti-flag antibodies (control). input control assays were performed in wcl using anti-ha or anti-baf antibodies. meanwhile, total mrna was extracted to examine the effect of baf and hbc combination on ifitm , , transcriptions by rt-pcr (c). β-actin was detected as control. ifitm transcription level was determined using rt-qpcr and normalized to the gapdh mrna level. (d) the flag-baf transfection with ifnα treatment was designated as . *, p < . ; **, p < . . nd = not detected. (e) hepg -ntcp cells were incubated with supernatants isolated from the supernatants of hepaad cells cultures (containing hbv) at geq for h or not (mock). co-ip assays were performed with baf antibodies, and immuno-complexes were detected by western blot assays using baf antibodies or baf antibodies. input control assays were performed in wcl using hbc antibody or baf antibody. finally, we evaluated the effect of hbc on the antiviral activities of iftim in hbv-infected cells. firstly, the impact of hbc on the anti-proliferation action of ifitm was investigated via sirnamediated knockdown in hepg cells and hepg . . cells. proliferation of indicated cells was assessed by mtt assays and viable cell counting. indeed, in hepg cells and hepg . . cells, the data from direct cell counting were consistent with those obtained from mtt assays (figure a, c, d, e) . in hepg cells, the cell viability increased with the time of ifnα stimulation (figure a) and decreased with the concentration of ifnα (figure c) . unter treatment of u/ml ifnα, baf enhanced the inhibitory effect of ifnα, and overexpression of hbc partially recovered the cell the pcmv-flag vector and pcmv-flag-baf were transfected into hepg cells, with or without ifnα ( u/ml) treatment for h. phosphorylation of stat was detected via western blot using antibodies against phosphorylated stat (pstat ). meanwhile, stat and baf were detected using anti-flag and anti-stat antibodies as control. (b) hepg cells were co-transfected with pgc-fu-flag-baf and pcmv-ha vector or pcmv-ha-hbc, with or without ifnα ( u/ml) treatment for h. co-ip assays were performed with anti-flag antibody, then baf -baf immuno-complexes were detected by western blot assays using anti-baf antibody or anti-flag antibodies (control). input control assays were performed in wcl using anti-ha or anti-baf antibodies. meanwhile, total mrna was extracted to examine the effect of baf and hbc combination on ifitm , , transcriptions by rt-pcr (c). β-actin was detected as control. ifitm transcription level was determined using rt-qpcr and normalized to the gapdh mrna level. (d) the flag-baf transfection with ifnα treatment was designated as . *, p < . ; **, p < . . nd = not detected. (e) hepg -ntcp cells were incubated with supernatants isolated from the supernatants of hepaad cells cultures (containing hbv) at geq for h or not (mock). co-ip assays were performed with baf antibodies, and immuno-complexes were detected by western blot assays using baf antibodies or baf antibodies. input control assays were performed in wcl using hbc antibody or baf antibody. finally, we evaluated the effect of hbc on the antiviral activities of iftim in hbv-infected cells. firstly, the impact of hbc on the anti-proliferation action of ifitm was investigated via sirna-mediated knockdown in hepg cells and hepg . . cells. proliferation of indicated cells was assessed by mtt assays and viable cell counting. indeed, in hepg cells and hepg . . cells, the data from direct cell counting were consistent with those obtained from mtt assays (figure a,c,d,e) . in hepg cells, the cell viability increased with the time of ifnα stimulation (figure a) and decreased with the concentration of ifnα (figure c) . unter treatment of u/ml ifnα, baf enhanced the inhibitory effect of ifnα, and overexpression of hbc partially recovered the cell viability (figure a) , however, the sirna mediated knock down of iftim increased the cell viability robustly, about two-four-fold compared to the control (figure c) . the results suggest that ifitm makes major contributions to ifnα inhibitory activities for hbv replication. next, we focused on the role of hb. in hepg . . cells, which contain a stably integrated hbv genome and produce endogenous hbc, overexpression of baf promoted the inhibition of ifnα (figure d ), however when hbc was silenced by sihbc, cell viability was reduced substantially (figure e) . consequently, the results demonstrate that hbc antagonizes the suppression of iftim and the proliferation of hbv-infected cells in vitro. next, the hbc effects on the inhibition of ifitm on hbv replication were measured. in figure e , phbv . , which contains . -fold hbv genome and can produce endogenous hbc, was co-transfected with empty vector flag-baf or ha-hbc into hepg cells. after treating the cells with u/ml ifnα for h, supernatants were collected to determine the hbv replication level by detecting the quantitation of hbsag, hbeag, and hbv dna copy numbers (figure f ). in the control, empty vector and phbv . were co-transfected, and it was observed that ifnα stimulation inhibited the hbv replication level significantly. in contrast to the control, when baf was overexpressed, the concentration of hbsag and hbeag in the supernatant was reduced about one-fold, and hbv dna copies were also suppressed up to five-fold. as expected, the ifnα inhibition was restored when hbc was co-transfected with baf . notably, hbsag and hbeag levels increased to similar levels as the control, with a slight elevation in the hbv dna copy numbers. collectively, the results illustrate that hbv evades ifnα mediated antiviral activity in vitro by downregulating the expression of ifnα effector. however, overexpressed hbc had little observed impact on the recovery of hbv dna levels, implying that ifitm is not the only primary ifnα-inducible isg that suppresses hbv dna replication. hbv replication is not directly cytotoxic to cells, while the host immune responses in infected hepatocytes are the main determinants of hepatocellular injury and hbv pathogenesis. innate , then treated with ifnα in indicated concentration for h. afterwards, the cell viability was measured using mtt assay and direct cell counting via trypan blue exclusion assay (a,c-e). (f) hepg cells were transfected with phbv . and pgc-fu-flag-baf , or co-transfected with pcmv-ha-hbc, then treated with u/ml ifnα for h. hbv replication levels were determined in the supernatants by measuring hbsag and hbeag concentration using elisa and hbv dna copy numbers using rt-qpcr. the empty vector and phbv . were co-transfected in the non-ifnα induced condition as control. the control without ifnα treatment was designed as % (a,c-f). *, p < . , **, p < . , ns = non-significant. hbv replication is not directly cytotoxic to cells, while the host immune responses in infected hepatocytes are the main determinants of hepatocellular injury and hbv pathogenesis. innate immunity is important in controlling viral spread immediately after infection and initiates efficient development of an adaptive immune response. the early phase of a viral infection is mainly characterized by the production of cytokines, type i ifn, and natural killer (nk) cells [ ] . type i ifns can be triggered directly by virus replication through the detection of viral rna or dna, and play a critical role in the immune recognition of hbv. they can be triggered directly by viral replication via detecting the presence of viral rna or dna and induce a large number of effectors working in combination to achieve a fully functional antiviral state [ , ] . ifn effectors target different steps of the viral life cycle, limiting the propagation and spread of the virus and restricting viral infection. therefore, interferon therapy remains one of the therapeutic strategies for hbv infection. in fact, the majority of chb patients treated with ifnα cannot acquire a long-lasting sustained response, especially those with a high viral load. data from animal models have shown that ifn effectors were rapidly induced upon infection with hbv [ ] . however, the hbv-induced ifn responses are weak [ ] , and that is why acute hbv infections usually show a lack of clinical symptoms. studies have shown that the clinical outcomes in chb patients are primarily determined by the interaction between hbv replication and host immune responses [ ] . as an important effector of type i ifns, mxa exhibits strong anti-viral activity against hbv, however, hbv down-regulates mxa expression significantly via the interactions between hbc and the interferon-inducible mxa promoter [ , ] . in addition, reports have indicated that under the stimulation of ifnα, the ifn-signaling pathway is generally blocked, however the formation of stat and isgf were evenly enhanced in hepg . . cells [ ] . here, our study shows that hbc inhibits the ifnα-induced ifitm transcription through disturbing the stability of pbaf complex in vitro, independent of the jak-stat pathway (figure a ). moreover, hbc exhibits anti-apoptosis activity by inhibiting trail-induced expression of the pathway-related death receptor in hepatocytes [ ] . hbc acts as a tumor suppressor as well, by blocking the transcription of the human tumorigenesis-associated genes, ifnβ and p [ , ] . notably, our data indicates that the inhibitory effect of ifitm on hbv dna replication seems weak. a potential explanation is hbv integration into the host genome. the hbv genome can present as non-integrated cccdna, which serves as a template for replication and can persist even after hbsag loss [ , ] . hbv manages to escape immunological recognition in early infection, remaining undetected and spreading for nearly five weeks [ ] . the use of cccdna as a transcriptional template in the nucleus likely contributes to hbv's capacity to limit detection in hepatocytes [ ] . in addition, hbv evades the host response through a complex combination of processes that include signaling interference, effector modulation, and continual viral genetic variation [ ] . reports have shown that hbv polymerase and hbx protein directly inhibit the cellular machinery that detects replication intermediates [ , ] . here, our study provides novel evidence that hbv counteracts ifnα antiviral activities through effector modulation, i.e., the downregulation of ifitm expression by hbc. the mechanism of the downregulation is illustrated as follows. under normal conditions, ifnα induction activates pbaf complexes, which consist of baf , baf , and other subunits. the activation of the pbaf complex triggers histone sliding on chromatin dna, leading to the exposure of interferon-stimulated response elements (isre). in this way, pbaf complexes facilitate the initiation of ifitm transcription (figure a) . actually, baf is essential for the stability of the pbaf complex. under hbv-infected condition, the interaction of hbc with baf competes against the binding between baf and baf , resulting in the loss in pbaf complex stability. thus, the efficient process of the pbaf complex is interrupted, leading to the suppression of ifitm transcription (figure b ). it provides details for the mechanism of hbv escape from ifnα-induced immune elimination. moreover, our future work will continue to explore the intrahepatic effect of hbc on hbv replication using hbv-transgenic mice. were evenly enhanced in hepg . . cells [ ] . here, our study shows that hbc inhibits the ifnαinduced ifitm transcription through disturbing the stability of pbaf complex in vitro, independent of the jak-stat pathway (figure a) . moreover, hbc exhibits anti-apoptosis activity by inhibiting trail-induced expression of the pathway-related death receptor in hepatocytes [ ] . hbc acts as a tumor suppressor as well, by blocking the transcription of the human tumorigenesis-associated genes, ifnβ and p [ , ] . taken together, our data demonstrate that hbc downregulates ifitm expression through interactions with baf , rather than the jak-stat pathway. the results also show that hbc partially restores ifitm antiviral effects on cell proliferation and hbv replication in infected cells in vitro by disturbing the stability of pbaf. it enriches the mechanism that hbv counteracts the innate immunity of ifnα, revealing a potential target for novel therapeutic strategies of hbv infection. nuclear import of hepatitis b virus capsids and genome genomic analysis of the host response to hepatitis b virus infection efficacy of nvr - , alone and in combination with pegylated interferon, vs entecavir in upa/scid mice with humanized livers and hbv infection innate immune responses in hepatitis b virus (hbv) infection full-length hepatitis b virus core protein packages viral and heterologous rna with similarly high levels of cooperativity morphological irregularities in dane particle cores hepatitis b virus core protein phosphorylation sites affect capsid stability and transient exposure of the c-terminal domain nuclear export and import of human hepatitis b virus capsid protein and particles the hepatitis b virus (hbv) core protein enhances the transcription activation of cre via the cre/creb/cbp pathway hepatitis b viral core protein activates the hepatitis b viral enhancer ii/pregenomic promoter through the nuclear factor kappab binding site hepatitis b viral core protein disrupts human host gene expression by binding to promoter regions exome sequencing identifies frequent mutation of the swi/snf complex gene pbrm in renal carcinoma pbaf chromatin-remodeling complex requires a novel specificity subunit, baf , to regulate expression of selective interferon-responsive genes selectivity of chromatin-remodelling cofactors for ligand-activated transcription expressing the human genome arid : a new tumor suppressor gene in hepatocellular carcinoma noncytolytic control of viral infections by the innate and adaptive immune response ifitms restrict the replication of multiple pathogenic viruses ifitm proteins restrict hiv- infection by antagonizing the envelope glycoprotein ifitm is a tight junction protein that inhibits hepatitis c virus entry distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus ifitm proteins mediate the innate immune response to influenza a h n virus, west nile virus and dengue virus hepatitis b and d viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis b and d virus a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding ifn-alpha inhibits hbv transcription and replication in cell culture and in humanized mice by targeting the epigenetic regulation of the nuclear cccdna minichromosome viruses and interferon: a fight for supremacy interferon-stimulated genes: a complex web of host defenses temporal analysis of early immune responses in patients with acute hepatitis b virus infection innate and adaptive immune responses in chronic hepatitis b virus infections: towards restoration of immune control of viral infection mxa inhibits hepatitis b virus replication by interaction with hepatitis b core antigen human mxa protein participates to the interferon-related inhibition of hepatitis b virus replication in female transgenic mice interferon-alpha response in chronic hepatitis b-transfected hepg . . cells is partially restored by lamivudine treatment hepatitis b virus core protein inhibits trail-induced apoptosis of hepatocytes by blocking dr expression transcriptional repression of the human p gene by hepatitis b viral core protein (hbc) in human liver cells persistence of cccdna during the natural history of chronic hepatitis b and decline during adefovir dipivoxil therapy hepatitis b virus infection and the immune response: the big questions viral clearance without destruction of infected cells during acute hbv infection this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we are grateful to r xiang for kindly providing hepg . . and phbv . plasmids, a francis and r dillard for their invaluable comments on the manuscript. the authors declare no conflict of interest. key: cord- -zml lbve authors: cuvelier, geoffrey d.e.; rubin, tamar s.; junker, anne; sinha, roona; rosenberg, alan m.; wall, donna a.; schroeder, marlis l. title: clinical presentation, immunologic features, and hematopoietic stem cell transplant outcomes for ikbkb immune deficiency date: - - journal: clin immunol doi: . /j.clim. . . sha: doc_id: cord_uid: zml lbve ikbkb immune deficiency is a rare but life-threatening primary immunodeficiency disorder, involving activation defects in adaptive and innate immunity. we present sixteen cases of a homozygous ikbkb mutation (c. dupg) in infants characterized by early-onset bacterial, viral, fungal and mycobacterial infections. in most cases, t- and b-cells were quantitatively normal, but phenotypically naïve, with severe hypogammaglobulinemia. t-cell receptor excision circles were normal, meaning newborn screening by trec analysis would miss ikbkb cases. although ikbkb immune deficiency does not meet traditional laboratory based definitions for scid, this combined immune deficiency appears to be at least as profound. urgent hsct, performed in eight patients, remains the only known curative therapy, although only three patients are survivors. ongoing infections after transplant remain a concern, and may be due to combinations of poor social determinants of health, secondary graft failure, and failure of hsct to replace non-hematopoietic cells important in immune function and dependent upon ikk/nf-κb pathways. in the canadian province of manitoba, our group has periodically managed young infants of northern cree first nations (aboriginal) descent presenting with early-onset and life-threatening viral, bacterial, mycobacterial, and fungal infections, clinically resembling severe combined immune deficiency (scid). infants were initially identified in the s from two small northern manitoba communities. in later years, infants from the neighboring province of saskatchewan were also identified. in , our group and collaborators identified the genetic defect in four patients as being due to a homozygous duplication mutation (c. dupg) in exon of ikbkb, the gene encoding inhibitor of nuclear factor kappa b kinase subunit beta (ikkβ, also known as ikk ) [ ] . the homozygous ikbkb duplication mutation seen in our population results in complete loss of ikkβ expression, a critical component of the canonical ikk-nuclear factor kappa b (nf-κb) pathway [ ] . normally, signaling through tumor necrosis family receptors, toll-like receptors, and antigen receptors on t-and b-cells induces activation of the ikk complex, which consists of the kinases ikkα and ikkβ, as well as the regulatory protein nuclear factor kappa-b essential modifier (nemo) [ ] . the active ikk complex, and specifically ikkβ, phosphorylates the inhibitory protein iκbα, resulting in release of cytoplasmic nf-κb. free from the influence of iκbα, nf-κb translocates to the nucleus to induce the transcription of a multitude of immune and inflammatory genes. given the central role of the ikk/nf-κb pathway in regulating both innate and adaptive immune responses, it is not surprising that patients with mutations in genes from this pathway manifest with primary immunodeficiency disorders [ ] . males with hypomorphic hemizygous mutations in ikbkg, which encodes for the nemo protein and results in reduced nemo expression, present with a combined immune deficiency that includes enormous allelic, immunologic and clinical heterogeneity [ ] [ ] [ ] [ ] [ ] [ ] . patients with hypermorphic gain of function mutations in nfkbia [ ] [ ] [ ] , resulting in reduced degradation of the iκbα protein, have also been shown to exhibit significant combined immune deficiencies. furthermore, the ikk/nf-κb pathway operates in both hematopoietic and non-hematopoietic cells, leading to the extra-immune findings seen in patients with defects in nemo and iκbα, such as ectodermal dysplasia and colitis. similar to patients with ikbkg and nfkbia mutations, the first four patients reported by our group with deficiency of ikkβ due to the c. dupg ikbkb mutation also presented with immune deficiency [ ] . since then, six additional patients from various ethnic backgrounds (including turkey, qatar, and the arabian peninsula) with different nonsense ikbkb mutations have been reported, with all patients presenting with early onset and profound combined immune deficiencies, but typically without findings of ectodermal dysplasia [ ] [ ] [ ] . in our initial report, patients had normal to near-normal numbers of t-and bcells of an almost exclusively naïve phenotype, severe hypogammaglobulinemia, and activation defects to a variety of stimuli in a number of immune cells, both innate and adaptive, more suggestive of a global functional defect as opposed to a quantitative lymphocyte deficiency [ ] . herein we describe the clinical presentation, immunologic features, and hsct outcomes for the largest cohort of infants with ikbkb immune deficiency resulting from complete loss of ikkβ expression published to date. the study protocol was approved by the health research ethics board of the university of manitoba. the authors identified all known (genetically confirmed) or highly suspected infants with the ikbkb mutation managed through the department of pediatrics, winnipeg children's hospital, cancercare manitoba and the manitoba blood and marrow transplant program dating to the s. data collected by retrospective chart review included clinical presentation, immunologic features, and hsct approaches and outcomes. although data was complete for patients diagnosed in a contemporary era (after ), data availability was variable for some patients before this . young infants of northern cree descent from the known affected communities presenting with a clinical picture consistent with a severe immune deficiency but without genetic confirmation of an ikbkb mutation were considered highly suspected if they also had a close familial relationship to a genetically confirmed infant with the mutation (e.g. sibling, or second-degree relative where there was known consanguinity within the family). we chose to report the decade of diagnosis (instead of year) to provide the reader with perspective on the immunologic investigations and treatments available at the time, while protecting against the potential for inadvertently identifying individuals, given that patients and their families reside in small communities. detailed family trees documenting consanguineous relationships within families have not been included for the same reason. patient laboratory values, including absolute lymphocyte count, cd , cd and cd t-cell counts, cd b-cell counts, cd nk-cell counts, and immunoglobulin levels were compared against published, age-adjusted variables [ , ] . laboratory parameters are described as low, normal, or high with respect to these ranges. maternal engraftment of t-cells was assessed by analysis of short tandem repeats using the ampf str® profiler plus™ pcr amplification kit from life technologies. chimerism analysis after hsct was performed on t-cells, b-cells, and myeloid cells labeled with monoclonal antibodies to cd , cd , and cd / b and separated using an automated immunomagnetic cell separation method with the robosep instrument from stemcell technologies. due to a potential paucity of cells with each chimerism test, purity of the various lymphocyte and myeloid subsets was not evaluated with each assay. from validation experiments, however, the purity of separated subsets is > % with a ± % error around lineage specific chimerism results. t-cell proliferative responses using pha were performed by hthymidine incorporation assay of peripheral blood mononuclear cells after stimulation with pha at : - : following a -day culture. percentage of normal pha response was measured by the stimulation index of the patient relative to the average stimulation index of two concurrently run healthy adult control samples. trec analysis, when performed at the time of diagnosis (n = ), was either done locally in our laboratory (n = ) [ ] or at a clinical laboratory (mayo clinical laboratories) (n = ). units of measure for trecs (copies/μl whole blood, copies/million pbmcs, copies/million cd cells) changed over time. sanger sequencing for the c. dupg in exon of ikbkb was performed locally on a genetic analyzer™ as per the method developed by pannicke [ ] . samples containing homozygous g insertions within the region of interest were classified as affected, and confirmed by sanger sequencing at an independent laboratory. sixteen patients with genetically confirmed (n = ) or highly suspected (n = ) ikbkb mutations were included (table ) . genetic confirmation for the five infants with highly suspected ikbkb immune deficiency was not possible since all had died, and dna was not available (all were born in years before the ikbkb mutation was known). one patient was diagnosed immediately after birth by direct ikbkb mutational analysis of a guthrie card, as part of a targeted newborn screening project started in november in two of the heavily affected communities. this patient's younger sibling was later diagnosed in-utero, due to the known family history, with confirmation of the mutation after birth. both siblings were placed in protective isolation and proceeded to hsct by - months of age. the other fourteen patients presented to health care facilities with severe infections at a median of . months of age (range: weeks - months). twelve patients came from the province of manitoba and four from the province of saskatchewan. four patients were previously reported in less detail in our initial report of ikbkb immune deficiency [ ] . we included these patients in order to describe additional clinical and immunologic characteristics and provide updates on long-term outcomes. details on the number and type of infections present at the time of initial presentation (before hsct) were complete for twelve and incomplete for two (patient who died of disseminated mycobacterium bovis-bacillus calmette-guerin (bcg) and patient who died of unknown sepsis). the two siblings identified by newborn screening and family history had no infections at the time of diagnosis of ikbkb immune deficiency and proceeded to urgent hsct without infections. for patients not identified by newborn screening, clinical presentation consisted of a multitude of severe bacterial, fungal, mycobacterial, and viral infections beginning at an early age (< months), usually in association with failure to thrive. mucocutaneous infection of the oropharynx and perineum with candida was common (n = ) with evidence of disseminated candida infection table clinical presentation and outcome of ikbkb immune deficiency. no. scid was suspected but since ikbkb mutation had not yet been described diagnosis was never confirmed. no. multi-organ failure present. not hct candidate. died after hct in the blood (n = ), urine (n = ), and cerebrospinal fluid (n = ) in three. severe, recurrent gram-negative and gram-positive bacterial infections were frequent and included bacteremia / sepsis episodes with escherichia coli (n = ), pseudomonas aeruginosa (n = ), klebsiella pneumoniae (n = ), enterococcus species (n = ), staphylococcus aureus (n = ), stenotrophomonas maltophilia (n = ), serratia marcescens (n = ), streptococcus pneumoniae (n = ), staphylococcus hominis (n = ), listeria monocytogenes (n = ), morganella morganni (n = ), leuconostoc lactis (n = ) and enterobacter cloacae (n = ). intracranial infections were present in six infants and included a combined staphylococcus aureus and candida albicans meningitis in one, and listeria monocytogenes meningitis, enterococcus meningitis, klebsiella pneumoniae meningitis, serratia marcescens intracerebral abscesses, and mycobacterium bovis meningitis with intracerebral abscesses in the other five. disseminated viral infections occurred with cytomegalovirus (n = ), adenovirus (n = ), varicella (n = ), and herpes simplex virus (n = ). severe pneumonia syndromes were documented in eleven as result of unknown pathogens (n = ), cytomegalovirus (n = ), respiratory syncytial virus (n = ), polymicrobial gram negative bacteria (n = ), adenovirus (n = ), rhinovirus (n = ), and parainfluenza (n = ). consistent with standard practice in the two affected communities at the time, four infants received the bcg vaccination after birth, all of whom developed disseminated bcg and died. biopsies (n = ) and autopsies (n = ) revealed mycobacterium bovis in multiple organs including the lungs, liver, spleen, lymph nodes, thymus, bone marrow, adrenal glands, kidneys, eyes, skin, brain and gastrointestinal tract. following these deaths, bcg vaccination in the two affected communities was discontinued. more recently, with the advent of targeted newborn screening for the homozygous ikbkb mutation in the two affected communities, bcg vaccination after birth is held until results of the newborn screen confirm that an infant is not affected. one patient met clinical and laboratory criteria for hemophagocytic lymphohistiocytosis (hlh), although it is possible that additional patients would have met hlh criteria but were unrecognized. despite the connection of ikbkb to the ikk/nemo complex, none of our patients exhibited ectodermal dysplasia. only one patient had documented organ abnormalities (dysplastic kidney at time of autopsy). of the sixteen ikbkb immune deficiency patients, eight died of overwhelming infection before hsct could be performed, and eight proceeded to hsct. detailed autopsy reports were available for three who died prior to hsct. aside from evidence of infection, all exhibited severe thymic hypoplasia with scant lymphoid cell populations, minimal to no lymph node and tonsillar tissue, and absence of germinal follicles in the spleen. a range of immunologic tests were performed over time, with the extent and type of testing dependent upon the year of presentation and initial suspicion of an immunodeficiency diagnosis. extensive immunologic investigations were more likely to occur in the contemporary era (after ). median total lymphocyte count at the time of diagnosis was . × /l (range: . - . × /l), with / patients exhibiting normal (n = ) to elevated (n = ) total lymphocyte counts for age. the three lymphopenic infants all had severe infections (disseminated bcg in two and klebsiella sepsis in one) at the time their initial blood work was performed, along with various lymphocyte subset and hematologic cytopenias (anemia, thrombocytopenia), more consistent with bone marrow suppression from infection. median cd t-cell count ( patients) was cells/μl (range: - cells/ μl), with only one patient having a cd t-cell count < cells/μl t-cell proliferative responses were available for patients (fig. ). relative to controls, three had pha responses < %, two had pha responses between and %, and eight had pha responses above % (range: % - %). no relation was evident between pha response and total cd t-cell count, although two of the patients with severe cd t-cell lymphopenia and disseminated bcg had no pha response. the two infants identified at birth without infections both had normal pha responses ( % and %). trecs were performed in six infants. all had normal trec expression levels ( copies/ pbmcs; , copies/ cd ; , copies/ cd ; copies/μl; copies/μl; copies/ μl). more extensive t-cell immune phenotyping was performed in three patients. all exhibited a preponderance of naïve cd t-cells (cd + cd ra + cd l + cd +) between %- % of the cd t-cell population, and naïve cd t-cells (cd + cd ra + cd l + cd +) between and % of the cd t-cell population. minimal to no central and effector memory t-cells ( - %) were present in any of the cases. one patient had tcr v-beta repertoire performed, revealing a normal polyclonal t-cell population. eight patients underwent hsct, using either bone marrow from a human leukocyte antigen matched parent or sibling (n = ) or unrelated umbilical cord blood (n = ) as the graft source (table ) . two patients not initially receiving a conditioning regimen (patients and ) due to critical illness from infections both failed to engraft, with a second transplant performed in one using myeloablative conditioning, resulting in subsequent engraftment. conditioning regimens were initially used in the other six. two patients ( and ), one of whom received a reduced intensity conditioning regimen followed by a cord blood transplant, initially engrafted but experienced secondary graft failure over two years post-hsct. four patients received a busulfanbased myeloablative conditioning regimen (with busulfan pharmacokinetic monitoring after ) with either full or mixed donor chimerism, and generally more favorable myeloid engraftment. three patients receiving transplant remain alive -months, -years and -years post-hsct. two patients died soon after hsct (days + and + ) from complications related to infections present at the time of transplant. three others died of new infections acquired post-hsct, including two (patients and ) who died of disseminated mycobacterium avium-intracellulaire infection in association with secondary graft failure at -and -years post-hsct. the third (patient ) died of streptococcus pneumoniae bacteremia and meningitis despite full donor chimerism at -months post-hsct. new clinically relevant infections, including viral, bacterial, fungal, and mycobacterial infections, were common in patients who survived at least -months after transplant, and were still occurring years after transplant despite full and mixed lymphoid and myeloid donor chimerism and normal neutrophil and lymphocyte counts. such infections included streptococcus pneumoniae bacteremia (n = ), meningitis (n = ), osteomyelitis (n = ), and septic arthritis (n = ); staphylococcus aureus bacteremia, cellulitis, and osteomyelitis (n = ); salmonella bacteremia and enterocolitis (n = ); and pseudomonas aeruginosa bacteremia (n = ). viral infections (e.g. influenza, parainfluenza, varicella, adenovirus) also occurred, with resolution in all patients. one patient developed mycobacterial tuberculosis pneumonia at -year post-hsct, was successfully treated with anti-tuberculosis medications, and remains a long-term survivor. all patients achieved normal numbers of cd +, cd +, cd +, cd +, and cd + cells post-hsct. immunoglobulin replacement was variable between patients and generally continued between -and -months post-hsct. pha responses were normal (above %) for all patients. despite these findings, specific antibody production to posttransplant vaccinations once immunoglobulin was discontinued was poor. for instance, patients and did not make anti-pneumococcal fig. . phytohemagglutinin responses at the time of ikbkb diagnosis by patient number. percent of control is defined as the stimulation index of the patient relative to the average stimulation index of two healthy controls. corresponding cd t-cell count (cells/μl) at the time of diagnosis is under each patient number. patients and both had disseminated bcg with bone marrow involvement and severe cytopenias, including lymphopenia, which may have impacted pha response. hematopoietic stem cell transplant outcomes for ikbkb immune deficiency. patient: detailed autopsy reports were available for patients after hsct. aside from evidence of infection, both exhibited severe thymic hypoplasia with scant lymphoid cell populations, minimal to no lymph node and tonsillar tissue, and absence of germinal follicles in the spleen. patient , who experienced secondary graft failure by -years post-hsct, developed disseminated mycobacterium avium-intracellulaire and was managed for -year with anti-tuberculous medications and splenectomy before death. the spleen was entirely replaced by atypical mycobacteria. at autopsy, performed -years after hsct, the thymus was small and atrophic, weighing only g, and lymph nodes were not readily apparent. this series presents the clinical features, immunologic presentation, and hsct outcomes for the largest cohort of ikbkb immune deficiency patients published to date. a number of important conclusions can be made. first, the clinical presentations in our case series indicate that ikbkb immune deficiency, as result of profound defects in innate and adaptive immunity, manifest as exceptional susceptibility to a variety of different but severe infections, including bacteria, viruses, fungi, and mycobacteria. we believe that ikbkb immune deficiency, although not meeting conventional laboratory-based diagnostic criteria for scid [ ] (due to higher cd t-cell counts, moderately decreased to normal pha responses, normal trecs, high proportions of naïve cd ra+ t-cells, and low cd ro+ t-cells in ikbkb immune deficiency), is as severe an immune deficiency as scid at a clinical level. we would suggest therefore, that inclusion of ikbkb deficiency under the descriptive category of "combined immune deficiencies generally less profound than scid" in the most recent international union immunologic societies classification [ ] , likely underestimates the severity of the immune deficiency and should be modified in future iterations. supporting a more profound immune deficiency are the other six reported patients with ikbkb mutations, who also presented with severe bacterial, fungal and viral infections as young infants. mycobacterial infection was also prominent, with disseminated bcg noted in four of the six [ ] [ ] [ ] . similar to our cohort, previously reported ikbkb immune deficient patients exhibited normal to elevated absolute t-cell and b-cell numbers, low cd ro+ memory t-cells, and absent or low class-switched memory b-cells. immunoglobulins were universally low or undetectable, but proliferative responses to pha were present and typically normal. only one patient was noted to have features of ectodermal dysplasia at an older age ( months) [ ] , a feature which we have not seen in any of our three post-hsct survivors. two of the other reported patients died from overwhelming infection prior to definitive therapy, and four received hsct, with only one long-term survivor. secondly, this data further expands the understanding of primary immune deficiencies involving aberrant ikk/nf-κb signaling. at least patients with germline gain of function mutations in nfkbia have been reported, with a severe but more variable clinical and immune phenotype that appears dependent upon the genotype (with missense mutations resulting in a more severe clinical phenotype) [ ] . like ikbkb immune deficiency, patients with hypermorphic nfkbia mutations that result in reduced degradation of iκbα, present with multiple and severe bacterial, fungal and viral infections starting at an early age, typically before -months. infections reported include invasive bacteria such as klebsiella, pseudomonas, haemophilus influenzae, and staphylococcus aureus, mycobacteria (including vaccine-strain bcg), as well as chronic mucocutaneous candidiasis, pneumocystis pneumonitis, and severe viral infections (rotavirus, norovirus, parainfluenza, rsv, cmv). similarly, patients with nfkbia mutations present with normal to elevated t-cell counts, an excess of naïve cd and cd cells, and normal mitogen proliferative response to pha in most. dysgammaglobulinemia (usually with one or more quantitatively abnormal immunoglobulin isotypes), impaired response to vaccine antigens, and when tested, absent or reduced memory b-cells and class switched memory b-cells have also been reported. unlike patients with ikbkb immune deficiency, however, almost all patients with iκbα defects show some degree of ectodermal dysplasia. by comparison, individuals with hemizygous hypomorphic ikbkg mutations with reduced nemo expression manifest a more variable clinical and immunologic phenotype when compared to patients with ikbkb immune deficiency and hypermorphic nfkbia mutations [ ] . in addition to infectious predisposition, patients with nemo deficiency may present with autoimmunity ( %) and inflammatory conditions (most notably inflammatory bowel disease in %), features not described in either our cohort of ikbkb immune deficient patients nor in those with hypermorphic iκbα defects. as in patients with ikbkb immune deficiency, patients with nemo deficiency commonly present with infections due to pyogenic bacteria ( %), mycobacteria ( %), viruses ( %) and fungal and opportunistic pathogens ( %) [ ] the median age at hsct for a large cohort of nemo deficient patients was . years old (range: . months - . years), an age that is older compared to our cohort, suggesting nemo deficiency may be a less severe immune deficiency relative to ikbkb immune deficiency [ ] . nemo deficiency also has a more variable immunologic phenotype, with hypogammaglobulinemia in only / ( %) and defects in specific antibody production in / ( %). similar to ikbkb immune deficiency, most patients with nemo deficiency have normal to elevated cd and cd counts, and % have normal mitogen induced proliferations [ ] . a third important finding of this study is that scid newborn screening by trec assays will not detect ikbkb immune deficiency. given the severe clinical phenotype of ikbkb immune deficiency, a pilot project of targeted newborn screening by direct genetic mutation analysis of guthrie cards for the ikbkb mutation was developed in select northern cree communities in manitoba. this investigation identified a carrier frequency of in . newborns, with a predicted mutation homozygosity of / births [ ] . over -years ( - ), newborns from two communities were screened, with one infant identified at birth (patient ) and a subsequent sibling (patient ) identified in-utero. both were placed in protective isolation after birth and received hsct at an early age, before the onset of serious infections, important variables associated with excellent outcomes in scid hsct [ , ] . in other populations with founder mutations or high rates of consanguinity, a similar genetic-based newborn screening strategy such as ours might be applicable. unfortunately, patient later died of invasive streptococcus pneumoniae infection in their home community, -months following an otherwise successful transplant. this illustrates a complex interplay between what we suspect are ongoing defects in immunity post-hsct, combined with poor social determinants of health. for familial and cultural reasons, our patients often have strong desires to return home soon after hsct, and the tertiary care hsct center is not easily accessible from their remote communities. with the death of patient , and with the recent transplant of patient , we have instituted a program of housing the family close to the tertiary care center for at least . years post-hsct, as well as regular immunoglobulin replacement and indefinite daily anti-pneumococcal antibiotic prophylaxis. much remains unknown about the immune abnormalities in patients with ikbkb immune deficiency, both before and after hsct. from our previous investigations, the broad immune defects relate to abnormal ikk/nf-κb signaling in t-cells, b-cells and innate immune cells, resulting in generalized activation defects to a variety of stimuli [ ] . with the exception of regulatory t-cells and gamma-delta t-cells, which are absent in ikbkb immune deficiency, other lymphocyte subsets appear to develop normally. despite thymic hypoplasia in five of our patients who died either prior to or after hsct, six other patients had normal trec levels at the time of diagnosis, suggesting no intrinsic defect in t-cell development and maturation within the thymus. unfortunately, none of the patients who had autopsies performed had detailed trec analysis, making direct correlation with the underdevelopment of the thymus and trec levels in a single patient impossible. detailed t-cell studies were not available for most of our patients pre-hsct, but when previously studied in one patient (and in one additional patient in this series), the t-cell receptor beta-chain variable region repertoire was polyclonal [ ] . we are unable to resolve this apparent contradiction in the autopsy and immunologic findings, and believe it warrants further study. likewise, when studied in two patients, kappa-deleting recombination excision circles (krecs) in dried blood spots from newborn guthrie cards of two patients were found to be normal, suggesting normal b cell development. in a mouse model involving a hypermorphic mutation of iκbα, nf-κb signaling in non-immune "architectural" cells were shown to be severely disrupted [ ] . although such cells are not derived from hematopoietic stem cells, they are nonetheless integral to lymphoid organogenesis, such as in the development of lymph nodes, peyer's patches, splenic marginal zones, follicular dendritic cells, and the formation of germinal centers in the spleen, which were absent. further, the immune defect in this model was not fully corrected by hsct. it is reasonable to suspect, therefore, that similar architectural cell dysfunction with resultant disruption of the immunologic niche could also play a role in ikbkb immune deficiency. failure to correct non-immune cells with hsct could explain the ongoing infectious complications seen years after an otherwise apparently successful, lymphoid engrafted transplant in ikbkb immune deficiency. the absence of splenic germinal centers and lack of tonsils and lymphoid tissues in the autopsies of patients who died post-hsct, and absent specific vaccine responses despite excellent donor t-and b-cell chimerism, supports that hsct does not fully correct all aspects of the immune deficiency in ikbkb. in years past, and in patients who were profoundly ill at presentation, full immune evaluations were not always available to further elucidate the nature of the immune deficiency in this population. at the moment, there are only three survivors in our cohort, for whom detailed t cell studies pre-and post-hsct (including serial measurements of t cell markers such as recent thymic emigrants, markers of naivety and memory cells and markers of activation or exhaustion) have been limited to date. moving forward, and with genetic-based newborn screening introduced for this condition in our province, we will now have this opportunity. despite these challenges, we believe that ikbkb immune deficiency is still a disorder that requires hsct. to our knowledge, no individual has been a long-term survivor of ikbkb immune deficiency without hsct. we are unaware of any reports of hypomorphic ikbkb mutations producing a milder clinical phenotype, allowing clinicians to take a wait and see approach. hsct for other disorders involving the ikk/nf-κb pathway have been previously described, including for hemizygous hypomorphic ikbkg mutations [ ] and hypermorphic nfkbia mutations [ ] . in the cohort of fourteen iκbα gain of function patients reported by boisson et al., eleven received hsct, with only five survivors. one patient died without receiving hsct, and two patients survived without receiving hsct. since most centers will not have genetically based newborn screening for ikbkb immune deficiency, clinicians would likely be faced with an infant presenting with severe life-threatening infections, given that trec based newborn screening will not detect the disorder. commercially available next-generation sequencing gene panels for immune deficiency disorders (including many scid panels) increasingly include ikbkb, which should facilitate diagnosis, although not always in a timely manner before hsct. in our series, two of the three patients surviving hsct had serious life-threating infections before transplant, including a case of multiple serratia marcescens intracerebral abscesses, where the long-term neurocognitive outcome was initially unclear. we have tended to be aggressive with antimicrobials and other supportive care measures, with the goal of reaching hsct as quickly as possible. this strategy has resulted in long-term survival of / infants receiving hsct with active infections. that being said, we suspect that survival will be significantly improved with current newborn screening strategies, in the absence of pre-hsct infection. unfortunately, and based on this limited dataset, we believe myeloablative conditioning may be required for engraftment in ikbkb immune deficiency. the largest and most recent study of hsct in nemo patients, however, did not find this to be necessarily true [ ] . in their cohort of fifteen patients receiving classical myeloablative conditioning and thirteen patients receiving reduced intensity conditioning, the global engraftment rate was %, with similar survival rates and secondary graft failure rates, independent of conditioning regimen intensity. our center currently uses once daily intravenous dosing of busulfan for days (aiming for an area under the curve approximating μmol/l*min), fludarabine . mg/kg/dose for days, and rabbit anti-thymocyte globulin . mg/kg/dose for days. we acknowledge other conditioning regimens might also be efficacious, although caution that late secondary graft-failure appears to be a concern in ikbkb immune deficiency. finally, and importantly, clinicians should be mindful that despite apparently reasonable basic laboratory immune reconstitution data for ikbkb immune deficiency patients post-hsct (e.g. t-cell counts, chimerism studies), late infections, including bacterial and mycobacterial infections in particular, appeared to be common. of interest, one of our long-term survivors who was fully engrafted developed primary mycobacterium tuberculosis pulmonary disease at -months post-hsct. with appropriate therapy, the infection resolved, suggesting hsct corrected enough of the immune deficiency. in contrast to our post-hsct patients with ikbkb immune deficiency, miot et al. found that bacterial infections only recurred at > -months post-hsct for nemo deficiency in / patients [ ] . we hypothesize that failure of hsct to improve all aspects of innate and adaptive immune function after transplant, as shown by the number of ongoing serious bacterial and mycobacterial infections and lack of protective specific antibody responses to vaccinations, could be because hsct does not correct cells of non-hematopoietic origin important in the immune response that are also dependent upon ikk/nf-κb signaling. a similar pattern of incomplete immune correction and ongoing bacterial and viral infections after hsct has been reported in a patient with the related autosomal dominant anhidrotic ectodermal dysplasia with immune deficiency due to hypermorphic iκbα defect [ ] . in summary, we present a relatively large cohort of a rare primary immunodeficiency disorder, ikbkb immune deficiency, characterized by the presence of naïve t-cells, evidence of a functional activation defect, and amenable to hsct, although with ongoing long-term infectious challenges. deficiency of innate and acquired immunity caused by an ikbkb mutation regulation and function of nf-kappab transcription factors in the immune system rare mendelian primary immunodeficiency diseases associated with impaired nf-kappab signaling x-linked susceptibility to mycobacteria is caused by mutations in nemo impairing cd -dependent il- production nemo mutations in unrelated boys with severe infections and conical teeth irak and nemo mutations in otherwise healthy children with recurrent invasive pneumococcal disease infectious diseases in patients with irak- , myd , nemo, or ikappabalpha deficiency a novel x-linked disorder of immune deficiency and hypohidrotic ectodermal dysplasia is allelic to incontinentia pigmenti and due to mutations in 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establishing diagnostic criteria for severe combined immunodeficiency disease (scid), leaky scid, and omenn syndrome: the primary immune deficiency treatment consortium experience international union of immunological societies: primary immunodeficiency diseases committee report on inborn errors of immunity human ikappabalpha gain of function: a severe and syndromic immunodeficiency hypomorphic nuclear factor-kappab essential modulator mutation database and reconstitution system identifies phenotypic and immunologic diversity hematopoietic stem cell transplantation in patients hemizygous for hypomorphic ikbkg/nemo mutations newborn screening for ikbkb deficiency in manitoba using genetic analysis immune reconstitution and survival of scid patients post-hematopoietic cell transplant: a pidtc natural history study transplantation outcomes for severe combined immunodeficiency defective lymphoid organogenesis underlies the immune deficiency caused by a heterozygous s i mutation in ikappabalpha successful allogeneic hemopoietic stem cell transplantation in a child who had anhidrotic ectodermal dysplasia with immunodeficiency we would like to thank all of the patients and their families; the nursing staff (debbie hanson, jennie pitura, shannon cisneros) who have taken care of the patients; drs. ulrich pannicke, stephan ehl, and klaus schwarz who were instrumental in the discovery of the ikbkb gene; drs. cheryl greenberg and paul van caeseele for their contribution to the newborn screening project for ikbkb deficiency; and luvinia kwan and agapito gentile for performing the ikbkb mutational analysis.this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. key: cord- -edliu it authors: zhou, hui; qian, qi; shu, ting; xu, jiuyue; kong, jing; mu, jingfang; qiu, yang; zhou, xi title: hepatitis c virus ns protein suppresses rna interference in cells date: - - journal: virol sin doi: . /s - - - sha: doc_id: cord_uid: edliu it rnai interference (rnai) is an evolutionarily conserved post-transcriptional gene silencing mechanism and has been well recognized as an important antiviral immunity in eukaryotes. numerous viruses have been shown to encode viral suppressors of rnai (vsrs) to antagonize antiviral rnai. hepatitis c virus (hcv) is a medically important human pathogen that causes acute and chronic hepatitis. in this study, we screened all the nonstructural proteins of hcv and found that hcv ns could suppress rnai induced either by small hairpin rnas (shrnas) or small interfering rnas (sirnas) in mammalian cells. moreover, we demonstrated that ns could suppress rnai via its direct interaction with double-stranded rnas (dsrnas) and sirnas, and further identified that the cysteine of ns is required for the rnai suppression activity through a serial of point mutation analyses. together, our findings uncovered that hcv ns can act as a vsr in vitro, thereby providing novel insights into the life cycle and virus-host interactions of hcv. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. hcv is a positive-sense, single-stranded rna virus that belongs to the genus hepacivirus of the family flaviviridae. its genome is approximately . kb in length and encodes a polyprotein that is cleaved by host and viral proteases to produce ten mature viral proteins, including structural core protein, envelope proteins e and e , the p ion channel protein, and nonstructural (ns) proteins ns , ns , ns a, ns b, ns a and ns b (yin et al. ) . hcv infection causes acute and chronic hepatitis in humans with a high propensity for chronicity. a large number of hcv-infected patients fail to clear this virus and remain asymptomatic for years to develop severe liver diseases such as cirrhosis and hepatocellular carcinoma (paboriboune et al. ) . the capability of establishing chronic infection by hcv is partly due to its ability to evade host innate immune responses (horner and gale ) . rnai is an evolutionarily conserved mechanism in all eukaryotes for post-transcriptional gene silencing. rnai serves as an important antiviral immune response in a wide range of organisms, including fungi, plants, and invertebrates (guo et al. ; maillard et al. ) . moreover, recent studies have demonstrated that rnai also exerts antiviral effects in mammals maillard et al. ; li et al. ; qiu et al. ; xu et al. ) . the antiviral rnai is triggered by the viral replicative intermediate double-stranded rnas (vri-dsrnas) produced in the course of viral replication. these vri-dsrnas can be recognized and cleaved by host endoribonuclease dicer into virus-derived small interfering rnas (vsirnas) of approximately - nucleotide (nt). argonaute (ago) protein within rna-induced silencing complexes (riscs) electronic supplementary material the online version of this article (https://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users. incorporates one of the strands of vsirna duplex, which directs the risc-mediated degradation of cognate viral rnas (maillard et al. ) . to counteract the antiviral rnai, viruses have evolved to encode viral suppressors of rnai (vsrs) targeting various steps of the rnai pathway (wu et al. ) . one common strategy utilized by vsrs, including nodamura virus (nov) b , ebola virus (ebov) vp , influenza a virus (iav) ns , and human enterovirus (ev-a ) a, is to prevent viral dsrna from cleavage by dicer (li et al. ; sullivan and ganem ; haasnoot et al. ; qiu et al. ) . alternatively, multiple vsr proteins have been shown to directly target the key components of the rnai pathway, such as dicer or ago proteins. for example, wuhan nodavirus (whnv) b can directly bind to drosophila dicer- to inhibit vsirna production (qi et al. (qi et al. , , while cricket paralysis virus a has been found to directly inhibit drosophila ago (nayak et al. ) . in the case of hcv, the core protein was found to suppress rnai by interacting with dicer (chen et al. ) . moreover, hcv structural e protein was also identified to antagonize rnai by targeting ago (ji et al. ) . consistent with the findings in hcv, a number of rna viruses were shown to encode more than one viral protein to counteract rnai. for instance, vp , vp and vp of ebov were found to suppress rnai through different mechanisms (haasnoot et al. ; fabozzi et al. ) . severe acute respiratory syndrome coronavirus (sars-cov) encoded a and nucleocapsid contain rnai suppression activities (karjee et al. ; cui et al. ) . besides, tat and nef proteins of human immunodeficiency virus- (hiv- ) were identified to suppress rnai through the dsrna-binding and ago interaction, respectively (bennasser et al. ; aqil et al. ) . the phenomena of viruses encoding multiple vsrs as well as vsrs targeting different steps of the rnai pathway highlight the importance of suppressing rnai during the viral life cycle. although the structural core and e proteins of hcv were shown to suppress rnai, it is unclear if any hcv nonstructural protein contains rnai suppression activity. in this study, we uncovered that hcv nonstructural ns protein possessed a potent in vitro vsr activity that suppressed the rnai induced by short hairpin rna (shrna) and sirna in mammalian cells. we also confirmed the conserved residue in hcv ns that could disrupt its activity to suppress rnai. overall, our findings demonstrated that hcv ns contains vsr activity, thereby providing insights into the life cycle of hcv. for expression of hcv nonstructural proteins including ns , ns , ns / a, ns a, ns b, ns a and ns b in hek t cells, their orfs were cloned into the prk-flag, respectively. the templates expressing hcv proteins were kindly provided by prof. ying zhu (wuhan university, china). the point mutations were introduced into the ns coding region by pcr-mediated mutagenesis with the appropriate primers. for expression of recombinant ns in sf cells, the orfs of ns and its mutant were constructed into the vector pfastbac htb-mbp as previously described (yang et al. ) . the resulting plasmids were subjected to the bac-to-bac baculovirus expression system to express the fusion proteins with an mbp tag at the n-terminus. the egfp-sirna was chemically synthesized by guangzhou ribobio co., ltd., china. all the primers and oligonucleotides used in this study are shown in supplementary table s . hek t, t-nodice (kindly provided by prof. bryan cullen, durham, nc, usa) and huh . cells were maintained in dulbecco's modified eagle's medium (dmem, gibco) containing % fetal bovine serum (fbs) (gibco), u/ml penicillin and lg/ml streptomycin at °c in an incubator with % co . cells were seeded in -well plates and grown overnight to reach % confluence. before transfection, the medium was changed to dmem containing without serum and antibiotic. cells then were transfected with the indicated plasmids by using fugene hd reagent (roche, basel, switzerland) according to the manufacturer's instructions. cells were harvested in lysis buffer [ mmol/l tris-hcl (ph . ), mmol/l nacl, % np , . % deoxycholate and a protease inhibitor cocktail (rhoche)]. then the lysates were subjected to % sds-page and western blotting analysis. the antibodies used in this study are as follow: anti-tubulin (proteintech group, : ), anti-flag (proteintech group, : ) and anti-myc (proteintech group, : ). total cellular rnas were extracted using trizol reagent (thermo) according to the manufacturer's instructions. for virologica sinica the detection of egfp mrna, lg of total rnas were subjected to denatured . % agarose gels with . mol/l formaldehyde. the separated rnas were transferred onto the hybond-a nylon membrane (ge healthcare), and fixed at °c for min. then the membranes were hybridized with dig-labeled probes in hybridization ovens at °c overnight. finally, the membranes were incubated with anti-dig antibody conjugated with alkaline phosphatase and exposed to the luminescent image analyzer las (fuji film). the probes for detection of egfp and gapdh mrna were complementary to their orf region of - nt and - nt, respectively. for detection of small rnas, lg of total rnas were subjected to mol/l urea- % page and transferred to hybond-a nylon membrane (ge healthcare). the membrane was chemically cross-linked in -ethly- -( dimethylaminopropyl) carbodiimide (edc) at °c for min. the dig-labeled rna probes targeting egfp sirna and u were synthesized by takara. the expression and purification of mbp alone and mbpfusion proteins were performed in sf cells using baculovirus expression system as previously described (yang et al. ) . briefly, cells were infected with mbp-tagged hcv ns expressing baculoviruses for h. infected cells were resuspended, lysed via sonication and then centrifuged at , g for min to remove debris. the protein in the supernatant was purified using amylose affinity chromatography (new england biolabs, ipswich, ma) according to the manufacturer s protocol and then concentrated using amicon ultra- filters (millipore, schwalbach, germany). all purified proteins were quantified with a bicinchoninic acid (bca) protein assay kit (cwbio, china) and stored at - °c in aliquots. proteins were separated on % sds-page and visualized by coomassie blue. we generated -nt dig-labeled dsrna and -nt sirna via in vitro transcription using dig rna labeling mix (roche). mbp-fusion ns or mutant proteins were reacted with dig-labeled rnas ( . lmol/l -nt dsrna or -nt sirna) in a binding buffer [ mmol/l hepes (ph . ), mmol/l nacl, . mmol/l mgcl , % glycerol and u of rnase inhibitor (promega)] at °c for min; the total volume was ll. then the reaction mixtures were separated on % (for dsrna) or % (for sirna) tbe-page and transferred to hybond-a nylon membrane (ge healthcare). the membranes were incubated for min with anti-dig antibody conjugated with alkaline phosphatase (roche). hek t cells were lysed in a lysis buffer containing mmol/l tris-hcl (ph . ), mmol/l nacl, . mmol/l mgcl , . % triton x , . u/ll rnase inhibitor (promega) and a protease inhibitor cocktail (roche). after centrifugation for min at , g, the supernatant was pre-cleared via incubation with protein-a/ g agarose beads (roche) at °c for h. then the precleared lysates were incubated with antibodies (anti-flag, anti-myc or anti-igg as a negative control) together with protein-a/g agarose beads (roche) at °c for h. the antibody-bound complexes were washed for five times with the same lysis buffer except that nacl concentration was raised to mmol/l. finally, rnas were extracted using trizol reagent (thermo) according to the manufacturer s protocol. to identify whether hcv has any nonstructural protein which works as a potential vsr, we screened them via the reversal-of-silencing assay in hek t cells (fig. a) . in brief, cells were co-transfected with the plasmids encoding egfp and egfp-specific shrna (shegfp), together with the vectors for divers hcv nonstructural proteins. nov b (nb ), a well-characterized vsr was used as a positive control. the expression of the hcv-encoded proteins and nb were detected by western blotting with anti-flag and anti-myc antibodies (fig. b) . at h post transfection (hpt), the mrna levels of egfp were detected by northern blotting with a digoxigenin (dig)-labeled rna probe targeting - nt of egfp orf. egfp-specific shrna can effectively eliminate the egfp transcripts (fig. a, lane ) . as shown in fig. a , hcv ns protein can effectively restore the expression of rnai-silenced egfp (lane ). expectedly, expression of nb or genetic ablation of dicer (nodice) suppressed shrna-induced rnai in hek t cells (fig. a, lanes and ) . importantly, hcv ns did not affect the transcription efficiency of egfp in the absence of shrna (fig. c) , excluding out the possibility that hcv ns can directly promote egfp transcription. we sought to examine whether the rnai suppression activity of hcv ns is dependent on the protein expression levels. thus, hek t cells were co-transfected with the plasmids for egfp and egfp-specific shrna, together with the increasing amount of ns plasmid as indicated. our findings showed that the reversal effect of egfp silencing increased progressively at the mrna levels, along with the growing amount of ns plasmid being transfected ( fig. a, b) . we further examined the vsr activity of hcv ns at different time points. our results showed that the reversal effect of egfp silencing could be observed at hpt (fig. c) , indicating that the vsr activity was dependent on the expression level of hcv ns protein. taken together, our findings showed that hcv ns displays vsr activity in mammalian cells. having established that hcv ns contains rnai suppression activity, we sought to examine the detailed mechanism through which hcv ns inhibits rnai. in the process of rnai, dsrna/shrna requires the dicer-mediated cleavage into sirna (maillard et al. ) . to investigate whether hcv ns can inhibit this step, small rnas harvested from hek t cells co-expressing egfp-specific shrna together with ns were subjected to northern blotting with a dig-labeled rna probe targeting egfp sirna produced from shrna by dicer. as shown in fig. a , the accumulation of dicer-cleaved sirna was reduced in the presence of ns compared to that in cells expressing empty vector, indicating that hcv ns can suppress dicer-mediated sirna production. these results are consistent with the previous findings that hcv ns effectively restored shrna-induced silencing of egfp transcript in hek t cells (fig. a) . in the process of shrna-induced rnai, shrna is cleaved by dicer into sirna. after identifying that hcv ns suppressed the sirna production in cells expressing shrna, we performed rna immunoprecipitation (rna-ip) assay to test whether hcv ns sequestrated dsrna in hek t cells. briefly, cells expressing flag-tagged ns , myc-tagged nb or empty vector, together with egfpspecific dsrna ( - nt of egfp orf) were lysed and immunoprecipitated with the indicated antibodies, respectively. rnas extracted from the rna-ip precipitates were examined via northern blotting with the rna probe targeting the -nt dsrna of egfp. as shown in fig. b , our findings show that ns can associate with dsrna in cells (lane ). we sought to examine whether hcv ns could directly bind to dsrna. to this end, we performed gel shift assays by incubating the recombinant mbp-fusion hcv ns (mbp-ns ; supplementary fig. s a ) and the in vitrotranscribed -nt dig-labeled dsrna. as shown in fig. c , the mobility of dsrna was inhibited in a dosedependent manner by the hcv ns protein (lanes - ) , compared to the control reaction with mbp protein alone. of note, mbp-fusion whnv b (mbp-wb ), another well-characterized vsr that contains the dsrna-binding activity, was used as the positive control (fig. c, lane ) . taken together, our findings indicate that hcv ns can suppress rnai by sequestrating dsrna. northern blotting with dig-labeled rna probe targeting the - nt of egfp orf. gapdh mrna was used as the loading control. b the expression of hcv proteins was detected by western blotting. arrow indicates the unrelated bands in the bot. c hek t cells were co-transfected with the plasmid encoding egfp, together with hcv ns or nov b plasmid, respectively. at hpt, egfp mrna levels were examined via northern blotting. we sought to examine whether hcv ns protein can inhibit rnai induced by sirna. to this end, hek t cells were co-transfected the plasmid for egfp and the chemically synthesized egfp-specific sirna (siegfp, nmol/l), together with the ns plasmid. egfp-specific sirna induces a significant reduction of egfp expression (fig. a, lane ) . our results showed that hcv ns efficiently restored the egfp mrna levels, indicating that hcv ns can inhibit sirna-induced rnai in mammalian cells (fig. a, lane ) . because hcv ns can directly bind to dsrna, we sought to determine whether ns suppressed sirna-induced rnai by directly binding to sirna. to this end, we conducted gel shift assay by incubating mbp-ns together with dig-labeled synthetic -nt sirna. as shown in fig. b , ns can directly bind to sirna in a dose-dependent manner. taken together, these results indicate that hcv ns can suppress rnai by sequestering sirna. after determining the vsr activity of hcv ns , we sought to identify the critical residues required for its rnai suppression activity. thus, we performed multiple sequence alignments of ns s encoded by multiple hcv isolates (supplementary fig. s b ). because the dsrna/ sirna-binding and protein dimerization are critical for vsr's activity (maillard et al. ) , the positivelycharged and highly conserved residues, including arginine (r), lysine (k), histidine (h), glutamic acid (e) and cysteine (c) were subjected to single-point mutation to alanine (a), and the resulting mutant ns proteins were then examined via the reversal-of-silencing assay in hek t cells (fig. a- c ). our findings show that the c a mutation (ns c a ) significantly suppressed the activity of hcv ns to suppress rnai (fig. c, lane and fig. d, lane ) . moreover, we further confirmed that the c is critical for the vsr activity of hcv ns via the reversal-of-silencing assay in huh . cell line (fig. e) , which is usually used for hepatitis c research. moreover, we found that c a abolished the dsrna-and sirnabinding activities of hcv ns in vitro (fig. a, b ). hek t cells were co-transfected with the plasmids encoding egfp ( . lg) and egfp-specific shrna ( . lg), together with the increasing amounts of the plasmid encoding hcv ns ( . lg, . lg and . lg, respectively). at hpt, egfp mrna levels were determined by northern blotting (a). gapdh mrna was used as the loading control. the mrna levels of egfp normalized to that of gapdh were quantified with bio-rad quantity one software and were graphed in (b) with that in the presence of the hcv ns ( . lg) defined as %. error bars represent sd values from the results of three independent experiments. *p \ . , as measured by two-way anova (graphpad prism). cell lysates were harvested and analyzed by western blotting with anti-flag, anti-myc and anti-tubulin antibodies. c hek t cells were co-transfected with plasmids encoding egfp ( . lg) and egfp-specific shrna ( . lg), together with either empty vector or the plasmid encoding hcv ns ( lg) or nb ( lg) as indicated. at , , and hpt, total rnas were extracted and the level of egfp mrna was examined by northern blotting. cell lysates were harvested and analyzed by western blotting with anti-flag, anti-myc and anti-tubulin antibodies. besides, we also found that ns c a failed to suppress the processing of shrna into sirna in hek t cells (fig. c ). rnai is an antiviral immune mechanism conserved in both invertebrates and mammals. as a counter-defense to rnai, viruses evolved to encode vsrs that were identified to target different steps of the rnai pathway. in the present study, we screened the viral nonstructural proteins of hcv for rnai suppression activity and found that hcv ns protein is a vsr. the gel shift and rna-ip analyses show that hcv ns possesses both dsrna-and sirna-binding activities that are required for rnai suppression. thus, we speculate that hcv ns may function as vsr by shielding virus-derived dsrna from dicer cleavage and binding to viral sirna to interfere with risc assembly. in addition to ns , hcv structural proteins core and e were also shown to contain rnai suppression activity by directly targeting the protein components of the rnai pathway (chen et al. ; ji et al. ) . hcv core interacts with dicer and inhibits the sirna production, while hcv e can block the risc assembly by interacting with ago . interestingly, we showed that hcv ns as a vsr can target the rna components of the rnai pathway by directly binding to dsrna and sirna. these results indicate that hcv can suppress antiviral rnai at different stages by encoding multiple vsrs. this phenomenon can also be found in other rna viruses, such as ebov, sars-cov, hiv- and denv (bennasser et al. ; karjee et al. ; fabozzi et al. ; aqil et al. ; kakumani et al. ; cui et al. ; kakumani et al. ) . thus, encoding multiple vsrs that target different steps of the rnai pathway may offer advantages in the mode of action required for efficient suppression of rnai, thereby highlighting the importance of the antiviral rnai in the hostvirus interactions. hcv ns protein is a * kda transmembrane protein that has multiple functions during the hcv replication. it contains a protease domain that functions as a cysteine protease to catalyze a cleavage between ns and ns (pieroni et al. ) . besides, ns colocalizes with e , e ns and ns a near the core protein and lipid droplet during the virion assembly . moreover, ns can work as a potent interferon antagonist and apoptosis inhibitor (erdtmann et al. ; kaukinen et al. ). here we found that hcv ns contains an rnai fig. c is critical for the vsr activity of hcv ns . a-d hek t cells were co-transfected with the plasmids encoding egfp ( . lg) and egfp-specific shrna ( . lg), together with either empty vector or the plasmids encoding different hcv ns mutants ( lg each) as indicated, at hpt, total rnas were extracted and the levels of egfp mrna were determined via northern blotting. t-nodice cells were used as a control. gapdh mrna was used as the loading control. cell lysates were also subjected to western blotting with anti-flag, anti-myc and anti-tubulin antibodies. e huh . cells were co-transfected with the plasmids encoding egfp ( . lg) and egfp-specific shrna ( . lg), together with either empty vector or a plasmid encoding ns or ns c a as indicated ( . lg each). at hpt, total rnas were extracted and the level of egfp mrna was examined via northern blotting. suppression activity, adding a novel function to its multiple roles in the viral life cycle. hcv ns is identified to possess dsrna-and sirna-binding activities, similar to many other vsrs such as nov b and sars-cov nucleocapsid. however, although these vsrs use the same strategy of suppressing rnai by sequestrating dsrna and/ or sirna, they do not share sequence identity or structural conservation, suggesting that vsrs of distinct virus families are evolved independently during the viral evolution. hcv ns forms a dimer with an n-terminal alpha-helical subdomain and a c-terminal antiparallel beta-sheet (lorenz et al. ). in addition, the conserved residues h , e and c have been found to important for hcv ns 's dimerization (lorenz et al. ) . our mutational analyses showed that mutation of c abolished the dsrna-and sirna-binding activities of hcv ns and eliminated its vsr activity, implying the dimerization of hcv ns is required for its rnai suppression activity. our findings are consistent with the previous observations that dimerization is important for vsr activities of many viruses. for example, ev-a a forms a dimer and disrupting its dimerization blocks the vsr activity (qiu et al. ) . in conclusion, our findings demonstrate that hcv ns can act as a vsr by sequestrating dsrna from dicer cleavage and interfering with risc assembly by sirnabinding. moreover, these results add hcv ns as a new member of hcv-encoded vsrs, which suppressing rnai by a different mechanism. and it provides insight into the life cycles and virus-host interactions of hcv. fig. the dsrna-and sirna-binding activities of hcv ns are required for its rnai suppression. mbp-ns wt and mbp-ns c a were incubated with . lmol/l -nt dig-labeled dsrna (a) or -nt sirna (b) at °c for min. complexes were separated on % (a) or % (b) tbe-page, respectively. c hek t cells were co-transfected with egfp specific shrna ( . lg) and either the plasmid encoding ns wt or ns c a ( lg of each). total rnas were subjected to small rna northern blotting. u was used as the loading control. the hiv- nef protein binds argonaute- and functions as a viral suppressor of rna interference evidence that hiv- encodes an sirna and a suppressor of rna silencing hcv core protein interacts with dicer to antagonize rna silencing the nucleocapsid protein of coronaviruses acts as a viral suppressor of rna silencing in mammalian cells the hepatitis c virus ns protein is an inhibitor of cide-b-induced apoptosis ebolavirus proteins suppress the effects of small interfering rna by direct interaction with the mammalian rna interference pathway small rna-based antimicrobial immunity the ebola virus vp protein is a suppressor of rna silencing regulation of hepatic innate immunity by hepatitis c virus suppression of short interfering rna-mediated gene silencing by the structural proteins of hepatitis c virus role of rna interference (rnai) in dengue virus replication and identification of ns b as an rnai suppressor dengue ns , an rnai suppressor, modulates the human mirna pathways through its interacting partner the a accessory protein of severe acute respiratory syndrome coronavirus acts as an rna silencing suppressor hepatitis c virus ns protease inhibits host cell antiviral response by inhibiting ikkepsilon and tbk functions interferon antagonist proteins of influenza and vaccinia viruses are suppressors of rna silencing rna interference functions as an antiviral immunity mechanism in mammals induction and suppression of antiviral rna interference by influenza a virus in mammalian cells structure of the catalytic domain of the hepatitis c virus ns - protease hepatitis c virus ns protein serves as a scaffold for virus assembly by interacting with both structural and nonstructural proteins antiviral rna interference in mammalian cells slicing and dicing viruses: antiviral rna interference in mammals a viral protein restricts drosophila rnai immunity by regulating argonaute activity and stability hepatitis c in laos: a -year retrospective study on patients in vitro study of the ns - protease of hepatitis c virus rna binding by a novel helical fold of b protein from wuhan nodavirus mediates the suppression of rna interference and promotes b dimerization targeting of dicer- and rna by a viral rna silencing suppressor in drosophila cells human virus-derived small rnas can confer antiviral immunity in mammals a virus-encoded inhibitor that blocks rna interference in mammalian cells viral suppressors of rna-based viral immunity: host targets zika virus infection induces rnai-mediated antiviral immunity in human neural progenitors and brain organoids cypovirus capsid protein vp has nucleoside triphosphatase activity nap l regulates hepatitis c virus entry and interacts with ns acknowledgements we wish to thank prof. ying zhu (wuhan, china) and prof. bryan cullen (durham, usa) for kindly providing materials. this work was supported by the strategic priority research program of chinese academy of sciences (xdb to x.z.), the national natural science foundation of china ( to y.q., to x.z. and to j.m.), the science and technology bureau of wuhan ( to x.z.), and the advanced customer cultivation project of wuhan national biosafety laboratory ( accp-ms to y.q.). x.z. is supported by the newton advanced fellowship from the academy of medical sciences, uk (naf \ ). author contributions hz performed the experiments, analyzed the data and drafted the manuscript; qq, ts, jx, jk and jm helped to perform the experiments; qq and ts helped to analyze the data; yq and xz designed the experiments, analyzed the data, and finalized the manuscript. all authors approved the final manuscript. conflict of interest the authors declare that there are no conflict of interest.animal and human rights statement this article does not contain any studies with human or animal subjects performed by any of the authors. key: cord- - gkdotvt authors: liu, william j.; shi, weifeng; zhu, wuyang; jin, cong; zou, shumei; wang, ji; ke, yuehua; li, xiaofeng; liu, mi; hu, tao; fan, hang; tong, yigang; zhao, xiang; chen, wenbin; zhao, yuhui; liu, di; wong, gary; chen, chengchao; geng, chunyu; xie, weiwei; jiang, hui; kamara, idrissa laybor; kamara, abdul; lebby, matt; kargbo, brima; qiu, xiangguo; wang, yu; liang, xiaofeng; liang, mifang; dong, xiaoping; wu, guizhen; gao, george f.; shu, yuelong title: intra-host ebola viral adaption during human infection date: - - journal: biosaf health doi: . /j.bsheal. . . sha: doc_id: cord_uid: gkdotvt the onsite next generation sequencing (ngs) of ebola virus (ebov) genomes during the – ebola epidemic in western africa provides an opportunity to trace the origin, transmission, and evolution of this virus. herein, we have diagnosed a cohort of ebov patients in sierra leone in , during the late phase of the outbreak. the surviving ebov patients had a recovery process characterized by decreasing viremia, fever, and biochemical parameters. ebov genomes sequenced through the longitudinal blood samples of these patients showed dynamic intra-host substitutions of the virus during acute infection, including the previously described short stretches of serial t>c mutations. remarkably, within individual patients, samples collected during the early phase of infection possessed ts at these nucleotide sites, whereas they were replaced by cs in samples collected in the later phase, suggesting that these short stretches of t>c mutations could emerge independently. in addition, up to a total of nucleotide sites spanning the ebov genome were mutated coincidently. our study showed the dynamic intra-host adaptation of ebov during patient recovery and gave more insight into the complex ebov-host interactions. the - epidemic of ebola virus disease (evd) in western africa is the largest evd outbreak to date, with , confirmed, probable, and suspected cases and , deaths as of june , (http:// who.int/csr/disease/ebola/en/). during this epidemic, the ability to perform next-generation sequencing of ebola virus (ebov) from patient specimens in the field [ , ] has generated ebov genomes, which helped to trace the origin, evolution, and transmission of ebov in west africa [ ] [ ] [ ] [ ] [ ] [ ] [ ] . based on the phylogenetic analysis, all of the ebov in this outbreak can be traced back to a few cases in guéckédou, guinea. subsequently, the virus spread to neighboring countries, such as sierra leone, liberia, nigeria, senegal, and mali [ , , ] . along with their geographical spread in west africa, ebov formed different lineages [ ] . these lineages are characterized with different single nucleotide polymorphisms (snps) emerging from different time points during the outbreak [ , [ ] [ ] [ ] , ] . these snps occur in both the non-coding and encoding regions, containing indispensable phylogenetic and evolutionary information [ , , ] . previous research demonstrated that the hotspots for non-synonymous substitutions are likely located in regions with a lower level of functional constraint of the encoded viral proteins [ ] . moreover, the intra-host selection for ebov to escape from a developing humoral immune response may drive the diversifying selection of glycoprotein (gp) mucin-like domain, as shown by the enrichment of mutations within the b-cell epitopes of gp [ ] , although this was not observed in ni's study [ ] . on the other hand, short stretches of intra-hostt to c (tnc) mutations were also observed [ , ] . this was speculated as a result of adenosine deaminases acting on rna (adars), which is yet unclear [ ] . it has been reported that viruses with the tnc mutations (genome positions - ) continued to circulate in the magazine wharf area, freetown, sierra leone, causing several infections [ ] . in particular, intra-host single nucleotide variations (isnvs) appeared during the course of the epidemic, within the b cell epitopes of gp and non-coding regions across the ebov genome [ , ] . interestingly, several isnvs were shared by two or more patients, which represented a combination of human-to-human transmission and recurrent mutations [ ] . these isnvs were used to estimate the effective viral population size within a single patient, during a transmission bottleneck [ ] , and to identify human-to-human transmission chains [ , , ] . furthermore, two isnvs were able to influence the transcription level of an adjacent gene of nucleocapsid protein (np) by two-fold [ ] . recent studies also showed that, during the epidemic, the ebov isolates from early in the outbreak with amino acid substitutions in the gp protein possessed increased tropism for human cells, indicating human adaptation of ebola virus during human-to-human transmission [ ] [ ] [ ] . however, only a few of the previous studies described longitudinal sequence data from a single patient [ , ] , and the intra-host dynamic evolution of ebov during disease progression is still largely unknown. at the sierra leone-china friendship biological safety laboratory (sle-chn bio-safety lab) [ ] , we cared for a cohort of ebov-infected patients (n = ) in the ebola treatment units (etus) in freetown, sierra leone, from mid-march to late june . the dynamic viremia [ ] and biochemical features during disease progression were characterized. by utilizing a deep sequencing platform in the field [ ] , we successfully generated virus genomes from longitudinally collected blood samples from some of the patients. surprisingly, we observed coincident emergence of serial nucleotide variations, including the previously defined short stretch of tnc mutations during the recovery process. in a single patient, genome sequences obtained from samples during earlier stages of the acute infection phase possessed ts at the tnc positions, whereas cs were found from samples collected during the recovery process. phylogenetic analyses showed that after the tnc mutations occurred, all strains possessing such mutations were grouped together, but were not clustered together with their earlier sequences without such tnc mutations from the same patient. our results suggested that such tnc mutations could arise independently within single patients, reflecting the host-adaptation of ebov during infection. we undertook a cohort study of patients admitted to jui ebola treatment centre (sierra leone-china friendship hospital) between march , and june [ ] . we used a standard case definition consistent with who guidelines. all patients were included in the study except for those who died upon arrival, or those who had no blood results within h of admission. primary survival outcome measure was collected from the ebov treatment centre. samples were tested at the on-site laboratory sle-chnbio-safety lab for the presence of ebov rna by real-timert-pcr against the glycoprotein (gp) and nucleoprotein (np) gene targets [ ] , after inactivation and manual rna extraction. positive results were reported as cycle threshold values. all patients received a rt-pcr test upon admission. the piccolo express system (abaxis, ca, usa) was used to generate metabolic and liver function profiles. amylyte was used to assay for blood biochemistry and liver function profile in the same day as the sample collection. all data were collected as part of routine patient care, and recorded on standardized forms, which were kept securely. the clinical data extracted for research purposes were anonymized and stored on a password-protected database. the sierra leone ethics and scientific review committee provided approval for the study. this work was conducted as part of the surveillance and public health response to contain the evd outbreak in sierra leone. blood samples from suspected individuals and oropharyngeal swab samples from corpses were collected for evd testing and outbreak surveillance, with a waiver to provide written informed consent during the evd outbreak under the agreement between the sierra leone and chinese governments. the activities were coordinated by the emergency operations centre in the charge of sierra leone ministry of health and sanitation during - epidemic of ebola virus disease (evd) in western africa, the ebola virus (ebov) genomes generated from the patient specimens based on next-generation sequencing platforms in the field helped to quickly trace the origin, evolution, and transmission of ebov. in particular, intra-host single nucleotide variations (isnvs) appeared during the course of the epidemic across the ebov genome. whether and how these isnvs could emerge from a single patient during the disease progression, are still largely unknown. evidence before this study some of the isnvs, such as short stretches of intra-host t to c (tnc) mutations were shared by two or more patients, which represented a combination of human-to-human transmission and recurrent mutations. furthermore, isnvs could appear in the key sites, such as the b cell epitopes of gp and non-coding regions across the ebov genome, which may influence the transcription level of an adjacent gene. in a cohort of ebov-infected patients in the ebola treatment units (etus) in freetown, sierra leone, the recovery processes of the patients were represented by the dynamic viremia and biochemical features. by utilizing longitudinally collected samples during the recovery process, we successfully generated series of virus genomes from the patients. we observed coincident emergence of serial nucleotide variations, including the previously defined short stretch of tnc mutations during the recovery process. phylogenetic analyses showed that after the tnc mutations occurred, all strains possessing such mutations were grouped together, but were not clustered together with their earlier sequences without such mutations from the same patient. our results suggested such tnc mutations could arise independently within single patients, reflecting the host-adaptation of ebov during infection. our data indicate that short stretches of tnc substitutions are part of the convergent evolution during the infection process of evd patients, shedding light on the dynamic intra-host genomic variation of ebov during the - epidemic. and who. all the information regarding individual persons has been anonymized in the report. the genome sequencing was performed as described previously [ ] . briefly, rna samples extracted from whole blood (two positive oropharyngeal swab samples with high ct values were not involved) from evd patients were reverse transcribed to cdna. pcr amplifications were performed with ebov-specific primer pairs with overlaps. amplicons from one patient were pooled for library preparation. ngs was performed using the bgiseq- (ion proton) platform. all sequenced reads were filtered to remove the low quality and short reads. the genome sequences of the viruses were assembled by mapping the filtered reads to the ebov consensus sequence (genbank: kt ) using roche newbler version . (roche), and the mutation site was manually checked with original sequencing data. clean reads were mapped to the ebov genome (kj ) by tmap . . . we then scanned the ebov genome site-by-site and determined the nucleotides for each genomic site according to mapping results. finally, the ratios of four nucleotides at each site were obtained. among the samples sequenced, full-length or nearly full-length ebov genomes were successfully generated in this study (table s ). the other samples with only short partial of the ebov genomes were not analyzed in this study. all of the mutation sites were counted for each released ebov fulllength genome, using the zaire ebolavirus isolate h.sapiens-wt/gin/ /makona-kissidougou-c (genbank accession no. kj ) as the reference sequence. the existence of substitutions within a genome was screened within the ebov genomes publicly available from genbank. a "c strain" was defined as the existence of or more t-c substitutions within a genome window width of nt. our dataset included full-length or nearly full-length ebov genomes sequenced in this study and ebov genomespublicly available from genbank. amaximum-likelihood phylogenetic tree was inferred using the software raxml, with the gtrgamma model and bootstrap replicates. the genomes and ngs data of the ebov viruses were deposited in genbank with the access numbers mf -mf (table s ). from march th to june th, , patients who had symptoms meeting the definition of suspected evd were admitted to the sierra leone-china friendship hospital in freetown. the blood specimens were collected and delivered by the sample center of ministry of health and sanitation (mohs), sierra leone. the sle-chn bio-safety lab tested the blood samples for ebov through a double-channelreal-time rt-pcr detection kit targeting both gp and np genes of ebov. a total of ( . %) patients were confirmed to have evd ( table ). the first blood samples collected from these patients after hospitalization were tested for laboratory confirmation, and the ct values from the gp and np channels matched well with a high linear correlation ( figure a ). of these patients, the most common clinical features at presentation included fever in patients ( . %; mean temperature, . °c) and gastrointestinal symptoms, e.g. abdominal pain, in patients ( . %) and anorexia/loss of appetite in patients ( . %) ( table ) . ocular signs were also common in the patients ( with conjunctivitis [ . %] and with pain behind the eyes [ . %]). the mean age of the patients was years (range, to ) and patients ( . %) were male ( table ). the case fatality rate of the evd patients was . % ( / ), similar to the overall ratio of . % ( , / , ) during this ebov epidemic. there was no significant difference (p = . ) in the average interval from symptom onset to presentation between survivors and non-survivors( figure b) . the mean body temperature on the day of hospitalization was . °c among survivors, significantly lower (p = . ) than that of nonsurvivors, . °c ( figure c ). the initial viremia of survivors and nonsurvivors was also significantly different, with non-survivors possessing lower mean ct values for both gp ( . for non-survivors vs . for survivors, p = . ) and np ( . for non-survivors vs . for survivors, p = . ) ( figure d ). .when there are two longitudinally-collected blood samples with ebola rna negative, the patient will be discharged. the corresponding temperatures (purple) during the blood sampling. blood samples from patients ( survivors and fatality ) were available for longitudinal collection during the hospitalization. as in the previous study [ ] , the viremia of the survivors ameliorated after the presentation, as revealed by increased ct values (figure ). interestingly, in our study, two different trends in the variation of body temperatures of the patients after hospitalization were observed. consistent with viremia, body temperatures of patients , , , and decreased after hospitalization. on the other hand, in patients , , , , , , and , a transient increase in body temperature after hospitalization was detected. especially in patient , the ct value had a dramatic decrease from . on day (hospitalization) to . on day ( days after hospitalization), and the body temperature of the patient elevated from . °c on day to . °c on day . on day , the viremia and fever of this patient decreased. in contrast to the survivors, the moribund patient had a continuous viremia and the body temperature increased from . °c on day to . °c on day . the patient died on day . blood biochemical parameters were also tested in the sle-chn biosafety lab in the field (figure ). hematological and biochemical abnormalities were observed in the patients upon hospitalization, which was also reported in the previous studies (biochemical testing in a laboratory tent and semi-intensive care of ebola patients on-site in a remote part of guinea: a paradigm shift based on a bleach-sensitive point-ofcare device. clinical, virological, and biological parameters associated with outcomes of ebola virus infection in macenta, guinea.). hyperglycemia occurred in patients , , upon hospitalization and ameliorated afterwards. abnormal concentrations of blood urea nitrogen, potassium, sodium, and chlorine were observed in the patients, especially in patients and . hematological abnormalities detected during the disease progression included reduced concentrations of hemoglobin and hematocrit. these biochemical abnormalities alleviated gradually during the recovery process. finally, full-length and four nearly full-length ebov genomes from patients were obtained based on the ngs platform in the field. through analysis of the ngs data of longitudinally collected blood samples from the same patient, we found that the previously reported short stretch of tnc mutations within positions - appeared in the late blood samples of the patients (day ) and (days and ), while these patients still possessed ts in early blood samples (day for patient and day for ) ( figure a ). in particular, in the day sample of patient , almost no cs were observed at the positions, whereas cs accounted for approximately % on day and~ % on day at all of the tnc nucleotide sites ( figure a ), indicating that day may be an intermediate status between days and . the similar ratios of t:c at the different tnc nucleotide sites on the same day may indicate that these tnc mutations occurred concurrently, though not all these mutations can be found on the same nucleic acid strands (reads). however, in patient , dominant percentages of cs (n %) were already observed on day , and continued increasing in percentage on day (~ %) ( figure a) . meanwhile, we found additional coincident substitutions distributed across the entire ebov genome (table and figure ) , which has the similar ratios for the substitutions as in the short stretch of the tnc mutations between positions and . this may also indicate that all these substitutions were correlated to each other. these nucleotide variations not only included tnc substitutions, but also other types of substitutions, e.g. cnt, gna, ang, ant, tna, and even one tndeletion mutation at site (table s ). these nucleotide variations occurred coincidently among different patients during the acute infection phase. the intra-host adaptation of ebov was also illustrated as the continuous change of isnv substitution ratios (the ratios of latter dominant isnv/former dominant isnv, for instance, ratios of c/t for the tnc nucleotide sites) across longitudinal sampling points in patients , , and ( figures b-d) . figure c ). for patient , substitution ratios remained n , indicating a dominant role of the latter isnv on both day and day ( figure d) , however, the increasing trend of the isnv substitution ratios could still be observed from day to day . however, there were still some exceptional sites which had consistent isnvs during the disease process (supplementary figure s ). all exceptional sites possessed the latter form at the target sites (e.g. site of viral genome had already mutated to c in patient on day ), indicating early substitution events at these sites. phylogenetic analysis of the novel and publicly released fulllength ebov genome sequences from genbank showed that the novel ebov sequences did not cluster together ( figure a ). alternatively, they formed four small independent clusters scattered across lineage sl , which was circulating during the late stage of the outbreak in . we further analyzed all ebov genomes available online and identified the strains with multiple tnc substitutions within a short genomic region in different positions of the genomes (termed as "c strains") ( figure b and table s ). a total of strains, including six sequenced in this study, possessed multiple tnc nucleotide substitutions (table s ). these "c strains" could be classified into at least different types according to the number of tnc substitutions and their positions ( figure b ). interestingly, apart from the well-known stretch with serial tnc mutations at genome position - , stretches with and serial tnc mutations were also found in the intergenic region between vp and gp in strains g . (genbank no. kr ) and libr (genbank no. kt ) (table s ) , respectively. to study the phylogenetic association of these "c strains", we mapped them onto the tree ( figure b and supplementary figure s ). however, these "c strains" scattered across the entire tree, with little phylogenetic association ( figure b ), indicating multiple independent origins of these tnc substitutions. however, some strains possessing the same tnc substitution type were clustered together (supplementary figure s ). based on current evidence, human-to-human transmission is the most plausible reason for these cases. in particular, ten strains possessed the tnc mutations within positions - ( figure c ). the prototype strain with the tnc mutations was j (kp ), sequenced from the magazine wharf area, freetown, sierra leone in november [ ] . it has been reported that j -like ebov continued to circulate in this region and had infected at least three additional patients by july . however, the strain sequenced from patient on days and the ratio of the reads of latter/former. c " " means that the corresponding data of the survivor was shown in figure b -d. d " " means that the corresponding data of the survivor was shown in supplementary figure s . e the ratio is termed as " " when the read of the former nucleotide is . f "n/a" means that the reading depths of the ngs was lower than . g "vp -vp " means that the substitutions locate in the non-coding region between vp and vp . possessed the tnc mutations and clustered together with other such "c strains". surprisingly, on day of patient , the virus possessed ts at all of the positions, and did not fall within this cluster. this was also observed with patient ( figure c ). virus samples collected on day from patient was located in a different cluster as the virus sequenced from the day sample. we further identified eight ebov strains sharing the previously described tnc mutations within positions - (the last column in table ). we called all the other viruses ( strains) as "t strains ( - )". interestingly, we found that almost all coincident substitutions that happened in our longitudinally sequenced ebov genomes (table ) had already mutated in these eight "c strains ( - )". in contrast, none of the other ebov "t strains ( - )" without the tnc mutations at position - have these substitutions. these data suggest that all substitutions distributed within the whole genome of ebov, including the short stretch of tnc mutations within position - , had a coincident evolution trend during the recovery process of the patients. in this study, we sequenced longitudinally-collected blood samples from ebov patients based on an in-fieldngs platform at the sle-chnbio-safety lab during the - ebov epidemic. although more a thousand of ebov genomes have been sequenced during the - ebov epidemic in west africa, most of the viruses were from different patients during the early days of the acute phase during the infection [ ] [ ] [ ] [ ] [ ] , , , ] . phylogenetic analysis showed that the genomes sequenced in this study were not grouped together. this suggested that in the late stages of the - ebov outbreak, the virus became diversified and various minor ebov lineages had been cocirculating in sierra leone. previous phylogenetic studies indicated that snps carried by different ebov lineages were important molecular markers to study the virus transmission among humans [ , , ] . short stretches of the tnc substitutions appeared sporadically without phylogenetic association as shown in our analysis ( figure b ). although the exact reason is yet unclear, this was speculated to be the result of adenosine deaminases acting on rna (adars) [ ] . thus, it is understandable that the tnc substitutions can occur in both the coding regions and the non-coding regions. in the present study, we found several related isnvs across the ebov genome occurring in the late acute phase of ebov infection, including a short stretch of tnc mutations within positions - . however, these mutations were not found in samples collected during the early infection phase. in one patient, we even observed the intermediate stage for these tnc mutations. these data suggest that the serial tnc mutations were emerged coincidently, and that the mutations could arise independently in different ebov patients. since only a few tnc mutations are able to change the transcription level of adjacent genes [ ] , such serial tnc mutations in our study, though some in non-coding regions, might be functional during ebov infection in humans, which should be a subject of further investigation. meanwhile,further analysis should be performed to investigate whether all these mutations can be found on the same nucleic acid strands (reads). phylogenetic analysis showed that earlier samples without the tnc mutations were not grouped together, whereas later samples with the tnc mutations were clustered together and formed a separate cluster. we . the schematic diagram for the dynamic intra-host substitution and inter-host transmission of ebov. base on the dynamic adaptation of ebola virus in the patients we described herein, we further proposed the schematic model for the relationship of the human to human transmission and the dynamic adaptation of the ebola viruses. panel a shows the disease process of one survived patient a from the preclinical period to symptom presentation, and then to recovery. during this process, the dominant viruses in the patient are the t strain which possess the former nucleic acids in the sites (table ), e.g. t in the tnc stretch ( - ). there also will be emergence of the c strains during the recovery process, which possess the latter nucleic acids in the sites (table ), e.g. c in the tnc stretch ( - ) due to unknown reasons as we indicated in our patients. t strain virus dominates in the acute detoxification period of patients and certain patients died during this period. thus in the general human to human transmission of ebola virus (patient a to patient c), the transmitted viruses are t strain virus, which takes the dominates in the ebov genomes of the west africa outbreak publicly available from genbank. however, we cannot exclude the possibility of the sporadic transmission of c strains in humans with close contact (a to b). patient b, who was infected by the c strain of ebola virus, may have a mixed quasi-species of the virus during the diseases process. this possibility requires further exploration. also identified a total of strains with serial tnc substitutions that belonged to different tnc mutation types, with different number of tnc substitutions and/or within different genomic regions. generally, strains possessing different tnc mutation types were not clustered together, but scattered across the tree. based on current evidence, there was no direct phylogenetic association between these different mutation types. this once again revealed that these various tnc mutation types could arise independently. however, some of the tnc substitutions were indeed shared by a few strains, which were clustered together in the tree. thus, the transmission hypothesis cannot be fully rejected yet [ ] . this study is the first showing that linked and coincident mutations observed in ebov could arise independently during human infections. it can be rationally proposed that the "t strains" are the dominant circulating ebovs during the epidemic ( figure ). most of the cases are infected via contact with the patients during the early stages of acute infection. during the late stages of the infection, the "c strains" emerge, although the virus titer has become lower during this period. transmission may also occur rarely between close contacts at this stage of disease. this may explain why the "c strains" could be found in such few patients in the acute infection phase. meanwhile, the tnc mutation happened at different time points of the patients after ebov infection, which may be related with disease progress or prognosis. for patient , the corresponding sites for these tnc were still t, while the tnc substitution was completed on day of the infection of patient . the fever duration and the viremia duration of patient were longer than . however, the relationship of the occurrence time of these mutations with disease progress or prognosis should be further investigated. overall, our data indicate that short stretches of tnc substitutions are part of the linked and convergent evolution during the infection process of evd patients, shedding light on the dynamic intra-host adaptation of ebov during the - ebov epidemic. supplementary data to this article can be found online at https://doi. org/ . /j.bsheal. . . . genetic diversity and evolutionary dynamics of ebola virus in sierra leone evolution and spread of ebola virus in liberia ebola virus epidemiology, transmission, and evolution during seven months in sierra leone genomic surveillance elucidates ebola virus origin and transmission during the outbreak mutation rate and genotype variation of ebola virus from mali case sequences diseases usamrioi, national institutes of h, integrated research facility-frederick ebola response t, monitoring of ebola virus makona evolution through establishment of advanced genomic capability in liberia distinct lineages of ebola virus in guinea during the west african epidemic ebola virus disease in west africa-the first months of the epidemic and forward projections emergence of zaire ebola virus disease in guinea the evolution of ebola virus: insights from the - epidemic non-coding regions of the ebola virus genome contain indispensable phylogenetic and evolutionary information intra-host dynamics of ebola virus during enhancement of replication of rna viruses by adar via rna editing and inhibition of rna-activated protein kinase genotypic anomaly in ebola virus strains circulating in magazine wharf area high-resolution genomic surveillance of ebolavirus using shared subclonal variants human adaptation of ebola virus during the west african outbreak ebola virus glycoprotein with increased infectivity dominated the - epidemic functional characterization of adaptive mutations during the west african ebola virus outbreak preliminary evaluation of the effect of investigational ebola virus disease treatments on viral genome sequences on the ground in sierra leone detection and analysis of ebola virus in sierra leone-china friendship biosafety laboratory from on the ground in western africa: from the outbreak to the elapse of ebola ebola viral load at diagnosis associates with patient outcome and outbreak evolution hundreds of chinese and sierra leonean staff on-site took part in the sample collection, transportation, detection, and data analysis. we especially acknowledge the excellent work of four previous detection teams of chinacdc (mobile and fixed labs) and the essential support (materials and reagents) of the national institute for viral disease control and prevention, china cdc. we also express our deep condolences to the family and associates of dr. abdul kamara of the ministry of health and sanitation, sierra leone, who contributed a lot to this project but succumbed after the successful control of ebola epidemic in sierra leone. the authors declare that there are no conflicts of interest. key: cord- - ciukd authors: jalloh, mohamed f; li, wenshu; bunnell, rebecca e; ethier, kathleen a; o’leary, ann; hageman, kathy m; sengeh, paul; jalloh, mohammad b; morgan, oliver; hersey, sara; marston, barbara j; dafae, foday; redd, john t title: impact of ebola experiences and risk perceptions on mental health in sierra leone, july date: - - journal: bmj glob health doi: . /bmjgh- - sha: doc_id: cord_uid: ciukd background: the mental health impact of the – ebola epidemic has been described among survivors, family members and healthcare workers, but little is known about its impact on the general population of affected countries. we assessed symptoms of anxiety, depression and post-traumatic stress disorder (ptsd) in the general population in sierra leone after over a year of outbreak response. methods: we administered a cross-sectional survey in july to a national sample of consenting participants selected through multistaged cluster sampling. symptoms of anxiety and depression were measured by patient health questionnaire- . ptsd symptoms were measured by six items from the impact of events scale-revised. relationships among ebola experience, perceived ebola threat and mental health symptoms were examined through binary logistic regression. results: prevalence of any anxiety-depression symptom was % ( % ci . % to . %), and of any ptsd symptom % ( % ci . % to . %). in addition, % ( % ci . % to . %) met the clinical cut-off for anxiety-depression, % ( % ci . % to . %) met levels of clinical concern for ptsd and % ( % ci . % to . %) met levels of probable ptsd diagnosis. factors associated with higher reporting of any symptoms in bivariate analysis included region of residence, experiences with ebola and perceived ebola threat. knowing someone quarantined for ebola was independently associated with anxiety-depression (adjusted or (aor) . , % ci . to . ) and ptsd (aor . % ci . to . ) symptoms. perceiving ebola as a threat was independently associated with anxiety-depression (aor . % ci . to . ) and ptsd (aor . % ci . to . ) symptoms. conclusion: symptoms of ptsd and anxiety-depression were common after one year of ebola response; psychosocial support may be needed for people with ebola-related experiences. preventing, detecting, and responding to mental health conditions should be an important component of global health security efforts. what are the new findings? ► to the best of our knowledge, the assessment was the first national survey that examined the impact of the devastating ebola epidemic on populationlevel mental health using globally validated scales, and conducted after more than a year of ongoing transmission of ebola in the country. ► we found that symptoms of ptsd and anxietydepression were common after one year of the outbreak, especially among those with ebolarelated experiences. ► furthermore, we have demonstrated the ability to rapidly administer brief mental health screeners at the population level to identify factors associated with mental health symptomology towards the end of an unprecedented infectious disease epidemic. recommendations for policy ► preventing, detecting and responding to mental health conditions should be an important component of global health security efforts. ► use of brief mental health screeners during outbreak response could increase the ability to identify and address the needs of at-risk groups. ► so doing could help avert the substantial short-term and long-term effects of mental health disorders on individual health and on national health systems, societies and economies. primarily in sierra leone, liberia and guinea. in sierra leone alone, there were reports of more than ebola cases, resulting in over deaths, and more than individuals were quarantined due to possible ebola exposure. little is known about the epidemic's effects on the mental health of the general population in the affected countries. numerous studies have examined the mental health effects associated with other infectious disease outbreaks including the severe acute respiratory syndrome (sars) epidemic [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and novel influenza a (h n ) pandemic. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the mental health impact of other emergencies, such as bioterrorism, have also been documented among survivors. psychological distress, anxiety, depression and post-traumatic stress disorder (ptsd) have been recorded among populations exposed to mass conflict and displacement including those affected by the civil conflict in sierra leone between and . known risk factors for anxiety, depression and ptsdincluding experience with ill individuals, perceptions of threat, high levels of mortality, food and resource insecurity, stigma and discrimination, and intolerance of uncertainty-may have been experienced by people in sierra leone during the ebola epidemic. adverse mental health outcomes could be expected in the general population given the magnitude of the epidemic. high levels of distress have been documented among ebola survivors in guinea and sierra leone and healthcare workers (hcws) in all three affected countries. there are few mental health resources in sierra leone; for example, when the ebola outbreak began, there was only one trained psychiatrist for the population of over million. assessments of mental health and of risk factors for mental illness can support policy efforts to improve resources to address mental health and inform how resources can be targeted most efficiently-especially in the aftermath of a devastating ebola epidemic. the sierra leone ministry of health and sanitation and the us centers for disease control and prevention collaborated with focus and other stakeholders to implement a national, household-based ebola knowledge, attitudes and practices (kap) survey in july . the survey assessed respondents' ebola-related kap, perceptions of ongoing ebola threat, ebola-related experiences, and anxiety-depression and ptsd symptoms. the present analysis aimed to estimate prevalence of mental health symptoms and factors associated with having symptoms in the general population. the national survey employed a multistage cluster sampling procedure with primary sampling units selected with probability relative to their size. in order to attain % confidence levels and cis of ± % estimates of the national population, individuals were approached across the regions and districts of sierra leone. using sierra leone's most recent census list ( ) of enumeration areas as the sampling frame, enumeration areas were randomly selected across all districts. within each cluster, households were selected using systematic random sampling. to generate reliable district-level estimates for key districts, we oversampled in the three districts still experiencing active ebola transmission. a weighting factor was applied to each record to adjust for the different sample sizes taken in different districts. within each household, the household head and another individual (aged between years and years) or a woman were approached for consent and interviewed. survey questions included sociodemographic characteristics, ebola experience, perceived ebola threat, anxiety-depression symptomology and ptsd symptomology (supplementary file ). ebola experience variables included whether participants knew someone who had died from ebola and whether they knew someone who had been quarantined due to ebola exposure. participants whose only reported experience with ebola-related death ( . %, n= ) or quarantine was related to public figures ( . % of sample, n= ), such as well-known medical doctors who died from ebola, were excluded from this analysis. these two variables were also combined into a two-level composite item which included: ( ) no experience with ebola-related death or quarantine; ( ) knowing others who had been quarantined or had died from ebola. participants' perceptions of ebola as a threat were measured by four items that asked whether they perceived that ebola was no longer a threat to ( ) sierra leone; ( ) their district; ( ) their community; and ( ) their household. participants responded using -point likert scale items ranging from (strongly agree) to (strongly disagree). responses were further dichotomised into 'agree' and 'disagree,' and the scores reversed so that higher scores represented more perceived risk. we also created a composite score across all four domains with representing 'any perceived ebola threat' and representing 'no perceived ebola threat. ' symptoms of anxiety and depression were measured by patient health questionnaire- (phq- ). phq- was developed by combining two ultrabrief screeners, the phq- and the generalised anxiety disorder scale, that have been demonstrated to reliably measure depression and anxiety symptoms. participants were asked to report their symptoms of depression and anxiety in the past weeks on a likert scale from (not at all) to (nearly every day) for a maximum score of . the sample was further dichotomised into those who expressed any symptoms compared with those who did not by creating a new composite variable. we also examined the prevalence of anxiety and depression using the established clinical cut-off total score of , which represents the proportion of people who would be considered as having clinical bmj global health levels of depression or anxiety if the screener were used for diagnostic purposes. symptoms of ptsd were measured by the impact of event scale- (ies- ), which is a validated, shortened version of the full ies-revised (ies-r). the full scale contains items (scored from to ) with demonstrated reliability and validity to measure ptsd symptoms across different cultures and settings. while ies-r is generally not used to diagnose ptsd in clinical settings, it is widely used for screening at-risk patients with ptsd. the ies- includes a total of six items-two items from each of the three subscales of the measure, namely intrusion, hyperarousal and avoidance. participants were asked to report their ptsd symptoms in the past days on a likert scale ranging from (not at all) to (extremely). we dichotomised the sample into those who expressed any symptoms versus those who did not by creating a new composite variable. we evaluated respondents for whom ptsd may be a 'clinical concern' using an inputted . mean item cut-off score (equivalent to / on ies-r). in addition, we assessed respondents who met 'probable diagnosis' of ptsd using an inputted . mean item cut-off score (equivalent to / total score in ies-r). data collection in june , focus recruited experienced data collectors, team supervisors and regional supervisors. they were trained for a week on overall assessment protocols and guidelines, informed consent, safety and security precautions, administration of questionnaire, and quality control and assurance. the training included oral translation of each item into local languages (krio, mende, temne and limba), back translations (orally), group discussions of the translations for accuracy in meaning, role plays to reflect possible range of responses, and group consensus on the final translations to ensure consistent and accurate use of each item. in july , the trained data collectors used open data kit for digital data collection at the household level. nearly all interviews (> %) were conducted in krio. in july , when the ebola kap was administered, % of the cumulative confirmed ebola cases in the country had been reported. control activities continued, including provision of prevention messages, case detection, contact identification, quarantine and monitoring, and management of cases and deaths. quarantine involved days of home-based isolation with armed uniformed police dispatched to enforce restriction of movement in and out of the household. quarantined individuals were clinically monitored, and if ebola was suspected, they were transferred to a holding centre for testing. the data were analysed using spss v. . statistical significance was defined as a two-tailed p-value less than . . for reliability, internal consistency was assessed by calculating cronbach's α values. for factorial validity, the factor structures of the phq- and ies- scales were examined with confirmatory factor analysis (cfa). the relationships between demographic variables (gender, age, education and region of residence), ebola experience, perceived ebola threat and mental health symptoms were examined. frequencies, proportions, % ci of proportions, as well as χ tests were generated to examine the relationships between sample characteristics and mental health symptoms. univariate and multivariate binary logistic regression analyses were conducted to examine the relationship between ebola experience, perceived ebola threat and mental health symptoms. we further examined the effect of ebola experience, perceived ebola threat and interaction between those two variables on mental health status by conducting a multivariable logistic regression controlling for potential confounders. to avoid multicollinearity, only composite scores were entered as predictors into the model. sex, age, education and region were included because they have been associated with mental health symptoms in other studies. goodness of fit index (gfi), comparative fit index (cfi) and root mean square error of approximation (rmsea) were calculated to measure the cfa model. weighted cell count, percentages and ors with % cis are presented in the logistic regression tables. of individuals approached, ( %) consented to participate in the assessment. sample characteristics by mental health symptoms are presented in table . the median age of respondents was years (sd= ); ( %) were male. the sample comprised respondents from all four geographical regions in sierra leone: ( %) from the west, north ( %), east ( %) and south ( %). boosted district samples in kambia and port loko, where cases were still being identified, resulted in a larger sample from the north. of all respondents, % had no formal education, % had some primary school education and % had secondary or higher education. nearly a third ( %) of respondents knew at least one person who died from ebola. similarly, participants ( %) knew at least one person who was quarantined. about a quarter ( %) of respondents knew someone who died from ebola and someone who was quarantined. nearly three quarters ( %) of respondents perceived an ebola threat at one or more levels: in sierra leone ( %), their district ( %), their community ( %) or their household ( %). prevalence of symptoms figure shows % ( % ci . % to . %) of respondents reported at least one symptom of anxiety or depression, with % ( % ci . % to . %) meeting the clinical cut-off definition. of all respondents, % ( % ci . % to . %) reported one or more ptsd bmj global health table a ,b describes respondents' experiences with ebola and the association with anxiety and depression and ptsd symptoms, controlling for age, gender, region and education level. the experience of knowing someone who died from ebola alone was not independently associated with anxiety and depression symptoms (adjusted or (aor) . % ci . to . , p= . ) but was independently associated with ptsd symptoms (aor . % ci . to . , p= . ). those participants who knew someone quarantined due to ebola exposure alone were more likely to report symptoms of anxiety and depression (aor . % ci . to . , p< . ) and ptsd (aor . % ci . to . , p< . ) than those who did not. respondents who had both experiences (that is, they knew at least one person who died from ebola and someone quarantined) were also more likely to report symptoms of anxiety and depression (aor . % ci . to . , p< . ) and ptsd (aor . % ci . to . , p< . ) compared with those who did not report both. those with any ebola experience were more likely to report anxiety and depression symptoms than those who had no ebola experience (aor . % ci . to . , p< . ) and were more likely to report ptsd symptoms than those with no ebola experience (aor . % ci . to . , p< . ). table presents the relationship between perceived ebola threat and reported symptoms of anxiety and depression and ptsd. respondents who perceived some ongoing threat of ebola were more likely to report symptoms of anxiety-depression (aor . % ci . to . , p< . ) and ptsd (aor . % ci . to . , p< . ) compared with those who did not. table presents multivariate analyses of the associations between ebola experience and perceived ebola threat and symptoms of anxiety and depression and ptsd, adjusting for gender, age, region and education levels. ebola experience and perceived ebola threat were independently associated with anxiety and depression symptoms as well as ptsd symptoms. in addition, the interaction between ebola related experience and risk perception was independently associated with both anxiety-depression and ptsd symptoms: participants who had ebola experience and also perceived ongoing ebola threat were more likely to report symptoms of anxiety-depression (aor · % ci · to · , p= . ) and ptsd symptoms (aor · % ci · to · , p= . ). in a national sample of sierra leoneans after more than a year of the unprecedented ebola epidemic, nearly half of all respondents reported at least one symptom of anxiety or depression and three quarters expressed ptsd symptoms. most respondents reported between one and four symptoms. after adjusting for sociodemographic variables, we found that persons with any level of ebola experience were more likely to report symptoms of anxiety-depression and ptsd. even though expression of one or more symptoms was widespread among our sample, a lower proportion of respondents met the clinical cut-off scores for anxiety-depression ( %- %) and probable diagnosis for ptsd ( %- %). the proportion of respondents who exhibited clinical level symptoms of anxiety-depression may be considered 'lower than expected' given the magnitude and duration of the epidemic, but may also point to a culture of resiliency among sierra leoneans. on the other hand, we documented substantial ptsd, which is a public health concern that may require targeted mental health interventions at the individual level and community level for those with some personal ebola experience. a national assessment of the mental health impact of the sars epidemic in taiwan, using a different scale than in our current study, found % prevalence of depression after the epidemic ended. another population-based survey in taiwan revealed % prevalence of psychiatric morbidity following sars. in singapore, a community-based sample detected that a quarter of all respondents had clinical levels of ptsd symptoms. other mental health assessments with sars survivors and hcws documented similar or higher clinical ptsd levels compared with our current assessment. one study found that hcws with a history of mental illness before sars were more likely to report new onset following the epidemic. in our assessment, we cannot determine how past mental health history of ptsd in sierra leone, especially due to the prolonged civil war from to , may have influenced the levels of clinical ptsd concern we detected. similar to sars, the h n pandemic was associated with psychological distress among the general population, family members of hospitalised patients with h n and hcws. in some instances, prevalence of h n -related anxiety was higher among those who had greater intolerance of uncertainty. additional research is required to better understand the relationship between intolerance of uncertainty and quarantine experience during large-scale infectious disease outbreaks. an assessment with hcws in china found that being quarantined and having perceived threat of sars were associated with high depressive symptoms several years after bmj global health the epidemic ended. in a separate study, h n quarantine experience did not predict elevated ptsd levels while dissatisfaction with control measures was a better predictor. to the best of our knowledge, no prior study has assessed the mental health impact of the protracted ebola epidemic at population levels in sierra leone, liberia or guinea. a limited number of studies have examined population-level mental health in other african countries. one such study in a predominantly rural community in ethiopia found that % of the population expressed clinical levels of mild depressive, anxiety and somatic symptoms. on the other hand, a wide variety of studies have examined anxiety and depression in highrisk populations in africa, including patients with tuberculosis in ethiopia and angola, rwandans who had experienced genocide, and nigerian prison inmates. findings of varying levels of mental health symptomology from these studies suggest that further investigations may be required to better understand specific mental health impact of the ebola epidemic on directly affected persons such as ebola survivors. in a systematic review, adverse mental health impact has been documented among conflict-affected persons. in sierra leone, during protracted civil conflict, exposure to traumatic events was associated with non-specific physical ailments. high prevalence of traumatic experiences and psychiatric sequelae has also been documented among sierra leonean refugees. among war affected youth in sierra leone, social disorder and perceived stigma contributed to both externalising and internalising problems. former child soldiers in sierra leone saw reliable improvement in ptsd symptoms over time, suggesting that a supportive environment may encourage resilience. a key recommendation in previous studies and who guidance is to integrate mental health into primary healthcare services. one study found global return on investments for scaling up treatment for depression and anxiety. an example of such effort is in progress in sierra leone wherein public health nurses are trained to screen patients for possible mental health needs. the who mental health gap action programme emphasises that scaling up mental health services is a joint responsibility that requires collaboration from governments, health professionals, donors, civil society, communities and families. limitations although a random national sample was obtained, our sample is not necessarily nationally representative. the sample had a higher proportion of respondents with any education compared with the general population. however, we did not find any association between education level and mental health symptoms, suggesting that this may not have influenced our findings. we acknowledge the necessity of validating survey instruments before using them in a new cultural context. although phq- and ies-r have been widely used globally, - neither has been validated nor used in sierra leone prior to this study. we therefore do not know the validity of clinical cut-off scores for our sample. to the best of our knowledge, phq- and ies-r (or the shortened form in this assessment) have not been used to measure population-level symptoms of mental health in any similar setting; making it impossible to compare our results to similar populations elsewhere. however, we found both had acceptable internal reliability and factorial validity. in the current survey, the phq- instrument demonstrated acceptable internal reliability (cronbach's α= . ) and good factorial validity (gfi= . , cfi= . , rmsea= · ). the shortened ies- scale used in the present study demonstrated acceptable internal reliability (cronbach's α= . ) and good factorial validity (gfi= · , cfi= · , rmsea= . ). in addition, the national sample was not designed to produce specific estimates for directly affected persons such as ebola survivors, families of ebola victims and quarantined persons. moreover, there are no baseline/historical data available for comparisons. we also did not measure the effects of exposure to sierra leone's civil conflict on long-term ptsd outcomes on the population prior to ebola. overall, our findings underscore the feasibility and importance of monitoring and addressing mental health during public health outbreaks as well as building capacity to do so as part of preparedness efforts. use of brief mental health screeners during outbreak response could increase the ability to identify and address the needs of high-risk groups. we have demonstrated the ability to rapidly administer phq- and ies- at a population-level to 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use disorders demographic and health survey psychometric evaluation of the indonesian version of the impact of event scale-revised the global burden of mental disorders: an update from the who world mental health (wmh) surveys global health security: the wider lessons from the west african ebola virus disease epidemic acknowledgements the authors thank the sierra leoneans who participated in this assessment and provided responses in the midst of an unprecedented epidemic. the authors also thank the data collection team from focus for their diligent efforts in ensuring data quality and the government of sierra leone and their national and international partners in the response. finally, we dedicate this article to the memory of our co-author, dr. foday dafae, the late director of disease prevention and control in sierra leone ministry of health and sanitation, in honor of his years of service to the people of sierra leone. contributors mfj, rb, aol and ps led the overall study design with substantial contributions made by the other coauthors. ps, mfj and mbj were responsible for training the data collectors and supervised all data collection and data management efforts. wl led all data analyses. all authors contributed equally to the iterative interpretation of the results and the writing and preparation of the manuscript.funding this study was funded by the centers for disease control and prevention ( . / ).disclaimer the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the us centers for disease control and prevention or the sierra leone ministry of health and sanitation.competing interests none declared. key: cord- - cjr rol authors: chan, marion m.; chen, rensa; fong, dunne title: targeting cancer stem cells with dietary phytochemical - repositioned drug combinations date: - - journal: cancer lett doi: . /j.canlet. . . sha: doc_id: cord_uid: cjr rol the tumor microenvironment is complex with the cancer stem cell (csc) as a member within its community. this population possesses the capacity to self-renew and to cause cellular heterogeneity of the tumor. cscs are resistant to conventional anti-proliferative drugs. in order to be curative, it is imperative that cscs must be eliminated by cancer therapy. a variety of dietary phytochemicals and repositioned drugs can act synergistically with conventional anti-cancer agents. in this review, we advocate the development of a novel approach, namely combination therapy by incorporating both phytochemicals and repositioned drugs to target cscs. we cover select dietary phytochemicals (curcumin, resveratrol, egcg, genistein) and repurposed drugs (metformin, niclosamide, thioridazine, chloroquine). five of the eight (curcumin, resveratrol, egcg, genistein, metformin) are listed in “the halifax project”, that explores “the concept of a low-toxicity ‘broad-spectrum’ therapeutic approach that could simultaneously target many key pathways and mechanisms” [ ]. for these compounds, we discuss their mechanisms of action, in which models their anti-csc activities were identified, as well as advantages, challenges and potentials of combination therapy. in the course of its lifespan, a somatic cell undergoes changes in genome and epigenome due to intrinsic (cell-developmental) and extrinsic (environmental) factors. when accumulated changes disrupt its regulation of cell growth and death, the resulting uncharted growth leads to cancer. carcinogenesis is a multi-step process: initiation, promotion, progression and metastasis. the "hallmarks of cancer" denote alterations in cancer cell physiology [ ] . indeed, only a very limited number of genes ( or so), covering a dozen signaling pathways on the processes of cell fate, cell survival and genome maintenance, are relevant and designated as "driver" mutations [ ] . histologically, a tumor is heterogeneous, consisting of cancer cells at various stages of differentiation, as well as other cell types (fibroblasts, immune cells, endothelial cells). these cells behave as members of a community; the tumor microenvironment (niche) is a complex interactive network [ ] . cancer stem cell (csc) is the cell within a tumor that possesses the dual capacity to self-renew and cause the heterogeneous lineages of cancer cells that comprise the tumor [ ] . conventional chemotherapy targets the bulk of proliferating cancer cells. examples are paclitaxel for several cancer types and imatinib (tyrosine kinase inhibitor) for chronic myelogenous leukemia (cml). however, cscs are resistant to these anti-cancer drugs [ ] . a curative cancer therapy must acknowledge csc plasticity and their complete elimination. many csc-targeting dietary phytochemicals can act synergistically with conventional anti-cancer agents. recently, investigators discovered csc-targeting repurposed drugs [ ] . we advocate here a novel approach: combination therapy with the pairing of csc-targeting phytochemicals and repositioned drugs. our interest in ovarian cancer led us to phytochemical-drug combinations [ , ] . anti-cancer studies have demonstrated benefits of specific phytochemical combinations over individual compounds [ ] . dietary phytochemicals and repositioned drugs should provide easier access towards clinical use, because of the former's "generally regarded as safe" (gras) status and the latter's prior food and drug administration (fda) approval. this innovative approach is advantageous: both compounds target cscs, an activity insurmountable by current anti-cancer agents. for csc targeting, we recommend four dietary phytochemicals (curcumin, resveratrol, egcg, genistein) and four repurposed drugs (metformin, niclosamide, thioridazine, chloroquine). (see fig. applications of csc-targeting phytochemical-drug combinations. furthermore, five (curcumin, resveratrol, egcg, genistein, metformin) are listed by an international task force of clinicians and scientists of "the halifax project", with dedicated objective to "explore the concept of a low-toxicity 'broad-spectrum' therapeutic approach that could simultaneously target many key pathways and mechanisms" for cancer treatment [ ] . block et al. ( ) stated: "among approaches, curcumin, genistein, resveratrol and egcg boast a wealth of fundamental research" [ ] . ( we are aware investigators may disagree with our selection of phytochemicals; others will also be cited.) all eight have efficacy, are low cost and free of intellectual property constraints. these attributes make sure the combinations can be widely affordable, and are economical compared to current chemo-and targeted therapies. different from conventional anti-cancer drugs with unique molecular targets, dietary phytochemicals and repurposed drugs are pleiotropic. each has a multitude of cellular targets (as shown later in compound description). multilateral targeting of molecular and cellular pathways will inhibit csc growth or induce csc differentiation, and hinder the csc's ability to develop resistance. in this case, "promiscuous drugs" is a virtue; "dirty" might be better [ , ] . a phytochemicaldrug combination is advantageous for synergistic effects. such pairing allows lower effective doses than using a single compound for activity. lower doses mean less potential toxicity and fewer side effects. for a particular cancer, various combinations can be explored to look for the ideal pair at optimal doses. for clinical use, the combination can be used alone (if both cscs and bulk cancer cells are eliminated), or as adjuvant to conventional cancer therapy (the combination targets cscs and conventional therapy eliminates the bulk cancer cells). if the approach of csc-targeting dietary phytochemical-repositioned drug combination is important, what are the reasons for its being to present a more comprehensive picture, we start with considerations to csc methodologies on which various observations were made, in the "evolving concept" of csc [ , ] . we follow with compound description, and end with challenges and the need of applying csc-targeting phytochemical-drug combinations in the current climate of cancer therapy. human cscs were first discovered in leukemia and then characterized in solid tumors. utilizing an array of mechanisms, cscs are insensitive to conventional cancer treatments, but these mechanisms can be targets of phytochemicals and drugs (as described later) [ ] [ ] [ ] [ ] [ ] . (see fig. for key mechanisms of csc resistance to therapy.) cscs may be quiescent and exhibit a slow proliferating nature. they may possess an efficient dna repair system, activated by genomic instability and regulated by epigenetic modifications via histone deacetylases. pro-survival signaling pathways may be activated (hedgehog, wnt, notch, pi k, nfκb). cscs may have high levels of anti-apoptotic molecules. for drug resistance, both the activation of drug transporters, with high level expression of atp-binding cassette (abc) membrane transporters for drug efflux, and the enzyme aldehyde dehydrogenase (aldh), for drug metabolic activities, have been detected. in addition, their niche has less reactive oxygen species (ros), thus less susceptible to radiation therapy. one notable csc-associated feature is epithelial-mesenchymal transition (emt), a process when epithelial cells lose polarity and become less adhesive [ ] . with these changes the cells become invasive, migratory and mesenchymal-like with "stemness" properties. the process involves the activation of transcription factors (snail, twist) and change in marker proteins (e-to n-cadherin). first seen in embryogenesis, emt has been viewed as reappearance of embryonic features in cancer cells. the process is reversible and has been linked to cscs for tumor heterogeneity and metastasis. emt involvement with cscs is under active research [ ] . cscs can be identified by in vitro and in vivo methods. they are isolated via fluorescence-activated cell sorting (facs) by their surface antigens or detoxification capacity. cscs express identifying cell surface antigens, for example, ovarian cscs express cd (prominin- ); these molecules can serve as biomarkers allowing their isolation by facs. aldh is a detoxification enzyme that oxidizes aldehydes to carboxylic acids, for further metabolism or excretion via the liver. its activity has been utilized for csc selection by facs, using a fluorescent substrate (aldefluor) [ ] . the "side population" (sp) assay identifies cscs apart from the main population of cancer cells in flow cytometry, by their fast expulsion of a fluorescent dye (hoechst ). however, cell surface markers for csc selection are also present in normal stem cells and other cell types, they are not unique to cscs [ ] . both sp and aldh selections were originally developed for hematopoietic stem cells (hscs) and then adapted to cscs. spheroid culture is another method for identifying cscs. a cancer cell (transformed due to mutations) exhibits anchorage independence, a characteristic allowing it to form a cell colony in the absence of substratum, as shown by soft agar colony formation assay (normal cells die in the absence of attachment via the process of anoikis). for serum-free spheroid cultures (spheres), cancer cells grow as spheroids in suspension (in untreated plastic ware) when provided with specific growth factors (insulin, epidermal growth factor, basic fibroblast growth factor), unlike ordinary tissue culture where cells usually grow as a monolayer (with fetal bovine serum for growth factors and other components). spheroids mimic the three-dimensional nature of tumors with limited oxygen available to the internal cells. this hypoxic condition favors expression of pluripotency maintaining transcription factors, thus promoting the differentiation to cscs and facilitating the state of "stemness" [ ] . spheroids can form secondary and tertiary spheres in subsequent cultures. cscs known as "neoplastic sphereforming cells" are measured by enumerating secondary spheres generated when re-plating the spheres, based on the assumption that the secondary spheres are clones [ ] . the technique of spheroid culture was originally developed for neurobiology and then adapted to cscs [ ] . apart from in vitro methods, the "gold standard" for csc is "neoplasia-initiating cells" that regenerate detectable neoplastic populations as xenografts in immunodeficient mice in vivo [ ] . cscs, isolated from either a fresh tumor or an established cell line, are transplanted to initiate tumor growth in immunocompromised recipient animals. different models are available: athymic nude mice, non-obese diabetic/ severe combined immunodeficiency (nod/scid) mice, and nod/scid interleukin- receptor gamma chain null (il rg −/− ) (nsg) mice. the abundance of cscs in a tumor sample can be estimated as the xenotransplantation yield, which confirms the presence of cscs by limiting dilution analysis [ ] . many compounds (natural, natural-derived, synthetic) have been reported as csc-targeting [ , , ] . two strategies are available for compound identification: a pathway-specific approach and a general high throughput, mass screening approach. the first yielded cyclopamine from the plant corn lily veratrum californicum; it targets glioblastoma cscs via the hedgehog pathway but can act as a teratogen that causes cyclopia. the second yielded salinomycin from the bacterium streptomyces albus; it is toxic to humans so its use is confined to the poultry industry, as a coccidiostat for treating a parasitic protozoan disease [ ] . for clinical uses, medicinal chemists must modify to remove teratogenicity or toxicity. identified compounds must target only cscs and not normal stem cells. even though cscs differ from the bulk cancer cells, they may share gene expression and signaling pathways with normal stem cells. csc-targeting phytochemicals and drugs are discussed in the following sections. dietary phytochemicals have advantages over other compounds [ ] : ( ) they usually have very low or no toxicity, in contrast to most chemotherapeutic drugs; ( ) they are present in commonly consumed food that is readily available to most people in daily life; ( ) they have shown potential as adjuvants to chemotherapy. originally, they were studied for cancer chemo-prevention [ ] . as initiation (chemoprevention) and growth (cancer chemotherapy) may share common molecular mechanisms, they were applied to cancer therapy. furthermore, phytochemicals can overcome drug resistance in ovarian and other cancers [ ] . they act on genes and non-coding gene regulatory pathways (microrna) [ ] . most importantly, phytochemicals target cscs [ ] [ ] [ ] [ ] [ ] [ ] . commenting on cscs and dietary phytochemicals, kim et al. ( ) wrote: "a diet-induced shift from deregulation to regulation in cancer stem cells could have profound influence on cancer relapses and therefore is of immense societal importance" [ ] . instead of diet, here we propose the use of csc-targeting phytochemical-drug combinations for cancer therapy. our laboratories have been studying the phytochemicals curcumin, quercetin and egcg. these polyphenols act synergistically with cisplatin (conventional anti-cancer drug) in growth inhibition of drugresistant ovarian cancer cell lines [ , ] . these compounds are active against cscs in vitro, as well as against cancer and other diseases in animal models. some are in clinical trials, though to date none has been approved for cancer treatment. here, we discuss four csc-targeting phytochemicals: curcumin, resveratrol, egcg and genistein. (see table for select cancer clinical trials of the four phytochemicals, from https://clinicaltrials.gov/.) curcumin, a diferuloylmethane, is the biological principle from the indian spice turmeric that gives curry powder the yellow color. turmeric is produced from rhizome of the plant curcuma longa; it has multiple utilities and health benefits in traditional indian medicine (from insect bites to wound healing). a widely studied property of curcumin is anti-inflammation. curcumin inhibits many pro-inflammatory gene products, including enzymes (inducible nitric oxide synthase, inos), transcription factors (nuclear factor kappa b, nfκb), cytokines (tumor necrosis factor, tnfα) and chemokines (interleukin- , il- ) [ ] . using the lipopolysaccharide (lps)-induced murine sepsis model, we reported curcumin ingestion by gavage down-regulated inos gene expression in the murine liver [ ] . curcumin may suppresses carcinogenesis by down-regulating inflammation, which supports cancer cell survival, proliferation and invasion [ ] . another widely studied anti-cancer property of curcumin is csctargeting [ ] [ ] [ ] [ ] [ ] . (see table for select cellular targets of phytochemicals and drugs in cscs.) cscs that resist conventional anti-cancer drugs are susceptible to curcumin. curcumin acts on stem cell signaling pathways implicated in the process of carcinogenesis, including wnt, notch, hedgehog, and signal transduction and activator (stat). for example, in hepatocellular carcinoma cscs, tsai et al. ( ) reported that curcumin inhibited sp, invasion, emt and reduced tumor size and lung metastasis in a nude mice xenograft model. immunoblots revealed that the sphingosine -phosphate receptor (sipr ) signaling pathway was inhibited [ ] . in cell culture, subramaniam et al. ( ) reported that curcumin-induced apoptosis of esophageal cancer cells and decreased esophageal csc spheroid size and number. the molecular mechanism was inhibition of the notch pathway, seen as a decrease in rna and protein expression of γ secretase, notch- protein and its ligand jaggard- [ ] . the investigators also found down-regulated oncomir mirna (mir- , mir- a) and up-regulated tumor suppressor mirna (let- a mirna) in the curcumin-treated esophageal csc spheroids. similar to the esophageal example, for colorectal cscs, ramasamy et al. ( ) reported curcumin-induced epigenetic modifications, including the methylation of epidermal growth factor receptor (egfr) promoter, expression of microrna oncomirs relevant to metastasis (for emt), as well as suppression of csc markers (cd , aldh + ) [ ] . thus, curcumin modifies epigenetically by inducing specific methylation changes and regulating microrna expression. huminiecki et al. ( ) have reviewed the functional genomic studies of curcumin from microarray, methylation array, microrna array to rna-seq and concluded that curcumin has "powerful effects" on gene expression, including "genes involved in cell signaling, apoptosis, and the control of cell cycle" [ ] . curcumin can induce csc differentiation [ ] . zhuang et al. ( ) reported that curcumin promoted differentiation of glioma cscs and induced autophagy, using immunofluorescence to show decrease in stemness markers (cd ) and increase in differentiation markers (βiii tubulin) [ ] . curcumin can alter chemo-sensitivity. it synergizes with conventional drugs and has specificity for cscs. in vitro, curcumin enhanced cisplatin effects on non-small cell lung csc [ ] . we found that curcumin ( μm daily for days via medium change) reduced sp of the c rat glioma cells, possibly via inhibition of the drug transporter [ ] . in vivo, curcumin enhanced paclitaxel effects on brain tumor cscs; it sensitized breast cscs to mitomycin c in a nude mice xenograft model [ ] . several reasons have been proposed to explain the csc-specific action and low adversity towards normal stem cells [ ] . curcumin uptake may be greater in cscs, and curcumin may target the csc microenvironment which obviously differs from those of normal stem cells. for example, buhrmann et al. ( ) reported that curcumin sensitized colorectal cscs towards fluorouracil ( -fu) and suppressed the crosstalk between cscs and stromal fibroblasts in the tumor niche, using co-culture and assaying emt markers by immunoblotting [ ] . resveratrol is a stilbene ( , ′, -tri-hydroxy-trans-stilbene) from grapes, peanuts and pine nuts, and is present in red, but not white, wine because it is associated with the skin of grapes. it is a pleiotropic agent with multiple cellular targets. for example, it inhibits the mammalian target of rapamycin (mtor) pathways and cyclooxygenase (cox) enzymes to play roles in inflammatory diseases, diabetes, obesity, cardiovascular diseases, neurological disorders and cancer [ ] [ ] [ ] . resveratrol also activates amp-activated protein kinase (ampk, a key nutrient sensor) and sirtuin deacetylases, leading to lifespan extension in yeasts, nematodes, fruit flies, fish and obese mice (but not normal mice) [ ] . it retards the ageing process in mammals, similar to caloric restriction. furthermore, resveratrol is a phytoalexin (compound produced by plants against pathogens, such as fungal infections to the grape plant) and we found this property can be harnessed for inhibiting the growth of human cutaneous fungal species, thus potentially useful for tinea (ringworm, athlete's foot) [ ] . in cancer, resveratrol can target cscs. park and pezzuto ( ) have reviewed the molecular alterations resulting from resveratrol intervention in different breast, prostate, lung and colorectal cancer models [ ] . the general approach in these studies is to isolate the cscs, assay the effects of resveratrol in vitro and in vivo (comparing cscs with the bulk cancer cells) and determine which molecular mechanisms are modulated. for example, fu et al. ( ) reported resveratrol cytotoxicity to breast cscs in vitro [ ] . mechanistically, immunofluorescence and immunoblot of microtubule-associated protein light chain (lc ) and β catenin proteins showed that it induced autophagy and suppressed the wnt pathway. resveratrol also inhibited spheroid formation, decreased aldh + cells and decreased tumor size in murine xenografts. shankar et al. ( ) reported resveratrol inhibited pancreatic csc spheroid formation via the induction of apoptosis, inhibition of mrna expression of the pluripotency maintenance transcription factors (oct , nanog), as well as interference with emt [ ] . similar inhibition was also observed in a transgenic mouse model of pancreatic ductal adenocarcinoma. with glioblastoma multiforme, found that cscs pre-treated with resveratrol in vitro were less tumorigenic, as the xenografted scid mice survive better than the untreated counterpart [ ] . the investigators also performed microarray analyses and shrna confirmation to compare the effects of resveratrol on cscs (cd + ) and non-cscs (cd -). resveratrol inhibited "stemness" gene expression and induced csc differentiation via suppression of the stat pathway. resveratrol, like curcumin, can be csc-specific and spare the normal stem cells. pandey et al. ( ) reported that resveratrol blocked gene expression of fatty acid synthase and inhibited breast csc growth, but the enzyme in normal human mammary epithelial cells was not susceptible [ ] . fu et al. ( ) reported resveratrol was cytotoxic to breast cscs but not normal breast epithelial cells [ ] . similarly, sayd et al. ( ) reported that resveratrol blocked enzymatic induction of sirtuin activity and inhibited the growth of glioma cscs, but had no effect on normal neural stem cells (from fetal brains) [ ] . epigallocatechin gallate (egcg), a polyphenol (flavone- -ol) from the plant camellia sinensis, is the major component in green tea. its many health benefits include cancer chemoprevention and treatment, reduction of atherosclerosis, hypercholesterolemia, alzheimer's and other ageing-related diseases. before cscs, egcg was first studied for its effect on normal stem cells. chen components, including egcg, inhibited normal rat neurosphere adhesion and migration [ ] . egcg has pleiotropic activity on multiple cellular targets, both genetic and epigenetic (including dna methyltransferase, nfκb, ampk signaling pathway) [ ] [ ] [ ] . it can act on its target with very impressive sensitivity, kd of . nm for the kd laminin receptor [ ] . in cancer, egcg can target cscs. it inhibited aldh activity, spheroid formation, and expression of "stemness" genes (oct and nanog) in human neuroblastoma cscs [ ] . similar results were observed in breast cscs. egcg treatment significantly decreased the expression of nanog, the number of aldh + cells, and the tumor volume in mouse xenografts [ ] . furthermore, chen et al. ( ) reported egcg inhibited the spheroid formation of colorectal cscs, the mechanism being inhibition of the wnt pathway, as shown by a decrease in β catenin in immunoblot [ ] . egcg works in combination with other phytochemicals and conventional drugs to enhance anti-cancer effects, targeting bulk cancer cells and cscs [ ] . it synergized with quercetin to decrease spheroids, inhibited activation of emt and increased apoptosis in prostate cscs [ ] . it synergized with temozolomide to decrease spheroids and pglycoprotein synthesis in glioma cscs [ ] . we found that egcg acted synergistically with cisplatin to inhibit the cisplatin-resistant ovarian cancer cell line c in cell culture [ ] . in vivo, the egcg and cisplatin combination decreased tumor formation in xenografts of head and neck, and nasopharyngeal cscs [ , ] . it induced chemo-sensitivity to cisplatin by enhancing apoptosis and inhibiting the phosphorylation of stat in nasopharyngeal cscs [ ] . these findings suggest its use as an adjuvant in cancer therapy [ ] . genistein, an isoflavone ( , , -trihydroxyisoflavone) from the legume plant glycine max, is found in soybeans. it has been classified as a phytoestrogen because it binds estrogen receptor. genistein is the active ingredient in soy-rich food that contributes to the lower rates of prostate and breast cancers in china and japan, as compared to western countries. besides as anti-cancer agent, genistein has other health benefits, including osteoporosis, heart diseases and cognition [ ] [ ] [ ] [ ] . genistein has multiple cellular targets and acts on a spectrum of protein tyrosine kinases and dna topoisomerase ii. it targets cscs in solid tumors. huang et al. ( ) reported genistein inhibited spheroids, "stemness" (oct and nanog gene expression) and reduced xenograft tumor volume of gastric cscs [ ] . genistein also inhibited drug transporter and extracellular signal-regulated kinase (erk) pathway in these cscs. in both breast and prostate cscs, genistein down-regulated the hedgehog pathway (reducing gli gene expression) and decreased spheroid formation in cell culture and tumor volume in xenografts [ , ] . in chronic myelogenous leukemia (cml), genistein reduced the leukemic progenitor cells by inhibiting expression of the tyrosine kinase coded from the breakpoint cluster region/abelson murine leukemia viral oncogene homolog (bcr/abl) fusion gene [ ] . however, as a protein tyrosine kinase inhibitor, genistein targets both leukemic stem cells and normal stem cells. genistein acts synergistically with conventional anti-cancer agents; it also targets the csc microenvironment. it overcame docetaxel resistance, and decreased the tumor size of docetaxel-resistant prostate cscs (than either compound administered separately) [ ] . breast adipose tissue contributes to breast cancer development. montales et al. ( ) found that genistein acted on the csc niche and inhibited the differentiation of mammary stromal fibroblast-like cells to adipocytes [ ] . the mechanism was inhibition of pparγ and fatty acid synthase gene expression. interestingly, the investigators discovered genistein's biphasic effect, only seen in low dose ( nm) but not high dose ( μm). montales et al. ( ) also found the effect of genistein to be transferrable [ ] . when fed to mice at concentrations present in soy protein isolate ( mg/kg food), their sera could inhibit the spheroid formation of human breast cscs when added at - % in cell culture. this suggests the presence of "csc inhibiting factors" in the circulation after genistein consumption. furthermore, genistein has transgenerational effect. maternal dietary supplementation with genistein leads to dna hyper-methylation in the embryo, and this methylation state is maintained until adulthood. the color change in the fur of genistein-fed agouti mice indicates an epigenetic biosensor for nutritional and environmental alterations on the fetal genome. phytochemicals such as genistein modify the epigenome and the effect starts early in embryogenesis [ ] . fda-approved small-molecule drugs for some diseases/disorders have been shown to target cscs by the process of drug repurposing. the nih national center for advancing translational sciences (ncats) defines: "repurposing generally refers to studying drugs that are already approved to treat one disease or condition to see if they are safe and effective for treating other diseases". the rationale is to expedite drug discovery. ncat estimates years for a new drug development. therefore, the strategy is "to reduce this time frame, decrease costs and improve success rates …. many agents approved for other uses already have been tested in humans, so detailed information is available on their pharmacology, formulation and potential toxicity. because repurposing builds upon previous research and development efforts, new candidate therapies could be ready for clinical trials quickly …" (https://ncats.nih.gov/preclinical/repurpose#learn-more). drug repurposing, also known as drug repositioning, drug re-tasking, drug reprofiling or therapeutic switching, is a way to obtain fast and cost-efficient drug discovery, since this strategy effectively enables the preclinical studies to be bypassed. resources (drugsurv, drug repurposing hub) are available for this "new tricks for old drugs" [ , [ ] [ ] [ ] . investigators can perform experimentation in silico, in vitro or in vivo in search for new targets of existing drugs. for cscs, drug libraries are screened in assays of csc inhibition, as shown below for niclosamide and thioridazine. here, we discuss four csc-targeting drugs: metformin, niclosamide, thioridazine and chloroquine. probably the most promising discovery is metformin, a synthetic biguanide derived from the herb french lily (galega officianalis) [ ] . it is an inexpensive and long-approved drug for type diabetes, prescribed to an estimated million individuals worldwide. it modulates energy metabolism by targeting ampk and other ampk-independent effects. besides diabetes, metformin has been shown to be applicable to cardiovascular and neurodegenerative diseases [ ] [ ] [ ] [ ] [ ] . it is in a clinical trial for longevity and ageing [ ] . metformin was explored for csc-targeting because it targets multiple signaling pathways (wnt, ampk, nfκb) [ , ] . metformin has been used in combination therapy. for example, it synergized with the conventional combination of -fluorouracil ( -fu), epirubicin and cyclophosphamide (fec chemotherapy) in inhibiting breast cscs derived from spheroids [ ] . it enhanced the effect of denosumab, an antibody against rankl (receptor activator of nfκb ligand, a cytokine), in inhibiting breast csc spheroids [ ] . it enhanced the sensitivity of pancreatic cscs to gemcitabine in vivo, significantly reducing the tumor volume in murine xenografts [ ] . when given in combination, metformin and doxorubicin showed the advantage of targeting both breast cscs and non-stem cancer cells, by reducing the overall tumor growth and tumor remission in nude mice [ ] . synergy was found with paclitaxel and carboplatin in prostate and lung cscs murine xenografts [ ] . for phytochemicals, montales et al. ( ) reported that metformin and genistein targeted colon csc spheroid formation and proliferation, and the combination increased the effectiveness of -fu [ ] . ning et al. ( ) reported that both curcumin and metformin individually inhibited pancreatic csc spheroids [ ] , the two compounds should be tested as a combination. these examples suggest a combinational strategy for metformin to target cscs and cancers [ ] . the next most promising existing drug is niclosamide, an anthelminthic against tapeworms [ ] . it is also active against viruses (severe acute respiratory syndrome, sars virus), possibly because it blocks proton carriers. niclosamide was explored for csc-targeting based on the general association that a developmental stage of tapeworm, the metacestode, resembles cancer in having uncontrolled proliferation, invasion and metastasis, and can be difficult to kill without collateral damage to the surrounding host tissues [ ] . niclosamide can block multiple csc signaling pathways, including wnt, notch, stat and nfκb [ ] . wieland et al. ( ) selected niclosamide as the lead for glioblastoma targeting after screening the member killer plate compound library [ ] . simultaneous inhibition of multiple pathways (wnt, notch, mtor, nfκb) was indicated when niclosamide pretreatment inhibited glioblastoma xenograft development in nude mice. from drugs in the lopac chemical library, [ ] and yo et al. ( ) [ ] selected niclosamide for its inhibition of breast and ovarian cscs, respectively. niclosamide inhibited spheroid formation and reduced tumor volume of xenografts in nod/scid mice. it acted on cisplatin-resistant ovarian cscs. spheroids from patient-derived cd + and aldh + ovarian cancer were susceptible to niclosamide, with inhibition of the wnt pathway (shown by β catenin immunoblot) [ ] . furthermore, it complements platinum drugs. niclosamide in combination with carboplatin inhibited primary ovarian cscs [ ] and in combination with cisplatin inhibited breast csc spheroid formation and reduced tumor size of xenografts in nude mice [ ] . thioridazine, a phenothiazine, is dopamine receptor antagonist originally prescribed as an anti-psychotic drug for schizophrenia in the s. withdrawn by norvatis in due to cardiac side effects, the generic version is still available. interest in thioridazine has recently been revived as anti-microbial against methicillin-resistant staphylococcus aureus (mrsa) and multidrug-resistant mycobacterium tuberculosis [ ] . thioridazine targets cscs. it can inhibit cscs from a spectrum of origins: myeloid leukemia, glioblastoma, lung, liver, ovarian and breast cancers [ ] [ ] [ ] [ ] [ ] . it inhibits csc spheroid formation and induces apoptosis in vitro, and the treated cells show reduced xenograft tumor volume in mice. sachlos et al. ( ) selected thioridazine in their screening for acute myeloid leukemia (aml) csc-specific but not normal pluripotent stem cell-reactive molecules from compounds of the nih clinical collection and canadian collection [ ] . thioridazine was found to induce aml differentiation; it also synergized with cytosine arabinoside (cytarabine, arac) in aml csc killing. in glioblastoma cscs, it induced autophagy (seen as expression of biomarker lc ) [ ] . in lung, hepatoma and breast cscs, it induced apoptosis in association with activation of caspases, inhibition of "stemness" gene expression (oct , cd ), and inhibition of the mtor pathway, respectively [ ] [ ] [ ] . synergy with conventional drug has been shown: thioridazine was co-delivered with doxorubicin in mixed polymeric micelles to target both breast cscs and bulk cancer cells [ ] . we found that thioridazine and curcumin may act synergistically in inhibiting spheroids from ovarian cancer cells, with the combination more effective than either compound alone. (see fig. for inhibition of spheroids by thioridazine and curcumin). chloroquine, a -aminoquinoline, is an anti-malarial [ ] . first discovered in b y hans andersag at bayer, it was ignored until rediscovery by us army during world war ii. highly effective and well tolerated, chloroquine remains the drug of choice for malaria. its accumulation within lysosomes raises organelles' ph and inhibits lysosomal function in the malaria parasite. chloroquine is also used for immunosuppression in rheumatoid arthritis. chloroquine, either independently or in combination with conventional drugs, has efficacy against several cancers. it inhibits cscs from a spectrum of origins: glioma, liver, pancreatic, urothelial, ovarian and breast cancer [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . it inhibits autophagy and spheroid formation; it induces csc apoptosis. these effects have been demonstrated in primary glioma cscs, cd + liver cscs enriched by deprivation of oxygen and nutrients, pancreatic cscs, urothelial carcinoma cscs, and breast cscs. the csc-targeting chloroquine shows synergistic effects with conventional therapy [ ] . it sensitizes radiation and chemotherapeutic agents in cancer [ , ] . for example, chloroquine increased the cytotoxicity of gemcitabine-mitomycin combination against urothelial carcinoma cscs [ ] . it synergized with gemcitabine to decrease tumor volume in pancreatic csc xenografts [ ] , and with carboplatin in breast csc xenografts [ ] . we discuss four phytochemicals and four drugs that target cscs, and view them as the most promising for a novel approach of phytochemical-drug combination. there are others. exploring csc-targeting phytochemicals is a growing field. other dietary phytochemicals include quercetin, a flavonol found in common fruits and vegetables such as apples, cranberries and onions; sulforaphane, an isothiocyanate found in cruciferous such as broccoli, cauliflower and kale; indole- -carbinol, another compound from cruciferous vegetables; and cucurbitacin i, from cucumber. non-dietary phytochemicals include parthenolide from herbal medicine feverfew plant (tanacetum parthenium), celastrol from traditional chinese medicine thunder god vine (tripterygium wilfordii), berberine from herbal medical plant chinese goldthread (coptis chinensis), oxymatrine from chinese herbal medical plant sophora flavescens, silybin (silibinin) from milk thistle (silybum marianum), and gossypol from cotton plant (genus gossypium) [ ] . similarly, the list of csc-targeting existing drugs is expanding. other repurposed drugs include anti-allergy drug tranilast, cholesterollowering statins (simvastatin and lovastatin), and anti-alcoholism drug disulfiram [ , , , , ] . we anticipate more to come. for challenges we start with the safety issue. with gras status dietary phytochemicals are apparently safe. for example, when curcumin was given to cancer patients at mg/day for months, only minor adverse effects were observed. daily oral doses of egcg, for weeks at mg/day in volunteers, caused only minor adverse effects. a single oral dose of resveratrol at g in volunteers caused only minor adverse effects [ ] . with prior fda approval, repurposed drugs are also presumably safe. although individual compounds are safe, for the selected synergistic csc-targeting phytochemical-drug combination, pharmacokinetics and pharmacodynamics features, toxicity profiles, are still needed because this information is lacking. current clinical trials may yield useful data. (see table for phytochemical clinical trials.) molecular targets of phytochemicals are under scrutiny. recent findings suggest that promiscuity (multiple targets) results from perturbation of cellular membranes, leading to alteration of multiple membrane protein functions [ ] . this multi-target nature of polyphenolic phytochemicals hamper therapeutic development because, in high throughput drug screening assays, phytochemicals (including curcumin, resveratrol, egcg and genistein) are known as "pan-assay interference compounds" (pains) and "invalid metabolic panaceas" (imps) for their non-specific inhibitions [ ] [ ] [ ] . whereas there may be truth to the designation, the fact that phytochemicals (such as curcumin) will cause global changes in gene expression shows there are more than mere non-specific protein interactions [ ] . nonetheless, multi-target interactions, specific or non-specific, at the protein or nucleic acid level, will be a plus in csc-targeting as long as normal stem cells are spared. additional concerns are phytochemical bioavailability in vivo and product quality. for some polyphenolic compounds, effects in vivo, although significant, are less prominent than ones observed in vitro [ ] . the bioavailability issue has been addressed with enhancing compounds, such as piperine from the spice black pepper (piper nigrum), which amplifies the effect of phytochemicals (including curcumin). bioavailability can be aided by formulations and special delivery systems, such as nanoparticles for curcumin and micelles for niclosamide [ , [ ] [ ] [ ] . curcumin is an example which we managed to devise a measure to enhance bioavailability. by oral gavage to mice on empty stomach, we demonstrated in vivo effect: curcumin inhibition of the inflammatory inos in murine liver (beneficial outcome) and exacerbation of murine visceral leishmaniasis, a protozoan parasitic disease (detrimental outcome) [ , ] . another concern is reliability of the source of phytochemicals. consistent and unadulterated phytochemicals are essential, for example, curcumin as curcumin c complex from sabinsa and egcg as polyphenon e from mitsui norin (as used in some clinical trials, see table ) [ ] . (repurposed drugs, as synthetic compounds, do not have this problem.) yet another approach is the synthesis of better analogs and derivatives (such as difluorinated-curcumin and various chloroquine analogs), but they are beyond the scope of this review [ , , , ] . discrepancies in phytochemical research results are another challenge. whereas we found curcumin inhibited sp in rat glioma [ ] , found curcumin induced human glioma cscs [ ] . kakarala et al. ( ) reported curcumin inhibited both breast cscs and normal human breast stem cells, thus affecting both cscs and normal stem cells [ ] . these differences can be attributed to variations in dosage, length of treatment periods, grade and stability of compounds used, or other experimental conditions [ ] . polyphenolic phytochemicals have antioxidant capacities against reactive oxygen species (ros), but under special conditions they exhibit pro-oxidant capacity [ ] . whereas ros are harmful in general, the removal of too much ros interferes with bodily functions, as seen in the warning presented by scientists to the food industry [ ] . related to dosage is the concept of hormesis [ ] , which suggests the fundamental nature of the dose-response curve is neither linear nor threshold, but u-or j-shaped. a low dose stimulatory response is the hormetic effect, representing overcompensation in response to disruptions in homeostasis. a biphasic dose response is observed. at high concentrations, phytochemicals can be toxic, whereas sub-toxic doses may induce adaptive stress responses. hormetic mechanisms of action have been proposed to underlie many of the health benefits of phytochemicals [ ] . protective effect of resveratrol is only observed in low dose but not high dose in a mouse colon cancer model [ ] . similar dose effects are seen in genistein [ ] . for repurposed drugs, most research results indicate csc-targeting, there are discordance. sancho et al. ( ) reported metformin-resistant pancreatic cscs [ ] and xin et al. ( ) reported metforminresistant liver cscs [ ] . in these studies, the effective dose of metformin is much higher than other drugs or phytochemicals. moreover, asiedu et al. ( ) reported heterogeneity in metformin response in both patient-derived and cancer cell lines [ ] . chloroquine is "a double-edged sword of autophagy", causing severe kidney damage [ ] . whether it is the compound's pleiotropic nature that leads to unexpected adverse effects is still unclear. for repurposed drugs, studies are needed to determine that they can reach the csc niche [ ] . combinational treatment has a long history in cancer chemotherapy. the first successful example is the treatment of childhood leukemia with "vamp", four drug combination (vincristine, amethopterin, -mercaptopurine, prednisone) in [ ] . our approach continues this tradition. we recommend four phytochemicals (curcumin, resverestrol, egcg, genistein) and four drugs (metformin, niclosamide, thioridazine, chloroquine). this choice is in sync with selections by the halifax project, aiming at multi-targeted compounds for cancer prophylaxis and treatment [ ] . we advocate a novel approach of csc-targeting dietary phytochemical-repurposed drug combinations and look forward towards clinical applications. in the united states, cancer drug prices are getting out of hand. the march report to president donald trump stated: "the president's cancer panel concluded that addressing the dramatic rise of cancer drug prices must be made a national priority. … innovative drugs offer new hope for patients to achieve long-term remissions-even cures-but virtually all new cancer drugs enter the market with a price tag that exceeds $ , per year and, increasingly, much higher" (https://prescancerpanel.cancer.gov/report/drugvalue). instead of new cancer drugs, the older repurposed drugs in combination with dietary phytochemicals are inexpensive and may work just as well. cancer is a worldwide problem affecting all nations rich and poor. on this topic, sullivan et al. ( ) warned: "treating cancer with the latest drugs and techniques is costly and will not improve survival globally" [ ] . they asserted: "cancer is on the rise" and concluded the focus should be "on building infrastructure, and delivering affordable, equitable and effective care". our idea of csc-targeting phytochemical-drug combination fits seamlessly in this scenario as effective and affordable cancer treatment for the global population. for world health organization (who), cancer is non-communicable chronic disease, and the increase in cancer is due to an aging population and lifestyle changes. there is an urgent need for affordable cancer treatment. whether independently administered or in combination (as adjuvant with current therapy), development of a csc-targeting dietary phytochemical-repurposed drug combination will be affordable, effective, and low toxicity. our goal is to increase the scientific community's awareness and build a momentum towards conducting well-controlled clinical trials, done under uniform experimental conditions and in a double-blinded manner, to fully validate this novel combinatorial approach. since we propose to combine dietary phytochemicals and repurposed drugs, i.e. non-patentable natural products and patent-expired older drugs, we envision financial supports for this endeavor have to come from governmental and philanthropic resources. the novel approach is ready for action. none declared. designing a broad-spectrum integrative approach for cancer prevention and treatment hallmarks of cancer: the next generation tumorigenesis: it takes a village cancer stem cells-perspectives on current status and future directions: aacr workshop on cancer stem cells dietary phytochemicals target cancer stem cells for cancer chemoprevention existing drugs and their application in drug discovery targeting cancer stem cells inhibition of growth and sensitization to cisplatin-mediated killing of ovarian cancer cells by polyphenolic chemopreventive 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treatment: friend, foe or foil? antioxidants in foods: state of the science important to the food industry hormesis: from marginalization to mainstream: a case for hormesis as the default dose-response model in risk assessment cancer chemoprevention: evidence of a nonlinear dose response for the protective effects of resveratrol in humans and mice myc/pgc- alpha balance determines the metabolic phenotype and plasticity of pancreatic cancer stem cells liver label retaining cancer cells are relatively resistant to the reported anti-cancer stem cell drug metformin patient-and cell type-specific heterogeneity of metformin response chloroquine in cancer therapy: a double-edged sword of autophagy a history of cancer chemotherapy cancer patients need better care, not just more technology we thank drs. david axelrod and dongming sun for helpful comments. we are grateful to claire chen, chase christensen, nana haruna, avery lee and tanjida tasmin for assistance in manuscript preparation. mmc received grant support from american institute for cancer research. rc was the recipient of undergraduate research fund from aresty research center at rutgers university. df received grant support from new jersey health foundation (pc - ). key: cord- -cv mkpzd authors: kim, jung heon; bae, wonjun; kim, jiyeon; hwang, eung soo title: an urgent need for global preparedness against the reemergence of “forgotten” infectious diseases in korea date: - - journal: j korean med sci doi: . /jkms. . .e sha: doc_id: cord_uid: cv mkpzd nan the authors have no potential conflicts of interest to disclose. . hav infection was not an important pathogen until the s- s because almost all persons were infected in early ages with low sequelae by poor hygienic environment. they had immunity to hav and there was no need for vaccination at those time. however rapid increases of hav from , cases in to , cases in were reported with an outbreak with , cases in . some of hav cases were imported from overseas by abroad travelers. what's next? measles, pertussis, and more? we should not make these infectious diseases as "never-ending stories," and comprehensive global preparedness for preventing outbreaks is needed urgently. we already know that vaccine program pursues high rate of vaccination. according to the report from korean centers for disease control and prevention (kcdc) in , vaccination rates between and -year-old children for national essential vaccination were over %; bcg . %, hepb . %, dtap . %, ipv . %, mmr . %, var . %, je . %. the vaccination rate in nip was reported in young ages but there is little nation-wide survey data of their appropriateness of immune formation to prevent infection after vaccination. we do not know why mumps and varicella are continuously and increasingly prevalent in spite of high vaccination rates. is it from a vaccine procedure failure or genetic changes of causative agents? it is impossible to answer this question immediately because there is little basic background data in korea. we habitually adopt the data of infection status, seroepidemiological data from other advanced countries when an outbreak occurs without our own continuous and routine investment for the basic infection data. if they were utilized without global understanding or analyzed without the comparison with our own data, it would not always help to resolve our need because the situation of each country is different from each other. we already know changes much in genetic characteristics of many causative pathogens, nutrition, individual and herd immunity, ways of domestic and international transportation, socioeconomical and environmental situation from the past ones. therefore, it is imperative that a global and comprehensive preparedness mechanism be implemented, not only for the emerging infectious diseases but also re-emerging, "forgotten" ones. the kcdc is the sole entity responsible for the control of legal reporting of communicable diseases in the country, but it has limited resources and faces an uphill battle in realizing complete preparedness for all infectious diseases. we have already learned a difficult lesson from the dearth of infectious diseases specialists during the influenza epidemic in and the middle east respiratory syndrome-related coronavirus (mers-cov) outbreak in . it is true that, in response to these crises, the korean government has reformed the structure of kcdc and increased its workforce to more effectively control emerging infectious diseases. however, old-fashioned infectious diseases, scarlet fever, mumps, hepatitis a, varicella, and zoster are in resurgence. it is necessary to gain a comprehensive understanding of the characteristics of pathogens, hygiene levels, immunity status and changes in each age group, environmental alterations, dietary nutrition, vaccine supply, treatment modalities, international relationship of diseases, so on. in order to make and keep korea safe from infectious diseases, we must expend every effort to go beyond the current fragmented approaches to institute a more balanced framework predicated on a mutually-reinforcing, cross-sectoral network of stakeholders. this would necessitate, inter alia, strengthening the planning and implementation capacities of korean government including kcdc, enhancing the participation of regional government bodies, supporting academic research at universities, infectious disease institutes and related entities. references . ministry of health and welfare national childhood vaccination coverage among children aged years in korea the research was performed using literature and services at the seoul national university college of medicine. key: cord- -z eh z authors: zhou, jie; li, cun; sachs, norman; chiu, man chun; wong, bosco ho-yin; chu, hin; poon, vincent kwok-man; wang, dong; zhao, xiaoyu; wen, lei; song, wenjun; yuan, shuofeng; wong, kenneth kak-yuen; chan, jasper fuk-woo; to, kelvin kai-wang; chen, honglin; clevers, hans; yuen, kwok-yung title: differentiated human airway organoids to assess infectivity of emerging influenza virus date: - - journal: proc natl acad sci u s a doi: . /pnas. sha: doc_id: cord_uid: z eh z novel reassortant avian influenza h n virus and pandemic h n (h n pdm) virus cause human infections, while avian h n and swine h n virus mainly infect birds and pigs, respectively. there is no robust in vitro model for assessing the infectivity of emerging viruses in humans. based on a recently established method, we generated long-term expanding d human airway organoids which accommodate four types of airway epithelial cells: ciliated, goblet, club, and basal cells. we report differentiation conditions which increase ciliated cell numbers to a nearly physiological level with synchronously beating cilia readily discernible in every organoid. in addition, the differentiation conditions induce elevated levels of serine proteases, which are essential for productive infection of human influenza viruses and low-pathogenic avian influenza viruses. we also established improved d monolayer culture conditions for the differentiated airway organoids. to demonstrate the ability of differentiated airway organoids to identify human-infective virus, d and d differentiated airway organoids are applied to evaluate two pairs of viruses with known distinct infectivity in humans, h n /ah versus h n and h n pdm versus an h n strain isolated from swine (h n sw). the human-infective h n /ah virus replicated more robustly than the poorly human-infective h n virus; the highly human-infective h n pdm virus replicated to a higher titer than the counterpart h n sw. collectively, we developed differentiated human airway organoids which can morphologically and functionally simulate human airway epithelium. these differentiated airway organoids can be applied for rapid assessment of the infectivity of emerging respiratory viruses to human. novel reassortant avian influenza h n virus and pandemic h n (h n pdm) virus cause human infections, while avian h n and swine h n virus mainly infect birds and pigs, respectively. there is no robust in vitro model for assessing the infectivity of emerging viruses in humans. based on a recently established method, we generated long-term expanding d human airway organoids which accommodate four types of airway epithelial cells: ciliated, goblet, club, and basal cells. we report differentiation conditions which increase ciliated cell numbers to a nearly physiological level with synchronously beating cilia readily discernible in every organoid. in addition, the differentiation conditions induce elevated levels of serine proteases, which are essential for productive infection of human influenza viruses and low-pathogenic avian influenza viruses. we also established improved d monolayer culture conditions for the differentiated airway organoids. to demonstrate the ability of differentiated airway organoids to identify human-infective virus, d and d differentiated airway organoids are applied to evaluate two pairs of viruses with known distinct infectivity in humans, h n /ah versus h n and h n pdm versus an h n strain isolated from swine (h n sw). the humaninfective h n /ah virus replicated more robustly than the poorly human-infective h n virus; the highly human-infective h n pdm virus replicated to a higher titer than the counterpart h n sw. collectively, we developed differentiated human airway organoids which can morphologically and functionally simulate human airway epithelium. these differentiated airway organoids can be applied for rapid assessment of the infectivity of emerging respiratory viruses to human. airway organoid | proximal differentiation | influenza virus | infectivity i nfluenza a viruses (iavs) can infect a diversity of avian and mammalian species, including humans, and have the remarkable capacity to evolve and adapt to new hosts ( ) . the segmented rna genomes of iavs and the low fidelity of rna polymerase allow antigenic shift and drift, which drive the viral evolution. thus, novel viruses from birds and pigs cross the species barrier and infect humans, leading to sporadic infections, epidemics, and even pandemics ( , ) . despite the tremendous progress made in virology and epidemiology, which subtype or strain of iav will cause the next outbreak remains unpredictable. a novel reassortant h n influenza virus from poultry has led to recurrent outbreaks of human infection in china since ( , ) . more than , laboratory-confirmed cases of h n human infections were reported by october , with a case-fatality rate higher than % ( ). in , the first influenza pandemic of the st century was caused by a novel pandemic h n (h n pdm), which originated via multiple reassortments of "classical" swine h n virus with human h n virus, avian virus, and avian-like swine virus ( ). while swine viruses infect humans only sporadically, this novel strain of swine-derived h n pdm virus has infected a large proportion of the human population, establishing sustained human-to-human transmission and circulating globally as a seasonal virus strain since then. proteolytic cleavage of viral glycoprotein ha is essential for iav to acquire infectivity, since only the cleaved ha molecule mediates the membrane fusion between the virus and the host cell, a process required for the initiation of infection. ha proteins of low-pathogenic avian iavs and human iavs carry a single basic amino acid arginine at the cleavage site ( , ), recognized by trypsin-like serine proteases. productive infection of significance influenza virus infection represents a major threat to public health worldwide. there is no biologically relevant, reproducible, and readily available in vitro model for predicting the infectivity of influenza viruses in humans. based on the longterm expanding d human airway organoids, we developed proximal differentiation and further established a d monolayer culture of airway organoids. the resultant d and d proximal differentiated airway organoids can morphologically and functionally simulate human airway epithelium and as a proof of concept can discriminate human-infective influenza viruses from poorly human-infective viruses. thus, the proximal differentiated airway organoids can be utilized to predict the infectivity of influenza viruses and, more broadly, provide a universal platform for studying the biology and pathology of the human airway. these viruses in the human airway thus requires serine proteases such as tmprss , tmprss , hat, and others ( ) . however, ha proteins of highly pathogenic avian viruses, such as h n , contain a polybasic cleavage site which is activated by ubiquitously expressed proteases. current in vitro models for studying influenza infection in the human respiratory tract involve shortterm cultures of human lung explants and primary airway epithelial cells. human lung explants are not readily available on a routine basis. in addition, the rapid deterioration of primary tissue is a major problem in infection experiments, since there is no protocol to maintain tissue viability in vitro. under air-liquid interface conditions, basal cells isolated from human airway can polarize and undergo mucociliary differentiation. however, this capacity is lost within two or three passages ( ) . collectively, these primary tissues and cells hardly constitute a convenient, reproducible model to study human respiratory pathogens. although various cell lines, e.g., a and madin-darby canine kidney (mdck) cells, have been commonly used to propagate influenza viruses and to study their virology, they poorly recapitulate the histology of human airway epithelium. in addition, due to the low serine protease activity, most cell lines do not support the growth of the influenza viruses with a monobasic ha cleavage site unless the culture medium is supplemented with an exogenous serine protease, trypsin treated with n-tosyl-l-phenylalanine chloromethyl ketone (tpck). thus, a biologically relevant, reproducible, and readily available in vitro model remains urgently needed for studying the biology and pathology of the human respiratory tract. recent advances in stem cell biology have allowed the in vitro growth of d organoids that recapitulate the essential attributes of their counterpart organs in vivo. these organoids can be grown from pluripotent stem cells (pscs) or tissue-resident adult stem cells (ascs) ( ) . asc-derived organoids consist exclusively of epithelial cells and can be generated from a variety of human organs, the first being the human gut ( ) . these human intestinal organoids represent the first model for in vitro propagation of norovirus and have allowed the study of other viruses ( , ) . we very recently described the initial conditions for expanding human asc-derived airway organoids (aos) for > y ( ) . of note, protocols also have been established for generating lung organoids from human pscs and embryonic lung ( , ) . characterization of the human aos. based on the recently reported protocol ( ) , we established several lines of aos from small pieces of normal lung tissue adjacent to the diseased tissue from patients undergoing surgical resection for clinical conditions. these aos, d cysts lined by polarized epithelium, accommodated the four major types of airway epithelial cells, i.e., ciliated cells (acctub + or foxj + ), goblet cells (muc ac + ), basal cells (p + ), and club cells (cc + ) (fig. a) . apical acctub clearly indicated the orientation of polarization. most organoids are orientated towards the lumen (fig. a , acctub left), while a small proportion of the organoids are inverted (fig. a , acctub right). beating cilia were visible (movie s ). no type i and type ii alveolar epithelial cells were present. thus, these organoids resembled the pseudostratified ciliated airway epithelium. the aos were inoculated with human iav h n pdm, the low-pathogenic avian virus h n /ah, and the highly pathogenic avian virus h n . the intracellular (cell lysate) viral loads of all three virus strains increased over log units (fig. b) . the extracellular (supernatant) viral loads, especially the viral titers, were elevated by - log units. ciliary beating plays an essential role in human airway biology and pathology, and - % of airway epithelial cells are ciliated ( ) . however, immunostaining (fig. a ) and flow cytometry indicated that ciliated cells were apparently underrepresented in these aos. therefore, despite the discernible multilineage differentiation and the ability to support replication of iavs, further improvements in morphology and differentiation appeared required. pd of the aos. to improve proximal differentiation (pd), we tried various protocols and variations thereof and settled on a medium described by konishi et al. ( ) for inducing ciliary differentiation in human psc-derived airway epithelial spheroids. the ascderived organoids in the original ao medium gradually enlarged, while those in pd medium became more compact ( fig. a) . after d of culture, the organoids in ao medium grew approximately two times larger, while the organoids in pd medium remained basically unchanged in size (fig. b) . from day , the number of ciliated cells increased markedly in pd medium. at day , beating cilia were observed in a minority (< %) of the organoids in ao medium, while abundant beating cilia were present in every pd organoid ( fig. c and movies s and s ). the synchronously beating cilia drove the cell debris within the organoid lumens to swirl unidirectionally (movie s ). the dramatically increased abundance of ciliated cells in the pd organoids was verified by immunofluorescence staining (fig. d) . consistently, the transcriptional levels of the ciliated cell markers foxj and sntn were strongly up-regulated in the pd organoids compared with the organoids in ao medium. the expression levels of the basal cell markers p and ck and the goblet cell marker muc ac also increased, whereas the levels of club cell markers cc and scgb a were substantially down-regulated in the pd organoids (fig. a) . importantly, we observed globally elevated expression of serine proteases including tmprss , tmprss , tmprss d (hat), and matriptase, which are essential for the activation and propagation of human iavs and low-pathogenic avian iavs ( ) . we also performed flow cytometry analysis to measure the percentages of the four cell types in the organoids cultured in two distinct media at day . it was shown that the percentage of ciliated cells increased remarkably, by about threefold, after pd, to over % in the pd organoids, while the ciliated cells invariably constituted a minority of the cells in the organoids in ao medium (fig. b and c). goblet cells also increased marginally, while club cells consistently decreased after pd (fig. b and c) . collectively, we successfully induced mucociliary differentiation and developed pd aos which morphologically and functionally simulate human airway epithelium. pd aos can identify human-infective virus. one of the most important and challenging issues for influenza research is to predict which animal or emerging influenza virus can infect humans. as mentioned above, the novel reassortant avian h n viruses have caused continuing poultry-to-human transmission since . in fact, other subtypes of avian iavs, including h n , h n , and h n , have been cocirculating with the h n viruses in domestic poultry. these viruses are highly similar in internal genes but differ in neuraminidase (na) or ha and na ( ) . however, very few human infections by h n , h n , and h n virus have been reported in the same territory and time frame, although people were exposed equivalently to these viruses and the h n viruses ( ) ; these findings suggest that these viruses are less infective to humans than the h n viruses. we isolated, plaque-purified, and genotyped these cocirculating viruses. we chose to compare the infectivity of h n with that of h n /ah in the pd organoids as a proof of concept, to verify that the differentiated aos can indeed simulate human airway epithelium in the context of influenza virus infection. fig. shows that viral loads in the cell lysate and medium of h n / ah-infected organoids increased gradually after inoculation; the viral titer increased more than log units within h, indicating a robust viral replication. the addition of the serine protease inhibitor aebsf significantly restricted the active replication of h n /ah virus, highlighting the importance of elevated serine proteases for viral replication. in contrast, h n propagated modestly, with viral titer - log units lower than h n /ah. thus, the distinct efficiencies of h n /ah and h n to infect and replicate in the pd aos can recapitulate the infectivity of these viruses in humans. establishing a d airway monolayer from aos to assess the infectivity of iavs. a limitation of d organoids for studying microbial infections is the inaccessibility of the apical surface to pathogens, since most organoids are orientated inwards, while receptors for most respiratory viruses are distributed in the apical surface. for virus inoculation, organoids must be sheared to enable sufficient apical exposure to virus inoculum ( ) . as reported previously, primary bronchial epithelial cells cultured in transwell inserts with the defined medium undergo mucociliary differentiation ( ) . this prompted us to transform d aos into a d monolayer using transwell culture. to this end, d aos were enzymatically dissociated into a single-cell suspension, seeded in transwell inserts, and then cultured in pd medium. the transepithelial electrical resistance (teer) in the d monolayers stabilized in the second week after seeding (si appendix, fig. s ). in addition, the dextran penetration assay performed at day indicated that an intact epithelial barrier was formed across the d monolayers (si appendix, fig. s ). the intense signal of acctub indicated that the d monolayers developed appreciable pd (fig. a) . the productive infection of h n /ah was clearly shown by the virus nucleoprotein-positive (np + ) cells at h post infection (hpi) in the d pd organoids (fig. a) . to verify the ability of d pd organoids to assess the zoonotic potential of animal viruses and identify the human-infective virus, we compared the relative replication capacities of h n /ah and h n viruses and of another pair of viruses, the highly human-infective h n pdm and a swine h n isolate (h n sw). the higher replicative capacity of h n /ah over h n virus was more pronounced in the d pd organoids than in the d pd organoids; the viral titer of h n /ah in the apical medium was - log units higher than that of the h n virus (fig. b) . consistently, h n pdm replicated dramatically, with a viral titer in the apical medium - log units higher than that of h n sw. due to the epithelial barrier formed in the d monolayers and the preferential release of virus from the cell apical side, the viral titers in the basolateral medium were consistently lower than those in apical medium at the corresponding time points. nevertheless, the differences in replicative capacity between h n / ah versus h n and h n pdm versus h n sw were even more prominent in basal medium than in apical medium at most time points. in this study, we describe the pd of human asc-derived ao culture for investigating a major animal and human pathogen, influenza virus. in particular, our differentiation conditions increase the numbers of ciliated cells (fig. ) , the major cell type in the human airway epithelium. the pd medium induces ciliated cell numbers to a nearly physiological level, with synchronously beating cilia readily discernible in every organoid (movies s and s ). in addition, the expression levels of serine proteases (fig. ) , known to be important for productive viral infection, were elevated after pd. among the up-regulated ha-activating serine proteases, the dramatically increased expression of hat in the differentiated aos can very likely be attributed to the increased abundance of ciliated cells, since ciliated cells are the main source of hat in the human respiratory tract ( ) . thus, the differentiated aos can simulate the human airway epithelium morphologically and functionally. as a further improvement, we developed d pd airway monolayers with an intact epithelial barrier to allow exclusively apical exposure to viruses (fig. a and si appendix, fig. s ), the natural mode of iav infection in the human respiratory tract. we then utilized two pairs of viruses with known infectivity to demonstrate, as a proof of concept, that these organoids indeed show significantly higher susceptibility to the human-infective viruses than to the poorly human-infective viruses. these d and d differentiated aos support the active replication of human-infective h n /ah and h n pdm. in contrast, the h n virus, which has been temporally and spatially cocirculating with h n viruses in domestic poultry and contains internal genes similar to those in h n viruses, replicated much less efficiently in both models. similarly, the swine h n isolate showed a substantially lower growth capacity than its counterpart, human-adapted h n pdm (fig. b) . the avian iav h n subtype viruses have been circulating in the bird market from - and have caused poultry outbreaks in the united states. sporadic human infections have been reported in the united states and europe. fortunately, all reported cases of human infection experienced mild influenzalike symptoms ( ) . while pigs are considered the intermediate hosts for interspecies transmission of iavs, swine influenza viruses lacking human adaptation markers rarely infect humans. sporadic human infections documented in the literature or reported by public health officials are generally mild or subclinical ( ) . the ability of the pd aos to differentiate avian h subtype virus and swine h subtype virus from the counterpart human-infective viruses suggests that these models could be used to assess the cross-species transmission potential of emerging influenza viruses in humans. in this study, only four strains of virus were assessed in the differentiated aos as a proof of concept. more virus strains should be evaluated to further understand the strengths and limitations of this pd ao model. in summary, we believe that these differentiated aos significantly extend the current armamentaria of the influenza research toolbox. asc-derived organoids, once established, can be expanded indefinitely while displaying remarkable phenotypic and genotype stability. they thus overcome the limitations in reproducibility and availability of the current in vitro model systems. aos can be easily established from human bronchiolar lavage material, allowing interindividual comparisons; aos can also be readily modified by lentiviruses and crispr technologies and can be single-cell cloned ( ) . in combination with the molecular toolbox of the influenza virologist, the human differentiated ao model system offers great opportunities for studying virus and host factors that define the characteristics of this major animal and human pathogen. establishing asc-derived human aos. the generation of four lines ascderived human aos was based on the previous protocol ( ) with a success rate of %. briefly, with ethical approval by the institutional review board of the university of hong kong/hospital authority hong kong west cluster (uw - ) and informed consent of the patients, small pieces of normal lung tissue (∼ . - . cm in size) adjacent to the diseased tissues were obtained from patients who underwent resection. the tissues were minced and digested with mg/ml collagenase (sigma aldrich) for - h at °c, followed by shearing using a glass pasteur pipette (drummond) and straining over a -μm cell strainer (falcon). the resultant single cells were embedded in % matrigel (growth factor reduced basement membrane matrix; corning) and were seeded in -well suspension culture plates (greiner bio-one). after solidification, matrigel droplets were maintained with ao culture medium (si appendix, table s ) at °c in a humidified incubator with % co . the organoids were passaged every - wk. the bright-field images of the organoids were acquired using a nikon eclipse ts inverted routine microscope. pd of human aos. the aos were split and maintained in ao medium for - d. the culture medium in half of the organoids was changed to pd medium, pneumacult-ali medium (stemcell technologies) supplemented with μm dapt (tocris). the organoids were then cultured in ao or pd medium for d. bright-field images were taken every d. diameters of individual organoids were measured with imagej (nih). the movies of organoids were acquired using a total internal reflection fluorescent (tirf) microscope (movie s ) and a nikon eclipse ti inverted microscope system (movies s and s ). at the indicated times, the organoids in the different media were harvested for detection of cellular gene expression or for flow cytometry analysis. establishing d differentiated aos with transwell culture. transwell culture of aos was performed as described elsewhere ( , ) with modifications. briefly, the organoids were dissociated into single-cell suspensions after digested with × tryple select enzyme (invitrogen) for - min at °c, sheared using a pasteur pipette, and strained over a -μm filter. approximately . × cells were seeded into each transwell insert (product no. ; corning). the cells were cultured in ao medium at °c in a humidified incubator with % co for - d. when cells reached > % confluence, the ao medium was changed to pd medium in both the apical and basal chambers. the medium was changed every other day, and the cells were maintained for d. teer was measured every other day using a millicell ers- volt-ohm meter (emd millipore). to assess the integrity of the d organoid monolayer as an epithelial barrier, at day after seeding, fitc-dextran with an average molecular weight of , (sigma aldrich) was added to the medium in the upper chamber at a concentration of mg/ml followed by incubation at °c for h. subsequently, the culture media were harvested from the upper and bottom chambers to detect the fluorescence intensity using the victor xiii multilabel reader (perkinelmer). eggs at °c for h. the eggs were chilled overnight at °c; then the virus-containing allantoic fluid was harvested, aliquoted, and stored at − °c. virus titer was determined by plaque assay. iav infection in human aos. the d aos were sheared mechanically to expose the apical surface to the virus inoculum. the sheared organoids were then incubated with viruses at a multiplicity of infection (moi) of . for h at °c. after washing, the inoculated organoids were re-embedded in matrigel and then were cultured in the pd medium. in the h n /ah and h n infection in the d pd organoids, one set of h n /ah-inoculated organoids was treated with the serine protease inhibitor aebsf ( . mm; merck millipore) during inoculation and after inoculation. at the indicated times, organoids, dissolved matrigel, and culture medium were harvested for detection of viral load. the cell-free matrigel and the culture medium from each droplet were pooled together as one sample, referred as the "supernatant." the supernatant samples were also used for viral titration. the d pd aos were inoculated with the indicated viruses at an moi of . from the apical side by adding the virus inoculum to the apical chamber followed by incubation for h at °c. at the indicated times, cell-free media were collected from the apical and basolateral chambers for viral titration. the membranes seeded with d organoids were incised from the transwell inserts and fixed, and immunofluorescence staining was applied as described previously ( ) . rna extraction and rt-qpcr. to evaluate the differentiation status of aos cultured in pd medium versus those in ao medium, the organoids were harvested at the indicated times, and rna was extracted using minibest universal rna extraction kit (takara). to quantify virus replication, the organoids and supernatant samples were lysed for rna extraction using the minibest universal rna extraction kit and minibest viral rna/dna extraction kit (takara), respectively. cdna was synthesized with the transcriptor first strand cdna synthesis kit (roche) with oligo-dt primer. qpcr was performed with lightcycler sybr green i master mix (roche) using genespecific primers (si appendix, table s ) to detect cellular gene-expression level and viral gene copy number. the mrna expression levels of cellular genes were normalized with that of gapdh. viral gene copy number was determined by absolute quantification using a plasmid expressing a conserved region of the iav m gene. plaque assay. a plaque assay was performed to determine titers of the virus stocks and supernatant samples as described elsewhere ( ) , with minor modifications. briefly, mdck cells were seeded in -well plates. confluent monolayers were inoculated with μl of -fold serial dilutions of samples and were incubated for h at °c. after the inoculum was removed, the monolayers were overlaid with % low melting point agarose (invitrogen) supplemented with eagle's minimal essential medium (mem) and μg/μl tpck-treated trypsin and were further incubated for - d. the monolayers were fixed with % paraformaldehyde (pfa) and were stained with % crystal violet to visualize the plaque after the agarose plugs were removed. virus titers were calculated as plaque-forming units per milliliter. immunofluorescence staining. to identify the indicated cell types and the virusinfected cells, immunofluorescence staining was applied to the d and d aos. briefly, the organoids were fixed with % pfa, permeabilized with . - % triton x- , and blocked with protein block (dako). then the organoids were incubated with primary antibodies (si appendix, table s ) diluted in antibody diluent buffer (dako) overnight at °c, followed by incubation with secondary antibodies (si appendix, table s ) for - h at room temperature. nuclei and actin filaments were counterstained with dapi (invitrogen) and phalloidin- (sigma aldrich), respectively. the confocal images were acquired using a carl zeiss lsm or confocal microscope. flow cytometry analysis. to assess the percentages of four types of cells, the aos were analyzed by flow cytometry. briefly, the organoids were dissociated with mm edta (invitrogen) for - min at °c, fixed with % pfa, and permeabilized with . % triton x- . subsequently, the cells were incubated with primary antibodies (si appendix, table s ) for h at °c followed by staining with secondary antibodies. a bd facscanto ii system was used to analyze the samples. statistical analysis. student's t test was used for data analysis. p < . was considered statistically significant. *p < . ; **p < . . ***p < . . influenza viruses en route from birds to man the emergence of influenza a h n in human beings years after influenza a h n : a tale of two cities human infections with the emerging avian influenza a h n virus from wet market poultry: clinical analysis and characterisation of viral genome human infection with avian influenza a(h n ) virus china novel swine-origin influenza a (h n ) virus investigation team ( ) emergence of a novel swine-origin influenza a (h n ) virus in humans proteolytic activation of influenza viruses by serine proteases tmprss and hat from human airway epithelium proteolytic cleavage of influenza virus hemagglutinins: primary structure of the connecting peptide between ha and ha determines proteolytic cleavability and pathogenicity of avian influenza viruses activation of influenza viruses by proteases from host cells and bacteria in the human airway epithelium rapid expansion of human epithelial stem cells suitable for airway tissue engineering modeling development and disease with organoids long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and barrett's epithelium replication of human noroviruses in stem cell-derived human enteroids human intestinal tract serves as an alternative infection route for middle east respiratory syndrome coronavirus long-term expanding human airway organoids for disease modelling a three-dimensional model of human lung development and disease from pluripotent stem cells human embryonic lung epithelial tips are multipotent progenitors that can be expanded in vitro as long-term self-renewing organoids airway epithelial cell cilia and obstructive lung disease directed induction of functional multi-ciliated cells in proximal airway epithelial spheroids from human pluripotent stem cells cocirculation of three hemagglutinin and two neuraminidase subtypes of avian influenza viruses in human infection with an avian influenza a/h n virus in guangdong in enteroviruses infect human enteroids and induce antiviral signaling in a cell lineage-specific manner human and avian influenza viruses target different cell types in cultures of human airway epithelium localization of human airway trypsin-like protease in the airway: an immunohistochemical study avian influenza a(h n ) virus in human exposed to sick cats swine influenza virus infections in man. swine influenza d g mutation in hemagglutinin of pandemic influenza h n ( ) virus enhances virulence in mice delayed antiviral plus immunomodulator treatment still reduces mortality in mice infected by high inoculum of influenza a/h n virus key: cord- -adbxzgvs authors: du, juan; zhu, teng; zhuang, lu; zhang, pan-he; zhang, xiao-ai; lu, qing-bin; liu, wei title: identification and complete genome characterization of human enterovirus from a child with pneumonia in china date: - - journal: arch virol doi: . /s - - -y sha: doc_id: cord_uid: adbxzgvs in this study, human enterovirus c (ev-c ) was detected in a -month-old boy diagnosed with pneumonia in china. a phylogenetic analysis showed that this strain was genetically closer to the lithuanian strain than to the usa strain. electronic supplementary material: the online version of this article ( . /s - - -y) contains supplementary material, which is available to authorized users. -month-old child [ , ] . since then, ev-c has been identified in four children from two other countries, china and mongolia [ ] . here, we report the identification and complete genomic sequence of ev- in a child with pneumonia in china, which may provide more information for understanding ev-c infection. a -month-old boy (patient cq ) from guizhou province was admitted to the children's hospital of chongqing medical university in june and diagnosed as having pneumonia. he had a -month history of phlegm in the throat, especially in the morning and at night, and tachypnoea. he occasionally regurgitated milk along with mucous sputum, and some medium and coarse moist rales could be heard in both lungs. transient rhinorrhoea, paroxysmal cough, and diarrhoea without fever were apparent during the course of the disease. laboratory examinations performed at admission revealed a normal white blood cell (wbc) count ( . × /l) and c-reactive protein level (< mg/l), with a mildly elevated platelet count ( × /l), and a high percentage lymphocytes ( %). two days before admission, an x-ray radiograph showed slight interstitial changes in both lungs, and routine blood examination revealed elevated wbc ( . × /l) and platelet ( × /l) counts. cephalosporin antibiotics for treating the infection for more than days had no obvious effect. a nasopharyngeal aspirate was collected from the child at admission, and the parents gave written informed consent for their child to participate in the study. we extracted rna and dna from the nasopharyngeal aspirate sample using aseptic techniques and tested the viral nucleic acids for evidence of respiratory viruses. using molecular methods described previously, we tested for influenza virus (a, b and c), ev, metapneumovirus, respiratory syncytial virus (rsv), adenovirus, human bocavirus, parainfluenza virus (types - ), and coronaviruses ( e and oc ) [ , ] . the study protocol was reviewed and approved by the ethics review committee of children's hospital of the chongqing medical university. the sample was positive for ev and parainfluenza virus type . igg antibodies against cytomegalovirus and epstein-barr virus were also detected, while the sputum culture was negative for these viruses. to determine the enterovirus type, we amplified a portion of the 'ncr-vp /vp region of the enterovirus by nested pcr as described previously [ , ] . the -bp amplicon was sequenced, and basic local alignment search tool (blast) analysis (http://www. ncbi.nlm.nih.gov) revealed a high degree of similarity to ev-c . using multiple primers (table ) , we determined the full-length viral genome sequence of the strain detected in patient cq (genbank accession no. mk ), which was found to consist of nucleotides, excluding the poly(a) tail. the 'utr contains nucleotides, and the 'utr consists of nucleotides. the cq genome contains a single open reading frame from nt to encoding a -amino-acid polyprotein. the base composition of the full genome is . % a, . % c, . % g, and . % u. the complete genome sequence was compared with those of hev-c strains available in the gen-bank database, which included ev- , ev- , ev- , ev- , and ev- . the cq isolate shares . %- . % nucleotide sequence identity in the coding region with other hev-c strains, including ev- ( . %- . %), ev- ( . %- . %), ev- ( . %- . %), and ev- ( . %- . %), and shares . %- . % nucleotide sequence identity with other ev-c strains ( table ) . after alignment using mega version . , we constructed a phylogenetic tree by the maximum-likelihood method, using the tamura-nei model and bootstrap replicates. the resulting tree showed that cq belongs to genotype ev-c (fig. a) . although it was genetically closer to the lithuanian strain than to the usa strain, the cq strain was separate from both strains, with at least % bootstrap support. the genome of the cq isolate has a typical enterovirus organization, encoding four structural proteins (vp , vp , vp and vp ) and seven non-structural proteins ( a, b, c, a, b, c and d). the vp sequence of the cq isolate is . %- . % identical to those of other ev-c isolates, and other genome regions displayed > % sequence identity to ev-c . conversely, all genome regions of cq showed less similarity to other ev-c strains (including ev-c , ev-c , ev-c , and ev-c ) ( table ) . a phylogenetic tree was constructed based on the vp gene nucleotide sequences using the maximum-likelihood method. this illustrated the differences among the cq strain isolated in this study, the lithuanian strain, the mongolian strains, and the usa strain (fig. b) . in other phylogenetic trees that were constructed based on the 'utr, 'utr, vp -vp region, a- c region and a- d region, more-pronounced genotype clustering was obtained, and the cq strain clustered with ev-c in all cases except for the 'utr tree (supplemental fig. ). in summary, we report the detection of ev-c in a child hospitalized with pneumonia. we demonstrated that the vp -vp , a- c and a- d gene regions of the cq strain were different from those of the other ev-c strains. because parainfluenza virus type and cytomegalovirus were also found in samples from this patient, the causal correlation between ev-c and respiratory disease could not be established. therefore, it is necessary to further investigate the role of ev-c in respiratory illnesses in children. conflict of interest the authors declare that they have no conflict of interest. the study protocol was reviewed and approved by the ethics review committee of children's hospital of chongqing medical university. we obtained informed consent from the child's parents. complete genomic sequencing shows that polioviruses and members of human enterovirus species c are closely related in the noncapsid coding region complete genome sequence of a novel human enterovirus c (hev-c ) identified in a child with communityacquired pneumonia novel human enterovirus c infection in child with community acquired pneumonia real-time rt-pcr detection of respiratory viral infections in four triplex reactions complete genome characterization of enterovirus circulating in northern italy shows recombinant origin of the p region development and implementation of a molecular diagnostic platform for daily rapid detection of respiratory viruses newly identified enterovirus c genotypes, identified in the netherlands through routine sequencing of all enteroviruses detected in clinical materials from respiratory infection with enterovirus genotype c , china and mongolia key: cord- -narre e authors: aziz, muhammad abdul; khan, amir hasan; adnan, muhammad; ullah, habib title: traditional uses of medicinal plants used by indigenous communities for veterinary practices at bajaur agency, pakistan date: - - journal: j ethnobiol ethnomed doi: . /s - - - sha: doc_id: cord_uid: narre e background: the pastoral lifestyle of indigenous communities of bajaur agency is bringing them close to natural remedies for treating their domestic animals. several studies have been conducted across the globe describing the importance of traditional knowledge in veterinary care. therefore, this study was planned with the aim to record knowledge on ethnoveterinary practices from the remote areas and share sit with other communities through published literature. methods: data was gathered from community members through semi-structured interviews and analyzed through informant consensus factor (fic) to evaluate the consent of current ethnoveterinary practices among the local people. results: in total, medicinal plants were recorded under the ethnoveterinary practices. most widely used medicinal plants with maximum use reports (urs) were visnaga daucoides gaertn., foeniculum vulgare mill., solanum virginianum l., withania somnifera (l.) dunal, glycyrrhiza glabra l., and curcuma longa l. new medicinal values were found with confidential level of citations for species including heracleum candicans and glycerhiza glabra. family apiaceae was the utmost family with high number ( species) of medicinal plants. maximum number of medicinal plants ( ) was used for gastric problems. high fic was recorded for dermatological ( . ) followed by reproductive ( . ) and gastrointestinal disorders ( . ). the main route of remedies administration was oral. conclusions: current study revealed that the study area has sufficient knowledge on ethnoveterinary medicinal plants. this knowledge is in the custody of nomadic grazers, herders, and aged community members. plants with new medicinal uses need to be validated phytochemically and pharmacologically for the development of new alternative drugs for veterinary purposes. the historical utilization of plants as health remedies both for human and animal is centuries old. it has been recognized that plants have the capacity to combat several types of diseases ethnoveterinary medicines, a term generally used for folk skills, beliefs, knowledge, practices, methods related to animals' health, and cure of various ailments in the rural areas [ ] . ethnoveterinary practices have achieved immense significance for the last decade owing to the discovery of some effective ethnoveterinary products [ ] . the utilization of traditional remedies poses a cheaper, easier, and sustainable alternative to synthetic drugs and pharmaceuticals [ ] . it has been reported that due to lack of proper animal husbandry practices, about - % of the losses occur in the animals' breeding sectors especially in developing countries [ ] , where the rural people are heavily dependent on livestock farming for their livelihood activities [ ] . the indigenous communities living in rural and mountainous territories of developing world consider livestock a vital source for economy, social security, and food and is thought to be a symbol of prestige for a particular family [ ] . livestock being as a subsector contributes around % of value addition in the agriculture sector and approximately % towards the gross domestic product (gdp). about million people living in the rural areas of the country are involved with the livestock subsector [ ] . hence, livestock raring plays a significant role in poverty reduction strategies. according to the report of economic survey of pakistan [ ] , the national herd of pakistan includes . million goats, . million cattle, . million buffalos, . million sheep, and . million camels. people residing in the remote areas utilize medicinal plants for livestock's health. particularly, the conventional lifestyle of nomadic and pastoralists makes it difficult for them to reach veterinary extension services due to high costs and less availability of allopathic medicines [ ] . in south asia, several ethnoveterinary studies have been conducted [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] including pakistan [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, scarce studies on ethnoveterinary medicines have been reported from the federally administrated tribal areas (fata) of the country. the tribal areas mainly comprised of mountainous territories where people use medicinal plants to treat livestock's diseases. traditional ethnoveterinary knowledge is mainly transmitted orally from one generation to another generation in the form of folk remedies, drawing stories, poems, drawing stories, folk myths, songs, and proverbs. this transmission of indigenous knowledge through oral way faces critical threats of extinction. therefore, it is necessary to record, document, and encourage the ethnoveterinary medication and integrate them into the existing animal health care services [ ] . bajaur agency is among one of the federally administrated tribal areas (fata) of pakistan having diversity of medicinal plants being used for the livestock's healthcare services. due to remote nature and lack of quality education, the area has been little explored for the scientific documentations of traditional knowledge. there is a dire need to explore the folk knowledge about the utilization of herbal remedies for veterinary practices prior to being extinct. hence, the current study was planned to investigate and document the traditional ethnoveterinary knowledge and practices and release it from the custody of knowledge bearers for sharing it with other communities through publish literature. bajaur (khar: headquarter) is the smallest agency of the fata having a total area of km . it shares km border with afghanistan, which is of great importance to pakistan and the region. the study area lays at an altitude of m above the sea level and geographically exists between °- °and °- °latitudes and °- °and °- °longitudes. the agency is surrounded to the west by kunar valley of afghanistan being separated by the rugged hindukush hills and other mountain passes known as nawa pass, ghakhi pass, and letai sar being the notable ones. the agency borders on south with mohmand agency, on east with lower dir district and the panjkora river, and on north with the watershed between bajaur agency and district dir. moreover, the agency is situated at the extreme end of the himalayan range. the areas dominated by agricultural lands are receiving about mm of average rain fall per annum. the two main tribes of bajaur agency known as tarkani and utman khel are mainly populated into seven tehsils including barang, nawagai, khar, mamund, salarzai, utman khel, and chamarkand. by profession, mostly, the people are farmers, teacher, drivers, and doing small scale businesses and jobs inside/outside the country. almost every household has a herd of domestic animals for socioeconomic gains. there are only three degree-level colleges and five higher secondary schools. moreover, there are only two government hospitals in the study area, while most people are deprived of modern health facilities, which justify their reliance on local herbalists (hakims). the study area consists of one veterinary hospital and small dispensaries to treat the domestic cattle. however, the local people still rely on traditional recipes due to larger distances from the aforementioned health centers. the dominant vegetation in the area is comprised of ailanthus altissimo, eucalyptus camaldulensis, ficus carica, melia azedarach, morus indica, morus nigra, olea ferruginea, pinus roxburghii, quercus baloot, and rumex hastatus. in the month of april, respondents were targeted based on their strong reputation in the field of ethnomedicinal knowledge while field survey was conducted from may to august . field visits were carried out prior to medicinal data collection in order to acknowledge the cooperation of the indigenous communities. mr. amir hasan khan, the local occupant of the area, visited different sites with his volunteer team including a taxonomist and a pharmacist. he arranged several meetings with the local representatives known as maliks, to whom objectives of the study were presented. a semistructured questionnaire was developed to gather knowledge on ethnoveterinary plants by following the method adopted by martin [ ] . mostly, the folk knowledge was gathered from nomads, farmers, and aged community members. the interviews were conducted at various places and in the local language called "pashto." each informant was acknowledged by presenting the main theme of the study to them in order to gain their consent and trust, which allowed the informants to talk more freely and openly. the recorded information was once again redisplayed to the informants to avoid errors and falsification. data was collected from different sites known as pashat, tali, inayat kali, ghar shamozai, loe sum, barang, mandal, khar, mamund, and salarzai. accordingly, the sites were categorized into foot hill villages and mountainous villages (fig. ) . a total of key respondents were selected belonging to different age groups, i.e., males and females ( table ). the selection of respondent was based on their high reputation with respect to traditional knowledge on ethnoveterinary plants. continuous relationships were maintained with the indigenous communities throughout the course of survey for the strong validation of traditional knowledge. surveyed ethnoveterinary medicinal plants were collected and identified by taxonomist at the department of botany, shaheed benazir bhuto university sheringal, district dir (upper), khyber pakhtunkhwa, pakistan. species botanical names and their family names were corrected and verified through the website www.kew.org/mpns. after collection, plants were pressed and dried under the shade, were poisoned ( % hgcl solution), and were mounted properly on the herbarium sheets for future reference. each herbarium sheet was labeled with a voucher number and submitted to the aforementioned department [ , ] . for each of the specie, use reports (urs) (citations) were counted. ur may be defined as the utilization of part of a plant species for a particular disease mentioned by an informant. to determine the informant consensus factor (fic), the reported species were arranged in various groups according to the ailment treated [ ] . ten ailment categories were prepared from the data. to calculate the fic, we used the formula, i.e., fic = nur − nt/ nur − . here, nur indicates the number of citations in each use category and nt represents the number of species cited. prospects and challenges to traditional ethnoveterinary knowledge indigenous communities play significant role in reporting traditional uses of medicinal flora. indigenous knowledge can be used as a tool to conserve and maintain the green diversity, and could be further utilized for scientific validation [ ] . during the nd session of united nations educational, scientific and cultural organization (unesco), traditional knowledge on ethnoveterinary medicines was declared an important part of cultural heritage, which is required to be brought under study, sustenance, and protection [ ] . indigenous communities at bajaur agency are dependent on livestock for supporting their livelihood. medicinal plants have a pivot role in the treatment of livestock's ailments in the area. usually, this treatment process depends either on the traditional knowledge being orally transmitted to the current generation of local people from their ancestors or through personal experiences. previous scientific literature has focused on the correlation of traditional medical expertise to ethnobotanical knowledge for the treatment of human ailments [ , ] , although the same plants may be used to treat livestock [ , ] . in our study, we have observed that the herders, farmers, and older community members are more equipped with traditional knowledge and familiar with veterinary medications, diagnosis process, and treatment. indigenous people of the study area are rich in traditional knowledge on veterinary medicines, which may be due to their close observation on domestic animals being considered as an important part of traditional lifestyle. most commonly, the male community member grazes herds of animal, while females take part in households' management. figures and showed some of the images of the grazed domestic animals, which are treated with medicinal plant in the area. other studies have explained this in a different way that men due to close proximity tend to know more about the animal behavior than women [ ] . people of the study area use plants not only for medicinal purposes to treat their domestic animals but also as a fodder. local community also prevents their animals from such nutrition, which is not healthy in certain conditions and seasons. one may consider this prevention to be a part of ethnoveterinary practices. nutrition is playing an important role in ethnoveterinary practices in both prevention and cure of domestic animals [ ] . livestock usually ingests some extra and non-important food substances in the green fodder, which could be termed as food medicines or medicinal food [ ] . studies have highlighted the importance of "food as medicines" in the context of local traditional knowledge; however, possible health advantages of food in ethnoveterinary methods need further attention [ ] . testing the nutritional status of each traditional ethnoveterinary remedy is not necessary; however, it is essential to evaluate the biological efficacy from the phytochemical, pharmacological, toxicological, and clinical perspectives for wider application. a considerable proportion of the documented uses of plant taxa in our study are in accordance with the established pharmacological effects [ ] . the prevailing indigenous ethnoveterinary knowledge in the study area is facing certain constrains leading it towards extinction. as an example, the nature of traditional knowledge is making it more difficult to learn and then transfer it in an accurate way. furthermore, practicing traditional therapies are not being respected by the new generation. other challenges include low literacy rate in the study area, no proper documentation of indigenous knowledge, and introduction of modern allopathic medicines, rapid technological advancement, and environmental degradation. similar kinds of threats have also been reported in other communities across the world [ ] [ ] [ ] . informants with little education were found less familiar to the traditional knowledge while people having no formal education were more responsive in this regard. some studies have found that education can be correlated with expertise either positively [ , ] or negatively [ ] , while others found no relationship [ ] . moreover, it is also ambiguous to determine the effect of "modernity" on the loss of ethnomedicinal knowledge. modernity has an established association with greater medicinal competence in dominica [ ] but appeared unrelated to variation in expertise among tsimane horticulturalists in bolivia [ ] . furthermore, it is also unclear whether correlation of expertise exists between ethnomedicinal knowledge and ethnoveterinary approaches; however, livestock keepers hold extensive knowledge related to disease prevention, diagnosis, and both traditional and novel biomedical treatments [ ] . in summary, despite maintaining knowledge on ethnoveterinary practices by the locals, the tendency to utilize modern pharmaceuticals is increasing day by day. hence, the conservation of ethnomedicinal knowledge by the local communities is extremely important for the livestock's health in the remote areas. the use of plants for medical purpose to treat a wide array of maladies emanates traces since the recorded history and even before. in our study, plant species belonging to families were documented. table presents details on the documented medicinal plants including their botanical names, vernacular names, family names, specimen numbers, parts used, medicinal uses, and use reports. family apiaceae ( species) has the high number of individual species used in ethnoveterinary practices followed by fabaceae ( species). other studies have also reported apiaceae as the dominant plant family being used in traditional medications [ , ] . the rationale of high use of apiaceae species in the current study, though based on traditional evidence, may be referred to their chemical constituents such as phenolics, poly phenolics, lectins, alkaloids, terpenoids, and essential oils, which carry antimicrobial potential [ ] . due to the predominance of sheep, goats, cows, and donkeys in the study area, we have specifically recorded the ethnoveterinary practices used for the treatment of these four types of domestic animals. key informants declared extensive uses of visnaga daucoides gaertn. table ) . medicinal plants with high urs strengthen the concept that such species are more significant to the local population and useful in sharing the traditional knowledge with one another in the area. in our study, v. daucoides is used to treat diarrhea, abdominal pain, and retained placenta in domestic animals. a whole plant is subjected to powder and is combined with flour and black tea to treat digestive problems especially in cow and buffalo. amaryllidaceae bulb of the herb is crushed and added milk for orally given to animals ( - days) for curing digestive complaints. bulb is crushed and mixed with way to administered orally for several days in order to rate of fertility in domestic animals. narcissus tazetta l. "sbbu- " gul-e-nargas leaves along with gurr and flour, fresh leaves (¼ kg) are boiled and orally given to livestock for the retained placental removal. apiaceae tea is prepared from its fruit (¼ kg), and then, it is combined with flour and given to cattle for days in order to treat gastric problems. cuminum cyminum l. "sbbu- " half kilogram of fruit is boiled in black tea and orally given for - days on daily basis for the expulsion of intestinal worms and treated gastric problems. eryngium biehersteinianum (m. bieb.) nevski "sbbu- " stem and leaves and stem powder of its stem and leaves is orally given for the treatment of liver problems up to the duration of - days. foeniculum vulgare mill. "sbbu- " fruit, leaves decoction is made from it fresh leaves and fruit ( - g), and t hen, it is combined with gurr, given orally to livestock for appetite and as sedative for the duration of to days. wall. ex dc. "sbbu- " fresh root of the plant ( g) is combined with wheat flour and made to paste which is orally given to goat, cow, and sheep as sexual tonic and to enhance the rate of fertility up to days. trachyspermum ammi (l.) sprague "sbbu- " seeds (¼ kg) of the plant, allium cepa, wheat flour, and foeniculum vulgare are thoroughly mixed. the resultant blend is then orally given ( days) and is considered as good appetizer. tea is made from its fruit and given orally to sheep, goat, cow, and buffalo while treating diarrhea, abdominal pain, and retained placenta. the remedy is constantly utilized for the duration of days. calotropis procera (aiton) dryand. "sbbu- " plants' fresh leaves are taken and decoction is made, and after that, the decoction is combined with "ajuga integrifolia" and is used for dermal parasites for to days. nerium oleander l. "sbbu- " to relieve the external parasite, the decoction of its leaves is used for animal bathing especially goat and cow. the decoction obtained from its bark and is combined with butter which is administered orally to all type of domestic animals to treat skin problems and as blood purifier. cassia fistula l. "sbbu- " amaltas fruit fruit of the plant is subjected to boiling along with milk and administered orally up to days to all sort of domestic cattle to relieve fever and gastric complexities. glycyrrhiza glabra l. "sbbu- " khwaga waly roots root (¼ kg) is subjected to paste which is mixed with flour and oil and then is given to goat, sheep, cow, and buffalo to increase milk production and enhance the rate of fertility. the remedy is used for the duration of to days. lotus corniculatus l. "sbbu- " stem and leaves, stem and leaves are crushed in weight of ¼ kg and orally given to cattle along with bread or dough for to days as sexual tonic and for urinary tract infections (uti). trigonella foenum-graecum l. "sbbu- " malkhoozi seeds seeds ( g) are crushed and given in dough to animals ( - days) against gastric disorders. decoction is made from the leaves and then gurr is added. this remedy is given orally to cattle for blood purification and as vormifuge. the water is applied topically to treat skin ailments. mentha spicata l "sbbu- " powder is made and decoction is made and then mixed with gurr and taken by animals to cure digestive problems. ocimum basilicum l. "sbbu- " kashmaly leaves seed plant leaves and seeds are subjected to decoction and used topically for skin problems. salvia moorcroftiana wall. ex benth. "sbbu- " kharghwag leaves decoction of its leaves is given orally daily for the treatment of digestive problems. gossypium arboreum l. "sbbu- " pomba kal about ¼ kg of its powder is mixed with gurr and used for to days. this remedy is administered orally on daily basis as galactagogue. grewia optiva j.r.drumm. ex burret "sbbu- " whole plant dried plant powder is subjected to oil ( ml), administered orally and topically twice a day for to days for wound healing process. the usage mode of ethnoveterinary plant species by one ethnic community is different from other communities due to difference in traditional knowledge [ , ] . previous literature has shown that decoction of the fruit of v. daucoides is used during abdominal pain, which is used to enhance body temperature in the study area [ , ] . in the same way, the f. vulgare is considered as a strong appetite and sedative. in other cultures across the globe, f. vulgare is used for various livestock problems. for instance, this plant is effective in digestion and diarrhea, when mixed with camellia sinensis, trachyspermum ammi, ghee, and sugar [ , ] . pneumonia is also being treated by giving its seeds to the animals [ ] , while other uses include galactagogue and ruminative [ ] . various parts of s. virginianum are taken for the treatment of cough, fever, milk production, and pain. there is scarce literature on the use of s. virginianum as galactagogue, which shows the unique use of this plant species in the study area and familiarity of local population through longtime experiences. published literature has indicated that the plant is also used for wound healing process [ ] fever, cough, and intestinal infections [ ] . roots and leaves of w. somnifera are given to sheep, cow, and buffalo for milk production and used as antipyretic and sexual tonic. indigenous populations comprising of various cultures residing in lesser himalayas (pakistan) use w. somnifera for bovine mastitis [ ] , while in ethiopia, this plant is being used to protect animals from bad evils [ ] . the plant has carminative effects and is used to remove the flatulence [ ] . additionally, this plant is used as refrigerant and for abdominal pain, digestion, jaundice, skeletonmuscular ailments, and wound healing against sunstroke [ ] ; for treating diarrhea [ ] ; for trypanosomiasis [ ] ; and for anorexia [ ] . informant reported g. glabra as galactagogue and enhances the rate of fertility. mussarat et al. [ ] reported that this plant is culturally used for the treatment of cough by the indigenous communities residing near the indus river, pakistan. however, from the literature, no conclusive evidence was found on the reported uses of g. glabra in our study. such evidence-based observations could justify the idea of cultural diversity across the regional level in plant remedies. previous studies related to the human's uses of g. glabra have demonstrates its effectiveness in the treatment of sex hormone imbalances and menopausal symptoms in women [ ] . in the current investigation, rhizome of c. longa is used as antiparasitic and treating genital infection and problems. in other cultures, across the country, the dried rhizome of c. longa is mixed with eggs and given for mastitis [ ] , jaundice, and skeleton muscular ailments [ ] . decoction of its leaves is mixed sugar, which is used as wound healing agent [ , ] . a root of c. longa is used for hoof problems and sore joints [ ] . in our study, the mustard oil is mixed with whey and is taken orally to relieve abdominal pain. the cultural ethnoveterinary uses from the lesser himalayas (pakistan) include that the oil extracted from b. rapa seeds is utilized for stomach disorders, eye infection, and skin diseases [ ] . furthermore, brassica rapa l. seeds are used for the retention of fetal membrane, while its oil is effective in treating genital prolepses and sores [ ] . this plant is also used in placental retention and mastitis and as antiparasitic [ ] ; myiasis, mange, and helminthiasis [ ] ; and flatulence [ ] . all these researchbased findings showed that the same medicinal plants are being used in different parts of the country; however, their uses differ from area to area and from culture to culture [ ] . the ethnoveterinary plants use by one ethnic community is almost different from other communities due to several reasons. to make a comprehensive comparative cultural diversity analysis of plant utilization in ethnoveterinary practices, we have selected a study conducted by aziz et al. [ ] in the fata region of pakistan. in comparison, we have found that most widely used medicinal plant species in our study are v. daucoides, f. vulgare, s. virginianum, w. somnifera, g. glabra, and c. longa. while according to aziz et al. [ ] , the ethnic communities in south waziristan agency are widely utilizing plant species such as b. rapa, punica granatum, capparis decidua, mentha longifolia, withania coagulans, and c. longa, during comparative analysis, it was found that only medicinal plants were commonly used in both regions for ethnoveterinary practices, which include acacia modesta wall, allium cepa l., allium sativum l., b. rapa, calotropis procera (aiton) dryand., cannabis sativa l., chenopodium album l. c. longa, f. vulgare, juglans regia l., nicotiana tabacum l., peganum harmala l., quercus oblongata d. don, trachyspermum ammi (l.) sprague, and v. daucoides. certain variations in the utilization of these plants and their parts were observed in both areas. for instance, the bulb of a. cepa is used as galactagogue by waziristanian communities while in bajaur, it is used to treat digestive problems. a. sativum is utilized for genital prolapsed while the same plant is used as sexual tonic for animals in bajaur agency. the seeds of b. rapa are widely used as appetizer and tonic and for cough, seasonal allergies, stomach disorders, and skin infections in south waziristan agency, while in the other region, it is used only against gastro-intestinal disorders. the indigenous communities at south waziristan agency consider the leaves of c. procera useful in joint pain while on the other side, the residents of bajaur agency used the latex against skin problems. c. album is used for wound healing and flatulence at waziristan while as stomachic at bajaur agency. j. regia is given for the retention of placenta at waziristan while gastric problems in bajaur. p. harmala is extensively used for gastrointestinal problems, as antiparasitic, and for skin diseases by waziristanian communities, while it is used only for the riddance of external parasites in bajaur. the possible reason for low consensus of the two regions in ethnoveterinary medicinal plants may be due to unique vegetation and distinct socio-cultural values. according to a survey, out of plant-derived pure compounds, % ( plant species) were having the same potential as indicated in traditional medications [ ] . as an example, galegine is obtained from galega officinalis l. and is used in the production of metformin and other bisguanidine-type anti-diabetic drugs [ ] ; khellin, extracted from v. daucoides., led to the development of cromolyn in the form of sodium cromoglycate, which is used as a bronchodilator; and papaverine isolated from papaver somniferum forms the baseline for verapamil, which is generally utilized for hypertension [ ] . survey participants did not describe the standardized dosage and recovery time like other previous ethnoveterinary documentations. the main problem highlighted in other studies is the lack of accuracy in such ethnoveterinary practices, which also push the locals towards modern allopathic drugs for livestock health maintenance [ , ] . the main reason that veterinarian has always complained is the non-standardized dosage in traditional medicines. though this is an accusation, one ethnomedicine does not mean that they lack efficacy but require standardization, which could benefit the traditional system by minimizing risks and toxicities. according to kearns [ ] , ethnoveterinary medicines are facing a great intellectual challenge from social theory and postmodernism, and this challenge was focused while detecting variations in animal health practices, beliefs, and experiences of various social groups. generally, it is not possible for all ethnoveterinary practices to be effective and, at the same time, they have certain weakness in terms of their efficacy as compared to modern medications [ ] . though it is convincing that most of the traditional veterinary medications have clear and sound health effects, many modern allopathic drugs are based on these medicines [ ] . certain plants in our study were used in single form for more than one disease. for example, cedrus deodara (roxb. ex d. don) g. don is used in a condition, in which milk obtained from the cattle gives bad smell, then the oil is given orally to the cattle. it is also used as a cooling agent and in treating digestive problems. in large quantity, the oil have the potential to depress the sexual power of male animals [ ] . monteiro et al. [ ] also reported similar findings from pakistan and brazil, respectively, where they described multiple uses of a single medicinal plant. utilization of certain plant species for multiple diseases is a widespread practice in ethnoveterinary medications. in contrast, some ethnoveterinary remedies (polyherbal formulations) are being made by combing two or more plants and additives such as whey, ghee, and sugar. this addition is generally followed in remedies to counteract the astringent taste, dilute, and reduce the relative potency of the remedy [ ] . in the study area, a total of plants were reported for gastrointestinal problems with maximum use reports of (table ) , which is regarded as the most common disease category in domestic animals being represented by abdominal pain, diarrhea, and digestive problems. these health issues can be easily detected by the respondents and may explain the fact that why the gastric problem category is high in ours as well as in others studies. different ailments were categorized into groups such as dermatological, gastrointestinal, galactagogue, reproductive, respiratory disorders, tonic, wound healing fever, and miscellaneous. those medical conditions, which were not fully described by the interviewees, were placed into the miscellaneous category. these include eye problems, weakness, and abnormal conditions related to various organ systems of animal bodies. highest fic values were recorded for dermatological problems ( . ) followed by reproductive ailments ( . ) and gastric disorders ( . ) ( table ) . fic value is an indicator of showing the consent of the local people on a specific plant species and efficacy of a certain taxa [ ] . sharma et al. [ ] declared that when fic becomes , it means that the local population is exchanging their view, ideas, and information about traditional medications, while on the other side, if the fic value is , then it is vice versa. fic value in the current study was recorded in between . and . for various livestock ailments (table ). these findings indicate the highest consent among the local people on traditional herbal therapies. previous research studies conducted in other areas also agreed to high consent of local people on traditional animal therapies. for instance, the reported fic values for dermatological problems were . , . , and . [ , , ] ; for reproductive disorders, . and . [ , ] ; for gastric problems, . , . , . , . , and . [ , [ ] [ ] [ ] ; for galactagogue, . and . [ , ] ; and for wound healing, . and . [ , ] . heinrich et al. [ ] has submitted the idea that high fic values can be used as a tool to target the plants for the isolation of biologically active components. in our study, most livestock's ailments were mentioned to be seasonal and epidemic due to change in fodder. furthermore, the concept of hot and cold food is also famous in order to prevent animals from diseases. the local residents change the relative fodder in different seasons in order to minimize the chances of various health problems in cattle. as an example, the seeds of the nigella sativa l. and kernels of q. oblongata are given to the cattle to energize them during the cold season. similarly, the fruits of the streblus asper lour. produce cooling effects and considered to be a better remedy during hot summer season. in the same manner, local communities tend to give the infusion of cannabis sativa l to their livestock in the summer season. quinlan [ ] and raziq et al. [ ] has also mentioned the concept of hot and cold food in traditional veterinary medications. drugs derived from plants or their extracts have certain therapeutic properties. to replace antibiotics by suitable therapeutic agents, plants can play an important role in combating with bacterial pathogens. there are several essential oils, which can be used as alternate of antibiotics. these oils can be easily isolated, having low toxicity on mammalian cells, and can be easily degraded in soil and water [ ] . in this section, we will analyze the pharmacological evidences of the most utilized studied medicinal plant species in order to check their therapeutic efficacy. in f. vulgare, phenols, phenolic glycosides, and volatile aroma compounds such as transanethole, estragole, and fenchone are reportedly the key phytoconstituents and responsible for its antioxidant activity. f. vulgare is pharmacologically validated (in vitro and in vivo) in demonstrating activities such as antibacterial, antifungal, antioxidant, antithrombotic, and hepatoprotective [ ] . by investigation, it was found that the leaf extracts of s. virginianum is more active against candida albicans, salmonella typhi, staphylococcus aureus, and nematodes [ , ] . for various extracts obtained in alcohol and water, it was found that w. somnifera has antibacterial potential, antihypercholesterolemic activities as well as diuretic potential [ , ] . it has been reported that alcoholic and aqueous extracts of c. longa have shown antibacterial activity [ ] while its ethanol, petroleum, water, and chloroform extracts are effective against certain strains of viruses, bacteria, and fungi and also have shown anti-inflammatory effects [ ] . researchers have claimed that plant-derived medicines used in traditional systems across the globe can be used as an indicator to consider them more effective than modern drugs [ ] . livestock keepers are using several plant-derived remedies for various acute as well as chronic disorders of cattle. plant-derived medicines have been used by physicians for hundreds of years in traditional systems, and most of the world population rely on these products for health care systems [ ] . there are several thousand plants across the globe being utilized for various therapeutic purposes both animals and humans [ ] . out of these medicinal plants, very low proportion has been investigated and proved scientifically for their indigenous uses [ ] . the essential oils in medicinal plants are having strong antimicrobial potential. as an example, essential oils of cinnamon, thyme, and oregano are therapeutically effective [ ] . antibiotic resistance is an emerging global concern related to veterinary and human medications [ ] . hence, it is necessary to search for new compounds to combat antibiotic resistant bacteria. improper therapeutic utilization of antimicrobial medicines in fishery, poultry, agriculture, and animal farming facilitates the emergence and production of drug resistant strains. additionally, poor prevention and control of unhygienic practices contribute in resistance emergence. the world health organization, food and agriculture organization, and world organization for animal health are stressing to promote best practices to avoid the emergence and spread of antibacterial resistance. continuous attempts are in progress to promote the moderate use of antibiotics in human as well as in animals to tackle the problem of antimicrobial drug resistance [ ] . in general, plants should be used as an alternative to synthetic drugs and investigated for their therapeutic efficacy. certain plants in our study including boerhavia erecta l., celtis australis l., chamaecyparis obtusa (siebold & zucc.) endl., eryngium biehersteinianum (m. bieb.), gossypium arboreum l., h. candicans wall. ex dc., narcissus tazetta l., opuntia littoralis (engelm.) cockerell, and s. asper need comprehensive phytochemical, pharmacological, and toxicological investigations. current study, one health concept, and changing environment current study reports that there are several ailments being treated with medicinal plants by the indigenous populations. most prevalent disease categories were dermatological, reproductive, and gastric problems. the dominance of these diseases not only poses threats to the domestic animals but also increases the chances of zoonoses. local population uses various animal products; hence, there are maximum chances of the migration of infectious diseases from these animals to humans. linkage of the ethnoveterinary studies with the researches of other disciplines may form an interdisciplinary approach to combat several types of health issues in both animals and plants. this approach mainly led to the concept of one health, which contributes towards understanding the complexities in health problems of living beings [ ] . a recent surge in emerging infectious diseases and their putative associated costs to society have reignited interest in the drive of disease emergence. a number of pathogens have emerged in the last years, including the severe acute respiratory syndrome virus, hendra virus, and nipah virus. however, there is a growing concern about the h n influenza virus, which fuelled much of the recent debate around emerging infectious diseases (eids) [ ] . one of the benefits that accrued from the attention on eids has been an increased recognition across a range of disciplines that the health of animals (including humans) and the health of the broader ecosystem are inextricably linked, which certainly given momentum to one health movement. one health is not all about eids, but it also covers important issues of food security and food safety [ ] . there is a strong consensus that the climate is changing now and that human activities are the primary cause [ ] . however, it is clear that climate change will alter the distribution and incidence of a wide range of diseases either directly or indirectly (e.g., diseases with a development stage outside the host) [ , ] . the pathways by which climate change can affect host pathogen vector interactions have recently been well described by gallana et al et al. [ ] . one health initiative task force (ohitf) [ ] defines one health as "the promotion, improvement, and defense for the health and well being of all species by enhancing cooperation and collaboration between physicians, veterinarians, and other scientific health professionals and by promoting strengths in leadership and management to achieve these goals". the one health approach plays a significant role in the prevention and control of zoonoses. approximately % of new emerging human infectious diseases are defined as zoonotic [ , ] . of the infectious diseases, approximately % are caused by multi-host pathogens, characterized by their movement across various species [ ] . this gives significant credence to the importance of examining health effects across species, in order to fully understand the public health and economic impact of such diseases and to help implement treatment and preventive programs. the application of one health approach has been recognized as a critical need by international organizations as well as the preferred approach to address global health issues. it is also noted that knowledge in veterinary medicine and animal nutrition and husbandry could provide insights into human nutrition and growth. it is a widespread phenomenon that natural resources including plants are always prone to threats in their natural habitat due to rapid human intervention and destructions of natural resources. the collection process of medicinal plants for ethnic practices and other anthropogenic practices is not only destructing the indigenous flora but also posing a threat to the traditional knowledge. unesco has emphasized on the documentation and preservation of traditional knowledge in south asia generally and pakistan and india particularly. however, efforts are going on but they are not sufficient for the conservation of traditional knowledge persistent since several centuries, which can lead to valuable discoveries in modern healthcare system. the local perception of indigenous communities regarding the threats being faced to the ecological resources especially the medicinal plants was examined in the current study. the lack of awareness has been observed as a major threat to the conservation of plant resources. it was also observed that different factors including time of collection, processing, storage, and herbal preparations are important and necessary steps to be considered for both economic returns and conservation. mainly, the local healers are involved in the collection of medicinal plants. a study in the swat region of pakistan has shown that higher economic outcomes can be obtained from proper harvesting of wild medicinal plants as compared to the standard cash crop [ ] . other studies are supporting our results by showing an enormous potential in improving the harvesting, storage, use, preparation, and marketing of the herbal product as a source of income [ ] . in the remote areas of the study region, local inhabitants obtained significant economic advantages from forest products. similar advantages have been reported for other mountainous communities in the northern parts of pakistan [ ] . there are certain other threats to the medicinal plant resources of the study area, which include deforestation, heavy grazing pressure, uncontrolled collection of fodder, and other non-timber forest products by the local people and traders. several studies have reported a decrease in the number of medicinal plants due to over exploitation and environmental degradation [ , ] . it is therefore a dire need to manage and design the overall grazing system to encourage the sustainable regeneration and protection of medicinal plants. keeping the observation and findings of the current investigation, proper management steps should be taken with the active participation from the indigenous communities to conserve this precious flora. it is also important to aware the local people about the market value and sustainable harvesting of medicinal plants. rapid modernization and urbanization is not only a threat for plant species' degradation but also a threat for the associated folk knowledge. that is why that the disappearance of folk knowledge has been declared more in danger than the natural resources themselves [ ] . therefore, we present a strong recommendation that ethnobotany as a subject should be included into the curriculum to help students in recognizing the endangered and medicinally important species of their respective regions. in addition, incentives may be given to farmers for the cultivation of medicinal plants on marginal lands and home gardens. indigenous communities at bajaur agency are dependent on medicinal plants for ethnoveterinary practices. knowledge about the traditional medicinal system is restricted to the herders, farmers, and elder community members. the younger generation is unaware of this traditional treasure and takes no interest due to modernization. hence, this study is an attempt towards the preservation of traditional ethnoveterinary knowledge from being extinct. there are several medicinal plants, which are being used in traditional herbal system of veterinary disorders. some of the important are v. daucoides, f. vulgare, s. virginianum, w. somnifera, g. glabra, and c. longa. new ethnoveterinary uses used at the study area were found for h. candicans and g. glabra. apiaceae is utmost plant family being in use for various livestock ailments. thorough phytochemical and pharmacological investigations are required by isolating the active compounds and testing the in vitro or in vivo efficacy of the abovementioned plants against the targeted veterinary diseases. furthermore, critical toxicological investigations are also required to ensure the safe and secure use of documented ethnomedicines. in order to share and further maintain this knowledge, it is direly needed to aware the rural population about the significance of traditional ethnoveterinary 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cultivation in the swat valley, north-western pakistan, for economic development and biodiversity conservation ethnomedicinal survey of important plants practiced by indigenous community at ladha subdivision, south waziristan agency ethnobotanical profile of plants of shawar valley, district swat, pakistan an ethnobotanical study of medicinal plants in high mountainous region of chail valley (district swat-pakistan) conserving indigenous knowledge as the key to the current and future use of traditional vegetables the authors extend their high appreciation and acknowledgment towards the local communities by providing moral support to the authors. this research study did not receive any grant from any organization. data gathered during the course of the study has been included in the article.authors' contributions ahk and hu conducted the field work. maa wrote the draft manuscript. ahk and hu helped in the compilation of data. ma gave technical comments on the draft and indicated the language and grammatical mistakes. maa and ma supervised all the stages. all the authors read and approved the manuscript.ethics approval and consent to participate not applicable the authors declare that they have no competing interests.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- - cm m ol authors: ku, therese; lopresti, natalie; shirley, matthew; mori, mattia; marchant, jan; heng, xiao; botta, maurizio; summers, michael f.; seley-radtke, katherine l. title: synthesis of distal and proximal fleximer base analogues and evaluation in the nucleocapsid protein of hiv- date: - - journal: bioorganic & medicinal chemistry doi: . /j.bmc. . . sha: doc_id: cord_uid: cm m ol abstract anti-hiv- drug design has been notably challenging due to the virus’ ability to mutate and develop immunity against commercially available drugs. the aims of this project were to develop a series of fleximer base analogues that not only possess inherent flexibility that can remain active when faced with binding site mutations, but also target a non-canonical, highly conserved target: the nucleocapsid protein of hiv (nc). the compounds were predicted by computational studies not to function via zinc ejection, which would endow them with significant advantages over non-specific and thus toxic zinc-ejectors. the target fleximer bases were synthesized using palladium-catalyzed cross-coupling techniques and subsequently tested against nc and hiv- . the results of those studies are described herein. for some time, the seley-radtke group has designed and synthesized various classes of flexible purine nucleos(t)ides, or "fleximers". [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] these novel nucleosides were designed to investigate how flexibility in the nucleobase could potentially affect receptor-ligand recognition and function. in addition, their flexible design allows them to overcome issues with binding site mutations thus retaining their activity. to date, fleximers have shown key advantages over the corresponding purine-base nucleosides. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] for example, the distal guanosine fleximer (flex-g, fig. ) proved to be an inhibitor of s-adenosyl-lhomocysteine hydrolase by adapting a syn conformation, thereby placing the exocyclic amino group such that it mimicked the amino group from an adenosine nucleobase. , moreover, the guanosine fleximer triphosphate (flex-gtp) was shown to be a superior substrate of human gdp-l-fucose pyrophosphorylase compared to the natural substrate gtp, likely due to the fleximer's ability to interact with amino acids in the active site not accessible by gtp. this also allowed flex-gtp to retain all activity when essential catalytic residues needed for gtp binding were mutated. , recently, a series of fleximers possessing acyclic sugars exhibited broad spectrum antiviral activities against coronaviruses, flaviviruses and filoviruses, further supporting their significance. , in an effort to explore the fleximer approach to other viruses such as hiv- , a virus with the ability to readily mutate and develop antiviral resistance, a series of fleximers were designed and studied in silico for their ability to inhibit the virus' nucleocapsid protein, nc. [ ] [ ] [ ] [ ] nc plays several key roles in hiv- replication. through non-specific binding, it acts as a chaperone protein, partially protecting the viral nucleic acids (nas). during reverse transcription, nc directs the annealing of cellular trna (lys, ) primer to the hiv- primer binding site, thus initiating the synthesis of the (−)-strong stop dna. , nc then facilitates the two strand transfers required for (−) and (+) strand synthesis. it is also implicated to be a vital element in vdna integration. , prior to encapsidation, nc discriminates viral from host na by selectively binding to the hiv- Ψ-encapsidation signal sequence. [ ] [ ] [ ] in addition, in vitro studies have shown that nc may chaperone the dimerization of the two copies of hiv- viral genomic rna by rearranging the kissing complex into an extended duplex through a series of stabilizing and destabilizing events, an important step prior to encapsidation. , because of nc's interaction with multiple highly conserved sequences of the hiv- genome, and being essential in all hiv- subtypes nc represents a powerful drug target for developing novel antivirals. [ ] [ ] [ ] [ ] more importantly, it is thought to be highly resistant to mutation due to its multifunctional role, thus providing a significant advantage over other protein targets. , thus, inhibitors of the interaction between nc and the viral nucleic acids could provide a new approach to antiretroviral therapy. , for this purpose, a series of fleximers were computationally designed with that goal in mind. herein we report the synthesis and biological results for a series of compounds that were predicted to interact with nc. in many structural studies done to date on nc, , , [ ] [ ] [ ] a guanosine residue was shown to consistently stack with the w residue, whether bound to dna (pbs-dna) or rna (Ψ-rna). as such, the inherent flexibility of the fleximer guanine analogue was predicted to affect binding and potentially result in inhibition. based on this hypothesis, a number of fleximer nucleosides were initially designed, however the early results showed that the sugar moiety on the fleximer nucleoside provided no benefit over the fleximer base itself. as a result, the corresponding fleximer base analogues were therefore pursued, since this would signfiicantly shorten the synthetic route. thus, the fleximer bases were then tested computationally against the nmr structure of the nc in complex with a small molecule inhibitor (figs. and ). to this end, a computational protocol was established and refined in the group of botta. , , several nc binding small molecules have already been discovered through this protocol, supporting its validity. , , , the docking results of the fleximer bases on nc revealed several key advantages for the proposed target compounds. the docking conformation of fleximer bases - ( fig. ) within the hydrophobic pocket that is located in correspondence of w showed an excellent structural overlay with respect to the guanine base. moreover, all of the fleximer bases were able to establish a network of h-bond interactions with the backbone atoms of key residues in the hydrophobic pocket (i.e. k , g , w , and m , fig. a -d) that is highly comparable to that established by the guanine base (fig. e) . additionally, - adopted a similar stacking conformation to w as is observed with the natural guanine. however the additional rotatable bond allowed for the pyrimidine moiety to extend and interact with the neighboring residues (i.e. k in the binding mode of , fig. d ) into the solvent area. the distal guanine fleximer base ( ) was predicted to bind stronger than the proximal base ( ) and, notably, slightly stronger than the guanine base (table ) , although scoring values calculated by the fred docking program with the chemgauss function were highly comparable to each other. , thus, the observed differences between guanine, and may not be significant. in addition, the docking study surprisingly showed that the bipyrimidine scaffold ( and , fig. ), would also be highly advantageous, although to a slightly lesser extent than the parent imidazole-pyrimidine scaffold (table ) , thus those targets were pursued as well. as a result of this molecular modeling analysis, compounds - ( fig. ) were selected as the best starting candidates as proof of concept. the intended route to achieve both sets of flexible purine and bipyrimidine bases utilized palladium-catalyzed cross-coupling, with the pyrimidine as the organometallic coupling partner and the imidazole as the halogenated coupling partner. the goal was to achieve two products from one reaction as the organometallic moiety has been shown to undergo homocoupling during cross-coupling reactions. [ ] [ ] [ ] [ ] [ ] as the distal compounds were predicted to be better binders to nc, the first goal was to install the organometal on the c- of to avoid protecting the exocyclic amine. to obtain the iodinated intermediate, commercially available -amino- -chloro- -methoxypyrimidine was iodinated using hydroiodic acid (scheme ). the reaction was then neutralized and filtered, and the precipitate recrystallized in ethanol to obtain . since the proximal intermediate -amino- -methoxy- -tributylstannylpyrimidine was easily achievable starting with -amino- -iodo- -methoxypyrimidine, [ ] [ ] [ ] similar methodologies were applied to obtain the -amino- -methoxy- -tributylstannylpyrimidine intermediate , using various palladium catalysts as well as a range of temperatures, but none of the conditions yielded the desired organostannane (scheme ). the organoborane was then considered for subsequent suzuki coupling (scheme ), however, the boronic ester could not be generated under any conditions. because the cross-coupling intermediates could not be obtained through palladium catalysis, harsher conditions were used to achieve the desired coupling partners. the exocyclic amine was protected (scheme ) with tms as it is immune to grignard and lithium reagents but can be cleaved easily in mildly acidic conditions. each tms group was added sequentially as opposed to simultaneously to form in situ. lithium halogen exchange using n-buli, followed by metalation with tributyltin chloride finally produced deprotected organostannane . characterization through h and c nmr in addition to ms confirmed the presence of the product however proved highly unstable and decomposed rapidly. as installation of the metal on the pyrimidine proved to be difficult, and literature showed that an organozinc intermediate could be generated in situ with the imidazole moiety for subsequent negishi coupling, the previous methodology was abandoned. a benzyl (bn) protecting group was initially chosen as bn's are robust against many conditions. the organozinc was generated in situ and subsequent negishi coupling with (scheme ) yielded the protected distal fleximer , albeit in poor yield ( %). moreover, the bn and methyl groups were extremely difficult to deprotect (scheme ). hydrogenation using pd/c in the presence of h at room temperature was thought to be sufficient to remove the bn group, however no reaction occurred. this was ultimately achieved by heating the reaction mixture of with ammonium formate and pd/c in etoh to °c. boron tribromide (bbr ) was employed to deprotected the methyl, however, a solubility problem arose when attempting to demethylate in dcm, and therefore deprotection of the methyl was more efficient when performed prior to deprotection of the bn (scheme ). unfortunately, these molecules proved difficult to purify via column chromatography, likely due to either their polar nature or their ability to stack efficiently from the presence of two heteroaromatic moieties and multiple hydrogen bonding elements. to bypass these challenges, a trityl protecting group was employed to protect the imidazole, and the exocyclic amine of the pyrimidine was protected with tert-butyloxycarbonyl (boc, schemes and ). negishi coupling using these two heterocycles proved more facile and the cross-coupling reaction proceeded at room temperature (scheme ). trityl deprotection was accomplished using acetic acid while boc deprotection required trifluoroacetic acid. as previously mentioned, the methyl protected fleximer guanine was insoluble in dcm, however, it was soluble in etoac, and final deprotection using bbr in etoac was successful. since was ultimately obtained through negishi coupling, a similar strategy was employed to obtain the analogous bipyrimidine (scheme ). unexpectedly, the amine-linked compound was produced instead. the compound was then subjected to aminolysis to convert the chloro group to an exocyclic amine, however, no starting material or product was recovered (scheme ). in hindsight, the presence of a palladium catalyst likely induced a buchwald-hartwig amination, leading to the synthesis of and the absence of compound . because the correct cross-couplings procedures could not be easily performed on the c- of , -bromo- , -dimethoxypyrimidine ( , scheme ) was pursued instead, which which was envisioned by first brominating barbituric acid with pobr followed by nucleophilic substitution using sodium methoxide to afford compound . to achieve the targeted bipyrimidine through stille coupling, was first converted to stannane . interestingly, was never synthesized, however the bipyrimidine was recovered at good yields ( %, scheme ). from there, two strategies were attempted to attain the desired bipyrimidine . first, removal of the methyl protecting groups followed by chlorination via pocl was tried, and intermediate was used crude as it was insoluble in various purification solvents. the tetrachlorinated intermediate could not be isolated as the crude reaction mixture was difficult to purify. the next approach was to directly convert the methoxy groups to amines ( ) and enzymatically convert the c -nh group to an eoh using adenosine deaminase. neither the starting material nor product were recovered, likely due to the harsh conditions used. since homocoupling of the pyrimidines had occurred with the dimethoxypyrimidines, it was speculated that the same conditions would likely produce a homocoupled product for the -amino- -methoxypyrimidines as well, which fortuitously proved true (scheme ). unfortunately, the product proved to be highly insoluble, and impossible to purify, and was only observed via ms. as achieving the distal analogues proved challenging, the focus then turned to synthesizing the proximal analogues. considering the synthesis of compound was facile, a straightforward stille with the halogenated pyrimidine produced the bipyrimidine (scheme ), however, similar purification and solubility issues as occurred. to complete this series, the proximal fleximer guanine was also pursued. instead of using stille cross-coupling techniques, the negishi method used for achieving the distal fleximer guanine was employed (scheme ). surprisingly, no product was observed when the reaction was allowed to stir at room temperature, and poor yields were found even after reflux. the failure of the reaction was speculated to be due to the placement of the halogen on the electron-rich carbon of the c- position of the pyrimidine. as palladium prefers to react with electron-deficient carbons, the oxidative addition reaction between the electron-rich halogenated coupling partner and palladium likely did not occur, which led to the absence of the desired coupled product. hence, the organozinc was placed on the pyrimidine in subsequent reactions. as predicted, the organozinc on the pyrimidine was successfully synthesized in situ, and negishi cross-coupling followed by deprotection of the trityl group was accomplished to yield (scheme ). regrettably, methyl deprotection to produce the proximal fleximer guanine was unsuccessful using bbr in either ch cl or etoac as the intermediate was insoluble in both. deprotection using catalytic sulfuric acid and tmscl in acetic anhydride also did not produce (confirmed through ms). the hypothesis to explain this phenomenon ties into the in silico results by bardon et al. that showed that the most thermodynamically stable conformation of the proximal fleximer bases is in a planar form. this conformation could be promoting the stacking of the bases that consequently does not allow to solubilize in usual solvents. the dimethoxypyrimidine series was also pursued and proved more facile to obtain as no additional protection steps were required. following the approach to realize the -amino- -methoxy series, the dimethoxy proximal compounds were synthesized using the pyrimidine as the organometallic moiety (scheme ). deprotection of the methyl groups were attempted, however, the resulting crude mixture was insoluble and thus purification was not possible. similar conditions were used to synthesize the dimethyl protected distal fleximer xanthosine as scheme (scheme ). the yield of ( %) was higher than that of ( %), with the major structural difference being the c- substitution (ome versus nhboc). with compound in hand, a h nmr experiment was performed with nc. if the fleximer did bind to the na binding site of nc where w resides, a shift should be observed in the aromatic region of the protein, similar to that shown in goudreau et al. after recording the h nmr spectra of nc in d o without , a one equivalent molar ratio of was added to the sample (dissolved in dmso-d ). both the aliphatic region and aromatic region (fig. ) were recorded but no significant changes in the nc signals were detected. this study does not necessarily exclude as an nc binder, but does prove that the interaction is at most very weak, thus undetectable via nmr experiments. unfortunately, none of the other products that were successfully purified would dissolve in the nmr solvent or in the presence of the protein, therefore the nmr studies were abandoned. finally, all of the target compounds were sent to the national cancer institute (nci) to be tested against hiv- by dr. eric freed. disappointingly, none of the analogues exhibited any meaningful antiviral activity. a number of fleximer bases were successfully synthesized via palladium-catalysed cross-couplings in this study, albeit with more difficulty than anticipated, especially during purification. the synthetic routes developed for the distal fleximer base bypassed the classic tricyclic route that has been used in the seley-radtke group for over a decade. if yields could be improved, this would potentially be useful for synthesis of distal fleximer nucleosides in the future. biologically however, these molecules were not recognized by nc based on the nmr experiment performed, and were inactive against hiv- . while the biological results were disappointing, the project did provide a better understanding of palladium-catalysed cross-coupling strategies for fleximer bases in terms of choosing the optimum crosscoupling partners. this which has proven advantageous for other projects ongoing in our group and others, the results of which will be published as they become available. all chemicals were obtained from commercial sources and used without further purification unless otherwise noted. anhydrous dmf, ch oh, dmso and etoh were purchased from fisher scientific. anhydrous thf, acetone, ch cl , ch cn, and ether were obtained using a solvent purification system (mbraun labmaster ). nmr solvents were purchased from cambridge isotope laboratories (andover, ma). all h and c nmr spectra were obtained either on a jeol ecx mhz nmr, operated at and mhz, respectively, or a bruker avance iii hd mhz nmr, operated at and mhz, respectively, and referenced to internal tetramethylsilane (tms) at . ppm. the spin multiplicities are indicated by the symbols s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), m (multiplet), and br (broad). reactions were monitored by thin-layer chromatography (tlc) using . mm whatman diamond silica gel -f pre-coated plates. purification was performed on a teledyne isco combiflash rf , and eluted with the indicated solvent system. yields refer to chromatographically and spectroscopically ( h and c nmr) homogeneous materials. mass spectra were recorded at the umbc mcac for nominal using bruker apollo™ ii esi/apci -maldi dual source for apex(r)-qe ftms or johns hopkins mass spectrometry facility for high resolution using vg analytical vg- se magnetic sector mass spectrometer. commercially available -amino- -chloro- -methoxypyrimidine ( . g, . mmol) was suspended in ml of wt% hi in h o at °c. the mixture was stirred at room temperature for h. the resulting sludge was diluted in ml h o and neutralized to ph - using sat. na co . the precipitate was filtered and recrystallized in etoh to yield a white solid ( . g, . mmol, %). ( ) ( mg, . mmol) was dissolved under n in ml of anhydrous thf and cooled to − °c. etmgbr ( . m, . ml, . mmol) was added dropwise and allowed to stir for min. tmscl ( . ml, . mmol) was added and allowed to stir for min. again, etmgbr ( . m, . ml, . mmol) was added dropwise and allowed to stir for min, then tmscl ( . ml, . mmol) was added and allowed to stir for min. n-butyllithium ( . m, . ml, . mmol) was added dropwise and allowed warm to room temperature and stirred for . h. tributyltin chloride ( . ml, . mmol) was added and the mixture was stirred for min. the reaction was quenched using ml nh cl and the solvent was removed in vacuo. the crude material was extracted into ch cl ( ml × ), washed with brine ( ml × ) and the organic layer was dried over mgso . the crude material was purified using silica gel column chromatography (hexanes/ etoac = : - : ) to yield a yellow oil ( mg, . -benzyl- -iodo- h-imidazole ( mg, . mmol) was dissolved under n in ml of anhydrous thf and cooled to − °c. etmgbr ( . m, . ml, . mmol) was added dropwise and allowed to stir for min. zncl ( . m in thf, . ml, . mmol) was subsequently added dropwise, stirred at − °c for min, warmed to room temperature and stirred for h. the organozinc was added dropwise to a mixture of ( mg, . mmol), pd(pph ) ( mg, . mmol), cui ( mg, . mmol) in ml of anhydrous thf and allowed to stir at room temperature for h. the reaction was quenched using ml sat. edta solution and thf was removed in vacuo. the crude material was extracted into ch cl ( ml × ), washed with brine ( ml × ) and the organic layer was dried over mgso . the crude material was purified using silica gel column chromatography (hexanes/etoac = : - : ) to yield a white solid ( mg, . mmol, % yield). r f = . , (etoac). synthetic procedure related to of scheme reported. ( mg, . mmol) was dissolved in ml trifluoroacetic acid and stirred for h. the solvent was removed and the crude material was purified using silica gel column chromatography (ch cl /ch oh = : - : ) to yield a white solid ( mg, . mmol, % yield). r f = . , (ch cl /ch oh = : ). ( mg, . mmol) was dissolved in ml anhydrous ch cl under n and cooled to − °c. bbr ( m, . ml, . mmol) was added dropwise and the reaction was allowed to warm to room temperature and stirred for h. the mixture was dripped slowly into ml iced water and stirred for min. the solvent was removed in vacuo and the crude material was purified using silica gel column chromatography (ch cl /ch oh = : - : ) to yield a white solid ( mg, . mmol, % yield). r f = . , (ch cl /ch oh = : ). -amino- -iodo- -methoxypyrimidine ( . g, . mmol) was suspended in ml ch cl under n . di-tert-butyl decarbonate ( . g, . mmol) and -dimethylaminopyridine ( . g, . mmol) were added. the reaction was allowed to stir at room temperature for h. tlc showed absence of starting material and two products. the solvent commercially available -iodo- -trityl- h-imidazole ( mg, . mmol) was dissolved under n in ml of anhydrous thf and cooled to − °c. etmgbr ( . m, . ml, . mmol) was added dropwise and allowed to stir for min. zncl ( . m in thf, . ml, . mmol) was subsequently added dropwise, stirred at − °c for min, warmed to room temperature, and stirred for h. the organozinc was added dropwise to a mixture of ( mg, . mmol), pd (pph ) ( mg, . mmol) and cui ( mg, . mmol) in ml of anhydrous thf and allowed to stir at room temperature for h. the reaction was quenched using ml sat. edta solution and thf was removed in vacuo. the crude material was extracted into ch cl ( ml × ), washed with brine ( ml × ) and the organic layer was dried over mgso . the crude material was purified using silica gel column chromatography (hexanes/etoac = : - : ) to yield a yellow oil ( mg, . mmol, % yield). r f = . , (hexanes/etoac = : ). ( ) ( mg, . mmol) was dissolved in ml acetic acid and stirred for h. the solvent was removed and the crude material was purified using silica gel column chromatography (ch cl / ch oh = : - : ) to yield an off-white solid ( mg, . mmol, % yield). r f = . , (ch cl /ch oh = : ). ( mg, . mmol) was dissolved in ml anhydrous etoac under n and cooled to − °c. boron tribromide ( . m, . ml, . mmol) was added dropwise. the reaction was allowed to warm to room temperature and stirred for h. the mixture was dripped slowly into ml iced water and stirred for min. the solvent was removed in vacuo and the crude material was purified using silica gel column chromatography (ch cl /ch oh = : - : ) to yield a white solid ( mg, . mmol, % yield). r f = . , (ch cl /ch oh = : ). . . synthesis of , ′-dimethoxy- , ′-bipyrimidine- , ′-diamine ( ) ( . mg, . mmol) was dissolved in ml degassed , -dioxane in a glass tube. bis(tributyltin) ( . ml, . mmol) was added, followed by pd(pph ) cl ( . mg, . mmol). the glass tube was sealed and heated to °c for h. the tube was cooled to °c, opened and warmed to room temperature. the crude content was filtered over a pad of celite and the solvent was removed. the crude material was purified using silica gel column chromatography (ch cl / ch oh = : - : ) to yield an impure mixture. another attempt at purification with silica gel column chromatography using the pharmasset conditions (etoac/ch ( mg, . mmol) was dissolved under n in ml of anhydrous thf and cooled to − °c. etmgbr ( . m, . ml, . mmol) was added dropwise and allowed to stir for min. tmscl ( . ml, . mmol) was added and allowed to stir for min. again, etmgbr ( . m, . ml, . mmol) was added dropwise and allowed to stir for min, then tmscl ( . ml, . mmol) was added and allowed to stir for min. etmgbr ( . m, . ml, . mmol) was added dropwise and allowed to stir for min followed by addition of zncl ( m in thf, . ml, . mmol) dropwise, stirred at − °c for min, warmed to room temperature and stirred for h. the organozinc was added dropwise to a mixture of -amino- -chloro- -methoxypyrimidine ( mg, . mmol), pdcl (pph ) ( mg, . mmol) and cui ( mg, . mmol) in ml of anhydrous thf and allowed to stir at reflux for h. the reaction was quenched using ml sat. edta solution and thf was removed in vacuo. the crude material was extracted into ch cl ( ml × ), washed with brine ( ml × ) and the organic layer was dried over mgso . the crude material was purified using silica gel column chromatography (hexanes/etoac = : ) to yield a white solid ( mg, . mmol, % yield). r f = . , (hexanes/ etoac = : ). h nmr ( mhz, dmso-d ) δ . (s, h), . (s, h), . (br, h), . (s, h), . (s, h), . (br, h). commercially available barbituric acid ( . g, . mmol) was suspended in ml of anhydrous toluene under n and cooled to °c. phosphorus (v) oxybromide ( . g, . mmol) was added and n,ndimethylaniline ( . ml, . mmol) was added dropwise. the mixture was heated to °c and stirred vigorously for h. the reaction was cooled to room temperature and quenched with ml iced water. the mixture was transferred to a separatory funnel and the remaining insoluble gum was washed with etoac ( ml × ). all organic layers were combined and washed with sat. nahco ( ml × ), then brine ( ml × ), and the organic layer was dried over mgso . the crude material was purified using silica gel column chromatography (hexanes/etoac = : - : ) to yield as a while solid ( . g, . mmol, % yield). r f = . , (hexanes/et o = : ). ( . m, . ml, . mmol) was added dropwise and the mixture was warmed to room temperature and stirred for h. the reaction was quenched using ml nh cl and the solvent was removed. the crude material was extracted into ch cl ( ml × ), washed with brine ( ml × ) and the organic layer was dried over mgso . the crude material was purified using silica gel column chromatography (hexanes/etoac . . synthesis of , ′, , ′-tetramethoxy- , ′-bipyrimidine ( ) ( mg, . mmol) was dissolved in ml degassed , -dioxane in a glass tube. bis(tributyltin) ( . ml, . mmol) was added, followed by pd(pph ) ( mg, . mmol). the glass tube was sealed and heated to °c for h. the tube was cooled to °c, opened and warmed to room temperature. the crude content was filtered over a pad of celite and the solvent was removed. the crude material was purified using silica gel column chromatography (hexanes/ etoac = : - : ) to yield a white fluffy solid ( mg, . mmol, % yield). r f = . , (hexanes/etoac = : ). ( mg, . mmol) and ( mg, . mmol) were dissolved in ml degassed dmf. pd(pph ) ( mg, . mmol), cui ( mg, . mmol) and csf ( mg, . mmol) were added. the reaction was allowed to stir at °c for h. the contents were cooled and filtered over celite. the solvent was removed, and the crude material was purified using silica gel column chromatography (ch cl / ch oh = : - : ) to yield contaminated . the contaminated sample was recrystallized in etoac, followed by ethanol, followed by methanol and finally dmso to obtain a white solid ( mg, . mmol, % yield). h nmr ( mhz, dmso-d ) δ . (s, h), . (br, h), . (s, h). ( mg, . mmol) was dissolved under n in ml of thf and cooled to − °c. etmgbr ( . m, . ml, . mmol) was added dropwise and allowed to stir for min. tmscl ( . ml, . mmol) was added and allowed to stir for min. again, etmgbr ( . m, . ml, . mmol) was added dropwise and allowed to stir for min, then tmscl ( . ml, . mmol) was added and allowed to stir for min. etmgbr ( . m, . ml, . mmol) was added dropwise and allowed to stir for min followed by addition of zncl ( . m in thf, . ml, . mmol) dropwise, stirred at − °c for min, warmed to room temperature and stirred for h. the organozinc was added dropwise to a mixture of ( mg, . mmol), pd(pph ) ( mg, . mmol), cui ( mg, . mmol) in ml of thf and allowed to stir at room temperature for h. the reaction was quenched using ml sat. edta solution and thf was removed in vacuo. the crude material was extracted into ch cl ( ml × ), washed with brine ( ml × ) and the organic layer was dried over mgso . the crude material was purified using silica gel column chromatography (hexanes/ etoac = : - : ) to yield a yellow solid ( mg, . mmol, % yield). r f = . , (etoac). ( ) ( mg, . mmol) was dissolved in ml acetic acid and stirred for h. the solvent was removed and the crude material was purified using silica gel column chromatography (ch cl / ch oh = : - : ) to yield an off-white solid ( mg, . mmol, % yield). r f = . , (ch cl /ch oh = : ). commercially available -bromo- , -dimethoxypyrimidine ( mg, . mmol) was dissolved under n in ml of anhydrous thf and cooled to − °c. etmgbr ( . m, . ml, . mmol) was added dropwise and allowed to stir for min. zncl ( . m in thf, . ml, . mmol) was subsequently added dropwise, stirred at − °c for min, warmed to room temperature and stirred for h. the organozinc was added dropwise to a mixture of ( )-iodo- himidazole ( mg, . mmol), pd(pph ) ( mg, . mmol) and cui ( mg, . mmol) in ml of thf and allowed to stir at reflux for h. the reaction was quenched using ml sat. edta solution and thf was removed in vacuo. the crude material was extracted into ch cl ( ml × ), washed with brine ( ml × ) and the organic layer was dried over mgso . the crude material was purified using silica gel column chromatography (ch cl /ch oh = : - : ) to yield a white solid ( mg, . mmol, % yield). r f = . , (ch cl / ch oh = : ). ( , -dimethoxy- -pyrimidinyl)- , , , -tetramethyl- , , -dioxaborolane ( ) commercially available -bromo- , -dimethoxypyrimidine ( mg, . mmol) was suspended in ml degassed dmf under n , followed by addition of bis(pinacolato)diboron ( mg, . mmol), potassium acetate ( mg, . mmol) and pdcl (dppf) ·chcl ( mg, . mmol). the mixture was heated to °c and stirred for h. the reaction was cooled and transferred to ml dh o. the mixture was extracted in etoac/toluene ( : , ml × ), washed with brine ( ml × ) and the organic layer was dried over mgso . the solvent was removed in vacuo and the crude product was used without further purification. . . synthesis of , ′, , ′-tetramethoxy- , ′-bipyrimidine ( ) commercially available -bromo- , -dimethoxypyrimidine ( mg, . mmol) and crude ( . mmol) were suspended in ml degassed , -dioxane/dh o ( : ) under n in a sealed glass flask. cs co ( . g, . mmol) and pdcl (dppf) ·chcl ( mg, . mmol). the glass flask was sealed, heated to °c and stirred for h. the flask was chilled to °c, opened, and warmed to room temperature. the crude content was filtered over a pad of celite and the solvent was evaporated in vacuo. the crude material was purified using silica gel column chromatography (hexanes/etoac = : - : ) to yield a white solid ( mg, . mmol, % yield). r f = . , (hexanes/ etoac = : ). -iodo- -trityl- h-imidazole ( mg, . mmol) was dissolved under n in ml of thf and cooled to − °c. etmgbr ( . m, . ml, . mmol) was added dropwise and allowed to stir for min. zncl ( . m in thf, . ml, . mmol) was subsequently added dropwise, stirred at − °c for min, warmed to room temperature and stirred for h. the organozinc was added dropwise to a mixture of ( mg, . mmol), pd(pph ) ( mg, . mmol), cui ( mg, . mmol) in ml of thf and allowed to stir at room temperature for h. the reaction was quenched using ml sat. edta solution and thf was removed in vacuo. the crude material was extracted into ch cl ( ml × ), washed with brine ( ml × ) and the organic layer was dried over mgso . the crude material was purified using silica gel column chromatography (hexanes/etoac = : - : ) to yield a white solid ( mg, . mmol, % yield). r f = . , (hexanes/ etoac = : ). ( ) ( mg, . mmol) was dissolved in ml acetic acid and stirred for h. the solvent was removed and the crude material was purified using silica gel column chromatography (ch cl / ch oh = : - : ) to yield a white solid ( mg, . mmol, % yield). r f = . , (ch cl /ch oh = : ). molecular modeling study was performed as described in previous studies. , briefly, the nmr structure of the nc in complex with a small molecule inhibitor was used as rigid receptor in molecular docking simulations, which were carried out by the fred docking program from openeye, version . . . , ligand conformational analysis was performed with omega from openeye. , the hiv- nc coding region in pnl - was pcr amplified using the ′-primer ccagctaccatacatatgcagaaaggc (ndei site underlined) and the ′-primer ggccggatcctccctaactaattagcct gtc-tctc (bamhi and stop codon underlined). the expression vector pet- a (novagen, madison, wi) was doubly digested with ndei and bamhi and treated with calf intestinal alkaline phosphate. the pcr product was purified by phenol-extraction and ethanol-precipitation and doubly digested with ndei and bamhi. the insert and vector were ligated using phage t dna ligase at °c for five hours and transformed into competent hms . dna from transformants were sequenced and found to be identical with the hiv- nc coding sequence in pnl - . a clone, designated as prd , overexpressed the -residue nc protein with the sequence m q k g n f r n q r k t v k c f n c g k e g h i a k n c r a p r k k g c w k c g k e g h q m k d c t e r q a n (the two zinc knuckles are underlined). ion-spray mass spectrometry confirmed the mass of the apoprotein to be ( ± ) da (calculated da) and ( ± ) da (calculated da) for the zn-bound protein. for protein expression of hiv- nc in escherichia coli, prd was transformed into bl (de ) plyse. the purification scheme for the recombinant hiv- nc was adapted from ji et al. and you & mchenry. culture media were supplemented with μg/l ampicillin and μg/l chloramphenicol. a starter culture of ml of zb inoculated from a single colony was grown at °c overnight. the starter culture was added to l of m zb supplemented with . mm zncl and grown at °c to an absorbance at nm of . to . before induction with mm iptg (isopropyl-β-d-thiogalactopyranoside). after three hours, the cells were harvested by centrifugation, resuspended in ml of lysis buffer ( mm tris-hcl (ph . ), % (v/v) glycerol, . m nacl, . mm zncl , mm dithiothreitol, mm edta), and stored at °c. to lyse the cells, the cells were thawed in ice-water, and ml of mm pmsf (phenylmethylsulfonyl fluoride), ml of mg/ ml pepstatin a, and . ml of % (w/v) sodium deoxycholate were added. the cells were sonicated by five bursts of seconds to reduce the viscosity. the nucleic acids were precipitated by adding % (w/v) polyethyleneimine (ph . ) dropwise to a final concentration of . % and stirred for minutes before centrifugation at , g for minutes at °c. the supernatant was collected ( ml), filtered ( . μm pore size), and loaded at ml/minute onto a ml q-sepharose and a ml sp-sepharose column (pharmacia) connected in series and previously equilibrated with ml of buffer a ( mm tris-hcl (ph . ), % glycerol, . m nacl, . mm zncl , mm bme (βmercaptoethanol)). after washing with ml of buffer a, the q-sepharose column was detached, and the sp-sepharose column was washed with . column volumes of buffer a. a ten column volume linear gradient from % to % buffer b ( mm tris-hcl (ph . ), % glycerol, . m nacl, . mm zncl , mm bme) was applied to elute the hiv- nc protein. the protein fractions were pooled ( ml) and loaded at . ml/minute onto a ml sephadex g- column (pharmacia) pre-equilibrated with two volumes of buffer c ( mm tris-hcl (ph . ), % glycerol, . m nacl, . mm zncl , mm bme). the nc protein eluted at ml and fractions were pooled ( ml) for concentration and dialysis into nmr buffer (see below). nmr buffer ( mm tris-hcl, ph . , mm kcl, mm nacl, mm mgcl ) was deoxygenated by sparging with argon for minutes and filter-sterilized ( . μm pore size). the protein sample was dialyzed using centricon- by adding nmr buffer five or six times (total volume - ml). the buffered protein sample was lyophilized for ease of storage. μm protein samples were made in μl of d o and loaded into a mm nmr tube. after taking the blank h spectrum, the test compound was titrated into the protein sample such that : ratio of compound/protein could be established. data for h nmr signal assignments were collected at a sample temperature of °c with a bruker dmx mhz ( h) nmr and bruker avance iii hd mhz nmr spectrometers. the authors have no conflicts to declare. fleximers" design and synthesis of two novel split nucleosides fleximers" design and synthesis of a new class of novel shape-modified nucleosides unexpected inhibition of s-adenosyl-lhomocysteine hydrolase by a guanosine nucleoside conformational properties of shape modified nucleosides -fleximers substrate discrimination by the human gtp fucose pyrophosphorylase identification of catalytic amino acids in the human gtp fucose pyrophosphorylase active site molecular chameleons". design and synthesis of c- -substituted imidazole fleximers design and synthesis of a second series of flexible nucleosides synthetic routes to a series of proximal and distal '-deoxy fleximers design, synthesis and evaluation of a series of acyclic fleximer nucleoside analogues with anti-coronavirus activity flex-nucleoside analogues -novel therapeutics against filoviruses carbocyclic '-nor "reverse" fleximers. design, synthesis, and preliminary biological activity reverse" carbocyclic fleximers: synthesis of a new class of adenosine deaminase inhibitors structure-based identification of hiv- nucleocapsid protein inhibitors active against wild-type and drug-resistant hiv- strains use of virtual screening for discovering antiretroviral compounds interacting with the hiv- nucleocapsid protein molecular dynamics and dft study on hiv- nucleocapsid protein- in complex with viral genome predicting the binding mode of known ncp inhibitors to facilitate the design of novel modulators dna condensation by the nucleocapsid protein of hiv- : a mechanism ensuring dna protection hiv- nucleocapsid protein zinc finger structures induce trna(lys, ) structural changes but are not critical for primer/template annealing genomic placement and the initiation step of reverse transcription in human immunodeficiency virus type nucleocapsid protein function in early infection processes analysis of ncp -dependent activation of hiv- cdna integration and its conservation among retroviral nucleocapsid proteins structure of the hiv- nucleocapsid protein bound to the sl psi-rna recognition element affinities of packaging domain loops in hiv- rna for the nucleocapsid protein effects of the nature and concentration of salt on the interaction of the hiv- nucleocapsid protein with sl rna flexible nature and specific functions of the hiv- nucleocapsid protein retroviral rna dimerization and packaging: the what, how, when, where, and why properties, functions, and drug targeting of the multifunctional nucleocapsid protein of the human immunodeficiency virus identification of hiv- inhibitors targeting the nucleocapsid protein targeting the viral nucleocapsid protein in anti-hiv- therapy nucleocapsid protein: a desirable target for future therapies against hiv- the nucleocapsid protein of hiv- as a promising therapeutic target for antiviral drugs synthesis and evaluation of bifunctional aminothiazoles as antiretrovirals targeting the hiv- nucleocapsid protein time-resolved fluorescence investigation of the human immunodeficiency virus type nucleocapsid protein: influence of the binding of nucleic acids probing the hiv- ncp nucleocapsid protein with site-specific gold(i)-phosphine complexes structure of the complex between the hiv- nucleocapsid protein ncp and the single-stranded pentanucleotide d(acgcc) discovery and structural characterization of a new inhibitor series of hiv- nucleocapsid function: nmr solution structure determination of a ternary complex involving a : inhibitor/nc stoichiometry identification of novel -benzoxazolinone derivatives with specific inhibitory activity against the hiv- nucleocapsid protein fred pose prediction and virtual screening accuracy fred . . openeye scientific software suppression of a palladium-mediated homocoupling in a suzuki cross-coupling reaction. development of an impurity control strategy supporting synthesis of ly preparation of , -diarylpropenes by phosphine-free palladium(o)-catalyzed suzuki-type coupling of allyl bromides with arylboronic acids highly efficient and accelerated suzuki aryl couplings mediated by phosphine-free palladium sources aryl couplings with heterogeneous palladium catalysts high yields of unsymmetrical biaryls via cross-coupling of arylboronic acids with haloarenes using a modified suzuki-beletskaya procedure greene's protective groups in organic synthesis synthesis and significant cytostatic activity of -hetaryl- -deazaadenosines applications of palladium-catalyzed c-n cross-coupling reactions metal-catalyzed cross-coupling reactions. nd, completely rev. and enl ed how flexible are fleximer nucleobases? a computational study a convenient synthesis of , -disubstituted imidazoles conformer generation with omega: algorithm and validation using high quality structures from the protein databank and cambridge structural database openeye omega . . . : openeye scientific software dynamical behavior of the hiv- nucleocapsid protein production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone effect of human immunodeficiency virus type (hiv- ) nucleocapsid protein on hiv- reverse transcriptase activity in vitro hiv nucleocapsid protein. expression in escherichia coli, purification, and characterization use of t rna polymerase to direct expression of cloned genes this work was supported by the national institutes of health t gm , r ai (ksr), and r gm , r gm (mfs). the authors also wish to thank the openeye free academic licensing program for providing a free academic license for molecular modeling and chemoinformatics software. supplementary data to this article can be found online at https:// doi.org/ . /j.bmc. . . . key: cord- -r sjv ih authors: antas, marta; woźniakowski, grzegorz title: current status of porcine epidemic diarrhoea (ped) in european pigs date: - - journal: j vet res doi: . /jvetres- - sha: doc_id: cord_uid: r sjv ih porcine epidemic diarrhoea (ped) is a highly contagious and devastating enteric disease of pigs caused by porcine epidemic diarrhoea virus (pedv), an enveloped, single-stranded rna virus belonging to the alphacoronavirus genus of the coronaviridae family. the disease is clinically similar to other forms of porcine gastroenteritis. pigs are the only known host of the disease, and the occurrence of ped in wild boars is unknown. the virus causes acute diarrhoea, vomiting, dehydration, and high mortality in suckling piglets reaching %. heavy economic losses in the pig-farming industry were sustained in the usa between and when pedv spread very quickly and resulted in epidemics. the loss in the us pig industry has been estimated at almost seven million pigs. the purpose of this review is a description of the current status of porcine epidemic diarrhoea in european pigs and the risk presented by the introduction of pedv to poland in comparison to the epidemics in the usa. coronaviruses (covs) cause a large variety of diseases in humans and animals. in pigs, coronaviruses affect various organs, including the gastrointestinal and respiratory tracts. covs have one of the largest genomes of all rna viruses, which in combination with their high genetic diversity causes mutation and recombination, resulting in new virus variants ( ) . besides porcine epidemic diarrhoea virus (pedv), the porcine coronaviruses comprise transmissible gastroenteritis virus (tgev), porcine respiratory coronavirus (prcv), porcine haemagglutinating encephalomyelitis virus (phev), and porcine deltacoronavirus (pdcov) ( ) . the clinical symptoms of ped are diarrhoea, vomiting, and dehydration which result in very high mortality among suckling piglets and large economic losses ( , , ) . various factors influence the clinical signs of ped, mainly the age of the animals, the herd's immune status, and the virulence of the strain ( ) . multiple pedv strains are circulating on different pig farms around the world and they differ in virulence. ped was observed for the first time in in the united kingdom. the virus only infected fattening pigs and sows. subsequently, the disease spread to other european countries. emergence of a new pedv strain in china in caused serious epidemics and this strain rapidly spread worldwide. in the usa, highly virulent strains infected pigs in states between and , resulting in significant economic losses ( , , ) . the disease was characterised by rapid diarrhoea onset in pigs of all ages and mortality in suckling piglets reaching % ( ) . in europe, ped re-emerged in in germany. subsequently, highly similar strains were also found in several european countries ( , ) . ped is not a notifiable disease in the european union and is not on the list of diseases reported to the world animal health organization (oie), therefore the status of this disease is not fully recognised. in the last years, only a few countries have reported clinical cases of ped and/or seropositive animals ( ). coronaviruses belong to the coronavirinae subfamily. currently, this subfamily is classified into the four genera alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus, which fall into distinct groups on the phylogenetic tree. ( , , ) . the taxonomy of swine coronaviruses is shown in table . the coronaviruses are pleomorphic and have one of the largest rna viral genomes and a unique replication strategy ( , ) . virions are spherical with diameter of approximately nm. the most prominent and the defining feature of coronaviruses are the spike structures on the surface of the virion, which give them the appearance of a solar corona ( ) . the pedv genome is single-stranded rna ( , nucleotides in length) of positive-sense polarity containing ′ and ′ untranslated regions (utr) at both ends. two thirds of the genome from the ′ end encodes proteins necessary for rna replication, while the one third on the ′ side of the genome comprises at least seven open reading frames (orfs) that encode four structural proteins: n (capsule), s (spike), e (envelope), and m (membrane), and three nonstructural proteins (replicases a and b and orf ). in the genome, they are arranged in the order ′-replicase ( a/ b)-s-orf -e-m-n- ′. the rna genome of the virus is associated with the n protein to form a long, helical ribonucleoprotein (rnp) complex. the virus core is enclosed by a lipoprotein envelope, which contains the s, e, and m proteins ( fig. ) ( , , , ) . the m protein is the most abundant component in the viral envelope, is necessary for the assembly process, and affects production of protective antibodies with virus-neutralising activity. the small e protein plays an important role during coronavirus budding. the n protein has multiple functions in viral replication, interacts with viral genomic rna, and associates with other n proteins to protect the viral genome. it disturbs antiviral responses by antagonising interferon production ( , ) . the s protein is encoded by a gene sequence which determines the virulence of a pedv strain ( ) . this protein is composed of , amino acids (aa), significant among which are a signal peptide ( - aa), neutralizing epitopes ( - , - , - , and , - , aa), a transmembrane domain ( , - , aa), and a short cytoplasmic domain. it consists of the s ( - aa) and s ( - , aa) domains, which are responsible for binding and fusion of the virus. the s domain is involved in a specific interaction with the cellular receptor and induction of neutralising antibodies ( , ) . only one serotype of pedv has been reported. the phylogenetic analysis of the s gene indicates that pedv could be genetically divided into two groups, these being the classical genotype (gi) and the field epidemic or pandemic genotype (gii). the early european cv strains (genbank accession number af ), vaccine strains, and strains adapted to cell cultures were classified to genotype , while genotype includes strains responsible for the epidemics in the usa and asia. strains identified in the usa have been ( , , , ) . all pedv pandemic strains circulating in china after were also clustered to genotype , and they were genetically separated from other global pedv strains and from earlier chinese strains ( ) . isolation of pedv from field samples is very difficult. the virus grows in vero cells (african green monkey kidney). however, its growth there requires the presence of trypsin-supplemented cell culture medium, because this protease plays an important role in cell entry and the release of pedv virions in vero cells. trypsin does this by cleaving the s protein into s and s subunits, and it enables efficient replication and spread of pedv in vitro ( , ) . pedv adapted to the vero cell line can be successfully propagated in other cell types. ped viruses cause cell death leading to lysis of infected cells, a change observable under the microscope as a cytopathic effect (cpe) which is characterised by cell membrane vacuolisation and syncytium formation ( , , ) . the ped virus can survive for variable periods outside the host, depending on the temperature and relative humidity. the virus is stable in temperatures ranging from °c to °c but loses its infectivity above °c ( ) . survival of the ped virus in different samples is presented in table . pedv is easily inactivated by heating to °c for min. at lower temperatures, ph is a factor in pedv inactivation. the resistance of ped virus in different ph values is given in table . pedv-inactivating disinfectants are oxidising agents, bleach, % phenolic compounds, % sodium hydroxide, formaldehyde and glutaraldehyde, % sodium carbonate, ionic and nonionic detergents, iodophors in % phosphoric acid, and lipid solvents such as chloroform ( , ) . direct transmission by the faecal-oral route may also occur through ingestion of virus-contaminated vomit. indirect transmission occurs through contaminated feed trucks, service vehicles, vehicles used for the movement of pigs, and people (pig owners and farm visitors). until the introduction of pedv to the usa, feed for animals was not considered to be a vector in the spread of the virus. recent studies have shown, however, that feed can be a potential vector for pedv transmission and contaminated feed can be a source of disease ( ) . it is widely acknowledged that the most important risk factors for spreading the disease are contaminated vehicles ( , ) . oral ingestion causes viral replication in the epithelial cells of the small intestinal villi and to a lesser extent in the colonic villi, which results in degeneration of enterocytes and shortening of the villi ( , ) . pedv can infect pigs of all ages, causing watery diarrhoea and vomiting with anorexia and dehydration, which is the major cause of death in young piglets. the clinical signs depend on the age of the pigs, immune status of the herds, and virulence of the strain ( , , ) . lesions are observed in the gastrointestinal tract and are characterised by a distended stomach filled with completely undigested milk dots and thin, transparent intestine walls ( ) . pedv infection is clinically indistinguishable from other forms of porcine gastroenteritis diseases such as those caused by tgev and pdcov, therefore pedv diagnosis cannot be made only on the basis of clinical signs and has to be confirmed by laboratory tests to make diagnosis final ( , ) . a variety of pedv detection methods are applied which include immunofluorescence (if) or immunohistochemistry (ihc) tests, in situ hybridisation, virus isolation, enzyme-linked immunosorbent assays (elisa), and various reverse transcription polymerase chain reaction (rt-pcr) techniques. samples which can be used in laboratory diagnosis are fresh faeces, oral fluids, small intestine tissue, and serum (to determinate the presence of antibodies). to detect pedv rna, rt-pcr can be used for diagnosis of acute outbreaks no longer than days after the onset of the disease. for surveillance and monitoring of ped, serological diagnosis is necessary. antibodies persist for more than one year in the serum of infected pigs ( , , ) . the regenerative ability of villi is instrumental in the recovery of the pig. regeneration speed depends on age of the animals; in adult and fattening pigs, villi are restored in three to four days, while in piglets the process is longer at six to seven days ( ) . the percentage of morbidity in the course of infection can climb to %. mortality is variable and also depends on the age of animals. in suckling piglets, mortality can attain %, in piglets older than days, it is less than %, and in adult and fattening pigs, it falls below %. there is no specific treatment other than symptomatic treatment of diarrhoea, however, most growing pigs recover without treatment within - days unless secondary infections occur. reinfection may occur when the immunity wanes ( , ) . ped was first observed in the united kingdom and belgium in early s ( ) . the disease caused mortality of about % in fatteners and adult pigs. suckling piglets were not affected and remained symptom-free even when the sows had watery diarrhoea for several days. at the beginning, the disease was incorrectly diagnosed as tge because the symptoms of these two diseases are almost identical. at a later stage, tge was excluded by laboratory diagnosis. in , new cases of the disease were described in the uk. this outbreak was different in that the virus affected pigs of all ages, including neonatal and suckling piglets, inflicting around % mortality. pedv was definitively identified for the first time in in belgium and was classified to the coronaviridae family, recorded as the cv prototype strain ( , ) . in the s and s, pedv was identified as the cause of severe epidemics in japan and south korea ( ) . in the same period in europe, outbreaks of ped appeared sporadically but the virus continued to spread and persisted in an endemic form in the pig population. outbreaks of ped were observed in the netherlands in - , in hungary in , and in the uk in . typical epidemic outbreaks of ped with high mortality in neonatal piglets were also identified in italy in [ ] [ ] and china in - ( , ) . the first symptoms of the disease in the usa were observed in april and were confirmed in the laboratory in may . the ped virus had escaped from biosecurity and control systems, spread very quickly to other areas, and infected hundreds of farms. by march , the presence of pedv had been confirmed in us states. pedv epidemics resulted in significant economic loss there, and the domestic pig industry lost almost % of its population (seven million pigs) ( , ) . the ped outbreak was characterised by watery diarrhoea, dehydration, and variable vomiting. all groups of animals were affected by the epidemics, but the highest mortality was % and occurred among suckling piglets ( , ) . the strains isolated in the usa were genetically related to the chinese pedv strains reported in . pedv was probably dispersed in the usa mainly through contaminated trucks, but other factors that assist the spread of the virus cannot be excluded ( ) . two strains of pedv have been identified in the usa: highly virulent non-indel (usa/kansas / ) and the milder variant s-indel (usa/oh / ) ( ) . both variants were classified to genotype . in experimental infections, s-indel strains showed lower pathogenicity and mortality (from % to %) than the non-indel strains, where the mortality rate was up to % ( , , ) . since the ped outbreak in the usa, detection of highly virulent strains has also been reported in canada, mexico, taiwan, south korea, japan, and ukraine. detection of s-indel strains has been reported in most european countries ( , , ) . ped is not notifiable in the european union and is not on the list of diseases reported to the world animal health organization (oie), therefore, it is not possible to accurately describe the occurrence of this disease in europe. in the last years, only a few countries have reported clinical cases of ped and/or seropositive animals ( ) . the ped outbreaks in europe were significantly different from epidemics in the usa or asia. after the epidemics in in the usa, ped cases were also confirmed in austria, belgium, france, germany, italy, the netherlands, portugal, and slovenia ( fig. ) ( , , ) . viruses identified in these european countries were very similar to usa/oh / strains. in germany ped emerged in , inflicting up to % mortality and typified by acute symptoms ( ) . in slovenia ped was confirmed in the laboratory in ( ) . at almost the same time, highly virulent strains were described in ukraine. these viruses were similar to usa/kansas / . so far, ukraine is the only european country where highly virulent strains have been identified. nevertheless, taking into account the occurrence of a virulent strain in ukraine and its rapid spread, it is likely that epidemics similar to those in the usa may also occur in europe ( , ) . considering the presence of pedv in countries neighbouring poland (germany, the czech republic, and ukraine) and the rapid spread of the virus, it is highly probable that pedv will also be introduced to poland. ped is a rapidly spreading global disease and causes large economic losses in pig production around the world. frequently appearing mutations cause variability in its viral genome and can change the pathogenicity of the new pedv variants. biosecurity is a central to the prevention or spread of pedv. applying the principles of biosecurity protocols, it is possible to reduce the risk of introducing the virus into swine herds. disinfection of vehicles entering farms is highly effective because contaminated faeces or vomit can be found on the wheels. in poland, the clinical symptoms of ped have been observed, but until no studies were conducted to confirm the presence of pedv. in - , the presence of the virus and/or of specific antibodies was confirmed on several farms in poland. samples of blood, faeces, slurry, and intestines were collected in herds in which the clinical symptoms of the disease had previously been observed. so far, virus isolation in vero cells has not been carried out in poland and nor has genetic material from positive samples been sequenced. it is foreseen that pedv might be transferred in the future to polish swine herds. the severity of ped which occurred in the usa has shown that this disease should be countered with timely preventive measures and early diagnosis. therefore, the role of the national veterinary research institute in puławy, poland, in the diagnosis of ped is of paramount importance in future pig production. the authors declare that there is no conflict of interests regarding the publication of this article. porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus pathogenesis comparison between the united states porcine epidemic diarrhoea virus prototype and s-indel-variant strains in conventional neonatal piglets isolation and characterization of porcine epidemic diarrhea viruses associated with the disease outbreak among swine in the united states scientific opinion on porcine epidemic diarrhea and emerging porcine 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coronaviruses. in: diseases of swine characterizing the rapid spread of porcine epidemic diarrhea virus (pedv) through an animal food manufacturing facility porcine epidemic diarrhea virus: a comprehensive review of molecular epidemiology, diagnosis and vaccines quercetin -rhamnoside reduces porcine epidemic diarrhea virus replication via independent pathway of viral induced reactive oxygen species investigation of three outbreaks of porcine epidemic diarrhea in germany in demonstrates age dependent differences in the development of humoral immune response a retrospective study detects a novel variant of porcine epidemic diarrhea virus in england in archived material from the year factors associated with farm-level infection of porcine epidemic diarrhea during the early phase of the epidemic in japan in discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus porcine epidemic diarrhea virus variants with high pathogenicity evolutionary insights into the ecology of coronaviruses distinct characteristics and complex evolution of pedv strains molecular characteristics of the spike gene of porcine epidemic diarrhoea virus strains in eastern china in key: cord- -ehr aaix authors: chang, jae c. title: acute respiratory distress syndrome as an organ phenotype of vascular microthrombotic disease: based on hemostatic theory and endothelial molecular pathogenesis date: - - journal: clin appl thromb hemost doi: . / sha: doc_id: cord_uid: ehr aaix acute respiratory distress syndrome (ards) is a life-threatening noncardiogenic circulatory disorder of the lungs associated with critical illnesses such as sepsis, trauma, and immune and collagen vascular disease. its mortality rate is marginally improved with the best supportive care. the demise occurs due to progressive pulmonary hypoxia and multi-organ dysfunction syndrome (mods) with severe inflammation. complement activation is a part of immune response against pathogen or insult in which membrane attack complex (mac) is formed and eliminates microbes. if complement regulatory protein such as endothelial cd is underexpressed, mac may also cause pulmonary vascular injury to the innocent bystander endothelial cell of host and provokes endotheliopathy that causes inflammation and pulmonary vascular microthrombosis, leading to ards. its pathogenesis is based on a novel “two-path unifying theory” of hemostasis and “two-activation theory of the endothelium” promoting molecular pathogenesis. endotheliopathy activates two independent molecular pathways: inflammatory and microthrombotic. the former triggers the release inflammatory cytokines and the latter promotes exocytosis of unusually large von willebrand factor multimers (ulvwf) and platelet activation. inflammatory pathway initiates inflammation, but microthrombotic pathway more seriously produces “microthrombi strings” composed of platelet-ulvwf complexes, which become anchored on the injured endothelial cells, and causes disseminated intravascular microthrombosis (dit). dit is a hemostatic disease due to lone activation of ulvwf path without activated tissue factor path. it leads to endotheliopathy-associated vascular microthrombotic disease (ea-vmtd), which orchestrates consumptive thrombocytopenia, microangiopathic hemolytic anemia, and mods. thrombotic thrombocytopenic purpura (ttp)-like syndrome is the hematologic phenotype of ea-vmtd. ards is one of organ phenotypes among mods associated with ttp-like syndrome. the most effective treatment of ards can be achieved by counteracting the activated microthrombotic pathway based on two novel hemostatic theories. acute respiratory distress syndrome (ards) is caused by severe pulmonary vascular dysfunction characterized by acute onset of dyspnea, tachycardia, hypoxemia associated with noncardiogenic pulmonary edema, and systemic inflammation. although the exact pathophysiologic mechanism causing pulmonary vascular dysfunction has not been determined yet, it is a circulatory dysfunction often associated with moderate thrombocytopenia [ ] [ ] [ ] and multi-organ dysfunction syndrome (mods). [ ] [ ] [ ] because its pathogenetic mechanism is not clearly recognized, no effective therapeutic agent targeting the underlying pathologic disease has been procured to date. ventilator support, fluid and electrolyte balances, and cardiopulmonary monitoring with the best supportive care have marginally improved the outcome of ards in several decades. mortality rate is still very high. it increases with disease severity. in a multicenter, international, prospective cohort study of patients with ards, unadjusted hospital mortality was reported to be % among those with mild ards, % for those with moderate disease, and % for patients with severe ards. recently, two proposed hemostatic mechanisms have opened the door in the understanding of ards from molecular pathogenesis associated with endotheliopathy that promotes inflammation and coagulation disorder in sepsis and other critical illnesses [ ] [ ] [ ] [ ] ; one is "two-activation theory of the endothelium" in which endothelial pathogenesis activates inflammatory pathway and microthrombotic pathway and the other is a novel "two-path unifying theory" of hemostasis in which hemostasis initiates thrombogenesis and promotes microthrombogenesis, leading to vascular microthrombotic disease (vmtd). , , , these two theories are congruous each other since the endothelium contributes to initial hemostasis and triggers molecular mechanism for thrombogenesis. in endotheliopathy, the pathologic nature of inflammation promoting inflammatory response is recognized and the character of "microthrombi" leading to multiple hematologic phenotypes is identified. in addition, the true mechanism of in vivo hemostasis in vascular injury and three different thrombogenetic mechanisms within hemostasis are uncovered. , through the recognition of endothelial molecular pathogenesis, enough evidences have been accumulated that ards is one of the phenotypes of mods occurring as a result of disseminated intravascular microthrombosis (dit), which is the underlying pathology contributing to endotheliopathy-associated vmtd (ea-vmtd). , [ ] [ ] [ ] the objective of this article is to analyze the clinical, pathological, and hematopathological features of ards and to account for involved pathophysiological mechanisms associated with endothelial dysfunction based on two hemostatic theories. in the end, this author will look into potential therapeutic option for the treatment of ards according to "theorybased medicine" instead of "evidence-based medicine" since clinical trials for ards have completely failed to find an effective therapeutic regimen. the most common underlying condition in ards is severe infection (eg, sepsis/septic shock with or without severe pneumonia) due to various microbic pathogens, which include bacteria, viruses, fungi, rickettsia, and parasites. ards also occurs in association with trauma to the chest/lungs and head/brain, , complications of surgery, pregnancy and transplant, [ ] [ ] [ ] [ ] [ ] certain drug, toxin, chemicals and venom exposure, and thrombotic thrombocytopenic purpura (ttp)-like syndrome. , [ ] [ ] [ ] in addition, it also has developed in association with disseminated intravascular coagulation (dic). [ ] [ ] [ ] some clinicians have interpreted dic was the cause of ards, but others proposed it was the result of complication of ards. regardless, enough evidences have been presented that ards is a clinical disorder of pathologic hemostasis associated with activated coagulation system such as dic. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] however, this author has placed quotation marks on "dic" because recent reinterpretation has identified the current concept of "dic" was ill-founded because it was based on the hemostatic mechanism of activated tissue factor (tf) path, , , , , , which will be discussed briefly later in this article. nonetheless, ards is one of the major organ phenotypic disorders among mods contributing to the death associated with microthrombosis in critically ill patients due to diseases such as sepsis, trauma, and immune disorders. as shown in table , although ards often occurs in association with a variety of sepsis, it can be preceded by pneumonia as seen in severe respiratory distress syndrome (sars) due to sars-cov and middle east respiratory syndrome (mers) due to mers-cov as well as bacterial, fungal, and parasitic pneumonia, especially pneumococcal in particular. the clinical feature of developing pneumonia before sepsis suggests organotropism plays an important role in certain pathogen as shown by sars virus possessing specific affinity to the lungs. , sometimes ards occurs following blood transfusion, which is called transfusion-related acute lung injury (trali) that is characterized by acute noncardiogenic circulatory disorder of the lungs following blood product transfusions. , sepsisassociated ards, notwithstanding the absence of pneumonia, not uncommonly develops with other organ dysfunction such as encephalopathy, hepatic failure, acute renal failure, and acute necrotizing pancreatitis. , this multi-organ involvement suggests ards may not be primary disease but is likely a part of ongoing systemic pathogenetic mechanism due to infection or other critical illnesses as illustrated in table . , now, the underlying physiologic alteration of mods in sepsis and other critical illnesses is identified as circulatory dysfunction occurring as a result of ea-vmtd. [ ] [ ] [ ] this is an extremely important concept in the understanding for the pathogenesis of mods as well as ards because we now know that the culprit of mods is dit, , [ ] [ ] [ ] [ ] [ ] which pathogenesis based on "two-activation theory of the endothelium" as shown in figure . , clinical and pathological features. the clinical features of ards are characterized by ( ) acute onset of noncardiogenic respiratory distress, ( ) bilateral pulmonary infiltrates, and ( ) evidence of diffuse circulatory obstruction of pulmonary vasculature. in addition to acute respiratory distress, hematologic features of ards include thrombocytopenia, , , - mods, , - , - dit, , , , "dic," , , [ ] [ ] [ ] and ttp-like syndrome, [ ] [ ] [ ] as presented in tables and . these manifestations seemed to be consistent with a hemostatic disorder, which is now recognized as ea-vmtd. , , until recently, it was debated whether ards was the cause of thrombocytopenia, inflammation, mods, and "dic," or was rather the result of another pathological condition producing those hematologic phenotypes. in later discussion, this dilemma will be further explored once the pathophysiological mechanism of ards is established. the pathological features of ards are characterized by ( ) diffuse alveolar damage associated with injury to alveolar lining and endothelial cells (ecs), ( ) exudative pulmonary edema, and ( ) hyaline membrane formation. [ ] [ ] [ ] these pathologic changes are very similar, if not identical, in each ards caused by different pathogens and insults, including pneumonia-initiated sars and mers. , one unique abnormality is hyaline membrane formation/deposits. this pathologic feature appears to be similar to that of vmtd associated with ttp and ttp-like syndrome, which microthrombi are characterized by hyaline thrombi composed of unusually large von willebrand factor multimers (ulvwf) and platelets. even though ards develops in association with divergent etiologies from sepsis to envenomation, its pathologic and clinical features are remarkably similar among different underlying diseases. [ ] [ ] [ ] diffuse alveolar damage was the histologic changes in most patients with ards and its progression included phases of exudative, proliferative, and fibrotic changes that correlated with the time rather than its specific causes. these findings are consistent with the hypothesis that pathogenesis of ards is not due to multifactorial processes primarily involving the lungs, but is the result of one pathophysiologic mechanism affecting the lungs and multiorgans. consumptive thrombocytopenia in critically ill patients. as in other critical illnesses, thrombocytopenia commonly occurs in ards during the course of the disease. - even after the known causes of thrombocytopenia such as heparin-induced thrombocytopenia, transfusion and drug-related thrombocytopenia, bone marrow suppression, and other identifiable thrombocytopenia are excluded, the mechanism of undetermined thrombocytopenia cannot be clearly accounted for in most of the cases. thus, this has been designated as thrombocytopenia in critically ill patients (tcip). , , recently, in critical illnesses such as sepsis and trauma, thrombocytopenia is suspected to be associated with endotheliopathy that initiates microthrombogenesis and forms microthrombi. the platelet consumption occurs when ulvwf released from endotheliopathy recruit platelets to form platelet-ulvwf complexes, , , which become microthrombi strings and platelets are consumed. this concept of tcip is direct and unequivocal evidence that endotheliopathy promotes in vivo hemostasis. thrombocytopenia in critically ill patient usually presents with mild to moderately decreased platelet count, and bleeding has not been a significant issue in the care of critically ill patients. according to hemostatic principles (table ) , blood vessel damage limited to ecs in endotheliopathy figure . endothelial molecular pathogenesis of ards and mods in critically ill patients. based on "two-activation theory of the endothelium. reproduced and modified from chang. endothelial molecular pathogenesis of ards as one organ phenotype among various mods is succinctly illustrated. the underlying pathologic nature of ards is a hemostatic disease due to endotheliopathy that promotes activation of two molecular pathways. one is inflammatory pathway, which releases cytokines and provokes inflammation, including fever, malaise, and myalgia. the other is microthrombotic pathway, which causes exocytosis of ulvwf and platelet activation and triggers much more deadly dit via microthrombogenesis, leading to eavmtd/dit. disseminated intravascular thrombosis orchestrates consumptive thrombocytopenia, maha, mods, and ttp-like syndrome. ards indicates acute respiratory distress syndrome; dit, disseminated intravascular thrombosis; ea-vmtd, endotheliopathy-associated vascular microthrombotic disease; ecs, endothelial cells; hc, hepatic coagulopathy; maha/amaha, microangiopathic hemolytic anemia/atypical microangiopathic hemolytic anemia; mods: multi-organ dysfunction syndrome; mof, multi-organ failure; tma, thrombotic microangiopathy; sirs, systemic inflammatory response syndrome; ttp, thrombotic thrombocytopenic purpura; ulvwf, unusually large von willebrand factor multimers activates ulvwf path, but tf path is not activated if subendothelial tissue (set)/extravascular tissue (evt) illustrated in figure is not compromised. , , as the hemostatic nature of microthrombosis associated with critical illnesses has not been recognized to date, tcip has been benignly neglected although bone et al in late s had observed thrombocytopenia was a significant component when he described thrombocytopenia in ards. now, tcip is found to be consumptive thrombocytopenia caused by microthrombogenesis that leads to ea-vmtd/dit. more recently, the significant role of the platelet has been recognized in the care of patients with critical illnesses and ards. the degree of thrombocytopenia in sepsis was associated with increased severity and higher mortality , and thrombocytopenia was an increased risk and predictive for patient mortality in ards. thrombotic thrombocytopenic purpura-like syndrome. when mild to moderate thrombocytopenia was present in ards, this author often found masked microangiopathic hemolytic anemia (maha) in hematologic evaluation. , microangiopathic hemolytic anemia was less prominent in ards with fewer schistiocytes than that in acquired immune ttp, and also with mild to moderate anemia. if, however, the evidence of hemolysis were evident with reticulocytosis, hypohaptoglobinemia, increased lactic acid dehydrogenase, and indirect hyperbilirubinemia, , , it was called atypical maha, which is more common in ttp-like syndrome (ie, ea-vmtd). in the literature, case reports described the association between ards and ttp-like syndrome. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in this author's experience, ards with coexisting ttp-like syndrome responded dramatically when therapeutic plasma exchange (tpe) was employed in very early stage of ards. , the reasons why the diagnosis of ttp-like syndrome has been masked in ards were due to inconspicuous schistocytosis and unsuspected diagnosis as well as major attention for the patient care directed to respiratory distress in real-time clinical practice. more likely, it was also due to diametrically different pathogeneses between ttp and ttp-like syndrome, in which clear distinction has not been recognized until recently. it was also caused by the fact that ards has never been considered to be a hemostatic disease, and further ttplike syndrome was unknown to be caused by endotheliopathy. table . three essentials in normal hemostasis. ( ) hemostatic principles ( ) hemostasis can be activated only by vascular injury. ( ) hemostasis must be activated through ulvwf path and/or tf path. ( ) hemostasis is the same process in both hemorrhage and thrombosis. ( ) hemostasis is the same process in both arterial thrombosis and venous thrombosis. in clinical medicine, all patients with ards should be evaluated with a high index of suspicion to look for atypical maha as well as thrombocytopenia. unexplained thrombocytopenia and firm evidence of hemolysis even with minimal or no schistocytes in repeated blood film examination may still be consistent with ttp-like syndrome. multi-organ dysfunction syndromes. in late s, vesconi et al suspected that ards was caused by pulmonary vascular microthrombosis following very elegant investigation demonstrating pulmonary microvascular occlusive lesions in balloon occlusive pulmonary angiography in patients with severe adult respiratory distress. in the study, multiple pulmonary artery filling defects were detected, which findings were interpreted to be consistent with vascular microthrombosis. however, the concept of microthrombosis was not defined yet in coagulation community. nonetheless, the thesis of microvascular thrombosis or vascular microthrombosis can explain the compromised vascular circulatory function of the lungs and occlusive lesions in pulmonary angiography better than any of other propositions. proposed theories for the pathogenesis of ards have included pulmonary vascular endothelial injury leading to endothelial dysfunction due to inflammatory cytokines or activated immune cells, upregulation of adhesion molecules such as soluble vascular adhesion molecule and e-selectin, underexpression of vascular endothelial cadherins, , interactions between neutrophils and cytokines promoting transendothelial migration of cytokine-primed neutrophils, and neutrophil extracellular traps (nets) provoking coagulation and microcirculatory failure. [ ] [ ] [ ] however, these theories could not define how and what molecular changes occur to lead to ards and produce increased capillary permeability that was considered to be the hallmark of ards. more importantly, these theories cannot answer why inflammation in ards is frequently associated with simultaneous hematologic syndromes and mods. , , the proposition of microthrombosis was a very important thesis because for the first time the potential hemostatic nature of ards was suggested. vascular microthrombosis or microvascular thrombosis has been well recognized as the underlying disease of ttp and more recently as that of ttplike syndrome, which occurs in ea-vmtd as well as "dic." although the molecular mechanism of ards has remained elusive, later khadaroo and marshall correctly understood that vascular microthrombosis could contribute its clinical expression not only as ards in the lungs but also as mods in other vital organs. in addition to common association of ards and mods, the similar, if not the same, pulmonary pathologic changes from different pathogens and noninfectious critical illnesses support the mechanism that each of clinical figure . schematic illustration of cross section of blood vessel histology and hemostatic components. the blood vessel wall is the site of hemostasis (coagulation) to produce hemostatic plug in vascular injury to stop hemorrhage from external vascular injury. it is also the site of hemostasis (thrombogenesis) to produce intravascular blood clots in intravascular injury to cause thrombosis. its histologic components can be divided into the endothelium, tunica intima, tunica media, and tunica externa, and each component has different function contributing to molecular hemostasis. as shown in the illustration, endothelial injury triggers exocytosis of ulvwf from ecs, set injury promotes the release of stf from tunica intima, tunica media, or tunica externa, and evt injury induces the release of etf from the outside of blood vessel wall. these depths of blood vessel injury contribute to the genesis of different thrombotic disorders such as microthrombosis, macrothrombosis, and fibrin clot disease/hematoma. this concept is important in the understanding of endotheliopathy leading to ards, which leads to lone activation of ulvwf to produce microthrombi strings in ecs. ards indicates acute respiratory distress syndrome; ecs, endothelial cells; etf, extravascular tf; evt, extravascular tissue; rbc, red blood cell; set, subendothelial tissue; stf, subendothelial tf; tf, tissue factor; ulvwf, unusually large von willebrand factor multimers. phenotypes of mods occurs as a result of the same systemic disease of vmtd, as displayed in figure . for example, similar to the organ phenotype of hemolytic uremic syndrme (hus) in the kidneys, the organ phenotype of ards in the lungs is caused by microvascular thrombosis, which is also characteristic of ttp, ttp-like syndrome, "dic," and thrombotic microangiopathy. in this context, it can be concluded that ards, hus, and every organ syndrome in mods are also the manifestations of ea-vmtd/dit, as illustrated in figure and further elaborated in figure . indeed, ards is just one phenotype among mods associated with increased microvascular permeability due to vmtd involving multiorgans. , , , , this is also true for the current biorgan designation syndromes such as hepatorenal syndrome, cardiopulmonary syndrome, pulmonary-renal syndrome, hepatic encephalopathy, cardiorenal syndrome, and others. it should be emphasized that ards is not the cause of mods, but all the organ phenotypes of mods as well as ards are collateral syndromes provoked by vmtd. next important question is how clinical expression of vmtd can be so variable in the development of mods among every individual patient as shown in figure . this will be discussed in the heading of tropism and endothelial heterogeneity within pathophysiological mechanisms involved in ards. systemic inflammatory response syndrome. the american college of chest physicians and the society of critical care medicine introduced definitions for systemic inflammatory response syndrome (sirs), sepsis, severe sepsis, septic shock, and mods in early s. the idea proposing the term sirs was to recognize it as a clinical response to a nonspecific insult of either infectious or noninfectious origin. however, sirs couldn't be defined as a disease entity and thus has remained to be just as a complex clinical syndrome associated with sepsis and noninfectious critical illnesses because the pathogenesis of sirs has not been clearly established. it generally has been considered to be expression of self-defense mechanism against overwhelming pathologic insults. over the past decades, it has become evident that endotheliopathy plays a major role in sepsis with inflammation and coagulation. in generalized endotheliopathy, sirs commonly occurs in association with ards, which is manifested by combined severe inflammation via cytokine release and microthrombosis via microthrombogenesis that often leads to mods. [ ] [ ] [ ] [ ] [ ] in severe ards, inflammation coexists with other the pathogenesis of mods seen with ards is summarized. any organ can be involved by vmtd in association with/without ards. however, mods is much more common in vital organs, especially in the lungs with ards, the brain with cnsd, and the kidneys with acute renal failure. please note that ards has shown to be associated with the every illustrated organ syndrome. aai indicates acute adrenal insufficiency; alf, acute liver failure; anp, acute necrotizing pancreatitis, ards, acute respiratory distress syndrome; arf, acute renal failure; cnsd, central nervous system dysfunction; dit, disseminated intravascular microthrombosis; ea-vmdt, endotheliopathy-associated vascular microthrombotic disease; fhf, fulminant hepatic failure; hcps, hantavirus cardiopulmonary syndrome; he, hepatic encephalopathy; hps, hantavirus pulmonary syndrome; hrs, hepatorenal syndrome; hus, hemolytic uremic syndrome; mods, multi-organ dysfunction syndrome; nomi, nonocclusive mesenteric ischemia; pdis, peripheral digit ischemic syndrome; rml, rhabdomyolysis; spg, symmetrical peripheral gangrene; wfs, waterhouse-friderichsen syndrome. organ phenotypes such as encephalopathy, acute renal failure, myocardial infarction, pancreatitis, fulminant hepatic failure, adrenal insufficiency, and others. , , thus, sirs can be best defined as combined syndrome of severe inflammatory response from activated inflammatory pathway and organ dysfunction from activated microthrombotic pathway as a result of generalized systemic endotheliopathy. however, we have to understand that inflammation and microthrombosis in endotheliopathy are two separate processes, although their crosstalk mechanism has been popularized. their molecular pathogeneses are independent, which is illustrated in two-activation theory of the endothelium ( figure ). this is the very reason why clinical trials based on antiinflammatory regimens have had no impact on coagulation system and has failed to improve the outcome as demonstrated in the management of sepsis-associated coagulopathy. in clinical practice, inflammation alone is not the major factor causing poor outcome of the patient, but clinical severity of mods caused by vmtd is the main culprit for the demise in severe sepsis. disseminated intravascular coagulation. "disseminated intravascular coagulation" has occurred with ards with or without sepsis. , , [ ] [ ] [ ] it has been considered be the most serious coagulopathy not only in sepsis but also in other human diseases, which is estimated to occur in about % to % of patients with sepsis. a study in japan found that a diagnosis of dic complicated about . % of admissions to university hospitals. according to nih national heart, lung, and blood institute, dic is a condition in which blood clots form throughout the body's small blood vessels. these blood clots can reduce or block blood flow through the blood vessels, which can damage the body's organs. generally clinicians and pathologists defined dic as a widespread hypercoagulable state that can lead to both microvascular and macrovascular clotting and compromise blood flow, ultimately resulting in multiple organ dysfunction and mods. the truth is the coagulopathy of "dic" couldn't be precisely defined because the concept of microthrombosis and its thrombogenesis had not been identified yet. it is because the physiological mechanism of in vivo hemostasis has been incompletely understood. for example, what is the role of von willebrand factor (vwf) in the thrombogenesis and coagulation? also, what is the role of the platelet in coagulation cascade? what is the difference between fibrin clots of activated tf path and thrombosis of deep vein thrombosis (dvt)? what is the difference between microthrombi and macrothrombus? why is microthrombosis disseminated, but is dvt localized? what is coagulation? and how is it different from thrombogenesis? all of these questions seem to be philosophical ones but are urgently needed practical questions in the patient care. there is no simple answer on the difference between dvt and dic. the former responds to anticoagulation, but the latter does not. why is it? current dilemma is that "dic" is found to be incorrect in its character and also in accepted contemporary pathogenetic mechanism according to this author's interpretation. [ ] [ ] [ ] [ ] [ ] the reinterpretation of "dic" based on "two-path unifying theory" of hemostasis and the mechanism of thrombogenesis clearly support that it occurs as a result of activated ulvwf path. [ ] [ ] [ ] [ ] [ ] the credibility of "dic," which coagulopathy has been blamed to microthrombi composed of platelet-ulvwf complexes via microthrombogenesis by some and to fibrin clots made of fibrin, platelet, and coagulation factors through uncontrolled activation of tf-initiated path by others, is seriously undermined because of the irreconcilable conflict between microthrombi and fibrin clots. [ ] [ ] [ ] [ ] [ ] in in vivo hemostatic process, microthrombi and fibrin clots can be easily differentiated as illustrated in figure a and b. the true character of blood clots in "dic" is the same microthrombi occurring in vmtd as seen in ttp and ttp-like syndrome. also, the pathophysiological mechanism of "dic" is not tf/fviia complex-activated coagulation cascade but instead is partial hemostasis due to lone activation of ulvwf path. since "dic" (ie, microthrombosis) occurs as the result of endotheliopathy alone without the damage of set/evt, , , tf path is not activated. this author has derived two theories of "two-activation theory of the endothelium" and "two-path unifying theory" of hemostasis from the analysis and interpretation of pathological, clinical, laboratory, and molecular characteristics between "dic" and "ttp-like syndrome" and elaborated these hypotheses in previous publications. [ ] [ ] [ ] [ ] [ ] therefore, i shall not repeat them again. in short, it can be affirmed that "dic" is exactly the same to ea-vmtd/dit, which hematologic phenotype is ttp-like syndrome. in summing up, the concept of "dic" has been built on the following faulty pathophysiologic mechanism of hemostasis. comments are followed after each statement. "dic" is uncontrolled "tf path" initiated coagulation disorder occurring in sepsis and other critical illnesses. (instead, it is "ulvwf path" initiated microthrombotic disorder.) "dic" is triggered by inflammation, leading to pathologic "fibrin clots" through "crosstalk" between inflammation and coagulation. (instead, it is triggered by microthrombogenesis, leading to pathologic "microthrombi strings" and "no crosstalk" is involved.) "dic" is caused by microvascular thrombosis initiated by "tf/fviia complex". (instead, it is caused by vascular microthrombosis initiated by "platelet-ulvwf complex.") "dic" "consumes coagulation factors and platelets in clotting process." (instead, it is the result of "released ulvwf from injured ecs that consume platelets in formation of microthrombi strings.") acute "dic" is characterized by thrombocytopenia, maha, mods, and severe hemorrhagic syndrome figure . a, normal hemostasis based on "two-path unifying theory." reproduced and updated with permission from chang. in normal hemostasis, two different thrombotic paths, microthrombotic (ulvwf) and fibrinogenetic (tf), are involved in normal hemostasis, but later the paths must unify to conclude normal hemostasis with passive role of nets; it stops the bleeding in external bodily injury and produces the thrombosis in intravascular injury. however, in the different level (depth) of intravascular injury, thrombogenesis takes two different paths. if the level of intravascular injury is confined to the endothelium, lone ulvwf path becomes activated and causes microthrombosis (ie, ea-vmtd) because tf path is not activated. on the other hand, if the level of intravascular injury extends from the endothelium to set/evt, tf path becomes also activated and causes macrothrombosis (eg, dvt). in another theoretical situation, if only set/evt is injured, available tf is supposed to activate lone tf path. however, in pathologic hemostasis, aberrant tf activation occurs and produces fibrin clots (ie, true dic) in apl due to tf expression in intravascular space from leukemic promyelocytes. acute promyelocytic leukemia causes consumption coagulopathy due to lone activation of tf path. this logic is based on "two-path unifying theory." please note different thrombotic disorders via microthrombogenesis, fibrinogenesis, macrothrombogenesis) in the figure, which are annotated in bold face. each pathogenesis occurs when ulvwf path, tf path, or combined paths are activated depending upon the levels of damage in intravascular injury (endothelium and set/evt). the characters of microthrombi, fibrin clots, and macrothrombus from different paths are very different and produce distinctly different clinical thrombotic disorders. b, three paths in thrombogenesis based on "two-path unifying theory." reproduced and updated with permission from chang. traditionally accepted hemostasis has been based on the concept of primary hemostasis in a local vascular injury followed by secondary hemostasis forming fibrin clots. however, this concept cannot explain microthrombi and thrombus formation. therefore, novel "two-path unifying theory" of hemostasis was derived from the vascular physiologic logic of hemostasis based on hemostatic principles and essential components participating in hemostasis and known works of many dedicated coagulation scientists. please note that there are different thrombogenetic paths in "two-path unifying theory" (macrothrombogenesis, microthrombogenesis, and fibrinogenesis) as annotated in bold face. each thrombogenetic path occurs when ulvwf path, tf path, and/or combined paths are utilized depending upon the vascular levels of damage in intravascular injury, which include the endothelium, set, and evt. the characters of the thrombus/blood clot from different paths are unique and produce distinctly different clinical thrombotic disorders. the pathogenesis ards is via microthrombogenesis due to lone activation of ulvwf path, which promotes microthrombi strings made of platelet-ulvwf complexes in pulmonary vasculatures. apl indicates acute promyelocytic leukemia; dic, disseminated intravascular coagulation; dvt, deep vein thrombosis; ea-vmtd, endotheliopathy-associated vascular microthrombotic disease; evt, extravascular tissue; set, subendothelial tissue; tf, tissue factor; ulvwf, unusually large von willebrand factor multimers. associated with "consumption coagulopathy" with depletion of fviii and fv. (instead, acute "dic" is characterized by "hepatic coagulopathy" with markedly increased fviii, markedly decreased fvii, and decreased fii, fv, fix and fx.) chronic "dic" is characterized by thrombocytopenia, maha, and mods without coagulopathy. (yes, the statement is true, but then it is the same picture to "ttp-like syndrome.") "dic", ttp, hus, ttp-like syndrome, and thrombotic microangiopathy are similar but "different diseases." [ ] [ ] [ ] [ ] [ ] [ ] (instead, all of them are the same disease called "ttplike syndrome" except ttp [ga-vmtd and aa-vmtd]. ) "dic" did not respond to any therapeutic agent utilized in clinical trials. but the reason is unexplained. (yes, the statement is true. the reason was the clinical trials were designed based on incorrect pathogenetic mechanism. but it is expected to respond to antimicrothrombotic therapy.) on the other hand, ea-vmtd/dit occurs due to microthrombogenesis as a result of lone activation of ulvwf path of hemostasis. from these statements, we can conclude as follows: "dic" is incorrect in its concept but is consistent with ea-vmtd/dit. , - chronic "dic" is incorrect term, which should be ea-vmtd/dit without hepatic coagulopathy. acute "dic" is incorrect term but is consistent with ea-vmtd/dit with hepatic coagulopathy. traditionally, dic has included ( ) "dic" that is associated with sepsis, trauma, and other critical illnesses and ( ) true dic that occurs in acute promyelocytic leukemia (apl) and rare cases of certain snake venom bite. the former is microthrombotic disease due to microthrombi strings (ttp-like syndrome) and the latter is hemorrhagic disorder due to fibrin clots (fibrin clot disease). since "dic" has been reappraised as ttp-like syndrome (ie, ea-vmtd/dit), once we move "dic" to the column of dit, the leftover is true dic that occurs in apl in which fibrin clots are formed by fibrinogenesis via extrinsic coagulation cascade from activated aberrant tf path. , finally, sepsis-associated coagulopathy (ie, microthrombopathy) seen in ards can be readily understood as ea-vmtd/ dit, which clinical phenotype is ttp-like syndrome with or without hepatic coagulopathy. on the other hand, aplassociated coagulopathy via fibrinogenesis can be understood as true dic (disseminated fibrin clot disease). this brief note on dic and "dic" seems to be a very complex conceptual issue at this time for readers, but the comprehension would become simple and clear once two theories (figures and a and b) are understood with the help of figure . it is no wonder why we could not unmask the true identity of "dic" term used more than years to date. succinctly speaking, ards is an organ phenotype of hemostatic disease occurring as a result of generalized endotheliopathy (ie, ea-vmtd/dit), leading to lone activation of ulvwf path that promotes microthrombotic pathway (ie, microthrombogenesis) and orchestrates consumptive thrombocytopenia, maha, mods, and ttp-like syndrome. , , generalized endotheliopathy also activates inflammatory pathway independent of microthrombotic pathway. the pathophysiological mechanisms involved in ards can be summarized as follows. complement activation in ards has been well recognized more than decades. [ ] [ ] [ ] however, its relationship between ards and complement activation has not been explored even though the role of c b- was suspected to contribute to its pathogenesis and c a in highly pathogenic viral infections was also implicated in acute lung injury. the activation of complement system is one of the key events in defense mechanism against sepsis. its protective function for host rapidly identifies and eliminates invading pathogen. opsonization of foreign surfaces by covalently attached c b fulfills major functions: cell clearance by phagocytosis, amplification of complement activation by the formation of a surface-bound c convertase, and assembly of c convertases. cleavage of c induces the formation of a multiprotein pore complex c b- (ie, membrane attack complex [mac]), which leads to cell lysis. even though its major role is protective function for host through innate immune defense, complement activation could promote destructive action to innocent bystander of the endothelium of the host, leading to endotheliopathy and neutrophil extracellular traps (netosis), which may impact the course of sepsis and other critical illnesses. membrane attack complex exerts deleterious effects to host's ecs unless cd glycoprotein is adequately expressed in ecs and protects them by inhibiting c polymerization from mac. , if cd is downregulated due to either gene mutation or acquired disease, perhaps activated complement could more readily exert destructive effect to the host's ecs causing endotheliopathy in critical illnesses. when mac attacks the membrane of ecs, channel (transmembrane pores) formation develops on the endothelial membrane and triggers endotheliopathy. [ ] [ ] [ ] [ ] considering the role of the complement in ards as well as in sepsis, endothelial dysfunction via activation of complement cascade is suspected to be the major component contributing to pathologic hemostasis of ards. the endotheliopathy in ards activates major molecular mechanisms; one is severe inflammation caused by inflammatory cytokines released from the endothelium and the other is hypoxic organ dysfunction caused by partial hemostasis via microthrombogenesis as illustrated in figure b . endothelial dysfunction has long been known to be the key modulator in the pathogenesis of ards as well as sepsis and critical illnesses. , [ ] [ ] [ ] [ ] the markers such as various cytokines and coagulation participants indicating endothelial damage were significantly altered in patients with critical illnesses compared with controls, which included vwf, fviii and endothelial procoagulants. recently, in view of the role of endotheliopathy and concept of vmtd based on novel in vivo "two-path unifying theory" of hemostasis, ards pathogenesis has been assured to be the result of pathologic hemostasis. [ ] [ ] [ ] , , in ards, heterogeneous expression of cell adhesion molecules by ecs was also noted in human pulmonary vasculatures. although e-selectin and vascular cell adhesion molecule were not expressed on ecs of normal lungs, immunochemical studies showed strong expression of both molecules on the larger vessels of the lungs supporting induction or upregulation in ards. , perhaps adhesion molecules could play a secondary role through endothelial heterogeneity and netosis in the phenotypes of ards-associated mods. it is now confirmed that ards is not the primary disease causing various organ dysfunction syndromes but is the secondary syndrome due to one of hypoxic organ dysfunction resulting from microthrombosis caused by endotheliopathy just like other mods as illustrated in figure . this concept of mods promoted by one pathogenetic mechanism (ie, microthrombogenesis) provoked by generalized endotheliopathy bespeaks of the following important implications in the understanding of ards and mods: acute respiratory distress syndrome is not the primary disease but is secondary clinical syndrome associated with one of different underlying causes (eg, sepsis, trauma, complication of pregnancy, surgery and transplant, cancer, drug/toxin, autoimmune disease, and others). both ards and other mods occur as a result of the same underlying pathophysiological mechanism, which is now identified to be microthrombogenesis due to generalized endotheliopathy, leading to vmtd. both ards and other mods are the phenotypes of ea-vmtd/dit. both ards and other mods would respond to the same treatment based on the same pathophysiological mechanism. certainly, the conceptual relationship between ards and mods guides us to the better understanding of endothelial molecular pathogenesis because the endothelium is distributed to the entire organ system and tissue of human body and protects from internal disease and external bodily injury through hemostasis and circulatory homeostasis. in endothelial pathogenesis of vmtd, the organ phenotype expression is variable among different hosts by the same pathogen or toxin as well as different pathogens or toxins, which variable expression in turn produces unusual exotic manifestations of mods. these phenotypes are likely to develop due to two main endowed biological mechanisms: endothelial heterogeneity of host - and organotropism of pathogen or toxin. [ ] [ ] [ ] [ ] variable clinical organ phenotypic syndromes occur as seen in the same type of the pathogen. examples are hanta virus, causing cardiopulmonary syndrome in the heart and lungs, shiga toxin-producing escherichia coli, presenting with encephalopathy and hus in the brain and kidneys, and neisseria meningitides, inciting waterhouse-friderichsen syndrome and meningitis in the adrenals and meninges. of course, a same organ phenotype can occur due to different types of pathogen. one of the interesting observations in ards is the common occurrence of combined syndrome of ards and acute necrotizing pancreatitis. , [ ] [ ] [ ] the character of this biorgan attraction of vmtd in a particular patient is similar as seen in combined syndromes of hepatic encephalopathy, cardiopulmonary syndrome, hepatorenal syndrome, pulmonary-renal syndrome, and others. the circulatory dysfunction and pathologic findings of diffuse alveolar damage in ards and necrotizing pancreatic damage in acute pancreatitis certainly support that biorgan syndromes are caused by the same pathogenetic mechanism associated with vascular microthrombosis. endothelial heterogeneity and/or tropism select the organ localization of microthrombi, but vascular microthrombosis inflicts physical damage to the organs. in clinical practice, oversimplified designation of organ phenotypes such as encephalopathy and rhabdomyolysis as well as ards might have interfered detecting the underlying etiology and mechanism of multi-organ syndromes as well as vmtd. some authors have claimed one organ phenotypic syndrome such as ards has caused several other organ dysfunctions, including pancreatitis, encephalopathy, renal failure, or hepatic failure. however, it should be understood that ards and additional organ syndromes begin with an equal footing in systemic vmtd, but the severity of selective organ damage is determined by the localization through selectivity of endothelial heterogeneity and tropism. since ards is not the primary disease, the term extrapulmonary manifestations or extrapulmonary phenotypes of ards are misrepresentation. it is this author's opinion that this conceptual misunderstanding has contributed to the delay in recognizing the pathophysiological mechanisms of ards as well as that of other mods such as hus, fulminant hepatic failure, acute pancreatitis, biorgan syndromes, and others. although ards was suspected to be associated with pulmonary vascular microthrombosis, it has taken several decades to recognize microthrombosis is a distinctly different disease from macrothrombosis seen in dvt and pulmonary embolism (pe). even though ards is different from dvt and pe, clinicians still might equate the character of microthrombosis in ards to that of dvt because we have known only one chang mechanism for thrombosis, which is the "blood clot" due to activated tf/fviia path. it is about time we accept the microthrombosis of ards is the product of different hemostasis from macrothrombosis of dvt or pe. this distinction certainly support new concept of mods, including encephalopathy, hus, acute necrotizing pancreatitis, diffuse myocardial ischemia, fulminant hepatic failure from dvt and pe. multiorgan dysfunction syndrome is caused by microthrombosis the same as in ards as a result of microthrombogenesis. , the term of microthrombogenesis is defined in previously mentioned two hemostatic theories. both theories are congruent to each other, although the "two-activation theory of the endothelium" represents endothelial molecular pathogenesis following exocytosis of ulvwf in endotheliopathy ( figure ) and the "two-path unifying theory" elaborates in vivo hemostatic process following intravascular injury via the release of ulvwf ( figure b ). in essence, microthrombogenesis in endothelial molecular pathogenesis and in vivo hemostasis is identical, but the former is the expanded version of ulvwf path illustrating how vmtd orchestrates clinical and pathological phenotypes via endotheliopathy. hemostasis based on "two-path unifying theory". both ttp and ttp-like syndrome are characterized by dit involving the vital organs. so is true with "dic," thrombotic microangiopathy, hus, and ards. therefore, all of them should be classified as vmtd. as discussed earlier in the reinterpretation of "dic", "dic" is the same disorder as ea-vmtd/dit, which hematologic phenotype is ttp-like syndrome. contemporary theory of tf/fviia-initiated coagulation cascade or cell-based coagulation theory cannot explain how microthrombi strings composed of platelet-ulvwf complexes is the same as fibrin clots within intravascular space. this conceptual conflict between activated tf path and activated ulvwf path has alerted this author with the insights that there must be at least two different paths of hemostasis. the existence of two utterly different characters of blood clots-microthrombi and fibrin clots-have contributed to the redrawing of the framework on two different thrombogenetic mechanisms: microthrombogenesis of ulvwf path and fibrinogenesis of tf path. the former assembles microthrombi strings as seen in vmtd such as ttp, ttp-like syndrome, "dic", hus, ards, and the latter generates fibrin clots as seen in apl and certain envenomation. then, next question is what is the character dvt and arterial thrombus seen in aortic aneurysm since they are neither microthrombi nor fibrin clots. instead, they are obviously macrothrombus, containing fibrin clots and platelets. therefore, it has to be concluded that both ulvwf path and tf path must be involved in the formation of macrothrombosis such as dvt and arterial thrombosis. through this elucidation, two-path unifying theory is borne out to explain not only "two-path unifying theory" of hemostasis but also three paths of thrombogenesis, which includes microthrombogenesis, fibrinogenesis, and macrothrombogenesis ( figure a and b) . finally, in vivo hemostatic mechanism, including the additional role of netosis at the unifying stage of activated ulvwf path and activated tf path, has been indirectly discovered. the present hallmark of ards is vascular microthrombosis (ie, vmtd) as seen with sepsis. sepsis is characterized by generalized endotheliopathy without compromise of set/ evt, which is also the same in ards. hemostatic involvement in sepsis is lone activation of ulvwf path on ecs, leading to formation of microthrombi, but bleeding does not develop because set and evt damage do not occur and tf path is not activated. unlike localized macrothrombosis (eg, thrombus of aortic aneurysm, acute ischemic stroke, and dvt), disseminated microthrombosis (ie, dit) presents with many intriguing features such as ards and hus as well as a variety of hematologic syndromes, including thrombocytopenia, maha, ttp-like syndrome, and "dic." [ ] [ ] [ ] , , according to "two-path unifying theory" of hemostasis, , two thrombotic/coagulation pathways, which are ulvwf and tf paths, are initiated in normal hemostasis but later the two paths must unify to conclude normal hemostasis with passive role of nets. hemostasis stops bleeding in external bodily injury but produce thrombosis in intravascular injury. in the different level (depth) of intravascular injury as presented in tables and , different paths of thrombogenesis take place depending upon what component(s) of vascular wall is damaged ( figure ). if the intravascular damage is confined to the ecs (level ), lone ulvwf path becomes activated and causes microthrombosis (ie, vmtd such as tia, ttp-like syndrome, ards, and mods) because tf path is not activated. on the other hand, if the intravascular damage extends from the ecs to set (level ), both ulvwf path and tf path become activated and cause macrothrombosis (eg, dvt; acute ischemic stroke) as illustrated in hemostatic theory ( figure a and b). , in addition, if the damage extends from the ecs to beyond vessel wall including set and evt (level ), both ulvwf path and tf path become activated and form macrothrombosis with additional evt bleeding (eg, thrombohemorrhagic stroke), which is summarized in tables and . for example, for stroke, this concept is very important in the understanding of thrombogenesis, not only in making the diagnosis but also in planning for the treatment. also, in another situation, if the ecs, set-and evt are damaged by obtuse external trauma, bleeding occur into evt in smaller vessels, but without bleeding into the damaged vascular lumen. tissue factor is released and mixed with blood in evt to activate fvii to trigger the activation of tf/vii path. it causes only "hematoma" without significantly breached ecs because ulvwf path is not activated. this logic is based on "twopath unifying theory." please see figure b , showing three different thrombogenetic processes: microthrombogenesis, fibrinogenesis, and macrothrombogenesis, which are annotated in bold/shaded face. each thrombogenesis occurs when ulvwf path, tf path, or combined paths are activated depending upon the levels (depth) of damage in intravascular injury. the characters of microthrombi, fibrin clots, and macrothrombus from different paths are very different and produce distinctly different clinical thrombotic disorders. among these characters, ards is a hemostatic disease made of "microthrombi strings." molecular pathogenesis based on "two-activation theory of the endothelium". the endothelial molecular pathogenesis triggering inflammation has been well known and documented in medical literature, but its molecular pathogenesis promoting microthrombogenesis has not been understood until recently. although the underlying pathology of ards is vmtd, the pathophysiological mechanism of endotheliopathy causing vmtd has remained in mystery, which is the very reason why progress has not been made in the treatment for ards. nor is the comprehensible pathogenesis of microthrombosis orchestrating hematologic expressions, including thrombocytopenia, maha, mods, and ttp-like syndrome. since sepsis is the initiating cause of microthrombosis and ards is the manifestation of organ phenotype of microthrombosis, sepsis and ards often coexist. further, thrombocytopenia, maha, mods, ttp-like syndrome, and sirs may occur simultaneously in both sepsis and ards. the "two-activation theory of the endothelium" , succinctly explains this pathogenesis of ea-vmtd/dit and identifies dit as a disease promoting all the clinical and hematologic syndromes. the proposed thesis of endothelial molecular pathogenesis in ards is endotheliopathy that initiates two important molecular events: ( ) release of inflammatory cytokines (eg, interleukin (il)- , il- , tumor necrosis factor a, adhesion molecules, and others) , , - and ( ) activation of the platelet and exocytosis of ulvwf. [ ] [ ] [ ] [ ] [ ] [ ] [ ] the former triggers inflammation, which process is called "activation of inflammatory pathway," and the latter mediates microthrombogenesis, which triggers "activation of microthrombotic pathway." these two independent pathways are the essence of the "two-activation theory of the endothelium." the manifestation of activated inflammatory pathway is fever, myalgia, arthralgia, and malaise, but that of activated microthrombotic pathway produces microthrombi strings composed of platelet-ulvwf complexes leading to vmtd. the activation of endothelial inflammatory pathway occurs due to cytokines in both septic and nonseptic critical illnesses. in sepsis-associated ards, the inflammation is accentuated, perhaps through additional loop of activated circulating immune cell pathway (eg, macrophages, monocytes, neutrophils, and lymphocytes) interacting with activated ecs. this pathway further upregulates the expression of inflammatory response, sometimes causing "cytokine storm." this additional mechanism may explain why more severe inflammation occurs in sepsis than in trauma. on the other hand, the activation of the microthrombotic pathway is promoted by excessively exocytosed ulvwf from injured ecs. following the endothelial release, ulvwf become anchored to injured ecs as long elongated strings. [ ] [ ] [ ] if in addition to the excess of ulvwf, the protease adamts , which cleaves ulvwf to smaller molecular weight vwfs, is underexpressed due to additional underlying heterozygous gene mutation, [ ] [ ] [ ] it is more likely to promote ea-vmtd/dit. this endothelial molecular pathogenesis through activation of inflammatory pathway and microthrombotic pathway clearly explains every hematologic feature and organ phenotypic syndrome occurring in ards. in the past several decades, many proposals for redefinition of ards have been forwarded to identify the pathophysiological mechanism and to improve the outcome of the disease with better classification and therapeutic design. [ ] [ ] [ ] [ ] [ ] [ ] however, medical community's task finding the answer on the pathogenesis of ards has been far from over. now, the recognition of ards as an expression of hemostatic disease that is characterized by vmtd has widely opened the door not only in the understanding of this lifethreating phenotype organ syndrome but also in redefining other clinical mods. additionally, with the identification of different thrombogenetic mechanisms of microthrombosis, fibrin clot disease, and macrothrombosis, various thrombotic disorders could be more precisely defined through the submechanisms of in vivo hemostasis. , acute respiratory distress syndrome is the most prominent organ phenotype syndromes developing in sepsis and other critical illnesses among mods. thus, once we understand ards as an organ phenotype syndrome of the lungs in vmtd, we should be able to understand the organ syndromes due to vmtd occurring in the brain, heart, liver, pancreas, muscles, adrenals, and others. it also affirms generalized ea-vmtd/dit is the underlying disease, and ards and other organ syndromes are the manifestations of each specific organ phenotype in ea-vmtd/dit. to make the matters simpler, the diagnostic evaluation and therapeutic approach are the same in every phenotype of mods. finally, we should be able to treat all the patients with every organ phenotype syndrome, combined biorgan syndrome and mods due to ea-vmtd/dit with the same regimen focused on microthrombogenesis. table summarizes the identity of ards defined through clinical, etiologic, pathogenetic and phenotypic features of ea-vmtd. the pulmonary physiologic alteration of hypoxemia, increased capillary permeability and circulatory failure, pathologic changes of diffuse alveolar damage, exudative pulmonary edema, and hyaline membrane formation are the result of pulmonary vascular microthrombosis. thus, the therapy for ards should directly target the pathogenesis producing vmtd. the berlin definition of ards addressed limitations of the american-european consensus conference definition, but poor reliability of some criteria may have contributed to underrecognition and antipathy by clinicians. no pharmacologic treatments aimed at the underlying pathology have been shown to be effective to date, and management remains supportive with lung-protective mechanical ventilation. in addition to cardiopulmonary evaluation for physiological changes due to respiratory distress as well as assessment of the underlying disease, the proper diagnostic approach of ards should start with hematological evaluation. first of all, every patient with ards should be evaluated for the potential of unrecognized ttp-like syndrome, which had been previously defined as "dic". , , unexplained thrombocytopenia, after the exclusion of known causes of thrombocytopenia, should be an initial clue suggesting ongoing microthrombogenesis, leading to ea-vmtd/dit. an additional finding of maha even with minimal degree of schistocytosis, if present, should confirm the diagnosis of ttp-like syndrome. , to look for schistocytes and evidence of hemolysis, blood films should be examined daily for several consecutive days by an experienced hematologist. unlike antibody-associated ttp (aa-vmtd, acquired ttp), schistocytes are fewer in ards, , perhaps due to difference in force of shear stress in the pulmonary vasculature. in critical care settings, in the past, its hemostatic nature could have been missed due to inattention to blood films and low index of suspicion even though an evaluation for unexplained thrombocytopenia and anemia could have been attempted. , , in ards, thrombocytopenia and intravascular hemolysis (ie, anemia, reticulocytosis, increased lactic acid dehydrogenase, indirect hyperbilirubinemia, and hypohaptoglobinemia with negative coombs tests) might be the sufficient criteria to establish the diagnosis of ttp-like syndrome to begin life-saving tpe at the earliest possible time. to solidify the concept that the underlying pathology of ards is ea-vmtd/dic, this author recommends to determine ( ) adamts activity and its autoantibody status, ( ) adamts gene mutation study, and ( ) fibrinogen quantitation, fviii and vwf activity in circulation, and coagulation factor assay for liver-dependent factors (ie, fii, fv, fvii, fix, and fx) to determine the cause of coagulopathy (ie, hepatic coagulopathy). the diagnostic assessments are summarized in table . since ards is one of mods, clinicians should stay vigilant with close clinical monitoring for developing additional organ phenotypes of mods as illustrated in figure . reflection on past clinical trials. more than a half century since the term ards coined, extensive controlled clinical trials have conducted for ards to evaluate the effects of pharmaceutical agents, such as statins, b agonists, anti-inflammatory agents, and corticosteroids, [ ] [ ] [ ] [ ] nutritional supplementation, such as glutamine, selenium, omega- fatty acid, [ ] [ ] [ ] and antioxidant therapy such as n-acetyl cysteine , in prevention and treatment. unfortunately, all of the trials failed to significantly benefit the patient with ards. the fact that the pathophysiologic mechanism of ards has not been clearly recognized and the failure of therapeutic regimens to restore the physiologic alteration of ards from endotheliopathy certainly indicates that the pathogenesis of ards is yet to be discovered. this author is confident that novel hemostatic "two-path unifying theory" and "twoactivation theory of the endothelium" uncover this long hidden mystery of the pathogenesis of ards and should yield effective therapeutic regimens sooner than later. therapeutic plasma exchange. in addition to the best supportive care with proper antibiotics, ventilator support, and appropriate fluid and electrolyte balance for ards, it is obvious that therapeutic approach should target the pathogenesis itself. since this newly recognized concept of the pathogenesis is a hemostatic disease called pulmonary vascular microthrombosis (ie, ttp-like syndrome as a result of ea-vmtd/dit) that is caused by the lone activation of ulvwf path, the therapeutic design should be utilizing the inhibition of vascular microthrombogenesis. at present, the only available antimicrothrombotic regimen is tpe. the rationale is microthrombosis produced by excessive production of ulvwf from endotheliopathy and relative insufficiency of adamts perhaps due to unsuspected gene mutation should respond to additional supply of ulvwfcleaving adamts from exchange of normal donor plasma. indeed, ttp-like syndrome associated with ards has shown excellent response to tpe when employed in very early stage. , , since the lungs are the very organ responsible for oxygen supply to other organs, ards is the most important organ phenotype among mods that could hasten the demise of the patient due to severe hypoxemia. at this time, the earliest intervention utilizing tpe is the only potentially effective treatment to save lives. otherwise, once the patient is entrenched in mechanical ventilation with volume overload following intravenous fluid and blood transfusions, the recovery from ards may become remote even with tpe. just as in sepsis and septic shock, tpe has been used sporadically in ards even without understanding of the concept of microthrombogenesis and vmtd and has shown significant benefit with safety in case reports and limited clinical series. [ ] [ ] [ ] [ ] [ ] [ ] antimicrothrombotic therapy. both ttp and ttp syndrome, including "dic," have shown the beneficial effect with tpe, which is a surrogate for replacement therapy of adamts despite its technical limitations and inconvenience. theoretically, the most efficient therapeutic regimen would be antimicrothrombotic agents, which could include recombinant adamts and possibly n-acetyl cysteine. , - both agents are neither approved nor utilized for human use as defined antimicrothrombotic agents, although adamts is in clinical trials for ga-vmtd. if we can prove their benefit for ards, the therapeutic potential to save so many lives for patients with ea-vmtd/dit in critical care medicine would be immeasurable. the worsening thrombocytopenia (ie, tcip) is the most important laboratory sign suggesting progression of ards. in this situation, platelet transfusion might be tempting, but it is contraindicated in ea-vmtd/dic because platelet transfusion would further promote microthrombogenesis and intensify mods associated with dit as well as maha and also may provoke trali syndrome. if hepatic coagulopathy coexist with ards, its coagulopathy could rapidly progress to combined microthrombo-hemorrhagic syndrome, which will demand a specialized care of coordination with a coagulation specialist. at last, the pathogenesis of ards is identified to be a hemostatic disease occurring due to lone activation of ulvwf path of hemostasis as a result of endotheliopathy in critically ill patients. its underlying pathology is ea-vmtd/dit and clinical phenotype is ttp-like syndrome. generalized endotheliopathy activates the inflammatory pathway and microthrombotic pathway, triggering ea-dit/vmtd. the former provokes inflammation, and the latter promotes consumptive thrombocytopenia, ttp-like syndrome, and hypoxic mods. systemic inflammatory response syndrome is combined syndrome from two independently activated endothelial molecular pathogeneses. acute respiratory distress syndrome is the pulmonary organ phenotype among various ttp-like syndromes. acute respiratory distress syndrome responds to the tpe if initiated in very early stage of the disorder. potentially effective targeted therapeutic strategy should be explored with antimicrothrombotic agents at the earliest possible time to save lives. the author expresses sincere appreciation to miss emma nichole zebrowski for her insights on the structural function of the blood vessel wall in in vivo hemostasis and excellent illustrative art works of figure . the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. the author(s) received no financial support for the research, authorship, and/or publication of this article. orcid id jae c. chang https://orcid.org/ - - - thrombocytopenia in critically ill patients due to vascular microthrombotic disease: pathogenesis based on "two activation theory of the endothelium 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malaria willebrand factor in experimental malaria-associated acute respiratory distress syndrome beyond plasma exchange: novel therapies for thrombotic thrombocytopenic purpura n-acetylcysteine reduces the size and activity of von willebrand factor in human plasma and mice key: cord- -w tn w authors: kim, hanbi; park, minseon; hwang, joonki; kim, jin hwa; chung, doo-ryeon; lee, kyu-sung; kang, minhee title: development of label-free colorimetric assay for mers-cov using gold nanoparticles date: - - journal: acs sens doi: . /acssensors. b sha: doc_id: cord_uid: w tn w [image: see text] worldwide outbreaks of infectious diseases necessitate the development of rapid and accurate diagnostic methods. colorimetric assays are a representative tool to simply identify the target molecules in specimens through color changes of an indicator (e.g., nanosized metallic particle, and dye molecules). the detection method is used to confirm the presence of biomarkers visually and measure absorbance of the colored compounds at a specific wavelength. in this study, we propose a colorimetric assay based on an extended form of double-stranded dna (dsdna) self-assembly shielded gold nanoparticles (aunps) under positive electrolyte (e.g., . m mgcl( )) for detection of middle east respiratory syndrome coronavirus (mers-cov). this platform is able to verify the existence of viral molecules through a localized surface plasmon resonance (lspr) shift and color changes of aunps in the uv–vis wavelength range. we designed a pair of thiol-modified probes at either the ′ end or ′ end to organize complementary base pairs with upstream of the e protein gene (upe) and open reading frames (orf) a on mers-cov. the dsdna of the target and probes forms a disulfide-induced long self-assembled complex, which protects aunps from salt-induced aggregation and transition of optical properties. this colorimetric assay could discriminate down to pmol/μl of bp mers-cov and further be adapted for convenient on-site detection of other infectious diseases, especially in resource-limited settings. t he point-of-care testing (poct) market of infectious disease represents promising and significant growth in the global in vitro diagnostics (ivd) industry. there are several factors that are stimulating the demand for infectious disease poct, including the increasing spread of human immunodeficiency virus (hiv), tuberculosis (tb), and malaria in developing countries, and the threat of emerging and reemerging infectious diseases such as the middle east respiratory syndrome (mers), severe acute respiratory syndrome (sars), zika, a variety of influenza strains, and the west nile virus. infectious diseases pose a significant risk to human health and has led to more than half of the deaths worldwide. additionally, widespread infectious diseases have caused a continuous increase in fatality rates in developing countries. the best way for containment of the epidemic is an early diagnosis, which is difficult using ordinary methods because of costly and large equipment, the necessity of experts, and slow data output. therefore, rapid poct methods are crucial for overcoming these limitations by miniaturizing and reducing the device cost and providing simple, fast, easy-to-use diagnostic tests without specialized training. colorimetric detection originating from gold nanoparticles (aunps) have been intensively studied because of their particular optical properties, i.e., localized surface plasmon resonance (lspr), which represent a color with maximal absorbance wavelength. aunps have been utilized in colorimetric assays for the detection of diverse biological molecules (e.g., proteins and nucleic acids ) in that the change in particle color is generated by sensitive reactivity of nanosized particles to external condition. in addition, the disperse state is adjustably modified from artificial electrostatic force control by ion, ph, biomacromolecules, and so forth. , for example, a positive electrolyte (salt) causes metallic nanoparticle aggregation, causing a significant color change as a red-shift in the lspr spectrum. , based on that, a modified aunp cross-linking method was demonstrated in mirkin's group for dna identification. in the research, the surfaces of aunps were conjugated with two thiolated single-stranded dna (ssdna) via strong au−s interactions. the hybridization of target dnas with the ssdnas on the surface of the aunps induces the formation of double-stranded dna (dsdna)-aunp networks, resulting in aunps aggregation and dramatic color changes. the target dna acts as a crosslinker between two ssdna-functionalized aunps. on the other hand, colorimetric dna sensing methods using functionalized aunps have been demonstrated using noncross-linking dna hybridization with the benefit of the powerful salting-out effect of dsdna compared to ssdna. since the ssdna density on the surface of aunps and the ionic strength of the solution affect the stability of functionalized aunps, control of the aggregated state by modification is quite difficult in practical applications. rothberg et al. suggested a label-free detection method based on the fact that ssdna in the solution could bind to citrate-capped aunps and electrostatically stabilize them at high ionic strength. in such a system, aunps are stabilized in the presence of ssdna, but aggregated with dsdna in a highly concentrated electrolyte solution. this strategy does not need the direct binding of ssdna on the surface of nanosized metallic particles, as the dna amplification step prior to detection is inevitable due to the low sensitivity of this method. since both ssdna and dsdna allow aunps to stabilize at low salt concentration, − cationic agents are mainly used to identify the target with probes. , in this regard, farhad rezaee et al. demonstrated a modified non-cross-linking aunp aggregation using disulfide self-assembly of terminal modified dna. long and flexible sulfur-rich, self-assembled products were well combined on the surface of aunps and effectively inhibit the particles from salt-induced aggregation. inspired by this research, we developed a colorimetric assay that relies on bare aunps that employs a disulfide-induced self-assembly. the bare aunp-based colorimetric method consists of two thiol modified probes at the ′ or ′ ends for targeting partial genomic regions ( bp) of mers-cov along with upstream e protein gene (upe) and encoding open reading frames (orf) a. this assay could be highly reliable for mers-cov diagnosis as we have followed who updated recommendations for infectious disease laboratory testing, which targets the two regions on mers-cov considered for potential preclinical screening and high sensitivity the developed assay platform was able to detect the target dna through optical properties of the gold nanoparticles such as color changes with the naked eye and spectral shifts on uv− vis wavelength. the specific thiolated probes form the complementary dsdna with the target and make a disulfideinduced long self-assembled complex due to continuous disulfide bond formation. the extended self-assembled complex can protect bare aunps for stability against saltinduced aggregation since sulfur-group at ends of dsdna are mediated covalent bond with gold surface. the interaction is known to generate stable conjugation within disulfide-induced self-assembled complex and aunps and intermolecular force between au−s attains about to kcal mol − . , otherwise, probes build a disulfide induced interconnection with each other in the absence of targeting dna. the probe dna terminal coupling is unable to cover the surface of aunps exposed to aggregation, leading to significant color changes as well as a broader red-shift of the lspr peak ( figure ). with this method, we can visually observe the results with the naked eye, not using costly equipment. this colorimetric assay is an important step to use of infectious disease poct across the developing world. moreover, this concept of a disulfide bond based colorimetric assay could be applied for diagnosis of other infectious diseases. preparation of the ideal gold nanoparticle upon a spectral centroid. we developed various sized gold nanoparticles for optimization as a colorimetric indicator. the citrate reduction method, i.e., turkevich-frens, is generally applied to synthesize stabilized gold nanoparticles under electrostatic repulsion. additionally, the ratio of the reducing agent and haucl allow for the preparation of a diverse size of aunps. we changed the volume of trisodium citrate acid from to μl to acquire different-sized aunps. the morphology of the prepared aunps and dls records are reported in figure a . according to the tem images and dls measurements, the average diameter of aunps decreased as the amount of trisodium citrate dehydrate increased. for instance, the addition of reducing agents ( , , , μl) allow us to synthesize -, -, -, and -nm-sized gold nanospheres, respectively. this was the result of changed citrate and gold ratio to control the reduction and stabilization of the nanoparticle surface. , we optimized an average of nm aunp through reduction with μl of trisodium citrate and called that formed particle "bare gold", which means "unmodified and inartificial conjugation with any molecules after synthesized process" and "citrate ion capped gold nanoparticle derived from reduction and stabilization effects of sodium citrate" for this experiment. the structural and spectroscopic properties are presented as (iv) in figure . to evaluate the effects of salt on as-prepared aunps, the absorbance spectra of the bare aunps and those with mgcl were measured using a conventional uv−visible spectrometer (figure b,c) . since the lspr spectral band of nanosized metallic particle is derived from dielectric factors, particle size and shape, the absorption peak shift indicated a changed environment surrounding aunps such as by conjugation of biomolecules. thus, the addition of mgcl induces a significant change in the absorption spectra. for example, the lspr band has a decrease in intensity and increase in bandwidth, as well as new bands at longer wavelength. these lspr band shifts represent the aggregation of aunps caused by the loss of interparticle repulsive force as salt disrupts the charge interaction surround aunps and promotes the interparticle van der waals attractive forces. as shown in the salt induced results, the solutions of gold nanoparticles, excluding nm aunps, became gradually transparent due to severe aggregation of metal particles and also a broader, flattened spectra appeared in the uv−vis measurement as figure c . therefore, we selected the -nm-diameter aunps that detected the lspr changes on the surface of the nanosized metallic particles in this experiment. furthermore, a spectral centroid was derived by calculating the centroid wavelength in each experiment spectrum to determine a credible and accurate point of the lspr band. the spectral centroid represents several parameters of the lspr band including the peak wavelength, bandwidth, and intensity that depend on the size, shape, concentration, and interparticle interactions of the aunps. , the shift of the centroid can be a comprehensive indicator for the overall transition of the spectral distribution toward shorter or longer wavelengths. for example, -nm-diameter aunps possess a spectral centroid at the orange-red wavelength nm, whereas larger -nmdiameter aunps exhibit a purple color with a spectral centroid at nm. optimization of disulfide-induced long self-assembly and the salting agent. in this report, bare -nmdiameter aunps were used, and we selected a target and control upstream of the e protein gene (upe) and open reading frames (orf) a on mers-cov, and the tobacco mosaic virus (tmv), respectively. furthermore, we synthesized forward and reverse thiol modified probes specifically binding the target dna and forming complementary base pairs. each of the thiolated probes were modified at the ′ site (right: ′r) and ′ site (left: ′l). once two probes simultaneously recognize a specific half of the target, the disulfide bonds at the ′ and ′ terminals readily form a sulfurrich self-assembled complex. specifically, target dna samples were mixed with thiolated probes ( ′ r, ′ l) in distilled water (d.w.) and then incubated at °c for a few minutes to establish disulfide-induced long self-assembled products. μl of the aunps solution was then add to the above-mentioned mixture ( μl). the solution was incubated at room temperature for min and various amounts of mgcl solutions were added. each test was evaluated in at least triplicate. we analyzed the color of the resulting solutions with the naked eye and uv−vis spectrometer. disulfide self-assembled products were visualized along with the gel electrophoresis, where the presence of the target (positive reaction) displayed bands with a characteristic ladder due to the formation of long assemblies of dsdna (figure a) . polymorphous bands appeared with the formation of disulfide self-assembly between the target and probes on original full-length native polyacrylamide gel electrophoresis (page) analysis image ( figure s ). additionally, we confirm the sizes of dna fragments with % agarose gel which is commonly used to separate and visualize the amplified dna outcomes depended on number of base pairs ( figure s ). furthermore, we confirmed the adsorbed components on the surface of gold nanoparticle via x-ray photoelectron spectroscopy (xps) (figure b ). in the case where thiolated probe met the incompatible dna, the s p signals mainly indicated the absorbed thiol component at s p / photoelectron binding energy (be) of . ev since a sulfur monolayer was formed on the surface of aunps from the probe solution (s̅ , sh̅ , or sh species) (figure b(i) ). , meanwhile, xps spectrum can be fitted at . and . ev of s p / binding energy when target dna and probes compose the disulfide-induced self-assembled multilayers on aunps (figure b(ii) ). the observed components at − ev of be are assigned to some s in multilayer which highly defect to the gold surface or domain boundaries. , although be of . appeared in figure b (i), the area in the xps spectrum was lower than thiol species ( : . ), and in page, the size of dna fragments only appeared close to bp ladder that indicates thiol containing short layers formed, not the disulfide based multilayer, and unbound thiol assigned that be of s p / when target dna is absent. also, the increased area at higher binding energy indicates higher coverage with absorbed polysulfides. based on results mentioned earlier from page and xps analyses, disulfide-induced self-assembled complex composed the multilayers on aunps, but thiolated ssdna probes form monolayer which is hard to cover the surface of gold nanoparticle from salt-induced aggregation. dls measurements also supported the size increasing of dna covered aunps which depended on extended layers ( figure s ). dynamic light scattering (dls) was used to monitor the distribution of the hydrodynamic size of the gold nanoparticles after immobilized ssdna or dsdna complex layer on the gold surface, respectively. as shown in figure s , the average hydrodynamic size of the gold nanoparticle protected by disulfide-induced self-assembly from the positive reaction was increased by approximately . nm compared to immobilized ssdna on gold nanoparticle from the negative reaction. the stability of the assay was investigated through the addition of various concentrations of mgcl into the bare aunps solution in the (i) absence or (ii) existence of the target (figure c ). mixed products with target dna and probe solutions shielded the particles, which tolerated the salting more than gold nanoparticles conjugated with thiol from disulfide-interconnected probes, since the disulfide-induced self-assembled complex was used to protect bare aunps against salt addition. the spectral peak of the aunps colloid was not quite different with bare aunps when the target dna is in the solution with . to . m mgcl , while aunps linked to disulfide-interconnected probes aggregated with any concentration of salt. however, the absorbance declined slightly, corresponding to an increased salt concentration whether extended disulfide assemblies were formed or not. therefore, the spectral centroid shifts depending on salt concentration were plotted and are shown in figure d . at all concentrations ( . to . m), the delta centroid of the negative control showed more shift than positive controls due to intergold nanoparticle aggregation, while the centroid shift of the positive control was the smallest at . m of mgcl . as previously mentioned, the absorbance declined conversely according to the increase in salt concentration. hence, . m of mgcl was chosen as the optimized concentration. verification of the colorimetric assay. to consider the disulfide self-assembled reaction dependence on target dnas and their concentrations, we measured the absorption change of the aunp solution before and after adding . m mgcl in the presence or absence of each target using a uv−vis spectrometer. when the target was absent, the wavelength was shifted more toward the red side of the spectrum compared to when the target was present. the wavelength peak shifts were , , and nm after complementary binding reactions between probes and (i) orf a, (ii) upe, and (iii) tmv, respectively ((i) to nm, (ii) to nm, and (iii) to nm) (figure a,b) . due to the broad spectrum of the negative control, delta centroid was estimated as . times greater than a positive reaction (avg. nm vs avg nm). these changes indicate that positive samples (orf a and upe) that are a mixture of thiol-modified probes and their target dna that hindered the aggregation of aunps through composition of the long self-assembled structures. negative samples (tmv) were unable to form long self-assembled structures due to mismatched targets. furthermore, we calculated a p-value to prove the reproducibility of this colorimetric platform (figure b ). the p-value is the probability that might yield the same results observed in the biological study, if the null hypothesis is true. all data groups were found to pass the null-hypothesis assuming a normal distribution was followed. therefore, the p-value of the delta centroid implies that this assay is considered statistically significant (p-value < . ). the limit-of-detection (lod) was determined in order to apply to the medical laboratory field. the lod of this colorimetric platform was pmol/μl, which was calculated by the equation mean blank + σ blank defined from the iupac standard ( figure c ). pmol/μl is about × copies/ μl, which requires only pcr cycles to diagnose mers patients who release . × to . × copies/μl in the sputum for days and have recovered without specific treatment for mers-cov. this assay can reduce the number of pcr cycles for diagnosis of mers even with a small amount of viral molecules, compared to conventional pcr reactions that require more than about cycles to detect mers-cov. − ■ conclusions a simple and fast colorimetric assay for detecting infectious disease that can be seen by the naked eye without costly equipment was developed. we proposed a colorimetric assay using disulfide bonds formed by hybridizing with thiolated probes and a target; this method inhibited the aggregation of aunps by salt and limits the color change for diagnosis of mers. this assay can confirm the presence of mers-cov within min without electrophoresis or other procedures. the assay could discriminate mers-cov with a potential detection limit of pmol/μl, which means it could distinguish a lower amount of target with a lower level of amplification, or even without amplification. a follow-up study will be conducted to apply this method to clinical samples with longer targeting regions. we anticipate that this method will provide rapid and accurate diagnostic results in epidemic areas, especially in resource-limited settings and will evolve by state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans emerging infectious diseases: threats to human health and global stability nanobioimaging and sensing of infectious diseases an operationally simple colorimetric assay of hyaluronidase activity using cationic gold nanoparticles electrostatic assembly of nanoparticles and biomacromolecules guided assembly of nanoparticles on electrostatically charged nanocrystalline diamond thin films localized surface plasmon resonance spectroscopy and sensing molecular linker-mediated self-assembly of gold nanoparticles: understanding and controlling the dynamics selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles gold nanoparticles for naked-eye dna 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colorimetric biosensing assays spontaneously formed sulfur adlayers on gold in electrolyte solutions: adsorbed sulfur or gold sulfide? sulfur-substrate interactions in spontaneously formed sulfur adlayers on au( ) sulfur multilayer formation on au( ): new insights from the study of hexamethyldisilathiane x-ray photoelectron spectroscopy sulfur p study of organic thiol and disulfide binding interactions with gold surfaces value of p-value in biomedical research limits of detection in spectroscopy comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity kinetic studies and infection control of respiratory viruses in infectious mers-cov isolated from a mildly ill patient middle east respiratory syndrome coronavirus (mers-cov) infections in two returning travellers in the netherlands key: cord- -svtdfeop authors: campos, angélica cristine almeida; góes, luiz gustavo bentim; moreira-soto, andres; de carvalho, cristiano; ambar, guilherme; sander, anna-lena; fischer, carlo; ruckert da rosa, adriana; cardoso de oliveira, debora; kataoka, ana paula g.; pedro, wagner andré; martorelli, luzia fátima a.; queiroz, luzia helena; cruz-neto, ariovaldo p.; durigon, edison luiz; drexler, jan felix title: bat influenza a(hl nl ) virus in fruit bats, brazil date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: svtdfeop screening of bats for influenza a viruses showed subtype hl nl in intestines of great fruit-eating bats (artibeus lituratus). high concentrations suggested fecal shedding. genomic characterizations revealed conservation of viral genes across different host species, countries, and sampling years, suggesting a conserved cellular receptor and wide-ranging occurrence of bat influenza a viruses. i nfluenza a viruses are major causes of human disease and are predominantly maintained in avian reservoirs ( ) . the segmented influenza a genome facilitates reassortment events in birds or intermediate hosts, such as swine and horses, leading to emergence of new variants potentially capable of causing zoonotic infections ( ) . bats are major sources of zoonotic pathogens ( ) . in pioneering studies from and , the first bat influenza a viruses, termed h n and h n , were discovered in bat species, sturnira lilium (little yellow-shouldered bat) and artibeus planirostris (flat-faced fruit-eating bat) ( , ) . bat-associated influenza a viruses are phylogenetically highly divergent from avian-associated influenza a viruses in their hemagglutinin (ha) and neuraminidase (na) genes, suggesting these viruses represent ancient influenza a strains ( ) . consistent with their genetic divergence, batassociated influenza a surface proteins lack typical hemagglutination and neuraminidase activities ( ) , leading to the terminology ha-like (hl) and neuraminidase-like (nl) for bat-associated influenza surface proteins. so far, only individual bat specimens yielded influenza a genomic sequences during the pivotal investigations ( , ) . hl nl has only been found in a. planirostris bat captured in peru in ( ) , challenging definite host assessments. to investigate bat influenza a virus epidemiology, we investigated bats in southern brazil during - . for this study, we sampled individual bats representing species and families across sampling sites (table ) . bats were captured using mist nets, euthanized, and necropsied and were identified on the basis of morphological criteria by trained field biologists as described previously ( ) . only intestine samples were available for virological analyses. the instituto brasileiro do meio ambiente e dos recursos naturais ( - ), instituto ambiental do paraná ( / ), and the ethics committee of the institute of biomedical science from the university of são paulo ( - - / ) authorized sampling. we tested intestine specimens from all bats using highly sensitive, broadly reactive nested reverse transcription pcrs targeting different regions of the influenza a polymerase basic (pb) gene ( , a. lituratus bats were the most abundantly sampled species ( table ) . the low overall influenza virus detection rate in this study ( . %, % ci . %- . %) was not significantly different by fisher exact test from the previous studies ( / bats for hl nl [ . %, % ci . %- . %; p = . ]; / bats for hl nl [ . %, % ci . %- . %; p = . ]). apparently low rates of acute influenza a virus infection in bats are not consistent with high seroprevalence of % in different bat species according to a preliminary investigation ( ) and may hint at seasonal variation in bat influenza virus infections, comparable to other batborne rna viruses ( ) . sanger sequencing of the screening pcr amplicons suggested close genetic relatedness of the strains circulating in brazil with the hl nl strain circulating in peru. virus concentrations in the positive intestine specimens as determined by strain-specific quantitative real-time reverse transcription rt-pcr (appendix table , https://wwwnc. cdc.gov/eid/article/ / / - -app.pdf) were high ( . × and . × rna copies/g of tissue). high hl nl concentrations in intestinal specimens are consistent with qualitative data from the pioneering study on hl nl ( ) and may suggest intestinal tropism and potential fecal shedding into the environment. we determined the full coding sequence of all segments of the viral genomes using primers aiming at amplifying overlapping regions of bat influenza a virus genomes (genbank accession nos. mh - ) (appendix table ). the hl nl variants in brazil differed by nt from each other across the combined genomic segments. four of those substitutions were nonsynonymous, causing amino acid exchanges in the pb (v i), pb (r k), nucleoprotein (g s), and na (v i) genes (table ; figure , panel b). this finding suggests recent common ancestry of the hl nl variants identified in the positive bats and was consistent with their detection in the same site days apart. comparison of the full coding sequence of the novel hl nl variants revealed high sequence identity between the peru and the brazil strains, . %- . % nucleotide identity across all genomic segments ( table ). the genomic relatedness of peru and brazil hl nl strains was surprising given a time span of years, a geographic distance exceeding , km, and different bat species that tested positive in our study and the previous study ( ) . all critical amino acid residues associated with influenza a virus replication and entry ( , ) were conserved between the brazil and the peru hl nl strains, including the ha monobasic cleavage site motif piketr/glf ( ) . thermodynamic modeling revealed that the amino acid exchanges observed between the brazil and peru hl nl strains did not alter the tridimensional structure of the hl and nl proteins, and neither mapped to the putative receptor binding site of the hl protein ( figure , panel c), nor to the putative active site of the nl protein ( figure , panel d) ( ) . this result suggests preservation of the biologic activity of these glycoproteins in different bat species and supported a broadly conserved cellular receptor of bat influenza a viruses that differs from sialic acid receptors used by avian-associated influenza a viruses ( ) . significantly fewer amino acid exchanges were observed between the hl proteins of brazil and peru bat influenza virus than between the respective nl proteins (p = . by fisher exact test) ( n t, k r, l m, i l, i v n t, k r, l m, i l, i v, g s nl . % i v, i l, v i, v i, l i, a t, v i, d e, v i, g e i v, i l, v i, v i, v i, l i, a t, v i, low rate of nonsynonymous substitutions in the hlencoding genes of bat influenza a virus variants was reminiscent of strong purifying selection acting on the hemagglutinin genes in avian-specific influenza a virus strains ( ) . this finding may suggest comparable evolutionary dynamics between chiropteran and avian reservoirs. definite assessments will require considerably larger datasets of bat influenza a virus strains. a. lituratus bats and a. planirostris bats, in which hl nl was originally detected in peru, represent closely related, yet genetically and morphologically clearly distinct bat species ( ) . the distribution of these bat species overlaps (figure , panel a) , potentially facilitating virus exchange across the populations. phylogenetic analyses confirmed the close genetic relationship between peru and brazil hl nl variants across all segments (figure ; appendix table ), suggesting lack of reassortment events according to the available data. our data thus suggest host associations of hl nl beyond the species level, comparable to genus-level host associations of other batborne rna viruses such as coronaviruses ( ) . the zoonotic potential of hl nl is unclear, yet humanderived cell lines were susceptible to infection by chimeric vesicular stomatitis virus pseudotyped with hl ( ) . the abundance of a. lituratus bats within latin america (figure bats are sources of high viral diversity and high-profile zoonotic viruses worldwide. although apparently not pathogenic in their reservoir hosts, some viruses from bats severely affect other mammals, including humans. examples include severe acute respiratory syndrome coronaviruses, ebola and marburg viruses, and nipah and hendra viruses. factors underlying high viral diversity in bats are the subject of speculation. the hypothesis is that flight, a factor common to all bats but to no other mammals, provides an intensive selective force for coexistence with viral parasites through a daily cycle that elevates metabolism and body temperature analogous to the febrile response in other mammals. visit our website to listen: http://www c.cdc.gov/podcasts/player.asp?f= global patterns of influenza a virus in wild birds chiropteran influenza viruses: flu from bats or a relic from the past? host and viral traits predict zoonotic spillover from mammals a distinct lineage of influenza a virus from bats new world bats harbor diverse influenza a viruses the neuraminidase of bat influenza viruses is not a neuraminidase genetic diversity of bats coronaviruses in the atlantic forest hotspot biome, brazil non-random patterns in viral diversity amplification of emerging viruses in a bat colony hemagglutinin homologue from h n bat influenza virus exhibits divergent receptor-binding and ph-dependent fusion activities evolutionary dynamics and global diversity of influenza a virus speciation dynamics of the fruit-eating bats (genus artibeus): with evidence of ecological divergence in central american populations ecology, evolution, and classification of bat coronaviruses in the aftermath of sars synthetically derived bat influenza a-like viruses reveal a cell type-but not species-specific tropism replication and shedding of mers-cov in jamaican fruit bats (artibeus jamaicensis) we thank mariana cristine pereira de souza, cairo monteiro de oliveira, and luciano matsumiya thomazelli for laboratory support. dr. campos is a postdoctorate researcher affiliated with the university of sao paulo and charité-universitätsmedizin berlin. her research focuses on emerging viruses from bats. key: cord- -l ghggu authors: hoang, minh; lin, wei-hao; le, van phan; nga, bui thi to; chiou, ming-tang; lin, chao-nan title: molecular epidemiology of canine parvovirus type in vietnam from november to february date: - - journal: virol j doi: . /s - - -z sha: doc_id: cord_uid: l ghggu background: canine parvovirus type (cpv- ) was first identified in the late s; it causes intestinal hemorrhage with severe bloody diarrhea in kennels and dog shelters worldwide. since its emergence, cpv- has been replaced with new genetic variants (cpv- a, cpv- b, and cpv- c). currently, information about the genotype prevalence of cpv- in vietnam is limited. in the present study, we investigated the genotype prevalence and distribution of cpv- in the three regions of vietnam. methods: rectal swabs were collected from dogs with suspected cpv- infection from northern, central, and southern vietnam from november to february . all samples were identified as parvovirus positive by real-time pcr, and further genotyping was performed using a simpleprobe® real-time pcr assay. results: of the vietnamese cpv- isolates, isolates ( . %) were identified as cpv- a, isolates ( . %) were identified as cpv- c and isolates ( . %) were untypable using the simpleprobe® real-time pcr assay. in northern vietnam, the percentages of cpv- a and cpv- c were . % ( / ) and . % ( / ), respectively. in central vietnam, the percentages of cpv- a and cpv- c were . % ( / ) and . % ( / ), respectively. in southern vietnam, the percentages of cpv- a and cpv- c were . % ( / ) and . % ( / ), respectively. cpv- b was not observed in this study. the vp genes of cpv- c in vietnam are more genetically similar to those of cpv- c strains in china and taiwan than to those of prototype cpv- c strains (fj ) or the first vietnamese cpv- c (ab ). conclusion: the present study provides evidence that cpv- c is the most prevalent variant in vietnam. phylogenetic analysis demonstrated that the recent vietnamese cpv- c isolates share a common evolutionary origin with asian cpv- c strains. electronic supplementary material: the online version of this article ( . /s - - -z) contains supplementary material, which is available to authorized users. canine parvovirus type (cpv- ) is one of the most dangerous enteropathogens, causing fatal disease in dogs and puppies worldwide [ ] . cpv- is a nonenveloped, small dna virus with a diameter of approximately nm and a single-stranded dna genome of approximately kb [ ] . cpv- belongs to the genus parvovirus in the family parvoviridae, which includes feline panleukopenia virus (fpv), mink enteritis virus, raccoon parvovirus, and porcine parvovirus [ ] . clinical manifestations of cpv- infection are characterized by intestinal hemorrhage with severe bloody diarrhea; other clinical signs include anorexia, depression and vomiting [ , , ] . cpv- is a rapidly evolving virus, leading to the mutation of novel variants since its first recognition in the late s [ ] . for example, cpv- a was approximately discovered in in the usa, and cpv- b and c were identified in and in the usa and italy, respectively, based on residue (asn in a, asp in b, and glu in c) in the vp protein of the parvovirus [ , ] . when a novel cpv- variant appears, it very rapidly replaces old variants [ , ] . in recent decades, cpv- c has been found to be widespread in european countries [ , ] , the usa [ ] , south america [ ] [ ] [ ] and africa [ , ] . the first reported occurrence of the cpv- c variant in vietnam was in [ ] ; however, since then, this strain has not been prevalent in asia [ ] . surprisingly, novel asian cpv- c isolates were identified in china [ ] [ ] [ ] [ ] , taiwan [ , ] , laos [ ] and thailand [ ] in the past few years. in vietnam, cpv- infection was first observed as sporadic cases in (unpublished data). subsequently, after its first emergence, there were widespread outbreaks of canine hemorrhagic enteritis with high morbidity and mortality occurring across the whole country [ ] . along with the increasing number of pet dogs in vietnam, cpv- infection has emerged as a veterinary public health concern that greatly affects puppies because of its high mortality and morbidity. however, current information related to the antigenic types of cpv prevailing in vietnam is poorly understood. thus, in the present study, we investigated the genotype prevalence and distribution of cpv- from naturally infected dogs in three regions of vietnam using a simplep-robe® real-time polymerase chain reaction (pcr) assay. rectal swabs were collected from dogs with suspected cpv- infection from northern, central, and southern vietnam from november to february . these dogs had displayed clinical symptoms of cpv- infection, including diarrhea or bloody diarrhea. fecal samples were collected from dogs with alimentary signs (diarrhea or bloody diarrhea) by inserting a swab into the rectum (~ cm), rotating the swab and then resuspending the swab in ml of phosphate-buffered saline. all specimens were transported on ice and stored at - °c at the key laboratory for biotechnology and animal disease, vietnam national university of agriculture, hanoi, vietnam. information including the sampling year, age, clinical history, and cpv- types of the sampled dogs is summarized in additional file . total dna was extracted using a genomic dna mini kit (geneaid biotech, ltd., taipei, taiwan) following the manufacturer's protocol. all of the clinical specimens were confirmed to be infected with parvovirus by real-time pcr, as described by lin et al. [ ] . parvovirus-positive samples were further characterized by a simpleprobe® real-time pcr assay, following the method developed by hoang et al. [ ] . first, the reaction was carried out in a final volume of μl containing mm mgcl , pm simpleprobe® (tib molbiolgmbh, berlin, germany), pm and pm each of primers parvo-f ( ′-aca cct gag aga ttt aca tat ata gca ca- ′) and parvo-a ( ′-att agt ata gtt aat tcc tgt ttt acc tcc- ′) , μl x light cycler genotyping master mix and μl x diluted template dna. real-time pcr was cycled under the same thermal conditions as those previously described for the simpleprobe® assay [ ] . to clone full length vp , we performed pcr amplification using the primer pair vp f ( ′-cggtgcagg acaagtaaaa - ′)/vp r ( ′-ggtgctagttgata tgtaata - ′) that amplified a bp fragment of the gene encoding the capsid protein. the pcr conditions were as follows: denaturation at °c for min; cycles of denaturation at °c for s, annealing at °c for min, and extension at °c for min; and a final extension step at °c for min. pcr products were electrophoresed, followed by the purification of electrophoresis products by a wizard® sv gel and pcr clean-up system (promega corporation, usa). the pcr products obtained from the previous step were cloned by a t&a® cloning kit before being sent for automated sequencing (mb mission biotech, inc., taiwan). the resulting sequences were compared with reference fpv (m ), cpv- (m ), cpv- a (m , and m ), new cpv- a (ay , ab , eu , jq , kf , and kr ), cpv- b (m and m ), new cpv- b (ab , ab , ab , ab , ab , ab , ab , ay , ay , jq , kr , and kr ), cpv- c (fj , ab , fj , km , kr , kt , kt , ky , mf , and ku ), cpv- a vaccine strain (fj ) and previous vietnamese strains (ab , ab , ab , ab , ab , ab , ab ). the clustal w method and megalign program (dnastar, madison, wi, usa) were used for multiple alignments of the nucleic acid and amino acid sequences. phylogenetic analyses were conducted by the maximum likelihood methods using mega based on the tamura-nei model [ ] . a total of specimens were collected from suspected parvovirus-infected dogs in three regions of vietnam, including northern, central and southern vietnam. all samples were positive for parvovirus dna. the age of the sampled dogs ranged from month to months old. the demographic summary of puppies that tested positive for parvovirus by real-time pcr is also presented in additional file . the number of puppies less than months old accounted for . % ( / ) of the sample, dogs of to months of age accounted for . % ( / ) of the sample, and dogs older than months accounted for . % ( / ) of the sample. for samples, the age group was unspecified. in terms of gender, . % ( / ) of the sample was male dogs, and . % ( / ) was female dogs. for samples, the gender was not reported. in the northern areas of vietnam, cpv- cases often peak in november and january, while the peak time for central areas is from december to march, and the peak time for southern areas is in june and july (fig. ). among the samples collected from the three regions in vietnam, samples ( . %) were clearly genotyped according to melting temperature analysis using a simpleprobe® real-time pcr assay (table ) . however, there were three cases ( . %) from northern vietnam for which differentiation was not possible by the simpleprobe® real-time pcr assay (additional file ). among samples, six isolates ( . %) were identified as cpv- a and isolates ( . %) were identified as cpv- c. in northern vietnam, the percentages of cpv- a and cpv- c were . % ( / ) and . % ( / ), respectively. in central vietnam, the percentages of cpv- a and cpv- c were . % ( / ) and . % ( / ), respectively. in southern vietnam, the percentages of cpv- a and cpv- c were . % ( / ) and . % ( / ), respectively (fig. ) . cpv- b was not observed in this study (fig. ) . our results showed that cpv- c is currently the most prevalent cpv- field strain circulating in vietnam. a total of dogs ( . %) were confirmed to be vaccinated against cpv- . among cpv- vaccinated dogs, were younger than months old, and were months old or older; the age of the remaining dogs could not be accurately determined. interestingly, all vaccinated dogs were identified as having cpv- c infection using the simpleprobe® real-time pcr assay. to investigate the association and diversity of cpv- strains circulating in vietnam, we sequenced and analyzed complete vp genes of cpv- strains collected from the three regions of vietnam. phylogenetic analysis based on the nucleotide sequences of the complete vp gene revealed two clusters that were prototype cpv- c strains and asian cpv- c clusters ( (fig. ) . the vp genes of cpv- c in vietnam are more genetically similar to cpv- c strains in china and taiwan than to prototype cpv- c strains (fj ) or first vietnamese cpv- c (ab ) (fig. ). in addition, one vietnamese strain (dn ) in this study was clustered with a cpv- a vaccine strain (fj ) (fig. ). there were several nucleotide mutations in the vp gene of all strains (table ), but only mutations (nucleotide position , , , , , and ) table . for the complete vp analysis, one sequence with an asn and sequences with a glu at position were classified as cpv- a and cpv- c, respectively. all cpv- c strains showed substitution at position ala gly, phe tyr, tyr ile, and gln arg ( table ) . thirteen of the cpv- c strains obtained showed a unique substitution at position (ile to met) caused by mutation of tat to tgt at nucleotide positions - of the vp gene (table ). cpv- c is currently the most prevalent genotype in asian countries [ ] [ ] [ ] [ ] [ ] [ ] . the first asian cpv- c case was detected in vietnam in [ ] . however, information regarding the current genotype prevalence of cpv- strains in vietnam is limited. this study is the first to investigate the genotype prevalence of cpv- in regions of vietnam. similar to other asian countries [ ] [ ] [ ] [ ] [ ] [ ] , the cpv- c variant appears to be the most prevalent genotype in the dog population in all regions of vietnam. surprisingly, no cpv- b cases were found in vietnam in the present study; however, according to previous reports, cpv- b had the highest detection rate in vietnam in [ ] ( table ). the reasons for this discrepancy may be as follows: i) current cpv- b vaccines are effective in preventing and controlling the disease in vietnam; or ii) there is limited or no protection from current vaccines against the cpv- c variant, and this variant rapidly replaced the previous circulation of cpv- strains. as such, we used a sybr green-based real-time pcr assay, which was sensitive, specific and reliable for the detection of cpv- , fpv and porcine parvovirus dna [ ] . however, there were three parvovirus-positive cases from northern vietnam for which differentiation was not possible by our simplep-robe® real-time pcr assay. this issue may be due to i) unusual changes in the probe sequence region [ ] ; or ii) the infection of vietnamese dogs by other parvovirus species for which the sequence analysis has not been investigated. the country of vietnam is divided into three regions with different climates. northern vietnam has a humid subtropical climate, while the central region has a tropical monsoon climate, and southern vietnam has a tropical savanna climate. another factor that may affect the study is the humidity in vietnam, which is - % on average. however, because of differences in latitude and topography, the climate tends to be markedly different across regions, which may cause the difference in the timing of parvovirus outbreaks in specific regions. in the northern part of the country, due to more seasons than other regions ( seasons in total), the seasonal periods of parvovirus outbreaks are usually from october to november (winter transition time), from december to january (spring transition time), and from march to may, with a peak in april (summer transition time) (fig. ) . northern areas show a disease period spread over many months, whereas outbreaks often occur at a more concrete, specific time in the central and southern parts of the country. in fact, in central vietnam, the period from december to march is the time when parvovirus infection increased rapidly in dogs. in the southern part of the country, small dogs are more likely to be affected during the period from june to august when the weather is beginning to switch from the dry to rainy season. thus, cpv- disease can occur in vietnam at any time of the year depending on the geographic location; however, when there is a change in climate, temperature and humidity make puppies susceptible to infection and cause cpv- to become widespread in the environment due to prolonged survival time. various contradicting studies emphasize the disease's seasonality in different specific geographical regions. some scientists have indicated that the highest incidence of this disease is detected in spring and summer [ , ] , while the opposite is true for other locations [ ] [ ] [ ] [ ] . dogs aged months to months accounted for the highest detection rate ( / ; . %), while this rate for the month to month age group was . % ( / ), and the rate for dogs over months old was only . % ( / ). for samples, the age group was not reported. the percentage of dogs under months of age who were greatly affected has been previously reported [ , ] . the reason dogs from months to months old are easily susceptible to cpv is due to the decrease in maternal antibodies [ , ] . in addition, the higher incidence of cpv- in dogs younger than months old might be due to quicker intestinal crypt cell multiplication with a higher mitotic index by changes in bacterial flora and daily diet during weaning time [ , ] . very few cases are recorded over year of age; the cause may be slight exposure to the virus, leading to antibody production in the host; previous vaccination of animals; or another reason not yet identified. dogs in the to month age group still exhibit cpv disease, which may be because of poor vaccine preservation or incorrect vaccination timing [ ] . we found that the proportion of infected females was less than that of males. of positive cases, were females ( . %), and ( . %) were males. coincidently, the current finding is consistent with studies by thomas et al. [ ] and gombac et al. [ ] , as well as other reliable publications [ , , [ ] [ ] [ ] . many assumptions could be analyzed; however, the most relevant cause could be that the majority of samples were collections from male dogs during the study. another explanation for this result could be that male dogs are more likely to be infected; in fact, more male dogs were admitted for treatment than female dogs. however, there is reportedly no influence of sex on the incidence of cpv [ , ] . the high incidence in male dogs may also be due to some behaviors and habits of pet owners or the hobby of selecting and keeping male dogs [ ] . a large number of dogs have been vaccinated against cpv- but still suffer from the disease, with most of the cases ( / ; . %) months of age and older. this issue may be related to failure to properly use the vaccine or to preserve the vaccine [ , ] . many studies have shown that cpv- and cpv- b vaccines provide protection against the cpv- a/ b virus and provide complete protection against the new cpv- c variant in dogs for up to years [ ] [ ] [ ] . however, there are some disagreements regarding the use of traditional vaccines against the new antigen variant cpv- c [ , ] . similar to a study conducted in taiwan, four of cpv- c-infected dogs died despite vaccination, including one adult dog that completed the vaccination program [ ] . this phenomenon has also been seen in vietnam where % of vaccinated positive cases were of genotype c. many cpv- vaccines are being used in vietnam; however, there is little documentation of the effects of cpv- vaccination against the current cpv- c variant. therefore, the efficacy of the current vaccine against the cpv- c variant remains to be evaluated. currently, there are many publications regarding the circulation of the cpv- c strain in many countries in asia [ - , - , , ] although the first publication on cpv- c was in vietnam in [ ] , the cpv- c variant has not been prevalent in asia. surprisingly, novel asian cpv- c isolates were reported in china [ ] [ ] [ ] [ ] , taiwan [ , ] , laos [ ] , and thailand [ ] . this finding suggests that novel cpv- c is now becoming increasingly prevalent in asian countries. based on the results of the phylogenetic analysis, the cause for the large amount of cpv- c detected in vietnam is the invasion of a foreign strain from to . in addition, there was no phylogenetical relation between vietnamese cpv- c and prototype cpv- c strains (european or american strains) or the past vietnamese cpv- c in , while these strains are similar to the recent asian cpv- c strains (fig. ) . phylogenetic analysis revealed that the recent vietnamese cpv- c strains share a common evolutionary origin with asian cpv- c strains. this finding could be explained by dog imports from china and other neighboring nations with a lack of oversight due to a long shared border with these countries and the effort needed from authorities to apply restrictions. amino acid substitutions of ala gly, phe tyr, tyr ile and gln arg of cpv- c have been observed in china [ , , ] and taiwan [ , ] . however, the functions of cpv- residues , , and are still unknown and remain to be elucidated. the substitution of ile met is unique to the vietnamese cpv- c strains. residue is not exposed on the capsid surface [ ] , and substitutions in this position may not affect the antigenicity of the virus. however, agbandie's study showed that the binding of dna within the internal surface of the parvovirus protein shell may cause specific conformational changes in the protein [ ] . therefore, the function of residue remains to be elucidated. this study is the first cpv- epidemiological survey in vietnam to document the presence and relative distribution of cpv- variants in three regions of vietnam over the past few years. phylogenetic analysis demonstrated that the recent vietnamese cpv- c isolates share a common evolutionary origin with asian cpv- c strains. it is important for veterinarians and dog owners to understand the current geno-prevalence of cpv- and effective prevention methods based on outbreak periods. availability of data and materials not applicable. canine parvovirus: a review of epidemiological and diagnostic aspects, with emphasis on type c nucleotide sequence and genome organization of canine parvovirus the emergence of parvoviruses of carnivores rapid antigenic-type replacement and dna sequence evolution of canine parvovirus isolation and immunisation studies of a canine parco-like virus from dogs with haemorrhagic enteritis natural variation of canine parvovirus evidence for evolution of canine parvovirus type in italy a canine parvovirus mutant is spreading in italy characterisation of the canine parvovirus type variants using minor groove binder probe technology occurrence of canine parvovirus type c in the united states first detection of canine parvovirus type c in south america evolution of canine parvovirus in argentina between years and : cpv c has become the predominant variant affecting the domestic dog population genotyping of canine parvovirus in western mexico molecular epidemiology of canine parvovirus in morocco molecular characterization of canine parvovirus- variants circulating in tunisia a novel antigenic variant of canine parvovirus from a vietnamese dog identification of a novel canine parvovirus type c in taiwan co-circulation of the rare cpv- c with unique gln arg substitution, new cpv- b with unique thr ala substitution, and new cpv- a with high prevalence and variation in heilongjiang province typing of canine parvovirus strains circulating in north-east china typing of canine parvovirus strains circulating in north-east china continuing evolution of canine parvovirus in china: isolation of novel variants with an ala gly mutation in the vp protein phylodynamic and genetic diversity of canine parvovirus type c in taiwan molecular characterization of canine parvovirus in vientiane emergence of canine parvovirus type c in domestic dogs and cats from thailand development of sybr green-based real-time pcr for the detection of canine, feline and porcine parvoviruses a simpleprobe® real-time pcr assay for differentiating the canine parvovirus type genotypereal-time pcr assay for differentiating the canine parvovirus type genotype mega : molecular evolutionary genetics analysis version . canine parvovirus in new zealand: epidemiological features and diagnostic methods risk factors associated with parvovirus enteritis in dogs: cases ( - ) canine parvovirus in a commercial kennel: epidemiologic and pathologic findings aspects of the diagnosis, pathogenesis and epidemiology of canine parvovirus parvovirus enteritis in dogs based on autopsy statistics - clinical and epidemiological aspects of canine parvovirus (cpv) enteritis in the state of molecular epidemiology of canine parvovirus in southern india incidence of canine parvovirus in diarrhoeic dogs by polymerase chain reaction frequency of cpv infection in vaccinated puppies that attended puppy socialization classes a modified live canine parvovirus vaccine. ii. immune response polymerase chain reaction based epidemiological investigation of canine parvoviral disease in dogs at bareilly region retrospective study of canine parvovirosis in slovenia. case report prevalence of canine parvovirus infection in assam occurrence of canine parvovirus in dogs from henan province of china in - dog ecology and demography information to support the planning of rabies control in machakos district epidemiological study of canine parvovirus epizootiological status of canine viral haemorrhagic gastroenteritis in bhubaneswar city epidemiology of parvovirus and coronavirus infections in dogs in assam evolution of canine parvovirus-a need for new vaccines? do two current canine parvovirus type and b vaccines provide protection against the new type c variant? vaccination with canine parvovirus type (cpv- ) protects against challenge with virulent cpv- b and cpv- c canine parvovirus type vaccine protects against virulent challenge with type c virus are licensed canine parvovirus (cpv and cpv b) vaccines able to elicit protection against cpv c subtype in puppies?: a systematic review of controlled clinical trials canine parvovirus: the worldwide occurrence of antigenic variants occurrence of canine parvovirus type c in the dogs with haemorrhagic enteritis in india the first detection of canine parvovirus type c in china structure determination of feline panleukopenia virus empty particles authors' contributions mh and whl performed the molecular data analysis and wrote the manuscript. bttn performed the cloning and sequence analysis. vpl and mtc managed the study, provided materials and reagents, contributed to the interpretation of the data, and cowrote the manuscript. cnl designed and analyzed the experimental data and wrote the manuscript. all of the authors read and approved the final manuscript. the study did not involve any animal experiments. the institutional animal care and use committee (iacuc) of vietnam national university of agriculture did not deem it necessary for this research group to obtain formal approval to conduct this study. not applicable. the authors declare that they have no competing interests. key: cord- -y g yak authors: choi, eunjin; ha, kee-soo; song, dae jin; lee, jung hwa; lee, kwang chul title: clinical and laboratory profiles of hospitalized children with acute respiratory virus infection date: - - journal: korean j pediatr doi: . /kjp. . . . sha: doc_id: cord_uid: y g yak purpose: despite the availability of molecular methods, identification of the causative virus in children with acute respiratory infections (aris) has proven difficult as the same viruses are often detected in asymptomatic children. methods: multiplex reverse transcription polymerase chain reaction assays were performed to detect common respiratory viruses in children under years of age who were hospitalized with ari between january and december . viral epidemiology and clinical profiles of single virus infections were evaluated. results: of , patients, viruses were identified in , ( . %), with the assay revealing a single virus in , cases ( . %). while major pathogens in single virus-positive cases differed according to age, human rhinovirus (hrv) was common in patients of all ages. respiratory syncytial virus (rsv), influenza virus (if), and human metapneumovirus (hmpv) were found to be seasonal pathogens, appearing from fall through winter and spring, whereas hrv and adenovirus (adv) were detected in every season. patients with aris caused by rsv and hrv were frequently afebrile and more commonly had wheezing compared with patients with other viral aris. neutrophil-dominant inflammation was observed in aris caused by if, adv, and hrv, whereas lymphocyte-dominant inflammation was observed with rsv a, parainfluenza virus, and hmpv. monocytosis was common with rsv and adv, whereas eosinophilia was observed with hrv. conclusion: in combination with viral identification, recognition of virus-specific clinical and laboratory patterns will expand our understanding of the epidemiology of viral aris and help us to establish more efficient therapeutic and preventive strategies. acute respiratory infections (aris) are a main cause of morbidity and mortality in children, with viruses being responsible for more than % of aris worldwide. , ) clinical manifestations of ari can hardly differentiate bacterial from viral etiologies, which can lead to the unnecessary use of antibiotics. recent development of the multiplex reverse transcription polymerase chain reaction (rt-pcr) assay makes noninvasive identification of respiratory pathogens possible. however, a more accurate diagnosis of causative ari pathogens does not decrease hospital admissions or antibiotic use in children with ari. ) since viruses are often detected in asymptomatic children, ) identification of a virus by rt-pcr does not always imply that it is the culprit for a current ari. because these viruses are sometimes asymptomatic carriers and sometimes pathogens, a diagnosis should not be based solely on viral identification in respiratory samples. moreover, new respiratory viruses have been increasingly recognized, but their clinical significance remains unclear. , ) thus, in addition to multiplex rt-pcr assays, korean j pediatr ; ( ) : - a better understanding of these viruses is required to improve clinical management. in this study, we assessed the viral epidemiology of ari in hospitalized children under age of years, and we characterized the virusspecific clinical and laboratory profiles as well as clinical outcomes. together with rt-pcr results, this data will help us understand the clinical course of viral ari and to establish more effective preventive and therapeutic strategies. this retrospective cohort study was approved by the institutional review board of korea university guro hospital (kugh). hospitalized patients under years of age with a discharge diagnosis of ari from january to december were enrolled. ari was diagnosed based on one of symptoms or signs; fever of more than . °c, cough, rhinorrhea, sore throat, tonsillar injection, wheezing, crackle, chest wall retraction. nasopharyngeal aspirates from all patients were obtained within hours of admission for multiplex rt-pcr assay to detect the following common respiratory viruses: influenza virus a and b (ifa, ifb), respiratory syncytial virus a and b (rsv a, rsv b), parainfluenza virus - (piv , piv , piv , piv ), human coronavirus e and oc (hcv- e, hcv-oc ), human rhinovirus (hrv), human enterovirus (hev), adenovirus (adv), human bocavirus (hbv), and human metapneumovirus (hmpv). only a single sample was taken from the patients during the admission. rt-pcr results were used to evaluate the incidence of respiratory viruses. laboratory parameters on the first day of admission, as well as overall clinical profiles including baseline characteristics, presenting symptoms and signs, treatments and clinical outcomes, were analyzed in patients with single-virus infections. any possible bacterial coinfections were excluded by sputum and blood culture and clinical course. mycoplasma coinfections were excluded by sputum pcr or by a serial increase of antibody titer or a high initial igm titer without history of recent respiratory disease. respiratory samples from children were collected by nasopharyngeal aspiration. the presence of each virus was determined using the seeplex rv rt-pcr assay (seegene, inc., seoul, korea). two sets of primers were designed based on conserved regions of genetic sequences for the respiratory viruses mentioned above. amplified pcr products were analyzed by electrophoresis on % agarose gel and were compared with the reference band size provided by the manufacturer. ) statistical analysis was performed using ibm spss statistics ver. . (ibm co., armonk, ny, usa). data were expressed as mean± standard deviation or as percentages, as appropriate. a multipleway analysis of variance was run to test differences between viral groups. then, each viral group was compared with the other viral groups using chi-square tests for categorical data and unpaired student t tests for continuous data. a p-value < . was considered statistically significant. from respiratory samples of a total of , patients, viruses were detected in , patients ( . %). after excluding patients with bacterial coinfections (mycoplasma in cases, other bacteria in cases, and mycoplasma with other bacteria in cases), , patients ( . %) were found to have viral aris. in those cases, the most common were hrv ( of , , . %), adv ( of , , . %), and rsv a ( of , , . %) (fig. viruses were detected more frequently in younger cases (≤ year old vs. > years old was . % vs. . %, p= . ). cases with multiple viruses tended to be male-dominant when compared to single-virus cases ( . % vs. . %, p= . ) ( table ) . a total of , single-virus cases were analyzed for their distribution at different ages (fig. ) . seventy-five percent of single-virus cases were identified in patients younger than years old. while rsv a, rsv b, hbv, and piv were major pathogens in patients younger than one year of age, ifa, ifb, hev, and adv were common in patients older than three years of age. hrv was a predominant single virus that was seen across all ages. although not as common as hrv, hmpv was also prevalent across all ages. during the observation period, many viruses showed seasonal patterns. ifa and ifb were most common from winter to spring, while hmpv was predominant in the spring, and piv and hev common from spring to summer. rsv a and b peaked during fall and winter biennially. although not common, hbv was prevalent during the spring and hcv was seen during fall and winter. hrv and adv did not show a discrete seasonal pattern (fig. ). although each virus prevailed throughout preferred seasons during the observation period, there was annual deviations. there were no differences in comorbidities such as premature birth, congenital heart disease and chronic lung disease among viral ari cases. rsv a, rsv b, and hmpv were more associated with lower ari compared with other viruses. croup was most commonly associated with piv and hcv ( . %, . %), while pneumonia was associated with rsv a, rsv b, and hmpv ( . %, . %, and . %). additionally, bronchiolitis was associated with rsv a and b ( . % and . %), while asthma exacerbations were associated with hrv ( . %, p< . ) only. most cases of ari presented with cough and rhinorrhea (data not shown). while fever was associated with many viruses, rsv a, rsv b, and hrv were not significantly associated with fever ( . %, . %, and . %, p< . ). adv and piv were associated with fever lasting more than days ( . %, headache was significantly associated with hev and adv ( . % and . %, p< . ). antibiotics were used more frequently in cases with adv and hmpv ( . % and . %, p< . ), and steroids were used more frequently in cases with hrv ( . %, p< . ). rsv was associated with greater nebulizer use, o supplementation and intensive care unit care, and it was also associated with a longer hospital stay ( table ). cases of adv, hev, and hrv had a greater tendency to show leukocytosis over , /l when compared to other viruses ( . %, . %, and . %, p< . ). although leukopenia less than , /l was observed more with ifa, ifb ( . %, . %, p< . ), an absolute neutrophil count less than , /ml was more associated with piv ( . %, p< . ). while neutrophil-dominant inflammation, represented by a neutrophil-to-lymphocyte ratio ≥ , was more associated with ifa, ifb, adv, hrv, and hev, lymphocyte-dominant inflammation with a neutrophil-to-lymphocyte ratio ≤ was more associated with rsv a, rsv b, piv, and hmpv. monocytosis greater than , /l was observed more with rsv a, rsv b, and adv ( . %, . %, and . %), while eosinophilia greater than /l was observed more with hrv than with other viruses ( . %, p< . ). although not common, thrombocytopenia less than , /l was observed more with hcv-oc ( . %, p= . ). ele vated levels of aspartate aminotransferase and alanine aminotransferase were associated more with piv and rsv a. c-reactive protein levels less than mg/l were associated with ifb, rsv a, rsv b, and piv ( . %, . %, . %, and . %, p< . ), and an erythrocyte sedimentation rate less than mm/hr were associated with ifa, rsv a, and piv ( . %, . %, and . %, p< . ). both c-reactive protein levels greater than mg/l and erythrocyte sedimentation rates greater than mm/hr were associated with adv (p< . ) ( table ). in this study, viruses were detected in . % of all ari cases, but when cases of bacterial coinfection were excluded, viruses were detected in . % of cases. bacterial infection may be erroneously estimated because we counted all possible cases, even if not confirmed. additionally, we did not routinely test for bacterial infection at presentation. nevertheless, viral detection rates in this study were comparable to those of other studies. , ) although hrv, adv, and rsv are the most prevalent viruses, rsv was more present as a single pathogen, whereas hrv and adv were more seen with multiple pathogens. rsv, hrv, and piv were the most prevalent single pathogens in patients younger than years old. after the age of three, the prevalence of rsv decreased while the prevalence of if and adv increased, making hrv, if, and adv the most prevalent after three years of age. the younger age group shows higher virus positivity compared with the older age group, which may reflect prolonged shedding of respiratory viruses in young children. ) cases with multiple detected viruses are more likely to be males, while the sex ratio in single-virus cases is concordant with that of cases examined. the incidence of infectious disease is also reported higher among males. , ) it is possible that after viral infection, viral clearance may be delayed in males, resulting in a greater chance for viruses to accumulate. the significance of multiple viral coinfections has been controversial. while it is unlikely to be associated with more severe illness, , , ) specific pathogen pairs such as rsv with influenza virus may be associated with increased severity. ) studies report strong causal attribution of rsv, if, and hmpv, less strong evidence for hrv and no attribution of adv, hbv and hcv, [ ] [ ] [ ] which is in agreement with the ratio of single to multiple detections of each virus in our study. quantitative viral analysis may help to distinguish active infection from viral shedding in cases with multiple viral coinfection. ) because the clinical significance of multiple viral infections remains controversial and hard to interpret, we compared single-virus cases to understand characteristics of each virus. seasonality of some respiratory viruses is an important clue for early recognition. in addition to the strong seasonal patterns observed with if, rsv, and hmpv, it is interesting that rsv a and b have an apparent biennial seasonality. while hrv and adv are prevalent throughout the year, seasonal patterns of viruses are discrete. each respiratory viruses differ in seasonal onset as well as activity, but this can slightly vary from year to year. knowing the seasonality of each virus will help to plan effective control strategies and to make epidemiological diagnosis. analysis of virus-specific clinical profiles showed that cases of rsv a, rsv b, hbv, piv, and hcv affect patients who are signifi cantly younger than those with ifa, ifb, adv, and hev aris. premature birth history, and presence of congenital heart disease and chronic lung disease are not associated with any specific virus. while fever is a common symptom of ari, cases of rsv and hrv are significantly afebrile. infants younger than months with rsv infection tend to be afebrile, which may be due to a lack of a pyrogenic cytokine response. , ) while patients with afebrile rsv cases are significantly younger than febrile rsv cases ( . months vs. . months, p= . for rsv a, . months vs. . months, p= . for rsv b), there is no age difference between afebrile and febrile hrv cases ( . months vs. . months, p= . ) in this study, suggesting that pyrogenic immune responses might be different depending on the virus. some viral respiratory infections are associated with asthma and asthma exacerbation. ) cases of rsv and hrv ari present more commonly with wheezing, but eosi nophilia and a diagnosis of asthma exacerbation are associated only with hrv infection in this study. a study performed in children with acute wheezing illness reports that rsv is the predominant virus in patients with no previous wheezing, whereas hrv is predominant in patients with a history of wheezing. ) whether rsv infection causes asthma is still debatable, but it is possible that lower ari with pathogens such as hrv and rsv could precipitate the development of asthma, especially in children with atopic features. ) the total leukocyte, neutrophil, lymphocyte and monocyte counts, and specific associations between blood cells, especially the neutrophil-to-lymphocyte ratio have been used as markers of inflammation and infection. , ) viral aris show leukocytosis rather than leukopenia and furthermore, this study shows that if, hrv, hev, and adv are associated with neutrophil-dominant inflammation whereas rsv, piv, and hmpv are associated with lymphocyte-dominant inflammation. of note, monocytosis is observed with rsv a, rsv b, and adv aris. other inflammatory markers such as erythrocyte sedimentation rate and c-reactive protein levels are not significantly increased in most viral aris except for adv infection. thus, adv ari present with neutrophildominant leukocytosis, a high erythrocyte sedimentation rate and a high c-reactive protein level, which mimics the response to bacterial infections. the decision to use antibiotics is determined by clinical and laboratory evaluation. in this study, antibiotics are used more often in cases of hmpv and adv, and they are also used more when infection in young infants who are potentially immune-incompetent, manifests as a lower ari. concerns of bacterial superinfection often cause us to continue the use of antibiotics, even after identification of a viral pathogen. application of a clinical rule without consideration of virus-specific clinical and laboratory characteristics may lead us to make a wrong decision. ) this study has some limitations. not all patients were examined for a bacterial etiology at presentation, and the use of antibiotics might affect bacterial detection. thus, it is possible that this study overestimates the number of viral aris. also, the rt-pcr assay used in this study did not quantify the viral load. it is still possible that even the single virus detected in a patient with a symptomatic ari might not be the causative pathogen. in conclusion, the epidemiology of predominant viral pathogens associated with aris among children differs seasonally as well as regionally. using routinely available hospital laboratory data will be a great addition to current virus surveillance and will help to identify the viruses circulating in the community and to predict their timing, trends and impact. ) in addition to viral identification, recognition of the virus-specific clinical and laboratory patterns presented in this study will expand our understanding of the epidemiology of viral ari and will help us to improve management and prevention of viral infections. respiratory virus infections aetiology of acute respiratory tract infections in hospitalised children in cyprus clinical impact of rt-pcr for pediatric acute respiratory infections: a controlled clinical trial respiratory pathogens in children with and without respiratory symptoms new respiratory viral infections the role of infections and coinfections with newly 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viruses in acute lower respiratory infections in children under five years: a systematic review and meta-analysis respiratory viral detection in children and adults: comparing asymptomatic controls and patients with community-acquired pneumonia fever and the thermal regulation of immunity: the immune system feels the heat epidemiology, clinical characteristics, laboratory findings and severity of respiratory syncytial virus acute lower respiratory infection in malaysian children role of viral respiratory infections in asthma and asthma exacerbations a molecular epidemiological study of respiratory viruses detected in japanese children with acute wheezing illness rhinovirus and the initiation of asthma the neutrophil-lymphocyte count ratio in patients with community-acquired pneumonia are there standardized cutoff values for neutrophil-lymphocyte ratios in bacteremia or sepsis? development and internal validation of a clinical rule to improve antibiotic use in children presenting to primary care with acute respiratory tract infection and cough: a prognostic cohort study a new laboratory-based surveillance system (respiratory datamart system) for influenza and other respiratory viruses in england: results and experience from no potential conflict of interest relevant to this article was re ported. key: cord- - o k og authors: lin, chun; chen, huanzhu; he, ping; li, yazhen; ke, changwen; jiao, xiaoyang title: etiology and characteristics of community-acquired pneumonia in an influenza epidemic period date: - - journal: comp immunol microbiol infect dis doi: . /j.cimid. . . sha: doc_id: cord_uid: o k og purpose: the etiology of community-acquired pneumonia (cap) in hospital patients is often ambiguous due to the limited pathogen detection. lack of a microbiological diagnosis impairs precision treatment in cap. methods: specimens collected from the lower respiratory tract of cap patients, viruses were measured by the single-plex real-time pcr assay and the conventional culture method was exploited for bacteria. results: among the patients, there were ( . %) pure bacterial infections, ( . %) yeast infections, ( . %) pure viral infections, ( . %) viral-yeast co-infections, and ( . %) viral-bacterial co-infections. the two most abundant bacteria were acinetobacter baumannii and klebsiella pneumoniae, whereas the most common virus was influenza a. conclusions: non-influenza respiratory microorganisms frequently co-circulated during the epidemic peaks of influenza, which easily being ignored in cap therapy. in patients with bacterial and viral co-infections, identifying the etiologic agent is crucial for patient’s therapy. community-acquired pneumonia (cap) is a common disease and a significant cause of morbidity and mortality worldwide [ ] . the severity of the disease can be affected by various factors including age, immune status of the host, and the single or mixed infection. the etiology of cap is essential because most respiratory illnesses lead to similar clinical presentations. currently, the ambiguous of etiology and lack of a microbiological diagnosis in cap impairs pathogen-directed antimicrobial therapy [ ] . in the last decade, the long-term implications of respiratory viral infection among susceptible hosts have increasingly been recognized [ ] . although approximately million cases of viral cap occur every year, the incidence of viral pneumonia still has been underestimated. in hospital, bacteria remain the first consideration in pulmonary infections, and antibiotic therapy is still the cornerstone of the majority of cap. influenza virus infection (ivi) causes annual epidemics that result in estimated billion infections, including - million severe cases and , - , mortality cases worldwide each year [ ] . ivi can cause primary viral pneumonia, which may progress to a potentially fatal outcome [ ] . it is difficult to distinguish the cause of cap induced by flu, other respiratory viruses or bacteria based solely on clinical symptoms, and the conventional lab biomarkers of viral and/or bacterial infections do not differ in influenza-positive compared with influenza-negative patients [ ] . influenza can temporarily suppress host immune defenses, leading to bacterial complications [ ] . secondary bacterial pneumonia is a frequent cause of excess mortality during influenza epidemics, but the epidemiology remains unclear [ ] . hospital admissions during the influenza epidemic season showed a moderate to strong association between influenza and bacterial pneumonia, whereas the interaction is modest or non-existent during nonepidemic periods [ ] . bacteria including streptococcus pneumoniae, hemophilia influenzae, and staphylococcus aureus, often interact with respiratory viruses (esp. the influenza virus), and shape the outcome of respiratory infection [ ] . studies have shown the evidence of bacterial invasion is more than % in influenza-infected cases, and the high rates of co-infections exist in these patients, indicating that viral infection may enhance both susceptibility and severity of subsequent bacterial infection [ , , ] . in vitro studies have shown that influenza infection enhanced susceptibility to pneumococcal pneumonia by about -fold in a week, but the interaction between influenza and bacteria is not limited in pneumococcal pneumonia [ ] . in the hospital, viral testing among patients with respiratory symptoms is uncommon [ ] , and the determination of the microbiological etiology is severely hampered in cap by the difficulty of obtaining specimens from the infected area (esp. from lower respiratory tract), samples could be easily contaminated by respiratory conditioned pathogens. at present, the epidemiological history of influenza-like illness (ili) and the incidence and clinical presentation of cap caused by viruses other than influenza during an influenza epidemic season were limited [ ] [ ] [ ] . in this study, we report on the surveillance of respiratory microorganisms, and its laboratory biomarkers, in cap patients admitted to a hospital during the january to august . the specific microbiome patterns, their clinical significance, and the antibacterial/antiviral treatment were analyzed in these patients simultaneous. we collected sputum specimens from cap patients who were admitted to the first affiliated hospital of shantou university medical college, shantou city, guangdong province in china from january to august . pneumonia was diagnosed as an acute illness with fever, cough, or dyspnea / tachypnea, and at least one new focal chest sign, which was confirmed by finding lung shadowing on the chest radiographs that were likely to be new and without other obvious causes [ ] . patients with cystic fibrosis or hiv infection were excluded from our study. the sputum specimens were collected and stored in a swab storage solution (copan, italy) and stored at − °c. the nucleic acid was extracted using a beads viral dna/rna extraction kit (tianlong, china) and np nucleic acid extractor (tianlong, china). respiratory viruses were detected using an agpath-id™ one-step rt-pcr kit (applied biosystems, usa) and using a real-time pcr system (applied biosystems, usa). negativecontrols (depc-treated water) were included in each analytical procedure. fourteen respiratory viruses including influenza a, influenza b, parainfluenza viruses - (piv - ), respiratory syncytial virus (rsv), coronaviruses e, oc , hku , and nl , human metapneumovirus (mpv), human rhinovirus (hrv), bocavirus (bov), and adenovirus (adv) were measured. all the above procedures were followed the manufacturer's protocol. all sputum specimens were cultured according to china national standard protocols to detect respiratory bacteria and yeast. specimens were inoculated in blood agar, eosin methylene blue agar, and chocolate agar and incubated for - h at °c. colony identification was undertaken using a vitek compact full automatic identification system (biomérieux, france data were analyzed by spss . . categorical variables were summarized and compared by χ test or fisher's exact test, the comparisons between the two groups were made by using the bonferroni test level adjustment method. continuous variables with normal distributions were used mean ± standard deviation(sd) to describe and compare using multiple variance of multiple samples. medians and interquartile ranges (iqr) were used to describe non-normal distribution of continuous variables, with comparisons based on the nonparametric kruskal-wallis h test for significance of the differences among more than two groups and the mann-whitney u test was used for significance of the differences between two groups. in table , patients included males and females. fifteen cases were children with a mean age of - yrs, and cases were adults with a mean age of ( - yrs). twenty patients ( . %) previously had a pulmonary-associated disease, which included cases of copd, cases of tuberculosis, cases of pulmonary heart disease, and cases of emphysema. a total of patients ( . %) had at least underlying disease, including high blood pressure, diabetes, heart disease, epilepsy, tumors and hepatitis. after admission, broad-spectrum antibiotics were prescribed to patients ( . %) as an empiric or definitive therapy, and patients were given antiviral treatment. patients had been given antiviral plus antibiotic treatment. the most frequently used antibiotic were quinolones, cephalosporins and β-lactams, and the most frequently used antiviral agents were oseltamivir, ribavirin, recombinant human interferon α- b. unfortunately, of the patients died, all were adults (male: ; female: ), aged - yrs, with an average age of . yrs. a single-bacterial infection was found in patients ( . %), a single-yeast infection was found in patients ( . %) and a singleviral infection was found in patients ( . %). co-infections (virus + bacterium and virus+ yeast) was found in patients ( . %), and the pathogens could not be unidentified in cases ( . %) ( table ) . bacterial infection was identified in ( . %) of the patients. the two frequently detected bacteria were a. baumannii and k. pneumoniae, which accounting for . % of the bacterial infection. in addition, there were cases infected by p. aeruginosa and s. aureus, respectively, and cases were e. coli (table ). three cases had more than one bacterial infection, which was s. aureus plus k. ornithinolytica, a. baumannii plus p. aeruginosa and s. pneumoniae plus e. aerogenes, respectively. viral infection was identified in / ( . %) of the patients. influenza a, influenza b, piv and rsv were the most common viruses among all cases, followed by e, mpv, oc , and piv (table ) . specifically, patients had more than one virus, accounting for . % of the virus-positive group. dual viral infection was observed in rsv coinfected with hrv, piv and hku , respectively, piv co-infected with bov, hku and e, respectively, and influenza a co-infected with mpv and hrv, respectively. patients had co-infections, including a single virus and a single bacterial/yeast co-infections in cases, two viruses and a single bacterium /yeast co-infections in cases. in these patients, bacterial/yeast co-infections with non-influenza viruses were more frequent than bacterial /yeast and influenza co-infections (table ). fever, cough, and shortness of breath were the most common clinical signs observed in %, . %, and . % of the bacterial cases, in %, . %, and . % of the viral cases, in %, %, and % of the yeast cases, and %, % and % of the co-infections cases, respectively. the bacterial group was significantly different from the viral group and the co-infected group in cough (p＜ . ), at the same time, the viral group and the yeast group in cough also has significant differences(p＜ . ) ( table ) . patients with pure non-influenza infection and patients with non-influenza and bacterium / yeast co-infection were admitted to the icu, and patients died in the non-influenza co-infected with bacterium / yeast group (table ) . in these patients, pharyngeal discomfort was only found in the pure influenza group. bacterium / yeast co-infected with non-influenza viruses were more common than bacterium/ yeast and influenza co-infections. wbc counts in the non-influenza groups were significantly higher than in the influenza groups, and the highest wbc counts were observed in the non-influenza and bacterium / yeast co-infected group. the plt count (p < . ) also had the similar trend. in the flu season, the cases of a. baumannii 's infection was greater than those in the non-flu season ( cases vs. case). it was noteworthy that bacteria such as h. influenzae, b. catarrhalis, s. maltophilia, e. cloacae, k. ornithinolytica, s. marcescens, s. haemolyticus, s. pneumonia were not detected in the non-flu season. the emergence of influenza viruses (flu a and flu b) was more common in the flu season, other virus like mpv and e were smaller than flu in the influenza season (table ). pathogen-directed therapy avoids unnecessary antibiotic or antiviral use, facilitating more timely and hence more effective use of drugs, help prevent secondary spread of infection, all of which shorten hospital stays and have a major impact on patient management and disease prognosis [ ] [ ] [ ] . accurate and rapid etiologic diagnosis is crucial to pathogen-directed therapy. to date, the gold standard for cap diagnosis is still based on the radiographic findings [ ] . the radiological findings of multilobar infiltration or pleural effusion generally indicate greater severity [ ] . although etiological studies have been performed, current microbial diagnostic tests frequently do not allow clinicians to rule out bacterial infection with certainty [ ] . obtaining an qualified sample may be a challenge as specimens were easily contaminated by the bacteria located in upper airways [ ] . for increasing the detection rate of pathogens, esp. for infection occurred in the lower respiratory tract, alveolar lavage fluid may be better in reflecting infectious status than the sputum. however, alveolar lavage fluids require semi-invasive surgery, which is certainly not routine in non-intubated patients, esp. to children [ ] . under this condition, taking sputum as the sample is more practical. therefore, the relevant cap guidelines recommend sputum examinations for patients with moderate to severe cap. in this study, a high microbial yield can be achieved when real time pcr assays plus the conventional diagnostic methods, such as bacterial cultures [ , ] . the single-plex real time table the clinical symptoms of bacterium group, virus group, yeast group and co-infected group. pcr assay were exploited for detecting respiratory viruses, which could improve diagnostic efficacy, particularly in diagnosing respiratory viral infections [ ] . etiologic diagnosis in the cap patients reached . %, we still had . % cap patients without clearly microorganisms diagnose. for patients with comorbidities, the use of antibiotics prior to sampling may obscure a potential bacterial detection [ ] . previous studies revealed that etiology remains unknown in approximately one-half of the cases [ , ] , indicating that establishment of a precise pathogen diagnosis for cap patients is challenging. better diagnostic methods are warranted to distinguish viral from p＜ . when the influ group is compared with non-influ group ** p＜ . when the influ group is compared with non-influ + bacterium/yeast group; Δ p＜ . when the non-influ group is compared with influ + bacterium/ yeast group; ▲▲ p＜ . when the influ + bacterium/yeast group is compared with non-influ + bacterium/ yeast group. the number of bacterial infections and viral infections in the influenza seasons and non-flu seasons in . bacterial cap, which will clarify if these viral infections cause cap on their own or merely predispose the patient to bacterial co-infections [ ] . differences in epidemiology make the knowledge of local etiology crucial for the appropriate choice of empirical antimicrobial treatment [ ] . even there was a severe influenza outbreak during our study, other respiratory viral infections were more than influenza infection ( . % vs. . %). in all pathogens that cause cap, bacteria occupied the majority of pathogens, and the most common bacterium was a. baumannii. our results was not in keeping with the previous studies, in which the most frequently identified pathogens were burkholderia pseudomallei ( %), s. pneumoniae ( %), k. pneumoniae ( %) and h. influenza ( %), respectively [ ] . although a. baumannii is an important pathogen of nosocomial infections, we often isolated it from infectious patients who is first admitted to the hospital, indicating that a. baumannii has a certain popularity in this area. most communityacquired cases of a. baumannii pneumonia are from tropical or subtropical countries in the asia-pacific region. in thailand, severe cap is caused by a. baumannii, and its mortality is times higher than hospital-acquired a. baumannii pneumonia [ , ] . in another aspect, most of our patients had underlying diseases, we could not exclude the possibility that patients had the bacterial infection in last hospital admission. the pathogen remains in the respiratory tract, causing clinical symptoms when the body's immunity is reduced. the outbreak of a. baumannii infection is also associated with multidrug resistance, and carbapenems have been considered to be a major factor in resistance to multidrug-resistant a. baumannii infection [ ] . moreover, the mortality of carbapenem-resistant a. baumannii pneumonia patients is higher than that of carbapenem-sensitive a. baumannii pneumonia patients [ , ] . s. pneumoniae and h. influenza, as co-pathogens, often seem to be part of a mixed infection (virus and bacterium) in adults with cap [ ] , representing the most common combination with respiratory viruses [ ] . the incidence of mixed infections was significant among patients admitted to the hospital with cap [ ] . in our study, . % of cap patients had single virus, and the co-infections (virus + bacterium and virus+ yeast) were found in . % cap patients. the incidence of coinfections was in agreement with a previous study [ ] . the most common bacterium co-detected with a virus was k. pneumoniae, as well as the yeast in our study, indicating that geographical variation exists in the prevalence of bacterium or yeast strains expressing factors that enable efficient disease potentiation during viral epidemics, which should be considered one explanation for regional differences in severity [ ] . the diversity in etiology revealed that understanding the local etiology of cap is essential to inform clinical management decisions. respiratory viruses usually followed seasonal patterns of activity [ ] , and epidemic influenza was noted in our studied area, which was active throughout the study period, and there was a high detecting rate of influenza a/influenza b was from january to may. during an influenza epidemic season, a high percentage of samples from cap patients contained at least one virus, and dual infections were very common [ ] . the respiratory virus other than influenza accounted for . % viral cap in our study, revealing that respiratory viruses can be cocirculating even during the highest epidemic peaks of influenza that showed a seasonal distribution. bacterial or fungal infections secondary to influenza viral infections were common [ ] , though we could not exclude that the increasing co-infections cap was due to secondary viral infection. as the clinical signs and symptoms overlapped between influenza and non-influenza viruses, pathogen-directed therapy is difficult if only based on clinical symptoms. studies have shown that delayed antiviral treatment is associated with high risk of progression to severe disease required icu admission or prolonged hospital stay, and even causing death [ ] . the microbiome's characterization and diversity are closely linked to the host status, and the host immune response may play a determining role in the infectious exacerbation of cap. the intrinsic complexity of the host-microbiome relationship currently limits the clinical indications of microbiome analysis [ ] . diabetes and high blood pressure were the most common underlying disease, which were known to be associated with an increased risk of complications and death among critically ill patients [ ] . detection of respiratory secretions by pcr method has provided a rapid and definitive diagnosis for the ilis in hospitalized adults [ ] . unfortunately, it is not routine test in most clinic labs. appropriate empiric therapy is likely to be suboptimal without narrowing diagnostic possibilities to the most likely possibilities pending definitive pathogen identification [ ] . effective, targeted therapy for cap requires an understanding of the heterogeneity of etiology and the individual host response to infection [ ] . this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. this study was approved by the ethics committee of shantou university medical college and informed consents were obtained from the patients. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. swedish guidelines on the management of community-acquired pneumonia in immunocompetent adults-swedish society of infectious diseases , scand comprehensive molecular testing for respiratory pathogens in community-acquired pneumonia comparison of the luminex xtag rvp fast assay and the idaho technology filmarray rp assay for detection of respiratory viruses in pediatric patients at a cancer hospital influenza (seasonal) fact sheet no metabolomics investigation reveals metabolite mediators associated with acute lung injury and repair in a murine model of influenza pneumonia relationship of influenza virus infection to associated infections in children who present with influenza-like symptoms the pathology of influenza virus infections do specific virus-bacteria pairings drive clinical outcomes of pneumonia? viral and bacterial interactions in the upper respiratory tract bacterial complications of respiratory tract viral illness: a comprehensive evaluation the co-pathogenesis of influenza viruses with bacteria in the lung bench-to-bedside review: bacterial pneumonia with influenza -pathogenesis and clinical implications identifying the interaction between influenza and pneumococcal pneumonia using incidence data viral etiology of influenza-like illnesses in viral etiology of medically attended influenza-like illnesses in children less than five years old in suzhou, china the epidemiology and etiology of influenza-like illness in chinese children from etiology and clinical characteristics of influenzalike illness (ili) in outpatients in beijing guidelines for the management of adult lower respiratory tract infections-summary comparison of the filmarray respiratory panel and prodesse real-time pcr assays for detection of respiratory pathogens the effect of rapid respiratory viral diagnostic testing on antibiotic use in a children's hospital community-acquired pneumonia standardized interpretation of paediatric chest radiographs for the diagnosis of pneumonia in epidemiological studies infectious diseases society of america/american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults diagnostic tests for agents of community-acquired pneumonia microbiome, biofilms, and pneumonia in the icu etiology of community-acquired pneumonia: increased microbiological yield with new diagnostic methods community-acquired pneumonia in chile: the clinical relevance in the detection of viruses and atypical bacteria etiology of community-acquired pneumonia and diagnostic yields of microbiological methods: a -year prospective study in norway bacterial coinfections in lung tissue specimens from fatal cases of pandemic influenza a (h n ) -united states microbial etiology of community-acquired pneumonia in the adult population of municipalities in eastern finland british thoracic society standards of care c. bts guidelines for the management of community acquired pneumonia in adults respiratory viruses associated with community-acquired pneumonia in children: matched case-control study community-acquired acinetobacter baumannii pneumonia acinetobacter baumannii: evolution of a global pathogen pneumonia caused by extensive drug-resistant acinetobacter baumannii among hospitalized patients: genetic relationships, risk factors and mortality comparison of pneumonia-and non-pneumonia-related acinetobacter baumannii bacteremia: impact on empiric therapy and antibiotic resistance pneumococcal coinfection with human metapneumovirus british thoracic society standards of care c. british thoracic society guidelines for the management of community acquired pneumonia in childhood viral pneumonia the use of a multiplex real-time pcr assay for diagnosing acute respiratory viral infections in children attending an emergency unit critical illness from pandemic influenza a virus and bacterial coinfection in the united states predictors and outcomes of respiratory failure among hospitalized pneumonia patients with h n influenza in taiwan early blood glucose control and mortality in critically ill patients in australia adult human metapneumonovirus (hmpv) pneumonia mimicking legionnaire's disease genomic landscape of the individual host response and outcomes in sepsis: a prospective cohort study this study was supported by the shantou science and technology project (grant numbers. ).this study was made possible by the generous support of the american people through the united states agency for international development (usaid) emerging pandemic threats program- (predict- ) (cooperative agreement no. aid-oaa-a- - ). the contents were the responsibility of the authors and do not necessarily reflect the views of usaid or the united states government. key: cord- -b tj x authors: giersing, birgitte k.; vekemans, johan; nava, samantha; kaslow, david c.; moorthy, vasee title: report from the world health organization’s third product development for vaccines advisory committee (pdvac) meeting, geneva, – th june date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: b tj x abstract the third meeting of who’s product development for vaccines advisory committee (pdvac) was held in june , with a remit to revisit the pathogen areas for which significant progress has occurred since recommendations from the meeting, as well as to consider new advances in the development of vaccines against other pathogens. since the previous meeting, significant progress has been made with regulatory approvals of the first malaria and dengue vaccines, and the first phase iii trials of a respiratory syncytial virus (rsv) vaccine candidate has started in the elderly and pregnant women. in addition, pdvac has also supported vaccine development efforts against important emerging pathogens, including middle eastern coronavirus (mers cov) and zika virus. trials of hiv and tuberculosis vaccine candidates are steadily progressing towards pivotal data points, and the leading norovirus vaccine candidate has entered a phase iib efficacy study. who’s immunization, vaccine and biologicals (ivb) department is actively working in several pathogen areas on the recommendation of pdvac, as well as continuing horizon scanning for advances in the development of vaccines that may benefit low and middle income countries (lmics), such as the recent licensure of the enterovirus (ev ) vaccine in china. following on from discussions with who’s strategic advisory group of experts (sage) on immunization, pdvac will also look beyond licensure and consider data needs for vaccine recommendation and implementation to reduce the delay between vaccine approval and vaccine impact. who's pdvac was established by the department of immunization, vaccines and biologicals (ivb) in , following a review of who's process for strategic priority setting for vaccines. the need for a group to advise who specifically on vaccine product development was highlighted, to accelerate vaccine availability and ensure accessibility of vaccines to low and middle income countries (lmics). pdvac's remit is to advise on the product development strategy of vaccine candidates at phase ii of clinical evaluation or earlier, and to report its proceedings to the who's principal committee on immunization policy recommendations: the strategic advisory group of experts on immunization (sage). the pdvac committee has a critical role in assessing the evolving vaccine development landscape and in helping to define where and how who can be most impactful, according to three criteria: likelihood of a product emerging from the pipeline, as defined by probability of technical and regulatory success, and the extent of awareness, activity and investment in a given area, a clear role for who with perceived added value for engagement in the pathogen area. typically, who engages in a pathogen area by working with a broad set of key vaccine development stakeholders to develop consensus on pivotal clinical trial design, vaccine roadmaps, or guidance documents on desired vaccine properties, referred to as preferred product characteristics (ppcs). ppcs define who preferences for the properties of vaccines to be used in lmics that are - years from licensure, and inform target product profiles in use by manufacturers and funders for vaccines. pdvac also encourages developers to be aware of the process and requirements for who prequalification (pq). who prequalification is a service to unicef and other un agencies that purchase vaccines once they have been licensed, to determine the acceptability, in principle, of vaccines from different sources for supply to these agencies. it aims to ensure that diagnostics, medicines, vaccines and immunizationrelated equipment and devices for high burden diseases meet global standards of quality, safety and efficacy, and are appropriate for use in lmics contexts in order to optimize the potential benefit of these interventions [ ] . the third pdvac meeting was held in geneva from - th june . dr. jean-marie okwo-bele, director of ivb, opened proceedings with a synopsis of the significant milestones in vaccine development in the nine months since the previous meeting in september : the first dengue and malaria vaccines have been licensed or achieved the equivalent of licensure, respectively, the first rsv vaccine candidate has entered phase iii studies in the elderly and pregnant women, the most advanced hiv vaccine candidate has met its endpoints in the interim analysis of a phase ii study, and preparations to commence an efficacy study are underway, who convened the mers-coronavirus r&d community, and a phase i clinical study is now underway (nct ), ebola virus vaccines are under review and have progressed to the point of consideration for licensure in record time, there are co-ordinated efforts to develop a zika virus vaccine as expeditiously as possible. a pdvac working group has overseen the development of a zika virus vaccine target product profile (tpp), and developed regulatory considerations towards phase i and emergency use authorization. in addition to these significant advances in vaccine development, the uk government published in may the report on 'tackling drug-resistant infections globally' that it commissioned in collaboration with the welcome trust [ ] . the report highlights the urgent need to reduce reliance on currently available antimicrobials, without which today's , deaths per year from drug resistant microbes is forecasted to increase to million, by . the cost in terms of lost global production due to infections that are not controllable due to antimicrobial resistance (amr) is estimated to be $ trillion by if no action is taken [ ] . the development of vaccines against pathogens that are currently controlled by antimicrobials has become an imperative, as they have the potential to reduce the prevalence and spread of drug resis-tance, as well as to reduce the use of antimicrobials more broadly [ ] . the decade for vaccines' global vaccine action plan (gvap) mid-term review, required an assessment of progress against objectives since its inception in , and strategic planning to achieve the stated targets within the remaining years. part of pdvac's remit is to review the vaccine development pipeline and consider the priority activities for ivb, within this context. during the remaining timeframe of gvap, a number of vaccines could reach licensure, and who needs to ensure early engagement with policy makers regarding potential vaccine implementation, as well as alignment with gavi's vaccine investment strategy. to facilitate information sharing, and tracking of progress within the global vaccine development community, the who has established and maintains an online 'vaccine pipeline tracker' in which information regarding all current clinical studies in several different pathogen areas can be found [ ] . in addition, landscape analyses for pathogens from the meeting have been collated within a special issue of the journal 'vaccine' and all are available through open access [ ] . these documents are authored by independent subject matter experts and review the status of vaccine candidate development, as well as assessing possible pathways to regulatory approval. pdvac reports progress on the global vaccine development pipeline to who's strategic advisory group of experts (sage) on immunization. at the meeting in april , advances in the development of interventions (vaccines and monoclonal antibodies) for respiratory syncytial virus (rsv) were presented for information. the reports from the october and april sage meetings are available online [ , ] . much of the discussion focused on the need to better understand the key factors for early for implementation, as well as safety and efficacy data to support the assessment of a vaccine for policy recommendation. as emerging vaccines are likely to require new vaccination platforms, such as maternal immunization, or visits outside of the current vaccination schedule, such as for the recently licensed malaria vaccine rts,s, cost-effectiveness data informing their optimal use and potential impact must be generated in line with conventional clinical data required for regulatory approval, to minimise the delay between vaccine licensure and uptake [ ] . the goals of this third pdvac meeting were to revisit the pathogen areas where there has been significant progress to report since recommendations from the meeting, as well as to: review status of vaccine development in new pathogen areas where there has been significant vaccine development progress, or where there is significant disease burden but r&d has stalled, refine the workplan and strategic directions for ivb in specific pathogen areas, identify cross-cutting issues that accelerate vaccine development or prepare for policy decisions, where appropriate, consider how to better align pdvac's vaccine development activities and strategies with other areas of research, to inform the vaccine development community regarding steps to be considered beyond vaccine licensure, and who processes for vaccine policy recommendation. the gvap is a -year strategic framework derived from the decade of vaccines collaboration [ ] to prevent millions of deaths by through more equitable access to existing vaccines for people in all communities. within this framework is a specific objective that supports research and development of innovations that will maximise the benefits of immunization, with indicators for progress towards development of hiv, malaria, tuberculosis and influenza vaccines. the gvap has just completed its midterm review stage, and following the recommendation from sage [ ] , the gvap assessment will highlight advances made in these areas. these four pathogen areas are standing agenda items for discussion at pdvac. in , mycobacterium tuberculosis (mtb) killed . million people ( . million of whom were co-infected with hiv) and is now the world's most deadly infectious disease [ ] . approximately , cases/annum are multi-drug resistant (mdr) or extensively drug resistant (xdr) and some strains are untreatable. in , six million new cases of mtb were reported to who, fewer than two-thirds ( %) of the . million people estimated to have contracted the disease. this means that % of new cases were not detected or reported. a vaccine is imperative to achieving the end tb goals [ ] , particularly through reaching the population who are undiagnosed and continue to transmit disease. as such, the tb vaccine development community has turned its focus to the development of vaccines targeted to adolescents and adults as the age-groups with highest burden of active disease and the source of mtb transmission. modelling studies suggest that prevention of pulmonary disease in this population from primary infection and from reinfection or reactivation of existing infections is the most effective strategy to prevent mtb infection and disease in infants and children [ ] . the most advanced vaccine candidates are targeting this indication, including current neonatal bcg replacement candidate vaccines that are also undergoing evaluation as a booster in later life. several of these candidates are in proof-of-concept clinical studies and are approaching key endpoints through prevention of infection or disease, or prevention of disease due to reinfection in this these target populations in the next - months [ ] . with this in mind, pdvac recommended that who prioritize and facilitate consensus building with respect to the development of strategic goal(s) and ppc(s) for vaccines targeted to adolescents and adults, in the first instance. there are several candidates and platforms in the pipeline that target this goal in this population, as well as other important target populations [ ] . pdvac acknowledged the significant need for development for these vaccines in parallel, as well as continued efforts to understand the biological mechanism of disease to support the immunological rationalization of candidates. the pox-protein public private partnership (p ) consisting of sanofi, glaxosmithkline (gsk), bill & melinda gates foundation, the us military hiv research program (mhrp), and the hiv vaccine trials network (hvtn) have been collaborating with the us national institutes of allergy and infectious disease (niaid) to optimize and assess the efficacy of the alvac/heterologous prime boost approach, following the demonstration of partial efficacy in the rv trial in thailand [ ] . the interim data from a phase i/ ii study (hvtn ) met its humoral and cellular immunological 'go' criteria, exceeding the rv responses against sub-saharan clade c antigens. extrapolation of these responses to those observed with rv , suggest that the optimised vaccine could offer at least % protection following a month booster. based on these data, a randomised placebo controlled phase iib/iii efficacy trial (hvtn ) enrolling subjects was initiated in late in south africa, and will evaluate alvac (clade c) prime/ bivalent recombinant gp protein with mf adjuvant as a heterologous boost, as well as the effect of a booster at months [ ] . futility analyses will be undertaken early in the year followup period. correlate of protection studies and assessment of crossreactivity to other regional clades are included in the study design. discussions with the south african medicines control council (mcc) are ongoing, and licensure in south africa could be as early as . other vaccine candidates are in development, including janssen's heterologous prime boost approach with ad /gp , currently undergoing dose regimen selection in phase i/iia trials. antibody-mediated prevention using broadly neutralizing, potent monoclonal antibody (bnmabs) approaches are also undergoing phase i/iia clinical evaluation. the niaid/vaccine research centre's vrc broadly neutralising mab is the most advanced candidate which has been shown to neutralise cd binding of % of viral isolates. hvtn /hptn and hvtn /hptn are phase iib studies to evaluate the efficacy of vrc in reducing acquisition of hiv- infection in high risk populations in the americas and sub-saharan africa, and started enrolment in . if shown to be effective, administration of vrc could be positioned as a long-acting supplement to increase effectiveness of anti-retrovirals. pdvac commended the advances in hiv vaccine development, and requested to be kept informed about progress with hvtn . currently, there are no known intentions for global studies with the p candidate vaccine, or to seek who prequalification. pdvac encourages the p partners and the south african hiv vaccine development community to keep who fully informed about progress with the trial. concerns were expressed regarding the lack of follow-on studies in thailand, given that the initial landmark rv trial was performed there. despite the substantial reduction over the last years (over % for global malaria mortality in children aged < years), mainly due to greater investments in malaria control, the who estimates there were million malaria cases in , % of which were in africa. of the , people who died from the disease in , % reside in africa [ ] . given the increase in multi-drug and insecticide resistance, there remains an urgent need for a vaccine to combat malaria. as reported in the pdvac meeting summary [ ] , the european medicine's agency (ema) provided a positive scientific opinion, indicating a favorable assessment of the risk-benefit balance of rts,s/as from a regulatory perspective. in october , two advisory bodies to who, namely sage and the malaria policy advisory committee (mpac), recommended pilot implementation studies of the -dose schedule of the rts,s/as vaccine in - distinct epidemiological settings in sub-saharan africa, at sub-national level, covering moderate-to-high transmission settings, with three doses administered to children between and months of age, followed by a fourth dose - months later. the intent of these pilot studies is to assess: the feasibility of providing all four doses of rts,s to the target age group through existing health services; the impact of rts,s on child mortality; whether there are any safety issues, particularly evidence of any causal relationships between rts,s administration and either meningitis or cerebral malaria (both signalled in the phase iii trials), whether introduction of the vaccine impacts positively or negatively on existing country immunization programs and on the use of currently recommended malaria control measures. in , the malaria vaccine technology roadmap was updated to include licensure of vaccines targeting plasmodium falciparum and plasmodium vivax by , with protective efficacy of at least % against clinical malaria, and that reduce transmission of the parasite and thereby substantially reduce the incidence of human malaria parasite infection [ ] . the vaccine candidate pipeline is robust, and includes novel antigens and platforms [ ] . second generation vaccines are expected to provide higher protection than rts,s in the longer term. optimised tools are needed to measure incremental improvements and predict potential cost effectiveness of new candidates. the development of controlled human malaria infection (chmi) models, efforts to harmonize elements of clinical trial design and standardization of various assays continue. pdvac stressed the importance of the development of nd generation malaria vaccines in parallel to the pilot implementation program for rts,s, and proposed that the current version of the vaccine roadmap be updated, potentially in , in light of the rts,s pilot implementation. in , pdvac noted that development of universal influenza vaccines will be challenging and protracted, particularly due to the lack of a regulatory pathway for novel antigens that operate through induction of t-cell immunity. rather, pdvac recommended that there be a focus on the definition of, and the collection of data to support implementation of 'improved' seasonal flu vaccines that would offer more immediate impact in lmics. pdvac advised who to develop strategic public health goals and ppcs for improved seasonal influenza vaccines, and to provide guidance on data requirements that would be needed to establish improved performance of such vaccines. a working group has been established, and has proposed a draft statement of unmet public health need: 'safe and well-tolerated influenza vaccines that are effective at preventing severe influenza illness, that provide protection beyond a single year, and that are programmatically suitable for use, are needed for low-and middleincome countries.' draft -and -year strategic goals for development of influenza vaccines that induce broader and more durable protection against severe illness caused by influenza a strains have been developed. these strategic goals and the draft ppc for nextgeneration influenza vaccines were presented at the upcoming eighth who meeting on development of influenza vaccines [ ] . pdvac reaffirmed the value of ppcs based on the two different approaches. there is a public health need to develop improved performance of currently available seasonal vaccines to offer protection over multiple seasons, and against drifted strains, with a view to generating shorter timelines to achieving availability and access in lmics. as part of this effort, it will be necessary to define the criteria needed to demonstrate clinical benefit, and additional data requirements to support policy recommendations. efforts to develop 'universal' vaccines that target conserved antigens, or conserved components of antigens, should continue in parallel, with a focus on identifying correlates of protection to support a regulatory pathway for this novel class of vaccines. diarrheal disease remains the second leading cause of death in children under years of age. although mortality has declined over the past four decades, morbidity has not declined significantly, despite improvements in water and sanitation and benefits from oral rehydration therapy. there are nearly . billion cases of diarrheal disease every year, many with acute and chronic effects such as growth stunting and cognitive impairment. these long term sequelae significantly impact quality of life and economic potential, and are estimated to affect one-fifth of children globally. in , pdvac recommended that who expand its remit to include support for enteric vaccine development, particularly against enterotoxigenic escherichia coli (etec) and shigella. one of the main objectives of the planned who engagement in this area will be to ensure that the design of the phase iii efficacy study, including definition of primary/secondary endpoints and long-term follow up, and the data generated, will be relevant to support a policy recommendation from sage. another key objective is to develop a who preferred product characteristics document which outlines who preferences, including considerations for development towards a potential combined vaccine. several vaccines are in development, with two etec candidates and seven shigella candidates currently in clinical studies. for etec, the most advanced vaccine is etvax adjuvanted with dmlt, which is being developed for both a pediatric and traveller's indication. a phase i/ii dose escalation, age de-escalation study in children is currently ongoing in bangladesh, with intent to further age deescalate into week-old infants in late . in parallel, a phase iib study in travellers is planned to begin in . based on an encouraging phase iib immunization and challenge study and additional positive protection studies in non-human primates (nhps), an adhesin-based subunit etec vaccine (fta) is moving forward with an accelerated clinical program designed to move a complete multi-valent vaccine into descending age field trials in . the most advanced shigella candidate is trivalent shigella killed whole cell (tswc) composed of formalin-inactivated s. flexneri a, s. flexneri a, and s. sonnei, expected to offer coverage across about % of isolates. a phase i study has been completed and a challenge trial with s. flexneri a prototype will begin in , followed by a study that will assess co-administration with etvax. both etvax and tswc are being developed for oral administration. other promising shigella vaccines in early stage clinical testing include two live attenuated vaccines, wrrs and shigetec. wrrs is in a descending age study in bangladesh, while shigetec, which is a combination shigella-etec combination vaccine, will begin a phase i study in early . three subunit approaches for shigella are also in phase i/ii studies; the prototype s. flexneri a bioconjugate vaccine (flexyn a), invaplex and the generalized module for membrane antigens (gmma). one of the critical strategic issues is whether to prioritize the licensure and approval of an etec vaccine, or to focus on the development of a combination with shigella that will likely delay the timeline to vaccine availability. epidemiologic data suggest that both intra-and inter-country disease heterogeneity is likely to exist and this may drive vaccine preferences, and presentation optimization. these data are critical to inform decision-making by country policymakers. for this reason, development of who derived preferred product characteristics for etec and shigella vaccines, alone and in combination is needed. in april , plos released a collection on 'the global burden of norovirus & prospects for vaccine development' [ ] , which includes the most current estimates on global norovirus disease burden of over , deaths in low resource countries, and a global economic burden of more than $ billion [ ] . recent molecular analyses of samples from the community based longitudinal birth cohort mal-ed study suggest that norovirus is the most common diarrheal pathogen in the first year of life, and the second most common in the second year of life. there are vaccine candidates in development, including three strategies to develop a combination vaccine against other enteric pathogens. however, only one candidate, which is composed of two vlps based on the gi.i and gii. norovirus genotypes, has entered clinical studies, a phase iib study began recently [ ] . the advent of cell culture methods for norovirus will facilitate many advancements, including the optimization of a neutralization assay and enable the assessment of antisera against this vaccine to block binding of a diverse genotypes. in addition, in response to the pdvac recommendation to consider incorporating norovirus surveillance within the who global rotavirus surveillance network, a survey of the capability and capacity at representative global sites has been performed to support a pilot study proposal. the recently published epidemiology and burden of disease data indicate that norovirus fulfils the pdvac criterion of unmet public health for a vaccine in lmics. however, the ability of the candidates in the pipeline to offer protection over the range of circulating and emerging viral genotypes, and therefore the duration of protection of these vaccines, is currently unknown. it is conceivable that the vaccine will need to be periodically re-formulated, to include emerging genotypes. in addition to infants as a priority target population, adults and particularly the elderly are at risk, requiring the potential need for two vaccine formulations and/or presentations. fortunately, at the current time, development of a norovirus vaccine that may offer efficacy in the context of low and middle income countries is proceeding with investment from the private sector, however an assessment of vaccine programmatic suitability and applicability to prequalification is needed, prior to phase iii trials to ensure the vaccine is appropriate for use in lmics, assuming it is demonstrated to offer coverage over circulating genotypes within lmics. rotavirus is the leading cause of severe diarrhea among all children below years of age worldwide, causing - % of severe diarrheal hospitalisations, and is associated with significant mortality, with the latest mortality estimates at , deaths in [ ] . the introduction of the live-attenuated oral rotavirus vaccines, rotateq and rotarix, in has had significant direct and indirect impact in countries where they are in use, including saving lives and reducing hospitalizations. however, in gavieligible and lmic countries in asia and africa the vaccine effectiveness is lower, with protective efficacy observed from to % against severe rotavirus diarrhea over the first year of life. waning of protection has also been observed in these settings, with lower protection rates ( - %) in the second year of life. in comparison, in high-income countries protection is higher ( - %) and persists into the second year of life. thus, despite the enormous success of the live oral rotavirus vaccines, several challenges and issues remain such as the lower protection in gavi-eligible and lmic countries in africa and asia, together with the high cost of available vaccines. despite an overall acceptable safety profile, the intussusception rate seems to be slightly increased by vaccination (occurrence - / oral rotavirus vaccine recipients) in high income countries. several new oral, live-attenuated vaccines, composed of alternative strains, are in mid-to late-stage clinical development. the current who guidance document for the quality, safety and efficacy of oral live attenuated rotavirus vaccines [ ] would be applicable for these next generation oral, live-attenuated vaccines. of these new oral rotavirus vaccines, rotavac c (developed by bbil) is the only vaccine currently licensed for use in children, having been approved for use in india in . this vaccine is available on the private market in india and staged roll out in public health system is planned in four states in india. another live rotavirus vaccine is being evaluated in a randomised placebo controlled trials in india (nct ) and in niger (nct ). efforts are underway to develop non-replicating rotavirus vaccines (nrrv) as second generation rotavirus vaccines, which may avoid the risk of intusseseption. the most advanced candidate is p -vp ⁄ , a trivalent truncated vp ⁄ of rotavirus genotypes p [ ] , p [ ] and p [ ] , currently in phase ii clinical testing with a parenteral route of administration (nct ). for both nrrvs and additional oral, live-attenuated vaccines in development, pdvac encouraged the rationalization of target product profiles for these new candidates, to clearly articulate the distinguishing/advantageous features over the existing vaccines, i.e. cost, safety, efficacy in lmic, stability, breath of protection, etc. the potential for any of these vaccine candidates to be included in combination with other emerging enteric vaccines will clearly be advantageous and should be encouraged and explorations of combination with ipv could be considered. clostridium difficile is the leading cause of healthcare-associated diarrhoeal disease in the high-income countries, and is strongly associated with increasing age and frailty, immunodeficiency and in particular, modification of the normal flora through antibiotic use. the results of infection range from asymptomatic carriage through mild infection to severe diarrheal disease, with complications including pseudomembraneous colitis and toxic megacolon. in the us alone, it is believed to have caused approximately . million infections and , deaths in [ ] . current interventions include antibiotic treatments, but their use can trigger relapse on withdrawal. data on the burden of disease in lmics is lacking, however hospital based studies in india, thailand and south korea suggest that the c. difficile infection is widespread, and global (douce, manuscript in preparation). there is a correlation between toxin neutralising antibody in human serum and disease protection; antibodies against toxin a are associated with protection against acute diarrhea, whilst immune responses to toxin b appear to be effective against severe disease and relapse. toxin-mediated disease is recapitulated in the syrian golden hamster, which is the standard preclinical model for demonstration of proof of concept. currently there are three vaccines in clinical development. a toxoid vaccine candidate (containing toxins a and b) recently completed a phase ii study in healthy adults and demonstrated induction of high levels of neutralizing antibodies [ ] and a phase iii study has been initiated. a genetically modified, detoxified whole cell vaccine has also completed phase ii, alhough results have not yet been reported. in phase i, the vaccine was shown to be safe and induced toxin-specific neutralizing antibodies that were sustained for months [ ] . the third candidate is an adjuvanted recombinant protein encoding binding domains of both toxins, and the results of a phase i trial has been reported [ ] , and a phase ii study has been completed. passive immunity by administration of a monoclonal antibody is also in phase iii evaluation (nct and nct ). pdvac agreed that the role for who in facilitating c. difficile vaccine development is not clear given the lack of data regarding the disease burden in lmics. however, it would be useful to understand the potential effectiveness of a vaccine in low resource contexts, and pdvac raised the possibility of testing existing samples from the gems and mal-ed studies for the presence of c. difficile. in addition, it would be helpful to assess the impact that these vaccines many have on reducing the use, and cost of antibiotics, and to consider this in the value proposition for vaccine decision-making. h. pylori is a highly motile, gram-negative bacterium that infects the mucus layer lining the stomach. infection typically occurs in childhood, although symptoms and clinical disease develop in only a minority of infected individuals during their lifetime. h. pylori is associated with gastritis, which causes several pathologies including gastric peptic and duodenal ulcer disease. most significantly, long term infection can result in gastric adenocarcinoma (ga) in later years of life; - % of ga cases are due to h. pylori infection. ga is the rd leading cause of death due to cancer, globally ( , deaths in , . % of all cancers) [ ] . the global prevalence of h. pylori is believed to be approximately % with the highest mortality rates in east asia and eastern europe. the route of transmission is poorly characterised but the oraloral route appears to be a common mechanism, as well as vertical transmission from mother to child. if untreated, most h. pylori infections are sustained for life, and % of those infected are thought to develop an associated pathology. if diagnosed, h. pylori infections are currently treatable with combination antimicrobial therapies. however antibiotic resistance is increasing, with % of patients in some countries currently failing first treatment and % failing two rounds of therapy. antimicrobial treatment offers no protection against reinfection. the choice of indication for an h. pylori vaccine is challenging: a prophylactic vaccine would likely need to be given to children in the first few years of life (to reach the maximum number of the target group while uninfected) but would need to offer long term protection to demonstrate clinical benefit against ga. an effective therapeutic vaccine however could be given at almost any age and would ideally be given by the th decade of life, prior to the peak of ga development which typically occurs from years of age. the most advanced candidate is a urease toxin fusion approach and has completed phase iii trials in children, in china, and demonstrated . % efficacy against natural acquisition of infection [ ] . however, protection appeared to wane to % over - years and next steps for this vaccine are not clear. several other candidates are in preclinical development with one close to phase i studies. pdvac concluded that the burden of h. pylori is significant, and that a vaccine that is able to protect against infection, with sufficiently long duration of protection, would be of public health benefit. therapeutic candidates are currently too upstream in development for there to be a role for pdvac. maternal immunization is increasingly considered as a strategy to prevent maternal and/or neonatal disease. this approach has been proven to protect against maternal and neonatal tetanus and has been in place for decades. who recommends influenza and pertussis vaccination of pregnant women to prevent disease in mothers and newborns, respectively. however, for the first time there are now vaccines in development, specifically indicated for immunization of pregnant women as the target population. respiratory syncytial virus vaccines are most advanced in this area followed by group b streptococcal vaccines. since the pdvac meeting, a special journal issue dedicated to the issues regarding the maternal immunization vaccination strategy has been published and a great deal of work is underway to strengthen the maternal immunization platform [ ] . due to the advanced stage of rsv vaccine and monoclonal antibody development, rsv was presented to sage for information in april . rsv causes . million episodes of lower respiratory infection (lri) annually in children and approximately , deaths, % of which are in lmics [ ] . recently updated estimates for rsv acute and severe lri (community based and hospitalized) disease and deaths will be published by the rsv global epidemiology network (rsv-gen) in early . in addition, the pneumonia etiology research for child health (perch) study will present and publish results on the etiology of severe and very severe pneumonia in hospitalized infants and children in sites in africa and asia. preliminary data analyses indicate that rsv was the leading pathogen in infants with severe pneumonia in this study. there are four rsv intervention strategies currently in development: ( ) maternal immunization to enable passive transfer of maternal antibodies to the foetus in utero, ( ) birth or early infant passive immunization with a long-acting monoclonal antibody, ( ) active pediatric immunization and ( ) vaccination of the elderly. the most advanced maternal immunization candidate begun phase iii efficacy testing in late following the demonstration of induction of palivizumab-competing antibodies (measured by elisa) in women of childbearing age (pmid: ) and pregnant women. this efficacy trial has a group sequential design and will enroll - participants in a randomised placebo controlled trial across multiple sites in both the northern and southern hemispheres, and is expected to take - years to complete. monoclonal antibody development for the prevention of rsv in pediatrics is the next most advanced, with an extended half-life candidate (medi ) that has been shown to be more potent in vitro than the currently licensed palivizumab. one dose may offer protection for up to months. a phase iib clinical study in infants born at - week gestation is planned, and the fda recently granted fast-track designation for this product. since the palivizumab patent recently expired, who in collaboration with the university of utrecht will develop a 'biosimilar' of palivizumab and reduce costs for lmic markets through high yield production and a novel financing plan [ ] . the estimated price is $us per child for the full month dose series and the first market authorization is expected in late . pediatric rsv vaccine candidates are the least advanced, however two adenovirus-based approaches have entered the clinic since the last pdvac meeting. a chimp adenovirus (chad) candidate is currently in phase i testing in adults, to be followed by age de-escalation into seropositive, and ultimately seronegative infants. ad is also being evaluated as a heterologous primeboost regimen, currently in phase i testing in adults. a number of pediatric vaccine candidates developed by the laboratory of infectious diseases, nih are in phase i trials in infants and children. of note, a vaccine containing a deletion of the m - gene showed evidence of diminished replication, enhanced immunogenicity, and asymptomatic 'boosting' (anamnestic response) following naturally acquired rsv infection (pmid: ). two vaccine candidates are in clinical development for the elderly with a post-fusion f-based adjuvanted nanoparticle in phase iii efficacy testing, with data expected in early . pdvac fully supported the following sage recommendations and called for who and partners to develop plans to support global policy-making for rsv maternal immunization as well as passive immunization with long-acting mab, following licensure. particular areas of emphasis include: ( ) rsv surveillance to determine seasonality and age-stratified rsv disease burden and community morbidity and mortality, especially in africa and south-east asia ( ) assessment of the long term effects of rsv interventions and the potential impact of vaccination on reducing recurrent wheeze, which, if demonstrated, would substantially increase the costeffectiveness and impact of rsv preventive interventions ( ) generation of cost-effectiveness and impact data. sage also emphasized the need for strengthening of the maternal immunization platform in collaboration with the influenza, tetanus and pertussis vaccine communities, along with preparations for potential country introductions of rsv vaccine. there is an urgent need to establish a who prequalification pathway for monoclonal antibodies, which does not currently exist. as a rsv vaccine or extended half-life monoclonal ab may become available in the next years, it will also be imperative to initiate early discussions with financing bodies, and to align with the gavi vaccine investment strategy (vis) to avoid delay in achieving the potential major public health impact of rsv immunization if recommended for use by who. globally, gbs remains the leading cause of sepsis and meningitis in young infants, with its greatest burden in the first days of life. intrapartum antibiotic prophylaxis (iap) for women at risk of transmitting gbs to their newborns has been effective in reducing the young infant gbs disease burden in many high income countries, but iap uptake is limited and difficult to implement in lmics. immunization of pregnant women with a gbs vaccine represents an alternative pathway to protecting newborns and young infants from gbs disease, through prevention of gbs colonization and transplacental antibody transfer to the fetus in utero. pdvac prioritized gbs in and encouraged who to engage on developing guidance on the development pathway for gbs vaccines, including development of a ppc guidance document and a vaccine roadmap. in april , who convened its first consultation on gbs vaccine development [ ] . the focus was on gbs maternal immunization development programs targeting lmic with the ultimate goal of reducing global newborn and young infant deaths. the major knowledge gaps about the disease burden characterization were identified. recent data suggesting that gbs is an under-reported cause of stillbirth may have profound implications on the estimate of the global public health impact of a future gbs vaccine. the relationship between gbs colonization and prematurity should also be clarified. disease surveillance in hic also suggest an important residual unmet medical need, despite implementation of iap. two major pharmaceutical companies are currently developing a multivalent polysaccharide conjugate vaccine, based on the available evidence of an association between trans-placental maternalfoetal transfer of antibodies targeting polysaccharides of the gbs envelope, acquired as a consequence of natural exposure, and a reduced risk of invasive infant disease. a vaccine incorporating five of the eleven described gbs serotypes is predicted to cover over % of the global circulating serotypes, but the risk of serotype replacement is unknown. an alternative approach is targeting surface expressed proteins, in an attempt to confer broad protection across all serotypes. epidemiological studies evaluating the role of maternal antibodies acquired following natural exposure will determine whether a protective threshold at birth can serve as an acceptable vaccine-induced correlate of protection. until additional epidemiological and immunological data are available, estimating vaccine efficacy against invasive gbs disease in neonates and young infants in a double blind placebo-controlled vaccine trial remains the gold standard for generating the evidence required to determine potential public health impact and inform policy decision-making. pdvac endorsed the consensus-based prioritization of future activities including the development of a ppc and vaccine development technology roadmap. efforts should be made to raise awareness of the burden of gbs disease and potential public health value of a gbs vaccine, particularly in countries that lack local epidemiological data. as with rsv, efforts must be made to leverage and strengthen the maternal immunization platform by alignment with other vaccines that are administered in pregnancy, including the brighton collaboration's considerations for safety monitoring through the global alignment of immunization safety assessment in pregnancy (gaia) [ ] . antimicrobial-resistant infections currently claim at least , lives each year across europe and the us alone, but amr affects many hundreds of thousands in other areas of the world [ ] . in european countries, more than % of bloodstream staphylococcus aureus infections are caused by methicillin-resistant strains (mrsa), with several of these countries seeing resistance rates closer to %. emerging resistance to treatments for other diseases, such as tb, malaria and hiv, have enormous impacts in lower-income settings, and by , the death toll due to amr infections in africa is predicted to be approx. , , per year. as mentioned above, each year almost . million cases of drugresistant tb are reported, and these are extremely costly to treat; an mdr case costs - -fold more to treat than drug a sensitive case, while an xdr case is - -fold more expensive [ ] . the who estimates that approximately $ billion per year is required to support tb care and control efforts in lmics. this is significantly more than the current investment in tb vaccine development programs. the o'neill review on antimicrobial resistance estimated that by drug-resistant infections could be claiming million lives per year and at an economic cost to the global gdp in excess of $ trillion. at the sixty-eighth world health assembly in may , a global action plan to tackle antimicrobial resistance, including antibiotic resistance, was adopted [ ] . its goal is to ensure continuity of successful treatment and prevention of infectious diseases with effective and safe medicines -including vaccines -that are quality-assured, used in a responsible way, and accessible to all who need them. the amr global action plan (gap) is based on work streams ranging from national plans, stewardship of antibiotics, encouraging r&d through developing new business plans and assessing environmental drivers. one work stream focuses on vaccines to prevent amr. the who gap workstream on vaccines to prevent amr is based on three complementary approaches: increasing the use of existing vaccines; developing vaccines against high burden diseases currently treated systematically with antibiotics; and prioritizing the development of vaccines for diseases where antibiotic resistance is significant. these three approaches, and the challenges associated with implementing them are summarised below: a). increasing use of existing vaccines: while it is logical that increasing the use of existing vaccines would reduce infections and result in reduced use of antibiotics, it is not always clear which vaccines, in which populations, would have the greatest impact on reducing antibiotic use and potentially amr, and should therefore be prioritized. for example, it has been shown that the use of pcv- in children results in a roughly % reduction in antibiotic-resistant strains of s. pneumonia, and the use of pcv- reduces outpatient antibiotic purchase, leading to the suggestion that global pediatric coverage with pcv could prevent million days of antibiotic use in children annually. however, it is thought that the bulk of pneumonia, and antibiotic use for s. pneumonia infections, is in older adults. this would suggest that demonstrating efficacy of pneumonia vaccines to reduce antibiotic use in older adults and an expanded use of these vaccines in that population group where very few countries have a vaccination policy may have a substantial impact on reducing amr. other existing vaccines which could impact antibiotic use include pertussis, haemophilus influenza, neisseria meningitides, typhoid, as well as influenza which, although not directly susceptible to antibiotic treatment, does result in bacterial super-infections and accounts for up to % of excess (winter-related) antibiotic prescriptions in some countries. in order for a rational evidence-based policy on expanding the use of existing vaccines a prioritization exercise needs to be performed, taking into account the disease burden in different populations, the antibiotic use associated with that disease burden, and an evaluation of how many days of antibiotic use would be avoided with each dose of vaccine administered. this exercise is particularly challenging since in most of the world antibiotics are taken in response to a symptom, rather than an identified infection. this means that, for example, preventing salmonella typhi-induced infection with vaccines may have minimal impact on antibiotic use for severe diarrhea. the prioritization exercise therefore needs to consider not only the disease burden, but the symptom burden and the proportion of that burden due to the vaccine-preventable infection. b). developing vaccines for diseases that are consistently treated with antibiotics, where amr is not currently an issue, but where the vaccine could reduce antibiotic use. one such example is group a streptococcus (gas). while gas is not directly associated with antibiotic resistance (there is little evidence of resistance to date) it has a high disease burden and is a source of extensive antibiotic use. in addition, it is thought that vaccine development is feasible. however to date there has been no significant effort from industry, possibly because of the weak market assessment since it can be treated with antibiotics, and such treatment is cheap. however the indirect costs from such antibiotic use, increased environmental exposure to antibiotics and expansion of amr have not been considered. taking these costs into account may contribute to the value proposition for developing and using such a vaccine. other such candidates could include group b streptococcus, and m. catarhalis and non-typeable haemophilus influenza, both responsible for otitis media which is another source of significant antibiotic prescription. while including potential impact on reduction of antibiotic use, prioritization of these candidate vaccines also needs to consider technical feasibility, whether the antibiotic use is appropriate, and whether alternative non-antibiotic approaches may make vaccine use less attractive. for example: while there are over million cases of otitis media per year in under - year olds, for which antibiotic treatment is usually prescribed which could justify development of a vaccine, most otitis media resolves and new guidelines recommend limiting antibiotic treatment. another example is urinary tract infections which are frequent in elderly patients and a cause of significant antibiotic use, yet there is little supporting evidence that these infections could be effectively reduced by vaccination. a prioritization exercise is therefore required for vaccines that are considered technically feasible, takes into account the potential amr impact, and therefore could contribute to the cost effectiveness of the vaccine if they were developed. to achieve this, the evaluation of impact on amr is recommended to be included in the review of vaccine conducted by pdvac. c). the third and most challenging approach is developing vaccines against pathogens that are frequently antibiotic resistant and becoming increasingly difficult to treat, the so-called eskape pathogens [ ] . this list includes staphylococcus aureus, pseudomonas aeruginosa, klebsiella pneumonia, as well as clostridium difficile and tuberculosis. there are numerous challenges in this approach. the first is that although infection with these may result in significant morbidity, the current global disease burden of many of these infections remains relatively low so prophylactic vaccination of the entire population would not be cost-effective. tuberculosis is however an example with significant disease burden and rapidly expanding multi-drug resistance. secondly many of these infections are associated with ageing, where immune decline may make immune interventions poorly effective, or are associated with penetrative medical interventions where the time available to induce a protective immune response may be insufficient. and finally, despite significant efforts to make effective vaccines against some of these has so far proven to be difficult. of these, tuberculosis appears to have the greatest global burden and public health impact, and tb vaccines are also the subject of extensive research. the amr gap activity in this area, to be conducted by ivr is firstly to promote tb vaccine research and development through facilitation of preferred product characteristics and the establishment of a roadmap for vaccine use, and highlighting the impact that the vaccine will have on antibiotic use and antibiotic resistance. additional activities involve monitoring the state of development of vaccines against the pathogens that are becoming antibiotic resistance, and facilitating their development. gas is a ubiquitous human pathogen that causes a broad disease spectrum, from mild to severe, the most serious of which is rheumatic heart disease (rhd). rhd affects approximately million people globally, of whom million experience heart failure and an estimated , die. gas is also a major cause of invasive disease, with a case fatality rate of - % in high income countries, and as high as % in lmics [ ] . on the milder end of the spectrum, gas causes approx. million cases of pharyngitis per year, resulting in - % of cases being treated with broad spectrum antibiotics, rather than penicillin ( % of cases), to which gas is universally susceptible. this extensive use of unnecessary and inappropriate antibiotics increases the likelihood of amr emergence against antibiotics that are used to treat a range of pathogens. previous human challenge studies, as well as preclinical animal models suggest that it is feasible to develop a vaccine against gas, and since the previous pdvac meeting, phase i studies for one candidate has been initiated in adults, and two additional candidates are expected to enter phase i studies in the next months. despite this encouraging progress, significant debate remains as to the appropriate indication and optimal clinical endpoints, and the regulatory pathway for a vaccine to prevent or reduce rhd is unclear. in addition, there is a perception that increased prescription of penicillin would be an equally as effective and a significantly more cost effective method of reducing conditions that result from gas infection. these issues are likely major stumbling blocks in incentivising investment in gas vaccine development. gas has been prioritized by pdvac previously, with a recommendation to develop a business case for both a global market, and also specifically for lmics which would focus on prevention of severe outcomes in resource poor settings. despite significant effort, it has been very difficult to engage stakeholders in this activity. on the recommendation of pdvac, who convened a consultation in december to examine the value proposition for gas vaccines, considering its potential impact across both high income and lower income settings -including the consideration of how current antibiotic treatment practices may increase amr, as well as to investigate the perceived regulatory obstacles. s. aureus is a bacterium that is found as both an asymptomatic colonizer of the skin and nares of human hosts, as well as a frequent cause of human disease. it causes a spectrum of clinical manifestations of varying severity, and is the most commonly isolated pathogen from skin and soft-tissue infections, septic arthritis, pneumonia, endovascular infections, osteomyelitis, catheter/other foreign-body infections, septicaemia, and toxic shock syndrome. methicillin-resistant s. aureus (mrsa) has been documented to be emerging at a rapid and increasing rate since the antibiotic was first introduced in , and hospital-associated mrsa (ha-mrsa) clones are now recognized to be the leading cause of nosocomial infections both in the united states and around the world, in high income as well as lmics. the emergence of communityassociated mrsa (ca-mrsa) in the past several decades is of concern, as is the emergence of highly resistant vancomycin-resistant s. aureus (vrsa). to date, active and passive immunization approaches have been based on increasing the concentration of opsonic antibodies to single surface antigens, and all have failed to demonstrate protection. antigenic variation, the multiple invasion pathways and lack of a surrogate of protection all present significant obstacles to vaccine development. following the failure of single antigen vaccine approaches, most development efforts are now focused on multiple antigens, and a number of candidates are in preclinical development. one multi antigen approach, comprised of antigens including two capsule polysaccharides, clumping factor a and a manganese transport protein, is the most advanced [ ] . current efforts are also focused on further characterizing the immunopathology and immunity of s. aureus infections to identify new antigenic targets, and developing more representative preclinical models in which opsonising and/or neutralising immune responses are measured. to date, none of the vaccine candidates in development have contemplated target populations or indications that are prevalent in lmics. focus has been on development of a vaccine that will protect against life-threatening s. aureus infections in high income countries, but it is hoped that such a vaccine would also protect against all s. aureus infections including more commonly encountered skin and soft tissue infections, and therefore be applicable in lmic contexts. since the pdvac meeting, a new global health sector strategy on sexually transmitted infections has been developed for - and adopted by who member states at the th world health assembly. within this strategic framework, sti vaccine development was highlighted as key need for future sti control [ ] . in addition, the global roadmap for vaccines against stis has been updated and included in the who special issue on pipeline vaccines published in vaccine [ ] . currently, the only sti vaccine candidates that are undergoing or approaching clinical development are against herpes simplex virus (hsv) and chlamydia trachomatis, and as such discussion was limited to these pathogens. hsv is the leading cause of genital ulcer disease, and a particular concern for lmics as it increases both acquisition and transmission of hiv infection. hsv type and type disease burden estimates were recently updated [ , ] , and it is estimated more than half a billion people live with genital hsv infection, worldwide. pdvac previously recommended that improved global estimates of neonatal herpes burden be generated, and assessment of available data has recently been completed with preliminary estimates of > , new cases globally, an incidence rate of approx. / , births, which is concerning because of a case fatality rate of % (looker, submitted) . the incidence is likely to be under-estimated in lmics where hsv infection rates are highest and poor healthcare infrastructure means that neonatal herpes cases are likely to be undetected, but primary data are lacking. ongoing evaluation of hsv infection as part of the child health and mortality prevention surveillance (champs) network will help to address this burden gap. at the meeting, the advance of therapeutic vaccine candidates for hsv- was highlighted, and the role of these types of vaccines in modulating the interaction between hsv and hiv acquisition was discussed as an important consideration for these vaccines in lmics. in consideration of this, and with who support, a systematic review/meta-analysis of hsv- and risk of hiv acquisition including studies will inform modelling of the potential impact of an hsv- vaccine on hiv incidence, and is expected to be published in late . a review of biological mechanisms of hsv-hiv interaction and implications for vaccine development has also been drafted. the pipeline for therapeutic vaccines remains robust, with candidates in clinical development, the most advanced of which now has data demonstrating significant reductions in hsv shedding ( %) and days with genital lesions ( %) over months [ ] . in response to these positive data, niaid has formed an hsv working group to propose desired characteristics for therapeutic and prophylactic vaccines for hsv, including indication, priority target populations, clinical trial endpoints, and safety and efficacy criteria. this document could form the foundation for a who consultative process to generate a guidance document on preferred product characteristics (ppc). pdvac encouraged who to actively collaborate and support development of ppcs for hsv vaccines. chlamydia trachomatis is a gram-negative bacterium that can infect genital, ocular and lung epithelium. it includes three sets of serovars: -serovars ab, b, ba, or c -cause ocular trachoma, which can lead to blindness -serovars d-k -cause sexually transmitted infection resulting in urethritis, cervicitis, pelvic inflammatory disease (pid) (and associated infertility, ectopic pregnancy, and chronic pelvic pain), neonatal pneumonia, and neonatal conjunctivitis -serovars l , l and l -cause lymphogranuloma venereum c. trachomatis can ascend to the upper genital tract and cause pelvic inflammatory disease (pid), which can in turn lead to long-term sequelae including tubal factor infertility, ectopic pregnancy, and chronic pelvic pain. other adverse outcomes of chlamydia include preterm birth, neonatal conjunctivitis and pneumonia, and increased hiv risk. currently management is through screening programs in some high income countries that are not feasible in resource constrained settings, where most cases are likely never diagnosed. who estimates that there were million new cases of chlamydia in [ ] with most cases among adolescents and young adults. the global burden of chlamydia-associated pid, infertility and other sequelae has not been well characterised and estimates of the proportion of infertility presumed to be associated with genital infection (e.g., have a fallopian tube etiology) in africa are outdated [ ] , but are thought to be approx. - % in women seeking fertility care. there are several vaccine candidates currently in preclinical development, with a subunit vaccine based on the chlamydial major outer membrane protein (momp) and live-attenuated (plasmid-deficient) approaches being the most advanced. the momp candidate entered phase i clinical testing in late , and a phase i study with the live attenuated candidate will commence in . the intended goal of a chlamydia vaccine is to decrease upper genital tract sequelae, however pid is challenging to use as clinical endpoint as it is difficult to definitively diagnose and the causes of pid are multi-factorial (typically the result of c. trachomatis in / of cases). the chlamydia vaccine community is seeking guidance and consensus building on clinical endpoints for clinical studies, including evaluating the potential role of biomarkers, radiologic, and other measures of upper tract ascension, infection, inflammation and damage. improved global burden of disease data and vaccine impact modelling on long term sequelae are also needed to define the investment case for these vaccines. pdvac commended the progress towards the first vaccine study against chlamydia since the s and look forward to discussing the path ahead once the early clinical data are available. this section refers to vaccines that have been licensed, or are approaching licensure in some areas of the world, but are currently limited in their use outside any single who region. in some instances, the vaccines may have the potential of offering broader public health impact by expanding approval and use in other geographical regions, and pdvac is seeking to understand the perspective in this regard. ev is one of the most common causes of hand-foot-andmouth disease (hfmd). sporadic ev outbreaks have occurred globally since it was first isolated in , but from the late s a series of large hfmd epidemics caused by ev have been reported in the asia-pacific region. in china alone, . million cases were reported between and , of which ( . %) were fatal [ ] . children less than years of age have the highest risk of disease, and although infection is unusually mild and selflimiting, severe infections can result in neurological and cardiopulmonary complications, and death. several ev vaccine candidates are in development, and it was stated at the meeting that the chinese national regulatory authority has licensed two ev vaccines, with another in progress. the first licensed vaccine was developed by the institute of medical biology, chinese academy of medical science, and has been approved for use to prevent ev disease in - month olds, based on a phase iii study that demonstrated % efficacy over month [ ] . sinovac has also licensed in activated vaccine, with supportive phase iii data in - month olds [ ] . beijing vigoo is in the process of licensing its inactivated ev vaccines in china (nct ). all three vaccines are adjuvanted with aluminum hydroxide. given that ev outbreaks occur in other areas of the world (recently reported in spain [ ]), further discussions are warranted in the international health community about how to assess the role of the chinese vaccines during outbreaks outside china. in the wake of the - ebola outbreak, various strategies were proposed to avoid such crises from reoccurring. key to improving r&d preparedness and response is determining which pathogens are likely to be the greatest threat, creating consensus with respect to product development strategies and coordinating global funding for complementary r&d efforts going forward. to tackle these questions, and at the request of its member states, who convened a broad global coalition to develop the r&d blueprint [ ] as a sustainable platform for accelerated r&d, with two complementary objectives: to develop (and implement) a roadmap for r&d preparedness for known priority pathogens, and to enable roll-out of an emergency r&d response as early and as efficiently as possible the main approaches underpinning the improvement of preparedness within the r&d blueprint include: as reported in the meeting summary, a consultation to initiate work towards a mers cov roadmap was held in december with aims of defining the key basic and applied research activities, identifying the priority technologies and capacities to support vaccine development, and finally understanding the financing/procurement opportunities. following this meeting, a draft roadmap was developed and underwent public consultation prior to finalisation and publication [ ] . it is well accepted that the extraordinary rate of ebola virus vaccine development was as a result of unprecedented collaboration and co-ordination of global vaccine r&d activities, and the availability of a number of candidate vaccines that could enter clinical phase evaluation [ ] . in the face of another pheic so soon following ebola virus disease (evd) outbreak, the global vaccine community is rallying, and reflecting on lessons learned from the experience in west africa only years ago. at the time of responding to the evd emergency, the availability of well-characterised pre-clinical models and robust data was essential for the comparative evaluation and selection of candidates to move into clinical studies. novel recombinant viral vector platforms, in combination with recombination proteins have been validated by evd experience and it could be argued are now less risky for development of vaccine against future pathogens, but manufacturing feasibility and scale-up capabilities still need to be confirmed for the most novel platforms. critically, sustainable public sector push and pull investment mechanisms beyond the initial emergency response phase need to be created, to incentivise manufacturers to engage in the long term commitment to developing and licensing vaccines that may only be used in outbreak or emergency scenarios. pdvac noted that a target product profile for a second generation ebola virus vaccine is under development that will likely cover ebola zaire, ebola sudan, and marburg filoviruses, and will need to demonstrate longer duration of protection. this tpp will provide guidance about whos preferences and minimally acceptable criteria for vaccines in this area. during the discussion it was clarified that who tpps include minimally acceptable criteria, whereas preferred product characteristics specify only preferences. the status of zika virus epidemiology and the understanding of its pathogenesis and associated sequelae are evolving so rapidly that publications on these issues are almost immediately out of date. pdvac's role has been to oversee a working group that has developed a target product profile (tpp) for use in an emergency, or future outbreak scenario. the tpp was made available for public consultation, after which subject matter experts, global regulators, developers and manufacturers were convened to discuss the regulatory considerations for developing a vaccine with the characteristics described in the tpp. the finalised tpp and position paper are publically available [ ] . in addition to reviewing the status of vaccine development against pathogens, pdvac considered a number of cross-cutting issues that could better integrate and therefore facilitate product development efforts for vaccines and other interventions. in addition to the significant morbidity and mortality that drives the development of vaccines against pathogens for which vaccines are currently not available, the who estimates that there are approximately . million deaths per year in children under from vaccine preventable diseases [ , ] . one of the reasons for this striking immunization gap is the cost and logistical challenges of delivery of these vaccines, over and above the cost of their manufacture. the remit of who's immunization practices advisory committee (ipac) is to provide strategic advice on immunization practices, tools, and technologies intended to improve the delivery of immunization programs at the country level. it oversees the recently formed delivery technologies working group (dtwg) composed of public health organizations, funders and procurement agencies as well as vaccine developers to evaluate r&d in novel delivery technologies and devices, for example the microarray patch, and compact, pre-filled auto-disable injection technologies (cpad). of particular focus for this group is the development and evaluation of a framework to analyze high-level trade-offs between important variables such as development, procurement and supply chain costs, coverage, efficacy, and safety in order to facilitate investment decisions by product developers, vaccine manufacturers, global policy makers, in-country decision makers and procurement agencies. this framework is referred to as total systems effectiveness (tse). the intent of this delivery technology working group is to offer a platform for discussion and guidance regarding vaccine preferences for lmics, early on in development, so that ultimately the vaccine is suitable for programmatic use. the dtwg reports directly to ipac, but has potential overlap with activities that are overseen by pdvac, particularly in consideration of second generation vaccines or new vaccines that may be developed with an alternative presentation to that of a needle and syringe. pdvac was supportive of the dtwg and encouraged continued communication between vaccine development and device/delivery technology development to identify potential opportunities for novel combination product development. there are several pathogen areas where mab are being developed as vaccine-like interventions, as their single dose regimen and long half-life render them amenable for lmics contexts, where they could offer significant public health benefit. candidates for rsv and rabies are approaching licensure within the next years, and a who procedure for who prequalification is urgently needed to avoid delay implementation. this gap has been recognized and will be addressed. the scope of pdvac overlaps with several other research agendas such as gvap, amr, new delivery technologies and development and consolidation of maternal immunization platforms. the pdvac research agenda needs to be clearly communicated, and pdvac and ivb will strive to be well-informed of efforts in other research areas, to help shape and align strategy where appropriate. future pdvac meetings will consider these potential overlaps in more detail, as well as how pdvac and ivb can facilitate development of integrated product development approaches. since its inception in , pdvac has reviewed the pipeline and vaccine development status of different pathogens. pdvac will continue to review new pathogen areas as candidates progress into clinical studies, providing that who engagement will likely facilitate the use of vaccines to reduce disease burden in lmics. one such pathogen is cytomegalovirus (cmv), which is a leading cause of congenital infections worldwide, resulting in - % of infants developing permanent sequelae including hearing loss and neurodevelopmental disabilities. there are little data on cmv infection in lmics, but a recent systemic review suggests that birth prevalence ranges are higher in lmics than in europe and north america [ ] , and several clinical trials of vaccine candidates are ongoing. as such, an assessment of cmv vaccine development will be undertaken by pdvac . with rsv, tb, hiv and enteric candidates approaching pivotal data points, understanding what data are needed to support earlier policy implementation and outcomes will be key, as well as understanding the potential impact of vaccines within the broader control strategy -including diagnostics and other preventatives -for these pathogens. vaccine impact modelling, and understanding the composite set of cost drivers through to vaccine delivery will be important. interaction with who's ipac and pq teams will increase going forward, to strengthen the link between product development and programmatic requirements. under the recommendation of pdvac, who will seek to broaden its role to support development of value propositions for vaccines against pathogens for which there is a poorly defined business case, for example gas and hsv. raising awareness of lmic disease burden and requirements/procedures for access to lmics markets may help to incentivise financing development of these vaccines. of key consideration may be the potential for these vaccines to reduce the emergence of amr, and pdvac recommended that this be considered as a criterion in future landscape analyses and ppc guidance documents. pdvac and who will continue to align activities with the priorities within the who r&d blueprint. pdvac is aware that several other organizations which are responsible for emergency preparedness have been through a process to prioritize their r&d agendas, with some commonality and some complementarity to the pathogens listed in the who blueprint. in this arena, pdvac will continue its horizon scanning role, and will advocate for commitment to product development of vaccines for emerging diseases to progress through robust preclinical proof of concept to generation of phase i data, as a minimum. pdvac strongly recommends the collaboration with other groups to co-ordinate advocacy and funding for vaccine development to prepare for the inevitable future emergencies. procedure for assessing the acceptability, in principle, of vaccines for purchase by united nations agencies tackling drug resistant infections 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age who guidance document for the quality, safety and efficacy of oral live attenuated rotavirus vaccines current status of clostridium difficile infection epidemiology defining the optimal formulation and schedule of a candidate toxoid vaccine against clostridium difficile infection: a randomized phase clinical trial a phase , placebo-controlled, randomized study of the safety, tolerability, and immunogenicity of a clostridium difficile vaccine administered with or without aluminum hydroxide in healthy adults safety, immunogenicity and dose response of vla , a new vaccine candidate against clostridium difficile, in healthy volunteers efficacy, safety, and immunogenicity of an oral recombinant helicobacter pylori vaccine in children in china: a randomised, double-blind, placebo-controlled, phase trial brewinski isaacs and a. sobanjo-ter meulen advancing maternal immunization programs through research in low and medium income countries global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis moorthy vasee s. who consultation on group b streptococcus vaccine development: report from a meeting tb vaccine research & development: a business case for investment clinical relevance of the eskape pathogens invasive group a streptococcus infection among children development of a multicomponent staphylococcus aureus vaccine designed to counter multiple bacterial virulence factors the global roadmap for advancing development of vaccines against sexually transmitted infections: update and next steps global estimates of prevalent and incident herpes simplex virus type infections in global and regional estimates of prevalent and incident herpes simplex virus type infections in global estimates of the prevalence and incidence of four curable sexually transmitted infections in based on systematic review and global reporting the pill, chlamydia and pid hand, foot, and mouth disease in china, - : an epidemiological study an inactivated enterovirus vaccine in healthy children efficacy, safety, and immunogenicity of an enterovirus vaccine in china a roadmap for mers-cov research and product development: report from a world health organization consultation on a path to accelerate access to ebola vaccines: the who's research and development efforts during the - ebola epidemic in west africa meeting report: who consultation on considerations for regulatory expectations of zika virus vaccines for use during an emergency global, regional, and national causes of child mortality in : a systematic analysis systematic review of the birth prevalence of congenital cytomegalovirus infection in developing countries we gratefully acknowledge the pathogen specific landscape analyses that were prepared by the following individuals: this report contains the collective views of an international group of experts, and does not necessarily represent the decisions or the stated policy of the world health organization, nor of the u. s. government.bsg is an employee of the u.s. government. this work was prepared as part of his official duties. title u.s.c. § provides that 'copyright protection under this title is not available for any work of the united states government'. key: cord- -jobd s l authors: tulgar, serkan; ahıskalıoğlu, ali; kök, abdulaziz; thomas, david terence title: possible old drugs for repositioning in covid- treatment: combating cytokine storms from haloperidol to anti-interleukin agents date: - - journal: turk j anaesthesiol reanim doi: . /tjar. . sha: doc_id: cord_uid: jobd s l nan until recently, haloperidol was the most commonly used agent in the prevention or treatment of delirium that developed in intensive care units ( , ) . herein, we would like to consider a completely different purpose for its use. in , milbrandt et al. ( ) reported that haloperidol decreased mortality of patients on mechanical ventilation and hypothesized that this was due to its effect on lowering cytokine levels and, therefore, aided in preventing a cytokine storm associated with severe disease. in line with this data, although certain texts claim the opposite, the hypothesis that haloperidol has inhibitory effects on proinflammatory cytokines may be an essential step in preventing the progression of the disease and reducing its severity. we accept that there is no certainty to recommend starting haloperidol for every patient diagnosed with covid- whose treatment had commenced. however, we recommend that haloperidol be considered as an option to treat patients who develop agitation during the treatment process or agitation/delirium during the intensive care treatment process and continue to be administered routinely until proven otherwise. of course, drug interactions must be considered. there is still insufficient evidence for the use of either tocilizumab or other treatment agents in the treatment of covid- . tocilizumab is available as an effective option in combating cytokine storm. however, this drug is not readily available all over the world. therefore, different options must be sought, and alternatives must be put forward. our current strategy is to gain time by using existing drugs outside of their known indications until a definitive treatment or vaccine is found in the treatment of covid- . in addition to clinical studies (nct ) related to the use of anti-il agents such as anakinra, siltuximab, and tocilizumab in combating cytokine storms, studies are also being conducted with old drugs (nct ) such as sildenafil, used indirectly outside of their indications. haloperidol should also be the subject of a similar study as soon as possible. use of spironolactone in sars-cov- ards patients hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial the fda-approved drug ivermectin inhibits the replication of sars-cov- in vitro covid- : consider cytokine storm syndromes and immunosuppression induction of pro-inflammatory cytokines (il- and il- ) and lung inflammation by coronavirus- (covi- or sars-cov- ): anti-inflammatory strategies the cytokine release syndrome (crs) of severe covid- and interleukin- receptor (il- r) antagonist tocilizumab may be the key to reduce the mortality tocilizumab treatment in covid- : a single center experience tocilizumab, an anti-il receptor antibody, to treat covid- -related respiratory failure: a case report interventions for preventing intensive care unit delirium in adults haloperidol prophylaxis in critically ill patients with a high risk for delirium haloperidol use is associated with lower hospital mortality in mechanically ventilated patients key: cord- -ynionj r authors: hwang, mihyun; bergmann, cornelia c. title: alpha/beta interferon (ifn-α/β) signaling in astrocytes mediates protection against viral encephalomyelitis and regulates ifn-γ-dependent responses date: - - journal: j virol doi: . /jvi. - sha: doc_id: cord_uid: ynionj r the contribution of distinct central nervous system (cns) resident cells to protective alpha/beta interferon (ifn-α/β) function following viral infections is poorly understood. based on numerous immune regulatory functions of astrocytes, we evaluated the contribution of astrocyte ifn-α/β signaling toward protection against the nonlethal glia- and neuronotropic mouse hepatitis virus (mhv) strain a . analysis of gene expression associated with ifn-α/β function, e.g., pattern recognition receptors (prrs) and interferon-stimulated genes (isgs), revealed lower basal mrna levels in brain-derived astrocytes than in microglia. although astrocytes poorly induced ifnβ mrna following infection, they upregulated various mrnas in the ifn-α/β pathway to a higher extent than microglia, supporting effective ifn-α/β responsiveness. ablation of the ifn-α/β receptor (ifnar) in astrocytes using mgfapcre ifnar(fl/fl) mice resulted in severe encephalomyelitis and mortality, coincident with uncontrolled virus replication. further, virus spread was not restricted to astrocytes but also affected microglia and neurons, despite increased and sustained ifnα/β and isg mrna levels within the cns. ifn-γ, a crucial mediator for mhv control, was not impaired in infected mgfapcre ifnar(fl/fl) mice despite reduced t cell cns infiltration. unexpectedly however, poor induction of ifn-γ-dependent major histocompatibility complex (mhc) class ii expression on microglia supported that defective ifn-γ signaling contributes to uncontrolled virus replication. a link between sustained elevated ifn-α/β and impaired responsiveness to ifn-γ supports the novel concept that temporally limited early ifn-α/β responses are critical for effective antiviral ifn-γ function. overall, our results imply that ifn-α/β signaling in astrocytes is not only critical in limiting early cns viral spread but also promotes protective antiviral ifn-γ function. importance an antiviral state established by ifn-α/β contains initial viral spread as adaptive immunity develops. while it is apparent that the cns lacks professional ifn-α/β producers and that resident cells have distinct abilities to elicit innate ifn-α/β responses, protective interactions between inducer and responder cells require further investigation. infection with a glia- and neuronotropic coronavirus demonstrates that astrocytes mount a delayed but more robust response to infection than microglia, despite their lower basal mrna levels of ifn-α/β-inducing components. lethal, uncontrolled viral dissemination following ablation of astrocyte ifn-α/β signaling revealed the importance of ifn-α/β responses in a single cell type for protection. sustained global ifn-α/β expression associated with uncontrolled virus did not suffice to protect neurons and further impaired responsiveness to protective ifn-γ. the results support astrocytes as critical contributors to innate immunity and the concept that limited ifn-α/β responses are critical for effective subsequent antiviral ifn-γ function. v iral infections of the central nervous system (cns) are rare but can lead to rapid mortality or long-term neurological disabilities even if acute encephalitis is resolved ( ) . early essential host defense mechanisms involve induction of alpha/beta interferon (ifn-␣/␤) and signaling through the ifn-␣/␤ receptor (ifnar) to upregulate ifn-stimulated genes (isgs). isg expression is associated with numerous biological activities, including antiviral and immunomodulatory pathways, e.g., interference with translation, apoptosis, and enhanced major histocompatibility complex (mhc) class i antigen (ag) presentation ( ) ( ) ( ) . while the ifn-␣/␤ response is critical in stemming cns viral replication and spread, it is rapidly downregulated and insufficient to eliminate virus in the absence of subsequent adaptive immune responses ( ) ( ) ( ) . moreover, the efficiency of the innate response in limiting viral spread sets the stage for the effectiveness of subsequent adaptive immunity ( ) ( ) ( ) ) . detection of viruses and induction of ifn-␣/␤ are initiated by pattern recognition receptors (prrs), which differ in subcellular localization and are specialized to recognize distinct molecular entities in virus structural components, genome, and/or replication intermediates ( , , ) . prrs which recognize rna viruses include endosome-associated membrane-bound toll-like receptors (tlrs), mainly tlr , and the cytoplasmic retinoic acid inducible gene (rig-i)-like receptors (rlrs) and melanoma differentiationassociated antigen (mda ). specialized prr adapter proteins transmit signals to activate ifn response factor (irf ), irf , and irf , which translocate to the nucleus to induce ifn-␤ and a subset of ifn-␣ genes ( ) . secretion of ifn-␣/␤ and signaling through ifnar subsequently activate isgs and the "antiviral state." prr activation also activates nuclear factor b (nf-b)-regulated genes, thereby inducing proinflammatory cytokines and chemokines to facilitate recruitment of innate and adaptive effector cells ( , ) . virtually all nucleated cells are able to induce and respond to ifn-␣/␤, including cns resident cells ( , ) . the cns does not harbor plasmacytoid dendritic cells, which are potent peripheral ifn-␣/␤ inducers ( , ) , and therefore has to rely on intrinsic induction of ifn-␣/␤. cns resident cells have been shown to differ widely in the repertoire as well as magnitude of basal and inducible transcripts encoding prrs and factors associated with the ifn-␣/␤ pathway ( ) ( ) ( ) ) . as ifn-␣/␤ signaling can rapidly induce genes involved in virus sensing and their signaling components, cell types which are not effective initial ifn-␣/␤ inducers may nevertheless contribute to the overall innate antiviral activity by efficient ifn-␣/␤ signaling and amplification of the ifn-␣/␤ response ( , , ) . the interdependence of cns cells in achieving an ifn-␣/␤-dependent antiviral state thus requires further investigation. microglia and astrocytes are the cns resident cells most prominently involved in early innate responses to injury, autoimmune attack, or infection ( , , , ) . in infections these early responses are essential to attract peripheral immune cells and limit viral dissemination ( , , ) . importantly, not only productively infected but also abortively infected astrocytes mount innate responses that contribute to protection ( , , ) . cns infections by several neuronotropic rna viruses, including la crosse virus, rabies virus, vesicular stomatitis virus (vsv), and theiler's murine encephalomyelitis virus (tmev), suggested that astrocytes, not neurons, are the main source of ifn-␤ expression and contributors to virus control through both tlr and rlr activation pathways ( ) ( ) ( ) . both microglia and astrocytes express various prrs, and their sentinel function is supported by their rapid response to microbial infection ( , , , , , ) . in addition to participating in direct antiviral functions, astrocytes regulate cns homeostasis, support neuronal function, and participate in formation and maintenance of the blood-brain barrier (bbb) ( , ) . both astrocytes and ifn-␣/␤ actively participate in bbb function to restrict cns entry of molecules, cells, or pathogens ( , ) . ifn-␣/␤ signaling specifically to astrocytes promotes bbb integrity in the cerebellum and protects from west nile virus infection ( ) . thus, both astrocyte ifn-␣/␤ induction and signaling regulate the outcome of infection within the cns at different functional levels. the essential role of ifnar signaling in limiting viral dissemination within the cns is supported by neurotropic virus infections of ifnar-deficient (ifnar Ϫ/Ϫ ) mice ( , , , ). however, caveats for ifnar Ϫ/Ϫ mice are the reduced basal levels of factors involved in the ifn-␣/␤ pathway as well as loss of innate myeloid cell dynamic activity in response to infection ( , , ) . further, while much information on cns innate responsiveness is derived from primary glial and neuronal cultures, the in vivo studies of ifn-␤ induction highlight how unsuspected players, such as abortively infected astrocytes, contribute to pathogen control ( ) . based on the prominent role of astrocytes in participating in innate responses, this study set out to assess how ifnar ablation in astrocytes affects pathogenesis in a gliaand neuronotropic coronavirus infection. the murine coronavirus mouse hepatitis virus (mhv) strain a infects microglia, astrocyte, neurons, and oligodendroglia following intracranial (i.c.) administration ( , ) . although mhvs are at best poor inducers of ifn-␣/␤ ( ) ( ) ( ) , they do induce ifn-␤ in microglia/macrophages ( ) . importantly, even the low levels of ifn-␣/␤ are essential to prevent viral dissemination and mortality ( , ) . the studies here reveal distinct patterns of basal and inducible levels of mrnas encoding components of the ifn-␣/␤ pathway in astrocytes and microglia isolated from naive and infected adult mouse brains. despite expressing lower baseline mrna levels, astrocytes upregulated ifn-␣/␤ pathway gene expression to a greater extent than microglia, supporting effective ifn-␣/␤ responses. ablation of ifn〈r in astrocytes using mgfapcre ifnar fl/fl mice resulted in severe encephalomyelitis and mortality by days postinfection (p.i.). this contrasted with mild clinical symptoms and no fatalities in infected control ifnar fl/fl mice. uncontrolled viral spread throughout the cns parenchyma of mgfapcre ifnar fl/fl mice not only was associated with increased astrocyte infection but also affected neurons and microglia, despite overall elevated and sustained levels of mrnas for ifn-␤ and ifn-␣ genes and isgs. ifn-␥, a crucial mediator of mhv control in the cns, was not impaired, despite reduced t cell cns infiltration. unexpectedly however, defective ifn-␥ signaling was implicated by impaired induction of ifn-␥-dependent mhc class ii expression on microglia. overall our results imply that ifn-␣/␤ signaling in astrocytes not only is critical in limiting cns viral spread but also promotes lymphocyte-derived protective antiviral ifn-␥ function. mhv strain a induces type i ifn in the cns coincident with viral replication. to evaluate the kinetics of mhv a replication relative to ifn␣/␤ and ifn␥ mrna levels in the cns, brains from uninfected and infected wild-type (wt) c bl/ mice were harvested out to day p.i. virus replication was monitored by expression of viral rna encoding the n protein (a n), which is present on genomic and all subgenomic rnas ( ) . viral n mrna was most abundant between days and p.i. and declined by day p.i. (fig. ), but it remained detectable out to day p.i. ifn␤, ifn␣ , and ifn␣ mrnas, the most abundant ifn␣ mrnas expressed following mhv a infection ( ) , also reached maximal levels between and days p.i. and dropped significantly by day p.i. (fig. ). while ifn␤ mrna was still elevated at day p.i., ifn␣ and ifn␣ mrnas had declined to basal levels by day p.i. ifn␥ mrna was already detectable at day p.i. but did not peak until to days p.i. expression levels declined by day p.i. but remained elevated above background out to day p.i. (fig. ) . ifn␣/␤ mrna levels thus peaked together with viral mrna, whereas the ifn␥ mrna peak was delayed and coincided with t cell infiltration ( ) . astrocytes exhibit distinct induction of and responsiveness to ifn-␣/␤ compared to microglia. although mhv a replicates in glia and neurons, it induces ifn-␣/␤ only in microglia, not astrocytes, using primary cell cultures ( ) . to assess the relative induction of and responsiveness to ifn-␣/␤ in astrocytes and microglia in vivo, we used infected gfap-gfp mice to isolate cd -negative, green fluorescent protein (gfp)-positive (cd Ϫ gfp ϩ ) astrocytes and cd int cd b ϩ microglia by fluorescenceactivated cell sorting (facs) ( fig. a ). measurement of viral n relative to glyceraldehyde- -phosphate dehydrogenase (gapdh) mrnas in either glia population revealed viral replication in both microglia and astrocytes at days and p.i. and a decline by day p.i. (fig. b ). while increased viral n mrna in microglia relative to astrocytes at days and p.i. did not reach statistical significance, overall viral n mrna levels mirrored those in total brain. the same populations of purified cells were used to measure transcript levels of the ifn␣/␤ genes, genes regulating ifn-␣/␤, signaling and selected antiviral isgs (fig. c) . in naive mice, constitutive expression of these genes was lower or undetectable in astrocytes compared to microglia. following infection, ifn␤ mrna was upregulated by day p.i. in microglia but was not upregulated until day p.i. and was less robust in astrocytes. in contrast, ifn␣ and ifn␣ mrnas were not significantly upregulated in microglia but were increased prominently in astrocytes by day p.i. consistent with the decline in viral rna, ifn␣/␤ mrnas dropped to baseline levels by day p.i. (fig. c ). transcripts for components required for ifn-␣/␤ signaling showed no significant differences between astrocytes and microglia throughout days to p.i. (fig. c) . while infection mediated a decline in ifnar mrna relative to basal levels in microglia by day p.i., it did not alter expression levels in astrocytes. stat mrna levels varied between cell preparations and showed no significant changes throughout infection in either cell type. irf transcripts were not affected by virus infection in microglia but increased modestly in astrocytes. in contrast, individual isgs were regulated distinctly not only between the glia populations but also within each glia type over time (fig. c) . mhv cns infection has been shown to strongly induce ifn-induced protein with tetratricopeptide repeats (ifit and ifit ) and =, =-oligoadenylate synthetase (oas ) transcripts ( , ) . although ifit mrna was increased in both populations by day p.i., the relative induction was significantly higher in astrocytes than in microglia at all time points. in contrast, ifit mrna was induced more prominently in microglia at day p.i., reached similar levels in both cell types at day p.i., and dropped thereafter in both populations. lastly, oas mrna showed peak upregulation in microglia by day p.i. and modest induction in astrocytes (fig. c) . under the assumption that viral mrna levels reflect similar replication in both glial populations, these data support microglia as superior initiators of ifn-␤ production relative to astrocytes following mhv a infection in vivo; these findings are consistent with results from primary neonatal cell cultures ( , ) . moreover, delayed ifn␤ upregulation in astrocytes suggested that their sensitivity to viral replication is enhanced once viral sensors are elevated via ifn-␣/␤ signaling. this notion was supported by delayed, yet more extensive, upregulation of the ifn␣ as well as the ifit and ifit genes compared to that in microglia. isg upregulation further appeared to be independent of direct changes in ifnar, stat , or irf transcript levels. many prr-encoding genes are themselves isgs and are upregulated during cns infections ( , , , ) . mda , rig-i, and tlr have all been established as prrs contributing to ifn-␣/␤ induction in mhv-infected microglia/macrophages and oligodendrocytes ( , , , ) . basal mda , rig-i, and tlr transcript levels were all significantly lower in facs-purified astrocytes than in microglia (fig. ) . nevertheless, astrocytes rapidly upregulated mda , rig-i, and tlr mrnas to levels observed in microglia as early as day p.i. (fig. ) , when total ifn␣/␤ mrna peaked in the brain (fig. ). relative to basal levels, the fold increase in astrocytes exceeded that in microglia. all three prr transcripts declined by day p.i., reflecting the decline in ifn-␣/␤. with the exception of mrna encoding ikk, a kinase involved in both induction and ifnar signaling, constitutive levels of transcripts for prr signal transduction factors represented by irf and irf were also lower in astrocytes (fig. ) . expression of the cellular kinase ikk mrna was gradually increased in both glial populations over time up to day p.i., with increases more pronounced in microglia (fig. ) . irf mrna expression was decreased in microglia following infection and remained unchanged in astrocytes, contrasting with upregulation of irf mrna in both cell types between days to p.i. and downregulation thereafter (fig. ) . these data are consistent with constitutive irf expression but inducible expression of both ikk and irf following infection or ifn-␣/␤ treatment of non-cns cells ( ) . overall, the data suggest that initial production of ifn-␤ in microglia results in upregulation of select isgs in astrocytes, thereby amplifying ifn-␣/␤. however, it remains unclear whether astrocytes induce ifn-␤ directly via virus-derived prr activators or via mediators released from necrotic or apoptotic cells in the inflamed cns environment. regardless, the results demonstrate that astrocytes are sensitive responders to ifn-␣/␤ and active participants in the ifn-␣/␤-induced amplification loop. ifnar signaling in astrocytes is essential to control mhv a infection. given the prominent response of astrocytes to ifn-␣/␤ following mhv a infection in vivo, we determined the protective role of astrocyte-mediated ifnar signaling against infection using mgfapcre ifnar fl/fl mice. disease onset was similar in mgfapcre ifnar fl/fl and ifnar fl/fl mice starting at day p.i. however, in contrast to mild clinical symptoms in ifnar fl/fl mice, mgfapcre ifnar fl/fl mice developed severe encephalitis and succumbed to infection by day p.i. (fig. a ). the -to -day-delayed mortality compared to that of ifnar Ϫ/Ϫ mice infected with a similar virus dose ( ) supported astrocytes as critical, but not the only, contributors to early ifnar-dependent protection. to evaluate whether disease severity and mortality in mgfapcre ifnar fl/fl mice were correlated with impaired viral control, infectious virus was measured in brain supernatants. the virus loads were comparable in both groups at day p.i. but reached ϳ -log -higher levels in mgfapcre ifnar fl/fl mice than in ifnar fl/fl mice by day p.i. (fig. b ). the anatomical localization and distribution of viral n protein were also evaluated by histology. compared to small isolated residual foci of viral ag-positive cells in brains of ifnar fl/fl mice at day p.i., mgfapcre ifnar fl/fl mice exhibited extensive dissemination with increased intensity of viral ag staining throughout the brain (fig. c ). high-magnification images indicated that virus was not limited to cells consistent with glial morphology but also was in cells with typical neuronal morphology in mgfapcre ifnar fl/fl mice (fig. c, insets) . as mhv a is hepatotropic following peripheral infection and can spread to the liver following i.c. infection of immunocom- by rt-pcr. data represent the means Ϯ sem from two experiments, each comprising or pooled brains per time point, and were analyzed by the unpaired two-tailed student t test and two-way anova. *, significance between microglia and astrocytes; #, significance compared to each respective naive population: * and #, p Ͻ . ; ** and ##, p Ͻ . ; *** and ###, p Ͻ . . promised mice ( ), we also surveyed liver for viral n mrna. the levels of viral n relative to gapdh transcripts were elevated in livers of mgfapcre ifnar fl/fl compared to ifnar fl/fl mice at day p.i. (values of Ϯ versus Ϯ ) (data not shown). however, overall viral n mrna levels were significantly higher in the cns ( , Ϯ versus Ϯ , respectively) (see fig. ). there was also no evidence of necrotic foci by gross visual examination (data not shown). mortality was thus attributed to overwhelming virus spread within the cns. mhv a infects multiple cns cell types, including microglia, astrocytes, and neurons ( , ) . to evaluate to what extent the absence of ifn-␣/␤ signaling in astrocytes predisposes astrocytes over other cell types to enhanced infection, brains from infected mgfapcre ifnar fl/fl and ifnar fl/fl mice were analyzed for viral n protein in combination with antibodies (abs) specific for neurons (neun), astrocytes (glial fibrillary acidic protein [gfap]), and microglia/macrophages (iba ). characteristic staining and colocalization patterns at day p.i. in wt mice are depicted in fig. a . microglia and neurons showed prominent perinuclear cytoplasmic n protein localization, with some extension into processes in microglia. in contrast, viral ag was only occasionally detected in the cytoplasm of astrocytes and was localized mainly proximal to, but not overlapping with, astrocytic processes. in this context it is critical to note that the anti-gfap ab used to identify astrocytes stains main stem branches but not the cytoplasm or fine processes of astrocytes and poorly stains gray matter astrocytes ( , ) , suggesting that viral protein may localize to astrocytic processes poorly detected by anti-gfap ab. total n protein staining revealed an ϳ -fold-higher positive area in mgfapcre ifnar fl/fl than in ifnar fi/fi mice as determined by pixels per observed field at day p.i.; the difference increased to ϳ -fold by day p.i., when virus already declined in ifnar fi/fi mice (fig. b) . keeping the limitations of gfap staining in mind, colocalization of viral ag with gfap was overall increased at day p.i. furthermore, when the relative proportion of the dual-positive area within the total viral ag-positive pixel area was assessed, percentages were similar in both mouse strains at day p.i. but were -fold higher in mgfapcre ifnar fl/fl mice than in controls at day p.i. (fig. c ). viral ag-positive microglia were also overall increased in mgfapcre ifnar fl/fl mice at day p.i. however, the relative proportion of the dually viral ag-and iba -positive area per total viral ag-positive area was comparable between groups at both days and p.i. (fig. d) . to evaluate the relative infection of astrocytes versus microglia, we also measured the proportions of viral ag-positive area relative to gfap-or iba -positive areas. while infection of microglia increased from ϳ % to ϳ %, it increased from Ͻ % to % in astrocytes in mgfapcre ifnar fl/fl mice between days and p.i. (fig. ) . although infected neurons were increased in mgfapcre ifnar fl/fl compared to ifnar fl/fl mice throughout the brain by day p.i., they declined significantly in both groups by day p.i. remaining elevated neuronal infection in mgfapcre ifnar fl/fl mice at day p.i. was still particularly evident in lower and distal parts of the brain (pons and medulla) (fig. e) . analysis of apoptosis to explain the decline in virus-infected neurons indicated and microglia in brain cortex of both mouse groups at day p.i. bar graphs show the proportion of n ϩ gfap ϩ or n ϩ iba ϩ per total n ϩ area, respectively. insets show infected astrocytes at higher magnification and arrows indicate infected microglia. (e) representative staining of viral n protein in neurons in brain cortex and brain stem at days and p.i. the bar graph shows average numbers of virus-infected neurons per brain section from or whole brain sections per mouse (n ϭ mice). arrows indicate virus-infected neurons. scale bar, m. (c to e) left and right panels indicate ifnar fl/fl and mgfapcre ifnar fl/fl mice, respectively. the data in panels b and c represent the means Ϯ sem from or fields in or slides from mice per group and were analyzed by the unpaired two-tailed student t test and two-way anova. *, significance between ifnar fl/fl and mgfapcre ifnar fl/fl mice; #, significance between days and p.i. in the same group: *, p Ͻ . ; ** and ##, p Ͻ . ; ***, p Ͻ . . overall sparse but more abundant neuronal apoptosis in mgfapcre ifnar fl/fl mice (fig. a ). occasional apoptotic cd ϩ lymphocytes were also observed with elevated frequency in mgfapcre ifnar fl/fl mice (fig. b ). while these results likely underestimate the extent of viral infection in astrocytes, they support that the absence of astrocyte ifnar signaling leads to increased viral dissemination most prominently within astrocytes but also to microglia and neurons. abrogated ifnar signaling in astrocytes does not impair overall expression of ifn-␣/␤, isgs, chemokines, and cytokines. to assess whether uncontrolled virus spread was attributable to overall impaired innate immune responses, we determined transcripts for ifn-␣/␤ and isgs in relation to viral n mrna levels in brains of both mouse groups (fig. ). viral n mrna levels were similar at day p.i. but increased significantly in mgfapcre ifnar fl/fl mice compared to ifnar fl/fl controls by day p.i. (fig. ) , consistent with increased infectious virus by day p.i. (fig. b) . elevated viral n mrna correlated with increased ifn␤, ifn␣ , and ifn␣ transcripts at day p.i. (fig. ) . to assess how elevated ifn-␣/␤ affected downstream ifnar signaling, we further compared mrna levels of the isgs (ifit , ifit , isg , mx , pkr, and oas ). while these transcripts were all similar in both mouse groups at day p.i., ifit , ifit , and mx mrnas were increased -to -fold in mgfapcre ifnar fl/fl mice by day p.i. isg , pkr, and oas mrnas were not affected or reduced relative to those in ifnar fl/fl controls (fig. ) . ifnar abrogation on astrocytes thus did not impair global ifn␣/␤ or select isg expression in the infected cns. however, innate immunity by ifnar-competent cells was clearly insufficient to stem viral dissemination throughout the cns in mgfapcre ifnar fl/fl mice. astrocytes are also potent inducers of chemokines and cytokines ( , ) , and a subset of chemokines, e.g., the neutrophil chemoattractant cxcl , can be negatively regulated by ifn-␣/␤ ( ). we therefore evaluated how the absence of ifnar on astrocytes modulates expression of select nf-b-dependent chemokines and the proinflammatory cytokine interleukin (il- ) ( , ) . transcripts for cxcl and ccl , potent chemoattractants for neutrophils and monocytes, respectively, were similar at day p.i. but were significantly upregulated at day p.i. (fig. ) , consistent with increased viral replication and ifn-␣/␤-mediated downregulation of cxcl by astrocytes ( , ) . increased il mrna expression at day p.i. (fig. ) further indicated that ifnar abrogation on astrocytes does not impact proinflammatory cytokine induction. in contrast, ccl mrna, which is prominently expressed by t cells during mhv infection ( ) , was significantly reduced compared to that in ifnar fl/fl mice at day p.i. dysregulation of these proinflammatory factors suggested that myeloid cells may be recruited in favor of lymphocytes in mgfapcre ifnar fl/fl mice, thereby contributing to morbidity and uncontrolled viral spread. infected mgfapcre ifnar fl/fl mice exhibit increased cns neutrophil accumulation and reduced t cell infiltration. flow cytometry revealed that altered chemokine expression patterns indeed affected the composition of inflammatory cells within the cns. consistent with elevated cxcl mrna, neutrophils in mgfapcre ifnar fl/fl brains were increased by ϳ -fold at day p.i. relative to those in ifnar fl/fl mice, although numbers in both groups were already reduced compared to those at day p.i. (fig. a) . differences in macrophages were not statistically significant (fig. b) . as both cd and cd t cells are essential to reduce infectious mhv, with ifn-␥ playing a prominent antiviral role ( , ) , we further assessed if impaired t cell recruitment and ifn-␥ production within the cns is associated with lack of virus control. indeed, both cd and cd t cells were reduced in the cns of mgfapcre ifnar fl/fl mice at day p.i. (fig. c) . however, similar ifn-␥ levels (fig. d) , despite reduced t cell numbers, suggested that increased viral replication leads to increased ag presentation, thereby triggering more or prolonged ifn-␥ production by t cells in mgfapcre ifnar fl/fl mice ( , ) . impaired access of t cells to the cns parenchyma was ruled out as an explanation of loss of viral control, as we found no differences in anatomical distribution of cd ϩ t cells within perivascular and parenchymal sites between the two groups (data not shown). uncontrolled viral replication in mgfapcre ifnar fl/fl mice despite similar ifn-␥ production thus implied that viral replication overwhelms the t cell response or that ifn-␥ signaling is impaired. determination of ifn-␥-specific responses in vivo is complicated by the fact that many factors upregulated by ifn-␥ are also induced by ifn-␣/␤ ( ). nevertheless, a prominent indicator of ifn-␥ signaling in microglia/macrophages is induction of the transcription factor class ii transactivator (ciita), a master regulator of mhc class ii expression ( , ) . interestingly, ciita transcripts were barely induced in brains of mgfapcre ifnar fl/fl mice, in contrast to robust upregulation in control mice, at day p.i. (fig. e) . analysis of mhc class ii expression on microglia by flow cytometry confirmed that the very modest ciita mrna induction impaired ifn-␥-dependent mhc class ii upregulation. only ϳ % of microglia expressed mhc class ii in mgfapcre ifnar fl/fl mice, compared to ϳ % in ifnar fl/fl mice, and the mean fluorescence intensity, reflecting expression levels, per cell was also lower (fig. f) . other ifn-␥-dependent factors include the chemokines cxcl and cxcl ( , ) . while cxcl expression is inducible mainly by ifn-␥, cxcl is inducible by ifn-␣/␤, ifn-␥, and tumor necrosis factor (tnf) ( , , ) . furthermore, cxcl is produced by macrophages/microglia and endothelial cells, but not astrocytes, whereas cxcl is induced prominently in astrocytes during inflammation ( , ) . assessment of cxcl and cxcl mrnas showed similarly low levels in both groups at day p.i. (fig. g) . however, unlike ifnar fl/fl mice, mgfapcre ifnar fl/fl mice failed to upregulate cxcl mrna by day p.i. in contrast, cxcl mrna levels were elevated by ϳ . -fold in mgfapcre ifnar fl/fl mice by day p.i. but did not increase in ifnar fl/fl mice. given that mgfapcre ifnar fl/fl mice do not express ifnar on astrocytes, cxcl mrna upregulation is likely driven by ifn-␥ and/or tnf. overall, these results implied that microglia, but not astrocytes, are significantly compromised in ifn-␥ responsiveness in mgfapcre ifnar fl/fl mice. preexposure to ifn-␣/␤ modifies ifn-␥ responsiveness in myeloid cells. one explanation for the differential responsiveness to ifn-␥ in microglia versus astrocytes in mgfapcre ifnar fl/fl mice is that prolonged ifnar signaling via elevated and sustained ifn-␣/␤ expression may skew subsequent ifn-␥ responsiveness. this is supported by an antagonistic effect of ifn-␣/␤ on ifn-␥ responses in macrophages following ifn treatment or listeria infection ( ) ( ) ( ) . we therefore used the viral rna mimic poly(i·c), a synthetic double-stranded rna (dsrna) analog recognized by tlr as well as rig-i/mda , to induce ifn␣/␤ mrna in bone marrow-derived macrophages (bmdm) and monitor ifn-␥ responsiveness. previous in vivo data revealed that poly(i·c) administration into the cns of mice induced ifn␣/␤ mrna by h, which was sustained out to h ( ). bmdm were thus pretreated with poly(i·c) for or h and subsequently treated with ifn-␥ for h. ifn-␥-only treatment was included as a positive control. poly(i·c) alone did not induce ciita mrna at h and induced it only sparsely by h. however, while ifn-␥ treatment alone effectively induced ciita mrna, it failed to induce ciita mrna in cells pretreated with poly(i·c) for either or h (fig. ) . similarly, poly(i·c) alone only sparsely induced cxcl mrna at h compared to induction by ifn-␥ treatment alone, and short poly(i·c) pretreatment suppressed cxcl mrna induction by ifn-␥. assessment of an inhibitory effect of prolonged poly(i·c) exposure was preempted by induction of cxcl mrna levels similar to those with ifn-␥. finally, treatment with poly(i·c) alone was superior to ifn-␥ alone in upregulating cxcl mrna, with saturation already achieved at h. these results are consistent with poly(i·c)-mediated upregulation of cxcl via ifn-␣/␤ and tnf and preempted use of cxcl as a target gene to assess suppressive functions on ifn-␥ signaling in this experimental setting. overall, these data indicate that preexposure of macrophages/ microglia to a virus-induced inflammatory milieu including ifn-␣/␤ has the potential to negatively regulate subsequent ifn-␥ signaling. however, the magnitude of ifn-␣/␤mediated suppression of ifn-␥-dependent gene induction depends on other regulatory elements in the respective target gene promoter. these results reveal that the duration and/or level of cell exposure to ifn-␣/␤ can substantially diminish responsiveness to temporally lagging ifn-␥, thereby reducing ifn-␥ protective functions. the ability to induce and respond to ifn-␣/␤ varies between cell types as well as between types of virus, but the contribution of different cns cell types to mount ifn-␣/␤-mediated protection in vivo are less well explored ( ) . our studies comparing the capacity of adult astrocytes relative to microglia to respond to mhv a infection revealed that astrocytes expressed lower basal levels of prrs and signaling components to induce ifn-␣/␤ and isgs than microglia, consistent with published gene array data ( ) . nevertheless, despite a delay in response to infection, astrocytes exhibited strong upregulation of prrs, ifn␣s, and select isgs, supporting that astrocytes are well positioned to respond to paracrine ifn-␣/␤ to amplify their innate antiviral response. the critical role of ifnar signaling in astrocytes to block viral spread was confirmed by rapid virus dissemination throughout the cns upon abrogation of ifnar signaling in astro-cytes. thus, although basal expression levels of ifn-␣/␤ signaling components have been shown to determine the responsiveness of cells to insults ( , ) , our data reveal that cells expressing low basal levels of ifn-␣/␤ pathway components can nevertheless trigger a protective innate immune response through paracrine ifn-␣/␤ signaling. delayed mortality of infected mgfapcre ifnar fl/fl mice compared to ifnar Ϫ/Ϫ mice implied that other cell types, including microglia, can produce and respond to ifn-␣/␤. this is clearly demonstrated by elevated and sustained ifn␣/␤ as well as select isg expression despite ifnar deletion on astrocytes. however, vast dissemination of virus throughout the cns clearly indicated that ifnar signaling specifically in astrocytes plays a dominant role in host protection, which could not be compensated by ifnarcompetent glia and neurons. while microglia appeared to exhibit some autocrine action as suggested by less overall infection relative to that in astrocytes, neurons in the brain stem appeared to be most susceptible and vulnerable to infection in the absence of astrocyte ifnar signaling. although we cannot exclude that inherently reduced activation of innate viral components in neurons may contribute to their susceptibility, our data indicate that overwhelming infection of ifnar-deficient astrocytes increases adjacent neuronal infection. west nile virus infection of neuronal cultures demonstrates that neurons have the capacity to mount innate viral responses via tlr and irf activations ( , ) . moreover, neurons have also been demonstrated to induce ifn-␤ following tmev and la crosse virus infection in vivo ( ) . nevertheless, these responses were very sparse and focal. in contrast, subsequent studies of la crosse virus infection in ifn-␤ reporter mice showed that apparently noninfected astrocytes and microglia in close proximity to infected neurons were prominent ifn-␤-producing cells, while ifn-␤ expression by infected neurons was rare ( ) . more recent data demonstrate that ifn-␤-producing astrocytes appeared to be transiently or abortively infected during various infections associated with neuronal tropism ( ) . the spatial extent of innate protection induced in an infected environment may thus depend on the prominent infected cell type. as both microglia and astrocytes have far-reaching processes, release of ifn-␣/␤ from these cells may provide more extensive protection than release from neuronal cell bodies. two independent studies with vsv indeed indicate that locally induced ifn-␤ in the olfactory bulb can trigger isgs in distal parts of the brain, although one indicates astrocytes and the other neurons as the prominent ifn-␤ inducers ( , ) . regardless, the vast increase of virus in mgfapcre ifnar fl/fl mice supports the notion that the inability of astrocytes to clear virus, rather than loss of direct isg effects on other cns cells, is responsible for loss of viral control. in addition to uncontrolled virus, elevated neutrophil accumulation may contribute to tissue damage and rapid morbidity of mgfapcre ifnar fl/fl mice. increased neutrophil cns infiltration correlated with increased mrna expression of the neutrophil chemoattractant cxcl ( ) and its downregulation by ifn-␣/␤ and ifn-␥ ( , , , ) . neutrophils are early innate responders, which contribute to bbb breakdown and tissue damage via secretion of matrix metalloproteinases (mmps) and a wide range of other degrading enzymes released upon degranulation ( , ) . during autoimmune-mediated cns inflammation, neutrophils have also been shown to release cytokines which mature antigen-presenting cells and subsequently reactivate t cells ( ) ( ) ( ) . elevated cxcl mrna as well as increased levels of mrnas encoding il- and ccl , two other proinflammatory factors prominently induced in astrocytes ( ) , further suggested that the nf-b pathway initiated by ppr activation was intact in infected mgfapcre ifnar fl/fl mice. finally, our results show that extensive virus spread in the absence of protective astrocyte ifnar signaling cannot be controlled by t cells. although previous studies of cxcl and cxcl monoclonal antibody (mab) blockade to reduce cxcr -dependent t cell recruitment showed a reduction in t cell migration and loss of viral control in a related mhv infection ( , ) , subsequent studies in cxcl -and cxcl -deficient mice indicated that cxcl is more critical for effective adaptive immunity ( ) . impaired t cell accumulation despite unimpaired cxcl mrna in mgfapcre ifnar fl/fl mice was thus unanticipated. while an overwhelming virus load may lead to aginduced t cell exhaustion and death ( ) , dysregulation of t cells requires future investigation. regardless of reduced t cell numbers, their initial activity as monitored by ifn-␥ secretion was not impaired. ifn-␥ signaling is essential for mhv control within the cns by both exerting direct antiviral effects and enhancing mhc class i and ii upregulation ( ) . however, reduced induction of ifn-␥-dependent ciita and cxcl mrnas showed that microglia were significantly impaired in ifn-␥ responsiveness. in contrast, increased cxcl mrna expression in the absence of ifnar on astrocytes implied that ifn-␥ signaling in astrocytes remained intact. the notion that elevated and sustained ifn-␣/␤ signaling acts as a negative regulator of ifn-␥ signaling was supported by earlier reports showing antagonistic effects of ifn-␣/␤ on ifn-␥-induced activation of macrophages ( - ). one possible mechanism may involve downregulation of the ifn-␥ receptor (ifngr), as shown during listeria infection ( ) . our in vitro studies further supported selected downregulation of ifn-␥ responsiveness by ifn-␣/␤ via pretreatment of bmdm with the dsrna mimic poly(i·c). ifn-␣/␤-dependent sensitization of microglia/macrophages to subsequent ifn-␥ responses may provide a mechanism to limit excessive immune responses leading to tissue damage. in this context it is of interest to note that both ifn-␣/␤ and ifn-␥ responses are tightly controlled by negative regulators, including phosphatases ( ) . cross talk between ifn-␣/␤ and ifn-␥ responses may thus provide additional levels to protect the host from exacerbated proinflammatory responses. overall, our data demonstrate a vital protective role of astrocyte-mediated ifn-␣/␤ signaling in host protection from neurotropic coronavirus-induced encephalomyelitis. further, the link between sustained elevated ifn-␣/␤ and impaired responsiveness to ifn-␥ supports the novel concept that temporally limited early ifn-␣/␤ responses are critical for effective antiviral ifn-␥ function. mes/j (gfap-gfp) mice were originally purchased from the jackson laboratory (bar harbor, me) and backcrossed to c bl/ mice for at least generations. b .cg-tg (gfap-cre) . mvs/j (stock number ; mgfapcre) mice were also purchased from the jackson laboratory (bar harbor, me). ifnar fl/fl mice engineered to contain loxp sites flanking exon of the ifnar gene were originally produced on the /ola background in u. kalinke's laboratory (paul-ehrlich-institut, langen, germany) as described previously ( ) and were backcrossed onto the c bl/ background in r. schreiber's laboratory (washington university school of medicine, st. louis, mo) as described previously ( ) . ifnar fl/fl mice were crossed with mgfapcre ϩ/Ϫ mice to generate mgfapcre ϩ/Ϫ ifnar fl/fl (mgfapcre ifnar fl/fl ) mice, which were crossed with ifnar fl/fl mice for experimentation. offspring were genotyped for both the presence of the cre recombinase gene and exon deletion from the ifnar gene by pcr using the following primers: cre, =-gtccaatttactgaccgtac acc- = (forward) and =-gttattcggatcatcagctacacc- = (reverse); ifnar flox, =-tgctttgaggagc gtctgga- = (forward) and =-catgcactaccacaccaggcttc- = (reverse). cre-negative ifnar fl/fl littermates were used as wt controls (ifnar fl/fl ). all mice were housed under specific-pathogen-free conditions at an accredited facility at the cleveland clinic lerner research institute. mice of both sexes were infected intracranially (i.c.) at to weeks of age with , pfu of the hepato-and neurotropic mhv a strain expressing enhanced green fluorescent protein (egfp), kindly provided by volker thiel (kantonal hospital, st. gallen, switzerland) ( ) . mhv a was propagated on delayed brain tumor (dbt) astrocytoma monolayers, and virus titers were determined as described previously ( ) . virus in the cnss of individual mice was measured by plaque assay on dbt cells using clarified supernatants from brain homogenates prepared as described previously ( ) . clinical disease severity was graded daily using the following scale: , no disease symptoms; , ruffled fur; , hunched back or inability to turn upright; , severe hunching/wasting or hind limb paralysis; , moribund condition or death ( ) . all animal procedures were approved by the institutional animal care and use committee of the cleveland clinic (phs assurance number a - ) and were conducted in compliance with the guide for the care and use of laboratory animals from the national research council. brains from mice perfused with cold phosphate-buffered saline (pbs) were homogenized in dulbecco's pbs (dpbs) (ph . ) using tenbroeck tissue homogenizers as described previously ( ) . homogenates were centrifuged at ϫ g for min at °c. cells were resuspended in rpmi containing mm hepes (ph . ), adjusted to % percoll (pharmacia, uppsala, sweden), underlaid with ml % percoll, and centrifuged at ϫ g for min at °c. cells were collected from the %/ % interface, washed with rpmi, counted, and suspended in fluorescence-activated cell sorting (facs) buffer ( . % bovine serum albumin in dpbs). fc␥ receptors were blocked with % mouse serum and rat anti-mouse cd / mab (clone . g : bd biosciences, san diego, ca) for min on ice prior to staining with fluorescein isothiocyanate (fitc)-, phycoerythrin (pe)-, peridinin chlorophyll protein (percp)-, or allophycocyanin (apc)-conjugated mabs specific for cd (clone -f ), cd (clone - . ), cd (clone gk . ), ly g (clone a ), cd b (clone m / ), and mhc class ii (clone m / . . ) (all from bd bioscience, mountain view, ca) and f / (serotec, raleigh, nc) in facs buffer. samples were analyzed on a bd accuri c plus instrument (bd biosciences). forward-and side-scatter signals obtained in linear mode were used to establish a gate containing live cells while excluding dead cells and tissue debris. data were analyzed using flowjo software (tress star inc., ashland, or). microglia and astrocyte isolation for gene expression analysis. brains from or naive or mhv a -infected gfap-gfp mice at days , , and p.i. were homogenized using a neural tissue dissociation kit (papain, miltenyi auburn, ca) following the manufacturer's protocol. brain homogenates were prepared for / % percoll gradients to isolate cells as described above. cns cells were blocked with mouse serum and anti-cd / mab as described above prior to staining with cd and cd b in facs buffer. microglia and astrocytes were sorted based on their cd int cd b ϩ and cd neg gfp ϩ phenotypes, respectively, using a facs aria iii (bd biosciences) and facs diva software (bd biosciences). sorted cells were resuspended in trizol (invitrogen, carlsbad, ca) and stored at Ϫ °c. gene expression analysis. rna from brains or sorted cells was extracted using trizol reagent (invitrogen, carlsbad, ca) according to the manufacturer's instructions. following treatment with dnase i using a dna-free kit (ambion, austin, tx), cdna was synthesized using moloney murine leukemia virus reverse transcriptase (invitrogen) in buffer containing mm deoxynucleoside triphosphate mix, ng random hexamer primers, and oligo(dt) ( : ratio) (invitrogen). rna expression was assessed using either sybr green master mix (applied biosystems, foster city, ca) or taqman fast master mix (applied biosystems, foster city, ca) as described previously ( ) . the following primers were used with sybr green master mix: for the gapdh gene, =-catggcct tccgtgttccta- = (forward) and =-atgcctgcttcaccaccttct- = (reverse); for cxcl , =-tgcac gatgctcctgca- = (forward) and =-aggtctttgagggatttgtagtgg- = (reverse); for cxcl , =-ga cggtccgctgcaactg- = (forward) and =-gcttccctatggccctcatt- = (reverse); for the viral nucleocapsid (n) gene, =-gccaaataatcgcgctagaa- = (forward) and =-ccgagcttagccaaaacaa g- = (reverse); for il , =-acacatgttctctgggaaatcgt- = (forward) and =-aagtgcatcatcgttgt tcataca- = (reverse); for oas , =-aaaaatgtctgcttcttgaattctga- = (forward) and =-tgtgcct ttggcagtggat- = (reverse); for ifnar , =-cccaaggcaagagctatgtc- = (forward) and =-tctgaa cggcttccagaact- = (reverse); for ikk, =-ccagaagattcagtgttgtttgg- = (forward) and =-tca ttgtagctgagccctg- = (reverse); for mda , =-gacaccagaattcaagggac- = (forward) and =-gc cacacttgcagataatctc- = (reverse); and for rig-i, =-gtcagcacaaaccacaacc- = (forward) and =-gtctcaaccactcgaatgtc- = (reverse). taqman fast master mix and taqman primers/probes gene expression in total tissue mrna or glial populations was normalized to the respective gapdh mrna expression in each mrna preparation and converted to a linearized value using the formula: e(c t gapdh Ϫ c t gene ) ϫ , . immunohistochemistry and immunofluorescence. brains from pbs-perfused mice were fixed with % neutral zinc-buffered formalin and embedded in paraffin for viral n protein analysis. the distribution of viral antigen (ag) was determined using mab j . specific, for the carboxyl terminus of the viral n protein, as the primary mab, biotinylated horse anti-mouse igg as the secondary ab, and streptavidinconjugated horseradish peroxidase and , =-diaminobenzidine substrate (vectastain-abc kit; vector laboratories virus-infected cells were identified using mab j . ( ) in combination with cell type-specific markers for microglia/macrophages using rabbit anti-iba astrocytes using rabbit anti-glial fibrillary acidic protein (anti-gfap all images were analyzed using fiji version . . quantification of viral n protein staining areas and the relative proportion of colocalization with gfap or iba reactivity were calculated per field of interest using image-pro plus version . . infected neurons were enumerated by counting entire brain sections. for immunohistological analysis, representative data are presented from or separate fields per mouse with or mice per group per time point. ifn-␥ elisa. ifn-␥ in brain supernatants was measured by enzyme bone marrow was collected from femurs and tibiae of -to -week-old mice, passed through a cell strainer, and centrifuged at ϫ g for min at °c. cells were suspended in bmdm medium (dulbecco modified eagle medium [dmem], % fcs, % l conditioned medium as a source of colony-stimulating factor , . % gentamicin, % sodium pyruvate) and seeded at a density of ϫ cells in ml bmdm growth medium into -mm tissue culture dishes. an additional ml bmdm medium was added days later viral diseases of the central nervous system regulation of type i interferon responses antiviral type i and type iii interferon responses in the central nervous system regulation of antiviral t cell responses by type i interferons type i interferon response in the central nervous system brain heterogeneity leads to differential innate immune responses and modulates pathogenesis of viral infections immune surveillance of the cns following infection and injury pathogen recognition and inflammatory signaling in innate immune defenses pathogen recognition and innate immunity innate antiviral signalling in the central nervous system oligodendroglia are limited in type i interferon induction and responsiveness in vivo basal expression levels of ifnar and jak-stat components are determinants of cell-type-specific differences in cardiac 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antagonistic functions tnf activates an irf -dependent autocrine loop leading to sustained expression of chemokines and stat -dependent type i interferon-response genes astrocytederived cxcl drives accumulation of antibody-secreting cells in the central nervous system during viral encephalomyelitis induction of ifn-alphabeta enables listeria monocytogenes to suppress macrophage activation by ifn-gamma agonist and antagonist effects of interferon alpha and beta on activation of human macrophages. two classes of interferon gamma receptors and blockade of the high-affinity sites by interferon alpha or beta contrasting effect of alpha/beta-and gamma-interferons on expression of macrophage ia antigens an rna-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex constitutive type i interferon modulates homeostatic balance through tonic signaling measure and countermeasure: type i ifn (ifn-alpha/beta) antiviral response against west nile 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defense following viral infection of the central nervous system the t cell chemoattractant ifn-inducible protein is essential in host defense against viral-induced neurologic disease high antigen levels are the cause of t cell exhaustion during chronic viral infection perforin-mediated effector function within the central nervous system requires ifn-gamma-mediated mhc up-regulation shp- tyrosine phosphatase functions as a negative regulator of the interferon-stimulated jak/stat pathway type i interferons directly regulate lymphocyte recirculation and cause transient blood lymphopenia type i interferon is selectively required by dendritic cells for immune rejection of tumors mouse hepatitis virus liver pathology is dependent on adp-ribose- Љ-phosphatase, a viral function conserved in the alpha-like supergroup pathogenicity of antigenic variants of murine coronavirus jhm selected with monoclonal antibodies distinct cd t-cell effects on primary versus recall cd t-cell responses during viral encephalomyelitis we thank michael diamond (washington university school of medicine, st. louis, mo) for kindly providing ifnar fl/fl mice on the c bl/ background. we also sincerely thank jennifer powers for exceptional facs purification and the cleveland clinic lerner research institute imaging core and john peterson for assistance with confocal microscopy. key: cord- -j q pcfa authors: zhan, xiu-xiu; liu, chuang; zhou, ge; zhang, zi-ke; sun, gui-quan; zhu, jonathan j.h.; jin, zhen title: coupling dynamics of epidemic spreading and information diffusion on complex networks date: - - journal: appl math comput doi: . /j.amc. . . sha: doc_id: cord_uid: j q pcfa the interaction between disease and disease information on complex networks has facilitated an interdisciplinary research area. when a disease begins to spread in the population, the corresponding information would also be transmitted among individuals, which in turn influence the spreading pattern of the disease. in this paper, firstly, we analyze the propagation of two representative diseases (h n and dengue fever) in the real-world population and their corresponding information on internet, suggesting the high correlation of the two-type dynamical processes. secondly, inspired by empirical analyses, we propose a nonlinear model to further interpret the coupling effect based on the sis (susceptible-infected-susceptible) model. both simulation results and theoretical analysis show that a high prevalence of epidemic will lead to a slow information decay, consequently resulting in a high infected level, which shall in turn prevent the epidemic spreading. finally, further theoretical analysis demonstrates that a multi-outbreak phenomenon emerges via the effect of coupling dynamics, which finds good agreement with empirical results. this work may shed light on the in-depth understanding of the interplay between the dynamics of epidemic spreading and information diffusion. the interaction between disease and disease information on complex networks has facilitated an interdisciplinary research area. when a disease begins to spread in the population, the corresponding information would also be transmitted among individuals, which in turn influence the spreading pattern of the disease. in this paper, firstly, we analyze the propagation of two representative diseases ( h n and dengue fever ) in the real-world population and their corresponding information on internet, suggesting the high correlation of the two-type dynamical processes. secondly, inspired by empirical analyses, we propose a nonlinear model to further interpret the coupling effect based on the sis (susceptible-infected-susceptible) model. both simulation results and theoretical analysis show that a high prevalence of epidemic will lead to a slow information decay, consequently resulting in a high infected level, which shall in turn prevent the epidemic spreading. finally, further theoretical analysis demonstrates that a multi-outbreak phenomenon emerges via the effect of coupling dynamics, which finds good agreement with empirical results. this work may shed light on the in-depth understanding of the interplay between the dynamics of epidemic spreading and information diffusion. recently, understanding how diseases spread among individuals has been an increasing hot research area of nonlinear studies [ ] . generally, epidemic spreading is considered to be a dynamic process in which the disease is transmitted from one individual to another via physical contact in peer-to-peer networks. to date, there is a vast amount of research tries to understand the epidemic spreading phenomenon, which could be mainly categorized into three types: (i) epidemic spreading on various types of networks [ ] , such as the scale-free network [ , ] , the small-world network [ , ] and the interdependent network [ , ] ; (ii) propagation mechanisms that describe the dynamic spreading process, such as the table illustration of parameters used in the spreading processes. susceptible-infected-recovered (sir) model for influenza [ , ] , the susceptible-infected-susceptible (sis) model for sexually transmitted disease [ , ] and the susceptible-exposed-infected-recovered (seir) model for rabies [ , ] ; (iii) data-driven modeling approaches that tackle the epidemic transmission [ ] by analyzing the available real datasets, such as the scaling laws in human mobility [ , ] , individual interactions [ , ] , and contact patterns [ , ] . the majority of the aforementioned studies focused on epidemic spreading independently, ignoring the fact that information diffusion of the diseases themselves may also have significant impact on epidemic outbreaks [ ] . for example, the outbreak of a contagious disease may lead to quick spreading of disease information, through either medias or friends. conversely, the information shall also drive people to take corresponding protective measures, such as staying at home, wearing face masks, and getting vaccinated [ ] . such behavioral responses may further impact epidemic outbreak in large population [ ] . therefore, studies on the coupling effect between epidemic spreading and information diffusion have attracted much attention from various disciplines. theoretical models have been proposed to explain how both disease and information simultaneously spread in the same population [ ] [ ] [ ] [ ] [ ] . in particular, the nonlinear influence of coupling parameters on the basic reproductive number ( r ) is studied to show the interplay between the two spreading processes [ ] . theoretical results indicate that the coupling interaction could decrease epidemic outbreak size in a well-mixed population [ ] . in some cases, enough behavioral changes would emerge in response to the diffusion of a great deal of disease information so that the severe epidemic would vanish completely, even the epidemic transmission rate was higher than the classical threshold initially [ ] [ ] [ ] [ ] [ ] . in addition, the interplay between information diffusion and epidemic spreading is elucidated on multiplex networks, where each type of dynamics diffuses on respective layers (e.g., information diffusion on communication layer versus epidemic spreading on physical layer) [ ] [ ] [ ] . as a consequence, the epidemic threshold, as related to the physical contact layer, can be increased by enhancing the diffusion rate of information on the communication layer. therefore, the effect of behavioral changes arises in three aspects [ ] : (i) disease state of the individuals, e.g., vaccination [ ] [ ] [ ] [ ] [ ] ; (ii) epidemic transmission and recovery rate [ , ] ; (iii) topological structure of contact network, e.g., the adaptive process [ ] [ ] [ ] [ ] . besides researches from physical discipline, scholars from mass communication share similar views on the causal linkages of the two diffusion processes. the outbreak of severe diseases usually attracts heavy media coverage, subsequently resulting in massive responses from the public: (i) cognitive responses, such as the attention to the information and increased awareness of the situation [ ] ; (ii) affective responses, such as anxiety, fear, or even panic [ ] ; (iii) behavioral responses, such as the adoption of new practices in order to replace undesirable habits [ ] . however, those assumptions are just theoretical hypotheses rather than empirical facts as it is difficult to find relevant data of one-to-one relationship in the spreading process. even when the data is available, it is also difficult to separate the unique effect of information on the control of epidemics from interference factors, such as variation of virus, seasonal factors and improved medical treatments, etc. present studies on the coupling dynamics mainly focus on the suppression effect of epidemic spreading by information diffusion. the occurrence of a disease prompts the sharing of corresponding information, leading to preventive measures that inhibit further epidemic spreading [ , ] . researchers have also pointed out that when the epidemic outbreak is under control, people shall not be very vigilant in discussing or sharing relevant information. it will lead to a consequent decrease in protection actions and may result in a recurrence of epidemics in future. for example, the spread of sars (severe acute respiratory syndromes) is alleviated in early march , however, a sudden increase appear later that month (as indicated in the evolution curve of the probable cases of sars, see fig. in ref. [ ] ). in this work, firstly, we demonstrate a similar outbreak pattern using data on the spread of two representative diseases, i.e., avian influenza a ( h n ) [ ] [ ] [ ] and dengue fever [ , ] , along with the diffusion of respective disease information. secondly, a nonlinear mathematical model is proposed to describe the coupled spreading dynamics as an sis spreading model. results show that information diffusion can significantly inhibit epidemic spreading. finally, both empirical analysis and the proposed model find good agreements in revealing a multi-outbreak phenomenon in the coupled spreading dynamics. to better illustrate this work, we collected data of two representative diseases, h n and dengue fever. each disease has two time series datasets: (i) daily number of individuals infected by the corresponding disease in china, which are collected from the chinese center for disease control and prevention ; (ii) online diffusion messages discussing or forwarding the information of the corresponding disease during the same period of epidemic spreading. the message diffusion data was crawled from the largest micro-blogging system in china [ ] , sina weibo ( http://www.weibo.com/ ). we have essentially obtained one-year data for the disease h n from the year to , and two-year data for dengue from the year to . we assume that individuals who post or retweet messages about the observed diseases are considered to be aware of the disease. empirical analysis of h n : fig. (a) shows the spreading processes of both disease and disease information of h n . it can be seen that the evolutionary trend of two processes are highly correlated, with pearson correlation coefficient of . . when the epidemic broke out in apr. and feb. ( fig. (a) ), it shows that many people were discussing it online simultaneously. actually, public responses to h n , such as staying at home or wearing face masks, can also affect the spread of the epidemic. the peaks of the disease spreading and the information diffusion shown in fig. (a) suggest that the mutual influence of these two spreading processes could be significant. interestingly, the size of the first epidemic peak (apr. ) is smaller than the second one (feb. ), which is inversely correlated with the information amount. that is to say, the number of individuals discussing the disease during the first outbreak is much greater than that of the second one. this might imply that the awareness of epidemics and the physical epidemics could influence each other. empirical analysis of dengue fever: fig. (b) describes the spreading processes of both disease and disease information of dengue. similar to the analysis of h n , the evolution trend of the two processes is also consistent with each other, with even much higher correlation coefficient of . . according to the two largest peaks (in sept. and sept. , respectively) of disease spreading, we find that the first epidemic peak is also smaller than the second one, while the corresponding information peaks show a contrary trend. considering the two small peaks of information in fig. (b ) and (b ), we can also find the same relationship between the the two dynamic processes as that of two largest peaks, suggesting also the possible coupling effect of the awareness of epidemics and the infected cases of dengue. in the aforementioned section, we empirically showed that the spread of disease and disease information has a coupling effect with each other by analyzing the data from two contagious diseases. inspired by the empirical results, we propose a network based nonlinear model to describe the interaction between epidemic spreading and information diffusion in this section. in this model, we assume there are two states for disease spreading: susceptible ( s ) and infected ( i ), and two states of information diffusion: aware (+) and unaware (-). as a consequence, each individual will be at one of the four states during the model evolution: • initially, one arbitrary individual is randomly picked from the given network as the spreading seed ( i + state). the rest individuals are set to be s − state. • at each time step, the infected individuals ( i + and i − states) will spread epidemics to their susceptible network neighbors ( s + and s − states) with given spreading probability. the infected individuals ( i + and i − states) could recover to the susceptible state with given recovery probability. • at each time step, individuals that are aware of the disease ( i + and s + states) will transmit the information to their unaware neighbors ( i − and s − states) with probability α. in addition, the informed individuals ( i + and s + ) could become unaware of the disease with the probabilities of λ and δλ, respectively. beyond the parameters given in table , we define σ as the probability of individuals taking protective measures. thus, σ s < is defined as that a susceptible aware individual ( s + ) will take protective measures to avoid becoming infected, and σ i < is defined as infected aware individuals ( i + state) will reduce contact with their susceptible neighbors or adopt medical treatments. in addition, we assume the infected probabilities for these two different populations are independent with each other, hence σ si = σ s σ i is defined as the probability of the i + state individuals infecting the s + state ones. when an i + individual is aware of the epidemic, s/he will take positive measures, leading to an increased recovery rate, which is represented by the factor ε > . furthermore, i + state individuals, which could be assumed to better understand the seriousness of epidemics, would be less likely to neglect relevant information, leading to δ < . in this work, since the spreading processes of information and disease are primarily determined by the corresponding transmission probabilities, we fix other parameters and mainly investigate the effects of α and β. in the following analysis, we set σ s = . , σ i = . , δ = . , ε = . , λ = . and γ = . . table . subsequently, the proposed model is performed on an er network with a total population n = , and average degree k = . to measure the spreading effects, we denote the infected level ( i ) as the fraction of infected individuals (both i + and i − ), and the informed level ( info ) as the fraction of individuals who are aware of the disease (both s + and i + ). fig. shows the simulation results by fixing the infection probability β = . . in this model, the parameter α can be considered as the information diffusion capability, hence larger α indicates that information diffuses much easier, resulting in a monotonically increase in the number of informed individuals (see the inset of fig. ). in fig. , it also shows that the increase in α will inversely hamper the speed of epidemic spreading, hence diminish the overall epidemic outbreak size. as a consequence, appropriate publicity might be an effective strategy to inhibit further spreading of epidemics, which is also consistent with the empirical analysis shown in fig. . in fig. , the model also indicates that there is mutual influence between information diffusion and epidemic spreading. a high prevalence of epidemic would lead to a small information fading probability δ, consequently resulting in a high infected level i . it in turn inhibits the epidemic spreading ( σ { i , s , si } < ). this coupling effect can be clearly described by the full set of differential equations (see appendix ). in addition, the equations are solved by mean-field and pairwise approaches, respectively. fig. shows the results of simulation, theoretical analysis of both mean-field and pairwise analysis. we find that the pairwise approach can better fit the model than the mean-field method. therefore, we use pairwise approach to perform further studies in the following analysis. in order to investigate the effect of the mutual interaction between α and β on the spreading process, we explore the phase diagram showing the fraction of infected individuals caused by combination of such coupling effects (see fig. ). the . that is to say, epidemic outbreak will occur if the parameter combination is larger than the critical value, otherwise the epidemic will die out. the results also clearly show that more individuals will be infected with large β and small α, suggesting that the information diffusion can impede the disease spreading. it is noted that the process degenerates to the standard sis model if α = , where there is no information diffusion in the system. thus, the epidemic outbreak threshold is β c = γ k = . [ ] , which is also consistent with the results of pairwise analysis and simulation shown in fig. . in addition, fig. (c) shows a detailed view of pairwise analysis for α, β ∈ [ , . ] in order to better observe the threshold changes. the threshold value of β is around . when α → , as the epidemic information cannot spread out in this case according to the inset of fig. . when α > , the epidemic threshold can be significantly increased because of the effect of information diffusion. on the contrary, fig. shows that the informed level only slightly ascends when α is large enough (e.g., α > . ), which leads to an obscure change in the epidemic threshold. this result additionally indicates that abundant information would not always work for obstructing epidemic spreading. for example, in the case that a disease with a strong infectiveness (corresponds to large β in fig. ), enhancing the public awareness alone is insufficient to control the large outbreak of epidemics. in order to obtain better understanding of dynamics of the critical phenomenon, we observe the evolution of infection density for various values of β in fig. . from the differential equation, di dt we can obtain i ∝ t − at the critical point, which shows a power-law decay. in addition, the inset of fig. also presents a power-law decay of the infection density when β ≈ . . by contrast, the infection turns to break out as an endemic, namely steady state, for β > . ( β = . in fig. ), otherwise the epidemic will be eliminated, so-called healthy state for β < . ( β = . in fig. ). therefore, it can be inferred that β c is approximately . in this case, which is consistent with the results in fig. , where β c is around . for α = . . interestingly, the empirical analysis also demonstrates that a multi-outbreak phenomenon emerges for both epidemic spreading [ , [ ] [ ] [ ] and information diffusion [ ] , in which there are several outbreaks during the dynamic process of epidemic spreading. generally, there are many complicated factors that might contribute to this phenomenon, including seasonal influence, climate change, and incubation period, etc. in this model, the periodic outbreaks can be interpreted by the influence of information diffusion. as discussed above, there is a mutual interaction as the two dynamics are coupled with each other during the process. on one hand, a larger proportion of infected individuals should result in an increase in preventive behavioral responses [ ] due to the increased awareness of the disease, consequently leading to a steady decrease of further infected cases. on the other hand, when the spread of epidemic tends to be under control, people shall become less sensitive to discuss or share relevant messages, which leads to dissemination of information and simultaneously raises the possibility of a second outbreak. notably, there are also some cases where the size of the second outbreak is smaller than the first one. for example, the eight dengue outbreaks in thailand over years from to [ ] , and there are also some cases that the second outbreak is larger than the previous one, as in the case of sars in [ ] and dengue in taiwan in - [ ] . in order to better understand the underlying mechanism that drives the multi-outbreak phenomenon of the coupled dynamics, we set two thresholds, i high and i low , to represent different infected levels. that is to say, when the fraction of infected individuals is larger than i high , the information diffusion parameter α will be set as high as α = . so that the information will diffuse even more quickly. accordingly, when it is smaller than i low , the parameter will directly decay to α = . to represent the corresponding response to abatement effect of information. fig. shows the simulation results. it can be seen that the epidemic spreads very quickly at the beginning as there are very few people aware of it, and soon reaches the threshold i high and triggers the designed high information transmission probability α = . . as a consequence, as the information bursts out, the high informed level has a significant impact on inhibiting epidemic spreading (the decay period of the epidemic), which will be completely suppressed if the high informed level remains. however, when the epidemic spreading is notably controlled from the first outbreak (i.e. the infected density is smaller than i low ), people are less likely to consider the epidemic as a threat, hence ignore relevant information and no longer actively engage in taking protective measures, which will in turn lead to a subsequent epidemic outbreak in the future. two representative outbreak patterns are shown in fig. , where the first outbreak is smaller than the second one ( fig. (a) ) and vice versa ( fig. (b) ). moreover, fig. , where the size of the first epidemic outbreak is smaller than that of the second one, while the informed level shows to the contrary. it should be noted that, due to the difficulty in collecting data of patient-to-fans to precisely quantify the informed level in the empirical analysis, the number of messages that discuss the epidemic is alternatively used in fig. . different from the trend shown in fig. , a high informed level( info > . ) must be maintained during the period when the infected level decreases shown in fig. . based on the model analysis, it could be concluded that it is important to raise public awareness of epidemic occurrence, especially during when the epidemic seems to be under control, otherwise, there is a likelihood of subsequent outbreak in the foreseeable future. furthermore, we explore the evolution of the informed and infected density with different values of β in fig. . in fig. (a) , it shows that the infected density firstly achieves a small peak and then rapidly vanishes, resulting in a evolution pattern known as healthy , which means there is approximately no disease. in fig. (b) , an oscillatory pattern is revealed for . < beta ≤ . . similarly, for large β ∈ ( . , ], the infected density firstly achieves a large peak (almost close to one), then rapidly decrease to a low level (nearly zero) and gradually raised to a steady state, showing a unimodal pattern [ ] . in this paper, we have studied the coupling dynamics between epidemic spreading and relevant information diffusion. empirical analyses from representative diseases ( h n and dengue fever ) show that the two kinds of dynamics could significantly influence each other. in addition, we propose a nonlinear model to describe such coupling dynamics based on the sis (susceptible-infected-susceptible) process. both simulation results and theoretical analyses show the underlying coupling phenomenon. that is to say, a high prevalence of epidemic will lead to a slow information decay, consequently resulting in a high infected level, which shall in turn prevent the epidemic spreading. further theoretical analysis demonstrates that a multi-outbreak phenomenon emerges via the effect of coupling dynamics, which finds good agreement with empirical results. the findings of this work may have various applications of network dynamics. for example, as it has been proved that preventive behaviors introduced by disease information can significantly inhibit the epidemic spreading, and information diffusion can be utilized as a complementary measure to efficiently control epidemics. therefore, the government should make an effort to maintain the public awareness, especially during the harmonious periods when the epidemic seems to be under control. in addition, in this work, we only consider the general preventive behavioral response of crowd. however, the dynamics of an epidemic may be very different due to the behavioral responses of people, such as adaptive process [ ] , migration [ ] , vaccination [ ] , and immunity [ ] . this work just provides a starting point to understand the coupling effect between the two spreading processes, a more comprehensive and in-depth study of personalized preventive behavioral responses shall need further effort s to discover. mean-field analysis: according to fig. , we adopt mean-field analysis for the spread of epidemic and information in a homogeneous network as follows: where n is the number of individuals in the system, k is the average degree of the network and the other parameters are illustrated in table . pairwise analysis: pairwise models have recently been widely used to illustrate the dynamic process of epidemics on networks, as those models take into account of the edges of the networks [ ] [ ] [ ] . in this study, we consider a set of evolution equations which are comprised of four types of individuals and types of edges. using the well-known closure, (assuming the neighbors of each individual obey poisson distribution) [ ] , we can get a set of differential equations as follows: ( ) epidemic processes in complex networks 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mobility responses to the large-scale spreading of infectious diseases modeling human dynamics of face-to-face interaction networks small but slow world: how network topology and burstiness slow down spreading temporal networks how human location-specific contact patterns impact spatial transmission between populations? dynamics of information diffusion and its applications on complex networks epidemic spreading with information-driven vaccination world health organization. consensus document on the epidemiology of severe acute respiratory syndrome (sars) the spread of awareness and its impact on epidemic outbreaks endemic disease, awareness, and local behavioural response modelling the influence of human behaviour on the spread of infectious diseases: a review interacting epidemics on overlay networks epidemic dynamics on information-driven adaptive networks on the existence of a threshold for preventive behavioral responses to suppress epidemic spreading the impact of awareness on epidemic spreading in networks imitation dynamics predict vaccinating behaviour social contact networks and disease eradicability under voluntary vaccination imitation dynamics of vaccination behaviour on social networks dynamical interplay between awareness and epidemic spreading in multiplex networks competing spreading processes on multiplex networks: awareness and epidemics asymmetrically interacting spreading dynamics on complex layered networks vaccination and the theory of games effects of behavioral response and vaccination policy on epidemic spreading-an approach based on evolutionary-game dynamics vaccination and epidemics in networked populationsan introduction information cascades in complex networks statistical physics of vaccination the impact of information transmission on epidemic outbreaks epidemic dynamics on an adaptive network adaptive human behavior in epidemiological models information spreading on dynamic social networks roles of edge weights on epidemic spreading dynamics issue competition and attention distraction: a zero-sum theory of agenda-setting moral threats and dangerous desires: aids in the news media diffusion of innovations h n is a virus worth worrying about determination of original infection source of h n avian influenza by dynamical model global concerns regarding novel influenza a (h n ) virus infections controlling dengue with vaccines in thailand impact of human mobility on the emergence of dengue epidemics in pakistan how events determine spreading patterns: information transmission via internal and external influences on social networks mapping spread and risk of avian influenza a (h n ) in china transmission characteristics of different students during a school outbreak of (h n ) pdm influenza in china the effect of antibody-dependent enhancement, cross immunity, and vector population on the dynamics of dengue fever spatial behavior of an epidemic model with migration epidemic enhancement in partially immune populations representing spatial interactions in simple ecological models the effects of local spatial structure on epidemiological invasions pair approximation of the stochastic susceptible-infected-recovered-susceptible epidemic model on the hypercubic lattice key: cord- -i ayr authors: perez, andres m.; linhares, daniel c. l.; arruda, andreia g.; vanderwaal, kimberly; machado, gustavo; vilalta, carles; sanhueza, juan m.; torrison, jerry; torremorell, montserrat; corzo, cesar a. title: individual or common good? voluntary data sharing to inform disease surveillance systems in food animals date: - - journal: front vet sci doi: . /fvets. . sha: doc_id: cord_uid: i ayr livestock producers have traditionally been reluctant to share information related to their business, including data on health status of their animals, which, sometimes, has impaired the ability to implement surveillance programs. however, during the last decade, swine producers in the united states (us) and other countries have voluntarily begun to share data for the control and elimination of specific infectious diseases, such as the porcine reproductive and respiratory syndrome virus (prrsv). those surveillance programs have played a pivotal role in bringing producers and veterinarians together for the benefit of the industry. examples of situations in which producers have decided to voluntarily share data for extended periods of time to support applied research and, ultimately, disease control in the absence of a regulatory framework have rarely been documented in the peer-reviewed literature. here, we provide evidence of a national program for voluntary sharing of disease status data that has helped the implementation of surveillance activities that, ultimately, allowed the generation of critically important scientific information to better support disease control activities. altogether, this effort has supported, and is supporting, the design and implementation of prevention and control approaches for the most economically devastating swine disease affecting the us. the program, which has been voluntarily sustained and supported over an extended period of time by the swine industry in the absence of any regulatory framework and that includes data on approximately % of the sow population in the us, represents a unique example of a livestock industry self-organized surveillance program to generate scientific-driven solutions for emerging swine health issues in north america. although porcine reproductive and respiratory syndrome (prrs) is one of the most important economic constraints to pork production in the us ( , ) , reporting of prrs outbreaks is not mandatory in the country. in the absence of a regulatory framework, prrs control and elimination actions are voluntary. at the field level, producers and veterinarians make decisions that seek to maximize profit while keeping the necessary standards for animal health and welfare. however, individual-level decisions may lead to complex and diverse epidemiological scenarios at a regional level. because of the epidemiological features of prrs virus (prrsv) transmission, such as high levels of disease incidence, high variability and rapid mutation of the virus, intensiveness of production, and, in some cases, vertical integration of the industry, and limitations of current preventive and control methods, there is not much hope for disease control if programs are not simultaneously implemented at local (i.e., production system level) and regional levels (i.e., state or county levels) ( ) ( ) ( ) ( ) . the perception that regional approaches are required to control the disease has led to the implementation of at least voluntary regional prrsv control or elimination projects in the us and canada ( ) . none have succeeded in eliminating prrsv regionally, and, arguably, most seem to have had limited success on significantly controlling the disease. part of the limited progress on those regional projects may be attributed to inconsistencies in regional biosecurity compliance and suboptimal biosecurity of swine operations, incomplete regional producer participation, poor standardization, and availability of information on pathogen monitoring and surveillance systems, including lags in detection and communication about outbreaks ( ) . lack of progress of regional control and eradication projects may also be at least in part associated with limited funding for disease and insufficient coordination of control activities. in contrast, one may argue that it is unknown what the epidemiological situation of the disease would be in the absence of those voluntary programs, and thus, the effectiveness of their implementation is debatable and subject to speculation. nevertheless, veterinarians and producers from individual farms and production companies, generically referred to as "production systems, " still need to make decisions intended to maximize their results ( ) using the information available to them, which may lead to scenarios that are not compatible with disease control at a regional level. the tension between "individual" and "common" good leads to a complex relation of behaviors and attitudes, resembling a "game." in this "game, " certain "players" (production systems) make decisions that may be conditional to the decisions made by other "players, " which, in turn, may result in a change of decisions taken by other "players." this "game, " which in social economics corresponds to a concept referred to as "game theory" ( , ), soon becomes dynamic, and the conditions required to control the disease at a regional level become, at minimum, difficult to reach. thus, prrsv control at the regional level becomes challenging, and often depends on regional leadership. if short-and long-term values of participation are not quantified and clear, it is unlikely that progress will be made. many stakeholders have perceived the social nature of prrsv control at the regional level in the us. spontaneous initiatives intended to voluntarily share knowledge have emerged in the country with the objective of promoting a greater good, i.e., control of a disease that affects the industry as a whole, even if such sharing may represent a potential risk or loss to their individual interest. here, we review the largest voluntary initiative for data sharing among us swine producers, including a summary of its design and governance, and highlighting a number of epidemiological features of the disease that the project helped to elucidate over the last years. the ultimate objective of this voluntary program is to build the capacity to respond in the event of an emerging disease, while supporting the prevention and control of endemic diseases of swine, such as prrsv. since the late 's, prrsv has been consistently generating losses in the us swine industry. thanks to an effective collaboration between researchers, swine veterinarians, and swine producers, epidemiological characteristics of the disease were uncovered and preventive and control measures were implemented. despite important improvements from a bioexclusion, biomanagement, and biocontainment standpoint, the virus continues to persist in us swine herds. because there has historically been no documentation of disease occurrence metrics through space and time, the industry could not systematically assess whether the current situation was better or worse compared to previous years, generating uncertainty and speculation. based on this knowledge gap and the need to further understand the epidemiology of the disease at larger spatial and temporal scales, a group of producers and practitioners decided to voluntarily share breeding herd prrsv status for their respective production systems. through weekly reporting, practitioners updated their respective prrsv breeding herds' status, making the estimation of weekly cumulative incidence reports possible. the product of this effort was then shared back to participants in the form of weekly reports. reports included information on disease prevalence, disease incidence, and proportion of herds in each prrsv status. reports also included benchmarking comparing numbers of participants to aggregated results from other participants in the database. the program's name changed through time and it is currently referred to as the morrison's swine health monitoring project (mshmp), in recognition of the late dr. robert morrison, who was the driving force leading the inception and organization of the program. compared to regional control projects, the mshmp is larger, including information from reproduction farms (sow farms, multipliers, genetic nuclei) in a number of regions, whereas regional control projects are typically smaller, limited to a geographical area, and including also information nurseries, growers, and finishers. mshmp participants have agreed that prrsv incidence graphs generated by their voluntary collaboration would be shared with the industry for benchmarking, disease monitoring, and promoting participation. in , the project included production systems that provided data related to prrsv breeding herd status, location, and prrsv interventions on a regular basis. those systems represented approximately one million sows, which at the time, accounted for roughly % of the total us breeding herd based on usda estimates ( ) . in , porcine epidemic diarrhea virus (pedv) and porcine delta-coronavirus (pdcov) emerged in the us swine population ( , ) . this dramatic and dynamic situation provided an opportunity for producers to continue working together, and therefore pedv and pdcov were added to the list of diseases being monitored and reported on a voluntary basis ( ) . over the years, others were prompted to join the project, which ultimately increased the representativeness of the database and thus provided a more accurate benchmark for the industry. senecavirus a and pathogens associated with central nervous system disease were later added to the list of pathogens reported to mshmp. at the time when this manuscript was written in february , production systems, accounting for ∼ % of the us sow population, continue to provide their data for the benefit of the industry (figure ). briefly, after each participant has voluntarily agreed to participate, a participation form and a data privacy agreement are signed prior to data acquisition. then, farm-level information, such as location, herd size, farm type (e.g., farrow-to-wean, farrow-to-feeder, farrow-to-finish), genetic level (e.g., multiplier or commercial herd) and air filtration status is provided by each participant and stored in a central database housed at the university of minnesota. one person within each participating system serves as point of contact for the project, becoming responsible for data sharing in direct communication with the mshmp's data coordinator. each week, participants share the list of sow farms that have changed their status, either worsening (e.g., outbreaks) or improving (e.g., ceased shedding or completed elimination). once the information is received by the mshmp data coordinator, it is reviewed for quality control and entered into the main database. data are then used to estimate measures of disease occurrence such as incidence and prevalence. for incidence, a graph featuring the weekly and yearly cumulative incidence is presented. incidence data are also used to create an exponentially weighted moving average graph, in which the magnitude and duration of the outbreak is graphed through time. additionally, that figure depicts a threshold level (upper confidence limit of the two lowest seasons in the previous years) that marks the start and end of epidemic periods. the prevalence graph for prrsv is based on a classification from the american association of swine veterinarians (aasv), in which each prrsv sow herd status is defined ( ) . the mshmp report is comprised of pages including participant logos and supporting/funding sources (page ), the aggregate incidence and prevalence graphs for prrsv (page ) and pedv (page ), the seneca valley virus and atypical central nervous system case counts (page ), a space for sharing the latest developments in swine related research referred to as "science page" (page ), and the names and affiliations of all the individuals that receive the public report (page ). additional pages are shared with the project participants referring only to their own systems, and including incidence and prevalence graphs for both prrsv and pedv. since its inception in , the mshmp has played a critical role in providing data that scientists translated into science-driven solutions to help the us swine industry mitigating prrsv impact. here, we summarize some important swine disease features that the mshmp has helped to elucidate and that promoted engagement and participation among producers. for the last years ( - ), prrsv has maintained stable incidence levels on an annual basis, with an increase in the number of outbreaks, colloquially referred to as "prrs season, " consistently starting between mid-october to mid-november ( ) . the only period that showed a substantial different incidence, compared to other periods, was in - , when pedv was first detected in the us ( ) . however, seasonal dynamics seemed to differ across different states, with minnesota, north carolina, and nebraska having more consistent seasonality than iowa, and illinois. furthermore, there seems to be a secular pattern in the southern and southeastern regions of the country, with large epidemics occurring every - years ( , ) . prrsv impact may be mitigated by implementing certain management strategies mshmp investigators developed herd-level metrics of success of prrsv control and elimination programs, such as timeto-stability (tts), time to baseline productivity (ttbp), and total loss per thousand sows. those metrics were used to compare the effect of multiple aspects or interventions on breeding herds affected with prrsv. prrsv-infected breeding herds achieved stability (i.e., producing prrsv-negative piglets) significantly sooner, compared to other strategies, when they had reported a prior infection (i.e., existing partial herd immunity) and implemented herd closure (i.e., temporary interruption of replacement breeding stock introduction), and/or used wild type live virus inoculation as part of load-closeexpose programs. specifically, tts was weeks sooner for breeding herds adopting live-virus inoculation (lvi) as part of a load-close-expose program compared to those that used mlv vaccination protocols. conversely, herds using mlv achieved ttbp weeks sooner, and lost , piglets/ , sows less than herds using lvi. altogether, economic analysis revealed an advantage for herds using mlv compared to lvi ( , ) . more recently, mshmp data have been used to estimate the breeding herd prrs time-to-stability of a subset of breeding herds from six production systems that used similar testing criteria. a total of breeding herds in the midwestern us participated in this study, which accounted for ∼ , sows. these herds reported prrs outbreaks between and . breeding herds that had prrsv outbreaks during the spring and summer had a significantly longer tts than herds that had outbreaks during the fall and winter. in addition, there was a significant difference between tts between production companies suggesting that there are particular factors within production systems that may drive viral persistence in breeding herds ( ) . the mshmp has been used to provide incidence data that helped to estimate the cost of prrs, as part of various projects aimed at monitoring prrs impact over time. those data have helped in part to demonstrate that the annual cost of prrs to the us swine industry has decreased $ . million from october ($ . million) to october ($ . million) ( ), partially possibly due to increased number of breeding herds constantly immunizing sows with attenuated prrs virus vaccines (figure ) , which has been associated with reduced production losses ( ) . some have suggested that, coincidently with the dissemination of research results demonstrating the impact of control measures on tts, the proportion of sow farms in the mshmp that have implemented vaccine-based control strategies substantially increased, suggesting that producers may have seen some value in the adoption and implementation of those research findings in the field (figure ) . noteworthy, however, it is also possible that the shift may be explained, at least in part, by other factors that were not formally assessed such as, for example, ped emergence in the country. in any case, data provided by participants has allowed mshmp to compile and visualize the rate at which different control strategies have been adopted by participants. swine farm density has long been recognized as an important indicator for the risk of farms becoming infected with prrsv and pedv, even though the relative contribution of different routes of infection remain debated among swine practitioners and producers. through the use of historical mshmp data along with publicly available datasets, insights on environment-related factors that could affect the risk of prrsv outbreaks were assessed. swine sites located in regions with higher land slopes, and swine sites surrounded by trees and herbaceous coverage were protected from reporting a prrsv outbreak compared to sites located in regions with lower land slopes and regions characterized by cultivated areas, respectively ( ) . however, the effects of slope may not be consistent across all regions, and may depend on the specific topography of the area ( ) . precipitation, temperature, and land cover were all contributors for prrsv spatial risk, with specific contribution amounts varying according to "subregion" in the us ( ) . for pedv, temperature, wind speed, and vegetation have been identified as important modulators of risk, even in swine-dense areas ( ) . taken together, these results demonstrate that, even for highly intensive pig production, environmental factors play an for the past several decades, epidemiological modeling has been an important tool for understanding and predicting the spread of infectious diseases. the availability of farm-level outbreak data through mshmp, combined with data on animal movements between farms ( ) , has enabled some of the first data-informed epidemiological models in the us swine industry. the availability of prrsv genetic sequences from farms that became infected [see for example, ( ) ] allowed for the model to be fit to the observed spatiotemporal dynamics of the unfolding ped epidemic in ( ) . data on which and when farms become infected also has opened the possibility of using this rich database to develop predictive models that could be used to forecast when a farm is expected to be at high risk. the potential for forecasting was initially explored using ped incidence data ( ) . animal movement data available within the mshmp was combined with environmental risk factors within a km radius around breeding farms to identify major drivers of ped outbreaks, which included the total numbers of pig movements into neighboring farms, regional hog density, environmental, and weather factors such as vegetation, wind speed, temperature, and precipitation, and topographical features such as slope. results suggest that ped occurrence may be predictable with an acceptable (i.e., > %) level of accuracy, which eventually may lead to the design and implementation of a near real-time forecasting system for these diseases. the us swine industry continues to reach important production levels together with expanding their breeding and growing herds. infectious diseases are one of the most important limiting factors as they deteriorate performance and increases production costs. furthermore, foreign animal diseases are of concern for the industry as they could potentially affect the industry's export market. therefore, the us swine industry continues to work closely with researchers to seek answers and implement procedures to mitigate the burden of endemic diseases, while building capacity to respond against emerging and foreign animal diseases. a program like mshmp, which its foundation is trust and voluntary participation, has had an important impact on the surveillance of pathogens for the national industry. an academic institution-led program has motivated producer and veterinarian collaboration for the greater good of the industry. reasons why producers and veterinarians have been willing to share their data are difficult t to assess. initially, there was an intention to standardize data collection and sharing, to improve situational awareness and facilitate the decision-making process considering the epidemiological situation of the disease in near real time and in the entire region, rather than on their own farms and systems only. however, motivation declines with time, and it is important to demonstrate the added value of the effort. in that regards, even though shared data are private and managed only by the mshmp team, producers and veterinarians still obtain a benefit because important outputs, that help inform their decisions, are routinely shared with them. prrsv has been used as a way to bring the industry together and build the methodology to create a program that has the potential to be adaptable to other diseases. mshmp has demonstrated over the years that voluntary organization of swine producers and practitioners toward a common goal resulted in a powerful initiative that outweighed initial concerns about own data protection. most importantly, such voluntary collaboration has also led to collaborative research involving a number of higher education institutions in the us that helped to elucidate some of the most important epidemiological features of endemic swine diseases in the us, and ultimately, provided a substantial support to the mitigation of disease impact in the country. the evolution of analytical capabilities for big data analysis is expected to result in novel and innovative tools that will allow for the routine analysis of big datasets, such as that collected in the mshmp. the promotion of social initiatives, intended to promote data sharing and self-organization of producers, along with the application of those novel analytical techniques, may help to reshape in the near future the landscape of coordinated activities to promote the prevention and control of food animal diseases worldwide. assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states assessment of the economic impact of porcine reproductive and respiratory syndrome virus on united states pork producers results of a control programme for the porcine reproductive and respiratory syndrome in the 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the u land altitude, slope, and coverage as risk factors for porcine reproductive and respiratory syndrome (prrs) outbreaks in the united states predicting outbreaks of porcine epidemic diarrhea virus through animal movements and spatial neighborhoods surveillance of porcine reproductive and respiratory syndrome virus in the united states using risk mapping and species distribution modeling characterization of swine movement in the united states and implications for disease control porcine reproductive and respiratory syndrome virus outbreak role of animal movement and indirect contact among farms in transmission of porcine epidemic diarrhea virus the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © perez, linhares, arruda, vanderwaal, machado, vilalta, sanhueza, torrison, torremorell and corzo. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -hbpfz rt authors: glingston, r. sahaya; deb, rachayeeta; kumar, sachin; nagotu, shirisha title: organelle dynamics and viral infections: at cross roads date: - - journal: microbes infect doi: . /j.micinf. . . sha: doc_id: cord_uid: hbpfz rt viruses are obligate intracellular parasites of the host cells. a commonly accepted view is the requirement of internal membranous structures for various aspects of viral life cycle. organelles enable favourable intracellular environment for several viruses. however, studies reporting organelle dynamics upon viral infections are scant. in this review, we aim to summarize and highlight modulations caused to various organelles upon viral infection or expression of its proteins. a unique feature of eukaryotic cells is the presence of distinct membrane bound structures called organelles. this subcompartmentalization of eukaryotic cells is very essential for its optimum function. organelles achieve this due to the presence of a unique set of proteins and distinctive lipid composition that determines their function. they are dynamic and interact with the surrounding organelles to regulate their biogenesis and function [ , ] . association of organelle dysfunction with human diseases/ disorders is reported and widely acknowledged [ ] . it is interesting that not only organelles are required for proper functioning of cells, but are also required for the successful infection of a virus [ , ] . it is well established that viruses develop alternate strategies to survive in the cells. the steps of the viral life cycle include entry, translation, replication, assembly and egress [ ] . moreover, viruses have developed remarkable ways to complete their life cycle by targeting specific cell organelles and processes. organelles like mitochondria, er, peroxisomes play an important role in innate immunity and host defence [ ] . recently lipid droplets have also been reported to be essential for innate response against viral infection [ ] . the viral life cycle is also a dynamic process that leads to the extensive host cellular reorganization. characterization of this spatiotemporal reorganization is a key step in order to understand the molecular mechanism of viral infection. localization of the viral proteins to an appropriate sub-cellular compartment is part of the strategy to hijack the host machinery and necessary pathways leading to establishment of an infection [ , ] . with the advent of several modern microscopy and proteomics methods, it is now clear that the localization and function of many host proteins are altered due to viral infections. not only the host proteins but the intracellular localization of the viral proteins and their interactions with the host proteins are also extensively studied using these methods [ ] . however, currently our understanding of organelle dynamics during viral infections is still at its infancy. current review briefly summarizes our knowledge of the various cell organelles/compartments following virus infection. this topic at the interjection of virology and cell biology represents an emerging research area of molecular investigations in virology. presence of nucleus that houses the genome is what distinguishes eukaryotic cells from prokaryotes (fig. ). it is a double membrane organelle and is the site for gene regulation and transcription. nuclear envelope comprising of nuclear pores is the barrier between the nuclear content and the rest of the cell. nucleolus is the region where ribosomal subunit assembly takes place in the nucleus. sub nuclear structures comprising of several proteins which regulate many cellular processes like apoptosis, dna damage, etc are called pml-nbs. many dna and rna viruses depend on the host nuclear proteins for their replication [ ] . several strategies are used by viruses in order to deliver its genome to the host cells [ ] . usually, when cells undergo mitosis, there is a temporary disassembly of the nuclear envelope (ne) which allows some viruses to enter into the nucleus [ ] . entry and integration of murine leukemia virus (mlv) into host nucleus depends on the ne breakdown during mitosis [ ] . various human immunodeficiency virus (hiv) preintegration complex proteins, such as the matrix [ ] , vpr [ ] , integrase [ , ] were found to contain nuclear localization signal (nls) in their genome sequence. this nls interacts with the nuclear transport receptors, which transports the viral proteins through the nuclear pore complex (npc). similarly, the influenza virus nucleoprotein (np) present in the viral ribonucleoprotein complex (vrnp) contains nls , nls and nls , which helps in its binding to cellular importins resulting in transportation of the vrnp to the nucleus [ e ] . in another strategy, the capsid of certain viruses gets attached to the cytoplasmic side of the host npc either directly or with the help of importins. this interaction acts as a signal for capsid disassembly followed by entry of the viral genome along with their proteins through the npc into the nucleus [ ] . studies on the herpes simplex virus- (hsv- ) infection on vero, bhk- and ptk cells reported transportation of viral tegument-capsid by dynein to the cytoplasmic side of npc [ , ] . the hsv- capsid binds to npc with the help of importin b and subsequently releases dna into the nucleus through the npc [ ] . similarly, the capsid protein of adenovirus (ad) on binding with the nuclear transport protein nup attaches to the cytoplasmic side of npc and releases the genetic material into the nucleus [ ] . some small viruses such as hepatitis b virus (hbv), baculovirus, etc were found to cross the npc and release their genome at the nuclear side of npc or within the nucleus [ ] . the capsid protein of hbv was reported to interact with npc via the nuclear receptor nup in xenopus laevis oocytes [ ] . furthermore, this binding depends on importin a, b and phosphorylated core protein present on its capsid [ ] . upon infection, hbv capsid enters the nucleus through the npc and subsequently releases its genome into the nucleoplasm [ ] . similarly, capsid proteins of simian virus (sv ) were found to enter the nucleus through npc [ ] . it has been reported that the disruption of the ne and nuclear lamina temporarily aid the nuclear entry of viruses such as parvoviruses [ ] . disruption of ne in xenopus oocytes and both ne and nuclear lamina in mouse fibroblast cells upon parvovirus infection and minute virus of mice (mvm) was reported using electron microscopy analysis [ , ] . presumably, viruses evolved different mechanisms to enter nucleus in order to facilitate their replication cycle and survivability inside the host cells. after the successful entry into the nucleus, both dna and rna viruses exploit various nuclear components such as nucleolus, promyelocytic leukaemia nuclear body (pln) and/or nuclear proteins in order to facilitate their replication (fig. ) [ ] . nucleolin, fibrillarin and b (nucleophosmin) are examples of nucleolar proteins reported essential for various functions like post transcriptional process, ribosome assembly, etc [ ] . these multifunctional host nucleolar proteins were reported to be incorporated into the replication and translation complex of many viruses [ ] . many dna viruses like hsv and ad were reported to be involved in relocalization or disruption of the host nucleolar proteins [ ] . for instance, the transfection of the recombinant ul protein of hsv- , tagged with an n-terminal hemagglutinin in vero cells resulted in the redistribution of nucleolin and b [ , ] . another nucleolar protein fibrillarin was also found to be redistributed in spots throughout the nucleus but in an ul independent manner [ ] . the core protein of ad was found to be associated with the nucleolus in hela cells [ ] . further studies showed that this association results in the relocation of the nucleolin to the cytoplasm of the infected cells [ ] . the ad-induced relocation of nucleolin was proposed to be a strategy used by virus to suppress its activity [ ] . additionally, infection of hela cells with ad resulted in inhibition of synthesis and maturation of rrna [ ] . nucleolar localization of the viral capsid and rna binding proteins of many rna viruses has been reported [ ] . for example, the encephalomyocarditis viral (emcv) proteins a and bcd and human rhinovirus hrv c protease were found to localize to the nucleoli resulting in inhibition of cellular rna transcription [ , ] . it has been reported that the expression of tat and rev proteins of hiv- in cos cells resulted in their accumulation in dense fibrillar component in the nucleolus [ ] . moreover, rev protein was reported to be involved in deforming the nucleolar architecture [ ] . similarly, the viral n protein was found to be localized to the nucleolus in addition to the cytoplasm upon infection of coronavirus resulting in delayed cell cycle to facilitate viral assembly [ ] . poliovirus (pv) or rhinoviruses interact with nucleolin and blocks the nuclear import leading to accumulation of nucleolin in the cytoplasm of the infected cells [ ] . the accumulated nucleolin interacts with the internal ribosome entry site (ires) element present in the upstream end of the viral genome to stimulate its translation [ ] . on the other hand, these changes result in the shutdown of cellular transcription leading to the downregulation of the host defence mechanism [ ] . infection of human respiratory syncytial virus (hrsv) in a cells resulted in depletion of nucleolin [ ] . although, both dna and rna viruses behave differently towards acquiring the nuclear niche however the goal being to establish control over cellular transcription and favour its genome over the host. pml nb is another subnucleolar component, which contains proteins such as pml and sp . these proteins are induced by interferons and hence pml nb can act as a target for viruses to escape the antiviral signalling response [ ] . redistribution of the pml nb has been reported upon infection of hsv- to bhk- cells [ ] . similarly, ebna lp protein of epstein-barr virus (ebv) was found to displace sp from pml nbs in burkitt's lymphoma and hep cells [ ] . the viral e -orf protein induced reorganization of pml nbs into elongated track like structures upon infection of cv cells with human ad [ ] . later, it was found that this reorganization was responsible for the downregulation of the host antiviral response [ ] . another example is the infection of human foreskin fibroblasts (hffs) with human cytomegalovirus (hcmv) resulting in the accumulation of pp at pml nbs in the infected cells [ ] . further studies showed that pp was responsible for the proteasomal degradation of pml-nb protein daxx, which is important for inducing intrinsic immune response against hcmv infection [ ] . some rna viruses also result in the redistribution and degradation of host pml nbs in order to neutralize the antiviral response. the ring finger protein z of lymphocytic choriomeningitis virus interacts with pml protein and leads to the redistribution of pml-nbs from the nucleolus to the cytoplasm [ ] . the expression of c protease of emcv in cho cells was reported to target pml-nbs and promote its degradation in proteasome and by sumo-dependent mechanism [ ] . interestingly, cho cells infected with rabies virus were reported to contain enlarged pml nbs [ ] . disruption of the nucleocytoplasmic trafficking in the host cells is another major alteration caused due to viral infections ( fig. ) [ ] . two conserved proteins pul and pul of hcmv were reported to remodel the nuclear lamina of helfs (primary human lung fibroblasts) for the budding of virions [ ] . interaction between human papillomavirus (hpv) and host mitotic chromosomes has been documented [ ] . infection of porcine bone marrow cells with african swine fever virus (afsv) resulted in fragmentation of the nucleus [ ] . the organelle found in continuation with the nucleus in a cell is the er (fig. ) . protein synthesis and folding (rough er), lipid synthesis, calcium regulation (smooth er), etc are some of the important functions of the er. multiple structural domains of the er are reported which enable it to serve as a site for various important functions in a cell. several morphological alterations of er such as vesicle formation, invagination of the membrane, zippered appearance, etc have been reported upon viral infection in different cell lines (fig. ) . the large malleable surface of the er aids viruses to form protective compartments to set up their replication machinery. these compartments known as viroplasm not only concentrate viral and host proteins required for viral genome replication but also protect the viral genome from cellular nucleases [ ] . in order to construct these compartments, viruses alter host's fatty acid metabolism, induce rearrangement of the membrane constituents and also recruit cellular machinery to produce proteins essential for its replication [ , ] . infection of vaccinia virus (vacv) in bs-c- , hela and rk- cells leads to the formation of vacv membranes derived from the er membrane [ ] . in case of dengue virus (denv- ), the genomic rna was observed to localize over the rough er in c / aedes albopictus cells [ ] . denv infection in human hepatoma (huh ) cells leads to invagination of er membrane resulting in the formation of spherules or vesicles containing double stranded rna [ ] . hela cells and cos- cells on infection with pv cause formation of single membrane tubules which later mature into double membrane vesicles (dmvs) [ , ] . similarly, severe acute respiratory syndrome corona virus (sars-cov) induced formation of er derived dmvs upon infection of vero cells [ , ] . zippered appearance of er was observed upon infection of infectious bronchitis virus (ibv) on mammalian, avian and tracheal epithelial cells [ ] . studies with the plant viruses like the potato virus x (pvx) reported tgbp protein induced reorganization of the er and appearance of vesicular structures in tobacco plants [ ] . effect of the viral protein expression on sterol biosynthesis apart from the alterations in er morphology have also been reported [ , ] . several viral proteins need to be glycosylated at their n-terminal to ensure proper folding and for the incorporation into virions [ ] . hence, a common phenomenon observed upon viral infection is interference with the host post translational machinery and competition with the cellular proteins to undergo these modifications [ ] . for example, protein porf of hepatitis e virus (hev) gets glycosylated in the er upon its expression in cos- cells and huh mammalian cells [ , ] . this increased biosynthetic load is proposed to increase in accumulation of malformed proteins resulting in er stress (fig. ) . er gets relieved from this stress by inducing a pathway called unfolded protein response (upr) pathway [ ] . upr serves to enhance the cell's degradation ability in order to establish er homeostasis [ ] . transport of misfolded, misassembled proteins from the er to the cytosol and clearance by the ubiquitin proteasome system takes place via the er associated degradation (erad) pathway [ ] . however, some viruses use erad pathway for their advantage by co-opting erad to disassemble and gain access to the cytosol of the host cell [ ] . upon sv infection of cv- , hela and t cells, induction of erad factors in turn induce er membrane reorganization into distinct subdomains called foci and the accumulation of sv in these foci was observed [ ] . later, sv particles travel across the er membrane to reach the cytosol [ ] . many enveloped and non-enveloped viruses that exploit er functions like upr, erad to facilitate their replication have been discussed elsewhere [ ] . in addition to the above listed mechanisms, er mediated nlinked glycosylation plays a very important role in the survival of the viruses. it has been very elegantly demonstrated that the biogenesis of influenza could modulate the host immune response [ ] . similarly, the role of n-glycans in the host immunity against hiv has been documented [ ] . these studies corroborate to a common point where the level of glycosylation in the viral surface glycoproteins could alter their antigenicity. mitochondria are double membrane bound organelles that comprise their own genome ( fig. ). they are involved in various functions like fatty acid metabolism, apoptosis, calcium homeostasis, etc. considered evolutionarily the oldest organelle of a eukaryotic cell, they are indispensable as power house of the cell. mitochondrial morphology is maintained by a series of interlinked events namely mitochondrial fusion, fission and mitophagy [ ] . mitochondrial fusion helps in exchanging matrix metabolites and intact mitochondrial dna while mitochondrial fission helps in sorting impaired mitochondria from the healthy population which are further eliminated by a process called mitophagy [ ] . all the above dynamic events are altered upon viral infection in order to facilitate its replication (fig. ) [ ] . for example, it was reported that hbv infection in the cell induces mitochondrial fission followed by mitophagy in order to attenuate apoptosis [ ] . on the other hand, expression of orf- b protein of sars-cov virus promoted mitochondrial fusion in hek cells [ ] . upregulation of mitophagy and degradation of the mitochondrial antiviral signalling protein (mavs) in order to attenuate the antiviral immune response in non-small cell lung cancer (nsclc) cells was reported upon measles virus infection [ ] . the expression of matrix protein (m) of human parainfluenza virus type (hpiv ) in hek t and hela cells was reported to induce mitophagy resulting in the suppression of type interferon response [ ] . hbv induces mitophagosome formation which upon fusion with lysosomes leads to mitophagy and prevents apoptosis, thus facilitating persistent infection in the huh cells [ ] . similarly, newcastle disease virus (ndv) was reported to induce mitophagy, which promotes ndv replication by preventing caspase dependent apoptosis in human non-small cell lung cancer a cells [ ] . viruses can alter the intracellular distribution of mitochondria either to prevent the release of mediators of apoptosis or to meet their energy requirement during replication by concentrating them near their viral factories [ ] . hbv x protein induces the perinuclear clustering of mitochondria in huh cells [ ] and afsv leads to the transport of mitochondria to viral assembly sites in vero cells [ ] . mitochondrial dna codes for proteins essential for respiratory functions of a cell. certain viruses evade the mitochondria associated antiviral response by damaging the host cell mitochondrial dna, which is essential for synthesizing enzymes for optimum mitochondrial function [ ] . for example, mitochondrial dna degradation is induced in mammalian cells upon expression of ul . , an amino-terminally truncated ul isoform of hsv- [ ] . raji cell lines infected with hepatitis c virus (hcv) also exhibit mitochondrial dna depletion [ ] . maintenance of mitochondrial/cellular ca þ homeostasis is vital for various cellular functions. many viruses are involved in altering the mitochondrial calcium homeostasis in order to meet their needs during replication [ ] . hcmv upon infection causes calcium influx into mitochondria from er [ ] . on the other hand, expression of b protein of coxsackievirus resulted in reduced signalling of ca þ between the er-golgi and the mitochondria in hela cells resulting in suppression of apoptosis [ ] . rotavirus was also reported to alter the calcium homeostasis in the host cell throughout its life cycle [ ] . mitochondria are the major source of ros in a cell and a balance between ros production and scavenging is crucial for optimum functioning of the cells. upon viral infection mitochondria undergo oxidative stress and result in an increased production of ros which in turn reduces virus replication [ ] . interestingly, ros is also involved in activating many host cellular pathways favourable for viral replication and pathogenesis. several viruses like hiv, hcv, adv, ebv, etc result in increased oxidative stress upon infection [ ] . on the other hand, both increase and decrease of oxidative stress is employed as a survival strategy by hbv [ , ] . many viral proteins reach mitochondria through the mitochondria-associated membrane [ ] a sub-compartment of the er or are targeted directly from the cytosol and result in an altered mitochondrial permeability transition pore (mptp) [ ] . mptp is also responsible for the maintenance of mmp and provides energy for atp synthesis. altering mptp leads to passive swelling, outer membrane rupture, osmotic water flux, and release of proapoptotic factors leading to cell death [ ] . in general, increased mmp induces apoptosis, while decreased mmp prevents apoptosis [ ] . viruses are proposed to decrease mmp to prevent cell death in order to promote their replication. however, in later stages, they may trigger an increase in mmp to release the progeny virions by apoptosis [ ] . the m l protein of myxoma pox virus prevents the loss of mmp in cos- monkey kidney cells, hela cells, thp- human monocytes and jurkat t lymphocytes [ ] . on the other side, the r protein of hiv- induces the loss of mmp in cem-c and jurkat cells and results in apoptosis [ ] . viruses alter the host mitochondrial metabolic pathways in order to maintain cellular energy homeostasis essential to ensure efficient replication and to avoid mitochondrial antiviral response particularly in case of slow replicating virus [ ] . some viruses modulate the normal cells to increase aerobic glycolysis and use glucose biosynthetically, which helps especially enveloped viruses to increase their available pool of fatty acids, lipids and nucleotides during their replication [ , ] the feline leukemia virus infection on lung fibroblasts (flf- ) resulted in a e % increase in glucose uptake and lactic acid production [ ] . an increase in the lactic acid production upon infection of the chick embryo cells with rous sarcoma virus was reported [ ] . hcmv upon infection of hffs, human retinal pigment epithelial cells (arpe ), human embryonic lung fibroblasts (mrc ) and vero cells enhances glycolytic flux and directs the supply of carbon from glucose to tca cycle. this helps to facilitate fatty acid biosynthesis while hsv- upon infection of same cell lines directs the central carbon metabolism in order to induce the production of pyrimidine nucleotide components [ ] . hcv core protein suppresses mitochondrial complex activity and impaired function of electron transport cycle leading to ros accumulation in hepatoma cells of transgenic mice [ ] . mitochondrial involvement in virus survival is quite relevant to the fact that viruses require a source of energy to favour the active processes involved in their life cycle. peroxisomes are single membrane bound dynamic organelles required for b-oxidation of fatty acids and ros metabolism (fig. ) . they have developed diverse functions which are organism and environment dependent. they are unique with respect to proliferation, as they can increase in number by both growth and division of pre-existing organelles and formation of new organelles from the er. many viruses or viral proteins are reported to localize to peroxisomes and/or exploit their functions to facilitate their replication in the host cells [ ] . presence of a peroxisome targeting signal (pts) in the protein sequence is reported to be essential for targeting proteins into the peroxisomes [ ] . however, it was also proposed that viral proteins without pts may get associated with host peroxisomal proteins in the cytosol which then ferry the viral protein into the peroxisomes in a piggyback fashion [ ] . for instance, hcmv encodes a protein called vmia reported to be localized to peroxisomes in hffs and hepg cells [ ] . the vmia interaction with the host pex protein aids the viral protein localization to the peroxisomes. fragmentation of peroxisomes upon vmia expression in hepg was also reported [ ] . in addition, peroxisomal localization of two cymbidium ring spot viral proteins p and p was reported in yeast [ ] . a role for peroxisomes in the replication of tbsv has also been reported. the viral replication protein p interacts with the host peroxisomal protein pex for its targeting to the peroxisomal membrane and subsequent replication [ , ] . the host hsp protein was reported to promote the localization of the viral replication proteins to the peroxisomes in yeast cells [ ] . n-terminal protease n pro of pestivirus is another example of a viral protein that is associated with peroxisomes and facilitates its survival and replication [ ] . some members of the family tombusviridae are involved in remodelling the peroxisomal membrane resulting in the formation of vesicular structures called "profoundly modified peroxisomes" or "peroxisome derived multivesicular bodies" [ ] . for example, the infection of cymbidium ring spot virus resulted in the formation of small vesicles at the periphery of the peroxisomes in plant leaf tissues [ ] . at the later stage of infection, the entire peroxisomal matrix is occupied by these vesicles [ ] . similarly, the cucumber necrosis virus (cnv) induces the formation of peroxisomal vesicles in which the viral rna replication occurs in yeast cells [ ] . studies revealed upregulation of peroxisomal biogenesis in endothelial cells upon latent infection of kaposi's sarcoma associated herpes virus [ ] . it was also reported that proteins involved in peroxisomal lipid metabolism were essential for the survival of latently infected human dermal microvascular endothelial cells and lymphatic endothelial cells [ ] . interestingly, hiv infection reduces the number of peroxisomes in infected cells due to upregulation in the levels of micrornas that inhibit production of peroxisome biogenesis factors [ ] . it has been reported that the west nile virus (wnv) and denv infection on a and hek t cells result in the degradation of peroxisomes [ ] . the capsid proteins of both denv and wnv were reported to interact with the peroxisomal protein pex required for peroxisome biogenesis. degradation and redistribution of pex from perinuclear region to juxtanuclear region was reported in the infected cells. in addition, reduced level of the antioxidant enzyme catalase was observed in the infected cells. these alterations resulted in an impairment of early antiviral signalling of the peroxisomes. expression of the pts containing vp protein of the rotavirus, in ma cell lines resulted in peroxisomal localization [ ] . the role for such a localization was speculated to utilize the peroxisomal lipid metabolism for the supply of cholesterol for lipid raft synthesis, required for the viral replication. peroxisomal b-oxidation metabolism leads to the production of myristoyl-coa by shortening the fatty acids chain which could be exported to the cytosol for nmyristoylation of the viral proteins vp and vp of rotavirus [ ] . studies on hiv suggested an interaction between viral nef protein and the peroxisomal enzyme thio-esterase using yeast two hybrid system [ ] . further studies reported an increase in the enzymatic activity of human acyl-coa thio-esterase on binding with the nef protein [ ] . the increased activity of the peroxisomal enzyme was speculated to contribute in alteration of the subcellular morphology and in downregulation of the host antiviral response [ ] . extensive modifications of proteins for proper functioning and targeting in a cell takes place at the golgi apparatus. other functions of golgi inevitable for the cell are carbohydrate synthesis and lipid transport. the unique stacked structural organization of the golgi is essential for these functions (fig. ) . the golgi apparatus is composed of three regions namely cis golgi network (cgn), medial and trans golgi network (tgn). various viruses and viral proteins have been identified to localize to these golgi regions during their life cycle. for example, hela cells upon infection with adeno-associated virus type (aav- ) led to an accumulation of the viral particles in the tgn and in the golginetwork associated coated vesicles [ ] . in sars-cov, the transmembrane domain of the orf b protein contains the retention signal required for accumulation of the protein in the cgn and tgn [ ] . reports suggest bunyaviruses assemble in the golgi as a result of its retention signal in the glycoprotein (gn) of the virus [ , ] . fragmentation of golgi body is a common phenomenon that occurs as a result of various viral infections (fig. ) . orf virus (a parapox virus) causes disruption and fragmentation of golgi in vero cells. this structural modification affects the late vesicular export machinery and results in downregulation of the host immune response [ ] . similar phenomenon was also reported upon expression of c protease of foot-and-mouth disease virus in vero cells [ ] . infection of hrv a on wi- , t, and h -hela cells was reported to induce fragmentation of golgi body, rearrangement of the golgi membranes into vesicles which are utilized as sites of rna replication [ ] . another incidence of the virus induced golgi body fragmentation was observed upon infection of sars-cov that resulted in death of vero cells [ ] . many rna viruses were also found to alter the integrity of golgi complex of the host cells. for example, the expression of n-terminal non-structural protein of norwalk virus in crandell-rees feline kidney (crfk) and hela cells co-localizes to golgi complex and induces its disassembly into discrete aggregates [ ] . another example is the pv infection on vero cells which results in complete disruption of the cgn into fragments scattered throughout the cytoplasm. the expression of pv protein b in vero, normal rat kidney (nrk) and cos- cells also resulted in golgi bodies disassembly [ ] . similarly, expression of protein a of avian encephalomyelitis virus (aev) in chick embryo brain (ceb) cells and cos- cells resulted in depletion of golgi stacks and in severe disassembly of golgi bodies [ ] . an interesting alteration in the morphology of golgi apparatus was observed upon infection of turnip mosaic virus (tumv) on n. benthamiana plant. tumv infection was observed to induce amalgamation of golgi apparatus, er and chloroplast [ ] . mccoy mouse fibroblast cells led to accumulation of golgin- a tgn resident protein in the viral factories [ ] . similarly, a protein of some picornaviruses were also found to interact with a golgi apparatus resident protein namely golgi adaptor protein acyl coenzyme a (acyl-coa) binding domain protein (acbd /gpc ), acts as an adaptor to recruit phosphatidylinositol -kinase class iii beta (pi kiii) in infected cells [ ] . evidence shows that the tomato spotted wilt virus (tswv) glycoprotein gn localizes to golgi membranes and induces deformation of the membranes into pseudo-circular and pleomorphic structures in tobacco plant cells [ ] . upon infection of hela cells, rabbit kidney cells and mouse monocytes-macrophages cell lines with vacv, viral progeny becomes enwrapped in the membrane derived from tgn cisterna to form the enveloped virus [ ] . several viral proteins localize to the golgi body and mature by undergoing post translational modifications such as glycosylation, phosphorylation, etc. rubella virus was reported to undergo a golgi dependent maturation upon infection of bhk- and vero cells [ ] . the inner tegument protein pul of the dna virus hsv was identified to be responsible in directing the viral capsids to the tgn in order to undergo secondary envelopment in different cell lines [ ] . the glycoproteins of bunyamwera virus undergo primary maturation by modifying their sugar composition in the tgn upon infection of the bhk- and vero cells [ ] . lipid droplets are single membrane bound organelles with a lipid core that primarily consists of neutral lipids like triacylglycerols (fig. ) . they are essential for lipid storage and metabolism in a cell. these stored lipids can be used to generate and maintain energy homeostasis and hence they are central to the cellular function. lipid droplets are dynamic intracellular organelles which are required for storing lipids in a cell. they play a major role in energy homeostasis and membrane trafficking [ ] . many rna viruses exploit this energy storing capacity of lipid droplets to facilitate their replication [ ] . upon expression, various viral proteins are reported to localize to the lipid droplets [ ] . for example, upon hcv infection, the hcv ns a protein localizes to the surface of the lipid droplets with the help of a host factor diacylglycerol acyltransferase (dgat ) in huh and hek t cells [ ] . another study reported that hcv core a protein is involved in downregulating the expression of phosphoinositide -kinase (pi k)/phosphatase and tensin (pten) which in turn induces the accumulation of enlarged lipid droplets in human huh and hepg cells [ ] . in another example, denv c protein was reported to bind and interact with perilipin a protein present on the surface of the lipid droplets in hepg cell lines [ ] . a similar localization of denv c protein was also observed upon infection of denv in bhk- , hepg , and c / ht mosquito cells of a. albopictus [ ] . additionally, the denv c protein localization on lipid droplets was also found to be essential for the denv replication [ ] . denv induces autophagy and results in a reduction in lipid droplet area in huh . cells, a subline derived from huh cells [ , ] . further analysis of the infected cells showed reduced levels of triglycerides in the lipid droplets and suggests that atp generation by b-oxidation of fatty acids is essential for robust replication of viral rna [ , ] . another study reports that rotavirus recruits lipid droplets into their viroplasm compartments upon infection of ma , caco- , bsc- , and cos- cell lines [ ] . confocal microscopy studies reported that two ld-associated proteins namely perilipin a and adrp colocalize with rotaviral proteins present in the viroplasm [ ] . overexpression of the hbv x (hbx) protein was found to induce lipid accumulation in hepatic cells [ , ] . na and colleagues found that hbx induces a pathway that involves the expression of liver x receptor and its associated genes result in accumulation of lipid droplets in hepg cell lines [ ] . lysosomes are single membrane-bound organelles which house enzymes involved in the degradation of various extracellular and intracellular macromolecules such as proteins, pathogens, etc through phagocytic or autophagic pathway. plant and fungal vacuoles have similar degradative and storage functions like the lysosomes (fig. ) . the specialized acidic lumen is the unique feature of these organelles which is needed to keep the enzymes active. viruses utilize lysosomal enzymes in order to facilitate their replication and release within the host cell [ ] . it has been suggested that lysosomal enzymes are involved at different stages of vacv and mouse hepatitis virus replication [ ] . they proposed a possibility for viruses to recruit lysosomal enzymes inside phagosomes in order to uncoat and release their genome in the host cell. a possible role for the lysosomal enzymes in enhancement of glycolysis in viral infected cells was also proposed. recent studies reported the accumulation of hav progeny in the lysosome of the host hepg cells. the maturation of these viral particles was reported to be catalysed by the lysosomal protease [ ] . sv infection in bsc- and t cell lines resulted in swelling up of lysosomes followed by the release of lysosomal enzyme into the cytoplasm [ ] . since lysosomes play a major role in the host's antiviral response, they are likely to become a target for certain viruses. the x protein of the hbv leads to inhibition of lysosomal acidification leading to loss of functioning [ ] . however, the lysosome's ability to fuse with autophagosomes was found to remain unaffected. this virus induced accumulation of immature lysosomes resulting in the suppression of autophagic degradation was followed by development of hbv-associated hepatocellular carcinoma [ ] . deglycosylation of the lysosome-associated membrane proteins by neuraminidase (na) of h n influenza virus resulted in destruction of lysosomes. this was followed by cell death due to the release of hydrolytic lysosomal enzymes to the cytoplasm [ ] . shubin and colleagues studied the expression of c protease of hav in a and human lung epidermoid carcinoma (calu- ) cell lines and found that hav c protease induces the development of non-acidic cytoplasmic vacuoles which originate from several types of lysosomal/endosomal organelles [ ] . similarly several viruses like hiv, adenovirus, pv have been reported to cause lysosomal rupture [ ] . tobacco plant when infected with cucumber mosaic virus (cmv), the viral replicase complex that constitutes the replicaseassociated protein cmv a and rna dependent rna polymerase protein cmv a was reported to localize on the vacuolar membrane [ ] . singapore grouper iridovirus (sgiv) infection on grouper embryonic cells (gecs) from the brown-spotted grouper epinephelus tauvina results in the formation of a large intracellular vacuole for viral accumulation. later, the virus recruits the host cytosolic membrane-bending proteins in order to induce tubulation of vacuolar membrane [ ] . the individual vacuoles fuse together to form a large vacuole which in turn fuses with the cell membrane. these events aid in the release of virions [ ] . semliki forest virus (sfv) targets the vacuolar atpase in order to cause acidification of vacuoles resulting in low intra luminal ph [ ] . upon infection with the sfv the endocytic vacuoles with low ph trigger membrane fusion essential for the viral pathogenesis in bhk- cells [ ] . vacuolar proton atpase activity leading to low intra endosomal ph was reported to be required for the entry of reovirus into the host cells [ ] . acidification of vacuoles by cellular v-atpase in human dermal fibroblast cells upon infection of hcmv was reported to be required for the formation of the specialized compartment for virion assembly [ ] . apart from the above discussed well defined organelles of a cell, viruses also modify and hijack various vesicular structures and protein complexes of the host cell to ensure efficient infection. we discuss these essential compartments/structures in this section. endosomes are required for internalization of extracellular material into the cells and lead them to lysosomes for degradation or recycle back to the plasma membrane. multi vesicular bodies are vesicular compartment of the endocytic pathway and contain intraluminal vesicles. autophagosomes are double membrane vesicular structures that sequester the cytoplasm containing proteins, organelles, etc and direct them to degradation (fig. ) . efficient cellular entry of most viruses is via endocytic pathway that comprises of various endosomes. several viruses like influenza, sfv, vsv, sv , ebola, etc use different endocytic pathways like clathrin, caveoli or micropinocytosis to gain entry into the cells [ , e ] . viruses modify or induce the formation of vesicles like endosomes in order to facilitate their multiplication inside the host cells [ ] . sfv modifies the endosomal and lysosomal membrane of the infected bhk- cells for the construction of its replication site [ ] . further the endosomes and lysosomes were reported to fuse together resulting in the formation of cytoplasmic vacuoles [ ] . sv was reported to trigger the formation of endocytic vacuoles that migrate and fuse with the nuclear membrane upon infection of cv- cells leading to the migration of virions into the infected cell nucleus [ , ] . endosomal sorting complexes required for transport (escrt) catalyse the process of invagination of endosomal membrane and result in the formation of multiple vesicular bodies (mvb) in eukaryotic cells [ ] . mvb act as an intermediate for transporting ubiquitinated or misfolded protein to lysosomes [ ] . mvb are also reported to play an active role in endolysosomal transport and budding of the virus. several rna and dna viruses hijack the escrt machinery in order to facilitate their release from the infected cells [ ] . the matrix protein vp recruits tsg protein and escrt- complex constituting of vps , vps b and vps in order to direct the ebola virus into multivesicular bodies that assists in their budding [ ] . hoffmann and colleagues reported that upon infection of hepatoma derived cell lines by the dna virus hbv, cellular a-taxilin acts as an adaptor for the binding of large hbv surface antigen with escrt components. this aids in recruiting the escrt machinery for the release of hbv-dna containing particles [ ] . various rna viruses alter autophagosomes and induce or suppress autophagy in order to complete their life cycle or to escape from the host antiviral response [ ] . it has been reported that the coxsackie b infection triggers the formation of autophagosomes in hela and hek cells [ ] . prevention of lysosomal fusion with autophagosomes also enhanced coxsackie virus replication in the host cells. wild type mouse embryonic fibroblast (mef) and huh cells also exhibit enhanced autophagosome formation upon denv infection [ ] . the viruses modulating the phenomenon of cellular autophagy for their advantage have been reviewed elsewhere [ ] . proteasome is a complex of proteases that selectively degrades intracellular proteins. this complex is tightly regulated and identifies polyubiquitinated proteins for degradation process. recent studies identified that viruses hijack ups in several ways to enhance their infectivity [ ] . this is achieved by degradation of host proteins of ups, enhancing the function of viral proteins by modifications, hampering the modifications of signalling molecules of innate immunity, etc [ ] . virus induced ubiquitination and subsequent proteasomal degradation of p as a strategy is employed by dna viruses such as hpv, adv, etc [ ] . viral protein x of hiv- was reported to be responsible for the ubiquitination and proteasomal degradation of the host protein samhd that inhibits hiv infection [ ] . an interesting strategy by denv was reported where the viral protein ns stimulates proteasomal degradation of stat and blocks type ifn signalling and thus evades the host immune mechanisms [ ] . similarly selective degradation of ns (non-structural protein) of the zika virus via proteasome mediated pathway was recently reported [ ] . this proteasome dependant degradation of the viral protein was proposed as a strategy for the host antiviral mechanism. a role for ups has also been reported in plant viral infections. components of rna silencing such as argonaute are targeted to proteasomal degradation by viruses like potato virus x, enamovirus, etc for efficient infection [ , ] . this review summarizes the modifications that organelles encounter upon viral infection in a cell. understanding organelle dynamics under various conditions is a fundamental question that has attracted researchers for a long time. the importance of organelle dynamics and function is highlighted by many examples of diseases/disorders where it is affected. alterations in organelles such as shape, content, dynamics and eventually the function as a result of viral infections is observed. not only viruses but pathogenic bacteria have also been reported to alter organelles for their survival and infection. as emphasized in this paper, recent studies have shown that many viruses encode proteins that are targeted to various cellular organelles and control their functions. certainly there exists a close relationship between organelle dynamics and viral infections but thorough characterization will highlight their relevance to pathogenesis. advanced methods in microscopy and proteomics have enabled such characterization and the molecular details of virus-host interactions and viral replication in host cells is now understood in detail for few viruses. it is important to determine how various organelle proteins are temporally and spatially regulated upon viral infections leading to altered functions. organelles not as independent entities but a role for inter-organelle communication/inter-organelle cross talk in a cell for optimum functioning is now unequivocally accepted. this is another very interesting aspect to be explored to enhance our understanding of the virus-host interaction mechanisms to enable design of new antiviral strategies. our understanding on the organelle-virus interaction has been rapidly increasing with the advent of new molecular biology tools and advance imaging techniques. although we know the basic modus operandi of the viruses, there might be a novel virus that behaves different than the existing dogma with respect to host cellular architecture. the authors declare to no conflict of interest. ground control to major tom: mitochondria-nucleus communication the upsides and downsides of organelle interconnectivity an organelle-specific protein landscape identifies novel diseases and molecular mechanisms hiv- and the host cell: an intimate association virulence and pathogenesis virus entry by endocytosis 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experimental analysis reveals a new transport-competent nuclear localization signal in the nucleoprotein of influenza a virus strain importin alpha nuclear localization signal binding sites for stat , stat , and influenza a virus nucleoprotein function of dynein and dynactin in herpes simplex virus capsid transport microtubule-mediated transport of incoming herpes simplex virus capsids to the nucleus herpes simplex virus type entry into host cells: reconstitution of capsid binding and uncoating at the nuclear pore complex in vitro import of adenovirus dna involves the nuclear pore complex receptor can/nup and histone h nucleoporin arrests the nuclear import of hepatitis b virus capsids in the nuclear basket phosphorylationdependent binding of hepatitis b virus core particles to the nuclear pore complex nuclear import of hepatitis b virus capsids and release of the viral genome role of nuclear pore complex in simian virus nuclear targeting pushing the envelope: microinjection of minute virus of mice into xenopus oocytes causes damage to the nuclear envelope parvoviral nuclear import: bypassing the host nuclear-transport machinery nucleoli: composition, function, and dynamics involvement of ul in herpes-simplex-virus- -induced dispersal of nucleolin involvement of the ul protein in herpes simplex virus -induced dispersal of b and in nuclear egress adenovirus core protein v is delivered by the invading virus to the nucleus of the infected cell and later in infection is associated with nucleoli adenovirus protein v induces redistribution of nucleolin and b from nucleolus to cytoplasm effects of adenovirus infection on rrna synthesis and maturation in hela cells encephalomyocarditis virus (emcv) proteins a and bcd localize to nuclei and inhibit cellular mrna transcription but not rrna transcription rhinovirus c protease precursors cd and cd' localize to the nuclei of infected cells the post-transcriptional regulator rev of hiv: implications for its interaction with the nucleolar protein b localization to the nucleolus is a common feature of coronavirus nucleoproteins, and the protein may disrupt host cell division inhibition of nuclear import and alteration of nuclear pore complex composition by rhinovirus nucleolin stimulates viral internal ribosome entry site-mediated translation quantitative proteomic analysis of a cells infected with human respiratory syncytial virus ifn enhance expression of sp , an autoantigen in primary biliary cirrhosis hsv- ie protein vmw causes redistribution of pml mediation of epstein-barr virus ebna-lp transcriptional coactivation by sp adenovirus replication is coupled with the dynamic properties of the pml nuclear structure adenovirus e orf protein inhibits the interferon-mediated antiviral response inactivating a cellular intrinsic immune defense mediated by daxx is the mechanism through which the human cytomegalovirus pp protein stimulates viral immediate-early gene expression two ring finger proteins, the oncoprotein pml and the arenavirus z protein, colocalize with the nuclear fraction of the ribosomal p proteins sumoylation promotes pml degradation during encephalomyocarditis virus infection rabies virus p and small p products interact directly with pml and reorganize pml nuclear bodies viral interactions with the nuclear transport machinery: discovering and disrupting pathways remodelling of the nuclear lamina during human cytomegalovirus infection: role of the viral proteins pul and pul papillomavirus interaction with cellular chromatin phenotypic and cytologic studies of lymphoid cells and monocytes in primary culture of porcine bone marrow during infection of african swine fever virus wrapping membranes around plant virus infection inhibition of sterol biosynthesis reduces tombusvirus replication in yeast and plants endoplasmic reticulum: the favorite intracellular niche for viral replication and assembly direct formation of vaccinia virus membranes from the endoplasmic reticulum in the absence of the newly characterized l -interacting protein a . intracellular localisation of dengue- rna in mosquito cell culture using electron microscopic in situ hybridisation composition and three-dimensional architecture of the dengue virus replication and assembly sites cellular origin and ultrastructure of membranes induced during poliovirus infection remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagylike origin for virus-induced vesicles ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum infectious bronchitis virus generates spherules from zippered endoplasmic reticulum membranes the potato virus x tgbp movement protein associates with endoplasmic reticulum-derived vesicles during virus infection tombusviruses upregulate phospholipid biosynthesis via interaction between p replication protein and yeast lipid sensor proteins during virus replication in yeast opportunistic intruders: how viruses orchestrate er functions to infect cells arms race between enveloped viruses and the host erad machinery mutational analysis of glycosylation, membrane translocation, and cell surface expression of the hepatitis e virus orf protein cytoplasmic localization of the orf protein of hepatitis e virus is dependent on its ability to undergo retrotranslocation from the endoplasmic reticulum the expanding roles of endoplasmic reticulum stress in virus replication and pathogenesis bap and bip are essential for dislocation of sv from the endoplasmic reticulum to the cytosol global sitespecific n-glycosylation analysis of hiv envelope glycoprotein the interplay between mitochondrial dynamics and mitophagy mitochondrial fusion, fission, and mitochondrial toxicity mitochondrial dynamics and viral infections: a close nexus hepatitis b virus disrupts mitochondrial dynamics: induces fission and mitophagy to attenuate apoptosis sarscoronavirus open reading frame- b suppresses innate immunity by targeting mitochondria and the mavs/traf /traf signalosome mitophagy in viral infections the matrix protein of human parainfluenza virus type induces mitophagy that suppresses interferon responses mitophagy promotes replication of oncolytic newcastle disease virus by blocking intrinsic apoptosis in lung cancer cells viruses as modulators of mitochondrial functions hepatitis b virus x protein induces perinuclear mitochondrial clustering in microtubule-and dyneindependent manners migration of mitochondria to viral assembly sites in african swine fever virus-infected cells herpes simplex virus eliminates host mitochondrial dna hepatitis c virus triggers mitochondrial permeability transition with production of reactive oxygen species, leading to dna damage and stat activation human cytomegalovirus pul x induces the release of endoplasmic reticulum calcium stores the coxsackievirus b protein suppresses apoptotic host cell responses by manipulating intracellular ca þ homeostasis role of ca þin the replication and pathogenesis of rotavirus and other viral infections human hepatitis b virus-x protein alters mitochondrial function and physiology in human liver cells hbx sensitizes cells to oxidative stress-induced apoptosis by accelerating the loss of mcl- protein via caspase- cascade influence of cytosolic and mitochondrial ca þ, atp, mitochondrial membrane potential, and calpain activity on the mechanism of neuron death induced by -nitropropionic acid viral product trafficking to mitochondria, mechanisms and roles in pathogenesis a pore way to die: the role of mitochondria in reperfusion injury and cardioprotection the myxoma poxvirus protein, m l, prevents apoptosis by direct interaction with the mitochondrial permeability transition pore the hiv- viral protein r induces apoptosis via a direct effect on the mitochondrial permeability transition pore a renewed focus on the interplay between viruses and mitochondrial metabolism systemslevel metabolic flux profiling identifies fatty acid synthesis as a target for antiviral therapy dynamics of the cellular metabolome during human cytomegalovirus infection glycolysis during early infection of feline and human cells with feline leukemia virus glycolysis in chick embryo cell cultures transformed by rous sarcoma virus divergent effects of human cytomegalovirus and herpes simplex virus- on cellular metabolism hepatitis c virus core protein inhibits mitochondrial electron transport and increases reactive oxygen species (ros) production viruses exploiting peroxisomes import of peroxisomal matrix and membrane proteins peroxisomes are platforms for cytomegalovirus' evasion from the cellular immune response expression of the cymbidium ringspot virus -kilodalton protein in saccharomyces cerevisiae and molecular dissection of the peroxisomal targeting signal localization of the tomato bushy stunt virus replication protein p reveals a peroxisome-to-endoplasmic reticulum sorting pathway the host pex p plays a role in peroxisomal localization of tombusvirus replication proteins a key role for heat shock protein in the localization and insertion of tombusvirus replication proteins to intracellular membranes the pestivirus n terminal protease n(pro) redistributes to mitochondria and peroxisomes suggesting new sites for regulation of irf by n(pro.) the fine structure of cymbidium ringspot virus infections in host tissues. iii. role of peroxisomes in the genesis of multivesicular bodies the role of the p :p /p interaction domain in rna replication and intracellular localization of p and p proteins of cucumber necrosis tombusvirus integrated systems biology analysis of kshv latent infection reveals viral induction and reliance on peroxisome mediated lipid metabolism micro-rnas upregulated during hiv infection target peroxisome biogenesis factors: implications for virus biology, disease mechanisms and neuropathology flavivirus infection impairs peroxisome biogenesis and early antiviral signaling identification of a type peroxisomal targeting signal in a viral protein and demonstration of its targeting to the organelle binding of hiv- nef to a novel thioesterase enzyme correlates with nef-mediated cd down-regulation a novel acyl-coa thioesterase enhances its enzymatic activity by direct binding with hiv nef endocytosis of adeno-associated virus type leads to accumulation of virus particles in the golgi compartment the transmembrane domain of the severe acute respiratory syndrome coronavirus orf b protein is necessary and sufficient for its retention in the golgi complex a signal for golgi retention in the bunyavirus g glycoprotein orf virus interferes with mhc class i surface expression by targeting vesicular transport and golgi foot-and-mouth disease virus c protease induces fragmentation of the golgi compartment and blocks intra-golgi transport fragmentation of the golgi apparatus provides replication membranes for human rhinovirus a the open reading frame a protein of severe acute respiratory syndrome-associated coronavirus promotes membrane rearrangement and cell death norwalk virus n-terminal nonstructural protein is associated with disassembly of the golgi complex in transfected cells poliovirus infection and expression of the poliovirus protein b provoke the disassembly of the golgi complex, the organelle target for the antipoliovirus drug ro- membrane-association properties of avian encephalomyelitis virus protein a impact on the endoplasmic reticulum and golgi apparatus of turnip mosaic virus infection a trans-golgi network resident protein, golgin- , accumulates in viral factories and incorporates into virions during poxvirus infection the a protein from multiple picornaviruses utilizes the golgi adaptor protein acbd to recruit pi kiiibeta tomato spotted wilt virus glycoproteins induce the formation of endoplasmic reticulum-and golgi-derived pleomorphic membrane structures in plant cells assembly of vaccinia virus: the second wrapping cisterna is derived from the trans golgi network structural maturation of rubella virus in the golgi complex inner tegument protein pul of herpes simplex virus type is involved in directing capsids to the trans-golgi network for envelopment key golgi factors for structural and functional maturation of bunyamwera virus emerging role of lipid droplets in host/pathogen interactions lipid droplets and viral infections down-regulation of phosphatase and tensin homolog by hepatitis c virus core a in hepatocytes triggers the formation of large lipid droplets dengue virus capsid protein binding to hepatic lipid droplets (ld) is potassium ion dependent and is mediated by ld surface proteins dengue virus capsid protein usurps lipid droplets for viral particle formation dengue virus-induced autophagy regulates lipid metabolism dengue virus and autophagy rotaviruses associate with cellular lipid droplet components to replicate in viroplasms, and compounds disrupting or blocking lipid droplets inhibit viroplasm formation and viral replication hepatitis b virus x protein induces lipogenic transcription factor srebp and fatty acid synthase through the activation of nuclear receptor lxralpha hepatitis b virus x protein induces hepatic steatosis via transcriptional activation of srebp and ppargamma liver x receptor mediates hepatitis b virus x protein-induced lipogenesis in hepatitis b virusassociated hepatocellular carcinoma activation of lysosomal enzymes in virus-infected cells and its possible relationship to cytopathic effects lysosomes serve as a platform for hepatitis a virus particle maturation and nonlytic release lysosomal changes in lytic and nonlytic infections with the simian vacuolating virus (sv ) hepatitis b virus x protein inhibits autophagic degradation by impairing lysosomal maturation neuraminidase of influenza a virus binds lysosome-associated membrane proteins directly and induces lysosome rupture protease c of hepatitis a virus induces vacuolization of lysosomal/endosomal organelles and caspase-independent cell death lysosomal cell death at a glance a zinc finger protein tsip controls cucumber mosaic virus infection by interacting with the replication complex on vacuolar membranes of the tobacco plant visualization of assembly intermediates and budding vacuoles of singapore grouper iridovirus in grouper embryonic cells involvement of the vacuolar h(þ)-atpase in animal virus entry membrane and protein interactions of a soluble form of the semliki forest virus fusion protein the entry of reovirus into l cells is dependent on vacuolar proton-atpase activity cellular v-atpase is required for virion assembly compartment formation in human cytomegalovirus infection endocytosis via caveolae endocytosis of simian virus into the endoplasmic reticulum cellular entry of ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes membrane dynamics associated with viral infection biogenesis of the semliki forest virus rna replication complex fusion of sv -induced endocytotic vacuoles with the nuclear membrane interaction of endocytotic vacuoles with the inner nuclear membrane in simian virus entry into cv- cell nucleus escrt complexes and the biogenesis of multivesicular bodies involvement of vacuolar protein sorting pathway in ebola virus release independent of tsg interaction identification of alpha-taxilin as an essential factor for the life cycle of hepatitis b virus divergent roles of autophagy in virus infection autophagosome supports coxsackievirus b replication in host cells autophagic machinery activated by dengue virus enhances virus replication irhom is essential for innate immunity to dna viruses by mediating trafficking and stability of the adaptor sting identification of three functions of the adenovirus e orf protein that mediate p degradation by the e orf -e b k complex molecular determinants for recognition of divergent samhd proteins by the lentiviral accessory protein vpx ns of dengue virus mediates stat binding and degradation viperin restricts zika virus and tick-borne encephalitis virus replication by targeting ns for proteasomal degradation the silencing suppressor p of potato virus x interacts with argonaute and mediates its degradation through the proteasome pathway the enamovirus p protein is a silencing suppressor which inhibits local and systemic rna silencing through ago degradation we sincerely apologize to our colleagues for any research study or review publication not being cited in this review. this is only due to space limitation. research in the laboratory of sn is supported by grants from science and engineering research board ( key: cord- -cb u s s authors: bedford, juliet; farrar, jeremy; ihekweazu, chikwe; kang, gagandeep; koopmans, marion; nkengasong, john title: a new twenty-first century science for effective epidemic response date: - - journal: nature doi: . /s - - -y sha: doc_id: cord_uid: cb u s s with rapidly changing ecology, urbanization, climate change, increased travel and fragile public health systems, epidemics will become more frequent, more complex and harder to prevent and contain. here we argue that our concept of epidemics must evolve from crisis response during discrete outbreaks to an integrated cycle of preparation, response and recovery. this is an opportunity to combine knowledge and skills from all over the world—especially at-risk and affected communities. many disciplines need to be integrated, including not only epidemiology but also social sciences, research and development, diplomacy, logistics and crisis management. this requires a new approach to training tomorrow’s leaders in epidemic prevention and response. when nature published its first issue in , a new understanding of infectious diseases was taking shape. the work of william farr , ignaz semmelweis , louis-rené villermé and others had been published; john snow had traced the source of a cholera epidemic in london (although robert koch had not yet isolated the bacterium that caused it ). the science of epidemiology has described patterns of disease in human populations, investigated the causes of those diseases, evaluated attempts to control them and has been the foundation for public health responses to epidemic infections for over years. despite great technological progress and expansion of the field, the theories and practices of infectious disease epidemiology are struggling to keep pace with the transitional nature of epidemics in the twenty-first century and the breadth of skills needed to respond to them. epidemiological transition theory has focused mostly on the effects of demographic and socioeconomic transitions on well-known preventable infections and a shift from infectious diseases to non-communicable diseases . however, it has become clear that current demographic transitions-driven by population growth, rapid urbanization, deforestation, globalization of travel and trade, climate change and political instability-also have fundamental effects on the dynamics of infectious diseases that are more difficult to predict. the vulnerability of populations to outbreaks of zoonotic diseases such as ebola, middle east respiratory syndrome (mers) and nipah has increased, the rise and spread of drug-resistant infections, marked shifts in the ecology of known vectors (for example, the expanding range of aedes mosquitoes) and massive amplification of transmission through globally connected, high-density urban areas (particularly relevant to ebola, dengue, influenza and severe acute respiratory syndrome-related coronavirus sars-cov). these factors and effects combine and interact, fuelling more-complex epidemics. although rare compared to those diseases that cause the majority of the burden on population health, the nature of such epidemics disrupts health systems, amplifies mistrust among communities and creates high and long-lasting socioeconomic effects, especially in low-and middle-income countries. their increasing frequency demands attention. as the executive director of the health emergencies program at the world health organization (who) has said: "we are entering a very new phase of high-impact epidemics… this is a new normal, i don't expect the frequency of these events to reduce." . we have to act now but act differently: a broader foundation is required, enhancing traditional epidemiology and public health responses with knowledge and skills from a number of areas ( table ) . many of these areas have long been associated with epidemic preparedness and response, but they must now stop being seen as esoteric 'nice things to have', and instead become fully integrated into the critical planning and response to epidemics. this will require considerable changes by the global public health community in the way that we respond to epidemics today and how we prepare for and seek to prevent those of tomorrow. it will mean reshaping the global health architecture of the response to epidemics and transforming how we train new generations of researchers and practitioners for the epidemics of the future . the modern research culture-often shaped by the behaviour of funders-has required many researchers to specialize in narrow fields, with less emphasis on translation than on field-specific innovations. although this siloed landscape has brought major advances in global health, it is not fit for the transitional phase of epidemic diseases: rapidly evolving, high-impact events bring together communities, responders and researchers who do not routinely interact. different assumptions, cultures and practices, each of which may be widely accepted within a particular community, make working together in outbreak situations more challenging. fundamental to success is respect and understanding of the contribution each party brings. in a successfully integrated approach, we each have to realize that our knowledge and skills are a small part of a rapidly expanding toolkit (box ). we need to understand major trends in research and how and when they may influence the response to an epidemic, develop new research to strengthen the support that we can provide across other areas and learn to operate in multi-stakeholder situations-including, at times, as part of a critical debate to bring better practices to the fore. central to this approach must be the communities who are at risk and those affected by epidemics: local people are the first responders to any outbreak and their involvement in the preparation and response activities is essential. from communities, through local and regional health authorities, national public health institutes and international organizations-including many essential partners in sectors beyond public health-the integrated approach must be supported. the who, in particular, has a critical part to play, using its unique mandate not to lead every aspect of preparation, response and recovery, but to change its practices, facilitate integration with and among others, and ensure accountabilities are built in from the bottom to the top. a wave of cholera epidemics across europe in the s and s catalysed a new era of 'infectious disease diplomacy' globally. nations recognized that infections do not stop at borders and that therefore multilateral collaboration is essential to protecting citizens from lethal epidemics. the development of germ theory through the second half of the nineteenth century transformed ideas about the causes of infections, informing scientific research as well as clinical responses. scientific understanding translated into vaccines and antibiotics, while programmes for child health, hygiene, clean water and sanitation became common in the twentieth century. as a result, childhood diseases such as measles and mumps became rare, smallpox was eventually eradicated and polio was eliminated from all but a handful of countries . many people thought that infectious diseases would soon be history. sir frank macfarlane burnet is often cited for his remark in the s that, with the emergence of new diseases being a distant prospect, "the future of infectious diseases will be very dull" . although the focus in high-income nations turned to non-communicable diseases, which constituted a considerable and increasing burden on the health of their citizens, infectious diseases did not disappear. some endemic infections such as malaria and tuberculosis were not susceptible to elimination strategies, and new diseases with epidemic and pandemic potential emerged. ebola virus disease was first identified in the s, hiv/aids in the s, nipah virus in the s, sars and mers at the start of the twenty-first century, and many more have since been identified. far from becoming 'very dull', the field of infectious disease epidemiology has sometimes struggled to adapt: as late as , respected researchers used a nineteenth century 'law' of epidemiology to make predictions about the aids epidemic-these turned out to be vast underestimates . advances in other fields gave epidemiology the chance to evolve. in , when the editors of the international journal of epidemiology provocatively asked whether it was time to 'call it a day' given the putative power of genomics to explain diseases over the capacity of epidemiologists to describe them, their conclusion was that it had the potential to positively transform epidemiology as much as the rise of germ theory a century earlier. at least pathogens that affect humans have been identified as emerging, re-emerging or evolving since the s , while increasing rates of antimicrobial resistance threaten to make formerly controlled infections, such as malaria, untreatable -this also limits our ability to control their epidemic potential. the demographic transition is driving much of this: human society is becoming more urban than rural for the first time in our history, bringing large numbers of people (and often animals) together in densely populated areas . agricultural and forestry practices are changing the relationships between people, animals and our respective habitats . travel is more accessible around the world, advances in computer science and computing speeds have led to a number of applications of artificial intelligence across society . applications in epidemiology include tracking online searches about disease symptoms to aid early detection of epidemics, although more sophisticated methods may be required before artificial intellegence becomes a reliable detection tool . crystallography modern x-ray diffraction and electron microscopy can reveal structures of viruses and antibodies in such detail that it is possible to identify specific sites of vulnerability on the virus. a previous study showed how such techniques identified an antibody that was much more potent against respiratory syncytial virus than the only currently available intervention . developing vaccines for emerging infectious diseases has many challenges, including the time it takes, a limited market and strict regulatory requirements for products that will be given to healthy people . platform technologies use one underlying approach with standardized processes and some antigen-specific optimization to speed up both development and manufacture of vaccines. for example, vector-based platforms combine an antigen, or a gene for an antigenic protein or peptide, in a virus-like particle or liposome. such platform technologies have the potential to deliver vaccines a few months after an emerging pathogen is identified and sequenced, rather than years . review so migration, trade and tourism bring more people into contact and thus affect disease transmission . climate change has many effects on ecosystems and environments, not least in changing the habitats and migratory habits of disease vectors . states with weak health systems are far less likely to cope with or recover from multiple emergent demands without damaging routine services . inequalities , inequities and distrust in national structures and institutions compound people's vulnerabilities . conflict increases the risk of epidemics and makes responding to them close to impossible . since , there have been several outbreaks of ebola (including the two biggest in history), not to mention outbreaks of sars, mers, nipah, influenza a subtype h n , yellow fever, zika and the continued spread of dengue. epidemics overlap and run into each other, yet the world is not currently equipped to cope with this increasing burden of multiple public health emergencies. preparing for epidemics, therefore, requires global health, economic and political systems to be integrated just as much as infectious disease epidemiology, translational research and development, and community engagement. epidemics represent shared risks that cross borders and all of society. health systems, routine care, trust in governments, travel, trade, business-all are disrupted during an epidemic. with such broad risks, the preparation and response must be nationally owned and led, internationally supported and undertaken with a whole-of-society approach. some initiatives have started to build frameworks for this to happen in a coordinated way. for example, the who's pandemic influenza preparedness framework brings together nation states, industry, other stakeholders and the who to implement a global approach to pandemic preparedness and response . a focus must be building coordinated regional and country expertise, resources and capacity through national and regional public health institutions . this brings its own challenges-governance of institutions, leadership, collaborations and interventions have to be impeccable or misconduct can thrive . unwelcome in itself, misuse of funding, resources or people within efforts intended to support an epidemic response will also undermine trust in the organizations that respond to an outbreak and, in turn, prolong the outbreak. key governance components include drafting policies in advance and being willing to implement those policies for data collection and sharing during epidemics. they must be flexible enough to enable affected communities and nations to retain ownership of the response, while drawing on international expertise to find the best possible response. governance should also include processes for vaccine and therapeutic approvals during outbreaks. however, it is clear that the centre of gravity for leadership, governance and implementation must be where the need is greatest if these are to truly deliver. in , julian tudor hart proposed the inverse care law: "the availability of good medical care tends to vary inversely with the need for it in the population served." . an analogue of the inverse care law can be applied to public health and epidemiology. expertise in these fields has traditionally gravitated towards centres of excellence in europe and the united states. of course, high-income countries are not immune to the disruption associated with epidemics, especially in an era of misinformation and growing mistrust in authorities and public health initiatives. however, the centre of gravity must shift so that globally representative distributed networks of collaborating centres can jointly ensure coverage in the regions that urgently need these skills on the ground . international collaborations remain important; however, strengthening epidemiology, public health and laboratory capacity in low-and middleincome countries is essential . collaborative interventions should not be limited to when there is a major outbreak, but be integrated into regular interactions. capacity, resources, expertise and governance can be supported by the increasing role for regional and national centres of disease control. the us centers for disease control (cdc) lends its expertise all around the world in addition to protecting the us population. in , the european cdc started, followed by the china cdc in and by the africa cdc in . although more can be done to improve data sharing and access to laboratories, the networks and connections between these centres have strengthened all of their work, as well as having a positive effect on public health systems in low-and middle-income countries. during the pan-european wave of cholera in the s, there were riots across the continent: doctors, nurses and pharmacists were murdered, hospitals and medical equipment destroyed . similar reports today usually come from communities that have not had positive prior interactions with public health initiatives, and thus the encounter with national or international teams who arrive only in response to a 'new' disease means that trust can never be assumed and has to be earned on both sides. engagement needs to start before an outbreak-ensuring that patients, their families and their communities are at the centre of all public health is essential for the successful prevention and response to epidemics. there is no public health without the support of the community. for example, the early detection of disease events will be improved if more national and regional public health institutions establish community event-based surveillance systems. communities are the first to know when something unusual happens -therefore training and mobilizing community volunteers to report such occurrences is a costeffective way to rapidly detect diseases and contain them at the source. this will also help to sustain engagement between communities and the organizations that respond to outbreaks. furthermore, improved information flow between the community and the public health system should provide a better understanding of local social networks to complement other means of tracking chains of transmission between individuals and places. this can be the community themselves, or it might be veterinarians who see clusters of sick animals, or nurses and doctors who care for patients in primary care-or it may be teams that are often forgotten in public health initiatives, such as those working in critical care facilities; it is striking how the first cases of nipah, sars, mers and influenza a subtype h n were all first identified by clinical teams in critical care facilities. an inclusive, whole-of-society approach is challenging, and the challenges may be magnified in a conflict or post-conflict zone. wars and conflicts not only increase the risk of epidemics as people move to escape violence and health services become harder to maintain , but also make public health responses vulnerable to interruption, thus making them less effective. then, miscommunication, mistrust, disease and violence can fuel each other in a vicious cycle. engaging local communities remains the highest priority, even in unstable contexts such as north kivu and ituri provinces of the democratic republic of the congo (drc) , where an ebola epidemic started in august . it seems inevitable that responding to epidemics in politically unstable environments will become more common, and skilled negotiators and peacekeepers will have to be better integrated in response teams. equally essential, therefore, will be an improved understanding of these challenging operational contexts among affected communities and external responders alike. social scientists have long applied their skills and knowledge in epidemic responses, although their roles have become more visible in recent years . by focusing on communities, social science humanizes the epidemic response , helps to increase understanding of context and may uncover associations between the context or local practices and the risk of transmission. the social science in humanitarian action platform has successfully produced rapid reports and briefings on regions in which an epidemic has been identified, and the global research collaboration for infectious disease preparedness includes a social science research funders' forum to 'propel research in this area' , acknowledging that its integration in the preparation and response to outbreaks is often missing or added as an afterthought to solve a problem that could have been forseen. there is still much to learn about how epidemic responders and social scientists can make the most of each other's expertise and how data from social science can fit into the wider information architecture of epidemic response. as an example, behavioural surveillance will be critical in twenty-first century responses to disease outbreaks . just as behavioural surveillance to improve the understanding of hiv was crucial in identifying high-risk groups for hiv infection, so human behaviours will continue to be important as we respond to future infectious diseases. for instance, the ebola virus outbreak in west africa probably began before december , but it took several months before hospital transmission and traditional burial practices were found to be the leading causes of its rapid spread. the increasing prevalence of mobile phones, wireless internet connectivity and social media activity raises the possibility of using these tools to gather data for epidemiological studies, diagnostics , population mobility during an ebola epidemic or influenza incidence in real time . future developments in predictive technology, machine learning and artificial intelligence will bring more opportunities to move towards 'precision public health' (box ). the use of data from people is becoming strictly controlled, however, and it will be a challenge to persuade countries to invest in a new surveillance system, for example, before its general effectiveness has been demonstrated at a country level . even then, technology-based solutions should be integrated with community-based programmes and other existing epidemic preparedness and response systems because surveillance is more effective when standardized among different countries, districts and communities. to this end, suites of guidance and open-access standardized tools are being developed for reporting cases of disease, as well as consent forms, standard operating procedures and training materials , properly validated diagnostic assays and access to quality-assurance panels in public and veterinary health. the rising trend of engaging citizens in data gathering is also welcome-the use of mosquito-recognition apps enables the collection of data far beyond the capacity of routine mosquito surveillance . this way, citizens feed information into the public health system and the feedback loop offers a fast and direct way to provide citizens with details of potential actions that they can take. as well as potentially supporting diagnosis and surveillance , the fast-developing field of genomic epidemiology can yield information to track the evolution of a virus such as ebola during an epidemic , . there will be times when it can detect outbreaks better than traditional epidemiology, illustrating the need to have these tools available in the same toolbox. during the large lassa fever outbreak in nigeria in , real-time genomic sequencing provided clear evidence that the rapid increase was not due to a single lassa virus variant, nor attributable to sustained human-to-human transmission. rather, the outbreak was characterized by vast viral diversity defined by geography, with major rivers acting as barriers to migration of the rodent reservoir . these findings were crucial in containing the outbreak. developing and sustaining the capacity to conduct real-time sequencing with adequate bioinformatics analyses at regional and national levels will be challenging in low-and middle-income countries. moreover, investments in relatively high-tech capacity (such as real-time sequencing) are competing with other, arguably more fundamental needs, such as equipment and training in primary laboratories. political engagement must be nurtured between epidemics: it is not enough to offer technological and laboratory support during a crisis, even with the promise of building capacity, if the political will is not there. however, with proper preparation, and accessible and trusted data sharing and governance mechanisms, laboratories with limited resources may be able to leap-frog into the twenty-first century , . vaccination is one of the most effective public health interventions and innovative strategies for research and development of vaccines, such as using ring vaccination as a trial design during ebola epidemics since - , must be encouraged. at the start of the - epidemic in west africa, vaccine candidates were already in development, based on a long history of preclinical research, although a lot of work was still required to get clinical trials underway in time to be useful . in , when zika was first internationally recognized as a pathogen that could cause birth defects , there was hardly any research and no vaccines in late-stage development. two-and-a-half years later, results from three phase i clinical trials had been reported , although challenges remained for further development. the lack of a profitable market for such products means that pharmaceutical companies lack the incentives to push this work between epidemics. initiatives such as the coalition for epidemic preparedness innovations are attempting to positively disrupt financing models for vaccines against epidemic diseases , and stockpiles of meningococcal vaccine, yellow fever vaccine and oral cholera vaccine are maintained by the international coordinating group to minimize potential delays due to limited manufacturing capacity . similarly, if investigational treatments or vaccines are to be used as part of the response to an epidemic, ethical protocols for managing informed consent and introducing them in clinical settings must be planned in advance with at-risk communities (box ). trial designs precision medicine refers to the use of genomic sequencing to retrace the specific course of a disease in individual patients, with the aim of being able to choose the best treatment option for each person. in public health, the analogous idea of precisely directing the right intervention to the right population is equally appealing. the potential of such an approach has been illustrated by the identification of two areas in the united states in that were at risk of zika transmission . rather than the whole country, or even only florida, being declared at risk, these two areas each measured less than km , and the response focused only on these specific neighbourhoods. by contrast, a campaign against yellow fever, also in , defined risk 'at the level of entire nations'. a broad interpretation of precision public health incorporates many different types of data to increase the power of epidemiology . such data would not only include genomic information, but also satellite imaging, mobile phone data, social media use data and so on. for example, a study published in combined epidemiological surveillance data, travel surveys, parasite genetics and anonymized mobile phone data to measure the spread of malaria parasites in southeast bangladesh . a retrospective analysis of mobile phone call data in sierra leone from showed how it might have been used to assess the impact of travel restrictions on mobility during the ebola epidemic . the principle of selecting the most relevant information from all available data seems within the scope of good epidemiological practice already. the challenge is recognizing and incorporating new types of data when they become available. should be created as soon as the option becomes viable. the essential consideration is how the resulting data can add to previous trials and influence the approach to trials in future epidemics. for example, research during the - ebola epidemic enabled progress on therapeutic agents that are now being trialled in the ongoing outbreak in drc . scientific progress during and between epidemics must be matched by other workstreams, such as the preparation of supply chain logistics and communication with at-risk populations. plans have to be made for a series of future outbreaks, enabling adaptive, multi-year, multi-country studies . similar plans are needed for continual preclinical research to ensure that future vaccine and therapeutic pipelines will be filled. the term 'one health' is used to acknowledge that human, animal and ecosystem health are tightly interconnected and need to be studied in the context of each other (fig. ) . changes in the environment-whether natural or anthropogenic-affect interactions between pathogens, vectors and hosts in multiple and complex ways, making the emergence or decline of endemic, epidemic and zoonotic diseases difficult to predict, while epidemics of animal diseases can challenge a community's access to food. the fact that pools of viruses, bacteria and parasites are maintained in wild and domesticated animals makes surveillance of potentially zoonotic diseases an intrinsic part of one health epidemic planning. many agencies and nations around the world now use prioritization tools such as those developed by the us cdc or the united nations (un) food and agriculture organization (fao) to identify and prioritize zoonotic diseases of concern. an early precedent was a joint consultation on emerging zoonotic diseases by the who, the fao and the world organisation for animal health in . understanding disease ecology in the zoonotic reservoir could potentially lead to ways to predict the risk of human disease, thus providing the basis for smart early-warning surveillance systems. individual countries with limited resources for epidemiological studies and epidemic preparation and response must decide their own priorities. however, infectious diseases do not respect borders. similarly, the interdisciplinary nature of one health means there are several different lenses through which different sectors assess risks and priorities. for one health approaches to work, these multiple perspectives must be taken into account, whether human health or animal health, ecology or social sciences . epidemics do more than cause death and debilitation: they increase pressure on healthcare systems and healthcare workers and draw resources from services not directly linked to the epidemic. this can leave a legacy of distrust between people, governments and health systems, although more-positive outcomes have been found to strengthen relations between communities and public authorities. the full social and economic costs of the ebola outbreak in west africa have been estimated to be as high as us$ billion when including the effect on health workers, long-term conditions suffered by , ebola survivors, and costs of treatment, infection control, screening and deployment of personnel beyond west africa. as healthcare resources became increasingly allocated to the ebola response, hospital admissions fell and deaths from other diseases rose markedly, adding us$ . billion to the estimated cost. such pressure can be withstood in high-income countries with strong health systems, but in low-income countries the pressure can quickly reach a breaking point. ebola killed almost . % of doctors, nurses and midwives in guinea, . % in sierra leone and just over % in liberia . this is compared to mortality between . % and . % of the whole population of these countries. estimates of the effect of this loss on maternal mortality suggest that thousands more women may have died in childbirth each year since the epidemic ended. beyond the tragic deaths of so many healthcare workers, people were less likely to use health services for children or adults during the epidemic, suggesting decreased trust or even fear of healthcare settings . more recently, in some areas affected by the ebola outbreak in drc, the introduction of free non-ebola healthcare led to unprecedented demand. however, healthcare facilities box in , the prevent project received wellcome funding to provide ethics guidance "at the intersection of pregnancy, vaccines, and emerging and re-emerging epidemic threats" . this was in response to the newly recognized association between infection with zika virus during pregnancy and microcephaly in the newborn. developing a vaccine was an obvious route to explore, but many researchers felt that they could not conduct clinical trials with pregnant women because it is generally assumed that the risk to the woman, the fetus or both outweighs any potential benefit. however, as heyrana et al. argue: "preventing pregnant women from participating in clinical trials is well intentioned but misguided." . prevent rapidly developed guidance for including pregnant women and their babies in zika vaccine research , and has since extended their scope to "a roadmap for the ethically responsible, socially just, and respectful inclusion of the interests of pregnant women in the development and deployment of vaccines against emerging pathogens." . integrating ethics in the preparation and response to epidemics does not close off avenues of research; it opens up possibilities and expedites progress. were not given sufficient additional resources to care for the number of people, which may have contributed to nosocomial infections. survivors, too, need to be cared for long after the epidemic is declared over. a cohort of more than , children is growing up in brazil after being born with microcephaly because their mothers were infected with zika during pregnancy. tracking the development of these children increases understanding of the effects of zika infection and helps to define what medical and social support the affected families may need as many of the children will grow up with severe developmental delays . the challenges posed by twenty-first century epidemics are real and changing: future epidemics will be fuelled by conflict, poverty, climate change, urbanization and the broader demographic transition. in our response we must consider epidemics not as discrete events, but rather as connected cycles for which we can prepare, even if we cannot predict specific outbreaks. the challenge is then to choose the right response at the right scale in the right area at the right time. there needs to be a greater emphasis on absorbing and using positive lessons from each episode and avoiding those that led to negative outcomes . the way that we train practitioners and researchers working in all fields relevant to today's epidemic landscape has to change. a modern approach that is capable of characterizing epidemics and the best ways to control them must go beyond a narrow definition of epidemiology that sustains artificial barriers between disciplines. instead, it must be able to integrate tools and practices from a diverse range of established and emerging scientific, humanistic, political, diplomatic and security fields. we believe that such an approach needs to become the norm for the curriculums of schools of public health around the world. as well as training new generations of epidemiologists so that they have the skills, knowledge and networks to recognize and make use of every tool available to help them to do their work effectively, the entire architecture of the response to epidemics has to be adapted. only then will we be able to maintain the comprehensive and effective response-including prevention and research-needed to stop epidemics and protect people's lives, no matter what the circumstances. celebration: william farr ( - )-an appreciation on the th anniversary of his birth rediscovering ignaz philipp semmelweis ( - ) louis-rene villerme ( - ), a pioneer in social epidemiology: re-analysis of his data on comparative mortality in paris in the early th century john snow's legacy: epidemiology without borders this is a wide-ranging meeting report that places modern epidemiology in the context of the past two hundred years and highlights the importance of bringing in new disciplines, remaining open-minded and using those skills across a wider range of societal issues than are traditionally considered public health robert koch and the cholera vibrio: a centenary epidemiology for the uninitiated the epidemiologic transition. a theory of the epidemiology of population change large ebola outbreaks new normal, says who applied epidemiology and public health: are we training the future generations appropriately? managing epidemics: key facts about major deadly diseases ignaz semmelweis, carl mayrhofer, and the rise of germ theory history of vaccination the global eradication of smallpox from emergence to eradication: the epidemiology of poliomyelitis deconstructed natural history of infectious disease p farr's law applied to aids projections epidemiology-is it time to call it a day? global rise in human infectious disease outbreaks pandemics, public health emergencies and antimicrobial resistance -putting the threat in an epidemiologic and risk analysis context how urbanization affects the epidemiology of emerging infectious diseases microbial evolution and co-adaptation: a tribute to the life and scientific legacies of joshua lederberg travel, migration and emerging infectious diseases understanding the link between malaria risk and climate the ebola outbreak, fragile health systems, and quality as a cure health inequalities and infectious disease epidemics: a challenge for global health security historical parallels, ebola virus disease and cholera: understanding community distrust and social violence with epidemics war and infectious diseases: challenges of the syrian civil war pandemic influenza preparedness framework for the sharing of influenza viruses and access to vaccines and other benefits (who how africa can quell the next disease outbreaks the ability to prevent, detect and respond to any health issues will always depend on the local capacity and although international partners can bring complementary expertise and resources, it is the local capacity that is critical; in this article, the authors argue for national investment in public health, health systems, science and local leadership un health chief orders probe into misconduct the inverse care law agenda setting, research questions and funding for biomedical research has historically been led from northern hemisphere countries in an unequal northern-southern hemisphere relationship science granting councils in sub-saharan africa: trends and tensions international federation of red cross and red crescent societies. community-based surveillance: guiding principles conflict and emerging infectious diseases institutional trust and misinformation in the response to the - ebola outbreak in north kivu, dr congo: a population-based survey application of social science in the response to ebola towards people-centred epidemic preparedness and response: from knowledge to action anthropology in public health emergencies: what is anthropology good for? launching a new era for behavioural surveillance integrated biological-behavioural surveillance in pandemic-threat warning systems taking connected mobile-health diagnostics of infectious diseases to the field population mobility reductions associated with travel restrictions during the ebola epidemic in sierra leone: use of mobile phone data national and local influenza surveillance through twitter: an analysis of the - influenza epidemic social media and internet-based data in global systems for public health surveillance: a systematic review isaric. protocols & data tools enhancing early warning capabilities and capacities for food safety real-time, portable genome sequencing for ebola surveillance infection control in the new age of genomic epidemiology genomic surveillance elucidates ebola virus origin and transmission during the outbreak genetic diversity and evolutionary dynamics of ebola virus in sierra leone genomic analysis of lassa virus during an increase in cases in nigeria the integration of genomics and other types of data into the surveillance, prevention and response of epidemics is critical and can help to transform the ability to enhance public health; although the tools are now available, it will be key to ensure that these new approaches are fully integrated and not seen as esoteric ivory tower research, but instead as an essential component of twenty-first century epidemiology, public health and epidemics-the next generation of leaders need to be efficacy and effectiveness of an rvsv-vectored vaccine in preventing ebola virus disease: final results from the guinea ring vaccination, open-label, cluster-randomised trial (ebola Ça suffit!) a seminal study that shows that ring vaccination could be used in the midst of a devastating ebola epidemic and, furthermore, that innovation research can be conducted in an epidemic, trial designs can be adapted without compromising scientific integrity and that ebola can be prevented through vaccination ethical rationale for the ebola "ring vaccination" trial design ebola vaccination in the democratic republic of the congo insights from clinical research completed during the west africa ebola virus disease epidemic gone or forgotten? the rise and fall of zika virus current status of zika vaccine development: zika vaccines advance into clinical evaluation cepi: preparing for the worst the development of global vaccine stockpiles the ebola clinical trials: a precedent for research ethics in disasters it is an ethical imperative to consider and implement research in an epidemic setting as, for many epidemic diseases, it is the only time at which to conduct the research that will inform and improve the lives of the individuals affected during epidemic and to ensure that future generations are better prepared; however, such research is challenging at many levels and it is critical to have an ethical framework that guides the research, places individuals and communites at the heart of the research and facilitates the maximum benefit for the maximum number of people improving vaccine trials in infectious disease emergencies progression of ebola therapeutics during the - outbreak ebola therapies: an unconventionally calculated risk performance of different clinical trial designs to evaluate treatments during an epidemic the one health concept: years old and a long road ahead surveillance of zoonotic infectious disease transmitted by small companion animals prioritizing zoonoses: a proposed one health tool for collaborative decision-making evaluation of the emergency prevention system (empres) programme in food chain crises report of the who/fao/oie joint consultation on emerging zoonotic diseases (who european centre for disease prevention and control. towards one health preparedness the economic and social burden of the ebola outbreak in west africa epidemics cause enormous disruption to countries, regions and the world; however, the focus is often on the epidemic itself, the pathogen and its immediate effect rather than the much broader effect that the epidemic has not only on the healthcare system-which lasts long after the epidemic itself-as routine vaccination programmes often collapse, maternal-child health suffers, and malaria, hiv and tuberculosis clinics and surgery-all aspects of healthcare-are disrupted, but also on the wider society, as mistrust and tension occurs between citizens, authorities and governments, and education, investments, businesses, trade and tourism inevitablely suffer leading to an economic impact that can health-care worker mortality and the legacy of the ebola epidemic patterns of demand for non-ebola health services during and after the ebola outbreak: panel survey evidence from further pieces of evidence in the zika virus and microcephaly puzzle responding to the ebola virus disease outbreak in dr congo: when will we learn from sierra leone? artificial intelligence in medical practice: the question to the answer? artificial intelligence and big data in public health structure of rsv fusion glycoprotein trimer bound to a prefusionspecific neutralizing antibody vaccine platforms: state of the field and looming challenges platform technologies for modern vaccine manufacturing four steps to precision public health precision" public health -between novelty and hype offline: in defence of precision public health mapping imported malaria in bangladesh using parasite genetic and human mobility data pregnant women & vaccines against emerging epidemic threats: ethics guidance for preparedness, research, and response increasing the participation of pregnant women in clinical trials the ethics working group on zikv research & pregnancy. pregnant women & the zika virus vaccine research agenda: ethics guidance on priorities, inclusion, and evidence generation acknowledgements we thank m. regnier at wellcome for editing the manuscript.author contributions all authors developed the scope and focus of the review and contributed to the writing of the manuscript. the authors declare no competing interests. correspondence and requests for materials should be addressed to j.f. reviewer information nature thanks peter byass, sharon peacock and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. reprints and permissions information is available at http://www.nature.com/reprints. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -s ft authors: tatarelli, p.; magnasco, l.; borghesi, m. l.; russo, c.; marra, a.; mirabella, m.; sarteschi, g.; ungaro, r.; arcuri, c.; murialdo, g.; viscoli, c.; del bono, v.; nicolini, l. a. title: prevalence and clinical impact of viral respiratory tract infections in patients hospitalized for community-acquired pneumonia: the vircap study date: - - journal: intern emerg med doi: . /s - - - sha: doc_id: cord_uid: s ft prevalence and clinical impact of viral respiratory tract infections (vrtis) on community-acquired pneumonia (cap) has not been well defined so far. the aims of this study were to investigate the prevalence and the clinical impact of vrtis in patients with cap. prospective study involving adult patients consecutively admitted at medical wards for cap and tested for vrtis by real-time pcr on pharyngeal swab. patients’ features were evaluated with regard to the presence of vrti and aetiology of cap. clinical failure was a composite endpoint defined by worsening of signs and symptoms requiring escalation of antibiotic treatment or icu admission or death within days. patients were enrolled, mean age . ± . years, . % female. patients ( . %) had no viral co-infection while in patients ( . %) a vrti was detected; influenza virus was the most frequently identified ( . %). the two groups were similar in terms of baseline features. in presence of a vrti, pneumonia severity index (psi) was more frequently higher than and patients had received less frequently pre-admission antibiotic therapy (adjusted or . , % ci . – . , p = . ; adjusted or . , % ci . – . , p = . ). clinical failure and antibiotic therapy duration were similar with regards to the presence of vrti and the aetiology of cap. vrtis can be detected in almost a third of adults with cap; influenza virus is the most relevant one. vrti was associated with higher psi at admission, but it does not affect patients’ outcome. community-acquired pneumonia (cap) is a common cause of hospitalisation and one of the main infectious causes of mortality worldwide [ ] [ ] [ ] . in europe, up to % of patients with cap are hospitalized every year and the cap mortality rate reaches . % [ , ] . streptococcus pneumoniae is the main aetiologic agent of cap, being responsible for two thirds of cases, but other pathogens such as haemophilus influenzae, moraxella catarrhalis, chlamydophila pneumoniae, mycoplasma pneumonia, legionella spp. and staphylococcus aureus are frequently involved [ ] . respiratory viruses (which include rhinovirus, metapneumovirus, adenovirus, coronavirus, respiratory syncytial virus, influenza and parainfluenza viruses) have increasingly been recognized as causes of respiratory tract infections [ ] . whilst epidemiology and clinical features of viral respiratory tract infections (vrtis) were mainly explored in children and outpatients [ , ] , a few data show increasing prevalence rates also in hospitalized patients, particularly in elderly and in presence of comorbidities such as asthma, chronic obstructive pulmonary disease or immunosuppression [ ] [ ] [ ] . the recent development and diffusion of nucleic acid amplification tests (naats) on respiratory samples have improved the ability to diagnose multiple upper and lower vrtis, possibly affecting prevalence estimates [ ] . compared to classical microbiological techniques, naats allow earlier and more sensitive diagnosis [ , ] , providing clinical advantages in terms of length of hospitalization, infection control and antibiotics use [ , ] . however, in non-intubated hospitalized adults with cap, lower respiratory tract sampling is not easy to obtain and vrti diagnosis usually relies on nose-throat swabs, affecting the clinical significance of identified viruses [ ] . beyond viral cap, upper vrtis predispose to pulmonary bacterial infections as they cause bronchoconstriction, increase mucus production [ ] [ ] [ ] , decrease the efficiency of muco-ciliary clearance [ ] , damage respiratory mucosa and cause leukocytes dysfunction [ ] . data from both in vitro animal models and clinical experiences have demonstrated that influenza and other vrtis are risk factors for pneumococcal pneumonia, both in adult and paediatric population [ ] [ ] [ ] [ ] [ ] . in spite of improved viral diagnosis and well-known pathophysiology, data on the clinical impact of vrtis on cap are controversial and some authors reported higher mortality rates [ ] . the primary aim of this study was to investigate the prevalence of vrtis in a prospective cohort of adult patients hospitalized at medical wards for cap; the secondary aims were to describe patient's features and outcomes according to the presence of vrti and the aetiology of cap. we prospectively enrolled all adult patients consecutively admitted for cap and tested for vrtis at two medical wards (infectious diseases and internal medicine unit) of policlinico san martino-irccs, genoa, italy, from november to february . patients having nosocomial pneumonia (i.e. developed at least h after hospital admission) and those diagnosed with opportunistic pulmonary infection were excluded. cap was defined as acute lower respiratory tract infection characterized by the presence of two or more signs and symptoms (among fever, cough, dyspnoea, pleuritic pain, crackles or bronchial breath at pulmonary auscultation), associated with at least one among (a) radiological findings (opacity or infiltrate at radiography or computed tomography interpreted as pneumonia by the attending physician), (b) serum levels of inflammatory markers above normal values and (c) neutrophilic leucocytosis, in patients hospitalized no longer than h [ ] . the definitive bacterial aetiology for cap was defined as ( ) identification of an aetiological agent in the blood and/ or pleural fluid and/or bronchoalveolar lavage (with > colony-forming units/ml; ( ) detection of legionella pneumophila spp. or s. pneumoniae antigen in the urine; ( ) positive pcr for legionella spp., m. pneumoniae or chlamydia spp. on pharyngeal swab and/or bronchoalveolar lavage. presumptive bacterial aetiology was defined by positive culture on sputum or positive pcr for h. influenzae, bordetella spp. or s. pneumoniae on pharyngeal swab [ ] in case of vrti, cap was considered of possible viral origin (or mixed origin in the presence of concomitant bacteria identification). antibiotic treatment escalation was defined as enhancing the anti-bacterial spectrum switching to or adding another molecule after at least a full h-course of an initial, inhospital treatment, with the exclusion of targeted treatment. clinical failure was defined as ( ) antibiotic treatment escalation; ( ) worsening of signs and symptoms of pneumonia requiring icu admission; ( ) death within days. in addition, the total time of antibiotic exposure (tta) was calculated. for each patient, the following information was collected: demographic data (i.e. age, gender, country of origin), comorbidities (i.e. concomitant pulmonary or extrapulmonary diseases), chronic therapies for concomitant diseases, influenza and s. pneumoniae vaccination status, antibiotic therapy, results of radiological investigations (i.e. chest x-ray or computed tomography), biochemical tests (white blood cells, c reactive protein and procalcitonin) and outcome. for every patient, the presence of vrtis was ascertained with multiplex real time-polymerase chain reaction (rt-pcr) on pharyngeal swab (allplex tm respiratory panel assay, seegene, seoul, south korea) [ ] . on the same sample, rt-pcr for s. pneumoniae, h. influenzae, c. pneumoniae, m. pneumoniae and b. pertussis was performed. microbiological findings from conventional tests (i.e. blood culture, sputum and broncoalveolar fluid culture, s. pneumoniae and l. pneumophila urine antigen detection, serology for intra-cellular bacteria) were also analysed. given the non-interventional nature of the study, bronchoalveolar lavage for microbiological tests was not systematically performed, but nonetheless available results were collected. curb- score > and pneumonia severity index (psi) class iv-v (i.e. ≥ ) at admission [ , ] were evaluated to assess cap severity at clinical presentation. being an observational study, treatment was decided by the physician in charge according to the usual standard of care. clinical outcome, according to the aforementioned definitions, was recorded for each patient. before analysing the study results, all the cases were reviewed by independent observers who verified their correct classification in the study definitions. statistical analysis was mainly descriptive. patients' features, pneumonia severity scores were evaluated according to the concomitant presence of a vrti and aetiology of cap. categorical variables were compared with chi-square test or fisher's exact test, when applicable. continuous variables were compared with mann-whitney and anova tests. a value of p < . was considered statistically significant. variables significantly (p < . ) associated with the presence of a vrti and the aetiology of cap at previously mentioned tests were evaluated by multivariate logistic regression models. the same statistical tests were used to evaluate if the presence of a vrti, the aetiology of cap and patients' features were associated with outcome variables, such as clinical failure and tta. statistical analysis was carried out using spss. the study protocol was approved by the local (ligurian region) ethic committee. upon enrolment all patients signed a dedicated informed consent. during the study period, patients were admitted for cap. of them, were excluded because of the diagnosis of opportunistic pulmonary infection (n = ) and missed testing for vrtis (n = ). thus, patients with cap and tested for vrti by pharyngeal swab were finally included in our study ( fig. ). patients' characteristics at baseline are reported in table at least one bacterium was documented in patients ( %) with cap. the most frequently identified bacterium was s. pneumoniae (n = , . %), followed by h. influenzae (n = , . %) ( table ). bacterial diagnostic samples considered were sputum (n = ), blood (n = ), urine (n = ) and bronchoalveolar lavage (n = ). nine sputum samples were collected in patients without pre-hospital antibiotic treatment. of the definitive/presumptive bacterial cap, ( %) were found with a concomitant vrtis, thus they were possibly caused by mixed viral and bacterial infection. influenza virus was identified in (virus a = , virus b = ), rhinovirus in , parainfluenza virus in , rhinovirus and parainfluenza virus in case. in cases, viruses were the only pathogens identified and were thus considered as possible causes of cap. the remaining cases were considered of unknown aetiology because neither bacteria nor viruses were isolated. distribution of viral and bacterial pathogens identified is summarized in table . comparing patients with and without vrti, no statistically significant differences were found in terms of age, gender, origin, smoking and alcohol habits, influenza and s. pneumoniae vaccination status, number and type of comorbidities, modes of admission to hospital, curb- score at admission, presence of bacteraemia, pneumonia radiological features or interval between onset of symptoms and hospital arrival (table ) . vrtis were slightly more frequently diagnosed in autumn and winter, although difference was not statistically significant ( . % vs . %, p = . ). in presence of vrtis, psi at admission was more frequently higher than ( / , %, vs / , . %, p = . ) and patients received less frequently pre-admission antibiotic therapy ( / , . %, vs / , . %, p = . ). including both these variables in a multivariate model, they remained statistically associated to the presence of vrti (adjusted or . , % ci . - . , p = . ; adjusted or . , % ci . - . , p = . , respectively) ( table ) . patients' features were also evaluated with regards to the aetiology of cap after exclusion of those patients with unknown aetiology. bacterial (both diagnosed with definitive and presumptive criteria), possibly viral and mixed caps were similar in terms of demographic and radiologic features, immunosuppressive status, curb and psi score at admission (p > . ). overall, patients ( . %) received home antibiotic therapy before hospital admission, whilst the remaining patients received first-line antibiotic treatment only thereafter. in-hospital antibiotic treatment included a beta-lactam in cases ( %). of them, ( %) were treated with piperacillin/tazobactam. antibiotics active against intracellular bacteria were used in cases ( macrolides, of whom in association with beta-lactams, and fluoroquinolones, of whom as monotherapy). with regard to antiviral treatment, patients received empirical treatment with oseltamivir, that was early withdrawn in patients who had no confirmed influenza infection by pcr. ribavirin, after a thorough assessment of risk-benefit analysis, was not administered in any of the patients ( with underlying hematologic malignancies) with respiratory syncytial virus-b infection. overall, clinical success was recorded in patients ( %). among the patients who experienced clinical failure, required antibiotic escalation (involving an anti-mrsa agent in cases), of whom required invasive ventilation and intensive care unit admission. three patients died. of them, the first was a -year old man with s. pneumoniae infection who died in icu, the second a -year old hiv-positive man with respiratory and cardiovascular disease who developed pseudomonas aeruginosa pneumonia and the third a -year old man with myelodysplastic syndrome and chronic kidney disease with a lobar pneumonia and rhinovirus identified on pharyngeal swab. clinical failure was not influenced by the presence of vrti and aetiology of cap. the only variable associated to clinical failure was baseline psi higher than (or . , % ci . - . , p = . ). tta was similar irrespective to the presence of vrti (mean ± sd . ± . versus . ± . days, p = . , in patients with and without vrti, respectively) and aetiology of cap ( . ± . versus . ± . versus . ± . versus . ± . days in bacterial, mixed, viral and unknown aetiology cap, respectively, p = . ). no other collected variables were associated to tta. in the present study, we prospectively analysed the prevalence and the clinical impact of vrtis on patients with cap hospitalized at medical wards. vrtis' prevalence rate we found was as high as . % ( % ci . - . %), but fortunately vrtis did not negatively affect clinical outcome. a recent meta-analysis reported a % ( % ci - %) vrtis' prevalence rate in adults with cap that rose to % ( % ci - %) including only studies where molecular assays were used [ ] . our data confirm that in a real-life setting, including a mixed population of immunocompetent and immunocompromised adults hospitalized for cap, vrtis are highly prevalent. at admission, patients with vrti had more frequently psi score iv-iv than those without. three previous studies (of whom two were retrospective) found an association between vrtis and psi class iv/v, but none of them reported on curb- score [ , , ] . contrary to psi score, we did not find any relationship between curb- and vrtis. reasons why higher classes for risk of severe pneumonia at psi but no at curb- were associated with vrti are unclear. one possible explanation might rely on the higher sensitivity of psi compared to curb- [ ] , even if guidelines do not recommend one score over another for patient's discharge [ ] . additionally, patients with vrti received pre-admission antibiotic therapy less frequently than those without. this finding may be explained in two ways: first, local and national recommendations are against the use of any antibiotic at the onset of a clinically presumed viral respiratory syndrome; second, a viral respiratory syndrome may actually favour the subsequent onset of a cap. interestingly, in our study cap radiological features were not affected by the presence of vrti. this is consistent with observations that, in some instance, viral and bacterial pneumonia may have similar radiological features [ ] it is worth noting that, in spite of higher psi scores in presence of vrtis, we found no association between vrtis and need of antibiotic treatment escalation, icu admission and risk of mortality. other studies investigating clinical outcomes in the same setting found opposite results. yoon [ ] found that preceding vrtis do not affect mortality in adults with pneumococcal cap, while voiriot et al. [ ] reported that mixed bacterial and viral infections are associated with hospital death and/or prolonged mechanical ventilation in icu-admitted cap patients. moreover, some authors used different surrogate endpoints of clinical response, such as length of hospitalization or length of mechanical ventilation. however, these endpoints may be affected by a variety of factors, including the presence of any comorbidity and the development of superinfection, thus not sufficiently clarify the role of vrtis. on the other hand, as escalation of antibiotic treatment is usually the consequence of lack of response to previous treatment, we evaluated the impact of vrtis on the need of antibiotic escalation. the interplay between virus and bacteria, spanning from the damage of respiratory mucosa to affecting the normal immune response, has been well described, in vitro and in vivo, for either influenza virus [ ] [ ] [ ] or other viruses, in particular rsv and metapneumovirus [ , ] in addition, beyond favouring bacterial infection, some respiratory viruses may per se cause pneumonia [ ] . thus, we stratified patients according to the supposed microbiological aetiology and we found that mixed and viral cap had pneumonia severity at admission, radiological findings and outcomes similar to bacterial cap. however, the viruses detected in the pharynx may represent only infection of the upper respiratory tract, while lower respiratory tract specimens may be more reflective of the pathogen causing pneumonia [ ] [ ] [ ] . in our real-life cohort, bronchoalveolar lavage culture was performed only when clinically indicated. as only a few patients experienced clinical failure and required icu admission or antibiotic escalation, invasive procedures were deemed unnecessary in the majority of cases. additionally, % of patients included in our cohort had cap of unknown aetiology, a finding that could be at least partially explained with previous at-home antibiotic treatment before specimens collection. the limited sample size of patients diagnosed with bacterial/viral/mixed cap and the lack of lower respiratory tract sampling in almost all cases prevents any firm conclusion on the clinical presentation and outcome of cap according to the aetiology and deserves further studies. influenza virus was the most frequent viral infection we found in our cohort. while the presence of vrti did not impact on the tta, negative pcr for influenza virus was used for early antiviral treatment discontinuation. indeed, current guidelines recommend universal empirical antiviral treatment in patients with influenza-like illness, whose clinical presentation (i.e. fever plus cough started within the last days) overlaps with that of cap [ ] . on the other hand, vrti identification was not useful to guide antibiotic treatment, as demonstrated by the lack of association with tta. beyond the lack of low respiratory tract specimens, the other limitation of this study is that we enrolled both immunocompetent and immunosuppressed patients. as immunosuppression is a risk factor for more severe infections (depending on the type and intensity), our data may overestimate the risk of clinical failure. of note, two out of three patients who died within days from admission for cap had concomitant immunosuppression. in conclusion, in spite of high prevalence rates, vrtis do not negatively affect the outcome of cap. further studies are needed to evaluate the clinical presentation and outcome of mixed and viral cap with respect to bacterial ones in hospitalized patients who do not need intensive care. funding the authors declare no funding foundation. conflict of interest the authors declare that they have no conflict of interest. ethical approval all procedures performed were in accordance with the ethical standards of the institutional and/or national research committee and with the helsinki declaration and its later amendments or comparable ethical standards. the study was approved 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title: the role of respiratory viruses in children with humoral immunodeficiency on immunoglobulin replacement therapy date: - - journal: pediatr pulmonol doi: . /ppul. sha: doc_id: cord_uid: ej eibd background: the role of viruses in children with respiratory tract infections and humoral immunodeficiencies has hardly been studied. we have evaluated these infections in children with humoral immunodeficiencies who required immunoglobulin replacement therapy, considering their relationship with symptoms, lung function, bacterial co‐infection, and outcomes. methods: we conducted a prospective case‐control study during a ‐year period, including children with humoral immunodeficiencies receiving immunoglobulin replacement therapy. for each patient, at least one healthy family member was included. respiratory samples for viral detection were taken every ‐ months, and in case of respiratory tract infections. symptoms questionnaires were filled biweekly. spirometry and sputum culture were performed in every episode. results: sixty‐six episodes were analyzed in patients (median age years; iqr ‐ ), identifying respiratory viruses ( . %), being rhinovirus the most frequently isolated one ( / ; %). positive viral episodes were associated with clinical symptoms ( % vs %), more frequent antibiotic treatment ( % vs %) or hospital admission ( % vs %) than negative ones. patients with positive viral detection showed impaired lung function, with lower fev and fvc values. conclusions: in our experience, viral respiratory tract infections can cause significant respiratory symptoms and impaired lung function, in children with hid, despite immunoglobulin replacement therapy. these patients could benefit from the monitoring of viral infections, as these may be a gateway for ongoing lung damage. children with severe t-cell immunodeficiencies present impaired clearance of respiratory viruses, and pulmonary complications of viral infections are leading causes of morbidity and mortality in this group of patients. however, the role of respiratory viruses in children with other types of primary immunodeficiency (pid), mainly those with humoral immunodeficiencies (hid) or diseases of immune dysregulation, has hardly been studied. children with hid usually suffer from recurrent bacterial respiratory infections, resulting in progressive bronchiectasis and chronic lung disease. [ ] [ ] [ ] [ ] immunoglobulin replacement therapy (irt) reduces the frequency of these infections. however, despite adequate irt, recurrent respiratory infections are still one of the leading causes of morbidity and mortality in these patients. [ ] [ ] [ ] little data are currently available regarding the susceptibility to respiratory viruses of hypogammaglobulinemic patients receiving irt. , , however, some other viruses have been described which play significant roles in these patients. [ ] [ ] [ ] [ ] human herpesvirus (hhv- ) has been associated with granulomatous/lymphocytic interstitial lung disease (glild) in patients with common variable immunodeficiency (cvid), and enterovirus is a known cause of fatal meningoencephalitis in patients with x-linked agammaglobulinemia (xla). , in addition, there are several recent reports showing an increased susceptibility to viral respiratory infections in adults with cvid receiving irt, - that can contribute to chronic and persistent pulmonary inflammation. , we report, to the best of our knowledge, the first study that analyses respiratory viruses in pediatric patients with predominantly antibody deficiency who required irt, considering their relationship with clinical symptoms and pulmonary function, bacterial co-infection, treatment and outcomes. we conducted a prospective case-control single-center study during year (november to october ) in a national reference unit for pid in madrid, spain. we included patients less than years of age diagnosed with hid who required irt. patients with combined immunodeficiency and/or reduced proliferative responses to mitogens (pha, pwm, cona) were excluded. patients with hid who did not require irt and/or patients receiving immunosuppressive treatments other than rituximab were excluded. committee. informed consents were obtained from patients and parents. for each patient, a healthy control was included, usually one of their parents. respiratory samples (nasopharyngeal aspirate [npa] for patients and nasopharyngeal swab for controls) were obtained every one to three months, coinciding with intravenous immunoglobulin administration or outpatient clinic evaluations, both in the patient and in the control group. other investigations performed during the same visit in the patient group included: spirometry (in patients older than years), spontaneous sputum culture (in patients older than years), igg levels, full blood count (fbc), c-reactive protein, and erythrocyte sedimentation rate (esr). if patients developed respiratory symptoms between visits, additional sputum culture and nasopharyngeal aspirates were performed in the first days after symptom onset, as well as an additional spirometry. in children older than years of age who needed antibiotic for treating respiratory infections, the sputum samples were obtained before starting the therapy. every moment in which a respiratory sample was obtained from a patient was defined as an episode. symptoms questionnaires elaborated ad hoc were filled out systematically biweekly by patients and controls, recording fever, increased respiratory secretions, cough, respiratory distress, sputum three independent rt-pcr assays were performed to detect sixteen respiratory viruses as previously published by our group. [ ] [ ] [ ] influenza a, b, and c viruses were detected by using previously described primer sets only to amplify influenza viruses in a multiplex pcr assay. a second multiplex pcr was used to detect parainfluenza viruses - , human coronaviruses e and oc , enteroviruses, and rhinoviruses (rv). presence of respiratory syncityal virus (rsv) a and b types, human metapneumovirus, human bocavirus, and adenoviruses were investigated by a third multiplex rt-nested pcr-brq method. spontaneous sputum samples were collected using sterile specimen containers and immediately processed or stored at °c until benavides-nieto et al. | processing was feasible. samples were cultured on standard media, and potential respiratory tract pathogens were identified and tested for antimicrobial susceptibility. pulmonary function tests were performed on all patients at study entry and repeated with every sample collection. spirometry was performed according to established guidelines during the study period, patients with predominantly antibody deficiency were included ( girls and boys). their main immunological diagnoses are reported in table . the median age of patients was years (iqr - ), and only two of them were younger than years. eleven patients were receiving intravenous irt and three patients, subcutaneous irt. immunological status at inclusion is described in table . eighteen healthy adult family members (mother and/or father) of the patients were included as healthy control subjects (at least one control per case included). on the other hand, other authors have hypothesized that persistent and recurrent viral respiratory infections could adversely affect the microbiome, leading to an increased bacterial density in children's nasopharyngeal tract, predisposing to sinopulmonary bacterial infections. , as other authors, we recommend screening for viral infection in children with hid and acute respiratory symptoms to avoid unnecessary antibiotic treatment. however, viral testing is routinely performed only in children with severe combined immunodeficiency (scid). , regarding hid, protocols present a great deal of variation across europe in the management of lung complications. there is an urgent need for consensus guidelines on how to monitor lung complications and how to treat respiratory infections in hid patients. not all our patients were typical hid such as xla or cvid. three of our cases were scid patients who had received a hematopoietic cell transplant, but presented persistent b-cell lymphopenia and hypogammaglobulinemia. although most children with scid recover cellular immunity year after transplantation, many patients are likely to be on irt for a considerable period until full reconstitution of b-cell immunity has been achieved. , b-cell immune reconstitution and its consequences regarding viral respiratory infections have not been deeply investigated in these patients. our data support that these patients are also prone to viral respiratory infections, which impact lung function and lead to antibiotic consumption. another patient was an ad-hies. regarding humoral immunity, many patients with ad-hies require immunoglobulin infusions due to memory b-cell lymphopenia and decline in specific antibody titers. recurrent respiratory tract infections have been described in these children, and more than % of them develop pneumonia. although bacteria are detected in % of respiratory exacerbations, viral pneumonia has also been reported. lung sequelae in these patients are frequent, mainly as bronchiectasis. thus, monitoring viral infections in these children may be important. one patient in our series suffered from apds . while the disease is heterogeneous, respiratory infections and their complications are frequent and often severe. the spectrum of pathogens is highly reminiscent of other primary antibody deficiency syndromes such as cvid. coulter et al seem to play a role in children with hid, as has also been described in adults. , further studies are needed to improve our knowledge about the impact of respiratory viral infections, their prevention and management in children with irt, with the aim of avoiding short-and longterm consequences. clinical features before hematopoietic stem cell transplantation or enzyme replacement therapy of children with combined immunodeficiency lung disease in primary antibody deficiency subclinical infection and dosing in primary immunodeficiencies immune deficiency: changing spectrum of pathogens respiratory infections and antibiotic usage in common variable immunodeficiency virus shedding after human rhinovirus infection in children, adults and patients with hypogammaglobulinaemia recurrent and persistent respiratory tract viral infections in patients with primary hypogammaglobulinemia granulomatous-lymphocytic interstitial lung disease (glild) in common variable immunodeficiency (cvid) enteroviruses in x-linked agammaglobulinemia: update on epidemiology and therapy 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findings from a european survey and subclinical infection working group immune reconstitution and survival of scid patients post-hematopoietic cell transplant: a pidtc natural history study the kinetics of early t and b cell immune recovery after bone marrow transplantation in rag- -deficient scid patients autosomal dominant stat deficiency and hyper-ige syndrome: molecular, cellular, and clinical features from a french national survey respiratory manifestations of the activated phosphoinositide -kinase delta syndrome clinical spectrum and features of activated phosphoinositide -kinase δ syndrome: a large patient cohort study escmid study group for infections in compromised hosts (esgich) consensus document on the safety of targeted and biological therapies: an infectious diseases perspective (agents targeting lymphoid cells surface antigens [i]: cd , cd and cd ) the role of respiratory viruses in children with humoral immunodeficiency on immunoglobulin replacement therapy to claire marsden for the revision of the english language. this study has been partially supported by fis (fondo de investigaciones sanitarias-spanish health research fund) grant no. pi ciii/ . the authors declare no conflict of interest. key: cord- -ba wq t authors: sias, catia; salichos, leonidas; lapa, daniele; del nonno, franca; baiocchini, andrea; capobianchi, maria rosaria; garbuglia, anna rosa title: alpha, beta, gamma human papillomaviruses (hpv) detection with a different sets of primers in oropharyngeal swabs, anal and cervical samples date: - - journal: virol j doi: . /s - - -x sha: doc_id: cord_uid: ba wq t background: recent studies have shown a -fold increase of oropharyngeal cancer in the presence of hpv, while α-hpv detection seems to be rare in oral cavity in comparison to anal or cervical district, many novel β and γ types have been isolated in this anatomical site suggesting a wide tropism range. currently, there are no guidelines recommending hpv oral cavity screening as a mandatory test, and it remains unknown which hpv types should be included in hpv screening programs. our goal was to assess hpv prevalence in oropharyngeal, anal, and cervical swabs using different sets of primers,which are able to amplify α, β, γ hpv types. methods: we analysed the presence of hpv dna in oropharyngeal (n = ), anal (n = ), cervical specimens (n = ) from hiv positive and negative patients using fap / and my / primers. all untyped strains were genetically characterized through pcr amplification and direct sequencing of partial l region, and the resulting sequences were classified through phylogenetic analysis. results: hpv prevalence was . % in oropharyngeal swab samples, including infections with multiple hpv types ( . %). hpv prevalence in this anatomical site was significantly associated with serostatus: . %in hiv positive and . % in hiv negative patients (p < . ). unclassified types were detected in specimens. in our analysis, we did not observe any difference in hpv (α, β, γ) prevalence between men and women. overall, β species were the most frequently detected . %. when using anal swabs, for hiv positive patients, β genus prevalence was % and γ genus was . % including unclassified types. in cervical samples from hiv positive women ( hpv negative and positive by my / pcr), only one sample was positivite for β( ) species ( . %) using fap primers. six of the untyped strains clustered with sequences from species , , , , of γ genus. four sequences remained unclassified. finally, β and γ hpv prevalence was significantly lower than their respective hpv prevalence as identified by the luminex system in all anatomical sites that were analyzed in previous studies. conclusion: this study provides new information about viral isolates present in oropharyngeal site and it will contribute to improve the monitoring of hpv infection. electronic supplementary material: the online version of this article ( . /s - - -x) contains supplementary material, which is available to authorized users. human papillomavirus (hpv) persistent infections are considered the primary cause of ano-genital cancer, where greater than % of cervical cancer and more than % of anal cancer contain hpv dna [ , ] . throughout the past years, several studies have suggested the link between hpv infection and other epithelial cancers, including cutaneous and oropharyngeal cancers. oropharyngeal cancers are often referred to as "head and neck cancer" (hnc), and mainly include squamous-cell carcinoma which occurs in the oral cavity, base of tongue, tonsils, adenoids pharynx and larynx. recent studies on hpv infection in oral exfoliated cells have shown that there is a -fold higher risk for oropharyngeal cancer in the presence of virus [ ] [ ] [ ] . the highest prevalence (up to %) for these hpv associated cancers has been found in the tonsillar cancers sub-set [ ] [ ] [ ] . since hpv presented a global prevalence in oropharyngeal squamous cell carcinoma (opscc) and oral squamous cell carcinoma (oscc) of . and . % respectively [ ] , iarc categorized this genotype as a risk factor with for the pathogenesis of both cancers [ ] . additionally, other hpv types have also been linked with these cancers, including hpv , , and [ ] including β and ɣ types [ ] . furthermore, while α-hpv detection seems to be rare in oral cavity in comparison to anal or cervical districts [ ] , many novel β and γ types have been isolated in oral cavity [ ] suggesting a wide tropism range of these genera [ ] . at the same time, different studies on hpv prevalence provided contradicting result for β and ɣ types not only in oropharyngeal site, but also in cervical and anal anatomical sites [ ] [ ] [ ] [ ] [ ] [ ] . to date, there are no guidelines recommending hpv oral cavity screening as a mandatory test, and no hpv dna test has been approved for hpv detection in oral cavity. in addition, it remains unknown which hpv types should be included in oral cavity screening. agalliou [ ] highlighted the link between β, γ hpv types and oral cancers, assessing the presence of hpv types which are not generally detected with commercial assays in cervical cancer screening. even though different methods have been used to detect novel hpv types, the association between new β and γ hpv types and oral cavity cancer has not yet been established. in this study, we analyzed the presence of hpv dna in oral, anal, and cervical specimens collected from hiv positive and hiv negative individuals, living in the same geographic area (regione lazio) by using my / [ , ] fap / primers [ ] . these primers, both targeting highly conserved region within l orf, are considered broad range pv primers and allow the detection of the great majority of already known and officially recognized hpv types. my / had been used in α hpv detection in cervical sites, where they showed good sensitivity in alpha hpv types amplification [ ] , while fap primers officially recognized hpv from α-pv, β-pv, and ɣ-pv genera, and might detect potentially new types [ ] [ ] [ ] [ ] . this property is particularly relevant, since ɣ and β papillomaviruses have been already identified in several anatomic sites [ , ] . granted that that hpv prevalence is significantly higher among hiv infected people and at multiple anatomic sites [ ] [ ] [ ] [ ] we included both hiv positive and hiv negative people in our study in order to also verify whether β and ɣ hpv positivity is influenced by host immune status. this is a retrospective study carried out at laboratory of virology inmi l spallanzani on residual oropharyngeal samples collected for respiratory virus detection, anal and cervical swabs collected for hpv testing in diagnostic routine. the hospital ethics committee approved the protocol. date of birth, date of swabs sampling, hiv serostatus were recorded from institutional database. in hiv positive patients, the most recent cd t cell count (± month from the date of sample collection) and hiv rna viral load (± month from the date of sample) with a detection limit of cp/ml (abbott molecular inc., des plaines, il, usa) were used to correlate clinical features and hpv positivity. oropharyngeal swabs -oropharyngeal samples were collected as following: the nylon-flocked tip was rotated - -times against right and left buccal mucosa, palatine, tonsils, upper and lower pharynx area. the swab was then plunged and stirred in . ml dmem medium with streptomycin and ampicillin. specimens were refrigerated within - h after collection until processing. anal swabs -we retrospectively analysed anal swabs previously tested by my / primers and typed by clart hpv clinical array or sanger sequencing (see hpv detection and typing section, below). one hundred samples belonged to hiv positive women ( hpv positive by my / pcr and negative by my / pcr), anal swabs were collected from men who have sex with men (msm). all msm specimen resulted positive in my / pcr. cervical swabs -a total of cervical swabs ( hpv negative and hpv positive, assigned by my / pcr) were considered in this study. all samples belonged to hiv positive women. general characteristics of hiv infected patients are presented in additional file : table s . oropharyngeal swabs were removed from the medium which we divided in two parts: half part was used for detecting respiratory viruses panel (influenza-a, b, rsv, rhinovirus, coronaviruses, metapneumovirus, adenovirus) and the other half was employed in testing hpv dna, having been stored at − °c until use. before nucleic acid extraction, all specimens were pre-treated. briefly, μl of clinical material was digested with μl proteinase k solution (qiagen, hilden germany) and lysed with al lysis buffer (qiagen, hilden, germany) at °c for min. nucleic acid extraction was done with a magnetic bead-based automated platform (qiasymphony, hilden, germany) in accordance with the manufacturer's instructions. nucleic acids were eluted in μl of ave elution buffer (qiagen, hilden, germany). nucleic acid from anal and cervical swabs were extracted as described elsewhere [ , ] . samples that were β-globin negative were excluded from the study [ , ] . ten μl of eluted nucleic acids were employed for evaluating the presence of hpv types by using my ( 'cgtccmarrggawactgatc ') and my ( 'gcmcagggwcataayaatgg ') [ , ] pcr and faststart dna polymerase (roche diagnostics gmbh, mannheim, germany). the pcr assay conditions were: °c for min, then cycles (denaturation °c/ s, annealing °c/ s, and extension °c/ min). one last step for extension was employed at °c for min. fifteen μl of the pcr products were mixed with loading solution in . % agarose gel electrophoresis stained by ethidium bromide and run for min at v. hpv genotyping of positive samples was conducted using genomica clinical hpv array (genomica, madrid, spain). clart hpv clinical array hpv is able to detect: , , , , , , , , , , , , , , , , , , , all adequate samples were retested using fap ( 'taacwgtiggicayccwtatt ') and fap ( 'ccwatatcwvhcatitciccatc ') primers [ ] able to detect α, β and γ hpv types. faststart dna polymerase were used in this assay condition: min at °c, cycles (denaturation °c/ s, annealing °c / s, and extension °c min) followed by a final extension step at °c for min. pcr products were also run on agarose gel. positive samples with fap set of primers and those positive with my / pcr protocol, but negative in genomica typing assay were purified and sequenced on the automated abi prism instrument, by using a bigdye terminator cycle sequencing kit (applied biosystems, warrington, uk). hpv sequences were aligned using mafft [ ] using the l-ins-i method. after visual inspection using seaview [ ] we removed sample sequence q and q were excluded from the alignment because they represented mixed infection and the sanger sequence interpretation was not optimal. reference sequences hm _hpv and gu _hpv were also excluded from the alignment. then, we further implemented gblocks [ ] to remove ambiguous positions using the least stringent options. to infer a maximum likelihood tree, we implemented raxml [ ] using a gtrcat model, with bootstrap replicates. tree visualization was achieved using figtree [ ] . finally, pairwise similarity matrices were constructed using blast. oropharyngeal swabs-a total of oropharyngeal swabs with β-globin positive signal were considered in this study. hpv prevalence was . % ( / ), including infections with multiple hpv types ( / , . %) ( table ). my / pcr gave positive results in samples. however, a blast search against genbank indicated that amplified fragments were identical to human sequences ( / , %), thus true hpv positive rate was estimated at . %. out of , three samples were identified as α and ten as β types. alpha-types were detected in two hiv positive patients ( / , . %), and in hiv negative patient. hpv α-types, and , were detected in hiv positive subjects ( / , . %) after being typed by clart hpv clinical array, while hpv was amplified in hiv negative subject ( / , . %); it was typed by sanger sequencing. all β hpv types belonged to β species (table ) , and hpv was the most frequent type ( / ), both in hiv positive and hiv negative patients; samples were identified as positive by fap pcr. no specific amplicons were observed. seven fap-detected hpv types were found in samples that also gave positive results with my /my pcr (table ) . types β were the most represented (n = ), while one sample was identified as γ type (hpv , γ ). additionally, one mixed infection (q ) and untyped hpv strains were also detected. among β species, the most frequent types were hpv (n = , . %) and hpv (n = , . %). three multiple infections harboured β and β types, whereas β types and untyped strains were observed in other multiple infections. no multiple infection harboured α with β or γ species (table ) . overall, β species were most prevalent (n = / , . %). hpv distribution differed significantly when the number of hpv β, γ-types were considered in hiv positive and hiv negative subjects: ( / , . %) vs ( / , . %) respectively (chi square test, p < . ). hiv positive people with hpv in oropharyngeal swabs showed a mean cd t cell count of . ± . . among hiv positive patients with detectable hpv, / had no detectable hiv rna in plasma and the other patients (n = ) showed a mean rna copies/ml . ± . . hpv prevalence rates were compared for statistical significance, using a chi squared test, in men and women both hiv positive and hiv negative. however, no difference was observed (chi square test, p > . ). anal swabs-eighty-six anal swabs from msm resulted positive by my / pcr, and anal swabs from women ( hpv positive by my / pcr and negative by my / pcr) were retested with fap primers. among msm anal swabs, samples showed single hpv infection by clart array testing, and patients harboured multiple hpv infection with at least one high risk (hr) type. in . % of samples we observed a clinically evident anal pathology, including subjects with anal intraepithelial neoplasia (ain) ii, patients with ain i. among hpv positive women, harboured a single infection (ain ii, n = ; ain i, n = ); atypical squamous cells of undetermined significance (asc-us), n = ; normal, n = ) and samples showed hpv multiple infection, all with at least hr type ( %) (ain ,n = ; ain i, n = ; normal cytology, n = ). hpv type was undetermined in one sample. among single infection with ain i, one specimen was infected with a low risk (lr)type (hpv ). nineteen anal swab specimens were identified as hpv positive by fap primers detection (female anal swabs, n = ; male anal swabs, n = ) ( table ). overall α-type strains were detected in samples with fap primers which were not previously detected by my / primers. all but one α-type strains were also detected with α mucosal hpv multiple infection typed with clart hpv clinical array which included at least one hr type. hpv β types (hpv and hpv ) were found in two specimens co-infected with α hpv types; specimens harboured γ hpv types (hpv and hpv ), both associated with type α hpv multiple infections. all fap untyped strains (n = ) were found in anal specimens infected by α types with at least one high hr type. considering cytological aspects, / samples, which resulted positive with fap primer pcr, had ain (i, ii) lesions, while / samples showed normal cytology (table ) . among anal swabs from females (n = ), only samples ( . %), previously tested my / hpv positive, resulted hpv positive with fap primer (hpv , α species; hpv , α species; hpv , α species). no untyped hpv strains were observed among female samples. overall, β genus prevalence was % and γ genus was . % including untyped isolates. mean of cd t cell count was . ± . cells/μl. overall / patients ( . %) were under antiretroviral therapy (art). all patients without art had a cd t cell count > / μl. current art use was not associated with risk of β and ɣ infection (chi square test, p > . ). cervical swabs-twelve specimens showed a single hpv infection, and had multiple infections by clart hpv array; / samples harboured at least hr genotype. cytology findings were available for specimens already resulted positive by clart hpv clinical array: had normal cytology, were asc-us or low grade squamous intraepithelial lesion (lsil), harboured hpv single infections, showed hpv multiple infections, and were high grade squamous intraepithelial lesion (hsil). all my / hpv negative women (n = ) showed normal cytological findings. eleven/ cervical samples resulted fap positive ( . %). however, only hpv types were not previously detected by my / primers. five samples harboured α types (hpv , n = α ; hpv , n = , α ; hpv α , n = ), and only one specimen gave positivity for β (hpv ) ( . %). the mean cd t cell count was ± cells/μl. ninety-six percent of women were receiving art per who guideline of starting art at cd t cell count < cell/mm . the woman harboring β hpv strain was under art. she had a cd t cell count of cells/μl and hiv rna viral load was not detected. overall cervical and anal β and γ type positivity was not related to immune status as measured by cd t cell count (see table ) and art treatment. furthermore, the immune-re constitution could potentially prevent the persistence of β and ɣ types in anal and cervical sites or alternatively, β and ɣ types might show to have a less tropism to these anatomical sites. for the phylogenetic analysis of our untyped hpv samples we used partial cds sampled sequences from major capsid protein l fap pcr products and reference sequences. our reference sequences represent classified and untyped hpv sequences (see table ). according to our phylogenetic analysis, of our sampled sequences (q , q , q , q , q and q ) clustered with previously classified reference sequences from species , , , and -γ genus. sequences q , q , q and q remained unclassified while showing great similarity with unclassified genomic regions af (fams ), kp , and af . respectively (see fig. and table ). in hnc, particularly oropharyngeal cancer, the true prevalence and involvement of hpv in the carcinogenetic process is still unknown. in fact, many studies report low prevalence of hpv obtained with systems used for the determination of hpv in cervical cancer screening (specific for α types), while other authors found higher prevalence when they used a platform able to detect hpv types belonging to genera other than α genus [ ] both in oral cavity and in oral cancer [ , ] . agalliu recently found a correlation between the infection of some β, ɣ hpv types and oscc [ ] . furthermore, a recent meta-analysis provided additional evidence of the involvement of β hpv types in the development of scc in immunocompetent individuals [ ] . additionally, several mechanistic studies have consistently showed that e and e from several β hpv types are able to target cellular proteins, such as p and prb, and to deregulate fundamental events involved in cellular transformation [ ] . thus β and ɣ hpv types detection could be included in a hpv screening test for oral cancer prevention, while a correct and suitable detection of hpv infection is becoming a priority. currently, there are few studies that compare the hpv prevalence obtained using the protocols, which are normally for cervical cancer screening with those obtained using methods that are able to detect hpv types belonging to genera other than α or new ones. in this study, in order to assess the impact of pcr system in hpv prevalence determination, we carried out the detection of hpv with my / primer set, used in several genotyping molecular assays (for example linear array hpv genotyping test, roche molecular system, ca usa; clart hpv clinical arrays, genomica, madrid, spain,) and fap primers able to detect α, β, γ hpv types in several specimens. in oropharyngeal swabs samples, my / primer set was able to detect . % ( / ) of hpv positive samples, while fap set of primers detected the presence of hpv in / samples ( . %). beta hpv was the main genus detected with a prevalence of . % in hpv positive samples. this prevalence was in agreement with bottalico et al. that used fap primer and my / for detecting hpv in oral swabs [ ] . hpv was the most frequent β type and hpv the main type among β species as described by bottalico [ ] . hpv , which was reported as the main β type in forslund study [ ] , was absent in our samples. hr, namely hpv , was observed only in one specimen from an hiv positive subject, whereas hr hpv were frequently observed in bottalico study group [ ] . the low frequency of hr could be imputed to immune status of the patients. our patients were under haart therapy and most of them had a cd t cell count > /μl. no severe compromise of the immune-system could have limited a persistence of hr in this anatomical site. unfortunately, no information was given in the bottalico paper on patient cd t cell count, and this fact did not allow a correct comparison of these results. according to previous data reported by forslund et al. [ ] , no difference in hpv prevalence was observed among men and women suggesting that host specific factors could contribute to hpv persistence and neoplasia development. gamma genus represented . % of hpv positive samples. to note, / γ strains were untyped. phylogenetic analysis revealed that samples (q and q ) belonged to γ species, while q to γ species and q to γ species. interestingly, the oral untyped genotypes fell in different genera and cluster with the anal untyped strains, suggesting a specific tropism of these gamma types for oral mucosa. gamma and were the most representative γ species described by bottalico in oral rinse and agalliu in hnc, suggesting their potential involvement in oscc development. unlike forslund and hampras [ , ] , we did not identified any β , β , β hpv types. this may be due to the types of biological samples that were used or to differences in the efficiency of extraction methods that were applied, which could have influenced the outcome of pcr [ ] . finally, differences in results might also be explained by the geographical distribution of hpv types. in general, differences in hpv β and γ types and their prevalence were observed by the luminex platform. according to him study [ ] , among β genotypes, hpv and hpv were the prevalent genotypes. while, among β strains, hpv was the most represented,and it was never detected in our samples. interestingly hr hpv was also the main β type observed also in moscicki study, whereas hpv represented the main β type [ ] . a different pattern of β types could be related to a different cellular input as described in a previous study [ ] , where several β hpv types showed essentially increasing prevalence with increasing dna input. beta hpv types , , , , , , and showed increased prevalence only in higher dna input groups [ ] . this may impose compromises in comparisons between studies; for example, our hpv negative result could be explained by an insufficient dna input in the pcr assay. further research is needed to establish whether there is an influence between cell number input and hpv β detection in oropharyngeal anatomical site and which cut-off would be suggested to avoid false negative results. possible type-specific differences in sensitivity to cellular input have to be evaluated in wider studies. if they are confirmed, they may be explained by the different sensitivities in detection systems and/or in the fig. a maximum likelihood phylogenetic analysis was inferred on unknown collected samples (bold) and reference sequences using the gtrcat model. out of unknown samples, sequences were clustered within previously classified hpv species lineages. bootstrap supports lower than % were excluded from the tree. genbank accession number: mh -mh different viral load spectra. we detected α strains in anal site fap primers that had not been typed by genomica, and β types, hpv and hpv , that had not been reported among oropharyngeal swabs. Βeta types frequency was sensibly lower to that found by luminex system reported by torres et al. [ ] which found . % of β types in anal swabs from hiv positive msm and . % among hiv negative msm. in this study, hpv and hpv were the prevalent types among β species, while hpv and hpv were the most frequently observed β types. among γ genus, the γ species was the most prevalent in both msm hiv positive and -negative groups, while we observed only γ untyped strains. donà reported higher prevalence of β ( . %) and γ types ( . %) in a group of msm similar to ours using the luminex system [ ] . some hypothesis could put forth to explain these data: i) the luminex system could be much more sensitive than the fap system, or ii) that cross-reactivity could occur in β and γ types detection when α multiple infections are present. in literature, cross-reactivity was also observed among α genotypes. preisler observed cross-reactivity both in lr and hr genotypes using hc , cobas, and aptima assays, despite what manufacturer claims: about % of hpvdna results in primary screening accounting for cross-reactivity, regardless of the assay [ ] . to obtain improved analytical and clinical performance, cross-reactivity studies should be focused, since this aspect could influence the effectiveness in a future head neck hpv cancer screening. in cervical swabs the fap primers mainly showed the presence of α genotypes (hpv , α ; hpv , α ) which were not detected by clart hpv clinical array/ my - primers and β type (hpv ). this low prevalence of β types confirms the data reported by bottalico et al. [ ] , which found a prevalence of % of the β types and % of the γ types in the cervical samples, emphasizing a weak β and γ type tropism towards the female genital mucosa. however, the genotypes hpv and hpv , detected by bottalico in cervical specimens, were never observed among our cervical samples, suggesting a different geographic distribution of hpv β and γ type similarly to that described for α genus. conversely, these findings were in disagreement with those obtained by luminex system, as described in previous study [ ] . a quantitative detection of the viral load in hair follicles demonstrated that the β genotype copy number was considerably lower than that reported for mucosal high-risk types from α genus in cervical tissue [ ] . thus, only comparable viral load detection studies with different methods and the potential for multiple infections detection could explain these differences in prevalence of β types, which are reported in literature. overall, our results confirmed a prevalence of > % for hpv strains in the oropharyngeal anatomical district. the genus β and γ were predominant when the analysis was carried out with fap primers. the discrepancy on the prevalence and hpv types reported in other studies [ ] [ ] [ ] ] seems to be due to the detection system: highest prevalence was always obtained with the luminex system. different sensitivity and specificity of hpv detection methods were also a problem in the determination of α hpv types, as described in a systematic review where approximately - % of all positive results showed discordance [ ] . a global proficiency program like labnet for α types in cervical hpv infection surveillance programs should be planned also for α, β and γ hpv type detection in oropharyngeal samples [ ] . standardizing methods for oral sample collection and hpv detection would ensure comparability between different detection methods in oral cavity samples [ , ] . in addition, considering that γ genus has been growing rapidly (currently γ types have been identified) surpassing α and β genera, and that % of the new types deposited in the hpv center within belonged to γ genus [ - ], our data reinforces the relevance of using primer sets able to detect a wide spectrum of hpv strains including β and γ types as well as new genotypes for hpv detection in the oropharyngeal anatomical site. this seems to be a crucial point since the meta genomic approach applied in some analysis [ ] should be used with caution, taking in account the possibility of having an overestimation of hpv types [ ] and requiring a confirmation of positivity by the sanger method. additional file worldwide burden of cancer attributable to hpv by site, country and hpv type casecontrol study of human papillomavirus and oropharyngeal cancer associations of oral α-, β-, and γ-human papillomavirus types with risk of incident head and neck cancer detection and quantitation of human papillomavirus (hpv) dna in the sera of patients with hpv-associated head and neck squamous cell carcinoma hpv infections and tonsillar carcinoma human papillomavirus (hpv) in head and neck cancer. an association of hpv with squamous cell carcinoma of waldeyer's tonsillar ring incidence of human papillomavirus (hpv) positive tonsillar carcinoma in stockholm, sweden: an epidemic of viral-induced carcinoma? hpv dna, e /e mrna, and p ink a detection in head and neck cancers: a systematic review and meta-analysis preventable exposures associated with human cancers prevalence of microsatellite instability, inactivation of mismatch repair genes, p mutation, and human papillomavirus infection in korean oral cancer patients oral human papillomavirus in healthy individuals: a systematic review of the literature characterization of three novel human papillomavirus types isolated from oral rinse samples of healthy individuals the oral cavity contains abundant known and novel human papillomaviruses from the betapapillomavirus and gammapapillomavirus genera betapapillomaviruses in the anal canal of hiv positive and hiv negative men who have sex with men beta and gamma human papillomaviruses in the anal canal of hiv-infected and uninfected men who have sex with men prevalence of beta and gamma human papillomaviruses in the anal canal of men who have sex with men is influenced by hiv status cervical infection with cutaneous beta and mucosal alpha papillomaviruses prevalence and epidemiologic profile of oral infection with alpha, beta, and gamma papillomaviruses in an asian chinese population the use of polymerase chain reaction amplification for the detection of genital human papillomaviruses identification and assessment of known and novel human papillomaviruses by polymerase chain reaction amplification, restriction fragment length polymorphisms, nucleotide sequence, and phylogenetic algorithms a broad range of human papillomavirus types detected with a general pcr method suitable for analysis of cutaneous tumours and normal skin molecular methods for identification and characterization of novel papillomaviruses diversity of human papillomaviruses in skin lesions multiple oral carcinomas associated with a novel papillomavirus in a dog beta and gamma human papillomaviruses in anal and genital sites among men: prevalence and determinants prevalence and risk factors for anal human papillomavirus infection in human immunodeficiency virus (hiv)-positive and high-risk hiv-negative women relationship between prevalent oral and cervical human papillomavirus infections in human immunodeficiency virus-positive and -negative women high diversity of alpha, beta and gamma human papillomaviruses in genital samples from hiv-negative and hiv-positive heterosexual south african men an anal cancer screening program for msm in italy: prevalence of multiple hpv types and vaccine-targeted infections frequency and multiplicity of human papillomavirus infection in hiv- positive women in italy oral human papillomavirus dna detection in hiv-positive men: prevalence, predictors, and co-occurrence at anal site enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia mafft: a novel method for rapid multiple sequence alignment based on fast fourier transform seaview and phylo_win: two graphic tools for sequence alignment and molecular phylogeny selection of conserved blocks from multiple alignments for their use in phylogenetic analysis raxml-vi-hpc: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models beta-hpv types in patients with head and neck pathology and in healthy subjects association between β-genus human papillomavirus and cutaneous squamous cell carcinoma in immunocompetent individuals-a metaanalysis the biology of beta human papillomaviruses the nasal mucosa contains a large spectrum of human papillomavirus types from the betapapillomavirus and gammapapillomavirus genera prevalence and concordance of cutaneous beta human papillomavirus infection at mucosal and cutaneous sites analysis of the effect of dna purificationon detection of human papillomavirus in oral rinse samples by pcr prevalence and transmission of beta and gamma human papillomavirus in heterosexual couples prevalence and multiplicity of cutaneous beta papilloma viruses in plucked hairs depend on cellular dna input crossreactivity profiles of hybrid capture ii, cobas, and aptima human papillomavirus assays: split-sample study beta-papillomavirus dna loads in hair follicles of immunocompetent people and organ transplant recipients concordance of beta-papillomavirus across anogenital and oral anatomic sites of men: the him study concordant testing results between various human papillomavirus assays in primary cervical cancer screening: systematic review continuing global improvement in human papillomavirus dna genotyping services: the and hpv labnet international proficiency studies detection of oral hpv infection -comparison of two different specimen collection methods and two hpv detection methods oral human papillomavirus infection incidence and clearance: a systematic review of the literature international standardization and classification of human papillomavirus types towards quality and order in human papillomavirus research hpviewer: sensitive and specific genotyping of human papillomavirus in metagenomic dna we are grateful to all member of virology laboratory inmi l. spallanzani for sample collection. we thank dr. mariana badescu for english editing. the authors declare that they have no competing interests. key: cord- -wo vq authors: khanna, varun; li, lei; fung, johnson; ranganathan, shoba; petrovsky, nikolai title: prediction of novel mouse tlr agonists using a random forest approach date: - - journal: bmc mol cell biol doi: . /s - - - sha: doc_id: cord_uid: wo vq background: toll-like receptor is a key innate immune receptor involved in detecting infectious diseases and cancer. tlr activates the innate immune system following the recognition of single-stranded dna oligonucleotides (odn) containing unmethylated cytosine-guanine (cpg) motifs. due to the considerable number of rotatable bonds in odns, high-throughput in silico screening for potential tlr activity via traditional structure-based virtual screening approaches of cpg odns is challenging. in the current study, we present a machine learning based method for predicting novel mouse tlr (mtlr ) agonists based on features including count and position of motifs, the distance between the motifs and graphically derived features such as the radius of gyration and moment of inertia. we employed an in-house experimentally validated dataset of single-stranded synthetic odns, to compare the results of five machine learning algorithms. since the dataset was highly imbalanced, we used an ensemble learning approach based on repeated random down-sampling. results: using in-house experimental tlr activity data we found that random forest algorithm outperformed other algorithms for our dataset for tlr activity prediction. therefore, we developed a cross-validated ensemble classifier of random forest models. the average matthews correlation coefficient and balanced accuracy of our ensemble classifier in test samples was . and . %, respectively, with the maximum balanced accuracy and matthews correlation coefficient of . % and . , respectively. we confirmed common sequence motifs including ‘cc’, ‘gg’,‘ag’, ‘cccg’ and ‘cggc’ were overrepresented in mtlr agonists. predictions on randomly generated odns were ranked and the top odns were synthesized and experimentally tested for activity in a mtlr reporter cell assay, with of the selected odns showing high activity, confirming the accuracy of the model in predicting mtlr activity. conclusion: we combined repeated random down-sampling with random forest to overcome the class imbalance problem and achieved promising results. overall, we showed that the random forest algorithm outperformed other machine learning algorithms including support vector machines, shrinkage discriminant analysis, gradient boosting machine and neural networks. due to its predictive performance and simplicity, the random forest technique is a useful method for prediction of mtlr odn agonists. toll-like receptors (tlrs) represent an ancient evolutionary host immune defense system. there are expressed tlr genes in mice ( in humans), and each is devoted to recognizing a distinct set of pathogen associated molecular patterns (pamps) that are not found in healthy vertebrate cells, making them an important tool to help fight infections [ ] . tlrs , , , and are extracellular and are situated in the plasma membrane where they bind bacterial cell wall components such as lipoteichoic acids, lipopolysaccharides, lipoproteins, and flagella. tlrs , , , are located in endosomes where they recognize specific nucleic acid sequences expressed by various pathogens [ ] . the extracellular signaling domain of tlr forms a horseshoe shaped dimer that forms a sandwich that clasps two cpg oligonucleotides (odn) resulting in the cytoplasmic domains coming into close proximity thereby triggering downstream signaling [ ] . upon activation, tlr triggers an innate immune response characterized by the production of pro-inflammatory cytokines such as tnf-α, il- , il- , and il- . some synthetic single-stranded odns that contain unmethylated cpg motifs mimic bacterial dna and can bind and activate tlr leading to cytokine secretion and enhancement of adaptive immune responses. synthetic tlr -active odns have shown utility as vaccine adjuvants and anti-cancer immunotherapeutic agents. to identify a good tlr ligand, typically a large library of odns needs to be synthesized and screened on cell lines, which is a time consuming and expensive task. we hypothesized that modern in silico high-throughput screening (hts) methods may improve the ability to identify novel highly active tlr ligands. in silico screening, also known as virtual screening (vs), has been widely used to enrich datasets with compounds that have a higher probability of binding to the target of interest [ ] [ ] [ ] , and has an advantage over traditional screening or physical hts due to its massively parallel processing ability; hence millions of compounds can be assessed economically in parallel. this is particularly important when the search space for potential odns tlr ligands is taken into consideration. a typical singlestranded odn tlr agonist is nucleotides in length, which amounts to total number of possible odns. vs methods are of two major classes based on the availability of structural information. if the d structure of a receptor is known, structure-based virtual screening (sbvs) [ ] techniques such as homology modeling, molecular docking and molecular dynamics can be used. however, if the structural information of the receptor is lacking, then ligand-based virtual screening (lbvs) [ ] techniques such as quantitative structure-activity relationship and machine learning are more appropriate. sbvs involves molecular complex optimization to find the most favorable d binding conformation of the ligand. consequently, sbvs is unsuitable for highthroughput screening of ligands like -mer odns, which have over rotatable bonds. on the other hand, lbvs is computationally inexpensive, easy to use and might therefore be useful in the screening of tlr activating odns. in a recent review, murgueitio et al. [ ] discussed the use of various computational approaches to investigate the structure and function of tlr receptors. to discover potential tlr ligands. zatsepin et al. [ ] screened a library of . million commercially available compounds to discover tlr antagonists by using computational chemistry and cell-based assays. the authors reported potential tlr antagonists with ic lower than μm, with five of them having ic values below μm. zhou et al. [ ] constructed a d structure of human tlr ectodomains, complexed with cpg odns using homology modeling, then used molecular docking to study the interactions between tlr and the odns. they reported that leucine rich region (lrr)- was the main region in tlr responsible for odn binding. the authors further reported that five positively charged residues within lrr were specifically involved in the odn binding to tlr . nagpal et al. [ ] reported a support vector machine model to predict odns with tlr activity with the model achieving a maximum matthews correlation coefficient of . with an accuracy of %. tlr ligand prediction tools require availability of well-annotated odn datasets with experimentally determined tlr activity data. machine learning (ml) based techniques such as decision trees, random forest, support vector machines and neural networks can then be applied to such odn datasets. ml is an umbrella term for statistical models built to discover patterns in existing data to explain unseen data. ml models are very powerful tools that have been used in the past to predict and classify the pharmacokinetics or toxicological profiles of compounds [ ] , predict biological activities or toxicity [ ] and assist in screening and optimization of compounds [ ] . to our knowledge, this is the first report on the use of random forest-based approaches to predict novel mtlr ligands based on an in-house experimentally validated odn dataset, with % prediction accuracy shown by experimental validation. the main goal of this study was to build a ml model that could distinguish odns that have high activity for mtlr from odns with low activity. we used odns with known high mtlr activity, as positive examples while odns with low activity were used as negative examples. we first analysed the dataset to understand the occurrence of sequence motifs in mtlr activating odns. we observed an uneven distribution of motifs with a few motifs such as 'gg' or 'cc' present in % of the odns in the high activity group compared to only % of the odns in the low activity group. figure shows the percentage of odns in the top motifs arrange in a clockwise manner, based on the absolute difference in the percentage of occurrence in high and low mtlr activity groups of odns. all motifs having an absolute difference above % are shown in additional file . we further analyzed the effect of motif occurrence on the mtlr activity score in the high and low activity groups of odns in the dataset. using the mann-whitney u test we compared the median mtlr activity score of odns with a motif to those without the motif for the two classes and calculated the p values. the significance threshold was set at . . figure shows the effect of top motifs occurrence in high (fig. a) and low (fig. b ) mtlr active group of odns. the darker colored bars stand for a significant difference in the median mtrl activity score (p < . ) due to the presence of the motif in the odns. the dotted line is the median mtlr score of . and . for the high and low activity groups of odns, respectively. within the low activity group (additional file ), we found that presence of motifs such as 'cc', 'gg', 'ggc', 'gcc', 'cccg' and 'cggc' significantly increases the median mtlr activity score, while the presence of motifs e.g. 'tgt', 'cgcgt' and 'tct' further lowers the activity of odns. in contrast, we found presence of 'cgtt' motif to significantly improve while 'ag' motif to significantly decrease the median mtlr activity score of the odns in the high activity group (additional file ). since there was no single motif that could account for the mtlr activity score of the odns, we surmised that the combination of motifs and their interaction with the tlr receptor was responsible for determining overall mtlr activity. mean classification levels achieved by all algorithms in different k-fold cross validation schemes when applied to bootstrap test samples obtained using the down-sampling technique are shown in fig. . we found that overall rf model either outperformed or was on par with the other prediction algorithms in all four cross validation schemes. in five-fold cross validation the best rates were achieved by the rf and svm model with a maximum balanced accuracy of . % and mcc of . (additional file ). the mean balanced accuracy and mean mcc for rf model in five-fold cross validation was . % and . , respectively, with standard deviations of . and . , respectively (table ). in ten-fold cross validation, rf and gbm achieved the best results with the maximum balanced accuracy and mcc of . % and . , respectively (additional file ). the mean balanced accuracy and mcc for the rf model in ten-fold cross validation was . % and . , respectively, with standard deviations of . and . , respectively (table ). in fold cross validation the best results were achieved by rf and svm with the maximum balanced accuracy and mcc of . % and . , respectively (additional file ). the mean balanced accuracy and mcc for the rf model in -fold was . % and . , respectively with standard deviations of . and . , respectively (table ). in fold cross validation random forest achieved the best result with the maximum balanced accuracy and mcc of . % and . , respectively (additional file ). the mean balanced accuracy and mcc of rf model was . % and . , respectively, with standard deviations of . and . , respectively (table ) . overall, the rf algorithm outperformed in all other ml methods, for different cross-validation values. we therefore selected rf with the -fold cross-validation scheme, having maximum mean balanced accuracy and mcc and minimum standard deviation on both measures, to perform the fig. the effect of top motifs in the high (a) and low (b) mtlr activity group of odns in the dataset. the darker bars represent a significant difference in the median mtlr activity score due to the presence of motif in the odns. the dotted line shows the median mtlr activity of . and . for the odns in the high and low activity groups, respectively, in the dataset mtlr activity predictions for the randomly generated odn dataset. external validation is the final step to evaluate the realistic performance of any prediction model. in this technique, the performance of the model is evaluated on a new dataset not used in training or testing the model. to rigorously evaluate the performance of our model, we randomly generated -mer odn sequences using an in-house written python script and then screened and ranked these randomly generated odn for mtlr activity using our rf model. these odns were not present in our original dataset of odns used for model building or training, and as they were virtual we had no prior knowledge of their likely mtlr activity at the time of model prediction. our rf model predicted of these random odns to be of high activity and we selected the top for chemical synthesis, and then experimental tested them for mtlr activity using the raw-blue reporter cell line that expresses mtlr. ninety-one ( %) of the predicted high activity odns had a mtlr activity value above . , confirming the high accuracy of the model in predicting odn sequences with positive mtlr activity (fig. ). this demonstrates that our mtlr -specific rf prediction model is rigorous, with a strong performance on making predictions on a completely independent dataset. in this study we demonstrated the feasibility of using an rf model for in silico screening of synthetic odns to detect high activity mtlr agonists. multiple sequence features such as simple counts of nucleotides, the distance between motifs and graphically derived features like the moment of inertia were calculated before building the rf model. we observed higher occurrence of several motifs such as 'cggc', 'cccg', 'gcc', 'cgg', 'ggc', 'ccg', 'ccc', 'gg' and 'cc' in high activity as compared to low activity odns. this means that these cytosine and guanine rich motifs along with the key unmethylated cpg dinucleotide contribute to strong mouse tlr activation. interestingly, this is in contrast with the thymine rich motifs reported for tlr fig. mean and standard deviation of balanced accuracy rates of the five classifiers on the twenty bootstrap test samples using k-fold crossvalidation scheme. mean balanced accuracy rate of rf model was greater than all five algorithms in all the folds stimulatory odns by nagpal et al. [ ] . this may be due the fact that our odn training set was mouse specific whereas the dataset used by nagpal et al. [ ] was not specific to any organism. on further analysis we found and motifs which significantly increased, or decreased, respectively, mtlr activity in the low activity group (additional file ), whereas, we found only and motifs in the high activity odns which significantly (p value < . ) increased or decreased, respectively, mtlr activity (additional file ). furthermore, we discovered motifs which significantly decreased mtlr activity in both low and high groups. for example, 'cgcgtg' and sub motifs like 'gcgtg' and 'cgcgt', decreased the activity of odns in both the high and low groups. however, we were unable to identify motifs that increased mtlr activity for both groups of odns. this suggests that a combination of motifs might be required to increase activity of odns in the high group whereas the activity of low odns can be improved even by inclusion of a single motif. cooccurrence of motifs and their effect on mouse tlr activity can be analyzed in the future to discover combinations of motifs responsible for the increase in the activity of odns in both groups. the performance of the rf model was compared to other methods, which were trained on the same data. the average classification accuracy achieved by all the methods when applied to bootstrap test samples in four different cross-validation schemes is shown in fig. . the results demonstrated that the rf model had the superior performance on the test datasets in most of the scenarios. the gbm and svm classifiers also had reasonable classification accuracy rates, however, rf outperformed them in -fold cross validation scheme. the selected rf model on average correctly classified . % of the odns in the training set with high activity for mtlr and . % of odns with low activity. the rf thereby achieved an overall balanced accuracy of . %. finally, the rf model was used to virtually screen randomly generated odns from which it predicted odns to have high activity for mtlr . due to large number of predicted positive hits, the top odns were selected for synthesis and testing for mtlr activity in vitro. ninety one out of the synthesized odns were found to have mtlr activity above the cutoff of . for high activity odns confirming the prediction potential of the rf model. however, fig. shows that the majority of predicted ligands had an activity value ranging from . to . , which indicates that the model might need to be further fine-tuned to get even higher activity ligands, with a much larger dataset than the randomly generated oligonucleotides screened to find high activity ligands. in this study we found several sequence motifs that help explain the mtlr activity of cpg odns. motifs including 'cgtt', 'ggc', 'gcc' and 'cccg' significantly improved, whereas motifs such as 'ag', 'tct' and 'cgcgt' significantly decreased, the activity of mtlr odns. further, we developed and validated an rf model for predicting odns with mtlr activity. the results showed that the rf method was well suited for predicting high activity mtlr specific odns and outperformed various other learning algorithms such as svm, sda, nn and gbm. the model was used to screen a random library of odns and correctly identified out of odns that were subsequently confirmed to have mtlr activity. this shows the power of machine learning models for discovering novel tlr agonists. the lead mtlr active odn candidates from the above studies are now being tested as vaccine adjuvants and anti-cancer agents in relevant mouse models. the quality of the training dataset determines the quality of the resulting machine learning model. missing or insufficient data, mislabeling of the target variable, and irrelevant features may complicate the learning task and hinder the performance of the trained model. the sequences of odns with experimentally determined mtlr activity were obtained from in-house data we generated on synthesized odns that were characterized using a mouse tlr expressing reporter cell line (raw-blue cells, invivogen, usa). the dataset consisted of odns with mtlr activity values ranging from . (no activity) to . (high activity). the odns were grouped into two classes (fig. ) based on their respective activity value (i.e. . and above: high activity and below . : low activity), resulting in a high activity group (count ) and a low activity group (count ). to ensure data quality, it is customary to check and remove any outliers, impute the missing data, check, and assign the variables the correct datatype. our dataset had neither missing values nor outliers and therefore, no further action was required in cleaning the dataset. however, to avoid overtraining the model with similar odns, the diversity of the dataset was increased by limiting the similarity within the group. this was achieved by clustering the odns within a group using the binary fingerprint features we developed during this study and applying a clustering cutoff of . to remove similar odns. this resulted in the removal of five odns from the low activity group with remaining. all odns in the high group (count ) were dissimilar enough not to breach the similarity cutoff and were retained. in our training dataset, the number of odns with low mtlr activity was approximately . times more than the number of odns with high mtlr activity. therefore, we used the down-sampling technique to balance the dataset, so that % of the samples were derived from the set of odns with high activity and % from the set of odns with low activity. subsequently, the down-sampled dataset was subdivided into training ( %) and testing (also known as validation) sets ( %), using a random sampling technique and the odns in the test set were excluded from model training. in order to choose the best classifier with k-fold cross validation, the performance of our models were measured using down-sampled test sets. the overall methodology adopted in the study is shown in fig. . in table , we present the composition of the dataset used in this study. for each instance, the training dataset was composed of odns (derived from odns with high and low mtlr activity each). the test dataset used to evaluate the performance of a model was composed of odns ( each from the two groups of high and low mtlr activity). for the prediction set, we used an in-house python script to randomly generate -mer odns, to capture the diversity of the -mer cpg-odn universe. every odn in the prediction set was classified using the selected model and crossvalidation scheme in a loop. for the final prediction, a consensus of the predictions were taken for each odn in the prediction set. finally, the top high activity predicted odns were selected for synthesis and experimental testing using the raw-blue reporter cell line assay. the training and test set odns along with experimental activity information are available in additional file . the measured mtlr activity value of all the synthesized -mer odns in the dataset. the odns were divided into two groups of high (shown in purple) and low (shown in green) activity using a cutoff score of . , based on the optimal density (od) results from the raw-blue reporter cell assay it is possible to generate a large number of features for the odn sequence data that can be used to construct machine learning models. however, there are several problems in using all the possible features as (i) some of the features may be highly correlated (ii) some may not be relevant and may contribute to the noise in the model and (iii) using a large number of features may lead to overfitting. additionally, constructing models with many features is computationally demanding [ ] . therefore, one of the most important aspects of creating a good ml model is the choice of appropriate features that can help explain the behavior of interest based on occam's razor principle (i.e. simple models are more likely to be closer to reality than complex models.) [ ] . while there are a variety of features used in bioinformatics for sequence data, we used the binary fingerprint features and numerical features, including count and position of motifs, distance of the motifs with respect to the start position and graphically derived features such as the moment of inertia and radius of gyration, to train the model [ ] . to generate fingerprint features, a fasta formatted file containing all high activity odn sequences was analysed using an in-house perl subroutine, to chop each sequence into motifs of increasing length from two to six nucleotides and record the start positions of the motifs. for example, with a small hypothetical odn 'tcg' of three nucleotides, two dinucleotides motifs tc , cg and a trinucleotide tcg motif were generated. finally, a dictionary of the motifs with at least % difference in the occurrence rate in low and high group of odns (count ) was prepared. subsequently, the dictionary was used to generate the binary fingerprint pattern for each sequence, where showed the presence of a motif while indicated its absence. different patterns of nucleotide usage in odns may lead to varied mtlr activity. therefore, all nucleotide characters (a, t, g, c) were counted in a sequence and the perl built-in dictionary data structure, hash, was used to store the count of each nucleotide. ambiguous nucleotide characters or gaps were ignored if present. calculating the distance between motifs with respect to their start positions the most commonly occurring motifs were used to calculate the distance between motif features along with their specific location. to map the position of a motif in the odns, the sequence of each odn was scanned for the presence of a motif and all the positions where each motif occurs were recorded. using eqs. ( )-( ), the distance between the second and first, third and first and the third and second occurrence of the motifs were calculated for all the motifs. where d_motif is the distance, p , p and p are the position , position and position of the motif respectively, and 'n' is the number of nucleotides before the latter motif. in the case of the absence of a motif, was substituted in the equation. it is important to keep 'n' in the equation to provide the specific location of the motifs within an odn, because the calculated distance between motifs could be same in several odns. for example, in a sequence s = tatgcgttcg-tacttgatctgac, the distance between cg motifs is - = . similarly, for another sequence s = tgctttcttgtcgtgcgggctgt, the distance between the cg motifs is - = , again. however, the descriptor d_cg _ value for s and s are and , respectively, with the addition of n to the simple distance formula of d_motif. the graphical representation of dna sequences have been used for many applications including assessing phylogenetic relationships [ ] , characterization of neuraminidase gene in h n avian flu [ ] and for describing similarity/dissimilarity of dna sequences [ ] . in order to derive features, the -mer odn sequences were represented as a d-graph, as previously described [ ] . briefly, each base in the sequence is represented as a material point on the graph which is treated as a rigid body and follows the rules of newtonian dynamics. numerical features such as the center of mass (μ x , μ y ), the principal moment of inertia(i , i ) and radius of gyration (r g ) were calculated for each sequence as described in [ ] . there are several feature selection methods used in machine learning to remove redundant or irrelevant features. these can be broadly divided into filter methods (e.g. correlation matrix, information gain, chi-square score, principal component analysis, regression coefficients, variable importance) and wrapper methods (e.g. forward/backward selection, randomized methods that combine pls with the genetic algorithm or monte carlo algorithm) [ ] [ ] [ ] . filter methods are easy to implement because there is no learning involved and depend only on the application of a cut-off value to reject features due to the low importance in the model construction. in the wrapper methods, the performance of a learning algorithm is evaluated to select the optimum subset of features therefore, it is a very computationally expensive process [ ] and is best suited for a limited number of features. furthermore, filter methods work well for text mining [ ] , and are applicable for odn features, which are essentially nucleotide "words." due to the large number of fingerprint features available ( in total), we first filtered out the constant and near-constant features (features with < . standard deviation) also known as zero and near zero variance features using the caret package in r. constant or near constant features take a unique value across samples and are uninformative. this resulted in the removal of features. since these features are binary in nature, we also checked and removed any linear combinations of features if present. this resulted in the removal of features. to understand the distribution in the high and low group of odns we created a cricos plot using the circlize package in r [ ] . for all numerical features in addition to removing zero and near zero variance features we also calculated the correlation matrix and filtered out features that were highly correlated. the correlation coefficient was set at . and features with correlation above the cutoff value were removed. we then normalized the remaining features using centering and scaling techniques to make them unit independent. subsequently, we merged the fingerprint and numerical features to give us a merged set of features, listed in table . in the current study, five ml algorithms, i.e. random forest, gradient boosting machine, shrinkage discriminant analysis, support vector machine and neural network were compared, and the best performing model was chosen for the prediction of novel mtlr active odns. to have a non-biased assessment of the performance, kfold cross-validation was followed where one instance of the down-sampled training data was further divided into k partitions. the value of k varies from , , to . for each partition, odns not included in the training were considered part of the testing dataset. finally, the testing data of the instance was used to evaluate the classification accuracy of the model, with the best model selected for prediction on an independent validation dataset. a graphic representation of the general procedure is given in fig. . the random forest (rf) algorithm was introduced by breiman in [ ] and is one of the most powerful ensemble machine learning technique that make predictions by averaging over several independent base learners in order to identify the class label for unknown instances. the base learners are usually the classification and regression trees (cart) constructed using a sample with replacement from the training data with the [ , ] . briefly rf takes advantage of two powerful statistical techniques, bagging and random feature selection. in bagging each tree is trained on a bootstrap sample (sampling with replacement) and the predictions are made by the majority vote of the trees. furthermore, in rf instead of using all the features, rf randomly selects a set of features to split at each node when growing a tree. to assess the performance of the rf algorithm, rf performs a type of crossvalidation using the out-of-bag (oob) samples (samples which are not included in the training set). the concept of variable importance is inbuilt in the rf algorithm and the importance is measured by the gini impurity criterion index [ ] . we used the caret package in r to evaluate the performance and developed an ensemble of different rf models for final prediction. the mtry parameter was tuned using the tunegrid argument in the train function. the accuracy of the five ml algorithms was measured by presenting the prediction results in the form of a confusion matrix and the variety of performance measures were calculated based on the following statistical measures: tp, true positivesthe total number of correctly classified high activity odns. tn, true negativesthe total number of correctly classified low activity odns. fp, false positivesthe total number of low activity odns incorrectly classified as high activity odns. fn, false negativesthe total number of high activity odns incorrectly classified as low activity odns. using the measures above, a series of statistical metrics were computed including sensitivity (se), specificity (sp), balanced accuracy (ba), matthews correlation coefficient (mcc) and precision. the recall rate for the members of the positive class (high activity odns) is given by sensitivity, in eq. ( ): similarly, the recall rate for the members of the negative class (low activity odns) is given by specificity, in eq. ( ): the balanced accuracy of the model was calculated based on the eq. ( ): we then calculated the mcc from eq. ( ); the coefficient returns a value between + and − . the higher the value of the coefficient, the better the classification result. finally, the precision was computed to measure the reproducibility of the results, in eq. ( ): mouse raw-blue tlr reporter cell assay raw-blue™ cells are derived from the murine raw . macrophage cell line with chromosomal integration of a secreted embryonic alkaline phosphatase (seap) reporter construct inducible by nf-κb and ap- and were acquired from invivogen. the presence of agonists of mouse tlr activates downstream signaling pathways leading to the activation of nf-κb and ap- , and the subsequent secretion by the raw cells of seap. levels of seap in the culture supernatant are measured chromatographically using the detection medium quanti-blue™. raw-blue cells were cultured in dmem supplemented with % (v/v) heat-inactivated fetal bovine serum, penicillin-streptomycin , u/ml (gibco), and normocin μg/ml (invivogen). subsequently, raw-blue cells were seeded at a density of approximately × cells/well in a volume of μl/well in a flat-bottom well culture plate (greiner-one). odns were diluted in saline and added to the culture plate containing raw-blue cells to the total volume of μl. after culturing the cells for h, the levels of seap were determined in the supernatant with quanti-blue™ solution (invivo-gen) by reading the absorbance at wavelength of nm. toll-like receptors: activation, signalling and transcriptional modulation the structural biology of toll-like receptors in silico approach to screen compounds active against parasitic nematodes of major socio-economic importance graphical representation and similarity analysis of dna sequences based on trigonometric functions discovering highly selective and diverse ppar-delta agonists by ligand based machine learning and structural modeling computational methods in drug discovery use of machine learning approaches for novel drug discovery balancing inflammation: computational design of small-molecule toll-like receptor modulators computational discovery and experimental confirmation of tlr receptor antagonist leads toll-like receptor interaction with cpg odn--an in silico analysis approach vaccineda: prediction, design and genome-wide screening of oligodeoxynucleotide-based vaccine adjuvants applying machine learning techniques for adme-tox prediction: a review deeptox: toxicity prediction using deep learning the curse of dimensionality in data mining and time series prediction the problem of overfitting d-dynamic representation of dna sequences coronavirus phylogeny based on triplets of nucleic acids bases graphical representation and numerical characterization of h n avian flu neuraminidase gene sequence a review of feature selection methods with applications a review of feature selection and feature extraction methods applied on microarray data a review of variable selection methods in partial least squares regression circlize implements and enhances circular visualization in r random forests random forests: from early developments to recent advancements the origins of the gini index: extracts from variabilità e mutabilità ( ) by corrado gini publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations this article has been published as part of bmc molecular and cell biology, volume supplement , : th international conference on bioinformatics. the full contents of the supplement are available at https:// bmcmolcellbiol.biomedcentral.com/articles/supplements/volume- supplement- . supplementary information accompanies this paper at https://doi.org/ . /s - - - .additional file . sequence motifs in mtlr active odns having an absolute difference in the occurrences above % in high and low activity groups of odns, arranged in a clockwise manner. the width of the ribbon shows the average percentage composition of the motifs each group.additional file . the effect of odn motif occurrences on the median mtlr activity in the low activity group. the median raw-blue activity for all the odns in the low activity group was . . increase or decrease in the median activity values due to the presence of a motif are coloured green and red, respectively, with statistically significant values in bold. the significance threshold was set at p-value < . . the motifs are arranged in alphabetical order.additional file . the effect of odn motif occurrences on the median mtlr activity in the high activity group. the median raw-blue activity for all the odns in the high activity group was . . increase or decrease in the median activity values due to the presence of a motif are coloured in green and red, respectively, with statistically significant values in bold. the significance threshold was set at p value < . . the motifs are arranged in alphabetical order.additional file . results of five-fold cross-validation.additional file . results of ten-fold cross-validation.additional file . results of fifteen-fold cross-validation. additional file . odns used as test and training sets for building the prediction model, along with activity information.authors' contributions vk and np designed the research; vk, ll and jf performed the research; vk, ll, jf. np, and sr analyzed data; vk, ll, np, and sr wrote the paper. all authors have read and approved the final manuscript. this work was supported by funding by the national institute of allergy and infectious diseases of the national institutes of health under contract hhs-n c. the content is solely the responsibility of the authors and does not necessarily represent the official views of the nih. all data reported in this study are available as tables and supplementary data. the cell line used in the assay is commercially available from invivogen inc. [ ] .ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests. key: cord- -cob ur authors: sharma, vaneet kumar; sharma, ity; glick, james title: the expanding role of mass spectrometry in the field of vaccine development date: - - journal: mass spectrom rev doi: . /mas. sha: doc_id: cord_uid: cob ur biological mass spectrometry has evolved as a core analytical technology in the last decade mainly because of its unparalleled ability to perform qualitative as well as quantitative profiling of enormously complex biological samples with high mass accuracy, sensitivity, selectivity and specificity. mass spectrometry‐based techniques are also routinely used to assess glycosylation and other post‐translational modifications, disulfide bond linkage, and scrambling as well as for the detection of host cell protein contaminants in the field of biopharmaceuticals. the role of mass spectrometry in vaccine development has been very limited but is now expanding as the landscape of global vaccine development is shifting towards the development of recombinant vaccines. in this review, the role of mass spectrometry in vaccine development is presented, some of the ongoing efforts to develop vaccines for diseases with global unmet medical need are discussed and the regulatory challenges of implementing mass spectrometry techniques in a quality control laboratory setting are highlighted. as the target populations for the vaccines are healthy individuals, pregnant women, or infants, vaccine safety is of paramount importance. appropriately, the vaccine landscape is changing from traditional vaccine approaches to cost-effective, highly scalable, and safe recombinant vaccines. , using recombinant dna technology, antigens are expressed in yeast, escherichia coli, baculovirus expression vector system (bevs), or mammalian cell lines. [ ] [ ] [ ] recombinant antigens are engineered to mimic the first step of virus attachment to the cell surface which is mediated by specific glycoproteins. the expressed recombinant antigens undergo multiple purification cycles to produce highly purified vaccines. , in order for recombinant vaccines to be acceptable to regulatory authorities, in-depth analytical characterization needs to be performed in this review, we focus on the expanding role of mass spectrometry in vaccine development, irrespective of the route of production. we also highlight the regulatory challenges and limitations of mass spectrometry-based techniques which constrain its further implementation as a quality control batch release assay in cgmp manufacturing. the role of mass spectrometry ( alternatively, an o-linked glycan can be formed by linking the oligosaccharide to a serine or threonine residue. glycosylation play a fundamental role in antigen conformation, folding, stability, solubility, and importantly, immune response. , plants, yeasts, and non-human cell lines generate glycans that are not compatible and bioactive within human hosts. thus, about % of all recombinant glycoproteins are produced in mammalian-based expression systems such as chinese hamster ovary (cho) cells. analytical characterization of a glycoprotein is challenging as there is inherent unpredictability associated with the glycans; they are either macroheterogeneous (potential glycan site in the protein not glycosylated) or microheterogeneous (different glycan structures found on the same site in the expressed protein). glycosylation analysis is performed to understand the nature of structural heterogeneity of glycans, quantify them, and more importantly to determine where glycosylation occurs (site specific analysis). mass spectrometry based techniques are valuable tool for detecting and investigating glycosylation ( figure ). although ms-based glycosylation analysis is not without limitations, the advances in ms instrumentation and glycan analysis software have led to increased resolution, automated identification, quantitative determination, and accurate structural characterization. , , another critical quality attribute (cqa) of glycoprotein is intramolecular disulfide bonds (s-s linkages); they ensure correct folding, functional activity and stability. incorrect formation of disulfide bonds can cause protein misfolding which tends to promote aggregation which could result in an unwarranted immune response. confirmation of correct disulfide bond formation in the recombinant first study analyzing the site-specific nglycosylation of gp . this report describes the structural characterization of the expressed gp . reversed phase hplc of the tryptic digest. peptides collected from rp-hplc were further identified by amino acid analysis (aaa) or nterminal sequencing analysis. leonard et al hiv- gp expressed in cho cells. mass spectrometric characterization of the glycosylation pattern. of hiv- gp . maldi and nanoesi-lc-ms/ms using a hybrid quadrupole-time-of-flight tandem mass spectrometer (q-tof) was used to assign glycosylation sites. zhu et al hiv gp jr-fl and con-s env expressed in cho and hek cells. a glycopeptide-based mass mapping approach was used to characterize the glycosylation of two env protein vaccine candidates in a glycosylation site-specific fashion. mass spectrometry based approach was used to map the complete glycosylation profile at every site in eleven hiv- env trimers. high-resolution lc-ms/ms was performed using an orbitrap velos pro hybrid mass spectrometer (thermo scientific) equipped with etd and coupled to an acquity uplc system (waters). go et al hiv gp monomers of the bg . sosip. global n-glycan site occupancy of hiv- gp by metabolic engineering and high-resolution intact mass spectrometry was performed. released n-glycan analysis was carried out using synapt g si ion mobility mass spectrometer. native highresolution mass spectrometry was performed on q exactive hybrid quadrupole-orbitrap mass spectrometer. as illustrated in the following section and in table , mass spectrometry-based techniques have been used to perform the structural characterization, glycosylation profiling and antigen quantitation during the development of the hiv, influenza, dengue, ebola, meningococcal, and other vaccines. the review also highlights that mass spectrometry-based methods such as glycan analysis has been used to analyze a specific envelope glycoproteins (env) and has broad applicability to any other glycoprotein-based vaccines. the quest for a safe and effective vaccine to protect against human one of the hypotheses being explored for protective immunity against hiv is to induce broadly neutralizing antibodies (bnabs) that target the highly glycosylated hiv- virion-associated envelop. a number of approaches are being pursued in the hiv- vaccine development field to establish protective immunity, including monomeric gp subunits and oligomeric gp and trimeric envelope glycoprotein to elicit bnabs. [ ] [ ] [ ] each of these envelop (env) immunogens have multiple exposed epitopes and are heterogeneously glycosylated molecules, with more than % of their mass consisting of glycans (∼ n-linked glycans). it is critically important to monitor this hiv env glycan shield during vaccine development. crispin and collaborators also extensively investigated the native glycosylation profile of envs, in particularly, trimeric immunogen bg sosip. . [ ] [ ] [ ] [ ] their studies led to an increased understanding of the glycan shield of the env and it is now largely accepted that to induce bnabs, the hiv vaccine candidate should have a glycan profile similar to the one present on the native env trimers. [ ] [ ] [ ] in a thorough study on trimeric env glycosylation, behrens et al influenza virus is a segmented, enveloped rna virus and is among the ic-idms-mrm method is truly an alternative to the approved srid method as it has dual purpose "potency and content determination", was found to be equivalent to the srid method and can also be used in response to a pandemic influenza threat. , additionally, a direct ultra-performance liquid chromatography (uplc)-idms method was reported for the rapid and accurate quantification of influenza na. label-free ms-based methods have also been reported for the simultaneous identification and quantification of ha and na in influenza vaccine with samples analyzed by lc-ms e on a waters synapt g mass spectrometer. quantification of proteins by labelfree lc-ms e is a powerful tool, in this method, alternating scans of low collision energy and elevated collision energy during lc-ms analysis to obtain both protein identity and quantity in a single experiment. quantification based on the experimental data showed that the signal intensity was proportional to concentration which allowed for the amount of any protein in the mixture to be estimated. lc-ms e utilizes parallel, multiplex fragmentation where all peptide precursors are simultaneously fragmented throughout the chromatographic the srid assay has been used for over years as a quantitative and potency method throughout the world despite issues regarding variability and availability of standard reagents. thus, even though new methods like hplc and mass spectrometry are being developed, it will take some time for these methods to be adopted worldwide. as the next influenza pandemic cannot be predicted, the health authorities' pandemic preparedness efforts include efforts to ensure expedited availability of pandemic vaccines. methods such as hplc and ms, with their ability to quantitate antigens without standard reference reagents, can become a cornerstone of pandemic influenza preparedness. the viral genus flavivirus, includes dengue virus, yellow fever virus, and and were glycosylated and, predominately, the n-glycan at asn was a high mannose-type and at asn was mainly a combination of complex-and hybrid-type glycans. this study provided important new insights for the role of glycans in the dengue virus-host cell interactions. in a separate study, accurate quantitation of the expressed four viral particles in the tetravalent dengue vaccine (cyd) was performed using targeted ms in selected reaction monitoring (srm) mode. the study described an orthogonal quantitation strategy (targeted ms in srm mode) and demonstrated that the variability of the ms method was low (between % and %) and the assay was linear between . and nmol/l. based on the reported method performance, it could be used to release future batches of the tetravalent dengue vaccine. ebola the data presented demonstrated that the n-glycan patterns were similar between gp , but o-glycan patterns were remarkably different on gp , the five ebola viruses. this study should serve as the foundation for future ebola viral entry and immunogenicity studies. to improve on the conventional approaches for absolute quantitation of gp in ebola virus-like particles (evlps), an isotope dilution full-scan liquid chromatography-high-resolution mass spectrometry method was developed using an ultimate hplc and an orbitrap elite hybrid ion trap-orbitrap mass spectrometer. the reported ms quantitation method provided not only a means to rapidly determine evlp batch quality based upon quantitation of antigenic gp but also ensured adequate preclinical/clinical dosing. chikungunya is a mosquito-borne viral disease, endemic in africa and southeast asia and has also recently emerged in the caribbean. currently there are no drugs or vaccines available for treatment or prevention. the name "chikungunya" derives from a makonde word meaning "to become contorted," and describes the stooped appearance of sufferers with joint pain (arthralgia ms-based approaches have been used to perform the physicochemical characterization for all of these vaccines. in one of the studies, researchers used lc-ms to characterize glycosylated lysine residues in menveo®. in another study a lc-ms method was used to determine the relative reactivity of lysine residues in crm to determine which of these amino acids were more susceptible to conjugation. lc-ms was also used to quantify the bexsero® vaccine which was the first vaccine developed by reverse vaccinology; a genome-based approach to vaccine development. in the field of vaccines, vera-velasco et al not only quantified the viral f, g and m proteins present in the viral particles but also analyzed the cellular proteomic composition in the niv vaccine candidate. traditionally, viral particles have been described as pure entities carrying only viral-derived proteins but the authors successfully analyzed the ratio between cellular and viral proteins in the niv vaccine candidate using lc-ms/ms. west nile fever is a flavivirus that causes a viral infection typically spread by mosquitoes. to date, no vaccine is available to prevent the west nile infection. partially purified virus-like particles were resolved by sds-page, and the coomassie blue-stained band corresponding to env protein was excised from the gel, destained, and analyzed by ms. microcapillary lc-ms was performed using a lcq deca ion-trap mass spectrometer (thermo finnigan) to identify the presence of env protein in virus-like particles to help in developing a potential vaccine. in should be a supplemental batch release test to ensure that the product is within specifications and without any unwarranted modifications. using ms techniques will ensure improved quality and increased safety for clinical trial participants. in conclusion, the evidence suggests that the emergence of ms-based techniques has advanced the field of vaccine development. history of vaccination vaccines, 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characterization of nglycosylation profiles from mammalian and insect cell derived chikungunya vlp label-free quantitative mass spectrometry for analysis of protein antigens in a meningococcal group b outer membrane vesicle vaccine physicochemical characterisation of glycoconjugate vaccines for prevention of meningococcal diseases defined conjugation of glycans to the lysines of crm guided by their reactivity mapping quantification by lc-ms e of outer membrane vesicle proteins of the bexsero ® vaccine the dual role of lipids of the lipoproteins in trumenba, a self-adjuvanting vaccine against meningococcal meningitis b disease robust manufacturing and comprehensive characterization of recombinant hepatitis e viruslike particles in hecolin ® hepatitis e virus: a renewed hope with hecolin molecular and structural characterization of the l virus-like particles that are used as vaccine antigens in cervarix™, the as -adjuvanted hpv- and- cervical cancer vaccine viruslike particles in vaccine development virus-like particle-based human vaccines: quality assessment based on structural and functional properties status of vaccine research and development of vaccines for nipah virus proteomic composition of nipah virus-like particles production of immunogenic west nile virus-like particles using a herpes simplex virus recombinant vector characterization of human enterovirus virus-like particles used for vaccine antigens mass spectrometric characterization of the glycosylation pattern of hiv-gp expressed in cho cells glycan profiles of the n-glycosylation sites of the hiv envelope protein cn gp comparative analysis of the glycosylation profiles of membrane-anchored hiv- envelope glycoprotein trimers and soluble gp mapping the complete glycoproteome of virion-derived hiv- gp provides insights into broadly neutralizing antibody binding haemagglutinin quantification and identification of influenza a&b strains propagated in per. c ® cells: a novel rp-hplc method hplc-based quantification of haemagglutinin in the production of egg-and mdck cell-derived influenza virus seasonal and pandemic vaccines optimization and qualification of a quantitative reversed-phase hplc method for hemagglutinin in influenza preparations and its comparative evaluation with biochemical assays characterization of a recombinant influenza vaccine candidate using complementary lc-ms methods comparative glycomics analysis of influenza hemagglutinin (h n ) produced in vaccine relevant cell platforms glycosylation analysis of engineered h n influenza a virus hemagglutinins with sequentially added historically relevant glycosylation sites particle quantification of influenza viruses by high performance liquid chromatography design and expression of a qconcat protein to validate hi protein quantification of influenza vaccine antigens evaluation of haemagglutinin content by rp-hplc to generate pandemic influenza vaccine topological n-glycosylation and site-specific n-glycan sulfation of influenza proteins in the highly expressed h n candidate vaccines glycosylation characterization of an influenza h n hemagglutinin series with engineered glycosylation patterns: implications for structurefunction relationships cross reactive material glycoconjugate vaccines contain privileged conjugation sites quality by design approach in the development of an ultra-high-performance liquid chromatography method for bexsero meningococcal group b vaccine proteomics-driven antigen discovery for development of vaccines against gonorrhea production and purification of filovirus glycoproteins in insect and mammalian cell lines key: cord- - dz bu authors: zhai, bintao; niu, qingli; liu, zhijie; yang, jifei; pan, yuping; li, youquan; zhao, hongxi; luo, jianxun; yin, hong title: first detection and molecular identification of borrelia species in bactrian camel (camelus bactrianus) from northwest china date: - - journal: infect genet evol doi: . /j.meegid. . . sha: doc_id: cord_uid: dz bu comprehensive epidemiological surveys for lyme disease have not been conducted for the bactrian camel in china. in this study, a total of blood specimens collected from bactrian camels from zhangye city in gansu province and yili and aksu in xinjiang province, china, were examined for the presence of borrelia spp. species-specificity nested pcr based on the s- s rrna, ospa, flab and s rrna genes revealed that the total positive rate of borrelia spp. was . % ( / , % ci = . – . ). these results were confirmed by sequence analysis of the positive pcr products or positive colonies. this is the first report of borrelia pathogens in camels in china. two borrelia species that cause lyme disease and one that causes relapsing fever were identified in the camel blood samples by sequencing. the findings of this study indicate that the bactrian camel may serve as a potential natural host of lyme disease and/or relapsing fever in china. borrelia species are distributed throughout the world and are maintained in nature within various arthropod vectors and mammalian, avian or reptilian hosts (brisson et al., ; vollmer et al., ) . in humans, borrelia spp. are the causative agents of a major disease: lyme borreliosis (lb) (mainly caused by b. garinii, b. afzelii, and b. burgdorferi sensu stricto). lb is also an important disease of domestic animals and wildlife worldwide. lb-group spirochetes, commonly known as b. burgdorferi s.l., cause one of the most significant natural zoonosis diseases that is carried and transmitted by ixodes spp. ticks (wodecka et al., ) . there are at least genospecies of b. burgdorferi s.l., which are classified based on their genetic differences. more than six of these genospecies have been reported in china (b. burgdorferi s.s., b. garinii, b. afzelii, b. valaisiana, b. sinica and b. yangtze) (yu et al., ) . b. garinii is the main genospecies and is distributed mainly in northern china, while b. burgdorferi s.s. is widely distributed in south china (chen et al., ; wan et al., ) . b. burgdorferi s.l. has been detected in more than mammalian species and seven genera of birds (li, ) . studies have shown that in addition to humans, at least six other taxa of mammals (sheep, cattle, horses, dogs, cats and mice) and two types of birds (seabirds and migratory birds) can be infected by borrelia spirochetes in china (keesing et al., ) . previous research used antibodies to detect the borrelia spp. antigen within camel sera in egypt and reported a positive rate of . % (helmy, ) . the genus camelus contains three species: camelus dromedaries (onehump dromedary), camelus bactrianus (two-hump bactrian camel), and camelus bactrianus ferus (two-hump bactrian camel). dromedaries are mainly found in the arabian peninsula, the middle east, and parts of africa, whereas bactrian camels are mainly located in central and northeast asia, northern china, and mongolia . camelus bactrianus ferus is a new species of camel, and it is mainly distributed in china and mongolia (guo, ) . currently, there are an estimated , camels, which are mainly distributed in xinjiang and the inner mongolian autonomous region in china (feng, potential spreaders of lyme disease in china. the exposure of camels to b. burgdorferi s.l. complex was investigated using pcr assay and sequencing. the sequences of the s- s rrna, ospa, flab and s rrna genes obtained from positive dna samples were analysed, and the ticks that potentially act as vectors were discussed. the bactrian camel is free-choice grazing animal that inhabits desert regions. a total of blood specimens from bactrian camels were collected during may and november in two lb-endemic localities at three sites in northwest china (gansu (zhangye) and xinjiang (ili and aksu)) ( fig. ) . each blood sample was collected from the jugular vein of the camel into a sterile tube containing an anticoagulant (ethylene diamine tetraacetic acid, edta). genomic dna was extracted from individual specimens using a commercial qiaamp dna blood kit (qiagen, maryland, usa) and a qiaamp dna mini kit (qiagen, hilden, germany) according to the manufacturer's instructions. the extracted genomic dna samples were then stored at − °c until use. a nested pcr for the detection of b. burgdorferi s.l. was carried out using four independent sets of species-specific primers to amplify the s- s rrna, ospa, flab and s rrna genes, as described in previous studies (zhang et al., ; postic et al., ; guy and stanek, ; wodecka, ; ni et al., ; zhai et al., ) . the primer sequences are shown in table . first-round pcr reactions were performed in a thermocycler (biorad, hercules, ca, usa) with a total volume of μl containing . μl of × pcr buffer (mg + plus), . μl of each dntp at . mm, . u of taq dna polymerase (takara, dalian, china), . μl of template dna, . μl of each primer ( pmol), and . μl of distilled water. the pcr conditions were as follows: min of denaturation at °c; cycles of °c for s, annealing for s (annealing temperatures of primers are listed in table ), and °c for - s (depending on amplicon size); with a final extension step at °c for min. nested pcr reactions included μl of the first-round pcr product as template for another cycles with the same parameters and annealing temperature profile as described above and in table . to avoid cross-contamination and sample carryover, pre-and post-pcr processing and pcr amplification was performed in separate rooms. b. garinii sz genomic dna (from lanzhou veterinary research institute) was used as a positive control, while distilled water was used as a negative control. pcr products were separated by . % agarose gel electrophoresis, and some positive amplicons from the second round of pcr amplification were directly used for sequencing (genewiz, inc. beijing china) or cloned into pgem-t easy vector (promega, madison, wi, usa). positive colonies were then selected for pcr amplification and sequencing. all the sequences obtained in this study were subjected to blast search via the ncbi website (https://blast.ncbi.nlm.nih.gov/blast.cgi) using the blastn program. multiple sequence alignment was executed in florence corpet (http://multalin.toulouse.inra.fr/multalin/). phylogenetic analysis was performed using mega . software (tamura et al., ) . phylogenetic trees of borrelia spp. strains were constructed using all the sequences generated in this study and related sequences previously deposited in genbank to show the relationships between different strains. a % confidence interval ( % ci) for the overall prevalence value of each borrelia spp. strain was calculated using ibm spss statistics version . . all sequences of borrelia spp. s rrna (including sequences fig. . phylogenetic tree of the s rrna gene sequences of borrelia species obtained in the present study and those deposited in genbank from different countries; accession numbers are shown after isolate names. the s rrna gene sequences obtained in this study are indicated by bold triangles. the tree was inferred using the neighbour-joining method of mega . ; bootstrap values are shown at each branch point. numbers above the branch reflect bootstrap support from replications. all sites of the alignment containing insertions-deletions or missing data were eliminated from the analysis (option "complete deletion"). from ili city and sequences from aksu city) were deposited in genbank under the following accession numbers: ky -ky (among them, aku = aku - , aku = aku - , aku = aku - , and aku = aku - ). the sequences of the borrelia spp. s- s rrna gene, including sequences from zhangye city and sequences from aksu city, were deposited in genbank under the following accession numbers: ku -ku . two sequences of the borrelia spp. flab gene from aksu city were deposited in genbank with the following accession numbers: ku -ku . two sequences of the borrelia spp. ospa gene from zhangye city were deposited in genbank with the following accession numbers: ku and ky . the blood samples collected from a total of bactrian camels in the field two chinese provinces were screened for the presence of borrelia spp. by nested pcrs based on four gene loci. the samples were amplified, and the pcr products had lengths of - bp for the four genes. the results of the nested pcr amplification for positive sample screenings are summarized in table . of these specimens, tested positive for s- s rrna, tested positive for ospa, tested positive for flab and tested positive for s rrna, with positive rates of . % ( % ci = . - . ), . % ( % ci = . - . ), . % ( % ci = . - . ) and . % ( % ci = . - . ), respectively. (the sample zy was detected from both s- s rrna and ospa, the samples aku - and aku - were detected from both s- s rrna and s rrna, and the samples aku - and aku - were detected fig. . phylogenetic tree of the s- s rrna gene sequences of borrelia species obtained in the present study and those deposited in genbank from different countries; accession numbers are shown after isolate names. the s- s rrna gene sequences obtained in this study are indicated by bold triangles. the tree was inferred using the neighbour-joining method of mega . ; bootstrap values are shown at each branch point. numbers above the branch reflect bootstrap support from replications. all sites of the alignment containing insertions-deletions or missing data were eliminated from the analysis (option "complete deletion"). from both flab and s rrna). at least one b. burgdorferi s.l. gene was detected in of the blood samples examined ( . %, % ci = . - . ). sequence analysis of the positive pcr products of the genes assayed in this study revealed that the sequences were most similar to those of b. garinii based on the s rrna, s- s rrna and flab gene sequences ( %, - %, and - % identity, respectively) (genbank accession numbers: cp , dq and cp ) and were also similar to b. burgdorferi ( %, %, and - % identity, respectively) (genbank accession numbers: af , kp and cp ). interestingly, a novel borrelia genospecies (genbank accession number: ky ) was identified from the ili region, which had a high identity with b. theileri kat ( . %, genbank accession number: kf ), which was detected from rhipicephalus geigyi, and with borrelia sp., with . % identity (genbank accession number: ab ), which was detected from haemaphysalis ticks collected from wild sika deer (cervus nippon yesoensis) from hokkaido, japan, based on s rrna. phylogenetic trees were constructed based on the identified borrelia spp. s rrna (n = ), s- s rrna (n = ), flab (n = ) and ospa (n = ) gene sequences by the neighbour-joining method using the software mega . ( fig. - ) . the phylogenetic tree based on s rrna sequences indicated that the s rrna gene sequences ( from aksu, from ili) from our study formed three distinct clades. the five aku (aksu) strains cluster within a sub-clade of one of three main clades, forming a sister clade with the b. garinii s rrna sequences from china. interestingly, the strains from the ili area belonged to two different clades, with two sequences of b. garinii and b. burgdorferi belonging to the ld borrelia spp. group and one sub-clade of b. theileri belonging to rf borrelia spp. group. in general, the results show the presence of high heterogeneity among the s rrna sequences of the different borrelia species strains (fig. ) . the s- s rrna sequences formed two distinct clades: strains ( from aksu and from zhangye) formed two distinct sub-clusters in the borrelia s- s rrna phylogenetic tree (fig. ) . the s- s rrna sequences from the aksu strains (aku - = aku of s rrna, aku - = aku of s rrna) and one zhangye borrelia spp. strain belong to one clade of the same branch, which are sister to b. garinii s rrna. one zhangye strain of the s- s rrna sequence was located within the same branch (fig. ) . a phylogenetic tree was constructed based on all the borrelia flab sequences deposited in genbank and two sequences (aku - = aku of s rrna and aku - = aku of s rrna) obtained in this study. the flab sequences from borrelia spp. formed two main clades. the fig. . phylogenetic tree of the flab gene sequences of borrelia species obtained in the present study and those deposited in genbank from different countries; accession numbers are shown after isolate names. the flab gene sequences obtained in this study are indicated by bold triangles. the tree was inferred using the neighbour-joining method of mega . ; bootstrap values are shown at each branch point. numbers above the branch reflect bootstrap support from replications. all sites of the alignment containing insertions-deletions or missing data were eliminated from the analysis (option "complete deletion"). sequences of the two aksu strains clustered together with the b. garinii flab sequences (fig. ) . two distinct clades were formed from the sequences of the borrelia spp. ospa phylogenetic tree. the sequences of two strains from zhangye clustered with the b. burgdorferi ospa sequences (fig. ) . in china, lyme disease is caused by various borrelia spirochetes. many of these agents are highly pathogenic to both humans and animals (liu et al., ) . previous studies reported the prevalence of borrelia in field-collected blood samples from cattle, sheep, dogs, rabbits and rats from different areas in china. these studies primarily used serological detection methods and showed that the distribution of borrelia varied considerably in the different areas (hou et al., ; wan et al., ) . the areas of zhangye city, gansu province, and of ili city and aksu city of xinjiang province all include desert regions and are located along the old silk road, halfway between eastern asia and europe, in areas where international livestock trade and travel were frequent (takada et al., ) . bactrian camels were important transportation for trade and travel in the desert within these regions. the bactrian camel can harbour and spread many zoonoses, such as middle east respiratory syndrome coronavirus (mers-cov), anaplasma, toxoplasma gondii, onchocerca, trypanosoma evansi, and parabronema skrjabini luo, ; wang et al., ; yang et al., ; yang et al., ) . ticks are one of the most significant vectors of borrelia burgdorferi s.l. and rf (relapsing fever). domestic animals, rodents, and many other wild animals host ticks, and animals bitten by infected ticks can acquire the pathogen and serve as natural reservoirs. the detection of b. burgdorferi s.l. using pcr is an alternative method that can be used to improve the control and prevention of lyme disease. according to the nested pcr results, field-collected blood samples assayed with primers targeting the s rrna, s- s rrna, flab, and ospa genes revealed ( . %), ( . %), ( . %) and ( . %) positive samples, respectively, from three regions of two provinces in china where these camels live. according to our knowledge, this is the first report of borrelia spp. infection in camels in china, indicating their reservoir role in the maintenance of this organism in the environment. the s rrna gene sequences of borrelia spp. detected from the aksu region had the highest infection rate ( . %), followed by the ili region ( . %). the genetic identity of b. burgdorferi fig. . phylogenetic tree of the ospa gene sequences of borrelia species obtained in the present study and those deposited in genbank from different countries; accession numbers are shown after isolate names. the ospa gene sequences obtained in this study are indicated by bold triangles. the tree was inferred using the neighbour-joining method of mega . ; bootstrap values are shown at each branch point. numbers above the branch reflect bootstrap support from replications. all sites of the alignment containing insertions-deletions or missing data were eliminated from the analysis (option "complete deletion"). spirochetes can be clarified by their differential reactivity with genospecies-specific pcr primers targeting the s- s rrna intergenic spacer amplicon gene. genetic heterogeneity should be further classified by analysing longer sequence data among b. burgdorferi strains that have been previously identified as the same genospecies of atypical strains of borrelia spirochetes (mathiesen jr et al., ; postic et al., ) . moreover, the s rrna gene was detected at a higher positive rate in blood examined for borrelia spirochetes than other genes in previous research (wodecka et al., ) . our study showed that the infection rate of these genes decreased in the order s rrna > s- s rrna > flab > ospa. two borrelia species, b. garinii and b. burgdorferi s.s., were identified, and b. garinii was found to be widely distributed in camels in china. in the present study, b. garinii and b. burgdorferi s.s. were identified in camels from aksu and ili in xinjiang province. sequence and phylogenetic analysis revealed that those isolates were closely related to the corresponding genotypes based on s rrna gene with high sequence similarities ( . %- %, genbank accession numbers: cp ; %, genbank accession numbers: ay ), although the bootstrap values of the phylogenetic tree were relatively low. this finding suggested the genetic diversity of b. garinii and b. burgdorferi s.s in different hosts and geographic locations. interestingly, the sequencing of cloned pcr products from the s rrna gene of borrelia spp. from the ili region showed the presence of a new borrelia species belonging to the relapsing fever group. the s rrna gene sequence of borrelia sp. obtained from camel has a . %, . % and . % similarity to the gene of b. theileri kat strain, borrelia sp. d and borrelia sp. _ _hjf, respectively (genbank accession numbers: kf , ab and ab ). b. theileri belongs to the rf borrelia spp. group and is the causative agent of bovine borreliosis. it was initially identified in cattle and subsequently in goats, sheep and deer from africa, south america, mexico and australia (l, ; mathiesen jr et al., ) . most of the rf borrelia spp. are transmitted by soft-bodied ticks, but b. theileri is found in hard-bodied ticks and is transmitted by rhipicephalus spp., including r. annulatus, r. decoloratus, r. microplus and r. evertsi (barbour et al., ; smith et al., ; trees, ) . this study provides the first report of b. theileri in camel blood samples in china. at present, b. burgdorferi has been isolated from nine ixodes ticks: i. acutitarsus, i. persulcatus, i. granulatus, h. longicornis, h. bispinosa, h. concinna, h. formosensis, boophilus microplus and d. silvarum (niu et al., ) . a previous study reported that the borrelia isolates were isolated from d. marginatus collected from camels in xinjiang, china, and these isolates were genetically identified as b. burgdorferi sensu stricto . the blood samples from bactrian camels in this study were donated by dr. li, who reported that there are ticks available to be collected from these bactrian camels that have been identified as belonging to h. asiaticum, h. dromedarii, r. sanguineus group, and d. niveus . thus, these tick species might act as potential vectors to carry and transfer borrelia spp. that cause camel borreliosis in china. further study is required to determine whether these ticks are competent vectors for borrelia spp. in conclusion, we successfully identified infection with borrelia spirochetes from camel blood samples from different geographic locations of gansu province and xinjiang province in china. b. garinii and b. burgdorferi s.s. were highly prevalent in the sampling areas of the two provinces surveyed. further studies concerning the prevalence of borrelia spp. groups for both lyme disease and relapsing fever should be performed to confirm the presence of these different borrelia species in camels within china. our findings suggest that borrelia infection in camels could potentially present a concern for public health. more detailed and widespread monitoring of tick populations and the screening for borrelia in a greater variety of hosts are warranted in future studies. horizontally acquired genes for purine salvage in borrelia spp. causing relapsing fever genetics of borrelia burgdorferi tick-borne pathogens and associated coinfections in ticks collected from domestic animals in central china. parasites vectors the prospects on camel milk industry development in china origin and evolution on camel detection of borrelia burgdorferi in patients with lyme disease by the polymerase chain reaction seasonal abundance of ornithodoros (o.) savignyi and prevalence of infection with borrelia spirochetes in egypt rats, the primary reservoir hosts of borrelia burgdorferi, in six representative provinces hosts as ecological traps for the vector of lyme disease sur la spirillose des bovidés research progress on animal of lyme disease anaplasma infection of bactrian camels (camelus bactrianus) and ticks in xinjiang studies on relation between lyme disease infection and human, livestock, rodents absence of middle east respiratory syndrome coronavirus in bactrian camels in the west inner mongolia autonomous region of china: surveillance study results from genetic heterogeneity of borrelia burgdorferi in the united states lyme borreliosis caused by diverse genospecies of borrelia burgdorferi sensu lato in northeastern china progress on lyme disease in china diversity of borrelia burgdorferi sensu lato evidenced by restriction fragment length polymorphism of rrf ( s)-rrl ( s) intergenic spacer amplicons expanded diversity among californian borrelia isolates and description of borrelia bissettii sp. nov. 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investigation of parabronema skrjabini disease of camels in inner mongolia region lamp for detection of trypanosoma evansi in the camels research progress on lyme disease identification and molecular survey of borrelia burgdorferi sensu lato in sika deer (cervus nippon isolation of borrelia burgdorferi in ixodes from four counties, in north xinjiang key: cord- -zyw aukk authors: wong, ho him; sanyal, sumana title: manipulation of autophagy by (+) rna viruses date: - - journal: semin cell dev biol doi: . /j.semcdb. . . sha: doc_id: cord_uid: zyw aukk autophagy is an evolutionarily conserved process central to host metabolism. among its major functions are conservation of energy during starvation, recycling organelles, and turnover of long-lived proteins. besides, autophagy plays a critical role in removing intracellular pathogens and very likely represents a primordial intrinsic cellular defence mechanism. more recent findings indicate that it has not only retained its ability to degrade intracellular pathogens, but also functions to augment and fine tune antiviral immune responses. interestingly, viruses have also co-evolved strategies to manipulate this pathway and use it to their advantage. particularly intriguing is infection-dependent activation of autophagy with positive stranded (+)rna virus infections, which benefit from the pathway without succumbing to lysosomal degradation. in this review we summarise recent data on viral manipulation of autophagy, with a particular emphasis on +rna viruses and highlight key unanswered questions in the field that we believe merit further attention. rna viruses have evolutionarily constrained genome sizes. at the same time they have co-evolved efficient means to manipulate host cellular processes to acquire nutrients while evading immune detection. multifunctional viral proteins, molecular mimicry of host components, and the intrinsically high mutagenicity of their rna genome converge to dysregulate host cellular pathways, and exploit metabolic processes to their advantage. one such target is the autophagy machinery. while this cellular degradative process has been historically described to restrict intracellular pathogens including bacteria, parasites and viruses, many have evolved mechanisms to circumvent and even actively benefit from it. autophagy is initiated by sequestration of cytoplasmic proteins and damaged organelles into crescent-shaped double-membrane vesicles known as isolation membranes, long-debated on their membrane source [ ] . the best understood trigger for induction of autophagy is amino acid deprivation, whereby autophagy related proteins (atgs) are recruited to nucleate the isolation membrane, which forms a cup-shaped phagophore. current consensus on the source of autophagosomal membranes is the endoplasmic reticulum [ ] . once contents are captured, the immature isolation membranes expand to forming autophagosomes, which subsequently fuse with lysosomes, thus forming autolysosomes. the contents undergo degradation within the autolysosomes to enable recycling during starvation. about genes have been reported to participate in the process of autophagosomal degradation. the core autophagy proteins are broadly categorised into five complexes: (i) the unc- like kinase (ulk ) complex, (ii) atg , (iii) the class iii pi k complex, (iv) wd repeat domain phosphoinositideinteracting proteins (wipi), and (v) ubiquitin-like atg and atg complexes. although a mechanistic understanding of the process is currently incomplete, formation of phagophore is believed to involve a cooperative activity of the ulk and pi k complexes, along with local phosphatidylinositol synthesis. these activities are followed by recruitment of atg -containing vesicles to phagophore assembly sites, which results in membrane expansion to form the autophagosomes (fig. ). detailed analyses of the known regulatory mechanisms have been reviewed elsewhere [ , ] . apart from turnover of organelles and primarily long-lived proteins, autophagy operates to defend host cells against intracellular pathogens -delivering trapped bacterial or viral products to lysosomes for degradation. besides, it is equipped to clear invasive pathogens through induction of cd + t-cell responses, and also initiates a primordial innate immune response by cooperating with pattern recognition receptor signalling to induce interferon production. this was recently described in the context of dna virus infections, where cyclic gmp-amp (cgamp) and sting-dependent activation of autophagy was necessary to remove viral dna from the cytosol [ ] . however, in an ongoing evolutionary arms race, most pathogens have acquired the ability to hijack and subvert autophagy to evade degradation through this pathway. remarkably, +rna viruses have adapted to not only protect themselves from autophagic elimination but even harness the machinery to their own benefit, as will be covered in more detail in the subsequent sections. among the +rna viruses, data on favourable versus detrimental impact of autophagy is particularly confounding [ ] . a link between autophagosomes and virus-induced vesicles was proposed by george palade by em imaging of poliovirus containing vesicles that resembled autophagosomal membranes [ ] . over the past few decades, a growing body of research has defined the critical role of this pathway in facilitating infection by numerous +rna rna viruses, including poliovirus (pv) [ , ] , coxsackievirus b (cvb ) [ , ] , cvb [ ] , enterovirus (ev ) [ ] , human rhinovirus (hrv) [ ] , foot-and-mouth disease virus (fmdv) [ ] , encephalomyocarditis virus (emcv) [ ] , dengue virus (denv) [ , ] , zika virus (zikv) [ , ] , hepatitis c virus (hcv) [ ] , mouse hepatitic virus (mhv), newcastle disease virus (ndv) [ ] , severe and acute respiratory syndrome coronavirus (sars-cov) [ ] , chikungunya virus (chikv) [ ] , and japanese encephalitis virus (jev) [ ] . in many of the above cases, pharmacological or genetic manipulation of autophagy in vitro confirmed an inhibition in replication and/or spread of these viruses, whereas induction of autophagy resulted in increased production of progeny virions [ , ] . current evidence indicates that many, if not all +rna rna viruses depend on the initiation of the autophagic pathway for their optimal production. this is counterintuitive, since these viruses replicate in the cytosol, and autophagy serves to promote degradation of cytosolic contents. therefore, it is evident that +rna rna viruses in particular have evolved sophisticated mechanisms to circumvent or exploit this pathway. not surprisingly, most +rna rna viruses also trigger massive membrane remodelling within infected host cells to create membrane delineated structures, often referred to as replication organelles, vesicle packets, convoluted membranes or double membrane vesicles, depending on their morphology and ultrastructure [ , ] . whether these replication organelles are pseudo-autophagosomes, has long been a point of contention. several genome-wide screens, e.g with crispr/cas libraries, haploid kbm cells, and shrna depletions, as well as proteomic studies have universally indicated the involvement of the autophagy pathway in +rna rna virus infections [ , , ] . however, among the odd autophagy-related genes, the functional contribution of the individual components in virus infection is far from clear. a recent targeted crispr/cas screen uncovered a fairly diverse range of involvement among autophagic components in three +rna rna virus infections -pv, denv and zikv [ ] . the authors reported that all three viruses employed multiple proteins of the autophagy pathway while bypassing others, and each virus used a unique set of initiation components. a common feature among the tested viruses underscored the requirement of the lc protein but not its canonical cellular lipidation process, where lc was recruited to virally induced membranes by alternative means. this study highlights the importance of assessing the pathway in its entirety when seeking to understand how pathogens co-opt it for purposes of genome replication and spread, as well as to identify universal drug targets. many different mechanisms have been proposed on how autophagy is manipulated to facilitate infection while preventing degradation of +rna rna viruses (fig. ). while in no way exhaustive, the following sections cover the salient features that are recurrent among several viral genera: the physical hallmark of the autophagy pathway is formation of lc + cytosolic double-membrane vesicles, also often observed in +rna rna virus infections. one of the long-running debates is whether these replication organelles are themselves immature autophagosomes or take advantage of the same machinery for their biogenesis. however, canonical autophagic vesicles are part of a degradative pathway, where they fuse with lysosomes for their contents to be hydrolysed by proteases and lipases. viruses from different families appear to possess a diverse set of strategies to prevent this from happening. flaviviruses, such as denv and zikv have been reported to trigger autophagy on the one hand, while utilising the er as a focal point for generating their replication organelles and assembly of progeny virions. consequently, both viruses have evolved means to suppress er-turnover via reticulophagy. the er-localised reticulophagy receptor fam b was identified as a restriction factor for both denv fig. . induction of the autophagy pathway. autophagy is initiated typically from cellular stress, such as starvation, whereby unc- -like kinase (ulk ) complex (comprising ulk , autophagy-related protein (atg ), fip and atg are activated. this complex triggers nucleation of the phagophore by phosphorylating components of the class iii pi k (pi kc ) complex i (consisting of class iii pi k, vacuolar protein sorting (vps ), beclin , atg , activating molecule in beclin -regulated autophagy protein (ambra ) and general vesicular transport factor (p ). this in turn activates local phosphatidylinositol- -phosphate (pi p) production at discrete er sites often referred to as omegasomes. wd repeat domain phosphoinositide-interacting proteins (wipis) and zincfinger fyve domain-containing protein (dfcp ) are then recruited to these phagosome assembly sites followed by recruitment of atg ˜atg -atg l complex that enhances atg -mediated conjugation of atg family proteins, including microtubule-associated protein light chain (lc ) proteins to membrane-resident phosphatidylethanolamine (pe), thus forming the membrane-bound, lipidated form lc -ii -the characteristic signature of autophagic membranes. atg s are required for elongation and closure of the phagophore membrane, and in selective autophagy, are involved in sequestration of specific cargo into autophagosomes. several cellular membranes, most likely the er, contribute to elongation of the autophagosomal membrane by serving as membrane reservoir -delivered by atg -containing vesicles. once sealed, autophagosomal membranes give rise to double-layered vesicles called autophagosomes, which mature and fuse with the lysosomes. autophagic cargo is hydrolysed and recycled back to the cytoplasm. and zikv. rnai-depletion of fam b significantly enhanced denv and zikv replication at an early stage of the viral life cycle. the virusencoded ns protease from several flaviviruses directly cleaved fam b at a single site within its reticulon homology domain to selectively suppress er degradation [ ] , underscoring a sophisticated mechanism to differentially regulate specific arms of autophagy. coxsackievirus b (cvb ), an enterovirus belonging to the picornaviridae family, is known to rely on autophagosome formation for optimal replication [ , ] ; however, both in vitro and in vivo evidence suggest that during infection, amphisome maturation and autophagic protein degradation are inhibited. an increase in autophagosomal abundance concomitant with a decrease in autophagic flux was reported with cvb , prompting the hypothesis that infection selectively triggers autophagosome formation while preventing the terminal stages in degradation [ , ] . the molecular determinants and mechanism by which cvb limits autophagic degradation is currently unknown. interestingly, treatment of cvb -infected cells with inhibitors of autophagosome maturation resulted in increased virus production, indicating that canonical autophagy was not completely blocked in virus-infected cells, and at least a population of the virus remained sensitive. a similar finding was reported more recently with rotavirus where virus replication benefited from induction of autophagy while blocking degradation [ ] . as with cvb , the mechanism by which rotaviruses specifically inhibit autolysosomal degradation has not been elucidated, emphasising the importance of identifying the specific virus or host components that prevent degradation to provide fundamental insights on autophagic regulation in general. the case with hcv infection is more convoluted on account of contradictory data: whereas gfp-rfp-lc expressing cells infected with hcv displayed a complete maturation of autophagosomes followed by fusion with lysosomes [ , ] , atleast one other study reported that hcv replication restricted autophagosomal fusion [ ] . yet another study reported that the autophagy pathway in its entirety was necessary during hcv-infection for optimal replication; however, the advantage derived from it was primarily due to suppression of innate immune responses [ ] . blocking fusion between autophagosomes and lysosomes has also been reported to increase denv yield [ ] . however, this effect may be viral serotype-specific, since inhibiting lysosome fusion reduced denv production [ ] . the mechanisms by which autophagy favors denv production was recently described where rather than replication, assembly and release of progeny virions was affected by blocking autophagy mediated lipid droplet hydrolysis [ , , ] . with coronaviruses, initiation of autophagy appears to be through the er-derived, ptdlns p-enriched omegasomes that normally operate during starvation. infectious bronchitis virus (ibv) -an avian coronavirus responsible for major losses to the poultry industry, is one such example where a significant portion of the genome encodes nonstructural proteins (nsp) dedicated to virus replication. expression of nsp proteins resulted in increased levels of ptdlns p on er membranes, recruitment of ptdlns p effector protein wipi and the generation of autophagosomes directly from the er [ , ] . however, when compared to the properties of starvation-induced autophagosomes, those generated by coronavirus infection or nsp proteins presented significant differences. nsp -induced autophagosomes displayed limited ability to undergo expansion, preventing formation of large autolysosomes, and hence circumvented degradation of viral particles through the lysosomal pathway [ ] . results obtained with ibv, sars and mhv nsp was recapitulated with middle eastern respiratory syndrome coronavirus (mers-cov), where a similar phenomenon was observed for nsp [ ] . a distinctive feature shared by +rna rna viruses is to assemble and replicate on intracellular membranes, which have been proposed to offer a two-fold advantage: (a) scaffold for anchoring and concentrating schematic illustration of the different pathways of selective autophagy that are triggered upon +rna rna virus infections. initiation of autophagosomes is through formation of an isolation membrane most likely derived from the er. depending on the molecular composition and function, they may form either omegasomes, edemosomes or amphisomes. flaviviruses such as zikv non-structural protein a (ns a) and ns b activate autophagy by inhibiting akt and mtorc ; autophagosomes generated are subverted to specialized functions to prevent viral degradation. turnover of organelles occur through convergence of specialised autophagosomes with lysosomes for their selective degradation: er via reticulophagy; mitochondria via mitophagy; lipid droplets via lipophagy and virions or viral proteins via virophagy. viruses that are known to upregulation specific autophagosomal pathways are depicted in black, those that suppress specific types or steps of autophagy are depicted in red. seminars in cell and developmental biology ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the replication complexes and (b) to insulate dsrna intermediates from innate sensing by cytosolic pattern recognition receptors [ ] . the replication complexes are typically composed of the viral rna-dependent rna polymerase, accessory non-structural proteins, viral rna, and host factors. specialised autophagosomes, sometimes referred to as amphisomes (formed upon fusion with endosomes), omegasomes, and ede-mosomes (both er-derived) have been hypothesised to function as replication organelles [ , , ] . given the resemblance of virustriggered double membrane vesicles with that of autophagosomes, it is plausible that generation of replication organelles are a result of mechanisms similar to autophagy [ ] [ ] [ ] [ ] . like autophagosomal membranes, many virus-induced vesicles are believed to be er-derived. recent data from several different studies have provided direct evidence of the association between viral replication complexes and autophagosome structures as summarized in table . poliovirus (pv) vesicle clusters were found to contain lc and lysosomal markers, reminiscent of autolysosomes, and colocalised with the pv replication complex [ , ] . furthermore, formation of infectious pv progeny virions was reported to depend on vesicular acidification, prompting the hypothesis that particle assembly, genome replication and virion maturation occurred in bona fide autophagosomal vesicles [ ] . among the flaviviridae family, several non-structural proteins have been observed in lc + vesicles. denv non-structural protein ns and dsrna were reported to co-localise with lc and ribosomal proteins [ , ] . another study described the induction of lc + vesicles, which colocalised with ns a in infected cells, or when transfected with a combination of denv ns a and ns b [ ] . this was independently corroborated by zikv infection in human fetal neural stem cells where expression of ns a and ns b were sufficient to block neurogenesis and promote autophagy, displaying partial colocalisation with lc + vesicles [ ] . ultrastructural analysis of chikv virions also suggested their location in the lumen of autophagome-like vacuoles [ ] . similar to other members of this family, hcv infection induces massive intracellular membrane rearrangements. competing hypotheses have been proposed as to whether autophagosomes themselves serve as sites for hcv replication. by sucrose gradient analysis, lc -ii was found to co-sediment with hcv rna and non-structural proteins ns and ns a [ ] . however, in a separate study confocal microscopy showed little evidence of co-localisation of lc or atg with hcv proteins [ , ] . along the same lines, depletion of either lamp or rab , which allowed accumulation of autophagosomes by preventing fusion with lysosomes inhibited hcv viral replication, also suggesting that they are not the major sites for hcv genome replication [ ] , while multiple reports indicate that lipid droplets are the more likely sites for replication and assembly, as reviewed elsewhere [ , ] . conflicting evidence also exists for mhv-induced replication compartments. on the one hand, mhv replication complexes were found to be associated with lc and atg in embryonic stem cell lines [ ] . on the other hand in primary macrophages and murine embryonic fibroblasts mhv replication did not require the autophagy gene atg [ ] . differences in permisiveness to infection often exists between primary cells and transformed cell lines, as does viral tropism towards distinct cell types, either of which can account for these experimental discrepancies. among other +rna rna viruses, immunoelectron microscopy demonstrated co-localisation of ev capsid protein vp with autophagosomes in virus-infected mouse neurons [ ] . similarly, during emcv infection, colocalisation of non-structural protein a and capsid protein vp was visualised by confocal and immunoelectron microscopy [ ] . colocalisation of non-structural proteins b, c, and a with lc , and structural protein vp with atg were also reported in fmdv-infected cells [ ] . although direct evidence of the association of viral replication complexes with autophogasomes is lacking for cvb , impaired maturation of autolysosomes brought about through pharmacological or genetic inhibition increased the accumulation of autophagosomes in virus-infected cells resulting in enhanced viral replication [ , ] . these data implicated autophagosomes as virus anchoring and replication sites during cvb replication. delineating the process of viral assembly from replication is technically challenging, especially since both processes would very likely induces formation of autophagosome-like double-membrane liposomes [ ] summary of interactions between proteins from positive strand rna viruses and host autophagy machinery. seminars in cell and developmental biology ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] occur in concert at the same sites. although utilisation of autophagosomes as assembly sites has been recorded for some dna-viruses e.g., hepatitis b virus (hbv) [ ] , experimental data with +rna rna viruses are scant. however, atleast for denv, recent evidence indicates that autophagy might actually assist in assembly of progeny virions, without them serving as replication organelles. one of the initial studies describing the induction of autophagy in flavivirus infections was performed by lee et al [ ] . the authors demonstrated that denv infection in hepatocytes induced autophagy; targeting with either the inhibitor -methyladenine ( ma) or sirnas against autophagy genes compromised infection. denv-induced autophagosomes colocalised with lamp , a marker of lysosomal fusion, which was independently validated by immunofluorescence assays and pharmacological inhibition. following this initial characterisation, a more mechanistic study emerged describing the role of selective autophagy facilitating hydrolysis of lipid droplets in an infection-specific manner [ ] . apart from the relatively non-specific bulk macroautophagy, cellular organelles are turned over through several types of selective autophagy. this phenomenon occurs under normal physiological conditions and is hypothesised to initiate a physiological response to appropriately address a specific stress. in the context of denv infection, a type of selective autophagy of lipid storage organelles (lipid droplets) referred to as lipophagy was described that hydrolyses neutral fat deposits to free fatty acids and cholesterol, and supplements cellular energy reservoirs [ , , ] . heaton et al performed a targeted sirna screen to identify cellular cofactors of denv replication in hepatocytes, which revealed, among others, genes involved in the induction of autophagy [ ] , and were further characterised to reveal that denv induced autophagosomes not only acquired lamp , but underwent complete maturation to become autolysosomes [ ] . these did not colocalise with markers of the viral replication complex, suggesting that they may play an indirect, non-structural role in denv replication. a subsequent study described the involvement of aup , a type-iii membrane protein, in the initiation of virus-induced autophagy. aup was regulated by monoubiquitin modification, where infection or a combined expression of viral ns a and ns b were necessary and sufficient to generate the unmodified form of aup -a step that was critical in induction of this pathway. interestingly, loss of aup did not affect viral replication; however, impaired autophagy was accompanied by degradation of viral proteins through the proteasomal pathway resulting in significantly reduced production of progeny virions, supporting a specific role of aup -dependent lipophagy in assembly of virus particles [ ] . these results were in agreement with an independent report demonstrating expression of dengue ns a was sufficient to trigger autophagy and protect against cell death. a growing body of evidence indicates that mitochondrial function is altered during flavivirus infections. although the mechanistic underpinnings are currently not well understood, atleast for hcv, parkindependent mitophagy has a significant effect on virus propagation. this was verified by silencing parkin and pink , which inhibited hcvtriggered mitophagy and in turn blocked virus replication. ultrastuctural analyses by electron microscopy and immunoelectron microscopy also confirmed the presence of damaged mitochondria in double-membrane vesicles in hcv-infected cells [ ] . whether this pathway is activated during infection by other flaviviruses is currently not known. several reports on mechanisms of secretion and cell-to-cell transfer of intracellular pathogens indicate non-degradative autophagic vesicles as an efficient mode of transport. an recent study with mycobacterium demonstrated that autophagosomes chaperone an organelle referred to as the "ejectosome", facilitating cell-to-cell spread of cytosolic bacteria [ ] . secretory autophagy is a newly discovered pathway in which autophagosomes fuse with the plasma membrane instead of lysosomes and release single membrane vesicles containing cytosolic content into the extracellular milieu [ ] . non-degradative autophagy has been suggested to facilitate nonlytic egress of some +rna rna viruses. the initial characterisation was with enteroviruses, which appear to exploit this pathway to exit cells, and are released into the extracellular environment as particle populations contained within vesicles [ ] . clusters of enteroviral particles were packaged with phosphatidyl serine into autophagic vesicles, which enabled efficient transfer to primary macrophages, significantly enhancing viral infectivity. this revealed a novel mode of transport where viral genomes were transferred en bloc to recipient cells, facilitating genetic cooperativity and enhancing infection. this mode of transfer had previously also been noted for cvb , where a recombinant fluorescent virus was released into the extracellular medium in microvesicles containing autophagic markers [ ] . poliovirus is often considered a lytic virus; however, non-lytic release of poliovirus has also been reported [ ] . reduced levels of viral particles in extracellular medium in autophagy-deficient cells correlated with inhibition of non-lytic release of autophagic vesicles. more recently, an important role of the secretory autophagy pathway was implicated in zikv vertical transmission as well as cell-to-cell spread of denv. zikv-induced autophagic activity in human trophoblasts restricted by pharmacological inhibition, or by deficiency in an essential autophagy gene, atg l , limited zikv vertical transmission and improved placental and fetal outcomes, which supported a role for autophagic secretion in the process [ ] . along the same lines, it was hypothesised that denv might evade neutralising antibodies and increase viral spread by exploiting autophagic vesicles for delivery to the extracellular medium [ ] . double staining of denv e antigen and lc in a close-contact co-culture experimental set-up verified secretion of denv-containing autophagic vesicles from donor cells, which were subsequently taken up by recipient cells. in a parallel study, maturation of infectious denv virus particles was attributed to this process, when cleavage of pr peptide from prm by the furin protease was prevented upon blocking autophagy [ ] . further investigation is needed to provide more direct evidence on the mechanism, regulation and molecular determinants of secretory autophagy in facilitating viral release. activation of autophagy represents a fairly ubiquitous response to eliminate intracellular pathogens. several studies have described mechanisms where pathogen recognition receptors trigger this response upon detection of microbe-specific pathogen associated molecular patterns. a diverse set of pathogens including bacteria, viruses and parasites have provided corroborating evidence supporting this phenomenon. whether this process occurs in parallel to non-degradative autophagy, or the cross-talk that might exist between the two flavours of autophagy during +rna rna viral infection merits further investigation. recent studies have shed light on how autophagy offers an advantage to hcv infection by suppressing innate immune responses [ , ] . this contrasts with previous data on hcv-induced incomplete autophagy and defined a pathway where the entire process from initiation through lysosomal degradation is necessary for hcv replication largely to suppress anti-viral innate immune response. in hcv-infected cells, interferon-β (ifn-β) production could be modulated by uprmediated autophagy; activation of this pathway reduced ifn-β production and vice-versa [ ] . inhibition of autophagy by suppressing beclin- or atg reduced hcv replication, which was accompanied by the activation of ifn signaling. a similar phenomenon was also proposed for denv where activation of autophagy not only facilitated viral h.h. wong and s. sanyal seminars in cell and developmental biology ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] replication, but also suppressed ifn-i production, suggesting that both viruses may share the same mechanism to evade innate immune responses. interestingly, in west nile virus infections, perturbation of intracellular cholesterol levels dictated ifn-i responses [ , ] . although the role of autophagy in wnv infection has been contested, replication was reported to occur independent of autophagy by mutiple groups -a clear difference from other flaviviruses; however, whether it plays a role in regulating free cholesterol and fatty acid levels is yet to be determined. together, these studies indicate a critical mechanism by which flaviviruses may avoid innate immune responses through activating the host autophagy pathway. the link between autophagy and apoptosis has been defined extensively in various contexts including cancer, neurodegenerative disorders, and infectious diseases [ ] . a more comprehensive understanding on circumventing cell death has been recorded for bacterial rather than viral infections, amongst which most are dna viruses. premature cell death can function as an anti-viral host mechanism by providing an unfavorable environment and shorter timeframes for viral propagation. induction of autophagy has often been linked to inhibition of apoptosis [ , ] . both denv and a murine flavivirus-induced autophagy was reported to prevent apoptosis mediated via the viral ns a protein [ ] . knockdown of autophagy-related gene expression abolished the protective role of autophagy against cell death and resulted in reduced viral replication. apart from denv, cross-talk between autophagy and apoptosis was also reported in cvb infection, where suppression of autophagy by ma triggered caspase activation and vice-versa [ ] . an interesting question that arises from virus-triggered induction of autophagy in the context of flavivirus infections is the process of antigen presentation. particularly in major histocompatibility complex (mhc)-ii positive cells, autophagosomes are constitutively generated to deliver viral antigens on mhc-ii molecules for adaptive immune responses. virus infections therefore frequently impede maturation of antigen presenting cells and subsequent adaptive immunity as reviewed in detail elsewhere [ ] . monocytes and monocyte-derived cells are a major target of flaviviruses, where antigen presentation is facilitated by autophagy. this implies that while autophagy favours production of viral progeny, it should simultaneously increase viral antigen presentation and t-cell responses, thus generating neutralising antibodies and promoting cytotoxic t-cell killing. information on these seemingly contradictory processes is currently limited. however, denv-infected human monocyte-derived dcs fail to upregulate mhc and co-stimulatory molecules and have an impaired ability to polarize cd + th type (th ) effector properties [ ] , contributing to inefficient adaptive immune responses observed in patients. in bulk cultures of dendritic cells, exposure to denv augments mhc-i and mhc-ii expression in non-infected bystander cells; however, infected monocyte-derived dendritic cells display an inhibition in this process within the same cultures [ ] . in clinical studies gene expression analyses of denv patients revealed that severe cases expressed lower levels of genes linked to antigen processing, presentation and t-cell activation compared to mild cases. another related flavivirus, japanese encephalitis virus, inhibits expression of mhc-i and induces functional impairment of dcs, resulting in poor cd + t cell responses [ ] . thus impaired antigen presentation and functionality of virus-infected dcs may reflect a viral immune escape strategy to dampen t-cell responses and impact disease severity. one study reported that denv activated autophagy only during the early infection stage, suggesting a biphasic response of autophagy to denv infection, where it shifted from a supporting to an antiviral role at later time points [ ] . these results might enable us to reconcile how flaviviruses have evolved strategies to manipulate this pathway while subverting t-cell based immune responses. a quantitative and time resolved analyses of this process in virus-infected cells might shed light on its utilisation in the benefit versus detriment towards virus production. activation of autophagy in the presence of intracellular pathogens is a fairly universal cellular response. xenophagy as an intrinsic defence mechanism was first described through electron microscopy studies upon visualising hsv- and cytomegalovirus inside autophagosomes [ ] . among viruses, autophagic protection has been recorded in a wideranging species and genera [ ] [ ] [ ] . the mechanism of autophagymediated restriction of +rna rna viruses is less well-documented on account of its proviral influence in most cases. however, there are instances where autophagic degradation of virions (virophagy) or viral proteins has been observed, especially in neurons where it is a critical form of antiviral defence. during sindbis virus (sinv) infection, beclin and p -dependent degradation of the capsid protein protects against sinv-mediated encephalitis [ , ] . moreover, atg deficiency results in delayed sinv clearance and accumulation of the autophagy receptor p . more recently, fanconi anaemia group c protein (fancc) was found to interact with the sinv capsid protein and facilitate virophagy [ ] . picornaviruses, such as pv and hrv permeabilise endosomes to release their genome into the cytosol. this step is detected by galectin , which restricts viral infection by initiating degradation of the viral rna genome [ ] . as counterstrategy, the host protein hras-like suppressor (pla g ) is exploited by the virus to enable genome delivery. cvb , also belonging to the same family, undergoes p -dependent degradation and uses the viral protease a to cleave p and inhibit virophagy [ ] . interestingly, although hcv has been demonstrated to induce autophagy to its own advantage by multiple groups, one study demonstrated that an er transmembrane protein, scotin, interacted with the viral protein ns a, resulting in its autophagic degradation to suppress viral replication [ ] . among flaviviruses, autophagosomal degradation of neurotropic viruses has been recorded. in the drosophila brain, zikv infection triggered nfκb-dependent inflammatory signaling, inducing expression of dsting which subsequently restricted infection by upregulating autophagy. defective or absence of autophagy resulted in increased infection in the fly brain and death [ ] . sting-dependent induction of protective autophagy was independently reported for dna viruses and sea anemone, supporting an evolutionarily conserved role for sting in microbial autophagy [ ] . despite major advances in elucidating molecular determinants of the autophagy pathway, the rules that govern its utilisation during infections are far from obvious. a complex interplay between viral manipulation and host innate immunity dictates disease outcomes. autophagy is expected to restrict viral infections at multiple levels by eliminating viruses, regulating inflammatory responses and promoting antigen presentation. however, +rna viruses have co-evolved to manipulate autophagy for immune evasion, replication, assembly and release from infected cells. the repertoire of universal and distinct mechanisms that these viruses draw on to interfere with autophagy are striking, often targeting the same pathway in unique ways with different functional implications. distinct viral strategies fine-tune the process to simultaneously escape destruction while capitalising on the structural and nutrient benefits that autophagy provides. several gaps remain in our understanding within the remit of viral manipulation of autophagy. first and foremost, more advanced strategies of isolating autophagic vesicles will be imperative for better characterisation of this process. emerging data indicate that many lc -positive vesicles that h.h. wong and s. sanyal seminars in cell and developmental biology ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] are induced upon virus infections are not autophagosomes. a combination of ultracentrifugation, density gradient separations and enrichment techniques will be necessary to characterise these vesicle populations. furthermore, in the context of virus infection, the equilibrium between degradative and secretory autophagy versus biogenesis of exosomal vesicles will need to be quantitated to arrive at firm conclusions regarding the functional outcome of autophagy. along the same lines, differences between infected host cells and neighbouring cells will become important to develop a more complete picture of its impact in viral pathogenesis versus immune responses. further understanding of the contribution of autophagy to the different stages in the viral lifecycle, subversion of its antigen presentation function and innate immune responses is therefore necessary to delineate the diverse functions of autophagy in virus pathogenesis. a current perspective of autophagosome biogenesis endoplasmic reticulum and golgi complex: contributions to, and turnover by mechanism and medical implications of mammalian autophagy autophagy during 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virus subverts cd + t cell responses dengue virus inhibition of autophagic flux and dependency of viral replication on proteasomal degradation of the autophagy receptor p herpes simplex virus and human cytomegalovirus replication in wi- cells. iii. cytochemical localization of lysosomal enzymes in infected cells autophagy is an essential component of drosophila immunity against vesicular stomatitis virus hsv- icp . confers neurovirulence by targeting the beclin autophagy protein autophagy pathway intersects with hiv- biosynthesis and regulates viral yields in macrophages protection against fatal sindbis virus encephalitis by beclin, a novel bcl- -interacting protein autophagy protects against sindbis virus infection of the central nervous system pla g represents a switch between entry and clearance of picornaviridae calcoco /ndp and sqstm /p differentially regulate coxsackievirus b propagation interferon-inducible protein scotin interferes with hcv replication through the autolysosomal degradation of ns a inflammation-induced, sting-dependent autophagy restricts zika virus infection in the drosophila brain amino acid substitutions in the non-structural proteins a or b modulate the induction of autophagy in west nile virus infected cells independently of the activation of the unfolded protein response the role of secretory autophagy in zika virus transfer through the placental barrier japanese encephalitis virus replication is negatively regulated by autophagy and occurs on lc -i-and edem -containing membranes rab and class iii phosphoinositide -kinase vps are involved in hepatitis c virus ns b-induced autophagy hepatitis c virus upregulates beclin for induction of autophagy and activates mtor signaling the autophagy elongation complex (atg - / l ) positively regulates hcv replication and is required for wild-type membranous web formation coronavirus membraneassociated papain-like proteases induce autophagy through interacting with beclin to negatively regulate antiviral innate immunity coronaviruses hijack the lc -i-positive edemosomes, er-derived vesicles exporting short-lived erad regulators, for replication coronavirus nsp proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate cleavage of sequestosome /p by an enteroviral protease results in disrupted selective autophagy and impaired nfkb signaling protein b of coxsackievirus b induces autophagy relying on its transmembrane hydrophobic sequences enteroviruses remodel autophagic trafficking through regulation of host snare proteins to promote virus replication and cell exit foot-and-mouth disease virus capsid protein vp activates the cellular eif s -atf pathway and induces autophagy via hspb viroporin activity of the foot-and-mouth disease virus non-structural b protein modification of cellular autophagy protein lc by poliovirus double-membraned liposomes sculpted by poliovirus ab protein this work was supported by health and medical research funds ( and and ), and partially by research grants council-general research funds ( ). ss is supported by the croucher foundation. key: cord- - rsk g l authors: kinast, volker; burkard, thomas l; todt, daniel; steinmann, eike title: hepatitis e virus drug development date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: rsk g l hepatitis e virus (hev) is an underestimated disease, leading to estimated million infections and up to , deaths annually. infections are mostly asymptomatic but can reach mortality rates up to % in pregnant women or become chronic in immunocompromised patients. the current therapy options are limited to the unspecific antivirals ribavirin (rbv) and pegylated interferon-α (pegifn-α). rbv leads to viral clearance in only % of patients treated, and is, similar to pegifn-α, contraindicated in the major risk group of pregnant women, emphasizing the importance of new therapy options. in this review, we focus on the urgent need and current efforts in hev drug development. we provide an overview of the current status of hev antiviral research. furthermore, we discuss strategies for drug development and the limitations of the approaches with respect to hev. with approximately million infected people per year, hepatitis e virus (hev) leads to more cases of acute hepatitis than any other human hepatotropic virus, such as hepatitis a virus (hav), hepatitis b virus (hbv), hepatitis c virus (hcv) and hepatitis d virus (hdv). hev is a quasi-enveloped positive strand rna virus ( figure a ) that is classified as a member of the genus orthohepevirus within the family of hepeviridae [ ] . the genotypes (gt) and of hev are obligate human pathogens and are primarily transmitted via contaminated drinking water. recent outbreaks of acute hepatitis linked to hev have, amongst others, been reported in nigeria [ ] , chad [ ] , and bangladesh [ ] . by contrast, the zoonotic gt and , which are endemic especially in europe and the americas, can cause in addition to acute infections also chronic infections in immunocompromised individuals. the most common infection routes are thought to be the consumption of undercooked meat of or contact with infected animals, such as pigs, wild boars, and deer, which constitute the virus reservoir. furthermore, the transfer of contaminated blood products is a man-made safety hazard, especially for risk groups such as immunocompromised patients [ ] . recent cases of human infections with gt [ ] and rat hev [ , ] extended the spectrum of hev gts, which are capable of jumping over the species barrier and are able to infect humans. acute hev infections are self-limiting in most cases, but especially for gt , they are linked to high mortality rates up to % in pregnant women [ ] . it is hypothesized that immunological and hormonal changes are responsible for the high mortality [ ] . hev has also been reported to cause a variety of extrahepatic manifestations, for instance, guillain-barré syndrome or pancreatitis (reviewed in [ ] , see figure b ). in total, . million estimated cases of acute illness and , - , deaths per year make this pathogen a non-negligible health burden. however, the current therapeutic options against hev are limited to the off-label use of the unspecific antivirals ribavirin (rbv) and pegylated interferon-α (pegifn-α). the treatment algorithm for figure . schematic representation of hepatitis e virus (hev) particle and the major clinical manifestations. (a) hev particle and genomic organization. the hev genome is composed of a single-stranded rna genome of~ . kb and is encapsulated in an icosahedral capsid. hev virions can occur in both a non-enveloped and in an enveloped form. the viral rna, which is capped with -methylguanosine ( mg) at the ´noncoding region and polyadenylated at the noncoding region, comprises three open reading frames (orf). furthermore, gt is believed to contain an additional orf (orf ). orf encodes the replicase proteins, including a methyltransferase (mt), cysteine protease (pro), helicase (hel), and rna polymerase (pol), as well as three regions without a reported enzymatic function (y, hypervariable region (hvr), and x). orf encodes the capsid protein, whereas orf encodes a viroporin. (b) major clinical manifestations. the majority of hev infections are asymptomatic. gt and gt infections can become chronic in immunosuppressed individuals, with high risk for developing severe complications, such as liver cirrhosis. hev has also been reported to cause a variety of extrahepatic manifestations, like guillain-barré syndrome. infections with hev gt cause acute hepatitis, with high mortality rates up to % in pregnant women. identification of novel therapy options can encompass several strategies. while some rely on de novo identification of compounds, another approach is to reuse already existing compounds, a process which is termed drug repurposing. drug repurposing can generally be described as the idea of using drugs or drug candidates for another than their primary indication. the pharmacokinetic and pharmacodynamic profile, including undesirable effects, is often already elucidated in vivo in animals and humans. the current first choice of anti-hev treatment, rbv, is an example for a repurposed drug that was originally developed for treatment of respiratory syncytial virus (rsv) in infants [ ] . rbv has not only been administered for antiviral therapy against rsv but also against hcv, influenza, viral hemorrhagic fevers [ ] and hev [ ] . recently, drug repurposing has gained increased attention in the scientific community with reviews covering either specific viruses [ ] or viral diseases in general [ , ] . surprisingly, there are only a few publications, in which this approach is used as a strategy to find an antiviral against hev. however, given the availability of public fda-approved compound libraries and the anticipated benefits of this approach over de novo development, publications screening for antiviral activity of already approved drugs are anticipated. de novo drug development can rely on screens, where compound libraries are tested for their capacity to interfere with the viral life cycle. both the target and the mode of action of the substance do not need to be identified. for structure-guided development, the target and ideally its crystal structure is already identified, which enables to specifically design antivirals. independent from the approach used to identify a prospect compound, the candidates have to be validated in vitro and in vivo. there are several in vitro models available, including different cell culture models as well as primary human hepatocytes and induced pluripotent stem cells. for a detailed overview, please see a recent review by meister et al. [ ] . similarly, there are several small animal models as rabbits, rats, ferrets, and birds, which are used for the different hev strains (reviewed in [ ] ). this review aims to give an overview over the current state of efforts to establish additional treatment options against hev. antiviral candidates are grouped according to the strategy that was used to identify them. before the concluding remarks, vaccination is also briefly discussed in terms of its potential to replace antiviral therapy options. '-c-methylcytidine ( -cmc), also known as nm , is a nucleoside analogue (na) originally developed against hcv. when first applied against hev in vitro, the compound demonstrated an inhibitory effect against hev replication (ic of µm/l) [ ] . furthermore, it inhibited the replication of both a luciferase replicon based on kernow c as well as the full-length virus without showing signs of resistance upon prolonged incubation [ ] . the effect could be reverted by addition of cytidine triphosphate, but not guanosine triphosphate to the cells. applying it together with rbv yielded in a moderate antagonistic effect, suggesting a convergent mechanism of inhibition. when assessed for treatment of hcv, insufficient oral bioavailability was reported. therefore, a prodrug termed nm was developed [ ] . however, the development was discontinued due to adverse toxic effects, which has been a problem for several 'methyl nucleosides [ ] . the toxicity correlates with the property to serve as a substrate for the mitochondrial dna polymerase and thus leading to the termination of mitochondrial rnas [ ] . the example of nm emphasizes that -cmc, although efficacious against hev in vitro, will probably need modifications to rule out undesired effects, in particular against the mitochondrial dna polymerase. recently, netzler and colleagues tested a collection of compounds with a reported inhibitory effect on the rna-dependent rna polymerase (rdrp) of different classes of viruses [ ] . both the class of nas as well as non-nucleoside inhibitors (nni) were considered in their study, covering compounds from late-preclinical stage to fda-approved drugs. they identified two compounds, gpc-n and nitd , inhibiting a gt derived subgenomic replicon. gpc-n had a half maximal effective concentration (ec ) of . µm and a therapeutic index (ti) of > (gpc-n ) whereas nitd had an ec . µm and a ti > . gpc-n has been described as an nni of the rdrp of multiple genera within the picornaviridae family [ ] . nitd has been reported to act as an antiviral on numerous viruses, amongst others hcv [ ] , west nile virus [ ] or zika [ ] . nitd has shown toxicity in vivo [ ] . so far, no clinical trials have been registered for both of these compounds. nishiyama and colleagues used an fda approved drug library for an in vitro screen against a gt strain containing a gaussia luciferase reporter [ ] . ciprofloxacin (cpfx) and ifn-λ - showed the best activity against the reporter genome. they also used an infection model, where cells infected with a full-length gt strain at a plateau phase were co-cultivated with naïve cells. all ifn-λ subtypes, but not cpfx, showed robust reduction of hev rna. sofosbuvir is a nucleotide prodrug, which can be incorporated into hcv rna by the ns b rdrp acting as a chain terminator [ ] . its antiviral potential against hev was demonstrated using gt hev replicons in huh and hepg cells. the ic values were in a low micromolar range, to orders of magnitude less potent than against hcv replicons [ ] . sofosbuvir showed an additive effect together with rbv. the inhibitory effect was confirmed by the same group in induced pluripotent stem-cell derived hepatocyte-like cells (ipsc-hlcs) for all four common gts [ ] and in cell culture for gt [ ] as well as gt [ ] by other groups. however, it was also described that both gt and gt replicons as well as full-length gt virus were not inhibited in hek t, u- mg, and huh cells [ ] . very recently, li et al. reported that a gt strain was moderately inhibited at µm by an isopropyl ester of sofosbuvir, psi [ ] . similar to the situation in vitro, the data on sofosbuvir's efficacy in vivo have been inconclusive. three case studies stated that sofosbuvir failed to clear hev in patients when administered together with rbv [ ] [ ] [ ] , while three reported successful treatment together with rbv [ ] [ ] [ ] . a multicenter phase ii study addressed whether sofosbuvir monotherapy is efficacious against hev. a total of mg sofosbuvir was administered once daily over a course of weeks to nine patients who had previously failed rbv therapy [ ] . none of the patients cleared the virus, although it reduced hev rna levels by at least one order of magnitude in five out of nine patients during the study period and significantly reduced alanine aminotransferase (alt) levels. with its antiviral efficacy being only moderate, it is probably not suited for use as monotherapy. however, it could further be investigated in combination with rbv. compound screening allows for the rapid testing of a large number of chemical substances and extracts with the aim of identifying new compounds. in the context of viral infections, important drugs identified with this method include maraviroc and etravirine [ ] , both of which target hiv as well as daclatasvir, which targets hcv [ ] . prerequisite for compound screening is a suitable assay, reflecting the physiological conditions of infection. importantly, the screening method has the benefit of not necessarily requiring profound knowledge about the viral lifecycle or specific targets. assays using purified enzymes, subgenomic replicons or full virus already have successfully been used in hcv research. there have been two reports about the antiviral activity of ethanol extracts of plants against hev. one extract was prepared from the plant lysimachia mauritiana and showed activity against a pshev -luc replicon in huh . cells, as well as while a full-length gt virus on rna and protein level in a cells [ ] . however, identification of the compound(s) responsible for the antiviral effect was not performed. the same group reported the antiviral effect of an ethanol extract of another plant, liriope platyphylla, against hev [ ] . both a pshev -luc-replicon as well as the c full-length genome were inhibited by the extract in huh . cells. by performing activity-guided fractionation and multicolumn chromatography, spicatoside a could be identified as the active compound, and its activity as a pure compound was demonstrated. no testing for toxicity, resistance induction or in vivo efficacy was conducted. zinc is an essential micronutrient that has been reported to reduce replication of, amongst others, hiv [ ] and coronavirus [ ] in high concentrations. in a study investigating the influence of different salts on hev replication, it was demonstrated that zinc salts inhibited replication of both gt and replicons as well as a gt clinical isolate in porf -huh cells [ ] . the effect was likely due to an inhibition of the rdrp, as an inhibitory effect on the protein was observed in vitro. a mild effect could be observed at µm, being strongest at µm. it will be of interest to investigate if zinc supplementation will prove an effective strategy against hev in patients. zinc levels in plasma have been reported to be usually around - µm depending on the study, as described in a meta-analysis [ ] . the authors also found that zinc supplementation increased plasma levels, with every doubling of the dose leading to a % increase [ ] . these data imply that zinc levels are highly regulated, suggesting that effective plasma levels in patients might be at best challenging to reach. there has been no evaluation of zinc monotherapy against hev in vivo so far. however, a case study [ ] investigated the influence of intra-erythrocyte zinc levels on the outcome of rbv treatment. they showed that treatment successes could not be attributed to increased zinc levels. although only comprising four patients in total, this suggests that it might not lead to viral clearance in immunocompromised individuals when combined with rbv. e is part of a small molecule compound library belonging to the diversity set ii of national cancer institute developmental therapeutic program. it was initially identified as an inhibitor of the rdrp of hcv, inhibiting hcv a replicon with an ec of . µm [ ] . the compound also inhibited hev, both the replicon p -luc as well as full-length p in huh or huh s - cells. interestingly, it did not show inhibitory effect on purified rdrp for both hcv and hev, suggesting that it does inhibit replication either by a affecting a host factor or that it does undergo modification within the host. no testing resistance induction or in vivo efficacy was performed. different from screening approaches, target-or structure-guided development aims at identification of suitable targets for an intervention first. prerequisite is that the function of a target is characterized. both the virus itself and the host factors necessary for the viral life cycle can be targeted ( figure ). only one protein of hev has been crystallized so far, which is a truncated version of the capsid protein porf [ , ] . although some conclusions regarding the involvement of certain domains in cell binding could be drawn, and heparan sulfates seem to be important for hev attachment to the host cell surface [ ] , this knowledge has not led to the establishment of a therapy concept yet. the scarcity of antiviral candidates directly acting on the virus can be explained by the lack of knowledge about hevs molecular virology. due to their small genomes, viruses depend on host factors for completion of their life cycle. this dependence on the host is a potential point to intervene. host-acting antivirals have the potential to interfere with the distinct steps of the viral life cycle, from blocking the entry receptor over preventing the formation of the replication complex to hamper the viral maturation by inhibiting cellular proteins. a detailed functional analysis of the host factor and its role in the context of hev infection is desirable and would help to optimize chemical intervention. additionally, direct acting antiviral specifically can target viral enzymes (e.g., helicase, polymerase, protease) without affecting host components. notably, the nucleoside analog ribavrin is reported to exert antiviral effects by targeting both the virus and the host [ ] . a hammerhead ribozyme is a small catalytically active rna molecule, capable of cleavage or self-cleavage [ ] . hammerhead ribozymes have been designed to target, amongst others, severe acute respiratory syndrome coronavirus (sars-cov) [ ] or hcv [ ] . however, there has only been one report so far describing the use of this technique in vivo [ ] , which dates back already more than a decade. in , it was reported that a hammerhead ribozyme could be designed to specifically cleave rna of hev gt in the 'cis-acting element [ ] . when expressed from a vector in hepg cells, they could decrease replication of a psgi-hev- 'luc construct. questions about delivery of the agent to target site in vivo in patients, as well as potential off-target effects, might remain unsolved, considering that there has been no follow-up study so far. peptide conjugated phosphorodiamidate morpholino oligomer (ppmo) are uncharged nucleic acid analogs containing a morpholine backbone connected by phosphorodiamidate linkage, rendering them resistant to many enzymes like nucleases, esterases, and proteases [ ] . ppmos bind rna via watson-crick base pairing and interfere with viral translation by steric blockage. ppmos complementary to sequences in sar have been shown to inhibit replication of a psk-e replicon in huh s - cells. they also reduced porf levels in hepg /c cells infected with kernow c and huh s - cells infected with psk-e [ ] . so far, there has been no follow-up on this study. mg , an inhibitor of the proteasome with low nanomolar inhibitory constant [ ] has been suggested as an antiviral against hev. it reduced rna and protein levels of a hev gt replicon in huh s - cells [ ] . however, when others reproduced the experiment in huh cells, they also found reduced expression of several housekeeping genes as well as overall rna levels [ ] . it is therefore assumed that mg inhibitory effect on hev is unspecific. some viruses can bind the cellular protein tumor susceptibility gene (tsg ) to hijack the escrt machinery for egress, for instance, hiv- [ ] . it was demonstrated that porf of hev can bind tsg via a psap motif in the viral protein and does colocalize with it in hev-transfected cells [ ] . consequently, porf is essential for viral egress, while having a neglectable effect on cell binding and replication [ ] . the interaction with tsg is mediated via the psap motif in the porf [ ] , the same motif that p gag of hiv- uses to engage with the cellular protein [ ] . cyclic peptides (cp) that had been developed to abrogate interaction of p gag and tsg and inhibited viral release of hiv virus like particles (vlps) [ ] were tested for their activity against hev [ ] . one of the inhibitors, cp , inhibited interaction of tsg and porf both in a yeast-three hybrid screen as well as in a pulldown assay. viral release of both gt and gt hev from huh porf or huh cells, respectively, was inhibited without showing significant toxicity. inhibition of viral release by targeting porf /tsg interaction is an interesting mechanism for antiviral development, with cp providing a starting point for further compound development and characterization. in general, targeting production of nucleotide synthesis to deplete or imbalance nucleotide pools is a strategy employed against several viruses [ , ] . the compounds rbv and mycophenolic acid (mpa), both of which target enzymes involved in nucleotide synthesis, are either already used as treatment against hev or have been reported for their potential to inhibit the virus. as its ester mmf, mpa inhibits the inosine- -monophosphate dehydrogenase (impdh), which is a pivotal enzyme in the purine nucleotide synthesis and is part of immunosuppressive regimens in organ transplant recipients. in a german study, a tendency was identified for its use in heart-transplant recipients being linked to clearance of hev infection without development of chronicity [ ] . in huh cells, mpa inhibited replication of a p -luc replicon, an effect that could be reverted by supplementation of guanosine, suggesting inhibition of impdh as inhibitory mechanism [ ] . in the same study, an additive effect with rbv was found. these results could not be confirmed in patients. a french study assessed the effect of immunosuppression by mmf on rbvs' potential to lead to a sustained virological response (svr) [ ] . they did not find evidence for an additive effect of mmf on rbv treatment. because of its immunosuppressive effect, mmf monotherapy to treat hev infections seems unlikely. given the lack of evidence for an additive effect with rbv, it will probably also not be used to treat hev infections together with rbv. although mmf seems unsuited as a drug against hev, targeting the impdh or enzymes involved in the purine synthesis pathway might still be a starting point for further studies. in a proof-of-concept study, wang and colleagues [ ] tested custom designed inhibitors of the impdh. all inhibitors decreased replication of a p -luc replicon, emphasizing the potential of this target. furthermore, inhibitors of pyrimidine synthase like brequinar and leflunomide inhibited the replicon. this is especially interesting, since both drugs have been tested in clinical trials and are therefore better characterized than newly developed inhibitors. targeting nucleotide synthesis, especially of pyrimidines, might therefore be an interesting approach to tackle hev, with brequinar and leflunomide already providing starting points for testing in vivo. it would be of interest to characterize these compounds better to evaluate their potential as hev antiviral. silvestrol is a natural compound belonging to the class of cyclopenta[b]benzofuran compounds, which are exclusively found in plants of the aglaia genus [ ] . silvestrol has been shown to target the translation initiation factor a (eif a) to rna, thereby preventing ribosome loading onto mrna and blocking translation [ ] . originally described in the context of cancer treatment, silvestrol has been reported to inhibit several viruses in vitro [ ] [ ] [ ] . silvestrol reduced viral titers, the number of infected a cells, as well as viral protein levels in infected cells in vitro [ ] . another study confirmed the inhibitory effect of silvestrol on gt replicons in hepg cells with an ic of around nm for gt hev [ ] . the inhibitory effects were consistent for different patient isolates covering the hev gt - , which were evaluated in ipsc-hlcs. silvestrol reduced viral titers in feces in xenograft mice infected with hev gt , therefore demonstrating its effectiveness in vivo. importantly, silvestrol was effectively inhibiting a p -luc replicon harboring the g r mutant, which confers rbv resistance [ , ] . these data demonstrate that silvestrol might provide a therapy option for otherwise untreatable rbv resistant cases. characterization of potential resistance barriers and a structure-activity relationship of the compound will be important next steps for further development of a promising candidate. vaccination may be an important strategy to reduce the global burden of the virus. in china, there is a vaccine licensed under the name hecolin ® . it consists of the amino acids - of the capsid e protein of hev gt , produced in e. coli [ ] . three doses at , , and months were tested in healthy patients - years of age [ ] . long-term studies following the same cohort up to months after vaccination showed an efficacy of . % in the intention-to-treat-analysis as well cross-protection against hev gt [ ] . there are currently several open questions about the vaccine, thereunder its efficacy and safety in risk groups, the cross-reactivity against other hev gts, and long-term protection after more than months. it is unknown whether hecolin ® is safe for pregnant women and their fetuses, which are a population especially at risk of fatal outcomes of hev gt infection. results obtained from pregnant women that had unintendedly been enrolled by mistake in the phase iii study suggest that the vaccine might be safe for them [ ] . however, because the study was not designed for that purpose, this finding is not a definitive conclusion. a study that does not exclude pregnant women is currently running in bangladesh and expected to finish in (clinicaltrials.gov identifier: nct ). it is also unknown if the vaccine is protective in the second risk group of immunosuppressed or immunocompromised patients. a french study investigated re-infections with hev in organ transplant recipients seropositive for anti-hev igg before transplantation [ ] . they showed that an igg titer up to . who units/ml does not guarantee protection from a re-infection up to one year after transplantation. the data from the long-term study on hecolin ® [ ] indicate that the geometric mean of igg titers months after the first dose is already below that value in individuals seronegative at baseline. notably, the igg titers of individuals, which were anti-hev igg positive before vaccination, fell below the threshold of . who units/ml after months. this raises the question whether chronic hev infections in immunocompromised patients can be attacked with the current vaccine. there are also no data on safety and efficacy in the risk group of patients with chronic liver disease. it is unknown if the vaccine confers cross-protection against hev gts , , or rat hev, although the cross-reactivity of the vaccine against gt hev is promising. the vaccine schedule requires six months; it would be to clarify if the vaccine can be used short-term to combat an outbreak. these limitations and uncertainties make research on antiviral therapy an important issue, regardless of the vaccine's benefits. the recently evolving attention on hev also led to an increased focus on finding a satisfactory therapy. however, sofosbuvir is the only candidate in clinical trials to date (figure ) . preliminary results indicate that it will not be a breakthrough in hev therapy options. apart from that, the nature of repurposed drugs might make them appealing to use for clinical trials. however, a -cmc derivative was discontinued due to toxic effects, while gpc-n or nitd never moved to clinical trials, which might indicate that they have adverse effects that could hinder development to an antiviral. the hits identified from other screens are either not well characterized yet, including toxicity, or are difficult to dose in vivo. poor characterization is also an issue for several of the target-based compounds, while one is an immunosuppressant. inhibition of porf and tsg might be an interesting target for drug development, as demonstrated using cp . silvestrol is a promising candidate, since it is comparably well characterized and shows efficacy in vivo and an additive effect with rbv. despite some challenges like scaling up its production, silvestrol is the most promising candidate at the moment. although the vaccine can be of great use in reducing hev burden, there are too many open questions, especially about their efficacy in risk groups and outbreak scenarios to solely rely on it to combat hev. an ultimate goal would be to discover specific agents only targeting the viral enzymes as, for instance, the hev protease or polymerase. these so-called direct acting antivirals (daas) are highly specific and were a breakthrough in hcv therapy with high cure rates and enhanced efficacy. being virus-specific, they might also be much more suited for the use in immunocompromised patients and especially pregnant patients, for whom there is no therapy yet. of note, none of the drug candidates presented in this paper has an approval for use in pregnant women. integral for the discovery of daa was in the case of hcv and will be in the case of hev the crystallization of the respective enzyme structures. this will enable structure-guided design of potent inhibitors by fitting the compound and the viral enzyme to complementary surfaces. as mentioned above, the structure of porf has been determined, but without further implications for drug development yet. regarding the polyprotein from orf , it is still debated whether it is cleaved into fragments upon translation or not. some studies argue that it is not cleaved [ ] [ ] [ ] [ ] , while others report cleavage into several fragments [ , ] . recently, a study was published suggesting that the proteases factor x and thrombin cleave the polyprotein and that silencing of these reduced psk-hev -luc replication [ ] . the assignment of functional domains to the porf polyprotein was done in , according to similarity to proteins in related viruses that had known functions [ ] . of these seven domains, the functionality could be confirmed with biochemical assays for four: methyltransferase [ ] , papain-like cysteine protease [ ] , helicase [ ] , and rdrp [ ] . the exact function of the other domains as well as the exact borders of each functional domain are not known to date. until it is not completely understood how the polyprotein is processed and how this does affect the structure and enzymatic activity of the functional domains, studies to identify antivirals based on structural insights seem at best unlikely. to date, the de novo development of candidate structures into a potential licensed drug is only a future perspective, underlined by the fact that none of the anti-hev candidates has been designed based on a structure of an hev protein. there are also only a few host factors known; therefore, the identification of novel host factors will be another cornerstone in combating hev. all -omics approaches to decipher the altered cellular environment during infection, as well as functional studies using cdna, shrna and sirna libraries to overexpress or silence host proteins, are powerful and successful tools to discover novel host factors. this will both give new starting point for drug discovery as well as potentially refine in vitro and in vivo models. overview of molecules/extracts with antiviral activity against hev. the depicted molecules/extracts are classified according to the strategy that was used to identify them. so far, the antiviral activity against hev of only four drugs (sofosbuvir, pegifn-α, ribavirin and silvestrol) was approved in experimental settings beyond in vitro cell culture systems. both drugs in the clinics are used off-label and therefore have not been tested in clinical trials against hev. funding: e.s. was supported by the german ministry of education and research (bmbf) through a ginaico grant gw and a silvir grant ggnatm . proposed reference sequences for hepatitis e virus subtypes a new hepatitis e virus genotype strain identified from an outbreak in nigeria a large outbreak of hepatitis e virus genotype infection in an urban setting in chad likely linked to household level transmission factors an outbreak of hepatitis e in an urban area of bangladesh abravanel, f. hev and 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hepatitis e virus produced by a reverse genetics system has the potential for zoonotic infection sofosbuvir and daclatasvir anti-viral therapy fails to clear hev viremia and restore reactive t cells in a hev/hcv co-infected liver transplant recipient chronic hepatitis e in a heart transplant patient: sofosbuvir and ribavirin regimen not fully effective combination therapy of sofosbuvir and ribavirin fails to clear chronic hepatitis e infection in a multivisceral transplanted patient sofosbuvir shows antiviral activity in a patient with chronic hepatitis e virus infection autochthonous acute hepatitis e: treatment with sofosbuvir and ribavirin sofosbuvir and ribavirin eradication of refractory hepatitis e in an immunosuppressed kidney transplant recipient efficacy and safety of sofosbuvir monotherapy in patients with chronic hepatitis e-the hepnet sofe pilot study high-throughput screening (hts) for the identification of novel antiviral scaffolds impact of high-throughput screening in biomedical research an ethanol extract of lysimachia mauritiana exhibits inhibitory activity against hepatitis e virus genotype replication spicatoside a derived from liriope platyphylla root ethanol extract inhibits hepatitis e virus genotype replication in vitro inhibition of hiv- infection by zinc group metal compounds +) inhibits coronavirus and arterivirus rna polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture zinc salts block hepatitis e virus replication by inhibiting the activity of viral rna-dependent rna polymerase the relationship between zinc intake and serum/plasma zinc concentration in children: a systematic review and dose-response meta-analysis failure to respond to ribavirin despite elevated intra-erythrocyte zinc level in transplant-patients with chronic hepatitis e virus infection a screen for novel hepatitis c virus rdrp inhibitor identifies a broad-spectrum antiviral compound biological and immunological characteristics of hepatitis e virus-like particles based on the crystal structure structure of the hepatitis e virus-like particle suggests mechanisms for virus assembly and receptor binding heparan sulfate proteoglycans are required for cellular binding of the hepatitis e virus orf capsid protein and for viral infection the ubiquitous hammerhead ribozyme development of a chimeric dna-rna hammerhead ribozyme targeting sars virus berzal-herranz, a. inhibition of hepatitis c virus replication and internal ribosome entry site-dependent translation by an rna molecule reduction in severity of a herpes simplex virus type murine infection by treatment with a ribozyme targeting the ul gene rna targeted cleavage of hepatitis e virus ' end rna mediated by hammerhead ribozymes inhibits viral rna replication antisense phosphorodiamidate morpholino oligomers as inhibition of hepatitis e virus replication by peptide-conjugated morpholino oligomers proteasome inhibitors: valuable new tools for cell biologists hepatitis e virus replication requires an active ubiquitin-proteasome system inhibition of hepatitis e virus replication by proteasome inhibitor is nonspecific tsg and the vacuolar protein sorting pathway are essential for hiv- budding enhanced alpha microglobulin secretion from hepatitis e virus orf -expressing human hepatoma cells is mediated by the tumor susceptibility gene orf protein of hepatitis e virus is essential for virion release from infected cells a psap motif in the orf protein of hepatitis e virus is necessary for virion release from infected cells structure of the tsg uev domain in complex with the ptap motif of the hiv- p protein inhibition of hiv budding by a genetically selected cyclic peptide targeting the gag-tsg interaction potent inhibition of hepatitis e virus release by a cyclic peptide inhibitor of the interaction between viral open reading frame protein and host tumor susceptibility gene inhibition of dengue virus through suppression of host pyrimidine biosynthesis anti-hiv- activity of leflunomide: a comparison with mycophenolic acid and hydroxyurea chronic hepatitis e in heart transplant recipients calcineurin inhibitors stimulate and mycophenolic acid inhibits replication of hepatitis e virus an early viral response predicts the virological response to ribavirin in hepatitis e virus organ transplant patients cross talk between nucleotide synthesis pathways with cellular immunity in constraining hepatitis e virus replication rocaglamide, silvestrol and structurally related bioactive compounds from aglaia species therapeutic suppression of translation initiation modulates chemosensitivity in a mouse lymphoma model silvestrol inhibits chikungunya virus replication broad-spectrum antiviral activity of the eif a inhibitor silvestrol against corona-and picornaviruses the natural compound silvestrol is a potent inhibitor of ebola virus replication the natural compound silvestrol inhibits hepatitis e virus (hev) replication in vitro and in vivo a mutation in the hepatitis e virus rna polymerase promotes its replication and associates with ribavirin treatment failure in organ transplant recipients in vivo evidence for ribavirin-induced mutagenesis of the hepatitis e virus genome a bacterially expressed particulate hepatitis e vaccine: antigenicity, immunogenicity and protectivity on primates efficacy and safety of a recombinant hepatitis e vaccine in healthy adults: a large-scale, randomised, double-blind placebo-controlled long-term efficacy of a hepatitis e vaccine safety of the hepatitis e vaccine for pregnant women: a preliminary analysis hepatitis e virus reinfections in solid-organ-transplant recipients can evolve into chronic infections cloning, sequencing, and expression of the hepatitis e virus (hev) nonstructural open reading frame (orf ) expression of the hepatitis e virus orf lack of processing of the expressed orf gene product of hepatitis e virus early secretory pathway localization and lack of processing for hepatitis e virus replication protein porf the in vitro-synthesized rna from a cdna clone of hepatitis e virus is infectious expression and processing of the hepatitis e virus orf nonstructural polyprotein activities of thrombin and factor xa are essential for replication of hepatitis e virus and are possibly implicated in orf polyprotein processing computer-assisted assignment of functional domains in the nonstructural polyprotein of hepatitis e virus: delineation of an additional group of positive-strand rna plant and animal viruses virus-specific mrna capping enzyme encoded by hepatitis e virus hepatitis e virus (hev) protease: a chymotrypsin-like enzyme that processes both non-structural (porf ) and capsid (porf ) protein ntpase and ' to ' rna duplex-unwinding activities of the hepatitis e virus helicase domain the ' end of hepatitis e virus (hev) genome binds specifically to the viral rna-dependent rna polymerase (rdrp) this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we would like to thank yannick brüggemann and patrick behrendt for insightful comments. the authors declare no conflict of interest. key: cord- - x on authors: rumlová, michaela; ruml, tomáš title: in vitro methods for testing antiviral drugs date: - - journal: biotechnology advances doi: . /j.biotechadv. . . sha: doc_id: cord_uid: x on abstract despite successful vaccination programs and effective treatments for some viral infections, humans are still losing the battle with viruses. persisting human pandemics, emerging and re-emerging viruses, and evolution of drug-resistant strains impose continuous search for new antiviral drugs. a combination of detailed information about the molecular organization of viruses and progress in molecular biology and computer technologies has enabled rational antivirals design. initial step in establishing efficacy of new antivirals is based on simple methods assessing inhibition of the intended target. we provide here an overview of biochemical and cell-based assays evaluating the activity of inhibitors of clinically important viruses. viral pandemics remain serious threats to humankind. every year, known viruses such as hiv- and hepatitis b virus newly infect millions of people across the globe. in addition, recent outbreaks of ebola virus, influenza virus, severe acute respiratory syndrome (sars) coronavirus and middle east respiratory syndrome-coronavirus (mers-cov) serve as a reminder of the silent danger. the world health organization reported in that over . million causalities per year are connected with hepatitis b and almost , with hepatitis c. occasional epidemic outbreaks of other viruses are also striking. these include outbreaks of noroviruses, flaviviruses (zika and dengue viruses), new strains of influenza viruses, the re-emergence of west nile virus in italy and the united states. despite relatively long pauses, viruses such as influenza virus can re-emerge and cause global pandemic health problems. unlike cellular genomes, which consist of double-stranded (ds) dna, viral genomes can be formed by a broad variety of nucleic acids (nas), including ds or single-stranded (ss) circular or linear dna or positive-, negative-or sometimes ambi-sense rna. as viral life cycles are dependent on cellular factors and cellular metabolic and signaling pathways, the number of possible antiviral drug targets is limited. however, almost all viruses encode unique proteins and enzymes that may serve as specific targets for antiviral therapy. the overarching goal of modern drug development efforts is to design compounds that specifically inhibit viral targets or cellular targets essential for virus replication. the purpose of this review is to provide insight into the broad variety of cell-based and biochemical assays used for identification and evaluation of antivirotics, including high-throughput screening (hts) methods. an overview of available inhibitors and vaccines, which has been reviewed elsewhere [e.g. in (de clercq and li, ) ], is beyond the scope of this paper. to package their genomes into particles of very limited size, viruses encode few genes. virions consist of an rna or dna genome that is protected by an outer shell called a capsid (also nucleocapsid) formed by a lattice of capsid proteins. in enveloped viruses, the capsid is additionally surrounded by a lipid bilayer spiked with viral proteins. the size of animal viruses ranges from approximately nm to over nm (cohen et al., ) . the capsids and virions of rna viruses adopt various shapes. viral capsids may be icosahedral (e.g. picornaviridae, astroviridae, reoviridae, togaviridae), bullet-shaped (rhabdoviridae), helical (coronaviridae), helical filamentous (filoviridae, orthomyxoviridae, paramyxoviridae), or filamentous (arenaviridae, bunyaviridae). the retroviral capsid core may be conical, spherical, or rod-shaped. the morphology of dna viruses is similarly diverse, ranging from icosahedral (enveloped -hepadnaviridae, herpesvridae; nonenveloped -adenoviridae, parvovirdae, polyomaviridae and papillomaviridae) to rodshaped (baculoviridae) or pleomorphic (poxviridae). the viral life cycle is the process of viral replication in a host cell. first, viruses enter the host cell and replicate their genomes. following translation of viral proteins by the host cell machinery, viruses package their genomic material into protective proteinaceous capsids and exit the cell to infect another host cell. nonenveloped viruses consist only of a protein capsid shell enclosing the viral genome and enzymes, while the capsid shell of enveloped viruses is enclosed in a lipid envelope derived from the host cell membrane. regardless of whether the virus is enveloped, its surface must display (glyco)proteins suitable for specific interactions with host cell receptors. in contrast to the surface proteins of nonenveloped viruses, those of enveloped viruses usually serve another function in addition to host cell recognition and attachment. for example, they may enable fusion of the viral and cellular membranes, usually through an interaction with a secondary receptor(s) or co-receptor(s). the fusion domains of viral surface glycoproteins thus can lower the kinetic barrier for the energetically demanding membrane fusion. the viral particle must be sufficiently stable to protect the genome until its delivery into the host cell. simultaneously, it must be metastable, to allow its disassembly and release of the genome for replication in the infected cell at the appropriate time. the energetic barrier that prevents viral disassembly outside the cell is lowered upon infection by structural changes in the viral components. these changes may be induced by binding of a cellular ligand or by changes in the environment, such as ph change upon entering a specific cellular compartment. numerous viruses preassemble immature particles that undergo irreversible (usually proteolytic) transitions into mature structures of fully infectious virions. mutual interactions of viral capsid proteins are typically different from those of cellular proteins, which predominantly create binary interactions. viral structural proteins interact with multiple neighboring partners to form multicomponent macromolecular structures [reviewed in (cheng and brooks, ; jayaraman et al., ) ]. the economy of the packaged viral genomes due to the limited capsid size implies formation of only a few types of structural proteins, which are usually symmetrically organized [reviewed in (prasad and schmid, ; raguram et al., ) ]. in the initial stage of infection, viruses must overcome several obstacles. the first is the cellular plasma membrane with an actin cortex. then, they must traffic through dense cytoplasm to reach their final destination for replication and assembly. these pathways are specific for different viruses and often are dictated by the size of the particle and its structure. the viral life cycle can be divided into several common stages, including entry, uncoating, genome replication, genome packaging and assembly, release, and maturation ( fig. ) . in general, the first phase of viral infection is specific recognition of the target host cell and binding to a surface receptor displayed on the cell membrane. this process is common to both enveloped and nonenveloped viruses. in enveloped viruses, the binding is mediated by viral surface components, typically oligomers of integral glycoproteins. nonenveloped viruses bind receptors through sites or projections on the capsid surface. viruses can use either a single receptor (e.g. tim- for hepatitis a virus, gm for sv , cd for poliovirus, low-density lipoprotein receptor for human rhinovirus) or multiple receptors with equivalent roles (e.g., nectin- / or hvem for herpes simplex virus / , ace or l-sign for sars coronavirus). for other viruses (e.g. hiv, hcv, adenoviruses, rotaviruses, picornaviruses, and some herpesviruses), the presence of at least two cytoplasmic membrane components is required. for example, hiv- binds to the primary receptor cd and one of two co-receptors (ccr or cxcr ) [reviewed in (cossart and helenius, ; grove and marsh, ) ]. to infect a cell, viruses must overcome the plasma membrane to deliver their genetic material into the cytosol. viruses enter cells by two main mechanisms. a majority of animal viruses, both enveloped and nonenveloped, enter cells by one or two types of endocytosis, such as clathrin-mediated endocytosis (e.g. vsv, influenza a virus, rhinovirus), caveolin-mediated uptake (echovirus, polyoma virus), clathrin/caveolin-independent endocytosis such as caveolar or lipid raft-mediated (e.g. sv , polyomavirus mouse), or macropinocytosis (e.g. vaccinia virus, respiratory syncytial virus, ebola virus, hiv- ) (blaas, ; cossart and helenius, ; fields and knipe, ; kirkham and parton, ; marechal et al., ; mayor and pagano, ; mercer and helenius, ; parton and simons, ; rasmussen and vilhardt, ; saeed et al., ) . these endocytic mechanisms enable the virus to be transported by the cell's machinery through the plasma membrane and to pass through the dense actin cortex. cellular entry of some viruses is coupled with receptor-mediated signaling, resulting in activation of molecules that facilitate virus entry by cytoskeleton reorganization, induction of long-distance transport of the virus-containing vesicles, or actin cortex disassembly. the second type of entry is used by some enveloped viruses (paramyxo-, herpes-, and retroviruses), which upon cell surface receptor binding, infect the cell by direct fusion of viral and plasma membranes at neutral ph. upon internalization, most viruses become trapped and enclosed in vesicles that pinch off the inner side of the plasma membrane and transport their cargo into the cytoplasm. on their way through the cell, these transport vesicles undergo maturation by fusing with other vesicles. to fulfill their task of genome replication, viruses have to escape from these endosomes. the "great escape" is triggered by activation of a fusion or penetration mechanism, such as changes in conditions in the endosomal interior [e.g. ph, ionic environment (e.g. calcium ion concentration), oxido-reducing conditions], changes in membrane composition, and other physico-chemical cues (blaas, ; cossart and helenius, ; inoue and tsai, ) . depending on the requirements for a particular virus, these events can occur in early endosomes (ph . - . ; e.g. hepatitis c virus, vesicular stomatitis virus), late endosomes (ph . - . ; e.g. influenza a virus, dengue virus, sars coronavirus), recycling endosomes, macropinosomes, the endoplasmic reticulum, the golgi apparatus, or lysosomes (ph . - . ) (blaas, ; cossart and helenius, ; grove and marsh, ; inoue and tsai, ) . for the majority of animal viruses, the activation of these fusion or penetration mechanisms occurs through conformational changes and structural rearrangements in viral surface proteins and/or the whole virion shell that may destabilize the capsid core. structural rearrangements in enveloped viruses usually mediate fusion of viral and endosomal membranes. in nonenveloped viruses, the structural changes uncover amphipathic or hydrophobic domains that may induce pore formation or disruption of endosomal membranes. to deliver genetic material to the replication site, these mechanisms ultimately release viral capsid structures from endosomal vesicles into the cytosol either by fusing with the endosomal membrane (enveloped viruses) or by penetrating the endosomal membrane (nonenveloped viruses). uncoating is the partial or complete disassembly of the protective capsid shell and/or lipid envelope to liberate the viral genome. for many viruses, this process is closely connected with conformational changes induced by the virus binding to the cell surface receptor; a low ph environment; or changes in oxido-reducing conditions, ion concentrations, or other factors. for enveloped viruses, uncoating involves a loss of the viral membrane by fusion either with the plasma membrane or with intracellular vesicles, followed by stepwise uncoating of the protective capsid shell. in nonenveloped viruses, the uncoating process typically involves conformational changes that result in the weakening of intermolecular interactions, loss of structural proteins, proteolytic cleavage, and so on (fields and knipe, ) . depending on the virus, uncoating can take place at the plasma membrane, in the cytoplasm, during endocytosis in early or late endosomes, in lysosomes, in the nucleus, or at the nuclear pore complex (npc). the ssrna genome of retroviruses, which is fully enclosed inside a protective capsid shell, must be reverse transcribed into dsdna and released from the capsid. although it is accepted that hiv- uncoating is linked to reverse transcription and nuclear import (ambrose and aiken, ) and is controlled by host factors (e.g. cyclophilin a, trim α), the precise molecular mechanisms that trigger the uncoating remain unknown. several hypotheses have been proposed, including breakage of the capsid shell due to increased inner pressure caused by accumulation of the reverse transcription product (rankovic et al., ) , the requirement of intact microtubules and dynein and kinesin motors (lukic et al., ) , and phosphorylation of capsid shell protein by the host cell kinase melk (takeuchi et al., ) . in contrast to the majority of rna viruses, which replicate in the cytoplasm, most dna viruses (with the exception of large dna viruses including poxviridae, asfarviridae, and mimiviridae) and several negative stranded rna viruses enter the nucleus to replicate their genomes (kobiler et al., ; koonin and yutin, ) . passive diffusion into the nucleus is suitable for molecules smaller than nm in diameter, but larger structures must enter through the nuclear pore complex (npc), which can accommodate molecules of up to nm (pante and kann, ) . translocation through the npc is tightly regulated and requires the presence of a nuclear localization signal (nls) on the passing molecule and nuclear import receptors (importins or karyopherins) (cautain et al., ) . due to the diversity of viral particle sizes (from nm to over nm) and structures, viruses have evolved several strategies to export their dna or rna genome into the nucleoplasm. with regard to the npc, these pathways can be divided into npc-dependent and npcindependent mechanisms. due to size limitations, only a few, very small simplified scheme of common stages of viral life cycle targeted by antiviral drugs. these stages including: ) attachment and entry, ) uncoating, ) genome replication, ) genome packaging and assembly of viral particle and ) virus release and maturation. m. rumlová, t. ruml biotechnology advances ( ) - viral capsid particles, such as hepatitis b virus (hbv; - nm diameter), can pass through the npc (cohen et al., ; rabe et al., ) in an importin-dependent manner. hbv then disassembles at the nuclear side of the npc (the nuclear basket), releasing its genome into the nucleoplasm (fay and panté, ) . the capsid shells or ribonucleoprotein complexes (rnps) of some larger viruses, such as influenza a virus and hiv- , disassemble within the cytoplasm. the viral genome, released from the shell and complexed with nls-containing components, is then translocated through the npc (ambrose and aiken, ; campbell and hope, ; cohen et al., ; fay and panté, ; hutchinson and fodor, ) . another mechanism, used by herpes simplex virus (hsv) and adenoviruses, involves cellular (importins, nup) or viral protein-mediated attachment (docking) of the capsid shell at the cytoplasmic side of the npc. this facilitates the passage of genomic dna into the npc either by ejection from an almost intact particle (through the capsid portal in hsv- ) or upon complete disassembly of the capsid shell (in adenoviruses) (kremer and nemerow, ; ojala et al., ; pasdeloup et al., ) . npc-independent mechanisms are used by some retroviruses (e.g. mlv) that enter the nucleus during the mitotic phase of the cell division cycle when the nuclear envelope is dissolved (matreyek and engelman, ; roe et al., ) . another mechanism, described for parvoviruses, involves partial disruption of the nuclear envelope (cohen and pante, ; fay and panté, ) . viral genomes can be encoded by various types of nas, as summarized in table for dna viruses and table for rna viruses. by convention, ssdna of equivalent polarity to mrna is designated as the positive (+) strand. the complementary ssnas are of minus polarity (−). the majority of dna viruses replicate in the nucleus, where cellular dna replication and transcription also occur. in contrast, rna viruses usually replicate in the cytoplasm. rna viruses are the only "organisms" that store their genetic information in the form of rna. replication of their genomes is accomplished either by rna-dependent rna synthesis ( fig. a , b) [reviewed in (ferron et al., ; lu and gong, ; menéndez-arias and andino, ; pietilä et al., ; tao and ye, ) ] or through rnadependent dna synthesis (reverse transcription) followed by dna integration, replication, and transcription ( fig. c ). as some of these enzymatic activities are not commonly found in uninfected host cells, rna viruses must encode enzymes to aid replication (table ). rna viruses are divided according to the baltimore classification into dsrna viruses (birna-and reoviridae); positive-sense ssrna viruses (corona-, flavi-, and picornaviriade); negative-sense ssrna viruses (filo-, rhabdo-, paramyxo-, and orthomyxoviriade), and ambisense rna viruses (arena-and some members of bunyaviridae) with both positive-and negativesense rnas (nguyen and haenni, ) (see table ). the nature of the genome not only dictates the mechanism of replication, but also has other important consequences. the genome of (+)rna viruses may serve directly as mrna for production of viral proteins. therefore, the mere introduction of genetic material (e.g. in exocytic vesicles) may result in productive infection. the rna polymerases that copy the genetic material of rna viruses are error-prone, which provides considerable genetic flexibility and the propensity to evolve drug-resistant mutants. these features are amplified in viruses with segmented genomes that undergo reassortment. in viruses with segmented genomes (orthomyxoviridae, arenaviridae and bunyaviridae), each segment is transcribed in an autonomous transcription-replication unit by viral rna polymerase that binds to the ′ end cap structure. of note, the genomic rna (grna) is capped by a unique mechanism called cap-snatching (ferron et al., ) , in which the cap is cleaved from cellular mrna and transferred onto the viral grna by a subunit of viral rna polymerase (pflug et al., ) . some dsrna viruses (birnaviridae and reoviridae) also contain segmented genomes. upon replication, these viruses must ensure stoichiometric incorporation of single copies of each grna segment into new particles. this is guided by specific packaging signals on each segment of grna that interact with positively charged domains in the capsid proteins [reviewed in (isel et al., ; pohl et al., ) ]. although different models of packaging have been proposed for various segmented genome viruses, a common feature is co-assembly of the capsid proteins with the grna and rna polymerase. the genomes of dna viruses come in a considerable variety of sizes and shapes, from small ss to large ds molecules that may be linear or circular. the size range of these genomes (from . kb to kb) reflects the necessity for some viruses to encode specific proteins required for viral replication. small-genome dna viruses (polyoma-, papilloma-, and parvoviruses) use only host cell enzymes for replication and transcription. the only exceptions are some hepadnaviruses (e.g. hepatitis b virus) that despite having small genomes (approximately kb), encode their own specific dna polymerase/reverse transcriptase that reverse transcribes pregenomic rna (pgrna) into genomic viral dna (fig. d , ii) (beck and nassal, ) . viruses with intermediate-size genomes (up to kb) (e.g. adenoviruses) encode their own dna polymerase for genome replication, but they usually utilize cellular rna polymerases ii and iii for transcription (fields and knipe, ) . viruses with large genomes (larger than kb) (e.g. herpesviruses) encode proteins for replication, including dna polymerase and dna helicase-primase, as well as some enzymes necessary for biosynthesis of deoxyribonucleotide triphosphates (dntps) and several transcription factors (boehmer and nimonkar, ) . poxviruses (e.g. vaccinia virus), are another type of virus with large dsdna genomes, and their replication, transcription, and translation take place entirely in the cytoplasm within discrete juxtanuclear sites called virus factories (moss, ) , (fig. d , iii). these viruses encode all enzymes and specific factors necessary for genomic replication and transcription. with their own replicative machinery, large-genome dna viruses can replicate in nondividing cells. in contrast, the replication of small-genome dna viruses, which depends on cellular dna polymerases, must occur simultaneously with the s-phase of the cell cycle (e.g. parvoviruses), or must express some viral protein/ oncogene to re-program the host cell-cycle regulatory proteins p or retinoblastoma protein (prb), triggering entry into the s-phase (e.g. polyomaviruses and papillomaviruses). by affecting the g /s checkpoint (controlled by p and prb), the viruses ensure the production of host enzymes required for viral replication (fields and knipe, ) . production of viral proteins of dna viruses with intermediate and large size genomes is divided into early and late phases. in the early (prereplicative) phase, nonstructural proteins required for dna replication are translated. the late phase, during which the late structural proteins needed for assembly are translated, begins after viral dna replication ( fig. d , i). depending on the species, viruses assemble either in contact with the cellular membrane or independent of the membrane in either the nucleus or cytoplasm. the membrane-independent route is used by nonenveloped viruses and a few enveloped viruses (e.g. orthomyxoviruses, herpesviruses, and some retroviruses) that acquire the membrane envelope after intracellular assembly during budding from the cell. the limiting step for nuclear assembly is the size of npcs, which are large enough to transport rna and import proteins required for assembly into the nucleus; however, npc size limits the transport of larger assemblies. therefore, some viruses form assembly intermediates in the nucleus. these structures are then exported to the cytoplasm, where they come together to form viral particles. nuclear export is specific and depends on the presence of nuclear export signals (nes) in the transported proteins. one example of a nonenveloped icosahedral virus that assembles in the nucleus is adenovirus, the assembly of which has been studied intensely due to its potential use in gene therapies [reviewed in (ahi and mittal, ) ]. recent data suggest that upon accumulation of multiple copies of adenoviral dsdna genomes, coordinated assembly and packaging occur by two interlinked mechanisms that involve both capsid proteins and core components (condezo and san martín, ) . the assembly occurs in the so-called peripheral replicative zone with the assistance of scaffolding proteins that facilitate formation of adenoviral particles but are excluded from mature viruses. adenoviruses are finally released upon lysis of the infected host cell. orthomyxoviruses and herpesviruses are enveloped viruses that assemble their nucleoproteins in the nucleus. herpesviruses package their dsdna genome as head-to-tail concatemers and assemble icosahedral procapsids on scaffold proteins in the nucleus [reviewed in (heming et al., ) ]. however, subsequent steps of herpesvirus assembly proceed in the cytoplasm. the preassembled procapsid is too large to pass through the npc, but it exits the nucleus by viral proteindriven vesicular transport across the nuclear inner membrane leaflet. thus, the herpesvirus acquires an initial envelope from the inner nuclear membrane [for review see (fields and knipe, ; hellberg et al., ) ]. next, the herpesviral membrane fuses with the outer nuclear membrane, and the naked particle is released from the nucleus into the cytosol. here, the virus acquires tegument (a protein layer between the capsid and the envelope) and other proteins. final herpesvirus envelopment occurs at the golgi membrane containing the viral glycoproteins (fields and knipe, ) . preassembled orthomyxoviral ribonucleoproteins, upon their export from the nucleus, are driven to the plasma membrane to which they attach through electrostatic interactions of the matrix protein m with membrane phosphatidylserine. the virions then assemble simultaneously with budding during which they also acquire ha, na and m membrane proteins. poxviruses undergo an even more complicated pathway. they are enveloped with multiple membranes acquired from er/intermediate compartments and golgi or early endosomes (moss, (moss, , . these membranes also provide the poxviruses with their membrane proteins. most viruses assemble upon interaction of their structural proteins with cellular membranes. the target membrane for assembly differs according to the virus type. flaviviruses assemble at the surface of the er and then upon their budding into the er lumen, the immature particles are then transported into the tgn. some viruses, such as coronaviruses, assemble at the er-golgi intermediate compartment [reviewed in (ujike and taguchi, ) ]. the assembly of bunyaviruses occurs concurrently with replication of grna segments in virus factories located along the golgi (guu et al., ; strandin et al., ) . the presence of membrane glycoproteins at the golgi membrane determines the sites where assembling bunyavirus particles bud into the golgi lumen, similar to other enveloped viruses. numerous viruses, including paramyxoviruses (cox and plemper, ) , orthomyxoviruses (pohl et al., ) , alphaviruses (jose et al., ) , rhabdoviruses (okumura and harty, ) , and most retroviruses (freed, ) , assemble underneath the cytoplasmic membrane, which facilitates assembly by providing a scaffolding function. the virus then acquires a lipid envelope through budding across the plasma membrane. during this process, it also gains the envelope glycoproteins (env) that are anchored in the plasma membrane by hydrophobic transmembrane domains. env reaches the plasma membrane by a cellular secretory pathway upon synthesis in the er and subsequent glycosylation in the golgi. usually, a specific interaction between viral structural proteins and glycoproteins is required. this may be either direct or mediated through the interaction with matrix protein (e.g. in (-)rna viruses such as ortho-and paramyxoviruses or rhabdoviruses). most retroviruses, including hiv (so-called morphogenetic c-type), also assemble at the plasma membrane of the host cell. the interaction of the structural polyprotein precursor (gag) with the plasma membrane is usually facilitated by a bipartite signal in the n-terminal domain of gag (i.e. matrix protein) comprising both the basic patch and n-terminally linked myristoyl residue (added co-translationally to gag). in contrast to c-type assembly at the membrane, morphogenetic b/d-type retroviruses assemble at pericentriolar sites where gag polyproteins are transported along microtubules by dynein molecular motors (sfakianos et al., ; vlach et al., ) . for both morphogenetic pathways, the packaging of grna facilitates assembly. (+)rna viruses have adopted a mechanism of extensive rearrangement of intracellular membranes to provide a milieu for virus replication and assembly. this mechanism effectively protects dsrna intermediates from degradation by the host cell machinery (delgui and colombo, ; jackson, ) . this process has been well-documented for poliovirus, in which the newly formed membranous structures exclusively serve for virus production. various types of vesicles that are formed upon viral infection have different roles in viral replication (rossignol et al., ) . some nonenveloped mammalian viruses, such as reoviruses, assemble in the cytosol in so-called viroplasms or viral factories into icosahedral particles consisting of three concentric layers encircling segments of genomic dsdna (benavente and martinez-costas, ; jones, ; shah et al., ) . rotavirus seems to be the only exemption in the reovirus family, as it enters the endoplasmic reticulum to gain its outer protein shell (trask et al., ) . numerous viruses assemble from polyprotein precursors that must be specifically cleaved by a viral protease to generate infectious particles. this mechanism, which is an irreversible step in the virus life cycle, ensures equimolar packaging of structural proteins and proportional co-assembly of viral enzymes in the form of precursors. upon proteolytic release, the liberated proteins may undergo different trafficking pathways or fulfill various functions in the virus. maturation changes the energy of interaction forces among those interfaces required for intracellular assembly of immature particles to those suitable for viral stability in the environment and for disassembly and uncoating of genetic material for replication [reviewed in (veesler and johnson, ) ]. in poliovirus, the autocatalytic and subsequent viral proteasemediated cleavage of p precursor protein allows formation of pentamers that interact with grna. additional cleavage of vp , yielding vp and vp , is required to form infectious poliovirus particles. in retroviruses, proteolytic processing is initiated by the autocatalytic liberation of viral protease, which subsequently cleaves the polyproteins to trigger major structural rearrangements in the virus and release of other viral enzymes (reverse transcriptase and integrase) and structural proteins. in herpesviruses, maturation involves proteolytic processing of the scaffolding protein and recruitment of tegument proteins that stabilize the particle and mediate interactions with the membrane during envelopment (gibson, ; tandon and mocarski, ; tandon et al., ) . adenoviruses undergo a maturation process that involves processing of six structural components of the core and one non-structural precursor that initiates replication of gdna (gaba et al., ) . one interesting feature of adenoviral maturation is that dna is required as a co-factor for protease activity. this means that maturation occurs only in particles that have packaged gdna (mangel and san martín, ) . flaviviruses form icosahedral particles upon budding into the neutral milieu of the er. the particles then translocate to the more acidic environment of the trans-golgi network. this ph shift results in disassembly of the immature lattice and extensive rearrangement of the flaviviral particle. this is connected with the exposure of a viral structural glycoprotein (prm) that is specifically processed by the , retroviruses (c) and dna viruses (d) (the schemes a-c were modified from: (ahlquist, ) . a: following endocytosis, the genome of (+)rna viruses may directly serve as mrna for translation of virus encoded proteins. among them, there are proteins of rna-replication machinery that recruit (+)rna into a membrane-associated replication complex. the genomic rna is replicated by using (-)rna template, which is transcribed in a low copy number amplified (+)rna is then packaged into newly assembled virions that exit the host cell either through secretion or cell lysis. b: upon the virus attachment, a core containing both grna and virus encoded rna polymerase is delivered into the cytoplasm by endocytosis. cytoplasmic transcription of the (-)rna template provides (+)rna that serves as mrna for translation of viral proteins. in dsrna viruses, the (+)rna is packaged into new cores which undergo maturation by synthesizing (-) rna (dotted strand) and acquiring surface proteins. in the (-)rna viruses, the (+)rna strand is transcribed into genomic (-)rna in the cytoplasm and then packaged. the new virions egress the cell either through secretion or cell lysis. c: following fusion of viral and cell plasma membrane, the retroviral core is released into cytosol, rna genome is transcribed into dsdna by viral reverse transcriptase concomitantly with uncoating of the viral core, ds dna is transferred into nucleus where it is integrated into host cell genome by viral integrase, following translation of retroviral structural and enzymatic polyproteins, the unspliced grna is packaged into the immature particles that usually assemble at the plasma membrane and the viral particles bud from the cell. d: three mechanisms (i.-iii.) of dna viruses replication are shown: (i): following entry and uncoating, the dna genome is transported to the nucleus; products of early genes (regulatory proteins, transcription factors) regulate the synthesis of viral enzymes (e.g. dna polymerase) required for genome replication; expression of late genes encoding structural capsid proteins in the cytosol, they are then transported into nucleus where packaging and pre-assembly take place; preassembled procapsids exit the nucleus and leave the cell (e.g. herpesviruses). (ii) unique replication cycle of hepatitis b virus (hbv): following entry, the viral particle is internalized by endocytosis and the nucleocapsid is released into the cytoplasm; the genome (relaxed circular rcdna) is transported into the nucleus, where it is converted to a covalently closed circular form (cccdna); which serves as a template for transcription of pregenomic rna (pgrna) for translation of the core protein and the viral polymerase and three subgenomic mrnas used for translation of regulatory and envelope proteins; following viral transcription and translation, the hbv core proteins self-assemble in the cytoplasm into viral nucleocapsid with concurrent incorporation of pgrna and hbv polymerase, pgrna is reversely transcribed into a rcdna within the nucleocapsid; nucleocapsid containing rcdna can either re-enter the nucleus to amplify cccdna, or can be enveloped by hbv envelope proteins in the endoplasmic reticulum. the particles are then secreted from the infected hepatocyte by exocytosis. (iii) upon entering the cell, the replication, transcription and translation take place entirely in the cytoplasm, within discrete juxtanuclear sites called virus factories (e.g. poxviruses) adapted from: ahlquist, p., . parallels among positive-strand rna viruses, reverse-transcribing viruses and double-stranded rna viruses. nat rev microbiol ( ), - . cellular protease furin in the trans-golgi network. the liberated globular heads of prm remain attached at the low ph, but are released when the virus enters neutral body fluids (rey et al., ) . one frequently used mechanism of release of nonenveloped and some enveloped viruses is lysis of the infected cell. this is the simplest release mechanism, although the transition to lysis from latent infection is delicately regulated (aneja and yuan, ; levings and roth, ; schmiedel et al., ) . however, viruses that are usually lysogenic may also use alternative release pathways (bird and kirkegaard, a) . these include non-lytic spread of viruses mediated by vesicles, which has been observed for poliovirus (bird and kirkegaard, b; jackson et al., ) , coxsackievirus (alirezaei et al., ) , and hepatitis a virus . another possibility is that vesicles released from a cell infected with (+)rna viruses contain naked viral grna. this (+)grna-containing vesicle functions itself as an infectious agent as it is transferred to another cell (bird and kirkegaard, a) . the standard way for enveloped viruses to leave the host cell is budding, which includes membrane extrusion and separation of the viral and cellular membranes (so-called pinching off). the lipid envelope layer acquired during viral particle budding through the plasma membrane protects the virus particle. there are three basic mechanisms of budding: i) mediated by envelope glycoproteins, such as the e protein of coronaviruses; ii) independent of glycoproteins, in which the viral structural proteins trigger the extrusion of the plasma membrane, such as retroviral budding in which strong interactions between the gag n-terminal domain (matrix protein) and the plasma membrane induce bulging of the membrane; and iii) requiring interaction between the viral glycoproteins and the capsid for membrane extrusion, as in alphaviruses. the final step that results in the separation of virus from the cell surface is pinching off the particle. this is a controlled process that involves both viral protein domains and cellular factors. during this process, viruses apparently use cellular machinery similar to that used during the last step of cell division (the release of the daughter cell) called endosomal sorting complex required for transport (escrt) [reviewed in (campsteijn et al., ; hurley, ; olmos and carlton, ; scourfield and martin-serrano, ) ]. direct interactions of numerous viral domains with escrt complex subunits have been identified (bieniasz, ) . in retroviruses, short specific amino acid sequences (ptap or psap) interact with the escrt components, and the interaction of hiv with the escrt proteins nedd and alix is wellknown (freed, ; fujii et al., ; gomez and hope, ) . however, this mechanism has also been adopted by other viruses that interact with the same escrt components, such as filoviruses (han et al., ; jasenosky and kawaoka, ; liu and harty, ) . interactions with escrt proteins have also been reported for vesicular stomatitis virus luyet et al., ), rhabdovirus (taylor et al., , arenaviruses (ziegler et al., ) , picornaviruses , paramyxoviruses (park et al., ) , and hepatitis c virus, a representative of the flavivirus family (falcon et al., ) . the typical release pathways used by viruses may vary under some conditions. for example, during chronic infection, numerous viruses use alternative cell-to-cell transmission that may help the virus avoid host neutralization (hulo et al., ) . syncytium formation, an hivinduced cell fusion, was recognized in the early s (callahan, ) . another type of cell-to-cell transmission through tight junctions was shown for hiv (hübner et al., ; wang et al., ) and murine leukemia virus (sherer et al., ) . receptors on tight junctions that specifically recognize hepatitis c virus (carloni et al., ; ploss et al., ) and reovirus (barton et al., ) have been identified. some viruses are able to modify cellular pathways to reprogram both the synthesis and metabolism of lipids and membrane compartmentalization for their transmission and to evade cellular defense mechanisms (mazzon and mercer, ) . despite a general understanding that poliovirus spreads through cellular lysis, it was recently found that it may also be transferred between cells by an autophagy-dependent mechanism, called autophagosome-mediated exit without lysis (bird and kirkegaard, b; bird et al., ; lai et al., ; richards and jackson, ) . similar mechanisms have been described for varicellazoster virus and human cytomegalovirus (grose et al., ; meier and grose, ) . poxviruses encode several proteins that block the apoptotic cellular response to the presence of their dsdna in the cytoplasm. this allows cell-to-cell passage of poxviruses by a mechanism known as apoptotic mimicry (amara and mercer, ; nichols et al., ) . in this process, enveloped viruses expose surface phosphatidylserine to mimic apoptotic bodies. these cells are then macropinocytosed by either dendritic cells or macrophages (mercer and helenius, ) . among the plethora of compounds designed to inhibit infectious viruses, only a few (< ) have been approved for clinical use (de clercq, ; de clercq and li, ) . nevertheless, some effective antiviral drugs have been on the market for several decades, such as the anti-influenza a virus drug amantadine, marketed under the trade name symmetrel (by dupont), which was approved in . in , burroughs-wellcome introduced acyclovir as an inhibitor of herpesviruses. its remarkable specificity is connected with its activation by viral thymidine kinase-catalyzed phosphorylation. however, due to development of drug resistance in a number of viruses, especially rna viruses, there is a continuous need to design and test new inhibitors, preferably targeting different steps of viral life cycles. here, we provide insights into the world of biochemical and cell-based assays that were developed to test antivirotics targeting various steps in the viral life cycle. different types of assays, including cell-cell fusion assays, cell-virus fusion assays with pseudotyped viral particles, and in vitro biochemical assays have been developed to screen inhibitors of viral entry. in enveloped viruses, entry is initiated by fusion of the viral envelope with the target cellular membrane. the entry mechanism has been well-described for hiv- , in which it is mediated by env glycoprotein, consisting of transmembrane gp and surface gp subunits. binding of gp to its cellular receptor, cd , and to one of two co-receptors, cxcr or ccr , triggers a refolding of gp that promotes fusion of the viral and cellular membranes. the refolding involves oligomerization of the extracellular n-and c-terminal heptad repeat (hr) domains of gp , which leads to the formation of a -helical bundle [reviewed in (melikyan, ) ]. jiang et al. established an in vitro system to quantify formation of the hiv- gp -helical bundle (jiang et al., ). in their system, the bundle is formed by mixing equimolar concentrations of peptides derived from the n-and c-hr regions of gp . elisa using the monoclonal antibody nc- , which specifically recognizes and binds an epitope formed on the gp -helix bundle but not the individual peptides, enables screening for compounds that prevent formation of fusion-active gp . for hts of hiv- fusion inhibitors, this method was modified to use a fluorescence-linked immunosorbent assay (flisa), in which the c-hr peptide was replaced with fitc-conjugated c-hr peptide (boyer-chatenet et al., ) . cell-based assays are routinely used to screen viral entry inhibitors. high throughput formats have been developed for screening of hiv- fusion inhibitors. cell-cell fusion assays involve two types of cells: effector cells that stably (bradley et al., ) or inducibly (herschhorn et al., ; ji et al., ) express hiv-env glycoprotein and target cells that express cd and either cxcr or ccr . co-cultivation of these cells leads to hiv- env-mediated cell membrane fusion, resulting in formation of multinucleated syncytia. membrane fusion induces expression of a reporter protein such as luciferase (herschhorn et al., ; ji et al., ) or β-galactosidase (bradley et al., ) . one such assay enables determination of both the efficiency and specificity of fusion inhibitors (herschhorn et al., ) . this approach uses effector cells that express both hiv- env and the renilla luciferase (r-luc) reporter protein using inducible tetracycline-controlled transactivator (tta) and target cells that express the hiv- receptor (cd ) and coreceptor (ccr ) and contain the firefly luciferase (f-luc) reporter gene under the control of a tta-responsive promoter. upon fusion of the effector and target cells, tta enters the target cells and activates the expression of the f-luc reporter. the inhibition of fusion of cellular membranes is determined as a decrease in f-luc luminescence, and the inhibitor specificity is measured as the r-luc activity (herschhorn et al., ) . based on an hiv- cell-cell fusion method that uses a computercontrolled digital image analysis system for automatic quantitation , a modified method was developed to screen inhibitors targeting mers-cov s protein (lu et al., ) . to test potential fusion inhibitors, effector cells stably expressing the mers-cov spike protein s s and egfp were used to mediate fusion with target cells expressing the dpp receptor (lu et al., ) . virion-based fusion assays are another category of cell-based fusion assays. one such approach is based on production of chimeric hiv- virions carrying β-lactamase-vpr chimeric protein (blam-vpr). chimeric hiv released into the cell culture media is isolated by ultracentrifugation and used to infect target cells. entry of the virions into the cytoplasm is detected by cleavage of a fluorescent substrate by βlactamase. the fluorescence shift corresponds to the fusion efficacy and is measurable by fluorescence microscopy, flow cytometry, or fluorometric plate reader (cavrois et al., ; cavrois et al., cavrois et al., , . modification of this assay by constructing pseudotyped hiv- virions in which hiv- env was replaced with ebola virus glycoprotein (gp) has also been described (yonezawa et al., ) . tscherne et al. developed an approach to monitor viral entry using the blam reporter (tscherne et al., ) . their assay uses influenza virus-like particles (vlps) bearing the influenza membrane proteins hemagglutinin and neuraminidase. blam tagged with influenza matrix protein (m ) is incorporated into the vlps and delivered into target cells. upon release, blam can be detected by flow cytometry, microscopically, or fluorometrically. a rapid cell-based hts method was developed to assess sars-cov entry inhibitors (zhou et al., ) . this dual envelope pseudovirion (dep) assay employs two hiv pseudoviruses: one encodes an envelope protein from the target virus and firefly luciferase and the second encodes a control, unrelated viral envelope protein and renilla luciferase. reporter expression is determined with the dual-glo luciferase assay system (promega). inclusion of the unrelated envelope protein greatly reduced false positive hits (zhou et al., ) . the method was further used to screen compounds that inhibit entry of filoviruses, including ebola virus . a similar approach employing four pseudotyped hiv viruses, carrying marburg virus glycoprotein, hemagglutinin and neuraminidase isolated from a/goose/qinghai/ / (h n ) influenza virus, ebolavirus zaire envelope glycoprotein, and lassa virus envelope glycoprotein, has been used for entry inhibitor screening . this screening identified multiple compounds with potent inhibitory activity against entry of both marburg and ebola viruses in human cancer cell lines, and confirmed their anti-ebola activity in human primary cells . other pseudotyped viral assays have been used to screen entry inhibitors of sars-cov, ebola, hendra, and nipah viruses. to establish infection, the glycoproteins of these viruses must be processed by the host intracellular lysosomal protease cathepsin l (catl). hts resulted in identification of several inhibitors that block the cleavage of viral glycoprotein but not catl itself (elshabrawy et al., ) . until recently, the development of anti-hbv therapeutics had been limited by the lack of an in vitro infection system. several aspects of the hbv life cycle have been elucidated using in vitro production of hbv particles after transfection of human hepatoma cell lines (hepg ) with recombinant hbv dna and by establishment of hepatoma cell lines with the entire hbv genome integrated, such as the hepg . . cell line (ladner et al., ; sells et al., ; sureau et al., ) . in addition to differentiated human (phhs) (gripon et al., ) and tupaia belangeri (glebe et al., ) hepatocytes, the heparg cell line, which exhibits hepatocyte-like characteristics, also supports hbv replication (gripon et al., ) . the identification of sodium taurocholate cotransporting polypeptide (ntcp) as an hbv receptor by yan et al. opened possibilities to use ntcp-complemented hepg cells not only for studies of hbv replication mechanisms but also for hts of inhibitors (yan et al., ) . in an infection competition assay, hbv particles were isolated and used to infect heparg and phhs cells that had been pre-incubated with hbv envelope protein-derived peptides to test their potential activity to block hbv infection. twelve days after infection, viral rnas were quantified by northern blot (gripon et al., (gripon et al., , . using this assay, researchers identified a peptide that specifically prevents hbv and hdv entry into heparg and phhs cell lines (gripon et al., ) , primary cultures of tupaia hepatocytes (glebe et al., ) , and cells in vivo (lütgehetmann et al., ; petersen et al., ; volz et al., ) . recently, a phase iia clinical trial of a first-in-class entry inhibitor (myrcludex-b) that functions as an ntcp inhibitor was promisingly completed (bogomolov et al., ) . development of cell culture systems producing hcv pseudoparticles (hcvpp) and infectious hcv particles (hcvcc) has shed light on hcv interactions and enabled discovery of antiviral drugs and vaccines (colpitts et al., ) . hcvpp consist of hcv e and e glycoproteins enveloping a retroviral particle that packages gfp mrna during assembly (bartosch et al., ) . the entry efficiency of hcvpp can be quantified by facs analysis as the number of gfp-positive cells. use of this screening system led to discovery of a triazine derivative that blocks the entry of hcv (baldick et al., ) . production of hcvcc is a robust system to generate infectious hcv in naïve cells (kato et al., ; zhong et al., ) . the anti-hcv activity of hundreds of compounds approved for a wide variety of indications was determined immunochemically with anti-hcv e antibody in -well plate format. over thirty compounds displayed anti-hcv activities, most of which were directed against hcv entry (gastaminza et al., ) . the majority of viruses enter cells by endocytosis. unfortunately, there are no suitable experimental techniques for endosome handling, making it difficult to study early steps in the viral life cycle such as uncoating and capsid disassembly. viruses that enter cells by direct membrane fusion, such as hiv- , are an exception. there are several methods available to monitor and quantify hiv uncoating. as some of these were reviewed recently by campbell and hope (campbell and hope, ) , we will discuss them very briefly. two main techniques are used in these assays: ultracentrifugation or utilization of hiv- specific cellular restriction factors. the "in vitro core-stability assay" is based on ultracentrifugation of released hiv- virions through a detergent layer, where the viral membrane dissolves, into a sucrose gradient, where the viral cores are concentrated (shah and aiken, ) . the "fate of capsid" assay uses ultracentrifugation through a sucrose cushion to separate the hiv- core from a whole cell lysate prepared shortly after infection (stremlau et al., ) . the "csa-washout assay" involves specific cellular factors (trim-cyclophilin) that restrict hiv- infection by binding to the capsid core and cyclosporine a (csa), which can effectively turn off restriction (hulme et al., ) . recently, a novel entry/uncoating assay (eurt), an alternative to blam-vpr (described in section . ), was reported (da silva santos et al., ). it quantifies the protein product of a virion-packaged mrna reporter upon uncoating. a method to monitor the uncoating/disassembly of the capsid of influenza a virus, which enters the cell by endocytosis, also is based on ultracentrifugation (stauffer et al., ) . purified virions are separated using velocity gradient centrifugation through a two-layer glycerol gradient. similar to the "in vitro core stability" assay for hiv, the sedimenting viruses migrate through the detergent-containing layer of the gradient, which dissolves the membrane to release the viral core. moreover, modification of the detergent-containing glycerol layer-for example, by lowering ph, changing ionic strength, or adding putative viral uncoating factors-enables study of the factors or compounds that affect viral uncoating in vitro. depending on the virus type, either dna or rna polymerases replicate the viral genome. thus, these enzymes play a key role in viral life cycles. reverse transcriptase, an rna-dependent dna polymerase of retro-and hepadnaviruses, is unique among nucleic acid polymerases. despite the different mechanisms of viral replication, polymerases, which are essential for all viruses, are excellent targets for antiviral therapies. polymerase inhibitors represent the vast majority of clinically approved antiviral drugs, followed by protease inhibitors, immunostimulators, entry inhibitors, and integrase inhibitors [reviewed in (de clercq and li, ) ]. polymerases continue to be a preferred target for newly designed inhibitors (clercq, ; de clercq and li, ) . polymerase inhibitors may be nucleoside and nucleotide analogs, pyrophosphate analogs, or nonnucleosides, such as allosteric inhibitors (de clercq and li, ; Öberg, ) . non-specific approaches such as plaque assays were initially used to monitor the effectiveness of polymerase inhibitors (tino et al., ; tisdale et al., ) . the activity of these inhibitors also can be evaluated based on their ability to prevent the cytopathic effects of the virus (zhang et al., ) . more straightforward assays involve measurement of incorporated radio-labeled nucleotides, which directly reflects the polymerase activity (coates et al., ; hirashima et al., ; joyce, ) ; these include hts methods using homopolymeric polycytidylic acid and [ h]-gtp (amraiz et al., ) . alternatively, the pyrophosphate released during the polymerase reaction can be measured luminometrically by combining the primer extension with the commercial piper assay (malvezzi et al., ) . the pyrophosphate anion also can be detected with dna-attached magnetic nanoparticles (tong et al., ) or quantum dots (chai et al., ) . frequently used fluorescence methods avoid the use of radiochemicals. these methods exploit a fluorescent label, such as dsdna binding dyes like sybr green or pi-cogreen (driscoll et al., ; holden et al., ; zipper et al., ) , or fret between two nucleotides (schwartz and quake, ; weiss, ) . numerous kits for quantification of products of both dna and rna polymerases, including reverse transcriptase, are commercially available (bustin, ) . quantitative real-time pcr is a preferred method to monitor the activity of dna polymerases (cellular as well as viral) in the presence of inhibitors (beadle et al., ; zweitzig et al., ) , and quantitative real-time reverse-transcription pcr has become standard for screening inhibitors of viral rna polymerases okon et al., ; pelliccia et al., ; zhang et al., ) . white et al. described a hts method using a microfluidic apparatus combined with digital pcr for single-cell analysis (white et al., ) . in their method, a fluorescently labeled template also serves as a primer, due to stem formation. it emits a measurable polarization signal both upon binding of the polymerase and extension (mestas et al., ) . the assay has been used for hts of inhibitors of poliovirus rna polymerase (campagnola et al., ) . when available, viral genomes modified with reporter genes can be used for cell-based luminescent or fluorescent screening of viral inhibitors (beadle et al., ; edwards et al., ; fenaux et al., ; feng et al., ; lo et al., ; madhvi et al., ; wang et al., c) . this approach can be extended to screen inhibitors of other enzymes. in the aptamer-based approach, a dna template encodes rna of interest joined to a fluorescence module and ribozyme sequence. when transcribed, the fluorescence module is released and detected (höfer et al., ) . hcv uses an rna-dependent rna polymerase (rdrp). a unique cell-based assay for monitoring hcv rna polymerase (ns b) activity, based on the innate immune signaling molecule retionic acid-inducible gene i product (rig-i), has been developed (ranjith-kumar et al., ) . the rig-i-like receptors are cytosolic proteins that recognize viral rna and induce production of proinflammatory cytokines and interferon-activated genes (jensen and thomsen, ) . rig-i triggers cytokine production stimulated by various viral rnas from different families, including flaviviridae, paramyxoviridae, rhabdoviridae, orthomyxoviridae, and arenaviridae, as well as ebola virus rna (jensen and thomsen, ) . the assay is based on recognition of hcv rna produced by active ns b by rig-i, followed by rig-i-mediated activation of firefly luciferase expression controlled by the interferon b promoter. renilla luciferase expression is used for normalization of transfection efficacy. the viral grnas of some rna viruses are modified at the ′ ends with cap structures, which may be either acquired from the host cell mrna (e.g. in influenza virus) or newly synthesized (e.g. flaviviruses). the methylation of viral grna mimics that of cellular mrna cap structures to enhance the chances of the virus to escape from cellular defense mechanisms and also to increase the efficiency of translation of viral proteins. virus-specific methyltransferases are thus a promising therapeutic target. virus-encoded methyltransferases have been identified and characterized in flaviviruses such as zika virus coutard et al., ; duan et al., ; munjal et al., ; zhao et al., ) , west nile virus, and dengue virus (dong et al., ) ; rhabdoviruses such as vesicular stomatitis virus (vsv) (rahmeh et al., ); coronaviruses such as sars (wang et al., b) ; and roniviruses (zeng et al., ) . in flaviviruses, the n-terminal part of ns methyltransferase catalyzes cap formation via both guanine n- and ribose ′-oh methylations at the ′ end of grna, and the c-terminus of the enzyme is responsible for the rna polymerase activity (ray et al., ; zhao et al., ) . the c-terminal part of the sars nsp protein exhibits guanine n -methyltransferase activity, forming the grna cap, while the ′- ′ exoribonuclease activity of the n-terminus enhances the fidelity of viral replication (case et al., ; minskaia et al., ) . in vsv, the methyltransferase activity resides in the conserved region vi of the multifunctional large polymerase protein (li et al., ; ma et al., ) . some cellular methyltransferases regulate viral infections, as shown for herpes simplex virus, for which epigenetic control is involved in viral latency (cliffe and wilson, ) . inhibition of human histone methyltransferases such as histone-lysine n-methyltransferase (ezh / ) (arbuckle et al., ) or lysine-specific demethylase- (lsd ) induces antiviral signaling pathways (liang et al., ) . the inhibitory effect of histone demethylase activity has been demonstrated for human cytomegalovirus (gan et al., ) and herpes simplex virus (hill et al., ; liang et al., ) . the activity of purified recombinant methyltransferases can be determined by measuring the methylation by-product s-adenosyl homocysteine (sah) using commercially available kits. in one such assay, conversion of s-adenosyl methionine (sam) to sah is monitored luminometrically via luciferase reaction, in which measurable atp is generated through a sequence of reactions with mtase-glo™ reagent (promega). the epigeneous™ methyltransferase kit (cisbio bioassays) is based on competition of produced sah with fluorescently labeled sah (sah-d tracer) for binding to a terbium cryptate-labeled anti-sah antibody. the decrease in fret between the tracer and antibody is then evaluated. elisa using anti- -methylcytosine antibody can be used to quantify methylation of immobilized cytosine-rich dna substrate (e.g. epiquik™ dna methyltransferase activity/inhibition assay kit; epigentek). this method was modified with homogenous time resolved fret (degorce et al., ) to screen a library of inhibitors against sars-cov nsp . flaviviral and human cap n -mtases have been screened with radioactive assays using h-labeled sam and gpppac or m gpppac synthetic rnas. the h methyl transferred onto deae filter-bound rna can be measured by scintillation after multiple washings to remove unincorporated h-sam . alternatively, in vitro transcription can be carried out using h-sam. the radioactively labeled products are then resolved by thin-layer chromatography and developed using a phosphorimager (li et al., ) . a yeast cell-based method was established based on the finding that coronavirus methyltransferase can functionally replace a methyltransferase essential for yeast viability sun et al., ) . in this method, sun et al. constructed recombinant yeasts producing the viral methyltransferase instead of the yeast one (sun et al., ) . this strain was used for a hts of methyltransferase inhibitor activity that negatively correlated with the cell density after h incubation. virus-encoded atpase-driven helicases have been identified in numerous human pathogens. helicases from several (+)rna viruses have been characterized, including ns helicases from flaviviruses such as dengue virus, hcv, west nile virus, yellow fever virus, and japanese encephalitis virus (cao et al., ; gu and rice, ; jain et al., ; lin et al., ; nedjadi et al., ; wu et al., ) . sars and mers coronaviruses encode nsp with helicase activity (adedeji and lazarus, ; hao et al., ; seybert et al., ) . in semliki forest virus, a representative member of the togavirus family, helicase activity is encoded by the n-terminal domain of nsp protein. helicases are also common in some dna viruses, including poxviruses. these include the vaccinia virus helicase-primase d (bayliss and smith, ; hutin et al., ) , e protein of bovine papilloma virus (yang et al., ) , and the large tumor antigen of sv (stahl et al., ) . helicases appear to be attractive targets for antiviral drugs (briguglio et al., ; frick, ; reynolds et al., ) , but development of such compounds is challenged by cytotoxicity and bioavailability issues (kwong et al., ) . traditional methods for monitoring the activity of rna helicases use radioactively labeled dsrna substrates and follow the unwinding reaction by electrophoretic separation (nondenaturing page) of the ss reaction products, which are detected autoradiographically (adedeji et al., ; utama et al., ) . to determine whether the inhibitor affects binding of helicase to nucleic acid, a standard gel mobility shift assay is usually used (adedeji et al., ) . helicase activity is fueled by atp hydrolysis; thus, inhibition of atpase activity became another possible antiviral strategy. atpase activity can be determined either by the decrease in atp or formation of adp (using commercially available fluorescent anti-adp antibodies) or inorganic pyrophosphate. phosphates released by atp hydrolysis can form a molybdophosphate complex that can be measured colorimetrically using malachite green, quinaldine red, or rhodamine b (baykov et al., ; debruyne, ; miyata et al., ) or by lightscattering (oshima et al., ) . the colorimetric methods can be miniaturized for hts (zuck et al., ) . the absorbance-based assay was also converted into a hts method based on fluorescence quenching by a colored quinaldine red complex ). an alternative method employs a europium-tetracycline complex for luminescent determination of inorganic phosphate (schäferling and wolfbeis, ) . a more complex coupled enzyme colorimetric assay (with maltose phosphorylase, glucose oxidase, and horseradish peroxidase) was used for hts of atpase inhibitors (avila et al., ) . assays for luminescent and fluorescent screening of atpase activity, including an immunochemical method using fluorescently labeled anti-adp antibodies, have been reviewed by shadrick and colleagues (shadrick et al., ) . in a radioactive method using [γ- p]atp, the amp product was separated from unreacted atp by thin-layer chromatography on polyethyleneimine-cellulose and visualized autoradiographically (adedeji et al., ) . several research groups have described fluorescence assays to identify inhibitors targeting sars-cov helicase (nsp ) (adedeji et al., ; cho et al., ; Özeş et al., ) . the substrate is usually a dsdna oligonucleotide consisting of a fluorescently labeled strand annealed to the complementary strand carrying a quencher. this approach was also adapted for real-time determination of the rna helicase activity of hcv (tani et al., ) . in this assay, an ssdna capture strand complementary to the strand carrying the quencher was used to prevent reannealing of the unwound duplex. recently, a colorimetric assay for monitoring helicase activity using dna-conjugated gold nanoparticles was developed (deka et al., ) . this method is based on shifts in the optical properties of nanoparticles due to dna unwinding and allows simple screening of inhibitor activity. the dsdna melting curves can be determined spectrophotometrically (at nm and nm) or even by the naked eye. another fluorescence hts of dengue virus ns helicase inhibitors measures the unwinding of a double-labeled molecular beacon (basavannacharya and vasudevan, ) . other approaches involve graphene oxide-based fluorescence monitoring of viral helicase activity [reviewed in (jang et al., ) ]. a gquadruplex-based method for label-free determination of hcv helicase ns activity measures changes in the luminescence of transition metal complexes with dna upon helicase-mediated quadruplex melting (leung et al., ) . both protein tyrosine kinases and protein serine/threonine (s/t) kinases have been found in viruses. tyrosine kinase function is wellunderstood in connection with oncogenic retroviruses, [for review on retroviral oncogenes, see (vogt, ) ]. in contrast to tyrosine kinases from oncogenic viruses, such as rous sarcoma virus src tyrosine kinase, viral s/t kinases share little homology with cellular enzymes. they are exclusively encoded by large dna viruses (e.g. herpesviruses), in which they play important roles in viral virulence, helping the virus to escape defense mechanisms such as those regulated by cytokine signaling pathways (jacob et al., ; sato et al., ) . their autophosphorylation and transphosphorylation activities mimic those of cellular cyclin-dependent kinases such as cdc . for example, viral kinases can phosphorylate translation elongation factor delta (ef- δ) (jacob et al., ; kawaguchi and kato, ) . all herpesviruses encode the s/t protein kinase ul , and us s/t kinases have been described in the alphaherpesvirus subfamily (kato, ; kawaguchi and kato, ) . in addition to protein kinases, hsv encodes a thymidine kinase. unlike cellular thymidine kinase, hsv thymidine kinase has a wide substrate specificity that includes pyrimidine and purine phosphonate analogs (e.g. acyclovir, ganciclovir, penciclovir) (de clercq and li, ) . in the body, ganciclovir is phosphorylated by cellular kinases and penciclovir and acyclovir by virus-encoded thymidine kinases to the active nucleoside triphosphate forms of the drugs that inhibit viral dna synthesis (de clercq and li, ; kokoris and black, ) . some cellular protein kinases appear to support viral replication. for example, polo-like kinases induce early stages in the influenza virus life cycle (pohl et al., ) , and human protein kinase c regulates the assembly of the ribonucleoprotein complexes in influenza virus (mondal et al., ) . inhibitors of abelson tyrosine-protein kinase are active against sars and mers coronaviruses (coleman et al., ) . several in vitro approaches can be used to determine kinase activity as well as the activity of kinase inhibitors. analyses of the cellular phosphoproteome, sometimes accompanied by phosphopeptide enrichment, have become standard to determine kinase activity (lea and simeonov, ; meyer et al., ; vyse et al., ) . these assays can be used to assess the impact of inhibitors on the overall phosphoproteome in mammalian cells. although these methods provide complex information about the overall array of kinases and phosphatases in the cell (olsen et al., ) , they are not applicable to screening inhibitors of a single viral kinase. for these purposes, in vitro assays with recombinant kinases have been developed [for review see ], including some hts fluorescence methods (zegzouti et al., ) that can replace radioactive techniques using [γ p]atp (sanghera et al., ) . mass spectrometric analysis also can be used to identify in vitro kinase inhibitors. for these analyses, synthetic peptides, proteins, or phosphatase-and heat-treated tissue samples (to dephosphorylate the proteins and inactivate all enzymes, respectively) are subjected to kinase treatment in the presence of inhibitors (huang et al., ; meyer et al., ; xue et al., ) . recombinant kinase activity in the presence of inhibitors can be quantified as atp consumption or adp production in the phosphorylation reaction by numerous commercial kits. other methods monitor binding of inhibitors to phage-displayed kinases in ligand competition assays (fabian et al., ) . fluorescence methods including fret, fluorescence polarization or intensity endpoint measurement, and lifetime imaging of fluorescence including fluorescence biosensors (zhang and allen, ) have been reviewed elsewhere. sulfonamido-oxine labeled peptides can be used as chromophores that bind mg + upon phosphorylation and emit chelation-enhanced fluorescence (devkota et al., ; luković et al., ) . kinase-catalyzed phosphorylation of fluorescent peptides promotes their binding to metal-coated nanoparticles, which decreases their mobility and enhances measurable fluorescence polarization (lea and simeonov, ; sportsman et al., ) . tbiii complexes, in which phosphotyrosine induces fluorescence emission (wang et al., a) , may be used to evaluate protein tyrosine kinase activity (akiba et al., ; sumaoka et al., ) . fluorescence polarization methods also can be useful for drug screening [reviewed in (hall et al., ) ]. other alternatives are immunochemical methods that use antibodies specific to the phosphorylated amino acids, such as phosphotyrosine (li et al., ; youngren et al., ) or phosphoserine/ phosphothreonine. these antibodies can be used to detect protein/ peptide phosphorylation by western blotting, elisa, or immunoprecipitation of phosphorylated proteins for further mass spectrometry-based analysis (grønborg et al., ; zhang et al., ) . an elegant approach that limits the false-positive hits in screening of specific kinase inhibitors is based on an in situ proximity ligation assay using both an antibody against the target protein and an anti-phosphotyrosine antibody (leuchowius et al., ) . both antibodies are coupled with oligonucleotides, which when brought together due to antibody binding, can be enzymatically ligated and replicated through rolling circle amplification to form a long linear tandem repeat of sequences detectable by a complementary fluorescent oligonucleotide. in addition to protein kinases, some lipid kinases have been targeted by antivirals. one example is sphingosine kinase , which affects replication of dengue virus (aloia et al., ) . its activity can be determined by measuring the production of p-labeled sphingosine- phosphate from sphingosine and [γ p]atp (clarke et al., ; pitman et al., ) . retroviral integrase inhibitors are a new type approved new type of inhibitors imposed by the emergence of drug-resistant mutants. hiv integrase activities, integrase inhibitors, and drug resistance have been discussed in detail elsewhere (andrake and skalka, ; anstett et al., ; hajimahdi and zarghi, ; liao et al., ; podany et al., ; thierry et al., ) . methods to assess the two major activities of integrase-end processing of the reverse transcription product and its joining to target chromosomal dna-have been reviewed in detail by several groups (engelman and cherepanov, ; marchand et al., ; merkel et al., ) . initial methods used radioactively labeled dna oligonucleotides comprising the terminal cis-acting sequences of linear viral dna required for integration. the joining of the processed strand to the other strand (self-integration) or to supplemented target dnas can be analyzed by page (katz et al., ; katzman et al., ) . a less time-consuming, non-radioactive method involves timeresolved fluorescence anisotropy measurement using a -meric oligonucleotide fluorescently labeled on the terminal gt dinucleotide. this assay monitors the binding of integrase to the substrate as well as the subsequent ′-processing reaction, which both change the anisotropy (guiot et al., ) . alternatively, the yields of both the processing and joining reactions can be measured upon separation of the radioactively labeled product from the rest of the dna molecule using adsorption to pei-cellulose (muller et al., ) . a real-time hts method measures fluorescence emission resulting from removal of the ′-terminal dinucleotide, labeled with a quencher, by integrase (he et al., ) . han et al. described a fluorescence method to screen molecules that inhibit binding of integrase to viral dna . methods evaluating the integrase strand transfer reaction have been modified to a high-throughput format using magnetic beads (he et al., ) or streptavidin-coated microplates (john et al., ) . a method to assess strand transfer by time-resolved fret with a europium-streptavidin-labeled substrate has been optimized for -and -well plate formats (wang et al., ) . the hbv capsid protein is the building block of the viral core, surrounding the viral nucleic acid (pre-genomic rna, pgrna) and reverse transcriptase. the hbv core is icosahedral, formed by copies of capsid protein dimers. in vitro hts of hbv core assembly inhibitors using a modified hbv capsid protein has been described (stray et al., ) . the capsid protein was modified by deleting the nucleic acid binding domain, which is dispensable for capsid assembly, and the nterminal assembly domain alone was used in the assay. to fluorescently label the hbv capsid protein, all cysteine residues dispensable for assembly were replaced with alanines. a unique cysteine residue (c ) was c-terminally joined to the assembly domain and labeled with fluorescent bodipy-fl dye (c bo). during assembly, capsid proteins dimerize, bringing c bo residues close together and resulting in c bo fluorescence self-quenching. following incubation of the labeled hbv protein with inhibitors, fluorescence was measured in black -well microtiter plates. development of in vitro assembly systems has contributed greatly to current understanding of the structure of retroviral particles and mechanisms of virion formation. these systems also became the base for several high throughput assays for screening assembly inhibitors. during the last years, a number of in vitro assembly assays have been established, mainly for hiv- (campbell et al., ; campbell and rein, ; ehrlich et al., ; gross et al., ; lanman et al., ) , mason-pfizer monkey virus (bohmova et al., ; klikova et al., ; rumlova-klikova et al., ; rumlova-klikova et al., ; ulbrich et al., ) , rous sarcoma virus vogt, , ; purdy et al., purdy et al., , , and murine leukemia virus (dolezal et al., ; hadravova et al., ; cheslock et al., ) . hts assays for inhibitors of hiv- assembly include several methods using purified hiv- ca or ca-nc proteins. one of them, the turbidimetric assay, is based on the observation that direct dilution of the hiv- capsid protein (ca) into a high-salt solution ( . - . m nacl) leads to the formation of tubular structures. as the tube formation is accompanied by an increase in light scattering, assembly can be detected as an increase in turbidity, and the rate of turbidity change is proportional to the rate of the assembly (lanman et al., ) . other method published by lemke et al., exploits the affinity of nucleocapsid (nc) to a short tg-rich deoxyribooligonucleotide, d(tg ), which is used as a scaffold (lemke et al., ) . this arrangement enables ca to assemble at much lower protein and salt concentrations than in the turbidimetric assay (lanman et al., ) . biotin-labeled d(tg ) bound on the surface of neutravidin-coated microtiter well plates nucleates assembly of complexes of ca-nc and soluble fluorescein-labeled d(tg ). fluorescence is measured after washing to remove the unbound and unassembled material from the captured assembly products (lemke et al., ) . similarly, the faith assay uses a dually labeled oligonucleotide (tqon). however, in this case, the ssdna oligonucleotide tqon is labeled with the reporter dye fluorescein (fam) as well as black hole quencher (bhq); thus, it does not emit any fluorescence. the assembly reaction is triggered by mixing hiv- ca-nc or a gagtruncated assembly-competent version with tqon. following incubation, during which tqon is incorporated into the particles, exonuclease is added to degrade unbound tqon, while co-assembled tqon is protected from cleavage. degradation of free unbound tqon with exoi results in separation of fam from its quencher, and the emitted fluorescence is measured (hadravova et al., ) . phage display has been employed to screen peptide inhibitors of hiv- assembly (sticht et al., ) . a commercial library of m -derived phages presenting random -amino-acid peptides was analyzed for specific binding to purified ca or ca-nc proteins. the specifically bound phages were sequenced, and corresponding peptides were chemically synthesized and re-tested in an in vitro assembly assay (gross et al., ) . late in the retroviral life cycle, grna is incorporated into the nascent particle during assembly at the plasma membrane. nc contains two zinc-finger domains that are responsible for specific binding of grna. to screen for compounds that would prevent nc-rna/dna interactions, a hts system consisting of two sequential screens was developed (breuer et al., ) . the primary screen uses fluorescence polarization (fp), while the secondary one uses differential scanning fluorimetry (dsf). the combination and order of these two techniques were selected to first identify compounds that disrupt interactions between dna and nc, and then identify the compounds binding to nc during the secondary screen. a rapid and simple turbidimetric method was developed to screen inhibitors of assembly of hcv core protein (fromentin et al., ) . for the in vitro assembly reaction, two components, an n-terminal part of the hcv core protein corresponding to the minimal assembly competent domain and rna corresponding to the full-length ′utr of hcv, were used. assembly of hcv nucleocapsid-like particles was initiated by mixing the purified protein and nucleic acid, and the assembly process was monitored by measuring turbidity at nm. numerous viruses, including picornaviruses, retroviruses, alphaviruses, and flaviviruses, encode proteases that are essential for their virulence. the majority of viral proteases specifically cleave viral polyprotein precursors to liberate the functional proteins of the virion. some viral proteases, such as the papain-like proteases of coronaviruses, also reprogram cellular signaling pathways, including ubiquitination mechanisms and interferon controlled responses, to prevent degradation of viral components (clementz et al., ; frieman et al., ; randow and lehner, ; xing et al., ) . numerous viruses, including papillomaviruses (bronnimann et al., ; buck et al., ) and retroviruses (hallenberger et al., ) , use also host cell proteases, mainly furin, to trim their envelope and surface proteins. this triggers conformational changes required for interaction with cellular receptors and membrane fusion in enveloped viruses. some viruses use proteases other than furin to modulate their infectivity, as shown for viruses entering airway epithelial cells [reviewed in (laporte and naesens, ) ] such as influenza virus (böttcher-friebertshäuser et al., ; kühn et al., ) , newcastle disease virus (gotoh et al., ) , and respiratory syncytial virus (sugrue et al., ) . extensive research efforts have yielded detailed information about hiv- protease and its inhibitors [reviewed in (de clercq, ; konvalinka et al., ; midde et al., ) ]. large amounts of data also are available for hcv (foote et al., ; pawlotsky et al., ; razonable, ) and sars-cov cl protease inhibitors (pillaiyar et al., ) , although there is not yet an inhibitor of the latter target approved for clinical use. numerous approved drugs are synthetic peptides derived from the natural proteolytic substrates of target viruses modified to improve the in vivo effects related to bioavailability, stability, and so on. numerous in vitro assays to monitor the activity of proteases and their inhibitors, including commercial kits, have been developed. classical methods use synthetic peptides that mimic the target sites of the protease. although some of the methods described here were not originally designed to screen the activity of viral proteases in the presence of inhibitors, they can be adapted for this purpose by simply changing the peptide sequence to the target site of the protease of interest. the cleavage yield is usually monitored either colorimetrically (ding and yang, ; zhou et al., ) or as a change in fluorescence triggered by the release of fluorescent labels such as -amido- -methylcoumarin (amc) or rhodamine (grant et al., ) . fluorogenic substrates have been used to determine the activity of coronavirus proteases and screen inhibitors (kuo et al., ; lee et al., ; park et al., ; song et al., ; tomar et al., ; wang et al., ; yang et al., ; zhao et al., ) . some recently described arrangements employ nanoparticles (feltrup and singh, ; khalilzadeh et al., ; udukala et al., ; wang et al., ; zeng et al., ) or quantum dot bioconjugates (lee and kim, ; li et al., ; medintz et al., ) with immobilized fluorescently or luminescently labeled peptide substrates. alternatively, cleavage products may be monitored by analysis of proteolytic products by mass spectrometric methods (hu et al., ; joshi et al., ; lathia et al., ; rumlová et al., ) , analytical hplc (teruya et al., ) , or electrochemical methods based on the difference in penetration of substrate and cleavage products through the membrane of a polyionselective sensor (gemene and meyerhoff, ; han et al., ) . to study the specificity of inhibitor binding and to extend the research to rational design of inhibitors, x-ray or nmr structures of proteases in complex with the inhibitor may be determined, as reported in numerous cases for the proteases of hiv- [reviewed in (ghosh et al., ) ], hcv (yilmaz et al., ) , and mers . cell-based assays can provide additional information, including the capability of the inhibitor to pass through the cell membrane and its stability in the cytoplasm. a general determination of infectivity, such as a plaque cytotoxicity assay in the presence of protease inhibitor, may be used for confirmation. one elegant example exploits the cytotoxicity of hiv protease. cells are transfected with a protease precursor fused to gfp. in the absence of inhibitor, hiv- protease is autocatalytically activated and cleaves a broad variety of cellular proteins, resulting in activation of apoptosis and cell death (cummins and badley, ; rumlova et al., ) . this toxic effect, when suppressed by active inhibitors, results in production of a gfp signal in surviving cells (lindsten et al., ) . another elegant approach for hiv and coxsackievirus b proteases, which both undergo autocatalytic cleavage, employs constructs in which the protease gene is inserted between sequences encoding the dna-binding domain and the domain that activates transcription of the gal -lacz reporter gene (dasmahapatra et al., ; murray et al., ) . the protease-mediated cleavage separates the dna-binding domain from the trans-activating domain and results in failure of reporter gene transcription. this approach has also been modified with gfp as a reporter gene (hilton and wolkowicz, ) . co-expression of cleavage-activated luciferase substrate and mers-cov protease permits both live-cell imaging and quantification of the enzyme activity (kilianski et al., ) . the need to develop new antiviral compounds will likely persist over the long term, although there has been enormous progress in molecular biology methods, especially rna silencing, bioinformatics, imaging, and structural biology techniques. viruses present challenging targets for drug development due their flexibility and adaptability caused by the error-prone copying of their genomes, which can result in emergence of drug-resistant mutants. viral integration into the host genome and inhibitor toxicity are other obstacles. here, we provide an overview of in vitro methods, including cell-based assays, that may be suitable for screening of antivirotics that interfere with the key steps of viral life cycles and target either virus or cell-encoded proteins required for the infectivity. biochemical characterization of middle east respiratory syndrome coronavirus helicase severe acute respiratory syndrome coronavirus replication inhibitor that interferes with the nucleic acid unwinding of the viral helicase components of adenovirus genome packaging click conjugation of a binuclear terbium(iii) complex for real-time detection of tyrosine phosphorylation pancreatic acinar cell-specific autophagy disruption reduces coxsackievirus replication and pathogenesis in vivo investigation of sphingosine kinase in interferon responses during dengue virus infection viral apoptotic mimicry hiv- uncoating: connection to nuclear entry and regulation by host proteins development of robust in vitro rnadependent rna polymerase assay as a possible platform for antiviral drug testing against dengue retroviral integrase: then and now reactivation and lytic replication of kaposi's sarcoma-associated herpesvirus: an update hiv drug resistance against strand transfer integrase inhibitors toward the identification of viral cap-methyltransferase inhibitors by fluorescence screening assay inhibitors of the histone methyltransferases ezh / induce a potent antiviral state and suppress infection by diverse viral pathogens highthroughput screening for hsp atpase inhibitors a novel small molecule inhibitor of hepatitis c virus entry junction adhesion molecule is a receptor for reovirus infectious hepatitis c virus pseudoparticles containing functional e -e envelope protein complexes suramin inhibits helicase activity of ns protein of dengue virus in a fluorescence-based high throughput assay format a malachite green procedure for orthophosphate determination and its use in alkaline phosphatase-based enzyme immunoassay vaccinia virion protein i r has both dna and rna helicase activities: implications for vaccinia virus transcription -phosphonomethoxy)ethyl]guanine (ode-bn-pmeg), a potent inhibitor of transient hpv dna amplification hepatitis b virus replication early steps in avian reovirus morphogenesis late budding domains and host proteins in enveloped virus release escape of non-enveloped virus from intact cells nonlytic spread of naked viruses nonlytic viral spread enhanced by autophagy components viral entry pathways: the example of common cold viruses herpes virus replication treatment of chronic hepatitis d with the entry inhibitor myrcludex b: first results of a phase ib/iia study effect of dimerizing domains and basic residues on in vitro and in vivo assembly of mason-pfizer monkey virus and human immunodeficiency virus cleavage of influenza virus hemagglutinin by airway proteases tmprss and hat differs in subcellular localization and susceptibility to protease inhibitors development and automation of a -well cell fusion assay to identify inhibitors of ccr /cd -mediated hiv virus entry identification of hiv- inhibitors targeting the nucleocapsid protein inhibition of rna helicases of ssrna(+) virus belonging to flaviviridae, coronaviridae and picornaviridae families furin cleavage of l during papillomavirus infection: minimal dependence on cyclophilins maturation of papillomavirus capsids absolute quantification of mrna using real-time reverse transcription polymerase chain reaction assays hiv- virion-cell interactions: an electrostatic model of pathogenicity and syncytium formation high-throughput screening identification of poliovirus rna-dependent rna polymerase inhibitors hiv- capsid: the multifaceted key player in hiv- infection in vitro assembly properties of human immunodeficiency virus type gag protein lacking the p domain self-assembly in-vitro of purified ca-nc proteins from rous-sarcoma virus and human-immunodeficiency-virus type- in vitro assembly of virus-like particles with rous sarcoma virus gag deletion mutants: identification of the p domain as a morphological determinant in the formation of spherical particles modulation of hiv-like particle assembly in vitro by inositol phosphates novel escrt functions in cell biology: spiraling out of control? molecular mechanism of divalentmetal-induced activation of ns helicase and insights into zika virus inhibitor design hcv infection by cell-to-cell transmission: choice or necessity? mutagenesis of s-adenosyl-l-methionine-binding residues in coronavirus nsp n -methyltransferase m demonstrates differing requirements for genome translation and resistance to innate immunity components and regulation of nuclear transport processes a sensitive and specific enzyme-based assay detecting hiv- virion fusion in primary t lymphocytes fluorescence resonance energy transfer-based hiv- virion fusion assay hiv- fusion assay a reversible fluorescence nanoswitch based on carbon quantum dots nanoassembly for detection of pyrophosphate ion functional screen reveals sars coronavirus nonstructural protein nsp as a novel cap n methyltransferase tsg : a novel anti-hiv- drug target protein-protein interfaces in viral capsids are structurally unique identification of a coumarin-based antihistamine as an anti-filoviral entry inhibitor charged assembly helix motif in murine leukemia virus capsid: an important region for virus assembly and particle size determination identification of a novel small molecule inhibitor against sars coronavirus helicase reduction in sphingosine kinase influences the susceptibility to dengue virus infection by altering antiviral responses deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases emerging antiviral drugs restarting lytic gene transcription at the onset of herpes simplex virus reactivation (-)- '-deoxy- '-thiacytidine is a potent, highly selective inhibitor of human immunodeficiency virus type and type replication in vitro pushing the envelope: microinjection of minute virus of mice into xenopus oocytes causes damage to the nuclear envelope how viruses access the nucleus abelson kinase inhibitors are potent inhibitors of severe acute respiratory syndrome coronavirus and middle east respiratory syndrome coronavirus fusion structures of ns methyltransferase from zika virus highthroughput approaches to unravel hepatitis c virus-host interactions localization of adenovirus morphogenesis players, together with visualization of assembly intermediates and failed products, favor a model where assembly and packaging occur concurrently at the periphery of the replication center endocytosis of viruses and bacteria zika virus methyltransferase: structure and functions for drug design perspectives structure and organization of paramyxovirus particles mechanisms of hiv-associated lymphocyte apoptosis: a novel entry/uncoating assay reveals the presence of at least two species of viral capsids during synchronized hiv- infection a genetic system for studying the activity of a proteolytic enzyme antiviral drugs in current clinical use approved antiviral drugs over the past years inorganic phosphate determination: colorimetric assay based on the formation of a rhodamine b-phosphomolybdate complex htrf: a technology tailored for drug discovery -a review of theoretical aspects and recent applications dna-conjugated gold nanoparticles based colorimetric assay to assess helicase activity: a novel route to screen potential helicase inhibitors a novel mechanism underlying the innate immune response induction upon viral-dependent replication of host cell mrna: a mistake of + srna viruses' replicases. front high-throughput screens for eef- kinase quantitative serine protease assays based on formation of copper(ii)-oligopeptide complexes functional and structural characterization of novel type of linker connecting capsid and nucleocapsid protein domains in murine leukemia virus ′-o methylation of internal adenosine by flavivirus ns methyltransferase a quantitative fluorescence-based steadystate assay of dna polymerase the crystal structure of zika virus ns reveals conserved drug targets high-throughput minigenome system for identifying small-molecule inhibitors of ebola virus replication assembly of recombinant human immunodeficiency virus type capsid protein in vitro identification of a broad-spectrum antiviral small molecule against severe acute respiratory syndrome coronavirus and ebola, hendra, and nipah viruses by using a novel high-throughput screening assay retroviral integrase structure and dna recombination mechanism a small molecule-kinase interaction map for clinical kinase inhibitors ultrastructural and biochemical basis for hepatitis c virus morphogenesis nuclear entry of dna viruses development of a fluorescence internal quenching correction factor to correct bont/a endopeptidase kinetics using snaptide antiviral nucleotide incorporation by recombinant human mitochondrial rna polymerase is predictive of increased in vivo mitochondrial toxicity risk a pathogenic picornavirus acquires an envelope by hijacking cellular membranes inhibition of hepatitis c virus replication by gs- , a potent c-nucleoside monophosphate prodrug transcription and replication mechanisms of bunyaviridae and arenaviridae l proteins fields virology boceprevir: a protease inhibitor for the treatment of chronic hepatitis c viral late domains hiv- assembly, release and maturation helicases as antiviral drug targets severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of irf and nf-κb signaling a method for in vitro assembly of hepatitis c virus core protein and for screening of inhibitors beyond tsg : the role of alix in 'escrting' hiv- proteolytic cleavage of bovine m adenovirus -encoded pviii epigenetically repressing human cytomegalovirus lytic infection and reactivation from latency in thp- model by targeting h k and h k histone demethylases unbiased probing of the entire hepatitis c virus life cycle identifies clinical compounds that target multiple aspects of the infection detection of protease activities by flash chronopotentiometry using a reversible polycation-sensitive polymeric membrane electrode recent progress in the development of hiv- protease inhibitors for the treatment of hiv/aids structure and formation of the cytomegalovirus virion pre-s antigen-dependent infection of tupaia hepatocyte cultures with human hepatitis b virus mapping of the hepatitis b virus attachment site by use of infection-inhibiting pres lipopeptides and tupaia hepatocytes the ins and outs of hiv replication mammalian subtilisin-related proteinases in cleavage activation of the paramyxovirus fusion glycoprotein: superiority of furin/pace to pc or pc / pc development of novel assays for proteolytic enzymes using rhodamine-based fluorogenic substrates hepatitis b virus infection of adult human hepatocytes cultured in the presence of dimethyl sulfoxide infection of a human hepatoma cell line by hepatitis b virus efficient inhibition of hepatitis b virus infection by acylated peptides derived from the large viral surface protein a mass spectrometry-based proteomic approach for identification of serine/threonine-phosphorylated proteins by enrichment with phospho-specific antibodies: identification of a novel protein, frigg, as a protein kinase a substrate varicella-zoster virus infectious cycle: er stress, autophagic flux, and amphisome-mediated trafficking a conformational switch controlling hiv- morphogenesis the cell biology of receptor-mediated virus entry the spring α-helix coordinates multiple modes of hcv (hepatitis c virus) ns helicase action 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protein ppxy late domain drives the production of defective interfering particles investigations on dna intercalation and surface binding by sybr green i, its structure determination and methodological implications miniaturization of absorbance assays using the fluorescent properties of white microplates characterization of a novel dna polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes this work was supported by ga Čr (cz) ga - s to mr, ga - s to tr and the ministry of education, youth and sport of the czech republic(oppc cz. . / . . / ), through its "national program of sustainability i" npu lo . key: cord- -wzsrqc p authors: xu, bo; tian, huaiyu; sabel, clive eric; xu, bing title: impacts of road traffic network and socioeconomic factors on the diffusion of pandemic influenza a (h n ) in mainland china date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: wzsrqc p the pandemic influenza virus caused the majority of the influenza a virus infections in china in . it arrived in several chinese cities from imported cases and then spread as people travelled domestically by all means of transportation, among which road traffic was the most commonly used for daily commuting. spatial variation in socioeconomic status not only accelerates migration across regions but also partly induces the differences in epidemic processes and in responses to epidemics across regions. however, the roles of both road travel and socioeconomic factors have not received the attention they deserve. here, we constructed a national highway network for and between cities in mainland china and extracted epidemiological variables and socioeconomic factors for each city. we calculated classic centrality measures for each city in the network and proposed two new measures (sumratio and multicenter distance). we evaluated the correlation between the centrality measures and epidemiological features and conducted a spatial autoregression to quantify the impacts of road network and socioeconomic factors during the outbreak. the results showed that epidemics had more significant relationships with both our new measures than the classic ones. higher population density, higher per person income, larger sumratio and multicenter distance, more hospitals and college students, and lower per person gdp were associated with higher cumulative incidence. higher population density and number of slaughtered pigs were found to advance epidemic arrival time. higher population density, more colleges and slaughtered pigs, and lower multicenter distance were associated with longer epidemic duration. in conclusion, road transport and socioeconomic status had significant impacts and should be considered for the prevention and control of future pandemics. the influenza a (h n ) pandemic posed one of the most serious global public health challenges in recent years [ ] . with the ease and speed of global travel in the st century, the world is now a global village in terms of epidemic transmission [ ] . the influenza a (h n ) virus can transmit from humans to humans by direct body contact or respiratory droplets. the infectious disease can spread widely as people carrying pathogens travel and commute between cities by multiple means of transport. thus, intercity travel is important for the diffusion of viruses [ ] [ ] [ ] . in this study, we constructed a national highway network including prefecture-level cities in mainland china and extracted three epidemiological variables (cumulative incidence, onset week, and duration of epidemics) and socioeconomic factors (related to population, income level, medical condition, livestock breeding, and school education) in each city. we calculated four classic centrality measures (degree, betweenness, closeness, and eigenvector centrality) for each city in the network and proposed two new measures (sumratio and multicenter distance). we first evaluated the correlation between the centrality measures and epidemiological features, and then used spatial autoregressive models to investigate the impacts of road network and socioeconomic factors on the diffusion of the influenza pandemic. the suspected and laboratory-confirmed cases of influenza a (h n ) used in this study were obtained from the surveillance system of the chinese center for disease control and prevention, with dates ranging from week (may) in to week (december) in . every data entry represents a case with attributes, such as the date of onset, date of birth, and address together with a corresponding administrative area number. matlab [ ] and arcgis desktop [ ] were used to extract information concerning the cumulative incidence which is the cumulative number of daily reported cases during the above time period divided by the population at risk, the onset week which is defined here as the week when the first case in a city was reported, and the duration which is the time from onset to the first epidemic peak, all in a certain prefecture-level city. statistical data for prefecture-level cities in were collected from the china economic and social development statistical database (http://tongji.cnki.net), including highway passenger capacity, urbanization ratio (denoted as urban ratio), population density (popdensity), per person gross domestic product (pgdp), average wage of employees (income), number of hospitals per , population (hospital), number of hospital beds per population (hos-bed), number of doctors per , population (doctor), number of colleges (college), number of middle schools (midschool), number of primary schools (prischool), number of college students per population (collegestu), number of middle school students per population (midschoolstu), number of primary school students per population (prischoolstu), and number of slaughtered pigs (pig). the network of national highways in china consists of nodes (representing prefecture-level cities) and links (representing national highway lines). every link is connected to two nodes, to which the two corresponding prefecture-level cities are connected by a section of national highways. there are seventy national highway lines in china. we calculated the actual distance between any pair of adjacent prefecture-level cities along the national highways and assigned a unique number to each city. two symmetric matrices (matrix a and d ) were constructed. matrix a is an adjacency matrix. if city i and j are adjacent along a national highway, then the value of element a (i, j) is one, else it is zero. matrix d is an adjacent distance matrix and element d (i, j) is the actual distance between city i and j if they are adjacent to each other along national highways (if not, d (i, j) equals zero). an unsymmetrical matrix d was generated from matrix d using the floyd algorithm [ ] to store the shortest distances between cities along the national highways. we calculated four common network node centrality measures for each city node, including the degree, which is the number of nodes directly adjacent to it; the betweenness, which is the number of shortest paths that pass through the node; the closeness, which is the sum of the shortest distances (geodesic paths) between that node and all other nodes in the network; and the eigenvector centrality, which is a measure of the influence of a node in a network [ ] . these node centralities are calculated with the igraph package [ ] in the r program [ ] . inspired by the method of dominant flow analysis [ ] , we propose a new node centrality measure called sumratio, which is a synthetic indicator combining the number of passengers and the spatial distance, measuring the global importance of a node to all others in a network. higher values of sumratio relate to more frequent exchanges of people and economic trade, which makes it easier for viruses to spread among individuals. sumratio can be calculated as follows. where g(i, j) represents the gravity coefficient between cities i and j; n is the outflow highway passenger capacity; d is the shortest distance between two cities along national highways; and m, β, δ, and γ are constants. gp(i, j) represents the ratio of gravity coefficient between cities i and j to the sum of gravity coefficients between city i and any other cities in the network and measures the relative importance of city j to city i. sumratio(j) is the sum of the gravity coefficient ratio, which measures the integrated importance of city j to all other cities in the network. the two matrices (matrix g and gp) are generated using a gravity model [ , ] based on the demographic data. matrix g is a symmetric matrix while gp is unsymmetrical [ ] . n is the total number of cities in the national highway network (n = ). m, β, and δ are set to be , , and , respectively, as estimated by reference [ ] . γ is set to be according to the result of reference [ ] . n can be estimated as follows: because the "highway passenger capacity" figures found in the statistical yearbook consisted of three parts, including the highway passenger capacity within a city, the inflow passenger capacity, and outflow passenger capacity of a city, we assumed that the inflow and outflow passenger capacities of a city are equal to each other and that they both could compose "the passenger capacity between cities". among the , nonzero values of the distance stored in matrix d , more than % were larger than km. hence, we assumed that the highway passenger capacity within a city can be represented by the number of passengers transported within the range of a circle whose center is a certain city with a radius of km. it has also been reported that the highway passenger volume occurred within a distance shorter than km accounted for more than % of the total highway passenger volume. therefore, we assumed that the highway passenger capacity within a city accounted for % of the total highway passenger volume and that the outflow highway passenger capacity accounted for %. we propose another new node centrality measure called multicenter distance, which refers to the shortest distance along national highways from a certain city to one of the eight center cities (chengdu, jinan, beijing, guangzhou, shanghai, fuzhou, wenzhou, and changsha city) where the epidemics arrived the earliest (before may , ). all the first cases in these eight cities were imported into china by planes from other countries. the spatial locations of the eight cities are shown in figure . the multicenter distance of city i (sd i ) is calculated as follows. sd i = min {d ij , i = all cities, j = center cities}, where i = , , , . . . , n; n is the total number of cities; j = , , , . . . , m; m indicates the number of central cities; and d ij is the distance along the national highways between a certain city i and a center city j. a correlation analysis was performed to assess the associations between disease variables (cumulative incidence, onset week, and duration from onset to the first epidemic peak) and highway network node centralities (adjacent degree, betweenness, closeness, eigenvector centrality, sumratio, and multicenter distance) using the pearson correlation coefficient and to figure out the significant explanatory variables for the following spatial regression analysis. to investigate the quantitative relationships between epidemic characteristics (e.g., cumulative incidence, onset week, and duration) and socioeconomic factors, including urban ratio, popdensity, pgdp, income, hospital, hos-bed, doctor, college, midschool, prischool, collegestu, midschoolstu, prischoolstu, and pig, as well as the network structure parameters of the city vertices calculated above and to quantify the contribution of road transportation and the spatial distance to the spread of the influenza virus, we used spatial autoregressive models [ ] performed in matlab. in the models, epidemic characteristics were set to be response variables, while the selected network node centrality measures and socioeconomic factors were included as explanatory variables. we were able to collect the records of all these variables in cities where influenza a (h n ) cases had been reported. before the regression analysis, we calculated the variance inflation factor (vif) of each explanatory variables to detect collinearity. a general version of the spatial model including both the spatial lag term and a spatially correlated error structure is shown as where y denotes an n × vector of response variable observations collected at n cities; x is an n × k matrix representing explanatory variables; ε is an n × vector of normally distributed (with constant a correlation analysis was performed to assess the associations between disease variables (cumulative incidence, onset week, and duration from onset to the first epidemic peak) and highway network node centralities (adjacent degree, betweenness, closeness, eigenvector centrality, sumratio, and multicenter distance) using the pearson correlation coefficient and to figure out the significant explanatory variables for the following spatial regression analysis. to investigate the quantitative relationships between epidemic characteristics (e.g., cumulative incidence, onset week, and duration) and socioeconomic factors, including urban ratio, popdensity, pgdp, income, hospital, hos-bed, doctor, college, midschool, prischool, collegestu, midschoolstu, prischoolstu, and pig, as well as the network structure parameters of the city vertices calculated above and to quantify the contribution of road transportation and the spatial distance to the spread of the influenza virus, we used spatial autoregressive models [ ] performed in matlab. in the models, epidemic characteristics were set to be response variables, while the selected network node centrality measures and socioeconomic factors were included as explanatory variables. we were able to collect the records of all these variables in cities where influenza a (h n ) cases had been reported. before the regression analysis, we calculated the variance inflation factor (vif) of each explanatory variables to detect collinearity. a general version of the spatial model including both the spatial lag term and a spatially correlated error structure is shown as where y denotes an n × vector of response variable observations collected at n cities; x is an n × k matrix representing explanatory variables; ε is an n × vector of normally distributed (with constant variance σ ) stochastic disturbances; n is the total number of cities; k is the number of explanatory variables incorporated into the regression model; w and w are n × n spatial weight matrices, usually containing contiguity relations; the parameter ρ is a coefficient on the spatially lagged response variable (w y); β represents the regression coefficients to be estimated to reflect the influence of the explanatory variables x on the variation of the response variable y; the parameter λ is a coefficient on the spatially correlated error µ; and i n is an n × n unit matrix. the earliest onset was week and the latest was week in . the earliest epidemic peak in all cities arrived in week , and the last one arrived in week . as shown in figure a , the weekly incidence curve rose slowly from may to august . then, it ascended quickly and arrived in the first peak ( cases) on september . during the period from october to december , the weekly incidence was larger than cases and reached the second peak ( , cases) on november . as shown in figure b , the cumulative number of cities where cases had ever been reported increased moderately before august . thereafter, it climbed up quickly and decelerated after september . it reached a plateau in december , when nearly all chinese cities had been exposed to the emerging influenza virus. the national highway network had a mean degree of . , an average clustering coefficient of . , a diameter of , and an average path length of . . in figure a -c, histograms of the road passenger volumes, sumratio, and multicenter distance of all the cities in the national highway network are displayed, respectively. as shown in table , each of the two newly proposed centrality measures (sumratio and multicenter distance) was significantly correlated with all three epidemiological features (cumulative incidence, onset week, and duration). while the correlations between classic centrality measures (degree, betweenness, and eigenvector centrality, except for closeness centrality) and any of the three epidemiological features were not statistically significant. in addition, sumratio and multicenter distance performed even better than closeness centrality. multicenter distance was positively correlated with the cumulative incidence (r = . , p < . ) and onset week (r = . , p < . ) but negatively correlated with the duration from onset to the first epidemic peak (r = − . , p < . ). a positive correlation between closeness centrality and onset week (r = . , p < . ) was identified, as well as a negative association between closeness and duration (r = − . , p < . ). sumratio was positively associated with the cumulative incidence (r = . , p < . ) and duration (r = . , p < . ), but negatively associated with the onset week (r = − . , p < . ). the pearson correlation coefficients between the number of slaughtered pigs and cumulative incidence, onset week, and duration of epidemics were − . (p = . ), − . (p < . ), and . (p < . ), respectively. therefore, the number of pigs would not be taken as an explanatory variable of the regression model when the cumulative incidence was taken as the response variable. as shown in table , each of the two newly proposed centrality measures (sumratio and multicenter distance) was significantly correlated with all three epidemiological features (cumulative incidence, onset week, and duration). while the correlations between classic centrality measures (degree, betweenness, and eigenvector centrality, except for closeness centrality) and any of the three epidemiological features were not statistically significant. in addition, sumratio and multicenter distance performed even better than closeness centrality. multicenter distance was positively correlated with the cumulative incidence (r = . , p < . ) and onset week (r = . , p < . ) but negatively correlated with the duration from onset to the first epidemic peak (r = − . , p < . ). a positive correlation between closeness centrality and onset week (r = . , p < . ) was identified, as well as a negative association between closeness and duration (r = − . , p < . ). sumratio was positively associated with the cumulative incidence (r = . , p < . ) and duration (r = . , p < . ), but negatively associated with the onset week (r = − . , p < . ). the pearson correlation coefficients between the number of slaughtered pigs and cumulative incidence, onset week, and duration of epidemics were − . (p = . ), − . (p < . ), and . (p < . ), respectively. therefore, the number of pigs would not be taken as an explanatory variable of the regression model when the cumulative incidence was taken as the response variable. based on the results of correlation analysis, we incorporated three network node centrality measures (closeness, sumratio, and multicenter distance) into spatial autoregressive models as explanatory variables. the vif of each explanatory variable except for closeness centrality ( . ) was less than ( table ) , so all the explanatory variables but closeness were able to enter the regression model. w c , w w , w w, w a cuminc is the same as that in table . b the closeness centrality was not included in the models as an explanatory variable because its vif was larger than . c w is the adjacent matrix a in the materials and methods section. *, **, and *** the regression coefficients are significant at the . level, at the . level, and at the . level, respectively. when we took the cumulative incidence as the response variable in the spatial autoregressive model, the spatial dependence coefficient ρ of the response variable was statistically significant (ρ = . , p = . ). the cumulative incidence can increase . %, − . %, . %, . %, . %, . %, and . %, with a % increase in popdensity, pgdp, income, hospital, collegestu, sumratio, and multicenter distance, respectively (table ). when we took the onset week as the response variable in the spatial autoregressive model, the spatial dependence coefficient of the response variable was statistically significant (ρ = . , p < . ). onset week can be delayed by . % and . % when popdensity and pig decreased by one percent, respectively (table ).when we took the duration from onset to the first epidemic peak as the response variable in the spatial autoregressive model, the spatial dependence coefficient of the response variable was also statistically significant (ρ = . , p < . ). duration can be lengthened by . %, . %, . %, and − . %, with a % increase in popdensity, college, pig, and multicenter distance, respectively (table ). in this study, we evaluated the impacts of road traffic and other socioeconomic factors on the nationwide spread of influenza a (h n ) across china in . we constructed a national highway network and proposed two new centrality measures: sumratio and multicenter distance. both new measures were significantly correlated with all three epidemiological features in question (cumulative incidence, onset week, and epidemic duration). based on the positive association between onset week and multicenter distance, we understand the transmission process of influenza a (h n ) as follows: at the very beginning, internationally infected individuals quickly arrived in the eight center cities and other cities in eastern china with frequent international communications [ ] , most of which are spatially distant from each other, after long distance travel by plane [ ] . by this means the virus was quickly introduced into new regions. this pattern was important for virus spread in the early stage of an epidemic, especially before strict control measures were taken [ ] . the virus then diffused over short-range connections as infectious individuals moved to neighboring areas by ground-based transport along highways [ ] . the epidemic followed the classic distance decay theory, starting earlier in the cities which are closer to their nearest center cities, and this was partly supported by the findings in reference [ ] . to verify our discoveries and explanations quantitatively and to acquire quantitative relationships between road traffic, as well as socioeconomic factors and the spread of influenza a (h n ), we conducted spatial regression analyses. when the cumulative incidence was taken as a response variable in the spatial autoregressive model, the results could be interpreted as follows: people had more chances to contact each other and transmit infectious diseases with a higher base probability in cities of a higher population density [ , , ] , which resulted in a higher cumulative incidence at the end of epidemics. the income level in these cities was also higher [ ] , and more workers would be attracted to come. it has been previously reported that the density of medical facilities was not significantly associated with the arrival time of the first confirmed influenza a (h n ) case in each chinese county in [ ] . our findings elaborate on this, showing that the density of hospitals has a significant effect on the cumulative incidence at the city level. as we know, cities with more (high-level) hospitals would attract more patients seeking medical treatment [ ] . moreover, many symptomatic patients gathered in hospitals where the space was confined, and cross infection and nosocomial infection by means of virus droplet and aerosol could be facilitated [ ] , which would also result in a higher incidence. previous research [ ] reported that people between - years old were most affected by the influenza a (h n ) in china. therefore, our positive association between the proportion of college students and cumulative incidence is consistent with previous works. cities with a higher sumratio were more likely to be traffic hubs in the national highway network, and the cumulative incidence would be higher. the larger the multicenter distance of a city, the lower the medical treatment level would be, and the cumulative incidence could not be reduced quickly and would remain relatively high. as for cities with a high gdp, they would tend to conduct stronger intervention measures to control the infectious disease, reducing the cumulative incidence. when the onset week was taken as a response variable in the spatial regression, we found that the population density and the number of pigs had negative effects on the epidemic onset in cities. a higher population density meant that the corresponding city was more central and it might have been more likely to have a large airport or train station, and then, the imported cases which could start an epidemic in the city might arrive earlier. the "first" reported case may not be the true first person who was infected by the influenza virus in a given city. it is possible that an infected individual arrived in a city and infected others around him/her, but the public health authority did not receive an infection report, and then, the epidemic started silently and developed freely. thus, the "first" case was not announced to the public until the underlying infected population had taken place. as the reservoir of the pandemic h n influenza virus, pigs can carry this virus, can transmit it to human beings, and may be a magnifier of the population of virus and potentially infected people. hence, the "first" case would be reported earlier in a city where more pigs were fed. when the duration from onset to the first epidemic peak was taken as the response variable in the spatial regression, the results can be interpreted as follows and be supported by the finding in reference [ ] that the periods of epidemics in smaller cities are shorter than those in larger cities. a city with a high population density would have a high proportion of susceptible people who are fuel for epidemics [ , ] , which would prolong the duration of an epidemic. it was reported that school students were more susceptible to the influenza a (h n ) than other age groups in china [ ] and that schools were high-risk areas for influenza outbreaks and should be the focus for prevention [ , ] . therefore, the result that the number of colleges was positively associated with the epidemic duration is consistent with the real situation. moreover, cities with a larger value of multicenter distance would have a smaller population, and the number of susceptible people would also be less. as a result, the epidemic duration would be shorter. intervention measures of public health authorities in china could shorten the epidemic duration, but these measures were taken upon human beings instead of pigs, which played a role of viral reservoir. the virus might spill over from pigs to the human population continuously, which would prolong the epidemic period. it may be an explanation for the significantly positive association between the number of slaughtered pigs and epidemic duration. in addition, it has been reported recently that the presence of airports and railway stations in prefecture-level cities did not have significant impacts on epidemic duration [ ] . our results complement previous findings and reveal that both road transport and socioeconomic factors had a significant influence on epidemic duration. among all means of transport, herein we emphasize the role of road transportation in the spatial diffusion of the influenza a (h n ) virus in mainland china. it has been reported recently that both aviation and road travel have a significant association with the epidemic arrival day in each prefecture-level city during the whole viral diffusion period, but the role of rail travel was only significant after august st [ ] . although air travel is a significant factor in the global spread of the influenza virus, short-range daily travel on the ground is more important at the regional scale [ ] , which our results support. to the best of our knowledge, most chinese people do not travel by air very often (at least in , except for businessmen). airplanes bring virus carriers to areas that are far from each other and disease-free before landing, which may lead to a spatially random dispersal of viral pathogens. in daily commuting, an infectious individual is likely to infect anyone with whom he/she is in contact, with the probability of infection associated with spatial distance and time length of contact, which may result in a spatially structured viral population [ ] . in our spatial regression analyses, the spatial dependence coefficient ρ of three epidemiological features are all significant and positive, which means that the epidemic in a city was positively correlated with those in neighboring cities and indicates that road travel would be more important than air travel for the spatial transmission of the influenza a (h n ) virus in china. unlike some countries in europe and america which experienced two infection peaks of influenza during , china had only one autumn-winter wave in , which could be attributed to the strict prevention and control strategies conducted by china at the early stage of the pandemic [ ] . for example, fever screenings performed in airports and railway stations could be expected to detect most symptomatic passengers with a fever, but the expected efficiency of fever screenings in long-distance highway passenger stations should be much lower because a bus is allowed to pick up passengers along the way and they need not gather in a specifically designated station before boarding and because a considerable number of highway passengers would choose private vehicles. therefore, it is much more difficult to restrict the diffusion of influenza virus by road travel. in this respect, the role of highway passenger transport in dispersing viruses spatially is more important than air and railway modes. we also suggest that the socioeconomic conditions themselves not only have direct impacts on the development of an influenza pandemic but also indirectly exert an influence by stimulating human domestic travel and by changing the patterns of human aggregation, which is congruent with previous research [ , , ] . it has been reported that people in a wealthy region travel farther than people in a relatively poor region in china due to the difference in basic facilities or people's living standard, and a pandemic emerging in less developed regions might diffuse more slowly [ ] . interestingly, health disparities of people in less developed and developed areas in china might be reduced by the interaction of epidemiological and socioeconomic factors in the face of a newly emerging influenza pandemic, especially in the early stages. for instance, although medical treatment in developed areas is better than that in less developed areas, the epidemic arrival time would be earlier in developed areas with a longer epidemic duration. with the reduction of airfares, the opening of more domestic airlines, and the promotion and popularization of high-speed trains in china, the time cost of the journey is greatly decreased in recent years, and passengers tend to think more about the attractiveness of certain places which are mostly determined by the places' socioeconomic and environmental conditions and think less about the length of trip distance when they choose destinations for travel. therefore, the impact of spatial distance on the diffusion of infectious diseases may become smaller in the future, while that of socioeconomic factors will be more and more important. it is interesting to note that the number of newly reported cases peaked at a time point later than the peak of the number of newly affected cities ( figure ). our explanations of this finding is that before week (until august) in , the number of cases was quite small and increased very slowly ( cases per week); the number of affected cities increased steadily ( cities per week), which was most likely to be driven by the sporadic importation of overseas cases into china, because the majority of new cases before week (until july) were imported cases [ ] . from week to week (until november), the increase in the number of new cases sped up significantly ( cases per week) and reached its first peak on september, and the large amount of existing cases greatly facilitated the invasion of the virus into unaffected cities; hence, the number of affected cities increased more quickly ( cities per week) than in the prior period. this could be explained by two possible reasons. the first may be that almost all the new cases were local cases after week (until september), which meant the community transmission of the virus within a city had started, so the number of weekly newly affected cities arrived in its peak on september and the rate of increase dropped afterwards. the second reason could be that the chinese government took strict prevention and control measures before september , but the measures were relaxed from september [ ] , which could also partly explain the sharp rise in the number of new cases and newly affected cities at the end of august and the beginning of september. the curve of new cases did not rise continuously but dropped to a local minimum in early october, which may be explained by the reduced case reporting during chinese national holidays [ ] and could further enlarge the time delay between its peak and the peak of the number of newly affected cases. from week to week (until december), no city was newly affected because almost all prefecture-level cities (except for yushu city in qinghai province) in mainland china had already reported cases in the prior period, and the increasing velocity of the number of new cases was nearly twice of that in the earlier period. there are some limitations in this study. although the trend of case reporting was consistent with that of the percentage of respiratory specimens tested positive for the influenza virus, the number of reported cases was much less than the real size of infected individuals [ ] . it was observed globally in many influenza outbreaksthat not all cases were reported to the medical services. therefore, data from virological and serological surveillance should be taken into consideration for further information. additionally, even though there were several socioeconomic factors which were not significantly associated with all three epidemiological features, there still existed other effective factors that were not included in this research, such as climate conditions. as an ecological study, the results of this study cannot provide evidence for causal relationships but merely suggest probable associations. in future research, a meta-population epidemiological model driven by road travel should be constructed to investigate the diffusion process of the influenza a (h n ) under the effects of socioeconomic factors. in summary, our study provides two major contributions to the literature. first, we propose two new node centrality measures for a national highway network: sumratio and multicenter distance, and both perform better than classic node centralities for statistically significant correlations with epidemiological features. second, we conclude that road transport network and socioeconomic factors had significant impacts on the diffusion of the influenza a (h n ) virus at the city level in mainland china and that both should be candidates for the prevention and control of future influenza pandemics. data accessibility: the data and analysis code for this study have been 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in u.s. cities closure of schools during an influenza pandemic roles of different transport modes in the spatial spread of the influenza a(h n ) pandemic in mainland china travel patterns in china we would like to thank jun cai, ruiyun li, zhe sun, sen zhou, and yao pei for their assistance in the epidemiological data preparation. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of the data; in the writing of the manuscript; or in the decision to publish the results. key: cord- - bxvenue authors: ashwell, m.; freire, m.; o’nan, a.t.; benito, j.; hash, j.; mcculloch, r.s.; lascelles, b.d.x. title: characterization of gene expression in naturally occurring feline degenerative joint disease-associated pain date: - - journal: vet j doi: . /j.tvjl. . . sha: doc_id: cord_uid: bxvenue degenerative joint disease (djd) associated-pain is a clinically relevant and common condition affecting domesticated cats and other species including humans. identification of the neurobiological signature of pain is well developed in rodent pain models, however such information is lacking from animals or humans with naturally occurring painful conditions. in this study, identification of housekeeping genes (hkg) for neuronal tissue and expression levels of genes considered associated with chronic pain in rodent models were explored in cats with naturally occurring osteoarthritic pain. fourteen adult cats were evaluated — seven without clinical signs of osteoarthritic pain, and seven with hind limb radiographic djd and pain. expression of an investigator-selected set of pain signaling genes (including asic , atf , cox , cx cl , nav . , nav . , nav . , ngf, nk r, tnfα, trka) in lumbar spinal cord dorsal horn and lumbar dorsal root ganglia tissues from clinically healthy cats and cats with djd were studied using quantitative rt-pcr (qpcr). hkg identified as the most stable across all tissue samples were many of the ribosomal protein genes, such as rpl and rps . qpcr results showed atf and cx cl up-regulated in djd-affected dorsal root ganglia compared to clinically healthy controls. in spinal cord, cx cl was up-regulated and ngf was down-regulated when djd-affected samples were compared to healthy samples. further work is needed to understand the neurobiology of pain in naturally occurring disease and what rodent models are predictive of these changes in more heterogeneous populations such as domestic cats. feline degenerative joint disease (djd) can be associated with mobility impairment in feline patients (gruen et al., (gruen et al., , lascelles, ) . this impairment is believed to be due to pain associated with the djd as mobility appears to be increased when pain relief is provided (gruen et al., (gruen et al., , lascelles, ) . the approach to pain management can be divided into two basic approachesto try analgesics known or thought to be effective in other conditions or other species, or to base the analgesic selection on a rationale evaluation of the neurobiological changes present in the target disease state in the target species. this latter approach could be described as making rationale analgesic choices on the neurobiological signature of the pain. presently, nothing is known about the neurobiology of feline djd pain. one approach to understanding the neurobiology of pain is to look at differences in gene expression between normal and phenotypically well-defined diseased states. limited work has been performed along these lines in dogs. hegemann et al. ( ) measured gene expression levels of interleukin (il)- α, il- ß, il- , il- , il- , il- , il- , il- , il- , interferon gamma (if-g), transforming growth factor beta (tgfß), and tumor necrosis factor alpha (tnfα) in synovial fluid collected from dogs with osteoarthritis and immune-mediated polyarthritis using semi-quantitative real-time pcr methods. they reported differences in gene expression levels for some of these genes when they compared affected and unaffected dogs, but did not relate the findings to the presence of painan important consideration given that common measures of disease, such as radiographic features, do not correlate well with pain levels. other investigators have evaluated the changes in gene expression in canine osteoarthritic cartilage (clements et al., (clements et al., , , but again, the results were not evaluated against whether or not there was a pain state present. quantitative real-time pcr (qpcr) studies have evaluated expression levels of various cytokines in feline allergic skin disease (taglinger et al., ) , feline chronic gingival stomatitis (harley et al., ) and feline coronavirus infection (gelain et al., ) . however, no studies have been performed in cats with djd to determine gene expression changes in relation to pain. we were interested in understanding the gene expression changes at crucial points in the nociceptive signal (pain) generation and transmission pathway in the dorsal root ganglia (drg) and the dorsal horn (dh) of the spinal cord. in this study, we first evaluated a panel of potential housekeeping genes to identify the most stable reference genes in djd and healthy cats in drg and dorsal horn as this had not been previously done. after the most stable reference genes were identified, a selection of genes previously associated with nociception in rodent models, and of interest to the authors, were examined using qpcr in the same samples to allow us to start characterizing the neurobiological signature of pain associated with djd in cats. identification of stable reference genes in pain-related tissues in felines is important because of the cat's use as a potential animal model for human conditions and the prevalence of aging cats in veterinary medicine. this study was approved by the ncsu institutional animal care and use committee (iacuc; approval no. - -o; approval date: october ). fourteen domestic cats euthanased at a local animal shelter, and of known pain status via pre-euthanasia examinations, were used for sample collection. the investigators evaluated the cats prior to euthanasia to determine whether they had pain associated with hind limb joints. cats were euthanased with an overdose of barbiturates for reasons unrelated to this study (population control) and the investigators had no input into which cats were euthanased. immediately after euthanasia orthogonal digital radiographs of the lumbar axial skeleton and appendicular joints of the hind limbs were performed and evaluated for the presence of radiographic signs indicative of naturally occurring djd as described previously (lascelles, ) . the tarsus, stifle and hip joints were opened and visually inspected for evidence of macroscopic lesions indicative of djd as described previously (freire et al., ) and scored as described previously . the spinal canal from the last thoracic vertebrae to the sacrum was opened and evaluated for macroscopic lesions indicative of intervertebral disc degeneration with disk prolapse and spinal cord compression. in summary, cats were considered 'pain positive' if there were signs of discomfort on manipulation of hind limb joints; there was gross evidence of djd in those joints; there was muscle atrophy in the hind limbs that was not seen elsewhere in the body; and there were no grossly visible indications of intervertebral disk prolapse or spinal cord compression. seven cats were considered free of musculoskeletal pain on pre-euthanasia evaluation, and of free of naturally occurring appendicular joint djd and spinal djd, and were included in the normal group (no abnormalities seen on digital radiographs and on macroscopic inspection of appendicular joints and axial skeleton). seven cats were included in the hind limb djd group and showed radiographic signs indicative of moderate-severe djd in one or multiple appendicular joints of the hind limbs. no evidence of spinal compressive lesions was present. the lumbar spine from mid body of third lumbar vertebrae to mid body of the fifth lumbar vertebrae (lumbar intumescence) was harvested and drg of fourth, fifth and sixth spinal segments from both sides were dissected and individually stored. right and left dh of the spinal cord segment harvested were dissected and individually stored (after initial exposure to rnalater for at least h). dissection was performed using a dissecting microscope (olympus szx , ). looking at the transverse cut surface of the spinal cord, the white and grey matters can be differentiated and the dorsal horn can easily be identified. the dorsal and ventral aspects of the spinal cord are identifiable by location of the ventral median fissure and the cranial and caudal aspects are determined by looking at the orientation of the nerve rootlets. the dura mater was opened and the spinal cord was divided into right and left segments following the ventral median fissure. another cut placed just dorsal to the central canal allowed the dorsal horn to be separated from the ventral. this cut was performed carefully to assure separation by identification of the grey matter corresponding to the dorsal horn on the transverse cut surface of the spinal cord (supplementary figs. s -s ). the same drg and dh segments of the spinal cord were sampled in normal cats for comparison. all tissue samples were stored in rnalater (qiagen) at À c for h and at À c thereafter until sample processing. total rna from drg and dh was extracted using the qiagen rneasy kit and an on-column dnase digestion to remove genomic dna. the manufacturer's protocol was followed through the chloroform extraction but instead of precipitating the rna in % ethanol, % ethanol was added and the mixture applied to the rneasy (qiagen) column, following the rneasy kit instructions through the end of that manufacturer's protocol. quantity of the extracted rna was measured on a nanodrop spectrophotometer (thermo scientific, wilmington, de) and quality was evaluated by running the extracted rna on a . % agarose gel to determine the integrity of the s and s ribosomal subunits. only samples showing a s: s rrna band intensity ratio of approximately (as determined visually) were used in this study. a high capacity cdna reverse transcription kit (applied biosystems inc., foster city, ca) was used to reverse transcribe ng of total rna per the manufacturer's protocol. subsequently, reactions were diluted : to provide enough template for all genes to be evaluated. primer sequences for gusb, hmbs, rpl , rps , rps and ywhaz were obtained from penning et al. ( ) . the remaining housekeeping and target gene primers were designed using beacon designer software (premier biosoft intl., palo alto, ca) to be compatible with sybr green i and to span an intron in order to detect genomic contamination. pcr primer sequences for the remaining housekeeping genes and pain-related target genes are given in table . quantitative pcr was performed in a volume of ml, consisting of ml of diluted cdna, nmol/l of forward and reverse primers, nmol/l fluorescein, and  power sybr green master mix (applied biosystems). a three-step amplification protocol was performed in an icycler iq (bio-rad). each reaction included one cycle at c for min, following by cycles at s at c, s at c- c for annealing, and extension for s at c. specificity of each reaction was assessed by melt curve analysis ( cycles starting at c with an increase of . c every cycle, with a dwell time of s) and one amplicon from each primer pair was dna sequenced to confirm identity. reactions were performed in duplicate, c t values were averaged for the replicates and negative controls were included to detect possible contamination. any duplicate measurements more than . c t apart were repeated for that sample or removed from the analysis. standard curves were evaluated for each primer pair by combining equal amounts of cdna from each specimen into a pool. the pool was then diluted : , : , : , : and : . dilutions were evaluated in duplicate to calculate amplification efficiencies which ranged from % to %, depending on the type of tissue and primer pair. bestkeeper (pfaffl et al., ) , genorm (vandesompele et al., ) and normfinder (ohl et al., ) were used to evaluate the potential reference genes and select the most stable in these particular tissues. fold changes between healthy and djd affected samples were computed via normalization to the geometric mean of the selected housekeeping genes. the fold change calculations incorporated corrections for reaction efficiency using the method of pfaffl et al. ( ) . changes in the expression of healthy and djd samples were evaluated for statistical significance using a linear mixed model derived from the work of steibel et al. ( ) . bayesian information criteria was used to select the best parameters to include in the model (those contributing to the lowest bic score). fixed factors included health status (healthy vs. djd), reproductive status (sterilized vs. intact), and radiographic score for djd. random factors included variables for position of the sample (left vs. right), age, and sex of the cats. the model was established with duplicate samples from a given cat nested within the cat. all analysis was conducted with spss software (version . , ibm) and an alpha (α) of . was used for all analyses. cats included in the djd-pain group were two castrated males, three intact males and two spayed females. mean age (aestandard deviation, sd) was ( . ) years old, and mean weight (aesd) was . ( . ) kg; median body condition score was / . the characteristics of the cats are detailed in table s (supplementary data), and all were designated 'pain positive'. bilateral hind limb appendicular joint djd in one or multiple joints were present in five of seven cats and samples from right and left dh of the spinal cord and right and left drg were analyzed. in the other two cats in the djd-pain group, only one hind limb had one or multiple joints affected with djd and samples from the nervous tissue ipsilateral to the affected side were collected. cats studied included in the healthy group were two castrated males, three intact males, one spayed female and one intact female. mean age (aesd) was . ( . ) years, and mean weight (aesd) was . ( . ) kg; median body condition score was / . samples were collected from right and/or left nervous tissues in these cats to match with the samples collected in the cats with djd. ten potential housekeeping genes for drg and dh collected from djd-affected and unaffected cats were examined using the three gene expression reference gene programs. the ten genes evaluated in this study were selected based on previous work by penning et al. ( ) that examined the stability of these genes in feline liver, kidney, dental roots, heart and mammary gland tissues but not in any central nervous system tissues. the genes examined were beta actin (actb); beta- -microglobulin (b m); beta glucuronidase (gusb); hydroxymethylbilane synthase (hmbs); tyrosine -monooxygenase/tryptophan -monooxygenase activation protein-zeta polypeptide (ywhaz); and ribosomal proteins l (rpl ), l (rpl ), s (rps ), s (rps ) and s (rps ). due to dissection errors, two drg were missing from the sample set; however drg representing all cats were used to evaluate the reference gene panel using bestkeeper and norm-finder, as those programs allow for missing data. only paired samples from cats were able to be evaluated using genorm as that program requires complete data. all genes amplified in the majority of the samples examined but b m had a sd > and was removed as a potential reference gene based on the recommendation from the bestkeeper software. based on bestkeeper results, actb, rps and rps were the most stable housekeeping genes in the drg samples. using results from genorm, rps , rps and actb were the most stable. and norm-finder produced results that differed from both genorm and bestkeeper, finding rpl , hmbs and gusb as the most stable genes in the drg samples (table ). an additional feature of genorm is the determination of the optimum number of reference genes needed for accurate normalization (fig. s a and s b ). the number of reference genes associated with an expression stability value of . or lower is considered the optimum number of housekeeping genes needed. for each of the tissues examined in the feline samples, the variation in normalization factor with two vs. three reference genes was < . , indicating the optimum table reference gene rankings for each tissue type using bestkeeper, genorm and normfinder. reference gene ranked order (most stable to least stable) bestkeeper number of genes needed was two. however, since the three programs did not always agree on the two most stable genes, three were selected for each tissue type. based on these results, the geometric mean of actb, rps and rpl was used as the reference value in each of the drg samples. a total of dh samples ( from cats with djd and from healthy cats) were used to evaluate potential housekeeping genes in this tissue. all ten reference genes amplified and had sd < so all were included in the reference gene analyses. determination of the most stable reference genes in dh was very consistent across the three evaluation programs, with hmbs being the most stable gene ( table ) . actb was the next most stable using bestkeeper and normfinder and this gene was placed as the third most stable using genorm. based on these results, the geometric mean of actb, hmbs and rpl was used as the reference value in each of the dh samples. after a set of stable reference genes were identified for each tissue type, genes associated with pain in rodents were selected (based on current knowledge of genes involved in pain states (foulkes and wood, ) and their expression levels compared in the drg from djd-affected and healthy samples. we hypothesized that these genes would show increased expression in djd cats. the selected genes were as follows: acid sensing ion channel (asic ); activating transcription factor (atf ); calcitonin gene related peptide (cgrp); cyclooxygenase (cox ); chemokine (c-x -x motif) ligand (cx cl ); sodium channel, voltage-gated, alpha subunit types ix (nav . ), x (nav . ) and xi (nav . ); nerve growth factor (ngf); tachykinin receptor (nk r); substance p (tac ); tumor necrosis factor alpha (tnfα); and tropomyosin receptor kinase a (trka). although two primer pairs were designed for each target gene, no amplification was detected for cgrp and tac , so they were excluded from this study. when djdaffected cats were compared to healthy control cats for the remaining target genes, atf and cx cl were significantly upregulated in the drg ( . -fold and . -fold, respectively; table ). ten target genes were selected for examination in dh samples-asic , cox , cx cl , nav . , nav . , nav . , ngf, nk r, tnfα, and trka. asic , nav . and trka were excluded because their pcr efficiencies were outside the acceptable range of - % and two genes (nav . and nav . ) did not amplify in any of the dh samples. of the five remaining genes, cx cl was up-regulated ( . -fold) and ngf was down-regulated ( . -fold) in the djd affected samples (table ). with quantitative pcr, multiple target genes may be evaluated to measure changes in expression. however, to accurately determine the relative expression levels, a reference is used to normalize the expression results for differences in cdna quantity between different samples, enabling comparisons between target genes across disease states. bestkeeper, genorm, and normfinder provide three different approaches for examining potential genes to select as the most stable genes for a given set of conditions. at least three other studies have identified suitable gene expression housekeeping genes in various feline tissues. penning et al. ( ) found that most of the ribosomal genes they tested appeared to be suitable reference genes in the different feline tissues. wensman et al. ( ) tested six potential housekeeping genes in different brain tissues and found that hprt, ywhaz and rps were the most stable. kessler et al. ( ) tested potential reference genes in neoplastic, endocrine, blood, liver, intestine and lymphoid tissues in healthy cats and found that rps , actb and abl were the most stably expressed housekeeping genes. while none of these other feline studies examined central nervous tissues, our results are similar, with several of the ribosomal genes and actb being stable reference genes. in rat dorsal root ganglia and dorsal horn samples piller et al. ( ) found that actb and hprt and also rpl and rpl a were suitable housekeeping genes in drg and dh samples, respectively, and wan et al. ( ) found rpl , rpl and actb most stable in drg samples isolated from a rat model of neuropathic pain. based on these other studies, our findings that many of the ribosomal genes and actb are the most stable in feline drg and dh samples are reasonable. the target genes selected in this study were chosen based on results from work on skeletal pain conducted in rodents (e.g. mantyh, ) and knowledge of genes involved in pain states (foulkes and wood, ) in an attempt to characterize the neurobiology of djd-associated pain in the cat. they were also chosen because of therapeutics that are available, or in development, or because of their involvement in neuropathic pain states. mrna levels of cx cl , also known as fractalkine, were higher in the drg of djd-affected cats, suggesting that these cats were experiencing a neuropathic pain state (clark & malcangio, ) . in studies examining changes in gene expression in drgs, the expression of activating transcription factor (atf ) is increased in the drg after peripheral nerve injury (shortland et al., ) and more recently has been shown to be increased also in models of oa in rodents (thakur et al., ) . atf is considered indicative of neuronal-damage and neuropathic pain. similar results were identified in the drgs isolated from the djd-affected cats used in this study, where the expression of atf was increased . -fold, and further suggests that cats with djd may be experiencing neuropathic pain like states. very little other work has been performed in drg from nonrodent species, but in horses with laminitis, immunohistochemical analysis of drg from hind limb laminitic horses, comparing lumbar drg with cervical drg showed an increased expression of atf , and the authors suggested this may be indicative of neuropathic pain (jones et al., ) . in many experimental pain models the expression of cox has been found to be increased in the spinal cord in response to inflammation and injury (vardeh et al., ) and contributes to pain, but we did not observe a significant difference in cox when we compared djd and healthy samples. the transcription factor atf has been shown to negatively regulate cox levels during acute inflammation in mice (hellmann et al., ) and our results may indicate the same negative regulation is occurring in cats with djd-pain. however, it is important to remember that our results reflect a single crosssectional chronic time point. the mechanisms of pain likely change over time, and the time course of the rodent models tends to be very short, and may not reflect mechanisms at later stages of the pain state. however, considering again our findings around cox , investigators have struggled to show clinical benefit of nsaids in cats with djd-associated pain (gruen et al., ) . in the djd-affected cats, we found significant increased expression of cx cl in dh, similar to what we detected in the drg samples. studies in rodents have shown that cx cl and its receptor cx cr are an important signaling pathway involved in microglial contributions to chronic pain. interestingly the fractalkine protein is membrane-bound until it is cleaved by different proteases, including cathepsin s. the cleaved, soluble form of fractalkine is associated with neuropathic pain, not the membrane-bound form (clark & malcangio, ) . we also found ngf to be significantly differentially expressed in dh, but not in the expected direction of that reported in other pain models. interestingly, in a study of a monoclonal antibody to nerve growth factor (anti-ngf mab) (gruen et al., ) , the anti-ngf mab produced robust and long-lasting improvements in activity and mobility, presumably due to a decrease in pain. this may illustrate how simply evaluating the expression of genes may not give the whole picturefor a comprehensive understanding of the neurobiology of pain, gene expression, translation into protein and post-translational modifications need to be evaluated. taken together, our results do point to increased expression of genes considered to be involved in neuropathic pain, and may be evidence of a neuropathic like pain state in cats with djdassociated pain. we expected that several of the genes we selected (which have all been shown to play a role in pain in rodent models of arthritis) would show altered expression between the djd-pain and healthy groups. there are several reasons for the relative lack of change across the genes we selected. rodent models are just thatinduced models, and these models may not reflect the neurobiology of pain in naturally occurring pain states. indeed, this is one of the reasons that has been suggested for the lack of translation of basic pain research into effective new therapeutics (lascelles et al., ) . another aspect of our study that should be factored into the interpretation of the results is that the tissues we collected contained varying types of cells and indeed tissue, and so we cannot directly infer that changes or lack of changes are due to any particular tissue type. we made every attempt to only use cats that definitely had djd-pain or were healthy, however, we did not have detailed history on each cat, and our pain status was simply designated as 'yes' or 'no'. future studies should endeavor to collect tissues from more highly phenotyped animals (with respect to pain status). this is particularly important as the neurobiology of pain probably varies over time and with features of the phenotype (e.g. excessive sensitivity present or not). the lack of changes in our study may reflect an underpowered study and the heterogeneity of the samples assessed. despite the shortcomings, we believe our study points to the need to look more closely at the naturally occurring pain states and such investigations may lead to novel therapeutic targets. however, there is a danger of missing novel, relevant targets if the starting point is already biased to what is known from rodent models, as our study was. selection of appropriate stable housekeeping genes is extremely important and has not been previously done for feline nervous tissues. as rodent species continue to illustrate they are not appropriate models for different human conditions, other animal models with naturally occurring disease, such as the cat, will become more and more prevalent and appropriate reference genes will be needed for accurate gene evaluation studies. our approach of evaluating neurobiological changes (gene expression) in nervous system tissue from cats with naturally occurring pain appears feasible. the results of this small study point to increased expression of genes considered to be involved in neuropathic pain, and may be evidence of a neuropathic like pain state in cats with djd-associated pain. further work should be undertaken to confirm our results, and expand on these studies. funding for this work was provided by private donations to translational research in pain program from individuals concerned about the 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monoiodoacetate model of osteoarthritis accurate normalization of real-time quantitative rt-pcr data by geometric averaging of multiple internal control genes cox in cns neural cells mediates mechanical inflammatory pain hypersensitivity in mice identification and validation of reference genes for expression studies in a rat model of neuropathic pain development of a real-time rt-pcr assay for improved detection of borna disease virus supplementary data associated with this article can be found, in the online version, at https://doi.org/ . /j.tvjl. . . . key: cord- - bhg t l authors: al-nour, mosab yahya; ibrahim, musab mohamed; elsaman, tilal title: ellagic acid, kaempferol, and quercetin from acacia nilotica: promising combined drug with multiple mechanisms of action date: - - journal: curr pharmacol rep doi: . /s - - -w sha: doc_id: cord_uid: bhg t l the pharmacological activity of acacia nilotica’s phytochemical constituents was confirmed with evidence-based studies, but the determination of exact targets that they bind and the mechanism of action were not done; consequently, we aim to identify the exact targets that are responsible for the pharmacological activity via the computational methods. furthermore, we aim to predict the pharmacokinetics (adme) properties and the safety profile in order to identify the best drug candidates. to achieve those goals, various computational methods were used including the ligand-based virtual screening and molecular docking. moreover, pkcsm and swissadme web servers were used for the prediction of pharmacokinetics and safety. the total number of the investigated compounds and targets was and , respectively. according to the results, the pharmacological activity was attributed to the interaction with essential targets. ellagic acid, kaempferol, and quercetin were the best a. nilotica’s phytochemical constituents that contribute to the therapeutic activities, were non-toxic as well as non-carcinogen. the administration of ellagic acid, kaempferol, and quercetin as combined drug via the novel drug delivery systems will be a valuable therapeutic choice for the treatment of recent diseases attacking the public health including cancer, multidrug-resistant bacterial infections, diabetes mellitus, and chronic inflammatory systemic disease. acacia nilotica is a tropical and sub-tropical medicinal plant belonging to the fabaceae family [ ] . no doubt, medicinal plants play a vital role in drug discovery, since they are affluent with bioactive phytochemical constituents that are valuable in the treatment of various diseases, particularly those causing recent threats attacking the public health including cancer, multidrug-resistant bacterial infections, diabetes mellitus, and chronic inflammatory systemic diseases [ , ] . the higher incidence of cancer and mortality rate [ ] , the emergence of bacterial resistance with the declining in the antibacterial research at several pharmaceutical companies [ ] , the huge prevalence and complications associated with diabetes mellitus [ ] as well as the long-term suffering associated with the chronic inflammatory systemic diseases such as rheumatoid arthritis and multiple sclerosis [ ] are leading forces that encourage us to participate in fighting against the probable threats. such an issue is attained via the discovery and development of efficient innovative anticancer, antibacterial, antidiabetic, and anti-inflammatory drugs. unfortunately, drug discovery is a time-consuming, costive, as well as difficult process [ , ] ; hence, it necessitated to involve sophisticated techniques in the drug discovery process in order to overcome those limitations. recently, one of the promising sophisticated techniques is the computational tools (computer-aided drug design) that have a valuable impact in the discovery and development of newer drugs with a reduction in time and cost [ ] . they include the ligand-based virtual screening that is based on the searching for the compounds having the highest probability in pharmacological activity [ ] and molecular docking that relies on the energy-based scoring function to identify ligand-target complex lowest energy [ ] . moreover, they involve the software of pharmacokinetics, toxicity, and the drug-likeness prediction that work by many algorithms [ ] including the graph-based signature [ ] . many studies concerning the application of the computational tools in the discovery of natural-derived drugs were conducted [ ] [ ] [ ] [ ] . a. nilotica is opulent of many phytochemical constituents including tannins, alkaloids, terpenoids, and flavonoids. many studies were conducted in it resulting in an evidence-based pharmacological data that revealed the potential pharmacological activities of the phytochemical compounds including anticancer, antibacterial, antidiabetic, anti-inflammatory, and other activity making the plant as a promising source for the development of innovative, safe, biodegradable drugs with great activity. the chemical structure of active a. nilotica's phytochemical constituents was elucidated, the correlation between the responsible phytochemical constituents for treatment and the diseases were conducted [ ] , but the determination of exact targets that phytochemical constituents bind and the mechanism of action were not performed; consequently, based on established literature and studies, we aim to identify the exact targets that phytochemical constituents bind to exert the pharmacological activity by utilizing the computational methods as a tool for the study so as to understand the mechanism of action. within the current drug design pipeline, drug target identification is a very important step in the understanding of the probable mechanism of action, increasing the confidence and reducing the attrition in clinical trials [ ] . furthermore, we aim to predict the pharmacokinetics (adme) properties and safety profile with the intention of identifying the best drug candidates. the qsar-based virtual screening is characterized by great and fast throughput with respectable hit rank [ ] . molecular docking is valuable to predict the stability of the ligand-target complex that reflects the biological activity [ ] . the pharmacokinetics, toxicity, and drug-likeness prediction are helpful to identify the best drug candidates [ , ] . to our knowledge, such a study was not conducted before. the chemical structure of the reported a. nilotica's phytochemical constituents ( compounds) [ ] was drawn via marven sketch software version . [ ] (fig. ) . the d structure was generated in a mol format with open babel software [ ] , minimized and optimized with cresset flare software [ ] at the accurate type calculation method. the screening for the exact target that the phytochemical constituents bind was performed via similarity ensemble search tool [ ] and targetnet web servers [ ] . the compound structures were submitted in smile format. the targets with higher probability score were selected for further validation via molecular docking study ( targets) . the linkage between predicted targets with the diseases was attained via uniprot [ ] , pharos [ ] , and therapeutic target databases [ ] . the results are listed in tables , , , , , and . the d structure of selected targets from virtual screening was obtained from the rcsb protein data bank [ ] . the structure with better resolution and validation scores was selected for the study. in order to validate the docking results, multiple d x-ray crystallographic structures for the same target were downloaded in pdb format. for the structures that have no practically determined d structure, phyre [ ] , swiss-model web server [ ] , and raptorx [ ] web servers were used for d structure modeling, then downloaded in pdb format. the target preparation was carried out in cresset flare software [ ] according to the default settings. after preparation, the targets d structures were minimized via cresset flare software [ ] at the normal type calculation method. the targets were input to the software in pdb format. the preparation of reported a. nilotica's phytochemical constituents for molecular docking study was carried out as described above. the docking calculations were carried out in cresset flare software [ ] in normal mode and default settings. the grid box was defined according to the co-crystallized ligands, but in the absence of co-crystallized ligands, the grid box was defined via picking of active site amino acids. beside the a. nilotica's phytochemical constituents, drugs that are well known to bind with the predicted targets (selected randomly from therapeutic target [ ] and pharos [ ] databases) and the co-crystallized ligands were used as positive controls. the compounds and the targets were input in mol and pdb format, respectively. the results are listed in tables , , , , , , and and figs. , , , , and . the intestinal absorption, volume of distribution, bloodbrain barrier, p-glycoprotein and cytochrome-p enzymes inhibition, the renal oct substrate probability, and total clearance were predicted via pkcsm [ ] and swissadme web servers [ ] . moreover, the hepatotoxicity, skin sensitization, the herg potassium channel inhibition, ames toxicity, human maximum tolerated dose, carcinogenicity, oral rate acute, and chronic toxicity were predicted via pkcsm web server [ ] at the default settings via submitting of the chemical structures in smile format. the results of pharmacokinetics are listed in tables and and toxicity in table . the probability of a. nilotica's phytochemical constituents to be as drug candidates was carried via applying of lipinski, ghose, veber, egan, and muegge filters. in addition, lead likeness and synthetic accessibility were used to predict medicinal chemistry friendliness. the prediction was carried out via swissadme web server via submitting of the chemical structures in smile format [ ] . the results are listed in table . the consistency and the reproducibility of the used tools including the molecular docking were validated by the resubmission of the compounds for many times. the anticancer activity of a. nilotica's was attributed to the suppression of the oncogenic transformations, progression, and development, dna replication, and transcription. moreover, the prevention of cancer cells proliferation, invasion, angiogenesis as well as the suppression of drug resistance and the induction of apoptosis. the anti-breast cancer activity was due to the inhibition of the aromatase enzyme and estrogen receptor beta. in contrast, the anti-prostate cancer activity is due to the control of metastatic behavior of prostate cancer via the interaction with nuclear receptor ror-alpha and the inhibition of steroid alpha-hydroxylase (table and fig. ) . . the antibacterial activity of a. nilotica was attributed to the prevention of fatty acids, peptidoglycans biosynthesis as well as the prevention of bacterial resistance to the beta-lactam antibiotics. the fatty acid biosynthesis inhibitory activity was against different types of bacteria including mycobacterium, pseudomonas aeruginosa, and vibrio cholera. the antiviral activity was attributed to the action on toll-like receptor . the anti-hiv activity is due to the inhibition of hiv integrase enzyme. the anti-coronavirus activity is due to coronavirus replicase polyprotein ab enzyme. the antiplasmodial activity was attributed to the inhibition of enzymes mo -related protein kinase pfmrk and m aspartyl aminopeptidase as well as the prevention of fatty acid biosynthesis via inhibition of the enzymes: β-hydroxy acyl-acp dehydratase fabz and hydroxyacyl-[acyl-carrier-protein] dehydratase (table ). the antidiabetic activity was attributed to the interaction with the insulin receptor, glycogen phosphorylase enzyme, sodium/glucose co-transporter as well as the aldose reductase enzyme ( table ). the anti-inflammatory activity was attributed to the inhibition of enzymes: arachidonate -lipoxygenase, cyclooxygenase- (cox- ), phospholipase a , receptor-interacting serine/ threonine protein kinase , and xanthine dehydrogenase/ oxidase as well as the interaction with macrophage migration inhibitory factor ( table ). the antidiarrheal, anti-platelets, and anticholinesterase targets the antidiarrheal activity was attributed to the interaction with the opioid receptors mu-type delta-type. the anti-platelets activity was attributed to the interaction with the p y receptor. the inhibition of the enzyme acetylcholinesterase is contributed to the anticholinesterase activity. according to the results, acacetin, γ-sitosterol, kaempferol, flavone, lupenone, lupeol, niloctane, and quercetin had the highest gastrointestinal absorption, tissue distribution (vd), and respectable total clearance. moreover, flavone, nilobamate, and niloctane were permeable to the blood-brain barrier (bbb). besides, acanilol- , acanilol- , γ-sitosterol, flavone, lupenone, and lupeol were found to be subjected to the metabolism via cyp a enzyme (table ) . moreover, (+)-mollisacacidin, catechin, chalconaringnen- -o-beta-glucopyranoside, epicatechin, niloticane, kaempferol- glucoside, leucocyanidin, and nilobamate were free from drugdrug interaction via the inhibition of cytochrome-p (cyp) or pglycoprotein (p-gp) i and ii enzymes (table ). w z* aua* -acacetin --− . -cyclin-dependent kinase enzyme bit is involved in regulation of transcription^ [ ] bcg* gzh* -acacetin . -death-associated protein kinase bit regulates type i apoptotic, type ii autophagic cell deaths^ [ ] auv* auu* -quercetin . -focal adhesion kinase enzyme bit is essential in angiogenesis, cell migration and apoptosis^ [ ] k y* d * -ellagic acid cell survival, and proliferation^ [ ] ayd binvolved in tumor transformation, progression, survival, angiogenesis and metastasis^ [ ] esm* cuh* -quercetin --− . -nuclear receptor ror-alpha bit is involved in cell growth, differentiation, and control of metastatic behavior of androgen-independent prostate cancer^ [ ] n * b w* -acacetin bit regulates centrosome separation, bipolar spindle formation in cell mitosis^ [ ] xnn* wqo* -quercetin . -p-glycoprotein and bthey involved in multi-drug resistance^ [ ] xwk* cbz* -acacetin . -platelet-derived growth factor receptor bit has a pro-angiogenic action [ in compounds, the numbers , , , … indicate a. nilotica's phytochemical constituents, letters a, b, … indicates positive controls, • indicates the cocrystallized ligands, and the italic emphasis indicates compounds with the higher scores. at ligand-based virtual screening score (lbvs sco.), en dash (-) means that the compound was not screened. asterisk (*) indicates the pdb id. swiss means that the d structure of the target was modeled using swiss-model web server [ ] the predicted toxicity according to the results, , -di-o-galloyl-beta-d-glucose, ellagic acid, kaempferol, and quercetin were non-toxic as well as non-carcinogen (table ) . according to the results, (+)-mollisacacidin, acacetin, catechin, epicatechin, kaempferol, naringenin, niloctane, and quercetin were found to be the best lead and drug candidates with good synthetic accessibility, followed by digallic acid, ellagic acid, leucocyanidin, and melacacidin (table ). despite the enormous conducted studies on the pharmacology activity of a. nilotica's [ ] , the determination of the target that contribute to its activity and the understanding of the mechanism of action as well as to assess the pharmacokinetics, safety, and the drug-likeness probability are important issues that were not conducted yet. such studies are required to bring the plant in the drug discovery pipeline so as to design a novel drug with broad-spectrum of therapeutic activity and safety. to identify the targets, targetnet web servers that utilize a qsar model based on the chemogenomic data as a predictive algorithm [ ] and similarity ensemble search tool [ ] were used. to validate the predicted target from the web servers, a molecular docking study was performed using cresset flare software [ ] that uses the lead finder program [ ] for bit is an essential bacterial enzyme in peptidoglycan biosynthesis^ [ ] dgi* c p* r * -quercetin bit is responsible for hydrolysis of beta-lactams, with substrate specificity toward cephalosporins, has an important role in cephalosporins resistance^ [ ] hdq* pu * r w in compounds, the numbers , , , … indicate a. nilotica's phytochemical constituents, letters a, b, … indicate positive controls, • indicates the cocrystallized ligands, and the italic emphasis indicates compounds with the higher scores. at ligand-based virtual screening score (lbvs sco.), en dash (-) means that the compound was not screened. asterisk (*) indicates the pdb id. phyre and raptor x means the d structure of target was modeled by phyre [ ] and raptorx [ ] web servers, respectively docking calculation. moreover, pkcsm [ ] and swissadme web servers [ ] were used to predict the pharmacokinetics (adme: absorption, distribution, metabolism, and elimination), toxicity, and the drug-likeness probability. the total predicted targets form the virtual screening with the highest probability that was validated by the molecular docking were targets. the interaction of acacetin with the cell division control protein homolog (cdc ) will prevent the oncogenic transformations. the inhibition of enzymes-anaplastic lymphoma kinase by quercetin, cyclin-dependent kinases , , and by ellagic acid and acacetin, aurora a and b by ellagic acid and quercetin, serine/threonine protein kinase nek by quercetin, proto-oncogene tyrosine-protein kinase src by ellagic acid, tankyrase and by acacetin as well as m phase inducer phosphatase by digallic acid, epicatechin, and kaempferol-will prevent the cancer progression and development. moreover, the inhibition of the enzymes-cell division cycle- -related protein kinase by ellagic acid, serine/ threonine-protein kinase pim- by quercetin, ellagic acid, and kaempferol as well as dna topoisomerase by in compounds, the numbers , , , … indicate a. nilotica's phytochemical constituents, letters a, b, … indicate positive controls, • indicates the cocrystallized ligands, and the italic emphasis indicates compounds with the higher scores. at ligand-based virtual screening score (lbvs sco.), en dash (-) means that the compound was not screened. asterisk (*) indicates the pdb id bit is a pro-inflammatory cytokine counteracts the anti-inflammatory activity of glucocorticoids^ [ ] . bit is responsible for the release of the arachidonic acid from arachidonyl phospholipids, thereby involved in the initiation of the inflammatory response^ [ ] . in compounds, the numbers , , , … indicate a. nilotica's phytochemical constituents, letters a, b, … indicate positive controls, • indicates the cocrystallized ligands, and the italic emphasis indicates compounds with the higher scores. at ligand-based virtual screening score (lbvs sco.), en dash (-) means that the compound was not screened. asterisk (*) indicates the pdb id kaempferol- -glucoside and , -di-o-galloyl-beta-d-glucose-will suppress the dna replication; the inhibition of enzymes-cyclin-dependent kinase by acacetin and telomerase reverse transcriptase by leucocyanidin, quercetin, ellagic acid, and kaempferol-will suppress the transcription; the inhibition of tyrosine-protein kinase lyn enzyme by ellagic acid will prevent the cancer cells proliferation; as well as the inhibition of angiopoietin- receptor, proto-oncogene tyrosine-protein kinase src by ellagic acid, and protein kinase c epsilon by kaempferol and naringnen will suppress the cancer cells invasion. furthermore, the inhibition of ephrin type b receptor and platelet-derived growth factor receptor by ellagic acid, vascular endothelial growth factor receptor by quercetin, as well as focal adhesion kinase enzyme ellagic acid and quercetin will suppress the angiogenesis. the inhibition of p-glycoprotein , transporters by kaempferol- -glucoside, chalconaringnen- -o-betaglucopyranoside, kaempferol, and quercetin as well as atp binding cassette sub-family g member by (+)-catechin- , digallate, chalconaringnen- -o-beta-glucopyranoside, in compounds, the numbers , , , … indicate a. nilotica's phytochemical constituents, letters a, b, … indicates positive controls, • indicates the cocrystallized ligands, and the italic emphasis indicates compounds with the higher scores. at ligand-based virtual screening score (lbvs sco.), en dash (-) means that the compound was not screened. asterisk (*) indicates the pdb id leucocyanidin, quercetin, ellagic acid, and kaempferol will suppress the cancer cells resistance. the interaction of lupeol, quercetin, ellagic acid, and kaempferol with the enzyme inducible nitric oxide synthase on macrophage will promote a tumoricidal action. the inhibition of the enzymes-bcl- -related protein a by leucocyanidin, quercetin, ellagic acid, and kaempferol, the induced myeloid leukemia differentiation protein mcl- by acacetin, quercetin, ellagic acid, and kaempferol as well as the interaction with enzymes: caspase by (−)-epigallocatechin- -gallate, quercetin, ellagic acid, and kaempferol, death-associated protein kinase by kaempferol and quercetin-will induce cancer cell apoptosis. consequently, those compounds show substantial anticancer activity ( table ) . the interaction of kaempferol and quercetin with the enzymes -oxyacyl-[acyl carrier protein] reductase fabg and the interaction of (−)-epigallocatechin- -gallate, (+)-catechin- , -digallate, and quercetin with enoyl-acyl carrier protein reductase will inhibit the bacterial fatty acids biosynthesis that is essential in the formation of bacterial membrane phospholipids [ ] leading to ban impairment in the cellular envelope structure and function, the ability to form biofilms as well as increasing the susceptibility to the environmental stress^ [ ] . moreover, the inhibition of the enzyme d-alanine d-alanine ligase by quercetin will suppress the peptidoglycans biosynthesis that is vital in bacterial cell structure causing loss of bacterial cell integrity [ ] . therefore, those compounds have significant antibacterial activity ( table ) . the interaction of leucocyanidin, ellagic acid, kaempferol, and quercetin with toll-like receptor will activate this innate immune receptor that helps in the recognition of microbial dna [ ] . the interaction of digallic acid, the italic emphasis indicates desirable prosperity bbb blood-brain barrier, vd volume of distribution, renal oct human organic cation transporter [ ] acacetin, ellagic acid, kaempferol, and quercetin with hiv integrase enzyme will inhibit the viral dna integration into host dna leading to the suppression of replication cycle [ ] . the interaction of quercetin with coronavirus replicase polyprotein ab will inhibit the transcription and replication of viral rnas [ ] . thus, those a. nilotica's phytochemical constituents exhibit considerable antiviral activity ( table ). the interaction of acacetin with the enzyme mo -related protein kinase pfmrk will disrupt the regulation of plasmodial cell cycle [ ] , and the interaction of quercetin and (+)-mollisacacidin with the enzyme m aspartyl aminopeptidase will prevent the invasion in host erythrocyte and the degradation of host hemoglobin [ ] . furthermore, the interaction of quercetin with the plasmodial enzymes β-hydroxy acyl-acp dehydratase fabz and hydroxyacyl-[acyl-carrier-protein] dehydratase will inhibit the fatty acid biosynthesis [ , ] that are important for plasmodial membrane [ ] . subsequently, those a. nilotica's phytochemical constituents show considerable antiplasmodial activity ( table ) . the interaction of ellagic acid with the insulin receptor will promote glucose uptake that lowers the blood glucose level [ ] . the interaction of quercetin with the enzyme glycogen phosphorylase will inhibit the glycogenolysis that reduces the hyperglycemia [ ] . moreover, the interaction of , -di-ogalloyl-beta-d-glucose and chalconaringnen- -o-betaglucopyranoside with the sodium/glucose co-transporter will inhibit the renal glucose reabsorption leading to a reduction in plasma glucose level [ ] , the interaction of dicatechin, kaempferol- -glucoside, leucocyanidin, ellagic acid, kaempferol, and quercetin with the aldose reductase fig. the d interaction between the best compounds with some of their predicted anticancer targets. a quercetin (violet) with anaplastic lymphoma kinase enzyme. staurosporine (turquoise) as control. b ellagic acid (dark yellow) with angiopoietin receptor. cabozantinib (turquoise) as control. c ellagic acid (dark yellow), kaempferol (pink), and quercetin (violet) with the aromatase enzyme. anastrozole (teal) and the co-crystallized ligand a asd (turquoise) as a control. d ellagic acid (dark yellow) and quercetin (violet) with aurora a kinase enzyme. the cocrystallized ligand a adp (turquoise) as a control. e ellagic acid (dark yellow), kaempferol (pink), and quercetin (violet) with caspase enzyme. the isatin sulfonamide (turquoise) as a control. ellagic acid (dark yellow), kaempferol (pink), and quercetin (violet) with steroid alpha-hydroxylase enzyme. galeterone (turquoise) as a control enzyme will suppress the development of the secondary diabetic complications [ ] , as well as the interaction of ellagic acid and quercetin with the beta-secretase enzyme will upregulate the insulin receptors in the liver [ ] . successively, those a. nilotica's phytochemical constituents have significant antidiabetic activity ( table ). the interaction of (−)-epigallocatechin- -gallate, ellagic acid, kaempferol, and quercetin with the enzyme arachidonate -lipoxygenase and the interaction of digallic acid, acacetin, ellagic acid, kaempferol, and quercetin with the receptor-interacting serine/threonine protein kinase will interrupt the inflammatory responses [ , ] . the interaction of (+)-mollisacacidin, naringnen, e l l a g i c a c i d , k a e m p f e r o l , a n d q u e r c e t i n w i t h cycloxygenase- enzyme (cox- ) will prevent the formation of inflammatory mediators prostaglandins [ ] , and the interaction of digallic acid, kaempferol, and quercetin with phospholipase a enzyme will prevent the initiation of the inflammatory response [ ] . moreover, the interaction of , -di-o-galloyl-beta-d-glucose, digallic acid, acacetin, ellagic acid, kaempferol, and quercetin with the xanthine dehydrogenase/oxidase enzyme will inhibit the formation of uric acid and reactive oxygen species [ ] . thus, those of a. nilotica's phytochemical constituents exhibit considerable antiinflammatory activity ( table ). the interaction of dicatechin with the mu and delta opioid receptors will lead to antisecretory and anti-transit action that will inhibit diarrhea [ ] . the interaction of , -di-o-galloylbeta-d-glucose with p y receptor will inhibit the platelet activation [ ] ; consequently, they have considerable antidiarrheal and anti-platelets activity, respectively (table ). the chemogenomic-based qsar models of targetnet web server were strictly evaluated and validated leading to respected screening results [ ] . furthermore, the lead finder program [ ] on cresset flare software [ ] is characterized by the combination between the genetic algorithm and different optimization strategies leading to great efficiency, robustness, accuracy, and speed of calculations [ ] . musab ibrahim et al. [ ] found the results of a molecular docking study about novel synthesized cox enzyme inhibitors conducted in cresset flare software were aligned with results of the conducted in vivo study. depending on that, the obtained results of the predicted targets could be with considerable accuracy. since the pharmacological activity does not depend only on the pharmacodynamic properties, but also on the pharmacokinetic properties. moreover, as the drug safety, the assessment of drug-likeness probability, and the synthetic accessibility are important issues [ ] , the identification of the best a. nilotica's phytochemical constituents will be attained by the assessment of those issues collectively. the pharmacokinetics is concerning the study of the entrance, movement, changing, and leaving of the drug to the body [ ] . the higher absorption from the gastrointestinal tract leads to higher drug concentration on the blood, the higher volume of distribution provides higher supply to the body tissues, and the adequate metabolism and elimination prevent the accumulation of the drug in the body, hence reduce the toxicity [ ] . consequently, the consideration of the pharmacokinetics in drug design is an essential task [ ] . fig. the d interaction between the best compounds with some of their predicted antiplasmodial and antidiabetic targets. a kaempferol (pink), and quercetin (violet) with β-hydroxy acyl-acp dehydratase fabz. the co-crystallized ligand b km (turquoise) as a control. b quercetin (violet) with hydroxyacyl-[acyl-carrier-protein] dehydratase. the cocrystallized ligand a emo (turquoise) as a control. c (+)-mollisacacidin (green), epicatechin (teal), and quercetin (violet) with m aspartyl aminopeptidase enzyme. d ellagic acid (dark yellow) with insulin receptor. ceritinib (turquoise) as a control. e quercetin (violet) with glycogen phosphorylase (muscle). the co-crystallized ligand avf (turquoise) as a control. f , -di-o-galloyl-beta-d-glucose (green) with sodium /glucose cotransporter . canagliflozin (turquoise) as a control for instance, lupenone is highly lipophilic; hence, it has higher absorption percent ( %); in contrast, the hydrophilic groups of ellagic acid reduce it absorption percent to . %, however, still it as a high absorption percent. the higher absorption will make lupenone is highly bioavailable. niloctane is permeable to bbb; therefore, its concentration that reaches the brain targets is more than ellagic acid that is not permeable to the bbb. the predicted volume of distribution (vd) of melacacidin ( . l/kg) is the highest one, meaning that it has the highest distribution in body tissues. in contrary, nilobamate has the highest predicted total clearance, meaning that it is the fastest one that eliminated from the body (table ) . moreover, the inhibition of cytochrome-p enzyme cyp a by ellagic acid will decrease the biotransformation of drugs that metabolized by it leading to increase in the concentration of them that may increase the side effects; consequently, the drug-drug interaction must be in consideration. the binding of dicatechin with the p-glycoprotein may decrease the transportation of drugs transported by this transporter and may involve in the drug resistance by the pumping out mechanism (table ) . furthermore, the predicted ames toxicity of epicatechin will lead to genotoxicity and mutagenicity [ ] , the predicted herg ii potassium channel inhibitory effect of acacetin bprolongs the qt interval in ecg that increases the risk for potentially fatal ventricular arrhythmias^ [ ] ; subsequently, such drugs will not be considered as drug-likeness candidate (table ) . besides, quercetin has no violation in lipinski rule of five; hence, it will a good candidate as an orally active drug as well as it has no violation in ghose, veber, and egan filters; therefore, it will be a good lead-likeness candidate [ ] (table ) . the d interaction between the best compounds with some of their predicted antiinflammatory, antidiarrheal, and anti-platelets targets and acetyl cholinesterase enzyme. a ellagic acid (dark yellow), kaempferol (pink), and quercetin (violet) with arachidonate -lipoxygenase enzyme. the co-crystallized ligand a c e (turquoise) as a control. b ellagic acid (dark yellow), kaempferol (pink), and quercetin (violet) with cyclooxygenase- enzyme. indomethacin (turquoise) as a control. c dicatechin with mu-type opioid receptor. eluxadoline (turquoise) as a control. d dicatechin with delta-type opioid receptor. eluxadoline (turquoise) as a control. e , -di-o-galloyl-beta-dglucose (yellow) with p y receptor. clopidogrel (turquoise) as a control. f digallic acid (yellow) and leucocyanidin (blue) with acetylcholinesterase enzyme. neostigmine (turquoise) as a control according to the results of pharmacodynamics, pharmacokinetics, safety, and drug-likeness predictions collectively, ellagic acid, kaempferol, and quercetin were the best a. nilotica's phytochemical constituents that contribute to the therapeutic activities. the d interaction with their predicted targets demonstrates marked ligand superimposing with the control compounds (e.g., figs. a, b, e, and b) ; however, it may at the same active site without ligand superimposing (e.g., figs. c and c). ellagic acid interacts with aurora a kinase enzyme with two binding modes (fig. d) . ellagic acid, kaempferol, and quercetin interact with steroid alpha-hydroxylase enzyme at a binding mode that differs from the binding mode of control galeterone (fig. f) . they were followed by (+)-mollisacacidin, epicatechin, and melacacidin, those of their predicted ames toxicity decreased their rank. the predicted herg ii potassium channel inhibitory effect of acacetin decreased its rank; however, it has good pharmacodynamics and pharmacokinetics profile. despite the efficient pharmacodynamics and the respectable safety profile of ellagic acid, kaempferol, and quercetin, practically, each compound suffers from the low bioavailability [ ] [ ] [ ] , albeit the predicted intestinal absorption of them is high ( table ) . the reduced bioavailability of ellagic acid is attributed to the poor absorption and rapid elimination from the body [ ] (the predicted total clearance of ellagic acid is high). the higher topological polar surface area (tpsa) ( table ) contributes to the poor absorption. the reduced absorption of kaempferol is attributed to the larger particle size and poor water solubility [ ] . the reduced bioavailability of quercetin is attributed to bthe poor solubility and crystalline form at body temperature^ [ ] . moreover, ellagic acid, kaempferol, and quercetin have many polar phenolic hydroxyl groups (structure l, o, and x); consequently, they are subjected to direct glucuronide conjugation with as phase ii metabolism. bkaempferol and quercetin are rapidly excreted in urine as glucuronides mainly^ [ ] . the reduced bioavailability affects pharmacological activity. hence, to maintain the pharmacological activity, the bioavailability must be enhanced. the nano-suspension form of kaempferol is increased its absorption and bioavailability [ ] . the administration of isoquercetin (quercetin- -glucoside) increases the absorption and bioavailability of quercetin [ ] . the bioavailability ellagic acid, kaempferol, and quercetin is increased bypassing the entero-hepatic phase ii conjugation (e.g., formation of ester derivatives) and by using novel drug delivery systems as the liposomes. furthermore, the co-administration of ginkgo biloba extract with kaempferol and quercetin increased the bioavailability of them [ ] . consequently, the combination of ellagic acid, kaempferol, and quercetin will be optimum treatment choice that maximizes the therapeutic activity and the safety profile as well as overwhelms the limits in the bioavailability. as they naturally are available in one plant, the combination of them at the therapeutic doses will be additive and will not induce drugdrug interactions. the design of multi-target drug is an effective promising approach for the treatment of complex disease [ ] . the computational methods including the virtual screening are not to substitute the in vitro and in vivo methods, however, to reduce the time, cost, and the difficultness in the drug target identification [ ] . therefore, this study is an attempt to identify the best a. nilotica's phytochemical constituents that contribute to its pharmacological activity as well as their targets. it is not meaning that this study alone will be sufficient to judge about the result; however, experimental studies are required to validate the results. according to the results of pharmacodynamics, pharmacokinetics, safety, and drug-likeness predictions collectively, ellagic acid, kaempferol, and quercetin were the best a. nilotica's phytochemical constituents that contribute to the therapeutic activities; consequently, we recommend the use of ellagic acid, kaempferol, and quercetin as a combined drug via the novel drug delivery systems for the treatment of recent diseases attacking the public health including cancer, multidrug-resistant bacterial infections, diabetes mellitus, and chronic inflammatory systemic diseases. moreover, we recommend wet lab studies to validate the results. of ligand-based virtual screening web tools and screening the synthetic accessibility is from (very easy) to (very difficult). the bold indicates desirable prosperity, the italic indicates undesirable prosperity as well as the bold-italic indicates the best compounds mw molecular weight, rotatb. bonds rotatable bonds, m r molar refractivity, h-don hydrogen bond donors, h-acc hydrogen bond acceptors, tpsa topological polar surface area a lipinski rule of five, ghose, veber, egan, and muegge describe the relationship between the pharmacokinetic and physiochemical parameters. the parameters including molecular weight, number of rotatable bonds, molar refractivity, number of hydrogen bond donors and acceptors, as well as the topological polar surface area. each parameter has a specific range that the structure must not be under or above it to be free from violation [ ] acacia nilotica (l.): a review of its traditional uses, phytochemistry, and pharmacology medicinal plants: future source of new drugs bioactive compounds from medicinal plants and their importance in drug discovery in pakistan cancer statistics. www.cancer.gov. accessed antibacterial drug discovery and structure-based design association. statistics about diabetes. www.diabetes.org chronic inflammatory systemic diseases an evolutionary trade-off between acutely beneficial but chronically harmful programs advances in molecular modeling and docking as a tool for modern drug discovery from drug target to leads-sketching a physico-chemical pathway for lead molecule design in silico a comparison of various optimization algorithms of protein-ligand docking programs by fitness accuracy swissadme: a 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potassium channel assays in drug development production, characterization, and evaluation of kaemferol nanosuspension for improving oral bioavailability bioavailability of ellagic acid in pomegranate (punica granum l.) juice bioavailability of quercetin bioavailability of ellagic acid after single dose administration using hplc disposition of quercetin and kaempferol in a human following an oral administration of ginkgo biloba flavonoid bioavailability and attempts for bioavailability enhancement comparative pharmacokinetics and bioavailability studies of quercetin, kaempferol, and isorhamnetin after oral administration of ginkgo biloba extracts, ginkgo biloba extract phospholipid complexes and ginkgo biloba extract solid dispersions in rats advancement of multi-target drug discoveries and promising applications in the field of alzheimer's disease qsar-based virtual screening: advances and applications in drug discovery conflict of interest the authors declare that having no conflict of interest. the italic emphasis indicates desirable prosperity, the bold emphasis indicates undesirable prosperity ames tox. ames toxicity key: cord- -rj dsib authors: schein, catherine h. title: polyglutamine repeats in viruses date: - - journal: mol neurobiol doi: . /s - - - sha: doc_id: cord_uid: rj dsib this review explores the presence and functions of polyglutamine (polyq) in viral proteins. in mammals, mutations in polyq segments (and cag repeats at the nucleotide level) have been linked to neural disorders and ataxias. polyq regions in normal human proteins have documented functional roles, in transcription factors and, more recently, in regulating autophagy. despite the high frequency of polyq repeats in eukaryotic genomes, little attention has been given to the presence or possible role of polyq sequences in virus genomes. a survey described here revealed that polyq repeats occur rarely in rna viruses, suggesting that they have detrimental effects on virus replication at the nucleotide or protein level. however, there have been sporadic reports of polyq segments in potyviruses and in reptilian nidoviruses (among the largest rna viruses known). conserved polyq segments are found in the regulatory control proteins of many dna viruses. variable length polyq tracts are found in proteins that contribute to transmissibility (cowpox a-type inclusion protein (ati)) and control of latency (herpes viruses). new longer-read sequencing methods, using original biological samples, should reveal more details on the presence and functional role of polyq in viruses, as well as the nucleotide regions that encode them. given the known toxic effects of polyq repeats, the role of these segments in neurovirulent and tumorigenic viruses should be further explored. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. mutations in human proteins that result in longer polyq repeat sequences have been linked to dementias and ataxias [ ] . their toxicity has been attributed, at the protein level, to aggregation of long polyq protein tracts, interference with autophagy [ ] and to their ability to bind rna in several model organisms, including marmosets [ ] , drosophila, and e. coli [ ] [ ] [ ] [ ] . proteins containing mutated longer repeats may also lose their function. for example, expanded polyq repeats in ataxin- may interfere with mirna function in machado-josef disease [ ] and expansion of the polyq tract in the androgen receptor reduces its dna binding capacity [ ] . the mechanisms of polyq toxicity are dependent on the protein encoded, and even alternative reading frames of the dna [ , ] . for example, aggregated polyq containing protein, huntingtin, is found in the brain of huntington's disease (hd) victims. however, a rare disease similar in clinical appearance to hd, huntington's disease-like (hdl ), has been linked to repeat regions in rna and alternative transcripts causing loss of expression of other proteins, such as junctophilin- [ ] . targeting such repeats at the protein or rna level may provide novel therapies for these diseases [ ] [ ] [ ] . while the mechanisms for the function and toxicity of extended polyq segments (or the nucleic regions that encode them) in eukaryotic proteins continue to be actively studied [ ] , there has been little exploration of their occurrence and possible roles, even in neurovirulent viruses. this is particularly curious, in light of the documented role of polyq tracts in transcription factors (tfs) and their abundance in eukaryotic genomes [ ] , even constituting a bpolyq interactome^ [ ] . the first goal of this work was to determine whether viral proteins contain polyq repeats at all. one might anticipate that longer polyq sequences, based on their tendency to aggregate electronic supplementary material the online version of this article (https://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users. and to interfere with transcription, would be selected against in rapidly replicating viruses under extreme environmental pressure. sequence selection in dna viruses during chronic infections, on the other hand, would favor compatibility with host cell transcription and translation mechanisms and immune evasion [ , ] , rather than rapid growth [ ] [ ] [ ] [ ] . as this study has found, polyq segments are indeed rare in the catalogued sequences of smaller rna viruses, but even very long repeats have been found in several large rna and dna viruses. the second goal is to suggest what functions, if any, such repeat sequences, at the protein or nucleotide level, could play in viral replication, chronic infection, or neuro-pathogenesis. clues for the potential role of the repeats could be gleaned from their roles in eukaryotic proteins, where they are present in many transcription factors. in addition, q-rich repeats in the n-terminus of the argonaute- protein of drosophila and other insects [ , ] are essential for antiviral activity [ ] and one in a cellular protein, tle , contributes to this protein's ability to control lytic reactivation of kaposi's sarcomaassociated herpesvirus [ ] . as discussed below, the polyq segments found in several viral proteins could indeed affect the ability of viruses to control the activities or transcription of their own or cellular proteins, while their possible role in neurovirulence remains to be established. while it may be surprising that polyq sequences in neurovirulent viruses have not been a major topic of study, it should be emphasized that the extent of very long repeat segments would be difficult to detect by short-read sequencing of the large viruses in which they have been found. in addition, cag triplet repeats are known to be unstable [ ] and may be specifically excised during the transition from latency to active growth, or after adaptation to cell culture. newer methods, designed to specifically determine repeat sequences in direct isolates from infected tissues, should reveal more details about the presence and roles of repeated sequences. searching for polyq tracts in viruses searching the published sequences of many different virus families revealed that while they are not present in smaller rna viruses, surprisingly long tracts of polyq have been found in larger rna and dna viruses. the search also suggested that repeats may be much more common in viruses than is indicated by the currently archived sequences. at the start of this work, the vipr database [ ] , which allows rapid access to the published sequences of over , viral genomes or genome segments, was used to determine which rna and dna viruses contain polyq repeats. a new resource, the influenza research database [ ] , was used to screen influenza virus sequences. once q-rich sequences were identified, blast searches starting from the viral proteins that contained them were used to determine the extent of their conservation in the same virus family and to find other virus proteins containing similar tracts. blast was also used to find viral proteins containing repeats similar to those of the argonaut- proteins. the vast majority of published rna virus genomes contain no extended polyq repeats if long polyq repeats are intrinsically toxic for the function of proteins, or stimulate aggregation [ ] , one would expect that rapid evolving rna viruses would selectively eliminate them. table summarizes searches of over , genome sequences of many families of pathogenic viruses, including single-strand rna viruses (flavivirus, reoviruses, picornaviruses, bunyaviridae, etc.), , segments of the dsrna reoviridae from the vipr database and over , strains of influenza from the influenza research database [ ] . this revealed that only a few rna viruses contain even a qqqq sequence. longer polyq sequences, which would be anticipated to cause aggregation of the viral proteins (or, as discussed later, interfere with autophagy), were not found. as long repeats of many other amino acids (especially d, t, l, e, p) and mixed basic or acidic residues occur very frequently, this would suggest that there is some selection against longer polyq tracts, either at the rna or protein level. literature searches have revealed sporadic reports of polyq segments in some small rna viruses, including potyviruses [ , ] , and even a coxsackie a isolate [ ] , whereby the lack of consistency among closely related viruses suggests these have no functional role. more meaningfully, blast searches beginning with a qrich sequence from dna viruses (see below) identified a polyq sequence in the first open reading frame of a nidovirus isolated from a python, representing a novel genus of torovirus [ ] . a similar polyq sequence is also found in the orf of the morelia viridis (boa constrictor) nidovirus, but not in that of a nidovirus isolated from lizards [ ] . nidoviruses (which include the coronaviridae) have the longest known rna virus genome, with continuous positive sense strands of - kbases [ ] . in contrast, other +-strand rna viruses range from . to kb, and negative-strand rna viruses have genome lengths ranging from to kb. bunyavirdae can be up to . kb in total length, but their longest (l) segments do not exceed kb. it is possible that these long polyq insertions may play a role, at the rna level, during genome replication or adapting to changing environments [ ] . as for other rna virus families [ , ] , several studies have indicated the importance of dsrna folding domains near the ′ end of coronavirus genomes [ ] . formation of dsrna intermediates [ ] , important for the interferon response [ ] [ ] [ ] [ ] , as well as viral enzymes that interfere with the oas/rnasel system that would target these [ , ] , are characteristic of infection by several different nidoviruses. these include the coronaviruses, mers and sars. the repeat rna sequence encoding the polyq region in the ′ region of the python virus may fold as an independent domain containing extended segments of dsrna (fig. s , [ , ] ), whereby the low energy of folding generates a dynamic, unstable structure [ ] . the cag repeat region might be excised completely during rapid growth, as cag repeats are known to be unstable [ , , ] . alternatively, it may be transcribed past during generation of subgenomic rnas, which in nidoviruses proceeds by selective transcription of parts of the open reading frames [ ] . polyq repeats in dna viruses searches within two diverse and well-studied dna virus families, poxviridae and herpes, indicated that several of these large viruses, known to cause chronic neurotropic infections, contain long polyq segments (tables , and fig. ). these dna virus genomes are - kbp, - times longer than those of the rna viruses. many herpes virus proteins contain variable length polyq repeats in conserved regions ( table shows some examples), and even longer polyq repeats have also been found (fig. ). in addition to direct polyq repeats, there are long, q-rich repeats in other viral proteins. for example, there is a long, variable length, q-rich repeat in the mc l protein of the pox virus, molluscum contagiosum [ ] . this virus causes the formation of wart-like blisters on the skin of infected individuals, and characteristic cellular inclusions. as discussed below, the longest repeats were found in dna virus proteins that function in enhancing transmissibility (cowpox ati) or contribute to viral latency (herpes viruses). the rna virus results, coupled with the fact that polyq expansions in human proteins can lead to disease, suggest that polyq segments are probably selected against in rapidly growing viruses. this leads to the question: what possible functions could they serve for the virus itself or interaction with host cells? this is an important question to answer as the repeats occur in proteins from viruses triggering hard-to-treat neuropathies and epilepsy [ ] and isolated from latently infected tissues and tumors. proteins although studied for their role in disease, polyq segments in mammalian proteins have important regulatory table ) functions. after a polyq repeat was found to be an activation domain [ ] of the tf, sp , the role of such segments in tfs was extensively studied [ ] long before they were documented to have pathogenic characteristics possibly related to folding and formation of aggregates in cells (e.g., [ ] ). more recently, the length of polyq inserts was directly related to their ability to enhance tf activity [ , ] . variable length q-rich repeats may also modulate tf activity in eukaryotic cells by modulating their solubility [ ] or by recruiting other factors to the dna binding complex. a polyq repeat in murine sry (sex determining region on the y chromosome) both stabilizes the protein and serves as a transactivation domain [ ] . however, the region is found only in rodent sryand can be replaced by an irrelevant protein (mcherry). consistent with a possible role for polyq tracts in viral proteins in controlling transcription, the first report of a polyq tract in a dna virus was in a baculovirus regulatory protein, where the authors noted the similarity of the amino acid repeats to those in sp [ ] . as table illustrates, polyq tracts are present in several regulatory proteins of herpes viruses. further evidence for a functional roles in controlling virus replication is that q-rich tetratricopeptide repeats are upregulated during bovine leukemia virus infection [ ] , as well as in human breast cancer cells [ ] . the q-rich n-terminal region of a cellular protein, transducing inhibitor of split (tle ), contributes to this protein's ability to control lytic reactivation of kaposi's sarcoma-associated herpesvirus [ ] . through interference with autophagy recent reports suggest that polyq segments may also serve to downregulate autophagy, which serves as a barrier to the growth of neurovirulent herpes viruses (whereby rna viruses may use the membranous structures characteristic of autophagy for their own replication). these examples suggest possible roles for the longer repeats in proteins of viruses that typically cause latent infections, including herpes simplex, epstein barr, βand ɣ-herpes viruses (fig. ) . mutations in beclin- , a protein which triggers the process, were previously linked to development of neurodegenerative diseases [ ] . neurovirulent herpes simplex virus produces a protein that specifically binds to and interferes with beclin- function [ ] , called neurovirulence factor icp . (or gamma . , ɣ . ). figure (based on [ , ] ) shows how an expansion of the polyq repeat in mutant ataxin- , as well as excess polyq from other cellular (or viral) proteins, could interfere with the interaction of ataxin- and beclin- to inhibit autophagy. the polyq region of wt-ataxin- , a deubiquitinase, is expanded in spinocerebellar ataxia type . the normal length polyq region mediates binding of ataxin- to beclin- , preventing its degradation and allowing it to stimulate autophagy (fig. , top line) . soluble, mutated polyq segments can inhibit this binding, thus preventing beclin- degradation and upregulation of autophagy, preventing efficient clearance of aging cellular, as well as viral, proteins. another indication that polyq sequences from the virus, or some other repeat in ɣ . , may also be involved in this regulation is that there is a discontinuity within the (otherwise well conserved) ɣ . sequence in many herpes isolates (see supplementary material). such discontinuities usually indicate repeat insertions [ ] . this finding ties in with many years of research on the effect of inhibiting autophagy on replication and neurovirulence of various viruses [ ] . while neurovirulent viruses such as herpes are indeed held in check by autophagy, some rna viruses subvert the process for their own replication (e.g., picornaviruses [ ] , dengue [ ] ). although poliovirus requires autophagy for non-lytic spread, its replication is not affected by beclin- inhibition [ ] , suggesting it uses other ways to trigger the process. as is probably the case with the polyq repeat in murine sry, polyq repeat regions in viral proteins are generally variable in length and may be unstructured or bdisordered^ [ ] . however, some of the examples where the repeats are found suggest they have important functions that would not be obvious during in vitro replication. once they have infected a cell, viruses enter different growth phases, ranging from almost no replication to rapid growth leading to cell lysis. a herpes virus-infected ganglion may contain less than copies of the virus/cell in the latent state and still successfully reactivate after stress (from heat, uv light exposure or infection with, for example, a rhinovirus) [ ] . although rna viruses are generally considered to be bhit and run^, with rapid clearance from the serum, recent experience with zika [ ] [ ] [ ] and ebola [ , ] viruses has shown that some may also persist within body compartments where they are protected from the immune response. this leads us to a complicated equation: a virus seeking to survive intracellularly must sacrifice rapid growth for its ability to evade immune detection. variable polyq repeats may allow a virus to adjust to changing levels of required cellular factors [ ] , and determine whether the virus is able to actively replicate, or assume a lysogenic state. herpes viruses in particular are known to incorporate genes from the cells they infect into their genomes that may aid in maintaining lysogeny. polyq insertions at the amino acid or rna level may directly contribute to viral latency by lowering the transcription or activity of the affected proteins. alternatively, their presence, or the rna tracts encoding them, could contribute to neurovirulence by mechanisms demonstrated for human proteins (e.g., huntingtin). (table gives a sampling), including regulatory proteins, an apoptosis inhibitor, and uracil-dna glycosylase (udg), all factors that may affect viral replication positively or negatively. it may also be pertinent that a long, q-rich repeat is present immediately after the catalytic domain of the deoxyuridine ′-triphosphate nucleotide hydrolase (dut) gene of the red deer parapox virus (rdpv). similar sequences have not been reported in the dut enzymes of other viruses, nor has the role of the polyq sequence been determined in rdpv. however, udg and dut enzymes, which remove or prevent insertion of u residues in viral dna, are found in all herpes viruses [ ] . their enzymatic activity is essential for neurovirulence, neuroinvasion, and escape from latency of herpes viruses [ ] . mutation of the virus encoded dut inhibits transcription of equine infectious anemia virus (eilv, a lentivirus and retrovirus). on the other hand, eilv can replicate in non-dividing cells [ ] if it allows incorporation of u into its dna [ ] . accordingly, insertion or amplification of the polyq segment in udg or dut could slow replication to help maintain a latent state. as single point mutations (d e in the active site, or those preventing phosphorylation of s [ ] ) are sufficient to reduce neurovirulence, dut may also be a target for antiviral drug design [ ] . however, such inhibitors must be very efficient, as residual low levels of the enzyme might have the negative effect of prolonging viral latency (analogous to antibiotic treatment selecting for slow-growing bacterial persister cells [ ] ). the long polyq repeats in other herpes virus proteins (fig. ) may also help to suppress virus growth during latency. these include the direct repeats of polyq that occur in the low complexity c-terminal regions of the tupaiid t protein (β-herpes group f, isolated from a lymphoma in a tree shrew [ ] ) and the rf protein of radinovirus type (ɣ-herpesvirus), isolated from a kaposi's sarcoma-like lesion in a macaque [ ] . it is possible that these polyq repeats were directly incorporated from the host cell genes, as their sequences are quite similar to some host proteins (fig. ) . further evidence that these polyq repeats were incorporated in an adventitious fashion from the host cell is that repeats are not found in the published sequences of the (otherwise similar) n proteins of radinoviruses type [ ] . longer repetitive regions could slow growth by decreasing transcription of an essential enzyme, making its rna more vulnerable to cellular nucleases, and at the protein level, reducing its solubility [ ] or enhancing its degradability. under growth conditions allowing the virus to resume lytic growth, where the enzyme activity is required to ensure efficient replication, the region fig. soluble polyq segments (of cell or viral origin) may prevent beclin- -induced autophagy, which depends on the dna binding ability of the polyq segment of wt-ataxin- (based on [ , ] ). scheme a shows that under normal cell conditions, ataxin- binding (mediated by its polyq region) to beclin- (becn) protects it from proteosomal degradation. this allows beclin- to stimulate autophagy, which eliminates both aging cellular proteins and those of viral invaders. scheme b suggests that viral proteins' polyq, similar to the extended polyq loop of mutant ataxin- , can interfere with this control by preventing ataxin- from binding. beclin- is now degraded and cannot stimulate autophagy, resulting in even more accumulation of polyq tracts, defective cellular, and viral proteins that will interfere with normal metabolism fig. the polyq region in the tupaiid t protein (herpes virus group f, isolated from a lymphoma in a tree shrew) is flanked by poly-prolines (p), similar to polyq expansions in huntingtn, and ataxins associated with neurological disease. proline residues may also affect protein solubility [ ] . two other mammalian proteins that also contain long polyq repeats are shown for comparison. the t repeat is encoded primarily by cag codons, as is the case with huntingtn, and the nidovirus repeat (fig. s ) encoding the polyq segment could be rapidly removed at the gene level. as with the rna viruses, the published sequences of only a few poxviridae proteins contain even a tetrad qqqq repeat. however, there is a conserved polyq insertion of variable length in the a-type inclusion proteins (ati) of cowpox (cwpx) viruses ( table ) . as with the q-rich repeat in the mc l protein of molluscum contagiosum (fig. ) , this repeat is a variable area in an otherwise well-conserved protein (supplementary material). the ati with the longest polyq segment is in strain fm , isolated from a lesion in a vole, which causes skin lesions and mild symptoms in its host. although the length of the polyq segment in the cwpx strains in table is not directly related to pathogenicity, ati plays a role in a more difficult to measure parameter: transmissibility. the ati protein, together with the p c protein [ , ] , allows cwpx to form protein inclusions that, when excreted from the animal, protect the virus from the elements. inclusions that sequester the virus (v + phenotype) contribute to the high transmissibility of cwpx in the wild. as jennings noted centuries ago, nearly all milkmaids had been infected with cwpx, as were probably most cows. it is significant that in a comparison of three cwpx strains, only the fm virus, which contained the longest polyq insertion in its ati, made v + inclusions containing virus particles [ ] . two strains with shorter polyq segments, the index strain brighton red and a similar strain from rat, formed inclusions that contained no internalized virus particles (v ). the atis of these three strains differ primarily in their polyq repeat region length (table and supplementary) . growth in cell culture alone does not indicate that ati is an essential gene [ ] , although it is one of the most abundant cwpx proteins, amounting to as much as half of all protein synthesis in the blate-late stages^of replication [ ] . deleting the ati gene leads to a faster growing virus [ ] . however, as discussed above, ati enhances transmissibility from animal to animal, as well as virus survival outside the host. the presence of a longer polyq sequence could reduce its transcription, synthesis, or solubility during restrictive growth in an organism, where ati's activity is not required. smallpox and vaccinia virus (vv) strains lack polyq segments in their atis and form only virus-free inclusions (v phenotype). cwpx and vv strains also differ in the ati interacting protein, p c, in that only cwpx strains contain long repeats (up to ) of aspartate (d) residues. these results suggest strongly that this amino acid repeat, together with the polyq segment in the ati, aid in sequestering virus particles into the v + inclusions, which further the extracorporeal survival of the virus. recent direct, deep sequencing of fresh cwpx isolates from diseased animals indicated diversity in both genome length and coding areas from the brighton red reference strain, including an additional bp orf [ ] . as table illustrates, freshly isolated strains have the longest polyq region in the ati, which makes it difficult to determine a bwild typel ength of polyq. it is, for example, possible that the polyq repeats in cwpx ati can be selected against during growth in tissue culture. in keeping with this, there is no polyq repeat in the (extensively passaged [ ] ) brighton red strain, first isolated in in england from human lesions. this strain would be expected to transmit poorly in the wild, thanks to its v phenotype. more recently isolated german strains ( and ) have shorter polyq regions than isolates from or , but it is unknown how often these have been transferred in cell culture [ ] . polyq repeats as a key to antiviral therapy as noted in the introduction to this article, a primary reason for documenting the presence of polyq segments in viruses is the role polyq sequences in human proteins have been shown to play in human neurological syndromes [ ] [ ] [ ] . considering the importance of glutamine metabolism for central nervous system function, it would be instructive to specifically test the role of the q-rich regions on virus latency or replication in neuronal cells. glutamine itself is extremely important in brain chemistry, and inhibitors similar to this amino acid have antiviral activity. a q analogue, -diazo- -oxo-l-norleucine (don), can delay encephalitis caused by alphaviruses, such as sindbis, by reducing the amount of glutamate synthesized from glutamine [ ] . adding polyq tracts to the antiviral agent zanamivir greatly enhanced its anti-influenza activity [ ] . as fig. shows, the viral proteins that contain long polyq segments are very similar to those implicated in huntington's disease and human ataxias, and may thus be targeted by protein- [ ] or gene-based [ , , [ ] [ ] [ ] therapies similar to those now being tested. going forward, diagnostics should, as much as possible, distinguish polyq sequences due to a latent virus from those indicating a mutation in a human gene. the flanking regions, which contain proline repeats (polyp), may also affect the solubility of the proteins [ ] . to date, there have been few investigations of a direct role for these polyq repeats in initiating neural damage. aiding in establishing a latent infection could, in itself, contribute to neurovirulence, due to the presence of viral products [ ] . polyq repeats in viruses could play important roles in controlling transcription, latency, transmissibility, and neurovirulence, whereby the latter three aspects of virus pathogenicity are independent of the ability of the virus to grow to high titer in cell culture. long polyq tracts in the protein products of neurotropic and cancer-related dna viruses could chronically disturb their host cells, by mechanisms similar to those identified for huntingtin and other ataxia-related proteins that contain similar repeats. just as b cells and other somatic cells may change their genome structure upon differentiation, it is probable that rapidly growing viruses (and those adapted to tissue culture) have different sequences than those in a latent state. serial cultivation can favor rapid growth and the loss of pathogenic characteristics, an attenuation process used since the first vaccines against yellow fever [ ] and poliovirus [ ] . the instability of repeated cag regions that encode polyq repeat sequences might be a mechanism for adapting virus replication to changes in environmental factors [ ] . this means that they may be selectively excised during generation of subgenomic rnas or resumption of active growth after latent periods. thus, rational reference sequences of viruses should be based on those obtained from direct isolates of diseased tissue or consensus sequences covering many isolates [ ] [ ] [ ] . as the brighton red example 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bansal, deepak title: hepatic sinusoidal-obstruction syndrome and busulfan-induced lung injury in a post-autologous stem cell transplant recipient date: - - journal: indian pediatr doi: . /s - - - sha: doc_id: cord_uid: yj r rwt veno-occlusive disease of the liver is mostly encountered as a complication of hematopoietic stem cell transplantation with myeloablative regimens with an incidence estimated to be . %. it is clinically characterized by tender hepatomegaly, jaundice, weight gain and ascites. strong clinical suspicion and an early recognition of clinical signs are essential to establish the diagnosis and institute effective regimen. another complication of cytotoxic drugs given for cancers, is development of busulfan-induced lung injury. a strong index of suspicion is needed for its diagnosis, especially in setting where opportunistic fungal and viral infections manifest similarly. we illustrate the clinical and autopsy finings in a ½-year-old boy who received autologous stem-cell transplantation following resection of stage iv neuroblastoma. he subsequently developed both hepatic veno-occlusive disease and busulfan-induced lung injury. the autopsy findings are remarkable for their rarity. volume __ september , jain, et al. hepatic sinusoidal-obstruction syndrome increased efforts, hypoxia (saturation on room air: %; increasing to % on % fio ). tachycardia was present (heart rate /minute); however, circulatory parameters were normal (bp / mm hg, normal capillary refill time and pulse pressure, warm extremities). pallor was present, with no evidence of skin or mucosal bleeding. systemic examination was consistent with pneumonitis as the patient had tachypnea, bilateral equal air entry, and presence of coarse crepitations in bilateral lung fields. abdominal examination showed dark brown pigmentation over abdomen with no tenderness, guarding or rigidity. hepatomegaly was present with liver palpable cm below costal margin (span cm). spleen was not palpable. there was no free fluid. cardiac and neurological examinations were essentially normal. the index case was managed as a case of pneumonitis post auto-sct. respiratory support was initially provided with nasal prongs-continuous positive airway pressure. intravenous antibiotics were started cefoperazone-sulbactam, amikacin and azithromycin. due to rapidly progressive respiratory distress, child was transferred to pediatric intensive care unit where mechanical ventilation was provided. there was single episode of fever on the day of admission ( . o c) with a subsequent afebrile period throughout the hospital stay. progressive respiratory distress worsened into acute respiratory distress syndrome (ards). ventilation strategy was modified accordingly. on day , there was development of hypotensive shock, initially responding to fluid boluses. on day , the shock necessitated inotropic support (dopamine, adrenaline, noradrenaline and pre-terminally, and vasopressin). there was development of left sided pneumothorax followed by cardiac arrest on the same day. the patient could be revived and pneumothorax was drained. multi-organ dysfunction developed with acute kidney injury (onset day ), requiring peritoneal dialysis. significant transaminitis with elevated bilirubin levels was documented. antibiotics were changed to vancomycin and meropenam on day ; azithromycin was continued. intravenous co-trimoxazole and gancyclovir were added in therapeutic, renal modified doses. platelet concentrates were transfused to maintain a platelet count above × /mm . there was development of refractory shock on day . the child suffered another cardiac arrest on day , and could not be revived. the fig. a-d) . with these chest radiographs, possible etiologies considered were infective, including fungal pneumonia and pcj pneumonia, cmv disease and miliary tuberculosis (tb). non-infective etiologies under consideration included pulmonary graft-versus host disease (gvhd) and pulmonary veno-occlusive disease (vod). this is a case of neuroblastoma, stage iv, day post auto-sct, presenting with fever, pneumonia, hypoxia, and investigations showing polymorphic leucocytosis with deranged liver function tests (lft). in a posttransplant patient, complications can be divided according to the duration subsequent to transplant. in the initial days, there is presence of neutropenia; between - days is the early post-engraftment phase and beyond days is late post-engraftment phase. the index case developed symptoms in the early post engraftment phase. common complications seen in early post-engraftment phase can be divided into infective and non-infective. infective etiologies include cmv which can explain both pneumonia and hepatitis. it is a common pathogen causing disease weeks post sct. india is an endemic country for cmv. in the index case, eta demonstrated polymerase chain reaction (pcr) positivity volume __ september , jain, et al. hepatic sinusoidal-obstruction syndrome for cmv along with radiological findings which were supportive of the diagnosis. however, the child deteriorated despite administration of gancyclovir from day onwards, which is unusual. typically blood pcr is positive in such cases, though not mandatory for diagnosis of cmv pneumonia. fungal infections are the next possibility, supported by the presence of candida in eta on two occasions. presence of normal neutrophil count and a normal serum galactomannan are odd points. galactomannan < . has shown a good negative predictive value for aspergillus infection [ ] . other viral infections that are important in post-sct scenario include respiratory syncytial virus (rsv), para-influenza virus, influenza, metapneumovirus, and coronavirus. multiorgan failure and lymphopenia is common in these patients. patients with rsv often require ventilation. associated co-infection with fungus, especially aspergillus can be seen. in the absence of investigations directed towards the myriad respiratory viruses, it is difficult to rule in or rule out these infections. tuberculosis should be considered in an immunocompromised patient in an endemic country; however, the rapid onset of disease, absence of a contact and negative evaluation make it unlikely. in our case other bacterial infections typically seen in an immunocompromised child are also unlikely in view of sterile cultures, complete absence of fever and normal creactive protein (crp).though this clinical presentation can be caused by infection with pcj, it is an uncommon infection. other atypical infections like nocardia and cryptococcus are rarer still. the non-infective etiologies causing respiratory symptoms in a post-transplant setting can be pulmonary gvhd, idiopathic pneumonia syndrome (ips), bronchiolitis obliterans syndrome (bos), cryptogenic organising pneumonia (cop) and sos. ips is a very common disease in this situation, but is typically seen post allo-sct and hepatitis is not an associated feature. on-going hepatic sos is unlikely as there was no weight gain or tender hepatomegaly. gvhd and bos are also typically diseases seen in allo-sct setting. pulmonary sos is very rare and normal echo findings negate this possibility. the clinical presentation is consistent with cop, though it is more common in females undergoing allogenic transplant. the final diagnosis is neuroblastoma stage iv, day + post auto-sct (bu-mel) with pneumonitis, ards and multi-organ failure; likely etiology being fungal pneumonia or cmv pneumonia and hepatitis secondary to ischemia with underlying sos. pediatric hemato-oncologist : ips occurs post sct day+ to . this child had typical bilateral basilar infiltrates and hypoxia. moreover, ips has a relationship with use of busulfan and pre-existing sos. presence of cmv positivity in eta is of questionable significance as it is a common organism. histopathological evidence from lung biopsy is essential to prove cmv pneumonia. liver dysfunction in the form of transaminitis was likely due to shock and ischemia. pediatric hemato-oncologist : bacterial and fungal infections cannot be excluded despite absence of fever, several sterile cultures and continued normal values of crp, though less likely. however, both cmv and pcj infections are possible with normal crp. absence of adventitious lung sounds at initial presentation, along with presence of hypoxia may be a pointer towards pcj pneumonia. immunocompromised state, lymphopenia and the fact that the child was not on pcj prophylaxis are important here. moreover, cmv is ubiquitous in our volume __ september , jain, et al. hepatic sinusoidal-obstruction syndrome pediatric population, and in pediatric oncology patients, we have seen a near % seropositivity. reactivation of cmv can occur at any point of time in these patients. important non-infective possibilities are ips and cryptogenic pneumonia. pulmonary sos is quite unlikely given the normal echo findings. adult hematologist: immune reconstitution posttransplant takes typically to months. this child was immunocompromised. adenovirus infection can be considered. it can be rarely seen in association with hepato-pulmonary syndrome. the excised tumor on histology was categorized as differentiating neuroblastoma ( fig. a-c) . autopsy revealed normal serous cavities. liver weighed g and revealed irregular areas of sinusoidal congestion, confluent at places with necrosis of adjoining parenchyma involving both right and left lobe (fig. d) . no thrombi were identified in right and left hepatic vein or inferior vena cava. microscopically, areas of centrizonal congestion were identified (fig. e) . furthermore, the dominant pathology was seen in the central vein and terminal hepatic venule (thv). there was varying degree of obliterative changes with subendothelial fibrosis and laying down of reticulin fibres and collection of extracellular matrix in subintimal zone. at places, the thv was completely obliterated with wipe out of centrizonal hepatocytes while the periportal hepatocytes were preserved (web fig. a-e) . in other regions, extravasated rbcs and areas of hemorrhage were noted in centri-zonal regions. besides acute obliterative changes, subacute changes in form of deposition of collagen around the hepatic venule and collection of hemosiderin laden macrophages were also noted. loss of hepatic parenchyma resulted in approximation of central veins structures (web fig. a) . both lungs weighed g with dull pleura. microscopy revealed features of busulfan-induced lung injury with marked prominence of type ii pneumocytes; many of them demonstrated nuclear atypia and hyperchromasia. marked thickening of interstitium with fibrosis was also noted (web fig. a-b) . other regions showed patchy acute bronchopneumonia and alveolar haemorrhages. features of pulmonary arteriopathy were also noted with prominence of intra-acinar arterioles. there were no features of vod in the pulmonary veins. an occasional focus of septic emboli with candida infiltration into parenchyma was noted. no cmv inclusions were noted in lungs. pcr carried out on lung tissue for adenovirus, rsv and metapneumovirus were negative. acute ulcers with candida infiltration were noted in stomach and small intestine. candida had disseminated to heart causing mural endocarditis, myocardial abscess and tiny ( - mm) vegetations on left atrial wall (webfig. c-f). both tricuspid and mitral valves were normal. dissemination with formation of fungal abscesses were also detected in psoas muscle and omental fat. subsequent to septic emboli, infarcts were detected in right kidney (upper pole) with thrombi within the branches of renal vessels and spleen. right kidney also revealed features of acute tubular necrosis in noninfracted regions. no residual tumor was detected in lymph nodes, thymus and bone marrow. the autopsy diagnosis is concluded as follows: in a known case-of neuroblastoma, undifferentiated (adrenal) post-autologous stem-cell transplant: • features of busulfan-induced lung injury with organizing bronchopneumonia and pulmonary arterial hypertension; • veno-occlusive disease in liver. • fungal (candida) ulcers in git with extensive dissemination to heart (mural endocarditis and myocardial abscess), lungs, skeletal muscle and omental fat producing embolic infarcts in right kidney and spleen. • no residual disease in bone marrow. hepatic sinusoidal obstruction syndrome (sos) is an obliterative venulitis of thv which occurs as a result of cytoreductive therapy prior to hematopoietic stem cell transplantation (hsct), ingestion of pyrrolizidine alkaloids, or radiation therapy [ ] [ ] [ ] [ ] . the primary pathogenetic event is the endothelial injury of sinusoids and small hepatic veins. following which, there is deposition of fibrin-related aggregates and oedema in the subendothelial zone [ ] . accumulation of these aggregates and entrapment of fluid and cellular debris progressively occlude the hepatic venous flow and leads to post-sinusoidal intrahepatic hypertension. this is accompanied by necrosis of perivenular hepatocytes. histologically, acute, sub-acute and chronic forms of sos have been described depending upon collagenization and fibrosis of terminal hepatic venule. incidence of sos varies from - %, as it depends on the conditioning regimen used as well as upon the patient's risk factors [ ] [ ] [ ] . sos occurs more often after allo-than after auto-hsct ( v/s %, respectively), suggesting a role of immune reactions in this disorder [ ] . few independent studies have documented increase in circulating levels of plasminogen activator inhibitor- (pai- ), a molecule released by the endothelial cells, in patients developing sos [ , ] . increased pai- levels might be of clinical utility in challenging clinical situations in patients with hyperbilirubinemia occurring after hsct. it forms one of the therapeutic targets for defibrotide, which reduces circulating pai- levels along with other actions. other endothelial markers, like intercellular adhesion molecule- (icam- ), e-selectin, von willebrand factor (vwf), and thrombomodulin may also be helpful in early identification of patients at risk of sos who may benefit from early introduction to therapies [ ] . diagnosis of sos is based on constellation of signs and symptoms and serum bilirubin levels. hepatic sos is clinically characterized by jaundice caused mainly by conjugated hyperbilirubinemia, tender hepatomegaly, fluid accumulation manifested as rapid weight gain and ascites [ ] . most commonly used diagnostic criteria for sos includes the seattle criteria [ ] , the modified seattle criteria [ ] , and the baltimore criteria [ ] . because of its high incidence and mortality, prophylaxis for hepatic sos is widely practiced, using different regimens in different centres. when hepatic sos is established, specific therapy is usually given in addition to general supportive care, especially in moderate or severe cases. hepatic sos is a formidable challenge both for patients undergoing stem cell transplantation and for their physicians. the second pathology in this child which significantly contributed to his downhill course was busulfan induced lung injury. intriguingly, in the present clinical setting, busulfan induced lung injury remains an diagnosis of exclusion, particularly with respect to considering usual and atypical infections. its clinical presentation includes a spectrum ranging from acute, rapidly progressive respiratory distress to chronic, interstitial lung disease with insidious onset [ , ] . the pathophysiology of drug-induced lung injury is not fully understood but direct toxicity of the drug to parenchymal cells, cell-mediated immune reactions and release of cytokines are believed to contribute to the lung injury. the pathologic findings consist of mainly diffuse interstitial pneumonitis, organizing alveolitis and cellular atypia within type ii pneumocytes. the injury pattern with busulfan is diffuse alveolar damage (dad) either in acute exudative phase with alveolar and interstitial oedema and hyaline membranes; or late reparative phase, which is characterized by proliferation of type ii pneumocytes and interstitial fibrosis [ ] . marked atypia of the type ii pneumocytes is a morphological clue in favour of busulfan induced lung injury in contrast to organizing bacterial pneumonia. moreover, pcr for cmv is helpful in excluding viral pneumonia. the prevalence of drug-induced pulmonary toxicity is increasing, and more than drugs are now known to cause lung injury. because this lung injury can be progressive and fatal, early recognition is important. the diagnosis of pulmonary drug toxicity should be considered in any patient with a history of drug therapy who presents with new or progressive respiratory complaints. the superadded fungal ulcer which developed preterminally with extensive dissemination to heart causing mural endocarditis and myocardial abscess eventually led to the demise of the child. hepatic sos contributes considerably to transplantation-related morbidity and mortality. recognition of this disease in the post-transplantation setting remains a challenge in the absence of specific diagnostic features as many other more common conditions can mimic it. a high index of suspicion is needed to identify patients with sos. while hepatic sos and busulfan induced lung injury are commonly reported as isolated findings following autologous sct, the coexistence of these are extremely rare and have not been documented in the literature thus far. the present case adds observational data to the existing literature and highlights the importance of keeping high index of suspicion for these two entities in patients following hsct, and early institution of effective therapy. veno-occlusive disease of the liver and multiorgan failure after bone marrow transplantation: a cohort study of patients serum galactomannan assay for the diagnosis of invasive aspergillosis in children with haematological malignancies sinusoidal obstruction syndrome (hepatic veno-occlusivedisease) hepatic veno-occlusive disease (sinusoidal obstruction syndrome) after hematopoietic stem cell transplantation sinusoidal obstruction syndrome vascular disorders of the liver. american association for the study liver diseases incidence and outcome of hepatic venoocclusive disease after blood or marrow transplantation: a prospective cohort study of the european group for blood and marrow transplantation. european group for blood and marrow transplantation chronic leukemia working party the relevance of plasminogen activator inhibitor (pai- ) as a marker for the diagnosis of hepatic veno-occlusive disease in patients after bone marrow transplantation endothelial dysfunction after bone marrow transplantation: increase of soluble thrombomodulin and pai- in patients with multiple transplant-related complications prediction of veno-occlusive disease using biomarkers of endothelial injury venocclusive disease of the liver after bone marrow transplantation: diagnosis, incidence, and predisposing factors venoocclusive disease of the liver following bone marrow transplantation busulphan lung in childhood lung function yrs after allogeneic bone marrow transplantation conditioned with busulphan and cyclophosphamide interstitial pneumopathies caused by busulfan. histologic, developmental and bronchoalveolar lavage analysis of cases key: cord- -reyx zo authors: vélez, juan; lange, malin k.; zieger, peter; yoon, ilkyu; failing, klaus; bauer, christian title: long-term use of yeast fermentation products in comparison to halofuginone for the control of cryptosporidiosis in neonatal calves date: - - journal: vet parasitol doi: . /j.vetpar. . . sha: doc_id: cord_uid: reyx zo the objective of this study was to compare the effect of non-gmo saccharomyces cerevisiae fermentation products (scfp) with that of a halofuginone treatment against cryptosporidium parvum infection in pre-weaned calves on a commercial dairy farm. a total of neonatal female calves, housed in individual hutches, were enrolled sequentially based on date of birth in blocks of animals each. calves within each block were allocated to one of treatments: remaining untreated, fed with scfp (diamond v smartcare(®) at g/d in milk and nutritek(®) at g/d in starter grain) for the first days of life, or treated with halofuginone ( . mg/kg/d) for the first days of life. fecal samples collected on days – post-partum were examined for both cryptosporidium oocysts and coproantigen. the presence and intensity of diarrhea were monitored by scoring daily for the first weeks of life. calves were weighed at , , and days of age. almost all calves were cryptosporidium-positive at least once during the study. halofuginone significantly reduced the number of cryptosporidium-positive fecal samples as compared to the two other groups. based on the coproantigen scores, both halofuginone and scfp feeding significantly reduced the intensity of cryptosporidium infection as compared to the untreated group. diarrhea was recorded in almost all calves at least once. neither the proportion of diarrheic calves nor the intensity and duration of diarrhea differed among the treatment groups significantly. the mean daily weight gain during the first weeks of life was significantly lower in halofuginone treated calves than in both other groups; however, at the end of the study period the total weight gain did not significantly differ among the treatment groups. in conclusion, the clinical results and weight gains of pre-weaning supplementation with the scfp were neither better nor worse than the -day halofuginone treatment suggesting that the scfp feeding may be from the clinical point of view a natural alternative measure, instead of halofuginone treatment, in bovine cryptosporidiosis. cryptosporidium species are minute protozoan pathogens of vertebrates (mammals including man, birds, reptiles, fish and amphibians) with a worldwide occurrence (fayer, ; deplazes et al., ) . they belong to the phylum apicomplexa and have been recently reclassified from coccidian to gregarine parasites (cavalier-smith, ; thompson et al., ) . more than cryptosporidium spp. are known at present, the genotype b ('bovine type') of the c. parvum complex being a major enteropathogen in neonatal calves (broglia et al., ; feng et al., ; holzhausen et al., ) . cryptosporidium spp. have a direct life cycle. in brief, infected hosts shed infectious oocysts with their feces, and new hosts become infected by oral ingestion of oocysts as host-tohost route, from their environment or via oocyst-contaminated drinking water or food. in mammals, cryptosporidium spp. primarily infect the intestinal epithelium parasitizing the microvillous brush border of enterocytes. the endogenous life cycle includes a first asexual multiplication (merogony) followed by a sexual development (gamogony) and a second asexual multiplication (sporogony) resulting in huge quantities of infectious oocysts which are shed with feces. oocysts can remain viable and infectious in the environment for months (fayer, ; deplazes et al., ) . cryptosporidium infections are very common and endemic in many cattle farms worldwide, especially in large dairy enterprises (joachim et al., ; trotz-williams et al., ; al mawly et al., ; delafosse et al., ; niine et al., ; urie et al., a, b) . c. parvum is one of the four main causes of diarrhea in calves within the first weeks of life (gillhuber et al., ; al mawly et al., ; anonymous, ) . calves become infected within the first day(s) of life. the infection may cause no clinical symptoms, but is often responsible for a watery diarrhea with acute onset, which can be accompanied by depression, weakness and anorexia. the disease results from a severe villous atrophy in the small intestines. the cryptosporidium-caused diarrhea generally lasts several days and is self-limiting because of the development of immunity (thomson et al., ) . cryptosporidium spp. are more or less completely insensitive to usual anticoccidial drugs, which are effective against eimeria spp. and other coccidian parasites in animals (stockdale et al., ; shahiduzzaman and daugschies, ) , possibly because of their gregarine character. there are two compounds used in the field that were found to have an anticryptosporidial effect in calves after oral administration for several days (treatment starting from the first day of life): halofuginone (silverlås et al., ; de waele et al., ; trotz-williams et al., ; almawly et al., ; niine et al., ) and paromomycin (fayer and ellis, ; grinberg et al., ) . halofuginone is the only drug approved against cryptosporidium for use in neonatal calves by the authorities in europe (anonymous, ) . however, in a meta-analysis of results from respective studies, it was concluded that "some beneficial effects were found for the use of halofuginone as prophylactic treatment against cryptosporidiosis, but the substance is toxic and should be used only with severe c. parvum-associated diarrhoeal problems" (silverlås et al., ) . in recent studies halofuginone given prophylactically resulted in reduced oocyst shedding, but did not improve clinical symptoms or bodyweight gain of calves as compared to untreated controls (trotz-williams et al., ; almawly et al., ) , or was even negatively associated with the weight gain (niine et al., ) . it was reported recently that -week feeding of neonatal cryptosporidium-infected calves with saccharomyces cerevisiae fermentation products resulted in significantly less fragmented and atrophied villi of the lower small intestines in comparison to untreated controls, suggesting a preventive effect of these products against the infection (vázquez flores et al., ) . therefore, the present longitudinal (cohort) study was performed to determine the parasitological, clinical and economical effects of s. cerevisiae fermentation products against cryptosporidium infections in neonatal calves in comparison with both a 'positive' (halofuginone treated) and 'negative' (untreated) control group. the study was performed on a commercial dairy cow farm with approximately cows in eastern germany from december to april . the farm was known to harbor cryptosporidium infection endemically, and halofuginone had been prophylactically used in the past. a total of female calves, all of holstein frisian breed and being healthy on the day of birth (respective study day ), was enrolled in the study. feeding and management practices were followed by the routine procedures of the farm. calves were immediately separated from their dams after birth, identified by numbered ear tags and housed outdoor in individual hutches on concrete floor supplied with straw bedding for the first weeks of life. each calf was given approximately l colostrum from its dam within the first h after birth. thereafter, they were fed with whole waste milk twice a day and, from the nd week of life, additional grain was offered for ad libitum consumption. the calves were from primiparous, secundiparous and multiparous cows ( , , and cows with , , or lactations, respectively). secundi-and multiparous dams had been vaccinated against rotavirus and coronavirus infection before birth of their calves, but primiparous dams did not get this vaccination following the farm practices. two s.cerevisiae fermentation-based non-gmo feed additives (scfp), smartcare ® and nutritek ® , were supplied by diamond v. both products were stored at room temperature and given according to the manufacturer's instructions: g smartcare/calf/day was mixed in milk and given orally, and g nutritek/calf/day were mixed with a small amount of starter feed and offered directly for feeding, both at the same time each day on days - . halofuginone (halo; halocur ® . mg/ml, msd tiergesundheit, germany) was given according to the manufacturer's instructions: calves of - kg and - kg birth weight received ml and ml halocur, respectively; lighter or heavier calves received ml halocur/ kg (corresponding to a dose rate of . mg halofuginone/kg). the drug was orally administered using the product´s dispenser once daily after morning feeding on days - . the study calves were sequentially enrolled in blocks of animals based on their date of birth, and calves within each block were allocated to one of treatments: ) remaining untreated (con); ) fed with scfp; or ) treated with halo. after allocation any calf suffering from a disease other than diarrhea during its first weeks of life as well as both calves in the same block were removed from the study and replaced by a new block of calves. the calves were weighed using a scale on the day of birth as well as on days , and . each calf was examined daily by inspection for any clinical symptoms in the morning throughout the study period. blood samples were collected from each calf by puncture of the vena jugularis on day ; serum was isolated by centrifugation and stored at − °c until examination. a total of fecal samples were rectally collected from each calf (in the morning of days - , , , , , and ), labeled and kept at + °c until examination. the consistency of fecal samples was estimated by scoring (solid = score , semi-liquid = , and watery = ; joachim et al., ) . two of the four calves that died during the study were necropsied by an external laboratory. to determine the efficacy of passive transfer of immunity serum total protein concentration (g/l) was estimated using a hand refractometer (weaver et al., ) . the negative carbol-fuchsin fecal smear staining method was used to detect cryptosporidium oocysts (heine, ; potters and van esbroeck, ) in fecal samples of days - . the intensity of oocyst shedding was estimated by scoring (scores - ) according to castro-hermida et al. ( ) . for the detection of cryptosporidium spp. coproantigen, a commercially available immunochromatographic in-situ rapid diagnostic kit (smartstrips ™ cryptosporidium-bio k in combination with smartstrips ™ app; both: biox diagnostics, rochefort, belgium) was used according to the manufacturer's instructions. this test allows a semiquantitative estimation of the intensity of cryptosporidium infection by scoring (scores - ). for the molecular characterization of the cryptosporidium species and c. parvum genotype present on the farm, dna was isolated from oocyst-positive fecal samples of different calves using qiaamp dna stool kit (qiagen gmbh, hilden, germany) according to the manu-facturer´s protocol. the small-subunit ribosomal rna (ssu rrna) gene was partially amplified by pcr, and restriction fragment length polymorphism (rflp) analysis was performed as described by xiao et al. ( ) . to identify the c. parvum subtype, dna was isolated, and a bp-fragment of the glycoprotein (gp ) gene was amplified by nested pcr using primers al and al in the first pcr and al and lx in the second pcr as described by sulaiman et al. ( ) . the amplicons were purified and directly sequenced using the primers of the second pcr through an external service provider (lgc genomics gmbh, berlin, germany). the gp sequences obtained were assembled and a blast search (https://blast.ncbi.nlm.nih.gov/blast. cgi) of the genbank database was performed. subtypes of c. parvum were designated according to the nomenclature described by sulaiman et al. ( ) , i. e., the number of the tandem trinucleotide repeats tca (a), tcg (g), and tct (t) coding for the amino acid serine and the number of the repeat acatca (r) immediately after the nucleotide triplets. for the qualitative detection of rotavirus, coronavirus and/or e. coli k coproantigens in all fecal samples of days - , a commercially available immunochromatographic rapid diagnostic kit (rainbow calf scours-bio k ; biox diagnostics, rochefort, belgium) was used according to the manufacturer's instructions. on day , a fecal sample of each calf was qualitatively examined for eimeria spp., giardia sp. and any other parasite stages, e.g., entamoeba cysts and strongyloides eggs, using the modified formalinether sedimentation technique (young et al., ) . additionally for giardia spp. coproantigen, a commercially available qualitative enzyme immunoassay (prospect ™ giardia microplate assay, virotech diagnostics gmbh, rüsselsheim, germany) was used according to the manufacturer's instructions. statistical analyses were performed using the statistical program packages bmdp (dixon, ) and bias (ackermann, ) . for the description of parameters the respective arithmetic mean, standard deviation, minimum and maximum were calculated. differences of the detection of any pathogen between the treatment groups were analyzed for significance using chi -test (dichotomous or nominal scaled variables) or kruskal-wallis test with bonferroni corrected dunn's posttest (ordinal scaled parameters). to get a global basis for evaluation the intensity of the cryptosporidium infection was estimated using the 'area under the curve' (auc) of both the oocyst shedding and coproantigen scores over the time. in a first step, the auc of the respective scores was calculated for each calf during days - . in a second step, a statistical comparison was done for the individual auc values using the one-way analysis of covariance with repeated measures with respect to the treatment group, having the grouped number of parturitions of cows as a covariate; in case of an overall significant difference, a pairwise comparison of treatments followed using the student-newman-keulstest. in addition to the auc analysis for oocyst and coproantigen scores, a two-way analysis of covariance with repeated measures with respect to the treatment group and day of observation, again having the grouped number of parturitions of cows as covariate was done (because the scores were not metrically scaled and normally distributed, the results must be seen as exploratory ones). the aim of this additional comparison was to examine the time effect. correlation analyses were done to analyze the relationship between the number of days with diarrhea and serum total protein concentrations, as well as auc values of cryptosporidium oocyst shedding or coproantigen scores. kappa statistic was calculated to assess the agreement between the results of the two methods used for the detection of cryptosporidium infection. additionally, the correlation of scoring results of both these methods was evaluated using spearman's rank correlation coefficient (r s ) with respect to the scale of the parameters. all the statistical tests were twotailed, and differences or effects with p-values ≤ . were considered as significant. the study calves were born from primiparous, secundiparous or multiparous cows. the average lactation number of cows was . ± . and did not differ significantly among the treatment groups (p = . ). the serum total protein concentration on day reached or exceeded the value of g/l in calves; in and calves it was g/l and g/l, respectively. it was not influenced by the number of parturitions of dams and did not differ significantly among the treatment groups (con: . ± . g/l; scfp: . ± . g/l; halo: . ± . g/l). the cryptosporidium species isolated from different calf fecal samples was identified by pcr-rflp analysis as c. parvum, genotype b, and based on the gp gene the isolates belonged to subtype iiaa g r . for all fecal samples examined the results (positive vs. negative) of the coproantigen immunochromatography substantially agreed with those of the carbol-fuchsin fecal smear staining (kappa value: . ; % confidence interval: . - . ). the relative sensitivity and relative specificity of the staining method was . % ( . - . %) and . % ( . - . %), respectively, employing the immunochromatography as relative gold standard. the scoring results of both methods were highly correlated (r s = . ; p < . ). almost all study calves, regardless of the treatment group, shed cryptosporidium oocysts ( . %) and were positive for coproantigen ( . %) at least once during their first weeks of life (table ). in total, ( . %) and ( . %) of the fecal samples examined on days - were positive for oocysts or coproantigen, respectively. the number of oocyst-and coproantigen-positive fecal samples in the treatment groups differed significantly (p = . and p = . , respectively): it was significantly lower in halo treated calves than in those of the con (p = . and p = . , respectively) and scfp fed calves (p = . and p = . , respectively). there was no significant difference between con and scfp (table ) . cryptosporidium oocysts and coproantigen were first detected in feces on day and , respectively. there was a trend of a later onset of oocyst shedding and coproantigen positivity in halo treated calves than in those of the other treatment groups ( table ). the individual duration of oocyst shedding or coproantigen positivity ranged from to days and to days, respectively, without significant differences among the treatment groups (table ) . the auc values of both the oocyst shedding and coproantigen scores over time were used to estimate the intensity of the cryptosporidium infection. the overall comparison of both auc values showed a significant difference among the treatment groups (oocyst shedding: p = . ; coproantigen: p = . ). in the subsequent pairwise comparison, the auc values of oocyst shedding scores were significantly lower in halo treated calves ( . times, p < . ) than in con ones, and the auc values of coproantigen scores were significantly lower in both halo treated ( . times, p < . ) and scfp fed calves ( . times, p < . ) than in con ones ( table ) . the two-way analysis of covariance did not show any significant effect of the age of cows (first parturition vs. second parturition, or first parturition vs. > parturitions) on the cryptosporidium oocyst shedding scores. however, the mean oocyst shedding score was significantly associated with the treatment (p < . ), and time (p < . ), and there was a significant treatment by time interaction (p < . ). this implies that the overall average score was different among the treatment groups, and the scores varied as calves aged. the score curves of con and scfp were similar and started earlier and ran higher than that of halo (fig. ) , however, overall means of auc between scfp and halo were similar (table ) . very similar results were obtained for coproantigen scores (data not shown). there was no statistically significant correlation between the auc values of cryptosporidium oocyst shedding scores (r = . , p = . ) or coproantigen scores (r = . , p = . ) of each calf and the serum total protein concentration measured on day . rotavirus, coronavirus or e.coli coproantigen was found at least once in . %, . % and . %, respectively, of calves and in . %, . % and . %, respectively, of the fecal samples examined on days - . there was no significant difference among the treatment groups (table ) . values of a parameter with the same superscript in a column are not significantly different (p > . . day and were the day of birth and last day of fecal examination, respectively. arithmetic mean (standard deviation). minimum-maximum. values with different superscripts in a column are significantly different (p < . and p < . for oocysts and coproantigen, respectively). arithmetic mean (standard deviation). four calves having diarrhea died during the study period ( . % mortality): one calf from each treatment groups con and scfp (on day and day, respectively), and two halo treated calves (on days and ). the scfp calf and one of the halo calves were post-mortem examined by an external laboratory; the pathological diagnosis was 'bacteria-and/or virus-caused enteritis' and 'candida-caused enteritis', respectively. diarrhea (diagnosed if a sample had not solid consistency) occurred in ( . %) of the calves at least once during days - . in total, . % of all fecal samples showed a semi-liquid or watery consistency, and the treatment groups did not differ significantly. diarrhea lasted for . - . days on average (table ). the majority of fecal samples with altered consistency was seen between days and (fig. ) . the number of days with diarrhea was not significantly correlated with its serum total protein concentration on day (r = . , p = . ). the auc values of the number of days with diarrhea did not differ significantly among the treatment groups (table ) . the intensity of cryptosporidium infection measured by the individual mean auc value of coproantigen scores was significantly correlated with the number of days with diarrhea during days - (r = . , p = . ): the higher the auc values the more days with diarrhea were observed in mean. for oocyst shedding this correlation was not statistically significant (r = . , p = . ). the individual birth weight of the calves ranged from . to . kg, but the mean values did not differ significantly (p = . ) among the treatment groups (table ). there was a highly significant group difference of the daily weight gain during the first weeks of life (p = . ): halo treated calves had a significantly lower average daily gain (adg) than scfp fed (p = . ) and con calves (p = . ). there was no significant difference between scfp and con calves. however, no significant difference in adg was observed among the treatment groups for the rest of the study period. at the end of the -week study period the individual total weight gain ranged from . to . kg, and the adg was ± g; it was slightly but not significantly lower in halo treated calves than in calves of the other treatment groups (table ) . on day , ( . %) of the calves examined were positive for eimeria (mainly e. bovis) oocysts, ( . %) for giardia (cysts or coproantigen) and ( . %) for entamoeba cysts without any significant differences among the treatment groups. no stages of other parasites were found. values of respective coproantigen with the same superscript in a column are not significantly different (p > . ). number of positive calves or samples/total number of calves or samples. fig. . course of mean fecal consistency scores (score : solid; : semi-liquid; : watery) of calves treated with halofuginone on days - (halo), daily fed with s. cerevisiae fermentation products on days - (scfp) or remaining untreated (con). currently, halofuginone is the only drug approved by the authorities in europe for the use in newborn calves against cryptosporidium infection. however, its prophylactic effect against cryptosporidiosis is equivocal (silverlås et al., ) . therefore, there is a demand for alternatives. the results of an experimental study had suggested a reduction of histopathological alterations in the small intestines caused by cryptosporidium in calves when scfp had been fed (vázquez flores et al., ) .the present longitudinal study was performed to evaluate, for the first time under field conditions, the effects of a long-term feeding with two non-gmo scfp against cryptosporidium infection in neonatal calves in comparison with both a prophylactically halo treated and an untreated group. as main results, the halo treatment showed a partial anticryptosporidial activity but not a significant clinical effect. the same findings were recorded for scfp feeding, which was, in other words, neither better nor worse than the halo treatment. the calves enrolled in the study had a very similar history: all were females, from the same herd, of the same breed, kept under same management conditions and received the same basic feed. the age of their dams did not differ significantly among the treatment groups. therefore, there was no impact of the age of dams on the serum total protein concentration, the intensity of cryptosporidium infection (estimated by auc values of oocyst shedding or coproantigen scores) nor the intensity of diarrhea (estimated by auc values of the number of days with diarrhea) of the respective calves. in almost all calves the serum total protein concentration reached or exceeded the value of g/l on day , regardless of the treatment group. this indicates a sufficient transfer of immunoglobulins by colostrum to calves (weaver et al., ) , which is principally necessary to protect calves from morbidity (urie et al., c) . it was, therefore, not surprising that the serum total protein concentration was not correlated with the intensity of cryptosporidium infection (measured by auc values of oocyst shedding or coproantigen scores) or the number of days with diarrhea in this study. a cryptosporidium infection was diagnosed in almost all calves in all treatment groups. in untreated control calves nearly half the fecal samples collected during the first weeks of life were positive for oocysts and coproantigen. in contrast, co-infections with other enteropathogens (rotavirus, coronavirus and e. coli) were detected in a smaller number of calves, and most of them were positive on one day only, resulting in only few positive fecal samples. this is in accordance with results from previous studies (e.g., gillhuber et al., ; al mawly et al., ; anonymous, ) . therefore, cryptosporidium was considered to be the main enteropathogen and responsible for the episodes of diarrhea in the present study. in untreated calves cryptosporidium oocyst shedding started . days after birth on average, which is in accordance with previous field observations (e.g., castro-hermida et al., ; niine et al., ; urie et al., a) . it is also similar to data from experimental infections (zambriski et al., ) showing that the study calves became infected shortly after birth. the species was identified as c. parvum genotype b, subtype iiaa g r that is commonly found in neonatal calves in germany (broglia et al., ; holzhausen et al., ) and elsewhere (feng et al., ) . the sensitivity of the direct fecal smear staining was lower compared with a coproantigen detection method used as gold standard, a finding already reported previously (e.g., chartier et al., ) . however, the results of both methods substantially agreed indicating that the fecal smear staining can be used as a simple, fast and inexpensive method for the cryptosporidium diagnosis, at least in endemically infected cattle farms. the -day prophylactic halo treatment neither prevented the cryptosporidium infection nor reduced the number of cryptosporidiumpositive calves. however, halo treatment resulted in a significant reduction of the number of cryptosporidium-positive fecal samples as compared with both the con and scfp fed calves. there was also a trend, being not significant, for a later onset of oocyst and coproantigen positivity in halo treated calves than in the two other groups. finally, the intensity of the cryptosporidium infection (estimated by auc values of oocyst shedding or coproantigen scores) was significantly reduced in the halo group in comparison with con but not with scfp. all in all, these results indicate some but not fully prophylactic anticryptosporidial effect of halo, as already observed in previous studies (e.g., silverlås et al., ; trotz-williams et al., ; delafosse et al., ; niine et al., ) . daily feeding with the two scfp for weeks reduced neither the number of cryptosporidium-positive calves nor the number of positive fecal samples. it did also not delay the onset or shorten the duration of oocyst and coproantigen positivity in comparison to con. however, it was interesting to note that the intensity of the infection estimated by auc values of coproantigen scores (but not of oocyst shedding scores) was significantly lower in scfp fed calves than in con ones. this finding may indicate some, although partial effect of the scfp on the endogenous multiplication of cryptosporidium, possibly resulting from a modified intestinal environment. assuming this is correct, a reduced endogenous multiplication of the parasite would result in less pathological alterations in the intestinal mucosa corresponding with the histological findings by vázquez flores et al. ( ) , albeit obviously without clinical improvement. in this context it is of interest to mention that scfp had been shown to have a positive effect on the intestinal development of preweaned calves (xiao et al., ) and a positive immunomodulatory effect in animals (park, ; chou et al., ) . of course, it warrants to further investigate the mechanism of action of scfp. in the present study relatively low daily weight gains, ranging from to g in group average, were recorded at the end of the -week period. this may be a negative consequence of the diarrheal episodes during the first several weeks of life in more or less all calves. the number of diarrheal days was significantly correlated with the intensity of the cryptosporidium infection indicating that the parasite was responsible for diarrhea in the present study. in contrast, a considerably higher adg ( g) at an average weaning age of days was reported for dairy calves in a large-scale us study; however, here clinical signs had been seen in only % of the animals, including % with digestive signs (urie et al., b) . although the halo treatment showed a partial anticryptosporidial activity, it had no clinical effect in the present study: it did not significantly affect the onset, nor the frequency, duration and intensity of diarrhea, the main clinical symptom of cryptosporidiosis. these results are consistent with observations in previous studies reporting a nonsignificant association between its prophylactic use and the incidence, duration or intensity of diarrhea (silverlås et al., ; trotz-williams et al., ; almawly et al., ; meganck et al., ) . the same findings were recorded for scfp feeding, which was, in other words, neither better nor worse than the halo treatment. a non-significant difference of weight gains between halo treated and con calves had been reported by others (trotz-williams et al., ) , and in a more recent study the halo treatment even had a negative effect on the daily weight gain after months (niine et al., ) . it should be stressed that in the present study the -day halo treatment clearly resulted in a negative impact on the weight gain during the first weeks of life as compared scfp or con calves. this temporarily insufficient weight development can be explained by the known toxicity of halo that can induce reversible gastro-intestinal inflammatory or necrotic lesions also at therapeutic doses (anonymous, ) . some calves from each treatment group were positive for eimeria spp. oocysts, giardia and/or entamoeba cysts at weeks of age. patent eimeria infections are occasionally seen at an age of weeks but still considered clinically inapparent (e.g., faber et al., ) . severe eimeria infection are first observed in older, group-housed calves (niine et al., ) and may then result in morbidity (bangoura et al., ) . giardia spp. infections are very common in cattle farms worldwide. calves usually become infected before the th week of life, and prevalence peaks were reported in -to -month old animals (geurden et al., (geurden et al., gillhuber et al., ; niine et al., ; urie et al., a) . giardia spp. seem to be able to induce diarrhea (gillhuber et al., ) and may reduce weight gain (urie et al., a) mainly in calves older than one month. entamoeba bovis infections are not uncommon in cattle but are considered less pathogenic (matsubayashi et al., ) . a pre-weaning supplementation with the two s. cerevisiae fermentation feed additives showed very similar clinical results and weight gains in cryptosporidium infected newborn calves as a -day halofuginone treatment under the conditions on this farm. this suggests that feeding with these s. cerevisiae fermentation products may be from the clinical point of view a natural alternative measure, instead of halofuginone treatment, in bovine cryptosporidiosis. this study was funded through a grant by diamond v, cedar rapids, iowa, usa. pz and iy are current employees of diamond v. bias für windows, version prevalence of 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fermentation products on dairy calves: ruminal fermentation, gastrointestinal morphology, and microbial community ethyl acetate as a substitute for diethyl ether in the formalin-ether sedimentation technique description of fecal shedding of cryptosporidium parvum oocysts in experimentally challenged dairy calves we gratefully acknowledge the owner of the dairy farm for allowing us to access to the facilities and to perform this study. we also thank the farm personnel for their helpful collaboration as well as dr. jörg hirzmann for his help and advice on the molecular characterization of the cryptosporidium species. key: cord- -xhpv y authors: land, kevin j.; boeras, debrah i.; chen, xiang-sheng; ramsay, andrew r.; peeling, rosanna w. title: reassured diagnostics to inform disease control strategies, strengthen health systems and improve patient outcomes date: - - journal: nat microbiol doi: . /s - - - sha: doc_id: cord_uid: xhpv y lack of access to quality diagnostics remains a major contributor to health burden in resource-limited settings. it has been more than years since assured (affordable, sensitive, specific, user-friendly, rapid, equipment-free, delivered) was coined to describe the ideal test to meet the needs of the developing world. since its initial publication, technological innovations have led to the development of diagnostics that address the assured criteria, but challenges remain. from this perspective, we assess factors contributing to the success and failure of assured diagnostics, lessons learnt in the implementation of assured tests over the past decade, and highlight additional conditions that should be considered in addressing point-of-care needs. with rapid advances in digital technology and mobile health (m-health), future diagnostics should incorporate these elements to give us reassured diagnostic systems that can inform disease control strategies in real-time, strengthen the efficiency of health care systems and improve patient outcomes. or in sequence. in case of discrepancies, a third test is used as a tiebreaker. an example of this approach is for hiv case detection. specificity. diagnostics should have low false positive rates. the ideal scenario is where the sensitivity and specificity achieved from the diagnostics used at the poc approach those of laboratory-based assays wherever possible. however, a lower specificity can be tolerated if the harm of overtreatment is much less critical than missing the diagnosis of an infection. screening syphilis during pregnancy is an example of this approach, as missing maternal syphilis can lead to stillbirths, preterm birth and congenital syphilis in a third of pregnant women compared to the harm of overtreatment with a single dose of penicillin . user friendliness. tests should be easy to perform in - steps and require minimal user training with no prior knowledge of diagnostic testing. typically, results should be available in - minutes after sample collection and enable patient management and treatment during the same visit. the benefits of a rapid test versus a more accurate test that requires patients to return for results have been previously demonstrated . robustness refers to the ability of the test to withstand the supply chain (temperature, humidity, time delays, mechanical stresses) without requiring additional (and often costly) transport and storage conditions (for example, refrigeration). ideally the test does not require any special equipment or can be operated in small portable devices that use solar or battery power. deliverable to end-users. delivery refers to the organizational structures and relationships established with the purpose of coordinating and steering the logistics of selecting, procuring, shipping, storing, distributing and delivering a new health technology to ensure it reaches the end-users in resource-constrained settings . compared to when assured was proposed, access to laboratories in resource-limited settings has improved dramatically both in numbers and in quality , , especially in areas such as hiv and malaria testing, cd counting and tb. however, there remains a critical need for a wider range of diagnostics that can be performed at the poc. from this perspective, we undertake a review of the assured benchmark over the last years, assess the factors contributing to the success and failure of the assured diagnostics over the past decade, identify areas that have remained difficult to address as well as lessons learned from implementation in resourcelimited settings, and highlight additional conditions that should be considered in addressing poc needs. since , several diagnostic tests that satisfy (or nearly satisfy) the assured criteria have been developed to identify major human pathogens. this includes tests for hiv, malaria, syphilis and tb ( table ) . the hiv rapid test is the first test to fulfil the assured criteria, followed closely by the malaria and syphilis rapid tests and, recently, a near-poc test for tb. much of this development was the result of advocacy by donors such as the bill & melinda gates hiv rdts. early detection of hiv infection has been a critical component of hiv control programmes worldwide since the early days of the epidemic. since most infected individuals do not show specific symptoms and the period of viraemia is short, screening for hiv antibodies in blood as a marker of exposure has been the most cost-effective means of identifying infected individuals. the who developed a process and criteria for validating the performance of hiv antibody detection assays more than a decade ago and showed that hiv rdts using finger-pricked whole-blood specimens has acceptable performance compared to laboratory-based immunoassays , . many donors -for example, global fund and implementation partners, including the who, unaids and pepfar -have helped affected countries with the procurement and introduction of rapid hiv tests and enable early detection and prevent onward transmission. as a result of funding from donors, increased accessibility of rdts, and the 'architecture' provided by national hiv/ aids control programmes, hiv tests successfully reached as many as million children and adults in low-and middle-income countries in (ref. ) . two major challenges remain: training and quality assurance. with thousands of poc testing sites across a country, even the most well-organized control programmes would find it difficult to provide adequate training on an ongoing basis. this problem is most acute in health facilities where there is high turnover of staff. the us centres for disease control and prevention (cdc) has developed a convenient means of external quality assurance by working with countries to generate thermally stable proficiency panels that can be shipped to every poc testing site to ensure competence of testing personnel. however, a study in south africa found that only % of hiv rdts were performed correctly . although not every error will result in an incorrect diagnosis, the alarming reality is that with million tests being performed annually worldwide, assuming a % accuracy rate, as many as . million incorrect results per year could potentially be generated. ongoing quality assurance efforts include developing key policy and quality documents for the implementation of hiv-related poc testing . for example, as poc technologies for hiv viral load and early infant diagnosis were being developed, there was tremendous emphasis on quality, given the complexity of the test and lessons learned from hiv rdts. malaria is estimated to be the cause of at least a million deaths a year worldwide, most of which are in sub-saharan africa. while microscopic identification of parasites in blood smears has been the traditional means of diagnosing malaria in patients presenting with fever, microscopy requires equipment, a source of electricity and trained laboratory technicians. malaria rdts were developed as many rural communities lack these resources, and to date there are over brands of malaria rdts made by approximately companies , which vary widely in performance, manufacturing quality and price. the who has set up a pre-qualification programme with the cdc and the foundation for innovative new diagnostics to evaluate these tests to inform test selection and procurement for national malaria control programmes . in countries that permit the sale of malaria rdts and medicines over the counter, the quality of these commodities cannot be guaranteed. the price of most rdt brands is between us$ . and us$ . per test , , and as with all diagnostics, it is important to caution that price pressure will ultimately affect quality. as with hiv, with the support of donors, selection of high-quality rdts and the architecture provided by national malaria control programmes, malaria rdts have reached millions of patients every year. this promising trend, coupled with the effectiveness of bednets for preventing transmission, has led to the call for malaria elimination in many parts of the world . however, challenges remain for the future of malaria poc testing and the global elimination of malaria. first, highly accurate tests are required for all malaria species affecting humans, but these panspecific tests are usually about % more expensive than rdts that only detect plasmodium falciparum. most p. falciparum rdts detect a malarial antigen, histidine-rich protein (hrp) , and the discovery of parasites that have a deletion in this gene has raised concerns about false negative results and ongoing transmission. also, the problems of providing adequate training and quality assurance of malaria tests and testing at remote poc sites are similar to those described for hiv rdts , . in the near future, as countries progress towards malaria elimination, funding for rdts may become an issue. as the intensity of transmission decreases due to lower parasite density in infected individuals, more sensitive tests will be needed, which can only be achieved with costlier amplification steps or with ultrasensitive platforms for antigen detection. also, decreasing numbers of cases mean that malaria tests are no longer cost-effective, and thus it is more difficult to justify funding in light of multiple competing priorities for limited health budgets. syphilis. syphilis, caused by the spirochete treponema pallidum, has a long latent period during which patients have no symptoms, but can remain infectious. syphilis in pregnancy can lead to adverse outcomes of stillbirths and miscarriage, and babies born with congenital syphilis in the developing world only have a % chance of survival during the first years of life , . despite the availability of simple diagnostic tests for antenatal screening and the effectiveness of treatment with a single dose of long-acting penicillin, syphilis is re-emerging as a global public health problem . it is estimated that , babies die each year as a result of syphilis-associated stillbirths and congenital syphilis, largely because of lack of access to antenatal screening , . rdts exist for detecting syphilis and are reported to have acceptable performance , and operational characteristics. the introduction of rdts was also acceptable to patients and health care providers and was shown to contribute to the improvement of antenatal care in low-resource settings . despite all this, syphilis rdts have not had the same success as hiv and malaria rdts. the main reasons for this are the lack of advocacy and political will to translate the evidence to national policy, lack of funding to make the tests affordable to those in need and the lack of a national control programmes to provide the architecture needed to coordinate all the different aspects of testing. ensuring adequate training for health care workers and supplies of commodities were cited as key implementation barriers in africa , . dual hiv and syphilis rdt testing in countries was prioritized by the who for the elimination of mother-to-child transmission (mtct) of hiv and syphilis by , , but a recent review showed that while prevention of mother to child transmission (pmtct) programmes for hiv have resulted in a dramatic decrease in the number of hiv-positive infants in sub-saharan africa, the rate of syphilis screening of pregnant women in the same countries has remained at unacceptably low levels of approximately % (ref. ). this is due largely to disparity in funding and lack of political will despite the global fund allowing countries to purchase syphilis rdts with hiv rdts since (ref. ). countries need to harness the architecture provided by hiv pmtct programmes to screen women for both infections using a single drop of blood in a single visit to a health care facility. dual rapid hiv-syphilis rdts with acceptable performance are now available and the who, as well as many countries, has adopted these hiv and/or syphilis rdts into their national guidelines , . tuberculosis. tb causes . million deaths a year, % of which occur in low-and middle-income countries. ending the tb epidemic by is among the health targets of the sustainable development goals . the tb lipoarabinomannan (lam) antigen test provides poc screening for active tb in hiv positive patients. this novel rapid test detects lam in urine samples, providing results in just minutes, enabling earlier treatment for patients . the lam test does not assess tb drug susceptibility, but multidrug-resistant tb is a threat to global health security with an estimated , new cases a year showing resistance to rifampicin (rif) -the most effective first-line drug . nucleic acid tests (nats) for tb are available and provide highly sensitive and specific means of diagnosis. the recent development of a sample-in-answerout automated testing device that allows for simultaneous detection of mycobacterium tuberculosis (mtb) and rif resistance in h min has improved case detection and could decrease transmission, though nats still require patients to make a return visit for test results and treatment. this test system is available in - modules, allowing for flexibility in throughput. the mtb/rif near-poc assay can improve time to diagnosis and treatment and increase the efficiency of the health system if introduced appropriately - , and as of june , two-thirds of high-burden mtb countries and half of countries with a high multidrug-resistant mtb had adopted this assay into their national tb programme guidelines. a wider adoption of such high-performing assays would allow countries to increase their case detection rates and potentially reach the milestones of the end tb strategy . although national tb programmes provide a robust architecture for the implementation of new technologies, challenges associated with the near-poc nat assay remain as barriers -affordability (molecular assays are device-based and costly, even with subsidy), expertise (more technically demanding than lateral flow rdts) and sustainability , in addition to power and per-test time. we expect that addressing these barriers would improve patient outcomes, but a true poc test is still needed to overcome remaining challenges such as sample preparation and demands on human resources . while culture has been a long-standing microbiological gold-standard and is highly specific, it is also the most technically demanding, costly and slowest of diagnostic options, requiring patients to return for another clinic visit to obtain their test results and treatment if necessary. thus, it does not conform well to the development of assured diagnostics. successful poc tests achieve high sensitivity, which is needed as screening tools to ensure that all true and suspect cases are brought to the attention of control programmes and appropriately managed. in particular, antibody-based detection tests, such as those for hiv and hepatitis c virus, are highly sensitive as antibodies are present in large quantities in blood, and blood samples can be collected with a finger prick and put directly into the test without any prior processing. also, antibody detection can be rapid and easy to perform with minimal training required (table ) . however, the choice of the antibody target affects a test's effectiveness -unless the diagnostic target is specific to the intended infection, there is the potential for false positive results due to cross-reactivity, which is the case with dengue and zika immunoassays , . therefore, antibody detection assays should be interpreted within the appropriate epidemiological context and findings from physical examination. the other major disadvantage of antibody-based tests is that antibodies are usually a marker of exposure to a pathogen and cannot be used to distinguish between those with active and past infection, which is the case of the rk tests for visceral leishmaniasis (vl) . a presumptive diagnosis of active vl can only be made using a combination of a positive rk test and more than weeks of fever and splenomegaly. the same is true of most syphilis rdts, which detect long-lived antibodies to treponemal antigens and thus do not diagnose active syphilis. antibodies to a non-treponemal antigen, which appears with infection and declines in the months after successful treatment, are used in a flocculation assay called the rapid plasma reagin assay, which can yield a result in minutes but requires electricity to operate, a centrifuge for processing serum from whole blood, a shaker and cardiolipin as an antigen. a combination treponemal and non-treponemal rapid test has been developed in an immunochromatographic test in a lateral flow format with acceptable performance characteristics . antigen detection poc tests, such as those for chlamydia and gonorrhoea, suffer from three major drawbacks (table ) . first, the specimen used for sexually transmitted infections are usually from urethral, cervical or vaginal swabs that require multiple steps to solubilize the bacteria and free antigen before reaction with the capture antibody. this sometimes requires a heating step and adds to the complexity and cost of the poc test. urine is the preferred specimen in men with such infections, and current poc tests require a urine centrifugation step to concentrate the bacteria before processing and reaction. another common drawback of antigen detection tests is the potential for false positive results due to cross-reactivity with antigens from closely related bacterial or viral species. this is especially true if polyclonal antibodies are used as capture antibodies. finally, a common disadvantage of current antigen detection poc assays is low sensitivity, often requiring to bacteria for the rdt to become positive. malaria rdts are an exception to these drawbacks in that the parasites are present in large quantities in blood and no pre-processing or concentration steps are required. for most antigen detection poc tests, the low sensitivity may be due to low concentration of target antigens, inefficiency of extraction or limited optimization of reagents, which has hampered chlamydia and gonorrhoea tests , . however, gift et al. showed that rapid chlamydia tests with a sensitivity of % can lead to more infected patients being treated compared to nats, which require patients to return for their test results . they called this the rapid test paradox, which was also recently seen with a p assay for early infant diagnosis of hiv, where a sensitivity of % can still lead to a higher 'diagnostic yield' than nats performed on dried blood spots sent from remote antenatal clinics . nats offer a more sensitive and specific alternative to antigen detection assays as nucleic acid targets can be selected from a genome sequence with high specificity, and the amplification process to increase sensitivity can be fairly rapid (table ) . for example, the near-poc tb diagnostic involves primer-based amplification of mtb dna specifically, including regions associated with rif drug resistance. thus, nat tests are generally highly sensitive and more specific than antigen-or antibody-based tests, and may help inform on drug susceptibilities. while instruments are available for highthroughput screening of patients and subsidies can bring test costs down to us$ , the equipment (capable of four tests at a time) costs more than us$ , , which may be prohibitive in high-incidence regions. thus, a truly poc diagnostic based on nats without needing any equipment is still a promise and not a reality as yet , . of all the criteria originally accepted, the requirement of equipment-free is perhaps the only one which is not as critical as originally defined. a wide range of near-poc nats have been developed for use outside of laboratory settings and most of these assays are automated, requiring only - minutes of hands-on time and minimal training. these near-poc devices come in sealed units with internal self-calibration making it easier to ensure quality of testing and most are also equipped with data transmission capabilities, which make these platforms very attractive in the context of disease surveillance and epidemic preparedness. most of these near-poc devices have a broad menu of diagnostic targets which makes the capital cost of purchasing the device less of an issue. reagents are pre-measured and dried ready for hydration with the introduction of the specimen. however, near-poc nat cartridges are expensive to manufacture and not affordable to most developing countries without subsidies. rapid advances in miniaturization, material science, electronics and data transmission in recent years have made minimal instrumentation a reality for new diagnostics, including the use of smart phones, which are widely available, custom developed and low cost. the use of a dongle to connect a smart phone to a microfluidic disc to detect hiv and syphilis antibodies in finger-pricked blood allows the phone to power the reaction, interpret the results and transmit the data to a central database. this smart phone-based poc assay is currently undergoing clinical trials . use of a smart phone to power nats is in development and will play an important role in future poc applications , . in particular, there has been considerable interest shown in utilizing mobile phones as readers and connectivity for rdts using rfid (radio frequency identification) to prevent errors in subjective interpretation and transcription [ ] [ ] [ ] [ ] . while phone-based diagnostics are attractive as an option, regulatory approval and rapid updates of phone software that can affect test performance are important challenges. defining and quantifying risks and benefits. while successes need to be celebrated, significant challenges remain. first, as no diagnostic is perfect, countries continue to struggle with defining acceptable risks of false positive or negative results when a novel diagnostic technology may provide incremental clinical benefits. in the developing world where over % of the population reside in rural areas, and the sustainable development goals urge countries to provide universal health coverage and leave no one behind, the trade-off between accuracy (sensitive and specific) and accessibility (user friendly, rapid and deliverable) becomes paramount . while it impossible to give absolute values across different tests, both sensitivity and specificity remain critical. in selecting an ideal test, the positive and negative predictive values, which are dependent on the prevalence of the disease as well as the characteristics of the diagnostic test, should be taken into account, but few studies have been performed to determine quantitatively what tradeoffs are acceptable. an analysis for syphilis screening compared the use of a laboratory-based immunoassay with treponemal rdts in tanzania . it showed that a test that has % sensitivity but can only be used in sophisticated laboratories can realistically only be accessed by - % of the population, while a rapid lateral flowbased antibody test that has only % sensitivity, but can be used at all levels of the health care system effectively gives a correct diagnosis to as many as % of the population, highlighting that tests should not be evaluated purely on technical performance, but rather on diagnostic performance and clinical impact. training and quality assurance. assuring the quality of tests and testing is the biggest challenge when testing is decentralized at poc. there are many rapid tests from various manufacturers available, and test quality may impact accurate and inconsistent diagnosis across different sites, with studies showing that high rates of errors were observed even in performance of simple rdts for hiv and malaria , . quality of testing requires proficiency panels be sent to all the poc testing sites by a national reference laboratory. including positive and negative controls with each box of tests will allow all tests to be approved once they arrive at their destination and before first use. recently, nine african countries developed a national system for assuring the quality of poc testing for hiv , . with data connectivity from each testing site, quality assurance results can be linked to test results at each poc testing site at a centralized database to trigger alerts for corrective action. supply chain software can also be linked through these connectivity solutions, avoiding stock-outs. demonstrating the value of a novel diagnostic technology. the value of a diagnostic goes beyond the technology, as each test needs to be matched to its 'testing environment' , which includes population characteristics, prevalence of target diseases, comorbidities/coinfections and health system characteristics. national programmes should take advantage of the rapid turnaround time of assured tests to streamline patient pathways and increase the efficiency of the health care system. the roll-out of poc diagnostics requires the use of implementation science to ensure success , taking into account the cultural, behavioural, socioeconomic and health systems contexts. this includes a careful assessment of acceptability and feasibility linked to possible increased stresses on the health system/provider when testing is introduced into settings where thus far no or limited testing was performed. studies using the genexpert mtb/rif tb test showed that new diagnostic tests would not have the expected impact on outcomes if test introduction is not accompanied by changes in patient pathways or practices , . these studies highlight the importance of programmatic monitoring of the impact of novel technologies beyond studies that are usually conducted under controlled conditions. likewise, same-day testing and treatment using a syphilis rdt strengthened health systems in the amazon forest and rural china when policy makers were involved and testing was introduced within the appropriate social and cultural contexts , . for example, in peru, women normally need to present six times to the largest maternal hospital in lima for their prenatal syphilis screening to be completed and treatment provided, but the introduction of the rapid syphilis test streamlined testing and treatment to one visit, reducing patient out-of-pocket expenses and increasing the efficiency of a busy health care facility . there are few detailed analyses which have been conducted to accurately determine the economic impact of many of the available tests, including the diagnostic cost versus the overall economic impact. while these benefits are intuitively important, they are often difficult to quantify . it would, therefore, be extremely useful to have economic analysis tools for each of the major diseases or conditions, so that developers have an understanding of cost implications and what cost structures would be acceptable to health service implementers. as an example, it may be necessary to accept cost trade-offs when addressing global health threats (for example, zika, ebola, and so on), where speed of intervention is critical. this having been stated, low-cost diagnostic tests remain critical in resource-limited settings. in the past two decades, the biggest drivers of diagnostic development were well-resourced diseases such as hiv, malaria and tb. in recent years, the increasing frequency and severity of global health emergencies caused by infectious diseases of epidemic potential, such as severe acute respiratory syndrome (sars) coronavirus, middle east respiratory syndrome coronavirus (mers) co-v, ebola and zika virus, have made the global community realize the need to accelerate assured test development, validation, manufacturing and deployment. there is a need for innovation in rapid detection technologies that are assured but allow multiplex detection of a panel of pathogens such as major causes of respiratory illness, haemorrhagic fevers or enteric infections in a single specimen. these tests should not only identify the cause of outbreak but also be used to process multiple specimens with high throughput throughout the outbreak. another major driver of test development is the need for cheaper, better and faster tests that can be used at poc to reduce inappropriate antimicrobial use. an estimated , people die from untreatable conditions due to antimicrobial resistance (amr) every year worldwide , and if amr continues to spread, by , million people may die from resistant infections annually at an economic cost estimated at $ trillion. at the primary care level, a simple rapid test that can be used to distinguish bacterial from nonbacterial causes of fever, respiratory and enteric infections would allow health care providers to reduce antibiotic use and preserve them for future generations. the o'neill report also pointed out that a test that can identify a pathogen and its antibiotic susceptibility at the poc would allow the safe use of first-line drugs or drug combinations with savings to the health care system. to stimulate interest in innovation in diagnostic tests that can be used at poc, several countries have set up challenge prizes. in , the united kingdom announced the longitude prize of £ million, which seeks an affordable, accurate, fast and easy-to-use test for bacterial infections that will allow health professionals worldwide to administer the right antibiotics at the right time. the challenge is currently ongoing, with final submission by . more information available at: https://longitudeprize.org. in september , the us department of health and human services announced a challenge prize competition in which up to $ million will be awarded for one or more novel and innovative poc diagnostics that would have clinical and public health value in combating the development and spread of antibiotic resistant bacteria. more information is available at: https://dpcpsi.nih.gov/amrchallenge. these prizes may catalyse new technologies, but there are still no guarantees to the sustainability of these tests in resource-limited countries given that cost is a critical factor woven into every step needed to bring about and sustain testing. donors and funders of diagnostics, such as the the bill & melinda gates foundation, global fund, unitaid and aid agencies from national governments such as pepfar and dfid, play a critical role in incentivizing diagnostic innovation and addressing market dynamics. technology advances to support innovation. technology , markets and medical devices have matured to enable connected diagnostics to become a useful tool for epidemiology, patient care and tracking, research, and amr and outbreak surveillance. the ability to digitize data from laboratory and poc platforms, including lateral flow rdt results, can standardize the interpretation of results and allows data to be linked to proficiency testing to ensure testing quality, reducing interpretation and transcription errors. remote monitoring of poc instrument functionality and utilization through connectivity allows programmes to optimize instrument placement, algorithm adoption and supply management. alerts can be built into the system to raise alarm at unusual trends such as outbreaks. the application of mobile devices and related technologies to health care is improving patients' access to health information and treatment, offering possibilities to diagnose, track and assess the impact of infectious disease interventions across the world , . smart devices can also be used to automatically add data to a central database and allow systems to assess likelihood of a diagnosis or predict disease outbreaks through machine learning, providing a route to smart diagnostics that incorporates historical or epidemiologic data to make diagnoses more accurate. finally, new engineering fields such as the internet of things (iot), industry . and printed electronics [ ] [ ] [ ] [ ] promise to add significantly to the technologies that can be chosen and incorporated into new diagnostic devices. many of these technologies are aimed at high-volume and low-cost distributable manufacturing, thus fitting in well with the goal of poc diagnostics. new detectors and light sources also suggest that incorporating these into tests could allow for more accurate result reading and the possibility of multiplexing. while it is clear that the original assured criteria remain relevant, opportunities exist to improve future diagnostics by incorporating new technological elements to provide real-time quality control for testing and treatment and overcoming the difficulties in specimen collection and/or processing , which currently limits scaling-up of diagnostics in resource-limited areas. we therefore propose two additional criteria of r (real-time connectivity) and e (ease of specimen collection and environmental friendliness) into the original assured, to create a new acronym of reassured (table ) . to use testing to effectively survey or treat patients, it is critical to obtain and analyse results at the poc . however, one of the main challenges to poc testing is ensuring that results are rapidly provided to the patient once testing has been completed ; this is formidable challenge when testing is decentralized at hundreds or thousands of different sites by health care workers who are not consistently skilled in reading test results, leading to risk that incorrect data is being recorded and/or transmitted. one solution would be to allow the analysis of test results at a centralized level for consistent diagnosis and epidemiological surveillance. many manufacturers are now embedding connectivity into poc and near-poc instruments (for example, the alere pima poc cd device ). other manufacturers have developed innovative connectivity solutions that can use mobile phones to read the results from lateral flow assays and provide electronic result exchange, while a third option would be to include connectivity directly onto the test . ideally, connectivity should not only include collection and transmission of test data, but also analysis to provide feedback for immediate patient treatment or for surveillance. with the addition of barcodes, two-dimensional codes or electronic storage into tests, a number of other data points can be collected, including manufacturing information (for example, lot numbers), dates, expiry dates, stock availability and possibly even environmental conditions such as temperature and humidity under which the tests have been manufactured, transported, stored and used. for instrumented tests, maintenance and other machine data can be collected. thus, connectivity solutions can increase quality assurance for poc tests and would allow for centralized and real-time decision-making, even across tiered laboratory systems and during outbreak investigations and global health emergencies. moving forward, it is critically important to make provisions for poor or non-existent internet availability to support sustainability standards and that systems are developed with compatibility so that they can be linked into central databases. ease of specimen collection. specimen collection and processing may need further in-depth development. first, it is ideal to have noninvasive specimen collection given that more tests become available as self or home tests. second, samples come in many different formats (blood, urine, sputum, stool, swab, breath, and so on) and require different preparation depending on the test to be performed. this may include concentrating the sample (or possibly titration), purification, lysing of cells, target amplification, and so on. while most of these processes can be easily performed in the laboratory, there remain few solutions for collecting and performing these tasks at the poc . oral tests for hiv and hepatitis c virus are good examples of advances in non-invasive sampling, although the collection device is more expensive than blood collection via disposable capillary tubes. related, given that rapid lateral flow antigen detection tests often have lower sensitivity, extra processing steps such as heating or other forms of extraction are required when using specimens other than blood. environmental friendliness. advances in technology have made the assured requirement for equipment somewhat less daunting. however, in light of more diagnostic tests now being performed in both urban and rural areas, there could be more future effort devoted to the environmental impact of these tests. the cumulative effect of many tens of thousands of tests performed at remote sites away from laboratory infrastructure may pose health and environmental risks and put a strain on limited resources. some examples include the plastics used in current rapid tests that cannot be recycled and give out toxic fumes when burned, and testing cartridges that may even contain harmful chemicals and need to be disposed of properly. recyclable materials should be used, when possible, for the housing, substrate matrix materials, and reagents used in tests. paper, an obvious choice of substrate material that has many inherent advantages over standard plastic materials, will see increased usage, not only in lateral flow formats but other paper based formats as well such as in micro paperbased analytical devices (μ pads) [ ] [ ] [ ] . and recently, cell-free synthetic gene networks for in vitro applications at poc, have been achieved by freeze-drying cell-free systems onto paper, which increases stability at room temperature and can be easily transported and stored, and simply re-activated by adding water . other factors also need to be considered, such as disposal of test reagents, clinical samples and materials used for active components such as electronic tracks and electrodes. the assured criteria have been a valuable framework for developing devices and methods to detect major human diseases in challenging poc contexts. however, over the last decade, new technologies, not envisaged or available at the time of the first assured criteria definition, have given rise to the possibility of including new considerations into the next generation of devices and tests. we propose the acronym reassured for the design of future diagnostic tests to address important priorities such as global health emergencies and amr. an ideal test would be one that combines the best of both existing diagnostic worlds (instrumented laboratory based tests with lateral flow tests) and technologies currently being developed (fig. ) , which would provide an important extension to diagnostic laboratory systems and fulfil the sustainable development goals of 'no one left behind' in terms of effective health care service delivery. such tests can only be created by forming strong collaborative partnerships across many disciplinary boundaries, and we look toward a future when data connectivity linking 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key: cord- -r j tnw authors: lim, jun hyeok; ryu, jeong-seon; cho, sang yong; kim, hyun-jung; jeon, sang hoon; kim, jung soo; nam, hae-seong; cho, jae hwa; kwak, seung min; lee, hong lyeol title: small-cell lung cancer presenting as fatal pulmonary hemorrhage date: - - journal: open med (wars) doi: . /med- - sha: doc_id: cord_uid: r j tnw small-cell lung cancer (sclc) is a lung cancer histological subtype unusual in its favorable response to cytotoxic chemotherapy. life-threatening manifestations at presentation are rarely reported and should be an important clinical concern. we report a case of a -year-old man presenting with rapid-onset refractory severe thrombocytopenia, development of massive hemoptysis, and death from respiratory failure. this case provides clinicians a reference for this unusual presentation and carries clinical implications for managing sclc patients. small-cell lung cancer (sclc) represents - % of all lung cancers [ ] . sclc differs from non-small-cell lung cancer in its rapid tumor doubling time, high growth fraction, early development of widespread metastasis, and better response to platinum doublets chemotherapy. thus chemotherapy is a treatment mainstay, even in poor eastern cooperative oncology group (ecog) performance status [ , ] . bone marrow involvement or paraneoplastic syndrome is common in patients with sclc [ ] . hematologic abnormalities such as anemia, leukopenia, and thrombocytopenia are reported to be occasionally accompanied by bone marrow metastasis or paraneoplastic phenomenon [ , ] . however, complications such as fatal hemorrhage are rarely reported. the clinical presentation can make diagnosis or treatment difficult. herein, we report an sclc patient who presented with rapid-onset, refractory severe thrombocytopenia and development of fatal pulmonary hemorrhage. a -year-old man visited an outpatient clinic complaining of cough and dyspnea (borg scale ). he was a current smoker of pack-years and denied histories of taking any medications or illness including cardiovascular, allergic, rheumatologic or respiratory diseases. a complete blood count revealed values within normal range, except for a lower value of platelet count, , /mm . increased haziness on the lower lobe of the right lung was noted on his chest radiography. when he returned after days, he was admitted with blood-tinged sputum and aggravated dyspnea (borg scale ). his ecog performance status was two. he was afebrile. an arterial blood gas study revealed ph . , paco . mmhg, pao . mmhg, hco mmol/l, and spo % on room air. complete blood count results were as follows: leukocytes , /mm (neutrophil . %, lymphocyte . %, monocyte . %, eosinophil . %, and basophil . %), hemoglobin . g/dl, hematocrit . %, and platelets , /mm . the serum lactate dehydrogenase level was , iu/l; c-reactive protein, . mg/dl. hepatic and renal function testing were within normal range. prothrombin time, activated partial thromboplastin time, and d-dimer were within normal range as well. a . cm sized mass in the lower lobe of the right lung and multiple lymphadenopathies in mediastinal and right supraclavicular areas were noted on chest ct scan ( fig. ) . a peripheral blood smear revealed leukoerythroblastosis with nucleated erythrocyte, left shifted neutrophils. anti-platelet antibody and anti-neutrophil cytoplasmic antibody were negative. anti-nuclear antibody was within pulmonary hemorrhage at sclc presentation normal range ( : ). because of the risk of bleeding due to severe thrombocytopenia, a bronchoscopic examination was not feasible and was postponed. a bone marrow examination was not performed because the patient was unable to maintain prone position due to dyspnea. platelet concentrates and packed red blood cells were started, given daily, and dexamethasone, mg was intravenously administered for four days. however, his platelet count remained stationary (fig. ) . on the fifth day after admission, cytological examination of his sputum yielded a diagnosis of sclc. metastatic lesions were not observed on brain mri and bone scintigraphy. on the seventh day, massive hemoptysis (≥ ml per day) abruptly occurred and his dyspnea was rapidly aggravated to of borg scale. a chest ct scan revealed diffuse ground glass opacities and consolidation in both lung fields. tranexamic acid and empirical broad-spectrum antibiotics including piperacillin-tazobactam, levofloxacin were initiated intravenously. sputum gram stain and culture for bacteria and fungus revealed no organism. sputum culture for adenovirus, parainfluenza virus, rhinovirus, respiratory syncytial virus, metapneumovirus, coronavirus, bocavirus, and enterovirus were negative. antibody studies for mycoplasma pneumoniae and rickettsia were negative, as was an antigen study for streptococcus pneumoniae. his arterial blood gas study showed ph of . , paco of . mmhg, pao of . mmhg, hco mmol/l, and spo % on oxygen supplied via reservoir mask flow rate l/min. on the tenth day, he was intubated and ventilated mechanically. he rapidly deteriorated and died of respiratory failure on the twelfth day. his family did not want to have a postmortem examination, against physician's recommendation. the research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance the tenets of the helsinki declaration, and has been approved by the authors' institutional review board or equivalent committee. informed consent: written informed consent was obtained from the patient for publication of this manuscript and any accompanying images. to our knowledge, this is the first report describing an sclc patient presenting with fatal pulmonary hemorrhage due to refractory thrombocytopenia. sclc is the most aggressive histological subtype of lung cancer. however, favorable response is expected in % to % of patients with extensive-stage disease when systemic chemotherapy is given [ , ] . life-threatening manifestations rarely present in sclc patients. when such manifestations do appear, as in the present case, the rapid-onset, unusual presentation may delay diagnosis or hesitation in deciding treatment, affecting survival adversely [ ] . therefore, prompt diagnosis and treatment is a critical issue [ ] . hematological abnormalities including anemia, leukopenia, and thrombocytopenia are commonly observed due to the toxicity of anti-cancer therapy. however, hematologic abnormalities resulting from bone marrow metastasis and paraneoplastic phenomenon are not as common as those resulting from anti-cancer therapy [ , ] . severe thrombocytopenia developed rapidly and was refractory to platelet transfusion and dexamethasone administration. in addition, tests for anti-platelet antibody and anti-neutrophil cytoplasmic antibody were negative and anti-nuclear antibody was within normal range. these findings indicate that autoimmune disease was not likely to be the cause of thrombocytopenia [ ] . bone marrow examination is a diagnostic process for evaluating hematological abnormality or staging the disease. however, it rarely changes the stage [ ] and bone marrow as an isolated metastatic site is found in fewer than % of sclc patients [ ] . leukoerythroblastic reaction on peripheral blood smear is suggestive of bone marrow metastasis in the present case [ , ] . although bronchoscopy may be considered for localization or treatment in massive hemoptysis, it is contraindicated in a patient with severe thrombocytopenia [ ] . a diagnostic and treatment plan should be determined after taking into account the benefit and risk on an individual patient basis. it is challenging to do in every possible case. the clinical presentation of the current case is unfamiliar to clinicians and rapid-onset, life threatening. however, prompt treatment with systemic chemo-therapy should have been considered because he was considered fit for systemic chemotherapy by virtue of his younger age, acceptable ecog performance status, and absence of comorbid diseases. cancer statistics national comprehensive cancer network. nccn clinical practice guidelines in oncology small cell lung cancer-version small-cell lung cancer thrombocytopenia in solid tumors bone marrow involvement in small cell lung cancer: prognostic significance and correlation with hematological and biochemical parameters delays in the diagnosis and treatment of lung cancer lactic acidosis with small cell carcinoma. rapid response to chemotherapy paraneoplastic autoimmune thrombocytopenia in solid tumors is bone marrow examination in small-cell lung cancer really necessary? leukoerythroblastosis and cancer frequency, prognosis, and physiopathologic significance systemic malignancies as a cause of unexpected microangiopathic hemolytic anemia and thrombocytopenia british thoracic society guideline for diagnostic flexible bronchoscopy in adults: accredited by nice acknowledgements: this work was supported by grants (hi c and hi c ) from the korea health technology r&d project, ministry of health and welfare, republic of korea. the authors have no conflicts of interest to declare. key: cord- -jtvez y authors: wu, xuan; song, zengxu; zhai, xiwen; zuo, lei; mei, xueran; xiang, rong; kang, zhuangzhuang; zhou, long; wang, hongning title: simultaneous and visual detection of infectious bronchitis virus and newcastle disease virus by multiple lamp and lateral flow dipstick date: - - journal: poultry science doi: . /ps/pez sha: doc_id: cord_uid: jtvez y abstract infectious bronchitis virus (ibv) and newcastle disease virus (ndv) are both important viruses seriously affecting poultry industry worldwide. in this study, reverse-transcription lamp (rt-lamp) was combined with lateral flow dipstick (lfd) forming a novel detection tool which could simultaneously detect ibv and ndv visually. primers targeted the ′-untranslated region ( ′-utr) of ibv genome and the conserved region of ndv large polymerase gene (lp). the specificity and sensitivity of this multiple reverse transcription-lamp-lfd (mrt-lamp-lfd) assay were compared with those of conventional rt-pcr, nested rt-pcr (nrt-pcr), quantification rt-pcr (qrt-pcr), and rt-lamp monitored by electrophoresis. no non-specific amplifications were observed when the assays were tested with unrelated viruses. according to the sensitivity study, when detecting ibv or ndv alone, the lowest detection limits of mrt-lamp-lfd were . ibv rna copies/reaction and . ndv rna copies/reaction. furthermore, when detecting ibv and ndv simultaneously, the lowest detection limit was the same as that of the single detection assays. in the clinical sample study, mrt-lamp-lfd performed the best among these assays. when tested with ibv or ndv single infected samples, the mean detection rates were . % and . %, respectively. in the ibv and ndv co-infected sample study, the mean detection rates of ibv and ndv were both %. this study showed that mrt-lamp-lfd was a promising qualitative detection tool suitable for field single or multiple ibv and ndv detection. infectious bronchitis virus (ibv) and newcastle disease virus (ndv) are of the most important viruses seriously affecting the poultry industry and causing huge economic losses worldwide (bande et al., ; brown and bevins, ) . ibv and ndv belong to the gammacoronavirus of the coronaviridae family and the avulavirus of the paramyxoviridae family, respectively (http://www.ictv.global). the genome of ibv is about . kb in length. it encodes non-structural proteins, and structural proteins: spike glycoprotein (s), small membrane protein (e), membrane glycoprotein (m), and phosphorylated nucleocapsid protein (n). at the and ends of the genome, there is an untranslated region (utr) each (armesto et al., ). ndv possesses a kb long genome comprising genes c poultry science association inc. received april , . accepted june , . corresponding author: whongning@ .com which individually encode the nucleocapsid (n), matrix protein (m), phosphoprotein (p), fusion protein (f), hemagglutinin-neuraminidase protein (hn), and large polymerase protein (lp) (de leeuw and peeters, ) . ibv and ndv both have high mutation rates, making their prevention and control difficult. quick and accurate detection of ibv and ndv is important for preventing the viruses from spreading. a wide variety of diagnostic assays for ibv and ndv have been developed, including virus isolation, and serological and molecular assays (bande et al., ; brown and bevins, ) . costs, requirements of stringent techniques, and time required limit the use of virus isolation as a routine virus detection assay (bande et al., ) . serological assays, such as hemagglutination inhibition and elisa, are faster and simpler than virus isolation, but tend to lack specificity and sensitivity, especially in the case of ibv, and poor crossreactions between serotypes makes serological tests less applicable (cavanagh, ; miller et al., ) . in view of their high sensitivity, specificity, and reduced flow time, molecular assays are the most commonly used methods for ibv and ndv monitoring. according to previous studies, both ibv and ndv quantification rt-pcr (qrt-pcr) detection methods were highly specific, and the lowest detection limits were - genome copies indicating that these qrt-pcr methods were highly sensitive (callison et al., ; farkas et al., ; wise et al., ) . another highly specific and sensitive molecular method is nested rt-pcr (nrt-pcr) which involves rounds of pcr amplifications. as previously reported, the lowest detection limits of ibv and ndv nrt-pcr assays were . and . eid /ml, respectively (nguyen et al., ) . while pcr assays are widely applied in pathogen detection, the conduct of pcr requires sophisticated laboratory equipment and observation of pcr product requires electrophoresis, making pcr assays unsuitable for point-of-care and visible detections, especially in some low-resource regions. loop-mediated isothermal amplification (lamp) amplifies dna under isothermal conditions by the bst dna polymerase large fragment (notomi et al., ) . numerous studies have demonstrated that the amplification efficiency of lamp is quite high (khan et al., ; zhang et al., ) . moreover, the specificity of lamp is also satisfactory as there are specially designed primers recognizing distinct regions on the target dna (asiello and baeumner, ; zhang et al., ) . furthermore, unlike conventional pcr assays, only simple devices are needed during lamp, such as a water bath or a heat block. lamp is thought to revolutionize molecular biology not only because of its excellent performance on dna amplification but also due to its diverse, simple, and intuitional reaction monitoring methods. several naked eye monitoring approaches have been applied, such as adding color indicators into reactions and combining with immunochromatographic techniques (parida et al., ; zhang et al., ) . lateral flow dipstick (lfd), an immunochromatographic technique, utilizes antibody capture followed by secondary antibody labeling (chen et al., ; zhang et al., ) . lamp combined with lfd (lamp-lfd) could be used for highly sensitive, simple, visual, and multiple pathogen detections (chen et al., ) . lamp products can be labeled by employing biotin/fitc modified fip/bip primers, and subsequently, these biotin-fitc double labeled lamp products can be captured by biotin-antibodies and immobilized at specific locations on lfd strips (test line). subsequently, fitc at the other end of the products can specifically combine with gold particles labeled with fitc-antibodies, thus making the results readable using the naked eye (nimitphak et al., ) . however, no previous studies have reported multiple detection of avian pathogens using lamp-lfd. both ibv and ndv are pathogens that cause avian respiratory diseases, and single or multiple infection by them may cause similar clinical signs. studies have shown that multiple conventional rt-pcr could be used for detecting and differentiating respiratory dis-ease pathogens in poultry diseases (pang et al., ; rashid et al., ) . however, the sensitivity of multiple conventional rt-pcr is not satisfactory. multiple nested rt-pcr is much more sensitive than multiple conventional rt-pcr but time-consuming (nguyen et al., ) . furthermore, these assays are not suitable for on-site pathogen detection, because products of rt-pcr need to be monitored by electrophoresis and qrt-pcr need to be conducted with highly accurate instruments. here, we developed a visual multiple rt-lamp-lfd (mrt-lamp-lfd) assay which could simultaneously detect ibv and ndv and be easily carried out and monitored by the naked eye. to evaluate this novel detection method, pcr assays (including conventional rt-pcr, qrt-pcr and nrt-pcr) and reverse-transcription lamp (rt-lamp) monitored by electrophoresis were also conducted and the specificity and sensitivity of the assays were compared with those of the mrt-lamp-lfd assay. a total of ibv strains, ndv strains, and the pcr and lamp target sequences of ndv and turkey coronavirus strains (tcov) synthesized by sangon biotech (shanghai, china) co, as well as other avian virus strains, were used for the determination of the specificities of rt-pcr and rt-lamp assays. the genbank numbers of ibv and ndv strains were labeled in figure s (supplementary information); the tcov strain and the other avian virus strains used in the specificity study were listed in table s (supplementary information). tissue samples used in this study were stored at − • c. rna in the samples was extracted with trizol (invitrogen, carlsbad, ca). subsequently, cdna was synthesized using primescript rt reagent kit (takara, beijing, china) following the manufacturer's instructions. briefly, the reverse transcription reaction mixture consisted of μl × primescript buffer, . μl primescript rt enzyme mix i, . μl oligo dt primer, . μl random mers, the rna of the virus, and rnase free dh o, thus creating a final volume of μl. complete genome sequences of ibv strains and ndv strains available in genbank were aligned using mega software. subsequently, to determine conserved regions in the ibv and ndv genomes, aligned results were used for similarity plotting analysis with the simplot program . . . primers for rt-pcr, nrt-pcr, qrt-pcr, and rt-lamp assays were designed tgcatgtgccacatgagact nd-b ctttcctctgtattctctctcc on base of ibv and ndv genome conservative regions: ibv primers targeted the -utr region, and ndv primers located in the conserved region of lp gene. primers for rt-pcr, nrt-pcr, and qrt-pcr were designed by primer premier software (premier inc., palm desert, ca); primers for rt-lamp assays were designed by primerexplorer v software (fujitsu, tokyo, japan). in mrt-lamp-lfd reactions, modified fip and bip primers were used: bio-ib-fip was modified by biotin on the -end, dig-nd-fip was modified by digoxigenin on the -end, and fitc-ib-bip and fitc-nd-bip were modified by fitc on the -end. the sequences of the primers are listed in table . the most appropriate annealing temperatures for each pair of primers were determined using gradient pcr and listed in table . conventional rt-pcr reaction mixture consisted of . μl × m pfu pcr mas-termix (mei , beijing, china), pmol of each primer, μl template and double distilled water (ddh o) creating a final volume of μl. the pcr parameters included an initial denaturation step for min at • c followed by cycles of denaturation at • c for s, annealing for s, extension at • c for to s depending on the sizes of the products and a final extension step at • c for min. nrt-pcr involved rounds of amplifications. the reaction mixture and parameters of each round of amplification were the same as that of conventional rt-pcr. the products of conventional rt-pcr and nrt-pcr were monitored by electrophoresis in % agarose gels. qrt-pcr assays were conducted with μl × sso-fast evagreen supermix (bio-rad, hercules, ca), pmol of each primer, μl template and ddh o making the final volume to μl. the parameters were: initial denaturation for s at • c followed by cycles of denaturation at • c for s, annealing/extension for s, and a final melting curve at to • c with increment . • c/ s. data was analyzed by bio-rad cfx maestro . software (bio-rad, hercules, ca). plate read was added during the extension and melting curve steps. to establish ibv and ndv qrt-pcr standard curves, fragments amplified using qib-f/r and qnd-f/r were individually cloned into peasy-t vector (transgen, beijing, china). plasmids were extracted using tianprep mini plasmid kit (tiangen, beijing, china). subsequently, ibv plasmids were -fold serial diluted from . × to . × copies/μl and ndv plasmids were -fold serial diluted from . × to . × copies/μl. diluted plasmids were used as standard samples during the establishment of figure . principle of mrt-lamp-lfd. ibv and ndv lamp amplify products were labeled with fitc/biotin and fitc/digoxigenin, respectively. gold particles modified with fitc-antibodies can combine with the labeled ibv and ndv products. subsequently, ibv products will be captured by biotin-antibodies immobilized on the test line , ndv products will be captured by digoxigenin-antibodies immobilized on the test line , and the free gold particles will be immobilized on the control line. thus, the products are visualized. ibv and ndv qrt-pcr standard curves. slopes and intercepts of standard curves, amplification efficiency (e) and corresponding correlation coefficients (r ) were generated using bio-rad cfx maestro . software (bio-rad, hercules, ca). the rt-lamp reactions were conducted under gradient temperatures ( to • c) to determine optimal reaction temperatures. the concentration of mgso ( to mm) and dosage of bst . warmstart dna polymerase (new england biolabs, ipswich, ma) ( to u) were also optimized. the optimal reaction mixture contains . μl × isothermal amplification buffer to evaluate the specificities of the assays, the phylogenetic analyses on pcr and lamp target sequences of ibv and ndv strains were conducted. according to the phylogenetic trees, ibv strains were grouped into clades, and ndv strains were grouped into clades. a total of ibv strains were used to determine the specificities of the assays distribute in all clades, likewise, ndv strains were used to determine the specificities distribute in all clades ( figure s , supplementary information) . in addition, cdna of avian reovirus (arv), infectious bursal disease virus (ibdv), and avian influenza virus (aiv), dna of gallid alphaherpesvirus (gahv- ) and fowl adenovirus (fadv), as well as synthesized sequence of tcov strain were used as templates for further evaluation of the specificities of the assays (table s , supplementary information) . to determine the lowest detection limits of the assays in terms of rna copy numbers, -fold serial diluted in vitro-transcribed rna of target regions was used as templates. briefly, fragments containing ibv -utr and ndv lp target regions were separately amplified using primer pairs -atcacactagccttgc gctaga- / -gcaaaagcatcagcgtaatcc- and -aatctgtattacatgtctagg- / -aga gagaatatatcctttcgc- . subsequently, the fragments were separately ligated downstream t promoter. in vitro-transcriptions were conducted using hiscribe t high yield rna synthesis kit (neb, beijing, china). the concentrations of the rna transcripts were measured using nano drop (thermo, shanghai, china) , and the copy numbers of ibv and ndv rna molecules were calculated following the formula reported previously (fronhoffs et al., ) . the copy numbers of ibv and ndv rna molecules were . and . copies/μl, respectively. ibv rna was -fold serial diluted into . to . copies/μl; ndv rna was -fold serial diluted into . to . copies/μl. whereafter, μl rna from each concentration was used in reverse transcription reaction ( μl reaction volume). after reverse transcription, μl cdna was used in the pcr and lamp assays. thus, the concentrations of serial diluted ibv and ndv rna, which was finally used as templates, were separately . to − . copies/reaction and . to − . copies/reaction. furthermore, serial diluted ibv and ndv rna was mixed to test the lowest detection limit of mrt-lamp-lfd when simultaneously detecting ibv and ndv. negative control reactions in specificity and sensitivity studies were conducted with total rna extracted from allantoic fluid of health specific pathogen-free (spf) chick embryo as templates. ibv-positive samples, including tissue samples ( tracheas and lungs), swabs ( oral swabs and cloacal swabs), ndv-positive samples, including tissues ( tracheas and lungs), and swabs ( oral swabs and cloacal swabs) were used to examine the performance of the assays in detecting ibv and ndv in clinical samples. in addition, to further investigate the specificities of the assays when detecting clinical samples, negative tissues (including tracheas, lungs, and kidneys), fadv positive livers, and aiv h n positive lungs were also tested by the assays. all these samples were collected from chicken farms distributed in provinces, china, during our routine monitor on avian diseases. to investigate whether the mrt-lamp-lfd assay could detect ibv and ndv in co-infected samples, both accurately and simultaneously, ten -wk-old spf chickens were inoculated with . eid ibv m and . eid ndv f e by the nasal route to mimic ibv-ndv co-infected chickens. oral and cloacal swabs were collected on and days-post-infection (dpi) from each bird. on dpi, all the chickens were sacrificed, and lungs and tracheas were collected. the animal experiment in this study was approved by the animal ethics committee of the college of life sciences, sichuan university (license: syxk-chuan- - ). all experimental procedures and animal welfare standards strictly followed the guidelines of animal management at sichuan university. statistical significance differences in the mean detection rates of the assays, when detecting different kinds of samples, were evaluated by one-way anova using graphpad prism version (graphpad software inc., san diego, ca). differences were considered to be significant at * p < . . under optimal annealing temperatures, rt-pcr and nrt-pcr amplified specific products of the expected lengths, and there were no non-specific bands observed in the negative controls ( figure a ). both in ibv and ndv qrt-pcr assays, fluorescent signals were detected with the target ibv or ndv templates, while no fluorescent signals were detected in negative control reactions ( figure b ). according to the standard curves, c t values (y), and log of copy numbers (x) were linearly correlated (ibv: y = − . x + . , e = . %, r = . ; ndv: y = − . x + . , e = . %, r = . ). the melting curve showed a single peak indicating no primer dimer formed ( figure c ). after ibv and ndv rt-lamp amplification, symbolic ladder-like bands were observed in a % agarose gel ( figure a ). as for mrt-lamp-lfd assays, ibvpositive reactions generated test line and the control line; ndv-positive reactions generated test line and the control line; and test lines , , and the control line appeared when both ibv and ndv were present; only the control line was generated when neither virus was present ( figure b ). as figure a shows, there were no positive reactions observed when ibv assays were tested with other pathogen templates except for tcov. the results are not unexpected, because tcov and ibv are very closely related in terms of both antigenic and genomic characterizations (guy, ) . target sequence ( -utr) nucleotide identities between tcov and ibv strains are . to . % (data not shown). moreover, according to the new taxonomy of viruses published by international committee on taxonomy of viruses, ibv, and tcov are classified as specie (http://www.ictv.global). ndv detection assays were specific to the ndv templates ( figure b ). conventional rt-pcr, nrt-pcr, and rt-lamp assays yielded specific products only when tested with target ndv templates. in mrt-lamp-lfd assays, test lines were observed when target templates were contained in the reaction mixture. as for qrt-pcr assays, fluorescent signals were detected only when tested with the ndv templates. the lowest detection limits of ibv conventional rt-pcr, nrt-pcr, qrt-pcr, rt-lamp, and mrt-lamp-lfd assays, when detecting ibv alone, were . , . , . , . , and . copies/reaction, respectively ( figure a ). the lowest detection limits of ndv conventional rt-pcr, nrt-pcr, qrt-pcr, rt-lamp, and mrt-lamp-lfd assays, detecting ndv alone, were individually . , . , . , . , and . copies/reaction ( figure b ). when simultaneously detecting ibv and ndv, mrt-lamp-lfd produced clear visible test lines at concentration of . ibv + . ndv copies/reaction, and the lowest detection limit was the same as that of mrt-lamp-lfd when detecting ibv or ndv alone ( figure c ). as figure a shows, mrt-lamp-lfd exhibited the highest mean detection rates in the detection of different types of clinical samples when conducting ibv or ndv single detection, . % for ibv and . % for ndv. statistical significance difference studies showed that the mean detection rates of mrt-lamp-lfd were significantly higher than that of conventional rt-pcr assays when detecting ibv or ndv alone (p < . ). no positive results were observed when the assays were tested with negative tissues, fadv positive livers, and aiv positive lungs. to further evaluate mrt-lamp-lfd, chickens were experimentally co-infected with ibv and ndv. results showed that mrt-lamp-lfd could not only detect pathogens simultaneously, but also showed higher mean detection rates than the other assays presented here. the mean ibv and ndv detection rates of different samples, detected by mrt-lamp-lfd, were both %, and were significantly higher than those detected by conventional rt-pcr and qrt-pcr (p < . , figure b) . . specificity study of pcr and lamp assays of (a) ibv and (b) ndv. in specificity study, cdna of ibv, ndv, arv, ibdv, and aiv and dna of gahv- and fadv, as well as the synthesized pcr and lamp target sequences including ndv and tcov were used as templates. in the ibv detection assays, positive results were detected with ibv and tcov templates. in ibv qrt-pcr, other viruses include ndv, arv, gahv- , ibdv, fadv, and aiv. as for ndv detection assays, positive results were observed only when ndv templates existed. in ndv qrt-pcr, other viruses refer to ibv, arv, gahv- , ibdv, fadv, tcov, and aiv. nc means negative control reactions which were conducted using total rna extracted from allantoic fluid of healthy spf chick embryo as template. markers in the electrophoresis were the same as that in figure a . timely and accurate diagnostic methods are very important for the control of infectious diseases, especially for ibv and ndv which are of the most important contagious viruses seriously affecting the poultry industry. furthermore, ibv and ndv produce clinical picture somewhat resembling each other, it is very much crucial not only to detect but also differentiate simultaneously. existing ibv and ndv diagnostic methods, including virus isolation and pcr assays, are specific and sensitive. however, they are not suitable for timely onsite pathogen detection. although portable pcr machines are gradually applied in the field, in most areas, especially in undeveloped and developing countries, these sophisticated equipments are too expensive to be popularized, and the sensitivity of pcr is not satisfactory. multiple rt-lamp-lfd developed in this study could detect and differentiate ibv and ndv, both simultaneously and accurately. when ibv and ndv cdna co-exist in the same reaction system, an ibv and ndv double-positive result was observed. to evaluate the sensitivity of mrt-lamp-lfd, conventional rt-pcr, nrt-pcr, qrt-pcr, and rt-lamp assays detecting ibv or ndv alone were also conducted to compare with mrt-lamp-lfd. when detecting ibv or ndv alone, mrt-lamp-lfd performed as sensitive as nrt-pcr and rt-lamp did, in a directly visual way. it is always thought that qrt-pcr methods provided high sensitivity during pathogen diagnoses. according to the result of detection limit study, nrt-pcr, and lamp assays, established in this study, possessed times higher sensitivity than qrt-pcr. in the first round of amplification of nrt-pcr, the original template was amplified with the outer primers. thus, the number of the fragments containing the inner primer target sequence is greatly improved compared with the original template. as a result, the number of templates in the second round of amplification of nrt-pcr is much higher than that in qrt-pcr. this is the reason why nrt-pcr could detect lower concentration of original templates than qrt-pcr do. similarly to our results, previous study conducted by weng and chen indicated that npcr showed higher sensitivity than real-time pcr when detecting phytophthora infestans (khan et al., ) . lamp is one of the most widely used isothermal nucleic acid amplification techniques (inats) . several studies on the diagnostic methods of other pathogens had showed that these inats possessed equal or even higher sensitivities compared with qpcr assays (gao et al., ; khan et al., ; yang et al., ) . our results indicated that lamp assays possessed higher sensitivities than qpcr when detecting ibv and ndv ( figure ). several multiple rt-pcr assays detecting avian respiratory pathogens have been developed in previous studies, while the sensitivity of these multiple assays was lower than single pathogen detection assays (nguyen et al., ; pang et al., ) . this may be due to the competition among different sets of primers. however, when detecting with ibv and ndv co-existing samples, the lowest detection limit of mrt-lamp-lfd was the same as that of mrt-lamp-lfd when detecting a single pathogen (i.e., . copies/reaction for ibv and . for copies/reaction for ndv), indicating that the sensitivity of mrt-lamp-lfd was not affected when the components of the reaction system became more complex. the purpose of this study was to develop a novel detection tool which could simultaneously accurately detect ibv and ndv on site. previous studies on pcr methods detecting ibv and ndv showed that these pcr methods are specific and sensitive, but the need for expensive thermal cycling equipments makes them not suitable for on-site ibv and ndv detection. portable pcr machines are gradually applied in the field. while, in most areas, especially in undeveloped and developing countries, these sophisticated to further evaluate mrt-lamp-lfd, chickens were experimentally co-infected with ibv and ndv. the mean detection rates of the assays, when detecting different kinds of samples, are labeled above the histograms. the fraction numbers under the x-axis represent (positive sample numbers detected by the assays)/(total sample numbers). * indicate p < . . equipments are too expensive to be popularized. thus, simpler and visible ibv and ndv detecting methods are urgently needed. high specific and sensitive single ibv and ndv rt-lamp assays have been developed in previous studies (chen et al., ; pham et al., ) . in these assays, products were visualized by electrophoresis or by adding color indicators. when detecting multiple pathogens, the products must be easily differentiated. when visualized by electrophoresis, lamp products could be distinguished by observing bands with different molecular weights, however, this method is not suitable for on-site pathogen detection. by adding color indicators, the change in color could be easily observed, but this change is non-specific. obviously, these two lamp monitoring methods are not applicable for on-site multiple pathogens detection. multiple lamp-lfd has been applied in the detection of some pathogens, and it showed great advances compared with common pcr and lamp assays, such as the requirement for little equipment, short reaction time, and the ability to detect multiple genes or pathogens (chen et al., ; lalle et al., ) . to our knowledge, rt-lamp-lfd has not been applied to ibv or ndv detection and no studies on an mrt-lamp-lfd technique that detects multiple avian respiratory viruses have been reported. in this study, the products generated in ibv and ndv rt-lamp were differentiated by biotin/fitc and digoxigenin/fitc labeling, respectively. the products could bind with biotin-or digoxigenin-antibodies fixed on different test lines on the lfd strip, and then products were visualized by combining with gold particles modified with fitc-antibodies (figure ). when tested with clinical samples, the mean detection rate of mrt-lamp-lfd was higher than that of the other assays. these results indicate that mrt-lamp-lfd is not only able to detect ibv and ndv simultaneously, but is also suitable for field testing in both technical (more sensitive than pcr and lamp assays) and practical aspects (more simple to put out and no specialized equipment needed). this study combined rt-lamp and lfd conducting a novel ibv and ndv mrt-lamp-lfd detection assay which is specific and sensitive in detecting ibv and ndv simultaneously. furthermore, mrt-lamp-lfd does not require specialized instrumentations, making it suitable for on-site detection. in conclusion, mrt-lamp-lfd is a promising qualitative detection tool, and is even applicable in some low-resource locations. supplementary data are available at poultry science online. table s . the tcov strain and other avian virus strains used in the specificity study figure s . phylogenetic analyses based on pcr and lamp target sequences of (a) ibv and (b) ndv. strains used in specificity study are in bigger font. wild strains detected during our avian virus monitoring, of which genome sequences have not been submitted to genbank, were labeled by solid circles; artificially synthesized sequences were labeled 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clinical samples evaluation of loop-mediated isothermal amplification for the rapid, reliable, and robust detection of salmonella in produce brief review of monitoring methods for loop-mediated isothermal amplification (lamp) this research is funded by the national key r&d program of china ( yfd ), the china agriculture research system (cars- ), the national system for layer production technology (cars- -k ), and the project for science and technology support program of sichuan province ( nz ). we declare that we have no conflict of interest. key: cord- -p pns r authors: malik, yashpal singh; verma, atul; kumar, naveen; deol, pallavi; kumar, deepak; ghosh, souvik; dhama, kuldeep title: biotechnological innovations in farm and pet animal disease diagnosis date: - - journal: genomics and biotechnological advances in veterinary, poultry, and fisheries doi: . /b - - - - . - sha: doc_id: cord_uid: p pns r the application of innovative diagnostic technologies for the detection of animal pathogens at an early stage is essential in restricting the economic loss incurred due to emerging infectious animal diseases. the desirable characteristics of such diagnostic methods are easy to use, cost-effective, highly sensitive, and specific, coupled with the high-throughput detection capabilities. the enzyme-linked immunosorbent assay (elisa) and polymerase chain reaction (pcr) are still the most common assays used for the detection of animal pathogens across the globe. however, utilizing the principles of elisa and pcr, several serological and molecular technologies have been developed to achieve higher sensitivity, rapid, and point-of-care (poc) detection such as lateral flow assays, biosensors, loop-mediated isothermal amplification, recombinase polymerase amplification, and molecular platforms for field-level detection of animal pathogens. furthermore, animal disease diagnostics need to be updated regularly to capture new, emerging and divergent infectious pathogens, and biotechnological innovations are helpful in fulfilling the rising demand for such diagnostics for the welfare of the society. therefore, this chapter primarily describes and discusses in detail the serological, molecular, novel high-throughput, and poc assays to detect pathogens affecting farm and companion animals. livestock, poultry, and aquaculture are among the fastest growing and expanding agriculture sectors to fulfill the need of the growing population of humans. however, the growth in this sector is under the continuous increasing threats of infectious diseases worldwide. this menace is further aggravated by globalization in animal trade for various purposes. the sudden entry of an infectious disease in a new country or geographical location could lead to delayed diagnosis and rapid spread into the susceptible animal population. in response to climate change, vector-borne diseases are also increasing worldwide. to prevent the spread of infectious diseases, one of the basic and critical requirements as prescribed by the world organization of animal health (oie) is the application of rapid, accurate, and highly sensitive identification of infectious agents. though the term "biotechnology" was coined in the year by karl ereky, the tangible biotechnological advancements in improving the human and animal health were started in the late th century. since then, biotechnological applications have been making significant contributions in the development of novel powerful diagnostic assays for the efficient diagnosis and control of animal infectious diseases. importantly, biotechnology has made the availability of pen-side tests for use at the field level to detect the causative infectious agent during a disease outbreak. in this chapter, we primarily describe and discuss the innovative biotechnological advancements made in the animal disease diagnosis in a step-wise manner. the impact of infectious diseases is immense and is felt all across the world. infectious diseases have affected the whole society, economy, and political system. vital sectors are under continuous economic loss and unrelenting development. the infectious diseases have taken a huge physical toll on animals and humans. this has pressed on humanity and has caused substantial economic, social, and mental losses. thus, it is a matter of animal health and economic interest to invest in strategies to give a blow to infectious diseases and put them under control. elimination of the pathogens and/or their vectors from their natural reservoirs would always be a first thought, but the removal is not easy, as they are constantly emerging and it is always very difficult to predict the emergence of infectious agents. evolution of pathogens is putting extra challenges, pressing on humanity to look at the new strategies and forcing the researchers to look for innovative ones. the newly evolved pathogens are always more advanced and deadly from the previous ones and put up a strong resistance. "from the evolutionary perspective, they [viruses and bacteria] are 'the fittest' and the chances are slim that human ingenuity will ever get the better of them." with the increase in the knowledge of infectious diseases and science, the degree of pace in pathogen discovery has increased. to keep pace and for better diagnosis, new tools and techniques need to keep on evolving. this is not only to quickly detect the pathogens, but also to make predictions with probable and possible outbreaks. to understand the scenario and to reach a definite conclusion, knowledge of epidemiology and pathogenic etiology also needs to be studied. this will facilitate in understanding the ancestry of the pathogen, and provide an insight and a mechanism for the epidemic, endemic, and even pandemic outbreaks. this system will also assist in understanding the interface transmission between and the directional flow of the zoonotic infectious diseases. thus, phylogenetic analysis and epidemiology would aim toward strategizing the challenges during pathogen surveillance and discovery. sars, a coronavirus was pandemic in . but epidemiology and microbiology mediated to stem its disastrous results and also the causative agent of sars was identified. bacteria, viruses, and parasites, present in feces, contaminate foodstuffs and cause disease in humans and animals, affecting the social set up and consumer demands. to increase the productivity and for the maintenance of good health of animals, antibiotics are frequently administered resulting in the growth and emergence of antibiotic-resistant bacteria. this further aggravates the condition, and makes the situation more appalling. overseas imported pets were also found to transmit and carry over the diseases to humans (smith et al., ) . even aquaculture is at risk of contaminants with the virus directly affecting the marine lives, as in the case of the new virus discovered in salmon (finstad et al., ) . even honeybees and other pollinators are transmitting pathogens like fungi, bacteria, and viruses through the contaminated food items (cox-foster et al., ) . an array of classical and conventional techniques have been developed and used for the laboratory diagnosis of infectious agents or pathogens. the techniques include serological, cell culture, and electron microscopyÀbased methods, which are either time-consuming or labor-intensive or both. however, with the advancement in the biotechnology field, new and robust diagnostic techniques are continuously evolving and taking over the conventional methods (caliendo et al., ) . presently, molecular detection-based methods such as polymerase chain reaction (pcr) or its variants, and serological methods such as enzyme-linked immunosorbent assay (elisa), are being used worldwide for the accurate diagnosis of many animal diseases. however, point-of-care (poc) and high-throughput novel assays have been developed recently. furthermore, we discuss the pros and cons of frequently used diagnostics assays for animal diseases in accordance with the following sections: serological methods were introduced in the early s for the diagnosis of pathogens. various serological diagnostics have been developed such as complement fixation, counter-immunoelectrophoresis, immunofluorescence in cell culture, elisa, radio immunoassay, immune adherence haemagglutination assay, reverse passive hemagglutination assay, latex agglutination (la), chemiluminescent immunoassay, and immunochromatography test (ict). among these, ict and elisa especially sandwich elisa and competitive elisa are used frequently in the commercial diagnostic kits for animal diseases worldwide. ict assays mostly utilized mammalian igg in commercial diagnostic kits, however, avian igy antibodies, with added advantages over the mammalian igg, have been employed for the detection of norovirus, rotavirus, and astrovirus in the fecal samples with good sensitivity and specificity ranging between % and % (khamrin et al., ) . modifications in elisa format or combinations with other diagnostic methods have proved novel ways to detect pathogens more efficiently and accurately. for example, recently a novel elisa for the detection of group a rotavirus antigen in the fecal samples of multiple host species has been developed . this assay utilizes the potential use of synthetic peptides and is based on the detection of conserved vp protein using anti-recombinant vp antibodies as capture antibodies and anti-multiple antigenic peptide (identified and constructed from highly immune-dominant epitopes within vp protein) antibodies as detector antibodies. another assay, which is simple to perform without the requirement of laboratory facilities, is dot-elisa. a highly sensitive and specific dot-blot assays for rapid detection of staphylococcal enterotoxin-a in food has been reported (singh et al., ) . dot-elisa has been employed in diagnosing various important poultry diseases (alam et al., ; dhama et al., ; he et al., ; majumder et al., ; manoharan et al., ) . immuno-pcr is another powerful assay that has been used for the immunodetection of viral nucleic acids. by combining elisa with pcr, sensitivity of detection can be increased up to times and is especially useful in detecting low quantity viruses in the stool samples (bonot et al., ) . the major advantage of immune-pcr is that several viral nucleic acids can be detected simultaneously. recently, a combination of nanoparticles with the immuno-pcr, also known as nanoparticle amplified immune pcr (npa-ipcr), has been reported which increases the sensitivity -folds compared to elisa and several folds to rt-pcr. antigen detection using an antibody bound to gold nanoparticle cofunctionalized with thiolated dna complementary to a hybridized dna has been developed (perez et al., ) . here, the presence of antigen/virus particles activates the formation of a "sandwich" complex of gold nanoparticle construct, virus, and an antibody functionalized nanoparticles used for extraction. now, this complex is heated to c, thus releasing dna tags followed by the detection through real-time pcr. npa-ipcr offers a viable platform for the development of an early-stage diagnostics requiring an exceptionally low limit of detection. nucleic acid-based detections are used through the amplification methods, hybridization methods, which could be in situ, in vitro, and in vivo. the most common and widely used hybridization-based method is in situ hybridization, which could utilize fluorescent (fish) or chromogenic (cish) molecules. the cish-based assays for the rapid characterization of microorganisms, such as mycobacterium species and the dimorphic fungi in positive culture samples have been described (louro et al., ; scarparo et al., ) . recently, a fish-based assay has been developed for the identification and differentiation of mycobacterium tuberculosis complex from nontuberculous mycobacteria (baliga et al., ) . nucleic acid amplification methods are amongst the best in detecting pathogens with high sensitivity and specificity in the clinical samples. various modifications in nucleic acid amplification methods have provided collectively robust methods to yield better and accurate results. these modifications could be categorized into two amplification methods viz pcr and its variants, and isothermal amplification methods. these are the most common tools used for the pathogen detection worldwide. the three basic variants include ( ) real-time pcr, which is a modified version of conventional pcr, where quantification of dna sequence is possible without any further step of running the amplified product on agarose gel; ( ) multiplex pcr, where multiple sequences can be detected in a single reaction mixture, and ( ) reverse transcriptase pcr (rt-pcr) where rna is transcribed to cdna and this cdna is used in the amplification as template. the real-time pcr can utilize different fluorescence chemistries such as sybr green, taqman, or molecular beacon probes. recently, a taqman real-time rt-pcr assay has been developed for rapid detection and quantification of japanese encephalitis virus in swine blood and mosquito vectors (pantawane et al., ) . in isothermal amplification, a number of target dna copies increase at a constant temperature in just one cycle without the need of a thermocycler. various techniques have been developed using isothermal amplification methods viz nucleic acid sequence-based amplification (nasba), transcription-mediated amplification (tma), signal-mediated amplification of rna technology (smart), strand displacement amplification (sda), rolling circle amplification (rca), loopmediated isothermal amplification (lamp), isothermal multiple displacement amplification (imda), helicase-dependent amplification (hda), circular helicasedependent amplification (chda), single primer isothermal amplification (spia), and strand invasion-based amplification (siba). in all these methods, isothermal temperature amplified products can be visualized on gel through the various structures visible on gel or by the incorporation of dyes in the special structures formed on amplification serving in real-time detection. among all these techniques, lamp is the most widely used isothermal amplification method which is in fact an autocycling strand displacement dna synthesis, which deploys four primers forming a stem-loop dna by self-primed dna synthesis and a dna polymerase with strand displacement activity (malik et al., ; parida et al., ) . recently, an improved strategy using a double-labeled probe to overcome the problem of false positivity of lamp together with target gene real-time quantification is devised for detection of avian orthoreovirus (kumar et al., ) and salmonella spp. (mashooq et al., ) . other potential isothermal amplification techniques are recombinase polymerase amplification (rpa) and nasba. the former has brought a breakthrough in the detection of nucleic acids as it does not require denaturation of the template. rt-rpa is an extension of the above method, in which bacterial rt is used in the amplification of rna. rt-rpa was developed to study the outbreak of footand-mouth disease (fmd) disease in egypt (abd el wahed et al., ) . high degree of fidelity, portability, cost efficiency, simplicity, sensitivity, and tolerance to inhibitors, put this method into the category of resounding techniques, and implementation is quite easy at quarantine stations (moore and jaykus, ); while the latter one requires initial denaturation of the template followed by temperature labile polymerase dependent isothermal amplification and was designed specially to detect rna (compton, ) . a multiplex real-time nucleic acid sequence-based amplification (qnasba) system for the simultaneous detection of rotavirus a, norovirus genogroup ii/astrovirus has recently been developed (mo et al., a) . rt-nasba proved as more efficient than the conventional rt-pcr and taqman rt-pcr assays (mo et al., b) . a microarray is a multiplex lab-on-a-chip test. it is an arrangement of the large amount of biological materials for high-throughput screening on a solid support generally a glass slide, through the detection-based assays. microarray has done wonders in the high-throughput screenings and for the breakthrough causes of the outbreaks. simultaneous detections of coinfections and other more phenomenal changes during the outbreaks are the crucial developments to study the infectious diseases in endemic regions. their easiness has brought the working systems onto the platform on global diagnostics. multiple diagnostics with hybridizing ability put it at more ease to strategize the control management programs. but, this technique comes at high expenditures. data management skills and their interpretations need off to the most important tasks to be worked on. with the advent, new kits for point-of-care detections, bioelectric arrays, and liquid microarrays are in the development process. this would be an easy and an improved hybridization method for individual probe and target combinations with accurate detections. this will reduce the effort from clinical diagnosis to the personal level. these all will help in understanding the proper and common pathogens with scaling down the time. peptide nucleic acids (pnas) are highly versatile synthetic oligonucleotides, in which the native sugar-phosphate backbone of dna is replaced with amino acids. pnas bind to complementary dna strands with higher specificity and strength. furthermore, they are resistant to nucleases and proteases, making them a highly stable diagnostic reagent. the pna-based assay has greater sensitivity than direct sequencing and is significantly more affordable and rapid (ray and nordén, ) . the potential diverse uses of pna have been exhaustively described in a recent review (gambari, ) . a rapid label-free visual pna-based assay for detection and pathotyping of newcastle disease virus has also been reported (joshi et al., ) . similarly, pna-based beacons have also been used in hiv genotyping with high specificity (zhang and apella, ) . aptamers are artificial nucleic acid ligands that are isolated from combinatorial libraries of synthetic nucleic acid by an iterative process of adsorption, recovery, and reamplification. dna aptamers in particular have many advantages over antibodies (brody and larry, ) . aptamers, first reported in , are attracting interest in the areas of diagnostics and offer themselves as ideal candidates for use as biocomponents in biosensors (aptasensors), possessing many advantages over state of the art affinity sensors (o'sullivan, ) . the aptamers have proved to be potential diagnostic assays, especially in the detection of toxins such as brevetoxin- , potent marine neurotoxins (shimaa et al., ) , marine biotoxinpalytoxin (shunxiang et al., ) , β-bungarotoxin (β-butx), and a neurotoxin from the venom of bungarus multicinctus (ye et al., a) . furthermore, aptamers have been used for the serological detection of mycobacterium bovis (fu et al., ) , cryptosporidium parvum (iqbal et al., ) , and prion disease (saijin et al., ) . biosensors are portable, easy to handle, ultrasensitive, quick, and may be quite specific with less probability of a false positive. biosensors work on various principles viz detecting the changes in the ph, the ion concentrations, mass by specific hybridization, enzymatic reaction, loss of functionality, change in the electrical potential, change in color, and temperature. based on these principles, many biosensors have been devised for the detection of animal pathogens; for example, an extended-gate field-effect transistor for the direct potentiometric serological diagnosis of the bhv- (tarasov et al., ) , nanowire-based immunosensor for bovine viral diarrhea virus (bvdv) (montrose et al., ) , luminescence resonance energy transferÀbased biosensors for the ultrasensitive detection of the h strain (ye et al., b) , quartz crystal microbalance (qcm)Àbased immunosensors to detect h n (li et al., ) , and spectrosenstm optical microchip sensors for foot-and-mouth disease virus (fmdv) (bhatta et al., ) . the limited methods for detection of microbial signatures and the advent of new technology for quick and parallel gene expression capacities have eased in the detection of microbial disease. next-generation sequencing (ngs) is now being increasingly applied in understanding the molecular epidemiology, transmission, and characterization of animal pathogens. instead of gene-by-gene analysis, large deposits of genes available in the clinical sample can be detected in a single test. applications of ngs are considered as more resourceful. thus, it is widely accepted as a diagnostic tool and speedily is being replaced with most other molecular diagnostic technologies and has brought revolution in the diagnosis of pathogens. various modifications and improvements have brought a huge change in the sequencing and identification of genomes. it all started with pyrosequencing on roche , with small read lengths and less efficiency. roche was followed by ion-torrent illumina platform. the ngs has made it possible to sequence the complete viral genomes of many viruses cost-effectively such as including an avian influenza virus (croville et al., ) , classical swine fever virus (leifer et al., ) , and bluetongue viruses (rao et al., ) . recently, nanopore technology, with the promising improvement has brought a wonderful efficiency with emerging science and technology (goodwin et al., ) . nanopore systems can sequence both dna and rna viral genome in real time. this technology is based on the principle that when a strand of dna/rna is allowed to pass through a nanopore, the current is changed as the based a, t, c; and c passes through the pore in different combinations. using these systems, sequencing can be performed on the portable minion device, the benchtop gridion and the high-throughput, high-sample number promethion. recently, nanopore sequencing has proved a revolutionary diagnostic tool in detecting the ebola virus (hoenen et al., ) , influenza viruses (keller et al., ; wang et al., ) and porcine viral enteric disease complexes (theuns et al., ) . overall, the biotechnological innovations have equipped us now to have high-resolution sequencing tools that are revolutionizing the ability of veterinary diagnostic laboratories to detect emerging animal pathogens. with the advent of many biotechnological advances in veterinary diagnostics, point-of-care diagnostics (pocd) are now available for economically important animal diseases. the pocd is basically a simple, rapid, and portable diagnostic device that can be applied at the field level in effective monitoring the disease status. most of the commercially available pocds utilize either antigen/antibody or nucleic acid detection technologies. the former is usually available in the format of lateral flow assays or immunochromatographic strip tests. these assays are simple to use, rapid, inexpensive, disposable, and thus make them the ideal assay for pocd for animal pathogens. the commercially available immunochromatographic strip tests for economically important animal diseases are summarized in table . . these assays are equally sensitive as compared to elisa (ferris et al., ) . furthermore, combining these assays with smartphones has made the increased sensitivity and quick reporting of results possible (yeo et al., ) . therefore, these assays offer a novel herd level surveillance tool, and provide immediate results to the farmers. however, these assays have less analytical sensitivity as compared to nucleic acid-based pocd. the real-time pcr (qpcr) is a well-established tool with high sensitivity of pathogens detection and recently, qpcr has been transitioned into pocd platform. these platforms are fully automated combining nucleic acid extraction, thermal cycling, and reporting of results on-site. for example, minilab (enigma diagnostics) is a platform ( À kg) which can be easily carried to field level and it combines silica paramagnetic-bead-based nucleic acid extraction with lyophilized qpcr reagents in a single cartridge. this platform has been validated for aiv, asfv, csfv, and fmdv (goldenberg and edgeworth, ) . however, this platform is still not available commercially. there are other platforms that do not include nucleic acid extraction step (need to be done separately), such as genesig (primerdesign ltd, united kingdom), genedrive (epistem ltd, manchester, united kingdom), cepheid smartcycler (cepheid), t-cor (tetracore), and r.a.p.i.d. (idaho technologies) (takekawa et al., (takekawa et al., , . the genesig is now supplying lyophilized qpcr assay kits for bovine, equine, porcine, avian, canine, and feline different pathogens. however, these kits are not yet licensed for diagnosis of animal pathogens and are for research purposes only. as per the agreement on trade related aspects of intellectual property rights (trips) under paragraph of article , many countries have excluded diagnostic, therapeutic, and surgical methods of humans or animals from the scope of patentable systems. however, the important patented technologies that are being used in the various formats of diagnostic assays are provided in table . . a high-speed reagent system for qpcr, full velocity technology has been developed by the stratagene which saves time in addition to highly reproducible results. this technology has been used for infectious diseases, cancer, and drug sensitivities testing and already granted five us patents, us , us , us , us , and us . besides, a pocd product, dual path platform (dpp) has been developed by the chembio diagnostic systems, on which tests to detect hiv and syphilis have already been developed. this company in collaboration with national institutes of health and the infectious disease research institute, united states is working constantly to use this platform for the detection of infectious diseases of humans and animals. farm animals reared all over the world for major agricultural and production purposes majorly include cattle, buffalo, sheep, and goats. since the last few decades, a number of infectious diseases have been found associated with farm animals, causing colossal loss to the livestock rearing community and few of them being zoonotic in nature, becoming a problem for the public health. highly contagious livestock diseases such as fmd, hemorrhagic septicemia, peste-des-petits ruminants, and surra cause irreparable economic losses. several other infectious diseases of dairy cows such as bvd, johne's disease, tuberculosis, infectious bovine rhinotracheitis, and liver fluke infestations are generally regarded as being widespread and endemic. the best known and arguably most important discovery of farm animal diseases in the last few decades is bovine spongiform encephalopathy (bse), and others include digital dermatitis, neosporosis and bovine abortion, bovine neonatal pancytopenia, arcanobacterium pluranimalium, and schmallenberg virus. among all, the world organization for animal health classifies fmd and bse as diseases of major interest in cattle. these diseases are known to have a significant effect on dairy production either directly due to death or indirectly due to effects on fertility or milk production, and subsequently, culling. the disease conditions are usually identified based on history and clinical profile of the affected population, but for affirmative diagnosis of the pathogens responsible, the identification of the causal agent is done on the samples or clinical specimens for submission to diagnostic labs. the clinical profiles of several diseases overlap, making diagnosis a little tricky and cumbersome, so initially, the isolation of the infectious agent in pure form using cell culture systems or growth on specific and selective medium became a chosen method for the diagnosis of many pathogenic diseases in farm animals. albeit their usefulness as most sensitive method of detection, they are not used routinely due to time-lapse in confirming illness (bursle and robson, ) . these methods may take hours to several weeks to obtain a confirmatory result. therefore, other approaches based on morphology/biochemical properties of pathogens took the lead and were favored for pathogen detection and identification. several infectious viral disease agents viz astrovirus, adenovirus, rotavirus, etc. were identified through electron microscopy (ong and chandran, ) . but, these also have some drawbacks like more time consuming, less sensitivity, and costly instrumentation. apart from these techniques, approaches like detection of pathogen-specific antibodies or detection of antigenic proteins of pathogens were adopted and categorized under serological assays. serological assays measure antigenÀantibody interactions for diagnostic purposes. these assays are continuously being improved with technologies like rapid strip detection, thus becoming the most preferred tools and are broadly referred to as immunoassays. enzyme immunoassays (elisa) have always been the field applicable diagnostic methods in the detection of various farm animal diseases caused by fmdv, clostridium perfringens, m. bovis, and escherichia coli. hitherto reports have shown the problem of false negative results and cross-reactivity in some of the serological methodologies. innovations including the use of synthetic biology by making highly reactive peptides help to increase the sensitivity and avoid the cross-reactivity issues to some extent. with the more recent advances in diagnostics with the availability of sequences, nucleic acid-based methods have complemented the established techniques as more specific and sensitive methods in detection of pathogens with lesser false positive results in comparison to serological-based methods (bursle and robson, ) . pcr and real-time pcr methods are regularly used in the detection of campylobacter, shigella, bovine respiratory syncytial virus, eimeria, salmonella species, and many other pathogens. likewise, seminested and nested pcr have been developed for the detection of babesia bovis and babesia bigemina. these nucleic acid-based techniques are amongst the standard detection methods and are routinely used for testing in diagnostic laboratories. to improve the efficacy and promote the simplicity, modifications in the form of isothermal amplifications, like lamp and polymerase spiral reaction (psr) have been adopted and are better in the application process, as these are easy to perform, portable, specific, sensitive, and most importantly, quick and cost-effective. lamp is established to be an apex, leading diagnostics for the detection of many farm animal-related diseases like fmd, brucellosis, bovine popular stomatitis, sheep pox, and goat pox (dukes et al., ; song et al., ; zhao et al., ) . likewise, psr has been developed for detection of brucella spp. (das et al., ) , bovine herpesvirus- (malla et al., ) , and canine parvovirus (gupta et al., ) . to further increase the sensitivity and specificity, the combination of elisa and pcr-like immune-pcr, proximity ligation assay, pcr-elisa, have been successfully discovered making the detection -fold more sensitive. the pathogen detected with these combinations includes low pathogenic strains of campylobacter (ding et al., ) . nasba, restriction fragment length polymorphism, amplified fragment length polymorphism, and random amplification of polymorphic dna, are various biotechnological tools that have been further advancing the diagnosis of various infectious diseases. in addition, ngs has brought a revolution in the diagnosis of many pathogens. it appears helpful in the identification of many pathogens, especially viruses in the fecal matter and those that could not be isolated in cell culture system. mining of sequences in samples gives varied genome sequences providing the clues of not only pathogens, but also new strains, genotypes, new viruses, and even the zoonotic efficiencies of the viruses. anis and coworkers demonstrated that targeted bovine ngs is a specific and cost-effective tool for diagnosis of major bovine pathogens in clinical samples (anis et al., ) . even pathogens with low pathogenicity such as bovine enteroviruses (bev), adenoviruses in wild captive animals, and hepatitis e viruses have been revealed in the mining of sequences in the sample. one of the approaches to disease diagnosis is the development of biosensors. assays based on biosensors uses the transducers to convert the biological interaction of pathogen with its specific antibodies to measurable signals. biosensors have been quite useful in the diagnosis in poc detection. biosensors with specific biochemical recognition helped in the identification of e. coli in cattle (dharmasiri et al., ) . colibacillosis is also seen in a variety of farm animals like cattle, pigs, and goats. in c. perfringens detection, epsilon-toxin-specific monoclonal antibody was immobilized onto single-walled carbon nanotubes and adjusted to detect relevant concentrations of toxin in nanomolars and were comparable to elisa-based results. many other methods like mass spectrometry, microarrays, and maldi-tof are also under employment for the detection of many farm animals-associated pathogens, like francisella tularensis, staphylococcus aureus, enterococcus faecalis, e. coli (demirev and fenselau, ; lundquist et al., ; van baar, ) . companion animals are the domesticated animals kept for company of human beings or for utilitarian purposes, that is, guarding, herding, military/police activity. they have grown along with the human civilization and evolution and have developed a good bond with humans. although there is a variety of species which are suitable as companion animals (dogs, cats, rabbit, ferrets, caged birds, fishes, and guinea pigs), dogs and cats are the most common companion species. their physical, behavioral, social, and emotional needs can be easily met at home. on the other hand, dogs and cats play a fundamental role in the life of human beings with many physiological and psychological benefits (wood et al., ) . also, living with companion animals makes human surroundings happier and prosperous. as these animals enrich our lives, it becomes our responsibility to take care of the companion animals and to protect them from any kind of harm. there is a spectrum of infectious diseases that occur in companion animals. lyme disease, psittacosis, hookworms, and salmonella are amongst the most common diseases in pet animals. (lembo et al., ; palatnik-de-sousa et al., ). since these animals share a close environment and are in direct contact with humans, hence they have a potential to spread these infections to human beings. chances of introduction of new diseases also arise on the import of animals from foreign lands. thus, maintenance of strict trade rules and regulations and hygienic conditions becomes a necessity. once the disease occurs, it is important to identify the causative agent to improve the effectiveness of treatment and to control the disease. since the clinical observations are not sufficient and can overlap with other diseases leading to misdiagnosis, several validated laboratory assays are used for confirmatory diagnosis. until years ago, these laboratory tests exploited cell culture (for isolation of specific pathogen) and serological assays (for detection of antibodies generated against a specific pathogen or antigenic proteins). few examples composed of vero cells and recombinant vero-slam cells used for culturing rickettsia rickettsia, and toxoplasma gondii, madin-darby canine kidney cells for canine adenovirus and canine herpesvirus. similarly, serological methods include a long list. immunodiffusion testing is most often used to detect antibodies to fungal pathogens in dogs, such as aspergillus fumigatus, coccidioides immitis, and blastomyces dermatitidis. agglutination tests include the microscopic agglutination test for serologic diagnosis of leptospirosis (agglutination of live leptospires) and the cryptococcal antigen la test (agglutination of antibody-coated latex beads). hemagglutination inhibition is used to determine antibody titers to cpv and canine influenza virus, and it evaluates the ability of serum to inhibit erythrocyte agglutination by these viruses. elisa is commonly used for the detection of feline retroviral, heartworm, giardia, leishmania, and tick-borne infections. indirect ifa for serologic testing in dogs and cats include quantitative serology for some tick-borne infectious diseases (e.g., ehrlichia canis, anaplasma spp.). direct immunofluorescence assay in veterinary medicine include diagnosis of giardia oocysts, felv within monocytes in peripheral blood or bone marrow, or canine distemper virus within epithelial cells from a conjunctival scraping. many of these assays involve the use of polyclonal antibodies, or, more commonly, monoclonal antibodies-dependent diagnosis. with the recent boom in the database of sequences for pathogens, new diagnostic tools like pcr, real-time pcr, and multiplex pcr have almost replaced the established techniques and are adopted as routine diagnostics for testing clinical samples. canine respiratory coronavirus, canine adenovirus- , canine herpesvirus, feline herpesvirus- , canine distemper virus, west nile virus, and encephalitis viruses are some of the examples, which are routinely diagnosed using these techniques. other biotechnological tools cover hybridization assays, pna, nanoparticles-based assays, etc. hybridization-based methods have also been found to be compatible with the diagnosis of many diseases and composed of taqman-based probes, molecular beacons, and fret-based probes. although not yet widely used for veterinary applications, pna probes are now increasingly available to detect target dna. fluorescent pna probes, followed by signal amplification were used to differentiate between m. tuberculosis complex and nontuberculous mycobacterium spp. (zerbi et al., ) . in another example, ngs has also been used for the comparison of the oral microbiome of canines with their owners as they are in direct contact with their pets and many diseases might get transmitted to them (oh et al., ) . gold nanoparticle-based immunochromatographic strip test using a combination of mab and pab was developed as an alternative for on-site and cost-effective diagnosis of cpv infection . another use of biotechnology has been observed for rapid and early detection of cpv using a qcm biosensor (kim et al., ) . also, for genotyping of cpv- , conventional methods are time consuming, therefore, a probe-based duplex fluorescence melting curve analysis (fmca) for genotyping six different cpv- variants (original cpv- , cpv- a, cpv- b, cpv- c, and vaccine strains of cpvpf and cpvint) using only two taqman probes has been developed (liu et al., ) . despite the fact that a wide range of diagnostic tools are available, there is a considerable chance for better advancement in diagnostics, in terms of speed and accuracy, to control and eradicate economically important diseases. in the near future, use of new biotechnological tools like biosensors and nanotechnology will pave the way. further, ngs platforms like minion (a portable, real-time ngs sequencer) coupled with nanopipe analysis are promising tools to perform bacterial and viral disease investigation in low throughput laboratories and specifically in the field (beato et al., ; shabardina et al., ) . although, yet not been adopted for animal disease diagnosis, but novel platforms such as smartphonebased diagnosis (which expands nucleic acid-based detection assays toward pocd) like rt-lamp and fluorescent lateral flow immunoassay (already developed for zika virus and dengue virus) provide exciting opportunities for veterinary diagnostics in the near future (rong et al., ) . biotechnological innovations have brought new generation diagnostic methods for rapid and sensitive diagnosis of various diseases of livestock and pet animals. infectious diseases entail remarkable economic loss, weak food production system, food insecurity, and high maintenance cost of the agriculture sectors including farm animals, poultry, and aquaculture. besides, these diseases carry a huge risk of transmission to humans as sporadic and endemic zoonoses. classical and conventional diagnostic methods are labor intensive, time consuming, less sensitive, and difficult to meet the needs of the emerging pathogen diagnostics. thus, new innovations have to be worked on and need to be practiced. over the long term, innovations will be helping in the diagnosis of pathogens with accurate, sensitive and specific detections. ngs, biosensors, and advanced amplification techniques will persist for longer periods in their constant modified forms. innovations will always be bringing the new applications in the diagnostics for the improved versions of techniques. new technique applications come with the cost and unbroken funding will be putting new prospective techniques into the trials. these techniques should be simplified in the innovations for their easy practices at the field itself, without looking for any skilled personnel/highly equipped laboratories. there is no conflict of interest. a portable reverse transcription recombinase polymerase amplification assay for rapid detection of foot-and-mouth disease virus dot elisa for newcastle disease, infectious bursal disease and mycoplasmosis evaluation 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human rotavirus using nsp gene specific reverse transcription loop-mediated isothermal amplification assay novel polymerase spiral reaction (psr) for rapid visual detection of bovine herpesvirus genomic dna from aborted bovine fetus and semen rapid serological profiling by an immunocomb-based dot-enzyme-linked immunosorbent test for three major poultry diseases development and evaluation of probe based real time loop mediated isothermal amplification for salmonella: a new tool for dna quantification rapid and simultaneous detection of three major diarrhea-causing viruses by multiplex real-time nucleic acid sequence-based amplification comparative detection of rotavirus rna by conventional rt-pcr, taqman rt-pcr and real-time nucleic acid sequence-based amplification novel single gold nanowire-based electrochemical immunosensor for rapid detection of bovine viral diarrhoea antibodies in serum development of a recombinase polymerase amplification assay for detection of epidemic human noroviruses comparison of the oral microbiomes of canines and their owners using next-generation sequencing identification of gastroenteric viruses by electron microscopy using higher order spectral features aptasensors À the future of biosensing? decrease of the incidence of human and canine visceral leishmaniasis after dog vaccination with leishmune in brazilian endemic areas taqman real-time rt-pcr assay for detecting japanese encephalitis virus in swine blood samples and mosquitoes loop mediated isothermal amplification (lamp): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases detection of respiratory syncytial virus using nanoparticle amplified immuno-polymerase chain reaction deep sequenc-ing as a method of typing bluetongue virus isolates peptide nucleic acid (pna): its medical and biotechnical applications and promise for the future smartphone-based fluorescent lateral flow immunoassay platform for highly sensitive point-of-care detection of zika virus nonstructural protein aptamer-based assay for prion diseases diagnostic direct identification of mycobacteria from mb/bact alert d bottles: comparative evaluation of two commercial probe assays nanopipe-a web server for nanopore minion sequencing data analysis development and evaluation of a gold nanoparticle-based immunochromatographic strip test for the detection of canine parvovirus aptamer-based competitive electrochemical biosensor for brevetoxin- enzyme-linked, aptamer-based, competitive biolayer interferometry biosensor for palytoxin development and evaluation of simple dotÀblot assays for rapid detection of staphylococcal enterotoxin-a in food establishment of loop-mediated isothermal amplification (lamp) for rapid detection of brucella spp. and application to milk and blood samples field detection of avian influenza virus in wild birds: evaluation of a portable rrtÀpcr system and freeze-dried reagents rapid diagnosis of avian influenza virus in wild birds: use of a portable rrtÀpcr and freeze-dried reagents in the field a potentiometric biosensor for rapid on-site disease diagnostics nanopore sequencing as a revolutionary diagnostic tool for porcine viral enteric disease complexes identifies porcine kobuvirus as an important enteric virus characterisation of bacteria by matrix-assisted laser desorption/ionisation and electrospray mass spectrometry minion nanopore sequencing of an influenza genome the pet connection: pets as a conduit for social capital? recognition of bungarus multicinctus venom by a dna aptamer against β-bungarotoxin upconversion luminescence resonance energy transfer (lret)-based biosensor for rapid and ultrasensitive detection of avian influenza virus h subtype smartphone-based fluorescent diagnostic system for highly pathogenic h n viruses amplified in situ hybridization with peptide nucleic acid probes for differentiation of mycobacterium tuberculosis complex and non-tuberculous mycobacterium species on formalin-fixed, paraffin embedded archival biopsy and autopsy samples advantages of peptide nucleic acids as diagnostic platforms for detection of nucleic acids in resource-limited settings development of loopmediated isothermal amplification assay for specific and rapid detection of differential goat pox virus and sheep pox virus all the authors of the chapter thank and acknowledge their respective universities and institutes. key: cord- -qduf kp authors: assane, dieng; makhtar, camara; abdoulaye, diop; amary, fall; djibril, boiro; amadou, diop; niokhor, diouf jean baptiste; amadou, diop; cheikh, loucoubar; ndongo, dia; mbayame, niang; lamine, fall; bouh, boye cheikh saad title: viral and bacterial etiologies of acute respiratory infections among children under years in senegal date: - - journal: microbiol insights doi: . / sha: doc_id: cord_uid: qduf kp acute respiratory infections (aris) are the leading cause of infectious disease–related morbidity, hospitalization, and morbidity among children worldwide. this study aimed to assess the viral and bacterial causes of ari morbidity and mortality in children under years in senegal. nasopharyngeal samples were collected from children under years who had ari. viruses and bacteria were identified using multiplex real-time reverse transcription-polymerase chain reaction and conventional biochemical techniques, respectively. adenovirus was the most prevalent virus ( %; n = ), followed by influenza virus ( . %, n = ), rhinovirus ( . %; n = ), enterovirus ( . %; n = ), and respiratory syncytial virus ( . %; n = ), whereas streptococcus pneumoniae ( %; n = ), moraxella catarrhalis ( . %; n = ), and haemophilus influenzae ( . %; n = ) were the most commonly isolated bacteria. virus pathogens seem more likely to be more prevalent in our settings and were often associated with bacteria and s. pneumoniae ( %; ) coinfection. respiratory tract infections (rtis) such as acute otitis media, sinusitis, bronchitis, and community-acquired pneumonia are a leading cause of infectious disease-related morbidity, hospitalization, and mortality among children worldwide, particularly in low-income countries. according to world health organization (who), the prevalence of hospitalized children under years with acute respiratory infections (aris) is estimated to be % and % of those were due to pneumonia. in addition, the number of childhood deaths annually related to aris is very important and is estimated between . and . million, and % of the deaths took place in africa and southeast asia which are the most. , bacteria and viruses have been reported as the main causes of aris. in children under years, aris are caused mainly due to viruses; respiratory syncytial viruses (rsvs), parainfluenza viruses, influenza virus a and b, and human metapneumovirus (hmpv) are the most common viruses isolated. , however, primary infections with viral pathogens can predispose to secondary bacterial infections, and the most frequently isolated bacteria in aris include streptococcus pneumonia and haemophilus influenzae. these bacteria were increasingly resistant to the most commonly used antibiotics for ari treatment, leading to increase in mortality rates, hospital durations, and health care-associated costs. in resource-limited countries, bacteria have been the main cause of aris. this could be explained by the inaccessibility of molecular diagnostic tools thus leading to inadequate antibiotics prescription and consequently contributed to a rapid increase in antimicrobial resistance among bacteria causing rtis. , in senegal, few studies on viral and bacterial etiologies of rtis are available in pediatric settings. respiratory tract infections are mainly empirically treated with antibiotics on a simple suspicion of bacterial infection. indeed, this could be one of the major causes of high morbidity and mortality rates. the aim of this study was to investigate the viral and/or bacterial infections associated with aris in children under years. this study has been approved by the ethics committee for research of the cheikh anta diop university of dakar. samples and information for questionnaires have been collected after patient's informed consent. bacteria were isolated from appropriate culture media and incubation conditions prior identification following microbiological standard procedures based on morphological, cultural, and biochemical or antigenic characters. strains were identified if the bacterial load was at least cfus/ml. data were analyzed using r statistical software version . . . over a period year, children with aris were included in this study including ( . %) men. overall, children were under months of age, were to months old, were to months old, were to months old, and were to months old. table shows the distribution of major viruses isolated during this study period, the frequency of single or coinfection, and the number of infections stratified by age group. among selected virus species, adenovirus was the most prevalent ( %, n = ), followed by influenza virus ( . %, n = ), rhinovirus ( . %, n = ), enterovirus ( . %, n = ), and rsv ( . %, n = ). single infection with adenovirus was very rare ( . %, n = ). however, adenovirus was associated with other viruses and bacteria in . % (n = ) and . % (n = ), respectively. in addition, adenovirus/other viruses/bacteria coinfections have been detected in . % (n = ) of children. influenza virus infections were associated with single-virus coinfections ( . %, n = ) or virus and bacteria coinfections ( . %, n = ). however, influenza virus single-infected children were not detected. low prevalence of both rhinovirus and enterovirus single infections was observed ( . %, n = ). these infections were most often associated with single virus or virus and bacteria coinfections. adenovirus, influenza virus, and enterovirus infections were more prevalent among children aged from to months. the prevalence of rsv infections associated with other viruses or other viruses and bacteria was . % and . %, respectively; however, no rsv single infection was detected. children aged from to months were more likely to be more affected with rhinovirus and rsv infections. the distribution of major bacterial clinical strains is shown in table . among isolated bacterial species, s. pneumoniae was the most prevalent bacteria ( %) followed by m. catarrhalis ( . %) and h. influenzae ( . %). bacterial single infections were also very rare: s. pneumoniae ( %), m. catarrhalis ( %), and h. influenzae ( %). no bacteria/bacteria coinfections were detected. however, coinfections with bacteria and viruses had been observed in children having aris and coinfection with s. pneumoniae ( . %) was most frequent followed by m. catarrhalis ( , %) and h. influenzae ( . %). the contribution of viral and bacterial pathogens to the clinical syndrome is depicted in tables and , respectively. acute respiratory infections may be caused by viruses, bacteria, or both. thus, determination of the causative agent is crucial to limit the abuse of antibiotics. in our study conducted over a period of year, we showed that viruses were the most common pathogens detected in aris. similar findings have also been reported in a study conducted in united states by obasi et al in . several studies conducted in other countries such as burkina faso, zambia, and niger provided evidence of the role of viruses in upper and lower airway diseases. in senegal, adenovirus was the most common pathogen detected from aris in children. this study confirms previous results reported between and in senegal by niang et al. the overall detection rate of adenovirus was % among the selected children. this adenovirus infection prevalence seems very high compared with rates documented from other countries, namely, ghana, burkina, and zambia. however, our results are in agreement with data reported in cameroon. overall, this confirms the overall high prevalence of adenovirus ari in africa. in addition to adenovirus, influenza virus, rhinovirus, and enterovirus are the most common viruses detected, as reported in other studies. , our study revealed viral codetection is frequent in aris. similar results were found in other countries. , , viral codetection in clinical settings is becoming more common since the introduction of molecular-based multiplex tests. although the clinical significance of these findings remains unclear, this seems to have no impact in disease severity. in this study, we also found that aris were associated with bacterial pathogens; among those, s. pneumoniae ( . %), m. catarrhalis ( . %), and h. influenzae ( %) were the most common. in contrast with our findings, high rate ( %) of s. pneumoniae detection was observed in niger among children having aris. in our study, prevalence of mono-infections with s. pneumoniae ( %), m. catarrhalis ( %), or h. influenzae ( %) was very low. thus, this low prevalence of bacterial pathogens in aris proves that antibiotics should no longer be systematically used in the treatment of aris. however, among low-prevalence bacterial isolates, codetections with viruses were more frequent and s. pneumoniae ( , %) coinfection was the most frequent in our findings. similar results were reported in other studies. , there results highlight the pivotal role of viruses in aris because primary infection with viral pathogens can predispose children to subsequent bacterial infections. our study has limitations; among others, the sample size which does not really allow to determine the direct association between viral or bacterial infection and disease severity. moreover, a case-control study would be more suitable to determine the exact role of viruses in aris pathogenesis. in conclusion, this study reports the profile of viral and bacterial pathogens among children under years hospitalized with aris in senegal. the high prevalence rates of viral infections and its clinical impact highlight the need to implement a systematic surveillance program for a better management of aris in children, particularly in resource-limited settings. estimates of wide distribution of child deaths from acute respiratory infections world health organization. acute respiratory infections in children rapid simultaneous diagnosis of infections with respiratory syncytial viruses a and b, influenza viruses a and b, and human para influenza virus types , and by multiplex quantitative reverse transcription polymerase chain reaction-enzyme hybridization assay (hexaplex) development of three multiplex rt-pcr assays for the detection of respiratory rna viruses acute bacterial sinusitis complicating viral upper respiratory tract infection in young children antibiotic resistance of bacteria responsible of acute respiratory tract infections in children attributable hospital cost and length of stay associated with health care-associated infections caused by antibiotic-resistant gram-negative bacteria identification of viral and bacterial pathogens from hospitalized children with severe acute respiratory illness in lusaka, zambia viral aetiology of respiratory tract infections in children at the paediatric hospital in ouagadougou (burkina faso) r: a language and environment for statistical computing. r foundation for statistical computing detection of viral and bacterial pathogens in acute respiratory infections viral and bacterial aetiology of severe acute respiratory illness among children < years of age without influenza in niger respiratory viruses in patients with influenza like illness in senegal: focus on human respiratory adenoviruses respiratory viruses in children hospitalized for acute lower respiratory tract infection in ghana viral etiology of severe acute respiratory infections in hospitalized children in cameroon viral aetiology of respiratory infections in children in south-western saudi arabia using multiplex reverse transcriptase polymerase chain reaction clinical disease severity of respiratory viral co-infection versus single viral infection: systematic review and analysis viral and bacterial detection in acute respiratory infection in children under five years bacterial and viral etiology in hospitalized community acquired pneumonia with molecular methods and clinical evaluation high nasopharyngeal pneumococcal increased by viral co-infection, is associated with invasive pneumococcal pneumonia the authors are grateful to cea-samef (dakar, senegal), micro csb system (dakar, senegal), and institute pasteur dakar (dakar, senegal) who have contributed to the success of this study. key: cord- -or zzjs authors: zhou, liang; zhang, ping; zhang, zhigang; fan, lidong; tang, shuo; hu, kunpeng; xiao, nan; li, shuguang title: a bibliometric profile of disaster medicine research from to : a scientometric analysis date: - - journal: disaster med public health prep doi: . /dmp. . sha: doc_id: cord_uid: or zzjs this study analyzed and assessed publication trends in articles on “disaster medicine,” using scientometric analysis. data were obtained from the web of science core collection (woscc) of thomson reuters on march , . a total of publications on disaster medicine were identified. there was a mild increase in the number of articles on disaster medicine from (n= ) to (n= ). disaster medicine and public health preparedness published the most articles, the majority of articles were published in the united states, and the leading institute was tohoku university. f. della corte, m. d. christian, and p. l. ingrassia were the top authors on the topic, and the field of public health generated the most publications. terms analysis indicated that emergency medicine, public health, disaster preparedness, natural disasters, medicine, and management were the research hotspots, whereas hurricane katrina, mechanical ventilation, occupational medicine, intensive care, and european journals represented the frontiers of disaster medicine research. overall, our analysis revealed that disaster medicine studies are closely related to other medical fields and provides researchers and policy-makers in this area with new insight into the hotspots and dynamic directions. (disaster med public health preparedness. ; : – ) n atural disasters, biological terrorism, nuclear leakage, public health emergencies, epidemic diseases, and other disasters directly threaten the survival and development of mankind. currently, a major disaster occurs almost daily in some part of the world. most population centers are concentrated in high-risk locales like metropolitan cities, which feature very frequent and multiple person-to-person contacts. high-risk occupations, international trade, and housing construction all increase the possibility of human exposure to disasters, leading to increased casualties after each disaster. the ever-increasing spiral of human populations, the rapid growth of technology, swift world-wide travel by millions of persons, and the exponential expansion of at-risk industries and residences combine to increase human exposure to disasters. in particular, the major casualties caused by the wenchuan earthquake, the nepal earthquake, and the indian ocean tsunami pose great challenges to disaster medicine. the wenchuan earthquake was one of the most devastating disasters in the past years and caused more than , casualties; the main causes of death were trauma and crush syndrome. in addition, there was a significant increase in the number of respiratory infections, enteritis, and skin diseases in the week after the earthquake. even a full year after the earthquake, some survivors began to suffer from posttraumatic stress disorder. disaster medicine has attracted global attention gradually by implementing emergency medical treatment, disease prevention, and health care science under the conditions of disastrous damage. after the september attacks, the united states made two major adjustments to the national disaster medical system (ndms) to form a high-efficiency operating mechanism called the national disaster medical rescue system. japan has also established a national rescue medical center in tachikawa city, tokyo. as a data transmission and command center for disaster medical care, it features a disaster medical information system used to determine damage for medical institutions. in addition, japan has enhanced its disaster emergency medical rescues by launching civil and community organizations. after the outbreak of severe acute respiratory syndrome (sars) in , china likewise began to attach importance to the establishment of an emergency medical system. then, after experiencing the wenchuan, lushan, and yushu earthquakes, and observing the actual rescue experience, it built an emergency medical rescue system, including a rescue command center and a medical rescue scene and rescue information platform. disaster medicine scholars have published a substantial amount of original research based on care during disaster rescues, emergency medical treatment, and disease prevention. however, the bibliometric profile of disaster medicine in the literature is still unknown. therefore, in this study, a scientometric analysis was conducted on disaster medicine to estimate the productivity of specific journals, countries, institutions, authors, and research areas, and to identify research hotspots and trends in this field. all of the data for this study were obtained from the web of science core collection (woscc) of thomson reuters on march , (incomplete data existed in ). the woscc, which includes the social sciences citation index, current chemical reactions, and index chemicus, is the most frequently used source of scientific information. the search term "disaster medicine" was used to retrieve titles, keywords, author information, abstracts, and references published from to . the following search string was used: (ts = (disaster medicine)) and languages: (english) and document type: (article or review). the impact factor of each journal was obtained from the journal citation reports science edition, accessed on march , . citespace iii ( bits) was used to analyze publication outputs and construct knowledge maps, to analyze the extracted records for citation characteristics, and to visualize the patterns and trends in disaster medicine. [ ] [ ] [ ] [ ] [ ] a total of studies on disaster medicine, published from to , were retrieved from the woscc (incomplete data existed in ). of these, ( . %) were original articles. although the number of publications increased only mildly from (n = ) to (n = ), the number of citations increased substantially from (n = ) to (n = ) ( figure ). of studies, ( . %) were cited at least once, with an average of . citations per article for , total citations. table shows the most frequently cited articles. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] among these articles, the most common topic was disaster rescue and post-disaster health effects ( of [ %]). the top-ranking paper ( citations) was published in chest and involved critical care treatment: "definitive care for the critically ill during a disaster: a framework for allocation of scarce resources in mass critical care -from a task force for mass critical care summit meeting, january - , , chicago, il." the article provided suggestions to perform triage while allocating scarce critical care resources during a disaster. the studies were published in different journals. the top-ranked journal, which published papers, was disaster medicine and public health preparedness, followed by two other journals with more than papers each: academic emergency medicine (n = ) and american journal of preventive medicine (n = ). disaster medicine and public health preparedness also had the greatest number of total citations (n = ), again followed by academic emergency medicine (n = ) and american journal of preventive medicine (n = ) ( table in the online data supplement). these research studies were published by countries. the top countries published of the studies, accounting for . % of the total number of publications. the country with the greatest number of publications was the united states (n = ), followed by japan (n = ) and china (n = ) ( table in the online data supplement). similarly, among the sponsoring institutions, the top institutions published literatures, accounting for . % of the total number of publications. new york university had the most publications (n = ), followed by the university of washington (n = ) and johns hopkins university (n = ) (table s ). a total of authors contributed to these studies; the top authors accounted for studies, . % of the total. three authors tied for first place, each with publications: m. d. christian, f. della corte, and p. l. ingrassia ( table in the online data supplement). a total of research areas were represented, with the majority of articles focusing on public environmental/occupational health (n = ), general internal medicine (n = ), and emergency medicine (n = ) (table s ). the visualization analysis for reference citations was conducted by citespace iii. the parameters in citespace were as follows: time span = years ( - ); time slicing = ; term type = burst terms; selection criteria (c, cc, ccv) = ( , , ) ( , , ) ( , , ) . the top most cited or occurring items from each slice were selected. the pathfinder network method was used to streamline the network and to map the visualization analysis. the network revealed nodes and lines. in figure , the thicker circle indicates a higher level of between-study centrality. in general, a study with a centrality value equal to or greater than . can be considered a key study; therefore, the key studies were [subbarao i, ] ( . ), followed by [einav s, ] ( . ) and [gillett b, ] ( . ). in addition, the red circles represent burst studies which represent the frontier of a period ; among them, the key burst studies were [subbarao i, ] and [walsh l, ] , which had the highest citation rates between and . figure presents the timeline view for hot keywords. the results show that the hotspots of disaster medicine during this period were spinal cord injury, conceptual framework, health professional, occupational medicine, medical surge capacity, oleic acid, lifesaving intervention, terrorist bombing, developing country, workforce, professionalization and west africa (figure ). in addition, the research papers published in journals represent the frontiers of certain subjects, and the references cited in these papers provide the knowledge base of the papers. in figure , the nodes of clusters # , # , # , # , and # were mainly distributed before (the knowledge base), while the nodes of clusters # , # , # , # , # , # , and # were mainly distributed between and (the frontiers). a total of papers on disaster medicine research were included in this analysis. the visualization was generated by the carrot system based on the first results of a search on regenerative medicine. there were results from the lingo clustering algorithm; the first ranked cluster was practices in disaster (n = ), followed by hospital disaster (n = ), disaster events (n = ), disaster setting (n = ), and earthquake disaster (n = ) (figure ). in addition, the time interval is depicted as a blue line, whereas the time period that represents a burst cited journal is depicted as a red line, indicating the beginning and the end of the time interval of each burst. the top burst references were public health preparedness ( . ), hurricane katrina ( . ), and european journal ( . ) ( table ) . to the best of our knowledge, this is the first scientometric analysis on the topic of disaster medicine. the results indicated a significant increase in the number of publications ( and ) . this increase may be due to several major disasters. the year was the first year after the haiti earthquake, and much of the related research focused on the implementation and development of international medical rescue. [ ] [ ] it was also the third year after the wenchuan earthquake, research on which mainly focused on disease classification and patient management of the patients. [ ] [ ] finally, was the th anniversary of the / attacks; the relevant research mainly focused on post-disaster effects, including respiratory and mental health problems, among survivors and rescuers. [ ] [ ] the united states, japan, and china published the most research on disaster medicine. this may be because the frequent occurrence of disasters in these countries has caused serious casualties and property losses. , [ ] [ ] [ ] likewise, these countries have participated in international humanitarian relief efforts many times, so they have a wealth of medical rescue experience. [ ] [ ] [ ] the top studies (by number of citations) all emphasized a lightweight map of major terms on disaster medicine. burst strength: representing the intensity of the frequency of a key word suddenly increasing over a short period of time. the burst-detection algorithm can be adapted for detecting sharp increases of interest in a specialty. in citespace iii, a current research front is identified based on such burst terms extracted from titles, abstracts, descriptors, and identifiers of bibliographic records. burst-detection algorithms can identify emergent terms; centrality: evaluating the parameter of the number of lines on a certain node, the larger the value, the more the number of lines, that is, the importance of the node in the whole network. disaster medicine and public health preparedness categories of disaster medicine, except for [leaning j, ] , which summarizes the challenges and pressures posed by natural disasters to public health. these categories are disaster rescue , [ ] [ ] [ ] [ ] and post-disaster health effects. [ ] [ ] [ ] [ ] overall, the most cited article was "definitive care for the critically ill during a disaster: a framework for allocation of scarce resources in mass critical care -from a task force for mass critical care summit meeting, january - , , chicago, il," which offers guidance for allocating scarce critical care resources, drawn from a task force on mass critical care. this task force provided several suggestions for managing the triage process when medical systems are overwhelmed. in addition, [devereaux av, ] was sponsored by new york university, which sponsored the greatest number of disaster medicine publications from to . among the authors, f. della corte, m. d. christian, and p. l. ingrassia were the most productive. in their papers, the most cited articles were all associated with the "task force for mass critical care." , [ ] [ ] among the studies of post-disaster health effects, the most cited article was "trends in respiratory symptoms of firefighters exposed to the world trade center disaster: - ," which described trends in post- respiratory and gastroesophageal reflux disease symptoms in wtc-exposed fire fighters. this study also contributed to the literature on public environmental/occupational health, the discipline that produced the most disaster medicine studies from to . based on the co-citation analysis, "a consensus-based educational framework and competency set for the discipline of disaster medicine and public health preparedness" had the highest centrality; it developed a new educational framework for disaster medicine and public health preparedness, based on consensus identification from an expert working group in the american medical association. a time-view map of the co-citation activities appears to the left of the column with the clusters' labels. new clusters include cluster # on spinal cord injury, # on conceptual framework, and # on health professionals; the landmark publications include [subbarao i, ] and [walsh l, ] . they were also the newest burst strength publications, which suggests that "public health preparedness" will become a hot topic in disaster medicine. terms analysis provides a reasonable description of research hotspots (areas of focused attention by a number of scientific researchers, addressing a set of related research problems and concepts), whereas burst words represent new research frontiers (emerging trends and abrupt changes that occur in a timely manner). as shown in figure , the top hotspots of disaster medicine research were: ( ) "practices in disasters." these papers focus on the practical elements of disaster medicine, including the treatment of wounded, effect evaluation, first aid, and disaster medical education. [ ] [ ] [ ] [ ] [ ] ( ) "hospital disasters." these papers also focus on practical elements of disaster medicine, including modular management, humanitarian relief, first aid management processes, and disaster emergency departments. [ ] [ ] [ ] ( ) "disaster events." these papers summarize the casualties of disasters and their impact on public health. - ( ) "disaster settings." these papers focus on the medical needs of the disaster, including medical personnel, medical equipment, and medical technology. - ( ) "earthquake disasters." these papers focus on medical rescues during an earthquake, including the medical decision-making, the rescue process, and the treatment of the special population. [ ] [ ] [ ] [ ] in addition, several burst terms were detected by cite space iii and are considered indicators of research frontiers over time. in the results, the time interval is shown as a blue line, and the time period that represents a burst term category is shown as a red line, indicating the beginning and the end of the time interval of each burst. therefore, the three newest frontiers were: ( ) "impact." these papers focus on post-disaster effects on the physiology and mental health of survivors, including their daily behaviors, physiological indicators, and mental states. [ ] [ ] ( ) "public health preparedness." these papers focus on the establishment, evaluation, and management of medical rescue systems. - ( ) "public health emergencies." these papers focus on the training of emergency personnel, the promotion of emergency technology, and the management of emergency procedures. , [ ] [ ] limitation in this study, although the noise data can be reduced by setting up the requisite statistical parameters in citespace iii, the source of data is limited by a generic search term strategy, which is likely to lead to some noise in the selection of articles. the major findings of the present scientometric study are helpful for all those involved in worldwide disaster medicine research. indeed, this study can help researchers better understand disaster medicine research worldwide and be useful, for example, in choosing appropriate journals for publication and collaborations. fellows choosing an institution for advanced work may also be interested in such an analysis. journals can determine where they stand in relation to other journals in publishing articles related to disaster medicine. governments and policy-makers can also ascertain the most effective countries and institutions in the world in this field, and this analysis may assist them to apprehend and predict the hotspots and dynamic directions of disaster developing health information documentation in disaster update in intensive care and emergency medicine medical action and reflections on china's rescue in nepal emergency medical rescue efforts after a major earthquake: lessons from the wenchuan earthquake the pandemic and all-hazards preparedness act: its contributions and new potential to increase public health preparedness development of the japanese national disaster medical system and experiences during the great east japan earthquake emergency medical rescue major earthquakes: lessons from the wenchuan earthquake emerging trends in regenerative medicine: a scientometric 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disaster medicine among health professionals, medical students, and local residents in building community disaster resilience: perspectives from a large urban county department of public health assessing disaster preparedness among latino migrant and seasonal farm workers in eastern north carolina association of community health nursing educators: disaster preparedness white paper for community/ public health nursing educators characteristics of effective disaster responders and leaders: a survey of disaster medical practitioners national differences in regional emergency department boarding times: are us emergency departments prepared for a public health emergency this study was supported by the third military medical university military doctoral program (no. jskt ).lz and pz conceived and designed the paper. lz, nx, and sl wrote the article. zz, lf, st, kh, lz, and pz collected and analyzed the data. sl provided critical revisions. all authors approved the final version of the paper. to view supplementary material for this article, please visit https://doi.org/ . /dmp. . key: cord- - zxztadu authors: zhao, ye; cheng, jinlong; xu, gang; thiel, volker; zhang, guozhong title: successful establishment of a reverse genetic system for qx-type infectious bronchitis virus and technical improvement of the rescue procedure date: - - journal: virus res doi: . /j.virusres. . sha: doc_id: cord_uid: zxztadu in this study, a pathogenic avian infectious bronchitis virus (ibv) qx-type strain yn was successfully rescued by vaccinia virus based reverse genetic technology. ten fragments contiguously spanning the complete ibv genome were amplified and cloned into the vaccinia virus genome by homologous recombination. the full-length genomic cdna was transcribed in vitro, and its transcript was transfected into bhk- /n cells that could stably express ibv n protein. at h post transfection, the culture medium was harvested and inoculated into -day-old specific-pathogen-free embryonated chicken eggs to replicate the rescued virus. this strategy was chosen to facilitate the rescue procedure and to ensure that the recombinant ryn virus will not require any cell culture adaptations. after only one in ovo passage, the recombinant yn virus (ryn) was successfully recovered and confirmed to possess the introduced silent marker mutation in its genome. biological characteristics of ryn such as the eid( ), tcid( ), replication in ovo, and replication kinetcs in vitro were tested and all were similar to its parental strain yn. our findings demonstrate the successful construction of highly-pathogenic qx-type ibv using a modified rescue procedure, allowing for future studies of the molecular biology and pathogenicity of ibv field strains. avian infectious bronchitis is a highly contagious disease caused by infectious bronchitis virus (ibv) that affects poultry production worldwide (cavanagh, ) . ibv belongs to the gammacoronavirus genus, in the coronavirinae subfamily, of the coronaviridae family, in the order nidovirales. the ibv genome is approximately . kb in size. all coronaviruses share a similar genome organization with gene located at the ʹ end of the genome. as the replicase gene, it has been proved to be a determinant of pathogenicity of ibv (armesto et al., ) . the structural genes cluster at the ʹ end and encode the spike (s), membrane (m), envelope (e), and nucleocapsid (n) proteins (cavanagh, ) . ibv also possesses two accessory genes, gene and gene , which have been shown to be dispensable for virus replication in cell culture youn et al., ; hodgson et al., ; bentley et al., ) . however, it seems that the deletion of accessory genes a, b, a or b from ibv could induce an attenuated phenotype both in vitro and in vivo (laconi et al., ; van beurden et al., ) . although ibv has been controlled by routine vaccination for a long time, it remains a significant threat to the global poultry industry due to the existence of many serotypes (capua et al., ) . the high evolution and recombination rates of ibv result in new serotype viruses emerging in the field and often causing great losses since current vaccines only provide partial protection against new strains (cavanagh et al., ; de wit et al., ) . specifically, a new ibv variant has been circulating in china since . this virus has been identified as the qx strain and has been primarily associated with various renal pathologies (wang et al., ) . phylogenetic analyses showed that the ibv isolates that clustered with qx were mainly chinese isolates (zhao et al., ) . these results further indicated that ibv isolates prevalent in china were evolutionarily distant from massachusetts (mass)-type strains (terregino et al., ; abro et al., ; yan et al., ) . understanding the pathogenic mechanism and molecular characteristics of circulating strains could significantly facilitate the development of vaccines that provide efficient protection against the highly variable ibv. since the initial development of a reverse genetic system for rna (taniguchi et al., ) , this technique has proven to be a powerful method for investigating the molecular biology of rna viruses and for studying the role of individual genes in pathogenesis. however, despite these successes, not all reverse-genetic systems of positivestranded rna viruses can be easily established. the large genome of the coronavirus poses a great obstacle to the construction of reverse genetic systems based on full-length cdna. cloning techniques using traditional plasmid dna cloning vectors cannot guarantee stability of large fragments of coronavirus cdna, and many studies have found that certain coronavirus gene sequences can have toxic effects in e. coli or cannot be cloned into conventional plasmid vectors or passaged in prokaryotic systems (gonzalez et al., ) . therefore, infectious fulllength clones of coronaviruses have only been established at the beginning of the st century (casais et al., ) . subsequently, reverse genetics was successfully applied to study the role of accessory genes in viral replication youn et al., ; laconi et al., ) , the potential of ibv as a vaccine vector (britton et al., ; bentley et al., ) , the virulence determinants of the beaudette and m strains (fang et al., ; maria et al., ; keep et al., ) , and the relationship between the s gene and tissue tropism of the virus (casais et al., ; britton et al., ; promkuntod et al., ; shan et al., ) . however, to the best of our knowledge, all existing reverse genetics systems for ibv were either based on vero cell-adapted beaudette strain, which is non-pathogenic in chickens (geilhausen et al., ) , or the mass-type strains, which are genetic quite distant from the prevalent qx-type strains. neither of these systems therefore served as an ideal virus model to study the replication and pathogenic features of qx-type strains. therefore, the application of reverse genetics techniques to qx-type strains may allow for modification of the viral genome and may provide a powerful tool for novel vaccine development. we previously isolated and characterized a virulent ibv strain from yun nan province, china, designated yn strain. this isolate is genetically similar to most of the prevalent qx-type ibv strains found in china, albeit with increased pathogenicity compared with previously characterized strains (feng et al., ) . in this study, we describe the establishment of a reverse genetic system of the virulent yn strain. we also developed a modified rescue strategy, which combines full length rna electroporation with in ovo virus amplification, thus avoiding cellculture adaption and providing improved rescue efficiency of ibv. these advances will enable us to modify specific gene regions and investigate their influence on gamma coronavirus replication and pathogenicity. ibv strain yn was isolated and propagated in the allantoic cavities of -day-old specific-pathogen-free (spf) embryonated chicken eggs (ece) (feng et al., ) . the complete genome of yn has been sequenced and submitted to the genbank database under accession no. jf . vaccinia virus vnoti/tk, cv- , and d r, and bhk- cells were described previously hertzig et al., ) . vaccinia virus vnoti/tk and vaccinia virus recombinants were propagated, titrated, and purified on monkey kidney fibroblasts (cv- ) by standard procedures . all cells were maintained in minimum essential medium containing % fetal bovine serum (fbs, gibco, grand island, ny, usa), u/ml penicillin, and mg/ml streptomycin. human embryonic kidney (hek) t cells (american type culture collection) were cultured in dulbecco's modified eagle's medium (dmem) (hyclone, logan, ut, usa) supplemented with % fbs, u/ml penicillin, and mg/ml streptomycin. viral rna was extracted from yn-infected allantoic fluid with a genmark rna purification kit (genmark, tai-chung, taiwan) according to the manufacturer's directions. reverse transcription was performed with the primescript™ rt master mix (takara, tokyo, japan). vaccinia y. zhao, et al. virus research ( ) viral dna was extracted from strain vnoti/tk according to a previously described method (bridgen, ) . primers used for amplifying the yn strain complete sequence (genbank, accession number jf ) were designed according to the intrinsic restriction enzyme sites in the genome. silent mutations were introduced into the genome by primers if no restriction sites were present, and also served as molecular markers of the recombinant viruses. each dna fragment was amplified from cdna templates by pcr using the cloneamp hifi pcr premix (takara). pcr primer pairs used to amplify genomic regions are listed in table . nucleotide (nt) changes and restriction sites have been presented as boxes and italicized typeface in table , respectively. pcr amplification of cdna fragments was performed under the following conditions: denaturation at ℃ for min, then cycles of ℃ for s, °c - ℃ for s, and ℃ for - s depending on the size of the products ( s/kb), and a final extension at ℃ for min. the pcr products were purified using a nucleospin® gel and a pcr clean-up kit (macherey-nagel, düren, germany) and cloned into pgpt- vector, which contains a dominant selective marker gene, escherichia coli guanine phosphoribosyltransferase (ecogpt) (mulligan and berg, ) , under the control of the vaccinia virus p . k early/late promoter. in order to construct the recombinant vaccinia virus containing complete ibv genome through vaccinia virus-mediated homologous recombination, the first step was to construct flanking region belonging to the vaccinia virus genome into the pgpt- plasmid. the left flank vvt was bp in size, an xbai and an bamhi restriction site were added at the ʹand ʹ end for the ligation into pgpt- , and an additional type iis restriction site bambi and five nucleotides 'actta' (shown in red in table ) from ibv ′utr beginning was added at the ʹ end of vvt for the further ligation with ibv fragements. between the vaccinia dna sequence and bsmbi site was a bacteriophage t rna polymerase promotor and an additional g nucleotide for the initiation of the in vitro transcription reaction. the pcr product vvt was digested with xbai and bamhi to ligated into pgpt- plasmid, next, we constructed the ibv segment f (corresponding to nt to in the yn genome) by rt-pcr. upstream of the segment f ′-end was a bsmbi restriction site, downstream of the segment f ′-end was a bamhi site, thus to allow insertion of the f cdna downstream of vvt in pgpt- by restriction endonuclease digestion and t -ligation. on the other side of pgpt- , a bp vaccinia virus flanking region vv- ′ was cloned into the plasmid following digestion with xhoi and ecori, and an additional noti restriction site was added before the ecori site to stop in vitro transcription after the poly(a) tail. the obtained plasmid, pgpt- f, was integrated into the vaccinia virus genome by homologous recombination under the positive selection pressure of mycophenolic acid (mpa) in the presence of xanthine and hypoxanthine, to which only recombinant vaccinia viruses with the ecogpt gene show resistance (falkner and moss, ) . ecogpt-selected viruses were purified and sequenced to ensure that ibv segment f and the ecogpt gene had been inserted into the correct position in the vaccinia genome, and verified virus was termed vv-yn- f. next, to generate a complete ʹ untranslated region (utr) with a -bp poly(a) tail in strain yn, a dna was synthesized and cloned in the puc plasmid containing a bp fragment f (ibv-yn nts - ) plus nts of the ibv-yn ʹutr sequence ( , - , ) followed by a -bp poly(a) tail and bp of vaccinia virus flanking sequence. we also incorporated several restriction sites for further modification in the order: ʹ-saci- f ( - )-bamhi-sali- ʹutr ( , - , )-poly(a) ( bp)-noti-vv ( bp)-xhoi- ʹ. this fragment was digested with saci and xhoi and ligated into the digested pgpt- plasmid to replace the ecogpt gene. next fragment r corresponding to the ibv-yn genomic region from nt to containing bamhi and sali sites was amplified and inserted between f ( - ) and the ʹutr ( , ) in pgpt- (fig. a) . the homologous recombination between pgpt- r- ʹutr and vv-yn- f was performed by exerting negative selection pressure on d r cells due to the presence of -thioguanine (hertzig et al., ) . after successful construction of the ibv-yn fragment f r containing the ʹ-t promoter and the ʹ-synthetic poly(a) tail in the vaccinia virus genome, the other eight remaining fragments ( f, r, , f, r, f, r, ) were inserted into the vaccinia virus genome by vaccinia virus-mediated homologous recombination using the method described above. the individual recombination steps are summarized in fig. b . the final recombinant vaccinia virus was purified and sequenced by deep sequencing. the vaccinia virus possessing the complete ibv-yn genome was amplified on a large scale in bhk- cells and designated vvibv-ryn. the recombinant vaccinia virus vvibv-ryn was then used to construct recombinant vvibv, vvibv yn- a/egfp, by two rounds of vaccinia virus-mediated homologous recombination. this virus consisted of direct replacement of ibv gene a sequence, beginning at the orf a initiation codon, and ending at the orf a termination codon in order to maintain the genomic sequence upstream of the b gene. the introduced reporter gene egfp, replacing orf a, was expressed utilising the existing gene trs. first, plasmid pgpt△ a was constructed to encode ibv-yn nt - and - upstream and downstream of the gpt gene. recombination of vvibv-ryn with plasmid pgpt△ a resulted in the isolation of the recombinant vaccinia virus vvibv-gpt△ a. vvibv-gpt△ a was used for recombination with the plasmid pibv-gfp, encoding ibv-yn nt - upstream and ibv-yn nt - downstream of the gfp sequence. the resulting recombinant vaccinia virus vvibv yn- a/egfp was obtained after gpt negative selection. to improve the rescue of recombinant ibv a cell line stably expressing the ibv n protein (bhk- /n) was generated using a lentivirus-based system. the construction procedure is shown in fig. . briefly, the complete n gene of ibv-yn was amplified by rt-pcr. the primers used were as follows: ibv-n-bamhi-f: ttaaggatc cgccac catg gcaagcagtaaggcat; ibv-n-not i-r: aaa agc ggc cgc tca aag ttc att ttc acc aag tg. two restriction sites, bamhi and noti, were introduced into the pcr product to digest and insert it into a plasmid with the hsv-tk promoter just upstream of the insertion site, and the obtained n gene plus tk promoter plasmid was designated ptk-n. then, the n gene plus tk promoter was amplified using the ptk-n plasmid as template with primers: tk-n-f-ecori: aacatacgctctcc atcaaaacaaaa; tk-n-r-bamhi: gatgcaatttcct cattttattag gaaag g. the psmpuw-sv -puro plasmid was cut with enzymes ecori and bamhi and the fragment containing the n gene plus the tk promoter was inserted by an in-fusion reaction. after the n gene and tk promoter were inserted into the plasmid expression system psmpuw-sv -puro (cell biolabs, san diego, ca, usa) ( fig. a) , the plasmid was cotransfected along with pcmv-dr . and pmd g-vsvg (cell biolabs, san diego, ca, usa)into t cell lines to package the lentiviral particles. lentiviral vector production was performed according to standard protocols. briefly, subconfluent t cells in a t flask were cotransfected with μg of psmpuw-sv -puro-n, μg of pcmv-dr . , and μg of pmd g-vsvg using the transfection reagent xtremegene (sigma-aldrich, st. louis, mo, usa). after h, the medium was changed and the lentiviruses were harvested after h and filtered through a . μm filter. stable transduction of the lentiviruses was carried out as follows: of bhk- cells were seeded in a -well plate and transduced at day with of the lentiviral vector. selection was performed from day using μg/ml puromycin until the parental cells from a parallel experiment died (fig. b ). the expression of n protein in the bhk- /n cell line was detected by immunofluorescence analysis (ifa). bhk- /n cells were cultured in -well plates for h at ℃ and % co , the medium was then y. zhao, et al. virus research ( ) removed, and cells were fixed with % (v/v) cold acetone for min in an ice bath. next, μl of chicken polyclonal anti-ibv positive serum diluted : in pbs was added and the reaction was incubated for h at ℃. the cells were washed, stained with anti-chicken fitc-labelled conjugate (sigma) for h at ℃, and directly examined by fluorescence microscopy (olympus, tokyo, japan). parental bhk- cells were treated in the same way as a control. to compare the rescue efficiency of our lentivirus transduced bhk- /n cell line with commonly used bhk- cells, we further constructed a recombinant vvibv strain (vvibv yn- a/egfp) expressing the reporter genes enhanced green fluorescent protein (egfp), in which gene a was replaced with reporter genes egfp. μg of yn- a/egfp full-length rna was used for electroporation of bhk- /n cells or bhk- cells respectively. afte h incubation at °c, the green fluorescence in both electroporated cell lines were observed under fluorescence microscopy. . . in vitro transcription, transfection, and generation of the rescued virus ryn ibv-yn full-length rna transcript was generated in vitro using purified genomic dna isolated from recombinant vaccinia virus (vvibv-ryn) containing the . -kb full-length ibv cdna, that was digested with noti and in vitro transcribed by the ribomax™ large scale rna production system-t kit (promega, madison, wi, usa) according to the manufacturer's instructions in brief, μg of ibv-ryn full-length rna was used for electroporation of bhk- /n cells as described previously (eriksson et al., ) . the transfected bhk- /n cells were incubated at °c for h in dmem supplemented with % fbs. after h, the virus-containing medium was collected and inoculated into six -day-old spf embryonated chicken eggs (ece). after incubation in ece at °c for h, allantoic fluid from three ece was harvested for further rt-pcr detection. the other three eggs were kept at °c until h post-inoculation to check for embryo curling caused by ibv. the allantoic fluid that cause embryo curling and rt-pcr detection positive was further passaged in ece for times to get the stable virus stock (p virus stock) that reproducibly induced embryo curling. this stock was then used for further analyses, such as determination of the % egg infective dose (eid ), the % tissue culture infective dose (tcid ) as well as replication in vitro and in ovo. to differentiate the rescued viruses from the parental viruses, viral rnas were extracted from allantoic fluid (ryn and parental yn) as described above. rt-pcr was performed with selected primer pairs designed around the silent molecular markers that had been introduced into the ryn genome ( ʹ-gtgtgctatgtaagg tggtg- ʹ, ʹ-ggagc atttttaactcgtagg- ʹ), and the pcr product was digested to verify the existence of the sali silent nucleotide change at position - . the pcr product was also sequenced for further confirmation. to determine eid of ryn, serial -fold dilutions ( − - - ) of the amplified virus were inoculated into -day-old spf ece via the allantoic sac route. for each dilution, . ml of virus solution was injected into each egg and five eggs were used for each dilution. inoculated eggs were incubated for h at ℃ to check for classical embryo curling caused by ibv. eggs that died within h of inoculation were discarded. eggs that died between - h were considered as infected. the viral titer was also assessed by determining tcid . for this procedure, chicken embryo kidney (cek) cells were prepared and seeded into -well plates two days before virus infection as previously described (hennion and hill, ) . serial -fold dilutions ( − - - ) of the amplified virus were added onto % confluent cek cells, and each dilution of virus was inoculated into four replicate wells on well plates. after h incubation at ℃, % co , the -well plates were assessed for ibv infection by an ifa assay as described above. the eid and tcid calculations were based on the reed and muench method (reed and muench, ) . to compare the in ovo replication of the rescued virus ryn and its parental strain yn in ece, μl pbs containing . eid of ryn or yn virus were inoculated into the allantoic cavities of -day old eces, and μl allantoic fluid of five eggs from each group were harvested at the time points of , , , , and h post inoculation (hpi) and pooled for the real-time pcr detection assay. in brief, the total rna of μl allantoic fluid were extracted using the viral rna isolation kit (foregene co., ltd, chengdu, china) according to the manufacturer's instructions. reverse transcription was conducted at ℃ for min, with μg of total rna using the primescript™ rt reagent kit (takara bio inc, beijing, china). the cdna samples were submitted for sybr green i real-time rt-qpcr to detect the viral load for ibv n gene as described before (zhao et al., ) . all quantitative pcr reactions were carried out in triplicate and repeated at least twice, and the expression of ibv was calculated according to a standard curve. to compare the in vitro replication of the rescued virus ryn and its parental strain yn on cek cells, μl pbs containing . tcid of ryn or yn virus were inoculated onto the cek cells in -well plates, and μl supernatants from three wells from each group were harvested at the time points of , , , , , and hpi for a real-time pcr detection assay for ibv n gene as described above. the virus copy numbers for each time point were detected in triplicate and calculated according to a standard curve. unless otherwise stated, all data are represented as the mean values ± standard derivations obtained from experiments performed at least in triplicate. data were analyzed using graphpad prism (graphpad software, san diego, ca, usa). the statistical significance of differences between groups was verified using one-way anova followed by bonferroni's multiple comparison tests. for all tests, the following notations are used to indicate significant differences between ryn and yn virus: * p < . ; ** p < . . to construct a cdna clone covering the complete ibv-yn genome we generated ten overlapping dna fragments by rt-pcr from ibv-yn rna and chemical dna synthesis. the fragment corresponding to the ibv-yn ′-end was engineered to contain a promoter for the t rna polymerase that was placed immediately upstream of the ibv-yn ′terminal a nucleotide. the fragment corresponding to the ibv-yn ′end contained a polya sequence of nts followed by a noti restriction endonuclease recognition site. this strategy has been successful for other full-length coronavirus cdna clones (casais et al., ; van den worm et al., ; tekes, ) . it allows to generate an in vitro transcribed rna containing one additional g nucleotide at the ′-end (required as transcription start site for the t rna polymerase) and a polya tail at the ′-end (fig. a) . the full-length cdna was sequentially assembled into the vaccinia virus genome by six rounds of homogenous recombination using the e. coli guanine phosphoribosyltransferase as selection marker for positive or negative selection as appropriate (fig. b) . the integrity of the resulting vaccinia viruses was assessed by rt-pcr and sequencing analysis of the recombined regions after each recombination step. the final recombinant vaccinia virus, vvibv-ryn, containing the complete ibv-yn genome was deep sequenced and the result showed % identity with the parental virus genome, with the exception of three silent nucleotide substitutions, t a, a c, and y. zhao, et al. virus research ( ) t c, which are diagnostic for the recombinant ibv genomic cdna (fig. a) . as demonstrated previously, the coronavirus n protein greatly facilitates the rescue of recombinant coronaviruses (yount et al., ; casais et al., ) . we therefore constructed a lentivirus-transduced bhk- /n cell line that stably expresses the ibv-yn n protein. the complete n gene of yn strain was amplified by rt-pcr and cloned downstream of the hsv-tk promoter. this expression cassette was then inserted into the lentivirus vector psmpuw-sv -puro and the resulting plasmid was used to generate lentiviral vector particles ( fig. a, b) . following lentivirus vector transduction and puromycin selection the resulting bhk- /n cells were assessed for n protein expression. as shown in fig. c , the expression of ibv n protein was readily detected by ifa, whereas no specific fluorescence was observed in parental bhk- cells. expression of reporter genes by ibv has been previously described and used to facilitate the studies of virus replication and transcription in target cells (bentley et al., ) . in this study we replaced the accessory a gene by genes encoding the egfp, resulting in the recombinant viruses vvibv yn- a/egfp (fig. a) . after electroporation of μg of yn- a/egfp full-length rna into bhk- /n cells or bhk- cells and incubation at ℃ for h, green fluorescence were observed in both electroporated cell lines under fluorescence microscopy, confirming the expression of gfp (fig. b ). however, we were able to detect cells displaying green fluorescence in bhk- cell lines, but only a very low number; the numbers of cells displaying green fluorescence in bhk- /n cell lines were obviously higher than in bhk- cell lines, indicating a higher rescue efficiency by using the bhk- /n cell lines. the recovery of infectious ibv-yn from cloned cdna was performed using a protocol that was successfully used for the recovery of recombinant hcov- e . briefly, vvibv-ryn dna purified from virus particles was cleaved with noti and used as a template for in vitro transcription with the t rna polymerase in the presence of a cap analog. the rna transcripts ( μg) of the ibv cdna were electroporated into bhk- /n cells, and the bhk- /n cell supernatant was harvested after incubation at ℃ in % co for h. the supernatant was subsequently inoculated into the allantoic cavities of ece for replication of the rescued virus. this strategy was chosen to facilitate the rescue procedure and to ensure that the recombinant ibv-ryn replicate efficiently in ovo. after h incubation in the allantoic cavities, the recovery of ibv-ryn was confirmed by rt-pcr amplifying and sequencing the genomic region that contained the molecular markers to distinguish ibv-ryn from ibv-yn. as shown in fig. a, b , the presence of a silent mutation that gave rise to a sali restriction site at position t c was verified by restriction enzyme digestion as well as sequence analysis. this analysis showed that the pcr product of ibv-ryn can be digested into two segments corresponding to the mutation introduced into the genome, whereas the pcr product of parental yn strain was resistant to cleavage by sali (fig. a) . accordingly, the t c mutation was confirmed by sequencing analysis (fig. b) . collectively, these results demonstrate the successful rescue of the pathogenic ibv strain yn from cloned cdna by using electroporation of full-length ibv in vitro transcripts into n-protein expressing cells and subsequent virus amplification in the allantoic cavities of ece. ece that have been inoculated with supernatants of ibv-ryn rnaelectroporated bhk- /n cells were also assessed for chicken embryo development, and as shown in fig. a , after only two passages in ece, typical embryo lesions such as curling, stunting, and dwarfing were observed. a similar phenotype was also observed when ece were inoculated with wild-type ibv-yn. to compare replication of ibv-ryn with the parental strain ibv-yn in ece, we first produced virus stocks that reproducibly induced embryo curling by passaging the viruses times in ece. μl of collected p ibv-ryn allantoic fluid stock or wild-type ibv-yn stock that has been proven to be positive for ibv has been serially diluted ( − - - ) and inoculated into -day-old spf ece. five eggs were used for each dilution. after h incubation at ℃, inoculated eggs were evaluated for classical embryo curling caused by ibv and the eid titer was calculated according to the reed and muench method (reed and muench, ) . ibv-yn and ibv-ryn were pathogenic to chicken embryos up to a dilution of , and the eid y. zhao, et al. virus research ( ) titer of ibv-ryn ( . / μl) was comparable to that of its parental virus ibv-yn, ( . / μl). we also assessed the virus titers of the same virus stocks in cek cells to determine the tcid . μl of collected p ibv-ryn allantoic fluid stock or wild-type ibv-yn stock that has been proven to be positive for ibv has been serially diluted ( − - - ) and added onto % confluent cek cells. four replicate wells on -well plates were used for each dilution. after h incubation at ℃, % co , the -well plates were assessed for ibv infection by an ifa assay as described above. titers were determined by observing infected cells under a fluorescence microscope and calculating the tcid per . ml. the tested tcid titer of ryn was almost equal to that of yn at . and . , respectively. finally, we assessed growth kinetics of ibv-ryn and ibv-yn in eces and cek cells. for the replication profile comparison in ece, μl pbs containing . eid of ibv-ryn or ibv-yn were inoculated into the allantoic cavities of -day old eces, and the allantoic fluid of five eggs from each group were harvested at the time points of , , , , and hpi and pooled for the real-time pcr detection assay (zhao et al., ) . as shown in fig. b , the recombinant ibv-ryn replicated in eces with similar growth kinetics as the parental ibv-yn virus. although the ibv-ryn had a relatively low replication speed at the first h, both viruses could reach the peak titers after h and stay at a high level as long as h. to assess the replication kinetics in cek cells, μl pbs containing . tcid of ibv-ryn or ibv-yn were used to infect cek cells in well plates, and the supernatants from three wells from each group y. zhao, et al. virus research ( ) were harvested at the time points of , , , , , and hpi. viral load was determined by a real-time pcr detection assay quantifying ibv n gene as previously described (zhao et al., ) . as shown in fig. c , growth kinetics and peak titers of the recombinant ibv-ryn were indistinguishable from those of ibv-yn (fig. c) . collectively, these results show that the recombinant ibv-ryn has the same in ovo and in vitro phenotype as the parental ibv-yn. in , the first full-length infectious clone of coronavirus was successfully rescued using the bac system (almazán et al., ) . the advantage of the bac system is that it can accommodate larger foreign genes or dna fragments, and methods for modification and screening are well established (almazán et al., ) . nevertheless, for certain coronaviruses stability of full-length bac clones remained a problem. an alternative system was described in the same year by yount and colleagues (yount et al., ) . they assembled a series of smaller subclones into an intact full-length tgev cdna by in vitro ligation (yount et al., ) . viral mrna was obtained by in vitro transcription, and then electroporated into bhk cells to successfully rescue recombinant tgev. notably this study also revealed that expression of the n protein facilitates the rescue of recombinant coronaviruses. one year later a third reverse genetic system for coronaviruses was described that is based on the use of vaccinia virus as eukaryotic cloning vector. this system has been first described for the generation of recombinant hcov- e and was subsequently applied to the generation of recombinant mouse hepatitis virus (mhv; coley et al., ) , ibv (casais et al., ) , feline infectious peritonitis virus (fipv; tekes et al., ) , and sars coronavirus (van den worm et al., ). as a large dna virus, vaccinia virus has the following advantages: ) vaccinia virus itself can accommodate large fragments of foreign genes (at least the size of coronavirus genomes), and the virus' own infectivity and replication ability are not affected. ) the inserted foreign dna can be stably maintained and amplified in the vaccnia virus genome without the need to rely on prokaryotic cloning systems. ) vaccinia virus can be modified by homologous recombination, making it simple and easy to introduce mutations, insertions or deletions into cloned coronavirus genomes. several studies have employed the reverse genetic system of ibv to study pathogenicity, tissue tropism, and functionality of accessory genes, as well as to explore the potential for a vector-based vaccine of ibv (casais et al., armesto et al., ; bentley et al., ) . however, to the best of our knowledge, all existing reverse genetics systems for ibv were either based on the non-pathogenic beaudette strain (casais et al., ) or the mass-type attenuated vaccine strains (zhou et al., ; beurden et al., ) . while according to previous researches, the prevalent serotype of ibv circulating in china and many other countries is qx type (monne et al., ; zhao et al., ) , and the qx-type strains typically share < % nucleotide identity with mass-type strains. therefore, the application of currently available mass-type ibv reverse genetics systems cannot provide full insight into the immunogenicity and pathogenicity of qx-type strains. in this study, we successfully established a reverse genetic system based on a highly virulent qx-type (gi- ) strain yn isolated in china. we could show that the recombinant ibv-ryn replicates with the same kinetics, reaches the same peak titers, and displays comparable pathogenicity as the parental ibv-yn in eces. this indicates that this system will be a powerful tool to elucidate crucial viral and host factors that impact the high pathogenicity of qx-type ibv strains, and it may also serve as a starting point for the generation of novel vaccine candidates directed against ibv-qx-type strains. we also developed a modified rescue strategy by combining full length rna electroporation into a bhk- /n cell line and subsequent virus amplification in ovo. this strategy was highly efficient for the rescue of field strains. by introduction of reporter gene egfp into the ibv-ryn genome, we could easily compare the rescue efficiency of bhk- and bhk- /n cell lines. after electroporation with same amount ryn-egfp/ a full-length rna, bigger numbers of green fluorescent cells were observed in the bhk- /n cell line than bhk- cell line, indicating the higher rescue efficiency of it. also, in our study, a remarkable difference of virus infectivity in ece (eid could reach . - . per . ml) was observed compared to cek cells (tcid could only reach . per . ml) for ibv-yn, suggesting that ece are more susceptible to ibv infection and thus greatly facilitate the rescue of recombinant viruses. by combining electroporation of ibv rna into bhk- /n cells and subsequent virus amplification in ece we could indeed reach a rescue efficiency of % and only one passage in ece was required for virus recovery, which makes the procedure for generating recombinant ibv field strains feasible and less labor intensive. in conclusion, we demonstrate the successful development of a vaccinia virus-based qx-type ibv reverse genetics system. this system allows for the generation of defined, genetically modified viruses to study ibv molecular biology and pathogenesis. we expect that the improved rescue procedure, involving rna transfection into bhk /n cells and virus amplification in ece, is readily applicable to other ibv field strains, and may lead to the development of vaccines to prevent or control infections of newly emerging ibv field strains. the authors declare that they have no conflicts of interest. characterization and analysis of the full-length genome of a strain of the european qx-like genotype of infectious bronchitis virus engineering the largest rna virus genome as an infectious bacterial artificial chromosome engineering infectious cdnas 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isolation and identification of glandular stomach type ibv (qx ibv) in chickens analysis of antigenicity and pathogenicity reveals major differences among qx-like infectious bronchitis viruses and other serotypes in vitro assembled, recombinant infectious bronchitis viruses demonstrate that the a open reading frame is not essential for replication strategy for systematic assembly of large rna and dna genomes: transmissible gastroenteritis virus model safety and efficacy of an attenuated chinese qx-like infectious bronchitis virus strain as a candidate vaccine evolution of infectious bronchitis virus in china over the past two decades establishment of reverse genetics system for infectious bronchitis virus attenuated vaccine strain h this study was supported by the national key research and development program of china ( yfd ). we thank kate fox, dphil, from liwen bianji, edanz group china (www.liwenbianji. cn/ac), for editing the english text of a draft of this manuscript. key: cord- -gmw gl r authors: saiz, juan-carlos; de oya, nereida jiménez; blázquez, ana-belén; escribano-romero, estela; martín-acebes, miguel a. title: host-directed antivirals: a realistic alternative to fight zika virus date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: gmw gl r zika virus (zikv), a mosquito-borne flavivirus, was an almost neglected pathogen until its introduction in the americas in , where it has been responsible for a threat to global health, causing a great social and sanitary alarm due to its increased virulence, rapid spread, and an association with severe neurological and ophthalmological complications. currently, no specific antiviral therapy against zikv is available, and treatments are palliative and mainly directed toward the relief of symptoms, such as fever and rash, by administering antipyretics, anti-histamines, and fluids for dehydration. nevertheless, lately, search for antivirals has been a major aim in zikv investigations. to do so, screening of libraries from different sources, testing of natural compounds, and repurposing of drugs with known antiviral activity have allowed the identification of several antiviral candidates directed to both viral (structural proteins and enzymes) and cellular elements. here, we present an updated review of current knowledge about anti-zikv strategies, focusing on host-directed antivirals as a realistic alternative to combat zikv infection. since the beginning of the st century, a number of infectious disease threats have emerged that demand a global response. among them, severe acute respiratory syndrome virus, avian influenza in humans, pandemic influenza a (h n ), middle east respiratory syndrome coronavirus, chikungunya virus, and ebola virus have been the most threatening ones. nonetheless, the emergency of a vector-borne virus, zika virus (zikv), which is responsible for congenital malformations and other neurological and ophthalmological disorders, was hard to predict. zikv is a mosquito-borne virus belonging to the spondweni serocomplex in the genus flavivirus of the family flaviviridae [ ] . the virus has been isolated from various mosquito species, although it seems that the natural transmission vectors are mosquitoes of the genus aedes [ , ] . besides mosquito bites, viral direct human-to-human transmission can occur perinatally, sexually, and through breastfeeding and blood transfusion [ ] . the zikv genome is a single-stranded rna molecule (≈ . kb) of positive polarity encoding a single open reading frame (orf) flanked by two untranslated regions at the and ends [ ] . zikv was first isolated from the serum of a monkey in , and one year later from aedes africanus mosquitoes caught in the same area, the zika forest [ ] . until it was detected in asia in the s, the virus had been confined to africa. later on, human outbreaks were reported in the pacific islands, micronesia in and, then, in french polynesia in [ ] . the natural course of zikv infection was usually asymptomatic or produce a relatively mild illness and an uneventful recovery [ ] , hence, the virus was considered an almost neglected pathogen until its recent introduction into the americas in , when it became a threat to global health, showing increased virulence, rapid spread, since the recent outbreak in in the americas, a quite high number of possible antiviral candidates are being tested in vitro and in vivo. however, until now, no specific therapy has been approved against any flavivirus [ ] , including zikv [ ] , and, thus, current treatments are mainly directed toward the relief of symptoms, such as fever and rash, by administering antipyretics, anti-histamines, and fluids for dehydration [ ] . nevertheless, it should be noted that some commonly used drugs, such as acetylsalicylic acid, are contraindicated in zikv-infected patients, since they increase the risk of internal bleeding, and other arboviruses (dengue or chikungunya viruses) that can co-infect the patients may produce hemorrhages [ ] . due to the natural course of zikv infection, which is usually asymptomatic or produce a relatively mild illness and an uneventful recovery, when facing anti-zikv strategies, a very important point to take into account is the main target population that would benefit from it, namely immunocompromised patients and pregnant women and their fetuses [ ] . in this sense, only for some of the tested drugs their safety profiles are known [ ] . however, in cases of food and drug administration (fda) (https://www.drugs.com/) category b compounds (animal reproduction studies have failed to demonstrate a risk to the fetus and there are no adequate and well-controlled studies in pregnant women), or even in those of category c (animal reproduction studies have shown an adverse effect on the fetus and there are no adequate and well-controlled studies in humans, but potential benefits may warrant use of the drug in pregnant women despite potential risks), or d (there is positive evidence of human fetal risk based on adverse reaction data from investigational or marketing experience or studies in humans, but potential benefits may warrant use of the drug in pregnant women despite potential risks), their use in pregnancy can be contemplated if the potential benefit outweighs the risks. even more, some of the assayed compounds cross the placenta and, thus, can also benefit the fetus. nonetheless, if used, this should be done in an individualized way, conditioning dosage and timings, and always under a clinician's control where the patient is informed of the pros and cons. current search for zikv antivirals is being conducted with different approaches: by screening of compounds libraries; by the repurposing of drugs of known active efficacy against other diseases now in use in clinical practice, many of which display broad-spectrum activity; and by testing natural products. two different strategies can be applied when pursuing for antivirals, those searching for compounds directed to viral targets (direct-acting antivirals) and those aimed to target cellular components needed for the viral life cycle (host-directed antivirals). among the virus-directed drugs tested [ , ] are those acting against the viral rna-dependent rna polymerase (non-structural protein (ns )) catalytic domain, including nucleoside analogs and polymerase inhibitors; the methyltransferase catalytic domain of the ns responsible for transferring the mrna cap; the ns b-ns trypsin-like serine protease needed for proper processing of the viral polyprotein; and the ns helicase. the crystal structures of all these proteins have already been resolved and will certainly help to find new antivirals [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in the same way, structures from other viral proteins are also available that could help to design zikv therapeutic alternatives, such as those of the capsid c protein [ ], whose destabilization may impair zikv multiplication, the ns [ , ], an immuno-modulator, or the envelope glycoprotein [ - ], which mediates cell binding and endosomal fusion, constitutes a major target for neutralizing antibodies, and could be also the target for virucidal compounds [ ] . on the other hand, it has also been reported that passive transfer of neutralizing antibodies to pregnant mice suppresses zikv multiplication, inhibits cell death, reduces the number of progenitor neuronal cells, and prevents microcephaly [ , ] . likewise, administration of monoclonal antibodies (mabs) recognizing the domain iii of the zikv-e protein protect mice of lethal zikv challenge [ , ] and other mabs are able to bind and neutralize zikv, including those directed against the e dimer epitope [ ] . human polyclonal antibodies produced in transchromosomal bovines also protect mice from zikv lethal infection, eliminated zikv induced tissue damage in the brain and testes, and protected against testicular atrophy [ ] . thus, administration of therapeutic antibodies seems to also be a potential strategy against zikv. nevertheless, it should be noted that, although still controvertial in the case of zikv infection [ ] , the well-known antibody dependent enhancement effect (ade) [ ] , of which dengue virus (denv) is the prototypic model, may potentiate the risk of disease exacerbation. flaviviruses have small rna genomes (around . kb in length) and thus require many host factors and co-option of cellular metabolic pathways to successfully infect host cells and propagate efficiently [ ] . this offers an opportunity to search for host targets as therapeutic tools that, in many instances, as they are shared by different members of the flaviviridae family, can be envisaged as pan-flaviviral antivirals [ ] [ ] [ ] . this strategy can be directed to host factors implicated in infection, pathogenesis, and in the immune response, as it has been shown for denv and the west nile virus (wnv) [ ] . in addition, their effect would be less prone to the emergence of mutants that will escape their action, as often occurs with drugs targeting viral components. consequently, this kind of approach could ideally lead to the discovery of broad spectrum antivirals that could provide low cost but effective tools for the control of flaviviral threats. different approaches are being used to identify potential host factors as therapeutic targets against flaviviruses including the analyses of transcript levels (e.g., next generation rna sequencing) for altered expression patterns during infection, proteome changes, kinases activities variations, and protein-rna interactions (e.g., two-hybrid screenings and affinity chromatography). likewise, functional analysis can be applied by overexpressing cdnas or by rnai-mediated loss of function screens using dsrna, sirna, or shrna libraries, although it should be noted that in some cases downregulation is inefficient and some genes have redundant functions [ ] . replicons may also be used to specifically assay replication activity [ , ] . theoretically, host-acting antivirals can be directed to any molecule or pathway implicated in the different steps of the viral life cycle, from early events (binding, entry, and fusion), to the formation of the replication complex, and the viral maturation and egress. the first step of zikv infection is its binding to the cellular receptor ( figure ). several molecules have been proposed as a zikv receptor (members of the tyro /axl/mer (tam) family of receptor tyrosine kinases, t-cell immunoglobulin and mucin domain (tim) and dendritic cell-specific intercellular adhesion molecule -grabbing nonintegrin (dc-sign)) that are expressed in different neuronal and non-neuronal permissive cell types. these molecules are also receptors for other viruses, including flaviviruses such as denv and wnv, regulate several cellular activities (adhesion, migration, proliferation, and survival, release of inflammatory cytokines, antigen uptake, and signaling), and play important roles in the host's response to infection [ ] . however, elimination of a known receptor does not necessarily result in complete protection from viral infection, since flaviviruses use different receptors and, thus, there is always redundancy and alternatives. for instances, inhibiting, downregulating, knocking-down, or ablating axl, although in some cases they reduce zikv infection, they do not completely abolish it, pointing to the use of different cell surface receptors on different cell types [ ] [ ] [ ] [ ] . different molecules have been shown to inhibit zikv infection at the entry step ( figure ). r (an axl kinase inhibitor) and myd (an axl decoy receptor) compromises, but do not completely abolish, zikv infection of glial cells [ ] . r , as well as cabozantinib, an inhibitor of axl phosphorylation, that are currently in clinical trials for anticancer activities, significantly impairs zikv infection of human endothelial cells in a dose-dependent manner by affecting a post-binding step [ ] . likewise, curcumin, a widely used food additive and herbal supplement, reduces zikv infection in cell culture inhibiting cell binding while maintaining viral rna integrity [ ] , as does suramin, an anti-parasitic that interferes with attachment to host cells and with virion biogenesis by affecting glycosylation and maturation [ , ] . once zikv binds to the cell receptor, like other flaviviruses, it is internalized through clathrin-mediated endocytosis and transported to the endosomes with the involvement of cellular actin and microtubules to establish a productive infection ( figure ) [ ] . after internalization, to start translation and replication, the viral genome is released inside the cytoplasm by fusing the viral envelope with the membranes of the cellular endosomes, a process triggered by acidic ph inside them [ , ] . nanchangmycin, an insecticide and antibacterial polyether, inhibits zikv multiplication and, although the exact mechanism of action has not been completely elucidated, it probably targets axl and blocks clathrin-mediated endocytosis [ ] . acid endosomal ph triggers rapid conformational changes on viral envelope protein that result in its fusion with endosomal membrane in a ph-dependent manner, thus allowing nucleocapsid release to the cytoplasm for genome uncoating ( figure ). the optimal ph for conformational rearrangements and viral fusion is . - . , and these processes are likely dependent on the presence of cholesterol and specific lipids in the target membrane [ ] . these processes can be potentially druggable, and in fact, arbidol, a broad-spectrum antiviral and immunomodulatory use for human influenza a and b infections, inhibits zikv multiplication in cell culture probably because it intercalates into membrane lipids leading to the inhibition of membrane fusion between virus particles and plasma membranes, and between virus particles and the membranes of endosomes [ ] . chlorpromazine, an antipsychotic drug that also inhibits clathrin-mediated endocytosis, reduced zikv infection, confirming the requirement for clathrin-mediated endocytosis of zikv [ ] . in addition, -hydroxycholesterol ( hc) is increased in zikv-infected human embryonic cells and brain organoids, and reduces viremia and viral loads without affecting viral binding, but blocking internalization and suppressing viral and cell membranes fusion [ ] . even more, hc reduces mortality and prevents microcephaly in zikv-infected mice, and also decreases viral loads in the urine and serum of treated non-human infected primates [ ] . daptomycin, a lipopeptide antibiotic that inserts into cell membranes rich in phosphatidylglycerol, which suggests an effect on late endosomal membranes enriched in this lipid, has also been described as a zikv inhibitor [ ] . the dependence on endosomal acidification for zikv infection also provides a host target suitable for antiviral intervention. for instance, obatoclax (or gx - ), an anti-neoplastic and pro-apoptotic inhibitor of the bcl- that targets cellular mcl- , impairs zikv endocytic uptake by reducing the ph of the endosomal vesicles in cell culture, and thereby most likely inhibits viral fusion [ , ] . however, obatoclax, which presents a low solubility, has not produced satisfactory results in clinical trials for hematological and myeloid diseases. saliphenylhalamide (saliphe), which targets vacuolar adenosine triphosphatase enzyme (atpase) and blocks the acidification of endosomes, inhibits zikv multiplication in human retinal pigment epithelial cells [ ] that are natural targets for zikv infection [ ] . similar results were found by adcock et al. ( ) with saliphe using a different screening [ ] ; however, they reported that, contrary to that described by others [ ] , other compounds that interfere with the endocytic pathway, such as dynasore, that blocks clathrin-mediated endocytosis, or monensin, a cation transporter, were either toxic for the cells used or did not show any anti-zikv activity, as neither did chloroquine (cq). these contradictory results are probably explained by the different methodologies, cell types, and, to a lower extent, viral strains used to analyze the antiviral activities of the compounds and suggest that compounds showing different activities should be carefully evaluated before going further with investigations. in this line, and contrary to above mentioned report [ ] , cq, an fda-approved anti-inflammatory -aminoquinoline and an autophagy inhibitor widely used as an anti-malaria drug that is administered to pregnant women at risk of exposure to plasmodium parasites, was shown to have anti-zikv activity in different cell types (vero cells, human brain microvascular endothelial cells (hbmecs), and human neural stem cells (nscs)), affecting early stages of the viral life cycle, possibly by raising the endosomal ph and inhibiting the fusion of the envelope protein to the endosomal membrane [ , ] . cq has been shown to reduce placental and fetal zikv infection [ ] , and also attenuate zikv-associated morbidity and mortality in mice and protect the fetus from microcephaly [ ] . even more, cq attenuated vertical transmission in zikv-infected pregnant interferon signaling-competent swiss jim lambert (sjl) mice, significantly reducing fetal brain viral loads [ ] . similarly, cq, and other lysosomotropic agents (ammonium chloride, bafilomycin a , quinacrine, mefloquine, and n-tert-butyl isoquine (gsk )) that neutralize the acidic ph of endosomal compartments, block infection of a human fibroblast cell line and vero cells [ , ] . additionally, by medicinal chemistry-driven approaches, a series of new , -bis(trifluoromethyl)quinoline and n-( -(arylmethylimino)ethyl)- -chloroquinolin- -amine derivatives have been proved to inhibit zikv replication in vitro with a higher potency than chloroquine or mefloquine [ , ] . more recently, by screening fda-approved drugs using a cell-based assay, it has been shown that amodiaquine, another antimalarial drug, also has anti-zikv activity in cell culture by targeting early events of the viral replication cycle [ ] . niclosamide, a category b antihelmintic drug approved by fda, was capable of inhibiting zikv infection, and although its antiflaviviral effect has been associated to its ability to neutralize endolysosomal ph and interfere with ph-dependent membrane fusion, in the case of zikv, it seems that it was affecting other post-entry steps [ ] . in addition, recently, it has been reported that niclosamide decreases zikv production, partially restores differentiation, and prevents apoptosis in human induced nscs; even more, it can partially rescue zikv-induced microcephaly and attenuate infection in a developed humanized zikv-infected embryo model in vivo [ ] . likewise, tenovin- , which represses cell growth and induces apoptosis in cells expressing p by inhibiting the protein-deacetylating activities of sirt and sirt and, thus, affects endosome functions, potently inhibits zikv infection in primary placental fibroblast cells [ ] . iron salt ferric ammonium citrate (fac) also inhibits zikv infection through inducing viral fusion and blocking endosomal viral release by promoting liposome aggregation and intracellular vesicle fusion [ ] . overall, these studies evidence the potential of targeting viral entry to combat zikv. once zikv-rna is released from the endosomes in the cytoplasm, it acts as mrna to synthesize the negative-strand viral rna that directs positive-strand rna synthesis (figure ) [ ] . silvestrol, a natural compound isolated from the plant aglaia foveolata that it is known to inhibit the asp-glu-ala-asp (dead)-box rna helicase eukaryotic initiation factor- a (eif a) required to unwind structured -untranslated regions and thus impairing rna translation, exerts a significant inhibition of zikv replication in a cells and primary human hepatocytes [ ] . n-( -hydroxyphenyl) retinamide (fenretinide or -hpr), an activator of retinoid receptors that inhibits the proliferation of cancer cells and can induce apoptosis, inhibits zikv in cell culture and significantly reduces both serum viremia and brain viral burden in mice by decreasing the rate of viral rna synthesis, though not via direct inhibition of the activity of the viral replicase [ ] . zikv relies on polyamines for both translation and transcription [ ] , so that, drugs targeting the polyamine biosynthetic pathway, such as difluoromethylornithine (dfmo or eflornithine), an fda-approved drug that is used to treat trypanosomiasis, hirsutism, and some cancers, as well as diethylnorspermine (denspm) limit viral replication in bhk- cells [ ] . zikv replication and particle morphogenesis take place associated with a virus-induced organelle-like structure derived from the membrane of the endoplasmic reticulum (er) (figure ) [ ] . de novo synthesized positive strand-rna, once packaged, form enveloped immature virions in the er, enter the secretory pathway and, then, in the trans-golgi network, the prm is cleaved before the virus is released from the infected cell ( figure ) [ , ] . er-membrane multiprotein complexes, such as the oligosaccharyltransferase (ost) complex, have been reported to be critical host factors for flavivirus multiplication. in this regard, it has been shown that the n-linked glycosylation inhibitor- (ngi- ) chemical modulator of the ost complex blocks zikv-rna replication in different cell types [ ] . similarly, the host er-associated signal peptidase (spase) is an essential, membrane-bound serine protease complex involved in cleavage of the signal peptides of newly synthesized secretory and membrane proteins at the er and also for processing of the flavivirus prm and e structural proteins [ ] . it has also been reported that cavinafungin, an alaninal-containing lipopeptide of fungal origin, potently inhibits growth of zikv-infected cells [ ] . nitazoxanide, a broad-spectrum antiviral agent approved by the fda as an antiprotozoan and with potential activity against several viruses in clinical trials (rotavirus and norovirus gastroenteritis, chronic hepatitis b, chronic hepatitis c, and influenza), also inhibits virus infection targeting a post-attachment step, most likely virus genome replication [ ] . likewise, brefeldin a, a penicillium sp. product that inhibits protein transport from the er to the golgi apparatus, inhibits zikv multiplication [ ] , as does emetine, an anti-protozoal agent that inhibits both zikv ns polymerase activity and disrupts lysosomal function [ ] . zikv infection leads to cell-death by inducing host caspase- and neuronal apoptosis during its propagation [ ] . thereby, bithionol, a caspase inhibitor, inhibits zikv strains of different geographical origin in vero cells and human astrocytes [ ] . similarly, by using a drug repurposing screening of over molecules, it was found that emricasan, a pan-caspase inhibitor that restrains zikv-induced increases in caspase- activity and is currently in phase clinical trials in chronic hepatitis c virus (hcv)-infected patients, protected human cortical neural progenitor cells (npc) in both monolayer and three-dimensional organoid cultures, showing neuroprotective activity without suppression of viral replication [ ] . additionally, bortezomib, a dipeptide boronate proteasome inhibitor approved for treatment of multiple myeloma and mantle cell non-hodgkin's lymphoma that regulates the bcl- family of proteins, has also been described as a zikv inhibitor [ ] . similarly, different cyclin-dependent kinase (cdk) inhibitors, such as (alphas)- -(acetylamino)-alpha-methyl-n-( -( -methylethyl)- -thiazolyl)benzeneacetamide (pha- ), reduced zikv-infection and propagation [ ] . however, cdk inhibitors should not be suitable for the treatment of pregnant women but could be useful for the treatment of other non-pregnant patients, preventing the complications associated with zikv infection. the need for specific host lipids for flavivirus replication and particle envelopment make lipid metabolism a potential target for an antiviral search [ , ] , and, even though manipulating a major metabolic pathway such as lipid biosynthesis can be envisaged as a dangerous antiviral approach due to the undesirable effects that could be detrimental for the host, current use of drugs such as ibuprofen and aspirin (cyclooxygenase- (cox- ) inhibitors) or statins ( -hidroxi- -metil-glutaril-coa (hmg-coa) reductase inhibitors) highlights the feasibility of lipid-based therapeutics [ , ] . accordingly, inhibition of key enzymes involved in fatty acid synthesis, such as acetyl-coa carboxylase (acc) [ ] , and fatty acid synthase (fasn) [ ] [ ] [ ] , are potential targets for anti-zikv therapy. in this line, we have reported that nordihydroguaiaretic acid (ndga) and its derivative tetra-o-methyl nordihydroguaiaretic (m n or terameprocol), two compounds that disturb the lipid metabolism probably by interfering with the sterol regulatory element-binding proteins (srebp) pathway, inhibit the infection of zikv and wnv, likely by impairing viral replication, as did other structurally unrelated inhibitors of the srebp pathway, such as -[(diethylamino)methyl]-n-[ -( -methoxyphenyl)ethyl]-n-( r)- -pyrrolidinyl-benzamide dihydrochloride (pf- ) and fatostatin [ ] . in the same way, the dependence on cholesterol for different processes during flavivirus infection also provides a suitable target for antiviral strategies. as mentioned above, hc reduces viremia and viral loads in vitro, and also reduces mortality and prevent microcephaly in mice, and decreases viral loads in the urine and serum in non-human infected primates [ ] . lovastatin and mevastatin are hypolipidemic agents (hmg-coa inhibitors) belonging to the family of statins that are widely used for lowering cholesterol in patients with hypercholesterolemia and have been previously shown to present antiviral activity against dengue and hepatitis c viruses. both agents have been proposed as therapeutic candidates against zikv [ ] . in fact, lovastatin attenuates nervous injury in animal models of gbs [ ] . likewise, imipramine, an fda-approved antidepressant, inhibits zikv-rna replication and virion production in human skin fibroblasts, probably by interfering with intracellular cholesterol transport [ ] . regarding sphingolipid metabolism, which has been involved in flavivirus infection [ ] , treatment with the neutral sphingomyelinase inhibitor gw reduced zikv production by affecting viral morphogenesis [ ] as described for other flaviviruses [ ] . finally, since adenosine monophosphate-activated protein kinase (ampk) is a master regulator of lipid metabolism, its activation by pf- or metformin reduced zikv infection by impairing viral replication [ , ] . thus, targeting lipid metabolism could provide therapeutic alternatives for the discovery of host-directed antivirals against zikv. the ns protein is the viral rna-dependent rna polymerase responsible for the rna synthesis that also inhibits interferon (ifn) signaling by acting over the signal transducer and activator of transcription (stat ) protein [ ] , being, thus, a major target for antiviral design. besides the proven antiviral activities of different nucleosides analogs and inhibitors of the zikv-ns [ ] , several inhibitors of the biosynthesis of nucleosides (purines and pyrimidines) also impair zikv replication (figure ). ribavirin is an inhibitor of the inosine monophosphate dehydrogenase (impdh) with antiviral activity to several rna viruses [ ] , but its mechanism of action is not entirely clear. it may act as a guanosine synthesis inhibitor, a viral cap synthesis inhibitor, a viral rna mutagen, and as an inducer of lethal mutagenesis [ ] [ ] [ ] . by using a cell-based assay, no antiviral activity of the drug was initially observed [ ] but, later on, it was reported that although no activity against zikv was detected in vero cells, the drug did inhibit virus multiplication in human cell lines, including liver huh- and rhabdomyosarcoma (rd) cells [ ] . further studies have confirmed an inhibitory activity of ribavirin against zikv strains of different geographical origin in various types of cells, such as human neural progenitor cells (hnpcs), human dermal fibroblasts (hdfs), human lung adenocarcinoma cells (a ), and even in vero cells [ ] [ ] [ ] . still more, the drug was shown to abrogate viremia in zikv-infected stat- -deficient mice [ ] , which lack type i ifn signaling, are highly sensitive to zikv infection, and exhibit a lethal outcome. two other inhibitors of impdh, merimepodib (mmpd or vx- ) [ ] and mycophenolic acid (mpa) [ , , ] also inhibit zikv-rna replication in different cell types, including huh- cells, human cervical placental cells, and neural stem and primary amnion cells. however, other authors [ ] have described that mpa have little effect on zikv replication and showed significant cell toxicity. likewise, azathioprine, another inhibitor of purine synthesis and immunosuppressant, impaired zikv replication in hela and jeg cells [ ] ; nonetheless, its use in pregnant women is not recommended. the above described contradictory results stress again the differences that drug treatments may have as a consequence of the different viral strains, cell types, and methodologies used to assess them. as with the inhibitors of purine biosynthesis, compounds inhibiting the synthesis of pyrimidines have also effect on zikv replication (figure ). so that, the virus was highly susceptible to brequinar and cid treatments in cell culture [ ] . however, it should be noted that it has been reported that brequinar, as well as dd , antiviral activity may not be due to pyrimidine deprivation, but rather to the induction of the cellular immune response [ , ] . similarly, other inhibitors of the pyrimidine synthesis, such as gemcitabine, an activator of cellular caspases [ , ] , and, although with a lower efficiency probably due to its lower solubility, -azauridine and finasteride, a -azasteroid analog of testosterone that inhibit type ii and type iii α-reductase and is being tested for benign prostatic hyperplasia and male pattern baldness, reduce zikv replication [ , ] . several other compounds have been shown to have anti-zikv activity by inhibiting viral entry and/or rna synthesis, although their mechanisms of action have not yet been fully elucidated. among them are antiparasitics such as ivermectin (used mainly against worms infections) and pyrimethamine (a folic acid antagonist that inhibits the dihydrofolate reductase and, thus, dna and rna synthesis, is classified as a pregnancy category c, and was initially used to treat malaria and now toxoplasmosis and cystoisosporiasis) [ ] ; antibiotics such as azithromycin that prevents infection, replication, and virus-mediated cell dead [ ] , and kitasamycin (a natural product from streptomyces narbonensis that inhibits protein biosynthesis) [ ] ; drugs used to prevent chemotherapy-induced nausea and vomiting as palonosetron (a fda-approved -ht antagonist) [ ] ; antidepressants like sertraline (a selective serotonin reuptake inhibitor) [ ] and cyclosporine (that is also use for rheumatoid arthritis, psoriasis, crohn's disease, nephrotic syndrome, and in organ transplants, is believed to lower the activity of t-cells, and is currently in clinical trials for tis possible use in ameliorate neuronal cellular damage) [ ] . similarly, after chemical screening, it was found that hippeastrine hydrobromide (hh), an active component of traditional chinese medicine, and amodiaquine dihydrochloride dihydrate (aq), an fda-approved drug for treatment of malaria, inhibit zikv infection of human pluripotent stem cell-derived cortical npcs and in adult mouse brain in vivo even when the infection was already ongoing but, again, their mechanisms of action are not known [ ] . besides drugs that act against host targets directly implicated in the viral cycle, there are compounds that can prevent undesirable effects of zikv infection. in this regard, zikv infection leads to massive neuronal damage, especially of neural progenitor cells, and neurodegeneration [ ] [ ] [ ] , via both direct replication in neuronal cells and possibly through increased excitotoxicity via over activation of n-methyl-d-aspartate receptor (nmdar)-dependent neuronal excitotoxicity in nearby cells. memantine, a pregnancy category b fda-approved drug widely used to treat patients with alzheimer's disease, as well as other nmdar blockers (dizocilpine, agmatine sulfate, or ifenprodil), prevents neuronal damage and death and intraocular pressure increase induced by zikv infection in infected mice, but it does not affect virus replication, pointing to its possible use to prevent or minimize zikv-related microcephaly during pregnancy [ ] . ebselen (ebs), an antioxidant that reduces oxidative stress and improves histopathological features in a testicular injury study model and is currently in clinical trials for various diseases, showed minor effects in reducing zikv progeny production and viral e protein expression and on overall survival and viremia level of challenged ag mice; however, it should be noted that ebs reduced some zikv-induced effects, such as testicular oxidative stress, leucocyte infiltration, and production of pro-inflammatory response, whereas, in a model of male-to-female mouse sperm transfer, the drug improved testicular pathology and prevented the sexual transmission of zikv [ ] . ifns play a key role in the elimination of pathogens and they are release upon the activation of the innate immune response by infecting viruses. in this way, zikv infection induces ifn signaling pathways and further activates cytoplasmic retinoic acid inducible gene protein (rig )-like receptors (rlrs) and several type i and iii ifn-stimulated genes, driving to the subsequent activation of the janus kinase (jak)/stat innate immune pathway that confer resistance to zikv infection [ ] . different studies showed that ifn-α, ifn-β, and ifn-γ inhibit zikv replication in cell culture [ , , ] , and that treatment of pregnant mice with ifn-λ reduced zikv infection [ ] . in addition, ifitm and ifitm , which are interferon-induced transmembrane proteins, impair early stages of zikv infection. even more, ifitm prevents zikv-induced cell death [ ] . likewise, it has been reported that an interferon-activating small molecule ( -( -fluorophenyl)- -( -isopropyl- , , -thiadiazol- -yl)- , -ihydrochromeno [ , -c] pyrrole- , -dione (avc) strongly inhibits replication of zikv in cell culture [ ] . however, it is also known that the virus is capable of evading type i ifn responses by acting over the jak/stat signaling pathway [ , [ ] [ ] [ ] , and that type i ifns might be mediators of pregnancy complications, including spontaneous abortions and growth restriction [ ] . in any case, use of ifn against zikv, alone or in combination with other antivirals, deserve further studies. by screening a library of known human micrornas (mirnas), small, noncoding rnas (sncrnas) that modulate gene expression post-transcriptionally and regulate a broad range of cellular processes, several mirnas were found to inhibit zikv by increasing the capability of infected cells to respond to infection through the interferon-based innate immune pathway [ ] . another alternative is intervening over epigenetic regulation by using epigenetics modulators. for instance, histone h k methyltransferases (ezh and ezh ) suppress gene transcription and it has been shown that inhibitors such as -[( s)-butan- -yl]-n-[( , -dimethyl- -oxo- h-pyridin- -yl)methyl]- -methyl- -( -piperazin- -ylpyridin- -yl)indole- -carboxamide (gsk- ) reduce zikv multiplication in cell culture through the activation of cellular antiviral and immune responses [ ] . in any case, further studies are needed to evaluate the potential therapeutic capability of these immunomodulators against zikv infection. a great effort is being lately made to find compounds to fight zikv infection by applying different approaches, from repurposing of drugs with known antiviral activity to the screening of bioactive molecules from different libraries, as well as natural products. however, most of the already tested drugs have been found to inhibit viral replication in vitro, and only a few have been tested in vivo. hence, since, in many instances, the results will be difficult to extrapolate to humans, it would be hard for most of the tested antivirals to complete the entire drug development pipeline. in 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infection inhibitors of the histone methyltransferases ezh / induce a potent antiviral state and suppress infection by diverse viral pathogens key: cord- -rpvl vmu authors: waltl, inken; käufer, christopher; gerhauser, ingo; chhatbar, chintan; ghita, luca; kalinke, ulrich; löscher, wolfgang title: microglia have a protective role in viral encephalitis-induced seizure development and hippocampal damage date: - - journal: brain behav immun doi: . /j.bbi. . . sha: doc_id: cord_uid: rpvl vmu in the central nervous system (cns), innate immune surveillance is mainly coordinated by microglia. these cns resident myeloid cells are assumed to help orchestrate the immune response against infections of the brain. however, their specific role in this process and their interactions with cns infiltrating immune cells, such as blood-borne monocytes and t cells are only incompletely understood. the recent development of plx , a specific inhibitor of colony-stimulating factor receptor that depletes microglia, allows studying the role of microglia in conditions of brain injury such as viral encephalitis, the most common form of brain infection. here we used this inhibitor in a model of viral infection-induced epilepsy, in which c bl/ mice are infected by a picornavirus (theiler’s murine encephalomyelitis virus) and display seizures and hippocampal damage. our results show that microglia are required early after infection to limit virus distribution and persistence, most likely by modulating t cell activation. microglia depletion accelerated the occurrence of seizures, exacerbated hippocampal damage, and led to neurodegeneration in the spinal cord, which is normally not observed in this mouse strain. this study enhances our understanding of the role of microglia in viral encephalitis and adds to the concept of microglia-t cell crosstalk. viral encephalitis is the most common form of brain infection and can cause severe and life-threatening consequences, including epilepsy (vezzani et al., ) . monocyte infiltration and activation of microglia, the resident macrophage population of the brain, are hallmarks of cns inflammation, including viral infection (prinz and priller, ) . however, the role of these cells in viral clearance and immunopathology is not well defined. at least in part this is due to the problems of differentiating invading monocytes from activated microglia in the brain and the lack of selective tools to manipulate these two types of myeloid cells. in several viral brain infections, activated microglia appears to be involved in inhibition of viral replication and in neurotoxicity, indicating the dual nature of microglia: they contribute to the defense of the cns but may also bear responsibility for cns damage (rock et al., ) . microglia expresses various pattern recognition receptors (prrs) to identify viral signatures called pathogen associated molecular patterns (pamps). upon stimulation by pamps microglia respond by releasing several pro-and anti-inflammatory cytokines such as monocyte chemoattractant protein (mcp /ccl ), interleukin (il)- β, type i interferon (ifn), ifnγ, and tumor necrosis factor (tnf)α (o'shea et al., ) . recently, a new compound, plx , which acts as specific dietary inhibitor of colony stimulating factor- receptor (csf r), a tyrosine kinase transmembrane receptor essential for the survival and activation of monocytes and macrophages in the periphery and microglia in the cns (erblich et al., ; hamilton and achuthan, ; elmore et al., ) , was shown to efficiently eliminate microglia in mice (dagher et al., ) . similar to the less specific csf r inhibitor plx , plx has been used to study the role of microglia in several conditions of brain injury (acharya et al., ; feng et al., ; beckers et al., ; hilla et al., ) . two recent studies https://doi.org/ . /j.bbi. . . received july ; received in revised form august ; accepted september reported that microglia depletion by plx increases virus replication and subsequent mortality in response to intracerebral infection with mouse hepatitis virus (mhv; wheeler et al., ) or flaviviruses (west nile virus and japanese encephalitis virus; seitz et al., ) . we used plx in a model of viral encephalitis in mice, in which theiler's virus (also termed theiler's murine encephalomyelitis virus [tmev] ), a naturally occurring enteric pathogen of the mouse, is used for intracerebral infection (vezzani et al., ; depaula-silva et al., ) . disease development following tmev infection depends on the mouse strain. the sjl/j mouse strain develops a multiple sclerosis-like progressive t cell-mediated autoimmune demyelinating disease, characterized by weakness of the hind limbs, which advances to a severe spastic paralysis, and inflammatory demyelination in the spinal cord, while the c bl/ j (b ) mouse strain develops hippocampal damage and acute seizures, which progress to epilepsy (depaula-silva et al., ) . the striking difference between the mouse strains seems to be partially due to the strong antiviral cytotoxic cd + t lymphocyte response observed in b mice, which is suppressed by the elevated induction of regulatory cd + t cells (tregs) in sjl/j mice, resulting in viral persistence and demyelination (depaula-silva et al., ) . we now report that elimination of microglia strikingly changes the disease phenotype of tmev-infected b mice, indicating that microglia is much more important in this model of viral encephalitis than previously thought. four-week-old female jax® c bl/ j (b ) mice were purchased from charles river (sulzfeld, germany) and housed in groups under standardized conditions, h/ h day-night cycle, - % humidity, - °c temperature and ad libitum tap water. environmental enrichment was ensured by provision of housing and nesting material. mice were randomly assigned into four experimental groups (vehicle vs. substance and subsequently into infected vs. mock-infected groups). according to a cooperative agreement between charles river and the jackson laboratory, the jax® c bl/ j mouse strain bred by charles river in europe is genetically equivalent to that bred by the jackson laboratory in the u.s. all animal experiments were conducted in accordance with the german animal welfare law and were authorized by the local government (laves oldenburg, germany, permission number . - - - / ). mice randomly assigned to the vehicle group received vehicle food chow, while mice randomly assigned to the treatment group had access to chow containing ppm plx per kilogram food chow (kindly provided by plexxikon inc., berkeley, ca, usa). the regular food uptake of a - g mouse is g per day, so the predicted overall intake of plx is . ppm per mouse per day. treatment started days prior to infection and continued until animals were sacrificed six to seven days post infection, resulting in a total treatment period of - days. this treatment protocol was established by plexxikon and was previously used by other research groups (dagher et al., ) . intracerebral infection with the daniels (da) strain of tmev was performed as previously described by bröer et al. ( bröer et al. ( , and waltl et al. ( ) . therefore, a deep isoflurane inhalation anesthesia was induced and either µl tmev solution ( . × pfu) or a mock solution were injected into the left parietal cortex of the mice using a free-hand method. the dose of tmev was based on previous experiments of our group in b mice, to ensure a high incidence (> %) of early seizures in this mouse strain (bröer et al., ) . development of acute seizures, weight, general appearance and behavior in the first week after infection was assessed by animal monitoring twice daily for one hour, one h in the morning (between and a.m.) and one h in the afternoon (between and p.m.), with at least h interval between the two recording sessions. care was taken to avoid any group differences in recording periods by randomly assigning animals to the recording sessions. recording was performed by experienced researchers, and the choice of recording periods was based on previous experiments with video-eeg monitoring of acute seizures in this model (for more details see bröer et al., bröer et al., , . on days or post infection animals were transcardially perfused with pbs followed by % paraformaldehyde. brains and spleens were removed and left in formaldehyde overnight before being trimmed and embedded in paraffin. additionally, segments of the spinal cord including the spinal column were removed, left in formaldehyde overnight, decalcified in edta for h and afterwards embedded in paraffin. animals used for flow cytometric analysis or rt-qpcr analysis were perfused with °c pbs only. brains for flow cytometry were harvested and stored in °c pbs until being further processed, whereas brains for rt-qpcr were embedded in tissue freezing medium (leica), frozen in liquid nitrogen and stored at − °c until further processing. for rt-qpcr analysis of the spinal cord, the spinal column was harvested, the vertebrae removed and the segments of the cord equally processed as brains for rt-qpcr analysis. edta-blood for flow cytometry was sampled prior to the start of perfusion analysis and stored at °c. histological analyses of the brain were mainly focused on the dorsal hippocampus, as this brain structure is mainly damaged due to a hippocampal tropism of tmev in b mice and is also associated with development of seizures and epilepsy (depaula-silva et al., ) . additionally, thalamic, hypothalamic and cortical regions were screened for morphological alterations and staining positivity. furthermore, paraffin embedded sections of the cervical, thoracic and lumbal region of the spinal cord were examined to assess alterations in these regions. inflammatory processes were assessed by histological staining with h&e and additional immunohistochemistry staining with mac- (bio-rad abd serotec gmbh, puchheim, germany), iba (wako chemicals gmbh, neuss, germany and abcam, cambridge, gb), cd (dakocytomation gmbh, hamburg, germany), foxp (ebioscience; antibody-clone-fjk- s-monoclonal/ - - ), tmem (abcam) and iba /mac- fluorescent double staining and were analyzed as described recently (bennett et al., ; bröer et al., ; ciurkiewicz et al., ; waltl et al., ) . h&e staining of spinal cord was used to examine neuronal necrosis, hypercellularity and perivascular infiltration and scored semi-quantitatively (gerhauser et al., ) . to assess the inflammatory signs in more detail, an additional mac- staining was performed and scored semi-quantitatively (bröer et al. ; waltl et al., ) as follows: score: , absent (no positive, activated cells within the region); , mild (single positive cells found in the region); , moderate (up to % of region populated with positive cells); , severe (> % of region populated with positive cells). data from both hemispheres were analyzed separately except for thalamus/hypothalamus and cerebral cortex, where mac- -positive cells were often in medial portions, so that these portions in both hemispheres were analyzed. cd -positive, foxp -positive and tmem -positive cells within the hippocampus and spinal cord were counted. neurodegeneration within the hippocampus was assessed using neun immunohistochemistry (merck millipore, darmstadt, germany) and scored semi-quantitatively (for details see polascheck et al., ; waltl et al., ) . also, fluoro-jade c (fjc) staining was performed to visualize the presence of degenerating neurons (gröticke et al., ; waltl et al., ) . glial fibrillary acid protein (gfap) was immunostained to evaluate gliosis and astrocytosis in a semi-quantitative manner (score: = absent, = mild, = moderate, = severe). virus antigen was stained using an anti-tmev (vp ) antibody, and virus-positive cells within the brain were scored in a semi-quantitative manner (score , no stained cells; , few positively stained cells (< %); , - % of cells stained; , > - % of cells stained; , > % of cells stained), whereas virus-positive cells within the spinal cord were counted (kummerfeld et al., ; waltl et al., ) . to assess demyelination within the spinal cord, luxol-fast-blue staining was performed and slides were screened for signs of demyelination (kummerfeld et al., ) . to assess alterations of the myeloid system in the periphery, spleens were stained for iba and tmev and analyzed using the positive pixel count of the imagej software . all analyses were performed by two experienced researchers blinded to the experimental groups. data of both researchers was averaged. immune cells were isolated from whole brain homogenates as recently described . briefly, in accordance to the manufacturer's specifications brain tissue was processed using the neural tissue dissociation kit (miltenyi biotec, bergisch gladbach, germany). afterwards, a percoll density gradient ( - - % percoll layers) was used for cell separation. following density centrifugation, immune cells were washed and stained for min at °c using the following antibodies: cd b apc-cy (bd biosciences; heidelberg, germany), and cd . pacific blue (biolegend, san diego, ca, usa). respective isotype control antibodies or unstained samples were used to determine gates. a slightly adjusted protocol was used to isolate t-, and b-lymphocytes from whole brain homogenates. therefore, the dissociation mix contained collagenase and dnase and the following antibodies were used: cd . pacific blue, cd ß percp-cy . , ly c af , b pe-cy , cd fitc, cd brilliant violet , foxp pe, ly g pe-cy , ccr af , mhcii pe (biolegend, san diego, ca, usa), and cd c bv and cd b apc-cy (bd biosciences; heidelberg, germany). for flow cytometric analyses of blood samples, edta-blood was stained at °c for min with the following antibodies: b pacific blue, cd af (biolegend), cd b apc-cy (bd biosciences), and csf r bv (biolegend) (for more details see waltl et al., ) . flow cytometry was performed using a lsrii flow cytometer (bd biosciences) and data was analyzed using flowjo software (tree star, ashland, or, usa). ribonucleic acid (rna) was isolated from frozen brain and spinal cord tissue using the rneasy lipid tissue mini kit (qiagen, hilden, germany) and subsequently transcribed into cdna with the omniscript rt kit (qiagen), random primers (promega, mannheim, germany), and rnase out (invitrogen, darmstadt, germany). rt-qpcr was performed for tmev, il- β, il- , il- , il- , csf r, tnfα, ifnγ, tgfβ , and three housekeeping genes (gapdh, β-actin, hprt) using standard protocols, the mx p qpcr system (agilent technologies deutschland gmbh, böblingen, germany), and brilliant iii ultra-fast sybr®qpcr master mixes (gerhauser et al., (gerhauser et al., , . forward (f) and reverse (r) primers designed for tmev and murine genes were as follows: tmev-f ′-gactaatcagaggaacgtcagc- ′, tmev-r ′-gtgaagagcggcaagtgaga- ′; il- β-f ′-agctacctgtgtcttt cccg- ′, il- β-r ′-agtgcagttgtctaatgggaac- ′; il- -f ′-gtt ctctgggaaatcgtgga- ′, il- r ′-ccagaggaaattttcaata ggc- ′; il- -f ′-actctccaccgcaatgaaga- ′, il- r ′-ctctca ggctccctcttcag- ′; il- -f ′-gccacctttgctgacctaag- ′, il- r ′-tttcccaaagccacgtcaag- ′; csf r-f ′-aggaggtgacagt ggttgag- ′, csf r-r ′-catggtcttgcacacgtagg- ′; tnfα-f ′-gcctcttctcattcctgctt- ′, tnfα-r ′-cacttggtggtttgcta cga- ′; ifnγ-f ′-cacggcacagtcattgaaag- ′, ifnγ-r ′-aatctg gctctgcaggattt- ′; tgfβ -f ′-ttgcttcagctccacagaga- ′, tgfβ -r ′-tggttgtagagggcaaggac- ′. tenfold serial dilution standards ranging from to copies/μl were used to quantify the results. experimental variations were corrected by using a normalization factor calculated from the housekeeping genes (vandesompele et al., ) . melting curve analysis was performed to control the specificity of each reaction. graphpad prism version (la jolla, ca, usa) was used for all statistical analyses. depending on the distribution of the data, either parametric or nonparametric statistical tests were applied. for comparisons of two groups, either student's t-test or mann-whitney u test were used; for more than two groups either one-way or two-way anova for parametric or nonparametric data were used, followed by a post-hoc dunnett's, tukey's or bonferroni's multiple comparisons test. the log-rank mantel-cox test was used for survival analysis. seizure frequencies were compared using fisher's exact test. a p ≤ . was considered significant. supplemental table s gives an overview of the various experimental results of this study. mice were treated with plx , an orally bioavailable selective csf r inhibitor that crosses the blood-brain barrier (dagher et al., ) , for a period of days prior to infection and treatment was continued until animals were killed - days after infection (fig. ). for this purpose plx was mixed into standard rodent chow at ppm. to verify the microglia depletion, we used a microglia-selective antibody (tmem ; bennett et al., ) . as shown in fig. a for the hippocampus and para-hippocampal tissue, treatment with plx completely depleted microglia in mock-infected control mice and mice infected with tmev, which was verified by counting tmem + cells in hippocampus (fig. b) . a similar lack of tmem -positive microglia was observed throughout the brain of plx -treated mice. in infected b controls, significant microglia accumulation was seen in hippocampus and para-hippocampal regions ( fig. a and b) . we also quantitated csf r mrna in the brain and spinal cord of mice with and without plx , showing a significant increase in csf r upon infection in mice not treated with plx ( fig. c and d). in non-infected mice, expression of csf r was almost completely abolished by treatment with plx , while a small but significant increase was seen in plx -treated infected mice (fig. d ). as described previously (howe et al., a; cusick et al., ; waltl et al., ) , flow cytometry was used to differentiate cd high cd b + cells, which are considered to be mainly brain-infiltrating blood-derived monocytes, from cd low cd b + cells, which are considered to be mainly resident microglia, in the brain (fig. a-d) . therefore, we will name these two cell populations monocytes and microglia in the following. we found that treatment with plx fig. . experimental protocol of the study. for details see the methods section. overall five experiments were performed with this protocol in groups of mice. first experiments were performed over days following infection. however, the poor health status of the infected plx treated mice at day , resulting in mortality or euthanasia (see results), led us shorten subsequent experiments to days. depletion of microglia via csf r inhibition by treatment with plx in theiler's virus-infected mice. in the representative photomicrographs shown in a, microglia were immunostained with tmem . infection increased tmem + cells in controls, which was completely inhibited by treatment with plx . quantification of data is shown in b (microglia in hippocampus). data are shown as boxplots with whiskers from minimum to maximal values; the horizontal line in the boxes represents the median value. in addition, individual data are shown. sample size: - mock-infected controls; - mock-infected mice with plx ; - infected controls; - infected mice with plx . significant differences to mock-infected mice are indicated by asterisks ( * p < . ; ** p < . ) while significant differences between infected mice treated with plx and infected controls are indicated by the hash sign ( # p < . ; ## p < . ; ### p < . ). (b) tmem + cells in the hippocampus. note the complete depletion of microglia in plx -treated mice. (c) csf r mrna quantitated by qpcr in the brains of mice. (d) csf r mrna quantitated by rt-qpcr in the spinal cord of mice. all other details in c and d are as described above for b. almost completely depleted microglia in mock-infected control mice and mice infected with tmev ( fig. b , d, e). as shown in fig. d and f, treatment with plx did not prevent the massive brain invasion of blood-derived monocytes after infection with tmev. in the blood, infected mice exhibited a significant increase in csf r + monocytes, which was significantly reduced by plx , but was still significantly higher than in mock-infected mice treated with plx (fig. g ). the number of iba + macrophages in spleen was not affected by treatment with plx (fig. h ). next we examined whether treatment with plx affects the adaptive immune response to infection with tmev. infiltration of cd + t-lymphocytes in the hippocampus was comparable in both groups (fig. s a ). in addition, significant invasion of cd + t lymphocytes (cd + t effector cells) was observed in the brain of both infected groups, but cd + cell invasion was significantly lower in the plx treated group (fig. a ). significant brain invasion of cd + cytotoxic t lymphocytes (cd + t effector cells), which are thought to play a significant role in viral clearance in the tmev model in mice (libbey and fujinami, ; depaula-silva et al., ) , was observed both in infected controls and infected mice treated with plx , without significant inter-group difference (fig. b ). the brain cd :cd ratio was . in infected controls, demonstrating that brain invasion of cd + t cells outnumbers invasion of cd + t cells in this model. as a result of reduced cd + t cell invasion in the plx -treated group, the cd :cd ratio was further reduced to . . we also determined cd + and cd + cells in the spleen of infected mice (data not shown), resulting in a cd :cd ratio of . in infected controls vs. . in plx -treated animals. interestingly, in addition to the reduced invasion of cd + t cells in the brain of microglia-depleted infected mice (fig. a ), the number of invading cd + cells expressing the adhesion receptor cd , which is upregulated after activation of naive t lymphocytes during their responses against viruses (baaten et al., ) , was significantly reduced in infected mice with depletion of microglia (fig. c ). the brain invasion of foxp + regulatory t cells (tregs) was increased in both infected controls and plx -treated infected mice to a similar extent without significant inter-group difference (fig. d) . however, in the hippocampus, which is the main target of tmev infection in b mice because of tropism of the virus to this region (libbey and fujinami, ) , a significant invasion of foxp + tregs was only observed in infected mice treated with plx (fig. e) . a fig. . microglia are indicated as cd low cd b + positive cells (black circle in each dot plot), while brain-infiltrating blood-derived monocytes are indicated as cd high cd b + cells (red circle in each dot plot). note the scarcity of infiltrating macrophages in mock control (a) and the marked infiltration of such macrophages in the brain following virus infection (c, d). also moderate microglia activation is seen in c, while treatment with plx almost completely depleted microglia in both mock-infected controls (b) and infected mice (d). quantification of flow cytometric data is shown in e (microglia) and f (invading monocytes). data are shown as boxplots with whiskers from minimum to maximal values; the horizontal line in the boxes represents the median value. in addition, individual data are shown. sample size: - mock-infected controls; mock-infected mice with plx ; infected controls; - infected mice with plx . significant differences to mock-infected mice are indicated by asterisks ( * p < . ; ** p < . ) while significant differences between infected mice treated with plx and infected controls are indicated by the hash sign ( # p < . ; ## p < . ). (e) microglia (cd low cd b + ) as analyzed by fluorescence-activated cell sorting. note the complete depletion of microglia in plx -treated groups. (f) brain-infiltration of inflammatory monocytes (cd high cd b + ) as analyzed by fluorescence-activated cell sorting. virus infection increased monocyte infiltration ∼ fold vs. mock, which was not affected by treatment with plx . (g) flow cytometric analysis of blood monocytes positive for colony stimulating factor receptor (csf r + ) indicated significant increases by infection in both groups. (h) in the spleen, macrophages were immunostained by iba , demonstrating a significant increase in both infected groups. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) similar finding was obtained for the spinal cord (fig. f ). since cd + t cell effector function is suppressed by elevated induction of tregs (richards et al., ) , the significant increase in tregs following csf r inhibition may lead to an unfavorable hippocampal and spinal cord ratio between tregs and effector t cells, thus reducing antiviral immunity in these regions. this possibility is substantiated by the high increase in brain mrna expression of the immunosupressive cytokine il- in the brain of infected plx -treated mice (see below), which is released by tregs and suppresses the activation of cytotoxic t cells (o'shea et al., ) . compared to mock-infected controls, a modest but statistically significant brain infiltration of neutrophils was observed in infected controls. in the plx -treated group only a trend (p = . ) for neutrophil infiltration was observed, but both infected groups did not differ from each other (fig. s b) . b mice typically survive the acute viral encephalitis and clear tmev within weeks after infection (libbey and fujinami, ) . furthermore, in this mouse strain the virus exhibits tropism to the ca and ca regions of the hippocampus following intracerebral infection (libbey and fujinami, ) . as shown in fig. a , c and d, in most b control mice theiler's virus was already eliminated from the hippocampus by day / , except for some staining in the ca in a few mice. in contrast, in mice treated with plx , virus antigen was present throughout the hippocampus (fig. b) , resulting in significant increases in virus antigen-positive cells in the ca /ca region of the hippocampus and other hippocampal regions (ca , dentate gyrus) in which normally the virus is not present (fig. c and d) . at higher magnification, localization of the virus in neuronal cell bodies was visible in the hippocampus. in addition, virus antigen was infrequently observed outside neurons, associated with cells (most likely macrophages) around small vessels in the hippocampus. furthermore, virus antigen was detectable in cerebral cortex, thalamus, hypothalamus (fig. e ), striatum and several other brain regions of plx -treated mice, demonstrating that treatment with this compound increased the brain distribution of the virus. this is also illustrated by the presence of virus antigen in the spinal cord, in which the virus was not present in infected b vehicle controls (fig. f ). in addition to tmev protein, we also determined tmev mrna, substantiating the significantly reduced elimination of the virus by treatment with plx ( fig. g and h) . immunohistochemistry with an antibody against mac- was used to determine neuroinflammation in the hippocampus of infected mice. mac- (also known as cd b and lamp- ) is a glycoprotein that is expressed at the plasma membrane of both activated microglia and fig. . infiltration of t lymphocytes into the brain of theiler's virus-infected mice. cd + and cd + t cells as well as cd + cd + and cd + foxp + t cells were analyzed by flow cytometry using the whole brain; in addition, foxp + regulatory t cells (tregs) were analyzed by immunohistochemistry in the hippocampus and spinal cord. data are shown as boxplots with whiskers from minimum to maximal values; the horizontal line in the boxes represents the median value. in addition, individual data are shown. sample size: - mock-infected controls; mock-infected mice with plx ; - infected controls; - infected mice with plx . significant differences to mock-infected mice are indicated by asterisks ( * p < . ; ** p < . ) while significant differences between infected mice treated with plx and infected controls are indicated by the hash sign ( # p < . ). (a) cd + t lymphocytes were significantly increased in the brains of both infected groups, but the increase was significantly lower in plx -treated mice. (b) cd + t lymphocytes were significantly increased in the brains of both infected groups. (c) cd + cd + cells were only increased in the brains of infected controls, but not the brains of microglia-depleted mice. (d) cd + foxp + cells were increased in the brains of both infected groups. (e) a significant increase in foxp + regulatory t cells in the hippocampus was only observed in infected plx -treated mice. (f) in addition to the hippocampus shown in e, a significant increase in foxp + regulatory t cells was also observed in the spinal cord of infected plx -treated mice. i. waltl et al. brain, behavior, and immunity ( ) - invading monocytes (prinz et al., ) . as shown in fig. , at one week after infection, only some infected b control mice exhibited mac- + cells in the hippocampus, whereas significant increase in mac- + cells was observed in the cerebral cortex, thalamus and hypothalamus. neuroinflammation was increased by treatment with plx in that significant increase in mac- + cells was also observed in hippocampal ca , ca , ca , and dentate gyrus (fig. ) . mac- labeling in the contralateral hemisphere was similar to that in the ipsilateral hemisphere shown in fig. . for further evaluating the activation status of myeloid cells in the brain, the amount of iba -and mac- -immunolabeled cells was compared in the hippocampus. ionized calcium binding adaptor molecule (iba ) is a calcium-binding protein that labels both resting and activated microglia and infiltrating monocytes, whereas mac- labels only activated microglia and invading monocytes (greter et al., ) . regarding iba -labeling, almost no such labeling was observed in mockinfected controls (fig. a) , while the number of iba + cells significantly increased in infected controls (fig. b) . in plx -treated infected mice, the number of iba + cells was lower in the ca /ca region compared to infected controls (fig. d) but not in other hippocampal regions (ca c shown as example in fig. e ). however, a difference in iba + cell shape was observed in that both round and ramified cells were observed in infected controls (fig. b) , representing monocytes and resting and activated microglia, whereas only round cells were observed in plx -treated mice (fig. c) , substantiating the depletion of microglia as indicated by flow cytometry. depending on the hippocampal sub-region and treatment, on average - % of the iba -labeled cells in infected mice were also positive for mac- . the number of iba /mac- double labeled cells in the hippocampus did not significantly differ between infected controls and plx -treated mice ( fig. f and g) . however, when the activation percentage was calculated (i.e., the percentage of iba /mac- double-labeled cells in percent of all iba -labeled cells), it was significantly higher in plx -treated mice than infected controls in the ca c (fig. i ) and perivascular area (data not shown). for further characterizing the neuroinflammation following infection, we determined perivascular cuffing and hypercellularity in he stained sections of the hippocampus. perivascular cuffing, the accumulation of lymphocytes and monocytes in a dense mass around vessels, is a typical sign of viral encephalitis (libbey and fujinami, ) . similarly, hypercellularity, which results from disease-associated infiltration of lymphocytes and monocytes but also from astrocytosis and microgliosis, is an indicator of neuroinflammation. as shown in suppl. fig. s c and d, infected b vehicle control mice only rarely exhibited perivascular infiltrates (pvi) at week after infection, whereas a significant number of such infiltrates were observed in the hippocampus of plx -treated mice. the same was true for hypercellularity (supplemental fig. s e ). in addition to microglia, astrocytes are activated in response to tmev infection in b mice, which can be labeled by glial fibrillary acidic protein (gfap); gfap + astrocytes are particularly prominent in the hippocampus of infected mice (libbey and fujinami, ) . in the present experiments, the number of gfap + astrocytes was only rarely different between mock-infected controls and tmev-infected b control mice, whereas a marked astrogliosis was observed in plx -treated mice (supplemental figs. s f and s ). within the hippocampus, gfap + astrocytes were primarily located in the tissue adjacent to the damaged cell layers (supplemental fig. s ). gliosis was also observed in the corpus striatum of all infected mice (not illustrated). when neurons were stained by neun, the most marked neuronal damage in infected b vehicle controls was observed in the ca region of the hippocampus (fig. a , e-h). treatment with plx significantly increased neuronal damage in the hippocampus (fig. b , e-h). significant neuron loss was also observed in parahippocampal cortex (not illustrated). for further analysis of neurodegeneration in the hippocampus and other brain regions, sections were stained with fluoro-jade c (fjc), a sensitive and specific fluorescent marker of dying neurons (schmued et al., ) . in mock-infected controls, no fjc-stained neurons were observed (not illustrated), while intense staining of pyramidal cells was observed in the ca and ca layers of some infected b vehicle controls (fig. c) . in infected mice treated with plx , fjc-labeled neurons were not only observed in ca and ca but all regions of the hippocampal formation, including ca , the granule cell layer of the dentate gyrus and the dentate hilus (fig. d) . furthermore, fjc-labeled neurons were observed in the cortex. counting of fjc-positive cells in the hippocampus substantiated the impression of visual inspections of sections in that infected mice treated with plx exhibited significantly more degenerating neurons in the hippocampus than infected controls (see fig. i and j as examples; significant differences also observed in ca a, ca c, and dentate gyrus). in contrast to sjl/j mice, b mice typically do not develop a chronic inflammatory demyelinating disease in the spinal cord, so b mice are considered to be resistant to the tmev-induced demyelinating disease (libbey and fujinami, ) . consistent with this view, infected b control mice did not show any indication of cell death or inflammation in the spinal cord (fig. c ) except for a moderate increase in cd + t lymphocytes and mac- labeling ( fig. k and l) . furthermore, myelin staining did not reveal any demyelination (fig. e) . in contrast, plx -treated mice exhibited tmev antigen (fig. a) , necrosis of motor neurons ( fig. b and g) and inflammation in the spinal cord (fig. d, h-j) , thus explaining the paralysis observed in these mice (see below). also, a significant increase in foxp + regulatory t cells was only observed in plx-treated mice (fig. d) . demyelination was not observed in plx -treated mice (fig. f) , which may be due to the fact that mice were killed - days after infection, because demyelination is known to develop slowly in sensitive mouse strains such as sjl/j (depaula-silva et al., ). data are shown as boxplots with whiskers from minimum to maximal values; the horizontal line in the boxes represents the median value. in addition, individual data are shown. sample size: mock-infected controls; mock-infected mice with plx ; infected controls; infected mice with plx . significant differences to mock-infected mice are indicated by asterisks ( * p < . ; ** p < . ; *** p < . ) while significant differences between infected mice treated with plx and infected controls are indicated by the hash sign ( # p < . ; ## p < . ; ### p < . ). in addition, individual data are shown. sample size: mock-infected controls; mock-infected mice with plx ; infected controls; infected mice with plx . significant differences to mock-infected mice are indicated by asterisks ( * p < . ; ** p < . ; *** p < . ) while significant differences between infected mice treated with plx and infected controls are indicated by the hash sign ( # p < . ). (c) scores for mac- labeling in the ipsilateral ca of the hippocampus. at the time chosen ( - days after infection), significant difference to mock controls is only observed for infected mice treated with plx . (d) scores for mac- labeling in the ipsilateral ca of the hippocampus. significant difference to mock controls is only observed for infected mice treated with plx . (e) scores for mac- labeling in the ipsilateral ca c of the hippocampus. significant difference to mock controls is only observed for infected mice treated with plx . (f) scores for mac- labeling in the ipsilateral dentate gyrus (dg) of the hippocampus. significant difference to mock controls is only observed for infected mice treated with plx . (g) scores for mac- labeling in the cerebral cortex. significant difference to mock controls is observed for both infected groups. (h) scores for mac- labeling in the thalamus and hypothalamus. significant difference to mock controls is observed for both infected groups. in infected b vehicle controls, animals did not show any signs of severe impairment due to infection and the following encephalitis as demonstrated by the total clinical score (tcs) and weight development during the first days after infection (fig. a ). however, this strikingly changed in the plx treated group starting from day five post-infection (fig. a ). animals showed severe paralysis of the hind limbs (fig. b) , bent-back posture, and a strong decrease in activity and food uptake. this health impairment was also displayed by severe loss of weight (fig. c) . hence, some animals, which strongly showed these symptoms, had to be sacrificed before day seven to avoid suffering. furthermore, one animal of the plx -treated group died following infection. survival analysis resulted in a highly significant difference between infected b vehicle controls and plx -treated mice (fig. d ). when determining the daily incidence of seizures across the days after infection, maximum seizure occurrence was reached significantly more rapidly in plx -treated mice than in vehicle controls, indicating that microglia depletion facilitated seizure induction (fig. a ). however, overall seizure incidence was not affected by microglia depletion: about % of the infected b vehicle controls exhibited seizures in the first week after infection compared to % of the plx -treated group, which was not significantly different (fig. b) . no seizures were observed in the mock-infected groups. most infected mice exhibited several seizures, but no significant intergroup difference in seizure frequency was observed (fig. c) . similarly, the maximum seizure severity was not different in the two infected groups (fig. d) . in a series of previous studies in tmev-infected b mice, release of the proinflammatory cytokines tnf-α and il- by invading monocytes was described as particularly important for the seizure phenotype of these mice (depaula-silva et al., ) . when these two cytokines were determined by rt-qpcr in the present study, significant increases in il- and tnf-α mrnas were observed in the brain (fig. a, supplemental fig. s a ) and spinal cord (fig. d, supplemental fig. s e ) of infected control mice. in infected plx -treated mice, the brain increase in il- was -fold more marked than the increase determined in infected controls (fig. a) , indicating increased release of this cytokine by invading monocytes. in addition to il- , a . -fold more marked increase in expression of il- was determined in the brain of infected plx -treated mice compared to infected controls (fig. b) . furthermore, a . -fold more marked increase in expression of ifnγ was determined in the brain of infected plx -treated mice compared to infected controls (fig. c ). in the spinal cord, the increase of ifnγ in infected plx -treated mice was -fold higher than the increase determined in infected controls (fig. f) . no marked differences between groups with or without plx were observed for il- β, il- , and tgf-β (supplemental fig. s ). il- was below detection level in both mock and tmev infected mice, including plx treated animals. supplemental fig. s summarizes the selective increases in cytokine and interferon mrna levels in infected plx -treated mice vs. infected controls, demonstrating the preferential increase of il- , il- and ifnγ in the brain and ifnγ in the spinal cord of plx -treated mice. since ifnγ is preferentially released by cytotoxic cd + t cells whereas il- is released by tregs in the infected brain (oleszak et al., ; depaula-silva et al., ) , we calculated the ratio between the increase in ifnγ and il- in the brain of infected controls vs. infected plx -treated mice. this ratio was . in infected controls, indicating higher functionality of t effector cells vs. tregs as regularly observed in tmev-infected b mice (depaula-silva et al., ) , whereas the ratio was . after in infected plx -treated mice, indicating suppression of t effector cells by activated tregs. intracerebral infection of b mice with theiler's virus is the first, and currently only, rodent model of viral encephalitis-induced seizures and epilepsy (vezzani et al., ; depaula-silva et al., ) . this model is ideally suited to investigate the mechanisms underlying the generation of seizures and hippocampal pathology developing during and after viral encephalitis. in contrast to b mice, which rapidly clear the virus, the virus persists in the cns of sjl/j mice, which do not develop seizures and hippocampal damage, but a slowly progressing inflammatory demyelination in the spinal cord, making the tmev infection in sjl/j mice a widely used model of multiple sclerosis (oleszak et al., ; procaccini et al., ; depaula-silva et al., ) . while tmev persistence in sjl/j mice is important to induce chronic demyelination, the trigger mechanism for demyelination is inflammation and the induction of autoimmunity (depaula-silva et al., ) . the persistence of tmev in the cns of sjl/j mice is thought to be related, at least in part, to the high induction of tregs during the acute stage of the infection, because tregs can secrete immunosuppressive cytokines (such as il- ), thereby blocking activation and effector function of cytotoxic cd + t cells that are important for viral clearance (richards et al., ) . in contrast to sjl/j mice, the lower number of tregs induced in b mice after tmev infection has been suggested to allow for rapid viral clearance by cd + t effector cells (richards et al., ) . fig. . neuroinflammation as indicated by iba and mac- labeling and double labeling. while iba labels both resting and activated microglia and infiltrated monocytes in the brain, mac- only labels activated microglia and infiltrated monocytes. (a) representative photomicrographs of a mock-infected control mouse. the first graph shows labeling of cell nuclei by dapi in blue, the second graph iba labeled cells in green, the third graph mac- labeled cells in red, and the fourth graph cells labeled for both iba and mac- . only few iba and no mac- labeled cells are visible, consistent with the lack of neuroinflammation in mock-infected mice. (b) representative photomicrographs of an infected control mouse. note the marked increase in both iba and mac- labeling. both ramified and round cell types are visible. iba and mac- double labeled cells are indicated in yellow. (c) representative photomicrographs of an infected mouse with plx treatment. note the lack of ramified cells, which is due to microglia depletion. scale bar in a, b and c = µm. (d-i) quantitative data for expression of iba , iba /mac- double labeled cells, and the "activation percentage" indicated by iba /mac- double labeling. data are shown as boxplots with whiskers from minimum to maximal values; the horizontal line in the boxes represents the median value. in addition, individual data are shown. sample size: mock-infected controls; - mock-infected mice with plx ; - infected controls; - infected mice with plx . significant differences to mock-infected mice are indicated by asterisks ( * p < . ; ** p < . ) while significant differences between infected mice treated with plx and infected controls are indicated by the hash sign ( # p < . ; i. waltl et al. brain, behavior, and immunity ( ) - indeed, tregs, characterized by expression of the transcription factor forkhead box p (foxp ), are known to modulate the immune response to viral infection, and numerous infection models have demonstrated that treg depletion increases viral clearance and ifnγ production by cd + t cells (groux et al., ; dittmer et al., ; he et al., ; eggena et al., ) . however, in tmev-infected b mice, ablation of foxp + tregs during the acute disease phase of tmev infection did not accelerate virus clearance (prajeeth et al., ) . in contrast, ablation of cytotoxic cd + t cells rendered b mice susceptible to develop chronic demyelinating neuroinfection comparable to the encephalomyelitis observed in sjl/j mice, which might partly be mediated by boosting the suppressive capacity of tregs on viral elimination (ciurkiewicz et al., ) . in addition to cd + t cells, it has been suggested that cd + t cells may confer protection against tmev persistence and demyelination in resistant mouse strains such as b (murray et al., ; kang et al., ; mohindru et al., ) . indeed, genetic deletion of either cd + or cd + cells from b mice resulted in viral persistence and demyelination during the chronic stage of disease (murray et al., ) . interestingly, neither cd + nor cd + cells are required for development of acute seizures following infection with tmev in b mice (depaula-silva et al., ) . the present study shows that depletion of microglia is an effective means to retard viral clearance and exacerbate the tmev-induced disease in b mice, thus defining the role of microglia in this model of viral encephalitis. previously, understanding the roles that microglia play in a neurotropic viral infection and how these roles affect the immune response to viruses has been complicated by differentiating microglia from extensively infiltrating monocytes with potentially overlapping functions during neuroinflammation (terry et al., ; prinz and priller, ; . furthermore, while microglia, the resident macrophages of the cns, are often touted as the first responders to a cns viral infection and are known to respond rapidly to injury (rock et al., ; carrithers, ) , there is little direct evidence to show that microglia are needed for an optimal host response to pathogens. selective and complete depletion of microglia by chronic treatment with the csf r inhibitor plx is an ideal means to study the functions that microglia use in responding to viruses. plx is a potent inhibitor of csf r tyrosine kinase activity (ki = . nm) with at least -fold selectivity over related kinases, and over -fold selectivity against a panel of kinases (dagher et al., ) . as shown here, by the plx treatment protocol used, csf r mrna expression in the cns was almost completely lost, which is a consequence of the depletion of microglia that express this mrna. recent studies in the tmev model in b mice have suggested that both seizures and hippocampal damage in this mouse strain are primarily due to infiltration of blood-derived monocytes and cytokines released by these cells (howe et al., a,b; cusick et al., ) , resulting in apoptotic death of neurons in the ca /ca area of the hippocampus (buenz et al., ; howe et al., b) . indeed, suppression of monocyte infiltration by deleting the chemokine receptor ccr prevented hippocampal damage following tmev infection, but did not prevent encephalitis-associated seizures , thus challenging previous assumptions with less selective approaches (cusick et al., ) . the present study shows that depletion of microglia by plx exacerbates hippocampal damage and increases progression of seizures following viral encephalitis, indicating that microglia may exhibit a protective role in this model, at least in the early phase following infection. furthermore, neuronal damage, associated with severe motor impairment, occurred in the spinal cord, which is typically not affected in b mice. the severe clinical impairment and enhanced mortality in infected mice treated with plx did not allow evaluating whether spinal cord demyelination occurs at later stages of the infection as observed in tmev-infected sjl/j mice. the first study that reported the effect of microglia depletion in b mice on viral encephalitis used intracranial infection with a neuroattenuated variant of the jhmv strain of mhv (wheeler et al., ) , which induces mild acute encephalitis and acute and chronic demyelinating disease (bergmann et al., ) . treatment with plx increased cns replication of mhv and subsequent mortality in b mice, which was explained by ineffective innate and virus-specific t cell responses resulting from microglia depletion (wheeler et al., ) . indeed, microglia depletion led to a markedly reduced brain infiltration of cd + t cells and foxp + tregs as well as ifnγ expression at days after infection (wheeler et al., ) . the present data show that these consequences of microglia depletion are virus specific, because we did not observe a decrease but a significant increase in infiltration of foxp + tregs in hippocampus and spinal cord and only a relatively small reduction in brain invasion of cd + t cells in tmev-infected, plx -treated mice compared to infected controls, although the invasion of cd + cd + t cells was markedly reduced by microglia depletion. furthermore, in apparent contrast to the study of wheeler et al. ( ) , ifnγ expression was significantly increased in brain and spinal cord of plx -treated mice in response to tmev infection. in a recent study using plx for determining the role of microglia in flaviviral pathogenesis in female swiss-webster mice (seitz et al., ) , depletion of microglia resulted in increased mortality and viral titer in the brain following infection with either west nile virus (wnv) or japanese encephalitis virus (jev). interestingly, the expression of several pro-inflammatory genes was increased in virus-infected, microglial-depleted mice compared to virus-infected, untreated controls. t cell responses were not examined in the study of seitz et al. ( ) . in both previous studies on depleting microglia in viral cns infection models, iba was used as a marker for microglia (seitz et al., ; wheeler et al., ) . however, iba is not selective for microglia but also labels monocytes that invade the brain during viral encephalitis (greter et al., ) , which is also shown here. for the present study, . data are shown as boxplots with whiskers from minimum to maximal values; the horizontal line in the boxes represents the median value. in addition, individual data are shown. sample size: mock-infected controls; - mock-infected mice with plx ; infected controls; infected mice with plx . significant differences to mock-infected mice are indicated by asterisks ( * p < . ; ** p < . ) while significant differences between infected mice treated with plx and infected controls are indicated by the hash sign ( # p < . ; ## p < . ). (e) shows the increased neurodegeneration in infected plx -treated mice in the ipsilateral ca layer of the hippocampus. (f) shows that neurodegeneration in the ca layer of the ipsilateral hippocampus does not differ between infected controls and infected plx -treated mice. (g) shows the increased neurodegeneration in infected plx -treated mice in the ipsilateral ca c layer of the hippocampus. (h) shows the increased neurodegeneration in infected plx -treated mice in the ipsilateral dentate gyrus (dg) layer of the hippocampus. (i) shows the increased number of dying neurons in infected plx treated mice in the ipsilateral ca /ca layers of the hippocampus. (j) shows the increased number of dying neurons in infected plx -treated mice in the ipsilateral dentate hilus of the hippocampus. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) data are shown as boxplots with whiskers from minimum to maximal values; the horizontal line in the boxes represents the median value. in addition, individual data are shown. sample size: mock-infected controls; mock-infected mice with plx ; infected controls; infected mice with plx . significant differences to mock-infected mice are indicated by asterisks ( * p < . ; ** p < . ) while significant differences between infected mice treated with plx and infected controls are indicated by the hash sign ( # p < . ; ## p < . ). all parameters shown in g-l demonstrated the increased neuroinflammation and neurodegeneration induced by treatment with plx in the spinal cord of c bl/ (b ) mice. all scale bars = µm. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) fig. . clinical data from infected mice. experiments were performed several times (see fig. ) and data summarized; sample size: mock-infected controls; mock-infected mice with plx ; infected controls; infected mice with plx . (a) illustrates the "total clinical score", describing the overall clinical impairment, ranging from (healthy) to (maximal clinical impairment) (see methods). infected controls did not differ from mock-infected controls (except for seizures, which were assessed separately as shown in fig. ), while infected plx -treated mice developed severe clinical symptoms, including paralysis of the hind limbs, bent-back posture, and a strong decrease in general activity and food uptake. data are shown as means ± sem and were analyzed by two-way anova; significant differences between infected plx -treated mice to all other groups are indicated by the hash sign ( # p < . ). (b) a representative picture of a plx -treated infected mouse with paralysis of hind limbs, which was never observed in infected controls. (c) body weight development. data are shown as means ± sem and were analyzed by two-way anova; significance between infected and mock-infected mice is indicated by asterisk ( * p < . ), while significance between plx -treated and vehicle-treated infected mice is indicated by the hash sign ( # p < . ). note the much more severe weight loss in infected plx -treated mice. (d) survical curves for infected controls and infected plx -treated mice. mice that had to be killed before day (the planned end of the experiment) because of poor health status were included in survival analysis. statistical evaluation of data by the log-rank (mantel-cox) test indicated a highly significant (p < . ) difference between groups. we chose the much more selective microglia marker tmem (bennett et al., ) , demonstrating that plx results in complete microglia depletion at the chosen dosing protocol. this was confirmed by flow cytometry. csf r, the receptor affected by plx , is also expressed on other myeloid cells, including brain perivascular macrophages and infiltrating hematopoietic monocytes (hamilton and achuthan, ) . as a consequence, we observed a significant decrease in circulating csf r + monocytes in the blood of mock infected mice after treatment with plx . after infection, blood csf r + monocytes increased in both controls and plx -treated groups, although to a slightly lower degree in the plx -treated mice. this was not associated with any obvious reduction in brain infiltration of blood-derived monocytes as both demonstrated by flow cytometry and immunolabeling by mac- and iba . instead, both mac- and iba indicated an increase of myeloid cells in the hippocampus and spinal cord of infected mice after depletion of microglia, indicating that infiltrating hematopoietic monocytes/macrophages try to compensate for the lack of microglia. in line with this idea, mrna expression of il- , which is released by both invading monocytes and activated microglia (oleszak et al., ) , was increased -fold in microglia-depleted brains of infected mice. interleukin- (il- ) plays an important role in the development and progression of inflammatory responses, in part by facilitating the generation of il- -producing th cells (kothur et al., ) . many viral infections, including tmev, result in the vigorous production of il- (kothur et al., ; depaula-silva et al., ) . b mice carrying an il- transgene, resulting in the production of excessive il- , develop a tmev-induced demyelinating disease accompanied by an increase in viral persistence and an elevated th cell response in the cns (hou et al., ) . il- producing th cells have been shown to promote fig. . insult-associated seizures monitored in the first week after infection. experiments were performed several times (see fig. ) and data summarized; sample size: mock-infected controls; mock-infected mice with plx ; infected controls; infected mice with plx . (a) occurrence of acute (early) seizures in the - days after infection. data are shown as percent of mice with seizures per mice infected with theiler's da virus for each experimental day. significant intergroup differences are indicated by the hash sign ( # p < . ; ## p < . ). (b) illustrates the incidence of seizures in the four groups, showing that none of the noninfected controls exhibited seizures whereas / infected controls and / infected plx-treated mice exhibited seizures, which both was significantly different from noninfected controls (indicated by asterisks; * p < . ). (c) frequency of seizures recorded in infected mice. data are shown as boxplots with whiskers from minimum to maximal values; the horizontal line in the boxes represents the median value; in addition, individual data are shown. no intergroup difference was found. (d) seizure severity as indicated by the maximum seizure score for each mouse with seizures. data are shown as boxplots with whiskers from minimum to maximal values; the horizontal line in the boxes represents the median value; in addition, individual data are shown. significant differences to noninfected controls are indicated by asterisks ( * p < . ). no intergroup difference was found in the experiment illustrated in d. persistent viral infection by inhibiting cytotoxic t cell function and apoptotic cell death (hou et al., ) . as shown by hou et al. ( ) , excessive il- promotes the generation of th cells, and the resulting il- and il- synergistically promote viral persistence by protecting virus-infected cells from apoptosis and cd + t cell-mediated target destruction. these data suggest that the -fold increase in il- following microglia depletion by plx may be involved in the altered responses to tmev infection observed in the present experiments. however, we did not observe an increase in il- in microglia-depleted mice following tmev infection, demonstrating low numbers of th cells in this early phase of the disease ( - days p.i.). instead, we observed a marked increase in brain mrna expression of the immunosuppressive cytokine il- in the brain of infected plx -treated mice compared to infected b controls. among other cell types, il- is secreted by tregs, which were significantly increased in the brain of infected plx -treated mice. thus, similar to tmev infection in sjl/j mice, microglia depletion in b mice seems to lead to an unfavorable ratio between tregs and t effector cells, resulting in suppression of t cell activation and effector function. this interpretation of the data was substantiated when calculating the ratio between ifnγ and il- in the brain of infected controls vs. infected mice treated with plx , demonstrating a reversal in this ratio from . to . . this indicates that il- suppressed functionality of ifnγ-releasing t effector cells following microglia depletion. in the spinal cord, however, a massive, -fold increase of ifnγ was observed. this increase in ifnγ may be critically involved in disease exacerbation. in neuroinflammatory cns disorders, ifnγ is elevated mainly in viral encephalitis, suggesting activation of lymphocytes in the cns which participate in viral clearance (kothur et al., ) . ifnγ, which is produced by activated natural killer (nk) and t cells, is important for virus clearance and induces many immunomodulatory effects, including activation of macrophages, promotion of leukocyte adhesion to allow trafficking of cells to the cns, direct antiviral and antiproliferative effects, and induction of the release of other cytokines, including tnf-α and il- (bowen and olson, ) . ifnγ has been implicated in the clearance of virus from cns following infection with a wide variety of viruses, including tmev (rodriguez et al., ) . however, at high concentrations as observed following microglia depletion here, ifnγ is neurotoxic through a neuron specific, calciumpermeable complex of ifnγ-receptor and the ampa subtype of glutamate receptors (mizuno et al., ) . thus, ifnγ in concert with other cytokines such as il- β may contribute to the increased degeneration of hippocampal and spinal cord motor neurons and clinical signs observed after microglia depletion in tmev-infected mice. this, however, does not necessarily explain the facilitation of seizures observed in infected plx -treated mice. here, a direct effect of microglia could be important. by debris clearance and continuously contacting dendritic spines to regulate structural synaptic changes, microglia plays a fundamental role in facilitating the reorganization of neuronal circuits and triggering repair in cns injury (neumann et al., ; kim et al., ) . proper synaptic pruning (or engulfment) by microglia has been shown to modulate seizure threshold (kim et al., ) , which could explain the facilitated seizure induction observed after microglia depletion in theiler's virus-infected mice. overall, based on the present data, the following scenario can be postulated. depletion of microglia reduces the local immune response to infection, resulting in increased virus proliferation. enhanced induction of tregs in hippocampus and spinal cord and resulting il- release seem to add to the reduced immune response to viral infection observed after microglia depletion. on the other hand, markedly fig. . tnfα, il- , and ifnγ mrna expression in brain and spinal cord of infected mice. data are shown as boxplots with whiskers from minimum to maximal values; the horizontal line in the boxes represents the median value. note that y-axis has a logarithmic scale. in addition, individual data are shown. sample size is for noninfected mice and for infected mice. significant differences to mock-infected mice are indicated by asterisks ( * p < . ; ** p < . ) while significant differences between infected mice treated with plx and infected controls are indicated by the hash sign ( # p < . ; ## p < . ). increased il- , and, in the spinal cord, ifnγ levels contribute to neurodegeneration and elevated seizure susceptibility. also, the increased astrogliosis observed in infected, microglia-depleted mice may contribute to neuroinflammation and related processes, although a neurotoxic subtype (a ) of astrocytes, which is induced by neuroinflammatory microglia (liddelow et al., ) , should be reduced or absent in microglia-depleted mice. our results demonstrate a critical role for microglia in the context of viral encephalitis, affecting virus replication and innate and adaptive immune responses as well as disease progression and severity. however, as indicated by the significant differences in response to microglia depletion between the study of wheeler et al. 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seizures but not hippocampal damage after acute viral encephalitis microglia are required for protection against lethal coronavirus encephalitis in mice we thank prof. robert s. fujinami for providing the da strain of tmev, plexxikon for providing plx , prof. andreas beineke for helpful discussion on a previous version of the manuscript, and edith kaczmarek, caroline schütz, danuta waschke, petra grünig, muneeb anjum, alina schidlitzki for skillful technical assistance. the study was supported by the niedersachsen-research network on neuroinfectiology (n-rennt) of the ministry of science and culture of lower saxony in germany and the helmholtz "zukunftsthema immunology & inflammation". chintan chhatbar was supported by a bilateral project of the german centre for neurodegenerative diseases and the helmholtz centre for infection research (to u.k.) and by the niedersachsen-research network on neuroinfectiology (n-rennt) of the ministry of science and culture of lower saxony, germany (to u.k.). iw and wl conceived the study; iw, ck, ig, cc and lg acquired and validated the data and executed the experiments; iw, ck, ig, cc, lg, and wl analyzed the data; wl wrote the manuscript and all authors revised the manuscript. the authors declare no competing financial interests. supplementary data to this article can be found online at https:// doi.org/ . /j.bbi. . . . key: cord- - g kb authors: takao, masaki; abe, hirohito; sakai, takashi; hamada, hidetoshi; takahara, shiro; sugano, nobuhiko title: transitional changes in the incidence of hip osteonecrosis among renal transplant recipients date: - - journal: j orthop sci doi: . /j.jos. . . sha: doc_id: cord_uid: g kb abstract background immunosuppressive therapy for renal allograft recipients has changed substantially since the introduction of the anti-cd monoclonal antibody, basiliximab. we hypothesized that recent improvements in immunosuppressive treatment may reduce the incidence of osteonecrosis of the femoral head (onfh). this study aimed to investigate transitional changes in the incidence of ofnh among renal transplant recipients by mri. methods participants comprised patients who had undergone renal transplantation from to , during which time basiliximab was in regular use at our institute (recent group), and patients who had undergone rt between and (past group). we compared onfh incidence between the two groups and evaluated risk factors for onfh, including immunosuppressants (calcineurin inhibitors, basiliximab, and/or steroids) and postoperative renal function. results incidence of onfh was lower in the recent group ( %) than in the past group ( . %; p = . ). in the recent group, age was greater, abo/human leukocyte antigen incompatibility was worse, while steroid dose was decreased and post-transplant renal function was improved. cumulative methylprednisolone dose at postoperative week and delayed graft function were identified as risk factors for onfh. conclusion risk of onfh after renal transplantation has fallen with the advent of regular use of basiliximab, although this agent does not appear to be a factor directly associated with the incidence of onfh. study design clinical prognostic study (level iii case control study). osteonecrosis of the femoral head (onfh) is well recognized as a musculoskeletal complication after renal transplantation (rt). with advances in immunosuppressive therapy, improved posttransplant renal function and reduced corticosteroid dose are reported to have led to a decrease in the incidence of onfh [ e ]. however, according to recent screening studies using magnetic resonance imaging (mri), this incidence ranged from . % to . % [ , e ] , inferring that onfh remains a significant complication among renal allograft recipients. immunosuppressive therapy for renal allograft recipients has changed substantially since the introduction of the anti-cd monoclonal antibody, basiliximab (simulect; novartis, east hanover, nj). when administered as induction therapy during the early postoperative period, improvements in postoperative renal function have been reported [ e ] . decreases in the doses of steroids and calcineurin inhibitors have also been reported [ ] and indications for rt have therefore been expanded to include higherrisk patients with older age and increased human leukocyte antigen (hla) mismatch. however, the impact of basiliximab administration on the incidence of post-transplant onfh has not yet been determined, and few mri screening studies have been conducted since its introduction [ ] . we hypothesized that recent improvements in immunosuppressive treatment may reduce the incidence of onfh even with wider indications for rt. the purpose of the present study was twofold: ) to investigate transitional changes in the incidence of ofnh among renal transplant recipients by mri; and ) to investigate factors related to onfh for the entire cohort. the ethical review board of our institution approved the design of this study (registration number: ). the recent group included patients ( men, women; mean (±standard deviation) age, ± years, range, e years); mean body mass index (bmi), ( . ± . kg/m ) who underwent mri of both hips after rt between april and june (i.e., during the period in which basiliximab was regularly used). during the period, patients underwent rt. the past group included patients ( men, women; mean age, ± years; mean bmi, . ± . kg/ m ) who had undergone rt between january and march (i.e., prior to the regular use of basiliximab). during the period, patients underwent rt. there was no significant difference in the rates of mri screening between the groups. ( . % ( / ) vs . % ( / ), p ¼ . , chi-square test). all patients were notified about the reported incidences of onfh after rt, benefits and risks of mri, and possible treatment options, and given the opportunity to object. mri was performed months after rt using either a . -t superconducting magnet system (signa horizon lx . t; general electric medical systems, milwaukee, wi), or one of two . -t superconducting magnet systems (smt x; shimadzu, kyoto, japan or achieva . t a-series; philips healthcare, amsterdam, the netherlands). osteonecrosis was defined as an area of normal intensity demarcated by a low-intensity band on t -weighted imaging [ ] . t -weighted spin-echo images were obtained using a . -t system in the coronal and sagittal planes, with the following settings: repetition time (tr), e ms; echo time (te), e ms; and slice thickness, e mm. spoiled gradient-recalled echo pulse sequences were obtained using the . -t system in the coronal plane with the following settings: tr, . e ms; te, . e . ms; flip angle, ; and slice thickness, . e . mm with no interslice gap. the following items were evaluated: onfh incidence; patient demographic and background factors, including gender, age, bmi, and abo and hla incompatibility; duration of preoperative dialysis; type of renal graft (living/cadaveric donor); preoperative immunosuppressant use, including calcium inhibitors (cyclosporine a/tacrolimus) administered initially or at the time of hospital discharge; concomitant basiliximab administration; steroid administration at , , , and weeks after rt, as prednisolone (psl), methylprednisolone (mpsl), and total steroid doses (converted to psl-equivalent doses); postoperative renal function, including delayed graft function (dgf), blood urea nitrogen (bun) and creatinine (cr) levels at weeks after rt, and acute transplant rejection; and duration of hospitalization. psl-equivalent doses were calculated by adjusting the mpsl dose on the basis of antiinflammatory potency, using a conversion factor of . . acute transplant rejection was diagnosed as follows: a % increase in bun or a % increase in cr level compared with those at a previous sampling, daily urinary output ml, and diagnosis by biopsy. dgf was defined as a patient requiring hemodialysis in the first week after rt. we compared these items between the recent and past groups using the mannewhitney u-test, chi-square test, and fisher's exact probability test for statistical analyses. in addition, we investigated factors influencing the incidence of onfh for the entire cohort. uniand multivariate logistic regression models were used to identify significant risk factors for onfh for the entire cohort. the adjusted odds ratio (or) for predicting onfh was determined for each factor identified by univariate logistic regression analysis. spss for windows version . j statistical software (spss, chicago, il) was used for all statistical analyses. a % significance level was applied to all tests. no cases of onfh were recorded in the recent group ( %; % confidence interval (ci), %e . %), although eight cases of onfh were recorded in the past group ( . %; % ci, . e . %; one-sided fisher's exact probability test, p ¼ . ). intergroup comparisons of background and demographic factors revealed significant increases in age, bmi, number of patients with abo and hla incompatibilities, and living kidney donors in the recent group ( table ) . comparison of postoperative immunosuppressant use showed increased frequency of tacrolimus use, increased number of patients with concomitant use of basiliximab, and decreases in mpsl, psl, and total steroid dose at postoperative weeks , , , and in the recent group ( table ) . regardless of the increase in risk factors for rt such as older age and increased number of abo/hlaincompatible transplants among patients, decreases in steroid administration and improvements in postoperative renal function have been observed in the recent group. in addition, significant decreases were seen in the number of cases with dgf, concentrations of bun and cr at weeks after transplantation, incidence of acute transplant rejection, and duration of hospitalization over time ( table ) . the overall results were surveyed for risk factors for onfh. according to the analysis of individual factors, total mpsl dose at postoperative weeks , , , and , total steroid dose at postoperative weeks , , , and and dgf were identified as significant risk factors (table ) . psl administration at postoperative weeks , , , and was not a risk factor. we performed multivariate analysis (logistic regression analysis) for age, gender, and total mpsl dose at postoperative week , and dgf. significant risk factors for onfh were total mpsl dose at postoperative week (or, . ; % ci, . e . ; p ¼ . ) and dgf (or, . ; % ci, . e . ; p ¼ . ) ( table ) . when the mpsl doses at postoperative weeks , , and were selected as a risk factor rather than that at week , the following were the adjusted values: for week , or was . ( % ci, . e . ; p ¼ . ); for week , or was . ( % ci, . e . ; p ¼ . ); and for week , or was . ( % ci, . e . ; p ¼ . ). mpsl administration at week showed the highest adjusted or. the types of onfh according to japanese investigation committee classification, their natural course and surgical treatment were summarized in table . improvements in post-transplant renal function and reductions in corticosteroid dose may have led to a decrease in the incidence of onfh [ e ]. renal graft function after rt has been improving with advances in immunosuppressive agents such as cyclosporine a [ , ] and tacrolimus [ e ] . several other immunosuppressive agents including sirolimus, everolimus, mycophenolate mofetil, and basiliximab have been introduced in combination with cyclosporine a or tacrolimus and have also improved postoperative renal function [ ] . in contrast, few reports have been published about the influence of these new immunosuppressive agents on the incidence of onfh after rt. basiliximab, an anti-cd monoclonal antibody, is a very strong immunosuppressive agent that exerts its effects through competitive interleukin- antagonism [ ] . when administered as an induction therapy during the early postoperative period, in conjunction with one or more conventional immunosuppressive drugs, decreases in the incidences of acute transplant rejection and dgf and improvements in survival of the transplanted kidney have been reported [ e ] . decreases in steroid and calcineurin inhibitor doses have also been reported [ , , ] . these marked changes in immunosuppressive treatments and postoperative renal function may have reduced the incidence of onfh. the proportion of onfh patients receiving steroid treatment for renal transplantation has reportedly decreased recently according to the multicenter hospital-based sentinel monitoring system for onfh in japan [ ] , although the reasons for these decreases in the proportion of onfh patients receiving renal transplantation remain unclear. in addition, only a small number of reports since have described mri screening for onfh after rt, and the impact of the introduction of basiliximab on the post-transplant incidence of onfh has yet to be determined. we therefore investigated the incidence of onfh since the advent of regular basiliximab use and found that postoperative renal graft function had improved, and the mpsl dose and incidence of dgf as risk factors for onfh had decreased. in addition, the incidence of onfh had decreased. basiliximab seems likely to indirectly reduce the incidence of onfh by reducing the incidence of dgf and allowing reductions in mpsl administration. three main limitations need to be considered when interpreting the results from the present study. first, the number of renal transplant recipients with use of basiliximab might be too small to evaluate its effect on the incidence of onfh. the number of patients included in previous mri screening studies ranged from to [ , e ] , so the number of patients in the present study is comparable with the numbers in those studies. second, mri screening was not performed before rt, so we could not be sure that onfh had not been present prior to transplantation. nevertheless, because the incidence of onfh in our study was lower than that in any previous study evaluated using mri, the incidence was presumed to have also been low even before transplantation. third, the timing of mri screening was earlier compared to other mri screening studies. this might be one reason the incidence of onfh at our institute was lower than described in other reports performed during a similar period. we did not encounter any symptomatic cases with onfh in which mri had been negative at months after rt, so we considered that the incidence of onfh occurring after months after rt was unlikely to have been particularly high. onfh did not occur in any of the patients who underwent rt between april and june at our institution, despite the wider indications for rt in high-risk patients, such as older age and more severe abo and hla incompatibilities. in previous mri screening studies of onfh after rt performed between and without the use of basiliximab, incidences of onfh after rt were within the range of . e . % [ , e ] . although a downward trend was observed in those studies, no mri screening studies for onfh have been reported since , and the present study showed the lowest incidence of onfh after rt among all relevant studies (table ) . to elucidate the risk factors for onfh, all rt recipients who underwent mri screening at our institute were analyzed. the results showed that dgf and cumulative mpsl administration up to weeks after rt were significant risk factors. other risk factors for onfh reported after rt include daily oral steroid dose, bun level months after rt, accumulated steroid dose at months, accumulated steroid dose at weeks, ethnicity (african-american), history of peritoneal dialysis, rejection, delayed graft function, and cyclosporine a administration [ , , , e ] . to date, cumulative steroid dose as of weeks postoperatively has been reported as the shortest-term risk factor for onfh [ ] . the risk factors for onfh after rt in the present study were in accordance with past findings. this result suggests that onfh occurs mostly within the first weeks after rt, suggesting the importance of controlling postoperative graft function and reducing cumulative mpsl administration in the first weeks after rt using immunosuppressive agents that are appropriate for the prevention of onfh. a recent meta-analysis of onfh occurrences among patients taking corticosteroids reported positive associations between mean daily corticosteroid doses and onfh across all diagnoses, including severe acute respiratory syndrome, systemic lupus erythematosus, bone marrow transplantation and renal transplantation, although the correlation between mean daily corticosteroid dose and onfh incidence was not significant among renal transplant recipients [ ] . no evidence of a significant correlation between cumulative corticosteroid dose and incidence of onfh has yet been provided [ , ] . in renal transplant recipients, daily steroid dose is gradually tapered according to the immunosuppressive regimen, so reflecting an overall picture of steroid treatment using a mean value for daily steroid dose is difficult. in addition, duration of steroid treatment and overall cumulative steroid dose vary widely among renal recipients, and steroid administration after onset of onfh might be included in the regression analysis. we therefore focused on the correlation between steroid administration including oral steroid administration and pulsed steroid therapy in the weeks after renal transplantation and onset of onfh at months. the risk of onfh after rt has fallen since the advent of regular basiliximab use, although this agent does not appear to be a factor directly associated with the incidence of onfh. factors related to onfh incidence were found to be renal graft function and mpsl administration for up to weeks. since the introduction of basiliximab, immunosuppressive therapy has changed markedly and renal graft control has also been enhanced, which may have led to decreases in the incidence of onfh. this research is supported by the practical research project for rare/intractable diseases from japan agency for medical research and development, amed. in accordance with icmje criteria, ns and st designed the study and mt wrote the initial draft of the manuscript. ha contributed to the collection and interpretation of data and the assistance of the preparation of the manuscript. the literature data were searched and analyzed by mt and ha. all other authors contributed to the data collection and interpretation. all authors approved the final version to be submitted for publication and agree to be accountable for all aspects of the work. none. all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee and with the declaration of helsinki and its later amendments or comparable ethical standards." informed consent was obtained in the form of opt-out on the web-site. those who rejected were excluded. maintenance immunosuppression use and the associated risk of avascular necrosis after kidney transplantation in the united states cyclosporin a and osteonecrosis of the femoral head incidence of hip osteonecrosis among renal transplantation recipients: a prospective study tacrolimus may be associated with lower osteonecrosis rates after renal transplantation early detection of avascular necrosis of the femoral head following renal transplantation initial changes of non-traumatic osteonecrosis of femoral head in fat suppression images: bone marrow edema was not found before the appearance of band patterns apparent avascular necrosis of the hip: appearance and spontaneous resolution of mr findings in renal allograft recipients initial mri findings of non-traumatic 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necrosis of the femoral head after renal transplantation rabbit-atg or basiliximab induction for rapid steroid withdrawal after renal transplantation (harmony): an open-label, multicentre, randomised controlled trial prolonged action of a chimeric interleukin- receptor (cd ) monoclonal antibody used in cadaveric renal transplantation monoclonal antibody basiliximab with low cyclosporine dose as initial immunosuppression a randomized, double-blind trial of basiliximab immunoprophylaxis plus triple therapy in kidney transplant recipients temporal trends in characteristics of newly diagnosed nontraumatic osteonecrosis of the femoral head from to : a hospital-based sentinel monitoring system in japan hospitalized avascular necrosis after renal transplantation in the united states corticosteroid administration within weeks after renal transplantation affects the incidence of femoral head osteonecrosis cyclosporine use and male gender are independent determinants of avascular necrosis after kidney transplantation: a cohort study high-dose corticosteroid use and risk of hip osteonecrosis: meta-analysis and systematic literature review etiologic classification criteria of arco on femoral head osteonecrosis part : glucocorticoidassociated osteonecrosis authors thank dr. hideki yoshikawa, dr. koji yazawa, and dr. naotsugu ichimaru for helpful advice. key: cord- - wptx j authors: kim, danbi; shin, ju ae; han, seung beom; chung, nack-gyun; jeong, dae chul title: pneumocystis jirovecii pneumonia as an initial manifestation of hyper-igm syndrome in an infant: a case report date: - - journal: medicine (baltimore) doi: . /md. sha: doc_id: cord_uid: wptx j rationale: pneumocystis jirovecii causes severe pneumonia in immunocompromised hosts. human immunodeficiency virus infection, malignancy, solid organ or hematopoietic cell transplantation, and primary immune deficiency compose the risk factors for pneumocystis pneumonia (pcp) in children, and pcp can be an initial clinical manifestation of primary immune deficiency. patient concerns: a -month-old infant presented with cyanosis and tachypnea. he had no previous medical or birth history suggesting primary immune deficiency. he was diagnosed with interstitial pneumonia on admission. diagnoses: he was diagnosed with pcp, and further evaluations revealed underlying x-linked hyper-igm syndrome. interventions: he was treated with trimethoprim/sulfamethoxazole for pcp, and eventually received allogeneic hematopoietic cell transplantation for hyper-igm syndrome. outcomes: twenty months have passed after transplantation without severe complications. lessons: pcp should be considered in infants presenting with severe interstitial pneumonia even in the absence of evidence of immune deficiency. primary immune deficiency should also be suspected in infants diagnosed with pcp. in children, pneumocystis pneumonia (pcp) was mostly reported as epidemics in premature and malnourished infants in the s and s. [ ] after then, most cases of pcp developed in children with immunocompromised states, and patients infected with human immunodeficiency virus (hiv) have comprised a majority of patients with pcp since the s. [ ] accordingly, the suspicion and diagnosis of pcp in children without any evidence of an underlying immunocompromised state are not easy. primary immune deficiency (pid), as well as hiv infection, malignancy, and transplantation, are known as risk factors for pcp, [ ] and pcp can be an initial clinical manifestation of pid. [ ] [ ] [ ] we diagnosed a -month-old male infant presenting with cyanosis and interstitial pneumonia with pcp. although he had no previous history consistent with pid, further evaluation revealed x-linked hyper-igm (higm) syndrome, and he received hematopoietic cell transplantation for the underlying higm syndrome. this case report was approved by the institutional review board of seoul st. mary's hospital (approval no.: kc zesi ). written informed consent for publication was obtained from the patient's parent. a -month-old male infant presented with cyanosis. his mother was diagnosed with rheumatoid arthritis months before his birth. pregnancy was identified at the gestational age of months, and medical therapy for rheumatoid arthritis had been discontinued since then. the mother experienced improvement in symptoms of rheumatoid arthritis without medication, and the patient was born via caesarean section at the gestational age of + weeks with a birth weight of . kg. he exhibited no medical problems at or after birth. two weeks prior, fever, vomiting, and diarrhea developed, and he was treated for acute gastroenteritis at a primary clinic. however, he visited the emergency room and pediatric outpatient clinic of our hospital with persisting vomiting and diarrhea and days before, respectively. his vomiting and diarrhea had continued since then, and moreover, peripheral and central cyanosis accompanied. on admission, he was afebrile and exhibited no respiratory symptoms including cough, rhinorrhea, and sputum. pulse oxymetry showed spo of % in room air, which rose to % with oxygen supplied at l/min. his vital signs were as follows: heart rate, beats/min; respiratory rate, breaths/min; and body temperature, . °c. physical examination revealed no accessory breathing sounds on chest auscultation and chest wall retractions despite tachypnea. chest x-ray showed bilateral diffuse haziness without definite cardiomegaly (fig. a) . echocardiography revealed an ejection fraction of . % without any anatomical and functional abnormalities. blood tests revealed a white blood cell count of , /mm , hemoglobin levels of . g/dl, platelet count of , /mm , and c-reactive protein levels < . mg/dl without any abnormal findings in blood chemistry. we suspected interstitial lung disease of non-infectious causes or afebrile viral pneumonitis, and a multiplex polymerase chain reaction (pcr) test for respiratory viruses was performed using a nasopharyngeal swab. although there were no family and individual histories consistent with pid, a pcr test for pneumocystis jirovecii was also performed using a nasal swab, considering interstitial pneumonitis accompanying severe hypoxemia without accessory breathing sounds. after admission, his respiratory rate increased to to breaths/min, and mechanical ventilator care was initiated on hospital day (hd) # . empirical intravenous trimethoprim/sulfamethoxazole (tmp/smx; mg/kg of tmp thrice a day) treatment for possible pcp was also initiated on hd # . methylprednisolone ( mg/kg twice a day) was also administered for possible interstitial pneumonitis of noninfectious causes. chest computed tomography showed diffuse homogeneous opacity occupying alveolar spaces throughout the whole lung fields (fig. ) . the multiplex pcr test for respiratory viruses revealed negative results for influenza virus, parainfluenza virus, respiratory syncytial virus, adenovirus, human metapneumovirus, rhinovirus, coronavirus, and human bocavirus. bronchoscopy was performed on hd # ; however, any findings of definite airway inflammation and increased pulmonary secretion were not observed. the results of the pcr test for p. jirovecii performed on admission were reported as positive on the evening of hd # . after then, negative culture results for bacteria, cytomegalovirus, and mycobacterium tuberculosis were reported in bronchial washing fluid samples; cysts of p. jirovecii were observed on gomori methenamine silver stains of bronchial washing fluids. weaning of ventilator care and tapering of methylprednisolone doses were initiated on hds # and # , respectively. chest x-ray findings showed improvement weeks since initiating treatment (fig. ) , and he was extubated on hd # . oxygen supply and methylprednisolone treatment were completed on hds # and # , respectively. a repeat pcr test for p. jirovecii showed a positive result weeks after initiating tmp/smx treatment. the pcr test showed a negative result weeks after initiating treatment, and the tmp/smx treatment was converted to prophylaxis ( mg/m /day of tmp, thrice a week on alternate days) on hd # . he was discharged from the hospital on continuing tmp/smx prophylaxis on hd # . a lymphocyte subset test was performed on hd # , and revealed the following results: cd + cells, . %; cd + cells, . %; cd + cells, . %; cd + cells, . %; and cd + cells, . %. igg ( mg/dl) and igm ( mg/dl) levels were within normal limits; however, iga and ige levels could not be measured. immunoglobulin levels measured month after admission showed similar results. genetic studies for higm syndrome were requested based on the repeated results of the immunologic tests. one month after discharge, a novel frameshift mutation on exon of the cd ligand (cd l) gene was identified using gene sequencing. the identical mutation was identified in his mother as a heterozygote, and the patient was eventually diagnosed with x-linked higm syndrome. tmp/ smx prophylaxis and intravenous immunoglobulin replacement therapy every weeks was continued. he underwent unrelated, matched peripheral blood stem cell transplantation months after the diagnosis of pcp, and months have passed after transplantation without severe complications. most cases of pcp in infancy were reported as epidemics in premature and malnourished infants around the time of world war ii. [ , ] after then, in developed countries, pcp has occurred sporadically in immunocompromised children rather than epidemically in infants. [ , ] hiv infection has been regarded as an important risk factor for pcp; however, immunocompromised patients who are hiv-negative have recently occupied a greater proportion of patients with pcp than patients with hiv infection. [ ] although most immunocompromised children who are hiv-negative are those with hematologic malignancies or solid tumors and hematopoietic cell or solid-organ transplant recipients, children with pid are also at risk for pcp. [ ] cd + t cells play an essential role in the host immune defense to p. jirovecii infection, [ ] and therefore, t cell impairment rather than b cell or phagocytic impairment, is thought to be a risk factor for pcp among children with pid. [ ] severe combined immune deficiency, wiskott-aldrich syndrome, and higm syndrome are the most common pids among children with pcp. [ ] higm syndrome is caused by a defect in the cd -cd l signaling pathway. [ , ] it causes a limitation in immunoglobulin isotype switching in b cells, and therefore, normal or elevated igm levels with low igg, iga, and ige levels are observed. [ , ] our patient showed normal igg levels, which were thought to be due to maternal antibodies transferred across the placenta. the normal igm levels and undetected iga and ige levels with an increased proportion of cd + cells raised the suspicion of higm syndrome. higm syndrome manifests as a t cell dysfunction rather than a b cell dysfunction, despite hypogammaglobulinemia, because most cases are caused by a mutation of www.md-journal.com the cd l gene located at the x chromosome which encodes cd l on the t cell surface. [ ] [ ] [ ] [ ] therefore, x-linked higm syndrome is the most frequent form of higm syndrome. [ , ] however, several other genes mutated and involved in cd -cd l signaling pathway or dna repairing system in immunoglobulin isotype switching can cause higm syndrome, namely: cd , activation-induced cytidine deaminase (aicda), uracil-dna glycosylase (ung), phosphatidylinositol -kinase catalytic delta (pik cd), phosphatidylinositol -kinase regulatory subunit alpha (pik r ), nuclear factor-kappa-b essential modulator (nemo/ikkg), inhibitor of kappa light chain gene enhancer in b cells, alpha (ikba), nuclear factor kappa-b subunit (nfkb ) genes participating in cd -cd l signaling pathway and post meiotic segregation increased (pms ), muts homolog (msh ), muts homolog (msh ), ataxia telangiectasia mutated (atm), nibrin/nijmegen breakage syndrome (nbs /nbn) genes involved in dna repairing system. [ , ] most forms of these mutations are expressed through autosomal recessive inheritance, although autosomal dominant inheritance has also been reported. [ , ] therefore, genetic evaluation should be performed in patients with higm syndrome phenotype to verify the exact disease mechanism and perform accurate genetic counseling. traditional therapies for higm syndrome include immunoglobulin replacement therapy, antibiotic prophylaxis for p. jirovecii infection, hygienic measures to prevent infection, and careful monitoring for other complications. [ , ] with these therapies, the median survival rate after diagnosis of higm syndrome was reported as years. [ ] however, hematopoietic cell transplantations (hcts) have been performed in patients with higm syndrome to improve their prognoses since . [ , ] although the overall survival rate was not significantly improved in patients undergoing hct compared to those receiving traditional therapies, the transplant outcome and overall survival rates tended to improve since . [ ] mitsui-sekinaka et al reported significantly higher overall survival in patients undergoing hct since the s than in those not undergoing hct. [ ] among hct recipients, children aged years or younger showed higher survival than those aged years or older. [ ] considering the improvement in hct practice and supportive cares for hct recipients, hct can be regarded as a main therapy for higm syndrome, especially in young children. about % of children with higm syndrome experience respiratory tract infection, and p. jirovecii has been the most common respiratory pathogen. [ ] [ ] [ ] [ ] especially, % of infants diagnosed with x-linked higm syndrome before year of age initially presented with pcp. [ ] primary infection of p. jirovecii occurs during infancy and early childhood. [ ] more than % of children acquire antibodies against p. jirovecii before years of age, and about % of them seem to experience asymptomatic infection. [ ] because respiratory symptoms and signs show no significant difference between children who test positive and negative for p. jirovecii and are diagnosed with respiratory tract infection, except for the frequency of apnea, [ ] clinical differentiation of p. jirovecii infection from respiratory tract infection due to other causes should be difficult. in particular, pcp is hardly suspected in children with pneumonia who have no evidence of an underlying immunocompromised state. however, pediatricians should consider that pcp can be an initial clinical presentation of pid, [ ] [ ] [ ] and therefore, pcp should be suspected in infants with severe interstitial pneumonia accompanying normal breathing sounds when common viral and bacterial pathogens are not identified, even though the infants may show no evidence of immune deficiency. in addition, underlying pid should be evaluated in infants diagnosed with pcp using recently developed molecular genetic techniques. in conclusion, we report an infant who presented with severe interstitial pneumonia and was diagnosed with pcp as the initial manifestation of underlying higm syndrome. pcp should be considered in infants presenting with severe interstitial pneumonia even in the absence of evidence of immune deficiency. pid should also be suspected in infants diagnosed with pcp to correct the underlying immunocompromised state fundamentally. what caused the epidemic of pneumocystis pneumonia in european premature infants in the mid- th century? pneumocystis jirovecii pneumonia clinical conditions associated with pcp in children pneumocystis jiroveci pneumonia revealing de novo mutation causing x-linked hyper-igm syndrome in an infant male. the first case reported from french guiana pitfalls of "hyper"-igm syndrome: a new cd ligand mutation in the presence of low igm levels. a case report and a critical review of the literature pneumocystis carinii pneumonia as the presenting infection in congenital hypogammaglobulinemia current insights into the biology and pathogenesis of pneumocystis pneumonia the hyper igm syndromes the hyper igm syndromes: epidemiology, pathogenesis, clinical manifestations, diagnosis and management hyper igm syndrome: a report from the usidnet registry clinical spectrum of x-linked hyper-igm syndrome long-term outcomes of patients with x-linked hyper-igm syndrome treated with or without hematopoietic cell transplantation clinical features and hematopoietic stem cell transplantations for cd ligand deficiency in japan search for primary infection by pneumocystis carinii in a cohort of normal, healthy infants key: cord- -qec cid authors: leber, amy l.; everhart, kathy; daly, judy a.; hopper, aubrey; harrington, amanda; schreckenberger, paul; mckinley, kathleen; jones, matthew; holmberg, kristen; kensinger, bart title: multicenter evaluation of biofire filmarray respiratory panel for detection of viruses and bacteria in nasopharyngeal swab samples date: - - journal: j clin microbiol doi: . /jcm. - sha: doc_id: cord_uid: qec cid the filmarray respiratory panel (rp ) is a multiplex in vitro diagnostic test for the simultaneous and rapid (∼ -min) detection of pathogens directly from nasopharyngeal swab (nps) samples. it contains updated (and in some instances redesigned) assays that improve upon the filmarray respiratory panel (rp; version . ), with a faster run time. the organisms identified are adenovirus, coronavirus e, coronavirus hku , coronavirus nl , coronavirus oc , human metapneumovirus, human rhinovirus/enterovirus, influenza virus a, influenza virus a h , influenza virus a h - , influenza virus a h , influenza virus b, parainfluenza virus , parainfluenza virus , parainfluenza virus , parainfluenza virus , respiratory syncytial virus, bordetella pertussis, chlamydia pneumoniae, and mycoplasma pneumoniae. two new targets are included in the filmarray rp : middle east respiratory syndrome coronavirus and bordetella parapertussis. this study provides data from a multicenter evaluation of , prospectively collected nps samples, with performance compared to that of the filmarray rp or pcr and sequencing. the overall percent agreement between the filmarray rp and the comparator testing was . %. the rp demonstrated a positive percent agreement of . % or greater for detection of all but three analytes: coronavirus oc , b. parapertussis, and b. pertussis. the filmarray rp also demonstrated a negative percent agreement of ≥ . % for all analytes. of note, the adenovirus assay detects all genotypes, with a demonstrated increase in sensitivity. the filmarray rp represents a significant improvement over the filmarray rp, with a substantially shorter run time that could aid in the diagnosis of respiratory infections in a variety of clinical scenarios. length of stay, better antimicrobial stewardship, and better patient cohorting to prevent nosocomial infections ( , ( ) ( ) ( ) ( ) . the filmarray respiratory panel (rp) was first introduced as a syndromic multiplex molecular test in for the detection of viruses; additional viral analytes and bacteria were made available with a software update in after fda clearance for these new indications. adenovirus inclusivity was improved with the addition of new primers after an additional fda clearance in (version . [v . ] ). all filmarray rp references henceforth in this article are to the current commercially available version of the device as of the publication of this paper, i.e., the filmarray respiratory panel v . . in order to ensure that a molecular diagnostic assay remains clinically relevant, and particularly for syndromic assays, it is important to periodically update the test to incorporate new sequence information and to accommodate emerging or previously unrecognized strains or pathogens. to this end, biofire diagnostics has updated the filmarray rp product again by adding new assays to broaden the test's detection capabilities (particularly for adenoviruses), modifying a subset of assays to reflect newly available genetic sequences of currently included analytes, improving chemistry to enhance sensitivity overall, and for the inclusion of new analytes. the new test also has a decreased run time (ϳ min versus ϳ min). the organisms detected by the note that results for mers-cov are masked in the filmarray rp product that is fda cleared for the u.s. market. this analyte is reported in the filmarray respiratory panel plus (rp plus) product, which is sold outside the u.s. for testing individuals demonstrating signs/symptoms of respiratory infection and has been cleared by the u.s. fda, with a modified intended use to aid in the differential diagnosis of mers-cov infections only in cases meeting mers-cov clinical and/or epidemiological criteria. the filmarray rp is identical to the current filmarray rp with respect to specimen type, handling, testing workflow, pouch controls, and analysis software. in this study, data are presented for a prospective multicenter clinical evaluation of the performance of the filmarray rp in residual nasopharyngeal swab (nps) specimens collected in viral transport medium (vtm). performance is compared to that of the filmarray rp for of analytes (all those in common between the two tests) as well as that of pcr followed by bidirectional sequencing for b. parapertussis. mers-cov was not circulating in the united states during the time of the study; therefore, all specimens were assumed to be negative, and no comparator testing was performed for this analyte. filmarray rp testing. approximately l of specimen was subjected to filmarray rp testing according to the manufacturer's instructions ( ) . all sample processing occurred in a biosafety cabinet with operators wearing gloves and other appropriate personal protective equipment. one sample was processed at a time, and the cleaning of work areas was done in accordance with the manufacturer's instructions ( ) . the filmarray rp test consists of automated nucleic acid extraction, reverse transcription, nucleic acid amplification, and result analysis in approximately min per run (i.e., per specimen). the filmarray software performs automated result analysis, with each target in a valid run reported as "detected" or "not detected." if either internal control fails, the software automatically provides a result of "invalid" for all panel analytes. there are targets, as shown in table , two of which are new to the filmarray rp . this study was conducted with an investigative-use-only (iuo) version of the filmarray rp that is identical to the final fda-cleared and ce-marked version. it is important to note that results for mers-cov are reported in this paper but are available only for the filmarray rp plus version of the product. comparator testing. comparator testing consisted of soc filmarray rp testing performed at the source laboratory for all analytes in common between the filmarray rp and the filmarray rp (all analytes except mers-cov and b. parapertussis). all specimens were assumed negative for mers-cov, as it was not circulating in the united states during the time of enrollment for the study. for b. parapertussis, two pcr assays targeting is (the same target identified by the filmarray rp ), followed by bidirectional sequencing, were used as the comparator method. nucleic acid was extracted from specimens using magna pure lc total nucleic acid kit-high performance (roche diagnostics, indianapolis, in). both real-time pcr comparator assays were validated and found to have a limit of detection (lod) that was equivalent to that of the filmarray rp assay. testing was performed at biofire in a blind manner. comparator assay results were considered positive only when a bidirectional sequencing result of adequate quality was found to match a sequence for the expected analyte with an e value of . eϪ or lower in the genbank nucleotide database (basic local alignment search tool [blastn] with default settings). a specimen was considered to be "positive" with a sequence-confirmed result from either assay. results and discrepant analysis. a filmarray rp result was considered a true positive (tp) or true negative (tn) only when it agreed with the result from the comparator method. discrepant analysis ensued when results were discordant, i.e., false-positive (fp) or false-negative (fn) results. when sufficient specimen volume was available, discordant specimens were investigated using a combination of retesting with the filmarray rp or comparator methods, as well as testing with additional, independent molecular assays. for additional analysis of adenovirus targets, specimens were also tested with a combination of pcr assays targeting the dbp, penton, and pol genes (combined with bidirectional sequence analysis) ( ) and the results of standard-of-care testing at one of the study sites (nch) using an adenovirus laboratory-developed test (ldt) pcr targeting the hexon gene as described previously ( ) ( ) ( ) . note that the performance data for positive percent agreement (ppa) and negative percent agreement (npa) presented in this paper consist of unresolved data as presented in the package insert for the fda-cleared test; discrepancy investigation is provided but was not used to recalculate performance data. statistical analysis. the exact binomial two-sided % confidence intervals ( % ci) were calculated for performance measures according to the wilson score method. a total of , prospective study specimens collected from geographically/demographically diverse subject populations were analyzed in this study. overall, the study included specimens from more male than female subjects ( % [ / , ] and % [ / , ], respectively). most specimens were from pediatric subjects: % of the specimens were from children aged years and under, % were from those aged to years, % were from adults over the age of years, and % were from adults aged to years. the majority of the specimens were obtained from hospitalized subjects and those visiting the emergency department ( % [ / , ] and % [ / , ], respectively), and % were obtained from subjects seen in an outpatient setting ( / , ). filmarray rp test performance. a total of , specimens met the inclusion criteria and were initially tested in the clinical evaluation. the overall success rate for the initial test of these specimens was . % ( , / , ); tests were unsuccessful ( due to an incomplete test, due to an instrument error, and due to control failures). eleven of these specimens were successfully retested. in addition, another specimens were later excluded for protocol reasons, resulting in a total of , specimens included in the data analysis. summary of filmarray rp findings. the filmarray rp detected at least one analyte in , of the , specimens tested, yielding an overall positivity rate of . %, as shown in table . the highest detection rate was seen in young children (Յ years of age). the relative prevalence of each analyte among the positive specimens detected by the filmarray rp is presented in table . the most prevalent organisms detected during this study were hrv/ev, rsv, adenovirus, and flua, which were found in ( . %), ( . %), ( . %), and ( . %) specimens, respectively. if taken together, coronaviruses (cov- e, -hku , -nl , and -oc ) were the third most prevalent target, with ( . %) detections. for flua h and the mers-cov targets, no positive analyte detections occurred in this prospective sample set. all other analytes were detected in fewer than (Ͻ . %) specimens. a summary of performance characteristics for individual filmarray rp targets is presented in table comparator analysis and discrepancy investigation. there were a total of , analyzable filmarray rp organism results for the , specimens. the overall percent agreement between the filmarray rp and the comparator testing was . % ( , / , ). there were , detected organism results with the filmarray rp ; the comparator methods were positive for , analytes. the overall ppa with respect to the comparator method was . % ( , / , ). there were , results not detected with the filmarray rp ; the comparator methods were negative for , analytes. the overall npa with respect to the comparator method was . % ( , / , ). using comparator testing results as the truth, there were fp detections and fn detections overall; additional discrepancy analysis was performed for these samples. for the fp cases ( %) and the fn cases ( %), there was supportive evidence for the filmarray rp result, bringing the adjudicated overall concordance for the positive and negative results to . % and . %, respectively. a summary of the discrepancy investigation is presented in table . for the viral analytes, the filmarray rp detected a total of , viral analytes. using the comparator results as the truth, the overall ppa and npa are . % ( , / , ) and . % ( , / , ), respectively. the results for several analytes of significant interest are further detailed below. for adenovirus, a significant increase in detections was observed in comparison to those by the filmarray rp, with a total of detections, of which ( . %) were fp. fp specimens with sufficient volume were retested with the filmarray rp to see if the original result had been an anomaly. when possible, specimens were also tested with a combination of pcr/sequencing assays targeting the dbp (n ϭ ), penton (n ϭ ), and pol (n ϭ ) genes and the results of the nch ldt assay (n ϭ ). combined, these investigations found additional evidence of adenovirus presence in of the fp specimens ( table ). all of these specimens had late amplification on the filmarray rp test, suggestive of low levels of analyte in these specimens. the four fp specimens for which the filmarray rp retest was positive also had late amplification, suggestive of a low level of analyte. there were also fn results for adenovirus. additional discrepant analysis for these specimens included retesting with the filmarray rp , a combination of pcr assays as described above, and any available nch ldt results for adenovirus. combined, these investigations found additional evidence of adenovirus presence in three of the four fn specimens. analysis of the fn specimen for which the filmarray rp retest was positive indicated late amplification, suggesting low analyte levels. all fn were adenovirus species c based on sequence analysis. a comprehensive summary of the adenovirus discordant analysis is provided in table . among the coronaviruses, all but one of the four targets demonstrated good performance, with a ppa of Ն % and an npa of Ն . %. the exception was cov-oc , which demonstrated a ppa of . %. the majority of fn specimens observed were due to a known cross-reactivity in the comparator method (see biofire filmarray rp package insert at https://www.online-ifu.com/iti ): a filmarray rpdetected result for coronavirus oc due to cross-reactivity with cov-hku is suspected whenever the filmarray rp reports detections for both cov-hku and cov-oc . this cross-reactivity has been corrected by the redesign of the cov-oc assay for the filmarray rp . six of eight filmarray rp fn specimens were tp for cov-hku , i.e., codetections reported by the filmarray rp and suggestive of this known crossreactivity. as stated previously, no mers-cov was detected in the cohort. of note is an npa of %, indicating a lack of cross-reactivity with other coronaviruses. data for some archived mers-cov specimens and contrived mers-cov samples are provided in the manufacturer's package insert for the filmarray rp plus ( ). a these data are presented based on a comparator assay only and do not reflect any discordant analysis. b the terms ppa (positive percent agreement) and npa (negative percent agreement) are used instead of sensitivity and specificity to indicate that a non-gold standard comparator (e.g., pcr) was used for the analysis. the flua targets showed no fp or fn detections; however, there were no positive detections for flua h during the study period, which was predominated by flua h - . for flub, there were two fp detections, which were confirmed on further investigation as tp. flua, flua h , flua h - , and flua h results were excluded from analyses for three specimens due to initial results of "influenza a equivocal" or "influenza a no subtype detected" by either filmarray rp or filmarray rp testing and insufficient specimen volume for retesting. detections for hrv/ev were numerous, with a total of , the highest of all detections in the trial. there were fp results and fn results. specimens with sufficient volume were retested with the filmarray rp or filmarray rp . when possible, specimens were also tested with a combination of five pcr assays targeting the = utr gene. for the fp samples, were positive with a filmarray rp retest; late amplification for of the specimens was suggestive of low levels of analyte. four more were positive with pcr assays. for the fn samples, four specimens were positive with the filmarray rp on repeat testing and one was positive with pcr assays. three of the four fn specimens for which the filmarray rp retest was positive had late amplification, suggestive of low levels of analyte. rsv detections totaled , making it the second most common analyte. eight of fp specimens were observed to contain rsv by independent molecular methods or retesting with the filmarray rp. these may have been missed by the soc filmarray rp test due to an estimated hundredfold difference in lod between the filmarray rp and filmarray rp ( ) . using the comparator results as the truth, the overall ppa and npa are . % ( / ) and . % ( , / , ), respectively, for all bacterial targets. the number of detections for each bacterial target was low (Յ ), with the exception of that for m. pneumoniae (n ϭ ) ( table ). the two bacterial analytes demonstrating a ppa of Ͻ . % were both low prevalence: b. parapertussis (n ϭ ), and b. pertussis (n ϭ ). the bacterial targets tended to be single analyte detections (b. pertussis, / ; c. pneumoniae, / ; and m. pneumoniae, / ) with no copathogen present. for b. parapertussis, all six detections were in the context of a codetection with one or more viruses. no sample had two bacterial targets detected. discordant analysis for the bacterial targets in shown in table . this study of the filmarray rp demonstrated the performance of the test in a large prospective study of , residual nps samples, with , results generated. these data are significant, as this is a substantial change from the results with rp, and the test will be adopted for use in a large number of clinical laboratories. the number of positive detections was relatively high for most organisms; notable exceptions were the results for mers-cov and flua h , which were not circulating in the study populations during the study period. the filmarray rp testing system was shown to be reliable, with very few failures ( . % success on the initial test attempt), and rapid, with results available in approximately min, which is shorter than that of the filmarray rp (approximately min run time). the data presented here along with testing of archived positive nps in vtm specimens and contrived specimens (not shown) ( ) were used as part of the regulatory submissions for the filmarray rp and rp plus, which received (k) clearance in the united states (rp ) and ce/ivd marking in the european union (rp plus) in june . the filmarray rp plus received de novo clearance in the united states in november . periodically updating testing that has been implemented is an important concept. the college of american pathologists covers this for laboratory-developed testing in its microbiology checklist, stating that laboratories should have written policies and procedures to evaluate nucleic acid tests for compatibility with currently circulating microbial strains ( ) . for testing cleared by the fda, the filmarray rp represents the fourth iteration of the multiplex panel since its introduction in , providing an update of the primer probes based on a reexamination of known sequences for the majority of the pathogens and adjustment of the assay conditions to maximize performance. as noted, there were a significant number of detections by rp that were not found by rp (n ϭ ). the overall design goal for rp was to increase the sensitivity for all analytes relative to that of rp, and this may account for a significant number of the observed fp detections. this is supported by lod studies reported in the product inserts ( , ) and the discordant analysis performed in this study. the increased inclusivity/sensitivity and decreased time to result to min for the filmarray rp may lead to improvements in outcomes, such as length of stay or proper stewardship, and warrant further study. viruses are a common cause of upper respiratory infections in both adult and pediatric populations, and this was also seen in our study cohort. viral detections were notably higher than those of the bacterial targets ( , viral detections versus bacterial detections). the filmarray rp showed an increased positive detection rate for all viral targets in comparison to that of the filmarray rp ( more detections, with supported by additional discrepancy investigation), with the exception of coronavirus oc (table ) , reflecting the increased sensitivity and inclusivity of the filmarray rp . the most common viral analyte was hrv/ev, with a total of detections versus detections with the filmarray rp. the increased number of hrv/ev detections may or may not be associated with true disease causation, as the majority ( . % [see table s in the supplemental material]) were in the context of codetection with other viral targets. rhinovirus has been reported as a commonly detected target among asymptomatic individuals, with rates ranging from to % depending on the study ( ) ( ) ( ) . while the filmarray rp was updated to broaden inclusivity, there was no change in specificity for hrv/ev, so there are still cross-reactions with enteroviruses and hence the rhinovirus/enterovirus designation. one of the more extensive modifications occurred in the detection of adenovirus. previous studies by leber et al. demonstrated a lack of sensitivity with the filmarray rp for adenovirus types a, d, and f ( ) despite an earlier redesign of the filmarray rp in ( ) . the redesign of the filmarray rp specifically targeted all genotypes to include genotypes a to f and not only those typically associated with respiratory infections (types b, c, and e). in our cohort, genotypes a, b, c, d, and f were demonstrated to be detected by the filmarray rp . detection of all genotypes is important, particularly in the immunocompromised, where the finding of adenovirus of any genotype in the nps in vtm may precede systemic infections ( , ) . in addition, the identification of species f in respiratory specimens has been reported in patients with respiratory illness ( ) as well as in . % of pediatric patient samples obtained after routine adenoidectomy/tonsillectomy ( ) . overall, there were relatively low numbers of bacterial detections with the filmarray rp (n ϭ ). the reasons for this are likely due to true disease prevalence differences during the study period. also, as seen in our data (table s ), the codetection of bacteria and viruses is not common, particularly with b. pertussis, as has been previously reported ( , ) . the target gene for b. pertussis in both the filmarray rp and the filmarray rp is the toxin promoter region. this single-copy gene is known to be more specific than the more commonly used insertion sequence (is ) gene that is a multicopy target present in several bordetella species. while having greater specificity, the toxin gene target may be less sensitive, as has been reported previously ( ) . the diagnosis of pertussis-like illness is improved with the inclusion of the insertion sequence element target for b. parapertussis in the filmarray rp . b. parapertussis is known to cause a pertussis-like illness and can cocirculate with other bordetella species ( , ) . the prevalence of b. parapertussis is uncertain, as it is not a reportable disease like b. pertussis and is not tested for as commonly ( , ) . m. pneumoniae was the most common of the bacterial analytes, with detections, more than with the filmarray rp. however, it should be noted that use of an nps specimen for the detection of m. pneumoniae may be suboptimal, particularly when diagnosing lower respiratory tract infection ( , ) . overall, the percentage of discrepant results was low ( . %, n ϭ ) ( table ), suggesting that the previous version of the filmarray rp had relatively robust performance. discrepancy analysis using filmarray rp retests and pcr and bidirectional sequencing confirmed of fp ( %), which is strong evidence that the filmarray rp has increased sensitivity compared to the filmarray rp. there are some limitations of this study. this was a prospective study; however, some samples were frozen at Ϫ °c or less prior to testing. however, data indicated that the frozen storage did not significantly affect performance ( ) . the study period bridges one calendar year ( ) and includes only two partial respiratory seasons, so variations in circulating strains, particularly flua, are limited. the comparator method for of the targets was the filmarray rp. data concerning filmarray rp performance compared to that of other amplified platforms or culture are not provided and will await other studies. finally, the lack of detection of mers-cov and flua h in the prospective study limited the data on the performance for these targets. a significant redesign of the filmarray rp has demonstrated excellent sensitivity and specificity in this multicenter clinical trial. this is an important step both for individual improvements in pathogen detection and as recognition by the manufacturer that continuous improvements in monitoring and inclusion of new or emerging strains or species are important. both have been incorporated into the design of the filmarray rp , improving its performance for the detection of infectious agents that are involved in respiratory infections. these changes include new targets (mers-cov and b. parapertussis), improvements to existing targets, and a decreased time to result. these improvements, combined with the simplicity of the testing process and a shorter time to result, make the filmarray rp a significant improvement in diagnostic testing. supplemental material for this article may be found at https://doi.org/ . /jcm . - . supplemental file , pdf file, . mb. the annual impact of seasonal influenza in the us: measuring disease burden and costs the economic burden of non-influenza-related viral respiratory tract infection in the united states impact of a rapid respiratory panel test on patient outcomes antimicrobial stewardship for hospitalized patients with viral respiratory tract infections impact of early detection of respiratory viruses by multiplex pcr assay on clinical outcomes in adult patients clinical utility of on-demand multiplex respiratory pathogen testing among adult outpatients efficacy and safety of withholding antimicrobial treatment in children with cancer, fever and neutropenia, with a demonstrated viral respiratory infection: a randomized clinical trial impact of multiplex polymerase chain reaction testing for respiratory pathogens on healthcare resource utilization for pediatric inpatients filmarray respiratory panel (rp ) instruction booklet rfit-asy- clinical and virologic characteristics may aid distinction of acute adenovirus disease from kawasaki disease with incidental adenovirus detection diagnosis of pediatric acute adenovirus infections: is a positive pcr sufficient? performance characteristics of filmarray respiratory panel v . for detection of adenovirus in a large cohort of pediatric nasopharyngeal samples: one test may not fit all filmarray respiratory panel (rp) instruction booklet rfit-asy- . biofire diagnostics llc rhinovirus detection in symptomatic and asymptomatic children: value of host transcriptome analysis frequent detection of respiratory viruses without symptoms: toward defining clinically relevant cutoff values identification of respiratory viruses in asymptomatic subjects: asymptomatic respiratory viral infections evaluation and implementation of filmarray version . for improved detection of adenovirus respiratory tract infection use of pcr to demonstrate presence of adenovirus species b, c, or f as well as coinfection with two adenovirus species in children with flu-like symptoms prevalence and quantitation of adenovirus dna from human tonsil and adenoid tissues bordetella pertussis and concomitant viral respiratory tract infections are rare in children with cough illness bordetella pertussis is an uncommon pathogen in children hospitalized with bronchiolitis during the winter season testing implications of varying targets for bordetella pertussis: comparison of the filmarray respiratory panel and the focus b. pertussis pcr assay molecular pathogenesis, epidemiology, and clinical manifestations of respiratory infections due to bordetella pertussis and other bordetella subspecies occurrence of bordetella species during an outbreak of cough illness in ohio: epidemiology, clinical features, laboratory findings and antimicrobial susceptibility patterns of bordetella parapertussis respiratory illnesses prevalence of bordetella pertussis and bordetella parapertussis infections in tunisian hospitalized infants: results of a -year prospective study comparison of oropharyngeal and nasopharyngeal swab specimens for the detection of mycoplasma pneumoniae in children with lower respiratory tract infection comparison of sputum and nasopharyngeal swab specimens for molecular diagnosis of mycoplasma pneumoniae, chlamydophila pneumoniae, and legionella pneumophila we thank the dedicated professionals across all the study sites, without whom this work would have not been possible.this study was designed and funded by biofire diagnostics. the filmarray rp was performed at the clinical study sites, while pcr for comparator and discrepant analysis was performed at biofire diagnostics.a.l.l. wrote and edited the manuscript. biofire employees (m.j., k.h., and b.k.) designed the study and wrote portions of materials and methods only; they edited the manuscript only for accuracy. all other authors edited the manuscript and provided input on the data presented.all authors received research funding from biofire for this study. additionally, a.l.l. has served on a biofire advisory panel. key: cord- -swxs pr authors: lacaille-dubois, marie-aleth title: updated insights into the mechanism of action and clinical profile of the immunoadjuvant qs- : a review date: - - journal: phytomedicine doi: . /j.phymed. . sha: doc_id: cord_uid: swxs pr abstract background vaccine adjuvants are compounds that significantly enhance/prolong the immune response to a co-administered antigen. the limitations of the use of aluminium salts that are unable to elicite cell responses against intracellular pathogens such as those causing malaria, tuberculosis, or aids, have driven the development of new alternative adjuvants such as qs- , a triterpene saponin purified from quillaja saponaria. purpose the aim of this review is to attempt to clarify the mechanism of action of qs- through either receptors or signaling pathways in vitro and in vivo with special emphasis on the co-administration with other immunostimulants in new adjuvant formulations, called adjuvant systems (as). furthermore, the most relevant clinical applications will be presented. methods a literature search covering the period – was performed using electronic databases from sci finder, science direct, medline/pubmed, scopus, google scholar. results insights into the mechanism of action of qs- can be summarized as follows: ) in vivo stimulation of th humoral and th cell-mediated immune responses through action on antigen presenting cells (apcs) and t cells, leading to release of th cytokines participating in the elimination of intracellular pathogens. ) activation of the nlrp inflammasome in mouse apcs with subsequent release of caspase- dependent cytokines, il- β and il- , important for th responses. ) synthesis of nearly qs- analogs, allowing structure/activity relationships and mechanistic studies. ) unique synergy mechanism between monophosphoryl lipid a (mpl a) and qs- , formulated in a liposome (as ) in the early ifn-γ response, promoting vaccine immunogenicity. the second part of the review is related to phase i-iii clinical trials of qs- , mostly formulated in ass, to evaluate efficacy, immunogenicity and safety of adjuvanted prophylactic vaccines against infectious diseases, e.g. malaria, herpes zoster, tuberculosis, aids and therapeutic vaccines against cancer and alzheimer's disease. conclusion the most advanced phase iii clinical applications led to the development of two vaccines containing qs- as part of the as, the herpes zoster vaccine (hz/su) (shingrix™) which received a license in from the fda and a marketing authorization in the eu in and the rts,s/as vaccine (mosquirix™) against malaria, which was approved by the ema in for further implementation in sub-saharan countries for routine use. vaccines are the most effective and least expensive methodology for preventing diseases caused by infectious pathogens. recently emerging or re-emerging diseases such as the severe acute respiratory syndrome (sars) in , h n influenza pandemic in or ebola virus in (meaning to help) are substances used in combination with a specific antigen (ag) to produce a more potent and persistent immune response against a specific disease than the ag alone. the aim is to confer longterm protection, and with the additional benefit that less antigen and fewer injections are needed (lee and nguyen, ) . adjuvants are used in many vaccines, but their mechanisms of action have not been fully elucidated. they include diverse classes of compounds such as mineral salts, microbial products, emulsions, saponins, cytokines, polymers, microparticules and liposomes (awate et al., ) . up to now, few adjuvants have been approved for use in humans (del giudice et al., ) . aluminium salts, which have been extensively used as adjuvants in vaccines for more than years, induced robust antibody responses but weak th cell type responses, which are instrumental for protection against many pathogens. the new generation of vaccines using purer components for safer vaccines (e.g.,subunits vaccines with highly purified recombinant antigens) are less immunogenic in contrast to live attenuated or inactivated whole-cell vaccines. therefore, they required the induction of strong cellular responses including cd + t-helper cells (th) and cd + cytotoxic t lymphocytes (ctl) in addition to antibody (ab) responses. thus, the development of new adjuvants is necessary for vaccines that require a cell-mediated immune response (cmi). among them, the triterpene glycosides represent an interesting class of clinically relevant adjuvants, particularly those of quillaja saponaria (qs). these amphiphatic plant glycosides possess a variety of pharmacological activities including immunoadjuvant, antitumor, anti-inflammatory, and antimicrobial properties which have been extensively studied in many in vitro and in vivo bioassays (lacaille-dubois and wagner, , , lacaille-dubois, , ). despite the apparent success of qs- as vaccine-adjuvant in the last years, there is an urgent need for a deeper understanding of its mode of action which could accelerate the development of new vaccine strategies. this requires an interdisciplinary approach between chemists, biochemists, molecular biologists and immunologists. the following review will summarize updated insights into the modes of action, the structure/activity relationships allowing detailed mechanistic studies, and the clinical status of the vaccine adjuvant qs- . with a growing understanding of the innate immune system (response first mediated by antigen presenting cells (apcs)) and its role in activation and modulation of adaptive immune responses (ag-specific b and t cell responses) di pasquale et al., ) , the mechanisms of action of adjuvants are being elucidated. they can act on one or more of the following targets to increase response to ags: ( ) sustaining release at the injection site (depot effect), ( ) transient secretion of cytokines and chemokines, ( ) recruitement of various immune cells (neutrophils, monocytes, eosinophils, macrophages and dendritic cells (dcs) at the injection site leading to a local immune-competent environment, ( ) expression by the recruited apcs of various pathogen recognition receptors (prrs) both on their surface (toll-like receptors, tlrs, c-type lectin receptors, clrs), and intracellularly (nucleotide oligomerization domain (nod)-like receptors (nlrs) and retinoic inducible gene- (rig)-like receptors (rlrs)), which are recognized and/or activated by adjuvants, ( ) maturation and activation of recruited apcs which up-regulate the expression of major histocompatibility complex (mhc)-i and/or mhc-ii and activation of co-stimulatory signals cd , cd / , ( ) increased capacity of apcs for ag processing and presentation by mhc, ( ) migration of the mature apcs to the draining lymph nodes (dlns) to interact with ag-specific b or t lymphocytes (through receptor-ligand interactions, mhc-t cell receptor (mhc-tcr), cd -cd l, cd / -cd ) which are activated to produce potent ab-secreting b cells and/or effector cd + t cell responses (awate et al., ) . researchers continue to characterize the adaptive immune response and to clarify the respective roles of vaccine-induced immune effectors ( fig. ) . they include abs produced by b lymphocytes that bind specifically to a toxin or a pathogen, memory b cells produced only when b cells received t cell help having the ability to respond rapidely on reexposure of the same antigen, cytotoxic cd + t lymphocytes that limit the spread of infectious agents by killing infected cells or secreting specific cytokines, and cd + t helper (th) lymphocytes. various th subsets have been defined through their cytokine profiles and ability to induce b cell and cd + t cell responses (reed et al., ; siegrist ). the th -type cd + t cells essentially release the awate et al., ; reed et al., ; lacaille-dubois and wagner, ) . phytomedicine ( ) proinflammatory cytokines interleukin- (il- ), interferon-γ (ifn-γ), tumor necrosis factor-α (tnf-α), which participate in the elimination of intracellular pathogens both directly (cytokine response) or indirectly via cd + t cell activation and differentiation in ctls (cellular immune response) and the generation in mice of the complement-fixing abs igg a and igg . the th -type cd + t cell response is characterized by secretion of il- , il- and il- providing b cell help and the preferential induction of igg , ige, and iga in mice, and ige and igg in humans (humoral response) (guy, ) . the saponin qs- has been isolated from quillaja saponaria tree bark (kensil et al., ) and is one of the most potent adjuvants known which is currently used in exploratory, and a few licensed, vaccines. qs- is a mixture of two isomeric molecules, qs- apiose (qs- api), and qs- xylose (qs- -xyl) ( : ), each having four domains: the triterpene quillaic acid, a branched trisaccharide linked at c- through an o-heterosidic bound, a linear tetrasaccharide linked at c- through an ester bound, and a glycosylated pseudodimeric acyl chain attached through a labile ester linkage at c- of the fucose unit of the tetrasaccharide (fig. ) . this compound showed a potent adjuvant activity against a wide variety of ags with low toxicity in preclinical studies in mice, guinea pigs, monkeys and baboons. it was shown to stimulate th and th immune responses, when added to vaccine (kensil, ) . this adjuvant is a candidate for many clinical trials of vaccines directed against infectious diseases, cancer, and others. however, the exact role of qs- , either through activation of receptors or signaling pathways, is poorly characterized. some hypotheses advanced that qs- may facilitate ag uptake into apcs by binding to cell surface lectins through its carbohydrate domains, leading to specific cytokine profiles that enhance the t and/or b cells' response (marciani, ) . an effective mechanism of action in which qs- apparently acts on both dcs and t cells was proposed (marciani, ) (fig. ) . exogenous ags and qs- enter dcs by cholesterol-dependent endocytosis. the high affinity of qs- for endosomal membrane cholesterol resulted in the destabilization of the membrane (lorent et al., ) leading to pore formation, thus facilitating the escape of the antigen to the cytosol for further processing inside the cell into peptides. they are then loaded to mhc-i and presented on the dc surface to naïve cd + t cells to yield ctls. the role of aldehyde in qs- was highlighted in the t cell by forming an imine with the amino group of the t cell surface receptor such as cd , providing a costimulatory signal to the t cell. this signal together with the mitogen-activated protein kinase (mapk) erk resulting from the activation of the tcr-mhc interaction, and the changes of the ionic balance na + /k + , stimulates t cell activation resulting in the secretion of th cytokines (marciani, ) (fig. ) . recently, studies in mouse apcs (dcs and macrophages) identified qs- as an activator of the nlrp inflammasome. qs- in combination with the tlr -agonist -o-deacyl- ′-monophosphoryl lipid a (mpl a), was shown to activate the acs-nlrp inflammasome, a multiprotein complex and cause subsequent release of caspase- dependent proinflammatory cytokines il- β/il- that can promote th cell maturation or drive inf-γ-mediated th responses, respectively (marty-roix et al., ) . thus, inflammasome signaling has the potential to direct the development of t helper subsets. however, the role of this signaling pathway in vivo remained to be clarified. although qs- has been used in many clinical investigations of vaccines, several drawbacks inherent to the natural product, such as its chemical instability, scarcity, heterogeneity, dose-limiting toxicity and poorly understood mechanism of action have limited its widespread clinical advancement, except for the recent malaria (mosquirix™) and shingles (shingrix™) vaccines developed by glaxosmithkline (gsk). to overcome these limitations, structural modifications of the natural product through chemical synthesis have been undertaken during the last decade, leading to the preparation of nearly saponin analogs, which have provided key insights into structure/activity relationships (sar) which are summarized in fig. . these achievements allowed the identification of new novel saponins probes with potent adjuvant activities, increased stability, and decreased toxicity, and the investigation into their mechanisms of action (fernández-tajeda et al., ; fernández-tejada, ) . firstly, the total synthesis of each isomeric qs- produced this potent adjuvant in high purity and homogeneous composition. then, some qs- variants (sqs- , sqs- , sqs- ) were chemically synthesized with more stable amide linkages in the acyl chain instead of the unstable native esters (fig. ). their immunological evaluation in mice demonstrated adjuvant activities comparable to qs- , as assessed by measuring ab responses by elisa one week after booster injection (day ) and significantly lower toxicity than the natural product, except for sqs- . further modifications in the acyl chain with a dodecanedioic acid and truncation of the tetrasaccharide led to a trisaccharide variant with potent adjuvant activity and whose toxicity issue is not completely resolved . this simplification led to further structural variations at the linker (ester linkage at c- ) between the triterpene and the linear oligosaccharide in order to modulate the distance and orientation of these two domains. the variants exhibit striking differences in adjuvant activity and toxicity in mouse vaccination models, yielding new insights into the structural requirements for adjuvant activity. molecular dynamics simulations of these compounds and qs- api have revealed distinctive conformational features correlating with adjuvant activity, which may help in the design of new analogs (walkowicz et al., ) . the modifications of the tetrasaccharide into a trisaccharide variant, and the acyl chain into a terminal amine variant enables the synthesis of novel acyl chain variants bearing fluorescent and iodinated substituents for subsequent imaging and in vivo biodistribution studies (fernández-tejada et al., ) . they were adjuvant-active in several mouse-vaccination models and less toxic than qs- . early studies of biodistribution and fluorescence imaging allowed investigations into the enigmatic mechanism of action of these saponins. preliminary results in vaccinated mice suggested the potential role of these fluorescent probes in ag ovalbumin (ova) trafficking by apcs from the site of injection to the lns for its presentation to the immune system and yielded some mechanistic understanding of the potentiation of the immune response (fernández-tejada et al., ) . extensive structure/function studies have shown that aryl iodide derivatives with a free c- oh display a good efficacy/ toxicity profile. it was concluded that the trisaccharide at c- was not required for adjuvant activity. modifications at c- aldehyde and c- hydroxyl have shown that echinocystic acid aryl iodide derivatives lacking the c- aldehyde but retaining the c- alcoholic function ( fig. ) exhibited potent adjuvant activity and no toxicity in a preclinical vaccination model using a four-component vaccine (muc -klh, ova, gd -klh) when tested at doses of and μg. these data indicated that the c- aldehyde previously suggested to participate in schiff's base formation with a presumed t cell surface receptor target (marciani, ) , is not required for adjuvant activity and revealed the previously unknown role of the c- alcohol in enhancing activity. this opens the door to semi-synthesis from easily available alternative precursors and allowed detailed mechanistic studies (fernández-tejada et al., ) . one of the promising approaches to improving efficiency of newly developed vaccines is given by the combination of several adjuvants into single formulations, such as the adjuvant systems as , as , as (garçon et al., ) . as , developed more than years ago, is a liposome-based adjuvant comprising two immunostimulants, mpl and qs- . liposomes are synthetic nanospheres consisting of phospholipid bilayers that can encapsulate antigens and act as antigen delivery vehicles (garçon et al., ) . mpl is a detoxified synthetic derivative of the lipopolysaccharide (lps) from the bacteria salmonella minnesota and induces activation of innate immunity via tlr- , stimulating nf-kb transcriptional activity. it stimulates the induction of ag-specific t cells producing interferon-γ (ifn-γ) and igg a antibodies. qs- from q. saponaria (licensed by gsk from antigenics inc., a wholly owned subsidiary of agenus inc., lexington, ma, usa) in the early evaluations as an adjuvant was suggested to promote high agspecific ab responses and cd + t cell responses in mice and high agspecific ab responses in humans (didierlaurent et al., ) . qs- , as an amphiphilic compound is known for its hemolytic properties, which can be abrogated through formulation in liposomes in the presence of cholesterol such as in as (beck et al., ; garçon and di pasquale, ) . this resulted in an acceptable tolerability profile for its use in human vaccines. as contains mpl/qs- in an oil-in-water emulsion. as , combining qs- , mpl, and cpg (an oligodesoxynucleotide and tlr- agonist), in a liposome is the most complex combination of adjuvants which has been used for applications in cancer immunotherapy. the mechanism of action of qs- in a liposome is poorly understood despite the clinical efficacy of as . therefore, some studies were carried out in order to tentatively elucidate the adjuvant effect of qs- in such a formulation. it was observed after an intramuscular (im) immunization in mice against two model antigens, hepatitis b surface antigen (hbsag) and ova that qs- , rapidly accumulated in cd + resident macrophages of the dln, where it induces caspase- activation. these early events then organized the recruitment of innate immune cells and activation of dcs and subsequent t cell priming. further analysis showed that the adjuvant effect of qs- depended on the integration of caspase- and myd pathways at least in part through local release of hmgb (high-mobility-group protein b ) (detienne et al., ) . a recent study reported that qs- directly activated human monocyte-derived dendritic cells (mo-dcs) and promoted a pro-inflammatory transcriptional program . in this model, qs- was endocytosed via a cholesterol-dependent mechanism and accumulated in lysosomes where it induces their destabilization through pore formation and potential release of their contents. this resulted in inflammasome activation, syk-(a tyrosine kinase) and cathepsin-b (a cysteine protease) dependent cell activation and cytokine production in mo-dcs. lysosomal disruption could also affect ag processing and translocation to the cytosol for presentation on mhcs. finally it was shown that cathepsin b contributes to the adjuvant properties of qs- on both cd + and cd + ag-specific t-cell responses in vivo. the adjuvant systems containing mpl/qs in combination with hbsag were shown to induce very strong and persistent humoral and cellular immune responses in healthy adults, as b inducing the strongest and most durable specific cmi after two doses, without serious adverse events. the cd + response was characterized in vitro by high lymphoproliferation, high ifn-γ and moderate il- production. this response was confirmed ex vivo by detection of il- and ifn-γ producing cd + t cells and in vivo by measuring increased levels of ifn-γ in the serum (vandepapelière et al., ) . therefore as b has been selected as lead as for the development of vaccines. experiments were conducted in mouse models with a recombinant varicella-zoster virus (vzv) glycoprotein e (ge) and the fluorescently labeled qs- incorporated in as (hz (shingle) vaccine formulation). as induces a rapid and transient activation of the innate immune system, both at the injection site and in the dln, leading to the generation of high number of efficient ag-loaded dcs in the dln to promote ag-specific adaptive immune responses (didierlaurent et al., ) . a comparative study of the immunogenicity of different formulations containing the subunit ge was carried out in a vzv-primed mouse model (dendouga et al., ) . this model was chosen in order to mimic the clinical setting, in which the large majority of persons at elevated risk for hz are vzv sero-positive. an hz candidate vaccine comprising ge ( μg) and as b ( μg each of mpl and qs- ) was shown to be highly immunogenic in mice, inducing higher responses of cd + t cells expressing il- and/or inf-γ and higher humoral responses compared to other ge/as formulations with different ag doses ( . , . , μg) or with lower doses of adjuvant ( . and . μg each of mpl and qs- , corresponding to as e and as f , respectively) (dendouga et al., ) . furthermore, the contribution of each component of as b to the induction of cellular and humoral response was assessed after immunization of mice with ge either unadjuvanted, or adjuvanted with qs- ( μg), mpl ( μg) (both in liposomes), and as . the results showed that the ge-specific cytokine (ifn-γ, il- ) producing cd + t cell responses induced by ge/liposome+qs and ge/liposome+mpl were of similar and low magnitude, whereas the response induced by ge/as b was significantly higher than ge/liposome+qs ( . fold difference) and ge/liposome+mpl ( . fold difference). this clearly showed that qs- and mpl acted synergistically to induce a high frequency of ge-specific cd + t cells. for the anti-ge antibody responses, an additive effect, rather than a synergistic effect was observed (dendouga et al., ; didierlaurent et al., ) . these results suggested that the ge/as b vaccine candidate was appropriate for further clinical assessment. an investigation of the molecular and cellular mechanism of as adjuvanted vaccines was undertaken in order to clarify how immunostimulants function in combination. . results from in vivo and clinical studies showed that the combination of mpl/ qs- in as resulted in the stimulation of de novo pathways that were not triggered by the individual components. mice were immunized twice, weeks apart, with the rts,s antigen of the malaria vaccine, formulated either without adjuvant, with mpl, with qs- (both included in liposomes) or with as . rts,s is a recombinant protein consisting of repeated sequences of the plasmodium falciparum circumsporozoite protein (csp-repeat region, (r)) and t-cell epitope domain (t) linked to the hepatitis b surface antigen (hbsag) (s), and hbsag alone (s) (moris et al., ) . the csp-specific immune response was measured after the second dose. the results clearly showed that mpl and qs- act synergistically to induce strong antibody and agspecific cd + t cell responses. in contrast, very low levels of cd + t cell responses were generated against csp. further investigations highlighted the key role of the early release of ifn-γ by innate resident cells (nk and cd + t cells) in the ln, draining the injection site, which is essential for the development of the cd + t cell response (th immunity). this was confirmed by measuring the levels of the cytokine after immunization of mice with hbs adjuvanted with as or with its components. as induced the early production of ifn-γ with a peak in concentration at h post immunization ( pg/ml), whereas mpl or qs- alone failed to significantly induce ifn-γ. this effect results from the mpl/qs- synergy and is controlled by macrophages, il- and il- . qs- containing adjuvants have been assessed in more than human clinical trials. as is a key component of the recently developed malaria and hz vaccines and other candidate vaccines under development against infectious diseases, and cancer didierlaurent et al., ; garçon and di pasquale, ) . recent clinical studies of prophylactic vaccines against malaria, hz, hiv, tuberculosis from an efficacy, immunogenicity and safety points of view (table ) and of therapeutic vaccines against melanoma, nsclc and alzheimer's disease, will be reported below. despite effective medicines, insecticide-treated nets and indoor insecticide spraying, malaria killed , people in , particularly sub-saharan african children (world malaria day, ) . the ag of the candidate malaria vaccine rts,s targets the csp which is expressed on the plasmodium sporozoite during the pre-erythrocytic stage of its lifecycle, the stage between mosquito bite and liver infection. rts,s /as , was the first malaria vaccine candidate, in which an adjuvant system was used (garçon et al., ) . however, rts,s/as was shown in a phase ii trial to be well tolerated, to induce strong humoral and cellular immune response and to improve protection against plasmodium falciparum challenge in comparison to rts,s/as (kester et al., ) . namely, it induced high levels of anti-csp igg titers and csp-specific polyfunctional cd + t cells expressing il- , ifnγ, tnfα or cd l which have been associated with protection in the experimental malaria challenge model in adults. other clinical trials in children and adults confirmed these observations and no serious adverse events were reported (agnandji et al., ; olotu et al., ; ansong et al., ; white et al., ; levast et al., ; leroux-roels et al., a) . therefore the development of rts,s/as was stopped and rts,s/s /as was consequently selected for phase iii development . analyses of data in term of efficacy, immunogenicity and safety of phase i-iii trials including adults, infants, and children from sub-saharan countries were reviewed (agnandji et al., ) . clinical trials in children with newly adjuvanted vaccines led to the conclusion of clinical safety except for some unexplained cases of meningitis in one phase iii as adjuvanted malaria vaccine trial, which warrant further safety evaluations (stassijns et al., ) . important insights into the molecular mechanism of protective immunity against malaria were achieved involving additional immunogenicity analyses into the potential contributive roles of csp-specific abs and csp-specific cmi to protection (moris et al., ; kazmin et al., ) leading to the identification of the nf-kb and ifn-γ pathways (van den berg et al., ). the first clinical large-scale phase iii trial (the rts,s clinical trials partnership, ) evaluating a malaria vaccine involving , infants and young children was completed in december at sites from african countries (burkina faso, gabon, ghana, kenya, malawi, mozambique, and tanzania). safety and efficacy studies of the rts,s/ as vaccine (mosquirix™) showed that it prevented many cases of clinical and severe malaria over the months after dose . vaccine efficacy was higher in children ( - months) than in infants ( - weeks), but even at modest levels ( . %). the final results in (the rts,s clinical trials partnership, ) from the same trial, showed the enhanced efficacy by administration of a booster dose in both age categories, months after the first three injections. with regards to risks, the safety of mosquirix is similar to that of other vaccines, the most common side effects being fever, pain and swelling at the injection sites. thus, the vaccine has the potential to make a substantial contribution to malaria control when used in combination with other effective control measures, especially in areas of high transmission. the ratio benefit/risk of mosquirix was found to be acceptable by the european medicines agency's committee for medicinal products for human use (ema/chmp) which delivered a positive scientific opinion in for use outside the european union (eu) (ema press release, ). furthermore, the world health organization recommended a large-scale pilot implementation of the vaccine in young children with a four-dose schedule in african regions of moderate to high parasite transmission (who, ) . three countries, ghana, kenya, and malawi were selected by the who in for this largescale pilot program which started in . it will be the first malaria table clinical trials involving newly adjuvanted prophylactic vacccines. vaccine provided to young children through routine immunization programmes (malaria vaccine implementation programme, ). the results of two phases ( - , and - ) providing insights on the feasibility of delivering the vaccine in real-life settings and on its safety profile in the context of routine use, will contribute to the future decisions on the wider-scale use of the vaccine. herpes zoster (hz), also known as shingles is a common, painful disease occurring principally in adults aged > years and immunocompromised individuals. hz is caused by the reactivation of the latent varicella-zoster virus (vzv) in the dorsal root or cranial ganglia usually many years after a primary vzv infection (chickenpox) that occurs during childhood . hz can lead to serious complications including post herpetic neuralgia (phn). a phase i/ii trial showed that a subunit vaccine containing the recombinant vzv glycoprotein e (ge) as ag and as b (called hz/su) was well tolerated and highly immunogenic with cd + t cell and humoral immune responses, being more immunogenic than a live attenuated vaccine (leroux-roels et al., ) . a phase ii trial in adults > years of age evaluating different formulations (containing , , or μg ge combined with as ) and different schedules showed that all formulations elicited robust humoral and cd + t cell immune responses that persisted for at least years after vaccination. two doses of ge/ as b were more immunogenic than one (chlibek et al., ) . in a follow-up study conducted on healthy adults, it was shown that the ge specific cd + t cell and anti-ge antibody responses persisted for years after two doses of μg ge/as b , without any safety concerns . therefore, the dose of μg ge was selected for other clinical developments. a two randomized placebo-controlled phase iii efficacy trials in countries evaluated the efficacy and safety of hz/su as compared to placebo in older adults (> and > years of age). participants received two im doses of the vaccine or placebo, months apart. hz/su has demonstrated high efficacy ( . % and . % for adults > years and > years, respectively) in preventing hz and phn in all age groups with an acceptable safety profile (lal et al., ; cunningham et al., ) . the robust immune responses remained for years after vaccination as assessed in subsets of participants of the phase iii trials. persistent anti-ge ab and ge-specific polyfunctional cd + t cell responses are likely important mechanisms by which hz/su confers the high efficacy against hz (cunningham et al., ) . in an extension study of the original phase ii trials reported by chlibek et al. ( chlibek et al. ( , , it was observed that hz/ su induces cell mediated and humoral immunity persisting up to years post initial vaccination in adults > years old, confirming statistical prediction models based on immune responses measured at earlier time points. immune responses are predicted to persist up to years after the initial vaccination (schwarz et al., ) . the acceptable benefit/ risks profile led to the licensing by the u.s. food and drug administration (fda) in of the hz/su vaccine (shingrix™) and a marketing authorization valid throughout the eu on march (shingrix, inn-herpes zoster vaccine. ema/ / ). shingrix is preferred to zostavax (a live attenuated vaccine), providing strong protection against shingles and phn. first introduced in , the bacillus calmette-guérin (bcg) vaccine is the only currently licensed vaccine available to protect against tuberculosis (tb), a disease caused by the bacteria mycobacterium tuberculosis (m.tb.). it protects children from meningeal and severe forms disseminated of tb and death, but offers limited protection against pulmonary tb in adolescents and adults, which is the form of the disease responsible for the vast majority of transmission and tb-related morbidity and mortality. therefore, there is an urgent need for a new and improved vaccine against tb for controlling this disease that continues to pose a global health threat with . million deaths in (van der meeren et al., ) . the m /as candidate vaccine contains the recombinant fusion protein derived from the two immunogenic m. tuberculosis ags (mtb a and mtb a) in as . a first phase i/ii randomized, controlled clinical study with m /as was carried out in belgium in healthy adults volunteers. the results showed that the vaccine was clinically well tolerated and induced high and persistent (up to three years) specific th cd + t-cell (co-expressing cd l, il- , tnf-α and ifn-γ) and ab responses, supporting its further clinical evaluation in tb-endemic settings (leroux-roels et al., ) . similar conclusions were drawn in a phase ii controlled trial conducted on healthy, hiv uninfected adolescents living in an area with high tb burden in south africa (penn-nicholson et al., ) . in a phase ii study m /as given to infants after or concomitantly with expanded-programme-on-immunization (epi) vaccines had an acceptable safety profile. these results suggested no interference of immunogenicity profiles occurred following co-administration of m /as and epi vaccines. two m /as doses elicited higher immune responses than one dose (ikodo et al., ) . in a large phase iib trial conducted on hiv-uninfected adults latently infected with mtb in three african countries, m /as e was shown to provide % protection for mtbinfected adults against active pulmonary tb, without evident safety concerns (van der meeren et al., ) . these results suggest further evaluations of m /as as a possible vaccination strategy against tb. there were approximately . million people living with hiv at the end of with . million people becoming newly infected in (hiv/aids, key facts, july ). while treatments are available to treat or to prevent hiv infections, there is no vaccine currently licensed. despite ongoing prevention efforts, there is an urgent need for a safe and effective prophylactic vaccine. prevention of infection through induction of neutralizing abs, induction of strong cellular immune responses in order to delay progression and reduce the transmission rate in high risk population, are the major aims for an hiv vaccine. although clinical attempts were disappointing, they contributed valuable insights into immune protective immunity. a randomized double-blind study conducted on healthy hiv-seronegative adults showed that a vaccine formulation containing gp , nef and tat ags and as b induced strong ab response and cd + t cell responses characterized by high lymphoproliferative capacity and il- production, that were maintained up to months after the last vaccine dose. a cd + t cell response was not observed (leroux-roels et al., ) . this study conducted with different adjuvants underlined the superiority of responses with as b in comparison with other adjuvants, confirming previous findings. another candidate hiv- vaccine consisting of a recombinant fusion protein (f ) encoding four hiv- clade b ags (p , p , reverse transcriptase (rt) and nef) adjuvanted with as was studied in a phase i/ii trials with healthy hiv-seronegative volunteers (van braeckel et al., ) . this vaccine was shown to be immunogenic with an acceptable safety profile. after two doses of μg, strong polyfunctional (cd l, il- , tnf-α, ifn-γ phenotype) and persistent cd + t cell responses were induced, suggesting that this vaccine merits further evaluation both in a prophylactic setting and as a potentially disease-modifying therapeutic vaccine in hiv- infected subjects. a phase i clinical study in such subjects has been initiated (harrer et al., ) . a phase ii study was conducted with the f /as b candidate vaccine on healthy adults in order to evaluate the effect of chloroquine on the specific cd + t cell responses and the safety profile of a booster dose (leroux-roels et al., b) . the results showed that a booster dose, administered alone or two days after chloroquine induced a strong ab immune response and robust f -specific cd + t cell response, but no significant cd + t cell response. this suggest that two primary doses of the f /as b vaccine induce long-lasting memory b cell and cd + t cell responses in healthy adults, confirming previous findings of the phase i/ii study (leroux-roels et al., b) . this vaccine was shown to be immunogenic with a clinically acceptable safety profile, but doesn't show significant viral efficacy in antiretroviral therapy-naïve hiv- -infected adults, results confirmed in a long term study (dinges et al., ; harrer et al., ) . the use of immunotherapeutic vaccines in cancer patients is aimed at inducing immune responses against existing tumors rather than protecting healthy individuals. they have been used in phase ii and iii trials against melanoma and lung cancer after surgical resection, and the disappointing results of the most recent studies will be shortly presented here. enhanced production of ganglioside gm is observed in different human tumor types including melanoma. anti-gm vaccines such as gm conjugated to a carrier keyhole limpet hemocyanin (klh) and administered with qs- induced high igm and igg ab responses but failed to show a beneficial effect in melanoma patients as it was shown in a phase iii clinical trial in term of relapse free survival, distant metastasis-free survival and overall survival (eggermont et al., ) . a five-years survival occurs for less than % of the patients with completely resected non-small-cell-lung cancer (nsclc). therapeutic cancer vaccination has the potential for eliminating remaining cancer cells after complete resection, thereby decreasing relapse rates and improving survival (vansteenkiste et al., ) . ag-specific immunotherapies enhance t-cell responses against specifically expressed tumor ags. in this context, the use of mage-a (melanoma antigen family a, ) as ag, which is expressed in a wide array of malignancies can be an application in cancer immunotherapy (esfandiary and ghafouri-fard, ) . the mage-a /as immunotheraputic vaccine was assessed for clinical efficacy in an international, multicenter phase iii study in surgically resected nsclcs. this study confirmed the acceptable clinical safety profile of the vaccine, but did not improve overall survival. therefore, its further development for use in nslcs has been stopped (vansteenkiste et al., ) . similar conclusions were drawn from a phase iii trial of a mage-a /as vaccine in patients with resected, mage-a -positive, stage iii melanoma, which led to the arrest of the clinical development of this vaccine for use in melanoma (dreno et al., ) . the pathogenesis of alzheimer disease (ad) is associated with the accumulation of amyloid β (aβ) and/or hyperphosphorylated tau proteins in the brain. active immunotherapy of ad is the most extensively studied approach, in order to evaluate its ability to elicite anti-aβ antibodies, to induce aβ clearance, and reduce aβ accumulation in the brain. vaccination with a full length aβ peptide (aβ - ) successfully elicited anti-aβ-ab in human subjects with ad, but adverse effects such as meningoencephalitis were observed, attributed to t cell activation (winblad et al., ) . to avoid this safety issue, a n-terminal (aβ − ) peptide conjugated to a carrier protein called vanutide cridificar (acc- ) was produced. in preclinical studies, it was shown to generate nterminal aβ abs in adult nonhuman primates without inducing aβ-directed t cell response, supporting further clinical studies. several phase ii trials in patients with mild to moderate ad were conducted in usa, europe, and japan by using doses of acc- ( , and μg) with and without the adjuvant qs- ( μg) or placebo to investigate doserange, safety, immunogenicity and long term treatment (arai et al., ; pasquier et al., ) . acc- +qs- elicited high, sustained anti-aβ igg ab titers compared with acc- alone, highlighting the role of qs- in this effect. no difference was observed among the three doses tested. furthermore, acc- at all dose levels with or without qs- was generally safe and well tolerated, but exploratory cognitive evaluations did not show differences between treatment groups and placebo. similar safety, tolerability and anti-aβ-igg immunogenicity profiles were observed in long term phase ii extensions studies, suggesting that side effects do not limit in principle anti-amyloid active immunotherapy (hüll et al., ) . however, larger trials of adequate duration, with optimized dosing may be needed to attest efficacy of anti-aβ vaccine therapy for ad. this overview provides a summary of recent reports on the mechanism of action of qs- , a promising triterpene glycoside vaccine adjuvant isolated from quillaja saponaria and the most relevant clinical applications. this compound showed a potent adjuvant activity stimulating humoral and cell-mediated immunity against a wide variety of ags and presented advantages over aluminium salts by inducing a th type immune response, necessary for controlling most intracellular pathogens. it is unlikely that a single mechanism of action could explain its effects as an adjuvant. this contribution highlights: ) the role of qs- acting on both apcs and t cells. the interaction between the aldehyde function and t-cell receptors such as cd , delivering co-stimulatory signals together with the presentation of the ag peptides by apcs to the t-cell resulted in the activation of t lymphocytes and secretions of th cytokines, an important immune effector mechanism for the elimination of infected cells. ) a novel mechanism of action based on the activation of caspase dependent nlrp inflammasome in mouse apcs, leading to the secretion of proinflammatory cytokine il- β/il- , which are important for th responses. ) the synthesis of nearly structural analogs of qs- and detailed sar studies leading to more easily available, and stable molecules with an improved activity/toxicity profile, among them a novel aryl iodinated simplified echinocystic derivative showing a potent adjuvant activity and considerably attenuated toxicity. ) the signaling pathways of qs- when formulated in a liposome, such as the rapid accumulation in dln resident cd + macrophages, where it induces caspase- activation and hmgb release. these events orchestrate the recruitment of innate immune cells and activation of dcs leading to t cell priming. a direct activation of dcs was also reported, resulting in release of cathepsin b which contribute to specific t cell responses in vivo. ) the development of adjuvant systems (as) including a combination of immunostimulants in a formulation, such as as (qs- / mpl in a liposome), in which they were shown to act synergistically in enhancing cmi responses. ) the high number of clinical applications in term of immunogenicity, efficacy, safety of qs- alone or in adjuvant systems in many prophylactic vaccines against complex pathogens, e.g. malaria, herpes zoster, tuberculosis, hiv, and therapeutic vaccines, e.g. cancer, ad. ) the licensing by the us fda in of a vaccine against shingles called hz/su (shingrix™) containing ge/as and its commercialization in the eu since march . ) the first malaria vaccine (mosquirix™) containing rts,s/as receiving a regulatory approval by the ema in for use outside of europe and being currently under exploration in sub-saharan regions for its use in routine. ) the disappointing phase iii clinical results in term of disease-free survival of patient with immunotherapeutic vaccines adjuvanted with qs- /as against nsclc and melanoma despite promising results of phase ii trials. ) the encouraging results of a phase ii trial confirming that the long term treatment with the immunotherapeutic vaccine vanutide cridificar (acc- ) adjuvanted with qs- against alzheimer's disease was well tolerated with an acceptable safety profile. the development of adjuvants in vaccines represents an area in constant evolution with substantial advances over the past two decades. qs- in ass is a key component of multiple vaccines targeting infections and endemic diseases in developing countries. recent research has led to the commercialization of shingrix™ and the approval of mosquirix™, two vaccines against hz in adults and malaria in children, respectively, which contain qs- as a part of their formulation. to ensure a continuous supply of this exciting molecule, the researchers aimed in the near future to find an alternative innovative process of producing this compound on a large scale based on plant cell-culture. this might overcome some limitations of the current traditional techniques of extraction with tedious purification processes and chemical synthesis involving many complex reactions. however, the synthetic technology initially used for the first synthesis of qs- , was subsequently applied to preparing novel structural analogs with potent activity and low toxicity, leading to sar studies and a better understanding of the mode of action. further work in this area could facilitate novel saponin design that will lead to the discovery of new adjuvants and improved adjuvant-antigen combinations for future vaccines, particularly against infectious tropical diseases such as dengue, leishmaniasis, and chagas diseases, which continue to cause significant morbidity and mortality in developing countries. evaluation of the safety and immunogenicity of the rts,s/as e malaria candidate vaccine when integrated in the expanded program of immunization clinical development of rts,s/as malaria vaccine, a systematic review of clinical phase i-iii trials t cell responses to the rts,s/as e and rts,s/as d malaria candidate vaccines 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dose in infants and children in africa: final results of a phase , individually randomised, controlled trial vaccines", section , general aspects of vaccination a systematic review and metaanalysis on the safety of newly adjuvanted vaccines amound children persistence of immune response to an adjuvanted varicella-zoster virus subunit vaccine for up to year nine in older adults an adjuvanted polyprotein hiv- vaccine induces polyfunctional cross-reactive cd + t cell responses in seronegative volunteers predicting rts,s vaccine-mediated protection from transcriptomes in a malaria-challenge clinical trial phase b controlled trial of m /as e vaccine to prevent tuberculosis vaccine adjuvant systems containing monophosphoryl lipid a and qs- induce strong and persistent humoral and t cell responses against hepatitis b surface antigen in healthy adult volonteers efficacy of the mage-a immunotherapeutic as adjuvant therapy in patients with resected mage-a -positive non-small-cell lung cancer (magrit): a randomized, double-blind, placebo-controlled quillaja saponin variants with central glycosidic linkage modifications exhibit distinct conformations and adjuvant activities lysosome-dependent activation of human dendritic cells by the vaccine adjuvant qs- the relationship between rts,s vaccine-induced antibodies, cd + t cell responses and protection against plasmodium falciparum infection active immunotherapy options for alzheimer's disease. alzheimer's res. ther. , . world health organisation, . malaria vaccine: who position paper first-malaria-vaccine-receivespositive-scientific-opinion-ema_en.pdf. ema press release . first malaria vaccine receives positive scientific opinion from ema there are no conflicts of interest associated with this publication. key: cord- -ahl ql authors: van buuren, nicholas; tellinghuisen, timothy l; richardson, christopher d; kirkegaard, karla title: transmission genetics of drug-resistant hepatitis c virus date: - - journal: elife doi: . /elife. sha: doc_id: cord_uid: ahl ql antiviral development is plagued by drug resistance and genetic barriers to resistance are needed. for hiv and hepatitis c virus (hcv), combination therapy has proved life-saving. the targets of direct-acting antivirals for hcv infection are ns / a protease, ns a phosphoprotein and ns b polymerase. differential visualization of drug-resistant and -susceptible rna genomes within cells revealed that resistant variants of ns / a protease and ns a phosphoprotein are cis-dominant, ensuring their direct selection from complex environments. confocal microscopy revealed that rna replication complexes are genome-specific, rationalizing the non-interaction of wild-type and variant products. no hcv antivirals yet display the dominance of drug susceptibility shown for capsid proteins of other viruses. however, effective inhibitors of hcv polymerase exact such high fitness costs for drug resistance that stable genome selection is not observed. barriers to drug resistance vary with target biochemistry and detailed analysis of these barriers should lead to the use of fewer drugs. in a recent triumph of modern science and medicine, patients chronically infected with hepatitis c virus (hcv) now receive multidrug regimens that are often curative and have low toxicity (lawitz et al., ; afdhal et al., ; dhaliwal and nampoothiri, ) . over the past two decades, researchers have developed and tested thousands of antiviral compounds with varying efficacies and toxicity profiles that have ultimately lead to the fda approval of powerful combination therapies (lawitz et al., ; scheel and rice, ) . several different direct-acting antivirals (daas) that target the ns / a protease, ns a phosphoprotein, or ns b rna-dependent rna polymerase of hcv have been approved for use in the clinic (afdhal et al., ; dhaliwal and nampoothiri, ; manns and von hahn, ; younossi et al., ) . ideally, the knowledge gained in developing hcv antivirals that are effective and not prone to the outgrowth of drug resistance will be applied to other viruses as well. the emergence of drug-resistant variants follows basic evolutionary principles, requiring spontaneous mutations as well as selective pressure, so that beneficial mutations increase the progeny size of genomes that bear them. the genetic diversity in rna viral genomes results from the high error frequencies incurred by rna-dependent rna polymerases, which occur at approximately  À errors for each nucleotide synthesized (sanjuán et al., ) . given the iterative copying of positive and negative strands, much higher cumulative error frequencies are observed, even during a single cycle of infection (sanjuán et al., ; acevedo et al., ) . when more than one mutation is required to confer drug resistance, the outgrowth of drug resistance can be delayed (bloom et al., ) . as a result, treatment with combinations of drugs can be extremely effective at suppressing siblings. if the drug target is, for example, a subunit of an oligomeric complex and subunits from different genomes have the opportunity to mix, chimeric oligomers often form. at the time of its creation, the drug-resistant genome will be a minority species, and such chimeras would be predominantly composed of the drug-bound, susceptible components thus incapacitating the entire oligomeric structure. such 'phenotypic masking' was originally invoked to explain the very low frequency of foot-and-mouth-disease escape variants following selection with neutralizing antibodies when passaged at high multiplicities of infection (mois) (holland et al., ) . our goal was to screen the hcv-encoded viral proteins that are current targets of antiviral compounds to determine the intracellular dominance relationships between drug-resistant and drug-susceptible genomes. the high cost to viral fitness of sofosbuvir-resistant variants is sufficient to explain its high barrier to resistance. there are currently no antivirals directed against hcv core protein; however, it is likely to be a dominant drug target. we used differential hybridization of rna probes to detect two different genomic rnas in a single cell by confocal microscopy and by flow cytometry. this analysis showed the cis-dominance of hcv viruses that are resistant to inhibitors of either ns / a protease or ns a phosphoprotein, consistent with the rapid outgrowth of drug-resistance in patients of these two inhibitor classes. newly mutated drug-resistant genomes first arise within cells that are pre-populated by drug-susceptible genomes. to mimic such mixed infections, we have previously employed co-infection of cultured cells with drug-susceptible and drug-resistant viruses at high mois to ensure mixed infection (tanner et al., ; mateo et al., ) . for hcv, it is not practical to use high mois to achieve co-infection due to the difficulty of obtaining sufficiently high-titer viral stocks. thus, we needed to develop an approach to distinguish between uninfected, singly infected and co-infected cells in relatively sparsely infected cell populations ( figure a) . to detect individual genomes in infected cells, a single-molecule fluorescence in situ hybridization (fish) approach was used. a recently developed branched dna probe technology allows the generation of sufficiently sensitive rna probes to identify single molecules within cells, but requires approximately nucleotides of differential probe hybridization to achieve specificity (affymetrix inc, ) . to create a viral strain with this extreme dissimilarity from wild-type virus, we tested the viability of three different codon-altered versions of the jfh variant of hcv ( figure b) . each mutated version contained - nucleotide changes that did not alter the protein sequence (figure -figure supplements - ). of these codon-altered (ca) variants, ca- was inviable, ca- showed reduced viral protein accumulation, and ca- showed accumulation of both viral protein and rna to abundances equivalent to those of the wild-type virus ( figure c) . recently, detailed analysis of covarying nucleotides within the hcv coding region has identified the location of several previously unknown functional rna secondary structures (pirakitikulr et al., ) . interestingly, ca- contains two such regions and ca- contains one, which correlates with decreasing viability, while ca- contains no such regions (figure -figure supplements - ) (pirakitikulr et al., ) . thus, subsequent experiments were performed only with ca- . this variant, now termed 'ca' virus, contains synonymous mutations over a -nucleotide region that spans the coding sequences for most of ns and the n-terminus of ns (figure to test the sensitivity of rna fish probes generated against the positive-and negative-strands of wild-type (wt) and codon-altered (ca) viruses, both confocal microscopy and flow cytometry analyses were employed. branched dna technology allowed the labeling of each target rna with as many as fluorophores ( figure a ) (affymetrix inc, ) . huh- . . cells were infected with either wt or ca viruses, subjected to fish and visualized by confocal microscopy. wt and ca probe sets specifically targeted either the positive-sense ( figure b ) or the negative-sense vrna ( figure c ) of their corresponding virus. additionally, we tested whether flow cytometry efficiently resolved cells transfected with different vrnas; transfection was used to maximize the yield of each population. we resolved cells transfected with wt vrna (figure di) , transfected with ca vrna (ii), a mixture of these two cell types (iii) and cells co-transfected with both wt and ca vrnas (iv). thus, figure . construction of codon altered jfh . (a) cell cultures coinfected with two strains of hcv result in four populations: uninfected, two types of singly infected, and coinfected cells. (b) three segments of the jfh genome, that were roughly nucleotides in length and had altered codon usage, were designed using geneart algorithms and synthesized. these genome fragments were then cloned into the jfh strain of hcv. specific rna probes could be used to monitor the fate of drug-susceptible and drug-resistant viruses in co-infected cells. to test the genetic properties of viruses that are resistant to ns / a inhibitors, we employed the original ns / a inhibitor, biln- ( figure a ) (lamarre et al., ) . like other ns / a inhibitors, biln- treatment rapidly allows the selection of drug resistant variants both in tissue culture and in patients (lamarre et al., ; lin et al., ) . given the ease of outgrowth of drug-resistant variants, we hypothesized that ns / a was not a dominant drug target and that drug resistance would be genetically dominant. ns -d a is the prototypic mutation associated with resistance to ns / a inhibitors. asp is in close proximity to the protease active site ( figure b ). the ability of the ns -d a mutation to confer resistance to biln- was confirmed in both the wt and ca backgrounds ( figure -figure supplement ). as shown schematically in figure d , the ability to track cells that are uninfected (u), singly infected with drug-susceptible virus (s), infected with both susceptible and resistant virus (s + r) and singly infected with drug-resistant virus (r), can reveal dominance relationships during co-infection. in the absence of a drug, all viral populations should be present. however, in the presence of a drug, three outcomes are possible depending on the genetic outcome within the r + s population. if drug resistance were trans-dominant ( figure e ), the drug-resistant virus would rescue the drugsusceptible genomes and all viruses in r + s cells would survive in the presence of the drug. s cells would drop into the u population, and r cells would survive. if drug resistance were cis-dominant ( figure f ), only the r viruses in the r + s cells would survive, because the drug-resistant proteins would be unable to rescue the s viruses in the same cell. consequently, the r + s cells would drop into the r population. if drug susceptibility were dominant ( figure g ), all viruses in the r + s cells would be cleared, and the r + s cells, like the s cells, would drop into the u population, and only the r cells would continue to replicate. to determine the dominance relationship between biln- -susceptible and the biln- resistant virus, huh- . . cells were infected with ca and wt-d a viruses ( figure c ). cells were infected for hr at mois such that all four populations were represented, followed by hr of continued incubation in the absence or presence of mm biln- . cells were then harvested, fixed, co-stained with wild-type and codon-altered rna probe sets and analyzed by flow cytometry. all four cell types appeared in the absence of biln- ( figure h ,i). in the biln- -treated samples ( figure j ,k), the susceptible s population shifted to the u cells as expected. the s + r cells, on the other hand, shifted to the r population upon drug treatment. thus, the drug-resistant viral genomes in the co-infected cells could replicate, but could not rescue the drug-susceptible ones. data from this and replicate experiments ( figure i ,k) confirmed the quantitative shift of s + r cells into the r population upon drug treatment. we conclude that, for the ns / a target, drug-resistant genomes are cis-dominant for the : ratio of s and r viruses tested here. we also tested whether over-expressed drug-resistant ns / a precursors could rescue biln- -susceptible virus ( proteins are drug targets, drug-resistant products will enhance the propagation of only those genomes that encode them, allowing powerful selection for drug resistance. for ns b polymerase inhibitor sofosbuvir, the few resistant viral variants that arise in patients are highly attenuated. to investigate whether a related compound, r (klumpp et al., ) , exacted a similar cost to viral fitness to drug-resistant variants, we attempted to recover r -resistant viruses for dominance testing. jfh was passaged for multiple rounds of infection in the . each mutation was introduced independently into the jfh genome and rna transfections were performed. the t a genome was the only variant to show any viral rna production by or days post-transfection. we noticed that f l and t a were always isolated together. to test whether these mutations could together increase viral fitness, jfh viruses were generated that contained both mutations. viruses with the mutations separate or together were passaged extensively in the presence of r . occasional resistant outgrowths were observed, but none conferred sustained growth ( figure -figure supplement c). thus, like sofosbuvir, the poor viability of mutant viruses resistant to r precludes the ability to perform further genetic analysis but provides an excellent paradigm for antiviral development. transmission genetics and phenotypic dominance of drug-resistant ns a variant y n ns a is highly oligomeric (sun et al., ; tellinghuisen et al., ) and we were curious as to whether drug resistance or drug susceptibility would be dominant during viral infections. this idea seemed promising because exogenously expressed ns a has a dominant-negative effect on the growth of hcv replicons (graziani and paonessa, ) . additionally, the ns a inhibitors, as a class, display ec 's in the low picomolar range (gao, ) , making them among the most potent antiviral compounds ever identified. assuming uniform inhibitor concentrations in cells and in medium, it has been estimated that only a small fraction of ns a molecules should be bound to drugs under inhibitory conditions (sun et al., ; gao et al., ) . thus, it seemed mechanistically likely that drug-bound ns a proteins from drug-susceptible viruses could be dominant inhibitors of ns a encoded by newly arising drug-resistant ones. however, ns a inhibitors have generally demonstrated low barriers to resistance in patients. our goal was gain mechanistic insight into this dichotomy. the structures of two such potent ns a inhibitors, sr (also known as bms- ) (lemm et al., ) and daclatasvir (gao et al., ) are shown in figure a . mutations of tyr to asp or his confer resistance to a broad array of ns a inhibitors (gao et al., ) . tyr is located near an ns a dimer interface shown in the crystal structure ( figure b ) . thus, this interface is postulated to be part of the binding site for the ns a inhibitor class. the y n and y h mutations were introduced into both the wild-type and codon-altered viruses. as shown in figure c , the y h mutation conferred resistance to both sr and daclatasvir while the y n mutation conferred resistance only to sr . to test whether susceptibility to ns a inhibitors was dominant in the context of viral infections, we analyzed u, s, s + r and r cell populations by flow cytometry as previously performed for the ns / a inhibitor in figure . huh- . . cells were coinfected with ca and wt-y n viruses for hr ( figure d ). cells were then treated with dmso or nm sr for hr, harvested, fixed, costained for wt and ca vrnas and analyzed by flow cytometry. in the absence of the ns a inhibitor, all four populations, u, s, r + s and r were observed ( figure e ,f). in the presence of sr , the s population of cells dropped into the u population as expected. as was the case in figure , the co-infected r + s population of cells dropped into the r population. thus, resistance to ns a inhibitor sr in the context of viral infection was genetically dominant and the lack of rescue of the s virus with which it was mixedly infected shows that drug resistance is also cis-dominant. hcv infected cells become resistant to superinfection upon expression of non-structural proteins (schaller et al., ; tscherne et al., ) . due to this superinfection exclusion, it is likely that all coinfected cells arise through nearly synchronous infection throughout the course of the experiment. to control for any effects on selection that may occur due to the differential timing of coinfections that occurs over the initial hr incubation period, we performed the same experiment with higher titer virus and a single cycle of infection in the absence of drug. huh . . cells were infected at an moi of focus-forming unit (ffu)/cell with ca and wt-y n viruses and incubated for only hr before drug treatment. cells were then incubated in the absence and presence of nm sr for an additional hr. in this case, we also observe cis-dominance of drug resistant wt-y n genomes, indicating that asynchronous coinfection has no effect on selection ( figure g) . finally, the cis-dominance of daclatasvir-resistant wt-y h was observed when coinfected with drug susceptible virus (s) in the absence and presence of daclatasvir ( figure h ). we conclude that ns a, despite being an oligomeric species is not a dominant drug target. instead, genomes resistant to ns a remain drug resistant in co-infected cells but do not rescue drug-susceptible viruses present in the same cell. this is consistent with the observed outgrowth of viruses that are resistant to ns a both in cultured cells and in patients, and with an earlier report that at least some functions of ns a act exclusively in cis (fridell et al., ) . one hypothesis that could mechanistically account for cis-dominant drug resistance is that ns a molecules expressed from different alleles may not freely associate in mixed oligomers. as previously demonstrated, two different ns as expressed from the same rna can associate, while ns a molecules expressed from different constructs could not (berger et al., ) . we were curious whether the dominance phenotypes were altered if we forced ns a alleles to mix. to test whether exogenously expressed drug-susceptible ns a proteins could co-assemble with drug-resistant ns a, we utilized the previously described hcv plasmid that expresses ha-tagged and gfp-tagged ns a within the same polyprotein but does not support genome replication ( figure a) . constructs that contained all combinations of drug-susceptible ns a (s) and the drug-resistant y n variant (r) were created. upon transfection, all tagged proteins were expressed and can be observed in figure (input, panels b,c). immunoprecipitation with anti-ha antibodies revealed that the gfptagged and ha-tagged ns a proteins were present in the same complexes in the presence or absence of sr . therefore, as has been shown previously, mixed oligomers can form upon coexpression within the same polyprotein (berger et al., ) . furthermore, these interactions are not disrupted by drug treatment or by drug-resistant mutations ( figure b ,c). to determine whether there were any functional consequences of mixed oligomer formation, we visualized cells that expressed mixed oligomers using confocal microscopy. all s and r combinations of ns a co-localized at discrete membrane-associated complexes characteristic of hcv infection in the absence of drug ( figure d , top panel). however, in the presence of sr , membrane-associated complex formation was inhibited in r:s and s:s expressing cells and observed only in r:r expressing cells ( figure d, bottom panel) . the dispersal of ns a signal upon drug treatment in the presence of s protein makes ns a protein appear less abundant ( figure d ). however, the immunoblots demonstrate that no such decrease in expression occurs as we observe equal levels of ns a protein independent of allele or the presence of drug ( figure b ,c). one hallmark of hcv infection is the accumulation of cytoplasmic lipid droplets (miyanari et al., ; romero-brey et al., ) . electron microscopy performed by high-pressure freezing and freeze-substitution, to preserve membrane structure, revealed many lipid droplets in the cytoplasm of cells expressing s:s, r:r and s:r combinations of ns as in the absence of inhibitor ( figure e,f) . however, in the presence of sr , only the r:r cells displayed the accumulation of lipid droplets ( figure e , g). therefore, using both assays, the presence of drug-susceptible ns a prevented drug-resistant phenotypes from being displayed, and thus drug-susceptibility was genetically dominant. this confirmed our original hypothesis that ns a had the potential to be a dominant drug target. one of the most likely mechanisms for cis-dominance, when the benefit of a particular gene product accrues only to the genome that encodes it, is physical isolation. we hypothesized that hcv genomes co-infecting the same cell might be physically isolated from each other. to test this possibility, confocal microscopy was used to identify and localize negative vrna strands in genome-specific rna replication complexes ( figure a ). the majority of negative strands of the two different viruses were found to be discrete. identification and quantification the vrna puncta in coinfected cells was determined computationally using volocity software. this program determined the number of negative strand puncta per cell per strain and quantified how many puncta overlapped ( figure b ). this value was low even for the positive stranded vrnas, which are present in the cytoplasm at much higher frequencies ( figure a,b) . as a positive control for colocalization, we performed a similar experiment but additionally stained for ns a or core in addition to minus strand vrnas. we would expect minus strand vrna and ns a to colocalize strongly, as ns a is present inside replication complexes. alternatively, core is not localized directly within replication complexes, but is present within packaging complexes and on lipid droplets, which are nearby. volocity was used to count negative strand vrnas, and then asked, how many of those puncta were touching ns a or core. representative images demonstrating each of the pairwise comparisons demonstrate that nearly % of all minus strand vrnas were touching ns a while fewer than half of the minus strand vrnas were touching core ( figure c,d) . these data support the hypothesis that, upon co-infection, drug-resistant and drug-susceptible rna genomes create independent membranous web structures, limiting the mixing of ns / a and ns a proteins and their precursors. this scenario is modeled schematically in figure e . failure of ns a proteins to mix during infection is a likely explanation for the cis-dominance of drug resistance observed in cultured cells (figure ) . these circumstances account for the ready outgrowth of drug resistance in patients (gao et al., ) , even though ns a is highly oligomeric. (berger et al., ) were created to co-express ha-and gfp-tagged ns a alleles. huh -lunet-t cells were transfected with ptm-dual-ns a constructs that contained two drug-resistant (r), two drug susceptible (s), or mixed alleles of ns a. proteins from transfected cell extracts that were incubated in the absence (b) or presence (c) of sr were subjected to sds-page without further fractionation (input) or after immunoprecipitation with anti-ha antibodies (ip aha). the gel was subjected to immunoblotting with gfp or ha antibodies as indicated. (d) cells were transfected with dual-ns a constructs in the absence or presence of sr for hr. cells were then fixed, stained with anti-ha antibodies and visualized by confocal microscopy. representative images from over cells are presented. (e) cells transfected with dual-ns a constructs that expressed drug-susceptible (s) and drug-resistant (r) alleles as shown were prepared for electron microscopy by highpressure freezing and freeze-substitution and visualized by transmission electron microscopy. even when drug-resistant ns a was overexpressed in a precursor form, no rescue of drug-susceptible virus was detected (figure -figure supplement c ). it has previously been shown that when ha-and gfp-tagged ns a molecules were expressed on different constructs such as those depicted in figure a , no mixed complexes were formed (berger et al., ) . thus, even though high-order ns a oligomers are formed in infected cells, it is unlikely that these are mixed-allele oligomers, preventing dominant inhibition of drug-resistant hcv. due to the highly mutagenic nature of rna viruses and the large number of genomes and antigenomes generated during infection, a high barrier to drug resistance is extremely difficult to achieve. this has led to abandoned usage and development of many otherwise promising antivirals. to decrease the frequency with which drug-resistant variants arise, combinations of antivirals that, individually, exhibit low barriers to resistance are often used. when drug-resistant variants are first formed intracellularly, through error-prone rna replication, they arise in a population that includes parental and sibling drug-susceptible viruses. several genetic relationships between drug-resistant and drug-susceptible genomes are possible. first, the drug resistance of the new variants has the potential to be genetically dominant, and rescue both resistant and susceptible viral genomes. alternatively, drug resistance can be cis-dominant, with the drug-resistant products rescuing only the genomes that encode them. finally, the drug-resistant genome can fail to benefit any genomes in the cell because the drug-susceptible products present in the same cell are dominant inhibitors. the daas targeting ns / a protease of hcv were the first to be discovered (lamarre et al., ) and the first to reach the clinic jacobson et al., ) . it was soon realized that, both during the growth of hcv replicons in cultured cells and in phase ii clinical trials, drug-resistant viruses were generated rapidly (lin et al., ) . nonetheless, in , further advances led to fda approval of telaprevir and boceprevir poordad et al., ) . the anticipation, which proved to be correct, was that inhibitors of ns / a would prove useful in combination therapies (feld et al., ; lawitz et al., ) . we have used flow cytometry to identify cell populations that are co-infected with hcv that is susceptible or resistant at a : ratio, to protease inhibitors. within these cells, the drug-resistant genomes replicated, but the drugsusceptible genomes did not. we therefore conclude that ns / a inhibitor resistance is cis-dominant (figure ) , which should allow the rapid and specific selection for outgrowth from its cell of origin. cis-dominance of drug resistance was also not originally anticipated while targeting ns / a. the original characterizations of the ns / a protease suggested that cleavage of the ns / a junction occurred in cis, but that cleavages at the a/ b, b/ a and a/ b junctions could all occur in trans (bartenschlager et al., ) . we felt that it was therefore, more likely, that drug-resistant ns / a could rescue drug-susceptible virus within the same cell. ns / a is not known to assemble into high-order oligomers in the same manner as ns a, and we therefore did not anticipate drug-susceptible ns / a would be trans-dominant. furthermore, a trans cleavage assay demonstrated that a ns b- b polyprotein could be cleaved by ns / a supplied in trans (romero-brey et al., ) . however, the trans-cleavage system does not result in membranous web formation that would accompany genome sequestration. other groups have reported a different result, that defective ns mutants cannot be rescued in trans by replicons with functional ns / a (kazakov et al., ; appel et al., ) . our interpretation of these studies is that ns / a is likely physically able to figure continued (c) huh . . cells were coinfected with ca and wt-y n at a moi of ffu/cell for hr. cells were costained to visualize core or ns a together with ca and wt vrnas. representative cells are displayed demonstrating all pairwise comparisons analyzed for colocalization. (d) volocity was used to quantify the number of vrnas per cell, the number of colocalized vrnas, as well as the number of vrna puncta touching ns a or core. (e) depiction of the clonal nature of individual rna replication sites (adapted from figure in zayas et al., ] ). membrane invaginations house either drugresistant (red) or drug-susceptible (blue) genomes. in the model, the rna replication sites are segregated and therefore only the rna from drugresistant virus is amplified in the presence of inhibitors of rna replication. it is visually suggested that ns a molecules that bring core protein to lipid droplets for viral assembly mix on this surface and that this could lead to genetic dominance of drug susceptibility at a packaging step. cleave in trans in cells, but requires access to the alternate precursor proteins in order for this to occur. therefore, cis-dominance of drug-resistance is likely the result of a lack of free-mixing of ns / a encoded by different vrnas within the same cell. ns a emerged as an hcv drug target through a chemical genetics screen for compounds that inhibited hcv growth but did not target the ns / a protease or the ns b polymerase (gao et al., ) . the ease with which resistant viruses were selected suggested that drug resistance was either dominant or cis-dominant. this was somewhat surprising, given that ns a is oligomeric and the ns a inhibitors are extremely potent, and have been postulated to function at sub-stoichiometric ratios to ns a protein (gao, ) . indeed, when drug-susceptible and drug-resistant ns a protein were co-expressed in the present study, hetero-oligomers formed and the biological phenotypes of the drug-susceptible protein were dominant ( figure ) . nonetheless, single-cell analysis of cells coinfected at a : ratio with ns a inhibitor-susceptible and -resistant viruses showed, as with the ns / a inhibitor, that resistance to both sr and daclatasvir was cis-dominant ( figure ) . what does cis-dominant resistance mean mechanistically? one potential mechanism is physical sequestration of the rna replication complexes of the two co-infecting genomes. genome-specific rna probing of co-infected cells revealed that both the negative strands and positive strands from the two viruses were present at physically distinct locations ( figure ). it is therefore highly probable that membrane-associated proteins such as hcv ns / a and ns a do not mix within individual rna replication complexes. however, not all mutations in a particular viral product should lead to the same defect with the same genetic properties. for example, we show here that viruses that are defective in a function of ns a in rna replication complexes are not rescued and have no effect on the outgrowth of drug-resistant variants. however, ns a also plays an important role in packaging and assembly of mature hcv particles on lipid droplets (miyanari et al., ; boson et al., ) . lipid droplets are large and form adjacent to rna replication complexes. in figure e , we have depicted the possibility that ns a molecules encoded by distinct rna replication complexes might mix on the surface of these lipid droplets. however, if replication of the drug-susceptible genomes is inhibited, contribution of their encoded proteins to any oligomers on the surface of lipid droplets should be minimal. in this vain, a hypothetical ns a inhibitor that allowed rna replication but inhibited the function of ns a in particle assembly might have different genetic properties than the ns a inhibitors currently in use. viral capsids have especially interesting genetic properties, often intermixing within co-infected cells. defective capsid proteins of poliovirus, hbv and hiv have been shown to be dominant inhibitors of wild-type viruses (crowder and kirkegaard, ; tanner et al., ; tan et al., ; tan et al., ; trono et al., ; pettit et al., ; lee et al., ; müller et al., ; checkley et al., ) . thus, when antiviral targets are capsid proteins, drug susceptibility can be genetically dominant by suppressing the outgrowth of drug-resistant virus within the cell in which it is first generated (crowder and kirkegaard, ; tanner et al., ; kirkegaard et al., ) . for hcv, very few inhibitors of capsid function have been identified, and their inhibition of viral growth is not sufficiently robust to make genetic experiments possible (kota et al., ) . it is therefore not yet possible to test if, as we hypothesize, drug-susceptible virus will prove to be a dominant inhibitor of drug resistance. consistent with this hypothesis, however, epitope-tagged hcv core protein can form mixed disulfide-bonded core oligomers (kushima et al., ) . the success of combination therapy for hcv and the efficacy of the individual constituents illustrate some of the weapons in the arsenal of antiviral strategies. future directions are likely to include, as well, the rational design of antivirals with high barriers to resistance such as those that hyper-stabilize oligomers and the prediction of daa targets that impart a high fitness cost to drug resistance. huh . . cells were a gift from dr. michael gale jr (university of washington) and were cultured in dmem (sigma, st. louis, mo) supplemented with % fetal bovine serum (omega, tarzana, ca), penicillin/streptomycin (invitrogen, grand island ny), non-essential amino acids (invitrogen), and glutamax (invitrogen). huh -lunet-t cells were a gift from dr. ralf bartenschlager (university of heidelberg) and were cultured in dmem supplemented with % fetal bovine serum, penicillin/ streptomycin, non-essential amino acids, glutamax and mg/ml zeocin (invitrogen). cell line identification was performed using str profiling services available through the stanford functional genomics facility. alignments were generated using huh as a reference. cell lines were screened for mycoplasma contamination using the mycoalert mycoplasma detection kit (lonza). the plasmid pjfh was a gift from dr. michael gale jr (kato et al., ) . this plasmid contains a synthesized genome length copy of the jfh strain of hcv (genotype a). to produce cell-culturederived hcv particles (hcvcc), pjfh was digested with xbai (new england biolabs). the linearized plasmid was then used as a template for in vitro transcription with the megascript high yield transcription kit (ambion). vrna was purified using trizol (invitrogen) and electroporated into huh . . cells as previously described to generate hcvcc cultures (wakita et al., ) . following a period of amplification, hcvcc cultures were converted to human serum media as described previously (steenbergen et al., ) . human serum media comprised dmem supplemented with % heat inactivated human serum (omega), penicillin/streptomycin, non-essential amino acids and glutamax. antibodies recognizing hcv core (abcam), gapdh (santa cruz biotechnologies), gfp (life technologies) and ha (genscript) were purchased from the individual suppliers. antibodies recognizing ns a were described previously (lindenbach et al., ) . to construct codon-altered strains of hcv, we subjected three approximately -nucleotide fragments of the jfh genome through the geneart codon optimization algorithm offered by life technologies. the genome fragments were composed of nucleotides - (ca- ), - (ca- ), and - (ca- ). all three codon-altered genome fragments were synthesized by life technologies and cloned into the pjfh plasmid by restriction digestion and ligation with t dna ligase (invitrogen). the resulting plasmids: pjfh -ca- , pjfh -ca- and pjfh -ca- , were used to produce hcvcc cultures as described above. to create drug-resistant hcvcc cultures, two subcloning plasmids were created by pcr by amplifying nucleotides - or - of the pjfh plasmid with taq polymerase (new england biolabs) and ligating the pcr products into pcr . (invitrogen). the resulting plasmids, pcr . - - and pcr . - - were used as templates for site-directed mutagenesis using the quikchange site-directed mutagenesis kit (agilent technologies). pcr . - - -y n was generated using the forward primer '-cctatcaattgcaatacggagggccag tgcgcgcc- ' and the reverse primer '-ggcgcgcactggccctccgtattgcaattgatagg- '. pcr . - - -y h was generated using the forward primer '-cctatcaattgccatacg-gagggccagtgcgcgcc- ' and the reverse primer '-ggcgcgcactggccctccgtatgg-caattgatagg- '. pcr . - - -d a was generated using the forward primer '-aaatccatcgccttcatcccc- ' and the reverse primer '-ggggatgaaggcgatggatttggc- '. these mutated hcv genome fragments were cloned into pjfh or pjfh -ca using restriction digestion and ligation with t dna ligase (invitrogen). hcvcc cultures were generated as described above. the plasmids ptm_ns - b_ns a-ha_ a_ns a-gfp_jfh (referred to as ptm-dual-ns a) and ptm_ns - b_ns a-gfp (referred to as ptm-ns - b) were the generous gifts of dr. ralf bartenschlager (university of heidelberg). the d a and y n mutations were cloned into the ptm-ns - b plasmid using the quikchange lightening mutagenesis kit using the primers described above. the ns a alleles of the ptm-dual- a plasmid were first separated by removing an rsrii fragment containing most of the ns a-gfp allele to create ptm-dual- a-drsrii and pcdna -ns a-gfp-rsrii. site-directed mutagenesis was performed using the quikchange lightening kit on ptm-dual- a-drsrii or on pcdna -ns a-gfp-rsrii independently. the rsrii fragments containing wildtype ns a or ns a-y n were then cloned back into the ptm-dual- a-drsrii vectors to create all combinations of wild-type ns a and ns a-y n ptm-dual- a. vrna was harvested from cells using trizol (invitrogen) or collected from hcvcc culture supernatants using the qiaamp vrna mini kit (qiagen). a standard curve was generated using in vitro transcribed hcv vrna. qrt-pcr was performed using the quantitect sybr-green rt-pcr kit (qiagen) and the qrt-pcr forward '-ctggcgactggatgcgtttc- ' and reverse '-cgcattcctccatc tcatca- ' primers. alternatively, the following ca-specific primers were used: forward '-gtggtg tccatgaccggca- ' and reverse '-ggtcacggggcctctcagt- ', or the following wt-specific primers were used: forward '-gtggtgagtatgacggggc- ' and reverse '-cgtgaccg-gaccccgtaag- '. samples were analyzed on a real-time pcr machine (applied biosystems). wt vrna target probes recognizing the ns region of either the positive or negative strand were designed and synthesized by affymetrix. these probes were specifically designed to avoid detection of codon-altered jfh viral rna. additionally, probes were designed to recognize the corresponding region of the negative or positive strand jfh -ca vrna. these ca target probes were specifically designed not to recognize the wt vrna. huh . . cells were infected with wt or ca hcvcc particles for hr. infected cells were fixed with % formaldehyde solution (sigma) and subjected to rna in situ hybridization (ish) using the viewrna cell assay kit (affymetrix) according to the manufacturer's protocol. cells were co-stained with both ca and wt vrna target probe sets in all experiments. cells were visualized on a leica sp confocal microscope. protein and vrna colocalization was performed on cells coinfected with jfh -ca and jfh -y n for hr. following infection, cells were fixed and stained using the view-rna cell plus assay reagents. core and ns a were visualized using the antibodies described above at a to dilution followed by the anti-mouse-alexaflour- secondary antibody at to dilution. quantification of colocalization was performed using volocity software (perkin elmer). briefly, we defined vrna puncta as objects larger than . mm . objects larger than . mm were broken into subunits based on total volume. objects sharing . mm of mutual space were quantified as mutual. due to the localization patterns of core and ns a, spot counting algorithms were not appropriate. total vrna objects and as well as the total number of vrna objects touching ns a or core were quantified. huh -lunet-t cells were transfected with ptm-dual-ns a constructs using branched polyethylenimine (sigma-aldrich) at a ratio of : . at hr post-transfection, cells were treated with nm sr or a dmso control. at hr post-transfection, cells were fixed with % paraformaldehyde, stained with anti-ha antibodies and dapi and visualized on a leica sp confocal microscope. a more detailed description of the rna fish microscopy methods are provided in bio-protocol (van buuren and kirkegaard, ) . huh -lunet-t cells were transfected with ptm-dual-ns a constructs using the polyethylenimine transfection reagent. at hr post-transfection, cells were treated with dmso or nm sr . at hr post-transfection, cells were harvested using an enzyme-free cell dissociation buffer (life technologies) and facs sorted for gfp-positivity on a facs aria cell sorter. gfp-positive cells were resuspended in % bsa in pbs then placed into a mm deep hat and high-pressure frozen using a leica empact . frozen samples were then freeze substituted in % osmium tetroxide and . % uranyl acetate in acetone using a leica emafs at À ˚c for hr, warmed to À ˚c in . hr at ˚c/ hr and held for hr then warmed to ˚c in hr at ˚c/hr and held for hr. the samples were then washed two times in acetone, then in propylene oxide for min each. samples are infiltrated with embed- resin (ems cat# ) mixed : , : , and : with propylene oxide for hr each, leaving samples in : resin to propylene oxide overnight rotating at room temperature. the samples are then placed into embed- for three hours then placed into taab capsules with fresh resin and placed into a ˚c oven overnight. sections were taken between and nm, picked up on formvar/carbon coated mesh copper grids, then contrast stained for s in . % uranyl acetate in % acetone followed by staining in . % lead citrate for min. cells were visualized using the jeol jem- kv microscope and photos were taken using a gatan orius k  k digital camera. huh . . cells were either transfected with wt and/or ca vrna as previously described or infected with wt and/or ca hcvcc particles. coinfections were performed by infecting with each virus for or hr followed by treatment with either mm biln- for hr or nm sr for hr. cells were harvested with trypsin and fixed with the flowrna fixation and permeablization kit. cells were then costained with ca and wt vrna target probe sets using the flowrna kit (affymetrix) and analyzed on the scanford facscan flow cytometer. data was analyzed and processed using flowjo software. mutational and fitness landscapes of an rna virus revealed through population sequencing ledipasvir and sofosbuvir for untreated hcv genotype infection quantigene flowrna assay. a novel rna ish multicolor application for flow cytometry efficient rescue of hepatitis c virus rna replication by transcomplementation with nonstructural protein a boceprevir for previously treated chronic hcv genotype infection kinetic and structural analyses of hepatitis c virus polyprotein processing daclatasvir-like inhibitors of ns a block early biogenesis of hepatitis c virus-induced membranous replication factories, independent of rna replication permissive secondary mutations enable the evolution of influenza oseltamivir resistance daclatasvir prevents hepatitis c virus infectivity by blocking transfer of the viral genome to assembly sites the capsid-spacer peptide gag processing intermediate is a dominant-negative inhibitor of hiv- maturation complete three-dimensional structures of picornaviral rna-dependent rna polymerases trans-dominant inhibition of rna viral replication can slow growth of drugresistant viruses coronaviruses resistant to a c-like protease inhibitor are attenuated for replication and pathogenesis, revealing a low genetic barrier but high fitness cost of resistance daclatasvir plus sofosbuvir for hcv infection general catalytic deficiency of hepatitis c virus rna polymerase with an s t mutation and mutually exclusive resistance towards '-modified nucleotide van oral direct-acting agent therapy for hepatitis c virus infection: a systematic review treatment of hcv with abt- /r-ombitasvir and dasabuvir with ribavirin combining oncolytic virotherapy and cytotoxic therapies to fight cancer distinct functions of ns a in hepatitis c virus rna replication uncovered by studies with the ns a inhibitor bms- chemical genetics strategy identifies an hcv ns a inhibitor with a potent clinical effect antiviral activity and resistance of hcv ns a replication complex inhibitors hcv ns a replication complex inhibitors dominant negative effect of wild-type ns a on ns a-adapted subgenomic hepatitis c virus rna replicon virus mutation frequencies can be greatly underestimated by monoclonal antibody neutralization of virions telaprevir for previously untreated chronic hepatitis c virus infection cell culture and infection system for hepatitis c virus hepatitis c virus rna replication depends on specific cis-and trans-acting activities of viral nonstructural proteins origins of combination therapy for tuberculosis: lessons for future antimicrobial development and application my cousin, my enemy: quasispecies suppression of drug resistance ná jera i. . the novel nucleoside analog r ( '-azidocytidine) is a potent inhibitor of ns b-dependent rna synthesis and hepatitis c virus replication in cell culture ml , a small molecule inhibitor of dimerization of the core protein of hepatitis c virus (hcv) a disulfide-bonded dimer of the core protein of hepatitis c virus is important for virus-like particle production an ns protease inhibitor with antiviral effects in humans infected with hepatitis c virus sofosbuvir for previously untreated chronic hepatitis c infection ribavirin, to treat chronic infection with hepatitis c virus genotype in non-responders to pegylated interferon and ribavirin and treatment-naive patients: the cosmos randomised study a strongly transdominant mutation in the human immunodeficiency virus type gag gene defines an achilles heel in the virus life cycle discovery of potent hepatitis c virus ns a inhibitors with dimeric structures in vitro resistance studies of hepatitis c virus serine protease inhibitors, vx- and biln : structural analysis indicates different resistance mechanisms complete replication of hepatitis c virus in cell culture novel therapies for hepatitis c -one pill fits all? suppression of drug resistance in dengue virus the r k substitution in hiv- subtype c is more deleterious for integrase enzymatic function and viral replication than in subtype b the lipid droplet is an important organelle for hepatitis c virus production hiv- gag processing intermediates trans-dominantly interfere with hiv- infectivity processing sites in the human immunodeficiency virus type (hiv- ) gag-pro-pol precursor are cleaved by the viral protease at different rates the coding region of the hcv genome contains a network of regulatory rna structures boceprevir for untreated chronic hcv genotype infection three-dimensional architecture and biogenesis of membrane structures associated with hepatitis c virus replication ns a domain and polyprotein cleavage kinetics are critical for induction of double-membrane vesicles associated with hepatitis c virus replication viral mutation rates analysis of hepatitis c virus superinfection exclusion by using novel fluorochrome gene-tagged viral genomes understanding the hepatitis c virus life cycle paves the way for highly effective therapies human serum leads to differentiation of human hepatoma cells, restoration of very-low-density lipoprotein secretion, and a -fold increase in hcv japanese fulminant hepatitis type titers resensitizing daclatasvir-resistant hepatitis c variants by allosteric modulation of ns a l f and v a sofosbuvir-associated hepatitis c virus ns b substitutions genetically altering the thermodynamics and kinetics of hepatitis b virus capsid assembly has profound effects on virus replication in cell culture the interface between hepatitis b virus capsid proteins affects self-assembly, pregenomic rna packaging, and reverse transcription dominant drug targets suppress the emergence of antiviral resistance exploiting genetic interference for antiviral therapy structure of the zinc-binding domain of an essential component of the hepatitis c virus replicase hiv- gag mutants can dominantly interfere with the replication of the wild-type virus detection and differentiation of multiple viral rnas using branched dna fish coupled to production of infectious hepatitis c virus in tissue culture from a cloned viral genome treatment with ledipasvir and sofosbuvir improves patient-reported outcomes: results from the ion- , - , and - clinical trials coordination of hepatitis c virus assembly by distinct regulatory regions in nonstructural protein a we thank drs. yury goltsev and garry nolan for advice on fluorescent cell sorting-based visualization of rna, drs. michael gale jr. and ralf bartenschlager for the generous donation of reagents and dr. peter sarnow for critical reading of the manuscript. we would like to acknowledge the work of drs. jeannie spagnolo and ernesto mendez for initiating hcv research in our laboratory. this work was supported by funding to kk from nih u ai (jeffrey glenn, pi), an nih director's pioneer award and the alison and steve krausz innovation fund. nvb was supported by the canadian institutes for health research ncrtp-hepc training program and the american liver foundation. electron microscopy was performed in a facility supported in part by arra award number s rr - from the national center for research resources (ncrr). the cell sciences imaging facility used for confocal microscopy was supported by arra award number s od from the ncrr. the contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views of the ncrr or the national institutes of health. key: cord- -pl d w authors: tapia, felipe; laske, tanja; wasik, milena a.; rammhold, markus; genzel, yvonne; reichl, udo title: production of defective interfering particles of influenza a virus in parallel continuous cultures at two residence times—insights from qpcr measurements and viral dynamics modeling date: - - journal: front bioeng biotechnol doi: . /fbioe. . sha: doc_id: cord_uid: pl d w defective interfering particles (dips) are a natural byproduct of influenza a virus (iav) replication. dips interfere with the propagation and spread of infectious standard virus (stv), reduce virus yields by competing for viral and cellular resources, and induce antiviral responses. these properties open exciting possibilities for the development of dip-based antivirals. exploring options for cell culture-based dip production, we have established a fully continuous cultivation process, where one bioreactor is used to grow cells that are fed to two bioreactors operated in parallel for virus production. this system allows head-to-head comparisons of stv and dip replication dynamics over extended time periods. cultivations were performed at two residence times (rt, and h) using mdck suspension cells grown in a fully defined medium. for infection, we used a virus seed generated by reverse genetics containing stvs and a known dip carrying a deletion in segment (dels ( )). four days post infection, dips achieved maximum concentrations of . · ( ) virions/ml and . · ( ) virions/ml for rts of and h, respectively. furthermore, oscillations in virus titers with two to three maxima were found for dip accumulation at and h rt, respectively. to complement the study, a basic mathematical model using simple kinetics and a reasonable number of parameters to describe dip-propagation in continuous cultures was established. upon fitting the model individually to each of the two data sets, oscillations in the viral dynamics and the cell population dynamics were described well. modeling suggests that both stv inactivation and virus degradation have to be taken into account to achieve good agreement of simulations and experimental data for longer rts. together, the high dip titers obtained, and the successful simulation of the experimental data showed that the combination of continuous bioreactors and mathematical models can enable studies regarding dip dynamics over extended time periods and allow large scale manufacturing of dip-based antivirals. defective interfering particles (dips) are a natural byproduct of influenza a virus (iav) replication. dips interfere with the propagation and spread of infectious standard virus (stv), reduce virus yields by competing for viral and cellular resources, and induce antiviral responses. these properties open exciting possibilities for the development of dip-based antivirals. exploring options for cell culture-based dip production, we have established a fully continuous cultivation process, where one bioreactor is used to grow cells that are fed to two bioreactors operated in parallel for virus production. this system allows head-to-head comparisons of stv and dip replication dynamics over extended time periods. cultivations were performed at two residence times (rt, and h) using mdck suspension cells grown in a fully defined medium. for infection, we used a virus seed generated by reverse genetics containing stvs and a known dip carrying a deletion in segment (dels ( )). four days post infection, dips achieved maximum concentrations of . · virions/ml and . · virions/ml for rts of and h, respectively. furthermore, oscillations in virus titers with two to three maxima were found for dip accumulation at and h rt, respectively. to complement the study, a basic mathematical model using simple kinetics and a reasonable number of parameters to describe dip-propagation in continuous cultures was established. upon fitting the model individually to each of the two data sets, oscillations in the viral dynamics and the cell population dynamics were described well. modeling suggests that both stv inactivation and virus degradation have to be taken into account to achieve good agreement of simulations and experimental data for longer rts. together, the high dip titers obtained, and the successful simulation of the experimental data showed that the combination of continuous bioreactors and mathematical models can enable studies regarding dip dynamics over extended time periods and allow large scale manufacturing of dip-based antivirals. viruses are a major threat to human health and significant efforts have been made over the last century to prevent and treat viral diseases. a huge success was the development of potent, safe and affordable viral human and veterinary vaccines using eggand cell-based production systems (aubrit et al., ; genzel, ; barrett et al., ; volz and sutter, ) . in addition, novel approaches toward vaccination are under development, i.e., dna and rna vaccines (kutzler and weiner, ; de gregorio and rappuoli, ; ahmed et al., ) . in addition, for various viruses such as hiv, iav, or herpes virus, potent antivirals are available for treatment [e.g., for influenza virus (samson et al., ; simonsen et al., ) ]. despite this, significant challenges remain. on the one hand, humans are constantly challenged by new viruses including zika virus, mers-coronavirus, or ebola virus. on the other hand, most viruses change rapidly in the face of selective pressure and emergence of antiviral drug resistance is of major concern [e.g., for influenza virus (samson et al., ; lackenby et al., ; shin and seong, ) ]. these challenges can only partly be addressed by existing technologies, since the development of new vaccines, the adaptation of existing manufacturing processes, and the identification of new antiviral targets are time consuming and costly processes. they involve complex decisions regarding the selection of efficient production systems, potency and safety aspects, and comprehensive clinical trials. moreover, conventional antiviral measures may be too slow to save lives in case of a rapidly spreading virus. an unconventional option to prevent virus spreading and cure infectious diseases is the use of viruses themselves. this concerns especially defective interfering particles (dips)defective viruses, which suppress the spread of their intact, replication competent counterparts, reducing infectious virus yields by up to five orders of magnitude (e.g., akkina et al., ; frensing et al., ) . dips were already identified in the early fifties by studies of von magnus (von magnus, ) , who performed undiluted serial passages of iav. dips are characterized by deletions in the viral genome preventing the synthesis of a protein essential for viral spread (nayak et al., ; dimmock and easton, ; frensing, ) . for replication, dips rely on the presence of homologous helper virus, further referred to as standard virus (stv), that supplies the missing viral protein(s) in trans. interestingly, the presence of dips has been demonstrated for almost every virus studied (huang and baltimore, ) making dips a viable option to prevent and/or treat a larger number of viral diseases. while initial studies suggested that dips suppress stv replication and may thus have the potential to protect against viral disease, it was not until the late eighties that researchers started to systematically exploit this approach (dimmock et al., ) . it was dimmock and colleagues (dimmock et al., ) who demonstrated that the use of molecular cloning technologies enables the generation of dips that have the potential to protect animals from iav infection. they also showed that the dip isolate named di , from influenza a/puerto rico/ / (pr ; h n ), which contains a single deletion in segment (s ; coding for pb ), is suitable to both therapeutically and prophylactically protect animals from a lethal challenge by the pandemic iav and, potentially, other respiratory viruses (duhaut and dimmock, ; easton et al., ; scott et al., ; dimmock et al., ; dimmock and easton, ) . they showed that production of the di antiviral candidate can be realized in eggs followed by an ultraviolet (uv) irradiation process to destroy stv infectivity (dimmock et al., ) . more recently, an in vitro and in vivo antiviral effect was demonstrated for a combination of three defective interfering genes of iav for avian and seasonal influenza using a dual-functional peptide vector (zhao et al., ) . despite the increasing interest in the potential use of dips as antiviral agents, relatively little is known regarding their spread and accumulation in cell populations. this also applies to large scale manufacturing of dips in biopharmaceutical industry where fertilized chicken eggs or animal cell culture technologies could be considered for efficient large scale dip production. while eggs have been successfully used for the production of dips in relatively small amounts (dimmock and marriott, ; dimmock et al., ) , cell culture-based technologies have been less explored and have several additional advantages for large scale dip manufacturing. firstly, animal cells are ideal for in-depth investigation of intracellular dip replication, their release and cell-to-cell spreading under controlled and welldefined cultivation conditions in bioreactors over an extended time period. secondly, cells could be specifically designed for dip generation (ozawa et al., ; bdeir et al., ; yamagata et al., ) using plasmids for reverse genetics (hoffmann et al., ) , which would allow to overcome the need of any infectious helper virus for dip replication. such dip preparations, in contrast to egg-based production systems, would not be contaminated with stv and would not need uv inactivation for use as antivirals (dimmock et al., ) . finally, there are various quantitative assays available for detailed characterization of the dynamics of virus titers and dip copy numbers. together with the use of specific staining methods and flow cytometry for monitoring the progress of infection in cells (frensing et al., swick et al., ) , mathematical models for dip and stv replication can be established to describe their basic dynamics in cell culture akpinar et al., a,b; laske et al., ; liao et al., ) . in this study, following the general ideas described by frensing et al. ( ) , we investigated dip production in a continuous cultivation system. frensing et al. demonstrated that continuous influenza virus production in a cascade of two stirred tank bioreactors showed oscillations in virus and cell concentrations due to the presence of dips. in contrast to their approach using only two vessels, we used one bioreactor for continuous cell production (cell bioreactor or cb) feeding two bioreactors for virus propagation (virus bioreactor or vb ; virus bioreactor or vb ) operated in parallel to allow for headto-head comparisons of virus seeds, media, cell lines, or changes in cultivation parameters under conditions as close to each other as possible. as a starting point, the impact of residence time (rt, and h) on dip and stv dynamics was investigated. with regard to the establishment of manufacturing processes for dip production, a mdck suspension cell line growing in a fully defined medium was used (lohr et al., ) . virus seeds containing known amounts of dips and stv were generated using a reverse genetics approach (hoffmann et al., ) . cultivations were performed over a period of days at two rts to allow for at least two oscillations in stv and dip replication dynamics . for process monitoring and model development, cell concentrations, infectious and noninfectious virus titers, as well as extracellular copy numbers of s for both stv, i.e., full-length (fl) s , and a known form of dip, dels ( ), were determined. based on the newly available quantitative data obtained for dip and stv concentrations, the mathematical model developed by frensing et al. ( ) was extended. after fitting, the basic process dynamics for both rts were described well by the simulations. in particular, these simulations capture the oscillations in process variables, e.g., virus titers. in addition, the model predicts the dynamics of cell subpopulations (uninfected, dip-only infected, stv-only infected, and co-infected cells). the canine cell line mdck.sus (through contact with prof. klaus scharfenberg, university of applied sciences emden-leer, germany) was cultivated in chemically defined smif medium (gibco, thermo fischer scientific, usa), supplemented with glutamine, and pyruvate (both mm final concentration, sigma, usa). mdck.sus cells were grown in shake flasks and passaged as described before (lohr et al., ) . influenza a/puerto rico/ / virus seed containing dips with a deletion in s , i.e., dels ( ), was generated by reverse genetics using an + plasmid-system (hoffmann et al., ; duhaut and dimmock, ) . this virus seed will be referred to as a/pr/ / -dels ( ). therefore, s rna was isolated from influenza a/puerto rico/ / (national institute for biological standards and control (nibsc), no. / ) and the respective cdna was cloned into the phw vector provided by erich hoffmann and robert g. webster from st. jude children's research hospital (memphis, tn, usa) (hoffmann et al., ) , resulting in the plasmid phw-s . the plasmid carrying the defective s sequence phw-a/pr/ / -dels ( ), previously described by dimmock et al. ( ) , was obtained by cloning the respective sequence into the phw vector. in addition, phw plasmids carrying fl segments - were used (also provided by erich hoffmann and robert g. webster). one day before transfection, · hek- t cells were seeded into mm dishes. prior to infection, cells were washed with pbs and ml opti-mem (gibco) were added. transfection was performed simultaneously with all nine plasmids, ng each, using lipofectamine ltx & plus reagent (life technologies) according to the manufacturer's instructions ( µl lipofectamine ltx per dish). to increase the amount of virus particles, · adherent mdck cells were added h post-transfection. after h at • c and % co , cells were washed with pbs and medium was replaced with dmem (gibco) containing units/ml trypsin (gibco, # - , sterile-filtered stock solution prepared in pbs with u/ml and stored at − • c). subsequently, the cells were incubated at • c for additional h. as a result, a virus seed containing a/pr/ / -dels ( ) and stv, with a tcid titer of . · virions/ml and an ha titer of . log (ha units/ µl) was obtained. note, that segment-specific reverse transcription-quantitative pcr (qpcr) to determine a/pr/ / -dels ( ) copy numbers in virus seeds is biased since it is contaminated with the plasmid carrying the di s sequence, phw-a/pr/ / -dels ( ). for low multiplicity of infection (moi) cultivations in continuous cultures, this is not relevant as plasmids are out-diluted (see below). in a next step, the virus seed for infection of suspension mdck cells in bioreactors was generated. therefore, the a/pr/ / -dels ( ) virus seed was passaged once in suspension mdck.sus cells with an moi of . . the virus was harvested h post infection (p.i.), clarified by centrifugation ( , g) and the supernatant was aliquoted. the resulting virus bank had an ha titer of . log (ha units/ µl), a tcid titer of . · virions/ml, a fl s content of . · copies/ml, and a di s (a/pr/ / -dels ( )) content of . · copies/ml. additionally, virus seeds were probed for remaining plasmids after reverse genetic preparation by qpcr, which could bias reverse transcription-qpcr data of the fl and di s measurements. quantitative pcr data showed, that initial plasmid contamination was . % for a/pr/ / -dels ( ) and . % for fl s . however, from zero h p.i. on the plasmid contamination was not detectable any longer, since the virus preparation was diluted due to the low moi applied for infection (moi . ). all experiments were performed in laboratories with a biological safety level (bsl ) certification following the respective safety regulations. a continuous bioreactor system consisting of three stirred tank bioreactors (str, dasgip) was used (figure ). the first . l str (with head space capacity for ml working volume) was used for cell propagation (cb), and the other two . l str were run in parallel and used for virus propagation (vb and vb ). after inoculation with mdck.sus cells at . · viable cells/ml, the cb was operated in batch mode the first h in ml working volume (wv). simultaneously, vb and vb were inoculated with . · viable cells/ml and grown in batch mode in ml wv and ml wv, respectively. working volumes were chosen to achieve rts of and h in vb and vb , respectively, using the flow rates (f) described in figure (rt = wv/f). the choice of h rt was motivated by similar experiments performed previously by our group . the h rt was selected based on scouting experiments performed in shake flasks and preliminary mathematical simulations (not shown). the cb was maintained at ml wv. cultivations parameters were • c, ph . , and a stirring speed of rpm. aeration was controlled to % dissolved oxygen partial pressure by pulsed addition with a mixture of air, oxygen and nitrogen through a dip tube. for infection, the dip-containing virus seed (a/pr/ / -dels ( )) was added to the vb and vb at an moi of . based on the viable cell count and the tcid titer of the virus seed at h of culture. additionally, the virus inoculum was supplemented with : ratio of trypsin (volume to culture volume). to avoid a fast wash-out of the virus seed, continuous mode was initiated at . h p.i. with the flow rates depicted in figure . during the run, trypsin ( . units/l) was present in the feed medium of the virus bioreactors. feed media of all three reactors were provided in l bottles chilled at - • c; only the feed medium of the cell bioreactor had to be refilled during the run-time. samples were taken twice a day from vb and vb and once a day from cb. about every h, virus harvests reservoirs were stored at− • c until further analysis. the experiment was terminated days p.i. the average cell concentration input for vb and vb was . · cells/ml as determined from the average viable cell count in cb with respect to str working volumes and feeding rates. rts were determined from the average volume and the average flow rate at the outlet of both str over the infection phase. viable cell concentrations were determined using a vicellxr (beckman coulter) and virus production was monitored by a hemagglutination (ha) assay (kalbfuss et al., ) and a tcid assay (genzel and reichl, ) . the maximum standard deviation of the ha assay was ± . log (ha units/ µl) and the dilution error of the tcid assay was ± . log (genzel et al., ) . furthermore, we determined the concentration of total virus particles c v in the supernatant based on the ha values using the following equation, c v = c ery · (log hau/ µl) , where c ery denotes the concentration of chicken erythrocyte solution added to the assay ( × cells/ml). to analyze viral genomes, bioreactor samples were centrifuged for min at g and viral rna was isolated from supernatants using the nucleospin rna virus kit (macherey-nagel) according to the manufacturer's instructions. reverse transcription of isolated rna to cdna and segment-specific amplification of iav genomes was performed as described previously (frensing et al., ) . reverse transcription-pcr products were directly analyzed on a % agarose gel using electrophoresis (see data sheet ; figure s ). transcription-qpcr for a/pr/ / -dels ( ) and fl s quantification absolute viral rna (vrna) copy numbers were determined as described previously (wasik et al., ) . in brief, -fold series dilutions of the corresponding vrna reference standards and rna samples were reverse transcribed with the tagged primer seg- -tagrt-for (atttaggtgacactatagaagcgagc-gaaagcaggtcaattatattc). subsequently, a qpcr was performed using the rotor-gene sybr green pcr kit (qiagen) following the manufacturer's recommendations and the primer pairs realtime-rev (ggaatcccctcagtcttc) and vrna-tagrealtime-for (atttaggtgacactatagaagcg) for quantification of a/pr/ / -dels ( ) and the primer pairs fl -realtime-rev (catttcatcctaagtgctgg) and vrna-tagrealtime-for for quantification of fl s . viral rna copy numbers were calculated based on the vrna reference standards with linear regression. the lowest concentrations, which allowed quantification, were . · copies/ml for a/pr/ / -dels ( ) and . · copies/ml for fl s , respectively (wasik et al., ) . the relative standard deviation for determining copy numbers by qpcr was and % for a/pr/ / -dels ( ) and fl s , respectively. quantification of contaminating plasmids was performed by dilution series of the corresponding plasmid as described above, but without the reverse transcription step. the plasmid phw-s was quantified with the primer pair fl -realtime-rev and phw-f (ctcactatagggagaccc). the plasmid phw-a/pr/ / -dels ( ) was quantified with the primer pair realtime-rev and phw-f. since number of genome copies do not directly translate into numbers of virus particles, we adjusted the qpcr measurements of a/pr/ / -dels ( ) and fl s . for this, we assumed that the sum of a/pr/ / -dels ( )-containing virions and the fl s -containing virions denotes the maximum number of virions present in a sample, which should equal the concentration of total virus particles c v . based on c v , we calculated the concentration of virions that contain either a/pr/ / -dels ( ) or fl s using equations ( ) and ( ), respectively. furthermore, a differentiation between infectious fl s containing virions (v s ) and non-infectious fl s -containing virions (v d ) is made. since we know the number of v s , i.e., the tcid titer, we can easily determine the number of noninfectious fl s -containing virions by solving equation ( ) for v d . based on quantitative data available from reverse transcription-qpcr for s encoding pb of iav that was established recently by our group, the segregated mathematical model describing this continuous virus production system established by frensing et al. ( ) was modified. the extended model version describes explicitly the dynamics of replication-incompetent virions (dips) containing a deletion in s (v ddi ) as described above (a/pr/ / -dels ( ), see materials and methods) in addition to noninfectious virions (v d ) and infectious virions (v s ) containing the fl s . as another modification, the infection of uninfected target cells (t) is considered separately for v s and v ddi to account figure | overview of the bioreactor setup for parallel continuous influenza a virus propagation in two vessels. mdck suspension cells were grown in a cell bioreactor (cb) feeding two virus production vessels (vb and vb ) operated at a residence time of and h, respectively. at time of infection, the suspension mdck cell-adapted virus seed containing a/pr/ / -dels ( ) and fl standard virus (stv) was added to vb and vb at a multiplicity of infection of . . subsequently, cells were constantly fed into both virus bioreactors (f, feeding rate). trypsin was added to the fresh medium reservoir of vb and vb . all green components refer to cb, all red components to the vbs; cb and vbs are connected via the purple tubing. for dip entry with subsequent dip replication in case of a co-infection. where µ denotes the specific cell growth rate, and kvi and kvidi are the specific virus infection rates for infectious stvs and dips, respectively. the last term in equation ( ) accounts for the continuous feed of cells with concentration t in at dilution rate d, which was set to adjust the two different rts for both virus reactors (see table ). with respect to the average concentration of cells observed in cb, we choose t in to be independent of time (data not shown). ideal mixing is assumed for all vessels. the population of infected cells is subdivided into cells infected with infectious stvs (i s ), dips (i d ), and both (i c ). the first term in equations ( ) and ( ) accounts for the infection of target cells by infectious stvs or dips, respectively. furthermore, the co-infection of i s and i d by dips and infectious stvs, respectively, yields co-infected cells i c in equation ( ). antiviral mechanisms, such as the interferon-mediated innate immune response, and virus-induced cell death can be triggered by the presence of intracellular viral rnas. in our experiments, the uptake of a high number of dips ( - dips per cell) results in a high number of intracellular viral rnas, comparable to levels reached during a conventional infection (e.g., frensing et al., ) . therefore, and in contrast to frensing et al. ( ) we assume that dip-only infected cells (i d ) do not continue to grow, but shut-off essential pathways for cell division, similar to the other infected cell populations i s and i c and, thus, die due to virus-induced apoptosis with the specific rate kcdv. note, that cane and colleagues have shown that dip-only infected mdck cells can continue to grow and give rise to dip-infected daughter cells (cane et al., ) . however, the experimental conditions applied in their study, i.e., the use of uv-irradiated virus seed, passaging of cells in a virus-free culture and a culture time of - days between passages, cannot be compared to the cultivation conditions of our system. the dynamics of infectious stvs (v s ), dips (v ddi ), and noninfectious virions containing a fl s (v d ) are described as similar to frensing et al. ( ) , we assume that stv-infected cells (i s ) produce infectious stvs (v s ) with the specific rate µvi and also have the potential to release dips (v ddi ) with the specific rate µvddi s . although in our analysis µvddi s is close to zero (see table ), the latter should not be ignored to allow for de novo generation of dips in case a dip-free virus seed would be used for the infection of bioreactors. co-infected cells release mainly dips containing the deletion in s (v ddi ) with the specific rate µvddi c . in addition, both stv-infected and co-infected cells release non-infectious virus particles (v d ) with rates µvd s and µvd c , respectively. furthermore, we assume that stvs are taken up by uninfected cells and diponly infected cells, while dips are either infecting uninfected cells or cells already infected by infectious stvs. finally, we assume that stvs can lose their infectivity with the specific inactivation rate kdvit contributing to the population of noninfectious virions (v d ), while both v ddi and v d deteriorate with the specific lysis rate kdvt. in contrast to the previous model , we neglected superinfection of i s and i c by stvs as well as superinfection of i d and i c by dips. to investigate the time course of dip formation based on reverse transcription-qpcr measurements, the following dip to stv ratio was determined from experimental data: where v ddi represent the reverse transcription-qpcr measurement of dels ( ) and v s the infectious virions with fl s , respectively. in addition, to describe overall cell growth dynamics, the concentration of all cells (cells total ) in the virus bioreactor was estimated. cells total = t + i s + i d + i c taken together, the model comprises seven ordinary differential equations. the set of ten parameters ( table ) was determined by minimizing the least-squares prediction error of the state variables v s , v ddi , v d and cells total , for which the error of each variable was weighted with its maximum measurement value. model equations were solved numerically using the cvode routine from sundials (cohen and hindmarsh, ) on a linux-based system. model files and experimental data were handled within the systems biology toolbox (schmidt and jirstrand, ) for matlab (version . . . r b). parameter values were estimated using the global stochastic optimization algorithm fssm (egea et al., ) . initial values were selected based on previous parameters determined for iav replication in animal cells (e.g., frensing et al., ) . the coefficient of variation of parameters was determined using copasi (hoops et al., ) . in order to reduce the risk of batch-to-batch variations for controlled experiments under different cultivation regimes, we established a parallel continuous production process for iav propagated in mdck suspension cells. in the set-up implemented, one cell bioreactor was used to feed two virus vessels (figure ) . as a starting point, we investigated the impact of rt ( and h) on the accumulation of dips and their impact on viral titers over a period of days. based on quantitative information available from qpcr and the mathematical model established, it is possible to describe the dynamics of the various cell populations as well as virus populations, i.e., infectious stvs, non-infectious stvs and replication-incompetent dips, and the dip to stv ratio for both cultivation conditions. the mathematical model used in the present study is loosely based on a previously published model with modifications as explained in section materials and methods. we fitted the parameters of the model to the two sets of experimental data and determined their coefficient of variation (cov) using copasi (table ) . for rt h, low cov (≤ %) were reached for parameter estimates of the specific growth rates for the different viral subpopulations v ddi and v s , v d , which are released by either co-infected cells i c (µvddi c ) or by stv-infected cells i s (µvi, µvd s ), respectively. similarly, the cov of estimates for µvddi c , µvi and µvd s for rt h were ≤ %. the specific stv infection rate kvi for rt h as well as the specific dip infection rate kvidi, and the specific apoptosis rate kcdv for both rt and h were estimated with higher cov values ( % < cov < %). interestingly, the specific infection rate of stvs kvi for rt h was estimated with a cov of %. the remaining parameters were estimated with cov > %. in particular, the specific de novo generation rate of dips µvddi s had a very high cov in both experimental scenarios. since dips are present in the seed virus, de novo generation could also be excluded from modeling assumptions and viral dynamics would still show oscillations due to the amplification of dips introduced at infection. in contrast, if a dip-free virus seed would be used to start infection, the oscillating viral dynamics could only be reproduced if de novo generation is accounted for. thus, we decided to keep this parameter to propose a model that covers more general cases. the specific production rate of non-infectious fl s containing virions µvd c could also be neglected, since these particles are most likely released only from stv-infected cells, which is supported by the reasonable cov values for the specific v d production rate (µvd s ) in both rt and h experiments. in addition, the high cov values for estimates of the specific virus inactivation and lysis rates (kdvit, kvdt) indicated that these mechanisms can probably be neglected, which we also analyzed in more detail with respect to the different rts (see section model reduction). overall, key parameter values estimated in this study, such as specific stv infection rate kvi, virus release rates, apoptosis rate kcdv and virus degradation rates, were in the same order of magnitude as determined previously for iav replication models proposed for adherent mdck cells (möhler et al., ; schulze-horsel et al., ; heldt et al., ) . at first, mdck suspension cells were seeded and grown in batch mode simultaneously in all three bioreactors cb, vb and vb (figure ) . cb was set to a working volume of ml and cells were grown in batch mode until a cell concentration of . · cells/ml (viability . %) was reached. the infected vessels vb and vb had a working volume of ml and ml, respectively. vb and vb were seeded at a similar concentration, however, they reached . · cells/ml and . · cells/ml at time of infection, respectively. the infection of vb and vb took place at h of culture at moi of . and left in batch mode for another . h before initiating the continuous mode (to avoid virus washout). the vb and vb vessels were operated at an rt of and h, respectively. those rts were adjusted using a constant feed of ml/h of cell broth from the cb and of . ml/h fresh medium into the virus bioreactors. furthermore (to maintain wv), . ml/h were harvested from each vbs. the average concentration of target cells transferred from the cb to both vbs t in was . · cells/ml. upon parameter estimation, we simulated the cell population dynamics in the virus vessels, which showed oscillations for uninfected target cells and the various infected cell populations for both rt and rt h (figure ) . although the total cell concentration in vbs remained stable with an average of . · cells/ml (standard deviation ± . · cells/ml) for rt and . · cells/ml (standard deviation ± . · cells/ml) for rt h, a tendency toward cyclic behavior was observed (figures a,b) . this is linked to the simulated dynamics of the various cell populations caused by virus propagation. as expected, addition of the virus seed causes a fast increase of stv-only infected cells, followed by a strong drop in their concentration and a simultaneous increase of the co-infected cell population through superinfection by dips (figures c,d) . the co-infected cell population reaches its maxima at ∼ days p.i. and days p.i., for rt and h, respectively. since co-infected cells release mainly dips, uninfected target cells fed into the vbs will be infected by dip only. while the dip-only infected cell population reaches a peak concentration at ∼ days p.i. (rt h) and days p.i. (rt h), the population of co-infected cells undergoes virusinduced apoptosis and ceases to a minimum (figures c,d) . however, the number of co-infected cells rises again quickly, since previously dip-only infected cells are now superinfected by stvs produced by the small sub-population of stv-only infected cells. this behavior is repeated for another to . cycles within the cultivation time. thereby, all subpopulations of infected cells reach their peak concentrations repeatedly, which are similar in both vbs and, thus, seem independent of the rt. overall, the variation in the measured cell concentrations in the vbs did not follow the oscillating trend described for similar experiments by frensing et al. ( ) using the avian age .cr cell line infected with iav (a/puerto rico/ / , moi . ) for a rt of h. indeed, there are qualitative and quantitative discrepancies between model simulation and measurement of the total cell concentrations. on the one hand, counting of mdck suspension cells required the addition of trypsin for dissociation of cell aggregates, which occurred occasionally and increased the risk of outliers in cell concentration measurements. on the other hand, our model is not as well-informed on the dynamics of the various infected cell subpopulations since measurements focused on viral dynamics (see following section viral dynamics). to better resolve this issue, follow-up studies are planned to investigate in detail the dynamics of the various cell populations using flow cytometry. ideally, this would not only involve the conventional monitoring of infected cells using iav-specific antibodies , but the use of fluorescence markers for specific intracellular labeling of di rnas, which are currently not available, but under development and will allow to distinguish co-infected from stv-only infected cells. the reverse transcription-qpcr measurements that enabled monitoring of both the fl s and di s (a/pr/ / -dels ( )) copy numbers together with the concentrations of the total number of virus particles (ha titer) and tcid titer (figures , ) , allowed discrimination of the various virus subpopulations. using these values, we calculated the concentration of virions containing either fl or di s based on the qpcr measurement given in "copies/ml" to "virions/ml" (see equations and ). overall, viral titers of fl and di s -containing virions showed the same trend for the two rts tested over the first days of culture (figure ) . maximum concentrations of fl s -containing virions with . · virions/ml and . · virions/ml were reached at ∼ day p.i. for rt of and h, respectively (figures a,b) . for rt h, titers slowly decreased to about virions/ml at ∼ days p.i. for the higher rt, the decrease in titers of fl s virions was delayed compared to rt and reached its minimum of about virions/ml at ∼ days p.i. at that time, the vb with rt h already reached its second peak, followed by a decrease in titers leading to a second minimum at about days p.i. the second minimum for rt h was reached once more about days later. the concentrations of di s -containing virions showed an initial delay in cycles of about days compared to their corresponding fl counterparts and reached their first maxima with . · virions/ml ( days p.i.) and . · virions/ml ( days p.i.) for rt and h, respectively (figures c,d) . the cyclic trend observed in both data sets is described clearly by the model fits. note, that some quantitative discrepancies between data and model simulation remain, such as underestimation of the fl s -containing virus titers for various time points (figures a,b) . although, measured and simulated virus titers show some discrepancies for these individual runs, simulations are mainly within the error range of the assays for ha (about ± . log) and tcid (about ± . log). regarding the error for the number of virions containing either fl or di s , it has to be considered that those are derived from both qpcr measurements (relative standard deviation of about %) and from ha titers (equations and ) . nevertheless, further investigations (e.g., next-generation deep sequencing) should be performed to elucidate those discrepancies in more detail. time courses for infectious virions v s measured by the tcid assay and for the sum of all viral subpopulations determined by the ha assay (v s + v d + v ddi ) also showed pronounced oscillations (figure ) . furthermore, the ha data indicate a trend for an overall decrease in the amplitude of titers toward later cultivation time points for both rts (figures c,d) . the tcid titers reached their maxima with the initial peaks, while the following peak titers were lower. however, no overall decrease in peak infectious titers was observed (figures a,b) . maximum tcid titers were achieved around days p.i. with . · virions/ml and . · virions/ml for rt and h, respectively. most likely, the higher infectious titer for the longer rt of h was related to the reduced wash-out (lower dilution rate) compared to rt h. in total, three cycles were observed for the short rt with a second and a third peak of ∼ · virions/ml at and days p.i., respectively (figure a) . a similar behavior was observed for rt h, however, only about . cycles were achieved during the cultivation time ( figure b) . while the tcid dynamics are captured well both quantitatively and qualitatively, simulated and measured ha values deviate to a certain extent (but within the assay error range) for data points after about days p.i. (figures c,d) . in particular, the decreasing trend of peak ha titers is not reproduced by the model (also in case the system is simulated for days p.i., data sheet ; figure s ). whether the decreasing trend of peak ha titers is an individual characteristic of these particular runs or if the model simulations still lie within the biological variation of these experiments needs to be addressed when more experiments become available. since the overall goal of this study was to establish and fit a basic model with a minimum number of parameters and simple kinetics, we decided to keep the model structure for now. likely, also more detailed studies regarding the dynamics of the individual cell populations will allow further improvement of the model (see discussion on cellular dynamics). as expected, oscillations were also observed in the dynamics of the dip to stv ratios (figure ) . in both cultivations, the ratio reached a maximum of about at ∼ days p.i. these maxima are similar for both rts and correlate with the lowest tcid titer. we may hypothesize that one infectious stv per dips is a critical ratio at which dips cause self-interference and start to hamper overall virus replication significantly. the impact of too high dip to stv ratios and related self-interference is supported by a modeling study of our group (laske et al., ) . as a consequence of self-interference, dip concentrations decrease, reaching a minimum around days p.i., and numbers of infectious stvs are rising again, initiating a new cycle (see also figures , ) . it would be interesting to test whether this critical ratio can be reproduced in other experiments of continuous two-stage cultivations. after fitting the present model ("model ") to both data sets, we tested if it could still describe the data if certain parameters were removed. first, we set the parameter for inactivation of v s (kdvit) and the lysis rate of virions (kvdt) to zero and re-fitted the model ("model "). for the data set of rt h, the goodness of fit was similar to the full model (model ), both visually ( figure ) and quantitatively as based on the objective function values ( table ) . this was expected, since both parameters, kdvit and kvdt, were already close to zero in model . for the second data set with rt h, the re-fitting with model resulted in a noticeable increase of the objective function value (table ). in addition, model was unable to describe the data qualitatively. in particular, the number of cycles in the viral dynamics could not be reproduced any more (figure ) . most likely this points to an important feature of cultivations with long rts, for which degradation and inactivation processes have to be taken explicitly into account to adequately describe the data. in contrast, in cultivations with short rts (higher dilution rates), the overall impact of the washout of virions on viral dynamics was probably higher than that of inactivation and degradation processes. next, we tested whether it is essential to account for separate specific rates of infection, for either stv (kvi) or dip infection (kvidi). for this, we used model and defined one joint infection rate (kvi in "model "). upon fitting model to the data of rt h, the overall dynamics were still described well. however, the model overestimated the tcid titer while underestimating the number of fl and di s -containing virions as well as the overall number of virions produced (figure ) . we observed a similar effect for the rt h data set, combined with a lower number of cycles compared to the fit with model (figure ) . this suggests that two separate infection rates are needed to achieve good agreement with experimental data. although, visually, goodness of fit with model in both data sets seems better than that of model , the objective function value has decreased only by % or by % compared to that of model , for the rt h ( table ) and rt h ( table ) , respectively. at a first glance, this decrease in the objective function value seems counterintuitive, however, since model still has a very good agreement with a majority of measurements for some of the state variables, e.g., dip concentration (figure d) , the objective function value might be comparable to that of model . while fitting oscillating data sets, we experienced that the objective function value can be misleading for some parameter regimes and that, for instance, optimizers may also yield "a good quantitative fit" by a straight line through the mean of the experimental data. finally, we also tested a fourth version of the model, which consisted of a combination of model and model , i.e., neglecting virus degradation processes and using a joint infection rate, simultaneously. in case of rt h, the model followed the same dynamics as model (figure ) . this was expected, since the analysis of model already showed that degradation processes are not highly relevant for rt h. thus, model and model are mechanistically identical, which also leads to similar parametrizations of these two models ( table ) . for rt h, the dynamics of model were similar to model , showing fewer cycles due to exclusion of virus inactivation and degradation processes (figure ) . in addition, model showed quantitative deviations similar to those of model . together, this underlines the importance of virus inactivation and degradation processes as well as the separation of dip and stv infection rates for rt h, which have to be taken into account to capture the experimental data. most likely the difference in kvi and kvidi is related to the underlying mass action kinetics of the model. since the dip concentration is, on average, about two orders of magnitude higher than the stv concentration, the model estimates a lower specific dip infection rate to yield an adequate amount of diponly and co-infected cells, and a high dip titer in the supernatant. whether this has any biological background still needs to be addressed experimentally. ideally, models including parametrization should allow the prediction of viral dynamics for different rt, e.g., to perform model-based process optimization. however, due to some mechanisms that seem dependent on the rt (explained above) none of the current model-parameter-combinations was able to predict viral dynamics for the other rt and vice versa (see also data sheet ; figures s , s ) . accordingly, we think that the model might still lack certain aspects or kinetics and therefore has room for further model extensions. for instance, including the eclipse phase, i.e., the time delay between virus infection and release of viral progeny, or taking into account the accumulation of other di rnas, except for a/pr/ / -dels ( ), might help to further improve model fits and enable predictions (see data sheet ; figure s ). in the present study, we introduce a two-stage continuous bioreactor setup for head-to-head comparison of iav dip production under different culture conditions. for infection, we used a virus seed generated by reverse genetics, which contained a known dip, a/pr/ / -dels ( ), which enabled the monitoring of di and fl virus replication based on qpcr measurements. we observed oscillations in viral titers, where the frequency was depending on the rt. i.e., an increase in the rt from to h caused a shift in cycles of about days. pcr analysis of iav segments - revealed that changes in the rt might also result in the accumulation of different dip subpopulations in long-term cultures. the mathematical model established allowed to describe the time courses of the various viral and cellular subpopulations. comparison of model fits to the two data sets obtained suggests that for the longer rt of h, inactivation of infectious stvs and degradation of virions has to be taken into account. interestingly, the goodness of fit was also affected by the additional assumption that dips and stvs infect cells at different rates. still, we observed some discrepancies between model and experimental data and found that the predictive power of the model needs improvement. this may be related to the fact that the model is still not fully informed, e.g., regarding the dynamics on the different infected cell subpopulations and the de novo synthesis and impact of other dips except for a/pr/ / -dels ( ). in addition, the model might be extended further by accounting for the eclipse phase of virus release. the use of reporter viruses and specific probes for di rna staining may help to elucidate those mechanisms in more detail. for parameter estimation, we have used the data set of only one parallel run and are, therefore, unable to evaluate goodness of fit with respect to the biological variation of these cultivations. this will be addressed in the future, when more cultivations performed at similar experimental conditions become available. in summary, the continuous cultivation system established is an excellent tool for detailed studies regarding dip replication in animal cells. based on the high dip concentrations obtained it also qualifies as a system for production of dips for animal trials and influenza antiviral therapy. all datasets generated for this study are included in the manuscript/supplementary files. ft, tl, mw, mr, yg, and ur contributed conception and design of the study. ft performed the experiments. tl, mr, and ur performed the mathematical analysis. ur wrote the first drafts of the manuscript. ft, tl, mw, yg, and ur wrote sections of the manuscript. all authors contributed to manuscript revision, read, and approved the submitted version. technologies for making new vaccines mechanism of interference by defective-interfering particles of influenza virus: differential reduction of intracellular synthesis of specific polymerase proteins spatial-temporal patterns of viral amplification and interference initiated by a single infected cell high-throughput single-cell kinetics of virus infections in the presence of defective interfering 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importance for contemporary pandemic response strategies visualizing infection spread: dualcolor fluorescent reporting of virus-host interactions chapter five -modified vaccinia virus ankara: history, value in basic research, and current perspectives for vaccine development propagation of the pr strain of influenza a virus in chick embryos. iii. properties of the incomplete virus produced in serial passages of undiluted virus cell culture-based production of defective interfering particles for influenza antiviral therapy generation of a purely clonal defective interfering influenza virus dual-functional peptide with defective interfering genes effectively protects mice against avian and seasonal influenza the authors would like to thank nancy wynserski for excellent technical assistance. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fbioe. . /full#supplementary-material key: cord- -rbp p s authors: zhou, ling; sun, yuan; lan, tian; wu, ruiting; chen, junwei; wu, zixian; xie, qingmei; zhang, xiangbin; ma, jingyun title: retrospective detection and phylogenetic analysis of swine acute diarrhoea syndrome coronavirus in pigs in southern china date: - - journal: transbound emerg dis doi: . /tbed. sha: doc_id: cord_uid: rbp p s swine acute diarrhoea syndrome coronavirus (sads‐cov), a novel coronavirus, was first discovered in southern china in january and caused a large scale outbreak of fatal diarrheal disease in piglets. here, we conducted a retrospective investigation of samples from swine farms with a clinical history of diarrheal disease to evaluate the emergence and the distribution of sads‐cov in pigs in china. our results suggest that sads‐cov has emerged in china at least since august . meanwhile, we detected a prevalence of sads‐cov ( . %), porcine deltacoronavirus ( . %), porcine epidemic diarrhoea virus (pedv) ( . %), rotavirus ( . %), and transmissible gastroenteritis virus ( %), and we also found the co‐infection of sads‐cov and pedv occurred most frequently with the rate of . %. we screened and obtained two new complete genomes, five n and five s genes of sads‐cov. phylogenetic analysis based on these sequences revealed that all sads‐cov sequences in this study clustered with previously reported sads‐cov strains to form a well defined branch that grouped with the bat coronavirus hku strains. this study is the first retrospective investigation for sads‐cov and provides the epidemiological information of this new virus in china, which highlights the urgency to develop effective measures to control sads‐cov. swine acute diarrhoea syndrome coronavirus (sads-cov) is a newly discovered coronavirus which is an enveloped, positive and singlestranded sense rna virus with a genome size of approximately kb (gong et al., ; pan et al., ; zhou et al., ) . sads-cov belongs to the family coronaviridae which contains four genera, alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus (woo, huang, lau, & yuen, ; woo et al., ) . so far, six coronaviruses have been identified from pigs, which include porcine epidemic diarrhoea virus (pedv), porcine respiratory coronavirus (prcv), sads-cov and transmissible gastroenteritis virus (tgev) that all belong to the alphacoronavirus genus, as well as one betacoronavirus, porcine hemagglutinating encephalomyelitis virus (phev) and one deltacoronavirus, porcine deltacoronavirus (pdcov) (lin, saif, marthaler, & wang, ; wesley, woods, & cheung, ; woo et al., ) . among these viruses, sads-cov is the most newly discovered coronavirus, which has been first reported in in china and is considered to be an hku -related coronavirus with a bat-origin (gong et al., ; zhou et al., ) . in january , sads-cov was detected in a swine farm and subsequently spread rapidly to three other farms in *these authors contributed equally to this work. guangdong province and caused the fatal swine acute diarrhoea syndrome (sads) characterized by the clinical signs with severe, acute diarrhoea and rapid weight loss of piglets. the symptoms of sads-cov are similar to those that caused by other swine enteric coronaviruses such as pdcov and pedv, but sads-cov is more harmful than these viruses because it has led to the death of almost , piglets in a short time and resulted in more significant economic losses (dong et al., ; sun, wang, wei, chen, & feng, ; zhou et al., ) . so, it is urgent to investigate the molecular epidemiology and transmission patterns of sads-cov for establishing effective controls for this new coronavirus. in the present study, we performed the retrospective pcr testing on diarrheal samples from swine farms in guangdong province to evaluate the emergence and the distribution of sads-cov in pigs in china. the prevalence and co-infection information of sads-cov from eleven sads-cov-positive farms was provided. the sequences of sads-cov, including two complete genomes, five nucleocapsid protein (n) genes and five spike protein (s) genes, were also identified and characterized to investigate the phylogenetic relationships of sads-cov. samples were homogenized in phosphate-buffered saline (pbs) ( % w/v), frozen and thawed three times, then centrifuged for min at , g. viral nucleic acid was extracted following the manufacturer's recommendations of axyprep tm body fluid viral dna/rna miniprep kit (axygen scientific, inc). the virus nucleic acid was stored at − °c until pcr was performed. a pair of primers (forward primer ′-ggtccctgtgaccgaagttttag- ′, reverse primer ′-gcgttctgcgataaagcttaaaactatta- ′) was designed to detected sads-cov based on the conserved n gene of this virus. one step rt-pcr using primescript ™ one step rt-pcr kit ver. with dye plus (takara, biotechnology, dalian, china) was carried out to amplify the target fragments by the following thermal profile of °c for min, °c for min, cycles of denaturation at °c for s, annealing at °c for s, an extension at °c for s, and a final step of °c for min. four other diarrheal pathogens including pedv, pdcov, rotavirus (rv), and tgev from sads-covpositive farms were also tested by rt-pcr according to the previously described methods (liu, zhu, liao, xu, & zhou, ; mai et al., ; stevenson et al., ) . specific primer pairs based on reported sads-cov strains (genbank accession numbers: mf -mf ) were designed for s genes, n genes and complete genome amplifications, respectively (table s ). pcr assays were performed with the following thermal profile: °c for min, cycles of °c for s, °c for s, and °c for min s, followed by a final min extension at °c. the products were purified following the manufacturer's the nucleotide sequences were assembled and aligned using the dnastar program (dnastar v . , madison, wi, usa). phylogenetic trees were constructed using the neighbour-joining method in mega . software with bootstrap analysis of , replicates. percentages of replicate trees in which the associated taxa clustered are shown as nearby branches (chenna et al., ; kumar, stecher, & tamura, ; tamura, nei, & kumar, ). the two complete genomes (accession number mg and f i g u r e phylogenetic analysis of the s genes of sads-cov and reference coronavirus species. the tree was constructed as per figure above. five new sequences of s genes studied in this work were indicated with "black solid circles" diarrhoea samples. our results showed that the first sads-cov positive sample was collected in august from the farm ls with a history of diarrhoea, as well as from other two farms tp and zw, which indicates that sads-cov has emerged in pigs in china at least since august . and this time point is months earlier than the first discovered time reported by our previous study (zhou et al., ) . as the same time, clinical signs of sads-cov during the retrospective investigation included sever and acute vomiting and diarrhoea, leading to death in piglets that were less than days of age with a mortality rate of around %. these clinical presentations were similar to those signs in the large scale outbreak of sads-cov reported by zhou et al. ( ) , except the mortality rate in piglets later increased to %. based on the rates of infection documented in our work, it revealed that pedv ( . %) was still the primary cause of the porcine diarrhoea, which is consistent with previous studies that pedv has been considered to be the major pathogen responsible for the porcine diarrhoea epidemic in china since (ge et al., ; sun et al., ; zhao et al., ) . the phylogenetic relationships of sads-cov sequences were also identified in this study. the results showed that all sads-cov sequences clustered together to form an independent branch and separated from other viral sequences in the genus alphacoronavirus. our results also indicated that both the complete genomes, n genes and s genes of all sads-cov strains shared the highest nucleotides identifies with those corresponding sequences of four bat coronavirus hku strains. in this work, the phylogenetic trees of full length genomes and s genes of sads-cov sequences showed that the sads-cov branch clustered with these four hku strains, which is same to previous results (gong et al., ; pan et al., ; zhou et al., ) . besides the genomes and s genes, the tree of n genes in our study revealed the identical result too. so far, a total of eight full-length genomes of sads-cov have been reported in guangdong province of china (gong et al., ; pan et al., ; zhou et al., ; this study). the two new genomes of sads-cov sequences in this work shared % nucleotides identities with the sequence mf published by gong et al. ( ) and our four previously reported sequences (zhou et al., ) , and shared . % nucleotides identities with the sequence mf studied by pan et al. ( ) . the results suggest that these eight sads-cov sequences may come from the same origin. only the phylogenetic tree of s genes in our work showed that sequences of the alphacov were divided into two sublineages, alphacov which contained all sads-cov sequences and alphacov , clustering together with sequences of the beltacov and the delatcov, respectively. and this result was consistent with the study of pan et al. ( ) . as a newly discovered coronavirus, the availability of sads-cov sequences data is limited which prevents better understandings of the molecular epidemiology of this virus. meanwhile, being a rna virus, sads-cov may mutate rapidly and exhibit high genetic differences (drummond, pybus, rambaut, forsberg, & rodrigo, ; kühnert, wu, & drummond, the authors declare no conflict of interests with any organization. ma https://orcid.org/ - - - x infectious diseases, , - . https://doi.org/ . /eid . porcine enteric alphacoronavirus gds | mf rhinolophus bat coronavirus hku ch/gd- / /p | mf bat coronavirus hku | nc bat coronavirus hku strain hku /hk/ bat coronavirus hku strain hku /hk/ human coronavirus e | nc camel alphacoronavirus isolate riyadh/ry / | nc e-related bat coronavirus strain btky e- | ky porcine enteric alphacoronavirus gds | mf rhinolophus bat coronavirus hku ch/gd- / /p | mf bat coronavirus hku strain hku /hk/ bat coronavirus hku strain hku /hk/ bat coronavirus a | nc bat coronavirus hku strain afcd | eu rousettus bat coronavirus hku | nc porcine epidemic diarrhoea virus strain gds | km porcine epidemic diarrhoea virus strain cv | kt porcine epidemic diarrhoea virus| nc human coronavirus nl | nc . nl -related bat coronavirus strain btkynl - a | nc e-related bat coronavirus strain btky e- | ky camel alphacoronavirus isolate riyadh/ry / | nc human coronavirus e | nc porcine hemagglutinating encephalomyelitis virus | nc human coronavirus oc strain sc | ky mouse hepatitis virus strain mhv-a c mutant | nc murine hepatitis virus strain jhm complete genome | ac middle east respiratory syndrome coronavirus | nc bat sars coronavirus hku - | dq bat sars-like coronavirus rsshc | kc bat sars-like coronavirus wiv | kf european turkey coronavirus d | kr bulbul coronavirus hku - | fj porcine deltacoronavirus isolate pdcov/chjxni / | kr . porcine coronavirus hku strain hku - | nc rhinolophus bat coronavirus hku ch/gd- / /p | mf bat coronavirus hku strain hku /hk/ bat coronavirus hku strain hku /hk/ human coronavirus oc strain sc | ky porcine hemagglutinating encephalomyelitis virus | nc mouse hepatitis virus strain mhv-a c mutant | nc murine hepatitis virus strain jhm complete genome | ac middle east respiratory syndrome coronavirus | nc bat sars coronavirus hku - | dq . sars coronavirus | nc bat sars-like coronavirus rsshc | kc bat sars-like coronavirus wiv | kf european turkey coronavirus d | kr porcine deltacoronavirus isolate pdcov/chjxni / | kr . porcine coronavirus hku strain hku - | nc bulbul coronavirus hku - | fj bat coronavirus hku strain afcd | eu bat coronavirus a | nc camel alphacoronavirus isolate riyadh/ry / | nc multiple sequence alignmentwith the clustal series of programs porcine deltacoronavirus in mainland china measurably evolving populations epidemiological survey of porcine epidemic diarrhea virus in swine farms in a new bat-hku -like coronavirus in swine phylogenetic and epidemic modeling of rapidly evolving infectious diseases mega : molecular evolutionary genetics analysis version . for bigger datasets evolution, antigenicity and pathogenicity of global porcine epidemic diarrhea virus strains the porcine microrna transcriptome response to transmissible gastroenteritis virus infection the detection and phylogenetic analysis of porcine deltacoronavirus from guangdong province in southern china discovery of a novel swine enteric alphacoronavirus (sea-cov) in southern china emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences outbreak of porcine epidemic diarrhea in suckling piglets epidemiology and vaccine of porcine epidemic diarrhea virus in china: a mini-review prospects for inferring very large phylogenies by using the neighbor-joining method genetic analysis of porcine respiratory coronavirus, an attenuated variant of transmissible gastroenteritis virus coronavirus genomics and bioinformatics analysis discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus the rate of co-infection for piglet diarrhea viruses in china and the genetic characterization of porcine epidemic diarrhea virus and porcine kobuvirus fatal swine acute diarrhea syndrome caused by an hku -related coronavirus of bat origin key: cord- -h smg w authors: takano, tomomi; yanai, yoshitomo; hiramatsu, kanae; doki, tomoyoshi; hohdatsu, tsutomu title: novel single-stranded, circular dna virus identified in cats in japan date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: h smg w we detected a novel feline stool-associated circular dna virus (fescv) in fecal samples from cats with diarrhea using consensus primers matching those of circovirus and cyclovirus. fescv is a circular dna virus containing a genome with a total length of , nt encoding open reading frames. phylogenetic analyses indicated that fescv is classified into a clade different from that of circovirus and cyclovirus. since the fescvs detected in several cats in the same household had genetically similar genomes, these viruses are most likely derived from the same origin. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. rcr iii) are commonly present [ ] . circoviruses include porcine circovirus , which causes post weaning multisystemic wasting syndrome in pigs [ ] , beak and feather disease (bfd) virus, which causes bfd in psittacines [ ] , and canine circovirus (cacv), which causes vasculitis and gastroenteritis in dogs [ ] . cycloviruses include human cyclovirus, which is associated with human acute flaccid paralysis [ ] , and bat cyclovirus isolated from bat feces [ ] . in cats, no circoviridae family other than feline cyclovirus has been reported. feline cyclovirus was identified by next-generation sequencing analysis in which the viral gene was detected in a pooled fecal sample collected from - healthy cats. its pathogenicity in cats has not been clarified [ ] . in this study, we performed nested pcr using circoviridae family consensus primers and detected novel cress dna viruses in several cats with diarrhea symptoms. the full genomic sequences of these viruses were clarified, and a phylogenetic analysis was performed. fecal samples were collected from cats at a private cattery in japan. these cats were maintained under conditions in which they had contact with each other. fourteen of the cats developed diarrhea and did not respond to anthelmintics or antibiotics. the remaining cats were healthy. the cats with diarrhea were separated from the other cats, and newly-collected feces were subjected to experiments. feline coronavirus was detected in fecal samples from healthy cats and cats with diarrhea. in addition, feline bocavirus was detected in fecal samples from cats with diarrhea ( we named the virus detected in this study feline stoolassociated circular virus (fescv). to determine the complete genomic sequence of fescv, inverse primers (cv f '-ttc tcc cga cct gga cat ag and cv r '-aca gag atg ata gcg tcc gg) were prepared based on a section of the base sequence of the acquired rep gene, and inverse pcr was performed. fig. s shows the schematic position of the inverse pcr primers and other primers used for the fescv genome. prior to inverse pcr, circular dna was multiplied using templiphi tm amplification kit (ge healthcare, usa). viral dna was amplified by inverse pcr, which was performed in a total volume of μl using the following mixture: μl of multiplied circular dna mixed with μl of x primestar buffer (takara, japan), μl of dntp mixture (takara, japan) containing . mm of each dntp, μl of μm primer mix (fc f), . μl of primestar hs dna polymerase ( . u/ml; takara, japan), and . μl of distilled water. using a thermal cycler, the dna was amplified at °c for min followed by cycles of denaturation at °c for sec, primer annealing at °c for sec, and synthesis at °c for min with a final extension at °c for min. from all samples, approximately , -bp amplicons were acquired. the sequences of the viral genomes were obtained by sequencing the overlapping pcr products (amplicons of and , bp in size). phylogenetic trees based on rep were analyzed using mega software (version ). phylogenetic relationships were evaluated using the neighbor-joining algorithm, and branching order reliability was analyzed by , replications of bootstrap resampling. all strains of fescv were circular dna viruses containing a genome (fig. b) . of the viruses that infect eukaryotic organisms, those most similar to fescv were rodent stool-associated circular viruses (rodscv). of note, the putative rep and putative cap of fescv were approximately % homologous with rna helicase and the hypothetical protein of giardia intestinalis, respectively. the fescv genomes were submitted to genbank under accession numbers lc (ku ), lc (ku ), lc (ku ), and lc (ku ), respectively. based on the base sequence of fescv clarified in this study, specific primers to detect the fescv gene (fescv f '-gct aag gtc tgc ctc agg tg and fescv r '-cta tgt cca ggt cgg gag aa) were prepared. pcr was performed as described above except the reaction temperature and time were modified as follows: dna was amplified at °c for min followed by cycles of denaturation at °c for sec, primer annealing at °c for sec, and synthesis at °c for sec with a final extension at °c for min. when an amplicon with the expected size of bp was detected, the sample was regarded as fescv-positive. of the cats with diarrhea, were fescv-positive ( . %). three of the healthy cats were fescv-positive ( . %) ( table ; see fescv-specific primer). to detect feline circovirus or feline cyclovirus in feline fecal samples, nested pcr using circoviridae consensus primers was performed. the degenerate primers used in table highly conserved motifs of rep detected in fescv based on the report by rosario et al. [ ] x: any amino acid u: hydrophobic amino acid (f, i, l, v, or m) this nested pcr assay were prepared based on the consensus sequence of the rep gene in strains of circoviruses and one strain of cyclovirus [ ] since we expected only circoviruses and cycloviruses to be detected. however, a novel cress dna virus, fescv, was detected in the fecal samples. phylogenetic analysis based on rep revealed that fescv belongs in a clade different from that of circoviruses and cycloviruses for which the reason is unclear. very few known cress dna viruses, including circoviruses, have been confirmed to have pathogenicity in the host. moreover, many studies on novel cress dna viruses analyzed only viral genes, that is, only a few studies analyzed whether the viral infection is manifested in the host. the fescv found in this study was detected in of the cats. based on this result, cats may be the natural host of fescv. furthermore, the fescvs (ku strain, ku strain, ku strain, and ku strain) are genetically similar and share approximately % of their genomes. this data suggests that these fescvs have the same origin. the fescv infection rates in healthy cats and cats with diarrhea were . and . %, respectively and demonstrate that although the infection rate was slightly higher in cats with diarrhea, half of the healthy cats were also infected with fescv. thus, it is unclear whether fescv is pathogenic. furthermore, the rate of co-infection with fescv and other viruses was approximately -fold higher in cats with diarrhea ( . %) than in healthy cats ( . %). in dogs, a relationship between cacv and gastroenteritis has been suggested. dowgier et al. [ ] reported that cacv infection and the development of acute gastroenteritis were correlated in dogs co-infected with other enteric viruses. the incidence of enteritis may increase when fescv-infected cats are co-infected with other viruses. further investigation of fescv pathogenicity in cats is necessary. the putative rep of fescv was % homologous with rna helicase of giardia intestinalis in blastx analysis, and the putative cap of fescv was approximately % homologous with a cysteine protease (hypothetical protein) of giardia intestinalis. we first suspected that the fescv gene identified in this study was derived from giardia intestinalis. however, we concluded that fescv is a circular dna virus based on the following: ) no giardia intestinalis was detected in the fecal test, and ) the complete genome of fescv was amplified using the rolling-circle amplification and inverse pcr assays. similar with fescv, rodscv is reported to have rep with < % homology with rna helicase of giardia intestinalis [ ] . moreover, both the putative rep and putative cap of fescv are similar to proteins derived from giardia intestinalis. to our knowledge, no study has reported a gene encoding cap or putative cap of cress dna virus generated by recombination with parasitic or bacterial genes. further investigation is necessary for the origin of the putative cap of fescv. a region presumed to be a classical nls was detected in the putative cap of fescv. as circoviruses lack dna polymerase, virus particles have to transfer into the nucleus for viral dna replication [ ] . in bfdv and duck circoviruses, viral dna may by transported into the host cell nucleus via nls-containing viral cap [ , ] . it is unclear whether the putative cap of fescv functions similar to the cap of bfdv and duck circoviruses. further studies regarding the intracellular localization of fescv putative cap is required. we detected a novel cress dna virus, fescv, in fecal samples from cats. although it was detected using consensus primers of circovirus and cyclovirus, fescv was phylogenetically positioned in a clade different from that of these viruses. fescv was detected in several cats that were housed together, suggesting that fescvs are derived from the same origin. in addition, fescv is considered to cause diarrhea in cats via co-infection with other enteric viruses. therefore, it is necessary to perform a large-scale epidemiological survey and clarify whether diarrhea is associated with fescv infection. consensus statement: virus taxonomy in the age of metagenomics time-dependent rate phenomenon in viruses characterization of the genomic sequence of a novel cress dna virus identified in eurasian jay (garrulus glandarius) identification and genetic characterization of a novel circular singlestranded dna virus in a human upper respiratory tract sample plasma virome of cattle from forest region revealed diverse small circular ssdna viral genomes pervasive chimerism in the replication-associated proteins of uncultured singlestranded dna viruses revisiting the taxonomy of the family circoviridae: establishment of the genus cyclovirus and removal of the genus gyrovirus molecular evolution of porcine circovirus type genomes: phylogeny and clonality evidence for specificity of psittacine beak and feather disease viruses among avian hosts circovirus in tissues of dogs with vasculitis and hemorrhage novel cyclovirus in human cerebrospinal fluid bat guano virome: predominance of dietary viruses from insects and plants plus novel mammalian viruses faecal virome of cats in an animal shelter multiple diverse circoviruses infect farm animals and are commonly found in human and chimpanzee feces a molecular survey for selected viral enteropathogens revealed a limited role of canine circovirus in the development of canine acute gastroenteritis the fecal viral flora of wild rodents replication of porcine circoviruses structural determination of importin alpha in complex with beak and feather disease virus capsid nuclear localization signal identification of two functional nuclear localization signals in the capsid protein of duck circovirus acknowledgements this work was in part supported by mext/jsps kakenhi grant number jp k . conflict of interest all authors declare that have no conflict of interest.ethical approval all applicable international, national, and institutional guidelines for the care and use of animals were followed by the authors. samples were obtained from an animal hospital, and this study does not include animal experiments. key: cord- -hn vmic authors: guo, jiahui; fang, liurong; ye, xu; chen, jiyao; xu, shangen; zhu, xinyu; miao, yimin; wang, dang; xiao, shaobo title: evolutionary and genotypic analyses of global porcine epidemic diarrhea virus strains date: - - journal: transbound emerg dis doi: . /tbed. sha: doc_id: cord_uid: hn vmic porcine epidemic diarrhea virus (pedv), which re‐emerged in china in october , has spread rapidly worldwide. detailed analyses of the complete genomes of different pedv strains are essential to understand the relationships among re‐emerging and historic strains worldwide. here, we analysed the complete genomes of strains from different countries, which were classified into five subgroup strains (i.e., gi‐a, gi‐b, gii‐a, gii‐b, and gii‐c). phylogenetic study of different genes in the pedv strains revealed that the newly discovered subgroup gii‐c exhibited inconsistent topologies between the spike gene and other genes. furthermore, recombination analysis indicated that gii‐c viruses evolved from a recombinant virus that acquired the ′ part of the spike gene from the gi‐a subgroup and the remaining genomic regions from the gii‐a subgroup. molecular clock analysis showed that divergence of the gii‐c subgroup spike gene occurred in april , suggesting that the subgroup originated from recombination events before the pedv re‐emergence outbreaks. interestingly, ascaris suum, a large roundworm occurring in pigs, was found to be an unusual pedv host, providing potential support for cross‐host transmission. this study has significant implications for understanding ongoing global pedv outbreaks and will guide future efforts to develop effective preventative measures against pedv. porcine epidemic diarrhoea (ped) is a devastating enteric disease in pigs that results in severe diarrhoea, vomiting, and dehydration, with very high mortality observed in suckling pigs (pensaert & de bouck, ) . porcine epidemic diarrhea virus (pedv), which is the causative agent of ped, belongs to the genus alphacoronavirus within the family coronaviridae and is an enveloped single-stranded positivesense rna virus (woo et al., ) . the pedv genome consists of seven open reading frames (orfs) organized in the order orf a, orf b, spike (s) glycoprotein gene, orf hypothetical protein gene, envelope (e) gene, membrane (m) gene, and nucleocapsid (n) gene . among the proteins encoded by the orfs, the s glycoprotein is located on the envelope of the virus in the large surface projections of the virion and plays an important role in the attachment of viral particles to host cell receptors (lee, park, kim, & lee, ) . thus, the s gene is considered important for understanding the genetic relatedness and epidemiological status of pedv field isolates, as well as for advancing vaccine development (chen, liu, lang, et al., ) . the ped disease was first discovered in pig farms in belgium and the united kingdom in (pensaert & de bouck, ) , with reports of its occurrence in china as early as the s (li et al., ) . with the emergence of new pedv strains, however, serious disease epidemics have been observed in china since october (sun et al., ) . beyond china, the disease has rapidly spread to more than states in the usa following its first outbreak in may , affecting more than , farms accounting for more than million piglets (cima, ) . japan, canada, mexico, and colombia have also experienced successive outbreaks, with considerable economic losses to the global pig industry (lara-romero et al., ; ojkic et al., ; takahashi, okada, & ohshima, ; valko et al., ) . major global outbreaks since have renewed concerns about the potential changes in the mode of pedv transfer (chen, liu, lang, et al., ; li et al., ; sun et al., ) . although increasing evidence suggests that pedv routinely undergoes significant changes, especially in spike proteins (lara-romero et al., ; stott et al., ) , the prevalence and evolution of pedv strains is not well-defined and limited knowledge is known regarding the ways in which pedv subgroups circulate among themselves and how they might influence the evolution of pedv. to better understand the molecular epidemiology and genetic diversity of pedv field isolates, we investigated the genetic characterization, origin, and evolution of emergent pedv strains worldwide, which will provide much needed information for the effective prevention and control of this disease. the complete pedv genome was selected for genetic analysis. to clarify the evolution of pedvs, we obtained four complete genome sequences of pedv from our own lab (i.e., zl , aj , hub - , and hub - ) (bi, zeng, xiao, chen, & fang, ) firstly, we performed multiple sequence alignment of the complete pedv genomes, as well as the orf ab, s, orf -e-m-n genes, and applied the bat coronavirus btcov/ / (genbank accession no. dq ) sequence as an outgroup (tang et al., ) . a maximum-likelihood (ml) phylogenetic tree was constructed using iq-tree v. . . (nguyen, schmidt, von haeseler, & minh, ) , with the best fitting evolutionary model suggested by the program following , bootstrap replicates. the phylogenetic tree was rooted against the pedv-related bat coronavirus, with removal of the long-branch leading to greater resolution of the viruses of interest. nucleotide and deduced amino acid sequences were aligned using mafft v. . (katoh & standley, ) . the resulting tree was visualized using itol v. (interactive tree of life, http://itol.e mbl.de/). we preliminarily screened the pedv sequence data set for recombination using rdp, geneconv, chimaera, maxchi, and seq, followed by secondary scanning and recombination using bootscan and siscan in recombination detection program version. . (rdp v. . ) (martin, murrell, golden, khoosal, & muhire, ) . sequences with significant signals for recombination determined by more than two methods were analysed in greater detail. nucleotide sequence similarity was assessed by simplot v. . . (lole et al., ) , with a sliding window size of bp, step size of nucleotides, and , bootstrap replicates, using gap-stripped alignments and the f (ml) distance model. all data were analysed using graphpad prism software (v. . , san diego, ca, usa). all s protein sequences from the pedv sample strains were analysed using the meta data-driven comparative analysis tool (meta-cats) (pickett et al., ) , with a p-value threshold of . (this threshold f i g u r e genotyping and origin of the pedv strains based on full-length genomic sequence analyses. (a) phylogram was tested by , bootstrap replicates, branch lengths were measured by the number of substitutions per site (see scale bars). names of strains, years, places of isolation, genbank accession numbers, genogroups, and subgroups are shown. (b) line chart shows the number of pedv sequences obtained by gene subgroup and year of sampling. yearly percentages of samples positive for pedv are indicated by different coloured lines respectively. data are indicated below sampling years. (c) subgroup distribution of all available complete or partial pedv genome sequences from countries reporting pedv infections (n.a., sequence not available). in the bar charts, counts are shown by country or region. data are indicated below bar charts [colour figure can be viewed at wileyonlinelibrary.com] was the maximum probability level for the likelihood that the position differed among groups simply by chance), to identify significantly different sites between the five subgroups. to characterize the genetic diversity of pedvs circulating globally, we constructed a phylogenetic tree using iq-tree based on the complete pedv genomes (see materials and methods . ). consistent with our previous research (wang, fang, & xiao, a) , the phylogenetic tree indicated that the complete pedv genomes evolved into two separate genogroups, gi (classical) and gii (variant), as presented in figure a . furthermore, genogroup gi evolved into two subgroups (gi-a and gi-b) and genogroup gii evolved into three subgroups (gii-a, gii-b, and gii-c). the gi-a subgroup mainly included the earlier pedv strains found in europe and belgium (virulent we identified the geographical and temporal distributions of the pedv strains to clarify the evolution of the virus. as shown in figure b , only sporadic outbreaks of pedv were reported before , with the pathogens involved in these outbreaks found within the gi genogroup. the pedvs were primarily located in the gi-a (virulent cv ) and gi-b (attenuated dr ) subgroups and were predominantly from asia (figure b ). however, considerable pedv outbreaks were reported in asia and the united states after , even for vaccinated piglets (lin, saif, marthaler, & wang, ) . based on our examination and assembly of public data, we identified that the pedv strains were primarily from the gii genogroup. interestingly, gii-b subgroup strains were reported predominantly in , whereas gii-a strains were reported more prevalently after and occupied a larger proportion of strains. moreover, reports of the newly discovered gii-c strains showed a significant increase after due to further sequencing from europe. the above results indicate that the epidemic strains from different periods were from the five different subgroups. based on the geographical distribution of pedvs (figure c) , the gi genogroup (classical and cell culture-adapted vaccine strains) largely originated from the earlier pedv-threatened areas, such as china, south korea, and europe. the different subgroups from the gii genogroup also showed characteristic geographical distribution. while most gii-a subgroup strains were from the americas, a small number were from china and japan or from sporadic outbreaks in a few other isolated areas. the gii-b subgroup strains were mostly endemic to asia, especially china, south korea, and japan. the gii-c subgroup strains were primarily from europe, with some from the usa and china. our study showed that pedv strains from different subgroups were prevalent within the same areas, implying that the coincident "hot spots" in pedv-endemic areas (e.g., china and south korea, figure c critical for determining the sources of some pedv variations. these "hot spot" areas have the potential to be important reservoirs for the genetic variation of pedvs, resulting in recombination between different pedv subgroups. we also examined the potential hosts of the pedv strains. results showed an unusual pedv strain (genbank accession no. kx ) hosted by ascaris suum (supporting information table s ) (shi et al., ) , a large roundworm in pigs, thus providing novel insight into the possible epidemiology of pedv infection. indeed, parasites have long been regarded as a harmful factor to the pig industry as sources for a variety of infectious agents (jesudoss chelladurai et al., ) . for example, metastrongylus larvae are considered a reservoir for various porcine viral pathogens, primarily swine fever virus and swine flu virus (sen, kelley, underdahl, & young, ) . however, whether ascaris suum plays a critical role as a pedv reservoir requires further investigation. to further explore the evolution of pedvs, we constructed three phylogenetic trees based on the orf ab, s, and orf -e-m-n gene sequences of the pedv strains. the orf ab and orf -e-m-n gene alignments confirmed that the gii-c subgroup was deeply nested within the gii-a subgroup (figure a and b) . strikingly, the phylogeny of the s gene suggested an entirely different evolutionary history for the gii-c subgroup compared to the other subgroups (figure c ). all gii-c subgroup strains showed inconsistent topology in the s gene phylogenetic tree, differing from the orf ab gene phylogenetic tree. this inconsistent topology, in which outlier sequences were found between two well-defined subgroups in a phylogenetic tree, was attributed to ( %- % genetic identity to the orf ab and orf -e-m-n genes). in contrast, the genetic identity between the s gene sequences of these viruses was only % and showed strong similarity with virulent cv ( % gene identity). in the s gene phylogeny, virulent dr did not cluster with kupe but instead with virulent cv , suggesting that the pedv s gene was subjected to relatively frequent recombination, even between divergent subgroups (figure c and supporting information figure s a ). moreover, the italy/ / pedv strain intragenogroup recombination provides a mechanism for amalgamation among these distinct subgroups and increases the genetic repertoire of co-circulating pedv strains. the s protein attaches to the cellular receptors of a host, resulting in virus entry by membrane fusion, and contains the domain that stimulates the production of neutralizing antibodies. variations in the s protein are important for understanding the genetic relatedness of pedv field strains (chang et al., ; lara-romero et al., ; wang et al., b) . to determine the significant s protein sequences among the five different pedv subgroups, meta-cats analysis was performed for spike protein sequences of all pedv strains. we identified amino acid positions with significant variation among the isolates from the five subgroups (supporting information table s ). as shown in figure , the gii genogroup contained distinct patterns of aa mutations (i t, i t, e q, t s, g s, n s, a v, s a, g d, s r, and r q), distinguishing it from isolates in the gi genogroup. although the gii-c subgroup s genes shared some aa substitutions with the gi-a and gii-a subgroups (two parents of recombination), they also exhibited three unique patterns (l , a/s , and h/t ), which clearly distinguished these isolates from those in the other subgroups, suggesting that the gii-c subgroup s gene evolved gradually through antigenic drift. it is well established that the s protein of coronaviruses induces high levels of neutralizing antibodies. four neutralizing epitopes ( - , - , - , and , - , amino acids) have also been identified in the pedv s protein (chang et al., ) . to explore whether the gii genogroup acquired substitutions in neutralizing epitopes characteristic of the gi genogroup, we mapped the significantly different positions to the equivalent positions in representative sequences (figure ) . results identified seven substitutions in the neutralizing epitopes of the s protein in the gii genogroup (l h, s g, v i, t s, g s, a e, and l f), which may explain why traditional inactivated vaccines and attenuated vaccines against the gi genogroup cannot effectively protect piglets threatened by a pandemic strain from the gii genogroup. in summary, this study revealed the genetic diversity and evolutionary dynamics of pedv strains. our genetic analyses showed that the pedv strains could be categorized into two groups, namely, gi (classical) and gii (variant) . we also discovered a new subgroup (gii-c) with novel genetic, molecular, and phylogenic characteristics. the gii-c subgroup evolved from a recombination event between the gi-a and gii-a subgroups, and we further found recombination in two relatively early strains: virulent dr and italy/ / . these recombination events occurred prior to the re-emergence of pedv in . additionally, to explore the potential link between s protein amino acid sequence variations and recombination, we performed a series of comparative analyses of the pedv s protein sequences. we found positions that were localized in a well-known neutralizing epitope and revealed several unique amino acids that could easily distinguish the different subgroups. this study provides critical information to help trace the sources of pedv variants and identify the evolutionary mechanisms involved. furthermore, this research will hopefully facilitate the development of diagnostic kits, vaccines, and new therapeutic strategies, which are expected to turn the tide in the prevention of pandemic outbreaks of pedv. none. http://orcid.org/ - - - baele, g., lemey, p., rambaut, a., & suchard, m. a. ( ) . adaptive mcmc in bayesian phylogenetics: an application to analyzing partitioned data in beast. bioinformatics, , - . https://doi.org/ . /bioinformatics/btx bi, j., zeng, s., xiao, s., chen, h., & fang, l. 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evolutionary and epidemiological analyses based on spike genes of porcine epidemic diarrhea virus circulating in thailand outbreak of porcine epidemic diarrhea in suckling piglets an outbreak of swine diarrhea of a new-type associated with coronavirus-like particles in japan. nihon juigaku zasshi prevalence and genetic diversity of coronaviruses in bats from china porcine epidemic diarrhoea virus with a recombinant s gene detected in hungary immunogenicity and antigenic relationships among spike proteins of porcine epidemic diarrhea virus subtypes g and g porcine epidemic diarrhea in china discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus key: cord- -z e pmk authors: chen, bin; zheng, zaiyu; yang, jinxian; chi, hongshu; huang, he; gong, hui title: development and characterization of a new cell line derived from european eel anguilla anguilla kidney date: - - journal: biol open doi: . /bio. sha: doc_id: cord_uid: z e pmk a new cell line derived from the kidney of european eel, anguilla anguilla, has been established and characterized. this cell line, designated as ek (eel kidney), has been maintained in leibovitz's l- supplemented with % fetal bovine serum for over months, and subcultured more than times. this cell line consists predominantly of fibroblast-like cells, and can grow at – °c under an optimum temperature of °c. the origin of this cell line was confirmed by polymerase chain reaction (pcr) amplification and s recombinant (r)rna sequencing. the chromosome analysis of ek cells at passage revealed an ananeuploid karyotype. the ek cells were successfully transfected with the pegfp-n plasmid, suggesting its potential in genetic studies. the susceptibility test showed a significant cytopathic effect (cpe) in ek cells for rana grylio virus, and the viral replication was evidenced with quantitative real-time pcr (qrt-pcr) assay. after poly (i:c) stimulation, the expression of the immune-related molecules including interferon regulatory factor- (irf ), interferon regulatory factor- (irf ) and cytochrome p (cyp ) were significantly upregulated in ek cells, while the expression of transforming growth factor (tgf-β) was downregulated. these results suggested the potential of ek cell line as a model in gene engineering, virus identification and environmental toxicology. european eel anguilla anguilla (l. ) was previously considered one of the most important aquaculture species in both europe and china, but is now listed as endangered due to the threats of overfishing, diseases, obstacles, ocean current changes, polychlorinated biphenyl (pcb) pollution, etc. (de boer et al., ; dekker, ; van ginneken, ; hendriks et al., ; knights, ) . both wild and farmed eels have suffered the attack of various viruses for over years, including herpesvirus, picornavirus and coronavirus (fichtner et al., ; ge et al., ge et al., , jakob et al., ; van beurden et al., ; yue et al., ; zhang and gui, ) . fish cell lines play an important role in the studies of aquatic virology, developmental biology, genetics, immunology, physiology, toxicology and pharmacology (baksi and frazier, ; bols, ; kohlpoth et al., ; ni shuilleabhaina et al., ) . since the setup of the first teleost cell line rtg- (wolf and quimby, ) , over fish cell lines have been established (fryer and lannan, ; lakra et al., ) . dozens of viruses have been isolated using fish cell lines, and explorations in emerging fields such as immunological signaling, aquatic oligodynamics, genetic engineering and environmental monitoring have shown enormous prospects (béjar et al., ; bryson et al., ; chen et al., ; dong et al., ; sahul-hameed et al., ; zhang et al., ) . to date, cell lines have been developed from only a few species of anguillidae fishes, or in other words, the anguillidae invitrome is small (bols et al., ) . the first cell lines were developed from the japanese eel, anguilla japonica (temminck and schlegel ) (chen and kou, ; kou et al., ) . more recently two cell lines, pble and eelb, have been described from the american eel, a. rostrata (lesueur ) (bloch et al., ; dewitte-orr et al., ) . however, few cell lines have been reported from the european eel a. anguilla (linnaeus ). in , we initially tried in vitro tissue and cell culture of multiple european eel organs (zheng, ) . in this study, we have developed and characterized a cell line derived from a. anguilla kidney, which proved to be susceptible to rana grylio virus (rgv). the responses of this cell line to regular immune stimulations were also investigated. after h of inoculation, cells were migrated outwards from the tissue explants (fig. a ) and the first subculture was conducted on day . the subculture was performed at a split ratio of : every h, and these cells were subcultured over times to date. the eel kidney (ek) cell line was anchorage-dependent, predominantly made up of fibroblast-like cells (fig. b) and was maintained in l- containing % fetal bovine serum (fbs) at °c. the ek cells recovered from liquid nitrogen storage at the th subculturewhose average viability was estimated to be %- %could reach confluency within days. the ek cells grew into a confluent monolayer at a temperature range between °c and °c, and at °c or °c several small colonies were formed. the maximum growth rate was observed at °c (fig. ) - days after inoculation, and the passage ek cells presented the logarithmic phase with a population doubling time (pdt) of . h. among the metaphase ek cells counted at passage , the chromosome numbers ranged from to (fig. a) . thirty-eight percent of samples presented normal diploid karyotype of n= ; of these, the metacentric, submetacentric and telocentric chromosomes were , and pairs, respectively ( n= m+ sm+ t) (fig. b) ; while the remaining % samples contained of or chromosomes. these results suggested that ek may be an aneuploid cell line. green fluorescence signals could be observed in the ek cells at h after transfection, and lasted for days or longer (fig. a ,b). the transfection efficiency was estimated to be about %. s rrna sequence analysis the species of the ek cell line was confirmed by s rrna gene analysis. an expected, pcr product of bp was obtained using specific amplification of s rrna from the extracted total genomic dna ( fig. s ), which was proved to be % identical to the published a. anguilla s rrna sequence (genbank: fm . ). cytopathic effect was first observed at h after infection, and was covered in over % of the monolayer at h ( fig. b ), while the monolayer in the controls stayed healthy (fig. a ). the qrt-pcr standard curve was plotted using linear-regression analysis according to the sequencing report of the pmd- t-mcp vector: y= − . x+ . , r = . , ≤x≤ (fig. c ), transcripts of mcp were increased significantly in the ek cells from to h after infection with rgv (fig. d ). immune-related gene expression after poly (i: c) exposure after poly (i: c) exposure, the expression of tgf-β transcripts reached a peak at h with an increased ratio of . -fold (p< . ), and then recovered to the initial level. the expressions of irf and irf were significantly upregulated at h post stimulation (p< . ), and showed the maximum increase ratio of . -fold and . -fold at h, respectively (p< . ). the expression of cyp was increased gradually and presented a significant difference at h, while the peak was observed at h with an increased ratio of . -fold (p< . ) (fig. ) . the internal control gene β-actin showed no significant change. ek is the first reported visceral cell line of a.anguilla (fryer and lannan, ; lakra et al., ) . as a european catadromous teleost (schmidt, ) , the artificial reproduction of a. anguilla is still an unresolved question to date, while the wild embryo or neonatal leptocephalus, or even the glass eels, are also extremely difficult to obtain (huang and chen, ) . establishment of new continuous cell lines should benefit the researches and protection of critically endangered (cr) animal. most of the fish cell cultures use mediums developed for mammalian cells, such as dmem, ham's f- , rpmi- , l- , etc. as an amino acid-rich nutrient medium forming a co -free system, l- has been used for successful application on fish cell lines and made co incubators unnecessary, which in turn significantly improved the stability and convenience of cell culture (leibovitz, (leibovitz, , . due to this advantage, more than % of the cell lines established after used leibovitz's l- medium (lakra et al., ) . this experiment has proved that l- was suitable for european eel tissue and cell culture as well, and this conclusion has been also evidenced by another study on pectoral fin cells of a. anguilla (mao et al., ) and the studies on a. rostrata cell lines (bloch et al., ; dewitte-orr et al., ) . fish cell culture has a convenient temperature range wider than mammalian cell culture, and the ek cells were accustomed to a wider temperature window ranging from - °c. the maximum growth rate was observed at °c, but the ek cells under this temperature usually were overgrown in h, and the confluent cell layer would be destructed by contraction and cell detachment in h (fig. s ). the flow cytometer records proved that suspension cells contributed to the total growth significantly at °c or higher temperatures. to meet the needs of regular physiological and pathological experiments, the optimal culture temperature of ek cells was designated °c, which was the same as for the a. japonica cell line ep- (kou et al., ) and the a. rostrata cell line eelb (bloch et al., ) , and simiar to the a. rostrata cell line pble ( °c) (dewitte-orr et al., ) . the appropriate temperature range for a. anguilla in the culture ponds is - °c (huang and chen, ). the karyotype analysis revealed that only % of the passage ek cells possessed the modal diploid chromosome number of n= (yang et al., ) . the ek strain showed the property to form neoplasms in vitro, and its aneuploidy suggested the possibility of immortality (chen et al., ) . this may be further tested by telomerase activity analysis . iridoviruses are common pathogens detected in a. japonica (sorimachi and egusa, ) and other aquaculture species in fujian province (yang, ) . compared with the former test using the eo cell line (ge et al., ) , in which only vesiculation was observed within week, the infection course took much less time on ek cells. the susceptibility of the ek cell line has made it an efficient tool for studying the local viral diseases. in recent years, cell lines have turned out to be viable tools for function analysis of fish innate immune genes (poynter and dewitte-orr, ) . although the rapid responding kinetics of the expression of various immune factors to in vitro or in vivo inflammatory stimulation has been confirmed repeatedly in the teleosts (haddad et al., ; huang et al., ; maehr et al., ; sudhakumari et al., ) , the mechanism of inflammation-induced immune modulation still remained ambiguous. the interferon regulatory factor (irf) family members played critical roles in cellular differentiation of hematopoietic cells, and the regulation of gene expression in response to pathogen-derived danger signals to regulate cell cycle and apoptosis (tamura et al., ) . irf was once reported to be upregulated in both peripheral blood leukocytes and in vivo after poly (i: c) stimulation in a. anguilla (huang et al., ) , and this has been confirmed in this study in association with the changes of irf levels. the cyp gene superfamily consists of a large number of genes encoding p enzymes involved in the detoxification of exogenous chemicals (e.g. drugs, chemical carcinogens, environmental pollutants) and the metabolism of various endogenous substrates (steroids, fatty acids, vitamins, prostanoids, etc.) (uno et al., ) . since the s, multiple forms of cyps have been considered common biomarkers in assessing the contamination of the aquatic environment (bugiak and weber, ; nilsen et al., ; winston et al., ) . in this study, the expression of cyp showed a significantly long-term upregulation after poly (i: c) induction, indicating the potential of ek cell line for toxicological and pharmacological analysis of aquatic pollutants. in summary, a continuous cell line from a. anguilla kidney has been established and showed its potential impact for studying infectious viral diseases of anguillidae fishes and for immune genetic, toxicological and pathological researches, benefitting the protection of a. anguilla. healthy a. anguilla (elvers) with an average weight of g from an eel farm in changle, fujian, china were kept in clean sea water at room temperature ( - °c) during transportation. before dissection, the fish were killed by anesthesia and then disinfected by % iodine tincture and % alcohol three times, respectively. the kidney tissue of the elvers was removed completely, washed five times with . mol l − pbs containing iu ml − of penicillin and μg ml − of streptomycin (sangon) at °c, and then minced thoroughly into pieces (c. mm ). the tissue fragments were rinsed three times with pbs, then attached to the bottom of cm culture flasks (corning), and wetted with serum-free leibovitz' l- (hyclone) at an interval of . cm. the full culture medium included l- supplemented with % fbs (gibco) and antibiotics as mentioned above. every single flask was incubated upside-down at °c for - h first. then ml full culture medium was dripped in before it was overturned to make the tissue explants soaked and ml additional culture medium was added every day till the total volume reached ml. all the flasks were then transferred to °c and the culture medium was half-changed every days. when the radial outgrowths surrounded the tissue explants, the culture was digested with . % trypsin solution (sigma-aldrich) at °c. after centrifugation at rpm for min, the cells were collected and suspended in ml full culture medium, then inoculated into a new . cm flask (bd falcon) incubated at °c until a cell monolayer was formed. when a confluent cell monolayer was observed at °c, the cells were washed and digested by . % trypsin solution for min, then inoculated to new cm flasks with a split ratio of : . from the sixth subculture, the antibiotics were no longer additives, and the concentration of fbs was reduced to % in the eighth subculture (lannan, ) . after h growth in vitro, the ek cells were harvested and re-suspended in l- containing % fbs and % dimethyl sulphoxide (dmso, sigma-aldrich) at a density of cells ml − . the cell suspension was dispensed into . ml cryogenic vials (corning) and kept initially at °c for min, then at − °c overnight, and finally transferred into liquid nitrogen (− °c). thirty days post cryopreservation, the frozen cells were resuscitated in the water bath at °c for min, and then suspended and inoculated into cm cell culture flasks at °c, wherein a full medium-change in h was necessary. the cell viability was evaluated by trypan blue staining (ott, ) . passage ek cells were inoculated to cm flasks ( × cells per flask) and incubated at °c, °c, °c, °c and °c, respectively. three flasks of cells from each group were harvested and counted by a flow cytometer every day until day . the cell pdt was calculated using the below formula (davis, ) : for chromosome analysis, ek cells at passage were transferred into a cm culture flask and kept at °c for h, and then transferred into l- medium containing % fbs and . μg ml − colchicine (sigma-aldrich). after incubation for h, the cells were collected with centrifugation and treated with ml of . % kcl for min. the cells were prefixed for min by dropping ml of carnoy's fixative (methanol:acetic acid= : , °c) into the suspension. after centrifugation at rpm for min, the cell pellets were fixed with ml carnoy's fixative for min. the fixed cells were centrifuged and re-suspended in ml carnoy's fixative, and then incubated at °c overnight. the suspension was dropped on cold glass slides, which were tapped to scatter the samples equally. the slides were then air dried and stained with % giemsa ( ph . ) for h. under a nikon eclipse te -s fluorescence microscope, metaphase cells were photographed and analyzed (levan et al., ) . the pegfp-n plasmid (takara) expressing a green fluorescent protein (gfp) was used for cell transfection. the ek cells at passage were inoculated at a density of × cells well − in a -well plate. after h, the cells were transfected with μg pegfp-n plasmid in μl lipofectamine™ reagent (takara) and incubated at °c for h. the cell culture was then transferred to l- supplemented with % fbs. after h, the fluorescence signals from the cells were observed by a nikon te s fluorescence microscope, the transfection efficiency was calculated by counting the ratio of gfp-positive cells to all cells in different optical fields. s rrna sequence analysis for authentication regarding the origin of the ek cell line, its s recombinant (r)rna gene was sequenced (englisch, ; frankowski and bastrop, ) . the total genomic dna of passage ek cells was extracted using the gene jet genomic dna purification kit (thermo fisher scientific). the fragments of s rrna gene were amplified using a pair of specific primers (table ) designed according to the published total a. anguilla s rrna sequence (genbank accession no: fm . ). the pcr amplification system was composed of a μl reaction mix containing . μl of ×buffer, . μl of deoxynucleotide triphosphate (dntp) mix ( . mm each), . μl of each primer ( μm each), . μl of ex-taq dna polymerase ( u μl − ; takara) and μl of the extracted genomic dna. the optimum conditions for pcr include: initial denaturation at °c for min, cycles at °c for s, °c for . min and °c for min, with a final elongation at °c for min. the pcr products ( μl per sample) were collected with high pure pcr product purification kit (roche) and analyzed in % agarose gels containing . mg ml − ethidium bromide, and then photographed using a fr- a gel image analysis system (furi the susceptibility of ek cells to rgv was investigated. purified viral samples were prepared according to the previous study (liu et al., ; ge et al., ) . the ek cells at passage were inoculated into cm culture flasks and incubated at °c for h, then washed with . mol l − pbs. ml of virus suspension (dilution= − ) was added to each flask respectively and removed after h incubation. the infected cells were kept at °c in l- supplemented with % fbs, and observed for a cytopathic effect (cpe) daily under a nikon eclipse te -s fluorescence microscope. total dna was extracted from the cells at , , , and h after infection with gene jet genomic dna purification kit, and used as the templates for qrt-pcr. the virus copy number was calculated with the ct standard curve. to define the responses of this cell line to immune stimulations, the changes of the expression of interferon regulatory factor- (irf , genbank accession no. kf . ), interferon regulatory factor- (irf , genbank accession no. kf . ), transforming growth factor-β (tgf-β, genbank accession no. aj . ) and cytochrome p (cyp a , genbank accession no. kf . ) in the ek cells caused by poly (i: c) induction were detected using qrt-pcr (wang et al., ) . poly (i: c) (final concentration= μg ml − ; sigma-aldrich) was added to the culture medium, and the cells were collected after , , and h incubation. the total rna was extracted using a trizol®plus rna purification kit (invitrogen), and then reverse transcribed into first-strand cdna as the template for qrt-pcr with superscript™ iii first-strand synthesis super mix for qrt-pcr (invitrogen). with the primers designed by primer premier . and beacon designer . (table ) , qrt-pcr was performed in the cfx touch™ real-time pcr detection system (biorad) using the power sybr® green pcr master mix reagent (applied biosystems). the conditions of reaction system were as follows: a μl reaction mix containing . μl of ddh o, . μl of power sybr ® green pcr master mix, . μl of each primer and . μl of first-strand cdna, while the cycling conditions were as follows: initial temperature at °c for min, then cycles at °c for s, °c for s. the −ΔΔct method was used to analyze the relative expression level. each experiment was repeated at least three times. the data are shown as mean ± s.e.m. , and the statistical significance was determined using one-way analysis of variance (dunnett's t test) (davis, ) . statistical analysis was done using spss (www.ibm.com/analytics/). isolated fish hepatocytes model systems for toxicology research an es-like cell line from the marine fish sparus aurata: characterization and chimaera production development of a cell line from 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metals, polycyclic (halogenated) aromatic hydrocarbons, and biocides in zebra mussel and eel from the rhine and meuse rivers exploring on the life cycle and artificial breeding of eel (anguilla) cloning and expression analyses of interferon regulatory factor (irf) and genes in european eel, anguilla anguilla with the identification of genes involved in ifn production monitoring of herpesvirus anguillae (hva) infections in european eel, anguilla anguilla (l.), in northern germany a review of the possible impacts of long-term oceanic and climate changes and fishing mortality on recruitment of anguillid eels of the northern hemisphere flow cytometric measurement of micronuclei induced in a permanent fish cell line as a possible screening test for the genotoxicity of industrial waste waters a cell line (ep- cell line) derived from "beko disease" affected japanese eel elver (anguilla japonica) persistently infected with pleistophora anguillarum development, characterization, conservation and storage of fish cell lines: a review fish cell culture: a protocol for quality control the growth and maintenance of tissue-cell cultures in free gas exchange with the atmosphere preparation of medium l- nomenclature for centromeric position on chromosomes characterization of an iridovirus isolate from rana catesbiana transforming growth factor-beta b: a second tgf-beta paralogue in the rainbow trout (oncorhynchus mykiss) that has a lower constitutive expression but is more responsive to immune stimulation the growth advantage of the pectoral fin cells of anguilla anguilla under different culture conditions cellular responses in primary epidermal cultures from rainbow trout exposed to zinc chloride induction of cytochrome p a (cyp a) in fish. a biomarker for environmental pollution tissue culture of fish cell lines length-dependent innate antiviral effects of double-stranded rna in the rainbow trout (oncorhynchus mykiss) cell line, rtg- establishment and characterization of india's first marine fish cell line (sisk) from the kidney of sea bass (lates calcarifer) breeding places and migrations of the eel a histopathological study of icdv infection of japanese eel anguilla japonica ontogenic expression patterns of several nuclear receptors and cytochrome p aromatases in brain and gonads of the nile tilapia oreochromis niloticus suggests their involvement in sex differentiation the irf family transcription factors in immunity and oncogenesis cytochrome p (cyp) in fish viral diseases of wild and farmed european eel anguilla anguilla with particular reference to the netherlands establishment and characterization of a head kidney cell line from large yellow croaker pseudosciaena crocea hepatic monooxygenase induction and promutagen activation in channel catfish from a contaminated river basin established eurythermic line of fish cells in vitro pcr detection of cage cultured large yellow croaker iridovirus in the luoyuan bay, fujian province comparison of karyotypes of anguilla anguilla and anguilla japonica cell isolation and culture of anguilla anguilla coronavirus like virus aquatic virology infection and propagation of lymphocystis virus isolated from the cultured flounder paralichthys olivaceus in grass carp cell lines preliminary establishment of the in vitro cell culture assay of different tissues of european eel (anguilla anguilla) the authors declare no competing or financial interests. supplementary information available online at http://bio.biologists.org/lookup/doi/ . /bio. .supplemental key: cord- -vx cypf authors: li, shi-fang; gong, mei-jiao; sun, yue-feng; shao, jun-jun; zhang, yong-guang; chang, hui-yun title: in vitro and in vivo antiviral activity of mizoribine against foot-and-mouth disease virus date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: vx cypf foot-and-mouth disease (fmd) is a highly contagious viral disease of cloven-hoofed animals, which has significant economic consequences in affected countries. as the currently available vaccines against fmd provide no protection until – days post-vaccination, the only alternative method to control the spread of fmd virus (fmdv) during outbreaks is the application of antiviral agents. hence, it is important to identify effective antiviral agents against fmdv infection. in this study, we found that mizoribine has potent antiviral activity against fmdv replication in ibrs- cells. a time-of-drug-addition assay demonstrated that mizoribine functions at the early stage of replication. moreover, mizoribine also showed antiviral effect on fmdv in vivo. in summary, these results revealed that mizoribine could be a potential antiviral drug against fmdv. foot-and-mouth disease (fmd) is one of the most economically and socially devastating diseases affecting cloven-hoofed animals [ ] . the infectious agent, foot-and-mouth disease virus (fmdv), is a member of the aphthovirus genus of the picornaviridae family, and contains single-stranded positive-sense rna genomes of about , nucleotides [ ] . as an antigenically variable virus, fmdv consists of seven serotypes (a, o, c, asia , and south african territories , , and ) and a large number of subtypes. in general, slaughtering fmdv-infected/exposed or fmdv-susceptible animals, restricting animal movement, and, in some cases, vaccinating against fmdv and then slaughtering these animals are used as control measures for potential outbreaks in disease-free areas [ ] . although inactivated fmd vaccines have been available since the early s and new novel vaccines are being continuously developed, they offer little or no cross-protection against various serotypes and subtypes of fmdv. in addition, these vaccines do not provide complete clinical protection until seven days post-vaccination. therefore, there is a need for developing effective and safe alternative antiviral strategies against fmdv [ ] [ ] [ ] . mizoribine, an imidazole nucleoside ( figure a ) [ ] , has been used as an immunosuppressive agent for the treatment of renal transplantation, autoimmune diseases, and steroid-resistant nephrotic syndrome in some countries owing to its antiproliferative activity against t and b lymphocytes [ ] . this drug could be phosphorylated by adenosine kinase and converted to mizoribine -monophosphate, replication of some dna and rna viruses, such as cytomegalovirus [ ] , respiratory syncytial virus [ ] , severe acute respiratory syndrome-associated coronavirus (sars-cov) [ ] , bovine viral diarrhea virus (bvdv) [ ] , vaccinia virus [ ] , influenza virus types a and b, and herpesviruses, in combination with acyclovir [ , ] . however, the antiviral activity of mizoribine against fmdv has not yet been investigated. hence, in this study, the antiviral effect of mizoribine against fmdv was evaluated in vitro using ibrs- cells and confirmed in vivo using suckling mice. figure b illustrates the results of the mts assay. as is shown in figure b , mizoribine presented little or no cytotoxicity to the cells. the cell viability was . %, . %, . %, . %, . %, and . % at mizoribine concentrations of , , , , , and μm, respectively, and the % cytotoxic concentration (cc ) of mizoribine was estimated to be more than μm on ibrs- cells. the inhibitory effect of mizoribine on fmdv infection in ibrs- cells was calculated by measuring cell viability using the results of mts assay. as indicated in figure a , the inhibition rates were approximately . %, . %, . %, and . % in cells treated with , , , and μm mizoribine, respectively, whereas other lower mizoribine concentrations demonstrated limited or no inhibitory effect on fmdv. the ic and si values were calculated to be . μm and . , respectively. interestingly, mizoribine also displayed activity against another fmdv strain, a/gd/mm/ , with an ic of . μm and si value of . ( figure c ). these data supported the potential broad-spectrum activity of mizoribine against rna viruses. figure b illustrates the results of the mts assay. as is shown in figure b , mizoribine presented little or no cytotoxicity to the cells. the cell viability was . %, . %, . %, . %, . %, and . % at mizoribine concentrations of , , , , , and µm, respectively, and the % cytotoxic concentration (cc ) of mizoribine was estimated to be more than µm on ibrs- cells. the inhibitory effect of mizoribine on fmdv infection in ibrs- cells was calculated by measuring cell viability using the results of mts assay. as indicated in figure a , the inhibition rates were approximately . %, . %, . %, and . % in cells treated with , , , and µm mizoribine, respectively, whereas other lower mizoribine concentrations demonstrated limited or no inhibitory effect on fmdv. the ic and si values were calculated to be . µm and . , respectively. interestingly, mizoribine also displayed activity against another fmdv strain, a/gd/mm/ , with an ic of . µm and si value of . ( figure c ). these data supported the potential broad-spectrum activity of mizoribine against rna viruses. the transcription of fmdv mrna was significantly reduced with , , , and µm mizoribine treatments relative to the dmso control group ( figure b ). consistent with mrna expression, immunofluorescence assay (ifa) revealed that mizoribine inhibited the expression of fmdv protein in a dose-dependent manner ( figure ). the concentration of µm, which showed more protection against cpe than other concentration, was chosen for the further study. the transcription of fmdv mrna was significantly reduced with , , , and μm mizoribine treatments relative to the dmso control group ( figure b ). consistent with mrna expression, immunofluorescence assay (ifa) revealed that mizoribine inhibited the expression of fmdv protein in a dose-dependent manner ( figure ). the concentration of μm, which showed more protection against cpe than other concentration, was chosen for the further study. subsequently, the antiviral efficacy of mizoribine was further evaluated at various intervals post-fmdv infection, we found that the viral b mrna and protein expressions were continuously inhibited at different time points ( , , , and h) after treatment with μm mizoribine; however, no significant differences were observed between h post-infection (hpi) and the control group ( figure ). taken together, these results suggested that mizoribine exhibited potent antiviral activity against fmdv in ibrs- cells at the early stages of viral infection. the supernatants were used for viral rna quantification using qpcr. the expression values relative to that of β-actin were calculated using the −∆∆ct method. cpe, cytopathic effect. statistically significant differences are indicated by asterisks (* p < . , *** p < . ). the transcription of fmdv mrna was significantly reduced with , , , and μm mizoribine treatments relative to the dmso control group ( figure b ). consistent with mrna expression, immunofluorescence assay (ifa) revealed that mizoribine inhibited the expression of fmdv protein in a dose-dependent manner ( figure ). the concentration of μm, which showed more protection against cpe than other concentration, was chosen for the further study. subsequently, the antiviral efficacy of mizoribine was further evaluated at various intervals post-fmdv infection, we found that the viral b mrna and protein expressions were continuously inhibited at different time points ( , , , and h) after treatment with μm mizoribine; however, no significant differences were observed between h post-infection (hpi) and the control group ( figure ). taken together, these results suggested that mizoribine exhibited potent antiviral activity against fmdv in ibrs- cells at the early stages of viral infection. subsequently, the antiviral efficacy of mizoribine was further evaluated at various intervals post-fmdv infection, we found that the viral b mrna and protein expressions were continuously inhibited at different time points ( , , , and h) after treatment with µm mizoribine; however, no significant differences were observed between h post-infection (hpi) and the control group ( figure ). taken together, these results suggested that mizoribine exhibited potent antiviral activity against fmdv in ibrs- cells at the early stages of viral infection. ''vc'' represents those cells treated with % dmso without mizoribine. values represent the mean ± standard deviation for three independent experiments. the asterisks indicate significant differences between mock-treated and drug-treated cells (** p < . ). inosine- ′-monophosphate dehydrogenase (impdh) is required for de novo purine nucleotide synthesis and its inhibition can lead to depletion of intracellular gtp pools. to investigate the effect of mizoribine on purine synthesis in fmdv, serially diluted guanosine was added to the infected cells treated with mizoribine. while guanosine had no effect on mizoribine, it significantly attenuated the anti-fmdv effect of mizoribine in a dose-dependent manner ( figure ). these data indicated that mizoribine activity against fmdv involved inhibition of impdh-dependent purine synthesis. inosine- -monophosphate dehydrogenase (impdh) is required for de novo purine nucleotide synthesis and its inhibition can lead to depletion of intracellular gtp pools. to investigate the effect of mizoribine on purine synthesis in fmdv, serially diluted guanosine was added to the infected cells treated with mizoribine. while guanosine had no effect on mizoribine, it significantly attenuated the anti-fmdv effect of mizoribine in a dose-dependent manner ( figure ). these data indicated that mizoribine activity against fmdv involved inhibition of impdh-dependent purine synthesis. ''vc'' represents those cells treated with % dmso without mizoribine. values represent the mean ± standard deviation for three independent experiments. the asterisks indicate significant differences between mock-treated and drug-treated cells (** p < . ). inosine- ′-monophosphate dehydrogenase (impdh) is required for de novo purine nucleotide synthesis and its inhibition can lead to depletion of intracellular gtp pools. to investigate the effect of mizoribine on purine synthesis in fmdv, serially diluted guanosine was added to the infected cells treated with mizoribine. while guanosine had no effect on mizoribine, it significantly attenuated the anti-fmdv effect of mizoribine in a dose-dependent manner ( figure ). these data indicated that mizoribine activity against fmdv involved inhibition of impdh-dependent purine synthesis. the suckling mice pretreated by subcutaneous injection of mizoribine or pbs in the neck were infected with fmdv to determine the antiviral activity of mizoribine in vivo. all the solvent-treated mice died within h after ld of o/my /by/ challenge. in contrast, a -h delay in death post-infection was noted in the mizoribine-treated group, and all the mice died within h after the viral challenge. the overall death time of mice treated with mizoribine was delayed by h, when compared with the control, and a significant difference in mouse survival was noted between the treatment group and control (p = . ) ( figure a ). the suckling mice pretreated by subcutaneous injection of mizoribine or pbs in the neck were infected with fmdv to determine the antiviral activity of mizoribine in vivo. all the solvent-treated mice died within h after ld of o/my /by/ challenge. in contrast, a -h delay in death post-infection was noted in the mizoribine-treated group, and all the mice died within h after the viral challenge. the overall death time of mice treated with mizoribine was delayed by h, when compared with the control, and a significant difference in mouse survival was noted between the treatment group and control (p = . ) ( figure a ). furthermore, significant histopathological damage was observed in the heart tissue of fmdv-infected mice, including considerable myocardial interstitial hemorrhage, myocardial fibronectin degeneration, and extensive inflammatory cell infiltration, as indicated by black arrows ( figure a ). in addition, myocardial fiber edema and incomplete fibrous structure were also observed. however, mice treated with mizoribine showed mild histopathological changes and only a small amount of inflammatory cell infiltration in the heart ( figure b ). intriguingly, although fmdv antigen was detected in the heart tissue of both mizoribine-treated and control mice, it was not statistically significant ( figure c,d) . these findings suggested that histopathological damage may be the main cause of death resulting from fmdv infection, and mizoribine can effectively alleviate these effects. furthermore, significant histopathological damage was observed in the heart tissue of fmdv-infected mice, including considerable myocardial interstitial hemorrhage, myocardial fibronectin degeneration, and extensive inflammatory cell infiltration, as indicated by black arrows ( figure a ). in addition, myocardial fiber edema and incomplete fibrous structure were also observed. however, mice treated with mizoribine showed mild histopathological changes and only a small amount of inflammatory cell infiltration in the heart ( figure b ). intriguingly, although fmdv antigen was detected in the heart tissue of both mizoribine-treated and control mice, it was not statistically significant ( figure c,d) . these findings suggested that histopathological damage may be the main cause of death resulting from fmdv infection, and mizoribine can effectively alleviate these effects. as fmdv exhibits high mutation rates and produces significant economic loss in affected countries, it is important to adopt effective measures to control this virus. it has been demonstrated that mizoribine does not produce tumorigenic and gonadal suppression effects, and exerts low bone marrow inhibition and hepatotoxic outcomes, and has been used for the treatment of renal diseases in humans [ , ] . thus, considering the safe, reliable, and acceptable efficacy of mizoribine in humans, it might also be employed for the treatment of animal diseases. as fmdv exhibits high mutation rates and produces significant economic loss in affected countries, it is important to adopt effective measures to control this virus. it has been demonstrated that mizoribine does not produce tumorigenic and gonadal suppression effects, and exerts low bone marrow inhibition and hepatotoxic outcomes, and has been used for the treatment of renal diseases in humans [ , ] . thus, considering the safe, reliable, and acceptable efficacy of mizoribine in humans, it might also be employed for the treatment of animal diseases. to the best of our knowledge, the present study is the first to report on the antiviral effect of mizoribine on fmdv both in vitro and in vivo. by using mts assay, the cytotoxicity of mizoribine was determined to be very weak, with a cc value higher than µm. furthermore, mizoribine showed significant anti-fmdv activity, not only against type o fmdv o/my /by/ strain, but also against type a fmdv a/gd/mm/ , with si values of . and . , respectively. these results indicated that mizoribine could be a better drug to prevent fmdv a/gd/mm/ infection. moreover, qpcr and ifa findings revealed that mizoribine can significantly inhibit the viral mrna and fmdv protein levels. to understand the preliminary antiviral mechanism of mizoribine, time-of-drug-addition assay was performed, and the results demonstrated that mizoribine mainly functions at the early stages of infection. it has been reported that the antiviral activity of mizoribine involves inhibition of impdh [ ] , an essential enzyme for the synthesis of guanosine monophosphate from inosine monophosphate through de novo pathway [ , ] . similarly, in the present study, the antiviral activity of mizoribine against fmdv was found to be attenuated by guanosine supplementation. the animal experiment demonstrated that mizoribine had an inhibitory effect on fmdv in vivo, and treatment with µg of mizoribine significantly prolonged the survival of fmdv-infected suckling mice. although the in vivo results did not show a significant increase in the survival rates of infected mice, the delay in death and alleviated histopathological changes suggested the inhibitory effect of mizoribine on viral replication. overall, the weak cytotoxicity and strong antiviral activities of mizoribine both in vitro and in vivo favored its further potential clinical applications in the treatment of viral infection. combination treatment strategies have been proposed to enhance the efficacy of antiviral agents, because of their advantages in overcoming viral mechanisms of resistance to antiviral treatments [ ] . it has been demonstrated that mizoribine enhances the anti-caprine-herpesvirus- activity of acyclovir, and the combination of mizoribine and acyclovir resulted in an almost complete inhibition of viral replication. thus, combined therapy of acyclovir and mizoribine could be exploited for the treatment of genital infection by herpesviruses [ ] . moreover, mizoribine has been reported to be active against the replication of bvdv in madin-darby bovine kidney (mdbk) cells, and a combination of interferons (ifns) and mizoribine had been noted to synergistically inhibit bvdv replication in bovine kidney cells [ ] . with regard to fmd, fmdv have been demonstrated to be very sensitive to ifns, and ifn-based strategies have been established to be an efficient biotherapeutic option against fmdv [ ] . similarly, the combination of antiviral agents, such as sirna, ribavirin, and ifns, has been determined to produce enhanced antiviral effect against fmdv [ ] . therefore, future studies must investigate whether a combination of ifns and mizoribine could produce increased inhibitory effect on fmdv replication both in vitro and in vivo. in conclusion, to the best of the authors' knowledge, the present study is the first to demonstrate that mizoribine can suppress fmdv replication in vitro as well as prolong the survival of suckling mice in vivo, suggesting the potential applications of this drug in antiviral regimens for fmd treatment. the findings of this study may warrant further investigations on the efficacy and safety of combined use of mizoribine and other antiviral agents in vitro and in vivo. thirty-two ( ) two-and three-day-old balb/c mice weighing - g were used to investigate the efficacy of mizoribine in vivo. all the animal trials were performed in a biosafety level- laboratory and approved by the animal ethics committee of lanzhou veterinary research institute, chinese academy of agricultural science (no. lvriaec - ). fmdv type o (o/mya /by/ ) strain was used for viral challenge. the cytotoxicity of mizoribine was evaluated using mts assay. briefly, × cells were seeded into each well of a -well plate containing µl of complete medium. on the following day, the cells were incubated with µl of mizoribine at various concentrations ( , , , , , and µm) for h. as a control, cells were treated with % dmso. after treatment, the supernatants were discarded, and the cells were washed three times. then, µl of dmem were added to each well along with µl of mts solution and incubated for an additional h at • c. the optical density of each well at nm was determined using a microplate reader (bio-rad, hercules, ca, usa). the cell viability was expressed as the percentage of absorbance of the treated cells to that of the control cells. the antiviral activity of mizoribine against fmdv was determined using an mts-based cytopathic effect (cpe) inhibition assay. briefly, the viral suspension ( tcid o/my /by/ ) was added to the ibrs- cell monolayers. after incubation for h, the cells were washed three times with dmem, and serially diluted mizoribine solution was added to the wells (eight wells for each concentration). then, the plates were incubated at • c in % co . after h, when maximum cpe was noted in the virus control group (vc), the cell viability was measured using mts assay as described earlier, and the percentage of inhibition associated with each mizoribine concentration was normalized with respect to the vc. the virus inhibition rate was calculated as follows: inhibition rate = (optical density of mizoribine group − optical density of vc)/(optical density of cell control group − optical density of vc) × %. the supernatant of each well was collected and the viral b mrna was analyzed using q-pcr. the % inhibitory concentration (ic ) values were calculated with graphpad software (version . , la jolla, ca, usa). the monolayers of cells were seeded in -well plates infected with tcid fmdv o/my /by/ at • c for h. then, a µm portion of mizoribine supplemented with serial dilutions of guanosine (from to µm) was added and incubated for h. after incubation, the viral protein and gene expressions were assessed by western blot analysis and q-pcr, respectively. the ibrs- cells were incubated with tcid o/my /by/ for h. subsequently, the viruses were removed and the medium was replaced. mizoribine ( µm) was added to the cells during infection (co) or post-infection ( , , and h). after h incubation, the fmdv b mrna and vp protein in the cells were determined by q-pcr and western blot analysis, respectively. the expression levels of fmdv b mrna and β-actin were determined by real-time pcr as previously reported [ ] . briefly, the total rna from the ibrs- cells was extracted using trizol reagent, and µg of rna was used in reverse transcription reaction using a primescript™ rt reagent kit containing gdna eraser, following the manufacturer's instructions. the reaction mixture for real-time pcr comprised diluted cdna ( µl), µm primers, and . µl of sybr green master mix to a final volume of µl. the amplification conditions were as follows: • c for s, followed by cycles of • c for s, • c for s, and • c for s. dissociation curves were generated to analyze the individual pcr products after cycles. the expression levels of fmdv mrna genes were normalized against those of porcine β-actin mrna. the analyses of the relative gene expression data were performed using the −∆∆ct method [ ] . for western blot analysis, the cells were lysed with pierce ripa, and the cell lysates were resolved and separated by % sds-page and transferred to polyvinylidene fluoride membrane. the membranes were probed with primary antibodies against fmdv vp protein (dilution, : ) to detect virus replication. a monoclonal antibody to β-actin (dilution, : ) was used as a loading control. the membranes were incubated with goat anti-mouse and anti-rabbit conjugated with horseradish peroxidase for h at room temperature and examined by pierce™ ecl western blotting substrate. to determine the antiviral activity of mizoribine, indirect immunofluorescence assay (ifa) was performed as previously described with minor modifications [ ] . the ibrs- cells were seeded into a -well plate at a concentration of × cells/well and incubated for one day. subsequently, the cells were incubated with tcid fmdv for h, and mizoribine diluted at indicated concentrations was added to the cells after removal of the viral inoculum. after h of incubation, the ibrs- cells were washed thrice with pbs and fixed with % paraformaldehyde for min. then, paraformaldehyde was removed and absolute methanol was added to the cells and incubated for min. subsequently, the cells were washed with pbs and blocked using blocking buffer (pbs supplemented with . % triton x- and % fbs). rabbit hyperimmune serum raised against type o fmdv (o/my /by/ ) was used to probe type o fmdv, and peroxidase-conjugated goat anti-rabbit igg (h + l) was employed as secondary antibody. finally, the cells were counterstained with , -diamidino- -phenylindole (dapi) and viewed under fluorescence microscope (nikon eclipse ts fluorescence microscope, yokagawa electric corporation, tokyo, japan). specific-pathogen-free three-day-old kunming suckling mice were inoculated with µg of mizoribine dissolved in µm dmso, % tween , and . ml of pbs by subcutaneous neck injection. the negative control was inoculated with pbs. after h, viral challenge was performed by intradermal injection with % lethal dose (ld ) fmdv serotype o o/my /by/ into the subcutaneous neck region of the mice. the animals were monitored for five days, and a log-rank test was performed for statistical analysis using graphpad software. at h post-infection, the suckling mice were euthanized and processed for histological and immunohistochemical investigations. for histopathological analysis, the heart tissues were fixed in % paraformaldehyde solution, embedded in paraffin, and cut into µm thick sections for standard hematoxylin and eosin (h & e) staining. with regard to immunohistochemical (ihc) studies, the fmdv antigen was detected as follows: µm thick paraffin-embedded tissue sections were deparaffinized and treated with methanol-hydrogen peroxide for min before heat-induced antigen retrieval in . m sodium citrate buffer (ph = ) for min. rabbit hyperimmune serum raised against type o fmdv (o/my /by/ ) was used as primary antibody. the tissue sections were processed with a splink detection kit for min and stained with dab for min at room temperature. after washing, the tissue sections were counterstained, mounted, examined under a digital microscope (ba digital, motic, xiamen, china), and photographed. the optical density of each tissue section was determined by image-pro plus . software (media cybernetics, rockville, md, usa). all the data are expressed as mean ± standard deviation (sd) for at least three independent experiments. one-way anova was used to analyze the difference between mizoribine and control groups using graphpad prism (graphpad software, inc., la jolla, ca, usa), version . , and significant differences were defined at p < . . selective index (si) = cc /ec . mutagenesis versus inhibition in the efficiency of extinction of foot-and-mouth disease virus antiviral activity of ovine interferon tau against foot-and-mouth disease virus development of vaccines toward the global control and eradication of foot-and-mouth disease foot and mouth disease vaccine strain selection: current approaches and future perspectives vaccination against foot-and-mouth disease virus confers complete clinical protection in days and partial protection in days: use in emergency outbreak response mizoribine and mycophenolate mofetil mode of action and effects in clinical use effects of cyclosporine, azathioprine, mizoribine, and prednisolone on replication of human cytomegalovirus recent progress in antiviral chemotherapy for respiratory syncytial virus infections kurane, i. inhibitory effect of mizoribine and ribavirin on the replication of severe acute respiratory syndrome (sars)-associated coronavirus inhibitors of the impdh enzyme as potential anti-bovine viral diarrhoea virus agents studies on bredinin. i. isolation, characterization and biological properties comparative inhibitory effects of various nucleoside and nonnucleoside analogues on replication of influenza virus types a and b in vitro and in ovo potentiating effect of mizoribine on the anti-herpes virus activity of acyclovir effects of mizoribine on mhc-restricted exogenous antigen presentation in dendritic cells inhibition of bovine viral diarrhea virus (bvdv) by mizoribine: synergistic effect of combination with interferon-alpha enhanced inhibition of foot-and-mouth disease virus by combinations of porcine interferon-alpha and antiviral agents in vitro inhibition of caprine herpesvirus by acyclovir and mizoribine antiviral activity of bovine type iii interferon against foot-and-mouth disease virus detection of all seven serotypes of foot-and-mouth disease virus by real-time, fluorogenic reverse transcription polymerase chain reaction assay analysis of relative gene expression data using real-time quantitative pcr and the (-delta delta c(t)) method. methods a novel type i interferon, interferon alphaomega, shows antiviral activity against foot-and-mouth disease virus in vitro the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. key: cord- - mqs mj authors: franco-martínez, lorena; tvarijonaviciute, asta; horvatić, anita; guillemin, nicolas; cerón, josé joaquín; escribano, damián; eckersall, david; kocatürk, meriç; yilmaz, zeki; lamy, elsa; martínez-subiela, silvia; mrljak, vladimir title: changes in salivary analytes in canine parvovirus: a high-resolution quantitative proteomic study date: - - journal: comp immunol microbiol infect dis doi: . /j.cimid. . . sha: doc_id: cord_uid: mqs mj the present study evaluated the changes in salivary proteome in parvoviral enteritis (pve) in dogs through a high-throughput quantitative proteomic analysis. saliva samples from healthy dogs and dogs with severe parvovirosis that survived or perished due to the disease were analysed and compared by tandem mass tags (tmt) analysis. proteomic analysis quantified peptides, and (corresponding to proteins) showed significantly different abundances between studied groups. ten proteins were observed to change significantly between dogs that survived or perished due to pve. bioinformatics’ analysis revealed that saliva reflects the involvement of different pathways in pve such as catalytic activity and binding, and indicates antimicrobial humoral response as a pathway with a major role in the development of the disease. these results indicate that saliva proteins reflect physiopathological changes that occur in pve and could be a potential source of biomarkers for this disease. parvoviruses are small icosahedral viruses that infect many animal species including humans worldwide. canine parvovirus type- is the causal agent of canine parvoviral enteritis (pve), a severe disease that causes nearby % morbidity and up to - % mortality in unvaccinated adults and puppies, respectively [ ] . the clinical presentation of pve ranges from mild to severe, based on clinical signs and physical examination [ , ] . the most common clinical signs in puppies include acute severe vomiting and bloody diarrhoea, fever, dehydration and lethargy [ ] . complications such as cardiac damage, which may result in death or permanent myocardial damage [ ] , and systemic inflammatory response syndrome (sirs) prior to sepsis have been described [ ] . in dogs, saliva has proven to be an useful biofluid for the diagnostic of several conditions such as leishmaniosis [ ] and helicobacter infection [ ] , as well as for evaluation of stress [ ] . the use of noninvasive specimens such as saliva provides several advantages compared to invasive methods, being safer for the personal and the patient, easier to collect, pain-free and causes reduced sampling stress [ ] . saliva can have potential advantages versus others non-invasive samples such as faeces, which usually need a laborious pre-treatment prior to its analysis. in particular, in the case of pve, it has been described a poor sensitivity of faeces when used in rapid antigen elisa test [ ] . for this, it is envisioned that saliva can play an increasingly important role in the early diagnosis and monitoring of diseases [ ] . however, to the author's best knowledge, there are no reported studies about changes in saliva composition in pve. the knowledge of the changes that occur in saliva in dogs with parvovirus could help to detect possible biomarkers in this fluid for pve disease. proteomics sciences focus on the study of the proteome content of cells, organs and organisms [ ] . proteomic techniques have become widely used with the aim of discovering disease-related biomarkers in diverse biological fluids that could be used in routine practice for early diagnosis, prognosis or treatment monitoring [ , ] . over the last several years, novel technologies such as the use of isobaric tags have greatly increase the sensitivity of the proteomic analysis [ ] [ ] [ ] . tandem mass tags (tmt) allow for relative simultaneous quantification of differentially labelled peptides [ ] since each sample is marked and distinguished by differences on their reporter ion masses [ , ] . however, there is still a scarcity of reports using tmt technology in saliva samples, and no reports have been found describing the use of tmt to detected possible biomarkers of diagnosis and prognosis in pve. the objective of the present study is to evaluate possible changes in saliva proteins associated to canine parvovirus infection. this would help to clarify if saliva components can reflect the physiopathological changes associated to the disease and to evaluate the possibility of identifying potential biomarkers of pve in saliva samples. for this, saliva samples were collected from healthy dogs (control group) and from dogs with pve that survived or died because the infection. saliva samples were analysed by quantitative high-throughput tmt-based proteomic approach enabling the identification of significantly varying proteins among different groups. subsequently, the differentially expressed proteins were used as a starting point for creating protein interaction networks. a total of client-owned dogs that were presented to the veterinary teaching hospital, small animal clinics, bursa uludag university, bursa, turkey, of different breeds were involved in this study (table ) . parvovirosis infection was diagnosed by compatible clinical (acute bloody diarrhoea, vomiting, anorexia, dehydration etc.) and haematological signs (leukopenia, neutropenia and lymphopenia) in combination with the positive test results of the commercially available faecal diagnostic test (antigen rapid pve kit, animal genetics, inc., suwon, korea). selected clinical (body temperature and heart and respiratory rates) and haematological (leukogram, eritrogram and thrombogram) findings were used to assess the general health status of the animal. dogs were excluded if they were co-infected with other viral (canine distemper or coronavirus), parasite (coccidiosis, giardiasis, ascaridiosis etc.), or vector borne diseases by use of faecal screening test, fecal microscopic examinations, or speed test for ehrlichiosis, anaplasmosis, lyme, and dirofilariasis (anigen rapid caniv- , bionote, korea), respectively. the dogs were divided into three groups according to their clinical condition and development of the disease. a group with healthy dogs was considered as control group. virus is shed for a few days before the onset of clinical signs. therefore, even if all dogs in control group were considered healthy based on the clinical, haematological and serum biochemistry findings (comprehensive profile, vetscan, abaxis), faecal screening test for cpv-ag were applied. dogs with negative test result were enrolled to study. all dogs in test group were treated as described previously [ ] . briefly, fluid replacement therapy (dextrose % with a balanced crystalloid solution -lactated ringer, and hydroxyethyl starch), anti-emetics (metoclopramid . - . mg/kg, bid, subcutaneously, and ranitidin - mg/kg, bid, intramusculary), parenteral antibiotics (ampicillin mg/kg, bid + amikasin - mg/kg, bid + metronidazol mg/kg, bid), immune stimulator (imunex), probiotics and vitamins (e and c) were used. dogs with clinical diagnosis of severe parvovirosis were divided into two groups according to the progress of the disease: dogs that survived (survival group) (age . ± . months, male and female) and that died because of the disease (non-surviving group) (age . ± . months, males and female). therefore, dogs were classified according to the severity of their clinical and haematological signs ( table ). there was a similarity of presented clinical signs and its duration ( - days) in dogs within test group. despite the treatment, three dogs died within days (one on day , one on day and other on day ). all the procedures were approved by the local ethical committee of the university of uludag. at least . ml of unstimulated saliva specimens were collected from each patient by placing small cotton swabs around the mouth. when the cotton swabs were thoroughly moist, they were placed in collection devices (salivette saliva collection tube / v-bottom, sarstedt, aktiengesellschaft & co, nümbrecht, germany), centrifuged ( x g for min, °c), and the supernatant was stored at − °c until analysis [ ] . due to dehydration and subsequent dry mouth, for some animals the sampling protocol was performed twice in order to obtain at least . ml of unstimulated saliva. from each sample, μg of acetone-precipitated proteins were subjected to reduction, alkylation, digestion and labelled using -plex tandem mass tag reagents according to manufacturer instructions (thermo scientific) with some modification, as described previously [ ] . in short, μg of sample and internal standards were reduced with mm dtt (sigma-aldrich), alkylated with mm iodoacetamide (sigma-aldrich) and precipitated with ice-cold acetone (vwr, pennsylvania, usa) overnight. samples were then centrifuged and acetone was decanted. pellets were resuspended with μl of mm teab buffer and digested with trypsin (promega) overnight at °c ( . μg of trypsin per μg of protein). tmt label reagents were equilibrated to room temperature, resuspended in anhydrous acetonitrile lc-ms grade (thermo scientific) and added to each sample. labelling reaction was incubated for h at room temperature and then quenched by adding % hydroxylamine (thermo scientific) for min. samples were then combined at equal amounts and μg of each mixed sample set was placed in a well of a microplate, vacuumdried and stored at - °c before further lc-ms/ms analysis. the lc − ms/ms analysis was performed on dionex ultimate rsls nano flow system (dionex, camberley, uk) and orbitrap elite mass spectrometer (thermo fisher scientific) as described elsewhere [ ] . in order to compare the abundances of peptides detected in proteomic analysis between groups in comparisons (control versus survival, control versus non-surviving and survival versus non-surviving), data were normalized by logarithmic transformation and student's t-test was used (one-tailed, unpaired) were performed. in all cases, values of p < . were considered to be significant. the data obtained in the proteomic study were used for the gene ontology (go) analysis. canine genes encoding proteins differentially expressed were converted to their human orthologs using the ensembl orthologs database and its biomart tool for data mining (www.ensembl. org). obtained genes were used to determine the go terms over-represented in two conditions (survival and non-surviving), by the utilization of the cytoscape (v . . ) plug-in cluego (v . . ) [ , ] on the homo sapiens go-biological process ( / / ). go terms overrepresented for each group were submitted to analysis by revigo (allowed similarity = . , simrel) to remove redundant go terms and groups related go terms based on their functional description [ ] . finally, pathways interactomes were designed in cytoscape using the radial layout. high-resolution quantitative proteomic analysis allowed the identification of canine peptides from the eleven non-depleted canine saliva samples. for the comparison control versus survival, peptides were differentially expressed, corresponding to unique proteins. for the comparison control versus non-surviving, peptides were differentially expressed, corresponding to unique proteins. for the comparison survival versus non-surviving, peptides were differentially expressed, corresponding to unique proteins. therefore a total of proteins were identified that differed between groups. of the differentially expressed proteins between survival and control groups, the proteins most down-regulated in the survival group were cathelicidin antimicrobial peptide (camp), rho-gdp dissociation inhibitor beta (arhgdib), apolipoprotein a- (apo-a ), neutrophil elastase (elane), matrix metalloproteinase- (mmp ), ef-hand domain containing protein d (efhd ), cd antigen (cd ), plastin- (lcp ), retinol binding protein (rbp ), and maltase-glucoamylase intestinal (mgam). epididymalspecific lipocalin- (lcn ), bpi foldcontaining family b member (bpifb ), lymphocyte antigen d (ly d), desmoglein (- and - , dsg and dsg , respectively), alpha- macrogloulin-like protein (a ml ), democollin- (dsc ), leucinerich-alpa- -glycoprotein (lrg ), polymeric immunoglobulin receptor (pigr), and l-amino acid oxidase (il i ) were the proteins most up-regulated in the survival group. of these, camp and arhgdib were the proteins most down-regulated (- . and - . -fold lower expression, respectively), while lcn and bpifb were the most up-regulated ( . and . -fold higher) in the survival group compared to the control group. when non-surviving and control groups were compared, proteins showed differences in abundance. the proteins most down-regulated were arhgdib, camp, apoa , hemoglobin subunit beta (hbb), rbp , cd , mmp , vitamin d binding protein (gc) and elane. on the other hand, immunoglobulin heavy chain (igh), lcn , clusterin (clu), trefoil factor (tff ), ly d, olfactomedin (olfm ), lrg , glyoxalase i (glo ), bpifb , and dsg were the proteins most up-regulated. finally, when the two groups of dogs with parvovirosis were compared, proteins showed different abundances. ezrin, allergen dog , lactoperoxidase, l-lactate dehydrogenase a, cystatin-m and macrophage-capping protein were higher in survival group, whereas alphaactinin- , peptidyl-prolyl cis-trans isomerase a, ribosyldihydronicotinamide dehydrogenase and lactoylglutathione lyase were upregulated in the dogs that died due to the disease. the go analysis performed using cytoscape plug-in cluego identified go terms between survival and controls, which were filtered for redundancies and then grouped into main groups using revigo. the functional groups included antimicrobial humoral response, entry of bacterium into host cell, tissue homeostasis, heterotypic cell-cell adhesion and endoplasmic reticulum mannose trimming (fig. ) . when comparing non-surviving and control groups, go terms were identified, which were grouped into main functional groups including negative regulation of cellular response to growth factor stimulus, antimicrobial humoral response, transition metal ion homeostasis, endoplasmic reticulum mannose trimming and glutathione derivate biosynthesis (fig. ) . comparison of survival versus non-surviving groups did not identify enough proteins significantly expressed to generate a reliable go analysis using only experimental results. finally, go terms most represented in this study after being filtered by revigo and depicted by cytoscape are shown in figs. - . when the network from control and survival group was analysed, a negative regulation of wound healing (related with the down-regulation of fibrinogen fga, fgb and fgg genes) and negative regulation of cellular response to growth factor stimulus (related to the up-regulation of the ubiquitin complex composed by rps a, ubc, ubb and uba genes) nodes demonstrated central roles in the development of the disease since they are involved in many pathways. the network obtained from comparison of non-surviving and control groups suggested that the over-expressed ubc gene (which expresses ubiquitin c protein) appears to have a central role by affecting or being affected by numerous pathways identified as related with nonsurviving group in parvovirosis. interestingly, this ubiquitin c protein is related only with go terms over-expressed in non-surviving group. this indicate that, in the present study, all cellular pathways which use ubiquitin form a pertinent group, which is positively correlated with the non-survival status. similar to the previously mentioned network, fibrinogen expression is down-regulated in saliva of dogs with parvovirosis. when merging fold changes data and over-represented pathways in both non-surviving and survival groups, the obtained network highlights that antimicrobial humoral response was shown as central term. this node is the only one which is over-represented in both parvovirus groups (survival and non-surviving) but with different expression when compared to controls: it was lower-expressed in the non-surviving group, but was not differentially expressed in survival group as compared to controls. antibacterial humoral response, negative regulation of response to external stimulus, and killing of cells of other organisms are the three down-regulated nodes which are directly linked with antimicrobial humoral response. the network also showed proteins surrounding this central node, of which were down-regulated (elane, kininogen [kng- ], fga, fgb, s calcium binding protein a [s a ], s calcium binding protein a [s a ], camp, lysozyme [lyz] and hrg), and up-regulated (beta- microglobulin [b m], deleted in malignant brain tumours [dmbt ], bpi fold-containing family b member [bpifb ], bpifb , and clu) when this paper was focused in the identification of the differentially expressed proteins in saliva and in describing the biological roles which may be affected due to pve. the present study identified for the first time differentially expressed proteins in saliva from healthy dogs and dogs with severe parvovirosis using tmt technology, of which were differentially expressed between animals that survived and perish due to the disease. these differences in proteins indicate that saliva can reflect several physiological pathways in canine parvovirosis. overall changes in protein expression in saliva from dogs with parvovirosis obtained in this study by high-resolution quantitative proteomic analysis suggested alterations in coagulation and inflammation systems, which are closely related pathways since the activation of one mechanism may lead to the activation of the other [ ] . coagulation products such as thrombin can promote inflammatory responses, while inflammation is known to suppresses anticoagulation mechanisms [ ] . the immune system provides protection against infection; however, its activation needs to be tightly regulated in order to prevent excessive inflammation and subsequent damage to the host [ ] . from all the proteins that change in saliva of dogs with parvovirosis, two that are most down-regulated and the two proteins that increased the most in the canine patients are worthy of specific mention. the two most down-regulated proteins in the dogs with parvovirosis that survived, cathelicidin antimicrobial peptide (camp) and rho-gdp dissociation inhibitor beta (arhgdib), have protective role against infections and are involved in the inflammatory response. cathelicidins are a group of short cationic antimicrobial peptides with an important role in the protection against infections [ ] [ ] [ ] . the main source of camps are the specific granules of neutrophils, which are released upon neutrophil activation [ , ] , although they can be produced by other cells such as epithelial cells at mucosal surfaces [ ] . camps are known to regulate the activation of a wide variety of toll-like receptors, which plays important roles in the modulation of the inflammatory response during infections [ , ] . in addition, antimicrobial activity has been described in camps, and the loss of cathelidicin expression has been reported to increase the susceptibility to escherichia coli infection in mice [ , ] . rho-gdp dissociation inhibitor beta (arhgdib) is primarily expressed in cells of a hematopoietic lineage and belongs to the rhogdi family, which are known to regulate rho-gtpases [ ] . arhgdib showed an ability to decrease human immunodeficiency virus type (hiv- ) replication in cell lines and can be used as a biomarker of hiv- infection [ ] . arhgdib expression has been also proposed as suppressor of metastasis and predictor of prognosis in human patients with bladder cancer, in which arhgdib positive tumours are related with longer disease-free and disease-specific survival rates [ ] . on the other hand, the two most up-regulated proteins in dogs with parvovirosis that survived, were lipocalin- (lcn ) and bpi fold-containing family b member (bpifb ). lipocalins are a family of proteins that usually carry lipids or other hydrophobic or amphiphilic compounds accommodated in a cavity within their conformation, including steroids, hormones and metabolites such as vitamins and cofactors [ ] . furthermore, lipocalins have been reported to be modulators of cell growth and metabolism, and regulators of the immune response [ , ] . although little is known about lcn , a related protein, neutrophil gelatinase-associated lipocalin, ngal, has been associated with bacterial infection in humans [ ] [ ] [ ] . bpifb belongs to a family of proteins considered as a key component of the innate immune response against gram-negative bacteria [ ] , but the physiological functions of the bpifb family still remain largely unknown [ ] . despite this, a previous study on bpifb has reported the involvement of bpifb proteins regulating enterovirus infection, with bpifb assisting the enterovirus replication by controlling secretory pathway trafficking and golgi complex morphology [ ] . in addition to these proteins, other proteins were significantly differentially expressed in saliva of parvovirus infected dogs compared fig. . over-represented go terms generated by the comparison of dogs that survived and dogs that died due to parvovirosis, grouped by revigo, based on their description. leading go term (n = ) for each group was defined as the highest significance inside the group. in silico inferred interactome network of identified go terms over-represented in canine parvovirosis (survival versus control groups). differentially expressed proteins interacting with at least term were added. radial layout was applied and the go group leader terms are in dark blue text. nodes colours represent the group of go terms (determined by revigo) and border are represented in green and red for over-expressed and lower-expressed proteins, respectively, when compared to control group. to healthy dogs. by analysing the over-represented cellular pathways defined by those proteins by gene ontology analyses, it could be observed that they are involved in other physiopathological pathways such as catalytic activity, binding, transporter activity, antioxidant activity, structural molecule activity, receptor activity and translation regulator activity. since more than % of the genes differentially expressed belong to the proteins related with catalytic activity and binding, the discussion will focus on these and related proteins. in regard to the proteins related to catalytic activity, some were down-regulated, including, apolipoprotein a- (apo-a ), neutrophil esterase (elane), complement c (c ) and histidine-rich glycoprotein (hrg). apo-a is the major protein component of high density lipoproteins in human and other mammals [ ] and the decreased levels apo-a observed in the present study are in concordance with other responses described in different canine diseases such as leishmaniosis [ ] or canine idiopathic dilated cardiomyopathy [ ] , suggesting that apo-a could be a feasible biomarker in dogs. lower expression of neutrophil esterase was observed in both groups of dogs with pve when compared to control group. neutrophil esterase is a serine proteinase with important antimicrobial effects that are released by neutrophils and enhances inflammatory responses. genetic mutations in its gene (elane) has been identified in forms of hereditary neutropenia in humans and dogs [ ] . there is a case reported of a mutation in elane gene as the cause of chronic parvovirosis in a woman [ ] . the activation of complement c supports local inflammatory responses against pathogens and initiates the humoral immune response, playing a central role in the activation of the complement system [ ] . increased c expression has also been reported in dogs with canine babesiosis [ ] . in our study, different hypotheses could explain the down-regulation of complement c in saliva of dog with pve such as a reduction due to cleavage of the protein in order to activate complement cascade, or as a consequence of an immunodeficiency [ ] . histidine-rich glycoprotein is a relatively abundant plasma protein with a wide range of targets and functions, such as regulating clotting and fibrinolysis, and angiogenesis [ , ] . hrg roles in anti-inflammatory effects and pathogen control have been proposed since, after experimental streptococcus pyogenes infection, hrg-deficient mice showed higher bacterial burden and proinflammatory cytokines than wild-type mice [ ] . thirty-four of the proteins differentially expressed in saliva between controls and dogs with parvovirosis have binding activity, including many of the proteins described above such as apo-a , a m, c and lcn . other important proteins differentially expressed in saliva between healthy and dogs with parvovirus with binding activity are desmoglein- and - (dsg and dsg , with . and . -fold higher in dogs with parvovirus, respectively), and clusterins (clu, . -fold higher). desmogleins are a family of calcium-binding transmembrane glycoproteins which play an important role in the desmosomes transmembrane component [ ] . in humans, salivary levels of dsg and dsg are correlated with serum anti dsg titres, proposing salivary dsg as suitable markers of diagnosis of pemphigus vulgaris [ ] . clusterins are secretory glycoproteins that are involved in multiple physiopathological processes, including lipid metabolism, tissue remodelling, reproduction, transport and cell apoptosis [ ] . these proteins have been classically considered as markers of cell death [ ] ; however, protective in silico inferred interactome network of identified go terms over-represented in canine parvovirosis (dead versus control groups). differentially expressed proteins interacting with at least term were added. radial layout was applied and the go group leader terms are in dark blue text. nodes colours represent the group of go terms (determined by revigo) and border are represented in green and red for over-expressed and lower-expressed proteins, respectively, when compared to control group. roles such as anti-apoptotic or prevention of complement attacking complex functions of clusterins have been suggested [ , ] . the bioinformatics analysis of the in silico inferred proteins network allowed the generation of more information from the experimental results, including data on intracellular pathways. this revealed the impact on processes such as an inhibition of humoral and cellular responses, and a positive effect in the entry of bacterium into host cell. these results are in concordance with the pathogenicity of pve in which, after the ingestion, the virus replicates in the lymphoid tissue in the dog's throat and then spreads to the bloodstream infecting rapidly dividing cells, resulting in depletion of lymphocytes in lymph nodes and necrosis of the intestinal crypts [ ] . these mucosal intestinal lesions may give rise to secondary bacterial invasions from the intestines, leading to systemic inflammatory response syndrome (sirs), sepsis and endotoxemia [ ] [ ] [ ] [ ] . bioinformatics allowed several pathways and proteins to be highlighted which could be useful in the diagnosis of pve with saliva samples. for example, the network created by comparison of survival and control groups revealed that fibrinogen and ubiquitin complexes could play important roles in the development of the disease. the down-regulation of fibrinogen observed in the present study is in contrast with other reports that showed increased levels of fibrinogen in dogs with parvovirosis [ ] , leishmaniosis [ ] or babesiosis [ ] ; although dogs with uncomplicated babesiosis showed higher levels of fibrinogen that those with sirs or multiple organ dysfunction syndrome (mods) [ ] . this could be explained by the fact that, although fibrinogen is considered as a positive acute phase protein in dogs, in disseminated intravascular coagulation (dic) and hyperfibrinolysis the concentration of fibrinogen falls due to fibrinogen cleavage to form soluble fibrin monomers [ , ] . progressive decrease of fibrinogen levels was also reported in dogs during acute haemorrhage [ ] , as occurs in parvovirosis. on the other hand, ubiquitins have been shown to be involved in numerous intracellular processes such as cell cycle control, apoptosis, dna repair, regulation of transcription, stress responses, and targeting cellular proteins for degradation by the s proteasome [ ] . as described in previous reports, viruses often make use of the ubiquitin conjugation to target for degradation of cellular proteins that might otherwise affect the replication of the virus [ ] . one of the limitations of the present study may be the sample size, which could have limited the potential to identify proteins that are reproducibly modulated in concentration in dogs with pve due to the biological inter-individual variations. therefore this could be considered as a pilot study, and further validation studies with a larger number of dogs should be made to confirm these findings. the changes in protein expression and the subsequent bioinformatics analysis described in the present study were based on the analysis of saliva and, therefore, further studies are desirable to discern if they reflect systemic alterations. nevertheless, despite these limitations, our results indicate that pve can produce changes in saliva proteome that can reflect an inflammatory response and changes in coagulation system and immune responseamong other biological processes. this paper was focused on the identification of the differentially expressed proteins in saliva and in the description of the biological roles of the proteins affected due to the disease. further large-scale clinical studies with larger populations would be desirable to elucidate if the proteins identified in this report can be of help in the earlier detection, fig. . in silico inferred interactome network of identified go terms over-represented in canine parvovirosis (survival and dead versus control groups). differentially expressed proteins interacting with at least term were added. radial layout was applied. nodes fill colour figure protein and go terms regulation (red for negatively and green for positively regulated proteins) and node border represent the presence of the term in dead only (black), survival only (white) or in both groups (grey). predicting the outcome, or giving information to the clinician to adjust the treatment. if these studies are successful, these proteins can be used for developing rapid diagnostic tests such as lateral flow kits in saliva for clinical practice. tmt-based proteomic analysis allowed the identification of proteins with different abundances between healthy dogs and dogs with pve, most of them not previously reported in canine parvovirosis. these proteins are involved in various physiopathological processes such as coagulation, inflammation and defence mechanisms, and could be considered as potentially suitable biomarkers of the disease. this study demonstrated that saliva proteome is a suitable biofluid for both study and diagnostic of canine parvovirosis. the authors report no other conflicts of interest. the authors alone are responsible 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and mods associated with canine babesiosis cardiopulmonary and inflammatory biomarkers in the assessment of the severity of canine dirofilariosis acute phase proteins in dogs and cats: current knowledge and future perspectives assessment of hemostatic changes in a model of acute hemorrhage in dogs crystal structure of a deubiquitinating enzyme (human uch-l ) at . angstrom resolution viral avoidance and exploitation of the ubiquitin system lfm was granted with predoctoral contract 'fpu' of university of murcia, spain. at has a post-doctoral fellowshisp "juan de la cierva incorporación" and de has a post-doctoral fellowshisp "juan de la cierva formación" supported by the "ministerio de economía y competitividad", spain. this work was supported by a grant from the program for research groups of excellence of the seneca foundation, murcia, spain (grant /germ/ ) and european commission era chair fp grant (vetmedzg # ). key: cord- - ipcdr authors: bouvette, jonathan; korkut, dursun nizam; fouillen, aurélien; amellah, soumiya; nanci, antonio; durocher, yves; omichinski, james g.; legault, pascale title: high-yield production of human dicer by transfection of human hek -ebna cells grown in suspension date: - - journal: bmc biotechnol doi: . /s - - - sha: doc_id: cord_uid: ipcdr background: dicer is a -kda protein that plays key roles in gene regulation, particularly as the ribonuclease iii enzyme responsible for cleaving precursor mirna substrates. its enzymatic activity is highly regulated by protein factors, and this regulation can impact on the levels of mirnas and modulate the behavior of a cell. to better understand the underlying mechanisms of regulation, detailed enzymatic and structural characterization of dicer are needed. however, these types of studies generally require several milligrams of recombinant protein, and efficient preparation of such quantities of pure human dicer remains a challenge. to prepare large quantities of human dicer, we have optimized transfection in hek - e cells grown in suspension and streamlined a purification procedure. results: transfection conditions were first optimized to achieve expression levels between and mg of recombinant dicer per liter of culture. a three-step purification protocol was then developed that yields – mg of purified dicer per liter of culture in a single day. from sec-mals/ri analysis and negative stain tem, we confirmed that the purified protein is monomerically pure ( ≥ %) and folds with the characteristic l-shape geometry. using an electrophoretic mobility shift assay, a dissociation constant (k(d)) of nm was measured for dicer binding to pre-let- a- , in agreement with previous reports. however, when probing the cleavage activity of dicer for pre-let- a- , we measured k(cat) ( . ± . min(− )) and k(m) ( . ± . μm) values that are much higher than previously reported due to experimental conditions that better respect the steady-state assumption. conclusions: the expression and purification protocols described here provide high yields of monomerically pure and active human dicer. cleavage studies of a pre-let- substrate with this purified dicer reveal higher k(cat) and k(m) values than previously reported and support the current view that conformational changes are associated with substrate binding. large quantities of highly pure dicer will be valuable for future biochemical, biophysical and structural investigations of this key protein of the mirna pathway. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. dicer is a multi-functional protein that plays a critical role in regulating several fundamental cellular processes, including rna interference, genome integrity, development, and antiviral immunity (for recent reviews see [ , ] ). it is a type iii endoribonuclease (rnase) that contributes to rna interference by processing specific hairpin structures and long double-stranded rnas into micrornas (mir-nas) and small interfering rnas (sirnas), respectively. in the mirna pathway, dicer is responsible for cleaving the precursor-mirna (pre-mirna) into a~ -nucleotide (nt) duplex, from which one strand will be loaded into the rna-induced silencing complex (risc) to repress mrna translation. currently, it is thought that dicer produces almost all mirnas in human cells (~ ), which together control over % of protein-coding genes [ ] [ ] [ ] [ ] [ ] . the pre-mirna processing activity of dicer is known to be highly regulated, and this likely contributes to precisely control mirna levels and thereby determine cell behavior and cell fate. the cleavage activity is regulated by protein cofactors, such as the tar rna binding protein (trbp) and the protein activator of the interferon-induced protein kinase (pact), as well as by several rna-binding proteins known to act either as activators or inhibitors (reviewed in [ ] ). moreover, high-throughput studies have identified additional non-coding rnas that are likely processed by dicer [ ] as well as several pre-mirna binding proteins that may regulate its cleavage activity [ ] [ ] [ ] . at this time, there is a need to integrate the available data into a coherent and detailed mechanistic understanding of dicer's activities and regulation via in vitro biochemical, biophysical and structural studies. however, for such research investigations to be carried out effectively, large quantities of highly pure and active recombinant proteins are needed. although bacterial overexpression systems generally provide a simple and fast method to obtain significant amount of recombinant proteins, it has not proven practical for human dicer ( kda). eukaryotic expression systems offer an important alternative for expression of such a large eukaryotic protein because they allow for proper folding and post-translational modifications (reviewed in [ ] ). over the past years, purification of recombinant human dicer following expression in insect cells (sf ) infected by baculovirus has been achieved with yields up to . - mg/l culture [ ] [ ] [ ] . this has allowed for the in vitro characterization of human dicer's enzymatic activity [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and provided the first descriptions of its three-dimensional structure by cryo-electron microscopy (cryo-em) at - Å-resolution [ ] [ ] [ ] . subsequently, the production method in insect cells was improved by systematic optimization of the overexpression and purification steps to yield milligram amounts ( - mg/l culture) of highly pure drosophila melanogaster dicer- (dmdicer- ) [ ] , which was used for its structure determination by cryo-em at -Å resolution. more recently, expression in hek cells grown in suspension was reported as part of a cryo-em study of human dicer that allowed structural reconstruction at . -Å resolution [ ] . this study provided unprecedented details into dicer's domain organization as well as its interaction with trbp and a pre-mirna substrate. however, the optimization of dicer production and the yields obtained were not reported. therefore, it is possible that large-scale expression from mammalian cells grown in suspension could be optimized to provide pure protein at higher yields than currently reported. such a procedure could be useful for future biochemical and structural investigations as there is still no x-ray or cryo-em structure of human dicer at atomic resolution and there are many questions that remain about its mechanism and regulation by protein factors. here, we describe an efficient and fast procedure to obtain milligram quantities of pure, monomeric and stable recombinant human dicer. to overcome the challenge of dicer over-expression, we optimized transfection and expression in an hek -ebna cell line. this allowed for purification of up to mg of dicer per liter of culture. a purification protocol was designed to minimize sample handling as well as to provide maximal recovery, purity and enzymatic activity. although we noticed that human dicer forms multimers at high concentration, our final three-step purification yields highly-pure monomeric dicer. the structure and activity of the purified dicer were assessed by size-exclusion chromatography coupled to multi-angle light-scattering and refractive index (sec-mals/ri), negative stain transmission electron microscopy (tem), binding assay and steady-state kinetics. our kinetic analysis for dicer cleavage of the pre-let- a- substrate were performed under strict steady state conditions and reveals k cat and k m values that are much higher than previously reported. overall, this new method will facilitate future biochemical, biophysical and structural characterization of dicer, including its interaction with rna substrates and protein regulators. to achieve high-level expression of recombinant human dicer, we relied on transient transfection of a suspensiongrown hek- cell line expressing the epstein-barr virus antigen- (hek -ebna or - e) with a ptt expression vector carrying a cmv promoter and the epstein-barr virus origin of replication orip [ ] [ ] [ ] . this mammalian expression system has been engineered for maximal expression of recombinant his-tagged proteins in serum-free media [ ] . linear polyethyleninime (pei) was selected as an efficient and cost-effective transfection reagent. for optimization of transfection conditions, small-scale suspension cultures were grown in serum-free media, either as -ml cultures in -well plates or -ml cultures in -ml disposable erlenmeyer flasks. in initial experiments where different quantities of plasmid dna ( , . and mg per l of culture) and pei:dna ratios ( : or : ) were tested, we found that optimal transfection efficiency could be reached when transfecting cells diluted to . × cells/ml h in advance using mg of dicer-expressing ptt plasmid with mg linear pei per l of culture. to estimate the transfection efficiency, we replaced % of the total transfected plasmid by a gfp-expressing plasmid and used fluorescence microscopy to count the percentage of gfp-expressing cells. transfection efficiencies of - % were typically reached at h post transfection (hpt) (fig. a) . such transfection efficiencies are considered very good especially when taking into account that they are underestimated compared to a transfection using % gfp-expressing plasmid [ ] . the cell cultures were monitored up to hpt to assess cell viability and viable cell density as well as the intracellular expression of recombinant dicer. it was found that cell viability decreases below % beyond hpt (fig. b, green) , whereas viable cell density is maintained at . × cell/ml (fig. b, black) and dicer expression levels are not significantly changed (fig. c) . similar results were obtained with large-scale transfections of up to l. thus, the optimum time for collecting the cells for protein purification was determined to be hpt, taking into consideration that additional time may lead to protein modifications that would adversely affect its activity. at hpt, volumetric expression of the intracellular dicer protein was ± mg per l of culture on average (fig. c) . an efficient multi-step purification protocol was developed to maximize purification yields (fig. b) . while optimizing this protocol, we noticed that dicer precipitated either at high concentration or when in contact with metallic surfaces such as stainless steel needles, fplc titanium alloy pump heads and the non-reactive hastelloy sample cell of the isothermal titration calorimeter. thus, the final purification protocol was optimized to avoid protein contact with metallic surfaces and minimize sample handling that could lead to protein aggregation and precipitation. large-scale expression of his-tagged dicer ( ml to l) was typically achieved in a . -l glass fernbach flask and harvested hpt. for cell lysis, the washed cell pellet was resuspended in a low salt buffer containing . % np- , allowing for lysis of the plasma membrane while leaving the nuclei intact. nuclei and cell debris were then removed by centrifugation and filtering of the resulting supernatant. the first step of purification consisted of an anion-exchange chromatography (q sepharose fast flow), which helped remove protein and nucleic acid contaminants (fig. c ). the addition of % glycerol in the elution buffers was found critical to purify dicer free of contamination with hsp (~ -kda intense band in lane of fig. f ), which is one of the most abundant protein in the hek- cell line [ ] . the dicer-containing fractions were pooled and directly loaded on a histrap hp column for purification by immobilized metal affinity chromatography (imac; fig. d ). this step, which was carried out in the presence of . m nacl and in the absence of glycerol, allowed for (fig. f ) . the dicer fractions were then loaded on a size-exclusion column (sec) to isolate the major monomeric population from multimeric contaminants. when this monomeric population of dicer was concentrated by ultrafiltration under standard conditions (e.g. mm tris ph . , mm nacl/kcl : , . mm mgcl and . mm tcep), it resulted in a significant loss of protein via precipitation. in addition, when dicer was concentrated prior to sec purification, substantial amounts of dicer multimers were detected in the sec elution profile. this led us to identify conditions that would allow for maximum protein recovery following concentration by ultrafiltration. we found that addition of sucrose and the non-ionic detergent n-dodecyl-β-dmaltoside (ddm) helped stabilize the monomeric form. thus, these reagents were added to the mobile phase during sec (fig. e , f ), and fractions containing monomeric dicer could be readily concentrated at up to . μm ( mg/ml) without precipitation. analysis of the purified protein by sds-page gels stained with coomassie blue shows greater than % purity (fig. f) , whereas western blot analysis indicate that~ % of the protein is recovered from the cytoplasmic fraction (fig. g ). yields of - mg of human dicer per liter of culture were typically obtained from large-scale preparations. size-exclusion chromatography coupled to multi-angle light-scattering and refractive index (sec-mals/ri) is a powerful tool to characterize purified biomolecules in solution; it allows accurate molecular weight determination as well as the determination of the oligomeric state in solution. in the sec-mals/ri experiments performed here, sec served only as a separation step, whereas the ls and ri detectors were used to calculate the molar mass of dicer after purification and concentration in a sucrose/ ddm-free buffer (fig. a) . although a small percentage of multimeric protein appears to be present in the void volume of the sec (elution volume~ ml), the purified dicer elutes predominantly as a single peak that corresponds to ≥ % of the total protein in the injected sample. the derived molecular weight for this peak based on light-scattering data is ± kda, which essentially matches the theoretical molecular weight of the histagged monomeric protein ( kda). the polydispersity index of . ± . over the entire eluted peak indicates that the main form of purified dicer is monodisperse, i.e. homogeneous with respect to molar mass. moreover, sec -mals/ri analysis of a purified dicer sample stored for months at − °c in sucrose/ddm-containing buffer shows that the purified protein remains almost exclusively monomeric ( ≥ %), indicating that the protein is intact after long-term storage (additional file : figure s ). is an efficient tool to evaluate the quality of a protein sample, providing information on the homogeneity and the three-dimensional shape of the biomolecule. tem micrographs of negative stain preparation of the purified dicer using uranyl formate illustrate particles of roughly equal size throughout the field of view, providing additional evidence of the homogeneity of the purified sample (fig. b) . model-free d class averages allowed us to observe the characteristic l-shape architecture of dicer, with two main unique subpopulations representing different forms of the enzyme, in agreement with previous studies [ , , ] . taken together, the sec-mals/ri and negative stain tem studies validate the monomeric l-shape architecture of the purified dicer and indicate that the conformational properties of the enzyme are preserved after long-term storage. binding and cleavage of pre-let- a- by dicer dicer is known to bind pre-mirna substrates and then cleave at two specific phosphodiester bonds to yield mirna- p/ p species. to evaluate the rna binding and cleavage activity of the purified recombinant dicer, we performed binding and kinetic studies using the pre-let- a- substrate, one of the most commonly used pre-mirna substrate for dicer characterization. binding studies were conducted via electrophoretic mobility shift assay (emsa) using p-labeled pre-let- a- and increasing concentration of dicer, either wild-type (wt) dicer supplemented with . mm edta to prevent cleavage or with the catalytically-inactive d / a variant (fig. ) . when fitting the binding data to the hill equation, dissociation constants (k d ) of ± nm (wt dicer) and ± nm (d a/d a dicer) were obtained with hill coefficient of . ± . and . ± . , respectively. to characterize the cleavage activity of the purified dicer enzyme, we determined the steady-state kinetic parameters k cat and k m under multiple turnover conditions. to ensure that the initial cleavage rates were measured under steady state conditions, the enzyme concentration ([e]; . nm to . nm) was varied along with the initial substrate concentration ([s]; . μm to . μm) to maintain [s]/[e] ≥ , and time points were collected after at least one turnover of the enzyme pool at up to % substrate cleavage, as previously reported for steadystate kinetic studies of e. coli rnase iii [ ] . for each substrate concentration, the cleavage reaction of p-labeled pre-let- a- was monitored by denaturing gel electrophoresis to quantify the percentage of cleavage (fig. a) . from this data, the concentration of product normalized against enzyme concentration ([let- a- ]/[dicer]) was plotted as a function of time, and the slope of the resulting time course was fitted by linear regression to derive the turnover frequency, expressed as v o /[e] t (fig. b) . by plotting and fitting to the michaelis-menten equation, a k cat value of . ± . min − and a k m value of . ± . μm were determined for cleavage of pre-let- a- by dicer (fig. c ). by optimizing transfection conditions, we have achieved high-level expression of recombinant human dicer using a - e cell expression system. in addition, a fast and efficient purification procedure was developed that yields up to mg of pure, monomeric and stable recombinant human dicer per liter of culture. to our knowledge this yield is higher than previously reported for human dicer, and it may be possible to obtain even higher yields by further optimizing transfection conditions, expressing dicer as a secreted protein, and/or developing an efficient stable transfection clone [ , , , ] . the reported purification protocol was designed to prevent protein aggregation and precipitation, since we observed that dicer tends to form multimers and precipitate at high-concentration or when it contacts metallic surfaces. nevertheless, the purified protein was shown to be monomerically pure and highly active. although affinity purification is often considered the best choice for the first purification step of a recombinant protein from a crude mixture, we selected an alternate approach to minimize sample handling that could lead to aggregation and precipitation, such as protein the relative signal of light scattering (red), refractive index (blue), and uv absorbance at nm (black) are represented by solid lines. the molar mass distribution is shown with green dots. a small amount of aggregated protein can be seen mainly from the light scattering signal. peak integration of the uv absorbance trace shows that the main peak contains ≥ % of the eluted protein. the molar mass calculated from mals analysis was normalized against bsa to give an average molecular weight of ± kda with a polydispersity index of . ± . over the entire eluted peak. (b) negative stain tem. left panel typical grid imaging of negatively-stained wt dicer. right panels main d class averages of wt dicer dialysis, desalting, and concentration. following cell lysis in low-salt buffer, the cytoplasmic fraction was first loaded on an anion-exchange column. the large volume and high salt concentration of the elution fractions from this first purification step is compatible with direct loading onto the histrap affinity column. subsequently, the small elution volume from the second purification step allowed for a single injection on the preparative size-exclusion column without any additional manipulations. overall, this strategy allows for multi-step purification of milligram amounts of recombinant dicer in a fast and efficient manner. in fact, the entire purification procedure can be conveniently completed in a single work day. it is important to note that the last step of purification can be performed either in the presence or absence of reagents (sucrose and ddm) that were found to prevent aggregation and precipitation at high protein concentration. these reagents are not required if the concentration of the protein eluted from the size-exclusion column, generally between . - μm, is sufficient for subsequent applications. however, if higher protein concentrations are required and/or long-term storage is desirable, these reagents should be included in the buffer used for size-exclusion chromatography and final protein concentration. the sec-mals results indicate that the purified dicer remains monomeric over six months of storage in sucrose/ddm-supplemented buffer. in agreement with this observation, similar thermodynamic and kinetic parameters (within two-fold) were measured over a period of nine months. in binding studies, we determined k d values of ± nm (wt dicer) and ± nm (d a/d a dicer) between dicer and pre-let- a- . these values are similar to previously reported k d values of . - nm obtained for the binding of wt dicer to pre-let- a- under similar conditions [ , , , ] . moreover, our studies confirm observations with other rnaseiii enzymes that the amino acid substitutions that disrupt magnesium binding in the rnaseiiia and rnaseiiib domains do not affect substrate binding to dicer [ ] . from kinetic studies of pre-let- a- cleavage by dicer, we obtained a k cat value of . ± . min − and a k m value of . ± . μm (k cat /k m = ± min − μm − ). these steadystate kinetic parameters are much higher than those derived from previous cleavage studies of the same pre-mirna substrate ( - nm) by dicer ( nm): k cat = . min − and k m = - nm (k cat /k m~ min − μm − ) [ ] . notably, we found similar values (k cat = . ± . min − and k m = nm ± nm; k cat /k m = ± min − μm − ) when performing kinetic experiments under similar conditions ( nm dicer and . - nm substrate; additional file : figure s ). however, the resulting curve did not fit a hyperbolic model, which is understandable given that the following condition, [s] ≫ [e], was not respected for every substrate concentration and, thus, did not agree with the steady-state assumption [ ] . by selecting experimental conditions that ensured the validity of the steady-state assumption ([s]/[e] ≥ ), much higher k cat and k m values are obtained, even though the k cat /k m is not substantially affected (additional file : table s ). these updated steady-state kinetic values provide insights into the kinetic mechanism of pre-mirna cleavage by dicer. given the k cat /k m of~ m − s − and the very low k cat value of~ . s − , the rate-limiting step may be product release as observed with other rnase iii enzymes [ , ] or possibly the slow formation and/ or conversion of the enzyme-substrate complex into a catalytically productive conformation. dicer is a complex and dynamic enzyme that is known to interact with several cellular proteins [ ] , and there is increasing evidence that conformational changes may be important for its cleavage activity. free dicer is known to adopt different conformations, as confirmed here from negative stain tem studies, and binding of pre-mirna or double-stranded rna substrates is linked to structural rearrangements [ , , , ] . furthermore, singlemolecule fluorescence studies have provided evidence for dicer adopting two different binding modes with a pre-let- a- substrate [ ] . more recently, cryo-em reconstructions of a pre-mirna/dicer/trbp complex captured two distinct conformations of a pre-dicing state, from which transition to the dicing state would require repositioning of the pre-mirna in the enzyme processing center [ ] . thus, additional studies are needed to better define the detailed kinetic mechanism of dicer and characterize the role that conformational changes play along the cleavage reaction pathway. we have achieved large-scale expression of recombinant human dicer in a human cell line. the expression in - e cells coupled with our streamlined purification protocol yields up to mg of protein per l of cell culture, which is higher than with previously reported protocols. the purification scheme was carefully optimized to minimize aggregation and maximize monomeric purity and stability of the final sample. to achieve these objectives, we minimized sample manipulation which, in turn, reduced the duration of the purification to a single work day. the purified enzyme adopts the typical l-shape architecture and binds a pre-let- a- substrate in the low nm range, as previously observed. steady-state kinetic studies yielded higher k cat and k m values than previously reported due to the use of experimental conditions that respected the steady-state assumption. together, these studies provide an efficient method for high-yield production of human dicer as well as revised kinetic parameters for dicer cleavage that should prove useful for future structural and mechanistic studies of dicer aimed at better understanding how the catalytic production of mirnas is achieved and regulated. for construction of the human dicer expression vector ptt -dicer, a pcr fragment encoding dicer was first generated from plasmid pfrt/to/flag/ha-dest dicer [provided as a gift from thomas tuschl (addgene plasmid # )] [ ] and inserted between the noti and hindiii sites of the ptt sh q vector to allow for expression of dicer with a c-terminal streptagii/his tag [ ] . the expression vector for the catalytically-inactive dicer d a/d a variant was prepared from ptt -dicer using the stratagene quikchangeii site-directed mutagenesis method. plasmids were amplified in escherichia coli (e. coli) dh α cells (invitrogen) grown in lb medium supplemented with mg/l amplicilin and purified using the qiagen plasmid giga kit. plasmids were stored at °c in water at a concentration of mg/ml. the puc -hh-pre-let- a- -hdv vector used for in vitro transcription of pre-let- a- was constructed using a puc vector, which allows for synthesis of pre-let- a- flanked by a ' hammerhead ribozyme (hh) and a ' hepatitis delta virus (hdv) ribozyme. plasmids were amplified in e. coli dh α cells grown in lb medium supplemented with mg/l ampicillin and purified using the qiagen plasmid maxi kit. plasmids were linearized with hindiii (new england biolabs) and stored as is at − °c at a concentration of mg/ml. all plasmids were verified by dna sequencing. hek- -ebna - e ( - e) cells were grown in suspension in freestyle (f ) medium (invitrogen) supplemented with mm glutamine, . % pluronic acid and μg/ml g at °c with % co . tryptone n was added to some of the cell cultures at a weight/volume ratio of . %, but was found to have minimal effect on cultures grown hpt. cultures were maintained under . × cells/ml in erlenmeyer flasks shaken at rpm in a humidified incubator at °c with % co . cell cultures were diluted to . × cells/ml h before transfection. the transfection mix was prepared in % of the final culture volume in f medium with μg/ml of dna plasmid and μg/ml of linear pei kda (polysciences inc.). the mix was agitated by vortexing cycles of s, incubated min at room temperature and incorporated in the culture [ ] , which was incubated for - h ( °c, % co ) with shaking ( rpm). for optimization of transfection conditions, small-scale cultures were used: -ml cultures were grown in -well plates (starstedt # . . ) and -ml cultures were grown in -ml disposable erlenmeyer flasks (corning # ). for dicer purification, large-scale cultures of ml to l were grown in thoroughly rinsed and sterilized -l glass erlenmeyer (corning # l) or . -l fernbach culture flasks (corning # ×l). the incubation time after transfection was fixed to h for large-scale cultures. at harvest time, cells were first counted and then pelleted by centrifugation at ×g and washed times with at least -pellet volumes of cold pbs. the washed cell pellet was either used immediately or frozen in liquid nitrogen and stored at − °c for later use. to optimize transfection conditions, small-scale transfections were monitored over a -day period by withdrawing a -μl aliquot from the culture every h to measure cell density, cell viability and recombinant dicer expression. from this aliquot, a -μl volume was mixed with an equal volume of trypan blue and loaded on a hemocytometer slide to determine the cell density and viability using a nikon eclipse ts microscope. the remaining μl of cell culture was pelleted, resuspended in μl of lysis buffer ( mm tris ph . , mm nacl, % glycerol, mm edta, mm egta, . % np- and . mm tcep [tris( -carboxyethyl)phosphine]) and spun for min at , ×g. the supernatant containing the cytoplasmic fraction was mixed with one volume of x laemmli buffer and stored at − °c. all samples collected during transfection were heated at °c for min, separated on a . % sds-page and blotted on a nitrocellulose membrane (bio-rad) for western-blot analysis. dicer was detected using a mouse anti-his tag primary antibody (medimabs #mm- -p), an hrp-conjugated anti-mouse secondary antibody (cedarlane # - - ) and the western lightning plus-ecl substrate (perkin-elmer #nel e ea). a chemidoc mp (bio-rad) imaging system was used for chemiluminescence detection, and band intensities were quantified using the imagelab software version . (bio-rad). transfection efficiency was monitored by substituting % of the total plasmid transfected by a ptt sh q -gfpq plasmid [ ] . after h, live cells were diluted with one volume of freestyle media supplemented with mm hepes ph . , and a thin layer of diluted cells was carefully deposited on a lab-tek-ii chamber slide. the slide was visualized by both phase contrast microscopy and by fluorescence microscopy on a nikon te u microscope ( × magnification). the ratio of cells expressing gfp over the total cells of at least cells was taken as the transfection efficiency. dicer-transfected cell pellets were resuspended in -pellet volumes of lysis buffer supplemented with one tablet of roche complete edta-free and incubated on ice for min. the in vitro transcription of human pre-let- a- with a '-hh ribozyme tag and a '-hdv ribozyme tag was carried out in mm hepes ph . , mm mgcl , mm dtt, mm spermidine, . % triton x- , mm of each ntp, μg/ml of linearized puc -hh-pre -let- a- -hdv plasmid, . u/ml of rnasin® plus rnase inhibitor and μg/ml of his-tagged t polymerase prepared in house [ ] . since both ribozyme tags were cleaved off during transcription, the pre-let- a- rna was generated with homogeneous ends directly from the transcription reaction. the rna was then purified by denaturing gel electrophoresis, as described previously [ ] . to convert the purified rna into an optimal dicer substrate with ′-phosphate and '-oh ends [ ] , a phosphorylation reaction was carried out with t polynucleotide kinase (neb #m s) according to the manufacturer protocol with either cold atp or atp-γ- p. the p-labeled pre-let- a- was further purified by denaturing gel electrophoresis, extracted from the gel by crush and soak, ethanol precipitated and resuspended in te ph . ( mm tris ph . and mm edta) [ ] . the cold phosphorylated rna was further purified by hplc at °c on a dnapac pa- × mm equilibrated with % dnapac-b buffer ( . mm tris ph . , m urea and . m naclo ) and % dnapac-a buffer ( . mm tris ph . and m urea). the sample was loaded and eluted with a linear gradient ( to %) of dnapac-b buffer. the fraction containing pure pre-let- a- was exchanged into te ph . and concentrated using a -kda mwco amicon® ultra- centrifugal filter unit (millipore). both the p-labeled and cold pre-let- a- samples were stored at − °c. human wt dicer final purification product was analyzed by sec-mals/ri using an akta micro fplc (ge healthcare) coupled to a multiple-angle light scattering and refractive index system (mals/ri; wyatt dawn heleos ii and optilab t-rex). a volume of μl ( - μg) of purified dicer was loaded on a superdex increase / (ge healthcare) column equilibrated in sucrose/ddm-supplemented storage buffer at a flowrate of . - . ml/min. the sec-mals/ri data was collected and analysed with astra chromatography software package, version . . . (wyatt technology). the mals detector was normalized using the monomeric and dimeric signals of bovine serum albumin (bsa; fisher scientific bp ). for the dicer sample purified in ddm-free buffer, the molar mass was determined by the dual detection method (ri/ls signals) implemented in the conjugated analysis mode of the astra software, using a refractive index increment (dn/dc) of . ml/g [ ] . a similar method was used for the dicer sample purified in ddm-containing buffer, however the dual detection method used the uv and ls signals, because the ri signal may be perturbed by ddm-dicer interactions. a -μl drop of nm human wt dicer in mm hepes, ph . , mm kcl, mm edta, mm dtt, and . % glycerol was applied onto a carbon-coated copper grid previously glow-discharged using an elmo glow discharge system (cordouan technologies, france). after min, excess liquid was blotted and stained for min with . % freshly prepared uranyl formate (electron microscopy sciences, pa). samples were imaged using a fei tecnai t (eindhoven, the netherlands) transmission electron microscope (tem) equipped with a lab filament and operated at an acceleration voltage of kv. micrographs were collected at defocus between and μm on a fei eagle k × k ccd camera at a magnification of~ , ×. from these micrographs, , particles were extracted and d align using xmipp from scipion [ ] . the particles were classified in a minimum of classes and the d volumes were measured using imagej [ ] . binding assays were carried using either wt dicer or the catalytically-inactive dicer variant (d a/d a). dicer d a/d a was first diluted in emsa buffer ( mm tris ph . , mm nacl and % glycerol), whereas wt dicer was diluted in emsa buffer supplemented with mm edta. in parallel, p-labeled pre-let- a- was refolded by heating at °c for min and snap-cooling at °c for a minimum of min. the rna was then diluted in . % np- and mm dtt to a concentration of pm. the binding reactions were initiated by adding μl of the rna sample to μl of the protein samples. standard binding reactions contained mm tris ph . , mm nacl, % glycerol, . % np- , mm dtt, and pm p-labeled rna ( . mm edta was added for reactions with wt dicer) with protein concentrations varying between . × and × of the estimated k d value. reactions were incubated for min at °c and loaded on a - % gradient polyacrylamide gel ( . : acrylamide:bis-acrylamide) in tris-glycine buffer ( mm tris-base and mm glycine), which was run for h at v in the cold room ( °c). gels were dried, exposed overnight to a storage phosphor screen (bio-rad) and visualized with a personal molecular imager (bio-rad). band intensities for the bound (b) and unbound (u) rna were quantified using the imagelab software version . (bio-rad). the fraction of bound rna [f = b/(b + u)] was plotted against protein concentration, and the binding data were fitted to the hill equation with the originpro software (originlab). the reported k d value and its errors represent the average and standard deviation from two independent experiments. for in vitro dicer cleavage assays, the rna and dicer solutions were prepared separately at twice their final desired concentration in μl of the cleavage reaction buffer ( mm hepes ph . , mm nacl, mm mgcl and . % np- ). the rna solutions, containing pm of p-labeled pre-let- a- and cold rna at concentrations ranging from . × to × the estimated k m , were heated at °c for min and snap-cooled at °c for a minimum of min to refold the rna. the concentration of the dicer solution was adjusted for each pre-let- a- substrate concentration to allow measurement of the initial velocity of the reaction ( - % substrate cleavage in min) under multiple turnover conditions (substrate/dicer molar ratio ≥ ). both rna and protein solutions were pre-heated for min at °c, and cleavage reactions were initiated by adding μl of the protein solution to μl of the rna solution. at specific time points, a -μl aliquot of the cleavage reaction was taken, immediately mixed with μl of stop buffer ( % formamide and mm edta). for each reaction, time points were taken at less than % reaction completion and over at least full turnovers of the enzyme pool to ascertain the possible use of the steady-state assumption. samples were analyzed by denaturating gel electrophoresis [ % acrylamide:bis-acrylamide ( : ) / m urea gel]. the amounts of substrate (s) and product (p) were quantified from radioactive bands as for the binding assay, and the fraction of cleaved product [f = p/ (s + p)] was plotted against time. the resulting time courses were fitted by linear regression and the slope was taken as the initial velocity additional file : an additional file named bouvette_additional_file .pdf is available that contain table s as well as figures s and s . table s . compares kinetic parameters obtained under conditions that respect or not the steady-state assumption. figure s . shows sec-mals analysis of purified wt dicer stored in sucrose/ ddm-containing storage buffer for months at − °c. figure s . molecular mechanisms of dicer: endonuclease and enzymatic activity dicer : mutations, micrornas and mechanisms annotating high confidence micrornas using deep sequencing data most mammalian mrnas are conserved targets of micrornas a dicer-independent mirna biogenesis pathway that requires ago catalysis a novel mirna processing pathway independent of dicer requires argonaute catalytic activity biogenesis of mammalian micrornas by a non-canonical processing pathway regulation of microrna biogenesis extensive terminal and asymmetric processing of small rnas from rrnas, snornas, snrnas, and trnas aribo pull-down for riboproteomic studies based on label-free quantitative mass spectrometry a compendium of rnabinding proteins that regulate microrna biogenesis systematic discovery of rna binding proteins that regulate microrna levels eukaryotic expression systems: a comparison ribonuclease activity and rna binding of recombinant human dicer human dicer preferentially cleaves dsrnas at their termini without a requirement for atp recombinant dicer efficiently converts large dsrnas into sirnas suitable for gene silencing the contributions of dsrna structure to dicer specificity and efficiency autoinhibition of human dicer by its internal helicase domain a role for the dicer helicase domain in the processing of thermodynamically unstable hairpin rnas substratespecific kinetics of dicer-catalyzed rna processing a comprehensive analysis of precursor microrna cleavage by human dicer differential roles of human dicer-binding proteins trbp and pact in small rna processing structure of precursor microrna's terminal loop regulates human dicer's dicing activity by switching dexh/d domain revealing a new activity of the human dicer duf domain in vitro in vitro reconstitution of the human risc-loading complex structure of the human dicer-trbp complex by electron microscopy substrate-specific structural rearrangements of human dicer structural insights into rna processing by the human riscloading complex overexpression and purification of dicer and accessory proteins for biochemical and structural studies cryo-em structure of human dicer and its complexes with a pre-mirna substrate high-level and high-throughput recombinant protein production by transient transfection of suspensiongrowing human -ebna cells transfection of hek -ebna cells in suspension with linear pei for production of recombinant proteins culture of hek -ebna cells for production of recombinant proteins purification of recombinant proteins from mammalian cell culture using a generic double-affinity chromatography scheme transient gene expression in suspension hek -ebna cells profiling core proteomes of human cell lines by one-dimensional page and liquid chromatography-tandem mass spectrometry the molecular architecture of human dicer pre-steady-state and stopped-flow fluorescence analysis of escherichia coli ribonuclease iii: insights into mechanism and conformational changes associated with binding and catalysis cost-effective gene transfection by dna compaction at ph . using acidified, long shelf-life polyethylenimine comparative study of polyethylenimines for transient gene expression in mammalian hek and cho cells trbp alters human precursor microrna processing in vitro coordinated activities of human dicer domains in regulatory rna processing defining the enzyme binding domain of a ribonuclease iii processing signal. ethylation interference and hydroxyl radical footprinting using catalytically inactive rnase iii mutants ribonuclease iii mechanisms of double-stranded rna cleavage single processing center models for human dicer and bacterial rnase iii trbp ensures efficient dicer processing of precursor microrna in rna-crowded environments molecular characterization of human argonaute-containing ribonucleoprotein complexes and their bound target mrnas the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme affinity purification of t rna transcripts with homogeneous ends using aribo and crispr tags role of slv in sli substrate recognition by the neurospora vs ribozyme dicer recognizes the ′ end of rna for efficient and accurate processing on the distribution of protein refractive index increments scipion: a software framework toward integration, reproducibility and validation in d electron microscopy nih image to imagej: years of image analysis -beyond protein family and domain annotations structure of the arabidopsis thaliana dcl duf domain reveals a noncanonical doublestranded rna-binding fold for protein-protein interaction a phosphatebinding pocket within the platform-paz-connector helix cassette of human dicer a structure-based model of rig-i activation we thankfully acknowledge alexandre desjardins and louise cournoyer for technical help with cell culture and transfection, geneviève di tomasso and pierre dagenais for technical assistance with protein and rna purification, aleksandr sverzhinsky and sébastien truche for technical assistance with sec-mals/ri data collection. the research was funded by a discovery grant from the natural sciences and engineering research council of canada (nserc) to pl and the canada foundation for innovation (cfi) for sec-mals/ri. these funding bodies did not play any role in the design of the study, the collection, analysis, and interpretation of data and in writing the manuscript. all data supporting our findings are displayed in the submitted manuscript.authors' contributions jb developed, performed and analyzed most of the experiments: the cloning, cell culture and transfection conditions, dicer purification, pre-let- a- synthesis and purification, dicer binding and processing assays as well as one sec-mals/ ri experiment (additional file : figure s ). dnk performed dicer purification and sec-mals/ri data collection and analysis (fig. a) . af performed all negative stain tem experiments and analysis (fig. b) . sa provided technical assistance to jb for cell culture, dicer purification and dicer binding assays (fig. c-g and fig. a) . an, yd, pl and jgo made substantial contribution to the conception and design, and/or analysis and interpretation of the data. jb and pl wrote the manuscript. all authors revised and approved the final manuscript.ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - ceqdzgc authors: minney-smith, c. a.; selvey, l. a.; levy, a.; smith, d. w. title: post-pandemic influenza a/h n pdm is associated with more severe outcomes than a/h n and other respiratory viruses in adult hospitalisations date: - - journal: epidemiology and infection doi: . /s x sha: doc_id: cord_uid: ceqdzgc this study compares the frequency and severity of influenza a/h n pdm (a/h ), influenza a/h n (a/h ) and other respiratory virus infections in hospitalised patients. data from adult hospitalised patients admitted to sir charles gairdner hospital, perth, western australia, with a respiratory illness between and were linked with data containing reverse transcription polymerase chain reaction results for respiratory viruses including a/h , a/h , influenza b, human metapneumovirus, respiratory syncytial virus and parainfluenza. of these, ( . %) had test results. multivariable regression analyses were conducted to compare the viruses for clinical outcomes including icu admission, ventilation, pneumonia, length of stay and death. patients with a/h were more likely to experience severe outcomes such as icu admission (or . , % ci . – . , p = . ), pneumonia (or . , % ci . – . , p < . ) and lower risk of discharge from hospital (indicating longer lengths of hospitalisation; hr . % ci . – . , p = . ), than patients with a/h . patients with a non-influenza respiratory virus were less likely to experience severe clinical outcomes than patients with a/h , however, had similar likelihood when compared to patients with a/h . patients hospitalised with a/h had higher odds of severe outcomes than patients with a/h or other respiratory viruses. knowledge of circulating influenza strains is important for healthcare preparedness. influenza infections have a large impact on human morbidity and mortality each year [ ] . this results in a substantial additional burden of hospital admissions each winter, both internationally and within australia [ ] . the severity and clinical impact of an influenza season varies from year to year and depends on a number of factors such as vaccination efficacy and circulating influenza strain. early evidence analysing the h n pandemic suggested that influenza a/h n pdm (a/h ) may have more severe clinical impacts than other seasonal influenzas [ , ] . this evidence continues to be supported in the post-pandemic years with several studies showing that patients with a/h have an increased risk of severe disease compared to patients with influenza a/h n (a/h ) [ , ] . in addition to influenza, there is also growing evidence of the importance of other viruses causing respiratory illnesses resulting in hospitalization [ , ] and for which there are currently no vaccines and few treatment options available [ , ] . prominent among these are respiratory syncytial virus (rsv), human rhinoviruses, human coronaviruses, human metapneumovirus (hmpv) and parainfluenza viruses (piv) on hospitalisation. most studies have been conducted in children, but it appears that these other viruses are likely to be of similar importance in adults [ , [ ] [ ] [ ] [ ] . describing the impact of specific influenza subtypes, as well as non-influenza viruses have on hospital admissions for adults will aid in assessing the expected population health benefits of influenza vaccines, and for any future vaccines and treatments for other respiratory viruses. this is particularly important in our aging population with an increasing prevalence of chronic diseases, as the impacts of respiratory virus epidemics will likely become more severe as the population ages [ ] . to the best of our knowledge, no study has investigated the frequency and severity of influenza subtypes as well as a broad range of other respiratory viruses in adults hospitalised in australia. this information is important in defining the health burden of all respiratory viruses, not only on the frequency of these admissions, but also for the clinical outcomes and the level of care required for these patients. the aim of this study was to investigate the role of different respiratory viruses in the severity of outcomes among patients admitted to an adult tertiary teaching hospital in perth, western australia. this was a retrospective cohort study analysing data from electronic records of patients admitted to sir charles gairdner hospital (scgh) from january to december . scgh is a large adult tertiary teaching hospital with over beds located in perth, western australia. it treats approximately inpatients and emergency department presentations annually [ ] . patients aged years and above who were admitted to scgh with a respiratory illness were included in this study. the hospital information system data from that time period were searched using the following codes from the international classification of diseases, tenth revision, clinical modification (icd- -cm) to capture all patients presenting with a possible respiratory illness: j -j (acute upper respiratory infections), j -j (influenza and pneumonia), j -j (other acute lower respiratory infections), j -j (chronic lower respiratory diseases), j (acute respiratory distress syndrome) and j (pulmonary oedema). icd- -cm codes were selected to capture all patients who may have received a respiratory viral test. data from patients meeting the search criteria in their primary diagnosis code, or in up to four further diagnosis/complication codes, were retrieved from the information system. these codes were also used to determine if patients had comorbidities such as chronic obstructive pulmonary disease (copd), cardiovascular disease (cvd), diabetes, compromised immunity, asthma or cystic fibrosis. although pregnancy and obesity are recognised risk factors for severe influenza, they were not included in this study due to very low numbers of patients identified through the icd- codes. available patient data also included patient demographics, admission and discharge dates, discharge codes and number of hours spent in icu or on ventilation. virological testing data were obtained from pathwest laboratory medicine wa (pathwest), who perform routine virological testing for scgh. during the study period samples were tested for a/h , a/h , inf-b, piv and rsv using a tandem multiplex reverse transcription polymerase chain reaction using primers and probes from a previously described method [ ] , with an additional target for the hmpv matrix gene [ ] . an scgh patient record was matched with a pathwest record if the patient received virological testing within days either side of their hospital admission date. records were matched using either unit medical record number and date of birth, or name and date of birth. any patients with multiple samples taken during the same admission were matched to the first sample collected that was positive for a virus. multiple admissions occurring within days of the first admission date were treated as a single admission for this study, otherwise they were analysed as separate admissions. data analysis was undertaken using stata (college station, texas, usa). age was categorised into three categories ( - years, - years and plus) to reflect current australian recommendations for influenza vaccination [ ] . for univariate analysis of categorical variables, relative risks and % confidence intervals (ci) were calculated and χ or fisher's exact test were used where appropriate to assess statistical significance of relative risks. we used the students t test or mann-whitney u test to assess statistical significance of differences in continuous variables between groups. severity was compared between a/h , a/h and the other viruses using seven outcome variables: diagnosis of pneumonia, admission to icu, requirement for assisted ventilation, time to discharge, time in icu, time on assisted ventilation and death. multivariable models (logistic or cox regression) were constructed for each outcome, comparing patients with inf-b, rsv, hmpv or piv to those with a/h or a/h . for the categorical outcome variables of icu admission, ventilation and pneumonia (all yes/no), logistic regression was used. we used cox proportional hazards regression to assess differences in the risk of discharge between patients with different viral infections at any particular point in time, as an indicator of length of hospitalisation. deceased patients were excluded from the cox regression models. patients with no virus detected, multiple viruses detected or influenza a unsubtyped, were excluded in all multivariable models. a p-value less than . was considered significant for all tests. the following variables were included in the initial model: virus type, age group, sex, copd, cvd, diabetes, immunocompromised, asthma and cystic fibrosis. variables were removed consecutively from the model in order of descending p-values, so that they were only retained if they were significant (p < . ) or showed evidence of confounding, with the exception of sex and age group which were retained for all models. a variable was determined to be a confounder if the difference between the adjusted and unadjusted or was greater than %. ethics approval was granted by scgh and curtin university human research ethics committees (approval numbers - and respectively). there were cases of possible respiratory illness admitted to scgh from january to december . of these, ( . %) were tested for respiratory viruses. patients who were tested were younger than patients who were not tested (median y.o. vs. y.o., p < . ). there was no significant difference in the presence of comorbidities between patients who were and were not tested ( . % vs. . % respectively, p = . ), however those that were tested were more likely to have been admitted to icu than those not tested ( . % vs. . %, p < . ). of the patients who were tested, ( . %) tested positive for at least one virus (table ) . a/h was the most common, being detected in ( . %) patients. the median age of patients with a virus detected was years. patients with a/h were younger than patients with a/h (χ = . , p < . ), inf-b (χ = . , p = . ), rsv (χ = . , p < . ), hmpv (χ = . , p = . ) or piv (χ = . , p < . ) with only . % of patients being over years (table ) , compared to between . % and . % for the other viruses. eleven patients ( . %) had two viruses detected and three patients had an influenza a virus which could not be subtyped. of the patients with a virus, ( . %) were tested either prior to, or within days of hospital admission, indicating detection of possible nosocomial cases to be uncommon. comorbidities were present in approximately half of the hospitalised patients, and this did not differ significantly between those with or without a virus detected ( . % and . % respectively, p = . ). the commonest comorbidities amongst the patients who tested positive for one of these viruses were copd in . %, and diabetes in . % (table ) . compared with patients with a/h , comorbidities were more common in those who had hmpv ( . % vs. . %, rr . , % ci . - . , p = . ) or rsv ( . % vs. . %, rr . , % ci . - . , p = . ). a higher proportion of admitted patients with hmpv had compromised immune function (rr . , % ci . - . , p = . ) compared to admitted patients with a/h and a higher proportion had asthma (rr . , % ci . - . , p = . ) compared to patients with a/h . a higher proportion of admitted patients with rsv had copd compared to admitted patients with a/h or a/h (table ; rr . , % ci . - . , p = . and rr . % ci . - . , p = . respectively). patients with a/h had a higher risk of being admitted to icu (rr . % ci . - . , p = . ), requiring ventilation (rr . , % ci . - . , p = . ) or developing pneumonia (rr . % ci . - . , p < . ) than did patients with a/ h . these patients also had longer hospitalisation time than patients with a/h (log rank χ = . , p = . ; table ). these differences remained significant in multivariable analyses for icu admission (or . , % ci . - . , p = . ; table ), pneumonia (or . , % ci . - . , p < . ; table ) and risk of discharge (hr . , % ci . - . , p = . ; table ). similarly, compared with a/h many of the non-influenza a viruses had less severe outcomes for icu admission, ventilation or pneumonia ( when compared to a/h , the non-influenza a viruses did not show any statistical differences in severity outcomes (tables - ), with the possible exception of piv which, in patients with pneumonia, had borderline higher odds of being detected than a/h (or . % ci . - . , p = . ) in multivariable analysis (table ). this study is one of the few to undertake a detailed comparison of admissions and clinical outcomes of the individual influenza virus types and subtypes and the non-influenza viruses in hospitalised adults. we used data retrieved from electronic health records to identify the respiratory viruses found in adults admitted to a tertiary hospital in australia during the period - , and used influenza a viruses as a comparator to assess the severity of illness due to the other respiratory viruses. overall a/h was the most frequently detected virus in adults admitted to our hospital for a respiratory illness. furthermore, either a/h or a/h was the dominant or equally dominant virus in every year of the study. that is consistent with the documented major role of influenza a viruses as causes of more severe influenza illness in adults, especially the elderly [ ] . however, across the study period, the total patients with non-influenza a viruses outnumbered those with influenza a viruses, and the numbers of hospitalised cases with rsv and inf-b were equal to that of a/h in . previous studies investigating the number of hospitalisations for non-influenza respiratory viruses are conflicting, with some studies showing the number of hospitalisations for influenza are greater than for rsv or hmpv [ , , [ ] [ ] [ ] , while others found that respiratory hospitalisations for rsv and/or hmpv are similar to influenza [ ] [ ] [ ] [ ] . this may vary according to the circulating influenza strains in each season. patients infected with a/h had more severe clinical outcomes than those infected with a/h , inf-b, rsv or hmpv. however, patients with a/h had similar clinical outcomes to the noninfluenza viruses. this indicates that these viruses contribute substantially to the healthcare burden of respiratory viruses. previous research has shown that for patients infected with respiratory viruses other than influenza who are admitted to hospital, the outcomes can be just as severe as for those with influenza, with similar rates of icu admission, ventilation and death [ ] . hospitalised rsv cases in particular have similar or greater odds of pneumonia, icu admission, ventilation and death compared to influenza [ , , , , ] . we showed that patients with a/h were more likely to be admitted to icu, develop pneumonia and be hospitalised longer than patients with a/h , indicating that a/h is more severe than a/h in this population, despite this virus affecting younger ages (mean age vs. years for h and h patients respectively). in , patients with a/h outnumbered those with [ ] [ ] [ ] [ ] ] , even though a/h was the dominant subtype causing hospital admission. in previous influenza pandemics, the pandemic strain has been associated with a high proportion of deaths in patients under years, which continues, albeit at lower proportions, in the decade following the pandemic [ ] . our study provides further evidence that post-pandemic influenza can still cause severe disease in young adults. however, in other populations a/h has been a larger contributor to severe illness [ ] , but this can vary with age [ , ] , so the impact of influenza a will differ from season-to-season [ , ] . in our multivariable analyses we adjusted for age, and therefore this impact may not be apparent. overall, comorbidities were common, being present in approximately half of the admitted patients. however, the presence of comorbidity was significantly less likely in patients with a/h , compared to those with rsv or hmpv. co-morbidities were also less frequently present in patients with a/h , however statistical significance was not reached (p = . and . for hmpv and rsv respectively). the lower number of comorbidities in patients with influenza may be due to a high proportion of people with comorbidities receiving the annual influenza vaccine, but vaccination status for the patients in our study was not available. another possible explanation for our findings is that influenza a is capable of causing hospitalisation in relatively healthy people, whereas the other viruses may cause less severe disease in patients without comorbidities. this study had a number of limitations. as this was a retrospective study, we could not investigate the impacts of viruses that were not routinely tested for. some patients received additional testing for other respiratory viruses such as adenovirus, rhinovirus and coronavirus, but as the number of patients with these tests was low, and there may be a selection bias for those who received extra testing, these were not included in the study. rhinovirus in particular has been implicated in previous studies as a possible cause of severe respiratory disease, having been detected in a large proportion of hospitalised patients [ , , , ] . in addition it has also been shown that influenza a and rhinovirus infections have similar measures of severity in immunocompromised patients [ ] . this study did not take into account the effect of influenza vaccination on our population. in , influenza vaccination coverage rates in australian hospitalised patients with acute respiratory illness was estimated to be . % in patients aged over , and . % in adults under with comorbidities, with the proportion of patients requiring icu being slightly lower in the vaccinated group than the unvaccinated group ( . % vs. . %) [ ] . influenza vaccine effectiveness against hospitalisation in the elderly and people with comorbidities varies according to the matching of the vaccine strains with circulating strains [ ] . while we found that non-influenza respiratory viruses can cause just as severe disease like influenza, we cannot say if this would be the case in an unvaccinated population. it is possible that influenza vaccination reduced both the frequency and severity of influenza hospitalisations, therefore this should be taken into account when interpreting the results of this study. we did not have information about the influenza vaccination status or antiviral treatment for the cases in our study. therefore we are unable to assess whether either treatment or vaccination attenuated the clinical picture of influenza cases. in addition, while attempts were made to minimise detections of nosocomial influenza, the presence of nosocomial cases in our study cannot be excluded. the study also did not consider the overall prevalence of influenza a viruses in the broader population during the study period. therefore, we are unable to determine whether the proportion of hospitalised cases with the different influenza a strains reflected the circulation of these strains in the population or severity of infection with each strain. also the relative impact of influenza vs. the other respiratory viruses will vary across time depending on the comparative levels of activity of the different viruses with the community. while our study involved hospital admissions, the proportion of patients tested for respiratory viruses was small ( %). the selection of icd- -cm codes used in this study to identify patients with respiratory illness was broad, which may account for the small proportion of testing. in addition, pathology testing for respiratory viruses was proportionally higher in winter compared to the non-winter months in our study (data not shown). infections during the non-winter months may have been missed due to lower rates of testing [ ] . in our study the rate of icu admission among tested patients was higher than that of untested patients, therefore we cannot discount that a selection bias may exist for testing of severely ill patients which could skew results. in addition insufficient power may have been a limitation of this study. additional studies are needed to further investigate the level of severity of disease in hospitalised cases with noninfluenza respiratory virus infections. we have found that hospitalised patients with influenza a/h had more severe clinical outcomes than patients with a/h , inf-b and the non-influenza respiratory viruses. knowledge of the influenza strains circulating and their associated clinical outcomes will aid in hospital preparedness during the influenza season. future research is needed to investigate the impact of a broader range of viruses associated with respiratory hospitalisations. supplementary material. the supplementary material for this article can be found at https://doi.org/ . /s x. estimates of global seasonal influenza-associated 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individual-level severity of seasonal influenza a h n compared with pandemic h n increased mortality in seasonal h n patients compared with those with pandemic h n in taiwan epidemiology and relative severity of influenza subtypes in singapore in the post-pandemic period from differences in the epidemiological characteristics and clinical outcomes of pandemic (h n ) influenza, compared with seasonal influenza concurrent comparison of epidemiology, clinical presentation and outcome between adult patients suffering from the c. a. minney-smith et al. pandemic influenza a (h n ) virus and the seasonal influenza a virus infection severity of human rhinovirus infection in immunocompromised adults is similar to that of h n influenza influenza epidemiology in patients admitted to sentinel australian hospitals in : the influenza complications alert network variable influenza vaccine effectiveness by subtype: a systematic review and meta-analysis of test-negative design studies respiratory viruses in adults hospitalised with community-acquired pneumonia during the non-winter months in melbourne: routine diagnostic practice may miss large numbers of influenza and respiratory syncytial virus infections acknowledgements. the authors would like to acknowledge jason fong for the provision of scgh data for this project and the staff in the pathwest molecular diagnostics laboratory for the pathology testing.financial support. no funding was received for this study. key: cord- - jhka wl authors: zhang, jialin; chen, jianfei; liu, ye; da, shi; shi, hongyan; zhang, xin; liu, jianbo; cao, liyan; zhu, xiangdong; wang, xiaobo; ji, zhaoyang; feng, li title: pathogenicity of porcine deltacoronavirus (pdcov) strain nh and immunization of pregnant sows with an inactivated pdcov vaccine protects ‐day‐old neonatal piglets from virulent challenge date: - - journal: transbound emerg dis doi: . /tbed. sha: doc_id: cord_uid: jhka wl in this study, the pathogenicity of porcine deltacoronavirus (pdcov) strain nh (passage , p ) was evaluated. we found that pdcov strain nh is enteropathogenic in ‐day‐old pigs. pathogenicity experiments provided a challenge model for studying the protection efficiency of passive immunity. in order to investigate the protective efficacy of passive immunity in newborn piglets, pregnant sows were vaccinated with either a pdcov‐inactivated vaccine at the houhai acupoint (n = ) or dmem as a negative control (n = ) using a prime/boost strategy and days before delivery. pdcov spike (s)‐specific igg and neutralizing antibody (na) responses were detected in immunized sows and piglets born to immunized sows. pdcov spike (s)‐specific siga was also detected in the colostrum and milk of immunized sows. five days post‐farrowing, piglets were orally challenged with pdcov strain nh ( ( ) tcid( )/piglet). severe diarrhoea, high levels of viral rna copies and substantial intestinal villus atrophy were detected in piglets born to unimmunized sows. only of piglets ( . %) born to immunized sows in the challenge group displayed mild to moderate diarrhoea, lower viral rna copies and minor intestinal villi damage compared to piglets born to unimmunized sows post‐challenge. mock piglets exhibited no typical clinical symptoms. the challenge experiment results indicated that the inactivated pdcov vaccine exhibited . % protective efficacy in the piglets. these findings suggest that the inactivated pdcov vaccine has the potential to be an effective vaccine, providing protection against virulent pdcov. states wang, byrum, & zhang, ) , followed by subsequent outbreaks in canada (ojkic et al., ) , south korea (lee et al., ) , thailand (janetanakit et al., ; saeng-chuto et al., ) and mainland china (dong et al., ) , exhibiting a global distribution trend. additionally, clinical reports have indicated that pdcov exhibits enteropathogenicity, causing severe diarrhoea and vomiting in roughly -to -day-old gnotobiotic and conventional piglets . pathological damage to the intestine, primarily in the jejunum and ileum, was characterized by intestinal villi atrophy and shortening and was confirmed by the pathogenicity experiments wang, hayes, sarver, byrum, & zhang, ) . such changes are clinically difficult to distinguish from the pathological changes caused by the porcine epidemic diarrhoea virus (pedv) and transmissible gastroenteritis virus (tgev) (jung et al., ; zhang, ) . pdcov infections have resulted in great economic losses for the global swine industry. therefore, fast and effective preventive measures are essential for the prevention and control of pdcov. currently, implementing vaccines remain the most effective means of disease control; however, there are no commercial vaccines available for pdcov. due to their immature immune system, neonatal piglets are highly susceptible to viral infection during their first few weeks of life. studies suggest that passive immunity is the most effective approach for protecting piglets from viral infection (langel, paim, lager, vlasova, & saif, ; leidenberger et al., ) . immunized sows can transfer antibodies against enteroviruses (e.g. pedv, tgev and porcine rotavirus) to neonatal piglets through their colostrum and milk. the protective efficiency of passive immunization has a high positive correlation with antibody levels in the colostrum and milk (sestak, lanza, park, weilnau, & saif, ) . therefore, passive immunity of newborn piglets can be achieved by immunizing sows that produce high levels of neutralizing antibodies (na) and transfer these antibodies through the colostrum and milk to the nursing piglets, which may be an effective means of controlling viral infection. in this study, a challenge model was established and the results indicated that pdcov strain nh (p ) is pathogenic to -day-old specific pathogen-free (spf) piglets. then, an inactivated pdcov vaccine was prepared and the immune responses and protective efficiency of the inactivated pdcov vaccine in pregnant sows was evaluated. after two doses of the vaccine, pregnant sows produced strong igg and na responses specific to pdcov s proteins. high levels of igg antibodies and na were also detected in the serum of neonatal piglets born to immunized sows, which suggests that the antibodies were successfully transferred through the colostrum and milk. five-day-old piglets were challenged with virulent pdcov to assess the protective efficacy of the vaccine, and the findings indicated that the inactivated pdcov vaccine provided . % protective efficacy. pdcov strain nh was isolated from pdcov-positive specimens with llc-pk cells (atcc ® cl- ™), and plaques were purified twice. pdcov strain nh was continuously passaged in swine testis (st) cells in dmem containing μg/ml tosylsulfonyl phenylalanyl chloromethyl ketone (tpck)-trypsin (invitrogen). at hr post-infection, both the supernatant and cells were harvested, titrated and stored at − °c for future analyses. the titre of pdcov strain nh ( th passage, p ) was tcid /ml, which was used for the challenge experiment. the inactivated pdcov vaccine was prepared using the th generation of viruses due to its low mutation rate and similar antigenicity compared to the parent virus. viral culture supernatants were inactivated with beta-propiolactone containing aluminium hydroxide adjuvant at a : ratio in order to prepare the inactivated pdcov vaccine. ten -day-old spf piglets were confirmed negative for pdcov, pedv, tegv and rpv by virus-specific pcr. pigs were maintained in germ-free isolation units of the animal facility located at the harbin veterinary research institute under standard conditions prescribed by the institutional guidelines. piglets were fed suckling piglet formula every hr. six of the spf piglets (piglets # , # , # , # , # and # ) were assigned to the pdcov-inoculation group, which were orally inoculated with the pdcov strain nh (p ) ( tcid /pig). the remaining piglets (piglets # , # , # and # ) were orally inoculated with volume-matched virus-negative culture medium and served as the negative control group. the clinical signs of vomiting and diarrhoea were evaluated every hr. the level of diarrhoea severity was scored for each piglet using the following criteria: = no vomiting or diarrhoea; = mild; = moderate; = severe. faecal swabs were collected for detecting viral rna. duplicate tissues for the duodenum, jejunum, ileum, caecum, colon and rectum were collected for viral rna detection and histological examinations. seven sows were confirmed to be negative for pdcov, pedv, tegv and rpv using virus-specific pcr and a serum neutralization test. all of the sows were randomly assigned to experimental groups: ( ) the immunization group (pdcov-inactivated vaccine; n = ); or ( ) the control group (dmem; n = ). sows from the immunization group were immunized with ml inactivated pdcov vaccine (pdcov strain nh p , equivalent to × tcid ) at the houhai acupoint (a concave part between the anus and tail). vaccination in the houhai acupoint was found to be helpful for inducing humoral and mucosal immune responses (li et al., ; liu, tan, wan, zuo, & liu, ) . all of the sows were immunized days prior to delivery, and a booster was administered days before delivery. sows from the control group were immunized with ml dmem as described above (table ) . serum was collected from sows (prior to the first immunization), , , , and days post-immunization. serum samples were inactivated at °c for min and stored at − °c for future analyses. milk was collected from the sows - days post-farrowing. three -day-old piglets from each pdcov-vaccinated gilts or mock gilts were permitted to continue drinking breast milk in order to detect the level of serum antibodies in piglets, while the other piglets were separated from the sows and housed in the experimental animal centre. separated piglets were fed suckling piglet formula every hr. newborn piglets ( or piglets from each sow were set as the control group) derived from pdcov-vaccinated gilts or mock gilts were challenged orally with tcid of pdcov strain nh (p ). the clinical signs of vomiting and diarrhoea were evaluated daily. diarrhoea severity was scored for each piglet using the following criteria: = no vomiting or diarrhoea; = mild; = moderate; = severe. faecal swabs were collected for the detection of viral rna. serum was collected from the breastfed piglets , , and days post-birth. the study protocol was approved by the institutional animal care and use committee of the harbin veterinary research institute. faecal swabs were centrifuged at , ×g for min at °c, and μl supernatant was collected for viral rna extraction. viral rna was extracted using a qiaamp ® viral rna mini kit (qiagen, hilden, germany) following the manufacturer's instructions. one hundred mg tissue samples from each piglet were ground in liquid nitrogen, and the total rna was extracted using an rnaiso plus kit (takara, kusatsu, japan) following the manufacturer's instructions. rna was then used to perform real-time (rt)-qpcr using specific primers and probes (pdcov-n-f: cgcttaactccgccatcaa; pdcov-n-r: tctggtgtaacgcagccagta; pdcov-n-probe: fam-cccgttgaaaacc-mgb) as previously described with minor modifications (ma et al., ) . briefly, μl rna was used in a μl pcr reaction system using a one step primescript™ rt-pcr kit (takara, kusatsu, japan) in a lightcycler (roche applied science, in, usa) under the following conditions: one cycle at °c for min and °c for s, followed by cycles at °c for s and °c for s. the duodenum, jejunum, ileum, caecum, colon and rectum tissues were cut into -μm-thick sections, mounted onto glass slides and blocked with % non-fat dry milk in pbs for min at °c. then, slides were incubated for min with a mouse polyclonal anti-pdcov s antibody followed by incubation with an alexa fluor ® donkey anti-mouse igg (sigma-aldrich, mo, usa) for min. nuclei were stained with dapi, and samples were observed with an inverted fluorescence microscope. tissues from intestinal tissues were fixed in formalin for hr and embedded in paraffin wax following standard methods. ihc detection of pdcov antigens was performed using an anti-pdcov-n monoclonal antibody prepared in the laboratory followed by an incubation period with horseradish peroxidase (hrp)-conjugated sheep anti-mouse igg (sigma-aldrich) for min at room temperature. reactions were developed with , '-diaminobenzidine (dab). distilled water was used to finish staining and stained with haematoxylin (he). dehydration, clearing and mounting were conducted with neutral gums. the extracellular domain of the pdcov spike gene was amplified and cloned into the pcagg vector with a c-terminal flag tag. recombinant s protein was expressed in t cells, purified using anti-dykddddk g affinity resin (genscript: l - ) and used as the coating antigens. pdcov s-specific igg, iga and siga antibody responses elicited by immunization with the inactivated vaccine were assessed by an indirect enzyme-linked immunosorbent assay (elisa). optimal assay conditions (i.e. antigen coating concentration, serum and sow milk dilutions, and secondary dilutions) were determined by a checkerboard titration. optimal coating concentrations for igg, iga and siga were . , . and . μg/ml, respectively. next, -well polystyrene microplates were coated with the optimal antigen in a bicarbonate/carbonate coating buffer overnight. plates were washed three times with pbst (pbs with . % tween ) and blocked with % non-fat dry milk at °c for hr. plates were washed times with pbst, diluted in either serum or sow milk ( : , μl/well) and incubated at °c for hr followed by incubation with a streptavidin-hrp-conjugated igg or iga antibody ( : , ) at °c for hr. after washing times with pbst, a mouse anti-fc fragment of siga molecule antibody ( : , ) ta b l e immune procedure collected then confirmed by an immunofluorescence assay (ifa) in order to determine the cut-off value. the levels of sow and piglet serum neutralizing antibodies were determined using pdcov strain nh (p ) with a virus neutralization test (vnt). in order to perform the assay, serum was heated at °c for min for complement inactivation. next, μl of twofold serially diluted serum was mixed with μl dmem containing data were analysed by student's t test. a threshold of p < . was considered to be significant. the antibody levels of the piglets are presented as box and whisker plots created with graphpad prism v . values are reported as the mean ± standard deviation (sd). gilts were immunized twice with the inactivated pdcov vaccine. after eating colostrum for days, piglets were divided into the challenge group, mock group and monitoring group in each sow the passive transfer of antibodies from immunized sows to piglets through their colostrum and milk was detected with an s-specific elisa. a high level of s-specific igg was detected in the serum of piglets born to immunized sows days post-farrowing (figure a ). in order to assess the protective effect of the vaccine, -day-old piglets ( total) born to immunized sows and -day-old piglets ( total) born to unimmunized sows were challenged with a high dose ( tcid ) of pdcov strain nh (p ). piglets ( total) born to immunized sows and -day-old piglets born to unimmunized sows ( total) were used as controls. piglets were monitored, and clinical symptoms were scored according to their level of diarrhoea (table ) . six piglets exhibited mild to moderate diarrhoea, in which the symptoms of four piglets from the challenge group and piglets from the mock group born to immunized group were due to indigestion. following oral inoculation with pdcov, two piglets born to immunized sows # ( / ) and # ( / ) exhibited mild diarrhoea days ta b l e pathogenicity of pdcov in piglets no obvious clinical symptoms were observed in the mock piglets from each group. the piglet faecal specimens were collected daily, and rna was extracted in order to detect viral rna copies by rt-qpcr. pdcov rna copies were detected in four piglets born to sows # , # and # , and low levels of viral rna were detected in piglet ( based on pcr data from the faeces and intestinal tissues, of pigs from the vaccinated sows were evidently infected after virus challenge. therefore, the passive immunity obtained from immunized sows induced . % protection against highly pathogenic pdcov challenge. in the present study, the pathogenicity of pdcov strain nh isolated in was assessed. the results suggest that the virus was enteropathogenic in -day-old spf pigs. a challenge model to study the protective passive immunity in neonatal piglets was conducted. the protective efficacy of passive immunity elicited by the inactivated pdcov vaccine against challenge with a highly pathogenic virulent strain in neonatal piglets born to immunized sows was investigated. results revealed that immunization with an inactivated pdcov vaccine could produce a strong antibody response in pregnant sows after the second vaccination days before delivery. high s-specific igg antibody and na responses were observed in the serum of sows post-farrowing. interestingly, a high level of s-specific siga was detected in colostrum and milk, although it lasted for only a short period of time. previous studies have indicated that siga was produced in the mammary gland by antibody-secreting cells and the recruitment of antibody-secreting cells into the mammary gland contribute to the production of siga (wilson & butcher, ) . therefore, it has been suggested that the oral route appears to be the most effective method for eliciting a strong siga response when vaccinating sows (gerdts & zakhartchouk, ) . however, the findings of this study suggest that vaccination with an inactivated vaccine by injection at the houhai acupoint in pregnant sows could also induce a strong siga response in the colostrum, which may subsequently provide protection for piglets against virulent challenge. results also confirmed that vaccination at the houhai acupoint could induce a mucosal immune response (liu et al., ) . the duration of antibody persistence until the end of the experiment was monitored, and the results indicated that a strong positive correlation existed between the igg antibody and na responses in the serum of immunized sows. the s-specific iga antibody response was also detected with an elisa; however, the results indicated that the serum, colostrum and milk of sows failed to produce an iga response after immunization with an inactivated pdcov vaccine. transferring antibodies (i.e. siga, igg and igm) from the sows to the piglets through the colostrum and milk has been considered to be the primary mechanism of protection mediated by passive immunity (poonsuk et al., ; saif & bohl, ; salmon, berri, gerdts, & meurens, ). the igg antibodies were absorbed by the piglets within the first - hr of life through the colostrum. in this study, a high level of igg antibodies was detected in the serum of piglets and, more importantly, such levels displayed longterm persistence (~ days) although piglets stopped consuming milk from immunized sows. these results suggest that igg from the serum of sows could be absorbed into the serum of piglets through colostrum. the level of igg antibodies in the serum of piglets ( piglets, days post-challenge) did not increase, which was due to the existing maternal antibodies in the serum of piglets interfering with an active immune response to infection. no s-specific siga antibodies were observed in the serum of piglets, which indicated that the function of siga was limited to the enteric cavity rather than the serum. the challenge assay suggested that antibodies transferred from sows through their colostrum and milk could provide protection for piglets. four piglets born to immunized sows displayed diarrhoea post-inoculation with pdcov strain nh, and viral rna copies were detected in the faeces. however, compared to the mock group, the four piglets exhibited mild to moderate diarrhoea (no severe diarrhoea) and lower viral rna copies in the faeces and intestinal tissues. low pdcov antigen levels were also detected in the jejunum and ileum by ihc, and minor intestinal villi damage was observed by he staining. collectively, these results suggest that the antibodies from sows could provide the piglets with partial, but not complete, protection. previous studies on the passive immunity of tgev suggest that viral inoculation by different routes induce different immunoglobulin isotypes (bohl & saif, ) . the production of iga in the milk of tgev antibodies was associated with an intestinal infection, while the production of igg was associated with parenteral antigenic stimulation. this study also indicated that the igg response in serum, colostrum and milk was predominantly detected after pregnant sows were inoculated with virulent tgev by intramammary injections. moreover, the passive immunity protection was good ( % mortality and morbidity) in -and -day-old newborn piglets born to immunized sows post-challenge. however, morbidity was % days post-farrowing with % mortality after tgev challenge. these results suggest that within the first week, igg antibodies in colostrum and milk of immunized sows could provide protection for piglets against tgev virulent challenge. in this study, iga antibodies were not detected in serum, colostrum or milk of immunized sows. the immunoglobulin in the antibodies was predominantly igg, and although the piglets were no longer receiving anti-pdcov igg antibodies from the milk, igg lasted ~ days in the serum of piglets. because of this, the immune protection response observed in pdcov-challenged piglets born to inactivated pdcov-vaccinated mothers was likely due to circulating anti-pdcov igg antibodies that were passively transferred through colostrum rather than anti-pdcov iga antibodies from milk, as observed previously in pedv research (poonsuk et al., ) . the siga and igg in colostrum and milk consumed by piglets for - days post-birth survived and were maintained until challenged to some extent in the gut. in the intestinal environment of piglets born to immunized sows, vnt antibodies might neutralize pdcov in gut and only a small amount of the virus might remain under the minimal infectious doses that could infect piglets (poonsuk et al., ) . another possible reason for this result may be due to the antibody-dependent cell-mediated cytotoxicity (adcc) effect, which promotes cell-mediated immune responses against pdcov, as previously studied in pedv (casadevall & pirofski, ) . in summary, the findings of the present study demonstrate that immunizing sows twice with an inactivated pdcov vaccine could induce igg, siga and na responses. piglets born to immunized sows could obtain protective antibodies by ingesting colostrum and milk. moreover, high levels of igg antibodies and na responses were detected in 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and phylogenetic analysis of porcine deltacoronavirus in korean swine farms virulence of current german pedv strains in suckling pigs and investigation of protective effects of maternally derived antibodies a flagellin-adjuvanted ped subunit vaccine improved protective efficiency against pedv variant challenge in pigs spinal segment distribution of neural innervation related to houhai acupoint and compared with zusanli and dazhui acupoints in domestic chicken origin, evolution, and virulence of porcine deltacoronaviruses in the united states the first case of porcine epidemic diarrhea in canada does circulating antibody play a role in the protection of piglets against porcine epidemic diarrhea virus? different lineage of porcine deltacoronavirus in thailand, vietnam and lao pdr in passive immunity to transmissible gastroenteritis virus: intramammary viral inoculation of sows humoral and cellular factors of maternal immunity in swine contribution of passive immunity to porcine respiratory coronavirus to protection against transmissible gastroenteritis virus challenge exposure in suckling pigs newly emerged porcine deltacoronavirus associated with diarrhoea in swine in china: identification, prevalence and full-length genome sequence analysis detection and genetic characterization of deltacoronavirus in pigs porcine deltacoronavirus: histological lesions and genetic characterization ccl controls immunoglobulin (ig) a plasma cell accumulation in the lactating mammary gland and iga antibody transfer to the neonate discovery of seven novel mammalian and avian coronaviruses in deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus porcine deltacoronavirus: overview of infection dynamics, diagnostic methods, prevalence and genetic evolution pathogenicity of porcine deltacoronavirus (pdcov) strain nh and immunization of pregnant sows with an inactivated pdcov vaccine protects -day-old neonatal piglets from virulent challenge the authors declare that there is no conflict of interest. the study's protocol was approved by the institutional animal care and use committee of the harbin veterinary research institute.this study does not contain research with human participants performed by any of the authors. https://orcid.org/ - - - key: cord- -wuegn jg authors: wang, liang; su, shuo; bi, yuhai; wong, gary; gao, george f. title: bat-origin coronaviruses expand their host range to pigs date: - - journal: trends microbiol doi: . /j.tim. . . sha: doc_id: cord_uid: wuegn jg infections with bat-origin coronaviruses have caused severe illness in humans by ‘host jump’. recently, novel bat-origin coronaviruses were found in pigs. the large number of mutations on the receptor-binding domain allowed the viruses to infect the new host, posing a potential threat to both agriculture and public health. liang wang, shuo su, yuhai bi, , gary wong, and george f. gao , , , * infections with bat-origin coronaviruses have caused severe illness in humans by 'host jump'. recently, novel bat-origin coronaviruses were found in pigs. the large number of mutations on the receptor-binding domain allowed the viruses to infect the new host, posing a potential threat to both agriculture and public health. the host range expansion of coronaviruses (covs) from wildlife to humans via genetic recombination and/or mutations on the receptor-binding domain in the spike (s) gene is well established and results in several diseases with high fatality rates, such as severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) [ [ ] . thus, pigs are regarded as mixing vessels for iavs. however, pigs were not known to be susceptible to bat-origin coronaviruses until recently, when two independent groups reported the detection of novel swine enteric alphacoronaviruses (seacovs) distinct from known swine coronaviruses (with one group successfully isolating live virus). the seacovs were found to be phylogenetically close to bat coronavirus hku [ , ] . this suggests that bat-origin coronaviruses may have 'jumped' the species barrier to infect pigs. in february , outbreaks of severe watery diarrhea of suckling piglets were reported in commercial pig farms in guangdong province, china. the disease had high case fatality rates (cfrs, over % for < -day-old suckling piglets), and none of the animals were positive for known pathogens responsible for porcine diarrhea [ ] . instead, two genomes of novel seacov were detected in the ill piglets by two independent groups, and preliminary analysis showed that the seacovs possibly originated from bat hku coronaviruses [ , ] . it is currently unclear whether seacovs had been circulating undetected in pigs, if the viruses had originated from cross-species transmission, or if the seacovs were the result of viral recombination. to understand the molecular origin and evolution of sea-covs, we performed a detailed phylogenetic analysis at the genomic level by using all known alphacoronaviruses and bat-origin coronaviruses which are known to cause severe diseases, such as sars and mers. a total of , , and complete genomes from mers-cov, sars-cov, and alphacoronaviruses, respectively, were used. phylogeny was reconstructed using conserved regions in genomes by the maximum likelihood method with replicates ( figure a) . consistent with previous studies, phylogenetic analysis shows that seacovs were closely related to the rhinolophus bat coronavirus hku isolated in southern china ( figure a ). seacov was found to share a common ancestor with human coronavirus e/nl , but these viruses are distant from other known swine alphacoronaviruses, indicating their different origins. further analysis on the s gene, which determines virus attachment, host cell entry, and 'host jump' of coronaviruses [ ] , showed that domain in the s subunit has structural similarity to that of nl . the rest of the domain on the s subunit was similar to that of murine hepatitis coronavirus (betacov) [ ] . there were residues of the receptor-binding domain (rbd, also called c-terminal domain, ctd) of seacov directly in contact with its receptor (angiotensin-converting enzyme ) that were mutated or deleted [ ] (figure b) . seven mutations (a deletion of four amino acids, and three substitutions) among the sites in seacov were also found in [ _ t d $ d i f f ] e/nl , indicating similarities in the receptor-binding mechanism between seacov and [ _ t d $ d i f f ] [ _ t d $ d i f f ] e/nl . therefore, seacov may be able to infect humans and should be closely monitored. infection of seacov in vero cells (a primate cell line) will provide experimental evidence to support this possibility [ ] . another requirement for the host range expansion of viruses is physical contact between different host species. the novel seacovs were both detected in guangdong province, whereas closely related bat coronaviruses were also isolated from guangdong or hong kong ( figure a) . in guangdong province, the high density of pig slaughterhouses and the wide distribution of bat species (figure a ) promote the possibility of viral cross-species transmission. additionally, bats have a wide geographical distribution in southern china, with extensive species diversity, unique behaviors (characteristic flight patterns, diet, roosting, and mobility) [ ] , and constant interactions with both pigs and humans (figure a ). pigs are well established as mixing vessels and as intermediate hosts for iavs, and coronaviruses have already been shown to possess potential for recombination in animals [ ] . given that pigs are in frequent contact with human and multiple wildlife species, and that pork is one of the most commonly consumed meats in non-muslim countries, it is important to assess whether pigs could be mixing vessels for the emergence of novel coronaviruses with high agricultural impact and risks to public health. it has already been reported that pigs are susceptible to infection with human sars-cov [ ] and mers-cov [ ] . additionally, the cd receptor sequence alignment of pigs and humans shows . % similarity, which is sufficient for potential cross-species transmission [ ] . in southern china, the unique climate, the high density of domestic as well as wild pigs, and extensive bat distribution, together with bats carrying large numbers of recombinant novel coronaviruses [ ] , could lead to the emergence of more novel coronaviruses in the future. the isolation of seacov from ill piglets expands our knowledge of the host range of bat-origin coronaviruses, and potentially poses a threat to public health. despite considerable progress in characterizing cross-species transmission for coronaviruses, several areas need to be addressed, including: (i) whether other unknown coronaviruses are circulating in pigs; (ii) whether pigs are mixing vessels for coronaviruses; (iii) whether seacov infects humans and causes severe disease; and (iv) whether seacov vaccines should also be developed to control the spread of this virus in pigs. in-depth epidemiological investigation and comprehensive analysis of these novel coronaviruses should be performed to answer these urgent questions. using metagenomics to characterize an expanding virosphere epidemiology, genetic recombination, and pathogenesis of coronaviruses molecular basis of binding between novel human coronavirus mers-cov and its receptor cd sars and mers: recent insights into emerging coronaviruses the pig as an intermediate host for influenza a viruses between birds and humans a new bat-hku -like coronavirus in swine discovery of a novel swine enteric alphacoronavirus (seacov) in southern china bat-to-human: spike features determining 'host jump' of coronaviruses sars-cov, mers-cov, and beyond bats: important reservoir hosts of emerging viruses co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia sars-associated coronavirus transmitted from human to pig livestock susceptibility to infection with middle east respiratory syndrome coronavirus key: cord- -yhed s authors: karelehto, eveliina; cristella, cosimo; yu, xiao; sridhar, adithya; hulsdouw, rens; de haan, karen; van eijk, hetty; koekkoek, sylvie; pajkrt, dasja; de jong, menno d.; wolthers, katja c. title: polarized entry of human parechoviruses in the airway epithelium date: - - journal: front cell infect microbiol doi: . /fcimb. . sha: doc_id: cord_uid: yhed s human parechoviruses (hpevs), a poorly studied genus within the picornaviridae family, are classified into genotypes of which hpev and hpev are the most often detected. hpev vp c terminus contains an arginine-glycine-aspartic acid (rgd) motif and has been shown to depend on the host cell surface αv integrins (αv itgs) and heparan sulfate (hs) for entry. hpev lacks this motif and the receptors remain unknown. hpevs can be detected in patient nasopharyngeal and stool samples, and infection is presumed to occur after respiratory or gastro-intestinal transmission. hpev pathogenesis is poorly understood as there are no animal models and previous studies have been conducted in immortalized monolayer cell cultures which do not adequately represent the characteristics of human tissues. to bridge this gap, we determined the polarity of infection, replication kinetics, and cell tropism of hpev and hpev in the well-differentiated human airway epithelial (hae) model. we found the hae cultures to be permissive for hpevs. both hpev genotypes infected the hae preferentially from the basolateral surface while the progeny virus was shed toward the apical side. confocal microscopy revealed the target cell type to be the p (+) basal cells for both viruses, αv itg and hs blocking had no effect on the replication of either virus, and transcriptional profiling suggested that hpev infection induced stronger immune activation than hpev . genotype-specific host responses may contribute to the differences in pathogenesis and clinical outcomes associated with hpev and hpev . parechovirus a species within the picornaviridae family comprises of genotypes, of which human parechovirus and (hpev and hpev , respectively) are the most often detected in clinical samples (harvala et al., ; romero and selvarangan, ) . hpevs are major human pathogens, typically causing respiratory, gastrointestinal, and febrile illness in young children (harvala et al., ; romero and selvarangan, ) . when compared to hpev , hpev infects younger patients and often causes more severe diseases, such as sepsis-like illness or central nervous system (cns) infections. the reason for these differences remains elusive (benschop et al., ; romero and selvarangan, ) . the hpev single-stranded positive-sense rna genome coding region, flanked by ′ and ′ untranslated regions, is translated into a single polyprotein and subsequently cleaved into three capsid proteins (vp , vp , and vp ) and seven non-structural proteins ( a-c and a-d). hpev contains a well-known integrin-recognition sequence, the arginine-glycineaspartic acid (rgd) motif, in the vp c terminus, while hpev lacks this motif (stanway et al., ; ito et al., ) . previous studies have identified three αv integrin (αv itg) heterodimers as hpev receptors (αvβ , αvβ , and αvβ ) (triantafilou et al., ; joki-korpela et al., ; seitsonen et al., ; merilahti et al., b) . heparan sulfate (hs) has been proposed to act as an attachment receptor for hpev (merilahti et al., a) . to date, no hpev receptors have been identified. analogous to virus species in the closely related enterovirus genus, hpevs are detected in patient nasopharyngeal and stool specimens, and are assumed to initiate their replication in the host respiratory and intestinal tracts (semler and wimmer, ) . however, it is not known whether the epithelium lining these anatomical sites can support hpev replication nor which cell types are involved. the air-liquid interface human airway epithelial (hae) cell culture system is a well-characterized three-dimensional organotypic model that has been shown to closely mimic the structure and function of the in vivo parent tissue (fulcher et al., ) . in the current study, we used this model to investigate hpev entry in one of the proposed primary replication sites, the airway epithelium. given that integrins and heparan sulfate have been reported to be expressed at the basolateral surface of polarized epithelium (erlinger, ; esclatine et al., ; lütschg et al., ) , and that hpev has been detected in respiratory patient samples more often than hpev (harvala et al., ) , we hypothesized that hpev replication efficiency would differ depending on the inoculation site and the hpev genotype. we determined the polarity of infection and replication kinetics of hpev and hpev in the hae model and performed immunofluorescence confocal microscopy analyses to characterize the hpev cell tropism. furthermore, we speculated that differences in the airway epithelium host response may contribute to the distinct clinical outcomes and performed transcriptome analyses to compare the hae gene expression profiles induced by hpev and hpev infection. hpev harris strain was obtained from the national institute of public health and the environment (rivm, bilthoven). hpev was cultured in ht cells (human colorectal adenocarcinoma; atcc, manassas, va). hpev strain, isolated from a dutch clinical specimen in , was cultured in llcmk cells (rhesus monkey kidney cell line, kindly provided by the municipal health services, rotterdam, the netherlands). enterovirus (ev ) - and enterovirus (ev ) were kindly provided by the national institute for public health and the environment (bilthoven, the netherlands) and by prof. van kuppeveld, utrecht university (utrecht, the netherlands), respectively. ev and ev were cultured in rd cells (human rhabdomyosarcoma; atcc, manassas, va). all cell lines were maintained in eagle's minimum essential medium (emem; lonza, basel, switzerland) supplemented with % heat-inactivated fetal bovine serum (fbs; sigma-aldrich, st. louis, mo), pen-strep ( u/ml penicillin and u/ml streptomycin; lonza bio whittaker, non-essential amino acids (neaa; sciencell research laboratories, carlsbad, ca) and lglutamine ( nm; lonza, basel, switzerland). the % tissue culture infective dose (tcid ) of virus stocks was determined using the reed and muench method (reed and muench, ) . nasal mucilair hae cell cultures from four individual donors ( , , , ) were purchased from epithelix sàrl (geneva, switzerland) (tapparel et al., ) . upon receiving the well-differentiated -well format transwell hae inserts, they were cultured for week at air-liquid interface before performing the infection experiments. the mucilair culture medium (epithelix sàrl) was refreshed every - days. donor hae culture infections were performed twice using technical duplicates. to confirm results in additional donors, infection experiments were repeated once using donor , , and hae cultures as biological replicates. apical surfaces of the hae cultures were washed once with hank's balanced salt solution (hbss; thermofisher scientific) and the culture medium refreshed prior to virus inoculation. hae cultures were inoculated by adding tcid of hpev or hpev (or hbss for mock) in a volume of µl on the apical or basolateral surface. for basolateral infection, hae inserts were inverted for the duration of inoculation. virus inoculum was removed after -h incubation at • c, % co , and the inoculation surface of the hae cultures was subsequently washed eight times with hbss. apical sampling was performed by adding µl of hbss on the hae culture apical surface followed by a -min incubation at • c, % co , and collection. basolateral samples were obtained by collecting µl of the hae culture medium. µl of fresh culture medium was subsequently added to maintain the total culture medium volume at µl per insert. ev was used as a control for apical infection and added only on the apical surface of the hae cultures. rna from µl of the apical and basolateral samples was isolated by automatic extraction using the magnapure lc instrument (roche diagnostics, almere, the netherlands) and eluted in µl. forty micro liter of the rna was reverse-transcribed and µl of the cdna was used for real-time quantitative pcr (rt-qpcr) targeting the ' untranslated region of the hpev genome . in addition, apical and basolateral hae samples were analyzed for the level of infectious hpev by tcid titrations using the reed and muench method (reed and muench, ) . hpev and hpev samples were titrated in the ht and llcmk cell lines, respectively. apical surfaces of the hae cultures were washed once with hbss and the culture medium was refreshed prior to the blocking experiments. host cell surface αv integrins were blocked by adding µl of µg/ml or µg/ml of function-blocking human αv itg mab clone l (alx- - -c ; enzo life sciences, inc. farmingdale, ny) on the apical or basolateral surface of the hae cultures. for basolateral blocking, hae inserts were inverted for the duration of the pretreatment. mab solution was removed after -h incubation at • c, % co , and replaced with mab solution containing the virus inoculum. virus binding to the host cell surface heparan sulfate was blocked by incubating the input virus with mg/ml excess concentration of heparin sodium salt (h ; sigma aldrich, st. louis, mo) for -h at • c, % co prior to inoculation from either apical or basolateral surface. subsequent virus incubation, washing and sampling steps were performed as described above. hae cultures from donor were used in duplo for µg/ml αv itg mab experiments. donors , , and were used as biological replicates for µg/ml αv itg mab and heparin experiments. as described above for hae cultures, identical αv itg and hs blocking experiments were performed against hpev in ht cell line (duplicate wells in -well microtiter plates). ev was used as a positive control for heparin experiments in rd cell line (tan et al., ) . hae donor cultures were basolaterally inoculated with hpev or hpev (or mock) as described above, excluding the post-inoculation washing, incubated at • c, % co for -h and fixed with % formalin in pbs for min at room temperature (rt). hae culture membranes were then excised from the inserts, submerged in . % triton x- for min at rt for permeabilization and in . % tween , and % bovine serum albumin (bsa) in pbs at • c overnight for blocking. hpev and hpev staining was performed using rabbit hyperimmune serum (kindly provided by dr. susi, university of turku, finland), which was raised against inactivated hpev virions and shown to cross-react with hpev (joki- korpela et al., ; karelehto et al., ) . additional immunofluorescent labeling was performed using the following primary antibodies; mouse β-tubulin-cy mab ( k , sigma aldrich), mouse mucin b mab (sc- ; santa cruz, dallas, tx), goat p pab (af ; r&d systems, minneapolis, mn), αv itg mab clone l (alx- - -c ; enzo life sciences), and hs mab clone f - e ( - ; amsbio llc, cambridge, ma). secondary antibodies used included donkey anti-rabbit igg alexa ab (a- ; life technologies, carlsbad, ca), donkey anti-mouse igg alexa ab (a ; life technologies), donkey anti-goat igg alexa (a- ; life technologies), donkey anti-rabbit igg alexa (a- ; life technologies) and goat anti-mouse igm alexa (a- ; life technologies). all antibodies were diluted in . % tween and % bsa in pbs. cultures were incubated with primary antibodies at • c overnight and with secondary antibodies for -h at rt and washed extensively after each step with . % tween in pbs. membranes were mounted on objective slides with prolong gold antifade mountant with dapi (life technologies). samples were examined by confocal immunofluorescence imaging using leica tcs sp x microscope with hc plan apochromat x/ . oil objective and leica las af software (leica microsystems, wetzlar, germany). confocal image stacks were deconvoluted by huygens software package (svi, hilversum, the netherlands) and subsequently compiled into d images and videos by leica las af. hae (donor ) cultures in five technical replicates were basolaterally inoculated with hpev or hpev (or mock) as described above and incubated at • c, % co . cultures were lysed and rna extracted by high pure rna isolation kit (roche lifescience, penzberg, germany) at the peak of replication days post infection. rna samples were concentrated to ng/µl using eppendorf concentrator plus (eppendorf, hamburg, germany), and the concentration and quality was determined by nanodrop (thermofisher scientific) and bioanalyzer (agilent technologies, santa clara, california). the extracted and concentrated total rna samples were labeled and hybridized using the human clariom s ht microarray platform according to the manufacturer's guidelines (affymetrix/thermofisher scientific). the technical quality of arrays was assessed by arrayqualitymetrics r package (kauffmann et al., ). all the samples, except one hpev infected replicate, passed the quality control assessments. the total number of samples used in the analysis included array replicates distributed as follows; mock, hpev -infected and hpev -infected. the brainarray (version ) custom chip description file (cdf) was used to re-map probesets on the arrays based on the latest genome and transcriptome information (dai et al., ) . the featurefilter function, implemented in the genefilter r package, was used to remove probesets which lacked their respective entrezid annotations (bourgon et al., ) . probesets normalization was achieved by robust multi-array average (rma) function implemented in the affy r package (irizarry et al., ; gautier et al., ) . the final expression set comprised genes and samples. for the hpev replication kinetics, the relative increase in hpev rna copy numbers and hpev tcid titers were calculated by subtracting the timepoint zero input values from all values. hpev rna copy number and tcid titer means of the technical and biological replicates between the apical and basolateral inoculation, or the mock and the mab or heparintreated samples, were compared per each virus and time point by two-way anova with tukey's multiple comparisons test using graphpad prism (graphpad software inc., la jolla, ca). differentially expressed genes were identified using the limma r package (ritchie et al., ) . in brief, ebayes moderated tstatistics was applied to test alternative hypothesis: ( ) that gene expressions of hpev -infected hae were different from mock, ( ) that gene expressions of hpev -infected hae were different from mock and ( ) that gene expressions of hpev infected hae were different from hpev -infected hae. for each test, we included in the empirical bayes estimation a mean-variance trend, to account for probes that are less reliable at lower intensities. nominal p-values of log fold changes were corrected for multiple testing using benjamini-hochberg false discovery rate (fdr). differentially expressed genes were considered significant if their absolute fold change was greater than . and the associated fdr adjusted p value was below e- . gene ontology over-representation analyses of biological processes described by the selected degs were performed using the topgo r package (alexa et al., ) . significance for each individual go-term was computed by means of the weight algorithm and fisher's exact test. to investigate hpev replication in the human airway epithelium, we inoculated well-differentiated nasal hae cultures, established from four individual donors, with hpev and hpev either from the apical or the basolateral surface (figure ). hpev and hpev could both replicate in hae cultures. apical inoculation resulted in low virus titers whereas basolateral inoculation led to significantly higher hpev and hpev titers at the apical compartment - days post infection (dpi; p-values < e- ) ( figure a) . infectious virus titers detected from the basolateral compartment were low with no significant difference between the apical and basolateral inoculation ( figure b) . tcid analyses showed that the viral replication peaked at day or ( figure a) . rt-qpcr analyses of the apical samples were in line with tcid titrations (figure c ). significant increases in the viral rna at the basolateral compartment was detected following apical inoculation ( figure d ). however, this was not reflected in the amount of infectious virus ( figure b) . tcid data could not be used to directly compare hpev and hpev replication efficiencies due to the different cell lines used in the assays. however, relative increase in the hpev rna copy numbers were significantly higher to dpi (basolateral inoculation and apical sampling, p-values < e- ) and to dpi (basolateral inoculation and basolateral sampling, p-values < e- ), whereas relative increase in the hpev rna copy numbers were significantly higher to dpi (apical inoculation and basolateral sampling, p-values < e- ). efficient apical infection in the hae cultures could be established with the respiratory picornavirus enterovirus (ev ; data not shown). to determine the hpev target cell type, we performed immunofluorescent labeling and confocal microscopy of the hae cultures. triple-staining of the hpev -and hpev infected hae cultures at -h post inoculation by ciliated cell marker β-tubulin, basal cell marker p , and hpev antibody (figure a ) or secretory cell marker muc b, basal cell marker p , and hpev antibody ( figure b ) revealed that while ciliated and secretory cells were localized on the apical surface of the hae culture, hpev-infected cells resided near the basolateral surface and were positive for the p basal cell marker (figure ) . d renderings of the confocal image stacks are provided in supplementary videos - . in addition, we observed that αv integrins were clearly localized to the basolateral surface of the hae whereas heparan sulfate was diffusely stained throughout the cultures (supplementary figure ) . as αv itgs and hs have been described to function as hpev receptors in vitro, we investigated the effect of blocking αv itgs on either apical or basolateral surface of the hae model prior to hpev and hpev inoculation. neither of the tested concentrations of the function-blocking αv itg monoclonal figure | human parechoviruses target p positive basal cells in the airway epithelium. confocal image stacks of hpev -and hpev -infected hae cultures triple-stained by (a) ciliated cell marker β-tubulin (red), basal cell marker p (purple) and hpev antibody (green) or (b) secretory cell marker mucin b (red), basal cell marker p (purple) and hpev antibody (green). nuclei stained by dapi (blue). scale bars, µm. antibody (mab) significantly inhibited hpev (figure a ) or hpev (figure b) replication. to block hs binding sites on the virus capsid, we incubated the viruses with heparin prior to either apical or basolateral hae inoculation but detected no significant inhibition of either hpev (figure a ) or hpev replication ( figure b) . hpev replication was inhibited in ht cell line following αv itg blocking, albeit this did not reach statistical significance (supplementary figure ) . however, we could not confirm the role of hs in hpev infection in ht (supplementary figure ) , while functionality of the same concentration of heparin in blocking was shown by complete inhibition of enterovirus (ev ) replication in the rd cell line (data not shown). due to the poor replication hpev in standard cell culture, we could not perform similar receptor blocking for hpev . next, we performed transcriptome analyses to compare the airway epithelium host response against hpev and hpev infection. virus replication in the hae was confirmed by rt-qpcr ( figure a ). the total numbers and overlap in significantly differentially expressed genes (degs; adj. p-value < e- , gene expression fold change > . ) are depicted in the venn diagram ( figure b) . as compared to the mock, hpev induced differential expression of genes whereas hpev induced genes. of these, genes were shared among the two viruses ( figure b) . of the shared genes, the expression profiles of genes did not significantly differ between hpev and hpev . thus, these genes represent a true common hpev deg signature. of the shared genes were found to be differentially expressed upon both hpev and hpev infection, but to a significantly different level. these genes represent the common hpev response. remarkably, only degs were specific to hpev in contrast to degs found to be induced exclusively by hpev . of the true common degs, were upregulated and downregulated when compared to the mock ( figure c ). common degs included upregulated and downregulated genes, while of hpev -specific degs were upregulated and downregulated. heatmaps of the true common, common, and hpev -specific degs are presented in figures d-f . direction of the gene expression among the true common and common degs was identical regardless of the hpev genotype (figures d,e) . overall hpev infection resulted in greater fold change of the common transcripts ( figure e ). hpev -specific degs were either upor downregulated upon hpev infection whereas expression of these genes remained at baseline in hpev infection ( figure f) . complete lists of all the degs induced by hpev and hpev , and a list of the degs different between hpev and hpev , are provided in supplementary tables - . gene ontology term over-representation analysis was performed to investigate which biological processes are associated with the observed degs. as expected the biological processes enriched in the true common set of upregulated genes were almost exclusively related to immune response and inflammation, whereas the few downregulated degs were associated with processes such as epithelial differentiation (cornification) and cytoskeleton remodeling (intermediate filament organization, actin filament capping) (figure a) . hundreds of common degs significantly more upregulated by hpev than by hpev were enriched for genes involved in immune responses and inflammation (type i and ii interferon responses, nf-κb signaling) ( figure b) . many of the common degs that were more downregulated by hpev than by hpev , were related to cell adhesion (leukocyte migration, extracellular matrix disassembly, heterotypic cell-cell adhesion, response to wounding). only six genes were found to be differentially expressed exclusively in hpev infection. three of these genes were downregulated (alpha kinase (alpk ), mss mitochondrial translational activator (mss ), biorientation of chromosomes in cell division like (bod l ) (fold changes − . , − . , − . and adj. p-values e- , e- , e- , respectively)), and three were upregulated (atpase phospholipid transporting b (atp b ), tensin (tns ), matrix metallopeptidase (mmp ) (fold changes . , . , . , and adj. p values e- , e- , e- , respectively)) (supplementary table ). hpev -specific upregulated genes were most notably enriched in the nf-κb signaling pathway activation ( figure c ). hpev infection increased the expression of key molecules involved in this pathway such as proto-oncogene relb, interleukin receptor associated kinase (irak ) and tnf receptor associated factor (traf ) (fold changes . , . , . , and adj. p-values e- , e- , e- , respectively) (supplementary table ). other hpev -specific enriched processes included for example tumor necrosis factor signaling and leukocyte adhesion ( figure c) . each of the viruses induced both up-and downregulation of a number of apoptosisassociated genes (supplementary tables , ) . however, hpev -specific upregulated degs were significantly enriched for apoptosis-related processes such as activation of cysteine-type endopeptidase and actin filament organization ( figure c ). for example, of the essential apoptosis effectors, caspases and were significantly more upregulated by hpev than by hpev (fold changes hpev vs. hpev . , . , and adj. p-values e- , e- , respectively) (supplementary table ) . the downregulated hpev -specific degs were annotated with various processes such as ones involved in protein translation (translational initiation, srp-dependent cotranslational protein targeting to membrane), rna degradation (nuclear-transcribed mrna catabolic process) and cell metabolism (oxidation-reduction process, mitochondrial atp synthesis coupled proton transport) ( figure c ). in this study we showed that the airway epithelium can support hpev replication, that infection occurs preferentially from the basolateral surface, and that the progeny virus is released from the apical surface of the epithelium. moreover, we identified p + basal cells as the hpev target cell type in the hae model and found that the magnitude of innate immune responses was greater following hpev than hpev infection. in contrast to what has been established previously for several respiratory viruses such as influenza virus, rhinovirus (rv), coronavirus, or respiratory syncytial virus (rsv) (matrosovich et al., ; johnson et al., ; griggs et al., ) , hpevs infected airway epithelium more efficiently from the basolateral rather than the apical surface. similarly, basolateral entry in the hae cultures has been reported for vaccinia virus, reovirus, some adeno-and arenaviruses and measles virus (sinn et al., ; vermeer et al., ; dylla et al., ; excoffon et al., ; lütschg et al., ) . in line with our hpev data, vaccinia, reo-, and measles viruses also exhibited apical shedding of the progeny virions (sinn et al., ; vermeer et al., ; excoffon et al., ) . we found that both hpev and hpev targeted the p + basal cells for replication. basal cells are the progenitor cell type of the airways and reside on the basolateral surface underneath a layer of differentiated ciliated and secretory cells (rock et al., ) . tropism toward the p + cells therefore explains the basolateral polarity of hpev infection. similar tropism has been reported previously for rv (a species) and rsv, but only upon exposing the basal cells to apical inoculation by mechanical or chemical damage (jakiela et al., ; persson et al., ) . since basal cells maintain the epithelial integrity and regulate the proliferation, extensive infection of these cells could result in epithelial damage and thus lead to respiratory symptoms in vivo. however, in contrast to the infection in immortalized cell lines, we did not observe major cytopathic effects in hpev -or hpev -infected hae cultures. here we examined the hpev cell tropism at -h post infection. further experiments investigating later timepoints are necessary to determine whether cells other than those positive for the p marker can be infected. previously reported transcriptome analysis of the airway basal cells has showed that while αv itg is not significantly more expressed in basal vs. the differentiated epithelia cells, β and β itgs do belong to the specific basal cell transcriptome signature (hackett et al., ) . these β itg subunits dimerize with αv itg and have been described to be the high affinity receptors of hpev in cell culture (seitsonen et al., ; merilahti et al., b) . hpev , but not hpev , contains the integrin-binding rgd-motif (stanway et al., ; ito et al., ), yet both viruses were found to infect the same cell type. reports of basolateral localization of the proposed hpev receptors, itgs and hs (erlinger, ; lütschg et al., ; johnson et al., ) , prompted us to study the polarity of hpev infection. however, we could only confirm basolateral localization of the αv itgs but not of hs in hae cultures. blocking these receptors on either apical or basolateral surface of the hae cultures had no inhibitory effect on either hpev or hpev replication. this is surprising since deletion of the rgd motif has been shown to abolish hpev infectivity in cell lines (boonyakiat et al., ) . while we cannot exclude the possibility that the efficiency of the function-blocking αv itg mab pretreatment of the hae or the heparin-pretreatment of the virus was insufficient, we did observe inhibition of hpev in ht cell line upon identical αv itg mab pretreatment and successfully used ev as a positive control in our hs blocking experiments. moreover, similar αv itg mab and heparin concentrations have been reported to inhibit hpev replication in monolayer cell cultures (triantafilou et al., ; merilahti et al., a,b) . we believe differences in the cell lines and read-out methods may explain why we could not validate the previously reported heparin-blocking of hpev in standard cell culture (merilahti et al., a) . in summary, we speculate that the hpev receptor-usage is cell line-dependent and may differ between monolayer cell cultures and polarized primary cell culture models such as hae. further research is needed to confirm this hypothesis. transcriptome analyses revealed that hpev -infection resulted in the expression of a considerable number of host genes whereas the hpev response was relatively modest. major difference between the responses was the over-representation of transcripts associated with immune and inflammatory responses following hpev infection. hpev and hpev induced genes involved in type i and ii interferon signaling and nf-κb pathways but the magnitude of these responses was higher in the hpev -infected hae cultures. as reported by us and others, in contrast to the humoral immunity against hpev , the maternal neutralizing antibody protection against hpev is low in women of childbearing age and lacking in hpev infected newborns (karelehto et al., pidj in press) (aizawa et al., ) . therefore, innate immune response at the virus entry site could contribute to the severe clinical presentation. two mechanisms may explain why hpev more often than hpev causes severe disease (romero and selvarangan, ) . (i) excessive release of proinflammatory cytokines following hpev infection could result in immunopathology involving increased vascular permeability and compromised blood-brain-barrier integrity (rochfort and cummins, ; danielski et al., ) . this is perhaps supported by the association of hpev and neonatal sepsis, a disease with various unspecific symptoms characterized by a systemic inflammatory response syndrome (shane et al., ) . (ii) conversely, neonates are known to have immature innate immune system, including downregulation of genes involved in nf-κb signaling and decreased cytokine production (raymond et al., ) , and thus may fail to elicit the necessary host response to control hpev infection. a detailed characterization of the host transcriptome by analyzing earlier timepoints and comparing different tissues from infants and adults, as well as validating the results at the protein level, will further elucidate the differences between hpev and hpev infection. for measles virus a model of infection has been proposed in which the virus is taken up by alveolar immune cells, transported across the airway epithelium and into the lymphoid tissue, the site of primary replication, followed by systemic spread and basolateral infection combined with apical egress in the airway epithelium (mühlebach et al., ) . recently, adenovirus (adv ) was shown to promote re-localization of its coxsackievirus and adenovirus receptor (carex ), via inducing il secretion of the immune cells, thus enabling apical entry (kotha et al., ) . similarly, we hypothesize that the airway-resident dendritic cells and macrophages play a role in facilitating hpev entry into the polarized epithelium. another explanation as to how hpevs might infect the airway epithelium in vivo involves m-cells, a specialized type of epithelial cells overlaying the peyer's patches in the intestinal epithelium (mabbott et al., ) . poliovirus, as well as the reovirus, has been shown to break the epithelial barrier by transcytosis through m-cells (morin et al., ; ouzilou et al., ; gonzalez-hernandez et al., ) . similar type of antigen-sampling cell has also been described in the airway epithelium (morin et al., ; mabbott et al., ) . while hae cell culture system is an elegant model, it contains only epithelial cells. underlying mesenchymal cells such as fibroblasts and immune cells can modulate the epithelial function and likely play a role in hpev pathogenesis (nowarski et al., ) . co-culture systems incorporating various cell types are more suitable to answer questions such as how hpevs access airway epithelium basal cells in vivo. in addition, as hpevs can invade the cns (romero and selvarangan, ) , and as many of the related enteroviruses gain entry in the human gut (semler and wimmer, ) , organoids represent attractive models to further study hpev infection of the cns and intestinal epithelium (iakobachvili and peters, ) . in conclusion, we showed that the hae is permissive to hpev infection, that hpev and hpev infect it in a polarized manner by targeting the basal progenitor cells, and that hpev , the genotype causing outbreaks of severe illness in neonates, induces an amplified immune and inflammatory host response when compared to hpev . this work represents the first step toward understanding the entry and genotype-dependent pathogenesis of human parechoviruses. the data discussed in this publication has been 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overview interaction of alphavbeta and alphavbeta integrins with human parechovirus molecular biology of picornaviruses neonatal sepsis measles virus preferentially transduces the basolateral surface of well-differentiated human airway epithelia molecular and biological characteristics of echovirus , a representative of a new picornavirus group enterovirus uses cell surface heparan sulfate glycosaminoglycan as an attachment receptor growth and characterization of different human rhinovirus c types in threedimensional human airway epithelia reconstituted in vitro human parechovirus utilizes integrins alphavbeta and alphavbeta as receptors vaccinia virus entry, exit, and interaction with differentiated human airway epithelia the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fcimb. . /full#supplementary-material key: cord- -w kl c authors: li, jin; tao, yue; tang, mingyu; du, bailu; xia, yijun; mo, xi; cao, qing title: rapid detection of respiratory organisms with the filmarray respiratory panel in a large children’s hospital in china date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: w kl c background: respiratory tract infections (rtis) are the most common illness in children, and rapid diagnosis is required for the optimal management of rtis, especially severe infections. methods: nasopharyngeal swab or sputum specimens were collected from children aged days to years who were admitted to a hospital in shanghai and diagnosed with rtis. the specimens were tested with the filmarray respiratory panel, a multiplex pcr assay that detects viruses, mycoplasma pneumoniae (m. pneumoniae), bordetella pertussis (b. pertussis) and chlamydophila pneumoniae (c. pneumoniae). results: among the children studied, ( . %, / ) tested positive for at least one organism, and multiple organisms were detected in ( . %). rhinoviruses/enteroviruses ( . %, / ) were detected most often, followed by respiratory syncytial virus ( . %, / ), parainfluenza virus ( . %, / ), influenza a or b ( . %), adenovirus ( . %), m. pneumoniae ( . %) and b. pertussis ( . %). the prevalence of organisms differed by age, and most of the viruses were more common in winter. of the children suspected of having pertussis, . % ( / ) tested positive for b. pertussis. conclusions: filmarray rp allows the rapid simultaneous detection of a wide number of respiratory organisms, with limited hands-on time, in chinese pediatric patients with rtis. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. acute respiratory tract infections (rtis) are the leading causes of outpatient visits and hospitalizations in all age groups, especially during winter and spring. for children under years of age, rtis are the second leading cause of death [ ] . most acute rtis in children are caused by respiratory viruses, such as respiratory syncytial virus (rsv), adenovirus (adv), rhinovirus (rv) and influenza viruses. in addition to viruses, atypical pathogens are major causes of pediatric rtis. one of the most common atypical pathogens is mycoplasma pneumoniae (m. pneumoniae), accounting for - % of hospitalized children with community-acquired pneumonia [ , ] . in addition to m. pneumoniae, the incidence of pertussis in china has significantly increased since . nevertheless, multiple epidemiological studies have suggested that the incidence of pertussis in china has been significantly underestimated [ , ] . the early diagnosis of the pathogen is beneficial for the precise selection of medication, which can largely avoid the overuse or even abuse of the antibiotics and improve the clinical care of patients. more importantly, the early diagnosis of contagious pathogens, such as bordetella pertussis (b. pertussis) and influenza viruses, can enable early isolation of patients, thus reducing the spread of pathogens. at present, the routine detection methods for respiratory pathogens in china are mostly based on immunological methods, which include the detection of m. pneumoniae and several major viruses, such as rsv, adv, rv, parainfluenza virus (para), influenza a virus (flua) and influenza b virus (flub). other respiratory viruses and atypical bacteria, such as chlamydophila pneumoniae (c. pneumoniae) and b. pertussis, are typically not routinely detected. given their poor sensitivity and long turn-around time (tat), immunological methods usually lead to broad-spectrum therapy and have been gradually replaced by molecular-based methods, such as conventional and real-time polymerase chain reaction (pcr), in developed countries [ , ] . however, most of these molecular tests are technically challenging and require independent spaces, such as pre-pcr and post-pcr rooms, to eliminate the potential risk of cross-contamination, and such requirement limits their applications in china. therefore, faster, more sensitive and easy-to-use assays for multiplex respiratory pathogen detection are urgently needed. filmarray (biofire diagnostics, utah, usa, owned by biomérieux) is a small, desktop, fully automated multiplex pcr device. the molecular system includes automated nucleic acid extraction, an initial reverse transcription step and multiplex nested pcr, followed by a melting curve analysis [ ] . the filmarray respiratory panel (filmarray rp) is both fda-approved and ce ivd-marked. the current version of filmarray rp (v . ) is able to detect viral and atypical respiratory organisms. the test is performed in a closed system that requires min of hands-on time and min of instrumentation time. several comparison studies between fil-marray and other tests for respiratory organisms showed comparable results [ ] [ ] [ ] . the aim of this study was to evaluate the application of filmarray rp for the detection of respiratory organisms, and to provide information about the seasonality and prevalence of these organisms in pediatric patients with rtis in a large children's hospital in china. the study population was enrolled according to protocol definitions and inclusion criteria. patients with respiratory infections, with or without fever (defined as body temperature ≥ . °c), were included if they had at least one of the following symptoms: ( ) cough; ( ) nasal obstruction; ( ) tachypnoea; ( ) nasal flaring; or ( ) hypoxia. patients admitted to the hospital had at least one of the following conditions: ( ) unabating high fever; ( ) dyspnea, tachypnea or hypoxemia; ( ) anorexia or dehydration; ( ) radiological confirmation of lung infection; or ( ) respiratory infection with underlying diseases, such as congenital heart disease, bronchopulmonary dysplasia, airway malformations, severe malnutrition. according to the chinese center for disease control and prevention (cdc), patients suspected of having pertussis should have a cough for more than weeks and have at least one of the following symptoms: ( ) paroxysmal cough; ( ) inspiratory whoop; or ( ) post-tussive vomiting. in the present study, patients suspected of having pertussis were diagnosed with pertussis when b. pertussis was positive by filmarray rp detection and were otherwise diagnosed with pertussis-like syndrome. nasopharyngeal swab (nps) or sputum specimens were obtained from patients with symptoms of rtis on the day of hospitalization at shanghai children's medical center (scmc) from december , to november , . demographic data and clinical features, as well as laboratory test and imaging results, were obtained for each enrolled patient. the study was approved by the institutional review board and the ethics committee of shanghai children's medical center (scmcirb-k ), and written informed consent was obtained from the parents of each patient. the filmarray rp v . targets organisms, including adv, influenza a viruses h , h , h (flua-h , flua- h , flua-h ) and flub, parainfluenza virus types to (para - ), coronaviruses e, hku , oc , and nl (cov-hku , nl , e, oc ), human metapneumovirus (hmpv), rsv, human rhinovirus/ enterovirus (rhino/entero), c. pneumoniae, m. pneumoniae and b. pertussis. the filmarray rp assay was performed according to the manufacturer's instructions. the principle of the assay has been previously described [ , ] . each pouch included internal run controls for every step, and results for the assay were only provided by the software if the quality control reactions showed appropriate results. spss software package v . was used for all statistical analyses. categorical variables were expressed as frequencies and percentages. the chi-square and fisher's exact tests were used to compare groups. continuous variables are expressed as the mean and standard deviation. student's t-test was used to assess the statistical significance between groups. p < . was considered to be statistically significant. a total of patients diagnosed with upper or lower respiratory tract infections, aged days to years, were enrolled in the present study between december , , and november , . congenital heart disease, congenital biliary atresia, malignancy and congenital immunodeficiency were the most frequently observed underlying diseases in these patients and contributed to % of the deaths observed in this study. the general characteristics of the patients enrolled are presented in table . overall detection rate of filmarray rp v . analysis of the positive rates and prevalence in different age groups all the patients were grouped by age as follows: infants (age: < year), toddlers (age: - years), preschoolers (age: - years) and school-aged children (age: - years) ( table ). the highest specimen positivity rate, at . % ( / ), was in the < -year age group, followed by . % ( / ), . % ( / ) and . % ( / ) in the - -year, - -year and - -year groups, respectively. there were no significant differences in the positivity rate of the different age groups. in contrast, the prevalence of organisms were different between the different age groups ( table ) . rhino/entero, para , rsv and b. pertussis showed the highest prevalence in the < -year age group, while adv, hmpv and flua showed the highest prevalence in the - -year age group. the most prevalent organism in the - -year age group was m. pneumoniae. no organism showed a notably high prevalence in the - -year age group. there was only one c. pneumoniae-positive patient during the study period, and this patient was in the - -year age group. among the specimens, ( . %, / ) were positive for more than one organism. the largest proportion ( . %, / ) of multi-organism-positive specimens had combinations with rhino/entero. rhino/entero plus para was the most common combination, making up . % ( / ) of all multi-organism-positive specimens, while the combination of rhino/entero plus adv was the second most common type ( . %, / ), followed by rhino/ entero plus rsv ( . %, / ). the multi-organism combinations are listed in additional file : table s . seasonal prevalence of respiratory organisms from december , to november , the number of positive specimens was determined during different months of the year to demonstrate the epidemiology of the respiratory organisms. regarding the atypical bacteria, m. pneumoniae was detected throughout the year, with the highest incidence occurring in september and three minor peaks in december, january and june (fig. a) . the highest incidences of b. pertussis were observed in march and may. only one case of c. pneumoniae was detected in july. the seasonal prevalence of viruses with high detection rates were as follows. both flua and hmpv had two peaks that occurred in january and march, and adv showed a peak in january (fig. b) . the prevalence of para remained high from february-august. the peaks in our study, specimens were collected from pediatric patients with rtis over a period of one year and analyzed with filmarray rp v . . the overall results yielded a positivity rate of . %, with multiple organisms detected in . % of specimens, which is in accordance with litwin and piralla's reports [ , ] . as in other studies, a notable variation in the pathogen prevalence with season and age was observed. most viruses had their highest positivity rates in winter, except that para positivity rate was well distributed through the spring and summer, and the epidemiologic peaks for hmpv occurred to months later than those for rsv [ , ] . the majority of respiratory viruses were observed in children younger than years old. notably, rsv was the most prevalent virus in the < -year age group, and the prevalence decreased with age; while the incidence of m. pneumoniae increased with age [ ] . multiple respiratory organisms were detected in . % of the specimens in our study, the largest proportion of which included rhino/entero. other studies in adults reported lower multi-pathogen detection rates of approximately . - . % [ , [ ] [ ] [ ] , suggesting that pediatric patients with rtis are more likely to be infected by multiple pathogens than adults. however, the clinical significance, including disease severity and hospitalization time, of multi-pathogen infection, especially rhino/entero combination infections, is not clear. a previous report indicated that dual-positive results with rsv and rhino/entero specimens might be due to viral shedding from a previous rhino/entero infection [ ] . nokso-koivisto et al. also found that rhinovirus was the most prevalent virus in asymptomatic carriers [ ] . the most unexpected result in our study is the high detection rate of b. pertussis, with an overall detection rate of . % in the group of patients, further demonstrating the value of filmarray rp in clinical application. at present, the diagnosis of pertussis in china is based on culture and serology results. however, both the cdc and world health organization (who) use positive pcr results as the criteria for diagnosis, suggesting that filmarray rp testing, in addition to culture, can be considered for patients with suspected pertussis in order to better monitor disease outbreaks. additionally, the early diagnosis of patients with b. pertussis, which is typically difficult to distinguish from pertussis-like syndrome, can also help to reduce unnecessary macrolide treatment. the limitation of the panel is the lack of b. parapertussis, which contributes to more than % of pertussis cases [ ] . however, it has been added to the second-generation panel, filmarray rp v . [ ] , and the prevalence of b. parapertussis in our patients is currently under investigation. as stated in the manufacturer's instructions, "filmarray respiratory panel (rp) is a multiplexed nucleic acid test intended for use with filmarray systems for the simultaneous qualitative detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swabs (nps) obtained from individuals suspected of respiratory tract infections". therefore, nps samples are recommended for filmarray rp, but there are also studies demonstrating a comparable or even higher detection rate in sputum [ , ] . however, the detection rate in sputum in our study was lower than that in nps samples. this might partially be attributed to the fact that most of the sputum samples ( . %, / ) were from icu patients, and the sputum-providing patients showed a higher positivity rate in their sputum culture than the nps-providing patients ( . % vs . %). in addition to sputum, bronchoalveolar lavage fluid (balf) is a common type of respiratory sample, and azadah et al. showed that detection in balf by filmarray rp can provide new and useful microbiological information within days after a negative nps result is obtained [ ] . therefore, the choice of the most appropriate sample type and time-point for each patient, particularly in specific clinical contexts, such as undergoing fiberoptic bronchoscopy or ventilator use, may require further investigation. as with other molecular methods, distinguishing whether the microbes detected in the filmarray analysis, especially those that are also detected in asymptomatic children, such as human rhinovirus, are causative pathogens or colonizers is not feasible [ ] [ ] [ ] . therefore, the clinicians should take caution when judging pathogens because the results are sometimes "false positive". on the other hand, despite the high detection rate of fil-marray rp, a negative result does not mean the patient is not infected; moreover, a positive result does not mean there is no other co-infecting agent, especially in critically ill patients, in whom a bacterial co-infection often occurs. for this "false-negative" limitation, biofire has a new pneumonia panel that also targets balf/sputum and covers common viruses, as well as bacteria, including klebsiella pneumonia, haemophilus influenza, streptococcus pneumonia and staphylococcus aureus. nevertheless, the filmarray panel only aims to rapidly provide results for potential pathogens as a reference. a more appropriate method is to comprehensively consider the results from other examinations, such as routine blood testing, c-reactive protein (crp), procalcitonin (pct), the erythrocyte sedimentation rate (esr), culture and radiography, as well as the patients' symptoms, including body temperature, breathing, blood oxygen, heart rate, and mental condition. our study also has several limitations. first, our study was performed in a single center and may not be representative of the entire chinese pediatric population. second, we did not have data from a more appropriate assay to evaluate the specificity of filmarray rp. additionally, we do not provide detailed information on the effects of filmarray rp on the use of antibiotics, clinical outcomes and health economics, which require further investigation. in conclusion, the filmarray rp assay significantly expands our ability to diagnose multiple respiratory infections caused by viruses and atypical bacteria. the array can detect respiratory organisms simultaneously, with a high detection rate, in min. our study provided the age groups and seasonal distributions of different organisms for pediatric rti patients. this study also provides new insights into the current status of pertussis infection in china. whether filmarray rp can enhance clinical decision-making and limit the unnecessary use of antibiotics in china as in other countries still requires further investigation. additional file : table s . combinations of multiple organisms detected with filmarray rp. (doc kb) additional file : table s . overall detection rates for the nasopharyngeal swab and sputum samples from the different age groups. epidemiology of viral respiratory infections community-acquired pneumonia requiring hospitalization among u.s. children epidemiology of acute respiratory infections in children in guangzhou: a three-year study seroprevalence of pertussis in china: need to improve vaccination strategies the seroepidemiology of immunoglobulin g antibodies against pertussis toxin in china: a cross sectional study routine molecular point-of-care testing for respiratory viruses in adults presenting to hospital with acute respiratory illness (respoc): a pragmatic, open-label, randomised controlled trial multiplex pcr and emerging technologies for the detection of respiratory pathogens an automated nested multiplex pcr system for multi-pathogen detection: development and application to respiratory tract infection charnot-katsikas a. comparison of cepheid xpert flu/rsv xc and biofire filmarray for detection of influenza a, influenza b, and respiratory syncytial virus comparison of the filmarray assay and in-house real-time pcr for detection of respiratory infection comparison of filmarray respiratory panel and laboratory-developed real-time reverse transcriptionpolymerase chain reaction assays for respiratory virus detection comparison of the filmarray respiratory panel and prodesse real-time pcr assays for detection of respiratory pathogens filmarray(r) respiratory panel performance in respiratory samples from neonatal care units seasonality and prevalence of respiratory pathogens detected by multiplex pcr at a tertiary care medical center human metapneumovirus subgroup changes and seasonality during epidemics seasonality, incidence, and repeat human metapneumovirus lower respiratory tract infections in an area with a high prevalence of human immunodeficiency virus type- infection etiologic spectrum and occurrence of coinfections in children hospitalized with community-acquired pneumonia performance of a novel microarray multiplex pcr for the detection of respiratory pathogens (symp-ari study) pcr for detection of respiratory viruses: seasonal variations of virus infections detection of respiratory viruses by molecular methods prospective evaluation of a novel multiplex real-time pcr assay for detection of fifteen respiratory pathogens-duration of symptoms significantly affects detection rate human picornavirus and coronavirus rna in nasopharynx of children without concurrent respiratory symptoms update on pertussis and pertussis immunization multicenter evaluation of biofire filmarray respiratory panel for detection of viruses and bacteria in nasopharyngeal swab samples detection of respiratory viruses in sputum from adults by use of automated multiplex pcr evaluation and implementation of filmarray version . for improved detection of adenovirus respiratory tract infection filmarray respiratory panel assay: comparison of nasopharyngeal swabs and bronchoalveolar lavage samples respiratory viral detection in children and adults: comparing asymptomatic controls and patients with community-acquired pneumonia interactions of respiratory viruses and the nasal microbiota during the first year of life in healthy infants etiology of severe pneumonia in children in developing countries we would like to thank the patients and their parents for the support and cooperation in publishing this work. availability of data and materials all data described in this manuscript is available upon request via email. authors' contributions qc and xm initiated the study. jl, yt, myt and bld performed the detection of respiratory organisms. jl, yt, myt, qc and xm wrote the manuscript and analyzed the data. yjx provided technical support and assisted in the data analysis. all authors read and approved the final manuscript.ethics approval and consent to participate the study was approved by the institutional review board and the ethics committee of shanghai children's medical center (scmcirb-k ), and written informed consent was obtained from the parents of each patient. not applicable. yijun xia is an employee of biomérieux. he was involved in the technical support and data analysis. all the other authors declare they have no conflict of interests to disclose. all the other authors declare that they have no competing interests. key: cord- - qh pblc authors: quah, jessica; jiang, boran; tan, poh choo; siau, chuin; tan, thean yen title: impact of microbial aetiology on mortality in severe community-acquired pneumonia date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: qh pblc background: the impact of different classes of microbial pathogens on mortality in severe community-acquired pneumonia is not well elucidated. previous studies have shown significant variation in the incidence of viral, bacterial and mixed infections, with conflicting risk associations for mortality. we aimed to determine the risk association of microbial aetiologies with hospital mortality in severe cap, utilising a diagnostic strategy incorporating molecular testing. our primary hypothesis was that respiratory viruses were important causative pathogens in severe cap and was associated with increased mortality when present with bacterial pathogens in mixed viral-bacterial co-infections. methods: a retrospective cohort study from january to july was conducted in a tertiary hospital medical intensive care unit in eastern singapore, which has a tropical climate. all patients diagnosed with severe community-acquired pneumonia were included. results: a total of patients were in the study. microbial pathogens were identified in ( . %) patients. mixed viral-bacterial co-infections occurred in ( . %) of patients. isolated viral infections were present in patients ( . %); isolated bacterial infections were detected in patients ( . %). hospital mortality occurred in ( . %) patients. the most common bacteria isolated was streptococcus pneumoniae and the most common virus isolated was influenza a. univariate and multivariate logistic regression showed that serum procalcitonin, apache ii severity score and mixed viral-bacterial infection were associated with increased risk of hospital mortality. mixed viral-bacterial co-infections were associated with an adjusted odds ratio of . ( % ci . – . , p = . ) for hospital mortality. conclusions: respiratory viruses are common organisms isolated in severe community-acquired pneumonia. mixed viral-bacterial infections may be associated with an increased risk of mortality. the microbial aetiology of severe community-acquired pneumonia (cap) remains varied throughout the world, with influences due to seasonal climate change, outreach of vaccination programmes and pathogen endemicity [ , ] . two decades ago, it was thought that viruses played a minor role in the pathogenesis of severe cap, notwithstanding influenza epidemics [ ] . recent literature contradicts this and suggests that viruses are frequently found in severe cap [ , ] . advances in molecular techniques have improved the sensitivity, accuracy and turnaround time of microbial diagnostic tests [ , ] . the availability of highly multiplexed commercial tests kits for common viral and bacterial pathogens has enabled these tests to be performed in large numbers of patients simultaneously, and across a variety of clinical settings [ , ] . multiple respiratory viruses may be present concurrently, or co-exist with bacterial pathogens to cause disease [ ] [ ] [ ] . the reported incidence of viruses in severe cap resulting in respiratory failure ranges . % to % [ , ] . this variation in the detection rate of viruses reflects potential limited availability of test assays and heterogeneity of physician practices in viral microbial diagnostic tests performed [ , ] . postulated prohibitive factors against the routine performance of viral diagnostics tests in patients with severe cap may include a lack of clear clinical guidelines, perceived low cost-effectiveness and the paucity of effective anti-viral therapies for respiratory viruses other than influenza. the primary aim of our study was to determine the risk association of microbial aetiologies with hospital mortality in severe cap, utilising a diagnostic strategy that incorporated molecular testing. our primary hypothesis was that respiratory viruses were important causative pathogens in severe cap and was associated with increased mortality when present with bacterial pathogens in mixed viral-bacterial co-infections. this was a retrospective cohort study performed in an -bed medical intensive care unit of a -bed tertiary teaching hospital, in singapore. ethics approval was obtained from the singhealth centralised institutional review board (cirb / /fp), with waiver of consent. adults above the age of admitted to the medical intensive care unit with severe cap from january to july were included. written and electronic medical records were reviewed. cap was defined as an acute infection of the lung parenchyma associated with acute chest radiographic infiltrates and or more of the following: fever of °c or higher or hypothermia of °c or less; a new cough; dyspnea not explained by other reasons; worsening cough or change in respiratory secretions in a patient with pre-existing chronic cough. these symptoms should have been present at the time of, or within h of, hospital admission. chest radiographs reported by the hospital radiologists were obtained to confirm the presence of radiographic pulmonary infiltrates. patients with shock requiring vasopressor support, mechanically ventilated or who have out of minor criteria for pneumonia severity as defined by the infectious diseases society of america/american thoracic society, are considered to have severe cap [ ] . the minor criteria include: respiratory rate of breaths per minute or greater; pao /fio ratio equal or less than ; multi-lobar pulmonary infiltrates; confusion or disorientation; blood urea nitrogen equal or greater than mg/dl; leukopenia with white blood cell count of less than , cells/mm ; hypothermia indicated by core body temperature less than °c; hypotension requiring aggressive fluid resuscitation. all patients with prior hospitalisation within days of study enrolment; on active chemotherapy for neoplastic diseases; receiving renal replacement therapy by haemodialysis and prisoners were excluded. immunocompromised patients were not excluded as they were likely to have cap microbial pathogens as well as opportunistic infections. the time of admission to the intensive care unit was verified with electronic records. all patients had at least set of aerobic and anaerobic blood cultures performed within h of admission. routine collection of endotracheal aspirates for gram stain and semi-quantitative bacterial cultures occurred within h of intubation. viral and atypical pathogen polymerase chain reaction (pcr) amplification tests were collected from endotracheal aspirates. sputum samples were sent for bacterial aerobic cultures, while nasopharyngeal swabs were performed for atypical pathogen and viral pcr amplification tests for subjects who did not require intubation. urinary samples were tested for the presence of urinary streptococcus pneumoniae antigen and legionella pneumophilia serogroup antigen in patients without anuria. where indicated, acid-fast staining and mycobacterial cultures of respiratory samples were performed. nucleic acid was extracted from swabs or respiratory samples using ez virus mini kit (cat no. , qiagen, germany) performed on a semi-automated system (ez , qiagen, germany), and stored at - °c for more detailed molecular testing. besides routine standard clinical testing, additional multiplex real-time pcr testing was performed for each extracted sample. anyplex written and electronic medical records were reviewed for clinical data and laboratory indices. the most severe value was recorded for analysis if any blood test was repeated more than once within h after intensive care admission. serum c-reactive protein was measured by particle enhanced immunoturbidimetry (cobas® crpl ) and serum procalcitonin was measured using sandwich immunoassay (cobas® elecsys brahms pct). variables such as the acute physiology and chronic health evaluation ii (apache ii) severity score, presence of shock, mechanical ventilation, intensive care unit length of stay, and mortality were captured prospectively as part of administrative clinical care audits. empirical antibiotics were deemed to be adequate if it adhered to the hospital antimicrobial guidelines. combination therapy with beta-lactams and macrolides were recommended. where melioidosis was suspected, carbapenems were preferred to other classes of beta-lactams. categorical variables were expressed as number (percentages) and normally distributed quantitative variables were expressed as mean (± standard deviation). categorical variables were compared with chi-square test or fisher's exact test, and quantitative variables were compared with t-test or mann-u whitney test. the primary outcome measure was all-cause hospital mortality. demographic and disease variables were included in the univariate and multivariable logistic regression model. pneumonia symptoms were excluded from regression analysis as there were no known associations with mortality in severe cap. the variable for number of co-existent pathogens was excluded from regression analysis due to significant overlap with mixed viral-bacterial co-infections, which was included in the multivariate model. the following severity indicators: shock, altered mentation and ventilator support were not included as these variables are incorporated in the apache ii severity score. mycobacterium tuberculosis was classified as a bacterium for the logistic regression. demographic and disease variables were then compared between patients with and without virus infections, for identification of potential risk factors associated with acquisition of respiratory viral infections. a p-value of < . was considered significant. missing data was excluded from univariate and multivariate analysis. stata special edition version . (statacorp llc, texas, usa) was used for statistical analysis. one hundred seventeen patients were admitted to the medical intensive care unit for severe cap within the study period. baseline characteristics of patients who suffered in-hospital mortality (n = , . %) were compared with patients who survived, in table . of note, the patients who did not survived were older compared to survivors ( . years vs . years, p = . ). other baseline demographic and co-morbidity variables were not significantly different. blood cultures, respiratory specimen cultures and respiratory specimen pcr testing for viruses and atypical bacteria were performed in all patients. results of microbiological tests are presented in table . causative microbial aetiologies were identified in . % of patients with specific organisms presented in table . majority of pathogens identified were respiratory viruses and bacteria. viruses were found in . % of patients (n = ) with the most common virus being influenza a. . % (n = ) of patients with influenza a received empirical oseltamivir on the basis on clinical suspicion. bacterial infections were found in . % of patients (n = ). . % of patients (n = ) had mixed virus and bacterial co-infections. patients ( . %) had only viral pathogens and patients ( . %) had only bacterial pathogens, found as the microbial aetiology for severe cap. using hospital mortality as the primary outcome, univariate logistic regression was performed (table ). patients who did not survive had a higher apache ii score and higher serum procalcitonin levels. the microbiological aetiology that was significantly associated with increased hospital mortality was the detection of mixed viral-bacterial co-infections. on multivariate analysis, apache ii severity score, serum procalcitonin and mixed viral-bacterial co-infections remained significantly associated with increased adjusted odd ratios for hospital mortality. while apache ii severity score is known to be predictive for mortality in severe cap, the study found that the presence of mixed viral-bacterial co-infections was associated with increased hospital mortality by an adjusted odds ratio of . ( % confidence interval . , . , p = . ). the patients with respiratory viruses detected (both isolated viral pathogens and mixed viral-bacterial co-infections) were compared with patients who did not have any respiratory virus infections, in table . the mean serum c-reactive protein was found to be greater ( . ± . mg/l vs. . ± . mg/l) in patients without respiratory viruses compared to patients with detection of respiratory viruses (table ). respiratory virus infection as a cause of severe acute respiratory distress syndrome is well-established in literature data was missing for patient [ ] [ ] [ ] . however, its significance as a contributory pathogen in the outcomes of severe cap is uncertain. in this study, we showed that respiratory viruses were as commonly found as bacteria ( . % vs . %), as an aetiological pathogen. mixed viral-bacterial co-infections occurred in . % of patients and was associated with an adjusted odds ratio of . for hospital mortality. the impact of respiratory viruses on the prognosis of severe cap remains unclear with recent studies demonstrating contradictory results. fisher et al., in a prospective -year microbiologic survey of nosocomial pneumonia and cap complicated by respiratory failure, showed that . % of patients had viral infections, which was associated with a hospital mortality of . % [ ] . however, siow et al., found that viral infections were independently associated with lower hospital mortality compared to other microbial aetiologies, with an adjusted odds ratio of . (ci . - . ; p = . ) [ ] . in light of these findings, accurate characterisation of the impact of microbial aetiology on the outcomes of severe cap is required to influence future development of rapid molecular diagnostics assays and novel antimicrobial therapies that would target both viruses and bacteria [ , ] . piralla et al. reviewed the microbiological data of severe cap in northern italy during winter-spring seasons over years and found that . % of patients had one or more respiratory viruses identified [ ] . the most common viruses isolated were influenza a and rhinovirus, similar with our findings. while our study was performed in a tropical country, local microbiological surveillance has shown that influenza epidemics occur twice annually [ ] . this would mean that influenza seasons occurred over the course of this study. the molecular mechanisms in the viral pathogenesis of severe pneumonia are most well studied in influenza a and streptococcus pneumoniae co-infections. viral infections alter host immune responses that increase susceptibility to bacterial infection through viral-induced interferons [ ] [ ] [ ] . on clinical suspicion alone, only . % (n = ) of patients with influenza a in this study received empirical oseltamivir. the authors postulate that incorporation of early influenza a diagnostic tests may decrease the time to effective anti-viral therapies. the second most common virus detected in our study was rhinovirus (n = ). the association of rhinovirus with severe pneumonia has previously been shown in a surveillance program for middle east respiratory syndrome coronavirus in saudi arabia [ ] . its genotypes a to c are associated with severe pneumonia, with in-hospital mortality rates from . to . % [ ] . patients who are immunocompromised or who have chronic lung disease are most at risk [ ] . there are several reasons why viral infections may have been less prominent as a cause of severe cap in prior decades. firstly, grève et al. performed a prospective observational study on physician practices in the use of respiratory virus diagnostics demonstrating that despite clinical guideline recommendations on testing of respiratory viruses during influenza season, less than half of patients admitted to the intensive care unit with pneumonia were tested for viral pathogens [ ] . this may have led to under-recognition of the true significance of viral pathogens and mixed viral-bacterial infections, on outcomes in severe pneumonia. other factors which may have contributed to underdiagnosis of viral pneumonias include the unavailability or cost of molecular diagnostic assays, and the lack of effective anti-viral therapies [ ] . however, the authors argue that in this age of globalisation, highly virulent respiratory viruses have the potential to spread rapidly. constant surveillance is required to detect outbreaks and for the implementation of isolation precautions in a timely manner [ , ] . understanding the significance of respiratory viruses in the pathogenesis of severe cap would guide administrators with resource allocation when implementing vaccination programs for at-risk populations. the high rates of compliance with performing respiratory aerobic cultures, blood aerobic and anaerobic cultures in this study, were in accordance with sepsis guidelines [ ] . the most common bacterial pathogen found was streptococcus pneumoniae ( . %), which is consistent with the known epidemiology of cap globally [ ] [ ] [ ] . gadsby et al., in a prior study, was able to demonstrate a bacterial yield of . % when respiratory specimens from patients with cap were tested with bacterial multiplex pcr [ ] . incorporating the use of bacterial multiplex pcr in future studies, may increase the rate of bacteria detection, and shed light on potential molecular synergisms between specific viruses and bacteria in the pathogenesis of severe cap. pulmonary tuberculosis is endemic in the region where this study was performed. in our study, patients had tuberculosis, one of whom had concomitant adenovirus infection while another had streptococcus pneumoniae. the initial presentation of pulmonary tuberculosis with clinical features consistent with severe cap has been described by tseng et al. [ ] , where % of patients with pulmonary tuberculosis presented with respiratory failure or septic shock. the authors postulate that pulmonary tuberculosis may play a role in increasing host susceptibility to severe infection with cap organisms. the authors recognise that there were several limitations to this study. firstly, while first-dose antibiotics would have been administered as soon as sepsis is identified, we are unable to accurately define the time between administration of antibiotics and collection of specimens for microbiological assessment. this may affect the yield [ , , ] . secondly, the inherent retrospective nature of this study increases the risk of bias in data collection. however, as part of pre-established intensive care unit clinical audits and with the availability of national health records, clinical data such as participant demographics, co-morbid illnesses and severity indicators such as apache ii were established at the time of intensive care admission and stored prospectively. thirdly, given the high population density of the country ( rd in the world) where this study was performed, the microbial epidemiology of severe cap may only be extrapolated to urban settings. the study is a single-centre survey conducted in of the acute general hospitals serving a population of . million in a land area of . km . hence, the epidemiology may lack generalisability when extrapolated to other tropical countries. the fourth limitation is that we included patients who were immunocompromised with typical cap organisms and survived. they may potentially have had opportunistic infections that were not detected, however, as they did not contribute to the mortality outcomes, we felt that the microbiological data contributed by these patients were useful in the understanding of the prevalence of various data are presented as number (%), mean ± standard deviation apache acute physiology and chronic health evaluation a data was missing for patients. b data was missing for patient classes of cap organisms. lastly, another potential limitation was that vaccination records could not be retrieved, and we were unable to ascertain its influences on microbial aetiologies of severe pneumonia in our study population. the main strength of the study is the characterisation of the epidemiology of microbial pathogens in severe cap. we were able to show that in a tropical environment, the viral and bacterial pathogens associated with severe cap were similar with regions with a seasonal climate. despite a lower-than-expected mortality rate for severe cap in our study ( . %) compared with international data [ ] [ ] [ ] [ ] , we were able to demonstrate that mixed viral-bacterial co-infections were independently associated with hospital mortality. respiratory viruses are important causative pathogens in severe cap and are associated with increased risk of mortality when present with bacterial pathogens in mixed viral-bacterial co-infections. abbreviations apache ii: acute physiology and chronic health evaluation ii; cap: community-acquired pneumonia; pcr: polymerase chain reaction etiology and outcome of severe community acquired pneumonia in immunocompetent adults etiology of community-acquired pneumonia and diagnostic yields of microbiological methods: a -year prospective study in norway epidemiology of community-acquired respiratory tract infections in adults the intensive care global study on severe acute respiratory infection (ic-glossari): a multicenter, multinational, -day inception cohort study viral-bacterial coinfection affects the presentation and alters the prognosis of severe community-acquired pneumonia molecular diagnosis of respiratory viruses the filmarray® respiratory panel: an automated, broadly multiplexed molecular test for the rapid and accurate detection of respiratory pathogens rapid viral diagnosis for acute febrile respiratory illness in children in the emergency department clinical utility of on-demand multiplex respiratory pathogen testing among adult outpatients burden of respiratory viruses in patients with acute respiratory failure community-acquired polymicrobial pneumonia in the intensive care unit: aetiology and prognosis clinical characteristics and outcomes in hospitalized patients with respiratory viral co-infection during the h n influenza pandemic management of severe communityacquired pneumonia: a survey on the attitudes of physicians in iberia and south america clinical practice of respiratory virus diagnostics in critically ill patients with a suspected pneumonia: a prospective observational study infectious diseases society of america/american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults acute management and long-term survival among subjects with severe middle east respiratory syndrome coronavirus pneumonia and ards virus-induced acute respiratory distress syndrome: epidemiology, management and outcome influenza a (h n ) vs non-h n ards: analysis of clinical course a prospective one-year microbiologic survey of combined pneumonia and respiratory failure the use of polymerase chain reaction amplification for the detection of viruses and bacteria in severe communityacquired pneumonia the past decade in bench research into pulmonary infectious diseases: what do clinicians need to know? laboratory diagnosis of pneumonia in the molecular age frequency of respiratory viruses among patients admitted to intensive care units in seven consecutive winterspring seasons ( - ) in northern italy characterization of influenza activity based on virological surveillance of influenza-like illness in tropical singapore the immunology of influenza virusassociated bacterial pneumonia integrated clinical, pathologic, virologic, and transcriptomic analysis of h n influenza virus-induced viral pneumonia in the rhesus macaque kinetics of coinfection with influenza a virus and streptococcus pneumoniae etiology of severe communityacquired pneumonia during the hajj-part of the mers-cov surveillance program clinical and molecular characterization of rhinoviruses a, b, and c in adult patients with pneumonia human rhinoviruses and severe respiratory infections: is it possible to identify at-risk patients early? viral pneumonia and acute respiratory distress syndrome burden of acute respiratory disease of epidemic and pandemic potential in the who eastern mediterranean region: a literature review global threat of animal influenza viruses of zoonotic concern: then and now surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock global and local epidemiology of communityacquired pneumonia: the experience of the capnetz network global changes in the epidemiology of community-acquired pneumonia community-acquired pneumonia requiring hospitalization among u.s adults comprehensive molecular testing for respiratory pathogens in community-acquired pneumonia empirical use of fluoroquinolones improves the survival of critically ill patients with tuberculosis mimicking severe pneumonia impact of antibiotic therapy in severe community-acquired pneumonia: data from the infauci study predictive factors of mortality in severe community-acquired pneumonia: a model with data on the first h of icu admission viral infection in patients with severe pneumonia requiring intensive care unit admission etiology of severe pneumonia in the very elderly assessment of prognosis in patients with community-acquired pneumonia who require mechanical ventilation the authors would like to thank the following: ms. carmen kam, the resident biostatistician for verification of the study statistical analysis; nurses ms. wang xi qin, ms. goh yuan xuan, ms. lim qian ru for assistance in data collection; senior medical technologist ms. heng ying xuan, ms. lee hui zi for assistance in performance of pcr tests; dr. tay tunn ren for her tutelage in manuscript writing. the study was performed with a grant awarded from changi general hospital research grant in (grant reference number chf . ), for an amount of $ singapore dollars (equivalent to usd , conversion usd = sgd . ). the datasets analysed during this current study are available from the corresponding author on reasonable request. authors' contributions jlq contributed to the design, analysis, interpretation of the study; drafting and revision of the manuscript. bj contributed to the design, interpretation and microbiological analysis of the study. pct contributed to the design and data acquisition of the study. cs contributed to the design of the work, drafting and revision of the manuscript. tyt contributed to the conception, analysis and interpretation of the study, drafting and revision of the manuscript. all authors have given final approval of the version to be published and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.ethics approval and consent to participate ethics approval was obtained from the singhealth centralised institutional review board, singapore. reference number: cirb / /fp. waiver of consent granted for this study. not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - wuttqw authors: pereira, e.p.v.; van tilburg, m.f.; florean, e.o.p.t.; guedes, m.i.f. title: egg yolk antibodies (igy) and their applications in human and veterinary health: a review date: - - journal: int immunopharmacol doi: . /j.intimp. . . sha: doc_id: cord_uid: wuttqw egg yolk constitutes a relevant alternative source of antibodies. it presents some advantages over mammalian serum immunoglobulins regarding productivity, animal welfare and specificity. the main immunoglobulin present in avian blood (igy) is transmitted to their offspring and accumulates in egg yolks, which enables the non-invasive harvesting of high amounts of antibodies. moreover, due to structural differences and phylogenetic distance, igy is more suitable for diagnostic purposes than mammalian antibodies, since it does not react with certain components of the human immune system and displays greater avidity for mammalian conserved proteins. igy has been extensively used in health researches, as both therapeutic and diagnostic tool. this article aims to review its applications in both human and veterinary health. antibodies are protein molecules produced in response to an antigen. due to their ability to bind to specific targets, they are widely used in research, diagnosis and therapy. most of the currently available antibodies are produced in mammals, especially in small rodents [ ] . however, the production of antibodies in mammals can be challenging due to the fact that some antigens elicit weak immune responses or are even completely non-immunogenic. moreover, the production of antibodies in mammals involves procedures that cause pain and distress to animals; such as immunization, blood sample collection and sacrifice [ ] . the search for more efficient and economical techniques, as well as for the reduction and refinement of the use of animals, has led to growing interest for egg yolk antibodies (igy). obtaining antibodies from egg yolk is a non-invasive method that eliminates the need for blood collection. hens produce a greater amount of antibodies compared to other animals, like rodents for example, considerably reducing the number of animals required for the production of antibodies [ , ] . this method presents several economical advantages over the use of other animals. for example, the cost of maintaining a hen is less expensive than maintaining animals like mice and rabbits; the amount of antibodies produced by chickens also corresponds to that of larger animals, such as goats and sheep. for instance, in one year, a hen lays about eggs and produces an average of , g of igy [ ] . moreover, the specific igy produced in chickens are about - % of the total amount of antibodies [ ] . nonetheless, the amount of antibodies produced is correlated to the quantity of antigen applied to the hen, its immunogenicity and molecular weight [ , ] . being a productive technique, as well as more refined from the point of view of animal welfare, igy technology has been used for several purposes in human and veterinary health, such as in immunodiagnostics [ ] , immunotherapy [ ] , neutralization of toxins from venomous animals [ ] and bacteria [ ] , and as functional food [ ] . considering the fast development of igy technology, this work aims to review its applications in human and animal health, in addition to increasing the potential use of these antibodies among researchers and consequently promoting the reduced use of non-invasive procedures on animals. the specific protective effect of egg yolk extracts from immunized hens, attributed to the transfer of serum chicken antibodies to eggs, was first described in [ ] . however, this knowledge remained without applications until it attracted the interest of the scientific community due to the search for animal welfare, which was driven by the works of russel & burch and the publication of the "principles of humane experimental technique" in . the use of igy increased in the s, possibly due to the development of commercially available secondary reagents, such as igy purification kits and anti-igy specific antibodies conjugated to alkaline phosphatase, fluorescein isothiocyanate and peroxidase markers [ ] . in , the european center for the validation of alternative methods (ecvam) workshop recommended the use of igy rather than mammalian igg, with the purpose of minimizing the pain generated by invasive collection of serum antibodies [ ] . igy is present in birds, reptiles, amphibians and lungfish and is the evolutionary precursor of igg and ige, present only in mammals [ ] . over time, igy was called igg due to the supposed similarity between the two. however, leslie & clem emphasized the distinct differences between igy and igg, such as antigenic differences and the major size of igy heavy chain, suggesting the use of this term instead of igg [ ] . the igy molecule has a structure similar to that of igg, with two heavy chains (h), each one with a molecular weight of to kda, and two light chains (l), with kda. the light chains have one constant region (cl) and one variable region (vl), similar to igg. the major difference between igy and igg is found at the heavy chains. igg has three constant regions at the heavy chains (ch -ch ), while igy has four constant regions (ch -ch ) (fig. ) . a further constant domain, with the corresponding carbohydrate chains, gives igy a higher molecular weight ( kda) in comparison to igg ( kda) [ ] . igy is less flexible than igg due to the absence of the hinge region between ch and ch [ ] , similar, in this aspect, to ige [ , ] . the presence of glycine and proline residues at ch -ch and ch -ch regions also limits igy flexibility [ ] . one advantage of this higher chain stiffness is that it may be associated with increased igy resistance to proteolytic degradation and fragmentation [ ] . also, igy is more hydrophobic than igg and has an isoelectric point between . and . . the half-life of purified igy is of months, retaining activity for up to six months at room temperature and one month at °c. moreover, affinity-purified and biotinylated igy retained high activity after five years of storage at °c [ ] . the structural and phylogenetic differences between igy and igg are manifested in different molecular and biochemical interactions. the fc portion of igy is unable to activate the human complement [ ] and to bind to rheumatoid factor [ ] and to protein g [ ] , it also does not react with human anti-mouse antibodies (hama) [ ] nor with the erythrocyte agglutinogens a and b [ ] . due to phylogenetic distance, chickens produce a stronger immune response against conserved mammalian proteins [ ] . although igy contains the variable (v), junction (j) and diversity (d) regions, the contribution of the v (d) j rearrangement to its immunogenetic diversity is small, due to the fact that only a single locus undergoes rearrangement [ ] . thus, the genetic diversity of igy is guaranteed by gene hyperconversion, a process in which pseudogenes donate homologous sequences to the functional gene (v) after v (d) j rearrangement [ ] . even though it is a protein molecule, igy is resistant to heat and ph, being stable between °and °c and active between ph . and . however, the affinity of igy to its antigen decreases with increasing temperature. nonetheless, the addition of sucrose, maltose and glycine protects igy from heat denaturation [ ] , while sorbitol stabilizes it at acid ph [ ] . igy is resistant to inactivation by the proteolytic enzymes trypsin and chymotrypsin, but is degraded by pepsin [ ] . the digestion of igy with papain leads to a fc fragment plus two monovalent fab fragments, like igg. nonetheless, igy digestion with pepsin leads to a pfc′ fragment and two monovalent fab fragments, unlike mammalian antibodies, which forms one bivalent fab fragment when cleaved by pepsin [ ] . the characteristics of igy, in comparison to igg, are summarized in table . several methods have been developed to protect igy from degradation by ph and pepsin present in the stomach, allowing it to arrive unscathed at the small intestine, which is especially important for the use of igy against enteric pathogens. among these treatments, chitosan and alginate microcapsules [ ] , β-cyclodextrin microcapsules [ , ] , copolymers of methacrylic acid [ ] , liposomes [ , ] , multiple emulsification [ ] , hydrogel [ ] and carbon nanotubes with hydrogel [ ] have been tested with different degrees of success. nonetheless, unprotected igy consumed as egg yolk in liquid [ ] and powder forms [ ] showed resistance to the gastrointestinal tract conditions in calves. the antibody titers are influenced by several factors, such as the antigen type and dose, the used adjuvant, the route of application, the inoculation frequency, age and stage of development in birds [ , ] . several protocols for obtaining igy, observing these different variables, have been applied [ , ] . basically, multiple immunization protocols were tested for different antigens and animals, in order to obtain the highest antibody titer for each case [ ] . several antigen types have been used to produce specific igy in birds, such as complex antigens (virus, bacteria and parasites) and single antigens (proteins, polysaccharides, peptides and nucleic acids) [ ] . different antigen concentrations can also be combined with adjuvant. however, it is common to use to μg of antigen per ml injected in two or three sites, with the age of hen ranging between seven and eight weeks [ ] . the induction of high antibodies titers depends on the use of adjuvants. though freund's complete adjuvant (fca) is the most potent to induce antibodies in laboratory animals, it can lead to severe inflammation at the injection site [ ] . although some studies have shown that the immunization with fca is more tolerated by birds than by mammals and seems to not generate tissue damage in chickens [ , ] , other studies using fca for bird immunization contradict these results [ , ] . since there is no consensus, one of the best substitutes for fca is freund's incomplete adjuvant (fia), which, unlike fca, does not contain mycobacteria. shade et al. recommended the use of fia, which can be used since the first immunization without any prejudice to the antibody titer [ ] . in spite of this, the first inoculation is generally performed using fca, while the subsequent inoculations are performed using fia, without the occurrence of inflammation at the injection site [ , ] . the most common route of antigen administration for igy production in chickens is the intramuscular route, where the inoculation is usually into the breast muscle. this technique is most suited for young hens, because subcutaneous injection into the neck is more difficult to perform and may cause more distress to the animal. the intramuscular injection into the leg must be avoided as it can lead to lameness [ ] . nonetheless, for a non-invasive method, the administration of antigen can also be performed orally [ ] . the number of inoculations required depends on the type and dose of the antigen, as well as the adjuvant used. at least two inoculations must be performed before the laying period, with an interval of four to six weeks. the titer of igy must be assessed days after the last immunization. if the antibody titer decreases, further immunizations must be done during the entire laying period to increase antibody titers year round [ ] . an increase in titer of egg yolk antibodies can be observed from the second week [ , ] or sometimes from the fifth week post antigen inoculation [ ] . after a steady increase, stabilization of the antibody titer occurs, reaching a plateau, and from there on it decreases progressively [ ] . through booster inoculations, it is possible to keep high titers of egg yolk antibodies for more than days [ ] . the igy extraction consists of the removal of lipids to form a water soluble fraction (wsf), followed by the precipitation of the antibodies contained therein. several extraction methods are available for obtaining igy from egg yolk and the choice of the suitable method depends on the purpose, which can require different purification degrees, as well as the extraction scale, cost and available technology [ ] . a frequently used method for the extraction of igy is the precipitation with polyethylene glycol (peg) . . the method consists on the delipidation by centrifuging egg yolk diluted in phosphate buffered saline (pbs) with . % of peg . next, the supernatant is twice centrifuged with % of peg to precipitate the antibodies [ ] . akita & nakai obtained highly purified igy by removing the lipids from the yolks by means of six-fold dilution in water at ph . , with an incubation time of h at °c, followed by igy precipitation with % of ammonium sulfate (nh ) so , which was supplemented by the addition of sodium chloride (nacl) or by ultrafiltration prior to gel filtration or ion exchange chromatography. the authors also achieved highly purified igy by means of ethanol precipitation at lower temperatures [ ] . nonetheless, a study conducted by araújo et al. showed that the most suited concentration of (nh ) so for igy's precipitation is %. in this study, higher concentrations of (nh ) so also caused the precipitation of non-specific proteins, while a lower concentration led to a greater loss of antibodies in the supernatant [ ] . since the fc region of igy does not bind to proteins a and g, which are commonly used in the affinity purification of mammalian igg, affinity chromatographic methods for igy purification requires other types of ligands. igy can be purified through the adsorption of the immunizing antigen to the solid phase of the affinity column, which generates monospecific antibodies that can be eluted under acid [ ] or alkaline conditions [ ] . the purification of igy can also be performed through thiophilic adsorption chromatography [ ] . in contrast to other affinity purification methods, it can be performed at mild conditions [ ] . hens are able to produce between and mg of igy antibodies by yolk, of which between and % are specific [ ] . in one year, a single laying hen produces between and g of total igy [ ] . although the production of igy for research is mainly done in chicken, other birds are also used for the same purpose, such as goose [ ] and quail [ ] , using an immunization protocol similar to that used for chickens. the production of igy and its possible applications in both human and veterinary health are summarized in fig. . the use of polyclonal igy against infectious diseases minimizes the risk of microbial resistance, since the antibody is directed to various antigens of the same microorganism, which requires multiple genes for its synthesis [ ] . therefore, specific igy antibodies are a relevant alternative to use as antimicrobials in human and veterinary health in the face of the emergence of resistant bacteria. igy antibacterial activity can be assessed by the inhibition of bacterial growth and biofilm formation in vitro, as well as adhesion ability, since they are prior conditions for successful colonization of a higher animal by bacteria and viruses [ ] . igy antibacterial activity against gastrointestinal pathogens has been widely investigated. nasiri et al. extracted igy from hens immunized with the recombinant protein fanc, from enterotoxigenic escherichia coli (etec) and these antibodies bound specifically to fanc in elisa, western blot and dot-blotting [ ] , demanding, thus, more investigations to evaluate its viability as a potential immunotherapeutic compound. li et al. evaluated the effect of specific and non-specific igy against salmonella typhimurium on the immune response of mice infected by the bacterium [ ] . specific igy reduced the expression of the pro-inflammatory cytokines tnf-α and inf-γ and elevated that of the antiinflammatory cytokine il- ; while non-specific igy also reduced the level of tnf-α, but did not alter the expression level of inf-γ and il- . although both specific and non-specific antibodies reduced the number of t lymphocytes in the lamina propria and of cd + , cd + and γδ t lymphocytes in the jejunum of mice, the animals treated with anti-s. typhimurium igy had a longer survival rate than those treated with nonspecific igy. igy activity against bacterial spores was also investigated. oral administration of igy against clostridium difficile spores both delayed the onset of diarrhea in rats treated prior to the infection and reduced its recurrence in infected rats. thus, specific igy could be used in the prophylaxis of c. difficile infection, as well as an immunotherapeutic to eliminate the remaining spores on the intestines of people who have already had the infection, which would reduce the recurrence of diarrhea [ ] . the therapeutic action of igy against helicobacter pylori has been largely studied as well, through antibodies directed to both the whole cell and to specific proteins. oral administration of anti-hpuc (h. pylori urease) igy inhibited the growth of h. pylori in vitro and reduced the inflammation of stomach in mice, with a significant reduction of neutrophils and lymphocytes infiltration on the tissue. a decrease in the serum level of antibodies against h. pylori was also observed in treated animals [ ] . similar results, as the reduction of serum levels of anti-h. pylori igg and the absence of inflammation signs on the stomach were observed in mice that received oral igy against the h. pylori vacuolating cytokine a (vaca) [ ] . specific igy against hp-nap protein, the main virulence factor of h. pylori, significantly inhibited the adhesion of the bacterium to an ags cells culture [ ] . solhi et al. evaluated the occurrence of cross reaction between strain-specific igy and four different h. pylori strains. the antibodies inhibited the cell growth and the urease enzyme not only of the strains for which they were specific, but also of other strains [ ] . the therapeutic potential of igy against bacterial respiratory tract infections has also been considered. igy against mycobacterium tuberculosis increased the proliferation of pbmc (peripheral blood mononuclear cells) in rats in a dose-dependent manner. an increased expression of cytokines that stimulate the cellular immune response was observed and an increase of mrna level of il- and ifn-γ was detected by rt-pcr, which indicates that anti-m. tuberculosis igy is a potential immunotherapeutic that stimulates t-helper immune response [ ] . shi et al. produced specific igy against multi-drug resistant strains of acinetobacter baumannii. the antibodies inhibited bacterial growth in vitro, significantly reduced the mortality of mice infected with the bacterium and attenuated the lung inflammation [ ] . igy has also been tested as a potential therapeutic resource in oral infections against prevotella intermedia. specific igy inhibited bacterial growth in liquid medium and showed therapeutic activity in rats with p. intermedia induced gingivitis. the reduction of gingival inflammation, bacterial plaque and bleeding, as well as the normalization of wbc (white blood cells) levels in the blood, were observed in treated animals. histopathological examination showed no signs of inflammation in the gums of rats treated with anti-p. intermedia igy [ ] . additionally, studies have shown inhibitory growth effect of therapeutic igy produced against fusobacterium nucleatum in liquid medium and in the formation of biofilm in polyestyrene plates. anti-f. nucleatum igy also reduced bone loss in mice with periodontal disease when applied after the infection, thus presenting therapeutic function [ ] . the therapeutic effect of igy against acne was also evaluated. revathy et al. produced and assessed the efficiency of specific igy against the bacterium propionibacterium acnes. the antibody inhibited bacterial growth and biofilm formation in vitro, proving to be a possible alternative to antibiotics in the treatment of acne [ ] . more recently, igy was tested for antibacterial effect in aquatic animals. gao et al. produced igy against vibrio spp., the major cause of death in white shrimp (litopenaeus vannamei), and demonstrated that powder egg yolk containing anti-vibrio igy significantly reduced the mortality of white shrimp infected with v. harveyi and v. parahaemolyticus [ ] , which indicates that therapeutic igy can also be applied in aquafarming. the prophylactic potential of igy against dental caries has been widely studied. an interesting study carried by bachtiar et al. evaluated the effect of soybean milk containing igy against the cariogenic bacterium streptococcus mutans in rats. the animals fed with this milk presented a decrease in the number of s. mutans in dental biofilm and fifteen days after milk intake igy could still be detected in the saliva [ ] . in other research, the effect of specific igy against the cell-associated glucosyltransferase enzyme (ca-gtf), of s. mutans, was evaluated. as a result, anti-ca-gtf igy inhibited bacterial adhesion in vitro and suppressed the oral cavity colonization [ ] . in addition, specific igy action against the comd protein, a quorumsensing signals receptor of s. mutans, was also explored. it inhibited, in vitro, the development of biofilm of s. mutans from the oral cavity of people with and without caries, and changes in the protein expression pattern were found in the bacteria treated with anti-comd igy [ ] . the protective effect of igy against opportunistic infections was also investigated by thomsen et al., where, by means of chemiluminescence, the production of reactive oxygen species (ros) in polymorphonuclear neutrophils (pmn) was evaluated as an indicator of the intensity of pmn mediated immune response against pseudomonas aeruginosas in vitro, as well as the influence of anti-p. aeruginosas igy in this process. the specific igy opsonized the bacterium and intensified the cellular immune response, probably due to the recognition of igy by receptors resembling avian igy receptors or physical-chemical changes in the bacterium, since igy does not activate mammalian fc receptors. anti-p. aeruginosas igy could be used in the prophylaxis of p. aeruginosas infection in patients with cystic fibrosis by increasing the cellular immune response [ ] . prophylaxis of bacterial infections in animals for consumption, using igy, was also considered. specific igy incorporated into hydrogel added to carbon nanotubes and chitosan (h-cnt) showed protective activity against enterotoxigenic escherichia coli (etec) in piglets [ ] . thus, anti-etec igy incorporated into h-cnt and orally applied could be used to prevent the intestinal infection in these animals. in order to generate antibodies against the bacterium campylobacter jejuni to be used as food additive aiming to control c. jejuni colonization in chickens, thibodeau et al. produced anti-c. jejuni igy in three different ways: oral administration of a pool of living bacteria from four c. jejuni strains, subcutaneous injection of extracts from the outer membrane of the bacterium or of formalin inactivated bacterium. the hens submitted to subcutaneous inoculation produced igy more intensely, although the antibodies obtained through oral inoculation also showed good activity. anti-c. jejuni igy, obtained from the three ways, presented bactericidal activity in vitro [ ] . igy against the bacterium aeromonas salmonicida, the causative agent of ulcers on fish skin, was added, in powder form, to the water in which koi carp (cyprinus carpio koi) ornamental fish were raised. anti-a. salmonicida antibodies showed good prophylactic activity against the infection, and a small amount of specific igy was enough to completely prevent the appearance of skin ulcers in healthy fish that cohabited with sick fish previously infected with a. salmonicida [ ] . the potential of igy technology to prevent bacterial infections in animals could be useful in the needful strategies to reduce the use of antibiotics in food animal husbandry, which has been linked to the selection and spread of resistant bacterial strains that threaten antibiotics as a therapeutic resource [ , ] . the therapeutic potential of igy against viral infections of the gastrointestinal tract in animals has been extensively investigated. oral administration of egg yolk powder rich in igy against bovine group a rotavirus (rva) was performed on calves infected by the virus. as a result, disease attenuation occurred in treated animals, with a reduction of the period of diarrhea and hyperthermia and the absence of other symptoms observed in untreated calves, such as anorexia, dehydration and depression. the egg yolk powder enriched with anti-rva igy retained the activity after two years, when kept at °c [ ] . in another study, igy against the s protein of porcine epidemical diarrhea virus (pedv) was produced and orally administered in piglets previously infected with pedv. anti-s igy reduced the severity of diarrhea and intestinal lesions caused by pedv infection and nullified the mortality of piglets due to the disease [ ] . another possible therapeutic use of specific igy is against dengue fever, as demonstrated by fink et al. anti-denv igy produced in goose was able to neutralize the virus in vitro and in vivo without binding to fcγ receptors on myeloid cells and generating ade (antibody dependent enhancement) in mice [ ] . the protective effect of igy against influenza has been widely studied. igy against the avian influenza a virus (h n ) were extracted from eggs available in supermarkets of vietnam, where the vaccination of chickens against h n virus is required. anti-h n igy was administrated intranasally in mice before and after their infection with h n and h n and completely prevented the disease onset [ ] . these results reveal that commercially available eggs that are produced in countries where anti-h n vaccination is mandatory constitute a considerable source of igy that could be used to prevent a potential pandemic of h n virus. following this rationale, wallach et al. produced igy against h n , h n and h n strains of influenza virus, which were tested for their ability to neutralize homologous and heterologous strains in vitro and to prevent the infection in vivo. the antibodies inhibited only homologous strains in the seroneutralization and in the haemagglutination inhibition assay, except anti-h n igy, which also inhibited h n . anti-h n and anti-h n igy were applied intranasally in mice h before the infection by influenza virus and showed prophylactic activity. thus, igy against influenza virus strains could be used in nasal, oral or spray applications to protect individuals and environments [ ] . igy against influenza b virus (ibv) was also tested and neutralized the activity of haemagglutinins and neuraminidase present in the virus and inhibited viral replication in vitro. when applied intranasally, anti-ibv igy prevented influenza development in mice treated prior to virus exposure and attenuated the disease in those treated post-infection [ ] . the prophylactic effect of igy against other viral infections of the respiratory tract was also evaluated. igy against andes virus (andv), the causative agent of the hantavirus pulmonary syndrome (hps), was obtained from geese eggs by inoculating these animals with the dna encoding the virus envelope glycoprotein. anti-andv igy was able to prevent hps development in recently infected mice but failed when applied after the onset of viremia, thus presenting prophylactic and non-therapeutic activity [ ] . the protective effects of igy were also considered for viral infections in poultry. aizenshtein et al. produced, in the same eggs, efficient igy against the newcastle disease virus (ndv), infectious bursal disease virus (ibdv), influenza and reovirus, which are pathogenic for birds [ ] , demonstrating that passive immunization with igy against several viruses is possible; nonetheless, it is a technology that must be further explored. a gel preparation for oral use containing igy against candida albicans was tested by tekeuchi et al. and caused a reduction in the number of colony-forming units (cfu) on the oral cavity of elderly people, showing promise for prophylactic use against c. albicans oral infection [ ] . in other research, specific igy inhibited the adhesion of candida albicans and candida glabrata to denture base material. anti-c. albicans igy was more effective against c. albicans than anti-c. glabrata igy, while both antibodies were equally effective in preventing the adhesion of c. glabrata [ ] . sampaio et al. produced a high avidity igy against the protozoan trypanosoma evansi. there was neither prophylactic action nor infection control in the treated rats, but anti-t. evansi igy increased their survival rate when used concomitantly to anti-hematozoa drugs [ ] . in another study, grando et al. inoculated trypanosoma cruzi trypomastigots in hens and extracted a high amount of anti-t. cruzi igy that cross-reacted with t. evansi antigens in western blot, showing that the anti-t. cruzi igy is not reliable as a diagnostic tool, but deserves more investigations as a possible therapeutic resource for trypanosomes infection [ ] . the phylogenetic distance between birds and mammals ensures a stronger immune response against mammalian antigens by birds [ ] . such a feature may be advantageous to produce igy against human tumor antigens. following this rationale, amirijavidv et al. produced highly specific igy against a sequence of amino acids present on the ectodomain of the trail (tnf-related apoptosis-inducing ligand) receptor trail-r (dr ). the antibodies bound to the amino acid sequence and activated the dr receptors in human breast cancer cells mcf , acting as a trail agonist and inducing apoptosis [ ] . igy against other receptors, such as the her receptor, was tested coupled to single walled carbon nanotubes (swnts) and specifically detected the her receptors on the surface of sk-br- cells. the binding of the complex to the receptors was measured by raman signals emitted by the nanotubes. swnt has a near infrared absorption (nir), which can be used for tumor ablation, and, coupled to anti-her igy, was able to kill sk-br- cells without needing internalization of the complex by the cell [ ] . these findings show that igy produced against tumor antigens is an attractive alternative for a more selective treatment of cancers and its use could, therefore, minimize the side effects of traditional chemotherapy. igy raised against porcine pancreatic lipase was used against the enzyme in vitro and in vivo. later, mice with obesity induced by high fat diet were orally treated with the antibody, which was given concomitantly with food, and a reduction of adipose tissue and liver fat level was observed, as well as an increase of fecal excretion of triglycerides and their decrease in blood plasma. anti-lipase igy inhibited the hydrolysis of diet fat and reduced its intestinal absorption, showing anti-obesity activity [ ] . wei-xu et al. evaluated the antiallergic effect of specific igy against the pro-inflammatory cytokines il-β and tnf-α in guinea pigs with induced allergic rhinitis. a reduction of the eosinophils number in the blood and in the nasal and bronchial lavages was found, as well as a decrease of eosinophils, neutrophils and lymphocytes infiltration into the nasal mucosa and the lungs of animals treated with anti-il-β and anti-tnf-α igy, alone or jointly [ ] . one of the side effects that occur in individuals receiving antivenom serum produced in goats, sheep and horses is due to the presence of serum proteins on the anti-venom serum derived from these animals, in which igg is not sufficiently purified [ , ] . one advantage of using igy in anti-venom serotherapy is that it is easily purified, which would minimize the occurrence of side effects due to nonspecific proteins. araujo et al. demonstrated this property when specific igy was produced as anti-venom of the snake genus bothrops sp. these antibodies neutralized a pool of venoms from five bothrops species, with an ed of μl/ ld , showing little to no side effects in mice [ ] . mendonza et al. also produced igy capable of neutralizing the venom of the peruvian snake bothrops atox. the anti-venom igy showed considerable cross reaction with the venom of bothrops brazili and could be used not only as b. atox anti-venom, but also as a tool for the research of cross reaction with venoms from different species [ ] . in another elegant work, andrade et al. produced igy against a pool of venoms from snakes of the genus bothrops and against the venom from the species crotalus durissus terrificus. anti-venom igy extracted from eggs was compared to the horse anti-venom igg in western blot. the results showed that specific egg's igy recognized the same antigens as the equine anti-venom [ ] . igy against coral snake venom was first produced in response to a pool of venoms from different species of micrurus. these antibodies recognized, by western blot, venom proteins from several snakes: m. isozonus, m. surinamensis, m. f. fulvius, naja kaouthia, n. pallida, bothrops colombiensis, crotalus durissus cumanensis and c. vegrandis and could, therefore, be used as a broad-spectrum snake anti-venom [ ] . zolfagharian and dounighi produced igy by inoculating the vipera lebetina snake venom, inactivated by γ radiation, in hens [ ] . these antibodies were effective in neutralizing the crude venom of vipera lebetina in mice. anti-venom igy were also obtained from eggs of hens immunized with the venom of the snake trimeresurus albolabris. these igy recognized, by western blot, most of the proteins present in the t. albolabris venom and neutralized it in mice in a dose-dependent manner [ ] . more recently, liu et al. extracted and purified igy from eggs of hens inoculated with the venom of the deinagkistrodon actus snake. these antibodies were able to neutralize the lethal effects of the venom, such as bleeding, edema formation and myotoxicity in a dose-dependent manner [ ] . da rocha et al. produced igy against ophidian toxins of crotalus durissus terrificus, bothrops jararaca and bitis arietans. the antibodies were able to bind to specific components of the venoms in western blot and protected % of the intoxicated mice when obtained after the ninth inoculation. the authors recommended the use of a small antigen dose ( μl) applied in successive inoculations for igy production, since this dose was enough to genetically alter the v(d)j segments on the naïve cells and to generate immunological memory [ ] . however, igy raised against the venom of the snake oxyuranus scutellatus was less effective than equine igg, being unable to neutralize the neurotoxic and coagulant effects of the venom [ ] . nonetheless, this result cannot be extended to igy produced against other venoms. igy against the tityus caripitensis scorpion venom, produced by alvarez et al., neutralized not only the venom of t. caripitensis, but also that of other tityus species (t. quirogae, t. discrepans and t. gonzalespongai), and inactivated the hyaluronidase, an enzyme that facilitates the toxin spread in the tissues, present in the t. serrulatus venom [ ] . thus, igy raised against t. caripitensis venom could be used as a broadspectrum anti-scorpionic serum. another application of igy technology, the prophylaxis of celiac disease, was demonstrated by gujral et al., who developed powdered egg yolk formula with protective sugars containing anti-gliadin igy, among which, the formula with mannitol (eyp-m) retained its activity after being submitted, in vitro, to chemical conditions analogous to those of the stomach and small intestine. the formula igy-epy-m neutralized in vitro both the isolated gliadin and that present in food matrix and inhibited its intestinal absorption in mice, showing promising for the prevention of celiac disease [ ] . bobeck et al. used igy against the human intestinal alkaline phosphatase (hiap) to assess the influence of iap on increased bioavailability of phytate phosphate in the presence of α-dihydroxycholecalciferol (vitamin d ) in chickens. anti-hiap igy was ingested by chickens and reduced the absorption of phytate phosphate, which suggests that although it performed less adequately than sevelamer chorhydrate, already used for the same purpose, anti-hiap igy can be optimized for the prevention of phytate phosphate toxicity induced by the consumption of the active form of vitamin d [ ] . the ability of igy to detect viral pathogens of the gastrointestinal tract in humans and animals has been widely studied. igy raised against canine parvovirus viral like particles (cpv-vlps) were used in elisa and immunochromatography, showing sensitivity and specificity in the detection of canine parvovirus in dog fecal samples [ ] . specific igy against the e protein of bovine viral diarrhea virus (bvdv) were used in elisa to detect pathogens that cause diarrhea in cattle. anti-e igy showed a high specificity, recognizing only bvdv. elisa and immunochromatography tests using these antibodies were efficient in detecting bvdv in serum samples of cows with diarrhea, showing a concordance of , % and % with rt-pcr, respectively [ ] . da silva et al. used igy against hepatitis a virus (hav) in a competitive immunoenzymatic assay to detect anti-hav igg in serum samples, showing satisfactory sensitivity and specificity [ ] . more recently, igy against hav was used to detect the virus in hepatic sections of infected rhesus monkeys by means of indirect immunofluorescence (iif). anti-hva igy was more efficient than the commercially available igg for the detection of the same antigen [ ] . igy was also used to detect viral pathogens in aquatic animals. specific igy against the soft-shelled turtle systemic septicemia spherical virus (stsssv) was used to compose a lateral flow assay to detect the virus in turtles. this assay was sensitive and specific, detecting stsssv in all infected turtles in serum and feces samples [ ] . a potential for diagnosis was presented by igy raised against the nucleocapsid protein (np) of coronavirus (cov). anti-np igy, used as capture antibody in elisa for detecting np, lowered its detection limit to a picogram level, which indicates that the antibody is promising for use in the diagnosis of acute respiratory syndrome associated to coronavirus (sars-cov) and is sensitive enough to detect small amounts of np [ ] . moreover, the ability of igy in diagnosing dengue fever was also evaluated. figueiredo et al. produced igy against the non-structural protein (ns ) of dengue virus (denv ). these antibodies were used to compose an immunosensor that was effective in electrically detecting the ns protein of denv in standard samples and could be used for dengue diagnosis in biological samples [ ] . one of the most studied diagnostic potential of igy is against staphylococcus aureus. the fact that the fc portion of igg reacts with the staphylococcal protein a makes igy a relevant resource for more specific detection of different s. aureus strains and their toxins, since, due to structural differences, the fc portion of igy does not react with protein a [ ] . following this rationale, walczak et al. produced igy against the fibrinogen binding protein (efb) of staphylococcus aureus and against a peptide epitope that encompasses the residues - of efb protein. anti-efb and anti-efb - antibodies presented high titers in elisa and strong avidity in western blot, showing promising use in the diagnosis of s. aureus infection [ ] . another elisa test using igy against the staphylococcal enterotoxin b (seb) of s. aureus as capture antibody and specific ssdna aptamers coupled to biotin as revealing was developed by mulidi et al. this assay was efficient in detecting seb, but also reacted with others staphylococcal toxins, such as staphylococcal endotoxins a (sea) and c (sec), toxic shock syndrome toxin (tsst) and α-hemolysin [ ] . igy raised against α-hemolysin was applied as capture antibody in elisa for detecting the toxin in the supernatant of s. aureus cultures. anti-αha igy showed high specificity against the toxin, without reacting with protein a, which is present in all s. aureus strains [ ] . the ability of igy to diagnose resistant s. aureus was also investigated. yamada et al. produced igy against the penicillin binding protein (pbp) a, which is present in methicillin resistant s. aureus (mrsa) strains. anti-pbp a igy was used in elisa, lateral flow and latex to detect mrsa and other s. aureus strains sensitive to methicillin and beta-lactams. the antibody was mrsa specific and did not react with the sensitive strains that express significant amounts of protein a [ ] . among the parasitic infections tested, cakir-koc evaluated the potential of igy in detecting the protozoan toxoplasma gondii by producing igy against its surface protein sag- , which reacted with the target antigen in elisa and western blot [ ] . therefore, this study revealed a potential diagnostic test for toxoplasmosis. more recently, anti-sag igy conjugated to fluorescein isothiocyanate detected t. gondii tachyzoites in a colony, by means of immunofluorescence, and may be used to detect the protozoan in other types of samples [ ] . in other interesting research, an indirect elisa using igy to detect cathepsin f (cf), a cysteine protease present in the helminth opisthorchirs viverrini, was developed. this assay showed good sensitivity in the detection of o. viverrini in fecal samples from humans living in endemics places; however, a cross reaction with taenia spp., echinostoma spp. and minute intestinal fluke (mif) was observed [ ] . therefore, improvements still have to be made before this technology is available. miura et al. produced igy against the gp protein, from the cryptosporidium hominis protozoan. these igy bound to gp in western blot and to c. parvum sporozoites in fecal samples by indirect immunofluorescence, suggesting that anti-gp igy could be used in the diagnosis of cryptosporidiosis caused by both c. hominis and c. parvum [ ] . several authors have been investigating the potential of igy in the detection of tumor markers. igy against the peptide antigen ca - , a commonly used breast cancer marker, was used as secondary antibody in a sandwich elisa aiming to detect ca - , showing potential for clinical use [ ] . sun et al. produced igy against two portions of the transmembrane glycoprotein her : her -a, proximal region, and her -b, distal region. anti-her -a and anti-her -b igy effectively detected the her glycoprotein in cultured breast cancer sk-br- cells by immunofluorescence and in sections of breast tumors over expressing her -b, using immunohistochemistry. in addition, the antibodies bound to the glycoprotein in elisa and western blot, which indicates that igy against her is promising for use in breast cancer diagnosis [ ] . in another study, Łupicka-słowik et al. developed a direct elisa test using a lysate of human malignant tumor cells and igy against bovine adenosine deaminase (c-ada). the assay was efficient in detecting human adenosine deaminase (h-ada) present in the tumor cells lysate, which was possible due to the high homology between c-ada and h-ada. the elisa test using anti-c-ada igy could be used to diagnose several types of malignant tumors in humans, as well as be an alternative to currently employed enzymatic methods for the detection and quantification of ada for pleural tuberculosis diagnosis [ ] . another igy developed to detect prostatic specific antigen (psa) and two peptide fragments of this protein demonstrated specificity and marked the antigen more strongly than the igg counterpart using western blot analysis. however, anti-psa igy had an unsatisfactory sensitivity when applied as secondary antibody in indirect elisa [ ] , thus deserving more investigation. in general, these findings suggest that the phylogenetic distance between birds and mammals, that ensures a stronger immune response against mammalian antigens by birds than by other mammals [ ] , makes the production of igy against tumor antigens advantageous not only for therapeutic purposes, as described above, but also for usage in several types of immunoassays for tumor detection in humans. hens immunized with umbilical cord sera produced specific igy against igg and the complement fractions c b and c d. these antibodies did not react with the c b fraction -which configures a higher specificity, since anti-c b antibodies often cross-reacts with the antigens of chido/rodgers rbc group -nor with erythrocyte antigens from abo group. these antibodies are, therefore, promising as a reagent for coombs test [ ] . igy immunoassays were used to detect the hepatic expression of cytochrome p e (cyp e ) in mice treated with medicinal herbs and products derived from plants rich in flavonoids. cyp e metabolizes a wide variety of chemicals with different structures, in particular small and hydrophobic compounds, including potential cytotoxic and carcinogenic agents. anti-cyp e igy was specific, without reacting with other cytochromes, and was able to detect the reduction of hepatic cype e expression due to the ingestion of natural products with hepatoprotective effects [ ] . the ability of igy in identifying harmful substances in consumer products has been evaluated. an elisa test was developed to detect the staphylococcal enterotoxin g (seg), using specific igy, and showed satisfactory sensitivity and specificity, reducing the interference of protein a that occurs in igg tests. this test was successfully used to detect seg in milk and dairy products samples and could therefore be used to identify the toxin in food [ ] . bittner et al. used igy in elisa to detect potentially allergenic proteins in commercially available latex gloves. this assay presented similar results to that of the gold standard test, which uses mammalian igg [ ] . igy can also be used in assays to identify antibiotic residues in food products of animal origin, as demonstrated in a study by he et al., in which produced anti-gentamycin igy specifically detected the target antibiotic present in animal origin products [ ] . following this rationale, li et al. used specific igy to detect kanamycin and gentamycin residues in milk and meat samples by means of competition elisa and fpia (fluorescence polarization immunoassay) [ ] . the potential of igy in identifying substances has also been used to evaluate the toxicity of a natural product employed in the alternative medicine. igy labeled with quantum dot were successfully used in a lateral flow assay for the detection of rhein; a toxic substance found in the plant rheum officinale, widely used in chinese traditional medicine; in plant samples and serum from users [ ] . igy raised against the bacterium listeria monocytogenes showed a significant inhibitory effect of bacterial growth in liquid medium and in fish samples stored between and °c in a dose-dependent manner, which indicates that anti-l. monocytogenes igy is a potential antimicrobial for use in the food industry [ ] . taking into consideration the versatility and the range of igy already tested against several bacteria, this result could be easily apply to other food poisoning bacteria and viruses. among the importance of this technology, leclair et al. demonstrated that igy produced against the staphylococcal enterotoxin b (seb), a potential biological weapon, can save individuals exposed to this material. the results with rhesus monkeys showed that animals that received anti-seb igy min before or h after exposure to a lethal seb aerosol survived [ ] , which indicates that anti-seb igy could be used to protect populations in a hypothetical context of bioterrorism involving seb [ ] . the latest findings using igy have clearly demonstrated the versatility of this technology. obtaining igy from birds presents several technical and economical advantages over mammalian igg, and as described in this review, igy technology has a broad spectrum of applications in human and veterinary health. it can be used in multiple types of therapies; it is useful in the prevention of various types of diseases and detects, by means of different techniques, several classes of antigens, such as microorganisms, tumor markers and substances. among the advantages of this technology, the replacement of invasive antibody collection by its extraction from eggs is one of the most interesting, considering the animal welfare benefits, with this technology it is possible to achieve great quantities of antibodies with a lower cost of production and less damage to animal welfare. due to its structural differences and phylogenetic distance, igy is more specific for diagnostic use and displays greater avidity for mammalian conserved proteins than igg, being, thus, an important alternative in the search for more effective diagnostics and therapies. in addition, in view of its proven ability to neutralize microorganisms, igy represents an important therapeutic resource in times of increasing resistance to antibiotics and emergence of viral diseases for which there is no treatment. there are no funding or competing interests to report. chicken egg yolk antibodies (igy) as an alternative to mammalian antibodies production of antibodies in chickens the production of avian (egg yolk) antibodies: igy. the report and recommendations of ecvam workshop igy technology: extraction of chicken antibodies from egg yolk by polyethylene glycol (peg) precipitation avian antibodies (igy) against trypanosoma cruzi: purification and characterization studies antibodies to proteins from yolk of immunized hens chicken egg yolk antibodies (igy) for detecting circulating antigens of schistosoma japonicum oral passive igybased immunotherapeutics: a novel solution for prevention and treatment of alimentary tract diseases eficacia experimental de anticuerpos igy producidos en huevos, contra el veneno de la serpiente peruana bothrops atrox protection against bacterial superantigen staphylococcal enterotoxin b by passive vaccination suppressive effect of functional drinking yogurt containing specific egg yolk immunoglobulin on helicobacter pylori in humans ueber natürliche immunität und ihre verwerthung für die immunisirungstherapie igy-technology: application and trends, epc - th european poultry conference igy: clues to the origins of modern antibodies phylogeny of immunoglobulin structure and function chicken immunoglobulin gamma-heavy chains: limited vh gene repertoire, combinatorial diversification by d gene segments and evolution of the heavy chain lócus avian igy antibodies and their recombinant equivalents in research, diagnostics and therapy chicken antibodies: a tool to avoid interference by complement activation in elisa use of chicken antibodies in enzyme immunoassays to avoid interference by rheumatoid factors protein g: a powerful tool for binding and detection of monoclonal and polyclonal antibodies chicken antibodies: a clinical chemistry perspective, ups extraction of a monospecific coombs-reagent from chicken eggs efficient production of chicken egg yolk antibodies against a conserved mammalian protein rearrangement of immunoglobulin genes in chicken b cell development avian b-cell development: generation of an immunoglobulin repertoire by gene conversion acid stability of anti-helicobacter pyroli igy in aqueous polyol solution oral passive immunization effect of anti-human rotavirus igy and its behavior against proteolytic enzymes production and purification of fab′ fragments from chicken egg yolk immunoglobulin y (igy) chitosan-alginate microcapsules for oral delivery of egg yolk immunoglobulin (igy) passive immune-protection of litopenaeus vannamei against vibrio harveyi e vibrio parahaemolyticus infections with anti-vibrio egg yolk (igy)-encapsulated feed microencapsulation protects immunoglobulin in yolk (igy) specific against helicobacter pylori urease microencapsulation for the gastric passage and controlled intestinal release of immunoglobulin y encapsulation of chicken egg yolk immunoglobulin g (igy) by liposomes protective effect of microencapsulation consisting of multiple emulsification and heat gelation processes on immunoglobulin in yolk ph-responsive hydrogels to protect igy from gastric conditions: in vitro evaluation in vitro toxicity evaluation of hydrogel-carbon nanotubes composites on intestinal cells egg yolk igy: protection against rotavirus induced diarrhea and modulatory effect on the systemic and mucosal antibody responses in newborn calves egg yolk igy antibodies: a therapeutic intervention against group a rotavirus in calves chicken egg yolk antibodies (igytechnology): a review of progress in production and use in research and human and veterinary medicine hen egg yolk antibodies (igy), production and use for passive immunization against bacterial enteric infections in chicken: a review antibody production in rabbits and chickens immunized with human igg. a comparison of titre and avidity development in rabbit serum, chicken serum and egg yolk using three different adjuvants freund's complete adjuvant in the chicken: efficient immunostimulation with severe local inflammatory reaction stable biocompatible adjuvants-a new type of adjuvant based on solid lipid nanoparticles: a study on cytotoxicity, compatibility and efficacy in chicken evaluation of igy capture elisa for sensitive detection of alphahemolysin of staphylococcus aureus without staphylococcal protein a interference generation and application of polyclonal igy antibodies specific for full-length and nicked prostate-specific antigen production and characterization of anti-campylobacter jejuni igy derived from egg yolks antibacterial activity of egg yolk antibody (igy) against listeria monocytogenes and preliminary evaluation of its potential for food preservation preparation and characterization of egg yolk immunoglobulin y specific to influenza b vírus highly sensitive detection of cancer antigen - using novel avian igy antibodies development of chicken egg yolk antibodies against streptococcus mitis -purification and neutralizing efficacy lmmunoglobulins from egg yolk: isolation and purification brazilian igy-bothrops antivenom: studies on the development of a process in chicken egg yolk isolation of immunoglobulin in yolk (igy) and rabbit serum immunoglobulin g (igg) specific against bovine lactoferrin by immunoaffinity chromatograph hen egg yolk antibodies purified by antigen affinity under highly alkaline conditions provide new tools for diagnostics. human intact parathyrin as a model antigen production and characterization of igy against canine igg: prospect of a new tool for the immunodiagnostic of canine diseases thiophilic adsorption chromatography dengue virus specific igy provides protection following lethal dengue virus challenge and is neutralizing in the absence of inducing antibody dependent enhancement development of anti-helicobacter pylori immunoglobulins y (igys) in quail production and characterization of egg yolk antibody (igy) against recombinant vp -s antigen chicken egg yolk antibodies (igy) modulate the intestinal mucosal immune response in a mouse model of salmonella typhimurium infection characterization of chicken igy specific to clostridium difficile r spores and the effect of oral administration in mouse models of initiation and recurrent disease treatment of helicobacter pylori infection in mice with oral administration of egg yolk-driven anti-urec immunoglobulin preventive effect of anti-vaca egg yolk immunoglobulin (igy) on helicobacter pylori-infected mice inhibitory effects of rhp-nap igy against h. pylori attachment to ags cell line in vitro evaluation of cross-strain inhibitory effects of igy polyclonal antibody against h. pylori, microbial pathog koerniasari, the activity of immunoglobulin y anti-mycobacterium tuberculosis on proliferation and cytokine expression of rat peripheral blood mononuclear cells effects of specific egg yolk immunoglobulin on pan-drug-resistant acinetobacter baumannii protective effect of an egg yolk-derived immunoglobulin (igy) against prevotella intermedia-mediated gingivitis effectiveness of egg yolk immunoglobulin (igy) against periodontal diseasecausing fusobacterium nucleatum in vitro evaluation of the efficacy of chicken egg yolk antibodies (igy) generated against propionibacterium acnes effects of soybean milk, chitosan, and anti-streptococcus mutans igy in malnourished rats' dental biofilm and the igy persistencyin saliva anticell-associated glucosyltransferase immunoglobulin y suppression of salivary mutans streptococci in healthy young adults anti-pseudomonas aeruginosa igy antibodies promote bacterial opsonization and augment the phagocytic activity of polymorphonuclear neutrophils igy against enterotoxigenic escherichia coli administered by hidrogel-carbon nanotubes composites to prevent neonatal diarrhoea in experimentally challenged piglets ulcer disease prophylaxis in koi carp by bath immersion with chicken egg yolk containing anti-aeromonas salmonicida igy medical consequences of antibiotic use in agriculture industrial food animal production, antimicrobial resistance, and human health immunoprophylactic effect of chicken egg yolk antibody (igy) against a recombinant s domain of the porcine epidemic diarrhea vírus spike protein in piglets prophylactic and therapeutic efficacy of avian antibodies against influenza virus h n and h n in mice cross-protection of chicken immunoglobulin y antibodies against h n and h n viruses passively administered in mice antiviral biologic produced in dna vaccine/goose platform protects hamsters against hantavirus pulmonary syndrome when administered post-exposure practical aspects in the use of passive immunization as an alternative to attenuated viral vacines effects of oral moisturising gel containing egg yolk antibodies against candida albicans in older people use of candida-specific chicken egg yolk antibodies to inhibit the adhering of candida to denture base materials: prevention of denture stomatitis production, purification and therapeutic potential of egg yolk antibodies for treating trypanosoma evansi infection apoptotic killing of breast cancer cells by igys produced against a small aminoacid epitope of the human trail- receptor, asian pac anti-her igy antibody-functionalized single-walled carbon nanotubes for detection and selective destruction of breast cancer cells antiobesity activity of hen egg anti-lipase immunoglobulin yolk, a novel pancreatic lipase inhibitor anti-interleukin- beta/tumor necrosis factor-alpha igy antibodies reduce pathological allergic responses in guinea pigs with allergic rhinitis a comparison of ovine and equine antivenoms the production and characterization of antibothropic and anti-crotalic igy antibodies in laying hens: a long term experiment coral snake antivenom produced in chickens (gallus domesticus) study on development of vipera lebetina snake anti-venom in chicken egg yolk for passive immunization anti-trimeresurus albolabris venom igy antibodies: preparation, purification and neutralization efficacy preparation and neutralization efficacy of igy antibodies raised against deinagkistrodon acutus venom development of igy antibodies against anti-snake toxins endowed with highly lethal neutralizing activity development of a chicken-derived antivenom against the taipan snake (oxyuranus scutellatus) venom and comparison with an equine antivenom antibodies anti-tityus caripitensis venom: purification and neutralization efficacy in-vitro and in-vivo binding activity of chicken egg yolk immunoglobulin y (igy) against gliadin in food matrix oral antibodies to human intestinal alkaline phosphatase reduce dietary phytate phosphate bioavailability in the presence of dietary α-hydroxycholecalciferol evaluation of chicken igy generated against canine parvovirus viral-like particles and development of enzyme-linked immunosorbent assay and immunochromatographic assay for canine parvovirus detection preparation of chicken igy against recombinant e protein of bovine viral diarrhea virus (bvdv) and development of elisa and ica for bvdv detection an immunoenzymatic assay for the diagnosis of hepatitis a utilising immunoglobulin y using immunoglobulin y as an alternative antibody for the detection of hepatitis a virus in frozen liver sections development of a colloidal gold immunochromatographic strip for the rapid detection of soft-shelled turtle systemic septicemia spherical vírus diagnostics of severe acute respiratory syndrome-associated coronavirus (sars-cov) nucleocapsid antigen using chicken immunoglobulin y electrical detection of dengue biomarker using egg yolk immunoglobulin as the biological recognition element the binding of staphylococcal protein a by the sera of different animal species method for generation of peptide-specific igy antibodies directed to staphylococcus aureus extracellular fibrinogen binding protein epitope a novel igy-aptamer hybrid system for cost-effective detection of seb and its evaluation on food and clinical samples detection of methicillin-resistant staphylococcus aureus using a specific anti-pbp a chicken igy antibody production of anti-sag- igy antibody against toxoplasma gondii parasites and evaluation of antibody activity by elisa method novel fitc-labeled igy antibody: fluorescence imaging toxoplasma gondii in vitro chicken igy-based coproantigen capture elisa for diagnosis of human opisthorchiasis evaluation of recombinant cryptosporidium hominis gp protein and anti-gp chicken polyclonal igy for research and diagnostic purposes highly sensitive detection of cancer antigen human epidermal growth factor receptor using novel chicken egg yolk immunoglobulin development of adenosine deaminase-specific igy antibodies: diagnostic and inhibitory application evaluation of igy antibody as a polyspecific coombs-reagent development of an igy antibody-based immunoassay for the screening of the cyp e inhibitor/enhancer from herbal medicines development of igy based sandwich elisa for the detection of staphylococcal enterotoxin g (seg), an egc toxin content of asthmagen natural rubber latex allergens in commercial disposable gloves development of indirect competitive elisa using egg yolk-derived immunoglobulin (igy) for the detection of gentamicin residues detection of kanamycin and gentamicin residues in animal-derived food using igy antibody based ic-elisa and fpia quantum dot-based lateral-flow immunoassay for rapid detection of rhein using specific egg yolk antibodies key: cord- -gcfx authors: ianevski, aleksandr; zusinaite, eva; kuivanen, suvi; strand, mårten; lysvand, hilde; teppor, mona; kakkola, laura; paavilainen, henrik; laajala, mira; kallio-kokko, hannimari; valkonen, miia; kantele, anu; telling, kaidi; lutsar, irja; letjuka, pille; metelitsa, natalja; oksenych, valentyn; bjørås, magnar; nordbø, svein arne; dumpis, uga; vitkauskiene, astra; Öhrmalm, christina; bondeson, kåre; bergqvist, anders; aittokallio, tero; cox, rebecca j.; evander, magnus; hukkanen, veijo; marjomaki, varpu; julkunen, ilkka; vapalahti, olli; tenson, tanel; merits, andres; kainov, denis title: novel activities of safe-in-human broad-spectrum antiviral agents date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: gcfx according to the who, there is an urgent need for better control of viral diseases. re-positioning existing safe-in-human antiviral agents from one viral disease to another could play a pivotal role in this process. here, we reviewed all approved, investigational and experimental antiviral agents, which are safe in man, and identified compounds that target at least three viral diseases. we tested of these compounds against eight different rna and dna viruses. we found novel activities for dalbavancin against echovirus , ezetimibe against human immunodeficiency virus and zika virus, as well as azacitidine, cyclosporine, minocycline, oritavancin and ritonavir against rift valley fever virus. thus, the spectrum of antiviral activities of existing antiviral agents could be expanded towards other viral diseases. dozens of viruses, such as fluav, hsv- , vzv, cmv and nov, constantly infect human population and represent substantial public health and economic burden (dalys and collaborators, ; disease et al., ) . emerging and re-emerging viruses, such as ebov, marv, lasv, chikv, zikv, denv, rvfv, mers-and sars-cov, surface from natural reservoirs approximately one each year and also represent global threats (howard and fletcher, ; who, ) . according to who, there is an urgent need for better control of these viruses, including drug-resistant and vaccine immunity escaping viral strains (bekerman and einav, ; de clercq and li, ) . antiviral drugs and vaccines are the most powerful tools to combat viral diseases. most drugs and vaccines, however, selectively target a single virus, thereby providing a "one drug-one bug" solution. by contrast, broad-spectrum antivirals (bsas) can cover multiple viruses and genotypes and reduce the likelihood of development of resistance. therefore, some bsas can be used for rapid management of new or drug-resistant viral strains, for treatment of viral co-infections reducing therapy complexity, as well as a first-line treatment or the prophylaxis of acute virus infections. thus, to overcome time and cost issues associated with the development of virus-specific drugs and vaccines, the development of bsas should be prioritized (bekerman and einav, ) . nucleotide and nucleoside analogues are excellent examples of bsas. they inhibit transcription and/or replication of different rna and dna viruses (de clercq, ) . in particular, valaciclovir inhibits replication of different herpesviruses and hbv (laube et al., ; vere hodge and field, ) . cidofovir and its lipid conjugate brincidofovir also inhibit replication of dsdna viruses, such as herpesviruses, adv, bkv, and hpv (andrei et al., ) . ribavirin blocks viral rna synthesis of fluav, hcv and rsv (hong and cameron, ) . favipiravir and bcx also inhibit replication of different rna viruses (mckimm-breschkin et al., ) . however, viruses are able to develop resistance to some of these nucleotide and nucleoside analogues. other examples of bsa agents include inhibitors of cellular pathways, which are exploited by different viruses for efficient viral replication (debing et al., ) . these agents overcome the problem of antiviral drug resistance. for example, lipid-lowering statins (atorvastatin, lovastatin, simvastatin, and fluvastatin) inhibit cellular hmg-coa reductase and attenuate replication of some enveloped viruses (bernal et al., ; enserink, ) . anti-malaria quinolones (chloroquine and hydroxychloroquine) inhibit acidification of endosomes, which is an essential process for uncoating of ssrna viruses (al-bari, ). anticancer kinase inhibitors dasatinib, imatinib, gefitinib, nilotinib, erlotinib and sunitinib impair intracellular viral trafficking and exert bsa effects (bekerman et al., ; schor and einav, ) . the anti-duchenne muscular dystrophy agent, alisporivir, targets cellular cyclophilin and inhibits the folding of hcv, hiv, mers-and sars-cov proteins, and, therefore, prevents formation of infectious virus particles (boldescu et al., ; de wilde et al., ; soriano et al., ) . thus, both host-directed antivirals and nucleotide/nucleoside analogues could possess bsa activity. here, we hypothesised that some of the identified safe-in-human bsas could possess novel antiviral activities and, therefore, could be used for treatment of many different viral infections. to prove this hypothesis, we reviewed safe-in-man approved, investigational and experimental antiviral agents. we identified compounds that target at least three viral diseases. we tested of the compounds against different viruses and found novel activities for of these agents. we conclude that the spectrum of antiviral activities for existing bsa agents could be expanded towards other viral diseases. information on the viruses and associated human diseases is summarized in table s . information on approved, investigational and experimental safe-in-human antivirals is summarized in tables s -s . this information was extracted from drugbank ( ), clinical trials websites ( ) and pubmed. information on approved, investigational, and experimental antivirals, which target ≥ viral diseases, is summarized in table s . eye diagrams and interaction network plots were created with javascript library d .js v ( ). a structural similarity plot for the drugs was constructed and visualized using a c-spade web application (ravikumar et al., ) . the compounds used in this study, their suppliers and catalogue numbers are summarized in table s . to obtain mm stock solutions compounds were dissolved in % dimethyl sulfoxide (dmso, sigma-aldrich) or milli-q water. the solutions were stored at − °c until use. bhk- cells (baby hamster kidney fibroblasts) were grown in glasgow's minimal essential medium (gmem) containing . % fetal bovine serum (fbs; gibco, paisley, uk), % tryptose phosphate broth (tpb), mm hepes, u/ml penicillin and . mg/ml streptomycin (penstrep, lonza basel, switzerland). ach- cells, a model for chronic hiv- infection, which possesses a single integrated copy of the provirus hiv- strain lai (nih aids reagent program), were grown in rpmi- medium supplemented with % fbs and penstrep. madin-darby canine kidney epithelial (mdck) cells, human embryonic kidney cells (hek t) and african green monkey kidney epithelial cells (vero) were grown in dulbecco modified eagle's medium (dmem; sigma-aldrich, st. louis, mo, usa) supplemented with mm l-glutamine (lonza; basel, switzerland), u/ml penstrep and % fbs. human telomerase reverse transcriptase-immortalized retinal pigment (rpe) cells were grown in dmem-f medium supplemented with u/ml penstrep, mm l-glutamine, % fbs, and . % sodium bicarbonate (sigma-aldrich). tzm-bl cells were grown in dmem supplemented with % heat-inactivated fbs and penstrep. human lung adenocarcinoma epithelial cells, a , were cultured in dmem containing . g/l nahco , mm hepes (euroclone, milan, italy), penstrep, and % fetal bovine serum (fbs, gibco) at °c. all cell lines were grown in humidified incubator at °c in the presence of % co . human influenza a/wsn/ (h n ) virus was generated using eight-plasmid reverse genetics system in hek and vero-e cells, as described previously (hoffmann et al., ) . ev strain was propagated in a monolayer of vero cells, as described earlier (myllynen et al., ) . hsv- was amplified in vero cells, as described previously (nygardas et al., ) . zikv fb-gwuh- strain was cultured in vero e cells, as described earlier (driggers et al., ) . for production of hiv- , × ach- cells were seeded in ml of full culture media, and hiv- production was induced by the addition of nm phorbol -myristate -acetate (viira et al., ) . the induced cells were incubated for h, and subsequently, the hiv- containing media was collected and filtered. the amount of hiv- in the stock was estimated by quantification of p protein in the media. quantity of p was measured using a reference recombinant purified p protein and anti-p -elisa, which was developed in-house. chikv- sg-nanoluc strain was generated from icdna clone of picres representing lr opy strain belonging to east/central/ south african genotype (utt et al., ) . rrv- sg-nanoluc strain derived from rrv-t strain (jupille et al., ) . sequence encoding nanoluc protein was placed between non-structural and structural regions of chikv or rrv genomes under the control of native subgenomic promoters of the viruses. to ensure expression of viral structural proteins, a copy of subgenomic promoter (residues − to + for chikv and residues − to + for rrv, positions given with respect to start site of subgenomic rna) was inserted immediately downstream of sequence encoding for nanoluc. recombinant rvfv expressing the far-red fluorescent protein katushka instead of the deleted nss protein (rrvfvΔnss::katushka) was used in this study (islam et al., ) . the virus stocks were stored at − °c. all the experiments with viruses were performed in compliance with the guidelines of the national authorities using appropriate biosafety laboratories under appropriate ethical and safety approvals. fluav virus was titrated in mdck cells using plaque assay as described previously (denisova et al., ) . ev titers were determined by plaque assay on a cells, as described earlier (marjomaki et al., ) . zikv titers were determined by plaque assay on vero-e cells, as described earlier (kuivanen et al., ) . hsv- titers were determined by plaque titration in vero cells in the presence of human immunoglobulin g ( μg/ml), as described earlier (paavilainen et al., ) . chikv- sg-nanoluc and rrv- sg-nanoluc were titrated in bhk- cells using plaque assay, as described previously (oo et al., ; taylor et al., ) . for testing the viability and death of compound-treated mock-, fluav-, ev -, chikv- sg-nanoluc-, rrv- sg-nanoluc-, zikv-and hsv -infected cells, approximately × rpe cells were seeded in each well of a -well plate. the cells were grown for h in cell growth medium. the media was replaced with virus growth medium (vgm) containing . % bsa, mm l-glutamine, . % nahco , and μg/ml l- -tosylamido- -phenylethyl chloromethyl ketone-trypsin (tpck)-trypsin (sigma-aldrich) in dmem-f . in the case of zikv, the media was replaced with dmem-f medium supplemented with u/ ml penstrep, mm l-glutamine, % fbs, and . % sodium bicarbonate. the compounds were added to the cells in three-fold dilutions at seven different concentrations starting from μm. no compounds were added to the control wells. the cells were mock-or virus-infected at a multiplicity of infection (moi) of one. when virus induced a cytopathic effect in cells (typically - hpi), celltox green express cytotoxicity reagent (ctxg, : dilution in the assay well, promega, madison, wi, usa) was added in vgm and the fluorescence was measured with a pherastar fs plate reader (bmg labtech, ortenberg, germany) or hidex sense microplate reader (hidex oy, turku, finland). the media was removed from the cells and stored at − °c. celltiter-glo viability reagent (ctg, promega, madison, wi, usa) containing firefly luciferase and luciferin was added ( μl per well). the luminescence was measured with a pherastar fs plate reader. for testing virus titers in compound-treated and non-treated fluav-, ev -, zikv-and hsv -infected cells the media was collected, serially diluted in pbs, and added to mdck (fluav), a (ev ), and vero-e (zikv and hsv- ) cells. the media was changed, and the cells were overlaid with plaque assay media. the cells were fixed, and viral titers were calculated. the titers were expressed as plaque-forming units per ml (pfu/ml). the presence of chikv- sg-nanoluc and rrv- sg-nanoluc in the media from non-or drug-treated rpe cells was evaluated by passaging the viruses in fresh rpe cells. the viruses expressed nano-luciferase from viral promoter, which was measured from lysed cells h post infection using renilla luciferase assay (promega, madison, wi, usa). for testing compound toxicity and efficacy against hiv- , approximately × tzm-bl cells were seeded in each well of a -well plate. tzm-bl cells express firefly luciferase under control of hiv- ltr promoter allowing quantitation of the viral infection (tat-protein expression by integrated hiv- provirus) using firefly luciferase assay. the cells were grown for h in cell growth medium. compounds were added to the cells in three-fold dilutions at seven different concentrations starting from μm. no compounds were added to the control wells. the cells were infected with hiv- (media that contained ng of hiv- p was used per well) or mock. at hpi, celltox green express cytotoxicity reagent (ctxg, : dilution in the assay well) was added and the fluorescence was measured with a pherastar fs plate reader. the media was removed from the cells, the cells were lysed, and firefly luciferase activity was measured using the luciferase assay system (promega, madison, wi, usa) and pherastar fs plate reader. to determine the efficacy of compounds against rvfv, the fluorescent intensity of individual infectious cell foci was quantified. briefly, a cells ( × /well) were grown in -well black plates with transparent bottoms (greiner bio-one international). compounds were serially diluted in dmso in two-fold steps from μm to . μm and mixed with pfu of rrvfvΔnss::katushka virus in a total volume of μl medium (moi, . ). the final concentration of dmso in the assay was %. the growth medium was removed from the wells and μl of compound and virus mixture was added to the cells for each compound concentration. at hpi the medium was removed, and cells were fixed with % paraformaldehyde (pfa) for h and washed with phosphate-buffered saline (pbs). μl pbs was added to each well and the plate was analyzed in the trophos plate runner hd (trophos, roche group) to count the number of virus infected cells per well, by identifying all individual cells expressing the far-red fluorescent protein katushka (islam et al., ) . the toxicity of compounds was analysed using ctg assay. the half-maximal cytotoxic concentration (cc ) and the halfmaximal effective concentration (ec ) for each compound were calculated after non-linear regression analysis with a variable slope using graphpad prism software version . a (graphpad software la jolla, ca, usa) or using hill curve fit with sigmastat . software (spss inc., chicago, il, usa). the relative effectiveness of drugs were quantified as the selectivity indexes (si = cc /ec ). there is limited information available on approved, investigational and experimental bsas. to identify all potential bsas, we reviewed approved, investigational and experimental safe-in-human antiviral agents in drug bank, clinical trials websites and pubmed, respectively. first, we searched for drugs, which have been approved for treatment of viral diseases in human. by excluding vaccines and discontinued drugs, we identified active antiviral substances. these compounds target viral, human and unknown factors and inhibit viruses (table s ). the approved antivirals include neutralizing antibodies, interferons, antisense oligonucleotide, and small molecules. only of the molecules target viruses or host factors associated with ≥ viral diseases. fig. shows bsas and other approved antiviral drugs linked to viral and host targets through viruses they inhibit. next, we reviewed all investigational antivirals and their safety profiles. by excluding vaccines from the search parameters, we found active compounds. these compounds target viral, human and unknown factors and inhibit viruses (table s ). the drug candidates include neutralizing antibodies, antisense oligonucleotides, interferons, enzyme and small molecules. only of the small molecules target ≥ viral diseases. fig. shows these and other investigational antiviral agents linked to cellular and host factors through targeted viruses. next, we searched for drugs, which have been approved for treatment of non-viral diseases, but for which antiviral activity has been reported. we found active compounds, which are all small-molecules. these compounds target viral, human and unknown factors, and inhibit human viruses (table s ) . thirty-nine of the antiviral agents target ≥ viral diseases. fig. shows these and other experimental antiviral agents connected to their host and viral targets through viruses they inhibit. altogether, we identified antiviral agents with available safety information in humans. these agents target host, viral and unknown factors and inhibit different viruses, belonging to viral families. fifty-nine agents have bsa activity (table s ). these agents target of the reported viruses (except andv, emcv and rabv) (fig. s ). structural analysis of bsa revealed that several drugs (such as, acyclovir, famciclovir and valacyclovir; imatinib and disatinib; minocyclin and doxycycline; ritonavir and lopinavir; hydroxychloroquine and chloroquine; esomeprazole and omeprazole) have similar scaffolds (fig. s ) . these drugs target a limited number of host and viral factors (fig. s ). in particular, statins (fluvastatin, lovastatin and simvastatin) target human hmg-coa; dalbavancin, oritavancin, teicoplanin and telavancin target human cyp a ; acyclovir, valacyclovir, famciclovir, azacitidine, brincidofovir, cidofovir, foscarnet, trifluridine, and vidarabine inhibit viral dna polymerases; bcx , favipiravir and ribavirin inhibit viral rna polymerases; and lamivudine and tenofovir disoproxil inhibit viral reverse transcriptases. we hypothesized that approved, investigational and experimental antiviral agents, which target ≥ viral diseases, could inhibit other viral infections. as a proof of concept, we tested of the identified bsas against fluav, ev , chikv, rrv and hsv- infections in rpe cells (alisporivir, cyt , sunitinib and thymalfasin were excluded; table s ). we also tested of the bsas against zikv infection in rpe cells (alisporivir, cyt , sunitinib, thymalfasin, dalbavancin, pentosan polysulfate and rapamycin were excluded). we evaluated viability and death of virus/mock-infected cells using ctg and ctxg assays, respectively. the ctg assay quantifies atp, an indicator of metabolically active living cells, whereas ctxg assay uses fluorescent cyanine dye that stains the dna of dead cells. after initial screening we found several hits, which kept infected cells alive or rescued infected cells from virus-mediated death. these hits are gemcitabine, gefitinib and vibarabine (fluav); gemcitabine, pirlindole dibucaine, fluoxetine and dalbavancin (ev ); gemcitabine, imatinib, ivermectin, lopinavir, lovastatin, ezetimibe, fluoxetine, bcx , chloroquine and hydroxychloroquine (zikv); chloroquine and mycophenolic acid (chikv); chloroquine, mycophenolic acid, dibucaine and itraconazole (rrv); as well as -azacitidine, gemcitabine, trifluridine and vidarabine (hsv- ). we repeated the ctg and ctxg assays with selected compounds and titrated fluav, ev , zikv and hsv- produced in drug-treated and non-treated cells. the presence of chikv and rrv viruses in the media collected from non-or drug-treated rpe cells was evaluated by infecting fresh rpe cells and measuring reporter protein expression from viral promoter (nanoluciferase activity). these experiments confirmed antiviral activity of gemcitabine against fluav (fig. s ) ; gemcitabine, pirlindole, dibucaine, fluoxetine, and dalbavancin against ev (fig. s ) ; gemcitabine, lopinavir, ezetimibe, bcx , chloroquine, and hydroxychloroquine against zikv (fig. s ) ; chloroquine and mycophenolic acid against chikv (fig. s ) ; mycophenolic acid against rrv (fig. s ) ; and gemcitabine against hsv- (fig. s ) . importantly, dalbavancin and ezetimibe demonstrated novel antiviral activities against ev and zikv, respectively. in particular, they rescued infected rpe cells from virus-mediated cell death, and lowered production of infectious virus particles without detectable cytotoxicity (table ) . we also examined toxicity and antiviral activity of of the bsa agents (excluding alisporivir, cyt , sunitinib and thymalfasin) against hiv- infection in tzm-bl cells. our primary screen identified ezetimibe, minocycline and rapamycin as anti-hiv- agents. validation experiment confirmed all three hits (fig. s , table ). interestingly, ezetimibe is a novel inhibitor, whereas minocycline and rapamycin are known anti-hiv- agents (heredia et al., ; singh et al., ) . in addition, we examined antiviral activity and toxicity of of the bsa agents (alisporivir, cyt , sunitinib, thymalfasin, dalbavancin, pentosan polysulfate and rapamycin were excluded) against rvfv expressing the far-red fluorescent protein katushka in a cells. our screen identified azacitidine, bortezamibe, cyclosporine, doxycycline, ezetimibe, fluoxetine, gefitinibe, minocycline, oritavancin, ritonavir and topotecan. azacitidine, cyclosporine, minocycline, oritavancin, ritonavirand and bortezamib remained after excluding compounds with si < ( fig. s ; table ). interestingly, azacitidine, cyclosporine, minocycline, oritavancin and ritonavirand are novel, whereas bortezamib is a known inhibitor of rvfv infection (keck et al., ) . thus, we tested several known bsa agents against (−)ssrna, (+) ssrna, ssrna-rt and dsdna viruses and identified novel activities for dalbavancin against ev , ezetimibe against zikv and hiv- , as well as azacitidine, cyclosporine, minocycline, oritavancin and ritonavir against rvfv. fig. shows known, validated and novel interactions between bsas and viruses. several approved and investigational agents, as well as safe in man chemical probes, were discovered for potential treatment of various viral diseases (bekerman and einav, ; de clercq, ; de clercq and li, ) . re-purposing such therapeutics from one viral disease to another could save resources and time needed for development of novel drugs. in this study, we tested approved, investigational and experimental bsa agents against fluav, rvfv, ev , zikv, chikv, rrv, hiv- and hsv- in vitro. we identified novel antiviral activities for dalbavancin (against ev ), ezetimibe (against hiv- and zikv), azacitidine, cyclosporine, minocycline, oritavancin and ritonavir (against rvfv) (fig. ) . dalbavancin is a lipoglycopeptide antibiotic, which is approved by the fda for the treatment of acute bacterial skin infections caused by staphylococcus aureus and streptococcus pyogenes. dalbavancin also inhibits mers-cov and sars-cov infections. it binds cathepsin l in the late endosomes/lysosomes and blocks the entry of ebov (zhou et al., ) . our study demonstrates that it also inhibits replication of ev , which also enters the cells via an endocytic route (krieger et al., ) . ezetimibe is an fda-approved medication that lowers plasma cholesterol by decreasing its absorption in the small intestine. ezetimibe also inhibits hbv and hdv infections by impairing viral entry mediated by pres -specific receptor hntcp (blanchet et al., ; lempp and urban, ; monrroy-bravo et al., ) . our study shows that ezetimibe also inhibited hiv- and zikv infections. however, the entry of these viruses into the cell is mediated by other receptors (hamel et al., ; wilen et al., ) . most probably, the anti-hiv- , ev , as well as hcv action of ezetimibe is associated with depletion of cholesterol which is required for entry of these viruses into the host cells (sainz et al., ) . importantly, this drug was successfully tested in combination with antiretroviral therapeutics to lower cholesterol levels in serum of hiv-infected patients (saeedi et al., ; wohl et al., ) . the question remains whether ezetimibe alone reduces hiv titers in these patients. azacitidine is a chemical analogue of cytidine, which is used in the treatment of myelodysplastic syndrome. it also inhibits fluav, adv, hiv- and hiv- replication by blocking viral rna or dna synthesis (beach et al., ; rawson et al., ) . cyclosporine is an immunosuppressive agent used for the treatment of rheumatoid arthritis, psoriasis, crohn's disease, nephrotic syndrome and keratoconjunctivitis sicca. in addition, cyclosporine is used to prevent graft rejection in organ transplant recipients. cyclosporine also inhibits hcv, fluav, wnv and zikv replication through blocking interaction of cellular cyclophilins with viral proteins and attenuating viral rna synthesis (barrows et al., ; firpi et al., ; qing et al., ) . minocycline is a broad-spectrum antibiotic and antiviral agent, which possesses activity against denv, hiv- and wnv (leela et al., ; quick et al., ; singh et al., ) . oritavancin is a semisynthetic glycopeptide antibiotic used for the treatment of gram-positive bacterial skin infections. it also inhibits ebov, mers-cov, and sars-cov infections (zhou et al., ) . ritonavir is an antiretroviral medication. it has also antiviral activity against mers-cov (chan et al., ) . we showed that azacitidine, cyclosporine, minocycline, oritavancin and ritonavir are active against rvfv. we also confirmed previously reported activities of chloroquine against chikv and zikv, mycophenolic acid against chikv and rrv, fluoxetine, pirlindole and dibucaine against ev , bcx , lopinavir and hydroxichloroquine against zikv, as well as gemcitabine against ev , fluav, zikv and hsv- (cao et al., ; delogu and de lamballerie, ; delvecchio et al., ; denisova et al., ; julander et al., ; kang et al., ; khan et al., ; kuivanen et al., ; lee et al., ; soderholm et al., ; ulferts et al., ; yuan et al., ) (fig. ) . the number of compounds with novel and confirmed antiviral properties may have been higher if we had used other cell lines, other viruses and viral strains, concentration ranges and purity of compounds, as well as endpoint measurements. also testing other compounds, which target < viral diseases, could reveal novel antiviral properties of these compounds and increase the number of potential bsas. for conducting this properly, a harmonized antiviral bioactivity data annotation, standardization, curation, and intra-resource integration is needed. excellent antiviral profiles from cell-line-based assays might not be reflected in vivo because systemic mechanisms may compensate the blocked target effect. therefore, the identification of bsa targets that are essential for viral replication but redundant for the cell is critical for reducing putative toxicities associated with blocking cellular pathways. thus, follow-up mechanistic studies and in vivo experiments are needed to validate our in vitro results. in conclusion, repositioning of safe-in-man agents from one viral disease to another could play a pivotal role in development of broadly acting antivirals. our study demonstrated the potential value of such approach with some examples, such as dalbavancin, ezetimibe, azacitidine, cyclosporine, minocycline, oritavancin and ritonavir, confirming a principle that "rich becomes richer". effective bsa treatment may shortly become available, pending the results of further pre-clinical studies and clinical trials. the most effective and tolerable compounds will expand the available therapeutics for the treatment of viral diseases. some of these compounds could be used as first-line therapeutics to combat emerging and re-emerging viral threats, having global impact by improving preparedness and the protection of the general population from viral epidemics and pandemics. the authors declare no financial and non-financial competing interests. half-maximal cytotoxic concentration (cc ), the half-maximal effective concentration (ec ) and minimal selectivity indexes (si) for selected broad-spectrum antivirals. ctxg -celltox green express cytotoxicity assay, ctg -celltiter-glo viability assay, otherplaque assay or reporter gene expression assay, n.a.not available. fig. . the interaction network between viruses and bsas, which are safe in man. drug-like shapes represent antiviral agents. blue spheres represent viruses. the diameter of spheres corresponds to the number of interactions between the viruses and the drugs. novel interactions between bsas and viruses are shown in red, validatedin blue, and known -in grey. 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vitro and in vivo glycopeptide antibiotics potently inhibit cathepsin l in the late endosome/lysosome and block the entry of ebola virus, middle east respiratory syndrome coronavirus (mers-cov), and severe acute respiratory syndrome coronavirus (sars-cov) this study was supported by the european regional development fund, the mobilitas pluss project mobtt (to denis e. kainov), jane and aatos erkko foundation grant # (to the vejo hukkanen). we thank qiuwei abdullah pan from the department of gastroenterology and hepatology, erasmus mc, university medical center rotterdam for advice. we thank ritva kajander for the hsv- culture. supplementary data related to this article can be found at http://dx. doi.org/ . /j.antiviral. . . . key: cord- -i bfuvq authors: macdonald-laurs, emma; koirala, archana; britton, philip n.; rawlinson, william; hiew, chee chung; mcrae, jocelynne; dale, russell c.; jones, cheryl; macartney, kristine; mcmullan, brendan; pillai, sekhar title: csf neopterin, a useful biomarker in children presenting with influenza associated encephalopathy? date: - - journal: eur j paediatr neurol doi: . /j.ejpn. . . sha: doc_id: cord_uid: i bfuvq purpose: neurological complications of influenza cause significant disease in children. central nervous system inflammation, the presumed mechanism of influenza-associated encephalopathy, is difficult to detect. characteristics of children presenting with severe neurological complications of influenza, and potential biomarkers of influenza-associated encephalopathy are described. methods: a multi-center, retrospective case-series of children with influenza and neurological complications during was performed. enrolled cases met criteria for influenza-associated encephalopathy or had status epilepticus. functional outcome at discharge was compared between groups using the modified rankin scale (mrs). results: there were children with influenza studied of whom / had encephalopathy and / had status epilepticus. only one child had a documented influenza immunization. the biomarker csf neopterin was tested in / children with encephalopathy and was elevated in / . mri was performed in all children with encephalopathy and was abnormal in ( %). treatment of children with encephalopathy was with corticosteroids or intravenous immunoglobulin in / ( %). in all cases oseltamivir use was low ( %) while admission to the intensive care unit was frequent ( / , %). clinical outcome at discharge was moderate to severe disability (mrs score > ) in the majority of children with encephalopathy ( / , %), including one child who died. children with status epilepticus recovered to near-baseline function in all cases. conclusion: raised csf neopterin was present in most cases of encephalopathy, and along with diffusion restriction on mri, is a useful diagnostic biomarker. lack of seasonal influenza vaccination represents a missed opportunity to prevent illness in children, including severe neurological disease. severe neurological complications from seasonal influenza, including influenza-associated encephalopathy/encephalitis (iae), cause considerable morbidity and mortality in healthy children, and those with pre-existing neurological disease. e recent estimates indicate the annual incidence of iae in australia is . per , , in children under years, with around % of hospitalized influenza cases associated with iae. other populations show similar or higher incidence, with japan's annual incidence of iae recorded as e / , , . , neurological complications attributed to influenza range from a mildly altered mental state, vertigo and brief febrile seizures to life threatening complications such as status epilepticus, meningitis, stroke, and demyelinating disease. antiviral agents, predominantly neuraminidase inhibitors, and immunomodulatory treatments (corticosteroids, intravenous immunoglobulin), are used to treat patients with influenza-associated neurological disease but there is limited evidence on their efficacy. while it is thought that more extensive changes on mri correlate with disease severity there are no other available biomarkers that predict outcome. the australian influenza season typically occurs between july and october. the season saw the highest levels of influenza reported since the pandemic year. the authors of this report noted an apparent increase in iae and other severe neurological complications during . here, we describe the clinical presentation, laboratory testing, neuroimaging, treatment and short-term outcome of these cases. in addition, we observed elevated cerebrospinal fluid (csf) neopterin e a marker of central nervous system (cns) inflammation e amongst children with iae that has not previously described. we compared the frequency of iae during the influenza season with previously published incidence estimates. materials and methods we identified children aged e years, with evidence of influenza and associated severe neurological disease including status epilepticus or moderate to severe encephalopathy, admitted to two paediatric hospitals which comprise the sydney children's hospital network, the largest paediatric network in australia. cases were ascertained between april st and october st, . at the children's hospital at westmead, cases were identified from those recruited under pre-existing surveillance studies: the australian childhood encephalitis study (ace), and the influenza complications network (flucan) surveillance study. , at sydney children's hospital, children were identified from neurology consultation databases. children were included if they required hospital admission and consultation from a paediatric neurologist for a neurological complication or worsening of a pre-existing neurological condition due to proven influenza. all children included either presented with status epilepticus (for min or longer) or reached level diagnostic certainty on the brighton encephalopathy score. children were excluded if: influenza was not confirmed, neurological symptoms were mild, hospital admission was not required, and when an alternative diagnosis could better explain the presentation. data were retrospectively collected from electronic medical records. we collected demographic data, presenting clinical characteristics, intensive care unit (icu) admission, and length of stay, laboratory results including csf testing, and influenza testing. csf analysis included cell count, protein, glucose, microscopy, lactate, oligoclonal bands, neopterin and influenza pcr. an elevated csf neopterin result was defined as > nmol/l. electroencephalogram reports and brain magnetic resonance imaging (mri) (t weighted, flair and diffusion weighted imaging) were assessed by a neurologist (s.p.) and neuroradiologist (c.c.h.). the neuroradiologist was blinded to diagnosis during review of the mri. influenza was most commonly acutely diagnosed through the detection of influenza rna in respiratory samples. both hospitals used multiplex pcr assays (seegene, south korea) which detected up to respiratory viruses. the assay has targets for both influenza a and b and was performed daily with a turnaround time of e days. at sydney children's hospital, the assay also had targets for subtypes of influenza a: h strains and the pandemic strain h n / . when influenza serology was requested to diagnose a recent influenza illness, this was performed using a complement fixation assay (virion, germany). treatment given including oseltamivir, empiric aciclovir, or rd generation cephalosporins, ivig, corticosteroids, and e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y ( ) e plasmapheresis were recorded. time from admission to commencement of oseltamivir was recorded. each case was assigned a modified rankin scale (mrs) based on the examination at presentation and discharge from hospital. the mrs ranges from (no symptoms) to (death). a poor outcome was an mrs score of > which indicates at least moderate disability requiring assistance. the relation between categorical variables was investigated using the two tailed fisher exact test. the mannewhitney u test was used to determine the relation between continuous variables. ethics approval was granted by the sydney children's hospitals network ethics committee (lnr/ /schn/ ). twenty two children were included in this case series; % ( / ) were female. the median age at presentation was five years (range: . e years). eight children ( %) had preexisting epilepsy and/or developmental delay. one child had an immunodeficiency (hypogammaglobulinemia) and receives monthly ivig. this child, who presented with status epilepticus, was the only child recorded as having received the seasonal influenza vaccination. eleven children ( %) met level brighton criteria for encephalitis and were designated as influenza associated encephalopathy/encephalitis (iae). two children with iae had previous episodes of acute disseminated encephalomyelitis (adem) with complete clinical and radiological recovery ( table cases & ). one child had mosaic tetrasomy x, while another had epilepsy and developmental delay. the remaining children in our series had status epilepticus. in contrast to the children with iae, over half of this group (n ¼ ) had preexisting neurological disease including two children with refractory genetic epilepsies (dravet syndrome and cdkl ). the majority of children had a fever ( %) and two-thirds had respiratory symptoms. half presented with neurological symptoms within two days of onset of their influenza illness. sixteen children ( %) presented with an altered level of consciousness. seizures occurred in children ( %) at any stage of illness and status epilepticus was frequent (n ¼ , %). other neurological findings at presentation were weakness (n ¼ , %), pyramidal signs (n ¼ , %), movement disorder (n ¼ , %) and ataxia (n ¼ , %). hallucinations, meningism, cranial neuropathy and pupillary changes were infrequent (< %). the majority of children (n ¼ , %) had influenza a. of those sub-typed (n ¼ ) half were h ( ) and half were h . four children had other respiratory pathogens co-identified on npa (rhinovirus, coronavirus, mycoplasma pneumoniae). enterovirus was detected in the npa of one child but was absent in csf. one blood culture was positive for staphylococcus epidermidis, and this was assessed to be a contaminant. lumbar puncture was performed in children where it was considered clinically indicated ( table ) . of those who did not have a lumbar puncture performed most were children who presented with status epilepticus alone, usually with known pre-existing epilepsy. one case, with acute necrotising encephalopathy (ane), who was deemed to be too unwell to undergo a second lumbar puncture for measurement of neopterin. where sampled, csf showed pleocytosis and elevated protein in only a third (each n ¼ ). influenza pcr on csf was positive in of children tested, in an immunocompetent previously well year old. the csf neopterin was elevated in of children tested; in seven children it was considerably elevated ranging from to nmol/l (normal < nmol/l), one had a borderline result ( nmol/l). csf neopterin was measured in one child presenting with status epilepticus and was . nmol/l (borderline result). while most children with iae had a raised csf neopterin ( / , %), only three had csf pleocytosis ( , , cells/mm ) and two had an elevated csf protein ( . g/l and . g/l). oligoclonal bands were measured on serum and/or csf in / children with iae and were not present in any. some children with iae had anti-neuronal antibodies testing performed on serum and/or csf, usually nmda and vgkc (table ). these were negative apart from two cases with mildly elevated anti-thyroid antibodies. other routine laboratory data were normal or only mildly abnormal in most children (table ) . mri brain was performed in children and showed new abnormalities in eight ( %), all with iae. the common acute mri abnormalities were the presence of t -flair hyperintensities, diffusion restriction (each, n ¼ ), and gadolinium enhancement (n ¼ ). the spectrum of radiological features are shown in fig. aei . diverse clinico-radiological syndromes were diagnosed including: ane (n ¼ ), acute encephalopathy with biphasic seizures (aesd) (n ¼ ), posterior reversible encephalopathy (pres) (n ¼ ), hemiconvulsion hemiplegia syndrome (hhs) (n ¼ ), and cerebellitis. genetic testing of ran-binding protein (ranbp ) was performed in the child with ane (table case ) and the child who died from an ane-like illness ( table , case ). both were negative. one child who met criteria for iae was subsequently found to have a mutation in the polymerase gamma (polg) gene (table , case ). fourteen ( %) children were admitted to the icu and nine ( %) required mechanical ventilation. thirteen ( %) children received oseltamivir. median time to commencement of oseltamivir from presentation was day (mean . days, iqr e days), but > days in five cases. two icu ventilated children were commenced on oseltamivir nine days after admission. in contrast, nineteen ( %) children were treated with a rd generation cephalosporin, while ( %) received aciclovir. nineteen ( %) received anticonvulsants and ( %) continued these on discharge. first-line e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y ( ) e immunomodulatory treatment (corticosteroids and/or ivig, plasmapheresis) was given to nine children with iae (corticosteroids (n ¼ ), ivig (n ¼ ) and plasmapheresis (n ¼ )) but none of those with status epilepticus alone. the median length of icu and hospital stay was four days (range e ) and days (range e days) respectively. children with iae were more likely to have both longer hospital (mean . days vs . days; p ¼ . ) and picu admissions (mean . days vs days; p ¼ . ) compared to children with status epilepticus. one child with iae died following an ane-like illness, although mri findings were atypical (table , case ). her post-mortem was inconclusive: showing generalised cerebral oedema and some features of acute hemorrhagic leucoencephalitis. ten out of children with iae had an mrs score of (normal) at baseline. at discharge from hospital / ( %) of children with iae had a higher in mrs score (mrs > ; moderate disability) compared to those with status epilepticus. the change in mean mrs was significant between the two groups: children with iae had a change in mrs of . points while those in the status epilepticus group had a mean increase of mrs of . points (p-value: . ). nearly all children ( / , %) with mri diffusion restriction had a poor outcome. the child with mri diffusion restriction and a good outcome had posterior reversible encephalopathy syndrome (pres). among the children with iae and a considerably elevated neopterin, four had an mrs > while three had mild deficits (mrs , and ). there was a mean increase in the mrs of . points the seven children with highly elevated csf neopterin ( e nmol/l). in this case series we observed two groups of children who presented with severe influenza related neurological disease. one group of children fulfilled criteria for iae, while the other group, most often with pre-existing neurological disease, presented with status epilepticus but otherwise did not fulfill criteria for iae. amongst cases of iae, elevated csf neopterin appeared to correlate with the presence of diffusion restriction on mri brain and adverse outcome. use of oseltamivir was infrequent among all cases, although use of antibiotics occurred in the majority. only one child had documented influenza vaccine, even amongst those with pre-existing neurological co-morbidity, despite the fact that this is recommended in australia. influenza associated neurological complications are thought to occur due to an inflammatory or immunemediated response to influenza infection rather than direct viral invasion. , among those with iae we observed, similar to previous authors , , that csf pleocytosis and detection of influenza in the csf occurred in a minority (n ¼ ; %, and n ¼ / ; %). , , , however csf neopterin, a biomarker of inflammation, was elevated in most children with iae (n ¼ , %). neopterin, a catabolic product of guanosine triphosphate (gtp), is synthesized by human macrophages upon stimulation from interferon gamma and can be measured in urine, serum and csf. while serum neopterin levels are useful in the diagnosis and monitoring of systemic infectious or inflammatory diseases, such as hiv, csf neopterin, reflecting intrathecal production by microglial cells, more accurately detects cns inflammatory diseases (infectious or immune mediated). , a recent review assessing biomarkers of csf inflammation found, among clinically available tests, that csf neopterin performed better than the presence of oligoclonal bands or csf pleocytosis in detection of cns inflammation. there is limited data regarding prognostically significant biomarkers in iae. pro-inflammatory cytokines may impair the blood brain barrier and induce apoptosis of neurons. elevated cytokines such as interleukin- (il- ) and tumour necrosis factor alpha have been demonstrated in children with iae and correlates with poorer outcome. , , testing csf il- outside the research setting is currently unavailable. clasmatodendrosis, abnormal morphological changes in astrocytes, occurring presumably due to the effect of proinflammatory cytokines, has recently been suggested to be a pathological feature of iae on autopsy. clasmatodendrosis was found in the cerebral white matter, thalamus, corpus callosum, cerebellum, thalamus and hippocampus of children with iae and may correlate mri changes commonly seen. previous authors have associated abnormalities on mri brain with poorer outcome. in our cohort mri brain abnormalities were diverse and common, particularly diffusion restriction in the subcortical white matter. diffusion restriction correlated with a poor outcome, apart from in the child who had pres, and was associated with an elevated csf neopterin in most cases. further studies of iae are required to evaluate whether significant elevations of csf neopterin, particularly in combination with diffusion restriction and other mri changes, could predict short and long-term outcome. oseltamivir, a neuramidase inhibitor which prevents release of influenza virus from infected cells has been shown to reduce influenza symptoms in otherwise healthy children by h ( % ci e h,p ¼ . ). only % ( children) were treated with oseltamivir and there was a significant delay in commencement in cases (> days in hospital). in contrast, empirical rd generation cephalosporin ( %) and aciclovir use ( %) was more frequent. this may be related to the perception among practitioners that antiinfluenza therapy has little benefit. we suggest in accordance with local guidelines, that children with encephalitis should be empirically treated with oseltamivir during the influenza season (may to october). the evidence for use of immunotherapy (ivig, corticosteroids) in iae, is limited , however, in our case series, most children with iae were treated with first-line immunotherapy with uncertain benefit. no serious side effects were reported. in , the burden of influenza in australia (particularly the eastern states) was the highest seen since the pandemic. based on iae incidence estimates published by britton et al. from the e influenza seasons in australia and the population coverage of our hospitals, we calculated that we would expect . ( . e . ) cases of iae in children (< years) per year. the iae case frequency observed in our cohort was twice the expected point estimate based on these previous incidence estimates but within the % confidence interval, and so contribute to validating the estimates from britton et al. the short-term outcome of our cohort, particularly those with iae, was alarming with % having a poor outcome. while there was a significant rate of icu admission among the group of children with status epilepticus ( %) this was not as high as children with iae ( %) and, most often, non-iae children did not experience a significant change in their mrs. this supports previous observations that survivors of iae during the h n pandemic, and in more recent nonpandemic influenza seasons in australia, experienced significant ongoing disability. , we have previously shown in a large retrospective encephalitis cohort study that icu admission, mri diffusion restriction and status epilepticus and were risk factors for a long-term abnormal outcome. these risk factors were common ( %, %, %) in children with iae from our cohort. the medium and long-term outcome in our cohort should be assessed including formal neuropsychological testing. further research is required to understand and modulate the cns inflammatory cascade present in iae in order to modulate long term neurodisability. the overall influenza immunisation rate during in australia was low at %, however a recently observed rate of vaccine receipt among children was even lower at . %. four age-specific quadrivalent influenza vaccines containing two strains of influenza a (h n [michigan] and h n [hong kong]) and two strains of influenza b (brisbane and phuket) were available in . children older than six months were eligible to be vaccinated and the vaccine was provided free to children with neurological disease. in our cohort just one child had a documented influenza vaccination, although a third of children were eligible for free immunisation and the remainder could have received an immunisation at the cost of around $ e aud. we emphasise that the severe syndromes and adverse outcomes observed here should be considered preventable. following high rates of influenza related morbidity in (including these cases), new south wales and other australian states have introduced universal funded seasonal influenza immunisation to all children aged months to years. our series has limitations. we describe children with severe influenza-associated neurological complications but did not include children with mild neurological complications. children with pre-existing epilepsy may not have always been tested for influenza and may be under-represented. the collection of clinical data was retrospective, and some electronic data were incomplete. seasonal influenza immunisation status was not always clearly recorded, although we reviewed the australian immunisation register to verify vaccination status where possible. influenza sub-typing from npa samples and csf influenza pcr testing was not routinely performed. in the se group, csf studies, including csf neopterin were performed infrequently and mri brain infrequently requested. due to this we were unable to use this group as a direct control for the finding of elevated csf neopterin the iae group. serial csf neopterin to assess treatment and clinical progress were not performed. this is the first series to demonstrate that elevation of csf neopterin, a marker of cns inflammation, occurs commonly in children with iae. csf neopterin may be a useful diagnostic marker for iae while its role as a prognostic marker e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y ( ) e requires further evaluation. mri diffusion restriction was associated with a poor outcome in iae. short-term outcomes of children with neurological complications of influenza, especially within the iae group, were alarming, with nearly two-thirds of children having a poor outcome despite receipt of icu support, anticonvulsants, first-line immunotherapy and, in some, anti-viral treatment. given the severity of influenza associated neurological complications, we recommend a "treat and test" approach to the use of oseltamivir in children presenting with acute encephalopathy/encephalitis during the influenza season. finally, seasonal influenza vaccination should be universally provided to children and those at risk of severe influenza, with better education and awareness to increase uptake in the paediatric population. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. neurological manifestations of influenza infection in children and adults: results of a national british surveillance study the spectrum and burden of influenza-associated neurological disease in children: combined encephalitis and influenza sentinel site surveillance from australia neuroinfluenza: evaluation of 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marker of active central nervous system inflammation recovery of motor function after stroke atagi) atagoi. the australian immunisation handbook australian government department of health acute encephalopathy and encephalitis caused by influenza virus infection evidence for influenza virus cns invasion along the olfactory route in an immunocompromised infant neurological and muscular manifestations associated with influenza b infection in children influenza virus and cns manifestations central nervous system manifestations in pediatric patients with influenza a h n infection during the pandemic neopterin in the diagnosis and monitoring of infectious diseases csf fluid neopterin: an informative biomarker of cns immune activity in hiv- infection utility of csf cytokine/chemokines as markers of active intrathecal inflammation: comparison of demyelinating, anti-nmdar and enteroviral encephalitis h n encephalitis with malignant edema and review of neurologic complications from influenza influenza-associated neurological complications clasmatodendrosis is associated with dendritic spines and does not represent autophagic astrocyte death in influenzaassociated encephalopathy prevention and treatment of influenza oseltamivir for influenza in adults and children: systematic review of clinical study reports and summary of regulatory comments immunomodulatory therapies in neurologic critical care intravenous immunoglobulin for the treatment of childhood encephalitis national survey of pandemic influenza a (h n ) -associated encephalopathy in japanese children influenza-associated encephalitis/encephalopathy identified by the australian childhood encephalitis study e infectious and autoantibody-associated encephalitis: clinical features and long-term outcome influenza season in australia. a summary from the national influenza surveillance committee influenza epidemiology, vaccine coverage and vaccine effectiveness in children admitted to sentinel australian hospitals in : results from the paeds-flucan collaboration influenza vaccine effectiveness against pediatric deaths seasonal influenza vaccination the authors have stated that they had no interests, which might be perceived as posing a conflict or bias. this manuscript has been contributed to, seen, and approved by all the authors. all the authors fulfill the authorship credit requirements. no honorarium grant or other form of payment was received for the preparation of this manuscript. supplementary data to this article can be found online at https://doi.org/ . /j.ejpn. . . . r e f e r e n c e s key: cord- -i lspg i authors: bashandy, samir a. e.; ebaid, hossam; abdelmottaleb moussa, sherif a.; alhazza, ibrahim m.; hassan, iftekhar; alaamer, abdulaziz; al tamimi, jameel title: potential effects of the combination of nicotinamide, vitamin b and vitamin c on oxidative-mediated hepatotoxicity induced by thioacetamide date: - - journal: lipids health dis doi: . /s - - -z sha: doc_id: cord_uid: i lspg i background: the liver disease is one of the most important traditional public health problems in egypt. oxidative stress is attributed to such pathological condition that further contributes to the initiation and progression of liver injury. in the present study, we have investigated if the strong antioxidant power of nicotinamide (na), vitamin b (vb ), and vitamin c (vc) can ameliorate taa-induced oxidative stress-mediated liver injury in the rats. methods: thirty-six albino rats were divided into six groups: control group; taa group (ip injection with taa at a dosage of mg/kg three times a week for two months); taa + na group (rats administered with na at a dosage of mg/kg daily besides taa as in the control); taa + vb group (rats administered with vitamin b at a dosage of mg/kg daily besides injection with taa); taa + vc group (rats administered with vitamin c at a dosage of mg/kg daily along with injection of taa). taa + na + vb + vc group (rats administered the with the three vitamins daily in taa pre-injected at the respective doses described above). results: treatment of rats with taa led to a significant elevation of aspartate aminotransferase (ast), alanine aminotransferase (alt), alkaline phosphatase (alp), lactate dehydrogenase (ldh), total bilirubin, cholesterol, triglycerides, low-density lipoprotein (ldl) and tumor necrosis factor-alpha (tnf-α) in the serum samples. moreover, malondialdehyde (mda), hydroxyproline and nitic oxide (no) were also significantly increased in the taa-treated rats, while reduced glutathione (gsh), superoxide dismutase (sod) and catalase (cat) were significantly compromised in the hepatic samples. rats administered with na, vb , and vc as individually or in combination ameliorated the deleterious effects of taa that was confirmed by histopathology. however, the combination of the three vitamins was found more effective as compared to each of the vitamins. conclusion: our work demonstrates that na, vb , and vc cross-talk with each other that act as a more potent biochemical chain of antioxidant defense against taa-induced toxicities in vivo. egypt has been a hot spring of academic research under tropical medicine programs. due to such medical problems, the confronting physicians have been facing the complications of hepatic fibrosis and cirrhosis because of schistosomiasis and chronic viral infections. it led many of the prominent hepatologists to establish clinical liver centers in entire egypt region [ ] . four over three decades, many investigators have been trying to find efficient and long-lasting clinical solutions for liver diseases. thioacetamide (taa) is a thio-sulfur-containing compound, which has been frequently used in leather, motor fuel, textile, paper, food, and beverage industries since long [ ] . administration of one dose of taa leads to acute hepatic toxicity, while chronic exposure causes hepatic cirrhosis and possible development of liver tumors. the metabolic activation of taa by cytochrome p generates taa-intermediates, and reactive oxygen species (ros) that can further covalently bind to biologically significant molecules and increase cellular oxidative stress, lipid peroxidation, and deplete glutathione [ ] . nicotinamide (na), is a precursor of essential coenzymes for numerous reactions in the body [ ] . although na is known to prevent or treat pellagra [ ] , it has other pharmacological actions as an antioxidant [ ] , and antinociceptive [ ] . nicotinamide, the amide derivative of vitamin b , has been shown to exhibit a number of anti-inflammatory properties including inhibition of inducible no synthase (inos) [ ] , free radical scavenging [ ] , suppression of mhc class ii expression [ ] and intracellular adhesion molecule icam- expression on endothelial cells [ ] . na is changed to nicotinamide adenine dinucleotide by the aid of enzymes. these cellular pathways are essential for energy metabolism and may directly influence normal physiology, as well as disease progression [ ] . for example, na has been shown to protect against bleomycin-induced pulmonary fibrosis [ ] , and doxorubicin-induced nephrotoxicity [ ] . riboflavin (vb ) is the central component of the cofactors flavin adenine dinucleotide (fad). vb plays a key role in cellular function, growth, development and energy metabolism [ ] . it is effective in preventing migraine [ ] and reduces hepatocellular injury following liver ischemia through its role in lowering hepatic inducible nitric oxide synthase and nitric oxide [ ] . vb can act against oxidative stress, especially lipid peroxidation and oxidative injury. it is reported that riboflavin protects the body against oxidative stress by glutathione redox cycling beside conversion of the reduced riboflavin to the oxidized form among other possible mechanisms [ , ] . vitamin c (vc) is the most important radical scavenging antioxidant in extracellular fluids [ ] vitamin c (ascorbic acid) is a six-carbon lactone is called an antioxidant because, by donating its electrons, it prevents other compounds from being oxidized [ ] . vc has been reported to attenuate hepatic damage and can act synergistically with other vitamins in the management of oxidative stress [ ] . vitamin c was reported to form a synergy with other agents to protect the liver. one of the obvious synergies is between vitamin c and vitamin e that ameliorated ethanol-induced hepatotoxicity via normalization of transaminases, lipid peroxidation [ ] and endogenous antioxidants [ ] . thus, this study aimed to investigate hepatoprotective activities of nicotinamide, vitamin b , and vitamin c, separately or in combination, against thioacetamide-induced liver damage, hyperlipidemia and oxidative stress in rats. the hepatoprotective activity was assessed using liver function tests, lipid profile, hepatic oxidative stress parameters, inflammatory markers and hydroxyproline as hepatic fibrosis marker. taa, na, vitamin b and vitamin c were purchased from sigma-aldrich corporation (st louis, mo, usa). remaining all chemicals were of analytical grade from authentic companies of international repute. the study was performed on six weeks' old male albino rats weighing - g, obtained from the egyptian holding company for biological products and vaccines, cairo, egypt. the animals were kept in an environment with controlled temperature ( °c), humidity ( - %), and photoperiod ( -h/ -h light/dark cycle). all the animals under study had free access to chow diet and water. all animal care protocols were by the research ethics committee, national research centre, egypt. the rats were sorted into six groups, each containing eight rats as follows: group i: control rats without any treatment. group ii: rats injected ip with taa at a dosage of mg/kg [ ] thrice a week for two months. group iii: rats administered with na at a dosage of mg/kg [ ] daily and injected with mg/kg of taa ip thrice a week for two months. group iv: rats administered with vitamin b at a dosage of mg/kg [ ] daily and injected with mg/kg of taa ip thrice a week for two months. group v: rats administered with vitamin c at a dosage of mg/kg [ , ] daily and injected with mg/kg of taa ip thrice a week for two months. group vi: rats administered with all three of the previous vitamins daily and injected with mg/kg of taa ip thrice a week for two months. the ratio of a multivitamin was performed as previously described [ ] . blood samples were collected from each group by puncture of the retro-orbital venous sinus, into heparinized tubes under light ether anesthesia weekly. the blood was centrifuged at ×g for min to separate plasma which was stored at − °c. after collection of the blood samples, the animals from all the groups were autopsied under light ether anesthesia. the liver was removed from its surrounding tissues, placed into tubes and washed with normal saline (cold) and kept at − °c. the liver tissues were homogenized in a cold potassium phosphate buffer ( . m, ph . ) and centrifuged at rpm for min at °c. the resulting supernatant of tissue homogenate was used for determination of the oxidative stress parameters. serum ast, alt, alp, ldh, total bilirubin, total protein, cholesterol, triglycerides, ldl and hdl concentrations were determined calorimetrically using the commercial kits (salucea company, netherlands). moreover, hepatic mda, gsh, cat, sod and no were determined calorimetrically using kits manufactured by bio-diagnostic, egypt. plasma tnf-α and hepatic hydroxyproline were determined by enzyme-linked immunoassay by the kits from r&d systems (usa) and koma biotechnology (seoul, korea) respectively. at the end of the experiment, a portion of a liver from each of the sacrificed rat was fixed in % buffered formalin and processed for paraffin sectioning after dehydrating them in various concentrations of alcohol, cleared with xylol and embedded in paraffin blocks. sections of about μm thickness were stained with hematoxylin and eosin (h&e) for histological study. data have been presented as mean ± s.e.m and analyzed by one-way anova followed by least significant difference (lsd) using spss software (version . , spss inc., chicago, il). the mean values illustrated in table showed a significant elevation of plasma ast, alt, alp, ldh and total bilirubin in rats treated with taa only, while total protein decreased compared to the control. these effects on liver function parameters associated with injection with taa were alleviated when the rats were also treated with na, vb or vc, with a significant decrease in the liver enzyme levels and bilirubin and an increase in total protein. administration of all three vitamins together had a more profound effect in mitigating the adverse parameters than when the rats were treated with just one of the vitamins. taa injection resulted in a significant increase in the plasma level of cholesterol, triglycerides, and ldl, while hdl decreased significantly (table ) . treating the rats with taa and na, vb or vc, however, resulted in the changes in these parameters being reversed to some extent. once again, a greater degree of improvement was noticed when the three vitamins were combined. the data relating to hepatic oxidative stress markers are summarized in table and show a significant decrease in hepatic gsh, cat, and sod in the taa group, but a marked increase in hepatic mda, no, hydroxyproline and plasma tnf-α. these changes were significantly reversed in rats treated with the three vitamins in combination. control hepatic tissue figure shows the typical structure of the hepatic tissues with normal polygonal hepatocytes and normal blood sinusoids. histopathological examination of hepatic sections from taa rats revealed the presence of vacuolated hepatocytes with degenerated nuclei and irregular and damaged cell membranes. disturbed and dilated blood sinusoids were also clearly observed along with fibroblasts distributed extensively through the hepatic tissue of this group. moreover, mitotic figures were also noted in some hepatic sections (fig. ). the sections from this group showed an increased number of irregular hepatocytes with degenerated nuclei. disturbed and narrowed blood sinusoids, frequently infiltrated with inflammatory cells, were also a prominent feature in the liver sections of these rats, along with fibroblasts extensively distributed through the hepatic tissue (fig. ) . the general structure of the hepatic tissue in this group was markedly improved. hepatocytes with faint cytoplasm but with healthy nuclei were arranged in hepatic cords creating narrow blood sinusoids (fig. ) . the effect of combining taa with treatment with na histological examination of different sections from rats of this group revealed that there was a marked improvement in the distribution of hepatocytes relative to the hepatic blood sinusoids. on the other hand, hepatocytes appeared with vesiculated nuclei showing partially degenerated chromatin materials. although there was an apparent improvement in the general hepatic architecture, many pathological signs could still be observed in the sections of this group. some hepatocytes appeared vacuolated, and the blood sinusoids were narrow and disturbed in comparison with those in the control sections (fig. ). liver disease has been one of the traditionally major health issues in egypt for a long time. the primary causes of liver disease are schistosomiasis (from the s until the s) and hepatitis c virus ( s). here, we used taa to induce an experimental model of liver damage to mimic the pathophysiological symptoms of acute human liver diseases. taa causes hepatic fibrosis through liver cell membrane injury, which in turn, causes a marked increase in enzymes levels. in the present study, taa caused a significant increase in alt, ast, alt, ldh and bilirubin levels versus to the control the ones. elevation of the activity of liver enzymes after taa administration indicates cellular leakage, loss of structural and functional integrity of the liver [ ] . we have previously reported several natural products with antioxidant and anti-inflammatory properties in vivo studies with promising results [ ] [ ] [ ] [ ] . the present investigation has shown that rats treated with taa have increased hepatic nitric oxide (no) and plasma tnf-α. although no is an important signaling molecule; yet its excess production results in the formation of peroxynitrite which is a very invasive cellular oxidant [ ] . it is interesting, therefore to report that both these parameters were effectively countered in the animals administered with na, vb or vc in the present study. lots of literature on anti-oxidative therapy and antioxidants have been proposed to prevent and treat liver diseases [ ] highlighting the curative properties of antioxidants in such ailments. specifically, treatment of taa rats with na improved the activities of liver enzymes and the histopathological structure of the liver. earlier, we have reported that na exerts a hepatoprotective effect against a high-fat ethanol diet and acetaminopheninduced acute liver injury [ ] . besides, it is documented that na benefits both dams and pups; hence it merits evaluation for preventing or treating preeclampsia complication in human pregnancies [ ] . furthermore, na has been effective in ameliorating many oxidative stressinduced pathological conditions [ ] . similarly, vc also normalized the increased activity of liver enzymes in the plasma samples confirming its hepatoprotective effects in the present investigation. studies have shown that this vitamin has hepato-protective properties due to its antioxidative properties, that gets better in synergy with other agents. the co-administration of ascorbic acid with alpha-tocopherol acetate and sodium selenate ameliorated ethanol-induced liver damage in rats [ ] . the vitamin is believed to normalize the liver function parameters like alt, ast, ldh, alp, some endogenous antioxidants as well as blood hydroperoxide and malondialdehyde in the livers of carbon tetrachloride intoxicated rats [ , ] . besides, it has shown restoration and reconstitution of the polyfunctional, long-lived t-cells in the diabetic rats [ ] . furthermore, the vitamin is the most important hydrophilic antioxidant with high efficiency of free radical scavenging in vivo [ ] . however, the mechanism involved appears to be ascorbyl radical reacting with other free radicals to stop their propagation [ ] . it is documented that vb significantly improves serum and the histological parameters of hepatocellular damage and neutrophil infiltration following liver ischemia in mice [ ] . recently, many studies have also demonstrated that irradiated riboflavin protects the cisplatin-induced oxidative stress-mediated hepatotoxicity and nephrotoxicity in rodent based studies [ ] [ ] [ ] [ ] . furthermore, vb and uv light together were able to reduce the titer of mers-cov in human plasma products to a non-detecting level suggesting that the combination may lower the risk of transfusion transmission of mers-cov [ ] . in this study treatment of rats with the three vitamins in combination led to a more pronounced alleviation of the effects of the liver injury induced by taa. we have previously found that folic acid and melatonin as a combination was superior to using them individually regarding recovering hepatic function and structure [ ] . in the present data, taa administration resulted in a significant reduction in the table effect of nicotinamide, vitamin b and vitamin c on hepatic mda (nmol/mg), gsh (μmol/g tissue), catalase (u/g tissue), sod (u/g tissue), no (nmol/g,hydroxyproline (ug/g tissue) and plasma tnf-α (pg/ml) plasma levels of a total protein that might be indicative of injury in the hepatocytes and excessive destruction of proteins including antioxidant enzymes and cellular reducing powers including sh-protein bond production or alterations in rna sequences in the target tissues. an increase in alp activity and bilirubin level after taa treatment reflects the pathological change in the biliary flow in the animals. likewise, their suppression in the rats treated with na, vb , and vc as combination suggests possible stabilization of the biliary dysfunction. furthermore, taa also enhanced the extent of lipid peroxidation, liver fibrosis and cirrhosis accompanied by the morphological and many other biochemical alterations resembling that of the human disease [ , ] . therefore, it is logical to say that taa caused the cellular damage by inhibiting the activity of the antioxidant enzymes in the present study [ ] . the combination of three vitamins significantly decreased liver hydroxyproline content in taa-treated rats, which may be due to a decline in the accumulation of fibrotic tissue in the liver. our findings are in accord with many earlier studies emphasizing the role of oxidative stress in the mechanisms of liver fibrosis and cirrhosis [ ] . the antioxidant enzymes (sod and cat) as well as reducing power gsh were found significantly improved in the liver homogenates of the rats treated with taa followed by the three-vitamins combination as compared to the control. the improvement of all these parameters seems to restore the structural and functional integrity of the target organs in the animals [ ] . hyperlipidaemia is a vascular risk factor associated with atherosclerosis, coronary artery diseases, cerebral vascular disease and peripheral disease. the present investigation showed that the three vitamin combination inhibits the increase in cholesterol, triglycerides and ldl levels in taa treated rats. however, the combination increased the hdl level. our study showed that taa toxicity stimulated hypercholesterolemia with hypertriglyceridemia. vc may hinder the development of atherosclerosis and prevent the onset of acute coronary events by several molecular mechanisms. the vitamin also helps in maintaining arterial wall integrity that can alter cholesterol metabolism by modulating the conversion of cholesterol into bile acids, while it affects plasma triglyceride levels via modulation by lipoprotein lipase activity [ ] . the vitamin has fig. showing putative mechanism of taa-toxicity amelioration by the proposed combination of the vitamins also been shown to protect hdl cholesterol from lipid oxidation. it, therefore, allows for reverse cholesterol transport involving the removal of unesterified cholesterol from extrahepatic cell membranes into esterified ones via lecithin: cholesterol acyltransferase [ ] . apart from these, another vitamin taken under the present study, nicotinamide has been reported to decrease serum cholesterol, triglycerides and free fatty acids in the fasting rat [ ] . moreover, na induced up-regulation of tumor necrosis factor-α (tnf-α) transcription and interleukin- (il- ). both the cytokines are with lipolytic properties, thereby increasing free fatty acid release [ ] . all the three vitamins have an excellent antioxidant and anti-inflammatory properties. upon their co-administration in taa pre-treated rodents, they decreased the oxidative stress that further improved cellular structure and functionality. the diminished liver function markers and cholesterol, ldl and tnf-α concomitant with enhanced activity of antioxidant enzymes (sod and cat) and replenishment of the cellular reducing power (gsh) are vivid indicators of recovery of tissue damage. the improvement in all these parameters indicates that the cellular, molecular and structural damage incurred by taa were alleviated by the combination of the vitamins. as each vitamin has a different degree of antioxidant and anti-inflammatory properties, their respective combination gives a different level of outcome after the treatment. however, when all the vitamins were administered together, they showed a synergistic ameliorative effect against the taa-induced toxic insults in vivo (fig. ). the present study entails the combination of na, vb , and vc has stronger antioxidant and anti-inflammatory power as compared to the individual potential of the vitamins against taa-induced toxicity. the three vitamins exert the protective effects through their antioxidant, antifibrosis and anti-inflammatory properties that consequently restore the structure and functionality of the target organs. hence, anti-oxidative therapy mainly comprising of natural antioxidants represents a reasonable therapeutic approach for the prevention and treatment of oxidative stressmediated liver diseases. liver disease in egypt: hepatitis c superseded schistosomiasis as a result of iatrogenic and biological factors bioactive food as dietary interventions for liver and gastrointestinal disease rat hmg-coa reductase activation in thioacetamide-induced liver injury is related to an increased reactive oxygen species content a review of nicotinamide: treatment of skin diseases and potential side effects pellagra: dermatitis, dementia, and diarrhea effect of combined fenofibrate and nicotinamide on oxidative stress and inflammatory cytokines involved in cisplatininduced nephrotoxicity in rats antinociceptive and antiinflammatory activities of nicotinamide and its isomers in different experimental models nicotinamide prevents interleukin- effects on accumulated insulin release and nitric oxide production in rat islets of langerhans oxygen radicals generated by the enzyme xanthine oxidase lyse rat pancreatic islet cells in vitro nicotinamide and -aminobenzamide reduce interferon-gamma-induced class ii mhc (hla-dr and -dp) molecule expression on cultured humanendothelial cells and fibroblasts inhibitory effects of nicotinamide on intercellular adhesion molecule- expression on cultured human thyroid cells elevation of cellular nad levels by nicotinic acid and involvement of nicotinic acid phosphoribosyltransferase in human cells evaluation of the effects of nicotinamide on the bleomycin-induced pulmonary fibrosis in rat doxorubicin induced nephrotoxicity: protective effect of nicotinamide modern nutrition in health and disease effectiveness of riboflavin in pediatric migraine prevention riboflavin (vitamin b- ) reduces hepatocellular injury following liver ischaemia and reperfusion in mice riboflavin (vitamin b ) and oxidative stress: a review ameliorative effect of riboflavin on the cisplatin induced nephrotoxicity and hepatotoxicity under photoillumination influence of vitamin c supplementation on oxidative stress and neutrophil inflammatory response in acute and regular exercise vitamin c as an antioxidant: evaluation of its role in disease prevention combined protective effect of vitamins c and e on cadmium induced oxidative liver injury in rats the hepatoprotective effect of vitamin c and e on hepatotoxicity induced by ethanol in sprague dawley rats combined effects of vitamin c, vitamin e, and sodium selenate supplementation on absolute ethanolinduced injury in various organs of rats the preventive and therapeutic role of curcumin in liver cirrhosis nicotinamide supplementation protects gestational diabetic rats by reducing oxidative stress and enhancing immune responses riboflavin attenuates lipopolysaccharide-induced lung injury in rats oral administration of vitamin c and histidine attenuate cyclophosphamide-induced hemorrhagic cystitis in rats single dose intravenous thioacetamide administration as a model of acute liver damage in rats the undenatured whey protein enhanced wound healing in mice whey protein enhances normal inflammatory responses during cutaneous wound healing in diabetic rats zinc improves the immune function and the proliferation of lymphocytes in cadmium-treated rats antioxidant potential of spirulina platensis mitigates oxidative stress and reprotoxicity induced by sodium arsenite in male rats wound healing of different molecular weight of hyaluronan; in-vivo study impairment of active avoidance learning and sensory motor reflexes in mice offspring induced by perinatal acute toxic exposure to selenium pathophysiological basis for antioxidant therapy in chronic liver disease hepatoprotective effects of vitamin c and micronized vitamin c against paracetamol induced hepatotoxicity in rats: a comparative study nicotinamide benefits both mothers and pups in two contrasting mouse models of preeclampsia role of nicotinamide (vitamin b ) in acetaminophen-induced changes in rat liver: nicotinamide effect in acetaminophen-damged protective effects of ascorbic acid, dl-alpha-tocopherol acetate, and sodium selenate on ethanol-induced liver damage of rats role of zinc as an antioxidant and anti-inflammatory to relieve cadmium oxidative stress induced testicular damage in rats carbon tetrachloride-induced hepatotoxicity and nephrotoxicity in rats: protective role of vitamin c vitamin c supplementation reconstitutes polyfunctional t cells in streptozotocin-induced diabetic rats determination of ascorbic acid and dehydroascorbic acid in biological samples by high-performance liquid chromatography using subtraction methods: reliable reduction with tris[ -carboxyethyl]phosphine hydrochloride antioxidants: characterization, natural sources, extraction and analysis scleral cross-linking using riboflavin uva irradiation for the prevention of myopia progression in a guinea pig model: blocked axial extension and altered scleral microstructure inactivation of middle east respiratory syndrome coronavirus (mers-cov) in plasma products using a riboflavinbased and ultraviolet light-based photochemical treatment vitamin b : a promising adjuvant in cisplatin based chemoradiotherapy by cellular redox management riboflavin ameliorates cisplatin induced toxicities under photoillumination folic acid and melatonin ameliorate carbon tetrachloride-induced hepatic injury, oxidative stress and inflammation in rats study of the effects of melatonin on experimentally induced hepatic fibrogenesis in rats reproducible production of thioacetamideinduced macronodular cirrhosis in the rat with nomortality hepatocytes are protected by herb phyllanthus niruri protein isolate against thioacetamide toxicity melatonin ameliorates carbon tetrachlorideinduced hepatic fibrogenesis in rats via inhibition of oxidative stress novel mechanisms of natural antioxidant compounds in biological systems: involvement of glutathione and glutathione-related enzymes regulatory role of vitamins e and c on extracellular matrix components of the vascular system vitamin c inhibits lipid oxidation in human hdl relationship of nicotinamide and nicotinic acid to hypolipidemia widespread effects of nicotinic acid on gene expression in insulin-sensitive tissues: implications for unwanted effects of nicotinic acid treatment tumor necrosis factor-alpha modulates human in vivo lipolysis the authors extend their appreciation to the deanship of scientific research at king saud university for funding the work through the research group project no. rg- - . we declare at this moment the role of the deanship of scientific research at king saud university in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.availability of data and materials all data generated or analyzed during this study are included in this published article. authors' contributions sb prepared the introduction and conducted the animal care, analyzed the biochemical data, prepared the results, figures, and the oxidative stress tests. he designed this study, supervised the practical work and finalized the manuscript. sm revised the manuscript. ia provided the chemicals and contributed to the final revision. ih designed the mechanism diagramme and checked english grammar, text, organization, and typesetting of the paper. all authors read and approved the final manuscript.ethics approval and consent to participate the study was approved by ethical committee of zoology department, king saud university, riyadh. the authors declare that there is no any individual person's data in any form (including individual details, images or videos). the authors declare that they have no competing interests.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -z bd a authors: sun, ning; wang, weiping; wang, jie; yao, xinyue; chen, fangfang; li, xiaojun; yinglei, yi; chen, bo title: reverse transcription recombinase polymerase amplification with lateral flow dipsticks for detection of influenza a virus and subtyping of h and h date: - - journal: mol cell probes doi: . /j.mcp. . . sha: doc_id: cord_uid: z bd a three reverse transcription recombinase polymerase amplification assays with lateral flow dipsticks (rt-rpa-lfd) were developed for identification of the matrix and hemagglutinin (ha) genes to detect influenza a virus and distinguish subtypes h and h . assessment of the assays’ specificity showed that there was no cross-reactivity with other targets. their limits of detection were . copies per reaction for the matrix gene, . copies per reaction for the h ha gene, and . copies/reaction for the h ha gene. of samples tested by rt-rpa-lfd assays, were positive for influenza a virus, were positive for h , and were positive for h . compared to the results obtained from real-time rt-pcr assays, the sensitivity of rt-rpa-lfd assays was %, . % and . % for the matrix, h , and h , with % specificity. the sensitivity of rt-rpa-lfd assays is lower than that of real-time rt-pcr, comparable or better than that of conventional rt-pcr, and much better than that of ridts. in conclusion, these assays offer an efficient and reliable tool for identification and subtyping of influenza a virus (subtype h and h ) in the resource-limited setting. influenza viruses are common and major pathogens that cause viral respiratory infections. these viruses are a serious threat to the health of people worldwide as the severe morbidity and mortality they cause. according to the world health organization (who), seasonal influenza viruses cause about , to , deaths each year. influenza viruses belong to the orthomyxoviridae family and are divided into four types: type a, b, c, and d [ ] . influenza a virus (iav) is the most virulent of these pathogens. based on two surface glycoproteins, hemagglutinin (ha) and neuraminidase (na), iavs are further divided into many subtypes, also known as serotypes. so far, serotypes of ha (h -h ) and serotypes of na (n -n ) have been described and verified [ ] . due to reassortment or mutation, iav subtypes evolve constantly and can cause pandemics, with a recent example being the influenza a(h n )pdm virus [ ] . among the iav subtypes, h n and h n are the most common subtypes causing disease in humans around the world. although vaccination is an effective countermeasure against infection by influenza virus, some limitations still exist [ , ] . clinically, na inhibitors and adamantanes are approved for treatment and prevention of influenza virus infection. however, some subtypes with highlevel antiviral resistance currently circulate which are resistant to all adamantanes or na inhibitors [ ] . moreover, effective antiviral treatment needs to be initiated within h of illness onset. hence, it is necessary to rapidly identify iavs for accurate treatment and infection control. infections of iavs can be clinically identified and diagnosed based on patient's signs and symptoms. however, approximately % of individuals infected are asymptomatic [ ] , and others infected by bacteria or other viruses, such as respiratory syncytial virus, parainfluenza virus, and rhinovirus, also show influenza-like signs and symptoms. compared to laboratory diagnostic technology, clinical diagnosis is estimated to have an - % positive predictive value [ ] . in the laboratory, the long turnaround time of viral culture is unfavorable for accurate treatment and control of iavs. rapid influenza diagnostic tests (ridts) are widely used to identify iavs in healthcare services because they are simple and swift [ ] [ ] [ ] to %, the defect is an inconsistent sensitivity ( - %) [ , , ] . more recently, nucleic acid amplification tests (naats), such as reverse transcription polymerase chain reaction (rt-pcr) [ ] [ ] [ ] [ ] , real-time rt-pcr [ , ] , and reverse transcription loop-mediated isothermal amplification (rt-lamp) [ , ] , have been used for rapid and sensitive diagnosis or subtyping of iavs. nevertheless, these methods require expensive equipment and/or skilled technicians, making them inappropriate for use in developing countries. recombinase polymerase amplification (rpa) is a low-resource diagnostic tool that depends on several enzymes running at a constant temperature [ ] . previously, rpa has been used for diagnosis of many pathogens, such as human immunodeficiency virus type [ ] , dengue virus [ ] , and brucella [ ] , with great success. therefore, detection of iavs and differentiation of their subtypes could be achieved with rt-rpa with lateral flow dipstick, providing an efficient and rapid assay for clinical diagnosis and epidemiological surveys. in this study, we developed three reverse transcription-rpa assays with lateral flow dipsticks (rt-rpa-lfd) for detecting iavs and distinguishing the h and h subtypes. one assay targeted to the matrix gene was used for diagnosis of iavs, and iav-positive samples were further subtyped using the other two assays. eighty-seven throat swabs were collected from children with influenza-like illness at jinling hospital (nanjing, jiangsu, china) using virocult swabs (yocon biotech. co., beijing, china) and stored at − °c within h. these specimens were collected between february and march . respiratory pathogens used in this study are listed in table . clinical isolates including h n , h n , influenza b virus (flu b), and respiratory syncytial virus (rsv) subgroup a and b were previously identified by rt-pcr and sequencing [ ] . h n , h n , and influenza b virus were designated as a/nanjing/ / (h n ), a/nanjing/ / (h n ), and b/victoria/ / , respectively. two subtypes of iav strains, a/michigan/ / (h n ) and a/hong kong/ / (h n ), were provided by shanghai institute of biological products co., ltd. staphylococcus aureus (atcc ), haemophilus influenzae (atcc ), streptococcus pneumoniae (atcc ), streptococcus hemolyticus, chlamydia pneumonia, mycoplasma pneumoniae were stored in our laboratory. human metapneumovirus (hmpv), herpes simplex virus (hsv- ), human coronavirus e (hcov- e), human adenovirus (hadv), parainfluenza virus - (piv - ), human rhinovirus (hrv), and twentyfour viral rnas extracted from nasopharyngeal aspirates were supplied by ningbo health biomed co., ltd (ningbo, zhejiang, china). this study was approved by the human use ethical committee at jinling hospital. the informed consent was obtained from all patients or guardians. since the matrix gene is conserved and usually used for detecting iavs in the previous reports [ , [ ] [ ] [ ] , primers and nfo (escherichia coli endonuclease iv) probe designed for the matrix gene were used to detect iavs. also, primers and probes were designed for subtyping h and h . due to antigen drift and genetic variability of iavs, many isolates are collected and identified in the influenza surveillance annually. this makes it difficult to align all the published sequences of iavs. consequently, we firstly classified the sequences in the database based on both the geographically country/region and collection date/ release date (influenza virus resource, https://www.ncbi.nlm.nih.gov/ genomes/flu/database/nph-select.cgi#mainform). secondly, a consensus sequence was obtained by alignment of the sequences isolated from the same country within a five-year span. finally, all the consensus sequences were aligned using the clustalw (http://www.ebi.ac. uk/tools/msa/clustalo/). the primers and nfo probes were designed according to the most conserved sequence, were synthesized by sangon biotech (shanghai, china), and are shown in table and table s . each nfo probe was modified with fluorescein isothiocyanate (fitc) at the ′ end, an internal abasic nucleotide analogue (tetrahydrofuran, thf) and a ′-polymerase extension blocking group c -spacer. opposing to nfo probe, the primer was labeled with biotin at the ′ end. viral nucleic acid was extracted from μl of viral transport medium and eluted into μl of elution buffer using the tianamp virus dna/rna kit (tiangen biotech co., ltd., beijing, china) as recommended by the manufacturer. the complementary dna (cdna) was synthesized using the reverse transcription system (a , promega, madison, wi, usa). briefly, in a . ml tube, μl of rna, μl of mgcl ( mm), μl of × reverse transcription buffer, μl of dntp mixture ( mm), . μl of recombinant rnasin ribonuclease inhibitor ( u/μl), μl of random primers ( . μg/μl), and . μl of amv reverse transcriptase ( u/μl) were mixed. the total volume was increased to μl by adding nuclease-free water. the mixture was incubated for min at room temperature and subsequently at °c for min. pcr was performed using ex taq version . plus dye kit (takara, dalian, china) in a total volume of μl, which contained μl of cdna, . μl of premix taq, . μl of μm forward primer (ampb, h f , or h f ), and . μl of μm reverse primer (ampc, h r , or h r ). amplification cycles were performed as follows: °c for min; cycles of °c for s, °c for s, and °c for s; cycles of °c for s, °c for s, and °c for s; and °c for min. pcr products were directly analyzed by gel electrophoresis on a . % agarose gel. real-time pcr was performed on applied biosystems real-time pcr system (thermo scientific, waltham, ma, usa) using premix ex taq™ (probe qpcr) kit (takara, dalian, china). the primers and taqman probes used for qpcr were reported in who information for the molecular detection of influenza viruses-update (table s ) [ ] . in a . ml tube, μl of cdna, . μl of premix ex taq, . μl of each forward and reverse primers ( μm), . μl of taqman probe ( μm), . μl of × rox reference dye ii, and . μl of nuclease-free water was added and vortex mixed. the reaction was run as follows: °c for min; cycles of °c for s, °c for s. ct values, which are defined as quantification cycle representing the crossing point between fluorescent value and threshold, were calculated automatically by the software v . . (applied biosystems, foster city, ca, usa). the positive sample was defined as its ct value less than , or a sample with ct value between and was confirmed again. the forward primer with a t promoter at the ' terminus was used to amplify the target gene (matrix, h ha, or h ha) in pcr. after gel purification, the linear products bearing t promoters were blunted with t dna polymerase, transcribed using the in vitro transcription t kit (takara, dalian, china), and digested using dnase i at °c for min. finally, the single-stranded rnas were purified using phenolchloroform extraction, and their concentration was measured by a nanodrop nano- spectrophotometer (thermo scientific, waltham, ma, usa). an lfd was prepared according to the previous reports [ , ] , which was composed of a sample pad, conjugate pad, nitrocellulose membrane, absorbent pad, and plastic adhesive backing. streptavidincoated gold colloid was dispensed onto the conjugate pad, and then dried at °c overnight. anti-fitc antibody (test line, abcam, cambridge, ma, usa) and biotinylated bovine serum albumin (control line, nanjing runyan biotechnology co., ltd, china) were striped onto the nitrocellulose membrane, and then dried at °c for h. finally, lfd was assembled and cut into -mm width strips using microcomputer automatic cutting machine (shanghai goldbio co., ltd., china). rpa was performed in a total volume of μl using twistdx nfo kit (twistdx, cambridge, uk). for each reaction, . μl of rehydration buffer, μl of each forward and reverse primers ( μm), . μl of nfo probe ( μm), μl of cdna, and . μl of nuclease-free water were mixed. finally, . μl of mm magnesium acetate was added. the mixture was incubated at °c for min. rpa products were analyzed using lfds. five microliters of rpa product was added into a tube containing μl of phosphate-buffered saline with tween- (pbst; mm nacl, . mm kcl, mm na hpo , mm kh po , ph . ; . % tween- ). lfds were inserted into the tube and incubated for min. images were recorded using a smartphone and analyzed using imagej software (national institutes of health, md, usa). the images were converted into -bit greyscale, and subsequently, the mean optical density of a fixed area was determined. relative optical density was calculated by dividing the mean optical density by the maximum grey value ( ). the sample was defined as positive when relative optical density was greater than the threshold ( . ), which was calculated by the average of twenty negative samples plus three times standard deviation. experimental data were analyzed by spss statistics (spssinc., chicago, usa), and the significance level was set at . . diagnostic performance of rt-rpa-lfd assays was evaluated by comparison with real-time rt-pcr. clinical sensitivity and specificity were calculated using standard formulas. clinical test results obtained from rt-rpa-lfd and real-time rt-pcr were analyzed with mcnemar's test. to optimize the performance of the detection and subtyping of iavs, we designed and synthesized eight sets of primer and probe (table s ). viral rnas extracted from two clinical isolates, h n and h n , were detected by rt-rpa-lfd assays. the results indicated that set (matrix), set (h ), and set (h ) show the best performance (fig. s ) . iav subtype h n and h n , influenza b virus (flu b), and other respiratory pathogens were used to evaluate the specificity of three rt-rpa-lfd assays (table ; fig. ). as shown in fig. , clear bands emerge on the test line for detecting both h n and h n by matrix rt-rpa-lfd assay, and no other visible bands are present on the test line for detecting other respiratory pathogens. the positive results obtained from detection of either h n or h n indicated that the other two assays were specific, and there was no cross-reactivity with other pathogens and between these two subtypes (fig. ) . although the positive and negative results are visible with the naked eye, the results of rt-rpa-lfd quantitatively analyzed using image software to diminish the influence of subjective factors (fig. b) . to determine the detection limits of rt-rpa-lfd assays, rna transcripts (matrix, h , and h ) were diluted by nuclease-free water and tested (table ; fig. ; fig. s ). by calculating the copy number of each transcribed rna in rpa reaction, probit analysis indicated that rt-rpa-lfd assays at % probability were able to detect . copies (table ; fig. ). the sensitivity of rt-rpa-lfd assays is lower than that of real-time rt-pcr [ , ] , comparable or better than that of conventional rt-pcr [ , ] , and much better than that of ridts [ ] . both eighty-seven throat swab specimens and twenty-four nucleic acids were tested using rt-rpa-lfd assays and real-time rt-pcr assays (table ; table s ). compared to real-time rt-pcr assays as the reference method, diagnostic parameters of rt-rpa-lfd assays, including sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv), were calculated as shown in table . overall, twenty-seven samples were positive for iavs by matrix rt-rpa-lfd assay, while thirty-six samples were positive by matrix realtime rt-pcr. we further found that ct values of nine discordant samples were greater than . fourteen samples were h -positive by rt-rpa-lfd, while fifteen samples were positive by real-time rt-pcr. only one sample with a ct value of . was negative by h rt-rpa-lfd assay. additionally, four of fourteen h -positive samples confirmed by real-time rt-pcr were negative by h rt-rpa-lfd assay. ct values of four discordant samples were greater than . mcnemar's test indicated that the difference between rt-rpa-lfd and real-time rt-pcr for distinguishing h and h subtypes is not significant (p > . ), but for detection of iavs (p = . ). agreement of rt-rpa-lfd and real-time rt-pcr was evaluated by kappa test. the correlation kappa values were . (matrix), . (h ) and . (h ), demonstrating that these assays had a good agreement for the detection of iav and subtyping of h and h . in order to comprehensively evaluate the performance of rt-rpa-lfd, positive throat swab specimens by matrix real-time rt-pcr were tested by ridts (rapid influenza a virus antigen test kits, guangzhou wondfo biotechnology co., ltd, china). although commercial antigen test kit is rapid, only fourteen samples are positive for iav (table ; table s ), and its positive rate is much lower than that of matrix rt-rpa-lfd assay. mcnemar's test suggested that there was a significant difference between matrix rt-rpa-lfd and ridts (p = . ). matrix rt-rpa-lfd assay could be sensitively used for diagnosis of iav in comparison to ridts. in this study, we developed three rt-rpa-lfd assays for detection and subtyping of iavs. the targets of these three assays were the matrix, h ha, and h ha genes. the matrix gene is considered to be conserved among iavs and was selected for identification of iavs [ , [ ] [ ] [ ] . primers and nfo probe were designed according to the alignment of matrix gene sequences. during the past several decades, subtypes h n and h n have been circulating worldwide [ ] . according to the latest influenza surveillance [ ] , h n and a(h n ) pdm accounted for the majority of circulating viruses. therefore, we designed another two assays for subtyping h and h , thus providing a rapid and convenient method for subtyping of h and h . specificity of the rt-rpa-lfd assays indicated that the expected signals were generated on the test line and no cross-reactivity was observed. the sensitivity of rt-rpa-lfd assays was evaluated using each single-stranded rna transcripts of the matrix, h , and h genes, and the limits of detection were determined to be . , . , and . copies per reaction, respectively. clinical samples were used to evaluate the performance of rt-rpa-lfd assay and to compare its performance with real-time rt-pcr. a total of samples were tested. although the sensitivity of rt-rpa-lfd assays was slightly worse than real-time rt-pcr, the former is a better choice for the point-of-care test in resource-limited settings. ridts are widely used for screening the influenza virus in official clinics or primary healthcare services. however, the detection limit of ridts is times higher than that of real-time rt-pcr [ ] . that induces a false-negative result, which can result in inappropriate antibiotic use, treatment failure for patients, and further viral spread. the negative result of ridts does not rule out influenza infection, particularly in the season of high influenza activity. when a negative result of ridt is inconsistent with clinical signs and symptoms, rt-rpa-lfd assays can be used as an effective supplement for iav diagnosis. in addition, rt-rpa-lfd assays can be used to identify the subtype for iav epidemiological investigations. although it is relatively rapid, specific and sensitive for diagnosis of iavs and subtyping of h /h by rt-rpa-lfd assays, there are two major drawbacks for these assays. one is that reverse transcription and rpa are two separate reactions. to further simplify the process, onestep rt-rpa was performed in a single tube containing twistdx nfo kit (twistdx, cambridge, uk), reverse transcriptase xl (amv, takara, dalian, china) and rnase inhibitor (takara, dalian, china), and used to detect iav and discriminate h and h . compared to two-step rt-rpa, the performance of one-step rt-rpa is unsatisfactory. this is probably because that there exists an inhibitory effect between the biotin-labeled reverse primer and reverse transcriptase, or between rt and rpa. this needs to try different strategies to make rt and rpa harmoniously run in a single tube. the other is that exists potential amplicons contamination. rpa product was pipetted to lfd for analysis by opening the reaction tube, which can cause amplicon contamination. this is a very prominent concern for quality control of these assays, especially which are used in a small hospital or rural areas with untrained staff. previous studies indicate that uracil-n-glycosylase (ung) and dutp can effectively eliminate amplicon contamination in pcr [ , ] or lamp [ ] . it is possible that the elimination of rpa amplicon contamination uses ung and dutp. importantly, many sealed devices are developed for analysis of rpa product, and this can also avoid amplicon contamination [ ] [ ] [ ] [ ] . therefore, the combination of rpa, ung/ dutp, and/or a sealed lf device to reduce amplicon contamination will be performed in the future. cost of the test per sample is approximately $ . (rt, $ . ; rpa reaction, $ . ; lfd, $ . ). a -μl reaction system and lfd assembled by ourselves greatly reduce the cost of test. with the improvement of availability and throughput, cost of rt-rpa-lfd will decrease in the further. for example, multiplexed recombinase polymerase amplification assay has been developed to detect intestinal protozoa reducing the cost of a lateral flow strip to $ [ ] . furthermore, using lfds can effectively save the cost of fluorescent detectors. overall, the rt-rpa-lfd assays developed in this study were advantageous for detection and subtyping of iavs. they are suitable for low-resource settings because they are performed at a relatively low temperature of °c and do not require expensive instruments. moreover, they are sensitive and specific enough for routine clinical diagnosis and epidemiological investigations. in conclusion, rt-rpa-lfd assays were described here for detection and subtyping of iavs, which offers relatively rapid, sensitive, and specific assays for diagnosis of iavs and subtyping of h and h . all procedures performed in this study involving human participants were in accordance with the ethical standards of jinling hospital of china and with the helsinki declaration and its later amendments or comparable ethical standards. the authors declare that they have no conflict of interest. isolation of a novel swine influenza virus from oklahoma in which is distantly related to human influenza c viruses new world bats harbor diverse influenza a viruses antigenic and genetic characteristics of swine-origin a(h n ) influenza viruses circulating in humans the evolving history of influenza viruses and influenza vaccines advances in the development of influenza virus vaccines implications of antiviral resistance of influenza viruses time lines of infection and disease in human influenza: a review of volunteer challenge studies influenza diagnosis and treatment in children: a review of studies on clinically useful tests and 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for simultaneous visual detection of both alleles phylogenetic analysis reveals the global migration of seasonal influenza a viruses update: influenza activity in the united states during the - season and composition of the - influenza vaccine quantitative rt-pcr evaluation of a rapid influenza antigen test for efficient diagnosis of influenza virus infection detection of respiratory viruses in sputum from adults by use of automated multiplex pcr design and performance testing of quantitative real time pcr assays for influenza a and b viral load measurement loop-mediated isothermal amplification using self-avoiding molecular recognition systems and antarctic thermal sensitive uracil-dna-glycosylase for detection of nucleic acid with prevention of carryover contamination development of recombinase polymerase amplification assays for detection of orientia tsutsugamushi or rickettsia typhi a rapid isothermal assay for the detection of hop stunt viroid in hop plants (humulus lupulus), and its application in disease surveys detection of root-infecting fungi on cool-season turfgrasses using loop-mediated isothermal amplification and recombinase polymerase amplification picoliter well array chip-based digital recombinase polymerase amplification for absolute quantification of nucleic acids we acknowledge the pediatrics outpatient department of jinling hospital, nanjing, china, for collecting pharyngeal swab specimens. we also acknowledge mr. wang zhixin for providing help and suggestions on the assembly of lateral flow dipsticks. supplementary data to this article can be found online at https:// doi.org/ . /j.mcp. . . . key: cord- -gm w s authors: aleksic sabo, verica; knezevic, petar title: antimicrobial activity of eucalyptus camaldulensis dehn. plant extracts and essential oils: a review date: - - journal: ind crops prod doi: . /j.indcrop. . . sha: doc_id: cord_uid: gm w s eucalyptus has become one of the world’s most widely planted genera and e. camaldulensis (the river red gum) is a plantation species in many parts of the world. the plant traditional medical application indicates great antimicrobial properties, so e. camaldulensis essential oils and plant extracts have been widely examined. essential oil of e. camaldulensis is active against many gram positive ( . – . %) and gram negative bacteria ( . – . %). the antibacterial effect is confirmed for bark and leaf extracts (conc. from . μg/ml to mg/ml), with significant variations depending on extraction procedure. eucalyptus camaldulensis essential oil and extracts are among the most active against bacteria when compared with those from other species of genus eucalyptus. the most fungal model organisms are sensitive to . – . % of e. camaldulensis essential oil. the extracts are active against c. albicans ( . – mg/ml leaf extracts and . mg/ml bark extracts), and against various dermatophytes. of particular importance is considerable the extracts’ antiviral activity against animal and human viruses ( . – μg/ml). although the antiprotozoal activity of e. camaldulensis essential oil and extracts is in the order of magnitude of concentration several hundred mg/ml, it is considerable when taking into account current therapy cost, toxicity, and protozoal growing resistance. some studies show that essential oils’ and extracts’ antimicrobial activity can be further potentiated in combinations with antibiotics (beta-lactams, fluorochinolones, aminoglycosides, polymyxins), antivirals (acyclovir), and extracts of other plants (e.g. annona senegalensis; psidium guajava). the present data confirm the river red gum considerable antimicrobial properties, which should be further examined with particular attention to the mechanisms of antimicrobial activity. the continuing increase in the degree of bacterial resistance to conventional antibiotics is a problem of global significance. the crisis of antimicrobial resistance has been ascribed to the misuse of these agents and today resistant strains are common, such as methicillin-resistant staphylococcus aureus, vancomycin-resistant enterococci, drug resistant streptococcus pneumoniae and mycobacterium tuberculosis, carbapenemresistant and extended-spectrum beta-lactamase producing enterobacteria, multidrug-resistant pseudomonas aeruginosa and acientobacter baumannii etc. it was estimated that the medical cost per patient with an antibiotic-resistant infection is up to $ , and infections are usually life treating (ventola, ; aslam et al., ) . similarly, fungi have become resistant to poliens, azoles and echinocandins, and the emergence of drug-resistant strains has been reported in all fungal species (robbins et al., ) . beside the noticeable emergence of resistant protozoa and viruses, there is a problem with camaldulensis is highly variable (table ). in the past, this character has been used to break up the group into different varieties or subspecies. the entire complex is currently under revision and new varieties or subspecies may be described or extant ones rationalised. until this work is completed, euclid ( ) decided to adopt a conservative view of e. camaldulensis. used for centuries as a traditional aboriginal herbal remedy, eucalyptus leaves and their essential oils have found various applications in everyday life due to their antiseptic, anti-inflammatory and antipyretic properties (jeane et al., ; kumar et al., ) . ancient aboriginal society in australia used e. camaldulensis plant in medicines to treat gastrointestinal symptoms (including colic, diarrhea, and dysentery), respiratory disease (colds, coughs, asthma, laryngalgia, laryngitis, pharyngitis, sore throat, trachalgia), arrest bleeding, open wounds, and cuts, as well as its decoctions for the relief of spasms, aches, and pains in muscles, but also pains in joints and even tooth (duke and wain, ) . as previously stated, e. camaldulensis plant is also known as red gum eucalyptus, murray red gum, and river red gum, because it produces red kino i.e. red gum, in significant amount. thus besides different plant parts aborigines used its secondary products as folk remedies. they made incisions in the tree trunks for obtaining the red kino and applied it directly to abrasions and cuts. except fresh kino, the dried, dehydrated kino was prepared and used in the same way as fresh, but after softening in water (williams, ; clarke, ) . another significant folk remedy is young leaves which were used for smoke bath, where burning leaves smoke surrounds patient. the smoking medicine was used for fevers, colds, flu and general sickness (williams, ; pennacchio et al., ; duke and wain, ) . being useful for treating various health conditions, e. camaldulensis and its folk remedies then were transferred and introduced to other parts of the world, such as africa. in sudan the red kino was used for sore throat and diarrhea, while the smoke of burnt leaves was inhaled in case of respiratory problems. in senegal for stomach-ache decoctions from leaves were prepared with sugar, while in zimbabwe for cough, flu, and fever a decoction of e. camaldulensis leaves were combined with citrus limon (l.) burm. f. fruits and psidium guajava l. leaves (doran and wongkaev, ; maroyi, ) . further more, to prevent tooth decay and periodontitis in nigeria teeth cleaning sticks were made from tree (bukar et al., ) , and in traditional medicine for healing wound infections, poultice of leaves containing eucalyptus oil have been used (adeniyi et al., ) . nowdays, e. camaldulensis has been the subject of numerous studies, to confirm plant's usefulness in traditional medicine for the treatment v. aleksic sabo and p. knezevic industrial crops & products ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] of various ailments (coelho-de-souza et al., ; ghani, ; ito et al., ) . its essential oils are reported to be anesthetic, antiseptic and astringent (jeane et al., ; kumar et al., ) . in addition, a decoction of the leaves is reported to be a remedy for sore throat and other bacterial infections of the respiratory and urinary tracts (bruneton, ) . due to numerous contemporary data regarding the antimicrobial activity of e. camaldulensis, this subject will be discussed in special section/chapter. except antimicrobial effects, e. camaldulensis plant extracts (pex) and essential oils (eos) and its constituents possess numerous other beneficial effects. one such effect is certainly gastrointestinal effect. in animal models, extracts of the leaves of e. camaldulensis and e. torelliana r. muell are reported to decrease gastric acid production and thus appear useful for the treatment of gastric ulcers (adeniyi et al., ) . it has been proven that e. camaldulensis leaves methanol extracts possessed ulcer-healing promoting effect when investigated in acetic acid induced-ulcer in rat (rattus norvegicus domesticus) (table ) . similarly, the poultice of the leaves is applied over wounds and ulcers (gill, ) . the antioxidant effect represents one more useful e. camaldulensis characteristic. the free radical scavenging activities of the essential oils is assessed by measuring their scavenging abilities for , -diphenyl- picrylhydrazyl (dpph) radicals. the scavenging activity for the eucalyptus camaldulensis was characterized as high ( . %) (ghaffar et al., ) . the results of e. camaldulensis eo antioxidant effect evaluation indicated that the eo had high potent ferrous ions chelating and total antioxidant activities comparing to ascorbic acid and bht (el-baz et al., ) . also, the e. camaldulensis var. brevirostris leaves ethanol extract possessed antioxidant activity, where the prevailing antioxidants in the extract were gallic and ellagic acid (el-ghorab et al., ) . there are several more studies dealing with antioxidant activity of e. camaldulensis (barra et al., ; salem et al., ; siramon and ohtani, ; olawore and ololade, ) , but it is interesting to mention here that e. camaldulensis flower eo inhibited melanogenesis through its antioxidant properties and by down-regulating both mitogen-activated protein kinases (mapk) and protein kinase a (pka) signaling pathways. this study indicated that the essential oil has the potential to be developed into a skin care product (huang et al., ) . thus, except the medical application, e. camaldulensis extracts are also currently used in cosmetic formulations, and leaf extracts have been approved as food additives (takahashi et al., ) . also, essential oils and their constituents have been used as flavoring agents in the formulation of different pharmaceutical products, cosmetics, and food industry (cowan, ) . except this, e. camaldulensis pex and eos have the potential to be used as antibacterial and antifungal agents in cosmetic and pharmaceutical products. it is interesting to mention here the activity of e. camaldulensis eos ( - mg/ml) on dental biofilm formation in vivo where detected inhibition was . - . % after four weeks, comparing to chlorhexidine ( mg/ml), which inhibited maximum . % of dental biofilm formation . extracts and essential oils of this aromatic plant can be used as food preservatives in order to reduce the dependency on synthetic chemicals in food preservation. in australia, it is also used as sources of wild honey, providing bees with good quality pollens and heavy yields of nectar (boland et al., ) . moreover, in industry, the wood of e. camaldulensis has been used for heavy construction, railway sleepers, flooring, framing, fencing, plywood, and veneer manufacture, wood turning, firewood, and charcoal production (boland et al., ) . eucalyptus camaldulensis wood burns well and make a good fuel, also its dense wood and coppicing ability make it an excellent species for fuelwood production used in several countries such as brazil (jacobs, ; eldridge et al., ) . furthermore, eucalyptus biomass residues from agro-forest and pulping industries represent a valuable source of highvalue compounds such as triterpenic compounds (domingues et al., ; ferreira et al., ) . due to the diversity of e. camaldulensis beneficial effects, all effects reported in the literature are summarized in table . beside the beneficial effects, essential oils and plant extracts can exert potentially unfavorable effects as complex mixtures of different compounds. a risk assessment of their hazard is always necessary before commercialization, so the estimation of eos toxicity has been already conducted resulting in human oral and dermal dose limit recommendations. for most eos the recommended dose is in range - %, but for cineole-rich eucalyptus sp. eos, including e. camaldulensis, the limit dose is %, indicating a generally low application risk (lis-balchin, ) . the safe daily oral dose in human adults is - mg, while semisolid preparations for topical use may contain - % of eucalyptus oil (blumenthal and busse, ; tisserand and young, ) . similarly, e. camaldulensis bark methanolic extract ld value for swiss albino mice (mus musculus) is very high - mg/kg, indicating its low host toxic effects (islam et al., ) . the low toxicity and natural origin of e. camaldulensis essential oil and extracts, in contrast to synthetic antimicrobials, favor their application as antimicrobial agents. eucalyptus camaldulensis leaves contain . - . % essential oil, of which % is , -cineole. there is considerable amount of cuminal, phellandrene, aromadendren (or aromadendral), valerylaldehyde, geraniol, cymene, and phellandral (council of scientific and industrial research, - council of scientific and industrial research (s.i.r, council of scientific and industrial research, - slee et al., ) . leaves contain - % tannin. the kino (a class of wood exudates), contains % kinotannic acid as well as kino red, a ebrahimi et al. ( ) repellency against the adult females of culex pipiens dried fruits essential oil two different treatment levels ( and μl) in six exposure times ( , , , , and s) erler et al. ( ) anti-diabetic effect albino rats oral mg/kg of body weight dawoud ( ) alloxan-induced diabetic rats leaves ethanol extract mg/kg of body weight in distilled water orally, e. camaldulensis leaves supplement incorporated into the diet g/kg/day dawoud and shayoub ( ) v. aleksic sabo and p. knezevic industrial crops & products ( ) - glucoside, catechol, and pyrocatechol. leaves and fruits test positive for flavonoids and sterols. the bark contains . - % tannin, the wood - %, and the kino . - . % (watt and breyer-brandwijk, ) . some of the reported phytoconstituents of the tree included essential oils, sterols, alkaloids, glycosides, flavonoids, tannins, and phenols. a considerable variation in the yield of leaf essential oil from e. camaldulensis has been reported (boland et al., ; shieh, ; moudachirou et al., ; farah et al., ) , depending on multiple biotope factors, and also genetic and/or epigenetic characteristics of the plant. the yields of e. camaldulensis leaves eo ( . - . %) originating from pakistan and morocco were similar (ashraf et al., ; farah et al., ) , while moudachirou et al. ( ) reported a variable oil content of . - . % from different locations of benin. the oil yield of e. camaldulensis from jerusalem was . % (chalchat et al., ) and significantly higher oil yield was reported for e. camaldulensis from taiwan: . - . % with respect to different seasons (shieh, ) . similar eos yield ( . - . %) has been reported for e. globulus, as one of the economically important plants for essential oil production (joshi, ; selvakumar et al., ; harkat-madouri et al., ) . the reported essential oil yield for other eucalyptus species is slightly higher, ranging from . % to % (w/w): the highest yield was obtained from e. cinerea f. (sebei et al., ) . all the e. camaldulensis essential oil single compounds belong to chemical class of hydrocarbons terpenes, further devided according to the number of isoprene units (c h ) to monoterepenes (c h ), sesquterepenes (c h ), and longer chains of isoprene units. oxygenated terpenes (oxygenated monoterepenes and oxygenated sesquterepenes) are called trepenoids. according to the literature, in e. camaldulensis eos dominates , -cineole (eucalyptol), trans-pinocarveol and terpinen- -ol from chemical class of oxygenated monoterepenes. the oils also contain considerable amount of monoterpene hydrocarbons (β-pinene, α-thujene, γ-terpinene, p-cymene), while sesquterepene hydrocarbons and oxygenated sesquterepenes are detected in significantly lower amounts (table ) . when the composition of five eucalyptus essential oils (eucalyptus camaldulensis, eucalyptus astringens maiden, eucalyptus leucoxylon, eucalyptus lehmannii and eucalyptus rudis endl) are compared, a high percentage of monoterpenes, mainly oxygenated compounds, with lower quantities of sesquiterpenes were recorded throughout the four seasons (ben jemaa et al., ) . in e. camaldulensis oil, monoterpenes were prevalent ( . - . % for all seasons) while sesquiterpene hydrocarbons ( . - . %) and oxygenated sesquiterpenes ( . - . %) were present in less extent. eucalyptus astringens oil had resembling chemical characteristic to e. camaldulensis oil, the abundant quantity of monoterpenes, which represented more than % of the total oil amounts for the all seasons ( . - . %). similarlly, e. leucoxylon essential oil, contained mainly oxygenated monoterpenes with a prevalence of , cineole ( . - . %), such as in e. camaldulensis oil ( . - . %). the essential oil of e. lehmannii also was made up largely of monoterpenes (hydrocarbons . - . % and oxygenated . - . %), with smaller amounts of sesquiterpene hydrocarbons and absence of oxygenated sesquiterpenes, unlike the oils of other eucalyptus species (ben jemaa et al., ) . the difference in the chemical composition of the e. camaldulensis eos may be due to many reasons, which can generally be classified in five main groups: ( ) the change in plant genes through generations and hybridizations (naturally and induced) may result in production a variety of volatile oils compared with those of different habitat; ( ) nutrients of different soils and their accumulation in the leaves may result in different plant metabolism and consequently production of different bio-products and also eos made of diverse compounds in variable amounts; ( ) acclimation of species to the australian environment in which it is growing in the past, compared with the introduced and/or worldwide planted trees on plantations; ( ) different ecotypes of the e. camaldulensis and ( ) differences may be due to plant part used for essential oils extraction and its stage of development (maturity). knowing that these factors express significant effect on the percent compositions of some eo components for this species, e. camaldulensis can be grown in the corresponding areas and in specific conditions to enhance eo production. the variations in the chemical composition of eucalyptus eos with respect to seasons have also been reported (tsiri et al., ) , but the most commonly detected as major components in the eucalyptus essential oil are , -cineole, β-pinene, γ-terpinene, and p-cymene. it is important to emphasize here that according to chemical composition the eucalyptus camaldulensis eo generally can be divided in two different types. type i is a cineole-rich essential oil containing - % , -cineole plus pinene, and type ii is a cineole-poor essential oil, containing significantly less cineol (williams, ) . plant genotype is important factor influencing the final chemical composition of eo (djilani and dicko, ) . due to genetic and epigenetic factors same plant species can produce a similar eo, but with different chemical composition and therapeutic activities. brophy and southwell ( ) examined essential oils of two variations, eucalyptus camaledulensis var. camaldulensis and eucalyptus camaledulensis var. obtuse, and the main compounds of eucalyptus camaledulensis var. camaldulensis were pcymene ( %), cryptone ( %), and spathulenol ( %), while eucalyptus camaledulensis var. obtusa had different main compounds: , cinole ( %), α-pinene ( %), and aromadendrene ( %), suggesting that these subspecies belong to different types of eucalyptus essential oils. the content of , -cineole was also variable but in the same range for essential oils reported from greece ( . - . %) (tsiri et al., ) , pakistan ( . - . %) (ashraf et al., ) , mozambique ( . - . %) (pegula et al., ) , nigeria ( . - . %) (oyedeji et al., ) , and taiwan ( . - . %) (shieh, ) . similarly, the major component of eucalyptus camaldulensis oils originating form burundi, morocco, and benin was , -cineole ranging from . to . % with no presence of cryptone (dethier et al., ; zrira and benjilali, ; zrira et al., ; moudachirou et al., ) . cryptone has been shown to be present in low-cineole varieties of eos from australia (bignell et al., ) , uruguay (dellacassa et al., ) and south florida (pappas and sheppard-hanger, ) . for example, the major constituents identified in the essential oil from south florida included p-cymene ( . %), cryptone ( . %), terpinen- -ol ( . %), spathulenol ( . %), and cuminaldehyde ( . %), with a very low amount of , -cineole ( . %) (pappas and sheppard-hanger, ) . it was proven that essential oils of different plant parts have different chemical composition (table ) . previous studies on the essential oil of e. camaldulensis flowers revealed the presence of , -cineole, βpinene, and spathulenol as the most abundant constituents (giamakis et al., ) . the essential oil of the leaves was found to contain pcymene, γ-terpinene, α-pinene, , -cineole, terpinen- -ol, α-terpineol, carvacrol, and thymol as the major components (siramon and ohtani, ) . the major components of the fruits essential oil were aromadendrene, α-pinene, drimenol, and cubenol (el-ghorab et al., ) . plant developmental stage and maturity of plant parts used for essential oil extraction also affect essential oil chemical composition. giamakis et al. ( ) analyzed the immature flowers and calli grown in athens (greece), and they found that the main monoterpenes produced in e. camaldulensis calli, cultured in darkness and under light conditions, were , -cineole, . and . % as well as β-pinene, . and . %, respectively. in lower amounts α-pinene ( . and . %, respectively) and the terpenoids, camphene, myrcene, isocineole, myrtenol, bicyclogermacrene, spathulenol, and trans-pinocarveol were present (less than %). apart from isocineole, these constituents were also determined in the essential oil from immature flowers. this is remarkable because giamakis et al. ( ) showed that undifferentiated calli are capable of producing high amounts of monoterpenoid compounds (approx. one third of that produced by the explant). this makes immature flowers from e. camaldulensis an interesting candidate for the development of calli able to produce high percentages of these two important monoterpenes, , -cineole and β-pinene (giamakis et al., ) . also, the influence of light conditions on essential oil chemical composition showed that light conditions did not considerably affect the production of monoterpenes and sesquiterpenes compounds by calli developed from immature flowers (giamakis et al., ) . furthermore, plant culture conditions can influence the essential oil chemical composition. soil salinity is a key ecological stress that severely influences plant productivity (williams, ) . ashraf et al. ( ) showed that salinity had a considerable effect on the percent compositions of some components of e. camaldulensis leaves essential oil. the mean values of , -cineole content of eo from saline and nonsaline provenances of pakistan were . and . %, respectively (ashraf et al., ) . therefore, they recommend stressing of this species by growing in the saline areas to enhance essential oil production for its various medicinal and pharmacological uses. extraction represents the primary step in obtaining the crude mixture of compounds from plants. quality and quantity of the extracts dependent of the target compound structures, natural sources, and type of processes (karacabey et al., ) , explaining the different phenolic composition in the extracts obtained with different procedures. the most commonly plant extract have been obtained by conventional solvent extraction methods (infusion, decoction, digestion, maceration, and percolation) (azwanida, ) using solvents such as water, ethanol, methanol, chloroform, dimethyl-sulfoxide etc. however, these techniques are demanding regarding the extraction process duration, organic solvent consumption, and lack of extraction automation. the potential and powerful alternative to conventional liquid solvent extraction methods are ultrasonic-assisted extraction (uae) and microwave-assisted extraction (mae), especially in the case of plant material (hao et al., ; eskilsson and bjrklund, ) . interest in mae has increased significantly over the past - years in particular medicinal plant research, as a result of its inherent advantages as special heating mechanism, moderate capital cost, and its good performance under atmospheric conditions (ballard et al., ; chan et al., ) . in addition, mae technique possesses many advantages compared with other methods for the extraction of compounds such as bioactive compounds (sanchez-aldana et al., ) saving in processing time and solvent, higher extraction rate, better products with lower cost, reduced energy consumption (up to -fold savings), and waste generation (yan et al., ; yemis and mazza, ) . this is confirmed for e. camaldulensis extraction of phenolic and flavonoid compounds, where the compounds were extracted with mae for min which was equivalent with uae ( min) and traditional extraction ( h) methods (gharekhani et al., ) . many authors reported the chemical composition of e. camaldulensis extracts. the leaves of e. camaldulensis from the zoo-botanical garden in giza (egypt) yielded major fractions (singab et al., ) . the major components of the first fraction (eluted with water) were identified as hhdp-glucopyranose, chlorogenic acid, and phloroglucinol derivatives. the second % methanol fraction was found to contain different galloyl-hhdp-glucopyranose positional isomers and pedunculagin as major components. the third % methanol fraction was predominantly composed of digalloyl-hhdp-glucopyranose (tellimagrandin i) α and β anomers, while the last % methanol fraction was composed of a mixture of ellagitannin dimers. the profiling of the obtained fractions by hplc-pda-esi/ms/ms indicated that ellagitannins were the most predominant components of all three methanol fractions (singab et al., ) . the secondary metabolites screening of e. camaldulensis leaf extracts from nigeria confirmed presence of tannin, saponins, and cardiac glycosides (ayepola and adeniyi, ) . analyzed n-hexane, chloroform, and methanol extracts of e. camaldulensis stem bark and leaf also grown in nigeria showed the presence of tannins and saponins in the stem bark and in the leaf of e. camaldulensis with absence of alkaloids in all extracts (adeniyi et al., ) . furthermore, the crude methanol leaf extracts also from nigeria contained in addition volatile oils and balsam (gum) (babayi et al., ) . similarly, the phytochemical screening of ethanol, methanol, and petroleum ether leaf extracts from nigeria contained in moderate to high amount secondary metabolites: alkaloids, saponins, tannins, flavonoids, steroid, carbohydrates, and cardiac glycosides, and not anthraquinones (chuku et al., ) . crude methanol leaf extracts of e. camaldulensis from iran had saponins, tannins, volatile oils, and balsam (gum), while the components such as anthraquinones, hydrolysable tannin, flavonoid, alkaloid, and glycosides were not detected (jouki and khazaei, ) ; crude methanolic leaves extract of e. camaldulensis from india contained anthraquinones, flavonoids, saponins, and terpenoids, while alkaloids, cardiac glycosides, and tannins were not detected (singh and thakur, ) . phytochemical screening of the crude stem barks methanol extract of e. camaldulensis from bangladesh indicated presence of saponins, flavonoids, tannins, and also volatile oils, while anthraquinones, hydrolysable tannins, alkaloids, and glycosides were not present (islam et al., ) . the polyphenolic composition (flavonoids and phenolic acids and aldehydes) also was studied in the soluble fractions of the methanolic extracts of eucalyptus camaldulensis originating from two spain provinces, huelva and pontevedra: gallic, protocatechuic, vanillic and ellagic acids, and protocatechic aldehyde were identified, along with eriodictyol, quercetin, naringenin, vanillin, naringin, quercitrin, luteolin, and kaempferol (cadahia et al., ) . eucalyptus camaldulensis extracts are generally rich in tannins which vary qualitatively and quantitatively according to the origin of the samples, and consequently protoanthocyanidin levels were influenced by the geographical origin (cadahia et al., ) . antimicrobial activity of e. camaldulensis eo and extracts are well documented against many microorganisms listed in the table . for easier comparison, all data commented here for eos were re-calculated from microliter per milliliter or microgram/milligram per milliliter to percentage (v/v or w/v), using the equitations presented in fig. . we considered here only minimal inhibitory and minimal bactericidal concentrations, while results obtained using disc or agar diffusion methods were not discussed. however, mic/mbc results vary between several micrograms to several milligrams. such high variation does not seem as real, even taking into account variation in oil composition, and are rather a consequence of erroneous equalization of one microliter and one microgram, or typographical errors. for instance, there are some nonsense mics, such as μl/ml, that is practically impossible to obtain (salem et al., ) . in some manuscripts even a species was not precisely indicated (harkenthal et al., ; karpanen et al., ; warnke et al., ; tadtong et al., ) and these results were not considered in the present review. eucalyptus camaldulensis plant extracts and eos were tested against wide range of bacteria. the most frequently included gram positive bacterium in screening is s. aureus. minimal inhibitory concentrations in most studies were in range . - . % indicating moderately high activity against this bacterium. beside s. aureus, antibacterial effect was confirmed against b. subtilis ( . - . %), m. luteus ( . - . %), and s. pyogenes ( . - . %) akin et al., ; knezevic et al., ; khubeiz et al., ; reda et al., ; ostad asiaei et al., ) . activity of eos was examined aginst l. monocytogenes in only one study and mic was not obtained with the highest used concentration of . %, and the same was reported for enterococcus durans (akin et al., ) . activity against gram negative bacteria has been documented in a greater extent and mics for the most frequently used model organism e. coli was in range . - . %. sensitivity of other enterobacteria is similar, with mics in range from . to . % for k. pneumoniae (khubeiz et al., ; ostad asiaei et al., ) , . - . % for salmonella enterica subsp. enterica serovar typhimurium (khubeiz et al., ) ; . - . % for serovars typhi, paratyphi, and . % for s. enteritis (ostad asiaei et al., ). other enterobacteria are similarily sensitive: shigella soneii was inhibited with . % of eo (ostad asiaei et al., ) , while proteus vilgaris was sensitive in a broad range from . % (ostad asiaei et al., ) to more than . % (khubeiz et al., ) . the published data for p. aeruginosa are in the same range as for p. vulgaris, which is not surprising, as both species are generally highly resistant to antimicrobial agents. on the contrary, a. baumannii which is also known to be multi-drug resistant, showed sensitivity to eo in a range . - . % . the lowest mic among gram negative bacteria was recorded for a gastrointestinal pathogen v. parachaemoliticus ( . %) (khubeiz et al., ) . although the gram positive bacteria are consider more sensitive to eos in comparison to gram negative, it cannot be applied for essential oils of e. camaldulensis, since the most sensitive bacteria are gram negative -a. baumannii and v. parahaemolyticus. the minimal bactericidal concentrations for most bacteria are equal to, or rarely up to times greater than mics. this low range in mic/mbc < indicates that e. camaldulensis eos act as bactericidal agents (pankey and sabath, ) . antibacterial effects of e. camaldulensis extracts have been examined in a wider extent than essential oils. inhibitory concentrations varied depending on extraction method, plant properties, and model organism, being in broad range from . μg/ml to mg/ml. crude aqueous leaf extracts show lower activity against various bacteria (range - mg/ml) (abubakar, ) , while bark aqueous extracts were more effective, with mic from . mg/ml for propionobacterium acnes to . mg/ml for p. aeruginosa (mabona et al., ) . the antimicrobial activity of methanol, ethanol or petroleum leaf extracts of e. camaldulensis showed significant variation; for instance, mics against b. subtilis varied from . up to mg/ml or against s. aureus . - mg/ml (chuku et al., ; ayepola and adeniyi, ) . for most examined gram negative bacteria mics were in range - mg/ml, while p. aeruginosa was even more susceptible (mics - mg/ml). similar or better antibacterial effect showed acetone leaf extract, being active against examined bacteria in range - mg/ml. the highest activity was obtained with dichloromethen extracts, with mics for staphylococcus spp. in range . - . mg/ml, and even lower for b. subtilis, p. acnes and b. agri (mics . - . mg/ml). interestingly, generally highly resistant m. tuberculosis was sensitive to methanol, n-hexane or chloroform extract with mics in range . - . mg/ml, while m. bovis was slightly less sensitive with mics . - . mg/ml (lawal et al., ; gemechu et al., ) . the progresses made on the investigation of essential oils mode of action, especially against bacterial cell targets, gave new perspectives in this combat. the knowledge about the essential oils and their target(s) on bacterial cell is crucial to understand which parts of bacterial cell are affected. the essential oils antibacterial action is linked to oil hydrophobicity that increases cell permeability and consequent leakage of cell constituents (dorman and deans, ; lambert et al., ; helander et al., ; turgis et al., ; ultee et al., ; faleiro, ) . it is important to perceive that a disturbed cell structures may affect stability of other cellular structures in a cascade type of action (carson et al., ) . essential oils have several target sites on bacterial cells; however it seems that all are directly or indirectly connected to the primary effect of essential oils on the bacterial envelopes. first of all, eos may cause cell wall and membrane disturbance (lambert et al., ; oussalah et al., ) , which further can lead to the significant loss of intracellular atp (oussalah et al., ; turgis et al., ) , induction the synthesis of heat shock proteins (burt et al., ) , ph disturbance (turgis et al., ; oussalah et al., ) , and intracytoplasmic changes (e.g. coagulation, periplasmic space enlargement) (becerril et al., ) . although the number of studies dealing with the eo mechanisms of action is increasing, there are still many questions to be answered before the precise mechanism is revealed. this is at the same time one of the main limitations for eos and extracts wide usage as antimicrobials. thus, the so far knowledge must be further improved enabling the combat bacterial pathogens and its resistance. contemporary research of eos and extracts are also focused on mechanism that allows bacteria to regulate some physiological activities, such as virulence, competition amongst populations, motility, sporulation, conjugation, antibiotic production, and biofilm formation (rodriguez-garcia et al., ) . this system of intercellular communication, quorum sensing (qs), is based on production of the signal molecules, called autoinducers (abraham et al., ) . this communication system is relative to cell density and some compounds interfere with the qs communication system and attenuating the bacterial pathogenicity, in the phenomenon known as anti-qs compounds (abraham et al., ) . there are reports of anti-qs activity of various plants species eos from genus eucalyptus, such as eucalyptus globulus l. (luís et al., ; cervantes-ceballos et al., ) , eucalyptus radiata d. (luís et al., ) , eucalyptus citriodora hook., eucalyptus smithii r. t. baker and eucalyptus staigeriana f. muell. ex bailey (luís et al., ) . unfortunately, there is still no data regarding e. camaldulensis anti-qs activity. in one study, antibacterial and in vivo biofilm preventive efficacies of e. camaldulensis oil were significantly higher than that of m. spicata oil and chlorhexidine, suggesting that e. camaldulensis eo is capable of fig. . schematic diagram for re-calculation from microliter or milligram per milliliter to percentage (v/v or w/v). affecting biofilm formation . considering the anti-qs activity of above mentioned eucalyptus species and also detected antimicrobial and anti-biofilm effect of e. camaldulensis it can be assumed that it possess similar or even higher anti-qs activity. however, future detailed studies of anti-qs and anti-biofilm e. camaldulensis activity are needed in order to confirm this assumption. the e. camaldulensis leaves extracts and eos have a potential as antifungal agents. they are able to act as a moderate antifungal agent against household molds, wood rot fungi (siramon et al., ) , and phytopathogenic fungi (gakuubi et al., ; mehani et al., ) . eucalyptus camaldulensis eo has been studied for antifungal activity and was active in concentration . - . % against most model organisms. the most sensitive seems to be f. sporotrichioides with mic . % (mehani et al., ) , while r. oryzae is the most resistant, as % of eo was ineffective against this fungus (siramon et al., ) . the best known human and animal pathogenic yeast candida sp. showed considerable sensitivity to e. camaldulensis eo, with mic approx. . % (siramon et al., ; nasir et al., ) . most eos from sardinia inhibited growth of a. niger and b. cinerea in concentration of μl/plate, while lower concentrations of the oils, such as μl/plate were ineffective (barra et al., ) . it is interesting to notice that liposomes containing e. camaldulensis eo were prepared (moghimipour et al., ) for antifungal oil activity. the particle size varied from . to nm for the different formulations, with approx. % of the essential oil entrapped. inhibition of microsporum canis, m. gypseum, trichophyton rubrum, and t. verrucosum was achieved with μl, and the liposomal gel formulation of the eo was proposed to improve antifungal activity. aqueous and organic extracts of e. camaldulensis have been reported to have antifungal activity (table ). methanol leaves extracts showed inconsistent activity against c. albicans: in one study it was in range - mg/ml (chuku et al., ) , while in another it was . mg/ml (babayi et al., ) , indicating difference in active concentration of one thousand times. the bark methanole extract was active in concentration . mg/ml. methanol leaf and bark extracts showed considerable activity against dermatophytes: . - . mg/ml against microsporum spp, . - . mg/ml against trichophyton spp., and . mg/ml against epidemophyton flocossum (takahashi et al., ; falahati et al., ; mabona et al., ) . beside the antifungal activity of eos and extracts of e. camaldulensis, it is worth noticing that this species, along with e. blakelyi m., e. gomphocephala a. dc, e. rudis endl., and e. tereticornis sm., is a reservoir of an emerging pathogenic fungus -cryptococcus gattii. this fungus is usually related to tropic and subtropic regions, affecting the respiratory and nervous systems of the immunocompetent humans and domestic animals (sorrell et al., ; chakrabarti et al., ; bielska and may, ; roe et al., ) . similarily, c. neoformans var. grubii can be isolated from flowers and bark of e. camaldulensis (gugnani et al., ) . according to the literature, e. camaldulensis is natural reservoir of cryptococcus and is considered responsible for occurring cryptococcosis worldwide. in this context, it is interesting to observe that c. neoformans is moderately sensitive to e. citriodora hook eos, with mic . % (wt/v) (pattnaik et al., ; luqman et al., ) or e. globulus labill. with mic . % (wt/v) (suliman et al., ) . unfortunately, data on cryptococcus sp. sensitivity to eos and plant extracts of reservoir species of eucalyptus, including e. camaldulensis, remains unknown. although there are no reports regarding e. camaldulensis mode of antifungal action, according to previous studies with other essential oils and fungi, the plasma membrane and the mitochondria are the probable antifungal targets of eos. according to recent studies, the antifungal activity of eos results from its ability to disrupt the permeability barrier of the plasma membrane and from the mitochondrial dysfunction-induced ros accumulation in a. flavus and c. albicans (tian et al., ; chen et al., ) . the exact mechanism of e. camaldulensis antifungal activity is unknown and should be elucidated in the future. infections caused by viruses are very common and sometimes life threatening, especially in immunocompromised patients and neonates (snoeck, ; khan et al., ) . despite the recent significant progress in antiviral drug development, viral infections are considered as one of the major causes of death worldwide (müller et al., ; meyers et al., ) . thus, novel natural antiviral agents need to be found urgently. many natural products possess antiviral activity and some of them are already in use for treatment of human viral infections with both rna and dna viruses (e.g. myricetin against coronavirus, linalool, urosolic acid, and apigenin against coxsackievirus, quercetin and narasin against denge virus, curcumin against hepatitis b and c virus) (lin et al., ; kitazato et al., ) . numerous secondary plant metabolites such as essential oils, flavonoids, saponins, tannins, alkaloids, lignans, terpenes, and phenolic acids express significant antiviral activity against different viruses (jassim and naji, ; chiang et al., ; sanchez palomino et al., ) . recently few studies confirmed the e. camaldulensis eos and plant extracts antiviral activity (table ) . eucalyptus camaldulensis eos reduce coxackie b and rotavirus wa multiplication for %, herpes simplex virus for %, but have no effect on adenovirus multiplication (el-baz et al., ) . similarly, methanolic extracts showed % inhibition of hsv and in concentration . - . μg/ml, and against varicella zoster virus at concentration . μl/ml (abu-jafar and huleihel, ). dimethyl sulfoxide (dmso) extracts inhbit multiplication of animal new castle virus in concentration range - μg/ml (al-hadid, ) . antiviral activity has been observed for e. camaldulensis methanolic extracts against polio virus, coxsackie b, and echovirus (adeniyi et al., ) . the data on antiviral activity of e. camaldulensis eo and extracts, although scarce, indicate their great potential, and necessity for further studies in this context. despite the fact that many plant extracts and essential oils were previously reported for their antiviral activities, the mechanism of action still remains poorly understood. there are many factors that influencing the eos mode of action, which should be taken in consideration when antiviral activity of plant antimicrobials is examined. one of such factors is the difference between enveloped and non-enveloped viruses, because the observed antiviral effect has usually been greater for enveloped viruses (yamada et al., ; siddiqui et al., ) . in majority of the studies dealing with antiviral mode of action, the focus has been on either the inhibition of viral adsorption to host cells or examination of the plant antimicrobials effectiveness against intracellular virus multiplication (gilling et al., ) . so, most commonly described modes of antiviral actions are virus inactivation and the impaired virus adsorption to host cells, which is often difficult to distinguish. like in other antimicrobial modes of action, antiviral mechanism is also dependent on eos or extracts compounds activity. this is one more factor that should be considered, because it affects the final antiviral mechanism of action. for some compounds the potential mechanism is reported, i.e. carvacrol acts directly upon the virus capsid and subsequently the nucleic acid (gilling et al., ) . when eos antiviral mechanism of action is considered, eos may cause the loss of the viral capsid integrity, ultimately leading to exposure of the viral genome. in addition, some eos subsequently act directly upon the viral nucleic acid (dna or rna). according to one study, e. camaldulensis eos may be promising antiviral agent against rna viruses with no effect against dna virus (el-baz et al., ) . also, with shorter periods of exposure to the antimicrobial, the virus is able to adsorb specifically to host cells; however, it may or may not be able to cause successful infection depending upon the integrity of the viral genome. on the other hand, after exposure to some eos virus capsid and genome remains intact. these antimicrobials appear to exert their antiviral effect by coating the capsid and thereby preventing specific adsorption of the virus to host cells (gilling et al., ) . although, there are still no reports regarding e. camaldulensis eos antiviral mode of action, there are some promising results about its antiviral effect (table ) . these promising results represent foundation for further research and potential application of e. camalulensis eos as potent antiviral agent. among all deverse benefitial activities, e. camaldulensis expresess also an antiprotozoal effect as well. the reports regarding this effect are listed in table . eucalyptus camaldulensis methanolic and aqueous extracts were active against leishmania major with ic values ( % inhibitory concentration) . ± . and . ± . μg/ml, respectively (nosratabadi et al., ) . this was characterized as moderate leishmanicidal activity, but considering fact that present therapy consist antimony compounds which are expensive, toxic, and drug resistance is prevalent, e. camaldulensis plant extract derivatives represent safe, inexpensive, and promising alterantive solution. there are also several reports regarding antiprotozoal activity against trichomonas vaginalis, a causative agent of trichomoniasis which is the most prevalent nonviral sexual infection. the first report on e. camaldulensis aqueous extract anti-trichomonas activity showed that extract was active at concentration mg/ml after h (mahdi et al., ) . however, recent studies reported better effect of the e. camaladulensis extracts (hassani et al., ; youse et al., ) . five different e. camaldulensis leaves extracts including total extract, diethyl ether, chloroform, ethyl acetate, and water fractions were active in concentration range . - mg/ml, with growth inhibiton achived after - h (hassani et al., ) . ethyl acetate fraction showed the highest percentage of growth inhibition with the lowest concentration ( . mg/ml) after and h (hassani et al., ) . even better antitrichomonas activity was detedted for wather and ethanolic extracts, where concentration - μg/ml killed trichomonas vaginalis for h (youse et al., ) . the mainstay medication for trichomoniasis is metronidazole; however some resistant strains to this treatment have been detected making these results of e. camaldulensis anti-trichomonas activity very promising as antiprotozoal agent. except extracts, significant antiprotozoal activity was reported for e. camaldulensis eos. the essential oils were found to possess antitrypanosomal activity in vitro in a dose-dependent manner in a short time. the decrease of trypanosoma evansi number over time was achieved in doses of mg/ml for min, mg/ml for min, and mg/ml for min. against trypanosoma brucei brucei eos was more potent in the concentration of mg/ml, decreasing the number of parasite for min, mg/ml for min, and mg/ml for min (habila et al., ) . such prompt decrease in parasite number in the in vitro tests for both trypanosoma brucei brucei and t. evansisuggests that the eos kill the parasites efficiently, but by an unknown mechanism. there are suggestions of some potential mechanisms of antiprotozoal eos action. the activity of eos could be due to the hydrophobic nature of the cyclic hydrocarbons, which allow eos to interact with the protozoans causing conformational changes in the parasite membrane structure, resulting in the loss of membrane stability (calsamiglia et al., ) . the essential oils also can act by inhibiting some key enzymes in the parasite glycolytic pathway (smith-palmer et al., ) . furthermore, some eos components inhibit acetylcholinesterase activity and act on other vulnerable sites, such as cytochrome p (maciel et al., ) . this multicomponent nature of plant eos is an advantage for several target sites on protozoans, which is of great importance. it was shown that eos of e. camaldulensis are more potent against b. subtilis, s. aureus, e. coli, p. aeruginosa, s. lutea, and p. carotovorum in comparison to eos from e. gomphocephala dc., but not against a. tumefaciens. furthermore, the e. camaldulensis var. obtuse showed even better effect than e. camaldulensis (salem et al., ) . among seven examine essential oils of various eucalyptus species, eo of e. camaldulensis was among the best against b. subtilis, a. niger, and r. solani, but not against e. coli and s. aureus (ghaffar et al., ) . similar activity of e. camaldulensis and e. torelliana f. muell. was recorded for extracts against six strains of h. pylori (adeniyi et al., ) . it is worth to notice that among extracts from plants growing in southern africa, e. camaldulensis bark extract showed considerable activity against bacteria and fungi, with an exception against p. aeruginosa and m. canis (mabona et al., ) . finaly, in many studies regarding antimicrobial activity of various eucalyptus species, e. camaldulensis was not always included (ashour, ; safaei-ghomi and ahd, ; mulyaningsih et al., ; elaissi et al., ; sebei et al., ) . due to rapid emerging of microbial resistance to conventional drugs, the necessity for efficient solution(s) is rising. the main strategy represents finding new antimicrobial agents; however another strategy goes in the direction of reducing degree of bacterial resistance and/or bacterial re-sensitization to conventional antibiotics. this can be achieved using combined therapies. the most of the tested combinations are dual combinations, but the combinations of three, four or more agents also can be efficient (lesjak et al., ) . this is in line with antimicrobial efficiency and potential of eos and plant extracts which are complex mixtures of numerous compounds. combination strategy could be very promising regarding the diversity of agents that could be tested: ( ) conventional non-antimicrobial agent (e.g. anti-inflammatory or anti-psychotic drug) + conventional antimicrobial agent (antibiotic); ( ) conventional antimicrobial agent (antibiotic) + natural antimicrobial agent (essential oil, plant extract, bacteriophage, antimicrobial peptide ect.); ( ) conventional antimicrobial agent (antibiotic) + single compound isolated from natural antimicrobial agent (with previously confirmed antimicrobial activity); ( ) combination of two or more single compounds isolated from natural antimicrobial agents (with previously confirmed antimicrobial activity). being a plant with already detected and evaluated antimicrobial activity, it can be assumed that e. camaldulensis also have a potential in combined therapy. this assumption is confirmed in some studies in vitro and in vivo (table ) . eucalyptus camaldulensis eos and extracts in vitro reduced resistance of mdr a. baumannii in combination with conventional antibiotics: β-lactams, ciprofloxacin, gentamicin, polymyxin b . similarily, the increase of β-lactamase produing mrsa and e. coli sensitivity to cephalexin, cefuroxime, amoxicillin, and ampicillin has been obtained through combination with e. camaldulensis eos (chaves et al., ) . synergistic activity has been recorded between e. camaldulensis plant extracts and gentamicin or ceftriaxone against mdr s. aureus and p. aeruginosa (reda et al., ; ibrahim et al., ) , as well as in combination with ampicillin against s. aureus (ibrahim et al., ) . furthermore, the combination of e. camaldulensis extract with another plant extract psidium guajava l. was also efficient against mdr bacteria (bala et al., ) . except antibacterial activity, other activities of e. camaldulensis extracts in combination were detected and characterized as efficient. antiviral activity was confirmed for the combination of e. camaldulensis % methanol leaves extract and acyclovir against herpes simplex virus - and - and varicella-zoster virus (abu-jafar and huleihel, ). similaily, the combination of annona senegalensis l. leaf methanol extract and e. camaldulensis extract efficiently cured in vivo albino mice infection with parasite trypanosoma brucei brucei (lafia strain) (kabiru et al., ) . all these data are very promising, but the methods used in different studies and results interpretation vary (table ) . as a consequence, the data should be taken with precautions, as can not be compared and properly discussed. to avoid this problem, the testing combinations of different agents should be conducted using standardized methods, such as time-kill method (clsi, ; verma, ) , checkerboard method (verma, ; eucast, ) , chou-talalay method (chou, ) or boik method (boik, ) . all these methods possess some shortfalls, such as timeconsuming, labor-intensive, limitations regarding the number of the agents in combination, etc. unfortunately, there is no one gold standard for synergy testing and prior the further application of different phytochemicals, this issue should be overcome. summarizing the available data on antimicrobial properties of eucalyptus camaldulensis essential oil and extracts, it is obvious that this plant is a valuable source of phytotherapeutics. the essential oil, as well as leaf and bark extracts are particularly valuable as antibacterial, antiviral, and antifungal agents, and their antiprotozoal activity should not be neglected taking into account current therapy cost, toxicity, and protozoal growing resistance. some e. camaldulensis plant characteristics such as easy cultivation, wide distribution by plantation, and rapid growth additionally support further examination of antimicrobial activity, in order to enhance the commercial production of e. camaldulensis based pharmaceuticals. the future studies should be focused on determination of the mechanisms of antimicrobial activity, paticularly potential anti-biofilm and anti-qs effects, as well as the activity enhancement in combination with other available agents. effects of eucalyptus camaldulensis and other antimicrobial agents in combination. two-dimensional checkerboard method (verma, ) synergism (fici ≤ . ) knezevic et al. 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essential oil and determination of its chemical composition study of the synergistic effect of antibiotics and plant extracts against clinical staphylococcus aureus strains antiviral drug resistance as an adaptive process growth inhibition and apoptosis of ehrlich ascites carcinoma cells by the methanol extract of eucalyptus camaldulensis cypellocarpins a-c, phenol glycosides esterified with oleuropeic acid, from eucalyptus cypellocarpa growth habits of the eucalypts eucalypts for planting. eucalypts for planting novel antiviral agents: a medicinal plant perspective analgesic and anti-inflammatory effects of essential oils of eucalyptus aroma profile of eucalyptus globulus: collected from north west karnataka the antimicrobial activities of methanolic extracts of eucalyptus camaldulensis against bacillus subtilis, staphylococcus aureus and escherichia coli evaluation of the efficacy of combination therapy in t. b. brucei -infected mice using extracts of annona senegalensis and eucalyptus camaldulensis anti-trypanosomal potential of eucalyptus camaldulensis modeling solid-liquid extraction kinetics of trans-resveratrol and trans-e-viniferin from grape cane antimicrobial efficacy of chlorhexidine digluconate alone and in combination with eucalyptus oil, tea tree oil and thymol against planktonic and biofilm cultures of staphylococcus epidermidis evaluation of eucalyptus essential oil against some plant pathogenic fungi extracts and molecules from medicinal plants against herpes simplex viruses chemical compositions and antimicrobial activity of leaves eucalyptus camaldulensis essential oils from four syrian samples antibiotc susceptibility testing; a standard single disc method viral infectious disease and natural products with antiviral activity antimicrobial activity of eucalyptus camaldulensis essential oils and their interactions with conventional antimicrobial agents against multi-drug resistant acinetobacter baumannii antimicrobial properties of different eucalyptus oils antimicrobial activity of diosquinone and plumbagin from diospyros mespiliformis (hostch) (ebenaceae) a study of the minimum inhibitory concentration and mode of action of oregano essential oil, thymol and carvacrol in vitro susceptibility of mycobacterium tuberculosis to extracts of eucalyptus camaldulensis and eucalyptus torelliana and isolated compounds ulcer-healing promoting activities of methanol extracts of eucalyptus camaldulensis dehnh. and eucalyptus torelliana f binary and tertiary mixtures of satureja hortensis and origanum vulgare essential oils as potent antimicrobial agents against helicobacter pylori essential oils composition and antibacterial activities of eucalyptus camaldulensis dehn antiviral natural products and herbal medicines aromatherapy science: a guide for healthcare professionals chemical composition, antioxidant, antibacterial and anti-quorum sensing activities of eucalyptus globulus and eucalyptus radiata essential oils antimicrobial activity of eucalyptus citriodora essential oil antimicrobial activity of south african medicinal plants with dermatological relevance: from a screening approach, to combination studies and bioactive isolation chemical composition of eucalyptus spp. essential oils and their insecticidal effects on lutzomyia longipalpi alternative drugs against trichomonas vaginalis traditional use of medicinal plants in south-central zimbabwe: review and perspectives phytochemistry and larvicidal activity of eucalyptus camaldulensis against malaria vector, anopheles stephensi. asian pac antifungal effect of essential oil eucalyptus camaldulensis plant on fusarium graminearum and fusarium sporotrichioide multicenter collaborative trial of intravenous acyclovir for treatment of mucocutaneous herpes simplex virus infection in the immunocompromised host preparation and characterization of liposomes containing essential oil of eucalyptus camaldulensis leaf chemical composition of essential oils of eucalyptus from benin: eucalyptus citriodora and e. camaldulensis. influence of location, harvest time, storage of plants and time of steam distillation antibiograms of faecal escherichia coli and enterococci species isolated from pastoralist cattle in the interface areas of the kafue basin in zambia evaluation of antiviral activity of south american plant extracts against herpes simplex virus type and rabies virus synergistic properties of the terpenoids aromadendrene and , -cineole from the essential oil of eucalyptus globulus against antibiotic-susceptible and antibiotic-resistant pathogens firewood crops. shrub and tree species for energy production antimicrobial potential of the ethiopian thymus schimperi essential oil in comparison with others against certain fungal and bacterial species in vitro antileishmanial activity of methanolic and aqueous extracts of eucalyptus camaldulensis against leishmania major eucalyptus camaldulensis var. nancy and eucalyptus camaldulensis var. petford seed essential oils: phytochemicals and therapeutic potentials evaluation of antimicrobial activity of eucalyptus camaldulensis essential oil against the growth of drug-resistant bacteria antimicrobial effects of selected plant essential oils on the growth of a pseudomonas putida strain isolated from meat antimicrobial characteristics of some herbal oils on pseudomonas aeruginosa with special reference to their chemical compositions antimicrobial activity of essential oils of five eucalyptus species growing in nigeria clinical relevance of bacteriostatic versus bactericidal mechanisms of action in the treatment of gram-positive bacterial infections the essential oil of eucalyptus camaldulensis dehn. from south florida: a high cryptone/low cineole eucalyptus antibacterial and antifungal activity of tan essential oils in vitro essential oil composition of eucalyptus camalddunensis uses and abuses of plant-derived smoke: its ethnobotany as hallucinogen, perfume, incense and medicine antimicrobial activity of eucalyptus camaldulensis and calotropis gigantea against various pathogens the effect of mentha spicata and eucalyptus camaldulensis essential oils on dental biofilm synergistic effect of combined antibiotic and methanol extract of eucalyptus camaldulensis leaf against staphylococcus aureus and pseudomonas aeruginosa molecular evolution of antifungal drug resistance gap, an aequorin-based fluorescent indicator for imaging ca + in organelles dating the cryptococcus gattii dispersal to the north american pacific northwest antimicrobial and antifungal properties of the essential oil and methanol extracts of eucalyptus largiflorens and eucalyptus intertexta chemotyping of diverse eucalyptus species grown in egypt and antioxidant and antibacterial activities of its respective essential oils screening of south american plants against human immunodeficiency virus: preliminary fractionation of aqueous extract from baccharis trinervis comparative extraction of pectic and polyphenols from mexican lime pomace and bagasse chemical composition and antibacterial activities of seven eucalyptus species essential oils leaves studies on the antidandruff activity of the essential oil of coleus amboinicus and eucalyptus globulus yields and chemical components of essential oils in eucalyptus camaldulensis leaves effect of essential oils on the enveloped viruses: antiviral activity of oregano and clove oils on herpes simplex virus type and newcastle disease virus phenolic constituents of eucalyptus camaldulensis dehnh, with potential antioxidant and cytotoxic activities antibacterial activity of eucalyptus camaldulensis against bacteria causing food-borne illness antioxidative and antiradical activities of eucalyptus camaldulensis leaf oils from thailand chemical composition and antifungal property of eucalyptus camaldulensis leaf oils from thailand river red gum. eucalyptus camaldulensis var. obtusa. centre for plant biodiversity research influence of subinhibitory concentrations of plant essential oils on the production of enterotoxins a and b and α-toxin by staphylococcus aureus antiviral therapy of herpes simplex natural environmental sources of cryptococcus neoformans var. gattii validating the in vitro antimicrobial activity of artemisia afra in polyherbal combinations to treat respiratory infections antimicrobial constituents and effects of blnded eucalyptus, rosemary, patchouli, pine, and cajuput essential oils antimicrobial activities of eucalyptus leaf extracts and flavonoids from eucalyptus maculata phenylpropanoid glycosides from lantana camara and lippia multiflora effects of supplementation of eucalyptus (e. camaldulensis) leaf meal on feed intake and rumen fermentation efficiency in swamp buffaloes the mechanism of antifungal action of essential oil from dill (anethum graveolens l.) on aspergillus flavus essential oil safety: a guide for health care professionals composition of fruit volatiles and annual changes in the volatiles of leaves of eucalyptus camaldulensis dehn. growing in greece antimicrobial activity of mustard essential oil against escherichia coli o :h and salmonella typhi the phenolic hydroxyl group of carvacrol is essential for action against the food-borne pathogen bacillus cereus the antibiotic resistance crisis: part : causes and threats methods for determining bactericidal activity and antimicrobial interactions: synergy testing, time-kill curves, and population analysis the battle against multi-resistant strains: renaissance of antimicrobial essential oils as a promising force to fight hospital-acquired infections the medicinal and poisonous plants of southern and eastern africa medicinal plants in australia volume : gums, resins, tannin and essential oils. mechanism of the antiviral effect of hydroxytyrosol on influenza virus appears to involve morphological change of the virus optimisation of the microwave-assisted extraction process for four main astragalosides in radix astragali optimization of furfural and -hydroxymethylfurfural production from wheat straw by a microwave-assisted process effect of echinophora platyloba, stachys lavandulifolia, and eucalyptus camaldulensis plants on trichomonas vaginalis growth in vitro in-vitro sensitivity pattern of some urinary tract isolates to carica papaya extracts the essential oil of the leaves and the fruits of e. camaldulensis essential oils of twentyseven eucalyptus species grown in morocco key: cord- -c wnr xe authors: gürsoy, elif; dincel, efe doğukan; naesens, lieve; ulusoy güzeldemirci, nuray title: design and synthesis of novel imidazo[ , -b]thiazole derivatives as potent antiviral and antimycobacterial agents date: - - journal: bioorg chem doi: . /j.bioorg. . sha: doc_id: cord_uid: c wnr xe a series of novel acyl-hydrazone ( a-d) and spirothiazolidinone ( a-d, a-d) derivatives of imidazo[ , -b]thiazole were synthesized and evaluated for their antiviral and antimycobacterial activity. the antituberculosis activity was evaluated by using the microplate alamar blue assay and the antiviral activity was evaluated against diverse viruses in mammalian cell cultures. according to the biological activity studies of the compounds, a-c displayed hope promising antitubercular activity, d was found as potent for coxsackie b virus, d was found as effective against feline corona and feline herpes viruses. consequently, the obtained results displayed that, a-d and d present a leading structure for future drug development due to its straightforward synthesis and relevant bioactivity. lately, much interest has been focused on the chemistry and the biological activity of fused heterocyclic systems because of their broad spectrum of physiological activities [ , ] . among these heterocyclic compounds carrying nitrogen atom, imidazo [ , -b] thiazole derivatives possess specific importance because of their diverse pharmacological activities. the imidazo [ , -b] thiazole derivatives have been reported in the literature as antibacterial [ , ] , antitubercular [ ] , antifungal [ ] , antitumoral [ ] , antiviral [ , ] , antihelmintic [ ] , analgesic [ ] , anti-inflammatory [ ] , antihypertensive [ ] , cardiotonic [ , ] , diuretic [ ] , herbicide [ ] and insecticide [ ] agents. levamisole [( s)- , , , -tetrahydro- -phenylimidazo[ , -b]thiazole] (fig. ) , which is the levogyre isomer of antihelminthic tetramisol and contains imidazo [ , -b] thiazole moiety, is a drug that has significant immunomodulatory properties [ ] in addition to its antihelminthic activity [ ] . besides the wide biological activity spectrum of imidazo[ , -b] thiazole derivatives, also the compounds bearing hydrazide, acyl-hydrazone and spirothiazolidinone moiety, have been reported in the literature with their various effects such as antibacterial [ ] , antifungal [ ] , antitubercular [ ] , antiviral [ ] , anticonvulsant [ ] and antidepressant [ ] . on the other hand, the two compounds bmy- and bms- , which are known in the literature for their antiviral activity and especially for their effects on influenza virus, have attracted the attention of researchers as compounds, that has a specific chemical character by an amide structure which connects an aliphatic cyclic system with an aromatic system [ , ] . in this study, we further explored the scaffold containing the imidazo [ , -b] thiazole ring as the aromatic moiety, that is linked by an amide to a spirothiazolidinone ring system as the aliphatic cyclic moiety and from this point forward, novel derivatives were synthesized (table ) , and broadly evaluated for their antiviral and antimycobacterial activity (fig. ) . two hit compounds were found to inhibit feline coronavirus or coxsackie b virus. besides, we observed some activity against mycobacterium tuberculosis, suggesting the versatility and relevance of this compound class to achieved anti-infective agents. compound was obtained according to the procedure described by robert et al [ ] . compound was obtained according to the procedure described by robert et al [ ] . compound was obtained according to the procedure described by harraga et al [ ] . general procedure for the synthesis of -( -bromophenyl)-n -(substituted/non-substituted cycloalkylidene)imidazo[ , -b]thiazole- -acetohydrazides ( a-d) , mol of was boiled in a water bath under reflux with ml of ethanol until a clear solution was obtained. . mol of cyclic ketone was added and heated for h. after cooling the mixture to room temperature, it was filtered and purified by crystallization with warm ethanol or washing. [ , -b] to a suspension of . mol of a-d in ml of anhydrous benzene is added ml of mercaptoacetic acid / -mercaptopropionic acid. the reaction mixture is heated under a reflux condenser using a dean-stark trap in a water bath for h. it is concentrated under reduced pressure and the excess acid is neutralized with nahco solution. the resulting product is kept in the refrigerator until solidified. the crude product is filtered, washed with water, dried and purified by crystallization or elution with ethanol. white solid, mp - °c, yield: . %. anal. calcd. for c white solid, mp, - °c, yield: . %. anal. calcd. for c white [ . ] [ , ] the antimycobacterial activity studies of the compounds were performed by the taacf (tuberculosis antimicrobial acquisition and coordinating facility by the national institute of health of the us government). primary screening was conducted at . µg/ml against mycobacterium tuberculosis h rv in bactec b medium using a broth microdilution assay the microplate alamar blue assay (maba) [ ] . compounds exhibiting fluorescence were tested in the bactec radiometric system. compounds affecting less than % inhibition in the primary screen were not generally evaluated further. compounds demonstrating at least % inhibition in the primary screen were retested at lower concentrations against m. tuberculosis h rv in order to determine the actual minimum inhibitory concentration (mic) using maba. rifampin was utilized as the standard compound in the assays and each assay was replicated four times. the mic was defined as the lowest concentration affecting a reduction in fluorescence of % relative to controls. concurrently with the determination of mics, compounds were tested for cytotoxicity (ic ) in vero cells at concentrations ≤ . µg/ml or times the mic for m. tuberculosis h rv (solubility in media permitting). after h exposure, viability was assessed on the basis of cellular conversion of -( , -dimethylthiazol- yl)- , -diphenyl tetrazolium bromide (mtt) into a formazan product using the promega celltiter non-radioactive cell proliferation assay. compounds for which the selectivity index ic :mic ratio) si > were assumed to possess in vitro activity confirmed in the bactec at . µg/ml. alamar blue susceptibility assay (maba). antimicrobial susceptibility testing was performed in black, clear-bottomed, -well microplates (black view plates; packard instrument, meriden, connecticut, usa) in order to minimize background fluorescence. outer perimeter wells were filled with sterile water to prevent dehydration in experimental wells. initial drug dilutions were prepared in either dmso or distilled deionized water, and subsequent twofold dilutions were performed in . cm of h gc (no tween ) in the microplates. bactec b-passaged inocula were initially diluted : h gc, and . cm was added to wells. subsequent determination of bacterial titers yielded × , . × and . × cfu cm − in plate wells for m. tuberculosis h rv. frozen inocula were initially diluted : in bactec b medium followed by a : dilution in h gc. addition of . cm to wells resulted in final bacterial titers of . x and x cfu cm − for h rv. wells containing drugs only were used to detect autofluorescence of compounds. additional control wells consisted of bacteria only (b) and medium only (m). plates were incubated at °c. starting at day of incubation, mm of x alamar blue solution (alamar biosciences/accumed, westlake, ohio, usa) and . mm of % tween were added to one b well and one m well, and plates were reincubated °c. wells were observed at and h for a color change from blue to pink and for a reading of ≥ , fluorescence units (fu). fluorescence was measured in a cytofluor ii microplate fluorometer (perseptive biosystems, framingham, massachusetts, usa) in bottom-reading mode with excitation at nm and emission at nm. if the b wells became pink by h, the reagent was added to the entire plate. if the well remained blue or , ≤fu was twofold drug dilutions were prepared in either dmso (sigma) or distilled deionized water and delivered via a . -cm insulin syringe in a -mm volume. drug-free control vials consisted of solvent with bacterial inoculum and solvent with a : dilution of bacterial inoculum ( : controls). vials were incubated at °c, and the gi was determined in a bactec instrument (becton dickinson) until the gi of the : controls reach at least . all vials were read the following day, and the gi and daily changes in gi (Δgi) were recorded for each drug dilution. the mic was defined as the lowest concentration for which the Δgi was less than the Δgi of the : control. if the gi of the test sample was greater than , the sample was scored as resistant even if the Δgi was less than the Δgi of the : control. the synthesized compounds ( a-d, a-d and a-d) were evaluated against diverse rna and dna viruses, using the following cell- to perform the antiviral assays, the virus was added to subconfluent cell cultures in -well plates, and at the same time, the test compounds were added at serial dilutions. appropriate reference compounds were included such as the virus entry inhibitors urtica dioica agglutinin lectin and dextran sulfate; broad virus inhibitors ribavirin and mycophenolic acid; antiherpetic drugs ganciclovir and cidofovir; and hiv inhibitor azidothymidine and nevirapine. after - days incubation at °c (or the target compounds a-d, a-d and a-d were synthesized from -( -( -bromophenyl)imidazo [ , -b] thiazol- -yl)acetohydrazide by a four and five-step synthesis through the pathways shown in fig. . -bromophenacyl bromide and ethyl -aminothiazole- -acetate were dissolved in acetone and mixed in room temperature. after standing for days, amino- -[( -bromobenzoyl)methyl]- -(ethoxycarbonylmethyl)thiazolium bromide ( ) was obtained [ ] . thereafter, compound was boiled under reflux in absolute ethanol and via the ring closure of compound , ethyl [ -( -bromophenyl)imidazo[ , -b]thiazole- -yl]acetate hydrobromide ( ) was obtained [ ] . by heating the compound and hydrazine hydrate in ethanol, -[ -( -bromophenyl)imidazo[ , -b]thiazole- -yl]acetohydrazide ( ) was obtained [ ] . the compound and cyclic ketones were heated under reflux in ethanol to yield -( -bromophenyl)-n -(substituted/non-substituted cycloalkylidene)imidazo [ , -b]thiazole- -acetohydrazides ( a-d). the reaction yields of compounds a-d were between the interval of %- . %. a-d were then reacted with mercaptoacetic acid / -mercaptopropionic acid in anhydrous benzene under reflux by using a dean-stark water separator. the obtained reaction mixture was eventually neutralized with nahco solution and by that way -[ -( -bromophenyl)imidazo[ , -b]thiazol- yl]-n-( -nonsubstituted/methyl- , , -nonsubstituted/alkyl/aryl- -oxo- -thia- -azaspiro [ . ] non/ [ . ] dec- -yl]acetamides ( a-d, a-d) were obtained. the reaction yields of the compounds a-d and a-d were between the interval of . %- . % and %- %, respectively. when the chemical structures of the synthesized compounds were examined, it was observed that the carbonyl group was not present in the compounds a-d whereas the same group was present in the spirothiazolidinone ring of the compounds a-d and a-d. also according to the ir spectroscopy results, the stretching band belonging to the carbonyl group was not visible in the spectrum of the compounds a-d whereas the mentioned band was able to be observed in the range of - cm − for a-d and a-d. the ir values belonging to the carbonyl groups of spirothiazolidinone ring of a-d and a-d were in agreement with the literature [ , ] . in the h nmr analysis, the nh protons of compound , which were observed as a broad singlet peak ( h) at . ppm, was disappeared in compounds a-d, whereas the peaks of aliphatic ch groups belonging to cyclic ketone arose as multiplet in the range of . - . ppm. when the results obtained from compounds a-d examined, it was determined that the peaks of s-ch protons were added to the spectrum in the range of . - . ppm as a distinctive indicator related to the formation of the mentioned compound [ , , ] . also from the h nmr analysis data obtained from compounds a-d, it was determined that the s-ch and ch-ch peaks of the spirothiazolidinone ring were in the range of . - . and . - . ppm, respectively and the values were in compliance with the literature [ , ] . the ir, h nmr, c nmr and ms spectra of the novel compounds are in agreement with the assigned structures. no unacceptable side reactions were observed, and products were obtained in moderate to good yields. the broad antiviral evaluation demonstrated that compound d was quite effective against feline coronavirus in crfk cells (table ) , with an antiviral ec value of . µg/ml and a superior selectivity index (ratio of cytotoxic to antiviral concentration) higher than . d was four-fold less active against feline herpesvirus. similarly, two compounds ( c and d) had weak anti-dna virus activity in hel cells, with antiviral ec values about µg/ml ( table ) . the most effective compound against feline coronavirus was d. when the sar analysis of d is evaluated by comparison with the other compounds, it can be understood that the existence of the structure of the spirothiazolidinone moiety is significant in the activity against the mentioned virus. it can also be observed that the spirothiazolidinone structure of d is separated from b-c by the -hydroxyphenyl substituent at the -position, and thus it can be concluded that the corresponding substituent is significant in the activity of the compound. additionally, the -position of the spirothiazolidinone structure separates d from b-d, indicating that the nonsubstitution of position is crucial for the antiviral activity against the feline coronavirus. the results also displayed that, the compounds bearing spirothiazolidinone structure did not show activity against dna viruses, whereas compound c and d, which are carrying acyl-hydrazone moiety, showed activity against dna viruses. the obtained result displayed the importance of the existence of the acyl-hydrazone residue for the activity against dna viruses. also, -phenylcyclohexyl and -( hydroxyphenyl)cyclohexyl derivatives of acyl-hydrazones have been found to further increase the activity against dna viruses. another hit compound, d, was somewhat active against coxsackie b virus (ec : µg/ml and selectivity index ≥ (table ) ). the antimycobacterial evaluation of compounds ( a-c, a-c) was performed. the results belonging in vitro primer analyses, that were performed against mycobacterium tuberculosis h rv strain by using maba in bactec b media, were given in table . according to the in vitro primer analyses, the compounds possessing any value where the mic value was equal to less than µg/ml were considered as active for antimicrobial activity and further analysis of the mentioned compounds were conducted. from this point forward compounds a-c were not detected as active and no further antimycobacterial activity was performed for them. in contrast to compounds a-c, compounds a-c were detected as active and further antimycobacterial activity of these compounds was conducted and the results were displayed in table . the obtained results displayed that, a-c, the acyl-hydrazone moiety containing imidazo[ , -b]thiazole derivatives, showed no or weak antitubercular activity and the ring closure raised the antitubercular activity of the compounds as can be seen from the results related to the compounds a, b, and c. the fact that the conversion of acyl-hydrazone derivatives to spirothiazolidinone derivatives by ring closure increased the activity, indicates the importance of the existence of spirothiazolidinone structure for antitubercular activity. antibiotic resistance is rising to high levels sharply all over the world and new kinds of resistance mechanisms are emerging worldwide. infections such as pneumonia, tuberculosis, gonorrhea, blood poisoning are becoming harder to treat and current antibacterials are losing their effectivity day by day. designing of new effectual compounds to deal with these resistant bacterias has become one of the most important issues today. similarly, mycobacterium tuberculosis remains a leading infectious cause of death worldwide today. especially the evolution of multi-drug-resistant (mdr) strains of mycobacterium tuberculosis, is the main reason for the increased incidence of tuberculosis, therefore, the development of new antimycobacterial agents has become an obligation. another important is that needs new drug candidates are antiviral therapy and it is definitely necessary to design and develop new effective antiviral agents. in the present study, in an attempt to find novel antiviral and antitubercular agents, diverse derivatives of imidazo[ , -b]thiazole were designed and synthesized. the compounds were screened for their antiviral and antitubercular activity. the antimycobacterial activities of the compounds were tested against the mycobacterium tuberculosis h rv strain and in the test method, each value, in which the mic value was equal to less than µg/ml, was actively accepted for antimycobacterial activity. the mic values of the compounds a, b, and c were determined as . µg/ml, . µg/ml and . µg/ml and the molecules were found as active. also, compound d was found as effective for coxsackie b virus. the antiviral activity and cytotoxicity of the compounds against feline corona and feline herpes viruses were investigated in crfk cell cultures and in comparison with hha, uda and ganciclovir references, compound d was found as highly effective. the findings revealed the promising antiviral and antitubercular activity of acyl-hydrazone, spirothiazolidinone and -methyl-substituted spirothiazolidinone derivatives of imidazo [ , -b] thiazole and these derivatives could be an interesting starting point for further structural optimization to obtain new promising and more potent antiviral and antitubercular agents. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. the levamisole story the authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discusses in the manuscript. this includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.no writing assistance was utilized in the production of this manuscript. the authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the declaration of helsinki for all human or animal experimental investigations. supplementary data to this article can be found online at https:// doi.org/ . /j.bioorg. . . key: cord- -m wodqr authors: yuan, lvfeng; zhang, shuai; peng, jie; li, yuchen; yang, qian title: synthetic surfactin analogues have improved anti-pedv properties date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: m wodqr surfactin has antiviral activity against various enveloped viruses by inhibiting viral membrane fusion. however, the potential utility of surfactin as an antiviral drug is limited by its cytotoxicity. in this study, surfactin analogues were obtained by chemical synthesis and evaluated to determine their anti-pedv activities, hemolytic activities, and critical micelle concentrations. the main goal of our study was to develop a safer drug; a surfactin analogue with high anti-pedv activity and low hemolytic activity. compared with surfactin, one of the analogues we developed, slp , has lower hemolytic activity, with the same antiviral activity. the selectivity index of slp is , while the si for surfactin is , in other words, the safe and effective concentration range of slp is times greater than that of surfactin. like surfactin, slp has a direct antiviral effect on pedv. structurally, slp is a linear lipopeptide with three carboxyl groups. surfactin derivatives similar to slp could be obtained by lactone bond hydrolyzation of surfactin, as well as total synthesis. surfactin has antiviral activity against a variety of enveloped viruses, including herpes simplex virus (hsv- , hsv- ), vesicular stomatitis virus (vsv), simian immunodeficiency virus (siv) and newcastle disease virus (ndv) [ , ] . we recently demonstrated that surfactin exerts its antiviral effects by inhibiting viral membrane fusion [ ] . membrane fusion between the viral envelope and the cell membrane is essential for enveloped viruses to invade host cells. surfactin can act directly on virus particles by insertion into the viral envelopes' lipid bilayer and thereby reduce the membrane fusion rate. in addition, since the lipid components of viral envelopes are provided by the host cell, their composition, structure, and function are widely similar in the enveloped viruses. surfactin has antiviral activity against numerous enveloped viruses, and it has promise as a broad-spectrum antiviral reagent, however, the effective dose range of surfactin is narrow, merely x the antiviral concentration causes hemolysis and cytotoxicity. in this study we compared chemically synthesized surfactin analogues to determine future directions for surfactin modification. a a a a a surfactin is a cyclic lipopeptide naturally produced by various strains of bacillus subtilis, the structure consists of a seven amino acid peptide loop and a hydrophobic fatty acid chain. the production of designer surfactins, made by changing the number and composition of amino acids and fatty acids has proven to be an effective strategy for screening large numbers of lipopeptides for biological activity, but most current research focuses on their anticancer [ ] , antimicrobial [ ] and insulin delivery [ ] properties but not on their antiviral potential. fatty acid chain length is critical to the antimicrobial effect of synthetic polymyxin analogues [ ] , while the specific antimicrobial spectrum of these analogues depends the amino acids in the lipopeptide [ ] . additionally, in synthetic daptomycin analogues, the introduction of aromatic groups in the fatty acid moiety also affects antibacterial activity [ ] . in this study, a series of similar lipopeptides were designed and synthesized using surfactin as a template. these analogues differ from each other in the number of hydrophobic amino acids, number of hydrophilic groups, charge properties, amino acid chirality, position of hydrophilic amino acids, and aromatic groups in the fatty chain. porcine epidemic diarrhea virus (pedv), a coronavirus, can infect pigs of all ages, but is especially virulent in newborn piglets, causing diarrhea, dehydration and even death [ ] . outbreaks of pedv have been reported in many countries [ ] [ ] [ ] [ ] , and have caused immeasurable losses to the global swine industry. recent research in our lab showed that surfactin acts directly on the pedv envelope and inhibits the fusion process with the host cell membrane. in this study, pedv was used as a target to screen chemically synthesized surfactin analogues for enhanced antiviral properties. our results provide a theoretical basis for the development of new surfactin-derivative antiviral drugs. all lipopeptides were synthesized by synpeptide co., ltd (shanghai, china). mass spectrometry was used to determination molecular weight and to confirm product sequences. the purity of all synthetic lipopeptide samples was over % by hplc. ten synthetic lipopeptide samples were named slp to slp . and their sequences as table . cercopithecus aethiops kidney epithelial cells (vero, atcc, ccl- ) were cultured in high glucose dmem (gibco, us), supplemented with % fetal bovine serum (fbs, gibco), at c in a % co humidified atmosphere. cells were routinely seeded at a density of × /ml vero cells were seeded at × cells/well in -well tissue culture plates and incubated - h at ˚c until approximately % confluency was reached. pfu of pedv mixed with an equal volume of slp in dmem or dmem alone were incubated minutes at ˚c, then added into the wells of the vero cells and incubated minutes at ˚c. cells were washed times with dmem then overlaid with dmem/ % agar and incubated hours at ˚c. the cells were fixed with % formaldehyde, then stained with . % crystal violet after the agar overlay was removed. the data are representative of biological replicates and each plaque assay was performed in triplicate and values are expressed as means ± the standard deviation. curve fitting and ec were calculated with graphpad prism using the log[inhibitor] vs. response equation. hemolytic activity was measured according to the methods described in jingdan [ ] , with some modifications. briefly, serial dilutions of slps were added to ul of a % suspension of porcine rbcs in pbs, followed by incubation for h at ˚c. cells were centrifuged at g for min, then ul of each supernatant was transferred to wells of a -well plate. the optical density at nm was measured using a microplate reader (infinite pro, tecan group ltd., switzerland) and % hemolysis was calculated using the formula: where od s , od b , and od p represent the optical density of the slp-treated samples, negative control and positive control, respectively. % triton x- was used for the positive control. each sample was run in triplicate. surface tensions were measured using the ring method [ ] . samples were freshly prepared in a testing flask and allowed to stand for minutes at ˚c. surface tension measurements were made using a surface tensiometer (hld-lst-ii, henlida co., ltd, china). linear regressions of the drop and the flat area were performed separately for the surface tension-concentration curve. the concentration at the intersection of the two lines is the critical micelle concentration (cmc). time of addition assay was performed according to the procedures described [ ] , with modifications. confluent vero cells in -well plates were infected with pfu of pedv and incubated for hr at ˚c to synchronize infection. the inoculum was removed and ml of ˚c dmem was added to each well, cells were then placed at ˚c in a humidified incubator. at the indicated time points, slp , surfactin, or dielaidoyl-phosphatidylethanolamine (depe, avantipolarlipids, alabaster, al) was added dropwise to cells or virus to a final concentration of μg/ml, μg/ml, or μg/ml and incubated a further hours at ˚c. viral nucleic acid and protein levels in the cells were measured by qrt-pcr and western-blot respectively. the data shown is representative of independent experiments. total rna was extracted from cells using trizol reagent (invitrogen) according to the manufacturer's instructions. cdna was generated by reverse transcription using hiscript tm qrt supermix for qpcr (vazyme) according to the manufacturer's instructions. pedv nucleic acid levels were assessed by measuring the viral nucleoprotein (n) using qrt-pcr with the takara sybr green qpcr kit (takara). primer sequences were as follows: gene level was calculated using the comparative ct method and normalized to the endogenous levels of gapdh. boiled cell lysates were subjected to sds-page then transferred to pvdf membrane (roche, basel, switzerland) using a semi-dry transfer apparatus (ge, little chalfont, buckinghamshire, uk). membranes were blocked in % non-fat milk in tbs containing . % tween- (tbst) then incubated overnight at ˚c with anti-pedv n-protein monoclonal antibody (median diagnostics, south korea) diluted in tbst with % bsa. membranes were washed min in tbst followed by incubation for h at rt with : hrp goat anti-mouse igg antibody (abgent, san diego, ca, usa) then thrice washed for min in tbst. membranes were incubated with ecl reagent (thermo, waltham, ma, usa) and imaged using a chemidoc™ system (bio-rad, hercules, ca, usa). native surfactin is an amphiphilic cyclic lipopeptide, it consists of a heptadine interlinked with fatty acid. using the native structure as a template, we designed surfactin analogues that were then commercially synthesized ( fig a) . slp consists of a heptadiene identical to the peptide of native surfactin, and a fatty acid chain the same length as in native surfactin. the n' of heptadine is linked to the fatty acid and the c'-is aminated. slp and slp have one and two fewer hydrophobic amino acids respectively, than slp . relative to spl , slp has one fewer hydrophilic amino acids, while slp has a free carboxyl at its c'-end that acts as an additional hydrophilic group. slp replaces the two acidic amino acids of slp with lysine (a basic amino acid). slp is a version of slp that contains only l-amino acids. the two hydrophilic amino acids of slp are distant from each other, but are in close proximity in slp . slp is identical to slp except for a diphenyl group in the fatty acid moiety. the mass spectra of the slps are shown in fig b and are consistent with the expected molecular weights. in all cases, slp purity exceeded %. the slps were tested for anti-pedv activity using a plaque reduction assay. serial dilutions of slps were incubated with an equal volume of pedv then aliquoted onto precooled cells in -well plates. after the virus has adsorbed to the cells at ˚c, slps were washed away. the adsorbed virus particles were then counted by plaque forming assays (fig a) . the results were fitted to a sigmoid-curve and plotted fig b. all slps have potent anti-pedv effects above μg/ml, but differences in their anti-pedv effect. slp , slp , slp , and slp have a higher potency than surfactin. hemolysis is commonly used to assess the biosafety of lipopeptides [ , ] . as shown in fig , slps have a range hemolytic activities. those of slp , slp , slp , and surfactin are similar, suggesting that the number of hydrophobic amino acids has little effect on hemolytic activity. however, other modifications significantly affect activity; slp , slp and slp have one, two and three carboxyl groups respectively, and the hemolytic activity is reduced in turn. in addition, the chiral differences that distinguish slp and slp at two amino acid residues, and the differences in the arrangement of amino acids between slp and slp also affect hemolytic activity. these results indicate that amino acid composition by itself does not completely determine the characteristics of slps. it is noteworthy that slp has an unexpected effect on porcine erythrocytes. although slp has a low hemolytic activity, it prevents the precipitation of red bloods under our assay conditions at concentrations above μg/ml. the selectivity index (si) was determined as the ratio of the % cytotoxicity concentration (cc ) to the % effective concentration (ec ). as shown in table , slp and slp have the highest and second-highest si values respectively. given the anti-sedimentation effect of slp on porcine blood cells, slp was selected as the most promising candidate. although slp and slp have stronger antiviral activity than surfactin, their si is lower due to their high hemolytic activity. the cmc was calculated by measuring the surface tension of each slp over a range of concentrations. as shown in fig , surface tension decreases as slp concentration increases, and as it approaches its lowest value, the curve exhibits an inflection point when the slp concentration reaches cmc. linear regressions were performed independently using the data on the descending part of the curve below the cmc and the equilibrium part above the cmc. the intersection of the linear regression lines corresponds to the cmc (table ) . in order to explore the relationship between cmc, anti-pedv activity, and hemolytic activity, scatter plots for pairs of assays (fig ) . each plot contains points representing the characteristic indices of slp to and surfactin. the distribution of points has no obvious regularity in any of the three plots, and the linear regression results are not significant. we conclude that the anti-pedv and hemolytic activities of the slps examined in this study are not related to their surfactant activity (represented by the cmc). in addition, there is no significant correlation between anti-pedv activity and hemolytic activity. synthetic lipopeptides that combine greater antiviral activity with lower cytotoxicity are still to be found, and the specific structure-activity relationship needs to be further elucidated. since slp was the most promising surfactin analogue, we chose it for further study. time of addition assays were performed to determine whether the slp exerts its anti-pedv effect at the same stage during infection as surfactin. since surfactin acts directly on virions, the experiment was designed to examine events early in the infection process. fig a summarizes the experimental plan and the eight treatments tested. briefly, slp , surfactin, or depe was added to virus alone, cells alone, or to cells and virus together prior to infection, during virus adsorption, during virus invasion ( h post infection at ˚c), or during replication ( - hpi). samples were harvested hours after infection, and analyzed to measure cellular levels of viral protein (fig b and c ) and viral rna ( fig d) . as expected for a normal component of the cell membrane, depe did not affect pedv replication at any stage, while slp and surfactin exhibited antiviral activity at specific stages. for example, when slp or surfactin are present throughout the experiment (treatment group ), little pedv replication occurs, as indicated by nucleic acid and protein levels. the same is true in treatment group , indicating that both compounds act directly on the virus. other antiviral mechanisms of slp and surfactin cannot be ruled out since pedv replication is inhibited to different degrees in some of the other treatment groups. wedge-shaped lipids in which the hydrophilic head has a larger cross-sectional area than the hydrophobic tail, are potential membrane fusion inhibitors [ , ] . in a recent study of rigid amphipathic fusion inhibitors, compounds with deoxyribose or acetate as hydrophilic moieties had anti-hsv effects [ ] . in addition, a recent study on antimicrobial activity of synthetic lipopeptides reported that lipopeptides with two to four positive charges and carbon atoms in the lipid chain have potent antimicrobial activity [ ] . fatty acid chain length from to carbon atoms is positively correlated with antimicrobial activity, but is also positively correlated with hemolytic activity and membrane selectivity [ ] . these factors must be considered in the design of new lipopeptides, and the structure-function relationships warrant further study. here we investigated lipopeptides containing two and three negative charges, and two positive charges, all with a fatty acid chain of carbon atoms. all of these lipopeptides had antiviral activity. in a previously published study we demonstrated that surfactin has antiviral activity as membrane fusion inhibitor [ ] . in order to improve the viral envelope selectivity of surfactin, we designed analogues with altered peptide amino acids, but kept the length of fatty acid chain of carbon atoms. compared with slp , slp has one additional carboxyl group at the end of the peptide, but the antiviral effect is similar. although slp has one less hydrophilic amino acid than slp , its antiviral activity increases about -fold. this result indicates that the hydrophilic portion of the wedge-shaped lipid needs to be sized appropriately for better antiviral function. in addition, slp differs from slp only in the order of two amino acids, but the antiviral potency of slp is . times greater, indicating the complexity of the relationship between molecular structure and antiviral effects. the cationic properties of synthetic lipopeptides are thought to be related to their antibacterial and hemolytic activities [ ] . slp , the only cationic lipopeptide examined in this study, has the strongest hemolytic activity, consistent with this hypothesis. cyclic lipopeptides are reported to more readily lyse bacterial membranes than linear or branched lipopeptides [ ] . in agreement, we found that the hemolytic activity of slp (a linear molecule, identical in amino acid sequence to surfactin) is slightly weaker than that of surfactin, which has a cyclic structure. it has also been reported that the total hydrophobicity of synthetic lipopeptides is inversely related to hemolytic activity, although this conclusion was from data on only related samples [ ] . our results do not support this conclusion. the relative hydrophobicity of slp , , and is slp >slp >slp , as indicated by their hplc retention times ( . , . , and . min, respectively). however, their hemolytic activity is not significantly related to the total hydrophobicity. in summary, we synthesized surfactin analogues and characterized them to identify those with enhanced anti-viral properties. slp equaled and slp exceeded surfactin's anti-pedv activity and both compounds had greatly reduced hemolytic activity, making their selection indexes and times larger respectively than surfactin's. however, slp exhibited a peculiar inhibition of red blood cell sedimentation. slp is therefore a more promising synthetic surfactin analogue. compared with surfactin, the structure of slp is distinguished by its linear lipopeptide and the additional carboxyl group at the c' of the peptide. slp also has two fewer hydrophobic amino acids than surfactin, this reduces the cost of synthesis while having little effect on antiviral activity. since surfactin has been shown to protect piglets from pedv challenge [ ] , the in vivo antiviral properties of surfactin analogues needs to be tested. furthermore, with respect to the future use of synthetic surfactins to control or ameliorate infection in pigs, the broader spectrum physiological activities of surfactin analogues needs further study and side effects beyond hemolytic toxicity need to be explored. our study suggests that slp , and other surfactin analogues, should be the object of in-depth study in order to develop a safer broad-spectrum family of surfactin antiviral agents. synthetic surfactin analogues have improved anti-pedv properties conceptualization: lvfeng yuan, qian yang. the pedv genome was detected by qrt-pcr. the experiment was repeated three times, normalized against group , and plotted as mean ± sd. � , p < . ; �� , p < . in two-tail t-test, for each reagent, compared with group . https://doi.org/ . /journal.pone. .g synthetic surfactin analogues have improved anti-pedv properties mechanism of inactivation of enveloped viruses by the biosurfactant surfactin from bacillus subtilis antiviral activity of antimicrobial lipopeptide from bacillus subtilis fmbj against pseudorabies virus, porcine 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activities key: cord- -oak lfmi authors: barratt, ruth; gilbert, gwendolyn l.; shaban, ramon z.; wyer, mary; hor, su-yin title: enablers of, and barriers to, optimal glove and mask use for routine care in the emergency department: an ethnographic study of australian clinicians date: - - journal: australas emerg care doi: . /j.auec. . . sha: doc_id: cord_uid: oak lfmi background: the risk of healthcare-acquired infection increases during outbreaks of novel infectious diseases. emergency department (ed) clinicians are at high risk of exposure to both these and common communicable diseases. personal protective equipment (ppe) is recommended to protect clinicians from acquiring, or becoming vectors of, infection, yet compliance is typically sub-optimal. little is known about factors that influence use of ppe—specifically gloves and masks—during routine care in the ed. methods: this was an ethnographic study, incorporating documentation review, field observations and interviews. the theoretical domains framework (tdf) was used to aid thematic analysis and identify relevant enablers of and barriers to optimal ppe use. results: thirty-one behavioural themes were identified that influenced participants’ use of masks and gloves. there were significant differences, namely: more reported enablers of glove use vs more barriers to mask use. reasons included more positive unit culture towards glove use, and lower perception of risk via facial contamination. conclusion: emerging infectious diseases, spread (among other routes) by respiratory droplets, have caused global outbreaks. emergency clinicians should ensure that, as with gloves, the use of masks is incorporated into routine cares where appropriate. further research which examines items of ppe independently is warranted. healthcare-associated infections are an ongoing threat to patients and clinicians, resulting in significant morbidity and economic cost [ ] . the risk of infection, for vulnerable hospital patients, their family members and healthcare professionals, increases during outbreaks of novel and re-emerging infectious diseases, such as the highest-risk healthcare professionals for exposure to bloodborne viral infections [ ] and respiratory diseases such as influenza [ ] . hunter et al. [ ] reported an estimated % rate of mers among ed clinicians in abu dhabi and, of those infected, % had been exposed before the diagnosis was made. personal protective equipment (ppe), including gowns, gloves, masks and protective eyewear, is crucial for protecting clinicians from acquiring, or acting as a vector of infection to other staff and patients [ ] . in addition to appropriate use of ppe, as part of transmission-based precautions (contact, droplet or airborne), standard precautions indicate use of ppe when there is a risk of exposure to pathogens: non-sterile disposable gloves if hands are likely to become contaminated and a surgical mask and eye protection when at risk of exposure to aerosols or direct splash with blood and body fluids. n /p masks are usually reserved for a few diseases (chickenpox, measles, tuberculosis) in which pathogencontaminated droplet nuclei (residue from evaporated droplets) or dust particles can remain suspended in air for long periods and enter the upper and lower respiratory tracts. sub-optimal use of ppe (i.e. contrary to the indications for standard and transmission-based precautions) by clinicians has been reported in different hospital settings [ ] [ ] [ ] . while gloves are the most frequently used item, masks are less appropriately used [ ] . although clinicians' use of ppe has been shown to increase when an outbreak is declared [ , [ ] [ ] [ ] , routine compliance is typically suboptimal [ , ] which increases the risk of occupationally-acquired infection and disease. there is limited recent literature examining the use of ppe in eds. following the introduction of universal precautions in the early s [ ] , a number of studies reported poor compliance with these measures in the ed [ ] [ ] [ ] . more recently, singh et al. used self-administered questionnaires to determine compliance with (what are now referred to as) standard precautions in the ed, and found that gloves were frequently used, but there was poor compliance with other ppe, especially eye protection [ ] . evanoff et al. [ ] observed video footage to assess compliance with ppe during invasive procedures in an ed and reported % glove use for trauma patient encounters, compared with % and %, for use of mask and protective eyewear, respectively. in a trauma centre study, the compliance rates for use of masks and eye protection, after an educational intervention, were % and %, respectively [ ] . ed clinicians are regularly at risk of facial contamination during invasive procedures, intubation and other resuscitative measures [ ] . they are also exposed to both seasonal and emerging respiratory infectious diseases. in one paediatric ed setting, only - % of clinicians reported that they always or usually wore a mask or eye protection, while assessing febrile respiratory patients during winter [ ] . although gloves are worn frequently, patient safety may be compromised by misuse, such as not changing them between dirty and clean tasks on the same patient or between different patients and/or failing to comply with hand hygiene before and after use, which often contaminates the clinician's hands [ ] [ ] [ ] . commonly cited factors contributing to sub-optimal compliance with ppe in healthcare include inadequate knowledge and training, perception of risk, organisational culture and environmental barriers [ , ] . reid et al. [ ] identified knowledge, access to ppe, patient diagnosis and unit culture, in the ed context, as factors influencing ppe compliance. healthcare transmission of novel infectious diseases can occur prior to recognition of an outbreak [ ] . while it is difficult to plan in advance for such a rare event, staff who are competent in the principles and practice of routine infection prevention and control (ipc) and ppe use are more likely to be better protected from the start and more prepared to implement high-level precautions rapidly and safely. in this area there is a paucity of literature which examines factors that facilitate or hinder the use of ppe during routine clini-cal care in the ed. most previous studies have focused primarily on compliance with standard precautions during procedures that pose a high risk of exposure to blood and body fluids [ , ] or on overall compliance with ppe use, without elucidating determinants of those behaviours [ ] . they have described 'how' clinicians use ppe, whereas the present study aimed to shown 'why' ppe is, or is not, used by exploring the factors that influence the use-specifically of gloves and masks-during routine care, in one ed. we employed methods that allowed close engagement with clinicians so as to understand their choices and behaviours and utilised the theoretical domains framework (tdf) [ ] to assess the relevant enablers and barriers. a better understanding of these practices in this context could assist managers, educators and clinicians to optimise enablers and address barriers, locally, and inform health policy and pandemic planning more widely. this qualitative study used ethnography to explore the use of gloves and masks by clinicians in an ed. this is a suitable methodology for the study of complex social and clinical interactions in the context of healthcare quality and safety [ ] as it involves direct observation of the behaviour of people and their social environment using varied data collection methods. the theoretical underpinnings of this research are grounded in behavioural science, in particular the tdf, which was used to inform the interview guide and subsequent data analysis. the tdf synthesises multiple theories of behaviour and behavioural change into domains which provide a framework for examination of cognitive, affective, social and environmental determinants and influences on behaviour [ ] . it has been used widely in patient safety research [ ] , including clinicians' ipc practices [ ] and is particularly useful for informing policy and planning practice improvement. the setting was a busy ed with over , presentations per year in a major tertiary hospital in sydney, australia. departmental staff were informed about the study through a staff e-newsletter and during several morning staff meetings which are attended by all staff on duty that day. a purposive snow-ball sampling technique [ ] was used to recruit clinical and non-clinical staff working in the department for semi-structured interviews, so as to obtain a crosssection of professional roles, experience and clinical expertise. approval for this study was given by the western sydney local health district human research ethics committee. written consent for interview was obtained by the researcher after negotiation with each participant in accordance with the approved study protocol. the researcher attended the ed during day shifts for one to two hours at a time, observing and taking notes on activities directly related to the aim of the study. local and hospital policies, signage, and other documentation relating to use of ppe in the ed context, were examined. reflexive review of field observations and documentation, was used to inform the interview guide [ ] but not included in the analysis reported here. twenty-two face-to-face, semi-structured interviews, lasting - min (average min), were conducted with clinicians (nurses and doctors) and non-clinical support staff at times and places convenient for them during the day. interviewees comprised five senior doctors (dr), seven nurses in senior roles (clinical nurse consultant [cnc]/ nurse manager [nm]/ nurse practitioner [np]), two registered nurses (rn), one enrolled nurse (en), two nurse graduates (ng), two support workers (sw) and two senior external clinicians from the ipc (cnc) and infectious diseases (dr) departments. the questions were guided by the domains of the tdf and focused on the desired behaviours of optimal compliance with glove and mask use. interviews were audio-recorded and subsequently transcribed verbatim. interviewees were invited to review their transcripts for accuracy. the data was analysed using a content and thematic approach in order to gather an in-depth understanding of factors affecting optimal glove and mask use. transcripts were reviewed independently by two researchers, the content was coded into tdf behavioural domains relating to the target behaviours [ ] and analysed thematically. no data were lost in the transcription or the interpretive analysis. thirty-one behavioural themes were identified that influenced participants' use of protective masks and gloves. these were mapped against the theoretical domains (table ) and further analysis allowed them to be classified as enablers and barriers to optimal use. the data revealed interdependency between some domains, resulting in natural grouping of the findings. for example, "participants' beliefs about the consequences" (tdf ) of glove and mask use were linked to "emotion" (tdf ) such as anxiety; therefore, the findings are described together. there was also mirroring of themes whereby one could be either an enabler or barrier within the same domain. for example, "knowledge" (tdf ), was an enabler of glove use but a barrier to appropriate mask use. findings are reported under tdf domain titles within the categories of enablers and barriers. in this study, enablers of optimal ppe use were represented in all domains; however, there were more enablers of optimal glove, than protective mask, use. enablers include a variety of factors that encourage, facilitate or are likely to increase glove or mask use (not necessarily appropriately) including internal/personal factors such as self-protection and/or external factors, as detailed next. participants' knowledge and skills, self-efficacy and confidence in the equipment, were interconnected as key enablers of optimal ppe use. all participants reported having received instruction in the use of ppe during either their professional or induction training. optimal use of gloves and masks was further enabled through education provided by the hospital ipc team or by some other clinicians with broader knowledge and/or interest in ipc. participants please cite this article in press as: barratt reported that high-level ppe skills had also been enhanced in recent years through simulation exercises for ebola virus disease. 'look, whenever there's attention to something, like the ebola, we had a lot of in-services regarding donning and doffing.' (doctor [dr] ) most clinicians' perceived knowledge of ipc policies supported their use of gloves as appropriate for standard and transmissionbased precautions. optimal ppe use had been further promoted recently through the introduction of an ed-specific poster that identified ppe required for specific diseases, which was attached to isolation trolleys and positively received by staff as helpful, particularly in choosing the correct mask. the majority of participants reported they were confident with the protection provided by the equipment and in their ability to correctly don and doff gloves and protective masks. 'i got taught that fitting of the mask, when the ebola . . .. was out. i remember being taught properly then how ppe should be worn.' (enrolled nurse [en] ) the participants' understanding and abilities in ppe use were consistent with their professional responsibilities as described in the next section. an important enabler of optimal glove and mask use was the professional responsibility some clinicians felt towards protecting patients from infections. for example: 'so yeah, the staff should also then be taking on some of that ppe responsibility, infection prevention responsibility.' (dr ) another associated professional responsibility that influenced appropriate glove use was the perception, by several doctors, that when there was no obvious risk of contamination, not wearing gloves facilitated a better doctor/patient relationship. this professional role identity was interconnected with the participants beliefs about the consequences of not using ppe, as outlined next. protecting themselves and not taking infection home to their family were reported to be strong motivators of ppe use. this belief in the negative personal consequences of not using ppe was often emotive: 'my concern is (a) infecting me and then taking it home to my family.' (dr ) glove use in particular was determined by the perception of personal risk, as summarised by this participant: 'personally, i will put gloves on if obviously there's blood, patient's got blood on them, so a trauma patient, i would generally put gloves on. patients who are a bit unhygienic, i'll put gloves on. so, both of these instances are to protect myself.' (dr ) for others, their use of ppe was influenced by previous experiences, such as working in the early days of hiv or having a urine splash to the face. the many participants who described a personal motivation for ppe use may have influenced the overall social culture within the department. the departmental norms and peer behaviour in the ed both reinforced and positively enabled clinicians' use of gloves, but less so for masks. glove use was reported to be embedded in routine tasks and patient encounters and clinicians would wait for, or remind, colleagues to don gloves when attending a patient: 'there's definitely a culture of these are the tools that we use to do our work. . . . and what i do notice is that people wait for you to put your gloves on.' (cnc ) during the winter respiratory virus season, visual signals such as patients wearing surgical masks or an increase in boxes of masks in clinical areas helped to reinforce mask use. staff were also expected to wear a mask when caring for a neutropenic patient: 'just the only other time when i think about wearing masks, is in patients who are in neutropenic. because that's the other setting where we say that it's required.' (dr ) the behavioural norms within the ed also influenced the individual's routine and habitual practices related to gloves and masks. although some medical staff reported using risk assessment to determine the need for gloves, as described above, the entrenched habit of most staff using gloves routinely for patient contact had the positive effect of facilitating their use when it was indicated as part of standard precautions. as this nurse explains: 'it's an autopilot thing, as soon as they go and get a new patient, straightaway grab a set of gloves and start doing what they need to do.' (registered nurse [rn] ) while glove use was almost automatic for the participants from the department, clinicians reported making a conscious decision to wear a mask. medical staff in particular reported making a risk assessment for mask use which was prompted by visual cues such as isolation trolleys and signs by the bedside or certain clinical information handed over about the patient: 'like the measles or something along those lines. that would prompt me to think, i need a mask and then let [the] nursing staff as well know. or a tb patient.' (dr ) support staff also chose to wear masks and gloves on occasions when they deemed there to be a risk of infection to themselves, as described by the following support worker: within the physical environment of the ed, staff were generally satisfied with the brand of gloves provided and noted that they were very accessible, which was an enabler of optimal use. the recent introduction of isolation trolleys, for patients in transmission-based precautions in curtained bed spaces, facilitated please cite this article in press as: barratt the support staff also found the isolation trolleys useful to alert them to the infectious status of a patient: 'if they go to enter a room and see the trolley outside they won't bother % of the time as not urgent enough to do so. or if they really have to they will put on the type of mask that is on the trolley.' (sw ) behaviour towards ppe was also influenced by the organisational ipc requirements for hospital accreditation. although no specific ppe monitoring was in place, annual training was encouraged. 'so we're trying to instil that they need to do an annual [ppe] competency. it's available, we're definitely not there yet.' (cnc ipc) the introduction of hospital-wide hand hygiene audits helped to promote correct hand hygiene behaviour around glove use and was reported to be an enabler. 'i am more compliant with hand washing prior to glove use than i probably was when i first trained.' (dr ) as illustrated, a range of factors were identified by participants as enablers of optimal ppe behaviour, primarily for glove use. within the same tdf domains, barriers to mask and gloves use were also described. unlike enablers, which mainly related to glove use, barriers to protective mask use were more frequently described by participants. as noted earlier, knowledge of policy was an important enabler of optimal ppe use. however, despite the ready availability of ppe policies and educational resources, participants described mask and glove practices that did not adhere to policy. thus, in this department, information resources and policy were sometimes a barrier because they were confusing. one clinician pointed to the various posters and guidelines as 'information overload', while others suggested that hospital-wide policies were not clear or did not work well in the ed context. 'i think that some of our bad practices, or some of our practices that, where you find someone wearing the wrong mask is all due to the fact that when we're educating and when we're following policy, the policy has been very, very ambiguous.' (cnc ) indications for which type of mask to use are described in the ipc policy relating to transmission-based 'airborne' and 'droplet' precautions. however, as the following participants describe, these terms were not always well understood and indicated a knowledge gap around the functionality and usage of the different types of masks. consequently, both medical and nursing staff reported choosing whichever mask was handy, not necessarily the one required, as the following nurse participants reported: 'if you said droplet or airborne you'd just mostly get a blank face and look at you and they might come up and go, well, maybe i need for the airborne the orange but i'm not sure . . . ' despite a knowledge of hospital policy towards masks, several participants preferred to apply their own professional autonomy in relation to mask use. professionally, some medical staff felt that using a mask restricted their ability to provide good clinical care, as it hindered communication and empathy with patients. the following participant felt that the mask interfered with their clinical assessment: 'the problem is, if you need to communicate with people, the mask can, particularly the n , can muffle your voice as well.' (dr ) another doctor perceived the mask as an obstacle to establishing a good understanding between themselves and the patient: 'but i don't want to be the one that's wearing the mask and making the patient feel like there's a barrier. (dr ) these aspects of the use of ppe that clinicians presented as barriers to their use, because it interfered with their professional role, were interconnected with their beliefs about the consequences of not using ppe, as outlined next. as described previously, an exaggerated perception of infection risk, leading to overuse, was a potential barrier to appropriate use of gloves: 'i'm probably not the best person because i think i probably overdo gloves. i do not even feel comfortable shaking hands with a patient without gloves.' (dr ) by contrast, minimal concern towards the risk of respiratory infection was a barrier to mask use. one clinician attributed this to her own immunity, while another suggested that he was as likely to get a cough or cold as a member of the general public as when working. 'i never wear a mask during the flu season unless obviously i felt like i had the flu. you know, my view of the flu is i get immunised. i catch a train and everyone coughs on me anyway. and i'm more likely to have immunity against things like that because i never get sick.' (dr ) 'so, yes, if they have a respiratory symptom, if they have a fever, there is a history of overseas travel and i'm suspecting some unusual organisms, yeah then i will . . . but if it's like cough and cold, just minor symptoms, probably not because we get exposed to it when we are out in public and in the shopping centre, anyway, and i wouldn't.' (dr ) another participant suggested that in the absence of visual reminders for infectious respiratory diseases such as a productive cough, they did not perceive enough risk to wear a mask. similarly, participants also felt a lack of personal risk if the patient was wearing a mask, although they acknowledged that it was often not worn correctly by the patient. however, potential consequences for other patients were not reported as a motivation for ppe use outside of caring for the immune-compromised patient. '. . . gloves are really more for our protection, especially, way more than they are for the patient's protection.' (rn ) these common perceptions of risk were re-enforced within the social setting of the unit. unlike glove use, there was no departmental norm for wearing protective masks, except when attending to immunosuppressed patients. although there was a general consensus that mask use could be improved, peer influence or role modelling was limited to a few senior nurses and doctors. 'i mean part of your ppe, you probably should put a mask on, but we generally don't.' (dr ) 'i guess, in general we don't use masks.' (rn ) the absence of a departmental culture of wearing protective mask impacted on the clinicians' intentions and decision-making, as described below. one of the barriers to optimal mask use was the lack of habitual mask use in daily care requiring the individual to make a conscious decision to use a mask as illustrated in this excerpt: 'but because it's not business as usual the only thing that would prompt me initially would be to think, oh i could get a splash here, so therefore i'll wear a mask.' (cnc ) conversely, although glove use was prompted by unconscious behaviours, this could lead to unnecessary glove use: 'but i've noticed that's something that happens a lot nowadays, that just to touch a patient, people will put gloves on, and i encourage them not to do that; that they don't need to, that the patient is not dirty.' (rn ) the individual's decision-making processes were also related to the environment within which they worked, as outlined next. the busy, chaotic context of an ed, was reported by many participants to be a barrier to optimal ppe use: 'and it's just so busy that sometimes you can see that, yeah, something might not be quite by the books because of the pressure and the stress of the environment and the amount of people coming in and out.' (enrolled nurse [en] ) participants cited urgency of care as barriers to performing hand hygiene prior to donning gloves (it took too long for the hands to dry) or mask. 'the fit test can be a bit of a deterrent in a busy environment, to have to make sure it's fitted properly.' (cnc ) '. . . the time to put it on and off, particularly if someone's sick.' (cnc ) there was also a belief that the differences between the ed environment and an inpatient unit allowed for different ppe practices. 'it's culturally acceptable in an emergency to doattend your cares of a patient without those precautions where it's not in the ward.' (np ) the lack of a designated place for boxes of masks -other than isolation trolleys -sometimes made it difficult to locate a mask and was a barrier to the use of masks for standard precautions. the open-plan layout of the department, with only two single isolation rooms, was also identified by several participants as a deterrent to implementing good ipc practices. 'so once they're not in those [isolation] rooms and they're just out in the general acute area, i think [staff are] much less so likely to adhere to those precautions.' (dr ) compared to general satisfaction with the gloves provided, participants described more undesirable qualities with using the masks. some participants reported that the n /p masks were more difficult to don, while others described discomfort and fogging of their glasses or protective goggles when wearing a mask. for one participant the discomfort of wearing a mask interfered with her ability to provide clinical care. 'like i really think it does make me abridge my assessment and examination because my desire to get the mask off is great.' (dr ) unlike hand hygiene audits, participants reported other external ipc monitoring as a barrier to optimal ppe use. this participant changed her behaviour with masks due to expectations of ppe audits: 'i think it's a bit of a throw-back from infection control. they will teach us about this mask and that mask, and then come and audit you, and then you're always afraid you're using the wrong one. so you just choose the higher one.' (cnc ) this ethnographic study explored the behaviour of clinical staff towards use of gloves and protective masks in a busy ed. analysis using the tdf elucidated factors that either promote or impede (occasionally both, in different circumstances) optimal ppe use, some of which have been identified previously in the literature [ ] . however, we also revealed ed-specific determinants of glove and mask use that have not been previously described. in addition to providing emergency care of patients, front-line clinicians play a central role in the initial screening, detection and ipc management of suspected but undifferentiated infectious diseases. this role inevitably puts them at personal risk of infection. therefore, protective barriers such as ppe are essential to minimise the risk both to themselves and to other patients. although occu-pational health and safety is important, clinicians should be aware of their professional duty towards patient safety. an important finding in our study was a significant difference, in use, between gloves and masks in that there were more reported enablers of use of the former and barriers to use of the latter. existing research has demonstrated that gloves are the most frequently used item of ppe, much more so than masks [ ] . a significant factor associated with frequent glove use idenitified in this study was some participants' motivation to use them for their own protection as a routine precaution. glove use was even more prevalent when there was a higher risk of blood and body fluid contamination, such as in the trauma and resuscitation areas. this aligns with the literature which reports compliance rates of - % for glove use during trauma encounters in ed [ , ] . less obvious contamination risks, such as an unrecognised mro-colonised patient, were also identified by participants as reasons for glove use. these patients present a significant risk in the ed for environmental contamination and staff acquisition [ ] . some participants argued that habitual use of gloves was a barrier to optimal use. it is difficult to ascertain whether the glove use was excessive as there is no published research that explores the indications for and use of gloves in an ed. a recent systematic review of glove use and transmission of infection in other inpatient departments concluded that gloves were often overused and misused [ ] . the published literature related to hand hygiene auditing provides some indication of ed rates for glove use. during a hand hygiene observational study in an ed, carter et al. [ ] reported that only % of hand hygiene opportunities, whether or not hand hygiene was performed, were associated with glove use, indicating that in this setting, the majority of patient encounters did not incur the use of gloves. nevertheless, when optimising behaviour for cross infection, attention should also be focused on hand hygiene practices associated with the use of gloves [ ] . in comparison, participants described fewer enablers of mask use, which reflects that they are used much less so than gloves [ ] . the apparent under-use of protective masks in this study reflects literature reports of low rates ( - %) for mask compliance in the ed setting [ , , ] . the optimal use of protective masks by healthcare workers has been shown to reduce transmission of sporadic and epidemic infectious diseases. during the global sars outbreak in , sars-cov transmission in a vietnamese hospital was significantly reduced when protective mask use among clinical staff increased [ ] . skowronski et al. [ ] attributes the prevention of sars transmission within a vancouver hospital to the prompt implementation of ipc measures, including ppe, in the ed for a traveller returning from asia with severe influenza-like illness. this is in contrast to the outcome for a similar case in toronto, when droplet and airborne precautions were not put in place in the ed for over h, resulting in further cases of cross infection [ ] . many participants blamed the chaotic, fast-paced ed environment, as a significant barrier to using a mask. while this argument has been reported previously [ ] , the same contextual reasoning could also apply to gloves, which are in fact regularly used and take longer to don and doff-at least if hand hygiene is included. thus, other factors may be more influential determinants of mask use, such as the team behavioural norms in the department or the individual's perception of risk of infectious diseases. one barrier to optimal mask use demonstrated in our research was the strong personal belief about ppe use of some senior medical staff, which overrode ipc policy. this is reflective of a recent study which found that the clinical autonomy of doctors was a significant factor in their ipc practice [ ] . in an ed where there are numerous 'leaders', different role models and aberrant behaviour can impact negatively on the ipc culture of the department. participants identified a lack of positive role modelling and leadership which has been shown elsewhere to influence individual behaviour towards ppe [ , , ] . in contrast to our findings, a recent qualitative study that utilised focus groups with nurses and assistants, reported a positive peer culture for encouraging respirator mask use [ ] . this may indicate a greater perception of risk associated with diseases that required an n /p respirator mask. in our study, a clinician's reduced perception of risk of infection from facial exposure was a barrier to wearing a mask. furthermore, clinicians perceived less risk to themselves when the patient was wearing a protective mask for a potential respiratory disease and felt protected enough not to wear a mask. public health guidelines recommend that symptomatic persons in hospital waiting rooms and other public spaces are given a mask to wear to prevent transmission of respiratory infection [ , ] . this measure is largely accepted by the public and has had some success in community settings [ ] [ ] [ ] . however, research is limited on its protective effect for clinicians engaging in direct patient care. the literature also reports the problem of noncompliance with mask use by the public [ ] . this risk may increase in the ed setting, where, as identified by participants in our study, patients are unwell and often non-compliant in correct mask use. to prevent early transmission of either routine or outbreak infectious diseases, frontline staff must be vigilant and adhere to routine ipc measures [ ] . this study identified the barriers to implementing effective protective mask use, which can be difficult in facilities with few isolation rooms or where staff rely on visual or verbal cues to instigate appropriate precautions [ ] . in addition, the placement of boxes of masks was a practical barrier. poor access to masks is also a common finding in the literature [ , , ] . in our study setting the introduction of ten isolation equipment trolleys addressed some of these barriers. applying human factors design principles is one method to address some of the contextual environmental barriers to optimal ipc behaviour such as difficult access to ppe [ , ] . it is worth noting that in this study an exclusively policy-driven approach to ppe use was not a consistent enabler of optimal practice. although normally viewed as a facilitator, policy in this setting was viewed as a barrier to optimal mask and gloves use. bouchoucha and moor [ ] suggest that deviating from ipc guidelines and policy can have serious consequences for patient safety. on the other hand, other authors have recognised that the unique complexity of an ed environment can challenge conventional ipc protocols and practices. for example, liang states that overcrowding, multiple clinician-patient encounters, limited isolation facilities and other factors unique to an ed are barriers to good ipc practice [ ] . chen et al suggests that, compared to inpatient settings, it is more difficult to implement ipc measures in an emergency or outpatient department [ ] . the study has some limitations. it reports participants' perceptions of the enablers of and barriers to optimal ppe use for routine care in one australia ed. this is a single-site study, and the findings are not expected to be representative in their totality of other eds. other eds will inevitably have characteristics which mediate enablers or, and barriers to, optimal ppe use, although it is expected that those identified in this study have resonance. the study design did not permit verification or otherwise of these findings beyond what was possible to observe during the field immersion. our findings have demonstrated that the determinants of ppe behaviour in an ed differed significantly between gloves and masks. the spread of emerging infectious diseases that have been responsible for global outbreaks recently, has included respiratory droplets. ed clinicians should therefore ensure that, as with gloves, the use of masks is incorporated into routine care where appropriate. these results support the need for further research which examines items of ppe independently. rb, glg, sh and mw conceived and designed the study and prepared the study protocol. glg supervised all data collection and study procedures. all authors contributed to interpretation of the results, preparation of the manuscript and approval of the final version. this work is supported by the australian partnership for preparedness research on infectious diseases emergencies (apprise) of which author glg is a chief investigator and author rb is recipient of a doctoral scholarship. the research presented in this article is solely the responsibility of the authors and does not reflect the views of apprise. rs is editor-in-chief of australasian emergency care but played no role in the peer review or editorial decision-making of the manuscript whatsoever. the authors declare no other conflict of interest. report on the burden of endemic health care-associated infection worldwide scope and extent of healthcare-associated middle east respiratory syndrome coronavirus transmission during two contemporaneous outbreaks in riyadh, saudi arabia healthcare-associated infections: the hallmark of the middle east respiratory syndrome coronavirus (mers-cov) with review of the literature responding to the severe acute respiratory syndrome (sars) outbreak: lessons learned in a toronto emergency department risks to healthcare workers with emerging diseases recognizing and managing emerging 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review infection prevention and control: who is the judge, you or the guidelines? infection prevention for the emergency department interpretation and clinical practice of regulation for prevention and control of healthcare associated infection in outpatient and emergency department in healthcare facilities key: cord- -j slaefb authors: silva, josé v.j.; ludwig-begall, louisa f.; oliveira-filho, edmilson f. de; oliveira, renato a.s.; durães-carvalho, ricardo; lopes, thaísa r.r.; silva, daisy e.a.; gil, laura h.v.g. title: a scoping review of chikungunya virus infection: epidemiology, clinical characteristics, viral co-circulation complications, and control date: - - journal: acta tropica doi: . /j.actatropica. . . sha: doc_id: cord_uid: j slaefb abstract chikungunya fever is a mosquito-borne viral illness characterized by a sudden onset of fever associated with joint pains. it was first described in the s during a chikungunya virus (chikv) outbreak in southern tanzania and has since (re-) emerged and spread to several other geographical areas, reaching large populations and causing massive epidemics. in recent years, chikv has gained considerable attention due to its quick spread to the caribbean and then in the americas, with many cases reported between and . chikv has further garnered attention due to the clinical diagnostic difficulties when zika (zikv) and dengue (denv) viruses are simultaneously present. in this review, topical chikv-related issues, such as epidemiology and transmission, are examined. the different manifestations of infection (acute, chronic and atypical) are described and a particular focus is placed upon the diagnostic handling in the case of zikv and denv co-circulating. natural and synthetic compounds under evaluation for treatment of chikungunya disease, including drugs already licensed for other purposes, are also discussed. finally, previous and current vaccine strategies, as well as the control of the chikv transmission through an integrated vector management, are reviewed in some detail. chikungunya virus (chikv) is the etiological agent of chikungunya fever (chikf), an arthropod-borne disease transmitted mainly by aedes genus species (weaver, ) . first described in in present-day tanzania, the early chikf cases were treated as dengue virus (denv) infection (lumsden, ) . following isolation of chikv from infected patients' sera, as well as ae. (stegomyia) aegypti (linnaeus, ) and culex spp. mosquitoes in , the virus was placed in the arbovirus group a (ross, ; calisher and karabatsos, ) . currently, chikv is grouped within the alphavirus genus, togaviridae family, and identified as member of the semliki forest virus (sfv) antigenic complex (van duijl-richter et al., ; ictv, ) . shortly after identification in east africa ( ), chikv was described in both central and southern regions of africa (uganda and sub- both urban and sylvatic chikv transmission cycles have been described (caglioti et al., ) . the sylvatic cycle (especially in africa) may involve the participation of some aedes species, such as aedes furcifer (edwards, ) , aedes taylori (edwards, ) , aedes luteocephalus (newstead et al., ) , aedes vittatus (bigot, ) and aedes fulgens (edwards, ) , and different non-human primate (nhp) species, possible reservoirs or amplifiers hosts for chikv [e.g. african green monkeys (chlorocebus sabaeus) (linnaeus, ), patas monkeys (erythrocebus patas) (schreber, ), guinea baboons (papio papio) (desmarest, ), guenons (cercopithecus aethiops) (linnaeus, ), bushbabies (galago senegalensis) (geoffroy, ), mandrills (mandrillus sphinx) (linnaeus, ), red-tail monkeys (cercopithecus ascanius schmidti) (matschie, ) and chacma baboons (papio ursinus) (kerr, )] (mcintosh, ; mccrae et al., ; diallo et al., ; chevillon et al., ; pruetz et al., caglioti et al., kading et al., ; althouse et al., ) . a vector role has also been suggested for culex and anopheles mosquitoes, which have been found infected in senegal from to (diallo et al., ) . in the urban cycle, ae. aegypti and ae. albopictus are known as the main vectors (weaver, ) . currently, they probably remain as the main vectors for chikv transmission in the americas, africa, europe, asia and oceania (horwood et al., ; vega-rúa et al., zeller et al., ngoagouni et al., . significantly, ae. aegypti can also be the main vector of other viruses, such as zikv and denv, and co-transmission events may be observed (carrillo-hernández et al., ) . however, it has been demonstrated that simultaneous infections by denv/chikv/zikv or denv/chikv in ae. aegypti does not compromise the vector competence (le coupanec et al., ; rückert et al., ) . it is known that horizontal transmission of chikv in aedes mosquitoes can occur and act positively in the maintenance of infection cycles ( fig. a ) (mavale et al., ) . vertical transmission of chikv into aedes has also been observed under natural and experimental conditions and has been pinpointed as a possible reason for viral persistence under harsh environmental conditions (fig. b) (agarwal et al., ; chompoosri et al., ; jain et al., ) . once inside the arthropod vector, chikv must replicate and reach the mosquitoes' salivary glands within roughly seven to ten days for transmission to a susceptible human (lim et al., ) (fig. c) . in human hosts, the intrinsic incubation time can vary from one to twelve days and infected individuals may present viremia of up to ten days (kam et al., ; simon et al., ; azevedo et al., ) . maternal-fetal transmission has also been reported in humans (fig. e ). neonatal encephalitis, as consequence of vertical transmission, was observed, for instance, during the brazilian epidemic in (bandeira et al., a; lyra et al., ) . however, no breast milk transmission has been evidenced (patterson et al., ) . despite the fact that chikv rna has been detected in semen even after days post symptom onset, indicating possible transmission via sexual intercourse, horizontal transmission between humans has not yet been reported ( fig. d ) (bandeira et al., b; patterson et al., ) . approximately - % of individuals infected with chikv develop clinical disease with fever and arthralgia (staples et al., ; ayu et al., ; nakkhara et al., ) . chikv infection has been associated with sudden onset of febrile illness (> . °c) ( % of patients), arthralgia ( % of patients), back pain ( % of patients), headache ( % of patients) and fatigue (who, ; thiberville et al., ) . the most common symptom in chikf is polyarthralgia, typically of bilateral polyarticular nature, affecting mainly peripheral joints (ankles, wrists and phalanges) and some large joints (knees and elbows) (who, ; morrison, ) . cutaneous manifestations are reported in circa % of acute cases. the lesions are characterized by macular or transitory maculopapular eruption, which can be edematous or itchy, often occurring in the body extremities, palms, soles of the feet, torso and face (simon et al., ; thiberville et al., ) . gastrointestinal symptoms, such as diarrhea, vomiting, nausea and abdominal pain, occur in - % of cases during the acute phase (thiberville et al., ) . other possible symptoms include erythema, asthenia, conjunctival effusion, persistent conjunctivitis and cervical lymphadenopathy (staples et al., ; staples and fischer, ; madariaga et al., ) . different studies have demonstrated that chikv infection can reach high viral loads, ranging from to copies of viral rna/ml, which seem to be correlated with the presence and severity of clinical signs and symptoms (chow et al., ; appassakij et al., ) . polyarthralgia and/or polyarthritis are hallmark symptoms of chronic chikungunya, mostly affecting small joints, such as phalanges and wrists, as well as large joints (e.g., ankles, knees and shoulders). the condition is usually severe and leads to mobility limitations of afflicted patients (hoarau et al., ) . polyarthralgia has been described to persist for varying periods of time, lasting from weeks to several months and, in some cases, up to five years, depending on the populations evaluated (borgherini et al., ; sissoko et al., ; manimunda et al., ; simon et al., ) . the persistence of polyarthralgia in some alphaviruses, such as sfv and sinv, seems to be associated with persistence of viral antigens and immune responses (inflammation) in the joints (atkinson et al., ; perri et al., ; hoarau et al., ; labadie et al., ; simon et al., ; poo et al., ; silva and dermody, ) . about the viral antigens, there is still no consensus whether they have replication competence (perhaps with mutations to promote their persistence) or are only the result of a delayed clearance of non-replicating viral antigen (atkinson et al., ; perri et al., ; poo et al., ; weaver and lecuit, ) . precisely on chikv, a study described the presence of macrophages with chikv genetic material and viral proteins in the synovial tissue of an -month-long chronically infected patient (hoarau et al., ) . experimental studies have shown chikv persistence in lymphoid organs, liver, joints, muscles and macrophages from nhps (labadie et al., ) . in addition, the presence of infiltrating cells, mainly macrophages, monocytes and lymphocytes, and specific proinflammatory mediators, such as il- , il- , and mcp- , within the synovial fluid probably also contribute to the chronicity of the inflammation in chikungunya disease (silva and dermody, ) . moreover, severe cases of chikungunya may be related to age and diverse underlying medical conditions, such as hypertension, respiratory conditions and diabetes mellitus (borgherini et al., (borgherini et al., , sankari et al., ; economopoulou et al., ; sissoko et al., ; tandale et al., ) . the pathogenesis of rheumatoid arthritis in chikf is still under debate. while certain studies have suggested that viral infection may trigger initiation of this chronic inflammatory disorder, other studies did not find inflammatory markers in infected individuals with chronic symptoms (staples et al., ; schilte et al., ) . chikv infections can also lead to atypical clinical manifestations. guillain-barré syndrome (gbs), for instance, has been associated with chikv infection (lebrun et al., ; oehler et al., ) . gbs comprises an acute inflammatory demyelinating polyneuropathy of global incidence, in which about two-thirds of cases occur after bacterial (e.g. campylobacter jejuni) (heikema et al., ) or viral infection (oehler et al., ) , such as by dengue- (simon et al., ) , west nile- (leis and stokic, ) , influenza- (choi and yeon, ) , cytomegalo- (steger et al., ) , human immunodeficiency- (girgin et al., ) , epstein-barr (phillips, ; kim et al., ) and zika viruses (rozé et al., ) . during the more recent chikv outbreaks, total or partial alopecia on the head or body, predominately in female patients, and ophthalmological alterations, such as uveitis and retinitis, were described during the chronic phase of infection (martínez-pulgarín et al., ; cunha and trinta, ) . in newborns, congenital infections may be accompanied by varying clinical signs, such as fever, lack of appetite, apnea, skin manifestations, distal and cerebral edema, encephalitis and hemorrhage (gopakumar and ramachandran, ; bandeira et al., a; lyra et al., ) . heart-and gastrointestinal disorders and cutaneous lesions are reported to manifest up to two days after the onset of fever in chikv-infected newborns and children (ernould et al., ) . bullous lesions associated to chikv infection have also been reported in four-month-old babies, who had % of their body surface affected on the second day after the onset of fever (robin et al., ) . deaths from chikv infection were previously considered a rare event. this perception, however, has changed since the latest epidemics, which presented a considerably increased mortality rate, probably due to neurological affections, mainly in neonates, immunocompromised and elderly (rampal et al., ; kee et al., chusri et al., bandeira et al., a) . it is challenging to differentiate clinical signs and symptoms of chikv infection from other pathologies, especially when zikv and denv are co-circulating in the same geographical region (hua and combe, ) . individuals infected by these arboviruses can present a wide range of similar clinical manifestations, such as rash, myalgia, exanthema, arthralgia, joint pain, headache, lymph node hypertrophy, neurological impairment and fever (brito and cordeiro, ) . in addition, it is difficult to determine the frequency and intensity of the symptoms and correctly assess pain (mild, moderate and intense) of afflicted patients (table ). in this context, variations in the clinical presentation of cases can give hints as to the viral etiology; for instance, the salient and prolonged polyarthralgia, often accompanied by rash, is typically more indicative of chikungunya, while hemorrhagic manifestations and myalgia are more commonly observed in denv infections (lee et al., ) despite the patients co-infected with chikv/denv, chikv/zikv and chikv/denv/zikv often do not show exacerbation of clinical signs, the co-infection presents as additional obstacle during differential diagnosis (furuya-kanamori et al., ; villamil-gómez et al., ; carrillo-hernández et al., ) . tables and summarize clinical and laboratory features that should be evaluated for efficient differential diagnosis of chikv, denv and zikv infections. since the variety and intensity of symptoms associated to chikv, denv and zikv infections are so similar and make clinical diagnosis difficult in areas of co-circulation, laboratory analysis is necessary to confirm the respective viral etiology. hematology findings associated to chikv infection are commonly either unspecific, however, lymphopenia and hypocalcemia were the most frequent observation, and severe thrombocytopenia is rare (borgherini et al., ; brito and cordeiro, ; paho, ) . moreover, the c-reactive protein is generally elevated in the acute phase illness (table ) (venugopalan et al., ; brito and cordeiro, ; paho, ) . elevation of hepatic enzymes, as well as elevated creatinine and creatine phosphokinase levels, have also been reported (danis-lozano et al., ) . the different laboratory patterns observed during chikv, denv and zikv infections, added to clinical findings, may be incorporated to support a correct diagnosis (tables and ) . laboratory tests for specific diagnosis of chikv infection are based on virus isolation, viral rna detection and serology (johnson et al., ) . despite not usually employed in routine diagnosis, viral isolation can be performed from sera collected up to seven days after onset of illness and inoculated into mosquito-or mammalian cell lines, in which cytopathic effects can appear between one to three days after inoculation. confirmation of results is possible via immunofluorescence or rt-pcr assays (panning et al., ; staples et al., dash et al., . recently, an immunochromatographic assay used anti-chikv e monoclonal antibodies to detect different chikv genotypes in samples from acutely infected patients. this highly specific and sensitive assay may also be an alternative method for chikv infection diagnosis (okabayashi et al., ) . molecular methods of chikv diagnosis, such as rt-pcr, rt-lamp, qrt-pcr, have gained increasing importance. they are more sensitive and faster than viral isolation, and permit rna detection from all chikv lineages with high specificity. usually, serum samples collected up to seven days of symptom-onset are suitable for chikv detection by molecular diagnostic platforms (edwards et al., ; litzba et al., ; sharma et al., ) . in addition, novel multiplex assays are capable of differentiating chikv from other infectious agents with a similar clinical spectrum. among them, rt-lamp assay has been shown to be capable of differentiating between zikv, chikv and denv infections (yaren et al., ) . a rt-qpcr capable of successfully differentiating between zikv-chikv-denv and chikv-denv-leptospira infections was also recently described (pabbaraju et al., ; giry et al., ) . in later phases of infection, chikv detection is usually based on serological methods, such as elisa and plaque reduction neutralization testing (prnt). elisa techniques are useful to distinguish between acute or convalescent infections via detection of anti-chikv igm or igg antibodies. igm can be detected from two/four days up to three months after the onset of illness, while igg can be detected for several years (grivard et al., ; pialoux et al., ; reddy et al., ) . moreover, elisa for chikv diagnosis are highly specific and have a high accuracy (johnson et al., ) . igm antibody-capture elisa (mac-elisa), via which igm antibodies can be detected in serum samples collected from four days after onset of symptoms, are the most table signals and symptoms that may contribute in the differential diagnosis between dengue, chikungunya and zika illnesses. commonly used tests for laboratory-based diagnoses (reddy et al., ) . prnt, used as a parameter to measure circulating neutralizing antibodies, is useful to establish immunoprotection levels based on the determination of serum antibody titers required to neutralize a known amount of infectious virus particles (azami et al., ) . alternative techniques for anti-chikv antibody detection include immunofluorescence and hemagglutination inhibition (staples et al., ). in general, any suitable etiological diagnosis should be reached through combined epidemiological, clinical and laboratory approaches performed by experienced health professionals. an algorithm-guided infographic with specific laboratory key findings to be used for differential diagnosis of chikv, denv and/or zikv infections is summarized in fig. . chikungunya dengue zika fever > °c ( - d a ) > °c ( - d) ≤ °c ( - d) rash ++ (d -d b ) + (d c ) +++ (d -d there is no licensed specific antiviral available for the control of chikv replication, thus therapeutic strategies must be supportive and symptomatic, including fluid intake (jain et al., ; who, ; kaur and chu, ) . in this context, non-steroidal anti-inflammatory drugs (nsaids), such as paracetamol, are indicated to reduce the fever and relieve arthralgic pain (who, ) . however, nsaids that interfere (at secondary level) with platelet aggregation or with other mechanisms of blood clotting (e.g. aspirin) should be avoided (goupil and mores, ) . the co-administration of nsaids with low-dose systemic corticosteroids has been recommended to reduce pain and improve quality of life in treating acute chikungunya cases with arthralgia (padmakumar et al., ) . past concerns that corticosteroid treatment may exacerbate alphaviral arthritides appear unjustified, since serodiagnosis has demonstrated antiviral immunity (mylonas et al., ) . a succinct overview of current strategies for inhibition of chikv infection has recently been published (subudhi et al., ) . briefly, among the anti-chikv drugs under evaluation, there are preparations that target the viral adsorption and fusion, translation of viral protein and genome replication (mainly in viral non-structural protein , nsp ), maturation of viral glycoproteins and immunological molecules (brighton, ; briolant et al., once the molecular test is negative, serological test must be performed. *perform serological tests for samples collected ≥ days after the onset of clinical signs and symptoms; **prnt is required due to the cross-reaction between denv and zikv; ***test also for other flavivirus (e.g. west nile and saint louis encephalitis viruses). reference: cdc ( ) and paho ( ). drugs and compounds under evaluation for treatment of chikv infection. licensed for malaria treatment antiviral activity has been already demonstrated against hiv a , sars-cov b and sfv. a pilot study shown that chloroquine could be employed in the treatment of arthralgia in chronic chikungunya. however, its use in the acute phase is still debated and some studies have also shown an increase of viral replication of sfv and ecmv after treatment with chloroquine. (brighton, ; de lamballerie et al., ; maheshwari, srikanta, bhartiyaet, ; khan et al., ) arbidol inhibition of the fusion between virus particle and plasma membrane, and between virus particle and the membrane of endosome ( silymarin reduction of both chikv replication efficiency and down-regulating of viral proteins involved in replication. silymarin interferes with post-entry stages of chikv infection, reducing, in a dose dependent manner, the nsp , nsp and e proteins production. (lani et al., ) ribavirin probable inhibition of the viral mrna polymerase by binding to the nucleotide binding site of the enzyme. antiviral licensed for treatment of rsv f and hcv g . patients in the drug group reported improvement in the joint pains and the soft tissue swelling also reduced. together with ifn-alpha b was able to inhibit chikv and sfv replication in vero cells (ravichandran, manian; palumbo, ; turner et al., ; briolant et al., ) decanoyl-rvkr-chloromethyl ketone (dec-rvkr-cmk) inhibition of the maturation of e glycoprotein by inhibition of furin. wichit et al., ) . examples of drugs and compounds under evaluation are available on table . in the absence of therapeutic strategies and licensed vaccines, efficient vector control plays a crucial role in chikv prevention (huang et al., ) . unfortunately, uncontrolled urbanization, lack of proper basic sanitation and increasing resistance to various classes of insecticides challenge the true impact of vector control measures for the reduction of arbovirus incidence (resnik, ; liang et al., who, . to overcome these obstacles, integrated anti-virus control is required and should include: a) epidemiological surveillance; b) environmental management focusing on educative actions to eliminate potential mosquito breeding sites and reduce standing water sites; c) chemical control using repellents (mainly for travelers and pregnant women) and insecticides, respecting the vectors resistance; and d) biological control against eggs, larvae and mosquitoes (fig. ) (hemingway and ranson, ; dumont and chiroleu, ; benelli, ; islam et al., ; benelli, ) . in this last context, larvivorous fish belonging to the genus gambusia (e.g. gambusia affinis) (baird and girard, ) and poecilia (e.g. poecilia reticulata) (peter, ) have been suggested in several countries and regions for mosquito control, mainly for ae. aegypti (hoy, ; das and prasad, ; cavalcanti et al., ; walton, ; chandra et al., ; seng et al., ; dumont and chiroleu, ; kweka et al., ; kamareddine, ; shulse et al., ; pereira and oliveira, ; chobu et al., ; . more natural mosquito predators, including copepods [e.g. mesocyclops thermocyclopoides (harada, ) and mesocyclops longisetus (thiébaud, ) ] and other invertebrate aquatic organism, have also been implemented successfully to reduce ae. aegypti and other culicidae populations (rawlins et al., ; manrique-saide et al., ; vu et al., ; schaper, ; mahesh kumar et al., ; benelli, ; pavela, ) . plant-borne molecules have shown activity against aedes, anopheles and culex larval instars . more recent approaches, based on "green"-synthesized nanoparticles, have been suggested for use against denv and ae. aegypti vector (madhiyazhagan et al., ; sujitha et al., ; . microbiological interventions, such as the use of bacillus thuringiensis var. israelensis (bti) (berliner, ) and entomopathogenic fungi [e.g. metarhizium anisopliae (metschnikoff, ) sorokin, and beauveria bassiana (balsamo) vuillemin, )], have shown effects on malaria mosquitoes, as well as on ae. aegypti and ae. albopictus (novak et al., ; blanford et al., ; armengol et al., ; knols et al., ; lam et al., ; ritchie et al., ; paula et al., a, b) . recently, bti was used to prevent zikv transmission in the continental united states (stoddard, ) . in another promising approach to arbovirus control, including also zikv, the release of wolbachia-infected male mosquitoes, sterile mosquitoes or insects carrying a dominant lethal gene can be used to reduce ae. aegypti and ae. albopictus populations (fig. ) (ferreira et al., ; alphey et al., ; walker et al., ; boyer, ; alphey et al., ; massonnet-bruneel et al., ; lee et al., ; zhang et al., a, b; dickens et al., ) . despite the theoretical basis for their effectiveness, additional studies are needed to verify the true risks and benefits of programs based on altered mosquitoes. this concern appears to be greater in relation to transgenic mosquitoes, especially on their efficacy, sustainability and impact on the environment and target and non-target species (wilke et al., ) . although many of these strategies have been initially directed against denv, their application to transmission control of chikv should be considered, mainly within an integrated approach, which rationally combines different measures. control programs for other insect-borne diseases, such as malaria (benelli and beier, ) , can also serve as model for chikv prevention. in a synergistic way, mass immunization against chikv would be an important tool for viral control and prophylaxis. several distinct vaccine approaches are currently under development, however, no vaccine has been licensed. anti-chikv candidates that have been already tested in humans and/or animals include inactivated-, attenuated-, virus like particle-(vlp), dna-and chimeric vaccines (eckels et al., ; levitt et al., ; muthumani et al., ; wang et al., ; tiwari et al., ; sharma et al., akahata et al., plante et al., ; wang et al., ; gorchakov et al., ; brandler et al., ; chang et al., ; garcía-arriaza et al., ; tretyakova et al., ; van den doel et al., ; erasmus et al., ) . in the past, viral inactivation strategies were the first to be tested in the course of anti-chikv vaccine development. chikv replicated in cell culture and subsequently inactivated by formalin, ether or , iodonapthyl azide (ina) was able to stimulate the production of neutralizing antibodies (eckels et al., ; tiwari et al., ; sharma et al., ) . particularly, the use of ina resulted in reduced binding capacity of anti-e neutralizing antibodies (sharma et al., ) , while formalin inactivation stimulated the cellular immune response with the production of anti-and pro-inflammatory cytokines (tiwari et al., ) . with the same aim of outlining the virulence of chikv infection, brandler et al. ( ) reported a recombinant live-attenuated measles vaccine expressing a vlp composed of the capsid (c) and envelope (e) proteins from the la réunion - chikv strain (ecsa lineage). in mice susceptible to measles virus (mv), immunization with mv-chikv induced high titers of chikv antibodies, specific cellular immune responses and protected all animals from lethal chikv challenge (brandler et al., ) . in phase i clinical trial (european clinical trials database, - -fig. . management for an integrated aedes vector control. *the use of insecticides and repellents should be carried out taking into account the mosquitoes' resistance profiles; ** it is important to consider and evaluate the influence of these interventions on ecosystem balance. ), a second vaccination resulted in % seroconversion for all participants. the immunogenicity of the mv-chikv was not affected by preexisting anti-mv immunity and no vaccination-related serious adverse effects were recorded (ramsauer et al., ) . also using the vaccine platform vlp-based, akahata et al. ( ) proposed the use of a vaccinal vlp expressing chikv structural proteins. the vlp-based vaccine, vrc-chkvlp - -vp, was obtained by transfection of a plasmid expressing c and e proteins of the chikv strain (wa lineage). the vaccine stimulated the production of neutralizing antibodies against the e protein of different chikv strains and was able to protect challenged monkeys. further tests in humans showed vaccine efficacy and immunogenicity, without reports of arthralgia as side effect (chang et al., ) . the vrc-chkvlp - -vp is one of the most developed strategies and is currently undergoing phase ii clinical trials (national clinical trials, ) . another approach to chikv vaccine development is the use of dna vaccines. in this context, a plasmid expressing consensus sequences of c, e and e chikv proteins elicited a robust cellular immune response and the production of high antibody titers capable of recognizing wild virus antigens (muthumani et al., ) . in another study, mice immunized with a dna vaccine containing the complete chikv genome of the / strain developed neutralizing antibodies and were protected from neurovirulent chikv (tretyakova et al., ) . these strategies involving inactivated viruses, vlp and dna vaccines often stimulate the humoral immune response, one of the main mechanisms for control and prevention of chikv infection (lum et al., ) . empirically-or reverse-engineered attenuated live vaccines, however, have been shown to be capable of inducing both cellular and humoral immune responses and have also been suggested to prevent chikv infection (levitt et al., ; wang et al., wang et al., , plante et al., ; gorchakov et al., ; garcía-arriaza et al., ) . an important example of attenuated chikv vaccine is the tsi-gsd- , a vaccine based on an empirically attenuated chikv strain (asian lineage) isolated from patient sera from the chikv outbreak in thailand (levitt et al., ) . by the end of its phase ii clinical trial evaluation, % of the vaccinated individuals presented neutralizing antibodies, % remained seroconverted after one year and . % presented temporary arthralgia (edelman et al., ) . in addition to classical attenuation via serial viral passage in cells, reverse genetics strategies have been employed as platforms for construction of recombinant attenuated viruses or vaccine chimeras (wang et al., ; plante et al., ; wang et al., ; garcía-arriaza et al., ; van den doel et al., ; erasmus et al., ). an anti-chikv strategy involves the development of an attenuated chimeric vaccine using the internal ribosome entry site (ires) of the encephalomyocarditis virus (ecmv). in this approach, the chikv subgenomic promoter from the lr -opy strain (ecsa lineage) is knocked via insertion of synonymous mutations and the ecmv ires sequence is added as new promoter for subgenomic rna transcription (plante et al., ) . briefly, the addition of the ires sequence to the attenuated viral genome prevents viral propagation in mosquito cells, thus restricting the target population. this vaccine strategy has been shown to lead to the stimulation of tcd and tcd responses in mice (plante et al., ) . in nhp, chikv/ires vaccine was also showed to be safe and immunogenically effective (roy et al., ) . finally, the preclinical safety of the chikv/ires vaccine was ensured in interferonα/β receptor-incompetent mice (a mice) (plante et al., ) . other viral chimeras have been proposed in the context of anti-chikv vaccine strategies. notably, attenuated strains of the eastern equine encephalitis virus (veev) or eastern equine encephalitis virus (eeev) were used as backbones to express chikv structural proteins, creating immunogenic chimeras able to stimulate production of neutralizing antibodies and protect mice against disease and viremia after chikv challenge (wang et al., ) . another chimeric construction, the modified vaccinia ankara expressing e glycoprotein or e -e - h-e proteins was shown to stimulate production of neutralizing antibodies in ifn-i (ifn α/β) and ii (ifn-γ) receptor knockout mice (ag mice), protecting against lethal infection (van den doel et al., ) . a replication-incompetent adenovirus vector also has been used to express the orf coding chikv structural polyproteins. a single dose of the chimera induced high antibodies titers capable of neutralizing asian and iol chikv lineages, protecting mice against viremia and arthralgia . most recently, a promising vaccine based on a chimera of eilat virus (eilv) and chikv has been reported (erasmus et al., ) . the eliv/ chikv possesses non-structural proteins of eilv and structural and accessory proteins of chikv strain (asian lineage). since the eilv is an alphavirus specific for insects, the eilv/chikv was unable to replicate in vertebrate hosts, thereby providing a high degree of safety. in nhp, eliv/chikv stimulated immune response and guaranteed protection against viremia (erasmus et al., ) . in the last decade, there has been an increase in the dissemination and co-circulation of several arboviruses. currently, areas that were endemic just to denv, for instance, are with autochthonous cases of chikungunya and zika diseases. these arboviruses have similar clinical spectrum and require an efficient laboratory diagnosis, especially for a rigorous epidemiological surveillance. in addition, chikv infections represent a serious public health problem. the high morbidity of chikungunya often results in absenteeism of afflicted individuals, incurring both psychosocial and economic impacts. in this context, the development of a specific anti-chikv drug is certainly an important demand. on the other hand, the ideal control strategy for chikv should combine an integrated vector management with mass immunization. although the different vector biocontrol strategies are promising, mainly within an integrated use, it is necessary to think about their sustainable use, assessing their real impacts on ecosystem equilibrium. the absence of chikv serotypes is considered a facilitative aspect in anti-chikv vaccine development, since a formulation developed from one strain will likely result in immunity against all chikv (smalley et al., ) . despite recent advances in vaccine strategies, the major challenge regarding chikv vaccine development remains the establishment of an equilibrium between immunogenicity and safety, notably a reduction of side effects, such as secondary arthralgia following immunization with attenuated virus. finally, recognizing the competence of vector and arboviruses control measures, we believe that the prevention of chikv infections should be planned within a global and multifactorial approach. this interdisciplinary strategy, currently framed within the one health concept, should thus integrate all aspects of health care for humans, animals and the environment (benelli and duggan, ) . the authors have no conflicts of interest to disclose. acknowledgments e.f. oliveira-filho and r. 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a chikungunya virus receptor protein assessment of flavaglines as potential chikungunya virus entry inhibitors point of sampling detection of zika virus within a multiplexed kit capable of detecting dengue and chikungunya chikungunya: its history in africa and asia and its spread to new regions in - combining the sterile insect technique with wolbachia-based approaches: ii-a safer approach to aedes albopictus population suppression programmes, designed to minimize the consequences of inadvertent female release combining the sterile insect technique with the incompatible insect technique: i-impact of wolbachia infection on the fitness of triple-and double-infected strains of aedes albopictus key: cord- -r on lpr authors: veggiani, gianluca; gerpe, maría carla rosales; sidhu, sachdev s.; zhang, wei title: emerging drug development technologies targeting ubiquitination for cancer therapeutics date: - - journal: pharmacol ther doi: . /j.pharmthera. . . sha: doc_id: cord_uid: r on lpr development of effective cancer therapeutic strategies relies on our ability to interfere with cellular processes that are dysregulated in tumors. given the essential role of the ubiquitin proteasome system (ups) in regulating a myriad of cellular processes, it is not surprising that malfunction of ups components is implicated in numerous human diseases, including many types of cancer. the clinical success of proteasome inhibitors in treating multiple myeloma has further stimulated enthusiasm for targeting ups proteins for pharmacological intervention in cancer treatment, particularly in the precision medicine era. unfortunately, despite tremendous efforts, the paucity of potent and selective ups inhibitors has severely hampered attempts to exploit the ups for therapeutic benefits. to tackle this problem, many groups have been working on technology advancement to rapidly and effectively screen for potent and specific ups modulators as intracellular probes or early-phase therapeutic agents. here, we review several emerging technologies for developing chemical- and protein-based molecules to manipulate ups enzymatic activity, with the aim of providing an overview of strategies available to target ubiquitination for cancer therapy. ever since the discovery of ubiquitin (ub) four decades ago (ciehanover, hod, & hershko, ) , this small protein has been linked to multiple cellular processes, including cell proliferation, development, immune responses, and numerous human diseases including cancer (calistri, munegato, carli, parolin, & palu, ; dantuma & bott, ; senft, qi, & ronai, ; zinngrebe, montinaro, peltzer, & walczak, ) . indeed, protein ubiquitination, the process of attaching ub and poly-ub chains to cellular substrates, is an integral and essential part of the cell machinery, and serves to modify, regulate, and degrade proteins ( fig. ) (swatek & komander, ) . ubiquitination initiates through the activation of ub by a ub activating enzyme (e ), which adenylates the ub c-terminus using its catalytic cysteine residue, resulting in a thioester bond. another thioester reaction is required for the transfer of ub onto a ub conjugating enzyme (e ). the ub-charged e enzyme is then recruited by a ub ligase (e ), which is responsible for the transfer of ub from its e -conjugated state to the substrate protein. to date, two ub-specific e activating enzymes have been identified in humans (groettrup, pelzer, schmidtke, & hofmann, ; mchugh et al., ; schulman & harper, ) , and n e s have been found in humans (morreale & walden, ; mulder et al., ) . humans also harbour approximately e ub ligases, which are classified into the homologous to e -associated protein carboxy terminus (hect) family, the really interesting new gene (ring) family and the u-box family (morreale & walden, ; mulder, harari, & simon, ; zhang & sidhu, ) . e ligases bind e enzymes thioesterified with ub (e ~ub) to mediate substrate ubiquitination through different catalytic mechanisms. the hect e ligases form an intermediate thioester bond with ub to its active site cysteine residue before transferring it to substrates. in contrast, lacking a catalytic cysteine, the ring and u-box e ligases facilitate ub transfer by an allosteric mechanism (chen et al., ; ozkan, yu, & deisenhofer, ; rotin & kumar, ; zheng & shabek, ) . single or multiple mono-ubiquitination and polyubiquitination at the n-terminus or lysine residues in ub are all possible modifications of protein substrates and will determine the fate of the protein (calistri et al., ) . ubiquitination at residue k of ub has been linked to endoplasmic reticulum-associated protein degradation (locke, toth, & petroski, ) ; k -linked ubiquitination has been associated with protein modification (nucifora jr. et al., ) ; k -linked ubiquitination is well studied for its role in proteasomal degradation of cellular substrates (calistri et al., ) ; and, k -linked ubiquitination results in protein recruitment, autophagy and degradation through the lysosomal pathway (erpapazoglou, walker, & haguenauer-tsapis, ) . on the other end of the spectrum, the process of ubiquitination can be reversed by deubiquitinating enzymes known as deubiquitinases (dubs) (zhang & sidhu, ) . humans encode seven different structural families of dubs, which comprise almost proteins in total (hermanns et al., ; wertz & wang, ) . ub-specific proteases (usps) represent the largest and best studied family, with close to usps currently described (yuan et al., ) . less research has been reported for the other families, with the next best studied being ovarian fig. . drug targets in the ubiquitin proteasome system. (a) schematic representation of the ubiquitination pathway. ubiquitination involves three steps. first, e activates ubiquitin (ub) with atp, forming a thioester bond. next, activated ub is conjugated to the e enzyme via a transthiolation reaction. finally, ring and u-box e ligases recruit the e enzyme and a substrate to facilitate transfer of ub from the e to the substrate by an allosteric mechanism, whereas hect e ligases directly ubiquitinate the substrate followed by the transfer of ub from e to the hect e ligase active site (chen et al., ; zhang & sidhu, ) . the diagram also illustrates drug development targets currently in the market (green dot), in clinical trials (blue dot), or still being tested in industry or academia (red dot). *pimozide is currently used in the clinic, but not for cancer therapy. # previously known as mln (tong et al., ) . ** identified within a small-molecule screen as an inhibitor of ubc -uev a e ub conjugating enzyme (pulvino et al., ) . (b) ub is conjugated through lysines once (monoubiquitination) or multiple times (multiubiquitination), as a single unit or as a chain (polyubiquitination). monoubiquitination and multiubiquitination processes have been linked to protein function modification, receptor endocytosis, nuclear export, transcription and histone regulation. lys -and lys -linked polyubiquitination has been shown to lead to proteasomal degradation as part of the ups. lys -linked polyubiquitination has been implicated in the lysosomal pathway, protein recruitment, membrane internalization, endosomal uptake, and autophagy. deubiquitination may also occur (swatek & komander, ) . tumor ub proteases (otu), followed by jab /mpn+/mov domain (jamm) metallo-enzyme proteases, ub c-terminal hydrolases (uchs), and josephin domain proteases (yuan et al., ) . targeting the ubiquitination pathway has been an attractive avenue for anti-cancer drug development, especially after the approval and success of proteasome inhibitor drugs (bortezomib and carfilzomib) ( fig. ) for treatment of relapsed or refractory multiple myeloma (richardson et al., ; richardson, et al., ) . this has propelled many studies and clinical trials to find new therapeutics targeting individual components in the ubiquitination pathway for the treatment of cancer, with the aim of providing better therapeutics with fewer side effects (table ) (chen, frezza, schmitt, kanwar, & dou, ; iida et al., ; yazbeck et al., ) . another important aspect to consider is the selective killing of cancerous cells rather than normal cells through targeting ubiquitination, as ubiquitination is upregulated due to the error-prone nature of cancerous cells and tissues (gallo, ko, & donoghue, ; ge et al., ) . indeed, bortezomib and carfilzomib have higher activity in malignant cells than in normal cells and tissues (berkers et al., ; codony-servat et al., ; parlati et al., ) . other inhibitors of ups components have also been shown to uniquely target cancerous cells (fiskus et al., ; li et al., ; pulvino et al., ; tsukamoto et al., ) . finally, the resistant, refractive and recurrent traits of cancer have inevitably resulted in the turnover of promising drugs like bortezomib, due to the development of resistance in response to treatment (hamouda et al., ) . consequently, finding new compounds that can target oncogenic pathways is crucial to establish effective cancer therapies. therefore, development of innovative strategies to validate and target ups proteins remains an important aspect of drug discovery in cancer research. before diving into recent advances in technology, we briefly describe the early stage of drug discovery in a historical perspective. two crucial types of screening methods were pioneered and developed in the s: target-based screens, which focus on a specific protein of interest reviewed by croston ( ) , and phenotypic screens, which concentrate on the disease state to be treatedreviewed by wagner ( ) . target-based high-throughput screening (hts) strategies for drug discovery kicked off in the early s (pereira & williams, ) (fig. ) . around the same time, researchers started to use phenotypic screens and genomic manipulation to paint a picture of cellular replication and the normal state of the cell, mainly in yeast as a model organism (fraczek, naseeb, & delneri, ; strynatka, gurrola-gal, berman, & mcmaster, ) . the combination of these approaches in the late s resulted in the birth of chemical genetics and led to many powerful developments such as directed evolution of enzymes, phage display of peptides and antibodies, and further refinement of target-based hts (e.g. combinatorial chemistry, split-and-pool synthesis and fragment-based discovery) (furka, asgedom, & diboa, ; smith, ; strynatka et al., ) (fig. ) . from the late s to the beginning of the s, the use of animal models shifted to cell-based screens that minimized the number of reagents required and allowed the output of screens to increase (strynatka et al., ) (fig. ) . research during these years also brought forth the generation of the first dna-encoded compound libraries (decls) (brenner & lerner, ; kinoshita & nishigaki, ) (fig. ) . within the span of years, high-throughput technologies further advanced to include feats in protein engineering such as proteolysis-targeting chimeras (protacs) derived from the ubiquitination pathway (zhou, bogacki, mcreynolds, & howley, ) , streamlined phage display approaches that include ub variants (ubv) to target protein-protein interactions in the ups (brown et al., ; ernst et al., ; ernst & sidhu, ; gabrielsen et al., ; ordureau et al., ; zhang et al., ; zhang et al., ; zhang et al., ; zhang & sidhu, ) and cell-based pharmacological hts assays to enhance oncolytic virus cancer-cell-killing efficiency through viral sensitizer screens (bourgeois-daigneault et al., ; diallo et al., ) (fig. ) . in less than ten years, from to , hts targetbased and phenotypic approaches were pivotal for drug discovery, yielding % and % of new molecular entities and next-generation drugs tested in clinical trials, respectively (swinney & anthony, ) . for instance, e ub conjugating enzyme inhibitors were discovered through a phenotypic and target-based small molecule screen for (chen, dexheimer, et al., ; davydov et al., ; dodge, ; gavory et al., ; gombodorj et al., ; lee et al., ; o'dowd et al., ; tong et al., ; yang et al., ; yang et al., ) (lebraud et al., ; sakamoto et al., ; sakamoto et al., ; xie et al., ) protein design and engineering zhang et al., ) tuberculosis therapeutics (darwin, ehrt, gutierrez-ramos, weich, & nathan, ) ( fig. a and table ). since the ubiquitination pathways and ups targets implicated in cancer drug development have been extensively reviewed (kar, keskin, fraternali, & gursoy, ; li et al., ; pal, young, & donato, ; shen, schmitt, buac, & dou, ; wertz & wang, ; zhang & sidhu, ) , here we focus on addressing emerging trends and applications that can rapidly identify modulators for proteins within the ubiquitination pathway. the topics discussed below will examine the use of high-throughput technologies, small molecule approaches, peptide mimetics, and protein engineering. in addition, we will discuss potential applications for these technologies in cancer therapeutic research. research on chemical space (jencks, ) stimulated the development of more ambitious target-based hts approaches (fig. ) . drugprotein interactions were simplified into binding events, where the probability of binding would increase with the size of the molecule (jencks, ) . previous hts relied on assaying natural or synthetic compounds, which typically originated from tedious and timeconsuming reactions of individual reagents (pereira & williams, ) (fig. ) . with the advent of combinatorial chemistry, mixtures (instead of individual reagents) began to be employed to generate many more different compounds (furka, ) (figs. and a) . later, furka and colleagues introduced split-and-pool synthesis (furka et al., ) , in which the notion of combinatorial chemistry was further exploited to include separation and ligation of compounds. these separation and ligation reactions increased the number of potential combinations (fig. a) , which were estimated to total approximately to million chemical compounds globally (hann & oprea, ) . this number has been exceeded now and million-to billion-compound libraries are routinely screened in industry (davis, plowright, & valeur, ; goodnow jr., dumelin, & keefe, ; litovchick et al., ) . however, since the potential drug-like chemical space is estimated at , these numbers still barely scratch the surface (davis et al., ) . although combinatorial chemistry and split-and-pool synthesis sampled larger chemical space and revolutionized the way hts was conducted for target-based drug discovery (potyrailo et al., ) , progress remains hampered by significant bottlenecks, including the expense of setting up an hts system, starting reagents with poor properties, and the presence of reactive and aggregator molecules (table ) (erlanson, ) . in fact, in the early s, compounds with poor properties like high lipophilicity were responsible for % of failed hts projects (keseru & makara, ) . starter reagents that lack qualities such as solubility result in inefficient downstream drug candidates that might be impractical in a clinical setting (keseru & makara, ). other problems such as reactive molecules and aggregators also delayed progress in target-based hts (erlanson, ) . reactive molecules or small impurities can oxidize proteins under the conditions used during hts, resulting in the generation of hydrogen peroxide, which can inactivate proteins and cause false positives. reactive molecules could arise from the possible combinations in split-and-pool synthesis, or simply as a side product of combinatorial chemistry (erlanson, ) . finally, aggregators are a side effect of high concentration assays. at high concentration, intermolecular interactions of a compound can result in non-specific inhibition of the target protein, but the compound itself might not be a high affinity binder (erlanson, ) . this effect has been documented in past studies, where as many as % of compounds tested were identified as aggregators (babaoglu et al., ) . while these pitfalls remain as intrinsic caveats for small molecule libraries, activity-based small molecule screens have identified ups inhibitors (chen, dexheimer, et al., ; davydov et al., ; gombodorj et al., ; ott et al., ; pulvino et al., ; yang et al., ; yang et al., ) . the cumbersome bottlenecks described above prompted the use of approaches to utilize chemical space in more effective ways (erlanson, ; yuen & franzini, ) . in the activity-based screen approach, small molecules are screened to identify high affinity binders. in these screens, small molecules are added to cells, cell lysates, or in vitro reactions with recombinant proteins to test for inhibition of activity of a target protein or a change in phenotype. this can be detected through the presence or absence of fluorescence or luminescence (janzen, ) . some of the most commonly used screening technologies include imaging or detection of: binding-or cleavage-based excitation of fluorescent probe-labeled proteins, fluorescence labeled antibodies targeting a specific protein, and fluorescence resonance energy transfer (fret) where one fluorophore emits energy and a proximal one absorbs this energy for excitation. screens can also be conducted with the use of flow cytometry, which can measure the light scattered through a cell to determine phenotype or expression of fluorescent-labeled proteins within the cell, and with luminescence-based assays, which are similarly designed to the fluorescent imaging assays mentioned above (janzen, ) . below, we present several representative studies utilizing these screening methods to develop chemical compounds targeting ups components of different protein families (table ) . one group was able to identify two molecules, pyr- and hli ( fig. ) , which inhibited the e activating enzyme uba (yang et al., ) and the ring-e ligase hdm (yang et al., ) , respectively, by first screening a commercial chemical library and then confirming the leads with purchased individual compounds (table ). this small-molecule library was previously developed by the vousden group to target autoubiquitination of e ligases (davydov et al., ) . in this assay, small molecules were incubated in ubiquitination reactions with recombinant e and e (ubch b), e (hdm ), and ub. an in vitro cellular assays (gavory et al., ; o'dowd et al., ) electrochemiluminescence (ecm) labeled antibody targeting ubiquitinated proteins was subsequently added. the authors proposed that reactions with significantly reduced ecm represented small molecule hits inhibiting hdm enzymatic activity (davydov et al., ; yang et al., ; yang et al., ) . during validation of these hits, pyr- , a pyrazone derivative (yang et al., ) , was found to target the e enzyme uba , and inhibit its activity with an ic of approximately μm (yang et al., ) . hli , a compound from a newly identified -nitro- -deazaflavin family (davydov et al., ; yang et al., ) , was shown to target hdm e ligase activity with an ic of approximately μm (yang et al., ) . to our knowledge, off-target effects and intracellular efficacy have yet to be thoroughly assessed for hli . the promiscuous nature of the assay in that it detects ubiquitinated proteins and the high ic value suggest that other cellular targets of hli may exist. another e -inhibitor, mln- or pevonedistat ( fig. and table ), is an adenosine sulfamate mimetic (chen, tsu, et al., ) . penovedistat was developed from a medicinal chemistry approach aiming to improve on a previously discovered inhibitor, n -benzyl adenosine, from a high-throughput screen (soucy et al., ) . pevonedistat was originally identified as an inhibitor of nedd activating e -ubiquitin activating enzyme (uba ) complex (soucy et al., ) and was later labeled as a pan-inhibitor of e activating enzymes (da silva et al., ; gavin et al., ; wertz & wang, ) . soucy et al. reported potent inhibition of uba in the single-digit nanomolar range with cross-reactivity against other e s in the low micromolar range (soucy et al., ) . pevonedistat is currently being tested in clinical trials of patients with acute myeloid leukemia, where the principal side effect seems to be liver toxicity and sepsis due to disruptions in the gtpase rhoa cytoskeleton protein and tumor necrosis factor (tnf)-α (swords et al., ; swords et al., ) . e inhibitors were also identified using a luciferase reporter cell line, in which inhibitor-mediated inactivation of the target protein resulted in loss of luciferase expression ( fig. and table ). in this study, a small molecule, the nitrofuran nsc , inhibited an e ub conjugating enzyme, ube n/ubc (in complex with ube v /uev a) by preventing thioester bond formation between the e and ub, and consequently, supressed nf-κb activity in active b-cell lymphoma cells (gombodorj et al., ; pulvino et al., ) (fig. ) . another study also identified nsc as an inhibitor of ubc in neuroblastoma cells (cheng et al., ) (fig. ) . nsc has been shown to inhibit ubc at concentrations of~ μm (hodge et al., ) . another group developed the first described fluorometric cell lysatebased assay to mirror physiological conditions for small molecule screens (ott et al., ) . they targeted uchl , a dub that is overexpressed in many cancers (bishop, rocca, & henley, ) , as a proof of principle for developing dub inhibitors with a cell lysate-based assay that they called alphalisa (ott et al., ) . ha-tagged uchl was labeled with an alpha-streptavidin donor bead and probed with biotin-tagged ub. binding of ub to the dub would result in fluorescence, and conversely, no fluorescence would occur in the presence of an inhibitor (ott et al., ) . the screen yielded a series of compounds that inhibited dub activity in the - μm range, and some had been reported previously, including isogambonic acid, celastrol, mangiferin and rifampicin (ott et al., ) . inhibitors of the dub usp , which has been implicated in the fanconi anemia signalling pathway in the dna damage response (bergink & jentsch, ; huang et al., ; nijman et al., ) , were identified with a small-molecule fluorometric assay that employed a ub substrate modified with rhodamine (chen, dexheimer, et al., ) , which emitted fluorescence upon cleavage (dang, melandri, & stein, ) . this simple in vitro assay could be used in a high-throughput fashion to screen for both small-molecule inhibitors and previously reported larger inhibitors (dang et al., ) . the study identified both novel small-molecule compounds as well as approved drugs, such as pimozide (a diphenylbutylpiperidine), as inhibitors of usp (chen, dexheimer, et al., ) (fig. and table ). in non-small cell lung carcinoma cells, pimozide was shown to reverse the chemo-resistance observed with cisplatin (chen, dexheimer, et al., ) , a commonly used chemotherapy drug (bloemink & reedijk, ; dasari & tchounwou, ) with known resistance in fanconi anemia (wang, ) . interestingly, pimozide is an anti-psychotic drug commonly prescribed to patients suffering from schizophrenia (seeman, ) , and although it has been postulated to act as a postsynaptic dopamine receptor blocker, its mechanism remains unknown (seeman, ) . another study confirmed that pimozide could inhibit usp and in turn decrease the clonal growth of glioblastoma cells as well as increase their sensitivity to irradiation, though they are commonly resistant to chemotherapeutic and irradiation treatments (lee et al., ) . notwithstanding this success, the potency of pimozide (ic ~ μm) is lower than those of clinically approved ups inhibitor s proteasome inhibitor bortezomib (ic ~ nm) ( fig. and table ), and another ups inhibitor vlx , which inhibited the dubs uchl and usp (ic ~ nm) and was previously studied in clinical trials but terminated due to limiting toxicities (study identifier nct ) (clinicaltrials.gov, ; wang et al., ) . while in general activity-and target-based small molecule screens have provided innovative approaches that make the process of drug discovery more economical and efficient, for most ups targets, these types of screens yielded compounds with sub-optimal pharmacological properties (e.g. biophysics, solubility, and cellular permeability) and fall short in terms of potency and specificity (lill & wertz, ) . we postulate that the intrinsic structural and catalytic properties of ups proteins pose an impediment to generation of active-site inhibitors. for example, hect e s contains a shallow active site and are subject to dynamic regulation including extreme inter-domain rotations accompanying catalysis (zheng & shabek, ) . on the other hand, the conserved nature of the dub catalytic pocket probably accounts for the observed low specificity of inhibitors targeting the cysteine active site . indeed, almost all of the ups small molecule inhibitors currently in the clinic or clinical trials function in an allosteric manner. for instance, nutlins disrupt substrate (p ) binding to the ring-e ligase mdm to stabilize p protein level and transcriptional activity (vassilev et al., ) , and smac mimetics compete with caspase binding to the bir domain of ring-e iap (inhibitor of apoptosis protein) to stimulate apoptosis in cancer cells (liu et al., ; wu et al., ) . finally, other than proteasome inhibitors like bortezomib and carfilzomib, there is only one family of drugs (thalidomide/lenalidomide) targeting ups components approved for treatment of cancer, specifically for multiple myeloma and other b-cell neoplasms. thalidomide was once well known for its adverse effects on fetus development before repurposing for cancer therapy through binding to an e ligase cereblon (crbn) and stimulating lymphoid transcription factor ubiquitination and degradation (ito et al., ; kronke et al., ; lu et al., ) . therefore, further development of allosteric modulators for ups components will likely lead to more effective and efficacious cancer therapeutics. what distinguishes fragment-based approaches from other smallmolecule hts assays is the further fine-tuning of small-molecule screens. once fragments are identified as binders of the target protein, researchers conduct medicinal chemistry to grow, ligate and merge the fragments into potent and selective inhibitors (erlanson, ; lamoree & hubbard, ) (fig. b) . in this approach, the focus is not on activity, but rather in discovering potential allosteric inhibitors. as such, the approach is not to detect the fragment binders in an environment where the protein might be active, but to first detect whether the fragment binds to the target protein, and customize the fragment via medicinal chemistry based on information gathered through biophysical analyses. in an elegant example, the fragment-based approach was used to develop inhibitors of usp (gavory et al., ) (table ) . first, surface plasmon resonance (spr) was used to screen a small library of molecules targeting the catalytic domain of usp , which is responsible for regulating mdm , the dominant ub ligase for p (gavory et al., ; iwakuma & lozano, ) . what distinguished this work from previous studies, which pioneered the first usp inhibitors (kategaya et al., ; pozhidaeva et al., ) , was the use of medicinal chemistry to tailor initial screening hits. the inhibitory fragments were used to grow six different compounds that retained the high affinity of the original fragments, setting these results apart from the poor pharmacological properties and low potencies previously obtained (kategaya et al., ; pozhidaeva et al., ) . the new usp inhibitors were also free from reactive species and were not aggregators. importantly, this work identified a new allosteric inhibitory site on usp , implying that affinity-based screens rather than activity-based screens may reveal more druggable sites. another group also used a similar approach to develop usp inhibitors with high potency (turnbull et al., ) . the work exemplified in these two studies is particularly relevant for producing compounds that are active against dubs, where other methods have proven unsuccessful (wrigley et al., ) . the researchers that developed the allosteric usp inhibitors followed up with a study in which they described in detail the fragment-based medicinal chemistry approach to identify novel usp inhibitors (o'dowd et al., ) . this study demonstrated that employing spr, a commonly used technology to detect binding (erlanson, ) , can be first used to identify potential allosteric binders of the protein, before continuing with x-ray crystallography to enable structure-based design (o'dowd et al., ) , an approach that has been responsible for many drugs developed to date (erlanson, ; lamoree & hubbard, ) . with both studies, the authors showed that structure-based design can aid with the elucidation of the mechanisms of action of novel inhibitors and can provide new insights into the functions of complex proteins with additional regulatory sites beyond the active site. we believe that this will have important implications for further development of inhibitors of other dubs. the innovation of decls may transform hit identification of allosteric binders of ups proteins into a faster and more manageable process thanks to the tagging of molecules with oligonucleotides. in hts assays where mixtures of small molecules are used, or when traditional combinatorial chemistry is employed, the disentanglement of hits is usually arduous and time consuming. however, with decl technology, inhibitor leads can be identified by rapid and efficient methods, including the polymerase chain reaction (pcr), deep dna sequencing, and mass spectrometry. ensemble therapeutics has presented data describing the use of decls to develop macrocycle compounds (mullard, ) that selectively and potently inhibit usp x (dodge, ) , a dub that is associated with many cancers (murtaza, jolly, gecz, & wood, ) . however, the results were presented in a conference proceeding, and without further information, the ultimate outcome of this work remains unclear. nonetheless, we predict that there will be more screens employing decls for inhibition of dubs and other ups targets. decls comprise combinatorial molecules, small molecules or fragments that are concatenated to oligonucleotides (fig. ) (mannocci, leimbacher, wichert, scheuermann, & neri, ; yuen & franzini, ) . the oligonucleotides can be dna or peptide nucleic acid (pna) (fig. a) . the greatest advantage of dna is its capacity for amplification. though not amplifiable, pnas have the advantages over dna of being more stable and compatible with solid-phase synthesis. pna libraries are also more amenable to use with combinatorial chemistry and split-and-pool synthesis methods (zambaldo, barluenga, & winssinger, ) (fig. b) . it has been reported that decls can accommodate up to compounds (goodnow jr. et al., ) . the hansen group described a yoctoliter ( − )-scale dna reactor to decrease the amount of material needed to deeply sample chemical space for highly efficient and unbiased molecular evolution (hansen et al., ). the combinatorial chemistry field continues to produce novel strategies for generating decls in a cost-effective manner. because decls are subject to the same processes described above for combinatorial chemistry and split-and-pool synthesis, they are also susceptible to some of the same pitfalls, such as the presence of side reaction intermediates and by-products that can affect interactions and lead to false positives. as such, the generation and downstream purification of these libraries prior to use in hts is of utmost importance. the chemistries applied in these processes are beyond the scope of this review, but they have been reviewed elsewhere (favalli, bassi, scheuermann, & neri, ; franzini et al., ; goodnow jr. et al., ; li, zheng, liu, & li, ) . decls are typically made as single or dual pharmacophores (fig. b) . single pharmacophores can be synthesized with dna-recorded or dnatemplated methods. in dna-recorded methods, compounds are coupled to single-strand (ss) or double-strand (ds) oligonucleotides, and after each coupling, additional dna barcodes corresponding to each individual compound are ligated, followed by amplification to generate dsdna. dna-templating methods involve coupled dnacompound moieties, which interact with each other via barcode complementarity; once in close proximity, coupling linkers are cleaved to release compounds that react with each other to generate cyclical compounds linked to dna barcodes. encoded self-assembled chemical (esac) libraries, also known as dual or complex pharmacophore libraries, initiate with compounds coupled to ssdna; other compounds are coupled to spacers that are used to hybridize two dna-encoded compounds, followed by amplification that results in the formation of dual pharmacophore compounds coupled to dsdna. decls are more cost-effective than fragment-based libraries, since many of the methodologies utilized for hts of decls can be performed on the bench without large, dedicated equipment (yuen & franzini, ) . a tagged target protein can be immobilized to a solid support, and following incubation with the decl, small molecules coupled to dna or pna that bind to the target can be enriched via tag-specific methodologies. following elution, the small molecules can be identified via pcr or sequencing in the case of dna-encoded libraries, or via mass spectrometry, spr or nmr, in the case of pna-encoded libraries (mannocci et al., ) (fig. c) . amplification can also be carried out in situ (yuen & franzini, ) . regardless of the particular approach, inhibitor development with small-molecule hts approaches depends on the solubility of chemical moieties (erlanson, ) . this has prompted the use of phenotypic screens to initialize screening (wagner, ) , and is one of the driving forces behind the field of peptidomimetics, which aims to utilize favorable qualities of peptides, including aqueous solubility, membrane permeability and internalization capacity, while maintaining the ability to interact with proteins and avoiding degradation through proteolysis (vagner, qu, & hruby, ) . peptide libraries have also been generated via solid-phase synthesis (bessette, rice, & daugherty, ; kenrick & daugherty, ; ryvkin et al., ; sidhu, ) , by covalently linking peptides to a resin and conducting the synthesis in the immobilized form (fields, ) . other than solid-phase peptide synthesis, peptide libraries have been typically generated with the use of bacteria or phage display. for example, antimicrobial peptides continue to be identified through screening peptides expressed in bacteria (tucker et al., ) . one study applied phage display to isolate potent competitive peptides for the e binding site of hect e ligases that inhibited hect e ligases by oxidizing the active site cysteine (mund, lewis, maslen, & pelham, ) . phage display, in particular, like decl libraries with fragments, can be harnessed to screen for highaffinity binding polypeptides of immobilized target proteins in a high-throughput manner (zhang, ben-david, & sidhu, ; zhang & sidhu, ) . a major obstacle to the development of small molecule inhibitors for ups components is the high structural similarity and functional redundancy among proteins in a particular family (renatus et al., ; vandemark & hill, ) . to overcome this limitation, knowledge of the ub pathway and phage display technologies were merged to produce ub variants (ubvs) as potent and highly selective inhibitors and activators of ups enzymes (ernst et al., ; ernst & sidhu, ; zhang, sartori, et al., ; zhang & sidhu, ) . ub interacts weakly but specifically with thousands of structurally similar proteins, via a common binding surface (husnjak & dikic, ) and its intrinsic conformational heterogeneity (lange et al., ) . in addition, ub is a highly stable protein that lacks cysteine residues and can be easily produced recombinantly, making it an ideal scaffold for generating inhibitors of proteins in the ups (job et al., ) . via protein engineering, several groups have generated ubvs with remarkable potency, specificity and cellular activity for the modulation of the enzymatic activities of ups proteins. indeed, given its a pna dna fig. . dna-encoded compound libraries (decls). approaches for using decls for drug discovery are similar to those developed for combinatorial chemistry and split-and-pool synthesis, as reviewed by (favalli et al., ; franzini et al., ; goodnow jr. et al., ; . (a) compounds are typically developed by combinatorial or other synthesis methods, and then coupled to specific dna sequences or pna chains. (b) schematic representation of decls. decls typically begin by split-and-pool synthesis and are assembled differently depending on whether they are single pharmacophores (dna-recorded and -templated) or dual pharmacophores [encoded self-assembled chemical (esac)]. dna-recording involves the coupling of small molecules to single-stranded (ss) or double-strand (ds) oligonucleotides prior to the ligation of dna barcodes and amplification. dna-templated approaches involve similar processes to dna-recorded, but this time already coupled dna-compound moieties interact with each other via complementarity; in proximity, linkers coupling the compound to the dna are cleaved, allowing the compounds to react and generate cyclical moieties. dual pharmacophore libraries consist of esac libraries. in these libraries, like in dna-recorded, compounds are first coupled to ssdna. hybridization occurs thanks to the spacer region of one moiety. amplification is then used to generate dsdna. (c) illustration showing a representative screen where target tagged-proteins are immobilized. tag-based affinity methods allow for washing off impurities and non-binders to enrich for hits. screening is conducted via amplification or mass spectrometry of barcoded compounds with dna or pna, respectively. small size, lack of disulfide bonds, and structural stability, ub represents an ideal scaffold for the next generation of protein therapeutics (job et al., ) . ubvs are capable of specifically inhibiting dubs in vitro and in vivo. by mutating residues in the ub core and on the surface, researchers at genentech developed a ubv that bound tightly to usp and inhibited activity in cells, resulting in enhanced mdm ubiquitination and p stabilization (zhang et al., ) . our group also developed an inhibitor of usp (ubv. . , ic values in the low nm range) by employing surface mutations only. notably, ubv. . proved to be more potent, and most importantly, much more selective than genentech's ubv, showing that core mutations may reduce specificity and thus should be employed with great care. ubv. . inhibited usp in cancer cells, and consequently, caused dramatic reductions of mdm levels and induction of apoptosis, in synergy with cisplatin, suggesting that ubvs could be used as enhancers of chemotherapeutic drugs (w. zhang, sartori, et al., ) . we also developed a ubv targeting usp , a second dub that deubiquitinates p , and showed that the ubv promoted export of p from the nucleus to the cytoplasm (zhang, sartori, et al., ) . recently, we developed ubvs for all four ub-binding domains of usp and engineered potent bivalent inhibitors of the enzyme by linking a ubv targeting the dusp domain to a ubv targeting the catalytic domain (teyra et al., ) (fig. a) . these dimeric ubvs exhibited enhanced specificity and inhibition of usp catalytic activity, and acted as inhibitors of the transforming growth factor β (tgf-β) signalling pathway in cells (teyra et al., ) . we also utilized ubvs to reduce the abundance and activity of cell surface receptors by inhibiting usp , an enzyme that regulates levels of epidermal growth factor receptor (egfr) and other receptors (fig. b) (ernst et al., ) . moreover, we recently established a yeast-two-hybrid (y h) screening platform (bruckner, polge, lentze, auerbach, & schlattner, ) for the in vivoselection of ubvs able to inhibit the cellular catalytic activity of usp (pascoe et al., ) , a dub involved in the protection of prostate cancer from apoptosis (priolo et al., ) . extending beyond usp dubs, we targeted otub , a member of the otu dub family, with a ubv that bound distal to the active site (fig. c ), but nonetheless, potently inhibited k -linked di-ub cleavage (ernst et al., ) . in the past, we have also employed ubvs as inhibitors of pathogenic dubs. in order to promote infection and replication in host cells, while evading the immune response, many human pathogenic viruses have evolved the ability to hijack and manipulate the ups (luo, ) . for example, the ub-specific protease of the herpes simplex virus antagonizes nf-κb activation, whereas the e oncoprotein of the human papillomavirus recruits host e ligases to promote ub-dependent degradation of p and consequent carcinogenesis (hoppe-seyler, bossler, braun, herrmann, & hoppe-seyler, ; ye, su, xu, & zheng, ) . we developed highly selective and potent ubvs targeting the dubs of the middle east respiratory syndrome coronavirus (mers-cov) and the crimean-congo hemorrhagic fever virus (cchfv), two emergent and globally important viruses (w. zhang, bailey-elkin, et al., ) . ubv.me. bound with subnanomolar affinity to the mers-cov dub by establishing a wide network of hydrophobic packing and hydrogen bond interactions (fig. d) . cell-based assays showed that ubv.me. efficiently suppressed the dub-induced activation of ifn-β promoter, thereby providing efficient blockade of viral replication and reducing mers-cov progeny titer by~ , -fold (w. zhang, bailey-elkin, et al., ) . moreover, han et al. recently highlighted the interplay between the hect e ligase wwp and the vp matrix protein of ebola virus in promoting viral budding from the host cell (han et al., ) . therefore, ub-based modulators of the ups may enable the generation of novel antiviral drugs. saturation mutagenesis scanning experiments (maynard, chen, georgiou, & iverson, ) performed with distinct ubvs targeting the structurally similar catalytic domains of usp or usp enabled elucidation of the interaction landscape of ub with the usp family (leung, dekel, shifman, & sidhu, ; pascoe et al., ) . thus, ubvs can be used not only as inhibitors of dubs, but also, as model molecules to identify alternative target sites for the development of small-molecule inhibitors that can target structurally conserved proteins with high specificity. hect e ligases, which play crucial roles in cancer, neurological disorders and viral infections (scheffner & kumar, ) , contain a hect domain that forms a covalent bond with ub (through a catalytic cysteine) before transferring it to a substrate protein. understanding of the diverse hect family has been limited by a paucity of selective and potent modulators. despite enormous efforts, development of synthetic molecules targeting hect e s has been hampered by large conformational changes accompanying catalysis, a shallow active site, and dynamic regulation of activity. to overcome this limitation, we systematically developed ubv inhibitors and/or activators for of the human hect e s (zhang et al., ) . structural analysis of ubvs targeting hect e ligases revealed that ubv inhibitors acted by blocking the binding site for e ligase rather than by blocking the active site (fig. e) (zhang et al., ) , further demonstrating that the plasticity of the ub fold is amenable for the development of ubvs with novel biochemical properties. surprisingly, a number of ubvs enhanced catalytic activity of hect e ligases (ernst et al., ; zhang et al., ) . for example, ubvs that bound tightly and selectively to nedd increased intracellular ubiquitination of the nedd substrate ying-yang (ernst et al., ) and inhibited the migration of cancer cells by promoting the ubiquitination of ras homolog gene family member b (rhob) (zhang et al., ) . ubvs also modulated the activity of membranebound ion channels by either activating or inhibiting nedd l (zhang et al., ) . ubvs that activated or inhibited nedd l reduced or increased cell-surface levels of the epithelial sodium channel (enac), respectively (zhang et al., ) . since elevated enac levels are associated with hypertension (ronzaud et al., ) , and suppression of this ion channel has been linked to liddle's syndrome, novel therapies for hypertension could be developed by targeting nedd l with molecules that modulate enac levels at the cell surface (aziz, memon, rahman, & ali, ) . with~ members, the ring family accounts for n % of human e ligases. in contrast to hect e s with an active catalytic cysteine, the ring domain acts as an inert scaffold that recruits e enzymes thioesterified with ub (e ~ub) and facilitates ub transfer to substrates. due to the lack of a catalytic cysteine, inhibition of ring function has been challenging. we identified a ubv targeting the ring domain of the e ligase cbl (ubv.pcbl), which reduced the internalization of egfr while sustaining akt pathway activation (fig. f) (gabrielsen et al., ) . remarkably, ubv.pcbl bound tightly to phosphorylated cbl (pcbl), but not to non-phosphorylated cbl, demonstrating that ubvs can recognize post-translationally modified proteins with high specificity (gabrielsen et al., ) . unexpectedly, we also isolated a dimeric ubv (ubv.xr d ) that recognized the ring domain of the e ligase xiap with high specificity and stimulated ligase activity in vitro and in cells (gabrielsen et al., ) . the structure of ubv.xr d in complex with xiap revealed that ubv.xr d forms a domain-swapped dimer that stabilizes the e -e ~ub conformation, resulting in enhanced activity of the e ligase (fig. g) (gabrielsen et al., ) . additionally, we utilized the ubv technology to target a ub-binding exosite on the ring domain of the anaphase-promoting complex subunit (apc ) (brown et al., ; yamaguchi et al., ) to probe the biochemical mechanisms of the large, -subunit cell-cycle regulatory anaphasepromoting complex (apc/c). in sum, this work provided a general strategy to inhibit or activate ring e ligases to modulate these enzymes in order to understand their catalytic mechanisms and biological functions. ubv inhibitors have also been developed for members of the multisubunit skp -cul -f-box (scf) e ligase family, and these have been shown to affect the mitotic cell cycle progression of cancer cells (fig. h) (gorelik et al., ; gorelik et al., ) . structural analysis of a ubv in complex with its target skp -fbw revealed extensive contacts with both skp and the f-box and showed that the ubv inhibited activity by blocking the binding site for cul (gorelik et al., ) . structure-based engineering of a loop on the ubv enabled the development of specific inhibitors for different members of the scf e ligase family (gorelik et al., ) . finally, the ubv technology has been extended to target non-catalytic ub-binding domains, exemplified by the small, helical ub-interacting motif of the yeast protein vps (fig. i) (manczyk et al., ) . engineered ubvs have also been developed to target proteins that are not known to associate with ub. hoffmann et al. used ribosome display (zahnd, amstutz, & pluckthun, ) to develop a ubv that bound to and inhibited the activity of tumor necrosis factor alpha (hoffmann et al., ) . similarly, we recently isolated ubvs that bound specifically to the human epidermal growth factor receptor (her ) and the growth factor receptor-bound protein (grb ) and showed that ubvs acted as potent antagonists of grb -mediated cell signalling (leung, jarvik, & sidhu, ) . we also developed a potent and highly selective ubv inhibitor of the tumor suppressor p -binding protein ( bp ), which recognizes ubiquitinated histone h a and acts as a central regulator of the double-strand break repair pathway (fradet-turcotte et al., ) . expression of the ubv in either human or mouse cells prevented the accumulation of bp at double-strand break sites, stimulating gene conversion and enhancing the efficiency of crispr-cas dependent gene editing (canny et al., ) . this work highlighted the robustness of protein design and engineering with the ub scaffold and the potential for targeting dna damage signalling proteins for applications beyond the ups. in summary, ubv technology represents a robust platform for the rapid development of intracellular probes for target discovery and validation. delivery of ubvs to cells represents a major obstacle for their direct use as therapeutics, but the constant advancements in delivery technologies for folded proteins has the capacity to unlock the potential of ubvs as drug candidates. drug discovery campaigns traditionally focus on the development of tight and specific small molecules that act as inhibitors by blocking binding pockets or active sites of disease-associated proteins. although this approach has proven successful (salami & crews, ) , it has been limited by the number of druggable protein targets, accounting for onlỹ % of the human proteome (dixon & stockwell, ; landry & gies, ; rask-andersen, almen, & schioth, ; santos et al., ; uhlen et al., ) . in fact, while nuclear receptors, enzymes, ion channels and g-protein-coupled receptors represent the majority of current drug targets, the human proteome is mainly composed of proteins involved in the organization, maintenance and assembly/disassembly of protein complexes that regulate signal transduction events and other critical functions. therefore, to expand the druggable protein space, researchers have employed alternative approaches that allow targeting of transient protein-protein interactions rather than deep grooves to which small molecules can bind. this paradigm shift focuses on the use of small molecules to control the cellular levels of drug targets through their precise degradation rather than the inhibition of their activity. this shift from target inhibition to induced target destruction is exemplified by proteolysistargeting chimeras (protacs), small molecules that recruit a ubiquitin e ligase for protein degradation (lucas & ciulli, ; neklesa, winkler, & crews, ; . in fact, protacs are heterobifunctional molecules composed of a ligand for a target protein fig. . methods for targeted protein degradation. (a) representation of the proteolysis-targeting chimera (protac) technology. protacs are heterodimeric molecules composed of a protein-targeting warhead connected to an e ub ligase binder via a chemical linker. protacs mediate the formation of a stable complex between the target protein and an e ligase, triggering ubiquitination and proteosomal degradation of the target protein. (b) click-formed proteolysis targeting chimeras (cliptacs) exploit the bio-orthogonal click reaction between a trans-cyclooctene-tagged binder for a target protein and a tetrazine-containing e ub ligase binder. the two molecules are administered individually and, upon cell penetration, form a complete protac that is able to induce protein degradation. (c) ubiquitibodies are constructed by fusing a scfv to the chip e ub ligase. the scfv enables specific recognition of a target protein and the e ligase facilitates ubiquitination to trigger degradation. (d) a target protein was fused to a halotag to achieve hydrophobic protein tagging via site-specific and covalent conjugation with a halotagged-adamantyl moiety (england, luo, & cai, ) . the adamantyl moiety displayed on the target protein mimics the partially unfolded state of proteins, triggering the activity of cellular quality control machinery, which recruits the ups for protein degradation. (e) a hydrophobic tag was developed by modifying an inhibitor of a target protein with an hydrophobic (boc arg) moiety (long et al., ) . upon binding to the target protein, the modified inhibitor enabled recruitment to the proteasome and subsequent degradation. (warhead) and a ligand for an e ub ligase ) that mediate the formation of a complex between the drug target and an e ligase promoting ubiquitination and subsequent degradation of the target (fig. a) . the first protac was built by linking target protein inhibitors to peptides that recruited the scf β-trcp e ligase (sakamoto et al., ) . although these protein degraders induced rapid depletion of targets such as the methionine aminopeptidase- , the estrogen receptor and androgen receptors (sakamoto et al., ; sakamoto et al., ) , the peptidic nature of the e ligase engager and its intrinsic instability posed limits for the application of the technology. a breakthrough in the protac technology occurred with the use of the non-peptidic inhibitor nutlin as the engager of the e ligase mdm , resulting in partial degradation of androgen receptors in cells (schneekloth, pucheault, tae, & crews, ) . however, due to the limited activity and complex chemical composition of this second generation of protacs, researchers were motivated to further improve stability and pharmacokinetic properties by decreasing size and identifying novel e ligase recruiters (buckley et al., ) .these efforts led to the development of current protac technologies. protacs rely on degradation rather than inhibition of target proteins, showing a remarkable efficacy at doses times lower than conventional drugs (bondeson et al., ) , a feature that has the potential to reduce systemic toxicity in vivo. recently, a systematic analysis of the mode of action of protacs revealed further benefits of this technology (bondeson et al., ) . this study showed that the stability of the target:protac:e ternary complex rather than the affinity of the target:protac interaction is the main factor responsible for activity (bondeson et al., ) . this finding opens the possibility of turning low affinity small molecules into potent protein degraders, potentially allowing the rapid development of new drugs. comparison between the degrading activities of protacs with different e ligase ligands demonstrated that the ub ligase engager not only dictates the efficacy of these molecules, but also controls the target specificity (bondeson et al., ) . in fact, protacs based on promiscuous kinase inhibitors (bondeson et al., ; h. t. huang et al., ) were rendered more selective simply by engaging different e ligases, suggesting that the specificity of degradation induced by a protac is mainly driven by the endogenous ligand specificity of the e ligase. therefore, protacs hold promise for the development of highly specific protein degraders even when using non-specific warheads to target proteins of interest. thus far, only a limited number of e ligases have been exploited for the generation of protacs. the technology can be further advanced by the development of novel ligands to recruit other ub ligases among the~ human e enzymes (nakayama & nakayama, ) . the importance of each protac component -warhead, linker and e engager -for achieving optimal target degradation has been described (bondeson et al., ; lai et al., ; maniaci et al., ; zengerle, chan, & ciulli, ) , but the prominent role played by the linker geometry has emerged only recently (burslem et al., ; gadd et al., ) . for example, the short linker connecting the promiscuous bromo-and extra-terminal (bet) jq ligand and the potent vh von hippel-lindau target's e ligase (vhl) engager, allowed for the formation of a stable target:vhl interface, ensuring the efficient and specific degradation of brd over the highly homologous brd and brd proteins (gadd et al., ) . protacs offer numerous advantages over conventional small molecules and provide an opportunity to expand the druggable protein target space. however, a number of questions remain to be addressed. despite the modular nature of protacs, the efficacy and specificity of new protacs needs to be assessed empirically, as changes in the protac chemical composition and geometry might affect activity and specificity. therefore, the development of high throughput methods for synthesis and characterization of protac molecules will accelerate the development of new inhibitors. limited in vivo data are available (qin et al., ; saenz et al., ) ; toxicity, pharmacokinetics, bioavailability, tissue distribution and metabolism of protac molecules will require extensive investigation. the large size of protacs (~ da) might affect cell permeability, potentially resulting in suboptimal presentation of a target to the e ligase; smaller protacs could improve cellular uptake and potency. to overcome this obstacle, lebraud et al. developed in-cell click-formed proteolysis targeting chimeras (cliptacs): protacs that are formed intracellularly upon click chemistry-mediated self assembly of the target warhead and an e ligase engager (lebraud, wright, johnson, & heightman, ) (fig. b) . similar to the protac technology, chimeric proteins formed by a single-chain variable fragment (scfv) of an antibody or a fibronectin type iii domain monobody fused to the e ub ligase carboxyl terminus of hsc -interacting protein (chip) have also been used to degrade target proteins (portnoff, stephens, varner, & delisa, ) . although this strategy enabled the efficient depletion of target proteins in cells, its application has been hindered by the correct folding of disulfide bondcontaining scfvs in the reducing environment of the cell cytoplasm (fig. c) . another method to trigger selective protein degradation via the ups is represented by the modification of proteins with hydrophobic tags (neklesa & crews, ) . covalent modification of proteins with hydrophobic tags induces the recruitment of molecular chaperones, which promote the ubiquitination and proteolytic removal of the target by the proteasome s (neklesa et al., ) (fig. d) . this approach achieved selective depletion of her and reduced cancer cell proliferation (xie et al., ) . similarly, linkage of small molecule inhibitors to a hydrophobic tert-butyl carbamate-protected arginine (boc arg) moiety induced selective degradation of target proteins (long, gollapalli, & hedstrom, ) (fig. e) . in contrast to hydrophobic tags, the (boc arg) moiety enabled the efficient depletion of both glutathione s-transferase (gst) and bacterial dihydrofolate reductase (dhfr) in a manner independent of ub and atp, suggesting that degradation was mediated by a direct interaction between the (boc arg) moiety and the proteasome subunit s (long et al., ) , without requiring target ubiquitination. these recent developments demonstrate that induced protein degradation has been significantly improved over recent years and is now well positioned to expand the druggable protein space. the ups provides a wide canvas for developing next-generation cancer therapeutic agents. while advances in hts technologies have decreased the time and cost needed to carry out drug discovery studies, activity-based small molecule screens have not yielded a satisfying number of viable drug leads. recent developments in fragment-based medicinal chemistry methodologies offer a promising approach to tackle the small molecule-based screen problems. the advent of ubvs also represents an exciting new technology that can rapidly provide highly potent and selective inhibitors and activators of enzymes that have resisted conventional approaches. combined with fragmentbased drug discovery methods and recent development in decls, small molecule screens for chemical compounds that can displace ubvs from target proteins could enable deep and effective sampling of chemical space to modulate the ups. finally, protac technology represents another promising means for cancer therapy. indeed, it seems likely that the most effective cancer therapies in the future could well rely on the combination of these diverse innovations to develop drug-like entities with activities well beyond the scope of current drugs. the authors declare no conflicts of interests. 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ubiquitin enzymes dna tags help the hunt for drugs peptide and small molecule inhibitors of hect-type ubiquitin ligases la fam fatale: usp x in development and disease ubiquitin ligases: cell-cycle control and cancer chemical biology: greasy tags for protein removal small-molecule hydrophobic tagging-induced degradation of halotag fusion proteins targeted protein degradation by protacs the deubiquitinating enzyme usp regulates the fanconi anemia pathway benzoxaborole compounds for therapeutic uses: a patent review ubiqutination via k and k chains signals aggregation and neuronal protection of lrrk by wsb identification and structure-guided development of pyrimidinone based usp inhibitors dynamics of parkin-dependent mitochondrial ubiquitylation in induced neurons and model systems revealed by digital snapshot proteomics cell lysate-based alphalisa deubiquitinase assay platform for identification of small molecule inhibitors mechanistic insight into the allosteric activation of a 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potent and efficacious proteolysis targeting chimera (protac) degrader of the bromodomain and extra-terminal (bet) proteins capable of inducing complete and durable tumor regression trends in the exploitation of novel drug targets structural basis of ubiquitin recognition by the deubiquitinating protease usp a phase study of bortezomib in relapsed, refractory myeloma assessment of proteasome inhibition for extending remissions, i ( ) renal tubular nedd - deficiency causes ncc-mediated saltdependent hypertension physiological functions of the hect family of ubiquitin ligases phage display peptide libraries: deviations from randomness and correctives novel bet protein proteolysis-targeting chimera exerts superior lethal activity than bromodomain inhibitor (beti) against post-myeloproliferative neoplasm secondary (s) aml cells protacs: chimeric molecules that target proteins to the skp -cullin-f box complex for ubiquitination and degradation development of protacs to target 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ubiquitin modifications how were new medicines discovered? pevonedistat, a first-in-class nedd -activating enzyme inhibitor, combined with azacitidine in patients with aml expanded safety analysis of pevonedistat, a first-in-class nedd -activating enzyme inhibitor, in patients with acute myeloid leukemia and myelodysplastic syndromes structural and functional characterization of ubiquitin variant inhibitors of usp mln (pevonedistat), a protein neddylation inhibitor, suppresses proliferation and migration of human clear cell renal cell carcinoma small-molecule protacs: new approaches to protein degradation leucettamol a: a new inhibitor of ubc -uev a interaction isolated from a marine sponge discovery of next-generation antimicrobials through bacterial self-screening of surface-displayed peptide libraries molecular basis of usp inhibition by selective smallmolecule inhibitors proteomics. tissue-based map of the human proteome peptidomimetics, a synthetic tool of drug discovery structural basis of ubiquitylation in vivo activation of the p pathway by small-molecule antagonists of mdm the resurgence of phenotypic screening in drug discovery and development emergence of a dna-damage response network consisting of fanconi anaemia and brca proteins the proteasome deubiquitinase inhibitor vlx shows selectivity for ubiquitin-specific protease- and induces apoptosis of multiple myeloma cells small molecule inhibitors reveal allosteric regulation of usp via steric blockade from discovery to the bedside: targeting the ubiquitin system enzymatic characterisation of usp deubiquitinating activity and inhibition structural basis of iap recognition by smac/diablo pharmacological targeting of the pseudokinase her cryo-em of mitotic checkpoint complex-bound apc/c reveals reciprocal and conformational regulation of ubiquitin ligation inhibitors of ubiquitin-activating enzyme (e ), a new class of potential cancer therapeutics small molecule inhibitors of hdm ubiquitin ligase activity stabilize and activate p in cells a phase ii trial of bortezomib and vorinostat in mantle cell lymphoma and diffuse large b-cell lymphoma herpes simplex virus ubiquitin-specific protease ul abrogates nf-kappab activation in dna sensing signal pathway inhibition of ubiquitinspecific proteases as a novel anticancer therapeutic strategy achievements, challenges, and opportunities in dna-encoded library research: an academic point of view ribosome display: selecting and evolving proteins in vitro that specifically bind to a target pna-encoded chemical libraries selective small molecule induced degradation of the bet bromodomain protein brd potent and selective inhibition of pathogenic viruses by engineered ubiquitin variants engineering cell signaling modulators from native protein-protein interactions generation and validation of intracellular ubiquitin variant inhibitors for usp and usp development of inhibitors in the ubiquitination cascade drug development: allosteric inhibitors hit usp hard system-wide modulation of hect e ligases with selective ubiquitin variant probes conformational stabilization of ubiquitin yields potent and selective inhibitors of usp ubiquitin ligases: structure, function, and regulation harnessing the ubiquitination machinery to target the degradation of specific cellular proteins ubiquitin in the immune system we sincerely apologize to the researchers whose work was not included in this review due to limited space. sss is supported by a genome canada disruptive innovation in genomics grant (ogi- ) and a cihr operating grant (mop# ). wz is the recipient of the cancer research society/ bmo bank of montreal scholarship for the next generation of scientists. key: cord- - xjmlwqr authors: draz, mohamed shehata; shafiee, hadi title: applications of gold nanoparticles in virus detection date: - - journal: theranostics doi: . /thno. sha: doc_id: cord_uid: xjmlwqr viruses are the smallest known microbes, yet they cause the most significant losses in human health. most of the time, the best-known cure for viruses is the innate immunological defense system of the host; otherwise, the initial prevention of viral infection is the only alternative. therefore, diagnosis is the primary strategy toward the overarching goal of virus control and elimination. the introduction of a new class of nanoscale materials with multiple unique properties and functions has sparked a series of breakthrough applications. gold nanoparticles (aunps) are widely reported to guide an impressive resurgence in biomedical and diagnostic applications. here, we review the applications of aunps in virus testing and detection. the developed aunp-based detection techniques are reported for various groups of clinically relevant viruses with a special focus on the applied types of bio-aunp hybrid structures, virus detection targets, and assay modalities and formats. we pay particular attention to highlighting the functional role and activity of each core au nanostructure and the resultant detection improvements in terms of sensitivity, detection range, and time. in addition, we provide a general summary of the contributions of aunps to the mainstream methods of virus detection, technical measures, and recommendations required in guidance toward commercial in-field applications. viruses are remarkable pathogens that are causing prominently increasing morbidity and mortality worldwide. their highly contagious nature and the absence of immediate and efficient control systems are the main reasons behind their potential health impacts. currently, viral infections and associated diseases are major causes of death in mankind, and under the present context of industrialization and immigration, they continue to emerge at a rapid pace, causing significant human, social, and financial costs [ ] . additionally, gaps in currently applied detection systems potentially contribute to increasing the likelihood of international incidences and outbreak of viral infections [ ] [ ] [ ] [ ] . the implementation of highly sensitive and specific diagnostic tools has the potential to rapidly identify viral infections, initiate and guide judicious controls, and subsequently curtail their dissemination. toward this endeavor and beyond the pitfalls of current immunological and molecular techniques commonly applied to virus detection, several new approaches based on nanoparticles (nps) have recently been developed. aunps are widely described to be suitable for numerous biosensing functions and applications. their unique photonic, electric, and catalytic properties, coupled with the molecular interaction specificity of various biomolecules (e.g., antibodies, single-stranded (ss) dna, and rna aptamers, among others), represent the design principles of a wide range of virus detection systems [ ] [ ] [ ] . the advantages of being simple, rapid, and sensitive and facilitating quantitative detection with excellent multiplexing capabilities have greatly promoted these systems to be envisioned as state-of-the-art technologies for virus ivyspring international publisher detection [ , ] . however, there are no available reviews of the applications of bio-aunp hybrid structures for sensing and detecting viruses. in this article, we provide a current review of aunp-based virus detection at the level of virus type, including the types and structures of the applied aunps. we highlight their role in enhancing sensory and detection performance in comparison with current techniques in terms of analytical sensitivity, detection range, and time. furthermore, this review specifically summarizes detection designs, formats, functions, and contributions, which are of special importance to both scientific and applied research. viruses are particulate in nature and usually exist in different morphological forms that generally range in size from to nm [ ] [ ] [ ] . their intact, mature infectious particles are typically composed of definite units of proteins and nucleic acid that self-assemble to form nanoparticulate structures called virions. the viral proteins are usually arranged in a surface layer called the capsid and sometimes in an outer envelope surrounding an inner core of nucleic acids. this core of viral nucleic acids can be single-or double-stranded (ds), dna or rna, one or several molecules, linear or circular in shape, and a few thousands of nucleotides to one million base pairs in size. the nanoscale size and the relatively simple structure of viruses tend to impose technical difficulties in establishing wide-use and long-term systems for virus detection. the small size of viruses increases the difficulty of their isolation and visualization compared with other microbes, such as bacteria and fungi that can be readily examined using ordinary light microscopes. only electron microscopy (em) with a high-magnification power of ~ , × can allow the direct visualization of viruses and the study of their structures [ , ] . therefore, em remains crucial for many purposes in virus research. however, em is certainly inappropriate for routine clinical diagnosis because of the required time and cost, as well as many safety concerns. furthermore, although the simple structure of virus particles allows the features of diagnostic relevance to be easily defined and tested, it presents limitations on the use of their characteristics for practical applications, especially with the increasing number of discovered viruses and recorded viral infections [ , , ] . in addition, this structural simplicity confers viruses with rapid rates of spontaneous adaptation and evolution that may occur through direct genetic mutation, genetic substitution, or recombination. in this way, viruses not only outpace our attempts to develop sustainable control strategies but also raise more questions about the appropriateness and validity of current diagnostic techniques for long-term use [ , ] . along with these limitations in virus detection and control, the possibility of viral infection emergence or reemergence has become increasingly prevalent, and severe pandemics and epidemics have occurred around the world. in the past, many outbreaks of viruses, such as human immunodeficiency virus (hiv), severe acute respiratory syndrome (sars), influenza virus (h n and h n ) and zika virus, started as local events and then expanded to have global consequences. hiv was discovered in central africa as a new virus causing acquired immune deficiency syndrome (aids) three decades ago, and it now represents one of the most significant public health threats worldwide [ , ] . the highly contagious respiratory virus sars has fueled global fears of pandemics since its first appearance in china [ , ] . in late , the world health organization (who) raised a global alert for a new sars-like respiratory coronavirus, which is now called middle east respiratory syndrome (mers) coronavirus. mers is now classified as one of the most prominent viral threats to the middle east and has caused hundreds of deaths and thousands of infections in a short time. moreover, in the past few years, the world witnessed the worst ebola outbreak ever that started to hit west africa in early and the pandemic reemergence of zika virus in [ , , ] . with these evident possibilities of massive outbreaks and implications, the situation is rapidly growing more serious, and the demand for the development of rapid diagnostics and effective control strategies is becoming more urgent. the first studies on virus isolation and detection were started early in the s when the first cell culture system and electron microscope were developed [ , ] . for decades, these techniques represented the main tools for studying and investigating the biochemical and morphological properties of viruses that remain the main foundation of all known classification and detection systems. however, their practical use in virus detection has remained greatly debated due to several considerations, including laboratory equipment expenses and time and safety concerns [ , ] . in the early s, the field of diagnostic virology was boosted with two other major developments: ) the birth of various immunoassays; and ) the invention of polymerase chain reaction (pcr). this was followed by the development of a very wide range of serological and molecular detection techniques, which rapidly evolved to constitute the mainstream approaches of both laboratory research and the clinical diagnosis of viruses ( fig. ) [ , ] . serology remains the standard method for virus detection. it primarily relies on testing for the presence of specific viral antigens or the corresponding antibody responses of the immune system. the most common types of serological tests include the neutralization assay, complement-fixation test, immunoprecipitation assay (ipa), hemagglutination-inhibition (hai) assay, enzyme immunoassay (eia), radioimmunoassay (ria), chemiluminescent immunoassay (cia), particle agglutination, immunostaining, immunofluorescence assay, single radial hemolysis, immunoblotting assay (iba), and immunochromatographic test (ict). the main working principle of these techniques is the use of specific antibodies in conjugation with different signal reporting systems, such as red blood cells, enzymes, and radioactive or fluorescent materials [ , ] . most of these techniques are relatively easy to perform and flexible in their timing of specimen collection, and most of the needed reagents are usually commercially available. furthermore, they are generally unrestricted by the practical limitations of virus isolation and propagation usually involved with other direct detection methods, such as cell culture and em [ , ] . therefore, serological methods are widely accepted and recognized techniques for simple, safe, and cheap virus detection [ , ] . in addition, they are usually described as the first choice for large-scale testing in epidemiological studies and for evaluating antiviral therapies and vaccinations. however, the accuracy and reliability of serological methods are usually challenged by the cross-reactivity of the used antibodies, and the risk of false-positive results is usually very high. in addition, most immune responses can only be detected for a period of time after the initial virus infection; thus, serological detection does not normally benefit patients. molecular techniques are attracting more interest and have found an increasing number of applications in virus detection. the discovery of the genetic enzyme systems involved in the cellular machinery of nucleic acid replication and the stunning invention of an in vitro nucleic acid amplification system, commonly called pcr, by mullis in the early s, opened new frontiers in nucleic acid-based detection [ ] . in addition, the known high specificity of nucleic acid hybridization and the absolute availability of its synthesis and modification guided the development of many figure . the onset of nanotechnology in virus detection applications compared with the development of the most common virus detection techniques. cell culture and electron microscopy techniques that are now commonly applied in the direct testing for and detection of viruses were discovered in the mid- th century [ ] . then, different serological and molecular techniques were developed. the serological detection of viruses with immunoassays was first reported in : a radioimmunoassay was applied for the detection of the australia antigen, later called hepatitis b virus surface antigen [ ] . pcr was discovered in the s and first reported in virus detection in for acquired immune deficiency syndrome detection [ ] . later, many molecular techniques, including amplification-and nonamplification-based techniques, were reported in virus detection. the concept of nanotechnology was envisioned as early as by the renowned physicist richard feynman [ ] . however, nanotechnology was only applied to virus detection in , when gold nanoparticles were employed for the detection of single-copy human papillomavirus [ ] . nanotechnology has recently come to represent one of the most outstanding trends in virus detection and diagnosis via the wide variety of assays described in this review. detection and genotyping techniques known for the rapid and specific detection of viruses. these techniques can be classified as amplification-or nonamplification-based molecular detection techniques. amplification-based molecular techniques usually employ one or more forms of nucleic acid amplification to allow the indirect detection of the target virus. these techniques represent the majority of molecular techniques applied in virus detection and include various types of target amplification techniques (e.g., pcr, loop-mediated isothermal amplification (lamp), transcription-mediated amplification, and nucleic acid sequence-based amplification), signal amplification techniques (e.g., branched dna and hybrid capture), and probe amplification techniques (e.g., ligase chain reaction and strand-displacement amplification). nonamplification-based techniques are mainly applied to the direct testing for the presence of a specific virus in clinical samples (e.g., in situ hybridization, southern blot hybridization, and dot blot hybridization). generally, molecular methods are relatively rapid and more sensitive than immunoassays and can be applied either in a simple form for the manual detection of viruses or as an embedded component of more advanced systems. numerous fully automated and high-throughput detection systems are now available and widely used in clinical settings. in fact, the development of advanced molecular detection systems has revolutionized the way in which diagnostic tests are delivered and greatly enhanced the control of many viral infections, whether in hospitals or communities. in addition, these systems have eliminated the biosafety and time concerns usually associated with the clinical and academic study of viruses. however, despite these wide and promising applications, most molecular techniques still have several potential limitations in repeatability, accuracy, sensitivity, and specificity that are mainly caused by the high genetic variability of some viruses. furthermore, these assays are expensive and time consuming and typically call for specialized laboratory instruments and skilled personnel [ , ] . the concept of nanotechnology was introduced in early [ ] . subsequently, nanotechnology was realized via various types of new materials that rapidly emerged as promising tools for biological and chemical analyses. nanomaterials are known to possess multiple unique optical, electronic, magnetic, and mechanical properties enabling very attractive applications, especially in the fields of biomedical imaging and clinical diagnosis [ , ] . the first reported application of nanomaterials in the detection of viruses was attempted in the late s: aunps were coupled with silver staining and applied for the detection of human papillomavirus in cervical carcinoma cells (fig. l) [ ] . currently, there is a very wide range of nanomaterials, including metal nps, carbon nanotubes, silica nps, quantum dots (qds), upconversion nps, and polymeric nps, that are being heavily investigated for virus detection [ , , ] . one of the most common approaches for exploiting these nanostructures in virus detection is the development of nanobio hybrid systems that contain one or more biomolecules derived from viruses (e.g., dna, rna, antibody, pentabody, antigen, or peptide) conjugated to the surface of different np forms. these systems leverage the significant labeling properties and signal transduction functions of nps and the specific activity of the conjugated biomolecules to act as multivalent-np probes [ , , ] . such virus-specific np probes have surprisingly been used to build up various optical, fluorometric, electrochemical, and electrical assays that have been extensively reported for single and multiple detection modes ( table ). the results of most of these studies clearly demonstrate the inherent potential of these probes, along with numerous advantages over traditional approaches, in terms of size, performance, specificity, signal sensitivity, and stability. additionally, these studies have extensively described their application to allow simple, rapid, highly sensitive and label-free detection. aunps are a leading class of metal nanostructures that is widely known for its chemical stability, water solubility, and broad size and shape controllability. aunps can range from to nm in size and have different morphological shapes, including spheres, rods, prisms, tetrapods, dog bones, cubes, shells and several hollow structures [ , ] . the synthesis of aunps can be performed using different methods such as chemical reduction of salts, ultraviolet irradiation, lithography, aerosol technologies, laser ablation, ultrasonic fields, photochemical reduction of au, and biological synthesis [ , ] . aunps possess a high surface density of free electrons that results in inherent optical, electrical, and catalytic properties. the excitation of aunps with light can cause these free surface electrons, i.e., "plasmons," to oscillate to one side away from the atomic core, which remains as a positive charge on the other side, thereby creating a dipole or plasmon polariton [ , ] . this dipole plasmon can change its direction in accordance with the frequency of incident light, and the resonance condition is reached when their frequency is approximately the same. this condition has been referred to as surface plasmon resonance (spr). the most widely used aunps exhibit intense spr bands that usually exist between - nm and are known to be weakly dependent on the size of the aunps and the refractive index of the surrounding media but very sensitive to both the shape of the nps and the interparticle distance [ ] . the spr of aunps has been observed to cause intense enhancing or quenching effects upon interactions with nearby photon emitters. these distance-dependent coupling effects are dipole-dipole interactions that usually include surface plasmon-mediated energy nanotransfer processes similar to fluorescence resonance energy transfer (fret) and may be either destructive (resulting in quenched emission) or constructive (resulting in enhanced emission). compared to other types of nanomaterials, metal nanoparticles, particularly aunps, constitute ideal tools in virus detection for numerous reasons, including the ease of synthesis, characterization, and surface modification, outstanding stability, biocompatibility, and exceptionally high absorption coefficients [ , ] . furthermore, as labeling agents, aunps are easily visualized due to their intense colors and are known to form stable and highly active bioconjugates with common targeting biomolecules, such as dna and proteins, thereby enabling highly sensitive and specific sensing and detection applications [ ] . aunps have been preferentially employed to perform numerous optical signal transduction functions in virus detection, such as resonance light scattering [ ] [ ] [ ] [ ] [ ] , color amplification [ ] [ ] [ ] [ ] [ ] [ ] , and fluorescence quenching or enhancing [ ] [ ] [ ] [ ] [ ] [ ] (fig. ) . resonance light-scattering-based detections usually involve measuring the amount of light scattered by different light spectroscopy techniques, including localized surface plasmon resonance (lspr) [ ] , raman spectroscopy [ , ] and dynamic light scattering (dls) [ , ] . the colorimetric detection of viruses using aunps usually relies on two main techniques: ) a color amplification technique in which aunps are applied to act as direct coloring labels with their characteristic, intense red color [ , , , ] ; and ) color change technique in which a color change from red to purple occurs in response to particle aggregation. aunp aggregation can be noncrosslinked aggregation caused by the target-triggered removal of stabilizing ligands from the surfaces of aunps [ , ] or interparticle-crosslinked aggregation caused by the binding of ligands on modified aunps with target analytes [ , ] . analogous to fret, aunp-based fluorescence quenching is a distance-dependent process in which aunps act to reduce the radiative rate of fluorophores in their proximity. thus, as efficient acceptors, aunps have been paired with dyeand qd-based fluorophores in different analyte-induced donor-acceptor crosslinking and coupling protocols [ - , , ] . aunps are widely described as electroactive and catalytic tags in various electrochemical assays applied to virus detection (fig. ). based on their redox and electrical properties, aunps can directly act as electrochemical tags detected either by their acid dissolution followed by electrochemical stripping measurements of gold ions [ ] or by their direct deposition on the surface of electro transducers, allowing an enhanced electrical conduction and resistance change [ ] [ ] [ ] [ ] [ ] . aunps can indirectly perform electrochemical transduction functions based on their catalytic activity toward some chemical reduction reactions of metal ions (e.g., silver and copper) [ , , ] or other species, such as h o [ ] [ ] [ ] . this potential to catalyze metal deposition is preferentially called np-metal enhancement amplification, and it represents the basic function of multiple scanometric [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , photoelectric [ ] , light-scattering [ ] , and colorimetric schemes [ ] for virus detection. additionally, aunp-based catalysis for the oxidation-reduction reaction of hydroquinone has been reported in another simple, colorimetricbased virus detection method [ ] . other studies have exploited aunps to enhance the detection sensitivity of some bioanalytical techniques, such as quartz crystal microbalance (qcm) [ ] , inductively coupled plasma mass spectrometry (icp-ms) [ ] , and atomic force microscopy (afm) [ ] in virus detection. through these schemes, aunps have been applied as effective nanoparticulate amplification tags to enhance mass, elemental, and topographic signal transduction, respectively (fig. ) . interestingly, the enhanced surface area-to-volume ratio of aunps has allowed them to further act as exquisite scaffolds for biorecognition and detection reactions (fig. ); in addition, it has granted these structures unprecedented capabilities for bioimmobilization, which is a basic principle of most aunp-based diagnostic designs. by coupling these bioimmobilization and signal transduction functions, aunps can achieve virus detection with highly improved analytical sensitivity and specificity. furthermore, the direct increase in loading efficiency allows new possibilities of controlled multifunctionalization with different biomolecules. based on this principle, developed aunp tags are characterized by enhanced reactivity and stability. increased loading efficiency also allows the design of new probes modified with multiple structures for biotargeting (e.g., antibodies and dna), together with other signal amplification structures (e.g., enzymes and dna oligonucleotides) [ , , , [ ] [ ] [ ] , both types of which are stridently included in the development of new diagnostic schemes and designs. for example, the aunp- figure . a generalized scheme for the basic characteristics and functions of aunps applied in virus detection. the size and metal nature of aunps are the key characteristics that enable them to directly transduce multiple types of signals, including optical, catalytic and electrical signals that can be detected by dynamic light scattering (dls), localized surface plasmon resonance (lspr), surface-enhanced raman scattering (sers), ultraviolet-visible (uv-vis) spectroscopy, fluorometry and other electrochemical analysis techniques. in addition, aunps act as nanoparticulate tags to enhance the signal detection of several analytical techniques, such as quartz crystal microbalance (qcm), atomic force microscopy (afm), and inductively coupled plasma mass spectrometry (icp-ms). their large surface area-to-volume ratio is known to permit them to function as an excellent scaffold for bioimmobilization and target-probe interactions with highly enhanced specificity and sensitivity. barcoding assay and its descendent technique of immuno-pcr universally rely on aunp tags modified with antibodies and dna oligonucleotides [ , , ] , and aunp-based enzymatic electrochemical assays are based on using aunps dually modified with dna and antibodies together with an enzyme to induce a chemical reduction reaction allowing electrochemical signal transduction [ , ] . several recent studies have attempted to harness the unique properties of aunps to develop various advanced schemes for virus detection. the developed assays are greatly variable in design and underlying principle. however, the utilization of aunps conjugated with specific virus-targeting biomolecules is a key component in most of these assays. various aunp bioconjugates have been widely employed in many colorimetric, scanometric, electrochemical, and fluorometric systems for the detection of many groups of well-known human viruses (table and fig. ). each of these groups will be discussed in more detail in the following sections. the bunyaviridae family comprises more than members that are primarily organized into four main genera: hantavirus, orthobunyavirus, phlebovirus and nairovirus [ ] . bunyaviruses are spherical, enveloped rna viruses - nm in size. their genome is composed of three segments of negative-sense, ssrna: a large segment (l, . - kb) that encodes rna polymerase, a medium segment (m, . - kb) that encodes viral glycoprotein, and a small segment (s, - . kb) that encodes nucleocapsid protein [ ] . these viruses are diverse in their host range and are frequently involved in a wide range of diseases in plants, animals, and humans. human pathogenic bunyaviruses, such as crimean-congo hemorrhagic fever virus, hantaan virus, la crosse virus, oropouche virus, rift valley fever virus, and toscana virus, continue to present increasingly important health concerns worldwide [ , ] . the genus hantavirus was recently expanded to include more than antigenically and genetically distinct htnvs [ ] . among them, rodent-borne htnvs can cause serious diseases in humans, including hemorrhagic fever with renal syndrome, and hantavirus cardiopulmonary syndrome, and hospitalizes more than , persons each year with a mortality rate reaching %. recently, the health impacts of htnvs are expected to dramatically increase in the near future due to the increasing number of reports on newly discovered htnvs [ ] [ ] [ ] . aunps were utilized to develop a novel immuno-pcr assay for the immunological detection of htnv nucleocapsid protein [ ] . this assay mainly relies on the enhanced surface area of aunps to prepare dually functionalized antibody-oligonucleotide conjugates that exceptionally carry specific monoclonal antibodies to label the target htnv antigen; the assay also involves barcoding dna for signal amplification (fig. a ). this assay could optimally detect concentrations as low as am of purified or spiked antigen samples, which is ~ orders of magnitude more sensitive than conventional elisa. the high detection sensitivity together with the targeting of htnv nucleocapsid protein [ ] , which is the most abundant viral component synthesized post virus infection, makes this assay a promising candidate method for the early diagnosis and control of htnv. rvfv is an arthropod-borne pathogen primarily known to affect animals and later discovered to infect humans. rvfv infection in humans can progress to serious hemorrhagic fevers that often lead to death, with prominent mortality rates of up to - %. infected persons can also develop other clinical manifestations, including retinitis, encephalitis, and paralysis [ , ] . rvfv is historically endemic to africa and has had a long history of outbreaks ranging from its reemergence in egypt in to the most recent outbreak in south africa in [ , ] . thus far, rvfv outbreaks are unpredictable and usually associated with very high socio-economic and public health consequences [ , ] . therefore, diagnostic methods that can rapidly identify the virus have become crucial not only for avoiding and preventing such outbreaks but also for monitoring cross-border dissemination. a novel immunoassay based on aunps coupled with a surface-enhanced raman scattering (sers) technique has been developed for the detection of rvfv capsid antigen [ ] . in this assay, aunps are applied as both immobilization scaffolds for the target-capturing antibodies and metal promoters to enhance the raman reporter dyes. the target antigen is first captured by antibody-magnetic particle conjugates and then labeled with dye/antibody-aunp conjugates, forming a three-component immunocomplex that is magnetically concentrated and finally detected by sers (fig. b) . this approach has shown an outstanding sensitivity level down to fg/ml of the target antigen. this high sensitivity with the possibility for the direct detection of rvfv in complex media or samples allowed by magnetic particles support the application of this assay for the rapid and sensitive detection of rvfv and the control of any future outbreaks. immuno-pcr assay for htnv detection using aunp probes dually functionalized with antibody (ab) and double-stranded (ds)dna. au-nanoprobes are directly applied to label virus antigens precaptured on a microplate. the aunps are carrying dsdna that includes barcode single-stranded (ss) dna for signal amplification. the barcode dna is separated, amplified and detected by gel electrophoresis [ ] . additionally, this assay can be modified in a more complex detection scheme that has been reported for human immunodeficiency virus detection [ ] . (b) surface-enhanced raman spectroscopy (sers)-based assay for detection of rvfv using raman reporter dye-coated aunps and magnetic nps (mnps). aunps and mnps are conjugated with a polyclonal ab specific for the target virus antigen forming aunp/virus antigen/mnp complexes; then, a nm laser excites the magnetically concentrated aunp/virus antigen/mnp complexes. the presence of the target antigen yields a reduction in the intensification of raman dye signature spectrum peaks, thereby providing an estimation of its concentration [ ] . this assay has been applied for the detection of wnv infection [ ] . the coronaviridae family comprises two subfamilies, coronavirinae and torovirinae, that were recently expanded to include many viruses. coronavirinae are classified into four genetically distinct genera (alphacoronavirus, betacoronavirus, gammacoronavirus and deltacoronavirus) [ ] . coronaviruses are enveloped rna viruses that are pleomorphic in shape (spherical and - nm or bacilliform and - nm by - nm). they have a relatively large, positive-sense, ssrna genome of - kb that encodes four to five structural proteins (s, the spike glycoprotein; m, the membrane glycoprotein; n, the nucleocapsid interrupt phosphoprotein; e, the envelope protein; and he, the hemagglutinin-esterase glycoprotein) and nonstructural polyprotein precursors (pp a, polyprotein a; and pp ab, polyprotein ab), which are later processed into several other nonstructural proteins and viral polymerase [ , ] . coronaviruses are clinically significant to humans and usually associated with different respiratory, intestinal, hepatic, or neurological diseases [ ] . hcov- e, hcov-oc , hcov-nl , and hcov-hku are among the most disseminating coronaviruses that usually infect the upper respiratory tract in humans, causing mild common cold-like diseases [ ] [ ] [ ] [ ] . other coronaviruses, such as sars-cov identified in china in and mers-cov discovered in the middle east in , are notorious pathogens able to cause widespread outbreaks of pneumonia and pneumonialike conditions with very high morbidity and mortality rates of up to % [ , ] . due to their high clinical significance and contagious nature, there has recently been great interest in testing and developing advanced sars detection methods. sars detection using aunps is primarily focused on developing rapid and specific molecular detection through two main assays: ) a colorimetric assay for pp ab gene detection; and ) an electrochemical assay for nucleocapsid protein gene detection ( table ). the colorimetric assay involves the ability of aunps to preferentially adsorb ssdna over dsdna and specifically sense the presence of the target dna [ ] . specific, short ssdna probes adsorb to the surface of aunps, resulting in an increased particle colloidal stability and an increased ability to withstand slightly elevated salt concentrations without significant aggregation or color changes. upon addition, the target dna specifically forms dsdna with the adsorbed ssdna probes, which then easily detach, leaving the aunps to aggregate due to the salt. the particle aggregation causes the red color of the solution to change to blue, indicating the presence of target sars nucleic acids; this change can be directly assessed by the naked eye or precisely quantified by ultraviolet-visible (uv-vis) spectroscopy in correlation to the target dna concentration (fig. a ). in the electrochemical assay, aunps are applied to enhance the electrode conductivity and increase the surface area available for detection probe immobilization [ ] . sars-specific dna-capturing probes are first immobilized on the surface of an electrode structured with aunps; then, they are allowed to hybridize with the biotinylated targets. then, streptavidin-labeled alkaline phosphatase is applied to catalyze the indirect reduction and deposition of silver ions, which are eventually measured by anodic stripping voltammetry in correlation with the target dna concentration (fig. b) . the aunp-based assays developed for the molecular detection of sars are relatively rapid and simple; especially the colorimetric assay, which completely eliminates the need for instrumentation or trained personnel and yields results exclusively in the liquid phase that can be rapidly identified as positive/negative by the naked eye within min. this technique can allow the detection of target sars nucleic acids with a sensitivity limit down to fm; thus, it can be very beneficial for the early diagnosis of the sars virus, which is critical for such a contagious pathogen. in addition, the simplicity and very high sensitivity of aunp-based colorimetric assays for nucleic acids have promoted the application of aunps for the detection of other viruses either using slightly modified procedures or in combination with other sensing characteristics of aunps [ ] . the family filoviridae can be classified into two main genera, ebolavirus [ , ] . filoviruses are filamentous, enveloped rna viruses nm in diameter and approximately - nm in length. their genome is nonsegmented, negative-sense, ssrna kb in size that encodes for at least proteins (nucleoprotein, viral proteins vp , vp , vp , and vp , glycoprotein, and polymerase protein) [ ] . aunps stabilized by single-stranded (ss)dna probes. in the presence of the target dna sequence, double-stranded (ds)dna is formed and desorbs from the surface of citrate-reduced aunps, leaving them to aggregate. this aggregation results in a color change from red to blue, indicating the presence of target nucleic acids [ , ] . (b) enzymatic electrochemical detection of sars by anodic stripping voltammetry using a aunp-screen-printed carbon electrode. the aunp-modified electrode is modified with capture dna probes that are allowed to hybridize with biotinylated targets. streptavidin-alkaline phosphatase (s-ap) is then applied for the indirect reduction of silver ions in the solution into a metallic deposit. the formed silver metals are then electrochemically measured to determine the target virus dna concentration [ ] . most ebovs primarily originate from africa, where they are frequently associated with large outbreaks of ebola hemorrhagic fever (ehf), which is now known to be one of the most deadly and virulent diseases affecting humans, with a case fatality approaching % [ ] . the early outbreaks of ehf were confined to sudan and its neighbor, democratic republic of the congo, from to , followed by several independent outbreaks in different countries: [ , ] . most of these outbreaks were caused by zebov and sebov, while some were caused by new species later named ciebov and bebov [ ] . these heavy series of ehf outbreaks are continuing, and most recently, multiple countries in west africa have struggled against a massive outbreak of ebov beginning in march . ebola is expected to remain a major global public health threat, especially with the current absence of effective treatments; therefore, it is necessary to have flexible rapid-detection schemes in place to diagnose and control ebov outbreaks [ ] . a very interesting aunp-based molecular assay has been developed for the bimodal scanometric and sers-based detection of ebov by using aunps to promote silver staining on their large surface area [ ] . the target ebov dna sequence is first captured on chip surfaces by specific, short dna probes and labeled with aunp-dna-cy probes. then, the formed -component hybridization complexes are subjected to a silver enhancement step. the deposited silver metal eventually enables scanometric detection (based on a silver-caused dark color) and sers detection (based on using cy as a raman-active tag) (fig. ) . this assay has additional benefits beyond its high specificity and sensitivity that reach down to fm of the target rna. because a very large number of probes can be designed based on using different raman tags, this assay can be readily adapted for multiplex detection, allowing the simultaneous detection of different diagnostic targets of the same virus or even completely different viruses. furthermore, it does not require dna amplification and eliminates the need for specific thermal cyclic equipment. in addition, the capability for bimodal detection simplifies evaluation of the results, as the output can be positive/negative either by traditional scanner systems or by sers when the probes are labeled with raman dyes. such simplicity based on bimodal detection without the need for complicated thermal amplification equipment, multiplexing, and high sensitivity supports the application of this technique for ebov, which is very contagious and usually necessitates the detection of several targets. it is a chip-based detection scheme in which the target dna is captured by immobilized, specific dna oligonucleotides and then directly labeled by aunp-dna probes, followed by silver enhancement. the aunps enhance the surface area available for silver deposition and dna immobilization to achieve highly sensitive scanometric detection signals. modification of the aunp probes with cy allows the additional sers-based detection of target dna. this scheme has been reported for the detection of a wide range of viral nucleic acids, including those of hcv, hbv, and hav, which belong to different virus groups [ ] . furthermore, the scanometric detection reported in this scheme has been considered a universal step in other detection schemes described for other viruses, such as hev [ ] and hiv [ , ] . this assay has been reported by the same authors for multiplex detection of hepatitis a virus (hav), hepatitis b virus (hbv), hiv, and variola virus (smallpox, vv), which confirms the broad capability of this assay for virus detection [ ] . flaviviridae is a large virus family comprising three main genera: flavivirus ( species, type species is yellow fever virus), hepacivirus (one species, hepatitis c virus; hcv), and pestivirus (four species, type species is bovine virus diarrhea) [ ] . flaviviruses are spherical, enveloped rna viruses - nm in size. their genome is nonsegmented, positive-sense ssrna approximately - kb in size and includes one open reading frame (orf) encoding three structural proteins (c capsid; prm, premembrane; and e, envelope), and seven nonstructural proteins (ns , ns a, ns b, ns , ns a, ns b, and ns ). several well-known human pathogens are included in this family, such as dengue virus (denv), hcv, yellow fever virus, and west nile virus (wnv), japanese encephalitis virus, and tick-borne encephalitis virus. these viruses have become increasingly serious and have been frequently involved in causing many endemics and epidemics around the world [ , ] . denv is the most rapidly spreading vector-borne virus in the world. denv is now known to be epidemic in more than subtropical and tropical countries, threatening up to . billion people. denv causes more than million new annual infections, including million with symptoms varying from flu-like, mild, undifferentiated fever to classic dengue fever (df) or df with hemorrhagic manifestations [ , ] . although many of these infections are self-limiting and resolve without hospitalization, some progress to severe disease that needs to be quickly identified and treated [ , ] . currently, there are no specific treatments or protective vaccines available against denv, and as a result of its rapid expansion, accurate and sensitive diagnostic techniques have become crucial for effective control and treatment applications [ ] . aunps have been combined with the well-known analytical techniques qcm and icp-ms to develop two novel molecular assays for denv detection. for the first time, these assays implemented aunps to increase the mass of target dna to allow its detection by qcm in a highly quantitative and sensitive manner (fig. a) or to amplify the detection signal by releasing many gold ions during icp-ms analysis of the target dna (fig. b ). in the aunp-based qcm assay, the presence of target dna initiates a layer-by-layer hybridization of the target dna and several specific multivalent aunp-dna probes, resulting in significant mass changes detected by qcm [ ] (fig. a) . whereas, in the icp-ms-based assay, two types of probes prepared from specific aunp-dna and mnp-dna conjugates are applied to specifically recognize and sandwich the target virus dna sequences. the mnp probes then help to magnetically separate the formed sandwich complexes, and the target dna concentration is indirectly estimated by detection of the au concentration existing in the sample [ ] (fig. b) . it is worth noting that both techniques are among the most novel applications of aunps in virus detection. the implementation of aunps extends the ability of qcm and icp-ms to detect dna. moreover, aunp-based icp-ms is among the most sensitive molecular dna assays applied for virus detection, with a detection limit down to zmol, which can greatly help to control denv and other viruses [ ] . the bio-barcode ssdna is released and employed to establish multilayered aunps over the electrodes to enhance the produced electric current [ ] . (b) dry-reagent strip-based dna assay using aunp-oligo (dt) reporters. aunps with poly (dt) are applied to label biotinylated target dna modified with a poly (da)-tail pre-captured by immobilized streptavidin in the test zone of the strip and generate characteristic red bands [ ] . (c) single-particle fluorometric detection of hcv using cy -tagged ssrna that is electrostatically adsorbed or covalently coupled through thiol (sh) chemistry to the surface of aunps. the coupling of aunps and ssrna probes brings cy in the vicinity of the aunp surface and eventually results in emission quenching; upon hybridization with the target rna, the quenched emission is released [ ] . hcv is one of the leading causes of chronic liver diseases in humans. approximately % of the global population has experienced hcv infection. currently, there are million chronic carriers, and more than three million new infections occur globally each year [ , ] . chronic hcv infection is frequently associated with marked hepatic injuries, including cirrhosis, liver failure or hepatocellular carcinoma. these types of injuries can be very severe and often end in death or liver transplantation [ , ] . hcv infection is generally resilient and usually requires treatment that combines numerous medications. the most popular treatment regimen for hcv is a combination of pegylated interferon alfa (peg-ifnα) and ribavirin (rbv) [ ] . other three-drug regimens based on the addition of the serine protease inhibitor telaprevir (tvr) or boceprevir (bec) to the standard peg-ifnα/rbv treatment have also been described [ ] . in addition, several recently developed oral drugs, such as simeprevir and sofosbuvir, have been approved by the fda for more effective care and shorter treatment duration [ ] [ ] [ ] [ ] . despite these numerous drugs and due to the lack of efficient vaccines against hcv, reliable and sensitive detection methods are essential for viral infection prevention and therapeutic responses. numerous aunp-based scanometric, fluorometric, electrochemical, and colorimetric assays have been reported for the molecular detection of hcv (table ). in these assays, aunps are utilized for different sensing functions: ) aunps have been modified with poly(dt)-tailed dna and directly applied to label target dna sequences terminally modified with a poly(da)-tail in a lfa assay to allow the colorimetric detection of hcv (fig. b) [ ] . ) aunps electrostatically or covalently modified with cy -tagged ssrna sequences have been applied for the fluorometric detection of target hcv sequences, in which the fluorophores are quenched in a process analogous to fret. the presence of cy in the vicinity of the surface of aunps eventually results in cy -emission quenching. upon hybridization with target viral rna, the electrostatically adsorbed probes labeled with cy are detached from the surface of the particles, releasing the quenched fluorescence. the covalently bound probes form rigid dsrna with a linear configuration, causing cy to move away from the particle surface and release the quenched emission [ ] (fig. c) . ) aunps have also been applied to enhance electrical conduction and catalyze a reduction reaction on the surface of detection electrodes. a specifically designed hairpin dna probe terminally modified with aunps and bound to the surface of a glassy carbon electrode is utilized to detect the amplified viral rna. the presence of target dna results in the formation of rigid dsdna, and the hairpin conformation consequently changes, moving the aunps away from the electrode surface, which eventually appears as a detectable decline in the current value in proportion to the target dna concentration [ ] . ) aunps decorated with a specific nucleic acid probe were used for colorimetric detection of hcv. the assay is based on using cationic aunps to induce the aggregation of aunps probes. in the absence of hcv, the cationic aunps electrostatically bind to negatively charged aunps-dna probes causing their aggregation and change of the solution color to blue. the presence of hcv nucleic acid protects the probes and prevents their aggregation by cationic aunps and the solution color remains red. this assay was specific and showed a sensitivity of . % and a detection limit of . iu/μl [ ] . in the immunological detection of hcv, aunps dually modified with capturing antibodies and barcoding dna, similar to those described for the immuno-pcr technique, in combination with magnetic np (mnp)-antibody conjugates, have been applied to capture and separate the target antigen. subsequently, the barcode dna is further allowed to act as a bridge, guiding the formation of a multilayer of aunp-dna probes over the surface of a nanogap electrode (fig. a) . the formation of such barcode dna-guided aunp multilayers significantly increases the detected electrical current and is utilized to quantitatively measure the target hcv antigen concentration in applied solutions. the results of this immunoassay indicate a detection limit of pg/µl [ ] . in a similar protocol, the barcoding dna was further applied to hcv core antigen detection based on an enzyme to release the barcode dnas to be quantified with rt-pcr. this assay showed a detection limit of fg/ml, which is one magnitude greater than the standard elisa ( ng/ml) [ ] . it is worth mentioning that the primary focus of the developed aunp-based assays was the detection of hcv rna rather than hcv antigens or antibodies. this is due to the broader potential of molecular assays for the detection and management of active hcv infections. hepadnaviridae comprises two main genera, orthohepadnavirus and avihepadnavirus. the genus orthohepadnavirus includes species (hbv, woodchuck hepatitis virus, ground squirrel hepatitis virus, and woolly monkey hbv) that infect mammals, while the genus avihepadnavirusincludes species (duck hbv and heron hbv) infect birds [ ] . hepadnaviruses are spherical, enveloped dna viruses - nm in size. they possess a unique gapped genome of . -kb circular dsdna, which contains partly overlapping orfs encoding the different viral proteins: the core protein (c), e antigen, polymerase protein (pol), envelope proteins, and transcriptional transactivator protein [ , ] . the prototype member of the family hepadnaviridae, hbv, is a health threat to ~ % of the global population [ , ] . hbv is the etiological agent of hepatitis type b in humans. it is estimated that nearly billion people around the world have been exposed to hbv infection, and there are more than million chronic carriers suffering from the serious risk of developing liver cirrhosis and cancer [ , ] . the availability of an effective protective vaccine against hbv has greatly reduced the number of new infections. in addition, there have been significant advances in the available treatments for chronic hbv infection. viral dna polymerase inhibitors and peg-ifnα are now well known to significantly control hbv infection and prevent its progression to chronic hepatitis b-associated liver failure and hepatocellular carcinoma [ , ] . however, chronic hbv infections remain highly contagious, and the virus can easily be transmitted to other persons through any close contact allowing the transfer of infected bodily fluids. the highly contagious nature of hbv and its ability to spread among people are the main reasons why this pathogen continues to threaten the public. therefore, diagnostic methods that are highly sensitive to low concentrations of virus are needed to curtail this virus and prevent its dissemination. hbv is by far the most common virus reported in the published literature regarding the applications of aunps in virus detection (fig. ) . interestingly, the developed assays cover most of the known diagnostic markers of hbv through many different electrochemical, scanometric, fluorometric, lightscattering, colorimetric, and sers detection schemes (table and fig. , - ) . several aunp-based electrochemical assays have been developed for the detection of hbv antigens and dna. two enzyme-amplified electrochemical assays have been reported for the detection hbv surface antigen (hbsag) using conjugates of aunps and horseradish peroxidase (hrp)-labeled antibodies against hbv surface antigen (hbsabs) [ , ] . the target hbsag is captured and labeled with aunp-hbsab/hrp as secondary antibody conjugates. then the reduction of h o catalyzed by the bound hrp is measured either by using an aunp/thionin/dna-modified au electrode coupled with a cyclic voltammeter [ ] or through using a nanoporous au electrode coupled with differential pulse voltammeter [ ] (fig. a) . alizadeh et al. reported aunps modified with horseradish peroxidase mimicking dnazyme to label hbsag magnetically captured and concentrated on the surface of a au sheet-like electrode. due to the efficient catalytic activity of hrp-mimicking dnazyme, the proposed immunoassay allowed quantitative detection of hbsag with a linear concentration range of . - pg/ml and detection limit of . pg/ml [ ] . other studies described the electrochemical detection of hbv based on aunp-metal enhancement amplification, including copper and silver metal enhancement [ , ] . using copper metal enhancement, hbsag is sandwiched by mnp-hbsab conjugates and aunp-hbsab probes to form a -component immunocomplex, which is magnetically separated and stained through a copper enhancement step (fig. b ). after copper acidic dissolution, the resulting ions are measured by anodic stripping voltammetry (asv) [ ] . using silver enhancement, hbv dna is detected using probes prepared of streptavidin-modified aunps. biotinylated hbsag gene sequences are magnetically pre-separated and concentrated using magnetic bead-dna conjugates. the deposited silver ions are then analyzed by an electrochemical stripping technique to detect the target hbv dna quantitatively [ ] . hbv detection through aunp-based scanometric assays mainly relies on the well-described aunp-promoted silver-staining protocol, in which aunp probes are applied to facilitate the reduction of silver ions into metallic silver that is deposited as visible black spots, indicating the presence of hbv. following this protocol, aunps modified with staphylococcal protein a (spa) [ ] or specific dna probes [ , ] have been utilized in multiple chip-based assays to permit the detection of different hbv antibodies and dna (fig. ). in addition, wang et al. combined this aunp-promoted silver-staining protocol integrated with a bio-barcode-amplification (bca) technique to allow an enhanced scanometric detection of hbv dna [ ] . this bca-based scanometric assay employs two different sets of dna conjugates to capture and detect a specific hbv sequence. the first set is a group of aunps modified with the short dsdna of a signal amplification dna strand (barcode dna) partially complemented with a detection probe strand, while the second set is a group of magnetic microparticles (mmps) modified with ssdna specific to the target hbv dna. the presence of the complementary target sequences of dna guides the assembly of aunp/dna/mmp sandwich hybrids. subsequently, the sandwiched hybrids are magnetically isolated and washed, and the barcode dna is eventually released from the aunps. the barcode dna is then added to the surface of a chip modified with specific dna-capturing probes and directly labeled with aunp-dna conjugates, followed by silver staining to further amplify the detection signal (fig. ) . the aunps-fluorometric assays for hbv are performed through different schemes that basically depict the quenching efficiency of aunps in a manner remarkably similar to the traditional fret protocol. zheng et al. developed a multiplex detection system based on applying gold nanorods (aunrs) as acceptors and qds of different colors as donors to simultaneously detect hbsag and hbv e antigen [ ] . this assay follows the direct sandwich immunoassay format, and the presence of target antigens is manifested by the fret-induced quenching of qd fluorescence in the formed sandwich nanostructure of aunr-ab /ag/qd-ab (fig. c ). draz et al. further coupled multiple aunp-peptide conjugateacceptors with single-core qd-antibody fab conjugate-donors, forming a preassembled hybrid nanocluster plasmonic resonator complex for hbsag and hbv particle detection (fig. c) . this scheme follows a competitive assay format, and the addition of hbv target surface antigen or particles to the preassembled nanocluster releases the quenched fluorescence signal of the qds in proportion to the in this assay, the target hbsag is captured and labeled with aunp-ab/hrp, and the detection signal is measured by using an ab-modified au electrode to assess the reduction of h o catalyzed by the bound hrp using differential pulse voltammetry (dpv) technique [ ] . (b) copper (cu)-enhanced electrochemical immunoassay. aunps serve as a scaffold for a horseradish peroxidase (hrp) enzyme that catalyzes a redox reaction in the first scheme or enhances metal deposition in the second scheme [ ] . (c) fluorometric detection by the fret-induced quenching of fluorophores on the surface of aunps. aunps and gold nanorods (aunrs) are applied to quench the fluorescence of fluorophores, such as (i and ii) quantum dots (qds) [ , ] and (iii) fluorescein amidite (fam) [ ] , through a fret-based interaction in the presence of the target virus. a similar aunp fret-based scheme has been reported for the detection of h n , which belongs to the family orthomyxoviridae [ ] . (d) light-scattering assays using aunp-antibody probes. (i) light-scattering immunoassay of virus antigen based on using aunps with different sizes ( nm and nm) conjugated with antibodies (abs) to sandwich target antigens and enhance the dynamic light scattering [ ] . additionally, this scheme has been reported with the use of aunps of the same size for h n detection [ ] . (ii) plasmonic immunoassay based on allowing aunr-ab conjugates to interact with the target antigen, thus changing the aunr localized surface plasmon resonance (lspr) behavior [ ] . target concentration [ ] . furthermore, lu et al. developed a fluorometric assay for hbv dna detection in which positively charged aunrs are employed to chelate fluorescein amidite (fam)tagged ssdna electrostatically onto their surface, forming fam-ssdna-aunr ternary complexes [ ] . the formation of these complex results in the fret-based quenching of fam fluorescence, and when complementary target dna is present in the system, a fam-ssdna/cdna duplex is formed. this dna duplex is comparatively more negative than the fam-ssdna and can mediate stronger electrostatic interactions with aunrs, leading to increased fret efficiency, which manifests as a further decline in the fluorescence intensity of fam. this change in fluorescence intensity is utilized to sense and estimate the exact concentration of the added target dna (fig. c) . aunp-based light-scattering assays have been described for the detection of hbsag through two main schemes: the first scheme applies spherical aunps of different diameters ( nm and nm) modified with hbsab to sandwich hbsag, and the presence of hbsag results in specific immuno-based aggregations that can be estimated by measuring the increases in hydrodynamic diameter using dls (fig. d) [ , ] ; the second scheme relies on the lspr characteristic behavior of aunrs to directly sense the binding of hbsag to the antibodies conjugated to their surface by measuring lspr peak shifts via uv-vis spectroscopy (fig. d) [ ] . a aunps-based colorimetric lateral flow assay (lfa) assay was developed for the detection of hbsag. this assay relies on the characteristic red color of different sized aunps to label hbsag on an immunochromatographic test strip. a strip composed of a sample pad, conjugation pad, control line, test line and absorbent pad was used to capture hbsag labeled with aunps and the test line becomes red in color. the results showed that the signal visibility of lfa could be improved by increasing the diameter of aunps, and ~ nm aunp enabled lfa assay with a detection limit of ng/ml [ ] . a aunps-based sers assay for hbv dna detection was developed following a sandwich assay format performed on a heat-responsive hybrid silicon substrate modified with aunps. the target hbv dna is captured on the surface of the silicon substrate and forms a sandwich complex with specifically designed aunps modified with dnaindocyanine green proximally to the surface, leading to high sers signals. this heat-responsive hybrid silicon substrate-based sers platform can detect remarkably low hbv dna concentrations of ~ . fm at °c and ~ . fm at °c [ ] . aunp-based fluorometric detection is the most sensitive among the reported methods for hbv detection, with a detection limit of particle/μl. due to the highly infectious nature of hbv, this limit of detection can be very useful for exploiting these assays for efficient control and management of hbv infection. in addition, the simplicity of scanometric and colorimetric detections is very interesting, allowing flexibility in the detection design and target, and was reported for single and multiple target detection for more efficient and accurate screening of hbv. on the other hand, the developed dls assay is very novel to hbv research, simple and sensitive; however, the need for bulky equipment for dls measurement remains the major challenge for its wide use and real application in hbv detection. the family hepeviridae is monogeneric with one exclusive member called hev, which is a spherical, small, nonenveloped rna virus with a ~ . kb ssrna genome [ ] . the viral rna is a positive-sense molecule organized into partially overlapping orfs flanked by short untranslated regions at both ends. the first orf encodes mature nonstructural proteins (methyltransferase, protease, helicase and replicase), the second orf encodes structural proteins (c and vp ), and the third orf encodes a small protein of unknown function [ ] . hev is known for causing human liver inflammation figure . aunp-based nucleic acid assay for hepatitis b virus (hbv) detection. hbv dna was detected using a scanometric detection that integrates magnetic separation and aunps-based bio-barcode-amplification method. aunps provide a large surface area for silver deposition and dna immobilization, thereby achieving highly sensitive scanometric detection signals through a chip-based silver deposition scheme [ ] . called hepatitis e, which is widely disseminated in developing countries with poor sanitation conditions and has recently become apparently endemic in some industrialized countries, including the usa [ ] . one aunp-based colorimetric assay has been reported for the molecular detection of hev [ ] . this assay targets a conservative fragment of hev in orf that is pre-amplified by one-step rt-pcr/pcr amplification, and the generated cdna is labeled by aunp-dna conjugates, forming three-component sandwich hybrids in a manner very similar to that previously depicted in fig. . this hybridization is followed by a silver-staining step to allow the scanometric detection of target dna either by the naked eye or using simple scanners, with a detection limit of fm of hev amplicon. one of the major advantages of this hev detection assay is its sensitivity, which can help control hev. however, this assay suffers from the same disadvantages of conventional pcr because of the requirements of the amplification step. herpesviridae is a taxonomically large family of viruses, including major subfamilies (alphaherpesvirinae, betaherpesvirinae and gammaherpesvirinae), which together comprise nearly genera with over viruses [ ] . repeats. the rearrangements of these repeated sequences result in different genome isomers. in epstein-barr virus (ebv) and kaposi sarcoma-associated herpes virus (kshv), from the subfamily gammaherpesvirinae, there are repeated sequences at the termini of their genomes that differ from those of other human pathogenic herpesviruses, including herpes simplex virus type (hsv- ) and type (hsv- ), human cytomegalovirus (hcmv), and varicella zoster virus (vzv); these viruses are characterized by the definite presence of us and ul regions in their genomes [ ] [ ] [ ] . herpesviruses can infect humans and many animals. they account for significant and recurrent human cutaneous diseases, including oral herpes, genital herpes, and many cancers. eight herpesviridae family members are commonly involved in human infection: hsv- (human herpesvirus ; hhv- ), hsv- (hhv- ), vzv (hhv- ), ebv (hhv- ), hcmv (hhv- ), hhv- , hhv- , and kshv (hhv- ). hsvs are among the most prevalent viruses in humans. there are two serotypically and genetically different hsvs, types (hsv- ) and (hsv- ) [ ] . hsv- is estimated to infect up to one-third of the world population, while hsv- infects nearly million people around the globe, with more than million new cases occurring every year [ ] [ ] [ ] . both hsv types are renowned for infecting epithelial cells of the skin or mucosal surfaces and then infecting the central nervous system, causing lifelong, incurable infections in humans. these infections are primarily asymptomatic but can progressively result in serious health complications. for example, hsv- infection is the main cause of infectious blindness in the world and has emerged as an important cause of genital disease in the developed world, and hsv- infection is the leading cause of genital ulcers worldwide [ , ] . nevertheless, these viruses are involved in many other clinical conditions, such as encephalitis, conjunctivitis, zosteriform skin lesions, pneumonia, and systemic infections that compromise vital organs in the body [ ] . therefore, desperate efforts have been directed toward developing an effective vaccine against hsvs. however, there are no protective vaccines against hsvs, and only a few anti-herpes drugs, such as acyclovir, valacyclovir, and famciclovir, are commercially available [ ] [ ] [ ] . these drugs are only helpful to control symptoms and signs of infection and do not eradicate the virus or even decrease the frequency or severity of infection recurrence and due to the drug resistance rapidly developed by hsvs during treatment, the efficacy of anti-herpes drugs is increasingly hampered over time. thus, the definitive diagnosis of hsv virus infection is essential for effectively controlling its severity and applying treatment regimens. with their characteristic red color, aunps have been exploited as colorimetric labels in a lateral-flow immunochromatographic assay (lfia) to allow the immunological detection of hsv antigen. in this assay, the lfia strip is preloaded with the labeling aunp-anti-human igg conjugates onto the conjugate pad, and the capturing igg- and igg- antigens, along with the anti-igg antibody, are immobilized onto separate test and control lines directly next to the sampling area (fig. a) . the sample is loaded and allowed to migrate across the strip with the chase buffer to react with the capturing antigens (igg- and igg- ) loaded on the test lines, as well as the anti-igg antibody loaded on the control line. as a result, hsv antibodies are captured on the test lines and labeled with the loaded aunp-dna conjugates, causing characteristic red lines to appear. according to the color of the test lines, the samples can be qualitatively determined to be positive or negative within min [ ] . lateral-flow immunochromatographic assay of hsv- antibodies using aunp anti-igg probes. sample addition followed by chase buffer addition causes gold nanoparticle (aunp)-anti-igg conjugates and the sample to migrate, contacting the test lines sequentially and resulting in the capture of type-specific antibodies to hsv- . the concentration of hsv- antibody-aunp complexes on the test line causes a corresponding pink color to form [ ] . this scheme is similar to that reported for the detection of h n [ ] and hiv- [ ] . (b) electrochemical detection of hcmv by the acidic dissolution of aunps. hcmv dna is directly labeled with specific aunp-dna probes. after the acidic dissolution of aunps, au iii ions are measured by anodic stripping voltammetry (asa) [ ] . (c) colorimetric one-pot np-based assay of kshv. this assay has been reported for the differential diagnosis of kshv dna and dna from a frequently confounding bartonella species. upon the addition of sample dna to a mixture of silver np (agnp)-dna and aunp-dna conjugates specific for kshv and bartonella dna, respectively, complementary dna hybridization triggers the aggregation of bare np conjugates and the subsequent decrease or disappearance of its corresponding color [ ] . hcmv/hhv- is the largest known endemic virus in humans. it persistently infects over % of the population worldwide [ ] . hcmv can infect a broad range of cells, including endothelial cells, epithelial cells, smooth muscle cells, fibroblasts, neuronal cells, hepatocytes, and many types of immune cells [ , ] . nearly all hcmv infections are asymptomatic, except in immunocompromised individuals and neonates, and can lead to severe symptoms. hcmv infection is the leading cause of congenital malformations in live births worldwide, while in immunocompromised individuals hcmv is usually opportunistic, causing very serious clinical manifestations, such as pneumonia, hepatitis, encephalitis, colitis, and retinitis [ , ] . moreover, hcmv has been described to be associated with several human malignancies, especially in the brain, breast, prostate, skin and colon [ ] . numerous hcmv vaccine candidates have been developed and tested, but a vaccine has yet to be licensed. antiviral therapy with several anti-hcmv drugs, such as ganciclovir, valganciclovir, foscarnet, and cidofovir, which target the viral dna polymerase, is currently the main strategy for controlling hcmv infection [ , , ] . unfortunately, these drugs result in substantial hematotoxicity and nephrotoxicity in patients, and hcmv remains a considerable public health threat without an efficient treatment or control strategy. in such conditions, the need for new diagnostic methods that can help to control the dissemination of this pathogen is very important. a novel aunp-based electrochemical assay for the molecular detection of hcmv dna has been reported [ ] . in this assay, aunps are applied to amplify the detected electrochemical signal by releasing many au ions upon exposure to acid dissolution. typically, the immobilized target hcmv ssdna is initially labeled with oligonucleotidemodified aunp probes followed by a metal dissolution step, releasing many gold ions. the concentration of solubilized gold ions is then indirectly assessed by asv (fig. b ). this assay allows the highly sensitive detection of target dna concentrations down to pm [ ] . kshv/hhv- is a leading cancer-causing virus. it is commonly responsible for inducing a considerable range of neoplastic diseases in immunocompromised patients, such as kaposi sarcoma, primary effusion lymphoma, and some kinds of multicentric castleman disease [ , ] . owing to the dramatically increasing rates of immunocompromised patients over the past two decades, the clinical significance of kshv is clearly increasing; additionally, kshv has a prevalence of - % in this class of patients worldwide [ , ] . while kshv is a relatively newly discovered virus, there are no standard diagnostic approaches, and the detection of this virus through traditional serological and molecular methods remains challenging. this difficulty is mainly due to the inconsistency observed among serological assays targeting different antigens, and the low levels of viral dna that are usually present in the peripheral blood of infected individuals [ , ] . hence, there is a continued interest in developing new reliable and sensitive methods for kshv detection. through using a mixture of aunps and silver nps (agnps), a colorimetric assay has been established for the multiplex, one-pot molecular detection of kshv and its frequently confounding disease bacillary angiomatosis [ ] . in this assay, two types of probes are prepared from aunps and agnps separately functionalized with kshv and bartonellaspecific dna-capturing dna probes, respectively, and mixed with the target dna. then, the color of the reaction mixture changes in a manner dependent on the type of target dna added to the reaction solution: the addition of kshv dna causes aunp conjugates to aggregate and the solution to appear murky yellow-orange, i.e., the specific color of the nonaggregated agnps, while the addition of bartonella dna causes agnp conjugates to aggregate and the solution to appear pink, i.e., the color of nonaggregated aunps (fig. c ). in addition to being simple, this multicolor-change system successfully allows the multiplex detection of targets present in nanomolar concentrations. the family orthomyxoviridae includes five genera: influenzavirusa, influenzavirus b, influenzavirus c, isavirus, and thogotovirus [ ] . the first three genera are widely known to infect birds, humans, and other mammals, causing influenza. the influenza-virusa genus contains many strains and can be further divided into several antigenic subtypes according to the antigenic properties of their envelope glycoproteins, i.e., hemagglutinin (ha) and neuraminidase (na). in contrast, the influenzavirusb genus has no distinct antigenic subtypes of hemagglutinin or neuraminidase. influenzavirusc is similar to types a and b but less common and lacks the na protein. the genus isavirus is known to infect salmon, while thogotoviruses usually cause infection in some vertebrates and invertebrates [ , ] . orthomyxoviruses can be spherical ( - nm) or filamentous ( nm in diameter and - nm long) in shape, and their genome contains or segments of linear negative-sense ssrna with a total length of - kb encoding different structural and nonstructural proteins, including nucleoprotein, three large proteins (pb , pb , and pa), matrix proteins (m and m ), the glycoproteins ha and na, and two nonstructural proteins (ns and ns ) [ , , ] . several strains of the genus influenzavirusa cause seasonal and occasional acute respiratory diseases called influenza, which is profoundly and rapidly spread in humans. influenza is reported to affect up to % of adults and % of children of the global population each year [ , ] . over the past decade, the world witnessed two major influenza outbreaks caused by newly emerging influenzavirusa strains: h n in (avian influenza), and h n in (swine influenza). both were severe enough to incite the who to issue a pandemic warning alert, which reached the highest level, i.e., "phase ," for h n in [ , ] . the severity of influenza infection depends on both the virus strain and the age of the infected person. in elderly persons, influenza can result in hospitalization or death, while in younger adults, influenza-associated morbidity usually results in restricted activity, impaired work performance, and increased health care use [ , ] . most people with the flu recover within one to two weeks without treatment. however, serious complications usually require medications to reduce the severity and duration of infection. some antiviral drugs, such as oseltamivir and zanamivir, can effectively treat or prevent influenza [ ] . on the other hand, immunization with vaccine preparations of the most-circulating virus strains (trivalent or quadrivalent vaccines) remains the key strategy for preventing both influenza and its serious complications [ , ] . in fact, both the treatment and management of influenza infection, either by antivirals or vaccination, depend on the influenza virus strains and the timing and severity of the infection. therefore, the rapid and accurate testing and typing of influenza is critical for treating infection and plays an important role in public health measures. the application of aunps in iav detection has primarily been focused on developing assays that enable the direct detection of whole iav particles [ , , ] or viral rna [ , , , , , , ] through numerous aunp-based fluorometric, sers, scanometric, light-scattering, colorimetric, and electrochemical detection schemes ( table ) . the aunp-based fluorometric assays developed for h n detection have simply exploited the intense plasmonic-based quenching properties of aunps through different detection formats. one of these assays targets the detection of the whole virus particle through a lateral-flow immunoassay format that is similar to that previously described for the detection of hsv- , as shown in fig. a . in this assay, the virus is first captured on a lateral-flow test strip by pre-immobilized antibodies; then, aunp-antibody conjugates are applied to label the captured virus particles. after washing, the aunps are collected and dissolved to release gold ions that are eventually utilized to quench the fluorescence of qds in an additional, separate step [ ] . in addition, ganbold et al. have developed a bimodal assay for the fluorometric and sers detection of viral rna using aunps modified with ssdna-dye probes [ ] . the surface ssdna-dye probes are weakly adsorbed to the surface of aunps by electrostatic interactions, and with the addition of the complementary target dna, perfectly matched dsdna forms and desorbs from the surface of aunps, resulting in measurable changes in the conjugated dye fluorescence and raman fingerprint that are more substantial than those resulting from either mismatched dsdna or ssdna (fig. a) . aunp-based colorimetric assays have been developed for iav detection through four main detection schemes: ) the first scheme involves the application of aunps conjugated with anti-digoxigenin as colorimetric labels to detect the target viral dna premodified with digoxigenin and electrophoresed through a membrane-based lateral-flow technique [ ] . ) the second scheme is simply based on the electrostatic interactions between the negatively charged phosphate groups of the target dna amplified with lamp and the positively charged ctab bilayer on aunrs, resulting in the aggregation of aunrs and a color change from red to purple [ ] . ) the third scheme relies on a magnetic-based hydroquinone oxidation reaction involving two metal np bioconjugates: iav-specific pentabody-mnps as capture probes and anti-aiv monoclonal antibody-aunps as detection probes. in the presence of virus particles, an immunocomplex forms and is separated by a magnetic field; after washing, aunps in the separated complex act to catalyze the oxidation of hydroquinone, which is detected optically in correlation with the aiv sample concentration [ ] . ) the fourth scheme relies on the peroxidase-like catalytic activity of au np films and colloidal aunps toward tmb-h o mixtures. the virus is captured on a -well plate modified with aunp film biofunctionalized with ha antibody. then it is labeled with aunps modified with na antibody for signal generation. tmb-h o is added, and rapid color changes are observed because of the oxidation of peroxidase substrate tmb. using h n , the response of the developed assay was linear in the range from pg/ml to μg/ml and the limit of detection was . pg/ml, while with h n , the optical density of the developed color was dependent on the virus concentration in the range of - , pfu/ml and the limit of detection was . pfu/ml [ ] . aunp-based electrochemical assays rely on using aunps to directly modify the target dna or electron transducer surfaces, allowing enhanced electrical conduction and increased surface area for probe immobilization and target interaction. following this protocol, liu et al. [ ] successfully detected aiv ha gene sequences using a dna aptamer immobilized onto an electrode; its surface was modified with a composite of carbon nanotubes, polypyrrole nanowires and aunps (fig. b) . furthermore, bonanni et al. [ ] developed an impedimetric electrochemical assay for the detection of aiv m gene sequences based on measuring changes in the impedimetric behavior of the electrode when the target dna hybridizes with the capture dna probes immobilized onto its surface and is subsequently labeled by aunps via streptavidin/ biotin interaction (fig. c) . aunp-based scanometric assays for detecting aiv are mainly performed using the well-described aunp-promoted metal staining protocol shown in fig. d . in this protocol, the target nucleic acid is recaptured on the surface of glass chips modified with specific capture dna probes [ ] . then, the captured probes are labeled with aunps either modified with specific dna probes or with the renowned dsdna intercalator daunorubicin [ ] to mediate the metal staining. following this labeling step with aunp probes, the presence of the target nucleic acids is visually determined with an additional silver-or gold-staining step. specific dna aptamers immobilized on a gold electrode (ge) modified with a layer of multiwall carbon nanotubes (mwcnts), polypyrrole nanowires (ppnws) and aunps are collectively applied as an electrochemical platform for the detection of dna hybridization using dpv [ ] . (c) impedimetric electrochemical assay using streptavidin-aunps probes. the direct/sandwich hybridization of biotinylated target dna with capture probes immobilized on the surface of a screen-printed electrode (spe) and additional conjugation with streptavidin-aunps results in impedimetric signal enhancement [ ] . (d) label-free scanometric detection using positively charged aunps. the electrostatic interaction between positively charged aunps and negatively charged dna is utilized to visualize the double-stranded (ds)dna formed by the hybridization of single-stranded probe dna and complementary target dna on the array of chips through aunp-mediated metal staining [ , , ] . the light-scattering detection of h n virus using aunps relies on dls to measure the aggregation of aunp-antibody conjugates induced by the addition of target virus particles (similar to fig. d ). the magnitude of aunp aggregation and the corresponding increase in their hydrodynamic size is correlated with the concentration of virus particles [ ] . finally, the immunological detection of h n hemagglutinin (ha) antigen has only been reported through a novel lspr-based immunoassay. in this assay, the presence of target antigen induces a sandwich immunoreaction between the fluorophorelabeled detection antibodies and the aunp-capturing antibody conjugates, directly resulting in enhanced fluorescence caused by the coupling of fluorophore emission with the localized surface plasmons of aunps in a manner similar to that shown in fig. c . the sensitivity of this method has been described to be -fold, improved over that of the conventional elisa technique, with a reported limit of detection of . pg/ml [ ] . papillomaviridae is a widely diverse virus family that includes hundreds of recognized viruses classified into taxonomically distinct genera designated with specific greek alphabet letters, from alpha to pi. the first five genera (alphapapillomavirus, betapapillomavirus, gammapapillomavirus, mupapillomavirus and nupapillomavirus) include the human papillomaviruses, while the remaining genera specifically include other mammalian and avian papillomaviruses [ , ] . papillomaviruses are spherical, nonenveloped dna viruses - nm in size. their genome consists of a single molecule of circular, supercoiled, dsdna . - kbp in size and encodes early expressed (e) and late (l) expressed proteins, in addition to a noncoding long control region [ , ] . hpv infections are the most epidemic sexually transmitted viral infections worldwide [ , ] . hpvs are highly epitheliotropic pathogens that usually infect the deepest layer of the skin or the genital surfaces. most hpv infections cause no symptoms and usually self-clear without causing clinical problems, but persistent infections lead to serious clinical complications [ ] . this persistence typically depends on the type of hpv causing the infection. among more than types of hpvs, approximately types infect the mucous membranes and can be either high-or low-risk hpvs. high-risk types, such as hpvs , , , and , are usually associated with many cancers of the cervix, vagina, vulva, penis, anus, neck, and head, as well as oropharyngeal cancer, while low-risk types, such as hpvs and , are mainly related to anogenital warts and recurrent respiratory papillomatosis [ , ] . currently, hpv is considered the most important cancer-causing virus, as it is associated with ~ . % of all human cancers [ ] . despite this high clinical significance of hpv, there are no available treatments, and vaccination using the bivalent vaccine against hpvs and or the quadrivalent vaccine against hpvs , , , and remains the first and only choice for controlling the global hpv disease burden [ ] [ ] [ ] . in fact, these vaccines are relatively expensive, and their efficacy is very restricted to specific hpv types. therefore, the development of accurate diagnostic techniques for hpv detection and typing is essential for acquiring surveillance data and initiating appropriate vaccination programs. aunp-based assays have been developed for the molecular detection of hpv through different photoelectric, fluorometric, electric and colorimetric schemes [ , ] . the photoelectric assay essentially leverages aunps to enhance the deposition of silver metal on their surfaces, as previously described for several other assays. however, the enhanced precipitation of silver metal in this assay is used to block the light generated from a photodiode array (pda), and the target dna concentration is measured in proportion to the decrease in light intensity as electrically detected using a bipolar integrated circuit pda chip [ ] . specifically, pcr-amplified biotinylated target dna is captured on the surface of the pda chip, and then labeled with aunp-biotin antibody conjugates; subsequently, a silver enhancement step decreases the light intensity, which is quantitatively translated into an electrical signal depending on the amount of target dna (fig. a) . on the other hand, a fluorometric assay integrating aunps and a microfluidic bead-based array has been developed as a rapid and controlled hpv dna detection system [ ] . in this assay, aunps dually functionalized with hrp enzyme and capture dna strands are applied to label the target dna captured on the surface of microbeads (fig. a) , causing hrp to be in proximity to biotin-tyramine loaded on the surface of the microbeads and catalyze their oxidation by hydrogen peroxide. this process eventually increases the deposition of biotin moieties on the surface of the beads, subsequently increasing the labeling efficiency with qd-streptavidin conjugates and yielding an enhanced fluorescence signal that varies significantly based on the target dna concentration (fig. b) . in the electrical detection of hpv, aunps were used as an immobilization scaffold for dna immobilization on a silicon substrate carrying nanogapped interdigitated electrodes. this aunps deposition technique was shown to reduce the size of the nanogaps, allowing enhanced electric detection of the target dna captured on the silicon substrate by specific hpv dna probes. the results demonstrated that this assay was able to specifically detect hpv dna within min and at concentrations down to pm [ ] . based on the color change of aunps upon aggregation, aunps carrying hpv dna probe were coupled with the lamp technique for colorimetric detection of hpv. lamp reaction was performed at a temperature of °c for min and the generated products were hybridized with aunp-dna probes for min. then the presence of the target dna was detected by the addition of magnesium salt. the addition of the salt causes aunps aggregation and the color changes from red to blue. the presence of lamp amplicons can protect aunps from aggregation and prevents aunps aggregation. the sensitivity of the developed lamp-aunp assay was greater than the lamp turbidity assay by up to -fold with detection limits of and copies for hpv and hpv , respectively [ ] . the family picornaviridae is taxonomically very diverse and has recently been expanded to involve genera: eight are originally identified genera (aphthovirus, cardiovirus, enterovirus, erbovirus, hepatovirus, kobuvirus, parechovirus, and teschovirus), and four newly proposed genera (avihepatovirus, sapelovirus, senecavirus, and tremovirus) [ ] . picornaviruses are spherical, relatively small ( - nm) , nonenveloped rna viruses. their genome is positive-sense ssrna that is . - . kb in size and terminally bonded to a noncapsid viral protein (vpg) at its ' end and to a polyadenylated tail at its ' end. the expression of this genome produces a single large polyprotein that undergoes a cascade of cleavage and processing reactions to form - final structural (vp - ) and nonstructural ( a-c and a-d) proteins [ ] . picornaviruses can infect a broad spectrum of hosts, including birds, humans, and other mammals [ , ] . the genera enterovirus, hepatovirus, parechovirus, kobuvirus, and cardiovirus include several important species that affect humans. particularly, the genus hepatovirus encompasses hav, which is the most likely causal agent of acute viral hepatitis in humans. hav infection is usually asymptomatic and self-limiting, and the related symptoms can be mild or sporadically progress to fulminant hepatic failure [ , ] . the availability of an effective vaccine against hav has substantially reduced the number of people who become infected with hav [ , ] . however, the ability of this virus to survive harsh environmental conditions and remain in seawater, fresh water, wastewater, and soil for extended periods increases the possibility of outbreaks, especially in developing countries where sanitation is not typically available [ ] . the transmission of the virus usually occurs through the consumption of contaminated materials. as such, the virus does not replicate in the environment, water, or foods, and traces of viral contamination are difficult to identify, which remains a challenge for controlling hav. the need for sensitive assays for hav remains an urgent public health demand. hav detection using aunps was accomplished through scanometric and sers-based assays. both assays rely on the extensively described virtue of aunps to enhance silver staining, thereby allowing the molecular detection of hav dna in a microarray chip format (table and fig. ). in the scanometric aunps modified with anti-biotin igg are applied to label biotinylated target dna captured on the pda chip. after a silver enhancement step, the precipitated silver metal at the surfaces of the aunps blocks the light irradiated from above by light-emitting diodes (leds) and decreases the resulting photocurrent [ ] . (b) microfluidic bead-based enzyme-aunp amplification method for the fluorometric detection of hpv. streptavidin-coated beads are dually functionalized with biotin-electron-rich protein and biotin-dna capture probes. the captured target dna first reacts with horseradish peroxidase (hrp)-functionalized aunp-dna labels, bringing hrp close to the surface of the microbeads. the oxidation of tyramine-biotin by hydrogen peroxide results in the deposition of multiple biotin moieties onto the surface of the beads. this deposition is markedly increased in the presence of immobilized electron-rich proteins. streptavidin-labeled quantum dots (qds) are then allowed to bind to the deposited biotin moieties and display amplified fluorescence signals in relation to the target dna concentration [ ] . detection scheme, aunp-dna conjugates are applied as specific detection probes to label target hav rna captured by specific vp probes on the surface of a microarray chip. a silver metal enhancement step is subsequently used for signal amplification, followed by the visual detection of nucleic acids [ ] . in the sers detection, the captured hav dna is labeled by aunp-dna probes loaded with the raman reporter dye cy ; this approach has been applied as previously described for ebov and depicted in fig. of this review [ ] . these assays are very simple and can detect target concentrations ranging from . × - - . × - m, with fm as a limit of detection, which can be superior to the relatively expensive pcr-based approaches currently used for hav detection and management. the family retroviridae is broadly classified into genera (alpharetrovirus, betaretrovirus, deltaretrovirus, epsilonretrovirus, gammaretrovirus, lentivirus, and spumavirus), which include nearly viruses [ ] . retroviruses are spherical, small ( - nm), enveloped rna viruses. their viral genome is typically bipartite and composed of two positive-sense ssrna molecules ( kb in total length). they comprise major genes that encode the different structural and nonstructural proteins of the virus: gag encodes the large protein form matrix, capsid, and nucleocapsid; pro encodes the protease enzyme; pol encodes the reverse transcriptase and integrase enzymes; and env encodes the envelope glycoproteins and transmembrane proteins [ ] [ ] [ ] . retroviruses infect a wide variety of vertebrates, including humans. their infection in humans is usually very serious and can potentially cause oncogenesis and variable hematopoietic and neurological conditions. among retroviruses, hiv is a well-known, formidable human pathogen responsible for the most devastating viral pandemic that has ever occurred in human history [ ] . hiv infection is usually correlated with the progression of what is clinically called aids, in which the immune system is harshly destroyed, and the entire body becomes vulnerable to terrible, life-threatening opportunistic infections and cancers. people with aids also suffer from other systemic symptoms, such as fevers, sweats, swollen glands, chills, weakness, and weight loss [ ] . currently, there is no effective vaccine against hiv, and most of the treatment strategies rely on using antiretroviral drugs, which in most cases maintain the viral load at low levels to prevent the formation of acute hiv infection rather than cure the virus [ ] . therefore, the accurate detection of hiv is a key determinant for monitoring hiv treatment and reducing its transmission and dissemination. currently, aunps have been extensively tested for the immunological and molecular detection of hiv through various scanometric, colorimetric, electrochemical, ipcr, and afm assays ( table ) . the scanometric assays target the detection of the hiv p antigen and hiv nucleic acid using the silver metal enhancement protocol. for hiv p , the target antigen is precaptured by magnetic particles [ ] or isolated in a microplate [ ] , followed by a silver enhancement step for the quantitative visual immunological detection of hiv (fig. a, b and fig. ). for hiv nucleic acid detection, aunp-dna probes specific to the hiv pol gene are applied to detect hiv nucleic acids precaptured and isolated by magnetic particles, followed by a silver metal enhancement step (fig. a) . the deposition of silver on the aunp surface increases the np size and scattering efficiency and additionally allows the target detection via dls [ ] . in aunp-based colorimetric detection of hiv, the large surface area of aunps was exploited to amplify the detection signal by enhancing the gold metal staining on their surfaces, allowing naked-eye detection of hiv nucleic acids (hiv gag gene) in a lateral-flow format, similar to the scheme presented in fig. a [ ] . on the other hand, the ability of aunps to enhance electron transfer has been applied to develop an electrochemical assay for hiv detection (fig. b) . in this electrochemical assay, the target hiv genome is detected in correlation with the change in electrical impedance of graphene sheets decorated with spherical aunp-dna probes specific to the hiv pol gene [ ] . the developed aunp-based rna detection of hiv using the aunp-lfa technique has a sensitivity limit of ~ log copies/ml, while the best dna detection can be achieved through the light-scattering scheme with a sensitivity limit of fm. furthermore, afm coupled with aunps as nanoparticulate labels has been applied for the detection of hiv p as follows: the target antigen is precaptured on the surface of a nanoarray specifically coated with anti-p and labeled with aunp-antibody conjugates that significantly changes the surface topography of the array, allowing the detection of p (fig. c) [ ] . so far the developed aunps-based assays for hiv are among the most innovative in virus detection and offer a significantly improved detection limit down to attomolar range, which is hundreds of times lower than that of conventional techniques used in hiv detection, including pcr and elisa. therefore, aunps assays can be very beneficial in detecting hiv early, preventing its dissemination, and treatment monitoring. furthermore, simple detection methods such as lateral flow and scanometric assays can be easily incorporated into the point-of-care detection of hiv, which is currently of great interest and more suitable for developing countries. aunps have made a tremendous impact in the detection of viruses, as demonstrated by the numerous assays for most of the clinically relevant groups of human viruses (fig. ) . coupled with different biomolecules, aunps have enabled the development of a completely new class of probes that are specifically composed of a aunp core (spherical or rod-like in shape) for signal readout and a surface layer of one or multiple types of biomolecules for target recognition and interaction. these probes have mostly been incorporated as a new set of functional and highly interactive components in different traditional techniques (e.g., eias and pcr) and have derived substantial improvements in design, detection performance, sensitivity, specificity and stability. this created a new trend in applying aunps to overcome the limitations of traditional techniques. aunp-based immunological and serological detection methods are the most common and have been accomplished through multiple scanometric, colorimetric, dls, immuno-pcr, electrochemical, electrical, sers, lspr, fluorometric, and afm-based approaches (table ) . aunp-based dls and lspr assays are especially novel, simple and among the most sensitive reported assays for the immunodetection of viruses with a detection limit that is hundreds of times lower than that of the standard elisa technique [ , ] . in contrast, the developed aunp-based molecular assays are not as sensitive as the conventional pcr technique or other pcr-based molecular techniques. however, the addition of aunps greatly simplifies the detection procedures of viral nucleic acids, and largely eliminates the need for complex procedures and expensive thermal cycling machines, which greatly reduces the detection time and total cost. in particular, colorimetric assays based on the intense red color of aunps represent an interesting qualitative shift in nucleic acid detection due to the complete elimination magnetic np (mnp)-dna probes scavenge target hiv sequences, which are then magnetically collected and labeled with aunp-dna probes. after repetitive washing, aunps are released into solution by a dehybridization step and subjected to silver metal enhancement, resulting in particle growth and increased scattering [ ] . (b) electrochemical impedance assay of hiv rna. graphene oxide sheets decorated with aunps are applied for the immobilization of dna detection probes. hybridization with specific dna sequence results in increased impedance [ ] . (c) atomic force microscopy (afm)-based immunoassay of hiv. aunp-antibody probes form a three-component sandwich immunocomplex with the target virus antigen captured on the surface of a specific antibody nanoarray; eventually, the increase in the surface height of the array is detected by afm [ ] . of the pcr amplification step and the possibility of direct and rapid detection of the target virus within a few minutes. other assays rely on well-known analytical techniques, such as icp-ms and qcm, which have been introduced to virus nucleic acid detection for the first time through the application of aunps and have achieved detection sensitivity down to femtomolar concentrations [ , ] . aside from these immunological and molecular assays, aunps have also been described for viral load testing by detecting intact virus particles, which are of special interest as a substitution to traditional cell culture technique [ , , ] . in this context, the small size of aunps probes that is comparable to virus particles, allows rapid, and highly dynamic interaction with viruses for gaining information about infectivity. the application of aunps, especially in biology and medicine, require specific structural and physicochemical characteristics, and any subtle alterations to the intended characteristics can compromise the function and performance of the whole system. for bioanalytic and diagnostic applications, aunps exhibiting homogeneous morphological and fine structural characteristics are important specifically for effective and accurate response to target detection. therefore, many pre-analytical factors related to np synthesis and surface modification are critical to the development of an efficient virus detection assay. the synthesis of aunps commonly relies on methods that basically employ wet chemistry reduction techniques, as presented in other reviews [ , ] . although these techniques are generally simple and fast and can support the large-scale production of aunps, the synthesis of uniform aunps with a discrete size and shape remains challenging [ ] . recently, some approaches have been reported for the controlled synthesis and modification of aunps. however, these methods remain unsuitable either due to the protocol complexity or difficulty of particle separation, which might require additional steps or specific surface modifications. therefore, the development of more stringent and advanced methods for the facile and controlled synthesis of uniform aunps with the option of performing multiple surface modifications is strongly recommended. because of the direct relationship between aunp size and shape and their function as labels, such characteristics are also of inherent importance to both the performance of au nanoprobes and their efficiency for diagnostic applications. for instance, small aunps are preferred in colorimetric and light scattering-based assays due to their spr band sensitivity. in contrast, large aunps are preferentially employed in assays such as bca, immuno-pcr, and enzymatic-based electrochemical detection techniques, which rely on the surface functionalization of aunps with targeting or signaling biomolecules. additionally, a large np size is highly desirable in assays that depend on aunps as metal labels, such as electrochemical stripping, qcm, and icp-ms detection methods. in addition, spherical aunps are more commonly applied than rod-shaped nps. however, the latter exclusively enable a very interesting class of lspr-based detection methods that have shown an unprecedented detection sensitivity range. since aunps by themselves are "blind" with respect to sensing the target, the addition of specific biomolecules to their surfaces is a key step for detecting viruses [ , , ] . unfortunately, most biomolecules have been reported to be very sensitive to harsh chemical modifications that can adversely affect their reactivity and specificity. thus, the type of targeting biomolecule and the surface conjugation reaction method applied are crucial and require optimization. short oligonucleotides and other dna sequences are physically rigid and more stable than protein biotargeting structures, including antibodies, which usually have several secondary and tertiary conformations that are important for their full, specific interaction with a target molecule. furthermore, the full reactivity of surface biomolecules via a suitable conjugation reaction is critical and should be retained and confirmed with additional assessment steps before use in virus detection. while the application of traditional conjugation techniques, such as electrostatic adsorption, is still widely reported, it is strongly recommended to be avoided due to the incident loss of conjugated biomolecule activity and large inequality in loading efficiency, which directly result in decreased assay sensitivity and reproducibility [ ] . in this regard, aunps have an enhanced surface area-to-volume ratio that greatly improves the efficacy of current conjugation techniques, and several directional and controlled conjugation methods based on covalent and noncovalent interactions have been thoroughly described in the literature [ , , ] . although aunps are widely employed in various sensing functions and formats, their use for monitoring of virus infection is challenged by long-term stability and reusability of the developed aunp-based sensors. the reusability of sensing platforms has a crucial role in developing applications that are reliable, economic and sustainable. nonetheless, the reusability of aunps in virus detection is limited to electrical and electrochemical assays, in which the main function of aunps is limited to enhancing the performance of the modified electrodes [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the long-term stability of aunps is reported and well investigated in numerous studies. however, their conjugates to different biomolecules (i.e., dna, rna, antibody, enzymes) are sensitive to storage time and conditions and the reusability of these conjugates is usually hampered by the loss of activity of surface molecules and irreversible aggregations. it was demonstrated that exposure to elevated temperatures, high salt concentrations, and reducing agents such as dithiothreitol (dtt), mercaptoethanol, and glutathione (commonly existing in biological fluids) results in the loss of stability and activity of aunp-conjugates [ , , ] . thus, the development of new chemistries and novel conjugation strategies for the synthesis of stable aunp-conjugates becomes very crucial for practical use of aunps in virus detection. considering these limitations and challenges, important analytical parameters, such as the use of positive and negative controls, follow-up patient specimens, and internal reference tests, are recommended to confirm the overall accuracy and practicality of developed schemes. a restricted set of measures related to the assay detection sensitivity, specificity, and performance is required for the quality assurance and assessment of these applications [ ] . due to the rapid development rate of aunp-based assays, international data analysis measures and standards are also critical to reflect the true assay sensitivity and specificity and to allow the evaluation of their ability and potential to substitute current detection techniques. furthermore, more efforts are needed to test the practicality and generalizability of these assays through clinical sample testing and in-field evaluation of the newly developed aunp-based methods along with the conventional methods using standard evaluation systems and protocols. aunps are a powerful addition to the range of techniques available for virus testing and control. when used as probes, aunps enable simple, rapid, and sensitive detection with an unprecedented multiplexing capability. importantly, aunp-based probes have matured from tools being added to improve traditional techniques, such as pcr, eliza, and 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using gold-dna probes we would like to thank frank fanqing chen the authors have declared that no competing interest exists. key: cord- -zlah u s authors: günther, sonja; felten, sandra; wess, gerhard; hartmann, katrin; weber, karin title: detection of feline coronavirus in effusions of cats with and without feline infectious peritonitis using loop-mediated isothermal amplification date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: zlah u s feline infectious peritonitis (fip) is a fatal disease in cats worldwide. the aim of this study was to test two commercially available reaction mixtures in a reverse transcription loop-mediated isothermal amplification (rt-lamp) assay to detect feline coronavirus (fcov) in body cavity effusions of cats with and without fip, in order to minimize the time from sampling to obtaining results. rna was extracted from body cavity effusion samples of cats, including samples from cats with a definitive diagnosis of fip, and samples of control cats with similar clinical signs but other confirmed diseases. two reaction mixtures (isothermal mastermix, optigene ltd.and pcrun™ molecular detection mix, biogal) were tested using the same primers, which were designed to bind to a conserved region of the fcov membrane protein gene. both assays were conducted under isothermal conditions ( °c– °c). using the isothermal mastermix of optigene ltd., amplification times ranged from and min with a sensitivity of . % and a specificity of . % for the reported sample group. using the pcrun™ molecular detection mix of biogal, amplification times ranged from to min with a sensitivity of . % and a specificity of . %. although the rt-lamp assay is less sensitive than real time reverse transcription pcr (rt-pcr), it can be performed without the need of expensive equipment and with less hands-on time. further modifications of primers might lead to a suitable in-house test and accelerate the diagnosis of fip. feline coronavirus (fcov), a member of the genus alphacoronavirus of the subfamily coronavirinae, family coronaviridae within the order nidovirales (de groot et al., ) , belongs to a group of enveloped, positive-sense rna viruses that cause diseases in several species, such as severe acute respiratory syndrome (sars) in humans or transmissible gastroenteritis (tge) in pigs. despite the high prevalence of fcov infections in the cat population worldwide, only - % of fcov-infected cats develop the fatal disease feline infectious peritonitis (fip) (addie and jarrett, ) . this change of virulence of a harmless fcov biotype that usually causes no clinical signs into the pathogenic variant is thought to be caused by mutations in the fcov spike protein gene (chang et al., ; vennema et al., ) . these mutations cause a change in tropism from enterocytes to macrophages, giving fcov the ability to infect and effectively replicate within cells of the macrophage lineage and cause a lethal systemic disease with multi-organ involvement (pedersen, ) . the median survival time of cats with effusive fip is only a few days (ritz et al., ) , and the diagnosis of fip commonly leads to euthanasia, since to date, no treatment has been proven to be effective. cats with fip show nonspecific clinical signs such as fever, weight loss and anorexia, often accompanied by body cavity effusions and/or ocular and neurological signs. a definitive diagnosis of fip ante mortem remains challenging, especially when no body cavity effusions can be detected (hartmann et al., ) . presently, the gold standard for the diagnosis of fip is considered to be immunostaining of fcov antigen in macrophages within tissue lesions, a technique that requires invasive tissue collection (kipar and meli, ) . in cats with fip, fcov can be detected by rt-pcr in cell-free body cavity effusions in more than % of the cases, while serum or blood samples often are negative (doenges et al., ) . for both immunostaining and rt-pcr, samples have to be sent to specialized laboratories, resulting in the delay of diagnostic results. this leads to further unnecessary testing for other diseases, to withholding necessary therapy of other treatable diseases, or to delayed euthanasia in cats suffering from severe signs of fip. therefore, a fast and simple point of care test would be very beneficial in the diagnostic process. loop-mediated isothermal amplification (lamp) is a simple, rapid, and cost-effective nucleic acid amplification method (notomi et al., ) and is already used for the detection of coronaviruses in humans and several animal species (hong et al., ; nemoto et al., ) . a set of four to six primers is used, that form products with self-hybridizing loop structures. by using a dna polymerase with strand displacement activity, no melting or annealing steps are required, and amplification products of different lengths are formed at a constant temperature of - °c (nagamine et al., ) . since lamp reactions only require a simple heat block with constant temperature, and dna amplification can be detected by fluorescence or color change, the method can be applied for point-of-care diagnostics (surabattula et al., ) . the aim of this study was to test specificity and sensitivity of two commercially available reaction mixtures in a reverse transcription lamp (rt-lamp) to detect fcov in body cavity effusions of cats with and without fip, and to minimize the time from sampling to obtaining results. this study included cats that were presented to the clinic of small animal internal medicine, lmu munich, germany. all cats included had body cavity effusions. in every cat presenting with body cavity effusions, fip is a potential differential diagnosis. an earlier study showed that fip is responsible for about % of effusions, while most of the remaining cases were caused by malignomas, cardiac insufficiency or purulent serositis (hirschberger et al., ) . the fip group (n = ) included cats with a definitive diagnosis of fip by one or more methods: all effusions of cats with fip tested positive for fcov by rt-pcr by a commercial laboratory, and in / samples putative disease-causing mutations could be detected. the rt-pcr detection method has been described previously (felten et al., ) . in / cats fip diagnosis was achieved by post-mortem examination, including full body necropsy with histopathological examination. diagnosis of fip was confirmed when typical histologic lesions where detected (surfacebound multi-systemic pyogranulomatous and fibrinonecrotic disease with venulitis with or without high-protein exudate). in / cats with full body necropsy immunohistological staining for fcov-antibody was done on tissue sections and returned a positive result. immunofluorescent staining of fcov antigen in macrophages of thoracic or abdominal effusion was done in / cats, and all samples returned a positive result. a summary of the cases in the fip group can be found in the supplementary table . cats were included in the control group (n = ) if they were definitively diagnosed with a disease other than fip that explained the effusion. cats of the control group suffered from neoplasia (n = ), decompensated cardiac diseases (n = ), inflammatory diseases (n = ), such as bacterial peritonitis and pleurisy, or other diseases (n = ). one cat had chronic thoracic chylous effusion of unknown origin and secondary fibroplastic pleurisy. in another cat, an end stage kidney disease caused effusion, and one cat had thoracic effusion after subcutaneous urethral bypass placement, which resolved after treatment. the diseases of the cats of the control group (n = ) were definitively confirmed ante-mortem (n = ) or at necropsy with histopathological examination (n = ). ante-mortem diagnosis was established by echocardiography for cardiac diseases (n = ), and by cytology for neoplasia (n = ). immunofluorescent staining of fcov antigen in macrophages of thoracic or abdominal effusion was done in / cats, with three positive and eight negative results. all effusions of the cats in the control group were tested for fcov by rt-pcr, and all results were negative. the rt-pcr detection method has been described previously (felten et al., ) . a summary of the cases in the control group can be found in supplementary table . body cavity effusion samples of all cats were obtained ante mortem with ultrasound guidance for diagnostic purposes. the use of samples for this study was approved by the institutional animal care and use committee ('ethikkommission des zentrums für klinische tiermedizin'), permission number - - - . samples were stored at - °c in a . ml eppendorf safe-lock microcentrifuge tube until assayed. all samples were centrifuged for s at , × g. the supernatant of centrifuged thoracic and abdominal fluids was used for rna extraction. when using fresh fluid samples, omission of the centrifugation step should be considered to include intact cells with a high viral burden (pedersen et al., ) . in thawed samples, cell integrity is lost and cell debris can be removed. viral rna was isolated using the commercial zr viral rna kit™ (zymo research corp.) following the manufacturer's instructions. briefly, μl aliquots of samples were mixed with a buffer that facilitates viral particle lysis and allows for rna adsorption onto the matrix of the zymo-spin™ column. then the rna was washed and eluted with μl of rnase free water. extracted rna aliquots were stored at − °c in an eppendorf . ml safe-lock microcentrifuge tube until further processing. the rt-lamp primer design was assisted by the software primerexplorer (https://primerexplorer.jp/e/). based on sequence analysis, the gene for the membrane protein (m) was selected as a target because it is highly conserved among fcov strains. the dna sequence from position , to , of the fcov strain black (genbank accession number: eu . ) was used to design the rt-lamp primers used in this study. a set of six primers, including two outer primers (forward primer f and backward primer b ), two inner primers (forward inner primer fip and backward inner primer bip), and two loop primers (forward loop primer loopf and backward loop primer loopb) were selected as the target sequence ( fig. and table ). detection of fcov was performed using rt-lamp. two different commercial reaction mixtures (isothermal mastermix by optigene ltd., uk, pcrun™ molecular detection mix by biogal, israel,) were compared using the same set of primers. for the amplification following the isothermal mastermix protocol, the total volume of μl per reaction tube included μl isothermal master mix, μl template, μl primer mix and . μl superscript® iii reverse transcriptase (thermo scientific). the primer mix consisted of pmol each of f and b primers, pmol each of fip and bip primers and pmol each of loopf and loopb primers. for negative control, μl water were added instead of μl template. the reaction mix was incubated at °c for min in a real-time pcr system (applied biosystems). during rt-lamp, fluorescence of dna products was measured once every minute (fam detection channel, λ max nm), and the time to threshold crossing was analyzed. a positive sample (positive in rt-pcr and sequenced for mutations) was included in every run. all samples run in the realtime pcr system were subjected to a melt curve analysis after the run. a single sharp peak in the melt curve analysis demonstrates amplification of a single pcr product. the positive samples all showed a single peak and the same melting temperature as the positive control sample. following the pcrun™ molecular detection protocol the reaction mix contained . μl of luminescent reagent, μl of template, μl of primer mix and additional . μl superscript iii reverse transcriptase (thermo scientific). the primer mix consisted of . pmol each of f and b primers, pmol each of fip and bip primers and pmol each of loopf and loopb primers. for negative control, μl water were added instead of μl template. the reaction mix was incubated at °c for min in a pcrun™ reader (biogal). amplification was detected by measuring bioluminescence twice every minute (fig. ) . pcr products of both methods were verified on an agarose gel showing a typical pattern of multiple bands of different molecular weights (fig. ) . the results of the samples in the fcov rt-lamp assays using two different commercial reaction mixtures are shown in table . two samples tested false positive using the isothermal mastermix and one sample tested false positive with the pcrun™ molecular detection mix. sensitivity and specificity for the reported sample groups of both rt-lamp assays are shown in table . the pcrun™ molecular detection mix performed better both in sensitivity and specifity than the isothermal mastermix. the amplification times for the isothermal mastermix positives ranged between and min and for the pcrun™ molecular detection mix positives between - min. in the present study, rt-lamp assays were evaluated as a diagnostic tool for detection of fcov, in order to distinguish cats with and without fip that are presented with body cavity effusions. time from sample to result was kept to a minimum by isolating rna in about to min using a simple rna extraction kit. both commercially available reaction mixtures allowed an easy and fast preparation of the amplification reaction. with lamp assays, direct detection methods built into the amplification device are preferred, since opening lamp reaction tubes after amplification is not advisable to decrease the risk of carry-over contamination (parida et al., ; zanoli and spoto, ) . in the present study, a portable device results was used for the pcrun™ molecular detection mix. positive and negative amplification reactions were indicated by the device with '+' and '-', making it compatible as a point-of-care instrument. the isothermal mastermix is intended for use with a portable device, which was not part of this study. the machine used instead replicates the reaction conditions with a constant block temperature and uses a comparable fluorescence detection system. both assays tested have similar demands concerning handling skills and preparation time. detection of positive samples took about half the time with isothermal mastermix compared to the pcrun™ molecular detection mix, yet both tests require less than min. the specificity of both methods for the reported sample group was comparable, with . % and . %, respectively. however, false positive results in a test that diagnoses a fatal disease are very critical and should not occur. a cross-reaction of the lamp-primers or carry-over contamination might be the cause of these false positive results. however, the negative controls without sample material did not show any indication for carryover contamination and stayed negative in the lamp assays. another possibility for false positive results is the detection of fcov in cats without fip, since systemic spread of fcov does occur, but does not inadvertently result in fip (porter et al., ) . this explanation is not very likely for the three samples of cats without fip that were positive by rt-lamp, since all three were negative by rt-pcr. the sensitivity of the pcrun™ molecular detection mix was superior for the reported sample group with . % compared to . % of the isothermal mastermix, using the same primers and pcr conditions. since the samples for the rt-pcr and for the rt-lamp had the same preanalytical treatment (frozen, thawed, and centrifuged), the results can be directly compared and showed that the rt-pcr for fcov performed much better than the rt-lamp in our sample group. this is in agreement with a study on other coronaviruses, where rt-lamp also exhibited a lower analytical sensitivity compared to rt-pcr (bhadra et al., ) . false negative results can occur in samples with a lower viral burden. the fcov viral load determined by rt-pcr in effusions has been found to be quite low in some samples of fip-suspected cats (lorusso et al., ) . in our study, the results for rt-pcr were only returned as 'positive' or 'negative' without quantification, leaving open the question whether only effusions with a high viral load resulted in a positive rt-lamp detection. another possible reason for the lack of sensitivity might be that sequences of current fcov strains show sequence variations compared to the sequences deposited in the genbank database, which were used to design the rt-lamp primers. although the primers were chosen to bind in highly conserved regions, variations can occur, which might impair binding and eventually lead to low or no amplification, resulting in poor sensitivity. reliable primers for rt-pcr target the ′ utr of the fcov sequence, but the rt-lamp primers that were suggested by the primerexplorer software in this region included more sequence differences than the m region that we selected. modifications of the primers might enhance binding and improve sensitivity. two studies on lamp-based identification of fcov have been published to date. a study from thailand tested samples of body cavity effusions from cats that were suspected to have fip both by rt-pcr and by rt-lamp. more samples tested positive with the rt-lamp than with the rt-pcr ( % vs. %) (techangamsuwan et al., ) . however, the inclusion criteria for cats to be suspected of having fip were not described in that study. their control samples consisted of plasma and fecal samples from healthy cats without any contact to other cats. the samples from this healthy control group also had more positive results by rt-lamp than by rt-pcr ( % vs %). the authors mention high rates of false positives in the negative controls (no template controls) when using rt-lamp, so it remains unclear whether their rt-pcr was less sensitive or their rt-lamp was prone to unspecific amplification. in the second study, different sample types of cats with a clinical suspicion of fip and fecal samples for screening for fcov-shedding cats were tested (stranieri et al., ) . in most sample types, including effusions, their rt-pcr had about twice as many positive results as their rt-lamp, and none of the rt-pcr-negative samples was positive in the rt-lamp method. in agreement with our findings, the sensitivity of the rt-lamp appears to be inferior to the rt-pcr. for detection of amplification, both studies used gel electrophoresis and one study (stranieri et al., ) additionally used detection of color change from violet to blue with hydroxynaphtol blue. while gel electrophoresis is quite time-consuming, the color change can be difficult to detect in samples with low amplification. amplification detection with fluorescence or luminescence as used in the present study is preferable, since the results can be read immediately and could be easily converted to a quantitative format with a standard curve. as a perspective for the future, rt-lamp reactions for virus detection could be run on new devices that integrate nucleic acid extraction, amplification and detection in a miniature format to achieve a true point-ofcare diagnosis (stumpf et al., ) . in conclusion, the rt-lamp in the present study was relatively specific but not very sensitive. the sample type was restricted to effusions of cats unequivocally diagnosed with fip and of cats without fip but with clinical signs indicative of fip. this is a realistic setting in which a veterinarian would use a test for detection of fcov in the effusion sample. the rt-lamp assay with the pcrun™ molecular detection mix can be used in a clinical setting to a certain extent. however, before it can replace conventional rt-pcr methods, sensitivity and specificity have to be enhanced by optimizing primers and amplification conditions. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. the pcrun™ molecular detection mix and the pcrun™ reader were provided by biogal free of charge. biogal was not involved in the study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. the fcov rt-pcr and subsequent mutation detection was done by idexx free of charge. idexx was not involved in the study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. a study of naturally occurring feline coronavirus infections in kittens real-time sequence-validated loop-mediated isothermal amplification assays for detection of middle east respiratory syndrome coronavirus (mers-cov) spike protein fusion peptide and feline coronavirus virulence virus taxonomy: ninth report of the international committee on taxonomy of viruses comparison of realtime reverse transcriptase polymerase chain reaction of peripheral blood mononuclear cells, serum and 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loop mediated isothermal amplification (lamp) development and application of reverse transcription loop-mediated isothermal amplification (rt-lamp) for feline coronavirus detection feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses isothermal amplification methods for the detection of nucleic acids in microfluidic devices we thank biogal for providing the pcrun™ molecular detection mix and the pcrun™ reader for this study. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.jviromet. . . . key: cord- -tjw t f authors: al awaidi, salah; abusrewil, suleiman; abuhasan, muslim; akcay, meral; aksakal, fatma n.b.; bashir, uzma; elahmer, omar; esteghamati, abdoulreza; gahwagi, milad; mirza, yusuf k.; grasso, cindy; kassianos, george; khris, moulud; mardani, masoud; maltezou, helena; nourlil, jalal; oumzil, hicham; osterhaus, ab; picot, valentina; pehlivan, tamer; saadatian-elahi, mitra; tali, İlham; tarraf, hesham; ugur, baris; zaraket, hassan title: influenza vaccination situation in middle-east and north africa countries: report of the th mena influenza stakeholders network (mena-isn) date: - - journal: j infect public health doi: . /j.jiph. . . sha: doc_id: cord_uid: tjw t f background: the middle east and north africa (mena) region faces a dual challenge with regard to influenza infection due to severe zoonotic influenza outbreaks episodes and the circulation of northern hemisphere human influenza viruses among pilgrims. methods: the mena influenza stakeholder network (mena-isn) was set-up with the aim of increasing seasonal influenza vaccination coverage by (i) enhancing evidence-based exchanges, and (ii) increasing awareness on the safety and benefits of seasonal vaccination. during the th mena-isn meeting, representatives from countries presented their influenza surveillance, vaccination coverage and actions achieved and provided a list of country objectives for the upcoming years. results: mena-isn countries share the goal to reduce influenza related morbidity and mortality. participants admitted that lack of knowledge about influenza, its consequences in terms of morbidity, mortality and economy are the major barrier to attaining higher influenza vaccination coverage in their countries. the cost of the vaccine is another key barrier that could contribute to low vaccination coverage. participants drew a list of strategic interventions to bridge gaps in the knowledge of influenza burden in this region. conclusions: participating countries concluded that despite an increase in vaccine uptake observed during the last few years, influenza vaccination coverage remains relatively low. priority areas should be identified and action plans tailored to each country situation set-up to investigate the best way to move forward. results: mena-isn countries share the goal to reduce influenza related morbidity and mortality. participants admitted that lack of knowledge about influenza, its consequences in terms of morbidity, mortality and economy are the major barrier to attaining higher influenza vaccination coverage in their countries. the cost of the vaccine is another key barrier that could contribute to low vaccination coverage. worldwide, influenza affects - % of the population and causes more than million deaths annually [ ] . this vaccinepreventable infectious disease belongs to the list of the world health organization (who) recommended vaccines, yet the vaccine uptake is in general low in both developed and developing countries [ ] . the finding of the who flu-net database showed that dynamic of influenza epidemics in the large majority of the middle east and north africa (mena) countries was in line with the northern hemisphere, with the largest peak observed between january and march [ ] . annual vaccination campaigns in the mena region target primarily specific high-risk groups i.e. pregnant women; individuals > months with underlying chronic diseases, elderly, residents of long-term care facilities, children aged - months and health care providers. the mena region faces a dual challenge with regard to influenza infection. first, the region tackled severe zoonotic influenza outbreaks episodes with lethal cases during the last years [ ] [ ] [ ] [ ] [ ] . second, the northern hemisphere human influenza viruses circulate in the region and are the most frequently detected respiratory viruses among pilgrims [ ] , increasing the risk for further spread of the disease. in addition, there is a risk of human infection from middle east respiratory syndrome coronavirus (mers-cov). indeed, since , outbreaks of mers-cov and very few sporadic travelrelated cases were recorded in saudi arabia [ ] and returning haji [ ] , respectively. although epidemiological analysis does not support human to human transmission, overcrowding, mass gathering and travel increase the fear about potential mers-cov international dissemination. it is very well known that diabetics have a significantly increased risk of flu-related hospitalization and death [ ] . with approximately million adults aged - living with diabetes, the mena region has the highest global prevalence of diabetes in the adult population [ ] . this point emphasizes on the commitment to increase influenza vaccination coverage in this region. the few available published literatures on influenza vaccination coverage in mena report low coverage in this region [ , [ ] [ ] [ ] . aligned with the objective of the who global influenza vaccine action plan to increase influenza awareness, the middle east and north africa influenza stakeholder network (mena-isn) has been initiated in . mena-isn is a network of regional experts with the global mission to increase seasonal influenza vaccination coverage by (i) enhancing evidence-based exchanges with national and international actors, and (ii) increasing awareness on the safety and benefits of seasonal vaccination. mena-isn support the who initiative in building laboratory capacity and surveillance in the region and urge the governments to give high priority to the establishment and continued support for influenza surveillance systems; identify the needs of countries for establishing or improving existing surveillance networks; disseminate surveillance and disease burden data through publications and develop actions to increase vaccination coverage rates in health care professionals (hcps), pregnant women, people at risk, elderly and children. the th mena-isn meeting was organized by foundation mérieux on - september, in riga, latvia. a total of participants from countries (egypt, iran, lebanon, libya, morocco, oman, pakistan and saudi arabia) attended the meeting. in addition to mena-isn country representatives, experts from the who global influenza program, the college of general practitioners (uk), the hannover veterinary university (germany), and hellenic centre for disease prevention and control (greece) were also present. each country representative summarized their current situation of influenza surveillance, influenza vaccination coverage and actions achieved, and provided a list of country objectives for the upcoming years. a panel discussion on the second day allowed the participants to discuss how to strengthen the network and its visibility and how to reinforce between country research and publications. herein, we report a summary of the country situation and actions to move forward. vaccination coverage in egypt remains low [ ] . however, the ministry of health (moh) makes all efforts to monitor influenza and make public recommendations to the benefit of influenza vaccination particularly in high risk groups or in face of epidemics. egypt is one of the countries that have experienced a large epizootic of highly pathogenic avian influenza in poultry caused by the influenza a (h n ) virus [ ] . an integrated national plan for avian and pandemic influenza was developed in response to the rapid spread of avian influenza in this country. national influenza centers equipped with tests such as virus isolation, polymerase chain reaction (pcr), serology and sequencing exist and are currently functioning. the surveillance of severe acute respiratory infection (sari) is enhanced and there is also a significant increase in the available number of influenza vaccines doses. furthermore, several social mobilization and advocacy campaigns have been recently conducted. progress achieved so far and the main mid-and long-term objectives of the country are detailed in tables and respectively. influenza surveillance system has been set-up in . the analysis of influenza a/h n pdm and a/h n viruses collected in iran during the - provided evidence of co-circulation of several influenza a virus strains [ ] . of the influenza typing studies specimens, were influenza a-positive, including a/h n pdm and a/h n , while were influenza b-positive [ ] . analysis of the a/h n viruses showed a genetic drift from the vaccine strain a/texas/ / with mutations [ ] . serological study among poultry workers from fars province of iran showed that exposure to avian h n viruses had occurred in this population [ ] . influenza surveillance system has been established since and an increased number of publications of influenza in peer-reviewed journals are among the most key actions achieved. progress achieved and country objectives for the upcoming years are listed in tables and respectively. influenza surveillance has continued through the - influenza season, during which nasopharyngeal swabs from influenza-like illness (ili) cases were analyzed. the results showed circulation of both influenza b yamagata and victoria lineage viruses in addition to influenza a/h n and a/h n pmd , emphasizing the importance of introducing the quadrivalents influenza vaccine. among the study population, only % were vaccinated (unpublished data). analysis of flu vaccination uptake overtime showed relatively high vaccination coverage ( %- %) until with a peak during the pandemic and decreased subsequently to reach % in - (unpublished data). however, these figures mostly reflect the vaccination coverage of the population served by one major hospital in beirut. it was noted that further studies to include more representative population from throughout the country are needed to accurately reflect the vaccination coverage in the country. based on vaccine dose distribution, vaccination coverage in lebanon is estimated at %. increased flu vaccine coverage and increased laboratory capacity to isolate and detect influenza are the main successes that have been so far achieved. although vaccine uptake is improving, there is still a long way to reach optimal vaccination coverage in high-risk individuals. the search for advocacy continues and includes representatives from academic, public and the government. the main achievements and objectives are listed in tables and respectively. the number of available doses of influenza vaccine has increased by more than folds since . also, good progress has been achieved in surveillance, social mobilization and advocacy. progress achieved and the main objectives are listed in tables and respectively. influenza is one of the moh priorities in morocco. sentinel surveillance exists since . however, influenza burden is not well understood. currently, health centers in all districts and private physicians in cities are involved in the ili surveillance system. overall, regional laboratories are in charge of detection and identification of influenza strains by reverse transcriptase pcr (rt-pcr.) the national institute of hygiene (rabat) and the pasteur institute (casablanca) have the capacity to perform rt-pcr, sequencing, virus isolation, and antiviral susceptibility screening. of the overall samples analyzed in casablanca during the - influenza season . % belonged to type a(h n ) and . % were victoria b lineage. the main objectives for the upcoming years are listed in table . oman sari surveillance has been launched at sentinel sites since and the central public health laboratory is recognized by the who as national influenza center (nic) since march . in , patients were enrolled in sari surveillance. the highest numbers of sari cases were recorded among children - years old followed by those - years of age. the overall mortality rate due to influenza was . % but no death was reported among pregnant women. influenza strains a (h n )pdm , a(h n ), and b viruses were detected during this season. influenza vaccine is recommended for hcps working in critical and non-critical units, administrative agents and educational institution staff. pre-vaccination awareness emails sent to hcps to inform them about the availability of the vaccine and the beginning of the vaccination campaign at the hospital. in addition, the awareness campaign includes presentations, person-person communication, pamphlet, banner, etc. vaccinators visit the wards to offer the vaccine. electronic and manual records are maintained at the institutional, governorate and national level. regarding pregnant women, influenza vaccination is integrated into the antenatal program and administered during antenatal visits. pre-vaccination counselling is also provided. furthermore, influenza advocacy campaigns provide awareness to the family and pregnant woman. the main progress achieved and future objectives are detailed in tables and respectively. currently, influenza is not considered as a health priority in pakistan. there is a distinct seasonality with peak activity levels observed in most regions during the winter season. available data indicates that there is a sizeable burden of influenza. however, influenza related morbidity and mortality estimates are not well known. in the key achievements include the implementation of sentinel influenza surveillance networks and the reinforcement of social mobilization and advocacy (table ) . country specific vaccination priorities must be determined for policy recommendations. the main objectives are listed in table . historically, vaccination coverage has been than % saudi arabia but higher rates are seen among hcps, pregnant women, elderly, haji and patients with chronic diseases. influenza surveillance system has been recently set-up in hospitals and public health centres in six regions. in order to increase vaccination coverage, a -phase strategic project ( p) has been launched in with the aim of reaching % vaccination coverage among at-risk population in years scope. several public awareness campaigns have also been conducted and the moh is funding flu vaccination. the main achievements and future objectives are detailed in tables and respectively. the panel discussion focused on exploring ways to increase influenza vaccination coverage in the participating countries and involving the moh in the network. lack of knowledge about influenza, its consequences in terms of morbidity, mortality and economy is the major barrier to attaining higher influenza vaccination coverage in the mena-isn countries. as stressed by the participating countries, under awareness about influenza could itself be related to communication gaps. tailored communication messages to the community to promote influenza vaccination and to increase awareness on influenza infection have been put in place in several countries. this includes advertising panels in the streets, lay public activities in shopping malls and airports, bulk messages, social media campaigns, etc., but more needs to be done. besides lack of knowledge, other factors such as misbeliefs and personal experiences could lead to low vaccine uptake. participating countries concluded that a better understanding of the factors underlying hesitancy and acceptability of the influenza vaccine is needed to devise evidence-based communication campaigns aimed at increasing vaccination coverage. the adaptation of who-sage available tools is each country has been suggested as an appropriate means to determine the underlying determinants of vaccine hesitancy in each country. the cost of the vaccine is another key barrier that could contribute to low vaccination coverage. country representatives concluded that depending on the economic situation, countries should primarily focus on increasing vaccination coverage among known high-risk groups (diabetics, pregnant women, people with underlying chronic diseases, etc.). some countries believed that even within the high-risk groups, a priority list should be drawn according to influenza burden in each group and each country. from country presentations, it was evidenced that the use of private health care facilities is more usual than the public facilities. nevertheless, a lack of communication between these two sectors was noted. strategic communication by mena-isn and the use of uniform terms of references for local stakeholders could help in enhancing the involvement of moh and the private sector thereby improving the situation for vaccine implementation. as reflected by individual country presentations, all mena-isn countries share the goal to reduce influenza related morbidity and mortality. vaccination coverage has slightly increased in all participating countries, yet more work should be done to reach the optimal vaccination coverage. the main challenges to increase vaccine coverage were: under awareness about influenza, financial and political issues, and limited collaboration between private and public health sectors. country representatives agreed on the need to design studies aimed at accurately estimating vaccination coverage among different populations and risk groups and assessing the impact of vaccination on hospitalization, mortality and herd immunity. this can be presented to the moh to develop evidence-based policies or policy updates. the data can be also communicated with the public to increase their awareness and willingness for influenza vaccination. pharmacies can play a fundamental role as they are easily accessible, but policies in some countries prevent pharmacies from administrating vaccines. in parallel, region specific information about vaccine efficacy, economic impacts of influenza (absenteeism, hospitalization, etc.) should be provided in order to provide more confidence about influenza vaccine. incentives for general practitioners (gps) could also be useful in mena-isn countries and should be considered [ ] . participating countries recommended the following steps to move forward • develop action plans tailored to each country situation by focusing on four main areas: (i) epidemiological and virological surveillance; (ii) vaccination; (iii) communication/awareness and (iv) advocacy • expand the influenza stakeholder network to involve decisionmakers including moh, gps, patients, industry, etc. • reinforce local influenza network and advisory groups • set-up research agenda to generate country and region-specific data • identify priority areas to investigate the best way to move forward • promote and increase collaborative research among the mena-isn countries • encourage publication and sharing of existing data • set-up a public mena-isn website to increase its visibility at national and international levels • improve/reinforce the link between mena-isn and who-emro pandemic influenza preparedness framework. the meeting was made possible thanks to fondation mérieux and unrestricted grants from sanofi pasteur. seasonal influenza vaccination coverage rates in countries in africa seasonal influenza vaccine dose distribution in countries epidemiology of seasonal influenza in the middle east and north africa regions, - : circulating influenza a and b viruses and spatial timing of epidemics pathogenicity of highly pathogenic avian influenza virus h n in naturally infected poultry in egypt cumulative number of confirmed human cases for avian influenza a(h n ) reported to who influenza research in the eastern mediterranean region: the current state and the way forward serological evidence of h n avian influenza virus exposure th mena-isn study group avian influenza outbreak in poultry in the lebanon and transmission to neighbouring farmers and swine cross-sectional survey and surveillance for influenza viruses and mers-cov among egyptian pilgrims returning from hajj during - . influenza other respir viruses occurrence of the middle east respiratory syndrome coronavirus (mers-cov) across the gulf corporation council countries: four years update middle east respiratory syndrome coronavirus (mers-cov) summary and literature update-as of benefits of flu vaccination for persons with diabetes mellitus: a review economic development and diabetes prevalence in mena countries: egypt and saudi arabia comparison global role and burden of influenza in pediatric respiratory hospitalizations, - : a systematic analysis influenza vaccination in turkey: prevalence of risk groups, current vaccination status, factors influencing vaccine uptake and steps taken to increase vaccination rate influenza a and b infection in children in urban slum seasonal influenza vaccine dose distribution in countries ( - ): little progress in estimated global vaccination coverage microevolution of highly pathogenic avian influenza a(h n ) viruses isolated from humans molecular characterization and phylogenetic analysis of human influenza a viruses isolated in iran during the - season mg, it, km, ma, bu, and tp are employee of sanofi pasteur. others authors do not have any conflict of interest to declare. key: cord- - eonuc x authors: siriprapaiwan, supatcha; moore, elvin j.; koonprasert, sanoe title: generalized reproduction numbers, sensitivity analysis and critical immunity levels of an seqijr disease model with immunization and varying total population size date: - - journal: mathematics and computers in simulation doi: . /j.matcom. . . sha: doc_id: cord_uid: eonuc x abstract an seqijr model of epidemic disease transmission which includes immunization and a varying population size is studied. the model includes immunization of susceptible people (s), quarantine (q) of exposed people (e), isolation (j) of infectious people (i), a recovered population (r), and variation in population size due to natural births and deaths and deaths of infected people. it is shown analytically that the model has a disease-free equilibrium state which always exists and an endemic equilibrium state which exists if and only if the disease-free state is unstable. a simple formula is obtained for a generalized reproduction number r g where, for any given initial population, r g < means that the initial population is locally asymptotically stable and r g > means that the initial population is unstable. as special cases, simple formulas are given for the basic reproduction number r , a disease-free reproduction number r d f , and an endemic reproduction number r e n . formulas are derived for the sensitivity indices for variations in model parameters of the disease-free reproduction number r d f and for the infected populations in the endemic equilibrium state. a simple formula in terms of the basic reproduction number r is derived for the critical immunization level required to prevent the spread of disease in an initially disease-free population. numerical simulations are carried out using the matlab program for parameters corresponding to the outbreaks of severe acute respiratory syndrome (sars) in beijing, hong kong, canada and singapore in and . from the sensitivity analyses for these four regions, the parameters are identified that are the most important for preventing the spread of disease in a disease-free population or for reducing infection in an infected population. the results support the importance of isolating infectious individuals in an epidemic and in maintaining a critical level of immunity in a population to prevent a disease from occurring. the seqijr model (see, e.g., [ , , ] ) is a generalization of the well-known seir model of an infectious disease in which a population at risk is separated into the four categories of susceptible (s), exposed (e), infectious (i ) and recovered (r) (see, e.g., [ , , , ] ). a susceptible person is an uninfected person who can be infected through contact with an infectious or exposed person, an exposed person is someone who has come into contact with an infectious person but is asymptomatic, an infectious person is symptomatic, and a recovered person is someone who has recovered from the disease. it is assumed that a recovered individual cannot become infected again. in an seqijr model, two extra categories of quarantined (q) and isolated (j ) are added to the seir model. a quarantined person is an exposed person who is removed from contact with the general population and an isolated person is an infectious person who is removed from contact with the general population, usually by being admitted to a hospital. quarantine and isolation are important in controlling epidemics because they are effective methods of reducing the contact between infected and susceptible populations. in this paper, we consider a generalization of the basic seqijr model developed by gumel et al. [ ] for the severe acute respiratory syndrome (sars) outbreaks of november -july . the seqijr model of gumel et al. for the sars outbreaks is useful for study for several reasons. firstly, gumel et al. suggested parameter values which can be used to obtain physically reasonable models for the four sars outbreaks in beijing, hong kong, singapore and toronto. secondly, considerable data are available on the sars outbreaks and mathematical models and the effectiveness of control measures have been studied by many authors (see, e.g., [ , , , , [ ] [ ] [ ] [ ] [ ] , ] ). in building their model, gumel et al. assumed that there was no immunization (no vaccine was available for sars), that susceptible people were born into the community at a constant rate and that the population was constant. they analyzed the effects of quarantine and isolation on the transmission of sars and showed that an effective isolation policy was more important than quarantine or a combination of quarantine and a less effective isolation policy in reducing the transmission of the disease. a similar conclusion was obtained by koonprasert et al. [ ] in a numerical study of the effectiveness of quarantine and isolation. however, as noted by a number of authors (see, e.g., [ , , , , , ] ), the effectiveness of isolation requires strict hospital hygiene to reduce the hospital-based (nosocomial) spread of a disease. the modifications introduced in our analysis include: ( ) the existence of an immunization program, ( ) changes in the total population due to births and deaths, ( ) the derivation of generalized reproduction numbers for disease-free states and endemic equilibrium states, ( ) a sensitivity analysis of the effect of variations in parameter values on the reproduction numbers for disease-free states and on the infected populations of the endemic equilibrium state, and ( ) a derivation of a simple formula for the critical immunization level required to prevent spread of the disease in an initially disease-free population. the organization of the paper is as follows. in section , we define the mathematical model. in section , we obtain analytical formulas for disease-free and endemic equilibrium points. section contains an analysis of the local stability of the equilibrium points and gives derivations of formulas for the generalized reproduction numbers. formulas are derived for sensitivity indices in section and for critical immunization levels in section . section and contain results of numerical simulations using matlab for the parameter values proposed by gumel et al. [ ] for four of the cities affected by the sars outbreaks of and , namely beijing, hong kong, singapore and toronto. in section , we give a discussion of results and conclusions. a flow chart of the dynamics of our seqijr model is shown in fig. . the system of equations for the seqijr model that we use is given in eqs. ( )- ( ) . definitions of the parameters in the model are given in table . for the immunization program, we assume that a vaccine is available and that a fraction ν of the susceptible population acquires immunity from vaccination per unit time. the effect of the immunization can then be modeled as a direct transfer of a fraction ν s of the susceptible people from the s to the r class per unit time. we also assume that people table parameters for the seqijr model (rates are per day). source: adapted from gumel et al. [ ] param definition rate of inflow of susceptible individuals into a region or community through birth or migration. µ the natural death rate for disease-free individuals ν rate of immunization of susceptible individuals β infectiousness and contact rate between a susceptible and an infectious individual ε e modification parameter associated with infection from an exposed asymptomatic individual ε q modification parameter associated with infection from a quarantined individual ε j modification parameter associated with infection from an isolated individual γ rate of quarantine of exposed asymptomatic individuals γ rate of isolation of infectious symptomatic individuals σ rate of recovery of symptomatic individuals σ rate of recovery of isolated individuals k rate of development of symptoms in asymptomatic individuals k rate of development of symptoms in quarantined individuals d rate of disease-induced death for symptomatic individuals d rate of disease-induced death for isolated individuals in the r class cannot be infected for a second time. where is an effective infectiousness rate of the s population due to contact with asymptomatic exposed and quarantined populations (e, q) and symptomatic infectious and isolated populations (i , j ), and where are decay rate coefficients for s, e, q, i , and j populations respectively. although the basic seqijr model only contains equations, one each for s, e, q, i, j, r, we have found it convenient (see, e.g., [ ] ) to add extra equations for easy computation of the total population size n (t) = s(t) + e(t) + q(t) + i (t) + j (t) + r(t) at time t and the death rate d(t) due to disease at time t. the equations of the gumel et al. model can be obtained from eqs. ( )-( ) by setting ν = and assuming that the total population is constant, i.e., that Π = d i + d j + µn . theorem . the system ( )- ( ) and ( ) has two equilibrium states: where proof. the equilibrium points are solutions of the system of nonlinear algebraic equations obtained by setting ( ) and ( ) . we begin by solving the system of nonlinear algebraic equations for s * , e * , q * , j * , r * and n * as functions of i * to obtain the equations in ( ) and ( ) . then, on substituting these solutions into ( ), we obtain therefore, two possible values for i * are i * = and the solution for i * in ( ) . the proof is complete. □ for an endemic equilibrium point, all populations (s * , e * , q * , i * , j * , r * , n * ) must be non-negative and the infectious population i * must be positive. proof. we first prove that if i * ̸ = then α s > α n . from ( ), we have then since µn * = Π − α n i * , we obtain and therefore therefore, if α s ≤ α n , then i * = and the endemic equilibrium does not exist. we now prove that if α s > α n and µα l − d s α s > , then all populations are positive. if α s > α n and µα l − d s α s > , then µα l − d s α n > . so we have therefore, the endemic equilibrium exists and all populations are positive. we next note that if µα l − d s α s = , then from ( ) i * = and the endemic equilibrium does not exist. the proof is complete. □ we consider three cases for generalized reproduction numbers r g with the property that a given population is locally asymptotically stable if r g < and unstable if r g > . these generalized reproduction numbers are useful because they give a simple test for the local asymptotic stability of an initial population. three useful cases are as follows: . basic reproduction number r . this number is defined for a population that is % susceptible so that if r < and infected people enter the population then the number of secondary infections is less than the number of primary infections, whereas if r > then the number of secondary infections is greater. . reproduction number r d f for disease-free equilibrium. . reproduction number r en for endemic equilibrium. the basic reproduction number r and disease-free reproduction number r d f of the seqijr model can be obtained from the next-generation method of van den driessche and watmough [ ] or by using lyapunov's first method, i.e., by checking the eigenvalues of the jacobian of the linearized system about populations with all infected populations equal to zero (see, e.g., [ ] ). however, as far as the authors are aware, the endemic equilibrium reproduction number r en can only be obtained by checking the eigenvalues of the jacobian of the linearized system about the endemic equilibrium point. for the next-generation method, we write x = (e, s, q, i, j, r) t and regard e as the next-generation "infected" population [ ] . then, (s, q, i, j, r) t can be regarded as the remaining populations. note that, in the seqijr model in this paper, an infected person always enters the e population first and then later enters the q, i , j or r populations. then the model can be written in the usual next-generation form as d x then, a disease-free reproduction number is defined as the largest eigenvalue of the matrix fv − , where f and v are the jacobian matrices of f(x ) and v (x ) at the disease-free equilibrium point ( . we have used the maple software package to find an analytic formula for the largest eigenvalue of fv − , i.e., for r d f , and obtained the result: from the next-generation method, if the disease-free reproduction number r d f < , then the disease-free equilibrium point is locally asymptotically stable and if r d f > then it is unstable. we will now prove that r d f can be rewritten in a much simpler form and that r d f > also corresponds to the condition that the endemic equilibrium exists. proof. from ( ) and using the definitions of α s , α e , α q and α j in ( ), we have then, the formula for the endemic infectious population i * in ( ) can be written as: therefore, i * > if and only if r d f > , i.e., if and only if the disease-free equilibrium point is unstable. to obtain the basic reproduction number r , we consider the special case that the initial population is the % susceptible population. for this case, we have s = n , which corresponds to ν = or d s = µ. thus the basic reproduction number r of the seqijr model is the proof is complete. □ in the previous section, we used the next-generation method of [ ] to obtain formulas for the reproduction number r d f for the disease-free equilibrium point and for the basic reproduction number r . we also showed that the endemic equilibrium point exists if and only if r d f > . in this section, we derive generalized reproduction numbers r g by using lyapunov's first method (see, e.g., [ ] ) of linearizing the system about an equilibrium point and checking that the real parts of all eigenvalues of the jacobian at the equilibrium point are negative for r g < and that the real part of at least one eigenvalue is positive for r g > . the linearized model of the system at an equilibrium point (s * , e * , q * , i * , j * , r * ) of ( )- ( ) can be written in the form: where x = (s, e, q, i, j, r) t − (s * , e * , q * , i * , j * , r * ) t and j * is the jacobian matrix at an equilibrium point given by: where (we omit the * on the populations in the matrix to save space) as usual for linear systems, we can assume that eq. ( ) has a solution of the form x(t) = e λt v, where λ is an eigenvalue of j * and v is the corresponding eigenvector. for the × jacobian matrix in ( ) it is not possible to obtain useful analytical expressions for all six eigenvalues either by a direct analysis or by solving the characteristic equation where i is a × identity matrix. it is also not possible to obtain useful analytical tests for the real parts of eigenvalues to be negative from the six routh-hurwitz conditions (see, e.g., [ ] ). however, it is possible to check two of the six routh-hurwitz conditions for the real parts of the six eigenvalues to be negative. these conditions are usually stated in terms of determinants derived from the coefficients of the characteristic polynomial. however, we will use the two equivalent necessary conditions for the real parts of the six eigenvalues to be negative that the sum of the six eigenvalues (trace (j * )) must be negative and the product of the six eigenvalues (det (j * )) must be positive. now, for the disease-free populations, we have l * since βε e α e < α l and µα l < d s α s , and therefore for the endemic population, l * > and then now, from eq. ( ), we have and trace(j * ) < . to evaluate the determinant of the jacobian j * in ( ), we first used the symbolic algebra toolbox in matlab to find the following analytic expression: then, after considerable straightforward algebra, we obtained the following equivalent expression for the determinant. where a generalized reproduction number can be defined as: formulas for the disease-free reproduction number (r d f ) and the endemic equilibrium reproduction number (r en ) can be easily obtained from eq. ( ). the results are summarized in the following theorem. theorem . necessary conditions for stability of the two equilibrium points of the seqijr model are: . disease-free equilibrium. . endemic equilibrium. further, the endemic equilibrium exists if and only if r en < . proof. for a disease-free population, we have l * = and , and therefore from ( ) the reproduction numbers for the disease-free cases are as stated in part of the theorem. these results are in agreement with the results from the next-generation method. a simple formula for the endemic equilibrium number can be obtained by substituting the expressions in eqs. ( ) , ( ) and ( ) for the endemic equilibrium populations into eq. ( ). we then obtain the result given in part of the theorem. since the endemic equilibrium exists only if µα l − d s α n > , it can be seen from eq. ( ) that r en < when the endemic equilibrium exists and that r en → when i * → , i.e., at the transition between the endemic and disease-free equilibrium points. □ note: in this example, it is possible to obtain the reproduction numbers from the trace and determinant of the jacobian at an equilibrium point because the change in stability occurs due to a single real eigenvalue changing value from negative to positive. a change in value of the determinant cannot be used to detect a change in stability if the change is due to a complex conjugate pair of eigenvalues crossing the imaginary axis, e.g., if an andronov-hopf bifurcation occurs (see, e.g., [ ] ). therefore, a negative trace and a positive determinant for the jacobian of a system of equations are necessary but not sufficient conditions for the real parts of all eigenvalues to be negative and for an equilibrium point to be locally stable. however, for any given set of parameter values it is very easy to check if r g < corresponds to asymptotic stability of an equilibrium point by numerically computing all eigenvalues of the jacobian matrix with any of the standard mathematical software packages such as matlab, mathematica, maple etc. sensitivity analysis is important as it can be used for determining the parameters which are of most importance in reducing the level of a disease (see, e.g., [ , ] ). the normalized sensitivity index for a quantity q with respect to a parameter h is defined by: the sensitivity indices can be calculated in at least three ways, namely, ( ) by direct differentiation of formulas for q, [ ] ). in this section, we will use the method of direct differentiation as it gives analytical expressions for the indices. however, as a check on our formulas we have compared numerical values from our formulas with numerical results we obtained using the method of chitnis et al. the normalized sensitivity indices can be computed from the formula in ( ) as we used the symbolic algebra package in matlab to derive normalized sensitivity indices for the fourteen parameters of the model from eq. ( ) and then simplified the expressions to obtain the list shown in table . the sensitivity indices for the basic reproduction number r can be obtained from the results in table by setting ν = , i.e., d s = µ. in this section, we derive formulas for the sensitivity indices for the endemic equilibrium populations. in particular, we derive indices for the endemic infectious population i * . the sensitivity indices for the other endemic populations can then be easily obtained from the sensitivity indices for i * using the formulas in eqs. ( ) and ( ). the formulas for the sensitivity indices for i * can be computed from the formula in ( ) as: where the formulas for the coefficients ξ h , ψ h , ω h of the sensitivity indices in ( ) are shown in table . the formulas for the natural birth rate Π and natural death rate µ are not included in table because these parameters are very difficult to change for human populations. as stated previously, the indices can also be computed using the method of chitnis et al. [ ] . as a check on our results, we have compared numerical results obtained from the formulas in table with results obtained from the method of chitnis et al. table formulas for endemic sensitivity coefficients in eq. ( ). from the disease-free reproduction number, we can easily find a formula for a critical immunization level required to prevent the spread of the seqijr disease. since, r d f = µα l d s α s < is required to stop the disease spreading and d s = µ + ν, we have the critical immunization level where r is the basic reproduction number. further, the critical ratio of immune (recovered) population to total population is r * for the models given in eqs. ( )- ( ) and ( ), we have computed the equilibrium points, generalized reproduction numbers, sensitivity indices, dynamical behavior and the critical immunization levels by using the parameter values estimated by gumel et al. [ ] for the sars epidemics in beijing, canada, hong kong and singapore. in this section, we will show the results for beijing. we will then briefly discuss any differences between the beijing results and the results for the other three regions in section . the meanings of the parameters in the seqijr model are defined in table and the parameter values that we use are shown in table . to examine the effect of immunization, we will use values of ν = . corresponding to a disease-free equilibrium, ν = . corresponding to an endemic equilibrium, and a critical value of ν corresponding to a value for the disease-free reproduction number of r d f ≈ . substituting the parameters for beijing in table into the system of differential equations for disease transmission in eqs. ( )- ( ) and ( ), we obtain eqs. ( ). d j dt = . i + . q − . j, table initial population values used for numerical integration. for the parameter values in table and an immunization rate ν = . , the disease-free equilibrium point is and the endemic equilibrium point does not exist as some populations are negative. the disease-free and basic reproduction numbers are: that is, the disease-free equilibrium is locally asymptotically stable and the population with % susceptible population will be unstable. these conclusions can also be checked by using matlab to compute the eigenvalues of the jacobian ( ) for the disease-free equilibrium and the % susceptible population. these eigenvalues are: since the real parts of all eigenvalues for the disease-free equilibrium are negative and the real part of one eigenvalue of the % susceptible population is positive, the disease-free equilibrium state will be locally asymptotically stable and the % susceptible population will be unstable. using the matlab program we have numerically integrated the system of eqs. ( ) for the four sets of initial conditions given in table to obtain the dynamical solutions shown in fig. . the graphs show that, for the four different sets of initial conditions, the four infected populations first decrease and then increase before finally converging to zero, i.e., the initial points are not locally asymptotically stable even though there is long-term convergence to the disease-free equilibrium point. however, it can also be seen that at the longer times, where the population values are close to the disease-free equilibrium point, the solutions converge asymptotically to this equilibrium state. this behavior for the longer times in fig. is in agreement with eq. ( ) and the values of the reproduction numbers and eigenvalues given above. using the analytical results given in table , we have computed the normalized sensitivity indices shown in table of the disease-free reproduction number r d f = . < for the parameter values of table and ν = . . for these parameter values, the sensitivity indices and Φ(r d f |k ) are positive and the remaining indices are negative. table normalized sensitivity indices Φ(r d f |h) and required % changes in parameter for % reduction in r d f for disease-free equilibrium state (beijing). for these parameter values, r d f = . < and the solution converges to a disease-free equilibrium point. then, a reduction in the value of r d f corresponds to a faster approach to equilibrium, i.e., to a faster disappearance of the disease. the values in column of table are the sensitivity indices and the values in column are % changes in parameter values required to give a % decrease in r d f . for example, in order to get a % decrease in the value of r d f , it is necessary to decrease the values of β, µ, ε j , k and k by . %, . %, . %, . % and %, respectively. also, in order to get a % decrease in the value of r d f , it is necessary to increase the values of ν, σ , d , σ , γ , d and γ by . %, . %, . %, . %, . %, . % and . %, respectively. since the natural death rate µ cannot be easily changed, the most effective methods of reducing r d f are to reduce the value of the infectiousness and contact rate β, increase the value of the immunization rate ν, reduce the value of the isolation modification parameter ε j and increase the value of the isolation recovery rate σ . for the beijing parameter values in table and ν = . , we obtain the endemic equilibrium point therefore, the disease-free equilibrium point is unstable and the endemic equilibrium point is locally asymptotically stable. as shown in gumel et al. [ ] (see also [ ] ), an endemic equilibrium point is also obtained for ν = . using matlab, we integrated the system of eqs. ( )-( ) for ν = . for the four sets of initial conditions given in table . since the dynamical behavior for the four infected populations are similar, we will only show the solutions for the infectious population (i ) and for the susceptible (s) and recovered (r) populations. the solutions for the s, i and r populations are shown in fig. first for a short integration time to show the initial behavior and then for a long integration time to show the convergence to an endemic equilibrium. it can be seen that the convergence to the endemic equilibrium is very slow and that many disease outbreaks occur during this period. this type of behavior was very common in "childhood" diseases such as measles, mumps, poliomyelitis, rubella, whooping cough etc. before vaccines became available for these diseases. although it is difficult to see in fig. , we found that epidemics can recur when the fraction of the susceptible population exceeds a certain critical level due to births or if the population of the immune recovered population falls below a certain critical level due to deaths. similar results for the dynamical behavior of populations in the endemic region have also been reported by koonprasert et al. [ ] in a study on the effectiveness of quarantine and isolation in an seqijr model. in the sensitivity analysis we do not consider variations in Π , µ, ε e and ε q . the parameter Π represents the natural birth rate and the parameter µ represents the natural death rate for the population. these parameters cannot be changed easily for human populations. also, we omit the modification parameters for infection from the exposed population ε e and the quarantined population ε q since these parameters have been assumed to be zero. we therefore only consider sensitivity indices for the remaining eleven parameters. using the analytical results given in table and eq. ( ), and the parameter values in table with ν = . , we obtain the values shown in table for the normalized sensitivity indices of the endemic equilibrium populations. as noted above, the values of r d f = . > and r en = . < show that the endemic equilibrium is locally asymptotically stable. since the sensitivity indices are functions of parameter values, the values in table will change if parameter values are changed. the interpretation of these indices is that a positive index means that i * increases if the parameter value is increased and a negative index means that i * decreases if the parameter value is increased with the magnitude giving the rate of change. in order to reduce the spread of the disease, it is necessary to reduce the infectious population (i * ) and isolated population (j * ) since these populations are capable of direct transmission of the disease to uninfected people. it is also desirable to reduce the number of deaths from the disease, i.e., to increase the total surviving population n * . from the indices in table , the most effective methods of reducing i * are, in order, to increase the isolation rate (γ ), reduce the transfer rate from exposed to infectious population (k ), increase the quarantine rate (γ ) and reduce the contact rate between infectious and susceptible people (β). the most effective methods of reducing j * are, in order, to increase the recovery rate of isolated individuals (σ ), reduce the contact rate β, and reduce the modification parameter ε j . it can be seen from table that increasing the death rates d and d of the infectious and isolated individuals would also reduce the infectious and isolated populations. this method is clearly not an acceptable control method for a human disease. however, this method was adopted as an effective control method in the case of the spread of avian flu among chickens in china and "mad-cow disease" in england, and it has also been used as an effective method of controlling some other diseases among animals and plants. the most effective methods for increasing the total surviving population n * , i.e., to reduce the death rate, are, in order, increase the recovery rate of infectious individuals (σ ), reduce the contact rate β and reduce the modification parameter ε j , i.e., to reduce the level of nosocomial disease as discussed in the introduction. using the parameter values given in table , we have computed and compared the disease-free and endemic results for the four regions. the overall behavior of the results for all four regions is similar, but with some differences in detail. we will compare the results for the reproduction numbers, the sensitivity indices and the critical immunity levels. table ranking for best parameters to reduce r d f . region ranking for best parameters to reduce i * . region beijing β, σ , ν . γ , k , γ . canada β, σ , ν . β, σ , γ . hong kong β, σ , ν . γ , β, k . singapore β, σ , γ . β, σ , γ . the results for the basic reproduction numbers and the disease-free and endemic reproduction numbers are shown in table . for each case, we have numerically integrated the differential equations and checked the asymptotic stability of equilibrium points by computing the value of the reproduction numbers and by computing the eigenvalues of the jacobian matrices. in all cases we have shown that the numerical results agree with the theoretical analysis. for each of the four regions, we have also computed the sensitivity indices of the disease-free reproduction number r d f and the endemic equilibrium populations for all of the parameters in the model. from these calculations, we can rank the indices in order of effectiveness in reducing a disease. the results for the disease-free equilibrium r d f are shown in table . the table shows the top three indices for each region for a disease-free case (r d f < ), and a critical immunization case r d f ≈ . for r d f < , the rankings for all regions are the same. the most effective methods of reducing r d f are, in order, to reduce the infectiousness and contact rate β between infectious and susceptible people, increase the value of the immunization rate ν and reduce the modification parameter ε j for the infectiousness and contact rate of isolated people. it can be seen from table that reducing (index is positive) the natural death rate µ is the third most effective method of reducing the value of r d f . however, since the natural death rate cannot be varied easily for human populations, this is not an acceptable control method for a human disease. for r d f ≈ , the most effective methods of reducing r d f are, in order, to reduce the infectiousness and contact rate β, reduce the isolation modification parameter ε j and increase the recovery rate of isolated individuals σ . table shows the rankings for the most effective methods for reducing the endemic infectious populations i * in the four regions for r d f ≈ and for r d f > . for r d f ≈ , the rankings for beijing, canada, hong kong are the same. the most effective methods of reducing i * are, in order, to reduce the contact and infectiousness rate β, increase the recovery rate of isolated individuals σ and increase the value of the immunization rate ν. for singapore, the most effective methods of reducing i * are, in order, to reduce the infectiousness and contact rate β, increase the recovery rate of isolated individuals σ and increase the isolation rate γ . for r d f > , the most effective methods of reducing i * for beijing are, in order, to increase the isolation rate γ , reduce the transfer rate from exposed to infectious k and increase the quarantine rate γ . for canada the most effective methods of reducing i * are, in order, to reduce the infectiousness and contact rate β, increase the recovery rate of isolated individuals σ and increase the isolation rate γ . for hong kong the most effective methods of reducing i * are, in order, to increase the isolation rate γ , reduce the infectiousness and contact rate β and reduce the transfer rate from exposed to infectious k . for singapore the most effective methods of reducing i * are the same as for the r d f ≈ case. the critical values of the immunization rate ν and the critical ratio of recovered to total population can be calculated from eqs. ( ) and ( ) and parameter values in table for the sars epidemics in beijing, canada, hong kong and singapore in and . the results for the four regions are shown in table . the results show that there is a big difference between the critical immunization levels and critical immunized ratio in the four regions. the difference is due to the different values of the basic reproduction number r = α l α s in the four regions. the difference in r values in turn is mainly due to differences in the values of the infection factor β and the modification factors for infection from the quarantined population (ε q ) and the isolated population (ε j ). we have studied an seqijr model for infectious disease transmission which generalizes the model developed by gumel et al. [ ] for the sars epidemics of and by including immunization and a varying total populations size. we have shown analytically that this model has a disease-free equilibrium state which always exists and an endemic equilibrium state which exists if and only if the disease-free equilibrium is unstable. we have defined generalized reproduction numbers r g for any given equilibrium state such that the state is locally asymptotically stable if r g < and unstable if r g > . as special cases, we have obtained simple formulas for the reproduction numbers for a % susceptible population (the basic reproduction number r = α l α s ) and for the disease-free equilibrium r d f = µα l (µ+ν)α s by both the next-generation method [ ] and from the eigenvalues of the jacobian of the linearized model about the disease-free equilibrium. in these formulas, α l can be interpreted as the rate of new infections from all infected individuals and α s as the rate of reduction of the susceptible population due to transfers to other population groups including to the recovered immune group. the effect of the immunization rate (ν) in reducing the reproduction number and increasing the stability of disease-free equilibrium states can be clearly seen by comparing the r and r d f formulas. we have also obtained simple formulas for a reproduction number r en for the endemic equilibrium state and shown that the endemic equilibrium is locally asymptotically stable if r en < and does not exist if r en > . we have also derived formulas for sensitivity indices of the disease-free and basic reproduction numbers, and the endemic equilibrium populations for changes in the values of model parameters. for the numerical study of the model, we have used values of model parameters estimated by gumel et al. [ ] for the sars epidemics in - in beijing, canada, hong kong and singapore. for each of the four regions we have considered parameter values corresponding to a disease-free equilibrium and an endemic equilibrium. from the computed sensitivity indices for each of the regions, we have shown that the most important parameters for reducing infection are, in order, ( ) for high immunization rates (ν ≈ . ) reduce the overall infection rate (β), increase the immunization rate, and reduce the modification factor for infection by isolated individuals (ε j ), ( ) for critical immunization rates with r d f ≈ , reduce β, reduce ε j and increase recovery rate of isolated individuals (σ ), ( ) for low immunization rates (endemic equilibrium) the most important for beijing and hong kong is to increase the isolation rate of infectious individuals (γ ) and the most important for canada and singapore is to reduce β. the numerical results for the reproduction numbers and the sensitivity indices clearly support the importance of isolation and to a lesser extent quarantine in controlling the spread of a disease. we have obtained very simple formulas in terms of the basic reproduction number r for critical values of the immunization rate and the ratio of the recovered (immune) and total populations that are required to stop the spread of a disease. our numerical results for the four regions considered in this paper clearly support the importance that is now widely accepted, except by anti-vax campaigners, of maintaining a critical immunization level of "herd immunity" to stop the spread of a disease. the differences in the critical values of the immunization rate in the four regions clearly show the importance of factors such as reducing infection rates, and increasing isolation and quarantine, in stopping an epidemic that can occur when the immunization ratio falls below a critical level. unfortunately, in and there was no known immunization available for sars. therefore, the immunization results in this paper are not relevant for the sars outbreaks of - . however, the inclusion of immunization in the study of an seqijr model is important as the seqijr model is a very general model which can be applied to a number of other diseases that are also mainly transmitted by person to person contact and for which immunization is available, e.g., influenza and "childhood diseases" such as measles, mumps, whooping cough etc. a similar model with the addition of a bird reservoir of the disease could also be used to analyze avian influenza. seir models have, of course, been studied by many authors and there is now an extensive literature on them. however, we would note that the formulas for generalized reproduction numbers, sensitivity indices and critical immunization levels for the seqijr model could easily be applied to seir models by removing the quarantined and isolated compartments and suitably redefining some of the parameters. determining important parameters 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transmission and control in china this research is supported by the centre of excellence in mathematics, the commission on higher education, thailand. this research was also supported by the science and technology research institute of king mongkut's university of technology north bangkok. we would also like to thank colleagues in the department of mathematics, faculty of applied science, king mongkut's university of technology north bangkok for encouragement and critical discussions. key: cord- -nt n p v authors: vissichelli, n. c.; miller, k.; mccarty, j. m.; roberts, c. h.; stevens, m. p.; de la cruz, o. title: bronchoalveolar lavage to evaluate new pulmonary infiltrates in allogeneic hematopoietic stem cell transplant recipients: impact on antimicrobial optimization date: - - journal: infection prevention in practice doi: . /j.infpip. . sha: doc_id: cord_uid: nt n p v summary background pulmonary complications cause significant morbidity and mortality after allogeneic hematopoietic stem cell transplant (ahsct). bronchoscopy with targeted bronchoalveolar lavage (bal) is often used in ahsct patients with suspected lower respiratory tract infection (lrti) to help guide management. aim to evaluate how positive bal results change antimicrobial management of ahsct recipients with suspected lrti. methods we performed a retrospective review of bal results from january to july for ahsct recipients. a positive bal was determined by culture, multiplex polymerase chain reaction (pcr), aspergillus galactomannan antigen (aga), and cytology. findings bal was positive for infectious etiologies in %, and antimicrobials were adjusted in / ( %) of patients. antibacterial escalation was predicted by a positive bal bacterial culture (or . , p= . ). antibiotic de-escalation was more likely with an elevated aga (or . , p= . ). antiviral initiation was more likely with positive bal multiplex pcr (or . , p= . ). antifungals were more likely to be escalated or changed with an elevated aga (or . , p= . ). the patients with a negative bal were more likely to be started on steroids (or . , p= . ). conclusions bal was helpful to determine the etiology of pulmonary complications and optimize antimicrobials. the addition of aga and multiplex pcr to standard bal significantly impacted de-escalating antibiotics and adjusting antifungals to provide adequate coverage. the association with an elevated aga with antibacterial de-escalation highlights a new role for bal in antimicrobial optimization. bronchoscopy with bronchoalveolar lavage (bal) is an important diagnostic tool to evaluate ahsct recipients with new pulmonary infiltrates or without clinical response to empiric therapy [ , ] . its utility is dependent on culture and molecular diagnostic results and the capability of changing medical management [ ] . in studies prior to the availability of polymerase chain reaction (pcr), antibiotics were withdrawn in up to % of ahsct recipients [ , ] . this study aims to determine how aspergillus galactomannan antigen (aga) and multiplex pcr added to traditional bal testing affects antimicrobial treatment in ahsct recipients with new pulmonary infiltrates. the first bal completed after ahsct in patients over age between january and july at the authors' institution were included. the decision to perform a bal was at the discretion of the attending physician caring for the patient. bal fluid was submitted for gram stain, bacterial, mycobacterial and fungal cultures, multiplex pcr, aga, and cytopathology. this study was approved by virginia commonwealth university's institutional review board. a positive bal was defined by a positive culture, multiplex pcr, elevated aga (optic density (od) > . ), and/or cytology (which includes detection of pneumocystis jirovecii). a positive bacterial culture required species isolation, excluding coagulase-negative staphylococcus and mixed respiratory flora. a positive fungal culture required the presence of fungal colonies, except non-cryptococcus yeast. the multiplex pcr used was the commercial kit biofire filmarrayÒ that can detect adenovirus, coronavirus, human metapneumovirus, human rhinovirus/enterovirus, influenzae a, influenzae b, parainfluenza (subtypes e ), respiratory syncytial virus (rsv), bordetella pertussis, chlamydophila pneumoniae, and mycoplasma pneumoniae. diffuse alveolar hemorrhage (dah) was diagnosed per guidelines [ ] . a change in management was defined by escalation or de-escalation of antibiotics, antifungals, or antivirals, adaptation of antifungals, or initiation of prednisone within days after bal since this is the time interval to obtain aga results at the authors' institution. deescalation was defined as cessation of one or multiple agents, conversion to oral therapy, or transition to a more narrow spectrum. escalation was defined as adding a new antimicrobial agent or transitioning to a broader spectrum. fungal adaptation was a transition to an alternative agent within the same class of antifungals. antimicrobial use within days of bal was considered prior therapy. ahsct was performed on the bone marrow transplant unit at the authors' institution. the conditioning regimen, immunosuppressive, and supportive therapies followed internationally accepted protocols. levofloxacin and fluconazole were used as anti-infective prophylaxis and prophylaxis for cytomegalovirus (cmv) and pneumocystis jirovecii pneumonia were used according to guidelines [ ] . a descriptive analysis performed using medians and interquartile ranges (iqr) for continuous variables due to nonnormal distributions and frequencies and percentages for categorical variables. we used a fisher's exact test to compare categorical variables and anova for continuous variables. a p-value of less than . was considered statistically significant. all analysis was performed using jmp pro [sas institute, cary, nc, usa]. bal was performed in ahsct recipients during the study period. procedures were excluded for patient age< (n¼ ), subsequent procedures on the same patient (n¼ ), and bal performed before transplant (n¼ ). patients who had a bal after ahsct were included. patient demographics, antimicrobials at bronchoscopy, and bal results are described in table i . most antimicrobial therapy was started within two days (n¼ ) or over days (n¼ ) before bal. eleven patients were not receiving antimicrobials. bal was positive for infectious etiologies in %, mostly with elevated aga ( / ), followed by multiplex pcr ( / ), positive bacterial ( / ), fungal ( / ) and afb culture ( / ). none of the patients with a positive bacterial culture were on levofloxacin prophylaxis. of the patients with a positive fungal result, / were on antifungal prophylaxis. only / patients with a positive bal aga also had a positive serum aga. all of the positive multiplex pcr were for viruses. nine patients had multiple infectious etiologies, all with a positive multiplex pcr. twentyeight patients had concomitant non-pulmonary infections. no patients were found to have pneumocystis jirovecicii, legionella, or cryptococcus by cytology or antigen testing. antimicrobials were adjusted in / ( %) of patients (table ii) . overall antibiotic escalation occurred in / , and was associated with a positive bal bacterial culture (or . , p¼ . ) (table ii) . antibiotic de-escalation was more likely with an elevated aga (or . , p¼ . ) (table ii) . antiviral initiation was more likely with positive bal multiplex pcr (or . , p¼ . ) (table ii) . antifungals were more likely to be escalated or changed with an elevated aga (or . , p¼ . ) ( table ii) . the patients with a negative bal were more likely to be started on steroids (or . , p¼ . ). bal was helpful to determine the etiology of pulmonary complications and optimize antimicrobials. this included escalating antibiotics to target organisms that were isolated. we believe it changes practice as bal is not only valuable when results are positive, but when an aggregate negative bal work up offers assurance for providers to address noninfectious etiologies. this is critical in intensive care settings when antiinflammatory therapy, such as high dose steroids, tumor necrosis factor inhibitors, or t-cell depleting agents are considered for pulmonary graft versus host disease, with suspected superimposed infection. only patients required initiation of targeted therapy for a microorganism isolated on bacterial culture that was not previously covered, including virulent organisms such as nocardia nova complex, pseudomonas aeruginosa, haemophilis influenzae, and stentrophomonas maltophilia. antibiotics were also escalated due to severity of illness despite negative culture data (n¼ ) and to cover an alternative infection (n¼ ), such as endocarditis. likely the use of antimicrobial prophylaxis effectively limited bacterial infections due to susceptible organisms, but may have added to the selection of more resistant bacteria such as stenotrophomonas and nocardia spp. bal was critical to recognize and treat invasive fungal infections, prompting antifungal escalation in % and initiation of targeted treatment for isolated rhizomucor spp. only two of the patients with an elevated bal aga also had an elevated serum aga. bal assisted in the diagnosis of these infections. this finding is important in that delays in treatment carry the potential for increased mortality [ ] . this is recognized in the american thoracic society guidelines, which suggest moving to bal aga and requesting bal aspergillus pcr when serum aga is negative. at a cutoff of . , the serum aga sensitivity is approximately e % and at . , it is e %. at a cutoff of . , sensitivity reduces to % and specificity increases to %. for bal aga with a cutoff of . , the sensitivity and specificity increases to % and % respectively [ ] . in addition, aspergillus spp. accounts for only % of invasive fungal infections in ahsct, and the remaining % includes deadly pathogens including zygomycetes ( e %), fusarium ( e % in usa), dematiaceous, and other rare molds that may require culture or tissue biopsy diagnosis. endemic mycoses only account for % of invasive fungal infections in ahsct and may be amenable to serologic or antigen testing without invasive procedures [ ] . a positive multiplex pcr was associated with antiviral initiation (ribavirin for parainfluenza or peramivir for influenzae), (table ii) . however, the results were similar on nasopharyngeal and bal multiplex pcr, as noted in prior studies [ ] . it is unclear if this multiplex pcr test is adequate for diagnosing lrti and more studies will need to be done to evaluate this. bal remained necessary to detect coinfections, as / patients with a positive multiplex pcr also had a positive bacterial culture (n¼ ) or elevated aga (n¼ ) ( table i) . reports of viral and bacterial coinfection, particularly with adenovirus or rhinovirus have been associated with higher morbidity/mortality but the understanding of this is limited [ , ] . it would be interesting to further investigate with larger studies the effect of viral illnesses on bacterial or fungal coinfections and how they affect treatment outcomes to determine if these more aggressive prophylaxis and diagnostic testing would benefit these patients. bal also had a major role in antimicrobial de-escalation. antibiotics were either stopped or converted to oral in patients with a negative bal bacterial culture. an elevated aga was predictive of antibiotic de-escalation (table ii) , which highlights a new role for bal in antibiotic optimization [ ] . a negative bal was associated with initiation of steroids for treatment of other conditions including graft versus host disease, pneumonitis, dah, and complications of engraftment (table ii) . even though the bal did not aid in these diagnosis (except dah), it mitigated the risk of starting steroids by lowering suspicion for pulmonary infections. study limitations include potential selection bias (only patients who had a bal were included) and the fact our results are from a single hospital. bal was performed at the discretion of the attending physician, and therefore the patient group only includes those deemed eligible for the procedure, which was likely urged by clinical status. a control group comparison (with or without available sputum cultures, nasal multiplex respiratory pathogen panel, serum aga and fungal urine antigen testing) would be likely biased to include those who were deemed less sick as well as those considered too high risk to be eligible for bronchoscopy, due to high oxygen requirement or bleeding risk. it would be unethical to randomly assign patients to bronchoalveolar lavage due to known risks and benefits. additional studies across multiple hospitals would be valuable to confirm our results. a multivariate regression analysis could not be performed due to low sample size. in summary, our study found that bal is a beneficial diagnostic tool to evaluate new pulmonary infiltrates in ahsct recipients. bal permitted targeted antimicrobial treatment and positively affected antimicrobial de-escalation and guided management of non-infectious diagnostic considerations. these data will inform local antimicrobial stewardship efforts. additional studies are needed to confirm our findings and to identify how bal can be used to improve antimicrobial prescribing. the authors declare that they have no conflict of interest or financial disclosures. diagnostic bronchoscopy in solid-organ and hematopoietic stem cell transplantation survey of academic pulmonologists, oncologists, and infectious disease physicians on the role of bronchoscopy in managing hematopoietic stem cell transplantation patients with pulmonary infiltrates role of bronchoalveolar lavage in the diagnosis of pulmonary infiltrates in immunocompromised patients bronchoscopic evaluation of pulmonary infiltrates following bone marrow transplantation the influence of diagnostic bronchoscopy on clinical outcomes comparing adult autologous and allogeneic bone marrow transplant patients diffuse alveolar hemorrhage guidelines for preventing infectious complications among hematopoietic cell transplantation recipients: a global perspective respiratory fungal infections in solid organ and hematopoietic stem cell transplantation microbiological laboratory testing in the diagnosis of fungal infections in pulmonary and critical care practice. an official american thoracic society clinical practice guideline filmarray respiratory panel assay: comparison of nasopharyngeal swabs and bronchoalveolar lavage samples severe adenovirus pneumonia followed by bacterial septicaemia: relevance of co-infections in allogeneic hematopoietic stem cell transplantation human rhinovirus infections of the lower respiratory tract in hematopoietic stem cell transplant recipients utility of early versus late fiberoptic bronchoscopy in the evaluation of new pulmonary infiltrates following hematopoietic stem cell transplantation the redcap software used in this effort is partially supported by ctsa award no. ul tr from the national center for advancing translational sciences. key: cord- - i twr authors: esposito, susanna; polinori, ilaria; rigante, donato title: the gut microbiota-host partnership as a potential driver of kawasaki syndrome date: - - journal: front pediatr doi: . /fped. . sha: doc_id: cord_uid: i twr kawasaki syndrome (ks) is a necrotizing vasculitis of small- and medium-sized vessels mostly affecting children under years of age; a host of clinical and epidemiological data supports the notion that ks might result from an infectious disease. however, many efforts have failed to identify a potentially universal trigger of ks. the contribution of the intestinal microbial community—called the “microbiota”—to ks has been evaluated by an increasing number of studies, though limited to small cohorts of patients. differences in the microbiota composition were found in children with ks, both its acute and non-acute phase, with abnormal colonization by streptococcus species in the intestinal tract and a wider presence of gram-positive cocci in jejunal biopsies. in particular, a higher number of gram-positive cocci (of the genera streptococcus and staphylococcus), eubacterium, peptostreptococcus, and hsp -producing gram-negative microbes have been found in the stools of ks children, and their effects on the antigenic repertoire of specific t cells and vβ t cell expansion have been assessed. conversely, lactobacilli were lacking in most children with ks compared with other febrile illnesses and healthy controls. all studies available to date have confirmed that an imbalance in the gut microbiota might indirectly interfere with the normal function of innate and adaptive immunity, and that variable microbiota interactions with environmental factors, mainly infectious agents, might selectively drive the development of ks in genetically susceptible children. further investigations of the intestinal microflora in larger cohorts of ks patients will provide clues to disentangle the pathogenesis of this disease and probably indicate disease-modifying agents or more rational ks-specific therapies. the most insidious primary vasculitis in childhood is kawasaki syndrome (ks), an acute multi-systemic illness which predominantly affects children under years of age ( ) . currently, this disorder of unknown etiology remains the main cause of acquired heart disease among children living in developed countries, where rheumatic fever has been surpassed ( , ) . this condition was originally called "mucocutaneous lymph node syndrome" by dr. tomisaku kawasaki, who was its discoverer, and was thought to be a benign children's disease; nowadays, the illness has been described worldwide in children of every ethnicity following the presence of fever persisting (at least) days together with (at least) of the following signs: bilateral conjunctival injection, oropharyngeal inflammation, abnormalities of hands and feet, polymorphous exanthema, and non-purulent cervical lymphadenopathy, usually unilateral ( ) . several years after the first description, fatalities occurred among children with ks younger than years living in japan, prompting clinicians to reconsider ks's long-term risks related to systemic and necrotizing effects on the vascular endothelium of small-and medium-sized arteries, which have been acknowledged in all guidelines related to the management of ks ( , ) . the most relevant sequelae of ks include variable degrees of damage within coronary arteries in combination with angina, myocardial infarction, ischemic cardiomyopathy, and sudden death; these complications should be preventable with a timely treatment of high-dose intravenous immunoglobulin (ivig), which is the recommended therapeutic strategy in ks ( ) . higher acute phase reactants and younger age at onset of ks are nodal points in determining, respectively, a failure in the response to ivig and an increased occurrence of coronary artery abnormalities ( ) . the prediction of ivig resistance is also crucial in ks patients, as recognizing these high-risk children should consent to start an intensified treatment protocol combined with ivig to prevent coronary injuries ( ) . ks incidence varies widely among different ethnic groups; for instance, in the united kingdom it has stabilized and remains low at . per , population under the age of years. however, general practitioners should be aware that the condition occurs throughout childhood and across the seasons, and-given the potential cardiovascular sequelae-ks should be considered in all children with persistent fever, even in older children and adolescents ( ) . ks is most prominently recognized in japan, korea, and taiwan, reflecting increased genetic susceptibility among asian populations. a recent study reported an incidence of ∼ per , children under years of age in japan ( ) . there is still much controversy about the etiology of ks, though epidemiologic and clinical data suggest that ks might originate from an abnormal response to undisclosed infectious diseases in genetically susceptible children ( ) . there is no agreement whether ks-related infectious agents are of viral, bacterial or fungal origin ( ) , and the underlying immune mechanisms behind ks have not been completely highlighted, remaining only partially known. the absence of a proven unambiguous cause of ks has induced the scientific community to pay attention to other environmental hypothetical triggers, and in particular the composition of the resident intestinal flora as a potential contributor to ks has been evaluated by different research groups. the main aim of this review was to analyze the relationship between the microbial community, or the "microbiota, " and the overall impact of bacterial or viral infections in the potential development of ks. scientific papers have been searched from the electronic databases of pubmed until january ; the retrieving words were "kawasaki disease, " "kawasaki syndrome, " "microbiota, " and "microbiome"; additional reports were identified and analyzed through the specific references cited in the retrieved papers. only papers published in english and those showing evidence-based data were included in our evaluation. the microbiota, a microbial community of trillions of microorganisms and at least , different bacterial species, some eukaryotic fungi and viruses, and which covers every surface of the human body, plays a contributory role in many infections, immune-mediated disorders, rheumatologic diseases, and disorders of the nervous system. the microbiome, on the other hand, is the collection of the whole genome sequences of those microorganisms, consisting of more than , , genes ( , ) . in particular, the gut microbiota is strictly linked to the chronological age of each individual and modulates host physiology and metabolism through different mechanisms. each stage of human life is characterized by a specific intestinal microbial composition: the microbiota that initially colonizes the fetus' intestinal tube consists of aerobic organisms such as enterococcus and streptococcus, is then gradually replaced by anaerobes such as bifidobacterium and lactobacillus and finally reaches the adult composition dominated by bacteroides and firmicutes ( , ) . the fetal microbiota is prone to be conditioned by the type of delivery; the mother's vaginal flora is a relevant source of lactobacillus, prevotella, and bifidobacterium. conversely, a cesarean delivery delays contact with these species, producing a similar-to-skin flora, dominated by staphylococci ( ) . the feeding regimens and food supplements also play a role in modifying the resident flora; a greater complexity is normally seen in infants fed with formula, rather than in breastfed babies who have an "adult-like" structured microbiota with a population rich with bifidobacteria, lactobacilli, and bacteroides ( ). the infant gut microbiota is variable in composition over time and highly changeable during the first year of life, being influenced by specific bacteria to which a baby happens to be exposed, as shown by the resemblance of infants' stool microbial community with mothers' milk and vaginal samples ( ) . thereafter, the infant's intestinal tract progresses toward an extremely dense colonization, ending with a mixture of microbes that is broadly very similar to an adult's intestine. during adulthood, the gut microbiota becomes stable and this intestinal homeostasis remains in equilibrium with the host. food habits influence the composition of the whole intestinal microbiota, as testified by the lower prevalence of bacteroides in those suffering from malnutrition and by different microbiota changes occurring in children with diet-related diseases, such as allergies and obesity ( ) . arumugam et al. identified three distinct enterotypes, namely bacteroides, prevotella, and ruminococcus, which reflect many individual alimentary profiles: bacteroides correlates to a highfat or high-protein regimen, whereas prevotella is associated with higher consumption of fibers and simple sugars ( ) . more recent data have consented to unify bacteroides and ruminococcus enterotypes due to the large similarity between the two ( ) . it is well-established that early events of birth, environmental factors during infancy, sex hormones, diet, body weight, and use of antibiotics can undoubtedly differentiate the composition of the microbiota ( , ) . there are no ideal culture methods to give us a real overview of the complete intestinal flora, though molecular methods, e.g., dna microarrays with comprehensive coverage of most bacterial taxa represented in the available database of small subunit ribosomal rna gene sequences, should allow the characterization of most taxonomic groups of the intestinal bacteria ( , ) . the ancient symbiosis between the human gastrointestinal tract and its resident microbiota involves diverse reciprocal interactions between the microbiota itself and the host, with relevant consequences for human health and physiology. the quality of the microbial flora has an impact on the maintenance of health and also on prevention of diseases. indeed, there are numerous roles carried out by the intestinal ecosystem, as the stimulation of angiogenesis, control of host fat storage and protection against other pathogens. in particular, the microbiota influences the formation and progress of regulation of both innate and adaptive immunity in close interaction with the intestinal mucosal immune system. the intestinal mucosa may be considered as an immunological niche as it hosts a complex immune-functional organ comprised by t cell subpopulations, neutrophils, macrophage-dendritic cells, enterocytes (that possess tight intercellular junctions) and their related anti-and pro-inflammatory cytokines as well as several other mediators of inflammation or antimicrobial peptides, defensins, and secretory immunoglobulin a (iga) ( ) . the intestinal microbiota may also have direct or indirect effects on the natural course of viral infections, interacting with viral particles and leading to differences in either pathogenicity or anti-viral immune response through recruitment and activation of several t cell subpopulations ( ) . a large amount of data has also depicted the relevance of gut microbiota-immune system cross-talk in several diseases, and indeed an "imbalance" of the intestinal flora has been shown in patients with atopic diseases and various noninfectious diseases, including metabolic disorders, chronic inflammatory bowel disease (ibd), irritable bowel syndrome, pancreatic diseases, atherosclerosis, and rheumatoid arthritis ( , ) . furthermore, the gut microbiota, interacting with pattern recognition receptors (prrs), signaling receptors that can recognize molecular structures of pathogens and activate the cascade of innate immunity, plays a crucial role in maintaining the homeostasis of the innate immunity responses in the gut, and leaks in the intestinal mucosal barrier lead to the translocation of bacterial products into portal circulation which promotes systemic inflammation ( ) . in addition, the microbiota can maintain a segregation between intestinal mucosa and bacteria via prrs, though pathogens might usurp innate signals to their advantage ( ) . several immunologic, metabolic and nutritional processes are normally controlled by the intestinal microbiota, and changes in the local microbial communities have been linked to chronic low-grade inflammation ( ) . alexander et al. demonstrated that the microbiota has specific effects on adaptive immunity, such as the induction of regulatory effector cd + cells and production of cytokines and antimicrobial factors, influencing the individual response to various environmental stimuli, as seen in ibd, crohn's disease and ulcerative colitis ( ) . in a recent work, schwiertz et al. have shown microbiota changes characterized by decreased numbers of faecalibacterium praunsitzii and increased numbers of escherichia coli in ibd ( ) . additionally, in celiac disease de palma et al. have described a difference in the microbiota composition with a reduction of bifidobacterium, clostridium histolyticum, clostridium lituseburense, faecalibacterium prausnitzii, and an abundance of bacteroides and prevotella strains ( ) . a significantly higher biodiversity in coeliac children's duodenal mucosa was demonstrated by schippa et al. who also highlighted that the possible pathophysiological role of such microbial differences needs further characterization ( ) . in addition, many immune phenomena were shown to deteriorate under the effect of changes in the microbiota, as revealed by the correlation among intestinal bacterial overgrowth, increased permeability, and development of non-alcoholic steatohepatitis ( ) . much interest has also been paid to the role of the microbiome in the development of "sterile" inflammation, and recent studies have proved that either depletion of the microbiota or changes in the diet and in the gut microbiome might lead to the improvement of inflammasome-mediated manifestations of autoinflammatory disorders, which are caused by dysregulation of specific components of innate immunity ( ) . these diseases can be subdivided into monogenic and multifactorial disorders, with the former being caused by mutations of genes involved in the regulation of the innate immune system and the latter by a combination of genetic background and environmental factors ( ) ( ) ( ) . given the evidence for the role of the intestinal microbiota in the inflammatory state of ibd, atopic diseases and numerous non-infectious diseases, it has been speculated that intestinal microbial agents might also play a trigger role in the development of other inflammatory disorders which do not have a clearly defined etiology, such as ks. the etiology of ks remains obscure, although clinical and epidemiological features suggest a primary infectious cause. indeed, a self-limited and generally nonrecurring illness that manifests itself by fever, rash, mucositis, conjunctival injection, and cervical adenopathy fits well with an infection. more precisely, clinical features of ks resemble some peculiar infectious diseases, such as streptococcal infections, staphylococcal toxic shock syndrome, and atypical measles ( , ) . additionally, age distribution, winter-spring seasonality, high rates of ks in siblings, and occurrence of community outbreaks are suggestive of a transmissible childhood disease, though many efforts with conventional bacterial and viral cultures and serological methods as well as animal inoculation studies have failed to identify a final unique infectious agent of ks ( ) . reported non-infectious factors associated with ks include carpet shampoo, preexisting eczema, environmental pollution, and house dust mites ( ) . the higher incidence of ks in asian populations, presence of familial clustering, and elevated risk of recurrence vs. the risk of a first episode in ks-naïve children are all strong evidence of a genetic contribution to ks susceptibility, probably in association with an infectious trigger ( , ) . however, ks does not appear easily transmittable and does not respond to any antibiotics: these characteristics should contradict a primitively infectious pathogenesis of the illness. the most current theory about the pathogenesis of ks is that the disease results from an exaggerated immune response toward environmental stimuli occurring in a genetically susceptible child ( ) . the prominent role played by the immune system in ks is confirmed by the high number of studies revealing the activation of neutrophils and multiple immune cells with overproduction of pro-inflammatory cytokines, including tumor necrosis factor (tnf)-α ( , ) . manlhiot et al. have very recently proposed a new pathogenetic model of ks in which the disease risk is determined by concurrent interacting processes: genetic susceptibility, habitual exposure to allergens, atmospheric biological particles, and infectious agents ( ) . a study exploring the immune responses during the acute phase of ks showed increased levels of lipopolysaccharide (lps) bound to the surfaces of circulating neutrophils via cd receptor and found markedly increased levels of the soluble cd in the plasma ( ) . furthermore, chen et al. ( ) demonstrated that nlrp inflammasome activation is associated with the development of coronary arteritis in a mouse model of ks, and that infusion of visfatin, a major injurious adipokine, can activate nlrp inflammasome and increase interleukin (il)- production, leading to enhanced endothelial dysfunction. these novel molecular mechanisms of vasculitis mediated by inflammasome activation open more specifically the road to innate immunity pathways in the interpretation of ks ( , ) . unchallengeable proof that an infection is the starting point of ks is not available. different epidemiologic studies have supported this hypothesis, based on documented infections by various microorganisms in many cases of ks. further clues are the occurrence of ks in epidemics, higher incidence during spring and winter and early age at which the disease might be acquired, that is months to years ( ). striking perturbations of many immune pathways occur during the acute phase of ks, which determines a multi-cytokine cascade in the vascular endothelium with focal disruption of small-and mediumsized vessels. however, the exact key steps leading to coronary arteritis are still far from being clarified, though endothelial cell activation; prolonged start-up of neutrophils, cd + monocyte/macrophages and cd + lymphocytes; production of oxygen intermediates and lysosomal enzymes; and oligoclonal iga response by plasma cells all appear to be involved ( , , ( ) ( ) ( ) . the contribution of viruses to ks has been suggested by ultrastructural studies which found cytoplasmic inclusion bodies containing rna of viral origin in the bronchial epithelia ( ) as well as by studies revealing the detection of intracytoplasmic inclusion bodies containing viral proteins and nucleic acid aggregates ( , ) . recent investigations have supported the hypothesis that immune responses in ks are oligoclonal rather than polyclonal (as found typically in superantigen-driven responses), and that iga plasma cells play a crucial role. indeed, higher levels of iga have been found in the vasculature of patients with previous ks, indicating an antigen-driven response against an etiologic agent which might have a respiratory or gastrointestinal portal of entry ( ) . about half of all ks patients might have one or more respiratory viruses detected by polymerase chain reaction, without any particular predominance, but a positive respiratory viral test or presence of respiratory symptoms at the time of presentation should not be used to exclude the diagnosis of ks ( ) . other studies have investigated a potential relationship of ks with coronaviruses, though without finding definite proof of cause and effect ( , ) . many viruses have been suggested to be implicated in the pathogenesis of ks, such as adenovirus, parvovirus b , rotavirus, h n influenza virus, epstein-barr virus, herpesvirus , coxsackie b virus, parainfluenza virus type , measles virus, dengue virus, human immunodeficiency virus, and varicella-zoster virus, but no significant differences emerged from case-controlled studies ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . one of the most difficult infections that are harder to distinguish from ks is caused by adenovirus, due to its frequent incidental detection in ks patients and frequent laboratory finding of increased inflammatory markers ( ) . in particular, adenovirus has been detected in . and % of cases with complete and incomplete ks, respectively, by jaggi et al. ( ) . there are reports that have emphasized the possible relationship of cytomegalovirus (in one child from turkey) and human bocavirus (in a french cohort of patients) with ks through serologic tests and molecular techniques, but this link might be only casual and misleading ( , ) . regarding the bacterial origin of ks, it is debated if the infectious agents might be conventional bacteria or bacteria with superantigen activity. superantigens (sags) are the most powerful t cell mitogens ever discovered, with potent immunostimulatory actions that simultaneously activate the major histocompatibility complex class ii molecules and t cell receptors, leading to massive activation of various immune cells. matsubara et al. conducted a case-control study with a serological approach based on enzyme-linked immunosorbent assay and measured serum antibodies against staphylococcal enterotoxins, including tsst- , and streptococcal pyrogenic exotoxin (spe), such as spe-a. they showed that ks patients had significant elevation of igm antibodies against one or more of five sags throughout the first to the fourth disease week ( ) . in a further paper the same authors analyzed the studies regarding the role of sags produced by staphylococcus aureus and streptococcus pyogenes in the pathogenesis of ks, finding numerous sags implicated, which brought the total number of the known staphylococcal sags to over and streptococcal sags to ( ) . the most recent work that analyzed the relationship between sags and ks examined different sag derived from streptococcus pyogenes in the stools of patients with ks. stool specimens were obtained from patients with ks during the acute phase and age-matched healthy children. the authors examined genes related to five sags-spe-a, spe-c, spe-g, spe-j, and tsst- using polymerase chain reaction; throat and stool cultures were assessed to evaluate the presence of streptococcus pyogenes and staphylococcus aureus in ks patients. they reported that at least one of the sag-related genes was detected more frequently in the stools of children with ks ( ). furthermore, among patients with ks, developed concurrent pneumonia and of these ( . %) had high titers of anti-mycoplasma pneumoniae antibody (> : ), suggesting a potential role of mycoplasma pneumoniae in ks and the importance of anti-mycoplasma treatment ( ) . another study has reported a possible role of fungal infections in the development of ks in japan, san diego, and the island of hawaii through an active role of tropospheric wind patterns leaving from central asia, in which toxins of candida species are aerosolized ( ) . in addition, due to the observation that candida albicans-derived substances, such as candida albicans watersoluble fraction (caws), induce a coronary arteritis in mice similar to that observed in ks, sato et al. evaluated the role of a free-β-glucan diet on caws-induced vasculitis and found that quality of diet might affect the progression of systemic vasculitis ( ) . caws should act as a pathogen-associated molecular pattern in mice and activate lectin pathway of complement by binding to mannose-binding lectin and inducing an acute inflammatory reaction in the vascular system ( , ) . there are three main reasons that led us to postulate a role of the microbiota in ks. the first is the unsatisfactory microbiological data that limited the association between infections and ks and a lack of evidence of any clear relationship between one or more pathogens with this illness. second, the most frequent bacteria or viruses associated with ks have a higher prevalence in the overall pediatric population, but only a limited number of children will develop the disease. third, the association of genetic predisposition and environmental factors in the pathogenetic process of ks makes ks itself similar to other multifactorial diseases. currently, the majority of data has found that the composition of the gut microbiota in ks patients differs from healthy subjects. lee et al. have postulated that the immune system should lose tolerance toward a part of the resident intestinal flora and that environmental factors, i.e., a western lifestyle or improved public hygiene systems, could transform the commensal flora into a pathogen one, as observed in different gastrointestinal disorders ( ) . a recent study about the resident flora and ks was focused on throat flora. horita et al. ( ) assumed that throat flora could be a reservoir of microorganisms triggering ks and, following this hypothesis, they investigated throat microorganisms of ks patients for their content and individual sag activity. in particular, they collected throat swabs at the start of ivig infusion in ks patients and compared them with non-ks controls (displaying other febrile illnesses). the results showed no difference in the throat flora between ks and febrile controls even in the mean mitogenic activity of bacteria isolated ( ) . the gastrointestinal tract could be one of the primary sites of entry of bacterial toxins in children with ks, and a perturbation in the intestinal microbiota has been linked to the disease's pathophysiology in another study by yamashiro et al. ( ) who investigated the microflora of the small intestine in japanese patients with ks. the range of bacterial species isolated from jejunal biopsies was characterized by a wider variety of gram-positive cocci in the acute phase of ks. notably, kinds of streptococci (streptococcus salivarius, streptococcus mitis, streptococcus oralis, streptococcus sangius, and gemella haemolysans) and kinds of staphylococci (staphylococcus capitis and staphylococcus hyicus) were isolated only from ks patients, suggesting that some antigens inducing a delayed-type hypersensitivity reaction in the mucosa might inundate the body by breaching the barrier of the small intestinal mucosa of ks patients ( ) . in fact, nagata et al. ( ) investigated cell surface phenotypes of mononuclear cells and enterocytes in the jejunal mucosa of japanese patients with ks and in patients with diarrhea due to cow's milk protein intolerance. both hla-dr+cd + and dr+cd + cells were significantly increased, and cd + cells significantly reduced in the lamina propria of ks patients during the acute phase compared with patients with cow's milk protein intolerance. these cell patterns returned to normal in the convalescent phase of ks. the authors concluded that a delayed-type hypersensitivity reaction was indeed present in the small intestinal mucosa of ks patients ( ) . due to the fact that the gastrointestinal tract is the largest interface between microbial factors and their host, containing the largest proportion of bacteria and the largest amount of lymphoid tissue in the body, it was hypothesized by eladawy et al. that the intestinal milieu could be altered in children with ks, and indeed ks patients have a higher incidence of gastrointestinal symptoms and complications ( ) . specifically, takeshita et al. ( ) evaluated patients with ks, patients with acute febrile diseases and healthy children, finding that the incidence of lactobacilli isolated from ks patients ( / , %) was significantly lower (p < . ) than in the other cohorts. moreover, no significant differences in the presence of staphylococcus, streptococcus, enterococcus, enterobacteriaceae, bifidobacterium, clostridium, veillonella, or bacteroides were found among the three groups. conversely, the presence of eubacterium and peptostreptococcus was significantly higher in ks patients than in patients with other febrile diseases (p < . and p < . , respectively), though no significant differences were observed between ks patients and healthy children ( ) . this observation confirmed that the majority of lactobacillus species are anti-inflammatory and beneficial for health, particularly during the first years of life, due to their action in protecting against colitis, reducing pro-inflammatory cytokines such as tnf-α, il- , or il- , and increasing the subsets of regulatory t cells ( ) . in fact, several studies have largely described the specific action of lactobacilli in maintaining the epithelial homeostasis of the gut and their striking anti-inflammatory potential ( , ) . nagata et al. ( ) also studied the role of the gut microbiota in ks pathogenesis via sags and heat shock proteins (hsps) produced by gut bacteria: the authors evaluated sags and hsps released by microorganisms isolated from the jejunal mucosa of children with ks compared with age-matched healthy controls, identifying strains of gram-negative microbes from patients with ks, which produced a large amount of hsp , having the power to induce an over-secretion of proinflammatory cytokines. they also identified strains of grampositive cocci with sag properties which induced the expansion of vβ t cells in vitro ( ) . this microbiological analysis disclosed different causative bacteria with a final common pathway of immune activation, which might contribute to the final development of ks. in order to evaluate the differential microbiota composition of ks patients, kinumaki et al. ( ) performed a metagenomic analysis using non-culture-based methods on feces. their study included ks patients ( males and females, aged - months, with a median age of months); the time of admission to the study was defined as the acute phase, while - months after the onset of ks was considered as the nonacute phase. the authors collected a total of samples− samples each for both acute and non-acute phases. it was demonstrated that the genera ruminococcus, roseburia, and faecalibacterium were mostly predominant during the nonacute phase, while a higher presence of streptococcus spp., including streptococcus pneumoniae, pseudopneumoniae, mitis, oralis, gordonii, and sanguinis, was detected in the fecal samples during the acute phase ( ) . this novel interpretation of a disease can be shared by other conditions, as liver cirrhosis and sjögren syndrome, in which major changes of gut microbiota with higher proportions of streptococcus spp. have been demonstrated ( , ) . these findings taken as a whole suggest that many other immune-mediated disorders are likely to be connected to an abnormal bacterial colonization of the intestinal tract, with a main role for streptococcus spp., and that changes in the gut microflora composition might promote systemic and extra-intestinal inflammation. therefore, all presented studies confirm the concept that an imbalance in the gut microbiota might directly or indirectly interfere with the normal functions of the immune system, and that the interaction with other environmental factors, mainly infectious agents, might lead to the final development of ks. table shows the characteristics of the microbiota in children with ks, as emerging by the most relevant studies dedicated to this issue. as no etiologic agent has ever been accused of being directly involved in the etiology of ks, basic research evaluating the pathogenic mechanisms of this disease will probably provide better therapies and probably consent to identify the most vulnerable hosts and protect them. a potential relationship between eubiosis and the protean functions of immunity has been contemplated by different studies, and conversely a relationship should exist between dysbiosis and immunity dysfunction shown by various diseases ( ) . it is strengthened that heterogeneity and abnormalities in the intestinal microflora composition may trigger or contribute to the development of specific diseases, and an increasing amount of research and microbiologic observations have led to a role of the intestinal microbiota for ks to be postulated. while we are becoming convinced that a role of the microbiota is likely, we do not know exactly the basic mechanism of how the microbiota should act. the principal obstacle of today's medical literature is to understand if the microbiota modification during ks can cause the disease or if it is a mark and a consequence of the disease itself, and if modulating the microbiota/microbiome might represent a future target of therapy in ks. this raises the hypothesis of targeting intestinal microflora in order to restore eubiosis through the rational use of antibiotics, xenobiotics, probiotics and nutrients. while multicenter trials and registries may allow us to improve the general outcome of ks, further in-depth analysis in larger cohorts of affected children will probably unravel the tangled pathogenesis of this mysterious disorder and find new targets for identifying disease-modifying agents or more specific therapies. se, ip, and dr contributed to all stages of the preparation of this manuscript, including conception and writing. all authors approved the final submitted version of the manuscript. viral infections associated with kawasaki disease epidemiology, clinical presentation, and outcomes of kawasaki disease among hospitalized children in an inner city hospital before and after publication of the american academy of pediatrics/american heart association guidelines for treatment of kawasaki 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heat shock proteins and superantigenic properties of bacteria from the gastrointestinal tract of patients with kawasaki disease characterization of the gut microbiota of kawasaki disease patients by metagenomic analysis altered mucosal microbiome diversity and disease severity in sjögren syndrome alterations of the human gut microbiome in liver cirrhosis eubiosis and dysbiosis: the two sides of the microbiota key: cord- -mbrw og authors: flego, michela; frau, aldo; accardi, luisa; mallano, alessandra; ascione, alessandro; gellini, mara; fanunza, elisa; vella, stefano; di bonito, paola; tramontano, enzo title: intracellular human antibody fragments recognizing the vp protein of zaire ebola filovirus inhibit the protein activity date: - - journal: bmc biotechnol doi: . /s - - - sha: doc_id: cord_uid: mbrw og background: ebola hemorrhagic fever is caused by the ebola filovirus (ebov), which is one of the most aggressive infectious agents known worldwide. the ebov pathogenesis starts with uncontrolled viral replication and subversion of both the innate and adaptive host immune response. the multifunctional viral vp protein is involved in this process by exerting an antagonistic action against the early antiviral alpha/beta interferon (ifn-α/β) response, and represents a suitable target for the development of strategies to control ebov infection. phage display technology permits to select antibodies as single chain fragment variable (scfv) from an artificial immune system, due to their ability to specifically recognize the antigen of interest. scfv is ideal for genetic manipulation and to obtain antibody constructs useful for targeting either antigens expressed on cell surface or intracellular antigens if the scfv is expressed as intracellular antibody (intrabody) or delivered into the cells. results: monoclonal antibodies (mab) in scfv format specific for the ebov vp were isolated from the eth- library of human recombinant antibodies by phage display technology. five different clones were identified by sequencing, produced in e.coli and expressed in cho mammalian cells to be characterized in vitro. all the selected scfvs were able to react with recombinant vp protein in elisa, one of the scfvs being also able to react in western blot assay (wb). in addition, all scfvs were expressed in cell cytoplasm as intrabodies; a luciferase reporter gene inhibition assay performed in a cells showed that two of the scfvs can significantly hamper the inhibition of the ifn-β-induced rig-i signaling cascade mediated by ebov vp . conclusion: five antibodies in scfv format recognize an active form of ebov vp in elisa, while one antibody also recognizes vp in wb. two of these scfvs were also able to interfere with the intracellular activity of vp in a cell system in vitro. these findings suggest that such antibodies in scfv format might be employed to develop therapeutic molecules able to hamper ebov infections. ebola hemorrhagic fever caused by the ebov is one of the most aggressive zoonoses affecting humans, leading to death within a few days of the exposure [ ] . six species of ebov are known to date, named after the geographical region in which they were first isolated: bundibugyo, reston, sudan, taï forest (formerly côte d'ivoire ebov), zaire and bombali ebov. sudan, taï forest, and zaire ebov are responsible for outbreaks in humans, whereas reston ebov infects non-human primates, and bombali virus was recently discovered in bats [ , ] . fatal ebov infections are characterized by rapid viral replication combined with an inadequate antiviral response. hallmarks of fatal cases are immune suppression with t cells levels below the normal level, no cd t cell activation, delay of antibody response in the blood, and high viremia ( genome copies/ml serum). the ebov pathogenesis starts with the subversion of both innate and adaptive immune response and the consequent induction of harmful inflammatory responses and tissue necrosis due to disseminated infections [ , ] . ebov has a linear, single-stranded, negative rna genome about , nucleotides in length. it is composed of seven genes coding for eight proteins in this order: np (encoding the nucleoprotein), vp , vp , gp (encoding the glycoproteins), vp , vp , l (encoding the polymerase). the gp gene codes for the two molecular forms gp and gp , generated by rna editing [ ] . vp is a conserved multifunctional protein which is a cofactor of the viral rna polymerase complex along with the np, vp , and l protein. its activity starts at an early stage of the ebov infection; it is also a doublestranded rna-binding protein shown to be implicated in hampering the innate immune response [ ] by blocking the ifn-mediated antiviral activity through multiple inhibitory effects which include disruption of the rig- pathway by preventing irf- phosphorylation [ , ] , and inhibition of activation of the ifn-inducible dsrna and dicer-dependent protein kinase r [ ] . antibody phage display technology makes it possible to select from an artificial immune system human antibodies in scfv format capable of specifically recognizing an antigen of interest. scfvs, consisting of the vh and vl chain regions of a whole immunoglobulin (ig), are the smaller portion still retaining the binding properties of the parental ig. this format is ideal for genetic manipulation in order to obtain antibody constructs potentially useful for diagnostic and therapeutic applications [ ] . furthermore, scfv antibodies can be expressed inside the cell as intrabodies so as to bind to their intracellular target antigen [ ] . the two main mechanisms underlying the efficacy of intrabodies are: ) knockdown of the activity of cytosolic antigens through cytosolic intrabodies [ ] [ ] [ ] ; ) diverting of antigens from their natural intracellular compartment by scfv binding due to an extra-signal for intracellular localization [ ] [ ] [ ] . intrabodies can be used to reveal the function of proteins by interfering with their function, although the possibility of targeting intracellular antigens gives them a therapeutic potential for a number of viral infections [ , ] , neurological diseases [ , ] and cancers [ , , , ] . this study reports the selection by phage display and characterization of different human scfv antibodies binding to an active form of the zaire ebov vp [ , ] . the ability of these scfvs, expressed as cytosolic intrabodies, to reverse the inhibition of type i ifn induction by intracellular expression of vp was tested by a luciferase reporter gene inhibition assay in a cells treated with dsrnas [ ] . in order to isolate antibodies specific for the recombinant vp expressed in e.coli and purified in an active form [ , ] , an approach based on the phage display technology was used. in the eth- library which we used, the diversity (about clones) has been introduced in the complementary-determining region (cdr ) of both the variable heavy chain (vh) and variable light chain (vl) domains [ ] . to recover antigen-specific antibody phages, an aliquot of the eth- antibody library containing cfu phage was used for the panning procedure as described elsewhere [ , ] . in fig. soluble scfvs derived from iptg-induced colonies were screened by elisa to find those specific for the vp protein. all the e. coli colonies corresponding to the clones exhibiting an od λ value in elisa higher than . , were grown and subjected to dna extraction and sequence analysis. several clones had identical nucleotide sequences and five different clones, namely b , a , e , f and h , were identified; the amino acid composition of the complete sequences of the vh and vl domains is shown in fig. along with the schematic representation of a scfv gene in the phagemid cassette. the scfv reactivity towards vp was further characterized by elisa (fig. , panel a) and wb (fig. , panel b) using vp recombinant antigen. the protein glucose oxidase (go) and an anti-go scfv for detection were used as negative controls. the anti-vp reactivity in elisa was confirmed for all b , a , e , f and h scfvs, while in wb the positivity was only observed for scfv a , which reacted with a kda protein identified as the recombinant his-tagged vp protein also detected by the anti-his mab. the scfvs b , e , f , h recognized their antigen in elisa but showed no reactivity in denaturing conditions of wb, suggesting that they probably recognize conformational epitopes. for its part, a is still reactive in wb and probably recognizes a linear epitope. the specificity of the anti-vp reactivity of b , a , e , f and h scfvs is confirmed by the observation that they did not react with the irrelevant go antigen either in elisa or in wb (fig. ) . in order to use the scfvs in the ebov vp luciferase reporter gene inhibition assay, the scfv genes selected were pcr amplified with opportune oligonucleotides and cloned into the ptarget vector for expression in the eukaryotic system. in view of the cytoplasmic vp localization, it was not necessary to provide the scfvs with signal sequences for expression in specific cell compartments. transfection experiments were performed in the cho cells to evaluate the functionality of the scfv ptarget constructs as described in methods. the a , h , b , f and e constructs were all able to express scfvs of the expected molecular mass in eukaryotic cells (fig. , panel a) . evaluation of the anti-ebov vp scfvs ability to restore the ifn-β activity by a luciferase reporter gene inhibition assay to evaluate the capability of the scfvs b , a , e , f and h to block the vp activity, the cell-based miniaturized luciferase reporter gene inhibition assay, previously described [ ] , was used. the assay measures the capacity of the vp expression to inhibit the ifn-β induced by dsrna treatment, in a cells. before performing the ebov vp luciferase reporter gene inhibition assay using the scfvs, it was crucial to exclude that the expression of an irrelevant scfv could influence the ifn-β induction, either in the absence or in the presence of vp expression. to this end, a cells, transfected with the pgl ifn-β luc and the pcdna -ebov-vp expression plasmid, were co-transfected with the irrelevant anti-go scfv expression plasmid. further, to exclude any non-specific effect due to the transfection procedure, the cells were co-transfected in parallel with the pgl ifn-β luc expression vector and an empty pcdna vector. the luciferase signal emitted in the case of pgl ifn-β luc and anti-go scfv concurrent expression, was comparable to that obtained with the ifn-β positive controls. also, in the case of ebov vp and anti-go scfv simultaneous expression, the measured ifn-β signal was comparable to that obtained with the ebov vp expressed alone (fig. , panel b) . the results confirmed that ifn-β induction was affected by neither the transfection procedure nor the irrelevant scfv either in the presence or in the absence of vp expression. next, we tested all the scfv ptarget constructs in the dsrna rig-i-mediated luciferase reporter gene inhibition assay. two of the five scfvs tested, f and e , showed a significant ability (p = . and p = . , respectively) to subvert the inhibition of the ifn-β production generated by dsrna, after the vp expression. by contrast, scfv h , a and b showed no ability to counteract the inhibition of ifn-β induction mediated by ebov vp in the cellular assay (fig. , panel c). competitive elisa using the scfv-expressing phage to verify whether the two different scfvs e and f , able to subvert the inhibition of the ifn-β production, targeted different epitopes, we performed a competitive elisa (fig. ). this relies on detection of the scfv-expressing phage particles that compete with soluble non-phage-fused scfvs for binding to the antigen immobilized on elisa plate. it was not possible to detect the soluble non-phage-fused scfvs using the anti-tag flag ab because the tag is also present on the phage. to determine the best scfv expressing-phage concentration for competition tests, identified as × tu/ml, we first performed a phage elisa experiment in which different scfv-expressing phage concentrations were tested on vp coated plates (data not shown). in a competitive assay, elisa plates coated with vp or with the control antigen go were blocked; they were then incubated with purified scfv-expressing phage both in the absence and in the presence of the competitor soluble non-phage-fused scfv at the maximum concentration of μg/ml. the binding of the scfv-expressing phages was measured. the detection step was via the phage coat protein using an anti-m mab conjugated to hrp. the anti-go scfv-expressing phage clone was assayed against the anti-go soluble nonphage-fused scfv as a positive control for competitive measurement (fig. , panel a) . it was found that μg/ml of soluble non-phage-fused scfv could inhibit the binding of the scfv-expressing phage in a dose-dependent manner. binding in the presence of specific anti-go soluble non-phage-fused scfv was compared: to binding in the presence of e anti-vp soluble non-phage-fused scfvs (p value = . ); to binding in the presence of f anti-vp soluble non-phage-fused scfvs (p value = . ); to binding in the absence of soluble non-phage-fused scfvs (p value = . ) using student's t-test. next, we assayed the binding of e scfv-expressing phages both in the absence and in the presence of the competitor soluble non-phage-fused scfv against itself as an intrinsic positive control, against anti-go soluble non-phage-fused scfv as a negative control and against f soluble non-phage-fused scfv for competitive measurement. the inhibiting activity was determined at a single fixed concentration of μg/ml. f soluble nonphage-fused scfv shows a clear competitive effect at the concentration used (fig. , panel b) . binding in the presence of f soluble non-phage-fused scfv was compared to the binding in the presence of anti-go soluble non-phage-fused scfv (p value = . ) and in the absence of soluble non-phage-fused scfvs (p value = . ) using student's t-test. as a further confirmation, we performed a one-shot experiment by detecting the competition suffered by the f scfv-expressing phages when co-incubated with e soluble non-phage-fused scfvs and with the control anti-go soluble non-phage-fused scfv at the concentration of μg/ml. also in this case we observed that the control anti-go soluble non-phage-fused scfv has no competitive binding effect. the e soluble non-phage-fused scfv competes with the binding of f scfv expressing-phages. binding in the presence of e soluble non-phage-fused scfvs was compared to the binding in the presence of anti-go soluble non-phage-fused scfv (p value = . ) and to binding in the absence of soluble non-phagefused scfvs (p value = . ) using student's t-test (data not shown). the urgency to find effective counter measures for the ebov disease (evd) was strengthened by the west africa outbreaks occurring in - , which resulted in , cases of ebola with , deaths [ ] , pushing the international scientific community to investigate the widest possible range of defense strategies to counteract the virus. however, the current ebola outbreak, which started in may in democratic republic of the congo (drc) and has caused cases and deaths to date [ ] , received only minor benefits from the use of new diagnostic assays, vaccines and drugs. the reason is that to control ebola outbreaks, transversal coordination between health facilities and communities is essential to achieve rapid isolation of the cases of the disease and to stop the transmission chain. detection of new cases at an early stage by rapid diagnostic tests and ring vaccination strategy with experimental vaccines on volunteers are therefore crucial for controlling ebov in the current drc outbreak [ ] . due to the variable onset of antibody response in the ebola-infected subjects, serology is not used in the acute evd diagnosis. conversely, virus and viral proteins accumulate in blood to detectable levels within a few days from disease onset. molecular tests based on the detection of viral proteins were developed and proved to be effective for diagnosis in acute infection [ ] . currently, in the case of suspected ebov infection, the world health organization (who) recommends a list of validated tests for detection of either viral rna by rt pcr or viral antigens by immunological tests [http://www.who. int/medicines/ebola-treatment/emp_ebola_diagnostics/en/]. most of the antigen-capture tests used in national reference laboratories [ ] utilize mabs generated in mice immunized with the recombinant np [ ] , vp [ ] or gp [ ] ebov proteins. during the recent outbreak, lateral flow immunoassays (lfis) emerged as powerful tools for rapid antibody-mediated antigen-capture practicable at the point of care [ ] . this confirmed the advantages of tests based on antigen-antibody reaction over rt-pcr methodology, which requires significant laboratory infrastructures often lacking in low-income countries. furthermore, in order to control the transmission-chain of the infection and limit virus spread, it is important to have tests that can be easily automated; as such, antigen capture tests meet this requirement. ebov vp is a validated drug target for which only a few small molecules have been reported to be active [ , , ] , albeit no drug has yet been approved. here, we present mabs in scfv format, which are able to react with the zaire ebov vp protein. this is a key viral protein whose action starts at an early stage of the infection and is based on interference with the host immune response by blocking the ifn-mediated antiviral activity. the antibodies presented here enrich the list of available anti-vp antibodies. they could be used individually or in combination to develop novel reagents for ebov vp detection and therapeutics. regarding therapy, pools of neutralizing antibodies have been used in passive immunization of individuals with acute infection [ ] ; nevertheless, antibodies specific for the vp would act with a different mechanism with respect to neutralizing antibodies targeting surface glycoproteins. recent studies showed that targeting vp by either nucleic acid mimics or sirnas, provides protection against the ebov infection in murine [ ] as well as in non-human primate models [ ] . however, the effectiveness of antibodies targeting the vp has not yet been demonstrated in vivo. here, we show that two out of five scfv antibodies selected against the vp significantly hindered the inhibition of the rig-i signaling cascade mediated by vp , in a cellular system. we characterized the binding of these two scfv clones to the recombinant vp antigen by competitive elisa, and found that their epitope is at least partly shared. a more detailed analysis of the binding epitopes could further define whether it is a total or partial sharing and which antigenic region of ebov vp is involved in the inhibition of the interferon pathway. regarding the other anti-vp scfvs selected, we cannot exclude that the observed lack of functionality is due to incorrect scfv folding in the cytoplasmic environment. as antibodies are usually produced in an oxidizing biochemical environment with the help of er-based chaperones, only a fraction of naïve antibodies can be folded correctly in the reducing cytoplasmic environment which prevents the formation of disulfide bridges. however, many examples of successful intrabodymediated proteins knockdown in vitro, obtained using cytosolic intrabodies, have been reported in literature [ ] . scfvs selected in the extracellular environment were previously reported to work intracellularly [ , , ] depending on intrinsic biophysical characteristics such as stability, mainly ascribable to the scaffold. however, several methods have been developed to address the issue of cytosolic intrabodies functioning [ ] . interestingly, anti-vp scfvs able to interfere with vp activity, were recently isolated from a phage library different from the eth- and were linked to a cell-penetrating peptide for intracytoplasmic delivery [ ] . therefore, although we used a different delivery system, our data strengthen the idea that intracellular antibodies in scfv format can be used to counteract ebov vp activity. scfvs against different intracellular ebov targets could be used either to develop a well-defined cocktail of antibodies with different specificities or also in combination with other drug molecules for therapeutic purposes, provided that an appropriate delivery system is developed. furthermore, the vp amino acid sequence is highly conserved among the zaire ebov isolated in several outbreaks (> . % amino acids identity). therefore, it can be hypothesized that the scfvs selected retain a broad-spectrum activity. nevertheless, the efficacy of these new antibodies should be further evaluated either in ebolavirus infected cells or in animal models of ebolavirus infection. five scfv antibodies against an active form of the zaire ebov vp were isolated and characterized. their specific reactivity in elisa and wb suggests the possibility of developing novel reagents for ebov vp detection during the virus life cycle. the two anti-vp scfvs f and e proved to be able to interfere with the vp -depending inhibition of ifn activity in a cell system, so suggesting that such antibodies also represent potential therapeutic agents. further investigations into an ebov infection system in vitro and in animal models are necessary to validate these reagents. the synthetic library of recombinant human antibodies (eth- ) consists of about scfv polypeptides displayed on the surface of the m phage. the library was built by random mutagenesis of the complementarity-determining region (cdr ) of the variable domains of both the heavy (vh) and light (vl) chain of immunoglobulins, using only three antibody germline gene segments (dp for the vh, dpk , and dpl for the vl). in the vh, diversity was created by randomizing four to six positions replacing the pre-existing positions to of the cdr ; in the vl, diversity was obtained by randomizing six positions ( to ) of the cdr [ ] . an aliquot of the eth- library, containing cfu phage, was used to isolate specific human antibodies in scfv format against the recombinant vp protein ( ) . immunotubes (nunc maxisorp; denmark) were coated overnight (on) at room temperature (rt) with purified recombinant vp protein ( μg/ml in pbs). after panning, phages were eluted with ml of mm triethylamine and the solution was immediately neutralized by adding . ml of m tris-hcl ph . . the eluted phages were used to infect an e. coli tg strain in a log phase, and amplified for the next round of selection, as described in flego et al. [ ] . three rounds of panning were performed to recover vp -specific antibody phages from the eth- library. for the preparation of soluble anti-vp scfvs, individual colonies were grown in flat bottomed wells (nunc) for h at °c in μl of . % glucose xyta medium and induced with μl of mm iptg/ xyta medium. the following day, the plates were spun down at g for min, and the supernatants containing soluble scfvs were recovered and tested for specificity with purified vp in elisa and wb, as described in flego et al. [ ] . plasmid dna from individual bacterial colonies clones was extracted using the quiaprep spin miniprep kit and subjected to enzymatic restriction; sequence analysis of the cdr regions was then performed with an automated dna sequencer (biofab, roma, italy) using the fdseq ( ′-gaa ttt tct gta tga gg- ′) and pelbback ( ′-agc cgc tgg att gtt att ac- ′) primers. the scfv gene clones reacting with the recombinant vp in elisa, were pcr amplified using the following primers: eth nco as a forward primer: ′ gcgc acc atg gcc gag gtg cag ctg ′. nhe i stop his as a reverse primer: ′ gcgc gct agc cta atg atg atg atg atg atg tgc ggc cgc gcc tag gac ′ containing the xhis tag sequence. for transient expression in eukaryotic cells, the amplimers were cloned in ptarget (promega) under the strong viral promoter (cytomegalovirus immediate-early enhancer). the clones obtained were sequenced to check for mutations possibly introduced by pcr, and used to transfect cho cells using jetpei dna transfection reagent, according to the manufacturer's instructions. transiently transfected cells were lysed after h with sds-loading buffer ( mm tris-hcl ph . , % sds, % -mercaptoethanol, % glycerol), loaded onto % sds-page, and then transferred to a nitrocellulose membrane using standard procedures. the membrane was blocked in % mpbs on at rt. blotted proteins were incubated for h in % mpbs with μg/ml of tet-ra·his antibody (qiagen), which was the only anti-his mab able to recognize the tag at the scfv cooh terminus, in our experimental conditions. after an additional incubation for h at rt in the presence of goat anti-mouse antibody hrp-conjugate ( μg/ml, dako), the reaction was developed and visualized with a chemiluminescence detection kit (pierce; il, usa). luciferase reporter gene assay ifn-β induction luciferase reporter gene assays a cells ( × per well) were transfected in -well plates with t-pro p-fect transfection reagent (t-pro biotechnology) with the construct pgl ifn-β luc, kindly provided by prof. stephan ludwig (institute of molecular virology, münster, germany). twenty-four hours after transfection, cells were additionally transfected using iav pr vrna and incubated for a further h at °c with % co . cells were harvested with lysis buffer ( mm na-mes ph . , mm tris-hcl ph . , mm dithiothreitol, . % triton x- ). the crude cell lysates were cleared by centrifugation and μl of cleared lysates were added to μl of luciferase assay buffer ( mm na-mes ph . , mm tris-hcl ph . , mm magnesium acetate, . mg/ ml atp) in a white -well plate. immediately after addition of μl of mm d-luciferin into each well, the luminescence was measured in victor luminometer (perkin elmer). the relative light units (rlu) were normalized as the fold activity of the unstimulated control. each assay was carried out in triplicate. the above described luciferase reporter gene assay was also performed for evaluating the ifn-β induction inhibition mediated by ebov vp . twenty-four hours after co-transfection with pgl ifn-β luc and pcdna or pcdna ebov wtvp expression vectors, cells were transfected with the ctrl anti-go scfv ptarget as an irrelevant scfv and with the different scfv p target vectors the next day, cells were additionally transfected with iav vrna. inhibition of luciferase expression was indicated either as the fold activity of the unstimulated control or as a percentage of the induced control. each assay was carried out in triplicate. competitive elisa using scfv-expressing phages for elisa competition assay, we used soluble nonphage-fused scfvs produced and purified as in gellini et al. [ ] . scfv-expressing phages were produced from a monoclonal bacterial culture grown to od = . - . and infected with m k helper phage in a ratio of around : phage/bacteria. one hundred ml of supernatant containing scfv-expressing phages were x concentrated by precipitation with peg and resuspended in pbs. phage titer was determined by plating of bacteria infected with phages at scalar dilutions. coating was performed with vp or go antigen as described in the elisa section. the following day, the plate was blocked with % mpbs for h at rt and washed with tpbs. twenty-five μl of soluble non-phage-fused scfvs at two-fold the desired final concentration were pre-incubated in % mpbs with the antigen for min prior to the addition of μl of the scfv-expressing phage mix at two times the desired final tu/ml in % mpbs, and incubated for h at rt. the plate was washed with tpbs and incubated with anti-m pviii coat protein mab conjugated to hrp (amhersham) diluted : , for h rt. a washing step was conducted followed by the addition of peroxidase substrate as described above. ebola haemorrhagic fever ebola virus disease the discovery of a new ebolavirus, bombali virus, adds further support for bats as hosts of ebolaviruses ebola virus: new insights into disease aetiopathology and possible therapeutic interventions how ebola and marburg viruses battle the immune system strategies of highly pathogenic rna viruses to block dsrna detection by rig-i-like receptors: hide, mask, hit the ebola virus vp protein functions as a type i ifn antagonist ebola virus protein vp impairs the function of interferon regulatory factor-activating kinases ikkepsilon and tbk- ebola virus vp antagonizes pkr activity through its c-terminal interferon inhibitory domain production technologies for monoclonal antibodies and their fragments expression and targetingof intracellular antibodies in mammalian cells generation and functional characterization of intracellular antibodies interacting with the kinase 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affinity against a marker of angiogenesis eluted from a two-dimensional gel design and use of phage display libraries for the selection of antibodies and enzymes generation of human antibody fragments recognizing distinct epitopes of the nucleocapsid (n) sars-cov protein using a phage display approach ebola: anatomy of an epidemic who regional office for africa. ebola virus diseases new ebola outbreak declared in democratic republic of the congo rapid diagnosis of ebola hemorrhagic fever by reverse transcription-pcr in an outbreak setting and assessment of patient viral load as a predictor of outcome identification of essential outstanding questions for an adequate european laboratory response to ebolavirus zairewest africa detection of ebola viral antigen by enzyme-linked immunosorbent assay using a novel monoclonal antibody to nucleoprotein development, characterization and use of monoclonal vp -antibodies for the detection of ebola virus production of monoclonal antibodies and development of an antigen capture elisa directed against the envelope glycoprotein gp of ebola virus strategies in ebola virus disease (evd) diagnostics at the point of care antiviral agents against ebola virus infection: repositioning old drugs and finding novel small molecules insights into ebola virus vp and vp interferon inhibitory functions and their initial exploitation as drug targets, infectious disorders -drug targets a randomized, controlled trial of zmapp for ebola virus infection vp knockdown inhibits ebola virus amplification and protects against lethal infection in mice postexposure protection of non-human primates against a lethal ebola virus challenge with rna interference: a proof-of-concept study specific in vivo knockdown of protein function by intrabodies human transbodies that interfere with the functions of ebola virus vp protein in genome replication and transcription and innate immune antagonism generation of human single-chain antibody to the cd cell surface determinant specifically recognizing ewing's sarcoma tumor cells publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors wish to thank philochem for the generous permission to use of the eth- antibody phage display library. we thank martin bennett for his help in revising the manuscript for grammar and style. authors' contributions mf isolated and characterized the scfvs, participated in the design of the research and drafted the manuscript. aa and mg performed wb analyses and scfv production. am worked on the design and the genetic construction of the anti-vp scfvs in ptarget vector and their expression in cho cells. pdb and la expressed the anti-vp scfvs in the ptarget vector and critically reviewed the manuscript. af and ef produced the recombinant vp , carried out ebov vp luciferase reporter gene inhibition assay and analyzed the data. sv coordinated the study design on the basis of the scfv isolation. pdb and et conceived, coordinated and supervised the study. all authors have read and approved the manuscript. this work was supported by sardinia regional government grants lr / (crp- /f i ), and intramural fund of istituto superiore di sanità. the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.ethics approval and consent to participate not applicable the authors declare that they have no competing interests. key: cord- -zgta ah authors: howard, evin; orhurhu, vwaire; huang, lisa; guthrie, barbara; phipatanakul, wanda title: the impact of ambient environmental exposures to microbial products on asthma outcomes from birth to childhood date: - - journal: curr allergy asthma rep doi: . /s - - - sha: doc_id: cord_uid: zgta ah purpose of review: asthma is a chronic respiratory condition with increasing domestic and worldwide prevalence that burdens individuals and the healthcare system with high costs associated with long-term treatments and acute emergency room (er) visits. it can be triggered by ambient microbes, including bacteria, viruses, and fungi. in this review, we examine the outcomes of asthma patients in relation to environmental exposures to ambient microbe products, focusing on whether exposure leads to asthma development from birth to childhood and if particular microbes are associated with worsened asthma exacerbations. recent findings: bacterial endotoxin is more prominent in homes with pets and may cause cytokine cascades that lead to asthma exacerbation. however, some studies have demonstrated a protective effect with early exposure. patients with positive aspergillus skin testing are more prone to moderate-severe or severe-uncontrolled asthma. fungal sensitization is also associated with earlier onset of asthma and demonstrates a dose-dependent relationship of symptom severity and duration. among viruses, rhinovirus has the greatest association with decreased lung function, severe asthma, and asthma-related hospital admissions. distribution of microbial products and associated asthma symptoms depends on the geographical climate. genetic variations among individuals also mitigate the effects of microbial products on asthma development and symptom severity. summary: microbial products of bacteria, fungi, and viruses are associated with the development of asthma, more severe asthma symptoms, and worse outcomes. however, some early exposure studies have also demonstrated a protective effect. bacterial and fungal products are related to decreased lung function and earlier onset of asthma. viral products are related to asthma-associated hospital admissions; and the climate and patient genetics can also temper or intensify the relationships between microbial products, asthma development, and asthma symptom severity. further research should focus on the effects of early microbe exposure and its interaction with human immune systems and asthma-related outcomes. every day, people in the usa die from asthma [ ] . in alone, there were , asthma-related deaths [ , ] . within the usa, the prevalence of asthma is in people, producing annual economic costs of over $ . billion, related to missed school and work, medical costs, and asthma-related mortality [ ] [ ] [ ] . ambient microbes have been shown to trigger asthma attacks [ ] [ ] [ ] . the most common microbes are bacteria, viruses, and fungi [ ] . though the literature has clearly demonstrated that microbes and allergens can trigger asthma exacerbations, controversy still remains whether or not microbes promote the development of asthma with early-life exposure or if they have a preventative effect against asthma symptoms in an individual with established disease [ ] [ ] [ ] . many longitudinal studies have demonstrated that early this article is part of the topical collection on allergies and the environment exposure to ambient microbial products increases the risk of the development of asthma [ •, , , •] . whereas, other studies, particularly related to human contact with animal microbes, have shown protective effects against the development of asthma [ •, , ] . with over . million asthma-related er visits each year, which may be potentiated by exposure to various ambient microbial products in the environment, earlylife exposure to microbial products and its effect on the respiratory system later in childhood warrants further investigation [ , , , ] . the purpose of this literature review was to specifically examine asthma outcomes related to environmental exposures to microbial products, pertaining to endotoxin from bacteria-( , )-β-d-glucan and ergosterol from fungus, and common viruses associated with worsening asthma morbidity (rhinovirus, respiratory syncytial virus (rsv), enterovirus, and the influenza virus) during infancy, and to assess the risk of asthma development later in childhood [ ] [ ] [ ] [ ] (see table ). while other reviews have focused on common routes of exposure and the concentration of microbial products (endotoxin, fungal spores, virus, etc.) in urban vs. suburban regions [ , ] , this review investigates if the exposure to microbial products during infancy leads to the development of asthma during childhood, and whether particular microbes have a higher severity for triggering asthma exacerbations over others. primary questions for this review consisted of the following: what microbes are associated with asthma severity and outcomes? what microbial products are known for triggering asthma symptoms and/or exacerbations? does the exposure of microbial products in the environment lead to the development of asthma? for this review, we selected original manuscripts in english, published in peer-review journals on the basis of bacterial, fungal, and viral products within the environmental matrix with children exposed to these microbial products. thus, we selected articles examining health outcomes for the development of asthma related to microbial product exposure during the time of infancy until childhood. however, we also highlighted factors in the literature that contribute to microbial exposure and their effect on asthma for people with an established diagnosis of asthma. only journal articles within the last years were chosen; however, there were no restrictions pertaining to specific populations or geographical locations. exclusion criteria consisted of animal studies, investigative procedures using only in vitro methods, studies not translated into english, articles focusing on the measurement of microbial products without their effect on asthma outcomes, and articles not assessing microbial exposure from the time of infancy. with this thought process in mind, the following word criteria was applied using pubmed as the primary search engine: ( the search returned a total of articles. after the removal of studies, consisting of reviews, duplicates, mouse studies, and irrelevant articles to asthma health outcomes, this then left studies. studies were pertaining to adults, which left a total of n = studies (see fig. ). the majority of the studies had a prospective cohort design ( / ), while the other two studies were retrospective cohort designs. environmental sampling consisted of different locations in participants' homes (living room, kitchen, bedroom, mattress), outside air, and settled dust in classrooms. all of the studies included examined asthma health outcomes related to ambient exposure to bacterial, fungal, and viral microbial products. considering bacterial products and asthma outcomes, the literature has discussed endotoxin, β-glucan, and lipopolysaccharide (lps). of these microbial products, when bacteria are destroyed or lysed, lps is dispersed into the environment or blood as endotoxin, which can lead to a cascade of inflammatory cytokines (il- , il- , il β, and tnf-α) and exacerbation of asthma symptoms [ , ] . the main route of contact to endotoxin is via inhalation, which has been demonstrated in multiple studies [ , , , ] . of the studies selected for review, there were two that specifically discussed endotoxin exposure from infancy [ •, ••] (see table ). tischer et al. conducted a prospective longitudinal study examining whether early exposure to microbial products in dust was associated with allergy and asthma later in childhood for children in suburban areas using the following three birth cohort studies for children born between and : [ ••] , dust samples were collected from children's mattresses, bedroom floors, and living room floors; and showed no association between endotoxin nor the fungal membrane lipid ergosterol in the development of asthma with exposure from birth to years of age. previous research has shown that endotoxin levels tend to be higher in homes with a pet such as a cat or dog in addition to homes of families that are low-income and/or may be in community projects that contain cockroaches [ , ] . similar results were seen in children by a study conducted by mendy et al. [ ] who found that overall for people who had a cat or dog, endotoxin levels were higher in the household with a pet and was associated with wheeze (or = . ; % ci ( . - . )) but not with asthma [ ] . co-exposure with dog and cat allergens modified the association with asthma, increasing the risk when exposed to endotoxin (or = . ; % ci ( . - . )) [ ] [ ] demonstrated that endotoxin exposure is associated with wheeze with exercise, office or er visits, and use of prescription medication for wheezing [ ] . studies have indicated that lung function and capacity can be decreased due to continuous exposure to endotoxin in patients with atopic asthma [ , ] . lipopolysaccharide has also been shown to promote eosinophil airway inflammation in patients with asthma despite maintenance treatment with inhaled corticosteroids (ics) [ ] . besides having a pet, there are other factors that could have contributed to the varying results between the cohorts within the tischer et al.'s study. the climate also plays a role in the concentration of endotoxin in the surrounding environment [ , ] . researchers have found a positive correlation between log -endotoxin and current asthma (or = . , % ci ( . - . ), p = . ) for hot-humid regions and a higher incidence of exercise-induced wheeze (or = . , % ci ( . - . ), p = . ) for subarctic/very cold/cold regions [ ] . high levels of endotoxin, regardless of being inside or outside the home environment, are associated with severe asthma [ , ] . a study conducted in japan by khan and colleagues showed that asthma-related visits to the er for patients -years-old or older was highest during the seasons of autumn and spring, when the environmental endotoxin was highest in the air [ ] . oluwole et al. [ ] showed that for to -year-olds, high concentrations of endotoxin within households, including mattresses, are associated with an elevated risk of moderate or severe asthma (adjusted or = . , % ci ( . - . )). tischer et al. [ •] also supported these • sleeping with animal fur was inversely associated with early wheezes at age and current asthma at age • sleeping on animal fur during the first months of life was associated with persistently stimulated interferon-γ response until age • animal fur exposure might act as an immune system stimulant and offer protective mechanisms against asthma and allergy, as observed in farm/rural environments not able to determine the microbial profile of the animal fur nor the intensity of the exposure. lifestyle factors also play a role in developing asthma bonnelykke et al. • after adjusting for total respiratory episodes and number of episodes per pathogen, only the total number of episodes were significantly associated with asthma development • risk factor for development is asthma is the number of respiratory episodes, not the specific viral trigger assumed that children who did not present to clinic did not have clinically significant respiratory episodes • maternal history of asthma and depression were associated within higher risk of asthma by age . • bacterial species kocuria, bifidobacterium, alloiococcus, and acinetobacter were found in homes of children without asthma, but not in homes of children with asthma. • endotoxin and ergosterol were not found to be associated with the development of asthma gastrointestinal and airway microbiome were not measured during early childhood results for children from infancy to years of age. in addition, these findings are also in accordance with previous investigations in the early s [ ] [ ] [ ] [ ] . non-environmental patient characteristics can also impact the development or severity of asthma. mendy et al.'s [ ] study determined that the combination of high levels of endotoxin exposure and low vitamin d levels are associated with an asthma diagnosis or recurrent wheeze (or = . , % ci ( . - . ), p = . ), current asthma (or = . , % ci ( . - . ), p = . ), wheeze over the last months (or = . , % ci ( . - . ), p = . ), and recurrent wheeze (or = . , % ci ( . - . ), p < . ) [ ] . nevertheless, this study was conducted in adults and the effect from infancy to childhood, regarding vitamin d levels in combination with endotoxin exposure, would need to be assessed. no studies on fungal spore exposure were found for the time period from birth to childhood to assess the development of asthma. [ •] . sensitization to fungal antigens is a key component to understanding asthma severity. chopra and colleagues showed that asthma patients with skin reactivity to the aspergillus skin test (ast) had more cases of moderate and severe asthma versus nonreactive ast patients ( . % vs. . %, p < . ) in a cohort of adult asthma patients over a -year period. sensitization to aspergillus has been found to have a more significant impact on asthma outcomes compared to other species of fungus [ , ] . for instance, vincent et al. [ ] demonstrated that adult asthma patients who were sensitized to penicillium chrysogenum, [ ] . masaki and colleagues' study in japan implicates a different species; their study investigated sensitization to candida and found that out of adults with asthma, more people had sensitization to candida than aspergillus ( vs. %) [ ] . the researchers also found that fungal sensitization leads to a higher rate of early-onset asthma (< years of age) compared to those without sensitization ( % vs. %) and sensitization increases with age [ , ] . however, further investigation is needed to examine early exposure of various fungal species during infancy and its effect on the development of asthma during childhood. the fungal genus alternaria has also provided more insight into fungal sensitization and asthma outcomes. recently, baxi and colleagues conducted a study in inner-city schools in new england with school-aged children with asthma and observed that children sensitized to alternaria had an association with asthma symptom days (or = . , % ci ( . - . ), p = . ); however, this did not apply to children not sensitized to alternaria (or = . , % ci ( . - . ), p = . ) [ ] . researchers found that asthmatic children who were sensitized to alternaria and exposed to high levels had . more symptom days per week period when compared to asthmatic and sensitized children exposed to lower levels of alternaria. this suggests a dose-dependent relationship between mold exposure and asthma severity [ ] . this finding is supported by oluwole et al. [ ] , who demonstrated that high mold levels in children's play areas are associated with current asthma (adjusted or = . , % ci ( . - . )). furthermore, additional studies have demonstrated that sensitization to alternaria increases with higher mold concentration exposure and may lead to a risk of hospitalization, especially in the urban setting [ ] [ ] [ ] [ ] . fungal exposure also impacts lung function [ ] . asthma patients with sensitization to aspergillus fumigatus have been found to have higher total serum ige levels and an increased degree of broncho-obstruction (as measured by fev /fvc), compared to asthmatic patients without sensitization [ , ] . the impact of fungal exposure on lung function has been evaluated in adults; however, this has not been thoroughly evaluated in infants and young children. the fungal product ( , )-β-d-glucan has been shown to have a low association with severe asthma (or = . , % ci ( . - . ), p = . ) in children [ , ] . ergosterol has also demonstrated a low affinity for the development of asthma [ ] . however, exposure to fungal spores has been shown by multiple studies in adults to be associated with asthma exacerbations; yet, there are very few studies involving children [ ] [ ] [ ] . in a study analyzing the fungal microbiome in exhaled breath condensate (ebc) of patients with asthma in italy, carpagnano et al. [ ] found that in adults with asthma, % of the people were colonized in the ebc by cladodosporium species, % by alternaria, and % by penicillium species [ ] . fungal colonization tended to be higher in asthmatics with severe and uncontrolled asthma with sensitization to fungal species such as alternaria [ ] [ ] [ ] . the differing levels of fungal species, with cladosporium being the most abundant, and alternaria and penicillium in lower quantities, was also found in manitoba, canada, by polyzoi and polyzois, examining mold within the bedrooms and basements of school-aged children [ ] . cladosporium was the most common mold found in homes ( . % of bedrooms and . % of basements), followed by alternaria ( . % of bedrooms and % of basements), and penicillium ( . % of bedrooms and . % of basements). in addition, visible mold in bedrooms and basements in participants' homes were associated with persistent asthma and colds ( . %, p < . ). other fungal studies conducted in europe, canada, and australia have also found similar results [ , , ] . viral exposure also plays a critical role in the development of asthma and asthma morbidity [ , •] . two studies were found that were conducted on early-life exposure to rhinovirus and asthma outcomes: one by rubner et al. [ ] and another by lukkarinen et al. [ •] . rubner et al. [ ] conducted a prospective cohort study with children, examining early-life rhinovirus exposure from birth to years of age. the researchers observed that wheezing with rhinovirus was associated with asthma at age (or = . , % ci ( . - . ), p = . ) and sensitization to allergens increased with age [ ] . fev % predicted has also been shown to be lower in asthmatic patients admitted to the hospital with an asthma exacerbation with concurrent rhinovirus infection, compared to children with asthma admitted for an exacerbation but negative for rhinovirus [ , ] . lukkarinen et al. [ • ] performed a -year long prospective cohort study following finnish children from birth to years of age and found that the risk factors for atopic asthma at study entry were sensitization to common allergens (dust, mold, etc.), and wheezing with rhinovirus infection (aor . , p < . ) [ •] . zheng et al. [ ] conducted one of the numerous studies that demonstrated that human rhinovirus (hrv) was the most common viral microbe to induce an asthma exacerbation in children [ ] [ ] [ ] . zheng and colleagues collected data from inpatient children with asthma exacerbations, outpatients, including patients with asthma exacerbations, and controls with stable asthma [ ] . researchers tested for common viruses including hrv, respiratory syncytial virus (rsv), parainfluenza virus type (piv ), influenza virus (ifv), human bocavirus (hb v ), atadenovirus (adv), human coronavirus (hcov), and hepatitis e virus. of all the viruses, hrv was the most prominent among inpatients with severe asthma exacerbations at . % and hcov was the least at % [ ] . the researchers found three variants of the hrv: hrv-a, hrv-b, and hrv-c, with hrv-c being the most virulent for severe asthma exacerbations [ ] . other studies conducted in japan, france, and australia have also identified rhinovirus as the most common viral culprit for asthma exacerbations [ , [ ] [ ] [ ] . white blood cells and neutrophil counts were significantly higher in patients with all hrv variants versus those who were negative for hrv ( [ ] . thymic stromal lymphopoietin (tslp), a cytokine that impacts t cell maturation, also plays a role in asthma severity in patients with viral illnesses. it has been demonstrated to have a positive outcome on patients during viral infections in patients with asthma [ ] . bjerregaard et al. [ ] performed a study in australia with virus-positive adolescent and adult patients (including rhinovirus, human coronavirus, parainfluenza virus, and human metapneumovirus) and controls. the researchers found that patients with tslp expression had lower levels of sputum eosinophils, lower fractional exhaled nitric oxide, higher blood neutrophils, lower asthma control questionnaire scores, and higher fev in comparison to patients with low tslp expression levels [ ] . again, these types of studies are rare for examining variances in lung function and asthma development from birth to childhood. in total, four studies were found on respiratory syncytial virus (rsv) that followed children from birth to childhood for the development of asthma [ , [ ] [ ] [ ] . two of the rsv studies showed that males from infancy to childhood have a high tendency of developing asthma after an infection, compared to females [ , ] (see table ). two out of the four studies were retrospective cohort studies and the other two were prospective cohort studies [ , [ ] [ ] [ ] . one of the retrospective studies showed that peak time for rsv-related hospitalizations was during autumn [ ] . the rsv studies showed a high risk for asthma with rsv infection and that the cytokines il- , il- β, and il- are elevated during infection, which leads to respiratory inflammation [ , ] . kitsantas et al. [ ] conducted a -year long study from birth to years of age, examining the risk of the development of asthma by years with early exposure of rsv infection during infancy. results showed that exposure to rsv did increase the risk of asthma by age (or = . , % ci ( . - . )). however, children who had a parent with a history of asthma posed a higher risk for childhood asthma by age (or = . , % ci ( . - . )) [ ] . a maternal history of asthma has also been demonstrated by o'connor et al. [ ••] to increase the likelihood of asthma in childhood by age (or = . , % ci ( . - . )). similar to what was discussed about fungi, asthma patients can also be colonized by viruses. nguyen-thi-dieu et al. [ ] conducted a cohort study in hanoi, vietnam, on hospitalized children under the age of with a diagnosis of asthma exacerbation and a control group of healthy children under the age of . patients underwent a clinical examination, blood analysis for a cytokine profile, pediatric asthma score (pas) for severity of asthma exacerbation, and nasopharyngeal aspirates to determine viral infection using real-time polymerase chain reaction (pcr). of the children diagnosed with an asthma exacerbation, there were . % with a mild pas, . % with a moderate pas, and % with a severe pas [ ] . nguyen-thi-dieu and colleagues found that . % of the asthmatic children were positive for hrv [ ] . the high number of asthma patients with a severe pas score ( %) was associated with a high number of children diagnosed with hrv [ ] . the researchers also found that the percentage of leukocytes in asthmatic children with hrv was significantly higher, compared to children without hrv ( . % vs. . %, p < . ) [ ] . no enterovirus longitudinal studies from birth to childhood were available, assessing asthma development; however, studies have been done in children with a diagnosis of asthma [ ] . smith-norowitz et al. [ ] studied patients in ireland, examining the levels of ige and igm antibodies (ab) for enterovirus with a mix of children and adolescents, and found that asthmatic children have higher ige ab levels, compared to non-asthmatic children, who have higher igm levels of ab (p < . ). the levels of enterovirus ige ab were shown to increase with age especially among the asthmatic group [ ] . another study analyzed data on a -year longitudinal study with , children from infancy to years of age in taiwan and found that children were diagnosed with asthma within . years after an enterovirus infection [ ] . there was also a higher incidence of asthma associated with enterovirus infection (hr = . , % ci ( . - . ), p < . ) [ ] . overall, the literature suggests that the rate of hospitalization due to enterovirus infection is low and does not increase asthma severity or length of hospital stay for asthma-related admissions [ , ] . the influenza virus is a major cause of acute exacerbation of bronchial asthma (aeba); however, rhinovirus has still been shown to be the number one viral cause of asthma exacerbations [ ] . yoshi et al. and other researchers have shown that obtaining the flu vaccine reduces aeba morbidity significantly when compared to individuals who are unvaccinated for the influenza virus ( . % vs. %), p = . [ , ] . influenza can worsen asthma morbidity; however, it has yet to be determined if there is a relationship pertaining to the development of asthma in association with an influenza infection [ ] . recent studies have found that certain bacteria play a role in preventative effects against the development of asthma. den hollander et al. [ ] performed a population-based prospective cohort study of pregnant women and their children in rotterdam, netherlands, and followed the children from pregnancy to years of age for evaluation of asthma. colonization of a european child with a cytotoxin-associated protein a (caga)-negative-helicobacter pylori (h. pylori) strain at age was associated with an increased prevalence of asthma overall (or = . , % ci ( . - . )) but was different for european children (or = . , % ci ( . - . )) and significantly less for non-european children (or = . , % ci ( . - . )) [ •] . there are also common bacteria within the environmental matrix that have been shown to have an effect on the development of asthma in children. teo et al. [ ] found that early childhood nasopharynx (np) colonization typically involved staphylococcus or corynebacterium, which was later replaced by moraxella or alloiococcus. staphylococcus was the dominant colonizing bacteria in the early healthy np microbiomes, but its presence declined rapidly with age. these researchers found that early asymptomatic colonization with streptococcus increased the risk of developing asthma [ ] . most infants were initially colonized with staphylococcus or corynebacterium before stable colonization with alloiococcus or moraxella [ ] . transient incursions of streptococcus, moraxella, or haemophilus marked virus-associated acute respiratory infections (aris) [ ] . o'connor et al. [ ••] found supporting results in a study examining early-life exposure to environmental factors, including dust, fungal products, bacterial taxa; cockroach, mouse, and cat allergen; from infancy to years of age. staphylococcus, haemophilus (pasturellaceae), several sphingomonas species members, and corynebacterium were among the taxa found in children's homes who had asthma [ ••] . however, for children who did not have asthma, there was a prevalence of alloiococcus (aerobacteriaceae), kocuria (micrococcaceae), acinetobacter (moraxellaceae) species, and bifidobacterium [ ••] . more research is needed to investigate the mechanism of transition of bacteria from one species to another in the infant microbiome and how this may possibly contribute to the development of asthma. in addition to the microbiome, children's genes affect how their bodies will react to their environment. like most illnesses, symptoms and severity can vary at the individual level based on unique genetic differences. carnes et al. [ ] found that there is a gene-by environment interaction for cd variant rs (p interaction = . ) but not for the tlr variants rs and rs . this research is also supported by an older study conducted by kljaic-bukvic et al. [ ] , who found that there are two genetic variants related to cd and endotoxin signaling that are highly related with asthma exacerbation hospital admissions. for cd single nucleotide polymorphism (snp) rs , carriers of the t-allele in children to years old had a decreased risk of repeated hospital asthma-related admissions, compared to homozygotes for the c-allele (or = . , % ci ( . - . ), p = . ) [ ] . c-allele carriers were at lower risk of asthmarelated hospitalizations in comparison with t-allele homozygotes (or = . , % ci ( . - . ), p = . ) [ ] . a human interleukin receptor alpha chain gene (il r) variant that results in a glutamine to arginine substitution at amino acid residue (il rα-q r polymorphism) has previously been associated with asthma diagnosis and severity [ ] . lai et al. [ ] have demonstrated that there is a gene-byenvironment interaction between the il rα-q r polymorphism and environmental endotoxin exposure in school-aged children with asthma. higher classroom endotoxin levels were associated with fewer asthma symptoms for children with the q/q genotype (or = . [ ] . in addition to genetics, exposure to animals, especially within the farm setting, has shown to play a role in asthma. the environment of a farm is a great example of exposure to all of the microbes and microbial products discussed in this review. carnes et al. [ ] found that high concentrations of endotoxin exposure at homes of people not born on a farm have a risk for the development of asthma (or = . , % ci ( . - . ), p = . ), compared to individuals who were born on a farm (or = . , % ci ( . - . ), p = . ). in addition, lampi et al. [ ] found that people born on a farm had a higher fev , compared to those not raised on a farm. the limitation here is that these participants in the lampi study could be more physically fit having to do the chores of farm work, compared to people not living on a farm. prior studies have attributed farm-specific protection from the development of asthma from endotoxin exposed in utero and during early childhood [ , , ] . an older study conducted on amish and hutterite communities whom participate in farming activities has revealed two very different results [ , ] . the amish farm children of indiana were found to have a lower prevalence of asthma and allergen sensitization ( . % vs. . % and . % vs. . %, respectively), compared to the hutterite farm children of south dakota [ , ] . the difference between these two farming communities was that the amish have constant direct contact with animals to do all of their farming traditionally, and the hutterite community relies on machinery and maintains the animals in one localized area away from the community homes [ , ] . this finding brings up a few questions. why is early farm exposure to endotoxin related to protective effects against the development of asthma and asthma symptoms; whereas, non-farm house endotoxin exposure is generally associated with asthma and asthma exacerbations? are there different microbes related to farm animals versus household pets, which are associated with increasing asthma symptoms [ , ] ? in a different study, tischer et al. [ ] examined the german lisaplus birth cohort and found that children who slept on animal fur during the first months of life had higher exposure to endotoxin in their mattress, compared to children who did not sleep on fur. more importantly, the researchers found a decrease in the risk of asthma development by age for children who slept on animal fur during the first months of life (aor = . , %, ci ( . - . ). in addition, for tnf-α, il- , and il- producing t cells, there were no significant changes found in blood levels associated with sleeping on animal fur the first months of life [ ] . this review has shed light on several different findings within the literature. based on the information provided, sensitization within the non-farming community for bacterial, fungal, and viral products increases with ige ab expression and over time with age [ , , ] . in terms of the fungal species, aspergillus fumigatus has shown to induce one of the highest sensitizations for moderate/severe asthma [ , ] . in addition, human colonization of the genera cladosporium, alternaria, and penicillium have been associated with severe asthma and their ambient environmental concentration is similarly proportional in various parts of the world [ , , , [ ] [ ] [ ] . for viruses, rhinovirus has been shown to be one of the most common triggers of a viral source for an asthma exacerbation [ , •, , , , ] . early exposure to endotoxin at a young age within the farm setting with direct animal contact may reduce the chances of developing asthma later in life [ , , , ] . for children not living on a farm, growing up with a cat or dog may have a protective effect against asthma symptoms [ •, ••] . the mechanism of this effect is yet to be understood and it appears that genetics plays a role in asthma severity especially among ethnic minorities [ ] . further research is needed to investigate the difference in microbes between farm animals and household pets and how early exposure effects the human microbiome and the immune system's response to inflammatory triggers. in addition, since studies have shown that people with severe asthma may be colonized with various genus of fungi, it would be worthwhile to further investigate the treatment of moderate/severe asthmatics colonized with fungi with other methods not associated with toxic side effects related to current medications for anti-fungals. in addition, more studies are needed examining the mechanism(s) for the transition of bacteria in the microbiome during childhood and how this may effect asthma outcomes. biomarkers such as dimethylamine, a metabolite produced by bacteria within the gastrointestinal tract, may pose a clue as it has been demonstrated to decrease in concentration from higher to lower levels with the development of asthma in children [ ] . further advances are also being made with dupilumab, a monoclonal antibody that blocks a common receptor for interleukin- (il- ) and interleukin- (il- ), involved in type inflammation [ ] . in adult patients with atopic asthma, it has been shown to reduce severe asthma exacerbations [ ] . this medication has recently been approved for children years old and up for the treatment of severe asthma [ ] . continued exploration of the human microbiome and its interaction with the environment hopefully may lead to more discoveries. all reported studies/experiments with human subjects performed by the authors have been previously published and compiled with all applicable ethical standards (including the helsinki declaration and its amendments, institutional/national research committee standards, and international/national/institutional guidelines). the authors declare no conflicts of interest relevant to this manuscript. human and animal rights and informed consent this article does not contain any studies with human or animal subjects performed by any of the authors. most recent national asthma data the economic burden of asthma in the united states airway microbiota in severe asthma and relationship to asthma severity and phenotypes endotoxin exposure: predictors and prevalence of associated asthma outcomes in the united states characteristics of severe asthma with fungal sensitization harrison's principles of internal medicine , )-β-d-glucan, and lps on each cohort from different geographical regions for the development of asthma in children from birth to age [ ]. it has been demonstrated that the climate and geographical region can affect asthma outcomes [ , ]; and this was observed as high endotoxin levels were associated with asthma at age for the piama cohort (dutch); an inverse relationship with the inma cohort (spanish), and no association with the lisaplus cohort (german). also, other studies have shown that early constant animal exposure reduces the risk of asthma early life rhinovirus wheezing, allergic sensitization, and asthma risk at adolescence association between respiratory infections in early life and later asthma is independent of virus type previous studies have demonstrated that early infant exposure to human rhinovirus (hrv) is associated with triggering asthma attacks this study demonstrated that early childhood exposure to rhinovirus significantly increased the risk for the development asthma, and found clinical markers that could be used to predict the development of atopic and non-atopic asthma in children [ ]. this study was different from previous studies farm environment during infancy and lung function at the age of : a prospective birth cohort study in finland exposure to farming in early life and development of asthma and allergy: a cross-sectional survey association of airborne particles, protein, and endotoxin with emergency department visits for asthma in kyoto epidemiological analysis and follow-up of human rhinovirus infection in children with asthma exacerbation association between enterovirus infection and asthma in children: a -year nationwide population-based cohort study ev-d infection in children with asthma exacerbation and pneumonia in mexico city during autumn. influenza other respir viruses high fractional exhaled nitric oxide and sputum eosinophils are associated with an increased risk of future virus-induced exacerbations: a prospective cohort study the indoor environment and inner-city childhood asthma environmental exposure to endotoxin and its health outcomes: a systematic review house dust endotoxin levels are associated with adult asthma in a u.s. farming population endotoxin tolerance: new mechanisms, molecules and clinical significance this study not only demonstrated this concept, following patients from birth to years of age, but also found that the bacterial species kocuria, bifidobacterium, alloiococcus, and acinobacter are found in the home of children without asthma furthermore, a previous study has demonstrated that the microbiome of infants' nasopharynx is initially colonized with alloiococcus when healthy and declines with acute respiratory infections and the development of asthma thus, this study shows key bacteria may have the potential to suppress or trigger the development of asthma from early childhood exposure sleeping on animal fur is related to asthma outcomes in later childhood the infant nasopharyngeal microbiome impacts severity of lower respiratory infection and risk of asthma development helicobacter pylori in children with asthmatic conditions at school age, and their mothers predictors of asthma following severe respiratory syncytial virus (rsv) bronchiolitis in early childhood association between respiratory syncytial viral disease and the subsequent risk of the first episode of severe asthma in different subgroups of high-risk australian children: a whole-ofpopulation-based cohort study effects of respiratory syncytial virus infection in infancy on asthma and respiratory allergy in -year-old children endotoxin predictors and associated respiratory outcomes differ with climate regions in the u exposure and sensitization to pets modify endotoxin association with asthma and wheeze relations of exhaled nitric oxide and fev to personal endotoxin exposure in schoolchildren with asthma lipopolysaccharide amplifies eosinophilic inflammation after segmental challenge with house dust mite in asthmatics the association between endotoxin and beta-( -> )-d-glucan in house dust with asthma severity among schoolchildren indoor determinants of endotoxin and dust mite exposures in hong kong homes with asthmatic children endotoxin exposure and eczema in the first year of life a longitudinal analysis of wheezing in young children: the independent effects of early life exposure to house dust endotoxin, allergens, and pets endotoxin exposure in asthmatic children and matched healthy controls: results of ipeadam study endotoxin exposure, serum vitamin d, asthma and wheeze outcomes relationship between mold exposure, specific ige sensitization, and clinical asthma: a case-control study evaluation of the association between sensitization to common inhalant fungi and poor asthma control clinical relevance of ige-mediated sensitization against the mould alternaria alternata in children with asthma association between fungal spore exposure in innercity schools and asthma morbidity indoor mold levels and current asthma among school-aged children in saskatchewan impact of occupational exposures on exacerbation of asthma: a population-based asthma cohort study fungal sensitization is associated with increased risk of life-threatening asthma indoor fungal diversity in primary schools may differently influence allergic sensitization and asthma in children the role of outdoor fungi on asthma hospital admissions in children and adolescents: a -year time stratified case-crossover analysis the relationship between biomarkers of fungal allergy and lung damage in asthma indoor microbial communities: influence on asthma severity in atopic and nonatopic children - , - )-beta-d-glucan, and ergosterol concentrations in dust are not associated with asthma, rhinitis, or eczema diagnoses in children analysis of the fungal microbiome in exhaled breath condensate of patients with asthma fungal sensitisation in severe asthma is associated with the identification of aspergillus fumigatus in sputum mould and grass pollen allergy as risk factors for childhood asthma in zaragoza presence of household mold, children's respiratory health, and school absenteeism: cause for concern identification of fungal candidates for asthma protection in a large population-based study associations between outdoor fungal spores and childhood and adolescent asthma hospitalizations influence of viral infection on the relationships between airway cytokines and lung function in asthmatic children characteristics associated with clinical severity and inflammatory phenotype of naturally occurring virus-induced exacerbations of asthma in adults the molecular epidemiology of respiratory viruses associated with asthma attacks: a single-center observational study in japan serum igg concentrations in adult patients experiencing virus-induced severe asthma exacerbations study of clinical characteristics and cytokine profiles of asthmatic children with rhinovirus infection during acute asthma exacerbation at national hospital of pediatrics increased seroprevalence of enterovirus ige antibodies in asthmatic compared with non-asthmatic children clinical characteristics of children infected with enterovirus d in an outpatient clinic and the association with bronchial asthma enterovirus d respiratory infection in a children's hospital in japan in detection of pathogens by real-time pcr in adult patients with acute exacerbation of bronchial asthma effects of inactivated influenza vaccine on respiratory illnesses and asthma-related events in children with mild persistent asthma in asia hospitalizations associated with respiratory syncytial virus (rsv) and influenza in children, including children diagnosed with asthma genetic variants in endotoxin signalling pathway, domestic endotoxin exposure and asthma exacerbations the r il- receptor alpha allele correlates with asthma severity gene-environment interaction between an il r variant and school endotoxin exposure contributes to asthma symptoms in inner-city children farm exposure in utero may protect against asthma, hay fever and eczema environmental exposure to endotoxin and its relation to asthma in school-age children amish children living in northern indiana have a very low prevalence of allergic sensitization rising prevalence of asthma is sex-specific in a us farming population staphylococcal enterotoxin ige sensitization in late-onset severe eosinophilic asthma in the elderly correlation of aspergillus skin hypersensitivity with the duration and severity of asthma. monaldi arch chest dis rhinoviruses significantly affect day-to-day respiratory symptoms of children with asthma absence of back to school peaks in human rhinovirus detections and respiratory symptoms in a cohort of children with asthma longitudinal urinary metabolomic profiling reveals metabolites for asthma development in early childhood dupilumab efficacy in patients with uncontrolled, moderate-to-severe allergic asthma targeted therapy for severe asthma in children and adolescents: current and future perspectives publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- - trs m authors: shin, minkyu; yoon, jinho; yi, chanyong; lee, taek; choi, jeong-woo title: flexible hiv- biosensor based on the au/mos( ) nanoparticles/au nanolayer on the pet substrate date: - - journal: nanomaterials (basel) doi: . /nano sha: doc_id: cord_uid: trs m an electrochemical flexible biosensor composed of gold (au), molybdenum disulfide nanoparticles (mos( ) nps), and au (au/mos( )/au nanolayer) on the polyethylene terephthalate (pet) substrate is developed to detect envelope glycoprotein gp (gp ), the surface protein of hiv- . to fabricate the nanolayer on the pet substrate, au is sputter coated on the flexible pet substrate and mos( ) nps are spin coated on au, which is sputter coated once again with au. the gp antibody is then immobilized on this flexible electrode through cysteamine (cys) modified on the surface of the au/mos( )/au nanolayer. fabrication of the biosensor is verified by atomic force microscopy, scanning electron microscopy, and cyclic voltammetry. a flexibility test is done using a micro-fatigue tester. detection of the gp is measured by square wave voltammetry. the results indicate that the prepared biosensor detects . pg/ml of gp , which is comparable with previously reported gp biosensors prepared even without flexibility. therefore, the proposed biosensor supports the development of a nanomaterial-based flexible sensing platform for highly sensitive biosensors with flexibility for wearable device application. flexible biosensors composed of polymer materials have recently attracted significant attention for their application in wearable devices and point-of-care (poc) diagnostic systems. polymer substrates such as polyethylene terephthalate (pet), polyimide (pi), polycarbonate (pc), and polydimethylsiloxane (pdms) are widely used as flexible substrates [ , ] . in addition, in order to fabricate flexible biosensors, various types of nanomaterials such gold nanoparticles (gnps), carbon nanotubes (cnts), and graphene oxide (go) have been introduced for granting the conductivity, enhancing the electron transfer, and biocompatibility [ ] [ ] [ ] [ ] [ ] [ ] . among these nanomaterials, carbon-based nanomaterials have been widely used due to their exceptional properties such as excellent electrical conductivity, high specific activated surface area, and chemical/biological stability [ , ] . recently, transition metal dichalcogenide (tmd) materials such as tungsten diselenide (wse ) and molybdenum disulfide (mos ) have been widely researched for their application in biosensors due to unique properties including electric charge effect and semiconducting property [ ] [ ] [ ] . in particular, mos has many advantages for biosensor development due to its electrochemical property and biocompatibility [ , ] . in addition, in order to maximize the benefits of mos on a large activated surface area, mos nanoparticles (mos nps) have recently been synthesized and applied to develop various sensors [ ] . however, most studies related to flexible biosensors composed of nanomaterials have been done through complex manufacturing methods. to replace the complex manufacturing water from a millipore milli-q water purifier operating at a resistance of mΩ·cm. myoglobin (mb) (≥ . %, sigma-aldrich, burlington, ma, usa), hemoglobin (hb) (sigma-aldrich, burlington, ma, usa), thioredoxin (trx) (sino biological, wayne, pa, usa), and prostate-specific antigen (psa) (abcam, cambridge, uk) were used to investigate the selectivity of the fabricated biosensor. ammonium molybdate tetrahydrate ( . g) and . g of thiourea were mixed with ml of di water. then . g of pvp was added to the mixture. the resulting solution was reacted at rpm for h at • c. the solution was transferred into a teflon-lined stainless-steel autoclave. the autoclave was set at • c for h. after the reaction was complete, the resulting black precipitate was cooled down to room temperature and washed with di water and ethanol using centrifugal filtration with rpm for min. the final product was air-dried at • c for h. the pvp-modified mos nps (concentration: mg/ml) were characterized by high-resolution tem using a jeol jem- operated at kv and by xrd (rigaku, tokyo, japan). nanolayer on the pet substrate, and immobilization of the gp antibody the fabrication of a flexible biosensor composed of ab/cys/au/mos /au nanolayer on pet substrate is shown in figure . to fabricate the au/mos /au nanolayer on the pet substrate, the rectangular pet substrate (size mm × mm) was cleaned in a sonication bath for min using acetone and di water, and then completely dried with n gas. after cleaning the pet, the au/mos /au nanolayer on the pet substrate was prepared by gold sputter and spin coater. the au layer was formed by sputter coating on the pet substrate. then, µl of the synthesized mos nps dissolved in di water (concentration: mg/ml) was dropped onto the au layer and spin coated (au/mos ) on the pet substrate at rpm for s; this process was done twice. after the spin coating process, the au layer was sputter coated once more on the mos layer. fabrication of the au/mos /au nanolayer on the pet substrate was verified by fe-sem, eds, and afm. to fabricate the ab/cys/au/mos /au nanolayer on the pet substrate, the gp antibody was immobilized on the au/mos /au nanolayer on the pet substrate. the fabricated electrode was washed with ethanol and di water in preparation for the gp antibody immobilization. cys ( mg) dissolved in ml of di water ( mm concentration) was then immobilized on the electrode by self-assembly for h at room temperature. the cys-immobilized electrode was washed with di water to remove any unbound cys. then, the gp antibody was attached to cys using the edc/nhs reaction. to achieve this, µl of the antibody solution (concentration: µg/ml) was mixed with µl of edc (concentration: mg/ml) and µl of nhs (concentration: mg/ml) for h at room temperature. then, the modified gp antibody with edc/nhs was dropped on the cys-immobilized electrode for h at room temperature. in addition, the gp antibody-immobilized electrode was washed with di water to remove any unbound gp antibody and dried with n gas. unbound cys. then, the gp antibody was attached to cys using the edc/nhs reaction. to achieve this, μl of the antibody solution (concentration: μg/ml) was mixed with μl of edc (concentration: mg/ml) and μl of nhs (concentration: mg/ml) for h at room temperature. then, the modified gp antibody with edc/nhs was dropped on the cys-immobilized electrode for h at room temperature. in addition, the gp antibody-immobilized electrode was washed with di water to remove any unbound gp antibody and dried with n gas. electrochemical properties of the fabricated biosensor were analyzed by electrochemical analyzer chi- e (ch instruments, inc., austin, tx, usa). cv and swv were conducted with a three-electrode system composed of the fabricated working electrode, a platinum (pt) wire counter electrode, and a silver/silver chloride (ag/agcl) reference electrode. the parameters for the cv experiment were as follows: sampling interval of mv/s, the scan rate of mv/s, and × − (a/v) sensitivity. the applied voltage range was from mv to − mv. the parameters for the swv experiment were as follows: amplitude of mv, frequency of hz, and × − (a/v) sensitivity. the applied voltage range was from mv to − mv. the electrochemical experiments were conducted with pbs solution (ph = . ) containing mm k fe(cn) and mm k fe(cn) which were used as the redox generator for electrochemical investigation. the flexibility of the fabricated biosensor was investigated using a micro-fatigue tester. to estimate the flexibility, the electrode was fixed to the tester and the tip of the tester was moved down to apply force on the electrode. the electrode was bent by the force applied to the center of the electrode. the elongation of the electrode by the applied force was measured to investigate the flexibility of the electrode. the fabricated biosensor was compared with a widely used rigid gold coated silicon electrode. the conformation and composition of the synthesized mos nps were investigated by tem and eds analysis. as shown in figure a , the tem images indicated that the synthesized mos nps were structured as well-dispersed nanospheres. the eds and eds mapping results are shown in figure b and figure s . eds and eds mapping images showed that the ratio of molybdenum (mo) and sulfur (s) was approximately : , which matched the theoretical composition of mos . additionally, the average size of mos (concentration: mg/ml) was investigated by dynamic light scattering (dls). the measured dls data indicated that . % of synthesized mos were about nm to nm, and . % of mos were over nm (figure c fabrication of the au/mos /au nanolayer on the pet substrate was confirmed by afm and fe-sem. figure a -c shows afm images of the pet substrate, au sputter coated pet substrate, and au/mos on the pet substrate, respectively. in figure a , the afm results of the pet substrate indicated a height of . nm. when au was sputter coated on the pet substrate, the height of the substrate increased to . nm due to the deposited au as shown in figure b . the mos nps immobilized on the au coated pet substrate by spin coating was shown in figure c , the height of the immobilized mos nps was about . nm. in addition, figure d -f shows fe-sem images of the fabricated au/mos /au nanolayer on the pet substrate. compared to the results in figure e , the existence of the sputter coated au particles on mos nps was confirmed in figure f . eds analysis of all the acquired sem images is shown in figure s . the amount of sputter coated gold in the au/mos /au nanolayer on the pet substrate was doubled to the au/mos on the pet substrate due to the twice gold deposition. in addition, the eds mapping result of the au/mos /au nanolayer on the pet substrate is shown in figure s . also, the cv result is shown in figure a for confirmation of the fabrication of the au/mos /au nanolayer on the pet substrate. in figure a , the cv results of the pet substrate showed no electrochemical signals, but signals were observed after au sputter coating on the pet substrate. also, the fabricated au/mos /au nanolayer on the pet substrate had higher electrochemical signals than the conventional bare gold electrode composed of gold ( nm)/cr ( nm)/sio (silicon dioxide). the current increase of the au/mos /au nanolayer on the pet substrate compared to conventional bare gold electrode was due to the large surface area and the efficient electron transfer by mos nps. fabrication of the au/mos /au nanolayer on the pet substrate was confirmed by afm and fe-sem. figure a -c shows afm images of the pet substrate, au sputter coated pet substrate, and au/mos on the pet substrate, respectively. in figure a , the afm results of the pet substrate indicated a height of . nm. when au was sputter coated on the pet substrate, the height of the substrate increased to . nm due to the deposited au as shown in figure b . the mos nps immobilized on the au coated pet substrate by spin coating was shown in figure c figure s . the amount of sputter coated gold in the au/mos /au nanolayer on the pet substrate was doubled to the au/mos on the pet substrate due to the twice gold deposition. in addition, the eds mapping result of the au/mos /au nanolayer on the pet substrate is shown in figure s . also, the cv result is shown in figure a for confirmation of the fabrication of the au/mos /au nanolayer on the pet substrate. in figure a , the cv results of the pet substrate showed no electrochemical signals, but signals were observed after au sputter coating on the pet substrate. also, the fabricated au/mos /au nanolayer on the pet substrate had higher electrochemical signals than the conventional bare gold electrode composed of gold ( nm)/cr ( nm)/sio (silicon dioxide). the current increase of the au/mos /au nanolayer on the pet substrate compared to conventional bare gold electrode was due to the large surface area and the efficient electron transfer by mos nps. nanomaterials , , x for peer review of cv and swv were performed to confirm the electrochemical properties of the fabricated biosensor with a [fe(cn) ] −/ − redox probe. figure b shows the cv result of the au/mos /au nanolayer on the pet substrate, the cys/au/mos /au nanolayer on the pet substrate, the ab/cys/au/mos /au nanolayer on the pet substrate. the reduction and oxidation peak currents of the au/mos /au nanolayer on the pet substrate was found to be . ma and − . ma, respectively. after cys immobilization, the reduction and oxidation peak potentials were changed from . v and . v to . v and . v, and the reduction and oxidation peak potentials were also increased to . ma and − . ma, respectively. this increase was due to the electrostatic interaction between the negatively charged [fe(cn) ] -/ -and the positively charged amine group of the cys immobilized on the au/mos /au nanolayer on the pet substrate. however, when the gp antibody was immobilized on the cys, the efficiency of electron transfer between the multilayer and the redox probe was reduced by the gp antibody, and the reduction and oxidation peak currents were lowered to . ma and - . ma, respectively. in addition, to confirm the reproducibility of fabricated biosensor, the average reduction peak current was investigated with four different measurements. as shown in figure s , the average reduction peaks of au/mos /au nanolayer on the pet substrate, cys/au/mos /au nanolayer on the pet substrate and ab/cys/au/mos /au nanolayer on the pet substrate were . ma, . ma, and . ma. in figure c , the cv results were obtained by increasing the scan rate from mv/s to mv/s to verify the relationship between the current peak and the scan rate of the fabricated the au/mos /au nanolayer on the pet substrate. figure d showed that the plotted reduction and oxidation peak currents of the measured cv showed a linear response to the increase in the scan rate. cv and swv were performed to confirm the electrochemical properties of the fabricated biosensor with a [fe(cn) ] −/ − redox probe. figure b shows the cv result of the au/mos /au nanolayer on the pet substrate, the cys/au/mos /au nanolayer on the pet substrate, the ab/cys/au/mos /au nanolayer on the pet substrate. the reduction and oxidation peak currents of the au/mos /au nanolayer on the pet substrate was found to be . ma and − . ma, respectively. after cys immobilization, the reduction and oxidation peak potentials were changed from . v and . v to . v and . v, and the reduction and oxidation peak potentials were also increased to . ma and − . ma, respectively. this increase was due to the electrostatic interaction between the negatively charged [fe(cn) ] −/ − and the positively charged amine group of the cys immobilized on the au/mos /au nanolayer on the pet substrate. however, when the gp antibody was immobilized on the cys, the efficiency of electron transfer between the multilayer and the redox probe was reduced by the gp antibody, and the reduction and oxidation peak currents were lowered to . ma and − . ma, respectively. in addition, to confirm the reproducibility of fabricated biosensor, the average reduction peak current was investigated with four different measurements. as shown in figure s , the average reduction peaks of au/mos /au nanolayer on the pet substrate, cys/au/mos /au nanolayer on the pet substrate and ab/cys/au/mos /au nanolayer on the pet substrate were . ma, . ma, and . ma. in figure c , the cv results were obtained by increasing the scan rate from mv/s to mv/s to verify the relationship between the current peak and the scan rate of the fabricated the au/mos /au nanolayer on the pet substrate. figure d showed that the plotted reduction and oxidation peak currents of the measured cv showed a linear response to the increase in the scan rate. nanomaterials , , x for peer review of the swv technique was performed to investigate the electrochemical detection performance and selectivity of the fabricated biosensor. to confirm its detection, the gp antigen dissolved in pbs solution was immobilized on the prepared ab/cys/au/mos /au nanolayer on the pet substrate for h at room temperature. the detection of the gp antigen at a concentration of . pg/ml to ng/ml was confirmed by swv. as shown in figure a , the electron transfer reaction between the redox probe and the biosensor surface was blocked by the antigen-antibody binding on the surface of the fabricated biosensor, and the current value was decreased when the concentration of the gp antibody was increased. in addition, the linearity of the current value for the gp antigen concentration was confirmed through the results obtained from swv. as shown in figure b , the gp antigen ranged from . pg/ml to ng/ml and showed excellent linearity with . of the coefficient of determination value (r ). in general, the concentration of gp in hiv-infected patients is approximately pg/ml to pg/ml, therefore the fabricated biosensor showed the possibility for gp detection in practice. the mean value and the error bars were plotted with the standard deviation (sd) from four different measurements. the detection limit of an electrochemical biosensor composed of the au/mos /au nanolayer on the pet substrate for hiv detection was . pg/ml based on the × (s/m) method, where s is the standard deviation of the blank signal and m is slope of the linear fitting curve, and this value was compared with other electrodes shown in table . to confirm the detection ability for the gp antigen prepared from a real sample, human serum was mixed with the gp antigen, and the detection of the gp antigen was confirmed by swv. for real sample analysis, the gp with different concentrations from pg/ml to ng/ml was mixed with serum. as shown in figure c , in the case of the gp antigen prepared in serum, the current the swv technique was performed to investigate the electrochemical detection performance and selectivity of the fabricated biosensor. to confirm its detection, the gp antigen dissolved in pbs solution was immobilized on the prepared ab/cys/au/mos /au nanolayer on the pet substrate for h at room temperature. the detection of the gp antigen at a concentration of . pg/ml to ng/ml was confirmed by swv. as shown in figure a , the electron transfer reaction between the redox probe and the biosensor surface was blocked by the antigen-antibody binding on the surface of the fabricated biosensor, and the current value was decreased when the concentration of the gp antibody was increased. in addition, the linearity of the current value for the gp antigen concentration was confirmed through the results obtained from swv. as shown in figure b , the gp antigen ranged from . pg/ml to ng/ml and showed excellent linearity with . of the coefficient of determination value (r ). in general, the concentration of gp in hiv-infected patients is approximately pg/ml to pg/ml, therefore the fabricated biosensor showed the possibility for gp detection in practice. the mean value and the error bars were plotted with the standard deviation (sd) from four different measurements. the detection limit of an electrochemical biosensor composed of the au/mos /au nanolayer on the pet substrate for hiv detection was . pg/ml based on the × (s/m) method, where s is the standard deviation of the blank signal and m is slope of the linear fitting curve, and this value was compared with other electrodes shown in table . to confirm the detection ability for the gp antigen prepared from a real sample, human serum was mixed with the gp antigen, and the detection of the gp antigen was confirmed by swv. for real sample analysis, the gp with different concentrations from pg/ml to ng/ml was mixed with serum. as shown in figure c , in the case of the gp antigen prepared in serum, the current value decreased with increase in concentration, the same as the gp antigen dissolved in pbs. the selectivity of the ab/cys/au/mos /au nanolayer on the pet substrate-based biosensor was measured by the addition of various types of antigens and proteins including hb, mb, psa, and trx prepared in pbs solution. the concentrations of the gp antigen, hb, mb, psa, and trx were fixed at ng/ml. as shown in figure d , the gp antigen had a low current value of . ma, while hb, mb, psa, and trx had high current values of . ma, . ma, . ma, and . ma, respectively. in addition, the rate of change of current value of gp antigen, hb, mb, psa, and trx were indicated . %, . %, . %, . %, and . %. the mean value and the error bars were obtained as the standard deviation (sd) of five measurements. as shown in the results of the selectivity test, the measurement of gp was confirmed since the current value of the gp antigen, a surface protein of hiv was smaller than that of other antigens and proteins, and the electrochemical signal decreased due to selective immobilization of the gp . value decreased with increase in concentration, the same as the gp antigen dissolved in pbs. the selectivity of the ab/cys/au/mos /au nanolayer on the pet substrate-based biosensor was measured by the addition of various types of antigens and proteins including hb, mb, psa, and trx prepared in pbs solution. the concentrations of the gp antigen, hb, mb, psa, and trx were fixed at ng/ml. as shown in figure d , the gp antigen had a low current value of . ma, while hb, mb, psa, and trx had high current values of . ma, . ma, . ma, and . ma, respectively. in addition, the rate of change of current value of gp antigen, hb, mb, psa, and trx were indicated . %, . %, . %, . %, and . %. the mean value and the error bars were obtained as the standard deviation (sd) of five measurements. as shown in the results of the selectivity test, the measurement of gp was confirmed since the current value of the gp antigen, a surface protein of hiv was smaller than that of other antigens and proteins, and the electrochemical signal decreased due to selective immobilization of the gp . the flexibility of the fabricated biosensor was confirmed by a micro-fatigue tester. figure a ,b show flexibility results of a conventional bare gold electrode, au sputter coated pet substrate, and the au/mos /au nanolayer on the pet substrate. in figure a , the flexure extension results of the conventional bare gold electrode were . mm due to the hardness of the electrode. sio -based rigid substrates were hard to bend using applied forces due to their lack of flexibility. however, au sputter coated pet and the au/mos /au nanolayer on the pet substrate showed excellent flexibility and flexure extension with . mm and . mm, respectively. these results were significantly higher than those of the conventional electrode because these electrodes were easily bent by the applied force due to their excellent flexibility. in addition, the au sputter coated pet and au/mos /au nanolayer on the pet substrate had flexure strength of . mpa and . mpa, respectively, which were lower than that of the conventional gold electrode due to the excellent flexibility. as shown in figure b , the conventional gold electrode was found to be rapidly damaged by strong forces due to the hardness of the electrode. however, the fabricated biosensor had a high flexure extension compared with the conventional gold electrode because of the small force applied to the substrate due to the characteristics of the flexible substrate. in addition, swv was performed to investigate electrochemical detection performance of the bent biosensor. to confirm the detection performance, ng/ml of gp antigen was immobilized on the ab/cys/au/mos /au nanolayer on the pet substrate. as shown in figure s , the current value of fabricated biosensor before bent was indicated . ma, and after the fabricated biosensor was bent, the current value was maintained to . ma. the flexibility of the fabricated biosensor was confirmed by a micro-fatigue tester. figure a ,b show flexibility results of a conventional bare gold electrode, au sputter coated pet substrate, and the au/mos /au nanolayer on the pet substrate. in figure a , the flexure extension results of the conventional bare gold electrode were . mm due to the hardness of the electrode. sio -based rigid substrates were hard to bend using applied forces due to their lack of flexibility. however, au sputter coated pet and the au/mos /au nanolayer on the pet substrate showed excellent flexibility and flexure extension with . mm and . mm, respectively. these results were significantly higher than those of the conventional electrode because these electrodes were easily bent by the applied force due to their excellent flexibility. in addition, the au sputter coated pet and au/mos /au nanolayer on the pet substrate had flexure strength of . mpa and . mpa, respectively, which were lower than that of the conventional gold electrode due to the excellent flexibility. as shown in figure b , the conventional gold electrode was found to be rapidly damaged by strong forces due to the hardness of the electrode. however, the fabricated biosensor had a high flexure extension compared with the conventional gold electrode because of the small force applied to the substrate due to the characteristics of the flexible substrate. in addition, swv was performed to investigate electrochemical detection performance of the bent biosensor. to confirm the detection performance, ng/ml of gp antigen was immobilized on the ab/cys/au/mos /au nanolayer on the pet substrate. as shown in figure s , the current value of fabricated biosensor before bent was indicated . ma, and after the fabricated biosensor was bent, the current value was maintained to . ma. in this study, the flexible biosensor based on a au/mos /au nanolayer on a pet substrate was developed to detect gp with high sensitivity. to develop the flexible biosensor, the au/mos /au nanolayer was fabricated by au sputter coating and mos nps spin coating onto a flexible pet substrate. the fabricated au/mos /au nanolayer on the pet substrate showed the well-oriented nps and uniform nanolayer formation on the pet substrate. the reduction and oxidation peak currents of the au/mos /au nanolayer on the pet substrate derived from the redox generator were . ma and − . ma, respectively, which were much higher than those peaks of the bare gold electrodes with . ma and − . ma due to the large surface area and effective electron transfer of the synthesized mos nps. the fabricated biosensor showed highly sensitive detection of gp with a detection limit of . pg/ml, which was more sensitive than previously reported electrochemical hiv biosensors. this biosensor showed a selective detection of gp added with various antigens and proteins such as hb, mb, psa, and trx. in addition, this biosensor showed excellent flexibility with flexure extension of . mm compared to sio -based conventional gold electrodes, and the fabricated biosensor maintained the detection performance after bending. in conclusion, the proposed flexible biosensor based on a au/mos /au nanolayer on a pet substrate can suggest the milestone for nanomaterial-based flexible sensing platform to develop the highly sensitive biosensors with flexibility for a wearable device application. in addition, because the gp concentration range of the hiv infected patient can be measured, it can be used in the commercial field. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , figure s : tem and eds analysis of mos nps. figure s : sem and eds analysis of (a) au sputter coated pet substrate, (b) the au/mos on the pet substrate and (c) the au/mos /au nanolayer on the pet substrate. figure s : eds mapping results of the au/mos /au nanolayer on the pet substrate. figure s : the reproducibility of reduction peaks of the au/mos /au nanolayer on the pet substrate, cys/au/mos /au nanolayer on the pet substrate and ab/cys/au/mos /au nanolayer on the pet substrate. error bars indicate the standard deviations of four different measurements. figure s : reduction peak currents of the ng/ml of gp antigen on the ab/cys/au/mos /au nanolayer on the pet substrate before bending and after bending. error bars indicate the standard deviations of four different measurements. a wrinkled ag/cnts-pdms composite film for a high-performance flexible sensor and its applications in human-body single monitoring direct delamination of graphene for high-performance plastic electronics h o biosensor consisted of hemoglobin-dna conjugate on nanoporous gold thin film electrode with electrochemical signal enhancement engineered gold-based nanomaterials: morphologies and functionalities in biomedical applications streptavidin-conjugated gold nanoclusters as ultrasensitive fluorescent sensors for early diagnosis of hiv infection gold nanostructures on flexible substrates as electrochemical dopamine sensors a facile and efficient protocol for preparing residual-free single-walled carbon nanotube films for stable sensing applications a flexible and highly sensitive nonenzymatic glucose sensor based on dvd-laser scribed graphene substrate nanostructured surfaces for analysis of anticancer drug and cell diagnosis based on electrochemical and sers tools enzymatic and non-enzymatic electrochemical glucose sensor based on carbon nano-onions a high-sensitive and fast-fabricated glucose biosensor based on prussian blue/topological insulator bi se hybrid film wse nanoparticles with enhanced hydrogen evolution reaction prepared by bipolar electrochemistry: application in competitive magneto-immunoassay biosensor based on ultrasmall mos nanoparticles for electrochemical detection of h o released by cells at the nanomolar level flexible electrochemical glucose biosensor based on gox/gold/mos /gold nanofilm on the polymer electrode shape-controlled gold nanoparticles supported on mos nanosheets: synergistic effect of thionine and mos and their application for electrochemical label-free immunosensing highly sensitive and flexible strain-pressure sensors with cracked paddy-shaped mos /graphene foam/ecoflex hybrid nanostructures highly flexible room temperature no sensor based on mwcnts-wo nanoparticles hybrid on a pet substrate coatings of indium tin oxide nanoparticles on various flexible polymer substrates: influence of surface topography and oscillatory bending on electrical properties a global perspective on hiv/aids massive covert infection of helper t lymphocytes and macrophages by hiv during the incubation period of aids brief but efficient: acute hiv infection and the sexual transmission of hiv quantum dot-based hiv capture and imaging in a microfluidic channel distribution and three-dimensional structure of aids virus envelope spikes plasmonic elisa for the ultrasensitive detection of disease biomarkers with the naked eye a simple and rapid dna extraction method from whole blood for highly sensitive detection and quantitation of hiv- proviral dna by real-time pcr polymerase chain reaction-free detection of hepatitis b virus dna using a nanostructured impedance biosensor sensitive electrochemical detection of gp based on the combination of nbd- and gp design and expression of a short peptide as an hiv detection probe towards hiv detection: novel poly(propylene imine) dendrimer-streptavidin platform for electrochemical dna and gp aptamer biosensors electrochemical sensor based on direct electron transfer of hiv- virus at au nanoparticle modified ito electrode this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest. key: cord- -ipe w y authors: evelyn, obando; jaime, fernández-sarmiento; david, montoya; lorena, acevedo; jenifer, arroyave; oscar, gamboa title: prevalence, clinical outcomes and rainfall association of acute respiratory infection by human metapneumovirus in children in bogotá, colombia date: - - journal: bmc pediatr doi: . /s - - -x sha: doc_id: cord_uid: ipe w y background: acute respiratory infections (aris) are one of the main causes of morbidity and mortality in children. viruses are the main etiological agents, and their behavior tends to be seasonal and vary by geographical location. human metapneumovirus (hmpv) has recently been described as a cause of severe acute respiratory infection and its prevalence and clinical behavior in children at moderate altitudes is unknown. methods: a cross-sectional study was carried out on patients seen at a university hospital in bogotá, colombia between october and december in a city at a moderate altitude above sea level. children with acute respiratory infections who had had a multiplex rt-pcr assay were selected. the prevalence of hmpv infection, its clinical outcomes and its relationship to rainfall were evaluated. results: out of a total of , discharged patients, multiplex rt-pcr was performed on and a virus was detected in children with acute respiratory infection (ari). the study group had a median age of months (iqr – ), with similar proportion of males and females ( . and . % respectively) and . % (ci . – . %) prevalence of hmpv infection. the group with hmpv infection showed a greater frequency of viral coinfection ( . % vs % p = . ) compared with ari caused by other viruses. the rate of bacterial coinfection (p = . ), presence of comorbidities (p = . ), length of hospital stay (p = . ), need for mechanical ventilation (p = . ) and mortality (p = . ) were similar for hmpv and other viral infections. a moderate correlation was established between hmpv infection and rainfall peaks (spearman’s rho . p = . ). conclusions: human metapneumovirus was the fifth most frequently isolated virus in children with ari, had similar clinical behavior and severity to other viruses but a higher rate of viral coinfection. its peaks seem to correlate to rainy seasons. acute respiratory infections (aris) are one of the main contributors to the burden of disease in pediatrics. it is estimated that children under years of age have an average of three to six episodes of ari per year, with ari being the second cause of death in this age group according to the world health organization (who) [ ] . viruses are the most commonly isolated microorganisms in acute respiratory infection, both in adults and in children. every year in the united states, between , to , children under years of age are hospitalized due to respiratory syncytial virus (rsv) and influenza virus infections [ ] . discovered in by dr. van den hoogen, human metapneumovirus (hmpv), belonging to the paramyxoviridae family, has circulated for more than five decades [ ] , but its importance as an etiologic agent of upper and lower ari, with the potential for developing severe disease is emergent. hmpv infection is responsible for approximately - % of ari hospitalizations in pediatrics [ ] , of those affected - % needs transfer to intensive care and - % requires mechanical ventilation [ ] . during - in the united states, carly et al. estimated that each pediatric patient hospitalized for acute respiratory infection due to hmpv may spend an average of dollars per hospitalization, which is much greater than the cost of other viruses and other chronic conditions [ ] . it has been suggested that the prevalence of hmpv has seasonal and geographical variations [ ] . colombia, located near the equator, is a tropical country with five natural regions; each region maintains an average temperature throughout the year so the rainfall is the most important climatic variable that affects viral circulation. bogotá is located in the andean region, is one of the coldest cities in the country, located at an altitude of m above sea level, acute respiratory infections have been thought to have a different behavior at higher altitudes in terms of higher frequency and severity. our objective is to estimate the prevalence and describe the clinical behavior of ari caused by hmpv in pediatric patients hospitalized in a fourth level hospital in bogotá and evaluate its association with the rainfall variations. a cross-sectional study was performed on children under years of age hospitalized at the fundación cardioinfantil-ic, a tertiary university hospital located in bogotá, colombia. a total of , patients were discharged between october and december , out of whom children had a multiplex rt-pcr (filmar-ray® biomériux). this city is located m above sea level with the characteristic weather and rains patterns of tropical countries and the andes mountains. it is characterized by an annual mean temperature of °c with variations between and °c, and peaks of rain from march to may each year, as well as from october to november. this study was approved by the institutional ethics committee (protocol: pm- - ). the data was taken from the institutional electronic charts of children who were hospitalized for acute respiratory infections and who received multiplex rt-pcr (filmarray® biomériux), the analyte used in this technique for hmpv detection was type , a ia - zeptometrix cf. patients who had had this test without experiencing respiratory symptoms and those who had been out of the city in the weeks prior to hospitalization were excluded. the respiratory multiplex rt-pcr assay (filmarray® biomériux) was ordered for patients with upper and lower respiratory symptoms at the attending physician's discretion, and samples were taken from nasopharyngeal aspirates by the respiratory therapy specialist and processed within min of collection, which is an institutional standard for sample handling. the rt-pcr detects viruses and bacteria and is currently considered to be the gold standard for detecting these microorganisms. clinical behavior was assessed in terms of the main complications previously described for hmpv, such as length of hospital stay, need for transfer to intensive care, need for ventilatory support and duration of mechanical ventilation. acute respiratory infections were classified as rhinopharyngitis, laryngitis, croup, bronchiolitis, tracheitis, pneumonia or acute respiratory distress syndrome (ards), according to the criteria of the attending physician. severe ari was defined as the need for oxygen with a fraction of inspired oxygen (fio ) greater than %; the need for non-invasive mechanical ventilation, high flow nasal cannula ventilation or invasive mechanical ventilation; and/or hemodynamic instability requiring vasoactive support. patients in whom hmpv was identified h after admission, and who did not have respiratory symptoms on admission, were considered to have a nosocomial infection. viral coinfection was established if two or more respiratory viruses were isolated on the rt-pcr assay, and bacterial coinfection was determined if blood cultures and/or orotracheal secretion cultures were positive and /or procalcitonin was > . μg/l. xadditionally, the ideam (institute of hydrology, meteorology and environmental studies) database was used. this is the governmental institution responsible for the analysis and detection of climate and meteorological changes in colombia. monthly rainfall patterns for the described study period were analyzed looking for an association with a higher or lower frequency of acute viral respiratory infections. statistical analyses were performed using stata . (statacorp lp, college station, tx), using descriptive statistics for the demographic variables and central tendency and dispersion measure for continuous variables, according to the distribution of the variable defined by the shapiro wilk normality test. absolute and relative frequencies were described for qualitative variables. the prevalence was estimated for the described observation period. for factors related to severity, a bivariate analysis was performed using student's t-test for independent samples, when the variable was quantitative and parametric. otherwise, a wilcoxon rank-sum test was used. for categorical variables, tests of independence were performed using chi-square. if this was not possible, due to the number of observations, fischer's exact test was performed. for seasonality analysis, the monthly number of infections due to human metapneumovirus was calculated, as well as the number of respiratory syncytial virus and rhinovirus/enterovirus infections, using a spearman's correlation analysis for non-parametric variables. out of a total of , pediatric patients seen during the research period, multiplex rt-pcrs were performed on hospitalized patients. altogether, were excluded because the test had been performed on a patient without a diagnosis of acute respiratory infection or the patient had traveled outside of bogotá during the weeks prior to being hospitalized. a total of patients were included; the median age in the analyzed group was months (iqr - ) in the hmpv positive group and (iqr - ) in hmpv negative group (p = . ). the proportion of affected girls and boys was similar between groups (p = . ) ( table ) . in ( . %) cases, at least one microorganism was detected, the most frequently isolated etiological agents being rhinovirus/enterovirus ( % (ci - %)), rsv ( % (ci - %)), parainfluenza ( . %), and adenovirus ( . %). a . % (ci . - . %) hmpv infection prevalence was found ( patients), making it the fifth most frequently isolated virus ( table ) . within this group, in out of cases, hmpv was the only virus detected. viral coinfection was documented in . % of cases. the most frequent viral association was with the rhinovirus/enterovirus complex in % of children, followed by influenza a/h - and parainfluenza . in this regard, the frequency of hmpv viral coinfection is greater ( . vs % p = . ) than the viral coinfection of the other viruses detected. bacterial coinfection was documented in . % of patients with hmpv and in % of children with other infections. of the patients studied, . % (n = ) were previously healthy and had no chronic medical conditions. the following comorbidities were found in the entire study group: congenital heart disease ( . %), prematurity ( . %), liver disease ( . %), bronchopulmonary dysplasia ( . %), cancer ( . %), transplant ( . %), kidney disease ( . %), and malnutrition ( . %). no statistically significant differences were found between those with or without metapneumovirus infection and the presence of any of the described comorbidities ( table ) . the most frequent diagnoses for both groups were pneumonia ( %), bronchiolitis ( . %), and rhinopharyngitis ( . %). altogether, . % of metapneumovirus infections were community-acquired, as well as . % of other respiratory infections ( table ) . the median hospital stay for the group with hmpv infection was . days (iqr - ) and for those without hmpv infection was days (iqr - ) (p = . ). of the patients with hmpv infection, . % required admission to the intensive care unit, and in the group without this infection, the rate was . % (p = . ). the need for mechanical ventilation showed a similar behavior between both groups. therefore, . % (n = ) of patients with hmpv infection required ventilatory support ( . % invasive and . % non-invasive) with a median duration of mechanical ventilation of days (iqr [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . similarly, . % ( ) of children without hmpv infection required mechanical ventilation ( . % invasive and . % non-invasive) with no significant difference in terms of need (p = . ) and duration of mechanical ventilation ( days, iqr - ) between the groups ( table ). the global mortality was . % of the study sample (hmpv . % vs non-hmpv . %, p = . ). out of the patients with hmpv, % were found to have severe respiratory infection (table ). this group with severe hmpv, . % were under years old, while only % of those without severe hmpv infection belonged to this age group (p = . ). viral coinfection was more frequent in the group with severe hmpv infection (n = ) ( % vs %, p = . ), compared to those who did not have it. likewise, the bacterial coinfection rate was considerably greater in patients with severe infection ( %) compared to those who did not have severe infection ( %) (p = . ). the rainfall in the city of bogotá was analyzed using the average rainfall reported by the ideam (institute of hydrology, meteorology and environmental studies) monitoring stations (fig. ) . typical tropical zone behavior was observed with a greater frequency of viral infections during the rainy season. the spearman rho correlation analysis showed a moderate correlation (r = . , p < . ) between the presence of hmpv infection and the highest rainfall peaks, just as occurs with rsv (r = . , p < . ) (fig. ) . however, this correlation was not seen with the rhinovirus/enterovirus complex, which was the most frequently detected co-infecting agent for hmpv (r = − . ). in this study, we investigated the prevalence of hmpv in pediatric patients hospitalized in bogota, colombia. we found a . % prevalence of hmpv infection (ci % - - . %). it was the fifth virus detected in hospitalized patients. in latin america, the prevalence in hospitalized patients is highly variable, being reported as high as % in the southern brazil [ ] , % in argentina [ ] and . % in uruguay [ ] . this behavior is most likely explained by the variable climate changes between the latin american countries and the techniques used for viral detection [ ] . hmpv has often been described as a coinfectant with other viruses. in children, viral coinfection is highly variable for hmpv [ ] . in spain, there was an estimated rate of % [ ] , in jordan it was . % [ ] , and in south china it was . % [ ] . the viral coinfection rate has not been described for hmpv in children in colombia, which in our study was %. however, we found a greater frequency of viral coinfection in the hmpv group compared with coinfection of the other respiratory viruses, and this difference was statistically significant ( . % vs %, p = . ). the most frequently described viral association in respiratory infection has been between rsv and hmpv. bear in mind that they belong to the same viral family and have a high genetic similarity, usually presented during the same epidemiological periods. in a recent article, schuster et al. found that rsv was the main coinfecting agent, with a global rate of coinfection of . % [ ] . in our study, rhinovirus/enterovirus was the main agent presenting simultaneously with hmpv in % of cases. contrary to the findings of this author, we did not find rsv coinfection in these children. with respect to bacterial coinfection, garcía-garcía et al. reported that . % of the patients in their study received concomitant antibiotic treatment due to suspected bacterial infection associated with hmpv infection [ ] , but the frequency of presentation is not documented. in our study, we found that . % of patients positive for hmpv had bacterial coinfection documented by pcr, cultures or positive procalcitonin techniques. regarding the clinical behavior of hmpv infection, it has been reported that . - % of affected patients require admission to intensive care [ , ] . in our study, [ ] , they found that the duration of ventilatory support in patients with hmpv was days and that % of their children required invasive ventilatory support. we found that children who required a picu stay had pneumonia more often than children with hmpv who did not need intensive care ( . % vs % respectively), which could partially explain these findings. inpatient outbreaks of rsv in children have been described on hospital floors and in intensive care units [ ] , but inpatient hmpv outbreaks are rarely reported [ ] . in this study, one out of five children acquired the hmpv infection during their hospital stay. nosocomial hmpv infections with molecular confirmation have been described in cancer patients, and outbreaks have also been described in geriatric institutions and on psychiatric wards [ , ] . contact seems to be required for viral transmission; the analysis of this situation forces us to look for strategies to decrease inpatient viral transmission by improving our isolation conditions and patient management [ ] . more studies are needed to determine the risk factors for acquiring nosocomial hmpv infection. considering that bogotá is located in a zone without seasons, it does not have the drastic temperature changes like other countries. the most important climatic variable that can affect viral circulation is rainfall. it has been postulated that viral transmission increases in colder seasons. also, that the antiviral immune response in the nasal epithelium is attenuated under these circumstances [ , ] . previous studies have shown that hmpv may have alternating epidemiological profiles in europe. it has been found that the percentage of annual hmpv infection may vary year to year, and may oscillate between . and . % in the same region [ , ] . the annual prevalence difference in our study was . % in and . % in . a greater frequency was noted in april-may , april-june and september-october , with a low positive correlation between hmpv infection and the highest rainfall peaks (r = . , p < . ). the differences in this circulation pattern, besides being related to changes in rainfall, may be associated with the circulation of different hmpv serotypes [ ] . four different hmpv lineages are known, designated as a , a , b and b . these may coexist in the same period or one may be predominant [ ] . the respiratory rt-pcr used in this study does not allow for serotype differentiation, and therefore no conclusion can be drawn regarding their differential behavior. our study has several limitations. the findings are from a single tertiary care center, and although multiplex rt-pcr was used, it was not performed on all hospitalized patients with acute respiratory infections due limitations of health system. likewise, this test does not identify serotypes (a , a , b , b ) which could partially explain some different seasonal behaviors of the virus. similarly, the study design allowed us to observe the behavior of the virus over a specific time period; this behavior may change according to serotypes and environmental variations, among others. additionally, our data only represents association because we cannot infer that hmpv was the only causal factor of the observed disease, especially in cases with viral coinfection. human metapneumovirus was the fifth most commonly detected acute respiratory infection virus in this study. its clinical behavior at moderate altitudes in terms of severity is similar to that of other respiratory viruses. these children frequently need to be transferred to intensive care and have viral coinfection. we observed that this infection presents more frequently during the rainy season. united nations children's fund (unicef) incidence, morbidity, and costs of human metapneumovirus infection in hospitalized children a newly discovered human metapneumovirus isolated from young children with respiratory tract disease human metapneumovirus: review of an important respiratory pathogen human metapneumovirus infections are associated with severe morbidity in hospitalized children of all ages population-based incidence of human metapneumovirus infection among hospitalized children human metapneumovirus in southern brazil evidence of human metapneumovirus in children in argentina investigación de metapneumovirus humano en pacientes hospitalizados: estudio multicéntrico relationship between meteorological conditions and respiratory syncytial virus in a tropical country rapid detection of respiratory organisms with the filmarray respiratory panel in a large children's hospital in china human metapnuemovirus infections in hospitalized children and comparison with other respiratory viruses. - prospective study human metapneumovirus infection in jordanian children. epidemiology and risk factors for severe disease epidemiological and clinical features of human metapneumovirus in hospitalised paediatric patients with acute respiratory illness: a cross-sectional study in southern china burden of human metapneumovirus infection in young children infección por metapneumovirus humano en niños hospitalizados por una enfermedad respiratoria aguda grave: descripción clínico-epidemiológica nosocomial respiratory syncytial virus infections in children's wards molecular epidemiological investigation of a nosocomial outbreak of human metapneumovirus infection in a pediatric hemato-oncology patient population serious outbreak of human metapneumovirus in patients with hematologic malignancies viral respiratory infections diagnosed by multiplex polymerase chain reaction in pediatric patients analysis of the thermal and ph stability of human respiratory syncytial virus temperature-dependent innate defense against the common cold virus limits viral replication at warm temperature in mouse airway cells biennial spring activity of human metapneumovirus in austria severe acute respiratory syndrome in children the role of human metapneumovirus in upper respiratory tract infections in children: a -year experience publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank the medical, nursing and respiratory therapy team of the pediatric icu for their kind cooperation, as well as the clinical laboratory of the fundación cardioinfantil-ic. authors' contributions drs. fj, oe, md and go contributed to designing and performing the study. drs. oe, al, md and aj participated in data collection. drs. fj, oe and go supervised study development and data collection. all the authors contributed to drafting the manuscript and reviewing the final article. all the authors approved the final manuscript and agreed with all aspects of the study. none of the investigators declare conflicts of interest. all authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work. our funding for research are researchers' resources. we did not have financing from any other company.availability of data and materials datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. the ethics committee of the fundación cardioinfantil-instituto de cardiología approved this study with committee's reference number ceic - and the parents of the children approved to participate in this study. not applicable. the authors declare that they have no competing interests.author details department of pediatrics and intensive care, universidad de la sabana, fundación cardioinfantil-instituto de cardiología, bogotá, colombia. key: cord- - nnyq p authors: imran, mudassar; usman, muhammad; malik, tufail; ansari, ali r. title: mathematical analysis of the role of hospitalization/isolation in controlling the spread of zika fever date: - - journal: virus res doi: . /j.virusres. . . sha: doc_id: cord_uid: nnyq p the zika virus is transmitted to humans primarily through aedes mosquitoes and through sexual contact. it is documented that the virus can be transmitted to newborn babies from their mothers. we consider a deterministic model for the transmission dynamics of the zika virus infectious disease that spreads in, both humans and vectors, through horizontal and vertical transmission. the total populations of both humans and mosquitoes are assumed to be constant. our models consist of a system of eight differential equations describing the human and vector populations during the different stages of the disease. we have included the hospitalization/isolation class in our model to see the effect of the controlling strategy. we determine the expression for the basic reproductive number r( ) in terms of horizontal as well as vertical disease transmission rates. an in-depth stability analysis of the model is performed, and it is consequently shown, that the model has a globally asymptotically stable disease-free equilibrium when the basic reproduction number r( ) < . it is also shown that when r( ) > , there exists a unique endemic equilibrium. we showed that the endemic equilibrium point is globally asymptotically stable when it exists. we were able to prove this result in a reduced model. furthermore, we conducted an uncertainty and sensitivity analysis to recognize the impact of crucial model parameters on r( ). the uncertainty analysis yields an estimated value of the basic reproductive number r( ) = . . assuming infection prevalence in the population under constant control, optimal control theory is used to devise an optimal hospitalization/isolation control strategy for the model. the impact of isolation on the number of infected individuals and the accumulated cost is assessed and compared with the constant control case. the zika virus is transmitted to humans primarily through aedes mosquitoes and through sexual contact. it is documented that the virus can be transmitted to newborn babies from their mothers. we consider a deterministic model for the transmission dynamics of the zika virus infectious disease that spreads in, both humans and vectors, through horizontal and vertical transmission. the total populations of both humans and mosquitoes are assumed to be constant. our models consist of a system of eight differential equations describing the human and vector populations during the different stages of the disease. we have included the hospitalization/isolation class in our model to see the effect of the controlling strategy. we determine the expression for the basic reproductive number r in terms of horizontal as well as vertical disease transmission rates. an in-depth stability analysis of the model is performed, and it is consequently shown, that the model has a globally asymptotically stable disease-free equilibrium when the basic reproduction number r < . it is also shown that when r > , there exists a unique endemic equilibrium. we showed that the endemic equilibrium point is globally asymptotically stable when it exists. we were able to prove this result in a reduced model. furthermore, we conducted an uncertainty and sensitivity analysis to recognize the impact of crucial model parameters on r . the uncertainty analysis yields an estimated value of the basic reproductive number r = . . assuming infection prevalence in the population under constant control, optimal control theory is used to devise an optimal hospitalization/isolation control strategy for the model. the impact of isolation on the number of infected individuals and the accumulated cost is assessed and compared with the constant control case. the zika virus spreads among humans primarily through an infected mosquito bite, which has been increasing at an alarming incidence rate worldwide over the past few years (dick et al., ) . it belongs to the family of flaviviruses which includes more than fifty viruses, such as dengue, yellow fever, and the west nile virus. this virus was first identified in the zika forests of uganda and east africa during the investigations on the ecology of the yellow fever (anderson et al., ) . the first isolation was made in april of from the serum of pyrexia rhesus monkey caged. the second isolation was made in in the same forest (dick et al., ) . just a year later, in , the virus was recovered from mosquito aedes africanus from the zika forest. the first human case of zika fever was reported in uganda in . the first outbreak of zika fever was reported in on the pacific island of yap, this outbreak caused symptomatic cases. another epidemic outbreak occurred in french polynesia between and . during this time, it was estimated that about , people were reported to have zika like symptoms (anderson et al., ) . epidemic, reporting the most cases of people infected with the zika virus worldwide. in , the state of rio de janeiro alone reported over , probable zika virus infections, however, this number dropped to only cases in . sexual transmission of the zika virus has also been reported from both males and females to their partners (cdc, ; ecdc, ; hastings and fikrig, ; summers and acosta, ) . zika virus can be sexually transmitted from a person who is infected by the virus, even while they are not symptomatic. furthermore, it has been suggested that zika virus can be transmitted from a pregnant woman to her fetus during pregnancy. zika virus can be transferred horizontally as well as vertically. during the zika epidemic, brazilian health officials reported an increase in the number of cases of microcephaly disease, a condition in which a baby's head is smaller than normal in zika affected areas. the main symptoms of zika fever include a fever, the maculopapular rash often spreading from the face to the body, joint and muscle pain, vomiting, or bilateral non-purulent conjunctivitis (ecdc). the first well-documented case of zika fever was reported in and started with a mild headache with later development of a maculopapular rash, fever, and back pain (hayes, ) . the symptoms of zika fever are thus quite similar to those of dengue fever and there is a strong possibility of misdiagnosis in regions where dengue virus is common. the incubation period for the zika virus is between and days (krow-lucal et al., ) . disease-related symptoms are developed within one week of infection for % of the infected individuals and within weeks among % (krow-lucal et al., ) of the infected individuals. the vast majority of infections are not contagious from person to person; however, it may be passed person to person during sex. the virus infection is usually diagnosed by a blood test. the disease symptoms are usually mild and short lasting ( days), and infection may go unrecognized or be misdiagnosed as dengue fever (ecdc). unfortunately, there is no vaccine, antiviral drug, or other modality available to prevent or treat the zika virus infection. zika fever is a preventable but not a curable disease. thus, the only means of controlling the zika virus is to control the mosquitoes that spread the disease and protection during sex. in the past several years, a number of deterministic models for the transmission dynamics of the dengue virus have been studied and analyzed (esteva and vargas, ferguson et al., ; garba et al., garba et al., , kautner et al., ; wearing and rohani, ) . after the zika outbreak, models for zika transmission have been developed (agustoa et al., ; maxiana et al., ; wiratsudakul et al., ) and analyzed. in these models, the authors included the effect of sexual transmission of the disease. in this work, we formulate and study a deterministic model for zika virus transmission including vertical and horizontal transmission of the disease. although esteva (esteva and vargas, ) discussed vertical disease transmission it was among vectors in a dengue transmission. our deterministic model for zika virus transmission includes horizontal and vertical transmission in both humans and vectors. as stated previously, it has been suggested that zika virus can spread to newborns from their mothers, and we therefore feel that an accurate model must include the vertical transmission. our work is an extension of our previous model (imran et al., ) by including a population group that is using controlling measures. in the previous work, we considered death due to the infection and the total human and vector populations were functions of time. the previous model did not possess global stability for both disease-free and endemic equilibriums. we showed a backward bifurcation phenomenon. the current model has constant population size. since death cases reported from zika fever were negligible, we take disease-induced mortality to be zero. there is no backward bifurcation and the steady states results are global. since the only way to control the disease is to isolate patients who have been infected with the zika virus, we included a new population compartment consisting of hospitalized individuals. we have calculated the basic reproductive number associated with our model that guarantees the elimination of the disease. finally, using optimal control techniques, we also propose and analyze the control strategies for decrease infected individuals while minimizing the costs and resources simultaneously. the rest of this paper is organized as follows, the proposed model is presented in section . basic properties and a detailed steady-state analysis of the model are presented in section . in section , we perform a sensitivity and uncertainty analysis of the model parameters and reproductive number associated with our model. section uses ideas from optimal control theory to propose various controlling strategies to overcome zika are proposed. finally, section presents our conclusions and contains a brief discussion of our results. we consider two types of populations in this model one for the humans and for the mosquitoes. the total human and mosquitoes populations at time t, denoted by n h and n v , are constant. the human population is divided into five mutually exclusive groups, susceptible humans s h (t), exposed humans e h (t), infected humans i h (t), isolated or hospitalized individuals h h (t) and recovered humans r h (t). the total vector population is divided into three mutually exclusive classes comprising of susceptible vectors s t the model assumes that the susceptible human population s h (t) has a recruitment rate μ h n h , where n h is total human population and μ h is the natural birth rate of humans. we assume that the birth rate of human population is same as the natural death rate. susceptible individuals get infected with zika fever virus (due to contact with infected vectors) at a rate λ h and thus enter the exposed class e h . in order to consider vertical transmission in our model, we make the assumption (see, e.g., li et al., ) that a fraction of newborn individuals from parents in the e h (t) and i h (t) classes will be infected, and thus remain in e h class before becoming infectious. we have assumed that the hospitalized individuals do not contribute to vertical transmission. population in each class is removed at the natural death rate μ h . we assumed a lifelong immunity for humans who recovered from zika virus. the exposed individuals who got an infection, move to infectious class at a rate ξ. the infected population recovers from the zika fever at a rate θ i and some infected individuals are transferred to hospitalized class at a rate τ. the hospitalized population recovered at a rate of θ h . the susceptible vector population s t ( ) v has a recruitment rate μ n v v , and μ v is a natural death rate of vector population. a fraction of offsprings in the e t ( ) v and i t ( ) v classes will be infected, and thus remain in e h class before becoming infectious. because of this vertical transmission, a fraction of susceptible individuals will enter the exposed class. susceptible vectors are infected with zika virus (due to effective contact with infected humans) at a rate of λ v and thus move to the exposed vector class e v . the susceptible, exposed and infected vectors have natural death rate μ . v in addition, exposed vectors develop symptoms and move to the infected vector class i v at a rate of σ v . it is assumed that infected vectors do not recover, and die at the natural death rate of μ v . as mentioned earlier there is no vaccine available for zika fever. the only way to control this disease is to reduce the contact rate either by killing the mosquitoes or using the protective measure like mosquito repellents, nets etc. the effective contacts will be further reduced by isolating the infected humans. isolation of individuals with disease symptoms constitutes what is probably the first infection control measure since the beginning of recorded human history (hethcote, ) . over the decades, these control measures have been applied, with varying degrees of success, to combat the spread of some emerging and re-emerging diseases such as leprosy, plague, cholera, typhus, yellow fever, smallpox, diphtheria, tuberculosis, measles, ebola, pandemic influenza and, more recently, severe acute respiratory syndrome (gumel et al., ; imran et al., ; lipsitch et al., ; lloyd-smith et al., ) . chavez et al. analyzed a saiqr model in detail, to investigate the effect of isolation on influenza (vivas-barber et al., ) . they used the isolation (quarantine) i-q model, where the infected population is isolated. we have included epidemiological factors like permanent or partial immunity after recovery as well as intervention control measures through the inclusion of a hospitalized (or isolated) class, h h . in this case both the total host population and the vector population are constant. the forces of infection λ h and λ v are given as (garba et al., ) : here we assumed that an individual in h class still can transmit the disease but with lower rate. the value of modification parameter ≤ η < . fig. presents schematic diagram of the model ( ). a description of the variables and parameters of the model ( ) is given in tables and respectively. the model ( ) will be studied on the closed set: is positively invariant and attracting with respect to the model ( ). it can be seen that the solutions are always positive. the right sides of ( ) are smooth, so that initial value problems have unique solutions that exist on maximal intervals. since paths cannot leave , solutions exist for all positive time. thus the model is mathematically and epidemiologically well posed. in this section, we will perform a detailed steady state and stability analysis of the zika fever model presented in section . the model ( ) has a disease free equilibrium (dfe) given by in order to investigate the local stability of the dfe ( ) , the next generation operator method (van den driessche and watmough, ) will be used. following the notation of van den driessche and watmough ( ), the matrix f (for the new infection terms) and the matrix v (of the transition terms) are given, respectively, by progression rate of humans from exposed to infected class τ hospitalization rate of infected individuals σ v progression rate of vectors from exposed to infected class c hv effective contact rate η modification parameter for relative infectiousness of hospitalized humans the basic reproduction number r for our model is given by ( ) when p = q = , vertical transmission is not present in the model; the above r reduces to the basic reproduction number r of a seir model for a vector disease (garba et al., ) . to get a better understanding of the basic reproduction number r in ( ), we rewrite it using taylor expansion about p and q: where ) and . note that r c is the basic reproductive number for horizontal transmission (khan et al., ) . the square root means that the two generations required for an infected vector or host to reproduce itself (van den driessche and watmough, ) . r p + r q is the sum of the number of infected individuals during the mean latent period and the number of infected individuals during the mean infectious period. similarly, r r + r s is the sum of the number of infected vector offsprings during the mean latent period and the number of infected vector offsprings during the mean infectious period. the expression r = r c r p + r c r q + r c r r + r c r s ) represents the total contribution to the infective class made by the exposed and infective individuals of first generation (li et al., ) . the local stability of the disease free equilibrium follows directly from van den driessche and watmough ( ) . we have following result about local and global stability of disease free state: lemma . . the dfe ( ) of the model ( . ), is locally-asymptotically stable if r < , and unstable if r > . ( ) is globally-asymptotically stable in whenever r < . be a solution of ( ) with x = x( ). a comparison theorem will be used for the proof. the equations for the infected components of ( ) can be written as (where the prime denotes the derivative with respect to time), lemma . established the local asymptotic stability of the dfe when r < , or equivalently, ρ(fv − ) < , which is equivalent to all eigenvalues of f − v having negative real parts when r < (van den driessche and watmough, ) . also, f − v has all off-diagonal entries non negative. then this means that the omega limit set of x , ω(x ), is contained in the disease-free space. but, on the other hand, it is straightforward to check that every solution with the initial condition in the disease-free space converges to the epidemiological implication of the above result is that the disease can be eliminated from the population if the basic reproduction number r can be brought down to a value less than unity (that is, the condition r < is sufficient and necessary for disease elimination) irrespective of the size of the initial populations in each class. the stability of the dfe is demonstrated in fig. a . if r > the dfe is unstable in this case and the solutions are attracted to an (apparently unique and stable) endemic equilibrium, as depicted in fig. b . in this section, the existence and stability of endemic equilibrium (ee) of the model ( ) will be discussed. we define endemic equilibrium to be those fixed points of the system ( ) in which at least one of the infected compartments of the model are non-zero. theorem . . the endemic state, , of the model ( ) exists whenever r > . denote an arbitrary endemic equilibrium of the model ( ). also, let solving the equations of the model ( ) for steady-state by setting right hand asides of the model ( ) . where and all k′s are defined above. substituting ( ) in ( ) and simplifying gives, also, substitute ( ) into ( ), we have the following equation in λ h . clearly the model ( ) has no endemic state if r < and one unique endemic state when r > . □ fig. shows the variation in r with the relative fraction of newborn exposed individuals (p) and newborn infected individuals (q). we notice that no less than % of newborn exposed individuals (p) and no more than % of the fraction of newborn infected individuals to bring r less than : fig. shows the effect of hospitalization rate τ of infected individuals on the basic reproductive number r . from this figure, we can see that effective isolation will help to reduce the basic reproductive number. about % of infected individuals should be effectively isolated to bring the basic reproductive number to less than . this plot shows that the effective isolation is helpful in controlling the epidemic of zika virus. ( ) showing the contour plot of r as a function of fraction of newborn exposed individuals (p) and newborn infected individuals (q). theorem . . if r > then the disease is strongly uniformly ρ-persistent: be a solution of model ( ). in this case there exists an endemic steady state. let (that is, x is the disease-free subspace). note that x, as well as + x ℝ \ , are positively invariant. also, all solutions originating in x converge to n as t → ∞. n is asymptotically stable in x. hence n is isolated in x. corollary . in salceanu ( ) )), together with proposition . and lemma . in salceanu ( ) , imply that {n } is also uniformly weakly repelling. then, from theorem . in smith and thieme ( ) we have that the semiflow generated by ( . ) is uniformly weakly ρ-persistent. from the positive invariance of , we have that ( . ) is point dissipative. then, according to theorem . in smith and thieme ( ) , there exists a compact attractor of points for ( ). this, together with uniformly weakly ρpersistent imply ( ) (see smith and thieme, , theorem . ) . in this case there exists an endemic steady state (smith and thieme, ) . □ the local stability of the endemic steady state of the model ( ) is given in the lemma below. theorem . . if r > , then the endemic state of the model ( ) is locally asymptotically stable in for model ( ). for the proof of this above local stability theorem see imran et al. ( ) . for the global stability of the endemic steady state , we consider the ( ) with no hospitalization and a small incubation period so that we can assume that susceptible individuals after infection move to infected class. in this case, it is easily seen that both for the host population and for the vector population the corresponding total population sizes are asymptotically constant. we assumed that in our model the total population is constant. previous results (thieme, ) imply that the dynamics of systems ( ) is qualitatively equivalent to the dynamics of system given by: theorem . . if r > , then the endemic state of ( ) is globally asymptotically stable in . o proof. we will use geometric approach to global-stability method given in li et al. ( ) . let x = (s, i h , i v ) and f(x) denote the vector field of ( ). the jacobian matrix and its second additive compound matrix j [ ] is (see li et al., ) : where now from the system ( ), we can write second and third equation, the variation in the values of the parameters of our model ( ) is a source of uncertainty and sensitivity. in this section, we carry out a parameter base global uncertainty and sensitivity analyses on r . there are a lot of reasons for the sensitivity of the parameters, for example, inadequate data, lack of information about the vertical transmission. we use the latin-hypercube sampling based method to quantify the uncertainty and the sensitivity of r as a function of the model parameters. we use the latin-hypercube sampling based method to quantify the uncertainty and the sensitivity of r as a function of model parameters, namely μ μ θ θ ξ σ p q r s c τ η , , , , , , , , , , , , . the partial rank correlation coefficient (prcc) measures the impact of the parameters on the output variable using the rank transformation of the data to reduce the effects of nonlinearity. the uncertainty analysis (figs. and ) yields an estimated value of r = . with % ci ( . , . ) for the zika fever. the sensitivity analysis suggests that r is highly sensitive to the parameters c θ θ μ τ , , , , . the accuracy and precision in the values of these parameters is vital for the accurate predictions of the model. the estimated parameters are presented in table . one of the goals of this study is to come up with a time-dependent hospitalization/isolation strategy that would minimize the infected population while keeping the costs to a minimum at the same time. m. imran et al. virus research ( ) - optimal control is a very useful mathematical technique that can help us to address these questions. here our goal is to put down infection from the population by increasing the recovered class and to minimize the required resources to control zika fever infection using isolation or hospitalization. the optimal control algorithm we use is based on pontrygain's maximum principle which appends the original model to an adjoint system of differential equations with terminal conditions. the optimal objective system, which characterizes the optimal controls, consists of differential equations of the original model (state system) along with the adjoint differential equations (the adjoint system). the number of equations in the adjoint system is same as that of in the state system. the detailed mechanism of forming the necessary conditions for the adjoint and optimal controls are discussed in fleming and rishel ( ) and pontryagin and boltyanskii ( ) . an important decision while formulating an optimal control problem is deciding how and where to introduce the control in the system of differential equations. the form of the optimal control primarily depends on the system being analyzed and the objective function to be optimized. in this paper, we will propose various strategies to eradicate zika fever using optimal control techniques. the first step is to find an optimal hospitalization schedule that minimizes the number of infectious individuals and the overall cost of hospitalization during a fixed time. we define the control set as u= {τ(t) : ≤ τ(t) ≤ ζ, ≤ t ≤ t, < ζ ≤ , τ(t) is lebesgue measur-able}. here ζ is a positive number and is defined as the maximum value attained during optimal control procedure. our aim is to minimize the associated cost function which is given as: here a i and a n are positive constants used to balance in the size of i(t) and n t ( ) v . further, we used a nonlinear cost function in order to accommodate the impact of variety of factors associated with hospitalization, documented widely in literature, see for instance kirschner et al. ( ) . w is weight associated with quadratic cost due to hospitalization. moreover a linear function has been chosen for cost incurred by infected individuals and the mosquitoes population. our objective is to find an optimal control for hospitalization rate τ * (t) such that . the lagrangian of the optimization problem is given the associated "hamiltonian" is given by where k i represents the right hand side of the ith equation in our original model. w depends on the relative importance of the control measures in mitigating the spread of the disease as well as the cost incurred (such as material resources and human effort) during the implementation of control measure per unit time. pontrayagin's maximum principal converts model ( ) and objective function ( ) into minimizing the hamiltonian ( ) with respect to τ. now we prove the following theorem to elicit the effect of optimal control of hospitalization. theorem . . there exists a unique optimal control τ * (t) which minimizes j over u. also, there exists an adjoint system of ϕ i 's such that the optimal treatment control is characterized as for some positive number ζ. the adjoint system is given as the above adjoint system also satisfies the transversality condition, proof. we can easily verify that the integrand of j is convex with respect to τ(t). also, the solutions of our model are bounded above. in addition, it is verifiable that the model has the lipschitz property with respect to the state variables. using the properties mentioned above along with corollary . of fleming and rishel ( ) , the existence of an optimal control is established. now using the pontryagin's maximum principle, we obtain . therefore on the set {t : < τ * (t) < ζ} we obtain now, we discuss the numerical solutions of the optimality system and the corresponding optimal control obtained using ζ = . . the optimal strategy is obtained by solving the optimal system consisting of both the state system as well as the adjoint system. since there are initial conditions present for the state equations, we start solving them with a guess for τ using the fourth-order forward runge-kutta method. the adjoint equations are then solved using the fourth-order backward runge-kutta method because of the presence of final conditions. then, the controls are updated by using a convex combination of the previous control and the value from the characterization given above. this process is repeated until we obtain a desired accuracy of convergence (fig. ) . fig. represents the optimal isolation(hospitalization) strategy to be employed to minimize the cost and the infected population. considering the practical constraints, an upper bound of . was chosen for the optimal hospitalization control. the figure shows that initially, the optimal level remains at the upper bound of . after which it declines steadily to . this implies that in the early phase of the endemic breakout, keeping the control at the upper bound would help in decreasing the number of infected individuals. fig. captures the dynamics of the infected population (i h ) by virtue of a comparison between the infected host population under optimal control and constant control. it can be seen that the decrease in the number of infected individuals is greater with optimal control as compared to that with constant control. furthermore, in contrast with a constant control, the infected population remains lower when an optimal control is applied. fig. shows a comparison between the costs associated with optimal and different constant control strategies. it is clear that the cost associated with different control strategies is higher as compared to that of optimal control. it is important to note that high constant isolation rate (τ = . ) incurs almost the same cost as the of optimal control. however, practically it is highly unlikely to implement these high constant controls primarily due to the lack of required resources and facilities. fig. captures the effect of the change in effective contact rate over the optimal control strategy. it is clear from the simulation that an increase in the contact rate may or may not lead to higher rates of hospitalization. in this paper, we have presented a zika fever epidemic model comprising of eight compartments consists of vector and human population. the dynamics of zika fever epidemic model have been considered. furthermore, using optimal control theory, we proposed control strategies to eliminate the infection from the population. a vertical and horizontal transmission model for zika fever is constructed in the form of a system of ordinary differential equations. this model features the study of zika fever by considering vertical transmission in both humans as well as vectors. the basic reproductive number r is formulated by using a next-generation matrix. this reproductive number is simplified in order to better understand the effect of vertical transmission parameters. it is shown that the disease-free steady state is globally asymptotically stable when the basic reproductive number (r ) is less than . the model has a unique endemic equilibrium when the reproduction number r exceeds unity. this equilibrium is shown to be globally asymptotically stable when the reproduction number r exceeds unity under the reduced model. it is locally asymptotically stable when we consider a full model. we performed a parameter based global uncertainty and sensitivity analysis on r . the uncertainty analysis yields an estimated value of the basic reproductive number r = . with % confidence interval ( . , . ). this estimated value of r is close to the calculated value of basic reproductive using real data (villela et al., ). our previous model had an estimated basic reproductive number r = . with % confidence interval ( . , . ) given in imran et al. ( ) . our sensitivity analysis on the zika model parameters showed that the most influential parameters are the effective contact rates, the recovery rate of the infected individuals and the birth rate of mosquitoes. we proposed an optimal controlling strategy to eliminate zika fever from the population. we observed that optimal control strategy is most effective in terms of eliminating infection as it minimizes our cost and resources at the same time. moreover, the control measures themselves may take time to implement, once the outbreak has been realized. despite these points, our analysis can help public health authorities to determine quasi-optimal strategies they might want to adopt, especially as our work highlights the relative effectiveness of different control strategies. mathematical model of zika virus with vertical transmission zika virus background zika virus (i). isolations and serological specificity estimated incubation period for zika virus disease analysis of a dengue 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clinical presentation, diagnosis, and prevention estimating the basic reproduction number for single-strain dengue fever epidemics optimal control of the chemotherapy of hiv global dynamics of an seir epidemic model with vertical transmission transmission dynamics and control of severe acute respiratory syndrome curtailing transmission of severe acute respiratory syndrome within a community and its hospital zika virus dynamics. when does sexual transmission matter the mathematical theory of optimal processes pan american health organization, zika cumulative cases robust uniform persistence in discrete and continuous dynamical systems using lyapunov exponents dynamical systems and population persistence zika virus in an american recreational traveler convergence results and a poincar bendixson trichotomy for asymptotically autonomous differential equations zika in rio de janeiro: assessment of basic reproduction number and comparison with dengue outbreaks dynamics of an saiqr influenza model ecological and immunological determinants of dengue epidemics dynamics of zika virus outbreaks: an overview of mathematical modeling approaches the authors declare that there is no conflict of interests regarding the publication of this article. we study the following feasible region of the new system ( ) key: cord- - og umiq authors: xin, shuyu; du, shujuan; liu, lingzhi; xie, yan; zuo, lielian; yang, jing; hu, jingjin; yue, wenxing; zhang, jing; cao, pengfei; zhu, fanxiu; lu, jianhong title: epstein-barr virus nuclear antigen recruits cyclophilin a to facilitate the replication of viral dna genome date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: og umiq epstein-barr virus (ebv) nuclear antigen (ebna )-mediated dna episomal genome replication and persistence are essential for the viral pathogenesis. cyclophilin a (cypa) is upregulated in ebv-associated nasopharyngeal carcinoma (npc) with unknown roles. in the present approach, cytosolic cypa was found to be bound with ebna into the nucleus. the amino acid - of the ebna domain was important for the binding. cypa depletion attenuated and ectopic cypa expression improved ebna expression in ebv-positive cells. the loss of viral copy number was also accelerated by cypa consumption in daughter cells during culture passages. mechanistically, cypa mediated the connection of ebna with orip (origin of ebv dna replication) and subsequent orip transcription, which is a key step for the initiation of ebv genome replication. moreover, cypa overexpression markedly antagonized the connection of ebna to ubiquitin-specific protease (usp ), which is a strong host barrier with a role of inhibiting ebv genome replication. the ppiase activity of cypa was required for the promotion of orip transcription and antagonism with usp . the results revealed a strategy that ebv recruited a host factor to counteract the host defense, thus facilitating its own latent genome replication. this study provides a new insight into ebv pathogenesis and potential virus-targeted therapeutics in ebv-associated npc, in which cypa is upregulated at all stages. epstein-barr virus (ebv) is a member of the gamma herpesviruses and was the first confirmed human tumor virus (ding et al., ; lu et al., ) . ebv ubiquitously infects more than % of the global population and is closely associated with the development of several malignancies, including burkitt's lymphoma and nasopharyngeal carcinoma (npc) (shair et al., ; yu et al., ; young, ; young and dawson, ; liu et al., ) . npc, which is primarily of epithelial origin, is a type of metastatic head-and-neck neoplasm that is highly prevalent in southern china and some other areas in east asia and africa (tang et al., ; tu et al., ; jiang et al., ) . ebv is able to establish life-long persistence in the human host (young and murray, ; . it contains a large genome approximately kb in size and has two phases in its life cycle (the latent and lytic stages) (yu et al., ; hammerschmidt and sugden, ) . ebv mainly spreads through the saliva of human host, infects b cells through the oral mucosal epithelium, and then transforms b lymphocytes into resting memory b cells through a series of viral latent transcription programs, thus establishing a lifelong latent infection pattern (thorley-lawson et al., ) . the majority of ebv infections in vivo are latent (thorley-lawson, ) . during ebv latency, the ebv genome exists in the form of episome dna, few viral genes are expressed, and no virion is produced. ebv nuclear antigen (ebna ) is the only viral protein that is expressed in all types of ebvassociated tumors (lu et al., ; tao et al., ) . determining how ebv is able to maintain its stable latent status in host cells is a topic of interest, because it may provide understanding about the pathogenesis of ebv and new targets to inhibit the persistence of ebv genome in the therapy of ebv-associated cancers. ebv replication is under the control of some host and viral factors that are not fully understood. ebna plays a key role in the replication and mitotic segregation of ebv dna episomes to daughter cells (yates and guan, ; frappier, b) . ebna -mediated s-phase episome replication depends on binding of ebna to the ebv origin of genome replication (orip) (reisman et al., ) . viruses are obligate intracellular parasites, and their replication cycles depend on some host cell factors. for example, some studies suggested that cellular origin recognition complex (orc) and minichromosome maintenance (mcm) complex are related to the ds element of orip, implicating them in the initiation of ebv dna replication (frappier, a; capone et al., ) . these host factors may also be potential targets for antiviral therapy. cyclophilin a (cypa) is a protein with multiple functions as a typical member of the cellular peptidyl-prolyl cis-trans isomerase (ppiase) family (braaten et al., ; bahmed et al., ) . cypa was discovered initially as an intracellular receptor of the immunosuppressive drug cyclosporin a (csa) (braaten et al., ; bahmed et al., ) . studies have shown that cypa can use il- to induce cell signal conversion, activate tyrosine phosphorylation and nuclear transport of transcription factor , and can bind and activate nf-κb . cypa is involved in the life cycles of multiple viruses and plays a critical role in their successful infectivity and replication, including human immunodeficiency virus type (hiv- ), hepatitis c virus (hcv), hepatitis b virus (hbv), vesicular stomatitis virus (vsv), vaccinia virus (vv), coronaviruses (covs), and feline coronavirus (bose et al., ; naoumov, ; jyothi et al., ; phillips et al., ) . the interaction between cypa and hiv protein promotes the replication and infection of hiv particles; cd is the main signal receptor of cypa, and the two interact to regulate the early steps of hiv replication (ciesek et al., ; tang et al., ) . conversely, cypa suppresses the replication of some viruses, such as rotavirus, infectious bursal disease virus and influenza virus (xu et al., ; liu et al., ) . however, the role and mechanism of cypa in the function of ebv remain unknown. our laboratory previously performed a proteomics study using npc tissues and found that cypa was upregulated from the early stages of npc (atypical hyperplasia and stage i) to the malignancy stages (yang et al., ) . since ebv infection also occurs during the early stage of npc (morrison et al., ) , we speculated that a potential relationship might exist between cypa and ebv, which initiated the present approach. recently, we have reported that the exosomal cypa level in npc is positively related that of exosomal latent membrane protein , which is another latent protein of ebv. the result suggests a relationship between cypa and ebv (liu et al., ) . ubiquitin-specific protease (usp ) is a type of deubiquitinase that is also known as herpesvirus-associated ubiquitin specific protease (hausp) and has been documented as a host factor that inhibits ebv replication (holowaty et al., a) . the usp -ebna interaction was selectively disrupted by deletion of ebna residues - just upstream of the dna binding domain, and the resulting ebna mutant exhibited significantly increased dna replication activity (holowaty et al., b) . usp also plays a role in the replication inhibition of kaposi's sarcoma-associated herpesvirus (kshv) by interacting with the latency-associated nuclear antigen (lana), which is the homology of ebna (jager et al., ) . as it is known, kshv also persists in infected cells mainly in a latent state, and lana, is expressed in all latently kshv-infected cells (rainbow et al., ) . however, the mechanism by which ebv overcomes host suppression to maintain its own pathogenesis remains to be investigated. here, we demonstrate that ebna binds to and recruits cypa to the nucleus to support the function of ebna in the replication of the viral latent genome. cypa overexpression could antagonize the host barrier, usp . the results revealed a strategy that ebv recruited one host factor to counteract another, thus favoring the viral dna replication and persistence in latent infection. the study provides novel insights into understanding ebv pathogenesis in epithelial cells. the p plasmid (maxi-ebv), which contains the complete ebv genome of the b - strain, was kindly provided by dr. w hammerschmidt (delecluse et al., ) . the human embryonic kidney hek cell line (hek ) was originally obtained from atcc and was used to establish latent infection of the whole ebv genome (p ) by using hygromycin for the screening, resulting in the c cell line that was previously described by our group (zuo et al., ) . subsequently, the cells were divided into groups of shrna (using prnat-u . -shrna-cypa treated cells), the nc shrna group (prnat-u . -shrna treated cells cypa nc), liposomal transfected cells are based on lipofectamine transfection reagent (invitrogen-thermo fisher; united states). the day after transfection, cells were treated with g was added (sigma-merck; germany) for cell selection. cells were subcultured every days. the cells were grown in dulbecco's modified eagle's medium (dmem; sigma) supplemented with % fcs. the c - cell line is an ebv-positive npc cell line (zuo l.l. et al., ) . all recombinant plasmids used for bimolecular fluorescence complementation (bimc) were constructed using standard cloning techniques according to the schematic diagram (supplementary figure s ) by using the pcaggs vector, which is a gift from dr. harty (lu et al., ) . cypa and cypb were amplified from cells by rt-pcr. cypa or cypb gene was fused with the n-terminal fragment of the yellow fluorescent protein (yfp), constructing the plasmids of pcaggs-flag-cypa-ny (cypa-ny) and pcaggs-flag-cypb-ny (cypb-ny), respectively. the ebna protein was expressed as a fusion protein with the c-terminal fragment of (yfp), constructing the plasmid pcaggs-myc-ebna -cy (ebna -cy). the expression plasmids pcaggs-flag-cypa and pcaggs-flag-cypb were also constructed respectively. the myc tag sequence was added to the n-terminal of ebna . myc-tagged ebna was generated by cloning into the sphi and nhei sites of the pcaggs vector to produce the pcaggs-myc-ebna plasmid. the truncations ebna (nt, - ) and ebna ( - ) were generated by pcr based on the plasmid pcaggs-myc-ebna using the primers listed in supplementary table s . the ebna deletion mutants ebna (nt, - ), ebna ( - ) and ebna ( - ) were generated by inverse pcr. for example, primer complementary to - bp (anti-sense) and - bp (sense) of ebna were used for amplification, and the resulting pcr. product was gel-purified and self-ligated, resulting in ebna ( aa - ). then a sphi-nhei fragment from ebna ( aa - ) was inserted into the sphi and nhei sites of the pcaggs-myc-ebna to produce the pcaggs-myc-ebna ( aa - ). the orip-sv -luc expression vector was obtained by introducing the flanking sequence of orip amplified from the pc -orip plasmid, which was a gift from prof. frappier (sivachandran et al., ) . the orip sequence was cloned into the saci and xhoi sites of the luciferase reporter vector, pgl -enhancer. the rt-qpcr was performed as previously described (yu et al., ) . briefly, total rna was isolated from cells using the trizol reagent (invitrogen). first, trizol was added to lyse the cells, then / volume of chloroform was added to the lysate to separate the dna, protein and rna, followed by the isopropanol and % absolute ethanol to precipitate the rna. for rt, µg of rna was reversely transcribed into cdna using the one-step gdna removal and cdna synthesis supermix kit (transgen biotech, beijing, china). real-time quantitative pcr (rt-qpcr) was then performed by using the kit of transstart r top green qpcr supermix (transgen biotech, beijing, china) according to the manufacturer's instructions. the following program was used for the rt reaction: • c for min, ice for min, followed by • c for min and then • c for s. the cfx multicolor detection system (bio-rad) was employed for the detection. primers for rna detection by qrt-pcr were designed based on rna sequences, and bate-actin was used as an endogenous control. the following procedure was used for the rt-qpcr: • c for min, followed by cycles of • c for s and • c for s. data obtained by the conventional method of calculating comparative method (− ct) . repeated three times for each sample in parallel in each experiment, and the results were expressed as the mean of three independent experiments. the sequences of the qrt-pcr primers are provided in supplementary table s . western blotting (wb) was performed using a standard protocol as described previously (zuo l.l. et al., ) . cells was lysed in ripa ( mm tris, ph . , mm nacl, % np- , . % sodium deoxycholate, . % sds, sodium orthovanadate, sodium fluoride, edta, leupeptin) followed by incubation on ice for min, protein quantification according to the specification of bca kit (beyotime, shanghai, china). the cells lysates were centrifuged at × g for min at • c. supernatant fractions were used for detection. samples with equivalent amounts of denatured protein were separated using % sds polyacrylamide gels (epizyme, shanghai, china). after electrophoretic separation, the proteins were transferred to a polyvinylidene difluoride (pvdf) membrane (millipore, danvers, ma, united states). the membrane was blocked with % skimmed milk for h at room temperature, followed by an overnight incubation with the primary antibody at • c. after washing three times, the membrane was incubated with a secondary antibody for h at • c. finally, the proteins of interest were detected using the luminata crescendo hrp substrate (millipore) and viewed with the chemidoc xrs + molecular imager (bio-rad). the results were analyzed using the image lab software (bio-rad). the following antibodies were used for the immune detection: anti-ebna (santa cruz biotech, de, united states, sc- ), anti-cypa (proteintech, chicago, il, united states, - -ap), anti-c-myc (sigma, st. louis, mo, united states, c ), and anti-flag (sigma, h ). gapdh (proteintech, chicago, il, united states, -i-ap) and bate-actin (proteintech, chicago, il, united states, -i-ig) were used as the loading controls. the secondary antibodies used for wb and if were as follows: hrp-conjugated anti-rabbit (cst, chicago, il, united states, # p ), hrp-conjugated antimouse (ge healthcare, united kingdom, na v) and alexa fluor donkey anti-rabbit igg (life technologies, united states, ). c cells were plated in a -well plate at a density of × cells per well. the next day, different csa concentrations as indicated were applied for treatment. at , and h posttreatment, the cell counting kit- (cck ) was used to test cell viability according to the manufacturer's instructions. the absorbance was detected at the nm wavelength using a microplate reader. cyclosporin a (dalian meilun, china) is a type of immunosuppressive preparation that targets cypa (chatterji et al., ) . the drug was dissolved in dimethyl sulfoxide (dmso). ebv-positive cells were cultured in medium containing µm of csa. after h of treatment, the cellular proteins and total rna were extracted and subjected to detection. bimolecular fluorescence complementation assay was performed as described previously (liu et al., ; lu et al., lu et al., , . the schematic diagram of bimc assay is shown in supplementary figure s . in brief, human hek t cells were grown on coverslips in six-well plates. the pcaggs-myc-ebna -cy plasmid or the mutant constructs and the pcaggs-flag-cypb-ny plasmid were cotransfected into the cells using the lipofectamine transfection reagent (invitrogen) according to the manufacturer's instructions. approximately . µg of each plasmid was used for the transfection. transfection of a single plasmid for each used in bimc assay was performed as a negative control. after h, the cells were gently washed for three times with pbs and fixed with cold % paraformaldehyde for min at room temperature. then, the cells were washed and subsequently stained with hoechst (sigma) for min at room temperature. finally, the cells were washed again, and the slides were observed for the specific yfp signal under a fluorescence microscope. a typical co-ip procedure was performed as described (liu et al., ; lu et al., lu et al., , . cell lysates were harvested directly or at h post-transfection as indicated. the lysed sample was centrifuged at , rpm for min at • c. the supernatant lysates of about mg were incubated with a primary antibody, such as an anti-flag monoclonal antibody (mab) (h , sigma, : ) or anti-mouse igg antibody (control) (sc- , santa cruz, : ), for h at • c. after centrifugation, the supernatant was transferred to a new tube containing µl of protein g beads (transgen biotech, beijing, china) and incubated for h at • c. after extensive washes with cold lysis buffer, the immunoprecipitated proteins were eluted in sds sample loading buffer (aurigene biotech, changsha, china), separated by sds-page, transferred onto polyvinylidene difluoride membranes (millipore), and detected by wb (zheng et al., ) . three small interfering rnas (sirnas) targeting cypa (genbank accession number: nm ) were designed and synthesized by guangzhou ribobio company. to evaluate the knock-down efficiency of the sirnas, pmol of each sirna was transfected into hek cells. the cells were lysed with ripa as described by wb, and lysates were used for western blotting with anti-cypa and anti-bate-actin antibodies. the sirna with the best knock-down efficiency was chosen for the subsequent experiments. a scramble sirna-nc was used as the control. hek t and vero cells grown on coverslips in six-well plates for h were washed gently three times with pbs, fixed with % formaldehyde in pbs for min, and then permeabilized with . % triton x- in pbs for min. subsequently, the cells were blocked with fresh % goat serum. the cells were incubated with an anti-cypa rabbit polyclonal antibody and anti-flag mouse mab overnight at • c, respectively. after washing five times with pbs, the secondary antibody was added for h of incubation at • c. then, hoechst was applied for nuclear staining for min at room temperature. the coverslips were finally mounted onto slides and observed under a florescent microscope (bx , olympus, japan). the chip assay was performed to detect the binding of ebna -orip according to previous reports (shen et al., ) . the immunoprecipitation kit (millipore) was used for the assay. hek cells were transfected with lipofectamine (invitrogen) according to the manufacturer's protocol. cells were collected after h and lysed with lysis buffer (beyotime, shanghai, china) [ mm tris (ph . ), mm nacl, % triton x- , sodium pyrophosphate, β-glycerophosphate, edta, na vo , leupeptin]. the immunoprecipitated nucleoprotein complexes were eluted by incubation twice for min at • c with µl of elution buffer ( % sds and mm nahco ), and the crosslinks were reversed by incubation at • c for h. the dna was extracted with phenol/chloroform and precipitated with ethanol. then orip dna was amplified by qrt-pcr using the primers listed in supplementary table s . the experimental procedure for the detection of orip transcription activation mediated by ebna was carried out according to previous description from other group . cells were seeded into -well plates h prior to transfection. the following day, ng of the orip-sv -luc reporter plasmid was transfected into the cells using lipofectamine . the sv -luc reporter plasmid was transfected as a control. cell lysates were collected at h post-transfection and assayed for luciferase activity using a luciferase assay kit (promega, madison, wi, united states) on the panomics luminometer. the renilla luciferase activity was also measured using an enzyme assay kit (promega). the results were normalized to the renilla activity. three parallel repeats were performed for each sample in each experiment, and the results were expressed as the mean of three independent experiments. the detection of ebv copy number was performed as described previously (zuo et al., ) . dna was extracted from the c -shnc and c -shcypa cells using the general allgen frontiers in microbiology | www.frontiersin.org kit (cwbio, hunan, china) and quantified. the relative ebv copy number was determined by rt-qpcr using the ebv dna quantitative fluorescence diagnostic kit (sansure biotech, hunan, china) according to the manufacturer's instruction. in this product, bamhi-w fragment in ebv genome is designed as the specific primers and probes. the ebv copy number concentration (copies/cell) of the samples was calculated according to the level of internal reference. the experiment was repeated for three times. the statistical analyses were performed using graphpad prism (graphpad software, ca, united states). differences between groups were determined using student's t-test or one-way analysis of variance (anova). the data are expressed as the means ± standard deviations (sds). single, double and triple asterisks indicate statistical significance ( * p < . , * * p < . and * * * p < . ). by immunofluorescence (if) assay, cypa was detected to be mainly localized in the cytoplasm in vero and hek t cells (supplementary figure s ) . to study the potential interaction between ebna and cypa, bimc assay was performed. the bimc assay is a useful tool for detection of protein-protein interactions in living cells, and the results can be visualized under a fluorescence microscope (hudry et al., ; yang et al., ) . a diagram of the basic principle of the bimc assay is shown in supplementary figure s . the ebna protein was expressed as a fusion protein with the c-terminal fragment of the yfp (pcaggs-myc-ebna -cy), and cypa was fused with the n-terminal fragment of yfp (pcaggs-flag-cypa-ny). these two plasmids were co-transfected into hek t cells, with the single pcaggs-flag-cypa-ny plasmid transfection as a negative control. the results showed that ebna and cypa interacted with each other in the nucleus (figure a , top). when the nuclear localization signal (nls) sequence of ebna was deleted, the proteins interacted in the cytoplasm ( figure a) . this translocation showed the specificity of the interaction mediated by ebna with an nls. cyclophilin b (cypb) is another member of the cyclophilin family, but we did not detect any binding signal for cypb-ebna in the bimc assay ( figure a) . there was no fluorescence signal in the cells transfected with only single plasmid which was a negative control (figure a , lower). ebna and cypa/b expression in the bimc assay was detected by wb ( figure b) . we further evaluated ebna and cypa intracellular localization in ebvpositive cells by if assays (figure c) . the result showed that cypa expressed in both cytoplasm and nucleus in ebv-positive c cells, while mainly in the cytoplasm of the ebv-negative hek cells (figure c) . the co-immunoprecipitation (co-ip) assay was used to verify the interaction of ebna with cypa. these results showed that both endogenous and exogenous cypa interacted with ebna (figures d-f) . the plasmids pcaggs-myc-ebna and pcaggs-flag-cypa or pcaggs-flag-cypb were transfected in hek cells, myc-ebna was immuneprecipitated with anti-flag antibody. as shown in figure g , the result of co-ip assay further validated that ebna interacted with cypa but not cypb. to identify the contribution of the functional domains of ebna to the cypa-ebna interaction, we constructed five deletion mutants of myc-tagged ebna named ebna - , ebna - , ebna - , ebna - , and ebna - . the plasmid structures are illustrated in figure a . the bimc assay revealed that yfp was reconstructed in hek t cells using wild-type ebna and four of the mutants (ebna - -cy, ebna - -cy, ebna - -cy and ebna - -cy) but not the ebna - -cy mutant (supplementary figure s a) . each negative control with single plasmid transfection is shown in supplementary figure s c . subsequently, we co-transfected pcaggs-myc-ebna or a pcaggs-myc-ebna mutant plasmid with pcaggs -flag-cypa into hek cells. a co-ip assay using a flag-tag antibody for the pulldown showed the same result (i.e., cypa did not interact with ebna - ) ( figure b ). this domain, which is also the region containing the usp binding domain in ebna (figure a) , was thus critical for the binding of ebna to cypa. co-expression of ebna - -cy (only this one domain of ebna ) and cypa-ny recovered their binding function based on the bimc assay results (supplementary figure s b) . the nls (amino acids - ) is also included in the domain containing amino acids - , and thus yfp was detected in the nucleus (supplementary figure s b) . these results indicated that amino acids - of ebna , which contain the usp -binding domain, were essential for the interaction between ebna and cypa. we designed three sirnas to observe their efficiency on cypa protein expression in hek cells ( figure a) . the results showed that the knock-down of sicypa- was best, and this sirna was used in the subsequent experiments, whereas a scrambled control sirna (sinc) had no effect on cypa expression. as shown in figures b,c , ebna protein and mrna expression decreased in response to sicypa in ebvpositive npc c - and c cells. conversely, ectopic cypa overexpression in the ebv-positive cell lines resulted in an increase in the ebna expression levels detected by wb and qrt-pcr (figures d,e) . as ebv episomal genome is easy to be lost with culture passages in vitro (frappier, b) , we investigated whether cypa depletion might expedite this process in consecutive passages. then we collected gdna from different generations figure | detection of cypa-ebna binding by the bimc and co-ip assays. (a) detection of the ebna -cypa interaction by the bimc assay. hek t cells were transfected with the indicated plasmids, including cypa-ny, ebna -cy, ebna nls-cy, and cypb-ny following by bimc analysis, and fluorescence was observed. transfection of a single cypa-ny plasmid did not lead to the production of fluorescence. scale bar, µm. (b) the proteins expressed from the plasmids used in the bimc assay were detected by wb. (c) the detection of cypa and ebna in ebv-negative and ebv-positive hek cells by if assay. scale bar, µm. (d) endogenous cypa interacts with ebna in hek cells. the plasmid pcaggs-myc-ebna was transfected into cells. an anti-myc antibody was used for the pull-down the cypa, and the wb assay was carried out for detection. (e) endogenous cypa interacts with ebna in c cells. ebna was immune-precipitated with anti-cypa. ebna was detected by wb (f) exogenous cypa interacts with ebna . hek cells were transiently transfected with flag-cypa alone or with myc-ebna . flag-cypa was immune-precipitated with anti-myc antibody. igg was used as a negative control for the pull-down in the co-ip assay. flag-cypa was detected by wb. (g) co-ip assay for comparison of the interactions of cypa and cypb with ebna . pcaggs-myc-ebna and pcaggs-flag-cypa or pcaggs-flag-cypb were transfected in hek cells. myc-ebna was immune-precipitated with anti-flag antibody. figure | identification of the ebna domain required for binding to cypa. (a) diagram of the ebna deletion mutants. the start and end amino acid residues for each fragment are indicated according to a previous report (young and murray, ) . (b) validation of the interaction between each mutant ebna and cypa by the co-ip assay. pcaggs-myc-ebna , pcaggs-myc-ebna mutants, and pcaggs-flag-cypa were transfected into hek cells. myc-ebna was immune-precipitated with anti-flag antibody. flag-cypa and myc-ebna were detected by wb. of cells and detected the copy number of each cell. as it shown in figure f , cypa depletion significantly facilitated loss of ebv copy numbers. ebna -orip binding has been validated to be necessary for the replication initiation of ebv genome (shen et al., ) . here, we investigated the effect of cypa knockdown on the orip luciferase activity in cells transfected with an orip luciferase reporter in c cells. ebv-positive c and ebv-negative hek cells were stably transfected with a lentivirus expressing a short hairpin (sh) of cypa and a scramble control (shnc) (figure a) , and the results showed that cypa was successfully knocked down. ebna protein expression was restored in the c -shcypa cells transfected with the wild-type cypa expression plasmid ( figure b ). in these cells, an orip luciferase reporter, orip-sv -luc was transiently transfected. after h transfection, luciferase activities were detected. the ebna activated orip luciferase was decreased by . -fold in the luciferase assay compared to that in the control cells (c -shnc) due to cypa depletion. in contrast, there was no effect on the transfection of an sv -luc reporter plasmid with cypa depletion (p < . , figure c ), ebna mrna was detected by qrt-pcr. when ebna was lacked, cypa knockdown did not affect luciferase activity of orip-sv -luc ( figure c) . the results indicated that cypa enhanced the ebna activation of orip transcription. the chip assay was used to further validate that cypa played a role in ebna -orip binding. pcaggs-myc-ebna and orip-sv -luc were co-transfected into hek -shnc and hek -shcypa cells. as shown in figure d , cypa interference (shcypa) reduced the ebna binding to orip dna by approximately % compared to that of the control (shnc) in hek cells (p < . ). cypa is an intracellular receptor of csa, which in turn inhibits the ppiase activity of cypa through binding to the hydrophobic pocket of cypa (chatterji et al., ). based on above data about cypa regulation in ebv genome transcription initiation, we further investigated the possibility of csa against ebv replication. the working concentration of csa was considered as µm according to its inhibition effectiveness and low cytotoxicity by the cell counting kit- assay ( figure a) . rescue experiments showed that ebna protein levels were restored compared to the csa treatment group (figure b ). ebna protein expression was evaluated by wb after treated or untreated with csa ( figure c, left) . rt-qpcr analysis showed that there was a significant reduction for the ebna following the decrease of cypa caused by csa (figure c, right) . subsequently, c cells were transiently transfected with the cypa expression plasmid together with orip-sv -luc. luciferase activities were increased at h post-transfection compared with that of no cypa overexpression, but there was no change for sv -luc (without orip), and ebna -orip-luc activity increased in cypa overexpression ( figure d) . the protein level of cypa and the mrna level of ebna are detected ( figure d) . we further investigate the role of csa in ebna -mediated orip transcription. after transfection of orip-sv -luc, the cells were treated with csa. the result showed that csa decreased ebna-orip-luc activity compared to the untreated group, with a decrease in cypa and ebna ( figure e) . however, overexpression of cypa and treatment of csa in hek cells did not affect the activity of orip-sv -luc ( figure f ). these data indicated that csa inhibited the ebna -mediated orip activation. chip assay followed by quantitative pcr (chip-qpcr) was used to evaluate the the effect of cypa depletion on loss of ebv copy numbers during passages. ebv-positive c was used for shrna stable transfection and selection. cell lines stably transfected with shrna-cypa or shrna-nc were established for the experiment. cell dispersal for each passage was performed at a ratio : . the copy number of the c -shcypa cells was decreased compared with the c -shnc, with passage th of c -shcypa set to . * p < . , * * p < . , * * * p < . . effect of cypa overexpression and csa treatment on ebna binding to orip in hek cells. myc antibody (for myc-ebna ) efficiently pulled down orip-dna from the above samples, and the results showed that csa treatment eliminates ebna -orip binding, while cypa overexpression increases its binding ( figure g) . the results suggested that the ppiase activity of cypa was required for the role of cypa in ebv latent replication. restoration of ebna protein expression in c -shcypa cells transfected with wild-type cypa expression plasmids. cypa and ebna proteins were detected by wb. (c) effect of cypa knockdown on ebna -orip-mediated transcription activity in the luciferase reporter assay. the ebna mrna was detected by rt-qpcr. (d) effect of cypa knockdown on binding of ebna to orip in the chip assay. cypa was depleted from hek with shrna. antibodies against myc and igg control were respectively used for the pulldown in chip assayshek . the precipitation of orip dna was quantitated by rt-qpcr. cypa and ebna proteins were detected by wb. * p < . , * * p < . , * * * p < . . ubiquitin-specific protease has been implicated in strong inhibition of ebv replication through its tight connection with ebna (holowaty et al., b) . in this study, as the above results demonstrated, the cypa binding domain was located within the domain containing amino acids - , which spanned the usp -binding domain in ebna . because cypa played an opposite role in regulating ebna function compared with usp , hek -shnc and hek -shcypa cells were transiently transfected with pcaggs-flag-cypa alone or with pcaggs-myc-ebna , a co-ip assay was carried out. the results showed that usp -ebna binding was strong in the hek -shcypa and hek -shnc cells. however, when cypa was overexpressed, the amount of usp bound with ebna was decreased remarkably (figure a) . in order to study the ppiase activity of cypa in this mechanism in ebv replication, a co-ip assay was performed to using the treatment of csa. the result verified that csa eliminated the antagonism of cypa on ebna -usp interaction ( figure b) . to investigate the ability of cypa to influence the ebna -orip connection through antagonizing usp , in hek cells, orip-sv -luc was transfected with pcaggs -myc-ebna , pcaggs-flag-cypa or pcaggs-myc-ebna mutant, a chip assay was designed based on ectopic cypa expression. as shown in figure c , the interaction of orip and the ebna mutant with deletion of amino acids was significantly enhanced. in contrast, cypa overexpression increased ebna -orip binding to a high level. the result demonstrated that the deletion of the binding site for both usp and cypa exhibited the enhancement of ebna -orip binding ( figure c ). this was a priority effect of the release of usp inhibition but not cypa facilitation. only when cypa was overexpressed, could the effect of usp inhibition be reversed. the results further showed that cypa overexpression was essential to overcome the usp suppression in regulating ebna replication function. ebv latent infection is an important causative factor in the development of related cancers such as npc (dittmer et al., ; zheng et al., zheng et al., , , although the mechanism is largely unclear. viral replication and genome maintenance in host cells are important for the pathogenesis. cypa was found to be highly expressed in npc in the previous work from our laboratory (yang et al., ; liu et al., ) and played an unknown role related to ebv. herein, we reveal that cypa, especially when increasingly expressed, contributes to the replication function of ebna . cypa first was recruited by ebna into the nucleus and then mediated ebna -orip binding and replication activity. the mrna expression of ebna measured by rt-qpcr. ebv-positive c cell lines were used for the test. (d) c cells were transfected with orip-sv -luc reporter plasmid and cypa expression plasmids. overexpressed cypa significantly increased ebna -orip-dependent luciferase activity, but had no effect on sv promoter dependent luciferase activity (left). ebna mrna was measured by rt-qpcr (right) . (e) orip-sv -luc reporter plasmid was transfected into c cells, following the csa treatment. csa treatment greatly reduced ebna -orip luciferase activity compared with untreatment. (f) csa and elevated cypa on ebna -orip-mediated transcription activity in the luciferase reporter assay in hek cells. (g) chip-qpcr was used to determine ebna -orip binding. csa treatment reduced ebna -orip binding while elevated cypa increased binding. cypa and ebna proteins were analyzed by wb. * p < . , * * p < . , * * * p < . . (c) effect of cypa overexpression and ebna - mutation on the ebna -orip binding detected by chip assay. * p < . , * * * p < . . figure | schematic for the mechanism of cypa in supporting the replication function of ebna . ebv genomic dna exists within the host genome in the form of extrachromosomal episomes. cytoplasmic cypa can be hijacked by ebna into the nucleus. overexpressed cypa can overcome the suppression of usp in binding to ebna . nuclear cypa mediates ebna -orip transcription, and thus contributing to the viral genome replication and maintenance. on the other hand, when cypa was upregulated, the usp suppression in ebna -mediated replication could be reversed. this interaction is a type of quantity-driven winning for cypa, because its rival usp is too powerful. it was reported that the affinity of usp -ebna was -fold higher than usp -p , implying the strong binding of usp -ebna (saridakis et al., ) . the csa treatment demonstrated that the ppiase activity of cypa was required for this function. the schematic working model is shown as in figure . cyclophilin a has been implicated in the life cycles of several viruses and plays a critical role in the successful infectivity and replication of these viruses, including some tumor viruses, such as hbv and hcv (bose et al., ; naoumov, ; jyothi et al., ; phillips et al., ) . it has not been reported for the relationship between cypa and kshv, another tumor virus in the same family of gamma herpesvirus as ebv. though cypa is involved in the regulation of several viruses, its function mode is different from that in other viruses depending on the different mode of viral infection and replication. for example, the interaction between cypa and hiv gag was proposed to facilitate disassembly of the viral rna containing core following virus entry and thus supports the efficient reverse transcription of the hiv- genome (luban et al., ; ott, ) cypa also enhances virus attachment to the host cell membrane through interactions with heparans (saphire et al., ) and after membrane fusion through interaction with cd , thereby promoting viral infection. in the present study, for the first time, we showed that cypa was also utilized by ebv in the modulation of viral replication function in epithelial cells. thus, cypa is involved in the maintenance of the virus during its latency in host cells. ebna mediates dna episome replication from orip (frappier, a) . our results revealed that cypa was recruited to influence ebna -orip-mediated transcription. cypa depletion significantly facilitated loss of ebv copy numbers (figure f) , suggesting that impairment of ebna-orip binding further weakened successful replication and maintenance of the ebv genome. epstein-barr virus can naturally infected b cells and latently maintained in resting b cells (thorley-lawson et al., ) . as cypa is a multi-functional protein, which can be expressed in all kinds of cells, and ebna is the only expressed protein of ebv in all latency types. therefore, we think that both cypa and ebna may be expressed in resting b cells. how cypa plays a role in the function of ebv in b cells remains to be further investigated. the deubiquitinase usp is also known as a hausp and has been found to be associated with several herpesviruses, including hsv- , ebv, and kshv (holowaty et al., b; jager et al., ; hammerschmidt and sugden, ) . a previous study demonstrated that usp suppressed ebv replication (holowaty et al., b) . here, we showed that ebv hijacked the host factor of elevated cypa to counteract usp , thereby adding to the definition of hausp. additionally, our data showed that deletion of the binding domain in ebna for both usp and cypa resulted in significantly increased ebna -orip binding activity (figure c) , mainly due to release of usp inhibition. the result was consistent with that of previous report (holowaty et al., a) , demonstrating that the suppressive role of usp was strong enough to greatly overshadow the improved role of cypa. in summary, the study reveals that cypa is a novel critical host factor utilized by ebna in the viral dna replication in epithelial cells. elevated cypa levels remarkably antagonize usp in the interaction with ebna . the results revealed a strategy that ebv recruited a host factor to counteract the host defense, thus facilitating its own latent genome replication and efficient persistence. our findings implied that ebv has evolved sophisticatedly. this study provides a new insight into ebv pathogenesis and potential virus-targeted therapeutics in ebvassociated npc, in which cypa is upregulated. the data used to support the findings of this study are available from the corresponding author upon request. we thank dr. wolfgang hammerschmidt (gsf-national research center for environment and health, germany) for kindly providing us the maxi-ebv system, which was used in the establishment of c cell line. we thank dr. lori frappier (university of toronto, canada) for the kind gift, plasmid pc -orip. we also thank dr. ronald n. harty (university of pennsylvania, united states) for providing us the pcaggs vector. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmicb. . /full#supplementary-material figure s | the schematic diagram of the bimc assay. figure s | cypa expression was detected by if assay in vero and hek t cells. scale bar, µm. after transfected the pcaggs-flag-cypa plasmid for h in vero cells, the flag antibody was incubated overnight in • c, followed by green fluorescent secondary antibody in • c for h, hoechst stained nucleus. hek t cells, cypa antibody incubated overnight, followed by red fluorescent secondary antibody, stained nucleus. extracellular cyclophilin-a stimulates erk / phosphorylation in a cell-dependent manner but broadly stimulates nuclear factor kappa b requirement for cyclophilin a for the replication of vesicular stomatitis virus new jersey serotype cyclophilin a is 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interfering with pattern recognition of tlr and tlr inhibition of epstein-barr virus infection by lactoferrin the copy number of epstein-barr virus latent genome correlates with the oncogenicity by the activation level of lmp and nf-kappab an update: epstein-barr virus and immune evasion via microrna regulation cadherin is activated by epstein-barr virus lmp to mediate emt and metastasis as an interplay node of multiple pathways in nasopharyngeal carcinoma key: cord- -xn j a authors: arruda, bailey l.; burrough, eric r.; schwartz, kent j. title: salmonella enterica i ,[ ], :i:- associated with lesions typical of swine enteric salmonellosis date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: xn j a salmonella enterica serotype i ,[ ], :i:- has been increasingly isolated from swine. however, its pathogenic potential is not well characterized. analysis of swine cases confirmed a strong positive association between isolation of i ,[ ], :i:- and lesions of enteric salmonellosis and suggested a similar pathogenic potential as that for salmonella typhimurium. salmonella enterica serotype i , [ ] , :i:-has been increasingly isolated from swine. however, its pathogenic potential is not well characterized. analysis of swine cases confirmed a strong positive association between isolation of i , [ ] , :i:-and lesions of enteric salmonellosis and suggested a similar pathogenic potential as that for salmonella typhimurium. o ver the past decade, salmonella enterica serotype i , [ ] , :i:-has emerged as a major public health threat in europe ( ) and the united states ( ) . as a monophasic variant of salmonella typhimurium, salmonella i , [ ] , :i:-was rarely identified before the mid- s ( ). however, it has now been detected in cattle ( ), poultry ( ) , and swine ( ) ( ) ( ) , and several human disease outbreaks associated with contaminated pork products have occurred ( ) ( ) ( ) ( ) . salmonellosis is also a major disease concern in swine. salmonella typhimurium has been considered the most common cause of enteric salmonellosis in swine ( ) . recent reports from of the largest veterinary diagnostic laboratories in the united states showed that there was a noted increase in the percentage of isolates of salmonella i , [ ] , :i:- ( , ) . despite the apparent increase in the isolation of salmonella i , [ ] , :i:-from swine and pork products, there is currently limited information regarding the pathogenicity of this serotype in swine. accordingly, this study assessed the pathogenic potential of salmonella i , [ ] , :i:-through the evaluation of microscopic intestinal lesions in swine enteric cases from which salmonella i , [ ] , :i:-was isolated compared with similar cases from which salmonella i , [ ] , :i:-was not isolated. the iowa state university-veterinary diagnostic laboratory (isu-vdl) is a national animal health laboratory network-accredited laboratory that receives > , case submissions annually, of which most are derived from swine production systems located throughout the united states. histopathologic analysis is performed by veterinary diagnostic pathologists on ≈ , cases from swine per year. the isu-vdl laboratory information management system provided the initial dataset for this analysis. to determine whether isolation of salmonella i , [ ] , :i:-from samples submitted from pigs was associated with microscopic lesions consistent with enteric salmonellosis, we compared cases from which salmonella i , [ ] , :i:-was isolated with cases from which neither salmonella i , [ ] , :i:-nor salmonella typhimurium were isolated; these samples were collected during july -december . we also reviewed cases from which salmonella typhimurium or of salmonella serogroup b serovars (salmonella derby, salmonella agona, and salmonella heidelberg) were isolated to determine the potential comparative pathogenicity of salmonella i , [ ] , :i:-. all of these cases met the following qualifying criteria: animals were - weeks of age, a salmonella serovar as outlined above was isolated by direct culture performed on enteric tissues, and histopathologic analysis was performed on the large intestine. to determine the association between the presence of salmonella i , [ ] , :i:-and lesions consistent with enteric salmonellosis, we also reviewed additional cases that met the previously stated inclusion criteria but from which neither salmonella i , [ ] , :i:-nor salmonella typhimurium were isolated; we randomly selected these cases from an excel (microsoft, https://www.microsoft. com) data file by using the rand() function. enteric samples submitted for isolation of salmonella were routinely processed and reported (appendix, https:// wwwnc.cdc.gov/eid/article/ / / - -app .pdf). microscopic lesions deemed compatible with salmonellosis in sections of the large intestine included erosion; ulceration; neutrophilic infiltration; crypt ectasia, crypt elongation, or both with associated neutrophilic exudation; goblet cell loss; luminal accumulation of neutrophils and fibrin; and submucosal accumulation of lymphocytes and macrophages. we used jmp pro (sas institute, https://www.sas.com) to perform all analyses. we used the pearson χ test and odds ratios to determine the association between isolation of salmonella i , [ ] , :i:-and pathologic diagnosis of enteric salmonellosis. we considered a p value < . significant. we isolated salmonella i , [ ] , :i:-from porcine cases that met all of the qualifying criteria. we isolated salmonella typhimurium from cases, and other potentially lesser pathogenic salmonella serogroup b serovars, including salmonella derby, agona, and heidelberg, from cases. a review of case data for clinical submissions to the isu-vdl confirmed a statistically significant positive association between histologic lesions consistent with enteric salmonellosis and isolation of salmonella i , [ ] , :i:-(odds ratio . , % ci . - . ; p< . ) (table) . we confirmed compatible histologic lesions of salmonellosis ( figure) for ( %) of cases from which salmonella i , [ ] , :i:-were isolated (appendix table ). histologic lesions consistent with enteric salmonellosis from which neither salmonella i , [ ] , :i:-nor salmonella typhimurium were isolated were observed for ( %) of cases (appendix table ). salmonella was isolated from of the cases, of which had lesions consistent with enteric salmonellosis. we confirmed compatible histologic lesions of salmonellosis in ( %) of cases in which salmonella typhimurium was isolated and ( %) of cases in which another serogroup b salmonella was isolated. other agents of enteric disease that were concurrently detected in some cases included rotaviruses, coronaviruses, coccidians, and hemolytic escherichia coli. however, none of these agents caused lesions consistent with those used to define salmonellosis in this report (appendix tables , ) . during an -month period and using the same qualifying criteria for cases, we identified salmonella i , [ ] , :i:from swine cases. however, salmonella typhimurium was isolated from only cases and another serogroup b salmonella as specified above was isolated from only cases. this finding represents nearly an -fold increase in isolation of salmonella i , [ ] , :i:-compared with salmonella typhimurium and is in concordance with findings of others who have identified an increase in isolation of salmonella i , [ ] , :i:-from swine samples submitted to veterinary diagnostic laboratories ( , ) . a major aspect of salmonella epidemiology is the variation in prevalence of serotypes or phage types over time in human and animal populations. the catalysts of such changes remain elusive ( ) . isolation of salmonella i , [ ] , :i:-was rarely reported before ( ), but this serotype has now become predominant in human clinical cases and has been isolated from food products, including pork, on different continents ( ). although increased isolation of salmonella i , [ ] , :i:-from swine samples has been documented, the pathogenic potential of this serovar in pigs had not been reported. in this study, we demonstrate a strong positive association between histologic lesions consistent with enteric salmonellosis and isolation of salmonella i , [ ] , :i:-. in most cases from which salmonella i , [ ] , :i:-or salmonella typhimurium were isolated, the severity of histologic lesions was similar. however, the percentage of cases in which histologic lesions consistent with enteric salmonellosis were present was lower for cases from which salmonella i , [ ] , :i:-( %) was isolated than for cases from which salmonella typhimurium ( %) was isolated. based on these data, we believe that salmonella i , [ ] , :i:-is a likely cause of enteric salmonellosis that has a similar or perhaps slightly lower pathogenic potential in swine than salmonella typhimurium. pathogenicity studies are needed to further characterize the pathogenic potential and s. enterica and swine enteric salmonellosis fitness of salmonella i , [ ] , :i:-compared with that of salmonella typhimurium in swine. we suspect that salmonella i , [ ] , :i:-has evolutionary advantages that have resulted in its predominance as one of the most common salmonella serotypes responsible for swine enteric salmonellosis. accordingly, it is essential to determine the putative attributes that facilitate its rapid spread and ecologic success. specifically, antimicrobial drug resistance genes and genes that encode resistance to heavy metal micronutrients should be evaluated, given their current and common use in us swine production. dr. arruda is an assistant professor and diagnostic pathologist at the iowa state university veterinary diagnostic laboratory, ames, ia. her primary research interest is infectious diseases of swine. the european union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in outbreak of multidrug-resistant salmonella i emergence, distribution, and molecular and phenotypic characteristics of salmonella enterica serotype serotypes and antimicrobial resistance in salmonella enterica recovered from clinical samples from cattle and swine in minnesota changes in the prevalence of salmonella serovars associated swine production and correlations of avian, bovine and swine-associated serovars with human-associated serovars in the united states nationwide outbreak of salmonella enterica serotype , :i: -infections in france, linked to dried pork sausage outbreaks of monophasic salmonella enterica serovar pork contaminated with salmonella enterica serovar salmonellosis and meat purchased at live-bird and animal-slaughter markets disease of swine key: cord- -db kb c authors: park, jun-gyu; Ávila-pérez, ginés; madere, ferralita; hilimire, thomas a.; nogales, aitor; almazán, fernando; martínez-sobrido, luis title: potent inhibition of zika virus replication by aurintricarboxylic acid date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: db kb c zika virus (zikv) is one of the recently emerging vector-borne viruses in humans and is responsible for severe congenital abnormalities such as microcephaly in the western hemisphere. currently, only a few vaccine candidates and therapeutic drugs are being developed for the treatment of zikv infections, and as of yet none are commercially available. the polyanionic aromatic compound aurintricarboxylic acid (ata) has been shown to have a broad-spectrum antimicrobial and antiviral activity. in this study, we evaluated ata as a potential antiviral drug against zikv replication. the antiviral activity of ata against zikv replication in vitro showed median inhibitory concentrations (ic( )) of . ± . μm and . ± . μm in vero and a cells, respectively; without showing any cytotoxic effect in both cell lines (median cytotoxic concentration (cc( )) > , μm). moreover, ata protected both cell types from zikv-induced cytopathic effect (cpe) and apoptosis in a time- and concentration-dependent manner. in addition, pre-treatment of vero cells with ata for up to h also resulted in effective suppression of zikv replication with similar ic( ). importantly, the inhibitory effect of ata on zikv infection was effective against strains of the african and asian/american lineages, indicating that this inhibitory effect was not strain dependent. overall, these results demonstrate that ata has potent inhibitory activity against zikv replication and may be considered as a potential anti-zikv therapy for future clinical evaluation. zika virus (zikv) belongs to the genus flavivirus within the flaviviridae family. zikv is an enveloped positive sense single-stranded rna virus with a genome size of ∼ . kb that encodes a single polyprotein, which is post-translationally processed by cellular and viral proteases into three structural (capsid, c; pre-membrane, prm; and envelope, e) and seven non-structural (ns , ns a, ns b, ns , ns a, ns b, and ns ) proteins avila-perez et al., ) . zika virus was initially isolated from uganda in and viral infections only occurred sporadically in africa and asia until . zikv appeared explosively as the first large-scale outbreak occurred in the yap island in and french polynesia in (weaver et al., ) . most recently, in , the first local transmission of zikv was found in territories of latin america and the caribbean, resulting in up to . million of zikv infection suspected cases (tang et al., ; tripathi et al., ) . like other members of the flaviviridae family, such as yellow fever virus (yfv), dengue virus (denv), japanese encephalitis virus (jev), and west nile virus (wnv), zikv is commonly transmitted by the bite of infected aedes mosquitos, but it can also be transmitted vertically from mother to child, through sexual contact, and in rare cases from blood transfusions (lessler et al., ; fink et al., ) . upon infection, zikv can be shed in blood, urine, semen, saliva, amniotic fluid, breast milk, and cerebrospinal fluid (nayak et al., ; colt et al., ; nazerai et al., ) . most people ( ∼ %) infected with zikv are asymptomatic or have mild symptoms such as fever, rash, joint pain, and conjunctivitis that can last for several days to a week (fink et al., ) . in rare cases, people with symptoms may have neurological guillain-barré syndrome complications (oehler et al., ; rivera-concepcion et al., ; nazerai et al., ) . in the case of pregnant women, zikv infection can lead to microcephaly and other fetal complications as occurred during the large-scale zikv outbreak in brazil in (lessler et al., ) . because of the significant outbreaks in south, central, and north america, zikv was declared a public health concern by the world health organization (who) in february (lazear and diamond, ; ramos da silva and gao, ; weaver et al., ; tripathi et al., ) . there are several vaccines and antiviral drugs currently under development for the prevention or treatment of zikv infection larocca et al., ; shan et al., ; fink et al., ) . dna-based larocca et al., ) , inactivated larocca et al., ; shan et al., ) , live-attenuated and mrna (richner et al., ) vaccines have been proposed for the prophylactic treatment of zikv infections. on the other hand, arbidol (arb) (fink et al., ; haviernik et al., ) , bortezomib, mycophenolic acid, daptomycin (barrows et al., ) , obatoclax, saliphenylhalamide, gemcitabine (kuivanen et al., ) , emetine (yang et al., ) , and sofosbuvir (bullard-feibelman et al., ) have been proposed for the therapeutic treatment of zikv infection. despite these tremendous efforts, there is currently no food and drug administration (fda)-approved vaccines and/or anti-viral drugs available for the treatment of zikv infection. since vaccination takes at least weeks to several months to show protective effects against zikv infection, vaccination is probably not the most appropriate prophylactic method for those who are traveling to areas where zikv is epidemic, endemic, or have already been infected. moreover, vaccination may cause an important issue, such as antibody-dependent enhancement (ade) priyamvada et al., ) . ade, which has been extensively described in denv (priyamvada et al., ) , is a phenomenon where preexisting antibodies facilitate binding and infection during subsequent exposure to infectious viruses, instead of neutralizing them, resulting in exacerbation of clinical signs priyamvada et al., ) . because of the structural similarities between denv and zikv, denv immunity-linked ade of zikv infection has also been reported priyamvada et al., ) . since vaccination for zikv could lead to denv ade, antivirals could represent a better choice for the control of zikv infection. aurintricarboxylic acid (ata), a polyanionic aromatic compound, has been shown to have inhibitory properties against several bacteria and viruses including, among others, yersinia pestis (liang et al., ) , cryptosporidium parvum (klein et al., ) , human immunodeficient virus (hiv) (mitra et al., ; de clercq, ) , hepatitis c virus (hcv) mukherjee et al., ; shadrick et al., ) , vaccinia virus (myskiw et al., ) , influenza virus (hung et al., ) , enterovirus (hung et al., ) and severe acute respiratory syndrome coronaviruses (sars-cov) (he et al., ) . mechanistic studies have suggested that ata has the ability to modulate various cellular enzymes such as activators of the janus kinase (jak ) and signal transducer and activator of transcription (stat ) families (rui et al., ) , inhibitors of nucleases (shadrick et al., ) , glucose- -phosphate dehydrogenase (bina-stein and tritton, ) , and topoisomerase ii proteins (catchpoole and stewart, ; benchokroun et al., ) as well as the enzymatic activity of the vaccinia virus ah l phosphatase (smee et al., ) . however, to date, the ability of ata to inhibit zikv infection has not been evaluated. herein, we investigated ata as a plausible prophylactic and therapeutic candidate against zikv infection. our results demonstrate that ata has a potent and effective antiviral activity against zikv in pre-and post-infection settings, including broadly antiviral activity against strains of the african and american/asian lineages with no toxicity up to , µm in cultured cells. these data support the feasibility of implementing ata for the treatment of zikv infection. african green monkey kidney epithelial vero (atcc ccl- ) and human adenocarcinoma alveolar basal epithelial a (atcc ccl- ) cells were maintained in dulbecco's modified eagle's medium (dmem; mediatech, inc.) supplemented with % fetal bovine serum (fbs) and % psg ( u/ml penicillin, µg/ml streptomycin, and mm l-glutamine) at • c in a % co atmosphere. paraiba/ zikv isolate was kindly provided by stephen dewhurst (department of microbiology and immunology, university of rochester). uganda/ (mr_ strain, catalog no. nr- ) and nigeria/ (ibh strain, catalog no. nr- ) zikv isolates were obtained from the biodefense and emerging infections research resources repository (bei resources). puerto rico/ (prvabc strain) and french polynesia/ zikv isolates were kindly provided from the centers for disease control and prevention (cdc). virus stocks were propagated in vero cells and titrated by plaque assay as previously described (marquez-jurado et al., ) . aurintricarboxylic acid (catalog no. a ) and arbidol (arb, catalog no. slm ) were purchased from sigma-aldrich, mo, united states. both compounds were prepared at mm stock solution dissolved in dimethyl sulfoxide (dmso) and kept at − • c until experimental use. each drug was diluted into infectious media (dmem % fbs, % psg) for the described experiments, where the maximum dmso concentration was . %. cell viability in vero and a cells was measured using the celltiter non-radioactive cell proliferation assay (promega) following the manufacturer's instructions. briefly, confluent vero or a cells ( -well plate format, × cells/well, triplicates) were treated with µl of dmem containing serially diluted (twofold dilutions, starting concentration of , µm) chemicals or . % dmso (vehicle control). plates were incubated at • c in a % co atmosphere for or h. samples were treated with µl of dye solution and incubated at • c in a % co atmosphere for h. next, cells were treated with µl of solubilization solution/stop mix and absorbance at nm was measured using a vmax kinetic microplate reader (molecular devices, waltham, ma, united states). viability of compound-treated cells was calculated as a percentage relative to values obtained with dmso-treated cells. non-linear regression curves and the median cytotoxic concentration (cc ) were calculated using graphpad prism software version . . confluent monolayers ( -plate format, × cells/well, triplicates) of vero cells were infected with plaque forming units (pfu)/well of paraiba/ , uganda/ , nigeria/ , puerto rico/ , and french polynesia/ at • c in infection media. after h of adsorption, virus inoculum was removed and cells were washed three times with infection media before adding fresh infection media containing % microcrystalline cellulose (avicel, sigma-aldrich) and the indicated concentration of compounds, or . % dmso as vehicle control. in case of pre-treatment experiments, the cell monolayers were treated with the indicated concentration of compound, or . % dmso, for the indicated times before zikv infection. infected cells were incubated at • c for - h, depending on virus strains. for immunostaining, cells were fixed with % paraformaldehyde for h, washed three times with phosphate buffered saline (pbs) and permeabilized with . % triton x- for min at room temperature. then, the plates were blocked with . % bovine serum albumin (bsa) in pbs (blocking solution) for h at room temperature, followed by incubation with µg/ml of the pan-flavivirus envelop (e) protein monoclonal antibody g (atcc, catalog no. vr- ) diluted in blocking solution for h at • c. after incubation with the primary antibody, cells were washed three times with pbs and developed with the vectastain abc kit and the dab peroxidase substrate kit (vector laboratory, inc., ca, united states) according to the manufacturers' instructions. stained plaques were analyzed using the ctl immunospot plate reader and counting software (cellular technology limited, cleveland, oh, united states). virus titers were calculated as pfu/ml (nogales et al., ) . non-linear regression curves and the median inhibitory concentration (ic ) were determined as described above. confluent monolayers ( -well plate format, . × cells/well, triplicates) of vero or a cells were infected (multiplicity of infection, moi, . ) with paraiba/ diluted in infection media for h at room temperature. after viral absorption, cells were incubated with infection media containing the indicated concentrations ( , , . , and µm) of ata. at , , , and h post-infection (h p.i.), tissue culture supernatants were collected and titrated on vero cells by immunostaining as described previously (marquez-jurado et al., ) . levels of apoptosis were measured using the caspase-glo r / assay (promega, wi, united states) following the manufacturer's instruction. briefly, vero and a cells ( -well plate format, . × cells/well, triplicates) were infected with zikv paraiba/ (moi of . ) and, at the indicated times post-infection, cells and tissue culture supernatants were collected and centrifuged. twenty five microliters of supernatants were mixed with µl of caspase- / reagent using a plate shaker, incubated at room temperature for h, and luminescence at nm was measured using a spectramax id (molecular devices, waltham, ma, united states) following the manufacturer's instructions. two-way anova was used to evaluate significant differences. data are expressed as the mean ± standard deviation (sd) of at least three independent experiments in triplicates using microsoft excel software. value were considered statistically significant when * p < . , * * p < . , * * * p < . , * * * * p < . . all data were analyzed with prism software version . (graphpad software, ca, united states). cc and ic were determined using sigmoidal dose response curves (graphpad software, ca, united states). the selective index (si) of each compound was calculated by dividing the cc with the ic . before examining the inhibitory effect of ata (figure ) against zikv infection, we first determined the cc of ata on vero and a cells (figure ) . for this, we treated both cell lines with serial (twofold) dilutions of ata and measured cell viability at and h post-treatment. as an internal control for these studies, we used arb, a drug that has been previously described to have antiviral activity against zikv in vero (haviernik et al., ) and a (fink et al., ) cells. we did not observe any toxicity with ata in vero (figure a ) or a ( figure b ) cells at or h post-treatment, even at the highest concentration tested ( , µm), while arb showed cc values of . ± . or . ± . µm in vero ( figure c ) and . ± . or . ± . µm in a ( figure d ) cells (table ) at or h post-treatment, respectively. to determine the ic of ata, vero and a cells were infected with pfu/well of paraiba/ and after h of viral absorption, virus inoculum was replaced with infection media with twofold serial dilutions (starting concentration of , µm) of ata or arb (figure ) and the ic calculated as described in the section "materials and methods." although the ic of ata ( figure a) and arb ( figure c) in vero cells were similar ( . ± . µm and . ± . µm, respectively), the selective index (si, cc /ic ) of ata (> . ) was significantly higher than that of arb ( . ) ( table ) . likewise, the ic of ata ( figure b ) and arb ( figure d ) in a cells were similar but with clearly different si values (> . for ata and . for arb) ( table ) . notably the cc , ic , and si of arb were similar to those previously described in the literature in these cell lines (fink et al., ; haviernik et al., ) . these data suggest that ata exhibited an effective inhibition of zikv infection with limited toxicity and si values better than those previously described for arb. we also observed that zikv replication was completely inhibited at a concentration of µm of ata in vero ( figure a ) and a ( figure b ) cells while . µm and µm concentrations of ata showed partial viral inhibition in vero and a cells (figures a,b) , respectively, demonstrating a dose-dependent inhibition of viral replication in both cell lines. we next evaluated the ability of ata to protect cells from the cytopathic effect (cpe) induced during zikv infection ( figure ) . to that end, vero and a cells were infected (moi . ) with paraiba/ and, after h of viral absorption, cells were treated with , . , , and µm of ata. at h p.i., cells were observed under a light microscope for evaluation of their morphology and cpe ( figure a ). as expected from our previous results, µm and more clearly µm of ata were able to prevent zikv-induced cpe in both cell lines (figure a) . to quantify the ability of ata to prevent zikv-induced apoptosis, tissue culture supernatants from zikv-infected vero and a cells were harvested at , , and h p.i. to measure the level of apoptotic signal as determined by caspase and activities ( figure b) . zikv-infected cells showed figure | aurintricarboxylic acid inhibition of zikv replication: vero (a) and a (b) cells ( -well plate format, . × cells/well, triplicates) were infected (moi . ) with paraiba/ . tissue culture supernatants were collected at , , and h p.i., and viral titer were calculated by immunostaining (fluorescent forming units, ffu/ml). dotted line indicates the limit of detection ( ffu/ml). data was expressed as mean and standard deviations (sd) from three independent experiments conducted in triplicates. statistical analysis was conducted by two-way anova, * p < . , * * p < . , * * * p < . , * * * * p < . , or no significance (n.s.). figure | aurintricarboxylic acid protects vero and a cells from zikv-induced cell death: vero and a cells ( -well plate format, . × cells/well, triplicates) were infected (moi . ) with paraiba/ . after h viral adsorption, cells were treated with the indicated concentrations ( , , . , and µm) of ata. at h p.i., cells were observed and imaged under an optical microscope. scale bar = µm. (a) caspase / levels were measured in the tissue culture supernatants at , , and h p.i. (b) data of each time point was compared to mock-infected control cells and expressed as mean of relative percentage and sd from three independent experiments conducted in triplicates. statistical analyses were conducted by two-way anova, * p < . , * * p < . , * * * p < . , * * * * p < . , or no significance (n.s.). increased caspase and levels up to eightfolds in vero cells and up to . -folds in a cells compared to mock-infected cells ( figure b ). levels of caspase and activation were dose-dependently reduced by ata with µm of ata showed only . -and . -fold induction as compared to mock-infected vero and a cells, respectively ( figure b ). we next determined whether ata is able to inhibit both ancestor african (uganda/ and nigeria/ ) and contemporary asian/american (puerto rico/ and french polynesia/ ) zikv lineage strains using our microplaque reduction assay (figure ) . we observed similar ic values of ata with uganda/ ( figure a , ic = . ± . µm), nigeria/ ( figure b , ic = . ± . µm), puerto rico/ ( figure c , ic = . ± . µm), and french polynesia/ ( figure d , ic = . ± . µm), compared to those observed with paraiba/ (figure and table ), demonstrating the broad antiviral activity of ata against different zikv strains, regardless of the year and place of isolation. to demonstrate the feasibility of using ata for the prevention of zikv infection, important for travelers to regions where zikv is endemic, we next evaluated whether pre-treatment with ata results in inhibition of zikv replication (figure ) . to that end, vero cells were pre-treated with ata for ( figure a) , ( figure b) , (figure c) , or ( figure d ) h prior to infection (moi . ) with paraiba/ . pre-treatment with ata for - h before zikv infection resulted in similar ic values ( . ± . µm, . ± . µm, . ± . µm, and . ± . µm; respectively) demonstrating that ata is stable and able to prevent zikv infection even when administered days previous to viral infection (figure and table ). the recent outbreak of zikv accompanied with severe pathology, including microcephaly in newborns, prompted many researchers to develop prophylactic vaccines and to identify therapeutic drugs against zikv infection larocca et al., ; lessler et al., ; shan et al., ; fink et al., ) . currently, there are no commercially available vaccines and/or antiviral therapies for the treatment of zikv infection. therefore, there is an urgent medical need for the development of effective counter measurements to control zikv infection. in this study, we demonstrated that ata (figure ) has limited toxicity (figure ) and an effective and dose-dependent antiviral activity against zikv infection (figures , ) in both monkey kidney epithelial vero and human alveolar a cells. notably, ata can prevent zikv-induced cpe and apoptosis in both cell lines ( figure ) and has broad anti-viral activity against representative zikv strains from the african (uganda/ and nigeria/ ) and the asian/american (puerto rico/ and french polynesia/ ) lineages (figure ) . moreover, ata can also prevent zikv infection even when administered days before infection (figure ) . aurintricarboxylic acid is a polyanionic aromatic compound that structurally relates to suramin (balzarini et al., ) and is believed to influence over host and viral enzymes (shadrick et al., ) . although the exact mechanism by which ata inhibits zikv infection was not identified in this study, there are several plausible mechanisms on zikv inhibition mediated by ata, including the targeting of viral and cellular proteins. in terms of inhibiting viral proteins, ata could bind to zikv ns helicase and prevent its binding to either atp or nucleic acids, as previously described for hcv (mukherjee et al., ; shadrick et al., ) . likewise, ata could inhibit the zikv rna-dependent rna polymerase (rdrp) ns protein, as described for hcv mukherjee et al., ; shadrick et al., ) and enterovirus (hung et al., ) . similarly, ata could inhibit the methyltransferase activity of ns involved in mrna capping processes, as previously described for other flaviviruses (denv and yfv) (milani et al., ; garcia et al., ) . because of the structural similarities between denv and zikv ns proteins, it is feasible that, similar to denv, ata binds to ns to inhibit zikv infection . moreover, it is possible that ata targets and has inhibitory activities against one or more of the viral proteins described above. in terms of targeting cellular proteins important for the efficient replication of zikv, it has been previously described that ata has anti-apoptotic properties in a variety of cells (chen et al., ) . it is possible that the anti-apoptotic activity of ata protects against zikv-induced cell death, as demonstrated in this study (figure ) . notably, it has been recently shown that zikv infection induced apoptosis through caspase and in a cells and through caspase in neonatal mice brain (huang et al., ; frumence et al., ) . these results suggest that inhibition of zikv replication results in a decrease in the level of apoptotic cells and that the anti-apoptotic effect of ata affects zikv replication. further research is guaranteed to yield a better understanding of the antiviral activity of ata on zikv infection, and other viruses, before the use of ata as an antiviral drug. during january to february , a total of residents from states in the united states were diagnosed with zikv infection (armstrong et al., ) . out of patients, ( %) traveled to areas of active zikv transmission before the infection and five ( %) did not travel but reported sexual contact with a traveler who had a symptomatic illness (armstrong et al., ) . for these reasons, preventive efforts are required prior to travel to areas of active zikv transmission. in this study, cells pretreated with ata for up to h prior to infection with zikv showed similar ic than those in post-treatment settings, potentially suggesting that ata might target a cellular protein required for zikv replication or that the concentration and stability of ata in pre-treated cells is sufficient to inhibit zikv infection, or both. nevertheless, these results demonstrate the feasibility of using ata for the prophylactic treatment of viral infection, including those traveling to areas where zikv is endemic. moreover, due the broad inhibition effect of ata against others viruses and parasites (liang et al., ; he et al., ; de clercq, ; myskiw et al., ; klein et al., ; chen et al., ; hung et al., hung et al., , mukherjee et al., ; shadrick et al., ) that are present in zikv endemic areas, treatment with ata could be used for the broad prevention of denv, yfv (milani et al., ; shadrick et al., ; garcia et al., ) , hcv mukherjee et al., ; shadrick et al., ) , and parasitic infestation (cryptosporidium parvum) (klein et al., ) for people traveling to these endemic regions. moreover, the broad spectrum antiviral activity of ata against different african and asian/american zikv strains further guarantees the feasibility of implementing ata to prevent zikv infection to travelers around the world. although ata has been amply evaluated in vitro, only few studies have assessed the activity of ata in vivo, including its use as a curative agent against thrombosis (strony et al., ) , apoptosis (roberts-lewis et al., ; heiduschka and thanos, ) , parasite infestations (klein et al., ) , bacterial (y. pestis) (liang et al., ) , and vaccinia virus (smee et al., ) infections. in the case of vaccinia virus, ata did not protect mice from a lethal challenge at a dose of mg/kg/day (smee et al., ) . further studies are needed to evaluate the anti-viral activity of ata in vivo for the treatment of viral infections, including zikv. our studies show limited toxicity, if any, of ata in cultured cells, including human a cells. the lack of knowledge about the use of ata in pregnant women requires future additional safety tests, including studies using validated animal models of zikv infection, before using ata for the treatment of zikv infection during pregnancy. based on the effectiveness of ata against zikv infection (si = . in vero cells and . in a cells) as compared to other previously described drugs, including emetine (si = . in snb- cells and . in env+ cells) (yang et al., ) , obatoclax [si = in human retinal pigment epithelial (rpe) cells] (kuivanen et al., ) , saliphenylhalamide (si > in rpe cells) (kuivanen et al., ) , gemcitabine (si > , in rpe cells) (kuivanen et al., ) , sofosbuvir (si > . in huh- cells and . in jar cells) (bullard-feibelman et al., ) and arb (si = . in vero cells and . in a cells) (fink et al., ; haviernik et al., ) and this study (ata, in vero cells, and . in a cells), it is possible that ata represents one of the most reasonable options of the treatment of zikv infection. protective efficacy of multiple vaccine platforms against zika virus challenge in rhesus monkeys travel-associated zika virus disease cases among u.s. residents-united 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influenza virus neuraminidase in vitro and in vivo activity of aurintricarboxylic acid preparations against cryptosporidium parvum obatoclax, saliphenylhalamide and gemcitabine inhibit zika virus infection in vitro and differentially affect cellular signaling, transcription and metabolism vaccine protection against zika virus from brazil zika virus: new clinical syndromes and its emergence in the western hemisphere assessing the global threat from zika virus aurintricarboxylic acid blocks in vitro and in vivo activity of yoph, an essential virulent factor of yersinia pestis, the agent of plague an alanine-to-valine substitution in the residue of zika virus ns a protein affects viral rna synthesis and attenuates the virus in vivo flaviviral methyltransferase/rna interaction: structural basis for enzyme inhibition hiv- upregulates fas ligand expression in cd + t cells in vitro and in vivo: association with fas-mediated apoptosis and modulation by aurintricarboxylic acid identification and analysis of hepatitis c virus ns helicase inhibitors using nucleic acid binding assays aurintricarboxylic acid inhibits the early stage of vaccinia virus replication by targeting both cellular and viral factors pathogenesis and molecular mechanisms of zika virus a 'furrytale' of zika virus infection: what have we learned from animal models? replication-competent influenza a viruses expressing a red fluorescent protein zika virus infection complicated by guillain-barre syndrome-case report, french polynesia humoral immune responses against zika virus infection and the importance of preexisting flavivirus immunity zika virus: an update on epidemiology, pathology, molecular biology, and animal model modified mrna vaccines protect against zika virus infection the zika virus: an association to guillain-barre syndrome in the united states -a case report aurintricarboxylic acid protects hippocampal neurons from nmda-and ischemia-induced toxicity in vivo activation of the jak -stat signaling pathway in nb lymphoma cells by an anti-apoptotic agent, aurintricarboxylic acid aurintricarboxylic acid modulates the affinity of hepatitis c virus ns helicase for both nucleic acid and atp a live-attenuated zika virus vaccine candidate induces sterilizing immunity in mouse models lack of efficacy of aurintricarboxylic acid and ethacrynic acid against vaccinia virus respiratory infections in mice aurintricarboxylic acid in a canine model of coronary artery thrombosis zika virus infects human cortical neural progenitors and attenuates their growth a novel zika virus mouse model reveals strain specific differences in virus pathogenesis and host inflammatory immune responses zika virus: history, emergence, biology, and prospects for control emetine inhibits zika and ebola virus infections through two molecular mechanisms: inhibiting viral replication and decreasing viral entry key: cord- -donflx w authors: khan, raymond m.; al-juaid, maha; al-mutairi, hanan; bibin, george; alchin, john; matroud, amal; burrows, victoria; tan, ismael; zayer, salha; naidv, brintha; kalantan, basim; arabi, yaseen m. title: implementing the comprehensive unit-based safety program model to improve the management of mechanically ventilated patients in saudi arabia date: - - journal: am j infect control doi: . /j.ajic. . . sha: doc_id: cord_uid: donflx w background: ventilator-associated events are common in mechanically ventilated patients. they are associated with more days on mechanical ventilation, longer intensive care unit (icu) stay, and increased risk of mortality. theoretically, interventions that prevent ventilator-associated events should also reduce associated morbidity. we evaluated the comprehensive unit-based safety program approach to improve the care of mechanically ventilated patients. methods: all mechanically ventilated patients admitted to the icu between october , , and october , , were prospectively monitored for the development of ventilator-associated events according to the national healthcare safety network criteria. a process care bundle (endotracheal intubation with subglottic suctioning, head-of-bed elevation ≥ °, target sedation scores, daily spontaneous awakening trials, spontaneous breathing trials), daily delirium assessment, and an early mobility protocol were instituted. the bundle compliance, ventilator-associated events rates, icu length of stay, and mortality rate were noted. the database allowed viewing of current rates, trends, and averages of all participating sites. results: in the study period, , patients were admitted to the icu, and , required mechanical ventilation ( , ventilator days). there were ventilator-associated events: ventilator-associated conditions, infection-related ventilator-associated conditions, and possible cases of ventilator-associated pneumonia. the icu mortality rate was . %, compared with . % for those mechanically ventilated patients with ventilator-associated events (p = . ). there was increased compliance for spontaneous awakening trials ( . %- . %, p = . ) and spontaneous breathing trials ( . %- . %, p = . ) and a decrease in infection-related ventilator-associated conditions ( . - . per , days), possible cases of ventilator-associated pneumonia ( . - . per , days), icu mortality ( . %- . %, p = . ), and ventilator-associated events associated mortality rates ( . %- . %, p < . ). physical therapy participation and mobility were . % and . %, respectively. conclusion: the implementation of a multipronged program like the comprehensive unit-based safety program could improve the care processes and outcomes of mechanically ventilated patients. in , the institute for healthcare improvement (ihi) , lives campaign introduced the concept of a "care bundle" for the prevention of ventilator-associated pneumonia (vap). a care bundle identifies a set of key interventions that, when implemented together as a best practice approach, are expected to improve patient outcomes. in recent years, approaches to the care of mechanically ventilated patients have evolved from fixating only on vap prevention to focusing on a more comprehensive strategy based on the recent finding of benefit from the combined approach of the abcde bundle (awakening and breathing trial coordination, delirium management and early mobilization) and the wake up and breathe collaborative trial. although previous improvement initiatives used vap rates as the primary outcome measure, it has been increasingly recognized that mechanical ventilation (mv) causes harm beyond just vap. hence, wider outcome measures were needed to determine the value and success of safety programs. the centers for disease control and prevention, together with several critical care societies, convened a group to address the limitations of the national healthcare safety network definition of injury caused by mv, and they proposed a new approach in . besides vap, the new algorithm uses objective criteria for the diagnosis of ventilator-associated events and conditions and infection-related ventilator-associated complications. this approach thereby broadens the definition of harm suffered by ventilated patients beyond pneumonia to include pulmonary edema, atelectasis, and acute respiratory distress syndrome. the comprehensive unit-based safety program (cusp) approach was developed by patient safety researchers at the johns hopkins hospital, baltimore, maryland. cusp is designed to improve teamwork and safety culture and to guide organizations to learn from mistakes by using a validated and structured framework. cusp involves a repetitious process that trains a multidisciplinary team about the science of safety, asking them to identify defects, learn from them, implement improvement tools, and establish a partnership with senior leaders. key components include identifying evidence-based interventions that improve the outcomes of interest, converting these interventions into behaviors, placing value on the wisdom of frontline staff, and empowering frontline staff to be actively involved in safety improvements. the cusp intervention has achieved great success in reducing vap, central lineÀassociated bloodstream infections, catheter-associated urinary tract infections, surgical care complications, mortality, and associated costs. , our hospital's infection control and intensive care departments implemented a vap prevention program in , which led to the reduction of vap rates from . to per , ventilator days in . despite our success in reducing the vap rates, our data indicated that intensive care unit (icu) length of stay (los) ( . vs . days) and mortality rates ( % vs . %) were both higher in the post-vap prevention bundles compared with the pre-vap prevention intervention group, implying that merely decreasing the rate of vap was not enough. we needed to implement other strategies to optimize patient care to improve outcomes. we joined the johns hopkins armstrong institute comprehensive unit-based safety program for mechanically ventilated patients and ventilator-associated pneumonia (cusp mvp-vap) project in october with the objective of improving the care delivery process and reducing the mortality of our mechanically ventilated patients. this article describes the impact of implementing the cusp mvp-vap project on patient care in our icus at the ministry of national guard health affairs in riyadh. this was a prospective quality improvement and patient safety study to describe the impact of implementing the cusp mvp-vap in a cohort of patients in our icus. this improvement project was performed at king abdul aziz medical city in riyadh, saudi arabia, for all adult patients who received invasive mv in the icu between october , , and october , . the icu had beds and was covered by onsite board-certified intensivists hours per day, days per week, with a nurse-to-patient ratio of approximately : and a respiratory therapist-to-patient ratio of approximately : . the hospital was a , -bed tertiary-care center accredited by the joint commission international, with an active infection prevention and control program that collaborated with the icu medical and nursing staff to ensure the implementation and monitoring of infection control practices. the institutional review board and king abdullah international medical research center ethics committee of national guard health affairs, riyadh, saudi arabia, approved this study and waived the requirement for informed consent. a multidisciplinary cusp mvp-vap team was created in september to implement evidence-based practices for all mechanically ventilated patients. the group was led by an intensivist but included other physicians, nurses, respiratory therapists, physical therapists, infection control practitioners, and quality management personnel. the team monitors (nurses and research coordinators) were trained on data collection and monitored compliance on a daily basis. they reviewed the electronic charts of all patients on mv in the icu daily. the implementation of each care process bundle element, along with the confusion assessment method for the icu (cam-icu) score and the maximum level of mobility for that day were recorded on a standard data collection form and entered into the johns hopkins armstrong institute database, which generated a compliance rate for our hospital. this compliance rate was compared with those of other institutions in the project, so that we could benchmark our performance. if a component of the bundle was not performed, the inspectors used this moment to elucidate any barriers to the implementation of the particular element. the first month (october ) was considered the baseline data point. we defined a ventilator-associated condition (vac) as an increase in fio . or positive end expiratory pressure (peep) cm h o sustained for calendar days in a patient on mv for > days with a baseline period of stability or improvement, defined by calendar days of stable or decreasing daily fio or peep values. an infection-related ventilator-associated complication (ivac) occurred on or after days of mv when a patient met the criteria for a vac plus both of the following: temperature > °c or < °c and white blood cell count , or , cells/mm , as well as a new antimicrobial agent(s) started and continued for calendar days. a possible vap (pvap) occurs in a patient with the criteria for an ivac and of the following: positive culture meeting quantitative or semiquantitative thresholds from endotracheal aspirate ( colony-forming units [cfu]/ml), bronchoalveolar lavage ( cfu/ml), lung tissue ( cfu/g), or protected specimen brush ( cfu/ml); purulent respiratory secretions (> neutrophils and < squamous epithelial cells per low-power field plus organism identified from sputum, endotracheal aspirate, bronchoalveolar lavage, lung tissue, protected specimen brush); or organism identified from pleural fluid, lung histopathology, legionella tests, or diagnostic test on respiratory secretions for influenza virus, respiratory syncytial virus, adenovirus, parainfluenza virus, rhinovirus, human metapneumovirus, or coronavirus. ventilator-associated events (vaes) are the sum of vac, ivac, and pvap. all patients on mv were reviewed prospectively and independently by physicians who confirmed the diagnosis. the incidence of vae, vac, ivac, and pvap was expressed as cases per , ventilator-days. the project had arms: daily care process, early mobility, and low tidal volume ventilation. participation could be in , , or all of the arms (fig ) . our hospital selected daily care process and early mobility, because low tidal volume ventilation was already a standard practice in our icus for all patients on mv. i. daily care process a. endotracheal tube with subglottic suctioning (sub-g eet) all patients anticipated to need mv for > hours were intubated with a taperguard evacuation oral tracheal tube (covidien, mansfield, ma). the data collectors inspected patients for the presence of sub-g eet when indicated and documented whether the subglottic drainage lumen was connected to the wall suction at the appropriate intermittent negative pressure. b. head of bed (hob) °t he hospital was equipped with hill-rom hospital beds (hill-rom, chicago, il). the angle of the hob was measured with an electronic device or built-in protractor present on the bed. elevation of the hob was the default order for all patients on mv. exceptions were hypotension; unstable physiological status; low cardiac index; recent cervical, thoracic, or lumbar surgery or instability; ventricular assist device; intra-aortic balloon pump; open abdomen; and patient refusal. this element required that the data collector directly observe the angle of the hob. a nurse-led sedation vacation protocol was implemented that allowed the nurse to stop all sedation at : a.m. if the patient fulfilled certain criteria. the sat was continued until either the patient was agitated or fully awake and could be assessed for delirium. for this element, the data collectors asked the bedside nurse whether sedation was interrupted. they then reviewed the patient's daily flow sheet to confirm the nurse's statement. if the chart did not reflect sedation interruption, then this bundle was considered noncompliant. d. sedation-minimized (sedation score target) sedation orders were entered via a standardized computer order set, with dosage adjusted based on the patient's weight and renal and hepatic functions. in addition, a target sedation score (richmond agitation-sedation scale [rass]) had to be assigned to the patient by the physician before the order could be completed. the targeted rass was addressed daily in rounds, and the nurse's documentation was examined to determine whether the patient's actual sedation score matched the planned target. a ventilator weaning protocol was drafted that allowed respiratory therapists to wean all patients on mv starting at : a.m., hour after the sedation was held. patients who met the following criteria were weaned to pressure support ventilation: awake or off sedation with rass or for > hour spontaneous inspiratory efforts oxygen saturation > % the spontaneous breathing trial was conducted by placing the patient on pressure support ( - cm h o) with or without cm h o peep. both the patient's icu flow sheet and respiratory therapist's notes were monitored to assess this element. the cam-icu advocated by the society of critical care medicine was used to evaluate for delirium. in our hospital the assessment tool was translated into arabic, and, after a validation process of several plan-do-study-act cycles, staff were trained to perform this appraisal. the cam-icu score was recorded at a.m. daily and documented : p if the patient is positive for delirium based on cam-icu assessment n if the patient is negative for delirium based on cam-icu assessment uta if unable to assess (ie, rass = ¡ or ¡ ) x if cam-icu assessment was not completed nk if cam-icu was completed, but results are not known nk was also used if it was not known whether the cam-icu was performed ii. early mobility a. mobility-tailor goals to maximize mobility all patients admitted to the icu had standing orders for physical therapy (pt) and occupational therapy as part of the admission order sets. the level of mobility ( to ) was recorded: : passively rolled or exercised; : transfer from bed to chair without standing; : sitting in bed/exercising in bed; : sitting at edge of bed; : standing with or without assistance; : transfer from bed to chair with standing; : marching in place; : walking at least steps; and : unknown what level of activity occurred. additionally, any perceived barrier to achieving a higher level of mobility was documented. there were bimonthly meetings among the cusp team and monthly webinars with the armstrong institute. frequencies and percentages were used for categorical variables, whereas means with standard deviations were presented for continuous variables. the fisher exact test was used to evaluate differences between categorical variables, and the t test was used to evaluate differences between continuous variables. comparisons for mortality, los, and mv days were made, with the first month (october ) used as the base line. the unadjusted risk of death from developing a vae was compared using the fisher exact test; for those patients receiving mv who did not develop a vae. p < . was considered statistically significant. during the study period , patients were admitted to the icu; , ( %) required mv, with , episodes of mv and , ventilator days. there were vaes, of which were vacs, ivacs, and pvaps (table , fig ) . the overall icu mortality rate was . % compared with . % for those with development of a vae (p = . ). the icu mortality rate for mechanically ventilated patients decreased from . % to . % (p = . ), whereas the mortality rate associated with vaes decreased from . % to . % (p = . ) over the study period (fig ) . there were significant increases in mv days and icu los for patients with vaes ( table ) . the mean care bundle compliance for all the elements was . %. the compliance rates for endotracheal intubation with subglottic suctioning, hob elevation °, daily sats, and sbts were . %, . %, . %, and . %, respectively. the greatest improvement was seen in sats, which increased from . % in november to . % in october ( . % absolute increase, p = . ). this was followed by sbts, which increased from . % to . % ( % absolute increase, p = . ). the compliance of endotracheal intubation with subglottic suctioning decreased by . % (p = . ), whereas for hob it remained around % throughout the project (fig ) . the target rass was achieved in . % of mechanically ventilated patients, whereas . % had a rass of ¡ to + . however, . % of patients had their sbt done off sedation, and the percentage of mechanically ventilated patients without sedation increased from . % to . % (p = . ). the delirium assessment compliance rate was . %, with . % reporting a negative cam-icu. the percentage of incorrectly reported cam-icu scores was . %, which significantly decreased from . % (november ) to . % (october ) (p = . ). the pt and occupational therapy participation rates were . % and . %, respectively. only . % of mechanically ventilated patients were moved from bed to chair. the most frequent level of mobility achieved was (passively rolled or exercised [ . %]). the other levels were (transfer from bed to chair without standing . the most common perceived barriers to mobilization were the following: patient weakness ( %), hemodynamic instability ( . %), low rass on sedation ( . %), low rass off sedation ( %), and the patient labeled comfort care ( . %). however, the most documented adverse event was circulatory or respiratory instability ( . %). in our study the implementation of the multifaceted cusp -mvp vap approach resulted in an increase in sat ( . %- . %, p = . ) and sbt ( . %- . %, p = . ) compliance; an increase in the number of mechanically ventilated patients without sedation ( . %- . %, p = . ); and a decrease in ivacs ( . - . per , mv days), pvap ( . - . per , mv days), icu mortality rates ( . %- . %, p = . ), and vae mortality rates ( . %- . %, p < . ). finally, we found that our compliance with pt participation and mobility were suboptimal. in , the centers for disease control and prevention replaced their vap surveillance definitions with vae objective criteria, in response to a series of concerns about the traditional vap definitions, including their complexity, subjectivity, burden on surveyors, lack of comparability between institutions, narrow focus, and limited association with adverse outcomes. furthermore, vap did not consistently identify patients at increased risk for poor outcomes, and interventions that reduced vap rates often had no effect on patient-centered outcomes, such as duration of mv or hospital mortality. this is demonstrated in our previous vap prevention project, in which, in spite of the rate of vap decreasing from . to per , ventilator days, the days on mv remained unchanged, whereas the icu los and icu and hospital mortality rates all significantly increased in the postbundle implementation group. the failure of most vap prevention strategies to yield better outcomes for ventilated patients raises the question of whether vap is the best target to drive surveillance and safety prevention programs, because quality improvement initiatives must focus on identifying and preventing objective complications that are unambiguously associated with poor outcomes. thus the explicit intent of the a vae criteria was to broaden the focus of quality surveillance beyond just pneumonia. klompas et al, in a retrospective study of , episodes of mv found that vaes were associated with more days to extubation (relative risk, our data also demonstrated that vaes are associated with increased mechanical ventilator days, icu los, and icu mortality, highlighting the point that a vae appears to be a clinically important event. the synthesis of the vae criteria has created a new opportunity for health care facilities to reexamine their approach to preventing complications and improving outcomes of mechanically ventilated patients. vae surveillance has a quality metric character and appears to identify potential safety opportunities to improve care and outcomes for patients. theoretically, interventions most likely to prevent vaes are those that help patients avoid intubation, minimize the duration of mv, or prevent the conditions that most commonly trigger a vae (pneumonia, volume overload, acute respiratory distress syndrome, and atelectasis). use of high-flow nasal oxygen for hypoxemic and noninvasive ventilation for hypercapnic respiratory failure may avoid intubation. minimizing sedation, performing daily coordinated sats and sbts, and perhaps early mobility are strategies to decrease the duration of mv. strategies to prevent pneumonia, volume overload, acute respiratory distress syndrome, and atelectasis include hob elevation, conservative fluid management, conservative blood transfusion thresholds, low tidal volume ventilation, and early mobility. these interventions are consistent with the best care practices advocated by the abcdef bundle, the surviving sepsis campaign, and the society for healthcare epidemiology of america's recommendations to prevent vap. [ ] [ ] [ ] growing data support that implementing and optimizing these practices can lower vae rates and improve patient outcomes. the cusp mvp-vap project was engendered to continue this wider focus of implementing an evidence-based practice bundle while caring for mv patients. our study showed that sat and sbt rates were . % and %, respectively. this is similar to data published from icus in maryland and pennsylvania with , ventilated patient-days in which compliance with sat and sbt was . % and %, respectively. our icus have a nurse-led sedation vacation protocol and target sedation scores for all sedated patients. furthermore, we have an sbt protocol that is respiratory therapist driven. our low compliance rates could highlight the difficulty of translating evidence-based practice to bedside, or they may represent a defect in our protocol design preventing meaningful change in the practice behavior and culture of our front-line staff. our data demonstrated that . % of mechanically ventilated patients were mobilized into a chair, and only . % were evaluated by a physical therapist while receiving mv. early and progressive mobilization has been demonstrated to be both safe and feasible for patients admitted to critical care. implementing early mobility programs has led to improvements in physical function and mobility levels, significant reductions in both icu and hospital los, and ventilation days and a reduction in both the incidence and duration of delirium. in fact, the abcde bundle is centered on approaches to implement the integrated pain, agitation, and delirium clinical practice guidelines to reduce delirium and weakness related to oversedation, prolonged mechanical ventilation, and immobility in mechanically ventilated critically ill patients. despite this, point prevalence surveys have shown that rehabilitation levels remain low. goddard and colleagues, using a theoretical domains framework of behavior change, found that the social influences domain (local champions, icu leadership, discord between team members and family members) and behavioral regulation domain (feedback and having a unit protocol) may act as barriers or facilitators to early rehabilitation. based on these findings, we formulated a multidisciplinary team and a mobility protocol to provide tools to standardize the care of our patients (appendix ). the strengths of our study include the use of prospective collected data, a large sample size with all patients observed daily until icu discharge, and a common surveillance system using standardized definitions with a web-based portal for real-time reports. despite its strengths, our study has several potential limitations. first, a reduction in vae rates might be beyond the effect of just implementing the bundle, because the vae rates might have decreased secondary to other simultaneous infection control projects in our icus. second, this was not a preintervention and postintervention study, so analyzing the full effect of the bundle is difficult. third, despite having standardized data collection techniques and sources, team members might be motivated to demonstrate improvement and potentially could bias results. fourth, we report vae rates per , ventilator days, and any intervention that decreases ventilator days may paradoxically increase vae rates and underestimate the impact of the intervention on vae outcomes. fifth, icu los may depend on multiple complex factors and not only vaes. finally, the data represent a cohort study from a single center, and our inventions might not be generalized to other institutions. sustaining a safety culture should be a public health priority of all health care facilities. a strategic framework for preventing vaes is to pair clinical bundle with practice behavior and culture change interventions. the implementation of a multipronged program like the cusp mvp-vap that places ownership on front-line staff, reduces appendix a early mobility protocol complexity, provides standardized tools, engages executives, and uses communication tools to strengthen teamwork could improve the care processes and outcomes of mechanically ventilated patients. the impact of a ventilator bundle on preventing ventilator-associated pneumonia: a multicenter study guidelines for severe infections: are they useful? liberation and animation for ventilated icu patients: the abcde bundle for the back-end of critical care the preventability of ventilator-associated events. the cdc prevention epicenters wake up and breathe collaborative search for direct top squark pair production in final states with one isolated lepton, jets, and missing transverse momentum complications of mechanical ventilation-the cdc's new surveillance paradigm observation of a new chi(b) state in radiative transitions to upsilon( s) and upsilon( s) at atlas creating high reliability in health care organizations targeted implementation of the comprehensive unit-based safety program through an assessment of safety culture to minimize central line-associated bloodstream infections introducing the comprehensive unit-based safety program for mechanically ventilated patients in saudi arabian intensive care units comprehensive unit-based safety program (cusp) to improve patient experience: how a hospital enhanced care transitions and discharge processes the impact of implementing multifaceted interventions on the prevention of ventilator-associated pneumonia ventilator-associated events years later ventilator-associated events and their prevention descriptive epidemiology and attributable morbidity of ventilator-associated events the clinical impact of ventilatorassociated events: a prospective multi-center surveillance study the impact of ventilator-associated events in critically ill subjects with prolonged mechanical ventilation should ventilator-associated events become a quality indicator for icus? potential strategies to prevent ventilator-associated events improving hospital survival and reducing brain dysfunction at seven california community hospitals: implementing pad guidelines via the abcdef bundle in , patients surviving sepsis campaign: international guidelines for management of sepsis and septic shock strategies to prevent ventilator-associated pneumonia in acute care hospitals: update two-state collaborative study of a multifaceted intervention to decrease ventilator-associated events safety of patient mobilization and rehabilitation in the intensive care unit. systematic review with meta-analysis earlier and enhanced rehabilitation of mechanically ventilated patients in critical care: a feasibility randomised controlled trial a feasibility study of a randomised controlled trial to examine the impact of the abcde bundle on quality of life in icu survivors barriers and facilitators to early rehabilitation in mechanically ventilated patients-a theory-driven interview study we thank dr. sean m. berenholtz (professor, johns hopkins armstrong institute for patient safety and quality) and the johns hopkins armstrong institute for patient safety and qualityÀcusp mvp vap team. key: cord- - vp fwv authors: simonsen, lone; higgs, elizabeth; taylor, robert j; wentworth, deborah; cozzi-lepri, al; pett, sarah; dwyer, dominic e; davey, richard; lynfield, ruth; losso, marcelo; morales, kathleen; glesby, marshall j; weckx, jozef; carey, dianne; lane, cliff; lundgren, jens title: using clinical research networks to assess severity of an emerging influenza pandemic date: - - journal: clinical infectious diseases doi: . /cid/ciy sha: doc_id: cord_uid: vp fwv background: early clinical severity assessments during the influenza a h n pandemic (ph n ) overestimated clinical severity due to selection bias and other factors. we retrospectively investigated how to use data from the international network for strategic initiatives in global hiv trials, a global clinical influenza research network, to make more accurate case fatality ratio (cfr) estimates early in a future pandemic, an essential part of pandemic response. methods: we estimated the cfr of medically attended influenza (cfr(ma)) as the product of probability of hospitalization given confirmed outpatient influenza and the probability of death given hospitalization with confirmed influenza for the pandemic ( – ) and post-pandemic ( – ) periods. we used literature survey results on health-seeking behavior to convert that estimate to cfr among all infected persons (cfr(ar)). results: during the pandemic period, . % ( . %– . %) of ph n -positive outpatients were hospitalized. of ph n -positive inpatients, . % ( . %– . %) died. cfr(ma) for ph n was . % ( . %– . %) in the pandemic period – but declined -fold in young adults during the post-pandemic period compared to the level of seasonal influenza in the post-pandemic period – . cfr for influenza-negative patients did not change over time. we estimated the pandemic cfr(ar) to be . %, -fold lower than cfr(ma). conclusions: data from a clinical research network yielded accurate pandemic severity estimates, including increased severity among younger people. going forward, clinical research networks with a global presence and standardized protocols would substantially aid rapid assessment of clinical severity. clinical trials registration: nct and nct . in , uncertainty about the emerging ph n virus' clinical severity hindered the early global response. although the rapid spread of the virus around the world fulfilled the traditional pandemic definition, its global mortality impact in the end proved to be smaller than any th century pandemic [ , ] . however, its relative mildness was not known in the early months of the outbreak. the earliest estimate of the case fatality ratio (cfr) was on par with the rating for the catastrophic pandemic, and a june assessment put it in the pandemic range (table ) [ ] . an evaluation of the pandemic response ordered by the world health organization's (who) director general [ ] found that a systematic way to assess both transmissibility and clinical severity-also known as its "seriousness" [ ] -is needed in the early phase of a future pandemic to assess the level of threat accurately and to mobilize resources appropriately. cfr is one important measure of clinical severity; others include the risk of admission to the intensive care unit (icu) and the need for mechanical respiratory support. a who task force is currently developing the data inputs and study designs needed to generate timely estimates of clinical severity [ ] . the centers for disease control and prevention has proposed a scheme for comparing pandemic and seasonal influenza graphically, plotting attack rates against clinical severity [ ] . in , uk public health england spearheaded what has become a standard first-line approach to assessing the clinical severity of a pandemic, known as the "first few hundred" (ff ) [ ] . these and similar studies gather data on the earliest cases that come to medical attention through outpatient facilities and hospitals and provide important descriptive data about symptoms, risk factors, and risk of progression to severe illness or death [ ] [ ] [ ] [ ] [ ] [ ] [ ] . these data can in turn be combined with other data on population attack rates to forecast national and global hospitalization and mortality estimates using a pyramid modeling strategy [ , ] . standard ff studies, however, lack historic controls in the form of a baseline from recent seasonal influenza seasons. they are also subject to selection bias, as the first cases that come to attention are likely to be more severe [ ] . unless an ff study is set in an existing surveillance system or ongoing clinical research data collection scheme, there is no obvious seasonal influenza baseline against which to compare the clinical severity of the pandemic virus. moreover, unless the pandemic is severe, an ff study in the outpatient setting alone will not have the statistical power to accurately estimate the cfr unless many thousands of patients are enrolled. global clinical research networks that study mild and severely ill influenza patients could be used to overcome many of these problems. two ongoing clinical cohort studies of influenza are conducted under the international network for strategic initiatives in global hiv trials (insight) umbrella, sponsored by the national institutes of health. since , insight has undertaken cohort studies- outpatient (flu ) and inpatient (flu )-specifically to address gaps in clinical research on the emerging influenza pandemic, including factors linked to disease progression and severe outcomes [ ] . insight annually enrolls hundreds of patients with suspected or confirmed influenza, with intake sites in countries. at these sites, experienced teams use a standardized protocol to collect extensive clinical data, perform long-term follow-up (at and days for inpatients, days for outpatients), and bank patient samples for further study. several articles on influenza have been published using insight data, including protocol descriptions and preliminary data [ ] , an exploration of biomarkers of influenza case severity [ ] , patient outcomes after ph n infection [ ] , and phylogeography of the ph n virus [ ] . we used insight data collected in the pandemic period ( - ) to retrospectively demonstrate how clinical research networks can provide essential early insights into pandemic clinical severity and other epidemiological parameters. to "leverage" the cfr computation, we multiplied the conditional probability of progression from outpatient to hospitalization by that of progression from hospitalization to death. to underscore the importance of having baseline data, we compared the estimated ph n clinical severity to that of seasonal influenza types and subtypes and noninfluenza respiratory patients in the post-pandemic period ( ) ( ) ( ) ( ) . our cfr estimates were in reasonable agreement with final global cfr estimates based on excess mortality estimates from time series of nationwide vital statistics data and seroepidemiology data-final estimates of a type that would only be available several years after the next pandemic emerges [ , , ] . here, we discuss what it would take to move a clinical research network like insight from routine research operation into emergency mode to generate timely and robust clinical severity assessments. the national institute of allergy and infectious diseases (niaid)-funded insight network initially focused solely on hiv but expanded first to include ph n and then all influenza types and subtypes and emerging respiratory pathogens such as middle east respiratory syndrome and severe acute respiratory syndrome. sites, located in of world regions (figure ), consecutively enroll adult patients aged ≥ years with suspected influenza. flu recruits patients who present at a physician's office or clinic with influenza-like illness (ili), defined as fever with either cough or sore throat. flu recruits patients with known or suspected influenza who require hospitalization. at enrollment, patient medical history and demographic information are recorded, and blood and oropharyngeal swabs are analyzed and stored. testing for influenza is done both locally and at an insight central laboratory. all patients are followed up, regardless of influenza test result, at days after enrollment in flu and at and days in flu . we extracted insight data on demographics, illness onset, medical history, and vital status at follow-up visit from the protocol databases. we defined the pandemic period as the to follow-up were treated as missing and removed from the analysis. we identified relevant case series in the literature reporting data on patients aged > years. after excluding studies with fewer than patients or with a specialty population (such as high-risk patients), we chose outpatient studies, set in the united states [ ] and in the united kingdom [ ] , and inpatient studies [ , ] , both set in the united states, for comparison with flu and flu ph n laboratory-confirmed patients during the pandemic period (table ) . we calculated the medically attended cfr (cfr ma ) from the probability that a medically attended ili (flu ) patient would progress to hospitalization by day and the probability that a hospitalized (flu ) patient would die by day : where h = hospitalization and d = death to estimate cfr among all infected persons (cfr ar ), we used findings from a uk health behavior survey that found that % of patients aged ≥ years with ili sought care for their illness [ ] and a uk serology study that found that % of influenza-infected adults aged - years were symptomatic [ ] . assuming that the nonmedically attended and asymptomatic influenza cases would not progress to severe illness, we have: , where "infection" is defined as a person who responded immunologically. the % confidence intervals (cis) on the cfr estimate were generated from the variance of the product of the proportions, p(h/ili) × p(d/h), using the delta method or a first-order taylor series expansion. we assumed the proportions were independent. in small samples with large variability, this may not be a good approximation. in some cases, negative values for the cis may be obtained. data analysis was done using sas, version . , and excel. the flu and flu protocols were approved by the institutional review boards or institutional ethics committees at the university of minnesota and at each of the participating clinical sites. all patients (or their proxies) gave signed informed consent prior to enrollment. during the pandemic period (october through september ), ili and hospitalized patients tested influenza ph n positive. of these, . % of ph n -infected flu outpatients were aged - years compared to only % of the flu inpatients. during the post-pandemic period (october through september ), ili and hospitalized patients were ph n positive; of these, % of ili outpatients and % of hospitalized patients were aged - years. in the pandemic period about / of outpatients and / of inpatients were from european sites, while during the post-pandemic period, after the network expanded to sites in world regions, these figures were / of outpatients and / of inpatients. we found that demographic and clinical characteristics of insight pandemic period ph n patients were similar to those described in published ff -like studies of adult ph n patients [ ] with respect to mean age, prevalence of symptoms and underlying diseases, mortality rates, and other characteristics ( table ) . five percent of ph n -confirmed ili patients were hospitalized, and . % of ph n -positive inpatients died (table , figure ). this yielded a ph n cfr ma of . % ( . %- . %) both for all adults and for adults aged - years. the cfr ma for patients aged ≥ years could not be established with confidence due to the small number of older outpatients in the study. as a nonhistoric control, the all-ages cfr ma of influenza testnegative patients was . % during the pandemic period, albeit with wide cis. it was not possible to establish a seasonal influenza comparison for the pandemic period because non-ph n influenza cases (h n , b) in the pandemic period were rare. the cfr ma for ph n cases in the post-pandemic period was . % for patients aged - years, -fold lower than the value for the pandemic period and comparable to the influenza-negative patients of the same age. we could not reliably assess ph n cfr ma for the ≥ years age group due to small numbers in the post-pandemic period; however, cfr ma was . % for seniors aged ≥ years positive for any influenza virus in the post-pandemic period vs . % for younger adults positive for any influenza virus. for the post-pandemic period (any subtype), we also estimated the conditional probabilities and the cfr ma by region (table ) . because the final who cfr estimate from the pandemic was based on attack rates as revealed by serology data, we sought to convert our medically attended cfr to one based on the attack rate. to do so, we used data from a study that indicated that approximately % of all cases are asymptomatic [ ] and from survey data that indicate that approximately % of adult ili cases sought medical attention [ ] . we found the cfr ar to be . % ( . %- . %; table ), or -fold lower than the cfr ma . who has recently expanded its pandemic definition to include clinical severity. this means that rapid and accurate estimates of pandemic clinical severity are needed to characterize the threat level and guide the global response. our analysis combining data from inpatient and outpatient insight cohorts demonstrates how preestablished global research networks could immediately begin rigorous studies to estimate the cfr, a key parameter of clinical severity of an emerging pandemic. assessments of the clinical severity in the pandemic became less dire as time passed [ ] . the earliest estimate of cfr, an ff -like case series of hospitalized patients in mexico, was a disturbing % of influenza-positive patients. however, as studies of the first (summer) wave in the united states, the complete southern hemisphere season in new zealand, and further studies from mexico were completed, it became clear that the pandemic would be relatively mild (table ) . several factors contributed to the early confusion in . the most important was probably selection bias toward sicker patients in the earliest ff -type case series studies [ ] . another factor was simply that studies reported on different types of cfr-either as a proportion of medically attended cases (cfr ma ) or as a proportion of all infected individuals (cfr ar ). most early assessments were of the cfr ma type, but these were not directly comparable. our method, retroactively applied to insight databases, yielded a cfr ma estimate of . %. using literature values that indicated that the probability of symptomatic people seeking medical treatment was % [ ] and that the probability of infected individuals being asymptomatic was also % [ ] , our cfr ma value would be equivalent to a cfr ar of . %, which data are for the pandemic and post-pandemic periods, computed as the product of the risk of flu influenza-like illness outpatients getting hospitalized and the flu hospitalized patients having died at day . abbreviations: p (d|h), probability of death given hospitalization; p (h|ili) , probability of hospitalization given influenza-like illness. *case fatality rate not calculated when fewer than outpatients or inpatients contained in any stratum. is in reasonable agreement with the final global who cfr ar estimate of . % [ , , ] . in addition to an absolute measurement of cfr, data from previous seasons can provide a relative comparison of pandemic to seasonal influenza severity; even if the absolute estimate of cfr is uncertain, it would be useful to know if an emerging pandemic has a cfr far higher than previous seasonal influenza experiences. thus, we also estimated cfrs for influenza patients from seasonal influenza epidemics - , as a surrogate for pre-pandemic baseline seasons. age greatly influences both seasonal and pandemic clinical severity estimates. in all influenza pandemics since , mortality was higher than normal in younger people and lower than normal in seniors, sometimes dramatically so [ ] . in the post-pandemic period ( - ) we found that the cfr ma of ph n for patients aged - years had fallen -fold from the pandemic period value, becoming similar to that of a/h n and b. this suggests that the emerging virus had settled into a seasonal epidemic pattern due to accumulated population immunity. moreover, in the post-pandemic period patients aged ≥ years with any influenza virus had a cfr ma approximately -fold higher than patients aged < years. these results corroborate a previous metaanalysis of ff studies that concluded that age is an important confounder of cfr estimates for ph n pandemic influenza [ ] . they also show how important it is to take into account both the age group and the type of cfr being calculated when comparing across regions and time. it is also possible that discrepancies in early assessments of cfr may in fact have reflected true geographical differences. for example, a comprehensive study of pandemic mortality that applied a uniform methodology to different regions found the mortality impact in central and south american countries table was approximately -fold higher than in europe [ ] . this indicates that early reports of higher severity in mexico than in new zealand may not solely have been the result of ascertainment bias. clinical severity can even increase substantially over time, as was seen in the influenza pandemic when a milder summer wave preceded the severe autumn waves [ ] . the best way around the measurement problems that occur early in a pandemic would be to compute the same type of cfr with the same protocol in multiple geographical settings. if possible, estimates should be stratified by risk factors, such as pregnancy and chronic illness, and baseline data should be collected during seasonal epidemics. while some countries have created ff protocols since the pandemic, a global standard along the lines we have outlined here would be helpful. we recognize limitations to our approach to computing cfr by multiplying conditional probabilities of disease progression. first, we used distinct groups of outpatients and inpatients who were recruited under different circumstances at different sites, often in different countries. it is therefore possible the cohorts differed in age composition, health status, or other important respects that could bias the result. however, we argue that the approach, while not ideal, would nonetheless supply timely and useful data, especially if it could be compared to baseline seasons. we also note that the characteristics of the insight ph n outpatients and inpatients in the pandemic period - are reassuringly similar in terms of age, symptoms, comorbidities, and outcomes to published uk and us ff studies of adult ph n influenza outpatients and inpatients ( table ) . a second possible caveat-that insight inclusion criteria might have varied over time and explained the drop in cfr ma over time-could be dismissed on the grounds that the influenza-negative patients did not have a significant drop in cfr ma between the pandemic and post-pandemic period. this means that the measured decrease in ph n clinical severity was real and not due to ascertainment or other bias. our retrospective analysis of pandemic clinical severity indicates that it is possible to use research networks to assess both the absolute magnitude of the clinical severity of a future pandemic and the relative increase compared to a seasonal influenza baseline. even if the seroepidemiology and health-seeking behavior surveys needed to convert cfr ma to cfr ar could not be done rapidly, comparison of cfr ma to previous seasons would reveal much about the relative magnitude of the emerging threat. to be useful in a prospective scenario, however, it would be necessary to ramp up the network's pace of operations from routine to emergency mode. for insight, that would mean, at a minimum, enhancing enrollment in sites located in areas initially affected by the emerging pandemic and increasing the tempo of laboratory processing of specimens and data analysis. in addition to assessing clinical severity, global research networks could play other key roles in pandemic response including studies of comorbidity patterns, risk factors, hospital and icu utilization, and mortality risk of hospitalized patients. moreover, protocols that enroll children could be used to understand the pathogen in this key age group. once a future pandemic outbreak begins, studies set in these networks could both characterize pathophysiology to optimize clinical management and provide a platform for rigorous clinical trials of new therapeutics. we suggest, therefore, that a specific role for clinical research networks carrying out ongoing rigorous research compliant with international standards be added to the international health regulations that govern international and national responsibilities for public health emergencies of international concern. notes acknowledgments. we thank and acknowledge all the patients who participated in this study. disclaimer. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. potential conflicts of interest. l. s. and r. j. t. report earning consulting fees from sage analytica, llc, during the conduct of the study. r. l. reports serving as co-editor on a book on infectious disease surveillance published by wiley blackwell, with royalties donated to the minnesota department of health. all remaining authors: no reported conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. global mortality estimates for the influenza pandemic from the glamor project: a modeling study estimated global mortality associated with the first months of pandemic influenza a h n virus circulation: a modelling study case fatality risk of influenza a (h n pdm ): a systematic review report of the review committee on the functioning of the international health regulations ( ) in relation to pandemic (h n ) infection fatality risk of the pandemic a(h n ) virus in hong kong novel framework for assessing epidemiologic effects of influenza epidemics and pandemics pandemic (h n ) influenza in the uk: clinical and epidemiological findings from the first few hundred (ff ) cases human infection with new influenza a (h n ) virus: clinical observations from mexico and other affected countries pandemic potential of a 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influenza: results of the flu watch cohort study preliminary estimates of mortality and years of life lost associated with the a/h n pandemic in the us and comparison with past influenza seasons epidemiologic characterization of the influenza pandemic summer wave in copenhagen: implications for pandemic control strategies flu clinical site investigators: argentina: laura barcan key: cord- - d o xk authors: choi, won hyung; lee, in ah title: the mechanism of action of ursolic acid as a potential anti-toxoplasmosis agent, and its immunomodulatory effects date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: d o xk this study was performed to investigate the mechanism of action of ursolic acid in terms of anti-toxoplasma gondii effects, including immunomodulatory effects. we evaluated the anti-t. gondii effects of ursolic acid, and analyzed the production of nitric oxide (no), reactive oxygen species (ros), and cytokines through co-cultured immune cells, as well as the expression of intracellular organelles of t. gondii. the subcellular organelles and granules of t. gondii, particularly rhoptry protein , microneme protein , and inner membrane complex sub-compartment protein , were markedly decreased when t. gondii was treated with ursolic acid, and their expressions were effectively inhibited. furthermore, ursolic acid effectively increased the production of no, ros, interleukin (il)- , il- , granulocyte macrophage colony stimulating factor (gm-csf), and interferon-β, while reducing the expression of il- β, il- , tumor necrosis factor alpha (tnf-α), and transforming growth factor beta (tgf-β ) in t. gondii-infected immune cells. these results demonstrate that ursolic acid not only causes anti-t. gondii activity/action by effectively inhibiting the survival of t. gondii and the subcellular organelles of t. gondii, but also induces specific immunomodulatory effects in t. gondii-infected immune cells. therefore, this study indicates that ursolic acid can be effectively utilized as a potential candidate agent for developing novel anti-toxoplasmosis drugs, and has immunomodulatory activity. recently, zoonotic diseases have been consistently occurring in different countries worldwide, causing serious threats in many countries and to humans globally. in this respect, the prevention and education of various zoonotic diseases are becoming a major issue. toxoplasmosis is a parasitic disease that is induced through the infection of toxoplasma gondii in many countries worldwide as a zoonotic parasitosis, causing serious diseases and chronic infection in various sites of the human body in all age groups, including adults and young children as well as pregnant women. in particular, because toxoplasmosis is a zoonotic disease that infects humans through cats, many people that have cats as pets, particularly pregnant women, need to take caution in this regard. in the biological aspect, t. gondii not only has intracellular organelles such as the golgi, endoplasmic reticulum, and mitochondrion, but also unique subcellular organelles such as the conoid, apicoplast, surface antigens (sags), dense granule proteins (gras), rhoptries, and micronemes [ ] . t. gondii has an inner membrane complex (imc) involving the plasma membrane, consisting of a unique double membrane structure which is combined with a cytoskeletal network. the imc acts as a major factor in the proliferation and growth for the survival of t. gondii, as well as in motility and cell division, which has been reported to include the t. gondii-infected cells treated with sf ( figure ). furthermore, it was clearly shown that there were significant differences between the untreated t. gondii-group and the experimental groups. in these aspects, it was demonstrated that ua effectively induced the selective anti-parasitic effect/action compared to the sf-treated group and the untreated group through a direct inhibitory effect on the viability of t. gondii. this shows that ua has the potential to be utilized as an anti-t. gondii candidate agent and/or a synergic compound with the existing drugs for developing novel anti-toxoplasmosis agents. therefore, these results indicate that ua strongly inhibits or blocks the survival of t. gondii by effectively inducing anti-t. gondii activity through the selective inhibitory activity and/or action against the viability of t. gondii. the t. gondii-infected cells treated with sf ( figure ). furthermore, it was clearly shown that there were significant differences between the untreated t. gondii-group and the experimental groups. in these aspects, it was demonstrated that ua effectively induced the selective anti-parasitic effect/action compared to the sf-treated group and the untreated group through a direct inhibitory effect on the viability of t. gondii. this shows that ua has the potential to be utilized as an anti-t. gondii candidate agent and/or a synergic compound with the existing drugs for developing novel anti-toxoplasmosis agents. therefore, these results indicate that ua strongly inhibits or blocks the survival of t. gondii by effectively inducing anti-t. gondii activity through the selective inhibitory activity and/or action against the viability of t. gondii. the normal lung cells were infected with t. gondii (cells: t. gondii = : ), and t. gondii-infected cells were treated with different concentrations ( . - µg/ml) of ursolic acid (ua) and sulfadiazine (sf) at • c for h, respectively; their survival rates were inhibited a dose-dependent manner. the results are presented as a percentage of the untreated normal group. * p < . was considered to be significant. t. gondii causes unique anti-apoptotic features and parasitic survival cycles or stages, such as the inactivation of apoptotic proteins during the parasitic life-cycle in host cells after infection, showing critical roles during the proliferation and survival of t. gondii. in particular, the pvm, that functions as a critical indicator during the proliferation stage of t. gondii, is activated in a time-dependent manner after cell invasion of t. gondii, which causes finally the proliferation and the growth of t. gondii. in this aspect, we investigated the inhibitory action of ua against subcellular organelles such as rhoptries, micronemes, and inner membrane complex that form its intracellular metabolic network as well as the homeostasis of t. gondii. as shown in figure , when t. gondii was treated with different concentrations ( and µg/ml) of ua and sf, respectively, the intracellular organelles (rhoptry protein (rop ), microneme protein (mic ), and inner membrane complex sub-compartment protein (imc sub- )) of t. gondii were markedly decreased in a dose-dependent manner, and their changes were clearly observed under electrophoresis. in particular, it is known that rhoptry plays as a critical factor when t. gondii enters into host cells during invasion stage [ ] [ ] [ ] [ ] [ ] . in these aspects, this result shows clearly that ua not only has the direct inhibitory effect against the survival of t. gondii but also induces the anti-t. gondii effect by strongly blocking or inhibiting the homeostasis and network structure of the intracellular organelles of t. gondii. therefore, these results demonstrate substantial evidence that ua consistently causes anti-t. gondii activity/action by effectively inhibiting the intracellular organelles of t. gondii. °c for h, respectively; their survival rates were inhibited a dose-dependent manner. the results are presented as a percentage of the untreated normal group. * p < . was considered to be significant. organelles of t. gondii t. gondii causes unique anti-apoptotic features and parasitic survival cycles or stages, such as the inactivation of apoptotic proteins during the parasitic life-cycle in host cells after infection, showing critical roles during the proliferation and survival of t. gondii. in particular, the pvm, that functions as a critical indicator during the proliferation stage of t. gondii, is activated in a time-dependent manner after cell invasion of t. gondii, which causes finally the proliferation and the growth of t. gondii. in this aspect, we investigated the inhibitory action of ua against subcellular organelles such as rhoptries, micronemes, and inner membrane complex that form its intracellular metabolic network as well as the homeostasis of t. gondii. as shown in figure , when t. gondii was treated with different concentrations ( and μg/ml) of ua and sf, respectively, the intracellular organelles (rhoptry protein (rop ), microneme protein (mic ), and inner membrane complex sub-compartment protein (imc sub- )) of t. gondii were markedly decreased in a dose-dependent manner, and their changes were clearly observed under electrophoresis. in particular, it is known that rhoptry plays as a critical factor when t. gondii enters into host cells during invasion stage [ ] [ ] [ ] [ ] [ ] . in these aspects, this result shows clearly that ua not only has the direct inhibitory effect against the survival of t. gondii but also induces the anti-t. gondii effect by strongly blocking or inhibiting the homeostasis and network structure of the intracellular organelles of t. gondii. therefore, these results demonstrate substantial evidence that ua consistently causes anti-t. gondii activity/action by effectively inhibiting the intracellular organelles of t. gondii. we evaluated the effect of ros and no production induced by ursolic acid in immune cells infected with t. gondii. the production of no and ros was measured in a dose-dependent manner when t. gondii-infected co-cultured immune cells were incubated with various concentrations ( - µg/ml) of ua and sf for h, respectively. in particular, the production of ros was increased in the t. gondii-infected group treated with ua compared with t. gondii-infected group. however, in µg/ml of ua, ros activity gradually indicated lower concentrations than other ua-treated groups (figure ), and these changes of ros activity show that ua acts as a modulator which consistently inhibits the proliferation and the growth of t. gondii by promoting the production of ros in immune cells after the parasite infection. even though sf-treated groups significantly increased ros production compared to the t. gondii-infected group, there were low ros-productive rates compared to ua-treated groups. furthermore, the rate of no production was strongly increased in t. gondii-infected group treated with ua compared with normal and t. gondii-infected groups, but in the range of µg/ml of ua, the rate of no production were low concentrations compared with other ua-treated groups ( figure ). however, the activities of both ros and no induced by ua were significantly increased more than normal and infection groups. interestingly, the activities of ros and no in t. gondii-infected cells treated with sf were lower than t. gondii-infected groups treated with ua. the activities of ros and no were significantly increased in t. gondii-infected groups treated with ua and sf, respectively, whereas their activities were gradually reduced in high concentrations. these results show that ua not only consistently induces the production of ros and no through the interaction between it and immune cells in t. gondii-infected immune cells, but may also be used as a synergic compound for the immunomodulation after parasitic infection. we evaluated the effect of ros and no production induced by ursolic acid in immune cells infected with t. gondii. the production of no and ros was measured in a dose-dependent manner when t. gondii-infected co-cultured immune cells were incubated with various concentrations ( - μg/ml) of ua and sf for h, respectively. in particular, the production of ros was increased in the t. gondii-infected group treated with ua compared with t. gondii-infected group. however, in μg/ml of ua, ros activity gradually indicated lower concentrations than other ua-treated groups (figure ), and these changes of ros activity show that ua acts as a modulator which consistently inhibits the proliferation and the growth of t. gondii by promoting the production of ros in immune cells after the parasite infection. even though sf-treated groups significantly increased ros production compared to the t. gondii-infected group, there were low ros-productive rates compared to ua-treated groups. furthermore, the rate of no production was strongly increased in t. gondii-infected group treated with ua compared with normal and t. gondii-infected groups, but in the range of μg/ml of ua, the rate of no production were low concentrations compared with other ua-treated groups ( figure ). however, the activities of both ros and no induced by ua were significantly increased more than normal and infection groups. interestingly, the activities of ros and no in t. gondii-infected cells treated with sf were lower than t. gondii-infected groups treated with ua. the activities of ros and no were significantly increased in t. gondii-infected groups treated with ua and sf, respectively, whereas their activities were gradually reduced in high concentrations. these results show that ua not only consistently induces the production of ros and no through the interaction between it and immune cells in t. gondii-infected immune cells, but may also be used as a synergic compound for the immunomodulation after parasitic infection. t. gondii proliferates rapidly in host cells through parasitic interactions and signals for its survival after cell invasion, which finally induces destruction of host cells. we evaluated the synergic effect and the activation of cytokines caused by ursolic acid in co-cultured immune cells (macrophages, t cells, b cells and basophil cells) infected with t. gondii. the production of the cytokines (interferon-β, granulocyte macrophage colony stimulating factor (gm-csf), transforming growth factor beta (tgf-β ), and tumor necrosis factor alpha (tnf-α)) in t. gondii-infected immune cells was notably changed in a concentration-dependent manner when the cells were incubated with different concentrations ( - μg/ml) of ua, and their changes were measured under an elisa reader. in particular, the production of interferon-β and gm-csf was effectively increased in t. gondii-infected cells treated with ua compared with t. gondii-infected cells, and these changes show that ua accelerated the production and release of interferon-β and gm-csf by stimulating the macrophage, b cells, and basophil cells ( figure ). moreover, the production of interferon-β was not changed in sf-treated groups, and the production of gm-csf was gradually decreased. furthermore, the expression of tgf-β and tnf-α was markedly reduced in t. gondii-infected cells treated with ua, whereas t. gondii-infected cells treated with sf did not cause the production of tgf-β and tnfα compared with other groups (figure ) . interestingly, t. gondii-infected cells treated with sf did not significantly produce the expression of interferon-β, tgf-β and tnf-α compared to the infected group. this shows that sf is not involved in immune response for expressing tnf-α, tgf-β , and interferon-β in immune cells. furthermore, the production of tgf-β was decreased in t. gondiiinfected cells treated with ua, but the result shows that ua reduced the expression of tgf-β in immune cells by directly inhibiting t. gondii. these results demonstrate that ua not only promotes defense against parasitic infection by effectively inducing the production of interferon-β and gm-csf from immune cells when the cells are infected with t. gondii, but also inhibits the overexpression of inflammatory cytokine by reducing the expression of tnf-α caused by infection. t. gondii proliferates rapidly in host cells through parasitic interactions and signals for its survival after cell invasion, which finally induces destruction of host cells. we evaluated the synergic effect and the activation of cytokines caused by ursolic acid in co-cultured immune cells (macrophages, t cells, b cells and basophil cells) infected with t. gondii. the production of the cytokines (interferon-β, granulocyte macrophage colony stimulating factor (gm-csf), transforming growth factor beta (tgf-β ), and tumor necrosis factor alpha (tnf-α)) in t. gondii-infected immune cells was notably changed in a concentration-dependent manner when the cells were incubated with different concentrations ( - µg/ml) of ua, and their changes were measured under an elisa reader. in particular, the production of interferon-β and gm-csf was effectively increased in t. gondii-infected cells treated with ua compared with t. gondii-infected cells, and these changes show that ua accelerated the production and release of interferon-β and gm-csf by stimulating the macrophage, b cells, and basophil cells ( figure ). moreover, the production of interferon-β was not changed in sf-treated groups, and the production of gm-csf was gradually decreased. furthermore, the expression of tgf-β and tnf-α was markedly reduced in t. gondii-infected cells treated with ua, whereas t. gondii-infected cells treated with sf did not cause the production of tgf-β and tnf-α compared with other groups (figure ) . interestingly, t. gondii-infected cells treated with sf did not significantly produce the expression of interferon-β, tgf-β and tnf-α compared to the infected group. this shows that sf is not involved in immune response for expressing tnf-α, tgf-β , and interferon-β in immune cells. furthermore, the production of tgf-β was decreased in t. gondii-infected cells treated with ua, but the result shows that ua reduced the expression of tgf-β in immune cells by directly inhibiting t. gondii. these results demonstrate that ua not only promotes defense against parasitic infection by effectively inducing the production of interferon-β and gm-csf from immune cells when the cells are infected with t. gondii, but also inhibits the overexpression of inflammatory cytokine by reducing the expression of tnf-α caused by infection. we evaluated the activation and interaction of various cytokines induced by ursolic acid through co-cultured immune cells (macrophage, t cells, b cells and basophil cells) infected with t. gondii. the changes of the cytokines (interleukin (il)- β, il- , il- , and il- ) in t. gondii-infected immune cells were measured in a dose-dependent manner when the cells were incubated with different concentrations ( - μg/ml) of ua, and their changes were evaluated under an elisa reader. in particular, the production of il- β and il- was reduced in the groups treated with ua compared with the infection group, and the expression of il- and il- was significantly increased in t. the changes of the cytokines (interleukin (il)- β, il- , il- , and il- ) in t. gondii-infected immune cells were measured in a dose-dependent manner when the cells were incubated with different concentrations ( - µg/ml) of ua, and their changes were evaluated under an elisa reader. in particular, the production of il- β and il- was reduced in the groups treated with ua compared with the infection group, and the expression of il- and il- was significantly increased in t. gondii-infected cells treated with ua (figures and ). these changes demonstrate that ua promotes the production and the expression of il- and il- by consistently stimulating the immune cells as well as inhibit the expressions of il- β and il- induced by t. gondii infection. interestingly, the production of il- β in t. gondii-infected cells treated with sf was significantly increased and the expression of il- was gradually decreased compared with the infection group. however, there were no significant changes of the production of il- and il- in t. gondii-infected cells treated with sf. in particular, significant differences in the cytokines were clearly measured between the t. gondii-infected group and ua-treated group, and these changes of cytokines were confirmed in all the groups. furthermore, the results show that this co-culture system through immune cells can be usefully utilized as a co-culture method for measuring various cytokines, and as an effective alternative to in vivo experiments. these results indicate that ua not only effectively induces the production of cytokines between the immune cells by increasing the expression of il- and il- in macrophage and b cells after t. gondii infection, but also suppresses or strongly reduces the expression of the inflammatory cytokines from the immune cells. gondii-infected cells treated with ua (figure and ). these changes demonstrate that ua promotes the production and the expression of il- and il- by consistently stimulating the immune cells as well as inhibit the expressions of il- β and il- induced by t. gondii infection. interestingly, the production of il- β in t. gondii-infected cells treated with sf was significantly increased and the expression of il- was gradually decreased compared with the infection group. however, there were no significant changes of the production of il- and il- in t. gondii-infected cells treated with sf. in particular, significant differences in the cytokines were clearly measured between the t. gondiiinfected group and ua-treated group, and these changes of cytokines were confirmed in all the groups. furthermore, the results show that this co-culture system through immune cells can be usefully utilized as a co-culture method for measuring various cytokines, and as an effective alternative to in vivo experiments. these results indicate that ua not only effectively induces the production of cytokines between the immune cells by increasing the expression of il- and il- in macrophage and b cells after t. gondii infection, but also suppresses or strongly reduces the expression of the inflammatory cytokines from the immune cells. in spite of constant public health efforts worldwide for the past decades, infectious diseases such as malaria, influenza, cholera, yellow fever, mers, sars, and tuberculosis have been consistently causing crises in public health as well as global concerns due to their pandemic potential. these serious infectious pathogens have enhanced adaptability to environmental variation and infectivity to host as well as resistance to existing drugs for their viability, evolving in a time-dependent manner. in these aspects, global nonprofit organizations and/or profit companies such as who and the bill & melinda gates foundation as well as various global leading pharmaceutical companies have continuously supported the study for blocking or treating chronic infectious diseases such as zika, aids, and tuberculosis as well as acute infectious diseases including influenza, malaria, cholera, yellow fever, and ebola, focusing on the development of novel drugs and effective vaccine against pathogens. furthermore, in spite of various efforts for developing anti-parasitic drugs against zoonotic parasitosis, effective novel drugs such as anti-malaria drugs have not yet been developed or launched. many people are living with animals such as cats and dogs. in particular, toxoplasmosis, a zoonotic parasitosis, is transmitted via physical contact with pets and companion animals, including cat and dogs. with respect to these aspects, we carefully focused on the parasitic infectious diseases that are consistently caused through various infection pathways and factors. among various parasites, t. gondii induces serious symptoms such as cerebral calcification and meningoencephalitis in the brain, while causing fetal infection through mother during pregnancy, as it is a zoonotic parasite which affects both humans and animals, resulting in parasitic diseases such as toxoplasmosis [ , ] . in spite of constant public health efforts worldwide for the past decades, infectious diseases such as malaria, influenza, cholera, yellow fever, mers, sars, and tuberculosis have been consistently causing crises in public health as well as global concerns due to their pandemic potential. these serious infectious pathogens have enhanced adaptability to environmental variation and infectivity to host as well as resistance to existing drugs for their viability, evolving in a time-dependent manner. in these aspects, global nonprofit organizations and/or profit companies such as who and the bill & melinda gates foundation as well as various global leading pharmaceutical companies have continuously supported the study for blocking or treating chronic infectious diseases such as zika, aids, and tuberculosis as well as acute infectious diseases including influenza, malaria, cholera, yellow fever, and ebola, focusing on the development of novel drugs and effective vaccine against pathogens. furthermore, in spite of various efforts for developing anti-parasitic drugs against zoonotic parasitosis, effective novel drugs such as anti-malaria drugs have not yet been developed or launched. many people are living with animals such as cats and dogs. in particular, toxoplasmosis, a zoonotic parasitosis, is transmitted via physical contact with pets and companion animals, including cat and dogs. with respect to these aspects, we carefully focused on the parasitic infectious diseases that are consistently caused through various infection pathways and factors. among various parasites, t. gondii induces serious symptoms such as cerebral calcification and meningoencephalitis in the brain, while causing fetal infection through mother during pregnancy, as it is a zoonotic parasite which affects both humans and animals, resulting in parasitic diseases such as toxoplasmosis [ , ] . furthermore, it induces serious complications in immune-deficient patients and hiv patients, which attenuates the interaction of the immune system that specifically responds to pathogens in the host [ , ] . t. gondii inhibits the interaction of cytokines such as il- and ifn-γ that are activated through the immune system, attenuating the production of cytokines from host cells. in addition, it is known that t. gondii blocks parasitic inhibitory system while inhibiting the signal pathways of cell cycle initiators or apoptotic mediators for the apoptotic stage in host cells after invasion [ , ] . t. gondii infection induces an imbalance of immune response by causing changes in cytokines such as tnf-α and tgf-β in the host, resulting in immune deficiency by breaking homeostasis and interaction of the immune system in the host. moreover, t. gondii induces the proliferative and growth cycle of t. gondii by rapidly forming pvm and by regulating cell cycle factors for its survival in host cells, maintaining the survival by accelerating the proliferation of t. gondii in host [ , ] . the subcellular organelles and granules of t. gondii, such as the conoid, gras, sags, the rhoptry, microneme proteins, and the inner membrane complex, were focused upon as major targets for blocking t. gondii infection, which was used as major recombinant proteins or factors for protective immunity including vaccination [ ] [ ] [ ] [ ] . for these reasons, in this study we not only evaluated the anti-parasitic effect of ua which inhibits the survival of parasite after t. gondii infection through a t. gondii-infected co-cultured immune system, but also confirmed its mechanism of action with respect to anti-t. gondii effects and immunomodulatory activity. in the present study, the survival rates of t. gondii were markedly inhibited when the parasites were treated with ua and sf compared with the non-treated t. gondii group, and the expression of subcellular organelles and granules of t. gondii in the infected cells was effectively decreased in t. gondii-infected group treated with ua compared with other groups. furthermore, the production of ros and no was significantly increased in the t. gondii-infected group treated with ua compared with both sf-treated and infection groups. interestingly, although ros production was activated in both t. gondii-infected cells treated with ua and sf, its rate was gradually decreased in the range of high concentrations of the compounds. in these aspects, no and ros production not only activates immune defense systems against various pathogens such as bacteria and viruses but also maintains effectively the changes of the homeostasis in the organism, promoting the production of cytokines induced by the interaction of immune cells. cytokines such as il- β, il- , and tnf-α were increased in the infected cells after t. gondii infection, and their production was significantly suppressed after ua treatment. in particular, although the production of tgf-β was reduced in t. gondii-infected immune cells treated with ua, it was shown that ua suppressed the expression of tgf-β in immune cells by inhibiting the survival of t. gondii. in addition, ua not only effectively increased the production of anti-inflammatory cytokines such as il- and interferon-β in t. gondii-infected cells but also activated the expression of il- and gm-csf, promoting the development of th cells and the production of interferon-γ that is induced by nk cells. taken together, the results of this study showed the anti-parasitic effects of ua against the survival of t. gondii as well as demonstrated its mechanism of action causing anti-t. gondii effect in t. gondii-infected co-cultured cells. furthermore, these results indicated that the expression of subcellular organelles and granules of t. gondii was specifically inhibited or blocked in a concentration-dependent manner after treatment with ua. this study provides evidence that ua not only decreases the inflammatory cytokines (il- β, il- and tnf-α) expressed from the immune cells after t. gondii infection, but also effectively increases the production of anti-inflammatory mediators such as il- , il- , gm-csf, and interferon-β, including the activation of ros and no production. interestingly, it was confirmed in this study that macrophage and other immune cells did not directly inhibit or rapidly decrease the survival of parasite as well as the proliferation of t. gondii through the bactericidal action such as phagocytosis of macrophage after t. gondii infection. this may be the result of the differences between the in vitro system and the in vivo experiment including rats and mice. however, the parasite may not be eliminated by the bactericidal action of macrophages and other immune cells in the infected body because t. gondii is found in various body sites of patients in the clinic. moreover, it may be changed or maintained to the parasite type that indicates characteristics of hibernation like t. gondii me strain (type ii) in the body. in summary, the results of this study demonstrate that ua induces anti-t. gondii effects/action by effectively blocking or inhibiting the survival of t. gondii, and also has anti-parasitic activity that consistently inhibits the proliferation/growth of t. gondii by strongly inhibiting the expression of subcellular organelles and granules of t. gondii, such as rop , mic , and imc sub in t. gondii-infected cells. in addition, these results show clearly that ua has the immunomodulatory action/activity which causes the change of cytokines in immune cells by decreasing the inflammatory cytokines (il- β, il- , and tnf-α) and/or by activating anti-inflammatory cytokines including il- , il- , gm-csf, or interferon-β through the interaction between ua and immune cells. furthermore, the results indicate that ua induces the death of t. gondii by promoting the production of ros and no, such as super oxides and hydroxyl radicals in t. gondii-infected immune cells. therefore, this study indicates that ua can be used effectively as a potent candidate agent or a synergic compound with existing drugs in the development of novel anti-t. gondii drugs of the next generation against toxoplasmosis, with potential as a modulatory substance against the immune response of the parasite in parasite-infected immune cells. moreover, ua requires further study for efficacy and safety against toxoplasmosis in preclinical stages through in vivo animal models in the near future. the anti-toxoplasmosis drug, sulfadiazine (sf), was dissolved in dmso, and ursolic acid (ua) was also dissolved in dmso to a concentration of mg/ml according to the manufacturer's instructions. sulfadiazine was used as a standard drug to evaluate whether or not ursolic acid has an anti-parasitic effect and activity against t. gondii. the compounds were filtered using . -µm membrane syringe filters (roshi kaisha, ltd., tokyo, japan) before use, and were stored at − • c deep-freezer until use. macrophages (raw . ), t cells (el ), b cells (fb ), basophil cells (rbl- h ), and normal lung epithelial cells (l ) were purchased from korean cell line bank at seoul national university and american type culture collection (manassas, va, usa). the cells were cultured in rpmi medium and dmem containing mm l-glutamine, supplemented with % decomplemented fetal bovine serum (fbs), penicillin ( units/ml), and streptomycin ( µg/ml) in a humidified atmosphere containing % co in air at • c. the t. gondii rh was suspended with × pbs, which was injected in the abdominal cavity of each balb-c/mouse. five days after the injection, t. gondii was collected from the peritoneal fluids of mouse kept in the abdominal cavities of the mice before use. in the in vitro study, host cells were infected with tachyzoite of t. gondii at a cell-to-parasite ratio of : . the inhibitory effect of ursolic acid against t. gondii was confirmed by directly evaluating the viability of t. gondii exposed to ua and sf through the mtt assay. briefly, after t. gondii was seeded in a -well plate ( × /well), t. gondii was incubated with different concentrations ( . - µg/ml) of ua and sf for h, respectively. the normal lung cells were infected with t. gondii (cells: t. gondii = : ), and t. gondii-infected cells were treated with different concentrations ( . - µg/ml) of ursolic acid (ua) and sulfadiazine (sf) at • c for h, respectively; then their viabilities were evaluated by mtt assay. it was to be determined whether or not ua has anti-parasitic activity and/or effect against t. gondii. the cells were divided into normal and experimental groups, and the survival rate (sr) of t. gondii was calculated as follows: % of sr = (od drug-tested wells − od blank )/(od control − od blank ) × . the optical density (od) was measured at a wavelength of nm using an elisa leader. the inhibitory effect of ursolic acid against the viability of t. gondii was evaluated by measuring the expression of subcellular organelles of t. gondii in t. gondii-infected cells exposed to ua and sf. after normal lung cells were seeded in a -well plate ( × /well), the cells were infected with t. gondii ( : , cells/parasite ratio), which was incubated with different concentrations ( - µg/ml) of ua and sf for h respectively, and their total rna was isolated from t. gondii strain using rneasy mini kit (qiagen, hilden, germany). total rna was obtained from untreated control and treated groups following the manufacturer's recommended procedure, and the total rna was quantified in an eppendorf biospectrometer (eppendorf, seoul, korea). one pair of primers was designed using the primer (version . ) and ncbi primer-blast according to the sequence of the selected specific gene in ncbi genbank as follows: t. gondii mic (accession number: af , bp), rop (accession number: am , bp), imc sub- (accession number: hq , bp), and β-actin (accession number: nm , bp). the specific genes of t. gondii were synthesized to complementary dna (cdna) through one-step rt-pcr kit (bioneer, daejeon, korea). the cdna was analyzed by electrophoresis in a . % (w/v) agarose gel containing µg/ml ethidium bromide at v for h, which was visualized under ultraviolet illumination. the β-actin was used as an internal standard for the amount of the expression of cdna present in each sample. the groups were divided into the untreated positive group (t. gondii-infected group) and experimental groups (ua and sf-treated groups). the effect of nitric oxide (no) and reactive oxygen species (ros) production of ursolic acid was confirmed by measuring no and ros production in t. gondii-infected co-cultured cells (macrophage and basophil cells) exposed to the compounds. the cells were divided into normal and experimental groups as described above. briefly, after the co-culture cells were seeded in a -well plate ( × /well), the cells were infected with t. gondii ( : , cells/parasite ratio), which was incubated with different concentrations ( - µg/ml) of ua and sf for h, respectively. the no and ros production were measured in the control and treated groups according to the manufacturer's instructions through no and ros production kits (cell biolabs, inc. san diego, ca, usa) respectively. the rates of no and ros production were measured using an elisa microplate leader (biotek instruments, inc., winooski, vt, usa). the effect of ursolic acid regarding immunomodulatory action was evaluated by measuring the expression of cytokines in t. gondii-infected co-culture immune cells (macrophage, t cells, b cells, and basophil cells) exposed to ua and sf, respectively. the cells were divided into normal and experimental groups. first, the macrophage and basophil cells were sequentially seeded in a -well plate ( × /well) in a humidified atmosphere containing % co in air at • c. second, t cells and b cells were additionally seeded in the plate ( × /well) after h. namely, the co-cultured immune cells were seeded in a -well plate ( × /well) and the cells were finally infected with t. gondii ( : , cells/parasite ratio) after h, and incubated with different concentrations ( - µg/ml) of ua and sf for h, respectively. the cytokines (tnf-α, tgf-β , interferon-β, gm-csf, il- β, il- , il- , and il- ) were respectively measured in normal, infected, and treated groups according to the manufacturer's instruction through cytokine elisa kits (thermofisher scientific, san diego, ca, usa) and inflammatory multi-cytokine kits (qiagen, hilden, germany), and the untreated t. gondii-infected cells were used as an positive group. all the results were expressed as mean ± standard deviation (sd) of three independent experiments. statistical analysis of the data was performed using student's t-test and analysis of variance (anova). a value of * p < . was considered to be statistically significant. the authors declare no conflict of interest. the following abbreviations are used in this manuscript: secretory traffic in the eukaryotic parasite toxoplasma gondii: less is more a novel family of toxoplasma imc proteins displays a hierarchical organization and functions in coordinating parasite division the inner membrane complex through development of 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attribution (cc by) license key: cord- -qxk wfk authors: yamin, mohammad title: information technologies of st century and their impact on the society date: - - journal: int j inf technol doi: . /s - - - sha: doc_id: cord_uid: qxk wfk twenty first century has witnessed emergence of some ground breaking information technologies that have revolutionised our way of life. the revolution began late in th century with the arrival of internet in , which has given rise to methods, tools and gadgets having astonishing applications in all academic disciplines and business sectors. in this article we shall provide a design of a ‘spider robot’ which may be used for efficient cleaning of deadly viruses. in addition, we shall examine some of the emerging technologies which are causing remarkable breakthroughs and improvements which were inconceivable earlier. in particular we shall look at the technologies and tools associated with the internet of things (iot), blockchain, artificial intelligence, sensor networks and social media. we shall analyse capabilities and business value of these technologies and tools. as we recognise, most technologies, after completing their commercial journey, are utilised by the business world in physical as well as in the virtual marketing environments. we shall also look at the social impact of some of these technologies and tools. internet, which was started in [ ] , now has . million terabyte data from google, amazon, microsoft and facebook [ ] . it is estimated that the internet contains over four and a half billion websites on the surface web, the deep web, which we know very little about, is at least four hundred times bigger than the surface web [ ] . soon afterwards in , email platform emerged and then many applications. then we saw a chain of web . technologies like e-commerce, which started, social media platforms, e-business, e-learning, e-government, cloud computing and more from to the early st century [ ] . now we have a large number of internet based technologies which have uncountable applications in many domains including business, science and engineering, and healthcare [ ] . the impact of these technologies on our personal lives is such that we are compelled to adopt many of them whether we like it or not. in this article we shall study the nature, usage and capabilities of the emerging and future technologies. some of these technologies are big data analytics, internet of things (iot), sensor networks (rfid, location based services), artificial intelligence (ai), robotics, blockchain, mobile digital platforms (digital streets, towns and villages), clouds (fog and dew) computing, social networks and business, virtual reality. with the ever increasing computing power and declining costs of data storage, many government and private organizations are gathering enormous amounts of data. accumulated data from the years' of acquisition and processing in many organizations has become enormous meaning that it can no longer be analyzed by traditional tools within a reasonable time. familiar disciplines to create big data include astronomy, atmospheric science, biology, genomics, nuclear physics, biochemical experiments, medical records, and scientific research. some of the organizations responsible to create enormous data are google, facebook, youtube, hospitals, proceedings of parliaments, courts, newspapers and magazines, and government departments. because of its size, analysis of big data is not a straightforward task and often requires advanced methods and techniques. lack of timely analysis of big data in certain domains may have devastating results and pose threats to societies, nature and echo system. healthcare field is generating big data, which has the potential to surpass other fields when it come to the growth of data. big medic data usually refers to considerably bigger pool of health, hospital and treatment records, medical claims of administrative nature, and data from clinical trials, smartphone applications, wearable devices such as rfid and heart beat reading devices, different kinds of social media, and omics-research. in particular omics-research (genomics, proteomics, metabolomics etc.) is leading the charge to the growth of big data [ , ] . the challenges in omics-research are data cleaning, normalization, biomolecule identification, data dimensionality reduction, biological contextualization, statistical validation, data storage and handling, sharing and data archiving. data analytics requirements include several tasks like those of data cleaning, normalization, biomolecule identification, data dimensionality reduction, biological contextualization, statistical validation, data storage and handling, sharing and data archiving. these tasks are required for the big data in some of the omics datasets like genomics, transcriptomics, proteomics, metabolomics, metagenomics, phenomics [ ] . according to [ ] , in alone, the data in the united states of america healthcare system amounted to one hundred and fifty exabyte (one exabyte = one billion gigabytes, or bytes), and is expected soon reach to and later . some scientist have classified medical into three categories having (a) large number of samples but small number of parameters; (b) small number of samples and small number of parameters; (c) large small number of samples and small number of parameters [ ] . although the data in the first category may be analyzed by classical methods but it may be incomplete, noisy, and inconsistent, data cleaning. the data in the third category could be big and may require advanced analytics. big data cannot be analyzed in real time by traditional analytical methods. the analysis of big data, popularly known as big data analytics, often involves a number of technologies, sophisticated processes and tools as depicted in fig. . big data can provide smart decision making and business intelligence to the businesses and corporations. big data unless analyzed is impractical and a burden to the organization. big data analytics involves mining and extracting useful associations (knowledge discovery) for intelligent decision-making and forecasts. the challenges in big data analytics are computational complexities, scalability and visualization of data. consequently, the information security risk increases with the surge in the amount of data, which is the case in big data. the aim of data analytics has always been knowledge discovery to support smart and timely decision making. with big data, knowledge base becomes widened and sharper to provide greater business intelligence and assist businesses in becoming a leader in the market. conventional processing paradigm and architecture are inefficient to deal with the large datasets from the big data. some of the problems of big data are to deal with the size of data sets in big data, requiring parallel processing. some of the recent technologies like spark, hadoop, map reduce, r, data lakes and nosql have emerged to provide big data analytics. with all these and other data analytics technologies, it is advantageous to invest in designing superior storage systems. health data predominantly consists of visual, graphs, audio and video data. analysing such data to gain meaningful insights and diagnoses may depend on the choice of tools. medical data has traditionally been scattered in the organization, often not organized properly. what we find usually are medical record keeping systems which consist of heterogeneous data, requiring more efforts to reorganize the data into a common platform. as discussed before, the health profession produces enormous data and so analysing it in an efficient and timely manner can potentially save many lives. commercial operations of clouds from the company platforms began in the year [ ] . initially, clouds complemented and empowered outsourcing. at earlier stages, there were some privacy concerns associated with cloud computing as the owners of data had to give the custody of their data to the cloud owners. however, as time passed, with confidence building measures by cloud owners, the technology became so prevalent that most of the world's smes started using it in one or the other form. more information on cloud computing can be found in [ , ] . as faster processing became the need for some critical applications, the clouds regenerated fog or edge computing. as can be seen in gartner hyper cycles in figs. and , edge computing, as an emerging technology, has also peaked in - . as shown in the cloud computing architecture in fig. , the middle or second layers of the cloud configuration are represented by the fog computing. for some applications delay in communication between the computing devices in the field and data in a cloud (often physically apart by thousands of miles), is detrimental of the time requirements, as it may cause considerable delay in time sensitive applications. for example, processing and storage for early warning of disasters (stampedes, tsunami, etc.) must be in real time. for these kinds of applications, computing and storing resources should be placed closer to where computing is needed (application areas like digital street). in these kind of scenarios fog computing is considered to be suitable [ ] . clouds are integral part of many iot applications and play central role on ubiquitous computing systems in health related cases like the one depicted in fig. . some applications of fog computing can be found in [ ] [ ] [ ] . more results on fog computing are also available in [ ] [ ] [ ] . when fog is overloaded and is not able to cater for the peaks of high demand applications, it offloads some of its data and/or processing to the associated cloud. in such a situation, fog exposes its dependency to a complementary bottom layer of the cloud architectural organisation as shown in the cloud architecture of fig. . this bottom layer of hierarchical resources organization is known as the dew layer. the purpose of the dew layer is to cater for the tasks by exploiting resources near to the end-user with minimum internet access [ , ] . as a feature, dew computing takes care of determining as to when to use for its services linking with the different layers of the cloud architecture. it is also important to note that the dew computing [ ] is associated with the distributed computing hierarchy and is integrated by the fog computing services, which is also evident in fig. . in summary, cloud architecture has three layers, first being cloud, second as fog and the third dew. definition of internet of things (iot), as depicted in fig. , has been changing with the passage of time. with growing number of internet based applications, which use many technologies, devices and tools, one would think, the name of iot seems to have evolved. accordingly, things (technologies, devices and tools) used together in internet based applications to generate data to provide assistance and services to the users from anywhere, at any time. the internet can be considered as a uniform technology from any location as it provides the same service of 'connectivity'. the speed and security however are not uniform. the iot as an emerging technology has peaked during - as is evident from figs. and . this technology is expanding at a very fast rate. according to [ ] [ ] [ ] [ ] , the number of iot devices could be in millions by the year . iot is providing some amazing applications in tandem with wearable devices, sensor networks, fog computing, and other technologies to improve some the critical facets of our lives like healthcare management, service delivery, and business improvements. some applications of iot in the field of crowd management are discussed in [ ] . some applications in of iot in the context of privacy and security are discussed in [ , ] . some of the key devices and associated technologies to iot include rfid tags [ ] , internet, computers, cameras, rfid, mobile devices, coloured lights, rfids, sensors, sensor networks, drones, cloud, fog and dew. blockchain is usually associated with cryptocurrencies like bitcoin (currently, there are over one and a half thousand cryptocurrencies and the numbers are still rising). but the blockchain technology can also be used for many more critical applications of our daily lives. the blockchain is a distributed ledger technology in the form of a distributed transactional database, secured by cryptography, and governed by a consensus mechanism. a blockchain is essentially a record of digital events [ ] . a block represents a completed transaction or ledger. subsequent and prior blocks are chained together, displaying the status of the most recent transaction. the role of chain is to provide linkage between records in a chronological order. this chain continues to grow as and when further transactions take place, which are recorded by adding new blocks to the chain. user security and ledger consistency in the blockchain is provided by asymmetric cryptography and distributed consensus algorithms. once a block is created, it cannot be altered or removed. the technology eliminates the need for having a bank statement for verification of the availability of funds or that of a lawyer for certifying the occurrence of an event. the benefits of blockchain technology are inherited in its characteristics of decentralization, persistency, anonymity and auditability [ , ] . blockchain, being the technology behind cryptocurrencies, started as an open-source bitcoin community to allow reliable peer-to-peer financial transactions. blockchain technology has made it possible to build a globally functional currency relying on code, without using any bank or third-party platforms [ ] . these features have made the blockchain technology, secure and transparent for business transactions of any kind involving any currencies. in literature, we find many applications of blockchain. nowadays, the applications of blockchain technology involve various kinds of transactions requiring verification and automated system of payments using smart contracts. the concept of smart contacts [ ] has virtually eliminated the role of intermediaries. this technology is most suitable for businesses requiring high reliability and honesty. because of its security and transparency features, the technology would benefit businesses trying to attract customers. blockchain can be used to eliminate the occurrence of fake permits as can be seen in [ ] . as discussed above, blockchain is an efficient and transparent way of digital record keeping. this feature is highly desirable in efficient healthcare management. medical field is still undergoing to manage their data efficiently in a digital form. as usual the issues of disparate and nonuniform record storage methods are hampering the digitization, data warehouse and big data analytics, which would allow efficient management and sharing of the data. we learn the magnitude of these problem from examples of such as the target of the national health service (nhs) of the united kingdom to digitize the uk healthcare is by [ ] . these problems lead to inaccuracies of data which can cause many issues in healthcare management, including clinical and administrative errors. use of blockchain in healthcare can bring revolutionary improvements. for example, smart contracts can be used to make it easier for doctors to access patients' data from other organisations. the current consent process often involves bureaucratic processes and is far from being simplified or standardised. this adds to many problems to patients and specialists treating them. the cost associated with the transfer of medical records between different locations can be significant, which can virtually be reduced to zero by using blockchain. more information on the use of blockchain in the healthcare data can be found in [ , ] . one of the ongoing healthcare issue is the eradication of deadly viruses and bacteria from hospitals and healthcare units. nosocomial infections are a common problem for hospitals and currently they are treated using various techniques [ , ] . historically, cleaning the hospital wards and operating rooms with chlorine has been an effective way. on the face of some deadly viruses like ebola, hiv aids, swine influenza h n , h n , various strands of flu, severe acute respiratory syndrome (sars) and middle eastern respiratory syndrome (mers), there are dangerous implications of using this method [ ] . an advanced approach is being used in the usa hospitals, which employs ''robots'' to purify the space as can be seen in [ , ] . however, certain problems exist within the limitations of the current ''robots''. most of these devices require a human to place them in the infected areas. these devices cannot move effectively (they just revolve around themselves); hence, the uv light will not reach all areas but only a very limited area within the range of the uv light emitter. finally, the robot itself maybe infected as the light does not reach most of the robot's surfaces. therefore, there is an emerging need to build a robot that would not require the physical presence of humans to handle it, and could purify the entire room by covering all the room surfaces with uv light while, at the same time, will not be infected itself. figure is an overview of the design of a fully motorized spider robot with six legs. this robot supports wi-fi connectivity for the purpose of control and be able to move around the room and clean the entire area. the spider design will allow the robot to move in any surface, including climbing steps but most importantly the robot will use its legs to move the uv light emitter as well as clear its body before leaving the room. this substantially reduces the risk of the robot transmitting any infections. additionally, the robot will be equipped with a motorized camera allowing the operator to monitor space and stop the process of emitting uv light in case of unpredicted situations. the operator can control the robot via a networked graphical user interface and/or from an augmented reality environment which will utilize technologies such as the oculus touch. in more detail, the user will use the oculus rift virtual reality helmet and the oculus touch, as well as hand controllers to remote control the robot. this will provide the user with the vision of the robot in a natural manner. it will also allow the user to control the two front robotic arms of the spider robot via the oculus touch controller, making it easy to do conduct advance movements, simply by move the hands. the physical movements of the human hand will be captured by the sensors of oculus touch and transmitted to the robot. the robot will then use reverse kinematics to translate the actions and position of the human hand to movements of the robotic arm. this technique will also be used during the training phase of the robot, where the human user will teach the robot how to clean various surfaces and then purify itself, simply by moving their hands accordingly. the design of the spider robot was proposed in a project proposal submitted to the king abdulaziz city of science and technology (https://www.kacst.edu.sa/eng/pages/ default.aspx) by the author and george tsaramirsis (https:// www.researchgate.net/profile/george_tsaramirsis). we have presented details of some of the emerging technologies and real life application, that are providing businesses remarkable opportunities, which were previously unthinkable. businesses are continuously trying to increase the use of new technologies and tools to improve processes, to benefit their client. the iot and associated technologies are now able to provide real time and ubiquitous processing to eliminate the need for human surveillance. similarly, virtual reality, artificial intelligence robotics are having some remarkable applications in the field of medical surgeries. as discussed, with the help of the technology, we now can predict and mitigate some natural disasters such as stampedes with the help of sensor networks and other associated technologies. finally, the increase in big data analytics is influencing businesses and government agencies with smarter decision making to achieve targets or expectations. the evolution of the internet: from military experiment to general purpose technology how much data is on the internet? science focus (the home of bbc science focus magazine) the deep web is the % of the internet you can't google. curiosity history on the internet . : the rise of social media tracking the evolution of the internet of things concept across different application domains integrated omics: tools, advances and future approaches medical big data: promise and challenges where healthcare's big data actually comes from. emerj large datasets in biomedicine: a discussion of salient analytic issues last accessed on / / from a brief history of cloud computing big data analytics: applications, prospects and challenges cloud computing in smes: case of saudi arabia. bijit-bvicam's bringing computation closer toward the user network: is edge computing the solution? managing crowds with wireless and mobile technologies. hindawi improving privacy and security of user data in location based services preserving privacy in internet of things-a survey towards integrating mobile devices into dew computing: a model for hour-wise prediction of energy availability enabling real-time context-aware collaboration through g and mobile edge computing finding your way in the fog: towards a comprehensive definition of fog computing minimizing dependency on internetwork: is dew computing a solution? addepalli s fog computing and its role in the internet of things rfid technology and its applications in internet of things (iot), consumer electronics, communications and networks (cecnet). in: nd international conference proceedings towards internet of things: survey and future vision internet of things (iot): a vision, architectural elements, and future directions blockchain technology in business and information systems research managing crowds with technology: cases of hajj and kumbh mela an overview of blockchain technology: architecture, consensus, and future trends blockchain technology for social impact: opportunities and challenges ahead blockchain for controlling hajj and umrah permits implementing blockchains for efficient health care: systematic review it applications in healthcare management: a survey application of service robots for disinfection in medical institutions service robots in hospitals: new perspectives on niche evolution and technology affordances key: cord- - i ld d authors: nefedeva, mariia; titov, ilya; malogolovkin, alexander title: molecular characteristics of a novel recombinant of porcine epidemic diarrhea virus date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: i ld d porcine epidemic diarrhea (ped) is a contagious viral disease in pigs, caused by the coronavirus porcine epidemic diarrhea virus (pedv). pedv infection results in significant mortality in piglets in unvaccinated herds. like many others rna viruses, pedv has high evolutionary rate and is prone to genetic mutations. in this study, we analyzed the complete genome sequence of the recently sequenced isolate pedv/belgorod/dom/ . a recombination event in s gene of pedv/belgorod/dom/ was detected. pairwise identity analysis of the whole genome sequences revealed that pedv/belgorod/dom/ is an intermediate between pedv and transmissible gastroenteritis virus (tgev) strains. these results can be used for further analysis of the evolutionary variability, prevalence, and epidemiology of the porcine epidemic diarrhea virus. the spike (s) protein of pedv is the viral protein that is subjected to the greatest immunological pressure and variability. deletions (s-indels) or small insertions have been observed in the s gene nucleotide sequences of many pedv isolates [ ] . the pedv strains that are currently circulating in the european union are similar to the american s-indel strains [ ] [ ] [ ] . the phylogenetic classification of the pedv strains is based on the analysis of complete genomes sequences obtained worldwide [ ] or individual genes such as s, m, n, or orf [ , , ] . in this study, we analyzed the genome sequence of recently sequenced pedv isolate, pedv/belgorod/ dom/ (genbank accession number mf ) [ ] . pathological samples from the intestine and stomach were taken from one-month-old sick piglets from the belgorod region of russia in [ ] . total rna was extracted from a % organ suspension using trizol reagent (ther-mofisher scientific) according to the manufacturer's instructions. next-generation sequencing was done using an illumina miseq instrument with a miseq reagent kit v in -× -bp pe mode (illumina, san diego, ca, usa) [ ] . the pedv/belgorod/dom/ isolate was subsequently isolated from the small-intestine tissue in vero cell culture. prediction of homologous recombination events was carried out using rdp (recombination detection program) and simplot [ , ] . pairwise identity analysis [ ] . phylogenetic trees were constructed based on pedv m and s gene sequences using the maximum-likelihood method in mega . . [ ] . bootstrap values were estimated for replicates. the complete coding sequence of the pedv/belgorod/ dom/ is , nucleotides (nt) in length (genbank accession number mf ) [ ] . two putative recombination sites were detected in the genome of recombinant pedv/belgorod/dom/ at nt in orf b and nt in the s gene ( fig. ). pedv strain lzc (ef ) and pedv strain slo/jh- / (ku ) were identified as the major and minor parental viruses, respectively. the recombinant event was identified by six modules (rdp, maxchi, chimaera, geneconv, bootscan, siscan) with high confidence (average p-value, . × − ). a similarity plot showed high overall sequence similarity between the pedv/belgorod/dom/ strain and the parental pedv strains, but with a marked drop in the nucleotide sequence similarity in the s gene region (fig. ) . phylogenetic analysis of the complete genomes showed that pedv/belgorod/dom/ has a distant relationship to known pedv strains. the pedv/belgorod/dom/ isolate does not belong to any groups formed by the american or chinese strains and forms a separate cluster together with the secov-ita recombinant strain isolated in italy (fig. ) . since only m gene sequences are available in the gen-bank database for the russian pedv isolates, we rebuilt the phylogenetic tree to refine the analysis. based on the phylogenetic analysis of the m gene, the pedv/belgorod/ dom/ isolate belongs to the same clade as other virulent russian pedv strains, indicating a high degree of sequence homogeneity in the m gene (fig. a) isolate is genetically distinct and does not belong to any group (fig. b ). this robust incongruence between the mand s-gene-based trees may be explained by a recombination event within the genome of the pedv/belgorod/dom/ isolate. such variability can lead to dramatic changes in viral virulence, pathogenicity, and antigenicity. pairwise identity analysis based on the spike amino acid sequences revealed that pedv/belgorod/dom/ is an intermediate between pedv and tgev and is also distantly related to other pedv strains (fig. ) . pedv/belgorod/dom/ has a unique spike protein sequence and low similarity to other pedv isolates. changes in the s glycoprotein gene play an important role, since this protein is important for tissue tropism and virulence [ ] . a preliminary animal study with pedv/belgorod/dom/ demonstrated that this recombinant is highly virulent in unvaccinated suckling piglets [ , ] . recombination events are possible and can sometimes be observed in cases where pigs have been vaccinated or infected with a mixture of tgev and pedv. such recombination events can potentially result in a loss of vaccine efficacy. boniotti et al. reported a virus possessing a tgev genome sequence in which the s protein sequence was identical to that of a pedv isolate (secov-ita ) [ ] . this chimeric virus was probably generated by recombination between tgev and pedv. similar chimeric viruses have been found by other research groups in germany [ ] and eastern europe [ ] . the genome sequence of one pedv isolate (ch/hnqx- / ) from china shows that this strain appeared due to naturally occurring recombination of the attenuated strains cv and dr with the circulating field strain ch/ zmdzy/ . the recombination events occurred in the s, orf , and n-structural protein-coding region and the replicase orf a region [ ] . the results of phylogenetic and recombination analysis revealed a discrepancy between the s gene sequence of pedv/belgorod/dom/ and the sequences of other isolates available in the genbank database. our results indicate that pedv/belgorod/dom/ is a new recombinant strain. interestingly, pedv/belgorod/dom/ and secov-ita (a recombinant strain from italy) form a unique phylogenetic group. pairwise identity analysis demonstrated that the amino acid sequence of the s gene of pedv/belgorod/dom/ is % identical to the s gene of other pedv strains and % identical to those of tgev strains. these data argue that pedv/belgorod/dom/ occupies an intermediate position between tgev and pedv. the identification of recombinant regions in pedv/belgorod/dom/ can be useful for further analysis of evolutionary variability, epidemiology, and development of a new diagnostic gene-based assay for porcine epidemic diarrhea virus. porcine epidemic diarrhea isolation and characterization of a variant porcine epidemic diarrhea virus in china nc_ . _secov_strain_italy/ / kr kr . _pedv_isolate_ v /bel/ mf . :pedv_isolate_kupe _ kx . :pedv_isolate_chsd kc . _pedv_strain_ch/fjzz- ef . _pedv_strain_lzc_from_china_ kc . _pedv_isolate_js jq . _pedv_strain_aƩenuated_dr _ jq . _pedv_strain_virulent_dr _ mf . _pedv_strain_pedv/belgorod/dom/ kc kx . _tgev_strain_tgev_ahhf_ dq . _tgev_strain_ts_ mf . :pedv_isolate_kupe _ kx . :pedv_isolate_chsd kc . _pedv_strain_ch/fjzz- mf . _pedv_strain_nw _ kp . _pedv_strain_ukraine kj . _pedv_strain_usa/minnesota af . _pedv_strain_cv _ ef . _pedv_strain_lzc_from_china_ kc . _pedv_isolate_js jq . _pedv_strain_aƩenuated_dr _ jq . _pedv_strain_virulent_dr _ mf . _pedv_strain_pedv/belgorod/dom kx . _tgev_strain_tgev_ahhf_ dq . _tgev_strain_ts_ new variants of porcine epidemic diarrhea virus, china emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences molecular characterization and phylogenetic analysis of membrane protein genes of porcine epidemic diarrhea virus isolates in china genomic and epidemiological characteristics provide new insights into the phylogeographical and spatiotemporal spread of porcine epidemic diarrhea virus in asia sequence analysis of the partial spike glycoprotein gene of porcine epidemic diarrhea viruses isolated in korea detection of porcine epidemic diarrhea virus using polymerase chain reaction and comparison of the nucleocapsid protein genes among strains of the virus porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis cell culture isolation and sequence analysis of genetically diverse us porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene new variant of porcine epidemic diarrhea virus comparison of porcine epidemic diarrhea viruses from germany and the united states genomic and evolutionary inferences between american and global strains of porcine epidemic diarrhea virus distinct characteristics and complex evolution of pedv strains complete genome sequence of a porcine epidemic diarrhea virus rdp : detection and analysis of recombination patterns in virus genomes full-length human immunodeficiency virus type genomes from subtype c-infected seroconverters in india, with evidence of intersubtype recombination sdt: a virus classification tool based on pairwise sequence alignment and identity calculation muscle: multiple sequence alignment with high accuracy and high throughput isolation and identification of porcine epidemic diarrhea virus in pigs under the outbreak at a large farm biological characteristics of an epizootic isolate bs- of porcine epidemic diarrhea virus porcine epidemic diarrhea virus and discovery of a recombinant swine enteric coronavirus new chimeric porcine coronavirus in swine feces characterization of a novel chimeric swine enteric coronavirus from diseased pigs in central eastern europe in genome sequencing and analysis of a novel recombinant porcine epidemic diarrhea virus strain from henan, china acknowledgements we thank olga strizhakova for providing pedv/ belgorod/dom/ . we also acknowledge yegor bazykin and alexey neverov for fruitful discussions of virus evolution and recombination analysis. we appreciate sandra blome and dennis hanke for pedv/ belgorod/dom/ complete genome sequencing. funding our work was supported by the ministry of science and higher education of the russian federation. the authors declare no competing interests. key: cord- -mxdlii authors: arji, goli; ahmadi, hossein; nilashi, mehrbakhsh; a. rashid, tarik; hassan ahmed, omed; aljojo, nahla; zainol, azida title: fuzzy logic approach for infectious disease diagnosis: a methodical evaluation, literature and classification date: - - journal: biocybern biomed eng doi: . /j.bbe. . . sha: doc_id: cord_uid: mxdlii this paper presents a systematic review of the literature and the classification of fuzzy logic application in an infectious disease. although the emergence of infectious diseases and their subsequent spread have a significant impact on global health and economics, a comprehensive literature evaluation of this topic has yet to be carried out. thus, the current study encompasses the first systematic, identifiable and comprehensive academic literature evaluation and classification of the fuzzy logic methods in infectious diseases. papers on this topic, which have been published from to and related to the human infectious diseases were evaluated and analyzed. the findings of this evaluation clearly show that the fuzzy logic methods are vastly used for diagnosis of diseases such as dengue fever, hepatitis and tuberculosis. the key fuzzy logic methods used for the infectious disease are the fuzzy inference system; the rule-based fuzzy logic, adaptive neuro-fuzzy inference system (anfis) and fuzzy cognitive map. furthermore, the accuracy, sensitivity, specificity and the receiver operating characteristic (roc) curve were universally applied for a performance evaluation of the fuzzy logic techniques. this thesis will also address the various needs between the different industries, practitioners and researchers to encourage more research regarding the more overlooked areas, and it will conclude with several suggestions for the future infectious disease researches. fuzzy logic approach for infectious disease diagnosis: a methodical evaluation, literature and classification show that the fuzzy logic methods are vastly used for diagnosis of diseases such as dengue fever, hepatitis and tuberculosis. the key fuzzy logic methods used for the infectious disease are the fuzzy inference system; the rule-based fuzzy logic, adaptive neuro-fuzzy inference system (anfis) and fuzzy cognitive map. furthermore, the accuracy, sensitivity, specificity and the receiver operating characteristic (roc) curve were universally applied for a performance evaluation of the fuzzy logic techniques. this thesis will also address the various needs between the different industries, practitioners and researchers to encourage . an infectious disease can be defined as a situation that is created by the incursion of a human body by harmful agents which will in turn hurt the body and may spread to other people as well [ ] . infectious diseases are the major cause of illness and death for any given population [ , ] . human immunodeficiency virus infection and acquired immune deficiency syndrome (hiv/aids), severe acute respiratory syndrome (sars), h n influenza and poliomyelitis are examples of an infectious disease [ , ] . while globally the incidence of the infectious disease varies greatly between countries, in the st century, the main incidences are hand-foot-mouth disease, hepatitis b, and tuberculosis [ ] . these diseases have a significant impact on the global health and economies [ ] . despite numerous valuable achievements regarding the procedures for preventing and controlling of various diseases, infectious diseases remain a massive threat to a population's well-being [ ] . aspects such as the increasing in antimicrobial resistance, increase in population and environmental changes are important in an infectious disease transmission [ ] . of million mortality reported per year throughout the world, . million were related to infectious diseases; which represent more than % of the overall deaths [ ] . additionally, clinical diagnosis and detection of the contaminated population are critical elements in controlling and supervising an infectious disease [ ] . for the diagnosis of different diseases, there are different essential criteria that should be interpreted by the caregivers [ ] . in a clinical situation, an analysis physician combines a patient's medical history, clinical symptoms, physical examination and laboratory findings [ ] . due to the elusive and complex nature of clinical decisionmaking, they may be accomplished with unwanted errors [ ] . in other word, medical diagnosis is an error-prone process, which occurs as the result of logical thinking [ ] . mathematical models are essential means for demonstrating the cause and effect relationship and evaluating the evidence for decisiveness regarding infectious diseases [ ] . computer-aided methods have the potential for examining the complexity of an infectious disease dynamics [ , ] . computational tools are essential for understanding epidemiological patterns in a disease outbreak [ ] . for handling the uncertainty of decision-making, studies have tried to explain the decision-making process in a medical setting by using the boolean or binary structure [ ] . fuzzy logic is considered as a vigorous technique for modelling ambiguity in medical practice [ ] . in medical field, most medical concepts are fuzzy [ , ] . these concepts usually are difficult to formalize and measure [ ] . fuzzy logic is making a decision in an inaccuracy, uncertainty and incompleteness environment [ ] . fuzzy methods deal with the classes whose boundaries are unclear and elusive [ ] . fuzzy logic concepts were introduced in [ ] . the medical field was one of the first fields in which fuzzy theory was implemented [ ] . fuzzy classes are given a degree of membership that is intermediary between and [ ] . some of the cases for a medical use of the fuzzy set theory are the myci, internist and doctormoon applications [ , ] . therefore, for these application methods, different types of research have been conducted in the disease diagnosis field. so, the major objective of the current study is to examine the researches in which fuzzy logic techniques have been applied in infectious diseases so to determining its trends and methods, through the processes of conducting a systematic literature review (slr). the current thesis is structured in the fallowing order. section exemplifies the procedures or the methodology of the current slr, section will present a complete report of the current systematic review. section will be dedicated to the discussion of this research. section will be dedicated to the implications of this study, its future research prospects and to the limitations of this evaluation and the last section will be the conclusion. we carried out an slr to define the influence of using the fuzzy logic methods in infectious diseases. the three main questions of this slr are as follow: (rq ) which domain of an infectious disease was more interesting in past researches? (rq ) which fuzzy methods were more dominant in infectious disease data analysis? (rq ) which performance evaluation methods were more frequently applied in previous reviews? for the gathering of the relevant information from all the eligible articles, a data extraction method was used for getting the detailed answer of the research questions. (rq ) which domain of an infectious disease was more interesting in past researches? (rq ) which fuzzy techniques were more dominant in an infectious disease data analysis? (rq ) which performance evaluation procedures were more frequently applied in the previous reviews? to answer the research questions and to produce the extracted data, numerous methods were used. generally, a narrative combining approach to answering different research questions was applied. furthermore, various visualization techniques such as tables and charts were used according to the research questions. current part is dedicated to the outcome of the current slr. first, we will demonstrate an overall description of the outcome of selecting the appropriate studies; and then, all the obtained outcomes will be categorized for each study questions independently. by searching for the four aforementioned databases, the candidate paper was removed as shown in fig. . then, based on using the exclusion conditions, studies were excluded. the other articles were investigated meticulously to choose relevant studies. by reviewing the title, abstract and the keywords, merely papers that have at least one of the inclusion measures were used. eventually, papers were used to get an answer to the research questions and data extraction for this methodical evaluation. fig. is showing the distribution of eligible papers per year. according to the diagram below, the frequency of papers relevant to the use of fuzzy logic in an infectious disease was roughly stable during the first years. however, as shown in the next chart, there is a persistent trend in the number of finally, the spread of the relevant articles based on a journal or conference, has been explained in table . based on the obtained results expert systems with applications journal was used by researchers for publishing their studies in the . percent of instances. rq : which domain of an infectious disease was more interesting in past researches? articles related to the precise domain of fuzzy methods application in an infectious disease, which have been considered from the past section, are summarized in the following section. fig. illustrates the distribution of numerous fuzzy techniques that have been used in an infectious disease data analysis. based on this figure, the results revealed that the critical application domain of the fuzzy methods in an infectious disease were relevant to dengue fever ( % of papers), hepatitis and tuberculosis ( % of papers). in addition, the other papers were related to diseases such as pulmonary infection ( . % of papers), urinary tract infections (utis), human immune deficiency viruses (hiv) and meningitis ( % of papers). for all the other diseases, there is one paper per each disease ( . % of papers). appendix a, table a lists eligible articles according to the specific type of disease, their objective, their inward and outward variables and findings. as shown in fig. , the distribution of the different fuzzy methods in various disease data analysis was as fallows. in articles published for dengue fever, . % of the papers applied fuzzy set theory and the adaptive neuro-fuzzy inference system (anfis) method in data analysis while . % of the papers used association rule mining. % of selected papers that related to hepatitis, employed neuro-fuzzy classifier as a fuzzy technique, while the other % were devoted to fuzzy inference system and fuzzy decision support system (fdss). furthermore, in tuberculosis studies, % of the papers used rule-based fuzzy logic and % of the papers applied the gaussian-fuzzy neural network method. rq : which fuzzy techniques were more dominant in an infectious disease data analysis? fuzzy inference systems are increasingly becoming more predominant in the area of fuzzy logic methods where % of the selected papers in the current slr are related to this particular method. fuzzy inference systems are used for the processes of mapping the inward variables to appropriate outward [ , ] . fuzzy inference process incorporates three key concepts: the membership functions, the fuzzy set operations, and the inference procedures [ ] . a fuzzy inference system can be divided mainly to four portions as follows: fuzzification, weighting, assessment of inference procedures, and the de-fuzzification [ ] . dragovit et al. utilized the fuzzy inference system to decide the probability of having peritonitis in patients. by using this system, the fuzzy rules enable the automation of the clinical decision making process in an imprecise and complicated conditions [ ] . urinary tract infections (utis) are considered amongst one of the utmost predominant bacterial infections. ibrisimovic et al. has suggested a fis fuzzy model, to provide the necessary support that the caregivers need for explaining the aftermath of a microscopic urine analysis. to create the model's various variables such as the colony forming units (cfu), white blood cells (wbc) and the red blood cells (rbc) as well as the turbidity of urine specimen used for inward variables, and the risk of a uti as an outward variable. the end result has revealed that the use of the fuzzy methods simplifies and secures the clarification of urine analysis [ ] . putra and munir proposed a method for diagnosis of measles, german measles and varicella. the data for those diseases were used because of the similarity of their infection mechanism and symptoms. a built fuzzy inference system b i o c y b e r n e t i c s a n d b i o m e d i c a l e n g i n e e r i n g ( ) - takes in the inward variables that represent the possible symptoms that may appear in each disease. cough, runny nose, sore throat, conjunctivitis, koplik's spot, diarrhea, headache, swollen neck or ear, loss of appetite, malaise, pimples/crust skin, joint pain used as inward variables. the application effectively identified out of accurate diseases throughout its testing stage [ ] . the anfis method was proposed by jang and its notions then were used in other fields [ ] . this method works by setting a list of features by using an amalgam of learning rules which will incorporate the back-propagation incline in error digestion and a tiniest squares method. this method can be implemented as the basis for creating a set of if-then guidelines with suitable association of functions to brand the inward and outward variables [ ] . anfis method has been notably successful in disease diagnosis in the past few years. as an example, the major groups of hepatitis in human beings are hepatitis a, hepatitis b and hepatitis c with the main symptoms being malaise (a common sick feeling), fever, and muscle pain, loss of appetite, nausea, vomiting, diarrhea, and jaundice. viral hepatitis disease can be diagnosed by blood test analysis and interpretations. dogantekin et al. developed an anfis system to be used for hepatitis diagnosis. the precision achieved in this automated system of diagnosis was at about . % [ ] . faisal et al. conducted a research using an adaptive neurofuzzy inference system to diagnose a dengue fever. the general precision of the developed method is . % with its sensitivity being at . % and its specificity being at . % [ ] . this method was used in the campisi research as well to determine the amount of the risk factors of an infection disease in relationship with oral candidiasis (oc) [ ] . additionally, shariati has recommended a method of diagnosis for both hepatitis and thyroid diseases. the researchers in this study compared the outcome of the anfis technique with the support vector machine (svm) and the artificial neural networks techniques. then, they demonstrate that this technique had an improved outcome in being a precise diagnosis in comparison with the previous techniques [ ] . the information in a fuzzy rule-based system is typically represented by using an if-then statement. rule-based fuzzy logic includes two portions: the precursor portion are the relevant conditions which are known as the inward variable(s), and the subsequent portion which expresses the outward variable(s). mamdani and sugeno are two different types of fuzzy rulebased systems. in the mamdani technique, the precursor, as well as the subsequent section, comprises fuzzy statements that reveal the value of the variables, while in sugeno method, the subsequent portion displays a nonlinear affiliation among both the inward and outward variables [ , ] . because of the complex nature of the numerous diseases, this technique can improve the infectious disease diagnosis and the treatment of such diseases as hiv and tuberculosis. based on this, sloot et al. efficiently used rule-based fuzzy logic to uncover the drug-resistance in hiv patients [ ] . furthermore, semogan et al. has created a clinical decision supporting system based on the fuzzy logic and the rule-based model that determine different classes of tuberculosis to assess respiratory diseases. in this system variables such as cough, cough duration, body temperature, fever duration, sputum discoloration, nose sputum, afternoon chills, night sweats, weight loss, and loss of appetite have been used in the diagnosis [ ] . b i o c y b e r n e t i c s a n d b i o m e d i c a l e n g i n e e r i n g ( ) - the fuzzy cognitive map (fcm) is a modelling technique that defines the connections between concepts such as variables, the inwards and the outwards by the methods of previous knowledge and experience. kosko has introduced fcm in . fcm is usually used to demonstrate the cause and effect relationship between the different concepts in a given system. fcms are used in numerous fields such as in engineering, error detection and medicine [ , ] . in this regard, mei et al. has applied fcm to describe how factors such as people's emotions and cognition functions influence each other to create epidemic infection [ ] . fcm was also used by the mago et al. research to develop the required knowledge-based structure used for recognizing the specific causes and the symptoms of meningitis disease in children. this system can be implemented as a dependable instrument in supporting the physician's to better their decision making processes [ ] . rq : which performance evaluation techniques were more frequently applied in the previous reviews? performance evaluation procedures are among the furthermost significant indicators for determining the quality of the artificial intelligence techniques [ ] . overall, performance evaluation procedures are classified into two main types; the first being the single scaler techniques and second being the graphical techniques. the sensitivity, specificity and the accuracy indicators are grouped into the single scaler techniques. receiver operating characteristic (roc) curve, cost-line, and lift graph are clustered together in graphical methods. anyhow, graphical methods cannot simply be clarified and analyzed as single scalar method [ ] [ ] [ ] . fig. indicates the most noticeable performance evaluation indicators used in eligible papers. for the current slr, most qualified studies did not use any kind of indicators for analyzing the performance of the fuzzy techniques. among the fuzzy techniques, anfis and fis techniques were evaluated by single scaler techniques and graphical evaluation techniques. the major objective of this slr was to select and examine the various studies relevant to the employment of the fuzzy logic techniques in an infectious disease. in this regard, studies were selected and analyzed from an original number of candidate studies. this section is dedicated to the discussion of the major findings of this study. rq : which domain of an infectious disease was more interesting in past researches? in last few years, it has been obvious from the results of analytic studies conducted in our research that there is a growing interest in studying the relevance of the fuzzy logic techniques for an infectious diseases diagnosis applications. the studies have shown that there is an increase in the number of published articles from % in up to . % of the evaluated papers in . this growing trend may prove the popularity of the fuzzy techniques between different academic papers and its valuable effects in identifying the infectious disease trends. additionally, different fuzzy logic techniques have been used in the evaluated papers. from a medical point of view, in fig. we found that the main application domain of fuzzy logic in infectious disease was related to dengue fever, hepatitis and tuberculosis respectively. the other noticeable outcome that we have found was the b i o c y b e r n e t i c s a n d b i o m e d i c a l e n g i n e e r i n g ( ) - fact that the evaluated papers were very diverse in terms of disease types. because of the big impact on global health by infectious diseases, determining the different features of these diseases are a valuable source for improving our knowledge and ability to predict how a disease will spread in a population [ , ] . the ability to comprehend and control an infectious disease can be obtained by the usage of mathematical models [ ] . the newly arising infectious agents such as hiv, the severe acute respiratory syndrome (sars), the mid-eastern respiratory syndrome (mers) coronaviruses; the west nile virus; the nipah virus; the drug-resistant pathogens; novel influenza a strains and the ebola virus outbreak were considered as big challenge to health in the recent century [ ] . using computeraided diagnosis techniques like the fuzzy logic technique can be useful in determining the main factors associated with the infectious diseases occurrence and epidemics. regarding this, in another field of research in relationship to the vaccination strategies for infectious diseases it has been shown that infectious diseases such as influenza have a seasonal pattern in which case mathematical techniques such as the fuzzy logic techniques is considered a vital instrument in the process of forecasting the viral development from one year to another. this technique can also deliver the required scientific confirmation which will help us decide the amount of vaccine treatment, its efficiency, its financial costs and its pattern of contact in any given population [ , , ] . it has been shown that the fuzzy based techniques have an essential part in the determination of infectious disease outbreaks. as an example for that, we have the hiv outbreaks, these techniques can identify at what time a viral transmission has occurred, its outbreak stage and the sexual behavior pattern of the specific population in which a disease is spreading. moreover, the infectious diseases annihilation and elimination techniques dictate that the transmission process itself has to be the target not the disease. regard this, the data sources are typically scattered and the collective effect of the numerous control techniques are complex and are dependent on the rate of transmission [ ] . rq : which fuzzy techniques were more dominant in an infectious disease data analysis? as shown in fig. , there is a various number of fuzzy techniques employed in the chosen papers. based on this studied section we can mostly classify the fuzzy techniques into four main classes; the fuzzy inference system, the rulebased fuzzy logic, anfis and the fuzzy cognitive map. the fuzzy inference system and the anfis technique were used as shown in . % and . % of candidate papers, respectively. fis is based on the 'if-then' rules and it can be used to predict the behavior of a various uncertain situations [ ] . as seen in the distribution of the studied articles, we can say that fuzzy techniques are efficient means for modelling unclear disease conditions such as in the case of transmissible disease diagnosis. additionally, in the last few years, there has been substantial interest amongst the researchers to apply the anfis technique in the case of an infectious disease. anfis combines the positive effects of both ann and fis in an influential tool for disease diagnosis. this technique does not require excessive knowledge in the modelling and training system. these techniques are usually valuable for the situation, which are in many cases complicated, with a nonlinear behavior pattern. this work approach has created a relationship between the inward and the outward features by the means of the neurons [ ] . rq : which performance evaluation procedures were more frequently applied in the previous reviews? performance evaluation procedures are usually employed as a valued method in determining the quality of the numerous fuzzy techniques [ ] . as shown in fig. in the current slr, amongst the single scaler techniques, the sensitivity, specificity and the accuracy are frequently utilized in the evaluation of a developed technique. additionally, in graphical evaluation techniques, the roc curve was the other measure of performance evaluation that was used in the adequate papers. these indicators are to be considered significant when reporting and assessing a diagnostic technique. scientists have to provide suitable information about the sensitivity, specificity, and the projected values when describing a computer based diagnostic technique end results and this information must contain how those metrics are concluded and also what are its appropriate interpretations [ , ] . although those indicators are promising in qualified studies, nonetheless, they typically generate an inadequate image of the indicators performance and thus it is possible to lose a certain amount of valuable information. moreover, it is possible that the employment of a single-scaler technique will not identify the full scope of a performance assessment. therefore, a comprehensive and dependable assessment must reflect all the numerous parts of performance distinctive quality. in this methodical review, the studies related to the employment of the fuzzy logic techniques in an infectious disease were assessed, and depending on the acquired outcomes, we can notice an interest amongst the researchers regarding this specific field of research. in last few years, a large number of the transmissible diseases that were supposed to be eliminated have made a comeback. certain factors such as manufacturing, agricultural practices, wars, changes in lifestyles, development and urbanization, and environmental change are all effective in the appearance and reappearance of an infectious disease [ ] . this slr's result demonstrates that even though these are the most of the infectious diseases that were investigated, but there is still more area that needs to be covered. nevertheless, more work should be carried out on the appearance and reappearance diseases domains such as h n , sars, zoonosis and the rift valley fever. internationally, there is a lack of an integrated framework for reporting infectious disease [ ] . additionally, an infectious diseases information system has an inadequate support for data analysis and generating predictive techniques centered on artificial intelligence. an integrated analytical framework that offers functions as in a progressive data analysis capability and a visualization support is of a critical importance [ , ] . there is serious need for creating an atmosphere for collecting, distributing, reporting, assessing, and picturing the infectious disease data and to provide support for decisionmaking tools regarding disease prevention, recognition, and controlling [ , ] . infectious disease observation and controlling has demanded an interdisciplinary work. to have the ability to achieve those objectives, the employment of geographic information systems (gis), three-dimensional information analysis, machine learning and visualization applications and techniques became a must. because of the significance of the infectious diseases at the global level, it is essential to simultaneously develop an incorporated infectious disease dataset and to make the specialized analytical and diagnostic methods all at the same time. additionally, there was slight conversation in the incorporated literature about three-dimensional information analysis for an infectious disease occurrence. three-dimensional information evaluation techniques may be useful in determining the concentration pattern of a disease occurrence and to make the required association from the determined patterns to the measureable procedures [ , ] . furthermore, the social networks information study has facilitated the evaluation of the association amongst the population in a particular social setting. methods that correlate spatial and social network data analysis are unusual but have the capacity to promote the determination of a spreading progress of an infectious disease [ ] . or we can say, the deployment of a unified spatial and social network structure to define the spreading of an infectious pathogens in a given populace will allow insights into both the understanding to the disease method of distribution and the possible process associated with the observed patterns. since there are various studies regarding social media information analysis, we recommend the use of this dataset for the prediction of an infectious disease epidemic. this research has its limitations. even with the use of a broad search approach, some of the publications regarding the fuzzy logic deployment in an infectious disease could not be recovered, as in the case of grey literature and reports that were not published in surveyed digital databases, which we have reviewed. thus, it is recommended that additional slr papers should be carried out to go through the other noticeable databases. additionally, some studies did not report clearly on the performance assessment technique. in conclusion, only the english language publications were included, therefore future studies could be expanded to incorporate relevant papers which are published in other languages. in this study we have identified, classified, and defined the use of the fuzzy logic techniques in infectious diseases. studies were scrutinized and the main conclusions can be briefed as follows: ( ) the key application field of the fuzzy logic in an infectious disease was related to dengue fever, hepatitis and tuberculosis, ( ) amongst the fuzzy logic techniques fuzzy inference system, rule-based fuzzy logic, anfis and fuzzy cognitive map are commonly used in many studies, and ( ) the major performance evaluation indicators such as the sensitivity, specificity, and the accuracy the roc curve is employed. in addition, this study highlights the absence of an integration of infectious disease information systems in order to provide a valuable datasets in this domain. additionally, using machine learning and visualization applications for information analysis is essential. even though in the current slr there is a diversity of infectious diseases that are investigated, there is only one article per each disease. there is additional need to use the fuzzy logic methods for infectious disease detection and prediction. it appears that one of the causes for a limited number of relevant articles to infectious diseases is in the difficulty in obtaining adequate research data. finally, we expect that a mounting number of infectious diseases datasets will be mostly obtainable in the forthcoming years because of raising collaboration amongst medical practitioners and researchers and that this would lead to additional studies of the machine learning techniques that can be useful in this regard. the generated results are more reliable for the prediction of tb in patients. langarizadeh [ ] meningitis rule-based fuzzy logic this system is used to distinguish between bacterial and aseptic meningitis, by using fuzzy logic. gram stain, white blood cell (wbc) count in cerebrospinal fluid (csf), percentage of polymorphonucleocytes in csf, csf protein, csf/ serum glucose ratio, wbc count in blood, percentage of blood neutrophils, blood c-reactive protein (crp), and platelet (plt) count. bacterial and aseptic meningitis this suggested system has shown great efficiency in regards to its ability to differentiating between the bacterial and aseptic meningitis. omisore [ ] tuberculosis genetic-neuro-fuzzy proposing a genetic-neuro-fuzzy for the diagnosis of tuberculosis. the study has proposed fcms for determining the symptoms and causes for meningitis patients. b i o c y b e r n e t i c s a n d b i o m e d i c a l e n g i n e e r i n g ( ) - biological sciences curriculum study nih curriculum supplement series. bethesda, md: national institutes of health how urbanization affects the epidemiology of emerging infectious diseases epidemiological features of and changes in incidence of infectious diseases in china in the first decade after the sars outbreak: an observational trend study clinical aspects of influenza a (h n ) in hivinfected individuals in são paulo during the pandemic of global trends in emerging infectious diseases modeling infectious disease dynamics in the complex landscape of global 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genetic-neuro-fuzzy inferential model for diagnosis of tuberculosis application of evolutionary fuzzy cognitive maps for prediction of pulmonary infections supporting meningitis diagnosis amongst infants and children through the use of fuzzy cognitive mapping key: cord- - rhgmtbo authors: bajpai, vijeta; gupta, ekta; mitra, lalita gauri; kumar, hemant; maiwall, rakhi; soni, kapil dev; gupta, amit title: spectrum of respiratory viral infections in liver disease patients with cirrhosis admitted in critical care unit date: journal: j lab physicians doi: . /jlp.jlp_ _ sha: doc_id: cord_uid: rhgmtbo background: clinical significance of respiratory viruses (rvs) as an etiology of pneumonia in liver disease patients with cirrhosis is usually underestimated. therefore, the aim of this study was to evaluate the spectrum of rvs in cirrhotic patients with pneumonia admitted in critical care units (ccus) and its impact on the clinical outcome of cirrhotic patients. material and method: a prospective study was conducted in a tertiary care ccu, and consecutive cirrhotic patients with pneumonia were included. bronchoalveolar lavage or throat swab/nasal swab was collected in viral transport medium for analysis of rvs by multiplex real-time polymerase chain reaction. a total of cirrhotic patients were included, viral and bacterial etiology of pneumonia was identified, and analysis was done with the clinical outcome. results: overall, rvs were detected in ( . %) cirrhotic patients and viral–bacterial coinfection in ( . %) cirrhotic patients. the most common virus detected was rhinovirus in ( %) patients. mortality in cirrhotic patients with rv infection was significantly higher in comparison to cirrhotic patients with no rv infection ( [ . %] and [ . %], respectively, p < . ). conclusion: respiratory viruses in cirrhotic patients with pneumonia are associated with poor clinical outcome. i nfectious diseases are common in liver disease patients with cirrhosis and exert one of the most important reasons for morbidity and mortality in critical care units (ccus). pneumonia is one of the most common infections in liver disease patients with cirrhosis admitted in ccu. [ , ] the prevalence of pneumonia is reported in about . % of cirrhotic patients with mortality rates as high as %- %. [ ] there are several consequences of pneumonia in cirrhotic patients which include acute on chronic liver failure, multiple organ failure, sepsis, prolonged hospitalization, and mortality. [ ] although pneumonia exhibits higher mortality in patients with cirrhosis, majority of the studies focused on bacterial pneumonia as the most common cause of pneumonia in cirrhotic patients; other pathogens especially are usually underestimated in such patients and are often not underreported. [ , ] there are very few studies which have shown the influence of respiratory viruses (rvs) in clinical outcome of cirrhotic patients in ccu. [ ] [ ] [ ] till now, there is no published study showing the prevalence of rvs and their bacterial coinfections in cirrhotic patients admitted in ccu care. with this background, the purpose of the current study is to investigate the prevalence of rvs in cirrhotic patients with pneumonia and its impact on the clinical outcome of cirrhotic patients with pneumonia admitted in ccus. this is a prospective study, done over a period of year from january to december , in ccus in liver center, and all consecutive cirrhotic patients who had developed clinical signs and symptoms of pneumonia were included. pneumonia was defined as a new infiltrate focus on chest radiological examination and one or more symptoms as follows: respiratory symptoms (i.e. cough, chest pain, and dyspnea), sign of infection (fever > °c, and/or white blood cell count > , /mm or < /mm . [ ] the exclusion criteria included: ( ) under years; ( ) pregnancy; ( ) patients with immunosuppression (including patients with chemotherapy and radiotherapy and patients with drug-induced immunosuppression as a result of cytotoxic or corticosteroids [defined as > mg/kg prednisone for > month]); ( ) bone marrow or solid organ transplantation; ( ) patients with hepatocellular carcinoma or with other types of carcinoma; and ( ) hiv infection. bronchoalveolar lavage (bal) or throat swab/ nasal swab (ts/ns) was collected from cirrhotic patients with pneumonia. bal sample was collected by trained pulmonologist patients on mechanical ventilator because the ts/ns could not be from them. ts and ns were collected from nonventilated patients with pneumonia. all samples were transported in viral transport media immediately to the virology laboratory on ice. all the samples were stored at − °c till further processing. complete clinical characteristics of the patient and other biochemical parameters were recorded from the hospital information system. the study was approved by the ethical committee (irb reference no: f. / / /ilbs/ ac/ / ) of the institute. a patient admitted to the intensive care unit (icu) with the diagnosis of pneumonia without prior contact of health settings was considered as having community-acquired pneumonia (cap).infection in a patient who developed clinical features of pneumonia after h of admission was considered as hospital-acquired pneumonia (hap). impact of clinical outcome due to viral pneumonia was assessed if there was a requirement for either invasive mechanical ventilation, extended duration of stay in icu (> days), or mortality of patients. in all the samples, nucleic acids (dna/rna) were extracted using commercial kit (rtp dna/rna virus mini kit, stratec molecular, birkenfeld, germany) according to the manufacturer's instructions. multiplex quantitative real-time polymerase chain reaction was done using commercially available kit capable of detection and quantification of different pathogens ( viruses and bacteria; ftd-rp , fast-track diagnostics, luxembourg) as per the manufacturer's protocol [ table ]. results were expressed as copies/ ml, and the linear range of the assay is - copies/ ml. data were presented as mean ± standard deviation or an absolute number and percentage. to compare categorical variables, chi-square test was used, and for continuous variables, student's t-test was used. p < . was considered statistically significant. data were recorded and analyzed using spss (version . ; spss). (ibm, armonk, ny, united states of america). a total of liver disease patients with cirrhosis were admitted in icu during the study period. diagnosis of pneumonia was made in ( . %) cirrhotic patients. eighty-six ( . %) patients had hap and ( . %) patients had cap. the baseline characteristics of the study population are depicted in table . a total of patients ( . %) underwent bronchoscopy bal for etiologic diagnosis of pneumonia, and ts/nss were collected from ( . %) patients. figure shows the workflow and distribution of respiratory pathogens in all patients. the overall prevalence of rvs was found in ( . %) cirrhotic patients with pneumonia, and both viral and bacterial coinfections were seen in ( . %) cirrhotic patients with pneumonia. the proportion of rv infections were little higher in hap group than in the cap group though not significant ( [ . %] and [ . %], respectively, p = . ). the comparison of various parameters of cirrhotic patients with and without rv infection is shown in table . overall, rhinovirus (rhv) was the most common identified virus in ( . %) cirrhotic patients, followed by influenza a (flu a) in ( . %) patients, human coronavirus (hcov) in ( . %) patients, respiratory syncytial virus (rsv) in ( . %) patients, human parainfluenza virus (hpiv) in ( . %) patients, and human metapneumovirus and human adenovirus (hadv) in one each of the patients. dual viral infections such as hadv + hpiv, hcov + rsv, and hadv + rhv were found in one each of the patients, respectively [ table ]. the seasonal distribution of various rvs is described in figure . rhv was present throughout the year. flu a virus and rsv were mainly present in winter months. hcov, hadv, and hpiv were mainly present in summer and early winter seasons. overall, mortality was seen in ( . %) cirrhotic patients with pneumonia in icu. mortality in cirrhotic patients with rv infection was higher than patients with viral-bacterial coinfection ( [ . %] to the best of our knowledge, this is the first prospective study representing the investigation of viral pneumonia in cirrhotic patients in ccus. in the present study, we evaluated the epidemiology, clinical characteristics, and prognosis of cirrhotic patients with viral pneumonia in ccus. specimens obtained from the upper airway as ts/ns are often used for rv testing in pneumonia patients due to ease of specimen collection in comparison to bronchoscopy collected bal sample. [ ] however, in the current study, the proportion of rvs detected from bal samples were more than ts/ns though not significant. therefore, here comes the importance to perform bronchoscopy and collection of bal sample in the most affected lung segment according to the radiographic signs to recover the responsible respiratory pathogen in patients with pneumonia, especially in patients having chronic liver disease. over the past decade, several studies have consistently demonstrated rvs to be the second most common etiological factor of cap, accounting for %- % of diagnosed cases of pneumonia in noncirrhotic patients in ccus. [ ] our study has demonstrated that the proportion of rv infections was little higher in the hcap group than in the cap group in cirrhotic patients, which is similar to one recent study done in nonliver adult patients. [ , ] a high index of suspicion of viral pneumonia is always necessary in patients with cirrhosis because there are no distinctive clinical features to differentiate between viral and bacterial pneumonia.; hence, a high index of suspicion is necessary for this infection. none of the previously done studies have shown the spectrum of different rvs in liver disease patients. only a few studies done in our center have shown the prevalence of influenza virus and its impact on the clinical outcome of cirrhotic patients. [ , , ] the current study has found that respiratory viral infections other than influenza virus infection are also an important etiology of pneumonia in liver disease patients with cirrhosis admitted in ccus. transmission dynamics and seasonal distribution of rvs are key importance in understanding and limiting burden of morbidity and mortality of pneumonia patients in ccus. [ ] in the current study, no clear seasonality is seen for human rhv. flu a virus and rsv were mainly present in winter months. this finding can inform the timing of flu a and rsv vaccination and the judicious use of antibiotics in cirrhotic patients admitted in ccus. mixed viral and bacterial infections are widely described in pneumonia patients in ccus; however, the presence of these coinfections on the cirrhotic patients' clinical outcome remains poorly studied. [ ] various studies suggest that viral and bacterial coinfections lead to a higher severity ongoing disease, especially in patients with chronic disease. [ , ] we did not find any significant correlation in mortality and median length of stay in ccus in patients with only rv infection and viral-bacterial confection which is not in concordance of previous done studies. [ ] our study had some limitations. first of all, our study was a single-center cohort and done in a small duration period. considering small number of patients, our observations should be confirmed in larger sample size with more detailed subgroup in future study. second, our study excluded cirrhotic patients with cancer and immunosuppression. therefore, exclusions may result in statistic bias and inexact conclusion. nevertheless, our investigation provided the first comprehensive study of the prevalence of rvs and its epidemiology in cirrhotic patients with pneumonia in ccus. with the high morbidity and mortality rates associated with rv infections and the lack of directed antiviral therapy for most of these infections, prevention remains the mainstay for reducing their incidence and controlling transmission of rvs, especially in ccus settings. rvs may contribute to significant cause of pneumonia in cirrhotic patients. therefore, there should be a high index of suspicion, and early diagnosis by rapid molecular test for rvs should be instituted in cirrhotic patients with pneumonia in ccus. pneumonia in patients with cirrhosis: risk factors associated with mortality and predictive value of prognostic models community-acquired pneumonia in patients with liver cirrhosis: clinical features, outcomes, and usefulness of severity scores high mortality of pneumonia in cirrhotic patients with ascites bacterial infections in cirrhosis: a position statement based on the easl special conference bacterial infections in end-stage liver disease: current challenges and future directions bacterial infections in patients with liver cirrhosis influenza a/ h n pdm infection in liver disease patients requiring icu care a/h n / influenza is associated with high mortality in liver cirrhosis clinical impact of a/h /n / influenza in patients with cirrhosis: experience from a nosocomial cluster of infection management of adults with hospital-acquired and ventilator-associated pneumonia: clinical practice guidelines by the infectious diseases society of america and the american thoracic society predictive value of testing nasopharyngeal samples for respiratory viruses in the setting of lower respiratory tract disease comparison of viral infection in healthcare-associated pneumonia (hcap) and community-acquired pneumonia (cap) incidence of respiratory viruses in patients with community-acquired pneumonia admitted to the intensive care unit: results from the severe influenza pneumonia surveillance (sips) project influenza a/h /n / infection in patients with cirrhosis has a poor outcome: a case series seasonality of respiratory viruses causing hospitalizations for acute respiratory infections in children in nha trang viral and bacterial co-infection in severe pneumonia triggers innate immune responses and specifically enhances ip- : a translational study viral-bacterial interactions in the respiratory tract secondary bacterial infections associated with influenza pandemics we would like to thank all the innocent patients for agreeing in the study and for donating their clinical samples. we feel privileged to offer the deepest sense of gratitude and respect to our teacher, dr. sk sarin, director of ilbs, for his invaluable guidance, supervision, and blessings, without which these works would not have seen the light of the day. nil. there are no conflicts of interest. key: cord- -zlr nwc authors: burimuah, vitus; sylverken, augustina; owusu, michael; el-duah, philip; yeboah, richmond; lamptey, jones; frimpong, yaw oppong; agbenyega, olivia; folitse, raphael; tasiame, william; emikpe, benjamin; owiredu, eddie-williams; oppong, samuel; adu-sarkodie, yaw; drosten, christian title: sero-prevalence, cross-species infection and serological determinants of prevalence of bovine coronavirus in cattle, sheep and goats in ghana date: - - journal: vet microbiol doi: . /j.vetmic. . sha: doc_id: cord_uid: zlr nwc cattle, goats and sheep are dominant livestock species in sub-saharan africa, with sometimes limited information on the prevalence of major infectious diseases. restrictions due to notifiable epizootics complicate the exchange of samples in surveillance studies and suggest that laboratory capacities should be established domestically. bovine coronavirus (bcov) causes mainly enteric disease in cattle. spillover to small ruminants is possible. here we established bcov serology based on a recombinant immunofluorescence assay for cattle, goats and sheep, and studied the seroprevalence of bcov in these species in four different locations in the greater accra, volta, upper east, and northern provinces of ghana. the whole sampling and testing was organized and conducted by a veterinary school in kumasi, ashanti region of ghana. among sampled sheep (n = ), goats (n = ), and cattle (n = ), the seroprevalence rates were . %, . % and . %. for cattle, seroprevalence was significantly higher on larger farms ( . % vs . %, comparing farms with > or < animals; p = . ). highest prevalence was seen in the northern province with dry climate, but no significant trend following the north-south gradient of sampling sites was detected. our study identifies a considerable seroprevalence for bcov in ghana and provides further support for the spillover of bcov to small ruminants in settings with mixed husbandry and limited separation between species. cattle, goats and sheep are among the major livestock species in ghana. the present numbers in based on the food and agriculture organization (fao) animal production database are estimated to range around . , . , and . million cattle, goats and sheep in the country, respectively. among livestock, only chicken outnumber these species ( million). while disease surveillance is in place, there are knowledge gaps concerning the laboratory-based prevalence of some major livestock diseases. among these is bovine coronavirus (bcov) that affects cattle and other livestock species including horses and camels. bcov (yang and leibowitz, ; oma et al., ; pfefferle et al., ) . while different strains may have some antigenic variability, all strains elicit cross-reactive seropositivity and thus form a single serotype (clark, ; el-ghorr et al., ) . the virus is an important livestock pathogen causing effects on animal welfare as well as the economy (lathrop et al., a) . it causes diarrhea and respiratory disease in calves, as well as winter dysentery in adult cattle (boileau and kapil, ; ksiazek et al., ) . transmission of bcov is mainly through respiratory or fecal-oral routes (clark, ) , infecting the respiratory (nasal, tracheal, and lung) and intestinal (villi and crypts of the ileum and colon) epithelial cells (park et al., ) . when infected with bcov, within-herd transmission is generally rapid and infected animals display diverse clinical signs including diarrhea with or without blood, fever, and respiratory signs, which range from none to severe (clark, ; boileau and kapil, ) . in many african countries including ghana, livestock species live in close contact and animals serve diverse purposes such as transportation, draught power, fuel, clothing and as a source of meat and milk. husbandry practices do not involve the same standards of species separation and hygiene as in other parts of the world. close and sustained interaction between different animals as well as between animals and humans pose a risk of interspecies spillover of pathogens. bcov is characteristically a cattle virus. however, reports indicate bcov infections also occur in small ruminants. previous studies in australia (pass et al., ) , new zealand (durham et al., ) , chile (reinhardt et al., ) , and scotland (snodgrass et al., ) reported bcov infection in small ruminants. eisa and mohamed, also detected bcov antigens in goats (eisa and mohamed, ) whereas tråvén et al., detected bcov antibodies in sheep (tråvén et al., ) . recently, gumusova et al., have also detected bcov antibodies in goats (gumusova et al., ) . studies regarding the prevalence of bcov and its associated risk factors are however limited in africa, and none have been conducted in ghana. this study evaluated the sero-prevalence of bcov infection and assessed its associated risk factors among cattle, sheep, and goats in ghana. this study employed a cross-sectional design and was conducted between january to december in five districts in four regions of ghana. ghana is located in the west of africa, sharing borders with togo to the east, cote d'ivoire to the west, burkina faso to the north and the gulf of guinea, to the south and lies on latitude . and longitude - . . ghana has a tropical climate with an average annual temperature of about °c and the annual rainfall of . mm/ ″. agriculture dominates the economy of ghana and extensive farming practices in the country increase the livestock-wildlife-human interface this study was approved by the wildlife division of the ghana forestry commission (approval number: ao ). a total of animals aged ≥ months, comprising cattle, sheep and goats were included in the study. animals aged < months were excluded due to the possibility of detecting maternal antibodies. sampling was done using a simple two-stage cluster sampling technique. the regional veterinary officers of the ministry of food and agriculture (mofa), ghana in the selected regions were contacted for information on animal populations in their respective regions prior to the study. the list provided served as the sampling frame. prior to the study, a survey was carried out and an inclusion criteria of animal population (cattle, sheep and goats) ≥ for districts to be eligible for selection for the study was upheld. as a result, five ( ) districts that fulfilled the criteria were randomly selected. secondly, farms within these districts with herd size ≥ animals were randomly selected. all cattle, sheep, and goats within the selected farms were included in the study. if a district meets the first criteria, but the individual farms fail to meet the second criteria, farms which were very close to each other were pooled together. sites included were: bongo district in the upper east; savelugu and wale wale in northern; ada west in greater accra, and north tongu in volta. the map with the locations for sampling in shown in fig. . a validated questionnaire was used to obtain data on possible risk factors of bcov. data collected include: age, sex, dietary changes, parturition, and lactation status of female animals. additionally, the body score, presence of ectoparasites, and signs of infections such as fever, diarrhoea, respiratory distress, neurological disorder, and icterus were also assessed. ten milliliters ( ml) of blood was collected through jugular puncture from each animal after disinfection of the site with % using vacutainer tubes (becton dickinson, nj, usa) with needles ( gauge). in the field, the blood was allowed to clot before transportation to the veterinary laboratory in the district. at the district laboratory, the samples were spun for min at rpm to obtain the sera. the sera were transferred into three separate aliquots in cryotubes for each animal. the tubes were subsequently placed in liquid nitrogen to minimize antibody degradation. these processes were undertaken under sterile conditions. upon obtaining representative samples per district, the frozen samples were transported in a cold-chain to the kumasi centre for collaborative research (kccr) for long term storage at − °c prior to laboratory analysis. sample collection and preparation in each district and transportation to the kccr lab took an average of - days. during laboratory analysis, frozen sera were thawed at room temperature, vortexed and aliquots of μl of each sample were prepared. the aliquots were incubated at °c for min in warm water prior to recombinant immunofluorescence assay (ifa) as previously described (hoye et al., ; el-duah et al., ; reusken et al., a) . briefly, vero b cells were co-transfected with pcg plasmids bearing human coronavirus oc spike proteins. after overnight transfection, cells were harvested by treatment with trypsin to detach them in a cell culture incubator at °c and re-suspended in dulbecco's modified eagle medium (dmem) (invitrogen, usa) in % foetal calf serum (fcs). aliquots of cells were pelleted at x g for min and washed twice with ml phosphate buffered saline (pbs). fifty microliters ( μl) of cell pellets were spotted on well slides by dispensing and immediately aspirating, allowing s interval between spotting. the cells were fixed using ice cold acetone/methanol ( : ), dried at ambient temperature, and kept at °c after drying for min. to conduct the assay, μl of protein-free blocking solution (roti®-block, carl roth, karlsruhe, germany) was first added to each of the spotted fields on the slide and incubated at room temperature in a humid box for min, followed by rinsing with x tween-free pbs. after inactivation at °c for min, sera to be tested were diluted : in a x concentration of the protein-free blocking solution. subsequently, μl of the diluted sera was applied on each of the spotted area and incubated at °c for h in a humid chamber, followed by rinsing with . % tween in x pbs. secondary antibody detection was done by the alexa fluorescent reporter-conjugated goat anti-bovine, donkey anti-sheep, and donkey anti-goat igg antibodies for cattle, sheep, and goat bcov igg respectively. test evaluation was done by microscopic examination under a fluorescent microscope and a positive outcome was determined by bright green cytoplasmic fluorescence as shown in fig. . data were presented as frequencies (percentages) and chi square test was used to test for association where applicable. univariate and multivariate logistic regression analysis were performed to determine the possible factors associated with bcov sero-positivity for cattle, sheep, and goats. a p-value < . was considered statistically significant. all statistical analyses were performed using ibm statistical package for the social sciences (spss) software (spss inc., chicago, il, usa), and graphpad prism version . (graphpad software, inc., la jolla, california usa). the proportions of sheep, goat, and cattle were . %, . %, and . % respectively. there were more adults than weaners ( . % vs . %) and more female animals ( . %) compared to male animals ( . %), with . % of the females being lactating females at the time of the study. majority of the animals had high rectal temperature ( . %) and . % had physical signs of disease of which . % was diarrhea, . % was respiratory distress, and . % of the animals were icteric. none of the animals had neurological disorders. additionally, a higher proportion of the animals had ectoparasites ( . %), were thin ( . %) and have not had any dietary change ( . %) ( table ) . the sero-prevalence of bcov in the entire animal population was . %. upon stratification by sheep, goats, and cattle, the prevalence was . %, . % and . %, respectively. cattles had significantly higher prevalence of bcov compared to sheep and goats. among the entire animal population, sero-positivity of bcov was significantly associated with farms with ≥ animals ( . % vs . %, p < . ). upon stratification by type of animal, this effect seemed to be explained by cattle ( . % vs . %, p = . ) but not sheep and goats that are normally kept in smaller groups ( table ). the sero-prevalence of bcov was highest in the northern region followed by the volta region (table ) . even though our sampling sites formed a north-south gradient, there was no latitude-dependent trend in seroprevalence. there was no statistically significant association between the possible risks factors assessed and bcov sero-positivity among all animals with the exception of dietary change, where a significantly lower odd of bcov was observed among cattle with recent dietary change [or = . , % ci ( . - . ), p = . ] ( (table ) . bcov is a ubiquitous infection and bcov-specific antibodies have been detected in cattle populations in numerous countries (hasoksuz et al., ; kapil et al., ; lathrop et al., b; yavru et al., ) . bcov shares recent common ancestry with human coronavirus oc (hcov-oc ) (vijgen et al., ) and the two are serologically closely related to the extent that hcov-oc is often used as a proxy in serological testing as previously described where specific proteins of bcov were not available for serological testing or when national legislation restricts the use of certain livestock pathogens (reusken et al., b; meyer et al., ) . in most african countries such as ghana, diverse farm animals live in close contact that poses a risk of cross-species infection. indeed, we found seropositivity against bcov not only among cattle ( . %) but also among sheep and goats at . % and . % prevalence rates, respectively. this prevalence was predominant among ontario feedlots, respectively based on enzyme linked immunosorbent assay (elisa) method. bidokhti et al. ( ); hasoksuz et al. ( ) , and yildirim et al. ( ) also reported a sero-prevalence of - %, . %, and . %, respectively among cattle. the discrepancies in the prevalence rates compared to that of this present study could be attributed, at least in part, to differences in geographical location, different management systems, source population size, method employed for bcov antibody detection, and the samples size used in the different investigations. in addition, the higher prevalence rate among cattle could be due to the fact that a higher proportion of the animals were cattle as well as the tropism of bcov to cattle few studies have been conducted to evaluate the prevalence of bcov among small ruminants. the most recent study was conducted by gumusova et al. in in turkey. in their study, they evaluated the sero-prevalence of bcov in goats by employing commercially available competitive elisa kits and reported a bcov sero-prevalence of . % (gumusova et al., ) . in a previous study, eisa and mohamed reported detection of bcov antigens in goats (eisa and mohamed, ) . additionally, tråvén et al., in a study among sheep from flocks in different parts of sweden, reported that % of the sheep were positive for bcov antibodies (tråvén et al., ) . prior to these recent studies, there had been reports of bcov infection in small ruminants in australia (pass et al., ) , new zealand (durham et al., ) , chile (reinhardt et al., ) , and scotland (snodgrass et al., ) . our finding in sheep and goats, thus, provides update information of spillover of bcov from cattle. granted the contagious nature of bcov, it is imperative that factors that influence exposure and the determinants of bcov infection be identified which would assist in the development of apt control and preventive measures against bcov and other infectious diseases. though there was no statistically significant association between the possible risks factors assessed and bcov sero-positivity, we found bcov sero-positivity to be significantly associated with farms with higher cattle density. this finding is in harmony with studies by beaudeau et al. ( ) ; hägglund et al. ( ) , and ohlson et al. ( ) who reported that large herd size is a risk factor for bcov infections in dairy cattle. this may be due to poor biosecurity especially among farms with larger herd size in ghana, and also due to the close contact between animals in these farms which could potentiate the transmission of bcov compared to farms with small herd size (beaudeau et al., ; ohlson et al., ) . infectious diseases surveillance can be greatly enhanced by research studies, as often there is close collaboration between governmental and academic institutions. restrictions in the movement of samples to prevent the spread of notifiable livestock diseases create a demand for domestic laboratory capacities. through this study we hope to demonstrate the value of capacity building in field-based research. sığırlarda coronavirus enfeksiyonunun epidemiyolojisi. ankara Üniv vet fak derg spatial patterns of bovine corona virus and bovine respiratory syncytial virus in the swedish beef cattle population reduced likelihood of bovine coronavirus and bovine respiratory syncytial virus infection on organic compared to conventional dairy farms bovine coronavirus associated syndromes bovine coronavirus rotavirus and coronavirus associated diarrhoea in domestic animals role of enteric pathogens in enteritis in lambs, goat kids and children and their zoonotic importance potential intermediate hosts for coronavirus transmission: no evidence of clade c coronaviruses in domestic livestock from ghana a serological comparison of bovine coronavirus strains first report of bovine rotavirus and bovine coronavirus seroprevalance in goats in turkey dynamics of virus infections involved in the bovine respiratory disease complex in swedish dairy herds detection of respiratory and enteric shedding of bovine coronaviruses in cattle in an ohio feedlot detection of respiratory and enteric shedding of bovine coronaviruses in cattle in northwestern turkey surveillance of wild birds for avian influenza virus excretion and persistence of bovine coronavirus in neonatal calves a novel coronavirus associated with severe acute respiratory syndrome association between infection of the respiratory tract attributable to bovine coronavirus and health and growth performance of cattle in feedlots antibody titers against bovine coronavirus and shedding of the virus via the respiratory tract in feedlot cattle antibodies against mers coronavirus in dromedaries the relationship between the occurrence of undifferentiated bovine respiratory disease and titer changes to bovine coronavirus and bovine viral diarrhea virus in ontario feedlots risk factors for seropositivity to bovine coronavirus and bovine respiratory syncytial virus in dairy herds bovine coronavirus in naturally and experimentally exposed calves; viral shedding and the potential for transmission dual enteric and respiratory tropisms of winter dysentery bovine coronavirus in calves intestinal coronavirus-like particles in sheep with diarrhoea distant relatives of severe acute respiratory syndrome coronavirus and close relatives of human coronavirus e in bats diagnosis of coronavirus in sheep in valdidia province middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study virus infections in cattle and sheep in scotland - serum antibodies to bovine coronavirus in swedish sheep evolutionary history of the closely related group coronaviruses: porcine hemagglutinating encephalomyelitis virus, bovine coronavirus, and human coronavirus oc the structure and functions of coronavirus genomic ′ and ′ ends bovine coronavirus (bocv) infection in calves with diarrhoea and their dams seroprevalence of the rotavirus and corona virus infections in cattle this work was supported by deutsche forschungsgemeinschaft under a grant to y. a. s. and c. d. (dr / - ). key: cord- -pd elo l authors: luo, wei; gao, peng; cassels, susan title: a large-scale location-based social network to understanding the impact of human geo-social interaction patterns on vaccination strategies in an urbanized area date: - - journal: computers, environment and urban systems doi: . /j.compenvurbsys. . . sha: doc_id: cord_uid: pd elo l abstract cities play an important role in fostering and amplifying the transmission of airborne diseases (e.g., influenza) because of dense human contacts. before an outbreak of airborne diseases within a city, how to determine an appropriate containment area for effective vaccination strategies is unknown. this research treats airborne disease spreads as geo-social interaction patterns, because viruses transmit among different groups of people over geographical locations through human interactions and population movement. previous research argued that an appropriate scale identified through human geo-social interaction patterns can provide great potential for effective vaccination. however, little work has been done to examine the effectiveness of such vaccination at large scales (e.g., city) that are characterized by spatially heterogeneous population distribution and movement. this article therefore aims to understand the impact of geo-social interaction patterns on effective vaccination in the urbanized area of portland, oregon. to achieve this goal, we simulate influenza transmission on a large-scale location-based social network to ) identify human geo-social interaction patterns for designing effective vaccination strategies, and ) and evaluate the efficacy of different vaccination strategies according to the identified geo-social patterns. the simulation results illustrate the effectiveness of vaccination strategies based on geo-social interaction patterns in containing the epidemic outbreak at the source. this research can provide evidence to inform public health approaches to determine effective scales in the design of disease control strategies. infectious diseases (e.g., influenza) have posed great challenges to society. dangerous infectious diseases, such as severe acute respiratory syndrome (sars), anthrax, h n flu, and ebola pandemics, have been exacerbated by increasing human density and population mobility seen in contemporary society (anderson et al. ; fraser et al. ; leung et al. ) . infectious disease outbreaks can spread worldwide via international transportation networks more rapidly than the distribution of vaccines (ferguson, fraser, donnelly, ghani, & anderson ; webby & webster ) . a key public health question is how to contain infectious diseases at the source or reduce transmission long enough to implement an effective response (longini jr et al. ) . network science has been used to study infectious disease transmission and to design effective control (germann, kadau, longini, & macken ; halloran et al. ; longini jr et al. ) . traditional network models are individual-centric approaches in which nodes represent individuals and links represent physical contacts (meyers ) . disease simulations based on network models primarily focus on health outcomes (e.g., the number of infected cases) (mao & bian ; ), but do not sufficiently consider the role of location in disease transmission and control (zhong & bian ) ; for example, what are the critical locations in the transmission? are location-based control strategies more effective than individual-based control strategies? how do the spatial flows of population mobility among locations influence epidemics and control strategy design? those are all critical public health questions in terms of spatially informed policies for infectious disease control and prevention (bian, , gao & bian, . to tackle the above questions, this research treats infectious disease spread as geo-social interaction patterns, in which locations interact with each other via viruses that transmit from one place to another because of human interaction and population movement (gushulak & macpherson ) . geo-social interaction patterns identified from human mobility data within cities have the potential to provide valuable insight for designing effective epidemic control measures (guo ) . luo ( ) empirically found that vaccination strategies considering geo-social interaction patterns have a high probability to contain pandemics at the source within a primary school environment. however, human interaction patterns at an urban scale, which are characterized by heterogeneous population interactions and movement in space, have not been used to inform prevention strategies. the spatial heterogeneity of population interaction and movement make the choice of appropriate containment areas for effective vaccination strategies challenging. this research, therefore, aims to understand the impact of geo-social patterns identified from human movement and interaction data on effective vaccination in the urbanized area of portland, oregon. we use a location-based network framework in which nodes represent locations and links represent population flows among locations. the framework explicitly represents spatial dynamics of disease transmission from one location to another as well as design control strategies according to population flows among locations. based on the location-based network, we simulate influenza transmission dynamics and evaluate the efficacy of different vaccination strategies according to the identified geo-social interaction patterns. our research questions follow: ( ) given geo-social interaction patterns, how does population distribution and movement impact the effectiveness of influenza control efforts? ( ) how does the size of the containment area impact the control effectiveness considering human geo-social interaction patterns? the remainder of this article is organized into the following sections. the second section introduces related work in agent-based epidemic models and prevention strategies. the third section introduces location-based network model and agent-based epidemic simulation model in the urbanized area of portland. the one section that follow present and discuss the simulation results. the last section concludes the article and discusses its implications. infectious diseases are transmitted from one individual to another through physical contacts. in many agent-based epidemic models, the concepts of networks are used to represent contacts among individuals (bian ; bian & liebner ) . each individual can have attributes, such as infection status and spatial locations, whereas each link can also have a set of attributes, such as the duration of the contact and the infection rate. based on human interaction networks, epidemiologists can use agent-based epidemic models to simulate disease transmission and design different control scenarios. recently, agent-based models for disease transmission at different spatial scales (e.g., city, nation) have been used to study spatio-temporal patterns of disease spread (bian et al. ; ferguson et al. ; ferguson et al. ; halloran et al. ; mao & bain ) . these models predict that interactions at homes and workplaces could cause local transmission followed by a long distance transmission to an areawide epidemic (eubank et al. ) . local transmission indicates that early disease transmission is concentrated around the infection sources. later, population movement and distribution determine the spatiotemporal spread of infection. prevention strategies for infectious disease can be classified into three broad categories: antiviral, vaccine, and non-pharmaceutical measures (ferguson et al. ) . antiviral measures require rapid identification of early infections for treatment in order to reduce their infectiousness. vaccination seeks to protect people from being infected by other infections via physical contacts. non-pharmaceutical measures (e.g., case isolation, household quarantine, school or workplace closure, restrictions on travel) aim to locally contain the spread of disease (ferguson et al. ; ferguson et al. ) . previous research has shown the effectiveness of the above prevention measures in reducing disease transmission with agent-based modeling (ferguson et al. ; ferguson et al. ; halloran et al. ), but those agent-based modelings are individual-centric without capturing explicitly spatial dynamics of the disease transmission for designing control strategy purpose. spatial locations, one attribute of each agent, are treated as spatial stamps of the transmission process in the individual-centric agent-based modeling (zhong & bian ) . such representations suffer from a number of drawbacks regarding transmission and control design. first, the importance of locations in understanding disease transmission and control has been missing. for example, antiviral "ring chemoprophylaxis" strategies aim to geographically targeted containment via applying a certain distance threshold (e.g., km) by means of prophylaxis to attempt to prevent spread to unaffected regions (lee et al. ) . in fact, infectious disease transmission is the mixing patterns of geographical mobility and social interactions that are much more complicated than a simple distance threshold. second, the design of control strategies based on individualbased networks (e.g., vaccinate the individual with a large number of contacts) is challenging because it is infeasible to keep track of all social contacts of infections (cohen, havlin, & ben-avraham ; gómez-gardenes, echenique, & moreno ; holme ), but the information to estimate population flows among locations based on intra-and inter-community travelers is widely available in the existing census data and travel survey reports (mao & bian ) . thus, location-based human interaction network models are required to easily represent explicitly spatial dynamics of the disease transmission and design control strategies to target certain critical locations first rather than prioritizing individuals. based on a location-based human interaction network model, luo ( ) proposed a new framework in terms of an effective disease control strategy that consists of identifying geo-social interaction patterns first, following by designing effective control measures according to those patterns, and ending with control measure evaluation. the work also showed that vaccination strategies considering the geo-social interaction patterns can reduce disease transmission within a primary school environment because they can prevent disease from spreading to other classes. though the above research has demonstrated the importance of locations considering human geo-social interaction patterns in designing effective control scenarios, little research has been conducted to evaluate the effectiveness of such approach in a larger scale (e.g., city). thus, this research develops an integrated two-layer location-based social network approach to investigate the impact of human geo-social interaction patterns on effective vaccination design in an urban environment. we use a previously built and parameterized network from a synthetic population for a normal day in the city of portland, oregon, usa (eubank et al. ) to construct the integrated two-layer locationbased social network framework. one layer is a location-based network for the vaccination scenario design and the other layer is an individualbased social network to simulate infectious disease transmission and control to evaluate the effectiveness of those scenarios. each is detailed next. the synthetic population data includes over . million individuals and more than . million locations. each synthetic individual has their demographic attributes such as income range and age, whereas each synthetic location has utm coordinates. based on the individual and location information, the simulated daily activity data also includes the purpose of the activities such as work, school, and shopping, in which the starting time and duration of activity are stored in seconds (fig. ) . the data also include the information representing dynamic personperson contact networks including contact hours, contact type, and location information. the total number of person-person contacts is approximately equal to millions. the simulated data are configured with social surveys, transportation simulations, and census data in the portland city. the data set is available at http://ndssl.vbi.vt.edu/ synthetic-data/. human interactions and population mobility show spatial and temporal co-occurrence that connect discrete events over the same or different locations to facilitate disease transmission (zhong & bian ) . spatial and temporal co-occurrence is the basis of the construction of location-based human interaction network, in which nodes represent locations and edges represent population flows between locations. the total number of travel activities by individuals between two locations (population flows) is recorded as the weight of the edge (fig. ).the total population flows among locations are more than millions. a spatially explicit agent-based epidemic model is built on the simulated daily activities of person-person contact network in the city of portland, oregon, usa (eubank et al. ) (fig. ) . the influenza transmission is simulated over the contact network with each individual as one agent changing their infection states over time. following the natural history of the influenza, each individual can take one of four statuses: susceptible, exposed, infectious, or recovered (seir) at a given time (anderson & may ) . all individuals in our model are initially considered as susceptible to influenza. after contact with an infection, susceptible individuals have a probability of infection. those newly infected change their statuses from susceptible to exposed. exposed individuals may progressively develop their infectious statuses, which further enable them to transmit the influenza viruses to susceptible contacts. finally, infectious individuals recover from influenza. first, the exposed and infectious periods are from the established literature (table ) . second, the infection probability is calculated based on the r (the basic reproductive number). r is defined as the average number of secondary cases produced by a single infection over the course of its infectious period in a completely susceptible population (diekmann, heesterbeek, & metz ) . the transmission probability is calculated in order to generate consistent values of r (the basic reproductive number) corresponding to the observed r of pandemic influenza ( . - . ) in previous studies (ferguson et al. ; mills, robins, & lipsitch ) . we use a monte-carlo method to randomize the infection probability of individuals to derive r . all parameters to describe the natural history of the influenza are in table . we implement three vaccination strategies: random-based, degreebased, and betweenness-based vaccination strategies over the influenza diffusion in the city of portland, oregon, usa. the three strategies are the most representative ones to compare the effectiveness of different vaccination scenarios in the established literature (mao & bian ; . the basic idea of vaccination strategies is first to rank the importance of individuals according to their network measures (e.g., degree, betweenness) and then target the individuals from the highest network measures to lowest. the randombased strategy treats all of individuals the same and randomly identifies a certain number of individuals for vaccination. the degree-based strategy prioritizes individuals who have the most contacts within the network for vaccination. the betweenness-based strategy ranks the importance of individuals according to their betweenness centrality that can capture the extent to which a particular node lies on those shortest paths that pass through the nodes (freeman ) . both degree-based and betweenness-based vaccination strategies have shown much more effective than random-based strategy. for example, betweenness-based vaccination strategy tend to prevent disease outbreak from one community to another via targeting individuals on the shortest paths, so it becomes more effective with stronger community structures methodology framework consists of the integrated two-layer location-based social network: location-based network to design control strategy considering geo-social interaction patterns and individual-based social network to evaluate effectiveness of the above control strategy with the implementation of agent-based epidemic model. the framework is implemented in a high performance computing environment. scale-free network (albert, jeong, & barabási ) . though identifying all targeted individuals with high degree or high betweenness may not be practical, information needed to identify these individuals is becoming available (e.g., gps, social media) . using the location-based human interaction network, the vaccination strategy is designed according to population flows among locations with infections and their neighboring locations. neighboring locations that have direct connections with infected locations are most likely to be infected, followed by locations that have indirect connections with those infected locations. thus, the vaccination strategy will target those direct neighboring locations first, followed by targeting indirect connected locations. in practice, it is much easier to target a small population size with direct neighboring locations instead of a large population size with indirect connected locations. thus, this study explores the impact of the size of containment areas on vaccination efficacy in order to provide evidence in public health practice. the strategy is implemented at the beginning of the influenza epidemic. the simulation will stop when there are no new infections for six consecutive days (length of exposed period + length of infectious period). we randomly picked one infectious individual as the infection source in the study area on the first day of the simulation. based on the strategy rationale, we considered the locations where the first infection visited as the original case cluster. the original case cluster and all of the rest locations which have the population flows to the original case clusters were used to determine the first level local containment scale. the second level geo-social local containment scale was determined by all of the locations in the first level local containment area and the locations which have the population flows to the locations in the first level local containment area, so were the third, the forth, and the fifth level local geo-social containment scales. though the location network was dynamic over time in a day, the strategy design was based on the total population flows among different locations within one day for a practice purpose. it is more practical to design vaccination strategy at a daily basis instead of at an hourly basis. in order to understand the impact of the spatially heterogeneous population distribution and mobility on the design of vaccination strategies, we randomly pick the first infection times with varying total number of individuals in the first level local containment area (i.e., , , and ). the larger number of individuals within the first level local containment scale is caused by the high local population density which potentially leads to more complicated population interaction and mobility to larger geographical regions. given that the vaccination results are sensitive to the number of available vaccines, we simulate results according to an increasing number of vaccines from , to , with an increment of , . there is one network, three different initial infection locations with times' randomness, three strategies (e.g., random-based, degree-based), four different local control regions (from the second to the fifth level local containment area), and twenty different possibilities for the number of available vaccines, yielding ( * * * * * ) combinations to simulate ( table ). the efficacy of vaccination strategies for each combination is estimated for , simulation runs, resulting in a total of , , epidemic simulation runs. because of the large number of individuals and locations in the study area and the massive number of simulations involved, the tasks in this research are extremely computation intensive. we tackle this issue by taking the advantage of a high performance platform of cpu clusters. parallel algorithms are developed to deal with the most computation intensive tasks: ) the calculation of the betweenness centrality and ) the simulation of influenza epidemics. for the former, it is widely understood that the definitions of betweenness centrality in very large networks have inherently high computational complexity. the parallel algorithm is adapted from the brande's algorithm (brandes ) , and the actual running time is about hours using computing nodes, each with cores and gb of ram memory. for the latter, the parallel algorithm is developed following the seir rules as discussed earlier, and the running time ranges from to hours for a given first infection and vaccination strategy using the same computing nodes. the parallel design does not only reduce the running time but also improve the scalability of this methodology framework, which are essential for analyzing extremely large network data. we use two metrics to measure the effectiveness of vaccination strategies in the city of portland: the number of infections and the success rate of local containment. for the first metric, a better vaccination strategy is expected to generate a lower number of infected cases. for the second metric, we measure how well we can contain the outbreak at the source. if the number of infections outside of the containment areas identified by geo-social interaction patterns is zero, we consider the local containment successful. otherwise we consider the local containment a failure. fig. show the containment effectiveness of three typical vaccination strategies in which the first initial infections occurring within the first level local containment scale with approximately , individuals in the city of portland according to the two metrics: the number of infections and the success rate of local containment. fig. illustrates that an increasing number of vaccines can produce a decreasing number of infections. the random-based vaccination strategy generates the largest number of infections, followed by the degreebased and betweenness-based vaccination strategies. the latter two vaccination strategies produce the similar control efficacy in which betweenness-based ones lead to a relatively faster decreasing number of infections. the explanation of such patterns is illustrated in the fig. . fig. shows that an increasing number of vaccines can produce a decreasing number of local containment failure rates. in other words, betweenness-based vaccination strategies can contain disease outbreak at the local geo-social scales more successfully than the other two vaccination strategies. one reason is that betweenness-based vaccination strategies target high risk individuals who lie on the bridge among different communities, so they can control epidemics more effective through preventing epidemic outbreak from the local geo-social containment areas to outside areas. from fig. (a-d) , there is an obvious trend that increasing geosocial scales of local containment areas result in an increasing number of infections. fig. suggests that increasing extents of local geo-social containment areas lead to difficulty in containing outbreaks at the local scale. for example, in order to successfully contain the disease locally larger than % with degree-based or betweenness-based approach, it cost , vaccines in fig. (a) , , vaccines in fig. (b) , , vaccines in fig. (c) and (d) . in addition to supporting the importance of reliable detection of early infections to contain outbreaks at the source, such patterns also illustrate that identifying an appropriate scale for the local containment is key for designing effective prevention strategies. fig. (c) and (d) show that when the local containment areas are larger than certain extent, there is no obvious difference in terms of containment efficacy. it is because that the number of available vaccines cannot effectively contain disease outbreak at the local scale in fig. (c) and (d). this study further explores the impact of spatially heterogeneous population distribution and mobility on the containment efficacy of local geo-social vaccination strategies. this section only displays the simulation results with the random-based vaccination strategies, because the other two strategies display the similar patterns. fig. and fig. show the results of the two metrics: the number of infections and the success rate of local containment, respectively. they both display consistent patterns: the initial infections occurring in the higher population density areas can lead to higher number of infections (fig. ) because they increase the local containment failure rates with the same number of vaccines (fig. ) . for example, the disease transmission caused by the first infection from locations with a medium population density can areas be confined locally at a very high percentage (i.e., %) with less than , infections using , vaccines ( fig. b and b ), whereas the same amount of vaccines can only successfully contain the disease outbreak from high population density locations less than percent with more than , infections. such patterns imply that we need more vaccine available in order to contain disease outbreak from critical locations with high human interaction density and large numbers of population flows from infected locations to others. thus, it is essential to place sensors in the hubs of the locations to allow highly efficient outbreak detection. fig. and show that increasing scales of local geo-social containment areas lead to decreasing efficacy of the same amount of vaccines. for example, the random-based control scenario with more than , vaccines can successfully contain the disease outbreak in a high population density area (fig. a) , but it generates less than , infections (fig. b) and more than , infections (fig. c and d) . from the both (c) and (d) of the fig. and , we can tell that there is no obvious difference in terms of containment efficacy when the local containment areas are larger than certain scales in both (c) and (d). one explanation is that a certain number of vaccines have its threshold of susceptible population pool. the number of local control failure increases after the susceptible population pool goes beyond the threshold with the increasing local geo-social containment areas. when the disease outbreak occurs outside of the containment areas in the most simulation runs, the number of total infections finally reach similar amounts. we also notice that it is unlikely to confine the disease outbreaks caused by the first infection from high population density areas, if the local geo-social containment areas are set too large (fig. c and d ). when the first infection is from low population density areas, the likelihood to contain disease outbreaks within a large local geosocial containment areas is still very high ( fig. c and d) . thus, we can reach the conclusion that an appropriate geo-social scale for the local containment and the population density where the first infection resides are two keys for prevention strategies to achieve their maximum control effectiveness. based on the comparison analysis above, broader scales of local geosocial containment areas lead to decreasing efficacy of the same amount of vaccines. we further examine and compare their spatial effectiveness through infection density maps (fig. ) . we choose degree-based vaccination strategies with the medium number of individuals (approximately equal to ) within the first level local containment scale. we pick the number of vaccines equal to , for the second level local containment scale (fig. a ) and the number of vaccines equal to , for the third, forth, and fifth level local containment scales (fig. b, c, and d) because they all have the local containment failure rates lower than % (fig. ) . fig. (a) induces an extremely low intensity of infections in the whole study area with a relatively higher intensity of infections in the central business district of the study area. it is caused by the densest residential population and business locations in the central business district. compared to the fig. (a), fig. (b) with a larger containment area greatly increases the infection intensities with an additional dense area: the city of vancouver on the north bank of the columbia river. vancouver is the largest suburb of portland, oregon with highly concentrated population density. the spatial effectiveness of fig. (c) and (d) lead to the wide spread of influenza over the study area with three of highest infection density areas. the similar spatial patterns and total infections in both fig. (c) and (d) indicate that a certain number of vaccinations have its upper limit of susceptible population pool. when the local containment scale is larger than the upper limit, there is no difference in terms of spatial effectiveness. the spatial effectiveness of fig. highlights the importance of early detection of infections with an appropriate local containment scale, which is capable of confining the wide spread of influenza over the study area. this research examined the impact of geo-social interaction patterns on effective vaccination strategies in the urbanized area of portland, oregon. to achieve this goal, we built a large-scale two-layer locationbased social network model including an innovative location-based network to design vaccination strategies and an individual-based spatially explicit disease model to evaluate the efficacy of the above vaccination strategies. we implemented the network model in parallel algorithms to take advantage of a high performance platform of cpu to tackle the challenge of extremely intensive computation with millions of nodes and edges. the simulation results suggested that geo-social interaction patterns can be used to design effective vaccination strategies to contain epidemic outbreaks at the source. increasing extents of local geo-social containment areas lead to decreasing control effectiveness because it becomes challenging to confine the spread of influenza within local containment areas. especially when the local containment scale is larger than the upper limit, there is little difference in terms of spatial effectiveness. vaccination designs should consider spatially heterogeneous population distribution and movement in an urban area. it is possible to confine disease outbreaks for early infections in the low population areas, even if local geo-social containment areas have been set too large. it is unlikely to confine disease outbreaks for early infections in the high population areas if local geo-social containment areas are set too large. this study highlighted the importance of identifying the population density for the early infections and an appropriate geo-social scale for local containment in order to achieve maximum control effectiveness with a limited number of vaccines. a successful vaccine strategy must meet a number of key criteria: ( ) early detection of the original case cluster, ( ) rapid delivery of treatment to targeted groups, and ( ) effective delivery of treatment to high risk individuals (eubank et al. ; ferguson et al. ) . the identified geo-social interaction patterns and their effectiveness in designing vaccination strategies provide valuable insights for helping meet the above challenges. they can help identify critical individuals, locations, and clusters of locations for disease control purposes. traditional prevention strategies, including ring vaccination and contact tracing, can only capture one aspect of infectious disease transmission (i.e., spatial, or social) and both of which have significant limitations. "contact tracing" strategies require the identification of people who may have had contact with infectious individuals (germann et al. ) , which is difficult to implement in real situations considering the limited resources and time before the disease outbreak. "ring vaccination" with a simple distance threshold (e.g., km) (lee et al. ) is easy to implement, but it may fail to capture the most likely and complicated disease transmission processes. many research have demonstrated that ring vaccination alone might not lead to containment of disease outbreak with a high r at the source (kucharski et al. ; wells et al. ) , but a combined intervention of contact tracing and ring vaccination might contribute increase the ability to contain outbreaks of emerging infectious disease threats (kretzschmar, van den hof, wallinga, & van wijngaarden ; longini jr et al. ; merler et al. ; diao and wang, ; . the proposed geo-social interaction patterns cannot only address the inherent challenges of contact tracing to identify contacts with infections, but also capture the mixed interactions of the social and spatial relationships among individuals that determine infectious disease transmission (luo & maceachren ) . in addition to taking advantages from both contact tracing and ring vaccination strategies, the proposed location-based network approach provides the potential solution to address the challenge that it is infeasible to identify individuals with high degree or high betweenness for the disease control purpose in critical and timely situations. the wide available data (e.g., sensor networks, twitter) can provide more reliable estimation on the critical locations than critical individuals with high degree or high betweenness. thus, public health workers should focus on those critical locations with their close neighbor locations first in a timely manner. the proposed location-based social network framework also suggests several directions for future research. first, the estimation of population flows among different locations is still based on the existing census data and travel survey reports. gao et al. ( ) found that large-scale social media data can provide reliable estimates of regional origin-destination trips on weekdays compared with the community survey data in greater los angeles area. it shows great promise to build location-based social networks with large-scale social media data to provide real-time estimation and prevention strategy design in a timely manner. second, the location-based social network framework is built on population interaction and movement patterns in a normal situation, but people may adjust their behaviors accordingly during an epidemic outbreak. thus, human preventive behaviors caused by inter-personal influence (mao & yang ) and mass media could be incorporated to refine the framework. third, this research focuses on the impact of vaccination strategies on geo-social interactions patterns, but has not evaluated such impact of other prevention strategies such as travel restrictions and case isolation. forth, this research also has implications for the control of other location-based spreading pheonmena, such as invasive species , diao & wang , and hiv transmission (luo et.al ) . to summarize, the proposed location-based social network framework can help geographically optimize the design of prevention strategies before epidemic outbreaks, especially for novel viruses with limited resources and time constraints. to understand advantages and disadvantages of the framework in a full picture, it is crucial to apply it for other regions with different population distributions and movements as well as epidemiological settings. the author declares that he has no competing interests. this material is supported in part from the nih/nichd r hd . error and attack tolerance of complex networks epidemiology, transmission dynamics and control of sars: the - epidemic infectious diseases of humans: dynamics and control spatial approaches to modeling dispersion of communicable diseases-a review a conceptual framework for an individual-based 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control strategy for ebola virus disease oseltamivir ring prophylaxis for containment of h n influenza outbreaks the epidemiology of severe acute respiratory syndrome in the hong kong epidemic: an analysis of all patients containing pandemic influenza at the source visual analytics of geo-social interaction patterns for epidemic control development of an agent-based model to investigate the impact of hiv self-testing programs on men who have sex with men in atlanta and seattle journal of spatial information science a dynamic network with individual mobility for designing vaccination strategies coupling infectious diseases, human preventive behavior, and networks-a conceptual framework for epidemic modeling agent-based simulation for a dual-diffusion process of influenza and human preventive behavior containing ebola at the source with ring vaccination contact network epidemiology: bond percolation applied to infectious disease prediction and control transmissibility of pandemic influenza immunization of complex networks dynamics and control of diseases in networks with community structure a high-resolution human contact network for infectious disease transmission are we ready for pandemic influenza? harnessing case isolation and ring vaccination to control ebola effects of immunization in small-world epidemics a location-centric network approach to analyzing epidemic dynamics support provided by the center for computational research at the university at buffalo. the authors appreciate shaohua wang's contributions to data processing at the early stage. the authors are grateful to the anonymous reviewers and editors, whose valuable comments and suggestions helped improve the quality of the paper. key: cord- -qk xb a authors: hanada, shigeo; pirzadeh, mina; carver, kyle y.; deng, jane c. title: respiratory viral infection-induced microbiome alterations and secondary bacterial pneumonia date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: qk xb a influenza and other respiratory viral infections are the most common type of acute respiratory infection. viral infections predispose patients to secondary bacterial infections, which often have a more severe clinical course. the mechanisms underlying post-viral bacterial infections are complex, and include multifactorial processes mediated by interactions between viruses, bacteria, and the host immune system. studies over the past years have demonstrated that unique microbial communities reside on the mucosal surfaces of the gastrointestinal tract and the respiratory tract, which have both direct and indirect effects on host defense against viral infections. in addition, antiviral immune responses induced by acute respiratory infections such as influenza are associated with changes in microbial composition and function (“dysbiosis”) in the respiratory and gastrointestinal tract, which in turn may alter subsequent immune function against secondary bacterial infection or alter the dynamics of inter-microbial interactions, thereby enhancing the proliferation of potentially pathogenic bacterial species. in this review, we summarize the literature on the interactions between host microbial communities and host defense, and how influenza, and other acute respiratory viral infections disrupt these interactions, thereby contributing to the pathogenesis of secondary bacterial infections. influenza and bacterial pneumonia are the leading cause of morbidity and mortality from infectious diseases worldwide. influenza and other respiratory viral infections predispose patients to secondary bacterial super-infections, which are frequently associated with a more severe clinical course. it is estimated that the so-called "spanish flu" pandemic of h n influenza a virus from to resulted in more than million deaths, with many caused by bacterial superinfection leading to secondary pneumonia ( ) ( ) ( ) ( ) ( ) ( ) ( ) . even in the antibiotic era, over half of patients with severe infections in the h n and h n pandemics had bacterial complications ( ) ( ) ( ) . bacterial co-infection was also detected in ∼ % of cases in the h n pandemic, with high mortality rates despite administration of appropriate antibiotics ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . thus, it is evident that a better understanding of the pathogenesis of secondary bacterial pneumonia following viral infections is needed in order to make therapeutic strides for this devastating complication. the mechanisms of post-viral bacterial infection are complex, comprising multifactorial processes mediated by interactions between viruses, bacteria, and the host immune system. the pathogenesis of super-infection has been attributed to direct mucosal/epithelial damage by influenza virus, increased bacterial colonization of the upper and lower respiratory tracts (urt and lrt, respectively), and dysregulation of immune responses, which all lead to increased susceptibility to secondary bacterial infections. however, emerging evidence suggests that our microbial communities residing on our mucosal surfaces likely shape the rigor of our immune responses and shape the ecological relationships between host and pathogens. over the past years, intense interest has focused on examining how the microbial communities which inhabit our bodies-which some consider to be a separate "organ system" given the sheer physical bulk, number of genes, and metabolic activities-govern the balance between health and susceptibility to diseases, including infections. this raises the possibility that disruptions in the normal microbial communities by an acute viral infection might contribute to the development of post-viral bacterial pneumonia. the recent development of culture-independent methods of microbial identification has enabled the study of microbial communities on mucosal surfaces of the human body, referred to as "microbiota." the microbial communities of mammalian hosts are diverse, comprised of bacterial, viruses, archaea, parasites, and fungi. the human microbiome project (hmp) and other similar large-scale sequencing projects worldwide have characterized the distinct microbial communities that have adapted to the unique environmental niches within our bodies, such as the gut, skin, airways, genitourinary tract, and oral cavity. the gut microbiome, in particular, has been shown to play an integral role in shaping the immune system starting early in life, with continued influence on priming the nature and robustness of immune responses throughout one's lifetime. the respiratory tract also harbors distinct communities of microbes, with multiple discrete ecological niches (e.g., nasal cavity, oropharynx, upper airways) that vary in terms of temperature, ph, oxygen tension, mucus production, and other factors. the effects of viral infections on both the gut and respiratory microbiome have recently undergone examination. surprisingly, influenza infection has been found to result in significant changes in the gut microbiome, despite the lack of detectable virions in the gi tract. by comparison, the effects of viral infection on the respiratory microbiome appear to be relatively modest, but detectable. while the effects of these alterations on risk of secondary bacterial pneumonia have not been studied, potential mechanisms by which these changes might modulate susceptibility to secondary bacterial infections include alterations in the nature and magnitude of the immune response in the host (microbiome on host effects) and facilitating growth of pathogens in the absence of normal commensals (inter-microbial effects). in this article, we review the current understanding of how alterations in the microbiome following viral infection might alter host immune responses and increase susceptibility to secondary bacterial infections. although the term "microbiome" encompasses all microbial communities, there is currently a paucity of studies on how the mycobiome (fungal microbiome) and the virome (viral microbiome) affect host defense against respiratory infections and vice-versa; thus, this review will focus on the bacterial microbiome literature. of the niches in the body, the gut microbial community has been the most intensively studied, with over , publications to date. while the virome and mycobiome (fungi) are also being analyzed, the bulk of the literature has focused on the bacterial component of the microbiome, and thus most of our understanding of the relation of the gut microbiome to host immunity and pathogenesis of chronic diseases comes largely from studies of the bacterial community. during health, the human gut bacterial community is diverse, with each individual harboring over trillion bacteria, comprised of over different species. the gastrointestinal microbiota is dominated by firmicutes (e.g., lactobacillus, bacillus, and clostridium) and bacteroidetes (e.g., bacteroides), with lower abundances of proteobacteria (e.g., escherichia) and actinobacteria (e.g., bifidobacterium) ( , ) . wild-living mice exhibit more diverse microbiomes, with significant abundance of proteobacteria as well as firmicutes and bacteroidetes ( ) . the gut microbiome, in addition to its metabolic functions in the host, plays an integral role in the development, instruction, and priming of the immune system. germ-free (gf) mice (which lack microbiota) have markedly underdeveloped gut-associated lymphoid tissues, decreased number and smaller-sized peyer's patches and mesenteric lymph nodes, and defects in antibody production, compared to specific pathogen free (spf) mice. not surprisingly, germ-free animals exhibit increased susceptibility to multiple types of infections, including viruses, bacteria, and parasites ( ) ( ) ( ) ( ) ( ) ( ) . however, compared to free-living mice or laboratory animals exposed to gut flora from wild mice, spf animals have a more limited microbial community and are also more susceptible to inflammatory diseases, with a reduced immune repertoire including deficits in memory responses ( , , ) . although an extensive discussion of the healthy gut microbiome and its impact on host immunity is beyond the scope of this review, we will highlight a few important aspects of how the intestinal bacterial community microbiome maintains a healthy host immune environment. first, bacterial metabolites generated by gut commensals contribute to the maintenance of intact epithelial integrity, regulatory t-cell development, and a relatively anti-inflammatory immune state. in particular, short-chain fatty acids (scfas) such as acetate, propionate, and butyrate are fermentation products of dietary fiber and carbohydrates by large intestinal bacteria ( ) . in addition to being a major energy source for intestinal epithelial cells, scfas promote the development of naive cd + t cells into regulatory t cells ( , ) , induce "tolerogenic" dendritic cells in the intestinal mucosa ( ), and limit autoimmity ( , ) . at the same time, microbial metabolites are integral for promoting immune responses in the gut against pathogens, including inducing secretion of il- ( ) and defensins ( , ) . thus, the products of microbiome metabolism are integral to the appropriate regulation of mucosal barrier integrity and immune homeostasis. in addition, specific members of the bacterial community have been shown to foster the proper maturation and development of the immune system. while this is still an area undergoing intense investigation, one notable example is the discovery that segmented filamentous bacteria are critical promoters of intestinal mucosal iga production ( , ) and th cell induction ( , ) . dysbiosis, or an imbalance in the normal composition of the microbiome, is associated with a variety of chronic diseases, many of which are characterized by chronic inflammation or abnormal metabolism, including inflammatory bowel disease, cardiovascular disease, and diabetes. thus, fostering appropriate levels of diversity and composition of the gut microbial community is critical for promoting health and immune homeostasis. during health, the composition of the microbiome is governed by a number of selective pressures unique to each anatomic niche, including temperature, nutrient availability, ph, oxygen tension, and the local immune environment. shortterm perturbations in the gut microenvironment caused by illness, antibiotic usage, or dietary changes (e.g., starvation) can alter the gut microbiome and subsequently lead to transient alterations in immune responses. thus, investigating whether influenza and other respiratory viruses alter the gastrointestinal microbiome could have mechanistic implications for viralmediated suppression of antibacterial immune responses. although the composition of the gastrointestinal microbiome is largely influenced by dietary patterns, respiratory viral infections could also contribute, along with other stress inducers such as broad-spectrum antibiotics exposure and chronic inflammation. using animal models of pulmonary infections by influenza and respiratory syncytial virus (rsv), multiple groups have shown that the gut microbiome is clearly impacted by respiratory viral infections, despite the lack of detectable respiratory virus in the gut ( ) ( ) ( ) ( ) ( ) . in a murine model of influenza infection, the investigators found that although the total numbers of bacteria in the gut did not decrease, there was a reduction in the quantities of segmented filamentous bacteria (sfb) and lactobacillus/lactococcus, accompanied by increases in enterobacteriaceae. interestingly, although sfb have previously been shown to induce th cells ( , ) , flu-infected mice had increased il- a levels and numbers of th cells in the small intestine and colon, which appeared to contribute to intestinal injury ( ) . in this study, antibiotic treatment prior to influenza infection ameliorated the degree of intestinal injury, but not lung injury, suggesting that gut dysbiosis contributed to local but not systemic inflammation. other groups have similarly reported increased proteobacteria (the phylum of which enterobacteriaceae are members) ( , ) , decreased firmicutes (which include sfb, lactobacillus and lactococcus species), and increased bacteroidetes ( ) following infection by flu or rsvs but not after administration of live attenuated influenza vaccine (laiv), indicating that live viral infection is required for these changes ( ) . the increase in proteobacteria appears to be mediated by type i interferons (ifns) ( ) , which not only depleted anaerobic bacteria but also increased susceptibility to secondary salmonella colitis. however, caloric restriction also figure | shifts in the mouse gut microbiome in the setting of influenza infection. during an acute respiratory viral infection, changes in the bacterial composition of the gut microbiome can be observed despite the absence of detectable virus in the gastrointestinal compartment. this suggests that systemic immune signals, physiologic changes (e.g., weight loss), and other still unknown factors are disrupting the normal ecology of the gut, thereby leading to dysbiosis. however, the majority of these studies have been conducted in laboratory animals housed under spf conditions. it remains to be determined whether human patients and mammalian hosts with more diverse baseline gut microbiota (i.e., mice in the wild), exhibit similar qualitative or quantitative changes. results in increased relative abundance of proteobacteria and increased bacteroidetes to firmicutes ratio, raising the possibility that decreased oral intake during influenza may contribute to changes in the microbiome ( , , , ) . it has also been shown that influenza infection alters intestinal microbiota composition through type ii ifn produced by lung-derived t cells recruited to the intestine ( ) . thus, changes in the gut microbiome appear to result not from direct viral effects but from systemic inflammatory signals that travel from the lung and trigger local inflammatory responses in the gut (figure ). interactions between respiratory tract infections and the gut microbiome are bidirectional. while respiratory viral infections can change the gut microbiome, the gut microbiome also shapes the adaptive immune responses against respiratory pathogens. mice pretreated with an antibiotic cocktail showed increased morbidity and mortality during influenza infection ( , ) . the severity of infection was associated with reductions in dendritic cell migration rate and the number of local t cells. mice given a week oral course of broad-spectrum antibiotics before respiratory viral infection mounted an attenuated anti-pr antibody response, were incapable of inducing cd + t cell-mediated ifn-γ response to pr antigen, and had fewer influenza-specific cd + t cells ( , ) . these mice also had higher viral titers in their lungs ( ) . germ-free mice and antibiotic-treated mice also exhibit impaired antibody responses to seasonal influenza vaccination, which was restored by oral administration of flagellated e. coli, demonstrating a dependence on tlr -mediated sensing of the host microbiota ( ) . the gut microbiome is essential for priming innate immune responses against pulmonary infections as well. during viral infections, the degree of macrophage response to respiratory viruses depends on the presence of gut microbes. macrophages from animals treated with antibiotics exhibited defective responses to type i and ii ifns and impaired capacity to limit viral replication, suggesting that intestinal microbiota provide immune stimulation that establishes an "activation threshold" for innate antiviral immune responses ( ) . a comparison of c bl/ mice from the jackson laboratory (which lack sfb in the stool) and taconic biosciences (which are sfb positive) revealed that sfb-deficient animals have increased lung bacterial burdens and more severe pneumonia when challenged with methicillin-resistant staphylococcus aureus (mrsa) ( ) , which was associated with decreased il- -mediated responses in the lung. another study using broad-spectrum antibiotic treatment followed by intranasal administration of s. pneumoniae in mice demonstrated that microbiome depletion led to decreased survival, increased lung bacterial burden, and increased systemic dissemination of bacteria ( ) . antibiotic-pretreated animals displayed altered cytokine profiles in the lung compared to untreated controls following s. pneumoniae infection, including significantly decreased tnf-α levels at and h after infection. additionally, in the microbiota-depleted group, alveolar macrophages and blood neutrophils exhibited decreased phagocytic activity, and decreased inflammatory cytokine production following ex vivo stimulation by toll-like receptor (tlr) ligands such as lipoteichoic acid (lta) ( ) . these effects might be mediated in part by decreased nod sensing of meso-dap (diaminopimelic acid)-containing peptidoglycan found in gut microbiota, which previously was shown to be essential for priming innate immune responses to s. pneumoniae ( ) . thus, antibiotic-induced disruptions in the normal gut microbial community alter multiple aspects of normal host defense against acute respiratory pathogens (figure ). collectively, the studies above suggest that modulation of the gastrointestinal tract microbiome plays an important role in acute respiratory infections, but precisely how the microbiome should be manipulated to promote appropriate immune responses during acute respiratory infections is unclear. currently, clinical studies have shown that although probiotics do not influence the incidence of respiratory tract infection, they do reduce the severity of symptoms and duration of the illness ( , ) . pinpointing which members of the gut microbial community are essential for proper immune priming is challenging, but necessary for guiding further microbiome-based therapies. clostridium orbiscindens, a member of the human gut microbiome, has been found to produce desaminotyrosine (dat) from metabolism of flavonoids and amino acids. antibiotic-treated mice exhibited markedly decreased fecal and serum dat levels, which was associated with attenuated type i ifn responses to influenza infection and increased mortality ( ) . thus, identification of dat-producing microbiota might serve as a modality for priming type i ifn responses against viral infections. another group demonstrated that oral administration of lactobacillus plantarum enhanced the type i ifn response and lowered viral titers in the lungs in a murine model of influenza infection ( ) . other lactobacillus strains are known to enhance tnf-α and ifnγ production by nasal lymphocytes upon influenza infection ( ) . oral administration of a probiotic cocktail containing lactobacillus restored the immune response and enhanced the activation of signaling pathways associated with recognition of single-stranded rna virus ( ) . an alternative approach to administering probiotics is to alter the local metabolic environment to regulate immune responses. a recent report demonstrated that animals fed a high fiber diet had increased generation of scfas, leading to enhanced antiviral cd + t cell immune responses and attenuated neutrophil-mediated lung injury during influenza infection, resulting in improved survival ( ) . thus, one strategy for decreasing the incidence of post-viral bacterial infections is to limit the severity of the primary viral infection. however, activation of antiviral immune responses, including type i and type ii ifns, have been associated with increased susceptibility to secondary bacterial pneumonia ( , ) . thus, another strategy is to enhance immune responses against common bacterial causes of pneumonia. one group re-colonized antibiotic-treated or germ-free mice with groups of cultivatable commensal bacteria, and found that administration of lactobacillus reuteri, enterococcus faecalis, lactobacillus crispatus, and clostridium orbiscindens, which are strong stimulators of nod (i.e., cytosolic receptor for muramyl dipeptide, which is found in cell walls of certain bacteria), are able to protect against bacterial pneumonia by enhancing gm-csf production ( ) . whether viral-induced changes in the gut microbiome is associated with immune defects that promote secondary bacterial pneumonia, or whether the impaired antibacterial defenses observed in virally-infected hosts can be restored by augmenting certain components of the microbiome are important areas to be investigated. the microbiome of the respiratory tract has also been investigated in the context of viral infections. its role in the development of secondary bacterial pneumonia following influenza and other acute respiratory viral infections is unclear. the respiratory tract is the main site of continuous contact with frontiers in immunology | www.frontiersin.org figure | effects of antibiotic pre-treatment on immune responses to influenza, streptococcus pneumoniae, and lipoteichoic acid (lta). the effects of the gut microbiome on immune responses to respiratory pathogens have been investigated by administration of oral antibiotics to generate alterations in the gut flora, followed by acute infection, and analyzing host immune responses compared to non-antibiotic-pretreated animals. multiple aspects of innate and adaptive immune responses are altered in antibiotic treated animals, including decreased antibody production, decreased phagocytic activity, and decreased inflammatory cytokine production by innate immune cells (e.g., alveolar and peritoneal macrophages) following ex vivo stimulation with tlr ligands. exogenous microbes. as is the case with the gut, immunity at the mucosal interface of the respiratory tract is a constant balance of tolerance of commensal and non-invasive microbes and immune activation against pathogens. the urt and lrt have similar microbial community compositions, although microbe densities are much higher in the former in healthy hosts. several factors are known to influence airway microbiome composition including infection history, age, genetics, and structural lung disease. the urt is an interconnected system consisting of the anterior nares, nasal cavity, nasopharynx, sinuses, eustachian tube, middle ear cavity, oral cavity, oropharynx, and larynx, each of which serve as distinct niches with their own microbial communities. in healthy adults, bacteria present in the nasal cavity are typically those associated with skin, predominantly members of the actinobacteria (e.g., corynebacterium spp., propionibacterium spp.), followed by firmicutes (e.g., staphylococcus spp.), and proteobacteria ( ) ( ) ( ) . the oropharynx contains members of firmicutes, proteobacteria, and bacteroidetes, including streptococcus, neisseria, haemophilus, and lachnospira spp. ( , , ) . skin and oral cavity lineages are represented in the nasopharynxe.g., streptococcus, staphylococcus, corynebacterium, and prevotella ( , , ) . a limited number of pathogens including streptococcus pneumoniae, neisseria meningitides, and haemophilus influenzae are commensal bacteria of the urt. in healthy individuals, the microbial community richness (i.e., the total number of bacterial taxa) is lower in the lrt than that in the urt ( , ( ) ( ) ( ) . contrary to dogma that normal healthy lungs are a sterile environment, a distinct, and somewhat dynamic lung microbiome can be identified using sequencing technology, with microaspiration serving as the primary route of microbial immigration from the urt to the lrt ( , ) . the major phyla in healthy lungs are bacteroidetes and firmicutes, which mainly include prevotella, veillonella, and streptococcus ( ) ( ) ( ) . individuals with chronic airway diseases (e.g., cystic fibrosis, copd) have increased bacterial populations in the lungs ( ) and differences in the relative abundance of certain species ( ) . impaired airway clearance due to intrinsic or extrinsic factors leads to the proliferation of bacterial species that can exploit this growth opportunity ( ) . how respiratory viral infection affects the diversity of microbial communities and whether viral-induced dysbiosis influences immune functions is being examined. nonetheless, bacterial colonization of the urt is generally considered as the first step in the development of invasive bacterial infections ( , ) , including secondary bacterial infections following respiratory viral infection. bacterial abundance, species diversity, and factors that shape the immune response to subsequent infections are discussed in greater detail below. respiratory viruses enter the human body through the urt and are the most common type of acute infections of the respiratory tract. one possible mechanism by which influenza and other viral infections might predispose infected hosts to secondary bacterial pneumonia is by altering the microbial composition of the upper respiratory tract, fostering enhanced growth of pathogens, and facilitating the subsequent entry of large bacterial loads into the lrt ( ) . this section will examine recent literature on how acute respiratory viral infections have changed the urt microbiome. given the effects of viruses on enhancing bacterial adherence to the epithelium ( ) ( ) ( ) , it is perhaps not surprising that multiple studies of human subjects as well as in animal models have shown that viral infections are associated with increased colonization by potentially pathogenic bacteria (known as "pathobionts"). a comparative analysis using qpcr to detect specific bacteria in adult patients with or without influenza a infection showed that staphylococcus aureus, s. pneumoniae, and h. influenzae were present in , , and % of infected patients, respectively as compared to , , and % of uninfected patients ( ) . in experimental in vitro models, viral infections increase the colonization rates of various bacteria in the urt ( - ), including s. pneumoniae and h. influenzae ( ) ( ) ( ) . in children, influenza is associated with a -fold increase in nasopharyngeal titer of s. pneumoniae ( ) . animal models have similarly confirmed that viral infection, particularly influenza, increases bacterial colonization rates in the urt, enhancing the risk of secondary bacterial infections ( ) ( ) ( ) . higher pneumococcal colonization density has been linked to respiratory virus coinfection and invasive pneumococcal pneumonia, after adjusting for age and sex ( ) . another case-control study comparing nasopharyngeal bacteria with and without pneumonia also found an association between nasopharyngeal load of s. pneumoniaebut not of h. influenza and m. catarrhalis-and viral coinfection and pneumonia ( ) . in addition, viral infections potentially may enhance transmission of bacteria. in a study of mice colonized with s. pneumoniae and then infected with influenza a virus days after, s. pneumoniae transmission occurred only when all mice were infected with influenza and was blocked by an influenza-neutralizing antibody ( ) . however, while specific bacteria might gain a competitive advantage during viral infections, this does not universally translate to all bacterial taxa. a recent study of subjects with and without respiratory viral infections demonstrated lower overall bacterial reads from nasopharyngeal samples in virally-infected subjects compared with uninfected controls ( ) . the relationship between acute viral infections and bacterial colonization appears to be bidirectional. bacterial carriage or their ligands can increase or decrease viral infectivity rate, thereby positively or negatively influencing the subsequent host immune response to viral infection. viral replication in the respiratory tract can be enhanced by exposure to s. pneumoniae ( ) . patients harboring s. pneumoniae are more likely to experience subsequent acute respiratory illness episodes than those without colonization ( ) . in addition, bacteria present in the airways can modulate host responses against viral infection. the presence of a nasopharyngeal commensal protected mice against rsv-induced airway hyperresponsiveness. rsv-infected mice who underwent antibiotic-mediated depletion of streptococcus viridans in the nasopharynx exhibited increases in number of inflammatory lymphocytes and airway hyperresponsiveness, and decreases in regulatory t cell number and transforming growth factor-β production ( ) . others have shown that colonization of the urt with s. aureus drastically reduced influenza-induced acute lung injury and mortality in mice by recruiting a c-c chemokine receptor type + cluster of differentiation (cd) b + monocyte subset to the lungs and inducing an m macrophage phenotype ( ) . with the availability of next-generation s rrna sequencing, microbiome-based studies have attempted to discern global patterns of change in the bacterial community of each anatomic niche during viral infections, such as changes in diversity. diversity can be assessed using a variety of indices, such as total number of unique species of the microbiome (i.e., richness) or other measures that account for both richness and the evenness of relative abundance of the members of the community (e.g., shannon index). results from microbiome analyses have not demonstrated consistent changes in diversity when comparing virally infected subjects with healthy controls. this is not surprising given the variability of the subjects sampled, differences in type and severity of viral infections, type and timing of sample collection, and analysis methodology. in some studies, increased bacterial diversity appeared to be associated with influenza severity. a french study of children admitted to the hospital with influenza revealed increased diversity of the nasopharyngeal microflora with increased influenza severity ( ) . children with severe influenza showed decreased relative abundance of s. aureus and increased abundance of prevotella, streptobacillus, porphyromonas, granulicatella, veillonella, fusobacterium, and haemophilus. a recent chinese study in patients with h n avian influenza demonstrated significantly increased diversity in the oropharyngeal microbiome of h n -infected patients compared to healthy controls, particularly h n patients with secondary bacterial pneumonia ( ) . conversely, a french study of nasopharyngeal samples and a south korean study of oropharyngeal samples from patients with acute respiratory viral infections both displayed decreases in diversity indices during viral infections compared to healthy controls ( , ) . both studies included subjects ranging from infants to adults > years of age, limiting conclusions about age-related effects. longitudinal studies conducted in healthy volunteers who underwent experimental self-innoculation with rhinovirus also failed to demonstrate significant changes in diversity of the urt microbiome, while administration of laiv vaccine to healthy adults led to increases in diversity measures following viral challenge ( , ) . thus, unlike other diseases where decreased diversity is considered deleterious to the host, the effects of viral infections on diversity per se are variable and not presently considered a good indicator of risk for complications, including secondary bacterial pneumonias. microbiome sequencing studies also enable investigators to identify changes in abundance among multiple bacterial taxa simultaneously, beyond just what can be cultured individually. this allows investigators to determine what groups of bacteria are changing in unison during viral infection and which are existing in competition with one another. this information may have implications for the development of probiotic therapies (as discussed below). a recent metagenomics-based study in france reported enrichment of s. aureus, s. pneumoniae, h. influenzae, moraxella catarrhalis and klebsiella pneumoniae in nasopharyngeal samples of subjects with confirmed respiratory viral infections compared to healthy controls ( ) . an examination of the oropharyngeal microbiome of pneumonia patients with and without influenza a h n pandemic viral infection showed that firmicutes (which include staphylococcus and streptococcus spp.) and proteobacteria (mainly pseudomonas amygdali, p. fluorescens, pseudomonas sp. uk , acinetobacter baumanii and a. junii)-were significantly enriched in patients with influenza ( ) . another study of patients with pandemic h n influenza infection revealed that the predominant phyla of the upper respiratory tract (nasal and nasopharyngeal samples) in patients harboring pandemic h n were actinobacteria, firmicutes, and proteobacteria although normal controls were not included; however, the authors suggested that flu is associated with an expansion of proteobacteria ( ) which is generally less abundant in healthy hosts. these findings are supported by another group who found that moraxella and enterobacter spp. (which are classified as proteobacteria) were the most highly represented bacteria in nasopharyngeal samples obtained from patients with pandemic h n influenza ( ) . however, these studies demonstrated that there was considerable inter-subject variability, highlighting the need for longitudinal studies to decipher changes following viral infection. investigators have also sought to determine whether specific viruses are consistently linked to enrichment of certain bacterial taxa. in the nasopharyngeal compartment of aboriginal and non-aboriginal children in australia, positive associations were detected between hrv and s. pneumoniae, h. influenza, and moraxella catarrhalis carriage as well as between adenovirus and m. catarrhalis ( ) . another study examining the presence of respiratory viruses by pcr panel and prevalence of bacterial carriage in the nasopharynx of children found a strong positive association between s. aureus colonization and influenza virus ( ). moreover, s. pneumoniae colonization was positively associated with the presence of hrv and enteroviruses; h. influenzae was positively associated with hrv and rsv; and m. catarrhalis colonization was positively associated with coronaviruses and adenoviruses. a s rrna sequencing-based study conducted in infants with acute rsv or hrv respiratory infections reported that infants with rsv had significantly higher abundance of staphylococcus spp. compared to hrv-infected infants ( ) . an analysis of the urt bacterial content of healthy asymptomatic individuals and patients with influenza virus, parainfluenza, hrv, rsv, coronavirus, adenovirus, or metapneumovirus by culture-independent pyrosequencing revealed six distinct bacterial profiles-i.e., streptococcus + prevotella + veillonella, streptococcus + haemophilus + neisseria, streptococcus, moraxella, haemophilus, and klebsiella. these profiles, however, were not associated with virus type but were linked to the age of subjects ( ) . given that many human studies are cross-sectional in nature, it remains unclear whether post-viral bacterial pneumonias might be the result of viral infections enhancing bacterial colonization or acquisition, colonizing bacteria influencing host susceptibility to respiratory viral infections, or a combination of both. another complicating factor particularly in cross-sectional studies examining the microbiome during viral infections is that the groups are not well-controlled and the sample numbers are relatively small considering the number of variables that could affect the respiratory tract microbiome-such as age, gender, oral hygiene and nose-picking habits, healthcare-based employment status, smoking status, medication use, exposure to small children, etc. the underlying type of viral infection, sampling timepoint after onset of infection, severity of infection, and concomittant antimicrobial usage are other confounding factors. this may underlie the highly variable and sometimes discrepant observations from microbiome studies in patients with viral infections. there have been few clinical studies comparing baseline pre-and post-infection microbiomes in otherwise healthy individuals with acute viral infections due to the difficulty of sampling before infection. however, the relatively few studies available provide insights into the dynamicity and stability of bacteria colonization patterns over time, and whether and how perturbations brought on by acute viral infections alter these patterns. in healthy children, the major phyla among nasopharyngeal microbiotas are proteobacteria, firmicutes, bacteroidetes, actinobacteria, and fusobacteria, with moraxella, haemophilus, streptococcus, flavobacteria, dolosigranulum, corynebacterium, and neisseria as predominant genera. changes in nasopharyngeal microbiome diversity were observed across seasons, with a predominance of proteobacteria and fusobacteria in fall-winter and bacteroidetes and firmicutes in spring; these differences were independent of recent antibiotics and viral co-infection ( ) . however, another analysis of two nasopharyngeal washes collected . - . months apart from children and adolescents with asthma showed no significant differences in nasopharyngeal microbiome diversity across seasons, although mean relative abundances of haemophilus, moraxella, staphylococcus, and corynebacterium varied significantly between summer and fall samples and between age groups. moreover, in . % of patients, operational taxonomic units (otus) in patients varied significantly between time points ( ) . an investigation of the frequency and seasonal variation in bacterial and viral load in asymptomatic healthcare professionals during the winter and summer months showed that of the subjects tested during the winter, were colonized with at least one bacterial species and tested positive for at least one virus. the most frequently detected pathogens were methicillinresistant staphylococcus aureus (mrsa), m. catarrhalis, and coronavirus. in contrast, of the subjects tested during the summer, harbored at least one bacterium (mainly mrsa and k. pneumoniae) and four tested positive for one virus ( ) . several larger scale surveillance studies of mainly pediatric populations have examined the natural temporal patterns in bacterial colonization during viral infections. one clinical investigation assessed the presence and density of s. pneumoniae, h. influenzae, and m. catarrhalis in the nasopharynx of children during urt infection and in the healthy state, and reported that the proportion of children colonized with these bacteria was higher during infection than during asymptomatic surveillance visits. mean density of all bacterial species was significantly higher at each visit when a virus was detected. interestingly, the percentage of colonized children and bacterial density were also higher at asymptomatic visits in which virus was detected than at those in which virus was not detected ( ) . another study of families with small children using longitudinal nasal swab sampling demonstrated that rhinovirus infection was associated with increased acquisition of s. pneumoniae from the community as well as increased transmission of s. pneumoniae within the family ( ) . other groups have examined the effects of experimental innoculation of hrv into the urt (nares) (figure ) . these studies reported no significant changes in total read counts or of the main phyla (e.g., actinobacteria, firmicutes, and proteobacteria) over time in nasopharyngeal samples ( ) or throat swabs ( ) . in the oropharyngeal compartment, rhinovirus infection was associated with a strong trend toward transient increases in the relative abundances of h. parainfluenzae, neisseria subflava and a weak trend toward an increase in s. aureus ( ) . by days, abundance of these bacteria had returned to baseline. nasopharyngeal sampling showed completely opposite results, with decreased relative abundance of haemophilus and neisseria spp., but an increase in the normal nasal commensal, propionibacterium, in subjects following hrv infection ( ) . no differences in staphylococcus were observed. however, the number of subjects were small in both studies, limiting the power to detect changes over time. nasopharyngeal microbiota composition has been shown to be altered by influenza vaccination (figure ) . administration of live attenuated influenza vaccine (laiv), which is nasally instilled, to healthy children increased the nasal colonization density of s. pneumoniae in subjects who harbored this bacterium at the time of vaccination, and transiently increased rates of colonization by h. influenza ( ) . in healthy adult volunteers, it was demonstrated that intranasal laiv administration induced an increase in the diversity of the nasopharyngeal microbiome, figure | changes in the human upper respiratory tract microbiome following viral exposure. given that bacterial pneumonia frequently arises as a result of aspirated bacterial pathogens, a potential mechanism by which viral infections might increase the risk of secondary bacterial infections is through increased colonization of the upper respiratory tract by bacterial pathogens. in human subjects, live attenuated influenza vaccine (laiv) and human rhinovirus (hrv) have been shown to disrupt the local host bacterial community, with increased relative abundance of potential pathogens (or pathobionts), such as staphylococcal and neisseria species. the major changes in the upper respiratory tract microbiome are highlighted here. as well as relative abundances of staphylococcus and bacteroides ( ). these changes were not observed in subjects given saline nasal spray. in a mouse model, bacterial density in the nasopharynx after laiv administration was increased as much as , times compared to influenza-naive hosts, and the duration of carriage of s. pneumoniae or s. aureus was also increased to -fold ( ) . however, systemic vaccination can also alter the urt microbiome. a longitudinal study of healthy subjects found a significant association between the presence of lactobacillus helveticus, prevotella melaninogenica, streptococcus infantis, veillonella dispar, and bacteroides ovatus and influenzaspecific h and h iga antibody response ( ) . thus, it is remarkable that a relatively mild viral stimulus such as flu vaccine can lead to detectable changes in the urt microbiome. although the data are still preliminary, animal studies have suggested that antiviral immune activation contributes to changes in the urt microbiome and facilitate colonization by potential pathogens, such as s. aureus. in a mouse model of s. aureus nasal colonization, the absence of type i ifn receptor was associated with decreased persistence of bacteria ( ) . type iii ifn, which is also induced during influenza infections, led to changes in the nasal microbiome, including increased numbers of culturable bacteria. increased upper respiratory tract persistence of s. aureus as well as increased risk of s. aureus pneumonia was observed in flu-infected wildtype mice compared to mice lacking the type iii ifn receptor ( ) . currently, however, is it unclear to what extent viral-induced changes in the urt microbiome alter subsequent immune responses against secondary bacterial infections. compared to studies of the urt microbiome, studies of the lrt microbiome following viral infections are relatively scarce due to the difficulty of obtaining uncontaminated samples from the lung. samples of convenience, such as sputum, suffer from oral contamination, but bronchoscopic samples are invasive and expensive to obtain on a regular basis. moreover, it is unclear whether outside of patients with chronic lung disease (e.g., copd), the lung microbial burden is of sufficient magnitude to exert robust effects on immune responses and risk of secondary bacterial infection during viral infection. data from a mouse model of influenza infection seem to indicate that flu infection has only a modest effect on bacterial counts, diversity and composition of the lung microbiome ( ) . in subjects with chronic obstructive pulmonary disease (copd) after hrv infection but not in healthy individuals, there was an increase in bacterial burden and growth of bacteria present at baseline, particularly h. influenzae ( ) . the researchers observed that the growth of bacteria seemed to arise from the existing community. s. pneumoniae intranasally inoculated into mice pre-infected with influenza virus first colonized the nose, followed by the trachea and lungs several days later with purulent inflammation. however, this effect was not observed in uninfected animals. this suggests that pneumococcal infection may sequentially develop from the urt to the lrt in influenza virus-infected subjects ( ) . thus, it is possible that some individuals with influenza infection might develop changes in their lung microbiome as a result of changes in their urt microbial communities. respiratory viruses not only alter the bacterial community in the urt, but also promote bacterial colonization of the lrt by a variety of mechanisms that impair bacterial clearance. first, mucus production in the respiratory tract is increased to facilitate viral clearance during infections. however, excessive mucus production can lead to airway obstruction by impeding mucociliary clearance ( ) . second, viral infections can also reduce ciliary beat frequency and the number of ciliated cells, disrupt the coordinated movement of cilia, and impede the repair of respiratory epithelial cells, further leading to reduced mucociliary clearance ( , ) . third, respiratory viral infections impair innate immune responses against bacteria ( ) ( ) ( ) . innate immune cells including macrophages and neutrophils are recruited to the lung by cytokines and chemokines for phagocytosis and bactericidal activity. prior viral infections dysregulate both alveolar macrophages ( , ( ) ( ) ( ) ( ) ( ) ( ) and neutrophils ( , , ) , thereby inhibiting bactericidal activity. thus, with multiple aspects of pulmonary host defense impaired, it would not be entirely surprising if a subset of influenza infected patients developed secondary bacterial pneumonia as a result of being unable to clear aspirated pathobionts from the urt. in addition to enabling us to determine what is present during states of health, large-scale sequencing-based microbiome analyses have also revealed who is not present during disease. it has long been appreciated that mechanisms have evolved in bacteria that confer competitive advantages, permitting them to survive in an otherwise inhospitable host environment. however, interspecies competition also maintains homeostasis of the microbial community, either through their abilities to capture scare resources (e.g., iron), or targeted killing of other bacteria (e.g., bacteriocins), preventing one microbe from dominating the community. thus, it is possible that the immune response incited by acute viral infections, changes in the host epithelial surface caused by the virus, or the virus itself might lead to elimination of a host commensal that is responsible for keeping pathobionts in check. for example, s. epidermidis and propionibacterium acnes abundance in the nares has been shown to be negatively associated with s. aureus carriage ( ) . understanding these interactions may create new avenues for therapeutic interventions aimed at reducing colonization by pathogenic bacteria during influenza epidemics or pandemics. one group of commensals that has been examined for its role in inhibiting nasal carriage by s. aureus and s. pneumoniae is corynebacterium spp. an early study in japan reported on the effects of introducing a corynebacterium strain into the nares of healthy adult hospital workers who were persistent carriers of s. aureus, with successful eradication in % of subjects ( ). the mechanism appeared to be bacteriocin-independent. in comparison, s. epidermidis implantation did not have an effect. whether the s. epidermidis strain used expressed the serine protease esp, which inhibits biofilm formation by s. aureus and nasal colonization ( ) , is unknown. subsequent studies by another group reported that c. pseudodiphtheriticum inhibited s. aureus growth, whereas c. accolens and s. aureus appeared to support each other's growth ( ) . conversely, other investigators observed that corynebacterium spp. were enriched in children who were not nasally colonized with pneumococcus, and demonstrated that c. accolens inhibit s. pneumoniae growth in vitro by expressing a lipase that releases free fatty acids from skin surface triacylglycerols, which inhibit pneumococcal growth. thus, painstaking identification and mechanistic interrogation of interspecies competition between commensals might lead to novel insights as to how viral infections might confer competitive advantage to pathobionts, and how to exploit natural strategies employed by commensals to restore homeostasis to the host microbial niche. interestingly, a recent preclinical study using a murine model of rsv and s. pneumoniae superinfection employed nasal priming by a c. pseudodiphtheriticum strain to augment host defense against the viral infection, which enhanced clearance of secondary bacterial challenge and reduced lung injury measures ( ) . finally, direct effects of the infecting virus on bacteria that comprise the microbiome may facilitate the transition from pathobiont to pathogen. a metagenomic analysis showed that ph n -associated airway microbiotas were enriched in genes associated with cell motility, transcriptional regulation, metabolism, and response to chemotaxis compared to the same bacteria in non-infected patients ( ) . these data imply that influenza infection perturbs the respiratory microbiome, leading to the production of secondary metabolites including immune-modulating molecules. viruses have also been found to impair bacterial biofilm formation and disrupt existing biofilm ( ) ( ) ( ) ( ) . influenza has been shown to affect the s. pneumoniae transcriptome in terms of downregulating expression of genes associated with the colonizer state and upregulations of bacteriocins ( ) . thus, direct effects of viruses on bacterial transcriptional patterns might be a mechanism by which colonizing bacteria acquire invasive potential, thereby leading to bacterial superinfections. there are several areas that must be addressed by future respiratory microbiome research. first, it is necessary to standardize protocols used to analyze the respiratory microbiome, including sampling, processing, and bioinformatics methodologies. for example, sputum may be an appropriate material for investigations of respiratory diseases since it contains components of the lrt and can be obtained easily. however, more reliable information on the lrt requires invasive samples such as bal or protected specimen brush frontiers in immunology | www.frontiersin.org or bronchial/lung biopsies. second, most studies are limited to experiments conducted in animal models. even in human studies, most analyses have been performed in a small number of patients and have described bacterial communities in the urt. the role of microbial communities outside of the lungs including gut, sinus, and skin should be considered in the context of airway diseases. third, most studies on the microbiome have focused on the bacterial component, and have largely omitted fungi and viruses. the role of viruses-including the vast number of phages that infect bacteria-and fungi in respiratory diseases cannot be examined through s rrna gene analyses, and there are no studies describing the composition and role of the respiratory virome due to the difficulty of comprehensive analyses for viruses. fourth, it is not sufficient to study microbial communities based on species composition; a functional characterization through transcriptome and proteasome analyses is necessary to understand mechanistic role of microbiome on outcomes of infection. finally, mucosal microbiome manipulations by vaccines, antibiotics, and probiotics in the gastrointestinal and respiratory tract niches represent novel approaches for the prevention, treatment, and management of acute and chronic lung diseases. however, given that antibiotic therapy could affect commensal bacteria and hasten the emergence of drug-resistant bacteria, more research is needed on the long-term effects of this therapy. animal models should be developed to study the influence of the urt and lrt microbiomes on immune responses to respiratory viral infections; only then will it be possible to consider the clinical application of microbiome modulation strategies. respiratory viral infections can initiate a cascade of host immune responses that alter microbial growth conditions in the urt, lrt, and the gut (supplemental table ). activation of influenzainduced antiviral interferon pathways can lead to inadequate innate immune cell responses during host defense against secondary bacterial infections, resulting in the proliferation of potentially pathogenic bacterial species. concomitant changes in the gut microbiome caused by the initial viral infection may also alter immune cell priming against secondary bacterial challenge, although this has not been examined to date. although the picture is incomplete, recent microbiome literature provides additional insights into the pathogenesis of dysregulated immune responses following acute viral infections, that may promote the development of secondary bacterial pneumonias (figure ) . clarifying the differences and dynamics of respiratory microbiota in healthy subjects and chronic lung diseases during acute respiratory viral infections can elucidate pathogenesis of viralbacterial interactions and provide a basis for developing novel approaches for the prevention, treatment, or management of acute respiratory infection and exacerbation of chronic lung diseases. sh and jd co-wrote the manuscript. sh, jd, and mp designed the figures and table. kc and mp edited and provided critical revisions of the manuscript. all authors approve the final version and agree to be accountable for the content of the manuscript. jd is supported by a research grant from the national institutes of health (grant no. r hl ). the views expressed in this article are those of the authors and do not necessarily reflect the position or policy of the department of veterans affairs or the us government. we thank cat meyer for her assistance with the figures. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material interactions between influenza and bacterial respiratory pathogens: implications for pandemic 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from colonization to invasive disease key: cord- -ntpm cg authors: otašević, s.; momčilović, s.; stojanović, n.m.; skvarč, m.; rajković, k.; arsić-arsenijević, v. title: non-culture based assays for the detection of fungal pathogens date: - - journal: j mycol med doi: . /j.mycmed. . . sha: doc_id: cord_uid: ntpm cg traditional, culture based methods for the diagnosis of fungal infections are still considered as gold standard, but they are time consuming and low sensitive. therefore, in order to overcome the limitations, many researchers have focused on the development of new immunological and molecular based rapid assays that could enable early diagnosis of infection and accurate identification of fungal pathogens causing superficial and invasive infection. in this brief review, we highlighted the advantages and disadvantages of conventional diagnostic methods and possibility of non-culture based assays in diagnosis of superficial fungal infections and presented the overview on currently available immunochromatographic assays as well as availability of biomarkers detection by immunodiagnostic procedures in prompt and accurate diagnosis of invasive fungal infections. in addition, we presented diagnostic efficiency of currently available molecular panels and researches in this area. the primary task of microbiological/mycological diagnostics is to provide adequate information of the infection cause and its antimicrobial susceptibility in optimal period of time. any delay in accurate pathogen identification and appropriate treatment initiation undoubtedly affect disease outcome. despite significant advances in medical technology in terms of rapid accurate diagnostic laboratory tests following by new therapeutics that have had a major effect in decreasing of morbidity and mortality of many fatal diseases, we are the witnesses that invasive fungal infections (ifis) are still one of the biggest problems in medicine [ ] [ ] [ ] . increasing the number of immunocompromised patients with t-cell mediated immunity deficiencies, mucosal-cutaneous barrier or metabolic dysfunction, neutropenia, aging, excessive use of antibiotics, cytotoxic therapy and transplantation, as predispose risk factors and conditions, significantly influence the higher incidence of ifis [ ] . moreover, the incidence and prevalence of nosocomial ifi are still high and candida species represent the third most common blood isolate from hospitalized patients. out of all ifi, % of invasive candidosis are from intensive care units (icu) . candida bloodstream infection is prolonging stay in the icu with mortality ranges from to %. also, it can cause complications during the clinical course of primary disease and increases treatment costs [ ] . as opposed to ifi, superficial fungal infections (sfi) of the keratin rich structures are rare systemic and serious. these infections are caused commonly by dermatophytes, yeasts or non-dermatophytic molds and represent one of the dominant infections worldwide with global prevalence ranges from - % [ , ] . the lack of rapid diagnostic methods for sfi consequently influence that only % of physicians in general practice and % dermatologists require mycological analyses before prescription of empiric treatment with systemic antifungals [ ] . additionally, these infections can affect the patient's quality of life as a potential cause of social, professional and emotional problems [ ] . thus, traditional, culture based methods for the diagnosis of fungal infections are still considered as gold standard, but they are time consuming and low sensitive. therefore, in order to overcome the limitations, many researchers have focused on the development of new immunological and molecular based rapid assays that could enable early diagnosis of infection and accurate identification of fungal pathogens causing superficial and invasive infection. in this brief review, we highlighted the advantages and disadvantages of conventional diagnostic methods and possibility of non-culture based assays in diagnosis of superficial fungal infections and presented the overview on currently available immunochromatographic assays as well as availability of biomarkers detection by immunodiagnostic procedures in prompt and accurate diagnosis of invasive fungal infections. in addition, we presented diagnostic efficiency of currently available molecular panels and researches in this area. c elsevier masson sas. all rights reserved. fungal infections of the skin, nail or hair could not be considered as a cosmetic problem of relatively minor significance. given the fact that deep infections and dissemination of pathogen can be expected in immunodeficiency, some genetic abnormalities such as card mutation, and leukemia [ , ] , the prompt diagnosis and treatment of sfis could prevent complications and unwanted clinical outcomes. so far, in the group of sfi, mucosal (oropharyngeal, vulvovaginal, intestinal) fungal infections have also been included. today, the prevalence of oropharyngeal and vulvovaginal mucosal candidosis and candida-overgrowth in intestines significantly rises up and the treatment of chronic/recurrent form is a big challenge for physicians. also, frequent changes of local therapy by systemic, with the goal to improve the treatment, probably influences the emergence of resistant candida species to azoles [ ] [ ] [ ] [ ] [ ] . new, highly specific and sensitive rapid tests will disable misdiagnosis and unnecessary proscribing of antifungals, which will be in accordance with recently initiate appeal that systemic antifungals have to be kept for ifi [ ] . historically, it is very often highlighted that traditional/ conventional methods for diagnosis of sfi and ifi are time consuming and low sensitive. the propagating/cultivating the causative agent is method with only deficiency in required time for isolation, but when we can conduct it, it is the unique analysis that provide antigen (ag) detection, molecular, biochemical identification of etiological agents and antimicrobial susceptibility testing. on the other hand, histopathological examination of the tissue samples that is considered as a ''gold standard'' for ifi has a major shortcoming that lays in the complexity of the invasive biopsy procedure [ ] [ ] [ ] . this review discusses currently available data on the advantages and disadvantages of some rapid assays for fungal detection, identification and the diagnosis of sfis and ifis. . rapid assays for diagnosis of superficial fungal infections (sfis) conventional microscopic examination (me) is cheap, easy-to use and fast method for detection of fungi in a patient's sample. although staining procedures offer additional information concerning morphology of microorganisms, me in a form of wet mount technique provides rapid detection of fungal blastoconidiayeast and pseudohyphal-hyphal forms in patient's material ( fig. ) [ ] . wet mount with chlorlactophenol or koh as reagents is still irreplaceable technique for screening of patients with suspected sfi of the skin, hair and nail. additionally, diagnosis of vaginal discharge by wet mount microscopy is useful for the diagnosis of candida vaginitis -one of the most prevalent genital infections in women [ , ] . nowadays, specific staining with chemicals or fluorescent dyes (acridin-orange, calcofluor wight, blankoflor) can improve visualization and detection of fungi [ , , ] . currently, there are efforts for establishing the imunochromatographic (ic) assays for the determination of dermatophyte [ ] and candida ag [ ] , which will represent significant progress in the diagnosis of sfis and genital candidosis, respectively. fast, easy to perform and easy to interpret, these assays can be a satisfactory replacement of me. so far, there have been reports of ic assays in which trichophyton antibodies (ab) have been used with colloidal gold particles to capture ag of different dermatophyte species such as t. rubrum, t. mentagrophytes, t. violaceum, t. tonsurans, microsporum canis, m gypseum, epidermophyton floccosum. comparing the diagnostic performances of ic for dermatophytes detection in skin and nail samples by me, higashi et al. reported that ic sensitivity and specificity were . % and . % for all specimens, . % and . % for scarified skin and . % and . % for samples taken from the nail plate, respectively [ ] . based on these results, authors recommended ic assay, as easy to use and rapid tool, for screening of dermatophyte infections, but for definitive diagnosis, me and culture are still recommended methods. in the study conducted by noriki and ishida, in-house developed ic assays showed very good diagnostic performances for dermatophyte detection in nail samples [ ] . similarly, another ic assay which used the colloidal gold-antimannan igg conjugate for candida detection in vaginal swabs was reported recently. advantage of this newly developed ic assay is the possibility of use in clinics and laboratories and detection of candida spp. in the concentration of cfu/ml of patients sample in min without expensive equipments. however, in comparison with culture, ic candida assay in vaginal swabs showed the high specificity . %, positive predictive value . , followed by lower sensitivity . %, and negative predictive value % for c. albicans. for the second most prevalent species c. glabrata (causing more-intensive and chronic form of genital candidosis), concentration has to be more then cfu/ml in patients sample [ ] . despite the few data in reference literature and the lack of commercial ic assays for sfi, further research, design and establishment of these assays for sfi will be of great significance for supporting conventional methods and facilitating the diagnosis of dermatophytosis and genital candidosis in women. during the last forty years, a number of molecular techniques have been developed for the identification of dermatophytes at species or strain level. these include fingerprinting methods, conventional pcr (employing selected genetic markers e.g. internal transcribed spacers (its), chitin synthase gene (chs ), topoisomerase ii gene, small and large ribosomal rna subunit and non-transcribed spacer/nts region), its restriction fragment length polymorphism (rflp) and total mitochondrial dna (mtdna) analyses which change the view to the taxonomy of dermatophytes [ ] [ ] [ ] [ ] . molecular diagnosis provides specieslevel identification of dermatophytes more accurately regarding their development in human, soil or animals. recent studies showed that the conventional pcr of its and chs regions might be helpful tool for a better understanding of dermatophyte identification, taxonomy, ecology and epidemiology [ ] . these findings revolutionized the diagnosis of sfi caused by dermatophytes and the use of ''in house'' or rare commercial molecular tools enable their rapid determination directly in patients sample within h. as for ''in house'' molecular tools, various types of pcr techniques have been developed in the last decade. these assays had evolution from conventional pcr, nested pcr, pan-nested pcr which was recommended as gold standard with higher sensitivity compared to conventional methods [ ] , followed by multiplex real-time pcr which provides detection of t. rubrum, t. interdigitale, t. violaceum and m. audouinii [ ] and later developed single-tube dermatophyte qpcr tool based on its sequences which design allows determination of species of three dermatophyte genera (trichophyton, microsporum and epidermophyton) directly in patients sample [ ] . besides, reported multiplex pcr for detection of t. rubrum and t. mentagrophytes in nail samples, which is based on chitin synthase i and its region, was proved as molecular tool with excellent diagnostic performance ( % sensitivity and % specificity) [ ] . few commercial assays have also been designed and some of them with high diagnostic performance such as duplex pcr which combines pan-dermatophyte pcr with a trichophyton rubrum-specific pcr. this assay provides rapid detection and identification of trichophyton rubrum in nail specimens and has shown specificity and sensitivity of % and %, respectively, in comparison with conventional methods [ ] . on the other hand, some multiplex pcr methods for dermathophytes or dermatophytes/candida species in clinical samples were also developed and clinical evaluation is still in progress [ ] . recently, published results [ ] showed that real-time pcr with specific pan-dermatophyte primer for detection of most agents from this group of fungi in clinical samples detected more infected patients compared with me. however, with unsatisfactory sensitivity of . % and positive predicative value of . %, this method cannot be suggested yet as sufficient replacement of conventional diagnosis. in addition, new data of tinea capitis in sub-saharan africa highlighted the importance of early detection of causative agents. the use of the pcr-elisa and rapid determination of antropophylic species (t. violaceum, m. audouinii, t. soudanense, t. rubrum) allow the effective and appropriate therapy, monitoring of possible source of infection and implementation of preventive measures with the goal of preventing the spread of causative agents and infection [ ] . by all means, the development, standardization, as well as the commercialization of molecular tools are of great importance for the perspective in the rapid diagnostics of sfi. prolong survival of high-risk patients (transplantation, immunodeficiency, immunomodulatory regimens, malignancy, longer stay in icu, etc.) with ifi primarily depends on timely, accurate diagnosis and antifungal treatment [ ] . rapid advances in the field of diagnostic methods for prompt diagnosis of ifi caused by candida spp., aspergillus spp., pneumocystis jirovecii (p. jirovecii) have been achieved in recent years [ ] . however, the standardization of currently available immunological, pcr and fluorescence in situ hybridization assays is still ongoing worldwide. besides, the development and utilization of highly specific and sensitive diagnostic tests for detection of ifi caused by other fungal species is also required [ ] . conventional methods and designed rapid assays for the diagnosis of ifi were summarized in the table . conventional me can be used in diagnosis of ifi but require a high level of expert knowledge, which represents the main disadvantage of this procedure [ ] [ ] [ ] [ ] [ ] . detection of invasive aspergillus hyphae by direct microscopic examination of samples obtained from primarily sterile regions is equated as proven invasive fungal disease [ ] . on the other hand, this infection can be ''probable'' in patients with positive microscopic finding of sputum or broncho alveolar lavage (bal) fluid in combination with the appropriate clinical criteria and predisposing factors. also, cryptococal meningitis can be rapidly diagnosed if encapsulated cryptococcus neoformans (cry. neoformans) yeast cells are visible by me of cerebrospinal fluid (csf) [ ] . adding of indian ink in wet mounts of csf sediment can improve cryptococcus detection and the diagnostic efficacy. specific staining of patients samples with giemsa, wright, wright-giemsa, fontana-masson, grocott-methenamins silver (gms) or fluorescent dyes (acridine orange, calcofluor whight or blankofor) can improve visualization of fungi and their detection by me [ , , ] . specific staining methods are most commonly used for the detection of p. jiroveci (carinii) in lower respiratory tract specimens [ ] or to identify the presence of intracellular forms of histoplasma capsulatum in bal, bone marrow, peripheral blood smears or touch preparations of lymph nodes and other tissues [ ] . acridine orange allows the detection of p. jiroveci in purulent specimens [ ] . these fluorescent stains can bind to the cellulose and chitin in the fungal cell wall [ ] and have become very useful tool in laboratory mycology. also, any fungi can be detected by this methods but their identification is difficult or impossible in many cases. the most important advantage of immunoassay is the possibility of performing directly on a clinical sample obtained from patients. ic assays for fungal ags are low-cost, easy to use, and applicable at the patient's bedside, and can be performed easy to a number of samples without special equipment (even without electricity). however, currently, there are only few commercially available immunochromatographic assays that enable the detection of surface or soluble yeasts or aspergillus ag in clinical samples. their detailed characteristics are presented in table . one of the most significant achievements in mycology is the development, evaluation and manufacture of immunoassays for diagnosis of ifi. with the introduction of immunoassays for ag detection in patients biological fluids such as serum, bal or csf, the diagnosis of ifis, previously diagnosed only by histopathological examination of tissue samples after biopsy, have become more rapid and non invasive. latex agglutination tests for cryptococcal gxm are chip, sensitive, specific and easy to perform. the polysaccharide capsular gxm that is produced in large quantities by the crytococcus can be detected in both csf and blood samples [ ] . also, detection of candida mn, aspergillus gm, , b-d-glucan (bdg) in serum, bal or other fluids significantly improved the diagnosis of ifi [ , ] . in the diagnosis of candidemia, mn and bdg are currently available. mn is a major component of candida cell wall, accounting for up to % of total dry cell weight, which is released in bloodstream during candida replication during infection. the platelia candida ag test (bio-rad laboratories, marnes-la-coquette, france) enables the detection of mn in blood (serum) samples in elisa format [ ] . several retrospective and prospective studies have evaluated the utility of mn detection for the diagnosis of invasive candidosis in haematological and icu patients with an overall sensitivity (sn) of % and specificity (sp) of % [ ] [ ] [ ] [ ] . the sn of the test seems to vary with the infecting candida species, being the highest in the case of c. albicans [ ] . a positive mn test occurs several days before radiological detection of hepatosplenic candidosis or positive blood cultures (bc) [ ] . in high-risk patients, the test should be performed two to three times per week since the presence of mn ag in blood is short-lived due to rapid clearance during each episode of candidemia with the concomitant appearance of anti-mn ab [ ] . another ag-based test detects the presence of bdg, the important component of the cell wall of most fungi, in sera samples and is approved by food and drug administration of the united states of america [ , ] . the test has been evaluated, mostly in icu patients, with an overall sn of % and sp of % for subjects with proven or probable ifi [ , ] . however, bdg is not species specific; therefore it is a pan fungal assay. results of many large studies have shown that with higher values of sn ( . %) and sp ( . %), extremely low value of diagnostic odds ratio ( . %) have been obtained and bdg concentration vary in ifi due to different fungal species. white et al. proved that bdg concentration for proven/probable ifi ( pg/ml) is significantly higher then in possible/suspeced cases and control population ( pg/ml) and it is different regarding the cause of ifi. bdg is useful for diagnosis of invasive aspergillosis, invasive candidosis and p. jirovecii pneumonia, but bdg concentration vary based on causative agents [ ] . additionally, positive bdg in the cases of p. jirovecii pneumonia cannot distinguish infection from coloniza- table summary of rapid assays for fungi identification and invasive fungal infections (ifis) diagnosis. tion, to promote the higher sensitivity bdg concentration of pg/ ml has to be considered as positive, but only concentration threshold of pg/ml increased specificity. the interpretation could be only with additional clinical, radiologic examination, immunoor molecular assays [ , ] . the test should be applied twice weekly and a single positive test is indicative of infection. true positive cases usually show falling bdg titter that eventually becomes negative in patients responding to antifungal treatment, a trend that cannot be observed in patients who does not respond to therapy. false positive bdg results may occur due to several reasons such as haemodialysis, abdominal surgery, treatment with blactam antibiotics and concomitant presence of lipopolysaccharide originationg from gram-negative bacteremia, which makes its application rather difficult in clinical settings [ , ] . however, based on excellent negative predictive value of this test (nearly %), lack of bdg detection is most useful for exclusion of ifi [ , ] . also, the colonization of individuals with candida sp. has no apparent effect on the bdg test outcome [ , , ] . as for the use of gm in diagnosis of invasive aspergilosis, recently published data suggest that detection of this aspergillus ag in blood and parallel pcr diagnostics provide very high sensitivity of % with specificity of % which influence % negative predictive value in high risk patients and enable the consideration of no-existing invasive aspergillosis in these patients and no need for antifungal therapy. contrary, positivity of both tests has positive predictive value of % that suggest probably occurrence of invasive aspergillosis [ ] . however, the cross-reactivity of fusarium spp. in the aspergillus gm elisa assay has been observed and needs to be taken into consideration in some patients, contraries or settings. the most significant progress in the diagnosis of infectious diseases, fungal infections included, has been made with the introduction of molecular biology. molecular detection and identification as well as nucleic acid testing (nat) have a great importance in the diagnosis of infectious diseases, especially in the presence of a suggestive patient history and clinical manifestations of the infection. these methods can be used as a sensitive and specific tool for the detection and identification of numerous microorganisms in patient specimens. the application of nat can enhance the speed of diagnosis. some of these tests can be completed in a few hours and have high sn and sp [ ] [ ] [ ] . however, the disadvantages include the high cost of reagents and instruments, appropriately trained staff and well-equipped laboratories that are able to fulfil the highest standards. additionally, possibility of samples or reagents contamination could be also the lack of these assays [ , , , ] . peptide nucleic acid fluorescent in situ hybridization (pna-fish) is a brand new, rapid, molecular diagnostic platform developed by advandx (woburn, massachusetts) based on proprietary of pna probe technology. the pna-fish uses a variety of currently approved probes, including ones for staphylococcus aureus, coagulase-negative staphylococci (cons), enterococcus faecalis and other enterococci, escherichia coli, klebsiella pneumoniae, pseudomonas aeruginosa, c. albicans, c. glabrata, c. parapsilosis, c. krusei, and c. tropicalis (advandx) [ ] [ ] [ ] [ ] [ ] . the pna-fish uses fluorescent-labelled probes to target species-specific rrna sequences in a high sn and sp fluorescence in situ hybridization assay. the probes bind to the target rna tightly, without electrostatic repulsion from the charged rna backbone and since rrna is amplified in viable cells of growing cultures, there is no need for its further amplification [ , , ] . cell lyses, usually used for isolation of genetic material, are also not necessary since the pna probes hybridize to rrna inside the bacteria or yeast enabling the whole cell analysis. the assay requires only limited sample preparation as cells do not need to be lysed to isolate genetic material, allowing a simple test procedure with visual results that match gram-stain morphology [ , ] . the major characteristics of pna-fish in a routine monitoring of analyses of sepsis are safety, sp, sn, the potential for highthroughput testing and economical feasibility. the pna-fish is easy to perform in clinical laboratory and does not require a significant capital equipment costs unlike microarrays or matrixassisted laser desorption ionization-time of flight mass spectroscopy (maldi-tof) [ , , ] . the pna-fish method requires only microscope equipped with a fluorescent lamp and dual band filters for results interpretation. the accuracy and sp of pna-fish can significantly affect antibiotic and antifungal utilization, allowing more targeted therapy, reduction of treatment duration, with an overall reduction in healthcare costs and increased benefit for patients [ , ] . recently, this methodology is additionally improved by introducing new quickfish, developed by advandx, that enables very fast ( minutes) identification of bacteria or yeasts from positive blood samples. . . molecular diagnosis of p. jirovecii pneumonia p. jirovecii is an opportunistic unicellular fungal pathogen that causes an acute and life-threatening pneumocystis pneumonia in immunocompromised hosts [ , ] , such as hiv-infected patients (especially those who do not know that they are hiv positive, do not comply with or respond to antiretroviral therapy or pneumocystis prophylaxis) [ ] , solid organ transplant and haematopoietic stem cell transplant recipients, patients with malignant diseases, non-hiv-infected patients receiving immunosuppressive medications (e.g. corticosteroids, monoclonal antibodies or cytokine inhibitors) for autoimmune or inflammatory diseases and subjects with congenital immunodeficiencies [ ] [ ] [ ] . the clinical course of infection is more acute and severe in hiv-negative immunocompromised patients than in hivinfected hosts with significantly higher rates of mortality ( - % vs. - %) [ ] . in some cases, however, patients may become colonized with p. jirovecii without signs or symptoms of acute disease [ ] . according to the results of the conducted studies, it has been shown that even - % of hiv patients and - % of non-hiv immunosuppressed patients are asymptomatic carriers of p. jirovecii [ ] . in patients with pneumocystis pneumonia, rapid and accurate establishment of the diagnosis and prompt treatment initiation are critical determinants for favorable clinical outcomes [ ] . considering the fact that p. jirovecii cannot be cultured in vitro, microscopic visualization of p. jirovecii cysts and/or trophozoites in lower respiratory samples such as induced sputum and bal fluids still remains the gold standard for the diagnosis of pneumocystis pneumonia [ ] . however, this method is largely subjective, nonspecific, low sensitive, and depends on the skill and experience of the observer, and the type of sample [ ] . despite all disadvantages, me is still frequently used in resource-limited laboratories for the detection of p. jirovecii in clinical specimens. this applies in particular to immunofluorescent assay (ifa) which is, compared with calcofluor white, grocott-gomori methenamine silver, and diff-quik staining method, significantly more sensitive ( . % vs. . %, . % and . %) but less specific ( . % vs. . %, . % and . %) [ , ] . in recent two decades, introduction of molecular detection methods into the clinical laboratory practice has led to significant progress in the diagnosis of pneumocystis pneumonia [ ] . nowadays, these methods have greatly expanded their scope of application and are also successfully used for investigation of potential outbreak scenarios, determination of epidemiology and transmission of pneumocystis pneumonia, and, as opposed to conventional microscopy, offer a high degree of objectivity [ , ] . pcr, as one of the most popular molecular diagnostic techniques in clinical microbiology at this moment, has been reported as a valuable tool for detection of p. jirovecii in different non-invasively and invasively collected clinical samples from the respiratory tract [ ] . in addition, a newly developed, improved version of the pcr assay -real-time pcr has contributed to a significant reduction in turnaround time (results are available after . h including dna extraction) and an overall increase in diagnostic sensitivity compared to ifa [ ] . moreover, as opposed to nested pcr, such one-step assays are easier to perform and decrease the risk of carry-over contamination [ ] . until today, various samples have been used for pcr detection of p. jirovecii pneumonia. pcr on bal sample has higher diagnostic sensitivity ( . - %) [ , ] in comparison with non-invasive samples such as sputum ( . %) [ ] or oropharyngeal wash fluid ( %)( ), but is difficult to obtain in some critically ill patients due to the increased risk of respiratory deterioration [ ] . also, in patients with non-productive cough suspected of having p. jirovecii pneumonia, induced sputum is not easy to obtain [ ] . it is important to emphasize that, although highly sensitive, negative pcr results of bal fluid do not provide reliable exclusion of p. jirovecii pneumonia given that the false negative results can occur when mutation at position (c t) of the mitochondrial large subunit ribosomal rna (mtlsurrna) gene of p. jirovecii is present [ ] . on the other hand, studies have shown that pcr analysis of oropharyngeal wash fluid is more specific ( %) than bal ( . - %) and sputum ( . %), which indicates that the pcr detection of p. jirovecii in the upper part of respiratory tract is a good indicator of p. jirovecii pneumonia [ , ] . in order to overcome insufficient specificity of pcr on bal fluid and sputum, a relatively new concept in the diagnosis of p. jirovecii pneumonia has been developed. this method is characterized by minimal invasiveness and includes pcr detection of cell-free dna, fragments of dna which are present extra cellular in different body fluids, in serum samples [ ] . the pioneering experimental studies have shown that diagnostic sensitivity of pcr detection of p. jirovecii dna in sera may vary from - % [ , ] . however, the most recent study has shown that, when compared with gms staining as the gold standard, the sensitivity of cell-free dna pcr is the same as bal fluid pcr and sputum pcr, while the specificity is much higher [ ] . when above mentioned studies are analyzed together, it can be concluded that diagnostic performances of pcr on serum dna depend largely on the amount of sample used for dna extraction, dna extraction method (magnetic bead methods are more efficient in serum cell free-dna extraction than proteinase k-phenol-chloroform) and the type of primers used for detection of pneumocystis [pneumocystis heat shock protein (hsp ) gene has higher sensitivity and specificity in pneumocystis detection than pneumocystis mt lsu rrna gene] [ , , ] . invasive pulmonary aspergillosis is a devastating opportunistic infection caused by molds belonging to the genus aspergillus. this infection mainly affects immunocompromized hosts, such as patients with cancer or hematologic malignancies, allogeneic hematopoietic stem cell and solid organ transplant recipients (especially lung transplant recipients), patients with advanced aids, and those presenting with chronic granulomatous disease [ , ] . in recent years, increase in incidence of invasive pulmonary aspergillosis has been noted in patients with lympho proliferative syndromes and in medical icu patients, particularly in those treated with corticosteroids. nowadays, despite the advances in understanding the disease and development of highly effective antifungals such as voriconazole and posaconazole, global mortality and morbidity rates are still high [ ] . timely and accurate diagnosis and early initiation of appropriate antifungal treatment significantly improve disease prognosis [ ] . diagnosis of invasive pulmonary aspergillosis is traditionally based on the isolation of fungi of the genus aspergillus in culture in combination with cme or radiographic findings [ ] . however, the sensitivity of culture is low and may take a long time to results. besides, cultures are incapable of differentiating between infection, colonization, or contamination. on the other hand, me, as inexpensive and easy-to-perform complementary diagnostic method, improves positive predictive value by confirming positive culture results and provides information about the infecting cell morphology, the state of infected tissues, and biofilm formation, but is not organism specific [ ] . immuno assays which are based on the detection of fungal cell wall biomarkers, such as bdg or aspergillus gm, have significantly reduced turn-around times compared with me, but it has been shown that they can be low sensitive and specific in certain patient groups [ ] . molecular techniques represent a novel approach to the diagnosis of invasive aspergillosis. currently, pcr is the most widely applied molecular diagnostic test, which enables amplification and detection of extremely low concentrations (from to pg) of aspergillus dna in various clinical specimens such as bal fluid, serum, plasma, whole blood and biopsy material [ , ] . also, pcr assays can quickly identify species within the genus aspergillus from cultures and non-cultured samples which is of great significance for epidemiologic studies and clinical practice given the fact that newly described species such as aspergillus lentulus and aspergillus calidoustus have been shown to have elevated minimum inhibitory concentrations (mics) to several antifungal drugs, including azoles [ ] . due to the lack of standardization and potential false-positive results, pcr was in excluded from european organization for the research and treatment of cancer/ mycoses study group (eortc/msg) definitions [ ] . in , a group of experts conducted an investigation in which aspergillus pcr was compared with gm and bdg and, based on obtained results; they concluded that ''pcr is now mature enough for inclusion in the eortc/msg definitions'' [ ] . studies have shown that performance of molecular tests varies depending on the specimen type and patient population [ , ] . pcr analysis of blood fractions is the most desired testing modality given that it requires minimally invasive sampling procedure [ ] . also, it is important to emphasize that this method is less exposed to accidental contamination and, in comparison with pcr on bal samples, offers higher diagnostic sensitivity when screening [ ] . moreover, it has been noted that the diagnosis of invasive aspergillosis can be excluded if consistently negative results are obtained during the testing of multiple blood samples [ ] . conducted meta-analyses have shown that aspergillus pcr on blood samples achieves sn ranging from to % and sp from to . % [ , , ] . when different fractions of blood are compared, meta-analysis showed that pcr assays of whole blood are more sn ( % vs. %), but less sp ( % vs. %) than those of sera. however, this difference is not statistically significant [ ] . on the other hand, another study which included hematology patients at risk of invasive aspergillosis reported the highest diagnostic sn ( %) of aspergillus pcr testing of plasma in comparison with pcr testing of serum ( %) and whole blood ( %) [ ] . this disagreement in the results of two above mentioned studies can be due to the differences in sample processing and prolonged specimen storage methods for the whole-blood specimens used in the second study [ ] . in high-risk patients, at least two positive whole-blood pcr specimens per patient may dramatically increase sp to % with positive predictive value of %, which is very indicative for invasive aspergillosis. however, this approach significantly decreases sn to %, which indicates that this diagnostic strategy is more valuable as a confirmatory tool than as a screening tool [ ] . another factor that may also affect the sn of pcr testing is current immunological status of the patient. in the study, which examined diagnostic performance of pcr analysis of sera obtained from neutropenic and non-neutropenic patients, it was shown that this testing modality has higher sn in neutropenic patients ( . %) than in nonneutropenic patients ( . %), but without statistical significance. likewise, the same study noted that the initial level of aspergillus dna is highly predictive of the -day mortality rate (below copies/ml-low probability; above copies/ml-high probability), which is of great significance for identification of patients who may benefit from more intensive care [ ] . finally, based on all of the above facts, it can be concluded that pcr analysis of serum or plasma seems to be diagnostic modality with acceptable sn and sp. also, these sample types, as opposed to whole blood, do not require preanalytical cell lysis steps and allow other biomarker diagnostic tests, such as gm, to be run [ , ] . bal fluid represents another commonly used sample for the diagnosis of invasive aspergillosis. compared with blood samples, the sp of aspergillus pcr bal testing is significantly higher [ ] . based on the results of conducted meta-analysis, the pooled sp of pcr on bal fluid specimens was even % for proven/ probable invasive aspergillosis, while sn was % [ ] . however, one of the major limitations of pcr testing of respiratory samples is the inability to differentiate airway colonization from invasive disease. also, the risk of sample contamination at the patient's bedside or in the laboratory by airborne spores is high which may lead to the occurrence of false positive results. furthermore, it is estimated that even % of bal samples from healthy subjects would be falsely positive after molecular testing which is significantly higher than in other diagnostic modalities (e.g. detection of gm) [ ] . quantitative pcr (qpcr) detection of high fungal burdens may be indicative of invasive aspergillosis, but this diagnostic modality has still many limitations [ ] . in recent years, resistance to azoles in a. fumigatus has emerged as a global health problem [ ] . based on published results of conducted studies, the resistance occurs as a result of tr /l h and tr /y f/t a mutations in the cyp a gene and its promoter region [ ] . these mutations were first found in the netherlands in (tr /l h) and (tr /y f/t a) [ ] and have since been reported in multiple european countries, middle east, asia, africa, australia and the united states [ ] . azole-resistant invasive aspergillosis is difficult to diagnose using traditionally culture-based methods given that aspergillus cultures are negative in the majority of patients. on the other hand, immunoassays, which provide detection of fungal cell wall biomarkers, are unable to identify patients with azole-resistant disease [ ] . consequently, two commercial multiplex real-time pcr assays (mycogenie (ademtech, pessac, france) and asper-genius (pathonostics, maastricht, the netherlands)) have been developed to enable the detection of both a. fumigatus dna and the most prevalent cyp a mutations responsible for azole resistance in clinical samples. these assays have been evaluated using bal fluid and serum samples. the aspergenius multiplex real-time pcr test includes two pcrs -the species pcr and the resistance pcr. in respiratory samples, the species pcr generated thesn and sp of . % and . %, respectively, in patients with hematological disorders, while the resistance pcr was able to detect resistanceassociated mutations in a culture-negative patient with invasive aspergillosis. as for the serum samples, species assay showed the sn and sp of . % and %, respectively in patients in whom fungal disease status had been previously defined using the revised eortc criteria [ ] [ ] [ ] . on the other hand, mycogenie showed thesn of . % and sp of . % for detection of aspergillus dna in respiratory samples, while in serum samples, the sn and sp were % and . %, respectively. it is important to emphasize that all isolates harboring the tr /l h mutations were accurately detected [ ] . recently, multiplex pcr and real-time mpcr molecular tests have been developed for detection and identification of various closely related pathogens causing respiratory, gastrointestinal and sexually transmitted infections as well as meningitis and sepsis. because of similar clinical symptoms, signs and possible simultaneous presence of several different agents, the differential diagnosis of these infections includes a large number of potential agents, which are clinically indistinguishable. use of molecular panels allows timely detection and discrimination of the infecting pathogens, which is important to optimize medication, prevent secondary spread of infection and unnecessary antibiotic use in viral infections, and may help in decisions regarding hospitalization and infection control measures. also, these panels include internal control for monitoring of every procedure step [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in medical mycology, the use of multiplex pcr for pathogens detection implies panels for detection of yeasts in blood of patients with sepsis and panels for cry. neoformans detection in csf of patients with meningitis/encephalitis. characteristics of these molecular panels are shown in the table . in recent years, different rapid molecular methods have been developed to speed up the identification of the microorganisms that grow in blood-culture (bc) bottles. on the first place, maldi-tof ms sepsityper kit tm can be used to directly analyze positive bc in real time and to provide definitive species identification within to minutes, with the identification rate of . % [ ] . the maldi-tof has the advantage of rapid turn-around time and ability to identify a large number of microorganisms directly. disadvantages include the high start-up costs associated with the purchase of mass-spectrometry equipment and training of personnel. as an alternative to maldi-tof, film array blood culture identification (bcid) panel (bio fire, usa), a multiplexed pcr based diagnostic test, can be used for positive bc. the bcid can detect bacteria, five candida spp. and three antimicrobial resistance determinants (mec a, van a/b, bla-kpc) in h from the time of bc positivity [ ] . this method has a several advantages: the large number of targets can be evaluated in a single test and all steps of the assay, from nucleic acid extraction to interpretation of amplification data, are performed in closed system and using a single pouch on a minimally processed clinical sample. the required laboratory procedures are not technologically complex and can be performed by persons who do not have training in molecular techniques [ ] . other methods such as nano particle probe technology (nucleic acid extraction and pcr amplification) and nano sphere's verigene can be used for identification. the platform consists of two assay panels: the bc-gp assay and the bc-gn assay. also it can detect drug resistance markers such as: mec a, van a/b, kpc, ndm, ctx-m, vim, imp and oxa genes [ ] . t magnetic resonance (mr) assay for the rapid diagnosis of candidemia in whole blood is one of tests in the new era of molecular diagnostics which can provide fast and species -specific (c. albicans, c. tropicalis, c. glabrata, c. krusei, c. parapsilosis) detection directly in the clinical samples. regarding the fact that t mr has % sensitivity and > % specificity, does not require cultivation as well as sample purification or preparation, it can be highlighted that this test represents example of point-of-care diagnostic test which can ensure the prompt and accurate results in . ae . hours with the possibility of treatment modification in cases of candidemia. this novel technology has . % negative predictive value in population of patients with candida invasive infection, disease with estimated prevalence of %, which will decrease the number of patients on empiric therapy and resistant strains occurrence [ ] . regarding the candidemia caused by c. glabrata, so far, it was considered that fluconazole (flc) resistance is common in c. glabrata and echinocandins are often used as first-line therapy. one of the major advantages of t mr is a possibility of rapid detection of c. glabrata in blood of patients with higher sensitivity (to > %) than culture (se = %) [ ] and its determination on low level of detection of cfu/ml. this is very important since that early initiation of appropriate treatment can improve prognosis and significantly decreases mortality. however, further improvement of diagnostic performances of t mr is required given that nearly % of patients had indeterminate t dx results (n = / ). also, the price of this method is high and does not enable day-to-day monitoring, contrarily to bc. in addition, only species of candida are detected by this assay [ ] [ ] [ ] . however, the resistance to echinocandin therapy associated with fks and fks gene alterations of c. glabrata has been noticed in recent years. these findings influence the establishment of a novel (allele-specific molecular beacon and dna melt analysis followed by asymmetric pcr) and highly accurate diagnostic assay for rapid identification of fks mutations associated with echinocandin resistance in this candida species. this platform can be diagnostic method for rapid detection of infections caused by c. glabrata resistant strains to echinocandin and will timely provide the choice of efficient treatment [ , ] . currently, there are efforts for development of mass spectrometryy (ms) procedure for fungal disaccharide (ds) detection in patient's sera for rapid diagnosis of ifi. applaing of maldi-tof ms for fungal molecules detection have shown very good diagnostic efficacy for early diagnosis of ic, ia and more important invasive mucor infection (imi). the first researches in this field have pointed out the fact that the sensitivity of ms-ds (with application of the higher cutoff) is higher than that of mn or bdg detection. in addition, neither neutropenia nor bacteriemia have been shown to influence the detection of fungal disaccharide dd in patients with ic. also, this method has provided earlier positive results than other tests on the first serum samples in the cases of ia. moreover, results of evaluation of this method suggest the possibility of panfungal analyses and diagnosis of imi for which currently there are no available commercial serological tests that are efficient for the diagnosis. however, it has to be highlighted that this biological test requires further evaluation and validation in multicenter studies [ ] . delays in the diagnosis of ifi and appropriate therapy application clearly affect the infectious diseases outcome in a negative fashion. on the other hand, non-established accurate identification of fungal pathogens that cause sfi can influence failure in implementation of appropriate anti-epidemic prevention measures. in the era of large number of commercial rapid tests, clinicians must decide which of them should be introduced to the routine work, based on an individual experience, consideration of the disease incidence in the population, the cost-effectiveness and finally adequate assessment and selection of patients who would have the most benefit from rapid treatment. the study was supported by the serbian ministry of education and science [the grant no and grant no iii ]. the authors declare that they have no competing interest. diagnostic performance of ! -b-d-glucan in neonatal and pediatric patients with candidemia candida bloodstream infections in serbia: first multicentre report of a national prospective observational survey in intensive care units medical 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negative gram stain using the biofire filmarray meningitis/encephalitis panel t magnetic resonance assay for the rapid diagnosis of candidemia in whole blood: a clinical trial rapid detection of fks-associated echinocandin resistance in candida glabrata increasing echinocandin resistance in candida glabrata: clinical failure correlates with presence of fks mutations and elevated minimum inhibitory concentrations application of mass spectrometry technology to early diagnosis of invasive fungal infections key: cord- -v q spmm authors: resende, talita pilar; medida, ramya lekha; guo, yue; vannucci, fabio a.; saqui-salces, milena; gebhart, connie title: evaluation of mouse enteroids as a model for lawsonia intracellularis infection date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: v q spmm lawsonia intracellularis, an obligate intracellular bacterium, is an important enteric pathogen in pig herds and horse farms worldwide. the hallmark feature of l. intracellularis infection is the proliferation of epithelial cells in intestinal crypts. a major limitation to the study of l. intracellularis infection is the lack of an in vitro model that reproduces the changes observed in proliferative enteropathy. here we investigated the suitability of mouse enteroids as a model to study l. intracellularis infection. mouse enteroids were microinjected with l. intracellularis, filter-sterilized l. intracellularis culture supernatant, or sterile cell culture media (dmem). l. intracellularis antigen was detected in mouse enteroids by immunohistochemistry and was located mostly in the basal region of the epithelium. there was no differential growth of enteroids among treatment groups, and cellular proliferation was not increased in l. intracellularis-infected enteroids in relation to non-infected enteroids based on immunofluorescence staining. l. intracellularis infection did not induce changes in gene expression of ki- (proliferation marker), sox (marker for transit amplifying cells) and muc (marker for goblet cells). these results indicate that although l. intracellularis antigen is detectable in mouse enteroids, indicating susceptibility to infection, mouse enteroids fail to replicate the cellular proliferation and gene expression changes observed in proliferative enteropathy. nevertheless, we have successfully demonstrated that mouse enteroids can be used to model days-long intracellular pathogen infection, serving as potential models for the study of other pathogens of interest in veterinary medicine. pig herds and horse farms worldwide are regularly challenged by proliferative enteropathy (pe). this disease is caused by lawsonia intracellularis and is characterized by the thickening of the small intestinal mucosa due to proliferation of intestinal epithelial cells. pe causes diarrhea and compromised weight gain in pigs [ , ] . in weaned foals, pe causes diarrhea and hypoproteinemia, occasionally resulting in death [ ] . other species are also affected by pe, including ratite birds, rabbits, non-human primates, rats and mice [ ] . lawsonia intracellularis is a gram-negative bacterium that requires an intracellular culture system and a specific atmosphere to be propagated in vitro [ ] . traditional single cell cultures (cell lines) are permissive to l. intracellularis propagation, but they have failed to reproduce the increased cellular proliferation observed in pe-affected animals [ ] . the lack of an in vitro model that represents the in vivo progression of pe has limited advancement in knowledge of the pathogenesis mechanisms utilized by l. intracellularis. hence, identification of new alternatives for control and prevention of pe has been difficult, and pig producers and horse owners have been relying on available commercial vaccines (whole cell vaccines) and antimicrobials to prevent and control pe [ ] [ ] [ ] [ ] . the costs associated with producing whole cell vaccines is reflected on the commercial price of the vaccines, hampering the access to some producers to the commercial vaccines, and, therefore preventing pe. in addition, the use of antimicrobials as growth promoters in swine production has raised concerns about the selection of antibiotic-resistant organisms in treated herds [ ] . therefore, a deeper understanding open access *correspondence: resen @umn.edu department of veterinary and biomedical sciences, college of veterinary medicine, university of minnesota, st. paul, mn , usa full list of author information is available at the end of the article of pe pathogenesis is necessary for the development of novel non-antimicrobial based prevention and treatment methods such as recombinant vaccines or antimicrobial alternative strategies [ ] [ ] [ ] [ ] . the currently held hypothesis is that l. intracellularis infects intestinal epithelial cells, especially in the crypt compartment, leading to cellular proliferation and decreased differentiation in goblet cells along with increased apoptotic events [ , , ] . the mechanisms involved in epithelial changes during the course of pe are still unclear. thus, knowledge about interactions between l. intracellularis and intestinal epithelial cells, determined using a controlled environment, could generate a better understanding of how l. intracellularis induces cellular proliferation. a promising alternative to single cell cultures are tridimensional multi-cell type cultures, also known as organoids. one of the most relevant advantages of organoids in relation to the traditional in vitro single cell culture is the similarity of the organoids to their respective tissue of origin [ , ] . enteroids, small intestinal organoids, for instance, possess not only enterocytes, but also enteroendocrine cells, transit amplifying cells, goblet cells and stem cells. all these cells form structures with cell distributions that are similar to crypts and villi found in the small intestine, and possess a lumen where cell debris and secretion are shed continuously [ ] . enteroids have been recently used to study host-pathogen interactions of important enteric bacteria in human medicine [ ] [ ] [ ] [ ] [ ] , but they have not been investigated as a model for bacterial infections in the field of veterinary medicine [ , ] . the objective of this study was to evaluate mouse enteroids as an in vitro model for l. intracellularis infection. infected enteroids were monitored over time by size along with changes in expression of genes that have been reported to change during pe. we found evidence of infection with l. intracellularis in enteroids followed for up to days post-infection (dpi). the genetic profile of infected mouse enteroids, however, diverged from the gene expression changes observed in pe. mouse enteroid preparation and maintenance were performed as described elsewhere [ ] (iacuc approval number - a). the formulation of the enteroid culture medium was as described previously [ ] . a l. intracellularis isolate (phe/mn - , atcc pta- , manassas, va, usa) at low (≤ ) passage [ ] was propagated in mccoy mouse fibroblast-derived monolayers (atcc ® crl- ™ ). mccoy cells were cultured as described elsewhere [ ] . cells were cultured in dulbeco's modified eagle medium (dmem, gibco thermofisher, waltahm, ma, usa) with % fetal bovine serum (fbs; heat inactivated, corning ™ cv, corning, ny, usa). mccoy cells at % confluency were infected with about l. intracellularis and incubated at °c under a controlled microaerophilic atmosphere created with a mixture of gas containing % hydrogen, % carbon dioxide, and % nitrogen [ ] . seven days after infection, cell monolayers were mechanically lysed by passage through a -gauge needle coupled to a syringe after immersion in sterile . % potassium chloride and then centrifuged at × g to separate cell debris from l. intracellularis organisms. the pellets were discarded and the remaining supernatant was then filtered through . µm sterile filters (millipore sigma, burlington, ma, usa) to remove any remaining mccoy cells and nuclei. after a final centrifugation at × g at °c the bacterial pellet was either used to re-infect mccoy cells, or to prepare the inoculum suspended in approximately µl of its own supernatant and used as inoculum. as l. intracellularis growth requires special atmospheric conditions [ ] , we first tested whether the atmosphere conditions required for l. intracellularis growth would impact growth and development of mouse enteroids. one plate with enteroids was maintained under atmospheric conditions specific for l. intracellularis i.e., % hydrogen, % carbon dioxide, and % nitrogen [ ] while another plate was maintained at % co as a control. both plates were incubated at °c. since l. intracellularis manipulation in vitro requires an antibiotic-free sterile system, antibiotics (penicillin/streptomycin) were excluded from both plates to assess sterility of the cultures. the plates were observed every h for weeks, with media replacement and enteroid passage as described previously. to standardize conditions for infection of mouse enteroids with l. intracellularis, we adopted the "incubation and seed" method [ ] . briefly, immediately after enteroid passage and before plating, enteroids were directly mixed with l. intracellularis suspended in µl of enteroid culture medium (resulting in an inoculum with ~ l. intracellularis organisms/ml), with occasional gentle agitation, and incubated at °c under special atmospheric conditions generated with a gas tank of % hydrogen, % carbon dioxide, and % nitrogen for min. the infected enteroids were briefly centrifuged at × g for min, mixed with - µl ice-chilled matrigel (corning, usa), and then seeded into culture plates. enteroids were harvested days post-infection (dpi) by removing culture media and suspending the matrigel in phosphate buffered saline (pbs). after centrifugation for min at × g, the pellet was suspended in % formaldehyde and incubated for h at room temperature. enteroids were then washed in pbs, centrifuged at × g for min, suspended in histogel (thermofischer, usa) and placed into a biopsy mold. histogel blocks were inserted into a cassette and maintained in % alcohol until they were processed for histological evaluation. four-micrometer sections were placed onto glass slides and used for hematoxylin and eosin (h&e) staining and immunohistochemistry [ ] . although l. intracellularis antigen was detected by ihc in the infected enteroids by this method (figure ), we opted to employ the microinjection approach described below to facilitate contact of l. intracellularis with the cellular apical membrane in an effort to improve the level of infection. mouse enteroids were cultured and passaged until a sufficient number of enteroids (average of enteroids per treatment group, per time point) with µm diameter were obtained for the trial. wnt a protein, which regulates the signaling pathways related to the cellular proliferation in the intestinal crypt compartment [ ] , was removed from the enteroid culture media at least days before infection to enable cells to better differentiate. twenty-four hours prior to infection, enteroids were divided into three treatment groups: dmem: sterile antigen is associated mostly with cell debris (arrow heads), × . b l. intracellularis antigen is associated with cell debris (arrow heads) and in close proximity to enteroid cells (black arrows), × . c l. intracellularis antigen is observed in close association with mouse enteroid cells (black arrows), × . d a few l. intracellularis antigens are observed in this image, some close to mouse enteroid cells (black arrow), and some associated with cell debris (arrow head), × . enteroid lumen is indicated by an asterisk (*). culture media; sn: sterile-filtered l. intracellularis-culture supernatant; l. intracellularis: suspension with about l. intracellularis organisms/ml. each enteroid received approximately nl of each respective treatment. treatments were placed directly into the lumen of the enteroids by microinjection using a microinjector (nanoject ii, drumond scientific, broomall, pa, usa) operated by a trained technician. experiments were performed in four independent replicates. microinjected enteroids were monitored for week, from the day prior to infection until dpi. images were captured at × magnification using an inverted microscope coupled with a digital camera immediately after injection ( dpi), and at , and dpi. the enteroid area was measured using an image software (nih imagej software . r, national institute of health, bthesda, md, usa). the relative enteroid area (enteroid growth) was measured by subtracting the enteroid area from the area recorded at its previous measurement. enteroids from each treatment group were harvested at , and dpi and prepared for immunofluorescence. matrigel was disrupted with a sterile pipet tip and the contents in each well were transferred to a . ml tube and centrifuged at × g for min. the supernatant was discarded, and the pellet suspended in ml of pbs. this step was repeated two times to remove matrigel. the resulting enteroid pellet was suspended and fixed in % paraformaldehyde for h at room temperature. after fixation, enteroids were washed three times in pbs and centrifuged at × g for min. fixed enteroids were embedded in histogel (richard-allan scientific histogel, thermofischer, usa) and then either placed in optimum cutting temperature (oct, tissue-tek; sakura finetek, beaver creek, co, usa) for preparing cryosections or dehydrated in % ethanol and embedded in paraffin. paraffin-embedded sections ( μm) on charged slides were used for ihc for detection of l. intracellularis using specific rabbit polyclonal antibodies and a previously described method [ ] . stained sections were evaluated by bright field microscopy. oct-embedded blocks were sectioned ( µm), placed on charged glass slides and stored at − °c until immunofluorescence staining. anti-ki- immunofluorescence was performed as described elsewhere [ ] . briefly, slides were hydrated with tris buffered saline (tbs) containing . % triton-x (tbs-t) for min and then blocked with % normal goat serum (zc , vector laboratories, burlingame, ca, usa) for min to prevent non-specific binding. sections were then incubated with anti-ki- antibody ( : in tbs-t, crm b, biocare medical, pacheco, ca, usa) for h, followed by a min wash in tbs-t and incubation with cy -labeled goat antirabbit antibody ( : , ab , abcam, burlingame, ca, usa) for min. all incubations were carried out at room temperature. slides were washed and mounted with prolongold (thermofisher, usa) and covered with glass coverslips. the number of ki- -positive cells and the total number of cells were counted in at least random fields at × using an olympus bx fluorescence microscope. enteroids were harvested at , , and dpi, released from matrigel as described above, lysed with . ml of trizol (thermofisher, usa) and stored at − °c until rna extraction. rna extraction was performed using chloroform and isopropanol precipitation following trizol protocols and then cleaning with the rneasy mini kit (qiagen, germantown, md, usa) according to the manufacturer's instructions. total rna was quantified using a nanodrop (thermo scientific, usa) and ng of rna were transcribed to cdna using the high capacity cdna reverse transcription kit (applied biosystems, foster city, ca, usa) with random hexamer primers. quantitative pcr reactions were performed using sybr green pcr master mix (applied biosystems) according to the manufacturer's instructions. amplification was performed with the following conditions: initial activation at °c for min, followed by cycles of denaturation at °c for s and annealing at °c for s. expression of the housekeeping gene gapdh was monitored as reference. relative gene expressions were normalized to gapdh using the primer efficiencies. the expression levels of ki- (marker for cellular proliferation), sox (marker for transit amplifying cells) and muc (marker for goblet cells) in enteroids of all treatment groups were measured at , and dpi. fold change in each case was calculated in relation to expression in enteroids from day (control). all primers used are listed in table . results are presented as a mean ± standard deviation of the mean (sem) for each group. two-way anova followed by geisser-greenhouse correction was used to verify statistical differences with p < . considered statistically significant. graphpad prism . software for windows (graphpad software, la jolla, ca, usa) was used to perform the analysis. there was no difference in growth or morphology between enteroids maintained for week in the microaerophilic atmosphere relative to those maintained under regular conditions, i.e., % co incubator (data not shown), indicating that growth conditions required for l. intracellularis do not impact enteroid viability or growth. mouse enteroids were infected with l. intracellularis by microinjection. enteroids were harvested and analyzed by ihc for l. intracellularis antigen at , , and dpi. l. intracellularis antigen presence was observed at all time points, indicating successful infection with l. intracellularis. unexpectedly, although l. intracellularis antigen was observed in the cytoplasm of enteroid cells, most of the antigen, especially at dpi, was found in the basal region of epithelial cells (figure ). this is in contrast to the location of l. intracellularis in vivo, which is in the apical region of the epithelial cells [ , ] . to determine whether l. intracellularis infection would lead to increased size or secretory activity of infected enteroids, the area of enteroids in each treatment group was measured at , , and dpi. l. intracellularisinfected enteroids did not have higher area compared with sn or dmem treatment groups ( figure a ). representative images of enteroids from each treatment group are shown in figure b . to determine whether l. intracellularis infection induces changes in the proliferation and differentiation of enteroid epithelial cells, as observed in the swine intestine, we evaluated expression of ki- , sox and muc in enteroids harvested at , and dpi relative to expression in enteroids at dpi. there were no significant differences in expressions of any of the genes analyzed in the treatment groups independent of the time point (figure ) . immunofluorescence staining confirmed that none of the treatment groups showed increased ki- expression overtime, although in the l. intracellularis infected group considerable variability in the in ki- expression throughout was noted ( figure ). classical single cell culture systems have been extensively used in studies to understand host-pathogen interactions. although they provide valuable information, these models are limited by their inability to represent the tissue organization observed in vivo since most of the single cell systems are either cancer-derived or transformed immortalized cell lines [ , ] . these limitations make it difficult to assess the proliferative effects of l. intracellularis infection [ ] . the mechanism by which l. intracellularis causes proliferation of intestinal epithelial cells remains unclear. recently, the effects of l. intracellularis infection on the proliferation of monocultures of non-intestinal, non-epithelial cells, as well as intestinal epithelial cells were investigated under various culture conditions [ ] and the findings supported previous observations that cell monocultures infected with l. intracellularis do not accurately represent proliferation observed in lesions in the intestine of affected animals. in vivo, l. intracellularis accesses the cell cytoplasm via the apical membrane and the organisms are observed in the cytoplasm throughout the course of pe. l. intracellularis is more frequently observed in crypt cells than cells of the villus [ , , ] . therefore, we hypothesized that enteroids would be a suitable model to further investigate l. intracellularis pathogenesis in vitro by providing polarized epithelial cell culture with all the crypt and villus epithelial cell types found in the mammalian intestine and similar cell exchange ratio to the in vivo intestine [ ] . enteroids were firstly developed in [ ] and have been used to study intestinal morphophysiology as well as pathogenesis of some enteric diseases [ , , , ] . human enteroids can be obtained by culture of intestinal crypts isolated from biopsies. mouse enteroids are more widely used in research as the ethical and biosafety regulations for human enteroids are more restrictive. additionally, mouse intestinal physiology is well-characterized and molecular tools to study intestinal cell proliferation and differentiation are available. the presence of l. intracellularis dna has been detected in feces of mice trapped in pig farms and in the surroundings of horse farms [ , ] , suggesting that mice are susceptible to infection. furthermore, mice experimentally infected with l. intracellularis develop intestinal lesions that resemble lesions in affected pigs and horses, although the lesions in mice are less extensive, less severe and mostly localized in the large intestine when compared to the lesions in pigs and horses [ ] [ ] [ ] . in addition, l. intracellularis has been successfully propagated in vitro using mccoy mouse fibroblast cells [ , , ] . based on this knowledge, we hypothesized that mouse enteroids may serve as a feasible model to investigate the progression of l. intracellularis infection and the mechanisms involved during cell proliferation. mouse enteroids in the tridimensional culture conditions, as spheres composed of polarized epithelium, have their cellular apical side oriented to the center of the enteroid. hence, l. intracellularis infection in this study was performed by microinjecting the bacterial suspension directly into the lumen of the enteroid. microinjection is a technique that has inherent limitations in controlling the bacterial inoculum, i.e., the exact number of bacteria each enteroid was exposed to. although we plated enteroids to obtain an average of enteroids per well, the actual number of enteroids per well and their size at the time of injection was found to be variable. these technical limitations may have contributed to the high variability observed in our results and constitute one of the limitations of using tridimensional enteroids as models for the study of luminal pathogens. others have used mouse enteroids [ , ] and human enteroids [ , ] to study bacterial pathogenesis using microinjection. however, none of these studies evaluated infection times longer than h [ , ] . because of the nature of l. intracellularis infection, longer durations, i.e., several days, are needed to observe the expected changes. to our knowledge, this is the first enteroid infection experiment reporting successful bacterial infection of enteroids for up to dpi. whether the length of the infection times had a significant effect on the number of bacteria present and variability on the evaluated parameters remains to be defined. one of the most intriguing findings in the present experiment was the unexpected location of l. intracellularis in the infected enteroids. while l. intracellularis antigen was observed at all time points tested ( , and dpi), the antigen was mostly observed localized in the extracellular space proximal to the basal region of the epithelium of the mouse enteroids. in vivo, l. intracellularis organisms are consistently observed on the apical side of the cytoplasm at similar infection time points to those used in this study [ , ] . a possible explanation for this difference in localization is that, in contrast to conditions in vivo, nutrients necessary for enteroid growth and maintenance are placed over the matrigel, in a region that corresponds to the basal lamina in vivo and not directly in the enteroid lumen. we speculate that the gradient of nutrients on the exterior of the enteroid could be causing the l. intracellularis to move towards the nutrients to support their growth. it is also possible that conditions of the enteroid culture environment could result in less permissible conditions for l. intracellularis closer to the lumen. another possibility is that the fibroblasts and immune cells in the basal region of the intestinal epithelium in vivo may have a regulatory effect on l. intracellularis traffic in the intestine, and the lack of those cells in the mouse enteroid culture would modify l. intracellularis traffic in vitro. immunohistochemistry for l. intracellularis is considered the gold standard method for diagnosis of proliferative enteropathy [ , ] . in the present study, we used immunohistochemistry to verify whether the mouse enteroids, microinjected with l. intracellularis suspension (~ l. intracellularis organisms/ml), were infected and to visually demonstrate the level of infection in these microinjected enteroids. our results were not only surprising in regards to the localization of l. intracellularis antigen, as discussed before, but also in regards to the amount and morphology of the stained antigen. generally, l. intracellularis antigen stained by immunohistochemistry is observed as small bacilli in the cytoplasm of cell cultures [ , , ] and in the apical cytoplasm of intestinal epithelial cells of naturally and experimentally-infected pigs. in the present study, instead of observing similar morphology of l. intracellularis antigen, we observed mostly the antigen as a "bundle" accumulated on the basal region of the enteroid cells. nevertheless, occasional well-shaped l. intracellularis antigens were observed in the cytoplasm of some enteroid cells ( figure d ), which is an indication that after the microinjection in the lumen of the enteroid, l. intracellularis organisms gained entrance into the cell cytoplasm. the events involving the changes in the morphology of the antigen and its final location in the basal side of enteroid cells remains unclear and may deserve more investigation. the homeostasis of the intestinal epithelium involves various signaling pathways of which canonical wnt signaling is responsible for maintaining a balance between the pool of undifferentiated cells with proliferation capacity, named transit amplifying cells, and differentiated cells [ , ] . in vitro, when enteroids are cultured in the presence of wnt a in the media, the transit amplifying cells stay undifferentiated for a longer period of time in relation to in vivo conditions [ ] , resulting in enteroids in a pro-proliferative state. in the present study, one of the main objectives was to verify the effects of l. intracellularis on cell proliferation, therefore we removed wnt a from the culture media days before microinjection and during the experiment to allow the epithelial cells to differentiate, and better reproduce the proportion of proliferative cells of the normal intestinal epithelium. however, we failed to detect increased proliferation in l. intracellularisinfected enteroids by rt-qpcr or immunofluorescence. it is possible that the removal of wnt a days before microinjection was not sufficient to reduce proliferation to a level where we could detect changes induced by l. intracellularis. another possible explanation for this lack of difference in proliferation between treatment groups was the small amount of l. intracellularis delivered to the enteroid lumen, thus requiring a longer time for the bacteria to replicate in numbers to produce a detectable change in proliferation markers. the low amount of antigen detected in the microinjected enteroids indicate that l. intracellularis did not encounter an ideal environment for propagation as it does in mccoy cells and in the intestines of mice and other rodents [ , , , , ] . the infection efficacy of enteroids derived from the intestines of more l. intracellularis susceptible species, such as pigs and horses, would help to confirm this hypothesis. changes in the area of enteroids can indicate an expansion on the number of cells or an increase in secretory activity. secretory activity of enteroids has been demonstrated by swollen enteroids as a result of accumulation of luminal secretions after stimulation with forskolin [ ] . in the present study, we expected to observe increased enteroid areas of infected enteroids that could be due to increased numbers of cells (proliferation) or secretory activity. in contrary to our expectations, we observed an increased volume from dpi to dpi on the sn group without changes in proliferation, suggesting the possibility of a secreted product with capacity to induce intestinal secretion. further confirmation of this phenomenon is required. l. intracellularis is a unique bacterium, requiring intracellular conditions and special atmospheric conditions for propagation in vitro. little is known about l. intracelllaris survival in the cellular cytosol, or the host-cellular pathways that are disrupted during l. intracellularis infection. by using mouse enteroids as an in vitro model for l. intracellularis infectioin, our aim was to develop a tool to further define several aspects of l. intracellularis pathogenesis. the localization of l. intracellularis more abundantly in outside the critical threshold of lawsonia intracellularis in pig faeces that causes reduced average daily weight gains in experimentally challenged pigs proliferative enteropathy lawsonia intracellularis infection and proliferative enteropathy in foals recent advances in understanding the pathogenesis of lawsonia intracellularis infections intracellular bacteria of porcine proliferative enteropathy: cultivation and maintenance in vitro effects of lawsonia intracellularis infection in the proliferation of different mammalian cell lines a novel inactivated vaccine against lawsonia 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institutional affiliations the authors acknowledge the technical support provided by marta ferrandis-vila, caitlin kupiec, ashley manicor, amanda g. de souza daniel, erika vasquez, and lacey marshall-lund. of the basal side of the enteroid epithelial cells may indicate nutrients or a microenvironment preferred by l. intracellularis and merits further investigation. likewise, little is known about mouse enteroids maintained with a pathogenic enteric bacterium for a period longer than h [ , ] . more information is needed to determine if long-term infection of enteroids mirrors the dynamics of epithelium in vivo in terms of secretion, differentiation and apoptosis observed in chronic enteric diseases. finally, yet importantly, although we were able to demonstrate infection of mouse enteroids, we did not observe the changes associated with l. intracellularis infection that have been noted in vivo in more susceptible species. testing l. intracellularis infection in enteroids from other more susceptible species will provide further insight to species susceptibility to pathogenesis. authors' contributions tpr contributed to the acquisition, analysis and interpretation of data and preparation of the manuscript. rlm contributed to execution of the experiments, data acquisition and interpretation. yg contributed in the execution of the experiments. cg, fav and mss contributed to the conception, design, data analysis and interpretation of the study. all authors read and approved the final manuscript. the authors disclose receipt of the following financial support for the research, authorship, and/or publication of this article: tpr was supported by the brazilian government sponsoring agency coordenadoria de aperfeiçoamento de pessoal de nível superior (capes). the project was partially supported by nifa min- - (mss). the data collected in this project is shown in our results section. further information can be available upon request. mouse manipulation was approved by the university of minnesota institutional animal care and use committee (iacuc approval number - a). the authors declare that they have no competing interests. key: cord- -fkg d ym authors: wang, leyi; eggett, therese e.; lanka, saraswathi; fredrickson, richard l.; li, ganwu; zhang, yan; yoo, dongwan; bowman, andrew s. title: development of a triplex real-time rt-pcr assay for detection and differentiation of three us genotypes of porcine hemagglutinating encephalomyelitis virus date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: fkg d ym porcine hemagglutinating encephalomyelitis virus (phev) is a single-stranded, positive-sense rna virus. phev mainly causes two types of clinical manifestations representing vomiting and wasting and encephalomyelitis in piglets. however, our recent findings provide strong evidence that phev can also cause respiratory disease in older pigs. genomic analysis of new phev strains identified in our former study further classifies phev into three genotypes. detection and differentiation of these new mutants are critical in monitoring phev evolution in the field. in the present study, we report the development of a triplex real-time rt-pcr assay for detection and differentiation of three phev genotypes, , , and . three sets of primers and probes were designed; one set of primers and probe targeting the conserved regions of the ′ end nucleocapsid for detection of all three genotypes and another two sets of primers and probes targeting the regions of ns with different patterns of deletions for detection of both genotypes and , or genotype only. genotype was positive when two probe dyes showed signals, genotype was positive when only one probe dye showed a signal, and genotype was positive when all three probes showed signals. the detection limit of the developed triplex real-time rt-pcr was as low as or dna copies for three sets of primers and probes. the specificity test showed no cross reaction with other porcine viruses. positive field-samples were correctly typed by this new assay, which was further confirmed by dna sequencing. the triplex real-time rt-pcr provides a rapid and sensitive method to detect and differentiate all three us genotypes of phev from clinical samples. porcine hemagglutinating encephalomyelitis virus (phev) is one of six known porcine coronaviruses (covs) causing diseases in pigs (gong et al., ; wang and zhang, ) . phev typically affects pigs less than three weeks of age and the clinical syndromes include the vomiting and wasting disease (vwd) and encephalomyelitis (quiroga et al., ) . phev was first isolated from suckling piglets suffering from encephalomyelitis in canada in (greig et al., ) . phev has since been identified in many countries of europe, asia, and america (cartwright et al., ; dong et al., ; forman et al., ; hirahara et al., ; mengeling, ; pensaert and callebaut, ; quiroga et al., ; rho et al., ) . although phev infection has been endemic in different countries for decades and the primary route of phev infection is through upper and lower respiratory tracts, only few reports suggest an association of the virus with respiratory disease in swine (cutlip and mengeling, ) . recently, we have demonstrated that phev caused an influenza-like illness (ili) in affected pigs and provided evidence for the role of phev as a respiratory pathogen (lorbach et al., ) . in that study, a significantly higher positive rate . % ( of pigs) was observed in pigs at the michigan fairs compared to . % ( of pigs) in indiana and ohio, indicating the phev-associated epizootic behavior in the michigan fairs (lorbach et al., ) . phev is a single-stranded positive-sense rna virus and belongs to the betacoronavirus genus of the coronaviridae family. the first two https://doi.org/ . /j.jviromet. . . received february ; received in revised form april ; accepted april thirds of the phev genome contain two large open reading frames (orf a and orf b) encoding the replicases, and the remaining part of the genome encodes six structural proteins (hemagglutinin-esterase protein, he; spike glycoprotein, s; envelope protein, e; membrane protein, m; nucleocapsid protein, n, and n ) and three non-structural (ns) proteins (ns , ns . , and ns . ). previous genomic characterization and analysis reveal that phev is closely related to bovine cov and human cov oc , and have identified truncated ns and ns . genes in the phev-vw strain (vijgen et al., ) . prior to our study (lorbach et al., ) , only one complete phev genome sequence (phev-vw ) was available in genbank. we completed the full genomic sequencing of phev isolates, and the analyses of complete genomic sequences showed that phev formed three distinct genotypes. interestingly, genotypes had deletions in ns and ns . , while genotype showed no deletion and genotype has a large deletion in the ns gene (lorbach et al., ) . the prevalence and distribution of three genotypes remain unknown in pigs in the united states. the traditional real-time rt-pcr (rrt-pcr) test currently used in diagnostic laboratories does not differentiate emerged phev from classical phev strains (lorbach et al., ) . the triplex rrt-pcr developed in the present study will fulfill this purpose and can be used to monitor phev of different genetic genotypes and differentiate between them. complete genomic sequences of all phev strains available in the genbank database were collected and aligned using clustalw of mega . . program. based on the conserved and variable regions of sequences, three sets of primers and probes were designed; the first set targeting the ′ non-coding region and n to detect all three genotypes (n-g ), the second set targeting the ns region to detect genotypes and (ns -g ) that have deletions for genotype , and the third set targeting the regions of ns for genotype (ns -g ) that there are deletions for genotypes and (fig. ) . the probes were labeled with different dyes for multiplexing purpose (table ) . rna was extracted using qiagen one-for-all vet kit (valencia, ca, usa) according to the manufacturer's instructions. the extracted rna was eluted in μl of elution buffer and stored at - ℃ until use. concentrations of rna samples were measured using qubit fluorometer. step rt-pcr kit (valencia, ca, usa) was used for amplification in the abi real-time pcr system (applied biosystems, ca, usa). the assay was performed in a -μl reaction mixture containing μl of × reaction buffer, μl of each primer ( μm), μl of each fluorogenic probe ( μm), μl of dntp ( mmol), μl of enzyme mix, . μl of purified rna, and an appropriate volume of rnase-free distilled water (dh o). the thermocycler amplification conditions were °c for min, °c for min, and cycles of °c for s, °c for s, and °c for s. rna was extracted from positive samples with known genotypes of sw (genotype ), sw (genotype ), and sw (genotype ), and then quantified using qubit fluorometer. the n gene was amplified using rna of sw (genotype ) and the primer set of phev n f pcr and phev ncr r, and the ns gene were amplified using rna of above three samples and the primer set of phev poly-f pcr and phev he-r pcr (table ). the pcr products were cloned into the pcr . vector (invitrogen, carlsbad, ca, usa). the detection limit of the triplex rrt-pcr assay was determined by testing -fold serially diluted positive phev rnas ( sw and sw ) and plasmid dnas (pcr . - sw -n, pcr . - sw -ns , and pcr . - sw -ns ) in duplicate. intra-specificity of the triplex real time rt-pcr assay was determined using a single probe of n-g , ns -g , and ns -g for detection of all three genotypes, both genotype and , and genotype only, respectively. inter-specificity of triplex real time rt-pcr assay was examined using various swine viruses available in our laboratory. these viruses include porcine reproductive and respiratory syndrome virus, swine influenza virus (h n ), transmissible gastroenteritis virus, porcine epidemic diarrhea virus, porcine deltacoronavirus, porcine circovirus , and senecavirus a. in the assay, . μl of rna or dna samples were used, . μl of genotype strain sw was used as a positive control, and . μl distilled water was used as a negative control. positive controls for each genotype were used in each run of the triplex real-time rt-pcr. nasal swab and nasal wipe samples were collected from exhibition swine at shows during − . seventyfive samples testing positive for phev were selected for this study and processed for rna extraction. rna samples were first tested for phev by a singleplex rrt-pcr as previously reported (lorbach et al., ) . if a test appeared positive, the triplex rrt-pcr was used to differentiate three genotypes of phev and a conventional pcr using a primer set of pehv poly-f pcr pehv he-r pcr (table ) was applied to amplify ns region. the amplicons were subjected to dna sequencing using a sanger method (acgt, inc.) for confirmation. based on the sequence alignment and analyses of all complete genome sequences of different genotypes, a conserved region in the ′ end of n gene was selected for detection of all three genotypes of phev, whereas ns gene with different patterns of deletions was used to design two sets of primers and probes to detect both genotypes and , or genotype only (fig. ). if only one fam signal is detected, it indicates genotype ; if two signals (fam and cy ) are detected, it indicates genotype ; and if all three signals (fam, cy , joe) are detected, it indicates genotype of phev (table ) . the triplex rt-pcr assay specifically detected genotype by the fam probe only, genotype by the fam and cy probes, and genotype by the fam, cy , and joe probes. by contrast, the triplex rt-pcr did not cross-react with any other swine viruses (porcine reproductive and respiratory syndrome virus, swine influenza virus (h n ), transmissible gastroenteritis virus, porcine epidemic diarrhea virus, porcine deltacoronavirus, porcine circovirus , and senecavirus a) used in the study. the sensitivity of the triplex rrt-pcr was determined through fold serial dilutions of rnas of known phev genotypes ( sw for g and g , and sw for g ) and plasmids pcr . - sw -n for g , pcr . - sw -ns for g , and pcr . - sw -ns for g in duplicate. the detection limit was . ng/μl and dna genome copies for n-g with ct value as a cutoff, and . ng/μl and dna genome copies for ns -g and -g with ct values and as cutoffs, respectively (fig. ). both replicates of n-g , ns -g , and ns -g produced positive results at or dna copies. there are strong linear correlations ( r > . ) between c t values and amount of viral rna and between c t values and corresponding amount of plasmid copy numbers for different targets ( fig. a, b , and c). a total of positive samples were tested by the singleplex rrt-pcr and were run again by the triplex real-time rt-pcr. fifteen samples appeared positive for genotype , samples were positive for genotype , and were positive for genotype . the sanger sequencing using pehv poly-f pcr-pehv he-r pcr primer set confirmed the genotyping results. to be noted, out of genotype strains have a primers and probes used in the study. different deletion pattern from that originally identified (fig. ) . in recent years, with the help of molecular technology, more novel coronaviruses have been identified in pigs. so far, there are six known porcine coronaviruses including transmissible gastroenteritis coronavirus (tgev), porcine epidemic diarrhea virus (pedv), porcine deltacoronavirus (pdcov), porcine respiratory coronavirus (prcv), bat hku -like porcine enteric alphacoronavirus (peav), and phev. these porcine coronaviruses cause neurologic (phev), digestive (tgev, pedv, pdcov, peav, phev), and respiratory diseases (prcv, phev). vwd and encephalomyelitis caused by phev are common and have experimentally been reproduced in pigs (mengeling and cutlip, ) . in contrast, only a few studies including ours report that phev causes respiratory problems in pigs. in our previous study, we reported that influenza-like illness in market-age pigs was strongly linked to phev (lorbach et al., ) . field and experiment infection studies are needed to elucidate the pathogenic mechanism of respiratory diseases caused by phev. in addition to the uncommon clinical presentation of phev, we show that there are three different genotypes cocirculating in the field in us. different deletion patterns (small, large, none) in ns are observed in genotypes , , and , respectively. genotype has a small deletion in ns . . genotype was predominant in pigs with respiratory disease from michigan fairs (lorbach et al., ) . it remains unknown if different genotypes have similar virulence. to further type samples positive for phev, we have developed a triplex rrt-pcr using one fam dye for genotype , the fam and cy dyes for genotype , and the fam, cy , and joe dyes for genotype . our data indicate that the triplex rt-pcr is highly specific with no cross-reaction with other known swine viruses and is sensitive with a detection limit ranging between . − . ng/μl of viral rna and - dna genome copies. furthermore, the developed assay correctly subtyped phev-positive samples, which was confirmed by sanger sequencing. it should be noted that genotype strains previously identified in our previous study have defective ns gene, and out of genotype strains detected in the present study have a larger deletion in ns , resulting in the complete deletion of ns . furthermore, out of genotype strains identified in the study have a different deletion pattern from other strains in genotype previously identified (fig. ) . similar to the finding that genotype was predominant in pigs of the michigan fair, genotype was also predominant ( %) in samples tested in the present study, whereas positive rates for genotype and were % and %, respectively. future studies are needed to address whether the large deletion in ns benefits virus replication or not. in contrast to conventional singleplex real time rt-pcr, the triplex real time rt-pcr that we have developed in this present study can be used to monitor phev genotypes and potentially to identify new variants if test results cannot be explained as expected. few limitations of our study need to be noted. for the specificity test, not all porcine coronaviruses were tested due to unavailability of prcv and peav. since prcv is a spike deletion mutant of tgev and the triplex assay developed did not show the cross reaction with tgev, it should cross react with prcv. for peav, blast search of sequences of primers and probes used in the triplex pcr showed no any hit to genome of peav, indicating that a cross reaction should not occur. the observed low efficiency of the triplex real-time rt-pcrs in the study was due to unknown factors including amount of pcr reagents and design of primers and probes. in summary, we have developed a triplex real time rt-pcr to detect and differentiate all three genotypes of phev. this triplex rt-pcr assay is highly specific and sensitive. the developed assay may be used to monitor different genotypes circulating in the field. vomiting and wasting disease of piglets lesions induced by hemagglutinating encephalomyelitis virus strain n in pigs identification and genetic characterization of porcine hemagglutinating encephalomyelitis virus from domestic piglets in china haemagglutinating encephalomyelitis virus infection of pigs a new bat-hku -like coronavirus in swine a hemagglutinating virus producing encephalomyelitis in baby pigs isolation of hemagglutinating encephalomyelitis virus from respiratory tract of pigs in japan porcine hemagglutinating encephalomyelitis virus and respiratory disease in exhibition swine incidence of antibody for hemagglutinating encephalomyelitis virus in serums from swine in the united states pathogenicity of field isolants of hemagglutinating encephalomyelitis virus for neonatal pigs characteristics of a coronavirus causing vomition and wasting in pigs hemagglutinating encephalomyelitis coronavirus infection in pigs detection and genetic analysis of porcine hemagglutinating encephalomyelitis virus in south korea evolutionary history of the closely related group coronaviruses: porcine hemagglutinating encephalomyelitis virus, bovine coronavirus, and human coronavirus oc animal coronaviruses: a brief introduction key: cord- -km qcmlh authors: fehr, anthony r.; jankevicius, gytis; ahel, ivan; perlman, stanley title: viral macrodomains: unique mediators of viral replication and pathogenesis date: - - journal: trends in microbiology doi: . /j.tim. . . sha: doc_id: cord_uid: km qcmlh viruses from the coronaviridae, togaviridae, and hepeviridae families ​all contain genes that encode a conserved protein domain, called a macrodomain; however, the role of this domain during infection has remained enigmatic. the recent discovery that mammalian macrodomain proteins enzymatically remove adp-ribose, a common post-translation modification, from proteins has led to an outburst of studies describing both the enzymatic activity and function of viral macrodomains. these new studies have defined these domains as de-adp-ribosylating enzymes, which indicates that these viruses have evolved to counteract antiviral adp-ribosylation, likely mediated by poly-adp-ribose polymerases (parps). here, we comprehensively review this rapidly expanding field, describing the structures and enzymatic activities of viral macrodomains, and discussing their roles in viral replication and pathogenesis. macrodomains were discovered in togaviruses, coronaviruses, and the hepatitis e virus over years ago. they were called the 'x' domain because they had no known function. about years later, several macrodomain structures were determined. they consist of central b-sheets surrounded by a-helices and bind to adp-ribose and its derivatives. they were named after the structurally homologous domain of the macroh a histone. originally described as adp-ribose- -phosphatases, both cellular and viral macrodomains enzymatically remove mono-and poly-adp-ribose from proteins, supporting the notion that protein adp-ribosylation is a component of the antiviral response. chikungunya virus and hepatitis e virus macrodomains are critical for replication, while the coronavirus macrodomain both blocks the innate immune response and separately promotes in vivo replication. recently, more defined roles for the marylating parps have been described, including a prominent role in the cellular stress response. parp regulates the er-stress response [ ] , and several parps are present in cytoplasmic stress granules (parps , , , , and a) [ ] . adp-ribosylation of argonaute proteins in stress granules by an unknown parp inhibits rna interference [ , ] . finally, parps have both antiviral and proviral activities (box ). as with any ptm, adp-ribose must be removed in a timely manner to prevent an untoward response and to recycle adp-ribose components ( figure ). the cellular enzymes that remove par from proteins are par glycohydrolase (parg), and to a lesser extent adp-ribosyl hydrolase [ , ] . the importance of removing par is demonstrated by the embryonic lethality of mice with genetic deletion of parg [ ] . however, parg only cleaves between the unique glycosidic bonds of par [ ] , and thus it was unclear how the terminal adp-ribose moiety was removed from the modified protein. this conundrum was resolved by the discovery that cellular macrodomain-containing proteins, including terminal adp-ribose glycohydrolase (targ ), macrod , and macrod removed this terminal adp-ribose [ ] [ ] [ ] . the human genome contains at least macrodomain-containing genes, but the substrate specificity and physiological roles of these proteins are largely unknown. almost three decades ago, bioinformatics approaches identified a unique conserved gene domain within three viral families, the togaviridae, coronaviridae, and hepeviridae [ ] [ ] [ ] . all of these families are positive-sense rna viruses and cause disease in humans and animals (box ). the domain was named the 'x' domain as its structure and function was unknown. these domains were defined as 'macrodomains' after crystal structures of both cellular and viral macrodomains revealed a core fold homologous to the nonhistone part of the macroh a protein [ ] [ ] [ ] . these macrodomains were shown to have phosphatase activity and were named adp-ribose- -phosphatases (adrps) [ , [ ] [ ] [ ] [ ] . despite the demonstrated adrp activity, it was unclear why viruses would devote a gene product to converting adp-ribose- -phosphate to adp-ribose. the discovery that macrodomains de-adp-ribosylate proteins strongly suggested that their major role was in reversing antiviral adp-ribosylation. however, this hypothesis has yet to be proven. as all three viral families that contain macrodomains include established human pathogens, understanding how box . the interplay between parps and virus infections several studies have found parps to have both proviral and antiviral activities. using parp inhibitors, several dna viruses, such as herpesviruses, poxviruses, and adenoviruses, have been found to require adp-ribosylation for optimal replication [ ] [ ] [ ] . this is not surprising given the importance of parps in cellular dna replication and repair. the hsv- icp protein enhances parylation by degrading par-glycohydrolase, which removes par from proteins [ ] . furthermore, tiparp was shown to adp-ribosylate tbk- and decrease ifn-b production, leading to enhanced replication of several rna and dna viruses [ ] . the best known antiviral parp, parp , also known as zap (zincantiviral protein) restricts the replication of several rna viruses by targeting viral or host rna for degradation [ ] . parp contains mutations in its parp domain, making it enzymatically inactive, indicating that its antiviral activity does not require adp-ribosylation [ ] . however, parp is known to be important for stress granule adp-ribosylation, and it also contributes to the adp-ribosylation of the overexpressed pa and pb proteins of influenza a virus which leads to their ubiquitination and proteasomal degradation [ , , ] . thus, while it does not directly adp-ribosylate proteins, it appears that parp participates in this process, possibly by binding to active parps and recruiting them to target proteins. similarly, parps , , and directly inhibit alphavirus replication, but in a parp-independent manner, as catalytically inactive mutants still blocked replication [ ] . overexpression of parp has also been shown to inhibit the replication of several negative-strand rna viruses, including vesicular stomatitis virus (vsv) [ ] . parp represses the transcription of integrated retroviruses in a chicken lymphoblastoid cell line, again in a parp-independent manner [ ] . in addition, multiple parps are interferon-stimulated genes (isgs), and in silico analyses indicate that parps , , , and are rapidly evolving, a hallmark of antiviral defense proteins [ ] . however, despite these data, antiviral adpribosylation of cellular or viral targets have yet to be identified. macrodomains contribute to replication and pathogenesis is important. here we comprehensively review the structures and enzymatic activities of these viral macrodomains, describe their potential functions, and discuss future challenges in understanding how macrodomains impact virus biology. macrodomains are conserved, $ amino acid protein domains present in all kingdoms of life. based on phylogeny, macrodomains are divided into different classes. most rna virus macrodomains fall into the macrod-like family, which includes human macrodomains macrod and macrod [ ] . the exceptions are the sars coronavirus (sars-cov) unique macrodomains (mac /mac formely known as sud domains), which we will not further discuss as they are distinct from the conserved macrodomain (mac ). macrodomains adopt a globular, mixed a/b/a sandwich fold, first shown when the structure of the archaeal macrodomain af [ ] hepatitis e virus (hev) is an emerging pathogen and is probably the most common cause of acute viral hepatitis in the world, with as many as million infections per year. mortality rates for acute infections are $ % for normal adults but as high as % for pregnant women in their third trimester, also often resulting in stillbirths of the fetus. hev, like hcv, can also lead to chronic hepatitis and cirrhosis [ ] . in developing countries, hev is spread by the fecal-oral route, while in developed countries hev is generally acquired through animal reservoirs, most likely pigs. treatment for hev infection includes either monotherapy with ribavirin or combination therapy of pegylated ifn and ribavirin. additionally, two vaccines have been developed for hev that have shown to be effective [ ] . the togaviridae are divided into two genera, the alphaviruses and the rubiviruses. rubella virus, a rubivirus, causes a congenital syndrome characterized by multiorgan birth defects. however, this disease has largely disappeared in the usa and other developed countries since an effective vaccine was developed. alphaviruses, which include chikungunya virus and ross river virus, are transmitted by mosquitoes and can infect both vertebrate and invertebrate species. in humans, these infections can lead to a number of different pathologies, the most common being severe arthritis or rash [ , ] . there are no alphavirus vaccines currently available. coronaviruses (covs) cause a wide variety of diseases in both humans and mammals. covs such as porcine epidemic diarrhea virus (pedv), bovine cov, and infectious bronchitis virus (ibv) cause severe disease in veterinary animals. while originally only thought to be a cause of the common cold in humans, recent epidemics have demonstrated that covs can also cause severe human disease. severe acute respiratory syndrome cov (sars-cov) and middle east respiratory syndrome cov (mers-cov) have emerged in the last years, causing serious respiratory disease in humans with high mortality rates ( - %). both viruses probably originated in bats and were transmitted to humans through animal reservoirs. mers-cov is endemic in camels in the middle east and will likely continue to cause lethal disease for many years [ ] . there are no approved vaccines or antiviral agents for human covs; however, several vaccines have been licensed for cov-mediated veterinary diseases [ ] . including those of alphaviruses and coronaviruses (covs), but not from hepatitis e virus, have been determined, with pdb entries available (www.rcsb.org). the globular macrodomain contains a conserved cleft that has been shown to bind adp-ribose [ ] , a feature that has been confirmed for viral macrodomains by numerous biochemical studies [ , [ ] [ ] [ ] . some of the most conserved residues of macrodomains are located at the surface and near the adpribose binding cleft ( figure ). based on adp-ribose-macrodomain complex structures, these conserved residues provide high specificity and affinity for adp-ribose. the distal ribose moiety is especially tightly coordinated by key residues found in loop (between b-sheet and a-helix ) and loop (between b-sheet and a-helix ) ( figure ). loop contributes backbone contacts to the adp-ribose a-phosphate and and oh groups of the distal ribose. in addition, the conserved asparagine residue (n in sars-cov) makes a hydrogen bond to the oh group. in cov macrodomains, the conserved histidine residue (h ) contributes to adp-ribose binding through a coordinated water. alphavirus macrodomains have a conserved cysteine (c ) that could fulfil a similar role, whereas human macrod contains an aspartic acid (d ) that makes a hydrogen bond with the oh group. loop contributes backbone contacts primarily to the b-phosphate of adp-ribose. in addition, isoleucine (i ) and phenylalanine (f ) residues provide van der waals contacts and are thought to direct orientation of the distal ribose [ , , , ] . in some macrodomains, tyrosine replaces phenylalanine and forms a hydrogen bond to the oh of adp-ribose. macrodomains with a threonine before the g residue contribute an additional hydrogen bond to the ʹ oh of the proximal ribose (e.g., t in chikungunya virus (chikv)). besides the two loops, other notable adp-ribose binding residues are d (n in sindbis virus (sinv) and s in hepatitis e virus (hev)), which contacts the amino group of adenine, and n (r in chikv and f in macrod ), which interacts with the adenine ring via p-charge or p-p interactions. depending on the adp-ribose contacting residues, the affinities for adp-ribose by different macrodomains vary, greatly with dissociation constants ranging from . to above mm [ , , , ] . binding of adp-ribose is not the sole property of macrodomains. the first enzymatic activity ascribed to macro-d macrodomains was the dephosphorylation of adp-ribose- -phosphate (a by-product of trna splicing reactions) identified through a biochemical-genomic screen in yeast [ ] . this phosphatase activity was also detected for viral macrodomains [ , , , , ] . however, the enzymatic activity of macrodomains is low, the activities vary across different virus species, and no easy explanation exists as to why adp-ribose- phosphate hydrolysis would benefit these viruses. therefore, other functions of viral macrodomains were considered. they include but are not limited to: binding adp-ribosylated proteins, poly-adp-ribose, rna or other nucleic acids [ ] . the discovery that macrod-like macrodomains are de-adp-ribosylating enzymes [ , ] suggested that viral macrodomains could have the same activity. indeed, li et al. were the first to show both de-mar-ylating and de-par-ylating activities for macrodomains from members of all three macrodomain-containing virus families [ ] . fehr et al. confirmed and additionally tested the effects of several mutations on the sars-cov macrodomain de-adpribosylating activity [ ] . eckei et al. demonstrated de-adp-ribosylating activity of macrodomains from different alphaviruses, addressing both de-mar-ylating and de-par-ylating activities [ ] ; however, it is still unknown which features of the macrodomain limit its activity to de-mar-ylation [ ] and which are required for de-par-ylation [ , ] . finally, mcpherson et al. showed de-mar-ylating activity for the chikungunya virus macrodomain and determined that this macrodomain removes adp-ribose from acidic residues but not from lysines [ ] . together, these four studies strongly suggest that de-adp-ribosylation is likely the biochemical function of viral macrodomains. adp-ribosylated proteins are linked to adp-ribose through the position, reminiscent of adp-ribose- -phosphate, explaining how macrodomains could have both activities. these latest studies have shifted the focus to de-adp-ribosylation, but other potential macrodomain functions should not be neglected, as the catalytic mechanism of de-adp-ribosylating macrodomains is not fully understood and the residues impacting de-adp-ribosylation often affect adp-ribose binding and other catalytic activities (e.g., adpribose- -phosphate hydrolysis). macrodomain mutations experimentally shown to disrupt catalytic function are summarized in table . the corresponding residues in different viral macrodomains are identified by sequence alignment and their relation to the adp-ribose substrate are visualized in figure . the mutational approaches interfere with macrodomain function by (i) reducing adp-ribose binding affinity (e.g., d a (sars-cov), t a (chikv), y f (chikv)), (ii) introducing steric hindrance (e.g., g v (sars-cov), g a (hev), g e (chikv)), or (iii) displacing potential catalytic water and reducing conformational strain on the distal ribose (e.g., g e+v r +y n (chikv)). the most common studied mutation is substitution of the highly-conserved asparagine to alanine (n a in sars-cov). this asparagine coordinates the oh of distal ribose and was proposed to be essential for catalysis. while the n a mutation abolishes the catalytic activity of sars-cov macrodomain [ , , ] , it reduces but does not abolish the catalytic activity of other cellular and viral macrodomains [ , , ] . such apparent discrepancy could be explained by reduced adp-ribose binding by the asparagine-to-alanine mutant since viral macrodomains, especially the covs, have a lower affinity for adp-ribose ( mm in sars-cov compared to . mm in human macrod ). the loss of a single hydrogen bond between the cov macrodomain and adp-ribose could reduce its affinity to a point where the macrodomain enzymatic activity is nondetectable. an alternative explanation is that different catalytic mechanisms exist among macro-d type macrodomains. despite these uncertainties, the strict conservation of this asparagine and the effects of its mutation on in vitro activity and virus fitness make it a valuable tool for studying viral macrodomains. the fact that viral macrodomains are generally required for virulence (see section below) suggests that small-molecule inhibition of macrodomains could be a novel therapeutic approach to preventing severe alphavirus, hepatitis e virus, or coronavirus-induced disease. one concern in considering the development of such molecules is the potential difficulty in specifically targeting the viral macrodomains but not the cellular macrod or macrod proteins. while such concern is warranted, there is potential for exploiting differences between human and viral macrodomains. residues contributing to distal ribose oh coordination differ between human and viral macrodomains. in addition, the space around the proximal ribose and the surface residues contacting the adenine-ring might be additional areas of interest for specific small-molecule design. the potential for virus escape by mutating at these differing structural information is available, uniprot retrieved sequences of the macrodomains were used and are indicated as (seq.). secondary structure elements are schematically depicted above the alignment, and the numbering is for a generic macrodomain (i.e., not including additional helices or sheets present in some but not all macrodomains). asterisks and red boxes indicate highly conserved, mostly substituted or key catalytic determinant residues of viral macrodomains. magenta-shaded boxes depict the degree of conservation. g e | d | q À reverted to wt (g e) [ ] g a | s + | +/À # virulence & replication [ ] v a | f +/À [ ] v e À [ ] residues could hamper the feasibility of inhibitor design and usability, but these concerns could be addressed experimentally. the observation that even a % reduction in viral macrodomain activity greatly reduced virus fitness suggests that viruses would struggle to escape potential inhibitors [ ] . if the risk of off-target effects in humans is too high, such inhibitors could be used in veterinary medicine. in recent years, significant progress has been made in deciphering the role of viral macrodomains, largely due to the advent of reverse genetic methods for these viruses. in this section, we review the known and potential roles for viral macrodomains. hev is a nonenveloped positive-sense rna virus with a genome of . kb that contains three open reading frames: orf , orf , and orf . the hev macrodomain is a part of the multidomain orf gene product of hev (figure ) [ ] . in a study on the orf gene products of hev, nan et al. found that orf inhibited poly i:c-dependent induction of interferon-b (ifnb) [ ] . using overexpressed proteins, only the papain-like cysteine protease and the macrodomain could block ifn-b expression. importantly, the hev macrodomain inhibited both ikke overexpression-induced ifn-b expression and interferon regulatory factor- (irf- ) phosphorylation. since the protein kinase ikke phosphorylates irf- , these data suggest that the macrodomain targets irf- . however, macrodomain inhibition of ikke-induced ifn-b expression was significantly less than its inhibition of poly i:c-induced ifn-b expression, suggesting that other mechanisms are involved. in a study by ojha and lole, the hev macrodomain was found to interact with the light-chain subunit of human ferritin (ftl), which led to a reduction of secreted ferritin [ ] . ferritin is an iron-binding protein that helps the cell store free iron. ferritin complexed with iron can activate nf-kb-dependent ifn production, suggesting that this interaction could be a mechanism of innate immune suppression [ ] . neither of these studies addressed whether the ability of the hev macrodomain to bind or hydrolyze adp-ribose was important for these observations. utilizing full-length replicon-based reverse genetic systems with either a gfp or luciferase reporter, parvez et al. and li et al. tested the role of the macrodomain in hev replication [ , ] . in these studies, several amino acids in the active site were mutated (n , n , h , g , g , g ) and tested for their ability to replicate. in the parvez study, all mutations produced wild-type levels of viral rna, but only n and g mutants were viable as measured by gfp expression. li et al. found that replication strongly correlated with the enzymatic activity of each mutation they created. these results demonstrate that the hev macrodomain is important for replication and acts after rna replication, likely prior to translation of viral proteins. another study found that the hev macrodomain interacted with the viral methyltransferase and orf using yeast- -hybrid and coimmunoprecipitation methods [ ] . it will be of interest to determine if these proteins are adp-ribosylated and whether these interactions are important for replication. togaviruses are positive-sense enveloped viruses with - kb rna genomes. the first twothirds of the genome encodes four nonstructural proteins while the final one-third of the genome encodes structural genes. the macrodomain is located at the n-terminus of nsp ( figure ) [ ] . in , park and griffin published a study focusing on two asparagine mutants (n a and n a) in the adp-ribose binding pocket of the sinv macrodomain [ ] . these mutant viruses replicated normally in bhk- cells but were modestly attenuated in neuronal cells and significantly attenuated in -week-old mice. upon replication in neurons, the n a mutation changed to threonine or aspartic acid, and a second site mutation of a glutamic acid at position to a glycine appeared. most macrodomains have a glycine at this position, suggesting that this mutation may enhance macrodomain activity. it was unclear how these mutations affected this protein, as neither mutant affected par binding and it was unknown whether alphaviruses' macrodomains had de-adp-ribosylating activity. however, recently it was demonstrated that chikv contains these activities [ , ] , making it likely that these mutations within the adp-ribose-binding pocket of the sinv macrodomain attenuated its enzymatic activity. as opposed to sinv, mutations in the active site of the chikv macrodomain had severe effects on virus replication [ ] . three recombinant viruses with mutations that severely affected both adp-ribose binding and hydrolase activity were unrecoverable as they quickly reverted to wild type (d a, g e, g e). the g e mutation even reverted when transfected into an aedes albopictus cell line, suggesting that antiviral adp-ribosylation also occurred in mosquito cells. other mutations that partially ablated hydrolase activity and adp-ribose binding (t a, g s, g a) replicated at to logs lower efficiency than wild-type virus in a neuronal cell line . finally, one mutation, y a, was identified that had enhanced adp-ribose binding but decreased hydrolase activity. this mutation was also significantly attenuated in nsc- cells, demonstrating that adp-ribosyl hydrolase activity alone is important for replication. in this study, de-adp-ribosylating activity, replication, and virulence did not perfectly correlate, suggesting a role for adp-ribose binding in virulence. of note, an additional mutant, r a, which had normal levels of adp-ribose binding and hydrolase activity in vitro, quickly reverted in tissue culture cells. this mutation could affect alternative functions of the macrodomain. for example, studies have found that mutations in the adp-ribose binding domain of the veev macrodomain could compensate for mutations in other regions of the genome; however, it is unclear whether these activities were related to de-adp-ribosylating activity or other functions [ ] [ ] [ ] . additionally, lulla et al. found that c-terminal amino acids of the semliki forest virus (sfv) macrodomain are required in the processing of the nsp / cleavage site, which could explain the defect of the r a mutation [ ] . covs are large, positive-sense enveloped rna viruses with $ kb genomes. the n-terminal two-thirds of the cov genome contains nonstructural proteins (nsps) while the c-terminal one-third of the genome contains the structural and accessory genes. the conserved macrodomain is located at the n-terminus of the multidomain nsp protein (figure ) [ ] . in sars-cov and possibly other covs, two additional macrodomains (mac /mac ) immediately follow the conserved macrodomain (mac ) [ ] . mac and mac do not bind or hydrolyze adpribose; in contrast, they bind g quadruplexes [ ] . using a sars-cov replicon system, kusov et al. found that mac was essential for replication [ ] . most studies of the conserved cov macrodomain (mac ) function have used recombinant viruses with the highly-conserved asparagine mutated to an alanine. this mutation has small to no effects on cov replication in cell culture [ , , , , ] . also, deletion of the macrodomain had no effect on replication of a sars-cov replicon [ ] . in marked contrast, several studies have found that the cov macrodomain is essential for viral pathogenesis. eriksson et al. first showed that the macrodomain was required for murine hepatitis virus (mhv) strain a -induced hepatitis [ ] , while fehr et al. demonstrated that mutation of the macrodomain of an encephalitic strain of mhv and of mouse-adapted (ma) sars-cov resulted in attenuation in vivo [ , ] . in all cases, the mutation was associated with reductions in viral load. it was also shown by both eriksson et al. and kuri et al. that the macrodomain could repress ifn production; however, it was unclear if this occurred in vivo and whether it was important for pathogenesis [ , ] . fehr et al. found that the sars-cov macrodomain repressed ifn and cytokine production both in mice and in a bronchial epithelial cell line [ ] . a coinfection with wild type and the macrodomain mutant sars-cov demonstrated that the enhanced interferon response was partially protective in mice. also, the reduced viral load observed in sars-cov-infected mice was independent of the ifn response, as viral loads were not increased in ifnar À/À mice infected with macrodomain mutant virus. in combination, these studies led to the conclusion that the sars-cov macrodomain promotes replication in vivo and independently suppresses the ifn response, with both functions playing a role in pathogenesis. despite the discovery that viral 'x' domains closely resemble cellular macrodomains, the function of these domains remained an enigma until recent discoveries showed that viral macrodomains remove adp-ribose from proteins. while this activity has not been demonstrated during infection, the publications discussed in this review strongly suggest that this is the case. as such, the primary focus of macrodomain research will likely shift from basic functional and biochemical studies to identifying antiviral parps and the protein targets of viral macrodomains (see outstanding questions). while identifying adp-ribosylated proteins has proven difficult in the past, new methods for detecting adp-ribosylation should lead to important discoveries in this field [ ] . these studies will significantly enhance our understanding of these proteins, which could lead to new approaches for treating and preventing disease caused by macrodomain-expressing pathogens. do viral macrodomains counteract antiviral adp-ribosylation? which parp(s) is responsible for antiviral adp-ribosylation? do invertebrates have antiviral parp activity that is counteracted by macrodomains? what are the adp-ribosylated protein targets of viral macrodomains? are targets mono-or poly-adp-ribosylated? how does adp-ribosylation impact these targets? what are the determinants of mono-versus poly-adpribose binding/removal? do all viruses require macrodomain enzymatic activity or is mar/par binding sufficient to promote virus fitness/virulence? what effects do mutations in viral macrodomains have on de-adp-ribosylation kinetics? how do viral macrodomains acquire specificity? do regions outside of the adp-ribose binding pocket contribute to interactions with targets? can a macrodomain from one virus family substitute for a different one? is the development of small-molecule inhibitors specific for viral macrodomains to repress viral pathogenesis feasible? could viruses escape these inhibitors? would these inhibitors also affect mammalian enzymes, leading to significant side effects? nicotinamide mononucleotide activation of new dna-dependent polyadenylic acid synthesizing nuclear enzyme adp-ribosylation: new facets of an ancient modification a family of killer toxins. exploring the mechanism of adp-ribosylating toxins toward a unified nomenclature for mammalian adp-ribosyltransferases family-wide analysis of poly(adp-ribose) polymerase activity readers of poly(adp-ribose): designed to be fit for purpose histone adp-ribosylation in dna repair, replication and transcription wnt pathway activation by adp-ribosylation parp is a tail-anchored endoplasmic reticulum protein required for the perk-and ire 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identification of rubi-and aphthovirus proteases and delineation of a novel conserved domain associated with proteases of rubi-, alpha-and coronaviruses computer-assisted assignment of functional domains in the nonstructural polyprotein of hepatitis e virus: delineation of an additional group of positive-strand rna plant and animal viruses the crystal structure of af a protein from archaeoglobus fulgidus with homology to the non-histone domain of macroh a structure and mechanism of adpribose- ʹʹ-monophosphatase (appr- ʹʹ-pase), a ubiquitous cellular processing enzyme structural basis of severe acute respiratory syndrome coronavirus adp-ribose- -phosphate dephosphorylation by a conserved domain of nsp a biochemical genomics approach for identifying genes by the activity of their products a highly specific phosphatase that acts on adp-ribose ʹʹ-phosphate, a metabolite of trna splicing in saccharomyces cerevisiae structural and functional basis for adpribose and poly(adp-ribose) binding 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identified antiviral activity of the parp-containing zapl protein interferon-stimulated poly(adp-ribose) polymerases are potent inhibitors of cellular translation and virus replication new parp gene with an anti-alphavirus function poly(adp-ribose) polymerase promotes transcriptional repression of integrated retroviruses rapid evolution of parp genes suggests a broad role for adp-ribosylation in host-virus conflicts molecular biology and infection of hepatitis e virus hepatitis e virus infection togaviruses: rubella virus alphaviruses (togaviridae) and flaviviruses (flaviviridae) middle east respiratory syndrome and severe acute respiratory syndrome porcine epidemic diarrhea: a review of current epidemiology and available vaccines key: cord- -dw sfzqx authors: van boheemen, sander; van rijn, anneloes l.; pappas, nikos; carbo, ellen c.; vorderman, ruben h.p.; sidorov, igor; van `t hof, peter j.; mei, hailiang; claas, eric c.j.; kroes, aloys c.m.; de vries, jutte j.c. title: retrospective validation of a metagenomic sequencing protocol for combined detection of rna and dna viruses using respiratory samples from pediatric patients date: - - journal: j mol diagn doi: . /j.jmoldx. . . sha: doc_id: cord_uid: dw sfzqx viruses are the main cause of respiratory tract infections. metagenomic next-generation sequencing (mngs) enables unbiased detection of all potential pathogens. to apply mngs in viral diagnostics, sensitive and simultaneous detection of rna and dna viruses is needed. herein, were studied the performance of an in-house mngs protocol for routine diagnostics of viral respiratory infections with potential for automated pan-pathogen detection. the sequencing protocol and bioinformatics analysis were designed and optimized, including exogenous internal controls. subsequently, the protocol was retrospectively validated using clinical respiratory samples. the developed protocol using illumina nextseq sequencing showed high repeatability. use of the national center for biotechnology information’s refseq database as opposed to the national center for biotechnology information’s nucleotide database led to enhanced specificity of classification of viral pathogens. a correlation was established between read counts and pcr cycle threshold value. sensitivity of mngs, compared with pcr, varied up to %, with specificity of %, dependent on the cutoff for defining positive mngs results. viral pathogens only detected by mngs, not present in the routine diagnostic workflow, were influenza c, ki polyomavirus, cytomegalovirus, and enterovirus. sensitivity and analytical specificity of this mngs protocol were comparable to pcr and higher when considering off-pcr target viral pathogens. one single test detected all potential viral pathogens and simultaneously obtained detailed information on detected viruses. viruses are the main cause of respiratory tract infections. metagenomic next-generation sequencing (mngs) enables unbiased detection of all potential pathogens. to apply mngs in viral diagnostics, sensitive and simultaneous detection of rna and dna viruses is needed. herein, were studied the performance of an in-house mngs protocol for routine diagnostics of viral respiratory infections with potential for automated pan-pathogen detection. the sequencing protocol and bioinformatics analysis were designed and optimized, including exogenous internal controls. subsequently, the protocol was retrospectively validated using clinical respiratory samples. the developed protocol using illumina nextseq sequencing showed high repeatability. use of the national center for biotechnology information's refseq database as opposed to the national center for biotechnology information's nucleotide database led to enhanced specificity of classification of viral pathogens. a correlation was established between read counts and pcr cycle threshold value. sensitivity of mngs, compared with pcr, varied up to %, with specificity of %, dependent on the cutoff for defining positive mngs results. viral pathogens only detected by mngs, not present in the routine diagnostic workflow, were influenza c, ki polyomavirus, cytomegalovirus, and enterovirus. sensitivity and analytical specificity of this mngs protocol were comparable to pcr and higher when considering off-pcr target viral pathogens. one single test detected all potential viral pathogens and simultaneously obtained detailed information on detected viruses. respiratory tract infections pose a great burden on public health, causing extensive morbidity and mortality among patients worldwide. e most acute respiratory tract infections are caused by viruses, such as rhinovirus, influenza a and b viruses, metapneumovirus, and respiratory syncytial virus. however, in % to % of the patients, no pathogen is detected. e this might be the result of diagnostic failures or even infection by unknown pathogens, such as the middle east respiratory syndrome coronavirus in . rapid identification of the respiratory pathogen is critical to determine downstream decision making, such as isolation measures or treatment, including cessation of antibiotic therapy. current diagnostic amplification methods, such as real-time quantitative pcr (qpcr), are sensitive and specific, but are only targeting predefined virus species or types. genetic diversity within the virus genome and the sheer number of potential pathogens in many clinical conditions pose limitations to predefined primer-and probebased approaches, leading to false-negative results. these limitations, combined with the potential emergence of new or unusual pathogens, highlight the need for less restricted approaches that could improve the diagnosis and subsequent outbreak management of infectious diseases. metagenomics relates to the study of the complete genomic content in a complex mixture of (micro) organisms. unlike bacteria, viruses do not display a common gene in all virus families, and therefore pan-virus detection relies on catch-all analytic methods. metagenomic or untargeted next-generation sequencing (mngs) offers a culture-and nucleotide sequenceeindependent method that eliminates the need to define the targets for diagnosis beforehand. besides primary detection, mngs immediately offers additional information, on virulence markers, epidemiology, genotyping, and evolution of pathogens. , e furthermore, quantitative assessment of the presence of virus copies in the sample is enabled by the number of reads. although original mngs studies typically aim at analysis of (shifts in) population diversity of abundant dna microbes, detection of viral pathogens in patient samples requires a different technical approach because of the usually low abundance of viral pathogens (< %) in clinical samples and the requisite of detecting both dna and rna viruses. hence, a low limit of detection for rna and dna in one single assay is essential for implementation of mngs for routine pathogen detection in clinical diagnostic laboratories. current viral mngs protocols are optimized for either rna or dna detection. , e consequently, detection of both rna and dna viruses requires parallel workup of both rna and dna pretreatment methods. in addition, to increase the relative concentration of viral sequences, viral particle enrichment techniques are often applied. , these techniques are laborious and not easily automated for routine clinical diagnostic use. moreover, during enrichment directed at viral particles, intracellular viral nucleic acids as genomes and mrnas are being discarded. after sequencing, the bioinformatic classification and interpretation of the results remain a major challenge. bioinformatic classifiers are often developed for use in either microbiome studies or classification of high abundant reads, whereas extensive validation for clinical diagnostic use in settings of low abundance is limited. after bioinformatics classification, the challenge remains to discriminate between viruses that play a role in disease etiology and nonpathogenic viruses. before considering mngs in routine diagnostics, there is a need for critical evaluation and validation of every step in the procedure. in this study, we evaluated a metagenomic protocol for ngs-based pathogen detection with sample pretreatment for dna and rna in a single tube. the method was validated using a selection of respiratory pediatric samples from the total positive and negative viral pcr results. the main study objective was to define a sensitive and specific method for mngs to be used as a broad diagnostic tool for viral respiratory diseases with the potential for automated pan-pathogen detection. twenty-five stored clinical respiratory samples (À c) from pediatric patients, sent to the microbiological laboratory for routine viral diagnostics in , were selected from the laboratory database (general laboratory information management system; mips, ghent, belgium) at the leiden university medical center (leiden, the netherlands). on the basis of previous pcr test results, a variety of positive and four negative respiratory virus samples with a wide range of quantification cycle (cq) values were included. the sample types represented routine diagnostic samples from pediatric patients that had been sent to our laboratory: nasopharyngeal washings, two sputa, two bronchoalveolar lavages, one bronchial washing, and one throat swab (in viral transport medium). the patient selection (age range, . months to years) represented the pediatric population with respiratory diagnostics in our university hospital in terms of (underlying) illness. total nucleic acids were extracted directly from ml of clinical material using the magnapure dna and viral na small volume kit (roche diagnostics, almere, the netherlands) with ml output eluate. clinical material was spiked with equine arteritis virus (eav) and phocine herpesvirus [phhv ; kindly provided by dr. h.g.m. (bert) niesters, umc groningen, the netherlands], as internal controls for rna detection and dna detection, respectively. to determine the optimal concentration of the internal controls, a -fold dilution series of phhv /eav was added to a mix of two pooled influenza a positive throat swabs (cq value, ) and read count and cq values were compared. concentration was based on the number of mngs reads. before sequencing, the dna input concentration was measured with the qubit (thermo fisher scientific, waltham, ma), to determine whether there was sufficient dna in the sample to obtain sequencing results. the range of dna input for library preparation was . ng/ml for throat the journal of molecular diagnosticsjmd.amjpathol.org swabs (see reproducibility experiment) up to ng/ml for bronchoalveolar lavages and sputa. to compare the effect of different dna fragmentation techniques, six pcr-positive samples (containing one to three viruses) and three pcr-negative samples were chemically fragmented using zinc ( minutes) as part of the new england biolabs library prep kit protocol, as described next in library preparation, and physically fragmented using sonication with the bioruptor pico (diagenode, seraing, belgium; on/off time, / seconds, cycli). three samples were also tested with the highintensity settings of the bioruptor pico (on/off time, / seconds; cycli). libraries were constructed with ml extracted nucleic acids using the nebnext ultra directional rna library prep kit for illumina (new england biolabs, ipswich, ma) using single, unique adaptors. this kit has been developed for transcriptome analyses. several adaptations were made to the manufacturer's protocol to enable simultaneous detection of both dna and rna viruses. the following steps were omitted: poly a mrna capture isolation (instruction manual new england biolabs number e s/l, version . , chapter ), rrna depletion, and dnase step (chapter . to . , . b, . a). the size of fragments in the library was to bp. adaptors were diluted -fold given the low rna/dna input and pcr cycli were run after adaptor ligation. sequencing was performed on illumina hiseq and nextseq sequencing systems (illumina, san diego, ca), obtaining million -bp paired-end reads per sample. to determine the detection limit of mngs, serial dilutions (undiluted, À , À , À , and À ) of an influenza aepositive sample were tested with both mngs and laboratory-developed real-time pcr. on the basis of run-off transcript experiments, the typical limit of detection of our real-time rna pcrs was estimated to be to copies/ reaction (data not shown). to estimate the reproducibility of metagenomic sequencing, an influenza aepositive clinical sample (throat swab) was divided into four aliquots, nucleic acids were extracted, and library preparation and subsequent sequence analysis on the illumina hiseq were performed in one run. all fastq files were processed using the biopet gears pipeline version . . , developed at the leiden university medical center (http://biopet-docs.readthedocs.io/en/stable, last accessed september , ). this pipeline performs fastq preprocessing (including quality control, quality trimming, and adapter clipping) and taxonomic classification of sequencing reads. in this project, fastqc version . . (https://www.bioinformatics.babraham.ac.uk/projects/fastqc, last accessed september , ) was used for checking the quality of the raw reads. low-quality read trimming was done using sickle version . (https://github.com/najoshi/sickle) with default settings. adapter clipping was performed using cutadapt version . with default settings. taxonomic classification of reads was performed with centrifuge version . . -beta. the prebuilt nucleotide index, which contains all sequences from the national center for biotechnology information's (ncbi's) nucleotide database, provided by the centrifuge developers was used (ftp://ftp. ccb.jhu.edu/pub/infphilo/centrifuge/data/old-indices, last accessed november , ) as the reference database. an overview of the bioinformatic process is shown in figure . in addition, a customized reference centrifuge index with sequence information obtained from the ncbi's refseq (accessed february ) database was built. refseq genomic sequences for the domains of bacteria, viruses, archaea, fungi, protozoa, as well as the human reference, along with the taxonomy identifiers, were downloaded with the centrifuge-download utility and were used as input for centrifuge-build. centrifuge settings were evaluated to increase the sensitivity and specificity. the default setting, with which a read can be assigned to up to five different taxonomic categories, was compared with one unique assignment per read, where a read is assigned to a single taxonomic category, corresponding to the lowest common ancestor of all matching species. kraken-style reports with taxonomical information were produced by the centrifuge-kreport utility for all (default) options. both unique and nonunique assignments can be reported, and these settings were compared. the resulting tree-like structured, kraken-style reports were visualized with krona version . . horizontal coverage (percentage) was determined using genomedetective website to determine the amount of reads needed, results of one million reads and million reads were compared. a total of one million reads were randomly selected of the million reads of one fastq file and analyzed. the random selection was performed with the fastqsplitter (https:// github.com/biopet/biopet/blob/v . . /docs/tools/fastqsplitter. md, last accessed september , ), which cuts a fastq file of million reads into pieces, of which one was selected. read counts were normalized by the total read count and target virus genome size. because ncbi's databases were lacking a complete phhv genome sequence, phhv was sequenced; and based on the gained sequence reads, the genome was built using spades. phhv assembly was done using the biowdl virus-assembly pipeline version . (https://github.com/ biowdl/virus-assembly, last accessed september , ). the quality control part of the biowdl pipeline determines which adapters need to be clipped by using fastqc version . . (https://www.bioinformatics.babraham.ac. uk/projects/fastqc, last accessed september , ) and cutadapt version . , with minimum length setting . the resulting reads were down sampled within bowdl to , reads using seqtk version . (https://github.com/ lh /seqtk, last accessed september , ), after which spades version . . was run to get the first proposed genome contigs. to retrieve longer assembly contigs, a reiterative assembly approach was used by processing the proposed contigs by the biowdl reassembly pipeline . . this preassembly pipeline aligns reads to contigs of a previous assembly, then selects the aligned reads, down samples them, and runs a new assembly using spades. subtools used for this consisted of bwa . . for indexing and mapping, samtools . it will be assigned to the lowest common ancestor figure the bioinformatic workflow of the metagenomic next-generation sequencing protocol studied. ncbi, national center for biotechnology information. the journal of molecular diagnosticsjmd.amjpathol.org contigs from the reassembly pipeline were then processed for a second using spades, with setting the cov-cutoff to five. the resulting contigs were then processed with the reassembly pipeline for the third and last time, setting the cov-cutoff in spades to . the contigs from the last reassembly step were then run against the blast nucleotide database using blastn . . of contigs, only five that showed the lowest percentage in identity matches with any other possible noneherpes virus species were selected. the final five contigs contained sequence lengths of , , , , , and nucleotides; the average coverage was , , , , and , respectively. the proposed almost complete genome of phhv was added to ncbi's genbank database (https://www.ncbi.nlm. nih.gov/genbank; accession number mh ). clinical sensitivity was analyzed using the optimized procedure, which in short consisted of total nucleic acid extraction, including internal controls ( : dilution); the adapted new england biolabs next library preparation protocol, including fragmentation with zinc, for combined rna and dna detection (see library preparation); and sequencing of million reads (illumina nextseq ). bioinformatic analyses were performed using centrifuge with ncbi's refseq database and unique assignment of the sequence reads. sensitivity and specificity of the metagenomic ngs procedure were compared with a published updated version of our laboratory-developed multiplex qpcr. the routine multiplex pcr panel consisted of respiratory target pathogens: influenza a/b viruses, respiratory syncytial virus, metapneumovirus, adenovirus, human bocavirus, parainfluenza viruses / / / , rhinovirus, and the coronaviruses hku , nl , e, and oc . thus, in total, pcr results were available ( targets  samples), of which were pcr positive and were pcr negative for comparison with mngs. the study design was approved by the medical ethics review committee of the leiden university medical center (reference b . ). serial dilutions of eav and phhv were added to an influenza a pcr-positive sample. serial dilution : , detected eav with a substantial read count in the presence of a viral infection and without a significant decline in target virus family reads (table ) . on the basis of these results, the concentration of internal controls was determined for further experiments. the eav cq value of the dilutions correlated with the number of eav reads from the centrifuge analysis. the comparison of fragmentation methods was done using a selection of samples with relevant target reads and performed on the illumina nextseq . the total reads were comparable among the three protocols ( figure ). the protocol with zinc fragmentation had higher yield in target virus reads for all rna viruses tested and adenovirus. the detection threshold of our ngs limit, deduced from serial dilutions of influenza a ( figure ) and eav (table ) , was comparable with a real-time pcr cq value of > , corresponding to approximately < to copies/reaction. the mngs results of an influenza aepositive sample tested in quadruple could be reproduced with only minor differences ( table ) : cv of . %: . log sd/ . log average. the centrifuge default settings, with ncbi's nucleotide database and assignment of sequence reads to a maximum of five labels per sequence, resulted in various spurious classifications ( figure ) [eg, lassa virus ( figure ), evidently highly unlikely to be present in patient samples from the netherlands with respiratory complaints]. the specificity could be increased by using ncbi's refseq database instead of ncbi's nucleotide database. the classification was further improved by changing the centrifuge tool settings to limit the assignment of homologous reads to the lowest common ancestor (maximum, one label per sequence). the centrifuge reporting of shared sequences between different organisms/subtypes differs, dependent of the classification and reporting algorithm. the default classification will assign a shared read to a maximum of five organisms (one read will be assigned five times); and with the lowest common ancestor classification setting, this read will only be assigned once (namely, to the lowest ancestor these organisms/subtypes have in common). classification with a maximum of five labels per read resulted in two different outcomes using the report with all mappings and the report with unique mappings, with the latter not reporting the reads assigned to multiple organisms. comparison of classification using these different settings shows the highest sensitivity and specificity using ncbi's refseq database with one label (lowest common ancestor) assignment, with both in silico prepared data sets containing solely eav sequence fragments ( figure ) and clinical data sets (with highly abundant background) ( figure ) . to determine the effect of the total number of sequencing reads obtained per sample on sensitivity, million and million total reads were compared by in silico analysis ( table ). one million total reads resulted in an approximate -fold decrease in target virus read count compared with million total reads, implicating a reduction of sensitivity. clinical sensitivity based on pcr target pathogens clinical sensitivity was analyzed using the optimized mngs procedure. the sample collection consisted of clinical specimens positive for at least one of the following pcr target viruses: rhinovirus, influenza a and b, parainfluenza viruses and , metapneumovirus, respiratory syncytial virus, coronaviruses nl and hku , human bocavirus, and adenovirus. fourteen samples were positive for one virus, six samples were positive for two viruses, and one sample was positive for three viruses with the laboratory-developed respiratory multiplex qpcr. cq values ranged from cq to cq , with a median of . with mngs, of the viruses demonstrated in routine diagnostics were detected (table ) , resulting in a sensitivity of % for pcr targets. if a cutoff of reads was handled, sensitivity declined to % ( / ) (table ) . a receiveroperating characteristic curve for mngs detection of pcr target viruses, depending on the cutoff level of the number of figure ; mngs target read count (log value) showed a correlation (pearson correlation coefficient, À . ; p z . ) with the cq values of the qpcr (figure ) . next to the viral pathogens tested by pcr, mngs also detected other pathogenic viruses, indicating additional viral sequences uncovered by mngs but not included in the routine diagnostics, with influenza c virus being the most prominent. out of a total negative target pcr results from these samples, results corresponded with the finding of target-specific reads by mngs. if a cutoff of reads was used, of the negative pcr targets were negative with mngs. the sample positive by mngs and negative by pcr was human parainfluenza virus ( reads). although no conclusive proof for either true-or false-positive mngs results could be found, specificity of mngs was % ( / ) when encountering all reads and ! % ( / ) with a -read cutoff (table and receiver-operating characteristic curve in figure ). in addition to subtyping (table ) , using the metagenomic sequence data, the nucleotide positions that conferred resistance to either oseltamivir or zanamivir were analyzed. sequence data of amino acids i , e , d , i , h , r , n , and i showed susceptibility to oseltamivir; and sequence data of amino acids v , r , e , q , d , r , r , e , r , and r revealed susceptibility to zanamivir. , data access the raw sequence data of the samples, after removal of human reads, have been deposited to the sequence read archive database (https://www.ncbi.nlm.nih.gov/sra; accession numbers srx to srx ). metagenomic sequencing has not yet been implemented as a routine tool in clinical diagnostics of viral infections. such the journal of molecular diagnosticsjmd.amjpathol.org application would require the careful definition and validation of several parameters to enable the accurate assessment of a clinical sample with regard to the presence or absence of a pathogen, to fulfill current accreditation guidelines. therefore, this study has initiated the optimization of several steps throughout the presequencing and postsequencing workflow, which are considered essential for sensitive and specific mngs-based virus detection. many virus discovery or virus diagnostic protocols have focused on the enrichment of viral particles with the intention to increase the relative amount of virus reads. however, these methods are laborious and intrinsically exclude viral nucleic acid located in host cells. herein, a sample pretreatment protocol was designed with potential for: i) automation, ii) pan-pathogen detection, and iii) detection of intracellular viral nucleic acids. consequently, any type of viral enrichment was excluded (filtration, centrifugation, nucleases, and rrna removal). the current protocol enabled highthroughput sample pretreatment by means of automated nucleic acid extraction and without depletion of bacterial or human genome, with potential for pan-pathogen detection. several adaptations in the bioinformatic script resulted in more accurate reporting of the classification output. addition of an internal control to a pcr is commonly used for quality control in qpcr. although the addition of internal controls in mngs is not yet an accepted standard procedure, eav and phhv were used as an rna and a dna control, respectively, to monitor the workflow in this diagnostic application. the amount of internal control reads and target virus reads has been reported to be dependent on the amount of background reads (negative correlation). in our protocol, the internal controls were used as qualitative controls but may be used as indicator of the amount of background. phhv showed less linearity in the dilution series, compared with eav, which may be indicative for a potential relative difference in efficiency of amplification of phhv viral sequences. because ncbi's databases were lacking a complete phhv genome, the centrifuge index building and classification was limited to classification on a higher taxonomic rank. to achieve classification of phhv at the species level, the whole genome of phhv was sequenced; and based on the gained sequence reads, the genome was built. the proposed nearly complete genome of phhv was submitted to ncbi's genbank database. sensitivity of the mngs protocol was maximum % based on pcr target viruses and depended on the cutoff level of reads for defining a positive result. five viruses, which were not recovered by mngs, had high cq values, > (ie, a relatively low viral load). this may be a drawback of the retrospective nature of this clinical evaluation as rna viruses may be degraded because of storage and freeze-thaw steps, resulting in lower sensitivity of mngs. a correlation was found between read counts and pcr cq value, demonstrating the quantitative nature of viral detection by mngs. discrepancies between the cq values and the number of mngs reads may be explained by unrepresentative cq values (eg, by primer mismatch for highly divergent viruses, like rhinoviruses/enteroviruses and differences in sensitivity of mngs for several groups of viruses, as has been reported by others). in addition, viral pathogens were detected that were not targeted by the routine pcr assays, including influenza c virus, which is typical of the unbiased nature of the method. in addition, although not within the scope of this study, bacterial pathogens, including bordetella pertussis (qpcr confirmed), were also detected. in the current study, only viruses were targeted because these could be well compared with qpcr results; bacterial targets remain to be studied in clinical sample types as sputum or bronchoalveolar lavages that are more suitable for bacterial detection. the analytical specificity of mngs appeared to be high, especially with a cutoff of reads. however, the clinical specificity, the relevance of the lower read numbers, still needs further investigation in clinical studies. sequencing using illumina hiseq with single, unique indexes resulted in rhinovirus-c sequences ( to reads) in all samples run on one lane, which appeared to be identical sequences. retesting of the samples with illumina nextseq resulted in disappearance of these reads. this problem could be attributed to index hopping (index misassignment), as described earlier. because of the chemistry, essential for the increased speed, the hiseq is more prone to index hopping between neighboring samples. although the percentage of reads that contributed to the index hopping was low, this is critical for clinical viral diagnostics, as this is aimed specifically at low abundance targets. , bioinformatics classification of metagenomic sequence data with the pipeline centrifuge required identification of the optimal parameters to minimize misclassified and unclassified reads. default settings of this pipeline resulted in higher rates of both false-positive and false-negative results. ncbi's nucleotide database includes a wide variety of unannotated viral sequences, such as partial sequences and (chimeric) constructs, in contrast to the curated and wellannotated sequences in ncbi's refseq database, which resulted in a higher specificity. in addition to the database, the journal of molecular diagnosticsjmd.amjpathol.org settings for the assignment algorithm were adapted as well. the assignment settings were adjusted to unique assignment in the case of homology to the lowest common ancestor. this modification resulted in higher sensitivity and specificity than the default settings; however, the ability to further subtyping diminished. this is likely to be attributed to the limited representation/availability of strain types within ncbi's refseq database. in consequence, this leads to a more accurate estimation of the common ancestor for particular viruses, but limited typing results in case of highly variable ones. to obtain optimal typing results, additional annotated sequences may be added or a new database should be built, with a high variety of well-defined and frequently updated virus strain types. to conclude, this study contributes to the increasing evidence that metagenomic ngs can effectively be used for a wide variety of diagnostic assays in virology, such as unbiased virus detection, resistance mutations, virulence markers, and epidemiology, as shown by the ability to detect single-nucleotide polymorphisms in influenza virus. these findings support the feasibility of moving this promising field forward to a role in the routine detection of pathogens by the use of mngs. further optimization should include the parallel evaluation of adult samples, the inclusion of additional annotated strain sequences to the database, and further elaboration of the classification algorithm and reporting for clinical diagnostics. the importance of both negative nontemplate control samples and healthy control cases may support the critical discrimination of contaminants and viral colonization from clinically relevant pathogens. optimal sample preparation and bioinformatics analysis are essential for sensitive and specific mngs-based virus detection. using a high-throughput genome extraction method without viral enrichment, both rna and dna viruses could be detected with a sensitivity comparable to pcr. using mngs, all potential pathogens can be detected in one single test, while simultaneously obtaining additional detailed information on detected viruses. interpretation of clinical relevance is an important issue but essentially not different from the use of pcr-based assays and supported by the available information on typing and relative quantities. these findings support the feasibility of a role of mngs in the routine detection of pathogens. global and regional mortality from causes of death for age groups in and : a systematic analysis for the global burden of disease study global and regional burden of hospital admissions for severe acute lower respiratory infections in young children in : a systematic analysis deaths due to respiratory tract infections in africa: a review of autopsy studies cdc epic study team: community-acquired pneumonia requiring hospitalization among u.s. adults the common cold aetiology of lower respiratory tract infection in adults in primary care: a prospective study in european countries isolation of a novel coronavirus from a man with pneumonia in saudi arabia exploring the potential of next-generation sequencing in detection of respiratory viruses a primer on metagenomics beer m: novel orthobunyavirus in cattle neurobrucellosis: unexpected answer from metagenomic nextgeneration sequencing genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans protocol for metagenomic virus detection in clinical specimens application of next generation sequencing for the detection of human viral pathogens in clinical specimens simultaneous virus identification and characterization of severe unexplained pneumonia cases using a metagenomics sequencing technique sequence analysis of the human virome in febrile and afebrile children diagnosis of human metapneumovirus and rhinovirus in patients with respiratory tract infections by an internally controlled multiplex real-time rna pcr validation of clinical application of cytomegalovirus plasma dna load measurement and definition of treatment criteria by analysis of correlation to antigen detection zincmediated rna fragmentation allows robust transcript reassembly upon whole transcriptome rna-seq cutadept removes adapter sequences from high-throughput sequencing reads centrifuge: rapid and sensitive classification of metagenomic sequences reference sequence (refseq) database at ncbi: current status, taxonomic expansion, and functional annotation interactive metagenomic visualization in a web browser genome detective: an automated system for virus identification from high-throughput sequencing data. bioinformatics spades: a new genome assembly algorithm and its applications to single-cell sequencing the sequence alignment/map format and samtools basic local alignment search tool performance of different mono-and multiplex nucleic acid amplification tests on a multipathogen external quality assessment panel detection of resistance mutations to antivirals oseltamivir and zanamivir in avian influenza a viruses isolated from wild birds assessing the oseltamivirinduced resistance risk and implications for influenza infection control strategies depletion of human dna in spiked clinical specimens for improvement of sensitivity of pathogen detection by next-generation sequencing rna and dna bacteriophages as molecular diagnosis controls in clinical virology: a comprehensive study of more than , routine pcr tests validation of metagenomic next-generation sequencing tests for universal pathogen detection quality control implementation for universal characterization of dna and rna viruses in clinical respiratory samples using single metagenomic next-generation sequencing workflow index switching causes "spreading-of-signal" among multiplexed samples in illumina hiseq dna sequencing estimating the rate of index hopping on the illumina hiseq x platform concerns over the origin of nih-cqv, a novel virus discovered in chinese patients with seronegative hepatitis we thank our project partners floyd wittink, wouter suring key: cord- -v m fzw authors: sun, yan-gang; li, rui; jiang, longguang; qiao, songlin; zhi, yubao; chen, xin-xin; xie, sha; wu, jiawei; li, xuewu; deng, ruiguang; zhang, gaiping title: characterization of the interaction between recombinant porcine aminopeptidase n and spike glycoprotein of porcine epidemic diarrhea virus date: - - journal: int j biol macromol doi: . /j.ijbiomac. . . sha: doc_id: cord_uid: v m fzw porcine epidemic diarrhea (ped) has caused huge economic losses to the global pork industry. infection by its causative agent ped virus (pedv), an alpha-coronavirus, was previously proven to be mediated by its spike (s) glycoprotein and a cellular receptor porcine aminopeptidase n (papn). interestingly, some recent studies have indicated that papn is not a functional receptor for pedv. to date, there is a lack of a direct evidence for the interaction between papn and pedv s protein in vitro. here, we prepared papn ectodomain and the truncated variants of pedv s protein in drosophila s cells. these recombinant proteins were homogeneous after purification by metal-affinity and size-exclusion chromatography. we then assayed the purified target proteins through immunogenicity tests, pedv binding interference assays, circular dichroism (cd) measurements, papn activity assay and structural determination, demonstrating that they were biologically functional. finally, we characterized their interactions by gel filtration chromatography, native-polyacrylamide gel electrophoresis (page) and surface plasmon resonance (spr) analyses. the results showed that their affinities were too low to form complexes, which suggest that papn may be controversial as the genuine receptor for pedv. therefore, further research needs to be carried out to elucidate the interaction between pedv and its genuine receptor. coronaviruses are enveloped single-stranded positive-sense rna viruses, comprising alpha-, beta-, gamma-, and delta-coronaviruses [ ] . they are critical causative agents, whose infections are often associated with respiratory, digestive, and neurological diseases in humans and animals. among these viruses, two beta-coronaviruses severe acute respiratory syndrome coronavirus (sars-cov) [ ] [ ] [ ] [ ] [ ] and middle east respiratory syndrome coronavirus (mers-cov) [ ] [ ] [ ] [ ] are lethal to humans, and an alpha-coronavirus transmissible gastroenteritis virus (tgev) has caused severe intestinal diseases to pigs [ ] . the infection of a coronavirus is mediated by its spike (s) glycoprotein and the s protein is further cleaved into s and s subunits by endogenous and/or exogenous proteases [ ] . the s subunit recognizes and binds to the corresponding host receptor, whereas the s subunit mediates membrane fusion between the virus and host cells [ ] [ ] [ ] . since intervention of s subunit binding to host receptor shows a great antiviral potential, there are increasing studies to elucidate their interaction mechanisms. angiotensin-converting enzyme (ace ) [ , ] and dipeptidyl peptidase (dpp ) [ ] [ ] [ ] have been identified as the receptor for sars-cov and mers-cov, respectively. the viral receptor-binding domains (rbd) are located in the c-terminal domains (ctds) of the s subunits (s -ctds) [ , , ] . human coronavirus e (hcov- e) [ , ] and tgev [ , ] utilize aminopeptidase n (apn) as the host receptor and their rbds are also located in the s -ctds [ , ] . porcine epidemic diarrhea (ped), characterized by vomiting, diarrhea and dehydration, has caused huge economic losses to the global pork industry [ ] [ ] [ ] [ ] [ ] [ ] . its causative agent, ped virus (pedv), was an alpha-coronavirus. previous studies showed that a porcine receptor apn (papn) mediated pedv infection [ ] [ ] [ ] [ ] and its density appeared to be an important factor in contributing to the virus efficient infection [ ] . pedv s -ctd (residues - ) was further demonstrated to interact with papn ectodomain (residues - ) [ ] . interestingly, a few recent studies show that papn is not the functional receptor for pedv infection [ , ] . however, there is a lack of direct demonstration of the interaction between papn and pedv s protein to clarify this discrepancy. in this study, we prepared the extracellular domain of papn, pedv s and its truncated variant (s t) in drosophila schneider (s ) cells. after purification, we assayed the target proteins for their biological functions. finally, we characterized their interactions by three canonical assays. ipec-j cell line (from porcine small intestines) was kindly provided by zhanyong wei of henan agricultural university, china. vero cell line (african green monkey kidney epithelial cell line), drosophila s cell line, pmt/bip/v -his a vector, pcoblast vector [ , ] and positive serum against pedv were kept in our laboratory. peasy-blunt-papn plasmid was constructed and kept in our laboratory. cellfectin ii reagent, blasticidin s, sf- ii serum-free medium (sfm) and trizol reagent were purchased from invitrogen (carlsbad, usa). escherichia coli strain trans α competent cells were purchased from transgen biotech (beijng, china). fetal bovine serum (fbs), dulbecco's modified eagle's medium (dmem), roswell park memorial institute- medium (rpmi- ), and antibiotics were purchased from gibco (grand island, usa). schneider's insect medium, tryptose phosphate broth (tpb), tpck-trypsin, and l-alanine -nitroanilide hydrochloride were purchased from sigma-aldrich (st. louis, usa). the mouse anti-his epitope tag antibody was purchased from proteintech (wuhan, china). horseradish peroxidase (hrp)-conjugated anti-mouse immunoglobulin g (igg) was purchased from santa cruz biotechnology (delaware ave, usa). pre-stained protein ladder ( to kda) was purchased from thermo fisher scientific (waltham, usa). mlu i, bgl ii and peptide-n-glycosidase f (pngase f) enzymes were purchased from new england biolabs (massachusetts, usa). enhanced chemiluminescence (ecl) plus reagent was purchased from solarbio (beijing, china). the cdnas encoding pedv s (residues - ) and its truncated variants (residues - , - and - ) were amplified by pcr using the codon-optimized s gene of the pedv ch/hnxc strain as template. the cdna encoding papn ectodomain (residues - ) was amplified by pcr from the peasy-blunt-papn plasmid. all pcr primers were listed in table . the pcr products were isolated and inserted into the pmt/bip/v -his a expression vector between the bgl ii and mlu i sites. all constructs were transformed into escherichia coli strain trans α competent cells and the recombinant expression vectors were verified by sequencing at shanghai sangon biotech co. ltd. (shanghai, china). the drosophila s cells were kept in schneider's insect medium supplemented with % heat-inactivated fbs at °c in a humidified incubator. the vero cells were grown in dmem supplemented with % heat-inactivated fbs at °c in a humidified incubator with % co . ipec-j cells were grown in rpmi medium with % heat-inactivated fbs at °c with % co . all cell cultures were supplemented with antibiotics ( u/ml penicillin, μg/ml streptomycin). the recombinant expression vectors and the pcoblast vector were co-transfected into drosophila s cells by cellfectin ii reagent according to the manufacturer's instructions. the stably transfected s cells were selected in schneider's insect medium supplemented with % fbs, antibiotics, and μg/ml blasticidin s. the stably transfected cell lines were then grown in sf- ii sfm and induced by . mm cuso for days. the culture supernatants were harvested by centrifugation at rpm at °c for min. expression of recombinant proteins was checked by mouse anti-his epitope tag antibody and hrp-conjugated anti-mouse igg using western blot. the supernatant containing papn ectodomain, pedv s or s t protein was adjusted to ph . by . m tris-hcl ph . , followed by centrifugation at rpm at °c for min. after filtration through . μm filter membranes, the supernatant was applied to histrap excel prepacked column (ge healthcare, fairfield, usa) or/and hitrap q hp prepacked column (ge healthcare) for purification. then, each target protein was subjected to size-exclusion column hiload / superdex pg (ge healthcare) and superdex increase / gl (ge healthcare) for further purification with mm tris-hcl ph . and mm nacl as the elution buffer. the purified proteins were treated with pngase f according to the manufacturer's instructions. the pngase f-treated and -untreated proteins were applied to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) under reducing (with β-mercaptoethanol) or non-reducing condition (without β-mercaptoethanol). . . function analyses of pedv s and s t proteins . . . immunogenicity tests of pedv s and s t proteins purified pedv s and s t proteins were subjected to sds-page and transferred to pvdf (polyvinylidene fluoride) membranes. the immunogenicity was then tested by positive serum against pedv and ecl plus reagent. the pedv ch/hubei/ strain was propagated on vero cells as previously described [ ] . briefly, when the confluence reached %, vero cells were washed three times and inoculated with tcid ( % tissue culture infective dose) pedv at °c for h. after that, the viruses not entering were removed and the cells were cultured with growth medium consisting of . % tpb, . % yeast extract and table primers utilized in this study. sequence a a the boldface letters indicate restriction sites. μg/ml of tpck-trypsin. when obvious cytopathic effects (cpe) appeared, the cells were frozen and thawed three times, and centrifuged at rpm at °c for min. finally, the supernatant was stored at − °c for further use. . . . interference of pedv binding by purified s and s t proteins the ipec-j cells were pre-incubated with indicated concentrations of the purified pedv s or s t protein for h at °c. cells were further inoculated with pedv ch/hubei/ strain at an moi (multiplicity of infection) of . for h at °c. then, the virus and protein mixtures were washed away. total rna was isolated using trizol reagent according to the manufacturer's instructions and the pedv binding was represented by the copies of the pedv nucleocapsid (n) gene using absolute quantitative pcr (the primers were listed in table ) [ ] . . . circular dichroism (cd) measurements of pedv s and s t proteins cd measurements ( - nm) were carried out on aviv sf spectrometer (lakewood, usa) at room temperature (rt) using a mm path-length quartz cell. the instrument was calibrated with mm phosphate buffer (pb) ph . , and freshly purified pedv s and s t proteins were adjusted to . mg/ml and . mg/ml in mm pb ph . , respectively. five scans were accumulated at a scan speed of nm/step with average time of . s. . . . activity assay of papn ectodomain nm purified papn ectodomain was mixed with μm to mm l-alanine -nitroanilide hydrochloride as substrate in μl mm kh po buffer ph . at °c for min. then, the product pnitroanilide was measured using a microplate reader (bmg labtech, offenburg, germany) at nm [ ] . the purified papn ectodomain was concentrated to mg/ml in mm tris-hcl ph . , mm nacl. crystallization of papn ectodomain was carried out by the sitting-drop vapor diffusion method with an equal volume of the target protein and various crystallization reagents from the crystallization screening kits (hampton, usa) at rt. the crystals were acquired with a reservoir solution containing % (wt/vol) peg , . m sodium fluoride and mm hepes ph . . the crystals were flash-frozen in liquid nitrogen using a crystal freezing buffer containing % ethylene glycol, % peg , . m sodium fluoride and mm hepes ph . . x-ray data sets of the crystals were collected at a wavelength of . Å on the beam line bl u at national center for protein sciences shanghai (ncpss) and shanghai synchrotron radiation facility (ssrf). the crystal structure was solved by molecular replacement [ ] using the mammalian apn structure (pdb code: hom) [ ] as the search model. the structure was refined by ccp program package [ ] and manually adjusted by the molecular graphics program coot [ ] until convergence of the refinement. solvent molecules were added using a fo-fc fourier difference map at . σ in the final refinement step. statistics of data collection and final model refinement were summarized in table . the coordinates of papn ectodomain were deposited in the protein data bank (pdb code z , http://www.rcsb.org). the final structures were analyzed by software pymol [ ] . papn ectodomain was mixed with excess pedv s or s t protein for h at rt in mm tris-hcl ph . , mm nacl. then, pedv s or s t protein, papn ectodomain and their mixtures were individually loaded on the superdex increase / gl column (ge healthcare) in the same volume and superposed for uv absorption peaks. the eluted proteins were analyzed by sds-page. the % native-page gel and running buffer were prepared without sds. the papn ectodomain and pedv s or s t protein were mixed in μl mm tris-hcl ph . , mm nacl at a molar ratio of : / : . after incubation at rt for min, pedv s or s t protein, papn ectodomain and their mixtures were analyzed by native-page. spr analyses were performed on biacore s (ge healthcare) to determine the equilibrium binding constants (k d , m), the association rate constants (k on , m − s − ) and the dissociation rate constants (k off , s − ) of papn ectodomain binding to pedv s or s t protein. pedv s or s t protein was coupled to the cm sensor chips (ge healthcare) and papn ectodomain was injected with the indicated concentrations ( . nm, nm, nm, nm and nm). the running buffer was hbs-ep ( mm hepes ph . , mm nacl, mm edta, . % surfactant p ). the binding kinetics were analyzed with the software biacore s evaluation . , and the k on , k off and k d were calculated using a : interaction model as previously described [ ] . the equations used for calculation are represented below with biacore spr terminology: where dr dt (response unit/s, ru/s) is the slope of the curve during association phase, c (m) is the concentration of injected papn ectodomain, rmax (ru) is the signal response for maximum absorption of pedv s or s t protein, and r (ru) is the signal response for papn ectodomain binding to pedv s or s t protein. all experimental data were presented as group means and standard errors of the means (sem). the experimental data were analyzed using the unpaired, -tailed student t-test using graphpad prism software (graphpad). differences at the % confidence level (p b . ) were considered statistically significant. the recombinant papn ectodomain (residues - ), pedv s protein (residues - ) and s truncated variants (residues - , - and - ) were produced in drosophila s cells. western blot analyses verified that papn ectodomain (fig. a) , pedv s protein (fig. b) and a pedv s truncated variant (s t, residues - , fig. c) were highly expressed. the expression of other pedv s truncated variants (residues - and - ) was too low to be detected (data not shown), which were consequently not considered in our subsequent experiments. the target proteins were purified through metal-affinity and sizeexclusion chromatography. after purification, these proteins achieved to a high purity (n %, fig. ). papn ectodomain (fig. , top) , pedv s (fig. , middle) and s t proteins (fig. , bottom) were eluted at . ml, . ml and . ml on the calibrated size-exclusion column, corresponding to approximately kda, kda, kda, respectively. as shown in fig. , there were differences between the bands of each target protein under reducing (lane ) and non-reducing (lane ) conditions, which implied that they contained disulfide bonds. in addition, the bands showed a significant change after de-glycosylation using pngase f (lane and lane ). therefore, the recombinant proteins were heavily glycosylated compared to their calculated molecular masses (papn ectodomain: kda, pedv s protein: . kda, pedv s t protein: . kda). based on the results above, recombinant papn ectodomain was obtained as dimers, and pedv s or s t protein existed as monomers, which showed similar natures of mammalian apn [ ] and other coronavirus s proteins [ ] [ ] [ ] as previously reported. . . immunogenicity tests, interference of pedv binding and cd measurements of purified pedv s and s t proteins we carried out immunogenicity tests and pedv binding interference assays to analyze whether purified pedv s and s t proteins were functional. as shown in fig. a and b, purified pedv s and s t proteins could be recognized by positive serum against pedv, indicating that these recombinant proteins had a comparable immunogenicity to the native virus. since porcine small intestines are the major target organ for pedv infection, ipec-j cells from porcine small intestines were applied in interference of pedv binding by purified pedv s and s t proteins. ipec-j cells incubated with pedv s or s t protein showed the viral rna reduction in a concentration dependent manner (fig. c ). especially at a concentration of μg/ml, pedv s or s t protein pretreatment caused a significant reduction compared to that incubated with control protein buffer (p b . ), demonstrating that purified pedv s and s t proteins were in functional form (fig. c) . we have taken efforts to crystallize functional pedv s or s t protein, and determine their crystal structures. unfortunately, crystallization of these proteins was unsuccessful (data not shown). cd measurement is usual for determination of protein secondary structures [ ] [ ] [ ] [ ] [ ] [ ] and we performed cd measurements to study their secondary structures. as shown in fig. , one minimum at about nm was observed for both pedv s and s t proteins, suggesting that the two proteins had β-sheets. these results were consistent with other coronavirus s proteins, which have a high content of β-sheets [ , , [ ] [ ] [ ] [ ] . mammalian apn is a member of zinc-dependent m metallopeptidases, which is widely distributed on the intestinal epithelia and the nervous system cells [ ] . it plays multifunctional roles in tumor angiogenesis and metastasis, signal pathway, and degradation of enkephalins [ ] . based on these functions, we performed activity assay for purified papn ectodomain. the enzymatic kinetics showed pedv s protein (residues - ). lane - : pedv s protein; lane : mock. (c): pedv s truncated variant (s t, residues - ). lane : pedv s t protein. lane m: thermo pre-stained protein ladder ( to kda). that purified papn ectodomain could degrade the substrate l-alanine nitroanilide hydrochloride into the product p-nitroanilide. the k m and k cat were . ± . mm and . ± . /s, respectively (table ) . furthermore, we determined the crystal structure of papn ectodomain. purified papn ectodomain was crystallized with one molecule in each asymmetric unit and its crystal structure was determined at . Å in a c space group (table ) . as described previously [ , , , , ] , papn ectodomain adopts a hook-like conformation or so-called seahorse shape ( fig. b and c) . it is consisted of four domains (i-iv) and a zinc ion in domain ii, characteristics of m metallopeptidases ( fig. a and b ). in addition, it is heavily glycosylated, and cys and cys in association with cys and cys bridge into two disulfide linkages in domain iv (fig. b) , which are consistent with our results stated above (fig. ) . a close structural comparison of our current papn ectodomain and that determined by others (pdb code: hom) reveals that the root mean square deviation (rmsd) differences are slight ( . Å for matching cα atoms), demonstrating that they share almost identical structural fold (fig. c ). taken together, purified papn ectodomain was shown to be biologically active on the basis of its enzymatic activity and crystal structure. papn was previously demonstrated as a functional receptor for pedv [ ] [ ] [ ] [ ] . however, the vero cell lines, possessing no apn fig. . cd spectra of functional pedv s (a) and s t proteins (b). one minimum at about nm was observed for both pedv s and s t proteins. fig. . immunogenicity tests and interference of pedv binding by purified s and s t proteins. purified pedv s (a) and s t proteins (b) were detected by western blot using the positive antiserum against pedv. (c) pedv binding interference with purified pedv s or s t protein. data represent means ± sem of three independent experiments. *significantly reduced viral rna (p b . ). [ , ] , are efficiently infected by pedv, and widely utilized for isolation and series propagation of the virus [ , [ ] [ ] [ ] [ ] . recently, no effects were reported on the susceptibility to pedv after knocking out apn in porcine swine testis cells and human cell lines (human hepatoma cells, huh cells and human cervical cancer cells, hela cells) by crispr/cas genome editing [ ] . moreover, soluble papn ectodomain neither interacted with pedv nor inhibited its infection [ ] . all these studies infer that papn may be not the receptor for pedv. to date, there are few direct evidences to clarify this discrepancy by characterization of the interaction between papn ectodomain and pedv s protein in vitro. we speculate that the lack of related studies is probably due to the difficulty in preparing the large pedv s protein, which contains many disulfide bonds and glycosylation sites as other coronaviruses [ , ] . in the current study, three canonical assays were carried out to characterize the interaction between papn ectodomain and pedv s or s t protein since these functional target proteins were successfully prepared. according to previous reports, coronavirus s-rbds and their host receptors have high affinities and yield stable complexes in vitro, which can be obtained by gel filtration chromatography [ , , , , , ] . therefore, we firstly characterized the interaction between papn ectodomain and pedv s or s t protein by gel filtration chromatography. the results showed that there were no complexes formed in these protein mixtures as no complex peaks appeared (fig. ) . additionally, we performed native-page to detect whether they formed complexes. as shown in fig. a , there were no bands of the protein complexes compared to single proteins. interestingly, pedv s t protein showed other forms in addition to monomers (figs. b and a) . in order to further corroborate the interaction between the proteins, the kinetics of papn ectodomain binding to pedv s or s t protein were measured by spr using biacore s . the profiles showed that there were no signal responses during their association and dissociation phases, which demonstrated that papn ectodomain neither interacted with pedv s nor s t protein under physiological condition (fig. b ). in contrast, hcov- e [ ] and tgev [ ] rbds had high affinities to their receptor apn. these results were similar to sars-cov rbd not binding to dpp or mers-cov rbd not binding to ace , while the k d of dpp binding to mers-cov rbd was about . nm (k on : . × m − s − , k off : . × − m − s − ) [ ] . in conclusion, our work characterized the interaction between recombinant papn ectodomain and pedv s protein in vitro. the results suggest that papn may be controversial as the genuine receptor for pedv because functional papn ectodomain neither binds to pedv s nor s t protein. our current work sheds some light on the invasion mechanism of the virus, and supports a molecular basis for prevention and control of ped. as s protein recognizing and interacting with the host receptor are crucial for pedv infection, further research needs to be carried out to identify the genuine receptor of pedv and elucidate the specific interaction between them. the authors declare that they have no conflicts of interest. papn ectodomain is colored as described for (a). the zinc ion is represented as red sphere and n-linked oligosaccharides are shown in stick representation. the n and the c termini are also labeled. (c) structural comparison of papn ectodomain in the current study with that determined by others (pdb code hom) in cartoon diagrams. the papn ectodomain in the current study is colored in green and the papn ectodomain previously determined is in cyan. their n and c termini are labeled. fig. . characterization of the interaction between papn ectodomain and pedv s or s t protein. (a): native-page analysis. lane : pedv s protein; lane : papn ectodomain and pedv s protein mixtures ( : ); lane : papn ectodomain; lane : papn ectodomain and pedv s t protein mixtures ( : ); lane : pedv s t protein. 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structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains cryo-electron microscopy structures of the sars-cov spike glycoprotein reveal a prerequisite conformational state for receptor binding cryo-em structure of porcine delta coronavirus spike protein in the pre-fusion state the moonlighting enzyme cd : old and new functions to target the structure and main functions of aminopeptidase n the x-ray crystal structure of human aminopeptidase n reveals a novel dimer and the basis for peptide processing allosteric inhibition of aminopeptidase n functions related to tumor growth and virus infection isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate cell culture isolation and sequence analysis of genetically diverse us porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene isolation and characterization of a korean porcine epidemic diarrhea virus strain knu- the dynamics of chinese variant porcine epidemic diarrhea virus production in vero cells and intestines of -day old piglets crystal structure of nl respiratory coronavirus receptor-binding domain complexed with its human receptor crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor we thank the staff of bl u beamline at national center for protein sciences shanghai (ncpss) and shanghai synchrotron radiation facility (ssrf), shanghai, china, for assistance during data collection. we also acknowledge qiang wei and hongfang ma from henan academy of agricultural sciences for their assistance on crystallization, and baolin xiao from henan university for his assistance on cd measurements. this work was supported by the key: cord- -hxxizipk authors: roberts, katherine e.; hadfield, jarrod d.; sharma, manmohan d.; longdon, ben title: changes in temperature alter the potential outcomes of virus host shifts date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: hxxizipk host shifts–where a pathogen jumps between different host species–are an important source of emerging infectious disease. with on-going climate change there is an increasing need to understand the effect changes in temperature may have on emerging infectious disease. we investigated whether species’ susceptibilities change with temperature and ask if susceptibility is greatest at different temperatures in different species. we infected species of drosophilidae with an rna virus and measured how viral load changes with temperature. we found the host phylogeny explained a large proportion of the variation in viral load at each temperature, with strong phylogenetic correlations between viral loads across temperature. the variance in viral load increased with temperature, while the mean viral load did not. this suggests that as temperature increases the most susceptible species become more susceptible, and the least susceptible less so. we found no significant relationship between a species’ susceptibility across temperatures, and proxies for thermal optima (critical thermal maximum and minimum or basal metabolic rate). these results suggest that whilst the rank order of species susceptibilities may remain the same with changes in temperature, some species may become more susceptible to a novel pathogen, and others less so. emerging infectious diseases are often the result of a host shift, where a pathogen jumps from one host species into another. understanding the factors underlying host shifts is a major goal for infectious disease research. this effort has been further complicated by the fact that host-parasite interactions are now taking place in a period of unprecedented global climatic warming. here, we ask how host shifts are affected by temperature by carrying out experimental infections using an rna virus across a wide range of related species, at three different temperatures. we find that as temperature increases the most susceptible species become more susceptible, and the least susceptible less so. this has important consequences for our understanding of host shift events in a changing climate as it suggests that temperature changes may affect the likelihood of a host shift into certain species. temperature is arguably the most important abiotic factor that affects all organisms, having both indirect and direct effects on physiology and life history traits [ ] [ ] [ ] . there is much to be learned about the impact of climate change on infectious diseases [ , , ] . changes in temperature can impact both host and parasite biology, leading to complex and difficult to predict outcomes [ , ] . host shifts, where a parasite from one host species invades and establishes in a novel host species, are an important source of emerging infectious disease [ ] . a successful host shift relies on a number of stages occurring [ ] . firstly, exposure of the host to the new pathogen species must occur in such a way that transmission is successful. secondly, the pathogen must be able to replicate sufficiently to infect the novel host. finally, there must be sufficient onwards transmission for the pathogen to become established in the new host species [ , , ] . some of the most deadly outbreaks of infectious diseases in humans including ebola virus, hiv and sars coronavirus have been linked to a host switch event [ ] [ ] [ ] [ ] and many others have direct animal vectors or reservoirs (e.g. dengue and chikungunya viruses) [ , ] . the potential for novel host shifts may increase with changing temperatures due to, fluctuations in host and/or parasite fitness, or changes in species distributions and abundances [ , ] . distribution changes may lead to new species assemblages, causing novel contacts between parasites and potential hosts [ ] [ ] [ ] . susceptibility to infection is known to vary with temperature, due to within individual physiological changes in factors such as the host immune response, metabolic rate or behavioural adaptations [ ] [ ] [ ] [ ] . thermally stressed hosts may face a trade-off between the resource investment needed to launch an immune response versus that needed for thermoregulation, or behavioural adaptations to withstand sub-optimal temperatures [ ] [ ] [ ] [ ] . temperature shifts could also cause asymmetrical or divergent effects on host and parasite traits [ ] . for example, changes in temperature may allow differential production and survival of parasite transmission stages, and changes in replication rates, generation times, infectivity and virulence [ ] [ ] [ ] . temperature is also known to impact vector-borne disease transmission through multiple effects on both vector life cycles and transmission behaviours [ , [ ] [ ] [ ] [ ] . host shifts have been shown to be more likely to occur between closely related species [ ] [ ] [ ] , but independently of this distance effect, clades of closely related hosts show similar levels of susceptibility [ , ] . thermal tolerances − like virus susceptibility − are known to vary across species, with groups of closely related species having similar thermal limits, with a large proportion of the variation in these traits being explained by the phylogeny [ ] [ ] [ ] [ ] . previous studies on host shifts have assayed the susceptibility of species at a single temperature [ , , , ] . however, if the host phylogeny also explains much of the variation in thermal tolerance, then phylogenetic patterns in virus susceptibility could be due to differences between species' natural thermal optima and the chosen assay temperatures. therefore, for experiments carried out at a single temperature, phylogenetic signal in thermal tolerance may translate into phylogenetic signal in thermal stress. any apparent phylogenetic signal in susceptibility could potentially be due to the effects of thermal stress, and may not hold true if each species was to be assayed at its optimal temperature. if this was indeed the case this would have implications for species distribution models that aim to use estimates of environmental conditions to predict host and pathogen ranges [ , , ] . here, we have asked how species' susceptibilities change at different temperatures and whether susceptibility is greatest at different temperatures in different species. we infected species of drosophilidae with drosophila c virus (dcv; dicistroviridae) at three different temperatures and measured how viral load changes with temperature. viral load is used here as a measure of dcv's ability to persist and replicate in a host, which has previously been shown to be tightly correlated to host mortality [ ] . we are therefore examining one of the steps ("ability to infect a novel host") needed for a host shift to successfully occur [ , , ] . we also examine how proxies for thermal optima and cellular function (thermal tolerances and basal metabolic rate) relate to virus susceptibility across temperatures, as increasing temperatures may have broad effects on both host and parasite [ ] [ ] [ ] . dcv is a positive sense rna virus in the family discistroviridae that was originally isolated from drosophila melanogaster and in the wild has been found in d. melanogaster and d. simulans [ ] [ ] [ ] . dcv infected flies show reduced metabolic rate and activity levels, develop an intestinal obstruction, reduced hemolymph ph and decreased survival [ ] [ ] [ ] [ ] . this work examines how temperature can influence the probability of host shifts, and looks at some of the potential underlying causes. we used drosophila c virus (dcv) clone b a, which is derived from an isolate collected from d. melanogaster in charolles, france [ ] . the virus was prepared as described previously [ ] ; briefly dcv was grown in schneider's drosophila line cells and the tissue culture infective dose (tcid ) per ml was calculated using the reed-muench end-point method [ ] . flies were obtained from laboratory stocks of different species. all stocks were maintained in multi generation populations, in drosophila stock bottles (dutscher scientific) on ml of their respective food medium at ˚c and % relative humidity with a hour lightdark cycle (table a in s text). each day, two vials of - day old male flies were randomly assigned to one of three potential temperature regimes; low, medium or high ( ˚c, ˚c and ˚c respectively) at % relative humidity. flies were tipped onto fresh vials of food after days, and after days of acclimatisation at the experimental temperature were infected with dcv. flies were anesthetized on co and inoculated using a . mm diameter stainless steel needle that was bent to a right angle~ . mm from the end (fine science tools, ca, usa) [ , , ] . the bent tip of the needle was dipped into the dcv solution (tcid = . × ) and pricked into the pleural suture on the thorax of the flies. we selected this route of infection as oral inoculation has been shown to lead to stochastic infection outcomes in d. melanogaster [ ] . however, once the virus passes through the gut barrier, both oral and pinpricked infections follow a similar course, with both resulting in the same tissues becoming infected with dcv [ ] . one vial of inoculated flies was immediately snap frozen in liquid nitrogen to provide a time point zero sample as a reference to control for relative viral dose. the second vial of flies were placed onto a new vial of fresh cornmeal food and returned to their experimental temperature. after days (+/- hour) flies were snap frozen in liquid nitrogen. this time point was chosen based on pilot data as infected flies showed little mortality at days post infection, and viral load plateaus from day at ˚c. temperatures were rotated across incubators in each block to control for incubator effects. all frozen flies were homogenised in a bead homogeniser for seconds (bead ruptor ; omni international, georgia, usa) in trizol reagent (invitrogen) and stored at - ˚c for later rna extractions. these collections and inoculations were carried out over three replicate blocks, with each block being completed over consecutive days. the order that the fly species were infected was randomized each day. we aimed for each block to contain a day and day replicate for each species, at each temperature treatment ( species × temperatures × experimental blocks). in total we quantified viral load in , flies over biological replicates (a biological replicate = change in viral load from day to day post-infection), with a mean of . flies per replicate (range across species = - ) . of the species, had biological replicates and three species had biological replicates. the change in rna viral load was measured using quantitative reverse transcription pcr (qrt-pcr). total rna was extracted from the trizol homogenised flies, reverse-transcribed with promega goscript reverse transcriptase (promega) and random hexamer primers. viral rna load was expressed relative to the endogenous control housekeeping gene rpl (rp ). rpl primers were designed to match the homologous sequence in each species and crossed an intron-exon boundary so will only amplify mrna [ ] . the primers in d. melanogaster were rpl qrt-pcr f ( '-tgctaagctgtcgcacaaatgg - ') and rpl qrt-pcr r ( '-tgcgcttgttcgatccgtaac - '). dcv primers were f ( '-gacactgccttt gattag- ') and r ( 'ccctctgggaactaaatg- ') as previously described [ ] . two qrt-pcr reactions (technical replicates) were carried out per sample with both the viral and endogenous control primers, with replicates distributed across plates in a randomised block design. qrt-pcr was performed on an applied biosystems steponeplus system using sensifast hi-rox sybr kit (bioline) with the following pcr cycle: ˚c for min followed by cycles of: ˚c for sec followed by ˚c for sec. each qrt-pcr plate contained four standard samples. a linear model was used to correct the cycle threshold (ct) values for differences between qrt-pcr plates. any samples where the two technical replicates had cycle threshold (ct) values more than cycles apart after the plate correction were repeated. to estimate the change in viral load, we first calculated Δct as the difference between the cycle thresholds of the dcv qrt-pcr and the rpl endogenous control. for each species the viral load of day flies relative to day flies was calculated as -ΔΔct ; where ΔΔct = Δct day -Δct day . the Δct day and Δct day are a pair of Δct values from a day biological replicate and a day biological replicate. calculating the change in viral load without the use of the endogenous control gene (rpl ) gave equivalent results (spearman's correlation between viral load calculated with and without endogenous control: ρ = . , p< . ) we carried out two assays to measure the thermal tolerances of species; a cold resistance measure to determine critical thermal minimum (ct min ) under gradual cooling, and a heat resistance measure through gradual heating to determine critical thermal maximum (ct max ). - day old males were collected and placed onto fresh un-yeasted cornmeal food vials. flies were kept for days at ˚c and % relative humidity and tipped onto fresh food every days. in both assays individual flies were placed in ml glass vials (st , ampulla, uk) and exposed to temperature change through submersion in a liquid filled glass tank (see fig a in s text). for ct max the tank was filled with water and for ct min a mixture of water and ethylene glycol ( : by volume) was used to prevent freezing and maintain a constant cooling gradient. five biological replicates were carried out for each species for both ct max and ct min . temperature was controlled using a heated/cooled circulator (txf , grant instruments, cambridgeshire, uk) submerged in the tank and set to change temperatures at a rate of . ˚c/min, always starting from ˚c (the rearing temperature for stock populations). flies were monitored continually throughout the assay and the temperature of knock down was ascertained by a disturbance method, whereby a fly was scored as completely paralysed if on gentle tapping of the vial wall the fly did not move any of its body parts. to examine how cellular function changes with temperature, we estimated the resting metabolic rate of each species at ˚c, ˚c and ˚c to examine if changes in general cellular processes were related to changes in viral load. following the same methods as the viral inoculation assay, groups of , - day old male flies from species were acclimatised at the three experimental temperatures for days (d. pseudoobscura was excluded as not enough individuals could be obtained from stocks for sufficient replication). every days flies were tipped onto fresh vials of cornmeal food. this was repeated in three blocks in order to get three repeat measures of metabolic rate for each of the species, at each of the three experimental temperatures. flies were collected in a randomly assigned order across the three blocks. closed system respirometry was used to measure the rate of co production (vco ) as a proxy for metabolic rate [ ] . flies were held in ml - airtight plastic chambers constructed from bev-a-line v tubing (cole-parmer instrument company, uk). all measures were carried out during the day inside a temperature controlled incubator, with constant light, that was set to each of the experimental temperatures that the flies had been acclimatised to. the set up followed that of okada et al. ( ) [ ] . compressed air of a known concentration of oxygen and nitrogen ( % o : % n ) was scrubbed of any co and water (with ascarite ii & magnesium perchlorate respectively) and pumped through a sable systems rm eight-channel multiplexer (las vegas, nv, usa) at ml/min - (± %) into the metabolic chambers housing the groups of flies. the first chamber was left empty as a reference cell, to acquire a baseline reading for all subsequent chambers at the start and end of each set of runs, therefore seven groups of flies were assayed in each run. air was flushed into each chamber for minutes, before reading the previous chamber. readings were taken every second for minutes by feeding the exiting air through a licor li- infrared gas analyser (lincoln, ne, usa). carbon dioxide production was measured using a sable systems ui analog-digital interface for acquisition, connected to a computer running sable systems expedata software (v . . ) [ ] . the metabolic rate was calculated from the entire -minute recording period by taking the co reading of the ex-current gas from the chamber containing the flies and subtracting the co measure of the incurrent gas entering the chamber. these values were also corrected for drift away from the baseline reading of the empty chamber. volume of co was calculated as vco = fr (fe co -fi co ) / ( -fi co ). where fr is the flow rate into the system ( ml/ min - ), fe co is the concentration of co exiting and fi co is the concentration co entering the respirometer. species were randomly assigned across the respiration chambers and the order in which flies were assayed (chamber order) was corrected for statistically (see below). to check for any potential effect of body size differences between species on viral load, wing length was measured as a proxy for body size [ ] . a mean of (range - ) males of each species were collected and immediately stored in ethanol during the collections for the viral load assay. subsequently, wings were removed and photographed under a dissecting microscope. using imagej software (version . ) the length of the iv longitudinal vein from the tip of the proximal segment to where the distal segment joins vein v was recorded, and the mean taken for each species. the host phylogeny was inferred as described in longdon et al ( ) [ ] , using the s, adh, amyrel, coi, coii, rpl and sod genes. briefly, any publicly available sequences were downloaded from genbank, and any not available we attempted to sanger sequence [ ] . in total we had rpl sequences for all species, s from species, adh from species, amyrel from species, coi from species, coii from species and sod from species (see www.doi.org/ . /m .figshare. full details). the sequences of each gene were aligned in geneious (version . . , [ ] ) using the global alignment setting, with free end gaps and a cost matrix of % similarity. the phylogeny was constructed using the beast program (version . . , [ ] ). genes were partitioned into three groups each with their own molecular clock models. the three partitions were: mitochondrial (coi, coii); ribosomal ( s); and nuclear (adh, sod, amyrel, rpl ). a random starting tree was used, with a relaxed uncorrelated lognormal molecular clock. each of the partitions used a hky substitution model with a gamma distribution of rate variation with categories and estimated base frequencies. additionally, the mitochondrial and nuclear data sets were partitioned into codon positions + and , with unlinked substitution rates and base frequencies across codon positions. the treeshape prior was set to a birth-death process. the beast analysis was run twice to ensure convergence for million mcmc generations sampled every steps. the mcmc process was examined using the program tracer (version . , [ ] ) to ensure convergence and adequate sampling, and the constructed tree was then visualised using figtree (version . . , [ ] ). all data were analysed using phylogenetic mixed models to look at the effects of host relatedness on viral load across temperature. we fitted all models using a bayesian approach in the r package mcmcglmm [ , ] . we ran trivariate models with viral load at each of the three temperatures as the response variable similar to that outlined in longdon et al. ( ) [ ] . the models took the form: where y is the change in viral load of the i th biological replicate of host species h, for temperature t (high, medium or low). β are the fixed effects, with β being the intercepts for each temperature, β being the effect of basal metabolic rate, β the effect of wing size, and β and β the effects of the critical thermal maximum (ct max ) and minimum (ct min ) respectively. u p are the random phylogenetic species effects and e the model residuals. we also ran models that included a non-phylogenetic random species effect (u np:ht ) to allow us to estimate the proportion of variation explained by the host phylogeny [ , , ] . we do not use this term in the main model as we struggled to separate the phylogenetic and non-phylogenetic terms. our main model therefore assumes a brownian motion model of evolution [ ] . the random effects and the residuals are assumed to be multivariate normal with a zero mean and a covariance structure v p � a for the phylogenetic affects and v e � i for the residuals (� here is the kronecker product). a is the phylogenetic relatedness matrix, i is an identity matrix and the v are × (co)variance matrices describing the (co)variances between viral titre at different temperatures. the phylogenetic covariance matrix, v p, describes the inter-specific variances in each trait and the inter-specific covariances between them. the residual covariance matrix, v e, describes the within-species variance that can be both due to real within-species effects and measurement or experimental errors. the off-diagonal elements of v e (the covariances) can not be estimated because no vial has been subject to multiple temperatures and so were set to zero. we excluded d. pseudoobscura from the full model as data for bmr was not collected, but included it in models that did not include any fixed effects, which gave equivalent results. diffuse independent normal priors were placed on the fixed effects (means of zero and variances of ). parameter expanded priors were placed on the covariance matrices resulting in scaled multivariate f distributions, which have the property that the marginal distributions for the variances are scaled (by ) f , . the exceptions were the residual variances for which an inverse-gamma prior was used with shape and scale equal to . . the mcmc chain was run for million iterations with a burn-in of million iterations and a thinning interval of , . we confirmed the results were not sensitive to the choice of prior by also fitting models with inverse-wishart and flat priors for the variance covariance matrices (described in [ ] ), which gave qualitatively similar results ( . /m .figshare. ). all confidence intervals (ci's) reported are % highest posterior density intervals. using similar model structures we also ran a univariate model with bmr and a bivariate model with ct min and ct max as the response variables to calculate how much of the variation in these traits was explained by the host phylogeny. both of these models were also run with wing length as a proxy for body size as this is known to influence thermal measures [ ] . we observed significant levels of measurement error in the metabolic rate data; this was partially caused by respiratory chamber order during the assay. we corrected for this in two different ways. first, we fitted a linear model to the data to control for the effect of respiratory chamber number and then used this corrected data in all further models. we also used a measurement error model that controls for both respiratory chamber number effects and random error. both of these models gave similar results although the measurement error model showed broad cis suggesting the bmr data should be interpreted with caution. all datasets and r scripts with the model parameterisation are provided as supporting information (s text). to investigate the effect of temperature on virus host shifts we quantified viral load in , flies over biological replicates, from species of drosophilidae at three temperatures ( fig ) . dcv replicated in all host species, but viral load differed between species and temperatures (fig ) . species with similar viral loads cluster together on the phylogeny (fig ) . measurements were highly repeatable (table ) , with a large proportion of the variance being explained by the inter-specific phylogenetic component (v p ), with little within species or measurement . we also calculated the proportion of between species variance that can be explained by the phylogeny as v p /(v p + v s ) [ ] , which is equivalent to pagel's lambda or phylogenetic heritability [ , ] . we found the host phylogeny explains a large proportion of the inter-specific variation in viral load across all three temperatures, although these estimates have broad confidence intervals due to the model struggling to separate the phylogenetic and non-phylogenetic components (low = . , % ci: . to examine if species responded in the same or different way to changes in temperature we examined the relationships between susceptibilities across the different temperatures. we found strong positive phylogenetic correlations between viral loads across the three temperatures (table ). our models showed that the variance in viral load increased with temperature, however the mean viral load showed no such upward trend (table ). this suggests that the changes in variance are not simply occurring due to an increase in the means, that is then driving an increase in variance. the high correlations suggest the rank order of susceptibility of the species is not changing with increasing temperature. however, the change in variance suggests that although the intercepts are the temperature-specific intercepts when the other covariates (e.g. wing size) are set to their temperature specific means. they can be interpreted as the expected viral loads at the root of the phylogeny at each temperature. v p is the variance in between-species effects, which are structured by the phylogeny, and v r is the variance in within species effects attributable to between individual differences and measurement error. reaction norms are not crossing they are diverging from each other as temperature increases i.e. the most susceptible species are becoming more susceptible with increasing temperature, and the least susceptible less so [ ] . for example, d. obscura and d. affinis are the most susceptible species at all three temperatures. the responses of individual species show that some species have increasing viral load as temperature increases (fig , e.g. z. taronus, d. lummei) , while others decease (e.g. d. littoralis, d. novamexicana). the changes we observe could be explained by the increase in temperature effectively increasing the rate at which successful infection is progressing (i.e. altering where in the course of infection we have sampled). however, this seems unlikely as at days post infection at the medium temperature ( ˚c), viral load peaks and then plateaus [ ] . therefore, in those species where viral load increases at higher temperatures the peak viral load itself must be increasing, rather than us effectively sampling the same growth curve but at a later time point. likewise, in those species where viral load decreased at higher temperatures, viral load would need to first increase and then decrease, which we do not observe in a time course at ˚c [ ] . to check whether this also holds at higher temperatures we carried out a time course of infection in a subset of six of the original experimental species at ˚c, where we would expect the fastest transition between the rapid viral growth and the plateau phase of infection to occur (fig b in s text) . this allowed us to confirm that the decreasing viral loads observed in some species at higher temperatures are not due to general trend for viral loads to decline over longer periods of (metabolic) time. we quantified the lower and upper thermal tolerances (ct min and ct max ) across all species with replicates per species. neither ct max nor ct min were found to be significant predictors of viral load (ct min - . , % ci: - . , . , pmcmc = . and ct max . , % ci: - . , . , pmcmc = . ). when treated as a response in models we found the host phylogeny explained a large proportion of the variation in thermal maximum (ct max : . , % ci: . , ) and thermal minima (ct min : . , % ci: . , . , see s text fig c) . we also measured the basal metabolic rate of flies from species, across the three experimental temperatures, to examine how cellular function changes with temperature. bmr was not found to be a significant predictor of viral load when included as a fixed effect in our model (slope = . , % ci = - . , . , pmcmc = . ). bmr increased with temperature across all species (mean bmr and se: low . ± . , medium . ± . , high . ± . co ml/min - , see s text fig d) . when bmr was analysed as the response in models, the phylogeny explained a small amount of the between species variation (low . , % ci: × − , . , medium . , % ci: × − , . , high . , % ci: × − - . , s text fig e) indicating high within species variation or large measurement error. consequently the mean bmrs for each species, at each temperature, were used in the analysis of viral load will be poorly estimated and so the effects of bmr will be underestimated with too narrow credible intervals. to rectify this we ran a series of measurement error models, the most conservative of which gave a slope of - . but with very wide credible intervals (- . , . ) . full details of these models are given in the supporting information (s text). we found that susceptibilities of different species responded in different ways to changes in temperature. the susceptibilities of different species showed differing responses as temperatures increased (fig ) . there was a strong phylogenetic correlation in viral load across the three experimental temperatures (table ) . however, the variance in viral load increased with temperature, whereas the mean viral load did not show the same trend. this suggests that the rank order of susceptibility of the species remains relatively constant across temperatures, but as temperature increases the most susceptible species become more susceptible, and the least susceptible less so. changes in global temperatures are widely predicted to alter host-parasite interactions and therefore the likelihood of host shifts occurring [ , , , , ] . the outcome of these interactions may be difficult to predict if temperature causes a different effect in the host and pathogen species [ , , [ ] [ ] [ ] . our results show that changes in temperature may change the likelihood of pathogens successfully infecting certain species, although they suggest that it may not alter which species are the most susceptible to a novel pathogen. the increase in phylogenetic variance with temperature is effectively a form of genotypeby-environment interaction [ , [ ] [ ] [ ] . however, it varies from the classically considered ecological crossing of reaction norms, as we do not see a change in the rank order of species susceptibly across the range of experimental temperatures. instead, we find the species means diverge with increasing temperatures and so the between species differences increase [ , ] . it is also important to note that temperature may not simply be causing a change in effect size when considering the biological processes occurring during host-parasite interactions [ , ] . for example, virus replication may plateau at higher temperatures due to resource limitation. the observed level of susceptibility may be the combined outcome of both host and parasite traits, which may interact nonlinearly with temperature. we also note that by using a limited range of temperatures for practical reasons we may have not captured all unimodal relationships between viral load and temperature. as temperature is an important abiotic factor in many cellular and physiological processes, we went on to examine the underlying basis of why viral load might change with temperature. previous studies that found phylogenetic signal in host susceptibility were carried out at a single experimental temperature [ , ] . therefore, the patterns observed could potentially be explained by some host clades being assayed at sub-optimal thermal conditions. we used ct max and ct min as proxies for thermal optima which, due to its multifaceted nature, is problematic to measure directly [ ] [ ] [ ] . we also measured basal metabolic rate across three temperatures to see if the changes in viral load could be explained by general increases in enzymatic processes. we found that these measures were not significant predictors of the change in viral load with temperature. this may be driven by the fact that all temperature related traits are likely to be more complex than what any single measure can explore. traits such as host susceptibility are a function of both the host and parasite thermal optima, as well as the shape of any temperature-trait relationship [ , ] . the host immune response and cellular components utilised by the virus are likely to function most efficiently at the thermal optima of a species, and several studies have demonstrated the outcomes of host-pathogen interactions can depend on temperature [ , , , ] . however, the mechanisms underlying the changes in susceptibility with temperature seen in this study are uncertain and a matter for speculation. our results show that in the most susceptible species, viral load increases with temperature; this may be due to the virus being able to successfully infect and then freely proliferate, utilizing the host cells whist avoiding host immune defences. in less susceptible species viral load does not increase with temperature, and in some cases it actually appears to decreases. here, temperature may be driving an increase in biological processes such as enhanced host immunity, or simply increasing the rate of degradation or clearance of virus particles that have failed to establish an infection of host cells. we have investigated how an environmental variable can alter infection success following a novel viral challenge. however, temperature is just one of the potential environmental factors that will influence the different stages of a host shift event [ ] . using a controlled method of viral inoculation allows us to standardize inoculation dose so we can ask, given equal exposure, how does temperature affect the ability of a pathogen to persist and replicate in a given host? however, in nature hosts will be faced with variable levels of pathogen exposure, infected through various modes of transmission and often by multiple strains or genotypes [ ] . such variables may have consequences for the establishment and subsequent infection success of any potential host shift event. it is known that oral infection by dcv is stochastic and immune barriers such as the gut are important [ , , ] , therefore establishing the relevance of infection in the wild in this system would require further study using different potential routes of infection. the geographical distribution of a host will also influence factors such as diet and resource availability [ , [ ] [ ] [ ] [ ] , and so further work on the role of nutrient and resource availability would therefore be needed to further explore the impact of these on potential host shifts. in conclusion, we have found changes in temperature can both increase or decrease the likelihood of a host shift. our results show the rank order of species' susceptibilities remain the same across temperatures, suggesting that studies of host shifts at a single temperature can be informative in predicting which species are the most vulnerable to a novel pathogen. changing global temperatures may influence pathogen host shifts; for example changes in distributions of both host and pathogen species may generate novel transmission opportunities. our findings suggest that increases in global temperature could increase the likelihood of host shifts into the most susceptible species, and reduce it in others. climate change may therefore lead to changing distributions of both host and pathogens, with pathogens potentially expanding or contracting their host range. understanding how environmental factors might affect broader taxonomic groups of hosts and pathogens requires further study if we are to better understand host shifts in relation to climate change in nature. climate warming and disease risks for terrestrial and marine biota global warming and temperature-mediated increases in cercarial emergence in trematode parasites climate change and evolutionary adaptation climate change and infectious diseases: from evidence to a predictive framework environmental-mechanistic modelling of the impact of global change on human zoonotic disease emergence: a case study of lassa fever. freckleton r, editor climate oscillations and the structure of natural communities emerging pathogens: the epidemiology and evolution of species jumps the evolution and genetics of virus host shifts host phylogeny determines viral persistence and replication in novel hosts population biology of emerging and re-emerging pathogens virus rna structure specialization facilitates host adaptation rapid spread of emerging zika virus in the pacific area ebola in west africa: the outbreak able to change many things impact of climate change on global malaria distribution chikungunya virus emergence is constrained in asia by lineage-specific adaptive landscapes the many projected futures of dengue rapid range shifts of species associated with high levels of climate warming host and parasite thermal ecology jointly determine the effect of climate warming on epidemic dynamics how will global climate change affect parasite-host assemblages? global temperature constraints on aedes aegypti and ae. albopictus persistence and competence for dengue virus transmission. parasit vectors evolution in action: climate change, biodiversity dynamics and emerging infectious disease thermal biology in insect-parasite interactions host thermal biology: the key to understanding host-pathogen interactions and microbial pest control? variation in the immune state of gammarus pulex (crustacea, amphipoda) according to temperature: are extreme temperatures a stress? environmental temperature variation influences fitness trade-offs and tolerance in a fish-tapeworm association the influence of ambient temperature on the course of myxomatosis in rabbits some like it hot: the effects of climate change on reproduction, immune function and disease resistance in the cricket gryllus texensis host-parasite and genotype-by-environment interactions: temperature modifies potential for selection by a sterilizing pathogen environmental stressors alter relationships between physiology and behaviour empirical evidence that metabolic theory describes the temperature dependency of within-host parasite dynamics parasites and global warming: net effects of temperature on an intertidal host-parasite system some (worms) like it hot: fish parasites grow faster in warmer water, and alter host thermal preferences global change, parasite transmission and disease control: lessons from ecology understanding uncertainty in temperature effects on vector-borne disease: a bayesian approach detecting the impact of temperature on transmission of zika, dengue and chikungunya using mechanistic models short title: temperature predicts zika, dengue, and chikungunya transmission impact of human mobility on the emergence of dengue epidemics in pakistan rethinking vector immunology: the role of environmental temperature in shaping resistance phylogenetic signal in plant pathogen-host range phylogenetic determinants of potential host shifts in fungal pathogens host phylogeny constrains cross-species emergence and establishment of rabies virus in bats the causes and consequences of changes in virulence following pathogen host shifts phylogenetic studies of coadaptation:preferred temperatures versus optimal performance temperature of lizards phylogenetic constraints in key functional traits behind species' climate niches: patterns of desiccation and cold resistance across drosophila species upper thermal limits of drosophila are linked to species distributions and strongly constrained phylogenetically upper thermal limits in terrestrial ectotherms: how constrained are they? fox c, editor infection success in novel hosts: an experimental and phylogenetic study of drosophila -parasitic nematodes spatial, seasonal and climatic predictive models of rift valley fever disease across africa global trends in emerging infectious diseases studies on drosophila c and a viruses in australian populations of drosophila melanogaster the discovery, distribution, and evolution of viruses associated with drosophila melanogaster twenty-five new viruses associated with the drosophilidae (diptera) physiological and metabolic consequences of viral infection in drosophila melanogaster drosophila c virus systemic infection leads to intestinal obstruction the novel genome organization of the insect picorna-like virus drosophila c virus suggests this virus belongs to a previously undescribed virus family the toll-dorsal pathway is required for resistance to viral oral infection in drosophila existence in drosophila of groups of picornavirus with different biological and serological properties host shifts result in parallel genetic changes when viruses evolve in closely related species a simple method of estimating fifty per cent endpoints measuring metabolic rates: a manual for scientists longevity, calling effort, and metabolic rate in two populations of cricket genetic architecture of metabolic rate: environment specific epistasis between mitochondrial and nuclear genes in an insect sexual size dimorphism in a drosophila clade, the d. obscura group geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data bayesian phylogenetics with beauti and the beast . mcmc methods for multi-respoinse generalized linear mixed models: the mcmcglmm r package r: a language and environment for statistical computing. vienna, austria: r foundation for statistical computing the phylogenetic mixed model maximum-likelihood estimation of evolutionary trees from continuous characters phylogenetic analysis and comparative data: a test and review of evidence inferring the historical patterns of biological evolution the role of genotype-by-environment interactions in sexual selection how will global climate change affect parasite-host assemblages? identifying climate drivers of infectious disease dynamics: recent advances and challenges ahead complex effects of temperature on mosquito immune function infection risk decreases with increasing mismatch in host and pathogen environmental tolerances the thermal mismatch hypothesis explains host susceptibility to an emerging infectious disease genotype-environment interaction and the evolution of phenotypic plasticity quantitative genetics and the evolution of reaction norms genotype-by-environment interactions and adaptation to local temperature affect immunity and fecundity in drosophila melanogaster coexistence of similar genotypes of daphnia magna in intermittent populations: response to thermal stress temperature checks the red queen? resistance and virulence in a fluctuating environment phenotypic variance, plasticity and heritability estimates of critical thermal limits depend on methodological context making sense of heat tolerance estimates in ectotherms: lessons from drosophila validity of thermal ramping assays used to assess thermal tolerance in arthropods the evolution of transmission mode costs and benefits of sublethal drosophila c virus infection entry is a rate-limiting step for viral infection in a drosophila melanogaster model of pathogenesis impact of environmental variation on host performance differs with pathogen identity: implications for host-pathogen interactions in a changing climate host nutrition alters the variance in parasite transmission potential measuring parasite fitness under genetic and thermal variation immunity in a variable world many thanks to darren obbard and frank jiggins for useful discussion and vanessa kellerman and johannes overgaard for discussion about thermal assay methods. thanks to dave hosken for use of bmr chambers and to the drosophila species stock centre for supplying flies. thanks to two anonymous reviewers for constructive comments. key: cord- -ju vuywm authors: rohde, rodney e. title: common myths and legends of rabies date: - - journal: rabies doi: . /b - - - - . - sha: doc_id: cord_uid: ju vuywm humankind has somewhat of a dark, yet almost fascinating, supernatural relationship with rabies. even after pasteur's rabies vaccine discovery, globally people continue to be stricken with it today. history has carried along the myths and legends that surround this diabolical virus. some still believe that rabies treatment requires or more shots to the stomach by some monstrously long needle. however, today's treatment regimen is typically only four vaccinations (five for immunocompromised) in the arm, plus human rabies immune globulin. this chapter explores the misunderstood concepts of rabies prevalence, signs and symptoms, exposures, and treatment protocols. rabies has been a part of human history for as long as it has been recorded in writing and art. from odysseus's story to achilles, the actual word (term) that provoked and transformed hector to a rage and frenzy, "lyssa," is closely linked to the word "lykos" or "wolf" and is used to invoke images and feelings of an animal's anger, madness, and wolfish rage. lyssavirus is a genus of rna viruses in the family rhabdoviridae, order mononegavirales. lyssaviruses are bullet-shaped, single-stranded, negative-sense rna viruses and the causative agents of the ancient zoonosis rabies. africa is the likely home to the ancestors of taxa residing within the genus lyssavirus, family rhabdoviridae. diverse lyssaviruses are envisioned as coevolving with bats, as the ultimate reservoirs, over seemingly millions of years ( fig. . ). through eons of time, a wide range of myths and legends have developed pertaining to rabies. many people still believe today that rabies treatment requires or more shots into the stomach by some monstrously long needle. while in fact, today's treatment regimen is typically only four vaccinations (five for immunocompromised individuals) in the arm, plus a dose of humane rabies immune globulin (hrig). this myth is but one of a long list of questions, untruths, or exaggerations about the disease known as rabies. • is there a way to vaccinate wildlife using a recombinant vaccine dropped from aircraft? [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] • can scientists actually determine the origin of a rabies virus variant by way of genetic testing and methodologies? [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] • are there special government laboratories that conduct specialized rabies testing? [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] • can international public health teams actually halt and remove a moving viral epizootic outbreak in a geographical area? [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] • is there a relationship between rabies and zombies? in this chapter, some of the more common misconceptions (and facts) about rabies will be described. these misconceptions are categorized under concepts of prevalence, signs and symptoms, exposure, treatment, and pop culture influence with respect to rabies. where exactly do most cases of rabies occur in the world? which type of animals are often more "at risk" for contracting rabies virus, otherwise known as highrisk animals? are there places in the world where one might travel without worrying about rabies? do coldblooded animals, insects, or birds have a risk for acquiring this deadly disease? there are many untruths to unpack with respect to the prevalence of rabies. learn more in the following accounts: • many people believe that rabies infections only occur in poor, third-world countries. however, the truth is that rabies or rabies-like viruses are present on nearly every continent, with the exception of antarctica. numerous and diverse variants of lyssaviruses are found in a wide variety of animal species throughout the world, all of which may cause fatal human rabies. rabies virus is by far the most common lyssavirus infection of humans. in the united states, only hawaii is rabies free. interestingly, in hawaii and other rabies-free areas that are often islands, there are very strict laws and restrictions on the transport of animals from rabies-endemic states or countries to rabies-free geographic locations. in the united states, rabies as a disease is most prevalent along the east coast (raccoon and bat rabies virus variants) from florida to maine and in southern arizona (arizona gray fox) along the mexican border. rabies is also prevalent in texas with south central skunk and bat rabies virus variants predominating. common myths and legends of rabies rodney e. rohde, phd, ms, sm(ascp) cm , sv cm , mb cm , facsc as a side note, one should always consider the risk of infectious disease when traveling to other countries by discussing travel plans with an appropriate person such as a travel physician or other health-care professional. there may be a need to plan for preexposure vaccinations (e.g., rabies, yellow fever, etc.) or medications (e.g., malaria, antibiotics) to take on their travels. • a frequent belief is that this disease kills many people in the united states. however, approximately % of human deaths from the disease occur in africa and asia, where access to health-care facilities and treatment protocols is limited. the estimated annual figure of almost , rabies fatalities in humans is probably an underestimate. almost all cases of rabies in humans worldwide result from bites from infected dogs. table ). • all warm-blooded animals can contract rabies, particularly mammals. a viral disease of the central nervous system, rabies transmits between animals, including humans, when saliva containing the virus enters an opening in the skin. usually, the rabies virus enters through the bite of a rabid animal, but transmission can also occur when infected saliva enters through mucous membranes or a break in the skin. viral familial relatives can be found with invertebrates and plants (distant hosts), but warmblooded vertebrates are the hosts for the rabies virus. among warm-blooded vertebrates, birds are susceptible to infection, but rabies predominates naturally among various mammalian populations. only rare accounts occur in nonmammalian hosts, but in the mammalia family, rabies cases have occurred from the armadillo to the zebra. rabies is a significant disease of domestic and wild mammals alike, yet its zoonotic aspect is the cause of major historical infamy. according to the centers for disease control and prevention (cdc), the first clinical signs and symptoms of rabies may be very similar to those of the flu including general weakness or discomfort, fever, or headache. these clinical signs and symptoms may last for days and are directly related to the difficulty of a differential diagnosis of rabies in the absence of an obvious animal bite (or other) exposure. there may be also discomfort or a prickling or itching sensation at the site of bite, progressing within days to signs and symptoms of cerebral dysfunction, anxiety, confusion, and agitation. as the disease progresses, the person may experience delirium, abnormal behavior, hallucinations, and insomnia. the acute period of disease typically ends after - days (see chapter ). once clinical signs of rabies appear, the disease is nearly always fatal, and treatment is typically supportive. disease prevention includes both passive immunity, through an injection of human rabies immune globulin, and active immunity produced via a round of injections with rabies vaccine. if a person has already begun to exhibit signs of the disease, survival is rare. to date, less than documented cases of human survival from clinical rabies have been reported and only two have not had a history of pre-or postexposure prophylaxis. one of the more bizarre, but not uncommon, signs and symptoms of rabies that has been reported is male patients exhibiting hypersexual behavior. the virus sometimes acts on the limbic system of the brain causing men to show this behavior: increased sexual desire, involuntary erections, and in some reports continuous orgasms occurring at a rate of one per hour! in general, most people think of dogs when the topic of animals that can carry or transmit rabies is mentioned. this may be due to the long-standing fascination with the popular media (and ancient writings) that target the canine as the primary vector for rabies. for example, if you have seen the movie old yeller or cujo, then you probably understand how the popular media can influence the public to fear rabies and scrutinize the family dog for any signs of "frothing or foaming" at the mouth. however, the disease affects both domestic and wild animals. early clinical signs of the illness include fever, pain, and/or a tingling sensation around the wound. however, unlike the popular notion of old yeller or cujo, the most typical clinical signs of rabies are unexplained paralysis and a change in behavior (refer to chapter for details). still, there are many reports of other strange clinical behaviors by a rabid animal. . exposure histories are categorized as bite, contact (eg, waking to find bat on exposed skin) but no known bite was acknowledged, or unknown (ie, no known contact with an animal was elicited during case investigation). † variants of the rabies virus associated with terrestrial animals in the united states and puerto rico are identified with the names of the reservoir animal (eg, dog or raccoon), followed by the name of the most definitive geographic entity (usually the country) from which the variant has been identified. variants of the rabies virus associated with bats are identified with the names of the species of bats in which they have been found to be circulating. because information regarding the location of the exposure and the identity of the exposing animal is almost always retrospective and much information is frequently unavailable, the location of the exposure and the identity of the animal responsible for the infection are often limited to deduction. ‡infection was not identified until . when an organ recipient developed rabies. one of the difficult decisions about rabies, if not the most difficult, is "what constitutes an exposure" when trying to discern whether to treat or not to treat an individual. likewise, there are important implications for the animal (domestic). for example, whether to quarantine the animal for observation or to euthanize an animal followed by laboratory testing for confirmation of rabies. the world health organization (who) has recommendations intended as a general guide. it is recognized that, in certain situations, modifications of these procedures are warranted. such situations include exposure of infants or mentally disabled persons and other circumstances where a reliable history cannot be obtained, particularly in areas where rabies is enzootic, even though the animal is considered to be healthy at the time of exposure. there are other myths and legends surrounding the question of a true rabies exposure. the following are a few of the common ones: • on a given warm summer evening in areas of texas just as dark approaches, bats start swooping in their feeding mode of eating insects, which is an ecologic and biologic benefit. this could initiate a warning from some folks about "bats getting tangled in your hair," leading to being exposed to rabies. of course, bats are not attracted to a person's head of hair and do not try to "nest" there, but if a bat did get tangled in someone's hair, they would receive pep if the bat could not be tested as its too close a contact to rule out a bite occurring. however, contributing to this misconception could be media and historical writings with respect to vampire lore surrounding bats. additionally, there were even popular television series like the andy griffith show that had an episode with barney fife discussing bats in caves and their potential for laying "their eggs" in a person's hair (fig. . ) . • can i get rabies if i handle blood, feces, or urine? the simple answer is no in this scenario. rabies is not transmitted through the blood, urine, or feces of an infected animal, nor is it spread airborne through the open environment. saliva provides the primary transmission medium when the animal is in the infectious stage of rabies. for the rabies virus to get to the salivary glands, it has to travel first from the site of entry (usually a bite wound) through the animal's nervous system, then to the brain. this is what causes most rabid animals to exhibit abnormal behaviors, depending on what part of the brain is infected. finally, the virus travels to the salivary glands during the terminal stage of rabies. it is this later stage of rabies when an animal is most infectious because the virus is in the saliva. treatment for rabies, one of the world's most diabolical viruses, has a long history of creative and bizarre origins. rabies is a terrifying disease that dates as far back as the beginnings of humankind. in a sense, we humans fear rabies because it crosses the line between humanity and animal. think about it, the disease is at the intersection of humans and animals. somewhere deep in the psyche, one can become terrified by the thought of a bite from a rabid animal because it symbolizes the very metamorphosis of a human becoming that very rabid animal-a vampire or werewolf if you let your brain take you to that place. it should not be a surprise that humans have a longstanding and deep-seated fear of diseases with animal origins, or as science calls them, zoonotic diseases. a majority of new diseases are zoonotic. for example, swine flu, west nile virus, anthrax, severe acute respiratory syndrome, tuberculosis, influenza, ebola, nipah, powassan, and plague represent diseases from a wide range of eras. it is not a far reach to realize that the collective conscience of the general public has been swayed and biased by diseases with animal origins. so, along with that understanding comes an almost guttural need to find ways to treat or cure those diseases. this is where some of the earliest misconceptions for treating rabies originate in our history. let us examine some of those treatments that seem to perpetuate in time regardless of the modern advances in medicine. • in many of the earliest recordings of the disease, the treatments centered on the wound. this makes sense when you consider that humans from early in history understood that the origin of rabies manifested in humans after a canid bite, often while the animal was drooling copious amounts of saliva (frothing at the mouth). without listing every single early thought on treatment of these wounds, the common thread is to "bleed and cauterize (burn) the wound." for example, in early traditional indian medicine, there is acknowledgment of the fatality of hydrophobia (fear of water in rabies patients) and a prescription of treatment for it-bleeding and cauterization of the wound. the process involved cauterizing the wound with clarified butter, which the patient is then asked to drink. it is accepted even today that after an animal bite, it is wise to let it bleed, followed by rigorous washing and flushing of the wound with soap and water. however, simply closing the wound or cauterizing it will not cure one from rabies. • interestingly, the very pathogenesis of rabies in how it takes the central nervous system hostage to spread its deadly virions is now being studied to treat and possibly cure disease in brain cancer patients. the rabies virus, which kills tens of thousands of people a year, has a rare ability to enter nerve cells and use them as a conduit to infect brain tissue. now, scientists are trying to mimic this strategy to ferry tumorkilling nanoparticles into brain tumors. in laymen's terms, this means using viruses to carry tiny cancer killing agents to the tumor. so far, the approach has been shown to work only in mice. if successful in people, these nanoparticles could one day help doctors send treatment directly to tumors without harming healthy cells. • alarmingly, homeopathic and neuropathic treatments have gained some traction in the world in a variety of applications for mild to deadly diseases. rabies is not immune to these "treatments." while there is truth that some modern day medicines and drugs come from nature (e.g., penicillin is derived from the fungus penicillium), in most instances it does not replace peer-reviewed and sound clinical trial-based medical treatments or procedures. homeopathy principles roughly state that substances that produce similar symptoms of a particular ailment can cure said ailment ("like cures like") and that diluting a substance increases its potency ("law of infinitesimals"), which brings to mind the "hair of the dog" remedy for some hangover sufferers. recently, there was a report in which diluted saliva from a rabid dog was used to "treat" a -year-old boy for aggressive behavior. the first question that comes to mind is "where did the united kingdom homeopathic pharmacy obtain rabid dog saliva?" regardless, the rabid dog treatment, called lyssinum (aka lyssin or hydrophobinum), is one of more than homeopathic products approved by health canada. there is cause for concern, including those at the us food and drug administration, that such homeopathic products can be harmful and/or delay actual medical interventions and treatments. the homeopath who administered the treatment admitted that "there is no common consensus about how the remedies work" and continued to claim that it was effective and safe, plus added that the saliva was diluted to the point that it would not contain any trace of rabies virus. most rabies experts would have concerns about this treatment. • in an earlier section of this chapter, it was mentioned that some individuals (including some in health care or other biological science majors or backgrounds) incorrectly believe that rabies treatment requires or more shots into the stomach by some monstrously long needle. actually, almost all modern treatment regimens for rabies include four key features: • generous cleaning and lavage of the bite wound with soap or topical antiseptic and warm water. • avoidance of wound repair. closed bite wounds from dogs (or other animals) often become bacterially infected with early closure. the recommendation is to use very few sutures early and wait - days to more definitively close the wound. additionally, tetanus immunization boosters are indicated. • human rabies immune globulin (hrig) is essential for bridging immunity until the response to rabies vaccination occurs - days later. the hrig is most effective when infiltrated in the wound and not intramuscularly (im) remote from the bite wound. • rabies vaccine should be a modern cell-based, inactivated vaccine. rabies vaccine is effective when given intradermally or im, although only the latter route is licensed in the united states. the who recommends several schemata for rabies immunization. in the united states, only four doses (from the original five) are now recommended ( , , , and days) because an immune response is invariably present by day . however, a fifth dose is still recommended on day for individuals who are immunocompromised. simply put, rabies usually kills its victims without early intervention. with the recent and increased attention from stories of survivors of rabies, there have been recent reports from india of natural survivors, but most often with poor functional outcomes. , the exciting new milwaukee protocol and its use globally has produced four survivors who have had excellent cognitive recovery and outcomes. however, two had spastic diplegia that has been described in animal survivor models of rabies. it really should not be a surprise that rabies, a disease that goes back to the dawn of human civilization, continues to influence our pop culture (movies, television, art and literature, and tales told over generations). perhaps it is because rabies has always been known to be the very transformation of a disease from animal to man that is easily observed. the cdc estimates that zoonotic diseases are very common, both in america and globally. they estimate that more than out of every known infectious diseases in people are derived from animals and out of every new or emerging infectious diseases in people are transmitted from animals. historically, humans just did not see the connection of infectious disease to animals. scientists and scholars blamed nearly everything but animals. even some of the nastiest culprits of disease like smallpox (albeit this infection is not zoonotic) or black death (bubonic plague), which spreads to humans via respiratory droplets or by way of hitching a ride on a rat or other rodents via a flea vector, were often misunderstood regarding transmission or cause. usually, this meant that things such as demons, bad air (literal meaning of malaria), heavenly bodies/stars, and even human behavior were the root of disease. almost all of them were blamed on nonhuman hosts, except for rabies and anthrax. regardless of how far one goes back in time, one did not need to look further than the consequences of what happened after a human was bitten by a "mad dog" or other animal. in addition, to add insult to injury (literally), it was often the owner's very own best friend-the dog. if one looks to the time of greek myth, we find lycaon, king of arcadia, transforming into a slavering wolf with rabid and foamy jaws. in fifteenth century spain, we read about witch-hunters called saludadores and they were known as healers of rabies. during the interim of the th and th centuries, our european counterparts were building two well-known legendsthe werewolf and vampire-which had the ability to be human and animal and pass on this ability via the bite. even as we approach the th century with viruses becoming more understood by science and the discovery of pasteur's rabies vaccine, people of france were still transfixed and terrified by the fantasy and horror that a rabies infection converted people into maddened animals. ironically, their horror (fantasy) had some foundation. even after pasteur's vaccine, which miraculously could save one from certain death by rabies if given prior to signs and symptoms, humankind's ongoing rabid fantasy about possible monster metamorphoses with rabies continues in the pop culture and is going strong to this day. one needs to look no further than even possibly american's most trusted media icon, walt disney, when he released old yeller or about years later when the novel cujo (and later film) transfixed audiences to fear rabies (figs. . and . ). vampire movie after vampire movie arrived followed by zombie movies (i am legend, world war z, etc.) and the more current television series (the walking dead, fear the walking dead, etc.) (fig. . ). rabies even shows up in our comedy with appearances in shows like king of the hill and beavis and butt-head, as well as in an episode of the office. in the popular seinfeld episode-classic tv-one of the main characters, elaine, gets bitten by a dog, the owner evades her, she has to get pep (the doctor tells her the shot will hurt very much), and she keeps thinking she is showing signs of rabies (spitting back water, frothing at the mouth, etc.). it seems that rabies will always have a place in shaping pop culture, especially in the realm of the horror and science fiction genre. one can assume that because of this ongoing fascination with rabies in the arts and in our passing on of stories, often handing them down from one generation to the next, that we will continue to be fed misconceptions (although often based on science) about this diabolical virus known as rabies. it is incumbent for all of us in the modern era to dispel these myths and legends so that we can move forward in our efforts to assist humankind and downplay the sometimes-misguided fascination with this ongoing threat. realistically, those in the rabies medical, public health, and research community have a long way to go for increased disease survivorship and increased health literacy between health-care professionals and the public. to give just one example about our need to learn and communicate in the world of rabies, one needs to look no further than the variances in virulence and possible better outcomes from rabies virus variant phylogeny differencesthe reasons for the overrepresentation of the silver-haired bat, lasionycteris noctivagans (fig. . ) rabies virus variant in human infections are unclear. the frequency of infection, shedding, and dissemination of rabies virus in l. noctivagans, compared with myotis lucifugus and eptesicus fuscus, suggests the discrepancy of human rabies cases may be due to increased infectivity in heterospecific hosts, human susceptibility, and/or behavioral factors. in the interim, misconceived notions will most likely continue to be generated regarding the prevalence, signs and symptoms, diagnosis, and treatment protocols pertaining to this notorious and ancient killer known as rabies. it is incumbent upon all of us to be better stewards in the art of science communication with respect to decreasing the misunderstanding and sometimes panic surrounding this ancient disease. a silver-haired bat, the type that has been responsible for numerous human rabies cases and deaths. (reprinted with pemission by source, fair use: https://com mons.wikimedia.org/wiki/file:silver-haired_bat.jpg.) a cultural history of the world's most diabolical virus lyssaviruses and rabies: current conundrums, concerns, contradictions and controversies the new rabies plague. college station molecular epidemiology of rabies epizootics in texas typing of rabies virus isolates by dna enzyme immunoassay rabies: methods and guidelines for assessing a clinical rarity bat rabies evaluation of oral rabies vaccination programs for control of rabies epizootics in coyotes and gray foxes bat-associated rabies virus in skunks rabies: an old disease for a new generation controlling rabies at its source: the texas experience -oral rabies vaccination program rabies in skunks in texas epidemiology of rabies in bats in texas molecular diagnosis and epidemiology of rabies invited interview for outbreak news today radio podcast -rabies: history, myths and diagnosis on outbreak news this week centers for diseases control and prevention -explore travel health with the cdc yellow book obscure and little known facts about rabies current status of rabies and prospects for elimination rabies surveillance in the united states during centers for disease control and prevention. what are the signs and symptoms of rabies the many faces of rabies things you may not know about rabies -but should world health organization. rabies -guide for post-exposure prophylaxis misconceptions about rabies how to stop brain cancer-with rabies treated" -yr-old boy's behavior problems with saliva from rabid dog rabies: rare human infection -common questions survival from rabies encephalitis atypical rabies encephalitis in a six-year old boy: clinical, radiological, and laboratory findings rabies: still a uniformly fatal disease? historical occurrence, epidemiological trends, and paradigm shifts overwintering of rabies virus in silver haired bats key: cord- - sxy spd authors: zhang, xiaojun; wang, fanfan; zhu, changwen; wang, zhiqiang title: willingness to self-isolate when facing a pandemic risk: model, empirical test, and policy recommendations date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: sxy spd infected people are isolated to minimize the spread of pandemic diseases. therefore, the factors related to self-isolation (si) should not be neglected, and it is important to investigate the factors leading the infected (or possibly infected) people to choose to self-isolate. in this paper, we tried to show that the theory of planned behavior provides a useful conceptual framework for si when facing a pandemic risk, and a regression method with chinese provincial (guangdong province) data was applied to investigate how attitude (att), subjective norms (sn), and perceived behavioral control (pbc) influence si when facing a pandemic emergency. the results and the robustness tests confirm that att, sn, and pbc have a significant positive influence on si when facing a pandemic emergency. att plays the most important role, followed by sn and then pbc. based on the factors of si, we found, through theoretical and empirical analyses, at least three important aspects that local governments need to consider to encourage citizens to self-isolate when facing a pandemic. alongside wars and natural disasters, plagues and epidemic (pandemic) diseases have had the highest death tolls in human history [ ] . the who reported that the next influenza pandemic "is a matter of when, not if" [ ] , and the world must prepare for the next inevitable flu pandemic. what can we do when the pandemic comes? outside pharmaceutical interventions, non-pharmaceutical interventions (npis) play an important role in delaying the first wave, reducing its peak and the spreading of new influenza cases across time [ , ] because a pandemic vaccine will not be in place when a pandemic starts. npis include more actions than just self-isolation (si), such as quarantining infected populations; the closing of borders, schools, and work places; hand-washing; the cleaning of surfaces; and so on [ ] . the isolation of infected people has been applied to minimize the spread of pandemic diseases at least since the old testament period as an instrument for controlling and quelling the spread of viruses and contamination agents [ ] . hence, isolation is seen as a critical part of public health interventions, as it protects people by separating those who have been infected by communicable diseases from the general population [ ] . many scholars believe that isolation has a great impact on preventing or delaying the spread of pandemics [ , ] . si means symptomatic individuals confine themselves to their homes [ ] . generally, isolation can take two forms: mandatory and voluntary [ , ] . voluntary si means that infected (or possibly infected) individuals choose to confine themselves to their homes; this intervention is generally considered capable of limiting the transmission of pandemic influenza [ , ] and is recommended by the european centre for disease prevention and control [ ] . the early initiation of voluntary si can overcome the negative effects of a delay in antiviral drug distribution when enough symptomatic individuals comply with home confinement at symptom onset [ ] . overall, si is of great importance for hampering the spread of pandemics and has been widely studied based on different methods. the effectiveness of voluntary si largely depends on public adherence to this intervention measure [ ] . unfortunately, voluntary si strategies may inconvenience individuals, lead to economic losses, or even contribute to moral conflicts; thus, voluntary si remains a controversial strategy [ , ] . to date, many scholars have demonstrated the relationship between si and the prevention of pandemics [ , , , ] . however, the factors involved in si should not be neglected, and it is important to investigate which factors will encourage infected (or possibly infected) people to choose self-isolation. surveys conducted in the united states (us) and australia during the pandemic showed that more than % of people were willing to stay home from work or school [ , ] , while - % of people were willing to self-isolate [ , ] . according to the self-reported behavioral intention regarding the h n influenza of university students in southwestern us, mas et al. ( ) claimed that an array of issues may influence students' decision to self-isolate, including interpersonal, academic, environmental, and social factors [ ] ; however, their analysis lacks an empirical basis. risk perception has been widely established as a significant predictor of engagement in preventive health behaviors, including si [ ] ; those who report being unfamiliar with the term "pandemic influenza," male respondents, and employed people who are not able to work from home have been found to be less willing to comply [ ] . a survey in two counties in north carolina showed that % of households with children under and % of working adults reported the ability to comply with si at home for - days if recommended to do so by the authorities [ ] . concomitantly, recent polls have shown that the willingness to comply with an si period strongly depends on the social condition and literacy of the individual [ ] . therefore, we seek an answer to the fundamental question about what factors affect the willingness to self-isolate. the remainder of the paper is organized as follows. section develops a conceptual model to explain the behavior. the data description, empirical results, and regional heterogeneity test are presented in sections and . finally, section presents our conclusions. several theories explain social behaviors [ ] . one is the theory of planned behavior (tpb), which is widely applied to explain many types of behaviors. the tpb is a cognitive theory that provides a useful framework for predicting and identifying health-related behaviors, which are usually found to predict behavioral intentions with a high degree of accuracy. the tpb proposes that the individual is influenced by three factors (see figure ): attitudes toward the behavior (att), subjective norms with respect to the behavior (sn), and perceived control over the behavior (pbc) [ , ] . the tpb has been explored in relation to behaviors such as counterproductive work behaviors [ ] , safe sexual behaviors among drug users [ ] , consumption of a low-fat diet [ ] , and healthy eating behaviors [ ] . for all of these reasons, it is appropriate to apply the tpb to the study of si intention when facing pandemic risk. in the present study, we sought to build upon the conceptual model and analyze factors correlated with si behavior based on the tpb. att refers to the degree to which a person has a favorable or unfavorable evaluation or appraisal of the behavior in question [ ] . the tpb is an extension of the theory of reasoned action [ , ] , and the individual's intention is a central factor of performing a given behavior: when the intention to engage in a behavior is stronger, the behavior is more likely to be adopted [ ] . consequently, the effect of att on si can be positive or negative. sn refers to the perceived social pressure to perform a behavior or not [ , ] . people are sensitive to the conformity pressures associated with real and perceived social norms as related to risk behavior [ ] . as many studies have noted, sn and att are suggested to exert their effects on behavior through intentions [ ] . pbc refers to the perceived ease or difficulty of performing the behavior, and it is assumed to reflect past experience as well as anticipated obstacles [ ] . pbc has been found to influence both behavioral intentions and actual behavior [ ] . the resources and opportunities available to a person must, to some extent, dictate the likelihood of behavioral achievement [ ] . influence both behavioral intentions and actual behavior [ ] . the resources and opportunities available to a person must, to some extent, dictate the likelihood of behavioral achievement [ ] . besides, several other factors may affect the willingness to self-isolate. gender differences are significant in the attitudes and behaviors related to pandemics [ ] [ ] [ ] . here, the factors that affect si are similar to those identified at the beginning of a pandemic, such as h n , hiv, and sars, suggesting that gender has an effect on si [ ] . a strong intention to comply with governmentadvised preventive measures in the future is associated with higher age [ , ] . education (edu) plays an important role in individual choice. people with less education receive less information about pandemics than people with higher education, which indicates that less educated groups are less likely to understand the importance of si [ ] . personal behavior during an epidemic depends on an individual's socioeconomic status (ss), as well as his or her perception of the epidemic in the community [ ] . married people enjoy better physical and mental health than people who are not married [ ] , so marital status may play a role in people's behavior. family members in need of special care (fnsc) are children, the elderly, those with disabilities, and those with chronic diseases. respondents who live with people with these conditions have a higher risk perception concerning health [ ] [ ] [ ] . therefore, we assume that respondents with family members in need of special care have higher perceived risks. furthermore, government trust (gt) [ ] [ ] [ ] , community resources (crs) [ , ] , and emergency services (es) [ ] are among the other variables affecting si. sars was first recognized in guangdong province, china, in november , and the virus quickly spread; more than patients were infected in countries [ ] . this situation prompted us to explore the factors affecting the willingness of patients to self-isolate who may be infected in guangdong province. a market research company, ltd, which has a professional computerized database, was entrusted to carry out an online survey of pandemic prevention from october to november in . the survey included the following steps. first, we determined the basic demographic characteristics such as gender, age, and education status of the respondents based on the guangdong internet industry development report, which was published by the guangdong communications administration. second, a questionnaire was designed, which involved many aspects of si. third, county-level units and county-level city units were selected according to the statistical yearbook of guangdong province . fourth, we transformed the designed questionnaire into a network questionnaire and sent it to the random sampling objects ( questionnaires) through a network link. each interviewee was asked to provide a contact phone number. finally, questionnaires were collected in guangdong province, which gives a gross besides, several other factors may affect the willingness to self-isolate. gender differences are significant in the attitudes and behaviors related to pandemics [ ] [ ] [ ] . here, the factors that affect si are similar to those identified at the beginning of a pandemic, such as h n , hiv, and sars, suggesting that gender has an effect on si [ ] . a strong intention to comply with government-advised preventive measures in the future is associated with higher age [ , ] . education (edu) plays an important role in individual choice. people with less education receive less information about pandemics than people with higher education, which indicates that less educated groups are less likely to understand the importance of si [ ] . personal behavior during an epidemic depends on an individual's socioeconomic status (ss), as well as his or her perception of the epidemic in the community [ ] . married people enjoy better physical and mental health than people who are not married [ ] , so marital status may play a role in people's behavior. family members in need of special care (fnsc) are children, the elderly, those with disabilities, and those with chronic diseases. respondents who live with people with these conditions have a higher risk perception concerning health [ ] [ ] [ ] . therefore, we assume that respondents with family members in need of special care have higher perceived risks. furthermore, government trust (gt) [ ] [ ] [ ] , community resources (crs) [ , ] , and emergency services (es) [ ] are among the other variables affecting si. sars was first recognized in guangdong province, china, in november , and the virus quickly spread; more than patients were infected in countries [ ] . this situation prompted us to explore the factors affecting the willingness of patients to self-isolate who may be infected in guangdong province. a market research company, ltd, which has a professional computerized database, was entrusted to carry out an online survey of pandemic prevention from october to november in . the survey included the following steps. first, we determined the basic demographic characteristics such as gender, age, and education status of the respondents based on the guangdong internet industry development report, which was published by the guangdong communications administration. second, a questionnaire was designed, which involved many aspects of si. third, county-level units and county-level city units were selected according to the statistical yearbook of guangdong province . fourth, we transformed the designed questionnaire into a network questionnaire and sent it to the random sampling objects ( questionnaires) through a network link. each interviewee was asked to provide a contact phone number. finally, questionnaires were collected in guangdong province, which gives a gross response rate of . % ( / ). after the survey, we made random checks of the respondents' urls and contacted some interviewees to ensure that every respondent was unique. finally, we cleaned the data and obtained respondents; some subjects were excluded due to missing values. the net response rate was thus % ( / ). all of the variables were measured by the survey questions (see table ). we divided sn into two kinds of social pressure, from neighbors and from the health department, which were measured by the two questions below. an additional three questions were used to describe pbc, which include the individual and family's risk perception of pandemics, and confidence to protect the family from a pandemic. ( ) absolutely not, ( ) gt "do you agree or disagree with local leaders?" ( ) strongly disagree, ( ) disagree, ( ) it does not matter, ( ) agree, ( ) strongly agree "do you agree or disagree that local government works well?" ( ) strongly disagree, ( ) disagree, ( ) strongly disagree, ( ) disagree, we first examined the descriptive statistics of the variables and mapped the average scores of the willingness for si using arcgis. then, a correlation matrix was determined to examine these relationships and influences, in which quantitative variables were included. third, multiple linear regression models were adopted to explore the effects of att, sn, and pbc on si. finally, we explored the effects of these influencing variables on si in different regions to test the stability of the results and identify the differences between regions. the statistical analysis was implemented using the statistical software stata/mp, version . (statacorp lp., texas city, usa). table provides descriptive statistics for the dependent and independent variables. overall, the average age of the participants was . , with most between the ages of and . of the participants, . % were female and . % were male. most of the participants had a college education or above, and . % of them were married or cohabiting. in terms of economic status, most of the participants were in the middle class. the average fnsc was . , which means at least two members per family were in need of special care. the participants were asked: "if you were advised by the health department to isolate yourself at home for days because of exposure to large-scale infectious disease patients, do you think you could do it?" of those sampled, . % said they could not isolate themselves, . % said that they could, and . % were unsure (see figure ). the participants were asked: "if you were advised by the health department to isolate yourself at home for days because of exposure to large-scale infectious disease patients, do you think you could do it?" of those sampled, . % said they could not isolate themselves, . % said that they could, and . % were unsure (see figure ). the mean score of si by municipality is mapped in figure . overall, the mean score of si in guangdong prefecture-level cities was between . and . out of , which indicates people have more willingness to self-isolate than they do in other regions. the cities exhibited some differences, with five cities (shantou, meizhou, shanwei, chaozhou, and zhuhai) having a mean score greater than . shantou city ranks first, with a mean score of . , and yunfu city ranks last, with a mean score of . . the mean score of si by municipality is mapped in figure . overall, the mean score of si in guangdong prefecture-level cities was between . and . out of , which indicates people have more willingness to self-isolate than they do in other regions. the cities exhibited some differences, with five cities (shantou, meizhou, shanwei, chaozhou, and zhuhai) having a mean score greater than . shantou city ranks first, with a mean score of . , and yunfu city ranks last, with a mean score of . . before a formal linear regression, the correlation matrix must be determined and the correlation of variables must be tested. these results are shown in table , and a significant association was observed for of the indicators of si. in particular, att, sn, and pbc were positively related to si. before a formal linear regression, the correlation matrix must be determined and the correlation of variables must be tested. these results are shown in table , and a significant association was observed for of the indicators of si. in particular, att, sn, and pbc were positively related to si. we first regressed on si with the control variables as a benchmark model ( ) . then, we added pbc, att, and sn sequentially to the model. table shows the crude and adjusted models for the multiple regressions of changes in the willingness to self-isolate when facing a large-scale infectious disease according to changes in pbc, att, and sn. the adjusted r of the benchmark model is lower than that of models ( )-( ), which means that those models are more appropriate than model ( ) and that pbc, att, and sn are important factors of si. overall, respondents with a higher education level had a higher willingness to self-isolate when facing a pandemic. pbc, att, and sn are the key factors influencing si (significant at p < . ), and they have different effects according to the estimated coefficients. att has the most important effect on si; when att increases by %, the willingness to self-isolate will increase by . %. when sn increases by %, the willingness to self-isolate will increase by . %. finally, when pbc increases by %, the willingness to self-isolate will increase by only . %. the social-economic status in guangdong province is unbalanced, which manifests as differences in the population, economy, industrialization, and lifestyle. guangdong province is often divided into four parts: the pearl river delta and eastern, western, and northern guangdong (the pearl river delta includes guangzhou, shenzhen, zhuhai, foshan, jiangmen, dongguan, zhongshan, huizhou, and zhaoqing; eastern guangdong includes chaozhou, jieyang, shantou, and shanwei; western guangdong includes zhanjiang, maoming, and yangjiang; and northern guangdong includes shaoguan, heyuan, meizhou, qingyuan, and yunfu) [ ] . regional differences may influence the willingness to self-isolate when facing a pandemic. some researchers have explored the different influences of cancer risk [ ] , enteroviruses [ ] , epidemics [ ] , and diabetes mellitus [ ] , among other health issue, in these four parts. therefore, we regressed on si across different regions. the coefficients and the significance levels of pbc, att, and sn in every model in table are similar to those in table (except the effect of sn is not significant in western guangdong). however, there are some regional differences. the control variables (including community resources, emergency services, and families with old people) have significant influences on si in the pearl river delta. in eastern guangdong, age, education, socioeconomic, trust in leadership, and families with children have important influences on si. however, only trust in leadership and families with old people have significant effects on si in western guangdong. moreover, si in northern guangdong is only affected by age. the positive and negative influences of these control variables are similar to those in table . no substantial changes in the independent and control variables can be detected, proving the robustness of our results mentioned above. in this paper, we built a conceptual model based on the tpb theory and regression method with chinese provincial (guangdong province) data to investigate how att, sn, and pbc influence si when facing a pandemic emergency. theoretically, att, sn, and pbc are very different concepts; however, each plays an important role in social and behavioral research [ ] . we ran multiple linear regression models to test the efficiency of the conceptual model we built based on the tpb. the coefficients of si on att, sn, and pbc passed the significance test, and all were positive; these results were confirmed by the robustness checks, indicating that the tpb can be used to explain si. of the three considered factors, att plays the most important role, followed by sn and then pbc. the influence of sn on forming intention proved to be generally weaker in previous studies than the influence of att [ ] . the coefficients of education on si in models ( )−( ) were significant and positive, even if the significance levels are different. therefore, when the participant's educational level was higher, so was his or her willingness to self-isolate. in models ( ) and ( ), marriage had a significant and positive effect on si, which means married individuals have a higher willingness to self-isolate. socioeconomic status also had a significant and positive effect on si in models ( ) and ( ), which means people of high socioeconomic status may have a higher willingness to self-isolate. as for government trust, trust leadership and trust government had significant positive impacts on si, indicating that a higher degree of government trust promotes people's willingness to self-isolate. emergency services also had a significant positive influence on si. conversely, community resources are not useful for si. one possible explanation is that if a community has more resources-such as money, food, and technology-the community will be more resilient, and people will be less willing to self-isolate because people believe that the community will address the risk of the infectious disease. for family members in need of special care (children, old people, those with disabilities, or those with chronic diseases), the effects on si of having a family member who is old or has a disability were significant and negative, which means such individuals may have less willingness to self-isolate. these people likely need to provide for the people in their family who need special care, and they have little chance to self-isolate. in contrast, the effect of having a family member with a chronic disease was significant and positive, indicating a higher willingness to self-isolate. reasons for this finding may include the following: vaccinations are recommended for those with chronic diseases, or those with a family member who has a chronic disease may have more medical knowledge and better understand the dangers of a pandemic. those in a family with a child were less willing to self-isolate, but the effect was not significant. there may be a conflict of interest concerning the use of isolation, as measures must be balanced against the potential to compromise individuals' liberty and autonomy [ ] . intention, pbc, att, and sn each reveal a different aspect of behavior and each can serve as a basis for attempts to change behavior [ ] . public health policies that encourage infected people to self-isolate can be beneficial to the community by lowering disease prevalence [ ] . behavioral science theory and research provide a perspective for understanding the factors contributing to people's behavior. consequently, the more we know about any given behavior, the more we can do to influence and change the behavior. interventions to strengthen the willingness to follow si instructions have timely relevance for the prevention and control of pandemic risk. based on the factors of si, we found that, through theoretical and empirical analyses, our study identifies at least three important aspects that local governments need to consider when encouraging citizens to self-isolate when facing a pandemic. our study has several implications for public health policy as well. first, extensive publicity in various forms is necessary to help residents better understand the pandemic and raise awareness about early treatment when facing a pandemic risk. greater understanding of pandemic influenza significantly increases compliance with public health containment measures [ ] . however, some studies have indicated that si does not solve all problems, and encouraging infected people to self-isolate does not always reduce the number of infections [ ] . therefore, we also need to classify the types of pandemic diseases and report to residents the correct solution to prevent a pandemic risk. it is of great importance to establish an early warning system to provide information about pandemics and to maintain communication with residents. second, the social norms of public health must be improved through joint efforts of the government, civil society, and the media. in contemporary political and legal life, law and policy play unique roles as two social norms and two means of social adjustment. on the one hand, relevant laws and regulations must be formulated to regulate behavior when facing a pandemic risk; on the other hand, public supervision and participation should be encouraged. third, although an influenza pandemic is perceived as a real risk in all countries, the level of self-efficacy appears to be rather low [ ] . therefore, when developing preparedness plans for an influenza pandemic, specific attention should be paid to risk communication and increasing perceived self-efficacy; otherwise, adherence to preventive measures may be low [ ] . therefore, residents should be given sufficient training to reduce the difficulty of applying health behavior through better public service. in addition to the application of tpb, we found that some participants' characteristics might also be factors related to si. from the perspective of social assistance, residents have different demands; however, the characteristics of different people must be classified to make future social assistance more precise. voluntary home isolation and quarantine are effective and acceptable measures [ ] ; however, various factors may affect individuals' willingness to self-isolate when facing a pandemic risk. in this article, we tried to show that the tpb provides a useful conceptual framework for si when facing a pandemic risk. using chinese provincial (guangdong province) data, we investigated how att, sn, and pbc influence the willingness of self-isolate when facing a pandemic emergency. the results confirmed by the robustness checks show that att, sn, and pbc have significant positive influences on si when facing a pandemic emergency, with att playing the most important role. furthermore, we found that family members in need of special care have different effects on the willingness to self-isolate. the effects on si of having a family member who is old or who has a disability are significant and negative, while the effect of having a family member with a chronic disease is significant and positive. these intentions, in combination with pbc, can account for a considerable proportion of variance in behavior. based on these findings, we provided several implications for public health policy. author contributions: conceptualization, x.z.; methodology, x.z. and f.w.; software, x.z. and f.w.; validation, z.w. and x.z.; formal analysis, c.z. and x.z.; investigation, x.z.; resources, c.z. and x.z.; data curation, c.z. and x.z.; writing-original draft preparation, x.z.; writing-review and editing, z.w. and x.z.; visualization, x.z.; supervision, z.w.; project administration, z.w.; funding acquisition, z.w. all authors have read and agreed to the published version of the manuscript. acknowledgments: the authors would like to thank the anonymous reviewers for their constructive comments regarding this article. the authors declare no conflicts of interest. probing into the effectiveness of self-isolation policies in epidemic control world must prepare for inevitable 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among high-risk groups in guangdong province estimation of cancer burden in guangdong province, china in human enterovirus surveillance in the slovak republic from an enterovirus epidemic in guangdong province of china, : epidemiological, clinical, and virogenic manifestations this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- - hxlx j authors: won, hokeun; lee, dong-uk; jang, guehwan; noh, yun-hee; lee, seung-chul; choi, hwan-won; yoon, in-joong; yoo, han sang; lee, changhee title: generation and protective efficacy of a cold-adapted attenuated genotype b porcine epidemic diarrhea virus date: - - journal: j vet sci doi: . /jvs. . .e sha: doc_id: cord_uid: hxlx j the recent emergence and re-emergence of porcine epidemic diarrhea virus (pedv) underscore the urgent need for the development of novel, safe, and effective vaccines against the prevailing strain. in this study, we generated a cold-adapted live attenuated vaccine candidate (aram-p -ca) by short-term passage of a virulent pedv isolate at successively lower temperatures in vero cells. whole genome sequencing identified amino acid changes in the cold-adapted strain with no insertions and deletions throughout the genome. animal inoculation experiments confirmed the attenuated phenotype of aram-p -ca virus in the natural host. pregnant sows were orally administered p -ca live vaccines two doses at -week intervals prior to parturition, and the newborn piglets were challenged with the parental virus. the oral homologous prime-boost vaccination of p -ca significantly improved the survival rate of the piglets and notably mitigated the severity of diarrhea and pedv fecal shedding after the challenge. furthermore, strong antibody responses to pedv were detected in the sera and colostrum of immunized sows and in the sera of their offspring. these results demonstrated that the cold-adapted attenuated virus can be used as a live vaccine in maternal vaccination strategies to provide durable lactogenic immunity and confer passive protection to litters against pedv. porcine epidemic diarrhea virus (pedv) is a member of the genus alphacoronavirus, belonging to in the family coronaviridae of the order nidovirales. pedv is a large, enveloped virus possessing a ′ capped, single-stranded positive-sense rna genome of approximately kb, with a ′ polyadenylated tail [ , ] . the pedv genome consists of seven canonical genes, including an open reading frame (orf) , flanked by ′-and ′-untranslated region (utrs). the first two large orfs comprise two-thirds of the genome and encode the replicase polyproteins, a and ab, which are then posttranslationally cleaved into mature non-structural proteins (nsp - ). the last one-third encodes four canonical coronavirus structural proteins in a fixed order, spike (s), membrane (m), envelope (e), and nucleocapsid (n), as well as a single accessory gene encoding orf between s and m [ ] [ ] [ ] . the virus can be phylogenetically divided into two genotypes comprising two sub-genotypes: genogroup (classical g a and recombinant g b) with low-pathogenicity, and genogroup (local epidemic g a and global epidemic or pandemic g b) with high-pathogenicity [ , , [ ] [ ] [ ] . pedv causes acute gastrointestinal symptoms characterized by a rapid onset of severe watery diarrhea, vomiting, fatal dehydration, and high mortality in newborn and suckling piglets [ ] . in , the virus was initially recognized in england and had since been geographically restricted and problematic in europe and asia during the past four decades [ ] . however, in early , pedv first emerged in the united states and rapidly spread to the adjacent north and south american countries, causing significant financial losses to their swine industries [ ] [ ] [ ] . soon after, the us prototype-like highly virulent g b pedv strains almost simultaneously invaded multiple asian nations, including south korea, taiwan, and japan, resulting in the recurrence of a massive nationwide ped epizootic [ , , ] . pedv has now emerged or re-emerged as one of the most devastating porcine viruses, posing a tremendous threat to the global pork business. the challenges of substantial monetary damage caused by pedv highlight the exigent necessity for the establishment of optimal immunization strategies. in most cases of porcine enteric viruses that produce clinical disease in newborn piglets before the development of active immunity, the neonates are protected by passive lactogenic immunity derived from the dams [ ] [ ] [ ] . prophylactic maternal immunization regimens with oral live viruses, including intentional infection and vaccination of sows during gestation, remain the most effective way to stimulate intestinal mucosal immunity that is subsequently transferred to piglets via mammary secretions [ , ] . although intentional infection or feedback through controlled oral exposure is extensively used to induce herd immunity, there are several potential risks associated with this practices such as the wide dissemination of other microbial pathogens and the uncertainty of immune induction [ , , ] . therefore, numerous research groups are attempting to develop safer and more effective live attenuated pedv vaccines to induce passive lactogenic immunity as an alternative to intentional infection. in south korea, a number of modified live virus (mlv) vaccines for classical g a pedv came to be widely used throughout the country; however, they were incapable of controlling the impact of the recent massive g b outbreaks in the domestic swine industry because of limited cross-protection between two genetic clusters [ , , , ] . considering this efficacy issue, there is a high priority for the development of a next-generation mlv vaccine against g b epizootic or related strains prevalent in the field, which can replicate to high titer in the gut and also boost lactogenic immune responses without giving rise to clinical illnesses. in general, the attenuated virus utilized to prepare the mlv vaccine can be achieved by traditional cell culture adaptation procedures of the virulent wild-type virus in non-host cell lines, but this process is impeded by difficulties in performing laboratory procedures, such as numerous time-consuming, and repetitive passages in non-host cell lines. in contrast, adaptation to growth at low (< °c) temperatures by short-term serial passages in vitro has been frequently used to generate several attenuated dna and rna viruses [ ] [ ] [ ] [ ] . in this study, we sought to create a cold-adapted attenuated g b pedv low-passage strain by progressively decreasing growth temperatures to °c in vero cells and then attempted to evaluate its protective efficacy on neonatal piglets against virulent pedv challenge. vero cells (atcc ccl- ) were cultured in alpha minimum essential medium (α-mem; invitrogen, usa) with % fetal bovine serum (fbs; invitrogen) and antibiotic-antimycotic solutions ( ×; invitrogen) and maintained at °c in a humidified % co incubator. the isolation of a highly virulent korean pedv g b strain, kor/aram/ , was conducted from clinical fecal samples on vero cells in the presence of trypsin (usb, usa). virus isolation was confirmed by cytopathic effects (cpes) observation, immunofluorescence assay (ifa), and nucleotide sequencing as described previously [ ] . a viral stock was prepared from the th passage in cell culture (aram-p ) and used as the parental and challenge virus in this study. pedv n protein-specific monoclonal antibody (mab) was obtained from choongang vaccine laboratories (cavac; korea). the pedv strain, aram-p , was plaque-purified twice in vero cells, and the purified virus was serially passaged in vero cells by gradually reducing the incubation temperature from °c to °c as described previously with some modifications [ ] . confluent vero cells grown in -mm diameter tissue culture dishes were washed with pbs and inoculated with pedv aram-p along with trypsin. after incubation at °c for h, virus growth medium [bmpro-v medium (cell science & technology institute, inc., japan) supplemented with μg/ml of trypsin] was added. the inoculated cells were maintained at °c under % co and monitored daily for cpe. when > % of the cells showed cpe, the inoculated cells were subjected to three rounds of freezing and thawing. the culture supernatants were then centrifuged for min at × g (hanil centrifuge fleta , korea) and filtered through a . -μm pore-size filter (millipore, usa). the clarified supernatants were aliquoted and stored at − °c as a viral stock (aram-p -ca) for the next passage. in the same manner, aram-p -ca was passaged times with step-wise descending temperatures of °c per passage in vero cells up to °c. subsequently, aram-p -ca was serially cultivated at °c for an additional passages for cold adaptation, and virus stock were produced at , , , and passages that were designated aram-p -ca, p -ca, p -ca, and p -ca. vero cells were infected with each aram virus stock in the presence of trypsin as described above. the culture supernatants were collected h post-infection (hpi) when % cpe commonly developed. virus titers were measured by end-point titration in -well plates using -fold serial dilutions of the supernatant samples in triplicate for each dilution to determine the amount of virus required to produce cpe in % of the inoculated vero cells. the % tissue culture infectious dose (tcid ) per ml of virus stock was calculated using the reed and muench method [ ] . the full-length genomic sequences of the parental aram-p and cold-adapted aram-p -ca strains were determined by traditional sanger methods. ten overlapping cdna fragments spanning the entire genome of each virus strain were amplified and sequenced as described elsewhere [ , , ] . the ′ and ′ ends of the genome of the strain were determined by rapid amplification of cdna ends (race) as described previously [ ] . the full-length genomic sequences were deposited in the genbank database under accession numbers mk through mk . the sequences of fully sequenced s genes and complete genomes of global pedv isolates were independently used in sequence alignments and phylogenetic analyses. multiple sequence alignments were generated using the clustalx . program [ ] and the percentages of nucleotide sequence divergences were assessed using the same software. phylogenetic trees were constructed from the aligned nucleotide or amino acid sequences using the neighbor-joining method and subsequently subjected to bootstrap analysis with replicates to determine the percentage reliability values of each internal node of the tree [ ] . all phylogenetic trees were generated using mega . software [ ] . the four in vivo swine studies described here were independently performed at the cavac animal facility under the guidelines established by its institutional animal care and use committee (iacuc, approval no. - ). all animals were obtained from a conventional breeding farm with a good health record and either vaccination against pedv or no known prior ped outbreak and were tested to confirm that they were not infected with any porcine enteric viruses. piglet challenge trials was conducted to determine the infectious dose and virulence of a pedv aram strain using -day-old conventional suckling piglets delivered from commercial crossbred sows (great yorkshire × dutch landrace). thirty pigs were divided into six groups (group -group ) of five animals and were fed commercial milk replacer frequently ( times daily) with ad libitum access to water for the study ( days). animals in groups - were allowed to acclimate for two days and then inoculated orally with ml of − , − , − , − , and − dilutions of the aram-p virus ( . tcid /ml) and cell culture medium at days of age, respectively. after inoculation, piglets were observed daily for clinical signs, including diarrhea and mortality. stool samples from pigs in all groups were collected prior to inoculation and daily with -inch cotton-tipped swabs and scored for fecal consistency for days post-inoculation (dpi): = normal; = pasty; = semi-liquid; = liquid or watery. mean pig diarrhea dose (pdd ) and lethal dose (ld ) were determined as the reciprocal of the virus dilution at which % of the pigs developed watery diarrhea (score ) or died at a given time point using the reed and muench method. to assess immunogenicity of cold-adapted derivatives, a total of -week-old conventional piglets delivered from commercial crossbred sows (great yorkshire × dutch landrace) were allocated into the cold adapted aram-p -ca-inoculated (n = ), p -ca-inoculated (n = ), p- -ca-inoculated (n = ), p -ca-inoculated (n = ), and sham-inoculated control (n = ) groups. animals were immunized orally with a ml dose of . tcid /ml of each virus or sham inoculated with cell culture medium. pre-immune sera were collected at the immunization, and antisera were collected at week intervals for weeks. pathogenicity tests were performed using a total of suckling piglets of days of age obtained from commercial crossbred sows (great yorkshire × dutch landrace). pigs were randomly assigned to three experimental groups: the parental aram-p -inoculated (n = ), cold-adapted p -ca-inoculated (n = ), and sham-inoculated control (n = ) groups. animals were fed commercial milk replacer frequently ( times daily) and had ad libitum access to water for the duration of the study ( days). after a -day acclimation period, piglets ( -day-old) in the virusinoculated groups received a ml dose of . tcid /ml (equivalent to ld as determined in this study) of one of the viruses orally. the sham-inoculated pigs were administered cell culture medium as a placebo. animals were monitored daily for clinical signs of vomiting, diarrhea, and mortality throughout the experiment. rectal swabs were collected from all pigs and diluted with pbs to % (w/v) suspensions followed by centrifugation. the clarified supernatants were subjected to conventional reverse transcription polymerase chain reaction (rt-pcr) using an i-tge/ped detection kit (intron biotechnology, korea) and real-time rt-pcr to detect the presence of pedv shedding. a clinical significance score (css) was determined with the following scoring criteria based on visual examination for dpi used to measure diarrheal severity: = normal and no diarrhea (mean cycle threshold [ct] values of > ); = mild and fluidic feces; = moderate watery diarrhea; = severe watery and projectile diarrhea (mean ct values of < ); = death. piglets were necropsied upon death after challenge throughout the study, whereas all surviving pigs from the challenge and control groups were euthanized at dpi for post-mortem examinations. swine vaccination and challenge experiments were conducted with a total of commercial crossbred pregnant sows (great yorkshire × dutch landrace) with the same parity and expected farrowing date. animals were allocated randomly into three experimental groups: vaccinated group (n = ), challenge control group (n = ), and strict negative control group (n = ). a multiple-dose ped homologous vaccination schedule at -week intervals starting prior to farrowing was applied in the present protection study. three sows in group were orally administered twice with a ml dose of the aram-p -ca with . tcid /ml at and weeks pre-partum. the remaining sows in groups and were unvaccinated and served as controls. all sows were monitored daily for clinical alterations and adverse effects following vaccination. to mimic the field conditions of nursing piglets, all sows were allowed to farrow naturally and nurse their piglets freely for the duration of the study. ten -day-old suckling piglets per litter (a total of newborn piglets) in groups and were challenged orally with a ml dose of . tcid /ml of virulent aram-p virus. the sham-inoculated piglets from group were administered with cell culture medium as a placebo. animals were observed daily for clinical signs, including vomiting, diarrhea, and mortality. fecal specimens were taken from all groups and examined for pedv shedding as described above. a css was determined for days postchallenge (dpc) as described above. blood samples were collected from sows before (at each vaccination), at, after farrowing (up to weeks) and also from representative piglets per litter from each dam at and dpc. colostrum was collected at the day of farrowing. viral rna was extracted from fecal suspensions prepared as described above using an i-tge/ ped detection kit according to the manufacturer's protocol. quantitative real-time rt-pcr was performed using a one step sybr primescript rt-pcr kit (takara, japan) as described elsewhere [ , , ] . a pedv isolate with a known infectivity titer was -fold serially diluted to generate a standard curve in each pcr plate. the virus concentrations (tcid /ml) in samples were calculated based on the standard curve. the mean ct values were calculated based on pcr positive samples, and the mean virus titers were calculated based on all pigs within the group. intestinal tissues and other major organs were grossly examined upon necropsy. small intestinal tissue specimens (< -mm thick) collected from each piglet were fixed with % formalin for h at rt and embedded in paraffin according to standard laboratory procedures. the formalin-fixed paraffin-embedded tissues were cut in - -μm thick sections on a microtome (leica, germany), floated in a °c water bath containing distilled water, and transferred to glass slides. the deparaffinized intestinal tissue sections were stained with hematoxylin and eosin (sigma, usa) to observe histopathological change or were subjected to immunohistochemistry (ihc) for the detection of pedv antigen using an mab specific for pedv n protein as described previously [ , ] . pedv antigen detection was semiquantitatively measured throughout the ihc-stained jejunal sections based on the following scoring criteria as described previously [ ] : = no signal, = %- % of villous enterocytes within the section showing a positive signal, = %- % of villous enterocytes showing a positive signal, and = greater than % of villous enterocytes showing a positive signal. the presence of pedv-specific neutralizing antibodies in serum and colostrum samples collected from pigs in all groups was determined using a serum neutralization (sn) test using pedv isolate aram-p as previously described [ ] . the neutralization titer was calculated as the reciprocal of the highest dilution of serum that inhibited virus-specific cpe in duplicate wells. all values are expressed as the means ± standard deviation of the means (sdm). all statistical significances were evaluated by a student's t-test by using graphpad prism software version . (graphpad prism inc., usa). the p values of less than . were considered statistically significant. pedv isolation in cell culture is the first step toward the generation of a live attenuated vaccine. thus, we initially attempted to isolate pedv from clinical fecal suspensions and successfully propagated one korean pedv strain, designated as aram, in vero cells. the aram strain produced obvious cpe typical of pedv infection, such as cell fusion and multinucleated cell (syncytium) formation, in infected vero cells, which was verified by detecting pedv antigens by the ifa using a pedv n protein-specific mab (fig. a) . growth kinetics analysis indicated that aram replicated efficiently in vero cells, reaching a maximum viral titer of > tcid by hpi (fig. b) virus titer (log tcid /ml) we subsequently determined the entire genomic sequences of the original fecal sample (aram-feces) and the cell culture-passaged aram-p virus using the sanger technology. both the identified genomes were identical in length [ , -nucleotide (nt)], except for the ′ poly(a) tails, and showed the typical genomic organization of alphacoronaviruses, consisting of the -nt ′ utr, the -nt orf a/ b (nt to for a and nt to for b), the -nt s gene (nt to ), the -nt orf (nt to ), the -nt e gene (nt to ), the -nt membrane (m) gene (nt to ), the -nt n gene (nt to ), and the -nt ′ utr. compared to the complete genome sequence of the aram-feces, the aram-p virus had no amino acid (aa) substitution throughout the entire genome, suggesting genetic stability during cell passages. interestingly, the genome size of the aram strain was -nt shorter than that of most g b field viruses; this was due to the presence of the unique -nt and -nt deletions (dels) at genomic positions , - , and , - , , respectively. these dels are completely absent in the genome sequences of the other global g and g strains available in the genbank database. the former -nt del resulted in a -aa del at aa positions - in the n-terminal domain (ntd) of the s protein ( fig. a) , while the latter -nt (thymine residues) del at nt positions - ( tattat ) in orf caused premature termination -nt upstream from the authentic stop codon, thereby possibly producing a truncated orf without c-terminal -aa residues (fig. b) . (fig. b) . phylogenetic analysis based on the complete s protein clearly classified the pedv strains into two distinct genogroup clusters, g and g , with two subgroups in each genogroup (fig. a) . the aram strains belonged to subgroup g b, as it clustered closely with the contemporary domestic and global g b isolates. subsequent whole-genome phylogeny showed the same grouping structure as that of an s gene-based phylogenetic tree (fig. b) . a pedv aram-p stock with a known cell culture infectious titer of . tcid /ml was serially diluted -fold with cell culture medium, giving rise to theoretical infectious titers of to − tcid /ml for the − to − dilutions, respectively, which were later used to determine the outcomes of infection in -day-old piglets ( table ) . each group (groups - ) of five piglets was challenged orally with the -fold serially diluted ( − - − ) aram-p virus, while control animals in group were fed cell culture medium. all five piglets in the groups to (inoculated with virus dilutions − to − ) and three piglets in group (inoculated with virus dilution − ) developed moderate to watery diarrhea (scores - ) by dpi and it lasted through the study period ( table ) . by dpi, % pigs of group , % ( / ) pigs of groups and , and % ( / ) pigs of group died from ped-related clinical signs. in contrast, all five pigs in groups (inoculated with virus dilution of − ) and the negative control group remained active and clinically unaffected throughout the -day experimental period. the pdd and ld of the aram virus were determined as . log pdd /ml and . log ld / ml, indirectly corresponding to theoretical cell culture infectious titers of . tcid /ml and tcid /ml, respectively. the cold-adapted g b pedv aram-ca strains were generated by a serial passage of aram-p at low temperatures commencing from °c to °c and additional adaptation for growth up passages at °c in vero cells. we initially aimed to evaluate the immunogenicity of the cold-adapted aram derivatives in the natural host. antisera were collected from the pigs at -, -, -, and -week after oral administration of each virus and were tested for neutralizing activity against the aram isolate. the serum samples obtained from pigs immunized with the cold-adapted aram-p -ca strain had higher neutralizing antibody titers, when compared with those from pigs in other ca virus-inoculated groups (fig. ) . furthermore, the immune sera of pigs inoculated with aram-p -ca or -p -ca strain showed little or no neutralizing activity against pedv. since our data indicated that only aram-p -ca can elicit robust antibody responses in immunized animals, this cold-adapted virus was selected for subsequent in vitro and in vivo studies. compared to °c, indicating impairment of growth at °c. this diminished growth at °c was confirmed by comparing growth kinetics in vero cells at both the temperatures. the aram-p -ca strain exhibited efficient growth and had an infectious titer of > . tcid / ml at °c by dpi, but slower replication rates with a maximum titer of . tcid /ml than that of the parental aram-p strain (a , -fold reduction) at °c, indicating an adaptation to low temperatures. to identify the mutation that occurred during the cold adaptation of pedv, the genome of the cold-adapted virus was sequenced and compared to that of the wild-type virus. no additional indels were found in the genome after cold adaptation, and the genome size of the aram-p -ca strain was determined to be , -nt, which was identical to the parental genome size. two genetic del markers of the aram strain were also completely conserved in s and orf of the cold-adapted aram-p -ca strain (fig. ) . the ′-and ′-utrs of the cold-adapted virus remained unchanged, whereas the coding region contained a total of nt substitutions (fig. ) . among these mutations, were non-synonymous, causing changes of -aa in nsp , -aa in nsp , -aa in s, -aa in e, and -aa in m. details in nt and aa differences between the parental aram-p and cold-adapted p -ca strains are summarized in table . further phylogenetic analysis based on the full-length s and the entire genome indicated that the cold-adapted aram-p -ca strain was still clustered into subgroup g b along with the parental and global epizootic strains (fig. ) . next, animal inoculation experiments were performed to examine the in vivo phenotypic alterations associated with serial in vitro passage at low temperatures (a cold adaption tool for virus attenuation) of the virulent aram strain. the pathogenicity of the parental aram-p and its cold-adapted derivative, aram-p -ca, was characterized in pigs. twelve piglets divided into three groups of four animals each were challenged orally with parental aram-p (group ) or cold-adapted aram-p -ca (group ), and the remaining piglets in a control group received cell culture medium. clinical signs were recorded daily, and fecal swabs were collected pre-and post-challenge for the duration of the study. during the acclimation period, all piglets were active, showed no clinical symptoms, and had normal fecal consistency that did not contain any pedv genetic material. after the challenge, none of the negative-control piglets developed clinical presentations typical of pedv throughout the study. in contrast, aram-p -challenged piglets (group ) displayed clinical signs including loss of appetite and diarrheic feces by dpi (mean css ± sdm of . ± . ) and underwent lethal watery diarrhea with vomiting thereafter (mean css of > . ) (fig. a) . all the inoculated animals in group finally died by dpi. remarkably, the cold-adapted aram-p -ca virus-inoculated piglets in group experienced neither pedv-related clinical symptoms nor mortality throughout the experiment. all animals in group were positive for pedv, as determined by rt-pcr, by dpi with a mean ct value of . (equivalent to . tcid /ml) and shed significantly higher amounts of pedv in feces with ct ranging from . - . ( . - . tcid /ml) until death (fig. b) . likewise, fecal shedding of pedv was detected in all the piglets in group by dpi with the mean ct values of . ( . tcid /ml), but thereafter decreased to the lowest levels thereafter followed by a slight increase at dpi. overall, the quantities of viruses in the feces of animals of group significantly declined compared to those in group , with wide ct ranges of . - . ( efficacy of cold-adapted attenuated pedv vaccine all animals in the parental aram-p -infected group were necropsied upon death at or dpi, while piglets in the remaining groups were euthanized at the end of the study for postmortem examinations (fig. ) . neither macroscopic nor microscopic intestinal lesions were evident in the negative control piglets (right panels c, f, and i). the virulent aram-p -inoculated pigs macroscopically showed archetypal ped-like gross lesions. their small intestines were dilated with accumulated yellowish fecal fluid and had thin transparent walls as a result of villous atrophy (panel a), whereas the other internal organs appeared normal. in contrast, all animals infected with cold-adapted aram-p -ca in group displayed no remarkable visible pathological lesions in their gastrointestinal tracts with normal bowel wall thickness, comparable to those in the negative control group (panel b). microscopic assessment revealed that the small intestines from all dead piglets in group were characterized by acute viral enteritis, with villous shortening and fusion, involving vacuolation of superficial epithelial cells, in the jejunum (panel d). furthermore, ihc staining showed intense antigen labeling in the cytoplasm of epithelial cells in atrophied intestinal villi with a immunosignal score of . ± . (mean ± sdm) in the jejunum (panel g). however, two out of four cold-adapted virus-inoculated pigs in group exhibited mild villous atrophy in the small intestines, while the remaining pigs showed normal intestinal histopathologies (panel e), analogous to those of the negative control group (panel f). pedv antigen was rarely present in the small intestines in animals of group with a significantly lower mean jejunal viral antigen score ( . ± . ) (panel h) than that in group (panel g). altogether, our results indicate that cold-adapted aram-p -ca showed noticeably weakened virulence with an attenuated phenotype in the highly susceptible piglets under experimental conditions. to evaluate the protective effectiveness of a cold-adapted pedv vaccine, our study employed an oral homologous prime-boost vaccination scheme at weeks apart before farrowing. three sows in group were orally primed and boosted with live vaccines at and weeks pre-partum, whereas positive (group ) and negative (group ) control animals were kept clinical significance score log tcid /ml unvaccinated. all sows in the vaccinated and unvaccinated groups experienced neither pedlike clinical symptoms, including pedv fecal shedding, nor adverse reactions to vaccines. at farrowing, there were no significant differences in reproductive performance between the vaccinated and unvaccinated sows. ten piglets per litter were allocated to each sow and were nursed by their dam. at day post-farrowing, all neonates in groups and were orally exposed to the virulent aram-p virus. in contrast, nursing piglets in group were mock-inoculated with mem. clinical signs were recorded daily, and fecal swabs were collected before and after the challenge for the duration of the study. during the pre-challenge period, all piglets were healthy and had no clinical symptoms showing normal fecal consistency without pedv shedding. following the challenge, pedv-exposed piglets from unvaccinated sows (group ) developed clinical symptoms, including anorexia and diarrhea by dpc (mean css of > . ) and suffered from severe watery diarrhea with vomiting thereafter (fig. a) challenge period. these results demonstrated that litters from vaccinated sows significantly mitigated diarrhea after the challenge compared to those from unvaccinated sows. pedv-associated mortality occurred in > % piglets ( of ) from unvaccinated sows by dpi, and additional fatalities occurred until the end of the study, indicating a rate of > % mortality in the unvaccinated challenge control group (fig. b) . in contrast, of piglets birthed by vaccinated sows (group ) died from pedv exposure, showing an approximately % survival rate in the piglets. pedv fecal shedding was detected in all piglets from unvaccinated sows (group ) by or dpc, and they persistently discharged high quantities (> . tcid /ml) of pedv in feces until death (fig. c) . however, all animals from vaccinated sows (group ) showed pedv shedding in feces by dpi with a peak mean titer of . tcid /ml but amounts of fecal shedding significantly declined thereafter. all piglets from the negative control sow (group ) remained alive and had no viral shedding in rectal swabs for the duration of the study. considering that neutralizing antibodies are associated with protective immunity in neonates against pedv infection, we tested serum and colostrum samples collected from sows and their neonatal piglets for the presence of anti-pedv antibodies by an sn assay. as shown in fig. a , all sows developed anti-pedv neutralizing antibodies after the live prime vaccination. thereafter, the neutralizing antibody titers in sera of vaccinated sows gradually increased, likely resulting from a homologous boost. all the three sows maintained high neutralizing antibody titers for weeks post-farrowing. consistent to the results from the serum samples, the neutralizing antibody level was significantly high in the colostrum of vaccinated sows (fig. b) . furthermore, the nursing piglets from each litter in group had pre-challenge neutralizing antibody levels similar to those of their own dam and retained antibodies up to dpi, indicating the protective effect of lactogenic immunity passively acquired from vaccinated sows (fig. c) . clinical significance score log tcid /ml (groups and ). taken together, these data demonstrate that oral vaccination with the coldadapted live attenuated virus efficiently elicits potent antibody responses in sows, which are subsequently transferred to the offspring via their milk to provide protection against pedv. a sow does not possess the means to transplacentally transmit maternal immunoglobulins to its fetus; hence, piglets are born without passive immune protection, being highly susceptible to a variety of infectious agents, particularly multiple enteric pathogens, such as pedv. as a substitute, the passive immunity is dependent on the supplement of maternal-derived immune components via mammary secretions (i.e., colostrum and milk) [ , ] . therefore, maternal vaccination is the only crucial and effective tool to confer passive protection to newborn piglets through lactogenic immunity against pedv infection. this objective may be accomplished by the selection of an ideal vaccine candidate, the antigenicity of which is close to that of an epidemic strain, and virulence is minimal or absent in piglets. in south korea, several g a pedv-based live vaccines have been extensively employed to prevent ped for decades. however, owing to partial protection of these historical vaccines against the contemporary g b strains prevailing since the - pandemic, a demand for the development of new effective vaccines based on the dominant epidemic strain has increased. although efforts to pioneer a new g b-based mlv vaccine have been made over the past several years, its success was delayed because of experimental hurdles such as a laborious long-term attenuation procedure in cell culture. it is acknowledged that cold adaptation of animal viruses at serially reduced temperatures for cultivation is the best method to derive a live virus vaccine line, as it is generally accompanied by loss of virulence [ ] . in this study, we used a cold adaptation approach with a stepwise lowering of the cultivation temperature in vitro for pedv attenuation and aimed to determine the immunogenicity and pathogenicity of the cold-adapted strain and its efficacy as an attenuated live vaccine candidate under experimental conditions. as the pandemic of the g b pedv strain ravages the global swine industry, the pedv field is moving towards the development of novel vaccines and vaccination strategies. to accomplish this task, we first isolated field g b pedv that can be efficiently propagated in vitro. the g b isolate, aram-p , exhibited comparable growth characteristics with other g b strains in regard to cytopathology, infectious titers, and replication kinetics in vero cells as reported previously [ ] . genetic and phylogenetic analyses showed that the aram isolates are most closely related to the recent g b strains prevalent worldwide. however, sequence comparisons with other pedv strains revealed outstanding genomic features in the aram strain, which naturally included two independent dels in s and orf , respectively. more interestingly, due to the -nt del in orf , aram is predicted to encode an alternative orf protein with a large -aa excised c-terminus. although the exact function of pedv orf remains unknown, a number of attenuated pedv strains contain their signature dels in several parts of orf , suggesting its critical role in viral pathogenesis [ , , ] . in particular, wang et al. [ ] reported an attenuated pedv that encodes a c-terminal truncated orf protein of -aa residues, which is form the most analogous to the orf of the aram strain. however, experimental oral inoculation of newborn pigs with aram-p induced severe clinical presentations and macroscopic and microscopic intestinal lesions typical of acute pedv infection as reported previously [ ] . in general, death rates average % in suckling piglets up to one week of age, often approaching % in neonates less than days of age, and decrease to % thereafter [ , ] . likewise, our study reproduced % mortality in -day-old conventional piglets inoculated orally with the − diluted aram virus (calculated dose of tcid per pig). the data demonstrated that the aram isolate was highly enteropathogenic in neonatal piglets, implying that the orf product would be irrelevant to pedv virulence. since a growing body of evidence proposes that a combination of multiple genetic mutations in s, orf , and functional nsps affects the virulence of pedv, we cannot exclude that the c-terminal defect in orf may be one of molecular and physiological factors that may simultaneously alter pathogenic mechanisms. however, consistent with previous works [ , ] , growth kinetics of the aram strain in cell culture supports the notion that the accessory orf gene is dispensable for pedv replication in vitro. collectively, despite a redundant role in virus propagation in vitro, conservation of the complete orf in pedv field isolates still suggests its importance in causing natural infection in the animal host. to serve as a novel mlv vaccine strain, we generated an aram-derived attenuated pedv strain using a cold adaptation attenuation method. our approach for viral attenuation was to obtain a cold-adapted strain with impaired growth at physiological temperature ( °c), which can sufficiently induce mucosal immunity in the natural host. the cold-adapted aram-p -ca strain was derived by serial passages of the aram isolate at a low temperature ( °c). the aram-p -ca strain developed unsuccessful in vitro infection at the physiological temperature, as a significant decrease in infectious units was observed. however, this virus showed significantly high immunogenicity in orally inoculated pigs. furthermore, when investigating the phenotype of aram-p -ca in highly susceptible day-old piglets, none of the inoculated animals became clinically sick and grossly abnormal. our experimental data indicate that the cold-adapted aram-p -ca virus is attenuated virologically in vitro and clinically in vivo. subsequently, we sequenced the entire genome of the aram-p -ca strain to decode the genetic mutations that emerged during sequential cell passages at °c. upon cold adaptation, a total of non-silent mutations arose in orfs through , except for orf , without extra indels throughout the genome, whereas the two s and orf del signatures of the aram strain were entirely maintained. intriguingly, -aa substitutions were accumulated in the s protein, among which -and -aa substitutions were distributed in the n-terminus of s and s domains, respectively, suggesting that some of these changes are associated with viral attenuation. since the pedv s protein is heavily glycosylated, altered glycan motifs may be involved in viral pathogenesis by modifying the protein conformation and function [ ] . however, no genetic changes were found in the putative n-glycosylation sites between the parental and cold-adapted strains. as the major antigenic determinant, the s glycoprotein possesses at least four neutralizing domains of pedv at aa positions - (ntd/s ), - , - , and - [ ] . the aa residues that comprise these domains remained nearly unchanged throughout the cold adaptation, except for one n y mutation in the ntd/s region. furthermore, no aa changes were found in additional discrete domains, including a c-terminal domain (residues - ) of s that can interact with cellular receptor(s) and a s fusion domain comprising of a hydrophobic fusion peptide (residues - ) and two heptad repeat regions (residues - and - ). although the polygenic traits are likely associated with pedv virulence, it is of utmost importance that future work using reverse genetics technology should aim to address whether genetic drift, especially in the s protein, influences the pathogenicity of pedv. in addition, attenuated viruses often have the propensity to revert to virulent form under certain circumstances, which is a main drawback for their use as an mlv vaccine. indeed, the aram-p -ca strain had an ability to replicate at °c since it denoted impaired, but not entirely halted, growth at °c, as the viral titer was determined to be . tcid /ml. this incompletely abolished growth of aram-p -ca at the physiological temperature implies a residual risk of reversion to virulence in vivo. thus, further safety studies will be necessary to evaluate the phenotypic stability of the attenuated aram-p -ca virus in neonatal piglets. to assess the effectiveness of the cold-adapted live attenuated vaccine, a homologous primeboost sow immunization trial consisting of oral administration of a double-dose of the p -ca virus (l/l vaccination) was implemented at intervals of weeks before delivery. newborn piglets born to vaccinated and unvaccinated sow groups were inoculated orally with the parental virulent pedv strain. the difference in the burden of diarrheic disease as measured by mortality and morbidity was investigated between the challenged litters from vaccinated and unvaccinated sows. piglets born to unvaccinated sows presented a much higher mean css, as determined by the intensity of diarrhea, than those of vaccinated sows. after dpc, the challenged animals born to vaccinated (group ) sows had mild-to-moderate diarrhea, whereas the piglets born to unvaccinated (group ) sows experienced fatal watery diarrhea. although nearly all the piglets in group died by dpc, group also exhibited more than % neonatal mortalities during the experimental period. on the contrary, our previous trial has shown that a multiple-dose parenteral immunization with inactivated killed g b vaccines (k/k vaccination) offered a more protective efficacy ( % survival rate) in piglets against g b virus than that in the present study [ ] . in contrast to piglets from unvaccinated sows that discharged high amounts of the virus in feces until death, pedv fecal shedding in animals from vaccinated sows was greatly reduced at dpc and more than % of the piglets that survived shed no virus in stools at the end of the trial. collectively, the maternal l/l vaccination described in the present study is unlikely to be more efficacious than the k/k treatment in terms of the protective rate; however, it could help alleviate diarrheal severity, including the duration and quantity of fecal shedding of pedv. although our l/l vaccination of sows did not entirely avert morbidity in piglets upon a pedv challenge, its advantageous effects on relieving pedv shedding would lessen the possibility of the environmental contamination in the farrowing barns, thereby breaking the chain of secondary spread of the virus to other animals and herds during epidemics. furthermore, pedv transmission via the l/l vaccination is unlikely, as no pedv genetic material was detected in feces from any of the pregnant sows vaccinated with p -ca. moreover, we were able to confirm the presence of satisfactory amounts of anti-pedv neutralizing antibodies in the sera and colostrum of l/l vaccinated sows at farrowing and post-farrowing stages, as well as in sera obtained from their offspring, indicating the piglet protection by transfer of maternal immunity via mammary secretions from the immunized dams. these data further strengthen the concept that an optimal vaccination regimen is associated with retaining high levels of neutralizing antibodies in the lactogenic secretions of vaccinated sows, which in turn are correlated with colostrum and milk iga antibody titers to offer protective immunity against pedv [ , , , ] . in summary, this is the first report describing the development of a cold-adapted mlv vaccine based on a virulent g b pedv strain. the present study demonstrated the loss of virulence of a highly pathogenic pedv strain, with genetic mutations throughout the entire genome, after cold adaptation. vaccination-challenge studies revealed that sows immunized with a homologous oral l/l maternal vaccination regimen provides protective lactogenic immunity to piglets, thereby reducing mortality, morbidity, and fecal shedding of the virus. in south korea, multiple-dose vaccination programs including l/k/k at -or -week intervals before delivery have been recommended nationwide in pregnant sows for decades [ , ] . due to the absence of a new safe and effective g b live vaccine, controlled oral exposure (feedback) for prime-boost vaccination strategies is mostly implemented as a countermeasure in the field since the - ped epidemics. despite certain positive contributions of feedback to naïve herds, intentional infection using an undefined inoculum may cause undesirable negative consequences, which include insufficient immunity induction, unexpected virus transmission, amplified viral recombination and diversity; and more importantly, dissemination of other microbial pathogens conceivably evolving to develop into a new disease agent. once the cold-adapted aram-p -ca-based mlv vaccine is supplied in the market, gradual withdrawal of the feedback practice will be imminent. since oral administration does not have the capacity to ensure that all animals are immunized with a proper dose, this demerit should be ameliorated to easily use the live vaccine under field conditions. with the availability of inactivated g b pedv vaccines in the domestic market, customized vaccination programs, such as the application of heterologous oral prime-parenteral boost l/k/k or l/l/k schemes, could be established based on various herd circumstances, including naïve, epidemic, and endemic statuses. it is noteworthy that maternal vaccination has to be implemented in conjunction with additional intervention strategies involving intensive biosecurity, husbandry, and hygiene practices for ped prevention and eradication. porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus porcine viruses: from pathogenesis to strategies for control fields virology heterogeneity in spike protein genes of porcine epidemic diarrhea viruses isolated in korea outbreak-related porcine epidemic diarrhea virus strains similar to us strains, south korea immunogenicity and protective efficacy of recombinant s domain of the porcine epidemic diarrhea virus spike protein porcine epidemic diarrhoea: new insights into an old disease a new coronavirus-like particle associated with diarrhea in swine deadly pig virus slips through us borders emergence of porcine epidemic diarrhea virus in the united states: 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cold-adapted influenza virus vaccine in an efficacy clinical trial development and characterization of cold-adapted viruses for use as live virus vaccines genetic characteristics, pathogenicity, and immunogenicity associated with cell adaptation of a virulent genotype b porcine epidemic diarrhea virus a simple method of estimating fifty per cent endpoints complete genome sequence of a novel porcine parainfluenza virus isolate in korea the clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools the neighbor-joining method: a new method for reconstructing phylogenetic trees molecular evolutionary genetics analysis (mega) software version . multiplex real-time rt-pcr for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus porcine reproductive and respiratory syndrome virus nucleocapsid protein modulates interferon-β production by inhibiting irf activation in immortalized porcine alveolar macrophages pathogenesis of porcine epidemic diarrhea virus isolate (us/iowa/ / ) in -week-old weaned pigs viral diarrhea of man and animals cloning and further sequence analysis of the orf gene of wild-and attenuated-type porcine epidemic diarrhea viruses pedv orf encodes an ion channel protein and regulates virus production manipulation of the porcine epidemic diarrhea virus genome using targeted rna recombination mutations in the spike gene of porcine epidemic diarrhea virus associated with growth adaptation in vitro and attenuation of virulence in vivo the s glycoprotein subunit of porcine epidemic diarrhea virus contains immunodominant neutralizing epitopes efficacy of an inactivated genotype b porcine epidemic diarrhea virus vaccine in neonatal piglets characterization of anti-porcine epidemic diarrhea virus neutralizing activity in mammary secretions key: cord- -tdswzj r authors: freitas, andré ricardo ribas; donalisio, maria rita title: excess of mortality in adults and elderly and circulation of subtypes of influenza virus in southern brazil date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: tdswzj r purpose: in the elderly population, the influenza infection and its clinical complications are important causes of hospitalization and death, particularly, in longer-lived age. the objective of this study is to analyze the impact of influenza virus circulation on mortality in the elderly and adults, in years with different predominant virus strains. methods: we performed a time trend study to evaluated excess of mortality for pneumonia and influenza, respiratory disease, and all-causes in southern region of brazil, from to . after considering other models, we opted for serfling regression. excess of death rates per , inhabitants were analyzed in specific age groups ( – , – , – , ≥ years) and by year of occurrence. mortality information were taken from brazilian mortality information system and etiological data were accessed in sentinel virological surveillance database, getting the weekly positivity of the immunofluorescence tests for influenza a (h n , h n ), and b. results: in southern brazil, there is an evident seasonal pattern of all death outcomes among different age groups in the dry and cold season (april–september). the highest excess mortality rates occurs among older, particularly in years of circulation of influenza ah n , especially among people ≥ years, in and —years of great severity of influenza activity. after , with the introduction of the pandemic influenza ah n , we observed a lower impact on the mortality of the elderly compared to < years. discussion: a cross reactivity antibody response from past exposure probably provided protection against disease in the elderly. despite not controlling for comorbidities, climate, and vaccination, for the > years, ratio of respiratory diseases excess mortality rates between ah n ( ) and severe year of h n ( ) shows protection in the pandemic year and great vulnerability during ah n virus predominance. conclusion: the reduced immune response to infection, and to vaccination, and presence of comorbidities recommend a special attention to this age group in brazil. besides medical assistance, the timeliness of vaccine campaigns, its composition, and etiological surveillance of respiratory diseases are some of the preventive and public health measures. introduction human influenza viruses can cause diseases through many direct and indirect pathological effects. consequences are destruction of infected cells, release of cytokines leading to fever, malaise, damage to respiratory epithelium and pulmonary parenchyma, and pneumonia. it includes secondary bacterial infections because of tissue damage and exacerbation of preexisting comorbidities such as cardiovascular and renal diseases, diabetes, or chronic lung disease ( ) ( ) ( ) . the rates of hospitalization and mortality associated with influenza are higher among patients with chronic diseases, children under year and after years of age ( , ) . with the aging population in recent decades, the raw number of hospitalizations and deaths related to pneumonia and influenza tends to increase ( ) , this phenomenon has been observed also in brazil ( , ) . however, the impact and severity of influenza virus circulation depend in part, on the strain that predominates in the season each year. due to the lack of laboratory confirmation, influenza-associated morbidity and mortality are often classified as pneumonia, other respiratory diseases, or other causes. given the difficulty of directly measuring influenza morbidity and mortality, time series models are used to elucidate disease patterns in various age groups. trends are usually determined by means of statistical inference, based on seasonal coincidence of the occurrence of certain diseases or death and laboratory confirmation of the viral circulation ( , ) . different approaches, with and without the quantification of the proportion of viral isolates, can produce average estimates of excess deaths associated with the circulation of certain viral variants ( ) ( ) ( ) . viral surveillance data, hospitalization, or death indicators are particularly useful for the study of influenza in the tropics, as seasonality may be less evident ( ) ( ) ( ) . serfling regression has been used to analyze excess of mortality related with respiratory virus circulation ( , ( ) ( ) ( ) . despite some limitations ( ) , the inclusion of sinusoidal terms in weekly regression may reduce spurious correlation between influenza occurrence and death ( , ) . it is particularly useful when no other covariables are available, and with small samples of viral sentinel surveillance data ( ) . poisson regression and the generalized linear model (glm) can produce more specific estimates and support adjustments for variables (temperature, humidity, comorbidities, other circulations of viruses), although they require a more robust and consistent virological surveillance and cannot be used for pandemics ( ) . in brazil, surveillance for influenza syndromes was implemented in , monitoring the occurrence of respiratory viruses (influenza a and b, parainfluenza , , and , respiratory syncytial virus, adenovirus). the brazilian ministry of health provides vaccination coverage annually since for seniors and some risk groups, with vaccine coverage of the elderly population at around % in southern brazil, the region with the highest coverage of the country. despite the adequate coverage, protective titers after vaccination (hi ≥ ) are consistently lower with poorer cell mediate and antibody responses in the elderly comparing to adults ( ) . considering the vulnerability of the elderly to influenza virus infection, and the lack of studies on its repercussion in brazil, the objective of this study was to analyze the impact of different strains of influenza a virus circulation. we analyzed particularly the most predominant variants (ah n and ah n ) on excess of mortality in the adults and elderly of different age groups in a region with marked seasonality of respiratory diseases in brazil. this is a time trend study to evaluated excess of mortality from to in southern region of brazil (states of paraná, santa catarina e rio grande do sul), total area is , , km , population is , , inhabitants with subtropical climate (köppen-geiger classification cfa). we choose these states for analysis because of the consistent seasonal pattern of influenza, as well as the availability and quality of etiological data from the virological surveillance system in that region. for the mortality rates of specific age groups ( - , - , - , and ≥ years) and death causes, we took data from brazilian mortality information system. causes are classified according to international causes of death icd- revision, pneumonia, and influenza (icd j to j . ), respiratory diseases (icd j to j ), and all-cause (excluding external causes of mortality). we obtained population of each year and age group from instituto brasileiro de geografia e estatística-ibge from the census- , and population estimates for the following years. etiologic information of flu-like syndrome was accessed in database of the national sentinel virological surveillance system. it has data from sentinel units distributed in all regions of the country-north ( units), northeast ( units), southeast ( units), south ( units), and central west ( units). surveillance is performed through the systematic collection of weekly samples of nasopharyngeal secretions from patients who present flu-like syndrome. reference laboratories process samples by using indirect immunofluorescence (iif), with tests for influenza a and b, parainfluenza , , and , respiratory syncytial virus, and adenovirus. a portion of the samples is submitted to polymerase chain reaction tests to identify the virus genotype. we calculated the laboratory positivity indicator using weekly positive results of iif divided by the total of weekly valid tests, i.e., excluding the results within inadequate samples (not enough biological material, improper storage, incorrect material in the sample) or inconclusive results (no valid results). influenza vaccination coverage (%) of southern region from to was obtained from brazilian national program of immunization data base (datasus). the criteria used to define the period of increase of influenza activity was when the positivity of the samples tested exceeded twice the annual mean of the weekly positivity of samples processed by surveillance, during two consecutive weeks. in the year , we consider the period officially recognized by the brazilian ministry of health as epidemic by the influenza ah n pmd strain, due to irregularity of the sample collection by the sentinel surveillance system at the end of epidemic. we calculated the weekly mortality rates by age group using the number of deaths per group of causes divided by the estimated population in the middle of the year multiplied by , . we constructed a serfling cyclical regression model ( ) for weekly data applied to each age group and causes of death (pneumonia and influenza, respiratory diseases, and all causes), as seen in others studies ( , ) , to estimate baseline of predicted deaths in the absence of influenza epidemics. to fit regression, we used period of years (from to ), excluding the weeks of epidemics periods. a cyclical linear regression was adjusted with the equation: where y is the mortality rate, β is the coefficients of regression, t is time in weeks, and t and t are variables for adjusting the secular trend of the disease. we used of sine and cosine for adjust of annual and semiannual periodic components. after adjusting a linear regression and define the expected mortality rate, we delimited % upper confidence limit of the baseline as the reference threshold in the absence of influenza epidemics. we calculated the excess of deaths as the observed mortality minus the expected mortality in the periods when mortality was above % of the confidence interval during epidemics periods. we also present ratios of excess mortality rates among years of predominant circulation of influenza strains ah n (mean and years of severity), ah n pre-pandemic, and ah n postpandemic for each age group. for data compilation, we used microsoft office excel , and for statistical analysis, spss for windows, version . . results table shows the proportion of positivity of the iif nasopharyngeal samples and the annual prevalence of strains of influenza in the period. before , the year of entry of the pandemic strain ah n pmd , there was a predominance of influenza ah n in the years to . after , there is alternation of strains in the southern brazil. annual elderly vaccination coverage in southern region is high and homogeneous, around %, and even higher in the recent years. there is an evident seasonal pattern of deaths from pneumonia and influenza, respiratory diseases, and all-causes among the elderly in different age groups in the dry, cold months (april-september) in southern region (figure ) . we note a progressive increase in the rates of excess deaths (of all outcomes) with increasing age, especially among those older than years. in the pre-pandemic years with dominance of the ah n strain, the excess of mortality rates associated with influenza were relatively low, compared to years of prevalence of ah n strain ( table ) . among those over years, the ratio of excess mortality rates between and the years with dominium of h strains was less than one. this ratio suggests that this age group was spared in the pandemic. however, in years of predominance of strain h , excess of mortality rate of all causes in this group were . per , (corresponding to , obits), , and . times greater than the same rate in years of circulation of h n in pre-and post-pandemic period, respectively. among adults ( - years), we observe a large excess of deaths rates during the pandemic ( obits), which correspond to . excess deaths from all causes, and excess mortality from respiratory diseases associated with viral infection in every , individuals of the age group. the ratio between excess mortality rates due to pneumonia/influenza in the pandemic year ( ) and the mean rate of the period was times higher among the youngest ( table ) . rates of excess mortality by pneumonia and influenza and respiratory diseases are lower than all causes in all age groups, but particularly high in older than years ( table ) . the results highlight the great vulnerability of elderly to influenza ah n , especially among older than years in severe years of influenza activity, like and . the study also shows the lower impact of influenza ah n pdm in this age group compared to younger. risk of dying among the elderly in years of circulating ah n influenza has been reported in several parts of the world ( , , , ) ; however, in brazil, there are no recent estimates available. few studies analyze the circulation and impact of influenza in tropical and subtropical regions ( , , ( ) ( ) ( ) . influenza b virus is also associated with severe disease ( ); however, this variant did not circulate with intensity during the study years in brazil. although the elderly are the most vulnerable group to viral respiratory infections, we found relative small excess of deaths in years of circulating ah n pre pandemic ( and ) . study comparing excess deaths from respiratory diseases in the elderly in latin america shows stable rates (mean of . per , inhabitants) in southern brazil between and (prepandemic flu a-h n ), although higher in brazil than in other countries ( ) . in the usa and in european countries, influenza seasons dominated by subtype ah n are typically associated with mortality two to three times higher than in seasons with predominance of ah n (prior to pandemic strain ) and of influenza b viruses ( , , , ) . when all causes of death are studied, the overall mortality associated with influenza among elderly exceeds that observed in younger age group. it should be considered that all causes mortality is a non-specific measure and a distant outcome of influenza infection. however, it is difficult to determine which group of causes of death could better characterize the influenza burden in mortality. by choosing only the respiratory causes, we may underestimate clinical complications of pulmonary viral infection (e.g., cardiovascular). therefore, in this study, we analyzed all causes, respiratory, and pneumonia and influenza deaths. the unfavorable evolution of infection in the elderly is possibly due to the prevalence of comorbidities, deficiencies in defense mechanisms, and poor antibody response to vaccination, as cellmediated and humoral responses limit severity of disease ( ) . patients with chronic diseases are more susceptible to infection due to decline of the immune function through inflammatory mechanisms, hindering the mucosal barrier, and the adaptive and innate immunological defense mechanisms ( ) . the immune response to infection in the elderly tend to be delayed and weak, with prolonged inflammatory responses, which involves different types of host reaction, mainly to clearance virus. the exacerbations of these mechanisms may induce immunemediated pathology causing tissue damage ( ) . cytokine high serum levels of il- , tnf-a, ifn-g and sil- r, chemokines ip- , mcp- , and monokine induced by ifn-g (mig), are associated with severe clinical cases and lung damage ( ) . immunological abnormalities in people with diabetes, chronic respiratory diseases, cardiopathy, or other chronic diseases have increased risk of severe infection and bad prognosis ( ) . for example, there is the consistent association of influenza infection with cardiovascular mortality, particularly acute myocardial infarction ( ) . in part, it is attributed to altering endothelial function due to an acute inflammatory and procoagulant stimulus during viral infection ( , ) . clinical complications of diabetes triggered by influenza infection cause impairment of leukocyte function and increase post-infection colonization rates resulting in poor prognosis in the elderly ( , ) . in young people and adults, in , the emerging influenza ah n strain had a notable impact on the mortality of people up to years in various parts of the world, including brazil ( , , , ) . excess mortality of individuals aged - years in the state of são paulo, brazil was identified during the pandemic ah n virus ( ) . pregnant women adults with metabolic conditions, including obesity, chronic respiratory disease, and other chronic diseases were significantly associated with severe acute respiratory syndrome and the lethality in brazil ( ) . our study showed a . -fold higher rate of mortality from pneumonia and influenza in adults ( - years) in the pandemic year ah n than the average of years with predominance of ah n circulation in southern region. in addition to the clinical severity and the large portion of the affected population, pandemics affect age groups in different ways ( ) . while only % of deaths from seasonal influenza occur among those under years of age, in the pandemics of , - , and , this proportion was , , and %, respectively ( ) . therefore, pandemics tend to affect a larger proportion of young people than seasonal influenza. in this study, higher rates of death due to pneumonia, influenza, respiratory, and all causes were observed among those aged - years in . one explanation for the higher mortality observed among the youngest is that they would be more prone to the situation known as "cytokine storm, " i.e., a dysfunctional overproduction of cytokines that would lead to diffuse damage to the respiratory tract with severe and potentially lethal systemic repercussions ( ) . viral replication and production of inflammatory mediators seem to be involved in the pathogenesis of infection with influenza a h n pmd , hindering the clearance of virus in lung tissue and leading to pathologic lesions ( ) . another explanation for the lower mortality in the elderly is that they were exposed previously to antigens of the pandemic virus. hancock et al. ( ) suggested a cross-reactive antibody response to pandemic ah n . similarities between ah n antigen from and were detected. this last virus strain has not circulated since ( ) , when the ah n strain was displaced by ah n (asian flu). at that time, ah n viral circulation occurred mainly in children, the current elderly of . the emergence of the ah n strain in the pandemic year (hong kong flu) affected several age groups. this new strain resulted from a large genetic mutation (shift) recombining virus material of the circulating ah n with the avian h , of asian origin, resulting in the new variant ah n ( ) . in - , under selective pressure an antigenic small mutation (drift), resulted in a/fujian/ / (h n ) a strains emerged after a "jump" in genes evolution of hemagglutinin and neuraminidase proteins of virus surface ( , ) . the circulation of the fujian strain had a great impact on the mortality from pneumonia in several parts of the world in - and - ( ) and in brazil ( ) . in , a new drift resulted in influenza ah n detected in south brazil ( ) also affecting hospitalizations and deaths in various parts of the world ( ) . we observed high rates of excess mortality in the elderly, in the years of and . limitations of this study refer mainly to the ecological analysis of pooled data. we did not analyze individual information regarding comorbidities and history of vaccination that could be important confounders influencing mortality ( ) . we just had the overall annual vaccination coverage which were in general, around % in the period. estimates of the number of deaths (all causes, respiratory, and pneumonia-influenza) supposedly related to influenza may be inaccurate in inferring the impact of respiratory viruses. correlations in time series studies may produce spurious associations, especially between all causes of death and influenza infection, due to the distance between cause and outcome, and to multiple components of the obits. serfling addresses part of this limitation by introducing sinusoidal terms in equation, since non-influenza mortality is not expected to coincide exactly with sinusoidal pattern ( , ) . moreover, excess mortality of pneumonia, respiratory diseases, and all causes can be considered as an alert to surveillance of viral respiratory diseases, such as a sentinel indicator to be investigated ( , ) . although all causes mortality is a non-specific indicator, it does not underestimate the complications of chronic diseases associated with influenza ( ). despite the influenza component in all causes mortality is small, the indicator can be considered an indirect measure, a warning, useful in epidemiological monitoring. another limitation is the lack of robust etiologic data from virological surveillance in the years - , which could lead to imprecision in the analyses; however, the data on the predominance strains in the southern region are reliable, and influenced the composition of the vaccine of each season. considering the option for the analysis model, serfling linear regression may produce different estimates when compared with other models (poisson, arima, and glm) ( , ); poisson and arima models produce higher mortality estimates than serfling, and serfling higher than glm, especially among the elderly ( , , ) . we chose serfling model because we do not have robust virological surveillance data, before , and the study period includes a pandemic year ( ) . besides, in this study, we did not analyze climatic variables (minimum temperatures and relative air humidity) that could also interfere with viral transmission and increase the impact of the disease, particularly in the elderly. in conclusion, probably previous exposures to influenza ah n in the past influenced the mortality of brazilian elderly in , despite the vulnerability of this age group to clinical complications. for the > years, we observe higher excess mortality rates (of all outcomes) in severe year of ah n circulation ( , ) . it is also worth noting that vaccination has been associated with the prevention of death particularly at age ( ) . therefore, the high elderly vaccination cover in southern brazil may have attenuated excess of mortality estimated, although the immune response is limited among those. more attention should be given to the circulation of influenza ah n in subtropical regions in brazil. the reduced immune response to infection and to vaccination, and associated comorbidities recommend a special attention to this age group. besides medical assistance, the timeliness of vaccine campaigns, its composition, and etiological surveillance of respiratory diseases in the region are some of the preventive and public health measures. both authors made contributions to the conception of the work, acquisition, analysis, interpretation of data, and writing the manuscript. center for disease control and prevention. prevention and control of influenza: recommendations of the advisory committee on immunization 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viruses collected from young children in uberlandia, brazil-from virus influenza detectados no estado do rio grande do sul durante center for disease control and prevention. influenza activity -united states and worldwide, - season the impact of influenza epidemics on mortality: introducing a severity index deaths averted by influenza vaccination in the us during the seasons / through / authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -rirbdhz authors: reddehase, matthias j. title: adverse immunological imprinting by cytomegalovirus sensitizing for allergic airway disease date: - - journal: med microbiol immunol doi: . /s - - -z sha: doc_id: cord_uid: rirbdhz cytomegalovirus (cmv) infection has a profound impact on the host’s immune system. immunological imprinting by cmv is not restricted to immunity against cmv itself, but can affect immunity against other viral or non-viral infectious agents and also immunopathological responses. one category is heterologous immunity based on molecular mimicry, where antigen recognition receptors specific for a cmv antigen with broad avidity distribution also bind with some avidity to unrelated antigens and exert effector functions against target structures other than those linked to cmv. another category is induction of cytokines by cmv infection that inhibit or drive immune responses to bystander antigens unrelated to cmv, and a third category is the activation of antigen-presenting cells by cmv from which unrelated antigens profit as “stowaways”. a striking example of the “stowaway” category, actually one that is of medical importance, has been published recently and will be discussed here for the more general reader. specifically, in a murine model, cmv airway infection and inhaled environmental antigen of poor intrinsic allergenic potential were found to sensitize for allergic airway disease (aad) only when combined. as to the mechanism, viral activation of cd b(+) conventional dendritic cells (cd b(+) cdc) that localize to airway mucosa facilitates uptake and processing of inhaled antigen. thus, cmv serves as a “door opener” for otherwise harmless environmental antigens that have no intrinsic property to activate dc. antigen-laden cd b(+) cdc migrate selectively to the airway draining lymph nodes, where they prime type- cd (+) t helper (th- ) cells. upon airway re-exposure to the inhaled antigen, th- cells secrete interleukins (il- , il- , il- , and il- ) known to induce goblet cell metaplasia, the lead histopathological manifestation of aad that is characterized by thickening of airway epithelia and increased numbers of mucus-producing goblet cells, resulting in enhanced mucus secretion and airflow obstruction. human cytomegalovirus (hcmv) is not a textbook example of a respiratory viral infection. nonetheless, hcmv infection has a respiratory phase that usually is not diagnosed because of its mild and unspecific symptoms in the immunocompetent host. so, it is subsumed under the popular term "common cold" that is used for symptoms of respiratory virus infections for which there is no medical indication to identify the cause and for which symptomatic treatment is sufficient, for instance infections with rhinoviruses, common coronaviruses, and parainfluenza viruses. historically, cmvs were named "salivary gland viruses" ( [ , ] , reviewed in [ , ] ), as they replicate in acinar glandular epithelial cells of the salivary glands, from where they are released into the salivary duct [ , ] . a frequent route of host-to-host transmission for primary infection is thus through saliva of a virus-shedding contact person, for instance in mother-to-child transmission. transmission probability is increased whenever very young children crowd together, such as in multi-child families and in daycare centers. as an example of real life epidemiology for lectures on medicine, toy sharing, that is the mouthing of toys that other children have mouthed, has been identified as a way by which hcmv is transmitted between peers (reviewed in [ ] , see also the contribution by adler and reddehase in this issue of mmim [ ] ). with the saliva, virus reaches upper airway mucosas, where it replicates persistently for a prolonged period, and from there it also reaches lower airway mucosas in bronchi, bronchioli, and the lungs, as shown long ago and revisited more recently in murine models of intranasal infection using murine cmv (mcmv) [ , ] . this topic is specifically addressed and more comprehensively referenced by zhang and colleagues in this issue of mmim [ ] . children who undergo a primary airway infection after transmission of hcmv are unavoidably at the same time exposed to a universe of inhaled environmental antigens, few of which are classical allergens, whereas by far most have lowto-no intrinsic allergenic potential, as otherwise all of us would suffer from allergy. clearly, allergic airway disease (aad) elicited by inhaled environmental antigens is not the rule. the development of aad requires a "sensitization phase", which, in immunological terminology, means "priming" of allergenspecific t cells, usually type- cd + t helper (th- ) cells, as well as re-exposure to the allergen for a recall response. in their effector phase, restimulated allergen-specific th- cells secrete cytokines that induce goblet cell (gc) metaplasia, such as the interleukins il- , il- , il- , and il- [ ] [ ] [ ] . remodeling of the airway mucosa by transdifferentiation of ciliated cells and clara/club cells to mucus-producing gc, rather than by proliferation of gc [ ] , associated with thickening of airway epithelia and chronic mucus hypersecretion and consequent airflow obstruction, is the lead histopathological manifestation of aad. mouse models, provided they are reasonably designed to meet a clinical correlate, have proven predictive value for human disease [ ] . as airway exposure to environmental antigens at the time of primary airway infection after hcmv transmission is a realistic scenario of medical interest, recent work modeled this scenario in the mouse with the aim to investigate a possible virus-allergen interplay [ ] . in the model (fig. , top and [ ] ), immunocompetent c bl/ mice were simultaneously exposed via the airways to mcmv and to purified ovalbumin (ova). ova was chosen to represent a protein antigen that has low allergenic potential on its own and fails to sensitize for aad. for tracking the fate of this low-potency allergen, ova was tagged with a red fluorescent dye. on three consecutive days (days , , and ) after sensitization, mice were re-exposed to ova aerosol for a recall response, and airway histopathology was evaluated days after the last challenge exposure (fig. , histological images) . mcmv airway infection alone did not trigger aad, nor did the combination of ova-sensitization and ova re-exposure. combined sensitization by ova and mcmv, however, followed by challenge exposures to ova, caused aad that is characterized by a massive increase in the number of mucus-producing gc. this histopathology is referred to in the literature as gc hyperplasia or metaplasia, depending on whether one focuses on the increase in gc numbers [ ] or on the remodeling of the mucosa by transdifferentiation of ciliated cells and clara/club cells to gc [ ] , respectively. it should be noted that aad was not induced when either ova sensitization or ova reexposure were missing. these controls verified the ovaspecificity of aad. depletion of cd + t cells shortly before the first ova challenge prevented aad, whereas depletion of cd + t cells did not. so, the effector cells in aad are ova-specific cd + t cells. based on the data by reuter et al. [ ] , fig. provides a graphical summary that illustrates the mechanism by which mcmv imprints the immune system for aad immunopathology. upon co-exposure of airway mucosa to inhaled antigen/low potency allergen, represented by ova in the specific case, and mcmv, the virus activates mhc-ii + cd c + dendritic cells (dc) that localize to the mucosa, specifically dc of the cd b + subset of conventional b low ly c low dc, briefly cd b + cdc. their activation is indicated by upregulation of cd ligands cd and cd , and is associated with enhanced uptake of ova. the ova-laden cd b + cdc then migrate selectively to the airway draining lymph nodes, where they present processed ova peptides by mhc-ii molecules to naïve ova peptide-specific th- cells for priming. upon re-exposure to inhaled ova, restimulated ova peptidespecific th- cells secrete effector cytokines il- , il- , il- , and il- , all known to be involved in the induction of gc metaplasia. in essence, the work by reuter and colleagues [ ] has shown that the key event in sensitization for aad is the efficient uptake and processing of an inhaled antigen by migratory dc that localize to the airway mucosa as sentinels. classical allergens and most pathogens have the intrinsic property to activate dc, whereas inhaled environmental protein antigens do not, and are thus ignored by the sentinels. this is a "healthy ignorance", as otherwise all environmental antigens would cause allergy. activation of dc by cmvs is beneficial for priming a protective antiviral immune response, though it can result in allergic disease when used by otherwise harmless "stowaways" for entering dc. the findings suggests that by this "door opener" function, cmv airway infection may broaden the spectrum of potential allergens. current experimental protocols did not lead to full-blown asthma. future work will address the question if repetitive allergen exposure will eventually lead to clinical asthma. another interesting aspect for future research is the finding that aad in the here discussed model was not accompanied by eosinophilia [ ] , unlike in established aad/asthma mouse models and in most clinical forms of asthma. so, is it a bad model? interestingly, there exists a clinical correlate, namely an asthma phenotype in humans known as non-eosinophilic asthma (nea). nea accounts for particularly severe cases of asthma, and its most relevant clinical trait is its poor response to asthma treatment with corticosteroids [ ] . testing if non-eosinophilic aad, reviewed here and described in greater detail in [ ] , qualifies as a model, or can be developed into a model, for clinical nea is therefore of top priority. uptake & processing fig. graphical abstract of the mechanism that leads to aad/gc metaplasia. for detailed explanation, see the body of the text. the waved symbol represents peptide(s) derived by antigen processing. cdc conventional dendritic cell. the graphics concerning the airway mucosa was inspired by artwork presented in [ ] propagation of salivary gland virus of the mouse in tissue cultures propagation in tissue cultures of a cytopathogenic virus from human salivary gland virus (sgv) disease the history of cytomegalovirus and its diseases margaret gladys smith, mother of cytomegalovirus: th anniversary of cytomegalovirus isolation mouse cytomegalovirus. necrosis of infected and morphologically normal submaxillary gland acinar cells during termination of chronic infection site-restricted persistent cytomegalovirus infection after selective long-term depletion of cd + t lymphocytes the epidemiology and public health impact of congenital cytomegalovirus infection pediatric roots of cytomegalovirus recurrence and memory inflation in the elderly interstitial pneumonia and subclinical infection after intranasal inoculation of murine cytomegalovirus murine cytomegalovirus (cmv) infection via the intranasal route offers a robust model of immunity upon mucosal cmv infection persistent viral replication and the development of t-cell responses after intranasal infection by mcmv airway goblet cell hyperplasia in asthma: hypersecretory and anti-inflammatory? the cytokine network in asthma and chronic obstructive pulmonary disease importance of cytokines in murine allergic airway disease and human asthma the airway epithelium in asthma mouse model of cytomegalovirus disease and immunotherapy in the immunocompromised host: predictions for medical translation that survived the "test of time coincident airway exposure to low-potency allergen and cytomegalovirus sensitizes for allergic airway disease by viral activation of migratory dendritic cells non-eosinophilic asthma: current perspectives publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations this article is part of the special issue on immunological imprinting during chronic viral infection.acknowledgements mjr is supported by the deutsche forschungsgemeinschaft sfb , individual project tp . conflict of interest the author declares that he has no conflict of interest. key: cord- -s wi t authors: basile, l.; torner, n.; martínez, a.; mosquera, m.m.; marcos, m.a.; jane, m. title: seasonal influenza surveillance: observational study on the – season with predominant b influenza virus circulation date: - - journal: vacunas doi: . /j.vacun. . . sha: doc_id: cord_uid: s wi t introduction: influenza is a common respiratory infectious disease affecting population worldwide yearly. the aim of this work is to describe the – influenza season and how it affected elderly population in catalonia despite moderate vaccine coverage among this age group. methods: influenza surveillance based on a primary care sentinel surveillance, virological indicators systematic sampling of ili attended and severe influenza confirmed cases (shlci) admitted to hospital. analysis of data by chi-squared, anova, multiple regression and negative control test or case to case for vaccine effectiveness assessment in primary care and shlci respectively. results: moderate-high intensity and early onset season with predominance of influenza b virus (ivb) ( %) followed by an increase of circulation of influenza a virus (iva). a total of iv from primary care samples. vaccine effectiveness (ve) in primary care setting was % ( %ci: – %). severe cases (adjusted cumulative incidence . / , inhabitants ( %ci: . – . )). the highest proportion of severe cases were in the > ( . %) (aor . ; %ci: . – . ; p < . ) followed by – yo ( . %) (aor . ; %ci: . – . ). ve in preventing intensive care unit (icu) admission was % ( %ci: – %). final outcome death while hospitalized occurred in shlci cases with a case fatality rate of . %. conclusions: – influenza season was an unusual epidemic season with an early onset, great predominance of influenza b (yamagata strain) virus with a high hospitalization rate of severe cases among elderly stressing the need to upgrade vaccine uptake in this age group. vigilancia de la gripe estacional: estudio observacional sobre la temporada - con circulación predominante del virus de la gripe b r e s u m e n introducción: la gripe es una enfermedad infecciosa respiratoria común que afecta cada año a una proporción importante de la población mundial. el objetivo de este trabajo es describir la temporada de influenza - y cómo afectó a la población anciana en cataluña a pesar de la cobertura moderada de vacunas en este grupo de edad. métodos: vigilancia de la gripe basada en la vigilancia centinela de atención primaria (ap), indicadores virológicos por muestreo sistemático semanal de pacientes con síndrome gripal (sg) atendidos en ap y casos graves de gripe confirmada grave ingresados ??en el hospital. las estadísticas utilizadas para el análisis fueron el test anova, la prueba de chi-cuadrado, el análisis de regresión múltiple y la prueba de control negativo y caso a caso para la evaluación de la efectividad de la vacuna (ev, por sus siglas en inglés) en ap y casos graves hospitalizados, respectivamente. resultados: la temporada - se caracterizó por presentar una intensidad moderadamente alta, con inicio temprano y de larga duración. el predominio del virus de la gripe b (vgb) ( %) seguido por un aumento de la circulación del virus de la gripe a (vga). un total de iv de muestras de ap. la ve para prevención de la infección en casos con sg en ap fue del % (ic %: - %). se registraron . casos graves (incidencia acumulada ajustada , / . habitantes (ic %: , - , )). la proporción más alta de casos graves fue en > años ( , %) (or: , : ic %: , - , ; p < , ) seguido del grupo de - años ( , %) (or: , ; ic %: , - , ). la ve en la prevención de ingreso en la uci fue del % (ic %: - %). se registraron defunciones con una tasa de letalidad del , %. influenza is a common respiratory infectious disease that affects an important proportion of population worldwide every year. clinical manifestations can vary from a relatively mild disease characterized by sudden onset of fever, headache, myalgia and cough to a severe disease due to complications such as pneumonia and death especially in people at high risk, mostly aged or older. , of the four types of seasonal influenza viruses (iv) (a, b, c and d), a and b viruses cause seasonal epidemics of disease. most cases of human influenza are clinically diagnosed as influenza-like illness (ili) making differentiation from other winter circulating respiratory viruses such as rhinovirus, respiratory syncytial virus, parainfluenza and adenovirus difficult. yet those cases requiring hospitalizations because of a more severe outcome are laboratory tested and confirmed as severe cases of influenza infection that need to be hospital admitted. surveillance of influenza is essential for the early detection of epidemics, annual updates of vaccine components and evaluation of new variants or subtypes of iv. although seasonal influenza vaccine does not display high effectiveness, especially when there is a high rate of mismatching between circulating and vaccine strain composition, it is the best preventive measure for preventing deaths and hospitalizations in at risk population. the - influenza season presented a predominant circulation of influenza virus type b during the first epidemic weeks with a high rate of severe influenza hospitalizations, especially among the elderly. the aim of this work is to describe the - influenza season according to the pidirac sentinel influenza surveillance system and how it affected elderly population in catalonia despite moderate vaccine coverage among this age group. the surveillance of influenza in catalonia started during the season - and was modified from season - , in order to achieve major geographical and major representativeness coverage, in the obtaining of samples for the viral study. primary care surveillance: the number of sentinel doctors participating in the season was of physicians ( general practitioners and pediatricians), located in primary care centers, distributed across several regions and covering . % of the population of catalonia. to achieve morbidity and virological indicators, these practitioners collect pharyngeal and nasal samples during the entire epidemic season, and forward data on attended morbidity. systematic sampling of the first weekly ili attended by each sentinel physician was carried out. a panel of respiratory viruses is investigated: influenza virus a (iva), influenza b (ivb) and c virus (ivc), and other respiratory viruses: respiratory syncytial (rsv), parainfluenza , , and (piv) viruses, adenovirus (adv), coronavirus (cov), enterovirus (ev), human rhinovirus (hrv), human metapneumovirus (hmpv) and human bocavirus (hbov). morbidity indicators were collected automatically on a daily basis from sentinel practitioners: home calls performed, number of ili cases diagnosed including data on gender, age and vaccination status. the thresholds for weekly incidence rates of ili to assess intensity of epidemic activity (baseline, low, moderate, high and very high) were obtained by the mobile epidemics method (mem) since , severe hospitalized laboratory confirmed influenza cases (shlci) were included in sentinel seasonal influenza surveillance to assess severity of influenza epidemics. shlci case definition is as follows: • anyone presenting with ili that requires hospital admission because of pneumonia, acute respiratory distress, multiorganic impairment, septic shock, icu admission or • anyone who develops the latter symptoms during their hospital stay having being admitted to the hospital for any reason other than influenza or the influenza epidemic activity in catalonia during the - season was moderately high, with an early onset by the end of and a long duration. the ili weekly incidence curve shows that the duration of the epidemic wave was longer that the former season, lasting weeks, with a maximum incidence of . / , inhabitants (fig. younger than years old the age group with the highest disease burden ( . / , h.). comparing with the previous season, the cumulative incidence rates were higher in all age groups as shown in table . the predominant virus was ivb virus followed by an increase of circulation influenza ah n and a(h n )pdm , after peak of epidemic activity was attained on week (fig. ) . a total of iv were isolated from the samples collected from the primary care sentinel network, . % of them were pediatric samples, with a positivity rate of . % ( / ) to respiratory viruses included in the panel for the virological assessment and of these . % ( / ) were positive to iv. the positivity rate for all respiratory viruses in adult samples (> yo) was . % ( / ); of these . % ( / ) corresponded to influenza iva. statistically significant difference was observed in the iv positivity in the > yo (or . : %ci: . - . ; p < . ). the distribution of iv according to type showed predominance of the ivb with % vs. iva ( %). the distribution of the type/subtype of iv is shown in fig. . according to the phylogenetic analysis of the iv circulating during the season, % of the ah n virus characterized belonged to the genetic group of the a/hongkong/ / like (also designated like a/bolzano/ / ) which was included in the seasonal anti-influenza vaccine. the remaining % belonged to the a/singapore/infimh- - / strain not included in the seasonal influenza vaccine. all a(h n )pdm strains belonged to a/michigan/ / (h n )pdm -like, included in the seasonal anti-influenza vaccine. an influenza a(h n )pdm (a/michigan/ / ) strain presented the h y resistance mutation to oseltamivir. the ivb strains identified correspond to the b/phuket/ / (lineage yamagata), strain not included in the trivalent anti-influenza vaccine. thirty four ( %) of the laboratory confirmed iv had been vaccinated. vaccine effectiveness (ve) to prevent infection ( ) cases may present more than one risk factor. before h of onset of symptoms vs. before h of admission. statistical difference was observed in icu admission for those patients who received antiviral treatment up to h after hospital admission ( . %) in contrast to those who did so more than h after admission ( . %) (or: . ; %ci: . - . ; p < . ) as to risk factors the most frequent was cardiovascular disease ( %) followed by chronic obstructive pulmonary disease ( . %) and diabetes ( . %). seven shilc cases were pregnant women, ( %) of whom were not vaccinated (table ) . a clear predominance of ivb was observed ( . %: cases) during the season, followed by a late increase in circulation of iva virus which accounted for . % ( cases) of shlci. by subtypes, shlci iva viruses were mainly a h n ( . %) (fig. ) . all ivb strains studied were identified as b/phuket/ / -yamagata lineage. of the influenza a h n isolates subtyped, . % were identified as a/singapore/ - / (h n ) strain not included in the seasonal vaccine. all a (h n )pdm strain analyzed belonged to the a/michigan/ / (h n ) strain included in the seasonal vaccine. ve in preventing icu admission rendered % ( %ci: , ) ( table ) . pneumonia was the main complication occurring in . % ( ) shlci cases, . % ( ) of which presented bacterial coinfection. acute respiratory distress syndrome accounted for % ( ) and multiorganic failure for . % ( ) cases. five influenza associated encephalitis occurred unvaccinated shlci patients aged -to years old. three were caused by ivb and by iva ( a(h n )pdm and not subtyped). final outcome death while hospitalized occurred in shlci cases with a case fatality rate of . %. the average age of the deceased was . years (sd . ) and the median age was years (rank inter-quartile - years), . % had at least one risk factor for developing severe complications and death from their influenza infection (not taking into account old age as a risk factor), . % of them were not vaccinated. ivb was identified in % ( ) influenza virus infection is still a topic of great relevance to global health. the high percentage of hospitalizations ( . %) and mortality in the age group of over ( . %), especially those over the age of , where mortality is higher ( . %), reflects the increase in the life expectancy of the population. this fact makes it necessary to deepen in the knowledge of how they affect aging, their interaction with the most prevalent chronic diseases in the elderly and their immune response in order to apply preventive measures that offer a better protection to this population group. the - influenza season showed a moderate, early-onset activity (approximately weeks) with longer epidemic duration than usual ( weeks). the pattern of atypical circulation with circulation of initial influenza b viruses and a rebound of influenza a virus could be responsible for this longer duration of the activity. the prevalence of virus b/phuket/ / -yamagata lineage not included in the trivalent vaccine of the - season and the contribution of virus circulation a/singapore/ - / (h n ), also not included in the vaccine as well as the high proportion of elderly cases ( % > years old) may have been the cause of the great number of shlci cases. this fact has also been reported in the spanish influenza surveillance network - report. at the primary care level, the most affected were those under years of age with a cumulative incidence of . in the - age group or . in the - age group. the ve has shown low values in > age group. however, it should be taken into account that vaccination has an impact on the reduction of hospitalization and admission to icu. in severe case hospitalizations due to severe influenza, > age group presented the highest hospitalization rates ( . / , ) compared to other age groups, which highlights the need to increase vaccine coverage in this age group to prevent mortality. the delay in the administration of antiviral drugs, from the onset of symptoms in those patients with a risk condition of complication identified such as elderly people or people with underlying chronic illnesses, is likely to be a bad prognosis of the flu vaccine. this fact evidences the need to administer early treatment (within h of admission) to be effective. most frequent influenza infection complication is pneumonia, yet in this season five influenza encephalitis were registered among adult shlci patients. acute encephalopathy syndromes as a complication of influenza infection has been previously described by other authors in children and adults, but our observation in unvaccinated adult population with risk factors to develop influenza complications underscores the need for higher vaccination coverage in at risk population despite age. [ ] [ ] [ ] there are some limitations to be taken into account, such as the fact that the total hospitalization burden including non-severe hospital admitted cases with laboratory confirmed influenza is not included. this data would be desirable to be included in influenza surveillance in order to have a broader assessment of influenza burden of disease in the population and in turn have a more exhaustive treatment and vaccine effectiveness estimation. in conclusion the - influenza season in catalonia was, as in the rest of spain, an unusual epidemic season with an early onset, great predominance of influenza b (yamagata strain) virus circulation followed by a second wave of influenza a (subtypes h n and (h n )pdm ) that resulted in a high hospitalization rate of severe cases especially among the elderly. the low vaccine coverage among > age group stresses the need to upgrade vaccine uptake in this age group as well as in those of any age with a risk factor for presenting influenza related complications. analysis of data and samples are performed under the epidemiologic surveillance of influenza and acute respiratory infections established for public health surveillance purposes, there is no need for individual signed consent. nevertheless verbal consent is requested by each physician upon sampling. no funding received. the authors declare there is no conflict of interest. vaccine effectiveness in older individuals: what has been learned from the influenza-vaccine experience estimates of us influenza-associated deaths made using four different methods. influenza other respir viruses influenza surveillance in europe: establishing epidemic thresholds by the moving epidemic method. influenza other respir viruses pla d' informació de les infeccions respiratòries agudes a catalunya (pidirac) european centre for disease prevention and control. severe influenza surveillance in europe estratègia de vigilància dels casos greus de grip hospitalitzats estimating influenza vaccine effectiveness with the test-negative design using alternative control groups: a systematic review and meta-analysis severe influenza in us hospitals, - : complications and risk factors for death in patients informe de la vigilancia d ela gripe en españa. temporada - factors associated with -day mortality in elderly inpatients with community acquired pneumonia during influenza seasons. hum vaccines immunother effectiveness of antiviral treatment in preventing death in hospitalized cases of severe influenza over six influenza seasons the spectrum and burden of influenza-associated neurological disease in children: combined encephalitis and influenza sentinel site surveillance from australia case report acute influenza virus-associated encephalitis and encephalopathy in adults: a challenging diagnosis neurologic complications of influenza b virus infection in adults key: cord- - fctxk authors: proudfoot, chris; lillico, simon; tait-burkard, christine title: genome editing for disease resistance in pigs and chickens date: - - journal: anim front doi: . /af/vfz sha: doc_id: cord_uid: fctxk nan for thousands of years, humans have used selective breeding to improve desirable traits in both livestock and companion animals. in livestock, targeted breeding has been common practice since the british agricultural revolution of the th century, with measurable production traits such as feed conversion in cattle or wool production in sheep actively selected for. in the late th century, genomic selection was added to the livestock breeding tool box; by reading specific locations in the genome and assigning them to measurable production traits, faster improvement in livestock production efficiency has been achieved. one of the inherently difficult production traits to measure is resistance to a specific disease, as animals with less severe symptoms or pathology may simply have been exposed to less pathogen. experimental infections guaranteeing equal pathogen exposures are expensive and require large numbers of animals for genetic association studies, making them ethically questionable. genome editing offers new opportunities to livestock breeding for disease resistance, allowing the direct translation of laboratory research into disease-resistant or resilient animals. made? genome editors are custom enzymes that allow scientists to cut the dna strands in the nucleus of a cell at a specific position. the researcher can then influence how the dna is repaired, introducing very precise genetic changes at a target locus in their species of interest. this technology has been revolutionary and provides exciting possibilities for the production of livestock resistant to viral diseases. such opportunities are particularly pertinent given state efforts to improve global food security and reduce food waste throughout the production chain. the most prominent editor technology today, crispr/cas, uses a nucleotide rna guide to target its enzyme component to a designated locus in the genome. the probability of off-target cutting with a high fidelity cas enzyme is very low, because with four potential base combinations at each of the nucleotides there are over one trillion unique guide combinations. once the enzyme has cut the dna strands, the predominant repair pathway in most cells is nonhomologous end joining, an error-prone process which often introduces small insertions or deletions into the genetic code at the break site. if the target is within a gene, such perturbations can result in a disruption to the function of that gene, potentially leading to a loss of protein function. this can be very useful to basic science as it allows researchers to discover functions associated with novel genes. for many applications, a more precise change to the genome is required. to that end, scientists regularly make an alternative dna repair process called homology-directed repair. to do this, researchers provide a novel dna sequence alongside the crispr/cas reagents, whereby the cellular repair machinery uses the new dna as a template when fixing the break. this approach facilitates the introduction of defined implications • genome editing technology enlarges the tool box of trait-selective breeding. • methods for genome editing have developed over the past decades, making the technology more efficient and specific. • technology to generate edited pigs and chickens is developing alongside genome editors to generate animals faster and more affordable. • for two major pig diseases, it has been shown that resistant animals can be generated that are refractory to infection. in chickens there are promising laboratory results but no genome-edited, resistant chickens yet. • genome editing allows us to overcome bottlenecks in trait-selective breeding and allows the incorporation of genetic traits from other breeds, related species, or laboratory results. • two major hurdles still to be faced prior to the implementation of this promising technology are consumer acceptance and the regulatory framework. changes at the genomic target locus and has sufficient refinement to alter a single nucleotide, allowing precise modification of gene function. finally, by introducing a pair of editors, it is possible to generate two concurrent dna breaks on the same chromosome. the cellular repair machinery then joins the ends of the cut sites, promoting the deletion of the intervening sequence. all the editor reagents introduced to the cell are rapidly degraded, with only the alteration to the genomic sequence remaining to be propagated following cell division. genome editing has been applied to a wide variety of agricultural species including salmonids, poultry, and ruminants. however, due to its global economic value, relatively short generation time, and multiparous nature, the most edited livestock species to date is the pig. there are two main methods widely used for the generation of edited pigs: cloning of edited fibroblasts or direct injection of the zygotes with editor reagents. both work well, and each has specific advantages. in cloning, fibroblast cells can be maintained in the lab for prolonged periods. this allows researchers to introduce editor reagents into the cultured cells typically by lipofection, electroporation, or microinjection. editing events in each cell of a population can be characterized and individual cells with the desired alteration to their genome selected for the cloning process, whereby the fibroblast cell is fused with an enucleated oocyte shell in a process called somatic cell nuclear transfer ( figure a ). the reconstituted "zygote" is then transferred to a recipient gilt or sow (carlson et al., ) . despite the benefit of being able to prescreen the donor cells, cloning is generally inefficient with hundreds of reconstituted zygotes being transferred to a single recipient. cloning also yields reduced litter sizes when compared with standard breeding and offspring often demonstrate reduced viability. as an alternative to cloning, newly fertilized zygotes can be directly microinjected with genome-editing reagents and transferred immediately back to the oviduct of a recipient animal ( figure b ). in contrast to cloning, this approach (lillico et al., ) results in the efficient establishment of pregnancies and robust litters. however, without the prescreening of cells that is routine in cloning, offspring from direct zygote manipulation inevitably encompasses a range of editing outcomes, since selection of a specific edit is not possible. porcine zygotes can also be generated by maturation of oocytes extracted from slaughterhouse-derived ovaries and in vitro fertilization. unfortunately, in vitro fertilization in pigs often results in polyspermy, rendering the resulting embryo inviable. however, in this controlled environment, editing rates can be increased and costs and animal use reduced. an emerging alternative to these proven methods could be the use of surrogate sires. as a first step towards this goal, pigs have been edited to remove a gene required for male fertility, generating an empty spermatagonial stem cell niche in the testis . spermatogonial stem cells can be isolated and cultured in vitro, opening the possibility to edit and characterize these cells before transfer to a recipient (park et al., ) ( figure c ). genetic modification of poultry poses unique challenges due to the very different physiology of the avian egg compared with a mammalian oocyte. as a result, isolation and transfer of a chicken yolk is not practical. one approach that has been taken is in ovo electroporation of editing reagents, which allowed the analysis of gene function in the neural crest (gandhi et al., ) . however, others reported that electroporation resulted in mosaicism with editing limited to a subset of cells as the chicken embryo is already much further developed when an egg is laid compared with a zygote (veron et al., ) ( figure d ). as a result, it is unlikely that this approach could be efficiently utilized to generate edited birds. an alternative approach involves sperm transfection-assisted gene editing, whereby sperm are lipofected with editing reagents before use in artificial insemination (cooper et al., ) ( figure e ). however, advances in chicken stem cell technology show the greatest promise for genome editing in chicken. primordial germ cells (stem cells that eventually develop into germ cells) can be isolated from the blood of developing chicks in ovo and cultured in vitro. as with mammalian fibroblasts, these cells can be edited and selected in vitro before transfer into the bloodstream of a stage-matched recipient where they migrate to and populate the developing gonad. the chicken embryo is accessed through an opening in the egg shell, which is sealed again until the chicken hatches. genome editing in primordial germ cells has been successfully demonstrated by a number of groups (park et al., ; taylor et al., ; idoko-akoh et al., ) and one group has generated modified birds (park et al., ) . the founder birds generated from this editing method are chimeric due to the presence of preexisting germ cells. the resulting offspring generated from breeding with the founders will be a mixture of edited or nonedited. recipient chicken embryos devoid of germ cells are currently being developed that will significantly increase the efficiency of this process (m. mcgrew, unpublished results) ( figure f ). genome editors will undoubtedly have a significant role on the generation of disease-resistant animals as exemplified below. it is important to note that currently the technology is limited to modifying a single gene or a snp with large effects; however, disease resistance in many cases is likely to be a polygenic trait. multiplexing technology is under development such that in the future polygenic traits could be altered in a single step. progress so far? porcine reproductive and respiratory syndrome virus. porcine reproductive and respiratory syndrome (prrs) is arguably the most economically important pig disease worldwide. the causative agent of prrs is an arterivirus, named prrs virus (prrsv), that affects pigs of all ages but most importantly causes late-term abortions and stillbirth in sows and severe respiratory disease in piglets with severe morbidity and high mortality. prrsv also incapacitates the pig's immune response, providing an ideal breeding ground for severe secondary infections, mostly by bacteria, which leads to increased use of antibiotics. prrsv exclusively infects cells of the monocyte/macrophage lineage and two macrophagespecific proteins, cd and cd , were identified as receptors for the virus: cd acting on the surface of the cells and cd inside the internalizing transport vesicles (calvert et al., ; van gorp et al., ) . the virus was thought to attach to cd to be taken up into the cells; however, genome-edited pigs lacking cd were not resistant to prrsv infection (prather et al., ) . cd on the other hand is thought to act through a key-lock interaction with the virus to allow it to escape from the internalizing transport vehicles into the cytosol where it replicates. cd consists of nine globular domains, organized like beads on a string, with domain determined to mediate the key-lock interaction allowing viral entry into pig cells (van gorp et al., ) . using genome editing to generate pigs lacking cd whitworth et al. showed for the first time that this approach could be used to produce livestock resistant to important viral diseases, in this case prrs (whitworth et al., ) . cd is known to have a range of important biological functions in homeostasis, inflammation, and immune responses. as a refinement on functional knock out of the entire cd protein, editing reagents were designed to remove only domain leaving the remainder of the protein intact. the resulting animals were completely resistant to prrsv infection and maintained the biological functions associated with the remaining domains of cd (burkard et al., ; burkard et al., ) . porcine epidemic diarrhea virus/transmissible gastroenteritis virus. the two coronaviruses porcine epidemic diarrhea virus (pedv) and transmissible gastroenteritis virus (tgev) both cause severe diarrhea in preweaned piglets and are associated with high morbidity and mortality. in vitro host-pathogen studies identified aminopeptidase n as the receptor for tgev and a potential receptor for pedv (delmas et al., ; li et al., ) . the use of genome editing to generate pigs lacking aminopeptidase n successfully showed that pigs resistant to tgev infection could be generated. however, the edited animals remained susceptible to pedv infection (whitworth et al., ) . aminopeptidase n is important for peptide digestion in the small intestine and knockout mice were shown to have delayed mammary gland development. in humans, aminopeptidase n defects are associated with different types of leukemia and lymphoma. therefore, further investigation into the potential consequences of the absence aminopeptidase n in pigs is warranted as it may affect the overall health and/or productivity of the animals. african swine fever virus. african swine fever virus (asfv) causes a severe hemorrhagic disease in domestic pigs (sus scrofa domesticus) and wild boars (sus scrofa ferus) with high mortality in pigs of all ages. asfv is highly contagious and can be transmitted by soft ticks of the ornithodoros genus. it was identified in and contained to africa with occasional transmission around the strait of gibraltar into portugal and spain. in an introduction of the virus into the caucasus region showed that the virus does not solely rely on ticks for transmission in the wild, as transport of contaminated material and direct contact between animals have been shown to be major routes of disease dissemination. since then, the virus has spread across eastern europe and russia and was recently found in western europe and china. asfv poses a huge risk to the pig industry worldwide and is a limiting factor to a sustainable pig industry in many parts of africa. interestingly, asfv also infects wild suids, such as warthogs (phacocherus africanus) and bushpigs (potamocherus porcus), without causing overt disease. such infected wild suids are thought to act as a reservoir of the virus in africa. in the late stages of asfv infection, a cytokine storm, i.e., an overreaction of the immune system, is observed, which is thought to strongly contribute to the lethal outcome of disease. a comparison of the warthog and domestic pig genomes identified differences in the rel-like domain-containing protein a (rela, also known as p ) protein, which is involved in nf-κb cytokine signaling, was thought to underlie the different responses of the related species to asfv infection (palgrave et al., ) . researchers used genome editing to convert a key region of the encoded domestic pig protein sequence to the warthog equivalent (lillico et al., ) . data on susceptibility of the edited animals to asfv infection have yet to be reported. in this instance, it is important to differentiate between disease resistance, the ability of an animal to suppress the establishment and/or development of an infection, and disease resilience, where an infected host manages to maintain an acceptable level of productivity despite challenge pressure. should these pigs prove to be resilient to asfv infection it is likely that their use may not be permitted in many jurisdictions, since they could act as reservoirs of infection. however, in environments where the disease is endemic use of such animals could be beneficial. avian leucosis virus. avian leukosis virus infection results in inappetence, diarrhea, weight loss, a reduction in eggs laid, and often causes tumor formation in the chicken. the virus is divided into six subgroups, with the avian leucosis virus subgroup j (alv-j) shown to be responsible for major disease outbreaks in china. the cellular receptor of alv-j was identified to be the chicken sodium/hydrogen exchanger protein on the cell surface. chicken somatic cell lines have been edited to introduce changes to this gene-conferring resistance to avian leucosis virus in vitro (lee et al., ) . despite cells showing resistance to alv-j infection, no edited chickens have been produced to date. in both mice and humans, a lack of the sodium/hydrogen exchanger protein is associated with severe neurological disease; however, targeted changes to single amino acids may retain biological functions of the protein in chicken while resulting in disease resistance. avian influenza virus. in chickens, disease resistance to avian influenza is at the top of the wish list due to the serious impact on chicken health but also the risk of transmission to humans. similarly, influenza a is also one of the diseases on the resistance wish list for pigs, as they can act as an intermediate host-aiding virus adaptation to humans. the acidic leucine-rich nuclear phosphoprotein- a (anp a) was found to play a key role in avian influenza virus replication in both chicken and water fowl. although the virus polymerase protein readily interacts with the avian anp a, the human version of the same protein supports only limited replication of the viral genome. it has been demonstrated in vitro that deletion of a small region of chicken anp a can prevent replication of avian influenza virus (long et al., ; long et al., ) . although the functional consequence of edited anp a has yet to be demonstrated in vivo, such approaches offer exciting opportunities that have the potential to benefit both industry and animal welfare. as exemplified above, currently many gene editing approaches focus on targeting host genes involved in mediating entry of the virus, with a special focus on receptors. however, as the example for avian influenza shows, host genes play an important role in other steps of the pathogen replication cycle and also provide editing targets for disease resilience or resistance. more in-depth host-pathogen interaction studies, including genome-wide editing studies in vitro, will no doubt produce a variety of further candidate genes for genetic disease resistance. an alternative antipathogen approach pursued for decades is the generation of transgenic livestock, expressing antiviral or antibacterial agents, such as enzymes or small interfering rnas. genome editing can be used to improve the integration efficiency of these transgenes at specific locations in the genome; however, the discussion of transgenic disease-resistant animals is beyond the scope of this review. how does genome editing fit within existing selective breeding structures and how will it be regulated? selective breeding has generated highly productive, robust animals that are adapted to a modern production environment. livestock production is dynamic, with evolving challenges such as climate change and disease outbreaks coupled with societal pressure to reduce antimicrobial use. selective breeding for disease resistance has proven difficult, as outbreaks are often sporadic and resistant/resilient animals often difficult to identify. in circumstances where a genetic trait for disease resistance can be identified in the breeding population, then selection through the selective breeding can be achieved. a good example of this is pigs with resistance to f type enterotoxigenic e. coli. association studies revealed that a polymorphism in the fucosyl transferase gene conferred resistance to these bacteria. there was initial concern that selection for the locus figure . genetic resistance to disease and how genome editing can help integrate traits into highly productive lines. (a) genetic resistance to disease may be present in a small percentage of production animals and genetic selection for these animals may be associated with the risk of inbreeding, productivity loss, or the risk of losing other desirable traits. genome editing allows integration of the disease-resistance trait into a wider selection of pigs, ensuring genetic variability and maintenance of desirable traits. (b) genetic resistance to disease may be present in an indigenous or less productive breed. crossbreeding would result in productivity loss and the risk of losing other desirable traits, such as fur color. genome editing allows for incorporation of genetic disease resistance into highly bred lines without losing productivity. (c) genetic resistance may be observed in a closely related species, e.g., wild boar or wild suids in the case of the domestic pig. integration into highly bred domestic pig lines would only be possible by genome editing. (d) resistance genes may be identified in laboratory research but not in highly bred lines, making integration into those productive animals only possible using genome editing. harboring this gene may counterselect for another gene associated with stress resistance. however, this proved not to be the case and genetic selection for the favorable fucosyl transferase allele has been integrated into many pig-breeding programs (coddens et al., ) . this was possible, in part, because the favorable allele was present at sufficient prevalence (in most studies between % and %) in the breeding population to allow for selection while avoiding inbreeding. in circumstances where an allelic variant associated with a resistant phenotype is present at a much lower frequency, it may prove difficult to incorporate effective selection into a standard breeding regime without the risk of inbreeding and related longer-term productivity loss (figure a) . genome editing has the potential to contribute in such circumstances, allowing the direct introgression of a beneficial allele into the offspring of diverse, highly productive animals. similarly, disease-resistance traits associated with less productive indigenous breeds are unlikely to be introduced to highly productive populations by standard crossbreeding as this would result in a significant set-back in productivity, abrogating decades or even centuries of advances made through genetic selection ( figure b ). in circumstances where resistance or resilience is observed in a related species, crossbreeding is simply not possible. genome editing could bridge these gaps. one example of this is resilience of wild suids to african swine fever virus while domestic pigs can suffer from severe disease. it is not possible to crossbreed these species, so introduction of the genetics underlying resilience is not possible by this route. genetic comparison can be used to identify the functional differences underlying such traits, and genome editing employed to introduce appropriate variants into domestic pigs ( figure c ). finally, with a good understanding of host-pathogen interactions, novel genetics that has not been observed in live animals can be created and tested for efficacy in a laboratory environment. this was the case for both the cd /prrsv and apn/tgev examples in pigs and would be the case for the anp a/ influenza and the alv-j resistance in chicken, described above. in such circumstances, integration through genome editing presents a practical route to benefit from the findings ( figure d ). it is imperative that in such circumstances thorough phenotypic characterization of the edited animals be carried out as deletion of all or part of a functional protein could result in a loss of (systemic) biological function. a second measure worthy of consideration before embarking on an editing project is whether the gene is located within a locus that has been actively selected in breeding programs. this could indicate whether a potential target is associated with known production traits. this approach has been taken for prrsv-resistant pigs, with evaluation as to whether the cd gene locus has been selected for in pig breeding programs (johnsson et al., ) . overall, genome editing holds vast promise for the future production of animals resistant or resilient to disease, improving productivity and animal welfare while reducing food waste throughout the production chain. through reduction of primary and secondary infections, it should also be possible to reduce antimicrobial use in livestock production. technical improvements in the generation of genome editing animals will undoubtedly reduce the cost implications of this technology. the two major hurdles still to be faced prior to implementation of this promising technology are consumer acceptance and the regulatory framework. approval of edited animals for human consumption relies on national and multinational legislation, which is currently at early stages. encouragingly, some international jurisdictions such as argentina and brazil have about the authors dr. chris proudfoot is research fellow at the roslin institute/university of edinburgh since . his work centers on generation of genome-modified livestock, with particular emphasis on genome editors, to improve disease resistance or to accurately model human disease. dr. proudfoot has worked extensively with zfns, talens, and crispr/cas to produce a variety of edited animals. he was a member of the team that produced the first edited livestock using this method. dr. simon lillico is a research associate at the roslin institute/university of edinburgh. he joined the institute on an industrial collaboration to produce highvalue therapeutic proteins in hens eggs and then applied his expertise in lentiviral transgenesis to generate livestock models of human diseases. the rapid expansion of the field of genome editors over the last yr has made practicable genome modifications which had previously been unattainable. dr. lillico has been at the forefront of application of these editors to livestock, creating either disease-resistant/resilient strains, or accurate models of human disease. dr. christine tait-burkard is an assistant professor at the roslin institute/university of edinburgh since in the departments of genetics and genomics and infection and immunity. her research focuses on understanding host-pathogen interactions on a cellular and genetic level, developing new in vitro tools for virus research, improving and developing easy-to-use diagnostics, and devising strategies to combat viral disease in livestock in general and pigs in particular. she employs genome editing and genetic selection to generate animals genetically resistant to viral disease. corresponding author: christine.burkard@roslin.ed.ac.uk already ruled that modifications, such as the prrsv-resistant pig, that do not have any new genetic information integrated into the animal, will be exempt from regulation. precision engineering for prrsv resistance in pigs: macrophages from genome edited pigs lacking cd srcr domain are fully resistant to both prrsv genotypes while maintaining biological function pigs lacking the scavenger receptor cysteine-rich domain of cd are resistant to porcine reproductive and respiratory syndrome virus infection cd expression confers susceptibility to porcine reproductive and respiratory syndrome viruses efficient talen-mediated gene knockout in livestock the possibility of positive selection for both f (+)escherichia coli and stress resistant pigs opens new perspectives for pig breeding generation of gene edited birds in one generation using sperm transfection assisted gene editing (stage) aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev optimization of crispr/cas genome editing for loss-of-function in the early chick embryo high fidelity crispr/cas increases precise monoallelic and biallelic editing events in primordial germ cells precise gene editing of chicken na+/h+ exchange type (chnhe ) confers resistance to avian leukosis virus subgroup j (alv-j) porcine aminopeptidase n is a functional receptor for the pedv coronavirus live pigs produced from genome edited zygotes mammalian interspecies substitution of immune modulatory alleles by genome editing species difference in anp a underlies influenza a virus polymerase host restriction avian anp b does not support influenza a virus polymerase and influenza a virus relies exclusively on anp a in chicken cells species-specific variation in rela underlies differences in nf-κb activity: a potential role in african swine fever pathogenesis successful genetic modification of porcine spermatogonial stem cells via an electrically responsive au nanowire injector generation of germline ablated male pigs by crispr/cas editing of the nanos gene targeted gene knockout in chickens mediated by talens an intact sialoadhesin (sn/siglec /cd ) is not required for attachment/internalization of the porcine reproductive and respiratory syndrome virus efficient talen-mediated gene targeting of chicken primordial germ cells sialoadhesin and cd join forces during entry of the porcine reproductive and respiratory syndrome virus identification of the cd protein domains involved in infection of the porcine reproductive and respiratory syndrome virus crispr mediated somatic cell genome engineering in the chicken gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus resistance to coronavirus infection in amino peptidase n-deficient pigs we acknowledge financial support from the biotechnology and biological science research council (bbsrc) (bb/ r / , bb/n / ) and the bbsrc institute strategic programme grant funding to the roslin institute (bbs/ e/d/ and bbs/e/d/ ). key: cord- -sh mrhoq authors: appak, Özgür; duman, murat; belet, nurşen; sayiner, ayça arzu title: viral respiratory infections diagnosed by multiplex polymerase chain reaction in pediatric patients date: - - journal: j med virol doi: . /jmv. sha: doc_id: cord_uid: sh mrhoq syndromic diagnosis by multiplex nucleic acid amplification tests is the most practical approach to respiratory tract infections since the symptoms are rarely agent‐specific. the aim of this study was to investigate the respiratory viruses in children admitted to a university hospital with acute respiratory tract infection during the last years by a multiplex polymerase chain reaction (pcr) assay. a total of respiratory samples collected from children between april and april tested by a multiplex real‐time pcr assay. two different commercial assays were used during the study period, "ausdiagnostics/respiratory pathogens (ausdiagnostics)" used between april and december , which changed to "fast track diagnostics/respiratory pathogens (fast track diagnostics)" after january to cover more viruses. nucleic acid extraction was done by ez advanced xl platform (qiagen). respiratory pathogens detected in of the ( . %) samples. the most prevalent viruses during the ‐year period were rhinovirus/enterovirus (rv/ev; . %), respiratory syncytial virus (rsv; %), and influenza virus a/b ( . %). rhinovirus was the main contributor to the rv/ev group as shown by the assay used during the ‐ period. rv/ev and adenoviruses detected throughout the year. influenza virus was most frequently detected during january to march when both rsv and metapneumovirus were also in circulation. the coinfection percentage was . %. rhinovirus was the most common virus in coinfections while rsv plus rhinovirus/enterovirus were the most frequent combination. rsv and metapneumovirus showed a similar seasonal distribution to the influenza virus, which made it necessary to use a virological diagnostic assay during the influenza season. the ausdiagnostics test is based on multiplex tandem pcr a pathogen was detected in ( . %) of the study samples. distribution of pcr positive samples according to age groups was found as follows; % ( of ) in less than years age group, % ( of ) in to years group, % ( of ) in the to years group, and % ( of ) in to years group. the positivity rate decreased significantly above years of age (p < . ). the mean positivity rate was . % during study period when the ausdignostics assay was used compared with . % of the second part of the study using the fast track diagnostics assay. the difference is statistically significant (p < . ; figure a ). rv/ev ( . %) and rsv ( . %) were the most commonly detected agents followed by iav ( . %; table ). when the distribution of the agents according to years were examined, rv/ev was the most common agent for each year. rsv and coinfections took the second and third place in the frequency order except in which they are replaced by piv- and adv. the study covered only the first months of where coinfections were the most frequent cause of the respiratory infections in the studied group and followed by rv/ev and influenza a/b, respectively ( figure b ). when the distribution according to age groups was determined, the first three pathogens were rv/ev, rsv, and coinfections for less than years; rv/ev, coinfections, and influenza a/b for to years; rv/ev, influenza a/b, and coinfections for to and to years (table ) . among the parainfluenza viruses, piv- was the most frequently detected subtype, followed by piv- , - , and - , respectively (table ) . table . the role and importance of respiratory viruses have become more easily detectable by the use of nucleic acid-based tests in clinical laboratories. thus it became possible to obtain detailed epidemiological data and to manage the patients better. our study retrospectively assessed the respiratory tract viruses detected in children with arti symptoms of to years between - . two different assays were used during the study period. ausdiagnostics assay covered a panel of rv as the number one etiologic factor. , , rsv ( . %) was the second most common viral agent after rv in this study. it was particularly detected during winter and spring months with a peak between january and march. consistent with our study, rsv is observed more frequently in winter months in many studies from different regions. rsv and hmpv were usually seen in children under five years of age as the cause of bronchiolitis and pneumonia. in our study, the age relation was significant for rsv and hmpv which were detected in % ( of ) and % ( of ) of children under years, respectively. similar to rsv, hmpv infections were frequently seen in winter and spring months as it was the case in our study while % of the hmpv cases were detected between january and april. iav and ibv were detected with a frequency of . % and . %, respectively, and peaked between january and march. the presence of rsv and hmpv in circulation during the same period was notable, which necessitates the use of virological methods for the differential there are four subtypes of pivs ( - ) which may cause croup, pharyngitis, laryngitis, tracheobronchitis, bronchiolitis, and/or pneumonia. piv- was the most commonly detected subtype in our study, followed by piv- , - , and - . piv- is detected more frequently in april, may, and october. it was not possible to detect the seasonal relation of other piv subtypes due to a small number of cases. in other studies, piv- was also reported as the most common subtype followed other pivs similar to our findings. , they were frequently detected during spring and early summer months. bocavirus, after being identified in has been isolated from . % to % of the respiratory tract samples of children who were admitted to the hospital due to respiratory viral disease. bocavirus causes coinfections up to % of the cases with other viruses such as influenza, rhinovirus, parainfluenza, rsv, and metapneumovirus. , the prevalence of bocavirus was . % in our study with a coinfection frequency of . %, mainly associated with rhinovirus/enterovirus, rsv, and influenza a/b. infections were seen throughout the year without a clear seasonal relation although % ( of ) of cases were between january and march which is parallel to the other reported studies. [ ] [ ] [ ] coronaviruses were identified nearly % of the patients, with oc being the most frequent subtype followed by nl , hku , and e. coinfections with rhinovirus or rsv were seen in almost half of the samples. these infections were detected in almost all months except june and august. it was reported that coronavirus infections peaked mainly in winter without any significant difference between the four subtypes in terms of seasonality and origin (community or hospital-acquired) of the infection. , bordetella spp is an agent that can be mistaken as viral infection since can lead to prolonged cough and severe infections in infants, and young children. there were only patients with bordetella infections in our study which were seen in patients less than -months old, except for one. although can be seen at any age, pertussis is more frequent and cause more severe disease in children younger than -months-old because of lack of primary vaccination. high vaccine coverage (at least %) is necessary to protect children against vaccine-preventable diseases with the herd immunity. the importance of herd immunity is nicely described by an italian study of three cases of b. pertussis, one -month old and two less than a month old children showing that reduced immunity of the childʼs family over the years increases the risk of infection especially in infants lack maternal immunity with a more severe clinical presenta- human metapneumovirus: review of an important respiratory pathogen epidemiology of acute respiratory infections non-influenza viruses in acute respiratory infections among young children. high prevalence of hmpv during the h n v. pandemic in poland molecular diagnosis of respiratory virus infections epidemiology characteristics of respiratory viruses found in children and adults with respiratory tract infections in southern china epidemiologic analysis of respiratory viral infections mainly in hospitalized children and adults in midwest university medical center after the implementation of a -virus multiplex nucleic acid amplification test ten year retrospective evaluation of the seasonal distribution of agent viruses in childhood respiratory tract infections rapid multiplex nested pcr for detection of respiratory viruses clinical impact of rt-pcr for pediatric acute respiratory infections: a controlled clinical trial comparison of three multiplex pcr assays for detection of respiratory viruses: anyplex ii rv , advansure rv, and real-q rv the role of multiplex pcr in respiratory tract infections in children extensive multiplex pcr diagnostics reveal new insights into the epidemiology of viral respiratory infections analysis of respiratory viral infections detected using multiplex real-time pcr in hwaseong global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis human metapneumovirus circulation in the united states influenza surveillance in five consecutive seasons during post pandemic period: results from national influenza center molecular epidemiology of adenovirus type in the united states molecular and clinical characteristics of adenoviral infections in taiwanese children in - parainfluenza virus infection seasonal trends of human parainfluenza viral infections: united states epidemiology of parainfluenza infection in england and wales, - : any evidence of change? human bocavirus frequent and prolonged shedding of bocavirus in young children attending daycare detection of human bocavirus type infection in panamanian children with respiratory illness the association of newly identified respiratory viruses with lower respiratory tract infections in korean children detection of human bocavirus in canadian children in a -year study human bocavirus infection in children with respiratory tract disease epidemiology and clinical features of human coronaviruses in the pediatric population seasonality and prevalence of respiratory pathogens detected by multiplex pcr at a tertiary care medical center pertussis (bordetella pertussis and b. parapertussis) report on three cases of pertussis in the urbino area (italy) systematic review and meta-analysis of respiratory viral coinfections in children respiratory viral coinfection and disease severity in children: a systematic review and meta-analysis viral respiratory infections diagnosed by multiplex polymerase chain reaction in pediatric patients the authors declare that there are no conflicts of interest. http://orcid.org/ - - - key: cord- -zkwiag a authors: balboni, andrea; bassi, francesca; de arcangeli, stefano; zobba, rosanna; dedola, carla; alberti, alberto; battilani, mara title: molecular analysis of carnivore protoparvovirus detected in white blood cells of naturally infected cats date: - - journal: bmc vet res doi: . /s - - - sha: doc_id: cord_uid: zkwiag a background: cats are susceptible to feline panleukopenia virus (fpv) and canine parvovirus (cpv) variants a, b and c. detection of fpv and cpv variants in apparently healthy cats and their persistence in white blood cells (wbc) and other tissues when neutralising antibodies are simultaneously present, suggest that parvovirus may persist long-term in the tissues of cats post-infection without causing clinical signs. the aim of this study was to screen a population of cats from sardinia (italy) for the presence of both fpv and cpv dna within buffy coat samples using polymerase chain reaction (pcr). the dna viral load, genetic diversity, phylogeny and antibody titres against parvoviruses were investigated in the positive cats. results: carnivore protoparvovirus dna was detected in nine cats ( . %). viral dna was reassembled to fpv in four cats and to cpv (cpv- b and c) in four cats; one subject showed an unusually high genetic complexity with mixed infection involving fpv and cpv- c. antibodies against parvovirus were detected in all subjects which tested positive to dna parvoviruses. conclusions: the identification of fpv and cpv dna in the wbc of asymptomatic cats, despite the presence of specific antibodies against parvoviruses, and the high genetic heterogeneity detected in one sample, confirmed the relevant epidemiological role of cats in parvovirus infection. parvoviruses are non-enveloped single-stranded dna viruses which infect a wide range of mammalian species, including several members of the order carnivora. the carnivore protoparvovirus , belonging to genus protoparvovirus, family parvoviridae, subfamily parvovirinae, includes several closely related autonomous viruses causing a range of serious conditions, especially in young animals: feline panleukopenia virus (fpv, the prototype virus of the former carnivore parvovirus), canine parvovirus (cpv), mink enteritis virus (mev), and raccoon parvovirus (rapv) [ ] . feline panleukopenia virus has been known to be a cause of disease in cats since the beginning of the twentieth century, although there are other similar parvovirus species affecting cats, such as mev and cpv. natural infections in cats with cpv have been reported but fpv remains the most prevalent parvovirus causing disease in cats [ ] [ ] [ ] . since cats are susceptible to fpv and cpv a, b, c variants, superinfection and co-infection with multiple parvovirus strains associated with high viral genetic heterogeneity can occur with relatively high frequency in feline hosts [ , [ ] [ ] [ ] . parvoviruses commonly cause acute infection with high levels of viral shedding which generally ceases within - weeks post-infection, after the development of high titres of virus-neutralising antibody [ , ] . nevertheless, parvoviruses can be detected in faeces forup to weeks after recovery, depending on the sensitivity of the diagnostic method used [ ] . cats experimentally infected with fpv shed the virus in both urine and faeces up to day - post-infection with parvovirus persisting in the lungs and kidneys for more than weeks in cats which have recovered [ ] . the detection of fpv and cpv variants in apparently healthy cats suggests that parvovirus infection may be common in some populations of clinically normal cats, and that asymptomatic cats may be able to shed parvovirus for prolonged periods of time [ ] [ ] [ ] . furthermore, the ability of fpv and cpv to persist in the peripheral blood mononuclear cells (pbmc) of cats irrespective of the presence of neutralising antibodies [ ] [ ] [ ] [ ] [ ] and the presence of parvoviral dna in the bone marrow of healthy cats [ ] , suggests that parvovirus may persist long term in the tissues of cats post-infection without causing clinical signs. the aim of this study was to screen a population of cats from sardinia (italy) for the presence of both fpv and cpv dna within buffy coat samples. the dna viral load, genetic diversity, phylogeny and antibody titres against parvoviruses were investigated in the cats testing positive to dna parvoviruses. this was a retrospective study, carried out on stored blood samples taken for routine diagnostic investigations undertaken at the department of veterinary medicine, university of sassari -uniss (sassari, no sardinia, italy). buffy coats from cats sampled between october and march were tested for the presence of feline and canine parvovirus dna using real-time polymerase chain reaction (pcr). the partial vp gene of the viruses identified was sequenced and used for statistical analyses and phylogenetic comparisons. in addition, the sera of the cats which were positive to dna parvoviruses were tested for the parvovirus antibody using the haemagglutination inhibition (hi) assay. samples of owned or stray cats with different life-style conditions (indoor or outdoor, living alone or in community) were tested to evaluate different exposure to parvoviruses. date of sampling, gender, age, breed, habitat and the clinical symptoms of the cats sampled are reported in table . according to these data, the animals were divided into two groups called a and b. the cats included in groups a were predominantly healthy cats from four multi-cat households. the cats in group b were sampled in the emergency room of the veterinary teaching hospital of the department of veterinary medicine (uniss) and were predominantly stray cats showing different clinical signs (sick cats). twenty-two of the ( . %) cats were clinically normal; / ( . %) showed clinical signs attributable to various diseases; / ( . %) showed gastrointestinal signs, such as vomiting and diarrhoea compatible with parvovirus infection; clinical status was unavailable for / ( . %) cats. the vaccination status of the cats included in the study was unknown, although it was hypothesised that the stray cats were unvaccinated. anti-coagulated peripheral blood samples in ethylenediaminetetraacetic acid (edta) and coagulated blood for serology were collected from each cat. the blood samples were stored a + °c and sera at − °c until use. buffy coat-containing mononuclear cells was isolated from ml of edta anti-coagulated peripheral blood samples using histopaque- (sigma aldrich, st. louis, mo, usa). the dna was extracted using the dneasy blood and tissue kit (qiagen, hilden, germany), according to the manufacturer's instructions. the extracted dna was eluted in μl of ultrapure rnasi and dnasi free water, and was stored at − °c after analysis. parvovirus screening was carried out using real-time pcr using two conserved primers (a-for and b-rev, table ) targeting a bp fragment of the vp gene. quantitative pcr (qpcr) was carried out using sybr premix ex taq ii (takara bio inc., shiga, japan) and the rotor-gene system (corbett research, mortlake, nsw, australia). the fluorescence signal was acquired on the fam channel (multi-channel machine, source, nm; detector, nm; gain set to ) with a fluorescence reading taken at the end of each elongation step. each run consisted of an initial incubation in order to activate the hot-start dna polymerase at °c for s followed by cycles of denaturation at °c for s, annealing at °c for s and polymerisation at °c for s. during the melt cycle, the temperature was increased by increments of °c from °c to °c. a pcr plasmid (invitrogen, carlsbad, california, usa) containing one copy of the vp target sequence was produced as the external standard for the construction of the assay standard curve for quantitative analysis. duplicates of six -fold dilutions of the standard plasmid, duplicates of the buffy coat dna extracts of the cats sampled and a no template control were simultaneously analysed. specimens were considered positive if the fluorescence curve in the amplification plot showed an exponential increase, and if a specific melting peak was observed. copies of viral dna were expressed per microlitre of dna extract. amplification and sequencing of the vp gene in order to differentiate between fpv and cpv variants a, b and c, a fragment of the vp gene coding for critical amino acid residues , , , and affecting the biological and antigenic proprieties was amplified and sequenced for each virus identified using real-time pcr. a hemi-nested pcr assay was developed by using three conserved primers (c-for, d-rev and e-rev, table ) for this purpose. both pcr reactions were carried out using taq dna polymerase (qiagen, hilden, germany) producing dna fragments of bp and bp in length for the first and the second reaction, respectively. the temperature cycling protocol of the first amplification consisted of °c for min, cycles with cycle at °c for s, at °c for min, and at °c for min, followed by a final elongation at °c for min. in the second amplification, the pcr conditions were °c for min, cycles with cycle at °c for s, at °c for min, and at °c for s, followed by a final elongation at °c for min. in both pcr reactions, fpv / [ ] was used as a positive control while ultrapure water was used in each experiment to avoid false positive results. the nucleotide sequences were obtained using both forward and reverse primers. direct sequencing of the pcr products of one virus identified (number / ) showed an unusually high number of ambiguities, suggesting a mixed viral population. therefore, the amplification product was cloned into the pcr /topo vector using the topo cloning kit (invitrogen, carlsbad, ca, usa), and was transformed into escherichia coli dh α-competent cells according to the manufacturer's protocol. ten recombinant clones were sequenced using both forward and reverse primers. the nucleotide sequences obtained were assembled and translated into amino acid sequences using bioedit sequence alignment editor version . . [ ] . the vp assembled nucleotide sequences were aligned with reference sequences of feline and canine parvoviruses available in the genbank (http://www.ncbi.nlm.nih.gov/genbank), including the modified live fpv vaccine strains available, using the clustalw method implemented with bioedit software. a variety of statistical analyses aiming to investigate nucleotide diversity, sequence variability and natural selection were carried out on the sequence data set using dnasp package version . . [ ] . statistical analysis was carried out on the subpopulations, grouping the sequence data in the fpv, cpv and / (all clones of sample / ) clusters. the following parameters were estimated for each cluster: total number of mutations (η), nucleotide diversity (π) and its standard error, total number of synonymous differences (syndif), and total number of non-synonymous differences (nsyndif). mutation frequency (total number of changes/total number of bases sequenced) and the percentage of mutated clones were used as indicators of genetic diversity of the viral population of virus / . phylogenetic relationships among the viruses detected and the parvovirus reference sequences were evaluated using mega version . . [ ] . the best-fit model of nucleotide substitution was determined using the find best dna/protein model function implemented in mega, and the tamura -parameter model was found to be optimal for all the sequence data (including reference strains). phylogenetic trees were constructed using the maximum likelihood method, and bootstrap values were determined by replicates to assess the confidence level of each branch pattern. the nucleotide sequences obtained in this study have been submitted to the genbank under accession numbers kt to kt . the parvovirus antibody titre was investigated using the haemagglutination inhibition (hi) test in the sera of the cats which tested positive to parvovirus dna by direct molecular diagnosis. a cpv- b isolate (number / ), recovered from the faeces of a dog with non-fatal enteritis, and isolated by the authors on the crandell rees felinekidney (crfk, cell line was obtained from the biobanking of veterinary resources of the istituto zooprofilattico sperimentale della lombardia e dell'emilia romagna izsler "bruno ubertini" http://www.ibvr.org/services/cellcultures.aspx) cell line, was used as the viral antigen. this strain was used in hi experiments because there are no significant differences in serum titres inhibiting the haemagglutination reaction when cats are infected by fpv or cpv, if cpv is used as an antigen [ ] . to calculate haemagglutinating units (hau) of the viral antigen, a haemagglutination (ha) assay was carried out on the cpv- b isolate following procedures described previously by carmichael et al. [ ] . porcine erythrocytes were washed three times in bis-tris buffered saline (btbs, ph . at °c) and suspended into . % (v/v) btbs containing % bovine serum albumin. to reveal the haemagglutination of both viruses, the test was conducted at a ph of - . and was incubated at °c [ , ] . the hi tests were carried out as previously reported by senda et al. [ ] . all sera were tested in duplicate on two different plates; two-fold dilutions of a commercial canine hyperimmune serum raised against canine distemper, canine hepatitis and canine parvovirosis (stagloban, ati, ozzano emilia, bo, italy), was used as positive controls. for all sera, the mean value of the results from the duplicate hi tests was calculated. if the mean value of the result from one sera was included between two successive dilutions, the lower titre was reported. cats with an hi titre ≥ : were considered positive and cats with an hi titre ≥ : were considered to be protected from infection. the buffy coat dna extracts of cats were tested in duplicate for the presence of parvovirus using a sybr green real-time pcr assay which showed a limit of detection of copy of the vp target dna per microlitre of extract. nine table ) ; no cat with gastrointestinal signs tested positive for parvovirus dna. in the positive samples, the amount of viral dna ranged from orders of magnitude to copies of dna/μl of extract. a melting curve analysis showed a solitary peak between . °and °c for standard plasmid dilutions and between °and °c for each positive cat sample. the nucleotide sequences of the partial vp gene obtained from positive samples were bp in length ( amino acid codons), and corresponded to residue - of the fpv reference strain cu- (genbank accession number m ). nucleotide sequences of the vp partial gene were also obtained for ten recombinant clones of sample / , numbered from c to c , respectively. clone c was an fpv which showed a change located in residue (lys → arg); clone c was an fpv which displayed two coding changes in thr -to-ala and pro -to-ser; c was a cpv- c with an amino acid change located at position (gly → arg); c was a typical cpv- c and clone / -c displayed amino acids identical to fpv in residues , , and while it showed glutamic acid typical of cpv- c in position . the predicted amino acid sequence changes are summarised in table . several synonymous and non-synonymous substitutions were detected by comparing the sequences as reported in table . feline panleukopenia viruses showed a total number of mutations slightly higher than cpv viruses, although synonymous changes predominated in the fpv sequences while non-synonymous changes were prevalent in the cpv viruses. sample / showed high genetic complexity generated within the host, and its sequence variability was higher than the variability of the other sequence data analysed as was seen by the values of parameter π. the non-synonymous fraction was greater for sample / , indicating clear prevalence of the number of non-synonymous mutations in the sample: non-synonymous mutations out of a total of mutations. the proportion of mutated viral clones was % for sample / and the mutation frequency was on the order of . × − (table ) . unrooted phylogenetic trees constructed from alignments of the nucleotide sequences obtained in this study with additional reference sequences showed two main clusters referable to fpv and cpv. the sequence of clone / -c formed a monophyletic branch inside the fpv clade (fig. ) . nucleotide sequences of the modified live fpv vaccine strains available from genbank were distinguishable from all the fpv and cpv sequences obtained in this study, and direct relationships between them were not evident from the phylogenetic tree. the hi titres obtained are reported in table . all the cats which tested positive for parvovirus dna were also positive for antibodies against parvoviruses; cats / and / showed a titre higher than : . the present study screened the occurrence of parvovirus dna and the feline host-immune status in a cat population. asymptomatic and symptomatic cats sampled in sardinia (italy) during - were tested for the presence of both fpv and cpv dna in wbc using qpcr, and the partial vp gene of the viruses identified was characterised. parvoviruses have commonly been titrated in the faeces of infected animals, insofar as the viruses shed in faeces reflect virus replication; the authors chose to analyse the wbc for the presence of viral dna as, in addition to the intestinal crypt cells, bone marrow and other lymphoid tissues are also a major target for parvoviruses in both dogs and cats [ ] . furthermore, since parvovirus can frequently be isolated in infected cats, even in the presence of high virus-neutralising antibodies [ , ] , antibody titres against parvovirus were established in the sera of positive cats using an hi assay. nine ( . %) out of a total of cats tested positive for parvovirus dna with viral dna quantities ranging from to copies/μl of extract, demonstrating that the viral genome was detectable, although at low levels, in the wbc of a relatively large number of cats. all the cats which tested positive for parvovirus dna had table change of amino acids to vp partial protein the presence of fpv and cpv dna in the faecal and peripheral blood samples of healthy cats has previously been reported [ , , [ ] [ ] [ ] , raising important questions regarding the role of cats in the epidemiology of parvoviruses. in our study, the absence of clinical signs consistent with parvovirosis in the cats which tested positive, together with the low amount of viral dna detected, suggested that the infection was asymptomatic or that residual viral dna remains in the organism after recovery from acute infection. furthermore, the detection of parvoviral dna in wbc reveals the presence of the virus in the bone marrow or in other lymphoid tissues which might reflect chronic or latent infection. the detection of cpv dna in apparently healthy domestic cats confirmed a previous survey in which a high prevalence ( %) of cpv in apparently healthy domestic cats living in rescue shelters was identified [ ] . canine parvovirus-like dna was also detected in the tissues of wildlife carnivores which had no fig. unrooted phylogenetic tree. the phylogenetic tree was constructed with the nucleotide sequences of the partial vp gene generated in this study and with feline and canine parvovirus reference sequences available on the genbank nucleotide database or analysed by the authors in a previous study (battilani et al., [ ] ). bootstrap values greater than %, calculated on replicates, are indicated on the respective branches. the feline and canine parvoviruses identified in italy not detected in this study but included in the phylogenetic analysis are designated by it. in bold: nucleotide sequences generated in this study. underlined: clone / -c clinical signs of active infection and, therefore, it was likely that this virus caused latent or persistent infection not only in domestic cats [ ] . animal parvoviruses, such as rodent protoparvovirus and aleutian mink disease parvovirus, have been shown to persist in their host [ , ] . persistence is a common feature also for human parvovirus b (b v) infection [ ] : b v parvoviral dna has been documented in a wide range of tissues and the bone marrow of asymptomatic adults, although the majority of people harbour parvoviral dna in a form which does not actively replicate [ , ] . of the nine cats testing positive for parvovirus dna, four showed fpv dna, four cpv dna (three cpv- b and one cpv- c), and cat / showed unusually high genetic diversity and evidenced dna belonging to two species of parvovirus in the same patient. the pathogenicity of cpv variants for cats is not fully understood. some studies have suggested that cpv had the same pathogenic potential as fpv in cats [ , [ ] [ ] [ ] [ ] ; in other studies, clinical signs were not observed in infected animals, with the exception of transient leukopenia [ , ] . these results led to the speculation that cpv, compared to fpv, can most frequently cause asymptomatic and persistent infection in cats, even if additional studies are clearly needed to fully understand the potential of cats as cpv carriers. in our study, an equivalent prevalence of fpv and cpv was found in the samples examined; this result might be related to the type of cat population sampled, which consisted mainly of healthy cats and cats showing clinical signs not related to parvovirosis. alternatively, it could be due to the biological matrix analysed since parvoviruses have commonly been investigated in the faeces of infected animals; instead, in our survey the wbc were analysed. the rates of variation of the fpv and cpv nucleotide sequences analysed in this study were similar, although genetic diversity in the fpv sequences was generated primarily by synonymous mutations which did not result in amino acid substitutions. this result was congruent with the evolutive behaviour of fpv which, since its emergence in , has not undergone significant changes in antigenic and biological properties. feline panleukopenia virus varied at a slow rate by random genetic drift and it maintained host-specificity [ ] . instead, in the cpv sequence data set, non-synonymous mutations were predominant. this finding is compatible with the pattern of evolution observed for cpv. since its emergence in the late s, cpv evolution has been driven by strong positive selection, giving rise to new antigenic variants which have replaced the original type [ ] . an unusual genetic complexity was reported for sample / , with six different viral dnas ascribable to two distinct species of parvovirus, fpv and cpv type c. although co-infection by more than one parvovirus species is a rare event, it has already been described in a cat simultaneously infected by fpv and cpv- a [ ] . carnivore protoparvoviruses show an estimated annual substitution rate on the order of − to − whereas the mutation frequency detected in sample / was on the order of . × − , a value which determines the quasispecies distribution in rna virus populations. carnivore parvoviruses are very prone to genetic evolution, showing substitution rates similar to those of rna viruses, with values of approximately − substitutions per site per year. this result, together with the detection of cpv- c, which has already been reported in multiple infections of high genetic complexity [ , , ] , confirmed that co-infection with different species of parvovirus in feline hosts led to a high genetic variability and to the potential emergence of new viruses [ ] . the presence of distinctive mutations between the fpv dna sequences detected and the sequences of modified live fpv vaccine strains available, together with the lack of information regarding the vaccination history of the cats sampled, allowed the authors to exclude the possibility that dna from vaccine strains was detected and did not allow speculation regarding the persistence of modified live vaccine strains in the cats sampled. however, viraemia persisting up to days post vaccination has been reported in dogs vaccinated with modified live canine parvovirus [ ] . additional studies are required to investigate the nature and clinical significance of the presence of parvovirus dna in the wbc of healthy cats and the potential of cats as parvovirus carriers. ideally, faeces for the examination of shedding should also be included in the sampling; viral isolation could be useful for testing viral vitality, and reverse transcription-pcr assay targeting international committee on taxonomy of viruses ictv genetic analysis of feline panleukopenia viruses from cats with gastroenteritis genetic complexity and multiple infections with more parvovirus species in naturally infected cats genetic analysis of feline panleukopenia virus full-length vp gene in domestic cats between high genetic diversity of the vp gene of a canine parvovirus strain detected in a domestic cat within-host genetic diversity of endemic and emerging parvoviruses of dogs and cats co-infection with feline and canine parvovirus in a cat infectious diseases of the dog and the cat infectious diseases of the dog and the cat canine parvovirus post-vaccination shedding: interference with diagnostic assays and correlation with host immune status immune carrier state of feline panleukopenia virus-infected cats antigenic and genomic variabilities among recently prevalent parvoviruses of canine and feline origin in japan feline host range of canine parvovirus: recent emergence of new antigenic types in cats canine parvovirus in asymptomatic feline carriers isolation of feline parvovirus from peripheral blood mononuclear cells of cats in northern vietnam predominance of canine parvovirus (cpv) in unvaccinated cat populations and emergence of new antigenic types of cpvs in cats characterisation of cross-reactivity of virus neutralising antibodies induced by feline panleukopenia virus and canine parvoviruses identification of parvovirus in the bone marrow of eight cats bioedit: a user-friendly biological sequence alignment editor and analysis program for windows / /nt. nucl acids symp ser dnasp v : a software for comprehensive analysis of dna polymorphism data mega : molecular evolutionary genetics analysis version . for bigger datasets hemagglutination by canine parvovirus: serologic studies and diagnostic applications canine parvovirus: strain difference in haemaglutination activity and antigenicity characterization of parvoviruses from domestic cats in brazil an improved hemagglutination test for study of canine parvovirus host-specific parvovirus evolution in nature is recapitulated by in vitro adaptation to different carnivore species pathogenesis of aleutian mink disease parvovirus and similarities to b infection minute virus of mice: antibody response, viral shedding, and persistence of viral dna in multiple strains of mice persistence of human parvovirus b in human tissues evidence for persistence of human parvovirus b dna in bone marrow tissue persistence of parvovirus b genotypes in asymptomatic persons isolation of canine parvovirus from a cat manifesting clinical signs of feline panleukopenia antigenic type distribution among canine parvoviruses in dogs and cats in germany characterisation of canine parvovirus strains isolated from cats with feline panleukopenia western european epidemiological survey for parvovirus and coronavirus infections in dogs efficacy of feline panleucopenia vaccine to prevent infection with an isolate of cpv b obtained from a cat pathogenic potential of canine parvovirus types a and c in domestic cats high rate of viral evolution associated with the emergence of carnivore parvovirus co-infection with multiple variants of canine parvovirus type (cpv- ) long-term viremia and fecal shedding in pups after modified-live canine parvovirus vaccination comparison of polymerase chain reaction with virus isolation and haemagglutination assays for the detection of canine parvoviruses in faecal specimens analysis of canine parvovirus sequences from wolves and dogs isolated in italy none. this research received no grant from any funding agency in the public or commercial sectors. the nucleotide sequences obtained in this study are available at genbank (no. kt -kt ). sequence alignment are available from the corresponding author (prof. mara battilani) upon request. alignment and phylogeny data were linked to the dryad repository via treebase search id: http://purl.org/phylo/treebase/phylows/study/tb :s . authors' contributions mb and aa designed the study. rz and cd collected the blood samples and the background information on the samples. preparation of buffy coat and dna extraction was performed by aa. the pcr and vp sequencing was carried out by aa, fb and sda under the supervision of mb. ab analysed the sequence data and drafted the manuscript. mb critically revised the manuscript for important intellectual content. all the authors read the manuscript and approved the final version. the study was carried out using stored blood samples which had been collected for clinical and laboratory purposes independent of the study with the agreement of the cat owners who presented their cats to the veterinary teaching hospital (university of sassari). as stored blood samples were used, no separate ethical approval was required for the study. all efforts were made to minimise the discomfort of the animals during sampling. not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- - sqovwb authors: li, hao; li, kai; bi, zhen; gu, jun; song, deping; lei, dan; luo, suoxian; huang, dongyan; wu, qiong; ding, zhen; wang, leyi; ye, yu; tang, yuxin title: development of a reverse transcription-loop-mediated isothermal amplification (rt-lamp) assay for the detection of porcine pegivirus date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: sqovwb a simple and accurate reverse transcription-loop-mediated isothermal amplification (rt-lamp) assay was developed and evaluated for the detection of porcine pegivirus (ppgv). the specific rt-lamp primers targeting the conserved regions of ns a genes were designed and used to detect ppgv. the optimal reaction parameter for rt-lamp assay was ℃ for min. the detection limit of the rt-lamp assay was copies of ppgv genome, which was times more sensitive than that of the conventional rt-pcr and comparable to nested rt-pcr and quantitative rt-pcr (qrt-pcr). there was no cross amplification with other related rna viruses. in the clinical evaluation, the rt-lamp assay exhibited a similar sensitivity with nested rt-pcr and qrt-pcr. the results indicated that rt-lamp assay developed in this study could be a highly specific, sensitive, and cost-effective alternative for a rapid detection of ppgv in field settings. porcine pegiviruses (ppgv) are small enveloped viruses with a single-stranded, positive-sense rna genome that have recently been assigned as the pegivirus genus in the flaviviridae family (berg et al., ) . ppgv genome contains a single large open reading frame (orf) encoding a polyprotein, which is cleaved into individual proteins including e , e , x, ns , ns , ns a, ns b, ns a, and ns b. pegiviruses are detected in diverse mammalian hosts including pigs, humans, horses, chimpanzees, bats, monkeys, and rodents (yang et al., ) . all known pegiviruses have been classified into species (a-k), and ppgv is a member of pegivirus k. ppgv was first discovered in germany in (baechlein et al., ) , and then reported in the united states in (yang et al., ) . in china, ppgv was first identified in guangdong province (lei et al., ) , where vesicular diseases broke out in a swine farm in . the serum samples were submitted to the veterinary diagnostic laboratory of jiangxi agriculture university, china for diagnosis. the tested results indicated that foot-and-mouth disease virus (fmdv) and seneca valley virus (svv) were negative but positive for ppgv by a rt-pcr assay. although it remained yet unknown about the association between ppgv infection and the disease, more epidemiological studies are needed to address the clinical effect of ppgv on swine health. currently, there are some methods available for ppgv diagnosis, including conventional rt-pcr, nested rt-pcr, and semi-quantitative rt-pcr (yang et al., ; lei et al., ; chen et al., ) . therefore, a rapid and accurate method to detect ppgv is urgently needed for monitoring its presence. in the past decade, loop-mediated isothermal amplification (lamp) has become an effective technique, which exhibits high sensitivity and specificity for diagnosing important pathogens in medicine and veterinary medicine (notomi et al., ) . lamp is a nucleic acid amplification-based method that generally requires a group of four specific external and internal primers (notomi et al., ) . besides, additional loop primers can be used to further accelerate the reaction (nagamine et al., ) . in this study, a reverse transcription lamp (rt-lamp) assay was established and evaluated for surveying the prevalence of ppgv in porcine serum samples. a set of three specific primers pairs was designed to amplify the ns a gene, which is suitable for rt-lamp and relatively conserved in ppgv genome. the developed rt-lamp assay showed high sensitivity and specificity for detection of the ppgv. the results tested by rt-lamp assay on clinical samples were % correlated to that of nested rt-pcr and qrt-pcr, indicating the assay could be used for the surveillances of ppgv infection. in this study, serum samples (n = ) of -week-old pigs with a vesicular disease from guangdong province, china in were collected. extraction kit (takara, china), and then stored at - ℃ until use. the extracted rna was converted to the first strand of cdna using primescript rt reagent kit (takara, china) following the manufacturer's instructions. for the construction of plasmid standards, an -bp fragment of the ns a gene of ppgv was initially amplified using a pair of primers (table ) with the st strand of cdna as template. afterwards, the purified fragment was cloned into the pmd -t vector (takara, china) according to the manufacturer's protocol. to assess the successful construction of the plasmid, designated as pmd-ppgv, dna sequencing was performed by sangon biotech company (shanghai, china). the concentration of pmd-ppgv plasmid was measured by a nanodrop spectrophotometer (thermo fisher scientific, usa). the copy number of the cloned gene was quantified as follows: [copy/ μl = plasmid concentration (g/μl) / [(plasmid length) × ) × ( . × )] (park et al., ) . serial dilutions of plasmid standards ( × - × copies) were used as templates for the determination of detection limit of pcr, which would be used as a positive control in the rt-lamp assay system. the three pairs of rt-lamp primers targeting the conserved regions of ns a genes were designed based on the sequences of the reference strain (ppgv_gd/ch/ , genbank: mg . ; ppgv_s / /ger/ , genbank: ku . ; ppgv_ /ger/ , genbank: nc_ . ; ppgv_ f/ger/ , genbank: ku . ; ppgv_ / nd/ , genbank: mg ; ppgv_ /mo/ , genbank: mg ; ppgv_ /ia/ , genbank: mg ) using the online software primer explorer v (http://primerexplorer.jp/e/), which included an outer pair (f and b ), an inner pair (fip and bip), and a loop pair (lf and lb). the inner primers fip and bip contained f and b sequences with the complementary sequences of f and b (f c and b c) in their ′-terminal, respectively (fig. ) . a pair of primers (f and r ) was used for amplifying the ns a gene in conventional rt-pcr and meanwhile served as outer primers for the nested rt-pcr (table ). the primer pair of f and b was used as inner primers for nested pcr. a pair of primers (f and r ) was used for qrt-pcr targeting the ns a gene which was designed using the online software primer (http://bioinfo.ut.ee/primer - . . /). the specificity of the primers was then examined by a basic local alignment search tool (blast) search (http://www.ncbi.nlm.nih.gov/blast) against the ncbi database. the results demonstrated that there was no hits, that is, no nonspecific primer binding site was observed. the final volume of μl reaction mixtures for rt-lamp was prepared, which contained μl of bst dna polymerase (neb, usa) ( u/ml), . μl of × isothermal amplification buffer, μl of betaine ( m), μl of mgso ( mm), μl of dntp ( . mm), μl of each inner primers fip and bip ( μmol), . μl of each outer primers f and b ( μmol), . μl of each loop primers lf and lb ( μmol), . μl of amv reverse transcriptase (takara, china) ( u/μl), μl of rna template, and the sterile distilled water was set as a negative control template. to determine the optimal reaction conditions, the rt-lamp reactions were incubated at different temperatures of , , , , , and ℃ for , , , , , and min, respectively. finally, the reaction was terminated by heat inactivation at ℃ for min. the amplified dna products from the rt-lamp were analyzed by . % agarose gel electrophoresis with the addition of . % ethidium bromide using a gel electrophoresis system (liuyi, china). the results were also observed directly by color change from orange to green with staining due to the presence of sybr green Ⅰ (thermo scientific, usa). after the amplification was completed, μl of coloring agent was added to each reaction tube, and the results were then examined visually. in rt-lamp assay with ppgv rna as template, the amplified dna products showed characteristic ladders of multiple bands on an agarose gel, indicating that the final products were a mixture of stem-loop dna with different stem lengths (khan et al., ) . the results of the rt-lamp reaction were also determined by direct visualization of the color change under daylight and uv light as shown in fig. . in contrast, lamp-specific dna bands and color changes were not observed in the negative control. meanwhile, the dna products of rt-lamp showed the highest intensity when the reaction was carried out for min (fig. ) . in regard to temperature, the optimal reaction temperature of rt-lamp was ℃. therefore, the optimized parameter for the established rt-lamp was ℃ for min. to determine the detection limit of the rt-lamp, -fold serially diluted plasmid standards of pmd-ppgv ( × - × copies) were used as templates in μl rt-lamp reaction system under optimized conditions. the sterile distilled water was set as a negative control template. nested rt-pcr was performed based on the protocol established in our laboratory (lei et al., ) . for the pcr step of the nested rt-pcr, the primer sets of f /r and f /b respectively served as outer ( st set) and inner primers ( nd set) ( table ). the reaction system contained μl of plasmid standards of pmd-ppgv ( × - × copies) or μl of the first pcr product, . μl of × pcr buffer (ta-kara, china), . μl of rtaq ( u/μl), μl of mgcl ( mm), μl of dntp ( . mm), and μl of each primer ( μmol). two rounds of nested pcrs were performed under the following conditions: ℃ for min; cycles at ℃ for s, ℃ for min, and ℃ for min; and a final extension of ℃ for min (wang et al., ) . the conventional rt-pcr was carried out under the same reaction condition as above in nested rt-pcr using the primer pair of f and r based on the procedure described previously (lei et al., ) . the products of nested rt-pcr and conventional rt-pcr were analyzed by . % agarose gel electrophoresis with . % ethidium bromide. for qrt-pcr, the primer set of f and r , targeting conserved region of the ns a gene of ppgv, was used ( table ). the qrt-pcr assay was performed by using a commercial qpcr kit (takara, china) based on the manufacturer's procedure. each μl reaction mixture consists of μl of tb green fast qpcr mix, μl of plasmid standards of pmd-ppgv ( × - × copies), . μl of ( μmol) each forward and reverse primers and . μl of rox. the amplification parameters included an initial denaturation at ℃ for min followed by cycles of ℃ for s and ℃ for min. the melting curve analysis was measured using the software supplied with abi fast real-time pcr system (applied biosystems, usa). the results demonstrated that the detection limit of the developed rt-lamp was copies/μl, which was much higher than conventional rt-pcr ( copies/μl), while it was comparable to that of the nested rt-pcr ( copies/μl) and qrt-pcr ( copies/μl), respectively (fig. ) . to assess the specificity of rt-lamp for ppgv, cdnas of seneca valley virus (svv), food and mouth disease virus (fmdv), porcine epidemic diarrhea virus (pedv), porcine deltacoronavirus (pdcov), and swine acute diarrhea syndrome coronavirus (sads-cov) were used, and the plasmid standards of pmd-ppgv was used as the positive control. the amplified products were analyzed by . % agarose gel electrophoresis with . % ethidium bromide and addition of sybr green Ⅰ staining solution. the results indicated that there was no cross amplification of the rt-lamp assay for these control viruses. using ppgv rna as a template, positive dna bands were amplified as expected (fig. ) , indicating that the rt-lamp assay developed was specific for ppgv. to further evaluate the developed rt-lamp assay, serum samples (n = ) from finishing pigs and sows collected in guangdong and jiangxi province in china during october to january were tested (lei et al., ) . total rna was extracted and used as a template for conventional rt-pcr, nested rt-pcr, qrt-pcr, and rt-lamp according to aforementioned methods. as shown in table , the positive sample rates of conventional rt-pcr, nested rt-pcr, qrt-pcr, and rt-lamp were . % ( / ), . % ( / ), . % ( / ), . % ( / ), respectively, suggesting these data were consistent with our previous findings (lei et al., ) . the detailed information is listed in table s . the results showed the rt-lamp had a similar sensitivity to nested rt-pcr and qrt-pcr, which was superior to conventional rt-pcr. pegiviruses have a broad host range and infect diverse animal species. ppgv is recently proposed as a novel species in the pegivirus genus. ppgv epidemics have been confirmed in germany in , the united states in , and china in . although several pigs positive for ppgv exhibited vesicle and lameness (yang et al., ) , there is limited evidence that infections were associated with a clinical disease. therefore, the clinical significance and epidemiological investigation of ppgv infection should be further studied. nevertheless, the establishment of a sensitive, cost-effective, and rapid assay for detecting ppgv is urgently needed. in this study, a rt-lamp assay was developed to monitor ppgv in pig populations. compared to other diagnostic methods, such as conventional rt-pcr, nested rt-pcr, and qrt-pcr (baechlein et al., ) , rt-lamp assay may eliminate a need for complex procedures and expensive instruments, which makes it suitable for poorly equipped laboratories and clinics (lopez-jimena et al., ) . rt-lamp is considered to be an alternative diagnostic tool in terms of its ability to rapidly amplify target nucleotide sequence(s) under isothermal conditions. lamp has been widely used to detect a variety of important swine pathogens, including pdcov (zhang et al., ) and pedv (mai et al., ) . in the present study, the rt-lamp reaction conditions were optimized in terms of reaction time and temperature. the detection limit of rt-lamp assay established is times higher than that of conventional rt-pcr, and had a similar sensitivity to nested rt-pcr and qrt-pcr. rt-lamp developed in the study showed a high specificity as it did not cross react with other related porcine viruses. in the clinical evaluation, the positive rate of rt-lamp were consistent with that of nested rt-pcr and qrt-pcr, indicating the ppgv-specific rt-lamp assay was a valuable tool for the rapid detection in field settings. in summary, we developed a rapid, simple, accurate, and cost-effective rt-lamp assay for detection of ppgv, and the assay could be used as an alternative for the clinical diagnosis of ppgv infection and epidemiological investigation. none. pegivirus infection in domestic pigs discovery of a novel human pegivirus in blood associated with hepatitis c virus co-infection semiquantitative duplex rt-pcr reveals the low occurrence of porcine pegivirus and atypical porcine pestivirus in diagnostic samples from the united states diagnostic accuracy of loop-mediated isothermal amplification (lamp) for detection of leishmania dna in buffy coat from visceral leishmaniasis patients detection and genetic characterization of porcine pegivirus from pigs in china development of a single-tube one-step rt-lamp assay to detect the chikungunya virus genome development of pooled testing system for porcine epidemic diarrhoea using real-time fluorescent reverse-transcription loop-mediated isothermal amplification assay loop-mediated isothermal amplification of dna loop-mediated isothermal amplification (lamp): principle, features, and future prospects loop-mediated isothermal amplification assay for the rapid and visual detection of novel porcine circovirus integrating nested pcr with high-throughput sequencing to characterize mutations of hbv genome in low viral load samples detection and genetic characterization of porcine pegivirus in pigs in the united states a simple and rapid identification method for newly emerged porcine deltacoronavirus with loop-mediated isothermal amplification rapid specific and visible detection of porcine circovirus type using loop-mediated isothermal amplification (lamp) this work was supported by the national key research and development program (grant number yfd ), the natural science foundation of jiangxi province (grant number acb and bab ), the graduate research & innovation projects in jiangxi (grant number yc -b ), and science and technology project of education department of jiangxi province (grant number gjj and gjj ). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.jviromet. . . . key: cord- -qm uw r authors: weerasuriya, a.u.; hu, z.z.; zhang, x.l.; tse, k.t.; li, s.; chan, p.w. title: new inflow boundary conditions for modeling twisted wind profiles in cfd simulation for evaluating the pedestrian-level wind field near an isolated building date: - - journal: build environ doi: . /j.buildenv. . . sha: doc_id: cord_uid: qm uw r the hilly topography of hong kong influences oncoming winds and gradually changes their wind directions along the profiles' height. the vertical variation in wind directions, or the twist effect, significantly influences the pedestrian level wind (plw) field in urban areas of hong kong, thus it is a topic demanding systematic investigations. in this study, a new set of inflow boundary conditions are proposed to model twisted wind flows in computational fluid dynamic (cfd) simulations. the new inflow boundary condition derived based on the horizontal homogeneous assumption, specifies a vertical profile of lateral wind speeds at the inlet boundary to sustain the twist effect in the empty computational domain. the proposed boundary conditions are used to simulate the plw fields near three isolated buildings with different height-to-width ratio using two cfd codes; openfoam, and fluent. the results reveal that openfoam is more reliable in simulating plw fields in twisted wind flows using the new set of boundary conditions. the three-dimensional flow field provided by the openfoam simulation shows sparse streamlines downstream the buildings, indicating lack of organized eddies in the building far wake, which negatively affects the dispersion of air pollutants in twisted winds. as both wealth and population amass in urban centers, the land becomes scarce and high-rise buildings fill up cityscapes [ ] . high-rise buildings are a major cause of wind nuisance at the pedestrian level: on the one hand, pedestrians could be in danger when exposed to intense downwash flows and sudden wind gusts induced by the high-rise buildings [ ] [ ] [ ] [ ] [ ] . on the other hand, when spaced closely together, highrise buildings obstruct natural breezeways, subsequently limiting the near-ground air circulation, potentially leading to undesirable low wind speed areas between buildings that form a cluster [ , ] . in a tropical metropolis such as hong kong, low wind speeds in urban areas may pose a serious threat to public health [ ] ; if winds do not have high enough speed, they cannot help regulating the body temperatures of pedestrians. poorly regulated body temperatures may rise, and once reaching a critical level, they may result in heat stroke or other medical conditions [ ] . also, low winds speeds cannot effectively carry air pollutants away and disperse them from the street level, resulting in poor air quality in city centers [ ] . unimproved poor air quality amplifies the risks of an epidemic such as the outbreak of the severe acute respiratory syndrome (sars) in . sars, having claimed many lives, prompted the government of hong kong and local scholars to appraise the state and quality of outdoor air ventilation. their combined effort has led to the introduction of the important protocol named air ventilation assessment (ava), whose aim is to prevent any further deterioration of the air quality in hong kong. the ava demands that any land developments and redevelopments in hong kong need to pass a wind tunnel test-based or computational fluid dynamic simulation-based assessment to ensure that they maintain a minimum wind speed at the pedestrian level no less than . m s − . given the importance of the pedestrian level wind (plw) environment in modern cities such as hong kong, many studies have focused not only on achieving acceptable wind environments for pedestrians but also on the factors that affect the wind conditions in built-up areas. the buildings in a built-up neighborhood are not the only factor that affects the plw field in that area. several other factors such as upstream terrain, atmospheric stability, and anthropogenic heat emissions may also have significant influence on the plw field. for example, the hilly terrain in hong kong is a major factor that governs the local wind climate [ ] . moreover, the properties of wind flows including velocity, direction, and turbulence modified by the hilly terrain should be considered when calculating wind loads on structures [ ] and assessing the urban wind environment in hong kong [ ] . tse et al. [ ] revealed how the hilly terrain of hong kong causes large variations in wind directions in a wind profile, subsequently modifying the plw fields around both isolated buildings and arrays of buildings. directional variations in a wind profile occur when winds detour as they are passing over hilly terrains. the influence of a hill on winds is maximum near the surface of the hill, gradually diminishing with height [ , ] . a wind profile containing wind directions that vary with height has a spiral shape, thus giving the profile the commonly seen term "twisted wind profile"; contrary to a twisted wind profile, a conventional wind profile has constant wind direction all along the profile's height. how much wind direction varies-also known as wind twist effect-at a given height z, can be expressed as yaw angle (θ) by equation ( ) . in equation ( ), v is the lateral wind velocity and u is the longitudinal wind velocity at height z. given that most urban areas in hong kong are located near mountains, and yaw angles have been observed to reach as much as °, tse et al. [ ] proposed to employ twisted wind profiles in wind tunnel tests when the urban plw field of hong kong is concerned. to follow up on this, tse et al. [ , ] conducted a series of wind tunnel tests to investigate the plw fields around isolated buildings and arrays of buildings with different dimensions. their results [ ] revealed noticeable flow modifications in the plw field under the influence of twisted wind flows. in a twisted wind flow, for example, the building's far wake was observed to deviate from the centerline of the building, and asymmetric corner streams and a new low wind speed zone attached to the wall on the left side of the building were observed. moreover, in a twisted wind flow, the plw fields around arrays of buildings showed noticeable modifications: for example, dissimilar wind speeds were found inside passages between the buildings [ ] . although these studies have shed light on how twisted winds influence the plw fields around both an isolated building and arrays of buildings, details of the flow fields have been obscured. this shortcoming is partly because of the data of the three-dimensional flow field were not obtained from the wind tunnel tests [ ] . without three-dimensional flow field data, tse et al. [ , ] made assumptions on possible flow mechanisms to explain the modifications in the plw field caused by the twist effect, but the explanation lacks the support of solid evidence such as simultaneous measurements of wind speed and flow field in the plw field. one of the main objectives of this paper is, therefore, to reevaluate the explanation proposed by tse et al. [ ] in a more direct and illustrative way using the computational fluid dynamics (cfd) technique. since the late s, cfd techniques have been applied to investigate plw fields in built-up areas [ ] . the main advantage of cfd simulation is its ability to provide simultaneous data on both wind speed and flow circulation at the pedestrian level, avoiding the hassle of the two-step approach common in wind-tunnel tests [ ] . cfd simulations also have proven accuracy in simulating plw fields around isolated buildings [ ] [ ] [ ] , around arrays of buildings [ ] [ ] [ ] , in idealized city models [ ] [ ] [ ] , and in real urban areas [ ] [ ] [ ] . all these studies highlighted the importance of proper inflow conditions for accurate results from cfd simulations. improper inflow boundary conditions, which likely create a non-equilibrium boundary layer, would cause a cfd simulation to deviate from reality [ ] . given the importance of an equilibrium boundary layer in cfd simulations of atmospheric flows, several sets of inlet boundary conditions have been proposed for reynolds average naiver stokes (rans) simulations, particularly for the standard k-ε turbulence model [ ] [ ] [ ] [ ] . in the derivation of profiles of u k , and ε for the equilibrium boundary layer, the commonly used shear stress model eventually leads to that u is a logarithmic function of heights [ ] . juretic and kozmar discussed the numerical set-ups to run a cfd simulation with vertically decreasing turbulent shear stresses [ ] ; yan et al. investigated the specification of k and ε which could create an equilibrium boundary layer with an arbitrary shear stress model [ ] . recently, these inlet boundary conditions have been extended to cover other k-ε turbulence models [ ] and the sst k-ω model (shear stress transport k-ω model) [ ] . other than the inlet boundary condition, the consistent top boundary condition is discussed by o'sullivan et al. [ ] , which facilitate the generation of the equilibrium and neutral boundary layer. in addition to the proposal of inlet boundary conditions, modifications are made to the two-equation turbulence model to create the equilibrium boundary layer [ , , ] . besides the specification of the boundary conditions and modifications to the turbulence models, dynamic approaches are also suggested, which include balanced forcing terms in the cfd simulations to produce an equilibrium boundary layer [ ] . this study aims to propose a new set of inflow boundary conditions to produce an equilibrium boundary layer with twisted wind profiles. in the present study, cfd techniques are employed to investigate the plw field around an isolated building, focusing on the flow modifications induced by the twist effect and flow mechanisms driving such modifications. in order to accurately and reliably simulate the plw field in the twisted wind flow, it is necessary to explore the inlet boundary conditions, which could sustain a twisted wind profile in a cfd simulation. although plenty of studies have been conducted to provide inlet boundary conditions in the simulation of the atmospheric boundary layer flow, no systematic investigations, to the best knowledge of the authors, have been carried out to show the sustainability of the twisted wind profiles in the cfd computational domain. the present study therefore carries the first attempt to include the twist effect in a cfd simulation of the atmospheric boundary layer flow. furthermore, the numerical simulation of the plw field around an isolated building reveals the three-dimensional flow field in a twisted wind flow in the vicinity of the building at the pedestrian level and above, which helps identify mechanisms leading to the flow modifications in the twisted flows observed from a corresponding wind-tunnel test. the findings made in examining the cfd simulation results, in turn, could provide suggestions for the implementation of the ava, and hence for the urban planning of a metropolitan in tropical or subtropical zone, such as hong kong. section of this paper presents the derivation of the new set of inflow boundary conditions, whose sustainability has been evaluated in an empty domain. section describes the wind-tunnel tests whose data have been used to verify the cfd simulations of the plw fields influenced by wind twist effects. section presents the numerical settings used to run the cfd simulations and discusses the simulated plw fields, putting particular focus on (a) the comparison between results of the cfd simulations and data from wind-tunnel tests, and (b) details of the flow field leading to plw field modifications. section offers some concluding remarks. the new inflow boundary conditions are developed based on the rans equations, in which the turbulent diffusivity is estimated according to the standard k-ε model [ ] . it is noted that the variants of the standard k-ε model have been widely used in cfd simulations of atmospheric flows, and therefore the proposed inflow boundary conditions provide a good starting point for developing similar inlet boundary conditions for other turbulence models. it is crucial that the flow conditions specified at the inlet should be maintained throughout the computational domain or until they reach the object of interest. in other words, the flow entering the computational domain through the inlet boundary should be in a horizontal homogeneous state. under this horizontal homogeneous assumption (∂ ∂x / = and ∂ ∂y / = ) and that assumption that = w , which has also been made as shown in the previous publications [ , ] , the rans equation and the transportation equations of turbulent kinetic energy (k) are reduced to equations ( )-( ). in the equations, k is the vertical turbulent diffusivity, u is the longitudinal wind velocity, v is the lateral wind velocity, w is the vertical wind velocity, z is the vertical coordinate, and k is the turbulent kinetic energy. the transportation equation of k should include the generation and dissipation of the turbulent kinetic energy, which are absent from equation ( ) . in fact, the local equilibrium assumption, which is expressed in equation ( ), is implied in the derivation of equation ( ) to formulate a close set of equations. the generation of turbulence, which can only originate from the shear of the mean flow, is assumed equal to the dissipation rate of the turbulent kinetic energy (ε) as shown in equation ( ) . other publications concerning the creation of equilibrium boundary layer in cfd simulations derived similar sets of governing equations for various turbulence models. it should be noted that in the derivations shown in the works of yang et al. [ , ] , richards and hoxey [ ] , the horizontal homogeneous assumption and the shear stress mdoel unavoidably leads to the logarithmic shape of u, ( ) and c μ is a model constant. a similar set of equations have been derived in previous studies focusing on the sustainable inlet boundary conditions [ ] [ ] [ ] . in contrast to the conventional cfd applications, in which the lateral wind velocity is often assumed zero at the inlet, the cfd simulation of twisted flows has the condition ≠ v z ( ) . hence, velocity inlet in fluent and fixed velocity patch in openfoam left velocity inlet in fluent and fixed velocity patch in openfoam rough wall modification with roughness height k s = . m and roughness constant c s = . . while equations ( ) and ( ) compute vertical profiles of k and ε, equation ( ) computes the vertical profile of v according to the profile of u. under the horizontal homogeneous assumption, the logarithmic function of heights is suggested to model the u profile [ ] [ ] [ ] . according to equation ( ), the v profile can also be described by a logarithmic function of heights if the u profile is logarithmic, and the influence of ground frictions, shown by the aerodynamic roughness length (z ), is included in both the profiles of u and v. in other words, the shears in the profiles of u and v are majorly induced by the ground friction and marginally induced by the vertical variation of wind directions if the logarithmic function is adopted to model the u profile. as illustrated in the derivation, equations ( )-( ) are the analytical solutions to the rans equations and the transportation equation of k under a series of assumptions. the transportation equation of ε is, however, not satisfied when v k , and ε are specified according to equations ( )- ( ) . in fact, the residual of the transportation equation of ε is controlled by the values of the constants c k and c k , and the influence of the residual on the sustainability of the inlet profiles is limited when turbulence intensities of the simulated wind field are within a reasonable range < ( %). the horizontal homogeneity of the proposed inflow boundary condition is evaluated in an empty domain using two cfd software; openfoam and fluent. openfoam is an open-source cfd simulation tool that has gradually gained popularity by virtue of its flexibility in adjusting the code for various purposes [ ] . fluent is one of the widely adopted commercial cfd codes used by researchers and engineers because of its user-friendly interface and embedded numerical models for different types of simulations. however, the lack of table lists the boundary conditions used for the simulations of the empty domain. it should be noted that the cfd codes, openfoam and fluent, share a set of boundary conditions and other numerical settings. in particular, the proposed inlet boundary conditions are employed to specify the profiles of u v k , , and ε at the inlet and lateral boundaries of the openfoam and fluent simulations. the parameters to be determined in equations ( )-( ) and the profile of wind velocities are specified according to the two twisted wind profiles simulated by tse et al. [ ] in a wind tunnel test. the two twisted wind profiles have the maximum yaw angle (θ) of° and ° at the lowest measurement height of . m and are hereafter referred to as twp and twp . fig. shows the profiles measured from the wind tunnel tests and the profiles used to specify the inlet boundary conditions for the cfd simulation. it is evident from fig. that the profiles employed in the cfd simulations, calculated according to equations ( )- ( ) , are similar to the profiles measured from the wind tunnel test. since the profile of u measured from the wind tunnel test is modulated using the logarithmic model as the target (u* = . m s − , z o = . m), it is reasonable that fig. (a) shows agreement between the profiles of u measured from the wind tunnel test and the cfd simulation. the profiles of v and k, on the other hand, measured in the wind tunnel test are not modulated using equations ( )-( ), and therefore deviate slightly from the profiles used in the cfd simulation. from comparing the profiles of u and v extracted from the cfd simulations at the center and near the outlet boundaries to the profiles specified at the inlet boundary (shown in figs. and ) , it can be discerned that the proposed inflow boundary conditions of v and k successfully model the twisted wind profile in both cfd simulations. in fact, there are no observable differences between the profiles of u and v at different locations in the empty domain. the profile of k, however, deviates slightly from the specification at the inlet boundary along the stream wise direction, and the maximum differences between k profiles at the inlet and near the outlet boundaries are under . m /s (< %). the differences in the k profiles presented in figs. and should be attributed to the near-wall treatments employed in the openfoam and fluent simulations. since the sustainability of the profiles of u and v is the main concern of the proposed inflow boundary condition and the deviation in the k profiles is within the acceptable range, both the empty domain simulations using openfoam and fluent substantiate that the proposed inflow boundary conditions have successfully maintained the profiles of wind velocities with twist effects in the cfd simulation. consequently, it is feasible to use the proposed inflow boundary conditions in a cfd simulation focusing on the influence of twisted wind flows on the plw environment around an isolated building. to quantitatively assess the accuracy and reliability of cfd simulations of the plw field around an isolated building, the wind-tunnel measurements are used as criteria in a systematic evaluation of the cfd simulation results. all the corresponding wind-tunnel tests were conducted in the clp power wind/wave tunnel facility (wwtf) at the hong kong university of science and technology (hkust). two types of wooden vanes with the maximum guide angles of° and° at the ground level, as shown in fig. , were installed in the low-speed section of the wind tunnel to simulate the two twisted wind profiles: twp and twp . the building models were installed at the center of the turntable, which was m downstream the vane systems ( fig. (a) ). the mean wind speeds measured at mm in the wind-tunnel, which corresponds to a m height in the full-scale according to the length scale of : , were taken as the wind speeds at the pedestrian level. the wind speeds were measured at more than locations using irwin sensors installed around the building models as shown in fig. (b) . in detail, the irwin sensors covered an area of . . m with the minimum resolution of . m . m near the buildings. three building models with the dimensions as listed in table are tested in the wind tunnel to present the plw fields around a tall and slender, a short and wide, and an intermediate isolated building. all the other details on the wind-tunnel experiments of the plw field around the isolated building can be found in the authors' previous publications [ , ] . the plw fields around the three building models; s , s , and s , under the influence of the two twisted wind flows (twp and twp ) are simulated using openfoam and fluent. in addition, a conventional wind profile, which is hereafter referred to as cwp, is adopted to conduct the cfd simulation as the control case. the cwp profile has similar wind speeds and turbulence intensities as the profiles of twp fig. (a) . the heights of cells close to the ground, on the other hand, are reduced (shown in fig. (b) ) to ensure that there are more than layers of cells within pedestrian level [ ] , which is . m for the simulations reported in the present study. the height of cells increases along the height of the building model and reduces when approaching the building top. the height of cells near the ground and close to the building top is around mm . , which makes the total number of cells exceeding . million for both openfoam and fluent simulations. the boundary conditions used to run the simulations are identical to the cases with the empty domain, which have been summarized in table . the quick scheme [ ] is used to discretize the momentum equation in both openfoam and fluent simulation, and the simple algorithm [ ] is employed to solve the governing equations. the simulations are kept for iterations to ensure that the converged results are obtained. the simulated wind velocity at the pedestrian level along the lines laterally penetrating the computational domain ( = − x m m and = x m m ) are monitored through the simulation process, and the fluctuations in the simulated wind velocities on the monitor during the last iterations for the openfoam and fluent simulations are under the levels of . % and . %, respectively, which substantiated that the converged results are obtained. before further verifying the cfd simulation results based on windtunnel measurements, it is crucial to check the independency of simulation results from both the meshes and numerical schemes used to discretize the momentum equation. therefore, a finer mesh, with the total number of cells exceeding . millions, is employed to run the simulation of the same domain using the same numerical setting as the simulation with coarse meth (containing . million cells). the finer meshes are produced through reducing the horizontal sizes of cells close to the building model from mm to mm , and increasing the number of cells in the vertical direction from to . the simulated wind velocities corresponding to the building model of s under the influence of the profile of twp at the lines cutting through the domain laterally ) are plotted in fig. for the fine mesh and coarse mesh. the x coordinate of the lines are determined through specifying the building center located at = x . it is evident from the figure that the simulation results, yielded by both the openfoam and fluent simulations, corresponding to the fine mesh is in excellent agreement with the simulation results of the coarse mesh, which substantiates that the simulations are grid independent. in addition to the independency of the grid, the independency of the simulation results from the numerical schemes is also checked. in detail, the sfcd scheme [ ] , muscl scheme [ ] and umist scheme [ ] , all in second order, are employed to discretize the momentum equations in the openfoam simulation in addition to the quick scheme used to run the control case. the simulated wind velocities, corresponding to different numerical schemes under investigation, along the lines cutting through the computational domain laterally ( fig. (a) ) and longitudinally ( fig. (b) ) are plotted. the x and y coordinates of the lines are determined through specifying the building center located at = x and = y . it is shown in the figure that the numerical schemes hardly influences the simulated wind field as the maximum difference among the simulated wind velocities at the same location is within the range of − ∼ − − ms ms . . . such differences are considered small in a independency check of the numerical scheme, and hence it is fair to conclude that the simulation results are independent from the numerical scheme used to discretize the momentum equation. fig. shows the distribution of wind speeds at the pedestrian level around the building model s as observed from the wind tunnel experiment and extracted from the cfd simulation. the results shown in fig. corresponds to the case in which the profile twp attacks the building model s at an incident angle of° . the white arrows shown in fig. indicate the direction of each approaching wind flow at the pedestrian level. as observed from fig. , the wind tunnel measurements show noticeable modifications in the plw field around the building model, including dissimilar corner streams, and the deviation of the far wake as reported by tse et al. [ ] . as it can be seen from fig. (b) and (c) that both openfoam and fluent acceptably replicate the flow modifications observed in wind tunnel experiments. there are, however, deviations in the plw field when comparing fig. (b) and (c) to fig. (a) . for example, on the leeward side of the building, cfd simulations tend to underestimate wind speeds. such a discrepancy may be attributed to, first, the low accuracy of irwin sensors in measuring wind speeds less than m/s [ ] , and, second, a wellknown drawback of cfd simulations in predicting wind conditions in the building wake [ ] . in addition to the overall comparisons, fig. shows wind speed variations at the pedestrian level in the wind flows of cwp and twp along four lines parallel to the y-axis. one of the four lines is at a . m distance upstream from the building center and the rest of the lines are at . m, . m and . m distances downstream from the building center. as shown in fig. (a) that both openfoam and fluent predict similar variations in wind speeds as observed in the wind tunnel experiment despite underestimations of wind speeds in the wake of the building. particularly, the mean wind speeds yielded from the cfd simulations are considerably smaller in an area bounded by the lines of = x , = x . m and the lines of = − y w , = y w downstream from the building (w is the width of the building). within this area, the mean wind speeds extracted form openfoam and fluent simulation results are, on average, . % and . % smaller than the measurements from the wind tunnel test. outsides this area, the cfd simulation results are in a better agreement with the wind-tunnel measurements as the discrepancies reduced to, on average, . % and . % for the openfoam and fluent simulations, respectively. similarly, fig. (b) shows that the wind speeds predicted by cfd simulations in the twp flow deviate from the wind-tunnel measurements, particularly in the wake of the building s . the differences in wind speeds between the cfd simulations of openfoam and fluent and the wind tunnel experiment are about . % and . % in the twp case. the largest difference in wind speeds is, however, observed away from the line = y for the twp case, in contrast to the results corresponding to the profile of cwp, where the largest difference is always found exactly on the line = y . the different locations of the largest difference are related to the location of the downstream far-field low wind speed (dflws) zone, which is deviated clockwise away from the line of = y in the twp flow. similarly, the simulated mean wind speeds are extracted along the lines parallel to the x-axis and compared with the corresponding windtunnel measurements in fig. . fig. (a) reveals that the results of fluent simulation overestimate plw speeds by about % in the cwp wind flow compared to the wind tunnel measurements. the predictions of the openfoam simulation, on the other hand, only moderately deviate from the wind-tunnel measurements. for instance, the deviation of openfoam simulation results from wind-tunnel measurements is less than % within the area bounded by the lines of = x and = x m . . in the twp flow, as shown in fig. (b) , fluent moderately overestimates the wind speeds, while the openfoam simulation produces wind speeds comparable to the data of wind tunnel experiment along the line of = speeds as measured from the wind tunnel experiment while the wind speeds extracted from the fluent simulation moderately deviate from the corresponding wind speeds measured in the wind tunnel test. fig. shows the longitudinal variations in wind speeds corresponding to the building models of s and s in the twp wind flow. fig. (a) implies that the simulation corresponding to a tall and slender building yields results that are in better agreement with wind tunnel measurements. the openfoam simulation, in particular, captures the longitudinal variations in the plw field observed in the wind tunnel test. the results of the fluent simulation are, however, not as accurate as those of the openfoam simulation. more specifically, the fluent simulated plw field shows small deviations from the windtunnel measurements along the line of = y m . and has large deviations along the line = y m . . in contrast, fig. (b) shows large deviations that are found for the cfd simulations. in particular, the cfd simulations present great deviations along the line of = − y m . when comparing to fig. (a) . it is believed that the large differences presented in fig. (b) are in the connection with the dflws zone, which spreads over a large area downstream the building s (see refs. [ , ] ). in addition, the plw field produced by the cfd simulations near the line of = x are noticeably smaller than the measurements of the wind tunnel test. the strong near-wall turbulence that reduces the accuracy of wind-tunnel measurements on the leeward side of the building s may be the reason for such noticeable discrepancies. the verifications presented in figs. - substantiate that open-foam outperforms fluent in simulating the plw fields around isolated buildings, except in the dflws zone, where the reliability of both the cfd codes and the wind-tunnel test are low. owing to its superior a plw field in a twisted wind flow as shown in fig. is noticeably different from that in a conventional wind flow. the latter has a symmetrical distribution of wind speeds as demonstrated by several previous studies [ , , ] in contrary to asymmetrical distributions of wind speeds in a twisted wind. tse et al. [ , ] assumed that the asymmetric distribution of wind speeds is a result a series of flow modifications induced by (a) an oblique wind attack angle at the ground level and (b) the vertical variation in wind directions. the study of tse et al. [ ] provided a tentative explanation on the flow modifications observed. using the detailed three-dimensional wind field produced by the openfoam simulation, the present study proposes a more systematic and theoretically sound explanation for the deviation of the dflws zone, which is considered as the most important flow modification in the twisted flow. in this study, the dflws zone is defined as the area in the far-field downstream the building with the wind speeds smaller than % of the approaching wind speed at the pedestrian height. fig. shows the location of the dflws zone in the simulation cases of the building s in the cwp flow and the two twisted wind flows, twp and twp . the dflws zone is symmetrical about the building centerline in the cwp flow ( fig. (a) ) but deviates clockwise about the centerline in the twisted wind flows (fig. b and c) . the deviation is dissimilar in the twp flow and in the twp flow. the deviation angle (α), as proposed by tse et al. [ ] , is employed to quantitatively assess the deviation of the dflws zone. the angle α, as shown in fig. , lies between the building centerline and a line connecting the building center and the center of the dflws zone. in the study of tse et al. [ ] , the deviation of the dflws zone is postulated as the displacement of the impinging location on the ground behind the building. owing to the streamlines parallel to the building centerline, the cwp flow impinges the ground exactly on the building centerline and subsequently creates the symmetric dflws zone. with a vertical variation in wind directions, the streamlines of the twisted wind flow are no longer parallel to the building centerline even when the incident wind direction is° . the oblique streamlines in the twisted wind flow pass over the building and impinge on the ground at a location deviating from the building centerline. the displacement of the impinging location, which is assessed by α, is therefore determined by the yaw angles of the twisted wind profile at the pedestrian level and at the top of the building as illustrated in fig. . fig. shows the streamlines in the twp flow at the pedestrian level and at the top of the building. it is evident that the direction of the streamlines at the top of the building s , whose direction is shown by the first arrow appeared clockwise, is less deviated from the building centerline (shown as the black dashed line) than the direction of the approaching wind at the pedestrian level (show as the arrow with dashed extensions). the deviation of the dflws zone, indicated by α contained in fig. , is, on the other hand, in between the lines indicating the directions of the flow at the building top and at the pedestrian level. in fact, owing to the interaction of wind circulations on the vertical and horizontal planes, the deviation of the dflws zone is neither equal to the yaw angles at the pedestrian level nor the angle at the top of the building but has a value in between the two yaw angles. in order to further investigate the influence of the vertical variation in wind directions, the plw fields around the building model s in the cwp wind flow striking the building model at° is simulated and compared to the simulation case completed using twp . given that the two wind flows strike the building from the same direction at the ground level, the comparison emphasizes the influence of the vertical variation in wind directions. fig. shows the variation in the vertical wind velocity (w) along two sampling lines of = y (line ) and = x m . (line ). it should be noted that the computational domain is rotated by° in the cwp case before the simulation results are extracted along the sampling lines. the variation of w along the sampling line , as shown in fig. (a) has two peaks for the cwp flow: a narrow peak around = x m . and a broad peak in the range between = x m . and = x m . . these peaks correspond to the two strong vertical circulations that occur next to the leeward side of the building and in the dnlws zone, respectively. the twp case has a comparable peak near the leeward side of the building but the peak at the dflws zone is considerably lower than that observed for the cwp flow. the low peak of the twp case evidently indicates a weaker momentum exchange in the vertical direction compared to the cwp flow. the variations in w along the sampling line for the cwp and twp flows, as shown in fig. (b) , have a phase difference of about m . in the range between = − y m . and = y m . the phase difference is gradually reduced to m . when the y coordinate approaches and eventually diminishes towards the point of = y m . . the phase difference implies that the plw field in the twp flow striking at° is not in exact agreement with the plw field in the cwp flow striking the building at° . in fact, the phase difference presented in fig. (b) suggests weak vertical advections in the dflws zone of the twp flow. as a result, the properties of the dflws zone such as its wind speeds, area and the location are less affected by the flow properties in the twp flow at higher altitudes, and hence the deviation angle of the dflws zone in the twisted flow is closer to the wind attack angle at the ground level. fig. shows the top view of the streamlines passing the building in the cwp and twp flows. the most distinctive difference is the absence of the organized vertical eddies from the right part of the wake (red elliptical area) in the twisted flow. in fact, twp has sparse streamlines at the right side of the wake than in the cwp flow. consequently, the wind velocity in the twp flow, especially its component in the vertical direction, is not as large as in the cwp flow on the right side of the dflws zone. the small vertical velocities lead to weak vertical momentum exchanges, which likely affect the air pollution dispersion in the dflws zone. in contrast, the organized vertical eddies in the cwp flow are advantageous for removing pollution from the pedestrian level. a new set of inflow boundary conditions is proposed to sustain twisted wind profiles in the cfd simulation that focuses on evaluating the plw fields around isolated buildings. the new inflow boundary conditions are developed assuming the horizontal homogeneity of the wind flow for the standard k-ε turbulence model. the proposed inflow boundary conditions have satisfactory consistency and the accuracy for modeling the twisted wind profiles with the maximum yaw angles of° and° using an opensource cfd code, openfoam, and a commercial cfd code, fluent. the new boundary conditions are used to specify the inlet boundary conditions in a series of cfd simulations for assessing the plw field around three isolated buildings, a tall and slender building (h/ w = : ), a short and wide building (h/w = . : ), and an intermediate building (h/w = . : ). the results of openfoam and fluent simulations were used for the assessment of the reliability of cfd simulations on the plw fields around isolated buildings under twisted wind profiles, and investigating the driven flow mechanisms for flow modifications reported by tse et al. [ ] . based on the three-dimensional flow field data extracted cfd simulations, following conclusions are drawn: both openfoam and fluent can successfully replicate the plw fields around isolated buildings, except in the dflws zone, in twisted wind flows. both cfd codes show large differences in wind speeds in the dflws zone compared to those measured in the wind tunnel test. the differences may be related to the accuracy of measurements of wind speeds in the dflws zone from wind tunnel tests and/or cfd simulations. outside the dflws zone, openfoam has superior performance in replicating wind speed and its variation compared with fluent. particularly, openfoam successfully predicts the variations in wind speed in the longitudinal direction as in the wind tunnel experiment while fluent regularly overestimates the wind speeds. the three-dimensional flow field data of twisted wind profiles depict that the deviation of dflws zone is resulted from the combined effect of the vertical circulation that passes over the building and the horizontal circulation that wraps the building at the pedestrian level. the vertical circulation in a twisted wind is found to be weaker than that in the conventional wind. thus, the deviation of the dflws zone is more affected by the yaw angle near the ground in a twisted flow. the vertical wind velocity (w) in the dflws zone is found to be considerably smaller in the twisted flow than that in the conventional wind flow. in particular, the twp flow has sparse horizontal streamlines in the dflws zone, indicating the absence of the organized vertical eddies to the right side of the wake, which negatively affects the removal of air pollutions at the ground level. policies and technical guidelines for urban planning of high-density citiesair ventilation assessment (ava) of hong kong problems of wind flow at the base of tall buildings amplification of wind speed at ground level due to construction of high-rise building in urban area wind environment around buildings, her majesty's stationery office the ground level wind environment in built-up areas wind environment around buildings: aerodynamic concepts air ventilation impacts of the "wall effect" resulting from the alignment of high-rise buildings wind tunnel study of pedestrian level wind environment around tall buildings: effects of building dimensions, separation and podium improving the wind environment in highdensity cities by understanding urban morphology and surface roughness: a study in hong kong urban thermal environment measurements and numerical simulation for an actual complex urban area covering a large district heating and cooling system in summer simulation of twisted wind flows in a boundary layer wind tunnel for pedestrian-level wind tunnel tests terrain characterisation and design wind profiles for hong kong wind direction field under the influence of topography, part i: a descriptive model wind direction field under the influence of topography: part ii: cfd investigations pedestrian-level wind environment around isolated buildings under the influence of twisted wind flows effects of twisted wind flows on wind conditions in passages between buildings years of computational wind engineering: past, present and future application of computational fluid dynamics in building performance simulation for the outdoor environment: an overview simulation of wind flow around three-dimensional buildings computational evaluation of wind effects on buildings numerical simulation of flowfield around texas tech building by large eddy simulation wind climate and urban geometry cfd evaluation of wind speed conditions in passages between parallel buildings-effect of wall-function roughness modifications for the atmospheric boundary layer flow cfd modeling of pollution dispersion in building array: evaluation of turbulent scalar flux modeling in rans model using les results effect of urban morphology on wind condition in idealized city models city breathability and its link to pollutant concentration distribution within urban-like geometries cfd simulation of outdoor ventilation of generic urban configurations with different urban densities and equal and unequal street widths prediction of wind environment and thermal comfort at pedestrian level in urban area large eddy simulation of wind field and pollutant dispersion in downtown macao cfd simulation and validation of urban microclimate: a case study for bergpolder zuid consistent inflow boundary conditions for modelling the neutral equilibrium atmospheric boundary layer for the sst k-ω model appropriate boundary conditions for computational wind engineering models using the k-ε turbulence model new inflow boundary conditions for modelling the neutral equilibrium atmospheric boundary layer in computational wind engineering improved k-ε model and wall function formulation for the rans simulation of abl flows rans simulation of the atmospheric boundary layer over complex terrain with a consistent k-epsilon model rans simulation of neutral atmospheric boundary layer flows over complex terrain by proper imposition of boundary conditions and modification on the k-ε model computational modeling of the neutrally stratified atmospheric boundary layer flow using the standard k-ε turbulence model appropriate boundary conditions for a pressure driven boundary layer consistent boundary conditions for flows within the atmospheric boundary layer a comprehensive modelling approach for the neutral atmospheric boundary layer: consistent inflow conditions, wall function and turbulence model equilibrium atmospheric boundary-layer flow: computational fluid dynamics simulation with balanced forces the prediction of laminarization with a two-equation model of turbulence rans simulation of abl flow over complex terrains applying an enhanced k-ε model and wall function formulation: implementation and comparison for fluent and openfoam on the use of the k-ε model in commercial cfd software to model the neutral atmospheric boundary layer aij guidelines for practical applications of cfd to pedestrian wind environment around buildings a stable and accurate convective modelling procedure based on quadratic upstream interpolation a calculation procedure for heat, mass and momentum transfer in three-dimensional parabolic flows a computer prediction for chemically reacting flows in stirred tanks towards the ultimate conservative difference scheme, v. a second order sequel to godunov's method upstream monotonic interpolation for scalar transport with application to complex turbulent flows cooperative project for cfd prediction of pedestrian wind environment in the architectural institute of japan authors of this paper would like to show their gratitude to the staff of the clp power wind/wave tunnel facility at the hong kong university of science and technology for their support for conducting the wind tunnel experiments. the work was also supported by national natural science foundation of china (grant no. , , ). key: cord- - zs qqo authors: alsultan, musaed abdulaziz; alhammadi, mohamed ali; hemida, maged gomaa title: infectious bronchitis virus from chickens in al-hasa, saudi arabia - date: - - journal: vet world doi: . /vetworld. . - sha: doc_id: cord_uid: zs qqo aim: this study aimed to isolate some of the currently circulating infectious bronchitis virus (ibv) strains from some broiler chicken farms in al-hasa and to do some molecular characteristics of these strains. materials and methods: we collected tissue specimens, including the trachea, bronchi, lungs, and kidneys from some four commercial chicken farms showing respiratory manifestations. we tested these tissue specimens by the real-time polymerase chain reaction (rt-pcr) and gel-based pcr. we selected some pcr positive samples for isolation in the embryonated chicken eggs (ece). we sequenced some pcr-positive samples and conducted phylogenetic analysis based on the obtained sequences. results: our molecular surveillance revealed that . % of the tested specimens were ibv positive by pcr. we selected some positive specimens showing low ct values by the qrt-pcr for virus isolation by the ece. the infected eggs showed hemorrhage, dwarfing, and death in some cases after three passages in the ece. we sequenced some of the positive pcr specimens and used the obtained sequences to draw the phylogenetic tree based on the partial ibv-orf- a, n, and s gene sequences. the phylogenetic trees based on the ibv-n and s gene sequences showed that the circulating ibv strains in al-hasa during was showing a high degree of identity to some strains from taiwan and italy. meanwhile, the grouping of these strains based on the ibv-s sequences revealed that the currently circulating ibv strains in al-hasa belonged to gr.i. along with strains from taiwan. conclusion: our results confirmed the continuous circulation of the ibv among the chicken population in al-hasa despite the intensive application of vaccines against this virus. infectious bronchitis virus (ibv) is a highly contagious respiratory viral disease of chickens of all ages. the ibv belongs to the order nidovirales, family coronaviridae, and genus gammacoronavirus [ , ] . the ibv genome is a positive sense rna molecule with a high tendency to frequent changes and recombination. there is a high degree of genetic diversity among ibv strains. until recently, there was no available system for the definite grouping and identification of various ibv genotypes. one recent study used the combination between the phylogenetic analysis and the pairwise similarities on both the nucleotides and the amino acid levels to do fine mapping of the ibv genotypes [ ] . this study used the full-length s gene sequences to develop a novel classification system for the ibv genotypes and lineages [ ] . based on the comparison of the coding sequence of the s gene from ibv strains, they were able to categorize the currently circulating ibv strains into genotypes and lineages [ ] . the ibv infection usually causes high economic losses among the poultry industry. it is quite possible to become endemic in the chicken industry in some regions of the world. the virus has wide tissue tropism, including the respiratory, digestive, renal, and reproductive systems of the affected birds. it may affect the oviduct and lead to low production and low-quality eggs or may cause severe renal complications and mortality among the affected birds [ ] . the ibv-infected birds usually shed the virus in their body secretions such as respiratory and the gastrointestinal tract secretions. these birds may remain active shedders of the virus for up to several weeks post-infection [ ] . secondary bacterial infections (escherichia coli and mycoplasma gallisepticum) always exaggerate the viral pathogenesis and auscultate the mortality rates among the infected chicken population [ ] . the ibv infections continue to be a major problem to the poultry industry worldwide. in spite of the availability of several ibv vaccines, the virus continues to cause many outbreaks among the chicken farms in both broiler and layer settings [ ] . efforts to control spreading of the ibv infections through vaccination resulted in some variable outcomes. many ibv strains and serotypes have emerged since its discovery > years ago; meanwhile, the misuse of the ibv vaccines complicates the evolution and emergence of new ibv strains [ ] . one possible explanation for the emergence of new ibv strains is the poor proofreading capability of the viral rna polymerase. this resulted in high mutation rates alongside the viral genome. this leads to the emergence of new ibv strains occasionally [ , ] . many ibv vaccines are commercially available, including inactivated, live attenuated, and recombinant. the live-attenuated vaccines are the most commonly used in all types of poultry; these provide a good immune response; however, there is a possibility of revert to virulence. meanwhile, the inactivated vaccines are usually administered to layers and breeder's chickens before the laying time as booster vaccines [ , ] . the ibv infection in chicken was first reported in saudi arabia in in field samples by the realtime polymerase chain reaction (rt-pcr) using n primers [ ] . another study reported the circulation of the ibv/ / serotype in saudi arabia in using the partial s gene [ ] . several ibv genotypes were previously reported in saudi arabia, including ch/ck/ldl/ , is/ / , isvariant / , and the d [ ] . we recently reported the circulation of very virulent ibv strains in chicken farms from eastern saudi arabia [ ] . several ibv variants and genotypes are currently circulating in the middle east, asia, and north africa such as iraq, egypt, libya, iran, and jordan [ ] . furthermore, many outbreaks were reported in saudi arabia, especially in the central region of the country [ , , ] . however, the full molecular characterization of these ibv strains and variants is not well reported yet. this study aimed for isolation and molecular characterization of the ibv circulating strains of the ibv in al-hasa in the eastern region of saudi arabia. this region is one of the major hubs for intensive poultry production in saudi arabia. we conducted this study according to the king faisal's university animal ethics protocols and the national committee of bio-ethics, king abdul-aziz city of science and technology, royal decree no. m/ (http://www.kfsh.med.sa/kfsh_website/ usersuploadedfiles% cncbe% regulations% english.pdf). the animal ethics committee of the king faisal's university approved this protocol. we conducted molecular surveillance for ibv on some chicken farms across al-hasa from november to april . birds in these farms were suffering from acute respiratory signs and high morbidity and mortality rates. these outbreaks were mapped around three major cities in the al-hasa province (table- ), saudi arabia (al-hufuf, mahasen, and almubarez). tissue specimens (trachea, lung, and kidney) representing four suspected ibv outbreaks were collected. these samples were stored at − °c for further processing. briefly, g per each tissue specimens was ground in a sterile mortar mixed with ml of phosphate-buffered saline (pbs) and sterile sands. tissues were then centrifuged at rpm for min. the supernatants were harvested and stored at − °c until use. isolation of the currently circulating ibv strains from some poultry farms in al-hasa was carried out by the inoculation of the ece. we used - days' chicken embryos from native breeds (non-ibv vaccinated) and proved to be ibv-negative antibodies by elisa. the inoculum was prepared as follows; ibv-suspected tissue suspensions were centrifuged for min at rpm. antibiotic mixture was added (penicillin and streptomycin) to each tissue suspension. we used µl per each tissue suspension to inoculate through allantoic route into five embryonated eggs [ ] . the inoculated eggs were incubated at °c. negative control pbs-inoculated eggs were done in parallel to the experimental infection of the ece. we observed the inoculated eggs daily by candling for or up to days post-inoculation. any early deaths within h post-inoculations were excluded due to the non-specific trauma-related death [ ] . the total viral rnas were extracted from tissue using the qiaamp viral rna mini kit (qiagen cat. no. qiagen, germany] as per the manufacturer's recommendations. the eluted viral rnas were b=broiler, l=layer, rs=respiratory signs, lu=lung, t=trachea, k=kidney stored at − °c for further testing. the positive control (the commercial live ibv-h vaccine for ibv, veterinary vaccine production centre, ksa) was processed in parallel to each batch of specimens as described above. the rt-pcr was performed using the ibv rt commercial kits (subang jaya, selangor de, malaysia) (qpcr/rt-qpcr kits kestrel bioscience llc, doc. no. cat# av ). the reactions were conducted as per the kit's instructions with some minor modifications. briefly, each reaction consisted of ×rt-pcr master mix ( µl, ibv ppm µl, ppm iec µl, nuclease-free water µl, and µl of rna template) in a total reaction volume of µl. meanwhile, we used µl of nuclease-free water as negative control. furthermore, a positive control reaction was carried out in parallel to each run. duplicate reactions were done per each specimen. the cycling parameters were °c for min, then °c for min, followed by cycles at °c for s, and °c for min. we designed some primers to amplify the partial ibv-n gene. we designed the ibv-n primers based on the ibv-n (accession no: kt ). the nucleotide sequences of the used ibv-n gene are ibv-nf ( '-cgctggagaatttcctcttg- ') and the ibv-nr ( '-ctagtccctagcagccatgc- '). meanwhile, we used the following primers, ibv-s f ( '-accggctgatggatggcat- ') and the ibv-s ( '-ttgcttacaaccaccctgtag- '), to amplify the partial ibv-s gene. the rt-pcr reactions were conducted in a µl reaction volumes using the one-step rt-pcr kit (qiagen, valencia, ca no. ). the reactions were conducted as per the manufacturer's instructions. the reaction mixture consists of µl of ×reaction buffer, µl of each primer, µl of enzyme mixture, µl of dntp mixture ( µm of each dntp), µl of the rna template, and µl of rnase-free water. reverse transcription was started at °c for min; initial taq polymerase activation was performed at °c for min, along with cycles of denaturation at °c for min, annealing at °c for min, and then extensions at °c for and min. for visualization of the amplified pcr products, electrophoresis of µl of each pcr products on % agarose gel was performed. this was done under the ultraviolet light and photographed by the bio-rad gel documentation system. µl of per each pcr product was mixed with µl of the loading buffer (blue/orange loading dye, roche). a -bp, ready-to-load dnamarker ladder was loaded in a separate, parallel lane. we processed a confirmed pcr-ece-p -ibv isolate for sequencing by the illumina next-generation sequencing approach. we used this approach to decode the partial-length genome sequences of this ibv isolate as previously described [ ] . we constructed the phylogenetic tree based on the obtained partial ibv-orf -a, ibv-n, and s gene sequences. we constructed the tree using the multiple alignments of these sequences with other ibv sequences retrieved from the genbank done using the mega- package, and phylogenetic analysis was done by the maximum likelihood method using the best-fit model determination and at least bootstrap replicates as previously described [ ] . meanwhile, we used the mega- software to calculate the pairwise distances between our ibv-s sequence and the available sequences from the genbank. we applied the non-probability sampling strategy for our specimen collection with incidental assignment approach as previously described [ ] . the ibv infection in chickens produces a wide range of clinical syndromes. some birds showed respiratory manifestation in terms of coughing, sneezing, and nasal discharges. necropsy examination of some ibv-infected birds revealed serous, catarrhal, or caseous exudate in the tracheal, nasal passage, sinus, and bronchi congestion (figure- a) . the edema of the lungs, cloudy or fibrinous inflammation of the air sacs, occasionally contains a yellow caseous exudate. edema and swelling of the kidneys were observed in some birds (figure- b) . we conducted molecular surveillance to check the prevalence of ibv across the al-hasa from october to april . we investigated four ibv outbreaks across the al-hasa province by the rt-pcr (table- ). we tested chickens' specimens from non-vaccinated chickens using tissue specimens from the trachea, lungs, and kidneys by the rt-pcr technique. our overall results showed that pooled chickens' tissue specimens were positive of tested samples ( . %). simply, specimens from farm no: is showing that out of specimens ( %) were positive. in the case of farm no: , of ( . %) were ibv positive. however, three specimens of nine tested from one particular chicken farm were positive in case of farm no: . interestingly, only two specimens from poultry farm on the farm no: were positive of tested specimens ( . %) ( table- ) . we already tested different tissue specimens, including the trachea, lungs, and kidneys, from some suspected ibv-infected birds. our results are showing that tracheal tissue specimens of were positive, while lung specimens of were positive; meanwhile, only two specimens from the kidneys were positive (table- ). we processed specimens (trachea, lungs, and kidneys) after pooling from three ibv outbreaks. we did the propagation for three passages. the ibv isolation was successful only in case of two specimens. the first specimen was collected from an rt-pcr-ibvpositive chicken trachea collected from farm no. . the second specimen was collected from positive ibv-rt-pcr lungs collected from chicken farm no. area (table- ). however, isolation from the inoculated kidney tissues from farm no. was not successful (table- ). the ibv infection of the ece resulted in many pathological changes on the inoculated embryos such as dwarfing, congestion, hemorrhage, and death at different time points. no pathological changes, alteration, or death was reported in the negative control group of embryonated eggs in the three subsequent passages. we used three tissue suspensions (trachea, lungs, and kidneys) from three specimens representing three different ibv outbreaks in some chicken farms from al-hasa region. we followed up these three inoculums on the ece for three subsequent passages. the ibvinfected tracheal specimen caused death and induced some pathological changes in two embryos from the inoculated embryos in the three passages. however, one of them showed no pathological changes or death at the end of the experiment. meanwhile, the ibvinfected lung tissue specimens showed that only one embryo was affected per each passage. we used commercial available rt-pcr kits to confirm the identity of some suspected ibv-infected chicken tissues, as well as some fluids from the embryonated egg passages. we carried out the rt-pcr amplification for three representative specimens from the ece passages p- - . meanwhile, we t lu k - - - total ibv=infectious bronchitis virus already included six original specimens from the ibvsuspected outbreaks from different farms, including some pooled tissue specimens (trachea, lungs, and kidneys). furthermore, we already included the ibv vaccine as a positive control of the reaction in addition to the kits positive control in the reactions. in addition to a negative control used a non-dna template. our results show that / ( . %) of the three tested specimens representing embryonated eggs passages - were positive. three specimens from selected ibv specimens were positive, as shown in figure- . based on the reported ibv-orf a sequences, these strains were closely related to ibv-l strains (figure- ) . the phylogenetic tree based on the partial ibv-s genes revealed that these strains were closely related to other ibv strains from taiwan and italy (figure- ) . the evolutionary relationship analysis based on the ibv-s sequences confirmed the s -based analysis (figure- ) . the phylogenetic analysis based on the generated ibv-n gene sequences clearly showed that the circulating strains were closely related to the ibv strain reported from taiwan and china (figure- ) . both the pairwise distance (table- ) and the phylogenetic analysis based on the generated ibv-s sequences of our isolates confirmed the blast results ( figures- and ). the geographical distribution of the suspected outbreaks represents different regions across the al-hasa. some chicken farms were selected around the al-hasa region as targets for our study. some of these farms were reporting ibv outbreaks based on figure- : phylogenetic analysis based on the partial s gene for the circulating infectious bronchitis virus (ibv) strains from some chicken farms in al-hasa region - . phylogenetic tree of the obtained partial saudi ibv-s gene isolated from al-hasa region - . the maximum likelihood phylogenetic tree was based on the partial ibv-s gene. the bootstrap is . the reported local ibv sequences clustered together with other ibv strains from italy and taiwan. a red triangle marks the reported sequence in this study. the obvious clinical signs and post-mortem lesions, while birds in some others were apparently healthy. the clinical examination of the affected birds showed depression, ruffled feathers, loss of the body weight, respiratory rales, nasal discharge, and lachrymal discharge (data not shown). necropsy examination of some affected birds revealed congested trachea, caseous and bloody plugs at the tracheal bifurcations ( figure- a) , congested lungs, and swollen kidneys ( figure- b) in some cases. these clinical signs are very much typical of the ibv pattern of infection in poultry farms [ ] . we conducted molecular surveillance for ibv among some ibv chicken outbreaks in al-hasa region - . we selected some tissue specimens to be tested by the commercial ibv-rt pcr kits. our results are showing that six specimens of nine ( . %) were ibv positive (table- ). we also selected some of these positive ibv specimens for the propagation and isolation of the currently circulating strains by the ece. we assessed the success of the ibv isolation in two different ways. first, the inoculated embryos showed pathological changes relevant to the standard ibv inoculation in the ece. these changes were in the form of congestion, dwarfing, hemorrhage, and death of the inoculated embryos. second, we were able to detect the ibv signal by doing rt-pcr assays on the ece tissues and fluids from different egg passages (data not shown). furthermore, we confirmed the propagation of the ibv on the eces by another method using the truncated ibv-n primers through the rt-pr technique. we conducted an ibv molecular surveillance among some selected chicken farms in al-hasa region by the rt-pcr technique using the conserved ibv-n primers. we tested specimens from layers, and broiler non-vaccinated chicken's flocks represented four suspected ibv outbreaks across the al-hasa. these tissue specimens were collected from various organs of the affected birds such as the trachea, lungs, and kidneys. we did pooling five organs per each tube considered as one specimen. we found that / specimens ( . %) were positive (table- ). our results also showed that / ( %) of the tested trachea were positive, while / ( %) lungs were positive. however, / ( %) tested kidneys were ibv positive (table- ) . we used the generated sequences of three independent genes (orf a, s , and n) in the ibv genome to do the phylogenetic analysis of the circulating strains in al-hasa region during . we found great overlapping and consistency of the phylogenetic analysis based on the ibv-s and n genes. the reported sequences were closely related to other ibv strains reported in taiwan, italy, and china ( figures- - ). this high identity to foreign ibv strains may be due to that saudi arabia does import chickens from different countries including uk, china, italy, and taiwan. however, the ibv-orf a sequences revealed that these strains were related to other ibv strains belonging to the ibv-qx strain (l ) (figure- ) . it is well known that there is a great diversity of s gene among ibv strains, which make it a strong candidate for the ibv classification [ ] . furthermore, novel variations were reported among most of the ibv-n gene [ , ] . this is suggesting the potential use of some ibv-s and maybe ibv-n genes for the classification of different ibv strains as described [ ] . our results confirmed recent reports about the circulation of different ibv strains in the kingdom and the eastern region in specific [ , ] . this indicates a wide distribution of ibv in the al-hasa in the kingdom in general. in spite of the massive application of ibv vaccines in chicken farms under study, ibv is circulating, and thus, the currently used ibv vaccines under field conditions did not provide enough protection against the ibv infection. another challenge in the context of this vaccination failure is the possibility of circulating of novel ibv strains that do not share any antigenic relationship with the currently used vaccines, and thus, protection cannot be achieved. we believe that ibv will continue to hit several chicken farms in the region and many other parts in the world. further studies are highly recommended to do thorough molecular characterization of the currently circulating and the possible emerging ibv strains; thus, protection was usually achieved through the preparation of the homologous relevant specific ibv vaccines. ibv continues to pose a great risk to the poultry industry in the al-hasa, saudi arabia. there is continuous circulation of the ibv between the broiler and layer chicken in this area. the currently circulating strains of the ibv belong to the gr.i- along with other genotypes from taiwan, china, and italy. table- : pairwise distance analysis of partial s gene for the circulating ibv strains in some chicken farms in al-hasa region gi. /it/kj in vitro and in ovo expression of chicken gamma interferon by a defective rna of avian coronavirus infectious bronchitis virus infectious bronchitis viruses with a novel genomic organization s gene-based phylogeny of infectious bronchitis virus: an attempt to harmonize virus classification avian infectious bronchitis virus detection of infectious bronchitis virus serotypes by reverse transcription polymerase chain reaction in broiler chickens phylogenetic distribution and predominant genotype of the avian infectious bronchitis virus in china during emergence of avian infectious bronchitis virus and its variants need better diagnosis, prevention and control strategies: a global perspective genetic diversity of avian infectious bronchitis viruses in japan based on analysis of s glycoprotein gene molecular epidemiology and evolution of avian infectious bronchitis virus in spain over fourteen years molecular evolution and emergence of avian gammacoronaviruses vaccination against infectious bronchitis virus: a continuous challenge recombinant live attenuated avian coronavirus vaccines with deletions in the accessory genes ab and/or ab protect against infectious bronchitis in chickens rapid detection and identification of avian infectious bronchitis virus variation in the spike protein of the /b type of infectious bronchitis virus presence of infectious bronchitis virus strain ck/ ch/ldl/ i in the middle east molecular characterization and phylogenetic analyses of virulent infectious bronchitis viruses isolated from chickens in eastern saudi arabia genotyping and pathotyping of diversified strains of infectious bronchitis viruses circulating in egypt detection of variant infectious bronchitis viruses in broiler flocks in libya molecular characterization of infectious bronchitis viruses isolated from broiler and layer chicken farms in egypt during isolation and propagation of coronaviruses in embryonated eggs infectious bronchitis disease. in: oie manual of diagnostic tests and vaccines for terrestrial animals. world organisation for animal health genetic characterization of the belgian nephropathogenic infectious bronchitis virus (nibv) reference strain b mega : molecular evolutionary genetics analysis version . for bigger datasets numerical taxonomy freeman. the principles and practice of numerical classification. freeman, san francisco pathogenicity study of iranian genotype of avian infectious bronchitis virus (ir- ) complete sequences of ' end coding region for structural protein genes of turkey coronavirus novel variation in the n protein of avian infectious bronchitis virus genetic grouping for the isolates of avian infectious bronchitis virus in taiwan molecular survey and phylogenic analysis of infectious bronchitis virus (ibv) circulating among chicken flocks in riyadh province, saudi arabia mgh designed and performed the experiments, oversee the entire work, and performed data analysis and interpretation. muaa conducted the fieldwork, performed some laboratory experiments, and drafted the manuscript. moaa helped in the data analysis and reading of the manuscript. all authors read and approved the final version of the manuscript. the authors declare that they have no competing interests. veterinary world remains neutral with regard to jurisdictional claims in published institutional affiliation. key: cord- -ri dy e authors: more, gd; dunowska, m; acke, e; cave, nj title: a serological survey of canine respiratory coronavirus in new zealand date: - - journal: n z vet j doi: . / . . sha: doc_id: cord_uid: ri dy e aims: to determine the seroprevalence of canine respiratory coronavirus (crcov) in new zealand dogs, and to explore associations with age, sex, breed, month, and geographical region of sampling and reported presence of clinical signs suggestive of respiratory disease. methods: a total of , canine serum samples were randomly selected from submissions to a diagnostic laboratory between march and december , and were analysed for crcov antibodies using a competitive elisa. logistic regression analysis was used to determine associations between seroprevalence of crcov and breed category, age, sex, sampling month, region, and reported health status of dogs. results: overall, / , ( . %) samples were seropositive for crcov, with / ( . %) positive dogs in the north island and / ( %) in the south island. age of dog, sampling month, region, and presence of abnormal respiratory signs were included in the initial logistic regression model. seroprevalence was higher in dogs aged ≥ compared with ≤ years (p < . ). the lowest seroprevalence was observed in july ( / ; . %) and august ( / ; %), and the highest in june ( / ; %). seroprevalence in dogs from auckland was higher than in dogs from the hawkes bay, manawatu, marlborough, and waikato regions (p < . ). abnormal respiratory signs (coughing, nasal discharge, or sneezing) were reported for / , ( . %) dogs sampled. seroprevalence for crcov tended to be higher among dogs with respiratory signs ( . ( % ci = . – . )%) than dogs with no reported respiratory signs ( . ( % ci = . – . )%). conclusions: serological evidence of infection with crcov was present in more than half of the dogs tested from throughout new zealand. differences in crcov seroprevalence between regions and lack of seasonal pattern indicate that factors other than external temperatures may be important in the epidemiology of crcov in new zealand. clinical relevance: our data suggest that crcov should be included in investigations of cases of infectious canine tracheobronchitis, particularly if these occur among dogs vaccinated with current vaccines, which do not include crcov antigens. canine respiratory coronavirus (crcov) is a large enveloped rna virus that is classified within a species betacoronavirus in the family coronaviridae (erles et al. ) . the virus was first isolated from dogs with respiratory disease in a rehoming shelter in the united kingdom (erles et al. ) . on the day of admission to that shelter % of dogs were seropositive, and nearly all seronegative dogs seroconverted to crcov after a -week stay at the shelter, indicating high transmissibility of the virus. the presence of crcov antibody on entry to the shelter was associated with decreased risk of development of respiratory disease, suggesting an aetiological involvement of the virus in infectious canine tracheobronchitis (ict). results of several further studies in the united kingdom and other countries also suggested that crcov contributes to development of respiratory disease in infected dogs (erles and brownlie ; ellis et al. ; kaneshima et al. ) . however affected dogs were often coinfected with several respiratory pathogens, and not all dogs with serological evidence of recent crcov infection developed respiratory disease, making the establishment of an aetiological link between crcov infection and disease challenging (erles et al. ; erles and brownlie ) . this is similar to the situation observed for other respiratory pathogens of dogs, underscoring the fact that the aetiology of ict is complex and factors other than exposure to a specific pathogen are likely to contribute to the outcome of infection (erles and brownlie ; mitchell et al. ) . canine respiratory coronavirus is closely related to bovine coronavirus (bcov) and human coronavirus-oc , but distinct from canine enteric coronavirus (cecov) , which belongs to the genus alphacoronavirus, and is an aetiological agent of enteric disease in dogs (erles et al. ; decaro et al. ) . antibodies raised to crcov are not cross-reactive with cecov (decaro et al. ; priestnall et al. ) . similarly, vaccines against cecov do not elicit protection against crcov infection (erles and brownlie ) . a high percentage identity was found between the amino acid sequence of the spike protein of bcov and crcov (erles et al. ) , enabling the use of bcov antigens for the detection of crcov antibody (priestnall et al. ; decaro et al. ; soma et al. ) . there are limited data on crcov epidemiology in new zealand. based on a single cross-sectional survey of dogs, knesl et al. ( ) reported that ( %) dogs were seropositive for crcov. in another new zealand-based study, / ( %) dogs sampled had antibody to crcov (sowman et al. ) . some of the dogs affected by ict seroconverted to crcov between acute and convalescent sampling, suggesting that crcov infection was associated with the development of disease in those dogs. however there was a poor match between diseased and healthy dogs in terms of age, breed, and use, so no conclusions could be made regarding aetiological involvement of crcov in development of ict (sowman et al. ) . the aim of the present study was to investigate the epidemiology of crcov in a large sample of dogs in new zealand, to explore the associations between seroprevalence for crcov and age, sex, breed, month, and geographical region of sampling, as well as the reported presence of clinical signs suggestive of respiratory disease. a convenience sample of canine sera was obtained from a commercial veterinary laboratory (new zealand veterinary pathology ltd., palmerston north, nz) on a monthly basis. approximately serum samples, representing % of monthly laboratory submissions, were randomly selected every month from march to december . this was done by physically pulling samples out of a bag containing all monthly laboratory submissions and checking these against an excel database containing available data for each sample. only those samples with information on sex, age, the region of sampling, and clinical history were included in the study. samples having incomplete information and duplicate sera having the same label numbers were excluded. in addition, samples collected from racing greyhounds that were sent directly to our laboratory were also included in the study. four were from dogs with respiratory disease and from healthy dogs. dogs were categorised as healthy or sick based on the information provided on the submission form. healthy dogs included those presented for procedures such as pre-anaesthetic work-up or pre-mating progesterone concentrations, while sick dogs included those presented for a variety of infectious or non-infectious diseases. the serum samples were also categorised into those that came from dogs for which no abnormal respiratory signs were listed on the submission form, and those for which at least one of the clinical signs commonly associated with respiratory disease (coughing, sneezing, or nasal discharge) was listed. sera were stored at − °c until assayed. presence of crcov antibody in canine sera was determined using a commercially available competitive elisa with bcov antigen (bio k -monoscreen abelisa bovine coronavirus/competition, bio-x diagnostics, rochefort, belgium). the test was performed according to the manufacturer's instructions using provided positive and negative controls. the results were calculated based on the optical density at nm (od ) and presented as percentage of inhibition (poi) according to the formula: the manufacturer's recommended cut-offs for bcov antibody were used. accordingly, samples with poi ≥ % were considered positive for crcov antibody. based on the results of the elisa samples were categorised as seropositive or seronegative and this was the dependent variable for analyses. independent variables included the dog-related variables of age (≤ , - , - , ≥ years), sex (female/male), breed group (pet dogs, working dogs, non-descript dogs), health status (healthy/not healthy), and presence of respiratory signs (yes/no), as well as the samplingrelated variables of month of sampling (march to december ) and geographical region (auckland, hawkes bay, manawatu, marlborough/canterbury, nelson/tasman, northland, otago, taranaki, waikato, wellington). for some analyses, geographical regions were categorised as south island and north island. there were dog breeds which were categorised as working dogs (huntaway, heading dogs, and greyhounds), pet dogs which included all other recognised breeds, and non-descript dogs which included unspecified breeds. associations between seroprevalence and categorical variables were initially examined using a two-tailed pearson's χ or kruskal-wallis tests. those variables that were associated (p ≤ . ) were included in a multivariable model, which was refined by a stepwise backwards selection process retaining variables with p ≤ . . the mean poi for dogs of different age categories or health status were compared using one-way anova and tukey's test. all statistical analyses were performed using r statistical software v . . (r development core team, ; r foundation for statistical computing, vienna, austria) or graphpad prism (version . , graphpad software, san diego, ca, usa). samples were analysed from a total of , dogs. the median age of dogs was years (min week, max years), and there were similar numbers of male and female dogs ( table ). the origin of the samples by geographical region is shown in figure : / , ( . %) were submitted from the north island and / , ( . %) from the south island. overall, / , ( . %) samples were seropositive for crcov, with / ( . %) positive dogs in the north island and / ( %) in the south island. the seroprevalence of crcov among dogs classified as healthy ( / ; . ( % ci = . - . )%) was similar to that among dogs classified as diseased ( / ; . ( % ci = . - . )%, p = . ). univariate analyses revealed associations between samples seropositive for crcov and sampling month, age, and geographical region (p < . ). no statistical association was found between crcov seroprevalence and health status, breed, or gender of the dogs sampled (p > . ). four variables satisfied the criteria for inclusion in the initial multivariable model (p ≤ . ), namely age, sampling month, region, and presence of abnormal respiratory signs (table ) . age, sampling month, and region were retained in the final model (p ≤ . ). seroprevalence for crcov was higher in dogs aged ≥ compared with ≤ years (p < . ), but mean poi in seropositive dogs was lower in older than in younger dogs ( figure ). abnormal respiratory signs (coughing, nasal discharge, or sneezing) were reported for only / , ( . %) dogs sampled. seroprevalence for crcov tended to be higher among dogs with respiratory signs ( . ( % ci = . - . )%) than dogs with no reported respiratory signs ( . ( % ci = . - . )%). in addition, seropositive dogs with abnormal respiratory signs (n = ) tended to have higher mean poi ( . ( % ci = . - . )%) compared to all other seropositive dogs (n = ) ( . ( % ci = . - . )%, p = . ), but mean poi was similar to that of seropositive healthy dogs (n = ) ( . ( % ci = . - . )%, p = . ). the results of this study provided evidence that infection with crcov was common among dogs in new zealand. overall, % of the sera tested were positive for crcov antibodies. this was higher than the % reported previously in new zealand (knesl et al. ). both studies used sera submitted to the same diagnostic laboratory as the source of diagnostic material. however in the study samples were tested in comparison to , in the current study. hence, the current estimation of crcov seroprevalence may be more accurate than the previous one. the population sampled in the study was somewhat loosely defined as representing a wide geographic area encompassing the central and lower north island of new zealand. although samples in the current study originated from a wider geographical area, including the south island, the majority of samples were from the central and lower north island, so of similar distribution to the previous study. the difference in seroprevalence is also unlikely to be attributed to differences in the serological tests used, as the sensitivity and specificity of bcov-based competitive elisa for detection of crcov antibody was reported to be . % and . %, respectively, when compared to a fluorescent antibody test with crcov antigen that was used in the previous study (priestnall et al. ) . the timing of sample collection may have contributed to the differences observed between the two studies. unfortunately, the timing of sample collection was not provided by knesl et al. ( ) , but in the current study the lowest seroprevalences were observed in july ( %) and august ( %), which were similar to the % reported by knesl et al. ( ) . this may also be supported by the fact that % of dogs tested as part of another new zealand-based survey were seropositive for crcov (sowman et al. ) . the number of samples tested in that study was only , but, as in the current study, the samples were collected over a period of several months from july to august . finally, we cannot exclude the possibility that the seroprevalence of crcov has increased in new zealand since . as measured by a blocking elisa using bovine coronavirus as antigen, in serum samples from dogs that were seropositive for crcov and aged ≤ (n = ), - (n = ), - (n = ) or ≥ (n = ) years. the median is indicated by the middle line of each box, the mean by a cross, the th and th percentiles are indicated by the upper and lower edges of the boxes and minimum and maximum values are indicated by the whiskers. the significance of differences are indicated by * (p < . ) and ** (p < . ). the new zealand data appear to be similar to the seroprevalence of crcov reported from other countries, including . % in canada, . % in the united states of america, % in the united kingdom, and . % in the republic of ireland (priestnall et al. ) , as well % reported from a multicentre study that included dogs from six european countries sampled over a period of years (mitchell et al. ) . similar to our results, other studies found regional differences in crcov seroprevalence, as can be exemplified by differences between various regions in the united states of america and in the united kingdom. it has been suggested that the crcov seroprevalence may be higher in areas with a higher density of humans, and therefore presumably canine, populations (priestnall et al. ). however crcov seroprevalence was similar in some of the less densely populated regions of the south island and the more densely populated region of auckland. on the other hand, regional differences were apparent, as seroprevalence in dogs from auckland was higher in comparison to dogs from the hawkes bay, manawatu, marlborough, and waikato regions. one possible reason for these differences is sample size and associated selection bias. in addition, population density in most parts of new zealand, possibly with the exception of auckland, is not uniform. hence it may have been of interest to stratify the samples by the size of town/city, in addition to the geographical region. this was not performed as we did not have access to addresses of the submitters beyond the region classification. overall, our data suggest that the epidemiology of crcov in new zealand is similar to that observed overseas, particularly in europe and in the united states of america. the crcov seroprevalence varied between different months, but there was no clear seasonal pattern observed. the lowest crcov seroprevalences ( % and %) were detected in the winter months of july and august, but the highest seroprevalence ( %) was also observed in winter (june). this suggests that temporal differences in crcov seroprevalence were more likely to be related to factors other than external temperatures. these could include the increased contact between infected and non-infected dogs through activities such as training, competitions, short-or long-term kennelling or travel, but the exact nature of such interactions and their influence on the spread of crcov remain to be elucidated. similarly to results from other surveys (priestnall et al. (priestnall et al. , knesl et al. ), the seroprevalence of crcov was higher in dogs aged ≥ compared with ≤ years. this is most easily explained by the increased likelihood of exposure to the virus for older dogs, which had more opportunities for contact with infectious dogs or contaminated environments than younger dogs. however we did not see a relative decline in the percentage of seropositive dogs among those older than years, as reported by priestnall et al. ( priestnall et al. ( , . those authors suggested that this could be related to the age-related fall in the efficiency of the immune response. in the current study, mean poi was lowest in seropositive dogs > years of age, which may support this conclusion. as it is currently unknown how long crcov antibodies persist in dogs, the lower poi detected in older dogs may also represent residual antibody due to past exposure as opposed to recent infection. no statistically significant difference was observed between the seroprevalence of crcov in healthy and sick dogs, although seroprevalence tended to be higher in dogs with abnormal respiratory signs compared to those with no reported respiratory signs. while this is consistent with the overseas data (erles et al. ; soma et al. ) , the number of dogs with respiratory signs in the current study was low, and thus these dogs were poorly matched to either healthy dogs or dogs with clinical problems other than respiratory disease. in addition, some dogs categorised as having abnormal respiratory signs may have been included due to non-infectious causes, as the group allocation was made based on limited clinical data provided on the submission form. finally, the retrospective nature of the study, combined with the samples being sourced from a diagnostic laboratory, was likely to introduce a selection bias towards dogs with a variety of health problems compared with the general population. hence field studies using similarly sized, age-matched groups of dogs with and without respiratory disease would be needed to further investigate the impact of crcov infection on the health status of affected dogs. the sampled population contained different dog breeds making the analysis of breed associations with seroprevalence of crcov impractical. therefore breeds were categorised into broad use categories instead. this may have introduced some confounding as it is possible that some dogs of breeds that are typically used as farm working dogs may have been kept as pet dogs, while some dogs that were classified as pets based on their breeds may have been used as working dogs. none-the-less, there appeared to be no difference in seroprevalence of crcov among dogs from different use categories, which was consistent with results of an et al. ( ) who reported no difference in crcov seroprevalence between farm dogs and pet dogs. also consistent with overseas findings (erles and brownlie ; soma et al. ) was the lack of association between the sex of the dog and seroprevalence of crcov, indicating that sex-related activities or behaviours are unlikely to be associated with the likelihood of exposure to the virus. in conclusion, we have shown serological evidence that more than half of the dogs tested from throughout new zealand were infected with crcov at some point during their lives. further studies into the virus-host interactions and the impact of crcov infection on the health status of dogs under local new zealand conditions are warranted. the importance of crcov in ict remains to be elucidated. however, considering the apparent high seroprevalence of crcov in new zealand, this virus should be included in investigations of cases of ict, particularly if these occur among dogs vaccinated with current vaccines, which do not include crcov antigens. a serological survey of canine respiratory coronavirus and canine influenza virus in korean dogs serological and molecular evidence that canine respiratory coronavirus is circulating in italy detection of coronavirus in cases of tracheobronchitis in dogs: a retrospective study from to detection of a group coronavirus in dogs with canine infectious respiratory disease investigation into the causes of canine infectious respiratory disease: antibody responses to canine respiratory coronavirus and canine herpesvirus in two kennelled dog populations canine respiratory coronavirus: an emerging pathogen in the canine infectious respiratory disease complex the prevalence of a group coronavirus in dogs in japan the seroprevalence of canine respiratory coronavirus and canine influenza virus in dogs in new zealand european surveillance of emerging pathogens associated with canine infectious respiratory disease serological prevalence of canine respiratory coronavirus serological prevalence of canine respiratory coronavirus in southern italy and epidemiological relationship with canine enteric coronavirus prevalence of antibodies to canine respiratory coronavirus in some dog populations in japan a survey of canine respiratory pathogens in new zealand dogs sincere thanks to raewynne pearson and adrienne french for supplying the serum samples for elisa and to janis bridges for help with statistical analysis. this study was partly funded by the new zealand greyhound racing association. key: cord- -yzc bosn authors: llano, manuel; peña-hernandez, mario a. title: chapter seven defining pharmacological targets by analysis of virus–host protein interactions date: - - journal: advances in protein chemistry and structural biology doi: . /bs.apcsb. . . sha: doc_id: cord_uid: yzc bosn abstract viruses are obligate parasites that depend on cellular factors for replication. pharmacological inhibition of essential viral proteins, mostly enzymes, is an effective therapeutic alternative in the absence of effective vaccines. however, this strategy commonly encounters drug resistance mechanisms that allow these pathogens to evade control. due to the dependency on host factors for viral replication, pharmacological disruption of the host-pathogen protein–protein interactions (ppis) is an important therapeutic alternative to block viral replication. in this review we discuss salient aspects of ppis implicated in viral replication and advances in the development of small molecules that inhibit viral replication through antagonism of these interactions. chromatographic resolution of human cell extracts subsequently analyzed by quantitative tandem mass spectrometry resulted in the identification of , physical interactions established by individual proteins. computational analysis of the physical interactions led to the mapping of complexes, suggesting an average of proteins per complex (havugimana et al., ) . these estimates are also supported by other analysis indicating that most of the mammalian complexes are formed by the association of three or four different proteins. importantly, some host proteins are found in more than one complex at a time (wong et al., ) . similarly, a study in human cells combining large-scale immunoprecipitation of bait proteins followed by high-throughput mass spectrometry analysis led to the discovery of interactions between unique proteins, suggesting an average of proteins per complex (ewing et al., ) . protein complexes are stable assemblies that carry out most of the biochemical activities in the cell. corum, a public database of mammalian protein complexes, compiles complexes reported in nonhighthroughput, physical interaction experiments. of the proteins annotated in corum, % belong to one protein complex. fig. represents the frequency of annotated complexes in the corum database. interestingly, protein complexes implicated in subcellular localization processes are notoriously overrepresented in this database, whereas others implicated in energy production, protein synthesis, and tissue differentiation are underrepresented. in these collection of experimentally characterized protein complexes, approximately % of all predicted human open reading frames are organized in protein complexes (ruepp et al., ) . however, this number is expected to be only a minor fraction of the human complexome considering that in yeast % of the encoded proteins reside in complexes (ruepp et al., ) . hypothesis-driven experiments conducted at small scale have notably contributed to our understanding of protein-protein interactions (ppis) implicated in viral replication. in addition, high-throughput technologies analyzing ppis, gene loss of function, or transcriptomic profiles have been utilized to define cellular complexes implicated in different steps of the viral life cycle. large-scale physical analysis of ppi is conducted by yeast two-hybrid (y h) screens (calderwood et al., ; de chassey et al., ; fossum et al., ; lee et al., ; rajagopala, casjens, & uetz, ; shapira et al., ; trigg et al., ; uetz et al., ) and tandem affinity purification followed by mass spectrometry (tap-ms) (germain et al., ; pichlmair et al., ; ramage et al., ; rozenblatt-rosen et al., ) . y h screens can analyze only binary interactions, whereas tap-ms allows for the identification of more complex interactors. then, the physical interactions identified with these methods are subjected to organization in functional networks by computational approaches. despite of their rate of success in ppis identification, both methodologies interrogating physical interactions of proteins fail to detect multiple types of ppis. for example, interactions involving transmembrane proteins, or transient/ weak interactions escape detection. similar to the physical methods, computational systems biology approaches successfully predict exploitation of biological processes by viruses (kitano, a (kitano, , b navratil, de chassey, combe, & lotteau, ; zak, tam, & aderem, ) . these methods use gene expression and gene loss-of-function systemic analysis to identify potential host factor networks implicated in viral replication (hirsch, ) . the main problem that the high-throughput functional approaches discussed earlier has is the poor data overlap resulting from these analyses. this is even though these methods have a high degree of similarity or complementary. therefore, validation of the discovered ppis is required (rozenblatt-rosen et al., ; shapira et al., ) . this validation involves the demonstration of the interaction by coimmunoprecipitation analysis, particularly during infection. prioritization analysis of ppis can be implemented by combining the findings from the direct interaction approaches with comprehensive gene loss-of-function screenings. an example of the effectiveness of this multifactorial approach for the identification of ppis is a study aimed to identify host-influenza ppis (shapira et al., ) . in this study, % of the genes initially found to be involved in viral protein interactions were demonstrated to influence viral replication by subsequent rnai screening. this high rate of positive validation may have been the result of the selection of the final candidate genes by a combined analysis of the results obtained in y h screens using viral and host proteins (direct interactors), in transcriptional profiling experiments to define genes regulated by viral infection, and in in silico predictions of genes implicated in the protein networks found in the first two experimental approaches. comparison of the cellular complexes exploited by different pathogens indicates an significant overlap, leading to the definition of cellular processes implicated in infection (brander & walker, ; davis et al., ; dyer, murali, & sobral, ; hirsch, ; hiscott, nguyen, arguello, nakhaei, & paz, ; pichlmair et al., ; rozenblatt-rosen et al., ; shapira et al., ) . therefore, shared cellular complexes could constitute broad-spectrum therapeutic targets potentially impairing more than one pathogen. an example of this strategy is discussed later when we consider the implication of protein translation in the mechanism of replication of multiple viruses. the overlap in the utilization of cellular complexes between different pathogens also brings support to the hypothesis that the innate immune system has evolved the ability of detecting patterns of pathogenicity. these patterns are generated by the activation of similar biological processes by different pathogens, indicating the existence of pathogen-induced processes (dyer et al., ; pichlmair et al., ; vance, isberg, & portnoy, ) . the viral manipulation of the activity of type i interferon (ifn)-stimulated genes (isg) illustrates this concept. protein kinase, rna-activated (pkr) is an isg that upon activation phosphorylates the α-subunit of the translation initiation factor eif- , inhibiting protein synthesis. viral dsrna is the main pkr activator in virus-infected cells, whereas the cellular protein pact (protein activator of pkr), that heterodimerizes with pkr, is an important activator of the kinase in stressed, noninfected cells in the absence of dsrna (li et al., ; patel & sen, ) . hiv- evades the robust type i ifn response mounted by plasmacytoid dendritic cells in infected individuals by rewiring the pact/pkr complex. in infected cells, hiv- tar nucleates a complex with the viral protein tat and the cellular proteins pact and adar (adenosine deaminase acting on rna ). in this complex, adar inhibits pact pkr activatory function by direct interaction with pact (clerzius et al., ) . another example of pathogeninduced host complex rewiring will be discussed later in relation to the role of hiv- accessory proteins in the targeting of the ubiquitin/proteasome system to degrade different restriction factors. importantly (chukwurah, handy, & patel, ) . therefore, the pact-adar -pkr axis is a cellular pathway commonly manipulated by different viruses that could constitute a therapeutic target with broad antiviral activity or pathogen-induced processes detected by the innate immune system. the hijacking of similar cellular pathways by different viruses also allows for the identification of broader biomarkers of viral infection. generally viral pathogens target cell cycle regulation, nuclear transport, and immune response processes (dyer et al., ; shapira et al., ) . ssrna(À) viral proteins tend to interact with processes implicated in the protection of rna from degradation and rna processing, whereas dsrna viruses are more connected to protein degradation processes. dna viruses, however, target proteins connecting the cell cycle with other processes such as chromosomal and transcriptional homeostasis (pichlmair et al., ; shapira et al., ). rnai-based screens have also been useful in identifying host factors implicated in viral replication. interestingly, the number of host factors identified for the different viruses analyzed in these screens outnumber by a factor of the proteins encoded by these viruses (hirsch, ) . this disproportion could illustrate the multifunctional character of viral proteins. in this case viral proteins are expected to establish multiple independent binary interactions with independent host proteins. alternatively, viral proteins could interact with host proteins organized in complexes. therefore, rnai-mediated downregulation of the subunits of these protein complexes will produce similar phenotypes. correspondingly, y h screenings have found that viral proteins establish a disproportionate number of binary interactions with the human proteome. these numbers of interactions registered for viral proteins exceed the predicted number of interactions estimated from the analysis of the human interaction network (shapira et al., ) . for example, assessment of the interaction of each of the viral proteins encoded by influenza with , human orf through y h screening found interactions with human proteins (shapira et al., ) . these evidences support a model indicating that viral proteins evolve multiple direct interactions. furthermore, computational analysis of host factors implicated in these pairwise interactions indicated that the host proteins occupy central positions, hub proteins, within the cellular interactome (shapira et al., ) . this higher than expected connectivity suggests that the direct interactions of viral proteins with host factors allow the access of the virus to cellular complexes. computational analysis of the interaction of human proteins binding to different viral proteins encoded by different viruses showed that approximately % ae . % of the unique targeted human proteins interact at least with one other human protein (dyer et al., ) . these data indicate that the majority of the host factors implicated in viral replication are in complexes. these conclusions are further supported by tap-ms and functional genomic studies. tap-ms studies of the virus-host interactome indicate that viral proteins tend to interact with proteins that have multiple interacting partners and participate in more cellular pathways (protein hubs), and also with proteins that are central to many pathways and, therefore, occupy a more central position within these networks (protein bottlenecks) (dyer et al., ; pichlmair et al., ; shapira et al., ) . for example, in a tap-ms experiment were mapped complex associations between viral proteins from different viruses and host proteins (rozenblatt-rosen et al., ) , highlighting the high degree of connectivity of the interacting proteins. as discussed above, some of the host factors predicted, by the combined transcriptional profiling and in silico analyses, to interact with host proteins implicated in direct binary contacts with influenza proteins (y h interactors) were demonstrated to influence viral replication in functional screenings (shapira et al., ). these findings demonstrate that influenza, and potentially other viruses, hijacks cellular networks by combining physical and regulatory (transcriptional level) interactions with these pathways. this multilayer regulation offers further alternatives of blocking physical or regulatory interactions aiming to impair viral replication. interestingly some of the host factors found in the networks of protein interacting with viral proteins are induced at the transcriptional level by infection in a type i ifnindependent manner (shapira et al., ). this indicates that viruses modulate, at the transcriptional levels, the abundance of proteins that will engage in physical binary or multiprotein complex interactions. therefore, computational analysis of the pathways discovered by functional genomics could lead to the identification of protein networks implicated in physical interactions with viral proteins. protein complexes established by viruses seem to determine the disease associated to the infection. for example, analysis of the interactome of e proteins from hpv types differing in their oncogenic potential associates with different subset of host proteins (rozenblatt-rosen et al., ) . therefore, disruption of these complexes could also prevent viral pathogenesis. almost % of the human-pathogen ppis reported belong to hiv- (dyer et al., ; fahey et al., ; ruepp et al., ) . generally host factors interacting with viral proteins tend to preserve the host protein interactions mapped in noninfected cells (rozenblatt-rosen et al., ; yu et al., ) , indicating that the normal host protein complexes rather than new virus-induced protein complexes are implicated in viral replication. this opens the opportunity to utilize the current body of knowledge on the human interactome to define the host complex hijacked by viruses. however, examples of virus-specific complex variants also occur. among them, one of the best characterized is the complex between hiv- vif and the transcriptional and protein degradation cellular machineries. vif removes apobec family restriction factors by targeting the ubiquitin ligase complex cul to this restriction factor, and inducing its proteasomemediated degradation. other hiv- proteins, vpu and vpr, also exploit cullin-ring e ligase to degrade other restriction factors. vif directly interacts with the substrate, the transcription factor cbf-β, the n-terminus of cul (cullin-ring e ligase), and the heterodimeric substrate adaptor elob/eloc. in turn, the c-terminus of cul binds rbx and recruits an e ubiquitin conjugation enzyme that mediates the transfer of polyubiquitin to the substrate proteins (jager, kim, et al., ; zhang, du, evans, yu, & yu, ) . cbf-β is required in this complex for the assembly of the vif-cul e -ubiquitin-ligase complex, but not for the binding of vif to the substrate . cbf-β normally heterodimerizes with the runx family of transcription factors preventing its degradation and vif competes with runx for binding to cbf-β (guo et al., ) . the surface area buried at the interface of the interaction of vif and cbf-β is large ($ a ) suggesting that is undruggable (salter, morales, & smith, ) , as we will discuss later for other surfaces of ppis. however, alanine scanning indicated that aa - of vif are a hot spot (defined in a later section of this review) in the interaction with cbf-β. furthermore, residues phe and ile of cbf-β establish important interactions with trp in vif through hydrophobic interactions (desimmie, smith, matsuo, hu, & pathak, ) , highlighting further opportunities for disruption of this complex with small molecules. therefore, these structural characteristics of the vif-cbf-β surface of interaction do not exclude its amenability to small-molecule interference, as we will show later for similar ppis. interfaces of ppi were considered undruggable for sometime, mainly because these surfaces of interactions are bigger than classical druggable protein surfaces, such as allosteric and catalytic sites in enzymes. the total area buried by the interactors in the binding site in ppis is in average (ae ) a , with some proteins extending to - a . in contrast, surfaces of interaction between proteins and small molecules are approximately from to a (arkin, tang, & wells, ; lo conte, chothia, & janin, ) . in addition, the surface of protein-protein interaction, although very variable in characteristics, was generally considered large patches of segmented complementary surfaces lacking small grooves or pockets (hopkins & groom, ; jones & thornton, lo conte et al., ) . furthermore, attempts to find small molecules interfering with ppis showed a very high failure rate ($ %) (brown & superti-furga, ) , therefore reinforcing the concept that surface of ppis is not druggable. in correlation with this, estimates using very stringent concepts for drugs indicate that only approximately % of the human proteome could be targeted by oral, drug-like small molecule. these predictions also indicate that of the druggable gene products only between % and % were implicated in diseases (hopkins & groom, ) . our understanding of the rules driving the ppis importantly changed with the discovery that the different residues buried in the large surfaces of interactions contribute differentially to the binding strength of the interacting proteins (clackson & wells, ) . this characteristic reduced considerably the area required to be targeted by small molecules. interrogation of the surfaces of binding in ppis by mutagenesis of individual residues (alanine scanning) indicated that mutation of a few residues implicated in these binding surfaces was sufficient to disrupt the ppis. residues importantly contributing to the binding free energy were defined as hot spots (clackson & wells, ) . these residues tend to cluster in tightly packed regions in the center of the surfaces of interactions and are called hot regions (clackson & wells, ; keskin, ma, & nussinov, ) . hot spots can be amenable to drug targeting due to their small surface area and important contribution to the total binding energy of the complex. however, not always hot spots are vulnerable to drugs, as additional topological constraints for drug binding limit the number of druggable hot spots (zerbe, hall, vajda, whitty, & kozakov, ) . in addition, in silico analysis indicated that interfaces could contain more than one hot region, and in some ppis the establishment of the complex depends on the cooperative interaction of different hot spots within the region (keskin et al., ) . by , more than ppis have been reported to be targeted by small molecules (morelli, bourgeas, & roche, ) . twenty-seven proteinprotein complexes were included in the release of the small-molecule orthosteric modulators of ppis database p idb. in this site are listed only those ppis in which there is structural information of the protein-protein and protein-inhibitor complexes (basse, betzi, morelli, & roche, ) . the majority of the most successful ppis inhibitors target hot-spot residues that cluster in small binding pockets ( - a ), and the surface of interaction of the binding proteins has short primary sequences (arkin et al., ) . structural analysis of protein-inhibitor complexes indicated that in some cases small molecules interrupting ppis bind to cavities not present in the surface of interaction of the protein-protein complex or in the interactor proteins when they are in isolation. these findings indicate some degree of adaptability in the surfaces of interaction initially considered lacking of druggable groves or pockets. according to in silico simulations the opening of some of these binding pockets is transient (wells & mcclendon, ) . most of the small molecules interfering with ppis bind directly to the implicated surfaces of interactions (orthosteric modulators) by targeting hot spot residues or by molecular mimicry of elements of secondary structures (arkin et al., ; basse et al., ; fry, ; wells & mcclendon, ; yin & hamilton, ) . however, several successful examples of small molecules disrupting ppis act through their binding to allosteric sites (crump et al., ; mcmillan et al., ; oswald et al., ; roche et al., ; szilagyi, nussinov, & csermely, ) . a well-characterized model of small-molecule disruption of a host ppi, deposited in the p idb database, is the mdm /p interaction. mdm binds to p , impairing its transcriptional activity and stability. the interaction surfaces are formed by a hydrophobic pocket (aa - ) in the n-terminus of mdm that is occupied by the hydrophobic side of an amphipathic α-helix in p (aa - ) . mutations at any of the four residues within the mdm -binding pocket or of three residues within the p α-helix block this ppi (moll & petrenko, ) . small molecules interrupting this interaction bind to the p -binding pocket in mdm (vassilev et al., ) . most of the data on small molecules targeting ppis in the p idb database correspond to host-host ppis and only two are relevant to viruses. that is the case of the interaction between the hpv proteins e and e , and of allosteric inhibitors of hiv- integrase activity (allinis) (engelman, kessl, & kvaratskhelia, ) . hpv e and e bind cooperatively to the viral origin of dna replication and are required for initiation of dna replication (berg & stenlund, ) . indandione derivatives that bind to the e transactivation domain block this interaction. one of these small molecules was shown to contact of the residues implicated in this interaction. allinis bind to the dimer interface of integrase to the same residues of the host protein ledgf/p (engelman et al., ) . ledgf/p is a chromatin-bound protein required for efficient hiv- cdna integration (llano et al., ; shun et al., ) , an essential step in the viral life cycle. ledgf/p binds to the dimer interface of the catalytic core domain of integrase. two residues in the integrase-binding domain of ledgf/p are essential in this interaction. asp in an interhelical loop of the host protein contacts, via hydrogen bonds, with residues glu and his in one integrase monomer, whereas ledgf/p residue ile interacts with a hydrophobic pocket (leu , ala , trp ) in the other integrase subunit (cherepanov, ambrosio, rahman, ellenberger, & engelman, ; cherepanov, sun, et al., ) . allinis bind to the viral integrase at the ledgf/p -binding site, preventing the binding of ledgf/p and affecting integrase catalytic core domain dimerization. this disrupts integrase assembly with viral dna and allosterically inhibits its activity (engelman et al., ) . -(quinolin- -yl) acetic acid derivatives (ledgins), and in particular compound , showed potent inhibitory activity of hiv- replication (christ et al., ) . this compound establishes hydrogen bonds with integrase residues glu , his , and thr that are hot spots in the ledgf/p -binding site in integrase (christ et al., ) . as allinis were more potent against hiv- in cells lacking ledgf/p , the contribution of ledgf/ p -binding inhibition to their mechanism of action is not clear (wang et al., ) . another successful strategy in the design of small molecules interfering viral replication has been the targeting of multimeric viral rna polymerases. influenza virus and vaccinia virus encode rna polymerases formed by the essential interaction of several subunits. in influenza virus each of the three viral subunits of the polymerase, pb , pb , and pa, is required to assemble in a complex for the polymerase activity. therefore, small molecules that interrupt the binding of pb into a hydrophobic groove in the c-terminus of pa (he et al., ) block replication of influenza a and b viruses (muratore et al., ) . similarly, vaccinia virus depends for replication on the formation of a trimeric complex between the dna polymerase e , the uracil dna glycosylase d , and the viral protein a (stanitsa, arps, & traktman, ) . small molecules disrupting this complex by impairing the interaction d -a inhibit replication of vaccinia virus and cowpox virus (schormann et al., ) . there are two types of physical interactions that can be targeted to interrupt the utilization of cellular complexes by the virus: the virus-host interface or the host-host interface of complexes hijacked by the virus. targeting protein interaction surfaces containing viral proteins is always associated with the selection of mutant viruses that escape control of the inhibitor (de clercq, ; strasfeld & chou, ) . this is the result of the highly mutagenic rate of viruses. however, targeting host complexes utilized by viruses at the host-host interface is expected to do not exhibit mechanisms of resistance, since host proteins are genetically more stable than their viral counterparts. therefore, disruption of the host complexes exploited by viruses rather than interference of the virus-host protein interface is an attractive alternative to avoid the selection of escape mutants resistant to the inhibitors. a major disadvantage of this strategy is the potential toxicity associated to the disruption of cellular complexes. potentially, this could be minimized if the interruption of the complex is transient. viral replication is a fast process that occurs in a few hours, and disruption of these complexes for a few hours could greatly affect the ability of the virus to replicate without affecting cellular viability. we will later discuss experimental findings supporting this view. in addition, the high degree of functional overlap in the human proteome, that is absent in viruses because of their relative smaller genomes, could also ameliorate the toxicity caused by the disruption of host-host ppis implicated in viral replication. the differences in functional redundancy suggest that the disruption of cellular complex will be more tolerable for the host than the pathogen. in support of this view, shrna-based studies have showed that many host factors required for viral replication do not carry essential cellular functions. for example, out of , transcripts permanently targeted with lentivirus-encoded shrnas, % were dispensable for the cell and deficient stable cell lines were developed but a fraction of them were required for efficient hiv- replication (yeung, houzet, yedavalli, & jeang, ). finally, targeting host complexes relevant for different viruses could allow the development of broad-spectrum antivirals. protein translation is essential for the virus and the cell. however, transient interference with protein synthesis at the initiation step has been demonstrated to be more detrimental for the pathogen than the host at a cellular level. both cellular mrna and mrna from different viruses carry a -methylguanosine cap (cap) on their terminal nucleotide. therefore, cap-dependent cellular translation is exploited by many different viruses. critical to this translation mechanism is the eukaryotic initiation factor f complex (eif f). this complex is required for the efficient recruitment of ribosomes to capped mrnas. the eif f complex includes a capbinding protein (eif e), an rna helicase (eif a), and a large scaffolding subunit (eif g). eif e protein binds to cap-mrna and then recruits eif g that binds eif a. therefore, inhibitors of eif e-eif g interaction (i.e., e rcat ) or the helicase activity (hippuristanol) drastically impair translation. e rcat completely blocks the replication of the human alphacoronavirus e (cencic, desforges, et al., ) or murine norovirus (royall et al., ) at doses that impair approximately % of the cellular protein synthesis (cencic, desforges, et al., ) or do not alter at all cell viability. at these doses e rcat affects eif e-eif g interaction as determined by coimmunoprecipitation (royall et al., ) . these results indicate that coronaviruses are more dependable on eif f for ribosome recruitment mrnas than the host. hippuristanol is a natural product that inhibits the activity of eif a by binding to its c-terminal domain. this compound impairs eif a rnabinding, atpase, and helicase activities. hippuristanol specifically impaired replication of poliovirus , caliciviruses (chaudhry et al., ) , human cytomegalovirus (lenarcic, ziehr, de leon, mitchell, & moorman, ) , and junin virus (linero, thomas, boccaccio, & scolaro, ) . despite the cell toxicity of hippuristanol at the doses and length of the treatment used in these reports, viral replication was affected specifically since this compound did not alter cellular viability. for example, -h treatment of raw . cells with hippuristanol reduced cell viability by %, but murine norovirus production was reduced by over -fold (chaudhry et al., ) . as described earlier, eif e interacts with eif g and assembles into the eif f complex together with eif a on the capped mrna. eif f complex formation leads to eif e phosphorylation at ser by the eif g-associated kinase mnk . inhibition of mnk by cgp specifically impairs protein synthesis in a variety of large dna viruses (walsh, mathews, & mohr, ) . for example, this compound reduced by -fold the replication of hsv- in quiescent primary fibroblasts without affecting cell viability (walsh & mohr, ) . in addition, cgp inhibits reactivation of kaposi's sarcoma-associated herpesvirus (walsh et al., ) . because mnk is not essential for cell growth or development, it is a potential therapeutic target (ueda, watanabe-fukunaga, fukuyama, nagata, & fukunaga, ) . there are other examples of small molecules inhibiting ppis in host complex that are not essential for the cell. some compounds targeting host-virus ppis implicated in the earlier steps of the hiv- life cycle are in this category. for example, the hiv- entry inhibitor maraviroc, that reached clinical use, acts through an allosteric mechanism. this drug binds to a hydrophobic pocket located in the transmembrane domain of ccr , altering the conformation of the extracellular domain of ccr and preventing in this manner the binding of env . in addition, the small molecule pf , that inhibits hiv- replication by triggering early uncoating (shi, zhou, shah, aiken, & whitby, ) , binds to a pocket encompassing the n-terminal domain-c-terminal domain interface of ca in the assembled capsid (bhattacharya et al., ) . cleavage and polyadenylation-specific factor (cpsf ) also binds to the same pocket in the capsid during hiv trafficking to the nucleus. this ppi delays uncoating, allowing hiv- to evade the innate immune activation through cytoplasmic nucleic acid sensor signaling (rasaiyaah et al., ) . we have presented literature evidences indicating that viruses interact for replication with cellular complexes. within these complexes, viral proteins bind to cellular proteins that are central (bottleneck proteins) in the interacting networks and establish multiple interactions in the human interactome (hub proteins). these ppis offer different opportunities for the development of small molecules to block viral replication. virusvirus, virus-host, and host-host ppis are amenable for pharmacological disruption. different viruses utilize similar host protein complexes during infection, showing overlap in the cellular processes hijacked. targeting these shared cellular processes opens the possibility to generate broad-spectrum antivirals and general biomarkers of viral infection. potentially, the innate immune system recognizes these shared pathogen-induced processes to counteract infection. a better understanding of this mechanism could greatly impact the development of stronger vaccine adjuvants. small-molecule inhibitors of protein-protein interactions: progressing toward the reality p idb v : update of a structural database dedicated to orthosteric modulation of protein-protein interactions functional interactions between papillomavirus e and e proteins structural basis of hiv- capsid recognition by pf and cpsf functional characterization of ireses by an inhibitor of the rna helicase eif a modulation of host immune responses by clinically relevant human dna and rna viruses rediscovering the sweet spot in drug discovery epstein-barr virus and virus human protein interaction maps blocking eif e-eif g interaction as a strategy to impair coronavirus replication reversing chemoresistance by small molecule inhibition of the translation initiation complex eif f caliciviruses differ in their functional requirements for eif f components structural basis for the recognition between hiv- integrase and transcriptional coactivator p solution structure of the hiv- integrase-binding domain in ledgf/p rational design of small-molecule inhibitors of the ledgf/p -integrase interaction and hiv replication adar and pact contribute to efficient translation of transcripts containing hiv- trans-activating response (tar) element a hot spot of binding energy in a hormone-receptor interface the pkr activator, pact, becomes a pkr inhibitor during hiv- replication structure of an allosteric inhibitor of lfa- bound to the i-domain studied by crystallography, nmr, and calorimetry global mapping of herpesvirus-host protein complexes reveals a transcription strategy for late genes hepatitis c virus infection protein network the design of drugs for hiv and hcv identification of a tripartite interaction between the n-terminus of hiv- vif and cbfbeta that is critical for vif function the landscape of human proteins interacting with viruses and other pathogens allosteric inhibition of hiv- integrase activity large-scale mapping of human protein-protein interactions by mass spectrometry gps-prot: a web-based visualization platform for integrating host-pathogen interaction data evolutionarily conserved herpesviral protein interaction networks protein-protein interactions as targets for small molecule drug discovery elucidating novel hepatitis c virus-host interactions using combined mass spectrometry and functional genomics approaches structural basis for hijacking cbf-beta and cul e ligase complex by hiv- vif a census of human soluble protein complexes crystal structure of the polymerase pa(c)-pb (n) complex from an avian influenza h n virus the use of rnai-based screens to identify host proteins involved in viral replication manipulation of the nuclear factor-kappab pathway and the innate immune response by viruses the druggable genome global landscape of hiv-human protein complexes purification and characterization of hiv-human protein complexes vif hijacks cbf-beta to degrade apobec g and promote hiv- infection principles of protein-protein interactions analysis of protein-protein interaction sites using surface patches hot regions in protein-protein interactions: the organization and contribution of structurally conserved hot spot residues computational systems biology systems biology: a brief overview an integrated approach to elucidate the intra-viral and viral-cellular protein interaction networks of a gamma-herpesvirus differential role for host translation factors in host and viral protein synthesis during human cytomegalovirus infection molecular basis for pkr activation by pact or dsrna junin virus infection impairs stress-granule formation in vero cells treated with arsenite via inhibition of eif alpha phosphorylation an essential role for ledgf/p in hiv integration the atomic structure of protein-protein recognition sites allosteric inhibitors of inducible nitric oxide synthase dimerization discovered via combinatorial chemistry the mdm -p interaction chemical and structural lessons from recent successes in protein-protein interaction inhibition ( p i) small molecule inhibitors of influenza a and b viruses that act by disrupting subunit interactions of the viral polymerase when the human viral infectome and diseasome networks collide: towards a systems biology platform for the aetiology of human diseases intracellular allosteric antagonism of the ccr receptor pact, a protein activator of the interferon-induced protein kinase viral immune modulators perturb the human molecular network by common and unique strategies the protein interaction map of bacteriophage lambda a combined proteomics/genomics approach links hepatitis c virus infection with nonsense-mediated mrna decay hiv- evades innate immune recognition through specific cofactor recruitment molecular gymnastics: mechanisms of hiv- resistance to ccr antagonists and impact on virus phenotypes murine norovirus (mnv ) replication induces translational control of the host by regulating eif e activity during infection interpreting cancer genomes using systematic host network perturbations by tumour virus proteins corum: the comprehensive resource of mammalian protein complexes- structural insights for hiv- therapeutic strategies targeting vif identification of protein-protein interaction inhibitors targeting vaccinia virus processivity factor for development of antiviral agents a physical and regulatory map of host-influenza interactions reveals pathways in h n infection small-molecule inhibition of human immunodeficiency virus type infection by virus capsid destabilization ledgf/p functions downstream from preintegration complex formation to effect gene-specific hiv- integration vaccinia virus uracil dna glycosylase interacts with the a protein to form a heterodimeric processivity factor for the viral dna polymerase antiviral drug resistance: mechanisms and clinical implications allo-network drugs: extension of the allosteric drug concept to protein-protein interaction and signaling networks cry h-seq: a massively multiplexed assay for deep-coverage interactome mapping mnk and mnk are essential for constitutive and inducible phosphorylation of eukaryotic initiation factor e but not for cell growth or development herpesviral protein networks and their interaction with the human proteome patterns of pathogenesis: discrimination of pathogenic and nonpathogenic microbes by the innate immune system in vivo activation of the p pathway by small-molecule antagonists of mdm tinkering with translation: protein synthesis in virus-infected cells phosphorylation of eif e by mnk- enhances hsv- translation and replication in quiescent cells hrp determines the efficiency and specificity of hiv- integration in ledgf/p knockout cells but does not contribute to the antiviral activity of a potent ledgf/p -binding site integrase inhibitor reaching for high-hanging fruit in drug discovery at protein-protein interfaces an evolutionary and structural characterization of mammalian protein complex organization a genome-wide short hairpin rna screening of jurkat t-cells for human proteins contributing to productive hiv- replication strategies for targeting protein-protein interactions with synthetic agents nextgeneration sequencing to generate interactome datasets systems-level analysis of innate immunity relationship between hot spot residues and ligand binding hot spots in protein-protein interfaces t-cell differentiation factor cbf-beta regulates hiv- vif-mediated evasion of host restriction we thank zachary s. martinez and angelica lopez, university of texas at el paso, for reviewing the manuscript. key: cord- -z vmhopa authors: ji, wei; niu, ling; peng, weiyu; zhang, yongli; cheng, hao; gao, feng; shi, yi; qi, jianxun; gao, george f.; liu, william j. title: salt bridge-forming residues positioned over viral peptides presented by mhc class i impacts t-cell recognition in a binding-dependent manner date: - - journal: mol immunol doi: . /j.molimm. . . sha: doc_id: cord_uid: z vmhopa the viral peptides presentation by major histocompatibility complex class i (mhc i) molecules play a pivotal role in t-cell recognition and the subsequent virus clearance. this process is delicately adjusted by the variant residues of mhc i, especially the residues in the peptide binding groove (pbg). in a series of mhc i molecules, a salt bridge is formed above the n-terminus of the peptides. however, the potential impact of the salt bridge on peptide binding and t-cell receptor (tcr) recognition of mhc i, as well as the corresponding molecular basis, are still largely unknown. herein, we determined the structures of hla-b* and h- k(d) in which two different types of salt bridges (arg -glu or arg -glu ) across the pbg were observed. although the two salt bridges led to different conformation shifts of both the mhc i α helix and the peptides, binding of the peptides with the salt bridge residues was relatively conserved. furthermore, through a series of in vitro and in vivo investigations, we found that mhc i mutations that disrupt the salt bridge alleviate peptide binding and can weaken the tcr recognition of mhc i-peptide complexes. our study may provide key references for understanding mhc i-restricted peptide recognition by t-cells. the presentation of viral peptides to host t lymphocytes is crucial for adaptive cellular immunity to clear viruses during the infection. in the structures of mhc i presenting these short peptides, the α and α domains of mhc i form a bed-like groove to comfortably accommodate the peptides: two α-helices as the bedrails and the β-sheets beneath as the mattress (bjorkman et al., ; niu et al., ) . the short peptides interact with the amino acids within the peptide binding groove (pbg) through different binding modes. first, the featured residues at the second position (p ) from the n-terminus and the last residue (pΩ) at the c-terminus of the peptides, termed primary anchor residues, protrude into the specific pockets in the pbg of the mhc i. though the positions of the primary anchors on the peptides are relatively conserved, the binding modes of the anchoring residues of the peptides to different mhc i molecules are diversified due to polymorphisms in the pocket-forming residues. second, the peptides also interact with the pbg via contacts between the backbone atoms of the peptides and the pbg residues (mitaksov and fremont, ) . third, a hydrogen bond network is formed between the two termini of the peptides and the conserved residues in the pbg. the latter two interaction modes are relatively conserved between different mhc i molecules (fremont et al., ; madden et al., ) . generally, mhc i binding peptides are located in the bottom of a ringent pbg, which leaves a solvent-exposed top surface of the entire peptide from the n-to the c-terminus, ready for the recognition of the t-cell receptors (tcr) (bjorkman et al., ) . however, based on the structures of a series of mhc i molecules, such as human hla-b* (madden et al., ) , rhesus macaque mamu-a* , and mouse h- k d (mitaksov and fremont, ; zhou et al., ) , there is a salt bridge positioned over the peptides formed by opposite charged residues from the α and α helices of mhc i, respectively. in humans and macaques, the salt bridge is formed by the positively charged arg on the α helix and the negatively charged glu on the α helix. meanwhile, in mice, the involved residues are arg on α helix and glu on α helix. both of these types of salt bridges overhang the n-terminal portion of the t-cell epitopes in the groove. obviously, the salt bridge forms a ligature to tightly fix the peptides into the pbg. however, thus far, it is still largely unknown how the formation of these salt bridges in mhc i molecules impacts peptide binding. further, considering that these salt bridges are also solvent-exposed, they are in the position to be recognized by tcrs. generally, t-cell activation by the mhc-peptide complex may occur in three correlated ways: ) direct interaction of the tcr with the mhc, termed mhc restriction; ) direct interaction of the tcr with the solvent-accessible peptide main chain and side chains; and ) interaction of the peptide with the tcr mediated by conformational perturbations in the mhcpeptide complex (baker et al., ; gras et al., ) . crystallographic studies clearly demonstrate that the polymorphic amino acids in the grooves of mhc i molecules from different mammals may influence the peptide conformation, subsequently impacting t-cell recognition (borbulevych et al., ; hulsmeyer et al., ; insaidoo et al., ) . nevertheless, to our knowledge, it has not been determined if the salt bridges in some mhc i molecules impact tcr recognition through a direct interaction with the tcr or indirectly through modulation of the peptide conformation. herein, by determining the crystal structures of human mhc i hla-b* complexed with a severe acute respiratory syndrome coronavirus (sars-cov) nucleocapsid (n)-derived t-cell epitope (oh et al., ) and mouse mhc i h- k d bound to an immunodominant t-cell epitope from human hepatitis b virus (hbv) core antigen (hbc) (li et al., ) , we clearly demonstrated the molecular features of mhc i molecules with two different salt bridges formed by the residues pairs arg -glu and arg -glu , respectively. we further investigated the impacts of the salt bridges on peptide binding and t-cell recognition by constructing a series of mhc i mutants in vitro and in vivo. our results elucidated the key features of mammalian mhc i molecules with salt bridges in the pbg and will benefit t-cell based diagnosis and vaccine development. the human hbv hbc protein-derived peptide hbc - (syvnt-nmgl) (li et al., ) and sars-cov n protein-derived peptide n - (getalallll) (oh et al., ) were synthesized with % purity by reverse-phase high performance liquid chromatography (scilight biotechnology, beijing, china). the peptides were stored at − °c as freeze-dried powders and were dissolved in dimethyl sulfoxide before use. the expression plasmid for mouse mhc i h- k d was constructed previously in our lab (zhou et al., ) . we also constructed three h- k d salt bridge mutants (r a, e a, and r a&e a) and three control mutants (s a, r a, a g) based on the wt plasmid (genewiz). for the hla-b* expression construct, the gene (genbank q ) was synthesized and ligated into the pet a vector (shanghai generay). the three hla-b* salt bridge mutants (r a, e a, r a&e a) and three control mutants (i a, r a, a g) were also constructed on the basis of the wt construct. six-to eight-week-old female balb/c mice were purchased from vital river laboratory animal technology company (beijing, china) and raised under specific pathogen-free conditions. all experiments were performed in strict compliance with the guide for the care and use of laboratory animals of the people's republic of china and approved by the committee on the ethics of animal experiments of national institute for viral disease control and prevention, chinese center for disease control and prevention. mice in the experimental group were subcutaneously injected at multiple sites with hbv peptide hbc - and the n-terminal fragment n ( - aa) of murine gp emulsified in complete freund's adjuvant for the first dose. mice were administered the mixture of peptide, gp fragment, and incomplete freund's adjuvant twice in -week intervals for the next two doses. mice were sacrificed to harvest splenocytes on day after the final immunization. mice in the control group were immunized with a mixture of phosphate buffered saline (pbs), gp fragment, and freund's adjuvant. antigen-specific t lymphocyte responses were detected with an ifnγ-secreting elispot assay by using splenocytes as previously described (tan et al., ) . briefly, -well plates were coated with μl/well of mg/ml anti-mouse ifn-γ antibody (bd pharmingen, san diego, ca) overnight at °c. after washing with pbs twice, the plates were blocked with culture medium for h at room temperature. mouse spleens were ground and filtered through cell strainers. cells were processed with red blood cell lysis buffer ( . m nh cl and μm tris, ph . ). we washed cells with pbs twice and centrifuged them to harvest lymphocytes. fresh mouse splenocytes ( . × ) in μl roswell park memorial institute (rpmi) medium with % fetal bovine serum (fbs) were seeded in each well. then, the peptides ( μg/ml) were added to the wells and incubated at °c in % co for h. phytohaemagglutinin (pha) was added as the positive control for nonspecific stimulation. cells incubated with medium alone were employed as a negative control that produced less than five spots in % of the experiments. finally, the cells were removed, and the plates were processed according to the manufacturer's instructions (bd). the colored spots, which represent epitope-specific t-cells, were counted and analyzed using an automatic elispot reader (ctl corp.). murine mhc class i h- k d and β m or human mhc class i hla-b* heavy chain and human β m were overexpressed in escherichia coli as inclusion bodies and subsequently refolded in vitro in the presence of a high concentration of peptide, as described previously (xiao et al., ) . briefly, the dissolved mhc i heavy chain and β m inclusion body and peptides were diluted at a molar ratio of : : , respectively, in refolding buffer ( mm tris−hcl, mm l-arginine, mm edta-na, mm glutathione [gsh], and . mm l-glutathione oxidized [gssg]). after h of slow stirring at °c, the mhc i/peptide complex was then concentrated and purified via superdex / g l (ge healthcare) chromatography. wt h- k d and the three h- k d mutant-restricted tetramers of hbvpeptide were prepared as previously described (liu et al., a; zhang et al., ; zhou et al., ) . briefly, recombinant h- k d /peptide complexes were purified and then biotinylated by incubation with dbiotin, atp, and the biotin protein ligase bira (avidity) at °c for h. the biotinylated h- k d was further purified by gel filtration to remove free biotin, and then the multimers were produced by using pe-streptavidin (sigma). cells from the subjects were stained with pe-tetramer and fitc-conjugated anti-cd antibody. all samples were analyzed with a facscalibur flow cytometer (bd biosciences) after staining. the conditions for the protein purification and the crystal growth of the complex formed by h- k d with hbv peptide hbc - was described previously (zhou et al., ) . human β m was used to generate the h- k d /hbc - complex. the renatured hla-b* /n - complex was further purified by resource q anion-exchange chromatography before crystallization. crystallization was performed using the hanging drop vapor diffusion technique. the concentration of protein was mg/ml, and the crystals grew in . m sodium citrate tribasic dehydrate (ph . ) and % (w/v) polyethylene glycol at °c. for cryoprotection, crystals were transferred to reservoir solutions containing % glycerol, flash-cooled, and maintained at k in a stream. x-ray diffraction data were collected at k at the ssrf beamline bl u (shanghai, china) at a wavelength of . Å ( table ). the structure of h- k d /hbc - and hla-b* /n - were resolved through the molecular replacement method using the mhc i structures with pdb codes fg and f m, respectively (liu et al., b) as the model in the crystallography and nmr system (cns) program. detailed model building was performed by hand using coot, and restrained refinement was performed using refmac . the stereochemical quality of the final model was assessed with the program procheck. structure-related figures were processed by pymol. to evaluate the thermostability of different mhc i complexes and also their mutants, we used cd spectroscopy as previously described (n. zhang et al., ) . we repeated at least three times for each protein. all complexes were prepared as described above and diluted to . mg/ ml in mm tris−hcl (ph . ) and mm nacl. thermal denaturation curves were determined by monitoring the cd value at nm using a -mm optical path-length cell as the temperature was raised from to °c at a rate of °c/min. the temperature of the sample solution was directly measured with a thermistor. the fraction of unfolded protein was calculated from the mean residue ellipticity (θ) by the standard method. the unfolded fraction (%) is expressed as (θ -θ n ) / (θ u -θ n ), where θ n and θ u are the mean residue ellipticity values in the fully folded and fully unfolded states, respectively. the midpoint transition temperature (t m ) was determined by fitting the data to the denaturation curves using the origin . program (originlab). for comparisons between multiple groups, two-way anova with bonferroni post-tests was performed. one-way anova analysis with bonferroni post-tests was used for comparison between multiple columns. statistical significance of differences between two columns was determined by student's t-test all tests were two-tailed with a significance level of . . all data analyses were performed with graphpad prism. the coordinates and structure factors of sars-cov peptide n - complexed to hla-b* and hbv peptide hbc - complexed to h- k d have been deposited in the pdb under accession numbers iex and vgk, respectively. the retrievement of the mhc i protein sequences available in the ipd-mhc database (https://www.ebi.ac.uk/ipd/mhc/) showed that %- % of the mhc i alleles available possess the paired residues for the potential salt bridge formation ( table ). the type salt bridge is formed by r on the α -helix and e on the α -helix, such as in h- k d , while the type salt bridge is constructed by r and e on the α -and α -helices, respectively, such as in hla-b* . moreover, we retrieved the allele frequency of mhc i molecules which are confirmed to possess salt bridge from the crystal structures in . the allele h- k d with type salt bridge is also common in mouse mhc alleles. thus, the impacts of the salt bridge formation overhead the pbg on the peptide binding and recognition by tcr would be a substantial phenomenon for different vertebrates, which has emerged in fish. to determine the potential role of the mhc i salt bridge in peptide binding, we utilized the peptide hbc - to facilitate the in vitro renaturation of h- k d and its salt bridge mutants h- kd-m (mutant r a), h- kd-m (mutant e a), and h- kd-m (mutant r a& e a) followed by size exclusion chromatography (gel filtration) analyses. the control mutants h- kd-c (mutant r a), h- kd-c (mutant a g), h- kd-c (mutant s a), which preserve the salt bridge were proceeded the same experiment as control mutants. compared to wild type (wt) of h- k d , all the three salt bridge mutants generated relatively lower yields of refolded products at the size expected for an mhc i monomer. however, the control mutants showed similar yields of renatured products as wt h- k d (fig. a and table s ). the binding stabilities of the peptide hbc - with h- k d and all the mutants were further analyzed by circular dichroism (cd) spectroscopy (fig. b) , with the t m s determined from melting curves. wt h- k d complexed with the hbc - peptide was stable, with a t m of . ± . °c. as expected, the h- k d salt bridge mutants h- kd-m , h- kd-m , and h- kd-m displayed significantly decreased stability with lower t m s ( . ± . °c for h- kd-m , . ± . °c for h- kd-m , and . ± . °c for h- kd-m ). moreover, the t m s of the control mutants have no statistically differences with wt h- k d ( . ± . °c for h- kd-c , . ± . °c for h- kd-c , . ± . °c for h- kd-c ). we also determined the binding capacity of the sars-cov-derived peptide n - with the human mhc i hla-b* , and its salt bridge mutants b -m (mutant r a), b -m (mutant e a), and b -m (mutant r a&e a) and its control mutants b -c (mutant a g), b -c (mutant r a), b -c (mutant i a). we found that three salt bridge mutants b -m ( . ± . °c), b -m ( . ± . °c), and b -m ( . ± . °c) displayed significantly lower binding capacity for the peptide compared to wt hla-b* ( . ± . °c) ( fig. c and d ), while the control mutants showed the identical binding capacity with the peptides as wt hla-b* ( fig. c and d) . thus, the salt bridge formation in both pbgs of h- k d and hla-b* contributed to the binding of the peptides, though breaking the salt bridge did not abolish peptide binding. to further investigate the role of the mhc i salt bridge above the pbg in t-cell recognition, we established a mouse model via three rounds of fortnightly in vivo injection of the hbv-derived peptide hbc - . elispot assays were then performed using freshly isolated splenocytes to assess the t-cell immune responses induced by the peptide. no specific reactivity of ifn-γ secretion could be detected in splenocytes separated from placebo-immunized mice (< sfcs/ splenocytes). in contrast, cd + t-cells from splenocytes of the mice immunized with peptide hbc - presented strong ifn-γ production ( fig. a) . the splenocytes from the peptide-inoculated mice were also stained with the h- k d tetramers prepared in the presence of peptide hbc - . the splenocytes from hbc - -inoculated mice contained . ± . % of peptide-specific cd + t-cells ( fig. b and c) . no peptide-specific cd + t-cells were identified from splenocytes of mice inoculated with placebo, as judged by hbc - tetramer staining. we also constructed tetramers based on a series of mutated h- k d mhc i heavy chains, including h- kd-m , h- kd-m , and the dual mutant h- kd-m . when compared to the wt tetramer, the three mutant tetramers stained a lower proportion of hbc - -specific cd + t-cells within splenocytes from mice inoculated with peptide hbc - ( . ± . %, . ± . %, and . ± . %, respectively). these results indicated that mutations of the residues forming the salt bridge positioned over the peptide may impact specific-t-cell recognition. though both h- k d and hla-b* contain the conserved negatively charged residue e , the structures of these two mhc i molecules clearly display two different types of salt bridges over the n-terminus of their complexed peptides (fig. ) . compared to h- k d , the salt bridge of hla-b* is located closer to the n-terminus of the pbg, and an angle of˜ °can be observed between the positions of the two salt bridges. this is due to the different positively charged residues in the salt bridges of the two mhc i molecules. however, in the structures of some mhc i available online thus far (e.g., h- l d , h- d b , and h- d d ), though they possess the same salt bridge-forming residues (r and e ) as hla-b* , no salt bridge is formed (pdb codes: ld , ce , bii) (supplemental fig. s ). the "broken bridge" is also found in several hla-b* structures (e.g. pbd codes: bst and jge) (supplemental fig. s ). particularly, h- d d possesses both r and r in its α helix, but no salt bridge is formed the balb/c mice were immunized with hbc - peptide (peptide group) or pbs (adjuvant group) together with adjuvants. elispot assays were performed using freshly isolated mouse splenocytes. the non-specific stimulant pha was used as a positive control, and mock indicates the negative control without any stimulant. (b) peptide-specific cd + t-cells stained by tetramers of h- k d and mutants. the hbc - peptide-specific cd + t-cells in the freshly isolated splenocytes from vaccinated mice were stained by h- k d tetramer, kd-m (mutant r a) tetramer, kd-m (mutant e a) tetramer, and kd-m (mutant r a&e a)tetramer, respectively. the hollow dots represent the peptide-immunized group, and the black dots represent the adjuvant group. the control was staining with unrelated tetramer (hla-a /influenza peptide gl ). error bars represent means ± sd. n = mice for per peptide group, n = mice for per adjuvant group. (c) the representative peptide-specific cd + t-cells stained by tetramers of h- k d and mutants. the statistical analysis between two groups used two-way anova with bonferroni post-tests. ** p < . , *** p < . . between e and either of these positively charged residues. next, we analyzed the hydrogen bonds within the two residues forming the salt bridge and the adjacent residues of both mhc i and the peptides (fig. ) . three different interactions contribute to the formation of the salt bridges: ) the electrostatic interaction between r or r in the α helix and the e in the α helix. the hydrogen bonds constructed between residues r and e in h- k d and between r and e in hla-b* are . Å and . Å, respectively. ) r or r in the salt bridge can form a hydrogen bond with the carbonyl group of the p residue of the bound peptide in the pbg (as in both the h- k d and hla-b* structures, and through a water molecule in mamu-a* ). ) for the mhc i-binding peptides with a hydrophilic residue at the p position, hydrogen bonds can be found between e and the side chains of the p residue of the peptide (as in the h- k d structure but not hla-b* ) (fig. ) . in hla-b * and hla-b* , a water molecule links the interaction between r and e (fig. ) . further analysis indicated that this water molecule is conserved in a series of hla-b* structures ( uxs, a , bsr, b s, bp , of , uxw, and w w) (supplemental fig. s ) . we further analyzed the structures of h- l d , h- d b , and h- d d , which contain the same residues for salt bridge forming (r and e ) as hla-b* but do not contain salt bridges. in the mhc i h- l d , h- d b , and h- d d structures, r is pulled away by e on the α helix, and r or k forms hydrogen bonds with e of the α helix. thus, no salt bridge is formed in these three mhc i structures despite the fact that they contain the corresponding residues (supplemental fig. s ). to investigate the potential influence of salt bridge formation on the structural conformation of the mhc/peptide complex, we superimposed the structures of the mhc i molecules with the two different salt bridges: r -e (h- k d , mamu-b* , and mamu-b* ) and r -e (hla-b* , mamu-a* , rt -a c, hla-b* , hla-b* , and hla-b* ) (fig. a) . compared to the structure of mhc i with the r -e salt bridge, the α -helix of mhc i molecules with the r -e salt bridge has an conformational shift (approximately . Å between h- k d and hla-b* ), using the α -helix of mhc i as the benchmark during the alignment (fig. b) . the peptides in the structures of mhc i complexes with r -e salt bridges also display conformational differences for the two residues at the p and p positions compared to the peptides under the r -e salt bridges (fig. c ). the cα of the p residues in the h- k d -binding peptides are closer to the c-terminus of the pbg and located lower and closer to the bottom of the groove (fig. d and e ). the r -e salt bridge acts as a "seatbelt" for the peptide, pushing both the conformational shift of the peptides and the α -helices. herein, we determined the crystal structures of hla-b* and h- k d molecules with two representative different salt bridges spanning the presented peptides. compared to wt, the peptide binding capacity of the salt bridge mutants was significantly lower. meanwhile, the control mutants showed no statistical difference. though the two salt bridges had distinct impacts on the conformational changes of the mhc i structures, both enhanced peptide binding and potentially contributed to tcr recognition. though the mhc i salt bridge-breaking mutants still bound to the peptides, their binding capacity were lower than wt. the impact on the thermostability of hla-b* by the mutants was not as much as that observed in the h- k d mutants. this is may be due to the intrinsic features of different salt bridges in the two molecules. using structural analysis, we found that the enhancement effect of peptide binding to mhc i was not only contributed by the direct formation of the salt bridge above the peptide main chain but also contributed by the conserved hydrogen bonds between the salt bridge-forming residues with the atoms on the main chain and the p residues of the peptides. a previous study indicates that a salt bridge (also called an ion pair in the study) formed by argα and aspβ in mhc class ii heterodimers is also critical for surface expression and peptide presentation (nalefski et al., ) . however, unlike the salt bridge overhanging the mhc i peptide, this argα -aspβ salt bridge is located under the main chain . the interactions between the two residues forming the salt bridge and the adjacent residues of both mhc i and the peptides. the interactions between r /r and e that form the salt bridge are denoted as red arrows. the cyan arrows represent the hydrogen bond between e and the side chains of the p residues of the peptides. the dark blue arrow marks the hydrogen bond between r or r and the carbonyl group of the p residue of the bound peptide in the pbg. in hla-b* , hla-b* and mamu-a* , the cyan dots represent water molecules. the black dashed lines in the structures represent the hydrogen bonds. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). of the mhc ii-presented peptide and may contribute to p binding and the c-terminal hanging conformation of the mhc ii-bound peptides. the salt bridge above the peptide both enhances mhc i-peptide binding and contributes to tcr recognition of mhc i-peptide complexes. birkinshaw et al. studied that a prototypical autoreactive bk tcr bound cd a when the presented ligands were permissive. tcr docked over the a' roof of cd a and a central cd a salt-bridge network (arg -glu salt-bridge) prevented the interaction between bk tcr and permissive ligands, and its autoreactivity was determined by contacts with cd a. nonpermissive ligands disrupted salt bridge interaction of cd a, disrupt the properties of a' roof and could hinder engagement of hydrophobic cdr β loop of the bk tcr with cd a (birkinshaw et al., ) . nurzia et al. indicate that the positive charging of arg is preferred for the tcr recognition of ankylosing spondylitis-associated b* complexes, as revealed by the r k and r a mutants with an overall reduced capability to present peptides to cd + t-cells (nurzia et al., ) . our data also demonstrate that tcell recognition was significantly alleviated when we broke the mhc i salt bridge with r a and e a mutations. previously determined complex structure between hla-b* : and kfj tcr showed that both the r and e of hla-b* : can form hydrogen bonds with the residues in the cdr α and cdr α loops of the tcr, respectively (chan et al., ) . this indicated that the residues in the salt bridge can be directly-recognized by the tcr (tynan et al., ) . meanwhile, our analysis also demonstrated that the salt bridge can lead to a conformational shift of the presented peptide and the relative locations of the α or α helices, which may subsequently and indirectly impact tcr recognition. further structural studies are required to investigate the accurate and detailed recognition of mhc i/peptide complex containing the salt bridge by the tcr. nevertheless, our current study still has limitations. the mutant mhc i tetramers used in this study including the h- k d mutants at r and/or e a can dismantle the salt breakage and then may indirectly alter t cell response. however, these residues can also be recognized by tcr, thus, the mutation of these residues may also directly influence the tcr response. we cannot exclude the possibility that the altered t cell response was due to altered contacts with the mutated residues, rather than the loss of the salt bridge interaction. in summary, our study indicates that the two different salt bridges in mammalian mhc i molecules, represented by hla-b* and h- k d , respectively, have different structural characteristics. and the mutations of the salt bridge-forming residues may impact both peptide binding and tcr recognition, directly or indirectly. our data is helpful for the understanding of the cd + t-cell recognition of mhc i-presented peptides. structural and dynamic control of t-cell receptor specificity, cross-reactivity, and binding mechanism alphabeta t cell antigen receptor recognition of cd a presenting self lipid ligands structure of the human class i histocompatibility antigen, hla-a t cell receptor cross-reactivity directed by antigen-dependent tuning of peptide-mhc molecular flexibility divergent t-cell receptor recognition modes of a hla-i restricted extended tumourassociated peptide crystal structures of two viral peptides in complex with murine mhc class i h- kb a structural voyage toward an understanding of the mhc-i-restricted immune response: lessons learned and much to be learned loss of t cell antigen recognition arising from changes in peptide and major histocompatibility complex protein flexibility: implications for vaccine design generation of murine ctl by a hepatitis b virus-specific peptide and evaluation of the adjuvant effect of heat shock protein glycoprotein and its terminal fragments diverse peptide presentation of rhesus macaque major histocompatibility complex class i mamu-a revealed by two peptide complex structures and insights into immune escape of simian immunodeficiency virus crystal structure of cell adhesion molecule nectin- /cd and its binding to immune receptor dnam- / cd cross-allele cytotoxic t lymphocyte responses against pandemic h n influenza a virus among hla-a and hla-a supertype-positive individuals the antigenic identity of peptide-mhc complexes: a comparison of the conformations of five viral peptides presented by hla-a the structure of hla-b reveals nonamer self-peptides bound in an extended conformation structural definition of the h- kd peptide-binding motif an ion pair in class ii major histocompatibility complex heterodimers critical for surface expression and peptide presentation structural basis for the differential classification of hla-a* and hla-a* into the a and a supertypes interaction pattern of arg in the a-pocket of differentially disease-associated hla-b subtypes suggests distinct tcr binding modes engineering t cells specific for a dominant severe acute respiratory syndrome coronavirus cd t cell epitope hemagglutininspecific cd (+) t-cell responses following -ph n inactivated split-vaccine inoculation in humans a t cell receptor flattens a bulged antigenic peptide presented by a major histocompatibility complex class i molecule diversified anchoring features the peptide presentation of dla- * : first structural insight into domestic dog mhc class i crystal structure of swine major histocompatibility complex class i sla- and identification of pandemic swine-origin influenza a h n virus cytotoxic t lymphocyte epitope peptides evaluation of zika virus-specific t-cell responses in immunoprivileged organs of infected ifnar -/-mice screening and identification of severe acute respiratory syndrome-associated coronavirus-specific ctl epitopes complex assembly, crystallization and preliminary x-ray crystallographic studies of mhc h- kd complexed with an hbv-core nonapeptide we thank prof. jianfang zhou (national institute for viral disease control and prevention, chinese center for disease control and prevention) for excellent assistance with flow cytometry. we thank dr. minghai zhou (the scripps research institute, scripps florida, usa) for his excellent work in crystallization of the proteins. we also thank dr. jianhui li (institute of biophysics, chinese academy of sciences) for instruction in circular dichroism spectroscopy. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.molimm. . . . key: cord- -mhfu e r authors: wang, wenling; huang, baoying; wang, xiuping; tan, wenjie; ruan, li title: improving cross-protection against influenza virus using recombinant vaccinia vaccine expressing np and m ectodomain tandem repeats date: - - journal: virol sin doi: . /s - - - sha: doc_id: cord_uid: mhfu e r conventional influenza vaccines need to be designed and manufactured yearly. however, they occasionally provide poor protection owing to antigenic mismatch. hence, there is an urgent need to develop universal vaccines against influenza virus. using nucleoprotein (np) and extracellular domain of matrix protein (m e) genes from the influenza a virus a/beijing/ / (h n ), we constructed four recombinant vaccinia virus-based influenza vaccines carrying np fused with one or four copies of m e genes in different orders. the recombinant vaccinia viruses were used to immunize balb/c mice. humoral and cellular responses were measured, and then the immunized mice were challenged with the influenza a virus a/puerto rico/ / (pr ). np-specific humoral response was elicited in mice immunized with recombinant vaccinia viruses carrying full-length np, while robust m e-specific humoral response was elicited only in the mice immunized with recombinant vaccinia viruses carrying multiple copies of m e. all recombinant viruses elicited np- and m e-specific cellular immune responses in mice. only immunization with rvj- m enp induced remarkably higher levels of il- and il- cytokines specific to m e. furthermore, rvj- m enp immunization provided the highest cross-protection in mice challenged with mld( ) of pr . therefore, the cross-protection potentially correlates with both np and m e-specific humoral and cellular immune responses induced by rvj- m enp, which expresses a fusion antigen of full-length np preceded by four m e repeats. these results suggest that the rational fusion of np and multiple m e antigens is critical toward inducing protective immune responses, and the m enp fusion antigen may be employed to develop a universal influenza vaccine. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. seasonal influenza is an acute respiratory infectious disease that can cause serious health problems. seasonal influenza epidemics caused by influenza a and b viruses result in - million severe cases and , - , deaths worldwide; %- % of adults and %- % of children suffer from flu annually (world health organization, who ). seasonal influenza viruses undergo antigenic drift, which makes the conventional seasonal vaccines ineffective when the vaccine strains mismatch with the epidemic strains. therefore, seasonal influenza vaccines must be updated regularly for effective prevention of influenza. the production time for conventional influenza vaccines can be lengthy. it takes - months to obtain vaccine strains using conventional reassortment technology or reverse genetics technology, followed by several more months for large-scale expansion of influenza vaccine using an ample supply of specific pathogen-free (spf) chicken embryos. generally, influenza vaccine production takes - months from the prediction of the epidemic strain to the ultimate production of the vaccine (emanuel and wertheimer ) . for more effective prevention of influenza, new vaccines are warranted to induce crossprotection and long-lasting immune responses (erbelding electronic (np) and extracellular domain of matrix protein (m e) can induce cross-protection against influenza a virus, showing promise as candidate antigens for the development of a broad-spectrum influenza vaccine (erbelding et al. ; zhang et al. ; kui et al. ) . previously, we expressed a fusion protein of np and m e (nm e) in escherichia coli and showed that immunization with nm e formulated with aluminum hydroxide gel protected mice from a lethal challenge with heterologous influenza virus . therefore, vaccination with recombinant nm e fusion protein is a promising strategy for the development of a universal influenza vaccine. however, a new expression system is required to further optimize the np-and m e-based vaccine. the vaccinia virus (tiantan strain) was developed in china as a vaccination agent against smallpox, as documented by who (fenner et al. ). this strain was used to inoculate several individuals for * years, playing a key role in the eradication of smallpox in china (ruan ) . the tiantan strain has favorable gene vector and vaccine characteristics, including a powerful multiplication capacity, wide host range, high capacity for insertion of a foreign gene, noncarcinogenicity, and induction of longlasting immunity. using the tk gene system of the tiantan strain, this vaccinia virus has been developed successfully as a viral vector for various vaccine applications (ruan ) , and severe acute respiratory syndrome coronavirus vaccines (yan et al. ). we have developed recombinant vaccinia viruses based on np, m , m , and pb of influenza a virus wang et al. wang et al. , . several of our np-based recombinant vaccinia viruses have induced a protective immune response in balb/c mice. to develop a vaccine with broader protection, we constructed recombinant vaccinia viruses expressing various combinations of np and m e to exhibit their maximal antigenicity. balb/c mice were immunized with the recombinant viruses to measure np-and m e-specific humoral and cellular immune responses as well as protective effect against lethal challenge with a heterologous influenza virus. materials b-gal ( - a) monoclonal antibody (sc- ) and np monoclonal antibody ( d ) (sc- ) were purchased from santa cruz biotechnology (santa cruz, ca, usa). m monoclonal antibody ( c ) (ab ) and alexa fluor Ò -conjugated goat anti-mouse polyclonal antibodies were from abcam (cambridge, ma, usa). bd cytometric bead array (cba) mouse th /th /th cytokine kit was purchased from bd (franklin lakes, nj, usa). peptides np - (rliqnsltiermvls; h- drestricted th epitope), np - (tyqrtralv; h- drestricted ctl epitope), and m e pooled peptides (peptides of residues - , - , and - ) were synthesized by beijing scilight biotechnology ltd. co. (beijing, china). vaccinia virus (tiantan strain) and influenza a virus a/puerto rico/ / (pr ) (h n ) were used in this study. vaccinia virus (tiantan strain) was amplified and titrated in primary chicken embryo fibroblasts (cefs). the pr influenza virus was propagated in -day-old chicken embryos at °c for h, and the allantoic fluid was then collected and stored at - °c until use. mouse % lethal dose (mld ) titer of the influenza virus pr was assessed in balb/c mice before conducting the challenge experiments. plasmid pjsc contains two dna fragments (tkl and tkr) derived from vaccinia virus tiantan strain to facilitate homologous recombination with vaccinia virus in host cells. between the two homology arms, there are p late promoter of vaccinia virus to regulate the lacz gene and p . early/late promoter of vaccinia virus to regulate the target genes. the pjsc was linearized at a bamh i restriction enzyme site immediately downstream the p . promoter and further treated with calf intestinal alkaline phosphatase. plasmid pet a-nm e contains the fusion gene of fulllength np ( amino acids) and the succeeding m e ( amino acids) from influenza a virus, a/beijing/ / (h n ) (bj ) . the np and m e fusion gene (referred to as npm e) was amplified from plasmid pet a-nm e by polymerase chain reaction (pcr) to introduce bamh i site at each end. the npm e was digested by bamh i, and inserted into the linearized plasmid pjsc . the resulting plasmid carrying npm e at the same orientation as the p . promoter was designated as pjsc -npm e. from plasmid pjsc -npm e, m e and np genes were amplified respectively, and fused (m e preceding np) by pcr to generate a fusion gene, m enp. dna fragment containing four copies of m e (from bj ) was amplified from plasmid, pet a-h m e, and fused to the terminus of full-length np gene by pcr, resulting in fusion gene, m enp. the dna fragment containing four copies of m e was also fused to the terminus of a np gene (from bj ) truncated at both ends (referred to as nps, encodes amino acids) that was amplified from plasmid pet a-npsm e by pcr. the resulting fusion gene was named m enps. as the fusion genes m enp, m enp, and m enps were engineered with bamh i site at each end through pcr, they were cloned into linearized pjsc as described above. the resulting plasmids were pjsc -m enp, pjsc - m enp, and pjsc - m enps, respectively. recombinant vaccinia viruses were generated via homologous recombination, as described by wang et al. ( ) . briefly, cefs were infected with vaccinia virus tiantan strain at a multiplicity of infection of . - . , followed by transfection with recombinant pjsc plasmids containing fusion genes of np and m e. subsequently, recombinant vaccinia viruses were screened by blue-white selection. viral dna was isolated from each recombinant vaccinia virus to confirm the presence of fusion genes (npm e, m enp, m enp, and m enps) by pcr and sequencing of pcr products. the confirmed recombinant vaccinia viruses were produced on a large scale in cefs. the control recombinant vaccinia virus rvj was generated using the same method with the empty pjsc vector. to confirm the expression of np and m e by the recombinant vaccinia viruses using indirect immunofluorescence, a cells grown in -well culture plates were infected with each strain of virus at - plaque-forming unit (pfu)/well. at h post infection, the cells were fixed with % paraformaldehyde, and the expression of b-gal, np and m e was confirmed using mouse monoclonal antibody (mab) against b-gal ( - a), mouse mab against influenza a virus m ( c ), and mouse mab against influenza a virus np ( d ). the signals were then visualized using alexa fluor Ò -conjugated goat anti-mouse. spf female balb/c ( - weeks old) mice were purchased from beijing vital river laboratory animal technology co., ltd. (beijing, china). all mice were bred under spf conditions at the institute of laboratory animal science, chinese academy of medical science and peking union medical college, beijing, china. mice were randomly grouped ( mice/group), inoculated intramuscularly with pfu recombinant vaccinia viruses (rvj , rvj-npm e, rvj-m enp, rvj- m enp, or rvj- m enps) at week and week in the bilateral gastrocnemius without the use of reagents or equipment. ten days after the second immunization, three mice from each group were retro-orbital bled and sacrificed. the spleens were removed aseptically and ground through a -mesh sieve. spleen mononuclear cells (smncs) were obtained as described by wang et al. ( ) . the cellular immune responses induced in mice were evaluated by enzyme-linked immunospot (elispot) assay ) and the bd cba mouse th /th /th cytokine kit according to the manufacturer's instructions. peptides np - , np - , or m e pooled peptides were used in the elispot and cba assays. the sera were separated by centrifugation, and serum igg titers were determined. two weeks after the second immunization, mice were anesthetized using sodium pentobarbital ( mg/ml) at a dose of mg/kg of body weight. subsequently, - mice in each group were challenged with times of mouse % lethal dose (mld ) of pr intranasally. weight loss and mortality were monitored daily for weeks after the challenge. mice that lost % of their initial weight were euthanized and recorded as having died. statistical analysis was performed using spss (ver. . ; spss inc., chicago, il, usa) and prism (ver. . a; graphpad inc., la jolla, ca, usa). log conversion was performed for antibody titers. differences in antibody titer, elispot, and cba results among groups were analyzed using one-way anova. the paired t test and log-rank test were used to analyze differences in weight loss curves and survival rate curves, respectively. differences with p values \ . were considered statistically significant. to generate fusion antigens with the highest potency for inducing cross-protection, we designed a variety of fusion antigen configurations by adjusting the fusion order of np and m e, increasing the copy number of m e ( m e), and truncating the length of nps. the np and m e gene fragments of influenza a virus a/beijing/ / (h n ) were joined to create npm e, m enp, m enp, and m enps using pcr (fig. ) . each amplified fusion gene fragment was cloned into plasmid pjsc under the regulation of p . early/late promoter. the resulting plasmids pjsc -npm e, pjsc -m enp, pjsc - m enp, and pjsc - m enps were confirmed by restriction endonuclease digestion and dna sequencing (data not shown). each confirmed plasmid was transfected into cefs infected by vaccinia virus tiantan strain to facilitate homologous dna recombination between plasmid and viral genomic dna. recombinant vaccinia viruses rvj-npm e, rvj-m enp, rvj- m enp, and rvj- m enps were screened by blue-white selection. the vaccinia virus dna was extracted to amplify the target gene fragments using pcr. the inserted target genes were confirmed by sequencing of the pcr products. a cells were infected with recombinant vaccinia viruses, and expression of the target proteins (np and m e) was identified by immunofluorescence. as a selective marker, b-galactosidase (b-gal) was expressed at similar levels in a cells infected by all recombinant vaccinia viruses. except the control virus rvj , all other recombinant vaccinia virus expressed m e and np in the infected cells. it seemed that rvj- m enps showed lower expression of truncated np than other recombinant vaccinia viruses carrying full-length np gene (fig. ) . flow cytometry analysis of the target protein expression showed similar results, and western blot analysis confirmed target protein expression at the expected molecular weights (supplementary materials). to characterize the immunogenicity of fusion antigens expressed by the recombinant vaccinia viruses, five groups of balb/c mice were immunized with pfu of recombinant vaccinia virus at week and . ten days after the second immunization, sera were taken to measure the antibody titer, and the spleens were removed aseptically to measure the cellular immune response. mice immunized with the recombinant vaccinia virus rvj-npm e and rvj-m enp showed strong antibody responses against np, with lower titers of antibodies against m e (fig. a) . rvj- m enp induced strong antibody responses against both np and m e. while rvj- m enps only induced a strong humoral immune response against m e but not np. elispot analysis (fig. b ) revealed that rvj-npm e, rvj-m enp, rvj- m enp, and rvj- m enps induced more np - -specific spot-forming cells (sfcs) than np - -specific sfcs. rvj-m enp induced significantly more np - -specific sfcs than rvj-npm e (p \ . ) and rvj- m enps (p \ . ). rvj- m enp also induced significantly more np - -specific sfcs than control virus, nevertheless there is no significant difference compared with rvj-m enp. except the control virus, all other recombinant vaccinia virus seemed to induce m e-specific cellular immune responses (fig. b) . however, only m e-specific sfcs induced by rvj-m enp are significantly more than those induced by control virus (p \ . ) and by rvj-npm e (p \ . ). cytokine measurements (fig. c) showed that m e pooled peptides stimulated smncs to secrete several cytokines including interleukin (il)- , il- , il- , and tumor necrosis factor. however, only immunization with rvj- m enp induced significantly higher levels of il- and il- cytokines than that with control (rvj ) when stimulation with the m e pooled peptides. we assessed the protective efficacy of the recombinant vaccinia viruses in the balb/c mouse model. two weeks after the second immunization, balb/c mice were challenged with mld of influenza a virus pr , and then weight loss and survival rates were monitored for weeks. the weight loss curve (fig. a) showed that mice immunized with rvj , rvj-npm e, and rvj-m enp experienced rapid weight loss, and all mice died by days post-challenge. mice immunized with rvj- m enps also experienced rapid weight loss; several of these mice died soon after the pr challenge, while others experienced maximum weight loss by day post-challenge followed by gradual recovery. mice immunized with rvj- m enp experienced the lowest average weight loss ( %) among the groups observed on day post-challenge, followed by rapid recovery to the initial body weight at day postchallenge. the survival rate results (fig. b) showed that mice immunized with rvj , rvj-npm e, and rvj-m enp died on days - , - , and - , respectively, post-challenge. in contrast, / mice in the rvj- m enp died on day post-challenge, showing a final survival rate of %. mice immunized with rvj- m enps died on days - post-challenge with a final survival rate of %. statistical analysis showed that mice immunized with rvj- m enp had significantly highest survival rate, and mice immunized with rvj- m enps also had significantly higher survival rate than the remaining three groups (fig. b ). the extracellular domain of m e is highly conserved among influenza a virus subtypes (muñoz-medina et al. ; zebedee and lamb ) ; thus, it was adopted as a target antigen in the development of a universal influenza vaccine. in the present study, a single m e induced a poor m e-specific humoral response (fig. a , rvj-npm e, rvj-m enp). four tandem repeats of m e induced robust antibody response against m e (fig. a , rvj- m enp, rvj- m enps), leading to a much stronger cross-protection than that of other groups against heterosubtypic h n influenza virus challenge (fig. b , rvj- m enp, rvj- m enps). it had been proved that more robust m especific immune response can be induced with multiple copies of m e (zhang et al. ; zhao et al. b; zhou et al. ) , and further enhanced by fusion with an appropriate protein carrier (ma et al. ; zhao et al. a; ebrahimi et al. ; alvarez et al. ; schotsaert et al. ). our data suggest that m e-specific antibody and cellular response played important roles in protection, and m e is more favorable than m e to induce protective immune response. m e specific antibody may play a protective role by antibody-dependent cell-mediated cytotoxicity (adcc), although it could not neutralize influenza virus (kim et al. ; lee et al. ) . besides, m e specific cellular immune response may stimulate cd ? t cells to protect animals (adlermoore et al. ). np has also been used as a target antigen to develop universal influenza vaccines li et al. ; wang et al. ; nahampun et al. ; lei et al. ; zheng et al. ) . in the present study, a truncated np protein lacking amino acids at the n terminal and * amino acids at the c terminal was designated as nps. although nps retaining the middle part of np with multiple conserved epitopes, it showed weaker immunogenicity compared with full-length np (fig. a , rvj- m enp, rvj- m enps), and less protection in this experiment (fig. b , rvj- m enp vs rvj- m enps, p \ . ), probably because of less np expression (fig. , middle panel) , suggesting that full-length np is critical toward inducing protective immunity at least in balb/c mouse. previous research proved that human mab against influenza np played an important role in fighting low-dose infection of influenza virus (fujimoto et al. ) , and np might exert a protective effect through adcc (kui et al. (hayward et al. ) . the present study showed that npspecific antibody and cellular response played important roles in protection. np-specific antibody could not neutralize influenza virus just as m e. however, it may protect animals against challenge by adcc, antibody-dependent cellular phagocytosis, or antibody-dependent complement deposition, and np cellular immune response may stimulate cd ? and cd ? t cells for protection (guo et al. ; lamere et al. ) ; the related mechanism is worthy of further investigation. humoral and cellular immune responses in balb/c mice immunized with recombinant vaccinia viruses. mice were immunized intramuscularly with plaque-forming units of recombinant vaccinia viruses at weeks and . group (g ) mice immunized with rvj were served as negative controls. a serum was obtained from three mice at days post the second immunization and analyzed by enzyme-linked immunoassay (elisa) for the presence of igg antibodies specific for np (left) or m e (right), as described in ''materials and methods''. the columns show geometric mean antibody titers, and the bars indicate the % confidence interval in each group (n = mice per group). comparative results between two groups are shown in the upper table (ns not significant, *p \ . ; **p \ . ; ***p \ . by one-way anova). b, c cellular immune response in mice immunized with recombinant vaccinia viruses. cytokines secreted from stimulated spleen mononuclear cells (smncs) were measured by enzyme-linked immunospot (elispot) assay (b) and mouse th /th /th cba kit (c). all mice in each treatment group were sacrificed at days post the second immunization. smncs were separated from mouse spleen samples, and lg/ml np - (left b), np - (middle b), and m e pooled peptides (right b, and c) were used as stimulants in the elispot and cba assays. after stimulation for h, the numbers of smncs producing interferon (ifn)-c (b) are presented as spotforming cells (sfcs)/ smncs. the columns show the average sfcs/ smncs, and the bars indicate the standard deviation of each group. lines above two or more groups indicate that there was no significant difference. *p \ . ; **p \ . ; ***p \ . by one-way anova. furthermore, we determined whether the immunogenicity of fusion antigens is influenced by the order of np and m e. our data showed that rvj-npm e and rvj-m enp induced similar antigen-specific igg response (fig. a , rvj-npm e vs rvj-m enp). however, rvj-m enp induced significantly more np - -as well as m e-specific sfcs than the former one (fig. b , rvj-npm e vs rvj-m enp), suggesting that m e at n terminal of the fusion antigen is a favorable configuration to induce np-and m e-specific cellular immunity. moreover, our data indicated that four copies of m e preceding np ( m e-np) is more robust toward inducing np-and m especific humoral immune response (fig. a , rvj-m enp vs rvj- m enp) and protection (fig. b , rvj-m enp vs rvj- m enp). in addition, we observed that only rvj- m enp immunization induced significantly higher levels of il- and il- cytokines in mice when stimulation with the m e pooled peptides (fig. c, rvj-m enp ). significant progress is made to develop influenza vaccine using viral vectors (kim et al. ; li et al. ; tutykhina et al. ; dhanwani et al. ) . several recombinant viral vaccines have been constructed based on modified vaccinia virus ankara expressing ha, np, m , and pb antigens (coughlan et al. ; mullin et al. ; di mario et al. ; altenburg et al. ) . vaccinia virus tiantan strain, developed in china as a vaccine against smallpox, has also been used as viral vector in numerous studies to develop recombinant viral vaccines. in this study, the vaccinia virus tiantan strain was used to construct recombinant vaccinia viruses expressing fusion antigens with different configurations. the recombinant vaccinia virus expressing m e and full-length np fusion antigen induced strong cross-protection ( %) against a lethal heterosubtypic pr challenge at mld and thus regarded as the optimal one among the four constructs. in the previous study, we expressed the fusion protein nm e in an e. coli system, and determined its immunogenicity in balb/c mice. however, it is inconvenient and time consuming to express and purify much more alternatives. moreover, it is impossible to express target antigens in an e. coli system successfully sometimes. compared with an e. coli system, the recombinant vaccinia virus system has characteristics such as high multiplication capacity, no requirement to purify proteins, and high-efficiency to determine the immunogenicity of target antigens in animal models (coughlan et al. ; mullin et al. ) . in fact, we succeeded in constructing and determining the antigenicity of several fusion antigens of np and m e efficiently. in summary, immunization with a m e and np fusion antigen not only induced np-specific humoral and cellular immune response, but also increased m e-specific immunity, which together led to superior cross-protection against heterosubtypic pr at mld . we concluded that the cross-protection potential correlates with both np and m e-specific humoral and cellular immune responses induced by rvj- m enp. however, the recombinant rvj- m enp containing m e and full-length np could not induce significant neutralizing antibodies, which help to reduce the severity and duration of the illness. therefore, it is necessary to introduce conserved b-cell epitopes of ha to induce an enhanced cross-neutralizing response and protective potency (koday et al. ) . future studies should focus on the development of universal influenza mice were monitored daily for days after pr challenge. a mice were weighed daily to monitor morbidity. average weights in each treatment group were followed for the duration of the study, and the percentage of the original body weight was calculated based on the average starting weight for each group at day . b survival rates were calculated and compared among groups. ns not significant, *p \ . ; **p \ . ; ***p \ . . vaccines containing multiple antigens to induce broad neutralizing responses, adcc, and broad cellular immune responses. protective immunity against influenza in hla-a transgenic mice by modified vaccinia virus ankara vectored vaccines containing internal influenza proteins hsp c fusion protein fully protected mice against lethal dose of h , h and h influenza a isolates circulating in iran who should get influenza vaccine when not all can a universal influenza vaccine: the strategic plan for the national institute of allergy and infectious diseases smallpox and its eradication. world health organization crossprotective potential of anti-nucleoprotein human monoclonal antibodies against lethal influenza a virus infection protection against multiple influenza a virus subtypes by intranasal administration of recombinant nucleoprotein natural t cell-mediated protection against seasonal and pandemic influenza: results of the flu watch cohort study influenza a virus nucleoprotein derived from escherichia coli or recombinant vaccinia (tiantan) virus elicits robust cross-protection in mice induction of h n -cross-reactive antibody-dependent cellular cytotoxicity antibodies by human seasonal influenza a viruses that are directed toward the nucleoprotein mucosal vaccination with recombinant adenovirus encoding nucleoprotein provides potent protection against influenza virus infection roles of antibodies to influenza a virus hemagglutinin, neuraminidase, and m e in conferring cross protection multigenic dna vaccine induces protective crossreactive t cell responses against heterologous influenza virus in nonhuman primates progress on adenovirus-vectored universal influenza vaccines contributions of antinucleoprotein igg to heterosubtypic immunity against influenza virus mechanisms of cross-protection by influenza virus m -based vaccines broadly protective immunity against divergent influenza viruses by oral co-administration of lactococcus lactis expressing nucleoprotein adjuvanted with cholera toxin b subunit in mice single-dose vaccination of a recombinant parainfluenza virus expressing np from h n virus provides broad immunity against influenza a viruses an m e-based synthetic peptide vaccine for influenza a virus confers heterosubtypic protection from lethal virus challenge influenza nucleoprotein delivered with aluminium salts protects mice from an influenza a virus that expresses an altered nucleoprotein sequence activation of cross-reactive mucosal t and b cell responses in human nasopharynxassociated lymphoid tissue in vitro by modified vaccinia ankara-vectored influenza vaccines silico identification of highly conserved epitopes of influenza a h n , h n , h n , and h n with diagnostic and vaccination potential expression of h n nucleoprotein in maize seeds and immunogenicity in mice healthy human subjects have cd ? t cells directed against h n influenza virus research and application of vaccinia 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tiantan strain: secreted sq protein induces robust neutralization antibody in mice influenza a virus m protein: monoclonal antibody restriction of virus growth and detection of m in virions vaccination with different m e epitope densities confers partial protection against h n influenza a virus challenge in chickens advancements in the development of subunit influenza vaccines induction of protection against divergent h n influenza viruses using a recombinant fusion protein linking influenza m e to onchocerca volvulus activation associated protein- (asp- ) adjuvant an h n m e-based multiple antigenic peptide vaccine confers heterosubtypic protection from lethal infection with pandemic h n virus cross-protection against influenza virus infection by intranasal administration of nucleoprotein-based vaccine with compound / adjuvant immunization with high epitope density of m e derived from pandemic h n elicits protective immunity in mice acknowledgements this work was supported by grant from the national key plan for scientific research and development of china ( yfc ). the authors gratefully acknowledge professor xiangmin zhang (wayne state university, detroit, mi usa) for the revision of the manuscript in english. key: cord- -o oauxex authors: fritzen, juliana t.t.; oliveira, marcos v.; lorenzetti, elis; miyabe, flávia m.; viziack, mariana p.; rodrigues, carlos a.; ayres, henderson; alfieri, alice f.; alfieri, amauri a. title: longitudinal surveillance of rotavirus a genotypes circulating in a high milk yield dairy cattle herd after the introduction of a rotavirus vaccine date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: o oauxex worldwide, neonatal diarrhea is one of the most important health issues affecting dairy calves, and rotavirus a (rva) is one of its primary causes. among the measures to mitigate the risk of diarrhea outbreaks, cow vaccination stands out as one of the most important. however, the immune pressure resulting from routine vaccination may be able to select specific g and p genotypes in rva field strains. this study aimed to determine the frequency and intensity of neonatal diarrhea and the incidence of rva and attempted to monitor the g and p genotypes present in the rva strains circulating in a high milk yield cattle herd vaccinated with rva g p[ ] strain. fecal samples (n = ) from holstein heifer calves between – days old that were born from rva-vaccinated cows were collected at different time points, regardless of the presence or absence of diarrhea. the presence of rva in fecal samples was determined by the polyacrylamide gel electrophoresis (page) technique and confirmed by reverse transcription polymerase chain reaction (rt-pcr). g and p amplicons from rva-positive fecal samples from calves of different ages and collections were subjected to nucleotide sequencing. the proportion of the calves and fecal samples that were positive for rva were . % ( / ) and . % ( / ), respectively. using sequence analysis, all rva field strains presented genotype g p[ ]. the protection of g p[ ] vaccination is clear, as this genotype was not detected in this study, and it is known that vaccination against rva reduces the incidence of diarrhea independent of genotype involved. this result demonstrates the importance of epidemiological monitoring of rva genotypes circulating in vaccinated dairy cattle herds to the early detection of new potential pathogenic rva strains. neonatal calf diarrhea (ncd) is the most common cause of calf morbidity and mortality, which can exceed % in certain instances (uetake, ) . the factors that influence the etiology of this syndrome include nutritional and sanitary management, immunological aspects, and infectious agents, such as bacteria, viruses, and protozoa (coura et al., ) . bovine rotavirus a (rva) is one of the most prevalent viral agents associated with ncd in dairy and beef cattle herds worldwide (al mawly et al., ; coura et al., ) . the non-enveloped virion of rva is composed of a triple-layered capsid that surrounds dsrna segments that encode structural (vp -vp , vp , and vp ) and nonstructural (nsp -nsp / ) proteins (greenberg and estes, ). based on their antigenic characteristics and sequence analyses of the vp gene, viruses within the rotavirus genus are primarily classified into distinct groups/species (ictv, ; matthijnssens et al., ; mihalov-kovacs et al., ) ; a newly-proposed species j has been described in the bat . the vp and vp genes encode structural proteins responsible for the induction of the immune protective response and are used for the binary classification of rva strains in terms of their g and p genotypes, respectively. currently, g and p genotypes are recognized by the rotavirus classification working group (rcwg, ) . calves aged to weeks have antibody levels from passive immunity decreasing to a level that is still sufficiently high to block responses to vaccines but is not sufficiently high to combat infection. thus, this period is considered a window of opportunity for the infection of microorganisms and, consequently, the occurrence of disease (chase et al., ) . therefore, the implementation of a cow vaccination program is an important strategy to protect the calves from rva infection and consequent neonatal diarrhea. the commercial vaccines that are available in brazil contain inactivated rva and other infectious agents that are implicated in ncd. there were different rva brazilian vaccine formulations: i) the bivalent vaccine g p[ ] and g p[ ]; ii) and monovalent g p[ ] genotype, which is the most prevalent genotype worldwide (papp et al., ) as well as in brazil (medeiros, ) . the aim of this study was to determine the frequency and intensity of neonatal diarrhea and the incidence of rva and to identify the rva g and p genotypes circulating in dairy calves born from cows that are regularly vaccinated with the rva g p[ ] strain in a high milk yield dairy cattle herd. this study was conducted in a brazilian dairy farm that is operated as a closed dairy cattle herd with holstein cows in lactation. the cows were managed using appropriate nutritional and health practices. this high milk yield dairy cattle herd produces an average of liters/ day/cow. the calves are reared in individual pens on elevated floors. the colostrum intake began shortly after birth and has taken place with adequate frequency and quantity for all newborn calves. sixty or days before calving, the cows were vaccinated against ncd. the commercial vaccine used in this study contained inactivated rotavirus a (uk-compton strain -g p[ ]), inactivated bovine coronavirus (mebus strain), and e. coli (k ) adesine f . the vaccination was performed according to the manufacturer's instructions. the fecal samples from heifer calves born from rva-vaccinated cows were collected at different time points ( , , , , , , , , , and days after birth), regardless of the presence or absence of diarrhea. the samples were collected between march and may , and a total of fecal samples were stored at - °c until analysis. at the time of collection, all of the fecal samples were classified according to their consistency using the following scale: , normal; , pasty; , soft; , watery. a score of or was considered non-diarrheic, and scores of or were considered diarrheic. the fecal samples from the same animal that were collected at different time points were processed at the same time to avoid any bias. the nucleic acid extraction was performed using a combination of the phenol/chloroform/isoamyl alcohol ( : : ) and silica/guanidinium isothiocyanate extraction methods according to alfieri et al. ( ) . the presence of rva dsrna in fecal samples was evaluated using polyacrylamide gel electrophoresis (page) (pereira et al., ) followed by silver staining (herring et al., ) . fecal samples with doubtful page results, as bands with low intensity or in anomalous positions, extra bands or undefined electropherotype were confirmed by reverse transcription polymerase chain reaction (rt-pcr) assay. the presence of rva in the fecal samples that were determined to be positive for rva via page was confirmed by rt-pcr using consensus primers to amplify a bp fragment from the vp gene (gouvea et al., ) and a bp fragment from the vp (vp *) gene (gentsch et al., ; martella et al., ) . the rt-pcr products were analyzed using electrophoresis with % agarose gels stained with ethidium bromide and observed under ultraviolet (uv) light. ten rt-pcr products of good quality from the rva-positive fecal samples from calves of different ages in the range of to days old and collections, including beginning, middle, and end of the experiment, were selected for sequence analysis. the rt-pcr products were purified using an illustra gfx pcr dna and gel band purification kit (ge ® , buckinghamshire, uk), quantified using a qubit ® fluorometer (invitrogen ® life technologies, eugene, or, usa), and sequenced using an abi genetic analyzer sequencer with a bigdye terminator v . cycle sequencing kit (applied biosystems ® , foster city, ca, usa). the phred and cap software packages were used for the nucleotide (nt) quality analysis and contig assembly of the rva sequences, respectively (http://asparagin.cenargen.embrapa.br). sequence similarity searches were performed using the basic local alignment search tool (blast) software (http://blast.ncbi.nlm.nih.gov/), and the genotype identification was performed using the rotac . tool (http://rotac. regatools.be/). phylogenetic trees were obtained using the neighbor-joining method with kimura -parameter model in mega v software. the bootstrapping probabilities were calculated using replicates. the bioedit software, version . . . , was used for the construction of the nt sequence identity matrix. the gene sequences described in the present study have been deposited in the genbank database under accession numbers: mh and mh . seventy-six ( . %) of the heifer calves were rva-positive according to at least of the fecal samples evaluated, and of these, ( . %) calves were determined to have excreted rva on more than one occasion. worldwide, the percentage of calves that are rva-positive varies based on the experimental design used in each study. in scotland and northern england (snodgrass et al., ) and new zealand (al mawly et al., ) , both using commercial enzyme-linked immunosorbent assay (elisa), rates of rva infection of . % ( / ) and . % ( / , ), respectively, were reported. in brazil, also using the page technique, coura et al. ( ) reported that . % ( / ) of dairy heifer calves that were - days old excreted rva during a longitudinal study, and in a transversal study only of ( . %) of dairy calves - days old were found to be rva-positive (alfieri et al., ) . the percentage of rva-positive fecal samples obtained during this study was . % ( / ). other longitudinal studies that have been conducted in brazil to determine the level of rva infection in calves born from vaccinated dairy cows had percentages of rva-positive diarrheic fecal samples that were . % ( / ) (coura et al., ) and . % ( / ) (rocha et al., ) . the distribution of the total samples and the rva-positive fecal samples for each fecal consistency score and time point at sample collection are shown in table . the frequency of diarrhea in calves during the experiment, shown in the table, was not high. fecal samples with scores of and (n = ) represented . % of the total samples, while samples with scores of and , indicating the presence of diarrhea, comprised . % (n = ) and . % (n = ) of the total samples, respectively. in a similar study conducted in brazil on rvaunvaccinated dairy cattle herds, the proportion of diarrheic fecal samples ( . %; / ) within the total number of samples was greater than that in this study (coura et al., ) . previous studies have indicated that the presence of rva infection in feces from diarrheic calves is significantly higher than that in nondiarrheic feces (al mawly et al., ; bartels et al., ; chitambar et al., ) . forty-eight of the ( . %) diarrheic fecal samples evaluated were rva-positive. on average, the frequency of rva-positive samples obtained from diarrheic feces in calves worldwide ranges between . and . % (alfieri et al., ; badaracco et al., ; barreiros et al., ; brito et al., ; buzinaro et al., ; caruzo et al., ; chitambar et al., ; falcone et al., ; malik et al., ; silva et al., ) . in this study, dsrna to from the rva was identified in . % ( / ) of the fecal samples with scores of or and in . % ( / ) and . % ( / ) of the fecal samples with scores of and , respectively. although rva was more frequent in diarrheic calves, the overall frequency of rva diagnosis was lower than that observed in unvaccinated herds (rocha et al., ) . the distribution of the fecal samples according to the age of the calves reveals that during the first and fourth weeks, the frequency of diagnosis of rva was . % ( / ) and . % ( / ), respectively. however, in the second and third weeks, the frequency of rva-positive fecal samples was higher, with the rate for the second week reaching . %. this finding suggests the occurrence of a decrease in passive immunity, corroborating the findings of previous studies that suggest that passive immunity protects calves during their first week and that their own natural resistance is not initiated until weeks of age (alfieri et al., ) ; suggesting the high susceptibility of these animals to disease during the period between the first and fourth week. meganck et al. ( ) and coura et al. ( ) reported that diarrhea caused by rva is most frequent in -to -week-old calves, and alfieri et al. ( ) suggested that animals from dairy cattle herds were most susceptible to rva infection at to weeks of age. in order to reduce the occurrence of neonatal diarrhea, the dairy farm utilizes vaccination with a commercial vaccine containing the rva uk-compton strain with the genotype g p[ ] . this vaccine is frequently used in dairy and beef cattle herds both from brazil and other countries around the world mainly in countries of the northern hemisphere such as canada, the usa, and the european union. to determine the g and p genotypes of rva strains present in the fecal samples collected during this study, rva-positive fecal samples were selected from calves of different ages and collections for nt sequence analysis of the vp and vp amplicons. the analysis enabled the identification of the genotypes, which were g p [ ] in all of the wildtype rva strains with a high ( . to %) nt similarity between them. viruses with the g and p[ ] genotypes are not included in the rva vaccine used by the dairy farm evaluated in this study. the sequence obtained of vp gene of rva had the highest ( %) nt similarity with that of the prototype b strain (genbank accession number: x (xu et al., ) ), which belongs to g lineage iv (fig. a) , and the vp gene had the highest similarity ( . %) to that from the turkish e tr strain (genbank accession number: fj (unpublished data)) of p[ ] lineage iii (fig. b) . a putative vaccine breakthrough associated with heterotypic rva infection in newborn calves was described in turkey, where the emergence of an rva strain with the genotype g p[ ] was reported in a cattle herd vaccinated with the g p[ ] rva strain, which demonstrated a failure of heterologous protection (karayel et al., ) . the occurrence of emerging heterologous rva genotypes is a phenomenon has been previously observed in brazilian dairy and beef cattle herds after the administration of a rv vaccine (medeiros et al., ; rocha et al., ) . similar occurrences have also been described in humans in brazil and other countries around the world (banyai et al., ; cowley et al., ; doro et al., ; khandoker et al., ; santos et al., ) . however, depending on other factors, such as the type of cattle (beef or dairy herds), the method of cow and calf management (intensive or extensive), and the inclusion of vaccination for neonatal diarrhea in the health program, different results can be found between studies. in france, kaplon et al. ( ) evaluated beef cattle herds subject to extensive management and found no differences in the frequencies of diarrhea and rva-positive fecal samples between vaccinated and unvaccinated herds; the majority of the genotyped rva-positive diarrheic fecal samples were found to have the same genotype (g p[ ]) as that of the vaccine strain. among the measures used to mitigate the risk of neonatal diarrhea outbreaks, the vaccination of cow herds appears to be the most important. in humans, the world health organization recommends the inclusion of the rva vaccination in all national immunization programs (who, ) . several studies have demonstrated that vaccination does not totally eliminate the risk of infection, but it certainly reduces both the frequency and severity of the episodes of neonatal diarrhea caused by rva infection in calves (kaplon et al., ; parreno et al., ) . considering that the immune pressure exerted by routine vaccination may have the potential to select strains containing specific g and p genotypes, it is important to monitor the genotypes present in the rva strains that are circulating among and within vaccinated cattle herds (cashman et al., ) . in summary, in this longitudinal study evaluating fecal samples ( calves), the low occurrence of rva in diarrheic fecal samples from calves born from regularly rva g p[ ]-vaccinated cows was determined. in addition, the genotype g p [ ] , present in the rva vaccine strain used on the farm, was not identified in any rva field strains identified in calves in this study. additionally, the presence of the rva g p[ ] genotype was verified, which was different from the vaccine strain, reinforcing the vaccine protection. the presence of rva in vaccinated herds may occurs due to immune pressure, individual factors, management procedures, nutrition, and environmental variations. the constant monitoring of circulating rva strains in neonatal diarrhea in dairy cattle herds is important to identify the 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for rotavirus species demarcation bovine neonatal diarrhea: epidemiology and molecular characterization of g (vp ) and p (vp ) genotypes of rotavirus a, brazil phylogenetic analysis of a g p[ ] bovine rotavirus strain isolated in a neonatal diarrhea outbreak in a beef cattle herd vaccinated with g p[ ] and g p[ ] genotypes advances in prevention and therapy of neonatal dairy calf diarrhoea: a systematical review with emphasis on colostrum management and fluid therapy candidate new rotavirus species in sheltered dogs review of group a rotavirus strains reported in swine and cattle modulation by colostrum-acquired maternal antibodies of systemic and mucosal antibody responses to rotavirus in calves experimentally challenged with bovine rotavirus electrophoretic study of the genome of human rotaviruses from rio de janeiro, são paulo and para, brazil rotavirus classification working group. newly assigned genotypes longitudinal study of bovine rotavirus group a in newborn calves from vaccinated and unvaccinated dairy herds rotavirus genotypes circulating in brazil before and after the national rotavirus vaccine program: a review molecular characterization of group a bovine rotavirus in southeastern and central-western brazil aetiology of diarrhoea in young calves newborn calf welfare: a review focusing on mortality rates world health organisation. rotavirus vaccines: an update sequence of the gene encoding the major neutralization antigen (vp ) of serotype rotavirus the authors thank the following brazilian institutes for financial support: the national council of scientific and technological development (cnpq), the brazilian federal agency for support and evaluation of graduate education (capes), and the araucária foundation (fap/pr). alfieri, a.a. and alfieri, a.f are recipients of cnpq fellowships. the authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. key: cord- - se wp o authors: chen, yi-shiuan; fan, yi-hsin; tien, chih-feng; yueh, andrew; chang, ruey-yi title: the conserved stem-loop ii structure at the ' untranslated region of japanese encephalitis virus genome is required for the formation of subgenomic flaviviral rna date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: se wp o flaviviruses accumulate abundant subgenomic rna (sfrna) in infected cells. it has been reported that sfrna results from stalling of host ’-to- ’ exoribonuclease xrn at the highly structured rna of the ’ untranslated region (utr). although xrn digestion of a ’-terminal -nt rna could stall at a position to generate the sfrna in vitro, we found that knocking out xrn had no effect on the accumulation of sfrna in japanese encephalitis virus (jev) infected cells. mutagenesis studies revealed that the stemloop ii (slii) at the ’ utr is required for the accumulation of sfrna. according to the results of an in vitro rna-dependent rna polymerase (rdrp) assay, the (-) - rna fragment, containing the putative promoter on the antigenome for the sfrna transcription, binds to rdrp protein and exhibits a strong promoter activity. taken together, our results indicate that the jev sfrna could be transcribed initially and then be trimmed by xrn or other unidentified exoribonucleases. japanese encephalitis virus (jev) belongs to the family flaviviridae and is a mosquito-borne zoonotic pathogen that causes human encephalitis and meningitis. the , nucleotides (nts) genome contains a single open reading frame (orf) encoding a polyprotein which is subsequently processed into three viral structural proteins and seven nonstructural proteins [ ] . the orf is flanked by ' and ' untranslated regions (utrs) which contain many important cis-acting elements involved in the regulation of viral translation, replication, and pathogenesis [ ] [ ] [ ] [ ] . during virus infection, all arthropod-flaviviruses generate abundant amounts of a noncoding subgenomic rna (sfrna) representing the '-terminal highly conserved region of the ' utr [ ] [ ] [ ] [ ] . the sfrna engages in multiple functions in order to control viral replication and antagonize host antiviral responses [ , , ] . these functions include (i) the involvement of cytopathicity and pathogenicity in mammals [ ] ; (ii) the down-regulation of plos antigenome synthesis and translation [ ] ; (iii) the dysregulation of host mrna stability [ , ] ; (iv) the antagonizing of host innate immune response [ ] [ ] [ ] ; (v) the suppression of rna silencing [ , ] ; (vi) the induction of apoptosis through the bcl- -mediated pi k/akt signaling pathway [ ] ; and (vii) the determination of infection and transmission rates of west nile virus (wnv) and dengue virus (denv) in mosquitoes [ , ] . how the sfrna is made in the infected cells is intriguing. several studies have shown that the sfrna is a product of the incomplete degradation of the viral genome by cellular '-to- ' exoribonuclease xrn ; a pseudoknot or a three-helix junction structure in the ' utr is responsible for stalling xrn degradation [ , [ ] [ ] [ ] [ ] . this cleavage happened within a few minutes when carried out in vitro, and there were no intermediate byproducts during the exoribonuclease degradation process of the viral genome to the sfrna in the virus-infected cells, which indicated that the degradation process is extremely efficient or that another biogenesis mechanism might be involved. the reason for these findings is not clear because degradation from the full-length genome seems uneconomic for viruses from an evolutionary point of view. furthermore, being infected with rna viruses induces double membrane vesicles to form replication/transcription complexes that are protease-and nuclease-resistant [ ] [ ] [ ] . the reason for such abundant levels of viral genome within the membrane-protected complex subjected to rna degradation remains unclear. although rdrp has no proofreading activity, considering the high molar ratio of the sfrna to the genome ( . - . in mosquito cells and . - . in mammalian cells) [ ] , the possibility that large portions of the synthesized genome are dysfunctional and subjected to degradation pathways is elusive. it may just be that the rna turnover by xrn exoribonucleases is quite normal but that sfrna accumulates because it is degradation resistant. in this study, we found that either knocking down or knocking out of exoribonuclease xrn had no effect on the accumulation of sfrna in jev-infected cells. in contrast, mutation of gdd motif in rdrp and disruption of the slii structure halted accumulation of the sfrna. furthermore, the minus-strand templates covering the putative promoter region used for an in vitro rdrp assay gave rise to synthetic products, suggesting that the jev sfrna could be initially transcribed from the antigenome and may be further trimmed by xrn or other unidentified exoribonucleases. bhk- and a cells were grown at ˚c in rpmi medium supplemented with % and % fetal bovine serum (fbs, gibco), respectively. a xrn knock-out cells were kindly provided by dr. bernard moss (niaid, nih) and were propagated as described previously [ ] . human embryonic kidney (hek t) cells were grown in dulbecco's modified eagle's medium (dmem, gibco) supplemented with % fbs (gibco). jev strain rp (genbank accession number af ) and dengue virus type (denv- ) strain were used in this study. detailed methods for the construction of clones are described in the supporting information s text. the majority of plasmids used for this study were constructed by rt-pcr using synthetic oligonucleotides, as listed in s table. the cdna encoded rdrp domain from amino acids at position to of the jev ns protein was amplified by rt-pcr and cloned into pet a (novagen). the lentiviral vectors expressing the short hairpin rna (shrna) (trcn- ) targeting human xrn gene (genbank accession no. nm_ . ) or a negative control shrna targeting gfp (shgfp) were obtained from the taiwan national rnai core facility. to knockdown xrn expression, × hek t and a cells were transduced with the shxrn or shgfp lentiviruses, respectively, and selected with puromycin ( μg/ml). at one-week posttransduction, cells were seeded on -mm plates (approximately × cells per plate) and infected with jev rp (or denv- as a control) at an moi of (for hek t) or (for a ). the cells were harvested with pbs at h post-infection, and the precipitates were then divided into two parts. one part was analyzed for western blotting and the other part was used for northern analysis. equivalent amounts of proteins ( μg/sample) were resolved by % sds-page, followed by western blotting using anti-xrn ( : , sigma) or anti-β actin ( : , sigma) antibodies. rna extraction and northern analyses were conducted as described previously [ , ] . in some cases, the blots were hybridized with the ird -labeled jev(-) - oligonucleotide, and the signals were scanned on a typhoon imaging scanner (ge healthcare); otherwise, the blots were hybridized with the strand-specific digoxigenin (dig)-labeling riboprobe detecting jev nt - or denv- nt - made by in vitro transcription (roche molecular biochemicals) and then visualized using luminescent image analyzer (las- , fujifilm), as described in a previous study [ ] . the relative intensities of rna bands on the membrane were estimated using multi gauge software (fujifilm). plasmid pgemt-jev- was linearized with sal i and transcribed in vitro using t rna polymerase (promega) according to the manufacturer's protocol. dna template used for runoff transcription to generate '-terminal nts of denv was amplified from ptight-denv plasmid [ ] using oligonucleotides f and r as shown in supplemental s table. dna template was removed by dnase i. approximately pmol of triphosphated rnas were incubated with units of rna ' pyrophosphohydrolase (rpph, neb) at ˚c for hours to generate '-monophosphated rna. the rpph-treated rna was purified by phenol-chloroform extraction and ethanol precipitation. next, ng of the monophosphated rna was digested with the indicated amount of xrn (neb) or rnase a (qiagen) for hour at ˚c. ng of rna was then separated in a formaldehyde-containing . % agarose gel and hybridized to a riboprobe as indicated. the procedure for dna-based infectious clone transfection has been described previously [ ] . briefly, bhk- cells ( × cells/well) in -well plates were transfected with . μg of ptight-jev (or mutant) plasmid, . μg of ptet-off (bd bioscience), and μl of lipofectamine (invitrogen) in μl of opti-mem medium per well at ˚c for hours, and the cells were refed with fresh medium. total rnas were extracted at days post-transfection (dpt), and the supernatant fluids were collected for measuring the virus titers. the transfected cells were passaged every days if no apparent cytopathic effect (cpe) was observed. rnas were extracted at the time when the cells showed a severe cpe. the titration of infectious virus was performed as previously described [ ] . briefly, serial -fold dilutions of virus were made in serum-free alpha-mem medium, and . ml of each dilution was added per well to a monolayer of bhk- cells in a -well plate. after hour of absorption at ˚c, the cells were washed with pbs and overlaid with growth medium containing . % le agarose (lonza), and the plaque was visualized with naphthol blue-black solution ( . % naphthol blue-black, . % sodium acetate and % glacial acetic acid) at days postinfection. viral titers were determined as plaque forming units per milliliter. riboprobe was made by in vitro transcription with t rna polymerase (promega) and dig-utps (roche molecular biochemicals). the dig-labeled rnas ( . pmol) were denatured at ˚c for min and then quick cooling on ice. binding reactions were set at room temperature in binding buffer ( mm hepes ph . , mm kcl, mm mgcl , mm dtt, units rnaseout) with purified recombinant rdrp protein or bovine serum albumin (bsa) at different concentrations ( . , . , , and . μm) in a volume of μl for min, followed by adding μl heparin ( mg/ml) for min. the rna-protein complexes were resolved by non-denaturing % polyacrylamide gel at ˚c, transferred to a positively charged nylon membrane (amersham), and then cross-linked using uv stratalinker (stratagene). detection of dig-labeled rna was performed using dig luminescent detection kit (roche) according to the manufacturer's instructions. to demonstrate the specificity of rna-protein complex formation, competition assays were performed using various amounts of unlabeled rnas added to the binding reaction. a recombinant jev rdrp was expressed in e. coli rosetta (novagen). protein expression was induced with isopropyl-β-d-thiogalactopyranoside (iptg) at a final concentration of . mm and induced culture was grown at ˚c for hours. expressed rdrp was purified by metal affinity chromatography on a nickel-chelate column according to the manufacturer's instructions (invitrogen). the rna templates used for the rdrp assay and the rna size markers were synthesized by in vitro transcription (promega) in the absence or presence of α- p-ctp, respectively. the in vitro rdrp assay was performed with ng of the recombinant rdrp protein and ng of rna templates or as indicated in a total volume of μl of the reaction buffer containing mm tris-hcl (ph . ), mm nacl, mm potassium glutamate, mm mgcl , mm dtt, mm mncl , % glycerol, units of rnase inhibitor (promega), mm (each) atp, utp, and gtp, . mm ctp, and mci of α- p-ctp ( , ci/mmole; mp biomedicals). the reaction mixture was incubated at ˚c for hours. rdrp reaction product was denatured in x loading buffer ( % formamide, . % xylene cyanol, . % bromophenol blue, mm edta, . % sds) and resolved on % polyacrylamide gel containing m urea. the gel was dried and visualized by autoradiography. to investigate whether the jev sfrna is an exoribonuclease xrn cleavage product, we analyzed the effect of xrn depletion caused by rna interference on sfrna generation in virusinfected cells. knocking-down (kd) the xrn expression by up to % in hek t cells continued to increase the abundance of sfrna throughout the -h infection period. more sfrna ( %) was observed in xrn knocked-down cells than in the wild-type (wt) cells ( fig a) . it should be noted that there were no decay intermediates between viral genome and the sfrna even in the xrn -deficient cells. similar results were also observed in a cells infected with jev (fig b) , whereas denv infection caused a slight reduction of sfrna accumulation in the xrn -kd cells (fig c) . statistical analysis of jev sfrna accumulation in xrn -kd cells among three independent experiments showed about . % ± . % at h post infection. no statistically significant differences in the amount of the sfrna were detected in the infected cells at any time in relation to the depletion of xrn . these results indicated that either the exoribonuclease xrn in jev-infected cells could be extremely active, such that the residual xrn ( - %) might be sufficient to digest the jev genome or that another nuclease or mechanism might be involved in the formation of sfrna. to rule out the possibility that the residual xrn may contribute to the formation of sfrna, we performed the experiments using xrn knock-out (ko) cells. as shown in fig d, sfrna accumulated abundantly in xrn -ko cells infected with either jev or denv indicating that xrn does not play a direct role for the formation of sfrna in vivo. to determine whether purified exoribonuclese xrn is able to stall at a position to generate the sfrna in vitro, a '-terminal -nt monophosphate rna derived from genome of jev or denv was incubated with . , . , or unit of xrn , respectively, and analyzed by northern blot analysis for sfrna production. the denv sfrna is readily formed when treated with xrn at concentration as low as . units (fig e, lane ) , whereas the -nt jev rna was relatively resistant to low concentrations of xrn ( . and . unit) (fig e, lanes and ) . in contrast, incubation with unit of exoribonuclease xrn resulted in the production of the sfrna (fig e, lane ) , indicating that a higher concentration of exoribonuclease xrn is required for stalling at a position to generate jev sfrna. the -nt rna treated with rnase a was rapidly and completely degraded (data not shown). several studies suggest the existence of xrn -resistant structure (xrrna) for the production of sfrna. to investigate the structure requirements for the production of jev sfrna in vivo, we performed mutagenesis studies in the context of a jev infectious clone (in the s text and s table) . the structure of the jev ' utr is diagramed in fig a. although equal amounts of cdna ( ng) for all mutants were transfected into bhk- cells, according to immunofluorescence assay (ifa) and northern hybridization results at days post-transfection (dpt), the replication efficiency of the cdna from different mutants varied (data not shown). these results suggest that the mutations made in these regions delayed the completion of the viral life cycle. we therefore collected the supernatant and measured the virus titers at the time when the cells showed strong cpe that varied from to dpt for different mutants (table ) . we then used equal amounts of moi at . for each recombinant virus to infect bhk- cells and analyzed the rna synthesis at h post-infection (hpi). a deletion that spanned nt - (Δ ) abolished the viral replication (fig c, lane ) , while the deletion of the au-rich region (Δau-rich) or the '-stemloop (Δ '-sl) slightly delayed viral replication but had no lasting effect on the viral replication overall or on the sfrna accumulation (fig c, lanes - and table ). genome cyclization has been shown to play an important role in viral replication [ ] . beside the canonical '-cyclization ( 'cyc) motif (cagcauauuga) at - that is complementary to 'cs (ucaauaugcug) at nt - , we found another motif located at the initiation site of jev sfrna that is very similar to the 'cyc motif (named cyc-like motif), the sequence of which is caugcauaugga, where the mismatches with the 'cyc motif are underlined). however, deletion of the cyc-like motif had no effect on the synthesis of the sfrna and the genome (fig c, lane ) , whereas deletion of the entire slii abolished the production of sfrna (fig c, lane ) . meanwhile, deletion of the lower left stemloop of the slii (Δslii. ), the upper stemloop (Δslii. ) or the lower left stem (Δslii. ) all impaired sfrna synthesis (fig c, lanes - ) , indicating that the slii structure is essential for sfrna accumulation. three point mutations disrupted base pairing of the first pseudoknot at the ' utr (pk ' or pk ") prevented sfrna formation (fig c, lanes - ) , while compensatory changes restoring the pk pairing (pk ' ") recovered the sfrna synthesis ( fig c, lane ) suggesting that pseudoknot structure is also required for the sfrna formation. to further demonstrate that the production of sfrna requires the pk structure but does not rely on the activity of exoribonuclease xrn , pk ' " and pk " mutants were used to infect wt or xrn -kd hek t cells, and then the resulting effects were compared. disruption of the pk structure (pk ") abolished the sfrna production in wt as well as in xrn -kd cells, while compensatory changes that maintained the pk structure (pk ' ") made no difference in the production of sfrna in xrn -kd cells or in wt cells (fig d) , indicating that the requirement of the pk structure for the production of sfrna does not rely on the activity of exoribonuclease xrn . all mutations made at the slii region apparently resulted in relatively smaller plaques ( fig e) , suggesting that the structure or sequences of the slii might play a role in determining the plaque size. viral titers measured at -h intervals showed about -to -fold reductions in comparison with the wt control for all the mutants except for the slii deletion mutant, which exhibited slow growth at earlier time points but eventually reached to the wt-virus growth level at hpi ( fig f) . mutations resulted in the reduction of virus titers, suggesting that these mutations delayed virus maturation and release rather than affecting viral replication because the rna levels of mutants measured at hpi by northern hybridization ( fig c) did not show apparent differences in comparison with the wt control. to test whether the formation of jev sfrna requires viral replication, the replication-deficient mutant, which had the essential polymerase motif gdd mutated (ns mt), was compared to the wild-type (wt) infectious clone. the sfrna in wt dna-transfected cells were visible at hpt and continued to increase to a very high level at hpt (fig , lanes - ) . the ns mt clone, on the other hand, completely abolished the viral genome replication while barely any sfrna accumulated, indicating that the formation of the jev sfrna correlates with viral replication (fig , lanes - ) . although efficient rna replication is required for the detection of any flaviviral rnas despite which mechanism used for the sfrna formation, our results were clearly different from the observations from wnv that bhk- cells transfected with replicon constructs containing various deletions had no effect on the accumulation of sfrna when compared to the wt [ ] . to explore the possibility that the jev sfrna could be a transcription product, we assumed that its promoter would be located on the antigenome adjacent to the ' initiation of the sfrna. to test whether viral rdrp binds to the putative promoter and initiates sfrna synthesis, we cloned the cdna fragment corresponding to the nucleotide at position - of the genome, and performed emsa experiments using the minus-strand rna as the riboprobe. purified rdrp protein ( . , . , , and . μm) retarded the migration of the (-) - rna in a dose-dependent manner, whereas no band shift was observed with non-specific rna or bsa control (fig a) . it should be noted that the additional band above the rna/protein complex (rpc) (fig a, lanes - ) is inversely correlated to the occurring of rpc suggesting that the highly ordered rna secondary structure may have intermolecular interactions when separated by non-denaturing gel electrophoresis. this band is an indirect evidence of rdrp binding at this region. the intermolecular interactions were disrupted with increasing amounts of rdrp (fig a, left panel) . in contrast, the band remained the same when incubated with increasing amounts of bsa control (fig a, right panel) . competition assays were performed to test the specificity of the interaction between the rdrp and the (-) - rna. increasing amounts of unlabeled rna out competed the rpc (fig b, lanes - ) . in contrast, neither trna (lanes - ) nor nonspecific rna (lanes - ) competed away the rpc. these results suggested that jev rdrp binds specifically to the (-) - rna. studies have shown that the first nucleotides containing the ' utr and 'cs of the plus-strand rna in denv [ ] and wnv [ ] contain promoter activity. we first tested the enzymatic activity of purified recombinant jev rdrp using (+) - rna as a template for an in vitro rdrp assay. the result showed that the (+) - rna is a functional template for rna synthesis whereas the ' -nt rna from bovine coronavirus (as a nonspecific template control) could not direct rna synthesis, nor the reaction without adding the rdrp (fig c) , indicating that the purified recombinant jev rdrp has specific enzymatic activity. we then used the (-) - rna as the template for the rdrp assay. two major synthetic products were detected, the larger band with nt in length same as the size marker driven by t rna polymerase, and the smaller band with nts in length presumably a transcription product initiated from the putative promoter (fig d, lane ) . we then constructed various templates for in vitro rdrp assays (fig e and f and s table) . interestingly, all constructs showed a de novo synthesis product same as the template size and a smaller band with approximately nts in length except for the (-) - rna (fig d and e ). although some nonspecific bands were observed, these results indicated that the recombinant jev rdrp could recognize the promoter and synthesize the corresponding sequences. several studies have demonstrated that the accumulation of the sfrna results from incomplete degradation of viral genome by host exoribonuclease xrn [ ] [ ] [ ] . the crystal structure of the xrrnas in the ' utr that resist the degradation process has been determined [ , ] , and the function of the stalling has been studied in several flaviviruses [ , [ ] [ ] [ ] . although stalling of xrn by the jev sfrna has been reported previously [ ] , detailed mechanism of the sfrna formation has not yet been elucidated. the results of this study showed that either knocking down or knocking out of xrn gave rise to more sfrna accumulation in virusinfected cells (fig a, b and d ). it has also been reported that xrn could be redundant with other exonucleases in producing zika virus sfrnas [ ] . these controversial results may be explained by several reasons including (i) exonucleases other than xrn can contribute to sfrna formation, (ii) depletion of xrn causes changes in total cellular rna level that may include products of endonucleolytic cleavage, and (iii) reduction in xrn may contribute to differences in susceptibility to viral infection [ ] . although we cannot vehemently rule out these possibilities, theoretically, exoribonuclease xrn depletion should reduce the production of the sfrna, resulting in a reduction of the molar ratio of the sfrna to the genome, yet an increase rather than a decrease in molar ratio was observed. furthermore, no intermediate degradation byproducts from viral genome to the sfrna were detected during infection even in the xrn -deficient cells (fig a- d) . morevoer, several unsolved mysteries including the unreasonably high rate of nonsense-mediated decay to generate the sfrna, the question of how the viral genome within the membrane-protected complex is subjected to rna degradation, and the lack of economy in degradation from the full-length genome from an evolutionary point of view, led us to reconsider other possible mechanisms of the jev sfrna formation. mutation of the gdd motif of rdrp in jev halted the accumulation of sfrna (fig ) , suggesting that abundant accumulation of sfrna depends on viral replication. by transfecting replicon constructs containing various deletions pijlman et al. demonstrated that sfrna does not require viral rna replication, viral proteins, or ' utr since all deletion mutants showed similar levels of sfrna accumulation [ ] . the sfrna was also detected in bhk- cells electroporated with nonreplicating replicon denv- containing the catalytic gdd motif of the ns replaced with aaa [ ] . the initial rna transcripts either from replicon or infectious clone are all driven by cmv promoter. if formation of the jev sfrna were not required for rna replication, the accumulation of sfrna could be detected in the gdd-mutant infected cells. although efficient rna replication is required for the detection of any flaviviral rnas no matter the sfrna is made either by degradation or by transcription, our results are clearly different from those of previous studies of the wnv and denv. although the wnv and jev belong to the same serotype, different viruses may have evolved to have different strategies for survival in infected cells. it is intriguing that, according to in vitro rdrp assay results, the ns protein binds to the putative promoter and produces a de novo synthesis product and a presumable transcription product of approximately nts in length (fig ) . we attempted to clarify the initiation site of the rdrp products by applying ' rapid amplification of cdna ends ( ' race) assays but failed to observe any specific sequences due to low amount of the radiolabeled products. efforts had also been made to demonstrate that the putative promoter is functional in infected cells by transfecting rna construct containing unrelated sequences under the control of the promoter. unfortunately, our numerous attempts to make such a construct failed probably because the transfected molecule may not be accessible as a template to the viral rdrp at the right place and right time. it has been shown that the recombinant jev ns exhibited a weak terminal transferase activity only when substrate ribonucleotides were limited [ ] . sufficient amounts of ribonucleotides were supplied in the in vitro rdrp assay (fig c and d) , thus the products are least likely resulting from the terminal transferase activity. furthermore, in vitro digestion of the ' utr containing fragment (a -nt rna) by xrn revealed that xrn could stall at a position to generate the sfrna (fig e) . these results let us to propose the model that the jev sfrna is likely made by transcription initially and then could be further trimmed by xrn or other unidentified exoribonucleases (fig ) . several sequences and structures were determined to define the cis-acting elements for sfrna formation. it has been shown in previous studies with wnv that the deletion of the ' half of the ' utr does not affect the genome replication but does halt the sfrna formation [ ] . however, a similar deletion at the ' utr of jev (Δ ) totally killed virus replication ( fig c, lane ) . whether a partial deletion of the dumbbell structure resulted in the lethal phenotype remains to be determined. it has been suggested that the subgenomic rna promoter sequences often form a stem-loop structure, which is proposed to facilitate the interaction with the transcriptase as well as other host factors [ ] . we found that deletion of the entire or any part of the slii all impaired sfrna synthesis (fig c, lanes - ) , indicating that the slii structure is essential for sfrna accumulation and could be a potential promoter for the sfrna synthesis. furthermore, pseudoknot structure on slii is required for the formation of sfrna in wnv, yellow fever virus (yfv), and zikv [ , , ] . our result also showed that pseudoknot structure is essential for the formation of sfrna in jev (fig c, lanes - ) . interestingly, mutations made at the slii region showed relatively smaller plaques and delayed viral maturation or release (fig e and f ). this observation may correlate with the report that sfrna-deficient wnv displays significantly decreased infection and transmission rates in mosquitoes [ ] . our results revealed from in vivo mutagenesis studies (fig ) and in vitro rdrp assays ( fig ) are quite consistent except for the template nt - . this template contains the entire slii region that is essential for the formation of sfrna in vivo (fig ) but has no product by the in vitro rdrp assay (fig d) . this difference may due to that there is not enough space on the template for rdrp binding because rdrp is considered as a large protein so both de novo products and transcripts vanished when performed the in vitro rdrp assay (fig d, lane ) . deletion of the slii in wnv led to downstream xrn stalling and the accumulation of smaller sfrna species (sfrna , sfrna and sfrna ) [ , ] . previous studies of yfv have shown that the sfrna is indeed truncated at the '-end [ ] . it has been reported that the sfrna only exists in certain isolates of the denv- [ ] . filomatori et al. reported that the accumulation of different species of sfrnas in denv- -infected vertebrate and invertebrate cells due to selective pressures act on the ' utr during host adaption [ ] . although smaller sfrna species were found in jev, sequences of these rnas have not yet been determined. nevertheless, deletion mutants made on the slii structure abolished all sfrna formation but did not give rise to smaller rna species (fig c) . it is still unclear whether the downstream sliv or the dumbbell structure on jev genome plays a role in stalling of the xrn . the mechanism for the formation of smaller jev rna species needs to be further characterized. genome cyclization has been shown to play an essential role in the replication of flaviviruses (for review see [ ] ). a promoter element known as stemloop a (sla) at the ' end of the genome has been well studied with regard to its function in denv and wnv [ , , ] . the initiation of minus-strand rna synthesis includes the rdrp binding at the sla at the ' end of the genome and the relocation of the rdrp at the ' initiation site by long-range rna-rna interactions [ , , ] . in this study, we identified the slii that showed promoter activity as good as the sla promoter (figs and ) . we also found that the initiation site of jev sfrna located at a '-cyclization-like motif. this cyc-like motif complementary to the ' cyclization sequences may be involved in genome cyclization during antigenome synthesis and may cause template switching to occur, as reported by maeda et al. [ ] . however, deletion of the cyc-like motif had no effect on sfrna accumulation in vivo (fig c, lane ) . a potential role of this cyc-like motif remains to be studied. although viruses in the same family share many common strategies for completion of their life cycles, some differences in those strategies make certain viruses unique within the given family. in the present study, we provide evidence that the jev sfrna is likely synthesized by transcription and could be further trimmed by exoribonuclease xrn and/or other unidentified enzymes (fig ) . the slii structure is responsible for sfrna synthesis. our observations expand the current knowledge about the mechanism of sfrna formation in jev-infected cells. supporting information s japanese encephalitis virus: from genome to infectome. microbes and infection rna structure duplications and flavivirus host adaptation specific requirements for elements of the ' and ' terminal regions in flavivirus rna synthesis and viral replication the ' and ' untranslated regions of the flaviviral genome ' cis-acting elements that contribute to the competence and efficiency of japanese encephalitis virus genome replication: functional importance of sequence duplications, deletions, and substitutions identification and characterization of small sub-genomic rnas in dengue - virus-infected cell cultures and tissues zika virus produces noncoding rnas using a multi-pseudoknot structure that confounds a cellular exonuclease a highly structured, nuclease-resistant, noncoding rna produced by flaviviruses is required for pathogenicity accumulation of a '-terminal genome fragment in japanese encephalitis virus-infected mammalian and mosquito cells inhibition of type i interferon induction and signalling by mosquito-borne flaviviruses functional rna during zika virus infection small noncoding rna modulates japanese encephalitis virus replication and translation in trans a noncoding rna produced by arthropod-borne flaviviruses inhibits the cellular exoribonuclease xrn and alters host mrna stability xrn stalling in the ' utr of hepatitis c virus and bovine viral diarrhea virus is associated with dysregulated host mrna stability japanese encephalitis virus non-coding rna inhibits activation of interferon by blocking nuclear translocation of interferon regulatory factor west nile virus noncoding subgenomic rna contributes to viral evasion of the type i interferon-mediated antiviral response dengue subgenomic rna binds trim to inhibit interferon expression for epidemiological fitness noncoding flavivirus rna displays rna interference suppressor activity in insect and mammalian cells flavivirus sfrna suppresses antiviral rna interference in cultured cells and mosquitoes and directly interacts with the rnai machinery. virology dengue virus subgenomic rna induces apoptosis through the bcl- -mediated pi k/akt signaling pathway noncoding subgenomic flavivirus rna is processed by the mosquito rna interference machinery and determines west nile virus transmission by culex pipiens mosquitoes dengue subgenomic flaviviral rna disrupts immunity in mosquito salivary glands to increase virus transmission the structural basis of pathogenic subgenomic flavivirus rna (sfrna) production an rna pseudoknot is required for production of yellow fever virus subgenomic rna by the host nuclease xrn rna structures required for production of subgenomic flavivirus rna rna structures that resist degradation by xrn produce a pathogenic dengue virus rna the endoplasmic reticulum provides the membrane platform for biogenesis of the flavivirus replication complex sars-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity in vitro architecture of the flaviviral replication complex. protease, nuclease, and detergents reveal encasement within double-layered membrane compartments opposing roles of double-stranded rna effector pathways and viral defense proteins revealed with crispr-cas knockout cell lines and vaccinia virus mutants a novel approach to propagate flavivirus infectious cdna clones in bacteria by introducing tandem repeat sequences upstream of virus genome defective interfering rnas of japanese encephalitis virus found in mosquito cells and correlation with persistent infection genome cyclization as strategy for flavivirus rna replication a ' rna element promotes dengue virus rna synthesis on a circular genome rna elements within the ' untranslated region of the west nile virus genome are critical for rna synthesis and virus replication new hypotheses derived from the structure of a flaviviral xrn -resistant rna: conservation, folding, and host adaptation functional non-coding rnas derived from the flavivirus ' untranslated region noncoding subgenomic flavivirus rna: multiple functions in west nile virus pathogenesis and modulation of host responses biochemical characterization of a recombinant japanese encephalitis virus rna-dependent rna polymerase analyses of subgenomic promoters of hibiscus chlorotic ringspot virus and demonstration of ' untranslated region and '-terminal sequences functioning as subgenomic promoters dengue virus genomic variation associated with mosquito adaptation defines the pattern of viral non-coding rnas and fitness in human cells functional rna elements in the dengue virus genome terminal structures of west nile virus genomic rna and their interactions with viral ns protein we thank dr. bernard moss for kindly providing the a xrn knock-out cells and dr. muthukumar nadar and dr. yu-pin su for constructive comments on this manuscript. conceptualization: yi-shiuan chen, yi-hsin fan, ruey-yi chang.investigation: yi-shiuan chen, yi-hsin fan, chih-feng tien. writing -original draft: yi-shiuan chen, yi-hsin fan, ruey-yi chang. yi-shiuan chen, yi-hsin fan, ruey-yi chang. key: cord- -vtgdqart authors: aston, emily j.; jordan, brian j.; williams, susan m.; garcía, maricarmen; jackwood, mark w. title: effect of pullet vaccination on development and longevity of immunity date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: vtgdqart avian respiratory disease causes significant economic losses in commercial poultry. because of the need to protect long-lived poultry against respiratory tract pathogens from an early age, vaccination programs for pullets typically involve serial administration of a variety of vaccines, including infectious bronchitis virus (ibv), newcastle disease virus (ndv), and infectious laryngotracheitis virus (iltv). often the interval between vaccinations is only a matter of weeks, yet it is unknown whether the development of immunity and protection against challenge when vaccines are given in short succession occurs in these birds, something known as viral interference. our objective was to determine whether serially administered, live attenuated vaccines against ibv, ndv, and iltv influence the development and longevity of immunity and protection against challenge in long-lived birds. based on a typical pullet vaccination program, specific-pathogen-free white leghorns were administered multiple live attenuated vaccines against ibv, ndv, and iltv until weeks of age (woa), after which certain groups were challenged with ibv, ndv, or iltv at , , , , and woa. five days post-challenge, viral load, clinical signs, ciliostasis, tracheal histopathology, and antibody titers in serum and tears were evaluated. we demonstrate that pullets serially administered live attenuated vaccines against ibv, ndv, and iltv were protected against homologous challenge with ibv, ndv, or iltv for at least weeks, and conclude that the interval between vaccinations used in this study (at least weeks) did not interfere with protection. this information is important because it shows that a typical pullet vaccination program consisting of serially administered live attenuated vaccines against multiple respiratory pathogens can result in the development of protective immunity against each disease agent. vaccination against respiratory viral disease is standard practice in commercial poultry operations. both live and killed vaccines are administered to poultry, and live vaccines are commonly used for a variety of pathogens because they are effective when mass applied and are relatively economical [ ] . in general, live vaccines induce local and cell-mediated immunity and provide a broader protective response than killed vaccines, whereas killed vaccines primarily induce humoral immunity and tend to be antigen-specific. the duration of immunity achieved following live vaccine administration depends on the age and type of bird, levels of maternal immunity, disease targeted by the vaccine, immunogenicity of the vaccine, method of vaccine application, number of and interval between boosters, virulence and similarity of the field challenge virus, interval between vaccination and challenge, and immunocompetency of the host [ ] [ ] [ ] . avian coronavirus infectious bronchitis virus (ibv) is an upper respiratory tract viral pathogen of poultry and leads to reduced weight gain and feed efficiency, drops in egg production and egg quality, stunted growth, and secondary bacterial infection resulting in airsacculitis [ ] . the virus initially replicates in the upper respiratory tract, followed by systemic replication in the reproductive tract and some strains can cause lesions in the kidney [ ] . infected birds may exhibit nasal discharge, coughing, sneezing, and tracheal rales [ ] . the disease is prevented by vaccination, and live vaccines are commonly used to induce local immunity and protection. live vaccines are generally administered to young birds to achieve early protection, and layers and breeders are also boosted with either live or inactivated vaccines, which vary based on their similarity to the circulating field viruses [ ] . newcastle disease (nd) is caused by virulent strains of avian paramyxovirus type , which has recently been reclassified as avian avulavirus (aavv- ) [ ] . depending on the strain of the virus, clinical signs of nd infection may be absent or may involve depression, inappetence, respiratory signs (nasal discharge, sneezing, coughing), reduced egg production and egg quality, and neurological signs (torticollis, circling, paralysis) [ ] . strains of newcastle disease virus (ndv) are characterized as lentogenic, mesogenic, and velogenic, according to their mean death time in embryos [ ] . lentogenic strains are of low pathogenicity causing mild respiratory or enteric infections, followed by mesogenic strains, while velogenic isolates are highly pathogenic often causing neurological signs and mortality [ ] . vaccination regimes against ndv vary and may utilize a combination of live, inactivated, and virus-vectored vaccines [ ] . in the united states, the most widely used traditional vaccine strains comprise lentogenic b (or virus clones of the b strain) and lasota strains [ ] . infectious laryngotracheitis (ilt) is a respiratory disease of poultry caused by gallid alphaherpesvirus i, and is economically important worldwide [ ] . clinical manifestations of ilt include increased mortality, reduced egg production, decreased body weight gain, conjunctivitis, tracheitis with expectoration of bloody mucus in severe cases, depression, severe dyspnea and susceptibility to other respiratory pathogens. live vaccines against ilt virus (iltv) may be of chicken embryo origin (ceo) or tissue culture origin (tco), in which they are passaged multiple times in eggs or tissue culture, respectively. although recombinant vaccines for ilt are commercially available, the ceo vaccine is the most widely used vaccine against iltv worldwide. because of the need to protect chickens against different viral pathogens from an early age, vaccination programs typically include multiple vaccines against a variety of pathogens. sample vaccination regimes in different poultry sectors are reviewed in the merck veterinary manual (www. merckvetmanual.com), in which the interval between vaccinations is often only a matter of weeks. however, there is little information showing that the intervals between vaccinations are sufficient for the birds to develop adequate immune protection against challenge for each virus. the literature shows that sequential viral infections may result in viral interference, in which one virus blocks the subsequent infection and/or replication of another virus in the host [ , ] , but until now it is unknown whether this phenomenon results in reduced protection from serially administered attenuated live vaccines in chickens. interestingly, it has been reported that simultaneous administration of viruses to chickens or turkeys does not result in viral interference [ ] . in this study, we investigate how a typical commercial vaccination schedule consisting of a combination of serially administered, live attenuated viral respiratory disease vaccines affects the development and longevity of immunity and protection against homologous challenge. a commercial ibv vaccine mildvac-ga- ® (merck animal health, summit, nj, usa) was used in this study. the vaccine was diluted according to the manufacturer's recommendations. the challenge virus used was ibv ga /cwl / and was prepared at a median embryo infectious dose (eid ) of . . virus titers were calculated by the reed and muench method [ ] . a commercial ndv vaccine b (newhatch-c , merck animal health, summit, nj, usa) was used for both vaccination and challenge and was reconstituted following the manufacturer's instructions. since mesogenic and velogenic strains of ndv require a biosafety level above bsl , which is not available in our laboratory, our experimental model for protection against ndv involves significantly reduced virus titers or sterile immunity against a second exposure (challenge) with the vaccine virus. an iltv commercial ceo vaccine (trachivax ® merck animal health, summit, nj, usa) and pathogenic iltv georgia broiler strain [ ] were used for vaccination and challenge, respectively. strain was propagated in chicken kidney cells obtained from -to -week-old specific pathogen-free (spf) chickens [ ] . the ceo vaccine was prepared following the manufacturer's recommendations. after inoculation, the median tissue culture infective dose (tcid ) was confirmed by titration of both viruses in chicken kidney cells as previously described [ ] . specific-pathogen-free eggs were obtained at days of incubation and hatched at the poultry diagnostic and research center, athens, ga. chicks were placed on fresh pine shavings in colony houses and pens. chicks were vaccinated with the manufacturers recommended dose in µl via the oculonasal route according to the following schedule; ibv at doa, ndv at weeks of age (woa), ibv at woa, iltv at woa, ndv at woa, and iltv at woa. in addition, a control group was not vaccinated. homologous challenges were conducted at , , , , and woa, and necropsies were performed five days post-challenge (dpc). at challenge, birds received one of four treatments: ibv ga , ndv b , iltv , or no challenge. the treatment groups for each challenge virus per time point were as follows: non-vaccinated, non-challenged (n = - ); vaccinated, non-challenged (n = - ); vaccinated, challenged (n = - ); non-vaccinated, challenged (n = - ). all ibv-challenged birds received an eid of × . per bird in µl intranasally. all ndv-challenged birds received the ndv b vaccine in µl intranasally, reconstituted according to the manufacturer's protocol. all iltv-challenged birds received the pathogenic strain at a dose of × . tcid per bird in µl split equally between eyedrop and intranasal routes. for ibv and ndv challenges, birds were observed at dpc for respiratory signs, as previously described [ ] : = absent; = mild; = moderate; = severe. for iltv challenges, birds were observed at and dpc for dyspnea, conjunctivitis, depression, and mortality, as described previously [ ] . the choanal cleft (ibv-and ndv-challenged and control birds at dpc) or trachea (iltv-challenged and control birds at and dpc) was swabbed for virus detection, and swabs were stored in pbs at − • c. at , , and woa, µl of tears was collected by adding granulated nacl to the eye. blood was collected by wing or cardiac puncture and added to a microcentrifuge tube to collect serum for antibody detection. birds were humanely euthanized, and the eyelid, harderian gland (hg), thymus, liver, spleen, cecal tonsils, and bursa of fabricius were collected and stored at − • c for virus detection and in % neutral buffered formalin. the trachea was removed, and one section was placed in % neutral buffered formalin, and the remaining portion of the trachea was submerged in tissue culture media for the ciliostasis test described below. the procedures were approved by the university of georgia institutional animal care and use committee (aup #: a - -r ). the ciliostasis test was performed on harvested tracheas collected in cell culture media (dulbecco's modified eagle's medium) at • c. for each trachea, five tracheal rings measuring approximately mm thick were cut and represented the proximal, middle, and distal portions [ , ] . cilia activity was observed using an inverted microscope (olympus, center valley, pa, usa). the scoring system follows: = all cilia beating; = % of cilia beating; = % of cilia beating; = % of cilia beating; = no cilia beating as previously described [ ] . the maximum possible score for each trachea is . each tracheal ring was scored by three individuals, and the average total score for each trachea was calculated. the ciliostasis protection score for each group was determined by the following formula: − [(total of the individual scores for the group)/(the number of individuals in the group × ) × ], and a score ≥ was considered protected. a section of each trachea was fixed in % neutral buffered formalin, processed, embedded in paraffin, and -µm sections were cut for hematoxylin and eosin staining. for ibv lesions, epithelial hyperplasia, lymphocyte infiltration, and epithelial deciliation were scored for each trachea. scores were determined as follows: = normal, = focal, = multifocal, and = diffuse, as described previously [ ] . for ndv lesions, a descriptive analysis was performed. for iltv lesions, microscopic lesions were scored on a scale of - (normal to very severe), as described previously [ ] . for ibv and ndv detection, viral rna extraction from µl of the pbs from each swab was conducted using a × magmax- viral isolation kit (thermo fisher, waltham, ma, usa) on a magmax™ express- deep well magnetic particle processor (thermo scientific, waltham, ma, usa), according to the manufacturer's instructions. the quantitative reverse transcription polymerase chain reaction (qrt-pcr) was performed with the agpath-id tm one-step rt-pcr kit (thermo fisher, waltham, ma, usa), following the manufacturer's protocol. each -µl reaction mixture contained . µl of × rt-pcr buffer, µm of each primer, µm of probe, µl of × rt-pcr enzyme mix, and µl of viral rna. the qrt-pcr reactions were run on the applied biosystems ® fast realtime pcr system (life technologies ltd., carlsbad, ca, usa) under the following conditions: one cycle of • c for min and • c for min, followed by cycles of • c for s and • c for s. the primers and probe for the ibv qrt-pcr were previously published [ ] , and are comprised of a forward primer ibv gu ( -gct ttt gag cct agc gtt- ), a reverse primer ibv gl ( -gcc atg ttg tca ctg tct att g- ), and a taqman ® dual-labeled probe ibv g probe ( -fam-cac cac cag aac ctg tca cct c-bhq - ). primers and probe for the ndv qrt-pcr were previously described [ ] and are comprised of a forward primer ndv m+ ( '-agt gat gtg ctc gga cct tc- '), a reverse primer ndv m- ( '-cct gag gag agg cat ttg cta- '), and a taqman ® dual-labeled probe ndv m+ ( '-fam-ttc tct agc agt ggg aca gcc tgc-bhq - '). the primers were obtained from integrated dna technologies (coralville, ia, usa), and the taqman probe was synthesized by biosearch technologies (novato, ca, usa). real-time rt-pcr components and thermocycler parameters were previously described [ ] . the data are expressed as the average cycle threshold (ct) value for all samples in each group, with positive ct values based on the limit of detection for this test associated with virus detection in eggs [ ] . each qrt-pcr reaction plate included a standard curve as an rna extraction control and as a positive control. ga ibv isolated from allantoic fluid was used as the template for the standard curve. negative controls were also included in each plate and consisted of pcr reagents with no rna. for iltv, total dna was extracted from the tracheal swabs using the megazorb ® dna extraction miniprep -well kit (promega, madison, wi, usa), as described previously [ ] . duplex real-time pcr assay that amplifies a fragment of the ul viral gene in iltv and a fragment of the chicken alpha -collagen gene was performed, as previously described [ ] . ibv-specific igg titers were detected using a commercial igg enzyme-linked immunosorbent assay (elisa) ibv antibody test kit (idexx, westbrook, me, usa). briefly, serum samples (stored at − • c) were diluted : , and the procedure was performed according to the manufacturer's protocol. tear ibv-specific iga was detected using a commercial igg elisa ibv antibody test kit (idexx, westbrook, me, usa). briefly, tears were serially diluted two-fold in pbs and incubated in duplicate in wells overnight at • c. all wash steps were performed using pbs-tween ( . % tween ). plates were incubated at • c for h in monoclonal mouse anti-chicken iga-biot ( : , clone a- , southern biotech, birmingham, al, usa), followed by hr in streptavidin-hrp ( : , southern biotech, birmingham, al, usa). final antibody detection steps were completed according to the manufacturer's instructions. endpoint titers were determined by reporting the lowest dilution at which the optical density (od), recorded at nm wavelength, was at least three standard deviations above the mean of control wells incubated with no tear samples. data from wells with a pinpoint color change due to residual substrate or air bubbles were excluded from analysis, and results were reported as log of the endpoint titer. the data were analyzed using prism v. . software (graphpad software, inc., la jolla, ca, usa; www.graphpad.com). for viral load data, a one-way analysis of variance (anova) with dunnet's posttest was used to compare treatment groups within each collection period. all other data were analyzed using a kruskal-wallis test with dunn's posttest to compare treatment groups within each collection period. significant differences were determined at p < . . at days following challenge with ibv ga , vaccinated/challenged birds had significantly lower rna loads compared to positive controls at all collection times and in all tissue samples, with the exception of cecal tonsil at woa (table ). in vaccinated controls, no ibv rna was detected at all collection times except in the cecal tonsils at and woa and in the choanal cleft at woa. ibv loads in negative controls in all tissues were below the limit of detection using the ct value of ≥ . as previously reported [ ] , at all collection times except the woa hg negative controls. clinical signs of ibv infection measured at days post-challenge were significantly reduced in vaccinated/challenged birds when compared to positive controls in all weeks except woa, but trends in clinical sign scores were numerically lower among vaccinated/challenged birds ( table ) . clinical signs were absent in non-challenged negative and vaccinated control birds, and signs in vaccinated/challenged birds were not significantly different from signs in non-challenged controls. notes: sem = standard error of the mean. table . clinical signs and microscopic lesions measured days following ibv ga challenge. letters (a-b) indicate significant differences among vaccine and challenge groups for each week (p < . ). histopathological examination of tracheas from all groups ranged from within normal limits to focal to multifocal minimal to moderate lymphocytic tracheitis; however, moderate lymphocytic infiltration was more frequently seen in positive controls. in all weeks, the proportion of vaccinated/challenged birds with deciliation or acute tracheal necrosis was significantly reduced compared to positive controls and was not different from negative and vaccinated controls (table ) . ciliostasis, defined as the cessation of tracheal ciliary movement, was measured at dpc, and the number of birds positive for ciliostasis and ciliostasis protection scores were calculated for each group (figure ). at all collection times, vaccinated/challenged birds were protected from ciliostasis (scores were > ), and positive controls were not protected (scores were < ). the non-challenged negative controls and vaccinated controls were protected (scores were > ) at all collection times. ibv-specific igg titers were measured in serum collected at dpc. at all times except at woa, vaccinated birds from both non-challenged and challenged groups exhibited significantly higher titers compared to non-vaccinated birds from both non-challenged and challenged groups (figure ). at woa, titers in vaccinated birds, regardless of challenge status, were significantly higher compared to positive controls. titers in vaccinated/challenged birds did not significantly differ from titers in vaccinated controls until woa, when vaccinated/challenged birds had significantly higher titers. compared to negative controls, vaccinated/challenged birds had significantly higher titers at all times. ibv-specific igg titers were measured in serum collected at dpc. at all times except at woa, vaccinated birds from both non-challenged and challenged groups exhibited significantly higher titers compared to non-vaccinated birds from both non-challenged and challenged groups (figure ). at woa, titers in vaccinated birds, regardless of challenge status, were significantly higher compared to positive controls. titers in vaccinated/challenged birds did not significantly differ from titers in vaccinated controls until woa, when vaccinated/challenged birds had significantly higher titers. compared to negative controls, vaccinated/challenged birds had significantly higher titers at all times. ibv-specific iga titers were measured in tears collected at dpc at , , and woa. at woa, titers in vaccinated/challenged birds were significantly higher compared to titers in non-challenged negative controls (figure ). at woa, vaccinated/challenged birds showed significantly lower titers compared to positive controls. in addition, the vaccinated/challenged birds had significantly ibv-specific iga titers were measured in tears collected at dpc at , , and woa. at woa, titers in vaccinated/challenged birds were significantly higher compared to titers in nonchallenged negative controls (figure ). at woa, vaccinated/challenged birds showed significantly lower titers compared to positive controls. in addition, the vaccinated/challenged birds had significantly higher titers than the vaccinated/unchallenged group at woa. no other significant differences were detected. at dpc with ndv b , vaccinated/challenged birds either had undetectable rna loads or significantly lower loads compared to positive control titers at all collection times and in all tissues sampled (table ) . non-challenged negative controls and vaccinated controls were negative for rna virus using the ct value of ≥ . as previously reported [ ] . at dpc with ndv b , vaccinated/challenged birds either had undetectable rna loads or significantly lower loads compared to positive control titers at all collection times and in all tissues sampled (table ) . non-challenged negative controls and vaccinated controls were negative for rna virus using the ct value of ≥ . as previously reported [ ] . table . average qrt-pcr ct values for ndv rna collected from choanal cleft, harderian gland, and conjunctiva days post-challenge with ndv b vaccine. letters (a-c) indicate significant differences among vaccine and challenge groups for each week (p < . ). clinical signs measured at dpc were significant at woa in positive controls, after which clinical signs in positive controls were no different from any of the other treatment groups (table ). no significant differences in clinical signs existed between vaccinated/challenged birds and non-challenged controls at any time. in all weeks, histopathological examination of tracheas was within normal limits or revealed focal to multifocal minimal to mild lymphocytic tracheitis, and there were no group-related differences. tracheas were also evaluated for ciliostasis, and no groups exhibited ciliostasis. ndv-specific serum igg titers in vaccinated/challenged birds and vaccinated controls were significantly higher than titers in both positive and negative controls at all collection times ( figure ). there was no significant difference in titers between vaccinated/challenged birds and vaccinated controls at any time. similarly, no significant difference in titers was detected between non-vaccinated/challenged and negative controls, except at woa in which titers from non-vaccinated/challenged controls were significantly higher. table . clinical signs measured days following ndv b challenge. letters (a-b) indicate significant differences among vaccine and challenge groups for each week (p < . ). clinical signs at dpc with iltv strain , vaccinated/challenged birds had significantly lower viral dna loads than positive controls at all collection times and in all tissues ( table ). the dna loads in the trachea and hg were undetectable or low and did not differ significantly from dna loads in nonchallenged negative and vaccinated controls, which were negative using the ct value of ≥ . as at dpc with iltv strain , vaccinated/challenged birds had significantly lower viral dna loads than positive controls at all collection times and in all tissues ( table ). the dna loads in the trachea and hg were undetectable or low and did not differ significantly from dna loads in non-challenged negative and vaccinated controls, which were negative using the ct value of ≥ . as previously reported [ ] . in the conjunctiva, dna loads in vaccinated/challenged birds were significantly higher than dna loads from both non-challenged negative and vaccinated controls, except at woa in which no significant difference was detected between vaccinated/challenged birds and vaccinated controls. clinical signs and viral dna were detected in challenged birds at dpc, but signs in positive controls had become more severe by dpc as tracheal viral load increased (table ). clinical sign scores measured at dpc were significantly reduced in vaccinated/challenged birds compared to positive controls, at all collection points except woa when clinical sign scores were numerically reduced. all negative controls and vaccinated controls had no clinical signs, and clinical signs in vaccinated/challenged birds were not significantly different compared to these controls, except at woa when clinical sign scores were significantly higher. table . clinical signs measured days following iltv challenge. letters (a-b) indicate significant differences among vaccine and challenge groups for each week (p < . ). clinical signs tracheal histological examination was within normal limits or revealed focal to diffuse minimal to mild lymphocytic tracheitis in tracheas from all groups. for the positive controls, / and / in weeks and , respectively, had acute focal necrotizing tracheitis with syncytia and intranuclear inclusions ( figure ). table . clinical signs measured days following iltv challenge. letters (a-b) indicate significant differences among vaccine and challenge groups for each week (p < . ). tracheal histological examination was within normal limits or revealed focal to diffuse minimal to mild lymphocytic tracheitis in tracheas from all groups. for the positive controls, / and / in weeks and , respectively, had acute focal necrotizing tracheitis with syncytia and intranuclear inclusions ( figure ). no ciliostasis was observed in the tracheas of birds from any of the groups. no ciliostasis was observed in the tracheas of birds from any of the groups. iltv-specific igg titers in serum collected days post-challenge were significantly higher in vaccinated birds from both challenged and non-challenged groups, compared to the positive and negative controls ( figure ). iltv-specific igg titers in serum collected days post-challenge were significantly higher in vaccinated birds from both challenged and non-challenged groups, compared to the positive and negative controls ( figure ). in the present study, we demonstrated that pullets serially administered live attenuated vaccines against ibv, ndv, and iltv were protected against homologous challenge with ibv, ndv, or iltv for at least weeks, as determined by challenge virus detection, clinical signs, histopathology and ciliostasis at days after challenge. additionally, our study showed that the age at vaccination and intervals between each vaccination did not interfere with the development of immunity to each virus and consequently protection against homologous challenge. we designed our vaccination protocol to represent a typical vaccination program for ibv, ndv, and iltv in commercial pullets. with the knowledge that live vaccine viruses can persist in flocks, it has been unclear, until now whether the immunity induced by a live vaccines could be compromised because of viral interference, a phenomenon in which one replicating virus blocks the infection and/or replication of another virus [ , ] . although the vaccines in the present study were administered at intervals of or weeks, it is feasible that virus from a previous immunization was still present at the time of the subsequent vaccination. ibv vaccines have been detected in the respiratory tract up to days post-vaccination [ ] , and ibv was isolated from tracheal and cloacal swabs collected at the point of lay and weeks of age in hens that had been virus-negative for several weeks following recovery from inoculation at one day of age [ ] . fentie, et al. [ ] reported that chickens vaccinated with ndv b shed vaccine virus days post-inoculation. in the present study, we demonstrated that pullets serially administered live attenuated vaccines against ibv, ndv, and iltv were protected against homologous challenge with ibv, ndv, or iltv for at least weeks, as determined by challenge virus detection, clinical signs, histopathology and ciliostasis at days after challenge. additionally, our study showed that the age at vaccination and intervals between each vaccination did not interfere with the development of immunity to each virus and consequently protection against homologous challenge. we designed our vaccination protocol to represent a typical vaccination program for ibv, ndv, and iltv in commercial pullets. with the knowledge that live vaccine viruses can persist in flocks, it has been unclear, until now whether the immunity induced by a live vaccines could be compromised because of viral interference, a phenomenon in which one replicating virus blocks the infection and/or replication of another virus [ , ] . although the vaccines in the present study were administered at intervals of or weeks, it is feasible that virus from a previous immunization was still present at the time of the subsequent vaccination. ibv vaccines have been detected in the respiratory tract up to days post-vaccination [ ] , and ibv was isolated from tracheal and cloacal swabs collected at the point of lay and weeks of age in hens that had been virus-negative for several weeks following recovery from inoculation at one day of age [ ] . fentie, et al. [ ] reported that chickens vaccinated with ndv b shed vaccine virus days post-inoculation. in addition, a study by hughes, et al. [ ] demonstrated intermittent shedding in the trachea from iltv-immunized chickens between and weeks post-vaccination. few experimental studies of sequential virus infections have been published, and fewer yet have been considered in the context of poultry viral respiratory pathogens. costa-hurtado, et al. [ ] demonstrated that chickens and turkeys serially infected with a mesogenic strain of ndv and highly pathogenic avian influenza virus (hpaiv) days apart resulted in an initial decrease followed by a subsequent increase in replication of the second virus. in a subsequent study [ ] , the same group found that low pathogenic avian influenza virus given days after a lentogenic strain of ndv did show viral interference whereas viral interference was not observed when the viruses were given simultaneously. we did not measure vaccine virus replication in the present study and, therefore, could not determine whether one vaccine virus compromised the infection and replication of a subsequent vaccine virus. however, our goal was to determine if chickens sequentially vaccinated with all three viruses were protected from viral replication and clinical signs following homologous challenge. thus, regardless of viral interference, our data shows that immunity to individual vaccine viruses was not compromised following sequential administration of multiple live attenuated vaccines targeting different viral respiratory tract pathogens. the detection of ibv rna in the cecal tonsils of vaccinated/non-challenged birds at and woa but not at subsequent times indicates that residual vaccine virus rna remained in the cecal tonsils until at least woa, following the second ibv vaccination. ibv has been isolated from the cecal tonsils at weeks post-infection and is known to persist for several months in various internal organs [ , ] . ibv rna was also detected in non-challenged negative control hg at woa but was absent from all other tissues collected from negative controls, which displayed no clinical signs of ibv infection. it is not clear why we detected ibv rna in samples from the hg in negative control birds but likely represents cross-contamination during processing of the samples since ibv was not detected in any other tissue. the tracheal histopathological lesions of deciliation in positive controls were consistent with previous reports of ibv-induced histopathology [ , ] , and were further confirmed by the presence of ciliostasis among positive controls. as expected, ibv-vaccinated/challenged birds were protected from ciliostasis. the european pharmacopoeia states that ciliostasis can be used to evaluate ibv vaccine efficacy, in which a lack of ciliostasis would indicate that the vaccine was efficacious [ ] . therefore, these observations further confirm that ibv-vaccinated birds were protected from homologous challenge. the observation of robust ibv-specific serum igg titers in vaccinated birds is consistent with previous studies showing that ibv infection stimulates a humoral response in chickens [ ] , but that circulating antibody titers do not correlate with resistance to infection [ ] . therefore, the presence of ibv-specific serum igg titers indicates only that the bird has been exposed to vaccine or challenge virus and should not be correlated with other measures of protection. the lack of significant titers in non-vaccinated/challenged birds can be explained by the early time of collection post-challenge. orr-burks, et al. [ ] found that significant changes in igg serum titer were not detected until days post-inoculation. orr-burks, et al. [ ] also demonstrated a lack of significance in ibv-specific iga titer in tears days after both primary and secondary exposure to ibv, but iga titer was significantly higher between and days after primary exposure to ibv. in this study, ibv-specific iga titers measured in tears at dpc did not reveal consistent trends and may be explained by the early time of collection post-infection. it was beyond the scope of this study to collect samples after dpc, but the production of ibv-specific iga in tears at later time points was demonstrated in a different experiment in which tears were collected between and dpc. in that experiment, naïve chickens challenged with ibv showed a higher trend of specific iga titer compared to non-challenged controls [ ] . the decision to use the b vaccine for ndv challenge was based on biosecurity regulations and the lack of appropriate biosafety level facilities needed for challenge with mesogenic or velogenic strains of ndv. since we used a lentogenic strain of ndv as challenge virus in our experimental design, protection was primarily based on significantly reduced or no virus detection at days after challenge, which is a measure of local immunity preventing virus infection and/or replication. presumably a bird protected from infection with a lentogenic strain of ndv would also show some level of protection from exposure to mesogenic or velogenic strains. vaccinated birds at each sampling time had significantly lower or no challenge virus rna compared to positive control groups, indicating that the vaccinated birds developed a local immune response and were indeed protected whereas the non-vaccinated positive controls were not. because lentogenic ndv only causes a mild respiratory or enteric infection [ ] , it was not surprising that respiratory signs and histological changes were mild or absent despite the presence of viral rna. we observed clinical signs of ndv infection only at woa in positive controls, while only a few birds showed mild clinical signs at and woa, and no clinical signs of disease were observed at later challenge times. this observation is consistent with existing knowledge that nd tends to be more severe in younger birds [ ] . the lack of ciliostasis observed in ndv b -infected positive controls contrasts with previous reports demonstrating that ndv caused ciliostasis in tracheal explants. butler, et al. [ ] demonstrated that ndv caused ciliostasis within to days after infection of tracheal explants, and malo, et al. [ ] reported that following vaccination of one-day-old chicks with lentogenic ndv, the peak of ciliostasis occurred at and dpv and waned by dpv. the discrepancy between our results and the previous studies may be explained by the use of a different lentogenic ndv strain; b in our studies and lasota in previous reports [ ] . it is well established that the lasota vaccine strain is more virulent than b [ ] , which may explain the ciliostasis observed in lasota-vaccinated chicks or lasota-infected tracheal explants, whereas ciliostasis was not observed in our study using b vaccine. robust ndv-specific circulating igg antibody responses developed following ndv vaccination and stayed elevated, which was consistent with prior research indicating that antibodies may be detected for up to one year in birds immunized multiple times against ndv [ ] . with the exception of woa, the igg titers in non-vaccinated/challenged birds were not significantly increased compared to titers in negative controls. this observation is not surprising given that ndv-specific antibodies are not detected in the serum until - days after exposure [ ] . for the iltv-challenged birds, vaccination prevented challenge virus replication in trachea and hg but did not completely block virus replication in the conjunctiva, especially at and woa. this may suggest a waning of the local immunity against iltv after the second vaccination at woa. in addition, the proximity of the conjunctiva to the inoculation site (eyedrop and intranasal) might explain why the local immune response was not able to completely block viral replication in the conjunctiva but successfully cleared the virus before it could replicate in hg and trachea. very few positive controls (non-vaccinated/challenged) demonstrated histological evidence of ilt infection in the trachea, though all positive controls tested positive for ilt dna by pcr. this finding is not surprising in light of a report by guy, et al. [ ] , which illustrated that histological detection of ilt is highly specific ( %) but poorly sensitive ( %). notably, intranuclear inclusion bodies are present only during the initial infection ( - days) and disappear following epithelial cell necrosis and desquamation [ ] , which may explain the observation that by dpc only / and / positive controls at and woa, respectively, had histological evidence of ilt infection despite the presence of ilt dna in the trachea. the absence of ciliostasis observed in both vaccinated and non-vaccinated, iltv-challenged birds at dpc was not surprising given that butler, et al. [ ] found that only some strains of iltv caused ciliostasis and did not correlate with virulence. moreover, the authors showed that ciliostasis rarely occurred before days, and sometimes even days, after inoculation. in addition, gerganov and surtmadzhiev [ ] also demonstrated ciliostasis in iltv-infected tracheal organ cultures at - days post-infection. therefore, our results combined with previous studies indicate that measuring ciliostasis may not be a reliable marker of protection from iltv infection and that ciliostasis in iltv infection studies may need to be evaluated at later times post-inoculation. the lack of significant iltv-specific serum igg titers in non-vaccinated positive controls may be explained by the early time of collection post-challenge, as iltv-specific antibodies do not become detectable until - dpi and peak at dpi [ ] . however, it is worth noting that antibody titers are not correlated to protection from iltv infection [ ] . igg titers were robust in vaccinated birds throughout the study and support previous data that antibodies may be detectable for at least a year [ ] . taken together, our data indicate that a typical pullet vaccination program consisting of serially administered live attenuated vaccines against ibv, ndv, and iltv does not interfere with immune responses to the individual vaccines, and the birds are adequately protected against homologous challenge until at least woa. this information is important because it shows that consecutively administered live attenuated vaccines against multiple respiratory pathogens can be an effective vaccination strategy for the development of protective immunity against each disease agent in long-lived birds. disease prevention and diagnosis infectious bronchitis virus variants: a review of the history, current situation and control measures the role of vaccination in risk mitigation and control of newcastle disease in poultry infectious bronchitis identification of vaccine strains of 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compare safety of live-attenuated respiratory newcastle disease vaccines in young chicks evaluation of the effect of live lasota newcastle disease virus vaccine as primary immunization on immune development in broilers rapid diagnosis of infectious laryngotracheitis using a monoclonal antibody-based immunoperoxidase procedure diseases of poultry cultivation of various avian viruses in pheasant trachea organ cultures and chick embryos studies on a serum neutralization test for the diagnosis of laryngotracheitis in chickens the authors declare no conflict of interest. key: cord- -iipyg ab authors: lee, john r.; huang, jennifer; magruder, matthew; zhang, lisa t.; gong, catherine; sholi, adam n.; albakry, shady; edusei, emmanuel; muthukumar, thangamani; lubetzky, michelle; dadhania, darshana m.; taur, ying; pamer, eric g.; suthanthiran, manikkam title: butyrate‐producing gut bacteria and viral infections in kidney transplant recipients: a pilot study date: - - journal: transpl infect dis doi: . /tid. sha: doc_id: cord_uid: iipyg ab background: the gut microbiome is being associated increasingly with development of infections besides clostridium difficile infection. a recent study found an association between butyrate‐producing gut (bpg) bacteria and less frequent development of lower respiratory viral infections in allogeneic hematopoietic stem cell transplant recipients (haak et al, blood ( ): , ). in this investigation, we examine the relationship between the abundance of bpg bacteria and the development of viral infections in a cohort of kidney transplant recipients. methods: we recruited kidney transplant recipients who provided fecal specimens in the first months after transplantation and profiled the gut microbiota using s rrna gene sequencing of the v ‐v hypervariable region. we classified the kidney transplant recipients into higher bpg bacteria group and lower bpg bacteria group using the same criteria of % relative gut abundance of bpg bacteria as the haak et al study. results: administration of antibiotics against anaerobes was associated with a significant decrease in the relative gut abundance of bpg bacteria. the higher bpg bacteria group was associated with less development of respiratory viral infections (hazard ratio [hr]: . , p = . ) but not with less development of cmv viremia (hr: . , p = . ) or bk viremia (hr: . , p = . ) at years post transplantation. conclusion: our pilot investigation supports future validation of the relationship between high relative gut abundance of bpg bacteria and decreased risk for development of respiratory viral infections. strains of escherichia coli and klebsiella pneumoniae that caused septicemia likely originated from the gut. the relationship between the gut microbiota and development of viral infections, however, is not well described. studies in mice have shown a relationship between the gut microbiota and impaired viral clearance. abt et al investigated antibiotic administration in a mouse model of lymphocytic choriomeningitis virus and found that antibiotic administration led to decreased innate viral immunity response as well as delayed clearance. further studies have revealed that butyrate, a product of certain gut anaerobic bacteria, can have an immunomodulatory role and contributes to overall health in distant sites such as the lung. haak et al investigated the role of butyrate-producing gut (bpg) bacteria on future development of viral infections. in a cohort of allogeneic hsct recipients, they reported that having a > % relative gut abundance of bpg bacteria is associated with -fold less future development of lower respiratory viral infections. based upon this study, we profiled the gut microbiota using s rrna gene sequencing of the v -v region in fecal specimens from kidney transplant recipients. we report that having a > % relative abundance of bpg bacteria is associated with less risk for development of respiratory viral infections in kidney transplant recipients, which provides further support for the findings from the haak et al study. kidney transplant recipients provided fecal specimens using the fisherbrand ™ commode specimen collection kit (thermo fisher science). fecal specimens were aliquoted into approximately mg aliquots and subsequently stored at − °c. the recipients were asked to provide the specimens at post-transplant week , , , and . dna extraction and s rrna gene amplification of the s rrna gene v -v region ( f and r) were performed as described in lee et al. sequencing of the pcr amplicons was performed on an illumina miseq platform ( base pair × base pair). bioinformatics and taxonomic classification were performed as described in lee et al. briefly, taxonomy was determined using nucleotide blast with the reference training set, ncbi refseq and a minimum e-value threshold of × − . the distribution of continuous variables was analyzed using the twotailed wilcoxon rank-sum test for unpaired values and the two-tailed wilcoxon signed-rank test for paired values; the distribution of categorical variables was analyzed using two-tailed fisher's exact test. a cox regression hazard model was used to estimate whether a relative gut abundance of bpg bacteria was associated with a decreased risk for development of respiratory virus infections, cmv viremia, or bk viremia. survival curves were constructed and compared using the log-rank test. all analyses were performed in r . . in rstudio . . . among the fecal specimens from the kidney transplant recipients, the mean ± standard deviation amount of high quality s rrna bacterial gene sequences was ± . the overall demographics and transplant characteristics of these subjects are presented in table . out of the bpg species, species were detected (table s ). the sum relative gut abundance of bpg bacteria was calculated for each fecal specimen (defined hereafter as relative gut abundance of bpg bacteria). the mean relative gut abundance of bpg bacteria among all of the fecal specimens was . % with a standard deviation of . %. the top bpg species were: faecalibacterium prausnitzii ( . %, mean), holdemanella biformis ( . %), eubacterium dolichum ( . %), coprococcus comes ( . %), subdoligranulum variabile ( . %), and eubacterium rectale ( . %). a heatmap of the relative gut abundance of these top species among all the fecal specimens is presented in figure . f i g u r e heatmap of the top abundant butyrate-producing gut bacteria in the kidney transplant cohort. the fecal specimens from the kidney transplant recipients are represented on the x-axis and the top butyrateproducing gut species are on the y-axis. the relative abundance of each species is represented in blue, log-scaled. there is marked variability in the top butyrateproducing species among the specimens signed-rank test; figure d ). we next evaluated the subgroup of subjects in the paired abx group who received anaerobic antibiotic coverage. anaerobic antibiotic coverage included the following antibiotics: metronidazole, carbapenems, clindamycin, combination of penicillin and beta-lactamase inhibitor, and oral vancomycin. , the rationale for evaluating antibiotics with anaerobic coverage was based on the strict anaerobic nature of many of the bpg bacteria (eg, faecalibacterium prausnitzii and eubacterium rectale). among the paired abx group, subjects had anaerobic antibiotic coverage and all had a significant decrease in the relative gut abundance of bpg bacteria from post-transplant week to post-transplant week (median . % vs . %, respectively, p = . , wilcoxon signed-rank test; figure e ). we also evaluated whether the relative gut abundance of bpg bacteria was associated with gender, age greater than years old, african american race, induction therapy, steroid maintenance therapy, and kidney function at month post transplantation. as antibiotics are associated with decreased abundance of bpg bacteria, we evaluated the paired specimens from the subjects who did not receive antibiotics from post-transplant week and post-transplant the abundance of butyrate-producing gut bacteria over time in the kidney transplant cohort. panel a. each point represents a single fecal specimen with the day the specimen was produced on the x-axis and the relative gut abundance of butyrate-producing gut (bpg) bacteria on the y-axis. the line represents a locally estimated scatterplot smoothing (loess) curve with % confidence intervals indicated by the shaded band. panel b. each point represents a single fecal specimen with the day the specimen was produced on the x-axis and the relative gut abundance of butyrate-producing gut (bpg) bacteria on the y-axis. a point in blue represents an individual fecal specimen from the specimens from the patients in the no abx group and a point in magenta represents an individual fecal specimen from the specimens from the patients in the abx group. the line represents a loess curve with % confidence intervals indicated by the shaded bands. panel c. box and whisker plot with the relative gut abundance of bpg bacteria on the y-axis and the fecal specimen posttransplant week on the x-axis for the subjects in the paired no abx group. in each box and whisker plot, the line represents the median, the edges of the box plot % and %, the whiskers . times the median, and points represent outliers. the p value was calculated using the wilcoxon signed-rank test. panel d. box and whisker plot with the relative gut abundance of bpg bacteria on the y-axis and the fecal specimen post-transplant week on the x-axis for the subjects in the paired abx group. in each box and whisker plot, the line represents the median, the edges of the box plot % and %, the whiskers . times the median, and points represent outliers. the p value was calculated using the wilcoxon signed-rank test. panel e. box and whisker plot with the relative gut abundance of bpg bacteria on the yaxis and the fecal specimen post-transplant week on the x-axis for the subjects in the paired abx group who received antibiotics with anaerobic coverage. in each box and whisker plot, the line represents the median, the edges of the box plot % and %, the whiskers . times the median, and points represent outliers. the p value was calculated using the wilcoxon signed-rank test week (paired no abx group). the relative gut abundance of bpg bacteria was significantly lower in the female group than in the male group (median . % vs . %, p = . , wilcoxon rank-sum test; figure s a ) and significantly lower in the steroid maintenance group than in the in steroid free group (median . % vs . %, p = . , wilcoxon rank-sum test; figure s e ). there were no significance associations between the relative gut abundance of bpg bacteria and age greater than years old, african american race, induction therapy, and kidney function at month post transplantation ( figure s b -d,f). we evaluated whether the relative gut abundance of bpg bacte- in this study, we report one of the first description of butyrate-producing gut bacteria over time in the kidney transplant population. our data reveal that antibiotics with anaerobic coverage is associated with a decrease in relative gut abundance of bpg bacteria. our data also support a relationship between high relative gut abundance of bpg bacteria and less risk for development of respiratory it is important to note the allogeneic hsct subjects had a much higher proportion of patients with a less than % relative gut abundance of bpg bacteria than the kidney transplant recipients in our study ( % vs. %, respectively). we speculate that this difference is likely because of the higher number of antibiotics given to the allogeneic hsct population. in our study, we found that an- analyses. in addition, we used the cutoff of % relative gut abundance of bpg bacteria, which was utilized in the allogeneic hsct recipient population. notably, there was a significance difference in the proportion of hsct recipients and in the proportion of kidney transplant recipients with less than % relative gut abundance of bpg bacteria and whether this cutoff is also relevant for the kidney transplant population is unknown. in order to address this concern, we evaluated both a bottom quartile cutoff as well as a median cutoff and we did find an association between high relative gut abundance of bpg bacteria and decreased risk for respiratory despite these limitations, our preliminary study provides one of the first descriptions of bpg bacteria in kidney transplant recipients early after kidney transplantation. we find that antibiotics with anaerobic coverage are associated with a decrease in the relative gut abundance of bpg bacteria and we also report an association between the relative gut abundance of bpg bacteria and the risk for development of respiratory viral infections in kidney transplant recipients. our results are preliminary in nature and require replication in a larger cohort of kidney transplant recipients. in particular, we did report a trend toward significance of the association between the relative gut abundance of bpg bacteria and cmv viremia, which would need further validation for the cmv viremia finding as well as for cmv disease. our study examined the relative gut abundance of bpg bacteria, but future studies examining the absolute gut abundance of bpg bacteria and/or fecal butyrate concentrations may provide more insight into the relationship between bpg bacteria and development of viral complications. nevertheless, if our findings are confirmed, our data support therapeutic options in the form of modulating the gut microbiota for decreasing viral infection rates. we thank the national institutes of allergy and infectious diseases for their research support via nih grant k ai (jrl) and r ai (ms). the authors of this manuscript have the following competing interests to disclose. egp has received speaker honoraria from bristol myers squibb, celgene, seres therapeutics, medimmune, novartis, and ferring pharmaceuticals and is an inventor on patent application # wpo a , entitled "methods and compositions for reducing clostridium difficile infection" and #wo a , enti- intestinal domination and the risk of bacteremia in patients undergoing allogeneic hematopoietic stem cell transplantation precision identification of diverse bloodstream pathogens in the gut microbiome commensal bacteria calibrate the activation threshold of innate antiviral immunity butyrate: a double-edged sword for health? impact of gut colonization with butyrate-producing microbiota on respiratory viral infection following allo-hct gut microbiota dysbiosis and diarrhea in kidney transplant recipients basic local alignment search tool refseq microbial genomes database: new representation and annotation strategy treatment of anaerobic infection in vitro activity of vancomycin and teicoplanin against anaerobic bacteria bifidobacteria and butyrate-producing colon bacteria: importance and strategies for their stimulation in the human gut body mass index differences in the gut microbiota are gender specific key: cord- - zlu g y authors: nan, yuchen; zhang, yan-jin title: antisense phosphorodiamidate morpholino oligomers as novel antiviral compounds date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: zlu g y phosphorodiamidate morpholino oligomers (pmo) are short single-stranded dna analogs that are built upon a backbone of morpholine rings connected by phosphorodiamidate linkages. as uncharged nucleic acid analogs, pmo bind to complementary sequences of target mrna by watson–crick base pairing to block protein translation through steric blockade. pmo interference of viral protein translation operates independently of rnase h. meanwhile, pmo are resistant to a variety of enzymes present in biologic fluids, a characteristic that makes them highly suitable for in vivo applications. notably, pmo-based therapy for duchenne muscular dystrophy (dmd) has been approved by the united states food and drug administration which is now a hallmark for pmo-based antisense therapy. in this review, the development history of pmo, delivery methods for improving cellular uptake of neutrally charged pmo molecules, past studies of pmo antagonism against rna and dna viruses, pmo target selection, and remaining questions of pmo antiviral strategies are discussed in detail and new insights are provided. researchers realized decades ago that antisense nucleic acids could be used to treat diseases. since then, antisense therapies using a variety of nucleic acids or nucleic acid analogs have been evaluated for use as therapeutic compounds in numerous applications. among these candidate treatments, morpholino oligos, also known as phosphorodiamidate morpholino oligomers (pmo), have demonstrated promising effectiveness in developmental biology research involving gene knockdown as well as clinical trials focusing on treatments of genetic disorders (summerton, ) . specifically, pmos are short single-stranded dna analogs that contain a backbone of morpholine rings connected by phosphorodiamidate linkages (summerton, ) . due to the neutral charged property, they are less likely to interact with proteins while maintain the binding to nucleic acids (moulton and jiang, ). more specifically, pmo bind to complementary sequences of target mrna by watson-crick base pairing and block mrna translation through sequence-specific steric blockade. this process is distinct from the rnase h-dependent mechanism for protein translation inhibition, as induced by other antisense compounds such as phosphorothioate dna (summerton, ) . importantly, pmo are resistant to a variety of enzymes in biologic fluids, which makes them highly suitable for in vivo applications (hudziak et al., ) . to date, pmo-based therapy for duchenne muscular dystrophy (dmd) has shown great success by bypassing effects of a gene mutation underlying a human disease. specifically, this pmo-based therapy restores production of functional dystrophin by altering rna splicing to remove the mutated dystrophin exon that disrupts downstream full-length dystrophin protein translation from the mutated mrna. this drug has been approved by the u.s. food and drug administration and is available in the market under the trade name exondys tm (eteplirsen). approval of exondys tm is a hallmark of pmo-based antisense therapy that demonstrates the potential safety and effectiveness of pmo technology, is driving future development of such therapies for treatment of other diseases. aside from genetic disorders, pmo therapies have also been evaluated as treatments for other broad categories of untreatable diseases, including viral infections, antibiotic-resistant bacterial infections and cancers (enterlein et al., ; warfield et al., ; cansizoglu and toprak, ; chen et al., ) . in this review, pmo background, as well as research focusing on intracellular delivery of pmo, pmo efficacy against both dna and rna viruses, and pmo target selection are outlined. finally, current challenges for development of successful pmo antiviral strategies are discussed in detail and new insights are provided. the concept of an antisense oligonucleotide (on) as a potential therapeutic agent was first demonstrated experimentally through on targeting of a translation initiation site within rous sarcoma virus rna. the on mechanism was initially explained by its participation in formation of an rna-dna duplex with viral rna that sterically blocks viral gene expression and ultimately prevents viral replication (zamecnik and stephenson, ) . subsequent studies revealed that on also participated in a second inhibitory mechanism involving rna:dna duplex recognition by the cellular enzyme rnase h, resulting in rna cleavage and abrogation of virus gene expression as well (tadokoro and kanaya, ). more recently, rnase h-mediated degradation of target rna has been shown to be the main rnainterference mechanism responsible for microrna (mirna), small interference rna (sirna), and dna-directed rna interference (saurabh et al., ) . due to their effectiveness and specificity, such technologies have subsequently attracted the attention of global researchers as tools for therapeutic purposes (davis et al., ; millington-ward et al., ; wittrup and lieberman, ) . the first antisense on tested in clinical trials beginning in was designed to target p for treatment of acute myelogenous leukemia (bayever et al., ) . as a medical milestone for the application of antisense technology, the first antisense drug (fomivirsen) was approved by the fda in for treatment of cytomegalovirus retinitis in immunodeficient patients (marwick, ; wikipedia, ) . since then, numerous antisense drugs have been tested in clinical trials for a variety of other human diseases. to date, five antisense compounds have received marketing authorization from the fda, including eteplirsen. currently, more than ongoing clinical trials of antisense compounds are listed on the website clinicaltrials.gov (godfrey et al., ) . however, compared with traditional drug development, industrial development of therapeutic sirna, dna-based on, or on analogs for control of gene expression have been less successful, as exemplified by the removal of the first commercially available antisense drug, fomivirsen, from the market. a key feature underlying the effectiveness of antisense on is that their nuclease resistance helps them to remain intact for hours in extracellular medium or within cells (summerton, ) . methylphosphonate-linked dna analogs developed in the late s constituted a major advance in the emerging antisense field, resulting in production of the first antisense drug exhibiting acceptable stability in biological systems (hudziak et al., ) . however, in addition to stability, the specificity of rnase-hbased antisense therapy is an important concern, since rnase-h cleaves dna/rna duplexes as short as -or -base pairs in length and is highly active against duplexes that are only - base pairs in length (monia et al., ; summerton, ) . therefore, rnase-h-independent steric blockage of antisense on may be a safer strategy. notably, the effective target region of the steric blocking agent on for inhibiting translation is generally limited to the -utr (untranslated region) and start codon regions of mrna, achieving a good specificity with fewer adverse off-target effects. furthermore, the binding of an rnase h-independent antisense on to a partially matched rna sequence is unlikely to have biological consequences (lebleu et al., ) . oligomers possessing a morpholino phosphorodiamidate backbone, also called pmo (figure ) , constitute a novel type of on analog that is synthesized from ribosides. the ribose ring is opened by oxidation, re-closed using ammonia, which forms a substituted morpholine moiety (summerton and weller, ) . next, the phosphodiester intersubunit bonds are replaced with phosphorodiamidate linkages (summerton and weller, ) . pmo demonstrate excellent resistance to nucleases, figure | comparison of chemical structures of pmo and dna. illustration of phosphorodiamidate morpholino oligomer (pmo) contains backbone of morpholine rings connected by phosphorodiamidate linkages. the ribose ring is opened by oxidation, re-closed using ammonia to form a substituted morpholine moiety. the phosphodiester intersubunit bonds are replaced with phosphorodiamidate linkages. proteases, esterases, and a variety of other enzymes present in biologic fluids (hudziak et al., ) . matrix-assisted laser desorption ionization-time of flight mass spectrometry (maldi-tof ms) analysis has demonstrated that pmo are completely resistant to different hydrolases in serum and plasma (hudziak et al., ) , bolstering pmo suitability for in vivo applications (hudziak et al., ) . furthermore, as uncharged molecules, pmo do not interact strongly with proteins, which minimizes hybridization-independent protein interactions since decreased effectiveness of on is likely due to their charged phosphorothioate backbone (moulton and jiang, ; hagedorn et al., ) . pmos bind to complementary sequence in target mrna by watson-crick base pairing to block translation through rnase h-independent steric blockade (figure ) (lebleu et al., ) . moreover, pmo targeting intron-exon junction sequence is capable to modulate pre-mrna splicing as well (figure ) (havens and hastings, ) . for above reasons, antisense pmos have been a revolutionary tool in developmental biology (eisen and smith, ) . by microinjecting pmos into eggs or single or pauci-cellular zygotes, pmos are apportioned into daughter cells during cell division to ensure their delivery to each cell during subsequent cell proliferation (heasman et al., ) . in addition to their use for studies of gene function during embryonic development, pmo have been also tested as treatments for a broad range of diseases in numerous clinical trials (mainly promoted by avi biopharma, inc., now sarepta biopharma inc.). however, application of unmodified pmo is greatly limited by inefficient in vivo delivery unless a relatively high doses are administrated (moulton and jiang, ). in fact, in the animal dmd model, high doses of unmodified pmo injection in dystrophic muscle were needed to induce functional dystrophin expression in skeletal muscle (moulton and jiang, ). thus, two forms of pmos with distinct chemical modifications were developed to facilitate intracellular delivery of pmos, including peptide-conjugated pmo(ppmo) and vivo-pmo. moreover, pmoplus, another novel form of positively charged pmo with a morpholino oligomer-based backbone, has been recently developed as the latest version of pmo. these novel pmo forms are discussed in detail below. initial techniques to deliver pmo into cultured cells were based on mechanical delivery methods such as microinjection or scraping (moulton et al., ) , with associated limitations. more recently, additional mechanical scraping methodologies, electroporation, or use of endosomal escape reagents have been evaluated to improve delivery of on into cytosolic or nuclear compartments in tissue culture (partridge et al., ; moulton and jiang, ) . currently, the most intensively developed and widely used in vivo pmo delivery strategy is based on the use of arginine-rich cell penetrating peptide (cpp) (lebleu et al., ; morcos et al., ) . cell penetrating peptide, also known as protein transduction domain (ptd) or tat-cpp (dietz and bahr, ) , was originally identified after discovery of an unexpected property of the human immunodeficiency virus (hiv) trans-activator of transcription (tat) protein (dietz and bahr, ) . the novel activity was revealed by observations that tat could transactivate a hiv- ltr promoter after crossing cellular and nuclear membranes (dietz and bahr, ) . subsequent structural analysis pinpointed a short peptide located in aa - of tat (sequence grkkrrqrrrppq) that is responsible for the membrane-crossing activity of the parent tat protein (vives et al., ; abes et al., a) . this novel peptide sequence, later named as cpp, was then evaluated for its ability to confer membrane-crossing abilities to other proteins and compounds, including pmo. after fluorescence microscopy or flow cytometry analysis of cells treated with fluoresceintagged pmo, cpp conjugation remarkably enhances cellular uptake of pmo by -to -fold compared with non-conjugated pmo (alonso et al., ) . moreover, other cationic peptides conjugated pmo were shown to be much less effective than pmo conjugated to tat-cpp, while tat-cpp significantly enhanced delivery of pmo to nearly all cells assayed (moulton et al., ) . furthermore, cpp-mediated delivery is a much simpler procedure to conduct than mechanical delivery methods. however, tat-cpp mediated pmos delivery required high pmos concentrations (above µm) to achieve therapeutic antisense activity with cytotoxicity observed. meanwhile, tat-cpp pmos conjugate studies established that the conjugate associated with cell membranes and that internalized conjugate localized to vesicles, cytosol, and nucleus (moulton et al., ) . therefore, cpp sequence optimization, to reduce cytotoxicity and increase uptake efficiency, should enhance pmo effectiveness. this concept has prompted a comparison of two types of cpp (rxr peptide and r pen peptide) (lebleu et al., ) . the most efficient cpp in this study was (r-ahx-r) r, in which ahx represents a -aminohexanoic acid spacer lebleu et al., ) . importantly, (r-ahx-r) r-pmo conjugates were shown to be effective in several murine viral infection models , as well as in treatment of duchenne muscular dystrophy. unfortunately, in some applications, effective doses have approached cytotoxic levels. this obstacle has limited their use in clinical settings (abes et al., b) . vivo-pmo exploits a non-peptide-based transporter to deliver pmo into cultured cells or tissues (morcos et al., ) . currently, gene tools llc (philomath, or, united states) is the major supplier of vivo-pmo for research and development applications. unlike cpp-conjugated pmos, vivo-pmo are covalently linked to a molecular scaffold that carries a dendritic structure assembled around a triazine core that holds eight guanidinium head groups optimally oriented for cell membrane penetration (morcos et al., ; ferguson et al., ) . vivo-pmo effectively entered in vitro cultured cells as well as a wide variety of mouse tissues in vivo to induce correction of a pre-mrna splicing error, as detected by using an experimental test system designed to detect such an event in cells and tissues (morcos et al., ) . compared with cpp-pmos, vivo-pmo have been less frequently investigated for inhibitory effects against target genes. however, available data suggest that at least a % knockdown of target genes can be achieved using vivo-pmo, with no adverse side effects both in vitro and in vivo (guo et al., ; kang et al., ; nazmi et al., ; reissner et al., ) . meanwhile, mouse model studies have also shown that intravenous (iv) and intraperitoneal (ip) administration of vivo-pmo were equally efficacious (reissner et al., ; sartor and aston-jones, ) . furthermore, it appears that vivo-pmo is less cytotoxic than cpp-pmo, since only one report has demonstrated cytotoxicity of vivo-pmo (ferguson et al., ) . it appears that the dendrimer of vivo-pmo is capable to induce red blood cell sedimentation, prompting ferguson et al. to recommend that oligonucleotide analogs should be analyzed for potential to base pair hybridization that may induce dendrimer clustering. moreover, supplementation of vivo-pmo with physiological saline or anticoagulation therapy holds promise for counteracting vivo-pmo toxicity (ferguson et al., ) . compared to cpp-conjugated pmo and vivo-pmo, pmoplus tm is newer type of charged pmo that contain positively charged piperazine groups within its molecular backbone . pmoplus is the most recently developed form of pmo and studies have demonstrated this type of pmo is well tolerated and exhibits improved efficacy in numerous in in vivo viral infection models relative to other pmo therapies (swenson et al., ; warren et al., warren et al., , warren et al., , meng et al., ) . however, pmoplus is still unavailable to most researchers due to its proprietary status as technology solely owned by avi. however, there are reports available to date suggest that pmoplus may be less cytotoxic than cpp-pmo. in a phase i clinical trial to evaluate pmoplus compounds against ebola virus, healthy male and female subjects between and years of age in six dose-expansion cohorts of subjects per dosage group, each received a single i.v. infusion of the active study drug ( . , . , . , . , , or . mg/kg pmoplus). results demonstrated that pmoplus treatments were safe and well tolerated at these doses studied (heald et al., ) , with safety superior to that of cpp-pmo or vivo-pmo. however, a systematic investigation is still needed to compare vivo-pmo and pmoplus with regard to efficiency, stability, and cytotoxicity. an illustration of chemical structures of cpp-pmo, vivo-pmo and pmoplus tm was listed as pmo have been explored as antiviral compounds against rna viruses, including ebola virus, flavivirus, coronavirus, picornavirus, and others. in this section, major advances in this research field are presented. the filoviruses, including marburg virus and ebola virus (ebov), are negative-sense, single-stranded rna viruses that are highly pathogenic, causing human outbreaks of viral figure | chemical structures of cpp-pmo, vivo-pmo, and pmoplus tm . pmo conjugated with cell penetrated peptide (r-ahx-r) r, in which ahx represents a -aminohexanoic acid spacer. vivo-pmo are covalently linked to a molecular scaffold that carries a dendritic structure assembled around a triazine core that holds eight guanidinium head groups optimally oriented for cell membrane penetration. pmoplus tm is charged pmo that contains positively charged piperazine groups within its molecular backbone. hemorrhagic fever with up to % fatality. the - epidemic of ebola virus disease caused , deaths of , total cases in west africa (fischer et al., ) . because currently no commercial vaccines or effective therapeutics are yet available for filovirus infections (fischer et al., ; reynolds and marzi, ; trad et al., ) , antiviral drugs are urgently needed. thus, both peptide-conjugated pmo (ppmo) and non-conjugated pmos have been tested against ebola virus infection in cultured cells and animal models. an earlier study showed that a mer ppmo targeting the translation start site region of ebov vp positive-sense rna exhibited sequence-specific, time-and dose-dependent inhibition of ebov replication in cultured cells (enterlein et al., ) . moreover, this ppmo provided complete protection of mice when administered before or after challenge with a lethal dose of ebov. interestingly, a corresponding non-conjugated pmo also provided protection of mice when administered prophylactically as well. meanwhile, another report in same year demonstrated that a combination of ebovspecific pmos targeting viral mrnas for vp , vp , and rna polymerase l protected rodents against ebov challenge when administered before and after exposure . in the same study, non-conjugated pmo were also tested in a prophylactic proof-of-principal trial in rhesus macaques whereby the same pmo formulation protected % of macaques from lethal ebov infection. more recently, pmoplus has also been shown to be effective against ebola infection in monkeys. when delivered - min after infection, pmoplus avi- (composed of avi- and avi- that target ebov vp and vp , respectively) (iversen et al., ) protected over % of rhesus monkeys against lethal infection with zaire ebola virus (zebov) (warren et al., ) . similarly, pmoplus avi- (composed of avi- and avi- that target vp and vp of marburg virus, respectively) protected % of cynomolgus monkeys against infection with lake victoria marburg virus (marv) when delivered after infection (iversen et al., ) . therefore, pmoplus holds great promise for treatment of patients infected with these highly pathogenic viruses. in another study, the same research group showed that pmoplus avi- targeting vp alone was sufficient to protect monkeys against lethal ebov infection, whereas pmoplus avi- , targeting vp alone, failed to do so (warren et al., ) . these results thus confirm that vp may be a key ebov virulence factor and may serve as a promising target for further development of effective anti-ebov treatment strategies. picornaviruses are non-enveloped positive-sense, single-stranded rna viruses belong to the family picornaviridae. this family includes more than genera and species (zell et al., ) , with rna genomes length ranging from . to . kb. many picornaviruses are important human and animal pathogens, including poliovirus, coxsackievirus b (cvb ), enterovirus (ev- ), and foot-and-mouth disease virus (fmdv). no vaccine yet exists for picornaviruses other than for poliovirus and fmdv. moreover, no effective antiviral therapy yet exists for treatment of infections caused by any pathogenic picornavirus. however, ppmo targeting conserved internal ribosome entry site (ires) sequences have been shown to be highly effective in protecting cultured cells against infection by human rhinovirus type , coxsackievirus type b , and poliovirus type (pv ) (stone et al., ) , with reduction of pv titers by up to log . this mer ppmo (enterox) targets an ires sequence that is identical for > % of all human enteroviruses and rhinoviruses which has been successfully used to treat poliovirus receptor (pvr) transgenic mice to prevent pv infection after challenge with three times the % lethal dose (ld ). this result also showed that mice receiving ppmo treatment exhibited an approximately % higher survival rate than controls, with significant reduction of viral titers in small intestine, spinal cord, and brain (stone et al., ) . coxsackievirus b (cvb ) is a primary cause of viral myocarditis, without any effective therapy. in study tested eight cvb -specific ppmo in cultured hela cells and hl- cardiomyocytes, as well as in a murine infection model (yuan et al., ). among eight ppmos tested, ppmo- , designed to target the portion of the cvb ires, was especially potent against cvb replication in cultured cells. when cells were treated prior to or shortly after cvb infection, virus proliferation was significantly inhibited, with approximate log decrease of viral titers. in a/j mice, ppmo- intravenous administration once prior to and once after cvb infection significantly reduced cardiac tissue damage, with notable decreases in myocardium virus titers than control (yuan et al., ) . enterovirus (ev- ) generally causes mild hand-foot-andmouth disease, but severe neurological complications with high mortality rates have been reported. in one study, testing of vivo-pmo designed to target the ev- ires and the rnadependent rna polymerase (rdrp) was performed (tan et al., ) . vivo-pmo targeting ev- ires significantly reduced ev- replication in human embryonal rhabdomyosarcoma rd cells. in contrast, vivo-pmo targeting ev- rdrp was less effective. the results suggest that ires-targeting vivo-pmo are potential antiviral candidates that can abrogate early ev- infection (tan et al., ) . fmdv causes a highly contagious viral disease of clovenhoofed animals that can lead to severe economic losses to the livestock industry. six ppmos were designed to target and utrs of the fmdv genome (strain a( ) cruzeiro/brazil/ [a( )cru]) and were evaluated in cultured cells (vagnozzi et al., ) . three of the ppmos, targeting domains including the portion of the ires and the two translational start codoncontaining regions, were highly effective in inhibiting fmdv replication. at low micromolar concentrations, ppmo led to a dose-dependent and sequence-specific virus titer reduction of over log , while three other ppmo that targeted other genome regions were less effective (vagnozzi et al., ) . the order nidovirales includes the families coronaviridae, arteriviridae, roniviridae, and mesoniviridae (cong et al., ) . the members of this group are positive-sense, single-stranded rna viruses. the coronaviridae and arteriviridae include groups of viruses infecting vertebrates (mainly mammalian species), whereas the other two families include viruses infecting invertebrates. pmo have been tested as antivirals against members of both coronaviridae and arteriviridae. members of coronaviridae include severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov), which are recently identified as potent human pathogens (cong et al., ) . mouse hepatitis virus (mhv), also belongs to this family, which has long served as a model for understanding viral hepatitis in humans and to assess pmo antiviral compounds (neuman et al., ; burrer et al., ) . ppmo targeting the mhv replicase exhibited low toxicity in dbt astrocytoma cells, a cell line for studying mhv infection (neuman et al., ) . a later study tested ppmos against several mhv strains in cell culture and in vivo using mouse models (burrer et al., ; moulton et al., ) . among ten ppmos against various viral genome target sites, ppmo term, which targeted the terminus of the rna genome, was found to be highly effective in inhibiting six different mhv strains. in mice, term ppmo treatment led to prevention of virus-induced tissue damage. prophylactic treatment with term ppmo also decreased mhv-induced weight loss and prolonged post-challenge survival. this study also showed that no weight loss or detectable histopathologic changes were observed after prolonged ppmo treatment of uninfected mice (burrer et al., ) . besides, ppmo have also been shown to inhibit sars-cov replication as well (neuman et al., ). among all ppmo tested, two ppmo targeting the viral transcription-regulatory sequence (trs) within the utr brought about the most significant inhibition of cpe and reduced cell-to-cell spread when administered prior to peak viral proliferation in cultured cells (neuman et al., ) . members of the arteriviridae include an economically important virus, porcine reproductive and respiratory syndrome virus (prrsv). prrsv causes a contagious swine disease characterized by reproductive failure in sows and respiratory disease in pigs of all ages. the disease has plagued the global swine industry since it was first reported in and current strategies used for prrs control are still inadequate (du et al., ) . research in our laboratory has focused on development of ppmos against prrsv. based on the genome sequence and viral protein function, a series of ppmos with various targets were designed that included three ppmo targeting the end utr of the prrsv genome, namely up , up , and hp, as well as six ppmo targeting the translation initiation regions of orfs - , namely p , p , p , p , p , and p han et al., ) . ppmo targeting the utr were highly effective for inhibiting prrsv replication in cell culture in a dosedependent and sequence-specific manner. specifically, ppmo up or hp caused a . log reduction of prrsv yield. moreover, up and hp also demonstrated broad inhibition of heterogeneous prrsv isolates . in addition, if ppmo up targeting the utr of prrsv genome was paired with ppmo p and p , which targeted translation initiation regions of orfs and , respectively, the combination enhanced inhibition of heterologous strains of the north american prrsv genotype more effectively than individually in vitro (han et al., ) . inhibition was verified at both prrsv rna and protein expression levels. since ppmo up , which targets a highly conserved sequence within the -terminal region of prrsv genome, effectively induces a multi-log inhibition of prrsv replication in vitro, we also conducted an in vivo evaluation of this ppmo (opriessnig et al., ) . prrsv-negative -week-old piglets received ppmo intranasally at h before infection as well as and h after prrsv infection. ppmo treatment was well tolerated in piglets, with no weight change observed across all piglet groups and ppmo administration significantly reduced prrsv viremia and interstitial pneumonia. moreover, in alveolar macrophages isolated at days post-infection, elevated expression of antiviral genes in ppmo-treated piglets was observed as well (opriessnig et al., ) . besides prrsv, antiviral effects of pmo were also evaluated against equine arteritis virus (eav), another member of the family arteriviridae (van den born et al., ) . similar to prrsv studies above, ppmo designed to target the terminal utr of the eav genome remarkably reduced virus replication in a sequence-specific and dose-responsive manner. however, ppmo that targeted -terminal regions of the viral genome or its anti-genome only resulted in moderate reduction of eav replication when relatively high concentrations of the ppmos were applied. moreover, ppmo targeting the eav trs, which is essential for subgenomic rna synthesis, were ineffective to achieve transcription interference (van den born et al., ) . however, ppmo targeting the utr of eav were able to cure viral infection of persistently infected hela cells (zhang et al., ) . flavivirus is a viral genus within the family flaviviridae. this genus includes west nile virus (wnv), dengue virus (denv), yellow fever virus, zika virus, and several other viruses which may cause encephalitis (coffey et al., ; musso and gubler, ) . except for yellow fever virus, no effective vaccine or antiviral drug exists against flaviviruses, prompting evaluation of pmo against several members of flavivirus. among a panel of ppmo against wnv, ppmo targeting the -and -termini of the wnv genome, designated end or csi, exhibited the greatest potency in blocking wnv replication (deas et al., ) . moreover, treatment of wnv-infected cells with either end or csi pmo led to a significant reduction of virus titers by approximately - log without apparent cytotoxicity. ppmo end inhibited wnv translation, whereas ppmo csi suppressed wnv rna replication. meanwhile, ppmo csi also inhibited other mosquito-borne flaviviruses when the targeted csi-like sequences which were relatively conserved to the respective wnv csi target sequence. therefore, ppmo targeting conserved cis-acting elements of flavivirus genomes should be explored as anti-flavivirus therapeutics (deas et al., ) . more recently, both ppmo end or csi were also tested in a mouse model of wnv infection and provided partial protection when administered at or µg/day. moreover, minimal to no ppmo-mediated toxicity observed, while toxicity was observed at a larger dosage of µg/day (deas et al., ) . a panel of ppmo was also tested against denv (kinney et al., ) whereby ppmo targeting -terminal nucleotides of the serotype denv (den- ) virus genome exhibited relatively poor suppression of den- virus titer. however, moderate reduction of titer was observed for ppmo targeting either the aug translation start site region of the single open reading frame or the cyclization sequence region. the most highly effective ppmo were sl and cs (targeting the -terminal nucleotides and the cyclization sequence region, respectively), which reduced viral titer by greater than . log compared to controls at days post-infection with den- virus. notably, treatment with µm cs inhibited replication of all four den virus serotypes by over log , in most cases to below detectable limits (kinney et al., ) . a third ppmo that was designed to target the top of the stem-loop ( slt) inhibited den replication in bhk cells (holden et al., ) . the inhibitory mechanism was studied using a novel den reporter replicon and a den reporter mrna. the results demonstrated that sl inhibited viral translation and cs blocked viral rna synthesis but not viral translation, whereas the slt inhibited both viral translation and rna synthesis (holden et al., ) . more recently, anti-denv ppmo sl and cs were also tested in ag mice before and/or after infection with denv- . intraperitoneal (ip) infection of ag mice with - pfu of denv- (strain new guinea c) shortened survival to - days. when sl or cs were administered before and after denv infection, the average survival time was extended by up to more days. this study also included pharmacokinetic and toxicology analysis of non-infected animals. the results showed that the mice had high concentrations of ppmo in liver following nine consecutive once-daily ip treatments of mg/kg ppmo, with little impact on overall mouse health . pmo and ppmo have also been studied for their ability to prevent or treat japanese encephalitis virus (jev) infection as well. a ppmo (p ) targeting the cyclization sequence ( csi) of jev exhibited significant antiviral activity in vero (epithelial), neuro a (neuronal), and j e (macrophage) cells at non-toxic concentrations (anantpadma et al., ) . addition of p to cells before infection decreased jev replication to undetectable levels in vero cells and resulted in a and % reduction in jev titer in j e and neuro a cells, respectively. in this study, antiviral effects of p were also assessed in vivo. when treated intracerebrally with a mg/kg dose of p every h for days, - % of -week-old mice were protected from a lethal dose of jev (anantpadma et al., ) . meanwhile, testing of vivo-pmo targeting the and utr of the jev genome have also been conducted in mice (nazmi et al., ) . administration of intraperitoneal injections of vivo-pmo ( mg/kg body weight) daily for up to days immediately after jev infection of mice prolonged survival, with reduced viral load and viral protein expression in brain. moreover, proinflammatory cytokine levels in brain, which normally increase after jev infection, were reduced following pmo treatment and align with observations of reduced microglial activation in brain as well (nazmi et al., ) . chikungunya virus (chikv) causes infection in humans that is associated with debilitating and persistent arthralgia and arthritis. two ppmo were designed to target highly conserved sequences present in chikv non-structural and structural polyproteins (lam et al., ) . cpmo , a ppmo that targets the orf aug region, significantly suppressed chikv replication in hela cells when administered before infection. notably, in neonatal mice, administration of µg/g cpmo before infection conferred % survival against chikv disease (lam et al., ) . ppmo have also been tested for antiviral effects toward group v (-)ssrna virus families that include pneumoviridae, paramyxoviridae, orthomyxoviridae, arenaviridae, and others. respiratory syncytial virus (rsv), a member of the family pneumoviridae, is a major cause of lower respiratory tract infections in infants, young children, and high-risk adults. currently, no vaccine exists to prevent rsv infection. however, two antisense ppmos designed to target the -terminal region and the translational start site of rsv l mrna have been tested and exhibited minimal cytotoxicity (lai et al., ) . ppmo aug- inhibited rsv replication by reducing viral titers by over log . when administered before rsv intranasal inoculation, ppmo aug- protected balb/c mice from infection, with reduced viral titers in lung tissue and attenuation of pulmonary inflammation (lai et al., ) . measles virus (mev) is a member of the family paramyxoviridae. mev is a highly contagious human pathogen that can be treated effectively with available antiviral compounds and prevented using a vaccine. five ppmos targeting mev genomic rna or mrna were tested in cultured cells (sleeman et al., ) . ppmo , targeting a conserved sequence in the translation start site of the mrna coding for viral nucleocapsid protein, was highly effective against multiple genotypes of mev (sleeman et al., ). influenza a virus, a member of the family orthomyxoviridae, is a relentless ongoing global public health concern. when delivered by intranasal administration, ppmo inhibited replication of equine influenza a virus fluav a/eq/miami/ / (h n ) in mice and exhibited no toxicity at effective antiviral concentrations in vivo (lupfer et al., ) . meanwhile, a ppmo panel was developed to target rna genome segments encoding polymerase subunits of a highly pathogenic mouse-adapted influenza a virus strain (sc m; h n ) (gabriel et al., ) . in this study, virus replication in mdck cells was significantly inhibited by three ppmo targeting either the translation start site region of pb or np mrna or the -terminal region of np viral rna (vrna). in a mouse model, when ppmo targeting the pb -aug region or np vrna were administered intranasally once h before and once days after intranasal infection with a lethal dose of sc m, treated mice exhibited significantly lower viral titers in lungs and % greater survival versus untreated controls over the -day duration of the experiment (gabriel et al., ) . besides targeting viruses, ppmo have also been tested for efficacy against host mrna encoding proteases crucial for viral infectivity. such ppmo can block host protease cleavage of the influenza virus hemagglutinin (ha) to inhibit viral infectivity. treatment of human calu- airway epithelial cells with a ppmo t-ex , designed to interfere with splicing of hacleaving protease tmprss pre-mrna, resulted in production of tmprss mrna lacking exon that resulted in production of an enzymatically inactive form of tmprss (bottcher-friebertshauser et al., ) . ultimately, t-ex ppmo was shown to prevent cleavage of has of various human seasonal and pandemic influenza a viruses, leading to significant reduction of viral titers by to log (bottcher-friebertshauser et al., ) . junín virus, a threat to human health and a member of the arenaviridae family, can cause meningitis and hemorrhagic fever. ppmo designed to interfere with translation have been shown to be effective in reducing junín virus replication (neuman et al., ) . in cultured cells, ppmo target sequences located at the termini of both genomic segments are highly conserved across the arenaviruses. consequently, these ppmo are effective against junín virus, tacaribe virus, pichinde virus, and also against lymphocytic choriomeningitis virus (lcmv) whereby they suppress viral titers in livers of lcmv-infected mice (neuman et al., ) . the genus alphavirus includes positive-sense rna viruses that threaten human health (paessler et al., ) . sindbis virus (sinv) is a member of the family togaviridae. ppmo targeting both the -terminus and aug translation start site of the sinv genome significantly suppressed sinv replication in tissue culture (paessler et al., ) . venezuelan equine encephalitis virus (veev) is another member of the togaviridae. ppmo targeting veev regions corresponding to sinv regions mentioned above inhibit several strains of veev in vitro. notably, mice pre-treated with pmo were protected from lethal veev infection, while only partial protection was observed for mice receiving only post-infection pmo treatment (paessler et al., ) . noroviruses, which belong to the caliciviridae family, are non-enveloped, positive-sense, single-stranded rna viruses with genomes of approximately . kb in length that encode three orfs. noroviruses cause non-bacterial epidemic gastroenteritis (bok et al., ) . ppmo targeting the first aug region of the orf near the -end of the murine norovirus (mnv) genome effectively inhibited mnv replication in cultured cells (bok et al., ) . moreover, a consensus ppmo targeting the corresponding -end of the genome of several diverse human norovirus genotypes also inhibited norwalk virus protein expression (a species of norovirus) in replicon-bearing cells in a cell-free luciferase reporter assay (bok et al., ) . similar to noroviruses, hepatitis e virus (hev) is a positivesense, single-stranded rna virus containing three orfs and is currently classified within the family hepeviridae (nan and zhang, ) . hev shares a similar genome structure with members of caliciviridae and was previously classified in that family (nan and zhang, ) . our study demonstrated that ppmo targeting the terminal of the hev genome at the start codon of orf are highly effective against both hev genotypes and infection in vitro (nan et al., ) . moreover, ppmo targeting the utr of the hev genome or the terminus of antisense hev rna can also block hev replication, but to a lesser extent than achieved by ppmo targeting the terminus (nan et al., ) . since the utr and terminus of antisense hev rna are generally considered binding sites for hev rnadependent rna polymerase (rdrp), it appears that ppmomediated steric blockade also applies to rdrp as well (nan et al., ) . as compared with pmos' applications against rna viruses, pmos use against dna viruses has been much less studied. to date, only members of herpesviridae have been tested for pmo-mediated inhibition. research from our lab evaluated pmos against kaposi's sarcoma-associated herpesvirus (kshv). kshv, also known as human herpesvirus (hhv- ), is a human oncovirus belonging to the gamma herpesvirus subfamily. kshv is associated with kaposi's sarcoma (ks) and two b-cell lymphoproliferative diseases: primary effusion lymphoma (pel) and multicentric castleman's disease (mcd) (purushothaman et al., ) . these diseases are aids-related malignancies in hiv-infected patients. kshv infection in humans exhibits either a lifelong immunologically silent and latent infection or a transient lytic infection with distinct viral gene-expression profiles (lagunoff et al., ; bechtel et al., ) . during the predominantly latent state of kshv infection, the kshv genome is maintained as circular, extra-chromosomal dna that replicates inside host cells in a cell cycle-dependent manner. expression of a few key viral regulators, such as latency-associated nuclear antigen (lana) encoded by orf , viral cyclin (vcyclin) encoded by orf , and viral flip (vflip) encoded by orf , are observed during latency (uppal et al., ) . upon reactivation to assume a lytic infection state, a full repertoire of lytic viral genes, including orf (transcription activator, rta), orf , orf , orf , orf , orf , orf-k , orf-k (virf- ), orf-k (viral interleukin- , vil- ), orf (viral g protein-coupled receptor vgpcr), and viral chemokines (orf-k /vccl-i and orf-k /vccl-ii) are expressed in a temporally-regulated manner (purushothaman et al., ) . based on the functions of kshv latent and lytic genes, a panel of ppmos was designed against a set of genes including lana, vil- , rta, and virf- (zhang et al., (zhang et al., , (zhang et al., , . treatment of kshv-positive pel cells with an rta-specific ppmo rp not only reduced rta expression but also caused down-regulation of several other early and late kshv gene products, including vil- , virf- , and orf-k . a. moreover, kshv dna copy numbers both in ppmo rp -treated pel cells and culture supernatants were reduced, demonstrating inhibition of kshv lytic replication (zhang et al., ) . furthermore, treatment of bcbl- cells with ppmo against lana reduced lana expression (zhang et al., ) . meanwhile, ppmo against vil- and virf- were also evaluated. the viral homologue of the proinflammatory cytokine il- , vil- , is believed to contribute to vascular permeability and formation of pel effusions (sakakibara and tosato, ) . this cytokine shares low but significant homology to human irf family members and acts as an oncogene to inhibit interferon induction (gao et al., ) . treatment of pel cells with ppmo designed against vil- mrna led to marked reduction in the proportion of vil- -positive pel cells and reduced both the growth of pel cells and kshv dna levels (zhang et al., ) . meanwhile, ppmo targeting virf- have also been shown to inhibit viral dna replication in addition to blocking virf- expression in bcbl- cells (zhang et al., ) . interestingly, reduction of virf- expression in kshv-infected cells resulted in higher expression levels of cellular irf- and of the signal transducer and activator of transcription (stat ) (zhang et al., ) . encouraged by these in vitro data, an in vivo evaluation of ppmo effects on expression of multiple viral genes was conducted (zhang et al., ) . however, in vivo results defied expectations drawn from in vitro studies. specifically, only ppmo against vil- demonstrated promising in vivo inhibition whereby scid mice treated with this ppmo exhibited no engraftment of kshv-infected pel cells and remained healthy throughout the -day study (zhang et al., ) . conversely, in scid mice receiving a combination of pmo against two virf- , there was a trend of less engraftment of kshv-infected pel cells, but the difference in results was not statistically significant between treated and control mice. therefore, ppmo targets selected using in vitro assessments may not achieve expected inhibition in vivo, emphasizing the fact that careful validation using adequate animal models is necessary. acyclovir (acv) is a nucleic acid analog of guanosine that is used to treat hsv. viral resistance to acv has become a recent concern. as a potential treatment for acv-resistant hsv, ppmo against resistant hsv- and hsv were evaluated both in vitro and in a mouse model as well. for hsv- , icp and icp were selected as virus ppmo targets. icp from both hsv- and hsv- acts as an e ubiquitin ligase to antagonize host innate immunity (halford et al., ; lanfranca et al., ) . icp is a multiple function regulator involved in pre-mrna splicing and the host innate immune response (christensen et al., ; tang et al., ) . when ppmo targeting translation start site regions of hsv- icp or icp mrna were applied before or soon after hsv- infection of cultured cells, a - % reduction of hsv- yield was observed, as assessed by reduced plaque formation. moreover, icp -specific ppmo also inhibited acvresistant hsv- plaque formation by - %, while an equivalent dose of acv only led to a - % plaque reduction (moerdyk-schauwecker et al., ) . in vivo data suggest that ppmo are well-tolerated in uninfected mice after days of administration of µg/day (moerdyk-schauwecker et al., ) . topical application of µg icp -specific ppmo into the eyes of hsv- -infected mice reduced the incidence of eye disease by . - % compared to controls. therefore, ppmo holds promise as an antiviral drug for use in treating hsv- ocular infection (moerdyk-schauwecker et al., ) . with regard to hsv- , ppmo targeting icp or icp mrna were also highly effective against either non-acvresistant or acv-resistant hsv- strains. in one in vivo study, ppmo were well-tolerated in balb/c mice and cotton rats. cotton rats receiving icp -specific ppmo h after hsv- inoculation showed a reduction in genital lesions and a . % reduction in mortality at days post-infection. mice receiving a combined regimen of µm of icp -and icp -specific ppmo before hsv- inoculation were completely free from genital viral infection at - days post-inoculation (eide et al., ) . a variety of studies suggest that pmo would be good candidate for antiviral therapeutics against emerging or reemerging viruses in the absence of other effective therapies. however, effective target sequences for pmo design need to be carefully validated. a list of previously published effective virus target regions used for pmo designed against rna viruses are briefly summarized in table . to date, for most pmos evaluated against positivesense rna viruses, targeting sequences of pmo are mainly located in the terminal ends of viral genomes (table ) . notably, it appears mrna-like properties of positive-sense rna virus genomes make them highly susceptible to pmo-mediated translation inhibition if the pmos pairing sequence are located at the terminal end of the viral genomes, with few exceptions. moreover, utr at either end of viral genomes are generally conserved among viral strains. conversely, for some positivesense rna viruses, pmo targeting of the ' terminal ends of the viral genome were tested as well. however, data appears to be mixed for terminal-targeting pmos. on the one hand, for members of flaviviridae, pmos targeting the ' cyclization sequence located within the terminal end are highly effective in inhibiting virus replication (deas et al., (deas et al., , kinney et al., ; holden et al., ; stein et al., ; anantpadma et al., ; nazmi et al., ) . on the other hand, the use of pmo targeting either the -terminal regions of the viral genome or of the negative genomic strand only resulted in moderate reduction of eav (van den born et al., ) . this result suggests that pmo targeting the terminal end are less effective for inhibiting eav replication compared to pmo targeting the terminal utr of the eav genome. therefore, the viral genome end is the preferred target region for antiviral pmo design against positive-sense rna viruses. in addition to pmo targets within the terminal end of positive-sense rna virus genomes, pmo targeting rna secondary structures should also be considered, especially for common conserved elements among rna viruses, such as ires sequences. generally, unstructured regions are more accessible to oligonucleotide binding than are structured regions, since internal structures within target rna can impede pmo binding (childs-disney and disney, ) . however, in some cases, direct targeting of ires sequences could inhibit replication of some viruses, including members of the order picornavirales (yuan et al., ; stone et al., ; tan et al., ) . therefore, a deep analysis of the secondary structures of target rna may aid pmo target selection. for pmos evaluated for antiviral activity against negativesense rna viruses, current reports mainly focus on blocking translation of individual viral genes rather than direct targeting the rna genome. for example, pmo targeting ebov vp alone was sufficient to protect monkeys against lethal ebov infection, whereas a pmoplus formulation designated avi- that targeted vp failed to do so (warren et al., ) . therefore, screening to find effective pmo targets of negative-sense rna viruses may require more careful consideration than needed for positive-sense rna viruses. although not yet extensively studied, pmo target selection appears to be more complicated for large dna viruses such as herpesviruses. due to their large genome size, such dna viruses encode many genes, including indispensable and dispensable genes. on the one hand, some indispensable genes studied so far do not appear to be good pmo targets. for example, hsv- early genes ul and ul encode the viral dna polymerase and the large subunit of ribonucleotide reductase, respectively, which are essential for viral dna replication (eide et al., ) . however, blocking hsv- ul and ul mrna translation by pmo did not significantly reduce viral replication or transmission from infected cells (eide et al., ) . on the other hand, dna viruses encode many dispensable accessory proteins with multiple functions, such as antagonist to host innate immunity. such genes need to be carefully validated both in vitro and in vivo before use as pmo targets. for example, pmo targeting of virf- of kshv demonstrated little protection against kshv-infected pel engrafts in scid mice, in spite of its effectiveness as a pmo target in vitro (zhang et al., ) . aside from challenges regarding target selection, the emergence of resistant virus after sequence-specific therapy is another common challenge faced by antivirals. indeed, a hcmv mutant with sequence-dependent resistance to the phosphorothioate oligonucleotide fomivirsen was discovered almost simultaneously with market approval of the drug (mulamba et al., ) . because pmo-based antiviral therapy is sequence-specific, pmo-resistant target mutations would abolish pmo inhibition, as has been already observed (neuman et al., ) . for west nile virus, the sequencing of ppmo-resistant wnv mutants has demonstrated that viruses resistant to -end ppmo treatments contained two to three mismatches within the ppmo-binding site, whereas csi ppmo-resistant viruses accumulated mutations outside the ppmo-targeted region (deas et al., ) . meanwhile, ppmo-resistant-wnv infection of mice was shown to antagonize pmo-mediated protection against virus (deas et al., ) . similar pmo-resistance has been reported for ebola virus after treatment with pmo targeting ebola virus vp and vp as well (kugelman et al., ) . therefore, mutations within or outside of pmo-targeting sequences might lead to pmo resistance and will be a future challenge. in our research of ppmo inhibition of prrsv, a multiple log reduction of viral rna copies was achieved when pmo sequence was complementary to a conserved region within the utr of the prrsv genome, although low level viral rna replication was still observed. this suggests that pmoresistant mutants might have arisen or that the effective one possible strategy to encounter pmo-resistance mutants would be incorporating of promiscuous bases such as inosine to compensate for predicted base-pair mismatches . however, it is a challenge to accurately predict the potential mutation nucleotides within a pmo-targeting sequence. therefore, the pmo target sequence against any specific rna virus must be carefully selected to minimize generation of mutants. moreover, use of multiple pmos against different targets of the same virus may help to avoid mutant virus generation. an alternative strategy to prevent pmo-resistant virus is indirect inhibition of a host factor essential for virus replication. currently, only one of such study has been conducted on influenza virus and exploits the fact that cleavage of viral hemagglutinin (ha ) by host proteases is crucial for viral infectivity. in this study, treatment of human calu- airway epithelial cells with a ppmo designed to interfere with pre-mrna splicing of tmprss (the host protease responsible for ha cleavage) resulted in tmprss mrna lacking exon , finally led to expression of an enzymatically inactive form of tmprss (bottcher-friebertshauser et al., ) . therefore, blocking of ha cleavage by this ppmo was confirmed in different human seasonal and pandemic influenza a viruses and resulted a significant reduction of viral titers (bottcher-friebertshauser et al., ) . in addition to above investigation, variety host proteins have been identified as essential factors required for virus replication, host defense and viral pathogenesis in recent years (rajsbaum et al., ; tripathi et al., ) . the putative ubiquitin ligase ubr was identified as a novel host factor involved in the budding of influenza virion (tripathi et al., ) , targeting such factor could block the release of influenza virus. although it is unknown if ubr could be employed by other enveloped viruses for budding as well, it is possible that targeting a commonly used host factor which is required for viral replication could broaden the antiviral spectrum of pmo. therefore, application of pmo to target host factor as antiviral strategy may not only avoid generation of pmo-resistant virus, but also expand the antiviral spectrum as long as the targeted host factor is employed by different virus for replication. it has been two decades since the first approval of an antisense-based therapy. pmos have become important on analogs that have driven development of antisense therapies with efficacy and safety demonstrated by the recent fda approval of eteplirsen. indeed, biological stability, neutral charge, and rnase h-independent mechanism of action are all unique pmo features. moreover, besides their development as antiviral compounds, pmo evaluation as anti-cancer or antibacterial agents is ongoing (enterlein et al., ; warfield et al., ; cansizoglu and toprak, ; chen et al., ; summerton, ) . since pmo have a good track record as useful tools in developmental biology and as therapeutic agents, three generations of pmo have been developed (unmodified pmo, conjugated pmo, and pmoplus) to improve intracellular delivery (daly et al., ) . however, several issues remain unresolved before pmo are adopted for widespread use. on the one hand, more studies are needed to establish a convenient route for pmo administration in vivo. in most in vivo studies, pmo are administrated via either intravenous or intramuscular injection. although reports have demonstrated that pmo administered via intranasal delivery can inhibit replication of viruses with a respiratory tract tropism (opriessnig et al., ; rajsbaum, ) , it is notable that in these in vivo studies employed pmos for as antiviral agents against two respiratory viruses, influenza virus and prrsv (opriessnig et al., ; rajsbaum, ) . since there is no investigation conducted to see if intravenous or intramuscular administration of pmo also effectively against influenza virus and prrsv, it is possible intranasal delivery of pmo is preferable for virus causes respiratory infection. moreover, it is also possible that virus-specific delivery routes based on the initial infection sites may offer a better antiviral efficiency rather than using intravenous route as universal way for pmo administration. on the other hand, although pmo is a highly adaptive platform for delivery of nucleic acid sequence-specific drugs, selection of an effective target and avoidance of the emergence of mutant virus also require more investigation. if these issues could be properly addressed, pmo should serve as a promising strategy for treatments of a variety of diseases, including difficult-totreat viral infections. as is generally true for antiviral drug development, high efficacy, low toxicity, good pharmacokinetics, good bioavailability, and low cost are all characteristics sought in a pmo compound. 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to reality in crop improvement inhibition of measles virus infections in cell cultures by peptideconjugated morpholino oligomers treatment of ag mice with antisense morpholino oligomers increases survival time following challenge with dengue virus a morpholino oligomer targeting highly conserved internal ribosome entry site sequence is able to inhibit multiple species of picornavirus morpholino antisense oligomers: the case for an rnase h-independent structural type morpholino antisense oligomers: design, preparation, and properties invention and early history of morpholinos: from pipe dream to practical products chemical modifications of antisense morpholino oligomers enhance their efficacy against ebola virus infection ribonuclease h: molecular diversities, substrate binding domains, and catalytic mechanism of the prokaryotic enzymes herpes simplex virus icp regulates alternative pre-mrna polyadenylation and splicing in a sequence-dependent manner ebola virus disease: an update on current prevention and management strategies meta-and orthogonal integration of influenza "omics" data defines a role for ubr in virus budding kshv lana-the master regulator of kshv latency antiviral activity of morpholino oligomers designed to block various aspects of equine arteritis virus amplification in cell culture structure-activity relationship study of the plasma membrane translocating potential of a short peptide from hiv- tat protein gene-specific countermeasures against ebola virus based on antisense phosphorodiamidate morpholino oligomers advanced morpholino oligomers: a novel approach to antiviral therapy advanced antisense therapies for postexposure protection against lethal filovirus infections delayed time-to-treatment of an antisense morpholino oligomer is effective against lethal marburg virus infection in cynomolgus macaques a single phosphorodiamidate morpholino oligomer targeting vp protects rhesus monkeys against lethal ebola virus infection knocking down disease: a progress report on sirna therapeutics inhibition of coxsackievirus b in cell cultures and in mice by peptide-conjugated morpholino oligomers targeting the internal ribosome entry site inhibition of rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide ictv virus taxonomy profile: picornaviridae curing of hela cells persistently infected with equine arteritis virus by a peptide-conjugated morpholino oligomer blockade of viral interleukin- expression of kaposi's sarcoma-associated herpesvirus inhibition of primary effusion lymphoma engraftment in scid mice by morpholino oligomers against early lytic genes of kaposi's sarcoma-associated herpesvirus inhibition of replication and transcription activator and latency-associated nuclear antigen of kaposi's sarcomaassociated herpesvirus by morpholino oligomers key: cord- -c nwygl authors: saldanha, i. f.; lawson, b.; goharriz, h.; rodriguez-ramos fernandez, j.; john, s. k.; fooks, a. r.; cunningham, a. a.; johnson, n.; horton, d. l. title: extension of the known distribution of a novel clade c betacoronavirus in a wildlife host date: - - journal: epidemiol infect doi: . /s sha: doc_id: cord_uid: c nwygl disease surveillance in wildlife populations presents a logistical challenge, yet is critical in gaining a deeper understanding of the presence and impact of wildlife pathogens. erinaceus coronavirus (ericov), a clade c betacoronavirus, was first described in western european hedgehogs (erinaceus europaeus) in germany. here, our objective was to determine whether ericov is present, and if it is associated with disease, in great britain (gb). an ericov-specific bryt-green(®) real-time reverse transcription pcr assay was used to test samples of faeces or distal large intestinal tract contents collected from casualty or dead hedgehogs from a wide area across gb. viral rna was detected in . % ( ) samples; however, the virus was not detected in any of the samples tested from scotland. the full genome sequence of the british ericov strain was determined using next generation sequencing; it shared % identity with a german ericov sequence. multivariate statistical models using hedgehog case history data, faecal specimen descriptions and post-mortem examination findings found no significant associations indicative of disease associated with ericov in hedgehogs. these findings indicate that the western european hedgehog is a reservoir host of ericov in the absence of apparent disease. the betacoronavirus genus includes multiple bat-associated viruses and several human pathogens, including severe acute respiratory syndrome coronavirus (sars-cov) and middle eastern respiratory syndrome coronavirus (mers-cov) [ , ] . in , a novel clade c betacoronavirus was discovered in the western european hedgehog (erinaceus europaeus) in germany [ ] . the virus was subsequently detected in the same species in france [ ] . characterisation of these erinaceus coronavirus (ericov) nucleotide sequences revealed high nucleotide identity to mers-cov [ ] , the cause of an acute respiratory syndrome in humans with high case fatality rates [ , ] . the structural spike protein is key to coronavirus virulence and host specificity [ ] and the gene that encodes it plays a pivotal role in coronavirus diversity [ ] . analysis of the receptorbinding region in the s protein of ericov revealed that it shared only . % amino acid identity with that of mers-cov [ ] . however, there have been no further published ericov whole genome sequencing attempts, and many questions remain over the origins and evolution of ericov [ ] . whilst there is no known threat to public health from ericov, further investigation of the virus is warranted to gain a fuller understanding of the impact of coronavirus infection on wildlife populations. bats are widely recognised and accepted as the natural reservoir hosts of a variety of coronavirus species, including sars-cov [ , ] . both bats and camels have been cited as possible reservoirs of mers-cov [ , ] . it is considered that bats are the likely natural reservoir of mers-cov [ , ] , and that dromedaries (camelus dromedarius), and potentially other species, act as an intermediate host for human transmission [ , , ] . mers-cov-like virus sequences have only been detected in one other species, e. europaeus [ ] . hedgehogs are members of the order eulipotyphla, which is closely related to chiroptera, the order of bats [ ] . this close association is what initially led to hedgehogs being investigated as a potential coronavirus reservoir [ ] . many animal species seem to have the capacity for coronavirus infection in the absence of apparent disease, including bats [ ] , aquatic birds [ ] and rabbits when inoculated with mers-cov [ ] . however, dromedaries infected with mers-cov develop pyrexia with mild upper respiratory signs, consisting of serous to purulent nasal discharge [ ] . lesions in infected dromedaries are comparable to those seen in the common cold in humans, including inflammation and loss of pseudostratified epithelial cells in the upper respiratory tract. coronaviruses that affect the gastrointestinal system, such as porcine transmissible gastroenteritis virus, typically cause similarly mild lesions, including destruction of enterocytes and villus atrophy [ ] . although still widespread throughout much of western and northern europe, the western european hedgehog population in great britain (gb) has seen a rapid decline in recent decades, with an estimated population decrease of % between the s and s [ , ] . the reasons for this decline are not fully understood, and it is likely to be multi-factorial (e.g. habitat loss and fragmentation, road traffic death and predation) [ , ] . the possible role of infectious disease also needs to be investigated, including the consequences of infection with newlydiscovered viral agents. here, we identify the presence of ericov in british hedgehogs, sequence the full genome, explore the spatial distribution of infection and investigate if there is any association of ericov with disease. a total of samples from individual hedgehogs were tested in this study. of these, were voided faecal samples collected from live hedgehogs that had been found injured, sick or abandoned juveniles and were consequently admitted to wildlife centres for treatment and rehabilitation in and . a total of nine wildlife centres across gb collected and supplied samples, using the methods described by sangster et al. [ ] . a fresh faecal sample was collected non-invasively shortly after admission and associated metadata, including clinical signs, signalment, location and history, were collected where available. faecal samples were stored at or − °c for a maximum of week at rehabilitation centres prior to submission. a subsample of each sample collected by sangster et al. [ ] was used in the current study. an additional four voided faecal samples were collected from the wild as part of a greater london-based hedgehog population survey conducted in to give a total of faecal samples from live animals. the remaining samples were of distal large intestinal tract contents collected from dead hedgehogs during post-mortem examination (pme) from to and comprised animals with infectious and non-infectious (e.g. trauma) causes of death. these hedgehogs had either died in wildlife centres within h of admission or had been found dead by members of the public. where possible, pmes were performed on fresh carcases, but frozen carcases that had been archived at − °c were also examined. systematic examination of external and internal body systems was performed using a standardised protocol, followed by microbiological, parasitological and histological investigations where indicated by macroscopic findings and when permitted by the state of carcase preservation [ ] . the contents of the distal large intestinal tract were sampled to most closely reflect freshly voided faeces, as the latter had contained the highest ericov viral titre in a previous study [ ] . faecal sample submission forms provided information on: admittance date, sample collection date, the location where the hedgehog was found, sex, live body weight, faecal characteristics (including colour and consistency) and reason for admittance. pme records were available for of hedgehogs from which samples of distal large intestinal contents were tested. these comprised data on the location where the dead hedgehog was found, the date the carcase was found, sex, age class, carcase weight, subjective assessment of body condition (based on muscle mass and body fat deposits), gross pme findings and the results of ancillary diagnostic tests. rna stabilising agent rnalater (ambion, life technologies europe, nl) was added to, and mixed with, a pea-sized amount of each sample (faeces or distal large intestinal tract contents) in a . ml microcentrifuge tube. each sample was then stored at either − or − °c. faeces were homogenised and pooled (five samples per pool). each µl of total pooled sample was added to µl buffer avl-carrier rna solution and extracted immediately following the spin protocol alongside negative extraction controls. a guanidine and column-based method (qiaamp viral mini kit qiagen, hilden, de) was used for rna extraction, according to the manufacturer's protocol. rna was then eluted in µl of buffer ave and stored at − °c. a specific oligonucleotide primer set (ericovr and ericovf, see supplementary table s ) was designed based on the ericov rna-dependent rna-polymerase (rdrp) gene sequence of the german virus sequence (kc ) with oligoarchitect™ (sigma-aldrich®, st louis, mo, usa) and used with the bryt-green based gotaq® -step real-time reverse transcription pcr (rt-pcr) system kit (promega corporation, madison, wi, usa) to detect ericov rna in an applied biosystems fast real-time system. a − dilution of extracted rna from a positive ericov sample (r / , previously confirmed through sequencing) was used as the positive control. nuclease-free water (promega corporation) was used as the negative control. following the amplification stages, melt curve analysis was performed in order to verify the specificity of the amplicons [ ] . in addition, a bp ericov ultramer oligonucleotide was used to quantify the limits of detection and assess the sensitivity of the designed assay. this was a sequence within the ericov primer rdrp target region, but with the removal of nucleotides at the centre to differentiate it from wild-type ericov (see supplementary table s ). a -fold dilution series was performed and a standard curve was generated using graphpad prism (graphpad software, ca, usa). to assess assay specificity, rna of the gammacoronavirus, infectious bronchitis virus (ibv), was also included as a control. to assess the primer dynamic range and potential rna degradation, a dilution series of rna extracted from faeces was tested. a 'spiked' dilution series was set containing % ( µl in µl) faecal rna extraction from an ericov-negative pool and a subset of negative samples (n = ) was tested using primers for the s ribosomal rna subunit. all samples extracted from faeces produced positive results confirming the presence of amplifiable rna (data not shown). real-time rt-pcr assays using the ericov primers were carried out for all pooled samples. single samples from positive pools were then extracted and tested individually. a subset of amplicons from individual samples (n = ) was also assessed by gel electrophoresis and compared to the positive control. sanger sequencing was conducted on three amplicons with ericovr and ericovf primers using standard protocols. amplicon sequences were i. f. saldanha et al. manually checked then aligned against the german ericov rdrp gene fragment sequence retrieved from genbank (accession number kc ) to confirm their identity (mega . ). whole genome sequencing was performed on rna extracted from one faecal sample collected in (r / ) which was identified as ericov-positive by real-time rt-pcr. sequencing was performed using synthesis technology (illumina, san diego, ca, usa). rna was extracted as before and dna was depleted from this sample using the on-column dnase treatment (rneasy® plus mini kit) following the manufacturer's instructions (qiagen); the resultant rna was then eluted in µl molecular grade water [ ] . cdna was synthesised from ng rna using a random cdna synthesis system (cdna synthesis kit, merck, de), according to the manufacturers' instructions. the cdna was purified using ampure xp magnetic beads (beckman coulter, high wycombe, uk), ng of which was processed for sequencing using the nextera xt dna sample preparation kit (illumina). a sequencing library was prepared according to the manufacturers' instructions and sequenced using an illumina miseq with × bp paired end reads following standard illumina protocols. for confirmation of the insertions and deletions of the resulting sequence, seven extra sets of primers were designed (available on request). the sequences of these short amplicons were compared with the sequence derived by illumina sequencing. the total reads ( ) were mapped to a reference ericov sequence from germany (genbank accession number kc ). a modified samtools/vcfutils script was used to generate an intermediate consensus sequence in which any indels relative to the original reference sequence were appropriately called. the intermediate consensus was used as the reference for subsequent iteration of mapping and consensus calling. the total number of assembled viral reads was ( . % of the total reads). despite the low proportion of viral sequence detected within the total data set, coverage of the entire genome was obtained (average coverage depth of reads). gross pme findings were categorised into whether respiratory abnormalities (including, but not limited to, pneumonia, consolidation, haemorrhage, congestion, granuloma and high crenosoma sp. burden) and/or gastrointestinal abnormalities (including, but not limited to, haemorrhage, inflammation, intussusception, rupture, abscess and faeces of abnormal colour or consistency) were detected. gross pme findings were available for both organ systems from of the hedgehog examined post mortem and were included in these analyses. a subset of hedgehogs (n = ) examined postmortem that were ericov-positive was selected for histological examination. selection criteria focused on carcases likely to provide the optimal state of tissue preservation from those available, prioritising tissues from carcases examined fresh or when frozen, from carcases with the least evidence of autolysis. a range of available tissue samples from each selected carcase was fixed in neutral-buffered % formalin and was prepared for histopathological examination and stained with haematoxylin and eosin using standard techniques. particular attention was paid to examination of the respiratory and alimentary tracts, when available, as these are the organ systems frequently targeted by mammalian covs. associations between ericov-positive status, case history signalment and pathological findings were explored. variables were first re-categorised and coded using microsoft excel before being exported to ibm® spss® version for windows (ibm®, chicago, il, usa) for statistical analyses. cross-tabulation and pearson's χ tests were undertaken for the binary dependent variable (ericov presence or absence) against all other categorical independent variables. in addition, where sample numbers were sufficient, binary logistic regression (blr) was performed to include consideration of continuous independent variables, such as age, and to quantify the strength of association for multiple variables simultaneously (year, region). a p-value of < . was considered statistically significant. arcgis pro version software (esri, redlands, ca, usa) was used to map the distribution of hedgehogs for which location data were available. of samples examined, ( faecal samples and distal large intestinal tract samples) were positive for ericov, giving a positive percentage of . %. the viral copies detected in positive faecal samples ranged from . × to . x per µl extracted rna. the limit of detection for the bryt® green i based ericov primer rt qpcr assay was calculated as . × copies per µl of extracted rna sample. ibv rna at − dilution was not detected by the ericov-specific assay. the complete genome of the hedgehog ericov from sample (r / , genbank accession number: mk ) is nucleotides (nt) in length, which is comparable to those sequenced from germany: kc and kc ( and nt, respectively). complete genome comparison with open reading frames (orfs) from the german ericov (kc ) gave identities ranging from % (orf b) to % (orf , encoding the envelope protein). the genes encoding the spike protein of the british and german ericov viruses shared % identity (table ) . the locations for all hedgehogs with available data (n = ) are displayed in figure , along with their ericov status. hedgehogs sampled for the study covered a wide area across gb, from devon in south west england to northern scotland. hedgehogs positive for ericov were found in southern england, east of england, north east england and wales. we found no evidence of ericov in hedgehogs from scotland, even though . % (n = ) of sampled hedgehogs were submitted from this country. there was a bias of submissions from the east of england, accounting for . % (n = ) of the total, with norfolk alone contributing . % (n = ) of the hedgehog samples examined, which is explained by a single, large participating wildlife centre in this region. although all regions of gb were represented, some appeared to be under-represented; for example, only . % (n = ) of submissions came from north east england. the majority of hedgehog samples were submitted in and . the proportion positive in was higher ( / , %) than in ( / , %) but there were too few submissions from other years to determine if there was an association with year of sampling. there was a higher number of juvenile hedgehogs included in the study in (n = ) than (n = ), but the proportion of juveniles positive in ( . %) was comparable to the proportion of adults positive ( . %). as coronaviruses are associated with diarrhoea in other species, we tested for the possibility of an association between ericov status and abnormal faeces. the proportion of samples from hedgehogs with reportedly normal faecal colour or consistency, which tested positive for ericov ( / ), was not significantly different from the proportion of all faecal samples with abnormal consistency and/or abnormal colour ( / ) (p = . ). however, a significant association was observed between ericov-positive hedgehogs and those with green faeces (p = . ). a significant association was also observed between ericov-positive hedgehogs and those with yellow faeces (p = . ). colour variation within a single sample of hedgehog faeces was not uncommon: . % (n = ) of hedgehogs with green coloured faeces were also reported to have faeces of brown, yellow or black colouration. no other statistically significant association was shown across the other independent variables, including body condition ( table ). a blr model testing all but the non-mutually exclusive variables also showed no statistical significance. the proportion of hedgehogs with respiratory abnormalities reported at pme that tested positive for eri-cov ( / ) was not significantly different from the proportion of those without respiratory abnormalities that tested positive ( / ) (p = . ). the proportion of hedgehogs with gastrointestinal tract (git) abnormalities that tested positive ( / ) was not significantly different from those that had no git abnormalities ( / ) (p = . ). a statistically significant association was shown between ericov status and geographical region of origin (p = . ) ( table ). the highest proportion of ericov-positive hedgehog samples were submitted from the south of england ( / , %); however, blr showed no significant association (p = . ) between ericov infection status and wider region when other factors including age and year were included. histopathological examination was conducted on available tissues from nine of the ericov-positive hedgehogs in this study that were examined post-mortem: results are presented with the signalment, macroscopic findings and other ancillary diagnostic tests for interpretation in supplementary table s . interpretation was complicated by the state of tissue preservation in the majority of cases, limiting the ability to appraise evidence of either a host inflammatory response or the presence of mild abnormalities (e.g. loss of pseudostratified epithelial cells in the respiratory tract or intestinal villus atrophy, as has been reported with some coronavirus infections in other species [ ] ). examination of the respiratory and gastro-intestinal tract identified frequent metazoan parasite infections of variable severity, sometimes in combination with bronchopneumonia. a range of other significant findings was detected, supported by ancillary testing, including trauma and yersiniosis. we detected ericov, a novel clade c betacoronavirus, in wild western european hedgehogs in gb. this virus had previously only been reported from germany and france; in both instances from this same species. our results, therefore, indicate that ericov is widespread in this european wild mammal. full genome sequence of ericov from gb showed % identity with a ericov previously detected in germany, and % identity with the virus responsible for mers [ ] . there was no evidence to indicate that ericov was causing clinical disease in hedgehogs. the lack of association with detection of ericov and abnormal faecal consistency, poor body condition (i.e. thin or emaciated), respiratory and digestive tract abnormalities suggests ericov is unlikely to be a primary pathogen in this species. this concurs with studies reporting natural cov infections in wild animal species, including bats [ ] and aquatic birds [ ] , which do not manifest as clinical disease. this is also a common feature of a reservoir or maintenance host [ , ] . coronavirus infections of man and other animals are known to predispose to secondary infections, for example, a study recently isolated feline coronavirus and several other enteropathogens from cats with diarrhoea [ ] , thus making any association difficult to detect should it occur with ericov infection in the hedgehog. in the current study, histopathological examination was complicated by concurrent parasitic and bacterial infections in several cases and limited by freeze-thaw artefact and the state of tissue preservation, therefore microscopic lesions caused by viral infection could have been missed or obscured. experimental infection studies and the development of in situ hybridisation to localise ericov in tissues would be worthwhile in the future to further elucidate the clinical significance of ericov infection. despite the lack of association between ericov and faecal abnormalities overall, there was an apparent association between ericov status and green or yellow faeces. green faeces is not an unusual finding in hedgehogs, and can often be associated with crenosoma sp., isospora or salmonella sp. infection [ ] . many of the hedgehogs reported to have green faeces also had faeces of another colour, suggesting that faecal colour change is not unusual. the colour change or the reporting thereof may not be consistent and therefore this result should be interpreted with caution. however, the possibility of a causative relationship between ericov infection and abnormal faeces colour cannot be excluded. significantly stronger amplification of cov rna has previously been described in juvenile and lactating female myotis sp. bats [ ] , and younger age has been shown to be associated with increased coronavirus shedding across various bat species [ , ] . although no evidence for such an association for ericov in the hedgehog was found in the current study, juveniles accounted for only . % of the hedgehogs sampled. examination of a larger sample size of juvenile animals would be worthwhile to further explore the possibility of age-structured prevalence. hedgehogs positive for ericov were detected over a wide region of england and wales, but not from scotland even though . % of all samples tested were submitted from this country (fig. ) . inferring population prevalence from these data is inappropriate due to the sampling strategy, relying on convenience sampling and the catchment area of rehabilitation centres. there is apparent sampling bias since % of all hedgehog samples were from the east of england. demonstrating regional differences in population prevalence and absence of infection would require a much larger, random sample of the population, and information regarding the population structure of hedgehogs in gb. annual variation in the proportion of ericov-positive samples was found. the proportion of hedgehogs sampled in that tested positive for ericov was higher than that in ; a difference that was consistent irrespective of sample type or hedgehog age class (table ). this temporal variability in detection rate is consistent with previous multi-year studies in bats where significant annual variation in coronavirus detection rate was reported [ ] . seasonal variation in contact rates, environmental factors and fluctuating levels of cov immunity in the population have all been postulated as causes for such fluctuations [ ] . proving viral persistence and establishing critical community size is challenging for wildlife populations [ ] . whilst there is now a wealth of studies investigating bat covs, research is lacking for other wildlife covs. there is evidence that the gregarious roosting habits of some species of bat facilitate cov transmission [ ] , but the transmission dynamics of ericov in the more-solitary hedgehog [ ] are likely to be very different. establishing whether infection is endemic in british hedgehogs will require longitudinal sampling, augmented by age-specific serology [ ] . overall, the percentage of ericov-positive hedgehogs detected, at . %, was lower than that reported previously, where . % ( / ) and % ( / ) of specimens tested positive for ericov in germany and france, respectively [ , ] . this difference could be due to assay sensitivity or sample degradation. although the limit of detection of the previously used assays was not given, average copy numbers in the faecal samples were well over the sensitivity threshold of the assay used here. all three studies included hedgehogs in wildlife centres. in the german study, no details were given for the timing of sampling. in the current and french studies, however, faecal samples were taken soon after admission (typically within h) in order to reduce the possibility of nosocomial transmission. dog kennels and shelters are recognised as important environments for maintaining enteric canine covs, with increased length of stay associated with increased probability of cov infection [ ] . however, nosocomial transmission seems to be an unlikely cause of the differences in sample percentage of positive samples in this study. another potential factor is that the relatively high percentage of positive samples in the french and german studies could be simply due to a much higher prevalence of ericov in the mainland europe hedgehog population: the recruited wildlife centres (from which the hedgehogs were sampled) were all located in north and north western france, with one located close to the border with germany [ ] . more studies are required to determine this. hedgehogs in northern europe hibernate for - months of the year [ ] . ericov was detected in both and , suggesting that the virus overwinters in the hedgehog population, but the method of viral persistence is not known. in the current study, no association was detected with the month of sampling but to explore this further, longitudinal sampling of individual hedgehogs could be conducted, including from animals before and after hibernation, to determine if there is persistence of infection in individuals. deriving full genome sequence is an important step in characterising this virus to assess relationships with other covs, including pathogens of domestic animals and man. overall identity to the previous coronavirus detected in hedgehogs from mainland europe was high ( %), but lower than the identity between two german ericov isolates ( %) [ ] . previous studies have demonstrated that ericov shares a common ancestor with mers-cov, and bat coronaviruses hku and hku , raising the possibility of a lineage that has been positively selected for and which has evolved with the potential for cross-species transmission. host tropism in the coronaviruses is largely determined by the spike protein and its ability to bind to host cell receptors. both the german and the gb ericovs have < % amino acid similarity with mers-cov at the crucial receptor-binding domain on the spike protein, suggesting that binding to the human receptor (dipeptidyl peptidase ) is unlikely. virus culture was not attempted in this study, but was previously unsuccessful and this might be due to the cell tropism of the virus [ ] . the detection of ericov in british hedgehogs is a notable finding, and the first report outside mainland europe. wildlife disease research in gb has greatly increased in recent years, particularly for popular wildlife species such as the hedgehog, where citizen science offers a cost-effective approach to achieving national disease surveillance [ ] . it is important that this continues if we are to gain a deeper 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intravenous immunoglobulin fails to improve ards in patients undergoing ecmo therapy date: - - journal: j intensive care doi: . /s - - - sha: doc_id: cord_uid: otf ruvj background: acute respiratory distress syndrome (ards) is associated with high mortality rates. ards patients suffer from severe hypoxemia, and extracorporeal membrane oxygenation (ecmo) therapy may be necessary to ensure oxygenation. ards has various etiologies, including trauma, ischemia-reperfusion injury or infections of various origins, and the associated immunological responses may vary. to support the immunological response in this patient collective, we used intravenous igm immunoglobulin therapy to enhance the likelihood of pulmonary recovery. methods: ards patients admitted to the intensive care unit (icu) who were placed on ecmo and treated with (ivig group; n = ) or without (control group; n = ) intravenous igm-enriched immunoglobulins for days in the initial stages of ards were analyzed retrospectively. results: the baseline characteristics did not differ between the groups, although the ivig group showed a significantly reduced oxygenation index compared to the control group. we found no differences in the length of icu stay or ventilation parameters. we did not find a significant difference between the groups for the extent of inflammation or for overall survival. conclusion: we conclude that administration of igm-enriched immunoglobulins as an additional therapy did not have a beneficial effect in patients with severe ards requiring ecmo support. trial registration: clinical trials: nct ; retrospectively registered. acute respiratory distress syndrome (ards) is characterized by pulmonary inflammation that can be caused by pulmonary and extrapulmonary origins. sepsis, bacterial pneumonia, polytrauma, and aspiration pneumonia are the most common causes of ards [ ] . predictors of survival include age, the type of underlying medical condition, the severity of pulmonary injury, the presence of extrapulmonary organ dysfunction, and ongoing sepsis [ ] . currently, clinical attempts to rescue ards patients include individualized ventilation and fluid management, adequate infection control, including early application of broad-spectrum anti-infectives, neuromuscular blockade using cisatracurium, sedation strategies, prone positioning, and finally extracorporeal membrane oxygenation (ecmo) to ensure oxygenation [ ] [ ] [ ] . although the incidence of ards is relatively high, with five to eight cases per , european inhabitants and even more in the usa, the various pathomechanisms are only partially understood, resulting in different experimental approaches to understand immune responses during early ards [ ] . one well-described issue is decreased immunoglobulin levels in patients with severe infection [ ] as an element of the immunological response in the initial phase of inflammation in response to sepsis [ ] . therefore, one approach to support critically ill patients is intravenous administration of igm-enriched immunoglobulins since this could potentially decrease the severity of inflammation. although this treatment was omitted in recent sepsis guidelines due to a lack of supporting evidence in high-quality trials [ ] , several studies, including one meta-analysis, describe beneficial effects of immunoglobulins in acute pneumonia induced by drug-resistant bacterial infections [ ] [ ] [ ] . furthermore, several case reports describe beneficial effects of antiviral therapy in combination with intravenous immunoglobulin therapy in immune-compromised patients [ ] [ ] [ ] . based on these data, we treated patients with ards requiring ecmo therapy with igm-enriched immunoglobulins immediately after intensive care unit (icu) admission. the objective of this study was to investigate whether intravenous immunoglobulin administration could improve the clinical course of ards in patients treated with ecmo. therefore, mortality, the duration of ecmo therapy, the incidence of renal replacement therapy, the duration of vasopressor and anti-infective therapy, length of stay in the icu, and length of stay in the hospital were analyzed retrospectively in ards patients requiring ecmo therapy. the study was approved by the local research ethics committee of the university hospital and the eberhard-karls university tübingen, germany. patients with severe ards treated with ecmo therapy between january and january at our institution were analyzed retrospectively. in all patients, ecmo therapy was required due to hypoxia and/or increased pulmonary resistance precluding protective lung ventilation. ecmo therapy was performed according to the guidelines of the extracorporeal life support organization (elso) using ila-active systems (novalung, stolberg, germany [ ] . fifty-seven patients were analyzed. twenty-eight patients were treated with igm-enriched immunoglobulins (pentaglobin®; biotest; dreieich, germany) (ivic-group). pentaglobin is an igm-enriched polyvalent immunoglobulin preparation derived from a plasma pool. it contains mg of igm, mg of iga, and mg of igg ( % igg , % igg , % igg , and % igg ) per millilitre. the indication for igm-enriched immunoglobulin (ivig) treatment was determined at the discretion of the treating intensivist. ivig was applied if a viral infection or an infection due to multi-resistant gram-negative bacteria (mrgn) was suspected. ivig dosing was performed according to the instruction of the manufacturer: . ml/kg/h (up to ml) as the initial dose, followed by . ml/kg/h for h until a total dose of mg/kg was achieved. twenty-nine patients did not receive immunoglobulins (control group). vasopressors (norepinephrine; arterenol® sanofi-aventis; germany) were used after volume resuscitation according to the sepsis guideline ( ) . renal replacement therapy was performed with citrate anticoagulation (multifiltrate, fresenius medical care, bad homburg v.d.h. germany) in patients with acute renal failure. data were retrospectively extracted from an ardsspecific database at our institution. since the electronic patient management system was changed during this time frame, not all required data were available for all ards patients. mrsa and vre screening was performed routinely in all patients admitted to the icu. to identify causal infections, bronchoalveolar lavage (bal) samples, blood cultures, urinary samples, and perioperative samples were analyzed. in patients at risk, pcr for atypical pathogens was also performed in bal samples. infection was defined as > colony-forming units (cfu) in bal and/or urinary cultures. for blood cultures, any replicate of bacterial growth was defined as infection. during flu season, influenza infection was detected by pcr. bal samples were also analyzed for other viral pathogens, such as cytomegalovirus (cmv) and herpes simplex virus (hsv). positive findings for these viruses were confirmed by cell cultures. patients with unknown pathogens and immune suppression were additionally screened for other pathogens. anti-infective therapy was applied according to the local guideline considering local resistance patterns. categorical data are reported as numbers and percentages, and continuous data are summarized with the median and range (i.e., the minimum and maximum values) unless otherwise indicated. to compare categorical variables or outcomes, such as the occurrence of infection or in-hospital death, between the ivig group (n = patients) and the control group, which included patients who did not receive ivig therapy, the chisquared test was used. for inter-group comparisons of continuous data, the two-tailed two-sample t test was generally performed. if the original data exhibited a lognormal distribution, e.g., icu length of stay (los), then raw data were log-transformed prior to analysis with the t test. a p value < . was assumed to indicate a statistically significant difference between the groups. the data analysis was performed using jmp® . statistical software (sas institute inc., cary, nc, usa). a total of patients with ards requiring ecmo therapy between and were analyzed retrospectively. twenty-eight patients were treated with igm-enriched immunoglobulins (ivig) for days according to the manufacturer's instruction (ivig group), and patients did not receive ivig therapy (control group). no adverse events were reported after ivig application. the median age of both groups was years, ranging from to years in the control group and from to years in the ivig group. the median simplified acute physiology score (saps) and acute physiology and chronic health evaluation (apache) score were comparable between both groups ( table ). the median apache score in the ivig group was vs. in the control group. in % ( / ) of the ivigtreated patients and % ( / ) of the control patients, the apache score was equal to or greater than . regarding preexisting disease, diabetes mellitus, cardiac disease, immune suppression, and malignancy were more common in the ivig group (table ) . five patients in the control group suffered ards of extrapulmonary origin (three patients with pancreatitis and two with polytrauma). the initial pao /fio ratio in the ivig group was significantly lower than that in the control group (ivig, median vs control, ; t test, log-transformed data, p = . ). seventy-five percent of the ivig-treated patients had severe ards, % had moderate ards, and no patient showed mild ards. in the control group, % of patients had severe ards, % had moderate ards, and % had mild ards. the ecmo duration was shorter in the control group ( - days; median days) compared to that in the ivig group ( - days; median days) and could be reduced earlier in the control group (median days) than in the ivig group (median days). the duration of ventilator support was longer in the ivig group ( - h; median h) compared to that in the control group ( - h; median h; t test, logtransformed data, p = . ). five patients in the control group and three patients in the ivig group were extubated before ecmo was discontinued. ten patients in the control group and seven patients in the ivig group suffered anemia due to bleeding complications, including cerebral hemorrhage (control: n = ; ivig: n = ) ( table ) . all patients suffered from septic shock requiring vasopressor therapy (control, - days vs. ivig, - days). renal replacement therapy was more often required in the control group ( % vs. %; p = . ; pearson's chi-squared test). hepatobiliary dysfunction was comparable between both groups (control, %; ivig, %). in % of the control patients and % of the ivig-treated patients, one or more pathogens could be identified as the cause of pulmonary inflammation. in ( %) patients in the control group, bacterial pathogens such as legionella (n = ; %), streptococcus pneumoniae (n = ; %), pneumocystis jirovecii (n = ; %), and e. coli (n = ; %) were identified in bal samples. none of the control patients showed multidrug-resistant bacteria in any samples. in % ( of ) of the control patients, a viral pathogen was detected in bal samples: influenza (n = ; %) and herpes virus (n = ; %). herpes virus infection included hsv (n = ; %) and cmv (n = ; %). herpes viral infection was confirmed by cell-based culture. in five of the control patients, fungal infection was diagnosed via blood culture (n = ), abdominal sample (n = ), and bal (aspergillus, n = ). in nine control patients, no causal pathogen could be identified ( %). two of the ivig patients were infected with resistant bacteria ( mrgn stenotrophomonas maltophilia and mrsa). in ivig patients, bacterial pathogens such as legionella (n = ; %), streptococcus pneumoniae (n = ; %), pneumocystis jirovecii (n = ; %), and mycoplasma (n = ) were identified in bal samples. viral infections were more frequent in the ivig group (p = . ; pearson's chi-squared test), especially influenza infection (p < . ). in more than half of the ivig patients, viral pneumonia was diagnosed according to the results of bal samples, radiological findings, and clinical symptoms. one or more of the following viruses could be detected in bal samples: influenza (n = ; %) and herpes virus (n = ; %). herpes virus infection included hsv (n = ; %), cmv (n = ; %), and hhv (in bal and blood; n = ). in of the ivig-treated patients, fungal infection was diagnosed in urinary tract (n = ) and abdominal samples (n = ). in addition, one patient suffered aspergillosis pneumonia. in three ivigpatients, no causal pathogen could be identified ( %). the duration of anti-infective treatment was significantly longer in the ivig group than that in the control group (control, median days; - days; ivig, median days; - days; t test, log-transformed data, p = . ). six patients in the control group and four patients in the ivig group underwent anti-infective treatment before admission to the icu. excluding these patients, the duration of anti-infective therapy relative to los in the icu was not significantly different between the groups (control median, % fraction of los icu; ivig, % of los icu). patients treated with ivig stayed for a median of . days in the icu ( to days) and . days in the fig. ). forty-three percent ( out of ) of these patients died; three patients died within the first week due to cerebral hemorrhage (n = ) or multiorgan failure due to septic shock (n = ). the control patients spent a median of days in the icu ( to days) and days in the hospital ( to days); % ( out of ) of these patients died. five patients died during within first week due to cerebral hemorrhage, heart failure, subarachnoidal bleeding (polytrauma), and hypoxic brain injury h after admission, and others died of multiorgan failure due to acute pancreatitis. lymphocyte levels were analyzed retrospectively when available. initially after admission, lymphocyte levels were not different between the groups (control alive . % ± . ; control dead . % ± . ; ivig alive . % ± . ; ivig dead . % ± . ). however, lymphocyte levels were higher in survivors compared to those in non-survivors (fig. ) . the increase in lymphocytes was more prominent in survivors compared to that in non-survivors (control alive . ± . ; ivig alive . ± . ; control dead . ± . ; ivig dead . ± . ; fig. ). in addition, in patients treated with ivig, this increase during treatment was more prominent than that in the control group in the first days (fig. ). since we did not evaluate differential blood counts routinely, limited data were available for determining significant difference. the purpose of this analysis was to systematically investigate the potential effect of igm-enriched immunoglobulins on the outcomes of ards patients requiring ecmo therapy. we analyzed patients; patients were treated with igm-enriched ivig, and patients did not receive ivig therapy. of all patients suffering from septic shock, % of the ivig-treated patients and % of the control patients had severe ards of various origins. however, although the ivig group had a lower pao /fio ratio and required longer ventilation support, longer vasopressor therapy, and longer anti-infective therapy and included mrgn-infected patients, outcome parameters such as los in the icu, los in the hospital, and mortality were not significantly different between the groups. in several studies, hypogammaglobulinemia and overexpression of genes encoding immunoglobulin segments were identified in patients with septic shock [ , , ] . therefore, ivig treatment could be a logical requirement in these patients. we included ivig therapy in our ards patients based on these considerations, although we did not measure immunoglobulin levels before treatment. indeed, in a recent retrospective analysis of patients, high igg levels were associated with high mortality in sepsis patients [ ] . pcr analysis showed an early molecular response to igm in the blood of patients with sepsis [ ] . igm levels remain low in non-surviving sepsis patients, whereas igm levels increase transiently in surviving patients [ ] . single studies and metaanalyses have shown beneficial effects of ivig therapy in patients with sepsis [ ] [ ] [ ] . however, ivig treatment remains controversial. randomized studies have not shown any beneficial effect of this intervention in patients with sepsis or sepsis-associated conditions [ ] . therefore, ivig therapy is not currently recommended in the latest sepsis guidelines [ ] . immunoglobulins interact with cd t-lymphocytes during bacterial eradication. in severe sepsis, b and t cells are depleted due to apoptosis [ ] . since our analysis is retrospective, we could not measure hla-dr expression or cytokine profiles in these patients. however, we found a trend of increased lymphocytes in non-survivors compared to survivors, which may reflect why we did not see any beneficial effect of ivig therapy. although immunoglobulins may play an important role in the innate immune response during ards, nonspecific immunoglobulins may not be as effective in a lymphopenia environment. lymphopenia has been described in ards patients and in animal models after viral infection or mycoplasma infection and affects all lymphoid tissue [ ] [ ] [ ] . however, in our small group of [ , ] , but whether this variability in response is due to genetic alterations [ ] , immune evasion by bacteria [ ] , or different stages of sepsis is unknown. this study has some limitations. it is a retrospective analysis of a limited number of ards patients. since we changed our ecmo system during the study period, we only included patients treated with the one system to exclude any impact of system differences. furthermore, since ivig was ordered by a physician primarily when a viral infection or mrgn infection was suspected, the ivig group included more patients with these infections. however, this may reflect true clinical circumstances in that the cause of disease is typically not determined at admission. ards is a multifactorial disease with a heterogeneous pathogenesis and variable timing and clinical presentations. therefore, one specific therapy is unlikely to improve outcomes. rather than excluding single therapeutic options, identifying patients' risk factors and the individual ards stage may be more important for successful outcomes. we report here in our retrospective analysis that intravenous igm administration in the initial stages of severe ards did not improve overall outcomes or the severity of disease. the acute respiratory distress syndrome the alien study: incidence and outcome of acute respiratory distress syndrome in the era of lung protective ventilation the standard of care of patients with ards: ventilatory settings and rescue therapies for refractory hypoxemia neuromuscular blockers in early acute respiratory distress syndrome prone positioning in severe acute respiratory distress syndrome transcriptomic evidence of impaired immunoglobulin 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plasmatic immunoglobulin g, a and m levels in septic shock patients serum igg levels and mortality in patients with severe sepsis and septic shock : the sbits data quantification of igm molecular response by droplet digital pcr as a potential tool for the early diagnosis of sepsis kinetics of circulating immunoglobulin m in sepsis: relationship with final outcome use of polyclonal immunoglobulins as adjunctive therapy for sepsis or septic shock a case of acute respiratory distress syndrome associated with novel h n treated with intravenous immunoglobulin g early therapy with igm-enriched polyclonal immunoglobulin in patients with septic shock surviving sepsis campaign: international guidelines for management of sepsis and septic shock sepsis-induced apoptosis causes progressive profound depletion of b and cd + t lymphocytes in humans clinical diagnosis of pandemic a(h n ) influenza in children with negative rapid influenza diagnostic test by lymphopenia and lower c-reactive protein levels. influenza other respir viruses clinical features and outcomes of severe acute respiratory syndrome and predictive factors for acute respiratory distress syndrome depletion of lymphocytes and diminished cytokine production in mice infected with a highly virulent influenza a (h n ) virus isolated from humans subphenotypes in acute respiratory distress syndrome: latent class analysis of data from two randomised controlled trials acute respiratory distress syndrome subphenotypes respond differently to randomized fluid management strategy the role of genetics and antibodies in sepsis immunoglobulins and their receptors, and subversion of their protective roles by bacterial pathogens none. this work was supported by a grant of the deutsche forschungsgemeinschaft to peter rosenberger dfg-ro / - . the datasets obtained and/or analyzed during the current study are available from the corresponding author upon reasonable request. authors' contributions sp prepared the manuscript and analyzed the data. as and ab are responsible for the data acquisition. ak is responsible for the data acquisition and analysis. gb is responsible for the data analysis. hah is accountable for all the aspects of the work and for ensuring that the questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. all authors read and approved the final manuscript. the ethics committee of the university hospital tuebingen (eberhard-karls universität tübingen; ethik-kommission; gartenstraße ; tübingen, germany) approved the study. the project number is / /bo . informed consent was not required from the patients involved into this retrospective analysis. this proceeding was approved by the local ethics committee (project number / /bo ) of eberhard karls university tuebingen since extraction of pseudonymous medical records provides structured data and therefore legally permits evaluation of the data for research. not applicable. the authors declare that they have no competing interests.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -j k oel authors: herrera-rodriguez, josé; signorazzi, aurora; holtrop, marijke; de vries-idema, jacqueline; huckriede, anke title: inactivated or damaged? comparing the effect of inactivation methods on influenza virions to optimize vaccine production date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: j k oel the vast majority of commercially available inactivated influenza vaccines are produced from egg-grown or cell-grown live influenza virus. the first step in the production process is virus inactivation with β-propiolactone (bpl) or formaldehyde (fa). recommendations for production of inactivated vaccines merely define the maximal concentration for both reagents, leaving the optimization of the process to the manufacturers. we assessed the effect of inactivation with bpl and fa on different influenza virus strains. the properties of the viral formulation, such as successful inactivation, preservation of hemagglutinin (ha) binding ability, fusion capacity and the potential to stimulate a toll-like receptor (tlr ) reporter cell line were then assessed and compared to the properties of the untreated virus. inactivation with bpl resulted in undetectable infectivity levels, while fa-treated virus retained very low infectious titers. hemagglutination and fusion ability were highly affected by those treatments that conferred higher inactivation, with bpl-treated virus binding and fusing at a lower degree compared to fa-inactivated samples. on the other hand, bpl-inactivated virus induced higher levels of activation of tlr than fa-inactivated virus. the alterations caused by bpl or fa treatments were virus strain dependent. this data shows that the inactivation procedures should be tailored on the virus strain, and that many other elements beside the concentration of the inactivating agent, such as incubation time and temperature, buffer and virus concentration, have to be defined to achieve a functional product. influenza virus is a threatening pathogen, that causes significant morbidity and mortality worldwide. it is responsible each year for epidemics that cause millions of cases of severe illness, and it is also a threat because of its potential to cross species barriers and generate pandemics [ ] . vaccination is the most cost-effective strategy to prevent infection and severe outcomes [ ] . currently marketed influenza vaccines include live-attenuated vaccines, whole inactivated virus (wiv), split vaccines and subunit vaccines (which can be derived from viral particles or recombinant antigens) [ ] . live virus vaccines rely on attenuation; e.g. use of reassortant strains with the backbone of cold-adapted viruses, but equal (or comparable) immunogenicity. for all the other types of vaccines derived from whole viral particles, a crucial step in production is virus inactivation. the general concept behind the inactivation procedure, stated by international guidelines as provided by ema, fda, and who [ ] [ ] [ ] , is that the process must inhibit the replication of the virus without destroying its antigenicity. thus, the inactivation process should cause minimum alteration of the main antigens, which for influenza virus are the viral surface glycoproteins hemagglutinin (ha) and neuraminidase (na). preparation of inactivated influenza vaccines is conventionally achieved by exposure of cell-or egg-derived live virus to betapropiolactone (bpl) or formaldehyde (fa). bpl reacts readily with nucleophiles, resulting in alkylated and acylated products. according to older literature, nucleic acids are the main targets of these modifications, which comprise nicks and cross-links between rna and viral proteins [ , ] . however, recent findings shed new light on the mechanism of bpl inactivation and show that it also alters viral proteins, resulting in loss of ha and na functionality and fusion ability [ ] . the stability of bpl is known to depend on the type of buffer and the ph of the mixture used, suggesting that the amount and nature of modifications in viral components will also be determined by these variables [ ] . in the case of formaldehyde, viral inactivation is achieved by the alkylation of amino and sulphydrilic groups of proteins and purine bases [ ] . as fa crosslinks the viral proteins [ ] , the fusion ability of the virus can be affected. since inactivation is a critical step during vaccine preparation, given that the manufacturing of not only wiv but also split and subunit vaccines depends on effective virus inactivation, the major pharmacopoeias specify quality standards for this process. recommendations in international guidelines regarding inactivation are, however, quite vague, and the establishment of optimal conditions is left to the manufacturers. specifications provided for the inactivation procedure include indications on the temperature of storage and the maximum amount of inactivating agents. nonetheless, the choice of variables such as the concentration of the virus at the time of inactivation, the buffer systems used to dilute the inactivators, the ph of the virus suspension, or the duration of the incubation with the agents remains yet unspecified. furthermore, the guidelines are not clear about whether these parameters are to be optimized on the basis of the vaccine strain, the concentration of the virus or any other criterion. more precise guidelines would be desirable in order to prevent the receptor binding sites and epitopes in the vaccine from being destroyed during the inactivation process. recent studies show that excessive inactivation with fa and bpl may cause unanticipated modifications to the vaccine antigens that result in diminished potency; lower hemagglutination titers and loss of na activity [ , [ ] [ ] [ ] [ ] [ ] . this suggests that chemical inactivation might affect the protein conformation leading to a loss of immunogenicity of the antigenic epitopes of the key surface proteins. the present study presents a systematic evaluation of different inactivation protocols (in accordance with the international guidelines) on several influenza a virus (iav) strains. our aim was to compare the effects of these procedures on the key properties, namely residual infectivity, receptor binding, fusion, and toll-like receptor (tlr ) mediated activation of innate immune mechanisms, and to determine whether these effects are similar for different virus strains. the strains used in this study were a/puerto rico/ / h n (pr ), a/new caledonia/ / h n (nc), a/perth/ / h n (h ), nibrg- a/turkey/turkey/ / h n (h ), and nibrg- a/anhui/ / h n (h ); all vaccine strains with a pr backbone produced through classic reassortment (nc, h ) or recombinant technology (h , h ). all seed viruses were obtained from the national institute for biological standards and controls, potters bar, united kingdom and were propagated in days old embryonated eggs. after inoculation of the virus and incubation for h, the allantoic fluid was harvested and clarified by low speed centrifugation, followed by two rounds of purifications of the virus on a sucrose gradient [ ] . protein and phospholipid determinations were performed on the purified virus using the micro lowry and the bligh and dyer method, respectively [ , ] . all virus preparations were used at a concentration of . nmol/ml of phospholipid for inactivation. the number of virus particles was calculated assuming that each virion contained about , phospholipid molecules. this number was derived using the reported composition ( - % lipid) and dry weight (  À g) of influenza virions and a mean mw for lipids of - g/mol [ ] . this calculation does not take into account a possible contamination of the virus preparations with exosomes which would also be assumed to contain lipids and would presumably co-purify with virus on the sucrose gradient. however, the amount of exosomes is not expected to vary among the virus strains and is therefore neglected here. all procedures and dilutions were performed in hne buffer ( mm hepes, mm nacl, . mm edta, ph . ). three standard inactivation protocols were selected: in the first one, beta-propiolactone % (acros organics, geel, belgium) was used at a final concentration of . % v/v and inactivation was done by overnight incubation (bpl). in the second protocol, % formaldehyde was used at a final concentration of . % v/v and incubation was for h (fa- ). the third protocol also involved the use of formaldehyde at a final concentration of . % v/v but incubation was done for h (fa- ). all incubations were performed at °c under constant stirring. after incubation with either bpl or fa, the preparations were dialyzed in , mwco dialysis tubes against hepes buffer overnight at °c to remove all traces of the chemicals. again, protein and phospholipid determinations were performed on the dialyzed samples, and all samples were normalized to a concentration of . nmol/ml of phospholipids. infectivity of both untreated and treated virus samples was tested by performing a tcid assay on mdck cells. briefly, confluent cultures of mdck cells in -well plates were incubated with ll of two-fold serial dilutions of virus or vaccine at an initial concentration of . nmol/ml of phospholipids at °c in % co for h. cells were then washed with pbs and incubated for days at °c with ll/well of episerf medium (gibco) supplemented with lg/ml tpck-treated trypsin. tcid titers were calculated according to the trimmed spearman-karber method [ ] . hemagglutination assays were performed using two-fold serial dilutions of culture supernatants in pbs in v-bottom plates ( ll/ well). subsequently, ll of % guinea pig red blood cells (harlan, the netherlands) were added to each well. the plates were incubated for h at room temperature and the hemagglutination or the absence of hemagglutination was determined visually for each well. membrane fusion was assessed by measuring leakage of hemoglobin during fusion of virus particles with erythrocytes [ ] . briefly, either guinea pig or chicken blood was diluted : with hne buffer ( mm hepes, mm nacl, . mm edta, ph . ). after centrifugation for min at g, erythrocytes were removed from under the layer of peripheral blood mononuclear cells, washed once again with hne buffer, counted and brought to a concentration of  / ll. virus-induced hemolysis was measured by mixing either live virus or vaccines ( nmol of phospholipid) with red blood cells (  ) and fusion buffers of different ph values, ranging from . to . in a final volume of ml. after min of incubation at °c the suspension was centrifuged at g for min and absorbance of the supernatant was read at nm. autohemolysis (occurring in fusion buffers of different ph values in the absence of virus or vaccine) and maximal hemolysis (in water) were used to set % and % of hemolysis. fusion was calculated as: %hemolysis ¼ à expod À autohemod at same ph maxod À autohemod at same ph % hek-blue tlr cells (invivogen) co-express human tlr and an nf-kb-inducible secreted embryonic alkaline phosphatase (seap) reporter gene that can be monitored using the detection medium quanti-blue tm . hek-blue tlr and hek-null (control, not expressing any tlrs) cells were cultured according to the manufacturer's instructions. . cells/well were plated in a -well plate and stimulated with serial -fold dilutions of untreated (starting dilution : v/v) or inactivated (starting dilution : v/v) influenza virus. initial concentration of stocks of live and inactivated virus was . nmol/ml of phospholipids. after h, ml of supernatant were added to ml of quanti blue. after h of incubation at °c, the plates were read in an elisa reader ( nm). results are expressed as relative activation of cells, in comparison to the activation level obtained upon stimulation with ng/ml of the tlr stimulant r (invitrogen) [ ] . in order to allow comparison of the effects of different inactivation processes, samples of different influenza virus strains (pr , nc, h , h and h ) were purified and diluted so that all the batches had the same virus concentration. since the amount of contaminating ovalbumin might differ among different virus preparations, phospholipid content was chosen over protein content for quantification of virus particles. each of the virus preparations at the desired concentration ( . nmol phospholipid/ml) was then divided in independent batches: of them were inactivated with bpl (bpl), were inactivated with formaldehyde with days incubation period (fa- ) and others were also inactivated with formaldehyde, but with days incubation period (fa- ). the last batch was kept untreated as a control. tcid assays on mdck cells were performed in order to determine the number of infectious particles in the virus samples before and after inactivation. the initial infectivity of the virus strains differed substantially: pr showed particularly high infectivity resulting in a particle/tcid ratio of about , while infectivity of h was low (particle/tcid ratio:  ). infectivity of the other strains was rather similar (particle/tcid ratios: between  and  ). after the inactivation process we observed that all treatments had reduced the virus titers by at least logs. in case of bpl treated virus, pr , h and h were found to have no residual particles in any of the independently treated batches while one batch of nc and all batches of h showed some (though very low) residual infectious particles ( - tcid /ml). fa, irrespective of the incubation period, was unable to completely inactivate the virus samples. residual titers varied from to tcid /ml with nc showing the lowest and h showing the highest titers. in the case of pr and h , we observed slightly improved reduction in the residual infectivity after longer incubation periods with fa, yet this effect was not observed for the other strains. hemagglutinin is an important determinant of the infectivity of influenza a virus. because the role of the hemagglutinin is to bind to the sialic acid receptors on the infected cell, we consider binding as a reflection of a functional protein and therefore of an antigen similar to the one found on the live virus. thus, we assessed the binding capacity of the viral hemagglutinin after the inactivation process. for untreated samples of the different virus strains the binding capacity in hau/ml was rather similar: .  for pr , .  for nc, .  for h , .  for h and  for h . for the inactivated samples the percentage of relative hemagglutinating capacity was calculated taking the obtained value of the untreated samples as %. as shown in fig. all treatments caused a reduction in the binding capacity of the virus. after inactivation with bpl there was a complete loss of binding ability for pr as well as for h , consistent among all batches. for nc, two batches retained a minimal amount of binding ability. h and h were the strains that retained most of the binding ability after inactivation with bpl, consistent among the batches ( - %). fa treatment had less effect on the hemagglutination ability of the virus strains than bpl, independent of the inactivation period. however, the effect of fa varied for the different virus strains. while pr lost almost its entire binding ability, nc retained most of its binding ability after inactivation with fa ( %). h , h and h retained some of the binding ability ( - %) with some variation among the different batches. next to being responsible for binding of influenza virus to a target cell, ha also mediates fusion of the viral with the endosomal membrane during the infection process. retaining this function after inactivation would be a further indication that ha is in its native state. moreover, it has been reported that fusion-active whole inactivated virus vaccines are better inducers of type interferons and allow better induction of cd t lymphocytes [ , ] . fusion ability was measured by incubation of viral or vaccine particles ( nmol of total phospholipids) with either guinea pig erythrocytes for pr , nc and h or chicken erythrocytes for h and h , following a protocol reported previously [ ] . as shown in fig. , all viruses fused with the respective erythrocytes with final rates of fusion varying from % for h and h to % for pr . the optimal ph for fusion was higher for pr (ph . ) than for the other virus strains (ph . or . ). bpl completely inhibited the fusion ability of the virus, except for h where some minimal fusion ability was retained (% %). in contrast, after fa inactivation, most of the virus particles retained some fusion ability. only the fusion ability of h was severely affected by fa. for pr , the inactivation process appeared to change the optimal fusion ph causing the inactivated samples to fuse at a lower ph. also for h , loss of fusion activity after fa treatment was more pronounced at higher than at lower ph. when comparing inactivation with fa for days and days, we noticed a further reduction in the fusion ability of the virus treated for days. this was most noticeable for h and h where we saw a reduction of around % of the fusion ability of the samples inactivated for days as compared to days. after infection, tlrs present on and in antigen-presenting cells of host cells sense the viral components; thus, they form an important constituent of the innate antiviral response [ ] . in particular, tlr is the sensor for the single-stranded rna present in live virus and whole inactivated vaccines; its activation is a good indicator of the ability of the virus to be internalized since the receptor is exclusively located on endosomal membranes [ , ] . in order to determine whether the inactivation procedure would affect the ability of the viruses to attach, fuse and activate tlr in the endolysosomal compartments, we stimulated hek blue tlr cells (expressing a reporter construct upon ligand binding to tlr ) with untreated and inactivated pr , nc, h , h or h . results are expressed as percentage of activation of the cells, compared to the response to a fixed amount of the tlr stimulant r (set as % activation). after h of incubation in a medium containing tpck-trypsin, the supernatants were harvested and presence of virus was determined by hemagglutination assay. tcid was calculated as described previously [ ] . stimulation of hek blue cells with untreated virus showed that the different strains varied largely in their ability to activate tlr and for the different virus optimal activation was observed at different dilutions (fig. , left panels) . inactivation had marked effects on the ability of the viruses to stimulate tlr . no activation was observed at dilutions found optimal for the different live viruses. for inactivated nc, h , and h even at the lowest dilution of : no or very little activation of tlr was found while pr and h showed some activation at low dilutions. bpl inactivation generally preserved the ability to activate tlr somewhat better than fa. thus, all inactivation protocols strongly reduced the ability of the viruses to stimulate tlr ; however, there were differences among the different virus strains. inactivation is a crucial but ill-defined step in influenza vaccine preparation. we therefore intended to systematically assess the effect of common inactivation procedures on different phases of the interaction of inactivated virus particles with cells in vitro. to this end, we used different virus strains in order to reveal possible strain-specific differences and performed the inactivation procedures for all viruses using a single buffer and the same virus concentration to eliminate possible variation, an approach also taken by others [ ] . our results show that, under the conditions used, bpl was quite efficient in inactivating the viral particles while formaldehyde, although reducing the infectious titer by - logs, was unable to completely abolish infectivity. all inactivation methods caused damage to the binding capacity of the viral particles, with bpl causing greater loss than fa. the ability of the virus to fuse with target membranes at low ph was especially affected by bpl treatment, while fa-treated virus still maintained some fusion ability. the stimulation of a tlr reporter cell line was also affected by the treatment, with fa inactivation leading to a greater loss of the ability to stimulate tlr than bpl inactivation. the magnitude of the effects caused by bpl or fa treatment appeared to be virus strain dependent, as different iav strains were affected to varying degrees. from our results, it is clear that chem-ical inactivation impacts on various properties of influenza virus in a treatment dependent and strain dependent way. our study revealed that bpl reduced the infectivity of pr , h and h virus to undetectable levels. nc and h virus showed some (although very low) residual infectivity (fig. ) indicating that these two strains might be more resistant to inactivation with bpl. previous studies show that bpl is capable of complete inactivation of influenza virus; however, the effectivity might vary depending on the incubation time and temperature. a review of the literature suggests that at a concentration of . % bpl is able to completely inactivate the virus if incubation is performed for - h at temperatures above °c (but below °c). however, if the same concentration of bpl is used but incubation is executed at °c like used in our study, an inactivation period ranging from h to a week is needed for complete loss of infectivity [ ] [ ] [ ] . in contrast to bpl, fa was not able to completely inactivate influenza virus in our study. again, there seem to be strainspecific differences in the sensitivity to fa since some strains were more effectively inactivated than others and for some strains (pr , h ) inactivation improved with longer exposure times to fa while for other strains this was not the case. a variety of different methods for fa inactivation has been described in the literature [ , , , [ ] [ ] [ ] [ ] . these methods vary with respect to the exact experimental conditions used, including concentration and incubation time. in some cases, incomplete inactivation is reported, mostly occurring when the inactivation was performed at low temperature [ ] , as was done in this study. inactivation procedures should not affect the immunological properties of the viral antigen. to determine the effects of the different inactivation protocols on the recognition of the virus by haspecific antibodies, we tried to perform a single radial diffusion (srid) assay on the virus samples before and after the inactivation procedure. however, the relatively low concentration of virus used for inactivation (chosen to allow optimal access of the inactivating agent to the viral proteins) was not suitable to render reliable readings. yet, functional properties of the viral ha, namely binding to sialic acid residues on the target cell and mediating fusion of the viral and the endosomal membrane, are also indicative of a func- fig. . effect of inactivation procedures on fusion ability. fusion ability was measured by incubation of viral or vaccine particles ( nmol of total phospholipids) with either guinea pig rbc (a-c) or chicken rbc (d-e) followed by the addition of fusion buffer to reach the desired ph. after min incubation, samples were centrifuged and the supernatants were collected. samples were read at nm on a spectrophotometer. percentage of fusion was then calculated as described previously [ ] . tional and unaltered protein. we therefore investigated the effects of the different inactivation protocols on hemagglutination and on hemolysis as surrogates for binding and fusion, respectively. as all our observations are based on in vitro tests, the correlation with the different vaccine potencies in vivo should be explored in future experiments. the use of single radial immunodiffusion assay as an estimate for protection has been shown to correlate both with in vivo responses [ ] and, more recently, with in vitro results of differently manufactured influenza vaccine [ ] . however, while the srid assay would have been an alternative method to measure ha integrity, it would have given information on antibody binding only. binding as measured in a hemagglutination assay was strongly reduced by bpl inactivation, with only h and h retaining some binding ability. fusion was even more deeply affected as all virus strains completely lost their ability to fuse after treatment with bpl. virus binding to the target membrane is a prerequisite for fusion (at least as measured in the assay used). thus, the loss of fusion ability was to be expected. previous reports have shown that h n and h n viruses had reduced agglutination capacity following bpl treatments [ , , , , ] -although in many cases the inactivation was performed at different temperatures, incubation times or concentrations than used in our experiments. for what concerns the effects of bpl on the fusion ability of iav, some studies show that h n and h n strains were almost completely inhibited in their fusion ability at bpl concentrations ranging from . % to . % [ , ] . however, budimir et al. [ ] managed to inactivate iav with bpl and retain the fusion ability of the virus; yet, the temperature discrepancy with our methods could have led to the observed differences in virus alterations. as for the effect of fa on the binding ability, fa treated virus strains were still able to bind to erythrocytes though with reduced effectivity, except for the pr strain which almost completely lost its ability to agglutinate erythrocytes. literature on the effect of fa on virus binding is contradictory. studies report that fa inactivation damaged the binding ability of the virus at conditions (incubation time, concentration and temperature) similar to those tested in our work [ , ] , while others found little effect of the fa treatment, though using different conditions as per the temperature at which the inactivation process was conducted [ , ] . all fatreated samples almost completely retained their fusion ability. geeraedts et al. [ ] and budimir et al. [ ] reported complete loss of fusion ability with fa treated virus, yet in these experiments iav was deliberately treated with fa at extremely high concentration or prolonged exposure to inhibit their fusion ability. it has been previously reported that the magnitude and the phenotype of the immune response induced by wiv influenza vaccines are superior to those induced by split or subunit vaccines [ , ] . the higher immunogenicity of wiv could be largely attributed to activation of tlr by ssrna present in the viral particles [ ] . furthermore, since the stimulation of an endosomal toll-like receptor depends on viral endocytosis (and thus on ha functionality) [ ] , the decreased stimulation of such a receptor can indicate possible modifications of the surface antigen induced by the inactivation procedure. we therefore investigated the effect of the different inactivation methods on tlr triggering. all inactivation procedures studied markedly reduced and often completely abolished tlr triggering, with effects of bpl inactivation being somewhat less severe than those of fa treatment. this was surprising considering that bpl is supposed to mainly affect nucleic acids while fa is supposed to mainly affect proteins [ ] . loss of tlr triggering did not necessarily correlate with loss of binding and was thus not only a result of less virus reaching the endosomal compartment. to our knowledge, the effect of virus inactivation on the capability of wiv to trigger tlr has not been studied previously. earlier studies demonstrating that wiv is capable of activating tlr were per-formed using much higher concentrations of virus ( mg/ml viral protein as compared to about . mg/ml used in this study) [ ] . in these studies, effects of the inactivation procedure might therefore have been obscured. several studies have reported the negative impact of chemical inactivation with bpl on virus characteristics; however, those studies have focused mainly on h n strains. bpl was found to affect binding [ , ] , fusion activity [ , ] and modify protein residues [ , ] , in line with our findings. though all the studies were in compliance with the established guidelines with respect to bpl concentration ( . %), all used different conditions with respect to buffer, virus concentration, incubation time and temperature etc. this might explain variations in outcome among the different studies. one of the initial effects of bpl addition is a decrease in ph caused by hydrolysis of bpl to b-propionic acid and hydracrylic acid derivatives [ ] ; this could then lead to all the other observed undesirable consequences of bpl treatment on ha and na functions. it is known that influenza virus strains vary in their sensitivity to low ph, with pr being one of the most labile strains [ , ] . our findings corroborate these earlier observations. on the other hand, fa has been linked to incomplete inactivation which can cause outbreaks of virus infections upon vaccination; this was reported for several viruses, such as foot-andmouth disease virus (fmdv) [ ] and venezuelan equine encephalitis virus (veev) [ ] . it has also been shown that temperature is an important factor in virus inactivation by formaldehyde: darnell me et al. [ ] reported that sars-coronavirus could not be inactivated at a low temperature of °c even after days of incubation. however, when using a higher temperature of or °c, formaldehyde could inactivate most of the virus after day. our results contribute to the increasing evidence that the inactivation protocols have to be adapted by virus strain and that many other important factors beyond the concentration of the inactivator itself, such as virus concentration, buffer, incubation time and temperature, have to be considered. novel inactivation protocols, such as uv and gamma radiation [ ] or the use of hydrogen peroxide [ ] , have already been mentioned in the literature but will need thorough testing and standardization before they can be employed in the context of influenza vaccine production. there are currently new emerging technologies to manufacture influenza vaccines that would not require an inactivation process, such as production of iav proteins on in vitro cultures or peptides derived from iav proteins, all showing promising results; yet, egg cultures are currently the cheapest and most efficient way to produce high amounts of vaccines in a relative short amount of time. therefore, until a new vaccine production method that can compete with the egg culture is developed, inactivation will be a standard procedure in vaccine manufacturing. our results are therefore a call for establishment of more detailed inactivation procedures for vaccine manufacturers, and for a search for different and more efficient inactivation methods to be included in the international guidelines. the authors declare that there are no conflicts of interest. pandemic influenza-including a risk assessment of h n advances in the development of influenza virus vaccines traditional and new influenza vaccines the european agency for the evaluation of medical products. note for guidence on harmonisation of requirements for influenza vaccines annex -recommendations to assure the quality, safety and efficacy of influenza vaccines (human, live attenuated) for intranasal administration summary minutes - nd vaccines and related biological products advisory committee meeting effect of formalin, beta-propiolactone, merthiolate, and ultraviolet light upon influenza virus infectivity chicken cell agglutination, hemagglutination, and antigenicity principles of selective inactivation of viral genome. vi. inactivation of the infectivity of the influenza virus by the action of fl-propiolactone treatment of influenza virus with beta-propiolactone alters viral membrane fusion reactions of betapropiolactone with nucleobase analogues, nucleosides, and peptides: implications for the inactivation of viruses inactivation of avian influenza viruses by chemical agents and physical conditions: a review identification of formaldehyde-induced modifications in proteins: reactions with insulin hydrogen peroxide inactivation of influenza virus preserves antigenic structure and immunogenicity effect of inactivation method on the cross-protective immunity induced by whole ''killed" influenza a viruses and commercial vaccine preparations evaluation of different methods of inactivation of newcastle disease virus and avian influenza virus in egg fluids and serum influenza virus inactivation for studies of antigenicity and phenotypic neuraminidase inhibitor resistance profiling development of a dried influenza whole inactivated virus vaccine for pulmonary immunization a simplification of the protein assay method of lowry which is more generally applicable bligh and dyer" and folch methods for solid-liquid-liquid extraction of lipids from microorganisms. comprehension of solvatation mechanisms and towards substitution with alternative solvents models of structure of the envelope of influenza virus determination of % endpoint titer using a simple formula induction of heterosubtypic cross-protection against influenza by a whole inactivated virus vaccine: the role of viral membrane fusion activity smallantiviral compounds activate immune cells via the tlr myd -dependent signaling pathway effect of viral membrane fusion activity on antibody induction by influenza h n whole inactivated virus vaccine the role of membrane fusion activity of a whole inactivated influenza virus vaccine in (re) activation of influenza-specific cytotoxic t lymphocytes toll-like receptor control of the adaptive immune responses th cells primed during influenza virus infection provide help for qualitatively distinct antibody responses to subsequent immunization evaluation of the effects of concentration, duration and temperature of incubation with beta-propiolactone on inactivation of cell-derived rabies virus evaluation of different inactivation methods for high and low pathogenic avian influenza viruses in egg-fluids for antigen preparation effect of physico-chemical factors on survival of avian influenza virus (h n type) whole influenza virus vaccine is more immunogenic than split influenza virus vaccine and induces primarily an igg a response in balb/c mice superior immunogenicity of inactivated whole virus h n influenza vaccine is primarily controlled by toll-like receptor signalling evaluation of virus inactivation by formaldehyde to enhance biosafety of diagnostic electron microscopy single-radial-immunodiffusion as an in vitro potency assay for human inactivated viral vaccines influenza vaccine manufacturing: effect of inactivation, splitting and site of manufacturing. comparison of influenza vaccine production processes effect of the b-propiolactone treatment on the adsorption and fusion of influenza a/ brisbane/ / and a/new caledonia/ / virus h n on a dimyristoylphosphatidylcholine/ganglioside gm mixed phospholipids monolayer at the air-water interface whole inactivated virus influenza vaccine is superior to subunit vaccine in inducing immune responses and secretion of proinflammatory cytokines by dcs influenza virus entry surface modifications of influenza proteins upon virus inactivation by b-propiolactone variations in ph sensitivity, acid stability, and fusogenicity of three influenza virus h subtypes stability of infectious influenza a viruses at low ph and at elevated temperature biochemical identification of viruses causing the outbreaks of foot and mouth disease in the uk review of accidents caused by incomplete inactivation of viruses inactivation of the coronavirus that induces severe acute respiratory syndrome, sars-cov analysis of antigen conservation and inactivation of gammairradiated avian influenza virus subtype h n we would like to thank sarah skeldon, national institute for biological standards and controls, uk, for running srid assays on the virus samples. key: cord- -p macofk authors: biezen, ruby; grando, danilla; mazza, danielle; brijnath, bianca title: visibility and transmission: complexities around promoting hand hygiene in young children – a qualitative study date: - - journal: bmc public health doi: . /s - - -x sha: doc_id: cord_uid: p macofk background: effective hand hygiene practice can reduce transmission of diseases such as respiratory tract infections (rtis) and gastrointestinal infections, especially in young children. while hand hygiene has been widely promoted within australia, primary care providers’ (pcps) and parents’ understanding of hand hygiene importance, and their views on hand hygiene in reducing transmission of diseases in the community are unclear. therefore, the aim of this study was to explore the views of pcps and parents of young children on their knowledge and practice of hand hygiene in disease transmission. methods: using a cross-sectional qualitative research design, we conducted in-depth interviews with pcps and five focus groups with parents (n = ) between june and july in melbourne, australia. data were thematically analysed. results: participants agreed that hand hygiene practice was important in reducing disease transmissions. however, barriers such as variations of hand hygiene habits, relating visibility to transmission; concerns around young children being obsessed with washing hands; children already being ‘too clean’ and the need to build their immunity through exposure to dirt; and scepticism that hand hygiene practice was achievable in young children, all hindered participants’ motivation to develop good hand hygiene behaviour in young children. conclusion: despite the established benefits of hand hygiene, sustained efforts are needed to ensure its uptake in routine care. to overcome the barriers identified in this study a multifaceted intervention is needed that includes teaching young children good hand hygiene habits, pcps prompting parents and young children to practice hand hygiene when coming for an rti consultation, reassuring parents that effective hand hygiene practice will not lead to abnormal psychological behaviour in their children, and community health promotion education campaigns. hand hygiene, including hand washing with soap and water, or the use of hand sanitizers, has been shown to reduce transmission of infectious diseases [ ] [ ] [ ] , especially gastrointestinal and respiratory tract infections [ ] . young children < years of age are most at risk, in particular those attending childcare or preschool [ ] [ ] [ ] . effective hand hygiene practice in community settings, has demonstrated a reduction of infections occurring in childcare [ ] [ ] [ ] [ ] , schools [ , [ ] [ ] [ ] , and in the home [ ] [ ] [ ] . according to aiello et. al's meta-analysis [ ] improvements in hand hygiene resulted in a % reduction in respiratory illnesses and a % reduction in gastrointestinal illnesses in community-based settings. the importance of hand hygiene practice in the prevention of infectious diseases was emphasized in all studies included in this meta-analysis. studies from europe, us, and the uk have also shown that hand hygiene interventions in the community can increase hand hygiene compliance among children [ ] [ ] [ ] . for example, interventions involving teacher modelling hand hygiene to school children [ ] , improving educator's knowledge and attitude towards hand hygiene [ ] , and the use of alcohol-based sanitizers [ , , ] have significantly reduced illness absenteeism in schools. however, factors such as lack of time to practice hand hygiene, poor adult modelling of regular hand washing, limited facilities including available sinks, soap and water, and the lack of knowledge regarding the importance of hand hygiene have hindered the compliance and sustainability of good hand hygiene practice [ , ] . despite wide promotion of hand hygiene in australia [ ] and good evidence that effective hand hygiene practice reduces infectious disease transmission, to date no studies have measured the efficacy and sustainability of hand hygiene practice in the australian primary care setting. thus, it is unclear whether primary care providers (pcps) and their patients follow recommended protocols to reduce infectious diseases, especially in young children. accordingly, the aim of this study was to explore the views of pcps and parents of young children regarding the practice of hand hygiene in the transmission of diseases in young children. data for this research were derived from a larger mixed methods qualitative study exploring pcps and parents' views, knowledge and attitudes towards their hand hygiene practice and reducing rti transmission in children < years of age. the methods applied have been previously described [ ] ; in summary, interviews were conducted with pcps and five focus groups with parents of young children (see table for schedules). pcps were defined as general practitioners (gps), practice nurses (pns), maternal child health nurses (mchns), and pharmacists (phs), and a diversified sampling strategy was applied to recruit them. the contact details of gps and pns were generated from an existing general practice database at monash university, victoria, australia. contact details for mchns and phs were generated from the maternal child health services directory [ ] and the local business directory respectively. recruitment was limited to one pcp per practice site across metropolitan melbourne, australia. purposive sampling via advertisements circulated to playgroups and mothers' groups was used to target parents and carers from the south east and east of melbourne, australia. five mothers' groups and play groups were initially approached to recruit the required number of parents and carers. if one site refused due to time or not enough willing participants then another would be approached until the total number of participants were reached. a total of five play groups (two accepted) and three mothers' group (all three accepted) were approached. interested participants were asked to contact the researcher (rb). all participants consented to up to an hour interview or focus group to explore their views, knowledge and attitudes towards management of respiratory tract infections, including prevention strategies such as influenza vaccination and hand hygiene in children < years of age. interviews and focus groups (each approximately h long) were conducted between june and july by rb. pcps' were interviewed at their work place or at a place convenient to them during practice hours; focus groups were conducted at play group centres or at scheduled mothers' group meetings. all participants gave written consent prior to data collection; pcps were provided with a aud$ and parents with a aud$ gift voucher upon completion. interviews and focus group discussions were digitally recorded and transcribed verbatim. data were analysed using a thematic approach [ ] to provide a flexible approach to identify, analyse and report themes or patterns within the data. initially, two researchers (rb and bb) read three transcripts independently to generate initial codes and themes, which were then compared and refined until consensus was reached. a further three transcripts were coded using the schemata and this process was repeated, three transcripts at a time, to incorporate emerging themes, until all transcripts were coded. data were managed using nvivo . study approval was obtained from monash university human research ethics committee (cf / - , , , ). a total of pcps ( females) and parents and carers ( females) participated in the study. the average years of experience for gps, pns, mchns and phs were . , . , . , and . years respectively. in the parents and carers cohort, % (n = ) were in the - years age group, with over % (n = ) having a graduate degree or higher. all participants revealed high levels of knowledge regarding hand hygiene and its importance. when asked, they gave their definition of hand hygiene, and discussed the importance of hand hygiene in reducing transmission of infection, including day to day practice. "washing hands frequently especially after sneezing, touching the nose, touching the mouth, coughing in the hands… the droplets in the transmission and what it means and even touching the handles of the doors, all of these can be a source of infection sometimes, and washing hands, i mean, they are important." gp "yeah i think it's [hand hygiene] quite important, because your hands touch anything. like your hands will touch the table and someone will come to the table your hands touched -without even realising, you're touching things. like you're touching your face all day. scratching your hair, everything, and then you go and touch things…" fg despite participants having good knowledge of hand hygiene, and recognising the importance in reducing disease transmission, many barriers such as variation in the practice of hand hygiene among pcps and parents, linking visibility to disease transmission, and doubts that hand hygiene practice was attainable in young children hindered good hand hygiene practice. we elaborate on these themes below. although pcps unanimously agreed that hand hygiene was important in reducing the transmission of diseases, there were large variations in practice. three types of hand hygiene practice were identified among gps and phs: some would wash hands between seeing patients irrespective of whether contact has been made, some would only wash hands if skin contact was made, while others would practice hand hygiene only if patients were visibly infectious. however, most gps commented that they would use alcohol sanitizers between patients if hand washing with soap and water was not possible. "… every time i examine the patient…" gp "not everyone, not if there's no skin contact…"gp "…if i'm handling something or i thought they are likely infectious..." gp "would be very rare. we don't try and touch… [we don't wash hands] not unless they are obviously sick…"ph pns on the other hand would often wash hands between patients as they were more likely to 'touch' patients during procedures, and rarely would mchns see babies/ children without skin contact. to the latter group, hand hygiene was habitual and 'routine'. alongside variations in hand hygiene practices among pcps, there were also divided views about whether to educate parents and patients on hand hygiene during a sick child consultation. some commented they would if time permitted; some would not as they assumed parents already had good knowledge of hand hygiene and transmission of infection. "i do talk to them and tell them it prevents a lot of cross infections…" gp "…it just doesn't come up, often there are other things to talk about, and we just don't have time." ph "look, parents… i don't know… but i can see most of the parents are quite… they know the hygiene.... they have the knowledge…" gp however, pcps commented they would not hesitate to discuss hand hygiene during a gastrointestinal tract infection consultation, but they did not always for an rti consultation. similar to pcps, parents also prioritised hand hygiene practice with gastrointestinal infections, which were seen as more infectious as they were more 'visible'. "just because i think of a cold as being non-severe… like, just a natural part of life. but gastro just would prefer to avoid." fg "gastro i would [discuss hand hygiene], but not respiratory tract infections." gp "but gastro, you're also vomiting and stuff, and go through places, institutions, like hospitals…" gp "… so when we triage… we do have a chat… like gastro… we have a chat to them about the transmission, and decreasing the spread of virus or whatever is causing the gastro, and what is going around..." pn "they [pharmacy staff] don't do it [wash hands] always, but if someone comes in with gastro, they would come straight up and (do the alcohol sanitising motion)…" ph pcps also commented that the interview process for this study gave them pause for thought making some gps realise that they need to talk to parents. while parents considered good hand hygiene as washing hands before meals, after meals and after going to the toilet, similar to pcps, parents also conflated 'dirt' with 'infectious' and dirt was a visual cue to prompt them to wash their hands. "just teaching her that if your hands are dirty you wash them, so even though i don't wash my hands every time i eat, i don't wash my hands if i've been out to the washing line, when she comes in [from outside] -"oh okay, we've got to wash our hands now"" fg "… if somebody has a cold or somebody has gastro or something like that then i'm really freaky about it and i clean everything within an inch of its life. but then other times, we're, kind of, more relaxed and pretty lazy about it." fg visual cues therefore determined behaviour such as when hands should be washed. gastrointestinal infections were seen as being 'visible' , therefore considered as more 'severe' than rtis, leading to the perception that disease transmission and infection control were visually based. although pcps demonstrated good knowledge of transmission of rtis -respiratory route and fomite transmissionthey still insisted that hand hygiene practice would not be effective in preventing or reducing rti transmission. "there is no prevention. i would have to stop sending children to crèche, and kinders, and schools because they get an infection … this is a part of life and growing up … it's not possible [to prevent]" gp pcps also believed that hand hygiene could not be achieved in young children as they presumed young children would not have hand hygiene awareness and good practice. in addition, prevention would not be achievable as parents and children have constant contact, especially as young children needed comforting when unwell. "yeah, well, probably not so much in the context of colds, kids are little anyway and they are not going to do it. i talk probably more in terms of gastro, we talk a bit about heightened domestic awareness and practice…" gp "they are going to kiss you, they are going to touch you… and they are going to kiss each other…" gp similarly, though parents acknowledged the importance of hand hygiene in reducing transmission of diseases, they also expressed reservations about 'over-surveying' their children and becoming 'germophobic'. over emphasising hand hygiene was perceived as leading to obsessive behaviours and psychological distress: "… i've actually had to pull it back because she was in there every five minutes… she got really quite ocd (obsessive compulsive disorder) about the whole thing…" fg "we sound like we're a bit paranoid… my daughter did say to me that i was turning her into a germ-a-phobe…" fg "i have seen a lot of quite obsessive hand washers at my new workplace." fg "i kind of figured i don't want to be too paranoid because you can't wipe your hand every two seconds…" fg while parents did not want to be 'paranoid' about being too clean and obsessive about hand hygiene, ultimately, they wanted to find that balance between good hand hygiene practice and not being paranoid about diseases. they did describe struggling to determine what was 'right' , the 'correct' hand hygiene practice, and what was considered as being 'too clean'. children being too clean was perceived as weakening immunity whereas being 'dirty' built immunity: "i also wonder about that whole cause [and] effect. because the people i know who wash their hands obsessively are always sick. and i just can't decide if they're always sick because they're obsessive hand washers or if they're obsessive hand washers because they're always sick…" fg "i worry about using the hand sanitiser too much… i don't know, i always think there's … almost too clean…" fg "i know some people that are clean, i don't know about too clean, but their kids get sick quite easily. i don't know whether it's because they're not getting immune to some dirt or something…" fg "we sound like we're a bit paranoid, but that's just us i think." fg even though barriers exist for both pcps and parents of young children when it came to good hand hygiene practice, they all agreed that hand hygiene training still needed to be taught early in life. "it really stems from the parents…"teaching hand hygiene when asked whose responsibility it was to teach hand hygiene practice to young children, pcps and parents commented that parents should be responsible. "… parents seem to talk to their kids about washing their hands…" gp "no, i haven't been telling them, no… i thought the mums would do it…" gp "so basically, it comes from the parents, if they set good examples…" fg the most effective approach to teaching young children good hand hygiene practice was identified by pcps and parents as role modelling. role modelling, the concept of washing hands in front of an audience so the behaviour can be imitated, was expressed as a good way to 'show' children how and when hands should be washed, allowing the behaviour to be 'copied'. hence developing their hand hygiene practice early in life, and eventually leading to sustained hand hygiene behaviour later in life. "i'm role modelling, so they can see me washing my hands… the most important thing i do (in the mother's group sessions)… that's hand hygiene." mchn "…having things down at the children's level, rolemodelling" fg this theme highlighted the general consensus that pcps and parents thought parents should be responsible for their children's hand hygiene practice, with prompting and role modeling as the most effective way to teach young children to start the good hand hygiene habit early in life. results from this study demonstrated the complex reasoning behind why a simple but important task such as hand hygiene is so difficult to consistently implement in everyday life. far from a benign, dispassionate process, there are inherent emotions invested in undertaking this task. while the world health organization 'my moments for health hygiene' recommends health-care workers to clean their hands before touching a patient, before clean/aseptic procedures, after body fluid exposure/risk, after touching a patient, and after touching patient surroundings [ ] , factors such as the pcp's own habitual hand hygiene behaviour; the expectation that parents themselves have good hand hygiene practice; scepticism that hand hygiene is effective in reducing rtis or achievable in young children contributed to the large variation seen in pcps' recommendations to promote hand hygiene. for pcps and parents of young children, hand hygiene practices were centered on visual cues such as gastrointestinal infections and 'dirt' as being 'visible' , rather than the transmission of diseases. while coughing and sneezing can be quite 'visible' , it is often not associated with being 'dirty' , hence it is less likely to result in a reflexive action resulting in hand washing. the risk that promoting hand hygiene practice could result in paranoia and the effect of being 'too clean' were overriding concerns for parents more so than the message itself. variations in practice stemmed from personal attitudes, perceived behaviour, control and subjective norms [ ] , leading to the intent to wash hands. some pcps thought parents were knowledgeable in hand hygiene practice and therefore did not feel the need to mention hand hygiene during an rti consultation. a recent study by barroso et al. [ ] found a counterintuitive inverse relationship between knowledge and hand hygiene behaviour: where medical students reported high hand hygiene behaviour yet had lower knowledge as compared with medical residents, suggesting that factors other than knowledge were important in determining hand hygiene behaviour in this cohort. furthermore, many pcps said they would not wash their hands if there was no patient contact and if the patient was not visibly 'infectious'. whitby et al. [ ] , describe how inherent hand hygiene practice drives the community where visibly soiled, sticky, or gritty hands would prompt hand hygiene behaviour. this 'perceived susceptibility' or 'personal risk' was also described in a study from eight mediterranean countries [ ] , where they found health care workers' hand hygiene compliance was significantly higher after patient contact compared to before patient contact, implying that self-protection was a major driver of hand hygiene performance in this cohort. our results indicated that while the importance of hand hygiene was undeniable, hand hygiene practice and passing hand hygiene knowledge to parents of young children varied considerably within and across pcp groups. the diverse situations each pcp face in different scenarios such as whether patients were seen as 'infectious' , or whether they believed parents have the knowledge as to whether they needed to talk to them about hand hygiene were contributing factors to the variations seen in these groups. parents also relied heavily on visual cues such as 'dirty' and 'infectious' , to determine the need to hand wash, as they did not always remind their children to wash their hands. however, the 'awareness' of hand hygiene practice might also explain that hand hygiene was often taken for granted, and not 'thought about'. therefore, behaviour change interventions might need to be regular and applied in small incremental steps. raising awareness of possible personal risk could improve practice and sustainability when it comes to hand hygiene behaviour [ ] . additionally, parents were reluctant to encourage hand hygiene practices in their child for fear their children would be 'too clean' , and that they needed to be visibly 'dirty' or 'infectious' to build their own immunity. this belief needs to be directly challenged by pcps during discussion in an rti consultation, and further educating parents on good hand hygiene practice should therefore be considered. a more concerning theme that emerged from our study resulting from the discussions emanating from the parents focus groups was parents' fear of their child developing abnormal behavior such as ocd. although studies have shown strong links between people with ocd and feelings driving them to engage repeatedly and excessively in behavior such as hand washing [ , ] , there is no evidence suggesting that hand washing 'triggers' ocd. these studies found that ocd was characterized by the reduced ability to terminate an action, such as hand washing, rather than a response to a perceive threat i.e. perceived susceptibility or personal risk. therefore, parents' fear of excessing hand washing leading to ocd was not valid. however, the fear was enough for parents to be vigilant with children's hand washing practice, therefore an important area for further research. perhaps one of the biggest barriers to good hand hygiene practice in young children was the skepticism displayed by parents and pcps that good hand hygiene practice was achievable in young children, and almost not worth pursuing. thus, while the 'intent' was there regarding hand hygiene, compliance did not always follow. even though successful interventions incorporating hand washing in young children have shown to reduce absenteeism due to infection [ ] , a recent study of childcare centres in the netherlands [ ] found that while hand hygiene opportunities were readily available for children, overall adherence to hand hygiene guidelines was only % in participating day care centres, which supports the publicly held view that hand hygiene practice is not achievable in young children. however, participants in the study also believed that hand hygiene behaviour should start early in life. a study in seoul, korea [ ] , conducted in an elementary school setting with year students, showed parents' handwashing practice, parent and child bonding, and shared time have a significant correlation with children's hand hygiene practice. our study also suggested that both pcps and parents thought hand hygiene practice should start with good role modelling in the home, with frequent reminders. our study was not without limitations. first, the research was conducted in metropolitan melbourne, and therefore our results may be not generalisable to other areas such as rural or remote sites, or developing countries where there might be reduced access to hand hygiene products and handwashing facilities. second, pcps and parents of young children who participated in the study were very interested in this area, potentially introducing selection bias. third, providing incentives to participants may have led to a possible source of bias, although these incentives are aligned with similar work with estimated earnings and average australian wage [ , ] . currently little is known regarding young children's hand hygiene practice in the australian community. our study has taken the first step in exploring pcps' and parents' attitude, views and practice of hand hygiene practice, thereby identifying barriers to hand hygiene practice for pcps and parents of young children, which potentially impact hand hygiene habit and behaviour of young children. to overcome some of these barriers to good hand hygiene practice, the following interventions targeting pcps and parents may help increase awareness of the importance of hand hygiene and encourage effective hand hygiene behaviour: ) introduce health promotion that will educate and remind the public that diseases are not always 'visible' and that whether or not one appears dirty, transmission is still possible; ) good hand hygiene habits should be taught early in a child's life to sustain effective hand hygiene behaviour; and ) the importance of role modelling as a way to develop good hand hygiene habit in young children. in addition, pcps should at least encourage parents and young children to practice hand hygiene when coming for an rti consultation, which may reduce the transmission of rtis, reinforce the message of the importance of hand hygiene compliance and result in healthy hand hygiene practice in young children. finally, parents should be reassured that effective hand hygiene practice will not lead to abnormal psychological behaviour in their children and that hand washing will not reduce a child's immunity. this study demonstrated that on the surface, both pcps and parents of young children thought hand hygiene practice was important. however, dissonance emerged in practice because hand hygiene is implicitly tied to beliefs such as washing hands only when 'dirty'; concerns that children need to build their immunity and are already too clean; and skepticism that hand hygiene can be achieved in young children. pcps should be made aware that hand hygiene can be part of the habit of washing hands between patients, due to fomite transmission of diseases in practice. parental education around the importance of hand hygiene, focused on the tangible goals of making hand hygiene a regular habit is paramount in teaching young children to develop good hand hygiene practice early in life. the decision to perform hand hygiene should not be based on 'dirt' or relating visibility of infection to transmission of infection. rather role modelling hand hygiene by parents as well as enforcing hand hygiene early in the child's life will help with better hand hygiene compliance leading to reduced transmission of infectious diseases. effect of handwashing on child health: a randomised controlled trial the impact of common infections on school absenteeism during an academic year effectiveness of alcohol-based hand disinfectants in a public adminstration: impact on health and work performance related to acute respiratory symptoms and diarrhoea effect of hand hygiene on infectious disease risk in the community setting: a meta-analysis incidence of acute respiratory infections in australia risk factors for acute respiratory infection in the australian community transmission of acute gastroenteritis and respiratory illness from children to parents hand hygiene to reduce community transmission of influenza and acute respiratory tract infection: a systematic review. influenza other respir viruses the effect of enhanced hygiene practices on absences due to infectious diseases among children in day care centers in helsinki enterococcus spp on fomites and hands indicate increased risk of respiratory illness in child care centers hand hygiene instruction decreases illness-related absenteeism in elementary schools: a prospective cohort study reducing absenteeism from gastrointestinal and respiratory illness in elementary school students: a randomized, controlled trial of an infection-control intervention hand sanitiser provision for reducing illness absences in primary school children: a cluster randomised trial a randomized, controlled trial of a multifaceted intervention including alcohol-based hand sanitizer and hand-hygiene education to reduce illness transmission in the home impact of the use of an alcoholbased hand sanitizer in the home on reduction in probability of infection by respiratory and enteric viruses an investigation of the effects of a hand washing intervention on health outcomes and school absence using a randomised trial in indian urban communities impact of an educational intervention upon the hand hygiene compliance of children enhanced performance feedback and patient participation to improve hand hygiene compliance of health-care workers in the setting of established multimodal promotion: a single-centre, cluster randomised controlled trial utilizing improvement science methods to improve physician compliance with proper hand hygiene inexpensive and time-efficient hand hygiene interventions increase elementary school children's hand hygiene rates the effect of a handwashing intervention on preschool educator beliefs, attitudes, knowledge and self-efficacy cluster randomized trial to evaluate the effect of a multimodal hand hygiene improvement strategy in primary care healthy hands: use of alcohol gel as an adjunct to handwashing in elementary school children factors influencing hand washing behaviour in primary schools: process evaluation within a randomized controlled trial hand hygiene of medical students and resident physicians: predictors of attitudes and behaviour welcome to hand hygiene australia (hha): hand hygiene australia why do we not want to recommend influenza vaccination to young children? a qualitative study of australian parents and primary care providers. vaccine maternal child health services: victoria state government using thematic analysis in psychology world health organization. my moments for hand hygiene. world health organization in the era of corona virus: health care professionals' knowledge, attitudes, and practice of hand hygiene in saudi primary care centers: a cross-sectional study behavioural considerations for hand hygiene practices: the basic building blocks self-protection as a driver for hand hygiene among healthcare workers using an analysis of behavior change to inform effective digital intervention design: how did the primit website change hand hygiene behavior across users? in the wake of a possible mistake: security motivation, checking behavior, and ocd when too much is not enough: obsessive-compulsive disorder as a pathology of stopping, rather than starting children's hand hygiene behaviour and available facilities: an observational study in dutch day care centres family factors associated with children's handwashing hygiene behavior general practice research. problems and solutions in participant recruitment and retention australian bureau of statistics. . -employee earnings and hours the authors would like to thank all the participants in this research. this study was part of a phd study, funded by the national health and medical research council (nhmrc), and the royal australian college of general practitioners (racgp). not applicable authors' contributions rb completed the background literature search and rb, bb, dg and dm contributed to the study design. rb conducted and transcribed all interviews. rb and bb performed the analysis of the data. rb drafted the manuscript. all authors revised all drafts and approved the final version of the manuscript.ethics approval and consent to participate all participants were provided with a plain language statement explaining the study and gave written consent prior to interview/focus group. the study was approved by monash university human research ethics committee (cf / - , , , ). the authors declare that they have no competing interest. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -itz cf authors: ni, qingyong; wang, yu; weldon, ariana; xie, meng; xu, huailiang; yao, yongfang; zhang, mingwang; li, ying; li, yan; zeng, bo; nekaris, k.a.i. title: conservation implications of primate trade in china over years based on web news reports of confiscations date: - - journal: peerj doi: . /peerj. sha: doc_id: cord_uid: itz cf primate species have been increasingly threatened by legal and illegal trade in china, mainly for biomedical research or as pets and traditional medicine, yet most reports on trade from china regard international trade. to assess a proxy for amount of national primate trades, we quantified the number of reports of native primate species featuring in unique web news reports from to , including accuracy of their identification, location where they were confiscated or rescued, and their condition upon rescue. to measure temporal trends across these categories, the time span was divided into three sections: – , – and – . a total of individuals of species were reported in news reports, mostly rhesus macaques (n = , . %, macaca mulatta) and two species of slow lorises (n = , . %, nycticebus spp.). during the same period, live individuals of rhesus macaques were recorded times ( , individuals) in the convention on international trade in endangered species of wild fauna and flora trade database, whereas slow lorises were only recorded four times (nine individuals), indicating that the species originated illegally from china or were illegally imported into china. due to their rescued locations in residential areas (n = , . %), most primates appeared to be housed privately as pets. a higher proportion of ‘market’ rescues during – (χ( ) = . , df = , p = . ), could be partly attributed to an intensive management on wildlife markets since the outbreak of severe acute respiratory syndrome (sars) in . more than half ( . %, individuals) of the primate individuals were unhealthy, injured or dead when rescued. thus, identification and welfare training and capacity-building should be provided to husbandry and veterinary professionals, as well as education to the public through awareness initiatives. the increase in presence of some species, especially slow lorises, with a declining population in restricted areas, also suggests the urgent need for public awareness about the illegal nature of keeping these taxa as pets. hundreds of wild animal species are traded both legally and illegally to satisfy the market for exotic pets (bush, baker & macdonald, ) . many of these species are native ; roberge, ) . public knowledge concerning wildlife conservation can be quantified by analyzing comments and associated data posted online. here, we aimed to measure the number of species of traded primates in news reports found by or surrendered to authorities in china, and examine trends over time and differences among regions. furthermore, we examined public statements in the reports to evaluate how well the public could identify species in comparison with official identification in these same reports; if members of the public knew whether or not species were threatened; and also evaluated health and welfare status of the rescued or confiscated animals. these data are critical to recognize the magnitude and diversity of illegally traded primates in china, and generate suggestions for management strategies and law enforcement. to reveal temporal variations in trade of native primate species in china, we used purposive sampling (newing et al., ) to collect rescue or confiscation related news online. we considered rescuing or confiscating to be descriptions of primates surrendered to or confiscated by the authorities, hereafter referred to as rescue events. we conducted the searches in february and limited the period from st january to st december , in three popular chinese web . search engines: baidu, and bing. baidu, especially, is by far the largest search engine in china, fulfilling a similar function to google. based on the chinese name of each species, we entered manually the simplified chinese key terms into each search engine (table ) . we used '新闻'(news) category to select news articles and filtered the articles related to rescue events using keywords '救护' (rescue) or '查获' (seize) or '没收' (confiscate). given the effects of search engine algorithms and previous search history on the results, we expected the potential bias could be reduced by cross validation of the three search engines. we combined all the news reports and excluded repetitive news based on date, site and media source. we identified the rescued animal in this news report as the pygmy slow loris based on the photograph, and thus the official recognition (the bengal slow loris) was considered to be incorrect. each report included various identification of the species included in the rescue event. these identifications were made by the public (public recognition), or an official who carried out the rescuing event, which was considered the official identification ( fig. ) . we categorized the public recognition as unrecognized; primate (but not to species); or species level identification (table ). based on information provided in the news reports, especially photographs, we assessed the taxonomic status to compare with the official identification. frequency in different categories of public recognition and accuracy of official identification were used as proxies for public knowledge. we followed the primate taxonomy as listed in the handbook of the mammals of the world, volume (primates), and original accounts for two taxa not included in that resource (m. leucogenys- li, zhao & fan, ; hoolock tianxing-fan et al., ) . we collected information in each news report on date of rescue or confiscation, number of individuals, location of rescue (e.g. field, market, residential area or transporting vehicles), physical condition of rescued individuals (e.g. healthy, unhealthy, injured or dead), and whereabouts of the individuals after being rescued (e.g. zoo, wildlife rescue centre, released into wild, or unreported) (table ; fig. ). china's primates are naturally distributed in of provincial-level administrative units (plaus), with four provinces in west and southwest china (yunnan = species, guangxi = eight species, tibet = eight species, guizhou = six species), containing the highest diversity ( fig. a) . we also recorded provinces where news was/had been reported to determine distribution of rescuing (fig. ) . for an overview of international trade, we examined data from the cites trade database, which provided all records of import, export and re-export of cites-listed species. the data were downloaded in may and 'year range' were limited from to with being the last year for which data were available. we searched live (liv) animals in order primates traded with all sources and purposes, and focused on (table ) . the data implicating china as importer and exporter were combined to obtain number of individuals traded per year for each species. we divided the time span from to into three sections: - , - and - , and used the kruskal-wallis non-parametric test to examine variations over periods in rescuing frequency. to measure temporal trends of public knowledge about primate conservation, kruskal-wallis test was also used to compare the proportion of each description category in three time sections towards those species which were reported in more than years. we calculated the shannon-wiener index (h ¼ À p s i p i ln p i ) and pielou index (e = h / ln s) for each year to evaluate diversity and evenness of primates reported, where s = total number of species recorded in a given year, p i = the proportion of individuals belonging to ith species. spearman's rank correlation coefficient was used to analyse annual variations of diversity and evenness. all the tests were two-tailed and a threshold for significance was p < . . we filtered valid news reports based on the topics of rescuing and confiscation, including individuals of primate species (table ; fig. ). the bengal slow loris (nycticebus bengalensis) was the most reported species with ( . %) individuals, followed by the rhesus macaque (m. mulatta, individuals, . %) and the pygmy slow loris (n. pygmaeus, , . %), while of primate species distributed in china were never reported (fig. ) . we recorded rescue events of bengal and pygmy slow lorises every year during the - period. rhesus macaque rescue events were reported in years ( , , ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) and the tibetan macaque (m. thibetana) in recent years ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the rescue news related to other species, however, was individually reported in no more than years. the diversity index increased significantly over time (spearman's rank correlation coefficient, ρ = . , p < . , n = ), as well as evenness (ρ = . , p = . , n = ). primate rescue frequency tended to increase during the last years from to (fig. a ) while number of news reports specifically on slow lorises fluctuated between years (fig. b) . comparatively, an average of , ± , live individuals of native primate species per year, including ten species in total, were recorded in cites trade database from to (fig. c ). the rhesus macaque contributed most of these internationally traded individuals ( , , . %, records), followed by the stump-tailed macaque (m. arctoides, , . %, three records). nine individuals (four records) of slow lorises and none of tibetan macaques and were reported in cites trade database over the studied period. among rescue events recorded, ( . %) reports were located in residential areas, followed by ( . %) in wild areas, ( . %) during transporting and ( . %) in the market. the proportion of market rescues was significantly higher in - than the other time sections but fewer individuals were rescued from markets in - based on the reports (v = . , df = , p = . ), especially for bengal slow lorises (v = . , df = , p = . ) and rhesus macaques (v = . , df = , p = . ). primate rescuing news covered more than counties in plaus throughout china (fig. b) , with a considerable proportion of rescuing events ( , . %) taking place in yunnan province, followed by guangdong ( , . %) and guangxi ( , . %). the rescuing news related to slow lorises occurred in plaus (fig. c) , while rhesus macaques rescues was reported in plaus, and other species were not individually reported in more than seven plaus. it is noteworthy that data from taiwan ( ), hong kong ( ) and macau ( ) were limited due to unpopular use of simplified chinese. we found that more than half ( . %, individuals) of the primate individuals were unhealthy, injured or dead when rescued. of individuals whose injuries were specified, most ( , . %) were suffering from leg wounds. the proportion of healthy individuals rescued was significantly lower in - than the other two time periods (v = . , df = , p = . ). the percentage of healthy individuals varied significantly over time in bengal slow lorises (v = . , df = , p = . ) and tibetan macaques (v = . , df = , p = . ), as well as the percentage of injured individuals in pygmy slow lorises (v = . , df = , p = . ), rhesus macaques (v = . , df = , p = . ) and tibetan macaques (v = . , df = , p = . ). the whereabouts of the individuals after being rescued were often unreported ( , . %), followed by 'wildlife rescue centre' ( , . %), field ( , . %) and zoo ( , . %). the individuals in nearly half of rescuing events ( , . %) were recognized as primates by the public, and the individuals in events ( . %) were recognized as a specific species (table ) . the public could not recognized the animals or referred to primates in events ( . %). the public recognitions of the bengal slow loris was consistent with the pygmy slow loris over the three time periods. the proportion of rescuing events in which the individuals could be recognized to a species level by the public varied over time for the rhesus macaque (v = . , df = , p = . ) and the recognition percentage of individuals identified as 'primates' varied significantly for the tibetan macaque (v = . , df = , p = . ). the official recognition was reported to species level in all of the news reports, but species in . % ( / ) of news including photos were incorrectly identified ( table ) . the pygmy slow loris, which was usually recognized as the bengal slow loris, contributed most ( / , . %) to these wrong identifications. all the other incorrect identifications ( ) were related to species of genus macaca (table ) , especially for rhesus macaques ( ) and tibetan macaques ( ). for certain native primate species in china, few individuals were traded internationally based on the cites trade database, whilst rescuing or confiscating news reports revealed that they were frequently traded in domestic areas. in addition, the lower frequency of rescuing or confiscating and a focus on web news mean that the number of individuals traded might be underreported. thus, it could be argued that a large amount of illegal trade at national level, especially for bengal and pygmy slow lorises, appeared to be underrepresented by official data. rhesus macaques composed a large proportion of rescuing reports, which was consistent with the fact that it is the most abundant primate species in china, and widely traded or housed for biomedical purpose (bontrop, ; fan & song, ) . given the extensive captive breeding throughout china (fan & song, ) , a large number of animals of rhesus and tibetan macaques rescued may be originally from captive populations. without any breeding centre in china, not to mention internationally, probably all slow lorises were wild-captured and trafficked illicitly. more than half of the primates in the rescuing news were located in residential areas, indicating that they had probably escaped from households where they were kept as pets. furthermore, in % of reports only a single individual was rescued or confiscated, underlining that the animals were the end-point of trade chain, and had presumably been trafficked several times before being housed (duarte-quiroga & estrada, ) . the chinese government has markedly strengthened management of wildlife markets since the outbreak of sars in , which was considered to originate from small wild animals (zhong, ) , likely explaining the significant increase of primates rescued from markets during - . illegal trade related to wildlife markets has declined during recent years, and large specialized traditional open markets tend to be replaced by underground trade networks, in particular, the booming online trade on social media (xiao, guan & xu, ) . eight online transactions of slow lorises, were detected and penalized based on web news from to , but only two reports before s. frequency of rescuing news on primate species varied remarkably between plaus, which indicates a significantly heterogeneous illegal trade distribution across china. a bulk of rescuing events took place in southwestern plaus, including yunnan, guangxi, guizhou, tibet and sichuan. this is consistent with the highest primate diversity in this area, where more than % of the total species in china are distributed and % are endemic. in addition, these areas are situated near southeast asia, which is a hotspot for global biodiversity (myers et al., ; sodhi et al., ) and wildlife trade (nijman, ) . yunnan and guangxi, in particular, share long borders with vietnam, myanmar and laos, and are considered as one of the major entrances for wildlife trafficking from neighbouring nations (li & li, ; shepherd & nijman, ; zhang, hua & sun, ) . guangdong province is one of the main destinations for smuggling and the largest wildlife markets (zhang, hua & sun, ; chow, cheung & yip, ) , making it another possible hotspot of primate trade. along with guangdong, beijing, shandong and zhejiang are among the most developed plaus in china and contributed a lot to the illegal wildlife trade (li & lu, ; yu et al., ) . zhang & yin ( ) found that consumers with higher income background had a higher wildlife consumption rate, suggesting that financial strength increases people's propensity to consume wild animals. to support this point, few primate rescues were reported in north-western plaus, the less developed regions in china. the lower primate trade rate observed in northwest and northeast may also result from a long distance from source areas. with the expansion of online trade in recent years, the trafficking sites have become increasingly extensive and scattered, and the distance between sources and the point of retail tend to be greater (zhang, hua & sun, ) . primate individuals were mostly sent to zoos, rescue centres or released into the wild after being rescued. for a considerable number of animals ( , . %), we were unable to extrapolate their final destinations from the news reports. given the scattered sites, it was not surprising that all the individuals were rescued by local forestry staff, who might encounter difficulties during rescuing, such as species identification. lack of discrimination in the trade, especially in morphology, combined with unresolved taxonomic issues, impedes assessing each taxon's potential vulnerability to trade (vonk & wüster, ; nekaris & jaffe, ; nekaris & nijman, ) . genetic, vocalization and behavioural analyses are essential for rescue and release programmes, yet may be beyond the capabilities for some facilities (mootnick, ) . as a consequence, the pygmy slow loris was usually confused with the bengal slow loris, and the two species were thus housed and released indiscriminately. limited understanding of slow lorises taxonomy throughout their distribution ranges confounded attempts to reintroduce these animals, or hold and breed them in captive facilities (nekaris & starr, ) . primates have specific physiological, physical, social and nutritional requirements, and it is unlikely that the welfare of pet animals can be adequately addressed in normal households (soulsbury et al., ) . captive primates, including those in zoos and rescue centres, have been commonly observed to suffer from incorrect diet, wounds or disease, unnatural environment, and fear or distress (duarte-quiroga & estrada, ; hevesi, ; nekaris et al., ) . the specialist needs of primate species also mean that they might experience elevated mortality and perish quickly in captivity (fitch-snyder, schulze & larsson, ) . more than one third of individuals of slow lorises, died within the first months in rescue centres in southern yunnan (q. ni, , personal communication) . hence, for many rescue institutions, immediate re-release is often considered as preferable (nekaris & jaffe, ) . the problem of rehabilitating captive animals without regard to genetic and ecological assessments, geographic distribution, and monitoring has become another difficult issue that remains to be resolved (duarte-quiroga & estrada, ; nekaris & starr, ) . the individuals in all the news reports related to release were reintroduced into wild directly during -year periods, by the local authorities without preparation and training involved, indicating that hard release has been rampant in primate rescuing throughout china. this might lead to high mortality of animals released (moore & nekaris, ) or endanger wild populations and other animals with disease transmission (wallis & lee, ) , thus becoming a useless conservation plan. accurate measures of wildlife trade are essential to devising sound conservation decisions, yet collection and quality control of such data are challenging (thomas & albert, ) . the database generated by cites offers an unparalleled opportunity to analyse international trade in species of conservation concern (foster, wiswedel & vincent, ) . however, the present official statistics have limited capabilities in representing species illegally harvested and traded (phelps & webb, ) , especially for those trafficked at national or regional level. therefore, taking these data at face value can sometimes distort the perceived risk of wildlife exploitation and lead to misallocation of resources and ineffective conservation efforts (thomas & albert, ; robinson & sinovas, ) . as a case study, the discrepancy between few cites trade records and massive rescuing news reports of slow lorises in china emphasize a need of more reliable and comprehensive understanding of some species, and calls for a harmonized mechanism in estimating national and regional wildlife trade. the success of wildlife conservation largely depends on public perspective, as well as assessment of causes that influence their outlook (wilson & tisdell, ; lindemann-matthies & bose, ) . media reports indicate that public knowledge towards primates in china varied between species. macaques, along with leaf monkeys and snub-nosed monkeys, represented the typical image of 'monkeys' (i.e. primates), in a broad sense, and were well known for the general public in china. the famous monkey king, sun wukong, for instance, was considered to be originated from these species (qin, ) . similarly, though mysterious in deep forests, gibbons historically occurred in many poems and paintings and were rich in symbolic meanings (geissmann, ; zhang, ) , whereas slow lorises played a minimal role in chinese culture. few relevant publications, combined with limited distribution areas in the southwest, has resulted in them being the least known primates within the country. chinese people usually judge animals by ethical standards and emphasize the creature's usefulness to humans, but ignore the physical characteristics of the animals (zhang, ) , leading to a series of misunderstanding on slow lorises. one of slow lorises' perceived uses to treat epilepsycalled 'mad sheep disease' by local communities-in traditional chinese medicine, as a result, was mostly attributed to confusing chinese common names with similar pronunciation (feng) among '疯' (mad)-, '风' (wind)-and '蜂' (bee)-猴 (monkey). given the clandestine illegal trade of primates based on web news reports, it can be concluded that monitoring systems of wildlife trade within china are insufficient, and there is an urgent need for initiatives to make regulatory mechanisms more effective (zhang, hua & sun, ; nijman, ) . a common problem in the enforcement of legislation to protect animals from illegal trade is the inability to identify species due to inadequate funding, education and staffing. recommendations to address these areas should include identification-training initiatives and capacity-building work (li & wang, ) . in addition, it is highly recommended that an approach concerning awareness initiatives and education programmes should be developed towards the public to make them more conscious about the illegal wildlife trade, with the final intention of discouraging the consumers to buy wildlife products. by , primate-breeding centres in china contained over , individuals, mostly rhesus macaques, and the number has been steadily increasing in recent years (jiang et al., ; cyranoski, ) . it is necessary to strengthen captive management and improve animal welfare, which was still inconsistent and rudimentary, since the concept has been introduced into mainland china only in the last few decades (lu, bayne & wang, ) . the authorities should accelerate the legislative process and provide animal welfare education to the public, as well as training to husbandry and veterinary professionals. the demand for pet primates, together with habitat loss and fragmentation, exerts a significant pressure on wild populations. in particular, slow lorises are perceived as suitable pets by both buyers and sellers due to their 'cute and cuddly' appeal, and have been one of the most popular primate taxa in wildlife markets (nekaris, ) . the widespread illegal trade in china seems to be incompatible to their restricted distributions, high threat category, and poor public knowledge. taking into account their small and declining wild population, it is urgent to take actions for conservation of this neglected and threatened primate taxon. we have presented a novel data on primate trade within china based on web news reports regarding rescuing or confiscating. the results indicate that some native primate species, particularly bengal and pygmy slow lorises, are threatened by domestic illegal trade, which appears to be 'unrecognized' in official channels, and lack of public knowledge impedes efforts to conserve these species effectively. in spite of potential bias in search results caused by search engine algorithms and manual filtering, and lack of the firsthand data from authorities, zoos or wildlife rescue centres, we expect that this study could facilitate the initial steps to raise public awareness on primate trade in china, especially for slow lorises. primates in traditional folk medicine: a world overview whose issue? representations of human-elephant conflict in indian and international media global trade in exotic pets wildlife markets in south china monkey kingdom: china is positioning itself as a world leader in primate research hunting, pet trade, and forest size effects on population viability of a critically endangered neotropical primate, sapajus xanthosternos (wied-neuwied, ) primates as pets in mexico city: an assessment of the species involved, source of origin, and general aspects of treatment impending extinction crisis of the world's primates: why primates matter description of a new species of hoolock gibbon (primates: hylobatidae) based on integrative taxonomy chinese primate status and primate captive breeding for biomedical research in china husbandry manual for asian lorisinae (nycticebus and loris spp.) opportunities and challenges for analysis of wildlife trade using cites data-seahorses as a case study. aquatic conservation: marine and freshwater ecosystems welfare impacts of the illegal wildlife trade in a cohort of confiscated greater slow lorises, nycticebus coucang investigation on wild animal resources involved in the border area of western yunnan gibbon paintings in china, japan, and korea: historical distribution, production rate and context welfare and health implications for primates kept as pets analysis on illegal trade of wild mammals in southern china china's mammal diversity and geographic distribution a complete guide to monkeys, apes, and other primates the dynamics of trade in live wildlife across the guangxi border between china and vietnam during - and its control strategies snow leopard poaching and trade in china wildlife trade in yunnan province, china, at the border with vietnam survey of illegal smuggles of wildlife in guangxi white-cheeked macaque (macaca leucogenys): a new macaque species from medog, southeastern tibet how many species are there? public understanding and awareness of biodiversity in switzerland current status of animal welfare and animal rights in china compassionate conservation, rehabilitation and translocation of indonesian slow lorises gibbon (hylobatidae) species identification recommended for rescue or breeding centers biodiversity hotspots for conservation priorities extreme primates: ecology and evolution of asian lorises media attention promotes conservation of threatened asian slow lorises unexpected diversity of slow lorises (nycticebus spp.) within the javan pet trade: implications for slow loris taxonomy cites proposal highlights rarity of asian nocturnal primates (lorisidae: nycticebus) exploring cultural drivers for wildlife trade via an ethnoprimatological approach: a case study of slender and slow lorises (loris and nycticebus) in south and southeast asia conservation and ecology of the neglected slow loris: priorities and prospects conducting research in conservation: a social science perspective in full swing: an assessment of trade in orangutans and gibbons on java and bali an overview of international wildlife trade from southeast asia primate conservation: measuring and mitigating trade in primates changes in the primate trade in indonesian wildlife markets over a -year period: fewer apes and langurs, more macaques, and slow lorises invisible' wildlife trades: southeast asia's undocumented illegal trade in wild ornamental plants exploratory research of images of chinese apes and monkeys captive conditions of pet lemurs in madagascar using data from online social networks in conservation science: which species engage people the most on twitter? challenges of analyzing the global trade in cites-listed wildlife an updated taxonomy and conservation status review of asian primates summarizing the evidence on the international trade in illegal wildlife an assessment of wildlife trade at mong la market on the myanmar-china border the state and conservation of southeast asian biodiversity the welfare and suitability of primates kept as pets disappearing in the night: an overview on trade and legislation of night monkeys in south and central america data on wildlife trade roles of cites in protecting new species primate conservation: the prevention of disease transmission how knowledge affects payment to conserve an endangered bird traffic wildlife cybercrime in china: e-commerce and social media monitoring in . cambridge: traffic briefing paper the list of wild mammals of illegal trade in china significant and timely ivory trade restrictions in both china and the united states are critical to save elephants good gibbons and evil macaques: a historical review on cognitive features of non-human primates in chinese traditional culture wildlife trade, consumption and conservation awareness in southwest china wildlife consumption and conservation awareness in china: a long way to go management and prevention of sars in china we are grateful to vincent nijman, marika roma, kathleen reinhardt, cristy jasso, miguel guinea and eleonora favilli for their comments on the manuscript. this study was supported by the national natural science foundation of china (no. ) and kadoorie farm & botanic garden. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. the following grant information was disclosed by the authors: national natural science foundation of china: . kadoorie farm & botanic garden.the authors declare that they have no competing interests. qingyong ni conceived and designed the experiments, performed the experiments, analysed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft. yu wang performed the experiments, analysed the data, contributed reagents/materials/ analysis tools, prepared figures and/or tables, approved the final draft. ariana weldon authored or reviewed drafts of the paper, approved the final draft. meng xie performed the experiments, analysed the data, approved the final draft. huailiang xu contributed reagents/materials/analysis tools, approved the final draft. yongfang yao contributed reagents/materials/analysis tools, approved the final draft. mingwang zhang contributed reagents/materials/analysis tools, approved the final draft. ying li contributed reagents/materials/analysis tools, approved the final draft. yan li contributed reagents/materials/analysis tools, approved the final draft. bo zeng contributed reagents/materials/analysis tools, approved the final draft. k.a.i. nekaris authored or reviewed drafts of the paper, approved the final draft. the following information was supplied regarding data availability:the raw data is provided in the supplementary files and . the data shows preliminary statistics of news reports regarding primate rescuing or confiscating in china, and cites reports on primate trade implicating china. supplemental information for this article can be found online at http://dx.doi.org/ . / peerj. #supplemental-information. key: cord- -e jd ac authors: remy, m. m.; alfter, m.; chiem, m.-n.; barbani, m. t.; engler, o. b.; suter-riniker, f. title: effective chemical virus inactivation of patient serum compatible with accurate serodiagnosis of infections date: - - journal: clinical microbiology and infection doi: . /j.cmi. . . sha: doc_id: cord_uid: e jd ac abstract objectives highly pathogenic viruses such as ebov are a threat to routine laboratory workers. inactivation procedures with triton x- . % and/or heat are currently recommended, but have unknown effects on the accuracy of serological testing. furthermore, virus inactivation by triton x- . % was shown to be ineffective in serum. this study aimed to demonstrate virus inactivation in serum by triton x- % and maintained accuracy of serological testing. methods a panel of serological tests was run on patient serum samples after treatment with triton x- %, . %, and . % + heat inactivation at °c for h. mean differences between measurements (bias) were calculated applying the bland–altman method. to determine effectiveness of virus inactivation, herpes simplex virus (hsv- ) was spiked into medium containing % or % serum, and treated with triton x- . % or %. infectious titres were then determined on vero cells. results serological measurements showed good agreement between controls and samples treated with triton x- . % and %, with an estimated bias of . ± . % (n = ) and – . ± . % (n = ), respectively. discordant qualitative results were rare. conversely, heat inactivation alone and combined with triton x- . % triggered a bias of . ± . % (n = ) and . ± . % (n = ), respectively. triton x- % completely inactivated hsv- in % and % serum while triton x- . % failed to do so in % serum. conclusions unlike heat inactivation, triton x- % enabled accurate serological testing and completely inactivated hsv- in serum. this simple method could allow safe routine serological diagnostics in high-risk patients. highly pathogenic viruses such as ebov or sars coronavirus are a continuous threat to clinical laboratory workers. the current epidemic of lassa virus in nigeria is one of the latest concerns [ ] and european hospitals must be prepared to deal with potentially infected returning travellers. while the microbiological diagnosis of such viruses should be performed under the required biosafety measures in specialized laboratories, blood samples from patients arriving at the emergency rooms are often sent directly to the routine laboratory where biosafety levels are low. early symptoms caused by highly pathogenic viruses are usually non-specific and require differential diagnosis of other infectious diseases such as malaria, dengue fever, hiv, hbv, or cmv infections. in confirmed cases, a potentially long stay in intensive care increases risks of secondary infections such as invasive fungal infections. diagnosis of these pathologies remains mostly based on serology. polymerase chain reaction (pcr) is an alternative diagnostic method that can be safely performed on infectious samples [ , ] ; however, it is only appropriate during the viraemic phase. to inactivate highly pathogenic viruses in serum the world health organization (who) and the us center for diseases control and prevention (cdc) recommend chemical inactivation with triton x- . %, heat inactivation at c for min, or a combination thereof [ , ] . triton x- is a non-ionic surfactant which can disrupt the lipid membrane of enveloped viruses without affecting proteins. it is therefore widely used in combination with tri-(n-butyl)-phosphate (tnbp) for solventedetergent inactivation procedures of plasma products for transfusion [ ] . besides, triton x- . % has been successfully evaluated for haematology and clinical chemistry [ e ] and for malaria rapid diagnostic tests (rdts) [ , ] . accuracy of immunoassays after triton x- treatment of serum was only partially assessed in and no decrease in sensitivity was reported [ ] . heat inactivation was compatible with detection of ebov-specific igg and igm [ ] and other viral antigens [ ] . however clinical chemistry studies showed that protein measurements and especially enzymatic activity measurements were mildly to severely affected by heat [ , e ] . other easily applicable inactivation methods such as sds treatment unfortunately denature proteins and are not suited for serological or clinical chemistry tests [ , ] . efficacy of heat inactivation was demonstrated for several enveloped viruses [ , ] , including haemorrhagic fever viruses [ ] . concerning detergents, complete inactivation of hiv was achieved with . % of triton x- [ ] whereas ebov treatment with . % of triton x- led to incomplete inactivation with a log infectivity reduction [ ] . besides, it has been recently claimed that the inactivation potential of triton x- . % was annulled in serum [ ] . inactivation tests were indeed performed on ebov and herpes simplex virus type (hsv- ) in . %, . %, or % serum, and viral inactivation by triton x- only succeeded in medium with less than % serum, suggesting that some factors in serum interfere with detergent effects. in practice, triton x- concentration is often increased to % to ensure a higher level of safety although no inactivation data support this choice. it thus remains to be determined if a higher concentration of triton x- can overcome efficiency issues in serum. the objective of our study was to assess the accuracy of a wide range of serological tests after chemical or thermal inactivation of serum. triton x- . % or % final concentration and/or heat inactivation at c for min were selected as promising inactivation methods. in addition, hsv- inactivation tests were performed with triton x- . % and % in medium containing % or % serum. we hypothesize that a tenfold increase of triton x- final concentration might allow complete virus inactivation in serum. all human blood samples were obtained for routine patient serological testing at the institute for infectious diseases, university of bern, switzerland. there was no suspicion of highly pathogenic virus infection in these patients. sera used in this study were stored at e c for less than years. they were retrospectively identified using the electronic database of the institute for infectious diseases of the university of bern according to the serological tests originally performed and to the results obtained. each selected serum sample was anonymized and tested for the same serological test as originally. informed consent for use of anonymized leftover patient material was waived. this procedure has been approved by the cantonal research ethics committee of bern, switzerland (basec-no req- - ). the chemical inactivation procedure consisted of mixing patient serum : with pbs or triton x- % or % working solution (please see supplementary materials). samples were incubated for min at room temperature. final concentrations of triton x- were . % and % (precisely . % and . %). some of the samples treated with pbs or triton x- % were additionally thermally inactivated by incubation at c for h in a table top thermoblock without shaking. samples were then kept a maximum of h at c before being used for different serological tests. pre-treated samples were analysed in parallel with the following methods. hiv, hcv, hbv, ebv, cmv, and toxoplasmosis markers (ag, total ig, igm and/or igg) were analysed on an archi-tect® system (abbott diagnostics), a fully automated immunoanalyser based on chemiluminescent microparticle immunoassays (cmias). antibodies to brucella species (igm and igg) were tested by indirect chemiluminescent immunoassay (clia) on another fully automated analyser (virclia®, vircell, granada, spain). for aspergillosis, a galactomanan test (platelia™ asper-gillus ag, biorad , hercules, ca, usa) was performed manually. antibodies to dengue virus (igm, igg) were detected by a manual elisa (euroimmun ei b- ). reactivity to francisella tularensis was evaluated by a rapid test (virapid tularemia, viracell microbiologists vr ) and by a microagglutination test using f. tularensis ag (becton dickinson , franklin lakes, nj, usa). all manual tests were performed according to the manufacturer's instructions. please see supplementary materials for further details. concentrated hsv- stock was spiked in mem containing % fbs or in pure human serum from an hsv- seronegative donor. it was then mixed : with triton x- working solution and incubated for h at room temperature. final concentrations of triton x- were . % and % (precisely . % and . %). final concentrations of serum were % for human serum and % for fbs. initial viral titre in these samples ranged between .  and .  tcid / ml. excess detergent was removed from inactivated viral samples (please see supplementary materials) and each sample was then titrated on vero cells (please see supplementary materials). each experiment was repeated twice. controls containing triton x- without virus were also filtered and titrated to evaluate cytotoxicity caused by residual detergent. graphpad prism (version . ) was used to plot and analyse data. for serological test results, blandealtman plots were constructed to evaluate differences between treated and untreated samples as percentage of differences from the mean (bias). serological results from chemically inactivated samples were additionally analysed by the deming linear regression model. spearman's correlation coefficients were calculated (data did not pass the d'agostino and pearson normality test). to assess agreement of qualitative items cohen's weighted kappa coefficients were calculated with graphpad quickcalcs. hsv- titres were log-converted to obtain a near-normal distribution for statistical analysis. one-way analysis of variance (anova) was performed followed by dunnett's post test to compare multiple groups to a control group. a p value > . was considered not significant (ns); p < . was considered highly significant (***). patient serum samples received for routine serological testing were chemically inactivated with triton x- at a final concentration of . % or % and compared with pbs-treated controls by several elisa-based serological assays (table ) . samples were carefully selected to cover a wide range of concentrations. qualitative results were assessed for each sample and a false result rate (frr) was calculated by computing false-negative and falsepositive results compared with controls for each test. the bias of test results compared with controls (mean differences versus average) was e . ± . % after treatment with . % of triton x- and e . ± . % after treatment with % of triton x- (table , figs. a,b) . this good agreement was confirmed by other statistical methods including deming linear regression with spearman correlation coefficient > . (table s ). automated chemiluminescent assays (cmia and clia) gave similar results to manually performed elisas. only one discordant qualitative result was observed for dengue igg after treatment with triton x- . % and concerned a sample which was around the borderline cutoff (table s ). the overall false result rate after treatment with . % of triton x- was . % ( e . %, table ). increasing concentration of triton x- to % led to a non-significant increase of this false result rate to . % ( . e . %, table ). accordingly, serology results were not substantially altered (fig. b) . false results concerned the same borderline dengue igg sample: one sample with an aspergillus test value around the positive cut-off and one sample for which hbv anti-hbc total ig changed from weak positive in the control to negative in triton x- % (table s ). in contrast, thermal inactivation by incubation of serum samples at c for h yielded aberrant serological test results with a mean bias of . ± . %, which further increased to . ± . % when combined with triton x- . % (table , figs. c,d) . since results did not fulfil the assumption of homoscedasticity (figs. c,d) , no regression analysis was performed. the most relevant changes were seen for cmv igg with biases of more than % (table ) . accordingly, high false result rates were observed for most of the assays with an average of . % ( . e . ) after heat inactivation and . % ( . e . ) after the combination treatment (table ) . hiv antigen þ antibody (agþab), hbv hbs ag, and igg test results, however, remained relatively unaffected by heat inactivation with minimal bias and no discordant qualitative results (table ). in addition to the elisa-based assays, we analysed the performances of a rapid test and a microagglutination test for the diagnosis of tularaemia after chemical or thermal inactivation of serum samples ( table ) . triton x- . % did not have an effect on the accuracy of both tests. triton x- %, however, yielded acceptable rapid test results but disturbed microagglutination results. end titres were affected and several prozone and partial agglutination effects were observed. heat inactivation yielded both poor rapid test and poor microagglutination results with many prozone effects. to evaluate the inactivation potential of triton x- on enveloped viruses we chose hsv- as a model virus. hsv is indeed a prototypic enveloped virus that grows easily to high titres and for which rapid and accurate infectivity assays are available [ ] . for these reasons it has been widely used for inactivation studies with various detergents [ , , ] . since it has been recently shown that serum annulled the inactivation effect of triton x- . % [ ] we performed our assays in % human serum or in medium containing % serum. as previously shown, triton x- . % could completely inactivate hsv- in medium containing % serum with a titre reduction of at least log but it had no effect on hsv- in % serum (fig. ) . on the contrary, a triton x- concentration of % led to complete hsv- inactivation in both % and % serum with a titre reduction of at least log (fig. ). recent events including ebola and lassa outbreaks in west africa [ , ] and two cases of andes hantavirus infection in a returning traveller at our hospital [ ] drew our attention to issues regarding biosafety for laboratory workers and optimal diagnostics for high-risk patients. in our routine laboratory we noticed a lack of protocols for virus inactivation and a lack of data regarding effect of inactivation on the accuracy of serological tests. here we demonstrate that heat inactivation is not a suitable method for the serological diagnosis of infectious diseases. on the contrary routine elisa-based-serological tests and rapid tests can be accurately performed after a treatment with triton x- . % or % final concentration. both quantitative and qualitative results remained relatively unaffected and false results mostly concerned borderline samples. these results should prompt further studies about the effect of triton x- % on clinical chemistry parameters, haematological tests, and malaria tests for which only triton x- . % has been tested so far [ e ] . as recently published [ ] , triton x- . % is not sufficient to inactivate hsv- in high concentrations of serum. increasing triton x- final concentration to % however led to complete inactivation of hsv- in % serum samples. this is of primordial importance for the safety of laboratory workers, considering that they receive and process undiluted serum samples, which are potentially infectious. since triton x- exerts its inactivation effect at the level of the viral envelope only, complete inactivation of hsv- with % of triton x- suggests that other enveloped viruses such as hiv, influenza, sars, or ebov could also be completely inactivated by the same procedure. this remains, however, to be tested especially for ebov, which is a rather stable virus [ , ] . preliminary results of ebov inactivation with triton x- % for min are promising [ ] . other proven inactivation methods such as the combination of triton x- and heat against ebov [ , , ] are unfortunately not compatible with serological diagnosis and should be limited to molecular diagnosis. moreover, although this combination allowed detection of ebov-specific igg and igm [ ] , our study suggests that inactivation procedures used for ebov serology should be further evaluated and compared to inactivation with triton x- alone. if proven efficient for enveloped viruses in general, this simple inactivation procedure based on a single pipetting step could be implemented in basic microbiology laboratories and in the field diagnostics during outbreaks of highly pathogenic viruses where a safe and rapid diagnosis is of primordial importance [ ] . the initial step of adding detergent to the serum sample does however require trained personnel, rigorous safety practices, and a biosafety cabinet of level . triton x- has a long history of usage in diagnostic procedures; nevertheless, it is unknown if high concentrations of detergent could potentially affect routine laboratory analysers. another limitation to the broad usage of triton x- consists in environmental concerns. this reagent will soon be forbidden in europe because of its endocrine disruption effects in aquatic organisms [ ] . restricted usage for well-defined procedures and appropriate waste treatment solutions will most probably still be possible in future. ecofriendly alternatives such as lauryldimethylamine n-oxide (ldao) [ ] have been shown to exhibit effective virus inactivation in a broad range of protein concentrations but need further evaluation with different viruses and conditions. hsv- (tcid /ml) +pbs, unfiltered +pbs, filtered +triton x- . %, filtered +triton x- %, filtered serum serum *** *** ns ns ns < fig. . influence of serum concentration on the chemical inactivation of herpes simplex virus type (hsv- ). reduction in hsv- titres in samples containing either % fetal bovine serum (fbs) or % human serum after treatment with . % or % triton x- for h at room temperature. controls received an equivalent amount of pbs. before titration, samples were filtrated through -kda membranes to remove residual triton x- . unfiltered pbs controls are shown to account for eventual virus loss through the filter. tcid /ml titres presented as mean ± sd were determined in three independent experiments with - replicates per experiment. abbreviations: hsv- , herpes simplex virus type ; tcid , % tissue culture infectious dose. in conclusion, we showed that addition of % triton x- to serum samples enables complete inactivation of hsv- and is compatible with accurate results of a wide range of routine serological tests. this study provides basis to the development of safe serological assays for the rapid and accurate differential diagnosis of infectious diseases in patients with suspected or confirmed infection with highly 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inactivation we wish to thank the laboratory staff of the serology and virology departments of the institute for infectious diseases and especially richard kamgang and sabine funez for excellent technical assistance. we are also thankful to johanna signer from spiez laboratory for her technical advices. a special thanks goes to d esir ee mayor who provided hsv- seronegative serum. some icons used for the graphical abstract are protected by a creative commons license. a previous version of this data was presented at the th european congress of clinical microbiology and infectious diseases (eccmid) in madrid, spain on april. supplementary data to this article can be found online at https://doi.org/ . /j.cmi. . . . key: cord- - kqtw s authors: toh, teck-hock; hii, king-ching; fieldhouse, jane k; ting, jakie; berita, antoinette; nguyen, tham thi; wong, see-chang; wong, toh-mee; lim, wei-honn; ha, siaw-jing; lau, chuet-zou; kong, sing-ling; bailey, emily s; warkentien, tyler e; husain, tupur s; gray, gregory c title: high prevalence of viral infections among hospitalized pneumonia patients in equatorial sarawak, malaysia date: - - journal: open forum infect dis doi: . /ofid/ofz sha: doc_id: cord_uid: kqtw s background: although pneumonia is a known cause of morbidity and mortality in sarawak, malaysia, the etiology and epidemiology of pneumonia are not well described in this equatorial region. routine clinical diagnostics for pneumonia etiology at government hospitals in sarawak had historically involved only bacterial diagnostics. viral diagnostics were only obtained through outside consultations. methods: from june , to may , , we collected nasopharyngeal swabs from patients of all ages older than month hospitalized with pneumonia at sibu and kapit hospitals. specimens were examined at our collaborating institutions with a panel of molecular assays for viral pathogens including influenza a (iav), ibv, icv, and idv, human adenovirus (adv), human enterovirus (ev), human coronavirus (cov), respiratory syncytial virus subtype a (rsv-a) or rsv-b, and parainfluenza virus (piv) types – . results: of samples examined, ( %) had molecular evidence of or more respiratory viruses. overall, the most prevalent virus detected was rsv-a ( . %) followed by adv ( . %) and iav ( . %), then rsv-b ( . %), ev ( . %), ibv ( . %), piv- ( . %), cov ( . %), piv- ( . %), piv- ( . %), and piv- ( . %). no specimens were confirmed positive for icv or idv. conclusions: the high prevalence of viruses detected in this study suggest that respiratory viruses may be responsible for considerable morbidity in equatorial regions such as sarawak. access to viral diagnostics are very necessary for medical staff to determine appropriate pneumonia treatments. as viral respiratory tract infectious diseases such as avian influenzas and severe acute respiratory syndrome coronavirus have emerged as increasing threats to global health security, international stakeholders such as the world health organization and the united nations international children's emergency fund (unicef) are calling for increased global surveillance for emerging and re-emerging respiratory viruses [ ] [ ] [ ] . locallevel surveillance systems are critical to understanding and monitoring the epidemiology of severe respiratory disease. understanding the etiology and epidemiology of pneumonia will guide antiviral interventions as well as enhance global emerging infectious disease preparedness. pneumonia is the second leading cause of death among children under the age of , resulting in more childhood deaths than diarrhea and malaria combined, with more than % of those cases occurring in low-and middle-income countries [ , ] . a meta-analysis of the global burden of childhood pneumonia found the greatest proportion ( %) of severe pneumonia cases occurred in southeast asia [ ] . however, because streptoccocus pneumoniae is the most common cause of vaccine-preventable severe pneumonia, much of the literature has focused on bacterial causes of pneumonia or antibiotic resistance, whereas fewer studies have prospectively investigated the viral etiology of pneumonia in southeast asia [ ] [ ] [ ] . one study of respiratory samples collected from children living in kuala lumpur under years of age between and found that . % of the samples were positive by immunofluorescence assays and viral cultures for viral pathogens, with a prevalence of . % for respiratory syncytial virus (rsv), . % for parainfluenza viruses (pivs), . % for influenza viruses, and . % for adenovirus [ ] . during the past years, the directors of government hospitals (sibu hospital and kapit hospital) in their namesake cities in sarawak, malaysia, have seen increasing pneumonia admissions (see supplementary figure kapit hospital serves a smaller population of approximately residents living along the rejang river [ ] , kilometers east and inland from sibu. the hospital has similarly experienced a recent increase in pneumonia admissions, estimated at more than per year. before this study, routine clinical diagnostics for pneumonia etiology at both sibu and kapit hospitals involved chiefly blood culture and gram stains of endotracheal secretion, if intubated, to detect bacteria. molecular and immunofluorescence laboratory diagnostics were only ordered for severe cases, for which clinicians had to send specimens to a specialized virology laboratory in kuala lumpur, malaysia or the university centre in kuching, sarawak. the overall objective of this study was to examine the viral etiology of and risk factors for pneumonia among patients admitted to sibu and kapit hospitals between june and may and, in doing so, to assist malaysian collaborators with setting up sustainable real-time molecular assays for viral respiratory pathogens. study enrollment took place between june and may at sibu and kapit hospitals. the major ethnic groups in sarawak are iban, chinese, malay, melanau, bidayuh, and orang ulu. as of , approximately . % of the population in sibu was iban and . % of the population in kapit was iban [ ] . as previously described [ ] , we adapted inclusion and exclusion criteria from united states, large and comprehensive, community-based pneumonia studies published in [ , ] . all patients older than days admitted to sibu or kapit hospitals and diagnosed with pneumonia by an attending physician were considered for study eligibility. a medical officer (mo) evaluated eligible subjects for inclusion and exclusion criteria, including confirmation by chest radiography within hours of hospitalization (see supplementary table ). adults years of age or older provided written consent, whereas children ages to provided written assent along with written parental or guardian consent. written consent was obtained from all parents or guardians of children under the age of . the study received a scientific review, and all procedures followed were in accordance with the ethical standards of the although this pilot study had sparse sarawak baseline virus prevalence data from which to calculate sample size, we approximated sample size based upon an estimated prevalence of influenza a (iav) causing % of pneumonias. hence, if we enrolled pneumonia patients, we could be confident to calculate an estimated prevalence within . % of the true prevalence of iav. from june to july , , the study team relied on convenience sampling to enroll as many patients as possible. for the remaining months of the study, mos enrolled patients on of randomly selected days of the week, which were communicated to them by a study coordinator. after obtaining written assent and/or consent, mos administered a brief questionnaire. the questionnaire captured demographic information (age, gender, ethnicity) along with household size (number of cohabitants) and contact with animals within the last days. in addition, all pre-existing health conditions and current medications were self-reported by the subject or their parent/guardian during the questionnaire, then confirmed by the mo through a review of the patient's medical records. the mo then used a flocked swab to collect nasopharyngeal (np) swab from the subject's nose, which was quickly placed into a viral transport tube containing ml sterile viral transport medium (bd universal viral transport; becton, dickinson and company, franklin lakes, nj). all specimens were stored at − °c at the sibu hospital clinical research center (shcrc) until ribonucleic acid or deoxyribonucleic acid extraction was performed using the qiamp cador pathogen mini kit (qiagen, hilden, germany). specimens collected in kapit were stored at − °c until they could be transported to sibu for processing. real-time reverse-transcription polymerase chain reaction (rrt-pcr) or real-time pcr (rpcr) was conducted on similar biorad cfx c touch thermal cycler real-time systems at shcrc, duke-nus in singapore and at duke university in durham, north carolina. all np swabs were examined for molecular evidence of influenza a (iav), ibv, icv, and idv, human adenovirus (adv), human enterovirus (ev), human coronavirus (cov), rsv subtype a (rsv-a) and rsv-b, and piv types - (piv- , - , - , and - ). primer and probes sequences for targeted pathogens are recorded in supplementary table . a positive control and a no-template control were included in each run. all specimens were first run at shcrc. one-milliliter aliquots of these specimens were later shipped on dry ice to either duke-nus or duke university for validation. laboratory staff at duke-nus and duke were blind to the results of the assays at shcrc. cycle threshold (ct) values < were considered positive, and ct values to were considered suspect to acknowledge that positivity could be the result of cross-reactivity or nonspecific amplification. cycle threshold values > were considered negative. all discrepant assays were noted and repeated at both shcrc and either duke or duke-nus, using the same original assays, and discrepancies were thus resolved. partial genome sequencing was also performed for several specimens to validate discrepant results. questionnaire data and molecular results were entered into redcap version . and verified at shcrc and at duke. data were imported into stata version . (statacorp, college station, tx), cleaned, and categorized for statistical analyses. we categorized continuous variables based on the distribution of the counts for household size (quartiles) and for age (quartiles). after examining age quartiles, we rounded age categories into approximate age quartiles so that age was treated as a whole number. demographic and clinical risk factors were examined for bivariate associations with the most prevalent positive molecular assays. pearson's χ test or fishers exact test were used for bivariate work. risk factors with a bivariate test statistic p ≤ . were included in stepwise, manual, backward-elimination, unconditional logistic regression models. risk factors with p < . were retained in final models, and odds ratios (ors) and % confidence intervals (cis) were calculated. a total of hospitalized pneumonia patients were enrolled at sibu and kapit hospitals between june , and may , , with of the subjects enrolled at sibu hospital ( . %) and enrolled at kapit hospital ( . %). of the enrolled subjects, ( . %) were male. a total of ( . %) enrolled subjects were children years of age or younger and ( . %) were of age years and younger. the majority of participants identified as iban, with ( . %) iban subjects enrolled at sibu hospital and ( . %) iban subjects enrolled at kapit hospital (table ) . of the np swabs collected, were run using rrt-pcr/ rpcr; np swab collected from a pediatric patient at sibu hospital was accidentally destroyed before molecular screening. one or more viruses were detected by rrt-pcr/rpcr in . % of the samples testing positive or suspect positive for either iav, ibv, icv, idv, adv, ev, cov, rsv-a, rsv-b, piv- , piv- , piv- , or piv- . the prevalence of each pathogen out of the total number of patients enrolled by month is shown in figure . we detected coinfections in of the patients with evidence of viral infection ( table ) . of the coinfected specimens, adv was detected in approximately % (n = ) with the most common coinfection combination being adv and rsv ( adv/ rsv-b coinfections and adv/rsv-a coinfections) followed by adv/ev coinfections. respiratory syncytial virus subtype b was the second most prevalent virus among coinfections (n = ); in addition to coinfections with adv-positive specimens, rsv-b was also detected in specimens found positive by rrt-pcr for rsv-a, piv- , ev, iav, and ibv. one pediatric patient's specimen was positive for viruses (rsv-b, adv, and ev). overall, the most prevalent virus detected was rsv (table ) , with samples testing positive for either rsv-a or rsv-b (overall prevalence of . %; prevalence of . % among children < years). a total of specimens tested positive by rrt-pcr for rsv-a for an overall prevalence of . %. an additional specimens were suspect-positive for rsv-a (ct values ranged . - . ); however, suspect-positive specimens were not included in the final analyses. eighty-two of the rsv-a-positive samples were from children ≤ years age , totaling a prevalence of . %. a total of ( . % prevalence) specimens were positive for rsv-b by rrt-pcr, with additional suspect-positive specimens (ct values ranged . - . ). thirtyfive of those specimens were collected from children < years ( . % prevalence). after rsv, adv and iav were the most prevalent, each with positive specimens detected by rpcr and rrt-pcr, respectively, for an overall prevalence of . %. fifty-seven of the adv specimens were collected from children < years ( % prevalence), and of the iav specimens were collected from children < years ( . % prevalence). a lower prevalence ( . %) of ibv was detected, with ibv-positive specimens, of which were collected from children < years of age. twenty-five specimens were positive for ev with a . % prevalence overall and . % prevalence among children < years. six specimens were positive by rrt-pcr for cov. among the piv types, piv- was the most prevalent, with specimens testing positive for piv- , followed by piv- detections, piv- detections, and piv- detection. no specimens were determined to be positive for icv or idv; however, there were suspect-positive idv samples (ct values ranged . - . ), which we were unsuccessful at isolating at duke university. one patient of the enrolled was pregnant and tested positive for iav. three severe adverse events were reported to the malaysian ethics board for participants who died after enrollment. all deaths were pediatric patients under year of age with negative blood cultures. human adenovirus was detected in the np swab specimens of of those patients, of whom had an endotracheal tube culture that grew enterobacter aerogenes before death. respiratory syncytial virus subtype a was detected in the third patient's np swab. no coinfections were detected among these patients. due to low counts, cov, icv, idv, and piv - disease outcomes were not included in the risk factor analysis. across the disease outcomes examined (rsv-a, rsv-b, iav, ibv, ev, and adv), gender was not found as a statistically significant risk factor in the initial bivariate screenings. additional potential risk factors such as pre-existing diseases, treatment history, and animal contact defined as touched or come within meter in the last days. c other animals include the following: cow, rats, rabbits, snake, and monkey. ethnicity were eliminated in the stepwise, backward-elimination multivariate modeling. the month of enrollment was a statistically significant risk factor for most virus outcomes, including rsv-a, rsv-b, iav, and adv (tables and and supplementary tables and ). respiratory syncytial virus subtype a was most prevalent during the first month of enrollment, june-july with an adjusted or of . ( % ci, . - . ) compared with rsv-a detection by rrt-pcr in november-december (table ). in contrast, there was a very low detection of rsv-b in june-july (see supplementary figure ). compared to june-july , rsv-b had an increased adjusted or beginning mid-february, with an adjusted or of . ( % ci, . - . ) between april and may , (table ) . we detected adv in over % of the specimens collected from april to may , . compared to the month of october-november , there was an increased adjusted or of . ( % ci, . - . ) of adv infection during the month of april-may. the only disease outcome for which location of hospitalization (kapit versus sibu) was statistically significant was ev; patients with specimens found positive for ev had a . higher or ( % ci, . - . ) of being enrolled at kapit hospital compared with sibu hospital (see supplementary table ) . age was a statistically significant risk factor for rsv-a, rsv-b, and adv, with pediatric patients ages month to year and to years having increased adjusted or compared with patients > years of age. detection of rsv-a and rsv-b was associated with quartiles of household size (number of additional cohabitants) (tables and ). the only animal exposure that was statistically associated with a disease outcome was cat exposure for ibv, with those patients reporting coming into contact with a cat within meter in the last days having an or . times higher ( % ci, . - . ) than those patients who did not have contact with a cat. sarawak, located on the northwest side of the island of borneo, has an equatorial climate with a high relative humidity that rarely drops below % and temperatures ranging from °c to °c (see supplementary figure ). both sibu ( . °n, . °e) and kapit ( . °n, . °e) are approximately degrees north of the equator (supplementary figure ) . the rainy season in this region typically falls between november and february, with december to march seeing the most rain. a drier season typically falls between may and september, with june to august seeing the least rain. our previous studies have investigated the relationship between relative humidity or absolute humidity and influenza virus transmission, suggesting that increased levels of humidity are linked with decreased transmission efficiency; however, the prevalence of iav in sibu and kapit hospitals suggest the high humidity associated with the equatorial climate in this region may not constrain iav transmission [ , ] . our findings regarding the prevalence of rsv support the existing literature that the antigenic subgroups a and b can cocirculate but typically differ by season [ ] . unlike iav, we saw seasonal variation of rsv-a and rsv-b during the drier months (see supplementary figure ) . the year-long data found a lower prevalence of rsv overall than an earlier analysis of the data collected during the first months of the study, june and july ; the logistic regression model from the pilot study also found that hospital location was a risk factor for rsv-a infection, with patients at kapit hospital having a higher adjusted or (adjusted or = . ; % ci, . - . ) of testing positive for rsv-a compared with patients hospitalized at sibu hospital [ ] . the finding that age was a statistically significant risk factor for rsv infection is consistent with the epidemiology of this virus. in the year-long study, we found a slightly elevated odds ratio of rsv-a and rsv-b infection among children ages to years (rsv-a adjusted or = . after the initial statistical analysis, pediatric data were stratified and analyzed for the outcome of rsv-a (see supplementary table ). in the stratified analysis, the same risk factors, including age quartile, household size, and month of enrollment, were found to be risk factors for rsv-a infection. we speculate that the high or ( . ; % ci, . - . ) observed for the association between cat exposure and ibv (see supplementary table ) may have occurred by chance. children < years adults ≥ years denominator is total number of specimens examined by rrt-pcr and rpcr for viruses, n = . when considering the number of cohabitants in the household of a subject testing positive for rsv-a, q (≥ ) had a slightly increased or compared with q ( to cohabitants). the same was true when the analysis was run on the stratified data for pediatric subgroups (see supplementary table ). in contrast, for rsv-b infection, q ( to cohabitants) and q ( to cohabitants) had slightly increased or compared with q ( . [ % ci, . - . ] and . [ % ci, . - . ], respectively). the mean household size among all enrolled subjects was . people with a range of to persons. household size was self-reported and did not capture the type of household (ie, single-family home or traditional longhouse). the study had a number of limitations. although the study offered the questionnaire in different languages to ensure comprehension, it is possible that questions about behavior were not accurately understood. after the first months of study, patients were supposedly recruited on randomly selected days of the week; however, we cannot eliminate the possibility that mos selected the sicker patients in the ward for this study. in addition, this study was limited to hospitalized patients and therefore excluded milder cases of pneumonia not requiring hospitalization. although we used rigorously validated real-time pcr assays as the gold standard for virus detection, the study was limited to a panel of viruses and we therefore likely missed other important pathogens present in specimens, including human metapneumovirus, rhinovirus, and bacteria. specifically, this study would benefit from an understanding of the prevalence of bacterial mono-or coinfections within the patient population. this study provides data on year of pneumonia admissions at district government hospitals in sarawak, malaysia; however, prevalence of the viruses investigated can vary between years. nevertheless, this study is one of the largest viral pneumonia etiology studies of its kind conducted in southeast asia. the findings led investigators to extend the study for an additional year, and a subset of samples will be analyzed at duke university for rhinoviruses a, b, and c. through this study we found a viral etiology for a relatively high proportion ( . %) of pneumonia cases, demonstrating the need for these hospitals to gain clinical diagnostics for viral pathogens. this will help hospital medical staff appropriately administer antiviral treatment for illnesses, such as iav and ibv, and avoid inappropriate use of antibiotics for pneumonia with a viral etiology. the findings from this study may assist the hospitals in their preparedness for future pneumonia admissions when new antivirals and vaccines are more readily available. whereas current therapies for rsv infection are largely supportive, as of there are more than ongoing rsv vaccine trials and rsv antiviral drug trials currently being evaluated, suggesting that new prevention and treatment mechanisms may be on the horizon [ ] . the risk factor analysis of age categories and rsv-a detection also provides evidence to support and encourage breastfeeding among newborns. in a commentary on pneumonia published in the lancet, watkins and sridhar [ ] describe the multifold factors limiting global action on pneumonia, including the challenges that arise from the complexity of pneumonia due to its multiple etiologies and consequentially diverse treatments. accurate diagnosis of pneumonia will not only allow clinicians to prescribe better treatment for patients, but it will also contribute to knowledge regarding the respiratory viruses most likely to cause illness in various age groups and communities. given the high prevalence of viruses detected among the patients enrolled in this study, routine surveillance and more sensitive molecular-based assays for respiratory viruses are recommended for hospitals in sarawak, malaysia and similar equatorial climates. supplementary materials are available at open forum infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. potential conflicts of interest. t one pediatric patient specimen destroyed, assay results out of n = . surveillance for emerging respiratory viruses global epidemiology of non-influenza rna respiratory viruses: data gaps and a growing need for surveillance addressing the public health burden of respiratory viruses: the battle against respiratory viruses (brave) initiative estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower respiratory tract infections in countries: a systematic analysis for the global burden of disease study global and regional burden of hospital admissions for severe acute lower respiratory infections in young children in : a systematic analysis global burden of childhood pneumonia and diarrhoea clinical and economic burden of community-acquired pneumonia amongst adults in the asia-pacific region the bacterial aetiology of adult community-acquired pneumonia in asia: a systematic review assessing the burden of pneumonia using administrative data from malaysia, indonesia, and the philippines epidemiology and seasonality of respiratory viral infections in hospitalized children in kuala lumpur, malaysia: a retrospective study of years surveillance for respiratory syncytial virus and parainfluenza virus among patients hospitalized with pneumonia in sarawak community-acquired pneumonia requiring hospitalization among u.s. children community-acquired pneumonia requiring hospitalization among u.s. adults humidity as a non-pharmaceutical intervention for influenza a absolute humidity modulates influenza survival, transmission, and seasonality respiratory syncytial virus-a comprehensive review pneumonia: a global cause without champions we thank the medical officers of sibu and kapit hospitals (namely, nga-hung ngu, khai-fatt chao, cheng-ing kong, zhen-hao chin, edmund kwang-yuen wong, tiana ti, and hilary hon-yun kueh) for enrolling patients. we thank the laboratory staff at kapit hospital (namely, cornelius jambol, goh hieng hua, and velarie bill) and tiing-tiing chua at sibu hospital for processing specimens. we thank dr. cheesan lee for helping us to begin this collaboration. we thank jessica choi, sarah philo, kerry mallinson, sarah paust, calvin wang, and christine wang for laboratory support. we thank dr. larry park for his support in the statistical analysis. this work was conducted in partnership with duke university, the duke global health institute, sibu hospital clinical research center, and segi university sibu clinical campus. we thank the director general of health, malaysia for his permission to publish this paper.disclaimer. the views expressed in this article are those of the author(s) and do not necessarily reflect the official policy or position of the department of the navy, department of defense, or the united states government.financial support. this work funded by the us naval medical research center-asia and vysnova partners (sc- -saber- - , sc- -saber- - ) and professor gregory gray's discretionary funds from duke university's global health institute. key: cord- -fxc zwr authors: inghammar, malin; borand, laurence; goyet, sophie; rammaert, blandine; te, vantha; lorn try, patrich; guillard, bertrand; buchy, philippe; vong, sirenda; tek chheng, eap; cavailler, philippe; mayaud, charles; tarantola, arnaud title: community-acquired pneumonia and gram-negative bacilli in cambodia—incidence, risk factors and clinical characteristics date: - - journal: trans r soc trop med hyg doi: . /trstmh/try sha: doc_id: cord_uid: fxc zwr background: in western settings, community-acquired pneumonia (cap) due to gram-negative bacilli (gnb) is relatively rare. previous studies from asia, however, indicate a higher prevalence of gnb in cap, but data, particularly from southeast asia, are limited. methods: this is a prospective observational study of patients ≥ y of age with cap from two hospitals in cambodia between and . the proportion of gnb was estimated. risk factors and clinical characteristics of cap due to gnb were assessed using logistic regression models. results: the prevalence of gnb was . % in all cap patients and . % among those with a valid respiratory sample. gnb infection was independently associated with diabetes, higher leucocyte count and cap severity. mortality was higher in patients with cap due to gnb. conclusions: we found a high proportion of gnb in a population hospitalized for cap in cambodia. given the complex antimicrobial sensitivity patterns of certain gnbs and the rapid emergence of multidrug-resistant gnb, microbiological laboratory capacity should be strengthened and prospective clinical trials comparing empiric treatment algorithms according to the severity of cap are needed. previous studies of community-acquired pneumonia (cap) have reported varying incidences of gram-negative bacilli (gnb). numerous studies from europe and north america have shown that gnb are not a common aetiology , and a significant prevalence mainly affects high-risk populations. [ ] [ ] [ ] although limited, previous studies from asia indicate that gnb is a more common cause of cap, particularly in southeast asia. , initial antibiotic treatment for cap is empiric. first-line treatment recommendations mainly target streptococcus pneumoniae, the major causative pathogen in most parts of the world. while gnb have repeatedly been associated with severe disease presentation and adverse prognosis, , , the choice of appropriate antibiotic treatment in cap is further complicated by the emergence of multidrug resistance among gnb, particularly in asia, since excessive use of broad-spectrum antibiotics for common infections can be harmful. it is therefore important to define the frequency of gnb in cap in southeast asia and to identify risk factors to provide support for clinicians in determining which patients merit deviation from the advocated first-line antibiotic therapy. in this prospective study we estimate the prevalence of gnb in patients admitted to hospital for cap, identify their risk factors and clinical predictors and assess cap-related mortality in cambodia, a low-income country in southeast asia. from april to july , a prospective surveillance study of acute lower respiratory tract infections was conducted in cambodia, in the takeo and kampong cham provincial hospitals. the study hospitals were located in takeo province (estimated population . million) and kampong cham province (estimated population . million), both located approximately km from the capital. the participating hospitals could provide supplementary oxygen, but mechanical or non-invasive ventilation and computed tomography (ct) scan were unavailable. details of patient recruitment and the methodology have been described elsewhere. patients were eligible if admitted to hospital with symptoms of acute lower respiratory tract infection, defined as onset ≤ d, fever ≥ °c or a history of febrile episodes within days and cough and at least one respiratory sign (dyspnoea, chest pain or crackles on lung auscultation) without any hospitalization in the previous weeks and without any known tuberculosis (tb), human immunodeficiency virus (hiv) or active cancer. the decision to hospitalize was left to the clinicians' judgement. the study was approved by the cambodian national ethics committee for health research ( -nechr). written informed consent was obtained from patients ≥ y of age or the parents/guardians of children. in the present study we included individuals ≥ y of age (the age cut-off for admission to adult wards in cambodia) with radiological and clinical signs compatible with cap. demographic, clinical and treatment data were recorded. on admission the following investigations were performed on all patients: chest x-ray, collection of blood and non-induced sputum for microbiological cultures and consecutive samples for direct sputum examination for acid-fast bacilli (afb) and nasopharyngeal swab for polymerase chain reaction tests for viral respiratory pathogens, including human metapneumovirus; respiratory syncytial virus (rsv); human bocavirus; influenza a and b viruses; coronaviruses oc , e, hku- , nl and sars; parainfluenza viruses - ; adenoviruses; rhinovirus and enteroviruses. pleural culture was taken if pleural fluid was present and drained. hiv was tested for according to national cambodian guidelines advocating routine hiv testing for patients with sexually transmitted infections or tb, pregnant women and hospitalized patients suspected of being infected with hiv. cultures (bacterial and mycobacterial) and molecular diagnosis were processed using standard microbiological procedures and performed at the institut pasteur du cambodge in phnom penh, cambodia. sputum samples were validated using may grünwald-giemsa staining by trained laboratory staff. only good quality samples (> polynuclear leucocytes and < squamous epithelial cells per low-power field) were sent for culture. antibiotic susceptibility testing (ast) was performed by disc diffusion according to guidelines from the antibiogram committee of the french society for microbiology (ca-sfm/eucast). as part of the national tuberculosis control program, repeated direct sputum examinations for afb were performed at the hospital laboratories. chest x-rays were reinterpreted by expert pulmonologists unaware of the final diagnosis and a review of the entire chart was done by a panel of external experts who assigned a final diagnosis to each patient, taking all clinical and diagnostic findings into account. pneumonia was defined as lobar consolidation and alveolar or interstitial infiltrates on chest x-ray. bacterial isolates were considered causative when either isolated from blood or pleural fluid or if significant growth (> colony-forming units [cfu]/ml) was documented in a validated sputum sample with no other positive blood or pleural cultures. gnb were defined as isolation of enterobacteriaceae or non-fermenters (escherichia spp., klebsiella spp., proteus spp., enterobacter spp., serratia spp., pseudomonas spp., burkholderia pseudomallei and acinetobacter spp.). the non-gnb group included all other pathogens and cap of unknown aetiology. haemophilus influenzae was included in the non-gnb group. patients with mixed gnb and non-gnb infections were excluded to reduce misclassification. underlying pulmonary disease was defined as reported chronic bronchitis or asthma, emphysema, bronchiectasis or other sequelae on chest x-ray. high alcohol consumption was defined as one or more glasses of rice wine or several daily beers. diabetes was defined by either self-reported diabetes or glycaemia > . mmol/l. an appropriate antibiotic treatment was defined as administration of at least one antimicrobial agent with in vitro activity against the identified pathogen. risk factors, clinical predictors and prognosis of gnb cap were assessed comparing gnb patients with all non-gnb individuals in the total cohort. in a sensitivity analysis, patients with positive afb were excluded. in a second sensitivity analysis, only patients with a valid respiratory sample sent for culture or who had a positive blood or pleural culture were included. this group is referred to as having a respiratory sample. student, wilcoxon rank sum, χ or fischer's exact test was used, as appropriate. logistic regression was used to assess predictors of gnb cap and predictors of death. variables were selected based on a priori knowledge. variables considered for inclusion were age, sex, comorbidities, previous hospitalization or treatment (< weeks before admission), clinical and radiological signs and severity of disease. in the multivariable models, the definition of severity was adapted to the cambodian setting and derived from the pneumonia severity index (psi). a severe case was defined by the presence of at least two of the following: systolic blood pressure < mmhg, heart rate > bpm, respiratory rate > /min, oxygen saturation < % and body temperature < °c or > °c. , missing data were assumed to be missing completely at random. variables were first assessed in univariable models and entered into multivariable models, retaining variables with p-values < . . the final model was derived from an initial explanatory model including all variables. variables were then withdrawn or re-entered m. inghammar et al. in a combined backward and forward selection manner. models were compared using likelihood ratio tests and aikaki's information criterion if not nested. the final model fit was assessed by the hesmer-lemeshow goodness-of-fit test. the area under the receiver operating characteristics curve was calculated to estimate the overall discriminatory power of the model. likelihood ratio tests were used to test for interactions. analyses were performed using stata version . (statacorp, college station, tx, usa). a total of patients > y of age were enrolled. of these, ( . %) patients were excluded: were assigned a final diagnosis of upper respiratory infection, a diagnosis of extrapulmonary or no infection and had a normal chest x-ray and were diagnosed as presenting acute bronchitis by the expert review panel. figure shows a flowchart of the cohort selection. in all, patients fulfilled the criteria for cap, among whom ( . %) were male, with a median age of y (interquartile range [iqr] - ). comorbid conditions were present in ( %) of the patients. bacterial aetiology could be assigned in ( . %) of the participants. among these, mycobacterium tuberculosis, h. influenzae, klebsiella pneumoniae, b. pseudomallei and s. pneumoniae were identified as the predominant pathogens, as previously reported. blood cultures were positive in ( . %) of the patients and ( %) had a viral co-infection. valid respiratory samples were obtained from ( %) of the patients. gram-negative bacilli were identified in / ( . %) of all cap patients and / ( . %) of the patients with respiratory sampling. the gnb pathogens identified were k. pneumoniae, %; b. pseudomallei, %; pseudomonas aeruginosa, %; escherichia coli, %; acinetobacter baumannii, % and various enterobacteriaceae (e.g., serratia, proteus, enterobacter spp.), %. mixed (gnb and non-gnb) infections were found in ( . %) of the patients and these patients were excluded, leaving patients with gnb for further analysis. among patients with gnb, / ( %) had positive blood cultures. of these, had no valid sputum samples, had negative sputum cultures (positive blood cultures for b. pseudomallei) and had sputum cultures that matched the results from blood cultures ( b. pseudomallei, a. baumannii). demographic and clinical characteristics are presented in table table s ). results from ast were available for / ( %) of the samples with bacterial growth. of the enterobacteriaceae tested, / ( %) were resistant against third-generation cephalosporins. were independently associated with gnb aetiology (see table ). in the sensitivity analysis, in which patients diagnosed with positive afb (n= ) were excluded, results were similar to the main analyses with the exception of cavitation on chest x-ray, which was associated with gnb aetiology (see supplementary appendix, table s ). patients with gnb had a higher in-hospital mortality or were more likely to be discharged to die at home ( table . gnb, severe cap and any underlying comorbidity were independently associated with a risk of death. in this prospective observational study from two provincial hospitals in cambodia, . % of all patients with cap had infection caused by gnb. this is likely an underestimation of the true incidence, since the proportion of gnb reached . % in the population with a valid respiratory sample. data from cambodia are scarce. in line with our findings, however, previous studies from neighbouring countries in asia have in general reported a high frequency of gnb regardless of age. a recent systematic review of cap aetiology based on studies from asian countries other than cambodia found an overall proportion of % in hospitalized patients. in southeast asia, the proportion was even higher, ranging from to %. although the reasons for the increased frequency of gnb are not fully understood, it can be explained in part by the contribution of b. pseudomallei (agent of melioidosis)-a highly pathogenic, still underrecognized agent of severe pneumonia that is endemic in southeast asia , -and the presumed high prevalence of a particularly virulent mucoid phenotype of k. pneumoniae that is prone to cause invasive disease. , k. pneumoniae has been shown to be among the most frequent cap pathogens in all age groups. - however, we and others have also found a high proportion of other gnb, such as p. aeruginosa, e. coli and a. baumannii. , many gnb are found in soil and humid environments. the higher incidence of gnb in cap might therefore reflect effects of climate conditions or exposure to an environmental reservoir analogous to b. pseudomallei. we are aware of very few publications on the gnb nasopharyngeal carriage rate. one recent study from indonesia reports a gnb carriage rate of %. two older studies comparing developing and developed countries found significant differences: - % vs - %, respectively. , it has been hypothesized that the widespread use of antibiotics (proper or substandard drugs) in asian countries could bias the incidence estimates, since prior antibiotic use promotes colonization by gnb. , prior antibiotic use or the use of counterfeit drugs can also favour selection of patients with gnb seeking medical care due to treatment failure. we found no statistical evidence of differences in gnb prevalence between the subgroups according to prior antibiotic treatment. however, half of our cohort reported treatment of unknown origin prior to inclusion. misclassification due to unawareness of whether or not antibiotics had been used could have biased our estimates. we found no association with underlying comorbidity, except for diabetes. in cambodia, access to quality health care is limited and comorbidities are likely to be underdiagnosed. a high degree of misclassification could therefore have biased estimates towards the null. nevertheless, the prevalence of diabetes found in our study was in line with previous prevalence estimates from cambodia. furthermore, due to the substantial financial burden hospitalization often imposes on the family, many families in cambodia choose to bring the patient home if they cannot afford care or when the patient is severely ill with little hope of recovery. the present study was based on transactions of the royal society of tropical medicine and hygiene hospitalized cases of cap, which could have biased our cohort towards previously healthy and younger age groups. while some previous studies have identified underlying pulmonary disease as a risk factor for gnb in cap, , others have not. , a large proportion of our cohort had an underlying pulmonary condition. the most frequent pathogens in this group were h. influenzae and s. pneumoniae, regardless of whether the underlying condition was bronchiectasis or chronic bronchitis/asthma. alcohol consumption was mainly associated with s. pneumoniae in our study. several gnb have been shown to cause necrotic pulmonary infections and cavitary lesions on the chest x-ray. , even with the large proportion of tb cases in our cohort-a well-known phenomenon in countries with a high burden of tb-we did not observe an association with gnb. importantly, when excluding afb-positive patients, cavitation was significantly associated with gnb aetiology. thus if direct sputum examination is negative, the likelihood of gnb is high in patients with cavitary lesions on the chest x-ray. gnb has repeatedly been associated with increased severity and worse outcome in cap. , in line with previous work, we found gnb to be associated with severe cap. inferences about disease outcomes such as death were limited by the large proportion of patients with missing information due to transfer to other health care units. despite this limitation, our results support previous findings that gnb are associated with worse outcome. since the ascertainment of comorbidities was insufficient and ast information was lacking for a large proportion of our cohort, we cannot conclude whether the excess mortality could be attributed to the gnb pathogens, to differences in underlying health status or to inappropriate antibiotic treatment. other limitations deserve to be mentioned. despite researchers' efforts, it was only possible to obtain valid sputum samples from half of the patients in our study; invasive procedures such as bronchoalveolar lavage or protected specimen brush were not available. however, numerous previous studies have shown that only - % of patients with cap can produce a valid sputum sample. , , chest x-ray was performed at admission, but no follow-up chest x-rays were performed and ct scan was not available. it was not possible to track patients who were transferred to other wards or who self-discharged against medical advice. determining the clinical relevance of a positive sputum sample in cap (i.e., whether it represents true infection or just colonization) is particularly difficult with gnb since specimen collection after antibiotic exposure and many factors related to the handling of specimens often favour the growth of gnb. , nevertheless, we believe that most of the gnb isolated represent true infection. first, % of the patients with gnb were bacteraemic. second, specimens were prospectively collected prior to any antibiotic treatment at the hospital. third, only validated sputum was sent for culture and we used a cut-off of cfu/ml of original sputum. our study was based on prospectively collected information from two separate geographical regions over a -y period. we therefore believe the results are generalizable to the general population in cambodia. in conclusion, as in other asian countries, cap due to gnb is more common in cambodia than in western settings. choosing the appropriate antibiotic is complex and further complicated by the large proportions of b. pseudomallei and by the rapidly increasing rates of antimicrobial-resistant enterobacteriaceae. prospective clinical trials comparing empiric treatment algorithms according to the severity of cap are therefore urgently needed. given the complex situation, microbiological laboratory capacity needs to be increased and blood and sputum culturing in cap should be encouraged. community-acquired pneumonia through enterobacteriaceae and pseudomonas aeruginosa: diagnosis, incidence and predictors community-acquired pneumonia due to gram-negative bacteria and pseudomonas aeruginosa: incidence, risk, and prognosis hospitalized communityacquired pneumonia in 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datasets generated and/or analysed during the current study are not publicly available but are available from the corresponding author on reasonable request.competing interests: none of the authors have any competing interests. philippe buchy is currently an employee of gsk vaccines in singapore.ethical approval: the study was approved by the cambodian national ethics committee for health research ( -nechr). informed consent was obtained for all participants. supplementary data are available at transactions online (http:// trstmh.oxfordjournals.org/). key: cord- -pwq gir authors: chiu, charles title: cutting-edge infectious disease diagnostics with crispr date: - - journal: cell host microbe doi: . /j.chom. . . sha: doc_id: cord_uid: pwq gir three recent science articles (chen et al., , gootenberg et al., , myhrvold et al., ) describe the use of crispr-cas technology to develop point-of-care diagnostics that directly detect viruses from clinical samples. these tests could radically transform approaches to diagnosing infectious diseases at the bedside and in the field. three recent science articles (chen et al., ; gootenberg et al., ; myhrvold et al., ) describe the use of crispr-cas technology to develop point-of-care diagnostics that directly detect viruses from clinical samples. these tests could radically transform approaches to diagnosing infectious diseases at the bedside and in the field. zika virus (zikv)-every couple of years, a new virus emerges from the hinterland and threatens to devastate the population. during each of these epidemics, we were woefully unprepared to meet the urgent need for field-deployable diagnostic tests to guide surveillance, treatment, and biocontainment efforts. the challenge faced by clinicians in accurately diagnosing infections extends not only to outbreaks in the field but also to routine patient care in community hospitals and clinics. individual molecular testing for the vast majority of pathogens is typically not available except as sendout assays to highly specialized reference laboratories. up until now, pointof-care diagnostic platforms for rapid, flexible, and accurate detection of pathogens directly from clinical samples did not exist. in three papers published in science (chen et al., ; gootenberg et al., ; myhrvold et al., ) , two groups report the use of crispr-cas-based technology for the development of molecular diagnostic assays, with a focus on infectious diseases. the crispr-cas system in bacteria has evolved as a mechanism to fight off viruses by cleaving dna or rna sequences from the invading phage (hille et al., ) . this technology, now widely repurposed for gene-editing applications, involves families of enzymes that are able to precisely cleave doublestranded dna (dsdna) or single-stranded rna (ssrna) at sequences complementary to a guide rna. for purposes of detection, the groups here leverage the ''collateral activity'' of either the cas enzyme in indiscriminately cleaving ssrna myhrvold et al., ) or the cas a enzyme in cleaving single-stranded dna (ssdna) (chen et al., ) . once the guide rna is activated by a complementary sequence, cleavage of a labeled ssrna or ssdna probe generates a signal that can provide a fluorescent readout in a number of portable formats, including the use of disposable paper strips ( figure ). the crispr-cas (sherlock)based myhrvold et al., ) and crispr-cas a (detectr)-based (chen et al., ) assays described in these papers carry several advantages over traditional microbiological diagnostics such as nucleic acid amplification testing (e.g., polymerase chain reaction [pcr]) or antigen-based testing (e.g., immunostrips) (drancourt et al., ) . nucleic acid amplification tests such as pcr are generally considered to be the most sensitive and specific tests available. however, these tests typically incorporate multiple steps to run the assay, including an upfront nucleic acid extraction step, and require dedicated instrumentation. in contrast, both groups demonstrate here that crispr-cas-based assays can be run directly on primary clinical samples as a single reaction and performed using minimal or no equipment. the assays are also amenable to the use of lyophilized reagents, foregoing the need for maintenance of a cold chain, and thus are attractive for use in the field. however, the crispr-cas assays are also shown to exhibit high sensitivity and specificity of detection, achieving a performance comparable to that of pcr. interestingly, this may be due to the fact that an isothermal amplification is still required as part of the crispr-casbased assay protocol to generate detectable fluorescent signal. at the same time, these assays are similar to antigen-based tests with respect to ease of use and speed, with a sample-to-answer turnaround time of - hr. in the paper by myhrvold et al., work deriving from the collaboration of two prominent investigators at the broad institute (feng zhang and pardis sabeti), the authors illustrate several applications for the sherlock assay, including ( ) zikv and dengue virus (denv) detection directly from bodily fluids such as saliva and urine in < hr, ( ) specific discrimination among the four different denv serotypes or identification of region-specific strains of zikv from the - pandemic, and ( ) detection of singlenucleotide polymorphisms such as a recent zikv mutation, s n, linked to cases of microcephaly in affected infants (yuan et al., ) . the last two examples are of particular relevance to public health epidemiology. however, the clinical utility of the assay can also be potentially extrapolated to individual patients, such as the determination of a particular hepatitis c virus (hcv) genotype to guide the choice of antiviral therapy (aasld/idsa hcv guidance panel, ). in the chen et al. paper from the laboratory of jennifer doudna at uc berkeley, the authors use the detectr assay to detect ''high-risk'' human papillomavirus types and , associated with invasive genital tumors, with % overall accuracy. this successful demonstration extends the potential application of crispr-cas technologies from infectious diseases to other fields. rapid detection of single-nucleotide polymorphisms from circulating tumor cells in blood, for instance, may usher in a new era of point-of-care diagnostics for early cancer. a crispr-cas-based assay can also be leveraged for use as both a companion diagnostic and treatment. for example, the broad institute group describes the repair of a mutation in the apc gene that has been linked to familial adenomatous polyposis, an inherited condition greatly increasing the risk of colonic and rectal cancers in affected individuals. using sherlock, they simultaneously genotyped and edited out the apc gene from a human embryonic kidney (hek ) cell line containing the insert. so, is this technology ready for prime time? at first glance, a crispr-casbased assay appears to have all of the characteristics desired in a point-of-care diagnostic: ( ) implementation as a single reaction; ( ) compatibility with lyophilized reagents; ( ) under -hr turnaround time; ( ) design flexibility, with generation of a new assay from scratch in under a week; ( ) portability without the need for electricity or expensive instrumentation; ( ) detection directly from clinical samples; and ( ) a colorimetric fluorescent readout. such an assay would have been tremendously helpful, for instance, if rapidly deployed and available for diagnostic testing early in the course of the west african ebov or zikv epidemics. however, it is unlikely that a crispr-cas approach by itself will completely solve the challenges of point-of-care diagnostic testing. fundamentally, the assay is still a targeted detection test using specific primers and probes and is thus subject to the same limitations. deploying the test for emerging outbreak investigation may not be useful if the cause of an outbreak is not known. for example, cryptic transmission and circulation of zikv had been ongoing in brazil for more than a year prior to its initial identification in may of (faria et al., ) , in part because zikv had never been seen in the americas and so had not been considered as a candidate outbreak pathogen a priori. in addition, the high specificity of crispr/cas cleavage, although of value for a targeted diagnostic, can be challenging with rna viruses such as zikv and ebov that rapidly mutate. indeed, mutations in the ebov genome likely resulted in decreased sensitivity and performance of existing pcr assays in detection of the makona strain that caused the ebov outbreak in west africa (sozhamannan et al., ) . the field of infectious disease diagnostics is also rapidly moving toward clinical syndromic-based multiplexing for detection, such as the use of targeted panels for diagnosing neurological, respiratory, and gastrointestinal infections (ramanan et al., ) . highly multiplexed or even fully untargeted methods (e.g., metagenomic next-generation sequencing) (schlaberg et al., ; somasekar et al., ) may be preferable for diagnosis of unknown acute febrile illnesses that may be caused by a variety of different pathogens. for instance, a significant proportion of patients during the west africa ebov outbreak had other infections, such as lassa fever or malaria, or were co-infected. of note, these diagnostic methods can be complementary. a crispr-cas-based assay may be used to initially screen for antibiotic resistance, for example, with reflex sequencing to characterize the specific genes and mechanisms involved. other foreseeable challenges to routine implementation of crispr-cas-based assays include performance, cost, and regulatory issues. since a target amplification guide rna molecules are constructed to target specific pathogens or tumor cells (for cancer screening). after collection of clinical samples in a point-of-care setting, such as the patient bedside, medical office, hospital ward, or in the field, the cas a-or cas -based assay can be performed directly from the sample in under hr, without the need for a separate dna or rna extraction step. note that the cas a protein targets dsdna, while cas targets rna. after activation of the cas a or cas protein, indiscriminate cleavage of fluorescent ssdna probes for cas a or ssrna probes for cas produces a detectable signal. there are numerous potential applications for crispr-cas-based assays, as listed on the right and described in the text. step is still incorporated as part of the assay, there remains the possibility of cross-contamination and/or contamination from the laboratory, reagents, or environment. in addition, the ''real-life'' performance and accuracy of crispr-cas-based diagnostic testing is not yet known, especially outside of the confines of a controlled laboratory environment. it also remains to be seen whether crispr-cas-based assays can be standardized for high-throughput diagnostic testing and how they will be viewed by regulatory agencies such as the u.s. food and drug administration. there may be issues regarding availability and cost of crispr-cas-based testing to the developing world, especially given that many of the intended-use cases for the technology have been patented. finally, a key consideration is how amenable the approach is to multiplexing. in the current papers, multiplexing of only one or a few targets in parallel was demonstrated, and it is unknown whether there is an upper limit to the number of targets that can be successfully incorporated in the assay. nevertheless, despite these concerns, crispr-cas-based technology has now provided us a powerful new tool in our perpetual fight against infectious diseases. hepatitis c guidance: aasld-idsa recommendations for testing, managing, and treating adults infected with hepatitis c virus crispr-cas a target binding unleashes indiscriminate single-stranded dnase activity the point-of-care laboratory in clinical microbiology establishment and cryptic transmission of zika virus in brazil and the americas multiplexed and portable nucleic acid detection platform with cas , cas a, and csm the biology of crispr-cas: backward and forward field-deployable viral diagnostics using crispr-cas syndromic panel-based testing in clinical microbiology professional practice committee and committee on laboratory practices of the american society for microbiology; microbiology resource committee of the college of american pathologists viral surveillance in serum samples from patients with acute liver failure by metagenomic next-generation sequencing evaluation of signature erosion in ebola virus due to genomic drift and its impact on the performance of diagnostic assays a single mutation in the prm protein of zika virus contributes to fetal microcephaly key: cord- -x avmtef authors: pérez‐rivera, claudia; ramírez‐mendoza, humberto; mendoza‐elvira, susana; segura‐velázquez, rene; sánchez‐betancourt, josé ivan title: first report and phylogenetic analysis of porcine deltacoronavirus in mexico date: - - journal: transbound emerg dis doi: . /tbed. sha: doc_id: cord_uid: x avmtef porcine deltacoronavirus has caused great economic losses in the swine industry worldwide. in this study, we carried out the first detection, sequencing and characterization of this virus in mexico. we analysed rectal samples by multiplex rt‐pcr to determine coinfections. in addition, the spike gene was amplified, sequenced and analysed phylogenetically. we found positive samples for porcine deltacoronavirus, representing . % of the total samples, and we determined that the most frequent coinfection was with porcine epidemic diarrhoea virus ( . %). four sequences of mexican isolates were most closely related to those of the united states. the antigenic regions and the glycosylation site of the strains obtained coincide with those previously reported. this relationship is probably related to the commercial exchange of pigs between the us and mexico and the geographical proximity of these two countries. porcine deltacoronavirus (pdcov) is an emerging virus that was recently described. this viral disease together with porcine epidemic diarrhoea (ped) have caused a significant economic impact due to the high mortality rate in piglets (jung, hu, & saif, ; zhang, ) . pdcov belongs to the recently classified subgenus buldecovirus of the deltacoronavirus genus (ictv, ) . the subfamily orthocoronavirinae is divided into four genera, alphacoronavirus, betacoronavirus, gammacoronavirus and deltacoronavirus. the first two genera originate in bats, while the third and fourth genera originate in birds (woo et al., ) . these viruses can affect several species of mammals and birds and cause various clinical conditions (neurological, digestive and respiratory) (wang, byrum, & zhang, ) . coronaviruses are unique within rna viruses because their genome is particularly large (~ . to ~ kb). these enveloped viruses, with a single chain in the ′- ′ direction and a helical nucleocapsid, contain at least seven open reading frames (orf), which code for four structural proteins (masters, ; sawicki, ). coronaviruses received their name due to one of their surface proteins, which is spicule-shaped, giving the appearance of a crown. this so-called s or "spike" protein is glycosylated and plays a fundamental role in the binding and entry of the virus into the cell (masters, ) . the genome organization of pdcov is in the following order: ′ untranslated region (utr), replicase (orf ab), spike gene (s), envelope gene (e), membrane gene (m), nonstructural gene (ns ), nucleocapsid gene (n), ns gene and ´utr. particularly, pdcov lacks orf and non-structural protein (lee & lee, ; woo et al., ; zhang & yoo, ) . in pigs, at least six coronaviruses are known to cause diseases. these belong to three of the four known genera. within the alphacoronaviruses are the transmissible gastroenteritis virus (tgev), the porcine epidemic diarrhoea virus (pedv), the porcine respiratory coronavirus and the hku -related bat coronavirus, which were described in china in related to acute diarrhea in pigs . in the betacoronavirus, there is the porcine hemagglutinating encephalomyelitis virus, and finally, in the genus of the deltacoronaviruses, there is the newly described porcine deltacoronavirus (pdcov) ictv, ) . kong in samples collected in . this study aimed to determine the variability of bat coronaviruses due to the importance that the coronaviridae family has in hong kong since the presentation of acute respiratory diseases in humans. in this work, pigs did not present an apparent clinical sign; however, pdcov was detected (woo et al., ) . subsequently, in early , pdcov was reported from the united states of america (usa) and canada, and it caused heavy economic losses to the swine industry due to the presentation of a clinical enteric disease song et al., ; wang et al., ) . the described infection was indistinguishable from that caused by pedv or tgev. however, the first reports in the usa mentioned that the mortality in piglets was lower ( %- %) than that normally observed in outbreaks of ped (marthaler, raymond, et al., ; wang et al., ) . experimental studies in piglets show that the most frequent signs related to pdcov are watery diarrhoea, vomiting and dehydration (ma et al., ) . the authors note that pdcov infections are common in pigs and that coinfections are frequent, especially with the porcine epidemic diarrhoea virus and rotavirus c (hu et al., ; marthaler, raymond, et al., ; song et al., ) . likewise, some authors speculated that coinfections are likely to cause higher mortality rates (song et al., ) . despite the impact that the disease has had in several pig-producing countries, until this year, we did not know about its presence or the genetic characteristics of the virus in mexico. the objective of this work was to identify the presence of this virus in mexico and to analyse the genomic sequence of the s gene (spike). to monitor the prevalence and sequence properties of pdcov in mexico, porcine rectal swabs were collected from five different regions of the country from to . the sampling locations are shown in figure . these samples were preserved at − °c until use, later they were diluted : with x pbs (ph . ) and then centrifuged at , rpm for min. once the sample was centrifuged, the supernatant was collected and the total rna was extracted using a commercial kit following the manufacturer's recommendations (kit qiaamp viral rna. qiagen cat. ). the resulting yield of rna extracted was µl at an average concentration of ng/µl. rt-qpcr was carried out to address the frequency of pdcov monoinfection and coinfection(s) with pedv and tgev. we used the vetmax™ pedv/tgev/sdcov kit (applied biosystems cat. a ) following the manufacturer´s recommendations. to amplify the s gene, rna from the samples that were positive by rt-qpcr, was reverse-transcribed (rt) using the reverse primer ′cactatgtctgacgcagaag ′ and superscript ii (invitrogen, san diego, ca). the rt products were then used to perform pcr using primers specifically targeting the s gene of the cdna obtained from the s gene amplification was subjected to sequencing using the ion personal genome machine platform (ion torrent thermo fisher scientific) following the supplier's specifications for dna sequencing. the obtained readings were filtered with the fastqc plug-in v . . . , selecting only those with a q score ≥ ( , , reads); the obtained depth was greater than ×. the alignment of the sequences obtained from the amplification of the s gene was performed using the clustal w program in mega software (kumar, stecher, & tamura, ) . for the construction of the tree, the neighbour-joining distance-based method was used, and the bootstrap analysis consisted of , repetitions using the same software. to predict the epitopes, the s protein was analysed with protean, dnastar v. . (madison, wi, ee. uu.). the jameson-wolf method predicted the potential antigenicity sites, which were compared with those reported by mai, k and collaborators based on the sequence hku - (genbank accession no. afd . ). likewise, the surface properties, including hydrophobicity, accessibility and flexibility, were analysed by the kyte-doolittle, plot-emini and karplus-schulz methods respectively also in protean software. furthermore, the positions of the epitopes were predicted using the online service http://www.cbs.dtu.dk/services/bepipred/. the modelling of the amino acid sequence of the protein was carried out in swiss-model (https://swissmodel.expasy.org/). the glycosylation sites were analysed with netnglyc . server (http://www.cbs.dtu. dk/services/netnglyc). pdcov infections in mexico have probably not been adequately addressed due to the high prevalence ped in the country since . pig farms have experienced sporadic outbreaks of diarrhoea in pigs less than weeks old, often without determining the causative agent, while assuming that the causative agent is pedv, possibly masking the presence of deltacoronavirus. a total of samples were analysed from five different regions of mexico, of which ( . %) were pdcov positive (table , figure ). the most common coinfection was pdcov/pedv, found in . % of the total deltacoronavirus-positive cases ( / ), a result that coincides with that reported by other authors (song et al., ; zhang, figure ). in addition, it is interesting that a considerable number of samples ( / ) were positive for the three viral agents studied (pdcov/pedv/tgev). the s protein has the same structure in all coronaviruses. it is the most studied protein due to the role it plays in binding to the receptor and thus determining tropism, in addition to mediating the fusion of membranes for entry of the virus into the cell shang et al., ) . within the coronaviruses, the genus deltacoronavirus is the one with the smallest s protein; however, the structure is the same; it is divided into two subunits, s ( - aa) and s ( - aa) (thachil, gerber, xiao, huang, & opriessnig, ) . and ohio / (genbank accession no. aib ), which have structures known by electron microscopy (shang et al., ; xiong et al., ) . we observed the mutation of amino acids at six sites ( e/d, n/k, ni, kn, il, av) . we built a phylogenetic tree using the neighbour-joining method with sequences from all countries that are available in genbank. three main groups were formed. in the first group are sequences from china (in green, figure ) and in the second, sequences from thailand, laos and vietnam are grouped (in orange). finally, in the third group, we found sequences from the us, japan and south korea we observed that at positions to , a amino acid insertion (n) increased the antigenic index, surface probability and hydrophilic level (figure c and figure s ) (mai et al., ) . likewise, glycosylations were located throughout the s region, similar those predicted by xiaoli, x in to with the illinois strain ( figure a ). knowing the glycosylation sites is important in the study of the antigenicity of the virus because the sites are part of a viral strategy to evade the host immune system (shang et al., ) . we know that the immune response of b cells is against the spike protein (mai et al., ) ; however, deeper studies would be necessary to evaluate whether these predictions coincide with the real antigenicity and pathogenicity of the protein. in conclusion, we determined that porcine deltacoronavirus occurs in mexico and that it is frequently associated with other pathogens, mainly pedv and tgev. to date, recombinations of pdcov with other coronaviruses have not been reported, but the recombinant capacity of these viruses is known, as demonstrated by the event reported in italy, where porcine enteric coronavirus (secov) was found. the genome of the s gene of this virus has greater homology with the ped virus, while the rest of its genome has homology with the tge virus (boniotti et al., ) . likewise, the mexican strain is phylogenetically closer to those strains reported in the us. this supports the theory of the current global distribution of porcine deltacoronavirus. further analysis of the structure and changes found in the s protein of the pdcov from mexico are necessary to determine its degree of pathogenicity and antigenicity. porcine epidemic diarrhea virus and discovery of a recombinant swine enteric coronavirus pathogenicity and pathogenesis of a united states porcine deltacoronavirus cell culture isolate in -day-old neonatal piglets isolation and characterization of porcine deltacoronavirus from pigs with diarrhea in the united states virus taxonomy: release ictv porcine deltacoronavirus infection: etiology, cell culture for virus isolation and propagation, molecular epidemiology and pathogenesis mega : molecular evolutionary genetics analysis version . for bigger datasets complete genome characterization of korean porcine deltacoronavirus strain kor/knu - broad receptor engagement of an emerging global coronavirus may potentiate its diverse cross-species transmissibility origin, evolution, and virulence of porcine deltacoronaviruses in the united states the detection and phylogenetic analysis of porcine deltacoronavirus from guangdong province in southern china porcine deltacoronavirus from the united states rapid detection, complete genome, and phylogenetic analysis of porcine deltacoronavirus (technical appendix) the molecular biology of coronaviruses coronavirus genome replication cryo-electron microscopy structure of porcine deltacoronavirus spike protein in the prefusion state newly emerged porcine deltacoronavirus associated with diarrhoea in swine in china: identification, prevalence and full-length genome sequence analysis development and application of an elisa for the detection of porcine deltacoronavirus igg antibodies detection and genetic characterization of deltacoronavirus in pigs discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavi glycan shield and fusion activation of a deltacoronavirus spike glycoprotein fine-tuned for enteric infections porcine deltacoronavirus: overview of infection dynamics, diagnostic methods, prevalence and genetic evolution immune evasion of porcine enteric coronaviruses and viral modulation of antiviral innate signaling fatal swine acute diarrhoea syndrome caused by an hku -related coronavirus of bat origin first report and phylogenetic analysis of porcine deltacoronavirus in mexico the authors declare no financial or commercial conflicts of interest. rene segura-velázquez https://orcid.org/ - - - josé ivan sánchez-betancourt https://orcid. org/ - - - susana mendoza-elvira https://orcid.org/ - - - key: cord- - sn d r authors: lee, yun ha; jang, yo han; kim, young-seok; kim, jinku; seong, baik lin title: evaluation of green tea extract as a safe personal hygiene against viral infections date: - - journal: j biol eng doi: . /s - - - sha: doc_id: cord_uid: sn d r background: viral infections often pose tremendous public health concerns as well as economic burdens. despite the availability of vaccines or antiviral drugs, personal hygiene is considered as effective means as the first-hand measure against viral infections. the green tea catechins, in particular, epigallocatechin- -gallate (egcg), are known to exert potent antiviral activity. in this study, we evaluated the green tea extract as a safe personal hygiene against viral infections. results: using the influenza virus a/puerto rico/ / (h n ) as a model, we examined the duration of the viral inactivating activity of green tea extract (gte) under prolonged storage at various temperature conditions. even after the storage for days at different temperatures, . % gte completely inactivated ( ) pfu of the virus ( log( ) reduction), and . % and . % gte resulted in log( ) reduction of the viral titers. when supplemented with % citric acid, . % sodium benzoate, and . % ascorbic acid as anti-oxidant, the inactivating activity of gte was temporarily compromised during earlier times of storage. however, the antiviral activity of the gte was steadily recovered up to similar levels with those of the same concentrations of gte without the supplements, effectively prolonging the duration of the virucidal function over extended period. cryo-em and dls analyses showed a slight increase in the overall size of virus particles by gte treatment. the results suggest that the virucidal activity of gte is mediated by oxidative crosslinking of catechins to the viral proteins and the change of physical properties of viral membranes. conclusions: the durability of antiviral effects of gte was examined as solution type and powder types over extended periods at various temperature conditions using human influenza a/h n virus. gte with supplements demonstrated potent viral inactivating activity, resulting in greater than log( ) reduction of viral titers even after storage for up to two months at a wide range of temperatures. these data suggest that gte-based antiviral agents could be formulated as a safe and environmentally friendly personal hygiene against viral infections. virus infections continue to pose major public health concerns with the possibility of epidemics and pandemics [ ] . a recent outbreak of ebola virus in and the global circulation of the pandemic influenza of swine origin (ph n ) in resulted in numerous deaths and hospitalizations [ , ] . middle east respiratory syndrome (mers) transmitted from camels to humans, and the human to human infections are being amplified in nosocomial setting in addition to direct household or community-wide transmissions [ , ] . in addition, although probably less deadlier than ebola virus, a foodborne noroviral infection causes gastroenteritis in humans, claiming millions of illnesses and hospitalizations, with occasional mortalities [ ] . most of emerging viral diseases are zoonotic in nature, posing unmet needs for personal hygiene and disinfectants for both humans and livestock. control and preventive actions against viral infections in human comprises vaccination, antiviral treatment, and personal hygiene. while vaccination and antiviral drugs are often considered very effective for preventing and controlling viral infections, personal hygiene provides much cost-effective alternatives to preventing such infections [ , ] . in general, personal hygiene usually involves hand washing with soap or a hand sanitizer, mostly alcohol-based, due to inhibitory activities of alcohol against viruses [ , ] . however, due to rapid evaporation upon air exposure, a short incubation with alcohol ( - %) was found ineffective in reducing the viral activity [ ] . in addition, there was a concern of fire safety and toxicity for the use of high alcohol content in hand sanitizers [ ] . furthermore, the united states center for disease control and prevention (cdc) reported that alcohol-based hand sanitizer use was a risk factor in long-term care facilities for norovirus [ ] . meanwhile, alternative hand sanitizers based on quaternary ammonium compounds have been used, but their toxicity and environmental concerns have been raised for its use in personal hygiene [ , ] . therefore, effective and environmental friendly compounds for sanitizers have been sought for safe human use to prevent microbial infections [ , ] . green tea (camellia sinensis) has been known to confer many benefits to human health [ ] [ ] [ ] [ ] . the polyphenolic catechins in green tea are composed of epigallocatechin- -gallate (egcg), epigallocatechin (egc), epicatechin-gallate (ecg), and epicatechin (ec) (fig. ) , among which, egcg is most abundant and exerts a wide range of physiological and pharmacological activities. most prominently, egcg exhibits antiviral activity in vitro against a variety of viruses of retroviridae, orthomyxoviridae, and flaviviridae, including important human infectious pathogens such as human immunodeficiency virus (hiv), influenza a virus, and hepatitis c virus [ ] . egcg potently exerts inhibition of influenza virus replication [ , ] , interferes with hcv entry [ , ] , and inactivates herpes simplex virus (hsv- ) and hsv- at acidic and neutral phs [ , ] . in addition, egcg was shown to block the enzymatic activity of the hiv- reverse transcriptase [ ] . one of proposed mechanisms for the antiviral activity of egcg is the covalent modification of proteins by egcg by autoxidation process, in which egcg is oxidized to form egcg quinone by autoxidation, which in turn can react with the nucleophilic thiol group of a cysteine residue to form egcg-protein complex [ ] . furthermore, it has been proposed that the autoxidation of catechins involves oxygen radical and molecular oxygen [ ] . although numerous previous studies have investigated antiviral activities of green tea extract or purified catechin components against viruses, there have been few reports with respect to developing disinfectants of public and personal hygiene. for practical use at home and in the field, a long-term stability at various working conditions is essential. toward finding a safe and effective hygiene agent against viruses, we evaluated the durability of antiviral effects of green tea extract (gte) as a powder type and a solution type over extended periods at various temperature conditions using human influenza a/h n virus. the effect of antioxidant additive such as ascorbic acid further suggests that the virucidal function is mediated by the prooxidant activities of catechins. mdck (madin-darby canine kidney) were obtained from atcc (american type culture collection) and cultured in mem (minimal essential medium, hyclone) supplemented with % fbs (fetal bovine serum, hyclone), at °c in % co . influenza virus a/puerto rico/ / (h n ) (pr ) viruses were propagated in day-old chicken embryos. the allantoic fluids were harvested and filtered by a syringe filter with a pore size of . μm and stored at a freezer (− °c) until use. green tea extract (gte) was provided as powder form from amore-pacific co, korea as described previously [ ] . briefly, green tea leaves were infused with °c distilled water in the ratio of : (w/w). after min of infusion, the tea extract was quickly separated from the tea leaves by filtration and the green tea extract (gte) was freeze-dried. the composition was analyzed by c reverse phase column chromatography (elution with % thf at the flow of ml/min). the gte was composed of caffeine ( . %), gallic acid ( . %), gc ( . %), egc ( . %), catechin ( . %), egcg ( . %), ec ( . %), and gcg ( . %), as determined by hplc. gte solution was prepared by adding water to the powder and filtering it through a . μm syringe filter. gte powder was dissolved in distilled water to make % ( mg/ml) solution and serially diluted to make . %, . %, . %, and . % solutions. the solutions were stored at various temperatures ( °c, °c, and °c), and aliquots were taken at predetermined time-points ( hours (h), h, day (d), d, d, d, d, d, and d) and immediately kept frozen at − °c until use. equal volume of pr virus ( pfu dissolved in pbs) was added to the gte solutions and incubated for six h at °c before determining the viral titers. residual viral titers were determined by plaque assay on mdck cells at °c, as described previously [ , ] . mdck cells in -well culture plates were infected with μl of -fold serially diluted virus samples and incubated for h on a shaker at room temperature (rt). the inoculums were removed and the cells were overlaid with medium containing % low-melting agarose, dmem, and μg/ml trypsin. the -well plates were incubated at °c in % co incubator. after three days of the incubation, the virus plaques were counted. the gte power was stored at °c and was taken at , , , and weeks and kept at − °c until use. the gte powders were dissolved in distilled water to make % ( mg/ml) solution and diluted to . % concentration. then, equal volume of pr virus solution ( pfu/ml) was added, and the mixture was further incubated for six h at °c. residual viral titers were determined by plaque assay on mdck cells at °c. antiviral effect and stability of gte with supplements gte solutions ( . , . , and . %) were supplemented with % citric acid, . % sodium benzoate, and . % ascorbic acid to make gte-mix. the gte-mix solutions were stored at °c and °c for up to days. the gte-mix taken at various time-points were incubated with pfu of pr virus at °c for six h for viral inactivation. residual viral titers were measured by plaque assay on mdck cells. to determine the chemical stability of green tea catechins, . % gte and gte-mix were stored at °c for up to days. aliquots were taken at pre-determined time-points and were kept frozen at − °c until use. the concentrations of catechins at various time-points were analyzed by gas chromatography (gc) with a hewlett-packard (hp) column equipped with a fid detector, using helium as a carrier gas. identification and quantification of the gc peaks was accomplished by gc/ms analysis with an agilent hp gc/mass spectrometer (agilent technologies, usa). the size distribution of influenza virus treated with gte or pbs was measured by dynamic light scattering (dls). × pfu/ml of pr virus was treated with . % gte or pbs for h at °c. after the treatment, the virus samples were applied to the particle size & zeta potential anlayzer (els- zs, otsuka electronics, japan). the size distribution was measured twice with accumulation time in pbs solvent at °c. the morphology of the virus treated with gte was analyzed by cryo-electron microscopy (cryo-em). purified influenza viruses were loaded onto the formvar film copper grid (electron microscopy sciences). after s of sample adsorption, the grid was negative stained with % uranyl acetate. sample vitrification was performed using vitrobot (fei), and the vitrified sample was imaged using a cryotecnai f transmission electron microscope (fei). first, we examined how long the viral inactivation activity of gte was maintained after storage in different conditions. various concentrations of gte solutions were stored at °c, °c, and °c for h to days, and the gte solutions stored for different times were tested for their viral inactivating activities against an influenza virus a/puerto rico/ / (h n ) (pr ). the results showed that . % and . % gte solutions, stored at °c and °c as long as days, maintained potent viral inactivating activities, completely removing the viral plaque-forming ability of pfu of the viruses (fig. a & b) . . % gte solutions stored for days at °c and °c also demonstrated potent viral inactivating activity, resulting in approximately log reduction of viral titers. . % gte solutions exhibited reduced viral inactivating activity following the storage more than days at °c and °c, but still could inactivate the viruses (> log reduction) even after days of storage ( fig. a & b) . . % gte solution stored at °c also showed potent inactivating activity, although the complete removal of pfu viruses was not achieved when the gte solution was stored for days (fig. c) . . % gte solutions stored for less than days and . % gte solutions stored for less than days at °c completely inactivated the viruses, but their activities decreased along storage time (fig. c) . the inactivating activity of . % gte solution was reduced by storage at °c, as compared to those at °c and °c, in which complete inactivation was observed by the same concentration of gte. . % gte stored at °c demonstrated constant levels of weak inactivating activities (< log reduction), irrespective of storage times (fig. b) . the data showed that gte was able to inactivate the influenza virus in a concentrationdependent manner. of note, the potency of inactivating activity was gradually weakened following the storage more than days, especially at lower concentrations of gte ( . % and . %). however,~ log reduction of viral infectivity was still maintained over days of incubation. therefore, it can be concluded that the viral inactivating activity of gte solution over . % were robust and stable for at least two months as a solution type disinfectant even at hot weather conditions like in summer. we further examined the maintenance of viral inactivating activity of gte stored in dried powder. gte powder was stored at °c for up to weeks, and the gte powder taken at different time-points was dissolved in distilled water to make a . % gte solution. the . % gte solutions were incubated with pfu of pr virus at °c for six h for viral inactivation. the results showed that, despite the storage for weeks, gte powder was able to inactivate the virus completely (fig. d) , indicating that the antiviral effects of gte powder could be stably maintained over prolonged storage. based on the previous report that ascorbic acid stabilized green tea catechins [ ] , we examined the effects of the addition of common food preservatives such as ascorbic acid, citric acid, and sodium benzoate on the viral inactivating activity of gte. . %, . %, and . % of gte solutions were supplemented with % citric acid, . % sodium benzoate, and . % ascorbic acid (gtemix), and the mixtures were stored at °c or °c for six h- days. the gte-mix solutions taken at various time-points were incubated with pfu of pr virus for six h at °c for viral inactivation. the results show that, on a short-term period up to one week, the potency of the antiviral activity was compromised, but still enabled the reduction of the viral titers by - log depending on gte-mix concentrations. the reduction of the antiviral potency was more apparent at lower temperature of °c (fig. a) , and at lower concentrations of gt-mix of . % and . % (fig. a, b) . the data also showed that, in the presence of an antioxidant such as ascorbic acid, the potency of gte-mediated antiviral activity was significantly reduced. for example, the antiviral activities of . % gte-mix solutions stored at both temperatures were not as strong as . % gte that completely inactivated the virus (fig. a-c) . likewise, . % and . % gte-mix solutions also demonstrated considerably compromised antiviral activities, as compared to the same concentrations of gte solutions (fig. b & c) . the reduced antiviral activities of gtemix solutions were more pronounced when stored for relatively short periods less than one week. interestingly, however, the antiviral activities of . % gte-mix stored at °c became complete after day of storage, and . % and . % gte-mix also steadily restored their antiviral activities at days to the similar levels with the same concentrations of gte (figs. b and a) . the similar phenomena were observed in the gte-mix solutions stored at °c. all the gte-mix solutions stored for less than days exhibited reduced antiviral activities but restored their activity to the similar levels to those of the same concentrations of gte solutions at days, and, notably, remained potent up to days (figs. c and b). thus, storage time-dependent inactivating activities of gte-mix solutions were in clear contrast to those of gte solutions, where the initial antiviral strength was high for up to two weeks but exhausted over prolonged storage (fig. ) . none of the individual supplements nor their mixture showed inactivating fig. maintenance of viral inactivation activity of gte solution with supplements. . , . , and . % gte solutions were incubated at °c (a) or °c (b) in the presence of % citric acid, . % sodium benzoate, and . % ascorbic acid (gte-mix). the gte-mix solutions incubated for different times were mixed with pf of pr virus for six h at °c for viral inactivation. residual viral titers were determined by plaque assay on mdck cells. as a control, the virus was incubated with pbs, and the viral titer was indicated by upper dashed lines. c as another controls, the virus was treated with the individuals of the supplement or with the mixture of three agents. data are the mean of two independent experiments. detection limit of plaque assay is , and error bars indicate the sd activity against the viruses (fig. c) , indicating that the inactivation was solely due to the gte. the results suggest that antioxidants such as ascorbic acid, although compromising the initial antiviral strength, could be used for prolonging the shelf-life of gte-based antiviral disinfectant. we further examined whether the addition of supplements (citric acid, sodium benzoate, and ascorbic acid) influenced the chemical stability of green tea catechins that were known to exert the antiviral activity. gte and gte-mix solutions were stored at °c for up to days, and the concentrations of catechins in the solutions were determined by gc/ms analysis. the catechins were stably maintained regardless of the presence of the supplements (fig. a) , showing that the addition of the supplements did not affect the catechins stability in the gte solution. next, we monitored the kinetics of viral inactivation of gte in the presence of the supplements. based on the result in fig. a , we further compared the inactivation kinetics between gte and gte-mix solutions. . % of gte and gte-mix solutions were stored at °c for day, and the solutions were then further incubated with pfu of pr virus at °c for - min for viral inactivation. the viral titers in the mixtures taken at different time-points were determined by plaque assay to observe the kinetics of time-dependent viral inactivation. viral inactivation by gte solution was rapid, resulting in greater than log reduction of viral titers within five mins after the incubation, and a complete inactivation was achieved after mins after the incubation (fig. b) . on the other hand, gte-mix solution resulted in only log reduction of the viral titers within mins, and complete inactivation was achieved after mins of the incubation (fig. b) , clearly showing that supplements added to the gte solution interfered with catechinmediated viral inactivation. taken together, the addition of the supplements to gte solution resulted in the delay of the exhaustion of the inactivating activity of gte, thus enabling a long-term maintenance of its antiviral function. finally, we examined whether gte treatment affects the morphological properties of the influenza virus. the pr virus was treated with . % gte or pbs and the virus particles were observed under cryoelectron microscope (cryo-em). as shown fig. a , the treatment of gte did not result in noticeable changes in particle sizes and the shape of the viruses, as compared with pbs control (fig. ) . furthermore, the size distribution of the virus particles was measured using dynamic light scattering (dls) system. pr virus treated with pbs showed the highest intensity at particle diameter of . nm (fig. b) . gte treatment resulted in the highest intensity at particle diameter of . nm, . nm, and nm for gte concentration of . %, . %, and . %, respectively, showing that gte treatment increased the viral particle sizes to . − . %. considering that the influenza virus particles typically have a size of − nm [ ] , the increase in the particle sizes by gte treatment does not appears to be significant. catechins may also interact with the viral membranes as well as the viral proteins [ ] . whether the observed changes in the particle size may be associated with the membrane fluidity/integrity, crucially important for viral membrane fusion for infection, remains to be further investigated. fig. effects of supplements on the kinetics of viral inactivation by gte. a chemical stability of green tea catechins. . % gte and gte-mix solutions were stored at °c for up to days. the samples were obtained at various time-points and catechins concentration was measure by gc. b kinetics of viral inactivation by gte and gte-mix. . % gte and gte-mix solutions were incubated at for h at °c. after the incubation, the gte and gte-mix solutions were mixed with pfu of pr virus and further incubated at °c for , , , , , , and mins for viral inactivation. residual viral titers at each time-point were determined by plaque assay on mdck cells. data are the mean of two independent experiments. detection limit of plaque assay is , and error bars indicate the sd the primary goal of the present study was to evaluate gte as potential virucidal disinfectant. we examined the temporal stability of virucidal activity of gte against the human influenza a/h n virus and the effects of antioxidant supplementation on its stability. it has been shown that green tea catechines or green tea extract exert potent antiviral effects against a variety of human infecting viruses including influenza viruses [ , ] . previous studies have mainly focused on the targets and inhibitory actions of green tea catechins at various stages of viral replication cycles [ , , ] . the present data showed that the virucidal effects of gte can be maintained up to eight weeks in a solution type and for weeks in a powder type, respectively, for the entire duration of experiments conducted. a long-term maintenance of virucidal activity could be related to the chemical stability of catechins [ ] . the concentrations of green tea catechins were measured over time and there were no significant differences in the concentrations between gte and gte-mix solutions (fig. a) . the data showed that the catechin concentrations remained constant over the tested period, implying that the additive antioxidant may not directly influence the chemical stability of catechins. the inactivating activities of . % and . % gte steadily decreased as storage time increased (fig. ) . given that the concentration of catechins remained constant over the prolonged storage as shown in fig. a , it is likely that crosslinking efficiency of the catechins to the viral proteins/membranes was decreased during the storage, for which the precise molecular mechanism remains to be elucidated further delicate studies. the potential effect of antioxidants was also evaluated, in the form of mixture (gte-mix) comprising ascorbic acid, citric acid, and sodium benzoate (common food preservatives). it is well documented that ascorbic acid is one of the strongest reductants and acts as an oxygen scavenger [ , ] . although our experimental condition using the ascorbic acid did not perfectly reflect anaerobic condition, our data clearly showed that the ascorbic acid delayed the gte-mediated viral inactivation, most likely through inhibiting the autoxidation of catechins mediated by molecular oxygen. the data suggest that the potency of virucidal activity was compromised for the initial period of a week; the reduction of infectious viral titer for gte and gte-mix was - log and - log , respectively. however, the virucidal activity of gte-mix persisted up to weeks (fig. a, b) , in clear contrast to gte without additives where the virucidal activity become exhausted after two weeks (fig. ) . thus, the combination of gte with ascorbic acid as antioxidant resulted in a sustained virucidal activity for extended period. it should be noted that contrasting activities, pro-oxidant and anti-oxidant, have been documented with green tea catechins [ ] . moreover, it has been proposed that the catechins in green tea crosslink with cysteine residues in a protein through autoxidation process [ , ] . thus, it is likely that a strong antioxidant such as ascorbic acid prevents the oxidation of polyphenolic oh groups of the catechins into quinone, a prerequisite for oxidative crosslinking to viral proteins [ ] . our data show that the viral inactivating activity of gte-mix recovered after days of storage despite the presence of ascorbic acid and other supplements (fig. a, b) . investigation of the chemical or biological status of ascorbic acid as an antioxidant especially in the mixture with gte during the storage could provide a possible explanation for this phenomenon. intriguingly, the antiviral activities of . % gte-mix and those of lower concentrations of gte-mix were differently affected by storage temperatures, as compared to those of gte. after storage at °c, the antiviral activity of . % gte-mix was reduced mildly, whereas those of . % and . % gte-mix were considerably dampened (fig. a) . conversely, after storage at °c, the reduction of antiviral activity was more pronounced in . % gte-mix than those in . % and . % gte-mix (fig. b) . although, the addition of the supplements including antioxidant obviously influenced the changes in the antiviral kinetics, further detailed analyses would be required to address the issue. a recent report suggested that urea supplementation of alcohol-based disinfectants in the presence of citric acid may increase their antiviral effects against a number of viruses [ ] . whether supplementing gte with such components may enhance the stability of antiviral effect of gte remains to be further tested. with promising outcome of long-term stability of virucidal effects of gte, it is likely that potent antiviral activity could be extended to other viruses, only to underscore a general and broader use of gte as an effective disinfectant against viral infections. the antiviral spectrum of gte includes not only enveloped viruses such as influenza virus but also non-enveloped viruses such as parvovirus as well [ ] . the present results could be usefully implemented for the development of the first-hand personal and public hygiene for lessening the medical burden associated with emerging and reemerging viral infections. the durability of antiviral effect of the gte against human influenza virus (h n ) was examined as a powder type and a solution type over extended periods at various temperature conditions. the data revealed that a potent antiviral activity of the gte with about log reduction of viral titers was maintained with wide range of temperatures ( - °c) for two months as solution type, and for prolonged period as power. these data suggest that catechin-based antiviral agents could be formulated as a safe and environmentally friendly personal hygiene against viral infections. when formulated with ascorbic acid, the potency of virucidal activity was temporarily compromised, while the exhaustion of antiviral activity was delayed, prolonging the duration of the virucidal function over extended period. in contrasts to most antiviral agents of synthetic chemicals, gte does not pose any safety concern, but even considered beneficial for human health. besides human use, it could also be used for protecting animals and livestock from viral infections and reducing zoonotic transmissions. the gte could be supplemented with other components for an extended use as environmentally friendly virucidal agents. cryo-em: cryo-electron microscopy; dls: dynamic light scattering; ec: epicatechin; ecg: epicatechin-gallate; egc: epigallocatechin; egcg: epigallocatechin- -gallate; fid: flame ionization detector; gte: green tea extract; gte-mix: gte solution supplemented with % citric acid, . % sodium benzoate, and . % ascorbic acid; pfu: plaque forming unit temporal and spatial analysis of the - ebola virus outbreak in west africa emergence and pandemic potential of swine-origin h n influenza virus unraveling the mysteries of middle east respiratory syndrome coronavirus mers outbreak in korea: hospital-to-hospital transmission norovirus gastroenteritis nosocomial spread of viral disease epidemiologic background of hand hygiene and evaluation of the most important agents for scrubs and rubs effectiveness of common household cleaning agents in reducing the viability of human influenza a/ h n analysis of alcohol-based hand sanitizer delivery systems: efficacy of foam, gel, and wipes against influenza a (h n ) virus on hands effectiveness of liquid soap and hand sanitizer against norwalk virus on contaminated hands virucidal activity of a new hand disinfectant with reduced ethanol content: comparison with other alcohol-based formulations use of alcohol-based hand sanitizers as a risk factor for norovirus outbreaks in long-term care facilities in northern new england sensitization prevalence for benzalkonium chloride and benzethonium chloride comparative study on toxic effects induced by oral or intravascular administration of commonly used disinfectants and surfactants in rats compounds derived from epigallocatechin- -gallate (egcg) as a novel approach to the prevention of viral infections evaluation of the antiviral activity of a green tea solution as a hand-wash disinfectant beneficial effects of green tea: a literature review beneficial effects of green tea-a review the role of tea in human health: an update a new function of green tea: prevention of lifestyle-related diseases anti-infective properties of epigallocatechin- -gallate (egcg), a component of green tea biological evaluation of anti-influenza viral activity of semi-synthetic catechin derivatives inhibition of the infectivity of influenza virus by tea polyphenols −)-epigallocatechin- -gallate is a new inhibitor of hepatitis c virus entry epigallocatechin- -gallate inhibits the replication cycle of hepatitis c virus epigallocatechin gallate inactivates clinical isolates of herpes simplex virus digallate dimers of (−)-epigallocatechin gallate inactivate herpes simplex virus epigallocatechin gallate inhibits the hiv reverse transcription step covalent modification of proteins by green tea polyphenol (−)-epigallocatechin- -gallate through autoxidation kinetic analysis and mechanistic aspects of autoxidation of catechins antiviral effect of catechins in green tea on influenza virus genetic analysis of attenuation markers of cold-adapted x- influenza live vaccine donor strain host defense mechanism-based rational design of live vaccine stabilizing effect of ascorbic acid on green tea catechins orthomyxoviridae: the viruses and their replication additional inhibitory effect of tea extract on the growth of influenza a and b viruses in mdck cells inhibition of epstein-barr virus lytic cycle by (−)-epigallocatechin gallate the green tea polyphenol, epigallocatechin- -gallate, inhibits hepatitis c virus entry esr study on the structureantioxidant activity relationship of tea catechins and their epimers effectiveness of ascorbic acid as an oxygen scavenger in improving viability of probiotic bacteria in yoghurts made with commercial starter cultures action of ascorbic acid as a scavenger of active and stable oxygen radicals the antioxidant and pro-oxidant activities of green tea polyphenols: a role in cancer prevention synthesis and structure identification of thiol conjugates of (−)-epigallocatechin gallate and their urinary levels in mice the effect of ascorbic acid on total antioxidant activity of black and green teas development and virucidal activity of a novel alcohol-based hand disinfectant supplemented with urea and citric acid disinfection efficacy against parvoviruses compared with reference viruses the authors acknowledge dr. byung min lee at korea research institute of chemical technology (krict) for gc analysis and yucheol cheong at yonsei university for technical assistance. availability of data and materials all data generated or analyzed during this study are included in this published article. authors' contributions jk and bs conceived and designed experiments. yl and yj analyzed and interpreted the data regarding viral inactivating activity of gte. yk performed dls analysis and obtained cryo-em images. yj, jk, and bs wrote the manuscript. all authors read and approved the final manuscript.ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- - syjr k authors: kanno, toru; ishihara, ryoko; hatama, shinichi; uchida, ikuo title: a long-term animal experiment indicating persistent infection of bovine coronavirus in cattle date: - - journal: j vet med sci doi: . /jvms. - sha: doc_id: cord_uid: syjr k a long-term animal experiment involving inoculation with bovine coronavirus (bcov) was conducted to verify its persistent infection in cattle. three colostrum-deprived holstein calves were housed separately in individual rooms of a high-containment facility and inoculated with the bcov strain kumamoto/ / . until the end of the experiment ( , , and days, respectively), viral rnas were detected sporadically by rt-pcr and nested pcr from plasma, nasal discharge, and feces. seroconversion and titer changes were validated by hemagglutination inhibition tests and neutralization tests. among the samples, nasal discharge showed a higher viral positivity than feces, which seemed to be associated with positive detection in the plasma. these data demonstrate the existence of persistent infection of bcov in the respiratory tissues of cattle. bovine coronavirus (bcov) is a member of the order nidovirales, family coronaviridae, subfamily coronavirinae, genus betacoronavirus, species betacoronavirus- . this species also includes human coronavirus oc , porcine hemagglutinating encephalomyelitis virus, equine coronavirus, and canine respiratory coronavirus [ ] . bcov infects the respiratory and digestive organs of cattle and causes neonatal calf diarrhea, bloody diarrhea in adult cattle (winter dysentery), and respiratory symptoms, including shipping fever in feedlot cattle [ , , ] . bcovs have spread widely across cattle farms all over the world, and thus, nearly all adult cattle have antibodies against the virus. outbreaks typically occur during autumn and winter [ ] and are associated with the housing period; however, several cases have also been reported during warmer seasons [ , , ] . although the mortality of this disease is low, it causes substantial economic losses owing to a reduction in milk production in dairy farms [ , ] and meat production in beef farms [ , ] . the main transmission route of bcovs is horizontal infection, i.e., ingestion or inhalation of virus from feces or nasal discharges into the mouth or nasal cavity [ , ] . there has been no report of vertical infection so far. bcov antigens are detected in the feces of clinically healthy cattle [ ] , and studies on bcov shedding in feces showed the virus to be detected over a long period on farms, even though the number of cattle shedding the virus decreased [ ] . this suggests the existence of persistently infected cattle, which might also be the origin of transmission. to verify this hypothesis, we conducted a longitudinal animal experiment. the bcov used in this experimental study was obtained from a clarified suspension of feces from a cow on a farm where a bcov case had been confirmed in by using hrt- g cells (isolate kumamoto/ / ) [ ] . the culture supernatant of the isolate was inoculated into cattle by the oral route. feces were collected when the cattle showed diarrhea. the isolate was passaged in colostrum-deprived holstein calves twice, and feces of the infected calves were harvested as inoculum for this study (data not shown). three colostrum-deprived holstein calves were used in this study. each animal was maintained in a separate room of a highcontainment facility at our institute. in this facility, the animal room area is separated from the preparation room by a shower room area. to enter the animal room area, the investigator undresses and puts on work clothes and boots in this area. the animal room area has independent animal rooms and a corridor. each animal room has a ventilator with hepa filers to prevent the doi: . /jvms. - reintroduction of the virus from outside to the experimental cattle. during the animal experiment, we adhered to the strict high hygiene and biosecurity measures to prevent reinfection from other cattle. to enter the animal room, boots were removed at the corridor, and the "inside" boots and waterproof clothes kept in the animal room were worn. to exit the room after husbandry and sampling, put off the boots and waterproof clothes were taken off after disinfection with sodium hypochlorite using a spraying device. the animal care and use committee of the national institute of animal health approved all animal procedures prior to the initiation of this study (authorization number: - ). one calf ( days old, referred to as "calf ") was inoculated orally using a catheter that delivered a sample of the centrifuged supernatant at , × g for min from a mixture of g feces as mentioned above and ml phosphate-buffered saline (pbs) containing µg/ml gentamicin into the esophagus. samples of nasal discharge, feces, plasma, and sera were collected daily until days post inoculation (dpi), followed by weekly collection until dpi and then twice-weekly collection until the end of the experiment ( , dpi). the nasal discharge samples were collected by inserting cotton into the nasal cavity for a few min and extracting the discharge using sterilized disposable syringes. feces were collected directly from the rectum and prepared as a % suspension in pbs containing µg/ml gentamicin and µg/ml trypsin for virus isolation and rna extraction. the same experiment was carried out on another calf ( days old, "calf ") using the centrifuged supernatant from a mixture of ml pbs and g feces of calf collected at dpi (after it showed diarrhea). samples of nasal discharge, feces, plasma, and sera were collected daily until dpi, followed by weekly collection until dpi and twice-weekly collection until the end of the experiment ( dpi). the same experiment was carried out on yet another calf ( days old, "calf ") using centrifuged supernatant from a mixture of ml pbs and g feces of calf collected at dpi, when it showed diarrhea. nasal discharge, feces, plasma, and serum samples were collected daily until dpi, followed by weekly collection until dpi and twice-weekly collection until the end of the experiment ( dpi). viral rna was extracted from plasma, nasal discharge, and % fecal suspensions in pbs using the high pure viral rna kit (roche diagnostics, tokyo, japan) according to the manufacturer's instructions. bcov-specific genes were detected from the extracted rna by the rt-pcr assay using a titan one tube rt-pcr kit (roche diagnostics), with the following thermal cycling profile: min at °c; min at °c; cycles of denaturation at °c for sec, annealing at °c for sec, and extension at °c for sec; and completion of amplification with a -min extension step at °c [ ] . the oligonucleotide primers used for nested pcr were designed from the nucleotide sequence of the kakegawa strain (genbank accession no. ab ) in this study. the primers were as follows: bcov-nu, ʹ-tgctacttctcagcaaccatcag- ʹ (nt , - , , sense primer), and bcov-nd, ʹ-ttggcatgcggtcctgttccaag- ʹ (nt , - , , antisense primer). the size of the amplification fragment is bp. nested pcr was performed by using takara ex taq (takara-bio., kusatsu, japan), and the thermal cycling profile was as follows: min at °c; cycles of denaturation at °c for sec, annealing at °c for sec, and extension at °c for sec; and completion of amplification with a -min extension step at °c. to prevent laboratory contamination in rt-pcr and nested pcr, we set up mixtures for these reactions in a laminar flow cabinet equipped with an uv lamp, wore fresh gloves at each step, used pipette tips with aerosol filters. negative controls were also included in every pcr experiment to detection of contamination. the rt-pcr and nested pcr products were visualized on . % agarose gels stained with ethidium bromide. the hemagglutination inhibition (hi) test was conducted by the microtiter method [ ] . sera were treated with kaolin and chicken erythrocytes and heat inactivated. as antigen for the hi test, the kakegawa strain was used in this assay because the hemagglutinating (ha) activity of the kumamoto/ / strain was low and unstable with chicken and mouse erythrocytes, similar to recent japanese isolates (kanno et al., unpublished data), whereas the ha activity of the kakegawa strain was sufficient. briefly, twofold dilutions of serum were prepared in duplicate and reacted with the virus. the hi titers were expressed as the reciprocal of the highest serum dilution that completely inhibited the ha activity of ha units of the virus. serum samples were also tested in a virus neutralization test by preparing serial twofold dilutions of serum were reacted in duplicate with of % tissue culture infective doses (tcid ) of kumamoto/ / , followed by incubation for hr at °c. after incubation, the serum-virus mixture was transferred onto monolayered human rectal tumor cells (hrt- g) cultured in microplates and incubated for days at °c. the neutralization antibody titers were expressed as the reciprocal of the highest serum dilution that completely inhibited the cytopathic effect (cpe). all calves showed clinical signs, such as loose or diarrheal stool, up to - dpi. viral rna was detected from the plasma during the period of onset (fig. a-c) . after recovery from the disease, viral rna was sporadically detected by rt-pcr and nested pcr from plasma. final detection days (last day when a sample showed a positive result) by rt-pcr were , , and dpi, and by nested pcr, , , , and dpi, respectively, in the calves. as with the plasma, viral rna was detected from nasal discharge on the basis of viral titers, and was detected sporadically by pcr-based experiments. final detection days by rt-pcr were , and dpi, and by nested pcr, , and dpi, respectively, in the calves. viral rna was not detected from the feces after the disease onset in calves and . however, nested pcr was positive for the virus at dpi in calf . in calf , viral rna from feces was detected sporadically by the pcr-based experiments. in this calf, the final detection day was dpi by rt-pcr and , dpi by nested pcr. virus isolation was conducted from the samples showing positive results by rt-pcr and nested pcr. the samples were inoculated into hrt- g monolayer cells in -well plates and incubated for days at °c and % co . if a cpe was not observed in the incubation period, passaging into fresh cells was conducted twice using the inoculum after freeze-thawing the cultures thrice. however, the virus was not isolated, except in the samples from the period of onset. the hi and neutralization titers in the calves increased several days after disease onset and peaked at , and weeks post doi: . /jvms. inoculation, and at , and weeks post inoculation, respectively, in the calves. thereafter, the titers gradually decreased. however, both titers showed a rapid rise at dpi in calf ( and , respectively) (fig. a ). ten days before the sampling, calf showed diarrhea at and dpi (data not shown). therefore, we surmised that the virus was reactivated in the cattle, probably in the respiratory tissues, causing diarrhea followed by this booster effect. the virus in the digestive tract might have been quickly inactivated and excreted; therefore, viral rnas were not detected from nasal discharge and feces at dpi, days after the onset. unfortunately, feces samples had not been collected at the onset, so no virus was isolated. a rapid rise in these titers was also found in calf at dpi, just after the detection of viral rna from the plasma at dpi by both rt-pcr and nested pcr (fig. b) . this finding might also be attributed to virus reactivation in respiratory tissues and quick inactivation by host immunity without clinical signs. this study showed that the bcov rna was long-lasting, having been detected from the nasal discharge of cattle that had been maintained in an isolated room of a high-containment facility to prevent virus intrusion from outside. this indicated the existence of persistent infection of bcov in the respiratory tissues of cattle, although further investigations are warranted because the virus was not isolated. other coronaviruses, such as transmissible gastroenteritis virus, canine coronavirus, and feline coronavirus, have also been reported to cause long-term infection [ , , ] . thus, coronavirus tends to cause persistent infection in host animals. often, calf diarrhea caused by bcov repeatedly occurs at the same farm every year. this might be explained by the presence of persistently infected cattle. conflicts of interest. the authors have no potential conflicts of interest to declare with respect to the research, authorship, and/or publication of this article. ratification vote on taxonomic proposals to the international committee on taxonomy of viruses bovine coronavirus shedding of enteric coronavirus in adult cattle monoclonal antibody capture enzyme-linked immunosorbent assay for detection of bovine enteric coronavirus severe outbreak of bovine coronavirus infection in dairy cattle during the warmer season isolation of bovine respiratory coronaviruses from feedlot cattle and comparison of their biological and antigenic properties with bovine enteric coronaviruses a longitudinal study of bovine coronavirus enteric and respiratory infections in dairy calves in two herds in ohio coronavirus-like particles in the feces of normal cats hemagglutination with nebraska calf diarrhea virus molecular analysis of the s glycoprotein gene of bovine coronaviruses isolated in japan from to phylogenetic studies of bovine coronaviruses isolated in japan association between infection of the respiratory tract attributable to bovine coronavirus and health and growth performance of cattle in feedlots detection and characterization of bovine coronaviruses in fecal specimens of adult cattle with diarrhea during the warmer seasons bovine respiratory coronavirus experimentally induced coronavirus infections in calves: viral replication in the respiratory and intestinal tracts coronavirus isolation from nasal swab samples in cattle with signs of respiratory tract disease after shipping morbidity in swedish dairy calves from birth to days of age and individual calf-level risk factors for infectious diseases risk factors for calf mortality in large swedish dairy herds experimental reproduction of winter dysentery in lactating cows using bcv -comparison with bcv infection in milk-fed calves experimental inoculation of adult dairy cows with bovine coronavirus and detection of coronavirus in feces by rt-pcr recovery of transmissible gastroenteritis virus from chronically infected experimental pigs astrovirus-like, coronavirus-like, and parvovirus-like particles detected in the diarrheal stools of beagle pups acknowledgments. we are grateful to mr. keiji itoh, mr. kazuhiko takase, mr. mitsutoshi noi, mr. yoshihiro himoro, and mr. kiyoshi tanaka for their skilled handling of the animals at the institute of animal health, hokkaido research center. key: cord- -beb k authors: zhang, shuai; hu, weiwei; yuan, lvfeng; yang, qian title: transferrin receptor is a supplementary receptor that assists transmissible gastroenteritis virus entry into porcine intestinal epithelium date: - - journal: cell commun signal doi: . /s - - - sha: doc_id: cord_uid: beb k background: transmissible gastroenteritis virus (tgev), the etiologic agent of transmissible gastroenteritis, infects swine of all ages causing vomiting and diarrhea, in newborn piglets the mortality rate is near %. intestinal epithelial cells are the primary target cells of tgev. transferrin receptor (tfr ), which is highly expressed in piglets with anemia, may play a role in tgev infection. however, the underlying mechanism of tgev invasion remains largely unknown. results: our study investigated the possibility that tfr can serve as a receptor for tgev infection and enables the invasion and replication of tgev. we observed that tgev infection promoted tfr internalization, clustering, and co-localization with tfr early in infection, while tfr expression was significantly down-regulated as tgev infection proceeded. tgev infection and replication were inhibited by occluding tfr with antibodies or by decreasing tfr expression. tgev infection increased in tgev-susceptible st or ipec-j cell lines and tgev-resistant caco- cells when porcine tfr was over-expressed. finally, we found that the tgev s protein interacts with the extracellular region of tfr , and that pre-incubating tgev with a protein fragment containing the extracellular region of tfr blocked viral infection. conclusions: our results support the hypothesis that tfr is an additional receptor for tgev and assists tgev invasion and replication. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. transmissible gastroenteritis virus (tgev), a porcine enteropathogenic coronavirus, can cause watery diarrhea, vomiting, and rapid dehydration. infection with tgev results in nearly % mortality in neonatal piglets [ , ] , and is responsible for enormous economic losses in the swine industry [ ] . tgev is an enveloped virus with a large positive-sense single-stranded rna genome that encodes structural proteins: spike protein (s), envelope protein (e), membrane protein (m), and nucleoprotein protein (n) [ , ] . the structure of the s glycoprotein changes radically as it binds to specific epithelial cell receptors and mediates the fusion of cellular and viral membranes [ ] [ ] [ ] [ ] . significant effort has been made to characterize the cellular receptors for tgev infection. porcine aminopeptidase n (papn), also known as cd , has been identified as a primary cell-surface receptor for tgev [ ] [ ] [ ] [ ] [ ] . our laboratory recently reported that epidermal growth factor receptor (egfr) also promotes clathrin-mediated endocytosis of tgev [ ] . tgev may use a third protein ( kda), currently uncharacterized, for cell entry [ , ] . identification of this receptor would provide additional insight into tgev-mediated disease and define a novel therapeutic target for protecting swine from tgev infection. tfr is a homo-dimeric type ii transmembrane glycoprotein with two apparent kda subunits (total kda). tfr contains a large extracellular c-terminal domain ( amino acids, includes a binding site for its ligand transferrin), a single transmembrane domain ( amino acids), and a short intracellular n-terminal domain ( amino acids) [ ] [ ] [ ] . in normal intestinal epithelium, transferrin receptor (tfr /cd ) is predominantly expressed in the crypt enterocytes (observed on the basolateral surface and in the cytoplasm of crypt epithelial cells) [ ] [ ] [ ] , with little expression in the epithelial cells at the apical of the villus. in contrast, under conditions of iron-deficiency anemia and low iron stores, the iron-responsive element/iron regulatory protein (ire/ irp) network prevents tfr mrna degradation [ ] [ ] [ ] [ ] [ ] and up-regulates tfr substantially, resulting in expression all across the intestinal epithelium [ , , ] . in addition to its functions in iron uptake and homeostatic maintenance of the intestinal epithelium [ , , ] , tfr had also been identified as a cellular entry receptor for several viruses, including the new world arenaviruses, new world hemorrhagic fever arenaviruses, machupo virus (macv) [ , ] , mouse mammary tumor virus (mmtv) [ , ] , the canine and feline parvovirus, feline panleukopenia virus [ , ] , and hepatitis c virus (hcv) [ ] . additionally, junín virus (junv) uses tfr as a cellular receptor for entry into susceptible cells by clathrin-mediated endocytosis [ ] . because tfr is distributed widely along the surface epithelium of newborns with anemia, and intestinal epithelial cells of newborn piglets are targets of tgev, it is possible that tfr is the as yet uncharacterized kda protein that mediates tgev infection. to test this hypothesis, we used cultured porcine intestinal columnar epithelial cells (ipec-j ), derived from the neonatal piglet mid-jejunum [ , ] , as a model to characterize the interaction between tfr and tgev in vitro. our results indicate that tfr serves as a receptor for tgev, contributes specifically to virion binding and endocytosis, and determines tgev tropism. rabbit anti-human tfr (abcam). mouse monoclonal antibodies to his ha, and gfp (cmctag, milwaukee, usa). rabbit monoclonal anti-tgev n protein was prepared in our laboratory. anti-gapdh monoclonal antibody, hrp-conjugated goat anti-rabbit and anti-mouse igg (h + l) (vazyme, nanjing, china). fitc-conjugated anti-tgev polyclonal antiserum (vmrd). anti-rabbit igg antibody (beyotime). ferristatin ii and lmp agarose (sigma-aldrich). protease inhibitor cocktail, enhanced chemiluminescence (ecl), and pierce bca protein assay kit (thermo scientific). protein a/g magnetic beads (b , bimake, usa). lenti-x htx packaging mix (clonetech). x-tremegene hp dna transfection reagent (roche, switzerland). pvdf membrane (millipore). np- lysis buffer (beyotime). trizol reagent (takara, dalian, china). ripa lysis buffer and sds-page sample loading buffer ( x) (fcmacs, nanjing, china). caco- and ipec-j cell lines (guangzhou jennio biotech co, ltd., china), st cell line (atcc,usa), and hek t cell line (atcc,usa) were cultured in dulbecco's modified eagle's medium (dmem from life technologies) supplemented with % fetal bovine serum (fbs, gibco), mm hepes (life technologies), and μg/ml penicillin/streptomycin (invitrogen) in a humidified atmosphere containing % co at °c. cells were routinely seeded at a density of × /ml in cm plastic tissue culture flasks (corning) and passaged every - days for a maximum of passages. tgev (strain shxb) was provided by the jiangsu academy of agricultural sciences and propagated in st cells. the complete tgev shxb genome sequence is available in genbank (kp . ) [ ] . viruses were labeled with the fluorescent probe dylight nhs ester (thermo scientific) according the manufacturer's recommended protocol. a dna fragment encoding tfr was amplified by pcr and then inserted into the plvx-dsred-monomer-n expression vector (ecori/bamhi) (clontech). similarly, tfr -out (encoding the extracellular region of tfr ) was cloned into pacgfp -c (using sali/bamhi) (clontech) and pet- a-c(+) (using ecori/sali). tgev spike (s ) was cloned previously into pcmv-c-ha (bamhi/xbai) in our laboratory [ ] . shrna sequences targeted against tfr were designed using tools available at http://rnaidesigner.lifetechnologies.com/ rnaiexpress/insert.do and block-it rnai designer. the best silencing efficiency was exhibited by clone nm_ . (sus scrofa transferrin receptor ). shrnas were cloned into the plvx-shrna vector (ecori/bamhi) (takara, dalian, china). all primers used in pcr are described in table . cells were seeded with or without treatment for the indicated times, inoculated with tgev at a moi of at °c for h, and then washed three times with cold phosphate-buffered saline (pbs) to remove unattached virus. the cells were shifted to °c in a % co incubator and maintained in dmem supplemented with % fbs and μg/ml penicillin/streptomycin. infected cells and supernatants were harvested at the conclusion of the incubation period. to determine whether tfr and tgev co-localize, ipec-j cells were seeded on cover slips in -well tissue culture plates, infected with tgev or -conjugated tgev at °c for h, and then incubated at °c for various times. cells were fixed in % paraformaldehyde for min and incubated with . % triton x- in pbs for min. after the cells were blocked with % bovine serum albumin (bsa), they were incubated with primary antibodies ( : ) overnight at °c. cells were then rinsed and incubated with fluorochrome-conjugated secondary antibodies ( : ) for min at room temperature, washed again three times with pbs and incubated with μg/ml dapi for min. images were captured using a zeiss lsm confocal microscope (carl zeiss, germany) and analyzed using zen (blue edition) (carl zeiss). at the indicated times post infection, cells were washed with pbs and lysed in ice-cold cell lysis buffer with protease inhibitor cocktail. total protein concentration was determined with a pierce bca protein assay kit, using the bicinchoninic acid spectrophotometric method. samples containing equal amounts of protein were separated sds-page and transferred to a pvdf membrane. the membrane was blocked with % nonfat milk in tris-buffered saline (tbs) containing . % tween , incubated overnight at °c with primary antibodies ( : ), and then incubated with the corresponding hrp-conjugated secondary antibodies ( : ) for min at °c. antibody binding was detected by autoradiography using ecl. ipec-j cells were incubated in ripa lysis buffer containing protease inhibitor cocktail and then centrifuged at , ×g for min. the cleared lysate was pretreated with μl anti-rabbit igg control and fresh protein a/g magnetic beads for h at °c to eliminate nonspecific binding to the beads. the beads were removed by magnetic separator and the lysate was incubated with μg of anti-tfr ab overnight at °c on a rocker platform. μl of fresh protein a/g magnetic beads were then added to the mixture, and incubated for h at °c on a rocker platform. the beads were washed four times with pbs, combined with page loading buffer, and incubated for min at °c to release the immunoprecipitated proteins. the proteins were analyzed by western blotting using anti-tfr ab. in parallel, magnetic beads loaded with tfr were incubated with purified tgev for h at °c on a rocker platform. beads were washed four times with pbs, and prepared for western blotting using the same methods. the blot was probed with anti-tgev-n ab and anti-tfr ab. to identify the tfr binding domain, we co-transfected plasmids expressing tfr and tgev-s (or tfr -out and tgev-s ) into hek t cells that had reached - % confluence. transfection was conducted using the x-tremegene hp dna transfection reagent following the manufacturer's instructions. after h, cells were lysed in np- lysis buffer containing a protease inhibitor cocktail then centrifuged at ×g for min. the supernatant was pretreated with protein a/g magnetic beads and igg (from the same species as the immunoprecipitating antibody) for h at °c to eliminate nonspecific binding to the beads. the beads were removed by centrifugation and the supernatant was then incubated on a rotary mixer overnight at °c with the immunoprecipitating antibody. fresh protein a/g magnetic beads were added to the mixture and incubation continued for additional hours at °c. the complexes were analyzed by western blotting using antibodies against ha, gfp, and tfr . to examine virus entry, ipec-j cells were infected with tgev at a moi of for h at °c. trizol reagent was used to extract total rna, following the manufacturer's protocol. rna was analyzed by rt-qpcr, as previously described [ ] . gene expression was calculated using the comparative ct method and results from three independent experiments. rna levels were normalized using endogenous gapdh rna as a reference. primer sequences used for qrt-pcr are listed in table . flow cytometry was used to detect tgev entry into cells as follows: fluorescently tagged tgev was incubated with cells for h at °c. the cells were washed with pbs and maintained in dmem for h at °c in % co , then harvested using . % trypsin. acquisition of the fluorescent cells done was by facs (becton dickinson) and the data were analyzed using flowjo software (version . . ). to produce lentivirus, we transfected the tfr expression construct or shtfr into hek t cells using the lenti-x htx packaging mix (plasmids plp , plp , and vsv-g) and the x-tremegene hp dna transfection reagent, following the manufacturer's instructions. h post transfection, the culture medium was renewed and incubation continued for and days. virus-containing supernatants were collected, filtered (pore size . μm), and stored at − °c. ipec-j , st, and caco- cells were infected with lentiviral particles (moi = ) containing the tfr expression construct. twenty-four hour post infection the culture medium was refreshed, and incubation continued for - h to allow for maximum protein expression. ipec-j and st cells were transfected with the tfr shrna (shtfr ) and the negative control (scrambled) vectors (shctrl). to generate cells that were stably transformed with ipec-j -shtfr and st-shtfr , lentiviral particles (moi of ) were added to the cells in the presence of μg/ml polybrene. after incubation for h, the cell cultures were expanded and maintained for weeks in dmem with μg/ml puromycin. surviving cells were maintained in medium supplemented with μg/ ml puromycin. lysates from transduced cells were analyzed by western blotting. expression and purification of his-tagged tfr -out recombinant protein tfr -out recombinant protein was expressed in escherichia coli bl- and purified using ni-nta resin, following the manufacturer's protocol as previously described [ ] . expressed a protein was used as a control. confluent monolayers of st cells in -well plates were inoculated with serial ten-fold dilutions of virus suspension and incubated for h at °c. the cells were then overlaid with . % low melting point agarose in dmem containing % fbs and incubated about h at °c. to visualize plaques, cells were stained with % crystal violet in methanol. data are presented as means ± standard deviation (sd) from three independent experiments. statistical analysis was performed using statistical program for social sciences (spss) . . differences between control and experimental groups were analyzed using student's t-test and one-way analysis of variance (anova). differences were considered statistically significant at * . < p < . , ** p < . . to investigate whether tfr is involved in tgev infection, we used confocal microscopy to visualize the subcellular locations of tgev and tfr during tgev infection. in the first experiment, tgev was directly labeled with dylight -labeled tgev, while tfr was labeled indirectly using a dylight -conjugated antibody (fig. a) . the results indicated that tfr co-localized with tgev. the experiment was repeated using a dual-label immunofluorescence strategy (fig. b) . the results were consistent with our initial observation, and also showed that tfr appeared to re-localize, internalize, and cluster early in tgev infection (fig. b and c) . based on fluorescence, tfr protein levels were significantly reduced compared to levels in uninfected cultures at h post infection (h p.i.) (fig. c) . western blotting analysis confirmed that tfr expression decreased significantly until h p.i. (fig. d) . the co-localization of tfr and tgev, and the decrease in tfr expression during tgev infection, suggest that tfr and tgev interact reciprocally. to test this hypothesis, endogenous tfr was enriched from ipec-j cell lysates using magnetic beads bound to anti-tfr ab. empty magnetic beads (i.e., without antibody) and magnetic beads bound to allogeneic anti-rabbit igg were used as negative controls (fig. a) . the beads were then tested for their ability to bind tgev. only beads that had previously captured endogenous tfr were able to bind tgev (fig. b) . importantly, pre-incubating tgev (moi ) with the precipitated endogenous tfr blocked viral replication at h p.i. (fig. c and d) . these data indicate that endogenous tfr interacts with tgev. to verify that tfr is involved in tgev infection, we assessed the ability of tgev (moi or ) to infect cells after blocking cellular tfr with anti-tfr ab. the cells were fixed, stained for tgev-n protein using a fluorescence-tagged antibody, and then examined by microscopy. fluorescence was significantly decreased at h p.i. in blocked cells (fig. a) . at h p.i., western blotting showed that expression of tgev-n protein was significantly lower in ipec-j cells that had been pre-incubated with anti-tfr ab (fig. b and c) . in contrast, tgev invasion was not affected by treatment with the tfr ligands holo-tf or apo-tf (additional file : figure s ). ferristatin ii, a tfr inhibitor that causes degradation of tfr ( ) , was used to confirm the role of tfr in tgev invasion. preliminary experiments showed that a μm dose of ferristatin ii did not result in detectable cytotoxicity (additional file : figure s ). tgev replication was significantly inhibited in ipec-j cells pre-incubated with this concentration of ferristatin ii at h p.i. (fig. d and e) . given the evidence that tfr functions in tgev infection, we performed analogous experiments using shrna to target tfr in ipec-j and st cell lines. both total tfr protein (fig. f ) and membrane tfr protein (fig. g ) decreased in the knockdown cells compared to normal cells. tgev-n mrna levels were also significantly lower in tfr knockdown cells at h p.i. (fig. h) . to determine whether tfr knockdown affects tgev replication in ipec-j cells, we used immunofluorescence staining to detect changes in tgev-n protein levels. tgev-n protein was obviously reduced in cells treated with the tfr knockdown shrna at h p.i., relative to cells treated with the scrambled shrna control (fig. i) . likewise, we observed a significant decrease in tgev replication in western blotting assays ( fig. j and k) (see figure on previous page.) fig. tgev co-localizes with tfr and decreases its expression. a ipec-j cells were infected with dylight -tgev (green), and cultured for h. the cells were then stained for confocal microscopy using a rabbit anti-tfr pab, followed by dylight -conjugated goat anti-rabbit igg (red). the panel shows a three-dimensional rendering of a representative field obtained using imaris . , and the arrows indicate co-localized signals (scale bar = μm). b ipec-j cells were infected with tgev (moi ) and fixed at h p.i. cells were then stained for confocal microscopy using a rabbit anti-tfr ab, followed by dylight -conjugated goat anti-rabbit igg (red) and fitc-conjugated anti-tgev polyclonal antiserum (green). the arrows indicate co-localized signals (scale bar = μm). c indirect immunofluorescence images of tfr in mock-and tgev-infected cells at h p.i. and h p.i. (moi ). fixed cells were stained for tfr (red) and the nucleus was stained with dapi (blue). scale bar = μm. d ipec-j cells were uninfected or infected with tgev (moi ) and harvested at different time intervals ( - h p.i.) . the cell lysates were analyzed by western blotting using anti-tfr , anti-tgev-n, and anti-gapdh antibodies as probes fig. tgev interacts with endogenous tfr . a endogenous tfr was enriched from ipec-j cell lysates using magnetic beads coated with anti-tfr ab. tfr immobilized on the beads was detected by western blotting. b the enriched endogenous tfr described in panel a was used as bait to bind tgev. empty magnetic beads and magnetic beads with allogeneic anti-rabbit igg served as negative controls. the n protein of tgev was precipitated using immobilized beads coated with anti-tfr ab but not anti-rabbit igg. c and d tgev (moi ) was pre-incubated with the precipitated tfr protein for h at °c before cells were infected. at h p.i., tgev replication was analyzed by western blotting. pre-incubation with tfr protein inhibited viral replication. the tgev-n to gapdh ratio was normalized to control conditions. data shown are means ± sd from three independent experiments. (* . < p < . , ** p < . ) and plaque assays (fig. l and m) at h p.i. similar results were also obtained in st cells (fig. n and o) . because the experiments described above indicate that tfr is involved in tgev infection, we investigated whether overexpression of tfr would enhance tgev infectivity. ipec-j and st cells were infected with lentiviruses (moi ) containing the tfr expression construct or the empty control vector. as expected, cells infected with the tfr-expressing lentivirus particles showed significantly increased tfr expression over cells infected with lentivirus containing the empty vector (fig. a) . to determine whether overexpression of tfr impacts the tgev infection process, we used flow cytometry to test the ability of dylight -labeled tgev to associate with ipec-j cells (fig. b and c) . tgev mrna levels were also were measured in ipec-j cells at h p.i. using rt-qpcr (fig. d) . both experiments demonstrated that overexpression of tfr protein significantly increases viral infectivity. tgev replication was also significantly higher in tfr -overexpressing st cells at h p.i. (fig. e and f ) . finally, when tfr was overexpressed i. for western blotting. tgev replication was inhibited by tfr knockdown. the tgev-n to gapdh ratio was normalized to control conditions. the data shown are means ± sd from three independent experiments. (* . < p < . , ** p < . ) in tgev-resistant caco- cells, they became susceptible to tgev infection (fig. g, h and i) . extracellular tfr interacts with tgev s protein in vitro tgev s protein binds to specific receptors on the membrane of susceptible cells [ ] . to examine the interaction between tgev s and tfr , we obtained lysates from t cells that had been co-transfected with plasmids expressing tgev s -ha and tfr , and then conducted a co-immunoprecipitation assay. the results confirmed that tgev s directly interacts with tfr (fig. a) . we then constructed a plasmid encoding the extracellular region of tfr (designated tfr -out) and transfected it into cells along with the tgev s plasmid described earlier. the co-precipitation assay confirmed that the extracellular region of tfr interacts with tgev s protein in vitro (fig. b) . finally, his-tagged tfr -out fusion protein was prepared using an e. coli expression system. protein quality was verified by sds-page (fig. c ) and western blotting (fig. d) . when cells were pre-incubated for h with tfr -out ( ng/ml) prior to infection by tgev, viral replication as reflected by tgev-n levels, was inhibited ( fig. e and f ) . this result was consistent with a plaque assay for virus particles present in the cell culture medium (fig. g and h ). tgev invades the epithelial cells of the intestine via a receptor-mediated fusion mechanism [ , ] . the species-specific virus tropism or host-range is usually determined by entry receptors [ ] . the intestinal epithelium of neonatal piglets is particularly susceptible to tgev [ ] . identifying the proteins that mediate the association between the host cell and the virus is therefore a crucial step for understanding virus-host interactions. in this study, we conducted experiments to determine if tfr can function as a receptor for tgev invasion. the results show that tgev induces the internalization, clustering, and down-regulation of cellular tfr . overexpression of tfr enhances tgev invasion, and infection by tgev can be inhibited if access to tfr is blocked, or if tfr levels are reduced. finally, we determined that tgev-s protein interacts with the recently, li et al. observed that the ability of tgev to bind mdck cells is enhanced when the cells express porcine aminopeptidase n (papn) [ ] . we found that overexpression of tfr in the refractory caco- cell line is sufficient to allow tgev entry, synthesis of viral rna and protein, and release of infectious tgev. papn has been shown to function as a receptor for tgev infection [ ] [ ] [ ] [ ] . however, this protein seems to be widely distributed on enterocytes and probably on other tissues, irrespective of age [ ] . an entry receptor or co-receptor usually mediates virus entry, as is the case for human immunodeficiency virus (hiv) and poliovirus [ ] [ ] [ ] [ ] . a kda protein that is restricted to the villous enterocytes of newborn pigs has been identified as a possible additional receptor for tgev, and may account for the age sensitivity of these animals to the virus [ ] . newborn piglets with iron-deficiency anemia usually present elevated levels of tfr on the entire surface of the intestinal epithelium [ , , ] . tfr may therefore be a extracellular tfr interacts with tgev s protein in vitro. a and b t cells were co-transfected with a ha-tagged tgev s expression plasmid together with plasmids expressing tfr or gfp-tagged tfr -out. cell lysates were immunoprecipitated with anti-ha antibody and anti-tfr antibody, or anti-ha antibody and anti-gfp antibody, respectively. the precipitates were examined by western blotting using anti-ha antibody and anti-tfr antibody (a) or anti-ha antibody and anti-gfp antibody (b) to examine the interaction between ha-tgev-s and tfr , or ha-tgev-s and gfp-tagged tfr -out, respectively. c his-tagged tfr -out was expressed in e.coli bl and purified using a ni-nta column. purified products were separated using sds-page and stained with coomassie brilliant blue. d purified tfr -out was verified by western blotting. e and f ipec-j cells infected with tgev (moi ) were pre-incubated with tfr -out ( ng/ml) for h at °c, and cell lysates were harvested for western blotting. the ratio of tgev-n to gapdh was normalized to control conditions, and culture supernatants were collected for viral plaque assays in st cells. plaques developed days after infection. normal cells and cells treated with pet- a-c(+) served as controls. data shown are means ± sd from three independent experiments. (* . < p < . , ** p < . ) major contributing factor to the high level of tgev susceptibility exhibited by anemic newborn pigs. tfr plays a role in hcv entry via the clathrin endocytic pathway, and the expression of tfr is down-regulated in hcv-infected huh cells [ ] . junv also uses tfr as a cellular receptor for entry into vero cells and is rapidly internalized by clathrin-mediated endocytosis [ ] , and canine parvovirus (cpv) utilizes limited tfr clustering on the surface to enter host cells through the clathrin endocytic pathway [ , ] . studies in our laboratories have demonstrated that egfr is another promoter for tgev entry via the clathrin-dependent endocytic pathway [ ] . upon binding to its ligand holo-tf (iron-bound transferrin), tfr that is associated with clathrin-coated pits of the plasma membrane is internalized by dynamin-mediated endocytosis. the small gtpase rab , and its upstream activator dennd , regulate membrane trafficking of tfr from recycling endosomes to lysosomes [ ] . we observed that a decrease in tfr expression follows tgev invasion. we hypothesize that the decrease occurs because tfr is degraded by lysosomes after binding to tgev, and that tfr plays a critical role in the clathrin-mediated endocytosis of tgev. one of the four structural proteins encoded by the tgev genome is the large transmembrane s glycoprotein. trimers of s molecules form the surface spikes on coronaviruses which mediate virus entry and is a primary determinant tissue tropism cells [ , ] . the ectodomain of the s protein consists of an n-terminal variable domain called s that is responsible for receptor binding, and a c-terminal conserved domain called s that is responsible for fusion. [ , , ] . the tgev-s protein interacts with papn and egfr [ , ] . in our study, we demonstrated that tgev interacts with endogenous tfr and that tgev-s protein interacts with tfr and the extracellular region of tfr . the extracellular region of tfr is located between amino acid residues - . our experiments confirmed that pre-incubating tgev with endogenous tfr or soluble tfr -out blocked viral invasion, supporting the hypothesis that these extracellular amino acids residues are important for the recognition of tgev s . in addition, the anti-tfr antibody used in our blocking experiments recognizes amino acid residues - of tfr . this reagent also blocks tgev invasion, presumably by occluding the site at which tgev binds to tfr . in contrast, neither holo-tf nor apo-tf interfered with the ability of tgev to infect ipec-j cells, indicating the use of non-overlapping binding sites. based on these results, we hypothesize that tfr amino acid residues - encode the binding site for s protein. our experimental results strongly support the conclusion that tfr functions as a receptor for tgev invasion. the results are also consistent with the hypothesis that tfr may be responsible for the high level of tgev susceptibility exhibited by newborn pigs. additional work will be required to verify this hypothesis. finally, this study will facilitate the development of vaccines to combat tgev infection. additional file : figure s . tgev invasion didn't affect with the treatment of tfr ligands, holo-tf and apo-tf. figure s . the molecular biology of coronaviruses development of protection against coronavirus induced diseases simulation of the economic impact of transmissible gastroenteritis on commercial pig production in australia complete sequence ( kilobases) of the polyprotein-encoding gene of transmissible gastroenteritis virus mechanisms of coronavirus cell entry mediated by the viral spike protein morphogenesis and proliferative rule of porcine transmissible gastroenteritis virus in porcine intestinal epithelial cells infection of porcine precision cut intestinal slices by transmissible gastroenteritis coronavirus demonstrates the importance of the spike protein for enterotropism of different virus strains human myeloid plasma membrane glycoprotein cd (gp ) is identical to aminopeptidase n aminopeptidase n is a major receptor for the enteropathogeniccoronavirus tgev binding of transmissible gastroenteritis coronavirus to cell surface sialoglycoproteins binding characterization of determinants in porcine aminopeptidase n, the cellular receptor for transmissible gastroenteritis virus further characterization of aminopeptidase-n as a receptor for coronaviruses the epidermal growth factor receptor regulates cofilin activity and promotes transmissible gastroenteritis virus entry into intestinal epithelial cells evidence for a putative second receptor for porcine transmissible gastroenteritis virus on the villous enterocytes of newborn pigs two amino acid changes at the nterminus of transmissible gastroenteritis coronavirus spike protein result in the loss of enteric tropism the transferrin receptor part i: biology and targeting with cytotoxic antibodies for the treatment of cancer the endocytosis of transferrin by rat intestinal epithelial cells crystal structure of the ectodomain of human transferrin receptor noncanonical role of transferrin receptor is essential for intestinal homeostasis transferrin receptors in the human gastrointestinal tract. relationship to body iron stores immunolocalization of transferrin and transferrin receptor in mouse small intestinal absorptive cells systemic iron homeostasis and the iron-responsive element/iron-regulatory protein (ire/irp) regulatory network iron regulatory proteins are essential for intestinal function and control key iron absorption molecules in the duodenum iron metabolism and related genetic diseases: a cleared land, keeping mysteries the transferrin receptor: the cellular iron gate current understanding of iron homeostasis secretory iga mediates retrotranscytosis of intact gliadin peptides via the transferrin receptor in celiac disease adaptive changes of duodenal iron transport proteins in celiac disease human cell surface glycoprotein related to cell proliferation is the receptor for transferrin transferrin receptor is a cellular receptor for new world haemorrhagic fever arenaviruses receptor determinants of zoonotic transmission of new world hemorrhagic fever arenaviruses mouse transferrin receptor is the cell entry receptor for mouse mammary tumor virus mouse mammary tumor virus uses mouse but not human transferrin receptor to reach a low ph compartment and infect cells cellular uptake and infection by canine parvovirus involves rapid dynamin-regulated clathrin-mediated endocytosis, followed by slower intracellular trafficking canine and feline parvoviruses can use human or feline transferrin receptors to bind, enter, and infect cells identification of transferrin receptor as a hepatitis c virus entry factor cell entry by human pathogenic arenaviruses the ipec-j cell line porcine ipec-j intestinal epithelial cells in microbiological investigations complete genomic sequence of the coronavirus transmissible gastroenteritis virus shxb isolated in china how viruses enter animal cells entry and release of transmissible gastroenteritis coronavirus are restricted to apical surfaces of polarized epithelial cells aminopeptidase n is not required for porcine epidemic diarrhea virus cell entry identification of a major co-receptor for primary isolates of hiv- hiv- entry into cd + cells is mediated by the chemokine receptor cc-ckr- hiv- entry cofactor: functional cdna cloning of a seven-transmembrane, g protein-coupled receptor a monoclonal antibody that blocks poliovirus attachment recognizes the lymphocyte homing receptor cd characterization of junin arenavirus cell entry limited transferrin receptor clustering allows rapid diffusion of canine parvovirus into clathrin endocytic structures small gtpase rab regulates constitutive degradation of transferrin receptor targeted recombination demonstrates that the spike gene of transmissible gastroenteritis coronavirus is a determinant of its enteric tropism and virulence major receptor-binding and neutralization determinants are located within the same domain of the transmissible gastroenteritis virus (coronavirus) spike protein epidermal growth factor receptor is a co-factor for transmissible gastroenteritis virus entry we thank dr. qinghua yu and dr. jian lin for helpful suggestions and comments on the manuscript, and dr. jie peng for her technical assistance. this work was supported by an award from the national natural science foundation of china ( ) and by the priority academic program development (papd) of jiangsu higher education institutions. we declare that materials described in the manuscript, including all relevant raw data, will be freely available to any scientist wishing to use them for non-commercial purposes, without breaching participant confidentiality. ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -p ondy authors: kautz, tiffany f; guerbois, mathilde; khanipov, kamil; patterson, edward i; langsjoen, rose m; yun, ruimei; warmbrod, kelsey l; fofanov, yuriy; weaver, scott c; forrester, naomi l title: low-fidelity venezuelan equine encephalitis virus polymerase mutants to improve live-attenuated vaccine safety and efficacy date: - - journal: virus evol doi: . /ve/vey sha: doc_id: cord_uid: p ondy during rna virus replication, there is the potential to incorporate mutations that affect virulence or pathogenesis. for live-attenuated vaccines, this has implications for stability, as replication may result in mutations that either restore the wild-type phenotype via reversion or compensate for the attenuating mutations by increasing virulence (pseudoreversion). recent studies have demonstrated that altering the mutation rate of an rna virus is an effective attenuation tool. to validate the safety of low-fidelity mutations to increase vaccine attenuation, several mutations in the rna-dependent rna-polymerase (rdrp) were tested in the live-attenuated venezuelan equine encephalitis virus vaccine strain, tc- . next generation sequencing after passage in the presence of mutagens revealed a mutant containing three mutations in the rdrp, tc- x, to have decreased replication fidelity, while a second mutant, tc- x displayed no change in fidelity, but shared many phenotypic characteristics with tc- x. both mutants exhibited increased, albeit inconsistent attenuation in an infant mouse model, as well as increased immunogenicity and complete protection against lethal challenge of an adult murine model compared with the parent tc- . during serial passaging in a highly permissive model, the mutants increased in virulence but remained less virulent than the parent tc- . these results suggest that the incorporation of low-fidelity mutations into the rdrp of live-attenuated vaccines for rna viruses can confer increased immunogenicity whilst showing some evidence of increased attenuation. however, while in theory such constructs may result in more effective vaccines, the instability of the vaccine phenotype decreases the likelihood of this being an effective vaccine strategy. due to the error-prone nature of the rna-dependent rnapolymerase (rdrp), rna virus replication is characterized by a high mutation rate that results in increased genetic diversity of progeny viruses (domingo et al. ) . this diversity can be utilized by the virus to evade and escape the immune system, as well as to adapt to new hosts. maintaining the correct amount of virus diversity of $ mutation per genome (nebot et al. ) is of utmost importance to rna virus survival within the host and during transmission. this is especially true for arboviruses (arthropod borne viruses), which utilize two distinct hosts to complete their transmission cycle. if the mutation rate of an rna virus is too high, more unfit progeny will be produced due to the increased number of mutations, most of which are deleterious by chance. this results in a substantial decrease in viable genomes, and possible extinction of the viral population due to error catastrophe (manrubia et al. ) . conversely, if the mutation rate is too low, little variation is produced, and the virus population becomes clonal and less able to adapt to diverse environments. both scenarios reduce the overall fitness of the virus and can lead to extinction (escarmís et al. ). this predicted lack of fitness with high-or low-fidelity replication has been demonstrated experimentally with a number of viruses, including poliovirus (pv) (pfeiffer and kirkegaard ; vignuzzi et al. ) , chikungunya virus (chikv) (coffey et al. ; rozen-gagnon et al. ), west nile virus (van slyke et al. ) , st louis encephalitis virus (griesemer et al. ) , human enterovirus (sadeghipour and mcminn ; sadeghipour et al. ; meng and kwang ) , foot and mouth disease virus (arias et al. ; zeng et al. zeng et al. , xie et al. ) , sars cov (eckerle et al. ; graham et al. ; graepel et al. ) , influenza virus (cheung et al. ; pauly et al. ) , norovirus (arias et al. ) and coxsackie b virus (gnä dig et al. ; levi et al. ; mcdonald et al. ) . these viruses were all subjected to treatment with nucleoside analogs such as ribavirin or 'fluorouracil, which increases the mutation rate and can lead to virus extinction, or by the insertion of mutations found to change fidelity in other viruses. virus populations circumvent extinction by developing resistance to the nucleoside analog, sometimes by developing mutations in the rdrp that either increase (high fidelity) or decrease (low fidelity/hypermutator) rdrp fidelity (i.e. the rdrp error-rate). when compared with unpassaged, wild-type (wt) viruses, fidelity mutants have similar growth kinetics in vitro, but are attenuated in vivo due to the alteration of diversity produced during replication, which hampers the ability of the virus to overcome bottlenecks in the host (pfeiffer and kirkegaard ; vignuzzi et al. ) . the alphavirus genus, family togaviridae, contains a number of important arboviruses that are distributed worldwide. alphaviruses are classified as either old world or new world viruses depending on their distribution. venezuelan equine encephalitis virus (veev) is a new world encephalitic alphavirus that circulates continuously in an enzootic cycle in the forests and swamps of northern south america, central america and mexico, typically between rodents and mosquitoes. spillover from this cycle is believed to cause tens of thousands of dead-end human infections annually (aguilar et al. ) . periodically, the virus emerges to cause epizootic cycles in equids, which can also spill over into humans, sometimes resulting in hundreds of thousands of infections (sudia et al. ; wang et al. ; brault et al. ; weaver et al. ; anishchenko et al. ). prior to the s, equine epizootics involving high fatality rates were frequently recorded in veevendemic areas. since horses remain important components of the local agricultural economies within these regions, veev has had a major impact on the economic output of at-risk areas (walton and grayson ) . recent outbreaks in both venezuela and mexico demonstrate the ability of veev to re-emerge periodically from its enzootic niche (rico-hesse et al. ; weaver et al. ; oberste et al. ) , and some earlier outbreaks spread as far north as texas (lord ; sudia et al. ) . independent of these natural cycles of infection, there is great concern for the use of veev as a biological weapon due to its high infectivity via the aerosol route. to address this threat, multiple vaccines have been developed to protect against veev infection. the first human vaccine described for vee, tc- , was produced following serial passages of the trinidad donkey veev strain (subtype iab) in guinea pig heart cells (berge et al. ). sequencing of tc- revealed eleven nucleotide changes, but attenuation was attributed to only of these mutations; one in the ' utr (untranslated region) and one in the e glycoprotein gene (kinney et al. (kinney et al. , . the reliance of tc- on only two point mutations for its attenuation increases the potential for reversion during vaccination, as well as instability during manufacturing. this concern has been supported by passaging in infant mice, where tc- reverted in a little as three intracranial passages (mckinney et al. ) . the retention of mosquito infectivity adds another safety concern for transmission from vaccinees to humans or equids, and the isolation of tc- from wild-caught mosquitoes in during an equine vaccination campaign underscores this risk (pedersen et al. ). due to these concerns, as well its incomplete immunogenicity (mckinney et al. ; alevizatos et al. ; engler et al. ) , tc- is only approved for equids, but it maintains investigational new drug status for at-risk military and laboratory personnel. the presence of a high mutation rate is thought to be one reason for the persistence of rna viruses in the environment. however, high mutation rates also have implications for liveattenuated vaccines (lav's) such as tc- , which typically generate long-lived immunity and are cost-effective. lav replication has the potential to restore a virulent, wt phenotype via one or more reversions or pseudoreversions. such events can lead to safety concerns and possible adverse events following immunization (roukens and visser ) . however, further increasing the natural mutation rate is also hypothesized to decrease the ability of the virus to successfully revert or pseudorevert through muller's ratchet (muller ; chao ) , by the accumulation of deleterious mutations in asexual populations (e.g. viruses) during replication in the absence of efficient recombination. additionally, deleterious mutations can also impair the ability of fitter viruses to replicate and increase in the population (duarte et al. ; escarmı s et al. ; escarmís et al. ) . thus, the rna virus error rate is finely tuned to allow for optimal virus population health while also allowing for the production of mutations, which are key for adaptation to new hosts and environments. the present study was designed to determine whether mutations in the tc- rdrp that alter virus fidelity, i.e. increasing the mutational spectrum, can attenuate the virus further, without sacrificing vaccine stability or immunogenicity. we discovered rdrp mutations that conferred a hypermutator phenotype. additionally, a variant with four mutations in the rdrp was found to have no evidence of a change in diversity after one cell culture passage, but shared many characteristics in common with the low-fidelity mutant. these mutants both resulted in increased phenotypic stability compared with wt virus, as well as an increase in immunogenicity and protection in a mouse model. to identify mutations in the rdrp gene associated with changes in fidelity, tc- rescued from an infectious clone was used to initiate serial vero cell passages in duplicate in the presence of ribavirin, 'flurouracil ( fu), or azacytidine (aza). viruses were titered using plaque assays following each passage, and diluted to an moi of plaque forming unit (pfu)/cell for each subsequent passage. following passages and , the rdrp gene (nsp ) was sequenced. three mutations were identified in both fu passages, and all were located in the ' end of the rdrp gene: a g c mutation resulting in the amino acid change g r, an a g mutation resulting in the amino acid change e g, and a g a mutation resulting in the amino acid change a t (amino acid numbers refer to nsp residues). the three mutations identified following the fu passages were cloned into the tc- backbone in conjunction with a high fidelity mutation that was previously identified in a related alphavirus, chikv (c y, which corresponds to position in tc- ) (coffey et al. ) . six mutants were created (fig. a) , four of which were individual mutants, and two with multiple mutations: a x mutant including g r, e g and a t and a x mutant including g r, e g, a t and c y. all clones were rescued by electroporation into baby hamster kidney (bhk) cells, and were titered using standard plaque assays on vero cells (beaty et al. ) . all rescued mutants were viable, producing between and log pfu/ml following electroporation (data not shown). as the mutants included amino acid changes in the rdrp of veev tc- , the viruses were subjected to standard replication curves on vero cells (fig. b) . no mutants showed significant differences from tc- by repeated measures anova. since vero cells are interferon-deficient, we used human embryonic kidney (hek)- cells to examine the effects of interferon on the replication of fidelity-altered viruses. tc- , the x mutant and the x mutant were subjected to one-step replication curves (fig. c) . as in vero cells, no significant differences were observed between the viruses and their tc- parent. to confirm the ability of the rdrp mutations to confer resistance to fu treatment (fig. d ), vero cells were treated with , , , , , or mg/ml of fu and infected with parent tc- or an rdrp mutant using an moi of . . repeated measures anova found no significant differences between the rdrp mutants and parent tc- when vero cells were treated with - mg/ml fu. however, treatment with higher fu concentrations caused titers for all single rdrp mutants to drop compared with parent tc- , demonstrating increased sensitivity to the treatment. of note, e g was significantly different from parent tc- for all of the high fu concentrations (p < . for the mg/ml treatment, p < . for the - ug/ml treatments). a t was slightly more resistant, with titers significantly reduced compared with parent tc- when cells were treated with - mg/ml (p < . ). g r and c y were the most resistant to fu treatment of the single mutants. titers were only significantly reduced compared with parent tc- for the - mg/ml treatments (p < . for g r mg/ml, p < . for all others). due to the increased sensitivity of these mutants to fu, it was unlikely that these single mutations caused the resistance observed during the initial fidelity mutant selection. unlike the single rdrp mutants, tc- x and x generally had similar or slightly higher virus titers compared with parent tc- , suggesting increased resistance to fu treatment. tc- x titers were significantly higher during the and mg/ml treatments (p < . and . , respectively), and tc- x titers were also significantly higher during the ug/ml treatment (p < . ). to determine if the rdrp mutants reacted similarly upon exposure to another nucleoside analog, tc- and the rdrp mutants were exposed to various concentrations of ribavirin (fig. e ). no significant difference was observed, except for the g r mutant, which displayed increased sensitivity (p < . for and mm, p < . for mm). to assess virulence, individual tc- mutants were injected intracranially into -day-old cd- mice at a titer of ca. log pfu (backtiters ranged from .  - .  per dose). mice were weighed daily and the time of death was recorded ( fig. a) . the tc- x mutant was significantly more virulent than the parent virus, with all animals succumbing by day compared with tc- , where mice began succumbing at day (p . , kaplan-meier test). the mean time to death for parent tc- and all mutants except tc- x was . - . days after infection, whereas tc- x showed a mean time to death of . days. fidelity mutants have an impaired ability to overcome host bottlenecks, which results in reduced mortality and titer in certain organs (pfeiffer and kirkegaard ; vignuzzi et al. vignuzzi et al. , graham et al. ; ) . to test this, individual mutants were injected subcutaneously into -day-old cd- mice to measure their ability to cause mortality (backtiters ranged from .  to .  per dose). tc- x and tc- x exhibited significantly reduced mortality as reflected in survival (kaplan-meier test, p . ) when injected subcutaneously, compared with the original tc- , and compared with intracranial injection in -day-old cd- mice (fig. b) . the remaining mutants caused similar mortality to original tc- , suggesting no change in their ability to traverse bottlenecks to cause fatal disease. to examine how different hosts in the arbovirus transmission cycle alter virus diversity, tc- , as well as the x and x mutants, were passaged once using primate (vero or hek- ) or mosquito (c / or u . ) cells and compared with the parental virus (fig. ) . tc- x and x were the only rdrp mutants chosen for this analysis, because they were most likely to have altered fidelity as measured by a preliminary illumina sequencing analysis measuring the diversity of one replicate of each rdrp mutant (data not shown), as well as the phenotypic tests examined in figs and . illumina sequencing showed that the x mutant produced a significantly higher amount of genetic diversity than parental tc- , independent of the cell type used (fig. a) . this, along with the previously discussed results, indicated that the x mutant is a low-fidelity variant. interestingly, the x mutant did not demonstrate a significant increase or decrease in diversity compared with tc- . cell type had no significant effect on virus diversity, at least not after one passage. when the number of nucleotide positions containing diversity was examined (fig. b ), the x mutant trended towards producing an increased number of positions with diversity, while the x mutant, again, looked much like the parent tc- . lastly, because tc- x and x only appear to be resistant to one nucleoside analog ( fig. d and e), it was important to determine if this resistance is due to an alteration in the types of mutations being made by these rdrp mutants (fig. c ). indeed, both tc- x and x produced much fewer u-c mutations. because fu is a uracil analog and ribavirin is not, this explains the difference in nucleoside analog susceptibility. when examining peaks of diversity across the virus genome, some hotspots were conserved between the parent virus and rdrp mutants (fig. , supplementary figs s -s ). these included nsp c a (asp-glu) and a del (met-stop), e t g (minus strand, val-gly), k c a (ala-asp) and e c a (minus strand, arg-ser). the parent tc- and, to a reduced extent, tc- x both produced diversity hotspots in e , but generally in different positions. in tc- x, these consisted of c t (ala-val), t c (val-leu) and c a (ala, minus strand, synonymous), while tc- had e hotspots at c t (val, synonymous), c t (ala-gly), c g (ala-gly), c a (ala, minus sense, synonymous) and t c (asn, synonymous). additionally, while there was no significant difference in diversity when different cell types were used (fig. ) , tc- x appeared to have an increased number of diversity hotspots in c / cells than the other cell types. immunogenicity of the mutants was tested in an adult murine model previously used to test the efficacy of tc- . adult cd- mice, weeks of age, were vaccinated with log pfu in a ll volume via the subcutaneous route. animals were bled on days - after vaccination and weighed daily for week and then again on days and (fig. a) . repeated measures anova was used to determine statistical significance, and bars indicate standard deviation. resistance of tc- mutants to treatment by fu (d). cells were treated with , , , , , or mg/ml of fu and infected using an moi of . ; all experiments were performed in triplicate. samples were titered by standard plaque assay. statistical differences were determined by repeated measures anova, and significance is detailed in the text. resistance of tc- mutants to treatment by ribavirin (e). cells were treated with , , , , or mm ribavirin and infected using an moi of . ; all experiments were performed in triplicate. samples were titered by standard plaque assay. statistical differences were determined by repeated measures anova, and significance is detailed in the text. animals exhibited no weight loss and viremia was sporadic with no significant differences among any of the viruses (repeated measures anova, table ). four weeks post-vaccination, the animals were bled to assay neutralizing antibody titers (fig. b ). all vaccinated mice exhibited a strong immune response as determined by a plaque reduction neutralization test (prnt ). the x and x mutants produced significantly higher mean neutralizing antibody titers compared with tc- (repeated measures anova, p ¼ . and . , respectively). following challenge at weeks post-vaccination with veev subtype ic strain , which was used previously for similar experiments rossi et al. ) , animals were weighed daily and monitored for survival ( fig. c and d) . all vaccinated animals showed complete protection from weight loss and death, whereas the sham-vaccinated animals all succumbed by day post-challenge. with the identification of the x and x viruses as potential fidelity mutants, it was important to determine if these viruses were consistently less virulent in infant mice when subjected to population bottlenecks. this was especially important to confirm for the x mutant, because of its identification as a lowfidelity mutant, which might make this strain less phenotypically stable. to test this, tc- , tc- x and tc- x clones were transcribed independently in duplicate. these constructs were rescued using bhk cells, and each produced between and log pfu/ml following electroporation. to measure virulence, -day-old cd- mice were injected intracranially or subcutaneously with pfu using these rescued viruses (fig. ) . intracranial survival curves (fig. a ) were similar to those in fig. a , with most mice succumbing - days post-infection. backtiters of the inocula ranged from . to .  pfu per dose. depending on the replicate group, tc- x or tc- x were slightly more or less virulent than the parental tc- . this suggests that the rdrp mutations do not alter virulence when administered intracranially, and that the significant differences in lethality observed in fig. a for tc- x are likely dependent on the mutant spectrum produced post-electroporation or due to host variation. subcutaneous survival curves (fig. b) were significantly different compared with fig. b . instead of half of the x and x mutants surviving subcutaneous inoculation, all individuals succumbed by days post-infection. inoculum back titers ranged from .  to .  pfu per dose. although all mice succumbed, there was variation between the replicates. for example, while one of the tc- x replicates survived an average of . days, the other replicate survived an average of . days. this and the intracranial survival curve results suggest that differences in the postelectroporation minority populations can result in variation in the degree of attenuation of low-fidelity viruses. to confirm that the fidelity-altering mutations did not revert upon repeated replication, tc- , tc- x and tc- x were subjected to five passages in vero cells. sanger sequencing of the rdrp revealed no mutations (data not shown). the x and x rdrp mutants and tc- were also subjected to serial intracranial passages in infant mice to determine if compensatory mutations accumulated in vivo. passage and virus was injected subcutaneously into -day-old mice to assess changes in virulence that occurred during passaging. tc- , tc- x and tc- x all gained virulence by passage compared with their unpassaged counterparts as shown in fig. b (kaplan-meier test, p . ) (fig. a) . at this point, the x and x mutants reached virulence equivalent to that of the original, unpassaged tc- with no significant difference in time to death. interestingly, no significant changes in virulence occurred between passage and for any of the viruses. this indicates that tc- x and x can only reach virulence equivalent to unpassaged tc- , and are are unable to increase in virulence past that point. additionally, while not statistically significant, mice inoculated with tc- x passage survived up to week longer than mice inoculated with tc- x passage . this suggests that additional passaging may result in decreased fitness for this construct. illumina sequencing following mouse brain passage showed an increase in diversity for both tc- x and tc- x (fig. b-f) . this suggests that tc- x may actually be low fidelity, and that multiple growth cycles may be required to truly assess fidelity changes. curiously, while diversity hotspots increased for both tc- x and x, the k hotspot observed after one cell culture passage was greatly reduced for all of the passage viruses (fig. d-f) . even though a different electroporation stock was used for infant mice passaging, tc- x and x still exhibited fewer u-c mutations (fig. c, supplementary fig. s ), suggesting that this is a stable phenotype for these two rdrp mutants. although tc- x and x were producing more diversity than the parent tc- , fewer revertents occurred at the e mutation required for tc- attenuation (fig. c ) (kinney et al. ). due to the position of the 'utr attenuation mutation, coverage was too low to assess true differences in the potential for this site to also revert, but no reads contained the revertent mutation for any virus. following passage in mouse brains, sanger sequencing was used to identify several snps (table ) . tc- and tc- x each produced two snps following passages, while tc- x had no consensus sequence changes. none of these snps reverted the tc- mutations that cause attenuation or the inserted rdrp mutations. additionally, there were no convergent mutations found in all passage series. infectious diseases transmitted by arthropod vectors are difficult to control, and protecting the population via vaccination tends to be more effective than trying to control or eradicate the vector population. although lavs tend to be cost-effective and immunogenic, lavs have a significant achilles' heel: the potential for reversion to wt virulence. this risk is particularly acute for rna viruses, which replicate without proofreading, resulting in an increased ability to revert to the wt sequence or restore virulence via compensatory mutations (pseudoreversion). however, altering virus fidelity can theoretically overcome this major drawback. this approach has only been examined with fidelity mutants of pv and a low-fidelity exon mutant of sars-cov (graham et al. ; vignuzzi et al. ). while the high fidelity x: blue; tc- x: green; tc- : red. repeated measures anova was used to determine statistical significance, p < . is represented by *, p < . is represented by **, p < . is represented by *** and p < . is represented by ****. pv (vignuzzi et al. ) and sars-cov exon (graham et al. ) mutant both demonstrated increased immunogenicity and an improved safety profile compared to the parent viruses, this was not observed for the low fidelity poliovirus (korboukh et al. ). however, fidelity mutants have only been generated using wt virus strains, which are unlikely to be sufficiently attenuated for vaccine use. we hypothesized that increasing the attenuation of a live-attenuated rna virus vaccine by altering the rdrp fidelity would further improve its safety and efficacy. we addressed this by validating a series of low-fidelity mutations in a model system using the veev vaccine strain tc- to determine how this affects the stability and attenuation. serial passaging of tc- in -fu identified three mutations in the rdrp, and validation showed that all three mutations were required to decrease virus replication fidelity. additionally, we identified a x mutant that acted much like tc- x, but showed no difference in diversity after one cell culture passage. at present, there is no atomic resolution structure for an alphavirus rdrp, which limits our understanding of their mechanisms of fidelity alteration. however, all the mutations in the x mutant (g r, e g and a t) were at the ' end of the nsp gene, similar to the placement of the pv high fidelity g s and low fidelity h r substitution, which are both on the periphery of the rdrp protein rather than near the active site (pfeiffer and kirkegaard ; campagnola et al. ; korboukh et al. ) . for pv, the presence of the g s mutation does not significantly alter the structure of the polymerase, but it does decrease the incorporation rate of new nucleotides (arnold et al. ; marcotte et al. ). in fact, the current theory is that this substitution simply stabilizes the polymerase rather than altering the active site. alternatively, low fidelity h r pv decreases fidelity by favoring an open state of the rdrp, which decreases the duration of this fidelity checkpoint, thus increasing the likelihood of ntp misincorporation (moustafa et al. ). the presence of the low-fidelity mutations outside of the predicted active domain of the tc- nsp gene suggests a similar mechanism, but confirmation will require an atomic resolution structure. there are two manners in which low-fidelity mutants have been isolated, either by mutating residues found to alter fidelity in other viruses or by passaging virus in the presence of a nucleoside analog. of the latter (arias et al. ; pauly et al. ; van slyke et al. ) , three low-fidelity mutants have been identified that are resistant to the nucleoside analog used for passaging, but are sensitive to other nucleoside analogs. in concordance with this, these low-fidelity mutants exhibit higher fidelity for the specific mutations selected by the nucleoside analog, but lower fidelity for other mutations. our x and x fidelity mutants appear to act similarly to these low-fidelity viruses, with resistance to 'fluorouracil, but not ribavirin. when examining the mutation frequency of different mutations, the fidelity mutants produced a much lower amount of u to c transition mutations, which would be one of the mutations expected to be selected against during 'fu passaging. this also may explain why the mutants are most resistant to 'fu when using moderate amounts of the nucleoside analog, but then increase in susceptibility again at higher . genome organization is color-coded using the following: nsp , blue; nsp , red; nsp , green; nsp , purple; capsid, orange; e , black; e , gold; k, navy blue; and e , maroon. concentrations, as there is no difference in the other uracil mutation frequencies. tc- x was clearly a low-fidelity mutant, as evidenced by the phenotypic tests, as well as the increased genetic diversity observed using illumina sequencing. the x mutant, while exhibiting phenotypic similarities with other altered fidelity mutants, had no significant difference in virus diversity compared with the tc- parent after one cell culture passage. however, tc- x may actually be a low-fidelity variant, as suggested by the increase in diversity that occurred during in vivo passaging, which exhibited gains in diversity nearly identical to tc- x. perhaps the c y mutation (coffey et al. ), a high fidelity chikv mutation, offers an additive effect to initially increase virus diversity to that of wt virus, while exerting additional attenuation via an unknown mechanism. however, it appears that the c y mutation exerts less of an effect over multiple virus replication cycles, resulting in increased virus diversity compared with parent tc- after passaging in vivo. alternatively, it could be that the cumulative effect of these mutations alters the ability of the virus to explore the most beneficial sequence space as the virus replicates, leading to muller's ratchet and decreased virus health. indeed, this may explain why the x mutant initially exhibited gains in virulence during in vivo passaging, but later became more attenuated. it would be interesting to continue passaging this mutant to determine if the mutation threshold is eventually crossed, leading to error catastrophe and virus extinction. if this hypothesis is validated, the x construct is likely to be a safer lav candidate than the x mutant. indeed, a mixture of these two hypotheses might explain the observed tc- x results. also of interest is the c y substitution that increased replication fidelity in chikv, but did not affect tc- fidelity, although it did attenuate tc- in the infant mouse model. this finding is the opposite of the outcome for sindbis virus, which exhibited low-fidelity features when a low-fidelity chikv mutation was inserted (rozen-gagnon et al. ). however, sindbis virus is more closely related to chikv than veev, so this may explain why the effects did not transfer to tc- even though the residue is highly conserved among alphaviruses (coffey et al. ) . illumina sequencing uncovered many noteworthy effects during rdrp mutant growth in vitro. in particular, the e diversity peaks identified in tc- were greatly decreased for the rdrp mutants. the e glycoprotein is responsible for the uncoating of the virus particle and escape of the genome into the cytosol. some substitutions in the chikv e glycoprotein (tsetsarkin et al. (tsetsarkin et al. , vazeille et al. ) . diversity in this region may be important for increased vector fitness and/or host jumps. when total genome diversity was analyzed for each construct, there was no significant difference in diversity when comparing cell types. this is in contradiction to the low-fidelity chikv mutants, which only showed increased diversity in vertebrate cells and not in mosquito cells (rozen-gagnon et al. ) . however, while no significant difference was observed in overall diversity between the cell types, tc- x produced much higher peaks in diversity in c / cells than was observed in other cell types or viruses. this is intriguing, because it is hypothesized that mosquitoes are drivers of diversity, likely due to the sequence specificity of the mosquito antiviral rnai response (i.e. sirnas), although this has only been demonstrated for west nile virus (brackney et al. (brackney et al. , grubaugh et al. grubaugh et al. , . this hypothesis does not appear to explain the increased diversity observed during the tc- x c / passages, because these cells lack a functional sirna response. additionally, no increased hotspots were observed for u . cells, which do produce antiviral sirnas. further work is needed to determine how these diversity hotspots correspond to small rna targeting in mosquito cells and examine the effects of the host immune response on virus populations. as previous studies have shown that high fidelity pv mutations cause attenuation in mouse models by reducing dissemination to the central nervous system (pfeiffer and kirkegaard ; vignuzzi et al. vignuzzi et al. , , we tested our mutants in a lethal mouse model for tc- . the x and x rdrp mutants initially displayed equivalent virulence to parent tc- when injected into a single tissue that can cause lethal damage (brain) and reduced virulence when injected via a multi-tissue model (subcutaneous). however, when this experiment was repeated with virus rescued from different electroporation pools, the x and x mutants displayed greatly increased virulence compared with the initial subcutaneous outcome. this suggests that minority variants randomly generated during the transcription preceding electroporation can have large effects on the overall virus population health, and thus virulence. importantly, tc- was always equal to or more virulent when injected subcutaneously compared with the rdrp mutants. while tc- is not lethal in an adult mouse model, it would be interesting to determine if the x and x mutants have consistently delayed and/or decreased dissemination compared with wt virus. this would allow for a greater understanding of how a low-fidelity vaccine would operate in a fully immunocompetent host. when it was established that different pools of virus may exhibit different degrees of virulence, it became important to test whether the mutants would remain attenuated when allowed to experience multiple growth cycles. perhaps counterintuitively, the x hyper mutator variant, while undergoing an initial increase in virulence during in vivo passaging, only reverted to the same level of virulence as unpassaged tc- . this is likely due to an increased amount of unfit virions (e.g. defective interfering [di] particles), which hamper wt virus virulence, as well as the inability of the x mutant to revert the inserted rdrp mutations. the passage results for each virus were very similar to the passage data, except for the x virus, which decreased in virulence so much that some infant mice were able to survive extra days. this passaging data demonstrates that both of our mutants exhibit decreased risk of reversion to wt virulence compared with the parent tc- . as our study was designed to evaluate the effectiveness of rdrp mutations that decrease fidelity in vaccines, the mutants were put through a standard challenge model previously used for different veev vaccines rossi et al. ) . mice showed no adverse effects from vaccination, viremia was consistent with that expected for tc- , and all mice were protected against lethal challenge. additionally, the mutants induced higher antibody titers than tc- . our current hypothesis for this observation is that the low-fidelity rdrp creates an increased number of di virus particles, which are well known to be strong immune stimulators (dimmock and kennedy ; mercado-ló pez et al. ; tapia et al. ; sun et al. ) . as veev first amplifies in the draining lymph node (macdonald and johnston ) , the presence of these di particles may restrict dissemination from the draining lymph node, allowing for a greater opportunity for antigen presentation and therefore an increased immune response. this needs to be explored in future work. here, we demonstrated that the presence of low-fidelity mutations in a lav resulted in reduced virulence in an infant mouse model and increased immunogenicity in an adult mouse model, both of which are desirable properties for lavs. however, we also observed different outcomes in low-fidelity mutant attenuation in vivo when different pools of virus stock were used, which suggests that the low-fidelity virus population might not be initially stable. this demonstrates that there is great need to understand the role that minority variants play in phenotypic changes in vaccine development, as changes in the mutant spectrum may significantly alter the phenotype of vaccines. however, the passaging results imply that the virus population stabilizes after multiple growth cycles, so low-fidelity rna virus vaccines may require passaging to ensure a consistent vaccination phenotype. overall, this study demonstrates that while low-fidelity mutations may be a potential tool to further develop, the initial phenotypic instability observed post electroporation suggests that this may not currently be a viable strategy for lav development. this study was carried out in strict accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health. the protocol was approved by the institutional animal care and use committee of the university of texas medical branch. vero (african green monkey kidney), hek- and bhk cells were obtained from the american type cell collection (bethesda, figure . survival of -day old cd- mice following subcutaneous injection with pfu in a ml volume of passages , or tc- , x or x from infant mouse intracranial passages (a). statistical differences were determined using kaplan-meier tests, and significance is detailed in the text. illumina-sequencing diversity analysis was used to determine changes in virus diversity of passage tc- , x or x in infant mice (b). mutation frequency of the e mutation required for tc- attenuation (c). illumina sequencing virus diversity hotspots for the coding regions of tc- (d), x (e) and x (f) genomes after five intracranial passages. genome organization is color-coded using the following: nsp , blue; nsp , red; nsp , green; nsp , purple; capsid, orange; e , black; e , gold; k, navy blue; and e , maroon. md) and maintained in dulbecco's minimal essential medium (dmem) supplemented with % fetal bovine serum (fbs), and penicillin and streptomycin (pen/strep) ( u/ml) in a c, % co incubator. c / cells were maintained in dmem supplemented with % fbs, % minimal essential medium non-essential amino acids, % trypose phosphate broth and pen/strep ( u/ml) in a c co incubator. u . cells were maintained in mitsuhashi and maramorosch media supplemented with % fbs, % sodium bicarbonate ( . %) and pen/strep ( u/ml) in a c co incubator. viruses were rescued from a tc- infectious clone as described previously and without further passage (smith et al. ) . viruses were passaged on vero cells unless otherwise stated. titers of virus were determined by standard plaque assay using vero cells (beaty et al. ) . parental tc- was passaged in the presence of mutagens, ribavirin (sigma aldrich, st louis, mo), 'fluorouracil ( fu) (sigma aldrich) or azacytidine (sigma aldrich) at concentrations of mm, mg/ml and . mm, respectively. cells were pretreated h prior to infection with the mutagens in duplicate, then infected with tc- at a multiplicity of infection (moi) of pfu/cell. following a -h incubation period the cells were fed with dmem supplemented with % fbs and pen/strep ( u/ml) as well as the mutagen from the original pretreatment. supernatants were harvested days post-infection and stored at À c after being supplemented with % fbs. viral titers were determined by standard plaque assay (beaty et al. ) . viruses were sequenced using sanger sequencing after passages , and to determine if any mutations had occurred in the rdrp. six tc- subclones containing mutations identified in the tc- rdrp or high-fidelity chikv rdrp were created by joining-pcr cdna fragments from the tc- genome using designed primers. the sequence of each construct was verified prior to cloning into the genomic infectious clone backbone. all viruses were rescued after electroporation into bhk cells as described previously in greene et al. ( ) . to ensure that the inserted mutations did not interfere with viral replication, standard replication curves were conducted. cells were infected at an moi of (hek- ) or . (vero) in -well plates and incubated for h before being washed twice with phosphate buffered saline (pbs) and overlaid with ml of dmem supplemented with % fbs and pen/strep ( u/ml). virus was harvested by removing the entirety of the medium and replacing it with ml of fresh medium. harvested virus was supplemented with fbs to a final concentration of %, and clarified by centrifugation at rpm for min. the supernatant was then removed and stored at À c. viral titers were determined by standard plaque assays (beaty et al. ) . rescued viruses were passaged five times on vero cells to ensure genetic stability. viruses were harvested days postinfection and diluted : before infection of vero cells in the subsequent passage. following passage , viral suspensions were placed into buffer avl (qiagen, valencia, ca) and rna extracted using the column method as per the manufacturer's protocol. cdna was produced using the superscript iii first round synthesis kit (invitrogen, carlsbad, ca) following the manufacturer's instructions. cdna was amplified for sequencing using the phusion enzyme (neb, madison, wi) in a ll volume following the manufacturer's instructions using primer sets that covered the rdrp region of the genome (primers available upon request). products were sequenced on the abi sequencer (abi, ). rescued viruses were tested for susceptibility to fu or ribavirin in triplicate. cells were pretreated with , , , , , or mg/ml of fu or , , , , or mm ribavirin for h. the cells were then infected with . moi of the rdrp mutants as well as tc- . medium containing the pretreatment amount of fu or ribavirin was added to the infected cells -h post-infection. virus was harvested -h post-infection and titrated in duplicate using standard plaque assays. to study virulence and attenuation, -day-old cd- mice (charles rivers, wilmington, ma) were inoculated intracranially (ic) with pfu of virus in a volume of ll, or subcutaneously (sc) with pfu in a volume of ll. animals were weighed daily for weeks and monitored for survival. in another experiment, -week-old cd- mice were vaccinated sc with the original tc- virus or one of the rdrp mutants with pfu in a volume of ll, or with pbs for unvaccinated controls (n ¼ per cohort). six-week postvaccination, animals were challenged sc with pfu of wt epidemic veev strain (suá rez et al. ) , with daily monitoring for signs of illness, survival and weight loss. blood samples were collected from alternating cages of vaccinated mice for days post-vaccination and days post-challenge for viremia detection, as well as weeks post-vaccination for antibody measurement by prnt. to assess genetic and phenotypic in vivo stability of the tc- rdrp mutants, each was subjected to serial, ic passages in six-day-old cd mice at a dose of ca. pfu in a ll volume per animal. animals were euthanized -h post-inoculation, and their brains harvested and homogenized to determine viral titer by standard plaque assay (beaty et al. ) . the homogenized brain was diluted to produce the same inoculum dose and used to initiate the subsequent passage. virulence of the mouse passages and viruses was compared with the parental strain by inoculating -day-old cd mice sc with pfu, as described earlier. stability of the genomic sequences was assessed by rt-pcr on rna extracted from mouse passages and viruses and sequenced using illumina or sanger sequencing, respectively. . next generation sequencing . . library preparation viral rna from cell culture extracts were fragmented by incubation at c for min in . ll of fragmentation buffer table . changes in nucleotide sequence following p in mouse brain as verified by sanger sequencing. tc- x tc- x tc- nsp c t x nsp t c x e a t d-l x 'utr c t x (illumina, san diego, ca). first and second strand synthesis, adapter ligation and amplification of the library were performed using the illumina truseq rna sample preparation kit as per the manufacturer's protocol. for virus isolates from animals, pcr was first done using sequence-specific primers tiled across the veev genome to provide amplified products of $ . kb in size that were used for library generation. products were fragmented using transposons, then adapter ligation and amplification of the library was performed using the illumina truseq rna sample preparation kit under conditions described by the manufacturer (illumina, san diego, ca). samples were tracked using the 'index tags' incorporated into the adapters as defined by the manufacturer. the quality for each sample/dataset was assessed using fastqc (hoffmann et al. ). the paired-end reads were merged for each sample and then filtered to exclude reads with unknown characters (anything other than a, t, c, g) and low quality (< quality score), so that only high quality reads were used during analysis. additionally, the first bases of each read were trimmed due to nucleotide bias. the analysis was performed using veev strain tc- , complete genome (genbank accession no.: l . ) (r. m. kinney et al. ). to analyze the variant hotspots, each sample was run through a novel rare variant pipeline (available upon request). the pipeline first maps each read to the reference veev genome with perfect match, then unmapped reads are re-mapped with mismatch and added to the final map. the base long reads used in the analyses were validated as viral sequences and not host sequences by analysis of the longest subsequences shared explicitly (no mismatches allowed) and longest similar ( mismatch allowed) between viral and host genomes. diversity per position in the viral genome was calculated by taking a ratio of the reads mapped with mismatches and all reads mapped at that position. positions in which the number of reads mapped with mismatches was higher than perfectly mapped reads or coverage was below were excluded from diversity calculations. mutation frequency per sample was calculated by summing per position diversities and normalizing by the number of positions for which diversity was calculated. positions with non-zero mutation frequency were considered to be variant positions. graphpad prism was used to perform all statistical tests, which are described in the text. endemic venezuelan equine encephalitis in the americas: hidden under the dengue umbrella live, attenuated venezuelan equine encephalomyelitis virus vaccine venezuelan encephalitis emergence mediated by a phylogenetically predicted viral mutation determinants of rna polymerase (in) fidelity revealed by kinetic analysis of the polymerase encoded by a foot-and-mouth disease virus mutant with reduced sensitivity to ribavirin remote site control of an active site fidelity checkpoint in a viral rna-dependent rna polymerase attenuation of venezuelan equine encephalomyelitis virus by in vitro cultivation in guinea-pig heart cells rnai targeting of west nile virus in mosquito midguts promotes virus diversification positively charged amino acid substitutions in the e envelope glycoprotein are associated with the emergence of venezuelan equine encephalitis virus structure-function relationships underlying the replication fidelity of viral rna-dependent rna polymerases fitness of rna virus decreased by muller's ratchet', nature generation and characterization of influenza a viruses with altered polymerase fidelity arbovirus high fidelity variant loses fitness in mosquitoes and mice prevention of death in semliki forest virus-infected mice by administration of defective-interfering semliki forest virus quasispecies dynamics and rna virus extinction rapid fitness losses in mammalian rna virus clones due to muller's ratchet infidelity of sars-cov nsp -exonuclease mutant virus replication is revealed by complete genome sequencing venezuelan equine encephalitis-specific immunoglobulin responses: live attenuated tc- versus inactivated c- vaccine repeated bottleneck transfers can lead to non-cytocidal forms of a cytopathic virus: implications for viral extinction multiple molecular pathways for fitness recovery of an rna virus debilitated by operation of muller's ratchet' coxsackievirus b mutator strains are attenuated in vivo a live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease proofreading-deficient coronaviruses adapt for increased fitness over long-term passage without reversion of exoribonuclease-inactivating mutations envelope glycoprotein mutations mediate equine amplification and virulence of epizootic venezuelan equine encephalitis virus mutagen resistance and mutation restriction of st. louis encephalitis virus experimental evolution of an rna virus in wild birds: evidence for host-dependent impacts on population structure and competitive fitness ires-driven expression of the capsid protein of the venezuelan equine encephalitis virus vaccine strain increases its attenuation and safety fast mapping of short sequences with mismatches, insertions and deletions using index structures the full-length nucleotide sequences of the virulent trinidad donkey strain of venezuelan equine encephalitis virus and its attenuated vaccine derivative, strain tc- rna virus population diversity, an optimum for maximal fitness and virulence fidelity variants of rna dependent rna polymerases uncover an indirect, mutagenic activity of amiloride compounds history and geographic distribution of venezuelan equine encephalitis role of dendritic cell targeting in venezuelan equine encephalitis virus pathogenesis pathways to extinction: beyond the error threshold crystal structure of poliovirus cd protein: virally encoded protease and precursor to the rna-dependent rna polymerase design of a genetically stable high fidelity coxsackievirus b polymerase that attenuates virus growth in vivo use of an attenuated strain of venezuelan equine encephalo-myelitis virus for immunization in man attenuation of human enterovirus high-replication-fidelity variants in ag mice highly immunostimulatory rna derived from a sendai virus defective viral genome structural dynamics as a contributor to error-prone replication by an rna-dependent rna polymerase the relation of recombination to mutational advance viral mutation rates association of venezuelan equine encephalitis virus subtype ie with two equine epizootics in mexico epistatic interactions within the influenza a virus polymerase complex mediate mutagen resistance and replication fidelity', msphere isolation of the vaccine strain of venezuelan equine encephalomyelitis virus from mosquitoes in louisiana a single mutation in poliovirus rna-dependent rna polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity emergence of a new epidemic/epizootic venezuelan equine encephalitis virus in south america ires-based venezuelan equine encephalitis vaccine candidate elicits protective immunity in mice yellow fever vaccine: past, present and future alphavirus mutator variants present host-specific defects and attenuation in mammalian and insect models ribavirin-resistant mutants of human enterovirus express a high replication fidelity phenotype during growth in cell culture venezuelan equine encephalitis virus in the mosquito vector aedes taeniorhynchus: infection initiated by a small number of susceptible epithelial cells and a population bottleneck postepizootic persistence of venezuelan equine encephalitis virus epidemic venezuelan equine encephalitis in north america in : vector studies immunostimulatory defective viral genomes from respiratory syncytial virus promote a strong innate antiviral response during infection in mice and humans defective viral genomes arising in vivo provide critical danger signals for the triggering of lung antiviral immunity chikungunya virus emergence is constrained in asia by lineage-specific adaptive landscapes sequence-specific fidelity alterations associated with west nile virus attenuation in mosquitoes two chikungunya isolates from the outbreak of la reunion (indian ocean) exhibit different patterns of infection in the mosquito, aedes albopictus quasispecies diversity determines pathogenesis through cooperative interactions in a viral population venezuelan equine encephalomyelitis virulence and viremia characteristics of epizootic subtype ic venezuelan equine encephalitis viruses and closely related enzootic subtype id strains re-emergence of epidemic venezuelan equine encephalomyelitis in south america foot-and-mouth disease virus low-fidelity polymerase mutants are attenuated ribavirin-resistant variants of foot-and-mouth disease virus: the effect of restricted quasispecies diversity on viral virulence the authors wish to thank antonio muruato and sasha azar for their assistance with animal work, maria (lola) alcorn for her aid with virus collections, and lark coffey for her helpful comments while reviewing the draft. research reported in this publication was supported by niaid of the national institutes of health under award numbers r -ai - a and r -ai - . tfk was supported by a predoctoral fellowship from the utmb mclaughlin endowment as well as a predoctoral fellowship from the nih/niaid emerging and tropical infectious diseases training program (t ai ). data are not publically available, but will be provided upon request. supplementary data are available at virus evolution online.conflict of interest: none declared. key: cord- -ko bdvo authors: vasilakis, nikos; tesh, robert b.; popov, vsevolod l.; widen, steve g.; wood, thomas g.; forrester, naomi l.; gonzalez, jean paul; saluzzo, jean francois; alkhovsky, sergey; lam, sai kit; mackenzie, john s.; walker, peter j. title: exploiting the legacy of the arbovirus hunters date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: ko bdvo in recent years, it has become evident that a generational gap has developed in the community of arbovirus research. this apparent gap is due to the dis-investment of training for the next generation of arbovirologists, which threatens to derail the rich history of virus discovery, field epidemiology, and understanding of the richness of diversity that surrounds us. on the other hand, new technologies have resulted in an explosion of virus discovery that is constantly redefining the virosphere and the evolutionary relationships between viruses. this paradox presents new challenges that may have immediate and disastrous consequences for public health when yet to be discovered arboviruses emerge. in this review we endeavor to bridge this gap by providing a historical context for the work being conducted today and provide continuity between the generations. to this end, we will provide a narrative of the thrill of scientific discovery and excitement and the challenges lying ahead. almost years have passed since walter reed, james carroll, aristides agramonte, and jesse lazear established that yellow fever is caused by a filterable infectious agent which is transmitted by the bite of a mosquito, then known as stegomyia fasciata (aedes aegypti). lazear, who like his colleagues, had been stationed by the us army in cuba to study the disease, died of yellow fever in september after being exposed experimentally to mosquitos that had fed on sick patients. at about the same time in south africa, james spreull and sir arnold theiler demonstrated that bluetongue disease of sheep is caused by an "ultravisible" agent that could be transmitted by the injection of an infected serum. epidemiological evidence suggested that the agent was vector-borne, and it was subsequently shown by r.m. du toit that the disease occurred in sheep inoculated experimentally with suspensions of wild-caught biting midges (culicoides imicola). these and other seminal discoveries precipitated a century of research into vector-borne and zoonotic viral diseases, resulting in the discovery and isolation of many hundreds of novel viruses from insects or vertebrate hosts. some were identified as important human or veterinary pathogens. many other viruses were archived in reference collections, with only basic characterization of their biological or molecular properties. in recent years, the advent of next generation sequencing (ngs) has transformed this situation. complete genome sequences are now available for many of the archived isolates, allowing more accurate taxonomic assignments, analysis of their phylogenetic and evolutionary relationships with other viruses, and evaluation of the potential risks they may present to humans and wild or domestic animal populations. ngs has also opened the door to viral metagenomics, which has greatly increased the pace of new virus discovery from a wide range of hosts, usually with complete or near-complete viral coding sequences, but no virus isolate and minimal biological data. this has presented both opportunities and challenges for virologists and epidemiologists, as well as viral taxonomists, evolutionary biologists, and bioinfomaticians. sadly, this technological revolution has been accompanied by a period of progressive disinvestment in training in classical virology. in this review, we recall the rich history of the discovery of arboviruses and other zoonotic viruses in various settings around the world and the many outstanding scientists who have contributed to the endeavour. we also consider the impacts of ngs and metagenomic analysis, and the implications of these new technologies for the future of this important field of research. the rockefeller foundation (rf) was organized in for "the well-being of mankind throughout the world" [ ] . at the time, yellow fever was still epidemic in many tropical and subtropical regions of africa and the americas, so in , the rf established the yellow fever commission, with the lofty goal of eradicating yellow fever from the world. during the next years, the rf supported an international group of scientists working on yellow fever in new york city in the united states (u.s.); rio de janeiro and salvador in brazil; bogota, colombia; yabba, nigeria; and entebbe, uganda [ , ] . much of the work and accomplishments of rf-funded personnel during this period was described in strode's classic book, entitled "yellow fever" [ ] . the major accomplishments included: confirmation of earlier work by the reed commission in cuba, demonstrating that yellow fever was caused by a filterable agent, yellow fever virus (yfv), that was transmitted by the bite of infected mosquitoes, aedes aegypti; . discovery that the rhesus monkey and the white mouse are susceptible to infection with yfv, providing models for subsequent studies on the pathogenesis, transmission, epidemiology, and control of the disease; . demonstration that convalescent sera of humans and animals infected with yfv neutralize the virus. this discovery led to the development of the mouse neutralization test, which allowed investigators to map the geographic distribution of the virus; . discovery of the forest or sylvan cycle of yfv and the importance of mosquitoes other than ae. aegypti in the transmission and maintenance of the virus; . development of the d vaccine strain of yfv and its first human trials. max theiler, son of south african bluetongue researcher sir arnold theiler, received a nobel prize in for this work. as a by-product of the overseas yellow fever investigations, rf-funded researchers also isolated a number of other previously unknown arboviruses, including west nile, zika, semliki forest, bunyamwera, bwamba, uganda s, ilheus, and anopheles a and b viruses [ ] . the threat and onset of world war ii changed the priorities of the rf, and most of its overseas yf research activities ceased during this period. many of the former american rf staff became involved in studies of diseases of military importance, such as typhus, malaria, sandfly fever, and dengue, some as civilians and others as members of the u.s. armed forces. in , after the end of the war, the rf decided to develop a major new program to study arthropod-borne viruses and to discover "what might be out there", as well as their disease associations, life cycles, and vectors. this led to the development of the rockefeller foundation virus program, a world-wide virus discovery program that was funded from to [ ] . over the next several years, field laboratories staffed by both rf and local scientists were established with foreign governments in pune, india; port of spain, trinidad; belem, brazil; johannesburg, south africa; ibadan, nigeria; cali, colombia; and cairo, egypt [ ] . in egypt, the cairo laboratory was associated with the u.s. naval medical research unit no. . scientists in these field laboratories were involved in the detection and investigation of human diseases in their respective geographic regions, surveying human and animal populations for serologic evidence of past viral infection, and searching for viruses in a wide variety of arthropods, mammals, birds, reptiles, and amphibians [ ] . all virus isolations were done on site, using the classic technique of intracranial inoculation of newborn white mice, but later, as vertebrate cell cultures became available, inoculation of cell cultures was also used. in addition, sentinel animals, such as non-human primates, mice, and hamsters, were also used in the field to detect virus activity. viruses isolated in the overseas laboratories were initially characterized locally, lyophilized for preservation and storage, and then aliquots of each new virus were shipped back to the rf central virus laboratory in new york for further characterization and study. it was during this period that jordi casals, delphine clarke, loring whitman, and others developed the sucrose-acetone method for preparation of viral antigens and began to adapt the hemagglutination inhibition (hi) and complement fixation (cf) tests to identify and group arboviruses [ ] [ ] [ ] . the rf virus program was extremely productive and many novel arboviruses, as well as non-arthropod-borne viruses (i.e., hantaviruses and arenaviruses), were discovered by rf-funded investigators during this period. the productivity of this search strategy in detecting novel viruses affecting humans was recently reviewed by rosenberg et al. [ ] . the virus discovery rate was highest in the period - , which coincided with the rf virus program. a similar approach was also practiced by the institut pasteur and other international groups involved in virus discovery during this period, as described in this article and in other publications [ ] [ ] [ ] [ ] [ ] [ ] , resulting in the detection of a high proportion of pathogenic arboviruses. the second advantage of the strategy was that it yielded actual virus isolates, whose pathogenesis could be studied experimentally in vivo and in vitro in vertebrate and arthropod models. in contrast, current search methods for new viruses, which generally use metagenomics and other sophisticated genetic techniques to detect novel viral agents, do not usually yield live viruses, only their nucleotide sequences. a complete or partial genetic sequence alone rarely provides insight into the epidemiology and ecology, host range, pathogenesis and disease potential, or transmission modes of the new viruses. in , the rf made a decision to phase-out its virus program and to devote more of its efforts and resources to other projects, such as population control and increasing food production ("the green revolution"). over the next years, the rf-funded investigators working in overseas laboratories were withdrawn and the respective laboratories were turned over to local institutions or governments. in , the rf made arrangements with yale university to transfer its arbovirus group to new since the establishment of the institut pasteur in paris in , louis pasteur sent collaborators to various countries, mainly in the french colonies of indochina and africa. in that early time, pasteur wanted to set up rabies centers where the disease was highly and dramatically prevalent; naturally, the research potential of these centers was rapidly extended to tropical infectious diseases [ ] . currently, spread among countries on five continents, there are institutions, of which bear the name of "institut pasteur" (ip). altogether, these institutions constitute a structure long called the "institut pasteur d'outre-mer", which in became the international network of pasteur institutes (réseau international de l'institut pasteur, riip) and associated institutes. from this emerging network, the first african laboratory for microbiology was created in saint louis, senegal in , and then transferred to dakar to become the pasteur institute of dakar (institut pasteur de dakar, ipd yellow fever was to play a key role in the research at ipd. in the s, ipd had developed a vaccine against yellow fever (strain fnv) and produced it commercially. the re-emergence of yellow fever in africa in the s occurred mainly in the savannah zone and revealed the lack of knowledge about the natural maintenance of the virus during the inter-epidemic period. a large study to understand the emergence and re-emergence of sylvatic yellow fever was established between the ipd, institute pasteur abidjan (ipa) in côte d'ivoire, and institute pasteur bangui (ipb) in the central african republic, in order to detect the circulation of the yfv and map its emergence in the different ecozones. permanent research stations were developed in rain forests and savannas to detect virus circulation in vectors and hosts from the tree canopies to the savanna ground. year-round mosquito and monkey sampling over a period of more than years made possible the detection of yfv in various monkey and mosquito species (e.g., aedes africanus, aedes opok, aedes furcifer-taylori, and aedes luteocephalus), thus elucidating the mechanism of yfv maintenance in nature. epidemiological observations during this period allowed max germain to formulate the concept of a "yellow fever zone of emergence" in west and central africa [ ] . these ecological transition zones constitute ecotones adjacent to sylvatic environments, where prevailing ecological conditions, such as vector abundance, presence of non-human primates, and closeness for human contact, enable the cross-species transmission of viruses into humans-a "zone of emergence", which clearly appeared as the main source of epidemics in west africa [ ] . thus, this specific ecosystem constitutes an ideal transition ecotype, where vaccination campaigns for the containment of epizootics and ultimately eradication ought to concentrate [ ] . lastly, virus isolation in male mosquitoes (ae. furcifer-taylori) allowed the documentation of vertical transmission, allowing for virus maintenance in the inter-epidemic periods [ ] . in the early s, research laboratories focusing broadly on arboviruses were established under the umbrella of the collaborating center for reference and research on arboviruses (crora), led by paul brés. crora laboratories were created at various ips throughout africa, including ipd, ipa, and ipb, as well as ip yaoundé (cameroon), and ip tananarive (madagascar). one of the major activities was to establish an inventory of arboviruses circulating in various ecosystems. although virus isolations were made at various ips, virus characterization and identification were carried out at the crora reference center at ipd, followed by confirmation at yaru, with final registration in the international arbovirus catalogue [ ] . at the end of the s, following an outbreak of the ebola epidemic in zaire in , ipb set up a research program on viral hemorrhagic fevers that lasted until [ ] . this research program was the result of an important collaboration between the various pasteur institutes in africa and researchers of orstom. in , orstom extended its field of research and expertise to tropical medical virology, thus supporting teams from the riip. the partnership between riip and ostrom was instrumental in expanding the scope of field research in africa, with seminal studies on the vertical transmission of arboviruses in mosquitoes [ ] or the role of environmental factors, such as climate and latitude, in arbovirus transmission from arthropod to vertebrate hosts, using yfv [ , ] and dengue virus (denv) [ ] [ ] [ ] as models. research at ipb was also critical in elucidating the etiology of exanthematous fevers, coined "congolese red fevers", that have long been attributed to rickettsia. a total of arboviruses (chikungunya, igbo-ora, o'nyong nyong, sindbis, bouboui, yellow fever, wesselsbron, west nile, zika, ilesha, bwamba, dugbe, tataguine, nyando, bangui, and rift valley fever viruses) were associated with these syndromes. symptoms, consisting of fever, diffuse pain, and exanthem, were present in more than % of the cases, with etiology dominated by four viruses-chikungunya, ilesha, bwamba, and tataguine [ , ] . between and , two field research stations were established in the central african republic, one located adjacent to the forest (bouboui) and the other in the wooded savanna (bozo) (figure ). during this period, more than , mosquitoes of identified species in , pools were preserved for virus isolation. the most common anthropophilic mosquitoes caught were aedes (stegomiya) africanus and ae. (st.) opok, inside the forest gallery, aedes vittatus, in the savannah, and anopheles gambiae and an. funestus, in the houses of the village of bozo. a total of viruses were isolated and assigned to different species. these included chikungunya, bagaza, bouboui, bozo, bwamba, ilesha, kamese, kedougou, middleburg, mossuril, m'poko, nyando, orungo, pata, pongola, simbu, sindbis, tataguine, wesselsbron, west nile, yellow fever, and zika viruses. altogether, six arboviruses were found in the forest gallery, including bouboui, bozo, chikungunya, orungo, yellow fever, and zika viruses, vectored primarily by ae. africanus. research at ipd and ipb was also instrumental in demonstrating the expanded range of the crimean-congo hemorrhagic fever virus (cchv) in west and central africa [ ] [ ] [ ] , followed by repeated isolation of the virus. this allowed a clear understanding of its eco-epidemiology in the region, including north-south migration of the infected ticks through the cattle traffic migration patterns in the sahel [ , ] . likewise, active circulation of the rift valley fever virus (rvfv) was also demonstrated in senegal [ ] , mauritania [ ] , upper volta (present day burkina faso) [ , ] , and the central african republic [ ] . support for riip laboratories located in africa was provided by the ipp and ostrom teams, as well as through collaboration with various research centers in the u.s., such as harvard university, supporting the initial study on yellow fever and fnv vaccine; yaru, as a partner for new arbovirus classification; the centers for diseases control (cdc) at fort collins colorado, for the study of arboviruses; the cdc in atlanta and the u.s. army medical research institute of infectious diseases (usamriid), for the initiation of viral hemorrhagic fever research in central and west africa. the era of virus discovery in australia can be traced to the summer of - , when a major epidemic of encephalitis swept through southeastern australia. there were clinical cases reported in victoria, new south wales, and south australia, of which ( %) were fatal [ ] . a similar epidemic of unknown etiology (named australian x disease) had occurred in eastern australia from until , with almost reported cases and an average case/fatality rate of % [ , ] . surprisingly, no further cases were reported in the intervening years. amongst those investigating the - epidemic were john a.r. miles and colleagues at the institute of medical and veterinary science in adelaide, and eric l. french of the walter and elisa hall institute of medical research in melbourne who, almost simultaneously, reported the isolation of a virus from the brain tissue of clinical cases [ , ] . the virus, named murray valley encephalitis virus (mvev), was shown to be closely antigenically related to japanese encephalitis virus (jev), a flavivirus (then designated group b arbovirus) known to cause fatal encephalitis in east and southeast asia [ ] . the subsequent development of arbovirology and the pathway to virus discovery in australia were linked intimately with the establishment of the queensland institute of medical research (now the qimr-berghofer institute) at herston in brisbane ( figure ). ralph l. doherty joined the staff and, in , established a program of virus isolation from mosquitoes, based at a field station at innisfail in the far north queensland. in , doherty isolated the ross river virus (rrv) from aedes vigilax mosquitoes collected in townsville [ ] . he subsequently showed that rrv neutralizing antibodies occurred commonly in human sera in eastern australia [ ] . he also showed that individuals suffering from a severe debilitating syndrome, known as epidemic polyarthritis, had high antibody titres to rrv, suggesting a causal relationship [ , ] . in , doherty and his colleagues isolated the virus from a boy from the edward river mission aboriginal settlement in cape york [ ] . rrv and the related alphavirus, the barmah forest virus (see below), are now recognized as important public health problems in much of australia and the pacific islands, causing arthritis, myalgia, and fatigue for six months or longer. several thousand cases of the disease are notified annually [ ] . , normanton, and cairns in far north queensland. these included four novel flaviviruses (kunjin, kokobera, edge hill, and stratford), three orthobunyaviruses (koongol, wongal, and maputta) and one orbivirus (corriparta) [ ] . the study also identified two alphaviruses previously unknown in australia (sindbis and getah) and the first isolations of mvev from mosquitoes [ ] . with support from the rockefeller foundation, a field station was established at kowanyama and further expeditions were undertaken to collect arthropods and potential mammalian hosts throughout queensland. anopheline mosquitoes and a swamp pheasant (centropus phasianinus) collected at kowanyama from to yielded three novel viruses (kowanyama, trubanaman, and alfuy viruses) [ ] . in - , three novel viruses were isolated at kowanyama (wongorr and mitchell river viruses from mosquitoes and the ngaingan virus from biting midges), three novel viruses were isolated from mosquitoes collected near charleville in western queensland (charleville, warrego, and wallal viruses) and two viruses (belmont and d'aguilar viruses) were isolated from mosquitoes and biting midges, respectively, collected near brisbane [ , ] . further expeditions to charleville to collect mosquitoes resulted in the isolation of two novel viruses in (facey's paddock and murweh viruses) and two novel viruses in (parker's farm and little sussex viruses) [ ] . leanyer virus was also isolated in from mosquitoes collected near darwin in the northern territory [ ] . viruses were also isolated from wildlife hosts; the almpiwar virus was isolated from a skink (cryptoblepharus virgatus) at kowanyama in [ ] and the mossman virus was first isolated from a rodent (rattus leucopus) captured near mossman in [ ] . in collaboration with doherty and his qimr team, expeditions were also conducted to isolate viruses from ticks associated with sea birds. in , harald n. johnson from yaru collected soft ticks (ornithodoros capensis) from sooty tern (onychoprion fuscatus) colonies on the great barrier reef near cairns, from which two viruses (upolu and johnston atoll viruses) were isolated [ ] . the saumarez reef virus was subsequently isolated by toby d. st. george and colleagues from australia's commonwealth scientific and industrial research organization (csiro) in , from the same species of ticks associated with sooty terns on a coral cay in the southern coral sea [ ] . in , m. durno murray from csiro undertook an expedition to the australian territory of macquarie island in the southern ocean to collect hard ticks (ixodes uriae) associated with sea birds, resulting in the isolation of two novel viruses (nugget and taggert viruses) [ ] . a second csiro expedition to macquarie island in yielded two additional novel viruses (gadget's gully and precarious point viruses) from hard ticks collected in royal penguin (eudyptes chrysolophus schlegeli) rookeries [ ] . other research groups in australia also joined the hunt for arboviruses during the late s and early s, including ian d. marshall at the australian national university in canberra and neville f. stanley at the university of western australia. from to , marshall and his colleagues conducted surveys for arbovirus activity, particularly rrv and mvev, in coastal regions of new south wales and in the murray river valley. in addition to these and other known arboviruses, marshall and colleagues isolated several novel arboviruses from mosquitoes, including gan gan, yacaaba, tilligerry and termeil, paroo river, picola, and barmah forest viruses [ , ] and a novel reovirus, nelson bay virus, from a fruit bat (pteropus poliocephalus) [ ] . like the related alphavirus rrv, the barmah forest virus was subsequently shown to be a cause of epidemic polyarthritis in humans [ , ] , with infections occurring commonly throughout australia [ ] . the gan gan virus also infects humans and is suspected of an association with epidemic polyarthritis [ ] . marshall also conducted a number of collecting trips to papua new guinea from to funded by the rockefeller foundation. during an expedition to the sepik river district of papua new guinea in - , he isolated the joinjakaka virus from a mixed pool of culicine mosquitoes and the japanaut virus from both culicine mosquitoes and a fruit bat (syconycteris crassa). in western australia, stanley and colleagues surveyed for arbovirus activity in the ord river valley from to [ , ] . from , mosquitoes of species, virus isolates were recovered, including isolates of mvev and isolates of the kunjin virus from cx. annulirostris, suggesting the region may be an endemic focus in australia [ ] . the study also identified eight novel viruses, including kimberly, parry's creek, ord river, and kunnanurra viruses, as well as four unknown isolates (or , or , or , and or ), which have yet to be characterized [ , ] . continuing surveillance in western australia by others has continued to reveal novel arboviruses, including oak vale, stretch lagoon, parry's lagoon, and fitzroy river viruses [ ] [ ] [ ] [ ] . in , csiro established a new virology laboratory at long pocket in brisbane, headed by toby d. st. george, to investigate endemic diseases of livestock in northern australia. in , doherty and colleagues had isolated bovine ephemeral fever virus (befv) from cattle during a major epizootic in queensland [ ] but, despite epidemiological evidence suggesting vector-borne transmission, the virus had never been isolated from insects. this led st. george and colleagues to attempt virus isolations from a location in northern australia, where serological monitoring of a sentinel herd of cattle indicated that befv was likely to be enzootic. for a continuous period from october until may , insect collections for virus isolation were conducted at beatrice hill southeast of darwin. from the , mosquitoes and , biting midges processed, one isolate of befv was recovered (from a mosquito pool). however, the collection also yielded other virus isolates from different serological groups, including four novel viruses (csiro village, marrakai, beatrice hill, and humpty doo virus) [ ] . most significantly, the collection also yielded a single isolate of a novel serotype of bluetongue virus (btv serotype , btv- ), a major pathogen of sheep and goats that had previously been regarded as exotic to australia [ ] . the isolation of btv- and the consequences for international trade dramatically changed the landscape with respect to virus discovery and characterization in australia. an immediate consequence was the approval of government expenditure for the establishment of the $ million csiro australian animal health laboratory (aahl) in geelong, victoria, providing high-level biosecure containment for laboratory work and live animal studies. the discovery also led to the establishment of a permanent veterinary virology capability at the berrimah veterinary laboratory in darwin under geoff p. gard, and a national program for serological monitoring of sentinel cattle herds and the collection of insect vectors. efforts to isolate viruses from arthropods and livestock intensified. in the northern territory, a second novel serotype of bluetongue virus (btv- ) was isolated from a healthy sentinel cow at victoria river station in [ ] , and four other novel arboviruses (coastal plains, berrimah, adelaide river, and koolpinyah viruses) were isolated from healthy cattle between and [ ] [ ] [ ] [ ] . in queensland, eight novel arboviruses were first isolated between and from biting midges (tibrogargan, tinaroo, peaton, wongabel, and walkabout creek viruses), healthy sentinel cattle (the douglas virus), and soft ticks (argas robertsi) (vinegar hill and lake clarendon viruses) [ , [ ] [ ] [ ] [ ] [ ] . surveillance activities by the berrimah veterinary laboratory have continued to the present, with regular reports of the isolation of novel arboviruses. continuing surveillance by others in northern australia has also resulted in the isolation from mosquitoes of the bamaga virus and new mapoon virus from cape york [ , ] . the s also saw several significant disease emergence events in australia, which drew particular attention to bats as reservoir hosts of highly pathogenic viruses. in september , an outbreak of a severe respiratory disease occurred in horses at a stable in brisbane. of the affected horses were euthanized or died of the disease. one of two severely affected humans who had contact with the horses also died. cooperation between the queensland government, the newly established csiro australian animal health laboratory, and others resulted in rapid isolation of the hendra virus, a novel paramyxovirus [ ] , and the identification of fruit bats as reservoir hosts [ , ] . the hendra virus has since re-emerged regularly in australia, with more than confirmed cases in horses and seven infected humans, four of whom have died. in , an injured female fruit bat (pteropus alecto) was found at ballina in new south wales. tissue homogenates from the euthanized bat were injected into mice, resulting in the isolation of a novel lyssavirus, subsequently named australian bat lyssavirus (ablv) [ ] . three fatal human cases of ablv infection have subsequently been reported [ ] [ ] [ ] . the virus is now known to occur at low prevalence in five of six families of bats endemic to the northern territory, queensland, and western australia [ ] . in april , another novel paramyxovirus, the menangle virus, emerged at a commercial piggery in new south wales, causing stillbirths with abnormalities of the brain, spinal cord, and skeleton [ ] . two humans exposed to the pigs also developed an influenza-like illness [ ] . fruit bats were again implicated as reservoir hosts [ ] . the role of bats in the ecology and emergence of pathogenic viruses has been a major focus of study in australia since that time, primarily involving research teams led by hume e. filed and linfa wang. in all, more than novel rna viruses representing families and genera have been isolated from humans, livestock, wildlife, and arthropods in australia and papua new guinea, and reported in the literature (table s ). many others have been isolated but remain uncharacterized and/or unreported. more complete characterization of these viruses will be facilitated greatly by the use of ngs. south and southeast asia have also been a fertile area for virus discovery, particularly novel mosquito-borne and tick-borne flaviviruses and viruses with reservoirs in bat species. early studies in india, partly funded by the rockefeller foundation, by telford work and his indian colleagues from the virus research centre in pune, including d.p. murthy, p.n. bhatt, h. trapido and k. pavri, led to the discovery of the kyasanur forest disease (kfd) virus [ ] . the virus was isolated from sera and tissues collected from a moribund black-faced langur (presbytis entellus). this followed reports of an epizootic of unknown etiology causing large numbers of deaths in non-human primates and a number of cases of severe febrile illness in villages close to forested areas where dead monkeys had been found. the virus was shown to be closely related to the russian spring-summer encephalitis (rsse) serocomplex of flaviviruses, now known as the tick-borne encephalitis (tbe) serocomplex of flaviviruses. the virus was also isolated from some larvae and nymphs of hemaphysalis spinigera ticks [ ] . kfd in humans followed a biphasic course, not unlike tbe, but with some hemorrhagic manifestations not seen in tbe, and without either meningitis or encephalitis. another tick-borne virus related to the rsse serocomplex, langat virus, had been isolated two years earlier from a pool of hard ticks, ixodes granualtus, collected from forest rats caught near kuala lumpur, malaysia, by c.e. gordon smith, then working at the institute for medical research in kuala lumpur [ ] , but it is not known to be a human pathogen. a number of mosquito-borne flaviviruses were first isolated in southeast asia. the most important with respect to human disease are three of the four dengue serotypes. although dengue serotype had been first isolated independently by hotta in japan in [ ] , and shortly after by sabin in cincinnati in with material collected in hawaii [ ] , the other three serotypes were first isolated from material collected in southeast asia. dengue serotype was also isolated by sabin in from material obtained from new guinea [ ] and dengue serotypes and were first isolated in from human sera and aedes aegypti and culex tritaeniorhynchus mosquitoes collected during a major outbreak of epidemic hemorrhagic fever in manila, philippines, by w.m. hammon, a. rudnick, and colleagues at the university of pittsburgh [ ] . other novel flaviviruses have been isolated in malaysia, thailand, and papua new guinea [ ] . the tembusu (tmuv) virus was isolated in kuala lumpur in from various mosquito species [ ] and was the first of several closely related viruses, including the thcar virus, which was isolated from a pool of cx. tritaeniorhynchus mosquitoes collected in chiang mai, thailand, in [ ] ; the sitiawan virus, from sick broiler chicks in malaysia [ ] ; and the duck tembusu virus, an infection of ducks and geese causing an egg-drop syndrome in china and southeast asia [ , ] . neutralising antibodies were found in humans in malaysia [ ] but the virus has not been implicated in human disease. two other mosquito-borne flaviviruses have been described, jugra virus and sepik virus. little is known about the jugra virus, which was isolated from aedes spp. and uranotaenaia spp. mosquitoes and from the blood of a cynopterus brachyotis fruit bat [ ] . the sepik virus was isolated in by ian d. marshall and colleagues from a pool of mansonia septempunctata mosquitoes collected in the sepik district of papua new guinea [ ] . it was associated with a hospitalized case of febrile illness of unknown origin, with rising neutralising antibody to the virus. the sepik virus is particularly interesting, as its nucleotide sequence analysis shows it to be the closest known flavivirus to yellow fever virus [ ] . a novel lineage of the west nile virus was isolated in sarawak, east malaysia, by d.i.h. simpson, e.t.w. bowen, and colleagues, from cx. pseudovishnui group mosquitoes [ ] . initially called kunjin virus, it was shown to differ significantly in genomic sequence from the australian kunjin viruses, which have been shown to comprise west nile lineage b viruses, and have been described as west nile lineage virus [ ] . two flaviviruses with no known vector have been isolated in southeast asia, both from cy. brachyotis fruit bats: the carey island virus was isolated from a bat in the jugra forest, malaysia, in by a. rudnick and colleagues from the institute of medical research, kuala lumpur and the international center for medical research, university of california [ ] , and the phnom penh virus was isolated by j.j. salaun and colleagues in from the salivary glands and brown of bats [ ] . a closely related virus, the batu cave virus, is considered to be a variant of phnom penh virus. a considerable number of novel bunyaviruses have been isolated from south and southeast asia, particularly by scientists from the national institute of virology (formerly the virus research centre) in pune, including p.n. bhatt, k. pavri, k.r. singh, c.n. dandawate, f.m. rodrigues, d.t. mourya, p.d. yadev, a.c. mishra, and many others. recently reviewed in [ ] , these viruses include the orthobunyaviruses, umbre, kaikalur, thimiri, and sathuperi viruses; a nairovirus, ganjam virus; phleboviruses, bhanja, and malsoor viruses; a hantavirus, thottapalayam virus; and two uncharacterized viruses, kaisodi and wanowrie viruses. additionally, novel bunyaviruses, batai and oya viruses, have been isolated in malaysia, the former from cx. gelidus mosquitoes [ ] and the latter from pigs [ ] , and the kaeng khoi virus was isolated from tadarida plicata bats in thailand. novel orbiviruses from south and southeast asia include the sathuvachari virus, isolated from starlings (brahminy myna) collected in vellore, tamil nadu, india, and most closely related to the mosquito-borne orbiviruses [ ] ; and the japanaut virus, isolated from a mixed pool of culicine mosquitoes from papua new guinea [ ] . new rhabdoviruses first isolated in south asia include the chandipura virus and joinjakaka virus-the former, a major human pathogen in india, was isolated from a human infection in near nagpur city [ ] , whereas the latter, isolated in from a mixed culicine pool in the sepik district of papua new guinea, is not associated with disease in humans or animals. chandipura infection is characterized by fever, chills, arthralgia, myalgia, vomiting, and weakness. two novel alphaviruses were reported in kuala lumpur-bebaru and getah viruses. bebaru was first isolated from cx. (lophoceraomyia) spp. collected in , but although neutralising antibodies have been found in human sera, it has not been associated with human disease [ ] . getah virus was first isolated from cx. gelidus mosquitoes collected in near kuala lumpur [ ] . it causes a mild disease in horses, characterized by pyrexia, edema of the hind limbs, swelling of the submandibular lymph nodes, and urticarial rash. it also causes a mild disease in pigs, with occasional reproductive problems, including abortion and neonatal infections. neutralising antibodies have been found in a number of animals and in humans. the important role of bats as reservoirs of a wide range of viruses was underlined by a number of the viruses described above, and particularly by the discovery of their role as the reservoir of the nipah virus in malaysia in [ ] and subsequently their probable role as the origin of severe acute respiratory syndrome coronavirus (sars-cov) [ , ] . the nipah virus, a virus closely related to the hendra virus in australia, was first isolated k.b. chua and s.k. lam during an outbreak of severe disease of humans and pigs in - in peninsula malaysia [ ] , resulting in human cases with a mortality of %, and the culling of over million pigs. transmission to humans was from infected pigs. the disease in humans was a rapidly progressive encephalitic syndrome, with a significant pulmonary syndrome in some patients [ ] . in pigs, the disease was spread via the respiratory tract, and the symptoms were either neural or pulmonary, or both. subsequent epidemics in bangladesh and india have substantially expanded our knowledge of the nipah virus, and have demonstrated that direct transmission of the virus from bats to humans can occur through the consumption of date palm juice, and possibly by other routes, and that mortality rates may often be significantly greater than in malaysia [ ] . furthermore, evidence of nipah-like and hendra-like viruses have been detected either by isolation or serology from other pteropid bats across the geographic range of the genus, and related viruses may be carried by other bat species on other continents. sars-cov was first isolated by m. peiris and colleagues in hong kong [ ] , and contemporaneously in the u.s. [ ] and europe [ ] . it was shown to be unrelated to other coronaviruses. transmission to humans is believed to have been via an intermediate host, such as the himalayan palm civets (paguna larvata), through wet markets in southern china. continued studies of the nipah virus in malaysia, and subsequently in bangladesh and india, and investigations of sars-cov in bats have resulted in an enormous explosion of knowledge of viruses carried by bats, with many examples from most viral families, although in many cases the information is from genomic fragments [ ] . virus isolations have been made from bats, especially frugivorous bats, from india and malaysia. one of the earliest isolations was a paramyxovirus of the genus rubulavirus, which was isolated from a rousettus leschenaultia bat collected near pune in india [ ] . a novel adenovirus from the genus mastadenovirus was also isolated from the same fruit bat species caught in maharashtra state [ ] . a number of viruses have been isolated from fruit bats in malaysia by k.b. chua, s.k. lam, l.f. wang, and their colleagues-tioman virus, a paramyxovirus in the genus rubulavirusi, isolated from pteropus hypermelanus and related to the australian menangle virus, but not known to cause human disease [ ] ; pulau virus, an orthoreovirus related to the nelson bay virus of australia, and not associated with human or animal disease [ ] ; melaka virus, an orthoreovirus, causing acute respiratory disease in humans [ ] ; and kampar virus, an orthoreovirus related to the melaka virus, and also causing acute respiratory disease [ ] . the importance of orthoreoviruses originating in pteropid bats was assessed in an outpatient clinic, where it was found that pteropine orthoreoviruses are among one of the common causative agents of acute upper respiratory tract infection (urti), with a cough and sore throat as the most common presenting clinical features [ ] . the history of arbovirus research in the ussr began in , when an expedition under the leadership of lev a. zilber (at the time, the head of central virological laboratory of narkomzdrav ussr in moscow) went to the russian far east to study seasonal epidemic encephalitis. this disease with high mortality rates affected forest workers and soldiers stationed in the taiga, mainly those who came from other regions of the ussr. the disease had a pronounced seasonality-the cases started being recorded at the beginning of may, reached the peak in early june, and declined by august. local doctors designated the disease "spring-summer encephalitis" (sse) and assumed that it had been caused by some kind of virus. it has also been suggested that there were some similarities between sse and "summer encephalitis" (japanese b encephalitis and st. louis encephalitis) described at the time, it was also assumed to be a toxic form of influenza [ ] . however, the etiology and transmission routes of sse remained unclear. during summer of , zilber and colleagues isolated at least strains of a new virus from the blood and cerebrospinal fluid of sick people, and from brain tissues of dead patients. the isolated virus had a weak antigenic relationship (in complement fixation tests) with japanese b encephalitis virus [ ] . based on comparative analysis of epidemiologic data and the seasonal abundance of ixodes persulcatus ticks in the taiga, zilber assumed that sse was transmitted by ticks, in contrast to "summer encephalitis", which is transmitted by mosquitoes [ ] . several strains of the virus were isolated from the ix. persulcatus ticks, and their ability to transmit the virus by biting laboratory animals was shown experimentally [ ] . one of the first isolated strains (sofjin) was used for infecting rhesus macaques, which developed the clinical symptoms with signs of central nervous system (cns) impairment, similar to those in sick people [ , ] . so, the etiological agent of sse, which was subsequently given the name tick-borne encephalitis, was discovered and is now known by the name tick-borne encephalitis virus (tbev). in - , subsequent expeditions under the leadership of pavlovsky and smorodintsev studied in detail various aspects of the ecology, epidemiology, and pathogenesis of tbev, as well as the protective properties of the first anti-tbe vaccine, obtained from the brain tissue of mice infected by the tbev strain sofjin [ ] [ ] [ ] . further studies showed that tbev is also prevalent in other regions of the ussr, including the european part, where the main vector of the virus is ix. ricinus ticks. at the same time, it was found that tbev is also an etiological agent of some seasonal encephalitis or febrile illnesses, such as central european encephalitis or biphasic milk fever [ ] [ ] [ ] . the strains of tbev were initially divided into two geographical subtypes ("far eastern" and "central european"). these subtypes differed in severity of the illness and had antigenic differences in virus neutralization tests with serum of convalescents [ ] . a third subtype of tbev ("west siberian") was described by pogodina and her colleagues in [ ] . genetic data that has been accumulating since the late s confirms the existence of three main tbev subtypes (or genotypes). the nucleotide difference between genotypes reaches - % when comparing complete genomes [ ] [ ] [ ] . tbe is the most important arboviral infection in russia. despite significant progress in the development of anti-tbev vaccines, thousands of cases are recorded annually in russia, mainly in siberian and far eastern regions [ ] . in the modern classification, tbev belongs to the species tick-borne encephalitis virus of the genus flavivirus (flaviviridae) [ ] . tbev is widely distributed within the area of its main arthropod vector-ix. persulcatus and ix. ricinus ticks, including russia, eastern and central europe, baltic and scandinavian countries [ , ] . the discovery of tbev as a causative agent of sse gave an impetus to studies of similar diseases throughout the ussr. during subsequent years, the major virological centres were established as parts of the academy of medical science of the ussr (ams ussr), such as the department of neurovirology at the institute of neurology ( ), the institute of virology ( ), the institute of poliomyelitis and viral encephalitis ( ), as well as departments of virology at regional medical institutes in siberia and the far east. scientists from these centres were actively involved in the study of various zoonotic viral infections distributed in the ussr. many participants of the first expeditions subsequently became famous virologists. one of the most notable ones is michael p. chumakov, who later headed the institute of virology ams ussr ( ) ( ) ( ) ( ) ( ) and the institute of poliomyelitis and viral encephalitis ams ussr ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) in moscow. chumakov organized numerous expeditions that aimed to study the etiology of zoonotic human infections. in the s, outbreaks of the disease, designated by local doctors as "atypical tularemia", "anicteric leptospirosis", and "omsk spring-summer fever", were recorded in several rural regions of the omsk district in western siberia. clinicians from the omsk medical institute, under the leadership of ahrem-akhremovich, described the disease in detail and named it omsk hemorrhagic fever (ohf), as the patients often developed hemorrhagic diathesis. they also suggested that ohf was transmitted by dermacentor reticulates ticks, which are highly prevalent in the region [ , ] . in , chumakov and colleagues investigated the blood of patients with ohf and isolated strains of a new virus, which was similar but different from tbev in serologic tests. the virus was named omsk hemorrhagic fever virus (ohfv). several strains of ohfv were also isolated from de. reticulates ticks collected in the natural foci of ohf [ , ] . in subsequent years, the ecology of ohfv was extensively studied by scientists from the omsk medical institute and the institute of poliomyelitis and viral encephalitis ams ussr. the de. reticulatus ticks and their host, a narrow-headed vole (microtus gregalis), are considered an original natural reservoir of ohfv. the european water vole (arvicola amphibius), the tundra vole (microtus oeconomus), and some species of shrews are also involved in the circulation of ohfv [ ] . however, the emergence of ohf outbreaks in the s was presumably a consequence of the introduction by humans of muskrats (ondatra zibethicus) to this region in - [ ] . muskrats are highly susceptible to ohfv and serve as an extremely effective amplifying host. the appearance and growth of the muskrat population in the natural foci of ohf led to an increase in infection rates in other animals and ticks [ , ] . in addition to transmission of ohfv by ticks, humans can get infected while hunting and skinning, by direct contact with blood and excretions of infected animals. such "muskrat outbreaks" among hunters and their family members have been registered in the region at different times of the year, including winter, which is the season of active hunting for muskrats [ , ] . based on antigenic relationships, ohfv was assigned to the tbe antigenic complex [ ] , and later was classified as a separate species, omsk hemorrhagic fever virus of the genus flavivirus (family flaviviridae) [ ] . genome sequence analysis confirmed the close evolutionary relationships of ohfv with tbev [ ] [ ] [ ] . in , virologists led by chumakov studied the etiology of an outbreak of a febrile illness which was accompanied with hemorrhagic manifestations ("acute infectious capillary toxicoses") in a rural area in the northwest part of the crimean peninsula. they designated the disease as crimean hemorrhagic fever (chf) and suggested that is transmitted by hyalomma (plumbeum) marginatum ticks. despite the absence of virus isolates from specimens from chf patients or from ticks, the viral etiology of chf and its zoonotic nature were proven experimentally by infecting volunteers with the blood of chf patients or a filtered suspension of ticks collected from a hare caught in the focus of the disease [ ] . sporadic cases and outbreaks of chf were subsequently recorded almost annually in southern regions of the european ussr and central asian soviet republics. the first strains of the chf virus were isolated by alexander m. butenko from chumakov's team in , from sera of chf patients and from hy. marginatum nymphs isolated in southern russia [ , ] . later, the chf virus was shown to be identical to the congo virus isolated from a patient with hemorrhagic fever in zaire (present day democratic republic of congo) and the virus received its present name, the crimean-congo hemorrhagic fever virus (cchfv) [ ] . cchfv is a one of the prototypic nairoviruses and today is assigned to the species crimean-congo hemorrhagic fever virus, of the genus orthonairovirus (nairoviridae: bunyavirales). during spring and summer , chumakov, together with libíková from the institute of virology in bratislava (former cžechoslovakia), investigated an outbreak of fibrile illness in the kemerovo district in western siberia. initially, it was assumed that the patients were affected by tbe, but the sera of the patients did not react with tbev-specific antigen in serological tests. on the contrary, a new virus, named the kemerovo virus (kemv), was isolated from the blood of patients. several strains of kemv were also isolated from ix. persulcatus ticks collected in the region where the outbreak occurred [ , ] . similar to kemv, the tribeč virus and lipovníc virus were isolated from ix. ricinus ticks in czechoslovakia in [ , ] . based on morphological studies, kemv was classified to the genus orbivirus (family reoviridae) [ ] . the ecology of kemv in russia has not been studied sufficiently, but recent research has shown that its prevalence in ix. persulcatus, ix. ricinus, ix. pavlovsky, and de. reticulatus ticks varies from zero to . % in different regions of the country [ , ] . from the above, it follows that in the period - , arboviruses in the ussr were studied mostly as causative agents of human disease. examinations of arthropods and vertebrates in the natural foci of important human disease often led to exploring some other arboviruses. for example, butenko isolated the west nile virus (wnv) and dhori virus (dhov) from hy. marginatum ticks for the first time in the ussr while studying the natural foci of cchfv in the southern region of russia in [ ] . in the late s, there was an ecological trend in virology developing in the ussr. the founder of the ecological approach to virology in the ussr was dmitry k. lvov, who established the department of the ecology of viruses at the d.i. ivanovsky institute of virology in moscow ( ) , and later headed the institute ( - ). under his leadership, an ecological and virological survey was organized, aimed to identify the arboviral diversity in hematophagous arthropods and wild animals of the entire ussr. the survey included collecting and examining mosquitoes, ticks, and vertebrate animals (mostly rodents and birds), as well as samples from humans, in different types of biocenoses located in different climatic zones of the ussr. lvov and colleagues isolated more than strains of different mosquito-borne viruses, including viruses of the california encephalitis antigenic group (species california encephalitis orthobunyavirus) and batai and batai-like viruses (species bunyamwera orthobunyavirus) in the genus orthobunyavirus, family peribunyaviridae [ ] [ ] [ ] [ ] . the other mosquito-borne viruses whose circulation was discovered and studied extensively, are the sindbis virus (sinv) and getah virus (getv) (genus alphavirus, family togaviridae) [ ] [ ] [ ] . one of the important subjects of d. lvov's research was ix. uriae ticks, which parasitize on colony-nesting sea birds. in - , more than virus strains were isolated from ix. uriae ticks collected in the nests of sea birds on the coasts and islands in the sea of okhotsk, the bering sea, and the barents sea [ ] . the isolated strains were mostly classified as novel bunyaviruses, flaviviruses, and orbiviruses, often based on morphological studies of the virion structure only, because their antigenic relationships with other viruses were not known at the time. among them, the sakhalin virus (sakhv) and paramushir virus (prv) were described as a novel bunyaviruses and later classified to the species sakhalin orthonairovirus (genus orthonairovirus, family nairoviridae) [ ] . several other new viruses (zaliv terpenia, comandory, and rucutama viruses) were discovered and now belong to the species uukuniemi phlebovirus (genus phlebovirus, family phenuiviridae) [ ] . the tyuleniy virus (tyuv) was isolated for the first time, which is one of the prototypic viruses of seabird tick-borne flaviviruses group (genus flavivirus, family flaviviridae) [ ] . the prevalence of the okhotsky virus (okhv) and aniva virus (aniv), two newly described viruses belonging to the species great island virus (genus orbivirus, family reoviridae), have been studied in detail [ , , ] . many new viruses were discovered by lvov and his colleagues while exploring the territories of central asia and transcaucasia. they isolated and studied the issyk-kul virus (iskv), which is associated with bats of the family vespertionidae and their argasid ticks [ , ] . new viruses, tamdy (tamv) and burana (burv), were isolated from hyalomma spp. ticks collected from sheep or cows in pasture lands [ ] . some novel viruses (artashat, chim, and geran viruses) were isolated from argasid ticks collected in rodent burrows [ ] . morphological studies of these viruses by electron microscopy identified them as bunyaviruses. recently, they were classified as different species of the genus orthonairovirus (family nairoviridae) [ ] . in total, during the ecological and virological surveys of the s to s, thousands of strains of different arboviruses were isolated, some of which remain to be classified. based on the studies conducted by soviet and russian virologists, we now know that at least zoonotic viruses, assigned to eight viral families, circulate on the territories of the former ussr [ ] . does this number reflect the true diversity of viruses circulating in the vast territory of northern eurasia? this question can only be answered by additional research aimed at finding new viruses, using new methods and approaches. the concept of viruses developed from the observations of ivanovsky and beijerinck of "filterable agents", with the discovery of the causative agent of tobacco mosaic in the s. yellow fever virus and dengue virus were the first two arboviruses to be isolated early in the th century [ , ] . the pioneering work of alexis carrel on the development of many cell and tissue culture methods in the s at the rockefeller institute, and later refinements by maitland, eagle, and enders, led to the widespread use of various culture systems as indispensable tools for virus studies [ ] [ ] [ ] . although these tools have since been used extensively for the in vitro characterization of viruses, they were inadequate for the identification and classification of a flood of novel viruses collected through the yfv surveillance program supported by the rockefeller foundation. jordi casals, among others, led the use of the complement fixation (cf) test in order to study viruses affecting the central nervous system. the cf test exploits the unique affinity of complement for antigen-antibody complexes. the original assay was developed in the s for the serologic study of yfv, was improved in the s [ , ] , and its sensitivity and specificity improved again in the s [ ] . using cf tests, casals and his colleagues were able to classify viruses into antigenic groups. however, the inherent complexity and labour consuming aspects of the assay (titrations of antigen, complement, and hemolysin for optimal outcomes), technical demands (accurate interpretation of outcomes) and the development of alternative assays (see below), have restricted its applicability in laboratories worldwide. hirst observed in that chicken erythrocytes agglutinated in the presence of the influenza a virus and that virus-specific antibodies inhibited agglutination, forming the foundation for the hemagglutination inhibition (hi) test [ ] . a decade later and on theiler's suggestion, casals showed that many arboviruses also agglutinated erythrocytes, establishing the hi test as a diagnostic tool for arbovirus infection and identification [ ] . the gold standard for arbovirus identification, the plaque reduction neutralization test (prnt), has its origins in the observations of stokes and colleagues, dating back in the s when monkeys could be protected against yfv by the inoculation of convalescent sera from patients who had recovered from the disease [ ] . by the early s, max theiler had adapted the assay for use in mice, in which mixed serum and virus was inoculated intracerebrally [ ] . the cell culture adaptation of the test was first demonstrated by itoh and melnick in for studying the seroconversion of chimpanzees infected with echoviruses [ ] , and a year later by henderson and taylor to detect antibodies to the eastern equine encephalitis virus [ ] . versions of this assay are now widely used, including the microprnt [ ] , the virus reduction neutralization test (vrnt) [ ] , the focus reduction neutralization test (frnt) [ ] , the rapid fluorescent inhibition test (rffit) [ ] , the flow-cytometry neutralization [ , ] , the colorimetric micro-neutralization assay (cmnt) [ ] , and the reporter virus particle-based neutralization assays [ ] [ ] [ ] [ ] . historically, these methods (virus isolation, hi, cf, and neutralization tests) served as the basis of arbovirus diagnosis for many years, augmenting electron microscopy (see section below), which allowed the visualization of viruses in infected tissues and cell cultures. however, an inherent limitation of the serology-based assays has been their inability to determine whether antibodies in the examined serum were the result of a recent or past infection. this conundrum was solved by determining whether antibodies were igm (recent infection) or igg (past infection), using the enzyme-linked immunosorbent assay (elisa) [ ] . the introduction of elisa revolutionized the field by also offering increased specificity and sensitivity for the accurate detection of many viruses. overall, although identification of pathologic agents through serologic assays is quite straightforward, there are instances where accurate identification may not be possible due to cross-reactivity. for example, cross-reactivity among flaviviruses poses a challenge in their identification, even when the "gold standard" of prnt for arbovirus detection is applied, especially in hyper-endemic settings of flavivirus circulation. several diagnostic labs faced this challenge during the recent emergence and explosive spread of the zika virus in the americas. a similar challenge is also common for the serologic diagnosis of bunyavirus infections, which is attributed to their ability to reassort. in this scenario, a novel bunyavirus may be misidentified as a known pathogen due to the presence of the m segment (contributed by the known pathogen), which encodes the immune-reactive envelope proteins (reviewed in [ ] ). since the beginning of modern virology in the s, transmission electron microscopy (tem) has been one of the most important and widely used techniques for the identification and characterization of new viruses. two tem techniques are usually used for this purpose: negative staining on an electron microscopic grid coated with a support film and (ultra) thin section tem of infected cells, fixed, pelleted, dehydrated, and embedded in epoxy plastic. negative staining can be conducted on highly concentrated suspensions of purified virus or cell culture supernatants. for some viruses, tem can be conducted on contents of skin lesions (e.g., poxviruses and herpesviruses) or concentrated stool material (rotaviruses and noroviruses). for successful detection of viruses in ultrathin sections of infected cells, at least % of cells must be infected, and so either high multiplicity of infection (moi) or rapid virus multiplication is required. viruses can be differentiated by their specific morphology (ultrastructure): shape, size, intracellular location or, for some viruses, from the ultrastructural cytopathology and specific structures forming in the host cell during virus replication. usually, ultrastructural characteristics are sufficient for the identification of a virus at the level of a family. in certain cases, confirmation can be obtained by immuno-em performed either on virus suspension before negative staining or on ultrathin sections. this requires virus-specific primary antibodies, which might be not available in the case of a novel virus. for on-section immuno-em, oso post-fixation must be omitted and the partially dehydrated sample must be embedded in a water-miscible acrylic plastic (usually lr white). the ultrastructure of most common viruses is well documented in good atlases and book chapters [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and many classical publications of the s, s, and s. several excellent reviews were recently published on the use of tem in the detection and identification of viruses [ ] [ ] [ ] . the advent of next generation sequencing (ngs) has expanded the tool kit of the virus hunter. for many years, sanger-based sequence analysis has been employed in the identification and characterization of viruses [ ] [ ] [ ] [ ] . however, with the completion of the human genome sequence, the necessity for a high throughput approach that provided a massively-parallel sequencing strategy was fully apparent [ , ] . the automated sanger method was considered a first-generation technology and newer methods are referred to as next generation sequencing (ngs). commercially available technologies from roche/ , illumina, life technologies/apg, oxford nanopore, and pacific biosciences offer unique ngs platforms, and all have been extensively reviewed [ ] [ ] [ ] [ ] [ ] . unlike sanger sequencing, ngs does not require prior knowledge of the viral sequence and thus can be used for viruses of unknown sequence. many viruses cannot be cultured in the cell culture systems currently in use. ngs has shifted the paradigm by removing the need for cell culture and so opening the door to the discovery of many new viruses. the first instruments for ngs were developed in the s and commercialized in the early s, and they were quickly adopted for the identification of novel viruses from a wide range of sources. the number of known viruses increased from about , in to approximately , viruses in [ ] , and that number has since increased dramatically. the technology has also been applied to sequence analysis of many previously known but poorly characterized viruses. this has significantly expanded the known virosphere and assisted in understanding the diversity and evolutionary relationships of viruses. the expansion of the known virosphere has also allowed the taxonomic assignment of an increasing number of viruses, with a total of orders, families, genera, and species of viruses currently approved by the international committee on taxonomy of viruses (ictv) [ ] [ ] [ ] [ ] [ ] [ ] . ngs has also allowed sequence analysis of hundreds or thousands of isolates known pathogenic viruses, facilitating epidemiological studies at scales extending from very local to global. this has generated a trove of new knowledge of significant public health importance. however, without the availability of isolates, ngs has not necessarily increased our understanding of the ecology of novel viruses, their host range, and the risks they may pose to public, veterinary, agricultural, or environmental health. the application of ngs to the unbiased mass sequencing and bioinformatic analysis of total nucleic acids extracted from biological samples obtained from a wide range of sources has led to an explosive increase in the number of complete or near-complete viral genomes. for example, a recent study using ngs to sequence the transcriptomes of arthropods representing species from four classes (insecta, arachnida, chilopoda, and malacostraca) identified novel viruses, many of which are represented by complete or near-complete genomes [ ] . the novel viruses encompass the entire taxonomic diversity of previously known families and/or genera of (-) ssrna viruses and include divergent viruses with entirely novel and unusual genome architecture. similarly, sequence analysis of the transcriptomes of animals of more than species sampled across nine metazoan phyla (arthropoda, annelida, sipuncula, mollusca, nematoda, platyhelminthes, cnidaria, and echinodermata), as well as chordates of the subphylum tunicata (salps and sea squirts), resulted in the discovery of rna viruses, mostly represented by complete or near-complete genomes [ ] . based on phylogenetic analysis of rna polymerase (rdrp) domain sequences, the novel viruses included clades representing many established families of plant and animal rna viruses, as well as at least five clades that are so divergent that they are considered as likely new virus families or orders. also, the sequencing of transcriptomes of gut, liver, and lung or gill tissue of fish, reptiles, amphibians, and birds identified novel vertebrate-associated viruses, representing every family or genus of rna virus associated with vertebrate infection, including those containing important human pathogens (orthomyxoviruses, arenaviruses, and filoviruses) [ ] . these and other similar studies have heralded a new era in virology, revealing new dimensions in viral biodiversity and providing largely unexpected insights into the deep evolutionary history of viruses. however, only a minor subset of newly discovered viruses has been subject to full phenotypic characterization which can provide critical and fundamental insights into their biology and virus-host interactions, ultimately transforming our understanding of the evolutionary forces that shape the virosphere and disease emergence. realistically, as important as these discoveries are to the advancement of science, very few of the viruses will ever cause human disease or influence the global economy. this mass sequencing approach, which has been called viral metagenomics, can also be applied in a more targeted way to identify viruses in clinical cases of diseases of unknown etiology or to survey for potentially novel zoonotic viruses that may represent a significant risk of transmission to humans. indeed, ngs is being used increasingly in conjunction with real-time pcr as a front-line tool in medical and veterinary settings for rapid detection and identification of exotic or unknown emerging viruses. for example, in , an outbreak of acute hemorrhagic fever occurred in mangala, democratic republic of congo (drc), involving three human cases, two of which were fatal. as no positive diagnosis was obtained using real-time pcr for known viral hemorrhagic fevers in africa, ngs was conducted on acute phase serum collected from the surviving patient, revealing the near-complete sequence of a novel rhabdovirus, bas-congo virus (basv; species bas congo tibrovirus) [ ] . although the disease was attributed by the investigators to the novel virus, no isolate was obtained and there was no evidence of neutralising antibodies in serum samples from undiagnosed hemorrhagic fever cases or random serum donors from the drc. subsequently, ngs of blood collected from healthy individuals from nigeria identified two related viruses, ekpoma virus (ekv- ; species ekpoma tibrovirus) and ekpoma virus (ekv- ; species ekpoma tibrovirus), and a serological survey indicated that antibodies to these or similar viruses occur commonly in healthy humans in nigeria [ ] . however, once again, neither virus was isolated. interestingly, several other tibroviruses had previously been isolated from healthy cattle or biting midges (culicoides spp.) in australia and florida [ , , , ] . none have been associated with either disease in livestock or the infection of humans [ , ] . these and other studies raise important issues regarding the significance of ngs data, even when providing complete or near-complete viral genome sequences, when investigating disease etiology. most viruses can cause asymptomatic infections and many newly discovered viruses may be benign in their natural host. therefore, in the absence of a virus isolate which can be used for experimental studies, establishing a causal association with disease based only on detection of the viral genome should be approached cautiously. the availability of a rapidly expanding number of novel viral genomes identified by metagenomic studies has also presented challenges for virus taxonomy-a system for classification of viruses that is administered by the international committee on taxonomy of viruses (ictv). should viruses detected only by their nucleotide sequence be classified and assigned to species and other higher taxa (genus, family order, etc.) alongside viruses for which we have a viable isolate? the ictv (then the international committee on nomenclature of viruses) was established in at the ninth international congress for microbiology in moscow, publishing its first report in [ ] . it operates under the auspices of the international union of microbiological societies (iums), with the authority to develop, refine, and maintain a universal virus taxonomy. historically, the description and classification of a new virus by the ictv required significant information, such as host range, serology, replication cycles, and structure, aspects that could be determined from the study of isolates. on the other hand, sequences alone provide a trove of information, including evolutionary relationships (e.g., phylogeny), genome organization (e.g., the number of genes and their order), presence or absence of distinctive motifs (e.g., protein cleavage sites, terminal sequences, internal ribosome entry sites), as well as genome composition (e.g., codon usage, gc content), which of course could be used to inform classification into species. these concerns framed the contents of a workshop of experts and members of the ictv, resulting in a seminal consensus statement in which viruses identified only from metagenomic data are considered to be bona fide viruses and thus candidates for taxonomic assignment [ ] . the expanding diversity of the known virosphere also presented challenges. a recent analysis of metagenomes of geographically and ecologically diverse samples led to the discovery of , new partial dsdna viral genomes encoding more than . million proteins, % of which had no sequence similarity to proteins from known virus isolates [ ] . other metagenomic studies have revealed similar diversity in ssdna and rna viruses, particularly in marine ecosystems [ ] . this has led ictv to consider a far broader framework for taxonomic assignment of viruses, recently approving the establishment of a taxonomic hierarchy that includes ranks (realm, subrealm, kingdom, subkingdom, phylum, subphylum, class, subclass, order, suborder, family, subfamily, genus, subgenus, and species), thus expanding the range even beyond those currently available for other organisms [ ] . the sheer volume of new virus genomes identified by metagenomic studies has also led to the development of new bioinformatic tools, that are increasingly being applied for automated virus classification of viruses, based almost exclusively on nucleotide sequence data [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . while the classic tools of virus discovery and characterization (e.g., em, serology, and tissue culture) are still widely used, ngs allowed for the rapid identification of an enormous repertoire of viruses, thus exponentially expanding the boundaries of the known virosphere. the resultant genomic sequences allowed for their accurate taxonomic assignments, analysis of their phylogenetic and evolutionary relationships with other viruses, and evaluation of the potential risks they may present to humans and wild or domestic animal populations. below are a few representative examples of how ngs transformed the known relationships of arboviruses within their respective families. rhabdoviruses contain negative-sense (-) single-stranded rna (ssrna) genomes. they are amongst the most numerous and diverse of rna viruses, naturally infecting mammals, birds, fish, reptiles, and amphibians, as well as insects, arachnids, crustaceans, nematodes, and a wide range of plants [ , ] . most (but not all) rhabdoviruses that infect vertebrates are transmitted by hematophagous arthropods. the rhabdoviridae currently comprises genera containing species and one unassigned species [ ] . of these, viruses assigned to seven of the genera (vesiculovirus, ephemerovirus, tibrovirus, hapavirus, ledantevirus, curiovirus, and sripuvirus) are considered to be arboviruses. viruses assigned to the newly characterized genus almendravirus appear to be insect-specific [ ] . viruses assigned to the genus tupavirus have been isolated only from vertebrates but may possibly have arthropod vectors. complete coding sequences are now available for more than other rhabdoviruses, many of which have been isolated from or detected in arthropods, but they have not yet been formally classified. while the classic tools of viral discovery (e.g., em, serology, and tissue culture) are still widely used, ngs has played a central role in recent genome sequencing efforts, revealing diversity, not only in the ecology of rhabdoviruses but also in the structural diversity of genome architecture [ , [ ] [ ] [ ] [ ] [ ] [ ] . in addition to the five canonical rhabdovirus structural protein genes (n, p, m, g, and l), it is now recognized that rhabdoviruses commonly contain multiple long open reading frames encoding putative accessory proteins, mostly of unknown function. ngs has also facilitated studies of the evolution of rhabdovirus genome organization [ ] and revealed the importance of arthropods in the evolutionary history of rhabdoviruses [ ] . according to the current international committee on taxonomy of viruses (ictv), classification of the order bunyavirales encompasses families of segmented (-) ssrna viruses-arenaviridae, hantaviridae, nairoviridae, peribunyaviridae and cruliviridae, mypoviridae, fimoviridae, wupedeviridae, phasmaviridae, and phenuiviridae-a classification based on structural, genetic, and antigenic characteristics [ ] . bunyavirales is a diverse order with a large number of viruses associated with human, veterinary, and plant disease, as well as being vectored by arthropods (mosquitoes, ticks, sandflies, and thrips) and infecting a wide range of other invertebrates. the recent discovery of gouléako (golv) [ ] and cumuto (cumv) [ ] viruses (the latter with ngs) which are evolutionarily related to but distinct from viruses in the genus phlebovirus, family phenuiviridae, suggesting they are to be assigned to the new genus goukovirus. in the past five years, ngs has revolutionized understanding of the vast diversity of this order, by allowing genetic identification of previously uncharacterized and unassigned bunyaviruses, which in turn provided a complimentary approach to the gold standard of classification (structural, genetic, and antigenic characteristics) for a more refined taxonomic classification. combined, these approaches will be instrumental for enhancing our understanding of their ecologic and geographic distribution, as well as public health impact. the family flaviviridae comprises positive-sense (+) ssrna viruses. the family contains several serious human pathogens, including dengue, yellow fever, zika, japanese encephalitis, west nile, and tick-borne encephalitis viruses (all arboviruses in the genus flavivirus) and the hepatitis c virus (a member of the genus hepacivirus). members of the genus flavivirus, like the alphaviruses (see section . ), are a diverse group of arthropod-borne viruses that are found on every continent except antarctica. several flaviviruses have been discovered and characterized recently, mainly through chance or increased surveillance efforts, and facilitated through ngs. newly characterized viruses include the mercadeo virus (mecdv), from pools of culex species mosquitoes in panama [ ] , sabethes flavivirus (sbfv), from sabethes belisarioi in brazil [ ] , the la tina virus (ltnv), from aedes scapularis in peru [ ] , the long pine key virus (lpkv), from anopheles crucians in the florida everglades, and the kampung karu virus (kpkv), from anopheles tesselatus in borneo [ ] , aedes flavivirus (aefv), from aedes albopictus laboratory colony that originated in thailand [ ] , xishuangbanna flavivirus (xfv), from aedes albopictus in china [ ] , and the cuacua virus (cucuv), from mansonia ssp. in mozambique [ ] . one unexpected finding was the discovery of segmented viruses that grouped in the family flaviviridae and were more closely related to flaviviruses than members of other established genera. the jingmen tick virus (jmtv) is unique in the family flaviviridae with a four segment positive-sense genome [ ] . traditional sanger sequencing generated the ns and ns gene sequences but could not connect these within a single genome segment. phylogenies based on either ns -like or ns -like sequences, which are encoded on two of the four segments, showed jmtv falling basal on the flavivirus tree and clearly distinct from the other viruses in this genus. the remaining two segments did not appear to have a flavivirus origin. jmtv has been isolated from cattle, monkeys, and ticks (rhipicephalus and haemaphysalis spp.) [ ] . this prototype virus has lent its name to the newly identified segmented flaviviruses, and other viruses have recently populated the jingmenvirus clade. similarly, the guaico culex virus (gcxv) consists of five segments, and initial studies suggested gcxv is insect-specific, based on its isolation from culex ssp. mosquitoes [ ] . additional potentially insect-specific four-segmented viruses include shuangao insect virus (saiv ), the wuhan flea virus (whfv), wuhan aphid virus (whav ), wuhan aphid virus (whav ), and the wuhan cricket virus (whcv) [ ] . interestingly, sequences that are similar to toxocara canis, a larva roundworm, and tentatively named t. canis larva agent virus (tclav), are distantly related to jmtv and appear to be the first evidence of a flavivirus-line organism in a member of the phylum nematoda [ ] . the discovery and characterization of a segmented genome flavivirus has significant implications, as it reveals that rna virus segmentation is an evolutionary process that has occurred in previously unanticipated circumstances. the alphaviruses are a diverse group of (+) ssrna arthropod-borne viruses that are found on every continent except antarctica [ ] . there were no known mosquito-only alphaviruses until , when analysis by ngs of a mosquito-pool from the negev desert in israel showed the presence of an alphavirus, the eilat virus (eilv) [ ] . further analysis showed that, although eilv could infect mosquito cells, it was unable to replicate in vertebrate cells [ ] . eilv is considered a vaccine candidate, as it can produce immunity without replication in the vertebrate host, illustrating the importance of such discoveries, which can lead to novel platforms for the prevention and/or treatment of disease [ ] . reoviruses are a diverse family of double-stranded rna (dsrna) viruses that infect a wide range of hosts and have a wide range of characteristics. of the genera, there are only three for which novel viruses have been described using ngs, and the majority of these are in the genus orbivirus. interestingly, because of the structure of the genomes of reoviruses and the conserved sequences at the ' and ' ends of the segments [ ] , it is relatively simple to sequence segments of reoviruses using more traditional techniques. this may be why there are so few novel reoviruses determined by ngs. novel viruses identified using ngs have been assigned to four other genera (seadornavirus, orthoreovirus, dinovernavirus, and cypovirus), all of which are shown in table . refers to year of the publication and not of the isolation of the virus. *not yet formally classified. newly recognized viruses containing a (+) ssrna genome have been proposed to form a new genus negevirus, related to genera of mite-infecting plant viruses (blunervirus, cilevirus, and higrevirus) in the new family kitaviridae [ , ] . originally, six viruses with restricted host range in insects (isvs), designated as negev (negv), ngewotan (nwtv), piura (piuv), loreto (lorv), dezidougou (dezv), and santana (sanv), were identified in and isolated from mosquitoes and phlebotomine sandflies, collected in brazil, the ivory coast, israel, indonesia, peru, and the usa, [ ] . their widespread geographic distribution was documented by several other groups, who reported the isolation and characterization of related viruses in the philippines (tanay virus; tanav) [ ] , trinidad and tobago (wallerfield virus; walv) [ ] , côte d'ivoire (goutanap virus; ganv) [ ] , portugal (ochlerotatus caspius negevirus; ocnv and culex univittatus negevirus; cunv) [ ] , brasil (brajeira and wallerfield viruses), colombia, and nepal. collectively, the close relationship of negeviruses with plant viruses of the genera cilevirus, higrevirus, and blunervirus, coupled with their heterogenous genome organization and architecture, provides support for the possibility that negeviruses are plant-like viruses that could eventually anchor a new virus family [ ] . members of the family mesoniviridae, a newly discovered family of (+) ssrna viruses assigned to the order nidovirales [ , ] , appear to have an extensive geographic distribution but a restricted host range within members of the family culicidae (flies) [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the mesoniviridae comprise of a single genus alphamesonivirus with nine recognized species: alphamesonivirus , including nam dinh (ndiv) [ ] , cavally (cavv) [ ] , ndiv a . [ ] , ndiv ngewotan, and ndiv houston [ ] viruses; alphamesonivirus , including the single isolate of the karang sari (ksav) virus, and the four bontag baru (bbav) isolates sampled in the early s in indonesia, but only recently characterized [ ] ; alphamesonivirus , including the dak nong virus (dknv), the three isolates of kamphaeng phet (kphv), sampled in indonesia and thailand in the mid- s [ , ] ; alphamesonivirus , including the casuarina virus (casv), isolated in australia from coquillettidia xanthogaster mosquitoes [ ] ; alphamesonivirus , the african hana virus (hanav) [ ] ; alphamesonivirus and , including ofaie (ofav) and kadiweu (kadv) viruses, isolated in the pantanal of brasil from mansonia sp. mosquitoes, respectively [ ] ; and alphamesonivirus- and , including the african nse (nsev), and meno (menov) viruses [ ] ; and the distinct yet unassigned species, the yichang virus [ ] , isolated from culex sp. mosquitoes in china. across several generations, virus hunters have left a profound legacy, both to science and to the broader global community. in the field, their work has often been conducted in difficult, demanding, and sometimes dangerous circumstances. in the laboratory, they have developed and applied tools and methodologies that led to improved diagnosis, prevention, and treatment of viral disease, and still lie at the center of virology today. others working at more fundamental scientific levels have used their virus isolates as scientific models, opening the door to a revolution in molecular and cellular biology. yet, their work continues. viral disease emergence remains one of the most serious threats to humanity, with potentially devastating social and economic consequences. recent examples, such as sars, mers, henipa-, ebola, and zika viruses, illustrate the threat that unidentified or poorly characterized viruses can, and almost certainly will, continue to present. equally devastating emerging diseases of livestock, fisheries, and crops threaten food production and livelihoods. as history has repeatedly shown us, technological revolutions have been often accompanied by periods of progressive disinvestment in training for the eclipsed technologies of the past, and in our case, classical virology. the recent emergence of the zika virus in the americas has reinforced the notion that traditional methods of virus discovery and new technologies offer a complimentary toolkit, especially in resource-poor settings that can be seamlessly integrated in the service of public health. it is our hope and expectation that new generations of virus hunters will appreciate the legacy of their predecessors and complement the power of ngs, metagenomics, and bioinformatics, not only to advance viral evolutionary biology, but to understand and limit the potential impacts of emerging viral disease. funding: this work was supported in part by nih grants r ai and u ai . the funding agencies had no involvement in the writing of the report or in 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mosquitoes in vietnam a new species of mesonivirus from the northern territory, australia genome sequences of five arboviruses in field-captured mosquitoes in a unique rural environment of south korea isolation and characterization of a novel mesonivirus from culex mosquitoes in china mesoniviruses are mosquito-specific viruses with extensive geographic distribution and host range novel viruses isolated from mosquitoes in pantanal, brazil the authors thank ashley rhame and dora salinas for excellent help in preparation of the manuscript. the authors declare no conflict of interest. key: cord- - dh authors: kumar, ashutosh; harjai, kusum; chhibber, sanjay title: a multiepitopic theoretical fusion construct based on in-silico epitope screening of known vaccine candidates for protection against wide range of enterobacterial pathogens date: - - journal: hum immunol doi: . /j.humimm. . . sha: doc_id: cord_uid: dh enterobacterial pathogens that have acquired antibiotic resistance genes are a leading cause of community and hospital acquired infections. in such a situation vaccination is considered as a better option to prevent such infections. in the current study reverse vaccinology approach has been used to select peptides from already known immunogenic proteins to design a chimeric construct. we selected yersiniabactin receptor of escherichia coli umn and flagellin of stenotrophomonas maltophila. b-cell linear epitopes were predicted using bepipred prediction tool. peptide binding with reference sets of alleles of mhc class i and class ii was also analyzed. the predicted peptides-mhc complexes were further validated using simulation dynamics. the in-silico construction of chimera was done by restriction mapping and codon optimization. chimera was evaluated using the immunoinformatic approach as done for the selected proteins. from the amino acids of fyua protein, a region from to was selected for containing more linear epitopes and the processing scores obtained were significant for mhc class i and class ii binding. similarly, from flagellin, a region between and amino acids was selected and the peptides present in the selected region showed lower percentile ranks for binding with mhc molecules. the simulation studies validated the predictions of peptide-mhc complexes. the selected gene fragments accommodating maximum part of these peptides were used to design a chimaeric construct of bp. from the immunoinformatic analysis, the chimera was found to be more immunogenic in terms of increased number of b-cell and t-cell epitopes along with increased coverage of global populations with allelic variability. communicable diseases caused by members of enterobacteriaceae family put a great burden on the society by affecting humans and their livestocks. these infections become quite severe when they are not controlled on time. some of the examples include pneumonia, pyogenic liver abscess, pyelonephritis and septicemia [ ] . the treatment has become difficult due to the emergence of antibiotic resistance among some of the pathogens. due to the existing challenge of treating these infections with antibiotics, it is inevitable for the research community to look forward to prophylactic means for the prevention of these infections. a large number of vaccine candidates have been proposed by various researchers for specific infections. however, evaluation of individual vaccine candidates under in-vivo infection conditions is an enduring task. in the recent years, "reverse vaccinology" (rv) has come to play an important role in scrutinizing the vaccine candidates by in-silico analysis [ ] thereby reducing the time required for ruling out ineffective candidates. this immunoinformatic approach is being frequently used by researchers to predict the epitopes on viruses. this has led to the finding of epitopes on nucleocapsid protein and ovarian tumour domain of crimean-congo hemorrhagic fever virus [ ] . another study investigated the variability among epitopes of hepatitis c virus (hcv) identified in genotype and also predicted the immunogenicity of their variants from other genotypes against south african human leukocyte antigen (hla) backgrounds [ ] . epitopes of e protein isolated from hcv have also been identified using the similar approach [ ] . effective immunogens of mers-cov have been discovered through immunoinformatics-driven genome-wide screening strategy [ ] . goodswen et al. [ ] have also used this technique for designing protein based vaccines against eukaryotic pathogens. reverse vaccinology has already been used against other bacterial pathogens like group b streptococcus, where genomic analysis has led to the development of a vaccine composed of four proteins giving protection against all serotypes [ ] . another in-silico study has found a protein bam a of acinetobacter baumanii to be a potential immunogen [ ] . rv has also been implied to predict the potential vaccine candidates from the proteome of burkholderia pseudomallei [ ] . the outer membrane proteins (omps) of these gram-negative bacteria are usually considered as potent vaccine candidates as they are exposed to the host immune defenses [ ] . these omps are not always conserved in different genus of bacteria but their lies a probability of presence of some conserved peptide sequence in these omps. in this study we have taken into account the proteins which have proved to be potential vaccine candidates on the basis of invivo research work on animal models. yersiniabactin receptor fyua is highly conserved protein prevalent in various members of enterobacteriaceae. as per the reports, fyua mediates the uptake of ferricyersiniabactin [ , ] confirming its role in the virulence of bacteria, which makes it an important vaccine candidate. moreover researchers have found it as a potential vaccine candidate against pyelonephritis in a murine model of urinary tract infection [ ] . it has also been found to be protective in murine model of pneumonia caused by k. pneumoniae in our laboratory (manuscript under communication). the flagellin protein is another potential vaccine candidate which has been included in this study. various studies have established the role of flagellin in inducing a systemic inflammatory response via intraperitoneal and intravenous administration [ , ] and a local inflammatory response with intraintestinal administration [ , ] . in our lab, flagellin of stenotrophomonas maltophilia has been shown to induce non-specific immune response which protected mice against subsequent bacterial challenge [ ] . in the current study both of these vaccine candidates have been analyzed using iedb server to design a novel insilico vaccine construct harbouring the properties of both these proteins. gene sequence of fyua accession no. nc_ . and flagellin nc_ . were taken from ncbi database [ ] . the obtained gene sequences were translated into the protein sequences using expasy-translate tool (swiss institute of bioinformatics). protein sequences of yersiniabactin receptor of escherichia coli umn and flagellin of stenotrophomonas maltophilia were analyzed for the presence of linear epitopes using bepipred portal of iedb server. both proteins were also analyzed on other algorithms, parker linear epitope prediction from the protein sequences fyua (a) and flagellin (b) using bepipred prediction portal of iedb server. the yellow peaks show the peptide sequences that are potential epitopes whereas the green peaks show the peptides that are not epitopic in nature. the encircled area on the graphs shows the region of protein having higher frequency of epitopic peptides. conformational epitopes on yersiniabactin receptor were predicted using discotope portal of iedb server by analyzing solvent-accessibility. in human population major histocompatibility complexes (mhc-i and mhc-ii) are encoded by human leukocyte antigen (hla) alleles and are required for the presentation of antigen to t cells. the peptides which could bind to mhc i molecules were predicted by mhc class i binding peptide prediction portal on iedb server using the consensus method. interactions were evaluated in terms of percentile ranks. similarly, peptides binding to mhc ii molecules were predicted by mhc class ii binding peptide prediction portal on iedb server using the consensus method. d structures of alleles were retrieved from rcsb pdb database [ ] . predicted peptide sequences and d structure of mhc class i and class ii alleles were submitted to cabs dock server for docking and simulation studies [ ] . secondary structures of peptides were generated from psipred [ ] . the simulation time was set to cycles. the results were clustered according to the distance between the residues of the peptide and mhc molecules. both gene sequences were analyzed on neb cutter for mapping restriction sites. the enzymes which could cut the dna at only single site were identified and the one that was common in both sequences was selected. the chimeric construct could be constructed after ligation of gene fragments excised from fyua and flagellin. briefly, both the genes could be digested with aatii to produce linear sticky ended fragments of . and . kb respectively. aatii will digest fyua at position bp and flagellin at position bp. these fragments could then be digested with xhoi to generate sticky ended fragments of . kb and . kb from fyua and . kb and . kb from flagellin. the . kb fragment from flagellin and . kb fragment from fyua could be ligated with t dna ligase followed by transformation, screening and sequencing. open reading frame for the sequence of chimeric gene was analyzed on expasy-translate tool (swiss institute of bioinformatics). the model structure of the chimera aksc was generated using modeller . [ ] . the structure was energy minimized and validated using molprobity [ ] server for its stereophysical characteristics. protein sequence of aksc was analyzed using bepipred portal of iedb server. the obtained linear epitopes were analyzed for changes in comparison to the epitopes predicted from the individual proteins. the epitopes were also analyzed for the presence of overlapping regions between individual proteins. protein sequence of aksc was analyzed to observe the peptides which could bind to mhc class i molecules. these peptides were predicted using mhc class i binding peptide prediction portal on iedb server. similarly, peptides binding to mhc ii molecules were predicted using mhc class ii binding peptide prediction portal on iedb server. interactions were evaluated in terms of percentile ranks. amino acid sequences of both the proteins were analyzed for the presence of linear epitopes using the bepipred prediction tool of iedb server. for fyua a threshold score of . for a window size of amino acids was generated in the portal. results in fig. (a) show that a large number of linear epitopes are present in fyua and most of the prominent epitopes were in the region from amino acid position to . the maximum score of . was obtained for the amino acids near position . similarly, linear epitopes were predicted for flagellin with the bepipred generated threshold value of . for a window size of amino acids. results in fig. (b) show a large number of linear epitopes on flagellin and most of the prominent epitopes were in the region from amino acid position to . the maximum value of . was obtained for the amino acids near position . the peptides that were significant linear epitopes are shown here in table . results of bepipred prediction as shown in fig. revealed the presence of a wide range of linear epitopes on both fyua and flagellin proteins. these results were further verified using other parameters like hydrophilicity, surface accessibility and antigenicity. similar results were obtained from all the above predictions as both the proteins were found to possess large number of linear epitopes (supplementary information si. .). the region accommodating the maximum number of epitopes was then selected to be taken further for theoretical construction of chimaera. further, evaluation of other parameters was done by keeping the selected regions into consideration. since, there lies a possibility that the chimeric protein may not take up the proper folding when over expressed due to physical constraint of large size. hence, it may be expressed as inclusion bodies when subjected to over expression. misfolding of protein would certainly not affect the t-cell dependent response since the generation of t-cell response depends on the processing and presentation of peptides on mhc molecules but it may affect the antibody response to conformational epitopes. however, results in table show table . therefore, the presence of linear epitopes can be considered as a very significant feature of the vaccine candidate protein as it could help in the generation of antibody response even if the protein is administered in denatured form. conformational epitopes of fyua were predicted using discotope tool on iedb server. results in fig. (a) as depicted by the green peaks is the region from amino acid position to that possess maximum conformational epitopes. these epitopes (yellow) were also shown on the -d structural image created by j-mol-pdb fig. (b) . since most of the conformational epitopes are present on the exposed surface, these epitopes may become a target of antibodies for effectively neutralizing the bacteria during infection. both the protein could prove to be good vaccine candidates if they are able to generate both b cell as well t cell responses. however, both the proteins qualified for the generation of b cell response therefore predictions were made for their ability to generate t-cell responses. this was done by predicting mhc class i and ii binding peptides. mhc class i binding peptides were predicted using the mhc class i binding prediction tool on iedb server. peptides with a percentile rank below were considered significant. results in fig. (a) show that significant interactions were obtained for fyua protein and among these, interactions were from the selected region of amino acid position to . similarly, for flagellin protein results in fig. (a) show about significant interactions and among these, interactions were from the selected region of amino acid position to (supplementary information si. .). therefore the results in fig. (b) show that % of the significant interactions were from the selected region of fyua and % of significant interactions were from the selected region of flagellin fig. (b) . the top peptides from the selected region having lowest percentile rank were shortlisted and are shown in table . data in table show that both of these proteins possessed significant number of peptidic regions that could bind to it is also seen that the peptides of both these proteins bind with different alleles which are present in different parts of world. this difference in the binding will turn out to be very beneficial when aksc will be used as vaccine as more the number of binding alleles more would be the coverage of human population. peptides binding to mhc class ii molecules were predicted using mhc class ii binding peptide prediction tool on iedb server. for mhc class ii binding, iedb recommended method and a reference set of alleles was used. results in fig. (a) show that significant interactions were obtained for fyua protein. out of which interactions with the reference set of alleles were from the selected region. similarly, for flagellin protein results in fig. (a) show that significant peptide allele interactions were predicted by the server and among these, interactions were from the selected region (supplementary information si. .). also the results in fig. (b) show that % of the significant interactions were from the selected region of fyua and % of significant interactions were from the selected region of flgellin fig. (b) . table depict the top peptides from the selected region having lowest percentile rank for fyua for flagellin respectively. alleles hla-drb * : , hla-drb * : and hla-drb * : are present largely in asian and russian populations (allelefrequencies. net). allele hla-dqa * : /dqb * : is found in asian, african, israel and french populations and hla-dqa * : /dqb * : is present in german, african and asian populations. results obtained after simulated -dimensional docking of predicted peptides with the predicted mhc molecules were presented in tables and . the data shows the average rmsd values of the each peptide-mhc complex. the average rmsd values between and are considered of medium accuracy whereas the values below are considered as highly accurate [ ] . along with the rmsd values the distance between the interacting amino acid residues of mhc molecules and that of the interacting peptide was also analyzed. the results in table and fig. show that the mhc class i and class ii binding peptide predictions made using the iedb server, were also found to be significantly accurate using the -d simulations for docking. this could be interpreted as the values of average rmsd were below in the simulated complexes. the interacting residues shown in the table were lying at a distance of < Å. these results of all the in-silico predictions helped us in choosing the regions from both the proteins, so that they could be combined to produce a single chimaeric construct. we therefore propose a strategy which could lead to the formation of the chimaeric construct. for designing the chimaeric gene construct, cloning strategy was adopted using genetic engineering tool for restriction digestion. gene sequences of both the proteins were analyzed on neb cutter tool of new england biolabs. restriction enzyme aatii (supplementary information si. .) was found to be present in both the protein sequences and cleaves fyua at position and flagellin at position and generates sticky ends. fig. shows the graphical representation of the cloning strategy. in-silico created chimeric construct was found to be consisting of base pairs (supplementary information si. .) which was further translated using expasy/translate tool and a protein of amino acids was formed to give a molecular weight of approximately kda (supplementary information si. .). this ruled out the presence of any stop codon within the whole sequence. the main objective of combining large gene fragments from both the proteins was considered so as to provide the cellular protein processing machinery with enough of proteasome cleavage sites. the possibility of a single peptide to act as a vaccine is usually a rare chance. hence a protein can be processed to in different ways to create epitopes whose presentation to t-cells may add to protection during pathogenesis. analysis of ramachandran plot of the chimera aksc generated using the molprobity server suggested that . % residues lied in the favoured and allowed region while only . % residues were in the outlier region (supplementary information si. .). the obtained values suggest that the model is structurally stable. further analysis of aksc showed an increase in the average threshold value in linear epitope prediction this probably resulted by combining the two proteins (fig. ). there were also the peptides in aksc that were epitopic and lied in the region joining both the proteins. this was another advantage of this chimaera as these overlapping peptides could increase the population coverage of vaccine. table show the peptides that are able to bind to mhc class i and class ii and are present in the region joining the two proteins. these peptides are able to bind to different alleles. results in table show the similarity of overlapping peptides which were generated as a result of fusion of the two proteins. the peptide analyzed using blastp showed its identity with peptides present in microorganisms other than the source of peptide. this also gave a hope that the chimaeric vaccine candidate may confer protection upon global populations from the infectious diseases caused by a wide range of pathogens. from the in-silico analysis it is concluded that reverse vaccinology can be used to create novel chimeric constructs from the already known vaccine candidates to make them more effective and to confer protection among diverse populations against a wide range of enterobacterial pathogens. for this research work no specific grant from any funding agency was provided. mr. ashutosh kumar was granted financial support in the form of senior research fellowship from indian council of medical computer-aided biotechnology: from immuno-informatics to reverse vaccinology epitope-based immunoinformatics and molecular docking studies of nucleocapsid protein and ovarian tumor domain of crimean-congo hemorrhagic fever virus sequence-based in silico analysis of well studied hepatitis c virus epitopes and their variants in other genotypes (particularly genotype a) against south african human leukocyte antigen backgrounds structural analysis and epitope prediction of hcv e protein isolated in pakistan: an in-silico approach epitope-based vaccine target screening against highly pathogenic mers-cov: an in silico approach applied to emerging infectious diseases enhancing in silico protein-based vaccine discovery for eukaryotic pathogens using predicted peptide-mhc binding and peptide conservation scores identification of a universal group b streptococcus vaccine by multiple genome screen in silico analysis of acinetobacter baumannii outer membrane protein bama as a potential immunogen in-silico analysis of burkholderia pseudomallei proteome to predict potential vaccine candidate proteins a multiepitope subunit vaccine conveys protection against extraintestinal pathogenic escherichia coli in mice the pesticin receptor of yersinia enterocolitica: a novel virulence factor with dual function reduced synthesis of the ybt siderophore or production of aberrant ybt-like molecules activates transcription of yersiniabactin genes in yersinia pestis immunization with the yersiniabactin receptor, fyua, protects against pyelonephritis in a murine model of urinary tract infection the innate immune response to bacterial flagellin is mediated by toll-like receptor flagellin, a novel mediator of salmonella-induced epithelial activation and systemic inflammation: iκbα degradation, induction of nitric oxide synthase, induction of proinflammatory mediators, and cardiovascular dysfunction role of flagellin in the pathogenesis of shock and acute respiratory distress syndrome: therapeutic opportunities humoral immune response to flagellin requires t cells and activation of innate immunity stenotrophomonas maltophilia flagellin induces a compartmentalized innate immune response in mouse lung induction of hepatitis a virus-neutralizing antibody by a virus-specific synthetic peptide the protein data bank cabs-dock web server for the flexible docking of peptides to proteins without prior knowledge of the binding site protein secondary structure prediction based on position-specific scoring matrices comparative protein modelling by satisfaction of spatial restraints molprobity: all-atom structure validation for macromolecular crystallography modeling of protein-peptide interactions using the cabs-dock web server for binding site search and flexible docking none to declare. supplementary data to this article can be found online at https:// doi.org/ . /j.humimm. . . . key: cord- -kxivgxbg authors: haverkamp, ann-kathrin; lehmbecker, annika; spitzbarth, ingo; widagdo, widagdo; haagmans, bart l.; segalés, joaquim; vergara-alert, julia; bensaid, albert; van den brand, judith m. a.; osterhaus, albert d. m. e.; baumgärtner, wolfgang title: experimental infection of dromedaries with middle east respiratory syndrome-coronavirus is accompanied by massive ciliary loss and depletion of the cell surface receptor dipeptidyl peptidase date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: kxivgxbg middle east respiratory syndrome (mers) represents an important respiratory disease accompanied by lethal outcome in one-third of human patients. recent data indicate that dromedaries represent an important source of infection, although information regarding viral cell tropism and pathogenesis is sparse. in the current study, tissues of eight dromedaries receiving inoculation of mers-coronavirus (mers-cov) after recombinant modified-vaccinia-virus-ankara (mva-s)-vaccination (n = ), mva-vaccination (mock vaccination, n = ) and pbs application (mock vaccination, n = ), respectively, were investigated. tissues were analyzed by histology, immunohistochemistry, immunofluorescence, and scanning electron microscopy. mers-cov infection in mock-vaccinated dromedaries revealed high numbers of mers-cov-nucleocapsid positive cells, t cells, and macrophages within nasal turbinates and trachea at day four post infection. double immunolabeling demonstrated cytokeratin (ck) expressing epithelial cells to be the prevailing target cell of mers-cov, while ck / and ck expressing cells did not co-localize with virus. in addition, virus was occasionally detected in macrophages. the acute disease was further accompanied by ciliary loss along with a lack of dipeptidyl peptidase (dpp ), known to mediate virus entry. dpp was mainly expressed by human lymphocytes and dromedary monocytes, but overall the expression level was lower in dromedaries. the present study underlines significant species-specific manifestations of mers and highlights ciliary loss as an important finding in dromedaries. the obtained results promote a better understanding of coronavirus infections, which pose major health challenges. in june a novel lineage c betacoronavirus (hcov-emc) was identified in a patient from the kingdom of saudi arabia who suffered from acute pneumonia and renal failure . subsequently, the virus was named middle east respiratory syndrome coronavirus (mers-cov) in accordance with the geographical area of its first description and main occurrence . until today, mers-cov represents an existential threat to global health since the virus spread to countries and caused more than laboratory confirmed cases in humans including fatal cases, which equals approximately one third of all affected patients (world health organization ( ) middle east respiratory syndrome coronavirus, available at http://www.who.int/emergencies/mers-cov/en/, accessed october , ) . the sequence of mers-cov was determined to be closely related to other betacoronaviruses isolated from bats and therefore a bat origin has been proposed early after genomic characterization [ ] [ ] [ ] [ ] [ ] [ ] . however, transmission of mers-cov to humans was suspected to occur via an intermediate mammalian host, since the majority of human middle east respiratory syndrome (mers) patients did not state any direct contact to bats prior to disease onset , . similarly, severe acute respiratory syndrome coronavirus (sars-cov), a betacoronavirus of the lineage b, originated from bats and spread from palm civets to humans in / . in , one year after the initial description of mers, serological investigations in livestock species suspected dromedaries (camelus dromedarius) to act as potential intermediate hosts for mers-cov . subsequent research on index cases , serological sampling , and virus isolation revealed a transmission from dromedaries to humans and confirmed dromedaries as the major reservoir for human infections . consequently, mers-cov infection in dromedaries was documented by serological surveys in large parts of the middle east, the canary islands, and africa, as previously summarized by hemida et al. and additionally in certain parts of southern asia and western africa . recently, mers-cov has also been detected in alpacas from qatar , but not in any other domestic livestock species . in recent animal trials with dromedaries, experimental mers-cov infection led to mild transient upper respiratory tract disease characterized by mild to moderate rhinitis with nasal discharge, tracheitis, and bronchitis accompanied by shedding of large quantities of virus from the upper respiratory tract. viral antigen was additionally detected in regional lymph nodes but not in any other extra-respiratory organ , . the serine exopeptidase dipeptidyl peptidase (dpp ; also known as cd ) has been identified as a major virus receptor in both humans and dromedaries , and is expressed in the lower respiratory tract of humans and the upper respiratory tract of dromedaries . more recently, carcinoembryonic antigen-related cell adhesion molecule (ceacam ) has been described as an additional cellular binding target for mers-cov that may facilitates virus entry . additionally, binding of mers-cov to sialic acids has been demonstrated in a newly published study by li and colleagues and is suggested to represent another important factor in viral host tropism . despite two recent animal trials , and the successful establishment of an orthopoxvirus-based vaccine against mers-cov in dromedaries , the virus infection triggered immune response and the exact cell tropism have not been evaluated so far, since valuable markers for immune cell phenotyping in dromedaries were hardly established. in order to facilitate such studies and other investigations in camels, a panel of antibodies for identification of different immune cell subsets has been recently tested . since dromedaries play a key role in transmitting mers-cov to humans, it was the aim of the present study to describe mers-cov associated lesions in the nasal epithelium and viral cell tropism of experimentally infected dromedaries in closer detail. experimental mers-cov infection of dromedaries leads to high virus load in nasal turbinates and trachea accompanied by necro-suppurative inflammation at day post infection. evaluation of hematoxylin and eosin (he) stained sections taken from respiratory epithelium of the nasal turbinates of mock-vaccinated animals at days post infection (dpi) revealed mild to moderate, segmental hyperplasia of the epithelium. moderate exocytosis of neutrophilic granulocytes, apoptosis and single cell necrosis of epithelial cells were observed within all layers of the epithelium and frequently accompanied by formation of small cavities in the apical epithelium (fig. a) . additionally, a partial loss of apical epithelial cells (erosion) was occasionally present within certain areas with marked inflammation (fig. b) . the epithelial surface was covered multifocally by large amounts of mucus, as well as viable and degenerated neutrophilic granulocytes and cellular debris. lamina propria and submucosa displayed mild to moderate edema and infiltration of moderate numbers of lymphocytes and macrophages as well as single neutrophilic granulocytes, which were predominantly located next to blood vessels. histology of trachea and bronchi revealed only mild lesions which were characterized by mild exocytosis of neutrophilic granulocytes and mild, segmental infiltration of lamina propria and submucosa by lymphocytes, macrophages, and low numbers of neutrophilic granulocytes (fig. c,d) . rarely epithelial erosion and accumulations of degenerated neutrophilic granulocytes within the apical epithelial layers (pustules) of the trachea were detected. the mva-s-vaccinated animals showed similar but less severe lesions at dpi in nasal turbinates, trachea, and bronchi which were rarely accompanied by degeneration, loss, and necrosis of single epithelial cells in the nasal turbinates. at dpi, very mild lesions were detectable in nasal turbinates, trachea, and bronchi of all investigated animals (mva-s-vaccinated and mock-vaccinated) characterized by mild, multifocal, lymphoplasmahistiocytic and neutrophilic infiltration of lamina propria and submucosa and rare exocytosis of neutrophilic granulocytes (data not shown). rarely, follicular aggregates of lymphoid cells were detectable in the lamina propria and submucosa of nasal turbinates and trachea in both groups and at all investigated time points. within pulmonary, tracheal and cervical lymph nodes as well as within tonsils mild to moderate follicular hyperplasia and single apoptotic lymphocytes were present. all other non-respiratory organs and the lungs did not show any significant lesions. in line with these observations, high amounts of mers-cov antigen were detected within the respiratory epithelium of the nasal turbinates of mock-vaccinated dromedaries at dpi by immunohistochemistry in areas scientific reports | ( ) : | doi: . /s - - - with most severe lesions ( fig. a) . rarely, mers-cov antigen was also present within single round cells located in the submucosa of the nasal turbinates, which were presumed to represent macrophages based on histological characteristics ( fig. a, insert) . in addition, single positive cells were detected in the epithelium of the trachea in both mock-vaccinated animals and in the epithelium of a large bronchus of one mock-vaccinated animal at dpi. in mva-s-vaccinated animals low numbers of positive staining cells were present in the epithelium of the nasal turbinates at dpi. no virus was detectable in trachea and bronchi of these dromedaries. at dpi, virus antigen was not detectable in the upper respiratory tract of mva-s-vaccinated animals but very rarely within the nose of mock-vaccinated animals (fig. b ,c). all these findings are in line with previous studies by the co-authors and other investigators . since high amounts of virus antigen were present within the nasal turbinates, and to a lesser extent also within the trachea of mock-vaccinated dromedaries at dpi, these tissues were further analyzed by additional immunohistochemistry using different antibodies. phenotyping of inflammatory cells revealed high numbers of cd + t cells in lamina propria and submucosa of nasal turbinates and trachea in mock-vaccinated dromedaries at dpi ( fig. a-c) . comparison of mock-vaccinated and mva-s-vaccinated animals at that time point revealed significantly increased numbers of t cells within the nasal turbinates, but not within the trachea of mock-vaccinated animals. at dpi, the numbers of t cells were scarce. similarly, increased numbers of cd + macrophages were detected at dpi compared to dpi in nasal turbinates and trachea, but no significant differences were present between both groups at the respective time points (fig. d-f) . the decrease of t cells and macrophages at dpi is in line with the significant reduction of virus antigen at the respective time point (fig. b,c) . immunohistochemistry for detection of cd + b cells did not reveal any differences between groups and time points in the nasal turbinates. however, at dpi numbers of b cells were significantly increased in the trachea of mva-s-vaccinated compared to mock-vaccinated animals and compared to the later time point (fig. g-i) . (fig. a,b) . to further distinguish epithelial subsets in the upper respiratory tract tissue of dromedaries, antibodies specific for certain ck of apical and basal cells have been established and were evaluated regarding their distribution (suppl. fig. s ). double immunofluorescence with these antibodies showed strong co-localization of mers-cov nucleocapsid with ck located in apical epithelial cells at dpi in nasal turbinates and trachea (fig. c,d) . there was no co-localization with ck / and ck , expressed by basal epithelial cells, in any localization (fig. e ,f). moreover, mers-cov nucleocapsid antigen was not identified in serous glands, located in the submucosa of the nasal turbinates, even if the apical proportion of these structures stained brightly positive for ck ( fig. c ). at dpi, localization of mers-cov nucleocapsid antigen in the nasal turbinates of mock-vaccinated remained basically identical to the distribution at dpi but the number of mers-cov positive staining cells was substantially reduced. in mva-s-vaccinated animals the amount of virus antigen detected by immunofluorescence was very low at dpi, but depicted similar co-localization with ck . (fig. c ). loss of dpp expression seems to be exclusively restricted to mers-cov-infected cells as adjacent cells staining negative for mers-cov antigen retained positivity for dpp (insert in fig. b ). for further characterization, staining of cilia was performed to evaluate whether multifocal lack of dpp was associated with concurrent ciliary loss. ciliary loss occurred frequently in association with infiltration of inflammatory cells into the epithelium but also in areas with rather mild inflammatory lesions restricted to lamina propria and submucosa, tunel assay was performed to determine whether the loss of cilia is directly related to virus-induced apoptosis of epithelial cells. since the tunel assay revealed some but not abundant apoptotic cells in the respiratory epithelium (suppl. fig. s ) cilia-specific alterations by mers-cov must be assumed. to finally elucidate the histogenesis of cells staining positive for mers-cov nucleocapsid antigen within the lamina propria of the nasal turbinates of mock-vaccinated dromedaries, additional double immunofluorescence labeling was performed. based on histological evaluation of he stained slides, mers-cov antigen was supposed to be located in macrophages or other inflammatory cells and double labeling with antibodies for detection of t cells, b cells, and macrophages was accomplished. staining of mers-cov nucleocapsid antigen with cd and cd antigen, respectively, did not reveal any co-localization, neither in the nasal turbinates nor in the trachea (fig. a-d) . however, single cells within the lamina propria of the nasal turbinates stained simultaneously positive for iba- and mers-cov nucleocapsid and were characterized by a macrophage-like morphology (fig. e) . in the trachea, double positive staining cell elements were absent (fig. f) . for further confirmation the reaction was repeated by use of another macrophage specific antibody. the anti-cd -specific antibody exhibited an intense co-localization of mers-cov nucleocapsid and cd in single cells in the same localization. overall, mers-cov nucleocapsid antigen was therefore not only detectable in ck + apical epithelial cells but also within the cytoplasm of single macrophages. however, co-localization of mers-cov nucleocapsid with iba- and cd , respectively, was not detected within the trachea, implicating that the presence of mers-cov in macrophages might be related to high virus loads in the respective localization. since dpp was only detectable within the apical brush border of the surface epithelium and submucosal glands, but not on the surface of inflammatory cells within lamina propria and submucosa of the nasal turbinates by immunofluorescence, dromedary and human lymphoid tissue were stained for comparison and control. whereas dpp was evident within lymphoid human tonsillar tissue (fig. a) , a positive signal was not observed in tonsils of mva-s-vaccinated and mock-vaccinated dromedaries using immunofluorescence. similarly, tonsillar tissue of the non-infected control animal did also not reveal any dpp expression. in human lymph node tissue dpp was rarely detectable by immunohistochemistry on round cells in the cortex and more frequently within the paracortex and medulla. in camels dpp was rarely demonstrable within the paracortex and medulla of lymph node but lacked expression in the cortex (fig. a) . in order to elucidate the subset of human immune cells expressing dpp , flow cytometry was performed and revealed dpp expression in human pbmc by cd + t cells and hardly by cd + b cells, cd + nk cells, and cd + monocytes (fig. b) . the s protein of mers-cov detected dpp + cells in both human and dromedary pbmc. lymphocytic and monocytic populations were differentiated based on their size and granularity. dpp was mainly expressed by lymphocytes in human pbmc and monocytes in dromedary pbmc even if the expression level was lower in dromedaries than in humans (fig. c) . it has recently been shown that dromedaries play a key role in the transmission of mers-cov at the animal-human interface . in addition, experimentally infected dromedaries serve as an important animal model for investigations on certain aspects of mers-cov pathogenesis , . the use of appropriate animal models is highly required, since human tissue samples from individual mers-cov cases are rarely accessible [ ] [ ] [ ] , which has in part been attributed to cultural reasons . two individual case descriptions detected viral particles via electron microscopy and immunohistochemistry in pneumocytes, pulmonary macrophages, renal proximal tubular epithelial cells, and macrophages within skeletal muscle. biopsies revealed necrotizing pneumonia, pulmonary alveolar damage, vascular disease, cardiac fibrosis, acute kidney injury, hepatitis, and myositis , . these reports from human tissue underline that the disease observed in dromedaries after natural and experimental mers-cov infection differs substantially from the human counterpart. whereas dromedaries develop only mild respiratory signs and lack overt pulmonary disease and systemic spread , , the disease in humans is often accompanied by acute respiratory distress syndrome, renal dysfunction, and lethal outcome . previous studies indicated that these differences are related to the fact that mers-cov predominantly replicates in the lower respiratory tract of humans but not of dromedaries that might, at least in part, be caused by differing expression patterns of the cell surface receptor dpp . whereas dpp is extensively expressed in the upper respiratory tract epithelia of dromedaries, its expression in the respiratory tract of humans is limited to alveolar epithelial cells and macrophages in the lower airways . in the present study, it has been shown that dpp is located on the apical brush border of ciliated ck expressing epithelia in the upper respiratory tract of dromedaries. in humans dpp dpp (red) is frequently detectable on lymphoid cells (white arrows) in the human tonsil but is completely absent in tonsillar tissue of dromedaries. immunofluorescence, x. in the lymph nodes of both human and dromedary, dpp (red) is detected in the paracortex and medulla (arrows), but in much smaller numbers in that of dromedaries (arrow). immunohistochemistry, x. in human pbmc, dpp is mainly expressed by cd + t cells and hardly expressed by cd + b cells, cd + nk cells, and cd + monocytes (b). s protein of mers-cov is used to detect dpp + cells in both human and dromedary pbmc. lymphocytes and monocytes population are differentiated based on their size and granularity. dpp is mainly expressed by lymphocytes in human pbmc while it is mainly expressed by monocytes in dromedary pbmc (c). data in figure b and c are visualized as the mean percentage. can be detected in the brush border of renal proximal convoluted tubules and enterocytes in the intestine but not within the upper respiratory tract . the present study demonstrates that acute mers-cov infection in dromedaries is accompanied by severe ciliary loss and concomitant lack of dpp on infected cells. adjacent cells in which mers-cov antigen is not detectable retain positive staining for dpp . ciliary loss and consequent disturbances of mucociliary clearance are a major issue in several viral infections and can foster the development of severe secondary bacterial disease . for instance, common cold in humans is accompanied by a massive loss of cilia and ciliated cells . similarly, human coronavirus infection of the upper respiratory tract has been described to be associated with migration of axonemes and basal bodies into the cell body (internalization) complemented by loss of cilia on the apical cell surface of infected cells. for the human disease it has been suggested that replicated virions are stored in apical vesicles before they are released. these vesicles may dislocate the basal body and withdraw the cilia into the cell . in dogs, canine respiratory coronavirus infection is also associated with loss and damage to tracheal cilia, accompanied by inflammation . similar mechanisms might also play a role in mers-cov in dromedaries and would at least explain the massive loss of cilia which appears not to be accompanied by massive cell death or other profound histological and ultrastructural alterations in the majority of affected epithelial cells. interestingly, ciliary loss is accompanied by lack of dpp , which serves as a cell entry receptor for mers-cov in dromedaries. therefore, the authors suggest that the mild and transient disease in dromedaries is, at least in part, attributable to the downregulation of its own cell entry receptor. further studies need to be performed to elucidate underlying mechanisms of dpp loss in mers-cov-infected ck positive staining cells of dromedaries. the remaining expression on adjacent mers-cov negative cells suggests a potential direct virus mediated mechanism. the detection of mers-cov nucleocapsid antigen in the cytoplasm of a limited number of cd /iba- positive staining cells within the lamina propria of the nasal turbinates of mock-vaccinated dromedaries at dpi is similar to previous investigations which detected viral antigen and rna in mononuclear cells and stellate cells of mediastinal lymph nodes in experimentally infected rhesus macaques and rarely within large mononuclear cells in the tracheal lymph node of infected dromedaries . moreover, infection of human monocyte-derived dendritic cells and monocyte-derived macrophages has been described in vitro and was accompanied by release of viral particles , . the infection of these cell types is supposed to be mediated by dpp , expressed on the cell surface of human macrophages , , and leads to suppression of the innate immunity by reduced expression of tumor necrosis factor (tnf) and interleukin- (il- ) . in contrast, it remains so far uncertain whether the intracytoplasmic detection of mers-cov nucleocapsid antigen in dromedary macrophages represents a true productive or abortive infection or whether it is related to phagocytosis of mers-cov fragments. since the number of positive staining macrophages was very low and dpp was not detectable on dromedary tonsils and hardly on dromedary lymphocytes and macrophages in lymph nodes it is not unlikely that the intrahistiocytic detection of mers-cov nucleocapsid antigen is related to phagocytosis of viral particles or infected cellular components. nonetheless the staining of the antigen within affected cells was brightly and diffusely distributed in the cytoplasm and the detection of viral antigen in phagosomes would be suspected to rather appear as discrete spots , . it might therefore been speculated that the viral antigen detection indeed represents virus infection of dromedary macrophages. however, further investigations have to elucidate whether it is a productive or abortive infection. the lack of detection of viral antigen in dromedary t and b cells is in contrast to the findings in humans where mers-cov is capable of infecting t cells derived from peripheral blood and lymphoid organs . on human t cells dpp is widely located on the cell surface and serves as a costimulatory molecule which contributes to t cell activation . contrarily, dpp has not been detected on the surface of dromedary lymphocytes in the tonsil and rarely on those located in lymph nodes of the upper respiratory tract in the present study. these results are in line with the lack of mers-cov antigen in infiltrated t and b cells within infected organs of dromedaries. in humans, abortive infection of lymphocytes induces apoptosis by activation of both the intrinsic and extrinsic pathway of apoptosis and is accompanied by massive downregulation of dpp on the surface of infected t cells . similarly, a loss of the dpp receptor has been detected on the surface of infected epithelial cells in the present study. taken together, obtained results suggest that the incapacity to invade t cells, alongside other factors, might contribute to the low pathogenicity of mers-cov in dromedaries. in summary, the present study highlights new and important differences between mers-cov infection in humans and dromedaries. most importantly, ciliary loss and reduction of dpp expression represent important features of the disease in dromedaries which will deepen our understanding of mers-cov. further investigations need to elucidate the underlying mechanisms of ciliary loss to gain insights into the pathogenesis of this emerging and life-threatening disease. just recently a novel alphacoronavirus has been detected in asian house shrews and future zoonotic transmissions of such novel and well-known coronaviruses will require a profound understanding of their pathogenesis in different host species to achieve better preparedness. animals and tissue sampling. all investigations were performed on archived postmortem tissue, which has been used in a formerly published animal trial pancreas, tonsil, palatum molle, intestine, abomasum, mesenteric lymph node and eyelid) were fixed in % neutral-buffered formalin and routinely paraffin-embedded for histology. serial sections were mounted on coated glass slides (superfrost plus ® , menzel co.) and stained with he or processed for immunohistochemistry and immunofluorescence, respectively. prior to embedding tracheal and nasal tissue were decalcified in % disodium-ethylenediaminetetraacetate (edta, serva electrophoresis gmbh) for h. as an additional control, nasal turbinate of a non-infected dromedary (collected during routine necropsy at the department of pathology, university of veterinary medicine hannover, germany) was used. euthanasia of the animal was performed due to non-respiratory disease. camel peripheral blood mononuclear cells (pbmc) were obtained from naïve animals from a previous experiment . human pbmc were obtained from healthy blood donors (sanquin bloodbank, rotterdam, the netherlands). the use of pbmcs for scientific research was approved by the sanquin bloodbank after informed consent was obtained from the blood donors. the human tonsillar tissue which was used for the establishment of the dpp -and ceacam -specific immunohistochemistry was kindly provided by one of the co-authors (is) and sampled during tonsillectomy. the human bronchiolar lymph nodes paraffin-embedded tissue samples were obtained from the erasmus mc tissue bank. these tissue samples were taken either from healthy donors or from patients with nonmalignant lung tumors. these tissue samples were residual human biomaterials that were collected, stored, and issued by the erasmus mc tissue bank under iso : standard operating procedures. use of these materials for research purposes is regulated according to human tissue and medical research: code of conduct for responsible use ( ). light microscopy, immunohistochemistry, immunofluorescence and tunel assay. he stained cross sections of all organs (mva-s-vaccinated and mock-vaccinated animals, and dpi) were evaluated by light microscopy for the distribution and characterization of lesions induced by mers-cov infection. in addition, all organs were screened for the presence of viral antigen by immunohistochemistry using a previously published monoclonal (mc) mouse antibody (sino biological inc.) . according to the results obtained by light microscopy and mers-cov-specific immunohistochemistry, nasal and tracheal tissues were further analyzed with respect to the immune response. immunohistochemistry was performed for the detection of cd + t cells (polyclonal rabbit anti-cd , dakocytomation gmbh), cd + b cells (polyclonal rabbit anti-cd , thermo fisher scientific), and cd + macrophages (monoclonal mouse anti-human macrophage scavenger receptor, human cd , biologo) as summarized in table . the used markers have been previously established for lymphoid dromedary tissue . additionally, epithelial cells were labeled by application of different antibodies (table ) directed against pan-cytokeratin (ck; monoclonal mouse anti-human cytokeratin, dakocytomation gmbh), ck / (monoclonal mouse anti-cytokeratin / , dakocytomation gmbh), ck (polyclonal rabbit anti-keratin , thermo fisher scientific) and ck (monoclonal mouse anti-cytokeratin , abcam). ceacam was investigated using a polyclonal rabbit anti-human ceacam antibody (abcam) and dpp was visualized using a polyclonal goat anti-dpp /cd -specific antibody (r&d systems). briefly, after dewaxing in roticlear ® (roth c. gmbh & co. kg) and rehydration in isopropanol and ethanol ( %, roth c. gmbh & co. kg), endogenous peroxidase activity was blocked by incubation of sections in % ethanol with . % h o (vwrtm international gmbh) for min at room temperature (rt). antigen retrieval was performed by incubating the sections in citrate buffer ( . g citric acid monohydrate in l distilled water, adjusted with naoh to ph = . ) for min in a microwave ( w). subsequently, sections were transferred to shandon coverplates tm (thermo electron gmbh) and nonspecific binding was blocked by inactivated % rabbit (dpp ) or goat serum (all other antibodies) diluted in phosphate buffered saline (pbs) including % bovine serum albumin (bsa; pbs/bsa) for minutes. afterwards sections were incubated with the respective primary antibody for min at rt. for appropriate negative controls, primary antibodies were replaced by ascites fluid from balb/c mice ( : ; cd , pan-ck, ck / , ck , mc mers-cov nucleocapsid), goat ( : ; dpp ) and rabbit serum ( : ; cd , cd , ck , ceacam ), respectively. subsequently, the appropriate secondary biotinylated antibody was added in a dilution of : with pbs (table ) . incubation for min at rt was followed by treatment with the avidin-biotin-peroxidase complex (vectastain abc kit standard, vectorlaboratories) according to the manufacturer's protocol. visualization of the reaction was achieved by the chromogen , -diaminobenzidine tetrahydrochloride (dab, . %, sigma aldrich chemie gmbh) and addition of . % h o . slides were finally slightly counterstained with mayer's hematoxylin (roth c. gmbh & co kg). subsequently, double immunofluorescence was implemented to determine the cell tropism of mers-cov in dromedaries. for this purpose, the two different mers-cov nucleocapsid-specific antibodies were used in combination with antibodies for detection of cd , cd , iba- (polyclonal rabbit anti-iba- , wako chemicals), cd , ck , ck , ck / , pan-ck and dpp , respectively. in addition, single immunofluorescence using anti-acetylated α-tubulin antibody (monoclonal mouse anti-α-tubulin, sigma-aldrich) was performed for visualization of the ciliary axoneme . after dewaxing and retrieval of antigens as described above, nonspecific binding was blocked by inactivated % horse (dpp ) or goat serum (all other antibodies), respectively, diluted in pbs/ . % triton x/ % bsa (pbs/triton x/bsa) for minutes. both primary antibodies were applied simultaneously in the respective concentrations (table ) diluted with pbs/triton x/bsa. for appropriate negative controls, primary antibodies were replaced by ascites fluid from balb/c mice ( : ; pan-ck, ck / , ck , cd , mc mers-cov nucleocapsid, α-tubulin), rabbit ( : ; ck , cd , cd , iba- , pc mers-cov nucleocapsid), and goat serum ( : ; dpp ), respectively. after incubation over night at °c both secondary antibodies were applied simultaneously ( : in pbs/triton x/bsa, table ) and were incubated for min at rt in the dark to visualize the respective antigens. nuclear counterstaining was performed with . % bisbenzimide (sigma-aldrich chemie gmbh) for minutes and sections were mounted with dako fluorescence mounting medium (dakocytomation gmbh). terminal deoxynucleotidyl transferase (tdt) dutp nick-end labeling (tunel) assay was performed to detect cells that undergo extensive dna degradation during the late stages of apoptosis on the camel nasal epithelium according to the protocol provided by the manufacturer (merck kgaa). tissues pre-treated with dnase were used as positive control while tissues stained with labelling solution only were used as respective negative control consistent with the manufacturer's recommendations. flow cytometry. in order to detect which subset of human immune cells expresses dpp , freshly isolated human pbmcs were incubated with a cocktail of fluorescence labelled monoclonal antibodies, i.e. anti-cd (clone sp - , bd biosciences); cd (clone mem , serotec); cd (clone b e , beckman-coulter); hla-dr (clone l , biolegend); cd (clone m e , bd biosciences); dpp (clone , r&d systems); and live/dead cell viability marker (thermo fisher scientific). appropriate ig isotype control antibodies were used as negative controls for each staining. s protein of mers-cov that recognizes dpp , used in a previous study , was applied to compare dpp expression in camel and human pbmc. cells were analyzed on a canto ii flow cytometer (bd biosciences) and using flowjo ® software (flowjo), then subsequently presented as mean percentage of positive cells. scanning electron microscopy. for scanning electron microscopy, formalin fixed samples of nasal mucosa, trachea, and bronchus of dromedaries infected with mers-cov were post-fixed in % glutaraldehyde (sigma-aldrich chemie gmbh) for hours. afterwards the samples were dehydrated in a series of graded ethanol, dried and coated in a sputter-coater (scd ; oerlikon balzers) with gold as described previously , . virus by immunohistochemistry five randomly selected images per section, antibody and animal were taken at x magnification from the epithelium and adjacent lamina propria and submucosa of nasal turbinates and trachea using an olympus bx- digital camera microscope (olympus optical co. gmbh) and the cell-d imaging software (olympus soft imaging solutions). afterwards, pictures were evaluated manually by counting immunopositive cells. statistical analysis was conducted using the statistical analysis software sas . and the enterprise guide . for windows (sas institute inc.). pair-wise comparison of groups (mva-s-vaccinated [n = high power fields] and mock-vaccinated [n = high power fields]) was performed by use of multiple mann-whitney-u-tests. results were considered statistically significant at p-value < . . column bars were generated with graphpad prism . (graphpad software, inc.). slides stained by immunofluorescence were evaluated for co-localization of antigens using an olympus ix inverted fluorescence microscope, the digital camera olympus dp (olympus life science) and the cell f imaging software (olympus soft imaging solutions gmbh, olympus optical co. gmbh). for visualization of the results of scanning electron microscopy a digital scanning microscope (dsm , carl zeiss jena gmbh) was used. per localization and time point post infection eight images were taken at x magnification and the percentage of ciliated area was estimated. data were analyzed using graphpad prism . (graphpad software, inc.). mann whitney-u-test was applied and results were considered statistically significant at p-value < . . table . antigen, clonality, species, source, antigen retrieval, dilution and secondary antibodies used for immunohistochemistry and immunofluorescence. ab, antibody; cd, cluster of differentiation; ceacam , carcinoembryonic antigen related cell adhesion molecule ; ck, cytokeratin; dpp , dipeptidylpeptidase ; gam-b, goat anti-mouse igg, biotinylated; gar, goat anti-rabbit, biotinylated; iba- , ionized calcium-binding adapter molecule ; immunoglobulin g (igg); mc, monoclonal; mers-cov, middle east respiratory syndrome coronavirus; n.a., not applied; pc, polyclonal; rag-b, rabbit anti-goat igg, biotinylated; rar, rabbit anti-rat igg, biotinylated. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group rooting the phylogenetic tree of middle 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coronavirus through its receptor, dipeptidyl peptidase differential expression of the middle east respiratory syndrome coronavirus receptor in the upper respiratory tracts of humans and dromedary camels carcinoembryonic antigen-related cell adhesion molecule is an important surface attachment factor that facilitates entry of middle east respiratory syndrome coronavirus identification of sialic acid-binding function for the middle east respiratory syndrome coronavirus spike glycoprotein evaluation of a panel of antibodies for the immunohistochemical identification of immune cells in paraffin-embedded lymphoid tissues of new-and old-world camelids mycobacterium bovis infections in domesticated non-bovine mammalian species. part : review of epidemiology and laboratory submissions in great britain clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates histopathology of middle east respiratory syndrome coronovirus (mers-cov) infection -clinicopathological and ultrastructural study middle east respiratory syndrome dipeptidyl-peptidase iv (cd )-role in the inactivation of regulatory peptides mucociliary clearance: pathophysiological aspects ultrastructural changes in human nasal cilia caused by the common cold and recovery of ciliated epithelium ultrastructure of human nasal epithelium during an episode of coronavirus infection tropism and pathological findings associated with canine respiratory coronavirus (crcov) productive replication of middle east respiratory syndrome coronavirus in monocyte-derived dendritic cells modulates innate immune response active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis a potential role for dendritic cell/macrophage-expressing dpp in obesity-induced visceral inflammation middle east respiratory syndrome corona virus spike glycoprotein suppresses macrophage responses via dpp -mediated induction of irak-m and pparγ evidence for phagocytosis of influenza virus-infected, apoptotic cells by neutrophils and macrophages in mice macrophage phagocytosis of foot-and-mouth disease virus may create infectious carriers middle east respiratory syndrome coronavirus efficiently infects human primary t lymphocytes and activates the extrinsic and intrinsic apoptosis pathways the costimulatory activity of the cd antigen requires dipeptidyl peptidase iv enzymatic activity discovery of a highly divergent coronavirus in the asian house shrew from china illuminates the origin of the alphacoronaviruses immunofluorescence staining of ciliated respiratory epithelial cells canine parainfluenza virus-induced encephalitis in ferrets intranasal infection of ferrets (mustela putorius furo) with canine parainfluenza virus the authors would like to thank bettina buck, petra grünig, kerstin schöne, caroline schütz, danuta waschke, and debby schipper for excellent technical assistance. the technical assistance of david solanes, xavier abad and ivan cordón and all animal caretakers from the cresa-irta biosecurity level laboratories and animal facilities is acknowledged. this research was performed as part of the zoonoses anticipation and preparedness initiative (zapi project; imi grant agreement no. ), with the assistance and financial support of imi and the european commission, and in-kind contributions from efpia partners. w. widagdo was supported by a top project grant ( ) funded by zonmw. cresa-irta thanks the funding through the cerca programme of the generalitat de catalunya (spain). supplementary information accompanies this paper at https://doi.org/ . /s - - - .competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -amv los authors: widagdo, w.; sooksawasdi na ayudhya, syriam; hundie, gadissa b.; haagmans, bart l. title: host determinants of mers-cov transmission and pathogenesis date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: amv los middle east respiratory syndrome coronavirus (mers-cov) is a zoonotic pathogen that causes respiratory infection in humans, ranging from asymptomatic to severe pneumonia. in dromedary camels, the virus only causes a mild infection but it spreads efficiently between animals. differences in the behavior of the virus observed between individuals, as well as between humans and dromedary camels, highlight the role of host factors in mers-cov pathogenesis and transmission. one of these host factors, the mers-cov receptor dipeptidyl peptidase- (dpp ), may be a critical determinant because it is variably expressed in mers-cov-susceptible species as well as in humans. this could partially explain inter- and intraspecies differences in the tropism, pathogenesis, and transmissibility of mers-cov. in this review, we explore the role of dpp and other host factors in mers-cov transmission and pathogenesis—such as sialic acids, host proteases, and interferons. further characterization of these host determinants may potentially offer novel insights to develop intervention strategies to tackle ongoing outbreaks. middle east respiratory syndrome coronavirus (mers-cov) is a novel pathogen that was isolated in late [ ] . since then, the virus has caused multiple outbreaks and infected more than individuals, [ ] who then develop a respiratory infection ranging in severity from asymptomatic to fatal [ , ] . severe-to-fatal mers-cov patients have a higher chance of transmitting this virus since they shed a higher amount of virus progeny in comparison to the asymptomatic-to-mild ones [ ] [ ] [ ] [ ] . identifying and quarantining these patients in healthcare facilities where outbreaks have occurred, together with implementing proper infection control, has been effective in reducing transmission and containing these outbreaks [ , ] . however, new mers-cov cases are still being reported, especially in the arabian peninsula [ , ] . this is partly due to the continuous zoonotic introduction of this virus to the human population in this region by dromedaries [ ] . the dromedary camel is the only animal species that has been reported to transmit this virus to humans [ ] [ ] [ ] [ ] . mers-cov infection in these animals merely causes mild upper respiratory tract infection [ , ] , but seroepidemiological studies showed that this virus has been circulating in dromedary camels for decades, suggesting the efficient transmission of mers-cov in this species [ ] [ ] [ ] [ ] . although the clinical manifestations, as well as transmission, are remarkably different in mers-cov-infected humans and dromedary camels, the viruses isolated from these two species are highly similar, if not indistinguishable [ , ] . this indicates that host factors play a significant role in mers-cov pathogenesis and transmission. however, the identity of these host factors and how they affect the pathogenesis and transmission of mers-cov are generally not well understood. dipeptidyl peptidase- (dpp )-the mers-cov receptor, sialic acids, proteases, and interferons are ; a cartoon representation of mers-cov s protein binding to dpp (pdb code l ) (b). the s protein consists of the s and s subunits. the α/β hydrolase domain of dpp is indicated in red, β-propeller domain in green, while part of the mers-cov s protein is shown in blue. other mers-cov-interacting host factors besides dpp are less extensively studied and have mostly been investigated in vitro. glycotopes of α , -sialic acids coupled with -n-acetylated neuraminic acid are recognized by the s protein of mers-cov during attachment [ ] . in the absence of these glycotopes, mers-cov entry is reduced but not abolished, indicating their function as an attachment factor rather than a receptor [ ] . besides α , -sialic acids, ceacam and grp have also been suggested to be attachment factors for mers-cov, but their roles in vivo during mers-cov infection are not clear at this moment [ , ] . post attachment, mers-cov uses the c-terminal part of its s protein-known as s ( figure a )-to interact with host proteases, such as furin, tmprss , and cathepsins [ ] [ ] [ ] [ ] . these proteases cleave the s protein and induce conformational changes, allowing fusion between viral and host cellular membranes, resulting in the release of viral rna into the cell cytoplasm [ ] . tmprss and dpp are held in one complex at the cell surface by a scaffolding protein, the tetraspanin cd , leading to a rapid and efficient entry of mers-cov into the susceptible cells [ ] . once fusion with host cell membranes has occurred, mers-cov subsequently replicates its genetic material and produces viral proteins in the cell cytoplasm to generate new virus progeny. during this stage, mers-cov uses its nsp - polyproteins to build its replication organelles as well as its accessory proteins such as the a and b proteins to inhibit host anti-viral defense mechanisms [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, the capacity of mers-cov accessory proteins to impede several pathways of host immune response in the lungs may be limited. mers-cov inoculation of macaques and genetically modified mice generally results in limited clinical manifestations; thus, adapting this virus through serial passaging or defecting the type i interferon pathway may be needed to enhance viral replication and pathogenesis in these animals [ , [ ] [ ] [ ] [ ] . these observations, together with studies showing type i interferon capacity to inhibit mers-cov infection in vitro [ , ] , highlight the importance of the innate immune response, especially type i interferon, as an inhibiting factor for mers-cov. so far mers-cov has been isolated from dromedary camels and humans [ , ] . both species are not only susceptible to mers-cov infection, but also capable of transmitting this virus [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] ]. however, current data indicate that virus spread is more efficient in dromedary camels than in humans [ , , [ ] [ ] [ ] ] . this difference in transmissibility could be partially due to the different tropism of mers-cov in these two species. in dromedaries, mers-cov has been shown to replicate in the nasal epithelium upon experimental in vivo infection [ ] , while in humans, mers-cov mainly replicates in the lower respiratory tract, particularly in the bronchiolar and alveolar epithelia [ , [ ] [ ] [ ] [ ] . higher viral rna levels in the sputum and lavage samples of mers-cov patients compared to nasal and throat swabs are consistent with the tropism of mers-cov in humans [ ] [ ] [ ] . this different mers-cov tropism in dromedary camels and humans is in line with the localization of dpp in the respiratory tract tissues of these two species. in humans, dpp is absent in the nasal epithelium but present in the lower respiratory tract epithelium, mainly in type ii pneumocytes [ , ] . in contrast, dpp is expressed in the nasal epithelium of dromedary camels [ ] . this difference in dpp localization between humans and dromedary camels therefore explains mers-cov tropism in these two species and highlights dpp as an essential determinant of mers-cov tropism. dpp localization has also been investigated in many other mers-cov-susceptible species. in gambian and egyptian fruit bats, dpp is expressed in the respiratory tract and intestinal epithelium, suggesting that mers-cov can target both tissues [ ] . in line with this finding, mers-cov inoculation via intranasal and intraperitoneal routes in the jamaican fruit bat led to viral rna shedding both in the respiratory tract and the intestinal tract [ ] . in contrast to frugivorous bats, dpp is limitedly expressed in the respiratory tract epithelium of two insectivorous bats, i.e., common pipistrelle and common serotine bats, but abundant in their intestinal epithelium [ ] . accordingly, sequences of mers-like-covs were mainly obtained from rectal swabs and fecal samples of insectivorous bats [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . these findings not only support insectivorous bats as the origin host of mers-cov [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , but also indicate the importance of intestinal tropism and fecal-oral transmission of mers-like-cov in these insectivorous bats. besides bats, humans, and dromedary camels, other animal species have also been proposed as potential hosts of mers-cov. remarkably, dpp of horses, llamas, alpacas, pigs, bovines, goats, sheep, and rabbits has been demonstrated to recognize the s protein of mers-cov [ , ] . in most of these species, there is a preferential upper respiratory tract expression of dpp observed. rabbits express dpp in the upper and lower respiratory tract epithelium, and thus may allow mers-cov to replicate in both compartments [ , ] . horses, llamas, and pigs mainly express dpp in the upper respiratory tract-particularly the nasal epithelium [ ] . upon intranasal mers-cov inoculation, llamas, alpacas, and pigs developed upper respiratory tract infection, while horses did not seroconvert and only shed infectious virus in a limited amount [ ] [ ] [ ] [ ] [ ] . the reason why horses seem to be less permissive to mers-cov remains to be investigated, but a chronic co-infection in the guttural pouch, a common disease among horses, might be one of the explanations. this guttural pouch infection results in excessive mucus production that might hinder mers-cov from attaching and entering the nasal epithelium [ , , ] . sheep, on the other hand, did not seem to express significant levels of dpp in their respiratory tract, and thus did not seroconvert nor shed infectious virus upon experimental mers-cov inoculation [ , ] . comparable to sheep, goats limitedly shed infectious virus upon experimental infection and did not transmit this virus to other naïve goats upon direct contact [ ] . the results of experimental mers-cov infection in livestock animals are in line with data from epidemiological studies. mers-cov seropositive llamas and alpacas are present in the field, while horses, goats, and sheep are generally found to be seronegative [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . given the fact that experimental in vivo infection studies and dpp expression analysis in different animal species revealed that dromedary camels are not the only animals in which mers-cov has an upper respiratory tract tropism [ , , , ] , it is then relevant to question whether other animals can potentially spread mers-cov as well. new world camelids, i.e., alpacas and llamas, are able to transmit the virus to respective naïve animals upon contact [ ] . pigs and rabbits, on the other hand, hardly transmit the virus-neither by contact nor airborne routes [ , ] . most likely, this is caused by the fact that pigs and rabbits, unlike dromedary camels, shed low levels of infectious virus upon mers-cov inoculation ( figure ) . this difference indicates that other host factors besides dpp could cause interspecies variation in mers-cov infection. indeed, several glycotopes of α , -sialic acids that function as attachment factors of mers-cov are present in the nasal epithelium of dromedary camels but absent in that of rabbits and pigs ( figure ) [ , ] . the lack of these glycotopes in pigs and rabbits might limit the susceptibility and transmission of mers-cov in these animals. although the role of these glycotopes in mers-cov transmission still requires further investigation, it remains plausible that an efficient transmission of this virus might require the presence of both dpp and mers-cov-recognized glycotopes of α , -sialic acids (figure ). . it has also been reported that the lysosomal proteases from bat cells support coronavirus spike-mediated virus entry more efficiently than their counterparts from human cells [ ] . these observations suggest that host proteases from different host species may determine the species and tissue tropism of mers-cov. because mers-cov has been circulating in dromedary camels for decades before emerging in the human population [ ] [ ] [ ] [ ] , it is plausible that this virus inhibits the immune response of dromedary camels more efficiently than that of other species, including pigs and rabbits. the difference in immune response among mers-cov-susceptible species is therefore another factor that might yield interspecies variation in permissiveness to mers-cov. characterizing the difference in host proteases and immune responses among mers-cov-susceptible species, as performed for dpp and mers-cov-recognized α , -sialic acid glycotopes (figure ) , has not yet been investigated. these data, however, may further explain interspecies variation in mers-cov infection and transmission. mers-cov causes respiratory infection in humans ranging from asymptomatic to severe pneumonia [ , ] . however, it is currently unclear what causes this intraspecies variation. epidemiology data indicate that individuals with certain risk factors are at higher risk of developing severe mers-cov infection [ , ] . this implies that some host factors may dictate the outcome of mers-cov infection, thus rendering intraspecies variation. two of the risk factors, i.e., smoking and chronic obstructive pulmonary disease (copd), have been shown to upregulate dpp expression in the lungs [ , [ ] [ ] [ ] , suggesting dpp as a possible reason for intraspecies variation observed among mers-cov patients. in healthy human lungs, dpp is almost exclusively expressed in type ii pneumocytes [ , ] . type ii pneumocytes are small cuboidal cells that can regenerate alveolar epithelium upon injury, and roughly cover % of the alveolar surface area. meanwhile, around % of the surface area of the alveolus is occupied by type i pneumocytes that are morphologically flat and responsible for gas exchange [ , ] . in the lungs of smokers and copd patients, unlike in healthy human lungs, dpp is prominently expressed in both type i and ii pneumocytes, indicating upregulated expression on type i pneumocytes [ ] . autopsy reports from fatal mers-cov patients showed that both type i and ii pneumocytes expressed dpp and became infected by mers-cov, proposing a role of dpp -expressing type i pneumocytes in mers-cov pathogenesis [ , ] . damage to type i cells in the lung alveoli during viral infection may lead to diffuse alveolar damage [ ] . in line with observations made in human mers cases, common marmosets that express dpp in both type i and ii pneumocytes have been reported to produce more infectious virus upon experimental mers-cov infection, compared to rhesus and cynomolgus macaques that merely expressed dpp in type ii pneumocytes [ , [ ] [ ] [ ] [ ] . accordingly, these common marmosets developed moderate-to-severe infection, while macaques generally developed mild transient pneumonia [ , , [ ] [ ] [ ] [ ] . similarly, in genetically modified mice that displayed mers-cov tropism for type ii pneumocytes, only mild clinical manifestations were observed upon mers-cov infection [ , ] . adapting mers-cov through serial passaging or upregulating dpp expression throughout the airway epithelium in mice, however, will induce severe clinical disease [ , ] . these data altogether support the role of dpp -expressing type i pneumocytes in the pathogenesis of severe mers-cov infection. the differential expression of host factors that limits the infection should also be taken into account. dpp in soluble form has been demonstrated to protect against mers-cov infection in vitro and in a mouse model [ , ] ; however, its presence in the lungs and role in mers-cov pathogenesis remain to be investigated. the host immune response also has the capacity to inhibit mers-cov infection. mers-cov has been shown to replicate to higher levels in immunocompromised rhesus macaques [ ] , consistent with the observation that immunocompromised individuals have difficulties clearing mers-cov upon infection [ , , ] . the survivors of mers-cov infection have been shown to develop virus-specific cd + and cd + t cell responses, implying the role of t cells in virus clearance [ ] . however, the depletion of t cells in mice can either lead to failure in mers-cov clearance or improvement in clinical outcome, depending on the type of mouse model used [ , ] . therefore, the role of adaptive immune response in mers-cov pathogenesis is currently unclear. on the other hand, one of the main components of the host innate immune response, type i interferon, inhibits mers-cov replication in susceptible cells, partly by inhibiting double membrane vesicles (dmv) formation [ , , , , ] . the absence of type i interferon signaling in mice also resulted in more severe clinical manifestations and histopathological lesions upon mers-cov infection [ ] . advance age, which can cause delayed type i interferon response upon viral infection, is a well-known risk factor for fatal mers-cov infection [ , , [ ] [ ] [ ] . collectively, these data highlight the role of host innate immune response as a potent inhibitor for mers-cov infection. it is indubitable that severe mers-cov infection is not solely driven by the pathogen. additional underlying conditions increase mers-cov replication and induce severe-to-fatal clinical manifestations [ , , , , ] . it is plausible that more than one underlying condition is needed to yield a fatal outcome. dpp upregulation in type i pneumocytes and insufficient type i interferon response might be crucial determinants for severe mers-cov infection (figure ). further investigation of the host determinants of mers-cov pathogenesis may offer insights for developing novel therapeutic measures. although mers-cov has been reported to undergo some genotypic changes since it emerged in the human population [ , [ ] [ ] [ ] [ ] , this has not resulted in distinct phenotypic changes so far [ , ] . therefore, host factors remain the most significant determinant in explaining inter-and intraspecies variations observed in mers-cov pathogenesis and transmission. dpp and mers-cov-recognized α , -sialic acids might partially explain these variations, since their localization has been demonstrated to be variable between mers-cov-susceptible species [ , , , ] . dpp expression in human lungs has also been shown to vary due to certain comorbidities [ , , ] . nevertheless, it is undoubtable that the inter-and intraspecies variation in mers-cov pathogenesis and transmission is a complex phenomenon influenced by more than one host factor. current data suggest proteases and interferons as other critical host factors, but how they instigate inter-and intraspecies variations, as well as their role in mers-cov pathogenesis and transmission, still remain to be further elucidated. characterization of the host determinants of mers-cov pathogenesis and transmission could potentially offer insight into this virus epidemiology and guide novel therapeutic development. it may also help to identify the most vulnerable individuals to protect against mers-cov infection-for example, by using vaccination. author 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transgenic mice to middle east respiratory syndrome coronavirus infection and disease pathogenicity and viral shedding of mers-cov in immunocompromised rhesus macaques atypical presentations of mers-cov infection in immunocompromised hosts recovery from the middle east respiratory syndrome is associated with antibody and t-cell responses cd + t cells and macrophages regulate pathogenesis in a mouse model of middle east respiratory syndrome interferon-beta and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cell-based assays antiviral innate immune response interferes with the formation of replication-associated membrane structures induced by a positive-strand rna virus ifn production ability and healthy ageing: mixed model analysis of a year longitudinal study in japan age-associated failure to adjust type i ifn receptor signaling thresholds after t cell activation dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice middle east respiratory syndrome the impact of co-infection of influenza a virus on the severity of middle east respiratory syndrome coronavirus mers coronaviruses from camels in africa exhibit region-dependent genetic diversity increase in middle east respiratory syndrome-coronavirus cases in saudi arabia linked to hospital outbreak with continued circulation of recombinant virus multihospital outbreak of a middle east respiratory syndrome coronavirus deletion variant deletion variants of middle east respiratory syndrome coronavirus from humans key: cord- - pkbbb authors: sunaga, fujiko; tsuchiaka, shinobu; kishimoto, mai; aoki, hiroshi; kakinoki, mari; kure, katsumasa; okumura, hanako; okumura, maho; okumura, atsushi; nagai, makoto; omatsu, tsutomu; mizutani, tetsuya title: development of a one-run real-time pcr detection system for pathogens associated with porcine respiratory diseases date: - - journal: j vet med sci doi: . /jvms. - sha: doc_id: cord_uid: pkbbb the etiology of porcine respiratory disease complex is complicated by infections with multiple pathogens, and multiple infections increase the difficulty in identifying the causal pathogen. in this present study, we developed a detection system of microbes from porcine respiratory by using taqman real-time pcr (referred to as dempo-pcr) to screen a broad range of pathogens associated with porcine respiratory diseases in a single run. we selected porcine respiratory pathogens (actinobacillus pleuropneumoniae, boldetella bronchiseptica, haemophilus parasuis, pasteurella multocida, pasteurella multocida toxin, streptococcus suis, mycoplasma hyopneumoniae, mycoplasma hyorhinis, mycoplasma hyosynovie, porcine circovirus , pseudorabies virus, porcine cytomegalovirus, swine influenza a virus, porcine reproductive and respiratory virus us strain, eu strain, porcine respiratory coronavirus and porcine hemagglutinating encephalomyelitis virus) as detection targets and designed novel specific primer-probe sets for seven of them. in sensitivity test by using standard curves from synthesized dna, all primer-probe sets showed high sensitivity. however, porcine reproductive and respiratory virus is known to have a high frequency of genetic mutations, and the primer and probe sequences will need to be checked at a considerable frequency when performing dempo-pcr from field samples. a total of lung samples from swine showing respiratory symptoms on six farms were tested by the dempo-pcr to validate the assay’s clinical performance. as the results, pathogens ( virus and bacteria) were detected and porcine reproductive and respiratory virus us strain, mycoplasma hyorhinis, haemophilus parasuis, and porcine cytomegalovirus were detected at high frequency. these results suggest that dempo-pcr assay can be applied as a screening system with wide detection targets. doi: . /jvms. bronchiseptica, haemophilus parasuis, mycoplasma hyorhinis, mycoplasma hyosynovie, pseudorabies virus (prv), porcine respiratory corona virus (prcv), porcine cytomegalovirus (pcmv) [ , , , ] . infection with each single pathogen does not necessarily result in appearance of symptoms, but complex infections with a variety of pathogens, including the indigenous agents, develop severe conditions. infections with such multiple pathogens make it difficult to rapidly identify the etiology of prdc. to adopt appropriate measures, such as vaccination or hygiene management, and to minimize the economic loss of prdc, it is necessary to quickly, accurately and comprehensively detect multiple pathogens present in varying proportions in each herd. serological tests [ ] , pathogen isolation [ ] and pcr-based tests [ , ] are currently available to diagnose prdc in laboratories. most tests are based on a one assay-one pathogen approach, and they are not enough for diagnosis of prdc in terms of comprehensiveness and rapidity. tsuchiaka et al. previously developed a system to detect microbes in bovine diarrhea by taqman real-time pcr, permitting the simultaneous screening of pathogens associated with diarrhea [ ] . taqman real-time pcr possesses the advantages of high sensitivity, high specificity, and simple operation. the objective of this study is to develop a system based on taqman real-time pcr that can detect pathogens, including viruses and bacteria, associated with porcine respiratory diseases in one run. a total of primer-probe sets were used to detect pathogens that certainly or possibly cause respiratory diseases on porcine. each primer-probe set was designed to detect a single target pathogen. new primer-probe sets were designed for pasteurella multocida and toxin, m. hyosynovie, pcv , pcmv, siv and phev using the primerquest software (integrated dna technologies, inc., coralville, ia, usa) based on consensus sequences of each pathogen obtained from the genbank database. primer and probe information and their target pathogens are summarized in table . genbank accession numbers, the reference sequence, country, host and first deposited year used for primers and probes design of each pathogen were shown in supplemental table . previously reported qpcr assays were used for pathogen species, including rna, dna viruses and bacteria [ , , , , , , , , ] . furthermore, as an internal control within the dempo-pcr reaction, primer-probe sets for β-actin were synthesized as previously reported [ ] . all hydrolysis probes were labeled with the reporter dye fam ( -carboxyfluorecein) at the ′ end and the fluorescent dye tamra ( -carboxytetramethylrhodamine) at the ′ end. primers and probes were purchased from sigma-aldrich (sigma aldrich, st. louis, mo, usa), and probes containing the mixed base were produced at integrated dna technologies (integrated dna technologies, inc.). step primescript rt-pcr kit (perfect real time) (takara bio, kusatsu, japan) was used to detect viral rna, and premix ex taq (perfect real time) (takara bio) was used to detect viral and bacterial dna. all reactions were performed in a total volume of µl, which contained the sample nucleic acid, primers, probes (the final concentration of all primers and probes was . µm) and all other components included in the kits, according to the manufactures' protocols. thermal cycling conditions were as follows: °c for min and °c for sec, followed by cycles of °c for sec, °c for sec, and °c for sec [ ] . fluorescent signal data were analyzed using an automatic quantification algorithm in lightcycler nano software . (roche diagnostics gmbh), and the parameters of analysis were as follows: exclude early cycle= , minimum relative amplifications= , and minimum amplification quality= . to verify the sensitivity, linearity, and efficiency of the real-time pcr assay, the limit of detection (lod), correlation coefficient (r ) and pcr efficiency (e) were determined from standard curves. standard curves were obtained, and the lod, r and e were calculated as described previously [ , ] . for the purpose of validation, real-time pcr reliability, sensitivity, and linearity of standard curves were verified by testing tenfold serial dilutions of synthesized dna, including each target genome sequence ( × to × copies/reaction). the synthesized dna was purchased from integrated dna technologies (integrated dna technologies, inc.). pathogen dilutions were repeated twice in separate runs, and a standard curve was constructed from the cq values. the pcr efficiency (e) was calculated using the standard curve slope according to the following formula: e=( − /slope (− ) ). the correlation co-efficient (r ) was also calculated. the limit of detection (lod) was defined as the lowest concentration at which a fluorescent signal could be detected in all reactions. reproducibility (inter-assay variance) was assessed using the coefficient value (cv) calculated based on quantification cycle (cq) values. the assay was applied to test clinical samples. a total of samples of porcine lung tissue submitted in - to azabu university for diagnosis of porcine respiratory pathogens were used to test. these pigs were to days old and belonged to farms (a to f), all showing respiratory symptoms (supplementary table ). lung tissues were minced by scissors, diluted : in phosphate buffered saline (pbs, ph . ), homogenized for sec at , rpm with the presence of three stainless steel beads (φ mm) by using the bead crusher µt- (taitec, inc.), and centrifuged at , g for min to obtain the supernatant. bacteria nucleic acids, viral dna, and viral rna were extracted from the supernatant using a qiaamp ® cador ® pathogen kit (qiagen, hilden, germany) with a sample volume of µl and elution volume of µl, as described by the manufacturer. the extracted dna and rna were stored at − °c until examination. the extracted nucleic acids were evaluated in triplicated by targeting respiratory disease complex pathogens in a single run of dempo-pcr. when the cq values were calculated by algorithm described above in more than two out of three runs, the samples were considered positive. in order to compare dempo-pcr assay with the classical method, the conventional pcr (cpcr) was performed under each condition using conventional primers (supplementary table ). a primescript tm rt master mix (takara bio) and gotaq ® green master mix (promega) was used. all reactions were performed in a total volume of µl, which contained the sample nucleic acid, primers (the final concentration of all primers was . µm) and all other components included in the kits, according to the manufactures' protocols. amplicons were detected by electrophoresing. the samples which showed the results of the dempo-pcr assay is not consistent with the cpcr assay did not match, were confirmed by direct sequencing of amplification products. all the experiments were conducted in accordance with the guidelines for the care and use of laboratory animals of azabu university. to evaluate the sensitivity, linearity, and efficiency of the pcr, -fold serial dilutions of synthesized dna were tested by realtime pcr. standard curves were constructed from cq values, then the lod, r , and e were evaluated ( supplementary fig. ). table shows the results for lod number and cvs of run-to-run variants. the lod, based on dna copy number, was ≤ copies/reaction for all primer-probe sets. the cvs were at most . %; this reproducibility was observed with prcv testing. in addition, the calibration curves of all assays covered a linear dynamic range of more than five orders of magnitude and showed r values of at least . . although the pcr efficiency for prrsv us strain and prv was slightly low ( . % and . %, respectively), the pcr efficiency in all detection assays was more than %, which was enough to quantify the target copy number. a total of lungs from different affected animals on six farms with respiratory disease outbreaks were applied to dempo-pcr. in addition, cpcr assay were also performed to compare the sensitivities of dempo-pcr assay. as the results, there were samples detected by dempo-pcr but not detected by cpcr. the sequences of these samples proved to be identical to the sequence of the target pathogens by direct sequencing of amplification products. to the contrary, there were no samples detected by cpcr but not detected by dempo-pcr (supplementary table ). the results are presented as the number and percentage of positive samples from each farm (table ). in samples from farm c and f, both viral and bacterial pathogens, including pcv ( % and %, respectively), prrsv us strain ( % and %, respectively) and m. hyopneumoniae ( . % and %, respectively), were detected at high frequency, whereas mainly bacterial pathogens, including b. bronchiseptica, h. parasuis, p. multocida, s. suis and m. hyorhinis were prevailed in farm a, and a. pleuropneumoniae and s. suis were prevailed in farm e. in samples from farms b, mixed infections of prrsv us strain, siv and bacterial pathogens; h. parasuis, p. multocida, m. hyorhinis, and m. hyopneumoniae were detected. pcmv was detected at high frequency from all farms, whereas p. multocida toxin, m. hyosynooviae, prv, prrsv eu strain, and phev were not detected. prdc is one of the most important health concerns for pig producers and involves multiple viral and bacterial pathogens. prdc is multifactorial, with both infectious and non-infectious factors contributing to respiratory disease seen in pigs between the ages of and months. the interaction of viral and bacterial pathogens, environmental factors, pig-specific factors and management conditions all contribute to the development and impact the severity of prdc [ ] . the most commonly isolated pathogens are prrsv, siv, pcv , and m. hyopneumoniae. the other pathogens associated with prdc are s. suis, a. pleuropneumoniae, p. multocida, p. multocida toxin, b. bronchiseptica, h. parasuis, m. hyorhinis, m. hyosynovie, prv, prcv, pcmv [ , , , ] . however, no single-reaction diagnostic test currently exists for the simultaneous detection of major pathogens commonly associated with prdc. routine diagnostic methods for detection of viruses implicated in prdc include virus isolation in cell culture, antigen detection by direct fluorescent antibody staining, and enzyme immunoassay [ ] and culture-based methods for bacteria [ ] . these methods are time-consuming and require independent tests for each pathogen. furthermore, the detection of bacterial pathogens typically depends on culture-based methods that can take several days to obtain results. due to their high sensitivity and ease of use, pcr and real-time pcr tests have been developed for several agents implicated in the prdc; however, these tests typically target single pathogens [ ] . a multiplex pcr assay capable of detecting five porcine viruses including two porcine respiratory viruses was developed [ ] . however, to date, there are no diagnostic tests capable of simultaneous detection of multiple major viral and bacterial porcine respiratory pathogens in a single reaction. recently lung et al. [ ] reported a novel prototype automated microarray that integrates and automates all steps of post-pcr microarray processing for the simultaneous detection and typing of four bacteria (m. hyopneumoniae, p. multocida, s. enterica serovar choleraesuis, s. suis) and four viruses (prrsv, siv, pcv , prcv) differentiation of the two prrsv genotypes and pathogenic versus non-pathogenic p. multocida strains. this electronic microarray assay can be completed in less than hr with little user handling plus approximately . hr for the rt-pcr. these methods are highly specific and sensitive, and easy to operate, but these are expensive to run and requires expensive equipment. on the other hand, dempo-pcr assay can be completed in less than hr, and easy operate. in this study, dempo-pcr has been developed, following the methods of diagnosis of bovine diarrhea developed by tsuchiaka et al. [ ] . since all primer-probe sets were optimized in the same temperature conditions, dempo-pcr can detect a total of pathogens, including viruses, bacteria, and toxin, in a single run of taqman real-time pcr. in sensitivity test by using standard curves from synthesized dna, all primer-probe sets showed high sensitivity. furthermore, the results of detection of target pathogens from clinical samples using this method showed similar results to the respective conventional pcr method. however, prrs virus is known to have a high frequency of genetic mutations, and the primer and probe sequences will need to be checked at a considerable frequency when performing dempo-pcr from field samples. the type of pathogens involved in prdc is specific to the regions and countries where production occurs [ ] . therefore, it may be necessary to change the inspect pathogens according to the regions. however, dempo-pcr is possible to detect many types of pathogens simultaneously. by dempo-pcr assay, multiple prdc pathogens can be detected comprehensively and simultaneously. this assay can quickly elucidate existence of pathogens in a sample. in this study, multiple viral and bacterial porcine respiratory pathogens were detected hyopneumoniae, m. hyorhinis) and four viruses (pcv , pcmv, prrs us strain, prcv) were detected on pigs of farm f. in this study, pcmv was detected in high proportion from pigs of all farms, and mixed infection with multiple pathogens was observed. pcmv has been documented worldwide, and shows high infection rates on pig farms in japan, europe, north america, and china [ , ] . hansen et al., reported that a significant association between pcmv and pcv was only found in the cases of prdc, and the role of pcmv in prdc needs to be elucidated [ ] . it is necessary to elucidate the combination of multiple pathogens for the elucidation of the etiology of prdc, and dempo -pcr will be a useful tool for that. phev is a subclinical infection, but its role as a respiratory pathogen was suggested since it was isolated from the acute respiratory disease in pigs in michigan in [ ] . the swine serological survey of phev also showed that it is widely and highly distributed in japan [ ] . therefore, phev was added to the target pathogens of dempo-pcr, but it was not detected from these samples. in conclusion, dempo-pcr can identify a wider range of existing pathogens quickly and easily compared to one assayone pathogen test. considering multiple etiology of prdc, screening by dempo-pcr would help us determine treatment and prevention measures. this detection system may provide an alternative testing method that is simpler, faster, and more comprehensive than existing assays. evaluation of ′ nuclease assay for detection of actinobacillus pleuropneumoniae detection of streptococcus suis in bioaerosols of swine confinement buildings porcine cytomegalovirus (pcmv) in early gestation development of a novel hot-start multiplex pcr for simultaneous detection of classical swine fever virus, african swine fever virus, porcine circovirus type , porcine reproductive and respiratory syndrome virus and porcine parvovirus detection of porcine reproductive and respiratory syndrome virus, porcine circovirus type , swine influenza virus and aujeszky's disease virus in cases of porcine proliferative and necrotizing pneumonia (pnp) in spain genomic organization and molecular characterization of porcine cytomegalovirus an investigation of the pathology and pathogens associated with porcine respiratory disease complex in denmark three cases of porcine respiratory disease complex associated with porcine circovirus type infection the use of oral fluids to monitor key pathogens in porcine respiratory disease complex survey of porcine hemagglutinating encephalomyelitis virus infection of pigs in japan development of a one-run real-time pcr detection system for pathogens associated with bovine respiratory disease complex simultaneous detection of north american and european porcine reproductive and respiratory syndrome virus using real-time quantitative reverse transcriptase-pcr multiplex method for simultaneous serological detection of porcine reproductive and respiratory syndrome virus and porcine circovirus type porcine hemagglutinating encephalomyelitis virus and respiratory disease in exhibition swine multiplex pcr and microarray for detection of swine respiratory pathogens development of real-time polymerase chain reaction assays for rapid detection and differentiation of wild-type pseudorabies and gene-deleted vaccine viruses herd factors associated with the seroprevalences of four major respiratory pathogens in slaughter pigs from farrow-to-finish pig herds development of a quantitative real-time taqman pcr assay for determination of the minimal dose of mycoplasma hyopneumoniae strain required to induce pneumonia in spf pigs of one-step real-time reverse transcriptase-pcr-based assays for the rapid and simultaneous detection of four viruses causing porcine diarrhea polymicrobial respiratory disease in pigs porcine respiratory disease complex after the introduction of h n / influenza virus in brazil swine influenza virus and association with the porcine respiratory disease complex in pig farms in southern brazil isolation, antimicrobial resistance, and virulence genes of pasteurella multocida strains from swine in china development of real-time pcr assay for differential detection of bordetella bronchiseptica and bordetella parapertussis multilocus sequence typing of mycoplasma hyorhinis strains identified by a real-time taqman pcr assay development of a novel detection system for microbes from bovine diarrhea by real-time pcr validation of a real-time pcr for haemophilus parasuis detection and typing of highly pathogenic porcine reproductive and respiratory syndrome virus by multiplex real-time rt-pcr development and validation of a triplex real-time pcr assay for the rapid detection and differentiation of wild-type and glycoprotein e-deleted vaccine strains of bovine herpesvirus type acknowledgments. this work was partially supported by the ministry of agriculture, forestry and fisheries of japan, award number: the research project for improving food safety and animal health of the ministry of agriculture, forestry and fisheries of japan ( ). key: cord- - vxqr mc authors: shi, ting; arnott, andrew; semogas, indre; falsey, ann r; openshaw, peter; wedzicha, jadwiga a; campbell, harry; nair, harish title: the etiological role of common respiratory viruses in acute respiratory infections in older adults: a systematic review and meta-analysis date: - - journal: j infect dis doi: . /infdis/jiy sha: doc_id: cord_uid: vxqr mc acute respiratory tract infections (ari) constitute a substantial disease burden in adults and elderly individuals. we aimed to identify all case-control studies investigating the potential role of respiratory viruses in the etiology of ari in older adults aged ≥ years. we conducted a systematic literature review (across databases) of case-control studies published from to that investigated the viral profile of older adults with and those without ari. we then computed a pooled odds ratio (or) with a % confidence interval and virus-specific attributable fraction among the exposed (afe) for common viruses: respiratory syncytial virus (rsv), influenza virus (flu), parainfluenza virus (piv), human metapneumovirus (hmpv), adenovirus (adv), rhinovirus (rv), bocavirus (bov), and coronavirus (cov). from the studies included, there was strong evidence of possible causal attribution for rsv (or, . [ % ci, . – . ]; afe, %), flu (or, . [ % ci, . – . ]; afe, %), piv (or, not available; afe, approximately %), hmpv (or, . [ % ci, . – . ]; afe, %), adv (or, not available; afe, approximately %), rv (or, . [ % ci, . – . ]; afe, %) and cov (or, . [ % ci, . – . ]; afe, %) in older adults presenting with ari, compared with those without respiratory symptoms (ie, asymptomatic individuals) or healthy older adults. however, there was no significant difference in the detection of bov in cases and controls. this review supports rsv, flu, piv, hmpv, adv, rv, and cov as important causes of ari in older adults and provides quantitative estimates of the absolute proportion of virus-associated ari cases to which a viral cause can be attributed. disease burden estimates should take into account the appropriate afe estimates (for older adults) that we report. acute respiratory tract infections (ari), including pneumonia, constitute a substantial disease burden in adults and elderly individuals. respiratory viruses are detected more frequently than bacteria in adults with pneumonia [ ] . the substantial contribution of viruses to ari hospitalizations among adults is being increasingly recognized [ , ] . although influenza virus (flu) is the most widely recognized viral infection associated with respiratory illness, > viruses have been linked to pneumonia, causing a substantial disease burden in adults and elderly individuals. these include common pathogens such as rhinovirus (rv), respiratory syncytial virus (rsv), flu, human metapneumovirus (hmpv), parainfluenza viruses (piv), and human coronaviruses (covs) [ ] . rsv is associated with a substantial disease burden in adults, especially among older adults (aged ≥ years) [ ] . moreover, adults hospitalized with rsv disease can develop severe respiratory complications [ ] . rv has been associated with severe respiratory disease outbreaks in adults in long-term care facilities in several settings [ ] . despite advances in diagnostic technology, defining the specific causes of viral pneumonia is challenging, particularly among older adults who may have lower viral loads and for whom viral diagnosis is frequently not considered and/ or testing is not performed [ ] . therefore, it is necessary to measure concurrently the background prevalence of nasopharyngeal viral infection in a control (asymptomatic) group, to investigate the etiological role of viruses in older adults with ari to help inform decisions on prevention and management strategies. previously, we have conducted a systematic review to understand the etiological role of common respiratory viruses, focusing on children aged < years [ ] . to the best of our knowledge, similar estimates for adults are lacking. therefore, we aimed to conduct a similar systematic review to identify all case-control studies since investigating the potential role of respiratory viruses in the etiology of aris in older adults aged ≥ years. we conducted a systematic review across databases (including chinese databases) following the approach detailed in the prisma (preferred reporting items for systematic reviews and meta-analyses) guidelines [ ] . tailored search strategies were developed and used to search the medline, embase, global health, lilacs, china national knowledge infrastructure (cnki), wanfang data, and chongqing vip databases (appendix). we further searched the reference lists of relevant articles for eligible articles. all searches were limited to between january and august . no publication status criteria or language restrictions were used. we included studies that fulfilled the following selection criteria (supplementary figure ) . three investigators (t. s., a. a., and i. s.) conducted independent searches of the english-language literature and extracted data by using standardized data extraction templates. one investigator (t. s.), whose first language is chinese, searched and extracted data from chinese-language databases (ie, cnki, wanfang, and cqvip). the protocol of this review was published in the prospero database (no. crd ). the case group was defined as older adults with ari or pneumonia aged ≥ years, adapted from world health organization integrated management of adolescent and adult illness [ ] . the details of the definitions are displayed in supplementary table . the control group was defined as older adults aged ≥ years who were either healthy or did not have any respiratory symptoms. we categorized countries as either industrialized or developing, on the basis of criteria from the united nations children's fund [ ] . we calculated odds ratios (ors) as the ratio of the odds of detecting each virus in older adults with ari or pneumonia to the odds of detecting each virus in healthy or asymptomatic controls, with accompanying % confidence intervals (cis). we used a continuity correction of . if a virus was detected in one group but not the other [ ] . this allowed calculation of an or for these instances and enabled inclusion in subsequent meta-analyses. using stata (version . ), we performed a meta-analysis of virus-specific ors and reported pooled meta-estimates with corresponding % cis, using the random effects model (ie, the dersimonian-laird method) because the included studies are heterogeneous in various aspects and are thus assumed to have different effect sizes [ ] . the virus-specific attributable fraction among the exposed (afe) was used to quantify the etiological role of each virus in patients with ari. this is an estimate of the percentage of aris that can be attributed to each virus, in absolute terms [ ] , and was calculated as * [or − ]/or, with a % ci (from the corresponding % ci of the or). moreover, for a specific virus, if all included studies did not report any virus detection in one group consistently (usually the control group), we assumed that a strong association indicating a possible causal role for this virus in ari could be concluded. in these circumstances, we considered that there was no need to run a meta-analysis that would only result in an extremely high or point estimate and an afe approaching %. we identified ( from chinese databases) records through the literature search and records from the reference lists of relevant articles. among them, only studies (including from chinese databases) fulfilled our inclusion and exclusion criteria ( figure ) [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . forty-three studies were excluded for a variety of reasons: no data specific to older adults ≥ years old were available (n = ), the case or control definitions were not fulfilled (n = ), no applicable data for cases and controls were reported (n = ), or serum was used as the clinical specimen (n = ). seven studies were conducted within developing countries, while were from developed countries (supplementary table ). although the search was performed for articles published since , all included studies were published since . all included studies were case-control studies with adults who had ari or pneumonia in the case group and asymptomatic or healthy adults in the control group. methods varied among studies. among the case definitions used, studies used ari or acute lower respiratory tract infection, while the others used (severe) pneumonia (n = ). all studies investigated a control group, which had no respiratory symptoms, and in studies healthy older adults (without acute illness) served as controls. of the case ascertainment methods used, articles recruited the cases from inpatients; , from outpatients; , from general practices; , from the community; and , from mixed settings (outpatient settings and the emergency department, and both outpatient and inpatient settings). in studies, controls were ascertained in hospital-based outpatient or clinic sites, whereas in studies, controls were identified from the community. all studies collected a mixture of nasopharyngeal swab specimens, nasopharyngeal aspirates, nasopharyngeal washes, oropharyngeal swab specimens, and nasal/throat swab specimens as the clinical specimen. all studies used polymerase chain reaction analysis (pcr; in some, pcr was combined with serologic analysis or culture) as the diagnostic test. meta-analyses of virus-specific ors are reported as well as the corresponding attributable fractions among the exposed (supplementary table ). rsv, flu (including flu a), hmpv, rv, and cov (also cov oc and e) were significantly more common in older adults with a diagnosis of ari or pneumonia than in asymptomatic or healthy controls (ors, these viruses had statistically significant positive afes, which showed clear associations between these viruses and ari or pneumonia in older adults. moreover, piv (including piv and piv ; data for piv and piv were not available), flu b, and adv were only identified in cases consistently across all included studies ( , , and studies, respectively) and absent in control groups. thus, these viruses were all assumed to have strong associations with ari. only studies had data available for bov, and although both reported virus detection in a greater proportion of cases than controls [ , ] , the association remains a question for further research. a subgroup analysis was performed to explore the roles of viruses in ari with respect to region: developing countries and industrialized countries. the meta-estimate or was higher in industrialized countries as compared to developing countries in the case of flu (with overlapping % cis), while it was similar for piv, adv, and rv. there were insufficient studies to conduct a similar subgroup analysis for other viruses. a sensitivity analysis was performed to investigate the roles of these common viruses in older adults admitted to hospitals with ari or pneumonia [ , , , , , , , ] . eight studies were included, and results are presented in supplementary table . the meta-estimate or did not differ significantly from the previous estimate, in which cases from other settings (ie, outpatient and general practice settings) were also included. and cov in ari and pneumonia in older adults, thereby indicating a potential for substantive reductions in the number of ari cases if older adults were vaccinated against these viruses or treated with antivirals. for the other respiratory viruses studied, the role of bov in ari and pneumonia was uncertain because of the limited evidence available from the published literature, requiring more research to clarify its role in older adults with ari. a sensitivity analysis focusing only on older adults who were admitted to hospitals with ari or pneumonia did not differ significantly from our estimate, in which patients from all settings were considered. this might result from the limited number of studies available to provide a more robust sensitivity analysis. no studies calculated adjusted ors to account for confounding effects from age or season, which might compromise the actual association and should be considered in the study design in future research. these findings should inform the results of studies that seek to estimate the global, regional, and national burden of disease due to these viruses in older adults [ ] . they show that rsv, flu, piv, hmpv, adv, rv, and cov are important causes of ari in older adults, and disease burden estimates should take into account the appropriate afe estimates (for older adults) that we report, rather than the afe estimates in children aged < years. there is considerable international attention on rsv-associated ari in older adults at this time, during which novel vaccine and antiviral strategies are being evaluated and prioritized [ , ] , and more-accurate disease burden estimates (using these results) would help to inform future policies and interventions. the prevalence of virus detection from etiologic studies of pneumonia in adults is substantially lower than the detection rate in studies of children. the epic (etiology of pneumonia in the community) study team showed that the viruses were detected in % of adults who had been hospitalized with community-acquired pneumonia, compared with % of children who were admitted to the hospital [ , ] . there are several reasons for such low levels of detection, such as the inability to obtain lower respiratory tract specimens, the use of diagnostic tests with insufficient sensitivity, the absence of appropriate diagnostic testing methods, the undetectability of the virus at the time of the study, and the presence of unknown pathogens that were not identified. the low rate of virus detection among adults who were hospitalized for pneumonia highlights the need for more-sensitive diagnostic approaches, innovative discovery of pathogens, and assessing viruses in the past history (weeks before the presence of disease) [ ] . moreover, chronic obstructive pulmonary disease (copd) exacerbation is a very important cause of aris and hospital admissions [ ] . only of studies included copd in the etiologic data, and this information was unclear in the remaining studies, which might have underestimated the role of viral infection in these patients. a previous etiological review focusing on young children aged < years [ ] showed that rsv, flu (including flu a), piv, hmpv, and rv were significantly more common in children hospitalized with acute lower respiratory tract infection than asymptomatic controls. the associations of these viruses (except rsv) with ari and pneumonia were stronger among adults. this is in part because, in comparison to young children, the detection of viruses in the control group (ie, among individuals without respiratory symptoms or healthy controls) was less common in older adults, with the exception of rsv. several methodological issues could affect our results: sample size, age group, case ascertainment, clinical specimen, and diagnostic testing. although a thorough search has been performed across databases, including chinese-language databases, only studies from the published literature were identified, which met our selection criteria. not every virus of interest was tested in each study. the number of studies available was even smaller when subgroup analyses and sensitivity analyses were performed. moreover, the sample size varied from to adults in the case group and from to adults in the control group. the small sample size undoubtedly contributed to the imprecise % cis around the ors. thus, we may have failed to detect clinically significant ari-virus associations, owing to small sample sizes. we aimed to stratify the association between common respiratory viruses in adults with ari or pneumonia by age. however, most articles did not stratify and report data by age group. instead, they summarized the result for the entire age group, usually in adults aged > years. therefore, some of our meta-estimate ors may not be representative of older adults who are aged ≥ years. since age might be a risk factor for ari in adults (the rate of severe ari increases as age advances), this could potentially affect the viral profile detected, introducing further heterogeneity [ ] . fifteen of studies used passive clinic or hospital based case ascertainment. among them, cases were recruited from inpatients, outpatients, emergency departments, or general practices, which might reflect different healthcare behavior and disease severity. also, since the episodes of ari and pneumonia were only diagnosed through routine care, this introduced bias, considering that testing was only done when the clinicians deemed it necessary to test. similarly, studies used community-based controls, while another studies recruited older adult controls from hospitals or general practices. hospital or clinical ascertained controls may not reflect the general population and may have other health conditions potentially affecting their viral carriage. moreover, recruiting controls who were selected as healthy or without respiratory symptoms could favor those who were not exposed to the respiratory virus (yielding a falsely high or). therefore, we consider that the ideal control group for these studies would be a random sample of an age-and sexmatched population of older adults who are from the same area of residence and studied at the same time as cases. all included studies obtained upper respiratory tract specimens (described as nasopharyngeal secretions). assays might have specimen-specific sensitivities and specificities for detecting viruses, which could lead to heterogeneity in the estimation of virus-specific rates. the sensitivity of using nasopharyngeal washes for detecting any virus in adults was found to be higher than that for using nasopharyngeal swab specimens, which in turn was higher than that for using oropharyngeal swab specimens ( . %, . %, and . %, respectively) [ ] . the limited use (due to ethical concerns and feasibility) of invasive procedures to obtain lower respiratory tract specimens directly from the lung also influenced the diagnosis of viral infection in adults with ari [ ] . pcr and serology-based diagnostic testing are more sensitive for detecting respiratory viruses than other methods, such as antigen detection and culture. high sensitivity is important for accurate assessment of etiological contribution, particularly in older adults who may have a lower nasopharyngeal viral load and an atypical clinical presentation [ ] . moreover, despite being uncommon, detection of viral coinfection (range across studies, %- %) may tend to overstate the contribution of individual respiratory viruses (although dual or multiple infections, in which both or several viruses have etiological importance, are possible). bacterial coinfections (range across studies, %- %) were also reported. with improving diagnostic methods, multiple etiological agents are increasingly identified simultaneously in older adults with ari, making the individual contribution of each agent difficult to define. viruses can be detected in individuals with no respiratory symptoms. this is often seen in volunteer challenge studies and in some community surveys [ , ] . the detection of viruses in control groups without respiratory symptoms might be due to a nascent infection or a persisting colonization from a previous infection [ ] . these factors will tend to result in true associations being attenuated. the fact that molecular detection of viruses in older adults with ari is higher than the detection rate in controls without respiratory symptoms may not necessarily indicate causation. alternative explanations should be considered first before causality can be concluded. these include the respiratory viral infection acting as a so-called innocent bystander, without a causal role, and serving only as a predisposing risk factor for ari. similarly, the absence of a positive association (and afe) does not mean that a virus is not a cause of ari. moreover, without establishing the temporal sequence of exposure and outcome, determinations of causality are less secure. therefore, the association between viruses and ari and pneumonia should be interpreted carefully. in conclusion, this review provides clear evidence that is suggestive of the potentially causal role of rsv, flu, piv, hmpv, adv, rv, and cov in older adults with ari and presents the first estimate of the proportion of ari cases that can be attributed to virus exposure. etiological studies, which simply report rates of viral identification as causal, should make attempts to interpret findings in terms of the proportion of ari cases among older adults in whom a respiratory virus is identified that can be attributed to this viral exposure. the resceu investigators are as follows national institute for public health and the environment) penta foundation) jeroen aerssens, veronique wyffels, and matthias cleenewerck (janssen) cdc epic study team. communityacquired pneumonia requiring hospitalization among u.s. adults viral pneumonia respiratory syncytial virus infection in elderly and high-risk adults modelling estimates of the burden of respiratory syncytial virus infection in adults and the elderly in the united kingdom high morbidity and mortality in adults hospitalized for respiratory syncytial virus infections rhinovirus outbreaks in longterm care facilities the diagnosis of viral respiratory disease in older adults aetiological role of common respiratory viruses in acute lower respiratory infections in children under five years: a systematic review and meta-analysis preferred reporting items for systematic reviews and meta-analyses: the prisma statement world health organization. integrated management of adolescent and adult illness (imai) the state of the world's children : reimagine the future: innovation for every child what to add to nothing? use and avoidance of continuity corrections in meta-analysis of sparse data introduction to meta-analysis attributable risk percent in case-control studies a case-control study of acute respiratory tract infection in general practice patients in the netherlands respiratory viruses in adults with community-acquired pneumonia prospective evaluation of a novel multiplex real-time pcr assay for detection of fifteen respiratory pathogens-duration of symptoms significantly affects detection rate microorganisms in respiratory tract of patients diagnosed with atypical pneumonia: results of a research based on the use of reverse transcription polymerase chain reaction (rt-pcr) dna microarray method and enzyme-linked immunosorbent assay viral respiratory tract infections in adult patients attending outpatient and emergency departments respiratory viral detection in children and adults: comparing asymptomatic controls and patients with community-acquired pneumonia aetiological role of viral and bacterial infections in acute adult lower respiratory tract infection (lrti) in primary care respiratory virus is a real pathogen in immunocompetent community-acquired pneumonia: comparing to influenza like illness and volunteer controls incidence and characteristics of viral community-acquired pneumonia in adults a prospective, community-based study on virologic assessment among elderly people with and without symptoms of acute respiratory infection viral etiology of acute lower respiratory infection in adult inpatients detection on non-bacterium pathogen in cases of acute respiratory infection human coronavirus infections in rural thailand: a comprehensive study using real-time reverse-transcription polymerase chain reaction assays human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand detection of respiratory syncytial virus and human metapneumovirus by reverse transcription polymerase chain reaction in adults with and without respiratory illness estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower respiratory tract infections in countries: a systematic analysis for the global burden of disease study accessed drug candidates and model systems in respiratory syncytial virus antiviral drug discovery cdc epic study team. communityacquired pneumonia requiring hospitalization among u.s. children better tests, better care: improved diagnostics for infectious diseases respiratory viruses in exacerbations of chronic obstructive pulmonary disease requiring hospitalisation: a case-control study identification of respiratory viruses in adults: nasopharyngeal versus oropharyngeal sampling lower respiratory tract virus findings in mechanically ventilated patients with severe community-acquired pneumonia time lines of infection and disease in human influenza: a review of volunteer challenge studies flu watch group. comparative community burden and severity of seasonal and pandemic influenza: results of the flu watch cohort study identification of respiratory viruses in asymptomatic subjects: asymptomatic respiratory viral infections we thank joris menten from janssen for reviewing the manuscript.financial support. this work was supported by the innovative medicines initiative joint undertaking (grant ), which in turn receives support from the european union's horizon research and innovation programme and efpia. is the elected president of the british society for immunology, which is an unpaid appointment but provides support for travel and accommodation at some meetings. all other authors report no potential conflicts. supplementary materials are available at the journal of infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. key: cord- -xpo zsub authors: mcgrath, james a.; o’sullivan, andrew; bennett, gavin; o’toole, ciarraí; joyce, mary; byrne, miriam a.; macloughlin, ronan title: investigation of the quantity of exhaled aerosols released into the environment during nebulisation date: - - journal: pharmaceutics doi: . /pharmaceutics sha: doc_id: cord_uid: xpo zsub background: secondary inhalation of medical aerosols is a significant occupational hazard in both clinical and homecare settings. exposure to fugitive emissions generated during aerosol therapy increases the risk of the unnecessary inhalation of medication, as well as toxic side effects. methods: this study examines fugitively-emitted aerosol emissions when nebulising albuterol sulphate, as a tracer aerosol, using two commercially available nebulisers in combination with an open or valved facemask or using a mouthpiece with and without a filter on the exhalation port. each combination was connected to a breathing simulator during simulated adult breathing. the inhaled dose and residual mass were quantified using uv spectrophotometry. time-varying fugitively-emitted aerosol concentrations and size distributions during nebulisation were recorded using aerodynamic particle sizers at two distances relative to the simulated patient. different aerosol concentrations and size distributions were observed depending on the interface. results: within each nebuliser, the facemask combination had the highest time-averaged fugitively-emitted aerosol concentration, and values up to . ± . mg m(− ) were recorded. the placement of a filter on the exhalation port of the mouthpiece yielded the lowest recorded concentrations. the mass median aerodynamic diameter of the fugitively-emitted aerosol was recorded as . ± . µm, lower the initially generated medical aerosol in the range of – µm. conclusions: the results highlight the potential secondary inhalation of exhaled aerosols from commercially available nebuliser facemask/mouthpiece combinations. the results will aid in developing approaches to inform policy and best practices for risk mitigation from fugitive emissions. aerosol therapy is a mainstay of drug administration in both home and clinical settings for acute and long-term treatments. a variety of aerosol generation technologies deliver drugs to the lungs through patient interfaces across the full range of neonatal to adult patients. the size distributions of generated medical aerosols are typically - µm [ ]. when released indoors, particles can remain airborne for times ranging from minutes to hours [ ] [ ] [ ] . exhaled aerosol is aerosol that may have been inhaled but did not deposit on the airway surface, or additionally, aerosol generated by the nebuliser during the inspiratory phase which was released before it could be inhaled. this exhaled fraction is regarded as a fugitive dose and is notable when considering possible unintended bystander inhalation, which includes caregivers or family members. exposure to fugitive emissions generated during aerosol therapy increases the risk of unnecessary inhalation of medication, as well as toxic side effects [ ] . however, the fundamental exhalation mechanism for previously-inhaled aerosol droplets during aerosol therapy has not been well described. secondary inhalation of medical aerosols is a risk factor for both caregivers and bystanders and has been highlighted as a significant occupational hazard in both clinical and homecare settings [ , ] . studies have raised concern surrounding potential secondary exposure of healthcare workers to aerosolised pentamidine, sustained release lipid inhalation targeting (slit) cisplatin, adeno-associated serotype vector containing cystic fibrosis transmembrane conductance regulator complementary dna (tgaavcf) and ribavirin [ ] [ ] [ ] [ ] . respiratory therapists have an increased risk of developing asthma after entering the profession, which may be partially explained by occupational exposure to a range of aerosolised substances [ , ] . in addition, there is growing global concern about acquired antimicrobial resistance (ami), with , people developing multi-drug resistant tuberculosis each year [ ] ; the use of nebulised antibiotics could potentially contribute to ami among long-term caregivers who may have inhaled fugitive antibiotic-laden aerosol. during the severe acute respiratory syndrome (sars) outbreak, anecdotal reports highlighted that infected persons receiving aerosol therapies may have facilitated the transmission of sars under certain circumstances [ ] . clinical guidelines recommended that the generation of aerosols should be avoided [ ] ; these included the use of non-invasive ventilation, high-flow oxygen (> l/min), and nebulisers. an in-vitro study examining the mechanical ventilation of a patient with/without expiratory filters reported that the drug deposited at the exhaust port without filtering exceeded the filtering case -fold. additionally, it was concluded that greater than % of the nominal dose could become 'second-hand medical aerosol' [ ] . high-definition video imaging studies used previously-inhaled saline aerosol and smoke particles to estimate the dispersion distance of exhaled air for various systems. exhaled droplets were observed exiting the side vents of a facemask, and substantial exposure to exhaled air was reported to occur within . m [ ] . nebuliser airflow, lung function and interface type were all reported to impact upon dispersion distance [ ] [ ] [ ] [ ] . a limitation of the visualisation approach is that the visible boundaries of exhaled flows could only be used as a guide to the real behaviour of droplets in exhaled air [ ] . in the respiratory therapy context, the secondary exposure pathway is not widely understood. limited evidence exists to explain fugitive emissions, and to date, research has primarily focused on infection control models. the current study aimed to enhance the understanding of secondary exposure to aerosol emitted during respiratory therapy with commonly-used equipment. this study examined the potential for secondary (caregiver/bystander) exposure in domestic and clinical caregiving settings, by examining some key factors that influence secondary exposure: caregiver position and device type. the design and operation features of two prevalent nebuliser technologies were assessed regarding their emission of exhaled aerosol. these were a vibrating mesh (vmn) used in combination with an aerosol chamber (aerogen solo/ultra, aerogen, galway, ireland) and a jet (jn) (cirrus , intersurgical, wokingham, united kingdom), both in clinical use today. this study incorporated two non-invasive ventilation interfaces (facemask and mouthpiece) in combination with the commercially available aerosol generators. the vmn used a valved facemask, while the jn used an open facemask. for the mouthpiece scenarios, both an unfiltered mouthpiece and filtered mouthpiece were used with each setup. a filtered mouthpiece refers to a filter placed on the exhalation port, while an unfiltered mouthpiece refers to the case where no filter is placed on the exhalation port. to replicate the ideal conditions achieved when a patient uses an interface correctly, each mask was glued to a plastic sheet to avoid a leak, while the mouthpieces were attached using a custom fixture. a supplemental gas flow rate of l/min was used with the vmn and the manufacturer-prescribed l/min was used for the jn. each nebuliser was individually assessed under simulated real use conditions. the laboratory room had a volume of m , three internal doors, but no external doors or windows; the room was mechanically ventilated with an air exchange rate of . h − . the ambient temperature and relative humidity in the test room were typically • c and %, respectively, during the test period. each aerosol generator/interface combination was connected to a breathing simulator (asl , ingmar medical, pittsburgh, pa, usa) via an absolute filter (respirgard ii , baxter, dublin, ireland). a simulated adult breath was used (breath rate bpm, tidal volume ml and inspiratory: expiratory (i:e ratio) : ). as a result, no institutional review board was required for this study. a nominal dose of . ml ( . mg) albuterol sulphate (ventolin, mg/ml, gsk, cork, ireland) was nebulised in each test run. aerosol delivery performance was evaluated by characterising the inhaled dose, that is to say, drug delivered to the patient. to determine the inhaled dose, each aerosol generator/interface combination was attached to an absolute collection filter ( figure ). albuterol is commonly nebulised as a medication, and also as a formulation used in the characterization of aerosol drug delivery systems, and is specified for use as a tracer aerosol [ ] . in this study, albuterol was eluted from filter or nebuliser components using a wash buffer. albuterol mass, expressed as a fraction of the nominal dose, at the end of each run was extracted and quantified using uv spectrophotometry (biochrom uv vis, cambridge, uk) at nm and interpolation on a standard curve of albuterol sulphate. the exhaled dose, the drug that was exhaled by the spontaneously breathing patient, was also characterised (mouthpiece testing only) by placing a collecting filter at the exhalation ports of each nebuliser/mouthpiece combination ( figure ). additionally, the residual mass, the drug that was available for nebulisation but remained in the device, was reported for both vmn and jn devices. all testing was carried out in triplicate. pharmaceutics , , x for peer review of flow rate of l/min was used with the vmn and the manufacturer-prescribed l/min was used for the jn. each nebuliser was individually assessed under simulated real use conditions. the laboratory room had a volume of m , three internal doors, but no external doors or windows; the room was mechanically ventilated with an air exchange rate of . h − . the ambient temperature and relative humidity in the test room were typically °c and %, respectively, during the test period. each aerosol generator/interface combination was connected to a breathing simulator (asl , ingmar medical, pittsburgh, pa, usa) via an absolute filter (respirgard ii , baxter, dublin, ireland). a simulated adult breath was used (breath rate bpm, tidal volume ml and inspiratory: expiratory (i:e ratio) : ). as a result, no institutional review board was required for this study. a nominal dose of . ml ( . mg) albuterol sulphate (ventolin, mg/ml, gsk, cork, ireland) was nebulised in each test run. aerosol delivery performance was evaluated by characterising the inhaled dose, that is to say, drug delivered to the patient. to determine the inhaled dose, each aerosol generator/ interface combination was attached to an absolute collection filter (figure ). albuterol is commonly nebulised as a medication, and also as a formulation used in the characterization of aerosol drug delivery systems, and is specified for use as a tracer aerosol [ ] . in this study, albuterol was eluted from filter or nebuliser components using a wash buffer. albuterol mass, expressed as a fraction of the nominal dose, at the end of each run was extracted and quantified using uv spectrophotometry (biochrom uv vis, cambridge, uk) at nm and interpolation on a standard curve of albuterol sulphate. the exhaled dose, the drug that was exhaled by the spontaneously breathing patient, was also characterised (mouthpiece testing only) by placing a collecting filter at the exhalation ports of each nebuliser/mouthpiece combination ( figure ) . additionally, the residual mass, the drug that was available for nebulisation but remained in the device, was reported for both vmn and jn devices. all testing was carried out in triplicate. the primary aerosol monitoring instrument used in this study was an aerodynamic particle sizer (aps) (aps, model tsi inc., st. paul, mn, usa) measuring aerosol size distributions from . to µm. throughout the experiments, the aerosol concentrations and size distributions of the aerosol were continuously measured in real time using two apss, which were located at respective distances of . m and . m from the simulated patient. these distances were chosen on the basis that . m is approximately one arm's length away, simulating, for example, a caregiver holding a the primary aerosol monitoring instrument used in this study was an aerodynamic particle sizer (aps) (aps, model tsi inc., st. paul, mn, usa) measuring aerosol size distributions from . to µm. throughout the experiments, the aerosol concentrations and size distributions of the aerosol were continuously measured in real time using two apss, which were located at respective distances of . m and . m from the simulated patient. these distances were chosen on the basis that . m is approximately one arm's length away, simulating, for example, a caregiver holding a nebuliser/facemask to a patient's face. a distance of . m was chosen on the basis that it is approximately the distance between beds in a primary care centre, simulating a patient situated in a bed next to the patient receiving the aerosol therapy. a -min baseline level of aerosol was established in the room pre-nebulisation. nebulisation was then initiated with the apss recording data for min, and at -s intervals. a duration of min was sufficient for nebulising the entire dose with a period for aerosol decay post-dose. this testing was carried out concurrently with the aerosol dose distribution testing described above. the statistical package iibm spss statistics (ibm corp., armonk, ny, usa, ) was used to analyse the data. summary and descriptive statistics were performed on aerosol concentrations. all data are expressed as mean, minimum and maximum. the collection filters were analysed in terms of the inhaled dose, exhaled dose and residual mass, and the results are summarised in table . while the residual mass and inhaled dose vary depending on the nebuliser type, results within each nebuliser group are substantially equivalent. prior to nebulisation, the mean and standard deviation of the ambient concentrations were . ± . ( . to . ) mg m − . table confirms that fugitive emissions were recorded during the nebulisation events for the facemask and unfiltered mouthpiece combinations. there was no recorded increase in aerosol concentration when using the vmn/filtered mouthpiece, while increased aerosol concentrations were recorded for the jn/filtered mouthpiece. it can be seen from table that the use of vmn/unfiltered mouthpiece and jn/unfiltered mouthpiece combinations resulted in reduced exhaled aerosol concentrations compared with the corresponding facemask. the temporal variations in the aerosol concentrations when using the jet nebuliser resulted in a larger difference between the two aps readings for the open facemask combination compared with the other configurations. the largest variations in aerosol concentrations between the two apss occurred during the nebulisation period; in all cases, there was less than a % averaged difference between the two sets of measurements in the final min after nebulisation, which indicates the diffusion of fugitive emissions throughout the room. table summarises the averaged mmad over the -min period recorded by the aps . metres from the facemask/mouthpiece. there is a noticeable shift in the mmad during the nebulisation event when using the facemask/mouthpiece compared with the initially-generated aerosol. table . thirty-minute averaged aerosol concentration during each nebulisation event (using different facemasks and mouthpieces); mean and standard deviations over the three events. the reduced size and restricted opening area of the valved facemask are assumed to result in an exhaled aerosol with a lower mmad than that exhaled in the open facemask configuration. the percentage of the original drug that could potentially be inhaled by a caregiver and a bystander was estimated. the averaged jn/open facemask time-series data were selected as these exhibited the highest level of fugitive emission. the five-minute baseline average ambient aerosol concentration was subtracted from the time-series data to focus solely on fugitive emissions. short-term exposure values for inhalation to represent light activity for the caregiver ( . × − m /min) and sedentary activity for the bystander ( . × − m /min), both in the age range of to < years old, were calculated [ ] . the caregiver and bystander were assumed to be at respective distances of . m and . m from the patient. in this scenario, the caregiver and bystander were estimated to be exposed to . % and . % of the original nominal drug dose placed in the nebuliser, respectively. this study highlights the potential secondary inhalation exposure to fugitive emissions for caregivers and other bystanders during a standard nebuliser treatment. this study provides real-time monitoring data of size-resolved indoor aerosol concentrations when an aerosol generator/non-invasive ventilation interface connected to a breathing simulator is used to replicate an adult breathing pattern. while the current approach did not explicitly remove the ambient aerosol contribution from the data, it is evident that the observed changes in aerosol concentrations were due to aerosol from the nebulisation events. several factors influence inhalation dose [ , [ ] [ ] [ ] , and all of these are likely to affect the quantity of released aerosol. environmental factors (including room dimensions and layout, air turbulence, ventilation, and temperature) also affect the released aerosol distribution [ ] [ ] [ ] . the current observations should therefore be extrapolated with caution, but nevertheless, it can be noted that this study provides compelling evidence that a caregiver or bystander receives an inadvertent drug dose which is intended for the patient. it can be noted that the respective inhalation exposures for a caregiver ( . %) and bystander ( . %) were based on fugitive emissions from a standard treatment using a standard compressor-driven jet nebuliser. however, it is common that higher nominal doses are administered ( - mg/h) for periods ranging from - h [ ] [ ] [ ] . aerosolised ribavirin has also been reported to be nebulised at a rate of g over - h per day [ ] . a caregiver/medical practitioner may be working in a hospital where several patients may receive aerosol therapy throughout the day, over many days/weeks or for several years. therefore, the potential for chronic low dose occupational exposure is of concern [ ] . in this study, medical nebulisers generated an aerosol with a size distribution within the respirable range of - µm. however, the mmad of the fugitive emissions ranged between . - . µm across all nebuliser combinations, indicating that a considerably smaller aerosol size fraction is released than originally generated by the nebuliser. this is a significant finding, as the lower size fractions remain airborne for extended periods of time [ ] . the vmn combinations had reduced fugitive emissions compared with the jn, likely due to the combination of lower airflow and the valve system on the vmn. in both cases, the unfiltered mouthpiece had reduced fugitive emissions compared with the facemask. since there was no appreciable change in the residual drug analysis for the different facemask/mouthpiece combinations, it can be concluded that the mouthpieces' shape and size do not influence inertial impaction and deposition [ ] . for the filtered mouthpiece combination, while there were some observed fugitive emissions from jn/filtered mouthpiece combination compared with the vmn, it is possible that the higher driving force required for the normal operation of the jn/mouthpiece caused additional system pressure, allowing aerosol to escape through the custom fixture; however, it is unclear if this would be replicated with a human patient. an earlier imaging study observed no emitted droplets when a respiratory filter was placed on the exhalation port [ ] . other research shows that attaching an exhalation filter to the nebuliser was % effective in capturing exhaled aerosol droplets [ ] . facemask design may also contribute to this. by necessity, compressor-driven jet nebulisers must be operated with an open facemask; i.e., one without valves. this, in combination with the continual positive pressure air flow, acts to force aerosol out of the device and into the environment throughout both the inhalation and exhalation cycles of the breath. this is not the case with the vibrating mesh nebulisers, where a driving air flow is not required for aerosol generation, and the valve system on the device described aids in building a reservoir of aerosol within the patient interface which is available for the next breath. in a previous study, the drug distribution between inhalation filters, exhalation filters, ventilator tubing and medication reservoirs was quantified for a vmn and jn [ ] , and some comparisons are possible with this study. the residual drug in the vmn was . % compared with . - . % (mean value for each interface) in the current study, while the residual for the jn was . % compared with . - . % in the present study, albeit with a different jn. inhalation filters recorded % and % [ ] , compared with the current study of . - . % and . - . % for the vmn and jn, respectively. in the respiratory therapy context, the secondary exposure pathway is not widely understood. an adult breathing pattern in combination with a facemask/mouthpiece was used to replicate a standard nebuliser treatment and monitor the consequent release of fugitive emissions. to the best of the authors' knowledge, this is the first study that quantifies time-series aerosol concentrations and the size distribution of fugitive emissions that result from standard nebuliser treatment. the findings confirm a potential exposure risk to caregivers and other bystanders to medical aerosols and highlight that variations in patient interfaces (facemask and mouthpiece) and aerosol generators (vmn and jn) influence fugitively-emitted aerosol concentrations. the purpose of this study is to aid in developing approaches for healthcare organisations to inform policy and best practices for risk mitigation from fugitive emissions. future studies should focus on investigating these factors in a real-life clinical scenario. respiratory tract model for radiological protection development of a probabilistic multi-zone multi-source computational model and demonstration of its applications in predicting pm concentrations indoors particle deposition rates in residential houses fill volume, humidification and heat effects on aerosol delivery and fugitive emissions during noninvasive ventilation availability, consistency and evidence-base of policies and guidelines on the use of mask and respirator to protect hospital health care workers: a global analysis evaluation of exposure and health care worker response to nebulized administration of tgaavcf to patients with cystic fibrosis phase i study of aerosolized slit cisplatin in the treatment of patients with carcinoma of the lung health care worker exposure to aerosolized ribavirin: biological and air monitoring exposure of health care workers to aerosolized pentamidine second hand (s)-albuterol: rt exposure risk following racemic albuterol evaluation of droplet dispersion during non-invasive ventilation, oxygen therapy, nebuliser treatment and chest physiotherapy in clinical practice: implications for management of pandemic influenza and other airborne infections who. implementation of the global action on antimicrobial resistance infection control precautions for aerosol-generating procedures on patients who have suspected severe acute respiratory syndrome (sars) aerosol generating procedures and risk of transmission of acute respiratory infections to healthcare workers: a systematic review secondhand aerosol exposure during mechanical ventilation with and without expiratory filters: an in-vitro study dispersal of respiratory droplets with open vs closed oxygen delivery masks: implications for the transmission of severe acute respiratory syndrome exhaled air and aerosolized droplet dispersion during application of a jet nebulizer exhaled air dispersion during noninvasive ventilation via helmets and a total facemask exhaled air dispersion distances during noninvasive ventilation via different respironics face masks noninvasive positive-pressure ventilation: an experimental model to assess air and particle dispersion airflow and droplet spreading around oxygen masks: a simulation model for infection control research anaesthetic and respiratory equipment-nebulizing systems and components; international organization for standardization exposure factors handbook aerosol drug delivery: developments in device design and clinical use advances in aerosols: adult respiratory disease in vitro determination of the main effects in the design of high-flow nasal therapy systems with respect to aerosol performance characterization of indoor aerosol temporal variations for the real-time management of indoor air quality using time-and size-resolved particulate data to quantify indoor penetration and deposition behavior a simulation study of the changes in pm . concentrations due to interzonal airflow variations caused by internal door opening patterns continuous bronchodilator therapy four hours of continuous albuterol nebulization comparison of two methods of delivering continuously nebulized albuterol prophylaxis and treatment of respiratory syncytial virus in adult immunocompromised patients assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: the challenge ahead aerosol technology: properties, behavior, and measurement of airborne particles assessing a system to capture stray aerosol during inhalation of nebulized liposomal cisplatin key: cord- -muybvyqa authors: fan, victoria y; jamison, dean t; summers, lawrence h title: pandemic risk: how large are the expected losses? date: - - journal: bull world health organ doi: . /blt. . sha: doc_id: cord_uid: muybvyqa there is an unmet need for greater investment in preparedness against major epidemics and pandemics. the arguments in favour of such investment have been largely based on estimates of the losses in national incomes that might occur as the result of a major epidemic or pandemic. recently, we extended the estimate to include the valuation of the lives lost as a result of pandemic-related increases in mortality. this produced markedly higher estimates of the full value of loss that might occur as the result of a future pandemic. we parametrized an exceedance probability function for a global influenza pandemic and estimated that the expected number of influenza-pandemic-related deaths is about per year. we calculated that the expected annual losses from pandemic risk to be about billion united states dollars – or . % of global income – per year. this estimate falls within – but towards the lower end of – the intergovernmental panel on climate change’s estimates of the value of the losses from global warming, which range from . % to % of global income. the estimated percentage of annual national income represented by the expected value of losses varied by country income grouping: from a little over . % in high-income countries to . % in lower-middle-income countries. most of the losses from influenza pandemics come from rare, severe events. few doubt that major epidemics and pandemics will strike again and few would argue that the world is adequately prepared. since the - ebola virus disease outbreak in western africa, the united states national academy of medicine and several other groups [ ] [ ] [ ] have pointed to gaps, and the need for greater investment, in preparation against epidemics and pandemics, of ebola virus disease and other infectious diseases. attempts to justify greater investment have mostly been based on estimates of the industrial and macroeconomic losses attributable to influenza pandemics. [ ] [ ] [ ] [ ] [ ] [ ] [ ] we have recently extended the loss assessment to include a valuation of the lives lost as a result of the increases in mortality resulting from influenza-pandemic risk. the inclusion of such a valuation increased the estimated loss attributable to modelled pandemic risk several fold. below, we discuss our method and summarize our findings. box presents the definition of several of the terms we are using in this paper. most previous economic studies on global influenza pandemics have focused on income losses, through reductions in the size of the labour force and productivity, increases in absenteeism and, importantly, as the result of individual and social measures that interrupt transmission, but disrupt economic activity. while measures such as the per-capita gross national income include the effect of pandemics on income, they also exclude the value of changes in mortality risk to individuals. if, in assessments of investments in pandemic preparedness and mitigation, we neglect this dimension of loss, we will underestimate the value of such investments, relative to alternative uses of public finances. the broader approach that we recently applied, to the assessment of economic losses attributable to pandemic influenza, factors in the intrinsic loss associated with increases in mortality. in effect, this approach assigns a dollar value to small changes in mortality probabilities, using values derived from empirical studies of how individuals and societies actually value changes in mortality risk. [ ] [ ] [ ] [ ] this approach has already been employed extensively in environmental economics , and has also been used in global health, by the lancet commission on investing in health. , past literature we searched google scholar and pubmed® for studies on the economic losses from influenza. almost all of the previous studies examined economic losses in terms of income and ignored the value of, and the loss associated with, mortality risk. the world bank, for example, generated estimates of global income losses under different influenza pandemic scenarios. , it found that a pandemic of the same severity as the influenza pandemic might reduce global gross domestic product by about % and that the disruptive effects of avoiding infection would account for about % of that reduction. another study of the consequences of a range of pandemic severities included an extremely severe scenario that would lead to income losses of over % of gross national income worldwide, including losses of over % of the gross national incomes of lower-income countries. we found other integrative estimates of the magnitude of pandemic risk in two partially proprietary sources. , several studies have examined specific dimensions of the economic impacts of annual influenza, such as direct costs, e.g. medical and hospitalizations costs, and indirect costs, e.g. lost earnings due to illness and productivity costs. there are examples of such studies based in the americas, , , [ ] [ ] [ ] [ ] asia , and europe. , other models have added an estimated value of the intrinsic undesirability of nonfatal illness or of pandemic fear, as seen in the population response to severe acute respiratory syndrome in asia. media coverage may also lead populations to overreact to mild pandemics. abstract there is an unmet need for greater investment in preparedness against major epidemics and pandemics. the arguments in favour of such investment have been largely based on estimates of the losses in national incomes that might occur as the result of a major epidemic or pandemic. recently, we extended the estimate to include the valuation of the lives lost as a result of pandemic-related increases in mortality. this produced markedly higher estimates of the full value of loss that might occur as the result of a future pandemic. we parametrized an exceedance probability function for a global influenza pandemic and estimated that the expected number of influenza-pandemic-related deaths is about per year. we calculated that the expected annual losses from pandemic risk to be about billion united states dollars -or . % of global income -per year. this estimate falls within -but towards the lower end of -the intergovernmental panel on climate change's estimates of the value of the losses from global warming, which range from . % to % of global income. the estimated percentage of annual national income represented by the expected value of losses varied by country income grouping: from a little over . % in high-income countries to . % in lower-middle-income countries. most of the losses from influenza pandemics come from rare, severe events. economic losses of pandemic risk victoria y fan et al. we found only two articles that included estimates of the loss from the elevated mortality associated with influenza pandemics. , of the studies included in a recent systematic literature review on the costs of influenza, only one took account of the value of mortality risks. one strand of economic research has examined the intrinsic value of mortality risks, which is commonly expressed as the so-called value of a statistical life. this value is derived either from questionnaires that canvass how much compensation an individual would demand, to accept a small increase in the probability of their death, or from quantitative studies of the labour market that investigate the trade-offs between small fatality risks and income. , beyond influenza, the value of mortality risks has been included in estimating the costs of vaccine-preventable diseases and in evaluating the economic burdens posed by rheumatic heart disease. far more studies have assessed the burden of specific environmental risk factors. , the value of a statistical life, which is sometimes expressed as the value of a standardized mortality unit (smu), i.e. an increase in the annual risk of death of in , varies by both the age and income of the individual involved. , , in general, the value of mortality is elastic to age and to income, i.e. younger individuals place a higher value on mortality than older individuals, and higher-income individuals generally value mortality more than lower-income individuals. the main findings of our recent study appeared consistent when, in robustness and sensitivity checks, we used estimates that were unconditional on age and estimates with varying income elasticity with respect to the value of mortality. given the uncertain nature of an influenza pandemic, in terms of both when it may occur and how large the mortality risks will be, we applied an expected-loss framework that accounts for the uncertainty over a long period of time. an expected-loss framework incorporates information on the risk of an uncertain event, e.g. a pandemic, with information on the severity or value of that event, e.g. the increase in mortality. although it has been estimated that the - ebola virus disease outbreak led to about deaths, the death toll from a severe influenza pandemic might be times higher than this. in any given year, however, the risk of a severe influenza pandemic is much smaller than that of an ebola epidemic. the use of an expected-loss framework allows policy-makers to compare the expected losses associated with events with relatively high annual probability, but low mortality, e.g. an ebola outbreak, with those of events with relatively low probability but high mortality, e.g. the influenza pandemic. expected-loss frameworks are commonly used, by actuaries in the insurance industry, to calculate the size of premiums, e.g. for flood or health insurance. to value the consequences of uncertain events appropriately, the insurance industry estimates so-called exceedance probability functions. these functions generate estimates of the probability that, over a specified time frame, losses from an uncertain event, e.g. an influenza pandemic, would exceed any specified level. for our analysis, we developed an exceedance probability function for a global influenza pandemic. to parameterize the function, we turned to historical data on global influenza pandemics since the s. [ ] [ ] [ ] [ ] six pandemics in this period led to excess mortality rates ranging between . % and . % of world population. in , this range would be the equivalent of between million and million excess deaths globally. a modelling exercise for the insurance industry concluded that the annual risk of an influenza outbreak on the scale of the pandemic lies between . % and . %. for more severe pandemics, we fitted a parametrized exceedance probability function to modelled data that had been previously reported. , following common practice in the insurance industry, we defined risk, r(s), in terms of the annual probability of a pandemic having a severity exceeding s smus and the return time for s as the expected number of years before a pandemic of at least severity s will occur. if t(s) is the return time, then t(s) = r(s) − . for example, if the annual probability of a pandemic of severity at least s is %, then its return time will be years. if we had access to a function r(s) showing exceedance probability as a function of severity, our analysis could proceed using the expected value of severity of all pandemics. because r(s) is the complementary cumulative of the density for s, we would have expected value of: we calibrated this model using historical estimates of the frequency and severity of influenza pandemics, which we obtained from our literature search on pubmed® and google scholar. for mortality data relating to the influenza pandemic, we also searched the libraries at harvard university and the university of hawai'i for historical documents and life tables. studies were restricted to those with abstracts in english. the consequences of a pandemic, in terms of lost income or lost lives. the expenditures made to prepare for -or recover from -a pandemic. excess death attributable to a given influenza pandemic (expressed in this paper in standardized mortality units or smus -a unit of per per year). the estimated probabilities that, in any given year, pandemics of varying degrees of severity will occur. defined in the probabilistic sense as the sum, across severities, of the losses associated with a pandemic of any given severity multiplied by the probability that a pandemic of that severity will occur in the coming year. note: much of this nomenclature accords with that of the insurance industry. policy & practice economic losses of pandemic risk victoria y fan et al. like other economic studies of pandemic influenza, we identified two main influenza pandemic scenarios in terms of aggregate mortality: moderate and severe. our review classified the pandemic as severe. as the world population in was about million and historical data indicate that there were at least million pandemicrelated deaths in that year, the excess death rate associated with the pandemic was at least . %. a closer examination of the data from india indicate that the true global rate was probably far higher than . %, the pandemic led to million deaths in india [ ] [ ] [ ] and it seems implausible that india accounted for % of all of the pandemic-related deaths at a time when it had % of the world population. however, to be conservative, we estimated an expected annual excess mortality rate of . smus. in the corresponding model for moderate pandemics, we used a global expected excess mortality rate of . smus, as seen in historical moderate pandemics. our calibration pointed to a very fat-tailed distribution. thus, compared with an exponential function, the hyperbolic family of complementary cumulative distributions provided more natural candidates for r(s). we parameterized the hyperbolic function in terms of its expectation and the fatness of its tail. thus: the estimated proportion of annual national income represented by the losses varied according to country income grouping, from a little over . % in high-income countries to . % in lower-middle-income countries ( table ) . the expected-loss framework distinguishes between the loss associated with a certain event that occurred, e.g. the mortality that occurred as a result of the influenza pandemic, and the expected loss associated with an uncertain event over a period of risk exposure. the expected loss combines both the risk of a moderate or severe pandemic and the losses from that event should the event occur. the expectedloss framework thus produces estimates of expected losses of an uncertain event, rather than actual losses of a certainly occurring event. we estimated the expected number of pandemic-related deaths to be about per year. this level of mortality is on a similar scale to that attributable to other, more certain, causes of death, including other major infectious causes of death. importantly, we concluded that most of the expected loss from influenza pandemics results from extreme events. another effort to estimate exceedance probability functions indicated that, among all pathogens that can cause a pandemic, influenza virus was likely to be the predominant cause of pandemic-related mortality. the implication is clear: any efforts at pandemic preparedness need to be most strongly focused on influenza and on preparation for a severe scenario. our results present losses much higher than those found in studies limited to income losses. income losses have been estimated to represent around % and % of the total economic losses associated with a severe pandemic and a mild pandemic, respectively. , in previous studies, across modelled pandemics of all severities, mean income losses were estimated to be us$ billion per year , , i.e. about % of our estimate of total pandemicrelated costs. in terms of the percentage of global income, our estimate of total pandemicrelated losses ( . %) falls within the corresponding intergovernmental panel on climate change's estimates of the costs of global warming ( . - . %). however, the magnitude of future global warming and the associated economic losses are still uncertain. , the same is true for future pandemics. many of the hundreds of studies on the potential costs of climate change have been hampered by the wide variation in estimates of the so-called social cost of carbon. if this cost is set at about us$ per tonne, the cost of the carbon dioxide emissions in would have been about % of global income. , as in many previous attempts to estimate the economic losses associated with a pandemic, many previous attempts to estimate the social costs of carbon have focused on national income accounts, without any explicit valuation of the increases in mortality resulting from climate change. the mortality-associated costs of climate change may be relatively small, however, since the slowness of climate change should allow for compensatory human adaptation. our study had several limitations. first, we ignored the intrinsic undesirability of nonfatal illness and/ or pandemic fear. intense media coverage may lead populations to overreact to mild pandemics. second, our estimates of future pandemic risk and severity, and the economic estimates based on these epidemiological estimates, are relatively crude partly because pandemics remain rare and uncertain events. future modelling should lead to improved estimates over time. third, the assignment of monetary value to small changes in mortality risk and, particularly the relationship between valuation of such risk and both individual income and age at death, remains controversial. however, the results of sensitivity analyses, in which we applied a range of assumptions on these parameters, indicated that our main findings were reasonably robust. in addition to pathogens of pandemic potential, an expected-loss framework may also be applied usefully to malaria and other diseases that have fluctuating incidence. as cases of the disease become rarer as the result of effective interventions, malaria becomes less visible politically and financially, and policy-makers in some countries may have responded by reducing control efforts prematurely. policy-makers, and the societies they serve, could benefit by using an expected-loss framework to estimate the losses associated with uncertain and rare events across the full range of potential outcome severities. this could lead to appropriate and beneficial adjustments to each policy-maker's sense of risk and sense of value and to improved national policies on epidemic and pandemic preparedness. a recent united states national academy of medicine report argued that, given the risks we estimated, policy attention has fallen short. il est nécessaire d'investir davantage dans la préparation contre les grandes épidémies et les pandémies. les arguments en faveur de cet investissement s'appuient en grande partie sur les estimations des pertes au niveau du revenu national que pourrait entraîner une grande épidémie ou une pandémie. récemment, nous avons élargi ces estimations pour y inclure la valeur des pertes faisant suite à des hausses de mortalité dues à des pandémies. cela a donné des estimations nettement plus élevées de la valeur totale de la perte que pourrait occasionner une future pandémie. nous avons paramétré une fonction de probabilité de dépassement pour une pandémie mondiale de grippe et avons estimé que le nombre escompté de décès dus à cette pandémie de grippe était d' environ par an. nous avons calculé que les pertes annuelles découlant du risque de pandémie représentaient environ milliards de dollars des États-unis, soit , % du revenu mondial par an. cette estimation rejoint (dans la fourchette inférieure) celles du groupe d' experts intergouvernemental sur l' évolution du climat quant à la valeur des pertes dues au réchauffement de la planète, qui vont de , % à % du revenu mondial. le pourcentage estimé du revenu national annuel représenté par la valeur escomptée des pertes variait selon la catégorie de revenu des pays: d'un peu plus de , % dans les pays à revenu élevé à , % dans les pays à revenu intermédiaire-tranche inférieure. la plupart des pertes découlant de pandémies de grippe sont dues à des événements rares et graves. Требуется увеличение инвестиций в подготовку к борьбе с крупными эпидемиями и пандемиями. Аргументы в пользу таких инвестиций в значительной степени основаны на оценках потерь в национальном доходе, которые могут возникнуть в результате крупной эпидемии или пандемии. Недавно авторы расширили эту оценку, включив в нее количество людей, погибших в результате увеличения смертности, связанной с пандемией. Это привело к более высоким оценкам полного ущерба, который может возникнуть в результате будущей пандемии. Авторы параметризовали функцию вероятности превышения смертности для глобальной пандемии гриппа и подсчитали, что прогнозируемое число смертей от гриппа и пандемии составляет около в год. Мы подсчитали, что ожидаемые ежегодные потери из-за риска пандемии составляют около млрд долларов США (или , % мирового дохода) в год. Эта цифра находится в пределах оценки (ближе к нижней границе), полученной Межправительственной группой экспертов по изменению климата для оценки риска глобального изменения климата, которая составляет от , до % от глобального дохода. Предполагаемый процент годового национального дохода, представленный ожидаемой величиной потерь, варьировался по группам стран в зависимости от уровня дохода: от немногим более , % в странах с высоким уровнем доходов до , % в странах с низкими и средним доходом. Большинство потерь от пандемии гриппа происходят по причине редких тяжелых явлений. hay una necesidad no satisfecha de invertir más en la preparación para grandes epidemias y pandemias. los argumentos a favor de dicha inversión se basan, en gran parte, en las estimaciones de las pérdidas en los ingresos nacionales que podrían darse como resultado de una gran epidemia o pandemia. recientemente, ampliamos el cálculo para incluir la valoración de las vidas perdidas como resultado del aumento de la mortalidad relacionado con la pandemia. esto dio como resultado unas estimaciones notablemente más altas del valor de la pérdida que podría resultar de una futura pandemia. hemos parametrizado una función de probabilidad de excedencia para una pandemia de gripe mundial y estimado que el número esperado de muertes causadas por una pandemia de gripe es de aproximadamente por año. calculamos que las pérdidas anuales esperadas del riesgo de pandemia son de unos millones de dólares estadounidenses, o el , % de los ingresos mundiales, por año. esta estimación se encuentra dentro, pero cerca del mínimo, de las estimaciones del panel intergubernamental del cambio climático sobre el valor de las pérdidas por el calentamiento global, que oscilan entre el , % y el % de los ingresos globales. el porcentaje estimado de los ingresos nacionales anuales representado por el valor esperado de las pérdidas varió según la agrupación de ingresos del país: de poco más del , % en los países con ingresos altos al , % en los países con ingresos medios o bajos. la mayoría de las pérdidas por pandemias de gripe provienen de casos raros y severos. assessment of economic vulnerability to infectious disease crises from panic and neglect to investing in health security: financing pandemic preparedness at a national level financing of international collective action for epidemic and pandemic preparedness in search of global governance for research in epidemics global macroeconomic consequences of pandemic influenza. sydney: lowy institute for international policy the economic impact of pandemic influenza in the united states: priorities for intervention. emerg infect dis economic losses of pandemic risk victoria y fan et al total economic consequences of an influenza outbreak in the united states. risk anal valuation of the risk of sars in taiwan. health econ on sars type economic effects during infectious disease outbreaks. washington: world bank world bank evaluating the economic consequences of avian influenza. washington: world bank the loss from pandemic influenza risk the cost of air pollution. health impacts of road transport valuing mortality risk reductions from environmental, transport, and health policies: a global metaanalysis of stated preference studies global health : a world converging within a generation the income elasticity of the value per statistical life: transferring estimates between high and low income populations boston: air worldwide pandemics: risks, impacts, and mitigation the annual impact of seasonal influenza in the us: measuring disease burden and costs. vaccine direct medical cost of influenza-related hospitalizations in children economic impact of influenza. the individual's perspective economic costs of influenza-related work absenteeism. value health the cost of influenza in thailand the macroeconomic impact of pandemic influenza: estimates from models of the united kingdom, france, belgium and the netherlands the possible macroeconomic impact on the uk of an influenza pandemic influenza cost and cost-effectiveness studies globally-a review the value of individual and societal risks to life and health during the ' decade of vaccines, ' the lives of . million children valued at $ billion could be saved. health aff (millwood) the economic impact of rheumatic heart disease in developing countries geneva: world health organization the next influenza pandemic: can it be predicted? jama a history of influenza the chronicle of influenza epidemics introduction to pandemic influenza through history modelling a modern-day spanish flu pandemic the population of india and pakistan influenza in india : excess mortality reassessed estimation of potential global pandemic influenza mortality on the basis of vital registry data from the - pandemic: a quantitative analysis observations on mortality during the influenza pandemic characterizing the amount and speed of discounting procedures expert judgments of pandemic influenza risks preparing for the next pandemic climate change temperature impacts on economic growth warrant stringent mitigation policy. nat clim chang updating estimation of the social cost of carbon dioxide global problems, smart solutions: costs and benefits using and improving the social cost of carbon economic aspects of global warming in a post-copenhagen environment the neglected dimension of global security: a framework to counter infectious disease crises the neglected dimension of global security -a framework for countering infectious-disease crises we thank peter sands and bradley chen. vyf has a secondary appointment with harvard t h chan school of public health, boston, united states of america. key: cord- -tu xrt x authors: li, cui; gao, fei; yu, lei; wang, ruoke; jiang, yisheng; shi, xuanling; yin, chibiao; tang, xiaoping; zhang, fuchun; xu, zhiheng; zhang, linqi title: a single injection of human neutralizing antibody protects against zika virus infection and microcephaly in developing mouse embryos date: - - journal: cell rep doi: . /j.celrep. . . sha: doc_id: cord_uid: tu xrt x zika virus (zikv) is a mosquito-transmitted flavivirus that is generally benign in humans. however, an emergent strain of zikv has become widespread, causing severe pre- and post-natal neurological defects. there is now an urgent need for prophylactic and therapeutic agents. to address this, we investigated six human monoclonal antibodies with zikv epitope specificity and neutralizing activity in mouse models of zikv infection and microcephaly. a single intraperitoneal injection of these antibodies conveyed distinct levels of adult and in utero protection from zikv infection, which closely mirrored their respective in vitro neutralizing activities. one antibody, zk b , showed the most potent neutralization activity, completely protected uninfected mice, and markedly reduced tissue pathology in infected mice. thus, zk b is a promising candidate for the development of antibody-based interventions and informs the rational design of zikv vaccine. zika virus (zikv) is a mosquitotransmitted flavivirus that can cause severe neurological defects in humans. li et al. have identified a human monoclonal antibody capable of protection against zikv infection and related diseases when tested in mouse models. this antibody serves as a promising candidate for clinical development against zikv. the recent, widespread neurological deficits caused by an emergent strain of zika virus (zikv) have caught the world off guard wikan and smith, ) . zikv was first identified in the forests of uganda, and infection was generally benign in humans (dick et al., ) . however, this new strain of zikv is far more virulent and causes a range of clinical anomalies rasmussen et al., ; wikan and smith, ) . most notable are microcephaly and other congenital defects in infants born to mothers infected with zikv during pregnancy (mlakar et al., ; petersen et al., ; rasmussen et al., ; wikan and smith, ) . although the exact mech-anism of neuropathogenesis remains uncertain, clinical abnormalities have been linked to the aberrant development and loss of neural progenitor cells (npcs) (cugola et al., ; gabriel et al., ; garcez et al., ; li et al., a li et al., , b tang et al., ) . the contemporary strain of zikv has enhanced replication capacity and a specialized tropism for npcs (cugola et al., ; dang et al., ; garcez et al., ; li et al., b; tang et al., ) , although other types of cells are susceptible (tabata et al., ; weisblum et al., ) . the infection inhibits npc proliferation and differentiation and can trigger apoptosis or autophagy. critically, the highest rates of birth defects occur in pregnant mothers who are infected during their first and second trimesters. this is presumably because, during the early stages of gestation, npcs have a greater susceptibility to zikv infection, and there is more viral transfer across the placental barrier (mlakar et al., ; petersen et al., ; rasmussen et al., ; wikan and smith, ) . to fully protect the developing fetuses, an intervention must occur before this period or, ideally, prior to infection (marston et al., ) . neutralizing antibodies are the essential mediator of immunity against viral infection (burton and hangartner, ; corti and lanzavecchia, ) . for zikv and other flaviviruses, human neutralizing monoclonal antibodies target the surface envelope glycoprotein (e) that facilitates infection (dejnirattisai et al., ; fernandez et al., ; fibriansah et al., ; heinz and stiasny, ; magnani et al., ; pierson and diamond, ; pierson and graham, ; robbiani et al., ; rogers et al., ; sapparapu et al., ; stettler et al., ; wang et al., wang et al., , b zhao et al., ) . we previously reported on a panel of monoclonal antibodies (mabs) derived from the longitudinal samples of a zikv-convalescent individual and characterized their neutralizing activities, epitope specificities, and development timeline over the course of infection . we also reported on mouse models of zikv infection and microcephaly, with enhanced specificity for neurological infection figure . experimental design to evaluate the prophylactic potential of six human mabs against zikv infection in developing fetuses and ag mice (a) timeline for mab injection, zikv inoculation, infant delivery, and the monitoring of a complementary set of clinical, virological, and neuropathological outcomes from embryonic day . to p . the six mabs tested are shown alongside their ic values and epitope specificities. zikv-inoculated fetuses and neonates are indicated by zikv + in red; those left unexposed are indicated by zikv À in blue. the cartoon mice on p include zikv + (small and large indicated in red) and zikv À (large indicated in blue); the size representations reflect potential body weight outcomes. each mab was tested, and the outcomes were monitored in three littermate neonates of three pregnant mice on p and p . (b) different levels of protection conferred by the six mabs, shown with the number (n) of neonates monitored for survival in each mab treatment group. (c) the body weight of neonates among the different mab groups. zikv + neonates indicated in red are presented as a percentage of the blue zikv À littermates. the zk b -treated group had no discernable differences between the zikv + and zikv À littermates and is, therefore, shown as a single red/blue checkered bar. the number of neonates for each analysis is indicated above the respective mab, with zikv + in red and zikv À in blue. (legend continued on next page) using a contemporary zikv asian strain (gz ). in the model of microcephaly, the virus was inoculated directly into the lateral ventricles of the fetal mouse brain (li et al., a) . zikv replicated in the fetal brain, with preferential infection of npcs. infection resulted in cell-cycle arrest, differentiation defects, and a large number of cell deaths, as well as clinical presentations of microcephaly (li et al., a) . here, we use the mouse models of zikv infection and microcephaly to analyze the in vivo protective activities of six human mabs and compare the findings with our reported in vitro neutralization activity, as measured by plaque reduction neutralization test (prnt). our results offer compelling evidence that the in vivo protection is directly associated with in vitro neutralization. one antibody, zk b , provides complete protection in pre-exposure treatment and partial protection in post-exposure therapy, with markedly reduced tissue pathology. we believe that zk b is a promising candidate for the development of antibody-based interventions and informs vaccine design specific to zikv infection. in vivo protection correlates with in vitro neutralizing activity of mabs we evaluated and compared the protective potential of six representative human mabs following the protocol highlighted in figure a . all these (zk - , zk - , zk f , zk c , zk c , and zk b ) were isolated by our team. two additional zikv domain iii (diii)-specific mabs (zv and z ) isolated by others were included for comparative analysis (robbiani et al., ; zhao et al., ) . a human antibody, middle east respiratory syndrome- (mers- ), targeting the middle east respiratory syndrome coronavirus (mers-cov), was used as a negative control. our mabs were initially isolated from the peripheral b cells of a convalescent chinese man who acquired zikv while traveling to venezuela during the outbreak in . specimens were derived from sequential blood samples collected on day (zk - ), day (zk - ), day (zk f ), or day (zk c , zk c , and zk b ) after symptom onset. they targeted the zikv e with varying degrees of binding and neutralizing activities and epitope specificities . for example, zk b and zk c were strictly zikv specific and demonstrated the most potent neutralizing activities, as measured by prnt. of these, zk b recognized diii, while zk c was specific for domain i/domain ii (di/dii). in comparison, zk - , zk - , zk f , and zk c were much less potent against zikv but cross-neutralized other members of the flavivirus family, such as dengue virus serotypes denv and denv . on the zikv envelope, they targeted either di/dii (zk - , zk - , and zk f ) or di/dii/diii (zk c ). in terms of genetic analyses, these antibodies demonstrated diverse heavy-chain variable regions. two, zk - and zk f , fell into the immunoglobulinheavy variable (ighv) - family, while zk - and zk c belonged to the ighv - family. finally, zk b and zk c belonged to the ighv - and ighv - families, respectively. to test prophylactic activity against zikv infection, mg/kg of each mab was administered intraperitoneally to a group of three pregnant mice on embryonic day . ( figure a ). the following day (e . ), the developing littermate fetuses in each pregnant mouse were either left untouched (zikv À ; figure a , blue) or inoculated with plaque-forming units (pfus) of zikv asian strain gz directly into the lateral ventricles of the brain (zikv + ; figure a , red). the neonates were closely monitored after delivery and analyzed for a complementary set of clinical, virological, and neuropathological outcomes on post-natal day (p) and for survival up to p . as shown in figure b , the levels of protection conferred by the different mabs varied considerably but clearly correlated with their neutralizing activities, as measured by half-maximal inhibitory concentrations (ics ) in the prnt . the most potently neutralizing mab, zk b , conferred complete protection. survival rates of infected fetuses were as high as % on p . the next potent, zk c , however, conferred only partial protection with median survival of days. the two antibodies (zv and z ) isolated by others, together with the rest of the mabs isolated by us, offered negligible protection and were virtually similar to the isotype control mers- . we also assessed impact on developing body weights and found weights measured on p closely correlated with the neutralizing activity. the zikv + neonates treated with the most potent mabs, zk b and zk c , had weight gain similar to that of their zikv À littermates. the zikv + neonates treated with zv , z , and the remaining mabs failed to do so. the control zikv + mers- group had the greatest loss in body weight. next, we studied whether the protection pattern observed in the pregnant mice also occurred in non-pregnant mice. we administered mg of each mab (zk - , zk - , zk f , zk c , zk c , zk b , zv , and z ) or isotype control mers- to a group of four -to -week-old ag mice via the intraperitoneal (i.p.) route. the following day, animals were challenged with pfus of zikv asian strain gz via the i.p. route. survival rates, body weight, and viral rna copies in the blood were monitored for up to days ( figure d ). consistent with the outcomes for pregnant mice, the level of protection in nonpregnant mice correlated with antibody neutralizing activities as measured by half-maximal inhibitory concentrations (ics ) in the prnt ( figure e ). for example, the most potent and protective mab in pregnant mice, zk b , provided complete protection in non-pregnant mice, with survival rates of % days after the challenge. the next potent and protective, zk c , conferred only partial protection, with median survival of days. the rest of the tested mabs, including the diii-specific ones (zv and z ) isolated by others (robbiani et al., ; zhao et al., ) , offered even lower levels of protection. zk - was virtually identical to the isotype control mers- . (d-g) shown here: (d) timeline for mab injection, zikv inoculation, and monitoring for (e) survival, (f) body weight, and (g) blood zikv rna up to days in ag mice. the body weight and zikv rna in the whole blood derived from a single measurement showed distinct results among the study groups. the number of animals used in each group was four. all data are presented as mean ± sem. *p < . ; **p < . ; ***p < . ; ns, no significant. similarly, changes in body weight measured over the -day follow-up also correlated with the neutralizing activities. the zikv + mice treated with zk b maintained relatively stable body weight throughout the study, although there was a noticeable decline after the first blood sample collection on day (figure f ). in contrast, zikv + mice treated with zk c lost substantial body weight beginning on day post-challenge. animals treated with the remaining mabs had severe and rapid losses in body weight that coincided with a clinical deterioration (figure e) . lastly, for zikv + mice treated with zk b , blood levels of viral rna were indistinguishable from those measured in uninfected animals on both day ( . ± . versus . ± . ) and day ( . ± . versus . ± . ) after challenge ( figure g ). however, rna levels measured in zk c -treated mice were, on average, log greater on day ( . ± . versus . ± . ) and more than logs greater on day ( . ± . versus . ± . ), compared to zk b . the remaining mabs failed to suppress viral replication. the measured zikv rna load on day ranged from . to . logs higher than that of zk b , depending on the mab used ( figure g ). taken together, these results demonstrated that the protective patterns of each mab were similar in both pregnant and non-pregnant mice and closely mirrored their respective in vitro neutralizing activities. the protective effects of each mab on body weight and overall survival mirrored their respective virological and neuropathological outcomes in treated mice. for instance, rna loads measured in zikv + neonates treated with zk b were suppressed in the blood ( . ± . log copies per milliliter) and the brain ( . ± . log copies per gram) to levels indistinguishable from those in zikv À littermates. in fact, zk b was able to reduce the zikv rna by . logs in the blood and . logs in the brain, relative to that in the zikv + mers- group (figures a and b ). in comparison, zk c was equally effective at suppressing replication in blood ( . ± . log copies per milliliter) but only moderately protective in the brain ( . ± . log copies per gram). the mabs zv and z only moderately suppressed zikv replication in the blood ( . ± . and . ± . log copies per milliliter, respectively) and the brain ( . ± . and . ± . log copies per gram, respectively). none of the remaining mabs notably suppressed replication in either blood or brain tissue. all of the zikv + neonates treated with these mabs had high levels of zikv rna, which were similar to levels in the zikv + mers- control group and significantly higher than those in their zikv À littermates (figures a and b ). importantly, low levels of zikv rna were associated with the healthier development of the neonatal brains. of the six representative mabs selected for in-depth neuropathological study, zikv + mice treated with either zk b or zk c maintained normal brain growth and structure. there were no notable differences in cerebral size, cortical thickness, or lateral ventricle area between these zikv + neonates and their zikv À littermates ( figures d- h ). however, zikv + mice treated with zv and z received only moderate protection, while zk f and mers- failed to provide any protection. low protection was measured by reduced cerebral size, thinner cortices, and enlarged lateral ventricles in zikv + neonates compared to their zikv À littermates ( figures d- h ). it is interesting to note that the e-specific human immunoglobulin g (igg) levels in the brain were higher in mice treated with zk b and zk c compared to the other mab groups. in particular, the levels of zk b ( . ± . mg/kg, which is translated into . ± . mg/ml) in zikv + neonates were well above the ic neutralization values measured in the prnt, whereas igg levels in the other mab groups measured less than or similar to those in the negative control pbs group ( figures a and c) . these results indicate that the tested mabs were able to transport across both the maternal-fetal placental barrier and the primitive blood-brain barrier of developing fetuses (saunders et al., ) . we also conducted immunohistochemical analyses of brain sections collected from p neonates in each group. as shown in figure a , in zk b and zk c groups, no zikv-infected cells (green) or apoptotic cells (cas + , red) were detected in the cortices of either zikv + neonates or their zikv À littermates. the mature neurons (neun; figure a , gray) and nuclei (dapi; figure a , blue) appeared healthy, with a tightly packed structure throughout the tissue sections. in distinct contrast, a large number of zikv-positive cells were identified throughout the cortices of the zikv + neonates in zv , z , zk f , and mers- groups. this high prevalence of infection was associated with significant cell death ( figure a , red) and degradation of the cortical structure ( figure a , gray and blue). we also quantitatively assessed the protective activity of zk b and zk c using marker cells from multiple tissue sections. as shown in figures b and c , treatment with zk b and zk c in zikv + neonates reduced zikv infection and cell death to levels indistinguishable from those of their zikv À littermates. furthermore, in the zk b and zk c groups, counts of mature neurons were equivalent between zikv + neonates and their zikv À littermates ( figure d ). however, for the zikv + neonates treated with either zv , z , zk f , or mers- , zikv-positive cell and apoptotic cell counts were as high as , cells and , cells, on average, per square-millimeter tissue section, respectively ( figures b and c) . counts of mature neurons were also significantly reduced in these groups ( figure d ). these results support the enhanced therapeutic activity of zk b and zk c at the cellular and tissue levels relative to zv , zv , zk f , and mers- and also offer an explanation for their impressive in vivo protection of developing fetuses. as zk b demonstrated the greatest prophylactic efficacy against zikv infection, we went on to evaluate its treatment efficacy after zikv infection of the developing fetuses. specifically, after the littermate fetuses were either left untouched (zikv À ) or inoculated with pfus of zikv gz in the lateral ventricle (zikv + ) on e . , we administered mg/kg zk b i.p. to the pregnant mice. this was done either on the same day (e . , day ), day later (e . , day ), or days later (e . , day ). the littermate neonates were closely monitored after delivery and analyzed for the same outcomes measured in the prevention experiments ( figure a ). as shown in figure a , delaying treatment from day to either day or day reduced the survival percentages. treatment on day offered the most effective treatment, with a median survival of about days. this is signif-icantly higher than that of mice treated on either day ( days) or day ( days). the survival advantage conferred by treatment on day corresponded with a clear reduction of viral load in the brain ( figure d ) and less damage to the cortical thickness and lateral ventricles (figures f and g) . however, the effect was less obvious when measured by body weight or each red dot represents a zikv + ; each blue dot represents a zikv À neonate. the red dots with blue coating represent no discernable differences between zikv + and zikv À littermates in zk b -and zk c -treated groups. the numbers of neonates for each mab analyses are indicated above each group, with zikv + in red and zikv À in blue. in (g) and (h), each dot represents the mean value of at least two slices from one neonate. all data are presented as mean ± sem. *p < . ; **p < . ; ***p < . ; ns, no significant. blood serum viral loads on p (figures b and c ). considering that, in our mouse model, the brain is the initial and active site of viral replication, we expected that antibody treatment effects would be more sensitive and immediate in this organ and would be apparent before effects on other organs and body weight as a whole. this hypothesis was supported by immunohistochemical analyses of brain sections derived from p neonates. as shown in figure a , zikv-positive cells (green) were only sporadically detected in the day- treatment group. however, they were detected throughout the cortices if treatment was delayed to day or day . a greater level of infection was associated with increased cell death ( figure a , red) and cortical abnormalities ( figure a , gray and blue). in particular, the tightly packed and well-organized structure of matured neurons in the cortices became loosely connected and disorganized. furthermore, a quantitative assessment of marker cells derived from multiple each red dot represents a zikv + neonate; each blue dot represents a zikv À littermate. red dots with blue coating represent no discernable differences between zikv + and zikv À littermates in zk b -and zk c treated groups. each dot represents the mean value of at least two slices from one neonate analyzed. the numbers of neonates for each analysis are indicated above each group, with zikv + in red and zikv À in blue. scale bar, mm. all data are presented as mean ± sem. *p < . ; **p < . ; ***p < . . tissue sections supported the evidence that the time of treatment administration impacted treatment efficacy. for instance, in the zikv + neonate group, treatment on day significantly suppressed the number of zikv-positive cells to ± and apoptotic cells to ± per square millimeter of tissue section. treatment on day , however, allowed widespread cell infection and cell death counts as high as , ± , and ± per square-millimeter tissue section, respectively ( figures b and c ). however, although tissue samples from all three temporal treatment groups showed structural abnormalities in the cortices, there were no obvious reductions in the total number of mature neurons ( figure d ). treatment with the isotype mers- control was worst among all groups, with no signs of figure . evidence of therapeutic potential of zk b against zikv infection in developing fetuses (a) decreased survival when treatment administration is delayed from day (e . ) to day (e . ) or to day (e . ). the number (n) of neonates monitored for survival is indicated. (b-d) after treatment with zk b on day , day , or day , we analyzed zikv + neonates (indicated in red) and zikv À littermates (indicated in blue) at p for (b) body weight, (c) zikv rna copies in the blood, and (d) zivk rna copies in the brain. the number of neonates analyzed for each treatment time point is indicated above each group. (e-g) shown here: (e) the representative coronal sections of the neonatal brains at p was visualized with nissl staining and analyzed for (f) cortex thickness and (g) ventricular area. in (f) and (g), each dot represents the value of one slice. the total numbers of slices analyzed for zikv À , day , day , day , and mers- were , , , , and , respectively. the number of neonates analyzed for each treatment time point is indicated above each group. scale bar, mm. all data are presented as mean ± sem. *p < . ; **p < . ; ***p < . ; ns, no significant. protection (figures and ) . these results highlight the critical role of zk b in attenuating disease progression by inhibiting cell infection and death at the cellular and tissue levels. furthermore, the time of administration relative to infection impacts efficacy. treatment has the greatest protective effect when initiated immediately after infection. we report here the systematic analyses of the prophylactic and therapeutic potential of a panel of human mabs against zikv infection in a mouse model of microcephaly and non-pregnant ag mice. we showed that different mabs demonstrated distinct protective activity against zikv infection and that the major determinant of efficacy is their intrinsic neutralizing activities. a single i.p. injection of pregnant and non-pregnant mice with the most potent mab, zk b , provided developing fetuses and adult animals with a complete protection against zikv infection. treatment with zk b markedly reduced fetal infection and tissue pathology and significantly delayed mortality. thus, zk b is a promising candidate for the development of antibody-based intervention and informs rational design of zikv vaccine. two unique aspects of our study are worth highlighting here. one is based in the tested mabs that are all derived from the same zikv convalescent individual at different time points in the course of natural infection. each has distinct neutralizing activity, epitope specificity, and lineage ancestry . this diversity not only allows us to pinpoint the major determinants of protection in the mouse model but also provides evidence for when protective potential arises during the course of a natural infection in humans. our results clearly show that neutralizing activity, rather than other features, is the critical biomarker for protection against zikv infection, although the role of fc-mediated antiviral functions still need further investigation (dejnirattisai et al., ; pierson and graham, ; sapparapu et al., ; stettler et al., ). among the mabs tested here, zk b is the most potent, and its epitope is located in the lateral ridge region within the diii of e . mabs that target diii with potent neutralizing activity have also been isolated by other groups, derived from either infected humans or mice, and have been shown to be effective in various models of zikv pathogenesis (fernandez et al., ; magnani et al., ; robbiani et al., ; stettler et al., ; wang et al., b; zhao et al., ) . some weakly neutralizing mabs have also been reported against the same domain (robbiani et al., ; sapparapu et al., ; stettler et al., ; zhao et al., ) . it would be hard to determine whether this convergence on diii is a purely random event or whether diii epitopes are somehow more exposed and, therefore, more accessible by the mabs. it would be interesting to compare the exact epitopes of the highly potent mabs to see whether the vulnerable spots are widely spread or confined to restricted regions on the diii. this information would undoubtedly contribute to our understanding of the immune recognition mechanisms and assist in the rational design of vaccines against zikv infection. similar to other reported diii-specific mabs, zk b was isolated late after the onset of symptoms ( days), when the production of diii-specific antibodies had become more prevalent the total numbers of slices analyzed for zikv À , day , day , day , and mers- were , , , , and in (b) and (d), and , , , , and in (c), respectively. the total number of neonates analyzed is indicated above each group. all data are presented as mean ± sem. **p < . ; ***p < . ; ns, not significant. . however, why the diii-specific response seems to be delayed relative to di/ii-specific responses, and whether a vaccine could induce the preferred diii-specific response ahead of those targeting di/ii, requires more in-depth study. nevertheless, the potent prophylactic and therapeutic activities demonstrated by zk b and other diii-specific mabs will serve as the key criteria for future antibody-based intervention against zikv infection. the other unique aspect of our study was the mouse model of microcephaly, in which the virus was directly inoculated into the lateral ventricles of the fetal brain. despite the surgical sophistication required with this technique, the model has been standardized with remarkable reproducibility and captures the phenotypic features that are key to zikv infection in humans (li et al., a wang et al., a; yuan et al., ) . direct inoculation eliminates the need for immune-deficient pregnant mice and allows for control over the developmental stage at which infection occurs. as a result, both pathogenesis and intervention strategies can be evaluated with more precision. critically, it is likely the most stringent model for brain infection, as it tests the immediate protective activity of mabs at the injection site, as well as their capacity in preventing subsequent dissemination throughout the body. in this regard, the levels of protection conveyed by zk b are, indeed, remarkable. furthermore, direct inoculation on e . also allows for the study of immediate and long-term impacts of zikv infection in the developing fetuses. in this study, neonates were followed up to p , but this could have been extended to investigate the long-term sequelae of zikv infection, be they physiological, cognitive, or behavioral. this feature is complementary to the existing model, in which zikv infection was initiated earlier in embryonic development and was frequently associated with fetal demise before or shortly after delivery (fernandez et al., ; morrison and diamond, ; sapparapu et al., ) . in the future, it would be interesting to test the panel of mabs identified here in the early infection model in order to evaluate their protective potential. in conclusion, our study provides compelling evidence that the protective potential of tested mabs against zikv infection in pregnant and non-pregnant mice was directly associated with their neutralizing activities measured in the prnt. the most potent neutralizing antibody, zk b , demonstrated impressive prophylactic and therapeutic activities and, therefore, could serve as a promising candidate for antibody-based interventions and inform rational vaccine design against zikv infection. we previously reported on the isolation and characterization of a panel of human mabs from longitudinal samples of a zikv convalescent individual and showed their distinctions in neutralizing activities, epitope specificities, and time of development during nature infection . we also reported on one of the most stringent mouse models of microcephaly, in which a contemporary zikv asian strain is inoculated directly into the lateral ventricles of the fetal brains (li et al., a) . the present study combines these methodologies in order to evaluate the protective potential of six representative human mabs against zikv infection and microcephaly. using a single i.p. injection of mabs in pregnant mice prior to zikv infection, we showed that these antibodies convey distinct levels of protection to the developing fetuses and newborns and that these levels closely mirrored their respective in vitro neutralizing activities. the most potent neutralizing antibody tested, zk b , provided a complete protection from zikv infection in pre-exposure treatment and partial protection in post-exposure therapy, as measured by markedly reduced tissue pathology. the tested mabs (zk b , zk c , zk f , zk c , zk - , and zk - ) targeted the zikv e and were initially isolated from a zikv convalescent chinese traveler visiting venezuela during the viral outbreak in . the details of their neutralizing activity, epitope specificity, lineage ancestry, and the time of isolation during natural infection were previously reported . two zikv diii-specific mabs, zv and z , were isolated by others (robbiani et al., ; zhao et al., ) and used here for comparative analyses. all mabs were in the human igg backbone, manufactured in f cells (atcc) by transient transfection, and purified by affinity chromatography using protein a agarose (thermo scientific). the mab concentration was determined using the bca protein assay kit (thermo scientific). we previously isolated the human mab mers- , which targets against the mers-cov and was used as a negative control (jiang et al., ) . approximately mg/kg of each tested mab or isotype control mers- was administered i.p. to a group of three pregnant mice at e . . all icr pregnant mice were purchased from beijing vital river laboratory animal technology. the experimental protocol and procedure were approved by the institutional animal care and use committee at tsinghua university ( -zlq ). the pregnant mice were kept in separate cages and maintained on a -hr/ -hr light/dark cycle throughout the experiment. for each tested mab, a group of three pregnant mice were included, and their littermate neonates were monitored for a complementary set of clinical, virological, and neuropathological outcomes on p and for survival up to p ( figure a) . specifically, the first (i) and second (ii) groups of littermate neonates were sacrificed on p . brain and blood samples were immediately collected, processed, and frozen at À c until use. group i was used to measure for viral loads in the blood serum and the brain, as well as human igg in the brain. group ii was used for blood serum viral loads, brain size, and section analysis. the third (iii) group littermate neonates were monitored for body weight at p and for survival up to p . non-pregnant c bl/ mice deficient in interferon (ifn)a, -b, and -g receptors (ag mice) were purchased from institute pasteur of shanghai, chinese academy of sciences. the mice were bred and maintained in a pathogenfree animal facility at tsinghua university. specifically, mg of each tested mab (zk - , zk - , zk f , zk c , zk c , zk b , zv , and z ) or isotype control mers- was administered to a group of four -to -weekold ag mice via the i.p. route. the following day, the animals were challenged with pfus of zikv (gz strain) via i.p. injection and monitored for survival, body weight, and viral rna copies in the blood up to days. on day and day after challenge, blood was collected from each animal for viral load measurement. microinjection of zikv into the brain ml zikv asian strain gz (genbank: ku and virus stock concentration . pfu/ml) was injected into the right side of the lateral ventricle of the embryonic mouse brains at e . , as described previously (li et al., a) . approximately half of the littermate neonates were injected with zikv (zikv + ), while the rest remained untouched (zikv À ). gz was derived from the same chinese traveler from whom the tested mabs were isolated . phylogenetic analysis of the complete coding sequences indicated that gz was tightly clustered with those circulating in the americas and belonged to the asian lineage, including those identified from french polynesia in . the whole blood ( ml) and right brain of the neonates were collected on p , immediately transferred to an eppendorf tube containing lysis buffer (qiagen), and kept in À c until use. each brain sample was weighted and homogenized using a stainless steel blender (next advance). total rna from the homogenized brain and the whole blood was extracted using an rneasy mini kit ( , qiagen) and reverse-transcribed into cdna using an iscript cdna synthesis kit ( - , bio-rad). as previously described, viral rna copies were quantified through taqman qpcr amplification of zikv envelope genes and expressed as log viral rna copies per gram for the brain or per millimeter for the blood samples calculated against the standard curve. the sequences for the primers and probes used for the analysis are shown as follows: zikv-fccgctgcccaacacaag, zikv-r ccactaa cgttcttttgcagacat, zikv-probe agcctaccttgacaagcartcagac actcaa ( fam, tamra). the neonatal right brains were collected on p and immediately frozen at À c until use. the frozen brain samples were weighted and homogenized with a stainless steel blender (next advance) before being mixed with ml pbs and allowed to stand at À c for hr. the tested mabs, suspended in the pbs, were collected by centrifugation at , g for min and quantified by serial dilution and application to elisa plates pre-coated with recombinant full-length e glycoprotein of zikv (gz , ku ), as previously described . bound mabs were detected using goat antihuman igg (fc specific) conjugated with horseradish peroxidase (promega, madison, wi, usa) and , , , -tetramethylbenzidine (tmb) substrate (cwbio, beijing, china). the mabs levels in the neonatal brains were calculated against a curve that was standardized using the corresponding mab suspended in pbs and expressed as milligrams per kilogram or micrograms per milliliter. brains were fixed in % pfa, dehydrated in % sucrose, and frozen in tissuefreezing medium before being sliced into -mm-thick tissue sections. for nissl staining, brain sections were stained with . % toluidine blue for min; dehydrated, in turn, by %, %, and % ethanol ( s, twice for each); and then hyalinized by xylene for more than min. for immunohistochemical staining, sections were blocked for hr at room temperature (rt) and incubated with the primary antibody for one night at c. after three rounds of extensive washing with phosphate-buffered saline with . % tween- (pbst), the secondary antibody was added and incubated at rt for hr. the section was then washed once more with pbst. the tissue sections were then imaged on an lsm (carl zeiss) confocal microscope and analyzed with imaris and imagej, as described previously (li et al., a) . the antibodies used for immunohistochemical analysis were caspase- (abcam, ab , : , ) and neun (abcam, ab , : , ). zikv serum ( : , ) was derived from the same convalescent patient. nuclei were stained with dapi (invitrogen). for experiments involving pregnant and non-pregnant mice, - mice were included in each assessment group to ensure representation and consistency of the data. all data were analyzed using prism software (graphpad). statistical evaluation was performed by student's unpaired t test. data were presented as mean ± sem. *p < . ; **p < . ; and ***p < . . broadly neutralizing antibodies to hiv and their role in vaccine design broadly neutralizing antiviral antibodies the brazilian zika virus strain 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of a newly emerging arbovirus delineating antibody recognition against zika virus during natural infection a single mutation in the prm protein of zika virus contributes to fetal microcephaly excretion of infectious zika virus in urine structural basis of zika virus-specific antibody protection we are grateful to the zikv convalescent patient for donating his blood samples and drs. jiang wang, wenxin hong, and lingzhai zhao for providing the patient with treatment and care. we thank drs. cheng-feng qin and gong cheng for providing the zika virus isolate gz and for assisting with evaluations of antibody protective activity in ag mice. we also thank ms. angela fan for editing the manuscript. all authors contributed to the present study, including intellectual input, results analysis, and actual writing and editing of the manuscript. specifically, c.l., f.g., r.w., y.j., l.y., and x.s. performed the experiments. c.y., x.t., f.z., z.x., and l.z. designed the study, and c.l., f.g., z.x., and l.z. analyzed the data and wrote the paper. patents have been filed for the isolated antibodies, and they are all available for collaboration in research and development through material transfer agreement. key: cord- -kvv fx n authors: barratt, ruth; shaban, ramon z.; gilbert, gwendoline l. title: clinician perceptions of respiratory infection risk; a rationale for research into mask use in routine practice date: - - journal: infection, disease & health doi: . /j.idh. . . sha: doc_id: cord_uid: kvv fx n abstract outbreaks of emerging and re-emerging infectious diseases are global threats to society. planning for, and responses to, such events must include healthcare and other measures based on current evidence. an important area of infection prevention and control (ipc) is the optimal use of personal protective equipment (ppe) by healthcare workers (hcws), including masks for protection against respiratory pathogens. appropriate mask use during routine care is a forerunner to best practice in the event of an outbreak. however, little is known about the influences on decisions and behaviours of hcws with respect to protective mask use when providing routine care. in this paper we argue that there is a need for more research to provide a better understanding of the decision-making and risk-taking behaviours of hcws in respect of their use of masks for infectious disease prevention. our argument is based on the ongoing threat of emerging infectious diseases; a need to strengthen workforce capability, capacity and education; the financial costs of healthcare and outbreaks; and the importance of social responsibility and supportive legislation in planning for global security. future research should examine hcws' practices and constructs of risk to provide new information to inform policy and pandemic planning. abstract outbreaks of emerging and re-emerging infectious diseases are global threats to society. planning for, and responses to, such events must include healthcare and other measures based on current evidence. an important area of infection prevention and control (ipc) is the optimal use of personal protective equipment (ppe) by healthcare workers (hcws), including masks for protection against respiratory pathogens. appropriate mask use during routine care is a forerunner to best practice in the event of an outbreak. however, little is known about the influences on decisions and behaviours of hcws with respect to protective mask use when providing routine care. in this paper we argue that there is a need for more research to provide a better understanding of the decision-making and risk-taking behaviours of hcws in respect of their use of masks for infectious disease prevention. our argument is based on the ongoing threat of emerging infectious diseases; a need to strengthen workforce capability, capacity and education; the financial costs of healthcare and outbreaks; and the importance of social responsibility and supportive legislation in planning for global security. future research should examine hcws' practices and constructs of risk to provide new information to inform policy and pandemic planning. preventing the transmission of infectious diseases in healthcare settings, and in society more broadly, is a core goal of contemporary public health and infection prevention and control (ipc). in recent years outbreaks of emerging infectious diseases caused by respiratory viruses have drawn considerable global attention, in particular severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers) and pandemic influenza a, h n (table ) . consequently, global and national planning for pandemic diseases is grounded in the expectation that a novel respiratory infection is most likely to be responsible for the next pandemic or infectious disease emergency [ ] . respiratory infectious diseases are transmitted via contact, droplet and/or airborne modes, necessitating healthcare worker (hcw) use of surgical masks or respirators and other personal protective equipment (ppe) together with appropriate hand hygiene. hospital-based transmission of respiratory infectious diseases of high consequence, such as influenza, can be minimised by limiting the part hcws play as vectors or victims of disease. hcws may continue to work with mild respiratory illness (presenteeism), which can be serious or life-threatening if transmitted to vulnerable patients, but they also may suffer serious effects from occupationally-acquired respiratory infections, leading to increased staff absenteeism, which will compromise patient care during epidemics. while policies and protocols for optimal use of ppe and other transmission-based precautions exist in the majority of healthcare facilities, hcw compliance with them is typically limited, particularly in non-outbreak situations or in the early stages before an outbreak is recognised [ , ] . in particular, hcws' use of protective masks when caring for patients with respiratory infections is an important and well-documented ipc measure [ ] . yet hcw use of protective masks, and ppe in general, during routine care is often suboptimal and can result in healthcare-associated acquisition of infection [ ] (table ) . while hcw compliance with the use of protective masks during infectious disease outbreaks has been well reported [ ] , there has been limited examination of hcw behaviours with respect to protective mask use during routine clinical care [ ] . consistent routine use of protective masks, based on relevant clinical indications, is important in preventing or delaying transmission from an unrecognised initial/index case [ ] . the appropriate use of ppe, including respiratory protection, and hand hygiene in routine care is critical to minimising pathogen transmission to staff and other patients; sub-optimal use exposes both hcws and patients to infection. compliance of hcws with wearing a protective mask may be related to their perception of risk and their risk-taking behaviours. the existing ipc literature primarily focuses on this topic in the context of sars or other pandemic respiratory diseases, with few papers investigating risk constructs for healthcare workers in routine care. the first and classic response to suboptimal behaviour is educative, with the provision of in-service and other training. we argue that the factors that lead to suboptimal use go far beyond knowledge and education, as well a subsequent national outbreak resulted in healthcare associated cases within the first month with over one fifth of these cases being hcws. one reason for so many hais has been attributed to sub-optimal use of routine protective equipment by hcws and the potential for infected hcws to act as vectors of infection [ ] . documented in other behaviours such as hand hygiene [ ] . interventions, and the research efforts used to generate evidence to support them, must take account of individuals' constructs and perceptions of risk and risk-taking behaviour. these perceptions are necessarily heterogeneous and vary between individuals and clinical settings. therefore, an understanding of the perceptions and behaviours regarding ppe use in different contexts is needed to inform successful behaviour change interventions [ ] . the importance and urgency of addressing suboptimal mask use by hcws is, in our view, based on a range of interconnected reasons all of which are critical to global health and security. these are as follows: the continuing burden of emerging infectious diseases for many centuries, since the age of the plague and smallpox epidemics to the th century outbreak of hiv/ aids, human infectious diseases of high consequence have presented a significant global public health challenge. these pandemics have resulted in deaths and disability of millions of people across the world, as well as causing social and economic disruption. despite improvements in communicable disease prevention and control, including effective sanitation, vector control, vaccines, and the international health regulations developed by the world health organization (who) [ ] , the new, emerging infectious diseases continue to threaten the well-being and economic stability of society and impose a significant burden on healthcare. although some infectious diseases, such as plague or smallpox, no longer present an active global pandemic threat, this century has seen both new and re-emerging infectious diseases give rise to widespread outbreaks. of particular current concern is re-assortment of rna in viruses such as influenza a which contributes to emerging pandemic influenza strains [ ] . furthermore, several zoonotic viral diseases that have infected humans through animal-to-human contact have also demonstrated human-to-human transmission, such as nipah virus [ ] . antecedents for the increasing burden of infectious diseases include a global population boom, changes in the use of land and environment, loss of wild life habitat, increased contact between wild and domestic animals and humans, the expansion in travel, an ageing population and developments in medical interventions. the latter two have led to an increase in the number of immunecompromised people who are susceptible to significant disease from emerging infections. many of these people attend, or are frequent inpatients of, healthcare facilities and therefore are at risk of healthcare-associated infections (hais). cheaper, easier and faster modes of travel, particularly by air, have enabled emerging infectious diseases to disperse more widely in short periods of time, than ever before. a clear example of this was sars, which spread from one "super-spreader" in a hotel in hong kong to numerous other countries via international guests who were infected, by contact, while staying in the same hotel [ ] . similarly a large outbreak of mers involving cases resulted from a single traveller returning to south korea from the middle east and attending several hospital emergency departments after he became unwell [ ] . the number of active outbreaks that are present around the world will vary on any given day; however at time of writing there were traveller alert notices for at least twelve different infectious diseases in more than countries [ ] and, on average, global infectious diseases emergencies are notified via the who each day [ ] . the use of protective respiratory masks has a human resource impact in healthcare organisations. clinicians are at a higher risk of acquiring influenza and other respiratory diseases than adults working in non-healthcare settings [ ] . sub-optimal protective mask use can increase this risk, which is exacerbated during high-risk periods such as the winter respiratory virus season. staff illness from respiratory infections has a direct impact on the workforce resulting in loss of productivity and associated economic burden within the healthcare setting, particularly with influenza [ ] . other respiratory viral diseases, such as the common cold, also contribute to a reduced work output [ e ] . productivity is affected if workers take leave to care for family members who are ill or children, because schools have been closed. although annual influenza vaccination is widely promoted as a means to reduce staff illness, average uptake by hcws is poor, unless is it mandatory. seasonal vaccine efficacy varies from year to year because of variable matching between vaccine and circulating strains, but is generally less than e % [ ] . even when hcw flu vaccine uptake is high the risk remains, because of vaccine mismatch with circulating strain, limited vaccine efficacy and/or mild or subclinical (but transmissible) infection in vaccinated subjects [ ] . consequently, hcws should still use respiratory protection when caring for patients with respiratory symptoms and/or patients at high risk of infection during outbreaks or high levels of respiratory infections in the community. not wearing a protective mask increases the risk of occupationally-acquired respiratory disease. hcw absenteeism due to influenza increases on average by two days per hcw, both during pandemic and a seasonal virus outbreaks [ ] . ip et al. [ ] examined overall sickness absences including sick leave due to acute respiratory infection (ari) for four distinct influenza periods between and including the influenza a(h n )pdm pandemic in hong kong. results showed that the daily hcw absenteeism rate for ari increased from the pre-pandemic in september a uk healthcare worker contracted monkeypox after caring for a patient with the disease prior to diagnosis. in a eurosurveillance report (add in ref) about the case, public health officials said that some hcws had been exposed as they were not wearing optimal personal protective equipment. baseline by . % and . % during the epidemic and pandemic periods respectively [ ] . similarly in canada, researchers demonstrated a significant increase in the rate of sick hours between the pre-influenza and / influenza period with only % of staff having zero sick hours productivity losses related to the common cold [ ] . a study examining the effect of influenza vaccination on emergency department workers' absentee rates reported that % of vaccinated and % of non-vaccinated workers required sick leave for influenza-like illness [ ] , although significant absenteeism during the h n influenza pandemic was not noted in the australian emergency workforce [ ] . staff illness compromises the quality and safety of patient care by loss of continuity of care through the requirement to employ agency staff in place of regular staff, who may be unfamiliar with the specialism of the clinical setting [ ] . staff absenteeism during outbreaks of emerging or high consequence infectious diseases, may also be due to hcws fear of acquiring the infection [ ] . similarly, presenteeism, or coming to work when ill, also results in a loss of productivity due to staff not working at full capacity [ ] . the health and safety of other staff are put at risk by hcws who continue to work while ill, while patient safety may be compromised through impaired clinical judgement. in a study undertaken in a children's hospital in philadelphia, ( %) of medical staff who were surveyed, reported that they would work with significant respiratory symptoms, despite acknowledging the infection risk to their co-workers and patients [ ] . in another study over % of us hcws who were surveyed worked with symptoms of influenza-like illness [ ] . whilst it is important to avoid presenteeism, it may be occasionally unavoidable e.g. because of significant or specialised staff shortages. if so, the risk may be mitigated by appropriate mask use. the hcws work capability may also be impaired by any physical and psychological consequences of wearing a mask, such as claustrophobia, respiratory distress, discomfort and skin irritation. the financial costs to society for respiratory infectious diseases can be significant. a us study estimated the annual economic burden of influenza, in , to be around us$ billion [ ] , while lost productivity due to influenza in france and germany was estimated at us$ e billion per year [ ] . sub-optimal mask use is likely to be associated with an increase in financial costs for individuals, the healthcare system and subsequently the wider society. although existing research has not examined the direct costs of not wearing a protective mask, van buynder et al. ( ) estimated the financial cost of hcws absenteeism due to influenza-like-illness to be greater than can$ million during the / winter season in a health district in british columbia [ ] . in addition, there are sick leave payments for staff and the costs incurred to replace them with casual staff. workers compensation fees may be driven up by hcws who take risks by not wearing masks. furthermore, there are significant monetary costs associated with patients acquiring a healthcare associated respiratory infection. the probability of a patient acquiring an influenza-like-illness increases when exposed to an infectious hcw, with one study reporting a relative risk of . when compared to no documented exposure [ ] . expenses for a hai include the overall cost of care for any additional inpatient bed days as a result of the infection, antiviral medication, other supportive therapy, radiology, laboratory and direct costs associated with the use of isolation and ppe measures. a korean study reported an average medical cost for a patient hospitalised with influenza in / was us$ . ae . [ ] . when a higher level of ipc measures is required e.g. mers or other emerging infectious disease, these costs can be excessive. veater et al. ( ) calculated an additional cost of pounds sterling per person per day, mainly due to staff time and ppe costs [ ] . third, sub-optimal mask use is associated with reductions in cost effectiveness of training methods in the use of ppe. effective training in ppe use is resource intensive and thus expensive to execute, whether delivered as demonstration learning by experts or technology-based education. inadequate training in ppe protocols is cited as one of the causes for poor compliance with ppe [ ] . these findings question the cost-effectiveness of current training methods. there is also a financial cost attached to the incorrect choice or unnecessary use of a mask, particularly in the case of the more expensive particulate respirator mask, or during a global outbreak event where stocks may be limited. the knowledge and skills of hcws are factors that affect protective mask use, therefore investigating how knowledge and cognition impacts on the hcw decision-making for mask use can inform the delivery of education and how policies are implemented. some of the aspects of knowledge related to mask use that may influence hcw behaviour include the source of knowledge, the indications for mask use, which type of mask to choose, how the mask functions to provide protection and how to put on and remove the mask safely. in the context of an emerging infection and limited available information, personal experience can influence hcws' perceptions of risk and behaviours related to protective mask use [ , ] . in contrast, a study undertaken in an outpatient paediatric setting, demonstrated that the use of ppe was not influenced by infectious risk perception [ ] . prior education and training will provide some of the essential information and skills required for optimal mask use but, in practice, routine training in the use of ppe is often cursory or non-existent. in a survey of healthcare workers in the us, % of doctors reported having received ppe training only as students (including clinical rotations) or not at all (c.f. % of nurses) [ ] . despite prior education, hcws may not apply their knowledge to the workplace [ ] . the method of training is therefore an important consideration for effective retention of knowledge and skills over time. several studies argue for improving the evaluation and training of hcws using ppe for infectious diseases and examining the effectiveness of various teaching approaches [ , ] . the recent ebola virus disease (evd) outbreak instigated intensive ppe training around the world, with a focus on donning and doffing protocols to maximise hcw safety. unsafe use of ppe has been blamed for some hcws becoming infected with evd or sars; subsequently several research studies have reviewed the effectiveness of different training techniques for the safe donning and doffing of ppe [ ] . these have included interactive online courses, and classroom teaching that incorporates fluorescent dye or harmless bacteriophages as surrogate markers of contamination [ ] . video-reflexive ethnography (vre) has been used as an interventional methodology to improve ipc practices [ ] . this method allows the hcw to view video footage of themselves making decisions around and subsequently using protective masks in every-day complex work. the clinicians can then reflect on their behaviour and suggest ways in which their own and colleagues' mask use can be optimised. although the techniques taught for donning and doffing protective masks as part of routine ppe are generally heterogeneous around the world, there are variations in mask design which may affect skills. there is also a lack of standardisation between and within institutions as to which clinical indications warrant a n or surgical mask. within society in general, individuals are not only motivated to protect themselves from infectious disease but often demonstrate a moral responsibility to protect others if they themselves are infectious [ ] . during periods of high-risk for respiratory infectious disease, such as the annual influenza season or a novel influenza pandemic, health departments have, and may, encourage or mandate the use of a protective respiratory mask by the general public to minimise the transmission from symptomatic people to others [ ] . in healthcare facility waiting rooms it is recommended that symptomatic patients be given a respiratory mask to wear to protect others as part of respiratory hygiene [ ] . this social behaviour may alter the perception of risk for staff towards mask use in two ways, particularly in the emergency department. firstly, hcws may take a view that it is the patient's, not their own, responsibility to abide by these infection prevention measures and purposefully choose not to wear a mask on the basis of responsibility. secondly, they may not perceive a risk of becoming infected if a patient is wearing a mask and so will not use one. there are several risks for hcws adopting this behaviour. the patient may not wear the mask correctly or remove it at any time, especially if they are kept waiting for long periods, thus exposing other patients and hcws. additionally, the patient may not be able to tolerate a mask for long if unwell and will then remove it. clinical examination may put the hcw at higher risk of exposure, even if the patient is wearing the mask correctly when they enter the room or cubicle and certain procedures, such as taking a swab for influenza testing, collection of an induced sputum specimen or intubation, require removal of the patient's mask. if an hcw fails to adequately explain why they are wearing a mask it can erect a social barrier between the hcw and patient. patients may feel stigmatised if staff wear a mask to care for them [ ] while staff may feel that wearing a mask in the ed can inhibit empathy and rapport with a sick patient [ ] . hcws working in paediatric units have expressed concern that ppe may frighten their patients [ ] . social interactions within the workplace can influence the health-related behaviour of workers. the safety climate and group norms at hospital unit level have been shown to influence the risk-taking behaviour associated with facial protective equipment [ ] . the use of protective masks in the healthcare setting is governed locally by policies in health and safety and ipc. as indicated earlier, adherence to such policies and guidelines is often poor. similar to other types of ppe and ipc measures, there is no strong culture of enforcement of policy relating to protective masks in the healthcare setting. this raises questions about the efficacy of mask policies, their awareness by hcws and how they are judged by clinical staff. in some countries, state-wide legislation mandates the use of a protective mask for various categories of clinical staff during the annual influenza season, if they have not received the influenza vaccination [ ] . this enforced measure has been resisted by some clinical staff because of its impact on personal choice [ ] and by others as illogical when considering the risk from all respiratory pathogens [ ] . although many countries provide national occupational safety and health policy direction, few enforce protective mask use in healthcare settings. nevertheless, sub-optimal mask use reinforces poor behaviour in the workplace and contravenes workforce health and safety responsibilities of employees [ ] . the behaviours of hcws towards protective mask use can affect the progression of a respiratory infectious disease outbreak and, if inappropriate, facilitate a pandemic. the consequences of a pandemic on a global scale are significant, with substantial negative societal effects. ease of access to international travel has been a significant factor in the worldwide spread of recent pandemics such as pandemic influenza a h n , sars and mers, therefore international travel and trade are often restricted [ ] . personal freedom of movement is also affected by public health quarantine measures and the prohibition of public gatherings. education is disrupted through school closures which results in parents taking time off work as a consequence. in addition to the consequences described above, the provision of healthcare to the general population can be disrupted. in , the influenza a h n pandemic impacted severely on the normal functioning of emergency departments in australia [ ] . more than three times the number of patients were seen, most with non-serious influenza symptoms. staff reported that heavy workloads, lack of infection control facilities and distraction from their core business compromised the care of non-flu patients. large numbers of patients requiring care will lead to bed shortages and hospital admission gridlock, probable loss of critical care beds which are blocked with long stay respiratory patients and the cancellation of routine surgical lists [ ] . furthermore, there will be fewer hcws available to provide the care due to their own illness or having to look after family members. in this paper we detail why we need to know more about hcws' decision-making and risk-taking behaviour in relation the use of masks for protection against infectious respiratory diseases. we argue that the value of such research would be its potential impact on the ongoing threat of emerging infectious diseases, workforce capability, capacity and educational needs, the financial costs of healthcare and outbreaks and the importance of social responsibility and appropriate legislation in planning for global security. specifically, research is required to determine whether hcws' perception of risk as it relates to the protection of themselves and others against transmission of infection influences their behaviour towards the use of a protective mask. there is also a need to determine the personal, professional and contextual factors that impact on hcws' perceptions of risk and their use of protective masks for infectious diseases. an exploration of the practices and constructs of risk by hcws will therefore provide valuable information to inform policy and pandemic planning. the sub-optimal use by hcws of protective masks for respiratory diseases has a significant impact at individual, organisational, societal and global levels. furthermore, the consequences of poor mask use will be exacerbated during a widespread outbreak or pandemic of a novel infectious respiratory disease, when pharmacological agents or vaccination are unavailable. minimising the transmission of respiratory disease through protective mask use leads to better outcomes for healthcare, workforce capability and economic stability. this paper has presented the background and justification for research into the attitudes and behaviour of hcws towards protective mask use for respiratory infectious diseases during non-outbreak situations so as to optimise the use of masks when indicated in every day practice. the research can provide insight into perceptions of risk and risk-taking behaviour in respect of mask use for respiratory infectious diseases and help to bridge the gap between theory and practice (see table ). rb and rs originated the concept for the paper and rb drafted the manuscript. glg and rs had critical review and input into the preparation of the manuscript. all authors approved the final version of the manuscript. rzs is a senior editor and glg a section editor of infection, disease and health but neither had a role in peer review or editorial decision-making of the manuscript. the authors declare no other conflict of interest. this work is supported by the australian partnership for preparedness research on infectious diseases emergencies (apprise) of which author glg is a chief investigator and author rb is recipient of a doctoral scholarship. this research presented in this article is solely the responsibility of the authors and does not reflect the views of apprise. not commissioned; externally peer reviewed. ethics approval is not required as this is a discussion paper. table tribute to the mask. there was a sick traveller in bed who had an airborne infection to spread the staff did their tasks, but didn't wear masks, and now many people are dead! development of framework for assessing influenza virus pandemic risk standard precautions but no standard adherence health care workers' perceptions predicts uptake of personal protective equipment australian guidelines for the prevention and control of infection in healthcare. national health and medical research council middle east respiratory syndrome coronavirus transmission among health care workers: implication for infection control evaluation of respiratory protection programs and practices in california hospitals during the e h n influenza pandemic scope and extent of healthcare-associated middle east respiratory syndrome coronavirus transmission during two contemporaneous outbreaks in riyadh, saudi arabia applying psychological frameworks of behaviour change to improve healthcare worker hand hygiene: a systematic review the behaviour change wheel: a new method for characterising and designing behaviour change interventions world health organisation. international health regulations rna virus reassortment: an evolutionary mechanism for host jumps and immune evasion in-depth assessment of an outbreak of nipah encephalitis with person-to-person transmission in bangladesh: implications for prevention and control strategies responding to global infectious disease outbreaks: lessons from sars on the role of risk perception, communication and management mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study travel health notices j travelers' health j cdc flu and ebola map j virus and contagious disease surveillance incidence of influenza in healthy adults and healthcare workers: a systematic review and meta-analysis annual burden of 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prevent pertussis exposures in a pediatric primary care network are health care personnel trained in correct use of personal protective equipment? healthcare workers' decision-making about transmission-based infection control precautions is improved by a guidance summary card personal protective equipment processes and rationale for the nebraska biocontainment unit during the activations for ebola virus disease the individual, environmental, and organizational factors that influence nurses' use of facial protection to prevent occupational transmission of communicable respiratory illness in acute care hospitals risk of self-contamination during doffing of personal protective equipment human factors risk analyses of a doffing protocol for ebola-level personal protective equipment: mapping errors to contamination beyond hand hygiene: a qualitative study of the everyday work of preventing cross-contamination on hospital wards prevalence of preventive behaviors and associated factors during early phase of the h n influenza epidemic clinical excellence commission. infection prevention and control practice handbook. principles for nsw public health organisations behind barriers: patients' perceptions of source isolation for methicillin-resistant staphylococcus aureus (mrsa) perceived barriers to adherence to tuberculosis infection control measures among health care workers in the dominican republic the cookie monster muffler": perceptions and behaviours of hospital healthcare workers around the use of masks and respirators in the hospital setting hospital unit safety climate: relationship with nurses' adherence to recommended use of facial protective equipment nurses' attitudes towards enforced measures to increase influenza vaccination: a qualitative study influenza vaccination of healthcare workers: critical analysis of the evidence for patient benefit underpinning policies of enforcement australian government. workplace health and safety domestic politics and the who's international health regulations: explaining the use of trade and travel barriers during disease outbreaks pandemic (h n ) influenza and australian emergency departments: implications for policy, practice and pandemic preparedness the practical experience of managing the h n influenza pandemic in australian and new zealand intensive care units key: cord- - jv em authors: alegbeleye, oluwadara oluwaseun; singleton, ian; sant’ana, anderson s. title: sources and contamination routes of microbial pathogens to fresh produce during field cultivation: a review date: - - journal: food microbiol doi: . /j.fm. . . sha: doc_id: cord_uid: jv em foodborne illness resulting from the consumption of contaminated fresh produce is a common phenomenon and has severe effects on human health together with severe economic and social impacts. the implications of foodborne diseases associated with fresh produce have urged research into the numerous ways and mechanisms through which pathogens may gain access to produce, thereby compromising microbiological safety. this review provides a background on the various sources and pathways through which pathogenic bacteria contaminate fresh produce; the survival and proliferation of pathogens on fresh produce while growing and potential methods to reduce microbial contamination before harvest. some of the established bacterial contamination sources include contaminated manure, irrigation water, soil, livestock/ wildlife, and numerous factors influence the incidence, fate, transport, survival and proliferation of pathogens in the wide variety of sources where they are found. once pathogenic bacteria have been introduced into the growing environment, they can colonize and persist on fresh produce using a variety of mechanisms. overall, microbiological hazards are significant; therefore, ways to reduce sources of contamination and a deeper understanding of pathogen survival and growth on fresh produce in the field are required to reduce risk to human health and the associated economic consequences. foodborne diseases are rife in many regions of the world, with at least in people falling ill yearly from consumption of contaminated food and , deaths occurring as a result, according to the world health organisation (who) ( ) . foodborne diseases have exerted pressure on medical services, contributed to economic and political distress, exacerbated malnutrition and led to human suffering. there are several agents such as chemicals, pathogens, and parasites, which may adulterate food at different points in the food production and preparation process (allos et al., ) . many of these agents have been extensively characterized and investigated by numerous studies (farber and peterkin, ; zhao et al., ; le loir et al., ; ehling-schulz et al., ; adzitey et al., ; botana, ) . strategies and protocols to prevent occurrence (and outbreak) of foodborne diseases have been devised and implemented by many researchers, regulatory bodies, and governments. however, despite the considerable progress achieved scientifically, foodborne diseases continue to occur, representing a significant cause of morbidity and mortality globally (mead et al., ; murray et al., ) . although foodborne diseases are more common in developing countries particularly in africa and south east asia with specific groups of people such as children, the immunocompromised, pregnant and aged being particularly at risk, foodborne diseases are not limited to these regions or groups of people (who, ) . for instance, according to the centres for disease control and prevention (cdc), between and , there were . million episodes of domestically acquired foodborne gastroenteritis caused by unspecified agents in the united states alone (cdc, ) . approximately . million acute gastroenteritis occurred, and there were at least , hospitalizations in the us each year and hospitalizations caused by the known gastroenteritis pathogens. an estimated persons died of acute gastroenteritis each year, of which deaths were caused by the known foodborne pathogens (scallan et al., ) . health canada ( ) estimates that e million cases of foodborne illnesses occur in canada every year. although the conventional notion is that foodborne diseases typically originate from meat and poultry products, vegetables and fruits have been implicated in various foodborne outbreaks (westrell et al., ; lynch et al., ; [european food safety authority (efsa), ] . a significant increase in foodborne disease outbreaks or cases associated with consumption of fresh produce has been reported. this increase has been largely due to a general increase in produce consumption, globalization of the produce industry and more effective surveillance (tauxe et al., ; lederberg et al., ; havelaar et al., ) . increased consumption of fresh produce is likely due to global government efforts to promote healthy eating, the associated health-promoting benefits of consuming fresh produce and ease of access to fresh local produce (pollack, ; regmi, ; berger et al., ; painter, ) . since fresh produce is mostly eaten raw or after minimal processing, pathogen contamination constitutes a potential health risk (callej on et al., ; li et al., ) . there are numerous factors capable of compromising the microbiological integrity of produce along the farm to fork continuum, all of which have potentially fatal outcomes. however, pre-harvest hazards to produce have been recognized as important because usually, once pathogen contamination is established in the field, it can be challenging to decontaminate produce. there are numerous circumstances that can undermine the safety of produce on farms. many of these arise because agriculture has grown more intensive over the years, and produce fields are often located near animal production zones thus entwining the ecological connections between wild animals, livestock and produce (strawn et al., a,b) . this, in many cases, predisposes fruits and vegetables to pre-harvest hazards. some important pre-harvest hazard sources to produce include the use of contaminated soil, irrigation water and manure for produce cultivation. wild animals and insects have also been implicated as vehicles of pathogens to produce. to ensure produce safety on a sustainable scale, it is imperative to correctly understand the routes of entry, fate, transport, establishment, and survival of pathogens in the agricultural environment such as soil, irrigation water and manure. the knowledge gap in this regard is being filled rapidly, as many studies have attempted to explain the behavior of foodborne pathogens in agricultural media and describe the associations among pathogens, produce and the agrarian environment. in this review, the extent of the produce contamination problem is discussed as well as the sources and routes of contamination of soil, irrigation water, fruits, and vegetables. also, the various mechanisms and strategies through which bacterial pathogens become established on fruits and vegetables are briefly examined. the nutritional and health benefits of consuming fruits and vegetables have been recognized and widely publicized. this has elicited changes in human dietary habits, with many consumers incorporating more fruits and vegetables into their meals. consequently, the global production of fruits and vegetables has surged exponentially in recent decades. the increased demand for produce has led to modifications such as increased use of soil amendments, utilization of alternative water sources and increased imports and exports in agriculture-spanning across agronomic practices, processing, preservation, packaging, distribution, and marketing . some of these modifications, however, have great potential to compromise the safety of fruits and vegetables. the biological hazards that are most relevant to fresh produce safety are either zoonotic or human in origin and can be classified into sporeforming bacteria, non-spore forming bacteria, viruses, parasites and prions (james, ) . most studies/surveillance efforts have identified bacterial contaminants in produce-borne illness outbreaks. there is, therefore, a disproportionately higher abundance of information regarding bacterial contamination in the literature. this may be because bacterial species have in fact caused many more outbreaks, but other microbial groups-viruses and parasites have been understudied. the most commonly implicated etiologic agents are presented in table . although data and information available on outbreaks associated with fresh produce are diverse and patchy, the available research evidence indicates that the foodborne illness burden due to contaminated produce has increased, in recent decades. in the united states, between and , approximately % of total foodborne illness outbreaks were produce related (jung et al., ) . in europe, from to , produce was linked with % of the outbreaks, % of the hospitalizations and % of the deaths (efsa, ) . in australia, fresh produce was linked to % of all foodborne disease outbreaks informed from to (lynch et al., . specific produce items are more commonly linked to foodborne illness incidents; for example, leafy greens such as lettuce and spinach, as well as fresh herbs such as parsley and basil are conventional sources of bacterial infections (who, ; berger et al., ; denis et al., ) . berries, green onions, melons, sprouted seeds, and tomatoes are similarly high-level priority produce items (olaimat and holley, ; denis et al., ) . in the us, between and , of multistate foodborne outbreaks were associated with vegetables (cdc, ) . a list of recent produce-related outbreaks is presented in table . most industrialized nations especially the united states have extensive and exhaustive datasets indicating the magnitude of outbreaks, the extent of severity and casualties incurred, the implicated pathogen and produce item as well as documented preventive protocols to avoid future outbreaks. unfortunately, however, the same is not true of many other countries especially african countries, the majority of which are still grappling with other challenges and hence, lack the resources to efficiently track and trace foodborne illness incidents (who, ) . many conventional foodborne detection methods are time consuming and laborious, and advanced techniques have therefore been developed and optimized as alternatives to or for use in combination with these traditional techniques. many of these are rapid, sensitive, reliable and standardized. they can be categorized into nucleic acid based, biosensor-based and immunological based methods (croci et al., ; adzitey et al., ; law et al., ) . typical examples include simple polymerase chain reaction (pcr), multiplex pcr, real-time pcr, nucleic acid sequence-based amplification (nasba), loop-mediated isothermal amplification (lamp) and oligonucleotide dna microarray. other examples are optical, electrochemical and mass-based biosensors, and enzyme-linked immunosorbent assay (elisa) and lateral flow immunoassay (law et al., ; gilchrist et al., ) . these advances in epidemiological investigation approaches and techniques have made it possible to explore the crucial associations between produce and pathogens. in spite of this, however, prompt identification of implicated produce vehicles, location or point of contamination in fresh produce associated outbreaks is still a significant challenge. one prime constraint is the relatively short shelf life of fresh produce, which is often discarded by the time an outbreak is identified (strausbaugh and herwaldt, ; lynch et al., ) . therefore, most of the time, the real source of contamination is not ascertained causing investigators to speculate or assume a source. this is, however, dangerous because, in addition to the possibility of being wrong, there is empirical evidence that once a particular transmission pathway is identified, repeated investigations are bound to be biased in causation (lynch et al., ) . another important consideration is that usually, outbreaks receive widespread attention if the event (i) has severe public health impacts (ii) is unusual or sudden (in that the etiological agent and/produce type are unanticipated; making the circumstances of the outbreak unique and (iii) poses a significant risk of international spread with consequences for international travel or trade. invariably, the smaller, 'less significant' outbreaks are never investigated. more importantly, foodborne illness incidents occur sporadically in populations, and these cannot be captured in routine epidemiological surveillance or outbreak investigations (scallan et al., ) . this means that the data available may not be a valid representation of the problem. it is likely that the foodborne illness burden related to consumption of contaminated produce is still largely underestimated. the possible routes and sources of produce contamination are numerous, and intensive efforts have been made to accurately understand the exact mechanisms through which pathogens are introduced into fresh produce (kotzekidou, ) . sources and routes of produce contamination vary for different production zones. this is because each farm has a distinct combination of environmental risk factors such as topography, land-use interactions, and climate. combinations of these peculiar environmental risk factors influence the frequency and transmission of foodborne pathogens and subsequently impact the risk of produce contamination (strawn et al., b) . primarily, pathogens may contaminate produce 'on-field' via various routes including; atmospheric deposition, uptake from contaminated soils and groundwater (harris et al., ; lynch et al., ; mei soon et al., ) , use of raw (or poorly treated) manure and compost, exposure to contaminated water (irrigation or flooding), transfer by insects, or by fecal contamination generated by livestock or wild table the most commonly implicated etiological agents in fresh produce borne illnesses (brackett, ; buck et al., ; heaton and jones, ; jung et al., ; callej on et al., ) . animals (cooley et al., ; uyttendaele et al., ) . a schematic representation of the main entry points for pathogens to humans via produce is provided in fig. . the use of organic materials such as livestock excreta, slurries, abattoir wastes, sewage sludge as well as municipal and industrial waste treatment residuals as soil amendments is widespread goss et al., ) . although these serve as a costeffective source of nutrients for agricultural purposes, research demonstrates that raw manure as well as contaminated (or improperly treated) manure constitute a significant risk of pathogenic contamination for produce (james, ; manyi-loh et al., ) . public health relevant bacteria, viruses and parasites such as e. coli o :h , salmonella spp., l. monocytogenes, campylobacter spp., porcine enteroviruses, bovine coronavirus, bovine virus diarrhoea cryptosporidium parvum and giardia have been isolated from raw/poorly treated manure (derbyshire, ; derbyshire and brown, ; sellers, ; strauch, ; pell, ; grewal et al., ) . pathogens may be spread through direct interaction of vegetable surfaces with manure, or by splashing of (contaminated) soil/manure particles from the soil on vegetables via rainfall and/ overhead irrigation or by vectors. additionally, manure piles stored next to growing areas may constitute contamination risk due to run-off (james, ; warriner et al., ) . manure application could be by broadcasting as a solid, semisolid or liquid throughout the field or by the introduction of livestock or wildlife feces at distinct locations (jung et al., ) . in many parts of the world, organic cultivation systems use more manure than conventional growers, and chemical treatment against pathogens is prohibited in organic farming. there have thus been some assertions that organic produce represents a more significant safety risk than its non-organic counterpart, although, there is no unequivocal research evidence supporting this claim loncarevic et al., ; warriner et al., ; ivey, ; maffei et al., ) . the survival of pathogens in manure and biosolids depends on factors such as the manure source, production process, and characteristics, treatment technique applied, physicochemical factors like ph and relative humidity, incidence of antagonists or predators, weather conditions, desiccation, aeration, soil type, degree of manure incorporation, amongst others (ingham et al., ; wood, ) (table ) . the manure composition, which is determined in large part, by the feed formulation, dictates the profile of pathogens occurring in manure as well as their ability to persist even posttreatment (franz et al., ) . certain workers have proposed that cattle diet may influence the incidence of representative bacterial species; e. coli o :h and salmonella in manure. these pathogens have been reported to persist longer in manure obtained from cattle fed diets rich in energy but low in fiber content such as high digestible grass silage and maize silage compared to animals that received diets with low energy and higher fiber content such as straw . it has also been suggested that feeding cattle with hay may significantly reduce shedding of acid-resistant e. coli (diez-gonzalez et al., ; . how effective these strategies are in reducing pathogen load in (animal-derived) manure, is however not clear. manure treatment techniques such as composting, aerobic and anaerobic digestion, pelleting, alkaline stabilization, conditioning, dewatering and heat drying have been used to treat manure before application as fertilizer for a long time. while many of them are reasonably efficient, concerns have been raised about their ability to satisfactorily eliminate pathogenic bacteria (day and funk, ; lu et al., ; lorin et al., ) . tailing of pathogen inactivation curves, as well as apparent regrowth or recontamination of bacteria after treatment, have been reported. many pathogens have been shown to be capable of withstanding manure treatment processes, thereby, constituting a major risk of contamination (brackett, ) . composting is a popular manure treatment and composting temperatures that exceed c for three days are considered sufficient to kill most pathogens (grewal et al., ) . however, few studies have demonstrated that the heat-induced death of bacteria in composted materials is a complex phenomenon (ingham et al., ; gupta, ) . bacterial regrowth and recontamination in cooled compost have been reported (hassen et al., ; ingham et al., ) . pelletizing is another common treatment available and is commonly applied to chicken manure (chicken manure pellets). pelletizing the manure reduces the off-odor and facilitates transport and storage. although the process usually involves a thermal procedure, more studies are required to validate whether the process efficiently inactivates clinically relevant pathogens (chen and jiang, ; jung et al., ) . the use of a fish emulsion as fertilizer has raised similar concerns; although most preparation methods available include a thermal process, the ability of this to inactivate enteric bacteria and viruses needs to be rigorously validated (jung et al., ) . due to the diverse range of variables associated with manure composition, treatment, pre-application storage, application and incorporation, regulatory bodies have stipulated minimum manureto-harvest time intervals necessary to ensure microbiological safety. the united states department of agriculture (usda) 'organic production and handling' specifies that unless composted, raw animal manure must be incorporated into the soil not less than days prior to harvest of a product whose edible portion has direct contact with the soil surface or soil particles, or days if there is no direct contact (usda, ) . canadian authorities specify , and months for tree fruits and grapes, small fruits and vegetables respectively as the minimum time delay between manure application and harvest for these crops (olaimat and holley, ) . irrigation water has been identified as a potential source of produce contamination (benjamin et al., ; uyttendaele et al., ; faour-klingbeil et al., ) . being a common and essential requirement for crop production, water must be supplied to plants when necessary, and irrigation water sources are used to supplement limited rainfall in many areas (kirby et al., ) . epidemiological investigations of food poisoning outbreaks, experimental studies examining pathogen contamination of fruits and vegetables as well as observations of increased incidence of disease in areas practicing wastewater irrigation with little or no wastewater treatment indicate that contaminated irrigation water might indeed be a source of foodborne pathogens on fresh produce (norman and kabler, ; hern andez et al., ; steele and odumeru, ) . for example, hepatitis a outbreaks associated with lettuce (seymour and appleton, ) and spring onions (josefson, ) were linked to sewage-contaminated irrigation water (heaton and jones, ) . various factors including irrigation regime (method and timing of irrigation), irrigation water sources, type of crop and land use practices in the farm influence the extent and frequency of pathogenic contamination of produce ( such as pathogen concentration, pathogen strain, weather patterns, plant state, and physiology also have significant implications for produce safety (marvasi et al., ; uyttendaele et al., ; decol et al., ) (table ) . there are several types of irrigation systems available, each of which is typically complex and has its own drawbacks. most irrigation systems create complicated ecological environments with multiple potential sources and routes of pathogenic contamination (pachepsky et al., ) . each irrigation subsystem: collection, replenishment, storage, conveyance, distribution off and on-farm, as well as on-farm application involve processes that have great potential to compromise the microbiological integrity of the irrigation water in unique ways. during transportation from the source to the field, water is susceptible to significant microbiological depreciation (pachepsky et al., ) . the prevailing deterioration dynamic will depend on the transportation mode. for instance, irrigation water transport via irrigation ditches and canals involves interaction with microbial reservoirs of bottom sediments, bank soils, algae and periphyton, whereas water transport via pipes involves interactions with biofilms in the transport pipes pachepsky et al., ) . this sort of contamination is particularly prominent in reclaimed water distribution systems . the method of storage for irrigation water can have a profound effect on pathogen transmission. for example, certain studies have demonstrated that water quality is rapidly degraded in storage ponds and tanks due to inputs from avian species or other wildlife (field and samadpour, ; mclain and williams, ; higgins et al., ) . other storage systems such as check dams, impoundments, inter-basin transfer schemes, abstraction schemes and reservoirs have been identified as places where indicator and pathogenic microorganisms can survive and proliferate (abbasi, ; kirubel, ) . the mode of application also has significant impacts on the risk of microbiological contamination (berger et al., ) . compared with furrow and subsurface drip irrigation systems, sprinkler irrigation poses a higher risk of microbiological contamination (kisluk and yaron, ; pachepsky et al., ) . surface furrow and drip irrigation systems minimize contact between edible portions of certain plants (leafy vegetables provide larger surface area for contact and possible microbial attachment) and irrigation water (directorate, ; fonseca et al., ; mei soon et al., ; uyttendaele et al., ) . hydroponic growing systems also offer this advantage (jung et al., ; allende and monaghan, ) . the irrigation application method has been determined to influence the internalization of some pathogens in produce such as spinach plants. according to some studies, the likelihood of internalizing pathogens increases when the organisms are introduced by water sprinkling systems as opposed to when the water is directly applied to the soil (solomon et al., ; stine et al., ; mitra et al., ; warriner et al., ; erickson et al., a; kisluk and yaron, ; zheng et al., ) . more details on pathogen internalization are provided in section (below). depending on the geographical location, the irrigation regime with respect to time of day, season and harvest time may influence the likelihood of pathogenic contamination. for example, kisluk and yaron ( ) in a study conducted in haifa, israel demonstrated that night-time irrigation and irrigation during the winter season is more likely to contaminate plants with enteric bacteria. contaminated irrigation water poses the most significant risk when crops are irrigated close to harvest time, because harvesting of produce containing viable pathogens is more likely. therefore, an adequate time interval between irrigation and harvest should be conscientiously followed. the microbial quality of irrigation water depends mostly on the source of the water. in order of increasing risk of microbial contamination hazard, irrigation water sources can be ranked as follows: potable or rainwater, deep groundwater, shallow groundwater, wells, surface water and raw or inadequately treated wastewater (james, ; pachepsky et al., ) . the microbial quality of rainwater or rain-harvested water is relatively good. the quality and safety of use, however, depends largely on the collection, transportation and storage means. this can be illustrated with roof-harvested rainwater, which may become contaminated with pathogenic bacteria and protozoan parasites because of the occurrence of animal droppings on roofs, particularly immediately after relatively long periods of drought . groundwater (or borehole water) is usually microbiologically safe, except if it has been contaminated with surface runoff or other sources of contamination close to the aquifer. certain farm operations such as intensive dairying and border-strip irrigation (a type of surface irrigation, which is a hybrid of level basin and furrow irrigation) (valipour et al., ) lead to leaching of pathogens such as e. coli and campylobacter to shallow groundwater, thereby contaminating it (close et al., ) . water from wells that are free from leaks and have sound casing are expected to be microbiologically safe. factors such as the design of wells, nature of the substrata, depth to groundwater and rainfall may affect the microbial quality of good water (james, ; gerba, ) . surface waters; which are the predominant source of irrigation waters in many countries, including open canals, ponds, lakes, rivers and streams are much more susceptible to pathogenic contamination compared to groundwater (allende and monaghan, ; uyttendaele et al., ) . sewage discharges, septic tank contamination, storm drains, wild and livestock defecation, run-off from contaminated fields, industrial and municipal effluents can all potentially contaminate surface waters (steele and odumeru, ; james, ) . wastewater is usually of poor chemical and microbiological quality. therefore, it requires extensive treatment before it can be safely used to irrigate crops. water sources (other than rain) used to irrigate produce is usually only minimally treated or untreated in many cases (steele and odumeru, ; jung et al., ) . it is expensive and time-consuming to treat irrigation water up to drinking water standards, which is the ideal recommendation (crook and surampalli, ; forslund et al., ) . although awareness of the potential dangers of using microbiologically compromised water for irrigation has increased in recent times, scarcity of water resources in certain regions has contributed enormously to the use of sub-optimal supplementary irrigation water sources. in such cases, irrigation water represents a greater microbiological risk to produce (sundstr€ om et al., ) . one of the most frequent pathogens implicated in water-related outbreaks is e. coli o :h (cdc, ; hilborn et al., ) . the organism can survive for a protracted period in water (even in deionized water) depending on temperature conditions (chalmers et al., ; islam et al., a) . it also exhibits a remarkable ability to withstand extreme environmental conditions such as high acidity and extremely low-temperature conditions. the ability of a pathogen to survive (or persist) in the environment (and on produce) is an essential determinant in the risk of human infection. the actual risks associated with pathogens occurring in irrigation water depend on numerous variables including environmental conditions such as temperature, ph and uv light (sant'ana et al., ) . other factors such as the excreted load of the pathogen, its latency period before it becomes infectious, its ability to efficiently multiply outside a mammalian host, its infectious dose for humans, inhibitory competition from the indigenous microflora as well as host response also play a relevant role (steele and odumeru, ) . bacteria and viruses survive for lengthier periods in groundwater compared to surface water because groundwater tends to be cooler, offers protection from sunlight, and has less biological activity (steele and odumeru, ) . these groups of microbes only typically last no longer than and days in surface water and sewage, respectively. conversely, parasites (eggs/cysts) may survive for as long as days or even several months in surface water and wastewater (lefler and kott, ; sagik et al., ; bihn, ) . this suggests that pathogenic microorganisms are capable of surviving for extended periods, which constitutes a profound threat to produce safety. regardless of the source or route of exposure, one potentially fatal consequence of pathogen contamination of irrigation water is the repeated inoculation of plants with the pathogens. the fate and transport of these pathogens once introduced into the produce vary widely (table ). some pathogens are capable of adhesion to surfaces of produce while some others can rapidly internalize into plant tissues under certain conditions, translocate and persist until consumed (wariner et al., ; bernstein et al., a; doyle and erickson, ) . this has rendered many conventional processing and chemical sanitizing methods ineffectual (hong and moorman, ) and is a growing public health concern. although the potential for produce contamination via irrigation water has been identified, it is difficult to estimate the magnitude of the problem (groves et al., ; antwi-agyei et al., ) . despite the fact that numerous studies have linked poor microbiological quality of irrigation water with the incidence of human pathogens on fruits and vegetables, direct evidence of irrigation water causing foodborne disease is relatively rare (harris et al., ) . this is because a substantive "cause-effect" relationship is yet to be established as it is required that the same pathogenic strain is isolated from the patient, produce, and irrigation sources (pachepsky et al., ) . furthermore, there must be a clear sequence of events connecting patient, produce, and irrigation source (steele and odumeru, ) . this is difficult to achieve due to certain limitations such as an inability to promptly identify the locations associated with produce contamination and delays inherent in foodborne outbreak investigations (pachepsky et al., ) . in the absence of direct confirmation, the "cause-effect" relationship can only be deduced based on circumstantial or subjective evidence (pachepsky et al., ) . also, it is apparent that there is no valid link between detected pathogen levels in irrigation waters and disease risk. some studies have demonstrated a lack of correlation between pathogen prevalence in waters used for irrigation and disease incidence due to consumption of irrigated produce (cooley et al., ; mcegan et al., mcegan et al., , . there is an abundance of laboratory studies elucidating potential mechanisms of produce contamination from waterborne pathogens. however, field studies showing the exact process of produce contamination via this medium are relatively scarce. it is thus expedient to generate more field data in this regard. soils typically harbor an abundant consortium of microorganisms, some of which are human pathogens such as b. cereus, clostridium botulinum, c. perfringens, listeria monocytogenes and aeromonas (nicholson et al., ; warriner et al., ; jay, ) . they may, therefore, serve as a medium of plant contamination through seeds, roots or surfaces. many soil resident pathogens have adapted to survival in soil with spores persisting indefinitely. however, since many agricultural soils are predisposed to point and nonpoint sources of pathogenic contamination, allochthonous pathogens may continuously be introduced into soil environments (santamaria and toranzos, ) . some of the primary sources of pathogens into soil include the use of contaminated irrigation water and manure, animal grazing, municipal solid wastes and other effluents (santamaria and toranzos, ; sant'ana et al., ) . the fate, survival and recalcitrance of pathogens in soil depend on factors such as soil type, soil moisture, ph, temperature, nutrient availability, agronomic practices, as well as soil biological interactions (table ) . soil matric potential (moisture levels) is determined by soil properties and water inputs through precipitation and/irrigation and has been demonstrated to be one of the most critical factors influencing microbial transport and survival in soil . cool, moist environments are favorable for the survival of bacteria and viruses. under dry soil conditions, a reduction in bacterial and viral population densities are usually observed (santamaria and toranzos, ; ghorbani et al., ) . escherichia coli survival has been reported to be highest in organic soils under flooded conditions, and peak populations recorded after a rise in the water-table accompanying significant rainfall events (tate, ; rochelle-newall et al., ) . some pathogens such as streptococcus faecalis have been proven to thrive poorly under low soil moisture conditions (kibbey et al., ; jamieson et al., ; cabral, ) . increased rates of virus inactivation at low soil moisture levels have been demonstrated (yeager & o'brien, ) . also, decreased recovery of viral (poliovirus type and coxsackievirus b ) infectivity in dried soils was attributed to evaporation of soil water in the same study by yeager & o'brien ( ) . in addition, experimentation by hurst et al. ( ) correlated inactivation of enteroviruses [echovirus type (strain wallace), coxsackievirus b (strain nancy) and poliovirus type (strain lsc)] in sludge-amended soils with moisture loss in the sludge piles. soil ph influences microbial diversity and the biogeochemical processes, which they mediate (fierer and jackson, ; nicol et al., ) . optimum ph for bacterial survival seems to be neutral, but fungi are known to be more tolerant of acidic conditions, compared to bacteria (leahy and colwell, ) . amino acids (most viruses behave as proteins) have different pk values and so the ratio of positive to negative charges on proteins vary with ph (yates et al., ) . in an experiment that lasted days using poliovirus type , echovirus , echovirus and coxsackie b , viruses were detected up till the th e th day at ph . while at ph . , the viruses died off between the th and th day depending on virus type (bagdasaryan, ) . soil types vary depending on organic matter content, water release characteristics, particle size distribution and moisture retention capacity. these variations significantly influence the survival of enteric pathogens in soil atkinson et al., ) . clay soils support the adsorption of microorganisms onto soil particles, and this reduces microbial die-off rates (reddy et al., ) . clays protect bacterial cells, and possibly viral particles, by creating a barrier against microbial predators and parasites (santamaria and toranzos, ) . viruses, which are mostly large proteins possessing various charges, are capable of forming numerous bonds with clay minerals (stotzky, ) . for example, the survival of e. coli is prolonged in clay soils where adsorption of cells to the soil particles protects it against protozoa (mosaddeghi et al., ) . escherichia coli can persist for up to weeks in clay and loam soils, but for much less ( weeks) in sandy soils (lang and smith, ) . results of a study that compared rotavirus survival in three soil fractions (whole soil, sand and clay) at temperatures , and c for days showed least survival in sand fractions (davidson et al., ) . in the absence of soil particles, rotavirus survived best at c with survival decreasing, with an increase in temperature, except in whole soil, where it survived better over the entire temperature range and for more than a week at c, indicating that whole soil offered some protective effect (davidson et al., ) . conversely, though, there is a report of shorter survival duration of enteroviruses (poliovirus type , echovirus , echovirus and coxsackie b ) in loamy soil than in sandy soil (bagdasaryan, ) . a link between higher organic matter content and enteric pathogen persistence has been established (jamieson et al., ; williamson et al., ; . there is overwhelming research evidence in this regard, seeing that many of the studies that compared the persistence of enteric pathogens in top and sub-soils recorded higher survival rates in topsoil (zhai et al., ; nyberg et al., ) . research has also shown higher pathogen levels in organic soils after manure application compared to sandy soils (tate, ; jamieson et al., ) . therefore, the rates of pathogen survival are lower in sandy soils, which have a low water-holding capacity (mubiru et al., ; erickson et al., a) . lower temperatures are more suitable for bacterial and viral survival. the ultraviolet radiation from the sun inactivates viruses on the surface of the soil, but viruses in deeper soil strata are protected from this (rodríguez-l azaro et al., ; zablocki et al., ) . in loamy soil samples, at ph . , poliovirus and echovirus were recovered after e days at e c compared to recovery e days at e c (bagdasaryan, ) . similarly, cfu/g amended with dairy manure persisted for e d in fallow soils, manure did not seem to affect persistence, clay seemed to improve pathogen persistence and activity gagliardi and karns, (continued on next page) poliovirus type and coxsackievirus b pfu were recovered for up to days at c whereas pfu were recovered from soil for up to days at c (temperature profiles tested were , and c) (yeager & o'brien, ) . the persistence of poliovirus in sludgeamended soil was assessed in a field study where appropriately cultivated and irrigated plots were treated with virus-spiked effluents by flooding. this was done for days spanning through spring, summer and winter seasons. poliovirus survived best during winter (when it was detected after days), but during summer, the longest survival period was days (tierney et al., ) . parasites seem to prefer warm temperature conditions. prevalence of hookworms have been correlated to warm temperatures, relatively high rainfall and low clay content (sandy soils with clay content of less than %) (mabaso et al., ) . nutrient availability is essential for the survival of microbes in the soil. the presence of organic matter promotes the survival, and in many cases, the regrowth of enteric bacteria looney et al., ) . organic matter improves nutrient retention, serves as carbon sources for bacterial species and enhances moisture retention (gerba et al., ; schoonover and crim, ) . apart from environmental stress responses, foreign enteric bacteria must compete with the endogenous microflora to become established in the soil environment (jiang et al., ) . some autochthonous soil organisms have been shown to be resistant to newly introduced microorganisms in their environment (ellis and mccalla, ) . also, certain bacteriophage, some protozoa, nematodes and free-living soil organisms such as bdellovibrio can parasitize non-indigenous pathogens, thereby limiting their survival (klein and cassida, ; goss and richards, ) . additionally, increased pathogen survival, and regrowth in some instances, in sterile soils and soils with relatively low biological activity has been reported (gerba et al., ; tate, ) . there is some research evidence that alien enteric pathogens compete poorly for nutrients and are thereby susceptible to inhibition by soil-borne bacteria (jiang et al., ) . the effects that this has on the persistence of pathogens (especially pathogens introduced via contamination) in soil is however not yet fully understood. the impacts that soil edaphic and biotic conditions have on the occurrence, fate and persistence of microorganisms in soils should not be underestimated. these factors can collectively or independently stifle or encourage foreign pathogens. for instance, members of listeria possess advantageous intrinsic factors such as an extensive repertoire of transport systems (like phosphotransferase system and transcriptional regulators) which makes them capable of successfully persisting in the soil ecosystem (newell et al., ) . however, these species are highly sensitive to extrinsic factors and this affects their ability to survive in soil environments (newell et al., ; locatelli et al., ) . although studies have been conducted on the occurrence of l. monocytogenes in various ecological niches, including soil, more emphasis has been placed on the occurrence of listeria spp. in fresh vegetables under storage conditions, food processing and packaging environments. the expression of genes and induction of proteins such as cold shock and cold acclimation rapid decline in cell numbers with no difference identified for soil type. increase in retention at both rainfall rates. campylobacter jejuni clay loam silt cfu/g cfu/g cfu/g proteins, as well as tolerance for low ph and high salt concentration in these environments have received much research attention. there is however, need for more research to understand the dynamics of listeria survival in soils. agronomic practices such as soil improvement and manure application method influence the survival of pathogens in the soil (table ) . soil improvement strategies (inorganic and organic fertilizer, compost, biosolids and other residuals application), significantly enhance the nutrient loads of soils (diacono and montemurro, ) . in varying degrees, these are important sources of primary nutrients such as n and p as well as secondary nutrients such as ca, mg and s to the soil. a ready supply of essential nutrients encourages the growth of pathogens. compost application modifies the long-term soil conditions by increasing the ph steadily, this, therefore, affects pathogen survival in soil (weller, ; sharma and reynnells, ) . bacteria tend to decline more rapidly when manure is applied superficially as opposed to when incorporated into the soil immediately after application (solomon et al., ; islam et al., a) . this is probably due to the elimination of drying conditions and exposure to uv at the soil surface (schulze-makuch and irwin, ) or because incorporation of manure disrupts macropores and boosts soil-bacteria contact . after manure application on land, if applied manure is contaminated, it is probable that the pathogens will move through the soil matrix, either vertically or horizontally. vertical movement of pathogens through the soil is influenced by the amount and intensity of rainfall, climatic conditions as well as the season of application. horizontal movement is known to be influenced by soil type, moisture levels, temperature, microbial activity, transport through plant roots, rainfall patterns, soil ph amongst other biophysical factors. it is, however, apparent that water flow is the most important dispersal factor for percolation of manure-derived pathogens in soils, regardless of type and structure although more quantitative information regarding this is desirable (mawdsley et al., ; jiang et al., ; jamieson et al., ; islam et al., b; you et al., ; semenov et al., ) . the extent of movement will affect the distribution and eventual fate of the pathogens. some will spread in soil and attach to roots. others may be washed off to surface waters or percolate to aquifers, potentially contaminating irrigation water sources (fig. ) vinten et al., ; avery et al., a, b; islam et al., b) . pathogens occurring in contaminated manure, therefore, can be rapidly transported within soil systems (gagliardi and karns, ; kisluk and yaron, ) . the success of conveyance and distribution, however, further depends on inherent survival capabilities of the pathogen as well as the presence and structure of plant root systems (fig. ) (kemp et al., ; mubiru et al., ; avery et al., a; . there is some evidence that pathogens may indeed survive longer in manure-amended soils than actual manure samples, and this has been illustrated for enteric species such as s. typhimurium and e. coli o :h . salmonella typhimurium, has, however, exhibited superior persistence capabilities compared to e. coli o :h in manure-amended soils (islam et al., b; you et al., ; fremaux et al., ; pornsukarom and thakur, ) . there is a paucity of data on the persistence of pathogens in manure amended-soils in the tropics (ongeng et al., ) . one interesting study provides an insight into the survival of e. coli o :h and salmonella typhimurium under tropical climatic conditions . the study showed that survival periods were mostly shorter than the observed record in temperate regions indicating that biophysical conditions in the tropics may be more injurious to these pathogens. it is, therefore, not prudent to predict the survival of e. coli and s. typhimurium in tropical soils from data obtained in temperate locations. the soil is the most important cultivation medium and represents a relevant risk for produce contamination. a myriad of studies regarding the behavior of pathogens in various kinds of soil ecosystems is available. however, validated consensus protocols for conducting and interpreting experimental studies as well as for evaluating the effects of environmental and soil characteristics on fate of pathogens in soils are not yet available. it is important to further understand the effects of soil types, environmental factors, biological processes and interactions, cultivation and management practices on the behavior of (indigenous and foreign) enteric pathogens in agricultural soils. apart from farm animals, whose roles as reservoirs of enteric pathogens has been established, wild animals such as birds, reptiles, rodents, amphibians, some helminths, and insects like flies and beetles can also serve as vehicles of pathogens to contaminate cultivation media and produce (beuchat, ; lim et al., ) . livestock and wild animals may gain access to cultivation areas either because of adjacent land use (livestock rearing) or by intrusion (jay-russel, ) . birds such as gulls, pigeons, chickens, starlings, canada geese, migratory ducks and sandhill cranes (pacha et al., ; hald et al., ; ekdahl et al., ; humphrey et al., ) have been determined to be carriers of pathogens such as e. coli, salmonella and campylobacter (wallace et al., ; schmidt et al., ; wani et al., ) . insects are typically ubiquitous in cultivation fields, and hence, have unrestricted access to produce. they are usually found in manure piles, feedlots and other habitats near cultivation fields, and so farms practicing mixed farming represent a more significant risk (martínez-vaz et al., ) . many bacterial species have evolved to exploit insects as hosts or vectors. filth flies, fruit flies, cockroaches and other insects act as mechanical and biological vectors to contaminate fruits and vegetables on the field (sasaki et al., ; mpuchane et al., ; alam and zurek, ; humphrey et al., ) . many pathogens use flies as vectors for cross-transmission. for example, the transient survival of pectobacterium carotovorum subsp. carotovorum in the gut of the fruit fly drosophila and subsequent transmission to other plants has been observed (nadarasah and stavrinides, ; lim et al., ) . under laboratory environment, direct bacterial transfer from contaminated flies to fruits or plant leaves was shown to occur (sela et al., ; talley et al., ; lim et al., ) . members of muscidae and calliphoride which are usually abundant in production fields adjacent to cattle rearing lots have been associated with the transmission of e. coli o :h . insects that feed on plants also play significant roles in produce contamination by providing direct routes for internalizing pathogens from manure to plants in the field . insect deterioration creates openings that aid the ingress of pathogens into inner plant tissues, thereby enhancing colonization of spoilage and pathogenic bacteria on produce (warriner and namvar, ; lim et al., ) . a seasonal trend to contamination by insects has been identified. there is increased insect and animal activity during the warmer months of the year. moreover, peak incidences of pathogens have been reported during the warmer months (liang et al., ) . reptiles including snakes, lizards, chameleons, turtles, as well as other ophidians, saurians and chelonians have been found harboring enteric bacteria like salmonella (corrente et al., ; beuchat, ) . many wild rodents are asymptomatic carriers of pathogens like salmonella and campylobacter. the occurrence of rodents on farms are often associated with infrastructural impairment, and although their destructive tendencies have been widely recognized, their zoonotic risks are often primarily underestimated. they are capable of amplifying the number of pathogens in the environment and transferring them to other farm animals and produce (meerburg and kijlstra, ) . commensal rodents (house mice and rats) pose a particular threat because of their ecology (they live close to livestock) and high fecundity (brooks and jackson, ; witmer et al., ) . foodborne illness resulting from the consumption of contaminated produce is dependent on specific factors. first, the produce must be contaminated with a pathogen, which must survive until the time of consumption at levels sufficient to induce illness (harris et al., ) . the dose required to cause illness in many cases, is very low, which indicates that the microorganism needs only to contaminate the food to survive without necessarily reproducing. for instance, pathogenic parasites and viruses are not capable of multiplying outside a human or animal host and only need to survive in sufficient numbers to cause illness (harris et al., ) . the survival and or growth of pathogens is influenced by the kind of organism, produce type, on-field environmental conditions, as well as the physiological state of the plant and pathogen. the possible routes of entry into plant tissues include: natural apertures (such as stomata, lenticels, sites of lateral root emergence), wounds caused by biotic or abiotic circumstances and following the flow of water from roots to leaves, where pathogens can efficiently survive and multiply (steele and odumeru, ; deering et al., ; hirneisen et al., ) . the popular opinion is that pathogens will survive but not thrive on intact (uninjured) outer surfaces of produce, primarily due to the protective effects of natural plant barriers (such as cell walls and wax layers) (mathews, ; heaton and jones, ) . survival and proliferation of enteric pathogens on produce is significantly enhanced if the protective barrier becomes compromised either by physical or biological damage (such as punctures or bruising), insect ruination or through degradation by plant pathogens. it is vital to understand the microbe-microbe and plant-microbe interactions that occur in the phyllosphere and rhizosphere which influence the adaptation, colonization, attachment is pre-requisite for the colonization and subsequent transmission of enteric pathogens throughout plants including the edible portions (berger et al., ) . it is important to note that attachment onto the surface of intact produce is limited in contrast to the attachment on other food commodities such as processed meat tissues . however, the attachment does indeed occur and is facilitated by stomata, lenticels, broken trichomes, as well as bruises and cracks occurring on produce surfaces. the incidence of scars and cracks (which may set in late in the growing season while the fleshy portion is enlarging rapidly) in certain fruits also aids pathogen attachment (bhagat et al., ) . cracks tend to occur in or on the weak areas on plant surfaces such as around lenticels and trichomes, and hence, these areas are more susceptible to invasion by pathogens. cavities within the epidermis may also develop from cuticular cracks as the fruit develops, thereby entrapping pathogens and shielding them from desiccation and disinfection. the initial phase of bacterial attachment is a rapid process initiated once the bacteria establishes contact with the plant surface (phyllosphere) (sant'ana et al., ) . the phyllosphere, also known as the aerial parts of plants pose challenges for microbial survival. exposure to high uv doses, temperature and relative humidity fluctuations sabotage viability (brandl et al., ; heaton and jones, ) . epiphytes that exist within the phyllosphere have, however, evolved specialized mechanisms to improve stress tolerance and nutrient acquisition. for instance, pseudomonas spp. produce pigments to insulate against uv and pectolytic enzymes to gain nutrients (heaton and jones, ) . the ability of the pathogen to persist on the phyllosphere improves the chances of a viable or infectious dose remaining post cultivation (heaton and jones, ) . the successful attachment on the phyllosphere also depends on the crop and pathogen type. a classic illustration is salmonella invasion of lettuce and tomatoes. salmonella contamination of lettuce and tomatoes via soil is usually quite low, implying that salmonella does not readily attach to or grow in the phyllosphere of these crops (critzer and doyle, ) . also, attachment of salmonella and e. coli o :h is observed more frequently with brassicaceae compared to lettuce, carrots, and tomatoes, which has generated the theory of selective attachment, suggesting that certain produce types are more prone to contamination than others (warriner and namvar, ). specific pathogens such as salmonella have surface epitopes that can bind to plant structures such as stomata to aid attachment (warriner and namvar, ). some also have higher capabilities to metabolize nutrients contained within the apoplastic fluid of plants (warriner and namvar, ) . these traits significantly enhance their attachment abilities. finally, hydrophobic interactions between a plants' epidermal layer and microbial cells are believed to play a major role in facilitating this initial phase of attachment (burnett and beuchat, ) . surface colonization is the final phase of attachment during which biofilms may be formed. biofilms are microbial colonies, which form when single microorganisms attach and aggregate on a hydrated surface and undergo a "lifestyle switch," giving up life as a single cell to live on a surface in an adhesive cell matrix with other microorganisms (lemon et al., ) . cells in a biofilm have a better chance of adaptation and survival (especially during periods of stress) as they are protected within the matrix (decho, ) and are usually resistant to antimicrobial agents (lemon et al., ) . naturally occurring biofilms are present in many fruits and vegetables, but the ability of foodborne pathogens to associate with them and persist is not yet fully understood (brackett, ; ferreira et al., ; larsen et al., ) . pathogen serovars that are strong biofilm producers have been shown to attach better to both intact and injured produce surface compared to strains that are weak biofilm producers (lindow and brandl, ; kroupitski et al., ) . the occurrence of biofilms improves the chances of transient occupants of leaf surfaces such as enteropathogens of becoming effectively incorporated into phyllosphere biofilms (heaton and jones, ) . bacterial appendages such as curli, pili, fimbriae, and flagella, as well as proteins in outer membranes and genes, may also facilitate the surface colonization by pathogens. increases in the expression of flic, flagellin-encoding gene have been observed in certain produce contamination studies. after attachment, it becomes very difficult to remove the pathogens from produce by surface washing (beuchat and scoutten, ) . overall, enteric soil pathogens may reach the edible portions of fruits and vegetables via numerous mechanisms and routes and these have been elucidated by several studies (natvig et al., ; johannessen et al., ; barak and liang, ; tyler and triplett, ) . some of these routes include germination of seeds in contaminated soils, which leads to bacterial colonization of roots and edible parts, direct transfer of pathogens within the soil to crops when heavy rain or water gun irrigation causes leaf splash, bacterial infiltration through roots, amongst others. attached pathogens can reach the interior of fruits and vegetables via a variety of pathways. the extent of internalization depends on factors such as the route and mechanism of entry, the type and age of the plant, the aerial and/ or root morphology and exudates, the soil type and biology and the strain and/serovar of bacteria (hirneisen, ; brandl et al., ; lim et al., ) . the mechanism could be either passive or active (sant'ana et al., ) . passive internalization involves the uptake of bacteria mainly through roots and seeds. mechanistically though, enteric pathogens may be internalized via the root system and transported to edible tissues, but the risk of contamination by this route is likely low . this is because in the environment, particularly areas that are not prone to contamination events, the levels of enteric pathogens are likely to be extremely low (cooley et al., ; matthews et al., ) . in contaminated zones, however, human pathogens may indeed invade root tissues and subsequently translocate to edible portions (solomon et al., ; solomon and matthews, ) . depending on the age of the plant, pathogens may invade external root surfaces (main and side roots, as well as root hairs) and subsequently internalize. the developmental stage of plant root systems when contamination occurs influences the capability of pathogens to interact with, penetrate plant roots and migrate to other tissues (mootian et al., ) . the physiological characteristics of the roots may also determine the success of internalization; for example, some root vegetables possess antimicrobial properties, which limits the growth and internalization of enteric bacteria . pathogens like e. coli o :h have been demonstrated to survive longer in the soil in the presence of rye and alfalfa roots (gagliardi and karns, ) . other work has demonstrated that pathogens enter root tissues at sites of lateral root emergence or through damaged roots (mendes et al., ) . salmonella and e. coli o :h have penetrated arabidopisis and lettuce plants' roots, while klebsiella pneumoniae have been detected on numerous plants' roots (tyler and triplett, ) . other examples include the invasion as well as (endophytic and systemic) colonization of barley roots by s. typhimurium, the shoots of black pepper stem cuttings by pseudomonas aeruginosa, as well as roots and shoots of tomato seedlings by p. aeruginosa (kutter et al., ; kumar et al., ) . it is, however, important to note that successful invasion of the root and shoot system may not guarantee translocation to the edible or foliar portions of produce. in some surveys, bacterial pathogens were detected in roots but not leaves of crops examined (watchel et al., ; warriner et al., ; bernstein et al., a; mitra et al., ; sharma et al., ) . a growing body of evidence suggests that seeds may serve as primary inoculum source in produce contamination. in the case of vegetables, seed sprouts have been implicated as the initial inoculum source, severally (warriner et al., ; deering et al., ; kumar et al., ) . in recent time, seeds have been recognized as a significant source of inoculum for foodborne illnesses associated with sprout consumption (mahon et al., ; national advisory committee on microbiological criteria for foods, ; buck et al., ; yang et al., ) . it is possible that enteric bacteria may be transmitted from contaminated seeds to sprout to mature plants, throughout entire plant life cycle up to consumption. the contamination may be transferred from seed again, thus persisting in produce cultivation cycles, for a long time. there is a record of e. coli :h adherence to outer surfaces and subsequent successful internalization of radish sprouts produced from contaminated seed during sprout growth (itoh et al., ) . rate and efficiency of uptake also depends on the type of produce, and the level of internalization varies widely among plants and even among different species of the same crop due to variations in intrinsic factors, which affect pathogen survival and proliferation (golberg et al., ) . for instance, certain produce items, like fully ripe tomatoes are typically in the ph range ( . e . ) which conventionally impedes growth of most enteric bacteria, whereas, the ph of numerous vegetables, melons, and soft fruits is usually . or higher, which is conducive for bacterial growth gagliardi et al., ) . therefore, gram-negative bacteria are more commonly associated with vegetables while molds and certain yeasts mostly occur on fruits, due to the differences in ph requirements of the respective groups of microbes (jay, ) . members of the brassicaceae family (radish, turnip and broccoli) were demonstrated to have a higher prevalence of salmonella contamination than lettuce, tomatoes and carrots when grown in contaminated soil (barak et al., ) . among leafy greens, radicchio and endive may be more likely to be contaminated than lettuce, spinach, parsley or cilantro (barak et al., ) . salmonella typhimurium has been demonstrated to internalize more efficiently in iceberg lettuce and arugula leaves compared to romaine, red lettuce, fresh basil, parsley and tomato leaves, which displayed only marginal internalization. listeria monocytogenes seems to exhibit a selective preference for certain vegetables like radishes and potatoes, as certain studies reported that although l. monocytogenes successfully invaded tissues of a wide variety of vegetables, radishes and potatoes appeared to be more often and severely contaminated (beuchat, ) . it is also apparent that l. monocytogenes does not survive and internalize satisfactorily on fresh carrot, in fact, low doses of raw carrot juice have been demonstrated to inhibit the growth of the pathogen (beuchat et al., ; farber and peterkin, ; oh, ; benkerroum, ) . internalization is believed to be a plant-pathogen specific interaction, and therefore, internalization success varies from pathogen to pathogen. a comparison of the internalization of l. monocytogenes to s. typhimurium on inoculated seeds of cress, radish, spinach, lettuce, mustard, carrots, and tomatoes showed significant variations in the rate and efficiency of internalization by the pathogens. under identical experimental conditions, s. typhimurium internalized into the roots of the vegetables, whereas, l. monocytogenes did not (jablasone et al., ) . similarly, while s. typhimurium was found to be associated with the internal portions of barley sprouts, l. monocytogenes, l. ivanovii and l. innocua were not (kutter et al., ) . furthermore, the degree of internalization is contingent on the serovar/strain (larsen et al., ) . gene expression, metabolic and antimicrobial capacities vary among strains. certain strains manifest up-regulation of peculiar genes like the pdu, cob-cbi, and out which improve carbon source utilization and may confer a competitive edge, thereby enhancing the survival and persistence of these strains (fox et al., ) . some e. coli strains possess metabolic capacities, which foster their survival in certain agroecosystems such as soils (franz et al., ) . in a bid to explain the strain-specific internalization dynamics, a five serovar salmonella cocktail (montevideo, michigan, poona, hartford and enteritidis) was inoculated into hydroponic growth substrates. serotypes montevideo and michigan were most prevalent, while enteritidis, hartford and poona were not detected in any of the tomato tissue samples (guo et al., ) . this is a quintessential illustration of internalization variation among serovars. likewise, salmonella serovars; cubana, infantis and typhimurium exhibited varying capabilities to internalize and colonize alfalfa sprouts when seeds were inoculated under identical environmental conditions (dong et al., ) . some scholars have endeavored to compare the survival of two arguably most prominent foodborne pathogens: e. coli and salmonella. serovars of both can proficiently adapt to environmental stress; -numerous strains are known to become habituated to low ph conditions and subsequently manifest remarkable tolerance to stress conditions. escherichia coli can perpetually evolve new varieties that have neither been previously reported nor characterized and which are capable of exploring and inhabiting previously unrecognized niches (newell et al., ) . both seem to be capable of long-term survival in the agricultural environment and on produce, but it is quite apparent that salmonella survives better than e. coli (brandl, ; mandrell, ; newell et al., ; schikora et al., ; ongeng et al., ) . many salmonella serovars bind to plants significantly better than e. coli strains. escherichia coli's inability to lower its metabolic rate to suit the low availability of accessible organic carbon and to competently cope with low nutrient conditions contributes significantly to its die-out in soils and on produce, and therefore, lowers its competitiveness (survival) compared to salmonella franz et al., , . internalization has been correlated with motility and chemotaxis. flagella mutants (flighi:tn , chey) deficient in motility and chemotaxis respectively have exhibited reduced attachment and penetration of lettuce leaves (kroupitski et al., ; lim et al., ) . it has also been hypothesized that products of photosynthesis serve as nutrients to aid internalization of pathogens (lim et al., ) . active internalization typically involves the penetration of bacteria through natural openings. the ability of foodborne pathogens to internalize in produce represents a significant public health risk because internalized pathogens are protected against optimized disinfection modes (meireles et al., ) except irradiation which seems capable of reasonably eradicating internalized pathogens in produce. the technique penetrates produce tissues to eliminate internalized pathogens, and gram-negative bacteria are very susceptible to even low doses (saroj et al., ; o'bryan et al., ) . however, treatment with irradiation may produce off flavors, colors and odors and may inactivate some of the nutrients (fan and sokorai, ) . it is, therefore, not accepted and endorsed for produce treatment. there are other relatively new technologies such as modified atmosphere packaging, ozone, ultrasound and ultraviolet treatments, which seem promising in ensuring the microbiological safety of fresh fruits and vegetable products (shayanfar and pillai, ) . however, limited commercial applications have been described for most of these new technologies. electron beam technology is another up-and-coming treatment option, which according to experts, can play a pivotal role in mitigating some of the contemporary microbiological risks facing the produce industry (shayanfar and pillai, ; lung et al., ) . it is an environment friendly, cost and time effective decontamination strategy that uses low-dose ionizing radiation to treat crops (-as well as other food items), to eliminate microbial contamination. it is capable of inhibiting the germination of crops and controls the rate of ripening of fruits and vegetables, thereby extending their shelf life (lung et al., ) . it inhibits a variety of enteric pathogens without compromising food sensory and nutritional qualities and can be used in combination with other traditional or non-traditional food processing technologies (lung et al., ) . regulatory authorities such as the us food and drug administration have approved it, but the full import of the safety of use is not yet conclusive. given the amount of evidence indicating that enteric pathogens (that are not plant pathogens) can invade and be internalized into plants, it is important to understand how such microbes establish access to plant tissues, as this may facilitate the development of strategies to reduce internalization. for successful colonization, major interactions take place between pathogens and their plant hosts that determine the success of the pathogenic attack (warriner and namvar, ). many enteric pathogens have devised mechanisms to overcome plants' basal defense mechanisms and innate immune responses (lim et al., ) . plants first line of response to foreign invasion is by the innate immune system. this consists of two main branches: pamp-triggered immunity (pti) and effector-triggered immunity (eti). in the first stage, microorganism associated molecular patterns (pamps or mamps such as flagellin, peptidoglycan, lipopolysaccharide) are identified by plant host receptors popularly known as pattern recognition receptors (prrs) (deering et al., ) . these batteries of receptors deployed by the host are designed to curb the growth and spread of the pathogen (ausubel, ) . pti response is broad-spectrum; sensitive to molecules familiar to many classes of microorganisms including non-pathogens. upon recognition, plant defense signal pathways are activated among which, jasmonate, salicylic acid and ethylene play essential roles. virulent plant pathogens may through diverse strategies, such as the production and secretion of effectors, efficiently override pti, for example, there are some 'effectors' that can overcome pti by interfering with mamp detection and subsequent defense signaling (kazan and lyons, ) . this results in effector-triggered susceptibility (ets). for susceptible interactions, effectors produced and released by the microorganism are transferred into the plant cell through the ttss (type iii secretion system). specific nucleotidebinding leucine-rich-repeat (nb-lrr) proteins encoded by resistance genes, resulting in eti and limitation of pathogen transmission to other tissues, recognize these effectors. while pti is considered the first line of defense against pathogenic infection, eti is an accelerated and amplified response, the outcome of which is often a hyper-sensitive response (hr) (spoel and dong, ) . the ability of pathogenic bacteria to colonize a plant may also be influenced by their interactions with other microorganisms either positively or negatively (deering et al., ) . if other microorganisms supply carbon sources (via degradation of cell wall polymers or induced secretion of sugars), or sequester antimicrobials, this can enhance pathogen colonization (bais et al., ; warriner et al., ; augimeri et al., ) . alternatively, plant pathogens that wound or destroy living tissue may create a microenvironment that is suitable for the survival and/replication of human pathogens (rashid et al., ) . pathogens are often associated more with plants whose tissues have been damaged by soft-rot pathogens compared to those with healthy tissues (brandl, ) . before pathogenic bacteria can colonize the surface or interior of a plant host, they have to contend with the naturally occurring microflora that is already established (deering et al., ) . the ability of the indigenous bacterial community to inhibit the growth of introduced enteric pathogens has been demonstrated by numerous studies (liao and fett, ; matos and garland, ; schuenzel and harrison, ; cooley et al., ; johnston et al., ) . there is direct evidence that the stomata play essential roles in internalization, host immunity and pathogen virulence of pathogens (kroupitski et al., ; zeng et al., ) . some researchers have reported that plant stomata close in response to plant pathogens and some human pathogens (melotto et al., ; roy et al., ) . escherichia coli o :h has been reported to trigger stomatal closure even under high relative humidity, a stressful environmental condition that generally weakens plant defenses against bacteria in field and laboratory conditions (roy et al., ) . stomata closure could be triggered by certain peptides such as flg produced by bacterial flagellin and lipopolysaccharides which are recognized by pamps or mamps in a salicylic acid-dependent manner. in the case of some plant pathogens such as xanthomonas spp. and pseudomonas syringae, virulence factors produced are capable of overcoming this innate immunity and counter stomata defense. for example, pst dc and several other pathovars of pseudomonas syringae, produce coronatine (cor), a phytotoxin which can reverse stomatal closure induced by bacteria or mamps (zeng et al., ) . stomatal immunity can diminish the penetration of human pathogens through the leaf epidermis, resulting in low bacterial titers in the plant apoplast (roy et al., ) . however, plant defense responses induced by pathogens vary and plants may recognize and respond to some human pathogens more effectively than others (roy et al., ) . for example, comparison of plant defense responses induced by e. coli o :h and s. typhimurium sl in arabidopsis thaliana and lettuce (lactuca sativa) revealed some variations. while e. coli o :h triggered stomatal closure, sl only induced a transient stomatal immunity. also, pr gene expression was significantly higher in arabidopsis leaves infected with e. coli o :h compared with sl (roy et al., ) . although, numerous studies have examined the intricacies of internalization in fresh produce, many of these are laboratory based. the few available field studies, which have mostly studied e. coli, indicate that internalization of pathogens may be not be very common in field settings erickson et al., b erickson et al., , erickson et al., , b . more field studies are therefore, required to properly understand the potential/likelihood of enteric pathogens to internalize in fresh produce as well as the actual factors that influence the success of internalization. to successfully achieve an acceptable level of microbiological safety for fresh produce, it is essential to control environmental contamination in the field by taking appropriate pre-harvest precautions. one fundamental factor to consider is the state or quality of the growing fields. fields on which wild or domestic animals have been recently grazed that have been subjected to flooding or may have been previously contaminated with manure constitute an unacceptable microbiological risk (turb e et al., ) . therefore, growers need to scrupulously investigate land history when selecting a location for produce cultivation (islam et al., a, b) . cultivation areas should be safeguarded from flooding, and fecal contamination and manure should be adequately treated (using popular methods like composting and aging) before application as fertilizer. also, suitable buffer zones (physical barriers) such as mounds, diversion berms, vegetative buffers as well as ditches should be erected between animal grazing regions and produce cultivation areas (james, ; olaimat and holley, ) . appropriate livestock waste disposal and farm general waste management should be adopted to ensure safety. numerous experts have highlighted the need for monitoring, regulation and control of the microbiological quality of irrigation water. several regional and international standards exist for irrigation water use and practices to prevent incidence of bacterial contamination. the use of potable water for irrigation (and other cultivation operations) is highly recommended. certainly, this is not economical in many instances and may increase production costs, which will raise prices; it is however, pertinent to public health safety. in developing countries, a myriad of safety regulations exists such as cessation of irrigation prior to harvesting, lowering of watering cans to reduce splashes from (contaminated) soil, adoption of furrow irrigation system over the use of sprayers which expose edible portions of leafy vegetables directly to irrigation water, and so on (keraita et al., ; amoah et al., ; uyttendaele et al., ) . in cases where surface water is the irrigation water source, drainage of contaminated water into the surface water reservoir may be prevented by constructing ditches, buffer strips, as well as retention and drainage systems. potential overflow points should be identified and eliminated. it is also important to determine (potential) points of contamination because control measures are bound to be more effective if focused on eliminating contamination at the source (madramootoo et al., ; pachepsky et al., ) . irrigation wells, functional septic, water and sewage systems should be installed and properly maintained especially during periods of excessive rainfall to prevent pathogen contamination (buck et al., ; olaimat and holley, ) . surface and groundwater resources should be protected from any potential sources of contamination including wildlife, animal waste, agricultural run-off, human activity, sewage, or industrial effluents. other management practices like; removal of riparian areas, erection of fences, and treatment of irrigation water (for example, using uv treatment) can be considered to enhance safety assurance of irrigation water. these precautions will minimize contamination risks on produce farms and should be applicable not just to supposed high-risk crops (such as leafy greens) but all produce (squash, and others) (strawn et al., b) . implementing some of these may, however, be costly and have negative impacts on landscape health. irrigation water sources should be routinely monitored to ensure microbiological safety (brackett, ; islam et al., b) . ideally, there should be more regular reporting on the microbiological quality of irrigation waters in different world regions. such surveys should reflect the true levels of actual pathogens rather than indicators, and bias should be avoided towards contaminated samples by intensively monitoring every irrigation source possible, and not just sites where extensive contamination has been known to occur (stoeckel, ) . as part of a total package of hygiene measures to prevent the transfer of foodborne pathogens, wild animals, birds, flies and rodents should be controlled in cultivation areas. interventions to mitigate wildlife intrusion of a farm may be costly and not entirely effective, especially if not done properly, thereby allowing certain animals direct access to crops. in many cases, it is not economical to fence large farms, but small farms can be fenced to restrict wild animals (jung et al., ) . other mechanical/biological control methods include the use of scarecrows, reflective strips, monitoring of animal tracks and field intrusion as well as gunshots to ward off pests and animals. mechanical traps and baits can be used to control mice and rodents. overall, practical, cost-effective methods should be adopted to mitigate wild sources and routes of produce contamination. considering that, in many important outbreaks, vegetable seed sprouts have been implicated as the initial inoculum source, the elimination of bacteria from seeds before planting has become crucial (buck et al., ) . chemical or physical treatment methods are usually used to decontaminate seeds, in a bid to reduce the risks of sprout borne disease outbreaks. however, this poses some challenges for growers, as the chosen decontamination method has to fulfill certain conditions. one important consideration is the preservation of seed viability. selected treatment dosage should be able to inactivate pathogens without adversely affecting seed viability (buck et al., ) . also, the treatment must be able to penetrate and access bacteria that may be residing in protected seed tissues, and finally, certain treatments may be inactivated by seeds, rendering them less effective (buck et al., ) . nevertheless, the efficacy of chemical seed treatments for sprout seed including chlorine compounds (commonly calcium and sodium hypochlorite), ethanol, hydrogen peroxide, calcium edta, -hydroxybenzoic acid, ozonated water and other commercial disinfectants have been extensively documented. it is also possible to use gaseous chemicals and thermotherapy (e.g., hot water treatment), although excessively high temperatures may affect sprout vigor. another potential issue with hot water treatment is that when treating large batches of seed, it is practically impossible to achieve temperature uniformity throughout the water bath. therefore, while a portion of the seeds receives the appropriate temperature-time exposure, some will still contain viable bacteria after 'treatment.' also, there is a potent risk of cross-contamination with this technique. other viable options include seed treatment with bacteriophage, combinations of thermotherapy with chlorine and the use of ionizing radiation. radiation is particularly appealing because it can penetrate seed tissues and possibly eliminate bacteria localized within protected tissues (buck et al., ) . however, it has been postulated that high levels of irradiation may distort the physiology and organoleptic properties of seedlings, more research is therefore, needed to evaluate the prospects and risks of this approach. other precautionary measures include testing seed lots for purity and germination rate prior to marketing, proper warehouse storage (in metal bins) until bagged, as well as ensuring general facility sanitation and employee hygiene (national advisory committee on microbiological criteria for foods, ). safety criteria and regulations are mostly region specific, it is however, critical to enforce these regulations, ensure that growers adhere to such and there is a need to constantly improve standards; if new information becomes available, regulations should immediately be updated (k€ opke et al., ) . most of the available data is from the developed world mainly from the us and certain parts of europe. it is necessary to develop surveillance and tracking systems and generate robust databases for other regions as well. more studies should be conducted under field conditions, rather than laboratory or greenhouse simulations, as this will provide a better understanding of how enteric pathogens behave in agricultural production environments. finally, and more importantly, it is necessary to ensure producers are mindful of their roles in assuring food safety. growers should be encouraged to adopt the best possible agricultural practices to ensure produce safety. it is also important to enlighten consumers about possible risks and appropriate mitigation strategies. there are wrong notions and misconceptions, which have to be corrected promptly, for example, many people believe it is not necessary to wash organically grown fruits and vegetables (leifert et al., ). it is evident that epidemiologic investigations are worthwhile as public health directives and policies based on investigation output have averted impending foodborne disease crises in many cases. the relevance of epidemiological surveys globally and regionally, therefore, cannot be overemphasized. this means that epidemiological investigation tools and systems need to be objective, updated, precise, flexible and timely. while significant progress has been achieved in the area of epidemiology, there are still certain cracks that need to be addressed. the use of routine, optimized clinical pathogen identification techniques may mean that new pathogens may likely be missed. this is a potentially grave issue, because periodically, since the development of foodborne disease surveillance, the list of foodborne pathogens has continued to expand. care should, therefore, be taken to avoid research bias since it is likely that produce items that have been previously associated with foodborne illness outbreaks and product recalls may receive particular scrutiny. new pathogens emerge due in part, to evolving ecology and technology while already recognized strains continue to evolve, potentially becoming smarter, evading and subverting detection, sanitization and plant host defenses. it is important to further understand the evolution dynamics and emergence of new pathogens, as well as develop and optimize methods to meet the emerging challenges. awareness and surveillance of viral and parasitic enteric pathogens need to be more robustly developed. although noroviruses, hepatitis a, rotaviruses as well as certain emerging viruses such as sars are well known, they are rarely routinely screened for in fresh produce in most countries. also, their ecology in fresh produce is poorly understood, for instance, the knowledge of the stability and persistence of human norovirus in foods has been garnered mostly from the study of surrogate viruses. more importantly, their significance in foodborne disease incidence remains undetermined. parasitic pathogens like ascaris, giardia, entamoeba, cyclospora, cryptosporidia and trichinella are recognized (newell et al., ; robertson et al., ) , but not all are routinely monitored in produce. the roles that livestock and wildlife play in pathogenic contamination of fruits and vegetables as well as their epidemiology through the food chain is poorly understood. it is difficult to compare the available studies because some have used naturally contaminated animals, while others used experimentally inoculated animals. the exact transport/transfer mechanisms of pathogens from animal fecal material or contaminated manure/soil to fruits and vegetables via splash are not yet properly understood. for example, it will be helpful to understand the specific spatial factors that influence the transfer of pathogens from fecal pellets to fruits and vegetables. the survival times for pathogens in fecal contaminants, manure, and manure-amended soils are inconsistent, reflecting the varying conditions under which many of the available studies have been conducted (these variations are demonstrated in tables e ). the fate of pathogens on the soil surface, the relationship between manure-derived pathogens and soil particles, as well as the states in which pathogens occur in soil slurry or manure mixtures, should be further explored. the exact mechanisms of uptake or (transmission) of pathogens from contaminated manure or manure amended soils to plants, particularly in field settings should be studied. this will facilitate the design of scientifically sound produce safety standards. the majority of studies available on pathogen transport in soils have been conducted using homogenized natural soils in laboratory designed soil columns. these may not be a true representation of field conditions and diversifying the experimental conditions will aid the development of efficient, grower-level interventions that will effectively reduce the likelihood of on-field contamination of produce. there are dissenting opinions among experts on a variety of issues pertinent to produce safety. with regards to the factors, mechanisms as well as principles that aid competent internalization and persistence of pathogens on produce, there are many variations. the available studies are difficult to compare largely because they have been conducted under varying physicochemical circumstances, types of microcosms, experimental conditions and used distinct strains (shown in tables e ). most studies were conducted under disparate environmental conditions, and accurate weather data necessary to interpret results from the varying sources is lacking. study results for one crop variety may indeed not hold true for other varieties, for instance, data for apples may not necessarily apply to all pome fruit and data for romaine lettuce may not apply to all leafy greens. when possible, varieties exhibiting greater potential for pathogen survival should be selected for experimental investigations. another relevant consideration for crop selection is preference for varieties that are indigenous to the region in question. some other seemingly trivial controversial issues include whether outer leaves are significantly more likely than inner leaves to become contaminated via splash and whether or not the potential for survival on the abaxial side of leaves is higher than on the adaxial side. the implications of dormant, non-dividing 'persister' cells occurring in certain plant pathogens on the ability to withstand environmental stresses and extensive survival as well as the issues surrounding linked resistance is still an important research debate. also, even though atmospheric deposition seems to be an uncommon route of pathogenic contamination for produce, it has been documented as a potentially important route (beuchat and ryu, ; harris et al., ; mei soon et al., ) . it will be worthwhile exploring how relevant this is for produce safety. while many of the available studies have made stringent efforts to simulate produce cultivation circumstances, it is extremely challenging to create precise/accurate environmental conditions in a laboratory setting. most studies are conducted under controlled laboratory conditions. factors like the biological activity of the soil, manure, water and crops, soil and water chemistries as well as meteorological elements such as wind, uv intensity, temperature, rainfall are simply impossible to replicate under laboratory conditions. laboratory scale model systems may provide important details about the roles of environmental variables on pathogen growth and survival in agricultural environments, but the slightest tweaks in experimental protocols can affect pathogen survival in agroecosystems. unfortunately, actual field-based studies are subject to disruption from unforeseen environmental events such as weather extremes and damage triggered by biological agents including insects or onset of plant diseases. more field studies (where typical agricultural practices and conditions are closely simulated) are therefore, highly desirable to further understand the persistence phenomenon. safety and ethical issues however restrict the use of pathogens in the greenhouse and field-based research. strategies to improve existing biocontainment and decontamination processes should be developed and optimized as soon as possible. another possible solution is to develop and optimize strategies that will cater for the experimental variations in model system development. an assessment and identification of environmental variables that influence the fate of test organisms should be included in experimental designs. despite meticulous planning however, a field trial may fail to yield serviceable results due to factors that are out of the researcher's control. consequently, more replicate trials may need to be conducted. furthermore, agronomic and farm management practices are not uniform in all regions, and production practices significantly differ from region to region depending on seasons and weather patterns within the same region. these often depend on operation scale, type of farming practices et cetera. the risks associated with conventional cropping systems are bound to differ from those of systems that combine intensive livestock farming with arable farming. in addition to general studies, a case-by-case approach should be considered where possible (if financial and technical resources, as well as other circumstances, permit) because farming operations vary widely from farm to farm and this influences the potential for pathogen occurrence, survival, proliferation and dissemination. the potential of fresh produce to harbor pathogens is now well recognized, and fresh produce has been established as a vehicle of foodborne disease. the diverse and complex sources and routes of enteric pathogens to fruits and vegetables have been widely researched. the interplay of land use, water management, weather patterns and specific pathogen properties and sources have been illustrated to have significant consequences for the microbiological safety of fresh fruits and vegetables. attempts have been made to understand the general microbial profile of fresh produce, the behavior, fate and transport of pathogens, as well as their location in and on plant parts. the facts gleaned from these studies have been the subject of many extensive reviews. there is abundant information about the factors that affect the contamination and persistence of pathogens on fresh produce. in light of the available evidence, significant effort must be made to efficiently monitor and illustrate recent trends in the occurrence of foodborne diseases associated with the consumption of fruits and vegetables. partnerships and collaboration among all relevant stakeholders; commercial growers, public health practitioners, veterinary and food safety experts and field biologists is necessary in order to ensure the safety of fruits and vegetables delivered to consumers. on a final note, the need to control all potential pathogen entry pathways has been established and is being continuously stretched by regulators and other specialists. there are numerous other factors along the food production chain that may predispose produce to microbial contamination. however, it is of utmost importance to avoid and control microbial contamination of produce at the preharvest stage. this is because contaminated manure, water and soil have been shown to indeed contaminate produce, and decontamination of produce, polluted arable soil and groundwater has proven to be a very challenging and expensive endeavour. the authors declare no conflicts of interest. . . effect of soil properties and environmental variables on the incidence of pathogens in soils water resources projects and their environmental impacts molecular techniques for detecting and typing of bacteria, advantages and application to foodborne pathogens isolated from ducks kinetics of pathogen destruction during storage of dewatered biosolids association of escherichia coli o : h with houseflies on a cattle farm irrigation water quality for leafy crops: a perspective of risks and potential solutions surveillance for sporadic foodborne disease in the st century: the foodnet perspective inactivation of ms- phage and poliovirus in groundwater lowcost options for reducing consumer health risks from farm to fork where crops are irrigated with a farm to fork risk assessment for the use of wastewater in agriculture in influence of temperature on salmonella survival in hog manure slurry and seasonal temperature profiles in farm manure storage reservoirs potential mechanisms for achieving agricultural benefits from biochar application to temperate soils: a review establishing a role for bacterial cellulose in environmental interactions: lessons learned from diverse biofilm-producing proteobacteria are innate immune signaling pathways in plants and animals conserved? escherichia coli o survival following the surface and sub-surface application of human pathogen contaminated organic waste to soil fate of escherichia coli originating from livestock faeces deposited directly onto pasture survival of e. coli o : h in organic wastes destined for land application persistence of a salmonella enterica serovar typhimurium dt clone in a piggery and in agricultural soil amended with salmonella-contaminated slurry effect of irrigation regimes on persistence of salmonella enterica serovar newport in small experimental pots designed for plant cultivation role of soil, crop debris, and a plant pathogen in salmonella enterica contamination of tomato plants occurrence of generic escherichia coli, e. coli o and salmonella spp. in water and sediment from leafy green produce farms and streams on the central california coast large multistate outbreak of norovirus gastroenteritis associated with frozen strawberries presence and public health implications of listeria monocytogenes on vegetables listeria monocytogenes: incidence on vegetables. food contr. 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the authors are grateful to the national council for scientific and technological development (cnpq) (grant # / - ) and the coordination for the improvement of higher education personnel (capes) (grant ## p ) for the financial support. oluwadara alegbeleye wishes to acknowledge the financial support of conselho nacional de desenvolvimento cientifico e tecnol ogico (cnpq) through award grant number / - . supplementary data related to this article can be found at https://doi.org/ . /j.fm. . . . key: cord- -b aqwubh authors: futas, jan; oppelt, jan; jelinek, april; elbers, jean p.; wijacki, jan; knoll, ales; burger, pamela a.; horin, petr title: natural killer cell receptor genes in camels: another mammalian model date: - - journal: front genet doi: . /fgene. . sha: doc_id: cord_uid: b aqwubh due to production of special homodimeric heavy chain antibodies, somatic hypermutation of their t-cell receptor genes and unusually low diversity of their major histocompatibility complex genes, camels represent an important model for immunogenetic studies. here, we analyzed genes encoding selected natural killer cell receptors with a special focus on genes encoding receptors for major histocompatibility complex (mhc) class i ligands in the two domestic camel species, camelus dromedarius and camelus bactrianus. based on the dromedary genome assembly camdro , we characterized the genetic contents, organization, and variability of two complex genomic regions, the leukocyte receptor complex and the natural killer complex, along with the natural cytotoxicity receptor genes ncr , ncr , and ncr . the genomic organization of the natural killer complex region of camels differs from cattle, the phylogenetically most closely related species. with its minimal set of klr genes, it resembles this complex in the domestic pig. similarly, the leukocyte receptor complex of camels is strikingly different from its cattle counterpart. with kir pseudogenes and few lilr genes, it seems to be simpler than in the pig. the syntenies and protein sequences of the ncr , ncr , and ncr genes in the dromedary suggest that they could be human orthologues. however, only ncr and ncr have a structure of functional genes, while ncr appears to be a pseudogene. high sequence similarities between the two camel species as well as with the alpaca vicugna pacos were observed. the polymorphism in all genes analyzed seems to be generally low, similar to the rest of the camel genomes. this first report on natural killer cell receptor genes in camelids adds new data to our understanding of specificities of the camel immune system and its functions, extends our genetic knowledge of the innate immune variation in dromedaries and bactrian camels, and contributes to studies of natural killer cell receptors evolution in mammals. due to production of special homodimeric heavy chain antibodies, somatic hypermutation of their t-cell receptor genes and unusually low diversity of their major histocompatibility complex genes, camels represent an important model for immunogenetic studies. here, we analyzed genes encoding selected natural killer cell receptors with a special focus on genes encoding receptors for major histocompatibility complex (mhc) class i ligands in the two domestic camel species, camelus dromedarius and camelus bactrianus. based on the dromedary genome assembly camdro , we characterized the genetic contents, organization, and variability of two complex genomic regions, the leukocyte receptor complex and the natural killer complex, along with the natural cytotoxicity receptor genes ncr , ncr , and ncr . the genomic organization of the natural killer complex region of camels differs from cattle, the phylogenetically most closely related species. with its minimal set of klr genes, it resembles this complex in the domestic pig. similarly, the leukocyte receptor complex of camels is strikingly different from its cattle counterpart. with kir pseudogenes and few lilr genes, it seems to be simpler than in the pig. the syntenies and protein sequences of the ncr , ncr , and ncr genes in the dromedary suggest that they could be human orthologues. however, only ncr and ncr have a structure of functional genes, while ncr appears to be a pseudogene. high sequence similarities between the two camel species as well as with the alpaca vicugna pacos were observed. the polymorphism in all genes analyzed seems to be generally low, similar to the rest of the camel genomes. this first report on natural killer cell receptor genes in camelids adds new data to our understanding of specificities of the camel immune system and its functions, extends our genetic knowledge of the innate immune variation in dromedaries and bactrian camels, and contributes to studies of natural killer cell receptors evolution in mammals. keywords: camelid, leukocyte receptor complex, natural killer complex, snp, microsatellites introduction camels (camelus spp.) represent an important genus for a number of reasons. due to their adaptation to desert or semidesert regions, old world camels tolerate harsh conditions, which are inhospitable for many livestock species, including extreme temperatures and prolonged periods without access to food and water (reviewed in jirimutu et al., ) . as a result, they are of socioeconomic importance across the middle east, northern africa, and much of asia, where they are used for meat, milk, hides, transportation, and sport. the significance of camels as a sustainable livestock species is likely to continue, as many regions face increased temperatures and desertification as a result of climate change (megersa et al., ; watson et al., ) . concurrently, recent trends towards intensive production and the movement of camel production to peri-urban settings are altering the pathogen pressures to which these animals are exposed (abdallah and faye, ) . camels are also of importance with respect to a number of specific infectious diseases. for example, dromedaries (camelus dromedarius) are a natural host of middle east respiratory syndrome coronavirus, and transmission of the virus from camels to humans has been confirmed (gossner et al., ; hemida et al., ) . interestingly, significant differences exist between dromedaries and bactrian camels (camelus bactrianus) with regard to their susceptibility to foot and mouth disease (fmd), one of the most costly diseases of production animals worldwide; i.e., dromedaries are not susceptible to fmd and do not transmit infection (wernery and kinne, ) . furthermore, the immune system of camels has several unusual features. notable among these is the presence of homodimeric heavy chain antibodies (hamers-casterman et al., ) , not known to occur in any other mammalian family, which have both potential and realized applications in a variety of research, diagnostic, and therapeutic settings (muyldermans et al., ; de meyer et al., ; steeland et al., ) . the persistence of uniquely organized ileal peyer's patches into adulthood of the dromedary (zidan and pabst, ) is another example. additionally, productively rearranged dromedary t-cell receptor delta variable (trdv) (antonacci et al., ) and t-cell receptor gamma variable (trgv) (vaccarelli et al., ) genes undergo somatic hypermutation to generate a diversified repertoire of these genes. this mechanism has not been documented for t-cell receptor genes in other mammalian species and appears to compensate the more limited repertoire of trdv and trgv genes found in camels relative to other artiodactyls (ciccarese et al., ) . a further atypical aspect of the camelid immunogenome is the unusually low genetic diversity of the major histocompatibility complex (mhc) of the three species of old world camels in both class i (plasil et al., ) and class ii genes (plasil et al., ) . the immunological characterization of cellular components of the camel immune system is scarce mainly due to the small number of cross-reacting monoclonal antibodies raised against leukocyte antigens of humans (hussen et al., ) , bovines, and/ or other related species (mossad et al., ) available. this is also one of the reasons why natural killer cells and their functions in camelids have not been studied so far. natural killer (nk) cells constitute a heterogeneous lymphocyte population (allan et al., ) involved primarily in innate immune responses against intracellular pathogens and tumor cells. they also influence adaptive immune responses via the production of cytokines (vivier et al., ) and crosstalk with dendritic cells (hamerman et al., ) , play a role in placentation (parham and moffett, ) , and contribute to the recognition of allogeneic cells. the diversity of the nk cell receptor repertoire is essential to the performance of these multiple functions. the integration of activating and inhibitory signals originating from various surface receptors determines the activation status of an individual nk cell, providing the capacity to discriminate between self and non-self or altered self (lanier, ) . characterization of genes underlying receptors on the nk cell surface can significantly contribute to our understanding of the functional heterogeneity of nk cells. among them, nk cell receptors (nkr) of several gene families bind polymorphic mhc class i or mhc class i-like molecules to mediate nk cell function. due to functional relationships between mhc and nkr molecules, the underlying genes and genomic regions represent an important biological model in terms of their co-evolution in the context of pathogen pressures, disease, and survival (guethlein et al., ; carrillo-bustamante et al., ) . however, the current knowledge of mammalian nkr genes, in comparison with that of mhc regions, is rather fragmentary. two major genomic complexes encoding nkr, the natural killer complex (nkc) and the leukocyte receptor complex (lrc), have been identified in mammalian genomes. genes in the nkc represent receptors with c-type lectin-like extracellular domains; genes in the lrc code receptors with extracellular ligand-binding domains belong to the immunoglobulin superfamily (trowsdale et al., ) . despite these structural differences, some receptor families of both complexes are able to fulfil the same functions in terms of mhc class i recognition, downstream signaling, and mediation of nk cell activation/inhibition. to accomplish these ends, different nkr gene families expanded and diversified in different mammalian species, representing an example of convergent evolution in mammals (kelley et al., ; guethlein et al., ) . two immunologically well-defined species, humans and mice, have expanded structurally unrelated receptor families: humans use the killer-cell immunoglobulin-like receptors (kir) and leukocyte immunoglobulin-like receptors (lilr), both encoded within the lrc, whereas in mice the killer-cell lectinlike receptor (klr) genes of one family (klra, formerly ly ) are expanded in the nkc. a common sign of these expanded gene families, along with allelic variation of members, is haplotypic variation in the number of genes and pseudogenes in populations/ strains of same species (marsch et al., ; schenkel et al., ) . the gene content of the lrc (human vs. primates) and nkc (mouse vs. rat) is known to vary even between closely related mammalian species and families; as a result, knowledge of nkr genes in a number of important species remains fragmentary or missing. significant differences have been shown to exist within artiodactyls, as for example between cattle and pigs (sanderson et al., ; schwartz et al., ; schwartz and hammond, ) , but knowledge of the genes underlying camel nkr is lacking. in the context of our work on the camelid immunogenome, the objective of this study was to characterize the genomic content of nkc and lrc with special focus on genes encoding natural killer cell receptors for mhc class i ligands in the two domestic camel species, c. dromedarius and c. bactrianus. based on our new assembly camdro of the c. dromedarius genome , we characterized the nkc and lrc genomic regions and three natural cytotoxicity receptor genes (ncr , ncr , and ncr ). their gene contents and organization were compared to the national center for biotechnology information (ncbi) reference genomes for c. dromedarius (ncbi genome accession code gca_ . ) assembly prjna _ca_dromedarius_v . , c. bactrianus (gca_ . ) assembly ca_bactrianus_mbc_ . , and vicugna pacos (gca_ . ) assembly vicugna_pacos- . . . the selected orthologous genes were searched in two genomes of domesticated artiodactyl species, cattle bos taurus (gca_ . ) assembly bos_taurus_umd_ . . and pig sus scrofa (gca_ . ) assembly sscrofa . . various individual genomes for c. dromedarius (ncbi bioproject accessions: prjna , prjna , and prjna ) and c. bactrianus (prjna and prjeb ) were searched in publicly available whole-genome shotgun contigs for candidate microsatellite markers. likewise, individual whole genome sequencing reads for v. pacos (prjna and prjna ) were used to estimate single-nucleotide polymorphism (snp) variability in selected genes of alpacas. alongside automatic computational annotation of genes in camdro (see elbers et al., ) , selected unrecognized genes for nk receptors were manually annotated in the nkc and lrc genomic regions. first, we searched the c. dromedarius ncbi reference genome by tblastn algorithm of ncbi's blast ® for orthologous protein sequences to killer-cell lectin-like receptors recently identified in cattle as klr genes (schwartz et al., ) . second, the ab initio messenger rna (mrna) models complementary dna (cdna) for corresponding genes of all klr gene lineages in the dromedary reference genome were inspected for completeness using ncbi's conserved domain database cdd search and tmhmm server v. . for prediction of transmembrane helices in predicted proteins. the cdna for klre was incorrect; thus, the cattle sequence (schwartz et al., ) was used instead. these cdna models were aligned against the camdro chromosome sequence using ncbi's splign algorithm and also blast ® searched in our full genomic assembly camdro . identified genes were annotated accordingly. the splign algorithm was also used on scaffolds for the dromedary, bactrian camel, and alpaca ncbi reference genomes. the killer-cell immunoglobulin-like receptor kir genes and leukocyte immunoglobulin-like receptor lilr genes were searched on camdro chromosome by the tblastn algorithm . individual immunoglobulin-like (ig-like) domains and the cytoplasmic tail of bactrian -domain receptor lilr (xp_ ) served as query sequences. orthologous and paralogous sequences were found. the corresponding genomic and predicted cdna sequences were retrieved from the ncbi reference genomes for both camel species. the full length kir and lilr cdnas were cross-aligned with adequate scaffolds of reference genomes using splign . the predicted protein sequences were screened with cdd and tmhmm server v. . search . the gene sequences were blast ® searched in camdro . identified full-length genes were annotated, and gene fragments were recorded. the natural cytotoxicity receptor genes ncr , ncr , and ncr were blast ® searched in the full assembly camdro based on annotation of the dromedary ncbi reference genome. although there exist numbers of alternatively spliced variants for each gene/protein, we focused on cdna models of the longest variant ( table ) . the predicted dromedary, bactrian camel, and cattle cdna for ncr were incomplete; therefore, we used the sequence predicted from the white-tailed deer odocoileus virginianus texanus instead. during the process of manual annotation, we also characterized the nkc and lrc regions, and comparisons were made between c. dromedarius, c. bactrianus, and v. pacos. to allow comparisons with other studies and identification of orthologues, a standardized systematic nomenclature of nkr genes (schwartz et al., ) was used. however, when referring to original reports on human or mouse genes, both the original and standard gene names and symbols were used. the gene-specific primers encompassing full-length genes with flanking sequences were designed on ncbi c. bactrianus gene sequences using primer-blast and checked for specificity against reference genomes of both camel species. the list of primer pairs used is available in supplementary table . various compositions of pcrs and adequate thermal profiles used are summarized in supplementary table . pcr products were checked by . % agarose gel electrophoresis and quantified by invitrogen ™ qubit ™ fluorometer using qubit ™ dsdna br assay kit (thermo fisher scientific, waltham, ma, usa). they were kept frozen at − °c until massive parallel sequencing. each individual's long-range amplicon of genes under study was indexed separately during library preparation using the nexteraxt dna library preparation kit (illumina, san diego, ca, usa) and sequenced on a miseq ™ system (illumina, san diego, ca, usa) platform using the miseq ™ reagent kit v ( cycles) according to manufacturer's protocol in different runs. the quality of the raw sequencing reads was checked using fastqc . low quality read ends were removed by trimmomatic (bolger et al., ) (slidingwindow: : ) . only reads longer than bp were used for the mapping by bwa-mem (li, ) . the alignment was post-processed using samtools (li et al., ) (sorting and conversions), gatk (depristo et al., ) (indel realignment), and picard (pcr duplicates removal). further, only mappings with the minimal mapped length of bp, a maximum of % soft-clipping, and a maximum of % mismatches were kept using ngsutils (breese and liu, ) and bbmap . the repetitive sequences of di-, tri-, and tetra-nucleotides were searched in the nkc and lrc of the bactrian camel ncbi reference genome by repeatmasker . candidate microsatellites (msats) were identified by blast ® search of repetitions flanked with bp sequences in whole-genome shotgun contigs from three dromedaries and two bactrian camels. the most diverse sequences with unique occurrence in genome were chosen, and primers were designed in oligo primer analysis software v. . (molecular biology insights, colorado springs, co, usa). primer specificity was verified against the ncbi reference genomes of both camel species using ncbi's primer-blast . the pcr conditions were optimized for six msats, finalizing in one -plex (czm -czm ) and one single (czm ) pcr protocol. reaction compositions were as follows: . μl × taq buffer (top-bio, prague, czech republic), . u combitaq dna polymerase (top-bio, prague, czech republic), µm each http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ http://broadinstitute.github.io/picard/ https://sourceforge.net/projects/bbmap/ http://repeatmasker.org/cgi-bin/webrepeatmasker dntp (thermo fisher scientific, waltham, ma, usa), . μl of each primer of concentration μm ( table ) , and ng of genomic dna. pcr reaction mix was supplemented with pcr grade h o (top-bio, prague, czech republic) to a final volume of . μl. the thermal cycler abi verity well (applied biosystems, foster city, ca, usa) was used for amplification. the thermocycling conditions consisted of initial denaturation at °c for min; cycles of denaturation at °c for s, annealing at °c ( °c for czm ) for s, and elongation at °c for s; and final elongation at °c for min and holding at °c. all markers were then tested by fluorescent fragment analysis using applied biosystems ® abi prism and sized with genescan ™ liz ® size standard (thermo fisher scientific, waltham, ma, usa). the data obtained from the fragment analyzer were evaluated using the genemapper ® software v. . (thermo fisher scientific, waltham, ma, usa). the dromedary dna samples in this study were transferred from previous projects [austrian science fund (fwf)p -b and p -b ; pi: p. burger] and originated either from plucked hair, ethylenediaminetetraacetic acid (edta) blood collected commensally on whatman fta ® cards (sigma-aldrich, vienna, austria) during routine veterinary controls, or from dna extracts sent by collaborators under bilateral agreements. samples were imported with permits from the austrian ministry of labour, social affairs, health and consumer protection. all the bactrian camel samples were collected commensally during veterinary procedures for previous research projects (gacr / / ; pi: p. horin). details about the samples are provided in the supplementary table . selected genes of the nkc and lrc regions were genotyped by targeted resequencing of long-range pcr amplicons in both camel species. for comparison, individual genotypes in the same batch of genes except lilr genes were acquired for alpacas by data mining. two panels of animals were created from collections of samples originating from various populations. the c. dromedarius panel encompassed individuals coming from jordan (irbid), iran, saudi arabia (magaheem and wadda), canary islands, uae (dubai), kenya, sudan, nigeria, and kazakhstan. the genomic dna was previously isolated by phenol-chloroform extraction and kept frozen at − °c. the c. bactrianus panel consisted of individuals from three mongolian regions (bayan ovoo, galshar, and norovlin). the genomic dna was isolated from frozen archived whole blood samples by nucleospinblood © kit (macherey-nagel, düren, germany) according to the manufacturer's protocol. the genes of interest were isolated by pcrs on genomic dna, obtained amplicons were indexed to track individual samples, and then were sequenced in multiple illumina next-generation sequencing (ngs) runs and mapped to adequate reference sequence for amplicon (see above). a panel of four alpacas was created from publicly available whole-genome sequencing projects. the raw data of illumina ngs runs were downloaded from the european nucleotide archive database (ena accession numbers srr - , srr , srr , and srr ). the quality was checked using fastqc and kraken package (davis et al., ) . adapter and quality (phred < ) trimming was performed by cutadapt (martin, ) . bwa-mem (li, ) was used for the alignment, and the alignments were post-processed by samtools (li et al., ) (sorting and conversions), gatk (depristo et al., ) (indel realignment), and picard (pcr duplicates removal). alignments were further filtered using ngsutils (breese and liu, ) and bbmap for maximum soft-clipping ( %), maximum number of mismatches ( ), minimum mapped length ( bp), maximum soft-clipping ( %), and minimum mapping quality (mapq ). the reference sequences for mapping were retrieved from the v. pacos ncbi reference genome. most v. pacos sequences were framed by the primer sequences used in camels. https://www.ebi.ac.uk/ena the alignments of reads to the reference sequence were inspected using igv software . the variable positions (variant in homozygous state) and confirmed sequence variants (variant detected in heterozygous state) were treated as snps. they were written to consensus sequences for each animal using iupac nucleotide ambiguity codes in bioedit, version . . . (hall, ) along with insertions/deletions, and sequences from same animal species were manually aligned. the number of snps was counted using dnasp version . program (librado and rozas, ) , and frequency was calculated as percentage. the cdna sequences were in silico extracted in bioedit v. . . , based on mrna models for each gene ( table ) and checked by splign for completeness. haplotypes of each diploid individual were reconstructed for every panel and gene (cdna) separately using phase (stephens and donnelly, ) algorithm in dnasp v. . . the coding sequences were extracted in bioedit v. . . . the number of snps was counted in dnasp v. . , and the percentage of coding sequence length calculated. amino acid sequences were deduced from coding sequences in bioedit v. . . . the manually aligned predicted protein sequences were compared. sequences differing in at least one amino acid position were numbered and designated as different alleles of a particular gene. a phylogenetic analysis of sequences obtained by long-range pcr or data mining was done separately for nkc (c-type lectinlike) and lrc (immunoglobulin-like) genes. the nucleotide coding region sequences were aligned by clustalw multiple alignment algorithm in bioedit v. . . . one haplotype per gene was chosen for each species to represent the respective loci of the dromedary, bactrian camel, and alpaca. corresponding cattle and pig sequences retrieved from ncbi's genbank were used for a comparison. the maximum likelihood phylogenetic trees were constructed in mega (tamura et al., ) based on the tamura -parameter model and the partial deletion method ( % cutoff) with bootstrap repetitions (tamura, ) . | characteristics of microsatellite markers in natural killer complex (nkc) and leukocyte receptor complex (lrc) regions. type of repetition c. dromedarius amplicon size forward primer reverse primer the general organization of the two genomic regions, the natural killer complex (nkc) and the leukocyte receptor complex (lrc), containing genes and gene families encoding the nk cell receptors annotated based on the dromedary genome assembly camdro , was established and is represented in figure . the phylogenetic trees of the genes analyzed are shown in figures and for nkc and lrc genes, respectively. a summary of the allelic variants of the predicted proteins for the genes genotyped in dromedaries and bactrian camels is given in the nkc region encompassing approximately . mbp was localized on chromosome of camdro . twenty-six genes encoding receptors with the c-type lectin-like domain (ctld) of different lineages were identified in this region. no expansion of any klr gene family was observed in the dromedary genome. most klr genes clustered at one end of the region. this cluster contains five functional genes (klra, klrd, klre, klri, and klrk), two functional members of the klrc family, and two pseudogenes (klrh and klrj). klrc is the only gene family with two members sharing the same ctld but signaling oppositely: klrc codes for an inhibitory receptor, while klrc encodes an activating receptor. members of three families (klrb, klrf, and klrg) are located at the opposite end of the nkc region, separated from each other by a group of c-type lectin (clec) genes. two members of each of these families were found in the camdro dromedary genome. the genes klrb and klrb b have the standard structure of inhibitory receptors with a cytoplasmic tail containing an immunoreceptor tyrosinebased inhibitory motif (itim). genes encoding their putative ligands (clec d and clec f, respectively) were found in the vicinity. similarly, the genes klrf (nkp ) and klrf (nkp ) encoding activating receptors are located in close proximity of genes coding for their predicted ligands, aicl and kacl (clec b and clec a, respectively). klrg encoding an inhibitory receptor marks the boundary of the nkc. klrg was found outside of nkc, on chromosome . while in the c. dromedarius ncbi reference genome the nkc is split into two scaffolds (nw_ , nw_ ), it is contained within a single scaffold (nw_ ) in the c. bactrianus ncbi reference genome. the gene content and gene orientations are the same in both genomes. the only exception is the presence of a premature stop codon in the bactrian klrc sequence, which thus seems to be a pseudogene. since no expansion of klr gene families was observed, we focused on the allelic variation of inhibitory receptors supposed to recognize mhc class i ligands. due to their poor quality, some samples were not successfully amplified by long-range pcrs. because of an apparent mixed ancestry (c. dromedarius x c. bactrianus) of one c. dromedarius, heterozygous sequences of mixed origin were removed. consequently, different numbers of genotypes were retrieved for different genes as indicated in supplementary table . despite polymorphisms existing in the genomic and predicted mrna sequences (supplementary table ) , none of the tested genes were found to have more than three protein variants. five of the eight tested genes in c. bactrianus were monomorphic on the protein level. klra encodes an inhibitory receptor with one itim signaling motif in its cytoplasmic tail and a relatively long extracellular stalk region (over amino acids). two variants of this receptor molecule were predicted in c. dromedarius, sharing the same ctld but differing by one amino acid residue in the cytoplasmic tail. a klra variant with the same ctld occurs frequently in c. bactrianus. a second variant of bactrian klra differs by nine amino acids (one in the cytoplasmic tail, four in the stalk, and four in the ctld). klrc codes for an inhibitory receptor with two itims. three variants of the klrc protein were identified in each camel species. one klrc variant was shared by both camel species, and two additional variants in each species differed by only one amino acid. only two different ctld variants were present in each species. klrc codes for an activating receptor with a charged amino acid residue (lysine) in the transmembrane domain. in c. dromedarius, klrc appears to encode two variants of a functional receptor. in c. bactrianus, this gene seems to be monomorphic with a premature stop codon shortly after the origin of translation. the klrd gene product consists of a ctld with a short stalk and a transmembrane domain with no signaling motif. for klrd, one protein sequence common to both camel species was observed, with two additional variants found in c. dromedarius, differing by one amino acid each. all three camel genes, klrc , klrc , and klrd, have codons for cysteine residues in the stalk region of the protein, allowing formation of disulfide links and of heterodimers klrd/klrc and klrd/klrc . another pair of presumably interacting receptors forming noncovalent heterodimers is klre/klri. the striking features of old world camelid klre are the presence of sequence for an itim in the cytoplasmic tail of the protein and the existence of a second variant in c. dromedarius with a duplication of six amino acid residues in the ctld. in c. bactrianus, klre encodes only one variant of the protein sequence. the klri gene showed very limited polymorphism in both camel species at the genomic level, encoding only one protein variant with only one functional itim (and one mutated motif) in the cytoplasmic tail of the molecule. the predicted protein product of the klrj gene was identical in both camel species. another sequence variant in the dromedary camel differed by only one amino acid in the stalk region. according to the adopted mrna model, all these sequences contain a premature stop codon in the ctld of the protein. figure | organization of genomic regions encoding nk cell receptors in dromedary camel. the nkc was delineated by styk and klrg genes on chromosome (cdr ) of camdro between . and . mb. klr genes are represented as solid color triangles, klr pseudogenes as empty color triangles, and lectin-like clec or non-lectin genes as solid grey triangles. the lrc was found between the oscar and il genes on chromosome (cdr ) in the region . - . mb. lilr genes are represented as solid orange triangles, lilr pseudogenes as empty orange triangles, immunoglobulin-like domains or cytoplasmic domain gene fragments as orange rectangles, kir pseudogenes as empty blue triangles, other types of ig-like genes as solid color triangles, and different types of flanking genes as solid grey triangles. green rectangles mark positions of newly developed microsatellite markers czm -czm . july | volume | article frontiers in genetics | www.frontiersin.org figure | phylogeny of nucleotide coding sequences for nkc c-type lectin-like genes analyzed by long-range pcr/data mining. the percentage of trees (out of bootstrap replicates), in which the associated sequences clustered together is given at branch nodes. branch lengths are expressed as the number of substitutions per site. clusters of genes are highlighted according to the color scheme used in figure . haplotypes generated in this study were chosen (one per gene) to represent loci of camelus dromedarius (dromedary), camelus bactrianus (bactrian), and vicugna pacos (alpaca). comparisons were made to bos taurus (cattle) sequences retrieved from genbank (accession numbers: kx , kx , kx , kx , nm_ . , nm_ . , nm_ . , and nm_ . ) and sus scrofa (pig) sequences (accession numbers: nm_ . , nm_ . , xm_ . , xm_ . , xm_ . , xm_ . , and xm_ . ). july | volume | article frontiers in genetics | www.frontiersin.org figure | phylogeny of nucleotide coding sequences for lrc immunoglobulin-like genes analyzed by long-range pcr/data mining. the percentage of trees (out of bootstrap replicates) in which the associated sequences clustered together is given at branch nodes. branch lengths are expressed as the number of substitutions per site. haplotypes generated in the study were chosen (one per gene) to represent loci (colored) of camelus dromedarius (dromedary), camelus bactrianus (bactrian), and vicugna pacos (alpaca). the nomenclature for the camelid lilr gene family is provisional and will change when a complete assembly of this region is available. some alpaca lilr genes were not included due to an incomplete resolution of this family in the reference genome. the alpaca's lilra -ig sequence was retrieved from genbank (accession number xm_ . ). a selection of bos taurus (cattle) sequences (accession numbers: nm_ . , nm_ . , nm_ . , nm_ . , nm_ . , nm_ . , xm_ . , xm_ . , xm_ . , xm_ . , xm_ . , and xm_ . ) and all functional sus scrofa (pig) sequences (accession numbers: nm_ . , nm_ . , nm_ . , xm_ . , xm_ . , xm_ . , and xm_ . ) were used for comparison. july | volume | article frontiers in genetics | www.frontiersin.org klrk in both camel species codes for a functional activating receptor with a charged amino acid (arginine) in the transmembrane region. two proteins with variant ctld were recognized in c. dromedarius, while two proteins in c. bactrianus share the same ctld as one of the dromedary klrk. in the phylogenetic trees obtained, all nkc genes sequences of both camel species clustered with their putative orthologues in alpaca, cattle, and pig (figure ) . the lrc region of approximate length . mbp was localized to chromosome of camdro . fifteen full-length genes encoding receptors containing immunoglobulin-like (ig-like) domains of various lineages were identified in the lrc region. besides two kir pseudogenes containing itim domains and an expanded lilr gene family, a singular ig-like receptor was found in the vicinity of fcar and ncr , located between nlrp and nlrp . it comprises two ig-like domains different from those of the lilr and the kir genes and has a long cytoplasmic tail with two itims. it is a novel type of lrc gene, similar to a gene recently identified in pigs (schwartz and hammond, ) . based on its structure, this inhibitory type of receptor gene may be functional, similarly to pigs. the expanded family of lilr genes is organized in two distinct clusters. the first region spanning approximately kb is located between the genes rps and cdc ep . this region contains three putatively functional genes, lilrb , lilrb , and a lilrb -like sequence. lilrb codes for an inhibitory receptor with three ig-like domains. lilrb and lilrb -like each encode four ig-like domains and a cytoplasmic domain with itims. several fragmented sequences containing ig-like domains were identified within this region as well. the second lilr region spanning approximately kb is located between lair and the two kir pseudogenes. three full-length lilr genes and a pseudogene were identified in this region. lilrb codes for an inhibitory receptor; it comprises four ig-like domains, a transmembrane region, and a cytoplasmic tail with two intact itims. lilrb also codes for an inhibitory receptor, but with only two ig-like domains. in addition, there are a potentially functional activating lilra gene and a lilrb pseudogene (containing an itim sequence) located in the same region. the predicted lilra contains four ig-like domains in its extracellular region but has no signal peptide sequence. the cytoplasmic domain is short and contains no itims. several fragments with complete or partial ig-like domains were also found in this region. in the current c. dromedarius ncbi reference genome, the lrc is split amongst at least four scaffolds (nw_ , nw_ , nw_ , and nw_ ). they matched our camdro assembly in terms of the number and orientation of orthologous non-ig-like genes recognized by automatic annotation. single ig-like genes and two kir pseudogenes, but not expanded lilr genes, were unraveled. the bactrian lrc of ncbi reference genome is contained within a single scaffold (nw_ ), but assembly of the expanded lilr genes is not resolved and lilrb -like is missing. the overall lrc organization in the c. bactrianus reference genome is the same as that of the lrc of dromedaries (figure ) . based on pcr and resequencing of representative panels of c. dromedarius and c. bactrianus, individual genotypes could be successfully identified for most of the genes analyzed. however, the amplification of kir dl sequences in bactrian camels provided only limited numbers of sequences (supplementary table ) . similar to nkc genes, some sequences from one c. dromedarius individual were removed due to their mixed origin. the kir dl gene contains a -bp deletion in the exon for the third ig-like domain, causing a frameshift and a premature stop codon. this deletion is identical in both camel species. the locus kirdp contained sequences with premature stop codons and frameshift mutations in both camel species. the same was found in ncbi reference genomes. therefore, we assigned kirdp and kir dl sequences provisionally as pseudogenes in both species. in contrast to the low polymorphism of the nkc receptors, higher numbers of variable amino acid sites were found within kir dl. an in silico -bp insertion resulted in three and seven predicted fulllength protein variants in bactrian camels and dromedaries, respectively ( table ) . lilrb encodes a protein of similar structure to kir dl, with three constant-type ig-like domains and two functional itims in its cytoplasmic tail. unlike other members of the lilr family coding for receptors with four ig-like domains, lilrb has no variable-type ig-like domain between its first and second domains. nevertheless, in c. dromedarius, only three variants with minor changes in their amino acid sequences were found, and in c. bactrianus, this gene appears to be monomorphic. the gene lilrb of c. dromedarius encodes a functional inhibitory receptor with four ig-like domains and two itims. six variants recognized in the panel of dromedaries share the same ig-like domains and differ only by two amino acids in the transmembrane region and one in the cytoplasmic tail. all lilrb -like sequences obtained by pcr were identical to lilrb sequences retrieved from the same dromedaries' dnas. in c. bactrianus, lilrb encodes two mrnas with a premature stop codon in the first ig-like domain and differs by - amino acid positions from its dromedary counterparts. no pcr products were obtained for lilrb -like from the bactrian camel panel. lilrb encodes a receptor with % identity ( % similarity) to lilrb in c. dromedarius. all four protein variants had only two different ig-like domains (the second and third) with one amino acid change each. five variants of the bactrian lilrb had inter-species specific positions with another amino acids differing within species. sequences of two activating lilra genes were retrieved by pcr. one of them, containing two ig-like domains, arginine in the transmembrane region, and a long cytoplasmic tail, but with the first itim deleted and the second mutated, was provisionally named as lilra -ig. both camel species shared one variant of the lilra -ig protein, and two additional variants were present in the dromedary. the second activating gene was designated lilra -ig as it contained four ig-like domains, arginine in its transmembrane region, and a short cytoplasmic tail with no signaling motif. three variants of the dromedary and four variants of the bactrian camel lilra -ig protein are very similar, differing in only six amino acid positions. one specific variant with an in-frame deletion of amino acids in the third ig-like domain was shared between species. the phylogenetic tree constructed for the coding regions of the camel lrc genes (figure ) and their homologs in alpaca, cattle, and pig revealed three main clusters of genes characterized by the overall structure of the encoded receptor. the first cluster grouped genes with four ig-like domains receptors (lilrs). the second group was a cluster of genes coding for receptors with three or two ig-like domains (kirs and lilrb ). the third cluster was formed by genes encoding receptors with two ig-like domains (ncr and lilra -ig). within the first cluster, three distinct camel genes, lilrb , lilrb , and lilra -ig, clustered with various cattle and pig lilr genes. likewise, various cattle and pig kir genes formed a cluster with camelid kir dl genes. this cluster was related with the cluster of camelid lilrb genes, while the cluster containing ncr gene sequences was related to the camelid lilra -ig genes. as no cattle and/or pig homologs of camelid lilrb and lilra -ig genes could be identified in the reference genomes of these two species, they did not appear in the trees constructed. the predicted proteins of the ncr , ncr , and ncr genes were studied as potentially activating immunoglobulin-like receptors for various ligands different from mhc class i. the ncr gene is located within the lrc, and the structure of camelid ncr is similar to the structure of this gene's products in other species, with two extracellular ig-like domains and a charged residue (arginine) in the transmembrane domain, which allows its interaction with activating adaptor proteins. two allelic variants were identified in each camel species that differed from each other by only one or two amino acids. c. bactrianus possessed one additional variant containing a premature stop codon in the first ig-like domain. the ncr gene is located on chromosome of camdro . it encodes a functional receptor with one extracellular ig-like domain and a charged residue (lysine) in the transmembrane domain. one allelic variant of the receptor is shared by both camel species. another variant, found only in c. dromedarius, has a premature stop codon in the stem region of the putative molecule. two additional variants were identified in c. bactrianus, differing by one amino acid each. the ncr gene is also located on chromosome , within the mhc region. all sequences in both camel species contained the same two premature stop codons; this gene thus seems to be nonfunctional in camels. the alpaca is evolutionarily the most closely related species to the old world camels. the v. pacos ncbi reference genome contains two scaffolds (nw_ and nw_ ) with clec and klr genes. the gene content and organization of the alpaca nkc region was found to be very similar to that of the camel nkc, with similarities of amino acid sequences ranging from % to %. based on publicly available alpaca genomes, the extent of polymorphism of genomic as well as of protein sequences was higher than in either of the camel species. five protein variants were predicted for klra, klrc , klrc , and klre. the amino acid changes were concentrated in the ctld and the stem of klra and in the ctld in klrc . in contrast, they were evenly distributed throughout klrc . klre coded for four functional protein variants with only three different ctlds, and amino acid changes were concentrated mostly in the cytoplasmic tail. one allele had a -bp insertion leading to frameshift and a premature stop codon in the ctld part of the receptor. the same itim motif as in camels was recognized in all five variants. the three variants of klrd identified differed by one amino acid each in the stem region of the receptor and thus shared the same ctld. klri also coded for three variants with the same ctld, differing only in the cytoplasmic tail. due to a non-synonymous substitution, a second itim was recreated in one of the variants. the five protein variants predicted for klrj differed in their ctlds, although all of them contained the same premature stop codon according to the mrna model. klrk haplotypes coded for a potentially functional activating receptor (arginine in the transmembrane region). only one amino acid difference was observed in two ctld types shared by the four klrk protein variants. the alpaca lrc region is fragmented amongst numerous scaffolds of the ncbi reference genome. one of them (nw_ ) contains a four-domain lilrb gene, kirdp, kir dl, fcar, ncr , gp , and a novel ig-like gene, while another scaffold (nw_ ) contains lilrb , comprising three ig domains, and lilrb, with four ig domains. only fragments of lilr genes could be found in the rest of the relevant scaffolds. like in camels, kirdp contains various frameshifts. in contrast, kir dl has retained an intact genomic sequence and is thus likely to encode a functional inhibitory receptor, but with only one functional itim. the second itim is mutated in all protein variants. seven variants of kir dl were identified, differing in amino acid positions in total. these sites are equally distributed throughout the molecule. lilrb also codes for a potentially functional inhibitory receptor with three ig-like domains and seven identified variants. they differed in positions located in two of the ig-like domains, the stem and the cytoplasmic tail. the protein homology in comparison with dromedary camel kir dl and lilrb counterparts reached %. the gene ncr of the alpaca encodes a functional activating receptor with a charged amino acid residue (arginine) in the transmembrane region. the three detected allelic variants differed only in the stem and transmembrane regions. ncr codes for a functional activating receptor (lysine in the transmembrane region) as well. three protein variants with minor changes in the signal peptide and the cytoplasmic tail not affecting their potential function were identified. all identified ncr sequences have premature stop codons in the ig-like domain. the amino acid sequence similarity to camel sequences was %. in contrast to the rather conservative organization of the mammalian major histocompatibility complexes, natural killer cell receptor genes and their complex genomic regions are evolutionarily flexible. several different types of genomic organization of the nkr regions have been recognized in mammals (martin et al., ; hao et al., ; guethlein et al., ) , and sometimes striking differences have been observed between related taxa (kelley et al., ; sanderson et al., ; schwartz et al., ; schwartz and hammond, ) . therefore, studies of genes encoding nk cell receptors may contribute to our understanding of the heterogeneity of nk cell functions in particular mammalian species. at the same time, these genes and especially complex genomic regions such as nkc and lrc represent a relevant model for evolutionary biology. characterization of nkr genes in so far poorly studied species and/or families can bring new information on evolutionary mechanisms governing this part of the mammalian immunogenome. despite the importance of camels as a model for immunogenetic studies (ciccarese et al., ) , virtually nothing is known about nk cells in camels in terms of their morphology, functions, their surface receptors, and/or underlying genes. in this context, this study represents the first report on the nkc and lrc genomic regions and on ncr genes in old world camels and their comparison with a new world camelid, the alpaca. whole genome sequences of old world camels, c. dromedarius, c. bactrianus, and camelus ferus (jirimutu et al., ; wu et al., ; fitak et al., ) and of the alpaca v. pacos (ncbi genome accession gca_ . ) are currently available. however, their annotation is rather fragmentary and largely composed of predicted sequences generated in silico, based on homologies and sequence similarities with other mammalian species. taking into consideration the quality of resources including the availability of biological material, we focused on the two domestic camel species, c. dromedarius and c. bactrianus. even for these species, however, the current status of whole genome assemblies proved to be insufficient for a correct annotation of nkr genes, especially of the lrc, containing multiple copies of sometimes highly similar sequences and exhibiting copy number variations. therefore, the major resource for our analyses was a new genome assembly of the c. dromedarius genome obtained by a combination of several long-read sequencing techniques and bioinformatic approaches . in all genes selected for sequence analyses, the genomic sequences of nkr genes were highly similar in both camel species studied as well as to available alpaca sequences. such a high sequence similarity was observed for a number of other genes and was also characteristic for mhc class ii sequences (plasil et al., ) . therefore, it seems that pcr failures observed in some cases probably do not indicate polymorphisms in the primer binding site. taking also into consideration the generally low polymorphism of the camel genomes (fitak et al., https://www.ncbi.nlm.nih.gov/genome ), the occurrence of a putative polymorphic variant on both chromosomes is not too likely. moreover, the pcr failures concerned mostly the loci klrc and kir dl in the bactrians, which seem to be both pseudogenes, so we have not explored them further for the purposes of this study. nevertheless, both loci merit to be further investigated in the future. information that the monomorphic status of the bactrian klrc could be explained by allelic drop-out or existence of copy number variation, i.e., partial/total deletion of klrc from some bactrian nkc haplotypes, and that polymorphic amino acid positions within the kir dl sequences were concentrated in the second immunoglobulin-like domain, known to interact with mhc class i ligands in mammals and in the stalk region of the molecule, need to be explored. another potential technical problem is the use of long-range pcrs for amplifying related members of a gene family, which may produce chimeric products. the nkc genes analyzed here were in majority single (not duplicated) genes with characteristic sequences. the lrc genes analyzed, with only lilr genes as members of expanded gene family/families, were different to such an extent that we could distinguish them. in addition, both types of phylogenetic trees clearly supported the individuality of each gene. moreover, sequences successfully amplified as a whole matched the reference sequences. the remaining ones were amplified in two pieces, and again, they matched the reference sequences. although we have checked the overlapping sequences of two-piece pcrs and they did not indicate more polymorphisms, we cannot strictly exclude the possibility that such a sequence could be composed of pieces of two different yet highly homologous loci, taking also into consideration numerous fragments of lilr genes observed in camdro . the genomic organization of the nkc region of camelids differs from cattle, the phylogenetically most closely related species, whose nkr genes have been studied so far. while in cattle an expansion of klrc and klrh genes was reported (schwartz et al., ) , the minimal set of klr genes observed in camelids resembles the genomic organization of the nkc of the domestic pig. similarly, the leukocyte receptor complex of camels is strikingly different from the cattle lrc containing expanded kir (sanderson et al., ) and lilr genes (hogan et al., ) . in camels, the lrc with non-expanded kir genes and several pseudogenes seems to be even less complex than in the pig (schwartz and hammond, ) . within the natural killer complex, all types of klr genes identified in mammals (hao et al., ) were found. none of them apparently expanded into a large family; the maximum number of members within a family was two. similar to other mammalian species, the klra gene codes for a homodimeric type ii inhibitory receptor (ly ) (dimasi and biassoni, ) , klrc encodes an inhibitory receptor with two itims motifs (nkg a) (vance et al., ) , klrc encodes an activating receptor (nkg c) , and klrg codes an inhibitory receptor for cadherin molecules (ito et al., ) . these data suggest that the function of these molecules could be very similar to human and other mammalian nk receptors, especially in terms of their capacity to form heterodimers cd / nkg a (klrd/klrc ), cd /nkg c (klrd/klrc ) (braud et al., ) , and/or klre/klri (saether et al., ) . in humans, the heterodimers cd /nkg a (klrd/klrc ) and cd /nkg c (klrd/klrc ) recognize a relatively low polymorphic non-classical mhc class i ligand hla-e, and their polymorphism is also rather low (braud et al., ) . contrary to rats, in which klrh recognize mhc class i ligand (daws et al., ) , camelid klrh sequences represent only remnants of a full gene sequence. although mrna for bovine klrj was described (storset et al., ) , the precise splicing of camelid klrj and possible expression as a functional receptor remains to be verified. the low polymorphism of camelid klrk is comparable to the limited polymorphism of this gene in humans and mice encoding an activating receptor nkg d for diverse ligands (reviewed in lanier, ) . the genomic organization of the nkc is similar in both domestic camel species and in v. pacos. the functionally important polymorphism of nkc genes is limited, with one monomorphic gene and six genes with two to three allelic protein variants in c. dromedarius. an even higher number of monomorphic klr genes (three functional and two potential pseudogenes) was observed in c. bactrianus, and its klrc seems to be a pseudogene. it is not clear how this low nkc variation can be related to the fact that no "hla-e-like" molecule has been found to date outside of simians and rodents and to our recent finding that the mhc gene cluster containing hla-e in humans has been lost in camels, similarly to cattle and pigs (plasil et al., ) . interestingly, v. pacos seems to be more polymorphic in nkc, at both the genomic and protein levels, despite the limited number of individual genomes analyzed. within the lrc region, no kir genes have expanded, while lilr genes expanded both activating and inhibitory family members. as for the nkc, the same overall organization of the lrc with fcar, ncr , kir, and lilrb genes, three lilrb genes encoding a -ig-like domain receptor, low variable kir dl and lilrb , and unresolved lilr gene fragments was observed in c. bactrianus. the polymorphism of kir dl was similar in the dromedary (seven possible protein variants) and the alpaca (seven functional protein variants), while the alpaca seems to be more variable in the lilrb gene (seven vs. three protein variants, respectively). unfortunately, we were unable to analyze further alpaca lilr genes, mainly due to a lack of correct fulllength gene reference sequences and/or to a low coverage of ngs reads available in public databases. ncr , ncr , and ncr are the major activating receptors on human nk cells (reviewed in koch et al., ) . the ncr gene is located within the lrc; ncr and are located on the human chromosome , with ncr mapping within the mhc (lanier, ) . the chromosome location of ncr , ncr , and ncr genes in the dromedary corresponds to the human homologues, suggesting an orthologous nature of the ncr sequences retrieved. however, only ncr and ncr have a structure of functional genes, while ncr appears to be a pseudogene. the ncr , ncr , and ncr genes of c. bactrianus and v. pacos are very similar in terms of their genomic locations, sequence homologies, and genomic variation. we are aware of the limitations due to the quality of the current assembly; however, the clusters of the lilr sequences identified in the phylogenetic trees indicated, similarly to nkc genes, the individuality of each of the genes. although the purpose of this study was to outline the general organization of the two nkr complexes in terms of major gene families represented, and their location within nkc and lrc, further work is needed to definitively resolve the complex structure of lrc region, and a detailed characterization of individual lilr genes and pseudogenes is needed. taken together, this first report on nkr genes in camelids revealed features characteristic for nkc and lrc of tylopoda. despite close phylogenetic relationships to cattle, important differences in the nkc and lrc genomic organization and their polymorphism were observed. on the other hand, many similarities with pigs were found. the data presented here increase our genetic knowledge of the innate immune variation in dromedaries and bactrian camels and contribute to studies of nkr evolution in mammals. the results of this project add to our understanding of specificities of the camel immune system and its functions and represent a prerequisite for future investigations on mhc/nkr interactions in health and disease. the camel datasets generated for this study can be found in the ncbi´s genbank ® . the sequences obtained for nkc genes have accession numbers mk -mk . the lrc gene sequences for both camel species have accession numbers mk -mk . the ncrs sequences have accesion numbers mk -mk . the dromedary dna samples in this study were transferred from previous projects [austrian science fund (fwf) p -b and p -b ; pi: pb] and originated either from plucked hair, edta blood collected commensally on whatman fta cards (sigma-aldrich, vienna, austria) during routine veterinary controls or from dna extracts sent by collaborators under bilateral agreements. samples were imported with permits from the austrian ministry of labour, social affairs, health and consumer protection. all bactrian camel samples were collected commensally during veterinary procedures for previous research projects (gacr / / ; pi: ph). all samples were collected by a licensed veterinarian in compliance with all ethical and professional standards. jf made nkc annotation, designed primers, carried out pcr for nkr genes, and analyzed data. jo made all ngs mappings. aj made lrc annotation and carried out pcr for lilrb . je provided camdro whole genome sequence and annotation. jw made microsatellite definition and analysis. ak designed the microsatellite project. pb designed the project. ph designed the typology of camel farming system in saudi arabia cattle nk cell heterogeneity and the influence of mhc class i expression and genomic analyses of camelus dromedarius t cell receptor delta (trd) genes reveal a variable domain repertoire enlargement due to cdr diversification and somatic mutation trimmomatic: a flexible trimmer for illumina sequence data hla-e binds to natural killer cell receptors cd /nkg a, b and c ngsutils: a software suite for analyzing and manipulating next-generation sequencing datasets the evolution of natural killer cell receptors characteristics of the somatic hypermutation in the camelus dromedarius t cell receptor gamma (trg) and delta (trd) variable domains kraken: a set of tools for quality control and analysis of high-throughput sequence data identification of an mhc class i ligand for the single member of a killer cell lectin-like receptor family, klrh nanobody-based products as research and diagnostic tools a framework for variation discovery and genotyping using nextgeneration dna sequencing data structural and functional aspects of the ly natural killer cell receptors improving illumina assemblies with hi-c and long reads: an example with the north african dromedary the de novo genome assembly and annotation of a female domestic dromedary of north african origin human-dromedary camel interactions and the risk of acquiring zoonotic middle east respiratory syndrome coronavirus infection co-evolution of mhc class i and variable nk cell receptors in placental mammals bioedit: a user-friendly biological sequence alignment editor and analysis program for windows / /nt nk cells in innate immunity naturally occurring antibodies devoid of light chains heterogenous but conserved natural killer receptor gene complexes in four major orders of mammals dromedary camels and the transmission of middle east respiratory syndrome coronavirus (mers-cov) characterisation of bovine leukocyte ig-like receptors reactivity of commercially available monoclonal antibodies to human cd antigens with peripheral blood leucocytes of dromedary camels (camelus dromedarius) killer cell lectin-like receptor g binds three members of the classical cadherin family to inhibit nk cell cytotoxicity genome sequences of wild and domestic bactrian camels comparative genomics of natural killer cell receptor gene clusters activating natural cytotoxicity receptors of natural killer cells in cancer and infection nk cell receptors nk cell recognition nkg d receptor and its ligands in host defense association of dap with activating cd /nkg c nk cell receptors aligning sequence reads, clone sequences and assembly contigs with bwa-mem. arxiv® the sequence alignment/map format and sam tools dnasp v : a software for comprehensive analysis of dna polymorphism data killer-cell immunoglobulin-like receptor (kir) nomenclature report leukocyte ig-like receptor complex (lrc) in mice and men cutadapt removes adapter sequences from high-throughput sequencing reads livestock diversification: an adaptive strategy to climate change and rangeland ecosystem changes in southern ethiopia identification of monoclonal antibody reagents for use in the study of immune response in camel and water buffalo camelid immunoglobulins and nanobody technology variable nk cell receptors and their mhc class i ligands in immunity, reproduction and human evolution the major histocompatibility complex in old world camelids and low polymorphism of its class ii genes the major histocompatibility complex of old world camelids: class i and class i-related genes klre/i and klre/i : a novel pair of heterodimeric receptors that inversely regulate nk cell cytotoxicity definition of the cattle killer cell ig-like receptor gene family: comparison with aurochs and human counterparts the ly gene family. a brief guide to the nomenclature, genetics, and role in intracellular infection the evolution of the natural killer complex; a comparison between mammals using new high-quality genome assemblies and targeted annotation the unique evolution of the pig lrc, a single kir but expansion of lilr and a novel ig receptor family nanobodies as therapeutics: big opportunities for small antibodies a comparison of bayesian methods for haplotype reconstruction from population genotype data natural killer cell receptors in cattle: a bovine killer cell immunoglobulin-like receptor multigene family contains members with divergent signaling motifs estimation of the number of nucleotide substitutions when there are strong transition-transversion and g + c-content biases mega : molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods the genomic context of natural killer receptor extended gene families generation of diversity by somatic mutation in the camelus dromedarius t-cell receptor gamma variable domains mouse cd /nkg a is a natural killer cell receptor for the nonclassical major histocompatibility complex (mhc) class i molecule qa- (b) innate or adaptive immunity? the example of natural killer cells camels and climate resilience: adaptation in northern kenya foot and mouth disease and similar virus infections in camelids: a review camelid genomes reveal evolution and adaptation to desert environments unique microanatomy of ileal peyer's patches of the one humped camel (camelus dromedarius) is not age-dependent project and the nkr study. jf and ph drafted the manuscript. jo, aj, and jw wrote paragraphs. je and pb edited the manuscript. all authors read, commented on, and approved the final version of the manuscript. key: cord- -v ci n authors: domingo, esteban title: introduction to virus origins and their role in biological evolution date: - - journal: virus as populations doi: . /b - - - - . - sha: doc_id: cord_uid: v ci n viruses are diverse parasites of cells and extremely abundant. they might have arisen during an early phase of the evolution of life on earth dominated by ribonucleic acid or rna-like macromolecules, or when a cellular world was already well established. the theories of the origin of life on earth shed light on the possible origin of primitive viruses or virus-like genetic elements in our biosphere. some features of present-day viruses, notably error-prone replication, might be a consequence of the selective forces that mediated their ancestral origin. two views on the role of viruses in our biosphere predominate; viruses considered as opportunistic, selfish elements, and viruses considered as active participants in the construction of the cellular world via the lateral transfer of genes. these two models have a bearing on viruses being considered predominantly as disease agents or predominantly as cooperators in the shaping of differentiated cellular organisms. to approach the behavior of viruses acting as populations, we must first examine the diversity of the present-day biosphere and the physical and biological context in which primitive viral forms might have arisen. evolution pervades nature. thanks to new theories and to the availability of powerful instruments, new experimental procedures, and increasing computing powerd which together constitute the very roots of scientific progressdwe know that the physical and biological worlds are constantly evolving. several classes of energy have gradually shaped matter and living entities, basically as the outcome of random events and darwinian natural selection in its broadest sense. the identification of dna as the genetic material and the advent of genomics in the second half of the twentieth century unveiled an astonishing degree of diversity within the living world that derives mainly from combinations of four classes of nucleotides. biodiversity, a term coined by o. wilson in and emphasized by t. lovejoy and others, is a feature of all living beings, be differentiated multicellular organisms, singlecell organisms, or subcellular genetic elements, among them the viruses. next-generation sequencing methods developed at the beginning of the st century allow thousands of sequences from the same biological sample (a microbial community in a soil or ocean sample, a tumor, or an infected host) to be determined. these procedures have documented the presence of myriads of variants in a "single biological entity" or in "communities of biological entities." differences extend to individuals that belong to the same biological group, be it homo sapiens, drosophila melanogaster, escherichia coli, or human immunodeficiency virus type . no exceptions have been described. diversity is extensive and not restricted to the genotypic level. it also affects phenotypic traits. during decades, in the first half of the twentieth century, population genetics had as one of its tenets that genetic variation due to mutation had for the most part been originated in a remote past. it was generally thought that the presentday diversity was essentially brought about by the reassortment of chromosomes during sexual reproduction. this view was weakened by the discovery of extensive genetic polymorphisms, first in drosophila and humans, through secondary analyses of electrophoretic mobility of enzymes, detected by in situ activity assays to yield zymograms that were displayed as electromorphs. these early studies on allozymes were soon extended to other organisms. assuming that no protein modifications had specifically occurred in some individuals, the results suggested the presence of several different (allelic) forms of a given gene among individuals of the same species, be it humans, insects, or bacteria. in the absence of information on dna nucleotide sequences, the first estimates of heterogeneity from the numbers of electromorphs were collated with the protein sequence information available. an excellent review of these developments (selander, ) ended with the following premonitory sentence on the role of molecular biology in unveiling evolutionarily relevant information: "considering the magnitude of this effect, we may not be overfanciful to think that future historians will see molecular biology more as the salvation for than, as it first seemed, the nemesis of evolutionary biology." the conceptual break was confirmed and accentuated when molecular cloning and nucleotide sequencing techniques produced genomic nucleotide sequences from multiple individuals of the same biological species. variety has shaken our classification schemes, opening a debate on how to define and delimit biological "species" in the microbial world. from a medical perspective, it has opened the way to "personalized" medicine, so different are the individual contexts in which disease processes (infectious or other) unfold. diversity is a general feature of the biological world, with multiple implications for interactions in the environment, and also for human health and disease (bernstein, ). viruses (from the latin "virus," poison) are no exception regarding diversity. the number of different viruses and their dissimilarity in shape and behavior is astounding. current estimates indicate that the total number of virus particles in our biosphere reaches , exceeding by one order of magnitude the total number of cells. viruses are found in surface and deep-sea and lake waters, below the earth surface, in any type of soil, in deserts, and in most environments designated as extreme regarding ionic conditions (i.e., hypersaline) and temperature (thermophilic) (breitbart et al., ; villarreal, ; lopez-bueno et al., ; box . ) . the viruses that have been studied are probably a minimal and biased representation of those that exist, with at least hundred thousand mammalian viruses awaiting discovery (anthony et al., ; epstein and anthony, ) . this is because high-throughput screening procedures have only recently become available, and also because prevention of disease has provided the main incentive to study viruses. disease-associated viruses are those most described in the scientific literature. current virology poses some general and fascinating questions, which are not easily approachable experimentally. here are some: • what is the origin of viruses? • did they originate before or after a cellular world was in place? • what selective forces have maintained multiple viruses as parasites of unicellular and multicellular organisms? • do viruses exist essentially as selfish parasites, or have they played constructive roles in the biosphere? • why have a few viral forms not outcompeted most other forms? or have they? • have viruses been maintained as modulators of the population numbers of their host species? • does virus variation play a role in the unfolding of viral disease processes? • is it quasispecies dynamics that characterize rna and many dna viruses, a remnant of an adaptive strategy that presided all life forms in the remote past? • is the behavior of present-day viruses at the population level only an inheritance of their origins or a present-day necessity? this book deals with some of these issues, mainly those that are amenable to experimental testing. topics covered include molecular mechanisms of genetic variation, with emphasis on high mutation rates, darwinian principles acting on viruses, quasispecies dynamics and its implications, consequences for virus-host interactions, fitness as a relevant parameter, experimental model systems in cell culture, ex-vivo and in vivo, long-term virus evolution, the current situation of antiviral strategies to confront quasispecies swarms, and conceptual extensions of quasispecies to nonviral systems. these subjects have as a common thread that darwinian natural selection has an immediate imprint on them, observable in the time scale of days or even hours. the capacity for rapid evolution displayed by viruses represents an unprecedented and often underappreciated development in biology: the direct observation of darwinian principles at play within short times. • total number of viral particles: w . this is times more than cells, and they are equivalent to  tons of carbon. • virus particles in cm of seawater: w . • virus particles in m of air: w  to  . • rate of viral infections in the oceans: w  /s. • a string with the viruses on earth would be about w  light-years long (w .  m). this is the distance from earth of the galaxy clusters centaurus, hydra, and virgo. evolution is defined as a change in the genetic composition of a population over time. in this book evolution will be used in its broader sense to mean any change in the genetic composition of a virus over time, irrespective of the time frame involved, and the transience of the change. we treat as evolution both the genome variation that poliovirus has undergone from the middle of the th century to present days and the changes that the same virus undergoes within an infected individual. it is remarkable that only a few decades ago virus evolution (or for that matter microbial evolution in general) was not considered a significant factor in viral pathogenesis. evolution was largely overlooked in the planning of strategies for microbial disease control. a lucid historical account of the different perceptions of virus evolution, including early evidence of phenotypic variation of viruses, with emphasis on the impact of the complexity of rna virus populations, was written by j.j. holland ( ) . despite having been largely ignored by virologists, the present book was partly stimulated by the conviction that the concept of complexity is pertinent to the understanding of viruses at the population level, having direct connections with viral disease and disease control. despite pleomorphism in cells and viruses (presence or not of envelopes, and viruses being spherical or even displaying a lemon-like shape, or being elongated), the size of viral particles and their host cells tends to be commensurate with the amount of genetic material that they contain and transmit to progeny ( fig. . ). the report from international committee on taxonomy of viruses (ictv) divides viruses into orders, and each of them is subdivided into several families, subfamilies, genera, species, and isolates (https://talk.ictvonline.org/), and each isolate includes a multitude of variants. the task of classifying viruses meets with considerable hurdles and requires periodic revisions by the ictv, an organization whose role has been essential to provide conceptual order in the vast viral world. one of its objectives is the assignment of newly discovered viruses to the adequate group. a remarkable number of isolates remain unclassified, an echo of the figure . representative average diameter values and genome complexity of viruses and some cell types. diameters are expressed in microns (m), length of dna in base pairs (bp), and of rna in nucleotides (nt). viral genomes can be linear, circular, diploid, segmented, or bipartite (multipartite in general; genome segments encapsidated in separate particles); in the latter case at least two particles, each with one kind of genomic segment, must infect the same cell for progeny production. the bottom boxes describe four groups of viruses according to the type of nucleic acid that acts as replicative intermediate. natural diversity of viruses, even among the limited subset that has been isolated and characterized. viruses can be divided into two broad groups: those that have rna as genetic material, termed the rna viruses, and those that have dna as genetic material termed the dna viruses. they can have linear, circular, or segmented genomes of single-stranded or double-stranded nucleic acid ( fig. . ). all evidence suggests that the polynucleotide chain (or chains) that constitute the viral genome has all the information to generate infectious progeny in a cell, as evidenced by the production of infectious poliovirus from synthetic dna copies assembled to represent the genomic nucleotide sequence (cello et al., ) . with regard to the concepts of genome stability versus variation addressed in this book, it is helpful to divide viruses into four groups, depending on whether it is dna or rna the type of genetic material, which acts as a replicative intermediate in the infected cell (bottom gray shaded boxes in fig. . ). the nucleic acids written in the four schemes are those involved in the flow of genetic information (indicated by arrows), not in gene expression since all of them use messenger rna (mrna) for virus-specific protein synthesis. mistakes in the form of misincorporation of nucleotides during the replication steps indicated by arrows are transmitted to progeny genomes. rnas produced by transcription to serve solely as mrnas are essential for gene expression and virus multiplication, but misincorporations in such transcripts are not transmitted to progeny. it could be considered that some mrna molecules when synthesized from the corresponding rna or dna template may acquire mutations and that these mutated molecules (e.g., an mrna encoding a viral polymerase), when translated, may produce a polymerase with lower copying fidelity that will evoke additional mutations; we will ignore this possibility since a single mrna molecule should have a rather limited contribution to the overall genetic variation of a replicating virus population with thousands of polymerase and template molecules in the replication complexes (or replication factories). group (with a replicative scheme abbreviated as rna/rna) includes rna viruses whose genomic replication cycle involves only rna. they are sometimes called riboviruses. examples are the influenza viruses, hepatitis a and c viruses, poliovirus, coronaviruses, ebola virus, foot-and-mouth disease virus, or tobacco mosaic virus, among many other important human, animal, and plant pathogens. their replication is catalyzed by an rna-dependent rna polymerase (rdrp) encoded in the viral genome, often organized as a replication complex with viral and host proteins in cellular membrane structures. group (rna/dna/rna) comprises the retroviruses [such as hiv- , the acquired immune deficiency syndrome (aids) virus, and several tumor viruses] that retrotranscribe their rna into dna. retrotranscription is catalyzed by reverse transcriptase (rt), an rna-dependent dna polymerase encoded in the retroviral genome. it reverses the first step in the normal flow of expression of genetic information from dna to rna to protein, once known as the dogma of molecular biology. this enzyme was instrumental in the understanding of cancer, and for genetic engineering and the origin of modern biotechnology. as a historical account of the impact of h. temin's work (codiscoverer of rt with d. baltimore), the reader is referred to cooper et al. ( ) . retroviruses include a provirus stage in which the viral dna is integrated into host dna. when silently installed in cellular dna, the viral genome behaves mostly as a cellular gene. group (dna/dna) contains most dna viruses, such as herpesviruses, poxviruses, iridoviruses, and papillomaviruses, and the extremely large viruses of amebae (i.e., mimivirus, megavirus, or pandoravirus, generically termed giant viruses) (la scola et al., ; colson et al., ) . their replication is catalyzed by a dna-dependent dna polymerase either encoded in the viral genome or in the cellular dna. cellular dna polymerases are involved in the replication of dna viruses that do not encode their own dna polymerase. finally, group (dna/rna/dna) includes viruses which despite having dna as genetic material, produce an rna as a replicative intermediate, the most significant examples being the human and animal hepatitis b viruses (hbvs) and the cauliflower mosaic virus of plants, termed hepadnaviruses. most viruses, from the more complex dna viruses [i.e., kbp ( base pairs) for the ameba mimivirus, kbp for some tailed bacteriophages, and up to kbp for poxviruses, iridoviruses, and herpesviruses], the virophages that are parasites of the giant dna viruses, the simplest dna viruses (the circular singlestranded residue dna of porcine circovirus), rna bacteriophages ( nucleotides of ssrna for bacteriophage qb), or subviral elements (viroids, virusoids, satellites, and helperdependent defective replicons) show remarkable genetic diversity. however, rna viruses that replicate entirely via rna templates (group in fig. . ); retroviruses (group ); and the hepadnaviruses (group ) display salient genetic plasticity, mainly in the way of a high rate of introduction of point mutations (chapter ). their mutability may be an inheritance of universal flexibility that probably characterized primitive rna or rna-like molecules, thought to have populated an ancestral rna world at an early stage of life on earth (section . . ). thus, the presence of rna at any place in the replicative schemes (group , , and in fig. . ) implies error-prone replication and the potential of very rapid evolution. "potential" must be underlined because high error rates do not necessarily result in rapid long-term evolution in nature (chapter ). the extent of genetic variation and its biological consequences have been less investigated for dna viruses than for rna viruses. the available data suggest that dna viruses are closer to rna viruses than suspected only a few years ago, regarding their capacity of variation and adaptation. this is particularly true of the single-stranded dna viruses of animals and plants. evolutionary theory predicts that highfidelity polymerase machinery is necessary to maintain the informational stability of complex genomes (those that carry a large amount of genetic information). this necessity accomplished by proofreading-repair and postreplicativerepair activities that assist the replicative dna polymerases and their cellular and viral dna progeny. our current capacity to sample many thousands viral genomes in short times (a trend that is continuously expanding) is revealing an astonishing number of slightly different viral genomes within a single infected host, and even within an organ or within individual cells of an organ! intrahost diversity of viruses can be the result not only of diversification within the host but also of coinfection with different viruses (or variants of one virus), or an infection that triggers reactivation of a related or unrelated virus from a latent reservoir, or combined effects of these mechanisms. in turn, interhost (long-term) virus diversification can result from selection acting on variants generated by mutation, recombination or reassortment, and random sampling events (independent of selection) within hosts and in host-to-host transmission, or their combined effects (chapters and ). a different picture of diversity is obtained by comparing the morphological characteristics of viral particles (termed virions). the hundreds of thousands of bacterial and archaeal viruses that have been recognized can be assigned to as few as morphotypes. the capsids of nonenveloped (naked) viruses display helical or icosahedral symmetry that determines the architecture of the virion. variation in size and surface protein distributions can be attained from limited protein folds and the same symmetry principles (mateu, , fig. . ) . divergent primary amino acid sequences in proteins can fold in closely related structures. the "structural space" available to viruses particles is much more restricted than the "sequence space" available to viral genomes (abrescia et al., ) . sequence space and its mapping into a phenotypic space are key concepts for the understanding of evolutionary mechanisms (chapter ). three-dimensional structures of entire virions or their constituent proteins can provide an overview of phylogenetic lineages and evolutionary steps in cases in which the information cannot be attained by viral genomics (ravantti et al., ). yet, minor genetic modifications that do not affect the phylogenetic position of a virus or the structure of the encoded viral proteins in any substantial manner can nevertheless have major consequences for traits as important as host range or pathogenicity. how such minor changes in viruses can have major biological consequences may relate to the historical role of viruses in an evolving, ancestral, pre-cellular biosphere. to further address this issue, we need to examine how viruses may have originated. this, in turn, begs the question of the origin of life and the possible involvement of viruses in early life development. an understanding of the mechanisms involved in the origin of life may help in penetrating into the origin of previral entities, defined as the precursors of the viruses we isolate in modern times. different notions on the origin of life have been held in human history, often linked to religious debate. opinions have ranged from a conviction of the spontaneous and easy generation of life from inanimate materials, or its beginning from a unique and rare combination of small prebiotic molecules, or being the result of a lengthy prebiotic process, or its inevitability as the outcome of the evolution of matter in our universe (or "sets" of universes with the adequate physical parameters, according to some cosmological models). as little as years ago (a time not distant from the discovery of the first viruses), there was a general belief in the spontaneous generation of life. this was somewhat paradoxical because chemists of the seventeenth century divided chemistry into mineral chemistry, vegetal chemistry, and animal chemistry. j.j. berzelius put together animal and plant chemistry and named the resulting discipline "organic" chemistry, which he distinguished from "inorganic" chemistry (berzelius, ) . he formulated what was known as the central dogma of chemistry: "the generation of organic compounds from inorganic compounds, outside a living organism, is impossible." the classical experiments of l. pasteur provided definitive proof that, at least under the prevailing conditions on present-day earth, "life comes from life" (pasteur, ) . he established what was considered the "central dogma of biology": "the generation of a whole living organism from chemical compounds, outside a living organism is impossible." the requirement of life to generate life was, however, extended to the belief that "living" and "nonliving" were two separate categories in the organization of matter, and that organic compounds could be synthesized only by living cells. this doctrine, called vitalism dominated biology for almost a century, and in a modified manner, it continues today regarding the interpretation of mental activity in humans (matter and spirit as "substance dualism"). historical views on the origin of life have been addressed in several publications (rohlfing and oparin, ; bengtson, ; de duve, ; eigen, eigen, , lazcano, ) . dogmas are generally not to stay. "vitalism" was shattered by the chemical synthesis of organic compounds from inorganic precursors (urea by f. w€ ohler in , acetic acid by h. kolbe in , hydrocarbons by d. mendeleev in , and several other compounds by m. berthelot in the second half of the th century). the evidence that no "vital force" was needed for such syntheses led f.a. kekul e to write in his classic textbook on organic chemistry published during e : "we have come to the conviction that . no difference exists between organic and inorganic compounds." from then on, organic chemistry became the chemistry of "carbon compounds." we know now that living entities are made of the same chemical elements . introduction to virus origins and their role in biological evolution found in the mineral world. of the elements in the periodic table (in ), are found in the human body. a key experiment carried out in by s. miller, working with h.c. urey showed that components of biological molecules could be obtained from inorganic precursors. he mimicked the conditions thought to be prevalent in the primitive earth, and mixed hydrogen (h ), ammonium (nh ), and methane (ch ) in a sealed reactor with an influx of water vapor. synthesis of a number of organic compounds occurred under the influence of electrical discharges. the de novo synthesized chemicals included amino acids (glycine, alanine, aspartic acid, and glutamic acid), formic, acetic, propionic and fatty acids, cyanide, and formaldehyde (miller, (miller, , . several researchers followed the miller's approach using other starting chemical mixes and confirmed that key components of the macromolecules that are associated with living materials (notably purines, pyrimidines, and amino acids) could be made from precursors, which were abundant in the primitive earth or its atmosphere. today, variant versions of miller's protocol (including additional starting chemicals, aerosol spread of chemicals, freezethaw cycles, different sources of energy, electron beams, etc.) produce interesting information on the synthesis of organic molecules (dobson et al., ; miyakawa et al., ; bada and lazcano, ; ruiz-mirazo et al., ) . intense ultraviolet (uv) irradiation may have contributed to the synthesis of compounds relevant to life: ammonia, methane, ethane, carbon monoxide, formaldehyde, sugars, nitric acid, and cyanide. complex organic compounds (notably aromatic hydrocarbons and alcohols) are found in interplanetary dust, comets, asteroids, and meteorites, and they can be generated under the effect of cosmic and stellar radiation. thus, many organic compounds could have been produced within the earth atmosphere or away from it, and be transported to the earth surface by meteorites, comets, or rain, to become the building blocks for additional life-prone organic molecules. places at which peptide bond formation and prebiotic evolution could have been favored are hydrothermal systems and the interface between the ocean and the atmosphere (chang, ; horneck and baumstark-khan, ; ehrenfreund et al., ; parker et al., ; danger et al., ; griffith and vaida, ; ritson and sutherland, ; nakashima et al., ) . a key issue from prebiotic syntheses is the degree of oxidation of the primitive earth atmosphere. records of an early surface environment dated . billion years ago were found in metasediments of isua, greenland. these materials suggest that the surface temperature of the earth was below c, with the presence of liquid and vapor water, and gases supplied by intense volcanism (co , so , and n ). the composition of primitive rocks, together with theoretical considerations, suggest a neutral redox composition of the earth atmosphere (with relative gas abundances: n , co > co [ ch , h o [ h , so > h s) around the time of the origin of primeval forms of life. the possible presence of a general or localized reducing atmosphere (n , co > ch > co , h o, w h , h s > so ) is still debated, but increasingly viewed as unlikely. in an oxidative atmosphere, yields of amino acids, nucleotides, and sugars would be lower. either these diminished yields were sufficient to attain critical levels of relevant building blocks, or an earlier reducing atmosphere may have accumulated them, among other possibilities (trail et al., ) . in spite of the validity of "life comes from life" in the current earth environment, the experimental facts suggest that there is no barrier for the generation of life from nonlife, provided suitable environmental conditions are met. in this line, a.i. oparin proposed that a "primitive soup" could well have been the cradle of life on earth, as described in his famous treatise on the origin of life (oparin, . origin of life: a brief historical account and current views ), a concept that had already been sketched by c. darwin. the "protein first" versus "nucleic acid first" for the origin of life is still a contended issue (falk and lazcano, ) . although during decades there was a preference for nucleic acids due to their superior capacity for selforganization to perpetuate inheritable messages through base pairings, recently both views have accumulated arguments in their favor. an integrative "metabolism first" view based on mutually catalytic networks of small molecules displaying the capacity to replicate and evolve is gaining adepts (lancet et al., ) . the building blocks of nucleic acids have been more difficult to obtain from primeval chemicals than the building blocks of proteins. peptides of about amino acids in length could have been easily formed under prebiotic conditions (fox and dose, ) , and peptides or their derived multimers had a potential to display catalytic activities at a proto-metabolic stage. peptide amyloids have been proposed as an alternative to nucleic acids for the origin of life in what has been termed the amyloid-word hypothesis (greenwald et al., ) . interestingly, amyloid aggregates display features of quasispecies, not in the way of mutant distributions found in viruses (chapter ) but as alternative protein conformations and conformation heterogeneity that confers functional diversity (see chapter on the collective behavior of prions). the protein-versus nucleic acid-first alternatives may not be incompatible. short nucleotide and amino acid polymers might have cooperated to cross a complexity threshold for self-sustained replication to arise and evolve (kauffman, ) . as soon as peptide-or protein-based catalytic activities developed, they had to be coordinated with oligo-or polynucleotide replication. this integration of genotypic information with its phenotypic expression may be achieved through hypercyclic couplings, as proposed by m. eigen and p. schuster (eigen and schuster, ; eigen, ) , to render incipient life forms sustainable. substantiating the abiotic synthesis of nucleotide-or amino acid-based polymers has been arduous relative to the synthesis of monomeric organic molecules. however, early work by l. orgel and his colleagues documented that polynucleotides could be synthesized from activated nucleotides in the absence of an enzyme (miller and orgel, ) , and recent work has established that there are multiple chemical pathways for abiotic and nonenzymatic nucleotide synthesis (adamala and szostak, ; ruiz-mirazo et al., ) . mineral surfaces (clay minerals, zeolites, manganates, hydroxides, etc.) have been proposed as key participants in the origin of life, by providing scaffolds for the synthesis of nucleotide and amino acid polymers (bernal, ; gedulin and arrhenius, ; ruiz-mirazo et al., ; nakashima et al., ; pedreira-segade et al., ) ). the relevant features of clays are their adsorption power, ordered structure, capacity to concentrate organic compounds, and ability to serve as polymerization templates. adsorption onto mineral surfaces may lower the activation energy of intermolecular reactions. minerals with an excess positive charge on their surfaces might have played a role in the evolution of rna-like or rna-precursor molecules. it has been suggested that mineral-organic complexes (chimeras between materials once thought to belong to unmixable categories) could have been the first living organisms endowed with a genetic program (cairns-smith, ) . bond formation and chain extension, which are needed to synthesize nucleic acids and proteins prebiotically, are inhibited in liquid water, thus favoring mineral surfaces as potential biogenic sites. polymer formation could have also been facilitated by heating and drying, with key involvement of the dried phases in catalysis (towe, ; horneck and baumstark-khan, ) . on different clay types, nucleotides and amino acid polymers of dozens of residues . introduction to virus origins and their role in biological evolution have been synthesized, providing a realistic scenario for a transition toward primitive selfreplicating entities (ferris et al., ) . one of the first episodes of darwinian positive selection could have operated prebiologically through differential surface binding, followed by the selection of autocatalytic heteropolymers (tkachenko and maslov, ) . we arrive at the paradox that while primitive polymerization reactions might have required dry surfaces, later forms of life evolved in a water-rich environment, with water being the major component of cells. "origin" and "establishment and evolution" of life have been considered either as linked processes that obey similar rules, or distinct events whose investigation requires dissimilar approaches. a distinction between "origin" and "establishment" will become pertinent also when addressing the origin versus the evolution of viruses later in this chapter, and the mechanisms of viral disease emergence in chapter . a simplified overview of a course of events that led to the origin of biological systems is depicted in fig. . . the prebiotic synthesis of potential building blocksdwhich might have been initiated earlier than million years agodrenders plausible the existence of a pre-rna era that was then replaced by an rna world in the late hadean early archean periods on earth. this stage should have been followed by one in which rna was complemented by dna as a repository of genetic information (bywater, ) . polymers other than dna and rna are also capable of encoding evolvable inheritable information (pinheiro et al., ; transitions. time (horizontal lines) is expressed as millions of years before present, counted from the big bang (estimated at w , million years ago). the time at which plants colonized land (green cone) and major mass extinctions (red triangles) are numbered: , end cretaceous extinction; , late triassic extinction; , end permian extinction; , late devonian extinction; , end ordovician extinction. a proposed sixth extinction presently underway is not indicated. illustration by c. perales and e. domingo from information retrieved from references given in the text. robertson and joyce, ) . heterogeneous nucleic acid molecules (including mixtures of ribo-and deoxyribo-polymers) can give rise to functional nucleic acids (gilbert, ; lazcano, a; gesteland et al., ; derr et al., ; szostak, ; trevino et al., ) . rna enzymes (ribozymes), such as rna ligases can evolve from random-sequence rnas (joyce, ; sczepanski and joyce, ) . the critical polymerization reaction involves the formation of a phosphodiester bond and release of pyrophosphatedanalogous to the reactions catalyzed by the present-day rdrpsdand represent an incipient, primitive anabolism (eigen, ; lazcano, a; orgel, orgel, , dworkin et al., ; joshi et al., ) . in support of a possible link between catalytic rna activities and solid mineral surfaces in the origin of life (as summarized in section . . ), the catalytic activity of the hammerhead ribozyme of the avocado sun blotch viroid was maintained when bound to the clay mineral montmorillonite (biondi et al., ) . minimum requirements for an rna world would be the presence of ribozymes and mechanisms for the intake of energy-rich molecules (orgel, (orgel, , . the inherently low copying fidelity of the putative ribozyme polymerases, estimated in À to À errors per nucleotide copied (wochner et al., ) should have ensured genetic variation for selection to act upon variant rna molecules. chirality (the existence of two mirror images or enantiomers of a molecule) poses a challenge for the chemical origin of biological molecules (caglioti et al., ; ruiz-mirazo et al., ; sczepanski and joyce, ) . present-day biological systems use only d-ribose (d from "dextro" or rotation of the plane of polarized light to the right) while chemical condensation reactions produce equal amounts of the d-and l-(levo) forms. nonenzymatic template-dependent reactions can be inhibited by the incorrect enantiomer. this led to the proposal that analogs devoid of enantiomeric forms, such as glycerol derivatives, could have been the basis of the most primitive genetic systems (schwartz and orgel, ) . theoretical studies support the notion that initial achiral conditions can evolve toward chirality, in what has been defined as an extension of punctuated equilibrium to prebiological evolution (gleiser et al., ) . probably, very little, if anything, remains in our present-day biosphere of a primitive rna world, let alone traces of the prolonged process that went from chemistry to the first replicating organizations, so profound have been the changes experienced by the earth and its surroundings for over four billion years (cantine and fournier, ) . contemporary catalytic rnas (found in the ribosome, as part of some protein complexes, and in some viroids), as well as nucleotide-like coenzymes, have been regarded as possible molecular remnants of a primitive rna world (lazcano, a) . some authors consider the possibility that cells whose genetic material is made of rna (forterre, ) may still hide in some remote sites of our planet (yarus, ) . for a transition from a purely rna (or rna-like) world to an extended scenario with the participation of proteins, the presence of a transfer rna (trna) quasispecies and the generation of a genetic code endowed with evolutionary potential must have been critical (eigen, ; koonin and novozhilov, ) . initial theories of how the genetic code might have arisen were put forward by f. crick, l. orgel, and c. woese in the middle of the twentieth century. main proposals included a stereochemical fit between some amino acids and the corresponding bases (or codons to be), progressive evolution from a one nucleotide to a three nucleotides code, gradual incorporation of amino acids in the coding system, and the frozen accident model of codon universality (for a review of early concepts, see crick, ) . new insights on the code origin have come from integrating the knowledge of the mechanisms of protein synthesis with likely events in the rna world. primitive trna quasispecies (existing around million years ago, late hadean, early archean, fig. . ) and trna aminoacylating ribozymes should have evolved to fit the genetic code, which was later expanded in coevolution with the translation machinery (szathmary, ; rodin et al., ; caetano-anolles et al., ) . the age of the genetic code has been estimated in . ae . thousand million years (eigen, ) , and the present code structure is remarkably redundant. effects of redundancy are to minimize the deleterious effects of mutations, and to contribute to flexibility of functional involvement of mrna secondary structure (shabalina et al., ; koonin and novozhilov, ; see also chapter ). the advent of dna as an informational macromolecule that was physically more stable than rna opened the way to an increase of complexity (in the sense of the amount of genetic information) of the genetic material. dna allowed integration of modules to form the first "chromosomes" and transcriptional regulation prior to protein expression. as in current evolutionary virology, perhaps the most challenging problem to understand early life is to identify the selective constraints that influenced the course of events. in contrast to the present-day environmental changes confronted by viruses (multiple and complex, but amenable to experimentation; chapters and ), the conditions that permitted primitive genetic entities to acquire expanded coding and signaling capacities defy our imagination. some aspects are considered next. the most salient attributes of living matter are reproduction, evolvability, energy conversion (metabolism), and compartmentalization. what selective forces might have led to the integration of these features? concerning reproduction, there must have been a critical transition from the absence of any inheritable instruction (despite the presence of primitive polymers) to the first molecules endowed with "inheritable" information, for example, a macromolecule capable of self-copying. such a molecule should have had an immense selective advantage over surrounding macromolecular peers devoid of the capacity for reproduction. the transition from "no inheritable information" to "inheritable information" is essential for the origin of life. current evidence suggests that the process that allowed such critical transformation was slow and inaccurate. slow because the catalytic rnas selected in the laboratory are about million-fold slower than most protein enzymes (jeffares et al., ; yarus, ) . inaccurate because preenzymatic nucleotide polymerization would rarely display error rates below À to À mutations per nucleotide copied (inoue and orgel, ) . no "predators" such as degrading enzymes were present to impede a slow accumulation of variant replicating molecules. the slow sequence exploration might have taken place in microenvironments shielded from damaging radiation until points in sequence space compatible with self-organization, replication, and adaptation were encountered. the adaptive potential of mutant distributions of polynucleotides is a key concept in the quasispecies theory of the origin of life (eigen and schuster, ) , and a signature of present-day viruses (chapter ). replicative inaccuracy and heterogeneity appear as recurrent requirements for the major transitions and adaptability of the forms generated in the course of prebiological and biological evolution. once a primitive molecular "memory" was implemented, in the words of m. eigen: " ..information generates itself in feedback loops via replication and selection, the objective being 'to be or not to be'" (eigen, (eigen, , . in those times, this was the only simple requirement: to be or not to be. this singular transition resulted in the first replicating entities that are also termed replicators or replicons. they were selected for replicability, stability, and evolvability with trade-offs . origin of life: a brief historical account and current views (acquisition of benefits for one of the three traits at some cost for another trait) likely play a role at this stage (see chapter for trade-offs in virus evolution). optimization of primitive replicons should have been facilitated by dual genotypic and phenotypic features in the same molecule (eigen, ) . that rna itself is both genotype and phenotype is a feature of present-day rna viruses. that is, the genomic rna determines phenotypic traits, independently of its proteincoding activity, for example, through its involvement in rna-rna and rna-protein interactions. "priming" of polynucleotide synthesis, in the sense as we know it today, should not have been a limitation since circular rna or rnalike molecules could fold partially to prime their own copying. the term "replicon" currently refers to any genetic element that encodes sufficient information to be copied (i.e., viruses, plasmids, etc.), even if the copying is carried out by (or in conjunction with) elaborate celldependent machinery. virtual replicons are used in computer simulations to learn about the dynamics of natural living systems (adami, ; eigen, ) . the environment in which primitive replicons had to self-organize about million years ago was very different from the environment we have today on earth. the sun was about % e % less luminous than the present-day sun, yet it produced more ultraviolet (uv) light. due to the absence of oxygen and ozone layer, the uv radiation that reached the earth was -to -fold more intense than today, with the difference being accentuated for the radiation in the e nm range. studies of the conversion of uv radiation into dna-damage equivalents suggest a two to three logarithm larger biologically relevant uv radiation during the time of the putative rna world as compared with today's radiation (canuto et al., ; chang, ; horneck and baumstark-khan, ) . in such an environment, radiationrelated mutational input could have had drastic effects on replicating entities in ways that can be only roughly anticipated from the present-day chemistry. even the simplest present-day rna genetic systems, despite their small target size, would undergo severe radiation damage. reconstruction of protein enzyme-free nucleic acid synthesis under the radiation conditions prevalent on earth during the rna world development, during late hadean and early archean eras, offers a fascinating challenge and opportunity of experimental research for the rising field of astrobiology. it has been considered that the time elapsed since the earth attained a life-friendly environment until protocells arose (from about million to about million years ago, fig. . ) was insufficient for life development. this led to the panspermia theory, which proposes that life has an extraterrestrial origin. panspermia in different forms has been defended by noted scientists, such s. arrhenius in the early twentieth century and later by f. crick and l. orgel (discussed by de duve, ) . in addition to the time estimates for life generation being arbitrary, our present understanding of how error-prone replication can facilitate evolvability and exploration of novel biological functions (chapters and ) converts a million-year time period in a long time for life to originate and initiate multiple branches for its development. energy conversion is an essential feature of life. the primitive precellular organizations might have obtained energy either from organic molecules captured from the external environment (heterotrophy) or from metabolites they synthesized endogenously using external energy (autotrophy). one line of thought considers that it is more likely that the first cells were heterotrophs, and that only later they evolved toward autotrophy, in the form of photosynthesis, which represented a major transition in the repertoire of biosynthetic pathways (nakashima et al., ) . fermentation reactions were likely the first ones exploited to break energy-rich bonds, as a source of energy for primordial biochemical reactions. an alternative view is that the first cellular organism was an autotroph, in particular, a chemoautotroph (also termed lithotroph) that used inorganic compounds to obtain energy. one of these proposals is that the formation of pyrite from hydrogen sulfide was used by primitive cells as an energy source, resulting from reactions such as fes þ h s / fes þ h þ þ e À (w€ achtersh€ auser, ). positive charges on pyrite crystals could accumulate negatively charged molecules (e.g., the products of co fixation) and undergo reductive reactions. the system might have selected surface-bound polymers rather than monomers, and given rise to biochirality (selection of one enantiomeric form over another) because of the chiral structure of pyrite (w€ achtersh€ auser, ). then, an evolution toward an archean carbon-fixation cycle would occur, a precursor of the metabolic cycles found in present-day archaeal and bacterial organisms (ruiz-mirazo et al., ) . the picture may have been more complex, as judged by the success of mixotrophic organisms which are capable of switching from phototrophy (lightmediated break-down of co for their metabolism) to heterotrophy in some extreme environments of the present-day earth (laybourn-parry and pearce, ). the integration of early replication systems and metabolism was probably favored by some compartmentalization of replicative-metabolic units through lipid bilayers (carrara et al., ; stano et al., ; ruiz-mirazo et al., ; hanczyc and monnard, ) . here a second decisive positive selection event might have entered the scene at the stage of formation of proto-cells and the first individual cells ( e million years ago, fig. . ; eigen, ) . lipid bilayers endowed with a splitting capacity should have been strongly selected at this stage of life development because of their power to spread. this underlines the concept that darwinian selection need not be associated exclusively with template-copying processes. membrane traffic and reorganizations are essential for the life cycle of many present-day viruses (huotari and helenius, ) . virus particle stability and capacity to spread, associated with membrane capture and interactions, might have derived from the early genomes that exploited membrane-based organelles to achieve functional diversification. compartmentalization is considered one of the key developments to initiate a cell-based life (morowitz, ; hanczyc and monnard, ). despite obvious difficulties in reproducing physical and chemical processes that were likely involved in the origin of life, there is sufficient evidence to render probable that the most primitive organizations that we would now consider as "living" resulted from the assembly of simple organic compounds that attained a required level of complexity (kauffman, ) . the various facets that distinguish living from inanimate matter are recapitulated in the definitions of life that scientists from different backgrounds in physics, chemistry, or biology have proposed (box . ). some definitions underline entropy requirements, while others emphasize self-organization, evolvability, metabolism, or regulated complexity. their disparity reflects the intricacies of a rather enigmatic unfolding of matter. a. lazcano summarized the current situation in the arduous search of the origin of life: "there are still unsolved problems but they are not completely shrouded in mystery, and this is no minor scientific achievement. why should we feel disappointed by our inability to even foresee the possible answers to these luring questions? as the greek poet konstantinos kevafis once . origin of life: a brief historical account and current views wrote, odysseus should be grateful not because he was able to return home, but on what he learned on his way back to ithaka. it is the journey that matters" (lazcano, b ; for a general overview of the history of life on earth, centered in paleontology, see cowen, ) . after this brief survey of the origin of life, we can now examine theories on how, when, and why viruses arose and became active actors in our biosphere. although not in a linear fashion, the number of nucleotides or base pairs in the genetic materialdthat presumably reflects the amount of genetic information relevant to confer phenotypic traitsdincreased as evolution led to differentiated organisms. the major theories of the origin of viruses are divided into two opposite categories: those that attribute virus origins to the early development of life, and those that propose that viruses arose when a cellular life was already in place ( fig. . ) . these two broad views that we may term "viruses without cells" and "viruses from cells" are not irreconcilable, although reconstruction of ancestral developments is challenging. they can be divided into five main theoriesdnot all independent or mutually exclusived which are summarized next. • life is the property of a system that continuously draws negative entropy (maintains orderliness), and delays decay into thermodynamic equilibrium (schr€ odinger, ). • life is an expected, collectively self-organized property of catalytic polymers (kauffman, ) . • life is a property of any population of entities possessing those properties that are needed if the population is to evolve by natural selection (maynard smith and szathm ary, ). this answer is not a tautology, as it allows many attributes to be excluded from the definition of life (de duve, ). • life is descent with modification. replication is life's ultimate chemical and physical survival strategy (yarus, ). • life is a self-sustained chemical system capable of darwinian evolution (nasa astrobiology institute). • living organisms are metabolic-replicating systems composed of molecules and cells which are subject to spontaneous changes in structure and function due to mutations (demetrius, ) . • life in three statements: ( ) life is not represented by any fundamental physical structure. ( ) life is an overall organization that is governed by functional rather than by structural principles. ( ) in order for life to come about, there must exist some physical principle that controls complexity (eigen, ) . • life, whatever else it may be, is certainly a regularity among material processes (eigen, ). • viruses were involved in the origin of life. some viruses are the descendants of primitive rna or rna-like replicons that preceded cellular forms. because of their limited genetic complexity, rna viruses and subviral rna elements have been considered possible descendants of the primitive replicating entities that predated cellbased life forms (upper diagram in fig. . ) . as early as the beginning of the twentieth century, h.j. muller, l.t. troland, and j.b.s. haldane suggested that viruses represented primordial life forms. influenced by the discovery of bacteriophages by f. d'herelle, j.b.s. haldane proposed viruses as intermediates between the prebiotic soup and primitive cells (reviewed in lazcano, ) . in those times, knowledge of viruses was superficial from today's perspective, and lent to daring proposals coherent with viruses being perceived as simple. despite simplicity and replication being features that could be also be attributed to primitive life forms, we have to distinguish the role that virus-like entities might have played in the establishment of early life from the possibility that present-day viruses reflect how early life might have been. an origin independent of cells is suggested by the presence of a number of "virus hallmark proteins" in viruses, which do not have counterparts in present-day cells, at least in the sequences represented in data banks. a number of protein folds (such as the double jelly-roll fold) found in the nucleoplasmic large dna viruses (and related structures) are also present in viruses that infect the archaeon sulfolobus, and in some single-stranded and double-stranded rna viruses that infect eukaryotic cells. these unique structures and other features of viruses, such as genome size and its composition, constitute a "virus innate self" (krupovic and bamford, ) , that has been taken as an indication that viruses were constructed from modules that were different from those captured to build cells. the fact that some viral proteins do not have cellular homologs (or are only distantly related to cellular homologs) has favored the view that a virus world preceded the origin of cells (forterre, ; villarreal, ; koonin et al., ; koonin, ) . in this line of thought, the present-day rna viruses are considered remnants of an rna world; no other biological group that has been identified uses rna as genetic material. likewise, transposable elements, repeat sequences of mobile elements (ltr-retrotransposons, lines, sines, alus) and of telomeres and centromeres are considered to be likely of a viral origin [reviewed in witzany, ) ]. figure . two possible courses of events regarding when viruses first appeared and participated in the evolution of the biosphere. the scheme of time frames and major biological events (rna world, first cells, and organisms) are those displayed in fig. . . according to the upper diagram, viruses (or previrus-like entities) arose together with the first (precellular) replicating entities. according to the second diagram, viruses (or previrus-like entities) arose when a cellular life had already been established. presence of virus is generically represented by the external, thick, black curves. the internal red, wavy lines represent generation, dominance, and extinction of multiple viral lineages whose numbers and true dynamics will remain unknown. illustration by c. perales and e. domingo from information retrieved from the different models of virus origin and references included in the text. other present-day rna genetic elements that include ribozymes as constituents [i.e., plant viroids, self-replicating rnas of about e nucleotides in length (hadidi et al., ) or the defective delta agent, also termed hepatitis delta virus (hdv)] might be a vestige of primitive genetic elements . hdv is dependent on hepatitis b virus (hbv) for the completion of its infectious cycle (taylor and pelchat, ) . the hdv genome is a mosaic rna consisting of a viroid-like rna and an rna region whose complementary rna (antigenomic strand) encodes two forms of a structural protein termed the delta antigen. both the genomic and antigenomic rnas possess a strong secondary structure with about % paired nucleotides. the delta antigen is encapsidated by the hbv surface antigen as a component of hdv particles. thus, hdv appears to be the result of an rna conjunction between a viroid-like rna and an mrna-coding region. such conjoined rnas might have been the precursors of the modern eukaryotic genome partition into coding sequences (exons) and intervening sequences (introns) (sharp, ; robertson, robertson, , chao, ; taylor and pelchat, ) . the structure of the hdv genome seems to echo processes that originated with primitive rnas selected for their ability to replicate that incorporated a protein-coding moiety through recombination with other rnas. replication-competent and protein-coding chimeras might have signaled a relevant intermediate step toward more complex rna and dna genomes. the advent of an enzyme that could copy rna into dna to carry out reverse transcription should have been instrumental in generating primitive dna viruses and other dna-based genomes of increased complexity (lazcano et al., ) . the discovery of the chimeric structure of the hdv genome illustrates how insights into the origin and early evolution of life can be gained from current genomics, despite lacking experimental approaches to recreate episodes that led to virus origins. even if experiments could be designed, the time frame involved would occupy several generations of scientists, which is not feasible given the current research grant system. • viruses originated from regressive evolution of microbes with a cellular organization and became parasites of cells. this theory is quite the opposite of the previous one because it presupposes that a cellular world was the source of viruses (lower diagram in fig. . ) . one of its conceptual pillars is that the intricacies of virus-host relationships unveiled by molecular virology render unlikely that present-day rna viruses are remnants of an ancestral rna viral world. also, the conditions prevailing in the rna world did not necessitate that a primitive replicon displays rapid replicationda trait of most present-day virusesdfor it to become established, because of the scarcity of predators. the theory of a cellular origin of viruses was already put forward in the twentieth century when it was evidenced that complex dna viruses encoded enzymes and immunomodulatory proteins that had cellular counterparts. the virus-generating cells could be autonomously functional from the onset (as autonomous as individual cells can be) or belong to a class of simple cells that parasitized functionally more advanced cells. one of the mechanisms by which viruses may have originated from cells is by regression or reduction. it implies that the number of genes of the virus-precursor cells diminished gradually or in a step-wise fashion to a minimum number compatible with replication at the expense of other cells that supplied basic components for gene expression and energy capture. the giant dna viruses (nasir et al., ; colson et al., ) have been regarded as a possible missing link between a primitive dna cell and the dna viruses, and some of them include part of the translation apparatus (abrahao et al., ) . the capacity to spread, so inherent to the concept of the virus, might have been first selected as a positive trait for cells, which then regressed toward a subcellular transmissible form. prokaryotic cells can spread effectively among differentiated eukaryotic hosts, and tumor cells have been regarded as transmissible parasites (banfield et al., ; murgia et al., ; pearse and swift, ) . the observation that some "infectious cells" can be disseminated by insects provides a model for an early origin of arthropod-borne viruses. it has been recognized that transmission of the virus from an infected cell into a recipient cell need not involve a prolonged stay of the virus in the extracellular environment. infection "synapses" allow intimate cell-to-cell contacts through which virus transmission takes place. it is estimated that synapsemediated transmission maybe -fold more efficient than the transmission of viral particles released into the extracellular environment. this is one of several mechanisms of bloc (multiple particles) transmission, which has several implications for viral quasispecies dynamics (chapter ). acquisition of capacity for long-range transmission in space and time should have provided a sufficient selective advantage for a "cell-toexternal environment-to-cell" transmission to coexist with "cell-to-cell" transmission in our biosphere. the possibility that rna viruses derive from some "organism" that used rna as genetic material was suggested initially by d. baltimore ( ) . the evidence that rna-dependent rna synthesis is rare in cells suggests that either rna viruses derived their replicates from a now probably extinct "rna organism" or that the viral replicates evolved from cellular dna polymerases (baltimore, ; forterre, forterre, , a yarus, ). • viruses originated from cellular dna or rna that evolved to embody autonomous replication, and an extracellular step in their replication cycle. an alternative to the regressive or reduction of virus origin from cells is the escape theory. it proposes that part of the genome complement of cells found a way toward autonomous replication in the sense that they could replicate on their own as parasites of cells. the capacity to replicate could come from the parental cell or be acquired externally. the kind of autonomy attained implied an intracellular phase of their multiplication cycle and an extracellular phase in the form of transmissible particles (virions). this new way of life should have been positively selected if an increased capacity of cell-to-cell transfer conferred an advantage to the cells regarding the acquisition of new traits for functional diversification while maintaining a capacity to sustain virus multiplication. decades ago, the view that viruses originated from subcellular organelles was a favored one (see e.g., joklik, ) . despite discernible sequence identity between some viral and mitochondrial dna sequences, no evidence of viruses having functions encoded in cellular organelles and not in chromosomal dna has been obtained, perhaps reflecting an earlier relationship between viruses and primitive freeliving cells rather than between viruses and modern (eukaryotic) cells (see also section . ) • viruses are as ancient as cells, and coevolved with cells or even with precellular genomic organizations, with which they shared functional modules. current genomics of viruses and their host organisms (bushman, ; mount, ; hacker and dobrindt, ) tends to favor a long history of coevolution between viruses and cells. the structure and functions of many viral proteins do not deviate in any salient way from cellular counterparts. this applies to proteins involved in genome replication, and in proteolytic processing of proteins and polyprotein precursors. in contrast, the "virus innate self" that groups viral proteins and protein folds not shared with cellular counterparts, and that provides a powerful argument for a virus origin independent from cells (section . . ), does not exclude a coevolutionary mechanism even if all modules were not common to cellular and viral entities over a several million years period (see section . ). comparative genomics suggests that the exchange of functional and structural modules through lateral gene transfers, together with fine adjustments mediated by mutation, have contributed to the coadaptation of cells and autonomous replicons over ancient evolutionary periods (gorbalenya, ; holland and domingo, ; jalasvuori and bamford, ; villarreal, ) . even mechanisms that prompt viral variation in cell tropism (chapter ) have parallels in differentiated organism as an example, a two amino acids insertion into ectodysplasinda member of the tumor necrosis-binding familydalters its receptor specificity, and the differential expression of the two protein versions plays a role in epidermal morphogenesis (yan et al., ) . furthermore, as the number of threedimensional structures for viral cellular enzymes has increased to reach thousands, structural similarities between key cellular and viral enzymes (polymerases, proteases) have become apparent. baltimore ( ) proposed that a limited number of "archetypal" proteins could be responsible for rna virus function. he named as "archetypal" a "positive virus polymerase," a "negative virus polymerase," a set of "surface" proteins, and several "proteases," among other proteins and regulatory elements. the argument that "archetypal" modules could spread among positive-and negative-strand rna viruses was based on features and mechanisms now recognized as much more profuse than in : the multifunctionality of viral proteins, their capacity to diversify by mutation, and the existence of rna recombination (chapter ). regulatory strategies were also likely shared by cells and viruses. small micro-rnas that now populate the cellular world can act as molecular switches for rna viral genomes to modulate their replication and gene expression (van rij and andino, ; diaz-toledano et al., ) . a likely course of events is that the long coevolution of protocells and primitive virus-like elements gradually shaped both cells at the individual and organismal level and the precursors of present-day viruses. • "protoviruses" might have originated in primitive vesicles. early protocellular communities probably lacked a cell wall or other compartmentalization barriers, an absence that allowed fluid transfers of metabolites and genetic material (woese, ) . the most primitive vesicles based on lipid structures inaugurated the distinction between "internal" and "external" milieu and might have evolved to contain self-replicating macromolecules (jalasvuori and bamford, ; adamala and szostak, ; hanczyc and monnard, ) . vesicles located in favorable microenvironments of a primitive earth had the potential to exchange small molecules between the "inside" and "outside," and export materials toward other vesicles. exchanges could assist in the coupling between genome replication and an incipient metabolism. depending on their composition, lipid vesicles could form and remain stable at temperatures of about c. their transfer to lower temperatures might have modulated their permeability prior to the stage at which peptide or protein transporters were inserted as membrane components. here again, "heterogeneities" became important: membranes made of mixtures of amphiphiles display increased thermostability, permeability, and tolerance to divalent cations. budding vesicles endowed with traits such as growth, division, and permeability should have been positively selected for their ability to spread favorable replicating molecules and mediators of protocellular functions. selected protoviral vesicles became gradually dispensable for the spreading of beneficial genes, and viruses evolved from being solely beneficial entities into also displaying a parasitic behavior that exploited cell resources (compare with sections . and . ). according to this model, when cells became independent units surrounded by lipid-based membranes, viruses promoted the selection of cells that expressed peptidoglycan molecules on their surface to decrease or prevent virus infection. this transition could mark the onset of an arms race behavior that has been associated with a survival strategy of many present-day viruses. a cell wall allowed vertical transmission of genetic information and rendered the cell metabolically independent. in addition, it provided an osmotic environment suitable for energy production. genetic information and molecular devices for energy capture, storage, and use were equally important for a sustainable cellular organization. thus, according to this theory, viruses originated from protoviral elements whose main function was to spread useful genes horizontally. the evolved viruses acted as selective agents to promote microbial evolution and then became established in an increasingly differentiated cellular world (villarreal, (villarreal, , hendrix, ; jalasvuori and bamford, ) . geological studies indicate that mass extinctions of a brief duration (less than , years, a short geological time!) occurred at several points when multicellular organisms populated the earth (some of them indicated in fig. . ) . the end-permian, end-triassic, and end-cretaceous extinctions rank among the most drastic, resulting in profound environmental perturbation (burgess et al., and references therein) . as depicted in the form of red upward and downward curved lines in fig. . , perturbations associated with viral extinctions (due to massive host extinctions) and severe bottleneck events have periodically blurred traces of many viruses unknown to us. mechanisms implied by each of the five theories summarized here might have participated in the origin of viruses as we know them today, and any model will remain tentative for several obvious reasons. viruses have not left a fossil record amenable to analysis with current technology. moreover, viral genomes can evolve at very high rates in response to environmental necessities (chapter ), and reconstructions of ancient earth environments are necessarily imprecise. for these reasons, viruses, independently on when they became active actors in the biosphere (fig. . ) , are unlikely to have maintained molecular signatures that could shed light on their remote past. the viruses that infect fungi, the mycoviruses, recapitulate key issues on virus origins and diversity (son et al., ; roossinck, ) . two main theories of their origin are that they evolved from plant viruses that invaded fungi or that they have coexisted and coevolved with their host fungi for a long time, in line with the long coevolution model of virus origins (section . . ). they are diverse regarding the genetic material that can be single-stranded or doublestranded rna or dna. they can be either asymptomatic, produce disease, or modify critical host traits, such as virulence, again reflecting a broad range of host interactions observed with . teachings from mycoviruses other viral groups. the presence of a dsrna mycovirus in a fungus is essential for the latter to confer heat tolerance to some plants; these findings have defined a three-way symbiosis required for an important phenotypic trait (marquez et al., ; roossinck, ) (see also section . . ). an often asked question is whether viruses are alive or not. the answer is debated, as reflected in the various definitions of virus (box . ). two opposite views coexist. one of them considers viruses as macromolecular aggregates, that is, viruses are regarded as "chemicals." a second proposal is championed by the "ribovirocell" concept of p. forterre, which implies that a viable, virus-infected cell contains two different organisms that coexist symbiotically: the cell that produces virus and the replicating virus itself. another facet of the same duality is manifested when viruses are considered merely as perturbing chemicals with no participation in the tree of life versus viruses being actually important players in the tree of life. the dual character of viruses as "alive" during intracellular replication and "not alive" outside the cell was stressed in some early literature (e.g., davis et al., ) . as discussed in section . , if life is best defined as a conglomerate of complementary features, viruses display two of these features: the capacity to replicate and to evolve. thus, although it is debatable whether viruses qualify as "alive," they are (and most likely have historically been) an integral part of life and the construction of life. viruses have influenced the tree of life, as we know it, with lateral gene transfersdsome mediated by virusesdbeing a key element in its architecture and differentiation of the different domains of life (ciccarelli et al., ; weiss et al., ) . because of the difficulties of defining "life" unambiguously, the reader might have noticed that in previous sections, the question addressed has been "how is life" rather than "what is life." the question of "why" have viruses persisted in the biosphere is addressed next. ( ) possessing one type of nucleic acid, ( ) multiplying in the form of their genetic material, ( ) unable to grow and to undergo binary fission, and ( ) devoid of a lipmann system (lwoff, ) . • viruses are entities whose genomes are elements of nucleic acid that replicate inside living cells using the cellular synthetic machinery and causing the synthesis of specialized elements that can transfer the viral genome to other cells (luria et al., ) . • viruses are replicating microorganisms that are among the smallest of all life forms (first two editions of fields virology). • viruses are transmissible deoxyribonucleic acid (dna) or ribonucleic acid (rna) genetic elements that require a cell for multiplication (domingo and perales, ). two general models have been proposed to explain the maintenance of viruses in the biological world: • viruses have persisted because they parasitized opportunistically any cellular niche which was compatible with their replication apparatus, and which provided adequate resources. in this view, viruses are "selfish" replicating elements that became successful when increasingly efficient polymerization activities became part of their life cycles. however, several observations indicate that what was once considered purely "selfish" (also referred to as "junk") cellular dna may not be that useless after all. given the intimate connections between cells and viruses, the view that viruses primarily exist because they are mere "selfish" entities seems unlikely. a different issue is that under some particular environments, a virus displays a "selfish" element-like behavior, as one of the outcomes of their having been positively selected. an example, is the blind replication at the expense of elimination of hosts in disease processes, although this is necessarily a transient behavior. • the presence of viruses was positively selected because they promoted cellular variation and functional diversification. this proposal relates to some of the theories of virus origins (section . ) and the early evolution of life that implied a role for virus-like entities. according to this view, viruses, together with other subcellular genetic elements (plasmids, retroelements, etc.) , penetrated the genetic material of ancestral cell forms, acted as agents of lateral gene transfers, and modified the expression profiles of the recipient cells. probably, there has been (and there still is) a constant flow of genes between cells and viruses and other mobile genetic elements. the abundance of endogenous retroviruses in the mammalian genomes (garcía-montojo et al., ) is a clear symptom of such a genetic flow. a nonfunctional viral infectivity factor (vif) [the hiv- protein that can counteract the mutagenic activity of the apolipoprotein b mrna editing complex (apobec) proteins; see section . in chapter ] was found in the remnant of a rabbit endogenous retrovirus termed rabbit endogenous lentivirus type k (katzourakis et al., ) . about % of the human genome is made of retroviral-like elements. present-day human endogenous retroviruses probably contribute to pluripotency of human cells (santoni et al., ) , and genome regulation (chuong, ) . the fusion of cells from the placenta is mediated by syncytin, a protein in herv-w endogenous retroviruses (chuong, ; villarreal, ) . in addition to the promotion of gene transfers to construct key cellular components, viruses probably acted as selective agents for cells to evolve defense mechanisms against viruses, and this may have originated new cellular functions. also, viruses could favor the survival of some cell types over others, based on differential cell susceptibility to virus infection, thus contributing to cellular diversification. the need to escape viral infection may have furnished novel cell surface receptor proteins through a selection of cellular escape mutants (buckling and rainey, ; saren et al., ) . some experimental systems consisting of persistently infected cells in which the cells and the resident virus coevolve (chapter ) illustrate how viruses could act as selective agents to promote cellular variation. such variation would not necessarily involve exchanges of genetic material between the virus and the cells, provided sufficient genetic variation of cells took place. multicellular organisms devoid of viral entities should have endured a long-term disadvantage over an alternative scenario with the coexistence of cells and viruses. selection by viruses need not be restricted to cells, and it can . being alive versus being part of life be extended to entire host organisms and their populations. an abundance of hosts may promote viral epidemiological fitness, which is a factor in viral disease emergence (chapters and ). host subpopulations may be selected by their resistance to epidemic outbreaks by highly pathogenic viruses, such as in the influenza pandemics or currently with the aids or zika pandemics, and ebola outbreaks in some parts of africa. traditionally, plagues decimated the human population and acted as selective agents for differential survival of individuals. selfish-opportunistic and selected-functional are not incompatible models of virus maintenance. once the instruction to replicate had been positively selected, selfish elements could ensue. as we learn about viral and cellular genomics, the current promiscuity and diversity of viruses (section . ) appear as complementary agencies to promote general biological evolution following darwinian mechanisms. viruses might have contributed to the dna replication machinery of cells, to the formation of the eukaryotic cell nucleus, and to a number of developmental processes (baranowski et al., ; bushman, ; bacarese-hamilton et al., ; mallet et al., ; villarreal, villarreal, , forterre, a) . cells are a necessity for viruses and viruses are promoters of cell diversity and as a consequence, of cellular differentiation (compartmentalization and functional specialization). present-day viruses reveal several mechanisms of exchange of genetic material that might have roots in early cellular evolution. temperate bacteriophages (the prototypic example being e. coli phage l) integrate their genomic dna in the dna of their host bacteria. the uptake of cellular genes by viruses has been amply documented in transducing bacteriophages (those that can transfer dna from one bacterium to another), as well as in rna and dna tumor viruses. even rna viruses that are not known to include a reverse transcription step in their replication cycle can incorporate host rna sequences. replication-competent, cytopathic variants of bovine viral diarrhea virus (a type species of the genus pestivirus of the important family of pathogens flaviviridae) can acquire cellular mrna sequences in their genome, via nonhomologous recombination (meyers et al., ) . insertion of s ribosomal rna sequences into the hemagglutinin gene of influenza virus increased its pathogenicity (khatchikian et al., ) . some defective-interfering particles of sindbis virus included cellular trna sequences at their -ends (monroe and schlesinger, ) . sequences related to some flaviviruses can persist in an integrated form into the dna of the insect vectors aedes albopictus and aedes aegypti (crochu et al., ) . endogenous hepatitis b viruses (ehbvs) have been identified in the genomes of birds and land vertebrates (amniotes), crocodilians, snakes, and turtles. the evidence is that ehbvs are more than million years old and that ancient hbv-like viruses infected animals during the mesozoic era (suh et al., , fig. . ) . the existence of alternative mechanisms for the integration of viral genetic material into cellular dna suggests an ancient origin and a selective advantage of exchanges of genetic information in shaping a diverse and adaptable cellular world (eigen, (eigen, , gibbs et al., ; villarreal, villarreal, , ). symbiosis is an important determinant of coevolutionary interactions between hosts and their parasites (vorburger and perlman, ) . frequent symbiotic and mutualistic interactions have been established with viruses. human endogenous retroviruses can protect human tissues and the developing fetus against infection by some exogenous retroviruses (ryan, ) . some bacteria require bacteriophage to express virulence determinants (tinsley et al., ) . symbiosis can be established between bacteriophages and animals (barr et al., ) . several plant rna viruses delay the symptoms of abiotic stress, such as those produced by drought and frost (dehydration, osmotic stress, and oxidative stress). protection is mediated by increased levels of osmoprotectants and antioxidants in the infected plants (xu et al., ) . symbiotic relationships represent a state of local equilibrium between viruses and hosts, triggered by compatibility and occasionally by mutual benefits. an arms race implied by the virus-host interactions described in previous sections might have been the modus vivendi for viruses, only interrupted by occasional armistices. alternatively, symbiotic and mutualistic interactions might have been the norm, only interrupted by occasional defections by killer personalities that have become the key actors of hospital wards and virology textbooks (roossinck, ; li and delwart, ) . as discussed in connection with natural counterparts of the transition toward error catastrophe in viruses (chapter ), cellular editing activities such as those displayed by some of the adenosine deaminase acting on double-stranded rna and apobec proteins are also part of the innate immune response against some viruses. in turn, viruses have evolved multiple functions to counteract the host immune response (chapter ). the recruiting of cellular functions to confront viruses attests of transient losses of an equilibrated coexistence. some middle-and long-term equilibrium between virus and host population numbers must be continuously restored by selective events; otherwise, this book would not have been written. the evolutionary origin of defense mechanisms against viruses can be regarded as a response to an excessive number of virus-cell interactions. superinfection exclusion is one of the mechanisms used by present-day cells to limit replication of a virus when another one is actively replicating (or incorporated) into the same cell. exclusion has a biochemical interpretation in the competition of two viral entities for cellular resources, but it might have been boosted by early cellular adaptation to limit viral invasions (chapter ). likewise, components of the intrinsic and innate immune response that prevent infection and disease might have also been endowed with activities that promoted cell variation for adaptability. the two opposite views of the activity of viruses in the biosphere (i.e., opportunistic occupation of any suitable cellular niche or intimate cooperative coevolution with host cells) would be expected to produce a different proportion of pathogenic viruses. opportunistic invasions should lead mainly to disease-prone viruses, while long coevolutionary periods should lead to a dominance of nonpathogenic viruses. not all viruses that have been characterized are pathogenic, and in fact, only a minority of those that exist might be. however, since only a limited number of the viral genomes predicted by metagenomic surveys have been characterized, it is not possible to adventure a proportion of beneficial or neutral versus harmful viruses. in the course of investigations on poliomyelitis, a search for related viruses was undertaken, and a number of new viruses later to be known as echoviruses were discovered. they were isolated because they caused cytopathology to cells in culture. the virus-containing samples were from individuals that did not show symptoms of a viral infection. the new isolates were designated as "orphans," meaning viruses without the disease. the term echovirus derives from the enteric cytopathogenic human orphan virus. some of them, or their close relatives, were later associated with disease syndromes, but others were not. viruses, as diverse as circoviruses, polyomaviruses, or herpesviruses colonize a . virus and disease considerable proportion of animals, and only some of the virus types are the direct cause of disease [e.g., postweaning multisystemic wasting syndrome (pmws) by porcine circovirus type , or cancer by some polyomaviruses, among many other examples]. disease potential is unrelated to viral genome size. pmws is associated with the smallest mammalian dna virus genome of only . kb, while the almost -fold larger herpes virus genomes can coexist with immunocompetent humans without the noticeable disease. the pathogenic character of a virus depends on the intricacies of virus-host interactions that are poorly understood. the more the knowledge of virus-host interactions, the border between the virus being pathogenic or nonpathogenic becomes fuzzier. viruses may not damage essential cell functions but may affect dispensable cellular functions. studies by m.b.a. oldstone and his colleagues on persistent infections of lymphocytic choriomeningitis virus in neuroblastoma cells demonstrated that the resident virus altered the expression of differentiated cell trait while preserving vital functions (oldstone et al., ) . this is one of many examples of virusinduced modifications of the so-called luxury functions of cells (oldstone, ) . disease manifestations depend on multiple host and viral factors (allelic forms of several genes, microbiome composition, immune status, coinfections with viruses or other agents, and so on). the dual infections with hiv- and hepatitis c virus (hcv) during the end of the th century accelerated the evolution of hcv-associated liver disease. in the following chapters, molecular mechanisms of virus variation are reviewed in connection with virus survival and host interactions. fig. . ) . in each box, the arrow indicates a succession of major developments, probably with overlapping phases. an evolving virosphere is portrayed as having influenced the establishment of the major cellular domains of life. two of several proposed cell differentiation routes into life domains are drawn. lgt (lateral gene transfer) are depicted as horizontal discontinuous lines, assumed to be more abundant during pre-cellular and early cellular evolution. illustration taken from domingo, e., perales, c., . virus evolution. in: microbial ecology and evolution. encyclopedia of microbiology. fourth ed. elsevier, amsterdam), with permission. a reasonable hypothesis is that mechanisms similar to those observed in the present-day biosphere and virosphere were also in operation during the multiple transitions from a primitive replicon-based biosphere to a cellular world differentiated into different domains of life ( fig. . ). although this point will again be examined as part of an overview in the closing chapter of the book (chapter ), here, some of the observations on the current biosphere dynamics that bear on the difficulties encountered in reconstructing the sequential transitions toward cellular differentiation are outlined. the following circumstances could have blurred the relationships among evolving previral and precellular entities and among viral genomes under construction, from the perspective of current genomics: (i) episodes of evolution at rates higher than average might historically have accelerated evolution and extinction of viral and cellular genetic elements. (ii) experimental evolution has documented that viruses often have available multiple mutational pathways to gain fitness in changing environments. this could well apply to other genome assemblies at different stages of complexity. the genome assemblies and linkages we observe are a set among many others possible that could have existed for prolonged time periods but that are now extinct. a related aspect is that the acquisition of genomic modules from other cells or viral elements could render prior acquisitions obsolete or even detrimental. in consequence, an observed absence of shared genomic regions among different viral or cellular lineages from different life domains does not exclude that the shared domains had been transiently (transiently meaning million years!), with no trace in present-day viruses or cells. (iii) bottleneck events could have erased biological organizations that had a similar degree of adaptation than others that survived. (iv) fitness variation associated with genomic changes (due to mutation, recombination or lateral gene transfers) is environment-dependent, and the changes in the environment over millions of years might have been dramatic. such changes might have affected a limited number of local environments or most environments globally. furthermore, most of these potentially perturbing events might have taken place many times in unpredictable successions, affecting local and few or general and many environments, thus rendering reconstruction of past events from current genomics problematic (domingo and perales, ) . again, these contingencies will be more apparent when different facets of viral dynamics are dissected in the following chapters. contrary to the usual practice in experimental virology, the contents of this chapter have forced a considerable degree of uncertainty, and at times, speculative argumentation. it was, however, a necessary exercise, since at least part of what we see today in viruses must have roots in the origin of life and the role of viruses in life development during epochs that humans have not witnessed. we are left with trying to reconstruct. this chapter is excitingly as close to physics and chemistry as it is to biology. many of the questions addressed are open to debate, and they will probably remain open for a long time. interestingly, the terms "heterogeneity" and "complexity" have appeared several times in this chapter, anticipating features of present-day viruses discussed in the upcoming chapters. complex populations, be they of viruses, cells, protocells, lipid vesicles, primitive replicons, or peptide soups, are the raw materials on which natural selection can act. primitiveness does not imply simplicity. complexity was likely a constant trait for early and modern life, both for the extinguished unknown viruses and for those we strive to understand and control (see summary box). • life is characterized by four integrated features: replication, evolvability, metabolism, and compartmentalization. it is debated whether replication or metabolism was the dominant triggering factor. • at least two ancestral positive selection events might have contributed to the development of life: the selection of replicating over nonreplicating polymers, and selection of splitting-prone versus nonsplitting-prone membrane vesicles. • present-day viruses may be descendants from the primitive replicons that participated in early life prior to cellular organizations, or from structured cells by escape or reduction. • key features of many present-day viruses may be an inheritance of primitive replicons, notably error-prone replication, spread through membrane structures, and tendency to engage in genome integration and transfers. • the geological record indicates multiple, brief, and drastic mass extinction events in earth's history. such events anticipate mass extinction of the resident viruses. a dynamics of emergences, reemergences, 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diversity in the near-surface atmosphere from molecular entities to competent agents: viral-infection-derived consortia act as natural genetic engineers ribozyme-catalyzed transcription of an active ribozyme on the evolution of cells virus infection improves drought tolerance two-amino acid molecular switch in an epithelial morphogen that regulates binding to two distinct receptors life from an rna world. the ancestor within key: cord- -trvleobp authors: hoy, carlton f.o.; kushiro, keiichiro; yamaoka, yutaro; ryo, akihide; takai, madoka title: rapid multiplex microfiber-based immunoassay for anti-mers-cov antibody detection date: - - journal: sens biosensing res doi: . /j.sbsr. . sha: doc_id: cord_uid: trvleobp on-site multiplex biosensors for innate immunity antibodies are ideal tools for monitoring health status of individuals against various diseases. this study introduces a novel antibody immunoassay testing platform incorporating microfiber-based arrays of antigens to capture specific antibodies. the fabrication and setup of the device revolved around electrospun polystyrene (esps) microfibers that act as three-dimensional membrane filters, capable of rapid and multifold analyte capture. in particular, the esps microfibers were patterned through localized oxygen plasma to create hydrophilic zones that facilitate fluid flows and immobilizations of antigens. the bulk of this robust antibody immunoassay platform could be installed into a compact syringe-driven cassette device, which could perform multiplex antibody immunoassay for antibodies specifically against middle east respiratory syndrome coronavirus (mers-cov) with rapid preparation amounting to a total of min, as well as high sensitivity and specificity for the mers-cov down to μg/ml. for the clinical use of immunoassays, these platforms are expected to be high-throughput, rapid and highly sensitive, as these are basic requirements for the necessary performance. thus, assays combining these characteristics are proposed and often realized by the use of microfluidics and nanotechnology [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . furthermore, many types of multiplex immunoassay platforms have been reported, including microelectromechanical systems (mems) [ , ] , paper-based [ ] [ ] [ ] [ ] , bead-based [ ] [ ] [ ] and array-based platforms [ ] [ ] [ ] [ ] . in recent years, one of the most prominent application of such immunoassay platforms from the perspective of healthcare has been virus detection [ , ] . since , mers-cov has become a prevalent issue affecting multiple countries [ ] [ ] [ ] . this coronavirus has affected individuals with a high mortality rate of about % which was reported by the world health organization (who) [ ] . furthermore, mers-cov being a relatively recent outbreak, there has been no effective vaccine which is clinically approved to treat an individual [ ] . therefore, a means for antibody diagnostics in a simple, robust and rapid manner is necessary to test vaccine or drug efficacies upon patients while also preventing the spread of this infectious disease. in the world of vaccination, blood antibody titres often in the form of enzyme-linked immunosorbent assays (elisa) are common methods to determine whether a patient currently has the necessary antibodies at adequate concentrations. elisa is a highly sensitive method that can achieve limits of detection (lod) below μg/ml for mers-cov, but the test can take days to produce diagnostic results due to the requirements for specialists and large equipment [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . thus, a more rapid and effective testing methodology that does not require large testing apparatus is desirable to test for vaccine or drug efficacy. in particular, it is important to emphasize the need for low-cost, simple and robust point-of-care (poc) device manufacturing in order to improve global healthcare. here, a rapid immunoassay methodology for antiviral antibody testing that can be conducted within minutes will be introduced. this work illustrates the construction of a novel multiplex antibody immunoassay test which leverages the esps microfiber mats as the capture membrane and pressure-driven convection for ultra-rapid testing. in addition to the previously established fluorescently-linked immunosorbent assay (flisa) microfiber platform [ , ] , the microfiber mats were pre-patterned with o plasma to create multiple hydrophilic zones with different antigens, and thus creating a multiplex detection system. as a proof of concept, two different antigens designed to individually capture their corresponding antibodies were tested: human https://doi.org/ . /j.sbsr. . received august ; received in revised form october ; accepted october serum albumin (hsa) and mers-cov. hsa is a representative protein that exists in human plasma samples, while mers is a viral agent currently lacking a means of vaccination [ , ] . the simplistic design, ease of fabrication and setup, the rapid speeds at which diagnostics can be performed for antibody detection with this system can be an integral step forward in creating a low cost commercial device for protecting the general public health in case of mers outbreaks. for the polymeric electrospinning processing, pelletized ps (ps japan) dissolved in a solution containing thf (sigma-aldrich) and dmf (sigma-aldrich). hsa full rapid flisa operations included goat anti-human albumin (bethyl laboratories, a - a), human reference serum (bethyl laboratories, rs - ) and fitc-conjugated goat anti-human albumin (bethyl laboratories, a - f), noted as 'anti-hsa', 'hsa' and 'fitc anti-hsa' in this study, respectively. for antibody testing both the hsa reagents mentioned and the following mers reagents: his-mers-np antigen protein (yokohama city university) and anti-mers-np (mab # , yokohama city university) were utilized and noted in this study as the 'mers' and 'fitc anti-mers' reagents, respectively. anti-mers-np was labeled by the fluorescein labeling kit -nh (dojindo inc.). phosphate buffered saline (pbs, ph . , gibco) with tween (pbs-t, ph . , . w/v% tween ) and skim milk (yukijirushi inc.) were utilized for different purposes like diluents, washes and blocking agents. additionally, fitcconjugated bovine serum albumin (bsa-fitc, sigma-aldrich, a - g) and bsa (sigma aldrich, a - g) were utilized in combination with esps fiber mats for fluorescence microscopy. pre-pelletized ps at vol%/vol% was dissolved into a solution of : thf/dmf. the solutions were left to stir mildly at room temperature for h to allow for the ps to dissolve entirely. the ps solutions were then loaded through a syringe to the electrospinning device. the environmental and processing conditions were set as noted in supplementary material table s . after electrospinning, a 'wet-press' technique reported by wu et al. was applied to the microfibers [ ] . this technique layered esps fiber mats by stacking layers of fibers atop one another, soaking them in ethanol (etoh) then sandwiching the stack between glass sheets and pressed by a kg weight for h before use. an o plasma cleaner (pdc- , expanded plasma cleaner, harrick plasma) paired with a gas flowmeter (pdf-fmg, plasmaflo gas flow mixer, harrick plasma) were utilized to treat the esps fiber mats with o plasma. a steel mask with holes (spot size = . mm) was custommade to create patterns of o plasma treatment on the esps fiber mats. esps wt% fiber mats that were wet-pressed at -layers were utilized for spot treatment experimentation. -layered wt% esps was used rather than -layer samples since the -layer samples showed a spreading of added reagents into non-hydrophilic spot regions over time. -layer samples showed minimal spread or leak of reagents beyond the scope of the o plasma treated hydrophilic spots. -layer esps fiber mats after spot treatment were tested for both protein adsorption and blocking preference by fluorescence microscopy. for protein adsorption esps fiber mats were submerged in a fitc-bsa solution ( wt% fitc-bsa to bsa in ml pbs) for h. samples were then dip-washed in pbs-t times. blocking effectiveness was tested by comparing bsa, blockingone and skim milk as blocking agents, where three separate esps fiber mats were soaked in the respective blocking solutions: bsa ( w/v), blockingone ( v/v), and skim milk ( w/v) for h before being dip washed in pbs-t times. then the samples were soaked in the same fitc-bsa solution as the previous experiment for h and dip-washed again in pbs-t times. all fluorescence images were measured under an inverted microscope (ckx , olympus) paired with a fluorescence light source and filter (u-hglgps, olympus). images were taken by the cellsens (olympus) image processing software and measured by imagej (national institutes of health) under the roi manager tool. a -mm monitor (advantec) was adapted as the cassette device by replacing the internal membranes with the -layered wt% esps samples together with an appropriately cut and shaped filter paper ( mm, no. b, kiriyama rohto) layered inside the -mm monitor housing environment. the setup had the lower layer as the filter paper with the upper layer as the o plasm-treated esps fiber mat. through the inlet, bulk reagents (pbs wash, antigen, and secondary antibody) were inserted and flushed out the opposite end outlet through syringe pressure. in all immunoassays, the -layered wt% esps membranes, which were treated with o plasma treatment for min at w, were utilized. rapid immunoassay testing using the cassette platform were performed in following steps: ( ) antigen immobilization, ( ) blocking, and ( ) antibody capture. first, hsa and mers concentrations were optimized independently on the treated esps membranes. hsa concentration in pbs was varied in the range from to μg/ml, using μl per spot. blocking by ml of skim milk at % w/v was then applied through the syringe directly into the cassette, where the solution was held for min then pulled through the outlet. finally, ml of fitcconjugated anti-hsa antibody solution at μg/ml was held in the cassette for min and also flushed through. similarly, mers was tested first by immobilizing his-mers-np at concentrations ranging from to μg/ml. after the skim milk blocking procedure, ml of fitcconjugated anti-mers np # solution at μg/ml was held in the cassette for min and flushed through. for both hsa and mers testing, washing was conducted with pbs-t with ml volumes flushed through three times through the cassette both after the blocking step and fitcconjugated antibody step. for the multiplex immunoassay, it was conducted identically to the above immunoassay protocol, but with different antigens patterned separately on a single esps membrane: spots were treated with mers ( μg/ml; μl), spots treated with hsa ( μg/ml; μl) and spots with no antigen immobilized. again, the blocking was performed with ml of skim milk at % w/v was held for min in the cassette and then flushed through. finally, three different test solutions of antibodies were prepared and tested through the multiplex esps membrane: ml of fitc-conjugated anti-hsa solution at : dilution, ml of fitcconjugated anti-mers np # solution at : dilution, and a ml mixture of fitc-conjugated anti-hsa ( : dilution) and fitc conjugated-anti-mers np # ( : dilution), where the concentrations for each antibody were identical in all cases. fig. and supplementary material table s illustrate the overall procedure as well as the final concentrations used for the test protocols. it is critical that as much antigens are immobilized to the surface to enhance the signal of the immunoassay system for the antiviral antibodies. therefore, the amount of protein immobilization was optimized through the microfiber surface hydrophobicity. the -layered wt% esps fiber mat samples were treated by o plasma spot treatment for different time lengths, then tested for protein adsorption to the surface. to determine the optimum condition for the largest antibody immobilization to the surface, bsa was used as the model protein. first, the contact angles were measured to confirm the effect of o plasma treatments (fig. ) . overall, it was confirmed that the hydrophilic areas treated by o plasma showed a decreasing trend in contact angle with increasing treatment time. on the other hand, the contact angles of hydrophobic zones were not significantly altered among the samples. next, the actual amount of bsa adsorption on plasma-treated samples were analyzed to estimate the antigen immobilization capacity. the results for varied o plasma treatment conditions are shown in fig. , where the samples were treated for , and min at w. it can be seen that the most amount of bsa was adsorbed to the -mintreated samples. the lack of sufficient o plasma time at min likely did not allow the oxygen to fully penetrate the fiber thus not yielding adequately-hydrophilic fiber surfaces for the bsa solution to penetrate the fiber and come in contact with the fiber surfaces. on the other hand, the excessive -min-treated samples over treated the samples to become super hydrophilic, preventing any hydrophobic bonding between the surface and protein samples. thus, for subsequent experiments, min was chosen as the optimal time for o plasma treatment for protein immobilization to the fiber surfaces. in addition, blocking agents were optimized for the esps samples and thus a similar protein adsorption test was conducted with three types of blocking agents: bsa, blockingone and skim milk (fig. ) . although all of them displayed efficient blocking of fluorescently- labeled bsa adsorption, it was demonstrated that skim milk performed the best. therefore, in subsequent experiments, skim milk was used as the blocking agent. to provide a proof of concept study for clinical application as to whether the fabricated esps platform together with the design rapid immunoassay protocol within a device-based environment could be applied to a rapid mers immunoassay, antibodies against hsa (control) and mers-cov (target) were tested using this platform. as mentioned in the introduction, by attaining a rapid detection of desired analytes, in this case antibodies to examine the immune system, a progressive step forward can be made towards preventative diagnostics. first, the optimization of the protocol for antigen immobilization to the esps platform was performed. here, both hsa and mers antigens were tested separately. optimization of hsa adsorption indicated a rapidly increasing trend up until about μg/ml to where increasing amounts of hsa immobilized to the surface has a plateaued effect of detection intensity (fig. ) . meanwhile, the mers adsorption test demonstrated that detection was limited at concentrations less than μg/ml, thus concentrations above this value were used for subsequent studies. lastly, multiplex rapid immunoassay using μg/ml of hsa and mers antigens was performed (fig. ) . three separate esps platforms, each with o -plasma treated spots, were all processed under the same conditions with the only difference being the antibody solution having: ( ) only fitc-conjugated anti-hsa antibodies, ( ) only fitc-conjugated anti-mers antibodies, or ( ) a mix of both fitc conjugate anti-hsa and fitc-conjugated anti-mers antibodies. it was successfully observed that, upon the esps platforms tested with fitc-conjugated anti-mers antibody and anti-hsa antibody solutions, each of mers and hsa antibodies could be selectively detected. additionally, when a mixed solution of fitc-conjugated anti-mers and anti-hsa antibodies was added, both antibodies could be simultaneously detected with the multiplex setup. it was also noted that the signals for both antibodies slightly decreased in the mixture test, in comparison to the individual tests, possibly due to the crowding. this multiplex microfiber-based immunoassay platform hinges on several key factors including the patterned o plasma treatment and efficient blocking that warrant some discussions. first, the rationale behind how proteins such as the immobilized antibodies/antigens preferentially adsorb to just the hydrophilic spots is of great importance. generally, the protein adsorption on flat surface is affected by the hydrophobicity and surface potential [ ] . in the case of epsp fiber mats, the nature of hydrophobicity arises from cassie-baxter regime consisting of two kinds of hydrophobic materials, which are the untreated ps and air [ ] . the o plasma treatment modifies the surface properties of ps rendering it hydrophilic. for flat ps plates, the surface wettability of water changed from . °± . °- . °± . °after treatment with o plasma for min. interestingly, in stark contrast to the flat plates, the o -plasma-treated ps microfibers resulted in greater levels of hydrophilicity ( . °± . °) after the same plasma treatment, as illustrated in fig. . this hydrophilic nature of the esps microfiber mat after o plasma treatment can be explained by the wenzel regime, describing the reduced apparent water contact angles for rough surfaces [ ] . indeed, the patterned o plasma treatment on the esps microfiber mats resulted in separate regions with these two different regimes, where only the hydrophilic surfaces promoted solution flow through the plasma-treated spots. in other words, o plasma treatment of ps was suitable for preventing air pockets within microfibers, and thus the protein solution could contact the fiber surface through wetting. on the contrary, the unexposed, hydrophobic surfaces of esps did not allow wetting, so the protein solution could not contact the fiber surfaces in these regions. thus, although the total amount of protein adsorption is regulated by the true contact angle of materials, the apparent contact angle arising of the porous structure of esps microfiber is also a crucial parameter in dictating protein adsorption. next, a brief discussion of skim milk as the blocking agent is of importance. often times, immunoassays are conducted with blocking agents commonly those of proteins which can adhere well to the surface without preventing any other adsorption of proteins to the surface. among the common choices, skim milk, bsa or bsa-based products, such as blockingone exist. as noted in fig. , skim milk had the best results for blocking in the case of the o -plasma-treated samples. the mix of variety of proteins that exist within skim milk could beneficially be penetrating the fiber and thus blocking the surface most effectively [ ] [ ] [ ] . it has been found that casein, within milk, can be an effective blocking agent due to its small protein size. together with other proteins within skim milk a closely packed surface of proteins can effectively prevent non-specific adsorption to the surface. in contrast, bsa has relatively large molecular weight components which can make random close packing of blocking to the surface difficult in comparison to that of skim milk. another interesting point is that skim milk is considered amphipathic having both hydrophilic and hydrophobic parts. as o treatment is introduced to the surface, the hydrophilic nature may have allowed milk to be effectively bound to the surface. lastly, the comparison of the multiplex microfiber platform used in this study to other conventional assays and devices is important. a comparable work to this study was conducted by sato et al. [ ] , where a combinatorial assay was conducted utilizing a microfluidic chip together with integrated polystyrene beads to measure iga antibody concentration. similar to how microfibers were utilized in this study to increase the surface to volume ratios, microbeads were packed into a microfluidic device with a filling factor of % (bead diameter of μm). although they were able to achieve a high level of sensitivity below μg/ml, their straightforward step-by-step immunoassay procedure of antigen immobilization and then capture of colloid gold labeled-iga antibodies took roughly h, compared to several minutes in this study. thus, sensitivity and speed is often a fine balance for these immunoassays, and priorities should be chosen based on the applications of these assays. the multiplex microfiber platform in this study is intended for antibody detection for poc monitoring of antibody levels. it has recently been reported that the antibody titers of patients infected by mers-cov range from : to : , depending on the time post infection, with the neutralizing antibody titer being more than : [ ] [ ] [ ] . considering that elisa is often used for determining antibody titers and its lod is around μg/ml [ ] , it is suggested that the required antibody concentration for neutralizing mers-cov would be above μg/ml. thus, the lod of μg/ml for the anti-mers-cov antibody by the multiplex microfiber platform, although not yet as low as other established platforms, would be sufficient to detect the necessary antibody production in mers-infected patients. the multiplex esps fiber system with a housing suited for poc was introduced as a means for rapid mers antibody detection. the capability of a rapid flisa was exhibited by utilizing -layered esps fiber mats treated with o plasma. the patterned o plasma treatment method with developed mask showed optimal protein adsorptive results at min at w. this in turn created a hydrophilic surface capable of antibody immobilization and, further, rapid flisa testing. in terms of the detection surface, effective detection could be achieved upon this platform within min of operation time. furthermore, the actual setup and preparation of the device could be completed in min, which includes capture molecule immobilization, blocking and washing steps. the results demonstrated the capability of the device for multiplex analysis by detecting antibodies for both hsa and mers-cov concurrently. this microfiber-based multiplex immunoassay platform serves as a stepping stone for the next-generation poc device that would enable preventative diagnostics by accurately and rapidly determining the existence of adequate antibodies and aid general healthcare through appropriate vaccination schemes. rapid automated high sensitivity enzyme immunoassay of c-reactive protein highly sensitive immunoassay of lung cancer marker carcinoembryonic antigen using surface-enhanced raman scattering of hallow gold nanospheres significance of antibody 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the mers-coronavirus spike protein and their application in species-independent antibody detection by competitive elisa an updated roadmap for mers-cov research and product development: focus on diagnostics fabrication and assessment of an electrospun polymeric microfiber-based platform under bulk flow conditions with rapid and efficient antigen capture a rapid, simple measurement of human albumin in whole blood using a fluorescence immunoassay (i) immunoassay on free-standing electrospun membranes understanding how charge and hydrophobicity influence globular protein adsorption to alkanethiol and material surfaces the rigorous derivation of young, cassie-baxter and wenzel equations and the analysis of the contact angle hysteresis phenomenon comparison of blocking agents for an elisa for lps non-specific adsorption of fish immunoglobulin m (igm) to blocking reagents on elisa plate wells phospholipids in milk fat: composition, biological and technological significance, and analytical strategies integration of an immunosorbent assay system: analysis of secretory human immunoglobulin a on polystyrene beads in a microchip viral shedding and antibody response in patients with middle east respiratory syndrome coronavirus infection antibodies against mers coronavirus in dromedary camels middle east respiratory syndrome coronavirus (mers-cov): infection, immunological response, and vaccine development we would like to thank dr. satoko matsunaga at the yokohama city university for her kind donations of mers reagents. this work was supported by the grant-in-aid for exploratory research [ k ] from the ministry of education, culture, sports, science and technology of japan. supplementary data to this article can be found online at https:// doi.org/ . /j.sbsr. . . key: cord- -p qchjva authors: alghamdi, abdulaziz; hassan, ahmed m; tolah, ahmed m; alamari, sawsan s; alzahrani, abdulrahman a; alsaaidi, ghaleb a; abujamel, turki s; azhar, esam i; hashem, anwar m title: molecular evidence of influenza a virus circulation in african dromedary camels imported to saudi arabia, – date: - - journal: open forum infect dis doi: . /ofid/ofz sha: doc_id: cord_uid: p qchjva little is known about influenza a viruses in dromedaries. here, we detected influenza a viral rna in specimens ( . %) out of nasal swabs collected from dromedaries between and in saudi arabia. positive samples were detected only in imported camels from sudan and djibouti but not local ones. partial genome sequencing indicates a close relationship to – human/swine influenza a h n isolates from different countries, suggesting possible interspecies transmission. taken together, dromedaries could represent a potentially unrecognized permissive host for these viruses, highlighting the need for enhanced surveillance in animals to aid implementation of one-health strategies. for centuries, camels have played a significant role as a major human companion and a key contributor to the livelihood of mankind. despite the known role of camels in zoonosis, many aspects of their health have not been studied before the emergence of the middle east respiratory syndrome-coronavirus (mers-cov) in [ ] [ ] [ ] [ ] . in fact, the possibility of spreading known and unknown zoonotic pathogens from camels to humans or other animals represents major public health and economic concerns [ ] [ ] [ ] . these concerns are exaggerated further because of the inevitable close contact between camels and humans during ranching, milking, feeding, and riding, raising the need for enhanced surveillance in these animals as a one-health approach. although data on influenza viruses circulation in camels are very scarce, some existing evidence suggests the viruses' ability to infect and cause disease in these animals. several outbreaks of severe respiratory diseases in bactrian camels have occurred between and in mongolia and were suspected to be due to influenza viruses [ ] . serum samples obtained from sick camels during these outbreaks had positive hemagglutination inhibition titers against influenza a/ ussr/ / (h n ) human vaccine strain [ ] . genetic and antigenic analyses of these isolates confirmed their relatedness to influenza a/ussr/ / (h n ) which is a reassortant vaccine strain obtained by reassortment between two h n strains (a/ pr/ / and a/khabarovsk/ ), possibly through transmission from vaccinated humans to camels [ ] . interestingly, experimental infection of bactrian camels with these isolates resulted in productive infections and antibody responses, indicating that camels are a potentially unexplored permissive host for influenza a viruses [ ] . further active and enhanced surveillance of bactrian camels in mongolia also resulted in the isolation of influenza a h n viruses that are phylogenetically related to equine influenza a viruses, suggesting possible interspecies transmission [ ] . although the existence of influenza viruses in dromedary camels has not been investigated properly, previous studies provided limited serological evidence of circulation of all influenza types (a, b, c, and d) among dromedaries in several african countries [ ] [ ] [ ] . it is clear that the potential role of camels in interspecies and zoonotic transmission of influenza as well as the ecology of these viruses in camels are poorly understood, highlighting the need for active and enhanced surveillance. here, we expanded our surveillance for influenza a viruses to include local and imported dromedary camels in saudi arabia, especially as thousands of camels are annually imported from african countries, such as sudan, djibouti, kenya, somalia, and eritrea, in addition to the large number of local camels. a total of and nasal swabs were collected from imported and local camels in saudi arabia, respectively. local camels were sampled form local farms in jeddah (western saudi) and riyadh (central saudi), whereas imported camels were sampled on incoming ships arriving from sudan ( samples) and djibouti ( samples) at jeddah islamic seaport (table ) before disembarking the vessels. all swabs were collected, immersed in viral transport media, transported in a cold container, and stored at - ˚c until studied. samples were collected upon ethical approval from the unit of biomedical ethics in king abdulaziz university hospital. extracted viral rna was screened for influenza a virus rna by one-step real-time reverse transcription polymerase chain reaction (rt-pcr) using single-step x quantifast rt-pcr master mix kit (qiagen, hilden, germany). targeting a bp fragment in the matrix (m) gene (infa-f: ′-gaccratcctgtcacctct gac- ′; infa-r: ′-agggcattytggacaaakcgtcta- ′; infa-p: ′-fam-tgcagtcctcgctcactgggcacg-mgb- ′), rt-pcr was conducted with consensus degenerate primers and probes according to world health organization protocol [ ] . viral rna from known influenza a isolates as well as no template controls were included in each run as positive and negative controls, respectively. samples with cycle thresholds (ct) values < were considered positive. precautions were taken to avoid cross-contamination of samples during all steps and all positive and some negative samples were re-extracted and retested independently to confirm the results. extracted rna from positive samples was used for one-step conventional rt-pcr using the superscript iii one-step rt-pcr high fidelity kit (invitrogen, foster city, ca) according to the manufacturer's instructions using the infa-f and infa-r primers. the resulting amplicons were analyzed on % agarose gel and bands with expected size (~ bp) were purified and sequenced using cycle sequencing on an abi automatic sequencer (applied biosystems, foster city, ca) using infa primers and the bigdye terminator v . reaction cycle kit (applied biosystems, usa) according to the manufacturer's instructions. sequences were assembled and analyzed for homology using the national center for biotechnology information (ncbi) blastn (nucleotide basic local alignment search tool), and the top hits with the highest alignment scores for each contig were kept and summarized in a tabular format. the viral genome was amplified directly from positive samples using the superscript iii one-step rt-pcr high fidelity kit according to the manufacturer's instructions by using the universal influenza a primers (fwuni and rvuni ) as previously described [ ] . purified rt-pcr amplicons were randomly fragmented and used for indexed metagenomic library construction utilizing the illumina truseq nano kit (illumina inc., foster city, ca between and , a total of nasal swabs were collected through active surveillance of imported and local camels ( from imported camels and from local camels). out of the tested samples, samples ( . %) were positive for influenza a virus by rt-pcr (table ) . viral rna was detected only in imported camels from sudan ( samples) and djibouti ( samples) but not local animals (table ). viral rna was detected in both male ( samples) and female ( samples) camels as well as from juvenile camels between to years ( samples) and those older than years ( samples) ( table ) . viruses could not be isolated from any of the positive samples in madin-darby canine kidney cells even after consecutive passages most probably due to the low viral load as shown by the high ct values, which varied from . to . (table ) . these results were confirmed for samples by conventional rt-pcr using the same primers used in the real-time rt-pcr targeting a conserved ~ bp fragment in the m gene of all influenza a viruses. query blastn of the obtained partial sequences of the fragments from these specimens further confirmed these results with > % sequence identity with at least or mismatches to multiple influenza a viruses subtypes (supplementary table s ). this analysis did not result in any definitive determination of the circulating subtypes as expected, because the used primers target a highly conserved region in all influenza a viruses (supplementary table s ). the obtained sequences were identical for samples spc , spc , and spc collected from djibouti in march with and mutations in the other samples (spc and spc ) obtained from camels from sudan and djibouti, respectively, at different time points (figure and table ). unfortunately, other trials to amplify other targets failed to generate products for sequencing possibly due to rna degradation and low viral load as well as the inability to use subtype-specific primers, especially as the exact subtypes were unknown. therefore, positive swab samples (spc and spc ) were analyzed by ngs for further confirmation. upon metagenomic sequencing of the samples, a total of out of and out of high quality reads that matched influenza a sequences in the influenza virus database by blastn were obtained from samples spc and spc , respectively. de novo assembly of these reads resulted in and contigs from samples spc and spc , respectively. query blastn of these contigs returned closely related viruses that belonged mostly to human and swine influenza a h n strains isolated between and from different countries (supplementary table s ). however, some strains from other subtypes such as h n , h n , and h n also were observed at lower frequencies (supplementary table s ). the pb contig ( bases) from both samples were identical and matched a fragment in the pb gene corresponding to the region between nucleotide to with > % identity and a minimum of mismatches. out of the top matches, were h n strains and were h n strains (supplementary table s ). blasting of the bases contig from segment in the spc sample showed > % identity match to residues to in the m gene of influenza a viruses with a minimum of mismatches with h n , h n , and h n strains out of the top matching sequences. the de novo assembled reads from segment of spc sample resulted in contigs ( and nucleotides) that matched regions corresponding to - and - in the ns gene of influenza a viruses, respectively. although the first contig ( nucleotides) matched h n , h n , and h n strains with > % identity, the second contig ( nucleotides) showed > % identity match to h n , h n , and h n strains as well as unknown or mixed influenza a isolates (supplementary table s ). emerging and re-emerging pathogens are potential pandemic threats that are often originate from animals and spread to humans [ ] . such threats could have a global impact on the public health and the economy. therefore, it is crucial to implement early detection, prediction, and risk assessment of pathogens in their animal reservoirs as a one-health approach for proper preparedness to minimize their potential spread to humans. we provide the first molecular evidence of influenza a virus circulation in dromedary camels imported from african countries (sudan and djibouti) but not in local camels from saudi arabia. our data suggest that dromedary camels could represent a potential unknown permissive host and zoonotic source for influenza a viruses. although the partial sequencing data obtained in this study point towards strains closely related to human and swine influenza a h n isolates obtained between and , it is clear that use of short fragments from highly conserved regions of internal genes are not definitive. unfortunately, we were not successful in isolating any virus from the collected samples for better characterization or able to obtain long genome sequences to accurately determine the subtype of these viruses. furthermore, the identical partial influenza rna sequences from different camels arriving from the same origin on the same day as well as the detection of viral rna from animals arriving from different countries at multiple time points over years reduces the possibility of environmental contamination or simple respiratory exposure to infected humans or animals. however, such evidence is not sufficient, and more studies are needed to confirm whether the detected viruses in camels are a result of a natural self-sustained infection or cross-species spillover from humans or pigs. nonetheless, previous productive experimental infection of camels with influenza a h n virus and their seroconversion, in addition to the detection of neutralizing antibody in farm and free ranging camels, clearly provide convincing evidence of the permissiveness of camels to influenza a viruses [ , , ] . although there is no report of human influenza infection due to contact with camels, the role of camels in human infections cannot be excluded, especially in high risk individuals who are in continuous contact with these animals directly or indirectly. the possible risks involved in interspecies transmission of influenza viruses and the odds of reassortment and coinfections in camels are also a big concern. the transmission of these viruses from camels to humans or pigs or vice versa cannot be confirmed and requires further studies to enhance our understanding of influenza ecology and epidemiology in camels and their role in influenza emergence. future studies should focus on virus isolation, full genome sequencing, and using hemagglutinin inhibition assays in order to determine accurately the possible circulating influenza a subtypes in dromedary camels. it is of note that the detected influenza a viruses were from asymptomatic animals, and the pathogenesis and transmission of this virus in camels are not known apart from previous experimental infection in bactrian camels [ ] . in conclusion, we provide the first molecular evidence of influenza a virus infection in dromedary camels, suggesting a possible role for dromedaries in influenza zoonosis or infection to other livestock. our data as well as previous reports suggest that influenza a virus could cause a sustained infection in these animals, highlighting the need for enhanced field surveillance for influenzas viruses as well as other pathogen in dromedary camels to help implementing better preventative one-health plans and programs. supplementary materials are available at open forum infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. trypanosomiasis of camels (camelus dromedarius) in algeria: first report camelus dromedarius brucellosis and its public health associated risks in the afar national regional state in northeastern ethiopia molecular and immunological characterization of hyalomma dromedarii and hyalomma excavatum (acari: ixodidae) vectors of q fever in camels middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia middle east respiratory syndrome coronavirus infection in non-camelid domestic mammals outbreaks of middle east respiratory syndrome in two hospitals initiated by a single patient in daejeon a reassortant h n influenza a virus caused fatal epizootics among camels in mongolia equine influenza a(h n ) virus isolated from bactrian camel detection of influenza antibody in animal sera from kassala region, sudan, by agar gel diffusion test preliminary survey for antibodies against respiratory viruses among slaughter camels (camelus dromedarius) in north-eastern nigeria serologic evidence for influenza c and d virus among ruminants and camelids world health organization. who information for the molecular detection of influenza viruses single-reaction genomic amplification accelerates sequencing and vaccine production for classical and swine origin human influenza a viruses public health. pathogen surveillance in animals we thank king abdulaziz city for science and technology for their generous funding and support.finanacial support. this work was supported by king abdulaziz city for science and technology through the mers-cov research grant program (number - ), which is a part of the targeted research program.potential conflicts of interest. all authors: no reported conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord- -lpxmmbpy authors: amini, rachid; gilca, rodica; boucher, françois d.; charest, hugues; de serres, gaston title: respiratory syncytial virus contributes to more severe respiratory morbidity than influenza in children < years during seasonal influenza peaks date: - - journal: infection doi: . /s - - - sha: doc_id: cord_uid: lpxmmbpy purpose: to compare the frequency and the severity of influenza and respiratory syncytial viruses (rsv) infections among children < months hospitalized with respiratory symptoms. methods: data from a prospective study conducted during the peak of five influenza seasons in the province of quebec, canada were used. results: we detected higher frequency of rsv compared to influenza viruses ( . % vs. . %). radiologically confirmed pneumonia was significantly more frequent in children with rsv ( %) than those with influenza ( %) and the clinical course was more severe in rsv than influenza-infected children, especially among infants < months. conclusion: even during peak weeks of influenza season, we found a higher burden and severity of rsv compared with influenza virus disease in hospitalized children < months. respiratory syncytial virus (rsv) and influenza virus infections are considered as leading cause of hospitalization among infants and young children [ , ] . in a recent study, the average hospitalization rate attributable to rsv infection has been reported to be . per children under years with seasonal variation from . to . per children [ ] . among the same age group, a systematic review has estimated influenza-associated hospitalization rates from to per children [ ] . for both viruses, the highest hospital admission rates were among infants < months with an average rate of . and . per children, respectively, for rsv and influenza [ , ] . most studies assessing the influenza and rsv-associated hospitalization burden focus on the entire respiratory viruses season. little information is available about the role of influenza viruses relative to rsv during periods of high influenza circulation among young children. the aim of this study is to compare the frequency and disease severity of influenza and rsv infections among children < months hospitalized with respiratory symptoms during the peaks of five influenza seasons. in the province of quebec, canada, a prospective surveillance study with virologic assessment for influenza and other respiratory viruses was conducted during the peak weeks of influenza circulation among patients admitted for acute respiratory symptoms to four acute-care hospitals since - . peak weeks are defined as consecutive weeks with ≥ % of weekly specimens testing positive for influenza as monitored by the provincial sentinel laboratory network. all patients hospitalized for fever/feverishness or cough or sore throat have a nasal specimen collected with a flocked swab. specimens are tested by the luminex xtag ® rvp fast multiplex, a polymerase chain reaction (pcr) assay which can detect respiratory viruses (rv) [ ] . patients admitted for ≥ h to the hospital floor are invited to participate in the study; those who give their informed consent are included. demographic and clinical information of patients including chronic conditions associated with increased risk of influenza complications is collected using a standardized questionnaire. in our study, patient's chronic conditions information was retrieved by nurses from medical records and we did not ascertain diagnosis criterions of the asthma condition identified through the chart review among the children < years. as it is difficult to diagnose asthma in these young children and confirm it as definite bronchial asthma, we used the term "asthma-like condition". additional details are presented elsewhere [ ] . the peak weeks of the - and - to - influenza seasons were included in the analysis. the - season was excluded, because only influenza viruses were tested during this season. proportions were compared with the chi-square test or the fisher's exact test where appropriate. continuous variables were compared using wilcoxon and kruskal-wallis nonparametric tests. during the peaks of five influenza seasons lasting from to weeks, children were enrolled in the study. nearly, a third were infants < months ( %, n = ), % were aged - months (n = ) and - months (n = ), and % were aged - months (n = ). except for the - , where the epidemic peak was characterized by the concomitant circulation of influenza a and b, all the other peak weeks of the study period corresponded mainly to the influenza a peak ( at least one respiratory virus was identified in . % of children. rsv was detected in more than half of children ( . %), and the presence of both influenza and rsv was observed in . % of cases. despite being during the peak weeks of the influenza epidemic, influenza viruses were detected in one of six children ( . %). influenza a (h n ) was the predominating subtype ( %) and influenza a (h n ) pdm and influenza b were detected in % and % of specimens, respectively. other respiratory viruses (orv) were detected in . % of children: entero/ rhinoviruses ( . %), coronaviruses ( . %), human metapneumovirus ( . %) and parainfluenza viruses ( . %), and other viruses (adenovirus and bocavirus) ( . %). among the young infants < months, the clinical course was less severe in human metapneumovirus (n = ) than in rsv-infected children based on the presence of radiologically confirmed pneumonia ( % vs. %), length of stay (median of days vs. days) and intensive care unit admission ( % vs. . %), although differences are not statistically significant due to the small sample size. rsv was more frequently detected among infants aged - months ( . %) than in those < months ( . %), - months ( . %), and - months ( . %) (p = . ). conversely, influenza was more frequent among older children aged - months ( . %) than in those < months ( . %), - months ( . %), and - months ( . %) (p = . ). there was no significant difference by age group in high-risk conditions for influenza complications, consultation delay, or influenza vaccine use between influenza and rsv-infected children ( table ). the distribution of the main reasons for admission was similar between influenza and rsv infections among children aged - months and - months. among infants < months, influenza-infected cases were more often admitted for fever than rsv patients ( % vs. %), while patients with confirmed rsv were more frequently hospitalized for dyspnea or bronchiolitis compared to influenza patients. the who severe acute respiratory illness (sari) case definition for influenza surveillance was met by more than % of children with influenza or rsv infections, with a higher proportion ( - %) among children ≥ months than youngest children and no significant difference between influenza and rsv-infected patients ( table ). fever (reported or measured) and cough were the two symptoms included in this case definition. infants < months with influenza had significantly higher rate of fever than those with rsv ( % vs. %, p < . ) and significantly less cough ( % vs. %, p < . ). we did not found in our analysis any specific sign or symptom discriminating between influenza and rsv infection in children < months. among these children, those with rsv had a significantly higher proportion of radiologically confirmed pneumonia than influenza patients ( % vs. %, p < . ) ( table ) . the median length of stay (los) in < -month-old children was similar ( days) for both influenza and rsvinfected children. among infants < months, median los was longer for rsv ( days) than influenza ( days, p < . ). while no child with influenza was admitted to the intensive care unit (icu), . % (n = ) of rsv-infected children required it and most of them ( %, / ) were aged < months (table ) . no death was reported among enrolled children. during peak weeks of influenza season with mostly no overlapping rsv peak weeks, we found a significantly higher burden of rsv compared with influenza virus disease in hospitalized children < months. our results underline the importance of testing young children with respiratory symptoms for a panel of respiratory viruses and the limited value of etiologic diagnoses based on clinical definition and epidemiologic data. in addition, our findings of a substantially greater rsv frequency among hospitalized young children even during influenza peak weeks underscore the need of prevention and control approaches that address rsv infections. to the best of our knowledge, this is the first study that describe the role of rsv relative to influenza viruses during the peak weeks of influenza epidemics among hospitalized young children. other prospective surveillance studies from the united states and europe documented the burden of influenza and rsv among enrolled children during whole influenza seasons or calendar years [ ] [ ] [ ] . conducted mainly among children < years in early s, these studies reported similar patterns of high rsv detection ( - %) relative to influenza ( - %). a recent study from south africa reported that during a -year period ( - ), rsv was more often detected ( %) than influenza viruses ( %) in children < years [ ] . we found the highest rate of rsv among infants aged - months with rsv detection in nearly three over four patients ( . %) followed by infants < months ( . %). similar to our results, other reports found that infants aged - months had the highest proportion of rsv detection [ , ] , while the highest rsv frequency in the south african study was among infants < months [ ] . the clinical presentation of rsv infection in our study revealed the predominance of cough over the fever in particular among infants < months. conversely, young infants infected with influenza had a significantly higher rate of fever than children with rsv infection. these results are consistent with previous reports on the rsv and influenza clinical features in young children [ , ] . we also found that a radiologically confirmed pneumonia was more frequent in rsv infection than in influenza infection, which is in line with other publications [ , ] . there was no difference in the median hospital duration ( days) between rsv and influenza-infected children < years, a finding similar to the south african study that reported a non-significantly different median los ( days) for rsv and influenza-infected children < years [ ] . however, there were differences by age group in our study: infants < months hospitalized with rsv infection had significantly longer hospital stay compared to those with influenza infection (median los of days vs. days). a longer average duration of hospitalization among rsv-infected infants has also been previously reported [ , ] . in this study, rsv infection was associated with higher but not significantly different icu admission rates than influenza infection ( . % vs. %, p = . ) among children < years. a comparable rate of . - . % of icu admissions associated with rsv has been reported in other prospective studies [ , ] . there were no deaths among children included in our analysis, which is consistent with recent studies reporting the relatively low case-fatality rate (< %) associated with pediatric influenza or rsv infections [ , ] . there are several limitations to this study. first, rsv seasonal epidemics overlapped differently with epidemic curves of influenza during the study period. both adults and children contribute to the quebec provincial sentinel data for respiratory viral surveillance. it is likely that children disproportionately drive rsv findings in the community and conversely that adults predominate influenza circulation data. even if rsv and influenza circulation in both adults and children may not coincide exactly with that of children, the overall rsv circulation relative to that of influenza is likely to be reflected in the sentinel surveillance curves we have presented. second, we defined influenza peak weeks as those during which the percentage of positive specimens tested was ≥ % as monitored by the provincial sentinel laboratory network. this definition was based on provincial data indicating that weeks with ≥ % of influenza-positive specimens represented more than % of total influenza-positive tests during entire influenza season, and this peak week definition was used in line with previously published works [ , ] . during the peak influenza weeks of the year study period, rsv and influenza had varying degree of seasonal overlap (fig. ) . relatively complete rsv and influenza peak weeks' overlap was evident during the two last seasons ( - and - ), with more than half ( %) of the total of rsv positive tests during the peak influenza included in our study. however, after excluding these two seasons, we found similar results of higher frequency of rsv compared to influenza infections (data not presented). third, there was no information about rsv risk factors for hospitalization such as history of prematurity, specific comorbidity (cystic fibrosis, neuromuscular diseases, etc.) as our original data focused on depicting the epidemiology of influenza infections. the low frequency of influenza detection is unlikely due to vaccination given the low rate of influenza vaccination among pregnant women (~ %) and children < years ( - %) in quebec [ , ] , and little evidence is available on effectiveness of current influenza vaccines for children < years [ ]. finally, influenza antiviral treatment data were not recorded systematically all seasons, preventing us from analyzing its impact on influenza duration of hospitalization. in conclusion, even during the peak weeks of influenza, more than half of hospitalizations for respiratory infections in children < years of age was due to rsv, with a clinical course more severe than influenza notably among infants < months. conflict of interest this work was a part of a study supported by the ministère de la santé et des services sociaux du québec. r. g. received research funding from ministère de la santé et des services sociaux du québec for this study and research grants from sanofi pasteur and pfizer for unrelated studies. all other authors report no potential conflicts. ethical statement institutional review board approval was obtained from all participating hospitals for the first two years; an exemption was obtained for the rest of years given the change of the design and objectives from a research study into a surveillance project. global burden of respiratory infections due to seasonal influenza in young children: a systematic review and metaanalysis global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in : a systematic review and modelling study respiratory syncytial virus-associated hospitalizations among children less than months of age the burden of influenza hospitalizations in infants from other respiratory viruses are important contributors to adult respiratory hospitalizations and mortality even during peak weeks of the influenza season population-based surveillance for hospitalizations associated with respiratory syncytial virus, influenza virus, and parainfluenza viruses among young children prospective population-based study of viral lower respiratory tract infections in children under years of age (the pri. de study) comparison of human metapneumovirus, respiratory syncytial virus and influenza a virus lower respiratory tract infections in hospitalized young children the role of hiv in influenza-and respiratory syncytial virus-associated hospitalizations in south african children trends in chronologic age and infant respiratory syncytial virus hospitalization: an -year cohort study respiratory syncytial virus-and influenza virus-associated hospitalizations in infants less than months of age defining the epidemiology and burden of severe respiratory syncytial virus infection among infants and children in western countries hospitalization for influenza a versus b. pediatrics epidemiology of influenza-associated hospitalization in adults when should a diagnosis of influenza be considered in adults requiring intensive care unit admission? results of populationbased active surveillance in toronto key: cord- -m xba g authors: macintyre, chandini raina; costantino, valentina; kunasekaran, mohana priya title: health system capacity in sydney, australia in the event of a biological attack with smallpox date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: m xba g planning for a re-emergent epidemic of smallpox requires surge capacity of space, resources and personnel within health systems. there are many uncertainties in such a scenario, including likelihood and size of an attack, speed of response and health system capacity. we used a model for smallpox transmission to determine requirements for hospital beds, contact tracing and health workers (hcws) in sydney, australia, during a modelled epidemic of smallpox. sensitivity analysis was done on attack size, speed of response and proportion of case isolation and contact tracing. we estimated clinical hcws and public hospital beds in sydney. rapid response, case isolation and contact tracing are influential on epidemic size, with case isolation more influential than contact tracing. with % of cases isolated, outbreak control can be achieved within days even with only % of contacts traced. however, if case isolation and contact tracing both fall to %, epidemic control is lost. with a smaller initial attack and a response commencing days after the attack, health system impacts are modest. the requirement for hospital beds will vary from up to % to % of all available beds in best and worst case scenarios. if the response is delayed, or if the attack infects people, all available beds will be exceeded within days, with corresponding surge requirements for clinical health care workers (hcws). we estimated there are public health workers in sydney with up to , contacts to be traced. at least million respirators will be needed for the first days. to ensure adequate health system capacity, rapid response, high rates of case isolation, excellent contact tracing and vaccination, and protection of hcws should be a priority. surge capacity must be planned. failures in any of these could cause health system failure, with inadequate beds, quarantine spaces, personnel, ppe and inability to manage other acute health conditions. smallpox is a category a bioterrorism agent, despite being declared eradicated in [ ] . the virus is retained in high security biosafety level laboratories in the united states and russia [ ] . the variola genome is fully sequenced and could be synthesized in a laboratory [ ] . this a a a a a to determine the capacity of the health system in sydney, a city of . million people in australia, during an epidemic of smallpox. specifically, we aimed to determine hospital bedcapacity for isolation, public health workforce capacity for contact tracing and health care worker (hcw) personal protective equipment (ppe) requirements under different attack scenarios. we also aimed to test a worst case scenario among the range of possible attack scenarios and identify modifiable factors which would prevent a worst case scenario. we constructed a modified seir model for smallpox transmission based on a model published in our previous study [ ] . model parameters and their estimation have been previously described [ ] . we assumed that the virus has not been genetically modified and that there is minimal residual immunity in the population from previous vaccination, as described in our previous study [ ] . we assumed an initial attack size of , or , infected. case isolation was assumed to reduce transmission to zero [ ] . given antivirals would be commenced after diagnosis and isolation, we assumed this effect would only apply in the healthcare setting and would not add to interruption of transmission above the effect of isolation alone, with the main transmission risk being in the community for undiagnosed or early cases prior to hospitalisation. we assumed that antivirals would therefore have no effect on community transmission, acknowledging that they would likely reduce morbidity and mortality for treated cases. we estimated number of hospital beds needed to control the epidemic, ppe requirements for clinical hcws and public health workers required for contact tracing, under different scenarios. we constructed a modified model for smallpox transmission. population residual immunity and contact mixing are based on assumptions used in our previous study [ ] . using ordinary differential equations as described in s file, the population moves through the disease compartmental epidemiological states of being susceptible, exposed, infected and recovered (seir) from smallpox. once infected, people move into the next state following disease duration rates. euler's approximation was used to estimate age-specific force of infection, assuming contact would be similar to observed patterns in the uk [ ] [ ] [ ] . different infectivity levels were based on the reproduction number for hemorrhagic, flat, ordinary and modified smallpox as previously described [ ] . the force of infection was multiplied by a parameter (α , α , α , α ) to account for different population susceptibility levels. model parameters and their estimation are described in our previous study [ ] . the model runs for days. the model assumptions are shown in table . to test the preparedness of the health system we assumed the base case response would be case isolation and treatment, contact tracing and ring vaccination. as the study was examining health system factors, ring vaccination was kept constant in the model at % with assumed adequate vaccine supply and trained vaccinators, and - % vaccine efficacy for uninfected people and - % for latent infected [ , [ ] [ ] [ ] as described in s file. hospital bed requirements. we estimate the number of hospital beds in sydney using data published for - in nsw [ ] . in nsw for - there were beds available from public hospitals ( . per population) and beds available from private hospitals ( . per population). this was resized for the sydney population. the number of hospital beds needed for case isolation was then modelled under different scenarios based on variation of response time (t), the percentage of infected cases isolated each day and how many contacts were traced. we tested if the number of available hospital beds in sydney will be enough to isolate up to % of all new infected cases every day, under different scenarios. clinical health workforce and ppe requirements. the clinical health workforce was estimated by the number of hcws in sydney for / , including aboriginal and tsi health practitioners, chinese medicine practitioners, dental practitioners, medical practitioners, radiation therapists, nurses and midwives, occupational therapists, pharmacists and ambulance services workers [ ] . the total estimated health workforce number was for nsw, with a total population of . million in [ ] . we applied the same percentage adjusted for the sydney population from the same year, . million [ ] . hcw distribution by age average number of contacts per case [ ] proportion of contacts traced around an infected case %, sensitivity analysis with % and % [ ] [proportion of cases that get isolated once infected and symptomatic %, sensitivity analysis on with % and % [ ] group was estimated using national and global health worker data [ , ] . we estimated, based on epidemic size and duration, the amount of respiratory ppe (n respirators) required for sydney clinical hcws assuming two respirators per shift per hcw. this is based on recommendations that disposable respirators should not be re-used, and the fact that a standard shift for a hcw would include at least one break, after which a new respirator would need to be used. public health workforce for epidemic control. there are no published data to estimate the public health workforce, comprising trained public health officers working in health departments and capable of conducting contact tracing and outbreak investigation, as public health workers are not registered health practitioners. the only uniform qualification in public health is a master of public health (mph) and similar degrees. whilst there are a large number of mph graduates in australia, the number working in government public health roles would be a minority. it should also be noted that a mph does not equip people with the skills for field response to an epidemic. there are approximately alumni of the national field epidemiology training program (fetp). in addition, there is a medical specialisation in public health medicine for a relatively small number of medical doctors, with an estimated full time equivalent public health physicians nationwide in [ ] . based on discussions with national experts we estimated there are approximately skilled public health officers in australia, although the actual number may be lower. the public health workforce was calculated using estimates of mph graduates currently working in government, fetp graduates or current fetp trainees and public health physicians. an optimistic assumption of public health officers nationally was used to estimate the number working in sydney. contact tracing requirements. in the base case, we assumed % of contacts would be traced and % infectious people would be isolated. we used age specific contacts rates, with an average of contacts per case based on european social mixing data [ ] . we estimated the number of public health workers required to conduct contact tracing under different scenarios. given contact tracing may require complex communications and travel over large geographic distances, we assumed one public health officer could trace contacts per day. australian guidelines for management of smallpox state that isolation is needed for nonimmune category a (high risk) contacts, in individual rooms with supervision by vaccinated staff [ ] . we conservatively assumed at least % of contacts traced would be category a, and would require supervised quarantine. data from tuberculosis studies [ ] as well as estimated social contact matrices suggest one person [ ] , on average, has - contacts at reasonable risk of infection. the closest of these contacts would include household contacts (about - people), plus - others in work or friendship circles. this would be about half of the - contacts. the number of contacts needed to be traced and managed was estimated based on attack size, time to response (t) and the percentage of infected cases isolated each day. a contact tracing day was defined as one day entirely spent tracing contacts per public health worker. sensitivity analysis. a sensitivity analysis was conducted on attack size, the proportion of infectious cases isolated and contacts traced, as well as time to commencing the response. to illustrate the difference in epidemic size between a single index case of smallpox imported from overseas, compared to a primary attack which results in or simultaneous firstgeneration cases, we modelled the epidemic resulting from , or initial first generation cases. the size of an attack is unknown, and would depend on the technical sophistication of aerosol dispersion of variola. to account for this uncertainty, we explored the influence of attack scenarios of , and initial infected as a wide range of possible attack sizes, to determine the impact of attack size on epidemic control. delays in diagnosis and time to obtaining laboratory confirmation could vary the time of onset of the response. we therefore varied the time of the response commencing between t = , and t = days following virus release. given an average incubation period of days for smallpox [ ] , this corresponds to day , and after the onset of symptoms of the index case. we estimated public hospital beds and private hospital beds in sydney. we estimated there are clinical hcws in sydney, the majority ( %) aged - years old, % nurses and % doctors [ ] . we estimated a public health workforce of nationally, with approximately public health workers in sydney. fig shows the relative epidemic size of a deliberate release scenario with or initial infected compared to a single importation of smallpox from an epidemic overseas, to illustrate the potential scale of the required public health response. the higher the initial number infected, the more rapid and severe the epidemic. without intervention, the death rate will reach an incidence (number of new infected people per day) rate of deaths per population per day. the overall reproductive number was estimated to be . . fig shows the influence on infections and deaths by varying time to response and case isolation rates. both timing and isolation rates of infected cases are highly influential in outbreak control. with infected initially, when isolation decreases, the deaths increase from a maximum of , and per day in the best scenario with % isolation (for t = , and respectively), to . , . and per day with only % of cases isolated (fig ) . fig shows the influence of varying time of starting the intervention and varying percentage of contacts traced on the incidence of infections and deaths with case isolation constant at %. with a high proportion of cases isolated, outbreak control can be achieved within days even with only % of contacts traced. fig shows the effect of varying both case isolation and contact tracing rates, with the intervention commencing at days-case isolation is more influential, with epidemic control severely impacted when isolation and contact tracing falls to %. in table we show the impact of the epidemic (total cases and contacts needed to be traced) by varying case isolation rates. the total number of cases range from to , ; and contacts that need to be traced between and in the best-and worst-case scenarios respectively. we estimated public hospital beds and private hospital beds in sydney. the modelled maximum number of people isolated at the same time is about . and . times the initial number of infected if we start intervention respectively at t = and at t = and peaks days after the response commences. therefore, if the initial number of infected is or , the available beds will not be completely exhausted, but treatment capacity for other illnesses may be impacted. in the case of initial infected, the maximum beds usage will reach . % and . % of sydney public available beds, if the response starts at time t = and t = days from the virus release respectively. if the initial number of infected is , the maximum beds usage will reach . % and . % of sydney public available beds days after the response commences, if the response starts at time t = and t = days from the virus release respectively. however, in the case of initial infected, the available hospital beds will be all used in the first few days of the response. fig shows the hospital bed usage in the worst-case scenario of initially infected, with varying start times of the response. maximum number of beds needed at the same time and day shown in the square windows. with initial infected if we start the intervention at t = from virus release, beds will be used the first day, the second day. at and days after commencing the intervention (at day and ) more than % and % of the total beds in sydney hospitals will be needed. at day post-attack, days after starting the intervention, % of all public and private beds will be used. if the intervention is delayed to day t = , almost % of the available beds will be used in the first days, % at day of response will be used and at day of the response (t = after the attack) all available public and private beds will be used. table https://doi.org/ . /journal.pone. .g results showed for % of new infected isolated. % % % maximum number of beds used in the same day (% of the total) initial infected shows the time to occupancy of all available hospital beds at levels of % or greater. whilst an attack of initial infections does not reach % of beds under any scenario, in the worstcase scenario (response commencing at day ) % of beds will be used by day . the number of hcws required and the ppe they need will be proportionate to the number of cases requiring treatment (table ). in scenarios described above where cases (beds) exceed % of available beds, staffing requirements will increase %, unless reduced staff/patient ratios are implemented. estimating a minimum of disposable respirators a day per hcws for days, over million respirators will need to be stockpiled for all clinical hcws in sydney. this number can be used to estimate requirements based on the estimated percentage of the clinical workforce needed for the epidemic, which will be proportionate to the number of cases requiring treatment ( table ). if % of clinical hcws are involved in care of smallpox patients, over million respirators will be needed. if the epidemic is not controlled within days this number will be doubled. the public health staff (phs) required to conduct contact tracing will depend on how many contacts one person can trace per day and the number of available phs, estimated to be in sydney. if one phs can trace contacts a day, then in the best-case scenario of contacts, over contact tracing days are required, based on the number of contacts in table above. in the worst-case scenario, , contacts and , contact tracing day are required. in the worst-case scenario, phs would work over days each doing contact tracing. if half of contacts are high-risk, quarantine spaces will be required for to , contacts. in the case of a smallpox release in sydney, a high-income, well-resourced city of over . million people, health system impacts may be substantial under some scenarios as shown in our model. we showed if smallpox arises overseas and is imported as a single case into australia by travel, control will be far easier than under an attack scenario. we showed that influential factors on epidemic impact are the size of the initial attack, time to commencing the response, case isolation rates and contact tracing for ring vaccination. whilst both are influential, case isolation is more influential than contact tracing. these public health interventions depend on physical and human resources, including clinical and public health workforce. whilst the size of an attack may not be within our control, other the influential factors are modifiable and potentially within our control. if the initial attack size is - and the response is rapid, an outbreak of smallpox can be controlled with case isolation, contact tracing and vaccination. however, if the response is delayed to days or longer (which equates to about weeks after the first symptoms occur), or if the attack infects people, epidemic control will be much more challenging, and the health systems impacts will be substantial. in the worst-case scenario, available hospital beds will be exceeded in less than days. the requirement for hospital beds for isolation of cases will vary from up to % to % of all available beds depending on the size of initial release and speed of response. even in the mid-range scenario of initial cases, up to % of all available hospital beds will be required for smallpox control. this does not account for the facilities required for quarantine of contacts, which must additionally be planned for, and in the worst-case scenario would require over , high risk contacts to be quarantined. quarantine and isolation capacity are critical to epidemic control. planning for surge bed capacity using available guidelines should be undertaken [ ] , and back up plans such as the use of community halls, school buildings, hotels or other large buildings should be made to ensure that that other viable isolation sites are pre-designated as smallpox treatment centres and available. during the pandemic of influenza, which was reportedly not as severe as expected, studies reported a tripling of patient presentations to hospital [ ] . plans for managing hospital bed capacity in the event of a large initial attack should also be made, including designation of specific treatment facilities, cancellation of elective surgery and decanting of patients with non-urgent other conditions into private hospitals or other facilities. the capacity for hospital beds for non-smallpox patients who require urgent hospitalisation must also be considered, and in some scenarios, the care of patients with urgent non-infectious conditions such myocardial infarction or stroke, may be compromised by lack of hospital capacity and staffing shortages. rapid response time is critical and becomes even more critical when the initial infected number is higher. responding more than days from the virus release (which means commencing the public health response within days of symptom onset, given an average day incubation period) will result in a more severe outbreak. whether it is feasible to commence response within the best-case scenario of days post-release (or days after symptom onset) is unknown, but unlikely. a rapid response depends on very early detection and diagnosis, as well as prompt commencement of case finding, isolation, contact tracing and vaccination. practically, the target for reducing the time to response is in early diagnosis, which depends on awareness first, followed by diagnostic test. delays may occur if the diagnosis is missed in the index case. recent examples of serious emerging infectious diseases where the diagnosis was missed include ebola in nigeria and the us, both of which occurred during the height of the west african epidemic when media reports were at a peak and awareness should have been high [ , ] . the largest epidemic of mers coronavirus outside the arabian peninsula occurred in south korea following a missed diagnosis and failure of triage in a patient with a relevant travel history and a respiratory clinical syndrome [ ] . the last european epidemic of smallpox in also involved a missed diagnosis, when a traveller to the middle east returned to yugoslavia, which had been free of smallpox for years. the patient had haemorrhagic smallpox, which was misdiagnosed as a severe adverse reaction to an antibiotic, and smallpox was not suspected until second generation cases began occurring, resulting in an outbreak of cases [ ] . excellent surveillance systems and triage protocols for early detection of low probability, high impact outbreaks such as smallpox is recommended. improving diagnosis requires triage protocols and rapid diagnostics, the latter being useful only if the diagnosis is suspected clinically in the first instance. other avoidable delays in response could include having pre-vaccinated first responder teams, pre-designated isolation and quarantine facilities, and rapid human resources and surge capacity scale up plans [ ] . we have showed that epidemic control is highly sensitive to case isolation rates, which need to be maintained at high levels. identifying and isolating less than half the new infected cases and tracing only half of all contacts will result in a blow-out of the epidemic. space and human resource requirements for case finding, isolation, contact tracing, vaccination and quarantine are therefore essential for preparedness planning. physical space requirements extend beyond isolation of smallpox cases, to quarantine of contacts. in the worst-case scenario, almost one million contacts need to be traced and there will be a lack of physical space for quarantine of high-risk contacts. plans for home quarantine and surveillance of contacts should also be undertaken and will require adequately trained personnel. the speed and effectiveness of contact tracing is also critical to the success of ring vaccination and will require an adequately trained critical mass of public health workers and epidemiologists, separate from the clinical workforce. in australia, as a federation, this will rely on state and territory capacity, and cross-border mobilisation of jurisdictional capacity in the event of a smallpox epidemic, and estimation of the current and required capacity for such an event. the fact that public health personnel are not registered as health practitioners or documented in any other centralised way, makes it more challenging to rapidly mobilise suitably qualified and experienced personnel for a large-scale epidemic response. this is a policy consideration that could be addressed as part of pandemic and health emergency planning, which may strengthen response. contact tracing may need to rely on community volunteers, as the available public health workforce will be inadequate in a large epidemic. staff surge requirements would track parallel to bed requirements and would be over % in some scenarios. the need for a clinical health workforce to treat smallpox will be high, with case numbers in the s to , s in many of the scenarios modelled, and just over , clinical hcws in sydney. limiting the number of hcws working in designated smallpox facilities is a sensible strategy. a possible approach to such a scenario would be a reduction of staff to patient ratios, as well as using trainee hcws. protection of these clinicians is key, with vaccination being the mainstay. up to , doses of vaccine will need to be reserved for clinical hcws and plans in place to commence vaccination. ppe will not be an alternative to vaccination, but an additional protective measure for hcws. today, work health and safety requirements would dictate that paprs or disposable respirators with a hood and coveralls be available to clinicians treating smallpox cases. perceived lack of protection during a serious emerging infection outbreak may result in refusal to work or industrial action by hcws [ ] . hcw may be well protected by ppe, but there is large uncertainty around effectiveness of ppe. studies of other viruses transmitted by the respiratory route suggests good effectiveness of respirators against smallpox [ ] . it should be noted that surgical masks are unlikely to offer protection to hcws based on available data [ ] . stockpiling may provide a short duration of supplies. the modelled epidemic may run for - days or more, depending on the scenario. a very large quantity of respirators may need to be stockpiled, depending on the percentage of hcw involved in direct care of smallpox patients. given the likely duration of an epidemic, plans should put in place for rapid procurement of ppe supplies beyond the stockpiled capacity. strategies to minimise the number of hcw treating each case of smallpox, including using designated smallpox hospitals, will reduce the quantity of ppe required. early identification of the epidemic, high rates of case isolation, excellent contact tracing and vaccination, and protection of hcws are the key influential components of epidemic control. failure in any of these could severely compromise the capacity of the health system. australia has a detailed plan for smallpox response, [ ] and we have outlined key influential parameters for disease control which can add further guidance on mitigating severe outcomes in both the planning and response stages. excellent surveillance systems and triage protocols for early detection of low probability, high impact outbreaks such as smallpox can make a difference, given the criticality of timing of the response and better prospects of epidemic control in the early stages. planning for the health system should consider rapid surge capacity for beds, strategies to create and staff make-shift designated smallpox treatment facilities, and protection of hcws at all levels of care. requirements for contact tracing are substantial and may require mobilisation of community volunteers and additional space for quarantine and surveillance of high-risk contacts. designated surge smallpox facilities and plans for management of other urgent health conditions should be considered. we have outlined several modifiable factors which, with good planning, can ensure adequate health system capacity in the event of a smallpox epidemic. general fact sheets on specific bioterrorism agents smallpox and its eradication: the pathogenesis, immunology, and pathology of smallpox and vaccinia the de novo synthesis of horsepox virus: implications for biosecurity and recommendations for preventing the reemergence of smallpox. health security how canadian researchers reconstituted an extinct poxvirus for $ , using mailorder dna biopreparedness in the age of genetically engineered pathogens and open access science: an urgent need for a paradigm shift development of a risk-priority score for category a bioterrorism agents as an aid for public health policy influence of population immunosuppression and past vaccination on smallpox reemergence who (world health organizazation). smallpox. geneva, switzerland: world health organization smallpox vaccines: past, present, and future public health and health reform in australia respiratory protection for healthcare workers treating ebola virus disease (evd): are facemasks sufficient to meet occupational health and safety obligations? planning for smallpox outbreaks modelling disease outbreaks in realistic urban social networks containing bioterrorist smallpox modeling the effect of herd immunity and contagiousness in mitigating a smallpox outbreak planned and unplannedfutures for the public health physician workforce in australia. a labour market analysis for the australasian faculty of public health medicine: australasian faculty of public health medicine:sydney hospital resources - : australian hospital statistics canberra: australian institute of health and welfare social contacts and mixing patterns relevant to the spread of infectious diseases transmission potential of smallpox in contemporary populations effectiveness of postexposure vaccination for the prevention of smallpox: results of a delphi analysis smallpox in europe ambulance service of nsw. workforce statistics staff turnover counting health workers:definitions, data, methods and global results. geneva: world health organization annual report / : melbourne. australian health practitioner regulation agency smallpox response plan and guidelines (version . ). archive-use only for research purposes preventability of incident cases of tuberculosis in recently exposed contacts. the international journal of tuberculosis and lung disease a survey of emergency department pandemic influenza a (h n ) surge preparedness office of the chief health officer. public health workforce surge guidelines transmission dynamics and control of ebola virus disease outbreak in nigeria ebola us patient zero: lessons on misdiagnosis and effective use of electronic health records hospital outbreaks of middle east respiratory syndrome smallpox as actual biothreat: lessons learned from its outbreak in ex-yugoslavia in . annali dell'istituto superiore di sanita exercise mataika: white paper on response to a smallpox bioterrorism release in the pacific uncertainty, risk analysis and change for ebola personal protective equipment guidelines the efficacy of medical masks and respirators against respiratory infection in healthcare workers. influenza and other respiratory viruses smallpox cdna national guidelines for public health units. communicable diseases network australia key: cord- -ukn hm x authors: sutherland, m. a.; lowe, g. l.; huddart, f. j.; waas, j. r.; stewart, m. title: measurement of dairy calf behavior prior to onset of clinical disease and in response to disbudding using automated calf feeders and accelerometers date: - - journal: journal of dairy science doi: . /jds. - sha: doc_id: cord_uid: ukn hm x abstract we determined if feeding and lying behavior, recorded by automatic calf feeding systems (acfs) and accelerometers, could be used to detect changes in behavior before onset of neonatal calf diarrhea (ncd) or in response to disbudding pain in dairy calves. at d of age, calves had accelerometers attached to their hind leg and were housed in pens with acfs. calves were examined daily for signs of illness or injury. of the calves monitored, were diagnosed with ncd; activities of calves with ncd were then compared with those of healthy controls (calves that had no symptoms of ncd, other illnesses, or injury). feeding (milk consumption and the number of rewarded and unrewarded visits to the feeder) and lying behavior during the d leading up to calves displaying clinical signs of ncd were analyzed. calves with ncd performed fewer unrewarded visits and consumed less milk than healthy calves during the - and -d periods before diagnosis with ncd, respectively. calves with ncd tended to perform fewer lying bouts than healthy calves over the -d period before diagnosis with ncd. at wk of age, a subset of healthy calves were allocated to of treatment groups: ( ) sham handling (sham, n = ), ( ) cautery disbudding (db, n = ), ( ) administration of local anesthetic (la) and db (la+db, n = ), ) administration of a nonsteroidal anti-inflammatory drug (nsaid) and db (nsaid+db, n = ), and ( ) administration of la, nsaid and db (la+nsaid+db, n = ). feeding and lying behavior were recorded continuously for h pre- and postdisbudding. we found no effect of treatment on the number of rewarded or unrewarded visits to the feeder and milk volume consumed h before administration of treatments. during the -h postdisbudding period, sham calves performed more unrewarded visits than db, la+db, and nsaid+db calves, but the number of unrewarded visits did not differ between sham and la+nsaid+db calves. during the first hour of the posttreatment period we noted a difference in lying times among treatments, with db and nsaid+db calves spending less time lying than sham calves and lying times being similar between sham, la+db, and la+nsaid+db calves. the acfs and accelerometers have the potential to automatically gather valuable information regarding health status and pain in calves. therefore, it may be advantageous to combine both of these measures (acfs and accelerometers) when evaluating ncd on farm or pain in calves in future research. automated techniques for measuring individual calf behavior on-farm are now available; feeding behavior can be detected remotely with automatic calf feeding systems (acfs; svensson and jensen, ; borderas et al., ) and activity can be recorded using accelerometers (e.g., hobo data loggers; bonk et al., ) . automated systems have the advantage of collecting data noninvasively with reduced labor input. feeding and lying behavior have been shown to change in response to sickness (svensson and jensen, ; borderas et al., ; szyszka and kyriazakis, ) and pain (graf and senn, ; heinrich et al., ) in calves. hence, automated measures of feeding and lying behavior could potentially be used to detect early signs of disease and pain in calves. neonatal calf diarrhea (ncd) is an enteric disease that is associated with severe diarrhea. once an animal displays clinical signs of ncd, much of the associated tissue damage to the intestinal submuscosa has already occurred (schroeder et al., ) ; therefore, early detection of ncd would enable calves to be promptly treated and moved to sick pens thereby reducing tissue damage and risk of disease transmission to other calves. previous studies using acfs and accelerometers have reported changes in feeding (svensson and jensen, ; borderas et al., ; lowe et al., ) and lying behavior (swartz et al., ) in response to ncd and respiratory disease. however, those studies predominantly focused on changes in behavior once calves became clinically ill or the time period just prior (svensson and jensen, ; borderas et al., ) , or calves that were diagnosed with respiratory disease (swartz et al., ) . to our knowledge, no studies have measured changes in feeding in conjunction with lying behavior in calves before diagnosis with ncd. it would be valuable to measure changes in feeding and lying behavior concurrently, several days before calves become clinically ill, to assess if information collected from acfs and accelerometers could be used for early detection of ncd on-farm. in the united states, % of dairy cattle producers routinely dehorn their cattle and of these % disbud using cautery (usda, ) . disbudding is often performed without pain relief; however, studies have shown that this procedure causes behavioral changes indicative of pain (reviewed by stafford and mellor, ) . for example, calves spent less time feeding and more time lying after being cautery disbudded than sham-handled controls (graf and senn, ; faulkner and weary, ) . therefore, a need exists to evaluate different pain-mitigation strategies to alleviate the pain caused by disbudding; to do this, however, laborintensive behavioral studies are often needed. it would be useful if automated measures of feeding and lying behavior were sensitive enough to detect behavioral changes in calves in response to a painful procedure such as disbudding. automated measures of feeding and lying behavior could then be used as a research tool to evaluate different pain-mitigation strategies for painful husbandry procedures in calves. disease and pain can negatively affect calf welfare, but these states are often difficult to detect and time-consuming to assess objectively. information on feeding and lying behavior collected from acfs and accelerometers could potentially be measured remotely on-farm and used by producers as a tool to help detect ncd early so that strategies could be promptly implemented to reduce the negative effects of this disease on calf health and production. in addition, automated measures of feeding and lying behavior could provide a useful and less labor-intensive research tool to evaluate different pain-mitigation strategies in calves. therefore, the objectives of our study were to determine if behav-ioral data collected from acfs and accelerometer data loggers could be used to detect changes in behavior before the onset of ncd or in response to disbudding pain in dairy calves. our study was conducted between july and october on agresearch's tokanui dairy research farm in south waikato, new zealand ( ° ′e longitude, − ° ′s latitude). all procedures involving animals were approved by the ruakura animal ethics committee (no. ) under the new zealand animal welfare act (ministry of primary industries, ). seventy-one friesian dairy calves (n = females) and friesian-hereford cross calves (n = females, n = males) were monitored in this study. calves were allocated to of pens (n = , , and calves/ pen respectively) at d of age according to their order of birth. one pen was filled at a time, which ensured pen mates were all of similar size and stage of development. all calves were monitored to obtain a sample of animals displaying clinical signs of ncd (as described below); a subset of calves that never displayed signs of ncd or other health issues or injuries were used in the pain-assessment study. calves were separated from their dams within h of birth and transported to the calf-rearing facility. upon arrival at the facility, all calves were weighed and individually identified using numbered (allflex, irving, tx) and electronic identification ear tags (allflex) placed in the left and right ears, respectively. at this time accelerometers were attached to the lateral side of the hind leg (as described below). the calf-rearing facility had a roof, solid dirt floors, and walls on all sides. calves were housed in indoor pens ( . × . m), located inside the calf rearing facility, with floors covered in wood chip bedding and post and rail fencing. each pen contained an acfs, water troughs, a hay feeder, and meal feeders attached to the side of the pen. water, meal (calf-pro %, seales winslow, morrinsville, new zealand), and hay were provided ad libitum. all calves were trained over a -to -d period to use the acfs from d of the trial. trained staff performed daily health checks to assess the calves' general health and to identify calves with signs of illness. a standard operating procedure developed in conjunction with a veterinarian was used. health checks assessed calves for the presence of diarrhea, high rectal temperatures ( . °c or higher), signs of dehydration (sunken eyes, or poor skin elasticity-assessed using the skin tent test), coat condition (shiny and smooth vs. dry and rough), position of the ears (erect vs. droopy), gut fill (full vs. empty stomachs), signs of navel ill (inflammation, swelling, or discharge from the navel), nasal or ocular discharge, swelling, abscesses, and injury. trained staff were also asked to comment if calves were coughing, had crackly breaths, or were breathing fast. in the present study, calves were diagnosed as having ncd when they presented with diarrhea and high rectal temperatures ( . °c or higher). a calf was considered to have diarrhea if it was observed passing feces with a loose to watery consistency and strong odor. for calves suspected of having diarrhea due to the presence of loose fecal matter on the top of the tail or hind legs, a fecal sample was taken to evaluate fecal consistency and to confirm whether or not that particular calf was diarrheic. only data collected from calves diagnosed as having ncd were considered as sick calves for further analysis. once calves exhibited signs of ncd, blood and fecal samples were collected for analysis to verify etiology of the disease. it is possible that any of the calves could have been experiencing subclinical disease during the course of the study, but it was beyond the scope of this study to test all animals for the presence of subclinical diseases; hence, animals were categorized as sick or healthy based on clinical signs of illness only. calf bw was recorded weekly. of the calves that were monitored, only data from a subset of calves [n = diagnosed with ncd (bw = . ± . kg, mean ± sd) and n = healthy calves (bw = . ± . kg)] were used in this component of the study (n = calves from group , n = calves from group , and n = calves from group ; n = females and n = males). each calf with ncd was matched with a healthy calf that was from the same group, was the same breed, and was approximately the same date of birth and birth weight as the calves with ncd. a calf was considered healthy if during the course of the study it presented no symptoms of ncd or other signs of illness or injury (e.g., coughing, rectal temperatures greater than . °c, navel ill, nasal or ocular discharge, swelling or abscesses, and so on, as defined in the daily health check standard operating procedure above). upon completion of the trial for the first groups, the experimental pens were cleaned in preparation for the final replicates (groups and ), which involved removing and replacing the top layer of woodchip with fresh wood chips. in addition, all surfaces were thoroughly sprayed using a broad-spectrum disinfectant (halamid, axcentive sarl; bouc-bel-air, france) and all water troughs and feed containers were also thoroughly cleaned and refilled. blood chemistry and hematology. blood samples were taken from all calves at , , and d of age and when a calf displayed signs of being clinically ill. blood samples were collected by jugular venipuncture into evacuated tubes that contained sodium fluoride, edta, or no anticoagulant (bd vacutainer, franklin lakes, nj). blood samples were stored at approximately °c until they were delivered to the new zealand veterinary pathology (hamilton, new zealand) laboratory for analysis. blood smear slides were performed on a sysmex xt- iv using veterinary software and sysmex reagents (sysmex corporation, kobe, japan) for estimation of total white blood cell, neutrophil, and lymphocyte counts. white blood cell differentials were performed using a standard cell count and the neutrophil-to-lymphocyte ratio was calculated by dividing the percent of neutrophils by the percent of lymphocytes. serum haptoglobin concentrations were measured using a commercially available colorimetric kit (phase haptoglobin assay cat. no. tp- ; tridelta development limited, maynooth, county kildare) in accordance with the manufacturer's instructions (http:// www .trideltaltd .com/ haptoglobin -main -page .html). the analytical sensitivity of this assay was . mg/ ml. fecal sampling. fecal samples were collected manually via gentle palpation of the rectum from any calves that were considered clinically ill and were tested for the presence of cryptosporidium, rotavirus, coronavirus, and salmonella. cryptosporidium was measured using an acid fast stain to visually detect the presence of cryptosporidium, which could be seen as round bodies measuring to µm in diameter and dark red or pink in color. if more lightly stained, the parasites showed internal bodies that were darker blue or brownish in color. the presence of rotavirus and coronavirus were determined using a commercially available elisa kit (pourquier elisa calves diarrhea; institut pourquier, montpellier, france). the -step, naked eye reading method was conducted in accordance with the manufacturer's instructions (https:// www .idexx .co .uk/ en -gb/ livestock/ livestock -tests/ ruminant -tests/ idexx -rota -corona -k -ag -test/ ). selective enrichment fecal cultures were used for the detection of salmonella. feeding behavior. the acfs (a&d reid, temuka, new zealand) used an electronic identification system to identify individual animals as they entered the acfs. each acfs had a single teat to deliver whole milk (at approximately - °c) on the following schedule: l, times per day with a minimum -min period with no milk access between each complete milk feed (as per normal farm practice in new zealand). if the calf did not consume the whole l during the visit, they could return at any point to consume the remainder of the allowance. the acfs recorded the total number of visits, rewarded visits (calf received part or whole milk allowance), unrewarded visits (calf received no milk), and milk volume consumed per visit. lying behavior. throughout the study period, lying and standing behavior was recorded continuously on all calves using hobo pendant g accelerometer data loggers ( k, onset computer corporation, bourne, ma) set at -min intervals recording the y and z axes. the accelerometers were placed in a durable fabric pouch and strapped onto the lateral side of the hind leg above the metatarsophalangeal joint. accelerometers were placed horizontally on the leg such that the x-axis ran parallel to the ground, pointing in the anterior direction, and the y-axis pointing toward the dorsal plane of the calf. the pouch was held in position using velcro patches, one sewn to the pouch, the other glued (ka-mar, livestock improvement corporation, hamilton, new zealand) to the leg of the calf. the pouch was further held in place by a strap around the leg of the calf. accelerometers were initialized and downloaded using onset hoboware pro software (onset computer corporation, version . . ). the g-force readings were converted into binary values (e.g., lying = , standing = ), and hourly (min/h) and daily (min/d) summaries of lying time and lying bout frequency (number of bouts/d) were calculated in sas . (sas institute inc., cary, nc) using a code designed for this purpose (awp ubc, ) and validated by bonk et al. ( ) for use in calves. at approximately wk of age ( ± . d of age, mean ± sd), female friesian dairy calves (bw = . ± . kg) were selected from the healthy animals and allocated to of treatment groups: ( ) sham handling (sham; n = ), ( ) cautery disbudding (db; n = ), ( ) administration of local anesthetic (la) and db (la+db; n = ), ) administration of a nonsteroidal anti-inflammatory drug (nsaid) and db (nsaid+db; n = ), and ( ) administration of la, nsaid, and db (la+nsaid+db; n = ). the combination of pain relief treatments were included to evaluate whether the automated systems were sensitive enough to measure varying degrees of pain. all calves were restrained in a calf crush with a head bail (front opening calf bail, te pari, oamaru, new zealand) during treatments. for db, la+db, nsaid+db, and la+nsaid+db calves, an electric cautery iron (quality electric debudder, v, w; lister gmbh, lüdenscheid, germany) was used to remove the calves horn buds. for la+db and la+nsaid+db calves, ml of local anesthetic ( % lignocaine hydrochloride; lopaine, ethical agents ltd., auckland, nz) was injected into the cornual nerve ( ml) and injection sites around the base of the horn bud ( . ml/site) min before disbudding. immediately before disbudding, a needle prick test was used to verify the success of the cornual nerve block. for nsaid+db-and la+nsaid+db-treated calves, meloxicam ( . mg/kg of bw; metacam, boehringer ingelheim ltd., auckland, nz) was injected subcutaneously min before disbudding. treatment groups were balanced for age and bw. feeding and lying behavior were recorded continuously for h before and h after administration of treatment, as described above. prior to analysis, all data were assessed for evidence of departures from the assumptions of normality and homogeneity of variance using the univariate procedures of sas version . (sas inst. inc.). the residuals from the log-transformed mixed model analysis showed good consistency with the normality assumptions of the model. for the calf health component of our study, data from calves that were identified with ncd were used (see description above). each calf with ncd was matched with a healthy control from the same pen taking into account sex, breed, birth date, and birth weight. healthy controls never presented with any symptoms of ncd or other signs of illness or injury during the study period. day was the day that calves showed signs of ncd and the equivalent day was used for d for control calves. we were interested in behavioral changes leading up to signs of ncd; therefore, we focused on the d before appearance of ncd. milk consumption, blood chemistry and hematology data were log-transformed to meet the assumptions of the analysis. milk consumption and lying (lying times and frequency of lying bouts) behavior data were analyzed using the mixed model procedure of sas. the model included the fixed effects of treatment (health status: ncd and healthy), day, treatment by day interaction, and the random effects of pen and calf. the model had a repeated structure on time allowing incorporation of heterogeneity of variances across time. for blood chemistry and hematology (cells counts and haptoglobin concentrations) data, only the blood results from d were compared; the model included the fixed effects of treatment (health status: ncd and healthy) and the random effect of pen. feeding behaviors (total number of visits, and number of rewarded and unrewarded visits) were analyzed using the glimmix procedure of sas with a specified poisson distribution. the model included the fixed effects of treatment (health status: ncd and healthy), day, treatment by day interaction, and the random effects of pen and calf. the model had a repeated structure on time allowing incorporation of heterogeneity of variances across time. for the analysis of the pain assessment component of this study, milk consumption was log-transformed to meet the assumptions of the analysis. milk consumption and lying times data were analyzed using the mixed model procedure in sas and the model included the fixed effects of treatment (sham, db, la+db, nsaid+db, la+nsaid+db), day/time, treatment by day/time interaction, and the random effects of pen and calf. the model had a repeated structure on time allowing incorporation of heterogeneity of variances across time. lying times were summarized over the entire -h pre-and posttreatment period and analyzed. in addition, hourly lying times for the first h after administration of treatments were analyzed, as this time period is representative of the cortisol response to cautery disbudding (graf and senn, ; stafford and mellor, ) . feeding behaviors (total number of visits and number of rewarded and unrewarded visits) were analyzed using the glimmix procedure of sas with a specified poisson distribution. the model included the fixed effects of treatment (sham, db, la+db, nsaid+db, la+nsaid+db), day/time, treatment by day/time interaction, and the random effect of pen. the model had a repeated structure on time allowing incorporation of heterogeneity of variances across time. the sample size was determined by a power analysis with % power and % significance level. data displayed in the graphs, tables, and text are actual data summarized by least squares means ± standard error of the means. statistical significance was determined at p ≤ . and . < p ≤ . were considered a tendency. fecal sampling. four calves tested positive for cryptosporidium, with rotavirus, with coronavirus, and with cryptosporidium and rotavirus. no calves tested positive for salmonella. eleven calves identified as having ncd according to the criteria (diarrhea and high rectal temperatures) did not test positive for either cryptosporidium, rotavirus, coronavirus, or salmonella, so the cause of ncd for these calves was unknown. blood chemistry and hematology. total white blood, neutrophil, and monocyte cell counts and the neutrophil-to-lymphocyte ratio were higher (p ≤ . ) and eosinophil cell counts tended (p = . ) to be higher in calves with ncd than healthy calves (table ) . health status of the calves did not affect any other hematology measures or haptoglobin concentrations (table ) . feeding behavior. calves with ncd tended (p = . ) to visit the feeder less than healthy calves over the -d period before signs of ncd (total number of visits to the feeder/d: control: . ± . ; sick: . ± . ). we observed no effect of health status (p = . ) or a health status × day interaction (p = . ) for the number of rewarded visits; however, the number of rewarded visits to the feeder changed (p < . ) over the -d study period (number of rewarded visits to the feeder/d: d − = . ± . ; d − = . ± . ; d − = . ± . ; d − = . ± . ; d − = . ± . ; d = . ± . ). the number of unrewarded visits was affected by health status (p = . ) and day (p < . ; figure ). calves with ncd performed fewer unrewarded visits than healthy calves on d − (p = . ), − (p = . ), and (p = . ). we noted a health status × day interaction for volume of milk consumed (p = . ; figure ); calves with ncd drank less on d − (p = . ), − (p = . ), − (p = . ), and (p < . ) than healthy calves. lying behavior. we found a health status × day interaction for time spent lying (p = . ; figure ). calves with ncd tended to spend more time lying on d − (p = . ) but less time lying on d (p = . ). moreover, calves with ncd tended (p = . ) to perform fewer lying bouts than healthy calves over the entire -d period before diagnosis with ncd (number of lying bouts/d: control = . ± . ; sick = . ± . ). feeding behavior. the total number of visits, rewarded visits, unrewarded visits, and milk volume consumed was similar (p > . ) among all treatments during the -h period before administration of treatments (table ) . during the to h posttreatment period, milk volume consumed was similar (p > . ) among sham and disbudded calves, with or without pain relief (table ) . however, sham calves visited the acfs more during the -h posttreatment period than db (p < . ) and la+db (p < . ) calves, but la+nsaid+db visited the feeder a similar (p = . ) number of times as sham calves (table ) . we observed no differences (p > . ) across treatments in the number of rewarded visits to the acfs (table ) . however, calves experiencing the sham treatment had more unrewarded visits during the -h posttreatment period than db (p = . ), la+db (p < . ), and nsaid+db (p < . ) calves, but the number of unrewarded visits was similar (p = . ) for sham and la+nsaid+db calves (table ) . lying behavior. the proportion of time calves spent lying was similar (p > . ) among all treatment groups over the -h pre-and -h posttreatment period (table ) . however, during the -h posttreatment period, we found a difference among treatments (figure ) . during the first hour, db (p = . ) and nsaid+db (p = . ) calves spent less time lying than sham calves. moreover, lying times were similar (p > . ) among sham, la+db, and la+nsaid+db calves. lying times were similar among all treatments during the -h posttreatment period. feeding and lying behaviors collected using acfs and accelerometers were affected by health status and pain in calves in the present study. the number of unrewarded visits declined before calves showed signs of ncd. differences in the number of unrewarded visits was apparent d before calves showed signs of ncd; however, we found no change in the number of rewarded visits before diagnosis. similarly, svensson and jensen ( ) found that the number of unrewarded visits was a more sensitive measure of sickness in calves than the number of rewarded visits. borderas et al. ( ) suggested that the effect of disease on feeding behavior would vary according to feeding motivation, as milk intake and frequency of visits to the feeder were more dramatically decreased in sick calves being fed a high (ad libitum or l/d) rather than a low ( l/d) milk allowance. in the present study, calves received a milk allowance of l/d, which is closer to the low than the high milk allowance provided in the borderas et al. ( ) study; hence, more dramatic changes in feeding behavior may have been observed if a higher milk allowance was provided to calves in the present study. therefore, milk allowance fed to calves needs to be taken into consideration when using behavioral data from acfs as an indication of ncd in calves. contrary to other studies (svensson and jensen, ; borderas et al., ; lowe et al., ) , milk consumption was reduced in sick calves up to d before displaying signs of ncd in the present study. hence, milk consumption in combination with the number of unrewarded visits maybe a good measure for early detection of ncd in calves. differences in feeding behavior among studies may relate to the pathogenesis of the disease. in other studies, calves were diagnosed with a range of illnesses, including gastrointestinal, respiratory, or a combination of these diseases (borderas et al., ; swartz et al., ) , or other signs of disease such as arthritis, fever, dull calf syndrome, and so on (svensson and jensen, ) . however, in those studies, samples were not taken to verify the pathogenesis of the diseases. in our study, only calves that were diagnosed as having ncd were used; however, even with fecal sampling we could only confirm the cause of ncd in out of calves. therefore, due to the a-c means within each row with different superscripts differ at p < . . sham handling (sham; n = ), cautery disbudding (db; n = ), administration of local anesthetic + db (la+db; n = ), administration of a nonsteroidal anti-inflammatory drug (nsaid) + db (nsaid+db; n = ), and la+nsaid+db (n = ). lying times (min/h; lsm ± sem) of calves during the first h after disbudding or control handling. treatments were sham handling (sham; n = ), cautery disbudding (db; n = ), administration of local anesthetic + db (la+db; n = ), administration of a nonsteroidal anti-inflammatory drug (nsaid) + db (nsaid+db; n = ), and la+nsaid+db (n = ). means within each hour with different letters (a-c) differ at p < . . differences in the pathogenesis affecting calves among our study and previous studies, it is difficult to know the reason for the difference in results. in addition to pathogenesis, age of calves at the time of diagnosis, severity of outbreak, and the time of diagnosis could potentially affect feeding and lying behavior in calves. further research focused on particular pathogens or disease severity would be of interest to assist in our understanding of the relationship between pathogenesis and sickness behavior in calves. calves challenged with a low dose of bacterial endotoxin spent more time lying and performed fewer standing bouts in borderas et al. ( ) , similar to the present study, and sick calves spent more time lying d before displaying clinical signs of ncd and tended to perform fewer lying bouts. a reduction in activity of sick calves may be part of the adaptive strategy adopted by mammals to help combat infections (hart, ) . however, on the day calves were diagnosed as having ncd in the present study their lying times were reduced. at the time calves were diagnosed with ncd, blood and fecal samples were collected and animals were moved to the sick pen and treated with electrolytes; this extra handling may account for the reduction in lying times. in the present study, disbudding treatment affected the total number of visits that calves made to the feeder during the -h posttreatment period. this difference was predominantly due to the number of unrewarded visits as disbudding treatment did not affect the number of rewarded visits. calves experiencing the sham treatment performed more unrewarded visits during the -h posttreatment period than db, la+db, and nsaid+db calves. the greater number of unrewarded visits suggests that even though there was no difference in the amount of milk consumed among treatments, calves that were not disbudded were more motivated to visit the acfs than calves disbudded with la or nsaid or without pain relief. fewer unrewarded visits to the acfs by disbudded calves could reflect a lack of appetite in these animals due to the pain or distress caused by disbudding. indeed, calves spent less time feeding after cautery disbudding (graf and senn, ) and amputation dehorning (sutherland et al., ) . it is also possible that disbudding caused the calves in the present study to become head shy and, hence, more hesitant to place their heads in the acfs due to the potential risk of knocking their head in the confined space. another possibility is that experiencing a painful husbandry procedure caused a change in the emotional state of the animals, which may in turn have affected their motivation to feed (neave et al., ) . interestingly, calves given local anesthetic and an nsaid before disbudding in the present study per-formed a similar number of unrewarded visits as sham calves, which suggests that the combination of la and an nsaid was the most effective pain-mitigation strategy evaluated in the present study. these results are in accordance with previous studies showing that the physiological and behavioral response to disbudding is markedly reduced when calves are given a combination of la and an nsaid before disbudding heinrich et al., ) . total milk consumption was not affected by disbudding, with or without pain relief, in the present study. in contrast, bates et al. ( ) found that when calves were given the same milk allowance as provided in the present study ( l/d), mean cumulative milk consumption over the -d postdisbudding period was greater for calves disbudded with pain relief compared with those without. therefore, it is possible that if milk consumption had been recorded for a longer period of time in the present study, treatment differences may have been detected. furthermore, milk allowance is an important factor to consider when using total milk consumption as an indicator of stress (e.g., pain or disease). borderas et al. ( ) found that sick calves consumed less milk at an acfs when provided with a high milk allowance in comparison to healthy calves, but total milk consumption was not affected when they were provided with a low milk allowance. calves provided only a low milk allowance are more likely to be motivated to feed. in the present study, calves were given access to l/d, whereas calves given ad libitum access to milk will drink up to kg/d (jasper and weary, ) . therefore, providing calves with a higher milk allowance is likely to increase the sensitivity of feeding behavior as a measure of stress (e.g., pain and disease) in calves. the proportion of time disbudded calves spent lying was similar across all treatments groups over the entire -h posttreatment period in the present study; however, during the -h period after disbudding, treatment affected lying behavior. during the first hour after disbudding, calves given local anesthetic showed similar lying patterns to control-handled calves, which is similar to the results reported by mcmeekan et al. ( ) h after amputation dehorning. these findings may suggest that calves given local anesthetic were experiencing less pain initially than calves disbudded without pain relief. moreover, calves given only an nsaid before disbudding showed similar lying times to calves disbudded without pain relief, which corresponds to the mechanism of action of nsaid; giving an nsaid (e.g., ketoprofen) reduces the inflammation-related pain associated with amputation dehorning in cattle, but not the initial pain caused by dehorning (mcmeekan et al., ) .these results suggest that lying behavior mea-sured using accelerometer data loggers maybe sensitive enough to assess pain in calves in response to an acute painful procedure such as cautery disbudding. in conclusion, automated measures of feeding and lying behavior could be used to detect changes in behavior before the onset of clinical signs of ncd or in response to acute pain in calves. the number of unrewarded visits to the acfs appeared to be a more sensitive measure of ncd and pain in calves than automated measures of lying behavior. it would be of interest to confirm these results using animals exposed to different conditions (e.g., different pathogens) or other painful procedures (e.g., castration, lameness). ubc animal welfare program: sop -hobo data loggers, sas code version . university of british columbia effect of analgesia and antiinflammatory treatment on weight gain and milk intake of dairy calves after disbudding technical note: evaluation of data loggers for measuring lying behavior in dairy calves behaviour of dairy calves after a low dose of bacterial endotoxin automated measurement of changes in feeding behaviour of milk fed calves associated with illness reduction in pain response by combined use of local anesthesia and systemic ketoprofen in dairy calves dehorned by heat cauterization reducing pain after dehorning in calves behavioural and physiological responses of calves to dehorning by heat cauterization with or without local anaesthesia biological basis of the behavior of sick animals. neurosci the effect of meloxicam on behavior and pain sensitivity of dairy calves following cautery dehorning with a local anesthetic effects of ad libitum milk intake on dairy calves brief communication: the use of infrared thermography and feeding behaviour for early disease detection in new zealand dairy calves effects of regional analgesia and/or a non-steroidal anti-inflammatory analgesic on the acute cortisol response to dehorning in calves effects of a local anaesthetic and a nonsteroidal anti-inflammatory analgesic on the behavioural responses of calves to dehorning accessed pain and pessimism: dairy calves exhibit negative judgement bias following hot-iron disbudding development and performance evaluation of calf diarrhoea pathogen nucleic acid purification and detection workflow addressing the pain associated with disbudding and dehorning in cattle effect of castration and dehorning singularly or combined on the behavior and physiology of holstein calves identification of diseased calves by use of data from automatic milk feeders short communication: automated detection of behavioral changes from respiratory disease in pre-weaned calves what is the relationship between level of infection and 'sickness behaviour' in cattle usda-animal and plant health inspection service (aphis)-veterinary services (vs)-center for epidemiology and animal health (ceah)-national animal health monitoring system (nahms) the authors thank agresearch staff, in particular suzanne dowling, tokanui farm staff (south waikato, new zealand), theresa topp from waikato university, and alvin reid (a&d reid, temuka, new zealand) for providing the automatic calf feeders. agresearch also gratefully acknowledges that this study was funded by the new zealand ministry of business, innovation and employment (wellington, new zealand). key: cord- -mr authors: oh, myoung-don; park, wan beom; park, sang-won; choe, pyoeng gyun; bang, ji hwan; song, kyoung-ho; kim, eu suk; kim, hong bin; kim, nam joong title: middle east respiratory syndrome: what we learned from the outbreak in the republic of korea date: - - journal: korean j intern med doi: . /kjim. . sha: doc_id: cord_uid: mr middle east respiratory syndrome coronavirus (mers-cov) was first isolated from a patient with severe pneumonia in . the korea outbreak of merscov involved cases, including fatalities. a total of % of transmission events were due to five superspreaders, and % of the mers cases were the patients who had been exposed in nosocomial transmission at hospitals. the epidemic lasted for months and the government quarantined , individuals for days to control the outbreak. this outbreak provides a unique opportunity to fill the gap in our knowledge of mers-cov infection. therefore, in this paper, we review the literature on epidemiology, virology, clinical features, and prevention of mers-cov, which were acquired from the korea outbreak of merscov. middle east respiratory syndrome coronavirus (mers-cov) was first isolated from a patient with severe pneumonia in september [ ] . since then, as of january , the world health organization (who) has been notified of , laboratory-confirmed cases of mers-cov infection, including at least deaths, from countries (fig. ) [ ] . most of mers-cov human infections have occurred in the arabian peninsula. saudi arabia reported , cases, including deaths (fatality rate, . %) [ ] . a recent article summarized the current knowledge on the epidemiology, virology, human pathogenesis, clinical management, and prevention of mers-cov infection [ ] . another comprehensive review article on risk factors and determinants of transmission of mers-cov from human to human in the community and hospital settings has been published recently [ ] . public health england also published an extensive review of literature for clinical decision-making support for treatment of mers-cov patients [ ] . the korea outbreak of mers-cov is the largest outside the arabian peninsula and provides a unique opportunity to fill the gap in our knowledge about mers-cov infection. since it has now been almost years since the mers outbreak in the republic of korea, we reviewed the literature to summarize the lessons we have learned from the korea outbreak. the korean journal of internal medicine vol. , no. , march fields. as of january , a total of articles were retrieved, and of them were written in english. the full texts of the english articles were reviewed and critically assessed. the korea outbreak of mers-cov resulted in cases including fatalities [ ] . the index case was a returning traveler from the middle east. the infection had spread within the hospital, and subsequently to other hospitals because of patient movement, resulting in nosocomial transmission at clinics and hospitals. the epidemic lasted for months, with the government declaring a "virtual" end to the epidemic on july , . in order to control the outbreak, the government quarantined , individuals for days, and the economic loss was estimated at . trillion korean won ( . billion us dollars) [ ] . the first patient (index case) with mers-cov infection was a -year-old korean man returning from the middle east. he ran a commercial greenhouse business in bahrain. he flew from seoul to bahrain on april , , and traveled through the united arab emirate and the kingdom of saudi arabia, and came back to seoul on may , . during his -day business trip, he did not visit a camel farm or a hospital. he did not eat camel meat or any other camel products. he did not remember meeting any persons with respiratory symptoms [ ] [ ] [ ] [ ] . seven days after returning home, on may , he developed fever and myalgia. he visited a local clinic on may , , and . his symptoms, however, did not improve and a new non-productive cough developed on th may. he visited pyeongtaek st. mary's (ptsm) hospital ( km south from seoul), a secondary hospital in gyeonggi province. his chest x-ray showed a consolidation in the upper zone of right lung, and he was admitted to the hospital for pneumonia on may . ceftriaxone and amikacin were started, but his pneumonia progressed despite the antibiotic treatment. he was transferred to , reported to who as of november , republic of korea other countries saudi arabia the chain of transmission was identified in all mers patients [ , ] . of the healthcare institutions that had taken in mers-cov-infected patients, nosocomial transmission occurred in hospitals and four clinics (plus two ambulances) [ , , ] . the epidemiological characteristics are summarized in table [ ] . the epidemic curve is shown in fig. . during his admission from may to at ptsm hospital, the first patient was unknowingly spreading mers-cov. patients, visitors, and healthcare workers (hcws) were exposed to mers-cov. therefore, by may , when the korea cdc noticed the importation of mers-cov into the republic of korea, individuals staying on the same floor as the first case had already been exposed to mers-cov. as a result, a total of mers cases occurred at ptsm hospital. of them, patients developed symptoms within days after exposure, suggesting they were first-generation cases; the remaining patients may be second-generation cases [ , ] . the mode of transmission of mers-cov at ptsm hospital remains uncertain. in addition to family members of the first patient, only one patient shared the same room (# ) with him; all the other cases stayed in other rooms, which were located > m apart from room # . all patient rooms were equipped with an air ventilating system except for room # . a recent study using a multi-agent modeling framework suggested the transmission occurred via a long-range airborne route or via a combination of a long-range airborne route and direct contact transmission [ ] . a chest computed tomography (ct) scan taken in the early stages (day or ) of illness showed very small, ground glass nodules in the periphery of the lungs, suggesting inhalation of airborne particles [ ] . many individuals had been exposed to the superspreader- at the er, and contact tracing identified patients, an estimated visitors, and hcws as contacts of superspreader- . of them, were quarantined and were under active monitoring. as a result of exposure to superspreader- , had laboratory-confirmed mers-cov infection, and an additional were secondarily infected from them [ , ] . the index case (superspreader- ) for the daejeon ( km south from pyeontaek city) outbreak was infected at ptsm hospital. he developed symptoms on may and was admitted to daechung hospital on may . as his pneumonia progressed despite the treatments, he was transferred to geonyang hospital on may . a total of secondary cases ( in daechung hospital and in geonyang hospital) were detected. although superspreader- caused the two hospital outbreaks, the me- dian time of incubation ( . days vs. . days), secondary attack rates ( . % vs. . %), and case fatality rate ( . % vs. . %) were different between the two outbreaks. the high fatality rate was associated with pre-existing pneumonia or poor underlying pulmonary function [ ] [ ] [ ] . a -year-old male, who had been exposed to the first index case on may , developed back pain on may . he is a son of the third mers case. his sister had also been exposed to mers- epidemiological characteristics have been reported by several groups (table ) [ , , , , , [ ] [ ] [ ] [ ] . the defining epidemiological characteristics were superspreading events at hospitals [ ] [ ] [ ] . early superspreading events generated a disproportionately large number of second-ary infections, and the transmission potential decreased sharply in subsequent generations [ , ] . a total of % of transmission events were due to five superspreaders, and % of the mers cases were in-patients who had been exposed within the hospitals [ ] . the spreaders transmitted mers-cov from days to of their illness (median, days), and the number of patients infected by each spreader ranged from to (interquartile range, to ) [ ] . the median incubation period and serial interval was days and . days, respectively [ , ] . r ranged from . to . , higher than the previous estimate of < [ ] . it was estimated at . for the outbreak cluster in ptsm hospital, and . for the outbreak in smc [ ] . a mathematical modeling study demonstrated that although r < overall, cluster sizes of over cases are not unexpected for mers-cov infection [ ] . compared with the non-spreaders, the spreaders had a higher frequency of fever (≥ . °c) and chest infiltrates in more than three lung zones, and longer non-isolated in-hospital days [ ] . the spreaders had higher viral load in sputum samples with the cycle thresholds for the upe and orf a genes of . vs. . and . vs. . , respectively. the spreaders with more than three transmissions had higher numbers of contacts and er visits [ ] . a brief exposure of a -minute stay and minutes of talking was enough for the transmission of mers-cov [ ] . the whole genome sequences of mers-cov isolated from the korea outbreak were reported [ , [ ] [ ] [ ] [ ] . the virus most similar to the korean isolates was a camel virus (camel/riyadh/ry / ) that belonged to lineage , a recombinant of lineage and [ , ] . viruses from lineage had been predominant in saudi arabian camels since november , but human viruses of this lineage were reported from february [ ] . a phylogenetic study of spike glycoprotein genes also showed the korean strains were closely related to the viral strains from riyadh, saudi arabia [ ] . in a molecular study, of mers-cov genome had an i t mutation and one d g mutation in the receptor binding domain of viral spike protein, resulting in reduced affinity to the human cognate receptor, cd [ ] . heterogeneity analysis revealed the combined frequency of i t and d g was high ( . %), although the frequency of both mutations varied greatly among specimens [ ] . another genome sequence study also reported deletions of and nucleotides between orf and the e protein, suggesting that the virus might be defective in its ability to package mers cov [ ] . a microevolution study found no evidence of changes in the evolutionary rate [ ] . however, whether the transmissibility and virulence of the korean isolates are different from those of the previous isolates from the kingdom of saudi arabia remains to be determined by phenotypic assays. a viral shedding study showed that the copies of mers-cov rna detected by real-time reverse transcription polymerase chain reaction (rrt-pcr) in respiratory samples peaked during week , and the median value was . log in the severe group and . log in mild cases [ ] . the study also showed that viral titers were higher in throat samples than in nasopharyngeal samples. another study also demonstrated higher viral loads were associated with severity and mortality [ ] . after mers-cov infection, antibody responses developed by the third week of illness in most patients [ ] . seroconversion rates increased with the increase of dis-ease severity. in a serologic study of mers patients, the seroconversion rate was % in asymptomatic infection, . % in symptomatic infection without pneumonia, . % in pneumonia without respiratory failure, and % in pneumonia with respiratory failure [ ] . the serological response remained detectable for > months in all survivors ( / ) who had severe disease. antibody titers were not detectable in four of six patients who had mild pneumonia, suggesting that mers-cov seroepidemiological studies may underestimate the extent of mild and asymptomatic infection [ ] . the korean society of infectious diseases compiled the clinical data of the mers patients [ ] . the median age was years (range, to ). the male to female ratio was : . the most common coexisting medical conditions were hypertension ( . %), diabetes ( . %), solid organ malignancy ( . %), and chronic lung disease ( . %). patients with mers-cov infection developed a spectrum of illnesses, ranging from asymptomatic to fulminant pneumonia with fatal outcome. the respiratory symptoms were similar to other acute viral respiratory infections. at the time of presentation, fever had developed in . %, cough in . %, and sputum in . % of patients. symptoms of upper respiratory-tract infections were infrequent: sore throat was present in . % and rhinorrhea in . % of patients. gastrointestinal symptoms were also observed: diarrhea was present in . %, nausea or vomiting in . %, and abdominal pain in . % of patients. diarrhea may be due to the side effect of lopinavir/ritonavir, as % of the patients received antiviral drugs for mers-cov treatment [ ] . in the early stages of illness, cough and sputum were not prominent even in patients who later developed overt pneumonia [ ] . at admission, % ( / ) of patients had abnormalities on chest radiographs, while . % ( / ) of them developed the abnormalities during the course of the disease [ ] . sudden progression of pneumonia occurred around day of illness [ , ] . predictive factors for development of pneumonia included older age, high fever, thrombocytopenia, lymphopenia, c-reactive protein (crp) ≥ mg/dl, and a high viral load www.kjim.org https://doi.org/ . /kjim. . in sputum (threshold cycle value of rrt-pcr < . ) [ ] . forty-five patients ( . %) were treated with mechanical ventilation and patients ( . %) needed extracorporeal membrane oxygenation [ ] . the typical course of pneumonia is shown in fig. . acute kidney injury (aki) developed in . % of the patients, . % ( / ) had proteinuria, and . % ( / ) had hematuria. fifteen patients were treated with renal replacement therapy [ ] . old age is an independent risk factor for the occurrence of aki [ ] . neuromuscular manifestations were not uncommon; hypersomnolence, weakness and tingling in the extremities were reported during the treatment of mers, suggesting guillain-barré syndrome or virus-related sensory neuropathy [ ] . serial changes in chest radiographs was reported in five patients [ ] . chest ct scans revealed rapidly developed multifocal nodular consolidations with ground-glass opacity halo and mixed consolidation, mainly in the dependent and peripheral areas [ ] . there are few laboratory findings specific to mers-cov infection, although monocytosis with normal white blood cell count and low crp level were more common in mers patients at initial presentation [ , ] . the guidelines for the molecular diagnosis of mers-cov infection have been published by the korean society for laboratory medicine [ , ] . specimen type and quality is important in the laboratory diagnosis of mers-cov infection. lower respiratory-tract samples, such as sputum and tracheal aspirates, have higher viral loads than upper respiratory-tract samples [ ] . however, mers patients may not shed the virus during the early stage of their illness. therefore, initial negative results should not rule out the possibility of mers [ ] , and patients suspected of having mers-cov infection should be retested using a lower respiratory-tract sample [ , ] . when sputum cannot be obtained, throat swabs may be an alternative source of diagnostic samples [ ] . special attention should be given to diagnosing mers-cov infection in immunocompromised patients, as they may present with atypical features, such as a longer incubation period, a longer period from initial pcr positivity to symptom onset, and persistent viral shedding [ ] . an antiviral treatment guideline has been published by the writing committee with support from the korean society of infectious diseases and the korean society for chemotherapy [ ] . the guideline recommended a tri- ple combination regimen of type interferon, ribavirin, and lopinavir/ritonavir for to days. however, the guideline was mostly based on expert opinions and further study for the optimal antiviral treatment in mers is warranted. the median duration of fever was days. the median time to negative conversion of mers-cov in sputum samples by rrt-pcr was days (range, to ) [ ] . the median time from symptom onset to death was days. the case fatality rate was . % ( / ) (fig. ) . the in-hospital mortality, -day mortality, and -day (from symptom onset) mortality were . % ( / ), . % ( / ), and . % ( / ), respectively [ ] . host factors associated with mortality were old age (> years), smoking history, pre-existing pneumonia, abnormal renal function, and comorbid conditions [ , [ ] [ ] [ ] . low albumin, altered mentality, and high pneumonia severity index score at admission were risk factors for mortality [ ] . mers-cov rna in blood samples was detected in % ( / ) of patients at presentation, and it was associated with a worse clinical outcome [ ] . a shorter incubation period was also associated with an increased risk of death [ , ] . a pregnant woman infected with mers-cov in her weeks of gestational age developed dyspnea and her chest radiograph showed diffuse infiltrates in the left lower lung zone. she recovered from the infection and delivered a healthy full-term baby without vertical mers-cov transmission [ ] . a patient developed organizing pneumonia days after the resolution of mers-cov infection. he was successfully treated with corticosteroids [ ] . in a mental health study, . % of , patients who were quarantined showed symptoms of anxiety, and . % reported feelings of anger during the weeks of quarantine [ ] . mental health support, accurate information, and appropriate supplies, including food, clothes, and accommodation, should be provided to isolated persons [ ] . a case of possible transfusion-related acute lung injury following transfusion of convalescent plasma was also reported [ ] . a comprehensive "mers infection prevention and control guideline for healthcare facilities" was drafted by the guideline development committee with generous support from korean academic societies [ ] . the guideline included practical aspects of infection control and prevention based on the experience from the korea outbreak, such as the composition of members for the mers emergency committee, a space plan for an anteroom for donning and doffing, isolation of in-patients and hcws in a hospital affected by an outbreak, and management of the deceased and autopsy [ ] . the hemodialysis unit may become an epicenter for mers-cov outbreak because hemodialysis patients must receive renal replacement therapy even during the outbreak, and the risk of exposure to mers-cov continues. during the korea outbreak, a hemodialysis unit was found to have a mers patient, and a total of patients and hcws were exposed to mers-cov. with support from the korean society of nephrology, the dialysis unit could continue to operate while the exposed patients were isolated in the unit. fortunately, no further transmission occurred at the unit [ ] . after this successful experience, the society published a clinical practice guideline for hemodialysis facilities dealing with mers patients [ ] . the korea outbreak was driven by the superspreaders who visited multiple healthcare facilities; thus, generating a large number of secondary cases. limiting unnecessary contacts with patients with respiratory symptoms in healthcare settings, especially in emergency departments, is of critical importance [ ] . mers-cov could survive for longer than hours at °c and % of relative humidity, suggesting contact or fomite transmission might occur in healthcare settings [ ] . mers-cov was detected by rrt-pcr in specimens taken from the medical equipment [ ] [ ] [ ] . mers-cov was also isolated by cell culture of the air and swab samples taken from a mers isolation unit, suggesting extensive contamination of the isolation unit [ , ] . of the cases, % were infected by undocumented contact between cases (i.e., indirect transmission of mers-cov via environmental contact) [ ] . therefore, fomites with possible mers-cov contamination should be sanitized, and a minimum room ventilation rate of six air changes per hour should be implemented to minimize recirculation of pathogen-bearing droplets. meticulous environmental cleaning may be important for preventing transmission in healthcare settings. there was a case of a -year-old female nurse who was infected with mers-cov during a cardiopulmonary resuscitation lasting hour for a mers patient who had pneumonia and hemoptysis [ ] . serological surveillance conducted after the mers outbreak for asymptomatic infection among hcws involved in the direct care of mers patients showed that . % ( / ) of them were mers-cov igg positive by an indirect immunofluorescent assay. among the hcws who did not use appropriate personal protective equipment (ppe), seropositivity was . % ( / ) compared with % ( / ) in hcws with appropriate ppe use [ ] . another serological survey also showed that none of the hcws were positive for mers-cov immunoglobulin g, although . % ( / ) of the hcws reported experiencing mers-like symptoms while caring for the mers patients [ ] . in a third study, of hcws showed seroconversion by a plaque reduction neutralization test, although % to % of hcws reported mers-like symptoms [ ] . during the outbreak in smc, all hcws assigned to mers patients were screened for mers-cov, regardless of the presence or absence of symptoms. of the hcws, three ( . %) asymptomatic hcws (two nurses and one physician) were found to be mers-cov (+), but they did not transmit the virus to others [ ] . in another study, an asymptomatic nurse without ppe contacted hcws, including close contacts who were exposed within m from the index nurse and were not using ppe. none of the exposed hcws were infected [ ] . however, the potential for transmission from asymptomatic rrt-pcr positive individuals is still unknown. therefore, asymptomatic hcws who are rrt-pcr-positive for mers-cov should be isolated and should not return to work until two consecutive respiratory-tract samples test negative on rrt-pcr [ ] . the government requested the korean society of infectious diseases to participate in the control of the mers outbreak. on june , , the society organized the rapid response team, consisting of infectious-disease doctors and two infection-control professionals [ ] . critical suggestions to prevent future epidemics were made regarding a rapid alerting system for index cases, provision of a sufficient airborne infection isola- the korean journal of internal medicine vol. , no. , march tion facility, education and training of hcws for important infectious diseases, and overcrowding and visitor control in the hospital [ ] . the korea outbreak was the largest outbreak outside of the middle east. researchers had a unique opportunity to compare the nature of mers-cov infection with the middle east experience. the outbreak unveiled the weak points of infrastructure in our medical system, especially of preparedness for emerging global infectious diseases. nosocomial transmission was one of the core features of mers-cov infection. the introspection for loose medical referral systems, overcrowding at the er, a lack of expert resources and infection control infrastructure, and lack of organized preparedness for medical crises prompted the government to reform the healthcare system, and healthcare sectors to invest further in infectious diseases and infection control. although the improvement is still ongoing, the speed and content are still currently insufficient. international cooperation to prepare for and defeat emerging infectious diseases should also be emphasized. lessons we learned from the outbreak are summarized in table . a single, missed case may trigger a huge, nationwide outbreak the first line of defense is not the thermal scanner at the airport. it is doctors in the community clinics/hospitals. due to sudden deterioration of pneumonia around day of illness, patients may visit emergency department, or be admitted to intensive care unit. superspreading events may occur in healthcare settings, especially at the emergency department. patients may transmit mers-cov as early as days after symptom onset. early detection and isolation is of critical importance. aggressive strategy for quarantine maybe necessary, especially when large number of individuals are exposed in the healthcare settings. huge socioeconomic impact with an estimated . billion us dollars isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) [internet]. geneva: world health organization ministry of health kingdom of saudi arabia. mers-cov daily update ministry of health kingdom of saudi arabia, c middle east respiratory syndrome middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission treatment of mers-cov: information for clinicians. clinical decision-making 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south korea serologic evaluation of mers screening strategy for healthcare personnel during a hospital-associated outbreak middle east respiratory syndrome coronavirus transmission in dialysis unit and infection control interventions in korea management of asymptomatic persons who are rt-pcr positive for middle east respiratory syndrome coronavirus (mers-cov): interim guidance [internet]. geneva: world health organization, c collaborative intervention of middle east respiratory syndrome: rapid response team institutional preparedness to prevent future middle east respiratory syndrome coronaviruslike outbreaks in republic of korea no potential conflict of interest relevant to this article was reported. key: cord- - syce n authors: domínguez-andrés, jorge; netea, mihai g. title: impact of historic migrations and evolutionary processes on human immunity date: - - journal: trends immunol doi: . /j.it. . . sha: doc_id: cord_uid: syce n the evolution of mankind has constantly been influenced by the pathogens encountered. the various populations of modern humans that ventured out of africa adapted to different environments and faced a large variety of infectious agents, resulting in local adaptations of the immune system for these populations. the functional variation of immune genes as a result of evolution is relevant in the responses against infection, as well as in the emergence of autoimmune and inflammatory diseases observed in modern populations. understanding how host–pathogen interactions have influenced the human immune system from an evolutionary perspective might contribute to unveiling the causes behind different immune-mediated disorders and promote the development of new strategies to detect and control such diseases. the evolution of mankind has constantly been influenced by the pathogens encountered. the various populations of modern humans that ventured out of africa adapted to different environments and faced a large variety of infectious agents, resulting in local adaptations of the immune system for these populations. the functional variation of immune genes as a result of evolution is relevant in the responses against infection, as well as in the emergence of autoimmune and inflammatory diseases observed in modern populations. understanding how host-pathogen interactions have influenced the human immune system from an evolutionary perspective might contribute to unveiling the causes behind different immune-mediated disorders and promote the development of new strategies to detect and control such diseases. infectious diseases are arguably the main source of evolutionary pressure that humanity has ever confronted. the dispersion of different human communities around the globe has exposed each population to different infectious agents, exerting a selective pressure (see glossary) on them; thus, adaptation to the new environment has favored the selection of the most beneficial genetic variants for the host. as a result, infectious agents have caused the expansion of alleles behind the induction of either protection or tolerance to these diseases; heritable variations, that increased the survival to deadly infectious agents, may have been naturally selected before the hosts had the opportunity to reproduce [ ] . natural selection driven by pathogens is probably more remarkable for those infectious agents that have been among us for a longer time, namely the causative agents of well-known diseases such as leprosy, smallpox, malaria, or tuberculosis. the genetic imprint of pathogen-driven selection depends on the length and the virulence of the infections and also the geographical distribution. the human genome presents more than genetic loci with traces of selective pressure [ ] . this group includes more than immune-related genes with functional variations between populations, which are probably behind the variability of responses to immune-related diseases reported nowadays [ , ] . besides natural selection, other evolutionary mechanisms, such as genetic drift, greatly influence the frequencies of the genetic variants found within diverse populations throughout the world [ ] (box and figure ). with the burst of next-generation sequencing and the development of cutting-edge technologies such as transcriptomics, proteomics, and systems biology, we are starting to witness the great impact of evolutionary processes on human immunity and how the interactions between microorganisms and humans that took place millennia ago might play a fundamental role not only in the response against modern pathogenic threats, but also in the emergence of autoimmune and inflammatory diseases observed in modern populations worldwide. in this review we offer a novel perspective on the role of infectious diseases as agents of natural selection and as forces behind the evolutionary pressure encountered by human ancestors and modern humans in their migrations around the globe. specific genetic variants selected throughout different periods of human history may have influenced immune responses of present-day populations against pathogenic microorganisms and may have played a role in the development of certain inflammatory and autoimmune diseases. the majority of experts agree that africa is where our species originated. genetic studies conducted in diverse contemporary populations suggest connections with ancestors that lived on the continent up to years ago [ ] . human evolution has been constantly influenced by pathogens; therefore, a great number of human genes linked to immune functions and immunity-related disorders have evolved along with humans. the heterogeneity in the immune response to infectious diseases across different populations is under genetic control and is the result of evolutionary processes. genetic variants that have been under evolutionary pressure can contribute to explaining the differences in the susceptibility to diseases observed across different populations. the ancestry of individuals from different populations across the globe greatly influences their possibility of developing certain autoimmune diseases and inflammatory disorders. the lifestyle of western societies affects the symbiotic relationships between humans, viruses, and other organisms and might contribute to the rise of certain autoimmune and inflammatory diseases. pathogens have played a central role as agents of natural selection from those very early days. among various infectious diseases, malaria has exerted the highest evolutionary pressure on the communities across the african continent ( figure ) [ ] . populations remaining in sub-saharan africa have been exposed to malaria for such long periods of time that their genetic structures have been shaped by the severity of malaria (plasmodium sp.) infections. in , allison described that sickle cell disease distribution was confined to africa and was associated with the geographical presence of malaria [ ] . this finding led to the more recent description of the existence of mutations in the hemoglobin-b (hbb) gene as a result of natural selection driven by evolutionary pressure for protection against malaria [ ] (table ) . similarly to hbb, some areas of west africa with a high incidence of malaria the gene variations that pass from one generation to the next are often transmitted as a random process known as genetic drift, while selection of advantageous variants tends to be preferentially transmitted. mutations, genetic drift, migration, and environmental selective pressure are among the fundamental processes behind the evolution of humans. the influence of these mechanisms in the diverse communities that were mobilized and then became isolated, as well as severe external factors such as epidemics, caused successive genetic bottlenecks in populations (see figure in main text) [ ] . human evolutionary studies are currently considered under 'modern synthesis', which merges darwin's ideas of natural selection with theoretical population genetics and mendelian principles, stating that evolution occurs via small genetic changes that are regulated by natural selection [ ] . these beneficial adaptations subsequently expand within the members of a population and become evident in the ancestral specificity and the geographical distribution of the advantageous alleles in the genomes of contemporary humans. genetic bottlenecks occur when the number of individuals in a population is reduced drastically due to a catastrophic event such as an earthquake, a flood, a famine, or the outbreak of an infectious disease. these events limit the genetic variation of a population and can lead to genetic drift. as a result, a smaller population, with a correspondingly reduced genetic diversity, remains to transmit genes to future generations through sexual reproduction. even if this reduction in the genetic diversity is temporary, it can lead to long-lasting effects on the genetic variation of the offspring populations. genetic drift: changes in the allele frequencies of a population over generations due to chance. genetic locus: fixed position on a chromosome (e.g., the position of a gene or a genetic marker). histocompatibility complex: region of approximately kb, located on human chromosome , that contains a large number of genes whose products are expressed as proteins on immune cells. of these genes, the best known are hla genes. introgressive hybridization: incorporation of genes from one species into the genetic reserves of another by interspecific hybridization and backcrossing with the parent species. present a high frequency of hemoglobin-c (hbc) in their populations, which is associated with a - % decrease in the possibility of developing the disease [ ] . this is also the case for the duffy antigen receptor gene (darc) in erythrocytes and single nucleotide polymorphisms (snps) in human leukocyte antigen (hla), which have been associated with protection against plasmodium vivax malaria in certain areas of africa where this disease is endemic [ , ] . another example of natural selection driven by evolutionary pressure for protection against malaria are thalassemia (a and b) pathologies, a group of hemoglobin disorders that presents an incidence of up to % among communities of west africa [ ] . the human casp t c snp, expression of which is restricted to the african subcontinent, south america, and certain areas of asia, can modulate immune and inflammatory responses to malaria by antagonizing interleukin (il)- b and nf-kb signaling in innate immune cells; moreover, caspase -deficient mice (casp -/-) exhibit decreased interferon (ifn)-g production and clearance of the parasite, relative to wild type (wt) infected mice [ ] . however, others have questioned these findings, given that caspase -deficient mice also lack caspase expression, so the effects observed might not be specific to caspase [ ] . mycobacterium tuberculosis (mtb) has caused infections in our species and ancestors for at least years [ ] . this long-standing relationship between humans and mtb probably underlies the large variety of immune-related factors that modulate susceptibility to mtb infection, including vitamin d receptor (vdr), natural resistance-associated macrophage protein (slc a ), tir domain containing adaptor protein (tirap), hla, monocyte chemoattractant protein (mcp- ), and cytokines such as il- and ifn-g [ ] ( table ) . patients with african ancestry present a higher frequency of mtb-related genetic variants than individuals from other populations, including variants in the gene encoding for toll-like receptor (tlr ), mediating cellular responses to bacterial malaria is one of the greatest causes of morbidity and mortality in the history of humanity. most human populations with a long history of endemic malaria have evolved genetic adaptations to malaria parasites due to the strong selective pressure that this infection has exerted. since the parasite infects erythrocytes, the evolutionary pressure has selected genetic variants that affect red blood cells and, therefore, the survival of the parasite as well. genetic variants conferring resistance to the disease have spread through human populations over time, including several abnormal hemoglobins that protect against malaria but usually cause erythrocyte-associated diseases in the populations where these adaptations are prevalent. these factors include the t c polymorphism in the caspase gene (casp ); the hemoglobin b (hbb) and hemoglobin c (hbc) variants; mutations in the duffy antigen receptor gene (darc); thalassemias (a and b); sickle cell disease; and polymorphisms in the human leukocyte antigen (hla) loci. dna nucleotide. for example, an snp may replace cytosine (c) with thymine (t) in a certain segment of dna. selective pressure: phenomenon that alters the behavior and fitness of living organisms within a given environment. it is the driving force of evolution and natural selection. thalassemias (a and b): inherited hemoglobinopathies characterized by a failure in the synthesis of the globin alpha or beta chains. toll-like receptors: family of transmembrane pattern recognition receptors expressed by immune and nonimmune cells that recognize conserved pathogenassociated molecular patterns. they play a pivotal role in innate immunity. transgenerational inheritance: transmission of traits from generation to generation. trends in immunology, december , vol. , no. lipoproteins [ , ] . selective pressure has also shaped the mechanisms that modulate the expression of genes implicated in immune responses against lassa virus, such as il- (il ) and the glycosyltransferase-like protein large (large), suggesting that the natural selection exerted by the virus drove the expansion of genetic variants that enhance immunity against lassa fever [ ] . these examples indicate that infectious diseases have contributed to shaping the genetic landscape of african populations and their descendants, and highlight the great impact of pathogens as an evolutionary force in humans. our homo sapiens ancestors were not the only species to venture out of africa, with other homo species performing a similar migration much earlier, such as homo ergaster, homo erectus, or homo heidelbergensis [ ] . from these early migrations, local populations such as the denisovans and neanderthals evolved [ ] . these lineages were not geographically isolated, but lived side by side with modern humans and interbred with them, leaving a genetic footprint in their common progeny. accordingly, - % of the genome of european and asian populations is thought to derive from these now-extinct hominid lineages [ ] . neanderthals spent close to years adapting to their environment and their immune systems were shaped by the infections they faced. by interbreeding with archaic humans, modern humans incorporated these advantageous adaptations in the genome of their descendants. this was highlighted by different studies that showed that the introgression of diverse genes related to immune functions, such as the oas cluster, tlr , or the histocompatibility complex from denisovans and neanderthals shaped the genetic landscape of present-day eurasian, but not african, communities. genomic sequences and expression data from lymphoblastoid cell lines from individuals of european and african ancestry confirmed that the tlr -tlr -tlr genetic loci, presenting signs of local positive selection and repeated introgression from both neanderthal and denisovan genomes [ ] [ ] [ ] , showed a significantly higher expression in individuals carrying archaic-like alleles than in individuals carrying the nonintrogressed modern human alleles [ , , ] . the expression of these genes has shaped human immune responses against different types of pathogens. for example, the gp protein of the hiv- virus has been recently recognized as a tlr ligand [ ] . in this regard, increased tlr expression has been correlated with higher il- production by the macrophage cell line thp- and higher titers of hiv- in the breast milk of hiv- -infected nigerian women relative to controls [ ] . tlr and tlr form dimers with tlr , triggering immune responses against different types of bacteria, fungi, virus, and parasites [ ] . variation in tlr -tlr -tlr is the major genetic determinant of human interindividual differences in tlr / -mediated responses, including cytokine production to a number of clinically relevant pathogens such as staphylococcus aureus and listeria monocytogenes [ ] (table ). this inheritance from archaic humans may have also left some human individuals more prone than others to developing asthma, hay fever, and other allergies (of snps associated with susceptibility to allergic disease, had a neanderthal or denisovan origin) [ ] , although these associations remain to be fully demonstrated [ ] . these reports demonstrate that by interbreeding with archaic humans, modern humans incorporated a group of advantageous adaptations to the genome of their descendants and contributed to shaping immune responses in modern human populations. the migration of our human ancestors out of africa implied the exposure to different types of infectious diseases (box and figure ). one study tested the responsiveness of human macrophages to pathogenic bacteria in vitro, finding that almost % of the genes present in human macrophages infected with the bacteria salmonella typhimurium or l. monocytogenes present different regulatory responses directly linked to the lineage of the donors and, also, that macrophages obtained from individuals of african origin display enhanced bactericidal activity compared with those from individuals of european lineage [ ] . the trend towards lower inflammatory responses in european populations is strengthened by the fixation of a tlr gene variant that results in lower proinflammatory gene expression in populations with a european ancestry compared with those with an african one [ ] . the largest population differences in gene expression between africans and europeans have been found in the macrophage receptor with collagenous structure (marco), a protein implicated in responses against viral infections and tlr-induced dendritic cell activation [ ] , the chemokine receptor cx cr , which mediates effector lymphocyte functions, and also several ifn-stimulated genes [ ] . west eurasian populations present a high frequency of tirap ser leu snps [ ] . tirap is an adaptor protein in tlr and tlr signaling pathways, involved in inflammatory responses and cytokine production. the heterozygous expression of the ser leu snp is protective against invasive pneumococcal disease, bacteremia, malaria, and tuberculosis, as shown in a case-control study of individuals from the uk, vietnam, and several african countries, and it is associated with lower tlr signaling in humans [ ] . this variant is considered to be a consequence of the natural selection that may have taken place in an early period following the migration of modern humans out of africa [ ] . european populations present a selective adaptation of the ifn gene that allows a high production of ifn-g in infectious scenarios due to the positive selection of ifng variants + g and + t; this suggests the existence of strong environmental pressures linked to higher ifn-g concentrations in plasma during mtb infection in european individuals relative to other populations [ , ] . in line with this, a database meta-analysis showed that individuals expressing the + t/a variant of ifng presented higher susceptibility to tuberculosis mtb infection than individuals without it, which might be considered a putative prognostic factor for the development of tuberculosis [ ] , although this remains to be robustly demonstrated. one of the most interesting aspects of humans is their ability to adapt to almost every ecosystem of the planet. the history of mankind is also the history of millions of individuals wandering around the world, looking for a better place to live. a glimpse to the migratory legacy of humanity around the globe reveals the great impact that the massive population movements defined the world as we know it today (see figure in main text). the distances ancient humans travelled are impressive, from the first hominids colonizing africa to the conquest of the americas in a time when the bering strait was not yet under water. the historical exodus of mankind started almost million years ago with the migration of homo erectus from africa through eurasia. from this event on, relatively isolated human populations evolved separately on different continents, leading to the emergence of different human species, such as neanderthals in europe, the denisovans in asia, and, later, modern homo sapiens in africa [ , ] . h. sapiens first colonized large areas of the continent around years ago [ ] , spread towards the middle east at some point between and years ago, and migrated through eurasia, reaching australia within years [ ] . asian human ancestors went through the frozen waters of the bering strait in two distinct waves to colonize the american continent approximately years before the present time [ ] . when humans ventured out of africa, they faced different types of pathogens than the communities that stayed in the african continent. with time, the series of events faced by diverse populations has generated differences in the immune responses to pathogens in the populations with an african or eurasian origin and which have spread throughout the world. the indigenous populations of south america are descendants of migrating populations of north-east asians that crossed the bering strait around years ago [ ] . five centuries ago, european settlers disembarked on the american continent, bringing a large collection of pathogens such as those causing measles, pneumonic plague, and influenza infections, which the indigenous populations had never faced before. these diseases rapidly spread and caused mortality rates above % [ ] . the consequences of these pandemics are still visible in current populations; one report studied dna from the bones of ancient humans from the tsimshian community, living in the british columbia region in canada until the th century, identifying marks of positive selection in a number of immune-related variants [ ] . specifically, the hla-dqa variant was present in almost the totality of tsimshian individuals, but only in one third of present-day humans studied; this suggested that ancient american genomes were evolutionarily selected to respond to local diseases but not to fight against pathogens brought by the europeans [ ] . another study compiled information on infectious diseases that have killed more than individuals among indigenous communities of the amazonia in the past two centuries, showing that the mortality rates and the incidence of infectious diseases rapidly decayed within the time following the first encounter with the pathogen, compatible with genetic adaptation [ ] . european colonizers underwent purifying selection in situations of intense pressure. such scenarios were documented when dutch colonists migrated to surinam and encountered epidemics of yellow and typhoid fever that caused a % mortality rate among settlers [ ] . variations in the frequencies of c , glo, and hla-b genes among the descendants were not likely caused by genetic drift, but rather, it has been proposed that these populations were probably selected through genetic control of survival to the epidemics [ ] (box ). africans and americans with an african ancestry present a much higher number of genetic variants related to robust inflammatory reactions, increased cytokine secretion, and bactericidal activities compared with the other populations [ ] , including more than genes with traces of recent selection, such as il a and il b gene variants [ ] . the degree of african ancestry, analyzed by fine-mapping analysis refined to the duffy-null allele of rs , was correlated with an increased amount of the proinflammatory chemokines ccl and ccl in plasma relative to controls [ ] . a study involving african american and hispanic american women found that the higher values of c-reactive protein (crp) in blood found in these populations compared with european americans were related to a crp-associated variant of triggering receptors expressed by myeloid cells (trem ) [ ] . moreover, comparison of health record data from individuals with connective tissue diseases, including rheumatoid arthritis and systemic lupus erythematosus (sle), as well as atherosclerotic cardiovascular disease from almost african american and european american adults was conducted; the study reported for the latter, a prevalence of atherosclerotic cardiovascular disease in . % african americans (particularly high in young individuals), relative to . % in european americans [ ] . these studies highlight certain genetic links to inflammatory predisposition/manifestation. however, increased proinflammatory activity is a double-edged sword. in the absence of regular pathogen challenges that require maintained modulation of the balance between inflammation and suppression of the immune response, the organism can overreact to inflammatory stimuli and trigger exacerbated responses. for instance, descendants of african populations generally present higher susceptibility to a variety of autoimmune syndromes such as inflammation-associated carcinomas, lupus, asthma, and multiple sclerosis (ms), the overall prevalence of which is up to three times higher in individuals with african ancestry relative to individuals with european ancestry [ , , ] . there are extensive differences in immune cell gene expression between americans with african and european ancestry. the increased proinflammatory responses observed in american individuals relative to other populations might be beneficial to combatting infections, but might also increase the chances of developing inflammatory and autoimmune disorders, which warrants further investigation. our gastrointestinal tract provides residence to both beneficial and potentially pathogenic microorganisms, harboring ten million different microbial genes in the human fecal microbiome [ ] . the microbiome has its own evolutionary scenario across different populations with divergent lifestyles, nutrition, and exposure to environmental agents, generating extraordinary heterogeneity. the ongoing process of 'lifestyle westernization' of different societies has an important impact on the mutualistic relationships between humans and commensal organisms worldwide. african tribes are adopting western subsistence patterns, leading to remarkable changes in the composition of their microbiota [ ] . the comparison of the intestinal flora of the baaka hunter-gatherers and the bantu agriculturalists (both from the central africa republic), with a group of us-born african americans showed a great example of the evolution of the human microbiome [ ] . specifically, the bantu, still engaged in hunting, have a greater bacterial gut diversity than their baaka neighbors, who left the jungle for agriculture, and even more than urbanized westerners (us african-americans) [ ] . this reduced microbiota diversity in western societies has been associated with a higher incidence of the so-called 'diseases of civilization' such as cardiovascular diseases, diabetes, obesity, and autoimmune disorders, which are very unusual in hunter-gatherer societies compared with communities living a western-type lifestyle [ , ] . although viruses are mainly seen as pathogenic agents, they also play a fundamental role in the evolution and maturation of the human immune system [ , ] . approximately % of the human genome is composed of endogenous retroviruses (ervs), sequences derived from past retroviral infections and permanently inserted into different regions of the human genome [ ] . one study showed that ervs played a central role in the induction of ifn-dependent immune responses and that the removal of one or more of these viral dna elements in the hela human cell line severely impaired the recruitment of transcription factors necessary to trigger the expression of ifng against vaccinia virus infection relative to controls [ ] . viruses can also influence the severity of infections caused by other viruses. for example, cytomegalovirus infection in hiv- seropositive humans can potentiate the effects of hiv- infection by expanding the pool of circulating regulatory t cells (immunosuppressive); these were shown to inhibit the proliferation of autologous peripheral blood mononuclear cell the origins of the hiv virus are still a matter of scientific discussion. the most accepted scenario argues that hiv originated in simians and was transmitted to humans in west africa in the s, likely due to local ingestion of ape meat infected with the simian immunodeficiency virus. around , the virus reached wide parts of the continent and finally spread overseas thanks to a group of haitian professors coming back from africa. in the following decades, the virus spread worldwide and generated the pandemic we now know. today, there are approximately million people worldwide living with hiv- /aids [ ] . ccr is a receptor of chemokines that plays a fundamental role in hiv- pathogenesis and it is also one of the most promising targets to restrict the infection, since mutations in this receptor turn individuals resistant [ ] . the ccr -d mutation results in a deletion that eliminates the hiv- co-receptor on lymphocytes, providing robust protection against hiv- and, therefore, aids [ ] . ccr -d allele frequencies reach % in northern europe populations, whereas it is not present in populations with different ancestry, such as east asian, native american, or african groups [ ] . this regional distribution of ccr -d variants is most likely related to a naturally selective episode that struck european populations around years ago and involved a strong infectious agent that also employed ccr [ ]. a mathematical model studying the changes in the european populations in the middle ages suggested yersinia pestis (bubonic plague) as the most probable infectious agent behind the pressure that selected this particular genetic variant [ ] . this is in agreement with the finding that european rroma populations, but not northwestern indian populations that inhabit the area where the rroma originally lived, present signatures of positive selection in tlr -tlr -tlr , which influence cytokine responses in y. pestis infections [ ] . (pbmc) in response to cytomegalovirus infection in vitro [ ] . in one study, patients with chronic hepatitis c virus (hcv) infection and hepatitis a virus (hav) superinfection presented lower titers of hcv rna than patients harboring only hcv, suggesting that hcv replication might be potentially suppressed during hav infection [ ] , although this will still require further investigation. the relationships between humans and pathogenic or nonpathogenic organisms are extraordinarily complex and include tripartite evolutionary interactions between humans and microbes competing with each other. this is the case of parasites that infect other parasites, such as bacteriophage viruses, that can influence the outcome of bacterial infections. for example, in a cohort of individuals with chronic wounds, a report showed that the phage pf, which coexists with pseudomonas aeruginosa in infected wounds, triggered the production of type i ifn, the inhibition of tumor necrosis factor (tnf) production, and the suppression of phagocytosis in human primary monocytes and mouse bone marrow-derived macrophages, dampening the antibacterial response and promoting the bacterial infection [ ] . however, bacteriophages can also provide protection to the human host by directly attacking pathogenic bacteria and by upregulating in human pbmcs the expression of proinflammatory genes such as il a, il b, il , tnfa, cxcl , and cxcl , as shown for several s. aureus and p. aeruginosa phages, including pnm, luz , - , and ge-vb_pae-kakheti [ ] . cooperative relationships between organisms are evolutionary processes themselves. the way microorganisms and their hosts associate can lead to interactions of mutualism, in which the interplay may be so intimate as to provide benefit for each party and influence immune responses against different types of pathogens. a great number of humans live far away from the original settlements of their ancestors and are subject to radically different environmental conditions. between two and three million people with european genealogy suffer from autoimmune diseases, the prevalence of which is also increasing in other populations across the globe [ ]. there is rising evidence that the emergence of autoimmune diseases is associated with the presence of a number of immune-related alleles that have been selected via evolutionary processes; and, furthermore, that the contrasting differences in the prevalence of autoimmune diseases between populations may be a result of different selective pressures [ ] . alleles associated with inflammatory diseases that present marks of modern positive selection include the risk allele fut at rs for crohn's disease (cd) or the risk variant sh b at rs for celiac disease [ ] ; such variants have been linked to the development of several human autoimmune diseases, such as type diabetes, ms, and celiac disease [ , ] . the analysis of the integrated haplotype score [ ] of loci associated with sle that might provide protection against infections, such as tnip , itgam, ptpn , tnfsf , uhrf bp , tet -dguok, and blk, has suggested that these loci exhibit robust signs of positive selection [ ] (figure , key figure and table ). african and asian human populations exposed to trypanosoma brucei or plasmodium sp. have presented positive selection of snps in the apol and fcgr b genes; indeed, by enhancing human macrophage-mediated phagocytosis of infected erythrocytes, despite their association with an sle predisposition, these snps have been associated with protective roles against sleeping sickness and malaria, respectively [ , ] . an analysis of human loci associated with inflammatory bowel disease (ibd) concluded that the majority of the loci associated with cd are also linked with a higher risk of developing ulcerative colitis [ ] ( table ). many of these loci were also associated with the development of other autoimmune diseases, namely psoriasis and ankylosing spondylitis [ ] . pbmcs from individuals carrying the sh b variant rs *a (associated with a risk for developing cd) [ ] presented higher production of il- and il- b after stimulation with lipopolysaccharide or muramyl dipeptide due to enhanced activity of the nod inflammasome pathway relative to controls [ ] ; this has suggested an immune-related role for sh b in the context of bacterial infections, which might help explain the positive selection of sh b approximately years ago [ ] . others found an association between genetic variants in nod , cd , and increased susceptibility to cd [ ] , in line with previous results showing that mutations in nod and tlr /cd are related to an increased risk of developing ibd [ ] . from another angle, changes in hygiene patterns seen in the past two centuries brought vast improvements in sanitation, drinking water, and garbage collection, which greatly reduced the exposure to many infectious diseases. however, these conditions may have reduced the exposure to viral and microbial agents that help the immune system to develop tolerance during childhood. the hygiene hypothesis proposes that the lack of exposure to microbial agents in the early stages of life is related to a higher risk of developing hypersensitivity reactions, based on the fact that children that are exposed to higher amounts of microbial stimuli (e.g., by growing on farms) are less prone to develop allergies and asthma [ , ] . moreover, reduced exposure to infectious agents can have a much wider effect than initially believed. lack of exposure to microbes in childhood can cause aberrant responses to infection and potentiate the effects of etv -runx mutations in the pathogenesis of acute lymphoblastic leukemia [ ] . by contrast, a meta-analysis of six observational studies, including participants, showed a correlation between low helicobacter pylori infection and ms, suggesting that low h. pylori prevalence might be a putative protective factor in ms, although this remains to be experimentally validated [ ] . one study also reported that antibodies against toxoplasma gondii were detected less often in patients with ms compared with healthy controls [ ] . however, these findings warrant further and robust investigation. overall, it is clear that evolutionary processes can drive the fixation of genetic variations that increase (or decrease) our defense against infections upon sensing microbial ligands, but can also lead to a greater risk of developing certain autoimmune diseases in which endogenous ligands can cause tissue damage and inflammation. a growing number of reports suggest that inheritance is not always governed by classical darwinian evolutionary processes. exposure to certain environmental stimuli can cause effects in the progeny of an exposed individual, even though the stimuli are no longer present. this type of transgenerational inheritance might be explained through the effects of epigenetic processes, which are hypothesized to be transmitted through the germline and passed on to the offspring [ ] . for example, the worm caenorhabditis elegans can transmit improved resistance to infections to pathogenic bacteria to their offspring through alterations of the histone landscape [ ] . indian meal moths exposed to low doses of virus are subsequently less susceptible to viral challenge, a protection offered to their offspring as well [ ] . transgenerational inheritance of diverse traits has also been observed in mice, in which the variation of the color of the coat is passed on the next generation [ , ] . offspring of male rats subjected to a high-fat diet present glucose intolerance and reduced insulin secretion, linked to reduced methylation at the il ra gene relative to controls [ ] ; and mice fed scorpions are more resistant to a challenge with scorpion venom than mice on a normal diet [ ] . since infections are one of the strongest factors impacting survival, it is conceivable that transgenerational transmission of traits in mammals, including humans, evolved to improve host defense. the number of studies of the potential role of epigenetic inheritance in shaping the human immune system is still scarce. however, different experiences undergone by certain communities indicate that these mechanisms might be important. for example, the babies of pregnant women who suffered during the early stages of pregnancy (the effects of the dutch hunger winter in ), years later showed reduced dna methylation marks in several genes that control metabolism and cell differentiation during development, such as igf , pim , txnip, abcg , pfkfb , and mettl , compared with their siblings [ , ] . this was related with higher rates of obesity, heart disease, cancer, and depression in individuals whose pregnant mothers suffered the effects of the famine [ , ] . some of these effects seemed to be present in the progeny of this group, that is, in the grandchildren of those who had passed the famine during pregnancy [ ] . the rapid growth in the number of reports covering the impact of epigenetic mechanisms in different human processes warrants further and robust studies on the impact of epigenetic inheritance in shaping the evolution of the human immune system. human immune responses have been shaped by the evolutionary pressure exerted by microorganisms and viruses throughout history. generating a broad range of genetic variations and immune functions in different populations favors the adaptation to new environments and increases the chance of survival of the human species against potential pandemics. much remains to be learned in this exciting field over the coming years in order to identify the main regulatory forces and the time window necessary for the fixation of an advantageous genetic trait in a population (see outstanding questions). the combination of the selective pressure caused by infectious diseases with other evolutionarily relevant processes, such as genetic drift, migratory events, bottlenecks, and introgressive hybridization, contribute to driving the expansion, fixation, or elimination of characteristic immune response-related traits in different populations around the world. these specific genetic variants are able to boost the host response against pathogens by improving the sensing of microbial ligands but can also lead to the development of autoimmune diseases, in which the immune system responds to endogenous ligands and induces abnormal responses targeted against the host's own tissues. of note, it is very difficult to assign certain variants, a specific role in the protection or induction of autoimmunity. to assign changes in the genetic landscape of human populations to certain diseases is an extraordinary challenge. moreover, our species is in constant evolutionary interaction with various microorganisms and viruses. populations of bacteria and their viruses (phages) undergo, under natural conditions, reciprocal evolution in terms of resistance and infection; this, in turn, also affects the evolutionary traits of our immune system. thus, an extraordinarily complex scenario exists in which organisms of different phyla interact, compete, and coevolve, to ensure their own survival. as novel tools, the development and refinement of methods that study large sections of the human genome, epigenome, and microbiota, will help to obtain genome-wide data in diverse human populations, allowing us to follow the evolutionary trails left from the encounters with different organisms, further unveiling the roots of human immunity. high-throughput biotechnology and an expanding computational capacity can enable the study of global population genomics and might contribute to decoding the origin and consequences of functional changes in adaptive alleles down to the single cell level. however, these methods also have limitations associated with the difficulty in linking gene variations to clinical phenotypes and disease, the generation of false positives, or the high number of samples necessary to reach reliable conclusions. expanding the heterogeneity of populations studied for immune gene association studies relevant to disease will be key, as generally, a large focus is placed on certain european or american communities, thus generating results that are difficult to extrapolate to other populations. the knowledge of the evolutionary and genetic basis of human immune traits and their impact on diverse pathologies (e.g., autoimmunity, infections, inflammatory diseases, cancer) increasingly suggests that the genetic basis of disease may be derived from a large number of rare variants of modest effect. the mechanisms described here acquire special importance in the current scenario of world globalization, in which the migration fluxes and the admixture of different populations are reaching unprecedented levels, allowing faster expansion of advantageous alleles. however, these processes may also accelerate the spread of new epidemics, as seen in the cases of hiv infection, or more recently, sars-cov, ebola, and chikungunya viruses; as well as the emergence of multiresistant bacteria and fungi, such as methicillin-resistant s. aureus or candida auris. this is just the starting point to unveil the evolutionary history of the relationships between pathogens, the immune system, and humans. further investigation of the functional adaptations of human populations is warranted to provide a broad picture of the functional consequences of evolution in human immunity. acknowledgments m.g.n. is supported by a spinoza grant of the netherlands organization for scientific research and an erc advanced grant (# ). the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. we thank laszlo a. groh for which are the strongest evolutionary forces that drive the evolution of the human innate immune system? how long does the genome of a given population take to adapt to a new infectious threat? are the mechanisms of resistance to infection transmitted only via genetic modifications, or can epigenetic adaptations to resistance also be transmitted to the progeny, and under what circumstances? the development of single-cell sequencing technologies has opened new fields of study. how does genetic variation of the expression of a specific variant vary between different cell subsets and how does it affect the overall phenotype of an individual within a population? are there specific immune processes that are preferentially impacted by evolutionary pressures? in western societies we enjoy a life expectancy vastly superior to that of our predecessors, but at the same time, we suffer diseases that they did not suffer. can some of the reasons for these changes lie among some of those bacteria that we have somehow lost in our microbiome? identifying these bacteria and understanding their effects on the human body might be the first step to developing putative therapies based on bacterial restoration. since many of the variants causing autoimmune diseases are linked to an enhanced responsiveness to pathogens that is no longer needed in developed countries, could these genes and their related pathways be employed as targets for new putative therapeutic approaches against inflammatory/ autoimmune syndromes? are other newly described genetic regulatory pathways, such as interfering rna or long noncoding rnas, also influenced by pathogen-driven evolutionary processes in different populations globally? proofreading and correction of the manuscript. we additionally thank srinivas agra for providing the figure icon 'bacteria' as deposited on https://thenounproject.com. natural selection and infectious disease in human populations genomic signatures of selective pressures and introgression from archaic hominins at human innate immunity genes genome-wide identification of regulatory sequences undergoing accelerated evolution in the human genome divergent selection of 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thomas; münch, jan title: the molecular tweezer clr inhibits ebola and zika virus infection date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: evk g ebola (ebov) and zika viruses (zikv) are responsible for recent global health threats. as no preventive vaccines or antiviral drugs against these two re-emerging pathogens are available, we evaluated whether the molecular tweezer clr may inhibit ebov and zikv infection. this small molecule has previously been shown to inactivate hiv- and herpes viruses through a selective interaction with lipid-raft-rich regions in the viral envelope, which results in membrane disruption and loss of infectivity. we found that clr indeed blocked infection of ebov and zikv in a dose-dependent manner. the tweezer inhibited infection of epidemic zikv strains in cells derived from the anogenital tract and the central nervous system, and remained antivirally active in the presence of semen, saliva, urine and cerebrospinal fluid. our findings show that clr is a broad-spectrum inhibitor of enveloped viruses with prospects as a preventative microbicide or antiviral agent. zika virus (zikv) was first isolated in from a febrile rhesus macaque in the zika forest of uganda (dick et al., ) . since then, sporadic zikv infections occurred in africa and asia (haddow et al., ; hayes, ) . until , zikv infection usually has been associated with mild symptoms and thus, the virus had not been considered a threatening pathogen. however, since the recent outbreaks it is evident that zikv causes drastic birth defects, most prominently microcephaly (mlakar et al., ; rasmussen et al., ) , and is associated with neurological disorders, such as guillain-barré syndrome (cao-lormeau et al., ; krauer et al., ) . since , countries and territories reported ongoing zikv transmission (http://www. who.int/emergencies/zika-virus/en/). in brazil alone, . million persons had been infected (shuaib et al., ) , prompting the who to declare zikv a public health emergency of international concern. currently, there are no specific treatments or vaccines against zikv making development of effective preventive measures an urgent public health need (barrows et al., ; paixão et al., ) . zikv is transmitted mainly via mosquito bites but cases of sexual transmission also https have been reported (d'ortenzio et al., ; moreira et al., ; petersen et al., ) . like other members of the flaviviridae family (e.g. dengue virus), zika virions are surrounded by a host-membranederived lipid bilayer containing envelope glycoproteins responsible for cell entry (sirohi et al., ) . ebola virus (ebov) was discovered in near the ebola river in the former zaire. since then, outbreaks sporadically occurred in africa, with the most severe in , reaching epidemic proportions and a death toll of more than , people (centers for disease control and prevention (cdc), ) . ebov belongs to the family of filoviridae, forms enveloped filamentous infectious particles, and causes hemorrhagic fever, a rare and deadly disease with a high fatality rate. in addition to being a global health concern, the virus also is considered a potential biological threat agent. ebov likely is transmitted from infected bats to humans where it may spread through personal contact, contaminated objects, or sexual intercourse (mate et al., ; pandey et al., ) . noteworthy, even after recovery, ebov is found in some body fluids, including semen, up to several months (deen et al., ) . as for now, no licensed vaccine or medicine is available to prevent or manage future ebov outbreaks (sharma and ketki jangid, ) . molecular tweezers are novel drug candidates for the treatment of amyloidosis and related conditions. a lead compound, clr , has been used in many in vitro and in vivo studies to date schrader et al., ) . binding of clr to lysine residues in polypeptides disrupts noncovalent molecular interactions that are important for the abnormal self-assembly that leads to the formation of toxic oligomers and amyloid aggregates (schrader et al., ; sinha et al., ) . in fact, clr prevents assembly and promotes disaggregation of amyloid fibrils that are associated with neurodegenerative diseases ferreira et al., ; prabhudesai et al., ; richter et al., ) . therapeutic effects of clr have been demonstrated in animal models of parkinson's disease (lulla, ; prabhudesai et al., ; richter et al., ) , alzheimer's disease malik et al., ) , familial amyloidotic polyneuropathy (fap) (ferreira et al., ) , and desmin-related cardiomyopathy and was found to be safe in mice at doses -times higher than those showing therapeutic effects . we have recently established that clr also disrupted the formation of amyloid fibrils in semen (lump et al., ) , which enhance hiv- infection (münch et al., ; usmani et al., ) . surprisingly, clr also inhibited hiv- infection through a direct virusinactivating mechanism: the tweezer preferentially bound to raft-rich regions in the viral membrane resulting in the disruption of the lipid bilayer and loss of infectivity. in line with the membrane targeting activity, clr is inactive against non-enveloped viruses such as human adenovirus but destroyed other enveloped viruses, including hepatitis-c virus, human cytomegalovirus, and herpes simplex virus, demonstrating that clr represents a broadly active antiviral compound with prospects for microbicide or drug development (lump et al., ) . thus, we evaluated here whether clr might also block infection of re-emerging enveloped viruses. we show that clr indeed inhibits infection of replication-competent ebov and zikv as well as marburg, rabies, and sars corona virus (sars-cov) pseudoparticles. clr lost antiviral activity in the presence of serum, but remained active in the presence of semen, urine, saliva or cerebrospinal fluid. thus, this broadly active compound represents a promising lead for further development as a microbicide to protect from the sexual acquisition of zikv or ebov, or as a topically applied drug for the therapy of viral infections of the skin, mucosa or the respiratory tract. clr and clr were synthesized as described previously (fokkens et al., ; talbiersky et al., ) and . mm stock solutions were prepared in pbs. zikv envelope protein had been ordered from fitzgerald industries international, acton, usa. vero e cells were used for propagation and infection with zikv as described . tcid values (tissue culture infectious dose ; tcid /ml) were determined according to reed and muench, multiplied with a factor of . to obtain pfu/ml (plaque forming units) and, by normalizing to the number of cells, used to calculate the respective moi (multiplicity of infection). sw (human epithelial colon carcinoma) cells were kindly provided by ninel azoitei (center for internal medicine i, university of ulm, ulm, germany). hff (human foreskin fibroblasts), human glioblastoma (a ) or neuroglioma (h ) cells were kindly provided by jens von einem and karin danzer (ulm university medical center, ulm, germany). hepatic huh- cells were kindly provided by s. pöhlmann (göttingen, germany). the reporter cell line tzm-bl was obtained through the nih arrrp. virus stocks of r -tropic hiv- nl - th were generated by transient transfection of t cells as described (münch et al., ) . methods describing the effect of clr on pseudotyped lentiviral particles ( . .), ebola virus infection ( . .), the detection of zikv infection by a colorimetric mtt assay ( . .) or by cell-based zikv immunodetection assay ( . .), flow cytometry ( . .) and confocal microscopy ( . .) as well as the rna release assay ( . .) and the antiviral activity of clr in body fluids ( . ) can be found in the supplement. we have shown previously that clr inhibits hiv- infection by disrupting the viral membrane (lump et al., ) , suggesting that the antiviral activity of the tweezer is independent of the presence of the viral glycoproteins. to test this hypothesis, we generated luciferaseencoding retroviral particles harboring glycoproteins derived from ebov, the closely related filovirus marburg virus, rabies virus (rhabdoviridae) or sars-cov (coronaviridae). these pseudoparticles were exposed to clr and then added to huh- cells. clr , which lacks hydrophobic side arms and has no anti-hiv- activity (lump et al., ) , served as a negative control. infection rates were determined three days later by quantifying luciferase activity and showed that clr , in contrast to clr , efficiently blocked infection by all tested pseudoparticles (fig. a) . the half-maximal inhibitory concentrations (ic ) of clr against the four pseudoparticles were similar and ranged between . μm for rabies and . μm for marburg virus (fig. a) . these data corroborate our previous findings that clr targets the viral membrane rather than a viral glycoprotein (lump et al., ) . we tested next whether clr blocked infection by the replication-competent bsl pathogen ebov. vero e cells were inoculated with gfp-encoding ebov (ebihara et al., ; hoenen et al., ) that had been exposed to clr or clr . after days, the number of virus-induced plaques revealed that clr , but not clr , reduced the infectious titer in a dose-dependent manner with an ic of . μm (fig. b) . next, we determined the effect of clr on zikv infection. vero e cells were inoculated with the african zikv mr strain that was isolated in from a sentinel rhesus macaque (dick et al., ) in the absence or presence of clr or clr and viral replication was monitored by light microscopy. in the absence of clr , zikv caused a profound cytopathic effect (cpe) as evidenced by large plaques caused by detachment of cells. clr had no effect on this phenotype ( fig. a) . however, exposure to μm clr prevented formation of the virus-induced cpe entirely. to quantitatively assess the antiviral activity, we used a colorimetric mtt assay measuring the virally induced cpe (müller et al., ) . in the absence of compounds or in the presence of clr , zikv resulted in more than % dead (detached) cells. in contrast, clr suppressed cpe formation with an ic of . μm (fig. b ). the mtt assay allows an indirect measurement of zikv infection as the tetrazolium dye is metabolized only in living, non-infected cells. to evaluate the effect of clr on zikv more directly, infected cells were stained for the viral e protein and analyzed by confocal microscopy (schandock et al., ) . upon treatment with or μm of clr , e protein-specific fluorescence could not be detected, whereas clr had no effect, as expected ( fig. c ). lack of e protein expression in cells infected with clr -treated virus was confirmed by flow cytometry (fig. d, supplementary fig. s a ). of note, clr was not cytotoxic at concentrations active against zikv ( supplementary fig. s b ), and did not cause alterations in cell morphology (compare confocal images and dot plots of uninfected cells vs. μm clr exposed cells in fig. c and d). thus, clr prevents zikv infection of vero e cells. we hypothesized that if clr inhibited zikv infection by a similar mechanism to other enveloped viruses, its activity would be directed against the viral membrane (lump et al., ) . indeed, when clr was added to cells, rather than to the virus, and washed out prior to infection, no antiviral effect was observed in a cell-based zikv immunodetection assay (fig. a) . we performed next a time-course analysis in which zikv was exposed to μm clr for to s prior to infection. already after exposure for only s, clr reduced infection by ∼ %. infection declined further with increasing incubation time and was nearly at the level of uninfected cells after s incubation (fig. b) . in agreement with a direct activity against the , rhabdoviridae (rabies virus) or the coronaviridae family (sars-cov), were exposed to indicated concentrations of clr or clr and then used to infect huh- cells. after three days, infection rates were determined by quantifying firefly luciferase activity and subtracting background activity derived from uninfected cells. values represent % reporter gene activities ± sd derived from triplicate infections, normalized to values obtained for cells infected in the absence of tweezers. (b) analysis of replication competent ebov was performed using confluent vero e cells in -well plates. rgebov-egfp was preincubated with clr or clr and mixtures were added to the cells. after days, samples were analyzed by counting the number of plaques per well and calculating the corresponding plaque forming units per milliliter (pfu/ml) for each inhibitor and dilution. displayed values represent the mean of three independent experiments ± sd. ic values were calculated by graphpad prism. one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare the different clr /clr concentrations to cells infected in the absence of compound (* denotes p < . ; ** denotes p < . ; *** denotes p < . ). light microscopy images of vero e cells infected with zikv mr in the absence or presence of μm clr or clr . images were taken days post infection (dpi). (b) zikv mr was incubated with . - μm clr or clr before these mixtures were used to infect vero e cells. after days, when significant cytopathic effects were visible, the number of adherent, viable cells were determined using the mtt assay (müller et al., ) . values represent mean ± sd of percentages derived from triplicate infections. ic was determined using graphpad prism. one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare the different clr /clr concentrations to cells infected in the absence of compound (*** denotes p < . ) (c) confocal microscopy images of vero e cells infected with clr -or clr -treated zikv at day post infection. cells were stained for zikv e protein (green) and nuclei (hoechst, blue) and imaged using confocal microscopy. scale bar: μm. (d) flow cytometry of infected vero e cells. virus was pretreated with pbs, clr or clr and added to cells. h later, cells were fixed, permeabilized, and stained with an anti e protein antibody, and quantified using an alexa coupled secondary antibody. percentages indicate the fraction of protein e positive cells. ic, isotype control. a.e. röcker et al. antiviral research ( ) - virion, the ic of clr increased from . μm to . μm when a fold higher multiplicity of infection was applied (supplementary fig. s ). clr was shown to destroy hiv- membrane integrity through interaction with raft-rich regions in the retroviral envelope (lump et al., ) . in contrast to hiv- , the zikv particle is relatively densely packed with glycoproteins (sirohi et al., ) , which might hamper the interaction of the tweezer with the zikv membrane. to determine whether clr might inhibit zikv infection through direct interaction with glycoproteins, increasing amounts of recombinant viral e protein were titrated to clr , and then these solutions were assayed for anti-zikv activity. as shown in fig. c , even e-antigen concentrations of μg/ml did not affect the antiviral activity of clr , suggesting that the tweezer did not reduce zikv infectivity through binding to the viral e protein. interestingly, we also observed that elevated e-antigen concentrations of and μg/ml reduced infection in the absence of clr (fig. c) , which is likely due to the competition between the virion-associated e protein and the recombinant e protein for cellular receptors. to test whether clr interaction with the viral particle results in loss of membrane integrity, as was shown in the case of hiv- , (lump et al., ) , we measured viral rna genome release. sucrose cushion purified zikv was incubated with clr , clr , pbs or triton x- , as a positive control, for min at °c and the amount of total rna in the solution was determined fluorometrically. as expected, no viral for virus treatment, zikv was incubated with clr for min at room temperature before the mixtures were added to vero e cells. for cell treatment, clr was added directly to the cells; after h, the medium was replaced and the cells were infected with zikv mr . dpi, cell-based zikv immunodetection assay was performed. values represent mean ± sd (n = ). one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare the different clr /clr concentrations to cells infected in the absence of compound (* denotes p < . ; *** denotes p < . ) (b) zikv was incubated for the indicated times with pbs or μm clr before the mixture was added to vero e cells. dpi, cell-based zikv immunodetection assay was performed. values represent mean ± sd (n = ). one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare cells infected with clr -treated zikv to cells infected with zikv that had been incubated with pbs for the same time period (*** denotes p < . ). (c) indicated concentrations of zikv e protein were titrated to μm clr or pbs before zikv was added. mixtures were used to inoculate vero e cells. dpi, the number of adherent, viable cells were determined using the mtt assay (müller et al., ) . the values shown are mean ± sd from triplicate infections. one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare cells treated with different concentrations of zikv e protein and clr to cells that had been treated with the same concentrations of e protein and pbs (*** denotes p < . ). (d) zikv mr was incubated with pbs, μm triton x- , . - μm clr or μm clr for min at °c. samples were inactivated by uv light of a laminar flow for min. then, μl of the samples were used to determine rna concentrations using the quantifluor ® rna system and a quantus fluorometer (promega). background values obtained from control samples using cell supernatant of uninfected cells were subtracted from the respective signals. rna levels of virus stock incubated with pbs were subtracted from these values. data points represent mean ± sd (n = ). one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare samples treated with different clr concentrations, clr or triton x- to pbs-treated virus (*** denotes p < . ). a.e. röcker et al. antiviral research ( ) - rna was detectable when zikv was incubated with buffer or clr (fig. d ). in contrast, incubation with triton x- resulted in readily detectable viral rna suggesting that the detergent lysed the zikv particle. similarly, elevated amounts of rna were detected upon treatment of zikv with clr , demonstrating the physical destruction of the viral particle by the tweezer. of note, clr itself did not act as detergent but targets the viral membrane (lump et al., ) . next, we analyzed whether clr was active against epidemic zikv strains. the fb-gwuh- isolate was derived in from the fetus of a pregnant finnish tourist returning from south america (driggers et al., ) , and the prvabc- isolate represents the current american epidemic strain, isolated in from a puerto rican patient (lanciotti et al., ) . both zikv strains were exposed to clr upon infection of vero e cells. cell-based immunodetection assay and flow cytometry experiments demonstrated that the tweezer suppressed infection of both strains with ic values of . μm for fb-gwuh- ( fig. a and supplementary fig. s ) , and . μm against prvabc- (fig. b) , respectively. as zikv can be transmitted via sexual intercourse, we studied whether zikv infects cells derived from the anogenital region and if so, whether infection can be blocked by clr . zikv effectively infected cell lines derived from cervix (fig. a, supplementary fig. s ) , colon (fig. b, supplementary fig. s ) , and primary foreskin cells (fig. c, supplementary fig. s ). viral infectivity was suppressed by clr but not clr , with ic values in the μm range ( fig. and supplementary fig. s ). because zikv is neurotropic (cao-lormeau et al., ; mlakar et al., ) and clr has been shown previously to penetrate through the blood-brain barrier (bbb) when administered systemically richter et al., ) , we also tested whether clr blocked zikv infection of brain-derived cells. both a glioblastoma and h neuroglioma cells were susceptible to zikv infection and clr entirely abrogated infection at μm (fig. a) . finally, we confirmed these findings obtained in human cell lines using primary murine cerebellar neurons. zikv infected neuronal bystander cells, and this was blocked by clr (fig. b) . there was no apparent toxicity of clr in the primary neurons at this concentration. . . the antiviral effect of clr is abrogated by serum but not urine, saliva, semen, or csf its broad antiviral activity makes clr an interesting lead compound for antiviral prevention or treatment. for a systemic application, clr must retain antiviral activity in blood. to test whether this was the case, clr was diluted in human serum, and then exposed to zikv. as shown in fig. a , serum concentrations of . % and higher abrogated the anti-zikv activity of the tweezer. similarly, serum also abrogated the anti-hiv- activity of clr ( supplementary fig. s ), precluding its use as a systemically applied antiviral drug. in contrast, clr remained active and inhibited zikv infection in the presence of up to % of urine (fig. b) , saliva (fig. c ), cerebrospinal fluid (fig. e ) and % human semen (fig. d) . thus, clr inactivates zikv in the presence of body fluids associated with virus transmission, and could be administered locally to halt virus replication, or applied topically as a microbicide to prevent e.g. sexual virus transmission. thereafter, vero e cells were infected and days later, infection rates were determined via a cell-based zikv immunodetection assay. data points represent mean ± sd (n = ). ic values were determined with graphpad prism. one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare the different clr concentrations to cells infected in the absence of compound (** denotes p < . ; *** denotes p < . ). zikv was incubated for min at °c with . - μm clr or clr . next, mixtures were used to inoculate hela (a), sw (b), or hff (c) cells. days later, infection rates were determined via quantification of the viral e protein using a cell-based zikv immunodetection assay. data points represent mean ± sem (n = ). one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare the different clr concentrations to cells infected in the absence of compound (* denotes p < . ; ** denotes p < . ; *** denotes p < . ). a.e. röcker et al. antiviral research ( ) - . discussion our results establish clr as a broad-spectrum antiviral agent, which not only inhibits infection of established viral pathogens (lump et al., ) , but also of emerging viruses, such as ebov and zikv. we characterized here mainly the effect of clr on zikv, as there is currently no specific antiviral therapy nor a preventive vaccine available. clr blocked zikv infection of primary brain-derived murine cells (fig. b) as well as human cell lines derived from the anogenital region (fig. , supplementary fig. s ) and the brain (fig. a) with ic values between and μm. these values are in the same range as ic values obtained against ebov ( μm, fig. b ) and hiv- ( - μm) (lump et al., ) . several lines of evidence demonstrate that the antiviral activity of clr is directed against the zikv particle itself. first, treatment of cells with clr prior to infection had no antiviral effect (fig. a) . second, the ic of clr increased with increasing viral concentrations ( supplementary fig. s ) . third, the integrity of the zikv particle was lost upon clr treatment, as shown by the release of viral rna from clr -treated virions (fig. d) . these findings were in agreement with our previous observations that clr selectively disrupted viral membranes (lump et al., ) . interestingly, the selectivity stems from clr 's preferential interaction with membranes containing high levels of sphingomyelin and cholesterol, two lipids that are enriched in membranes of enveloped viruses, such as hiv- or ebov (bavari et al., ; brügger et al., ; chazal and gerlier, ; lorizate et al., ) . the selective interaction of clr with lipids that are enriched in the viral but not the cellular membrane may also explain its minimal effects on cell viability ferreira et al., ; lopes et al., ; prabhudesai et al., ; sinha et al., ) (fig. c , d, supplementary fig. s b ). clr is slightly cytotoxic at concentrations of ∼ mm, corresponding to selectivity indices of - , which is in a reasonable range for drug development (buschmann and mannhold, ; food and drug administration, ) . . clr inhibits infection of human and murine brain cells. (a) human glioblastoma (a ) or neuroglioma (h ) cells were infected with zikv in the presence or absence of . - μm clr . two days later, cells were stained for zikv e protein (green) and nuclei (with hoechst; blue) and imaged by confocal microscopy. scale bar: μm. (b) primary murine cerebellum cultures were infected with zikv mr that had been incubated with . - μm clr for min at °c. dpi, cultures were fixed, permeabilized and stained for the neuronal protein βiii tubulin (red), zikv e protein (green) and nuclei (with hoechst; blue). scale bar: μm. clr lost antiviral activity in the presence of serum, precluding application as a systemic antiviral drug. this finding was unexpected because clr has been found previously to be stable in mouse and human plasma, with a half-life of ∼ . h in mice after sc or iv injection . moreover, clr showed therapeutic effects in animal models of alzheimer's and parkinson's diseases malik et al., ; prabhudesai et al., ; richter et al., ) . these data suggest that the loss of antiviral activity in the presence of serum likely was due to binding of clr to serum proteins. the concentrations of clr required to prevent amyloid formation in vitro and in vivo are in the sub-micromolar range, which is - orders of magnitude lower than those needed to block viral infection. presumably, the vast majority of clr was bound to serum proteins and hence unavailable to act on the virus, whereas the minority of the tweezer remains free at concentrations allowing to exert anti-amyloid activity. this interpretation also is supported by the observation that clr retained anti-zikv activity in semen, urine, saliva, and csf. the total protein concentrations in these body fluids are ∼ - orders of magnitude lower than in serum (hu et al., ; vibhakar et al., ) . thus, a larger percentage of clr molecules likely are free in these biofluids and hence antivirally active. clr has been described previously as a novel bi-functional microbicide counteracting sexual hiv- transmission both through directly targeting virus infectivity and by inhibiting the infection-promoting activity of amyloids in semen (lump et al., ) . we show here that the tweezer also inhibits pathogenic zikv infection of cells derived from the anogenital region in the presence of semen. interestingly, both zikv and ebov are present in semen of infected individuals (atkinson et al., ; deen et al., ; moreira et al., ) and can be transmitted by sexual intercourse, similar to hiv- (d'ortenzio et al., ; mate et al., ; moreira et al., ) . thus, application of clr as a microbicide in the vaginal or rectal tracts might protect from acquiring different viral pathogens. interestingly, clr has been shown to penetrate the bbb richter et al., ) , where it may also block replication of neurotropic viruses such as zika or rabies (cao-lormeau et al., ; ludlow et al., ; mlakar et al., ) . however, it needs to be considered that clr may only block cell-free virus infection but not cell-to-cell viral spread, which may limit its utility as therapeutic agent. therefore, the effect of clr on cell-tocell viral transmission needs to be explored. another means of application of clr is topical, e.g., by directly administering clr on virus-infected body surfaces, such as the skin or mucous membranes, to treat, e.g., hsv-induced herpes labialis or genitalis. topical medications may also be inhalational, such as medications against flu or respiratory syncytial virus (rsv), or applied to the surface of tissues other than the . after min of incubation, the mixtures were used to infect vero e cells. days later, infection rates were determined via a cell-based zikv immunodetection assay. data points represent mean ± sem (n = ), except for csf and serum: mean ± sd (n = ). one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare the different clr concentrations to cells infected in the absence of clr but in presence of the same respective body fluid concentration (* denotes p < . ; *** denotes p < . ). a.e. röcker et al. antiviral research ( ) - skin, such as eye drops applied to the conjunctiva, for example, in cmvinduced retinitis. to address the challenge of emerging and re-emerging viral pathogens, it is imperative to develop broad-spectrum classes of antiviral agents as the conventional one-bug-one-drug paradigm is insufficient (zhu et al., ) . current direct-acting antiviral drugs are highly successful but have a narrow spectrum of coverage and are only available against a very limited number of viral pathogens. moreover, drug development is slow and expensive, and it typically takes more than a decade to get market approval. currently, no specific antiviral treatment exists against most, if not all, emerging and re-emerging viruses. broad-spectrum antivirals may offer certain advantages as they reduce time and cost of drug development, allow off-label use, and could be applied even before a viral threat is diagnosed (bekerman and einav, ; zhu et al., ) . clr is broadly active against enveloped viruses as it disrupts lipid bilayers containing elevated levels of sphingomyelin and cholesterol (lump et al., ) , which are typically enriched in viral membranes (bavari et al., ; brügger et al., ; chan et al., ; chazal and gerlier, ; lorizate et al., ) . we have shown that clr inactivates major viral pathogens such as hiv- , hsv- , cmv, and hcv, as well as zikv and ebov. it is highly likely that clr also blocks other enveloped viruses, such as severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) corona viruses, influenza or rsv. most notably, resistance development against clr is very unlikely to occur, as this would require changes in the lipid composition of the cellular and the viral membrane, which is difficult to envisage. in conclusion, clr represents a promising prototype of a broad-spectrum antiviral agent. all data were analyzed using graphpad prism version . for windows, graphpad software, la jolla california usa (www.graphpad. com). the significance level was calculated using one-way one-way analysis of variance (anova) (non-parametric, grouped), followed by bonferroni's multiple comparison 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tweezer broad-spectrum antiviral agents a.e.r. is funded by a fellowship of the landesgraduiertenförderung baden-württemberg and is part of the international graduate school in molecular medicine ulm. j.a.m. is indebted to the baden-württemberg stiftung for the financial support of this research project by the eliteprogramme for postdocs. authors declare to have no conflict of interest. supplementary data related to this article can be found at http://dx. doi.org/ . /j.antiviral. . . . key: cord- -erofmh authors: yang, seung won; jang, yo han; kwon, soon bin; lee, yoon jae; chae, wonil; byun, young ho; kim, paul; park, chan; lee, young jae; kim, choon kang; kim, young seok; choi, seong il; seong, baik lin title: harnessing an rna-mediated chaperone for the assembly of influenza hemagglutinin in an immunologically relevant conformation date: - - journal: faseb j doi: . /fj. rr sha: doc_id: cord_uid: erofmh a novel protein-folding function of rna has been recognized, which can outperform previously known molecular chaperone proteins. the rna as a molecular chaperone (chaperna) activity is intrinsic to some ribozymes and is operational during viral infections. our purpose was to test whether influenza hemagglutinin (ha) can be assembled in a soluble, trimeric, and immunologically activating conformation by means of an rna molecular chaperone (chaperna) activity. an rna-interacting domain (rid) from the host being immunized was selected as a docking tag for rna binding, which served as a transducer for the chaperna function for de novo folding and trimeric assembly of rid-ha . mutations that affect trna binding greatly increased the soluble aggregation defective in trimer assembly, suggesting that rna interaction critically controls the kinetic network in the folding/assembly pathway. immunization of mice resulted in strong hemagglutination inhibition and high titers of a neutralizing antibody, providing sterile protection against a lethal challenge and confirming the immunologically relevant ha conformation. the results may be translated into a rapid response to a new influenza pandemic. the harnessing of the novel chaperna described herein with immunologically tailored antigen-folding functions should serve as a robust prophylactic and diagnostic tool for viral infections.—yang, s. w., jang, y. h., kwon, s. b., lee, y. j., chae, w., byun, y. h., kim, p., park, c., lee, y. j., kim, c. k., kim, y. s., choi, s. i., seong, b. l. harnessing an rna-mediated chaperone for the assembly of influenza hemagglutinin in an immunologically relevant conformation. molecular chaperones are involved both in the facilitation of protein folding and prevention of protein aggregation, which would otherwise lead to cytotoxic consequences ( , ) . conventionally, these pivotal functions have been considered unique properties of chaperones and chaperonins, which are proteins themselves ( ) . nonetheless, recent reports suggest that rna molecules can also function as chaperones and are extremely effective in executing the folding of a variety of proteins both in vivo and in vitro ( , ) . the potential role of rnas in modulating aggregation and amyloid formation has been reported for the p tumor suppressor ( ) . moreover, chaperone activity has been discovered as intrinsic to some ribozymes, which are rna molecules that function as catalysts: both in the m rna ribozyme that is responsible for the maturation of trnas and in the large rrnas that function as peptidyl transferase during ribosome-dependent protein synthesis ( ) ( ) ( ) . being distinct from the rna chaperone, which is a protein that facilitates structural alteration of rna molecules, the term for rna as a molecular chaperone (chaperna) refers to an rna molecule that serves as a chaperone for the folding of client proteins ( ) . nevertheless, neither the extent of its involvement in the folding and homeostasis of normal cellular proteins nor its efficacy in the folding of difficult-to-express proteins has been explored. it is noteworthy that the proper use of molecular chaperones for the in vivo folding of recombinant proteins in escherichia coli has been reported only in isolated cases, and no study of considerable size that has shown broad efficacy has been conducted ( ) . therefore, the possibility that the moonlighting activity of rnas as chaperones beyond the known canonical cellular functions could be used as an efficient vehicle for the folding and assembly of proteins requires dedicated exploration. with respect to recombinant vaccines, the assembly of monomeric antigens into oligomeric structures is crucially important for the ligation of b cell receptors to enhance immunogenicity and to induce relevant neutralizing (nt) antibody responses toward protection ( , ) . in the present study, we show that a chaperna is remarkably effective in the folding and trimeric assembly of influenza hemagglutinin (ha) into an immunologically activating conformation. a convenient avenue for harnessing the chaperone function is to fuse genetically the target protein of interest with an rna-interacting domain (rid) as a docking tag that enables interaction with cellular rnas ( ) . a judicious choice of the rid is required for a biomolecule to function as a chaperone without physically interfering with the oligomeric structure of the target antigen. therefore, the rid should preferably be small and sufficiently flexible not to interfere with the multimeric assembly of the target antigen, and moreover, it should be intrinsically nonimmunogenic ( ) . in this study, a small trna-binding domain of lysyl-trna synthetase (lysrs; aa positions - ), which was derived from the host being immunized, was selected as the transducer for the chaperna function of cellular rnas. the n-terminal domain is intrinsically disordered but can switch from an unfolded structure into an a-helical conformation in vitro after trna binding ( , ) . this domain has been shown to interact in vitro with nonspecific trnas ( ) . by exploiting the chaperna function, herein, we demonstrate, for the first time, to our knowledge, that influenza ha can be assembled into a soluble, trimeric, and immunologically relevant conformation. the influenza ha globular domain (hagd), including the host receptor-binding domain, was produced predominantly in the trimeric form, and remarkably, mutations that affect the trnabinding domain rendered the trimer assembly defective. immunization of mice with this purified ha elicited a high degree of hemagglutination inhibition (hi) and high titers of nt antibodies without crossreaction with the selfrid and provided a sterile protection against a lethal challenge. pandemics and annual influenza epidemics have been the causes of considerable mortality and a heavy burden on human health ( , ) . the speedy and timely delivery of vaccines is crucial for effective responses to pandemics. conventional systems that rely on virus culture, either from embryonated eggs or cell cultures, are not likely to meet the time requirements. taking advantage of the novel concept of protein folding, as presented herein, should facilitate the development of pandemic vaccines. rna viruses are responsible for most of the emerging and re-emerging viral infections ( ) , and the chaperna described herein may offer a novel prophylactic and diagnostic platform for the effective control and management of these infections. basically, different rna-interaction domains-rid, nterminal domain of lysrs (lysn), and lysrs-were used here for construction of expression vectors. n-terminal appendage ( aa long) of human lysrs, which is known to interact with nonspecific trna in vitro, served as rid of human origin (hrid). for comparison, lysrs from e. coli (elysrs; . kda) or lysn ( . kda) were also used ( ) . expression vectors of wild-type (wt) hrid and rna-nonbinding mutant hrid (hridmu) were constructed in versions. the first mutant has aa substitutions (k a and k a), the second mutant carries substitutions with alanine (k a, k a, r a, k a, k a, and k a), and the third mutant contains substitutions with alanine (k a, k a, r a, k a, k a, k a, k a, k a, and k a). subsequently, the hagd, originating from influenza a/puerto rico/ / (pr ; h n ) virus, was inserted between the kpni and hindiii restriction sites to construct the hridmu-hagd. promiscuous gene expression (pge)-rid, derived from the pge-lysrs vector, served as an expression vector in e. coli ( ) . first, the lysrs gene of the pge-lysrs vector was deleted by means of restriction enzymes ndei and kpni. the genes of rids of human, murine, and rabbit origins were amplified by pcr, resulting in hrid, mouse rid (mrid), and rabbit rid (rrid); each was inserted into pge-lysrs (separately). the rid expression cassette is composed of an rid, a d linker (hexaaspartic acid sequence) to aid soluble expression, an alternating serine/glycine space linker, a tobacco etch virus (tev) protease recognition cleavage site (enlyfq), a multiple cloning site (mcs; gtgsdivdkl: kpni/bamhi/ecorv/sali/hindiii), and a hexahistidine tag ( xhis) for nickel (ni)-affinity purification, under control of the t promotor ( ) . hagd cdna of the pr (h n ) strain was amplified by rt-pcr and was inserted between the kpni and hindiii sites of the pge-rid vector. all of the constructs were designed to yield fragments-rid and hagd-after cleavage with the tev protease. hrid served as a template for mutagenesis of amino acid residues involved in rna binding. all of the recombinant proteins were expressed in an e. coli strain bl star (de ) plyss (invitrogen, carlsbad, ca, usa) and were purified from the soluble fraction of cell lysates. the primary culture was grown overnight in ml of the luria-bertani medium containing mg/ml ampicillin and mg/ml chloramphenicol at °c. large-scale culture was implemented in ml of the luria-bertani medium that was inoculated with ml culture broth and incubated at °c until optical density at nm (od ) of . - . was reached. protein expression was induced by the addition of mm isopropyl b-d- -thiogalactopyranoside at various temperatures. cells were harvested at g when an od of . - . was reached. the cell lysates obtained by sonication were centrifuged at , g for min, and this procedure separated them into supernatant and precipitate fractions. proteins were purified by ni-affinity chromatography. each soluble fraction equilibrated in buffer a [ mm tris-hcl (ph . ), mm nacl, % glycerol, mm -me, . % triton x- , and mm imidazole] was applied to an ni-nitrilotriacetic acid resin (ge healthcare, little chalfont, united kingdom) in a column pre-equilibrated with buffer a. after a wash with buffer a, proteins were eluted with a linear gradient of imidazole - mm by means of buffer b (buffer a supplemented with mm imidazole). each fraction was analyzed by sds-page, and the fractions containing proteins of interest were pooled, concentrated by centrifugal filters (centriprep; emd millipore, billerica, ma, usa), and then dialyzed against buffer c [ mm tris-hcl (ph . ), mm nacl, . mm edta, and . % tween ] . after sds-page, followed by coomassie staining, the concentration of purified proteins was determined using bovine serum albumin (bsa; amresco, solon, oh, usa) of known concentrations as a standard. the hagd without rid fusion formed insoluble aggregates, which necessitated refolding in vitro. the e. coli lysate was centrifuged, and the inclusion bodies were washed with % triton x- . the pellets were denatured and solubilized with m guanidine hydrochloride, supplemented with mm dtt at a final protein concentration of mg/ml. the solution containing the monomeric hagd was dialyzed against mm tris-hcl (ph . ) to remove the denaturation agents. the extent of refolding into oligomeric assembly was next analyzed by size-exclusion chromatography (sec). to predict the potential trimerization capacity of the mrid-hagd recombinant protein, the crystal structures of influenza pr (h n ) ha (protein data bank id: ru ) and the nterminal domain of brugia malayi asparaginyl-trna synthetase (protein data bank id: kqr) were retrieved from http:// www.rcsb.org/. all heteroatoms and water molecules were eliminated from the structure. with the use of homology modeling by modeler (https://salilab.org/), the rid-hagd fusion version, including a -aa linker (ddddddsgen-lyfq), was built, and the asparaginyl-trna synthetase structure served as a template for mrid and rrid ( , ) . the trimerization of each mrid-hagd and rrid-hagd structure was confirmed by cluspro . protein-protein docking (boston university, boston, ma, usa) ( , ) . the lowest energy of an mrid-hagd trimer was . kcal/mol, and for the rrid-hagd trimer, it was . kcal/mol. the final structures were visualized by means of a surface model in the university of california, san francisco (san francisco, ca, usa) chimera software. to confirm the folding and stabilization effects of rna on soluble proteins, cell lysates with a recombinant protein, mrid-or rrid-hagd, were obtained by sonication or b-per lysis (bacterial protein extraction reagent, ; thermo fisher scientific, waltham, ma, usa). cell lysates (t) were centrifuged at , rpm for min to obtain soluble (s) and pellet (p) fractions. rnase a ( mg/ml; ; intron biotechnology, seongnam, korea) was added to the soluble fraction and incubated for min at °c to deplete this fraction of rna. the mixture (st) was centrifuged at , rpm for min and thus, separated into the supernatant of soluble fraction (ss) and the precipitate of soluble fraction (sp) fractions. the soluble fraction (s) without rnase a served as a control. all of the fractions obtained were identified by sds-page analysis in a % gel. the oligomeric status of purified recombinant proteins was analyzed by sec at °c on a superdex- analytical gel-filtration column (ge healthcare). the column was equilibrated with a buffer [ mm tris-hcl (ph . ), mm nacl, mm -me, and . % triton x- ] and was calibrated using broad-range mw markers: ferritin ( kda), aldolase ( kda), conalbumin ( kda), ovalbumin ( kda), and blue dextran ( kda; ge healthcare). for this assay, ml fetuin (sigma-aldrich, st. louis, mo, usa) per well at mg/ml was used for coating -well nunc plates (thermo fisher scientific) and was incubated at °c overnight. the plates were washed with pbs containing mm tris-hcl (ph . ), with . % tween (pbst), and were blocked with % bsa in pbst for h at room temperature. next, ml of each recombinant hagd protein at various concentrations was added into each well, and then the plates were incubated for h at room temperature, followed by washing with pbst. recombinant enhanced green fluorescent protein (egfp) (or rid) served as a control protein. after that, ml of an anti- xhis tag antibody conjugated with horseradish peroxidase (hrp; thermo fisher scientific) was added into each well at a dilution of : and was incubated for h at room temperature. after a wash with pbst, ml of the hrp substrate solution (bd biosciences, san jose, ca, usa) was added into each well, and then the plate was developed in the dark for min. the colorimetric reaction (blue to yellow) was stopped by addition of ml/well n h so , and od was measured on a microplate reader (fluostar optima; bmg labtech, ortenberg, germany). all of the animal experiments were carried out in strict accordance with the recommendations of the korean food and drug administration. immunization of rabbits was conducted at animal facilities of young-in frontier (gasandong, south korea). namely, . mg of an rrid fusion protein emulsified in freund's complete adjuvant was injected intradermally at multiple sites on the back of each rabbit. starting from wk after the first immunization, each animal was boosted twice with the same protein-adjuvant mixture every wk. rabbit blood was collected wk after each boost and was centrifuged to prepare serum and stored at °c. the experimental protocol was reviewed and approved by the institutional animal care and use committee (iacuc) of young-in frontier. mouse experiments were carried out on -wk-old female balb/c mice (orient bio, seongnam, south korea). every group of mice ( - /group) was immunized via the intraperitoneal route with - mg of a purified rid-fused recombinant protein or hagd without rid, along with mg imject alum adjuvant (thermo fisher scientific) on d (primary), (first boost), and (second boost). after the first immunization, each mouse was boosted twice, at wk apart. pbs-immunized mice served as a control. blood samples were collected via transorbital bleeding, d after each boost, and serum was prepared, aliquoted, and stored at °c. the protocol of the experiment was evaluated and approved by the iacuc of the yonsei laboratory animal research center two weeks after the secondary immunization, all of the mice were challenged intranasally with time ld [i.e., plaque-forming units (pfus), of the pr (h n ) virus under anesthesia with avertin or mock infected with pbs as a control]. the survival rate and changes in weight of the challenged mice were measured every day for wk. after the virus challenge, mouse blood samples were collected via transorbital bleeding and were centrifuged at °c for min to prepare serum samples. to this end, -well nunc plates (thermo fisher scientific) were coated with ng/well rid, an rid-fused protein, or pfu/well pr (h n ) virus and were incubated at °c overnight. the plates were washed with pbst and were blocked with % bsa in pbst for h at room temperature. next, ml serum samples, at various dilutions, was added into each well, and the plates were incubated for h at room temperature, followed by washing with pbst. one hundred microliters of a secondary goat anti-rabbit igg antibody or goat anti-mouse igg antibody conjugated with hrp (sigma-aldrich) was added into each well at a dilution of : , and was incubated for h. after a wash with pbst, ml of the substrate , , , -tetramethylbenzidine solution (bd biosciences) was added into each well, and then the plate was developed in the dark for min. the colorimetric reaction (blue to yellow) was stopped by addition of n h so ml/well, and od was measured on a microplate reader (fluostar optima; bmg labtech). this analysis was carried out by means of the final serum samples from immunized rabbits and mice. various dilutions of each serum were used for the analysis of the antibodies in the serum samples. as secondary antibodies, a goat antirabbit igg mab or goat anti-mouse igg antibody conjugated with hrp (sigma-aldrich) was applied. for the detection of antibodies against ha in serum samples of pr (h n ) virusinfected mice, mg of an rid-hagd protein was loaded onto a gel for sds-page, followed by western blot (wb), including mouse serum (dilution of : ). the protein gels were blotted in the trans-blot turbo transfer apparatus with pvdf midi transfer packs (bio-rad laboratories, hercules, ca, usa). the pvdf membranes were immediately transferred to a blocking solution- % skim milk in tris-buffered saline/tween (tbst)-and were incubated with gentle agitation for h at room temperature. after a wash with tbst, the membranes were incubated for h or overnight with gentle agitation at °c in ml of the primary-antibody solution with rabbit or mouse serum ( : dilution) and % bsa in tbst. the blots were rinsed times with tbst and were incubated in ml of a secondary-antibody solution containing an hrp-conjugated goat anti-mouse igg antibody (bio-rad laboratories; : , dilution) or an hrp-conjugated goat anti-rabbit igg antibody (bio-rad laboratories; : , dilution) in pbst for min with gentle agitation at room temperature. the membranes were washed with tbst, followed by development by means of an ecl detection reagent (bio-rad laboratories) for min. after that, images were captured on x-ray film using a developing and fixing solution in a darkroom. blood samples were collected from mice by transorbital bleeding and were allowed to clot at room temperature. the clots were removed, and the samples were centrifuged in a microcentrifuge at °c for min at rpm. the clarified serum samples were transferred to sterile microcentrifuge tubes and were heat inactivated at °c for min. the heatinactivated serum samples were subjected to -fold serial dilutions in -well plates with minimum essential medium (thermo fisher scientific). for the analysis of titers of nt antibodies against the influenza virus, equal volumes ( ml) of the influenza virus ( pfus) were added to the diluted serum samples and mixed. the -well plates containing virus and serum samples were incubated at °c for h. these mixtures were then adsorbed to -well plates containing confluent madin-darby canine kidney cells. the agaroverlaid plates were incubated at °c in a % co incubator. the cell monolayers were stained with crystal violet at d postinfection. in each assay, the serum samples were diluted and analyzed in duplicate, and each assay was performed at least twice. nt titers were calculated from dilutions that correspond to a % plaque reduction compared with the control. serum samples were treated with a receptor-destroying enzyme to remove nonspecific agglutination inhibitors and heated at °c for h. the serum samples were subjected to serial -fold dilutions with pbs in -well plates. equal volumes ( ml) of an influenza virus solution ( hemagglutination units) were then added to the diluted serum samples and mixed; next, the plate was incubated at °c for h. after that, an equal volume of a % chicken erythrocyte suspension was added with incubation at °c for h. the hi antibody titer of each serum was expressed as the reciprocal of the highest dilution of the sample that completely inhibited hemagglutination. six-week-old female balb/c mice (orient bio) were anesthetized before the intranasal infection with ml of a virus suspension and euthanized by cervical dislocation to minimize suffering. the viral replication in the respiratory tract was measured in the lungs. whole lungs were excised for viral titration. the whole lungs were homogenized with a homogenizer in pbs and centrifuged to remove cell debris. these supernatants were transferred to a new tube and stored at °c until analysis. schematic presentation of expression vectors is given in fig. . as initial screening for soluble expression, a variety of fusion vectors-pge-lysrs ( ), pge-rid , and pge-lysn -was tested (fig. a) . lysrs, which is composed of lysn and a c-terminal domain, was derived from e. coli (fig. b) . lysrs of eukaryotic origin carries an extra n-terminal domain (rid) that is absent in the e. coli counterpart (fig. b) . as a target antigen, the hagd of strain pr (h n ), containing the receptor-binding site, was used (fig. c, d) . as an rid, the most n-terminal trna-binding domain of ; aa from lysrs was derived from each of specieshumans, mice, and rabbits-to obtain hrid, mrid, and rrid, respectively, and hridmu (fig. b) . in addition to rids, the lysn (amino acid positions - ) and the whole lysrs from mice and rabbits were cloned as fusion partners for the soluble expression of hagd and other test proteins. the fusion linker contains the enlyfq sequence for the site-specific cleavage by tev protease, if needed (fig. d) . the schematic diagrams of ha trimers in the mature virion and of the recombinant rid-hagd fusion protein are shown in fig. e . according to some reports, the solubility of fusion proteins is somewhat influenced by the location of the fusion tag ( , ) . the n-terminal fusion of mrid increased water-soluble expression by ; % compared with the c-terminal fusion (supplemental fig. b ). in addition, the n-terminal fusion protein mrid-hagd was eluted mainly in a trimeric form, whereas the c-terminal fusion protein hagd-mrid was eluted at ; % in a dimeric state (supplemental fig. d) . therefore, all fusion tags in this study were placed in the n-terminal part of target proteins. expression at lower temperature increased the solubility of the proteins (fig. b-f) , in agreement with findings that the slowing down of translation enhances the proper folding and solubility of proteins ( ) . as a control, direct expression of gfp or bone morphogenetic protein (bmp ) without fusion to an rid yielded predominantly a precipitate of insoluble aggregates under all of the temperature conditions tested (fig. d ). initial screening of lysrs proteins for solubility revealed that the elysrs had superb solubility compared with the counterparts of mouse or rabbit origin ( fig. a) . parallel analyses showed that the rids of all species are produced predominantly in the soluble form (. % for hrid and mrid and ; % for rrid, respectively; fig. b ). of note, the rid equivalent is absent in e. coli lysrs (fig. b) and therefore, could not be tested. thus, the rids of human and animal origins were further explored as a docking tag for potential soluble expression of target proteins: bmp , gfp, and hagd (fig. d, e) . as shown in fig. e , unlike the hagd without rid, hagds as a fusion to an rid of varied origin were expressed as soluble proteins with a good yield ( - % of total protein). the solubility of all of the recombinant proteins was greatly enhanced (by ; - %) compared with direct expression without fusion (fig. d, e) . overall, the expression of fusion partners of mouse origin (mrid, mlysn, mlysrs) was higher than the expression of fusion partners of rabbit origin, but their solubility levels were similar ( fig. a , c, e, f). despite minor differences, each rid fusion showed a strongly increased total soluble yield of the respective proteins. because there were no significant differences between lysn and an rid in promotion of the solubility of a reporter protein, rids were chosen as a fusion tag for the rest of the experiments: an rid of small size (; kda) is not expected to interfere physically with trimeric assembly of hagd and as a docking tag, is less immunogenic than lysn (; kda) or lysrs (; . kda; fig. a-c) . thus, mrid-hagd and rrid-hagd were chosen as immunogens for inoculation of mice or rabbits, respectively. in parallel, egfp was used as a reporter for solubility as a fusion to rid, where its solubility ratio was ; % at °c (fig. g ). the proteins were purified from ml culture by -step ni chromatography. after cell lysis and centrifugation, the soluble fraction containing the bulk of recombinant proteins was loaded onto the ni column, and proteins were eluted with a linear gradient of imidazole, ranging from to mm. rid-hagd proteins with the xhis tag were purified effectively from the soluble fraction. the sds-page analysis revealed that the major protein band corresponds to the expected molecular mass of ; kda ( fig. e, g) . rid-fused egfp with the xhis tag was also purified in abundance from the soluble fraction as a control. fractions enriched in rid-hagd (fig. h ) or rid-egfp (fig. i ) were pooled and concentrated by centrifugal filters and then quantified. the final concentrations of purified proteins were ; and . mg/ml (mrid-hagd and rrid-hagd, respectively). the final concentrations of purified rid-egfps were ; and . mg/ml (mrid-egfp and rrid-egfp, respectively). likewise, the final concentrations of proteins without fusion to mrid after chemical refolding were ; . and . mg/ml for the hagd and egfp, respectively. all of the recombinant proteins were dialyzed against pbs. the oligomeric state of rid-fused proteins and of the hagd without rid was determined by sec at °c on a superdex- increase column (ge healthcare), and the mw of the eluted fractions was estimated by calibration with marker proteins of known size (fig. ) . based on the elution volume and partition coefficient (k av ), the mw of mrid-hagd was estimated to be . kda. likewise, rrid-hagd showed an elution volume of . ml and the estimated size of . kda (fig. a) . of note, most of the protein was eluted as a single peak, without any peaks in void volume, suggesting that the protein is predominantly in a defined conformation without any insoluble aggregates of ill-defined conformations. according to the elution pattern, the rid-fused hagd proteins were predominantly in the trimeric state, resembling the assembly status of the whole ha protein in an influenza virus. the estimated size of trimeric rid-hagd ( - kda) is slightly bigger than the calculated value ( kda = kda). the flexible nature of the fusion junction and the elongated nature of the rid are expected to cause slight overestimation of the overall size of a fusion protein ( , ) (fig. e) . sds-page analysis also confirmed that the bulk of rid-hagd proteins was present in the trimer fraction. apparently, the bound trnas dissociated and failed to be copurified with the ha proteins during the purification process, probably owing to relatively low affinity for rid ( ) . serving as a control, mrid-egfp was eluted at elution volumes . and . ml, indicating a mixture of oligomeric (; %) and monomeric (; %) conformations, with the estimated size of the monomer . kda, close to calculated . kda (fig. a) . elysrs-hagd ( . kda) was eluted as a mixture of oligomeric ( %) and dimeric ( %; estimated as . kda) conformations (fig. b ). according to the elution pattern, figure . determination of the oligomeric status of fusion antigens by gel-filtration chromatography and a fetuin-binding assay. a) mrid-hagd and rrid-hagd were eluted in trimeric form on the basis of k av , followed by analysis of rid-hagd by sds-page. mrid-egfp, as a control, was eluted as a mixture of oligomeric ( %) and monomeric ( %; estimated at . kda) forms. b) lysrs-hagd, as a control, was eluted as a mixture of oligomeric ( %) and dimeric ( %; estimated as kda) forms. c ) the refolded hagd without rid was eluted predominantly in monomeric form ( %; estimated at . kda). d) the glycoprotein (fetuin), which contains terminal sialic acid residues as a receptor for ha, was adsorbed to a microplate and analyzed for binding to mrid-hagd, hagd without rid, and elysrs-hagd. the mrid-egfp protein served as a negative control. all of the data were obtained in duplicate. elysrs-hagd protein was a mixture of oligomers and the dimer in solution. the hagd without rid, refolded from inclusion bodies ( . kda), was eluted mainly as a monomeric conformation (fig. c) . the results showed the efficacy of the rid in promoting the folding and assembly of its reporter ha protein into the native trimeric conformation. the recombinant hagd proteins were evaluated regarding their capacity for binding to terminal sialic acid residues by an elisa. trimeric ha glycoproteins are responsible for binding of the influenza virus to sialic acid on the host cell membrane for initiation of the infection cycle. fetuin is a glycoprotein that contains sialic acid and can be used in a convenient assay for evaluation of binding of a functional trimeric assembly of an ha protein ( ) . in this assay, the quality of ha folding could be assessed regarding receptor binding at the initial stage of viral infection. our results showed that the e. coli-derived recombinant ha, which is not glycosylated, binds to fetuin (fig. d) , suggesting that glycosylation is not important for the folding of ha ( ) . of note, the mrid-hagd trimer manifested stronger binding than did monomeric hagd (without rid) obtained by refolding (fig. d ). elysrs-hagd, which is predominantly in a dimeric form, failed to bind, as was the case for mrid-egfp, which was thus selected for negative controls (fig. d) . consistent with other reports ( ), the receptor-binding activity of mrid-hagd reflects its proper folding and assembly into the biologically relevant trimer conformation. the effect of rna on the solubility of antigens depletion of rna by rnase a was performed to test the potential effect of rnas on the solubility of proteins. the total e. coli lysate (t) was centrifuged to prepare two fractions: the soluble (s) and pellet (p). subsequently, the total of soluble fraction (st) was treated with rnase a and centrifuged again to obtain the supernatant of soluble fraction (ss) and the precipitate of soluble fraction (sp). solubility of the protein was greatly reduced by rnase a treatment for both mrid-hagd and rrid-hagd, according to coomassie staining (fig. a) . the results were further substantiated by the semiquantitation of protein bands in wb by densitometric scanning (s/p , %; ss/sp without rnase a , %; ss/sp with rnase a , %; fig. b ). this finding showed that rna plays an important role in the maintenance of the solubility of ridfused ha proteins. to address further the rna dependence of folding and assembly, mutations were introduced into key amino acid positions in hrid that are crucially involved in trna binding. the choice of mutations was guided by detailed reports on mutagenesis ( ) . cumulative mutations (lysine or arginine to alanine) were introduced into each site to generate three mutants-hridmu , hridmu , and hridmu -carrying mutations at , , and all sites involved in rna binding, respectively (fig. b) . all of these proteins were expressed in soluble form, purified by ni-affinity chromatography, and subjected to sec for the analysis of trimeric assembly. all mutants showed enhanced solubility compared with the wt hrid-hagd protein at °c (fig. c) , although the solubility levels were not noticeably different at °c. all of these proteins were purified by -step ni-affinity chromatography (fig. d) . remarkably, sec data revealed that as the number of mutations increased, the proportion of trimeric assembly decreased, with a corresponding increase in the amount of the oligomer in the void volume (fig. e) . these results strongly indicated that the interaction with rna is indeed required for the assembly of the ha antigen into the immunologically relevant conformation. the purified proteins were applied to antibody production in mice and rabbits. after immunization with an rid-hagd or rid-egfp fusion protein, antiserum was obtained, and an elisa was performed to estimate the amount of a specific antibody against the docking tag and the reporter proteins, respectively (fig. a-f) . elisa data confirmed that immunization elicited robust antibody responses against target reporter antigen hagd or egfp but much less toward the rid docking tag. first, irrespective of the reporter protein, the elisa signal was much stronger against fusion proteins than against the rid itself (for rrid, fig. a , b for egfp and hagd, respectively; for mrid, fig. c , e for the respective proteins). second, for the same reporter antigen, the elisa signal was much stronger for the reporter antigen regardless of the source of the docking protein (compare fig. e with f for the hagd and fig. c with d for egfp). third, the same conclusions were drawn, regardless of the animal species to be immunized (compare fig. a with b for rabbits and fig. c -f for mice). it should be noted that specificity of the antibody response to a reporter protein relative to the rid docking protein was not appreciably different regardless of the origin of an rid and the animal species to be immunized (compare fig. a with d for egfp and fig. b with f for the hagd), probably as a result of high homology (; %) in an amino acid sequence between the murine and rabbit counterparts. in contrast, data from the elisa involving elysrs-hagd as an immunogen showed a similar magnitude of the responses to fusion proteins elysrs and elysrs-hagd, suggesting that the immune response was directed predominantly against the elysrs docking tag with a negligible response to the desired hagd region (fig. g) . high immunogenicity of elysrs may be because of low sequence homology of lysrs between the mouse and e. coli (; %). in addition, wb analysis was carried out to examine the binding specificity of the antibodies produced against recombinant proteins. after treatment with the tev protease, the xhis tag and the peptides corresponding to the mcs were expected to be removed from an rid and rid-hagd (fig. d) . accordingly, the antibody against rid-hagd bound specifically to the hagd (cleaved) and rid-hagd (remained uncleaved) but not to the rid docking tag alone (cleaved; fig. a -f and supplemental fig. a-d) . the full-spectrum analysis of the antibody specificity is shown in wb data in supplemental fig. a -e. the rid fusion tags, rid-egfp and rid-hagd, were digested with tev, resulting in cleavage products of the expected fig. a ). for convenience, protein fragments, with or without the his tag + mcs peptide moiety, are indicated with black and white arrows, respectively. rrid was detected by rabbit antiserum raised against rrid fusion proteins but failed to be detected after tev treatment (supplemental fig. b ). these results suggested that the positive detection, albeit a weak one, by wb was a result of the immune response to the his tag + mcs moiety, rather than to the self-rrid docking tag. to determine whether anti-histidine (his) tag antibodies raised against protein could bind to the his tag attached to another (irrelevant) protein, we performed an additional wb analysis. serum samples from rrid-hagdimmunized rabbits showed an immunoreaction with the egfp protein, and conversely, serum samples from mrid-egfp-immunized mice showed an immunoreaction with the hagd protein (supplemental fig. e ). given that only the his tag was a common region between rrid-hagd figure . analysis of differences in antibody production levels by an elisa and wb. a) the difference in antibody levels (according to an elisa) between groups rrid and rrid-egfp relative to the serum samples from rabbits (n = ) immunized with rrid-egfp; (lower) wb analysis for the detection of antibodies in the rabbit serum samples. b) differences in antibody levels between groups rrid and rrid-hagd relative to the serum samples from rabbits (n = ) immunized with rrid-hagd; (lower) wb analysis for the detection of antibodies in the rabbit serum samples. c-f ) differences in antibody levels between groups rid and rid-fused protein relative to the serum samples from mice (n = ) immunized with an rid-fused immunogen; (lower) wb analysis for the detection of antibodies in the mouse serum samples. g) differences in antibody levels between groups elysrs and elysrs-hagd relative to the serum samples from mice (n = ) immunized with elysrs-hagd. error bars indicate the sd of each cohort. the x-axis indicates reciprocal serum dilution (log ). the initial serum dilution value was / and / for mice and rabbits, respectively. and egfp and between mrid-egfp and hagd, it is likely that the bands indicated by black arrows represent a specific interaction between the anti-his tag antibody and the his tag. likewise, in rrid-egfp or rrid-hagd, the predominant response was directed against the egfp or hagd reporter proteins, rather than rrid. likewise, mrid was weakly reactive with the murine antiserum against mrid-egfp (supplemental fig. c ), but the reactivity disappeared after tev cleavage (white arrow), suggesting that the self-mrid was not immunogenic in mice. the lack of crossreactivity between mouse antiserum and rrid of rabbit origin was probably a result of high homology of rids between the host species (supplemental fig. c, d) . therefore, the weak but distinct elisa response to the rid docking tag (fig. ) is not actually a result of immunogenicity of the rid but of the peptides artificially introduced for affinity purification and cloning purposes. this finding indicates that the rid docking tag itself is nonimmunogenic, and the immune response was aimed predominantly at the desired viral antigen after immunization. the wb analyses, along with the elisa, confirmed that rid can serve as an excellent enhancer of folding and assembly of a target antigen without compromising the specific immune responses. mice were infected intranasally with a sublethal dose ( pfus) of the pr (h n ) virus to obtain antiserum against the pr (h n ) virus. four weeks later, blood was drawn from each mouse via transorbital collection, and serum was prepared. all of the mice survived with a maximal weight loss of % by d postchallenge (dpc) and recovered slowly. furthermore, wk later, blood was collected from the recovered mice, and the resulting serum was subjected to elisa and wb analyses (fig. a, b and supplemental fig. a) . the results confirmed that rid-hagd (trimeric form; fig. a ), as a coating antigen, was detected by highly diluted antisera from infected mice (n = ), whereas the refolded hagd without rid (monomeric form; fig. c ) was detected at a much lower dilution of the antisera (; -fold lower) than rid-hagd was. the rid docking tag, as a negative control, failed to show any figure . detection of anti-influenza virus antibodies in mouse serum samples by means of the recombinant viral proteins and the pr (h n ) virus. a, b) elisa and wb data revealed that the recombinant proteins, rid-hagd, were detected by highly diluted antibodies against the pr (h n ) virus in serum samples from infected mice (n = ), whereas the refolded hagd without rid as a coating protein (compared with rid-hagd) was detected only at much lower dilution ( -fold or ; -fold lower) of the antisera, and the rid docking tag failed to show any immunoreactivity. c ) elisa data for detection of antibodies in serum samples from mice infected with various viruses (n = ). as a coating antigen, the recombinant mrid-hagd protein and coldadapted x- -based genetic reassortants (carrying internal genes from cold-adapted x- virus and the surface ha and neuraminidase genes from wt viruses) were used as the source of the ha antigen. nc, new caledonia; pa, panama; id, indonesia; kr, korea; b/y, influenza b virus yamagata. d) elisa data showing that according to the number of boosts, high-titer antibodies in serum samples from mice (n = ) immunized with mrid-hagd ( mg/mouse) bound to the pr (h n ) virus ( pfu/well). error bars indicate the sd of each cohort. the x-axis indicates reciprocal serum dilution (log ). the initial serum dilution value was / . e ) elisa data showing that regardless of the number of boosts, antibodies in serum samples from mice (n = ) immunized with the hagd without rid ( mg/mouse) hardly bound to the pr (h n ) virus ( pfu/well). error bars indicate the sd of each group. the x-axis indicates reciprocal serum dilution (log ). the initial serum dilution was / . immunoreactivity (fig. a, b) . wb then confirmed that the antibody response was directed against hagd, whereas rid, either before or after tev cleavage, failed to react with the antiserum (supplemental fig. a) . these results indicated that recombinant rid-hagd may serve as a diagnostic antigen with high specificity and sensitivity for influenza infection. in addition, we compared elisa reactivity among various antisera from mice infected with different subtypes of influenza strains. as expected, the highest reactivity was seen with the homologous pr (h n ) antiserum (fig. c) . potential crossreactivity with different ha antigens was examined using cold-adapted x- -based reassortant strains carrying internal genes from the cold-adapted x- virus and the genes encoding surface proteins ha and neuraminidase from wt viruses ( , ) . lower reactivity was observed with heterologous virus new caledonia (nc; h n ) or heterosubtypes, including panama (pa; h n ), indonesia (id; h n ), or korea (kr; h n ; fig. c and supplemental fig. b) . alternatively, to test whether the antibody elicited by the rid-hagd vaccine binds to the homologous pr (h n ) virus, we conducted an elisa with the antisera from mice immunized with mrid-hagd or hagd without rid (as a control; n = ), by means of the pr (h n ) virus as a coating antigen (fig. d, e) . the results confirmed that mrid-hagd elicited specific antibodies against the homologous virus and that repeated boosts with the fusion protein further amplified the antibody response, suggesting that the quality of the recombinant antigen is immunologically relevant. despite immunizations, the antisera to the hagd without rid rarely bound to pr (h n ) viruses, indicating that the hagd without rid could not induce antibody responses specific to the virus in mice (fig. e) . these results suggested that recombinant rid-hagd may serve as a good antigen for accurate diagnosis of infection or quantification of vaccine antigens. to determine whether the rid-hagd recombinant proteins were suitable as vaccine antigens and elicited protective antibodies, we conducted an hi assay and an nt assay using the antisera derived from the immunized animals. the hi assay against the pr (h n ) virus was carried out by means of antisera collected from the mice (n = ) immunized with rid-hagd ( mg/mouse) or rid-egfp (as a control, mg/mouse), along with an alum adjuvant (thermo fisher scientific) via the intraperitoneal route. the mouse antisera against rid-hagd showed detectable levels of hi titers and nt antibody titers, whereas those from the rid-egfp group failed to show such an activity (fig. b, c) . the hi activity appeared to be similar, regardless of the origin of the rid docking protein. therefore, with the use of mrid-hagd ( mg/mouse), hagd without rid ( mg/mouse), and pbs, detailed analyses of immune responses were conducted after prime-boost immunization (n = ) via the intraperitoneal route (fig. d, e) . as shown in fig. d , e, boost immunization with mrid-hagd ( mg/mouse) augmented the immune responses, and high hi titers (. ) were achieved by the second boost immunization, whereas the nt antibody levels (. ) correlated with the hi antibody levels. the multiple immunizations with the hagd without rid did not generate hi or nt antibodies to the pr (h n ) virus (fig. d, e) . antisera from rabbits (n = ) immunized with mg of the rrid-hagd recombinant vaccine (fig. f , g) showed higher hi titers (. ) and higher nt antibody levels (. ) than those shown by mouse antisera (fig. b, c) . the control rabbit antisera against the rid-egfp proteins or pbs showed neither the hi ability nor nt titers against the homologous pr (h n ) virus (fig. f, g) . these results clearly showed that the rid-hagd fusion proteins could be further optimized into effective vaccine antigens that can produce protective nt antibody responses against influenza viruses. all of the immunized mice were challenged intranasally with one time ld of the pr (h n ) virus ( pfus) under anesthesia. the survival rate and changes in weight of the challenged mice were monitored daily for d. mice immunized intraperitoneally with mg rid-hagd antigens (mrid-hagd: n = ; rrid-hagd: n = ) lost ; % of body weight by dpc (fig. a, b) . alternatively, immunization with mg mrid-hagd (n = ) via the intraperitoneal route resulted in ; % loss of body weight by dpc, but the weight fully recovered thereafter (fig. c, d) . the complete protection was achieved by vaccination with the rid-hagd recombinant antigens, whereas the mice (n = ) immunized with pbs as a control died within dpc. of note, immunization with elysrs-hagd even at a high dose ( mg), even at high reactivity in elisa (fig. g ), failed to provide protection, and all of these mice (n = ) succumbed within dpc (fig. c, d) . the immunization with the hagd without rid ( mg) resulted in less protective efficacy compared with the mrid-hagd, judging by weight changes and survival rates of the mice (n = ) after the challenge (fig. c, d) . the lung viral titers from the mrid-hagd-immunized group were below the detection limit at dpc, whereas persistent viral infection was detected in the pbs control group (fig. c) . to evaluate potential crossreactivity with heterosubtypes, the mice were challenged with the h n virus ( times ld ) after vaccination. all of the immunized mice died within dpc, and the viral titer remained high at dpc (fig. f) , indicating that rid-hagd provided strain-specific protection (with rapid weight loss) from a homologous infection. a novel protein-folding function of rna molecules has been recognized ( , , ) , and some of these rnas even outperform previously known molecular chaperone proteins in specific cases ( , ) . initially recognized in hiv infections ( ) , the chaperna activity is intrinsic to some ribozymes ( , ) and probably operational during the figure . immunogenicity of the rid-hagd recombinant vaccine. a) schematic illustration of the immunization and challenge schedule. b) an hi assay against the pr (h n ) virus was conducted with the antisera collected from the mice (n = ) immunized (intraperitoneally) with rid-hagd ( mg/mouse) or rid-egfp (as a control, mg/mouse), along with the alum adjuvant. c ) an assay of nt of the pr (h n ) virus was performed on the serum samples from mice (n = ) immunized with rid-hagd ( mg/ mouse) or rid-egfp (as a control, mg/mouse). detection limits are and for the hi assay and nt assay, respectively. d) an hi assay toward the pr (h n ) virus was performed on serum samples from the mice (n = ) immunized (intraperitoneally) with mrid-hagd ( mg/mouse), pbs, or the hagd without rid ( mg/mouse). the hi antibody titers increased ; -fold, depending on the number of immunization boosts with mrid-hagd ( mg/mouse). however, immunization boosting with pbs or hagd without rid did not yield immune responses. e) an assay of nt of the pr (h n ) virus was performed on the serum samples from mice (n = ) immunized with mrid-hagd ( mg/mouse), pbs, or hagd without rid ( mg/mouse), respectively. the nt antibody titers increased by the factor of ; , depending on the number of boosts. however, immunization boosting with pbs or hagd without rid did not yield immune responses. detection limits are and for the hi assay and nt assay, respectively. f ) antisera from rabbits (n = ) immunized with mg of the rrid-hagd recombinant vaccine showed higher hi titers than mouse antisera did (b). g) the rabbit antisera (n = ) obtained by immunization with mg rrid-hagd showed higher titers of nt antibodies than mouse antisera did (c ). the control rabbit antisera (n = ) after immunization with mg of an rid-egfp protein showed neither an hi ability nor nt antibodies against the homologous pr (h n ) virus. folding and homeostasis of normal cellular proteins ( , ) . whether a chaperna operates in combination with molecular chaperone proteins during the de novo folding of proteins remains an exciting possibility. nonetheless, the variety of guest proteins that are amenable to rnadependent folding is not yet known, and the identity of rna molecules among the rna-interacting proteins is not well documented. a convenient avenue for exploiting the mice immunized (intraperitoneally) with mg rid-hagd (n = ) or rid-egfp control (n = ), along with the alum adjuvant, were challenged with times ld of the pr (h n ) virus ( pfus), and the body weight changes (a) and survival rates (b) of the infected mice were monitored daily. immunization with the rid-egfp control (n = ) failed to provide protection, and all of these mice (n = ) succumbed within dpc. c, d) the mice vaccinated intraperitoneally with mg of the mrid-hagd immunogen (n = ) or mock infected (pbs control; n = ) were challenged with times ld of the pr (h n ) virus ( pfus), and the body weight changes (c ) and survival rates (d) of the infected mice were monitored daily. the mice immunized with mg lysrs-hagd (n = ) or mg of the hagd without rid (n = ) were also challenged with times ld of the pr (h n ) virus ( pfus), and body weight changes of the infected mice were monitored daily (c ). immunization with elysrs-hagd or pbs failed to provide protection, and all of these mice (n = ) succumbed within dpc. immunization with the hagd without rid provided partial protection, and only one mouse succumbed within dpc (d). e, f ) replication of the challenge virus in the lungs of the immunized mice. the titers of challenge virus in the lungs were measured by a viral plaque assay. dashed lines denote a detection limit, . . error bars indicate the sd of each cohort. the chaperone activity of rna for the folding of difficultto-express proteins is to use rids that interact with known, identified rna molecules for fusion with target proteins ( , ) . by taking advantage of the chaperna function herein, for the first time, we show that influenza ha can be assembled in a soluble, trimeric, and immunologically activating conformation. the influenza ha antigen could be produced in bacteria as a soluble trimeric structure that can induce the production of nt antibodies for effective protection against a lethal challenge. as revealed during the h n pandemic in , the supply of sufficient doses of vaccines within a short period is a prerequisite for mitigating the economic effect and addressing public health concerns during a pandemic. previously, all efforts toward the bacterial production of recombinant ha vaccines have been dependent on the chemical refolding of misfolded insoluble aggregates ( , ( ) ( ) ( ) ( ) . all of these processes have depended on the initial production of ha as insoluble aggregates and required subsequent chemical refolding procedures, involving solubilization with chaotropic agents, dilution, and concentration before an immunologically relevant conformation was obtained. therefore, the production of immunologically relevant antigens in mammalian or insect cells in baculovirus vector systems has been favored. nevertheless, these systems invariably require cell culture methods that are highly expensive and difficult to scale up. the production of ha in a soluble, trimeric conformation should greatly simplify the production process, ensuring the quality of the resultant vaccines and making them amenable to implementation of pandemic preparedness ( ) . in the proposed system, an rid serves as a docking tag for transducing the chaperone function of rna for the folding and assembly of the downstream reporter protein (ha) via interaction with resident cellular trnas. from the functional perspective, the coupled action of this chaperone during protein folding and assembly is similar to that of chaperone proteins ( , ) . our results also indicate that the rids that were derived from various animals can be used as immunologically tailored, antigen-folding vehicles for antigens, which could serve as diagnostic and prophylactic tools for viral infections. in addition to the de novo folding of monomers, the subsequent assembly of monomeric antigens into multimeric-ordered structures is crucially important, especially for augmentation of the immune response and proper presentation of epitopes by immune cells toward protection ( , ) . therefore, a judicious choice of an rid is required; while mediating the chaperna function, the docking tag should not physically interfere with the formation of oligomeric structures. in the present study, an n-terminalappended trna-interaction domain ( ) of a eukaryotic lysrs was chosen as the rid. this domain is small (; aa long) and belongs to the family of intrinsically unfolded domains; it is expected to be sufficiently flexible so that it does not physically interfere with the trimer assembly of ha located downstream ( ) . intrinsically, the influenza hagd proteins were expressed and purified predominantly as trimers (fig. a) . although the expression levels and solubility of elysrs-hagd containing elysrs as an alternative trna-binding domain were substantially higher, the fusion protein existed primarily as a dimer, as confirmed by gel-filtration chromatography under native conditions (fig. b) . possibly, the stabilization energy of dimerization of the relatively large lysrs (; . kda) may physically interfere with the trimerization of hagd (; . kda) ( ) . therefore, despite high immunogenicity, as demonstrated by an elisa (fig. g) , immunization failed to provide protection against a lethal challenge (fig. c, d) . however, the hagd, without an attached rid protein, yielded interesting results when administered to mice. even without eliciting antibody responses, three immunizations with the hagd without rid partially protected the mice from the lethal challenge. we assume that this partial protection may be because of the t cellmediated immunity against ha. there are several reports indicating the existence of t cell epitopes in the ha of influenza viruses ( ) ( ) ( ) . it is likely that multiple immunizations with the hagd without rid induced specific t cell responses, contributing to the protection. the results emphasize the importance of the assembly of antigens in a native conformation for induction of protective nt antibody responses. the process has distinctive advantages over the chemical refolding of insoluble aggregates ( , , ) , which require solubilization with chaotropic agents, dilution, and concentration before adopting an immunologically relevant conformation. the production of the hagd with a receptor-binding domain in a soluble, trimeric, immunologically activating conformation should ensure the quality of viral antigens and make them amenable to implementation of pandemic preparedness ( ) ( ) ( ) . it is worth comparing the mechanistic differences of the process described herein from other approaches to the oligomeric assembly of ha. the existing approaches involve fusion of ha with ferritin into nanoparticles or with a foldon that is derived from the natural trimerization domain of isoleucine zippers or t fibritin ( ) ( ) ( ) ( ) , wherein the major amount of stabilization energy is provided by the external trimerization domain. in the present approach, using a chaperna, the rid remains an independent monomer, and thus, it is not expected to contribute to intermolecular stabilization among hagds. rather, trimer formation is primarily driven by the intrinsic stabilization energy of mutual interactions among hagds. here, an rid, as a small, structurally independent domain, is not expected to interfere with the interactions within hagd trimers in space, as governed by the thermodynamic stabilization energy in the assembly of trimers. the rid that was tested in the present study is derived from human lysrs, which can switch spontaneously from an unfolded structure into an a-helical structure after trna binding ( ) . it is noteworthy that the unique ability of intrinsically unfolded domains to increase the solubility of target proteins has been reported, although its effect on biologic activities remains unknown ( ) . the rid that was used in this study is relatively unfolded, and therefore, the linking of the docking protein with the rid may increase its solubility, in agreement with other reports ( , ) . therefore, it is intriguing that the hridmu , hridmu , and hridmu proteins maintained high solubility (even higher than that of the wt hrid; fig. c) , and yet, the extent of the trimeric assembly of hagd was greatly lowered (fig. e) . these results confirm that the rna interaction is indeed crucial for the trimeric assembly of ha. the results also offer a discretionary note on the quality evaluation of recombinant antigens: solubility is necessary but not sufficient to represent a biologically or immunologically relevant conformation. the mechanism of the chaperna-mediated assembly of monomers should be explored further, although it is functionally similar to that of protein-based molecular chaperones. molecular chaperones are proven to be crucial for not only the de novo folding but also, the assembly of monomers into multimeric complexes ( , ) . moreover, in contrast to the foldase type of molecular chaperones, which consume atp for recycling chaperones from docking proteins, a chaperna is expected to function as a chaperone without the atp requirement, in a way similar to that of the holdase type of chaperones ( ) ( ) ( ) . here, the affinity of rna may be a determining factor for the capture and folding of the guest proteins. in addition, the potential effect on the trna-induced a-helical structural transition of an rid to folding of the downstream docking protein should be investigated further. overall, our results show that rna can affect the kinetic landscape of the folding pathway in favor of the productive folding and assembly of vaccine antigens ( , ) . the potential immunogenicity of the rid docking tag should be taken into account, especially in designing the folding vehicle for vaccine antigens. to ensure that specific antibody responses are directed against the hagd after immunization, the rid should remain nonimmunogenic. accordingly, an rid should originate, preferably from the host being immunized, and was of murine origin in this study, wherein immunization of mice and a mouse model of a lethal challenge were used. for human clinical trials, the rid can be of human origin to direct the elicited antibody response predominantly against ha. the chaperna function may be extended to a supramolecular assembly of viral antigens, which is required for augmenting immunogenicity. we recently confirmed that the present strategy can be successfully applied to the assembly of bacterially synthesized monomers of a norovirus particle, which comprises monomers, into virus-like particles (vlps; unpublished results). however, in vlp assemblies, prior cleavage of the rid tag was required to enable the compact packing into vlps. this system may also be applied to preparation of mab for diagnostic and therapeutic interventions. basically, the process involves isolation of spleen cells from an immunized mouse, followed by their fusion with immortalized cells and screening of reactive clones. in this case, an rid, originating from the host being immunized (murine or rabbit origin in this study), was used to ensure that most of the positive antibody clones were directed to the target antigens, for instance, the spike protein of the middle east 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surveill response j doi: . /wpsar. . . . sha: doc_id: cord_uid: q nzv d nan w hen an influenza pandemic swept the globe in , it was nicknamed the "spanish flu" despite evidence of circulation in other countries. this was because the spanish press were free to publish stories about the outbreak that peers in neighbouring countries were not due to wartime censors. other governments hid negative news about the pandemic and over-reassured the public. attempts to prevent panic backfired, and the resulting breakdown in trust "threatened to break the society apart". the pandemic illustrates the consequences of failing to transparently and effectively communicate risks to the public during a public health event. this article discusses the lessons learnt in risk communication during the response to recent outbreaks in the world health organization's western pacific region. these lessons can inform preparedness for pandemic influenza and other public health threats. risk communication is defined as "the real-time exchange of information, advice and opinions between experts, community leaders, or officials and the people who are at risk". the outbreak of severe acute respiratory syndrome (sars) in china in in particular highlighted the importance of open risk communication -a lesson that was reiterated once more during the outbreak of middle east respiratory syndrome in the republic of korea in . effective risk communication during a public health emergency can be difficult, especially in the early stages when many of the facts may be uncertain. health authorities can be reluctant to proactively communicate as they are apprehensive of saying the wrong thing, creating panic or looking like they do not have all the answers. however, delaying communication can result in the public listening to rumours or relying on less accurate sources of information, or can lead to the very panic authorities were trying to prevent. done correctly, however, risk communication can calm fears, facilitate the acceptance of containment measures, curtail the spread of unhelpful rumours and engage affected communities in control measures. in the wake of sars, risk communication was included as a core capacity required of member states under the international health regulations ( ). guidance on how to implement and build risk communication capacity has also been part of the asia pacific strategy for emerging diseases (apsed) since the first edition. it has long been recognized that national risk communication plans, supported by trained risk communication personnel, adequate financial allocations, clear internal procedures and mechanisms for coordination, are essential for effective risk communication and should be established before the onset of a public health emergency. advance preparation, including building an understanding of prevailing cultural practices and establishing relationships with community influencers, is central to ensuring that risk communication efforts are tailored to the local context. health capacities, such as surveillance and laboratory networks, than they do on risk communication. countries are encouraged to learn from recent outbreaks and emergencies and to invest in their internal capacity for risk communication as per the asia pacific strategy for emerging diseases and public health emergencies (apsed iii). this includes integrating risk communication into outbreak preparedness, planning and response, communicating quickly and transparently, using a mixture of channels to best reach their target audience (including social media, where appropriate) and actively engaging communities in the response. the vision laid out in apsed iii is one where risk communication moves from being purely an art to also a science, as risk communication becomes more professionalized and evidence-based. risk communication professionals should come to be recognized as social scientists conducting work that is as important to the success of emergency preparedness and response as the work of epidemiologists, laboratory experts and other public health personnel. in prioritizing and strengthening risk communication, countries will be better placed to limit the health, social and economic impacts of the next influenza pandemic. health was low, while others expressed a high level of concern about the virus. some people also had immense distrust in the vaccine, because of perceived conflicts of interest between pharmaceutical companies and health authorities. research published following the h n pandemic indicated that countries should be prepared to address rumours and misconceptions about vaccine safety, to carefully communicate the severity of disease and to enlist the support of trusted members of the community. the role of social media also needed to be considered. after the pandemic, it was recognized that risk communication approaches could be tested and honed during seasonal influenza outbreaks. ten years after sars, china proactively informed the public and international community about human cases of avian influenza (h n ), demonstrating the benefit of timely and transparent risk communication. chinese focus group participants were reassured by this increase in transparency, with one participant stating, "i am quite positive that our government absolutely has the capability to control this disease". another study found that discussion of h n on sina weibo, a popular chinese social media platform, dropped following several formal announcements, potentially indicating reduced public concern about the outbreak. while social media was used to listen to the public following the discovery of human cases of h n , the response to an outbreak of influenza-associated severe acute respiratory infections (sari) in fiji in showed that more traditional means of communication still have a place in effective risk communication. health authorities worked with religious leaders, women's groups and youth networks to engage vulnerable groups, particularly pregnant women, encouraging vaccination and adoption of protective behaviours. , for communities in remote areas and outer islands, where communication is often limited, authorities shared health messages via radio, reaching an estimated % of the population. unfortunately, while much progress has been achieved in risk communication under apsed, other core public health capacities for pandemic preparedness and response continue to be prioritized over risk communication. results from joint external evaluations of ihr core capacities in the western pacific region show that countries score far higher on traditional public the great influenza, the epic story of the deadliest plague in history communicating risk in public health emergencies: a who guideline for emergency risk communication (erc) policy and practice. geneva: world health organization middle east respiratory syndrome in the republic of korea: transparency and communication are key. west pac surveill response the sars epidemic and its aftermath in china: a political perspective learning from sars: preparing for the next disease outbreak: workshop summary pdf;jsessionid= af cb dff edf feea? sequence= ) manila: who regional office for the western pacific an outbreak investigation of paediatric severe acute respiratory infections requiring admission to intensive care units -fiji suva: fiji ministry of health and medical services health and nutrition cluster bulletin # . suva: fiji ministry of health who western pacific region: jee mission reports asia pacific strategy for emerging diseases and public health emergencies (apsed iii). manila: who regional office for the western pacific unresolved issues in risk communication research: the case of the h n pandemic australia's influenza pandemic preparedness plans: an analysis us department of health and human services early response to the emergence of influenza a(h n ) virus in humans in china: the central role of prompt information sharing and public communication. bull world health organ perceptions on the risk communication strategy during the avian influenza a/h n outbreak in humans in china: a focus group study. west pac surveill response key: cord- -bbbq ylr authors: tong, michael xiaoliang; hansen, alana; hanson-easey, scott; xiang, jianjun; cameron, scott; liu, qiyong; liu, xiaobo; sun, yehuan; weinstein, philip; han, gil-soo; bi, peng title: china's capacity of hospitals to deal with infectious diseases in the context of climate change date: - - journal: soc sci med doi: . /j.socscimed. . . sha: doc_id: cord_uid: bbbq ylr objectives: infectious diseases are a major cause of morbidity and mortality in china. the capacity of hospitals to deal with the challenge from emerging and re-emerging infectious diseases due to climate change is of great importance to population health. this study aimed to explore the capacity of hospitals in china to deal with such challenges. methods: a cross-sectional questionnaire survey was utilized to gauge information regarding capacity of hospitals to deal with infectious diseases in the context of climate change among clinical professionals whose roles pertained to infectious disease diagnosis, treatment and management in anhui province of china. descriptive analysis and logistic regression analysis were performed on the data. results: more than % of participants believed climate change would have an adverse influence on population health and infectious disease control in china. most indicated that their hospitals were well prepared for emerging infectious diseases at present, and they considered that logistical support in hospitals (e.g. administrative and maintenance services) should be strengthened for future capacity building. the majority of participants suggested that effective prevention and control measures, more interdisciplinary collaborations, more funding in rural areas for health care, and improved access to facilities enabling online reporting of infectious diseases, were extremely important strategies in building capacity to curb the population health impact of emerging and re-emerging infectious diseases due to climate change in china. conclusions: clinical professionals recognized that climate change will likely increase the transmission of infectious diseases. although rural health care and hospitals’ logistical support need to be improved, most professionals believed their hospitals to be capable of dealing with emerging diseases. they thought that interdisciplinary and cross-regional collaborations, together with necessary resource support (e.g. improved facilities for rural health care) would be important control strategies. currently there are roughly million cases of reported infectious diseases in china, which result in approximately , deaths annually (national health and family planning commission of the prc, ) . previous studies have found the incidence of infectious diseases has been sharply reduced due to great improvements in health care services (hipgrave, ) . however, the emergence and re-emergence of some climate-sensitive diseases, such as malaria, dengue and hemorrhagic fever with renal syndrome (hfrs) have occurred in recent decades in china (huang et al., ; xiang et al., ; zhang et al., b) . malaria was a serious public health problem during the s with an incidence of approximately per , population . in , the incidence was only per , with about , cases reported . however, malaria cases in china increased to , in (zhou et al., ) dengue cases were reported prior to , but frequent outbreaks have occurred during the last few decades in china (lai et al., ) . hfrs, a serious zoonotic disease caused by hantaviruses, can be transmitted to humans by contact with infected rodents or their excreta . the disease is frequently reported in china where roughly % of the world's cases occur (xiao et al., ) . although the incidence of hfrs significantly declined during the s, there has been an increasing trend since (xiao et al., ; zhang et al., ) . the possible reasons for the emergence and re-emergence of these infectious diseases may be linked to climate change, population movement, rapid urbanization and increased surveillance efforts (tong et al., ; wu et al., ) . it is widely known that the global average combined land and ocean surface temperatures have increased by . °c over the period from to (intergovernmental panel on climate change, ). the intergovernmental panel on climate change (ipcc) indicates that the global average surface temperature will increase by . - . °c by - compared to - , and other climatic variations, such as changes in rainfall patterns and relative humidity will also occur (intergovernmental panel on climate change, ). climate change has, and will continue to, impact the transmission of vector and rodent-borne diseases by affecting the growth and development of the vectors or hosts, shortening the incubation period of the pathogens within the vectors, and impacting human behaviour (e.g. more time spent outdoors) (world health organization, ). the projected temperature increase and change in rainfall patterns may bring about an increase in cases of infectious diseases such as malaria, dengue and hfrs zhang et al., a; zhou et al., ) . in china, annual average land surface air temperature has increased by . - . °c over the past years, and is projected to increase by . - . °c by as compared to (china national development and reform commission, ) . extreme climate events, such as extremes in temperature, drought in north china, and flooding in south china, may become more frequent and intensive (china national development and reform commission, ) . the variations in temperature, rainfall, humidity and extreme events posed by climate change could facilitate infectious disease transmission and result in the possible increase in cases of infectious diseases in china (tong et al., ) . in the chinese health care system, both the clinical health sector and preventive medicine (public health system) play important roles in the protection of population health. in the clinical health system, hospitals provide patient diagnosis, treatment and management. in the public health system, the centers for disease control and prevention (cdc) work to protect and improve public health and safety, and focus on disease prevention, control and surveillance; and cases of notifiable diseases are reported by clinical professionals to the local cdc (he, ) . therefore, hospital clinical staff and cdc public health professionals perform different roles, and may have different views on infectious disease control and prevention. given that the ability of the public health system (i.e. cdcs) to deal with emerging and re-emerging infectious diseases in the context of climate change has been extensively studied (tong et al., a; tong et al., ) , a similar investigation of the clinical health system where diseases are diagnosed and treated, is warranted. this study will contribute to a better understanding of infectious disease diagnosis, treatment, prevention and control in the face of climate change in china, and may also benefit disease control in other countries . the study employs a cross-sectional questionnaire survey among clinical professionals to explore china's capacity of hospitals to deal with infectious diseases in the context of climate change. further, the study explores participants' views on capacity building in the hospital sector to curb potential emerging and re-emerging infectious diseases due to climate change in china. a questionnaire was administered to clinical professionals in november . the questionnaire instrument design was informed by previous studies and relevant literature on climate-sensitive diseases (national development and reform commission of the prc, ; semenza et al., ; tong et al., ; wei et al., ) . the questions asked about clinical professionals' thoughts on climate change, disease occurrence, the capacity of hospitals to deal with infectious diseases, and strategies to build the capacity of the hospital sector and curb the health impacts of climate change related to infectious diseases. an open-ended question was included to explore in greater depth, participants' understandings of disease control, diagnosis, treatment and management in the context of climate change. the questionnaire is included in appendix a. the study site of anhui province was selected because it has a high incidence of infectious diseases, especially malaria and hfrs (bi et al., ; jiao et al., ; the people's government of anhui province, ). anhui province is located in east china (see fig. ), and has a warm-temperate, semi-humid monsoonal climate with an average annual temperature between and °c, and annual precipitation between and mm (the people's government of anhui province, ). participants were clinical professionals from three major hospitals in anhui. a total of clinical professionals were surveyed in two general hospitals in the capital city of anhui. a further were surveyed in an infectious diseases hospital in a prefectural-level city. the clinical professionals included both doctors and nurses whose roles pertained to infectious disease diagnosis, treatment, and management. investigators administered the questionnaire in the three hospitals. to maximize the response rate, the principal researchers selected six key senior contacts from hospitals to assist with the distribution of questionnaires to potential participants. the process of participation was voluntary, and no incentives were offered. there are clinical professionals relating to infectious diseases in the three major hospitals, and for ease of administration, the entire population was used as sample. in total, questionnaires were distributed, and after omitting incomplete questionnaires, were analyzed, with a response rate of %. all returned questionnaires were entered using epidata . software (lauritsen, ) to create a database. statistical analyses were performed with stata . (statacorp, ). participants' demographic characteristics were descriptively analyzed. binary logistic regression was used to explore the association between binary responses and demographic variables. ordinal logistic regression was used to explore the association between ordinal responses and demographic variables. the demographic variables were age, gender, professional level, length of employment, education, and occupation. the binary responses were "yes", and "no/unsure". the ordinal responses were "very concerned", "concerned", "slightly concerned", and "not concerned"; or "extremely important", "very important", "important", "less important" and "not important". data were analyzed with a two-sided test and p-values less than . were considered statistically significant. table shows the demographics of the participants. in this study, % of the clinical professionals who participated were doctors, and . % were nurses. while ages ranged from to years, . % of professionals were aged below years, with the mean age being . years. of the participants, . % were female. junior level professionals accounted for . %, intermediate level professionals . % and senior level . % of the total participants. over % of the participants had worked in hospitals more than five years. more than % held a university degree or higher qualifications, especially the doctors who were mostly highly educated with a bachelor of medicine degree or above (see supplementary table s in appendix b). as shown in table , more than % of the professionals were either very concerned or concerned about climate change. there was a statistically significant association between age group, professional level and the concern about climate change (see supplementary table s in appendix b). those who were over years (or = . , % ci: . - . , p = . ) and the senior staff (or = . , % ci: . - . , p = . ) were more likely to be concerned about climate change. furthermore, % of the professionals agreed with the statement that the weather was becoming warmer, especially nurses and the professionals who had been employed more than ten years (see supplementary table s in appendix b) . nearly all ( . %) believed climate change would have an adverse influence on population health. furthermore, . % and . % of professionals agreed that predicted increasing temperatures and changes in precipitation patterns would affect infectious disease transmission. specifically, . %, . %, and . % of professionals were either extremely or very likely to believe that there was an association between climate change and malaria, dengue, and hfrs, respectively. table shows that about % of participants thought that there had been an increasing number of patients with malaria, dengue, and hfrs, and roughly % and % of professionals thought that climate change and population migration, respectively, were contributing factors. more than % of participants believed that malaria and hfrs hospital-reporting protocols were in place, and . % believed so for dengue. in terms of the diagnostic capability of hospital laboratories, . % of professionals responded that their hospitals were 'always' or 'mostly' able to rapidly provide diagnostic tests for malaria, . % for dengue, and . % for hfrs, respectively. some % of participants rated diagnostic and treatment capacity as excellent/good for malaria and hfrs, while % thought this was the case for dengue. moreover, if an unusual cluster of cases was noticed, most professionals would take actions such as discussing with colleagues and laboratory technicians, informing the public health officer, and consulting with the cdc. in addition, there were no significant differences in these perceptions of infectious diseases between doctors and nurses. clinical professionals' perceptions of the current capacity of their hospitals to deal with infectious diseases are shown in table . specifically, . % of participants agreed that they had sufficient staff to deal with disease outbreaks. also, more than % believed that their staff were well informed about current infectious disease trends, and that the quality of reported data from their hospitals to the cdc was excellent. however, . % of professionals thought that logistical support in hospitals (for example administrative and maintenance services) needed to be strengthened. overall, . % either agreed strongly or somewhat that hospitals were well prepared for the threat of a serious emerging disease. there were no significant differences between perceptions of doctors and nurses regarding the current capacity of the hospital. additionally, . % agreed that more research on the health impacts of climate change was needed. to build capacity to meet the challenge of emerging and re-emerging infectious diseases due to climate change, . % of participants thought that prevention and control measures were extremely important strategies, and . % believed more collaboration with their local cdc was extremely important (table ) . furthermore, multivariate ordinal logistic regression analysis showed that compared to those under years, professionals aged over years were more likely to indicate that prevention and control measures are extremely important (or = . , % ci: . - . , p = . ). senior level staff were more likely to believe that more collaboration with the cdc was extremely important (or = . , % ci: . - . , p = . ); while doctors were less likely to believe so (see supplementary table s in appendix b). about % believed more funding was required for rural health care and that improving the accessibility of the online infectious disease reporting system for rural hospitals was also extremely important. more than % of those surveyed believed that the health impacts of emerging and re-emerging infectious diseases due to climate change could be addressed with strategies such as: better response mechanisms; strengthening the climate change poses a significant threat to global population health (intergovernmental panel on climate change, ). clinical professionals are at the frontline of health care provision to the population and are likely to witness firsthand the health impacts of a changing climate (blashki et al., ) . they, therefore, provide a unique perspective on the health impacts of climate change and an indepth understanding of local community health (blashki et al., ) . to the best of our knowledge, this study is the first of its kind to gauge clinical professionals' perceptions of the capacity of china's hospitals to manage infectious diseases under climate change and to provide insight for policymakers, practitioners and medical educators regarding climate change adaptation in the health sector. concern about climate change was acknowledged by most professionals, especially among senior staff and the group aged over years. the majority of professionals, particularly nurses and those with longer terms of employment indicated that the weather was getting warmer. nearly all respondents indicated climate change would adversely affect population health, and most indicated it would facilitate infectious disease transmission. less than % of participants thought climate change was unlikely to be associated with malaria incidence compared to % for dengue and hfrs. the results were consistent with our previous studies among cdc staff who indicated climate change was more likely to have an influence on malaria than hfrs, whilst the perception of an association between climate change and dengue was not as strong as in a previous study conducted in guangdong (tong et al., ) . this is likely due to guangdong having the highest incidence of dengue in china, whilst in anhui there are fewer dengue cases reported (lai et al., ) . moreover, compared with previous studies among cdc public health professionals in other provinces (tong et al., a) , the hospital clinical professionals in this study were more likely to indicate that climate change would have an influence on hfrs. this could be due to the relatively high number of hfrs cases in anhui province, and hence firsthand knowledge of the disease and its determinants. additionally, another study conducted among health experts in europe also indicated their perceptions of the negative impact of climate change on vector-borne, food-borne, water-borne and rodent-borne diseases (semenza et al., ) . despite . % of participants agreeing strongly or somewhat that hospitals were currently well prepared for the threat of a serious emerging disease, climate change may present new challenges for health sectors such as increased incidence in certain climate-sensitive diseases, or diseases either not previously seen in the area, or not seen for some time. such an influx in cases could stretch the coping capabilities of the present system. this may explain why the public health and clinical health sectors share concerns about potential emerging and re-emerging climate-sensitive diseases. the majority of professionals indicated that climate change and population migration were considered as the most significant factors associated with the increase of these infectious diseases. this is in line with other studies that indicated the impact of climate change and migration would affect infectious disease transmission (gao et al., ; mcmichael, ; sang et al., ) . higher temperatures and changing precipitation patterns can contribute to the increasing m.x. tong et al. social science & medicine ( ) - population of vectors/rodents and more frequent contact with humans. simultaneously, population movement can facilitate disease transmission from one region to another. in china, currently it is estimated that approximately million people have migrated from poor rural areas to gain work in the cities (national health and family planning commission of the prc, ). this "floating population" (highly mobile population) of internal migrants chiefly work in insecure lowwage jobs. moreover, they often lack health insurance and defer seeking timely medical treatment, which could have a negative influence on infectious disease control (qin et al., ) . future infectious disease control and prevention should be cognizant of climate change impacts and population movements on the transmission of these diseases. in addition, building a more comprehensive national health insurance system covering the internal migrant population's health expenses in any part of china may provide benefits for population health (qin et al., ) . most professionals claimed that there was a hospital protocol in place for the reporting of notifiable diseases and believed that the capacity for disease diagnosis and treatment was either excellent or good, especially for malaria and hfrs. as anhui historically has a high incidence of malaria and hfrs (chen and qiu, ; gao et al., ) , professionals are more likely to have experience and confidence in dealing with these diseases, whereas historically there has been a low incidence of dengue in the province. the majority of professionals believed that their hospital laboratories were able to provide rapid diagnostic test results for malaria, dengue and hfrs. additionally, most professionals purported they would take comprehensive actions if they detected an unusual cluster of cases. this indicates there is a strong likelihood that outbreaks of emerging or re-emerging diseases would be detected early by the clinical professionals or the cdc. this detection of unusual trends could aid in curbing transmission if preventive measures are activated early. regarding the capacity of hospitals to deal with disease risks, most participants believed they were well prepared for the threat of a serious emerging disease. of particular note, participants indicated there was a need to strengthen the hospital's logistical support. this is in line with another study conducted by xu and chu in focusing on logistics capacity in hospitals, which advocated that a reliable logistical support system is one of the most important components in modern health care systems and should be given higher priority to sustain and promote better health care services in the long-term (xu and chu, ) . currently, the main obstacles to improving hospitals' logistical support in china are the lack of high-quality staff and regulatory frameworks for logistics management within hospitals (lin, ) . promoting specialized training for logistics staff and implementing management and quality control guidelines would be important. moreover, the problems in rural areas were highlighted, especially regarding the funding for rural health care support and accessibility to an online reporting system. the severe acute respiratory syndrome (sars) outbreak demonstrated that rural health care was the weakest component of china's disease control and prevention system (knobler et al., ) . more resource allocation to rural areas would be a vital step in advancing china's capacity building in response to emerging infectious diseases due to climate change. in this study, older and senior staff were more likely to indicate that primary prevention measures and interdisciplinary collaboration were important, compared with the younger or non-senior staff. such differences could be due to the rich working experience of the groups, who may have a better understanding of the infectious disease control and prevention strategies. most participants also suggested a need for improved environmental health, more health education programs and financial support to improve vector/rodent-borne disease control, diagnosis, treatment and management in the context of climate change. some studies have made future predictions about the effect of climate variation on dengue and malaria (caminade et al., ; ebi and nealon, ) , although there are many confounding factors to consider. although some participants in this study indicated that changes have already been noted, it would be useful in future studies to gain an insight into stakeholders' perceptions of when global warming would reach a point where there is a marked effect on the burden of climate-sensitive diseases. some limitations of this study deserve mention. firstly, this study was conducted in one province. the results may not be generalizable to clinical professionals in other provinces of china. secondly, as dengue is an emerging and uncommon disease in anhui, participants in this region may be less likely to have hands-on experience in dealing with dengue cases, and feel less confident about dengue diagnosis and treatment. lastly, the study was conducted among three major hospitals in cities, and results may not be generalizable to rural areas, townshiplevel hospitals or village-level clinics. nevertheless, these findings may provide information for policymakers and clinical professionals pursuing initiatives to strengthen the capacity for hospitals to cope with a potential increase over time, in cases of climate-sensitive infectious diseases. this study revealed that most clinical professionals thought climate change would have an impact on infectious diseases, and believed climate change and population migration were significant factors associated with infectious disease transmission. health professionals in our study thought that the overall capacity of the hospital healthcare system to deal with infectious diseases was excellent. however, logistical support should be strengthened in hospitals and more climate change related research is needed. issues that could be addressed include prevention and control measures, collaboration with the cdc, inhouse staff training and improved healthcare systems in rural areas. the multiple direct and indirect impacts of climate change will continue to threaten the health of the chinese population. these findings may help health policymakers develop organizational adaptation policies to address the adverse impact of climate change on 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and meteorological factors in animal reservoir, natural and socioeconomic variations and the transmission of hemorrhagic fever with renal syndrome in adhere to scientific development to improve the overall logistics capacity in hospital historical patterns of malaria transmission in china climate variability and hemorrhagic fever with renal syndrome transmission in northeastern china spatiotemporal transmission dynamics of hemorrhagic fever with renal syndrome in china meteorological variables and malaria in a chinese temperate city: a twenty-year time-series data analysis malaria situation in the people's republic of china in . chin geographical, meteorological and vectorial factors related to malaria re-emergence in huang-huai river of central china this work has been funded by the department of foreign affairs and trade through the australian development research awards scheme under an award titled 'how best to curb the public health impact of emerging and re-emerging infectious diseases due to climate change in china' [project id: ] and the national basic research program of china ( program) [grant no. cb ]. the views expressed in the publication are those of the authors and not necessarily those of the department of foreign affairs and trade or the australian government. the commonwealth of australia accepts no responsibility for any loss, damage or injury resulting from reliance on any of the information or views contained in this publication. we thank the anhui medical university, anhui provincial hospital, the second hospital of anhui medical university and fuyang no. people's hospital getting involved in this study for their assistance in the distribution and return of questionnaires. all survey participants are greatly appreciated for their valuable contributions. supplementary data related to this article can be found at http://dx. doi.org/ . /j.socscimed. . . .m.x. tong et al. social science & medicine ( ) - key: cord- -qg hl ft authors: yoon, ji hye; lee, jihye; lee, jun young; shin, young sup; kim, dong eon; min, jung sun; park, chul min; song, jong hwan; kim, seungtaek; kwon, sunoh; jang, min seong; kim, hyoung rae title: study on the ‐phenylchroman‐ ‐one derivatives and their anti‐mers‐cov activities date: - - journal: bull korean chem soc doi: . /bkcs. sha: doc_id: cord_uid: qg hl ft study on the ‐phenylchroman‐ ‐one derivatives and their anti‐mers‐covactivities. [image: see text] middle east respiratory syndrome coronavirus (mers-cov) has been one of the most fearful diseases by its mortality and possibility of global outbreak. until january , laboratory-confirmed cases of mers-cov, including associated deaths (case-fatality rate: . %) were reported globally. in korea on may , confirmed cases including associated deaths ( . %) were reported by an outbreak caused by only one infected person from saudi arabia. at present, no effective vaccine or therapeutics are available for the prevention or treatment of mers-cov infection. [ ] [ ] [ ] however, various basic and clinical research are on-going. we screened a variety of natural products against mers-cov as an attempt of developing anti-mers-cov drugs, and many flavonoids showed anti-mers activities. among them, -phenylchroman- -one derivatives e.g., bavachin and bavachnin separated from dry seed of psoralea corylifolia l., korean medicinal herb exhibited comparatively good activities. bavachin derivatives have been reported as their diverse biological activities, including anti-cancer, ppar agonist, anti-inflammatory, anti-alzheimer, immunomodulatory, anti-osteoporosis, and anti-viral (against carp virus and influenza a ) activities. the synthesis of racemic mixtures of bavachin derivatives were reported , and these compounds have been proven to be ppar agonists and anticancer reagents. however, exchanges of phenol group to aniline group have not been tried. bavachin and bavachinin showed good anti-mers-cov activities of . and . μm respectively by phenotypic cellular screening with vero cell. as the small structural difference between two compounds (-oh vs. -ome) could change the anti-viral activity, we tried to synthesize the bavachin derivatives for structure-activity-relationship study (sar study). first, we synthesized racemic bavachinin ( a) according to the procedure reported by du et al. the double bond in the side chain of a was reduced by hydrogenation with pd/c to prepare a, and oxidation of chromane ring with i was performed to produce a (scheme ). a was synthesized by the modified procedure of that of a. , was reacted with n-boc-protected -aminobenzaldehyde by aldol condensation to give , which was cyclized by kf to produce a (scheme ). the further derivatizations of a, a, a, and a were performed by the substitutions of phenolic oh ( a, a, a) and nh ( a) groups. for example, racemic bavachnin, a was reacted with iodomethane, -bromopropane, and benzyl bromide in the presence of potassium carbonate to afford b, c, and d, respectively. the derivatizations of phenol group of a and a were performed by the similar procedures as shown in scheme to prepare b- d and b. b and c were prepared by acetylation and propargylation of the amino group of a as shown in scheme . total compounds of bavachinin derivatives in four core structures were evaluated to figure out their anti-mers-cov activity and cell-cytotoxicity by cellular phenotypic screening method as shown in table . the activity of synthesized racemic bavachin ( a) was around half of the natural bavachin but o-methylated a, racemic bavachnin ( b) showed similar activity of natural bavachinin (entry and in table ). the further alkylations of phenolic oh of a with isopropyl and benzyl group decreased the anti-mers activities (entry and in table ). a was prepared by hydrogenations of external double bonds of a, which showed the best anti-mers activities regardless of their o-substituents. interestingly, o-isopropyl note and o-benzyl derivatives ( c and d, respectively) showed similar activity with a (non-substituted) better than b (omethylated), but the cytotoxicity for vero cell also increased. a showed good activity similar to that of a, however its cytotoxicity was much less than that of a. considering activity and cytotoxicity, a was thought to be the best compounds for anti-mers drug. the pharmacokinetic study of a was performed on rats by iv and po. a could not be detected in plasma after min in both iv and po study by its rapid clearance, but its liver microsomal phase i stability (% of remaining after min) in rat was %. we decided to change the phenolic oh group to amino group ( a) based on the assumption that phenolic oh group was concerned with its instability in plasma. though bioavailability ( %) was not satisfactory and liver microsomal phase i stability was not improved ( %), the results from pharmacokinetic study of a afforded acceptable curves in both iv and po study as shown in figure . the anti-mers activity of a was a little decreased compared to a, however, the anti-mers activities of its derivatives were conserved. a also exhibited anti-viral activity against sars-cov ( . μm) in vero cell. as a conclusion, a series of -phenylchroman- -one derivatives were synthesized for the chemical modifications of bavachin, and they exhibited anti-mers activities in vero cell. a showed the best activity, but a was thought to be the best compounds for anti-mers drug in consideration of cytotoxicity. the modification of phenol group ( a) to amino group ( a) could much improve pharmacokinetic properties. we expect the study on bavachin derivatives can contribute to the development of anti-mers drug. scheme . o-alkylations of a. reagents and conditions: (a) iodomethane, k co , dmf, rt., overnight, % ( b); -bromopropane, k co , dmf, c, overnight, % ( c); benzyl bromide, k co , dmf, rt., overnight, % ( d). for immunofluorescence staining, the fixed cells (above) were permeabilized with . % triton x- (sigma-aldrich, st. louis, mo, usa) for min. then the cells were incubated with rabbit anti-mers-cov spike antibody for h at c. after three washes with pbs, the cells were incubated with alexa -conjugated goat anti-rabbit igg (h + l) secondary antibody and hoechst (life technologies, waltham, ma, usa) for h at c. images were acquired by perkin elmer operetta ( ×; waltham, ma, usa). the acquired images were analyzed with in-house-developed image-mining . (im . ) plug-in software as previously described (pmid: ). in the analyzed image, the total number of cells and the number of infected cells were determined by counting hoechst-stained nuclei and spike protein-expressing cells, respectively. the infection ratio of each well was normalized using the average infection ratio of mock control as % and the average infection ratio of negative control ( . % dmso) as %, in each assay plate. cell ratio was determined according to the number of cells of each well versus the average number of cells of mock control, in each assay plate. all work with mers-cov was performed in an enhanced biosafety level- (bsl- ) facility at institut pasteur korea. antimicrob key: cord- -b l epy authors: falsey, ann regina title: respiratory viral infections date: - - journal: genomic and precision medicine doi: . /b - - - - . - sha: doc_id: cord_uid: b l epy molecular analysis of respiratory viruses and the host response to both infection and vaccination have transformed our understanding of these ubiquitous pathogens. polymerase chain reaction for the rapid and accurate diagnosis of viral infections has led to a better understanding of the epidemiology and impact of many common respiratory viruses and resulted in better patient care. over the past decade a number of new respiratory viruses including human metapneumovirus and new coronaviruses have been discovered using molecular techniques such as random primer amplification, pan-viral array and next generation sequencing. analysis of the host transcriptional response during respiratory viral infection using in-vitro, animal models and natural and experimental human challenge have furthered the understanding of the mechanisms and predictors of severe disease and may identify potential therapeutic targets to prevent and ameliorate illness. respiratory viruses are a major cause of morbidity and mortality throughout the world and affect persons of all ages [ ] [ ] [ ] [ ] . in addition to > million office visits for upper respiratory infections each winter, hospitals fill to capacity with admissions due to community acquired pneumonia, acute exacerbations of chronic obstructive pulmonary disease, asthma and bronchitis and many of these illnesses are due to viral infection. "pneumonia and influenza" consistently ranks as the fourth most common discharge diagnosis, and each year, , to , hospitalizations and to , deaths in the united states are attributable to influenza [ , [ ] [ ] [ ] [ ] . due to their epidemic nature influenza and rsv are widely recognized as pathogens in adults and children, respectively. however, the true burden of disease and the contributions of other viruses such parainfluenza viruses (piv), human metapneumoviruses (hmpv), coronaviruses (cov), and rhinoviruses (hrv) now being more fully recognized using modern molecular detection methods [ ] [ ] [ ] [ ] [ ] . in addition to sensitive and rapid diagnostic testing, new molecular techniques allow an understanding of viral evolution, mechanisms and predictors of severe disease, interrogation of vaccine responses, improved bacterial and viral diagnostics and associations of viral infections with non-respiratory medical events. in this chapter the many ways molecular and precision medicine have impacted the field of respiratory viral disease will be reviewed. in the past defining the epidemiology and impact of viral respiratory pathogens was significantly hampered by slow and/or insensitive diagnostic techniques such as cell culture and antigen detection [ , ] . polymerase chain reaction (pcr) has revolutionized the study of respiratory viruses and provides extremely sensitive, specific and rapid means for the detection of fastidious and non-cultivatable respiratory viruses [ ] . pcr based epidemiologic studies now provide a more complete understanding of the clinical spectrum and age ranges of populations affected [ ] [ ] [ ] [ ] [ ] [ ] . in one study, conventional methods yielded a viral diagnosis in % of pneumonia cases, while use of pcr increased the yield to % [ ] . technology has rapidly evolved from single-plex pcr and gel electrophoresis to multiplex real time assays where products are detected by luminescent signals proportional to the target amplified [ ] . there are currently a variety of commercially available assays that detect from to viral respiratory pathogens and maintain excellent sensitivity [ ] . many clinical microbiology laboratories are now moving to primarily molecular methods for viral detection and pcr formats are becoming increasingly simple so that nucleic acid extraction and pcr is fully automated with little operator input. molecular point of care assays will soon be feasible [ ] . in addition to providing more sensitive means of detecting known viruses, molecular methods are extremely useful for viral discovery [ ] [ ] [ ] . over the past several decades a number of new respiratory viruses or variants have been identified including hmpv, novel strains of coronaviruses (hku , nl , sars-cov, mers-cov), rhinovirus c, human boca virus, parechoviruses and new strains of avian influenza viruses. molecular methods have been critical for the rapid identification of new viruses associated with dramatic lethal outbreaks but also for pathogen discovery for routine respiratory illnesses. despite intensive investigation, in - % of lower respiratory illnesses no pathogen can be identified suggesting additional agents may yet be discovered [ ] [ ] [ ] . several different genomic approaches for pathogen discovery have been used successfully and include random primer amplification, pan-viral dna microarray and next generation sequencing ( fig. ) [ ] . if a viral class of an unknown pathogen or variant virus is suspected, consensus pcr using degenerate primers to detect sequences broadly conserved between members of a group can be used as was done to identify two new coronaviruses, hku and mers-cov [ , ] . another technique for viral discovery is random primer amplification with conventional shotgun sequencing of pcr products [ ] [ ] [ ] . such was the case when van den hoogen discovered a new respiratory virus in , in young children with bronchiolitis who tested negative for rsv [ ] . after detecting paramyxovirus like particles in cell culture, rna was subjected to random primer pcr and viral sequences were compared to all known pathogens. the new virus most closely aligned to avian pneumovirus but was determined to be a unique human pathogen and named human metapneumovirus (hmpv). similarly, in peiris identified a novel coronavirus as the cause of severe acute respiratory syndrome (sars-cov) using degenerate/random primers pcr amplification [ ] . using pan viral micro array investigators at the center for disease control and prevention independently identified the same sars-cov [ ] . in this technique, after random primer amplification, pcr products are hybridized to microarrays consisting of mer oligonucleotides derived from every fully sequenced viral genome. hybridized sequences are scraped from the microspot, amplified, cloned and finally sequenced [ ] . identification of completely novel infectious agents requires unbiased and sequence independent methods for universal amplification [ , , ] . conventional sanger sequencing may have poor sensitivity for genomes at low quantity. next generation sequencing (ngs) involves the analysis of millions of sequences and can detect small amounts of novel nucleic acid sequences in clinical samples. continuous sequences are assembled, host sequences are subtracted and the residual sequences are analyzed for similarity to known microbial sequences. ngs has led to the discovery a numerous novel human and animal pathogens [ ] . a recent study of nasopharyngeal aspirates of thai children with respiratory illness using ngs identified a number of mammalian viral sequences belonging to newly described families of viruses such anelloviridae as well as novel strains of hrv, enteroviruses and hbov [ ] . a critical step in viral discovery is the availability of bioinformatic tools to efficiently identify unique viral sequences in complex mixtures of host, bacterial and fungal sequences. new computational tools for analysis of the virome such as "virusseeker" are being developed [ ] . of note, detection does equate with causation and after discovery further studies are necessary to infer more than association. the genetic and antigenic evolution of error prone rna respiratory viruses, particularly influenza, has been of interest for several decades [ , ] . understanding the selective pressure exerted by pre-existing immunity on viral evolution may help design more effective influenza vaccines and surveillance of animal populations can be critical for early identification of emerging influenza viruses [ ] . advances in deep sequencing make it possible to measure low frequency within host viral diversity and factors such as antigenic diversity, antiviral resistance, and tissue specificity can now be studied to understand the complexities of viral evolution [ ] . influenza evolution at a population level has been studied years, yet, new antigenic variants are initially generated and selected at the level of the individual infected host. within a host, influenza viruses exist as a "swarm" of genetically distinct viruses [ ] . sanger sequencing defines consensus sequences and cannot resolve minority variants below % of the viral population. deep sequencing has been used in natural infection and human challenge studies to characterize between and within host genetic diversity [ , ] . the identification of low frequency mutations in the hemagglutinin (ha) antigenic sites or near the receptor-binding domain in vaccinated and unvaccinated influenza infected persons highlight viral evolution within a host due to selective immune pressure [ ] . similarly, ngs can reveal the rapid evolution of drug resistant variants during therapy [ ] . using samples collected over time, the mutational spectrum of h n influenza a virus in an immunocompromised child was delineated [ ] . individual resistance mutations appeared weeks before they became dominant, evolved independently on cocirculating lineages. the within host evolution of antiviral resistance reflected a combination of frequent mutation, natural selection, and a complex pattern of segment linkage and reassortment. within host sequencing diversity has also been examined in an infant with severe combined immune deficiency with persistent rsv infection [ ] . ngs was performed on samples obtained before and after bone marrow transplantation. the viral population appeared to diversify after engraftment with most variation occurring in the attachment protein (g). in addition, minority viral populations with palivizumab resistance mutations emerged after its administration. deep sequencing of hrv during human challenge studies has shown that hrv generates new variants rapidly during the course of infection with accumulation of changes in "hot spots" in the capsid, c, and c genes [ ] . a genome-wide association study (gwas) involves rapidly scanning sets of dna, or genomes, of many people to find genetic variations associated with a particular disease. typically, the genomes of cases are compared to non-affected controls and search for single nucleotide polymorphisms (snps) or polygenic changes that are associated with risk or protection from susceptibility or severity of the condition. gwas have been useful to find genetic variations and risk for asthma, cancer, diabetes, heart disease and autoimmune illnesses with relatively limited studies relating to infectious diseases [ , ] . recent studies examining host genetic factors conferring susceptibility to respiratory viruses such as pandemic h n influenza a, sars-cov and rsv now provide some insight into host genetic factors for respiratory viral infections [ ] [ ] [ ] . previously most influenza research focused on viral genetics of novel viruses, yet experience with h n and h n clearly indicate host factors also influence disease severity [ , ] . a number of candidate genes influencing respiratory virus susceptibility have been identified in animal and human studies and involve host virus interactions, innate immune signaling, interferon related pathways and cytokine responses (table ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . over studies have evaluated genetic polymorphisms associated with severe rsv disease and none demonstrates dramatic results [ ] . most focused on one or a few candidate genes resulting in only modestly increased odds ratios of severe illness. a relatively large study of almost hospitalized children that examined snps in candidate genes demonstrated that susceptibility to rsv is complex with a several associations to a few innate immunity genes. these included a vitamin d receptor gene associated with down regulating interleukin (il- ), gamma interferon (ifn-γ), nitrous oxide synthase (nos a), the jun oncogene, an important transcriptional regulator for innate immune pathways, and ifn-α (ifna ) an antiviral cytokine [ ] . the host transcriptional response can be analyzed to investigate disease pathogenesis using a variety of methods including in-vitro studies of bronchial epithelial cells (bec), animal models and infection both natural and experimental challenge [ ] [ ] [ ] [ ] . in addition, two compartments, the respiratory epithelium and blood can be sampled in human studies and interrogated using different viruses or viral strains to develop gene signatures for prognosis, as indicators of severity and to identify potential therapeutic targets. most respiratory viral mechanistic studies have been performed using influenza viruses, rsv, hrv and coronaviruses [ ] [ ] [ ] [ ] . using bec, the common and (h n , h n , h n and h n ) and analyzed cellular responses using microarray [ ] . common proinflammatory cytokines and antigen presentation were identified although each viral response was unique and notably, h n responses were most similar to h n . the response of different clinical isolates of rsv in a cells, and monocyte derived human macrophages demonstrated that the pattern of innate immune activation was both host cell and viral strain specific [ ] . using rna seq, differences in il- and ccl were noted among the responses to different clinical isolates suggesting different rsv strains may vary in inherent virulence. human studies have shown significant differences in the blood transcriptional profiles which change over time and differ depending on the infecting respiratory virus. mejias and colleagues were able to differentiate rsv, hrv and influenza in young children based on the blood gene profile (fig. ) . hrv infection exhibited the mildest innate and adaptive responses compared to rsv and influenza and neutrophil gene expression was greatest in rsv infection with marked suppression of b and t cell and lymphoid responses [ ] . notably, gene expression changes persisted up to month after infection. similarly, studies of h n infected patients showed transcriptional profile changes persisting up to month with a transition from innate to adaptive immunity [ ] . because of the association of hrv and exacerbations of asthma, the host response to hrv has been of particular interest [ ] [ ] [ ] [ ] . studies using becs from asthmatic and healthy donors demonstrate different transcriptional profiles when infected with hrv [ ] . hrv, similar to other picornaviruses induces gene expression down regulation by the a and c proteins. in both asthmatic and healthy control derived cells the majority of genes were down regulated after exposure to hrv. however, some significant expression differences in inflammatory, tumor suppressor, airway remodeling and metallopeptidase pathways have been noted in asthmatic derived cells. asymptomatic hrv infection is quite common and its role in asthma pathogenesis has been questioned. interestingly, heinonen et al. did not find a difference in the blood transcriptome of asymptomatic hrv infected children compared to non-infected controls [ ] . whereas, wesolowska-anderson and colleagues demonstrated over differentially expressed genes in the nasal epithelium of asymptomatic infected hrv patients [ ] . thus, the blood transcriptome may not be as informative as the nasal epithelial transcriptional response for asymptomatic hrv infection. given the significant host response to asymptomatic infection, hrv may play a role in asthma exacerbations in the absence of clinically evident disease. lastly, it may be possible to identify patients with asthma who are prone to frequent hrv related exacerbations by examining the gene expression response of their pbmcs stimulated with hrv [ ] . gene expression studies focusing on illness severity may enhance our understanding of disease pathogenesis, can identify potential therapies to modulate harmful host responses and can be used to develop biomarkers for predicting life threatening disease [ , [ ] [ ] [ ] . a number of studies have been undertaken to understand the pathogenesis of severe rsv in young children and have identified a variety of gene expression patterns in blood including under expression of t cell cytotoxicity/nk cells and plasma cell genes, as well as upregulation of jak/stat, prolactin, il- signaling, cell to cell signaling, and immune activation pathways [ , ] . using nasal epithelial gene expression analysis, van den kieboom identified differentially expressed genes in children with mild, moderate and severe rsv infection [ ] . ubiquitin d, tetraspanin , mucin , β microseminoprotein, chemokine ligand were up regulated and differentiated mild from severe illness. lastly, nasal gene expression is complicated by interactions of the nasal microbiota and host cell gene responses [ ] . in nasal samples from children with rsv infection, h. influenzae and s. pneumoniae dominated microbiota, toll like receptors and neutrophil/macrophage signaling were over expressed and the presence of h. influenzae and s. pneumoniae along with age and sex were predictive of risk of hospitalization due to rsv. transcriptional profiling related to severity has been analyzed in seasonal influenza as well as emerging avian pathogens with a recognition that disease is not only due to an infection with a novel virus in a non-immune host but may also be due to an exaggerated host immune response [ , ] . in a study of primarily seasonal influenza (h n , h n ), influenza infection was associated with a significantly stronger antiviral, cytokine, attenuation of t/nk cell response compared to patients with respiratory illnesses of unknown etiology regardless of severity [ ] . notably, ifn and ubiquitination was significantly down regulated in those with severe vs. mild to moderate disease. in a study of the lethality of h n influenza and h n vietnam influenza virus in macaques, upregulation of key components of the innate immune response and cell death pathways were noted were noted with h n infection but were down regulated with h n [ ] . early up regulation of the inflammasome likely resulted in some of the severe tissue damage noted with the h n influenza infections. in vitro, animal and human challenge studies have been used to identify new strategies control or prevent symptomatic or severe infection [ , ] . in hrv challenge studies, virperin expression correlated with rhinorrhea and chilliness. knockdown of expression resulted in increased viral replication in becs suggesting virperin has antiviral actions and might have potential therapeutic use. influenza challenge studies clearly show a definable transcriptomic profile in the blood prior to the onset of symptoms offering the possibility of earlier and more effective oseltamivir treatment [ , ] . lastly, host gene expression studies may allow investigation into links between respiratory viral infections and specific non-respiratory events. there is ample epidemiologic evidence that influenza epidemics are linked with increased rates of strokes and myocardial infarction (mi) [ , ] . increased rates of falls and functional decline in nursing homes have also been associated with increased influenza activity [ , ] . however, direct links of events to viral infection are scarce in part due the event of interest may follow the infection by several weeks when the virus is no longer detectable by traditional testing. several gene profiling studies have identified viral infection signatures that may persist up to -month post infection [ , ] . thus, it might be possible to study patients with falls or cardiac events for evidence of recent viral infection using a host response viral signature. in addition, evaluating the host response can provide information on mechanisms of disease. a viral gene signature was used to evaluate patients undergoing cardiac catheterization [ ] . notably, % vs. %, p = . of those with a viral gene signature present vs. those without viral signatures, suffered an mi. furthermore, h n infected patients showed an increased gene platelet expression signature providing insight into how infection may induce a prothrombotic state. given the availability of rapid and accurate multiplex pcr for viral detection, host-based diagnostics might seem unnecessary. however, current pcr assays use conserved known viral sequences but can miss novel or significantly mutated viruses. this issue was seen in with pandemic h n when influenza pcr assays had to be adapted to optimally detect the new influenza strain [ ] . the emergence of novel respiratory viruses are a persistent threat and methods to detect a "viral signature" in the setting of clusters of severe pneumonia cases could be very useful. zaas and colleagues developed an acute respiratory viral gene signature using microarray analysis of the blood from volunteers experimentally infected with influenza a, hrv or rsv [ ] . the signature was subsequently % sensitive and % specific in classifying as viral influenza and hrv infected patients presenting to an emergency room. additionally, a distinct blood transcriptome signature was noted in patients with severe h n pneumonia [ ] . upregulated genes included those related to cell cycle, dna damage, apoptosis, protein degradation, and t helper cells. down regulated genes were primarily in immune response pathways suggesting immunosuppression as a mechanism of severe influenza pneumonia. investigators developed a gene classifier which predicted h n influenza a regardless of concomitant bacterial infection and such a predicator could guide antiviral therapy in the face of negative pathogen detection methods. in most cases of respiratory infection, the precise microbial etiology is unknown and antibiotics are frequently administered empirically [ , ] . although sensitive molecular diagnostics (pcr) now allow rapid diagnosis of a wide variety of respiratory viruses, their impact on patient management and antibiotic prescription has been modest primarily due to concern about bacterial co-infection [ ] [ ] [ ] . approximately % of adults hospitalized with a documented viral respiratory infection have evidence of concomitant bacterial infection and thus clinician concerns are reasonable [ ] . importantly, sensitive and specific diagnostic tests for bacterial lung infection are currently lacking [ , ] . although the site of infection is the respiratory tract, blood is a convenient sample comprised of components of the innate immune system (neutrophils, natural killer cells), as well as the adaptive immune system (b and t lymphocytes) [ ] . recent studies indicate that viral and bacterial infections trigger pathogen specific host transcriptional patterns in blood, yielding unique "bio-signatures" that may discriminate viral from bacterial causes of infection [ ] [ ] [ ] [ ] . in the largest study to date, tsalik et al. used gene expression in blood to discriminate bacterial from viral infection or non-infectious illness in subjects with respiratory illness [ ] . these investigators defined predictor genes in a model with an accuracy of % to discriminate clinically adjudicated bacterial, viral, and non-infectious illness. most studies to date have used micro array but recently rnaseq has been used to differentiate viral and bacterial respiratory illness and in one study genes were noted to be differentially expressed [ ] . three pathways (lymphocyte, α-linoleic acid metabolism, igf regulation pathways) which included genes as predictors for bacterial infection from non-bacterial infection (naïve auc = . ; nested cv-auc = . ). to date, a number of gene expression studies of adults and children have developed predictors with similar accuracy (auc ranging from % to %), yet there has been little overlap in classifying genes identified [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . diverse populations, types of infection, plus alternate analytic tools used, likely explain the different genes identified. more work needs to be done to refine predictive gene sets including patients with mixed viral-bacterial respiratory tract infection. most studies to date have focused on blood; however, analysis of the nasal respiratory epithelium which is the site of infection might offer advantages. although data are limited, several recent papers demonstrate that nasopharyngeal host response can also be used as a diagnostic for respiratory viruses [ , , ] . immune response to influenza vaccine is variable and influenced by a variety of factors including prior vaccinations and infections, age, the presence of underlying conditions and the type of vaccine administered. yet, even among a relatively homogeneous cohort of young healthy adults, antibody responses to vaccine can be variable [ ] . transcriptional profiling of whole blood provide insights into the mechanisms of variability, the effects of age, and vaccine types. the ability to predict vaccine response at baseline based on a transcriptomic signature would have significant clinical implications. to understand the biologic effects of live attenuated influenza vaccine (laiv) compared to trivalent inactivated vaccine (tiv) blood transcription profiles from young children were assessed by microarray at day post vaccination [ ] . many more genes were differentially expressed in children receiving laiv compared to tiv ( vs. , respectively) and many modulated type ifn. the efficacy of laiv has been problematic in recent years and assessing stimulation of type ifn genes could represent a potential biomarker for response to laiv [ ] . bucasas and colleagues evaluated gene expression at multiple time points after vaccination of healthy young men with tiv [ ] . they noted marked up regulation of gene expression of ifn signaling, il- regulation, antigen processing and presentation genes within h of vaccination and were able to define a gene expression signature that correlated with the magnitude of antibody response. in another study, a gene profile predictive of antibody response days after influenza vaccination of young and older adults was developed [ ] . notably, the predictive genes were the same in young and old as well as a subgroup of subjects with diabetes suggesting similar pathways were involved despite differences in age and underlying medical conditions. additionally, transcriptional profiling has been used to signatures in blood associated with b cell memory responses to vaccination. in a study of older and middle aged adults vaccinated with tiv including an h n antigen, metabolic, cell migration/adhesion, map kinase and nf-ᵏb cell genes correlated with peak memory b cell elispots [ ] . finally, in a study of over subjects vaccinated over several seasons, a predictive signature of nine genes and three gene modules were significantly associated with the magnitude of the serum antibody response (fig. ) [ ] . interestingly and in contrast to a previous study, the signature was distinct to the younger cohort. for example, inflammatory genes were associated with better response in the young but a worse response in the elderly. in summary, gene expression studies could be used to evaluate new vaccines and develop predictors of vaccine response in different subgroups of patients based on age and disease state allowing for individualized vaccine regimens. molecular analysis of respiratory viruses and the host response to both infection and vaccination have transformed our understanding of these ubiquitous pathogens. the ability to accurately diagnosis viral infections has not only impacted patient care but also changed our perceptions of the burden of disease and populations effected. transcriptional profiling of blood and nasal epithelium may provide therapeutic targets to prevent and ameliorate illness as well as offer predictors of severe disease. 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correlate with the humoral immune response to influenza vaccination in humans systems biology of vaccination for seasonal influenza in humans transcriptional signatures of influenza a/h n -specific igg memory-like b cell response in older individuals multicohort analysis reveals baseline transcriptional predictors of influenza vaccination responses key: cord- -ear cyri authors: bakker, craig; halappanavar, mahantesh; visweswara sathanur, arun title: dynamic graphs, community detection, and riemannian geometry date: - - journal: appl netw sci doi: . /s - - - sha: doc_id: cord_uid: ear cyri a community is a subset of a wider network where the members of that subset are more strongly connected to each other than they are to the rest of the network. in this paper, we consider the problem of identifying and tracking communities in graphs that change over time – dynamic community detection – and present a framework based on riemannian geometry to aid in this task. our framework currently supports several important operations such as interpolating between and averaging over graph snapshots. we compare these riemannian methods with entry-wise linear interpolation and find that the riemannian methods are generally better suited to dynamic community detection. next steps with the riemannian framework include producing a riemannian least-squares regression method for working with noisy data and developing support methods, such as spectral sparsification, to improve the scalability of our current methods. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. community detection is an important activity in graph analytics with applications in numerous scientific and technological domains (girvan and newman ) . given a graph g = (v , e) with weight function w : e → + , the goal of community detection (or graph clustering) is to partition the vertex set v into an arbitrary number of disjoint subsets of v called communities (or clusters) such that the vertices within a community are tightly connected with each other but sparsely connected with the rest of the graph. clustering on g can be represented as c(g), which is a unique mapping of each vertex to a community. we restrict our work here to undirected, unweighted graphs and to the disjoint partitioning of vertices into communities. for a detailed treatment of this topic, the reader is referred to the work by fortunato ( ) . the relationships between entities in domains such as sociology, finance, cybersecurity and biology are most naturally modeled with the use of graphs. the inherently dynamic nature of such data (fenn et al. ) leads to dynamic graph representations. a dynamic graph changes over time through the addition and deletion of vertices and edges. a snapshot of this graph, g n , consists of the vertices and edges that are active at a given time step n. modifications from time n to n + are represented by g n . clustering can be performed at each time step, c(g n ), and as the graph evolves, so do its communities. temporal communities can undergo several different transitions: growth via addition of new vertices, contraction via deletion of vertices, merging of two or more communities, splitting of a community into two or more communities, birth and death of a community, and resurgence or reappearance of a community after a period of time. efficiently detecting these transitions is a challenging problem. the problem of dynamic community detection has received significant interest in the academic literature (cazabet and amblard ) . current approaches for dynamic community detection broadly fall under two headings: incremental community detection and global community detection. the approaches in the first category focus on the systematic propagation of communities through time, whereas the approaches in the second category attempt to simultaneously optimize for multiple metrics on several snapshots of data. stability of computation and accuracy of results are the fundamental limitations of the incremental approaches, while memory (space) and computational requirements are the main limitations of the global approaches (cazabet and amblard ) . incremental approaches are fundamentally combinatorial in nature (tantipathananandh and berger-wolf ; nguyen et al. ) and involve methods to track communities through time. the stochastic nature of these algorithms makes these methods unstable leading to inaccurate results. mucha et al. ( ) build on the seminal work of lambiotte et al. ( ) for community detection in dynamic multiplex networks by specializing null models in terms of stability under laplacian dynamics. there is a well-developed suite of methods for community detection in static graphs, but it is not always clear how to extend those methods to dynamic graphs in a way that captures the time-varying nature of those graphs' communities. the challenge is to develop methods that vary continuously in time, like the graphs themselves, between snapshots. moreover, if existing methods are extended through time, it will be beneficial to do so in a way that provides new insight or analytical tools as well. with that in mind, we propose a riemannian geometry approach that views dynamic graphs (and thus dynamic communities) through the lens of laplacian dynamics on a matrix manifold. riemannian geometry provides ways of calculating quantities such as distances between laplacians and trajectory speeds on the matrix manifold. as such, it provides a clear and consistent way of representing graph dynamics. this framework is also modular with respect to existing static community detection methods. in this paper, we provide the background theory needed to describe dynamic graphs in terms of laplacian dynamics on matrix manifolds. the primary contribution of this paper is to bring existing theory to bear on a new application area -dynamic community detection. we use riemannian geometry to interpolate between snapshots of dynamic graphs (using geodesics) and to calculate averages of those snapshots; we explicitly show the formulae for performing these calculations. the interpolated and average graphs are then amenable to existing static community detection methods. this allows us to use a consistent approach to track community behaviour both between snapshots, via interpolation, and across snapshots, via averaging. simply transferring previously derived formulae would not allow us to consider disconnected graphs, however, so our contributions also include a way of transforming disconnected graphs so that they are amenable to the matrix manifold tools. using both synthetic and experimental graph data, we experimentally evaluate two different kinds of geodesics. we identify their strengths, as compared with entry-wise linear interpolation, and also discuss their weaknesses. finally, we derive interpolation and extrapolation error bounds for both geodesics (shown in the appendix) and identify promising avenues of future research in this area. our framework enables more accurate prediction of community transitions by building interpolated graphs between snapshots, global community detection through data aggregation, and prediction of future behaviour through extrapolation from given snapshots. we describe the basics of our framework in the "riemannian geometry and dynamic graphs" section, show how it can be applied to dynamic clustering in "a riemannian framework for dynamic community detection" section, and compare the riemannian methods with an entry-wise linear approach on synthetic and real network data in the "computational experiments" section. the novelty of our approach arises primarily from the application of riemannian geometry to dynamic graphs. when combined with existing spectral methods, this also provides a new interpretation of community splitting and merging as bifurcations in a gradient flow dynamical system (see the "dynamic spectral clustering" section). to the best of our knowledge, the riemannian framework presented in this paper is the first of its kind; it is our intent that the research community build from and extend this work to enable features of dynamic community detection not currently considered here. differential geometry deals with mathematics on manifolds; manifolds are spaces that are locally euclidean (i.e., flat), but generally non-euclidean globally (boothby ) . a riemannian manifold is a type of manifold that has a metric associated with each point on the manifold. the traditional methods for calculating angles and distances in flat spaces have to be modified on manifolds to account for manifold curvature, and the metric is an integral part of those modifications on riemannian manifolds. a key part of riemannian geometry, for the purposes of this paper, is the geodesic. geodesics are the equivalent of straight lines in curved spaces. a geodesic is (locally) the shortest path between two points. great circles on a sphere are examples of geodesics on a curved manifold. consider a flight from vancouver, canada to london, england: the two cities are at similar latitudes, so on a mercator projection map, the shortest flight would seem to be a straight west-to-east trajectory. in reality, however, flights between the two cities traverse the pole because that is a shorter route -it is the great circle route. the discrepancy is due to the curvature of the earth, which is distorted on a flat map. from another perspective, a geodesic is the path that a particle on a manifold would take if it were not subject to external forcing; a geodesic with constant speed has zero acceleration. riemannian geometry can be applied to matrix manifolds. the grassman and stiefel manifolds are perhaps the most frequently encountered matrix manifolds in differential geometry because they have closed-form solutions for quantities such as geodesics (absil et al. ). pennec et al. ( ) developed a metric for the manifold of symmetric positive-definite matrices with corresponding expressions for distances, geodesics, and tangent vector inner products in closed form. these formulae are valuable because even when there is a well-defined metric on a manifold, distances and geodesics between points do not usually have closed-form expressions. such quantities have to be solved for numerically. working on this matrix manifold, when appropriate, can be useful: matrix symmetry provides a reduction in effective dimension, and properties such as symmetry and positive-definiteness are automatically preserved. bonnabel and sepulchre ( ) extended this framework to include symmetric positive-semidefinite matrices. the extension essentially worked by decomposing a positive-semidefinite matrix into a nullspace component (a grassman manifold) and a positive-definite component, which could then use the existing metric. researchers have previously used non-euclidean geometries to investigate graphs (krioukov et al. . that work has then been applied to large-scale networks such as the internet (boguná et al. ) . the approach described in this paper differs in a subtle but meaningful way. in those papers, the mappings used treat graph nodes as points in a hyperbolic space. our present work, however, treats the entire graph as a single point in a non-euclidean space. the work of bonnabel and sepulchre ( ) combined with that of pennec et al. ( ) enables us to consider graph laplacians as points on a manifold of positive-semidefinite matrices. each graph is a point, and thus a time-indexed sequence of graphs forms a trajectory on the manifold. this, in turn, means that we can calculate quantities such as trajectory velocities, distances between graphs (represented by manifold distances between their respective points), and relevant geodesics. given that we are interested in dynamic community detection, the laplacian is a natural object to work with. the laplacian uniquely defines a graph (up to self-loops), and there is already a known connection between the laplacian spectrum and community structure (newman ) . previous work in dynamic community detection (e.g., mucha et al. ( ) ) has also worked with the laplacian. graph laplacians have a certain structure that make them amenable to the riemannian geometry techniques presented here as well: laplacians are symmetric (for undirected graphs) and positive-semidefinite. adjacency matrices, for example, are generally indefinite and thus would not be suitable for use with the matrix manifolds described here. we chose to work with the combinatorial laplacian, l = d−a, because it has a constant nullspace for connected graphs (newman ) . this constant nullspace makes the geometric calculations much simpler than they would be otherwise. it is possible to use other laplacians, such as the normalized laplacian. if these laplacians do not have constant nullspaces, though, the interpolation involves extra calculations (detailed by bonnabel and sepulchre ( ) ). assuming no self-loops, the combinatorial laplacian also has the virtue of being easy to convert into an adjacency matrix. that being said, as long as a laplacian is symmetric positive-semidefinite and has a constant nullspace dimension (for connected graphs), it is possible to calculate geodesic interpolations for that laplacian. there are two other relevant considerations we wish to address here. firstly, the laplacians of unweighted graphs constitute a discrete (and therefore sparse) subset of the matrix manifold. as such, any continuous trajectory will contain weighted graphs. secondly, directed graphs do not have symmetric laplacians, and thus they cannot be considered within this framework without symmetrizing them somehow (e.g., by ignoring the directionality of edges). for the purpose of community detection, though, edge direction may not be important. there are two primary components to our framework. the first involves modelling and analyzing the dynamic behaviour of the graph prior to any community detection. for this, we show how to calculate an average graph from a collection of snapshots (for use in a time-averaged community detection) and how to interpolate between time-indexed graph snapshots (for seeing how the graph evolves over time). in the appendix, we derive and analyze bounds on the interpolation error in terms of distance on the manifold. the second component consists of applying community detection methods to the dynamic graph. in this paper, we will focus on spectral methods, because they have convenient properties under continuous laplacian dynamics, and the louvain method (blondel et al. ) , because of its computational speed and ability to handle disconnected graphs. however, the riemannian geometry methods do not require using any one particular community detection method. we begin with interpolation between two snapshots. it is possible to do this using an entry-wise linear approach, l(t) = ( − t) l a + tl b , but there are good reasons not to use this approach. firstly, the laplacians for a given dynamic graph all exist on a matrix manifold. for the trajectory l(t) on that manifold, though, the trajectory speed is not constant, the trajectory direction is not constant, and it is not the shortest path from l a to l b . it is precisely analogous to the mercator projection map example given earlier -moving at a constant velocity (i.e., constant speed and direction) on the map would not correspond to moving at a constant velocity on the earth because of the earth's curvature. experimentally, we have observed that the linear interpolation begins and ends its trajectory moving very quickly while the bulk of its trajectory moves relatively slowly. the difference between maximum and minimum velocities can be orders of magnitude, depending on the size of the graph and the distance between the two graphs being interpolated. secondly, in connected graphs, the product of the laplacian's non-zero eigenvalues (i.e., the determinant of the positive-definite component) is concave along the linearly interpolated trajectory. if the two points are far enough apart, this product will go through a maximum between the two points. this maximum can, again, be orders of magnitude greater than the product at either endpoint; like the trajectory velocity, this variation will depend on the size of the graphs in question and their distance apart. the geodesic interpolation, however, provides a linear variation in the product of the eigenvalues. pennec et al. ( ) comment on this in more detail. for a graph, this product relates directly, by kirchoff 's matrix tree theorem, to the number of spanning trees in the graph (harris et al. ) . in other words, the linear interpolation increases the overall connectivity of the graph between snapshots. finally, the linear interpolation cannot always be used for extrapolation. all of the interpolated laplacians are positive-semidefinite, but it is easy to provide examples where the extrapolation quickly becomes indefinite. instead, we propose using geodesic interpolation. a geodesic interpolation trajectory has a constant velocity, produces an eigenvalue product that varies linearly between endpoints that are connected graphs, and can be extrapolated indefinitely without leaving the manifold of positive-semidefinite manifolds (with constant nullspace dimension). following bonnabel and sepulchre ( ) , we show how to calculate this geodesic between two snapshots of a given dynamic graph. consider the laplacian l at a point. it can be represented with its eigendecomposition: where the columns of α span the range of l. moreover, the nullspace, ξ , is always parallel to ( , , . . . , ), and thus span(α) is constant even though α may not be, in general. consider the geodesic between l a and l b . we can calculate the svd of α t b α a : the diagonal matrix σ ab has the principal angles between the subspaces spanned by α a and α b as its diagonal entries. since those subspaces are the same, σ ab = i for any two laplacians. we then calculate , and u is constant for all points on the geodesic; α and o are not constant, though. furthermore, we can use the same u matrix for any laplacian of a given dynamic graph without affecting our calculations, because the span of u is constant. we calculate r = u t lu for l a and l b . the geodesic from l a at t = to l b at t = is then if there are multiple time-sequenced snapshots, this method can be used to do a piecewise geodesic interpolation with t being shifted and scaled appropriately. note that the constant laplacian nullspace means that we can work solely with the r components of l and ignore the grassman component. we can also extrapolate with this geodesic simply by continuing the trajectory for t > . if we are interested in the average behaviour of a dynamic graph, we can calculate the least-squared-distance mean (the karcher mean) of a set of graph snapshots. to do this, we use the r matrices derived from the graph laplacians as before; each graph i has a matrix r i associated with it, and we want to determine the 'average' matrix s for n snapshots. we then list the sum-of-squared-distance function, the distance function itself, and the gradient of the squared distance (pennec et al. ) , respectively: we use iterated gradient descent to calculate the mean: according to pennec et al. ( ) , this usually converges quickly. riemannian geometry centers around the riemannian metric -changing the metric entails changing properties of the manifold (such distances and geodesics). the current metric can be described as affine-invariant (pennec et al. ), but it is not the only metric that could be used for the space of positive-definite matrices. we could also use a log-euclidean metric as described by arsigny et al. ( ) . the primary reason to consider using the log-euclidean metric instead of the affine-invariant one is computational cost: the formulae for distances and geodesics are simpler and easier to calculate for the log-euclidean metric. those distance and geodesic formulae are, respectively, another computationally beneficial feature of the log-euclidean metric is the closedform expression that it has for calculating the mean of a set of matrices: to utilize these formulae for interpolating between graphs, we would simply replace eq. with eqs. , with eq. , and the iterated process in eq. with a single evaluation of eq. . there are other expressions that are simpler to evaluate for the affine-invariant metric, but those quantities may not be needed, and the different invariance properties of each metric may be valuable in different circumstances. on a practical level, the two metrics generally produce similar interpolations (arsigny et al. ) : the spectrum of the affine-invariant interpolations tends to be slightly more isotropic than that produces by the log-euclidean interpolations, but both interpolate determinants linearly between interpolation points (see the "graph interpolation and averaging" section). for the rest of this paper, we will distinguish the geodesics and means calculated with the two methods as being either affine-invariant (ai) geodesics or log-euclidean (le). the methods described in this paper currently assume that the graph in question is connected and remains so at all points of interest. as they stand, they could potentially handle a graph with a constant number of disconnected components (which would correspond to the laplacian nullspace having a constant dimension), but this does not significantly improve the method's generality. in order to be widely applicable, the interpolation methods need to be able to handle changing connectivity. we can accommodate this by using a bias term with, potentially, a thresholding procedure. for a given adjacency matrix a, we add to each off-diagonal entry a bias term /n, where and n is the number of vertices in the graph, to produce a biased adjacency matrixà (which is now connected). we then construct a biased laplacian matrix from a, perform the interpolation on the biased laplacian and subtract /n from each offdiagonal entry of the adjacency matrices produced by the biased interpolation. if need be, we can then apply a threshold to the resulting adjacency matrices or round those matrices to an appropriate number of decimal places. this approach essentially replaces the laplacian's λ = eigenvalues with λ = . empirically, we found that this approach did not significantly change the interpolated trajectories for connected graphs while also producing reasonable results for disconnected graphs. if we consider the properties of the riemannian metrics discussed in this paper, we can see why adding this small bias would not significantly disturb a geodesic trajectory. with these metrics, matrices with zero or infinite eigenvalues essentially exist at infinity. for matrices with finite eigenvalues greater than zero, the distances between matrices are relative and directly tied to the matrices' spectra. for example, the distance from λ = − to λ = − is comparable to the distance from λ = to λ = . this means that a geodesic, which is a minimum-distance path between points, will not significantly alter the part of the spectrum associated with λ = values unless it is absolutely necessary to do so in order to reach the destination. moreover, adding a fully connected graph with edge weights of would not meaningfully change the community structure because of the separation of scales (presuming a very small value of ). in our computational experiments, we found that = − provided a good balance between avoiding ill-conditioning and keeping small, but even increasing to − did not change the interpolation significantly. as we increased , though, we found that the geodesic interpolations approached the trajectory of the linear interpolation; at, say, = , they were almost identical. this, too, makes sense: as the eigenvalues become uniformly larger, the manifold becomes flatter, and the differences between the data points become smaller. the flatter the manifold, the closer the geodesic is to the linear interpolation. however, the geodesic interpolation is still guaranteed to remain positive definite, and the linear interpolation is not. this suggests that if the linear interpolation were more desirable in a particular application but the application also called for the use of extrapolation, then using a geodesic with a large bias term could provide the desired capabilities. it is possible to use spectral clustering with the first non-trivial eigenvector for community detection, but this method can be improved upon by using multiple eigenvectors (boccaletti et al. ). this approach is convenient for continuous laplacian dynamics because as long as the eigenvalues are distinct, we can expect the eigenvectors and eigenvalues to vary smoothly with smooth changes in l. if the eigenvalues of the eigenvectors in question are not distinct, then the eigenvectors are not uniquely defined, and if eigenvalues whose eigenvectors are being used for spectral clustering cross during the course of a trajectory, the spectral clustering may experience a discontinuous jump. disconnected graphs can provide exactly this kind of behaviour (e.g., with multiple zero eigenvalues). moreover, if the number of disconnected components is not constant, then it will not suffice simply to consider the first m non-zero eigenvalues, for the set of such eigenvalues will not be constant. assume that the graphs are connected, that there is an ordering of the eigenvalues of l such that λ i ≤ λ i+ , λ = , and that eigenvector ξ (i) is associated with λ i . we can then plot each of the graph nodes in n , where node k has coordinates given by n+ ) , and use clustering techniques to identify communities. one way of identifying and tracking communities is through defining a kernel for the nodes. summing over all of the nodes then produces a density function. the maxima of that density function correspond to cluster centroids, and the separatrices between maxima define community boundaries in the (reduced) eigenspace. with a symmetric gaussian kernel, this density function would be . other kernels could be used, but this provides an easily differentiable density function, and the magnitude of the kernel is not very important -what matters is the relative changes in density, not the function's absolute value. see an example of this in the spectral plot shown in fig. . the format of fig. is used for all other spectral plots in this paper. changes in the graph's communities can then be seen as changes in the density function. the density of a cluster is proportionate to the magnitude of the density function at the peak (i.e., the cluster centroid). community growth and contraction can be seen by points traversing community boundaries (i.e., separatrices). birth and death correspond to the emergence or disappearance of a peak in the density function. merging and splitting correspond to the merging and splitting, respectively, of the density function peaks. this splitting and merging correspond very closely to pitchfork bifurcations fig. -d spectral plot of graph nodes. the graph nodes are plotted as points, the contours show the magnitude of the density function, and the horizontal and vertical axes correspond to the ξ ( ) and ξ ( ) components, respectively. this particular plot shows two distinct communities with one node at approximately (- . ,- . ) that does not belong very strongly to either community and a cluster of points around (- . , . ) that seems close to forming its own community in dynamical systems; more precisely, the pitchfork bifurcation happens to the gradient flowẋ = ∇f . birth and death also correspond to pitchfork bifurcations, but this is not as immediately obvious. it is a corollary of the poincaré-hopf theorem: creating a new maximum results in the creation of additional saddle points and/or minima (domokos et al. ) . to identify death, merging, or splitting, we can track the hessian of f. if it becomes singular at a point, that is an indication of a potential bifurcation there. birth may be identified in the same way, but searching the space for such a phenomenon may be more difficult than simply tracking known maxima and monitoring the hessian at those points. once the spectrum has been plotted, techniques such as k-means clustering can identify communities. this should produce a sufficient approximation of the separatrices between maxima. however, if two eigenvectors are used, it may even be easier to identify communities visually. to demonstrate our methods, we initially created a series of graph snapshots using a synthetic graph process. the dataset was created by generating two erdős-rényi (er) random graphs with nodes each, as representing distinct communities, with edge probabilities of p e = . for both. we then began connecting the nodes belonging to the two communities through an inter-community edge probability of p int p e ; we increased p int all the way to p e to simulate the distinct communities merging. once the merger was complete, we gradually decreased p int to simulate the splitting of a large community into smaller ones. to test our methods on real-world data, we used proteomics data produced by mitchell et al. ( ) . networks were produced by identifying subnetworks of upregulated proteins (p < . and fold change > . compared to uninfected mocks) from the overall human protein-protein interaction network (keshava prasad et al. ) . the network data indicates time-varying linkages between different proteins in human lung epithelial cells that have been infected by the severe acute respiratory syndrome corona virus (sars-cov). the proteomics network formed a relatively sparse, highly disconnected graph of nodes, and we used the data snapshots at t = , , , , , , and , where t is the number of post-infection hours. because this graph is disconnected (and severely so), we use the bias approach described in the "disconnected graphs" section. we implemented our methods in python, making particular use of the matrix exponential and logarithm functions in the scipy package. to evaluate the interpolation and averaging results for the synthetic network, we recorded connectivity measurements, spectral snapshots from interpolated and averaged laplacians, and the total number of communities in the interpolated and averaged laplacians. to measure connectivity, we used the logarithm (for scaling purposes) of the product of the non-zero laplacian eigenvalues as mentioned in the "graph interpolation and averaging" section. for the spectral snapshots, we used the eigenvectors corresponding to the first two non-trivial eigenvalue to produce plots as described in the "dynamic spectral clustering" section. these snapshots provided an evaluation that was more qualitative than quantitative. we then used the louvain method to perform community detection. the graph snapshots are provided in additional file , and the code implementing the methods is provided in additional file . the spectral snapshots and connectivity measurements were not as useful for the proteomics network because the proteomics network was highly disconnected, but the louvain method was still applicable for community detection. to investigate the interpolation and averaging of community structure for this network, we tracked the total number of communities, the total number of communities with at least five members, community similarity, and graph energy. because the network was highly disconnected, the louvain method produced many small or single-member communities. tracking the number of communities above a certain size helped to reduce the amount of noise due to that effect. by community similarity, we mean not just the number of communities but the composition of those communities as well. it can be difficult to measure the degree of similarity between two graphs' community structures when there are many communities and the community labelling is not consistent, but we can look at the pairwise similarity with the rand index (rand ) . the rand index works by using a baseline or ground truth case, considering every distinct pair of nodes, and determining whether or not they are in the same community. it then looks at these same pairs in another graph of interest. if, for a given pair of nodes, the nodes are either in the same community as each other in both graphs or not in the same community as each other in both graphs, that pair gets a score of ; otherwise they get a score of , indicating a dissimilarity between the community structures of the two graphs. summing the results over all pairs and dividing by the number of pairs yields a score between and , where indicates that the two graph's community structures are identical. the smaller the value, the less similar the structures are. given that we had no ground truth between the data snapshots, we instead looked at the changes in this metric from one snapshot to the next. ideally, there would be a steady change in this value between points -a sawtooth pattern over the course of the whole interpolation -as we measured how the interpolation differed from the most recent data snapshot. finally, to measure network connectivity, we used graph energy instead of a laplacian eigenvalue product. the energy of a graph, e, is defined as the sum of the absolute values of the eigenvalues of the adjacency matrix. given that it is bounded by the number of edges, m, in an unweighted graph (brualdi ), we can also use it to bound the number of edges: and thus it gives us information about both graph spectra and graph connectivity. for both sets of data, we used thresholding on the edge weights to get unweighted graph equivalents. this procedure, and especially the threshold value used, was more impactful on the proteomics data than on the synthetic data. the graph spectral snapshots are shown in fig. , and we can clearly see the expected merger and separation of two communities there. we can now interpolate from the third to the fourth data snapshot and then from fourth to the fifth data snapshot to further investigate this community merger and separation. snapshots from the ai geodesic interpolation are shown in fig. ; the results from the linear and le geodesic interpolations fig. synthetic graph spectral plots, frames - . the spectral plots of the synthetic data snapshots are presented in order from left to right, and top to bottom. they show two communities that are stable and separate except for the merger shown in the fourth frame. there are also nodes that do not associate closely with any community at various points in time were almost identical with these. increasing the temporal resolution would become increasingly cumbersome for presentation in a printed format. however, the method does lend itself well to video presentations of the dynamic community behaviour (see additional file for an example). synthetic graph interpolation. the interpolation's frames are presented from left to right, and top to bottom. at the top left, the first frame is the third data snapshot, the sixth frame is the fourth data snapshot, and the eleventh frame is the fifth data snapshot; the interpolated frames are taken at evenly spaced time intervals between the data snapshots. the interpolated frames show a clear progression of community merging and splitting as well as some outliers that do not seem strongly attached to any community in fig. , we can see how the graph connectivity changes over time. the geodesic curves both interpolate the eigenvalue product linearly between points, whereas the linear interpolation is slightly concave. for this dynamic graph, the data points are relatively close to each other, and thus the geodesic and linear interpolations are very similar. if we interpolate between t = , t = , and t = , we can see the distinction more clearly, as in fig. . thresholding gives us a piecewise constant graph. the graph dynamics consist of an edge addition phase followed by an edge subtraction phase, so the thresholding parameter simply determines when that entry flips from to (or vice versa). if we were to use a finer time resolution, we might see a slight difference between the linear and geodesic interpolations with respect to when this transition happens, but the basic behaviour would remain the same. in performing community detection, we found that the geodesic interpolations produced adjacency matrices with negative entries. almost all of these entries were on the order of . to . , and none were larger than . . negative edges need not be a barrier for community detection (e.g., see traag and bruggeman ( ) ), but they can cause problems for the louvain method, so in doing community detection, we simply set these entries to . this was only necessary for community detection on graphs that did not use thresholding. when using a threshold, any value equal to or below the threshold, including a negative value, was set to . the spectral plots showed two communities merging and splitting with some outliers along the way. we found that the louvain method split the merged community into four, and the outliers sometimes formed very small communities of their own (fig. ) . the difference in results between the two methods suggests that in community detection, it may be worthwhile to be able to assign an 'unaffiliated' status to some nodes -nodes that are not really part of any community. this kind of behaviour is what gives us, for example, the brief existence of a small community (of size ) in the le geodesic interpolation between logarithm of product of non-zero eigenvalues over time with longer interpolation window (no threshold), synthetic graphs. interpolating between graphs that are 'farther apart' leads to a more apparent distinction between the geodesic and linear interpolations. the ai and le geodesic are still indistinguishable with regards to the connectivity measure, however t = and t = . when we use a threshold, these behaviours cease, as we now have a graph that is piecewise constant in time for all three interpolations. finally, we consider the average behaviour of this graph using the mean graphs produced by each interpolation method. the spectral plots of these graphs are shown in fig. , and they clearly show two distinct communities. this indicates that the merging of the two communities was only a transient effect and that the same communities reemerged after the temporary merger. the averaging process preseved the structure that we designed the dynamic graph to have. if the second pair of communities were significantly different from the first, then the spectrum of the average graph would not display two distinct communities so clearly. table illustrates the similarities while highlighting the small differences between the results: the geodesic interpolations consistently have slightly higher modularity and slightly lower connectivity than the linear interpolations, but thresholding the resulting graphs reduces those differences. this is not surprising given both the propensity that linear interpolations have for increasing connectivity and the similarity of the geodesic and linear interpolations in this case. in interpolating the proteomics network data, we again obtained negative adjacency matrix entries (around % of the total entries). the ai geodesics produced far fewer such entries than the le geodesics (by an order of magnitude), and the ai entries were usually smaller. of the negative entries, the largest was - . , but less than % of the negative entries had magnitudes greater than . . as with the synthetic graphs, we simply set these negative entries to when using the louvain method. figure shows how the number of graph communities varied over time and how different thresholding levels affected those results. with no thresholding, we found that fig. communities in interpolated synthetic graphs. when no threshold is applied (top), the louvain method produces varying numbers of communities during the merger of the two original communities, and even the data snapshot of the merged communities shows not one but four communities; we also see some differences between the two geodesic interpolations. with a threshold (bottom), though, the piece-wise constant nature of the interpolation shows forth the results were too connected (i.e., not enough communities) for all three interpolation methods: after leaving a supplied data point, the number of interpolated communities would immediately drop, remain relatively constant, and shoot up upon reaching the next data point. thresholding produced better results. generally speaking, the ai geodesic produced too many communities while the linear interpolation produced too few, and neither produced a steady deformation from one data point to the next. the le geodesic showed an intermediate behaviour in this regard, and a threshold of . produced best performance. the number of communities produced by the interpolation did not vary smoothly, but there was a general progression from data point to data point. changing the threshold value had a small effect on the ai geodesic, but it did nothing to improve the linear interpolation, and using a threshold value of . actually produced an odd spike in the number of communities halfway between data points. we will return to this phenomenon later. in looking at fig. , though, we see that there are many communities relative to the size of the graph -most of these are communities of one or two nodes that are not connected to the rest of the graph. if we only consider communities of a certain size, we can get a more accurate picture of the true community dynamics. in fig. , we look only at communities that contain at least five nodes and consider how the results are affected by different threshold values. when using a threshold value, the results are somewhat similar to those in fig. . in fig. , there were too few communities because the graph was more connected, and we observe the effects of that increased connectivity here, too: there are fewer communities overall, but the communities that are present tend to be larger, and there are more large communities. the geodesic interpolations, on the other hand, were less connected. therefore, they had had many small communities and relatively few larger ones; the best results came from the le geodesic with a small threshold. the case without thresholds was more interesting. there, the linear interpolation still often produced too many communities, but the geodesic results did not uniformly produce too few communities. the le geodesic may have been slightly better than the ai geodesic, but they were both still producing results that looked much more reasonable than they had when we plotted the total number of communities. in fact, those results look even more regular and smooth than the thresholded results. with some analysis, we can see why using a threshold value of . produced odd spikes in the number of communities for the linear interpolation. let us assume that we are interpolating from adjacency matrix a to adjacency matrix a . let us denote the edges in a that are not in a with the adjacency matrix a add and the edges in a that are not in a with the adjacency matrix a sub . our linear interpolation from a at t = to a at t = would then be if we use a threshold τ such that matrix entries greater than τ are sent to and entries less than or equal to τ are sent to , we get two possible interpolation patterns, each with three interpolated values. if τ < . , then if τ ≥ . , then a −a sub will be less connected than either of the interpolation end points, and if τ = . , then a(t) = a − a sub only at t = . . that is why we see that spike in the number of communities. the community similarity results are shown in fig. . with no thresholding, the linear interpolation performs best. both of the geodesics tend to become even less similar to the previous snapshot than the snapshot they are progressing towards, resulting in a u-shape, whereas the linear interpolation has a more consistent decrease. all three interpolations, though, show a sharp decrease in similarity immediately after leaving a snapshot. surprisingly, the le geodesic also produces more extreme results than the ai geodesic. thresholding produces the best result, and it does so with the le geodesic and a threshold of . . the linear interpolation once again shows its piecewise constant behaviour, but fig. community similarity in interpolated proteomics network. when no threshold is applied (bottom right), the le geodesic displays more extreme behaviour than the ai geodesic. a threshold of . (top right) gives the best performance for the geodesics, a threshold of . (top left) produces excessive variation in the le geodesic, and a threshold of . (bottom left) produces almost piecewise constant behaviour in the geodesics. the linear interpolation produces reasonable results when no threshold is applied a threshold of . is no longer optimal for the le geodesic, and the ai geodesic performs reasonably well at that threshold value. plots of the energy of the interpolated graphs are shown in fig. . when no threshold is applied, the linear interpolation produces an almost linear progression, whereas both geodesic methods go through significant minima between data points. the geodesics are designed to interpolate laplacian eigenvalue products linearly, whereas the linear interpolation produces a linear variation in the eigenvalue sum. linearly changing the sum produces a concave change in the determinant, as we saw in fig. , and we can now see that linearly changing the product produces a convex change in the sum. the interpolations in question are being performed on the laplacian, not the adjacency matrix, but we can see a clear connection. when we look at the thresholded results, we see that the linear interpolation consistently produces graphs with high energy values, the ai geodesic produces graphs with low energy values, and the le geodesic is somewhere in the middle. for the le geodesic, the best threshold value is around . , where the interpolation produces a relatively steady change in graph energy from data point to data point (unlike the linear and ai geodesic interpolations, which basically plateau between points). this is consistent with what we saw in fig. and what we know about sparsity and the different interpolations. finally, we can look at the average graphs calculated using the three different methods. table shows the number of communities for each of the averaged graphs, and table shows the number of communities with at least five nodes in those graphs. the average graph without thresholding showed a much higher level of connectivity than any of fig. graph energy in interpolated proteomics network. applying thresholds of . (bottom left), . (top right), and . (top left) produced the same kind of trends in the interpolations' graph energy as was the case in considering the number of communities: low-energy (i.e., less connected) graphs with the ai geodesic, high energy (i.e., more connected) graphs with the linear interpolation, and graphs of varying energy with the le geodesic. interpolation without a threshold (bottom right) gave similar performance for the geodesics the data snapshots, and this was the case for all of the averaging methods. this would make sense if the community structure changed significantly from snapshot to snapshot. thresholding the average graph produced more reasonable results, though the ai average was highly disconnected, and the linear average showed a very large change in behaviour when the threshold dropped below . . table records results congruent with those in table . with the linear mean graph, we see more communities with at least five members than any of the individual graph snapshots have -again, the linear interpolation produces results with increased connectivity. the riemannian mean graphs without thresholds produce more reasonable numbers of communities, but applying a threshold to the geodesic means severely reduces those numbers. the most reasonable result with a threshold seems to be the le mean with a threshold of . or the linear mean with a threshold of . . next, we can look at the average difference in community assignment between the mean graphs and the data snapshots in table . the linear mean performs better than the others when no threshold is used, but with a threshold, the best results come from the riemannian means (which are almost identical). these values are quite high -both here and in the interpolation results shown in fig. -and this is likely due to the large number of unconnected nodes. the basic trends in the numbers of communities are reflected in the graph energies recorded in table : the linear averages have very high energy and the geodesic averages have very low energies, with the le averages' energies slightly higher than the ai averages' . what is somewhat surprising, though, is the difference in graph energies between the non-thresholded means -the numbers of communities in each are similar, but the linear average has an energy roughly an order of magnitude higher than the riemannian averages. the energy of the linear mean without thresholding or with a threshold of . seem to be the most reasonable values. in concluding our observations about these averages, we note that the weights on the linear average graph will all have weights that are multiples of / (because there are seven data points provided), and thus there will be no difference in results for any two thresholds that lie between n and n+ . this explains why the results for threshold values of . and . are the same for the linear average, for example. the geodesic interpolations provide no such structure, and our results here would suggest that low thresholds are generally required to get good results out of the geodesic interpolations. in the appendix, we have provided error bounds for each geodesic interpolation in terms of distance on their respective manifolds. the actual error incurred will depend on the problem in question, though. that kind of error, or even entry-wise error, may not be the most important kind of error to consider for our purposes here, however. rather, we may care most about the community structure. based on our community-related metrics (connectivity and similarity), the le geodesic, with a threshold for the proteomics data, performed the best. the ai geodesic was too sparse and disconnected, while the linear interpolation was too connected (as expected). the optimal choice of threshold value depended on the metric being considered: . was by far the best when considering community similarity, but . was better for the other metrics under consideration. in general, the optimal threshold value will likely depend on the problem in question and the quantities of interest, but we found that the le geodesic responded to changes in the threshold value more readily than the ai geodesic did. in this paper, we used the same bias value for all of the proteomics interpolations ( − ), but as mentioned in the "disconnected graphs" section, increasing the bias value caused the geodesic interpolation to approach the linear one. figure shows an example of this where increasing the bias term causes the le geodesic to behave more and more like the linear interpolation (compare with fig. ). future work may involve experimenting with different bias terms to find a happy medium between the linear and pure geodesic interpolations. one concern about the geodesic interpolations is the transient edges that they produceedges that do not exist in either end point but emerge and disappear during the interpolation process. the weights on these edges were small, but they could be positive or negative, and they arose in both the synthetic and proteomics data, so they are not simply an artefact of using the bias addition approach to deal with disconnected graphs. moreover, using a low threshold means that some of these edges may not disappear when that threshold is applied, and therefore they may affect the community structure of the graph. using a larger bias value to more closely approximate a linear interpolation may ameliorate the problem, but it would be valuable to look in more detail at why these transients occur and how to interpret them from a graph theoretic perspective. for example, does it make sense to say that the 'shortest' or 'least energetic' path from one graph snapshot to another might involve some transient edges? from the perspective of the manifold geometry, it clearly does, as the shortest path between two points is a geodesic, but it is not clear if the same holds true purely from a network perspective. in short, the geodesic interpolations are not perfect, and there are still unanswered questions, but it is nonetheless clear that linear interpolation is not well suited to graph interpolation if the ultimate goal is community detection. when using a threshold, linear interpolation will always produce a piecewise constant result consisting of three phases. without thresholding, the linear interpolation inflates overall graph connectivity, and the greater the difference between the two graphs, the greater the inflation. as an extreme example, consider interpolating between a graph with adjacency matrix a to a graph with adjacency matrix − a. the result 'halfway' between them would be a fully connected graph with edge weights of . . these issues are particularly prominent when calculating averages over multiple graphs. perhaps most saliently for our purposes here, the linear interpolation did not produce steady changes in the community structure between data points -the proteomics data showed that the linear interpolation almost always had markedly fewer communities than the data points it connected. the ai geodesic produced transient edges that were smaller in magnitude and fewer in number than the le geodesic, but it was also more expensive and produced graphs that were too sparse (e.g., too few communities); the le geodesic used a similar approach but produced better results when combined with a threshold. similar trends held true, generally speaking, for the mean graphs as well. currently, the computational cost of geodesic interpolation is high because it requires calculating matrix functions like the exponential and logarithm. the le geodesic is noticeably faster than the ai geodesic in calculating interpolated points, though, due to the fractional matrix powers used in the former but not the latter. furthermore, the average graph is significantly easier to calculate for the le geodesic because it has a closed-form expression, whereas the ai geodesic requires an iterated numerical solution. these computational costs are not prohibitive for graphs with hundreds of nodes, but for much larger graphs -say, on the order of nodes -the computational cost could render our methods infeasible. one possible approach would be to project the graph laplacians to a lower-dimensional space, perform the interpolation there, and then project back to the original space with some kind of low-rank or sparsity criterion; riemannian optimization on matrix manifolds could be useful for determining an optimal low-rank projection (vandereycken ) . another option would be to use graph spectral sparsification (batson et al. ) to produce sparse graphs that approximate the spectrum of the original graph. we would then perform the interpolation on those sparse graphs. given the close relationship between geodesics and spectral properties, this approach may be better-suited to the geodesic interpolations than to the linear interpolation. either way, it should be possible to come up with an error bound, in terms of the distance between the approximate and true solutions, that relates to the approximation used. as an alternative to thresholding, it may also be possible to identify the laplacians of unweighted graphs that are 'closest' to the geodesic trajectory and use them to define a kind of discrete trajectory of unweighted graphs that most closely approximates the geodesic between two unweighted graphs. this could potentially be more accurate than simply thresholding the adjacency matrix entries. our present interpolation methods match the supplied data points exactly, but the transitions from one interpolation to another are not smooth. it may be valuable to develop more sophisticated interpolation methods that will enforce smoothness, such as polynomial and spline interpolation, using the form of the geodesic interpolations. we may not want to match the supplied graph snapshots exactly, though. instead, we may need to come up with an approximating curve for noisy data. it is possible to define a geodesic that minimizes the sum of squared distances between it and a set of time-indexed data (much like a linear least-squares regression). we could then solve for the regression coefficients in a manner similar to the calculation of the geodesic mean. both higher-order interpolations and least-squares interpolations are possible for the ai and le geodesics, but they may be easier to derive and computationally cheaper for the le versions than the ai versions. regardless of which is used, though, the geometries in which the interpolations are embedded would ensure that the laplacians remain positive-semidefinite and thus representative of real graphs. there is also the option of using other laplacians (e.g., a normalized laplacian). some of these laplacians have spectral properties, such as bounded eigenvalues, that may induce better interpolation behaviour. if these laplacians also have non-constant nullspaces, though, that would add complexity to the interpolation procedure. this would not be a significant hurdle for piecewise geodesic interpolation, but it may be problematic for graph averaging and some of the interpolation expansions described in the paragraph above. we have not yet looked at this problem in detail, however. finally, as mentioned previously, the riemannian framework does not require any one particular community detection method, though it may have some natural connections to spectral clustering. future work with the framework could include comparing different static clustering methods (either analytically or computationally) to see if there are any that would be particularly well-or ill-suited to this kind of interpolation and averaging. we described and implemented riemannian methods for interpolating between and averaging dynamic graph snapshots. following that, we demonstrated the use of these methods on a synthetically generated dynamic graph and an experimentally produced proteomics network and compared them with entry-wise linear interpolation. the linear interpolation increased graph connectivity between interpolation points, and we showed that when a threshold is used to produce unweighted graphs from the interpolation, the entry-wise linear approach will always produce a three-phase piecewise constant result. the geodesic interpolations created using the riemannian methods produced graphs with linearly varying connectivity when applied to connected graph snapshots and produced decreased connectivity between interpolation points when applied to disconnected graph snapshots. we found that using a low threshold on the edge weights improved our results on the disconnected graphs. however, these interpolations produced transient edges (with small positive and negative weights). one area of future work will be to investigate why this behaviour occurs and interpret it in graph theoretic terms. choosing larger bias values when applying these methods to disconnected graphs may improve the quality of the interpolation, from the perspective of graph connectivity, and it may also reduce the presence of transient edges as well. other significant next steps for this work include developing techniques for applying our work to significantly larger graphs and expanding upon our current interpolation methods to produce the riemannian analogues of polynomial interpolation, spline interpolation, and least-squares regression. we cannot say that a linear interpolation will always have the smallest amount of error, but a linear interpolation would haveÿ = , so we would expect it to have a smaller error bound than an arbitrary nonlinear interpolation (i.e., one not using higher-order derivative information). we end up with a similar result in considering the distance between a dynamic graph trajectory x(t) through the positive-definite subspace of the laplacian and an ai geodesic interpolation y (t) between x( ) = r and x( ) = r : and commute with each other and with powers of each other (including negative powers) because they have the same eigenvectors. traces of matrix products are also constant under cyclic permutations of those products. we will use these properties to derive an expression for tr ˙ using this matrix commutivity and greene's results on traces of matrix products (greene ) : since c (and its powers) commute with exp(ct). therefore,Ẏ g y − g is constant in time. for the entry-wise linear interpolation, however, there is no closed-form expression foṙ y l or y − l ; y l is the positive-definite component of the interpolated laplacian. in general, though,Ẏ l y − l will not be constant in time. we can then consider the second derivative of the original distance function: d geometric means in a novel vector space structure on symmetric positive-definite matrices spectral sparsification of graphs: theory and algorithms fast unfolding of communities in large networks compex networks, structure and dynamics sustaining the internet with hyperbolic mapping riemannian metric and geometric mean for positive semidefinite matrices of fixed rank energy of a graph encyclopedia of social network analysis and mining the mechanics of rocking stones:equilibria of separated scales dynamical clustering of exchange rates community detection in graphs community structure in social and biological networks traces of matrix products human protein reference database- update curvature and temperature of complex networks hyperbolic geometry of complex networks random walks, markov processes and the multiscale modular organization of complex networks a network integration approach to predict conserved regulators related to pathogenicity of influenza and sars-cov respiratory viruses community structure in time-dependent, multiscale, and multiplex networks dynamic social community detection and its applications a riemannian framework for tensor computing objective criteria for the evaluation of clustering methods finding communities in dynamic social networks community detection in networks with positive and negative links low-rank matrix completion by riemannian optimization the authors would like to thank jason mcdermott for providing the proteomics data used in this study. this work was funded by the microbiomes in transition (mint) initiative at the pacific northwest national laboratory. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. for -d linear interpolation, there is a well-defined error bound for the interpolation: a linear interpolation of f (x) from x to x has an error bound ofwe can then consider the euclidean distance between a trajectory x(t) and its approximation y(t), t ∈ [ , ], from which we can calculate an error z(t):for the geodesic, d dt Ẏ g y − g = , but this will not be the case for the entry-wise linear interpolation. also note the recurrent x − x − term: x x is the vector from x to y (pennec et al. ) , so x − x − is essentially a measure of trajectory discrepancy rescaled by x.as with the vector trajectory previously, we cannot say that a given interpolation will always the most accurate one. however, one of the error terms disappears for the ai geodesic interpolation; all other things being equal, it is reasonable to expect that the error on the geodesic interpolation will be, at the very least, less variable than the error on the entry-wise linear interpolation. for extrapolation, the error estimate is no longer relevant for the entry-wise linear method because such an extrapolation is not guaranteed to remain positive-semidefinite. however, the error on the ai geodesic extrapolation is well-defined by the remainder formula in taylor's theorem. for example, extrapolating past r to t > using an ai geodesic built by interpolating from r to r would produce the following error bound:with the derivatives as previously calculated. for the le geodesic,in general, all of these derivative terms will be non-zero. however, for the le geodesic interpolationseveral of the terms in d dt tr ˙ will therefore be zero for the le geodesic. as such, we would expect the error from the le geodesic to be less than the error from the entrywise input interpolation for the same reasons that we would expect the ai geodesic error to be smaller than the entry-wise linear interpolation. we can then plug these results into eq. to get error bounds for the le geodesic. additional file : the synthetic data snapshot files begin with 'comm-fixed', and each snapshot file is suffixed with its time index. the proteomics snapshot data files begin with "ppn-numeric" and they are also suffixed with their time indices. the .edges files may be read with a text editor; we recommend textpad. (zip kb) the python implementation of the methods described in the paper. (py kb) additional file : a video of the spectral plots created with the ai geodesic interpolation on the synthetic graph data progressing through the data snapshots in order from the initial to the final frame. (mp kb) the datasets supporting the conclusions of this article are included in the article's additional files.authors' contributions cb proposed the riemannian framework, performed the mathematical derivations, implemented the methods, generated results, and wrote the main body of the paper. mh obtained the proteomics data, provided the community similarity metric, co-wrote the literature review, offered comments and corrections on the manuscript, and suggested reviewers. as proposed the bias method, wrote code to assist in the community detection, co-wrote the literature review, generated the synthetic data, offered comments and corrections on the manuscript, and suggested reviewers. all authors have read and approved the manuscript. key: cord- -ch a d authors: de conto, flora; conversano, francesca; medici, maria cristina; ferraglia, francesca; pinardi, federica; arcangeletti, maria cristina; chezzi, carlo; calderaro, adriana title: epidemiology of human respiratory viruses in children with acute respiratory tract infection in a -year hospital-based survey in northern italy() date: - - journal: diagn microbiol infect dis doi: . /j.diagmicrobio. . . sha: doc_id: cord_uid: ch a d acute respiratory tract infections (artis) are among the leading causes of morbidity and mortality in children. the viral etiology of artis was investigated over years (october –september ) in children in parma, italy, using indirect immunofluorescent staining of respiratory samples for viral antigens, cell culture, and molecular assays. respiratory viruses were detected in cases ( . %); ( . %) were single infections and ( . %) mixed infections. the highest infection incidence was in children aged > months to ≤ years ( . %). human respiratory syncytial virus ( . %) and human adenovirus ( . %) were the most common viruses identified. the virus detection rate decreased significantly between the first and third epidemic season ( . % vs. . %, p < . ). the simultaneous use of different diagnostic tools allowed us to identify a putative viral etiology in half the children examined and to provide an estimate of the epidemiology and seasonality of respiratory viruses associated with artis. acute respiratory tract infections (artis) are a persistent public health problem (lu et al., ) . artis affect the upper respiratory tract, leading to colds, rhinosinusitis, pharyngitis, laryngitis, tracheitis, and otitis media, as well as the lower respiratory tract, causing tracheitis, bronchitis, bronchiolitis, and pneumonia (bicer et al., ) . although the majority of artis remain confined to the upper tract (tregoning and schwarze, ) , they can cause severe manifestations when affecting the lower tract (zappa et al., ) . viruses are frequently the cause of artis characterized by high risks of morbidity and mortality (taylor et al., ) . the viruses most frequently detected are: influenza viruses a and b (iav, ibv), human parainfluenza virus (hpiv), human adenovirus (hadv), human metapneumovirus (hmpv), and human respiratory syncytial virus (hrsv). this list is constantly evolving along with improvement in the performance of diagnostic tests (ljubin-sternak et al., ; tregoning and schwarze, ) and the discovery of more recent viruses, for example, human bocavirus (hbov) (allander et al., ) , human coronaviruses (hcovs) nl and hku , and new human enterovirus (hev), parechovirus, and rhinovirus strains (berry et al., ) . the main diagnostic methods for respiratory virus infections are: virus isolation in cell culture, viral antigen/nucleic acid detection, and virus-specific serology. virus isolation is labor-intensive (landry, ) , making it impractical to determine a diagnosis during the acute infection phase. shell vial cultures and viral antigen detection reduce the time for diagnosis (engler and preuss, ; gardner and mcquillin, ) . nearly all these methods are being replaced by highly sensitive multiplex pcr (kim et al., ; tregoning and schwarze, ) . arti prevalence in children varies by geographic area, season, and year (goktas and sirin, ; moura et al., ; panda et al., ) . recent italian data are limited, apart from those relating to influenza (pariani et al., ; trucchi et al., ) . this three-year (october -september ) hospital-based survey in parma (northern italy) aimed to determine the prevalence of respiratory virus infections, their seasonality, and any patterns of mixed infections in children with artis by using indirect immunofluorescent staining of respiratory samples for viral antigens, cell culture, and molecular assays. the study was conducted at the virology unit of the university hospital of parma (northern italy), a -bed tertiary care center with more than , admissions per year from the city and surroundings with approximately , inhabitants. laboratory diagnosis was performed upon medical request. patients' identities and medical information were protected. the inclusion criteria were: patients ≤ years old, acute fever, respiratory symptoms, and illness onset within days. from october st, to september th, , samples from children ( females, . %, and males, . %; inpatients, . %, and outpatients, . %) were collected by bronchoalveolar lavages (bals), bronchial aspirates (bass), nasopharyngeal aspirates (npas), nasal swabs (nss), sputum (sp), and throat swabs (tss). npas, nss and tss were stored in viral transport medium (de conto et al., ) and all samples were kept at °c until submitted to laboratory (within h of sampling). age was available for children ( . %) and ranged from ≥ days to years (mean age: years months; median age: years month). children were divided into four age groups: to ≤ months ( . %), n months to ≤ years ( . %), n to ≤ years ( . %), and n to years ( . %). of note, children ( . %) were examined at least twice during the survey, with a time interval between care provider visits of days to months, presumably for either worsening symptoms of a current infection or infection recurrence. npas, nss, and tss were centrifuged ( rpm, min, °c) and the pellets resuspended in phosphate-buffered saline (pbs, ph . ; mm na hpo , . mm kh po , mm nacl, . mm kcl), before cytocentrifugation ( rpm, min). cell sediments on slides were dried in a laminar-flow hood and fixed in acetone ( min, − °c). these were then hydrated with pbs, blocked with % bovine serum albumin (bsa) in pbs, and the slides were incubated ( h, °c) with anti-hrsv monoclonal antibodies (mabs) ( : ; argene/biomérieux, france) in . % bsa in pbs. after washes with pbs, they were incubated with fluorescein isothiocyanate-conjugated anti-mabs ( : ; argene/ biomérieux) in . % evans blue in pbs ( min, °c). after further washes with pbs, the sediments were mounted in glycerol and analyzed by epifluorescence microscopy (dmlb leica, germany). madin-darby canine kidney cells with -linked sialic acids enhanced expression (mdck-siat ) came from sigma-aldrich (italy), with human lung fibroblasts (mrc- ) from lgc standards (italy). human larynx epidermoid carcinoma (hep- ), embryonic human intestine (intestine ), rhesus monkey kidney (llc-mk ), and african green monkey kidney (vero) cells came from the "istituto zooprofilattico sperimentale della lombardia e dell'emilia-romagna" (italy). cells were grown in earle's modified minimum essential medium (e-mem) supplemented with mm l-glutamine, % fetal bovine serum, and antibiotics ( u/ml penicillin, μg/ml streptomycin), and maintained at °c in a % co atmosphere. reagents were from life technologies (italy). after aspiration of the growth medium, mdck-siat , hep- , and intestine cells grown in -well chamber slides (fisher scientific, italy) were incubated with samples ( h, °c). after restoring the e-mem, incubation was carried out in e-mem for h before iif with mabs against hadv, hpiv, hrsv, and influenza virus (iv) (argene/ biomérieux), as described above. hadv, hev, hpiv, hrsv, and iv isolation was performed in llc-mk , mdck-siat , hep- , intestine , mrc- , and vero cells grown in -well plates (sigma-aldrich). after aspirating the e-mem, cells were incubated with samples ( h, °c). after restoring the e-mem, incubation in e-mem was continued until the appearance of cytopathic effect (cpe). if the cells showed no cpe after days, they were scraped, and cell suspension was collected for a second inoculation. when cpe was observed, cells were scraped in pbs and cytocentrifuged, before iif. when cpe suggested hev infection, neutralization assays were performed. hevs serotype identification was performed with type-specific antisera (statens serum institut, denmark), according to lim and benyesh-melnick ( ) . samples were screened for hadv, hbov, hcov, hmpv, hpiv, hrsv, and iv nucleic acids with real-time (rt)-pcr or reverse transcription (rt)-rt-pcr assays. nucleic acid extraction was performed with the nuclisens® easymag® kit on the easymag extractor (biomérieux, italy) and rt with the rt-kit plus (elitechgroup molecular diagnostics, france) on the geneamp pcr system thermocycler (applied biosystems, thermo fischer scientific-waltham, usa). extracted nucleic acids were tested with influenza a/b q-pcr alert amplimix kit (elitechgroup) on the rt-pcr system (applied biosystems). the respiratory multi well system (mws) r-gene™ assay (argene/ biomérieux) was used to detect adv/hbov, hcov/hpiv, and rsv/ hmpv on the rotor-gene q system (qiagen, germany). assays were performed according to the manufacturer's instructions. a chi-square test was performed using graphpad prism software. p b . was considered statistically significant. a total of respiratory secretions from children were analyzed as described in the methods section; samples ( . %) from ( . %) children ( males, . %, and females, . %) were virus-positive (table and supplementary table ) . single infections were detected in ( . %) samples from ( . %) children. mixed infections were detected in ( . %) samples from ( . %) children. there was a significant difference in the frequency of single vs. mixed infections among positive samples ( . % vs. . %; p b . ) as well as among total examined samples ( % vs. . %; p b . ). among mixed infections, two viruses were observed in ( . %) samples, three viruses in ( . %) samples, and four viruses in ( . %) samples. the percentage difference in the number of mixed infections with two viruses ( . %) and four viruses ( . %) was significant (p b . ). overall, viruses were detected. hrsv ( / : . %) and hadv ( / : . %) were the most common viruses identified, followed by hcov ( / : . %), iv ( / : . %), hbov ( / : . %), hpiv ( / : . %), hmpv ( / : . %), and hev ( / : . %). hrsv and hadv were found in single ( . % and . %, respectively) and mixed infections ( / : % and / : . %, respectively) ( table ) the virus detection rate decreased from . % ( / ) ( . ) ( . ) ( ) ( ) ( ) ( ) ( . ) hrsv + hcov ( . ) ( . ) ( ) ( ) ( ) ( ) ( . ) hadv + hbov ( . ) ( ) ( ) ( ) ( ) ( ) ( . ) hrsv + hbov ( . ) ( . ) ( ) ( ) ( ) ( ) ( . ) hrsv + iav ( . ) ( . ) ( ) ( ) ( . ) ( ) ( . ) hcov + iav ( . ) ( . ) ( ) ( ) ( ) ( ) ( . ) hadv + hpiv ( . ) ( . ) ( ) ( ) ( ) ( ) ( . ) hadv + hmpv ( . ) ( . ) ( ) ( ) ( ) ( ) ( . ) hcov + hmpv ( . ) ( . ) ( ) ( ) ( ) ( ) ( . ) hbov + hcov ( . ) ( . ) ( ) ( ) ( ) ( ) ( . ) hbov + iav ( . ) ( . ) ( ) ( ) ( ) ( ) ( . ) hrsv + ibv ( . ) ( ) ( ) ( ) ( ) ( ) ( . for the children with viral artis and age available, the mean and median age were years months and year months, respectively. the infection rate was highest in children between n months to ≤ years ( . %; . % among positive cases), followed by the to ≤ months ( . %; . % among positive cases) and n to ≤ years ( . %; . % among positive cases) groups (fig. a ). significant differences in the infection rate were evidenced among children aged to ≤ years and n to ≤ years (p b . ), among children aged to ≤ years and n to years (p b . ), and among children aged n to ≤ years and n to years (p b . ), but not among children to ≤ months and n months to ≤ years (p n . ). hrsv had the highest prevalence ( / : . %) in children aged to ≤ months, followed by hcov ( / : . %) and hadv ( / : . %), while hadv prevailed in children n months to ≤ years ( / : . %), followed by hrsv ( / : . %) and hcov ( / : . %) (fig. b) . in children n to ≤ and n to years, hadv prevailed ( / : . % and / : . %, respectively), followed by iv ( / of the virus-positive samples, were detected by molecular assays and by culture ( . % vs. . %, p b . ) ( table ) . most of the culture-positive samples ( , . %) were also positive by molecular methods, although different virus combinations were detected in mixed infections. in discordant culture-negative samples molecular assays detected hcov ( . %), hbov ( . %), and hmpv ( . %). finally, of the samples positive for hrsv by pcr, ( %) were also detected by iif ( % vs. %, p b . ). this survey carried out in parma (northern italy) from october to september describes the detection of viruses associated with respiratory samples from patients presenting with symptoms consistent with acute respiratory tract infections. overall, . % of cases were positive by the simultaneous employment of different diagnostic assays. other studies have reported similar rates, although they refer to a single epidemic season and fewer number of cases examined exclusively by pcr (annan et al., ; pratheepamornkull et al., ; zuccotti et al., ) . however, different results have also been described (do et al., ; venter et al., ) . it must be considered that many factors could lead to prevalence variations, such as the virus panel examined, diagnostic methods, study setting, and geographic area. hrsv ( . %) and hadv ( . %) prevailed; hcov ( . %), iv ( . %), and hbov ( . %) showed a higher frequency than hpiv ( . %), hmpv ( . %), and hev ( . %). single infections predominated over mixed infections ( . % vs. . %, p b . ), which was consistent with previous observations (martin et al., ; paranhos-baccalà et al., ) . of multiple infections, dual infection ( %) was predominant, in accordance with other authors (martin et al., ; peng et al., ) . hrsv, hadv, and hcov were mainly involved in mixed infections, as would be expected from their overlapping seasonal distribution. although hrsv and hadv are among the viruses most commonly involved in mixed infections (cantais et al., ; harada et al., ) , the relevance of rhinoviruses, hpiv, and iv has also been assessed (lu et al., ) . very complex combinations of mixed viral infections have been observed (lu et al., ; martin et al., ; paranhos-baccalà et al., ) and certain pathogens appear to have higher multiple infection frequency (brunstein et al., ) . in this scenario, superinfection exclusion mechanisms preventing a secondary infection with the same or a closely related virus should be envisaged (folimonova, ) . although mixed infections have been widely assessed in artis (moesker et al., ; seo et al., ; turner et al., ) , there is still no clear relationship with prolonged virus shedding, asymptomatic virus persistence, and disease severity. however, there is evidence that genome quantification could clarify the role of each virus in mixed infections (franz et al., ; martin et al., ) . accordingly, high viral loads have been related to severe diseases (franz et al., ; hasegawa et al., ; jansen et al., ) , but conflicting results have been reported (adams et al., ; wu et al., ) . in addition, some viruses have been detected in healthy children (advani et al., ; moe et al., ) . in this study, molecular assays showed greater sensitivity when compared to culture assays ( . % vs. . %, p b . ) and hrsv antigen detection ( % vs. %, p b . ). conversely, culture assays allowed the detection of infectious viruses in ( . %) of the positive samples, eventually providing a clue to discriminate the main causative agent in co-infections. nevertheless, cautious conclusions should be drawn, since the growth difficulties of some viruses may have decreased the number of those found by culture assays, while the long-term viral excretion after clinical recovery in children with frequent artis may have increased the mixed infection rate. the epidemic season of respiratory viruses ranged from to months, with peaks in february ( and ) or january ( ). the virus detection rate decreased significantly between the first and third seasons ( . % vs. . %, p b . ), but environmental factors may have influenced the frequency and severity of artis (nenna et al., ) . hrsv showed a sharp winter/early-spring seasonality. when compared to previous reports, in the parma area the hrsv epidemic season was shorter (medici et al., ) . hadv circulated persistently throughout the study with higher rates in fall and spring of the first two years of surveillance, eventually indicating a reduction of circulation due to the accumulation of immunity in the population. the iv infection seasonality was consistent with previous reports (del manso et al., a manso et al., , b . no definite seasonality was shown by hbov, hcov, hmpv, hpiv, and hev. a correct viral diagnosis allows highlighting virus seasonality, which could favor both preventive measures and reduction of overuse of antibiotics. accordingly, in children with hrsv or iv, bacterial infections are less prevalent than in those without a viral infection (purcell and fergie, ; smitherman et al., ) . the virus detection rate was significantly higher in children aged to ≤ years ( . %), when compared to the n to ≤ years ( . %, p b . ) and n to years ( . %, p b . ) groups, suggesting that immaturity of the immune system and absence of previous immunity could influence the severity of artis (hammond et al., ; monto, ) . hrsv prevailed ( . %) in younger infants ( to ≤ months), while hadv was prevalent in all other age groups studied. accordingly, hrsv causes severe diseases in young children (moe et al., ; nguyen et al., ; shi et al., ) . for all the remaining respiratory viruses the highest detection rate was among children aged n months to ≤ years. the attribution of an etiological role in artis to hbov has been extensively debated (debiaggi et al., ; schildgen et al., ) . in this study, hbov was most frequently found in single infections ( . %) than in coinfections, strengthening the idea that it can be considered a pathogen. in conclusion, although this study has certain limitations, such as the lack of rhinovirus detection, it does provide relevant information on respiratory virus circulation in children with artis in an area of northern italy with a temperate climate. the simultaneous use of different diagnostic approaches made it possible to carry out in-depth analysis of a large number of cases spanning over years. these findings contribute to estimate the disease burden associated with respiratory viruses, reinforcing the need for timely virologic diagnosis and continuous surveillance to optimize the prediction and control of artis in children. 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clinician participation in the detection and reporting of emerging respiratory infectious diseases date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: hptjqmrl background: we sought to assess reporting in china’s pneumonia of unknown etiology (pue) passive surveillance system for emerging respiratory infections and to identify ways to improve the pue surveillance system’s detection of respiratory infections of public health significance. methods: from february –may , , we actively identified and enrolled patients in two hospitals with acute respiratory infections (ari) that met all pue case criteria. we reviewed medical records for documented exposure history associated with respiratory infectious diseases, collected throat samples that were tested for seasonal and avian influenza, and interviewed clinicians regarding reasons for reporting or not reporting pue cases. we described and analyzed the proportion of pue cases reported and clinician awareness of and practices related to the pue system. results: of ari admissions in two hospitals, ( %) met the pue case definition; none were reported. of specimens tested, ( %) were seasonal influenza virus-positive; none were avian influenza-positive. < % pue case medical records documented whether or not there were exposures to animals or others with respiratory illness. most commonly cited reasons for not reporting cases were no awareness of the pue system ( %) and not understanding the case definition ( %). conclusions: most clinicians have limited awareness of and are not reporting to the pue system. exposures related to respiratory infections are rarely documented in medical records. increasing clinicians’ awareness of the pue system and including relevant exposure items in standard medical records may increase reporting. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. china established the pneumonia of unknown etiology (pue) surveillance system in for timely detection of emerging respiratory infectious diseases [ ] , and the system has played an important role in detecting human infections with novel avian influenza viruses including a(h n ), a(h n ), a(h n ), and a(h n ) [ ] [ ] [ ] . nevertheless, a evaluation identified persistent underutilization of the pue surveillance system [ ] . more recently, inconsistent reporting occurred during the initial outbreak of low pathogenic avian influenza (lpai) a(h n ) [henceforth a(h n )], prompting public health authorities to allow clinicians to report cases directly without expert consultation committee approval. this change resulted in the reporting of cases in weeks compared with cases in the previous years [ ] . laboratory and case investigation resources were quickly strained and reporting procedures reverted to those used prior to the outbreak [ ] . as a result, case reporting subsequently decreased. a assessment of clinician and health administrator knowledge, attitude and practices related to pue surveillance conducted within healthcare facilities revealed a willingness to report pue cases, but identified limited awareness of the pue system, lack of understanding of the reporting process, and failure to follow the case definition [ ] . to evaluate these gaps, we piloted a -month active surveillance program in two hospitals to ) quantify the number of cases meeting the pue case definition and the number reported and ) to identify ways to improve the pue surveillance system's detection of respiratory infections of public health significance. national guidelines [ ] require all inpatient and outpatient healthcare facilities to report cases meeting the pue case definition. clinicians should report cases to an expert consultation committee, which after review of clinical and laboratory data determines whether to report the case to the pue surveillance system [ ] . if a case is reported to the pue system, the local center for disease control and prevention (cdc) will conduct a field investigation, collect respiratory specimens and send them to a national influenza surveillance network laboratory for testing of avian influenza viruses and, if associated with clusters of respiratory disease or relevant travel history, testing of severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov). a pue case is defined as an illness of unknown etiology with ) axillary temperature > °c, ) radiographic pneumonia, ) low or normal leukocyte count or low lymphocyte count during the early stages of disease, and ) no improvement or worsening symptoms after - days of antimicrobial treatment per clinical guidelines [ ] . participating hospitals were selected based on four criteria: if the facility ) admitted at least patients per month with a discharge diagnosis of pneumonia during february through may - ; ) used an electronic hospital information system; ) demonstrated willingness and capacity to collaborate with both national and local cdcs and ) was located within one of china's of provinces with previously identified h n and/or h n human cases. we selected two tertiary hospitals in anhui province: the second people's hospital of fuyang city, a -bed facility, which from february through may - , admitted an average of pneumonia patients per month, and lu'an city people's hospital, a -bed facility, which over the same time period admitted an average of pneumonia patients per month. fuyang hospital, an infectious disease hospital, had experience treating human infections with avian influenza, while lu'an hospital, a general hospital, did not. after reviewing the hospital information systems, the evaluation team developed a list of admission diagnoses that captured the majority of acute respiratory infections (ari) (additional file ). every day (including weekends) from february through may , , a designated, trained surveillance officer in each hospital ) reviewed the hospital admission registry database to screen all admission diagnoses from the prior days for diagnoses from the screening list (additional file ); ) reviewed admission medical records with a matching diagnosis to identify and enroll patients with illnesses meeting the pue case definition; ) days later, conducted a second medical record review for patients not enrolled during the first review to identify and enroll patients with illnesses newly meeting the case definition (for example, patients with no improvement or worsening symptoms after - days of antimicrobial treatment per clinical guidelines); and ) days later, conducted a third review of records for patients not enrolled during the first two reviews to enroll any remaining patients meeting the case definition. [fig. ]. for patients with illnesses meeting the pue case definition, the surveillance officer used a standard questionnaire to collect information from the hospital information system related to demographics and, if available, epidemiological risk factors, including exposures to poultry, patients with similar symptoms, and travel history. surveillance officers followed the pue surveillance protocol [ ] to investigate enrolled pue case-patients. surveillance officers conducted face-to-face patient interviews using a standard questionnaire to collect the same information described in the medical records review section above to determine both accuracy and completeness of medical records. surveillance officers collected throat swabs from all identified pue case-patients per the surveillance protocol [ ] . specimens were transported to the local cdc laboratory per standard procedures and tested for influenza viruses using real time reverse transcription polymerase chain reaction (rrt-pcr). if identified pue case-patients were part of a cluster of epidemiologicallylinked respiratory illnesses, specimens would also be tested for sars-cov and mers-cov. if a case-patient reported travel history to the middle east, specimens would be tested for mers-cov. the pue surveillance protocol describes a three-step procedure for reporting cases to the pue system: ) clinicians report identified pue cases to their supervisor; if the supervisor concurs, the case is reported to the director; ) the director determines whether to report the case to an expert consultation committee which usually includes specialists from the respiratory medicine department, the radiology department, and infection control; and ) the expert committee determines whether to report the case to the pue system. [ fig. ]. in our evaluation, after patients with illnesses meeting the pue case definition were enrolled, surveillance officers interviewed all clinicians who had primary medical responsibility for these patients. if the case were reported to the clinician's supervisor, the surveillance officer also interviewed the supervisor and a representative member of the expert committee. [ fig. ] surveillance officers used a standard questionnaire to collect ) demographic and occupational information about the clinician being interviewed and, when applicable, ) demographic and occupational information about the senior clinicians who received the case report, and ) reasons for reporting or not reporting pue cases. first, we described the number, proportion, ward distribution, and testing results of pue cases identified by surveillance officers. wilson score was used to calculate % confidence intervals (ci) for proportions. second, we described and compared epidemiologically-linked exposures documented by clinicians in the medical records and used chi-square tests to compare differences in the frequency of exposure histories collected by surveillance officer interviews. third, we described the number of pue cases reported at each of the tiers of the three-step reporting process. finally, we described factors associated with clinicians' reporting or not reporting pue cases. during the evaluation period, surveillance officers reviewed , hospitalization registrations, and screened patients with ari admission diagnoses. of those were from lu'an hospital and were from fuyang hospital. among all ari patients, ( %) had illnesses that met the pue case definition. the proportions of ari cases meeting the pue case definition in each of the two hospitals were similar [ % ( / ) vs. % ( / ), p = . ]. among the pue case-patients identified, % were male, and % were aged - years; most were from the pediatrics ( %) and pulmonary ( %) departments. [ table ]. the proportion of ari admissions meeting the pue case definition was highest in the icu ( %), followed by the infectious disease ( %), tuberculosis ( %), pediatrics ( %) and pulmonary ( %) departments. oral throat swabs, collected within hrs of pue case enrollment, were tested from ( %) of the identified pue case-patients. of the specimens, ( %) were positive for seasonal influenza viruses and none were positive for avian influenza viruses. none met sars-cov or mers-cov testing criteria. of pue case-patients with laboratory results, % ( / ) had documented exposures; % had documented histories related to contact with water potentially infected with parasites which is recorded as standard practice within the "personal history" section of the medical record in china. other exposures which were intermittently documented in the medical records included ( ) ( ) internal medicine emergency room ( ) ( ) internal medicine ( ) ( ) other departments ( ) ( ) animal exposure ( %), contact with persons with respiratory illnesses ( %), exposure to healthcare facilities caring for patients with respiratory illnesses ( %), and any travel history ( %). in addition, ( %) records documented occupation, a required item in the demographic section of the medical history, and identified two pue case-patients as healthcare workers [ table ]. among the enrolled pue case-patients interviewed by surveillance officers, ( %) had at least one exposure relevant to respiratory infections of public health significance, including animal exposure, contact with similar cases of respiratory disease, travel to/living in areas of novel respiratory epidemics, and occupational exposures. in the days before illness onset, ( %) had occupational exposure to poultry/livestock, ( %) were medical staff, ( %) had animal exposure ("exposure to poultry, pigs, etc."), ( %) had close contact with persons with similar respiratory disease symptoms, and ( %) had exposure to a healthcare facility caring for patients with respiratory illnesses [ table ]. although the relevant respiratory infectious disease exposures identified in the medical record and the surveillance officer interview were the same in > % for all exposures analyzed, there were discrepancies. occupational exposures, exposures to persons with similar respiratory symptoms, and animal exposures were identified less frequently through medical records compared to surveillance officer interviews [ table ]. none of the clinicians interviewed from lu'an hospital reported knowledge of the pue surveillance system compared with ( %) of the clinicians from fuyang hospital. at fuyang hospital, knowledge was highest among clinicians with > years ( %) and - years of work experience ( %) and lowest among those with < years of work experience ( %). none of the patients meeting the pue case definition were reported to the surveillance system. during the interviews with clinicians, the most common reasons clinicians cited for not reporting included: being unaware of the pue surveillance system ( %), not understanding the pue case definition ( %), and not accepting the pue case definition ( %) [ table ]. although none were reported to the national system, a clinician reported one pue case with acute respiratory distress syndrome (ards) and possible viral pneumonia to his supervisor. due to disease severity, the lack of a diagnosed pathogen, and no improvement on treatment, the supervisor reported the case to the director, and experts within the ward concluded that the illness met the pue case definition. they reported the case to the hospital's expert committee, which reported to the hospital's department of disease control, which reported the case to the local cdc and sent specimens for laboratory from february through may , , we conducted active surveillance in two hospitals and found that % of all patients admitted with ari met the pue case definition. none of the respiratory specimens tested were positive for avian influenza. only one pue case was reported to the local cdc; however, it was not reported to the national system because the specimen tested negative for avian influenza virus. our findings raise questions about the feasibility of using the existing pue case definition to identify respiratory infections of public health significance. extrapolating our results, if clinicians reported all illnesses meeting the pue case definition from china's more than , hospitals, the number of pue cases identified would be in the hundreds of thousands per year. such numbers would overwhelm the public health system's capacity for laboratory testing and epidemiologic investigations. the impracticality of the existing pue case definition is supported by both a prior study which found that % ( / ) of community-acquired pneumonia diagnoses met the pue case definition [ ] and by the pue surveillance experience in when streamlined reporting procedures led to a surge in cases that quickly strained response efforts [ ] . modifying the system to decrease the number of cases that meet the pue case definition but are not emerging respiratory infections of public health significance would increase the system's feasibility, acceptability, and usefulness. the extent of under-reporting in this pue assessment far exceeds the estimated - % under-reporting of cases of notifiable diseases identified during - in evaluations of the china national notifiable disease reporting system for notifiable diseases, for which reporting is required by law [ ] [ ] [ ] . during our evaluation, none of the identified pue cases was reported to the national system. one clinician reported one pue case appropriately, but the expert committee did not appropriately follow final reporting procedures by reporting the case to the pue system. the two most common reasons clinicians cited for not reporting pue cases to the system were not having knowledge of the pue system and not understanding the pue case definition. knowledge varied by hospital, with > % of clinicians from fuyang hospital reporting knowledge of the pue system compared with none from lu'an hospital. this difference may be explained by fuyang hospital's specialization in infectious disease and its recent experience treating one avian influenza a(h n ) virus infection and one avian influenza a(h n ) virus infection in and respectively. lu'an hospital, a general hospital, had no recent experience treating infections with avian influenza virus. pue surveillance system knowledge also varied significantly by clinicians' years in practice. china cdc conducted intensive, national clinician training on the pue system when the system was established and on-going trainings during the h n outbreaks through . in , the ministry of health required training on the new protocol of surveillance, investigation and management for pue for clinicians at all medical wanted to treat the patient longer prior to reporting ( , ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( , - . ) ( , - ) the patient was transferred to another hospital or ward ( , - ) ( , - . ) ( , - ) when a case is reported, the laboratory results are returned too late such that the testing is not helpful for diagnosis and treatment *ci refers to confidence interval institutions [ ] , however, national pue-related training has not occurred since . therefore, clinicians with < years work experience have not received systematic, indepth training on pue surveillance. another recent study among hospital clinicians in beijing found that new clinicians knew little about key public health concerns, such as infection control within healthcare facilities, without having received formal training [ ] . these findings highlight the importance of training clinicians on public health surveillance systems, reporting requirements and other key public health topics both as they enter the workforce and as refresher courses to improve their capacity to identify and report emerging and re-emerging infectious diseases. this evaluation found that clinicians did not document respiratory infection-related exposures. during the interviews, % identified pue case-patients reported recent exposures to animals and % reported exposures to patients with similar illnesses, yet these exposures were rarely documented in the medical record. these findings suggest that clinicians may not routinely assess respiratory infection-related exposures. about half of the medical records from identified pue cases documented any contact with parasites-infected water, because asking about "any potential exposure to parasite infected water" is a routine practice when completing the "personal history" section of the medical record [ ] . this finding suggests that clinicians are more likely to ask about specific exposures when they are part of routine practice as opposed to exposures that may only be asked intermittently if not part of routine practice. the widespread use of electronic medical records in china provides an opportunity for prompting clinicians to ask about certain exposures relevant to infectious diseases that can be documented in a standardized way in patient's medical record. by developing a checklist within the electronic medical record with questions related to exposures relevant to emerging respiratory diseases such as live poultry and swine for priority use in inpatient wards in china, clinicians would be prompted to routinely ask about these exposures. this may in turn improve detection and reporting of emerging respiratory infections. first, increasing documentation of relevant exposures may facilitate the addition of more specific epidemiologic criteria to the pue case definition to reduce the number of cases meeting the case definition that are not infections of public health concern. second, clinicians who identify concerning exposures in patients with respiratory diseases may be more likely to report these cases to the pue system. finally, since the majority of hospitals in china now use electronic medical records system, if exposure data were collected systematically as part of these systems, it is possible that cases with relevant epidemiological data could be automatically flagged for reporting. further investigations would be needed to assess these modifications to see if they have a positive impact on case reporting without overwhelming the pue system. this evaluation is subject to several limitations. first, the assessment occurred when there were no local reports of human infection with avian influenza virus. pue case identification and reporting may increase during outbreak periods. however, in , during the th epidemic of influenza a(h n ) which had the largest number of human infections to date, fuyang hospital did not report any cases to the national system despite sending potential pue case specimens to local cdc for testing, of which two were positive for influenza a(h ). during this same period, lu'an hospital did not send any potential pue case specimens to local cdc for testing, nor did it report any cases to the national pue system. lu'an hospital did, however, report one confirmed h n case to china cdc directly. second, the reporting practices within these two hospitals may not represent reporting practices throughout china. finally, the screening admission diagnosis list may not have captured all pue cases [additional file ]. our findings suggest that most clinicians are not reporting cases to the pue surveillance system. if clinicians were to report all cases meeting the pue case definition, the large number reported would likely overwhelm the public health system's capacity for laboratory testing and case investigation. of those reported, the vast majority of cases would not be emerging infections of public health concern. our findings lead to several recommendations that may increase the specificity of the pue case definition, increase clinician participation in the pue system, and contribute to the early detection of emerging respiratory infections in china. ) modifying the existing pue case definition by adding relevant exposure history may improve the specificity, feasibility and utility of case reporting. ) including exposure items related to emerging respiratory infectious diseases in the standard infectious disease history documented in medical records may increase the likelihood that clinicians will assess exposure histories relevant for emerging respiratory infections. including these items in the respiratory diseases department, the pediatric department and the intensive care unit, where pue cases are more common, may be most useful. ) providing clinicians with frequent publichealth related training and communications will ensure that clinicians are aware of public health reporting requirements. a multi-pronged approach to incorporating public health practice into clinical settings may include: offering training to clinicians as they enter the workforce followed by annual refresher courses, posting publichealth related updates and notices in clinical areas, incorporating public health guidance into hospital policies, engaging clinicians in the development of clinically-appropriate and easy-to-apply case definitions, and regularly sharing local and national public health data of interest with clinicians to highlight the public health importance of their work. ministry of health protocol of surveillance, investigation and management for pneumonia of unknown etiology investigation and analysis on the first case of human infected with avian influenza (h n ) in yunnan province epidemiological on the first human case with avian influenza a (h n ) virus in hubei province analysis on reporting of unknown etiology pneumonia cases in china the evaluation of surveillance system of highly pathogenic avian influenza virus infection in china. beijing: chinese center for disease control and prevention use of national pneumonia surveillance to describe influenza a(h n ) virus epidemiology, china assessing clinicians' acceptance of the pneumonia of unknown etiology surveillance system current status and clinical study of suspected pneumonia cases of unknown origin in china an investigational analysis of missing reports of notifiable diseases in medical facilities all over china in evaluation on management and quality of communicable diseases network direct reporting in china reporting quality of notifiable communicable diseases in hospitals in china the notice for the intensive training of local clinicians in different level of medical institutions for detection and reporting of avian influenza an analysis on knowledge survey of hiv occupational exposure and the influencing factors in medical workers at two tertiary general hospitals in beijing the notice of the ministry of health on the issuance of basic rules of medical records publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank the colleagues from china cdc, anhui province cdc, lu'an prefecture cdc, fuyang prefecture cdc, lu'an city people's hospital and the second people's hospital of fuyang city who supported project implementation for this assessment, including case screening and investigation, clinician interviews, specimen collection, laboratory testing, and logistical administration. authors' contributions nx, ys, yw, jw, cm. g, zf and ql contributed to the design of this study; nx, yw, jw, zd, js, wy, gg, rw, pg, zr, lg, px and dl contributed to the implementation and data collection of this study; nx, ys, yw, aj. m, cm. g, sz, dn, and ql contributed to data analysis, manuscript writing and manuscript revision. all authors have read and approved the final version of the manuscript. dr. nijuan xiang is an epidemiologist and chief of the surveillance, alert and risk assessment (sara) branch, public health emergency center, chinese center for disease control and prevention. her research interests include surveillance for emerging infectious diseases, prevention and control strategies for emerging infectious diseases, and surveillance and risk assessment for public health emergencies. this work was funded by a us cdc cooperative agreement ( u gh - ) and supported by the china-us collaborative program on emerging and re-emerging infectious disease. the fund body did not participate in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. all data generated or analysed during this study are included in this published article [and its supplementary information files]. the datasets generated and/or analysed during the current study are not publicly available due to the datasets containing personally identifiable information used for public health surveillance purposes. requests for data can be directed to the corresponding author.ethics approval and consent to participate this evaluation was approved by the chinese center for disease control and prevention institutional review board (number: ) and was determined to be non-research public health practice by us cdc. all patients participating in this evaluation, or their parent or guardian for patients < years of age, provided oral informed consent prior to interview and written informed consent prior to throat swab collection. not applicable. the authors declare that they have no competing interests. key: cord- -hksmt i authors: mclean, rebecca k.; graham, simon p. title: vaccine development for nipah virus infection in pigs date: - - journal: front vet sci doi: . /fvets. . sha: doc_id: cord_uid: hksmt i nipah virus (niv) causes a severe and often fatal neurological disease in humans. whilst fruit bats are considered the natural reservoir, niv also infects pigs and may cause an unapparent or mild disease. direct pig-to-human transmission was responsible for the first and still most devastating niv outbreaks in malaysia and singapore in – , with nearly human cases and over fatalities. pigs can therefore play a key role in the epidemiology of niv by acting as an “amplifying” host. the outbreak in singapore ended with the prohibition of pig imports from malaysia and the malaysian outbreak was ended by culling % of the country's pig population with costs exceeding us$ million. despite the importance of niv as an emerging disease with the potential for pandemic, no vaccines, or therapeutics are currently approved for human or livestock use. in this mini-review, we will discuss current knowledge of niv infection in pigs; our ongoing work to develop a niv vaccine for use in pigs; and the pig as a model to support human vaccine development. nipah virus (niv) is an enveloped, single stranded, negative sense rna paramyxovirus, genus henipavirus. the natural hosts and wildlife reservoirs of niv are old world fruit bats of the genus pteropus ( ). both nipah and the related hendra virus possess a number of features that distinguish them from other paramyxoviruses. of particular note is their broad host range which is facilitated by the use of the evolutionary conserved ephrin-b and -b as cellular receptors ( ) . the niv attachment glycoprotein (g) is responsible for binding to ephrin-b /-b ( ). following receptor binding, the g protein dissociates from the fusion (f) protein. subsequently, the f protein undergoes a series of conformational changes which in turn initiates fusion of the viral and host membrane allowing entry ( ) . during viral replication, the f protein is synthesized and cleaved into fusion active f and f subunits. these subunits are subsequently transported back to the cell surface to be incorporated into budding virions, or facilitate fusion between infected and adjacent uninfected cells ( ) . this cell-to-cell fusion results in the formation of multinucleated cells called syncytia, and greatly influences the cyopathogenicity of niv as it allows spread of the virus, even in the absence of viral budding ( , ) . niv infection is currently classed as a stage iii zoonotic disease, meaning it can spill over to humans and cause limited outbreaks of person-to-person transmission ( , ) . niv outbreaks have been recognized yearly in bangladesh since as well as occasional outbreaks in neighboring india (figure ). these outbreaks have been characterized by person-to-person transmission figure | previous locations of henipavirus infection outbreaks. nipah and hendra virus distribution map highlighting the range of the natural wildlife reservoir, pteropus spp. bats [adapted from ( ) ]. and the death of over % of infected people ( , ) . in may , the first ever outbreak in southern india was reported. a total of niv cases, of which resulted in death, were reported in the state of kerala. pteropus giganteus bats from areas around the index case in kozhikode, kerala, were tested at the national high security animal diseases laboratory at bhopal. of these, % were found to be niv positive by rt-pcr ( ) . characteristics of niv that increase the risk of it becoming a global pandemic include: humans are already susceptible; many niv strains are capable of person-to-person transmission; and as an rna virus, niv has a high mutation rate ( ) . niv has been found to survive for up to days when subjected to various environmental conditions, including fruit bat urine and mango flesh ( ) . whilst survival time was influenced by fluctuations in both temperature and ph, the ability for niv to be spread by fomites could play a role in outbreak situations. the first and still most devastating niv outbreak occurred in peninsular malaysia from september to may ( , ) . the link to pigs in this outbreak was obvious as % of the infected patients had contact with pigs ( ) . if a niv strain were to become human-adapted and infect communities in southeast asia where there are high human and pig densities and pigs are a primary export commodity, infection could rapidly spread and humanity could face its most devastating pandemic ( , , ) . in september , there was an outbreak of severe febrile encephalitis among pig farmers in the state of perak, malaysia, that was associated with a high mortality rate. a total of cases of encephalitis, of which resulted in death, were confirmed. these deaths were initially thought to be due to japanese encephalitis (je), an endemic disease in malaysia. however, with most cases occurring in men who worked with pigs, the epidemiological characteristics of this disease were distinct from those of je, where ∼ % of cases occur in children aged - years ( ) ( ) ( ) . the epidemiological link was from fruit bats infecting pigs that then served as amplifier hosts, resulting in transmission to humans through close contact ( ) . as a result of movement of infected pigs and humans to other states in malaysia, by february similar diseases were recognized in both pigs and humans in new outbreak areas ( ). in the following month, there were cases of respiratory illness and encephalitis amongst singapore abattoir workers who had handled pigs imported from the outbreak areas in malaysia ( ) . due to this, the importation of pigs from malaysia ceased which in turn ended the outbreak in singapore. the outbreak in malaysia ended when . million pigs ( % of the country's pig population) were culled from outbreak and surrounding areas ( , ) . the niv outbreak incurred significant economic costs and long-term damage to the malaysian pig industry: us$ million in direct costs and lost market revenue, including us$ million in compensation to farmers for the . million pigs slaughtered and , jobs lost ( ) . to this date, malaysian pig farming is only permitted in "identified pig farming areas." pigs also suffered during the / malaysian outbreak, but this was only diagnosed as part of the investigation following the human cases. the severity of symptoms of niv infection in pigs varied with age. in suckling pigs (< weeks old), mortality could be high (up to %) and labored breathing and muscle tremors were evident. in growing pigs ( to months), an acute febrile (> . • c) illness was observed with respiratory signs ranging from increased or forced respiration to a harsh, loud non-productive cough, open mouth breathing, and epistaxis ( ) . in some cases these respiratory signs were accompanied by one or more of the following neurological signs: trembles, neuralgic twitches, muscle fasciculation, tetanic spasms, incoordination, rear leg weakness, or partial paralysis. pigs of this age had high morbidity and low mortality (< %) ( ) ( ) ( ) . some animals over months of age died rapidly (within h) without signs of clinical disease. respiratory signs were reported in adult pigs, as with younger animals, although these were less obvious (labored breathing, bloody nasal discharge, increased salivation) and neurological signs included head pressing, bar biting, tetanic spasms and convulsions. first trimester abortions were also reported ( ) ( ) ( ) . in an experimental infection study, pigs were inoculated subcutaneously with a niv isolate from the central nervous system of a fatally infected human patient. infection elicited respiratory and neurological symptoms consistent with those observed in naturally infected malaysian pigs, which included febrile illness, incoordination, mucosal nasal discharge, and persistent cough ( ) . pigs inoculated orally with the same dose did not show clinical signs although they still shed virus. in a second study, piglets were inoculated oronasally with a human niv isolate ( ) . all infected animals showed a transient increase in body temperature between and days post-infection. two of these animals developed transient respiratory signs, mild depression and a hunched stance. both these studies concluded that niv infection in pigs had no pathognomonic features i.e., the clinical signs observed were non-specific. this can make field diagnosis of niv infection in pigs difficult, as observed in the outbreak in malaysia ( , ) . the name proposed for the disease caused by niv infection of pigs was "porcine respiratory and neurological syndrome" (also known as "porcine respiratory and encephalitis syndrome"), or, in peninsular malaysia, "barking pig syndrome" ( ) . niv infection was included as the sixth pig disease notifiable to the oie world organization for animal health ( ) . the oie approve diagnostics and recommends preventative and control measures for a range of transboundary livestock diseases. despite the importance of niv as an emerging disease with the potential for pandemic, no therapeutics or vaccines are approved for use in humans or livestock species. due to the lethal nature of niv infection, producing a safe, live attenuated vaccine with no potential for reversion is difficult. however, recombinant niv mutants, attenuated in hamster and ferret models, have been shown to generate strong neutralizing antibody responses ( , ) . more commonly, niv vaccine approaches have focused individual candidate antigens delivered as subunit vaccines or using viral vectors. the most studied vaccine candidate is the soluble form of the g protein (sg) from the related hendra virus (hev). hev and the niv malaysia strain share between and % amino acid homology between their proteins; with f and g proteins sharing and % homology, respectively ( ) . both f and g envelope glycoproteins are regarded as vaccine candidate antigens since they are the targets of niv neutralizing antibodies ( ) . an adjuvanted hev sg protein subunit-based vaccine (equivac r hev, zoetis) has been licensed in australia to protect horses against hev and to reduce the zoonotic risk to humans ( ) . equivac r hev protects ferrets and african green monkeys (agms) after experimental challenge with niv, as well as hev ( , ) . surprisingly, this vaccine failed to protect pigs from experimental niv challenge ( ) . since the vaccine induced cross-neutralizing antibodies but not measurable t cell responses, the authors concluded that both arms of the adaptive immune response may be required for protection against niv and hev. these studies also potentially highlight that adjuvants can have species specific effects and tailoring of adjuvants to the target species may be required or considered in the context of preclinical models. the experimental viral vectored vaccine candidates for niv include vesicular stomatitis virus, rabies virus, canarypox virus (alvac strain), adeno-associated virus (aav), measles virus, newcastle disease virus (ndv) and venezuelan equine encephalitis virus ( ) . alvac expressing niv g or f (alvac-g and alvac-f) was found to protect pigs against niv challenge weeks after the second immunization ( ) . high titres of niv neutralizing antibodies were induced with the alvac-g vaccine, while despite the low levels of neutralizing antibodies induced by the alvac-f; all vaccinated pigs were protected against virulent niv challenge. recombinant attenuated ndv expressing niv glycoproteins have been shown to induce long lasting niv-specific nabs in pigs, with the vector expressing niv g performing better than niv f ( ). however, no challenge was performed in this study and it remains to be determined whether these paramyxovirus-based vaccine candidates are efficacious. compared to canarypox vectors, ndv-based vectors have a number of advantages including their high titer propagation in chicken eggs removing the requirement for cell culture ( , ) . despite these encouraging results and the continued threat posed by niv, no vaccine candidate has progressed toward market for either pigs or humans. the promising performance of experimental niv and hev vaccines in animal models and the licensure of equivac r hev, as a "one health" vaccine to safeguard animal and human health, strongly support the proposition that a safe and effective niv vaccine may be developed for pigs to reduce the severe economic consequences of niv outbreaks and the threat to public health. with partners, we have initiated a project that aims to develop such a vaccine. we are systematically analyzing the immunogenicity and protective efficacy of three niv vaccine candidates in pigs: ( ) an adjuvanted niv sg protein (orthologous to the equivac r hev vaccine), ( ) niv g protein delivered by a replication-deficient simian adenoviral vector (chadox niv g), and an adjuvanted, molecular clamp stabilized niv f (mcsf) protein. chadox is a multispecies vector with an established human and livestock safety profile ( ) . chadox offers the potential for both single dose efficacy and thermostabilization ( , ) . the molecular clamp is a proprietary stabilization domain that preserves the f protein in its native "pre-fusion" form, which should enhance immunogenicity and thermostability. in depth analyses of t cell and antibody responses are being conducted to identify correlates of vaccine-induced protection. we will examine the durability of niv-neutralizing antibodies and other immune responses associated with protection, including a comparison of a singleshot vs. homologous prime-boost immunization regimes. incontact animals will be introduced to assess transmission of challenge virus from vaccinates or unvaccinated control animals. the sporadic nature of niv outbreaks means that the commercial development of niv vaccines for use in pigs (other livestock or humans) is limited and animal health companies are of the opinion that niv vaccines will have limited marketability. our ongoing studies should help facilitate this by developing a safe and efficacious prototype niv vaccine that is amenable to "surge production" and discrimination of infection in vaccinated animals (diva) capability. subsequent development and licensure of this vaccine will require engagement with international, regional, and national agencies and the creation of dependable markets via the establishment of niv vaccine banks. the oie world fund manages vaccine banks and the delivery of vaccines for avian influenza, rabies, foot-andmouth disease, and peste de petit ruminants ( ) . vaccine banks ensure the procurement and delivery of high quality vaccines mass-produced in line with oie intergovernmental standards. critically these vaccine banks can be rapidly deployed when required and this model appears most appropriate in the context of reactive emergency vaccination programmes to aid niv outbreak control. vaccines can play a major component in an emergency response against emerging infectious disease, with the main aim to reduce virus spread between susceptible hosts ( ) . the precise decisions on control strategies will be complex and vary for different regions. factors such as: herd density, production systems, the presence of susceptible wildlife, the impact on export trade and current opinions on economic vs. ethical factors will likely play a role. one strategy to halt a niv outbreak would be to deploy a stockpiled vaccine for ring vaccination around the niv affected area. this approach was utilized in the ebola outbreak in guinea and showed great promise in terms of disease containment and elimination ( ) . for such a strategy, a vaccine with single-dose efficacy and a rapid onset of immunity preventing virus transmission would be preferential. this is likely to be best achieved with a viral-vectored ( ) or mrna vectored vaccine ( ) . the highly unpredictable nature of niv outbreaks means that it is highly unlikely that niv vaccines would be used routinely by pig producers. one strategy that could help ensure that immunity to niv is maintained in pig herds could involve the engineering of niv g into a live attenuated viral vaccine, such as pseudorabies, which are widely used in countries at-risk. the recent ebola and zika epidemics highlighted how poorly prepared we were to deal with these new and emerging diseases. there has therefore been a global drive to develop vaccines against these diseases and improve preparedness. the coalition for epidemic preparedness innovation's (cepi's) was established in with a mandate of financing and coordinating the development of new human vaccines to prevent and contain infectious disease epidemics. cepi selected niv, lassa virus and middle east respiratory syndrome-coronavirus, three pathogens from the who's list of priority diseases needing urgent r&d attention as its initial focus ( , ) . the who's list of priority diseases is part of the r&d blueprint, which identifies priority diseases and addresses gaps in the global scientific community to increase preparedness for future outbreaks. the main aim of the blueprint is to fast-track the availability of effective tests, vaccines, and medicines that can be used to save lives and avert large scale crises ( ) . in , the us food and drug administration (fda) established the "animal rule" for regulatory approval of vaccines and therapeutics for which efficacy testing in humans is impossible, therefore requiring relevant animal models that represent a disease model similar to that of the human disease ( ) . vaccine efficacy studies in animal models aim to identify specific vaccine-induced correlates of protection including neutralizing antibodies or cell-mediated responses ( ) . in , a vaccine to protect against anthrax was the first to be approved through the "animal rule" ( ) . the licensing pathway for the "animal rule" requires that immunogenicity results from clinical trials must be consistent with previously identified immune correlates associated with protection ( ) . therefore, identifying reliable markers of vaccine-generated immunity becomes critically important for pathogens such as niv. large animal models have been shown to more accurately predict vaccine outcome in humans in comparison to small animal models ( ) therefore defining correlates of vaccineinduced protection in pigs, may play an important role in supporting subsequent human vaccine licensure under the "animal rule." animal models can be validated for a particular disease according to a number of different criteria, which include "face" and "predictive" validity. for face validity there must be similarities in the pathology and clinical symptoms between the animal model and the human disease ( ). as discussed above, niv infection of pigs causes a similar respiratory and neurological syndrome as seen in human infections. although, disease severity in pigs may be considered lower than in humans. the predictive validity of a model means that clinically effective interventions demonstrate a similar effect in the animal model ( ) . no clinical trials of niv vaccine candidates have been reported to compare with vaccine performance in animal models, including the pig. as noted above, the success of the equivac r hev vaccine in horses and other animal models was not replicated in swine ( , ) , highlighting a potential issue of predicative validity when comparing niv vaccines between animal species, which may extend to humans. on the other hand, pigs have been used successfully as models to study many human infectious diseases ( ) ( ) ( ) ( ) ( ) ( ) ( ) , including niv infection ( ) . there is also a growing appreciation that pigs provide a superior animal model for influenza a virus infection and immunity and should play a more prominent role as a model for human influenza vaccine development ( ) . the success of the pig as an experimental animal model is partly due to their similarities with humans in terms of anatomy, immunology, and physiology, but also due to their manageable behavior and size, and by the general ethical acceptance of using pigs for experimental purposes instead of non-human primates ( , , ) . the niv outbreaks in malaysia and singapore demonstrated that pigs can play a key role in the epidemiology of niv by acting as an amplifier host. the region most at risk of niv infection has some of the highest pig population densities found anywhere in the world, which are rising fast due to the demand of a growing human population. this increases the risk of niv transmission to pigs and humans. the development of a niv vaccine for use in pig populations would decrease the major risk niv poses to the developing pig industries, as well as to the livelihoods of poor livestock keepers in southeast asia. the use of non-human animal models is crucial for vaccine development against diseases such as niv since efficacy testing in humans is impossible. the pig model may therefore contribute to human vaccine development, supporting human vaccine licensure under the animal rule. pteropid bats are confirmed as the reservoir hosts of henipaviruses: a comprehensive experimental study of virus transmission functional studies of host-specific ephrin-b ligands as henipavirus receptors structural basis of nipah and hendra virus attachment to their cell-surface receptor ephrin-b unraveling a three-step 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open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -jkjdglns authors: alotaibi, badriah; bieh, kingsley; yassin, yara; mushi, abdulaziz; maashi, fuad; awam, amnah; mohamed, gamal; hassan, amir; yezli, saber title: management of hospitalized drug sensitive pulmonary tuberculosis patients during the hajj mass gathering: a cross sectional study date: - - journal: travel med infect dis doi: . /j.tmaid. . . sha: doc_id: cord_uid: jkjdglns background: to document the management of drug-sensitive tb patients during the hajj and assess compliance with the saudi tb management guidelines. method: the study was conducted in hospitals in makkah during the and hajj seasons. structured questionnaire was used to collect data on relevant indices on tb management and a scoring system was developed to assess compliance with guidelines. results: data was collected from tb cases, . % ( / ) were saudi residents. sputum culture was the only diagnostic test applied in . % ( / ) of patients. most ( . %, / ) confirmed tb cases were isolated, but only . % ( / ) were tested for hiv and merely % ( / ) received the recommended four st-line anti-tb drugs. guideline compliance scores were highest for infection prevention and control and surveillance ( . / ) and identifying tb suspects ( . / ). the least scores were obtained for treating tb ( . / ) and diagnosing tb ( . / ). conclusions: healthcare providers training and supervision are paramount to improve their knowledge and skill and ensure their compliance with existing tb management guidelines. however, there may be a need for the introduction of an international policy/guideline for tb control and management during mass gatherings such as the hajj to guide providers’ choices and facilitate monitoring. tuberculosis (tb) remains a global public health problem with significant morbidity and mortality. in , the world health organisation (who) estimated that million people developed active tb causing up to . million deaths [ ] . the who's end-tb strategy aims to reduce the overall number of tb deaths by % and the tb incidence rate by % in compared with the baseline incidence and mortality figures [ ] . key to achieving these targets is the early diagnosis and appropriate management of tb cases worldwide according to national and international guidelines [ ] [ ] [ ] [ ] . this includes in the context of mass gatherings such as the annual hajj in makkah, kingdom of saudi arabia (ksa), where over million pilgrims, many originating from tb endemic areas, congregate in crowded settings and worship under conditions that increase the risk of tb transmission [ , ] . in the context of the hajj, respiratory tract infection including those caused by viruses have been researched in detail [ ] . however, while tb cases have been reported during the event [ , ] , tb management approaches during this unique event remained largely undocumented, and it is unknown whether these are consistent with the ksa and international tb management guidelines. hospitals in makkah serve both hajj pilgrims as well as local residents and the hajj workforce during the mass gathering free of charge. the additional stress on these health facilities during hajj may compromise the services provided for all patients during the event, including those diagnosed with tb. this study documents the management of drug-sensitive tb patients during hajj and explores the compliance of healthcare providers with the ksa tb management guidelines in the ministry of health (moh) hospitals in makkah during the mass gathering. this cross sectional study took place in makkah, saudi arabia, and https://doi.org/ . /j.tmaid. . . received january ; received in revised form june ; accepted july included hospitals comprising those serving pilgrims in hajj holy sites. the study was conducted during the hajj lunar month ( st- th dulhija) during the and hajj seasons, corresponding to nd sep- st oct and nd aug- st sep , respectively. all hospitalized adults (> years old) diagnosed with drug-sensitive pulmonary tb (ptb) during the study period were enrolled in the study if they consented. these patients are referred to in the manuscript as "suspected tb patients" until the time they were confirmed to have tb by the healthcare facility there were admitted to. the management of tb patients was documented using a specifically designed data collection form which included patients' demographics data, underlying health conditions and tb risk factors as well as clinical data including various aspects of tb management such as patients' screening, infection prevention and control (ipc), tb diagnosis and treatment and case notification and outcome. data was collected by the study team from patient's records, attending physician and through interviews with patients. all analyses were done using spss . (spss inc., chicago, usa) and sas . (sas institute inc., nc, usa) software program. variables were characterized using frequencies and mean for the respective categorical and continuous variables. a scoring system was developed, having identified four key themes from the questionnaire and literature review which were relevant to tb management in hajj. the themes were ) identifying tb suspect, ) ipc and surveillance, ) diagnosing tb and ) treating drug-sensitive tb. for each theme, relevant indicators were identified from the questionnaire (tables - ) . a score of was assigned for each indicator/variable that was consistent with the ksa tb management guideline [ ] (latest version during study period) and for inconsistency with the guideline. the indicator subscore (x) for guideline consistency was obtained as follows: x = number of cases consistent with guidelines (c)/ total number eligible of cases (d) * the eligible cases summed the consistent and inconsistent responses with tb guideline and strictly excluded missing data and unknown responses. the guideline consistency score for each theme was obtained by calculating the mean of the indicator sub-scores for each theme. the study was approved by the king fahad medical city ethics committee and the institutional review board (irb log: - e) and conducted in accordance with the ethics committee's guidelines. characteristics of the study population are presented in table . for the two-year period, confirmed drug-sensitive ptb patients were recruited for the study. the mean age of the study population was years (sd = . years, range - years). most ( . %, / ) were males and over half were over years old ( . %, / ), with only primary or no formal education ( . %, / ). the tb patients were nationals of countries but the majority ( . %, / ) had been residing in ksa for at least one year (table ) . over one third of the cases ( . %, / ) did not complete their hajj rituals while the status of % ( / ) of the cases was not known. three confirmed tb cases ( . %, / ) did complete their hajj rituals in individual ambulances. no mortality was recorded among the tb patients at the time when the study ended with half ( / ) having been discharged (table ) . in relation to tb risk factors, only . % ( / ) of respondents reported prior travel to high tb burden countries (pakistan and indonesia) and . % ( / ) stated that they had performed hajj or umrah within the past year. the majority ( %, / ) of the previous hajj or umrah pilgrims were saudi residents. a sizable proportion of tb cases declared that they had smoked or were current smokers of tobacco products ( . %, / ) or had chronic diseases ( . %, / ) especially diabetes and hypertension (table ) . upon registration, most ( . %, / ) suspected tb patients were admitted to an isolation room or ward. otherwise, the patients were either admitted into the emergency room (er [ . %, / ]), icu ( . %, / ), neurosurgery ward ( . %, / ), orthopedic ward ( . %, / ) or general ward ( . %, / ). while a proportion ( . %, / ) of suspected tb patients was separated from other patients during registration, the triaging status of . % ( / ) of suspected tb patients was not reported ( table ). the proportion of suspected tb patients put in isolation while waiting diagnosis was . % ( / ) and rose to . % ( / ) once drug-susceptible tb was confirmed. only ( . %) confirmed tb patient was managed in the er. generally, most ( . %, / ) confirmed tb patients spent less than day in the health facilities before they were isolated. in all cases, appropriate symptoms were sought from tb suspects, in particular, cough ≥ weeks ( . %, / ) and fever with chills/night sweat ( . %, / ). the history of contact with active tb cases was obtained from . % ( / ) of suspected tb cases, although the status of this variable was unknown in a further . % ( / ) of the cases (table ). in general, . % ( / ) of the tb suspects were questioned about other tb risk factors. specifically, their country of residence, hiv status and potential occupational exposure to tb (table ) . furthermore, in the majority of cases ( . %, / ) providers used recommended screening test for active tb (chest x-ray) in case management. in terms of diagnostic period, % ( / ) of tb suspected patients within known health facilities visits status had > visits to health facilities before appropriate screening/diagnostic tests were ordered (table ). however, the period between patient registration/arrival in health facilities and order of tb screening/diagnostic test(s) was ≤ h in . % ( / ) of cases. similarly, in most cases ( . %, / ), the period between ordering screening/diagnostic test(s) and confirmation of tb diagnosis was ≥ days. in general, . % ( / ) of cases were diagnosed within days of arrival/registration to healthcare facilities. sputum culture was the diagnostic test utilized in the majority ( . %, / ) of cases (table ). xpert mtb/rif assay was not utilized for tb diagnosis. in one instance, none of the recommended diagnostic tests were ordered for the tb suspected patient. only . % ( / ) of suspected/confirmed tb cases were screened for hiv. in over half of cases ( . %, / ), the tb suspected patients were questioned about their tb history, although for a further . % ( / ), response to this variable was unknown ( based on the duration of hajj season (around one month), the possibility of monitoring treatment completion is unfeasible in a hajj study. thus, appropriate post-hajj referral and provision of drugs to last during the referral period were proxies for estimating possible continuity of care. in most cases it was not known whether confirmed tb patients were referred for further treatment after completion of hajj ( . %, / ) or were given enough anti-tb drugs to last until they arrive in their country of residence ( . %, / ). in . % ( / ) of cases, the confirmed tb cases were reported to the ksa moh preventive medicine department. the reporting status of the rest of the cases ( . %, / ) was unknown. a proportion ( . %, / ) of the confirmed tb cases was reported to the appropriate country medical missions' office. this excludes the . % ( / ) of cases with unknown medical missions' reporting status. among the latter, . % ( / ) were saudi residents. the confirmed tb case status of two residents of pakistan and myanmar were not reported to their respective country medical mission's office. the tb management guidelines compliance scores across the identified themes are presented in table . out of a maximum possible score of , the overall guideline compliance score was highest for the themes ipc and surveillance ( . ) and identifying tb suspects ( . ). the least scores were obtained for the themes treating tb ( . ) and diagnosing tb ( . ). some notable variations in theme's sub-scores were observed. for instance, while the overall score for the identifying tb suspects theme was high, a low score was documented for obtaining history of tb risk factors from patients. inversely, while the overall score for treating tb was average, high score was seen in relation to not starting tb treatment for patient before tb diagnosis. this study exemplifies the compliance of tertiary healthcare providers with the saudi national guidelines for tb management during the hajj. the result showed high level of compliance with the assessed tb management guidelines indices for systematic screening of tb suspects as well as ipc and surveillance, but low compliance scores were obtained for prompt tb diagnosis and use of standardized treatment regimen for drug-susceptible tb. most tb cases in the current study were males and over half were above years old with primary or no formal education. this is in accordance with global and hajj-related data and established risk factors for tb [ , , , ] . however, the prevalence of coexisting chronic diseases ( %, / ) among tb patients, especially diabetes, was higher than that reported internationally as well as previous studies among hajj pilgrims with tb [ , [ ] [ ] [ ] . the presence of chronic diseases, increases the risk of tb disease, predisposes to severe illness and complicates tb treatment [ , ] . as such, the management of comorbidities is now a key focus of the integrated, patient-centered care and prevention strategy of global tb control [ ] . around % ( / ) of the tb cases reported being current smokers and a similar proportion ( / ) indicated that they did smoke in the past. this is higher than that reported in another study among hajj pilgrims with tb ( . %) [ ] but lower than figures from some international reports [ , ] . other risk factors for tb such as visit to, or residence in high-burden countries and occupational exposure were uncommon among tb patients in the study and so was previous hajj or umrah performance. while the latter events are not an established risk factor for tb transmission, hajj is a risk of tb infection and both clinically-recognized or undiagnosed active tb have been reported at the pilgrimage [ , , ] . the majority ( . %, / ) of tb patients in this study were ksa residents. this may be explained by the fact that the study included both pilgrims and non-pilgrims and that healthcare facilities in makkah provide healthcare to pilgrims and non-pilgrims during the hajj. although saudi arabia is not a high tb burden country, tb incidence in the country show significant regional variation with the makkah region showing much higher tb incidence rates than the rest of the country and rising trend [ , ] . in addition to the hosting of the hajj and umrah mass gatherings, this high tb incidence may also be related to the fact that around % of the makkah region population are non-saudis, many originate from and frequently visit high tb burden countries [ , ] . regardless, it is evident that in addition to strategies to control imported active tb [ ] , interventions to prevent transmission during hajj from locals and internal pilgrims with tb should also be developed and implemented. generally, home-based care for tb is preferred to methods of care that are based on strict hospitalization. in the ksa context, medical or mental instability and residence in congregate settings are among factors that may warrant hospitalization [ ] . in this study, most of the suspected and confirmed tb patients were admitted and isolated for tb management. to the best of our knowledge, there is no standardized global protocol guiding the choice of suitable models of care-whether home based or hospitalized care-during international mass gatherings. however, considering the potential of tb transmission in such crowded settings, the constant mobility of pilgrims and challenges in verifiable or stable residences for pilgrims during hajj, hospitalization, although undesirable, seems a logical and practical choice for tb management during the mass gathering. ipc in healthcare settings is one of the key strategies for tb control [ ] . however, implementation of ipc recommendations seems to be inadequate with several studies reporting poor tb infection control measures in health facilities [ ] [ ] [ ] [ ] . further, many hcws are practicing without adequate infection control training and often lack knowledge on tb infection control strategies and guidelines [ , ] . in the current study, we report high compliance with the aspects of tb ipc [ ] . early detection through systematic screening of tb suspects is key to improving tb case detection. the who recommends that persons with signs and symptoms consistent with tb should be evaluated for tb to ensure prompt diagnosis and treatment [ , ] . similarly, the saudi tb guideline recommends that healthcare workers (hcws) should be knowledgeable about tb symptoms to facilitate the efficient identification of tb suspects for diagnosis and treatment [ ] . in the current study, providers utilized presenting symptoms to correctly identify suspected tb patients in all cases. cough and fever with chills/night sweat were the most frequent symptoms among patients. this finding corroborates existing evidence that identifies cough as the most common symptom of ptb [ , ] . further, in majority of cases ( . %, / ), chest x-ray, a recommended screening tool for active tb, was conducted for the tb suspects. chest x-ray is particularly more sensitive for tb screening after a positive symptom screening [ ] . however, we also found that less than half of the tb suspected cases were questioned about tb risk factors. adequate knowledge of tb symptoms and risk factors among providers are prerequisites for correct and prompt identification of suspected tb patients for screening and diagnosis [ , ] . in view of the significant use of both symptom-based and radiological screening methods in this study, a total guideline compliance score of . out of was obtained for the prompt identification and screening of tb suspects theme for tb management. delayed diagnosis of tb can enhance the transmission of infection, worsen the disease and increase the risk of death [ , ] . in the current study, half of the tb cases had more than visits to healthcare facilities before tb screening/diagnosis tests were ordered for the patients. while studies from other settings reported similar findings [ , ] , our results are concerning, as delays in diagnosing tb during hajj may lead to significant transmission given the crowded setting during the event. similarly, sputum culture (which takes at least - weeks to produce results) was the only recommended diagnostic test applied in about % ( / ) of cases. the application of sputum culture as the singular diagnostic test is not consistent with approved standards for tb diagnosis [ ] . as % ( / ) of suspected cases were confirmed to have tb by the third day of arrival in the health facility, it appears that providers relied on screening tests, such as chest x-ray, for the confirmation of tb diagnosis. this practice is inconsistent with both national and international guidelines; chest radiography is only recommended for screening purposes. the ksa tb guidelines recommended the use of xpert mtb/ rif as an initial tb diagnostic test on a conditional basis [ ] . as such, the latter was not included in the scoring criteria for this study. nonetheless, xpert mtb/rif, which could detect tb and mdr-tb by proxy in the same day [ ] , was not applied for tb diagnosis in this study. although available in a number of saudi hospitals and reference labs, the roll out of xpert mtb/rif has been slow and its use for pointof-care testing is limited [ , ] . access to same day diagnosis of tb could prove valuable in a highly mobile hajj population where followup visits to the same health facility may not be guaranteed and where delays in diagnosis may increase the risk of transmission in such crowded settings. as such, ksa authorities should consider the provision of tb molecular testing capability in health facilities within the hajj areas to facilitate rapid (same-day) diagnosis of tb during the mass gatherings. due to the synergistic relationship between hiv and tb, it is recommended that all tb patients should be screened for hiv [ ] . yet, only a fraction of tb patients were questioned about their hiv status ( . %, / ) or tested for hiv ( . %, / ) in this study. this is much lower than what is reported globally [ ] . as a low prevalence setting, knowledge of hiv among healthcare workers is low in saudi arabia [ ] . yet, hiv could be a more frequent comorbidity among pilgrims who arrive with active tb from areas with high hiv disease prevalence [ ] . more so, a missed or delayed hiv diagnosis in a tb patient stalls the commencement of appropriate treatment and results in poor outcomes for the patient, community and health system [ ] . therefore, healthcare providers in ksa ought to be trained and guided to conduct screening for hiv and other comorbidities in all suspected tb patients irrespective of their nationality. in general, because of delays in diagnosis, infrequency of hiv testing and failure to utilize the appropriate diagnostic tests for suspect tb patients, the combined score for the tb diagnosis theme was out of a maximum of , the lowest score of all tb management themes in the current study. treatment of tb in ksa is free of charge for pilgrims and other patients and both the ksa and who guidelines for tb management recommend the use of four st-line anti-tb drugs in the treatment of drug-susceptible tb [ , ] . the guideline compliance score for tb treatment in this study was average; partly because % ( / ) of tb patients received fewer than four st-line anti-tb drugs. in general, inappropriate treatment of tb is common worldwide. in a systematic review that included studies from countries, inappropriate treatment regimens were prescribed in % of the studies and the percentage of patients on inappropriate regimens varied between . % and % [ ] . poor knowledge of national and international tb management guidelines contributes to inappropriate prescription of anti-tb drugs by healthcare providers, and the use of inappropriate regimen drives the occurrence of relapse and the emergence of drugresistant tb [ , ] . both the who and ksa tb guidelines recommend that all patients with ptb being treated with the st-line regimen should have their sputum samples tested by the end of the nd, th and th month of treatment [ , ] . in the current study, all confirmed tb cases with known notification status were reported to the saudi health authorities. however, it is unknown whether the continuum of care was maintained for tb cases who were international pilgrims and who had to return to their home countries soon after the pilgrimage (before the end of the treatment period). any travel-related treatment interruptions could breed treatment relapse and drug-resistance and propagate community spread of tb. both national and international tb guidelines fall short of providing guidance on tb control at international mass gatherings, including procedures for ensuring access to care and support services during travel. thus, the development and dissemination of a multinational hajj and umrah and/or mass gatherings-specific tb management protocols are needed. these protocols should also include pathways for the safe transfer across borders and follow up of tb patients involved in mass gatherings. the current study is among the foremost surveys of tb management at international mass gatherings. while the small number of cases and high proportion of unknown responses for some variables constituted limitations, the tb management indices obtained was a fair representation of the compliance of providers with national and international tb guidelines in moh hospitals during the hajj. the findings provides a basis for the review of existing practices across settingsprivate and public sector vs national and foreign health facilities-and serves as a reference for the development of appropriate guideline and protocol for tb management at the hajj and umrah, as well as other settings with similar health system resources and population dynamics hosting recurrent international mass gatherings. in the short term, availability of rapid molecular diagnostic techniques for tb as we all improving hcws' knowledge regarding tb management guidelines and monitoring compliance are needed to ensure tb patients are management appropriately during hajj and that tb transmission is prevented. no conflicts of interest to declare. none to declare. the study was approved by the king fahad medical city ethics committee and the institutional review board (irb log: - e) and conducted in accordance with the ethics committee's guidelines. all participants gave verbal consent before enrolment. world health organization. gear up to end tb: introducing the end tb strategy. world health organization world health organization. early detection of tuberculosis: an overview of approaches, 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meta-analysis world health organization. treatment of tuberculosis: guidelines. geneva: world health organization key: cord- - pxki y authors: huang, huanhuan; chen, suqing; zhang, xiaoyan; hong, linliang; zeng, yongbin; wu, bin title: detection and clinical characteristics analysis of respiratory viruses in hospitalized children with acute respiratory tract infections by a gexp‐based multiplex‐pcr assay date: - - journal: j clin lab anal doi: . /jcla. sha: doc_id: cord_uid: pxki y background: the information regarding viral epidemiology and clinical characteristics in hospitalized children with acute respiratory tract infection (arti) in central fujian is limited. in this study, we aimed at analyzing the viral epidemiology and clinical characteristics of arti in hospitalized children admitted to the first affiliated hospital of fujian medical university. methods: cohort of hospitalized children ( days to years) diagnosed with arti admitted to the department of pediatrics from january , , to december , , was enrolled in this study. nasopharyngeal swab or sputum samples on the day of hospitalization were tested for viruses via a gexp‐based multiplex‐pcr assay. the viral profiles and clinical characteristics were analyzed. results: the overall positive rate of the samples was . % ( / ). among the positive samples, ( . %, / ) had a single virus and ( . %, / ) had multiple viruses. there was a significant difference in the frequency of single vs mixed infections among positive samples ( . % vs . %; χ ( ) = . , p = . ) as well as among the total examined samples ( . % vs . %; χ ( ) = . , p = . ). human rhinovirus was the most prevalent virus ( . %, / ), followed by influenza a ( . %, / ) and human adenovirus ( . %, / ). there was no significant difference in the etiological distribution of viral pathogens between males and females (χ ( ) = . , p = . ). viral infections were more likely to occur in the winter‐spring months than in the summer‐autumn months ( . % vs . %, χ ( ) = . , p = . ). conclusions: the gexp‐based multiplex pcr is an accurate and high‐throughput assay allows us to quickly detect multiple respiratory viruses simultaneously in pediatric patients. our study provides information on the viral profiles and clinical characteristics in hospitalized children with arti, which would help better effective prevention strategies. acute respiratory tract infection (arti) is a leading cause of hospitalization, morbidity, and mortality in children in pediatrics throughout the world. [hadv] ) has been identified as a major cause in arti in children. identifying the pathogens of viral infection timely is especially important for early diagnosis and clinical decision-making for the pediatricians. presently, several diagnostic methods for the detection of respiratory virus, including virus culture, viral-antigen detection, and viral-antibody detection have been described. , virus culture is a gold standard method; however, this method is labor-intensive and time-consuming, making it impractical to be used in the clinical laboratory. antigen and antibody detection methods are easy-to-perform; however, they exhibit poor sensitivity and may have a false negative or positive reaction. in contrast, molecular techniques, such as polymerase chain reaction (pcr) and real-time fluorescent pcr assays, are sensitive and specific for virus detection ; however, only one virus can be detected by conventional pcr at a time. it should be noted that multiplex-pcr technology can simultaneously detect multiple pathogens at the same time, which is also easy-to-operate and need less workforce. it is well known that arti prevalence in children may vary in different geographic regions and different seasons. , however, information regarding viral arti in pediatric hospitalized children in fuzhou city (central fujian) is still limited. thus, to better understand the information about the epidemiology of the pathogens in pediatric hospitalized patients with arti and provide effective prevention strategies, we aimed in this study to investigate the epidemiology of respiratory viruses via a gexp-based multiplex-pcr assay in children under years of age in pediatrics. this study was conducted from january , , to december , , in the first affiliated hospital of fujian medical university. the inclusion criteria were as follows: patients under years old, acute fever, and symptoms of arti. the definition of arti was according to diagnostic criteria of "zhu futang practical of pediatrics". briefly, patients with arti appear at least one of the following symptoms: sore throat, cough, shortness of breath, or coryza as an acute onset of symptoms within two days. the exclusion criteria for all participants were as follows: antiviral, antibiotic, or hormonal drug treatment prior to admission; and patients receiving radiotherapy, chemotherapy, or immunosuppressive therapy. nasopharyngeal swab (nps) or sputum samples were obtained from patients with symptoms of arti on the day of hospitalization. total nucleic acid was extracted using a nucleic acid extraction kit following the manufacturer's instruction (ningbo zd biotechnology co., ltd). the -μl pcr amplification reaction mixtures contained μl of premix, μl of rt-pcr reverse transcriptase, and μl of nucleic acid. rt-pcr conditions were as follows: °c for minutes and then °c for minutes; the reaction was terminated by incubation at °c for minutes. multiplex-pcr conditions were as follows: step , °c for seconds, to °c touchdown pcr for seconds, and °c for seconds, repeated for six cycles; step , °c for seconds, °c for seconds, and °c for seconds, repeated for cycles; step , °c for minutes; and step , °c. were excluded for analysis in this study. the statistical analysis was performed using statistical analysis software spss package version . for mac os x (spss inc). continuous variables were expressed as means ± standard deviations. positive percentages in male and female patients and positive percentages among different age groups or different season groups were analyzed with the chi-square test. a p-value <. was considered to be statistically significant. rna-free water and the plasmid carrying the corresponding viral nucleic acid were used as a no-template control (ntc) and positive control (pc), respectively. as indicated in figure , no corresponding peak except the internal reference was observed in the ntc. whereas virus peaks appeared in the pc, and the peaks were well separated. no crossover reaction was found in pc. these indicated that the results of gexp-based assay were accurate and reliable. the overall positive rate of the samples was . % ( / ). the positive rates of viral infection in male and female patients were . % ( / ) and . % ( / ), respectively. there was no significant difference in the etiological distribution of viral pathogens between males and females (χ = . , p = . ). in this study, all the patients were grouped by age as follows: infants (age: < year), toddlers (age: - years), preschoolers (age: - years), and school-aged children (age: - years). the distributions of the viral etiologies of the four age groups were shown in table ; however, no significant difference among the different age groups was observed. the hospitalized children were divided into four groups according to table . overall, the positive detection rate was . %, . %, . %, and . % in spring, summer, autumn, and winter, respectively. viral infections were more likely to occur in winter-spring months than in the summer-autumn months ( . % vs . %, χ = . , p = . ). it should be noted that hadv occurred more frequently in summer. hrsv was detected almost throughout the year. other viruses occurred almost sporadically throughout the year without obvious seasonal trends. currently, nucleic acid amplification techniques (naats) (such as pcr and multiplex pcr) have been increasingly used for identification of pathogens in infectious respiratory diseases ; and one of the multiplex-pcr technology, genomelab gene expression profiler genetic analysis system (gexp)-based assay (developed by beckman coulter), has been adopted in the infectious pathogens diagnostics [ ] [ ] [ ] ; this detection technique has been applied in clinical for over years; however, more clinical application studies are still necessary, especially in southeast china. genomelab gene expression profiler genetic analysis system is a high-sensitive and specific analytical platform for high-throughput nucleic acid detection based on capillary electrophoresis separation technology, multiple gene analysis technology, and high-sensitivity laser-induced fluorescence technology. the gexp-based multiplex-pcr assay can easily detect and analyze up to genes simultaneously in a single analysis by converting amplification with multiple primers to amplification with a pair of universal primers. in this study, we used gexp-based multiplex-pcr assay to detect kinds of virus, as well as mycoplasma pneumonia and chlamydia pneumoniae in samples from hospitalized children with acute respiratory tract infection over a period of year. it was a rapid, accurate, and high-throughput analytical method which can finish the detection process within hours and may aid the pediatricians for early medical decision-making. overall, the positive rate for the detection of viruses in the present study was . %; hrv, ifva, hadv, and h n ranked top four in all the infected viruses. with the application of this multiplex-pcr assay, the detection of multiple co-infecting viruses had become common which reflected the high sensitivity of this multiplex-pcr assay. kouni s described that the prevalence of viral co-infections ranged between % and % in children seen in the hospital or emergency department. in the present study, the detection rate of in our study, gender had no influence on the detection rate of respiratory viruses, which was consistent with other reports. , , many studies documented that respiratory viral infection was characterized by seasonal distributions, with their peaks lasting from winter to early spring. , in the present study, the detection rate of respiratory viruses was also higher in winter and spring. one possible reason was school-aged children ( - y), n = all ages, n = no. however, there are several limitations to our study. first, all data for this study were obtained from a single center and may not be representative of the entire pediatric population. second, the sample size was relatively small, and more samples are needed to confirm these observations in future. in summary, the gexp-based multiplex-pcr assay allows us to quickly detect multiple respiratory infections caused by viruses. the array can detect respiratory viruses simultaneously in only hours. our study provides information on the viral profiles and clinical characteristics in hospitalized children with arti, which would help better effective prevention strategies. the study was supported by the grant from national natural science foundation of china ( ). the authors thank all the participants in this study for their cooperation in samples preparation. also, huanhuan huang wants to thank, in particular, the care and love in daily work and life from professor miaohui huang over the past years. huanhuan huang https://orcid.org/ - - - x yongbin zeng https://orcid.org/ - - - bin wu https://orcid.org/ - - - altered respiratory virome and serum cytokine profile associated with recurrent respiratory tract infections in children aetiological role of common respiratory viruses in acute lower respiratory infections in children under five years: a systematic review and meta-analysis respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology clinical characteristics and viral etiologies of outpatients with acute respiratory infections in huzhou of china: a retrospective study epidemiology of human respiratory viruses in children with acute respiratory tract infection in a -year hospital-based survey in northern italy rapid detection of respiratory organisms with the filmarray respiratory panel in a large children's hospital in china nucleic acid amplification-based diagnosis of respiratory virus infections viral etiology of acute respiratory infections in hospitalized children in novosibirsk city beijing: people's medical publishing house simultaneous differentiation of the n to n neuraminidase subtypes of avian influenza virus by a gexp analyzer-based multiplex reverse transcription pcr assay comparing the yield of oropharyngeal swabs and sputum for detection of common pathogens in hospitalized children with lower respiratory tract infection a comparison study between gexpbased multiplex-pcr and serology assay for mycoplasma pneumoniae detection in children with community acquired pneumonia evaluation of viral co-infections in hospitalized and non-hospitalized children with respiratory infections using microarrays use of multiple imputation to estimate the proportion of respiratory virus detections among patients hospitalized with community-acquired pneumonia the spectrum of viral pathogens in children with severe acute lower respiratory tract infection: a -year prospective study in the pediatric intensive care unit epidemiology and clinical characteristics of acute respiratory tract infections among hospitalized infants and young children in epidemiology and burden of influenza in healthy children aged to months: analysis of data from the placebo arm of a phase iii efficacy trial global role and burden of influenza in pediatric respiratory hospitalizations, - : a systematic analysis key: cord- -hxlebas authors: broekhuis, femke; madsen, emily k.; keiwua, kosiom; macdonald, david w. title: using gps collars to investigate the frequency and behavioural outcomes of intraspecific interactions among carnivores: a case study of male cheetahs in the maasai mara, kenya date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: hxlebas intraspecific interactions between individuals or groups of individuals of the same species are an important component of population dynamics. interactions can be static, such as spatial overlap, or dynamic based on the interactions of movements, and can be mediated through communication, such as the deployment of scent marks. interactions and their behavioural outcomes can be difficult to determine, especially for species that live at low densities. with the use of gps collars we quantify both static and dynamic interactions between male cheetahs (acinonyx jubatus) and the behavioural outcomes. the % home-ranges of males overlapped significantly while there was little overlap of the % home-ranges. despite this overlap, male cheetahs rarely came into close proximity of one another, possibly because presence was communicated through frequent visits to marking posts. the minimum distance between individuals in a dyad ranged from m to m but the average proximity between individuals ranged from , ± , m to , ± , m. possible interactions took place more frequently at night than by day and occurred mostly in the % home-range of one individual of a dyad or where cores of both individuals overlapped. after a possible encounter male cheetahs stayed in close proximity to each other for up to hours, which could be the result of a territory defence strategy or the presence of a receptive female. we believe that one of the encounters between a singleton and a -male coalition resulted in the death of the singleton. our results give new insights into cheetah interactions, which could help our understanding of ecological processes such as disease transmission. intraspecific interactions, or interactions between members of the same species, are an important component of population dynamics as they play a role in sociality [ ] , mating events [ ] , of movement behaviour and mortalities. based on previous research we predict that males will overlap spatially but that there will be little overlap of the core areas [ ] . we also predict that marking posts are frequently visited by both individuals in a dyad, i.e. pair of cheetahs, and that occasions where individuals of the dyad are in close proximity to each other are infrequent. because encounters between males can be aggressive [ ] we predict that the movement behaviour after a possible encounter would indicate avoidance behaviour (moving away from the encounter location, moving away from one another, increased distance travelled and decreased path tortuosity). the study was conducted in the maasai mara, in the southwest of kenya (centred at ˚s and ˚e), which is part of the larger serengeti-mara ecosystem. the study area (~ , km ) included the maasai mara national reserve and the surrounding wildlife conservancies. the area experiences one wet season spanning from november to june and one dry season spanning from july to october [ ] . after the wet season, the long grass attracts large numbers of migratory ungulates, including the white-bearded wildebeest (connochaetes taurinus) and the common zebra (equus quagga), from the serengeti in tanzania. throughout the year there is an abundance of cheetah prey including resident white-bearded wildebeest, thomson's gazelle (eudorcas thomsonii), grant's gazelle (nanger granti) and impala (aepyceros melampus) [ ] . the habitat in the maasai mara varies, ranging from open grasslands and shrubland, to riverine forests found along the major rivers and their tributaries [ ] . the open grassland plains, which are dominated by red oat grass (themeda triandra), are mostly found toward the south and west of the study area, while the north and north-east consist mostly of croton thickets (croton dichogamous) and vachellia woodlands (vachellia drepanolobium and v. gerrardii). global positioning system (gps) satellite collars (african wildlife tracking -www.awt.co.za) were fitted on four adult male cheetahs between th october and th february . in compliance with kenyan law, all immobilizations for deployment/removal of collars were performed by a kenya wildlife service veterinarian. cheetahs were free-darted and immobilized using a combination of ketamine ( - . mg/kg) and medetomidine ( . mg/kg), remotely administered by a dan-inject co rifle (dan-inject, denmark), and reversed with atipamezole ( . mg/ml; following [ ] ). sedation time was kept to a minimum, typically less than hour. after immobilization all cheetahs recovered fully, showing no signs of distress and no apparent side effects were observed in the short-and long-term. collars, which were only fitted on adults, weighed grams which is the recommended weight for cheetah collars [ ] . all collars were removed if they malfunctioned or if the batteries were low. the animal handling protocols used conformed to the standards of the american society of mammalogists [ ] and permissions to deploy collars were provided by the kenya wildlife service (permit no.: kws/ brm/ ) and the national commission for science, technology and innovation (permit no.: nacosti/p/ / / ). the collared males were all singletons, except for one male (m ), who was part of a fivemale coalition. over an month period, the five-male coalition were sighted on occasions and only on one occasion did the coalition separate for a period of < hours. we therefore collected data on a total of eight individuals in four social groups. while this is a relatively small sample size, it is a quarter of the entire population as there are approximately adults within the study area [ ] . the collars collected gps coordinates every three hours ( h, h, h, h, h, h, h and h) and when there was satellite communication, data were uploaded on a daily basis at h. on average, the collars were deployed for days ranging from to days (table ) . for each pair of cheetahs (hereafter referred to as a dyad), we only used simultaneously collected data for the analyses. static interactions. to determine the static interactions between male cheetahs we calculated their space use and the amount of overlap for each dyad to determine the possibility that individuals could encounter each other either directly or indirectly. space use for each individual per dyad was based on their utilisation distributions, which is the distribution of an individual's locations over time [ ] , using the adehabitat package [ ] in r [ ] . to calculate the utilisation distributions we used a fixed kernel density estimate using a bivariate normal kernel. we used the reference bandwidth parameter (h ref ) as the smoothing factor unless h ref > then we used % of the h ref to minimise over-smoothing of the data. using the resulting utilisation distribution, we determined both the % and % kernels which respectively represent an individual's total space use and their core areas. for each dyad we then calculated the amount of overlap of the % kernels and the % kernels. marking posts are used by male cheetahs to communicate their presence to conspecifics. using the methods described by [ ] , we located marking posts based on a cluster analysis using data from the gps collars and opportunistically when conducting fieldwork. for each dyad, we determined how many marking posts were found within the % kernel overlap. we used the recurse package [ ] to calculate ) how many marking posts were visited by each individual and ) how many marking posts were visited by both individuals in a dyad (hereafter referred to as mutual marking posts). we classified a visit when a cheetah came within m (half the average step-length, see dynamic interaction for details) of a marking post. for the mutual marking posts we calculated ) the time between individual visits and ) the time spent within m of a mutual marking post by each individual and tested whether there were differences between individuals. in addition, we calculated the time between visits from two different individuals i.e. we calculated how long it took for individual x to visit a mutual marking post once individual y had visited and vice versa. we then tested whether the time it took for an individual to visit a marking post once the other individual had been there differed between the individuals. we tested the data for normality using the shapiro-wilk test and used a t-test if the data were normally distributed and a wilcoxon test if they were not. if the data were not normally distributed then we provided the median ± median absolute deviation in addition to the mean ± standard deviation. dynamic interaction. we first explored the movement of the different individuals using the movevis package [ ] . then, to determine whether interactions between two individuals in a dyad were likely to occur, we calculated the proximity between simultaneous locations using the wildlifedi package [ ] . the hr resolution of the data is quite coarse so we used different proximity thresholds to group possible encounters based on the average -hour step-length which was , m ± , m (mean ± standard deviation). we used four proximity thresholds: < m, < m, < m and < m which correspond to . , , . and times the average step-length. we then determined whether these possible encounters took place at night (fixes at hr, hr, hr or hr) or during the day (fixes at hr, hr, hr or hr), whether they occurred within % kernels and the distance to the nearest known marking post. the half-way points between the two individuals were used as the estimated location where a possible encounter occurred. encounter outcomes. the gps data were examined once every few days. if any unusual behaviour was detected, such as no movement after a possible encounter, the field team would investigate to establish whether any injuries or deaths occurred as a result. in addition, for each possible encounter we compared the behaviour before to the behaviour after at four different time lags; hrs, hrs, hrs and hrs. first, we calculated the distance between two individuals before and after a possible encounter to determine whether males moved away from one another after an encounter. then, per dyad, we calculated ) the distance to the encounter location for each individual, ) the distance travelled per individual and ) the path tortuosity, or straightness. path tortuosity was calculated by dividing the net displacement by the total distance travelled. a value of around would indicate that the individual travelled in a straight line whereas a value < would be indicative of a tortuous path. we compared the proximity of the two individuals within a dyad, distance to encounter location, distance travelled and tortuosity before and after a possible encounter. we tested the data for normality using the shapiro-wilk test and used a t-test if the data were normally distributed and a wilcoxon test if they were not. in total, we secured simultaneous data on four male cheetah dyads ranging from to days per dyad. one individual (m ) was part of three of the four dyads, two individuals (m and m ) were part of two dyads and one individual (m ) was part of one dyad ( table ). across the four dyads the % kernels ranged from km to , km and the % kernels ranged from km to km ( table ) . for all the dyads, the % kernels overlapped but the amount of overlap ranged from % to % where in dyad the % kernel of individual m fell completely within the % kernel of individual m (fig ) . only the core areas ( % kernel) of dyad had an extensive area of overlap ( % and %), whereas the cores of the other dyads did not overlap or the area of overlap was minimal (< %). we found marking posts in the study area and the number of mutual marking posts per dyad ranged from to with the average number of visits to these marking posts ranging from to . (table ) . for the three dyads that had the most extensive spatial overlap and the largest number of mutual marking posts (dyads , and ) the average time that an individual was within m of a mutual marking post did not vary significantly between individuals in the same dyad, ranging from . ± . hours to . ± . hours (mean ± standard deviation; table ). the data were not normally distributed and the median for the same three dyads ranged from . ± . hours to . ± . hours (median ± median absolute deviation). for all the dyads, the average time between visits of a mutual marking post varied significantly between the individuals (table ). in the case of dyad and , individual m , who we classified as territorial, visited mutual marking posts more frequently compared to individuals m and m , neither of which were strictly territorial ( table ) . the time between different individuals visiting the same mutual marking post ranged from . ± . days to . ± . days and did not differ significantly across the dyads (table ) . for three of the four dyads (dyads , and ) the possibility of individuals within each dyad encountering each other was high as they overlapped extensively in space and had a large number of mutual marking posts. for these three dyads we explored their simultaneous movements and calculated the proximity between each individual within a dyad. the individuals within the three dyads did on occasions come into close proximity to one another as can be seen in the animation provided in the s movie. the minimum distance between individuals in a dyad ranged from m to m but the average proximity between individuals ranged from , ± , m to , ± , m (fig ) . possible encounters were classified according to four different thresholds and we detected four possible encounters with a proximity threshold of < m, with a proximity threshold of < m, with a proximity threshold of < m and with a proximity threshold of < m (table ). possible encounters were more likely to occur at night than during the day (χ = , df = , p = . ) and occurred most frequently at hr and midnight. of the possible encounters, % (n = ) occurred within the core area of one individual, % (n = ) occurred where the % kernels overlapped and two possible encounters in dyad did not occur in any of the core areas. eleven ( %) of the possible encounters occurred within m of a known marking post. for possible encounters with proximity thresholds of < m, < m and < m the distance between males was overall significantly less during the period to hours after a possible encounter, compared to the and hours before a possible encounter (table ). in other words, rather than moving away from each other, male cheetahs stayed in close proximity to each other for up to hours after a possible encounter. for the distance to the encounter location, distance travelled and tortuosity we wanted to determine whether there was individual variation within each dyad. however, because of the paucity in the number of possible encounters that were detected per dyad we were only able to carry out the analysis for possible encounters with a proximity threshold < m. in general, cheetahs were closer to the encounter location after a possible encounter compared to before for all four time lags, apart from individual m in dyad where the opposite trend was intraspecific interactions among carnivores: a case study of male cheetahs observed, however none of the results were significant (s table) . similarly, cheetahs travelled less after a potential encounter compared to before, apart from individual m in dyad where the opposite trend was observed. some of the results, especially at the hr and hr lag were significant for dyad and (s table) . on the th february the collar on m stopped transmitting but when the team visited the last location sent by the collar, neither cheetah nor collar could be found and the individual has not been seen since. on the st october the collar on m stopped transmitting data after the collar data showed that individuals m and m had come within m of each other. the team went to the last gps coordinate that was transmitted by the collar and found the remains of m m from the last gps fix sent by the collar. upon inspecting the carcass, a puncture wound was found on the left side of the skull. based on the circumstantial evidence, we believe that the death of m was either a direct or an indirect result of an aggressive interaction between him and the -male coalition (m ). interestingly, three months prior to this encounter m and his coalition mates started establishing a territory approximately km southwest from m 's territory. two days before the encounter the coalition travelled km to the encounter location, spent hours within m of m and then travelled km straight back to the core of their territory. m did not return to the vicinity of the encounter between the st october , when the encounter took place, and rd february , when m 's collar was removed. the closest they came to m 's territory during that time was approximately km (fig and the animation in s movie). using gps collar data we documented static and dynamic interactions between male cheetahs in kenya's maasai mara and investigated the outcomes of these interactions in terms of movement behaviour and mortalities. as we predicted, male cheetahs showed extensive spatial overlap of the % kernels. this high degree of overlap observed in the maasai mara could be related to the pattern of prey availability [ ] , although we do not have the data to test this. however, apart from one dyad, there was little overlap of core areas ( % kernels) and it could be that core areas are defended more intensively than the peripheral areas [ ] . similar to observations in other areas, marking posts were frequently visited by males [ , ] and this could indicate the mechanism that results, despite the extensive spatial overlap, in the rarity of occasions when members of a dyad were in close proximity [ ] . interestingly, our results show that possible encounters were most likely to take place in the core area of one individual of a dyad or where cores of both individuals overlapped. we also found that, similar to african wild dogs (lycaon pictus), possible encounters occurred more at night than during the day [ ] . while cheetahs, like african wild dogs, are predominantly diurnal they can be active at [ ] and nocturnal activity for males has been found to be considerably higher than for females [ ] . data from camera traps set at marking posts found that visits occurred more at night than during the day ( [ ] ; kk unpublished data) suggesting that male nocturnal activity is partly driven by patrolling behaviour which is probably why encounters predominantly took place at night. in some species, including african wild dogs and white-faced capuchins (cebus capucinus), avoidance behaviours, characterised by an increase in distance and speed travelled postencounter, were observed as a result of interactions between different groups [ , ] . however our results, in contrast to our predictions, did not show avoidance behaviour postencounter as males stayed in close proximity to each other - hours after a potential encounter. it is possible that males stayed in close proximity to each other, as part of a territorial defense strategy, if a recent scent of a conspecific was detected. this behaviour has been observed in dwarf mongoose (helogale parvula) groups, who moved slower and covered shorter distances in the hour following the encounter of rival faeces at a latrine site within their territory [ ] and red fox (vulpes vulpes) males who spent more time in scent-marked areas [ ] . alternatively, males could come into close proximity to one another if they are attracted to a resource, such as a female in oestrus [ , ] . cheetahs exhibit a high rate of multiple paternity [ ] so it is possible that multiple males stay in the vicinity of a receptive female with the hope of getting a chance to mate. these encounters could however result in fatalities if the removal of competition increases future mating opportunities [ ] . if encounters occur as a result of access to a receptive female rather than to a static, long-term resource such as a territory then this could explain why the five-male coalition did not take-over the territory of individual m after he died. aggressive interactions with fatal consequences are not uncommon in cheetahs. caro [ ] reported three cases in serengeti where singletons were killed by coalitions (all three-male coalitions). similarly, mills and mills [ ] found that % of male-male encounters recorded in the kgalagadi transfrontier park in botswana/south africa resulted in death. to our knowledge, fatal interactions have not been observed between female cheetahs. this could explain why male mortality is higher and life expectancy lower for males compared to females [ , ] resulting in a female biased sex ratio [ ] . for some species, such as voles (microtus oeconomus), lions and grizzly bear (ursus arctos), the removal of males, through either displacement or mortality, has a negative effect on population growth as a result of increased infanticide [ ] [ ] [ ] . infanticide has however not been observed amongst cheetahs [ ] possibly because it rarely occurs in predominantly solitary species [ ] . the removal of males could however have other population-level consequences [ ] but the impact of male mortality on population dynamics in cheetahs is unclear. static and dynamic interactions can play a role in disease transmission [ , ] . in the mara-serengeti ecosystem there is a relatively high prevalence of mange [ , ] and in southern africa cheetahs have been positively tested for feline coronavirus (fcov) and feline panleukopenia virus (fpv), which can be highly contagious and fatal [ , ] . pathogens such as these can easily spread through faeces and other bodily fluids, which are deposited and investigated by male cheetahs at marking posts. this could explain why in several males in the maasai mara, who overlapped spatially, died of a yet unknown disease within a short space of time [ ] . we suggest that future epidemiological research should investigate the role of scent marking posts and movement in disease transmission [ ] . here we give a descriptive analysis of the static and dynamic interactions between male cheetahs and the outcomes of these encounters. despite the clear patterns that were observed, there are several caveats that warrant discussion. firstly, we were only able to use data from four collared males, one of which was part of a -male coalition. it is therefore possible that other uncollared individuals, including the other members of the -male coalition, could have influenced the results. secondly, because of the resolution of the collar data we might have missed visits to marking posts and we inferred when interactions took place rather than being able to detect actual interaction (apart from one occasion). our results are therefore likely to be on the conservative side and we suggest that future studies use higher resolution data and/ or proximity loggers to investigate actual interactions between individuals (e.g. [ , ] ). however, even with a relatively coarse resolution of data and only a small number of individuals we managed to investigate interactions and subsequent outcomes between males giving a first detailed insight into intraspecific interactions in cheetah. table. summaries for each dyad of the distance to the encounter location, distance travelled and tortuosity before and after a possible encounters with proximity threshold of < m. 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free-ranging namibian cheetahs (acinonyx jubatus) disease outbreak thresholds emerge from interactions between movement behavior, landscape structure, and epidemiology wildlife contact analysis: emerging methods, questions, and challenges we would like to thank the mara cheetah project and mara lion project teams for assisting with fieldwork. key: cord- -w iqeayr authors: gallien, sarah; moro, angélique; lediguerher, gérald; catinot, virginie; paboeuf, frédéric; bigault, lionel; gauger, phillip c.; pozzi, nathalie; berri, mustapha; authié, edith; rose, nicolas; grasland, béatrice title: limited shedding of an s-indel strain of porcine epidemic diarrhea virus (pedv) in semen and questions regarding the infectivity of the detected virus date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: w iqeayr pedv is mainly transmitted by the oro-fecal route although pedv shedding in semen has already been shown for an s-non-indel pedv strain infection. the aim of this study was to determine if pedv can be shed in semen from spf (specific pathogens free) boars infected by a french s-indel pedv strain (pedv/fr/ / ) and in case of positive semen to determine the infectivity of that semen. both infected boars had diarrhea after inoculation and shed virus in feces. pedv genome was also detected by rt-qpcr in the sperm-rich fraction of semen ( . × ( ) and . × ( ) genomic copies/ml) from the two boars infected with the s-indel pedv strain but only once at dpi. in addition, pedv rna in peyer’s patches and in mesenteric lymph nodes was also present for the two inoculated boars. the pedv positive semen (s-non-indel and s-indel) sampled during a previous trial and in this boar trial were inoculated to six spf weaned pigs. the inoculated piglets did not seroconvert and did not shed virus throughout the duration of the study except for one pig at dpi. but, pedv could be detected in intestinal tissues such as duodenum, jejunum and jejunum peyer’s patches by rt-qpcr except for one pig. even if pedv genome has been detected in semen, experimental infection of piglets with positive semen failed to conclude to the infectivity of the detected pedv. porcine epidemic diarrhea (ped) was described for the first time in europe in the s (jung and saif, ; song and park, ) . this disease is characterized by a severe, profuse watery diarrhea with or without vomiting and dehydration and is caused by an etiologic agent called porcine epidemic diarrhea virus (pedv), a positive-sense, singlestranded rna enveloped coronavirus of -kb (jung and saif, ) . after several outbreaks in the 's, ped has been persisting in europe with sporadic cases until the late 's (jung and saif, ) . outbreaks of ped have also been described in asia in the s and from (jung and saif, ; wang et al., ) . currently, two genotypes are circulating in several regions in the world, namely the s-non-indel strains and the s-indel strains, showing insertions-deletions in the s segment of the s gene (jung and saif, ; vlasova et al., ) . from clinical reports, these two types of strains seemed to be different in terms of morbidity and mortality with the s-non-indel pedv strains associated with more severe clinical cases and higher case-fatality rate compared to the others (vlasova et al., ; wang et al., ) . in contrast to s-non-indel pedv strains, exclusively reported from america and asia, s-indel pedv strains have also been identified in europe since (efsa, ; stadler et al., ; vlasova et al., ) . pedv is transmitted mainly by the oro-fecal route, but also contact with contaminated equipment, vehicles used for pig transport or the staff (bowman et al., ; jung and saif, ; lowe et al., ) . airborne transmission of the virus was also shown for s-non-indel strains (alonso et al., ) . pedv transmission through contaminated milk from dam to piglets can also occur (sun et al., ) but vertical transmission of pedv through semen has never been shown. s-non-indel pedv strain shedding has recently been evidenced in the different fractions of semen (seminal and sperm-rich fractions) and in gelatin plug of specific pathogen free (spf) boars experimentally inoculated (gallien et al., b) . however, to the best of our knowledge, the presence of pedv rna in semen of boars infected by an s-indel pedv strain has not been studied so far. thus, the aim of this study was to determine if pedv can be shed in semen from spf boars infected by an s-indel french pedv strain (pedv/fr/ / ). subsequently, the infectivity of quantitative rt-pcr (rt-qpcr) positive semen was evaluated in an experimental infection of spf weaned piglets. two experimental trials were carried out in the air-filtered level biosecurity facilities of the french agency for food, environmental and occupational health & safety (anses) in accordance with the european and the french regulations on animal welfare. protocols used were approved by the ethics committee registered under number # by the french ministry of . the first trial (trial ) was performed to evaluate pedv shedding in semen from large white spf inoculated boars. two boars (boars i and i aged ½ and ½ years-old) were housed in the same room but in two separate pens. the two other boars (boars c and c ), of similar age to boars i and i , were used as negative controls and housed within the biosafety level- , air filtrated anses spf herd. this experiment lasted for days post-inoculation (dpi). at dpi, the inoculated boars were euthanized including anesthesia (zoletil®, virbac, carros, france, mg/kg) followed by bleeding before necropsy. the boars c and c were not necropsied. the second trial (trial ) was carried out for days to assess the infectivity of rt-qpcr pedv-positive semen. the positive semen fractions from two experiments were used and were collected from trial with the s-indel pedv strain and from a previously described trial involving an s-non-indel strain (gallien et al., b) . six spf weaned pigs of three week-old were housed in the same room in two different pens separated by a plastic partition. two pigs of the same age housed in another room were used as controls. at dpi, pigs were euthanized after anesthesia as already described and necropsied. inoculation procedures the two boars of the trial received orally ml of a homogenate of the pedv french s-indel strain pedv/fr/ / (genbank number: kr ) titrating viral genome copies/ml (equivalent to ≈ tcid /ml). the pedv french s-indel strain was amplified in a threeweek-old spf weaned pig inoculated with a homogenate of jejunum collected from ped-affected pigs belonging to a french farm in . the inoculum was prepared from the jejunum of this spf pedv-inoculated pig by homogenization in dulbecco's phosphate-buffered saline (sigma-aldrich, saint louis, mo, usa) ( % w/v). the homogenate was centrifuged at , ×g for min at °c and the supernatant filtered through a . μm filter. next-generation sequencing (ngs) was performed on the inoculum to obtain the pedv complete genome sequence and to ensure the absence of other viral rna sequences. the absence of porcine circovirus type (pcv ) and porcine reproductive and respiratory syndrome virus (prssv) in the inoculum was assessed by the absence of seroconversion against pcv and prssv in the inoculated boars at the end of the trial. the six spf weaned pigs of trial were orally inoculated with ml of contaminated semen: pigs # , # , # , # with the s-non-indel and pigs # , # with the s-indel (table ). the semen samples inoculated were stored at − °c until inoculation of the piglet. the semen were defrosted on ice just before the inoculation and reconstituted in cold dulbecco's phosphate-buffered saline to avoid thermal shock. for the two trials, clinical signs (lethargy, outward appearance, behavior, breathing, diarrhea and vomiting) were recorded daily. pedv shedding in fecal samples was evaluated daily the first week after inoculation, and then three times a week until dpi for trial and for trial , at . dpi, twice a day from to dpi, once at and dpi and then three times a week until the end of the trial. pedv shedding was also assessed in semen for trial . semen was collected before inoculation and every day the first week post-inoculation and then twice a week until the end of the trial. the belly and the sheath of the boars were cleaned with cleaning wipes before sampling in order to avoid any contaminations of the semen with positive pedv-feces/aerosols. a swab of the prepuce was also done before each collection to rule out the possibility of external contamination of semen. semen was collected manually without sexual stimulation with support of a collection dummy. the gelatin plug was also collected at the end of the semen ejaculate. blood samples were also collected before inoculation and at the end of trials for the two trials, and at dpi for trial and once a week until the end of the trial for trial to assess seroconversion and viremia by rt-qpcr ( fig. ) . during necropsy, organs of the digestive tract (duodenum, jejunum, ileum, colon, peyer's patches (jejunum and ileum)), spleen, liver, mesenteric and inguinal lymph nodes, lungs, were collected and stored in rna later tissue storage reagent (sigma-aldrich, saint louis, mo, usa) for the two trials. organs of the reproductive tract were also collected for the trial (vas deferens (right and left), testicles (right and left: apical pole, distal pole, median axis), epididymis (right and left: head, body, tail), prostate, cowper's glands (right and left), seminal vesicles (right and left: apical and distal pole) and spermatic cords (right and left)) and stored in the same tissue storage reagent. macroscopic lesions were also evaluated on necropsy for the two trials. all samples collected during the two trials were stored at − °c. fresh semen samples were centrifuged at ×g for min at °c (pal et al., ) . this centrifugation step allowed separating the seminal fraction from the sperm-rich fraction of the semen. these two fractions and the gelatin plug were stored at − °c. blood samples were centrifuged at ×g for min at °c and then sera were stored at − °c until use. one gram of feces (or ml of feces in case of liquid feces) was homogenized in ml of dulbecco's phosphate-buffered saline (sigma-aldrich, saint louis, mo, usa). the fecal homogenates were then centrifuged at , ×g for min at °c. tissues were homogenized in dulbecco's phosphate-buffered saline (sigma-aldrich, saint louis, mo, usa) at % v/w using a bead mill (retsc, haan, germany). these homogenates were centrifuged at , ×g for min at °c and the supernatants were stored at − °c. total rnas were extracted from the fecal and tissue homogenates, from the preputial swabs and from sera using the qiagen rneasy mini kit (qiagen, hilden, germany) according to the manufacturer's instructions. five μl of eluted rna were used as templates for pedv rt-qpcr. rna extraction controls were performed for every five samples to check for any pedv contamination by replacing sample with rnase free water. rna extracted from feces with the rneasy mini kit was diluted : to avoid any pcr inhibition. total rnas were extracted from the two semen fractions (seminal and sperm-rich fractions) and the gelatin plug using trizol® assay (thermo fisher scientific, waltham, ma, usa) (gallien et al., b) . the number of pedv genome copies was assessed by real-time rt-pcr using power sybr® green rna-to-ct ™ -step kit (thermo fisher scientific, waltham, united states of america) on an applied biosystems® real time pcr system (thermo fisher scientific, waltham, united states of america) as already described (gallien et al., b; kim et al., ) . the primers used were designed from the conserved regions of the pedv nucleocapsid gene for universal detection of both strains (forward, ′-cgcaaagactgaacccactaa- ′; reverse, ′-ttgcctctgttgttacttggagat- ′). for each pcr run, a positive control containing pedv rna extract from a pedv cell culture supernatant was included. two negative controls were also included on the plate, the rna samples were replaced by rnase free water. one negative control was placed close to the positive control and the second one at the end of the plate. all samples were processed in duplicate. sera were tested for pedv antibodies using a commercial elisa test, id screen® pedv indirect (id vet, grabels, france). the elisa test is validated if the mean value of the positive control optical density (od) is greater than . and if the ratio of the mean values of the positive and negative controls is greater than . for each sample, the s/p (sample-to-positive) ratio was calculated. samples with s/p ratios equal to or higher than % were considered positive for pedv antibodies (fig. ) . both inoculated boars demonstrated clinical signs. they were lethargic as soon as dpi and until dpi. a reduction of food intake was also observed for the two inoculated boars at dpi and until dpi. diarrhea was also observed at dpi. none of the control boars demonstrated clinical signs during the trial. the inoculated boars were pedv seronegative before inoculation and became seropositive at ± dpi (s/p% i = . % and s/p% i = . %). only boar i was still positive for pedv antibodies at the end of the trial (s/p% i = . %). the two control boars were seronegative before and at the middle of the trial. all boars were rt-qpcr negative for pedv in feces prior to inoculation. the two control boars remained pedv negative in feces until the end of the trial. however, pedv rna was first detected in the feces of boar i at dpi ( . × genome copies/ml of feces). at dpi, boars i and i showed . × and . × genome copies/ml of feces, respectively. the viral genome was continuously detected until dpi. maximum shedding was detected between . and dpi for the two boars ( . × genome copies/ml for boar i at . dpi and . × genome copies/ml for boar i at dpi). pedv rna was then sporadically detected for the two boars (at dpi for boar i and at and dpi for boar i ) (fig. ) . no pedv nucleic acid was detected in the seminal or the sperm-rich fractions of semen and in the gelatin plug collected from boars i , i , c and c before inoculation. the two control boars remained negative for pedv rna in all fractions of semen and in the gelatin plug throughout the trial. pedv rna was detected for both inoculated boars at dpi in the sperm-rich fraction of semen with . × and . × genome copies/ml for boars i and i , respectively. no pedv rna was detected by rt-qpcr thereafter in this fraction. the inoculated boars remained rt-qpcr negative for the seminal fraction and the gelatin plug throughout the trial. all swabs collected from the sheath tested negative for pedv rna, even when the virus was detected in the spermrich fraction ( dpi). pedv rnas were detected in the jejunum peyer's patches ( . × and . × genome copies/ml for boars i and i respectively), in the ileum peyer's patches ( . × and . × genome copies/ml for boars i and i respectively) and in the mesenteric lymph nodes ( . × and . × genome copies/ml for boars i and i respectively). pedv rna was not detected from the organs of the reproductive tract. no lesions were observed in the gastrointestinal or reproductive tract. none of the inoculated piglets showed clinical signs throughout the trial. no seroconversion of these pigs was observed at dpi. no pedv nucleic acid was detected in feces of pigs # , # , # , # , # and # during the trial except for pig # at dpi ( . × genome copies/ ml). some digestive tissue samples were found positive for pedv in rt-qpcr for pigs # , # , # , # and # at dpi (table ) but no lesion was observed in the gastrointestinal tract. oral inoculation of mature boars by an s-indel french pedv strain demonstrated less severe clinical signs compared to those observed in boars inoculated with a s-non-indel us pedv strain (gallien et al., b) . however, the infection induced profuse diarrhea and affectd growth performances similarly to weaned pigs inoculated with this s-indel french pedv strain (gallien et al., a) . the duration of virus fecal shedding determined in our study ( days) was shorter compared to weaned pigs inoculated with an s-indel pedv strain ( - days) (leidenberger et al., ; lohse et al., ) or for boars inoculated with an s-non-indel pedv strain ( - days) (gallien et al., b) . in this experimental challenge, the presence of pedv rna in the fig. . fecal pedv shedding detected from boar i and i (log(pedv-genome copies/ml)) in trial . pedv genome loads (genome copies/g) detected in the tissues collected at necropsy from spf weaned pigs inoculated with pedv rt-qpcr positive semen in trial . sperm-rich fraction of semen of both inoculated boars was detected at dpi only, which is much more limited than in the sperm-rich fraction of semen from boars inoculated with an s-non-indel pedv strain (gallien et al., b) . in the previous study, pedv rna could be detected for a longer period in the sperm-rich fraction of semen: i.e. transient detections of pedv rna during three distinct periods comprised between and days. pedv rna was also detected in the seminal fraction of semen and in gelatin plug in s-non-indel pedv inoculated boars contrasting to the observations from the present study. this difference regarding pedv rna detection in semen could be linked to the viral strain used for inoculation and its virulence. clinical signs induced with s-non-indel pedv were more severe compared to clinical signs observed in s-indel pedv challenged boars (gallien et al., b) . viral shedding in the sperm-rich fraction of semen has already been reported for prrsv which belongs to the same order as pedv, the nidovirales order, and differences in terms of shedding in semen between genotype and highly virulent genotype prrsv strains have already been shown (christopher-hennings et al., ; prieto and castro, ) . prrsv genotype strains could be detected during longer periods in semen compared to genotype strains. hence, the duration of detection of highly virulent genotype prrsv strains in semen was reported between to days (christopher-hennings et al., ) while the presence of genotype prrsv strains in semen could be observed more sporadically: only at one point after infection, at dpi (prieto et al., ) or during shorter periods comprised between and days (swenson et al., ) . detection of s-indel pedv rna in semen appeared sporadically after the occurrence of clinical signs and detection in feces in contrast to what we observed for an s-non-indel pedv strain. moreover, no s-indel pedv rna was detected in semen after cessation of clinical signs and fecal shedding in contrast to what was observed with the s-non-indel strain (gallien et al., b) . these data suggest that the risk of introduction of pedv shedding boars via imports should be more predictable from clinical exams or fecal shedding assessment for s-indel strains than for s-non-indel. absence of detection of pedv rna in the genital tract has been shown in boars inoculated with s-indel pedv strain as well as in boars inoculated with an s-non-indel strain at dpi (gallien et al., b) . mc carty and al., showed that pedv rna could be detected in penis and testicle at days post-infection (mccarty et al., ) . that presence was noticed at infection peak when a viremia could be noticed too. that could explain the absence in reproductive tract of pedv we noticed at days post infection, a very late date compare to the infection peak. during the second trial, no shedding was detected in feces of spf weaned pigs inoculated with s-indel and s-non-indel rt-qpcr pedv positive semen except for pig # at dpi. the presence of low pedv genomic loads could be noticed in different intestinal tissues. however, clinical signs and seroconversion were absent in all inoculted piglets. the results of the present study neither confirms nor denies if the rt-qpcr pedv positive semen contained infectious virus. additional trials with longer periods of observation, sequential slaughters or the use of a more sensitive model such as neonatal piglets should be conducted in order to determine if the pedv rna detected in the tissues were caused by pedv infection. the use of neonatal piglets could have been in fact a most sensitive model to evaluate pedv infectivity but to realize that kind of trial in our experimental facility, we should have infected two sows with their suckling piglets and for material reasons (place, cost, animal availability…), this option could not be selected. moreover, with these results, it is impossible to speculate whether pedv-positive semen could infect sows via natural insemination. in fact, it has already been shown for other porcine viral pathogens that contaminated semen could be infectious, but could not infect sows or gilts through artificial insemination. weaned pigs inoculated with pcv positive semen, for example, presented a viremia and anti-pcv antibodies but the same pcv positive semen could not infect sows through artificial insemination (madson et al., ) . the amount of virus contained in semen could explain the impossibility of viral transmission through artificial insemination (grasland et al., ; madson et al., ). an impact of the amount of virus contained in semen on the capacity of the transmission of viral pathogens has also been shown for prrsv. the prrsv can be transmitted through semen but only when it is present in this matrix at a given concentration. under experimental conditions, prrsv transmission was successful when semen contained × tcid /ml of virus i.e. on average genome copies/ml. however, when the amount of virus contained in semen was approximately × tcid /ml ( genome copies/ml on average), transmission through semen was limited. when the amounts were even lower ( × tcid /ml, genome copies/ml on average), transmission was no longer effective (prieto and castro, ) . a possible impact of the amount of genomic load of pedv contained in semen on the capacity of the transmission of pedv might be suspected in the present case. to conclude, a very transient shedding of pedv in semen was observed in case of infection by an s-indel pedv strain conversely to what was observed with s-non-indel pedv strain. however the infectivity of the virus present in pedv s-indel or s-non-indel positive semen was not evidenced. evidence of infectivity of airborne porcine epidemic diarrhea virus and detection of airborne viral rna at long distances from infected herds investigating the introduction of porcine epidemic diarrhea virus into an ohio swine operation persistence of porcine reproductive and respiratory syndrome virus in serum and semen of adult boars detection and duration of porcine reproductive and respiratory syndrome virus in semen, serum, peripheral blood mononuclear cells, and tissues from yorkshire, hampshire, and landrace boars collection and review of updated scientific epidemiological data on porcine epidemic diarrhoea better horizontal 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characteristics and complex evolution of pedv strains porcine epidemic diarrhea virus variants with high pathogenicity new variant of porcine epidemic diarrhea virus the authors are grateful to anses, inra and lncr for their financial support. we also would like to thank phillip gauger from iowa state university who provided the us non-indel strain and all the team members at the experimental laboratory for their contribution. all the authors designed and carried out the experiments. sg analyzed the data and wrote the manuscript. bg, nr and mb supervised the project. all the co-authors revised the manuscript. all authors read and approved the final manuscript. the authors declare that they have no conflict of interests. key: cord- -xxc vdnt authors: ahmed, anwar e.; al-jahdali, hamdan; alshukairi, abeer n.; alaqeel, mody; siddiq, salma s.; alsaab, hanan; sakr, ezzeldin a.; alyahya, hamed a.; alandonisi, munzir m.; subedar, alaa t.; aloudah, nouf m.; baharoon, salim; alsalamah, majid a.; al johani, sameera; alghamdi, mohammed g. title: early identification of pneumonia patients at increased risk of middle east respiratory syndrome coronavirus infection in saudi arabia date: - - journal: int j infect dis doi: . /j.ijid. . . sha: doc_id: cord_uid: xxc vdnt background: the rapid and accurate identification of individuals who are at high risk of middle east respiratory syndrome coronavirus (mers-cov) infection remains a major challenge for the medical and scientific communities. the aim of this study was to develop and validate a risk prediction model for the screening of suspected cases of mers-cov infection in patients who have developed pneumonia. methods: a two-center, retrospective case–control study was performed. a total of patients with confirmed pneumonia who were evaluated for mers-cov infection by real-time reverse transcription polymerase chain reaction (rrt-pcr) between september , and june , at king abdulaziz medical city in riyadh and king fahad general hospital in jeddah, were included. according to the rrt-pcr results, patients were positive for mers-cov and were negative. demographic characteristics, clinical presentations, and radiological and laboratory findings were collected for each subject. results: a risk prediction model to identify pneumonia patients at increased risk of mers-cov was developed. the model included male sex, contact with a sick patient or camel, diabetes, severe illness, low white blood cell (wbc) count, low alanine aminotransferase (alt), and high aspartate aminotransferase (ast). the model performed well in predicting mers-cov infection (area under the receiver operating characteristics curves (auc) . ), on internal validation (auc . ), and on a goodness-of-fit test (p = . ). the risk prediction model, which produced an optimal probability cut-off of . , had a sensitivity of . and specificity of . . conclusions: this study provides a simple, practical, and valid algorithm to identify pneumonia patients at increased risk of mers-cov infection. this risk prediction model could be useful for the early identification of patients at the highest risk of mers-cov infection. further validation of the prediction model on a large prospective cohort of representative patients with pneumonia is necessary. middle east respiratory syndrome coronavirus (mers-cov) was first identified in saudi arabia in . the diagnosis of this infection remains complex (al johani and hajeer, ; sung et al., ; ahmed, a) and it has a high fatality rate (ahmed, b, c; aleanizy et al., ; sherbini et al., ; kim et al., ) . the early detection and identification of individuals at high risk of a disease is the most effective strategy to improve patient clinical outcomes (ahmed, a) and reduce the costs of testing, both physical and economic (ahmed et al., (ahmed et al., , . the real-time reverse transcription polymerase chain reaction (rrt-pcr) has been found to be valid and accurate for the evaluation of respiratory swabs, sputum, and other endotracheal aspirate material (corman et al., a, b) . however, although rrt-pcr is the most accurate and sensitive test available at the authors' centers, absolute identification of mers-cov may require multiple clinical specimens from different sources and take days (corman et al., a, b; anon, a) . the saudi ministry of health (moh) has developed mers-cov visual triage guidelines to identify suspected cases (anon, b) . the current guidelines include fever, respiratory symptoms, gastrointestinal symptoms, chronic diseases, and risk of exposure to mers-cov. in clinical practice, identifying high-risk individuals can be challenging, since most laboratory-confirmed mers-cov patients report common clinical risk indices to patients with other respiratory conditions. for instance, respiratory and gastrointestinal symptoms are common for both mers-cov and non-mers-cov patients (mohd et al., ) . thus, further exploration must take place to reduce the risk of mers-cov infection. a risk prediction model is urgently needed to stratify patients with suspected mers-cov. this may decrease the risk of virus transmission to others who are in close contact, for example healthcare workers, patients, and hospital visitors, by allowing the careful management of those who are potentially infected at an early stage of infection. developing a mers-cov prediction model may more efficiently aid physicians in identifying individuals at high risk and selecting the necessary test(s) to improve prevention and control measures. several methodological studies have shown that combining demographic characteristics with clinical, radiological, and laboratory information can improve risk assessment and diagnostic accuracy (ahmed et al., (ahmed et al., , sidransky, ; etzioni et al., ) . these previous studies used a linear combination to develop an algorithm that combines demographic characteristics, symptoms, and clinical, radiological, and laboratory findings to identify the highly suspected mers-cov cases. mers-cov was initially identified in a patient being treated for pneumonia in (zaki et al., ) , and since then, pneumonia and its symptoms have remained common characteristics in mers-cov patients (saad et al., ; choi et al., ) . the aim of this study was to develop and validate a reliable risk prediction model for the screening of suspected cases of mers-cov infection in patients who have developed pneumonia. a two-center, retrospective case-control study was conducted utilizing data from king abdulaziz medical city in riyadh (kamc-r) and king fahad general hospital in jeddah (kfgh-jed), saudi arabia. the data were obtained between september , and june , . kfgh-jed experienced a mers outbreak between march and may (oboho et al., ) , and kamc-r experienced a large mers outbreak between june and august (al-dorzi et al., ) . both study centers applied the saudi moh case definitions (anon, b) to identify suspected individuals in the population studied, and rrt-pcr was used as the gold standard test to diagnose mers-cov in multiple and different clinical specimens when necessary. mers-cov-related symptoms were common at both centers. the project received ethical approval from two independent ethics committees: the saudi moh (irb log number - e) and king abdullah international medical research center (study number rc / ), riyadh saudi arabia. during the study, the medical records of subjects who were being assessed by rrt-pcr for suspected mers-cov were reviewed. the suspicion of mers-cov infection at both kamc-r and kfgh-jed was determined based on the saudi moh guidelines (anon, b) . two groups were compared: patients who were positive and patients who were negative for mers-cov infection. in an effort to reduce heterogeneity between the study groups, only subjects with a lung infiltrate on chest x-ray and/or computed tomography (ct) scan of the chest were included in the analysis. thus, subjects who had no available results of a chest x-ray or ct scan of the chest were excluded. the initial screening for suspected mers-cov patients includes pneumonia (anon, b) . most of the patients studied were evaluated for pneumonia immediately after presentation. the study excluded subjects who were less than years of age (pediatrics/children), as defined in the saudi moh guidelines (anon, b) , and excluded subjects who had upper respiratory tract infections (respiratory symptoms, positive or negative mers-cov rrt-pcr, and normal chest x-ray and ct scan of the chest). the final sample comprised a cohort of subjects who had a lung infiltrate on chest x-ray and/or a ct scan of the chest, who were classified according to the results of mers-cov rrt-pcr. the case group consisted of pneumonia patients who were positive for mers-cov infection, and the control group consisted of pneumonia patients who were negative for mers-cov infection. cases were defined as patients with mers-cov pneumonia who had positive mers-cov rrt-pcr on nasopharyngeal aspirate and/ or bronchoalveolar lavage in addition to a lung infiltrate on chest xray and/or ct scan of the chest. controls were defined as patients with respiratory symptoms, a lung infiltrate on chest x-ray and/or ct scan of the chest, pneumonia, and negative mers-cov rrt-pcr result of nasopharyngeal aspirate and/or bronchoalveolar lavage. nineteen predictive variables were included: age, sex, fever (temperature ! c), one composite respiratory symptom (the presence of cough, bloody cough, shortness of breath, or chest pain), one composite gastrointestinal symptoms (the presence of diarrhea, vomiting, or nausea), seven mers-cov potential risk factors (contact with sick patients or camels, severe illness (defined according to the patient's clinical status, 'yes/no', which is based on clinical judgment), diabetes, lung disease, liver disease, renal disease, and heart disease), and seven laboratory measurements (white blood cell (wbc) count ( /l), platelets ( /l), creatinine (mmol/l), bilirubin (mmol/l), alanine aminotransferase (alt; u/l), aspartate aminotransferase (ast; u/l), and albumin (g/ l)). the reference ranges for the laboratory measures were as follows: wbc count, -  /l; platelets, -  /l; creatinine, - mmol/l; bilirubin, . - . mmol/l; alt, - u/l; ast, - u/l; albumin, - g/l. no serological tests were available at the centers for these patients. stata statistical software release , (statacorp. llc, college station, tx, usa) was used for the data analysis. the sample characteristics were recorded as the frequency and percentage, or as the mean ae standard deviation (sd). laboratory measurements were summarized as the median and th- th percentiles. a subgroup analysis (chi-square test, independent samples t-test, or mann-whitney u-test) was used to identify unadjusted associations between demographic, clinical, and laboratory measurements according to mers-cov status. the performance of each bivariate predictor was further assessed by unbiased estimate, the area under the curve (auc), and its % confidence interval (ci). stepwise binary logistic regression was employed to develop a mers-cov risk prediction model and identify factors that could be used to estimate the probabilities of mers-cov infection. the goodness-of-fit of the final model was tested by hosmer-lemeshow procedure: a large p-value indicates that a model has a good fit. the discrimination ability between high and minimal risk of mers-cov infection of the final model was assessed by the auc and its % ci. a receiver operating characteristics (roc) curve was generated for the risk prediction model. two hundred random samples were drawn with replacements from the original study sample (n = ). the model internal validity was assessed in these samples by the auc and its % ci. optimal cut-off values of the probabilities for each model were determined using a generalized youden index (youden, ) . at these thresholds, it was possible to achieve the best balance between specificity and sensitivity. a total of pneumonia patients were included in the analysis: . % were confirmed mers-cov-positive and . % were confirmed mers-cov-negative. the mean age at presentation was . years, with a standard deviation of . years; age ranged between and years. of the total sample, . % had been in contact with a sick patient or camel, % had fever, . % had at least one respiratory symptom, and . % had at least one gastrointestinal symptom. the two groups were similar in the distribution of age (p = . ) and sex (p = . ). the characteristics of the sample can be found in table . subgroup analyses are presented in tables and . the chisquare test or the independent samples t-test revealed that sex (p = . ) and age (p = . ) were similar in the two groups. the risk of mers-cov infection was similar in patients with and without fever (p = . ), respiratory symptoms (p = . ), or gastrointestinal symptoms (p = . ). severe illness (p = . ), contact with a sick patient or camel (p = . ), diabetes (p = . ), heart disease (p = . ), and renal disease (p = . ) were significantly associated with an increased risk of mers-cov infection. the independent samples mann-whitney u-test revealed that the wbc count (p = . ) and platelet count (p = . ) were significantly lower in patients who were positive for mers-cov than in those who were negative for mers-cov infection. in contrast, creatinine (p = . ), bilirubin (p = . ), ast (p = . ), and albumin (p = . ) were significantly higher in patients who were positive for mers-cov than in those who were negative for mers-cov infection. alt (p = . ) was insignificantly higher in patients who were positive for mers-cov than in those who were negative for mers-cov infection. according to the individual roc curve analysis (table ) , severe illness, diabetes, wbc count, creatinine, bilirubin, albumin, and ast were the most important predictors of mers-cov infection. a risk prediction model was developed using stepwise binary logistic regression (p . ). the model retained seven independent variables that were associated with increased odds of mers-cov (table ). male sex (adjusted odds ratio (aor) . , p = . ), contact with a sick patient or camel (aor . , p = . ), diabetes (aor . , p = . ), severe illness (aor . , p = . ), low wbc count (aor . , p = . ), high ast (aor . , p = . ), and low alt (aor . , p = . ) were found to have a significant impact on the prediction of mers-cov. the hosmer-lemeshow test indicated that this model fitted the data well (p = . ). this model showed substantial discrimination, with an auc of . and a % ci of . - . ( figure ). the prediction model was validated using the bootstrap procedure. a total of random samples were drawn with replacements from the original sample, and the model showed a substantial ability to assess mers-cov infection in this study population (auc . , % ci . - . ). the findings in table were used to create a risk-probability model. the risk prediction for the model can be expressed by the following equation: predicted probability = [ + exp( . -( .  male) À ( .  sick patient or camel contact) À ( .  diabetes) À ( .  severe illness) + ( .  wbc count) À ( .  ast) + ( .  alt))] À . a calculator was developed to calculate the potential risk of mers-cov infection in pneumonia patients. we determined the optimal cut-off or threshold values of the probabilities to mark the differences between the high-risk and low-risk groups. when an equal weight was given for sensitivity and specificity (m = ), the optimal cut-off value (probability ! . ) produced sensitivity and specificity of . and . , respectively. when more weight was given for sensitivity than specificity (m = . ), the optimal cut-off value (probability ! . ) produced sensitivity and specificity of . and . , respectively. when more weight was given for specificity than sensitivity (m = . ), the optimal cut-off value (probability ! . ) produced sensitivity and specificity of . and . , respectively. based on data from the two largest hospitals in saudi arabia, a risk prediction model was developed for mers-cov infection in pneumonia patients. this model was generated from a retrospective study and should be assessed prospectively for external validation. seven variables were identified as having a great impact on the mers-cov risk assessment prediction. the risk prediction model highlights the strong potential impact of male sex, contact with a sick patient or camel, severe illness, diabetes, low wbc count, high ast, and low alt on mers-cov infection. these few important parameters could be part of routine medical examinations to be performed (for the purpose of identifying highly suspected individuals) in daily clinical practice in order to make a prompt and timely clinical decision. according to the model, high ast was associated with an increased risk of being infected with mers-cov. this finding is similar to that of mohd et al., who noted high ast levels in mers-cov patients (mohd et al., ) . unlike their findings, it was noted in the present study that the impact of alt became significantly negative after controlling for several confounders. however, this type of association should be evaluated further in a prospective study in the presence of other unmeasured confounders. although sex was found to have no impact on mers-cov infection in the unadjusted analysis, the multivariate analysis revealed that the risk of mers-cov infection was . % times higher in males than in females. this may be because other factors are playing a role in the development of mers-cov in males, such as camel contact, since males are more likely than females to have contact with camels. in agreement with the recent saudi moh mers-cov visual triage guidelines for the identification of suspected cases (anon, b) , it was found that the odds of being infected with mers-cov were higher in patients with diabetes as compared to those with no diabetes. this also supports the findings of previous studies (badawi and ryoo, ; assiri et al., ; al-tawfiq et al., ; alraddadi et al., ) in which researchers systematically recognized that diabetes is a risk factor for mers-cov infection. these findings indicate that more attention should be given to assessing the risk of mers-cov infection in diabetic patients and whether the risk depends on a specific diabetes type or condition in these patients. as asserted in the saudi moh mers-cov visual triage guidelines and many other studies (muhairi et al., ; younan et al., ; reeves et al., ; sabir et al., ; azhar et al., a, b) , contact with a sick patient or camel was identified as an independent predictor of mers-cov infection, according to the risk prediction model. it must be noted that the finding in the present study could have been limited by combining camel contact and sick patient contact due to the small sample size of each category. this study shows the importance of incorporating various types of information to improve the performance of the risk prediction. according to the linear combination model, it was found that several of the parameters highlighted in the saudi moh mers-cov visual triage guidelines were not able to distinguish between 'highrisk' and 'low-risk' groups, nor did they help in predicting mers-cov infection. for instance, fever, respiratory symptoms, gastrointestinal symptoms, heart disease, and renal disease were noted to have an insignificant impact on mers-cov infection. however, in agreement with the saudi moh mers-cov guidelines and two other reports (mohd et al., ; arabi et al., ) , the odds of being infected with mers-cov were associated with a significant risk reduction of . % for each unit increase in wbc count. these results suggest that demographic, clinical, radiological, and laboratory data should be used in routine practice to identify suspected mers-cov cases, as such data could serve as the first line of prevention strategies. it was found that the accuracy of prediction (figure ) was further improved when combining various medical and patient variables as opposed to relying on a single factor (table ). this has been proven theoretically and in application (ahmed et al., (ahmed et al., , (ahmed et al., , etzioni et al., ; shen, ; pepe and thompson, ; su and liu, ; thompson, ) , where a linear combination has been used to maximize the diagnostic accuracy of a disease of interest. the strength of this study lies in the fact that a simple and applicable predictive model was developed that incorporates demographic, clinical, radiological, and laboratory data, where these were functionally associated and contributed greatly to stratifying and distinguishing between a high and a minimal risk of mers-cov infection. this simple evaluation of suspected mers-cov cases appears promising and could be implemented easily in routine clinical practice. this model could be used as a risk stratification method or a triage tool to help physicians in making an informed decision and planning the next step when deciding whether an rrt-pcr or further investigation is necessary. it was possible to derive a risk probability algorithm (range - ), a generalized youden index (choi et al., ) was used to determine an optimal cut-off to stratify the risk, and a risk probability of ! . was identified as being the optimal cut-off, with a sensitivity of . and specificity of . . several limitations to the proposed risk prediction model were identified. the study findings were based on a retrospective design; therefore this prediction model should be interpreted with caution. limited information was available on patient variables, clinical variables, and transmission routes. for example, information on primary cases and secondary cases may be supplemented by the results of clinical, radiological, and laboratory data. in this study, 'contact with a sick patient' and 'contact with a sick camel' were combined into one variable due to the small number in each category. severe illness was based on a subjective judgment. an additional potential limitation was that the duration of symptoms was not available for these patients. this study investigated a very specific population (pneumonia) at only two centers, which could compromise the applicability and generalizability of the risk prediction model. moreover, the prediction model may not be generalizable to patients who do not fulfill the moh guidelines. further validation of the prediction model on an external sample and prospective cohort of representative patients with pneumonia is necessary, specifically in relevant settings: emergency, outpatient, inpatient, and community. despite these limitations, the model developed shows promise for the identification of suspected mers-cov cases in clinical practice. this model could be applicable in various healthcare settingsinpatient, outpatient, and emergency departmentsbecause no extensive laboratory testing is required and samples may be available within short turnaround times. this may allow rapid evaluation and improve clinical decision-making. in conclusion, this study provides a simple, practical, and valid algorithm to identify individuals at increased risk of mers-cov infection among patients who have developed pneumonia. this risk model is not only useful for risk stratification and decisionmaking in clinical practice, but it could also be useful in preventing and managing the possible spread of mers-cov. the usefulness of this newly developed tool most be validated in an external prospective study. the project received ethical approval from two independent ethics committees: the saudi ministry of health (irb log number - e) and king abdullah international medical research center (study number rc / ), riyadh saudi arabia. none. none declared. accuracy and cost comparison in medical testing using sequential testing strategies reducing cost in sequential testing: a limit of indifference approach believe the extreme (be) strategy at the optimal point: what strategy will it become diagnostic delays in symptomatic cases of middle east respiratory syndrome coronavirus infection in saudi arabia estimating survival rates in mers-cov patients and days after experiencing symptoms and determining the differences in survival rates by demographic data, disease characteristics and regions: a worldwide study the predictors of -and -day mortality in mers-cov patients mers-cov diagnosis: an update the critical care response to a hospital outbreak of middle east respiratory syndrome coronavirus (mers-cov) infection: an observational study 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pneumonia in saudi arabia the authors acknowledge the saudi ministry of health and king abdullah international medical research center for approving this research project. the authors would like to thank the leaders of king abdulaziz medical city in riyadh and king fahad general hospital in jeddah for their support and understanding. supplementary data associated with this article can be found, in the online version, at https://doi.org/ . /j.ijid. . . . key: cord- - cgwivt authors: baier, claas; haid, sibylle; beilken, andreas; behnert, astrid; wetzke, martin; brown, richard j. p.; schmitt, corinna; ebadi, ella; hansen, gesine; schulz, thomas f.; pietschmann, thomas; bange, franz-christoph title: molecular characteristics and successful management of a respiratory syncytial virus outbreak among pediatric patients with hemato-oncological disease date: - - journal: antimicrob resist infect control doi: . /s - - - sha: doc_id: cord_uid: cgwivt background: respiratory syncytial virus (rsv) is responsible for upper and lower respiratory tract infection in adults and children. especially immunocompromised patients are at high risk for a severe course of infection, and mortality is increased. moreover rsv can spread in healthcare settings and can cause outbreaks. herein we demonstrate the successful control and characteristics of a rsv outbreak that included patients in our department of pediatric hematology and oncology. methods: we performed an epidemiologic investigation and a molecular analysis of the outbreak strains. moreover we present the outbreak control bundle and our concept for rsv screening in the winter season. results: rsv a and b strains caused the outbreak. rsv b strains affected patients, of whom were co-infected with rsv a. exactly this rsv a strain was detected in another patients. our multimodal infection control bundle including prophylactic rsv screening was able to rapidly stop the outbreak. conclusion: an infection control bundle in rsv outbreaks should address all potential transmission pathways. in pediatric settings the restriction of social activities might have a temporal negative impact on quality of life but helps to limit transmission opportunities. molecular analysis allows better understanding of rsv outbreaks and, if done in a timely manner, might be helpful for guidance of infection control measures. respiratory syncytial virus (rsv) of the family pneumoviridae is a single stranded rna-virus with two antigenic different subtypes (a and b). it causes upper and lower respiratory tract infection (urti and lrti) in children and adults in a seasonal pattern [ ] [ ] [ ] [ ] . the median incubation period is . days [ ] , ranging from to days. human to human transmission takes place via droplets as well as direct and indirect contact (e.g. contaminated surfaces or hands of medical staff ). patients with hemato-oncological disease are at risk for severe rsv-caused infection -especially in the context of hematopoietic stem cell transplantation (hsct) [ , ] . in the literature varying rsv-related case fatality rates are reported in children with cancer to range from % to % [ ] [ ] [ ] . respiratory tract infection (rti) due to rsv is typically community/household-acquired. rsv is a member of the so called community-acquired respiratory viruses such as influenza virus. nevertheless, hospital (nosocomial) acquisition is possible as well and transmission may occur by other infected patients, staff or visitors [ , ] . rsv outbreaks in inpatient pediatric oncologic care facilities and in adult hematology and oncology units have been described [ , [ ] [ ] [ ] [ ] [ ] . an understanding of transmission pathways helps to guide adequate outbreak control measures and to implement prophylactic measures. finally, rsv-caused respiratory tract infections are a differential diagnosis worth considering in neutropenic cancer patients with fever [ , ] . therapeutic options for patients in hematopoietic stem cell transplantation and with intensive cancer therapy, who are severely infected by rsv, include the use of systemic or aerosolized ribavirin and polyclonal intravenous immunoglobulins (ivig) [ , ] . the rsvspecific monoclonal antibody palivizumab has been used for treatment and for passive immunization (prophylaxis) in high risk pediatric patient groups [ ] [ ] [ ] . here we describe the successful management and characteristics of a rsv outbreak in a single non-hsct pediatric hematology and oncology ward including patients in march and april . moreover, we show the results of the molecular strain analysis. an outbreak case is a patient with a positive rsv laboratory testing in samples from the upper or lower respiratory tract and a definite or possible nosocomial onset. a definite nosocomial case was defined as a positive rsv laboratory testing on day or later of the hospital stay. a possible nosocomial case was defined as a positive rsv laboratory testing on day to of hospital stay. patients who were admitted to the ward with a new positive rsv laboratory testing, and had been on the ward within days prior to admission were also considered a possible nosocomial case. all patients that were accommodated in the same room with cases were considered contacts. a combined nose/throat swab was taken for routine viral diagnostics. material from the lower respiratory tract was suitable as well. samples taken for diagnostic purposes were processed at the institute of virology using real-time rt-pcr or direct fluorescent antibody (dfa) staining. rna was extracted from the specimens using a qiaamp viral rna mini kit in a qiacube according to the manufacturer's instruction (qiagen, hilden, germany). cdna synthesis, amplification and detection of nucleic acid were performed in an applied biosystems® real-time pcr system (life technologies, carlsbad, california) by a commercially available one-step real-time rt-pcr kit (rsv/hmpv r-gene® pcr kit, biomérieux, nürtingen, germany) according to the manufacturer's instructions. for dfa staining a ready to use fitc (fluorescein isothiocyanate) -labeled monoclonal rsv antibody (light diagnostics, merck, darmstadt, germany) was used according to a protocol described before [ ] . pcr and dfa did not differentiate between rsv a and b. one diagnostic specimen was tested using a point-of-care test (poct) system (sofia, quidel, kornwestheim, germany), which is available in the pediatric emergency room. nasopharyngeal aspirates from outbreak patients (case , , , , , and ) were taken only for strain typing purposes on one occasion (march th ), which were exclusively processed at the institute for experimental virology, twincore -centre for experimental and clinical infection research. in addition, selected archived (frozen) samples taken for diagnostic purposes from outbreak patients (case , , , , , ) were provided by the institute of virology and processed at the institute for experimental virology, twincore -centre for experimental and clinical infection research. linearized acrylamide (ambion, thermo fisher scientific; μg/ml final concentration) was added to the sample and total rna was extracted from μl of aspirate according to the manufacturer's description (qiaamp viral rna mini kit, qiagen, hilden, germany). cdna synthesis was performed using the superscript iii kit from invitrogen (invitrogen, darmstadt, germany) and random hexamer primers. next, a nested pcr was performed first amplifying the rsv-g and f protein coding region and in a second round amplifying the g protein gene. pcr products were sent for sanger sequencing (gatc, konstanz, germany) and the sequences were analyzed using mega software and the highlighter analysis tool [ ] . we obtained ethical approval for this study from the ethics committee of the hannover medical school. the outbreak occurred in the clinic for pediatric hematology and oncology which is a tertiary referral center for children from - years with hematologic and solid neoplasia. the affected ward harbors singleand two-bed rooms. each single room has an anteroom and a high-efficiency particulate air filtration with the air flow directed to the hallway. the same floor houses the outpatient clinic and a recreation room for the hemato-oncological pediatric patients. exchange from patients between the ward and the outpatient clinic occurs regularly. moreover two recreation rooms for social activities are part of the ward. most patients receive antineoplastic therapy during their stay. a substantial number of patients have a neutrophil count below cells / μl. the ward is serviced by permanent health care workers (hcws) and house-keeping staff. external personnel (such as medical consultants or physiotherapists) enter the ward when necessary. autologous and allogenic hsct are performed on a separate ward with single rooms. parents are allowed to stay overnight with their children. patients with a positive rsv test are electronically marked in the hospital alert system. besides single room accommodation, contact and droplet precautions (surgical mask, gown, gloves) are used at any time by visitors and hcws when entering the room of a rsv positive patient. affected patients are encouraged to stay in their room and are trained in hygienic hand washing to minimize spread via contact. when leaving the room becomes necessary (e.g. for examination), patients wear a surgical mask. these measures also apply for patients with typical respiratory symptoms (e.g. cough or sneezing) before a pathogen is identified. measures are usually suspended when there are two negative rsv pcr-based test results at a minimum -day interval and therefore patients are not anymore considered infectious. hcws with symptoms of urti are suspended from direct patient care and wear a surgical mask while on the ward. visitors with symptoms of acute rti are not permitted to enter the ward. strict hand hygiene following who guidelines is implemented. targeted rsv diagnostics are performed in case of suspected viral rti. positive testing for respiratory viruses is regularly reported for epidemiologic and infection control reasons to the infection control staff. in march a total of patients (cases to ) were tested rsv-positive in respiratory samples. patient characteristics are shown in table . patients fulfilled criteria for nosocomial acquisition. the remaining patients had a possible nosocomial acquisition, as they had been on the ward within days prior to admission. epidemic curve and an outbreak timeline with the diagnostic results can be seen in figs and . case was neutropenic and developed severe lrti with a rsv positive bronchoalveolar lavage and a requirement for oxygen. case also suffered from lrti, which was less severe. cases to had respiratory symptoms of an urti such as cough, sneeze and a positive rsv testing in secretions from the upper respiratory tract. case was co-infected with influenza a virus . patients received oral ribavirin therapy (case , , , ) and patients (case to ) temporarily required supportive oxygen administration via nasal cannula. ivig or palivizumab was not administered. no direct rsvassociated mortality was observed. in addition, all patients were empirically treated by antibiotics presuming bacterial co-respectively superinfection according to inhouse standards (table ) . viral persistence (viral shedding) is defined as the time period from first positive diagnostic test to sustained negativity. duration of viral persistence was minimally days (case ) and maximally at least days (case )see also table . active outbreak management was started after detection of new rsv infected patients in calendar week (see fig. ). an outbreak control team consisting of the infection control unit and the physicians in charge was established. the head of the clinic for pediatric hematology and oncology, the medical director of our institution, and the public health authority were informed. in addition to the existing standard infection control measures described above, interventional measures were introduced. all hcws (including permanent staff members and external personnel), visitors and outpatients were required to wear a surgical mask at any time (patient care and non-patient care activities) when on the ward and in the outpatient clinic (preemptive barrier precaution). moreover, roommates of patients tested positive for rsv in their clinical course were moved to single rooms for days (typical maximum incubation period; so called quarantine). they were repeatedly tested for rsv using pcr. all newly admitted patients were tested for rsv (admission screening). twice weekly pcr rsv prevalence screening for all patients on ward was established (prevalence screening). if possible, elective patient admissions were delayed to reduce patientto-nurse ratio. moreover, only parents were allowed as visitors. all social activities for the patients and relatives were suspended. during outbreak, all two bed-rooms were occupied by one patient only, meaning single room isolation for all patients on the ward. repeated training sessions for staff were provided by the infection control team. they addressed rsv transmission pathways and underlined the importance of droplet precautions (e.g. masks and cough etiquette for hcws and visitors) as well as hand hygiene. intervention measures were fully implemented on nd of march. the last nosocomial case occurred on th of march (case ). however, the patient had been discharged from the ward on nd of march, and readmitted on th of march (fig. ) . thus, after intervention measures had been in place no further nosocomial rsv for phylogenetic analysis of the rsv genome we focused on the viral glycoprotein g of rsv as this gene is highly variable and shows the highest sequence variance between the rsv subgroups a and b ( % amino acid sequence identity [ ] ). from the total of patients, five of them were infected with a rsv a strain (case , , , , ), one was tested positive for rsv b (case ) and two patients were infected with rsv a and b (case and ) dependent on the time point of sampling. despite the coexistence of genetically definite genotypic strains with many nucleotide exchanges especially in the c-terminal variable region of rsv-g [ ] [ ] [ ] , we detected the very same nucleotide sequence for the coding region of the rsv-g protein for all patients infected with rsv a (fig. a) . only one single nucleotide exchange was detected in the intergenic region of the virus infecting patient c and only at one time point of sampling ( fig. a; c _ _ ). in the specimen taken days later (c _ _ ), this variation was no longer detectable. among the three patients infected with rsv b, we observed nucleotide differences in the coding region of the g protein (fig. b) . two additional variations were observed in the intergenic region of the g gene (fig. b) . the rsv b viruses infecting patients c and c were almost identical with merely three nucleotide differences between them. notably, the chromatograms of the sanger sequencing from these patients revealed sequence ambiguity at exactly these three positions: between g/a at position , between t/a at position and g/a at residue . while in case c , residues g, t and g were dominant, in patient c , residues a, a, a predominated. this result suggested that these two patients were infected with an essentially identical viral quasispecies and that merely the relative number of viruses with g, t, g residues compared to viruses with a, a, a nucleotides at these positions varied between these two patients. in contrast, patient c carried a rsv b virus with an unambiguous sequence at these three positions g, t, g (fig. d) . moreover, it displayed three additional polymorphisms in the g protein coding region relative to the virus infecting patients c and c , thus supporting the conclusion that this patient was infected with a different rsv b virus. noticeably, case was tested rsv negative in two samples taken and processed for routine diagnostic on march th and th . rsv strain sequences available from other, nonoutbreak pediatric patients (same season as the outbreak) were used for comparison. these strains show a predominantly polyclonal pattern for rsv a (fig. c) . interestingly, one patient (rsv_ _ ) was infected with the very same rsv b strain as cases and (fig. c , lower part), whereas there were clear sequence differences for the other rsv b strains isolated from non-outbreak pediatric patients. for our clinic of pediatric hematology and oncology this was the first actively managed rsv-outbreak. in the previous two winter seasons in total only rsv-positive patients were detected on the affected ward. we studied the epidemiologic and molecular background of this outbreak. considering bed and room occupancy on the ward during the outbreak, direct patient to patient transmission (e.g. via droplets or contaminated surfaces) in cases and as well as and seemed epidemiologically possible as each pair was accommodated in the same room before samples were tested positive for rsv. cases and acquired rsv at day and of their stay on the ward, respectively, suggesting nosocomial rsv acquisition. these two patients did not share rooms with other infected patients but were on ward during the outbreak. cases and were tested positive for rsv on admission. nosocomial acquisition was considered possible, as case and had been discharged and days, respectively, from the affected ward prior to re-admission. during this previous stay patients with symptoms of rti and a positive rsv test were already on the ward. nonetheless, community-onset still was an option for case and . based on this epidemiologic background, our main hypothesis was that direct and indirect patient to patient transmission (the latter for example via the hcws' hands) caused the outbreak. however, at this point transmission by an infected visitor or hcws acting as a point source could not be excluded. moreover, taking all epidemiological data into account, a random introduction of several different community-acquired strains seemed unlikely to us. we suspected ongoing transmission of a single rsv variant and sequencing was used retrospectively to test this hypothesis (see below). the standard, pre-outbreak infection control measures regarding rsv were mainly in line with previously made recommendations for hospitalized patients with hematooncologic disease [ , ] . the additionally implemented fig. highlighter plot depicting nucleotide mismatches comparing the sequence of the strain obtained from patient c to all other rsv a strains, and the sequence of the strain obtained from patient c to all other rsv b strains. a rsv a and (b) rsv b glycoprotein sequences were aligned using mega and depicted as highlighter plot using the highlighter analysis tool [ ] . nucleotide exchanges compared to a reference sequence (c _ _ ( ) for rsv a and c _ _ ( ) for rsv b) are depicted in color. absence of sequence information is depicted as grey bar. a schematic of the rsv g protein with the different domains is depicted on top [modified from [ ] ]. ( ) indicates samples taken exclusively for strain typing at the th of march and ( ) indicates samples collected for routine viral diagnostics. c rsv-g sequence alignment from other pediatric, non-outbreak patients compared to the references c and c , respectively. d sequencing chromatograms for rsv b cases. the depicted area is highlighted by * and ** in figure b . overlying sequence information from different quasispecies detected in the samples are highlighted in a box measures, in particular single room accommodation for contact patients (quarantine), suspension of all social activities, and surgical masks for all hcws and visitors at any time, addressed the postulated rsv transmission pathways during this outbreak. these postulated pathways were direct patient to patient transmission (e.g. roommate to roommate), but also transmission via hcws and visitors. direct patient to patient transmission as the most probable route of infection has been shown by lehners et al. in a large rsv outbreak in a german hematology and transplant unit [ ] . jensen et al. described direct patient to patient transmission, mixed with introduction of strains from outside, in an outbreak affecting immunocompromised adults [ ] . we therefore focused on patient to patient transmission early during the outbreak by strict isolation precautions for rsv infected patients and contacts. isolation for infected patients was also a key measure in a multimodal control bundle described by inkster et al. [ ] . contact patients were isolated for days and repeatedly tested in order to disrupt infection chains as described in literature [ ] . this so called quarantine concerned patients in our outbreak. one of them (case ) was eventually tested rsv-positive at day of quarantine while being negative at day and . this underlines the value of the measure. finally, we reemphasized in training sessions the need for preemptive isolation of patients with respiratory symptoms. as all these measures required more isolation capacity on the ward, we restricted elective admissions and located all patients in single rooms. as another measure we reduced direct patient to patient contacts on the ward by suspending community events, as active social behavior can be a risk factor for nosocomial rsv acquisition [ ] . even so this noticeably restricted the social life for the patients and their families during the outbreak, we enforced this measure. we further restricted social contacts by temporally limiting visits of infants to the ward, as (especially young) infants are known to be the main reservoir for rsv and as our outbreak was approximately concurrent (slightly delayed) to the rsv community peak. only parents were allowed to the ward, which is in line with an intervention done by kelley et al. [ ] . a restrictive visiting policy is as well described by singh et al. in a pediatric rsv outbreak [ ] . the use of surgical masks for everyone on the ward is an important measure to prevent droplet associated nosocomial rsv transmissions. this is even more rational as rsv may be transmitted via symptomless or oligosymptomatic persons (e.g. hcws or visitors) and the infectious period can in fact already begin to days before actual onset of symptoms. a literature review by french et al. concluded that personal protective equipment might be advantageous for reducing nosocomial rsv transmission [ ] . kelly et al. showed that five hcws showing only mild symptoms were involved in a rsv outbreak on an adult stem cell transplant unit [ ] . this underlines the necessity that hcws with respiratory symptoms should not participate in direct patient care activities, at least in a high risk patient care setting. we re-emphasized this issue in training sessions for the hcws. although staff screening is described in literature [ ] , we were able to terminate this outbreak without staff screening. a cohort of hcws to take care of solely rsv-positive patients as reported before [ ] had also not been established but would have been another option in case of an ongoing outbreak. temporal survival of respiratory viruses in general [ ] and specifically rsv [ ] on inanimate surfaces is described, thus contact transmission via the hands of staff was conceivable for nosocomial acquisition. this is especially of importance as cough etiquette and compliance to basic hygienic principles may be reduced for obvious reasons in pediatric patients, so a higher environmental rsv burden is probable. nonetheless we did not implement changes in the well established cleaning and disinfection procedures on the ward. we detected prolonged rsv persistence (virus shedding), which has been reported in patients with hematological disorders [ ] . this finding needs to be considered for efficient outbreak control and favors the practice of repeated testing in immunocompromised patients as we did. likewise, this is important as pediatric hemato-oncologic patients are often readmitted several times for cancer treatment cycles or fever in neutropenia. when symptoms are no longer present or mild but viruses are still being shed, rsv may be re-introduced to the ward. thus, for termination of isolation precautions during the outbreak, we required negative results as reported before [ ] . in fact, two subsequent negative results at a minimum -day interval were necessary. the usefulness of this requirement is supported by the longitudinal course of the samples from patient which were obtained in april and may. this patient produced positive specimens on two occasions, after one specimen had been tested negative (see fig. ). active rsv-surveillance by screening on admission and twice weekly for all patients on the ward insured rapid detection of rsv-positive patients. this is in line with successful infection control measures reported in literature [ , ] . we presume that a prophylactic admission and prevalence rsv screening for all patients in the winter season might be helpful as a preventive measure in high risk populations. therefore, one consequence of this outbreak was the implementation of an active rsv surveillance (admission and prevalence screening once weekly) in our clinic for pediatric hematology and oncology during the rsv season. the beginning and ending of this seasonal screening period is determined by in-house and regional/national rsv epidemiology [ ] . moreover, pre-rsv-season audits involving clinicians, infection control staff and the institute of virology take place to ensure timely beginning of screening procedures and adherence to the existing infection control practices. molecular characterization of rsv strains, for instance by whole genome sequencing [ ] or characterization of rsv g-protein [ , ] , has been used to investigate nosocomial rsv outbreaks. we were able to collect and examine selected outbreak strains by g-protein gene sequencing. we found that cases to were infected with an rsv a virus with identical g protein coding region. in case of patient c one nucleotide difference in the intergenic region of the g gene was observed in one of two samples collected five days apart (fig. a . it is possible that this change was due to natural drift of the infecting virus over time or that this polymorphism is indicative of the presence of two slightly different viruses replicating in parallel and dominating on the one and the other day of sampling, respectively. moreover we found that case had a rsv-b infection and that cases and were co-infected by rsv a and rsv b viruses. while sequence analysis of the earlier samples of case and revealed infection by the rsv a virus, the sequence analysis of the later specimen showed infection by an rsv b virus. with the available specimen, we were unable to distinguish if these two patients had a prolonged co-infection between these viruses or if they were sequentially infected by rsv a and rsv b. these findings became available only after the outbreak ended, as routine virological testing during the outbreak did not include molecular differentiation of rsv a and b. in retrospect, these results indicated the decision not to cohort rsv-patients during the outbreak, as we probably might have cohorted rsv-patients with different subtypes. detailed sequencing analysis suggests that cases and were infected by an almost identical rsv b virus population. we observed three nucleotide differences between these viruses; however, nucleotides of the viruses at these three positions were ambiguous in both cases (g,t,g versus a,a,a residues). thus, both patients were likely infected by a highly similar rsv b quasispecies which was characterized by two different nucleotide signatures varying in abundance between patients. in contrast, the rsv b virus infecting patient c differed in two key criteria. first, it did not show any sequence ambiguity at the three above mentioned residues that was characteristic for the rsv b virus population observed in patients c and c . second, it displayed three additional polymorphisms in the coding region of the g protein. taken together, this suggests that patient c was infected by another rsv b virus and that there was no transmission from patients c and c to patient c . outbreak strains of the subtype rsv a were highly similar and different from polyclonal strains from other nonoutbreak pediatric patients (fig. c) . we therefore conclude that a single rsv a strain was introduced to the ward and then spread within the ward. interestingly, the rsv b isolate c _ _ ( ) was identical to the community strain rsv_ _ , however further epidemiologic and clinical information are not accessible for the non-outbreak patient. taken together, the nucleotide analysis suggests independent introductions of at least different rsv b strains into the ward affecting patient c , c and c , and transmission of one rsv a strain on the ward between patients c to c . looking exclusively at the molecular analysis, it is not possible to disclose the exact transmission pathway of rsv a. rsv a might have been introduced to the ward by an infected patient (index patient) on the ward (maybe case ) and was then successively transmitted from patient to patient. alternatively, a point source, such as a rsv-positive hcw, may have caused the outbreak. however, in correlation with the epidemiologic observations such as overlapping patient stays on the ward, stay of case and , and case and in a double room, and social activity on the ward in the initial phase of the outbreak, we consider a direct and indirect patient to patient transmission most likely. rsv poses a significant infectious threat to pediatric patients with an underlying oncologic disease. this outbreak and other outbreaks reported in literature demonstrate the potential of rsv to spread in a hospital. we strictly enforced our existing infection control practices and implemented temporally additional measures to terminate the outbreak. according to our experiences an outbreak control bundle for rsv should include (preemptive) barrier precautions (especially masks), prevalence and admission screenings for all patients, and strict isolation procedures for infected patients and contact patients. quarantine for contacts should at least be for days, the usual maximal incubation period of rsv. in pediatric settings the restriction of visitors (especially siblings) and social activities on the ward can be helpful to prevent transmission and rsv introduction from outside, but definitely limits social life quality. as shown in other outbreaks with viral and bacterial pathogens restriction of admissions still is a very effective measure as it enables single room accommodation for all or the majority of the patients. moreover, a decrease of the patient to nurse ratio makes transmission more unlikely. in our case the molecular analysis was very helpful to verify the true outbreak character of the rsv cluster and revealed ongoing transmission of an unique rsv a strain on the ward, and an probable independent introduction of different rsv b strains into the ward. respiratory syncytial virus -a comprehensive review respiratory syncytial virus: the virus, the disease and the immune response defining the epidemiology and burden of severe respiratory syncytial virus infection among infants and children in western countries respiratory syncytial virus: how, why and what to do incubation periods of acute respiratory viral infections: a systematic review respiratory syncytial virus infection in patients with hematological diseases: single-center study and review of the literature respiratory syncytial virus infection in recipients of allogeneic stem-cell transplantation: a retrospective study of the incidence, clinical features, and outcome respiratory 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review how long do nosocomial pathogens persist on inanimate surfaces? a systematic review possible transmission by fomites of respiratory syncytial virus long-term shedding of influenza virus, parainfluenza virus, respiratory syncytial virus and nosocomial epidemiology in patients with hematological disorders community acquired respiratory virus infections in cancer patients-guideline on diagnosis and management by the infectious diseases working party of the german society for haematology and medical oncology defining the timing of respiratory syncytial virus (rsv) outbreaks: an epidemiological study investigation of respiratory syncytial virus outbreak on an adult stem cell transplant unit using whole genome sequencing molecular characterization of a respiratory syncytial virus outbreak in a hematology unit in heidelberg structure and function of respiratory syncytial virus surface glycoproteins not applicable. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.availability of data and materials all data generated or analysed during the current study are included in this published article. all authors contributed to the manuscript according to the icmje (international committee of medial journal editors) recommendations: all authors were involved in data acquisition, analysis and interpretation. sh, rb and tp carried out molecular analysis. cb, sh, tp and f-cb prepared the manuscript. cb organized the drafting process. asb and anb, cb and f-cb were involved in the active outbreak management. ts and cs carried routine rsv diagnostic. mw collected specimens for typing purposes. all authors critically revised the manuscript and account for accuracy and correctness. all authors have read and agreed to the final draft before submission. we obtained ethical approval for this study from the ethics committee of the hannover medical school (nr. - ). submit your next manuscript to biomed central and we will help you at every step: key: cord- -dlwph za authors: alshahrani, mohammed s.; sindi, anees; alshamsi, fayez; al-omari, awad; el tahan, mohamed; alahmadi, bayan; zein, ahmed; khatani, naif; al-hameed, fahad; alamri, sultan; abdelzaher, mohammed; alghamdi, amenah; alfousan, faisal; tash, adel; tashkandi, wail; alraddadi, rajaa; lewis, kim; badawee, mohammed; arabi, yaseen m.; fan, eddy; alhazzani, waleed title: extracorporeal membrane oxygenation for severe middle east respiratory syndrome coronavirus date: - - journal: ann intensive care doi: . /s - - -x sha: doc_id: cord_uid: dlwph za background: middle east respiratory syndrome (mers) is caused by a coronavirus (mers‐cov) and is characterized by hypoxemic respiratory failure. the objective of this study is to compare the outcomes of mers-cov patients before and after the availability of extracorporeal membrane oxygenation (ecmo) as a rescue therapy in severely hypoxemic patients who failed conventional strategies. methods: we collected data retrospectively on mers-cov patients with refractory respiratory failure from april to december in intensive care units (icus) in saudi arabia. patients were classified into two groups: ecmo versus conventional therapy. our primary outcome was in-hospital mortality; secondary outcomes included icu and hospital length of stay. results: thirty-five patients were included; received ecmo and received conventional therapy. both groups had similar baseline characteristics. the ecmo group had lower in-hospital mortality ( vs. %, p = . ), longer icu stay (median vs. days, respectively, p < . ), and similar hospital stay (median vs. days, p = . ). in addition, patients in the ecmo group had better pao /fio at days and of admission to the icu ( vs. , and vs. , p < . ), and less use of norepinephrine at days and ( vs. %; and vs. %, p < . ). conclusions: ecmo use, as a rescue therapy, was associated with lower mortality in mers patients with refractory hypoxemia. the results of this, largest to date, support the use of ecmo as a rescue therapy in patients with severe mers-cov. middle east respiratory syndrome (mers), which was first described in , is caused by a novel coronavirus (mers-cov). the world health organization (who) as of december reported confirmed cases of the mers-cov infection globally with an overall mortality rate of % [ ] . the majority of cases were reported in saudi arabia, wherein were confirmed cases, and of which ( %) died [ ] . human coronaviruses were first identified in the mid- s and usually cause mild upper-respiratory tract illness. in , the first confirmed case of mers-cov was reported from saudi arabia [ ] . mers-cov infection is associated with significant mortality related to the virulence of the virus, nature of the disease, and the lack of effective therapy. patients with mers-cov who develop acute respiratory distress syndrome (ards) are at a high risk of dying from refractory hypoxemia, multiorgan failure, and septic shock [ ] . current interventions such as lung protective ventilation, prone ventilation, and neuromuscular blocking agents have been shown in randomized trials to improve mortality in patients with ards [ ] [ ] [ ] . however, in some patients, these conventional measures fail to maintain adequate oxygenation; therefore, other rescue therapies are considered, such as different modes of ventilation, inhaled pulmonary vasodilators, and extracorporeal membrane oxygenation (ecmo). anticipated difficulties in patient recruitment, study design, and ethical concerns affect the feasibility of conducting randomized clinical trials that examine the efficacy of ecmo in this population. therefore, observational studies are a reasonable alternative. in this study, we aim to describe the effect of ecmo rescue therapy on patient-important outcomes in patients with severe mers-cov. in response to the large mers-cov outbreak, the saudi ministry of health implemented a national ecmo program in april . the saudi ecmo program provided a rapid transportation chain system (medevac system), isolated intensive care unit (icu) beds, and venovenous (v-v) ecmo machines in selected centers across the country. an ecmo team was created that was available h a day/ days a week. the team included an intensivist trained in ecmo, a cardiac surgeon, a perfusionist, and ecmo-trained nurses. the intensivist on the ecmo team triaged all calls from other centers based on predefined criteria, wherein patients were predetermined to be candidates to receive ecmo or not. criteria for eligibility to receive ecmo were based on the extracorporeal life support organization (elso) [ ] guidelines and are listed below. we retrospectively identified patients who would have been eligible for ecmo but did not receive it because the ecmo program was not available at that time (prior to april ). the intervention (ecmo) group was included from five main ecmo centers in three major cities in saudi arabia after the program initiation (april to december ). all participating hospitals were accredited by the joint commission international and had closed icus with -h coverage by trained intensivists. we obtained ethics approval from the saudi ministry of health ethics review board and from individual centers' ethics boards. patients were candidates to receive ecmo if they have met the following criteria: the ecmo group included patients who met the above criteria and received ecmo after implementing the ecmo program from april to december . we included all patients with mers-cov who received ecmo during that period. the control group were patients who met the above criteria but did not receive ecmo in the period prior to the introduction of ecmo program (prior to april ). weaning from ecmo was primarily based on clinical improvement demonstrated by adequate oxygenation and gas exchange shown in vital signs, blood gases, and chest x-ray. the decision for readiness of a patient to be weaned from ecmo was left to the judgment of treating clinician and the ecmo team. the weaning process followed the elso criteria as follow: weaning starts by decreasing the flow to l/min while keeping the sweep of % (to maintain spo > %). if spo remains within target, a trial of clamping the catheters and keeping the patient on the ventilator at appropriate settings was attempted. we designed an electronic pretested data abstraction forms; the forms were pilot tested prior to data collection to ensure accuracy and reproducibility. trained personnel collected the data at each participating center under the supervision of the local principal investigators. research personnel collected data on patients' demographics, comorbidities, acute physiology and chronic health evaluation ii (apache ii) score, laboratory results (hemoglobin concentration, white blood and platelets counts, kidney function, blood gases), ventilator modes and settings, interventions used to treat refractory hypoxemia (prone ventilation, use of neuromuscular blocking drugs, and pulmonary vasodilators), vasoactive support, antimicrobial and antiviral therapy, steroid use, and primary and secondary outcome data. data were tested for normality using the kolmogorov-smirnov test. a repeated-measures analysis of variance was performed. fischer's exact test was used for the categorical data. independent t test was used to compare the continuous variables in the two groups. the mann-whitney u test was performed to compare the nonparametric values of the two groups. data were expressed as median (interquartile range (iqr) [range]), number (proportion), or mean (sd) as appropriate. the volume of cases was not enough to allow a priori power analysis. however, a post hoc power analysis indicated that the current sample size of patients is powered to detect % absolute difference in mortality rate, with a type i error of . and a power of %. a value of p < . was considered statistically significant. eighty patients with confirmed mers-cov infection were admitted to the icus of participating centers from april to december . thirty-five patients met our eligibility criteria and were included in the analysis, in the ecmo group and in the control group. as shown in table , the baseline characteristics were similar in both groups; the median ages were ( vs. years), and mean apache ii score ( vs. ) were not statistically different. (p = . and p = . ; respectively). adjunctive therapies were used in both groups. ribavirin was used significantly more often in the ecmo group compared to the control group ( vs. %, p = . ), interferon was also used more in the ecmo cohort compared to controls ( vs. %, p = . ), and the use of steroids was similar in both groups ( vs. %, p = . ). at day one of eligibility to ecmo, more patients in the control group required hemodynamic support with norepinephrine compared to ecmo group; however, both groups had similar use of epinephrine and dobutamine, continuous renal replacement therapy (crrt), modes of ventilation, positive end-expiratory pressure (peep), and neuromuscular blocking agents (tables and ). alveolar recruitment maneuver was used in one patient in the ecmo group. none of the patients received prone ventilation. throughout days - , more patients in the control group developed renal impairment and had significantly lower pao /fio ratio (table ) . other laboratory values were similar between both groups (table ). however, due to the small sample size, it was not feasible to adjust for all confounding factors. in the ecmo group, the v-v mode was used in all patients via the percutaneous cannulation approach for vascular access. femoral-femoral access was used in % of patients, while femoral-jugular access was used in % of cases. ecmo access was inserted by a cardiac surgeon in % of cases and by a cardiac intensivist in the remaining %. chest x-ray was used to confirm successful cannulation in patients and transesophageal echocardiography (tee) in one patient. blood flow (l min − ), revolutions per minute, and sweep gas among ecmo patients had a mean (sd) of . ( . ), . ecmo-related mechanical complications occurred in ( %) patients; one patient developed pneumothorax that was treated with chest tube insertion, and two patients had major bleeding immediately after the initiation of ecmo. compared to the control group, the ecmo group had significantly lower in-hospital mortality ( vs. %; p = . ), longer icu stay ( vs. days; p = . ) ( table and fig. ). less use of norepinephrine at days and (p < . ), and better oxygenation (higher pao / fio ratio) throughout days - (table ). in this retrospective cohort study, we found that ecmo rescue therapy was associated with lower in-hospital mortality, better oxygenation, and fewer organ failures compared to historical control (usual care) in patients with severe mers-cov. however, the length of hospital stay was the same and a possible explanation is that during the crisis phase, patients were mechanically ventilated in the ward when icu beds are full, and it is possible that this could have contributed to similar stay in hospital in both groups. although elso issued guidelines on the use of ecmo in patients with ards, these guidelines do not address specific disease context, and are difficult to generalize to the heterogeneous ards population. therefore, we conducted this observational study to report on the efficacy and safety of ecmo in patients with severe mers-cov infection. there is a single case report in the literature looking at ecmo in mers-cov patients. guery et al. described the use of ecmo in two patients with acute respiratory failure secondary to mers-cov infection in france, where both patients developed severe hypoxia and increasing oxygen requirements, leading to mechanical ventilation and ecmo use. one patient died, and the other survived after approximately months in hospital [ ] . ecmo use in respiratory failure has been reported with variable survival rates. the first randomized clinical trials (rcts) failed to prove superiority of ecmo over conventional management [ , ] . however, the severe adult respiratory failure (cesar) trial showed improved -month survival in patients who were referred early to an ecmo center [ ] . this was the largest clinical trial to investigate the efficacy of early use ecmo in patients with ards. despite concerns about the trial design and possible differences in steroid use and ventilator strategies, these results contributed to the increasing use of ecmo worldwide. in this study, we observed no significant differences in the use of adjunctive therapies except for ribavirin use in the ecmo group. the benefit of antiviral therapy in mers-cov infection remains unclear. recent korean guidelines published during the mers-cov outbreak in south korea suggested the use of antiviral therapy in patients with severe mers-cov [ ] . in patients with respiratory failure from h n infection who required the use of ecmo, the survival rate varied considerably between studies ranging from to % [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . there was a large variation in survival rates, which could be explained by differences in patients' baseline characteristics and severity of illness. in one study, older, obese, diabetic, or immunocompromised patients were found to be at a higher risk of developing severe [ ] [ ] [ ] [ ] . in this study, the two groups were comparable at baseline, and there were no significant differences between groups in any of these variables. another large observational study examined the predictors of death in h n patients who underwent v-v ecmo and found that creatinine and bilirubin levels, systemic arterial pressure, hematocrit, and pre-ecmo hospital length of stay were associated with higher mortality [ ] . another important factor is the center experience and volume of cases; this could have contributed to the variability in survival rates with ecmo use. a recent study by barbaro et al. [ ] demonstrated that centers with > ecmo cases/year had better survival rates than centers with less than cases per year. in saudi arabia, ecmo was not available except in one center until the mers-cov crisis; thereafter, the ecmo program was implemented as a therapeutic option for patients with refractory hypoxemia. ecmo interventions were run in tertiary centers with equipped icus by most experienced intensivists and perfusionists who received training in ecmo prior to the start of the program. although more ecmo patients received ribavirin and interferon therapy, we do not believe that this difference has an impact to our findings. published reports on this therapy are limited, but none showed significant improvement with this combination [ ] [ ] [ ] . the largest study to date published in abstract format [ ] showed no reduction in mortality. therefore, we believe that the imbalance of co-interventions between the two groups is unlikely to affect the estimation of treatment effect. in regard to infection control issues, caregivers safety of ecmo patients was organized and maintained by aggressive measures which were applied strictly and monitored closely with all admissions were taken to airborne isolated rooms which impacted the containment of the virus plus applying the universal protective personal measures all the time during the patients encounter. because of these stringent measures, there were no reports by or about any caregiver of any ecmo patient being affected. to our knowledge, this is the largest study to describe outcomes in patients with mers-cov who received ecmo. there are several strengths to our study: the "before and after" design allowed us to compare ecmo cases to a control group with similar demographics and within the same institutions. we also collected data on important variables and confounders, and conducted adjusted analyses to assess the impact on the results. we adhered to the strengthening the reporting of observational studies in epidemiology (strobe) guidelines [ ] . despite the strengths of our study, it has several important limitations. first, the retrospective nature of this study renders it at risk of bias. all patients in the control group died, which may be explained by the severity of illness, as these were patients who had ards and were eligible otherwise. we cannot rule out the possibility of selection bias, as we were unable to track all transfer requests due to the outbreak and crisis at the time, leaving us with limited information. in addition, some patients were transferred from non-participating ecmo centers; therefore, baseline pre-ecmo data such as blood gases and ventilator settings could not be obtained. furthermore, due to insufficient documentation during the outbreak and crisis circumstances, we were not able to track the ecmo requests to the referral call center. there were differences in some co-interventions (e.g., antiviral therapy), and the influence of unmeasured confounders cannot be excluded. such concerns can only be addressed in rcts; however, conducting rct is likely to be challenging in the context of epidemics. this study was not designed to compare the cost of interventions; although it is an important outcome that could help the clinicians and stakeholders to make decisions. lastly, the small sample size limited our ability to perform an adequate multivariate analysis. similar to other ecmo studies, it is difficult to determine if the mortality was the result of refractory respiratory failure or other causes like septic shock or other organs failure. in summary, the use of ecmo was associated with lower mortality in patients with severe mers-cov infection and refractory hypoxia. future randomized trials, although challenging to conduct, are highly needed to confirm or dispute these observations. until more data are available, ecmo could be considered as a rescue therapy in selected mers-cov patients with refractory hypoxemia. 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network experience during the influenza a(h n ) pandemic: preparation for severe respiratory emergency outbreaks middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection epidemiological, demographic, and clinical characteristics of cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study hospital outbreak of middle east respiratory syndrome coronavirus predicting mortality risk in patients undergoing venovenous ecmo for ards due to influenza a (h n ) pneumonia: the ecmonet score. intensive care med association of hospital-level volume of extracorporeal membrane oxygenation cases and mortality. analysis of the extracorporeal life support organization registry ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome oronavirus:an observational study ribavirin and interferon alfa- a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study ifn-α a or ifn-β a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study effect of ribavirin and interferon on the outcome of critically ill patients with mers the strengthening the reporting of observational studies in epidemiology (strobe) statement: guidelines for reporting observational studies we wish to acknowledge the saudi ministry of health for implementing a national ecmo program in april who assisted us throughout the course of this research. we thank our colleagues from all participating icus throughout saudi arabia who provided insight and expertise that greatly assisted the research, and for their comments on an earlier version of the manuscript. the authors declare that they have no competing interests. all data produced and analyzed during this study are included and presented as tables in this manuscript. authors have no objection in granting and assigning the annals of intensive care journal unrestricted right to reproduce, publish, and distribute this manuscript in all forms including electronic form either offline or online media. no patients were involved neither in the design, recruitment, and conduction of this study nor in the development of outcome measures. we plan to disseminate the results of the study in lay language for patient interest groups. we had one institutional review board approval (irb-h- -j- ) for the ecmo from the saudi arabia ministry of health as all ecmo program during the outbreak was under the umbrella of the ministry. all authors declare that they receive no support from any commercial organization or company. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - x q f authors: astudillo, patricio; angulo, jenniffer; pino, karla; de carvalho, joseane biso; de morais, guilherme loss; perez, sebastián; de vasconcelos, ana tereza ribeiro; ferrés, marcela; lópez-lastra, marcelo title: correlation between female sex, il b genotype, and the clinical severity of bronchiolitis in pediatric patients date: - - journal: pediatr res doi: . /s - - - sha: doc_id: cord_uid: x q f background: single-nucleotide polymorphisms (snps) that impact on the differential expression of interleukin b (il b) are implicated in the progression of viral-induced diseases. in this prospective longitudinal cohort study, we evaluated the association between il b snps rs and rs and the clinical outcome of bronchiolitis in pediatric patients. methods: a total of infants suffering from bronchiolitis, categorized based on the final clinical outcome as mild or severe, were genotyped for il b snps rs and rs . results: when infants were categorized exclusively based on the final clinical outcome, no association was established between il b snps and the severity of bronchiolitis. however, when stratified by sex, the homozygotes for the minor alleles of rs (t) and rs (g) were associated with a mild disease in girls but not in boys. conclusion: snps rs and rs correlate with the severity of bronchiolitis and display a sex bias, where gg rs and tt rs genotypes are associated with a mild disease in girls but not in boys. these findings suggest that innate immunity and female sex links with the outcome of the diseases induced by respiratory viruses, such as rsv. bronchiolitis is the leading cause of hospitalization in children aged < years worldwide. the human respiratory syncytial virus (rsv) is the most prevalent etiological agent of bronchiolitis. even though several risk factors are expected to predispose an individual to severe bronchiolitis, most infants hospitalized with bronchiolitis exhibit no known risk factors that can be associated with the disease. by the age of years, nearly all children have been infected with rsv at least once, yet only - % develop a severe disease requiring hospitalization, , suggesting a genetic predisposition to develop a severe disease. [ ] [ ] [ ] [ ] [ ] [ ] in agreement with this possibility, a twin cohort study revealed a higher concordance in the hospitalization rates of identical twins when compared to fraternal twins, estimating that approximately % of the propensity to develop severe bronchiolitis upon rsv infection was attributable to genetic differences. the potential relevance of genetic background to the degree of severity of bronchiolitis is further reinforced by the differences in the rate of rsv hospitalization observed in populations from different racial and ethnic backgrounds living in the same geographic region or country. , together, these findings suggest that host genetic factors might likely contribute to the severity of virus-induced bronchiolitis. the single-nucleotide polymorphisms (snps) rs and rs are located near the interleukin b (il b) locus on chromosome and known to affect the expression of il b mrna and protein. the expression of il b, also known as interferon lambda (ifnλ ), is elevated in individuals with the cc rs and tt rs genotype, relative to individuals with an non-cc rs and non-tt rs genotype. [ ] [ ] [ ] [ ] noteworthy also is that cc rs and tt rs genotypes are the favorable il b genotype for hepatitis c virus (hcv) infection as the elevated expression of il b is associated with virus clearance, a positive response to therapy, and a better disease outcome. [ ] [ ] [ ] [ ] as ifnλs are the predominant ifns induced in epithelial cells by respiratory viruses, , we were intrigued by the possible association between genetic factors associated with ifnλ expression and disease outcome. thus we designed a prospective cohort study to specifically address whether a link exists between snps rs or rs and the severity of bronchiolitis in infants. patients a prospective longitudinal cohort study was performed on previously healthy, full-term infants aged < years admitted at the pediatric units of two tertiary hospitals (centro asistencial doctor sótero del río and hospital clínico uc-christus) and one ambulatory unit (centro médico san joaquín uc-christus) in santiago de chile during the winter season of years - with a clinical diagnosis of bronchiolitis. bronchiolitis was defined as an acute lower respiratory tract infection characterized by rhinorrhea, cough, and diffuse wheezes and/ or on auscultation. patients' records were followed until their discharge with all patients receiving standard treatment and fluid management according to clinical guidelines. the length of stay (los) in the hospital and pediatric intensive care unit (picu) admission, days of oxygen use, the requirement for non-invasive (niv) or invasive mechanical ventilation (ivm), and mortality were considered in the final outcomes. the criteria used to define clinical severity were based on a previously validated bronchiolitis score system ranging from to , which incorporates information such as days of hospitalization, admission to picu, maximum percentage of fraction of inspired oxygen (fio ), and required number of days of supplementation and mechanical ventilation (supplemental table s and ref. ). in agreement with ref., score values lower than seven were defined as "mild" illness (g group), while scores of seven and above were considered "severe" cases of bronchiolitis (g group). all individuals with comorbidities, including preterm, neuromuscular disease, chronic pulmonary disease, primary and acquired immune deficiency, down syndrome, prophylactic use of palivizumab, congenital heart diseases, immigrants, and tourists, were excluded from the study. all patients were evaluated for the presence of atopic comorbidities (atopic dermatitis, allergic rhinitis, food allergies) and other epidemiological variables (birth weight, sex, eutocic or cesarean delivery, and weight at presentation) as suggested in ref. clinical data were complemented with a chest x-ray and laboratory analysis. bacterial infection was suspected when c-reactive protein test was > mg/dl (normal value: mg/dl), the leucocyte count > , × mm , and based on chest x-ray examination. parents were requested to answer a questionnaire to enable the identification of any additional epidemiological factor associated with their infants' disease (breastfeeding at months of age, maternal smoking during pregnancy, tobacco exposure at home, parental history of asthma the distribution of the snps rs and rs within the chilean population was determined by us in a previous study. genotyping of snps rs and rs was performed as described. the amplification reaction was conducted in a stratagene mx p thermal cycler (agilent technologies, inc., santa clara, ca) and the correct assignment of alleles in each sample was attributed automatically by the mxpro software (agilent technologies, inc.) and manually confirmed by three independent investigators. positive and negative controls described in ref. were used in each genotyping assay. statistical analysis demographic, clinical, and laboratory data are described as frequency (%) for categorical variables and mean (±standard deviation) for quantitative variable data. odds ratios (ors) are reported with a % confidence interval (ci). disease outcomes and severity of bronchiolitis were compared among all patients, while the polymorphism frequency was compared between patients and non-infected controls. the clinical symptoms exhibited by patients were analyzed using a two-tailed fisher exact test for each variable performed in the statistical package for the social science software (version , spss inc). the association between polymorphisms and severity of infection was calculated using a two-tailed fisher exact test with a × contingency table as described elsewhere using the graphpad (version . ) and openepi (version . ) software as described in ref. the distribution of hardy-weinberg equilibrium (h-we) was verified by χ test. a wilcoxon matched-pairs test was used to determine differences between genotype frequencies in the bronchiolitis and control groups. the crude or values were calculated by univariate analysis using the spss v . software package. differences were considered significant at p < . . results were confirmed by the snpstats web tool (https://www.snpstats.net/). the association for each snp was analyzed in turn with five inheritance models (codominant, dominant, recessive, overdominant, and log-additive). an allele-recessive effect model for rs and rs was selected for the final analysis. data were fitted for the selected covariates (sex, hospitalized status, virus, and viral coinfection), including one variable of response (clinical severity). the analysis of association for each snp was performed with logistic regression analysis summarized with genotype frequencies, proportions, ors, and % cis. the two snps are included in the analysis and linkage disequilibrium (ld) and haplotype analyses were performed. the association analysis of haplotypes was similar to genotypes analysis, using logistic regression (or and % ci). a multivariate stepwise forward and reverse logistic regression analysis was conducted using the spss v . software package. differences were considered significant at p < . . a total of infants diagnosed with bronchiolitis between and were evaluated, of which were enrolled. infants not included in the study had previous comorbidities (n = ), had more than two hospitalizations (n = ), were not available for the study (n = ), were transferred to another hospital (n = ), or declined to sign the consent form (n = ). among the enrolled infants, showed a single infection by rsv ( . %), the most commonly detected virus, followed by hmpv ( . %), hpiv ( . %), iv ( . %), hrv ( %), hbov ( . %), ev ( . %), and cov ( . %) (fig. ) . the demographic and clinical features of the infants enrolled in the study are summarized in table . the mean age of patients was . months, and ( . %) were boys. upon recruitment, patients were categorized into two groups (g and g ) according to the severity of the respiratory illness at hospital discharge. , g included ( . %) infants who presented a severe respiratory disease, while g included ( . %) patients exhibiting mild bronchiolitis. when compared, the two groups showed no significant differences in sex distribution, gestational age, birth weight, type of delivery, nutritional status, maternal pregnancy diseases (preeclampsia, gestational diabetes, hypothyroidism, neurological or another diseases), personal history of atopy, or laboratory findings (table and supplemental tables s and s ). however, patients in group g tended to be younger; to present a lower weight at the time of enrollment; exhibited a significantly higher tobacco exposure risk at home; tended to have parents with a history of asthma; to be more likely to have been admitted to the picu; to present a clinical bacterial coinfection; to show an elevated use rate of bronchodilators, antibiotics, and systemic corticosteroids; and to require ventilatory support (or: . ( % ci: . - . )). patients from g also presented atelectasis in findings (or: . ( % ci: . - . ) and opacity (or: . ( % ci: . - . )) in chest x-ray analysis and had an increased hospital and picu los, increased supplemental oxygen days, and a higher clinical score at the time of medical evaluation at the emergency unit (p < . ; table and supplemental tables s and s ). clinical symptoms that significantly associated with a severe disease were fever, previous history of costal retraction, regular oral tolerance, cyanosis, or apnea. to the physical exam, infants in g exhibited a prolonged capillary refill, severe retraction, presence of dehydration, wheezing, crackles, and prolonged expiration relative to infants in g (supplemental table s ). analysis of the il b snps in confirmed bronchiolitis patients snp analysis of the bronchiolitis patients (global) revealed that the frequencies for the rs cc, ct, and tt genotypes were . , . , and . , while frequencies for the rs tt, gt, and gg genotypes were . , . , and . , respectively (fig. a) . the allele frequencies for each snp within the group of bronchiolitis patients were similar to those of the non-infected control cohort, where rs cc, ct, and tt genotype frequencies were . , . , and . and rs tt, gt, and gg genotype frequencies were . , . , and . , respectively. a multiple snp analysis revealed a positive ld value (d' = . ). a direct comparison of snps rs and rs genotype combinations between the non-infected control cohort and all bronchiolitis patients showed no significant variation in genotype distribution (fig. b) . however, the data do reveal a weighting for homozygote individuals for the major allele (c) in rs among the ill (fig. b) . within the control cohort, snp rs and rs genotypes were in h-we (χ test p > . ). in bronchiolitis patients, snp rs (χ test . ; p = . ) and rs (χ test . ; p = . ) genotypes were not in an h-we, suggesting a possible selection effect most probably associated with the risk of hospitalization. results further suggest that, when combined, il b rs /rs genotypes differ in their correlation with the risk of hospitalization due to bronchiolitis. while not statistically significant, there appears to be a trend toward hospitalization due to bronchiolitis for haplotypes cc/tt and ct/tt when compared to haplotypes tt/gg, tt/tg, and tt/tt ( fig. b and table ). link between il b snps and the severity of bronchiolitis next, we evaluated whether a link existed between il b snps and the severity of bronchiolitis in the global population of bronchiolitis patients. analysis of snp rs and rs revealed no correlation with the severity of bronchiolitis for the overall ill population (fig. a, b) . these findings are in full agreements with data reported for an italian cohort of infants suffering from bronchiolitis. however, infants homozygotes for the minor allele (t) in snp rs or homozygotes for the minor allele (g) for snp rs were significantly younger and weighed less at the time of hospital admission (table ) . we, then, stratified our study population by viral coinfection (with vs. without) or hospitalization status (hospitalized vs. non-hospitalized). results revealed no correlation between the severity of bronchiolitis and viral coinfection but being homozygotes for the minor allele (g) for snp rs linked with a mild bronchiolitis among hospitalized patients (or: . ( % ci: . - . ), p = ; table ). the effect of the il b genotype on spontaneous hcv clearance is associated with sex. - so next we stratified our population by sex (male vs. female). results show that our study population had more boys than girls, suggesting that the first have a . time higher incidence of bronchiolitis. in boys, we found no evidence of a link between the severity of bronchiolitis and either snp rs or snp rs (table ) . however, in girls, being homozygote for the minor alleles, in rs (t) and in rs (g), did correlate with mild bronchiolitis (or: . ( % ci: . - . ), p = . ) and or: . ( % ci: . - . , p = . ) ( table ) . correlation of il b snps rs and rs with the severity of rsv bronchiolitis rsv infections ( total infections) are associated with % of all bronchiolitis patients included in this study (fig. ). so next we explored whether there was an observable relationship between snps rs or rs and the severity of rsv bronchiolitis ( single-infected patients). the demographic and clinical features of the subgroup of rsv-infected patients are summarized in table . when classified according to the severity of the disease, infants with a single viral infection ( . %) exhibited severe disease symptoms (g -rsv) and ( . %) exhibited mild disease symptoms (g -rsv). the clinical observations that were significantly different between these groups included fever, cyanosis/apnea, prolonged capillary refill, the magnitude of retractions, and dehydration (supplementary table s ). laboratory findings showed a significant difference in bicarbonate levels (p = . ) between both groups, while chest x-ray analysis showed a difference in the presence of atelectasis (or: . ( % ci: . - . )) and opacity (or: . ( % ci: . - . )) (supplementary table s ). no differences were observed in sex distribution, gestational age, birth weight, type of delivery, nutritional status, maternal pregnancy diseases, and personal history of atopy ( fig. correlation between clinical severity of global bronchiolitis or rsv bronchiolitis and il b snps rs and rs . a, b a total of pediatric bronchiolitis patients were grouped according to the snp rs (a) and rs (b) genotypes and stratified according to the severity of the disease. c, d a total of pediatric patients diagnosed with rsv bronchiolitis were grouped according to the snp rs (c) and rs (d) genotypes and stratified according to the severity of the disease. genotypes were divided into those homozygous for the minor allele (tt rs and gg rs ) and those with heterozygous or homozygous status for the major allele (non-tt rs and non-gg rs ). the total number of patients in each group was defined as %, and the severity of the bronchiolitis was evaluated as a dichotomous variable using fisher exact test. the number within each box corresponds to the percentage of patients with mild or severe disease population, rsv-infected infants seemed biased toward the combined cc rs -tt rs haplotype. however, no significant correlation between rs and bronchiolitis was found (fig. c) , while being homozygote for the minor allele (g) in rs ( patients) correlated with mild rsv bronchiolitis (or: . ( % ci: . - . )) (fig. d) . consistent with a mild disease, a generally lower supplemental fio requirement was observed in infants homozygotes for the minor alleles, in rs (t) and in rs (g), during hospitalization. also, homozygosity for the minor allele in rs (g) associated with a lower hospitalization and picu los (table ) . when stratified by sex, no association between snps rs or rs and the severity of the illness could be established for boys (table ). however, for girls, being homozygote for the minor alleles, in rs (t) (or: . ( % ci: . - . )) and in rs (g) (or: . ( % ci: . - . )), correlated significantly with a mild rsv bronchiolitis (table ). to control the potential confounding effects of the various risk factors identified by univariate analysis, a multiple logistic regression model was constructed using the clinical outcome of rsv bronchiolitis as a response variable. the stepwise forward and reverse logistic regression analysis included demographical, clinical, and laboratory variables (age < months, newborn weight > kg, obesity, sex, fever, severe retractions, cyanosis/apnea, capillary refill, dehydration, antibiotics, systemic corticosteroids, chest x-ray findings, tobacco exposure at home, phototherapy, breastfeeding, parental history of asthma, maternal smoking, clinical bacterial coinfection), and tt or gg genotype of il b snps rs and rs . the clinical outcome of rsv bronchiolitis was dichotomized (mild vs. severe). table summarizes the crude ors for each variable processed by a univariate logistic regression model and the or obtained by a multivariate stepwise forward (model ) and reverse (model ) logistic regression analysis. models and this study describes the relationship between il b snps rs and rs and the severity of bronchiolitis in a population of previously healthy chilean infants. in spite of a previous study, conducted on a cohort of italian infants, that suggested no correlation between il b snps rs and rs and bronchiolitis, we decided to proceed with this project based on the particular genetic background of the chilean population. the impact of il b snps among diverse ethnic populations is well documented. , the chilean population, in contrast to the italian cohort above, is admixed with ancestral contributions from europe and native america, as well as africa. opposing to what was initially expected, no significant relationship was found between il b snps rs and rs and the severity of bronchiolitis when chilean subjects were categorized based on the severity of the disease (fig. a, b) . thus, when analyzing the total study population categorized only by disease severity, our findings are in full agreement with the previous study. importantly, however, in contrast to the preceding study, we extended our analysis by additionally stratifying patients by sex (table ) . stratifying by sex alone did not directly link to the severity of bronchiolitis (tables and ) unless the snps are included in the analysis. results indicate that being homozygote for the minor alleles in rs (t) and in rs (g) correlate with a mild disease in girls but not in boys (table ). this sex bias was also replicated in the subgroup of rsv bronchiolitis patients ( table ). the predominance of severe bronchiolitis in boys vs. girls as well a sex bias for other snps in bronchiolitis has been previously described by others. [ ] [ ] [ ] moreover, the association between snps in il b and sex is also known in the context of an hcv infection. [ ] [ ] [ ] however, to our knowledge, this study represents the first report to link snps in il b and female sex for bronchiolitis. interestingly, the snp genotype associated with a lower expression of il b, non-cc rs and non-tt rs genotypes, correlated with a mild bronchiolitis in girls. the precise mechanisms underlying the association between mild bronchiolitis and the il b genotype in girls are still obscure as this study was not designed to evaluate cytokine expression within the infant population. so, no direct conclusions can be drawn on any association between il b expression and disease progression. nonetheless, the direct correlation between il b snps rs and rs and the differential expression of il b has been well documented. [ ] [ ] [ ] [ ] consistent with our observations, a previous report shows that females exhibiting the poorly expressing il b non-tt rs genotype clear hcv as efficiently as males with the favorable highly expressing tt rs genotype. these findings are most probably attributed to known sex-based differences in immunity, which are most evident during the early stages of life. [ ] [ ] [ ] thus it is reasonable to suspect that the level of il b in girls possessing the tt rs /gg rs haplotype is sufficient to elicit efficiently response to the viral infection giving rise to a mild bronchiolitis as a final clinical outcome. if so, this would indicate that, in the context of both general and rsv bronchiolitis, the tt rs /gg rs haplotype is protective in girls. also, our findings support previous reports which suggest that innate immunity links with the outcome of the diseases induced by respiratory viruses, such as rsv. in spite of the intriguing possibility raised by this study, which suggests that the host's genetic makeup may play a role in the severity of bronchiolitis, we recognize that the present report has important limitations, primarily sample size. we concede that a larger study is needed to fully confirm the current findings. also, % of all cases included in this study corresponded to rsv bronchiolitis (fig. ) . based on results reported in table , we cannot discard that this factor alone biases our conclusion regarding the general bronchiolitis population ( table ). the number of bronchiolitis cases associated with other individual viral infection was insufficient to draw any statistically significant alternative conclusion. furthermore, the study population that was evaluated herein was sourced from two high complexity hospitals, which may have caused selection bias toward severe patients with respiratory distress. this could explain why the cc rs and tt rs genotypes were found enriched within ill individuals when compared to the non-infected control population. finally, this study was focused specifically on the chilean population, which is genetically distinct from other populations, even when compared with those of other south american neighboring countries. this might limit the generalizability of the findings to other populations with different ancestry composition. notwithstanding these biases, the results presented herein establish a clear link between female sex, tt rs /gg rs haplotype, and the clinical severity of rsv bronchiolitis in chilean pediatric patients. correlation between female sex, il b genotype, and the clinical severity. . . p astudillo et al. 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gobierno de chile, through grant conicyt-programa de investigación asociativa (pia) act to m. m.l.-l. conceptualized and designed the study, analyzed the results, drafted the initial manuscript, and reviewed the revised manuscript. p.a. conceptualized and designed the study, coordinated patients' recruitment, collected samples, extracted dna, genotyped, analyzed the results, drafted the initial manuscript, and reviewed the revised manuscript. j.a. designed the genotyping strategy, conducted genotyping, analyzed data, and reviewed and revised the manuscript. j.b.d.c., g.l.d.m., and a.t.r.d.v. conducted the bioinformatics and statistical analysis of the data. k.p. extracted dna and conducted genotyping and reviewed and revised the manuscript. m.f. and s.p. participated in patient recruitment, sample collecting, and reviewed and revised the manuscript. the online version of this article (https://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -vlszl i authors: chen, si; liu, dafei; tian, jin; kang, hongtao; guo, dongchun; jiang, qian; liu, jiasen; li, zhijie; hu, xiaoliang; qu, liandong title: molecular characterization of hlj- , a recombinant canine coronavirus strain from china with an orf abc deletion date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: vlszl i canine enteric coronaviruses (ccovs) are important enteric pathogens of dogs. ccovs with different variations are typically pantropic and pathogenic in dogs. in this study, we isolated a ccov, designated hlj- , from a dead -week-old male pekingese with gross lesions and diarrhea. interestingly, sequence analysis suggested that hlj- contained a -nt deletion in orf abc compared with reference ccov isolates, resulting in the loss of portions of orf a and orf c and the complete loss of orf b. phylogenetic analysis based on the s gene showed that hlj- was more closely related to members of the fcov ii cluster than to members of the ccov i or ccov ii cluster. furthermore, recombination analysis suggested that hlj- originated from the recombination of fcov - and ccov a , which were both isolated in the united states. cell tropism experiments suggested that hlj- could effectively replicate in canine macrophages/monocytes and human thp- cells. this is the first report of the isolation of strain hlj- in china, and this virus has biological characteristics that are different from those of other reported ccovs. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. canine coronavirus (ccov) is a member of virus family coronaviridae, order nidovirales. ccov is single-stranded positive-sense rna viruses with a genome that is between and kb in length [ ] . based on antigenic and genetic relationships, ccov consists of two distinct genotypes, ccov-i and ccov-ii [ , ] . ccov was first recognized as an enteric pathogen of dogs in , when it was observed to cause clinical signs including anorexia, lethargy, vomiting, and mild-to-severe diarrhea [ ] . the clinical signs usually lasted - weeks and were followed by recovery, or occasionally by dehydration that led to death, mainly in puppies [ ] . in recent years, researchers have reported an increasing number of cases of virulent ccov infection in dogs and the emergence of ccov variants with recombinant s genes. recombination occurs when different ccov types infect the same host simultaneously [ , , , ] . therefore, ccov has become a cause of concern as an emerging pathogen in dogs. in the present study, we report the emergence and molecular characterization of an, fcov-like recombinant ccov-hlj- , which was isolated from a fecal sample from a dead dog that exhibited enteritis. during the spring of , a dead dog, a privately owned -week-old male pekingese, was submitted for laboratory investigation. the clinical symptoms were lethargy, inappetence, fluid diarrhea, vomiting and dehydration with death after days. necropsy of the dog showed hemorrhagic enteritis and severe lesions in the liver, spleen and lung. hemorrhages of the liver and spleen were observed on their surfaces. multiple areas of congestion were found in the lung. a fecal sample was collected and subjected to virological investigations. rapid kits were employed to identify common canine viral pathogens, including canine distemper virus (cdv), canine parvovirus (cpv), canine adenovirus- (cav- ), cav- and ccov (bionote, hwaseong-si, gyeonggi-do, south korea). the primers p-f and p-r (table s ) were employed to confirm the above results [ ] . the crfk cell line was used for virus isolation. after three rounds of purification by plaque assay [ ] , the virus was propagated and titrated and then harvested by one cycle of freezing and thawing. viral rna was extracted from the original fecal sample and from crfk cells infected with ccov using trizol reagent (invitrogen, carlsbad, ca, usa). fifteen primer pairs were designed based on the conserved regions of ccov strain tn- (table s ). the rt-pcr assay was performed as described previously [ ] . the protocol for electron microscopy was described previously [ ] . an indirect immunofluorescence assay (ifa) was conducted using a standard procedure. an anti-ccov antibody (vmrd, wa, usa) and fitc-conjugated rabbit anti-dog igg (sigma, ca, usa) were used as primary and secondary antibodies, respectively. a ccov-n polyclonal antibody was prepared as follows [ ] : briefly, the complete n gene was amplified using a forward primer ( ' ttt gga tcc atg gcc aac cag gga caa cgc ') and a reverse primer ( ' ttt gcg gcc gctta gtt cgt tac ctc atc aat '). then, the products were cloned into the vector pgex p- . purified gst-n recombinant protein was used as an antigen to inject female balb/c mice. after three immunizations, serum was collected and stored at - °c. rna was extracted from fecal and organ samples using a previously established taqman-based real-time rt-pcr assay for rapid detection and quantification of ccov rna [ ] . ??the primers?? df f and orf cr (table s ) [ ] were used to detect sg mrnas from the orf abc region by rt-pcr, using a one-step rt-pcr kit (abm, richmond, bc, canada). a phylogenetic tree based on the sequences of the structural proteins was constructed using the neighbor-joining (nj) method of molecular evolutionary genetics analysis (mega) software (version . ). bootstrap values were estimated for , replicates. simplot . . was used for nucleotide sequence comparison of hlj- to the reference fcov strains and ccov strains. the hlj- sequence obtained in this study was assembled and submitted to the genbank database under accession number ky . canine blood monocytes originating from specific-pathogen-free (spf) dogs were isolated [ ] . phorbol -myristate -acetate (pma) was purchased from sigma-aldrich (st. louis, usa). the human monocytic cell line thp- was provided by the national institute for communicable disease control and prevention, chinese centers for disease control and prevention (beijing, china). thp- cells were induced to differentiate into a mature-macrophage-like state by stimulation with ng of pma per ml for h before treatment. we isolated a canine coronavirus with a deletion in the orf abc region from a dead dog in china. real-time rt-pcr assays were carried out to determine the viral load in the faeces, intestine, lung, liver, spleen, kidney and heart of the dead dog. all of the organs except the kidney were found to be positive for ccov (table s ), indicating that this strain is a pantropic ccov strain. next, after three rounds of plaque assay screening, a novel isolate, hlj- , was successfully isolated and purified. the presence of virus particles was confirmed by electron microscopy of negatively stained samples (fig. s ) . sequence analysis suggested that there was a -nt deletion in orf abc when compared to isolates - , tn- and bgf- . this deletion resulted in truncations in orf a ( nt) and orf c ( nt) and the complete loss of orf b ( fig. a and b) . to investigate the stability of the orf abc region in the hlj- genomic rna, the virus was propagated in crfk cells for nine generations. total rna was extracted from hlj- -infected crfk cells and from the original faecal sample. analysis by pcr confirmed that the orf abc deletion was present not only in the purified virus but also in the original faecal sample (fig. s ) , indicating that the deletion was not due to viral adaptation during cell culture passage. examination of subgenomic orf abc mrnas showed that the genome of hlj- contained a truncated orf abc (fig. c) . previous studies have demonstrated that this region is variable, and a natural cb/ strain that causes fatal infections and long-lasting lymphopenia has also been shown to contain a deletion in orf b [ , , ] . strains / and na/ are closely related to the prototype strain cb/ , which causes diarrhea and acute lymphopenia [ , ] . furthermore, strain bgf also has a deletion in the orf abc region [ ] . these results demonstrate that the orf abc region may be prone to being lost during the evolution of the virus. recombination plays a vital role in the evolution of ccov, allowing the emergence of new strains with altered virulence and immunogenicity. in recent years, ccovs with different genotypes and subgenotypes have been detected. ccov-ii is the oldest genotype, which has been known since [ ] . more recently, recombination between ccov and tgev has been detected, which has led to the emergence of ccov-iib isolates in which the n-terminus of the s gene is very similar to that of tgev, while the rest of the genome resembles that of the reference ccov-ii isolate [ ] . a ccov strain (ucd- ) that had potentially recombined with tgev was identified in the late s [ ] . genetic analysis of several ccovs circulating in italy first revealed a new canine genetic cluster in which recombination events within the s gene had increased the similarity of ccovs to the feline homolog [ ] . on the basis of their genetic relationship to fcov-i, the new genotype was initially designated "fcov-like ccovs". recently, an additional orf, named orf , located between the end of the s gene and the orf a gene, was also detected [ ] . in this study, phylogenetic analysis based on the s protein revealed that hlj- is closely related to members of the fcov-ii cluster and is distinct from ccovs ( fig. a) . analysis of the s (receptor-binding) domain showed that it clustered closely with fcov-ii - (fig. b) , while the s (fusion) domain clustered with ccov-ii, fcov-ii and tgev (fig. e) . the coronavirus s protein consists of two independent subdomains, the n-terminal domain (ntd) and the c-terminal domain (c-domain) [ ] . the ntd clustered closely with cb/ and - (fig. c) , and the c-domain clustered with - (fig. d) . in contrast, the m gene clustered with a and had a -amino-acid deletion compared with a , cb/ , / (fig. h) , and the e gene cluster with tgev and did not clustered with ccov and fcov (fig. g) . these data indicate that hlj- is probably a recombinant of tgev, fcov-ii and ccov-i/ ii, with recombination sites located between the s, e and m genes. simplot analysis was carried out to identify possible recombination events and breakpoints in the complete nucleotide sequence of the hlj- genome. the analysis the orf abc region of feline coronavirus has been shown to affect viral cell tropism. truncated orf abc of fipv df- has been shown to effectively replicate in macrophages/monocytes, while infection with fcovs with the complete orf abc region was confined to the intestinal tract [ ] . in the present study, we found that hlj- and fipv df- could also effectively replicate in canine macrophages/ monocytes (fig. a) . however, hlj- could replicate in thp- cells, while df- could not (fig. b) . these results suggest that hlj- has the ability to alter its tissue tropism from the intestinal tract to systemic infection and to alter its viral cell tropism from dogs to humans, which should be regarded as a potential threat to domestic dogs. accordingly, epidemiologic studies are required to determine whether domestic ccov isolates have similar characteristics. we report for the first time that ccov hlj- , with an orf abc deletion, has been isolated in china. hlj- represents a recombinant coronavirus with distinct host cell tropism. acknowledgements this work was supported by the state key laboratory of veterinary biotechnology foundation (sklvbp ). conflict of interest the authors declare that they have no competing interests. virus taxonomy, th report of the international committee on taxonomy of viruses molecular characterization of feline infectious peritonitis virus strain df- and studies of the role of orf abc in viral cell tropism recovery and characterization of a coronavirus from military dogs with diarrhea canine coronavirus highly pathogenic for dogs canine coronavirus highly pathogenic for dogs new enteric viruses in the dog an update on canine coronaviruses: viral evolution and pathobiology immunity after natural exposure to enteric canine coronavirus does not provide complete protection against infection with the new pantropic cb/ strain recombinant canine coronaviruses related to transmissible gastroenteritis virus of swine are circulating in dogs quantitation of canine coronavirus rna in the faeces of dogs by taqman rt-pcr a pantropic canine coronavirus genetically related to the prototype isolate cb/ molecular characterisation of the virulent canine coronavirus cb/ strain molecular characterization and phylogenetic analysis of transmissible gastroenteritis virus hx strain isolated from china feline and canine coronaviruses: common genetic and pathobiological features preparation and characterization of mouse polyclonal antibody against conserved region of human foxo . xi bao yu fen zi mian yi xue za zhi canine enteric coronaviruses: emerging viral pathogens with distinct recombinant spike proteins molecular characterization of a canine coronavirus na/ strain detected in a dog's organs crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor genetic diversity of a canine coronavirus detected in pups with diarrhoea in italy characterization of a recombinant canine coronavirus with a distinct receptor-binding (s ) domain molecular characterization of a virulent canine coronavirus bgf strain cocirculation of canine coronavirus i and iia/b with high prevalence and genetic diversity in heilongjiang province plaque assay for canine coronavirus in crfk cells the s gene of canine coronavirus, strain ucd- , is more closely related to the s gene of transmissible gastroenteritis virus than to that of feline infectious peritonitis virus key: cord- - yxurnev authors: green, manfred s; leduc, james; cohen, daniel; franz, david r title: confronting the threat of bioterrorism: realities, challenges, and defensive strategies date: - - journal: lancet infect dis doi: . /s - ( ) - sha: doc_id: cord_uid: yxurnev global terrorism is a rapidly growing threat to world security, and increases the risk of bioterrorism. in this review, we discuss the potential threat of bioterrorism, agents that could be exploited, and recent developments in technologies and policy for detecting and controlling epidemics that have been initiated intentionally. the local and international response to infectious disease epidemics, such as the severe acute respiratory syndrome and west african ebola virus epidemic, revealed serious shortcomings which bioterrorists might exploit when intentionally initiating an epidemic. development of new vaccines and antimicrobial therapies remains a priority, including the need to expedite clinical trials using new methodologies. better means to protect health-care workers operating in dangerous environments are also needed, particularly in areas with poor infrastructure. new and improved approaches should be developed for surveillance, early detection, response, effective isolation of patients, control of the movement of potentially infected people, and risk communication. access to dangerous pathogens should be appropriately regulated, without reducing progress in the development of countermeasures. we conclude that preparedness for intentional outbreaks has the important added value of strengthening preparedness for natural epidemics, and vice versa. the biological weapons convention prohibits the manufacture and use of biological weapons. it came into force in , and has undergone periodic reviews, the last being in . to date, countries are signatories to the convention. unfortunately, terrorist groups or rogue governments are unlikely to feel bound by international agreements. the potential for bioterrorism is of particular concern, since it can cause disease, death, and panic-in great disproportion to the resources expended. there have been a few well documented cases of bioterrorism. in , a religious sect in the usa deliberately contaminated restaurant salad bars with salmonella typhimurium, intending to disrupt local elections. the attack resulted in several hundred cases of salmonellosis and no deaths. the anthrax letters incident in in the usa resulted in cases of inhalation anthrax, with five deaths, and another cases of cutaneous disease. extensive circumstantial evidence strongly suggests that the perpetrator was a civilian employee of the us military. however, no evidence of a clear motive was found. thousands of workers received prophylactic or post-exposure therapy, and affected buildings were decontaminated at huge expense. , in , a cult in japan carried out an attack using anthrax spores with no physical casualties, but later, evidence of post-traumatic stress syndromes was found in victims of the attack. the perpetrators were apparently planning to use other agents such as q fever bacteria, botulinum toxin, and ebola viruses, but they were detained before they could implement further attacks. in this review, we discuss the threat of bioterrorism, potential perpetrators, and general preparedness principles. we examine the special characteristics of biological agents that could potentially be used for bioterrorism, advances in prevention and treatment of diseases caused by these agents, and the remaining deficiencies in the management and control of possible bioterrorist outbreaks. in all respects, the ways in which the resources developed for bioterrorism preparedness could be used for controlling naturally occurring epidemics remain a guiding principle. • preparedness for intentional outbreaks will strengthen the response to naturally occurring epidemics • high level leadership should be maintained with responsibility and authority • health-care providers should maintain awareness of biological agents with bioterrorism potential and consider the presence of unknown pathogens • emergency room and community physicians should be updated regularly about the clinical manifestations of diseases caused by potential bioterrorist agents and emerging infectious diseases. • personal protective equipment should be improved to become more user friendly • improved surge capacity (the ability to rapidly gear up the health system to cope with a sudden, large increase in patients with a serious, contagious disease) is required, particularly in peripheral areas • the capacity of general and reference laboratories should be increased, to keep developing faster, more reliable diagnostic tests • new and improved vaccines (pre-exposure and post-exposure) and treatment regimens should be developed • clinical and environmental surveillance needs to increase • syndromic surveillance systems can be maintained to register suspicious or confirmed cases reported by physicians, and the data can be used to improve risk communication programmes and to monitor the progress of an outbreak • an adequate stockpile of vaccines and medications should be maintained, both nationally and internationally • to improve preparedness for natural and bioterrorist outbreaks, international cooperation should include joint exercises involving multiple countries and constant improvement in the exchange of information on potential bioterrorism threats and management following the breakup of the former soviet union, there was concern that loss of control of their biological weapons programme could allow terrorist groups to gain access to both the weapons and scientific expertise. additionally, in the past few years, developments in the field of microbial genetics have heightened concern about the possible abuse of new technologies. since there are so many unknowns, it is extremely difficult to assess the risks and threats of bioterrorism. , the most likely perpetrators could be disgruntled individuals, terrorist organisations, or rogue countries that are believed to support international terrorism. whereas individual attackers are unlikely to cause mass casualties, terrorist organisations could pose a substantial threat if they gain access to sophisticated biological weapons, materials, or scientific expertise. although regulations and safeguards for securing dangerous pathogens in research laboratories now exist in most countries, the scope of these regulations and the extent of the safeguards vary. rogue countries have the necessary capabilities for a bioterrorist attack but might be restrained by the threat of the response of a unified global community. knowledge gained from legitimate research that could also be applied to bioterrorism is considered dual-use. as a result, the regulation of legitimate research on infectious diseases has increased. there will always be a risk of the "insider threat", , which typically involves a single individual, so it is important to assure that new regulations truly increase security and have minimal negative effect on legitimate research. the cost of regulations applied to research on infectious diseases, in terms of missed opportunities for international collaboration, exchange of pathogens, and sharing of novel agents, is often intangible and overlooked. it is essential to promote healthy organisational cultures to enhance both safety and security in laboratories. since a bioterrorist attack is a low-risk, high-impact event, effective and sustained preparedness is an essential component in both the deterrence and management of an attack. a bioterrorist attack has a lot in common with naturally occurring public health emergencies resulting from infectious diseases. however, there are some important differences. since it is a deliberate act to cause harm, there are the obvious security considerations. the resulting outbreak differs in some important ways from naturally occurring epidemics-for instance, it is more likely to be a point source outbreak initiated by simultaneous exposure to many people. the infectious agent used is likely to be uncommon and possibly not endemic to the region, might have been modified genetically to make it resistant to current medications and vaccines, and produced in a way that enhances its transmission or virulence. therefore, early clinical symptoms and signs after infection with a bioterrorist agent might be unusual, complicating both recognition and management of the disease. these factors could create greater public panic. despite the many similarities with naturally occurring outbreaks of infectious disease, preparedness for bioterrorist attacks is more complex. in many aspects, a bioterrorist attack has the characteristics of a mass casualty event, and thus preparedness involves strengthening of the specialised infrastructure that is required for treatment of seriously ill patients over a very short period of time. new prophylactic and treatment regimens for unusual diseases are required, to ensure their accessibility when needed, along with clear standards for the handling and study of dangerous pathogens. when the proportion of available resources poor international preparedness • delay before the who declared a public health emergency of international concern • delay in implementing coordinated international assistance • logistic challenges in delivering support to assist epidemic response • shortcomings in who's regional and country-level capacity exposed • lack of global plans to address an epidemic of a high-risk pathogen in the least developed urban centres • evaluation of promising vaccine and therapeutic interventions came too late and ebola virus. the local and international responses to the west african ebola virus epidemic revealed shortcomings that could allow highly contagious epidemics of infectious disease to spread widely before they are terminated (panel ). during the cold war, agents that could potentially be used as biological weapons were identified on the basis of the following characteristics: pathogenicity for humans, animals, or plants; ability to cause disability or death; stability and infectivity as small particle aerosols; and capability of being readily and rapidly produced and weaponised in munitions or delivery systems. more characteristics have been added, to include other features of biological agents such as the relative ease of medical prevention or treatment and the likelihood of harm to the perpetrator. the us centers for disease control and prevention (cdc) identified bacteria, viruses, and toxins that could potentially be weaponised (panel ). in , they categorised them into three groups-a, b, and cdepending on ease of dissemination, severity of illness caused, and ability to cause death. biological agents can be infectious and contagious, infectious but not usually contagious, or toxins if they are neither. category a agents were considered the greatest risk to public and national security. the more recent classification of tier select agents and toxins is similar to the category a classification (table ) . other agents, such as naturally occurring pathogens, produce diseases that are considered of intermediate risk to the public (eg, brucellosis, glanders, q fever). they are moderately easy to disseminate, and include emerging and re-emerging infectious diseases. however, genetic modifications could make them more virulent, produce uncharacteristic clinical signs, increase their resistance to treatment and vaccines, and even change their transmissibility or host range. genetic modifications could be made using the tools of synthetic biology; such activities might be an example of dual-use research. , for instance, in , the spanish influenza pandemic virus was reconstructed, and the poliovirus was synthesised nearly years ago. the addition of an immuno-modulatory gene to the mousepox virus genome in , rendered a mousepox vaccine ineffective, and this technology could potentially be applied to the smallpox virus. the recent synthesis of the extinct horsepox virus has been a reminder that the smallpox virus could be reconstructed, and that the regulations that have been put in place to prevent the misuse of powerful, cheap, and globally available tools must be reconsidered. this possibility has also raised the issue of whether research results should sometimes be censored, or even refused publication, if the potential to cause harm is too high. although bioterrorist agents could be disseminated through multiple routes, the aerosol route would likely maximise exposure. contagious agents could produce a large number of second and later generation cases, depending on the number of people initially exposed, the series average number of people who acquire the disease from one infected individual (r ), and the disease generation time in humans. for instance, the r of pneumonic plague has been estimated to be around · , whereas the r for smallpox is likely to be around . for diseases that are not contagious, such as inhalational anthrax, the number of cases of disease will depend almost entirely on the size of the population exposed and the timing of post-exposure antibiotic prophylaxis. aerosolised agents remain the threat of most concern, but safety and security of food [ ] [ ] [ ] and water supplies are also important components of primary prevention (panel ). new methods to detect toxins in food, such as antibody based assays, are being developed. rapid diagnostics take on additional urgency in a bioterrorist event, because of both health and security concerns. since the anthrax letters, there have been major advances in diagnostic capabilities. some of the greatest advances in the past decade have been in the speed and reduced cost of sequencing capabilities. , highly sensitive and specific pcr-based systems, coupled with modern sample preparation technologies, have enabled sequencing technologies to become less costly, more portable, and multiplexed. with fieldable patient-side diagnostics and sequencing outputs directly connected via cloud-based networks, health-care providers globally can make decisions more rapidly and respond more quickly for individual care or outbreak detection. a rapid, cartridge-based assay for francisella tularensis has been developed for use at point of care. a system that uses a sensitive microsphere technology to detect both antibodies and antigens is now available to diagnose infections with ebola virus and lassa virus. although diagnostic elisa tests are available for anthrax antibodies, , a compact system (genexpert) that includes both sample processing and pcr amplification can produce a result in about minutes. a rapid and sensitive method to detect smallpox virus has been developed for use at point of care, based on antibody immuno column for analytical processes (abicap) immunofiltration, that produces results in about minutes. however, diagnostic electron microscopy is also still considered a fast and efficient method to identify smallpox and other viral agents. ebola virus was rapidly sequenced during the outbreak in sierra leone to link sporadic cases with the transmission chains. advanced proteomics are also being developed as reference assays and a new method for simultaneous immunodetection of anthrax, plague, and tularaemia from blood cultures has recently been reported, using multiplexed suspension arrays. next generation safety and security of food [ ] [ ] [ ] and water supplies are important components of primary prevention: • intentional contamination of food should be considered, particularly during a large foodborne epidemic with a common source • salmonella and shigella species, enterohaemorrhagic escherichia coli (all serotypes), vibrio cholerae, cryptosporidium parvum, and noroviruses are all potential candidates for intentional contamination of food • contamination of water with biological agents should still be considered, even though it is unlikely to be the major target of bioterrorism, due to chlorination, dilution, and the need for large quantities of the agent to cause a substantial outbreak • c parvum and noroviruses are more resistant to chlorination than other agents, so can be a threat to the water supply • food-borne or water-borne dissemination of these biological agents might lead to higher rates of morbidity and case fatality than previously observed, if the population has been exposed to substantially higher infectious doses • algorithms could be developed to measure the likelihood that outbreaks of disease were a consequence of intentional contamination of food or water, using descriptive, analytical, and molecular epidemiologic tools (none are known to be available so far) increased networking and collaboration of laboratories will also improve the response to intentional outbreaks. effective global surveillance of infectious diseases is essential to control both intentional and naturally occurring epidemics. surveillance data can be used to monitor the progress of an outbreak, and for risk communication. to obtain information rapidly, the ongoing collection of health-related data (termed syndromic surveillance) has been introduced, to monitor patterns of symptoms and signs that are suggestive of an outbreak. [ ] [ ] [ ] [ ] although it was hoped that syndromic surveillance would be a more sensitive method for early detection of an epidemic, frequent reports of unusual increases in incidence of non-specific illnesses can desensitise and paralyse the system. in fact, early detection will depend largely on alert, prepared clinicians. for example, when the anthrax attacks occurred, an astute clinician identified the index case. emergency room and community physicians should be updated regularly on the clinical signs and symptoms associated with the most common bioterrorist agents. the syndromic surveillance system would be more useful after suspicious or confirmed cases have been reported by physicians. a focused analysis of the surveillance data against non-specific, background disease rates could detect changes and provide information about the dynamics of the disease. special legislation might then be necessary, to gain access to medical records. the internet facilitates other potential forms of surveillance and communication about infectious diseases. , one such system is promed, which was established by the user community and has proven effective in connecting clinicians and scientists around the world; it has already served as an early warning system standard contact and airborne precautions should be taken. supportive therapy and antibiotics can be provided for secondary infections. there is some evidence of the potential efficacy of thiosemicarbazones. cidofovir has shown in vitro efficacy against variola, and has shown efficacy against other diseases caused by human orthopoxviruses, notably diseases caused by vaccinia viruses. it has also shown efficacy in animal models of orthopoxvirus infections. since , the us food and drug administration (fda) has approved drugs and biologic agents developed under the animal rule. this rule allows for approval of a drug that cannot be tested for efficacy in humans, but is effective in animals and safe in humans. the first drug approved under this rule was the monoclonal antibody raxibacumab for treatment of inhalation anthrax. tecovirimat is a drug that inhibits all orthopoxviruses tested in vitro. it was found to be highly effective in treating monkeypox and rabbitpox in animals and is considered safe in humans. tecovirimat is being considered by the fda for approval for use in humans to treat smallpox under the animal rule. standard and droplet precautions should be taken. ciprofloxacin, levofloxacin, and doxycycline have been approved for the treatment of pneumonic plague. streptomycin and gentamicin have been found to be effective in treatment, although there is some evidence of the development of multiple resistance. , tularaemia isolation of the patients is not necessary and standard precautions should be taken. ciprofloxacin, levofloxacin, and doxycycline are all approved for the treatment of tularaemia. streptomycin and gentamicin have been found to be effective. , haemorrhagic fevers standard contact and airborne precautions should be taken until diagnosis is confirmed. subsequently, droplet precautions can be considered. supportive care and treatment of secondary infections can be provided. ribavirin is now approved for treatment of lassa fever and it also appears to be effective against new world arenaviruses and crimean-congo haemorrhagic fever. protective n respirators and clothing should be provided to health-care personnel. clothing of patients should undergo decontamination and thorough handwashing. supportive therapy is available, with antibiotics such as ciprofloxacin, doxycycline and ampicillin. if the bacteria are resistant to some of the antibiotics, the treatment regimen will depend on sensitivity testing. the regulations require immediate reporting of serious health risks by all member countries. additionally, who has established the global outbreak alert and response network, and the european union has a programme called bichat to improve cooperation between member states in preparedness and response to biological and chemical attacks. they operate the early warning and response system for outbreaks of communicable diseases. the world organisation for animal health has developed plans to identify and deal with a bioterrorism attack on populations of food-producing animals. canada has established the global public health intelligence network for worldwide monitoring of threats to public health. a major benefit of a less formal global collaboration is the development of networks of trust among knowledgeable scientists and clinicians, who are considered early warning posts for both natural and intentional outbreaks. the one health initiative encourages collaboration between health professionals and is as important for bioterrorism preparedness as it is for management of emerging infectious disease and the global spread of antimicrobial resistance. the management of patients that have been infected during incidents of bioterrorism can be challenging. precautions and treatment regimens for several bioterrorist agents are summarised in panel . although supportive care serves as the basis of management for all agents, treatments for some of the relevant diseases have substantially progressed. the management of inhalation anthrax has advanced since the attack, , , with improvements in critical care, and in treatment of acute lung injury and acute respiratory distress syndrome, severe sepsis, and septic shock. pleural effusions are routinely drained and there are more options for antimicrobial therapy. antibiotics are still recommended for days after exposure or diagnosis, together with the anthrax vaccine. if the vaccine is given concurrently with antibiotic treatment, the period of treatment could be shortened. antibiotics for treatment of other bacterial infection are usually given for shorter periods, since the causative agents do not sequester spores. tularaemia is treated with ciprofloxacin or doxycycline. for smallpox, antivirals such as cidofovir or a related acyclic nucleoside phosphonate analogue appear to be more effective than post-exposure vaccines in preventing mortality, according to experiments in non-human primates infected with monkeypox virus. this suggests that antivirals might play an important role when preparing for a smallpox outbreak. for viral haemorrhagic fevers, ribavirin might have some efficacy in post-exposure prophylaxis. a small-molecule antiviral drug, gs- , has been developed that appears to be effective in treating ebola virus infection. for intentional and other sudden outbreaks of contagious disease, isolation of patients and controlling risks to health-care workers remains extremely challenging, as was noted in the mers coronavirus, sars, ebola virus disease, and avian influenza epidemics. hospital units adequately equipped for isolation are needed, similar to those equipped to care for filovirus-infected patients, with negative pressure air filtration. if facilities are too small for the number of patients, a lower level of isolation with strict barrier nursing should be implemented, and in the event of a very large outbreak, there might be a need to set up isolation facilities in public buildings. in regions with poor infrastructure, it might be necessary to consider treating patients in their homes. people who have died should be regarded as infectious and handled with the same precautions used for patients. burial procedures might have to be modified, but every effort should be made to respect religious practices and traditions of the local culture. quarantining of people who might have been exposed to the infectious agent can be problematic, as was the case in the sars and west african ebola virus epidemics. since the quarantined population includes both people who were exposed and people who were not, the risk of disease transmission is higher. during the ebola virus outbreak in west africa, suspect cases were held until they could be cleared as negative, which took about days pending the pcr results. on a national level, reducing the movement of populations is a sensitive issue, potentially interfering with commerce. closure of schools is an important means of achieving social distancing to reduce spread. whether the public should use masks during an outbreak of a contagious disease is less clear, and the efficacy of the masks is extremely variable. the most important reasons for variable efficacy are differences in facial shape, incorrect application, and duration of use. a substantial proportion of the cases and fatalities in the sars and ebola virus epidemics were among healthcare workers. clear guidelines specific to each agent are available to health-care personnel, public health workers, and emergency workers for the use of masks and personal protective equipment. the national ebola virus training and education center has been established in the usa to train health-care workers and assist hospitals in preparing for patients infected with high hazard virus in the usa and other countries. laboratory workers must be trained to work with dangerous pathogens and wear protective gear. designated threat pathogens must be stored, handled, and transported under a different set the role of vaccines in pre-exposure and post-exposure prophylaxis measures should be in place to protect the population from biological agents likely to be used in an attack before an incident occurs. however, since a bioterrorist incident is likely to be caused by biological agents not covered by routine immunisation, pre-exposure prophylaxis is generally confined to vaccines for military forces, health-care workers, and emergency response personnel. to the majority of the population, only postexposure prophylaxis is relevant. post-exposure prophylaxis for an intentional outbreak would include both those people known to be exposed during the incident and those people who were infected by others. the prophylaxis itself might consist of both vaccines and antimicrobials. when using live, attenuated vaccines, the relatively large proportion of the population who has some form of immunodeficiency has to be taken into account. , monoclonal antibody preparations are now being considered for prophylaxis in select, high risk groups. table summarises pharmacological prophylaxis for tier pathogens. currently, the vaccines that would most likely be used for pre-exposure or post-exposure prophylaxis are the smallpox and anthrax vaccines. since routine vaccination against smallpox was stopped in the s, less than % of the world's population has been vaccinated, and antibody titres usually decline markedly after to years. residual cellbased immunity can persist for many years. , postexposure prophylaxis involves ring vaccination, which requires intensive tracing and vaccination of primary contacts, followed by vaccination of secondary contacts, and finally, vaccination of all people in a defined affected region. for post-exposure prophylaxis of those directly exposed during the incident, vaccination can be effective if given within to days of exposure. since it might take that amount of time to detect the first cases after exposure, the vaccine will generally only be effective for secondary and subsequent contacts. immune-boosting adjuvants and toll-like receptor agonists have the potential to improve the immune response to post-exposure vaccination. serious side-effects are relatively rare , but can affect com pliance. in those cases, lower doses of vaccine might be administered, to provide adequate protection with fewer side-effects. newer smallpox vaccines are in development, including those that could immunise people who have atopic dermatitis. because it is produced in small quantities by collecting antiserum from immunised humans, there might be a shortage of vaccinia immune globulin, used to treat people who would have serious side-effects with the vaccine. one possible solution to this shortage would be to use antibodies against other poxviruses, such as cowpox and monkeypox, because of their cross-protective properties. since naturally occurring inhalation anthrax is extremely rare, there have been safety and immunogenicity profiles of the anthrax vaccine in humans, but the efficacy has only been tested in animal models, and not in clinical trials. [ ] [ ] [ ] the current anthrax vaccine is made from culture filtrates of a toxigenic, avirulent, nonencapsulated mutant of the bacillus anthracis vollum strain, and is administered in five intramuscular doses, followed by annual boosters. the protective, antigen specific memory b cells persist for many years after vaccination and are associated with humoral immunity. serum igg response to the vaccine has been % after the fourth dose. for post-exposure prophylaxis in unvaccinated people, the vaccine should be administered as a three-dose subcutaneous series (at , , and weeks), in conjunction with a -day course of appropriate antimicrobial drugs. it has been given to thousands of us military personnel, and notable adverse events have been rare. the anthrax vaccine is not recommended for pregnant women, although one study with women in the us military, inadvertently vaccinated during pregnancy, did not show evidence of an increase in birth defects. in this study, women received the vaccine in the first trimester and in the second and third trimeseter. more effective anthrax vaccines that require fewer doses are constantly being tested, , such as the neat protein anthrax vaccine, a dual purpose influenza vaccine that protects against anthrax, and a combined anthrax-plague vaccine. apart from the d yellow fever live attenuated vaccine and the junin virus vaccine, no vaccines for haemorrhagic fevers have been licensed. since the west african ebola virus epidemic, new ebola virus vaccines that have been long under development are being used successfully in the epidemic in the democratic republic of the congo. essentially no other licensed vaccines are available for other tier select agents. the previously licensed, formalin-inactivated, whole-bacilli plague vaccine has not proven effective against primary pneumonic plague in non-human primate models, series us military. the vaccine has not been used widely for preexposure prophylaxis and has no place in post-exposure prophylaxis. new vaccines against tularaemia are under development , including one that might provide crossprotection between plague and tularaemia. the investigational pentavalent (abcde) botulinum toxoid vaccine was provided by the cdc for laboratory workers at high risk of exposure to botulinum toxin and it has also been given to military members that are at risk. the botulinum toxoid vaccine produces effective immunity after several months and has no value for postexposure prophylaxis because of the short latent period of the toxins. this vaccine was discontinued in , because of declines in immunogenicity and adverse events. new recombinant botulism vaccines are being developed, in addition to vaccines against glanders and rift valley fever. [ ] [ ] [ ] efforts are ongoing to greatly shorten the time required to develop and produce new vaccines and other immune approaches. improved technologies are important for rapid scale-up and production of new treatment regimens, particularly following an attack with a contagious agent. the largely unpredictable nature of an epidemic initiated intentionally is likely to increase uncertainty and reduce public trust in the authorities. public education and effective risk communication are essential to increase public confidence and improve cooperation and compliance with recommended medical counter-measures. the anthrax vaccine in military populations has caused considerable scepticism regarding the need for, safety, and efficacy of the vaccine. [ ] [ ] [ ] clinicians and public health personnel should have access to up-to-date information, and the general public should be provided with nontechnical information and simple instructions on how to act during an emergency. sandman has proposed that "one should not over-reassure, ack nowledge uncertainty, and share dilemmas". this behaviour would only cause overreaction or panic when new information about the risk is made public. risk communication will be necessary at all stages: before a bioterrorist incident occurs, when an incident is suspected, when it is confirmed, while it is taking place, and in the aftermath. credible and trusted spokespersons, including respected clinicians, scientists, and public servants for a country, should be adequately informed before an incident. during an outbreak, there could be unexpected events, such as atypical presentation of cases, varying responses to treatment (including unusual side-effects), and false positive and false negative diagnoses. the public might lose trust in the authorities if apparently unexposed people become ill. the advent and global distribution of social networking increases the risk of the dissemination of false or misleading information. lastly, a major infectious disease incident will also require flexibility and possible changes of established government policy. environmental detection of biological agents is another area of research that should be developed. to date, most systems of environmental detection have focused on anthrax, as a result of the anthrax attacks. , however, a sensitive and specific set of recombinase polymerase amplification assays for fast screening, detection, and identification of b anthracis in a field setting has recently been developed. the rare occurrence and likely small effect of an aerosol bioterrorist attack limits the practical use of environmental detection to special event venues, public transportation systems, and possibly some government buildings thought to be likely targets. many countries have national stockpiles of drugs and vaccines, for use in the event of a biological or chemical attack, or for serious outbreaks that might achieve epidemic proportions. the usa, for instance, maintains a strategic national stockpile of vaccines and other medical countermeasures. global stores of smallpox vaccines are held by who, in addition to stores held by individual countries. some countries have undertaken active vaccination programmes against smallpox and anthrax in the military and first responder populations. preparedness for bioterrorist incidents requires constant re-evaluation of policies. although there is no evidence that the h n influenza pandemic or the - ebola virus epidemic in west africa were initiated intentionally, the local and international responses revealed strengths and weaknesses in the current state of preparedness for bioterrorist incidents. the ebola virus epidemic spread to a number of countries, with more than cases reported worldwide and a case fatality rate of more than %. imported cases of ebola virus disease were identified in the usa and spain. locally, in the affected countries in west africa, % of the people who died because of ebola virus disease were health-care workers. various shortcomings in the response to the epidemic have been identified since (panel ). accurately predicting the intentional misuse of a biological agent to cause harm is difficult without intelligence data, but several attempts have been made to rationally predict the categories of risk: man-made, natural, accidental, contagious, and non-contagious. series risks. the risk of bioterrorism has called into question some of the dogmas related to eradication of diseases such as poliomyelitis and measles. for example, if polio is successfully eradicated, universal vaccination might have to continue because of the risk of poliovirus being used as a bioterrorist agent. [ ] [ ] [ ] emerging and reemerging infectious diseases will continue to be a threat, but preparedness for bioterrorism is, in many ways, similar to preparedness for naturally emerging disease. all countries should collaborate to address the root causes of terrorism, and develop appropriate preventive strategies. effective preparedness is, in itself, a deterrent to bioterrorism, since it reduces the incentive to use biological weapons by making a country or region a hard target. it is also the cornerstone of consistent and effective responses to naturally occurring epidemics. the abuse of biological agents can be further reduced or discouraged with reliable intelligence and an effective response if it does occur. national and regional resources and capabilities will vary, but all will require infrastructures that are capable of recognising and dealing with a variety of biological agents. the needs of specific populations, such as the paediatric population, pregnant women, elderly people, and people with immunological disorders, must also be addressed. funding for biodefence is crucial to adequate preparation and response to bioterrorist threats. international preparedness for bioterrorism has the dual benefit of strengthening the infrastructure for responding to naturally occurring epidemics of highly pathogenic organisms. lessons from the west african ebola virus epidemic show that health-care providers must always be watchful for unusual presentations of disease, and new and improved approaches must be developed for early detection and response. health-care providers need more effective means of isolating infected patients, and better methods to control the movement of potentially infected people outside of the affected areas. personal protective equipment should be inexpensive and effective, and available to use with minimal training and under harsh environments. protection of health-care workers against infection remains particularly prob lematic, and should be a focus of research and development. the ebola virus epidemic has highlighted the importance of improving the logistics of moving human and material resources in areas with relatively poor infrastructure. risk communication and public education before and during an outbreak need to be improved. more clinical trials should be fast-tracked during development of new vaccines and antiviral drugs. preparedness for a low-risk, high-impact event that is bioterrorism should be monitored constantly, tested in tabletop exercises, , and integrated into the routine functioning of the health system. here it would serve the dual purpose of ensuring that countries are prepared to meet the challenges of controlling epidemics of emerging and re-emerging infectious diseases. msg designed the review, did the literature search and was responsible for writing the manuscript. jld assisted in the literature search, revised the manuscript, and supplied technical expertise. dc assisted in the literature search and revised the manuscript. drf helped design the review, contributed source material, did the literature search, and helped write and revise the manuscript. we declare no competing interests. we searched pubmed and google scholar using the terms "bioterrorism", "sustainable bioterrorism preparedness", "all-hazards infectious disease preparedness", "biological threat agents", "bioterrorism preparedness", "emerging infectious diseases", "smallpox", "anthrax", "plague", "tularemia", "botulism", "hemorrhagic fevers", and "risk communication". we searched national and international reports from the who and us centers for disease control using the terms "bioterrorism", "bioterrorism preparedness", and "emerging infectious diseases". we also completed web searches for "disease surveillance", "infectious disease diagnostics", "medical countermeasures", "emergency healthcare delivery", and "risk management". we focused on academic literature in english and restricted most of our searches to documents published since , with an emphasis on those published after , and included mainly those published since . federal funding for health security in fy a large community outbreak of salmonellosis caused by intentional contamination of restaurant salad bars investigation of bioterrorism-related anthrax, united states, : epidemiologic findings remediation of bacillus anthracis contamination in the u.s. department of justice mail facility total decontamination cost of the anthrax letter attacks bacillus anthracis incident post-traumatic stress disorder symptoms in victims of tokyo subway attack: a -year follow-up study biological warfare and bioterrorism: a historical review the threat of bioterrorism: identifying the unknown. ffi focus biodefense in the st century department of health and human services (hhs). possession, use, and transfer of select agents and toxins-addition of bacillus cereus biovar anthracis to the hhs list of select agents and toxins. interim final rule and request for comments destruction of microbial collections in response to select agent and toxin list regulations implementing the select agent legislation: perfect record or wrong metric? gaps remain in china's ability to detect emerging infectious diseases despite advances since the onset of sars and avian flu health system resource gaps and associated mortality from pandemic influenza across six asian territories ebola: lessons learned and future challenges for europe public health assessment of potential biological terrorism agents responsible conduct by life scientists in an age of terrorism mitigating the risks of synthetic biology. council on foreign relations characterization of the reconstructed spanish influenza pandemic virus chemical synthesis of poliovirus cdna: generation of infectious virus in the absence of natural template expression of mouse interleukin- by a recombinant ectromelia virus suppresses cytolytic lymphocyte responses and overcomes genetic resistance to mousepox labmade smallpox is possible, study shows the de novo synthesis of horsepox virus: implications for biosecurity and recommendations for preventing the reemergence of smallpox epidemiologic determinants for modeling pneumonic plague outbreaks risk assessment and risk communication strategies in bioterrorism preparedness. nato security through science series a: chemistry and biology estimating time and size of bioterror attack threat of a biological terrorist attack on the us food supply: the cdc perspective a large food-borne outbreak of group a streptoccocal pharyngitis in an industrial plant: potential for deliberate contamination german outbreak of escherichia coli o :h associated with sprouts water and bioterrorism: preparing for the potential threat to u.s. water supplies and public health a proteomics assay to detect eight cbrn-relevant toxins in food index case of fatal inhalational anthrax due to bioterrorism in the united states rapid outbreak sequencing of ebola virus in sierra leone identifies transmission chains linked to sporadic cases real-time pcr to identify variola virus or other human pathogenic orthopox viruses pocket dna sequencers make real-time diagnostics a reality sensitive detection of francisella tularensis directly from whole blood by use of the genexpert system comparison of magpix assays and enzyme-linked immunosorbent assay for detection of hemorrhagic fever viruses the early humoral immune response to bacillus anthracis toxins in patients infected with cutaneous anthrax specific, sensitive, and quantitative enzyme-linked immunosorbent assay for human immunoglobulin g antibodies to anthrax toxin protective antigen rapid detection of bacillus anthracis bloodstream infections by use of a novel assay in the genexpert system rapid and sensitive point-of-care detection of orthopoxviruses by abicap immunofiltration rapid viral diagnosis of orthopoxviruses by electron microscopy: optional or a must? striking against bioterrorism with advanced proteomics and reference methods simultaneous immunodetection of anthrax, plague, and tularemia from blood cultures by use of multiplexed suspension arrays the changing face of pathogen discovery and surveillance public health surveillance and infectious disease detection evaluation of a syndromic surveillance system using the wsare algorithm for early detection of an unusual, localized summer outbreak of influenza b: implications for bioterrorism surveillance syndromic surveillance during pandemic (h n ) outbreak internet-based surveillance systems for monitoring emerging infectious diseases digital surveillance for enhanced detection and response to outbreaks if syndromic surveillance is the answer, what is the question? clinical recognition and management of patients exposed to biological warfare agents public health. ethics and the conduct of public health surveillance digital disease detectionharnessing the web for public health surveillance factors influencing performance of internet-based biosurveillance systems used in epidemic intelligence for early detection of infectious diseases outbreaks the internet and the global monitoring of emerging diseases: lessons from the first years of promed-mail social and news media enable estimation of epidemiological patterns early in the haitian cholera outbreak geneva: world health organization the global public health intelligence network and early warning outbreak detection: a canadian contribution to global public health with the changing biological threat…smart international engagement policy would lower cost and increase national security animals as sentinels of bioterrorism agents clinical management of potential bioterrorism-related conditions wisconsin department of health services. infection control and prevention-standard precautions oral tecovirimat for the treatment of smallpox plague: recognition, treatment, and prevention the sanford guide to antimicrobial therapy new therapeutic approaches for treatment of tularaemia: a review centers for disease control and prevention expert panel meetings on prevention and treatment of anthrax in adults antitoxin treatment of inhalation anthrax: a systematic review consequences of delayed ciprofloxacin and doxycycline treatment regimens against francisella tularensis airway infection a single cidofovir treatment rescues animals at progressive stages of lethal orthopoxvirus disease therapeutic efficacy of the small molecule gs- against ebola virus in rhesus monkeys risks to healthcare workers with emerging diseases: lessons from mers-cov, ebola, sars, and avian flu implementing a negative-pressure isolation ward for a surge in airborne infectious patients large-scale quarantine following biological terrorism in the united states: scientific examination, logistic and legal limits, and possible consequences ebola and fda: reviewing the response to the outbreak, to find lessons for the future assessment of the potential for international dissemination of ebola virus via commercial air travel during the west african outbreak transmission of ebola viruses: what we know and what we do not know estimating the size of the u.s. population at risk of severe adverse events from replicating smallpox vaccine influence of population immunosuppression and past vaccination on smallpox reemergence next-generation monoclonal antibodies: challenges and opportunities analysis of historical data suggests long-lasting protective effects of smallpox vaccination duration of neutralizing antibody persisting in thai individuals after childhood vaccination against smallpox a model for a smallpox-vaccination policy can postexposure vaccination against smallpox succeed? immune-boosting adjuvants tlr and tlr agonists improve postexposure vaccination efficacy of live smallpox vaccines risks of serious complications and death from smallpox vaccination: a systematic review of the united states experience adverse events following smallpox vaccination with acam in a military population factors associated with healthcare worker acceptance of vaccination: a systematic review and meta-analysis reducing the dose of smallpox vaccine reduces vaccine-associated morbidity without reducing vaccination success rates or immune responses comparing new-generation candidate vaccines against human orthopoxvirus infections long-term safety of replication-defective smallpox vaccine (mva-bn) in atopic eczema and allergic rhinitis cross-neutralizing and protective human antibody specificities to poxvirus infections randomized, double-blind, placebo-controlled, safety and immunogenicity study of formulations of anthrax vaccine adsorbed plus cpg (av ) in healthy adult volunteers select human anthrax protective antigen epitope-specific antibodies provide protection from lethal toxin challenge protective antigen-specific memory b cells persist years after anthrax vaccination and correlate with humoral immunity lethal factor antibodies contribute to lethal toxin neutralization in recipients of anthrax vaccine precipitated serum igg antibody response to the protective antigen (pa) of bacillus anthracis induced by anthrax vaccine adsorbed (ava) among u.s. military personnel an overview of adverse events reported by participants in cdc's anthrax vaccine and antimicrobial availability program safety of inadvertent anthrax vaccination during pregnancy: an analysis of birth defects in the u.s. military population progress and novel strategies in vaccine development and treatment of anthrax host immunity to bacillus anthracis lethal factor and other immunogens: implications for vaccine design progress toward the development of a neat protein vaccine for anthrax disease a dual purpose universal influenza vaccine candidate confers protective immunity against anthrax a bivalent anthrax-plague vaccine that can protect against two tier- bioterror pathogens, bacillus anthracis and yersinia pestis experimental treatment of ebola virus disease with tkm- : a single-arm phase clinical trial plague vaccines: current developments and future perspectives plague vaccines: status and future intranasal delivery of a protein subunit vaccine using a tobacco mosaic virus platform protects against pneumonic plague protective immunity against lethal f. tularensis holarctica lvs provided by vaccination with selected novel cd + t cell epitopes francisella tularensis live vaccine strain deficient in capb and overexpressing the fusion protein of igla, iglb, and iglc from the bfr promoter induces improved protection against f. tularensis respiratory challenge yopp-expressing variant of y. pestis activates a potent innate immune response affording cross-protection against yersiniosis and tularemia notice of cdc's discontinuation of investigational pentavalent (abcde) botulinum toxoid vaccine for workers at risk for occupational exposure to botulinum toxins recombinant botulinum neurotoxin hc subunit (bont hc) and catalytically inactive clostridium botulinum holoproteins (cibont hps) as vaccine candidates for the prevention of botulism burkholderia pseudomallei and burkholderia mallei vaccines: are we close to clinical trials? safety and immunogenicity of a mutagenized, live attenuated rift valley fever vaccine, mp- , in a phase dose escalation and route comparison study in humans bioterrorism risk communication policy why do uk military personnel refuse the anthrax vaccination? a longitudinal study of uk military personnel offered anthrax vaccination: informed choice, symptom reporting, uptake and pre-vaccination health notes from the field: compliance with postexposure prophylaxis for exposure to bacillus anthracis among u.s. military personnel-south korea rapid detection of bacillus anthracis spores using immunomagnetic separation and amperometry rapid detection of viable bacillus anthracis spores in environmental samples by using engineered reporter phages sensitive and specific recombinase polymerase amplification set of assays for fast screening, detection and identification of bacillus anthracis in a field setting the pitfalls of bioterrorism preparedness: the anthrax and smallpox experiences pandemic preparedness and response-lessons from the h n influenza of global catastrophic biological risks: toward a working definition expert views on biological threat characterization for the u.s. government: a delphi study the risk of bioterrorism re-analysed risks of paralytic disease due to wild or vaccine-derived poliovirus after eradication eradicating polio: a balancing act vaccination against polio should not be stopped vaccines should be kept even if polio is wiped out ebola and zika: cautionary tales medical countermeasure development since : a long way yet to go preparing for biological threats: addressing the needs of pregnant women safety of live vaccinations on immunosuppressive therapy in patients with immune-mediated inflammatory diseases, solid organ transplantation or after bone-marrow transplantation-a systematic review of randomized trials, observational studies and case reports biosecurity: assessing the bioweapons threat a plague on your city: observations from topoff shining light on "dark winter key: cord- -dde nlhh authors: antabe, roger; ziegler, bianca rosa title: diseases, emerging and infectious date: - - journal: international encyclopedia of human geography doi: . /b - - - - . - sha: doc_id: cord_uid: dde nlhh emerging and infectious diseases have persisted as leading causes of global morbidity and mortality. caused by pathogens including bacteria, viruses, parasites, or fungi, they are known to pose serious health threats to the world's population dating back to ancient egypt. in the th century alone, infectious diseases were responsible for decimating – % of the world's population. the discovery of vaccines, coupled with improved sanitation, hygiene, and health care, witnessed the eradication of several infectious diseases, although some have resurfaced or are resurfacing since the latter part of the th century. while geography partly define hotspots for emerging and infectious diseases, low socioeconomic development, poverty, and underfunded health care systems remain driving forces for the reoccurrence of these diseases among vulnerable populations who experience material deprivation. to eradicate infectious diseases, a global response will have to prioritize the allocation of resources by way of expertise and technology to areas that are most affected. furthermore, an effective surveillance system, and a rigorous vaccine deployment regime targeting vulnerable persons and regions is desirable in mitigating the impacts of these diseases. infectious diseases are illnesses, which are spread directly or indirectly, from person to person. infectious diseases can be classified under one of the following origins: zoonotic, vector-borne, or drug-resistant. the majority (over %) of emerging and reemerging infectious diseases are caused by bacteria, viruses, parasites, or fungi, which were passed from animals to humans, known as zoonotic pathogens. zoonotic pathogens, which originated in wild life, are the causative agents for diseases such as severe acute respiratory syndrome (sars) and ebola. as a result, zoonotic infectious diseases have led to epidemics that drew attention to the interactions between humans and wildlife, including the domestication of animals, food production and intensive farming, moving animals and plants to foreign environments, urban sprawl and migration, and climate change and deforestation. vector-borne infectious diseases are spread between humans and animals by vectors such as mosquitoes and ticks. these diseases, including malaria and lyme disease, impact specific geographic regions. tropical and subtropical areas are disproportionately affected, and the poorest and most vulnerable tend to face the highest burden of these diseases. the history of these diseases includes widespread illness and high mortality rates affecting populations on a global scale. the history of infectious diseases goes back as far as smallpox being found on egyptian mummies and the plague infecting and killing - % of the world's population in the th century. however, by the end of the s, most infectious diseases were considered a health issue of the past. with improved public health measures including sanitation processes and sewage treatments, as well as improved healthcare services and the introduction of antibiotics and vaccination protocols, the prevalence of infectious diseases appeared to be declining. as a result, health care professionals and researchers turned their attention and available resources toward the rising issue of chronic disease. yet, during the last quarter of the th century, infectious diseases began to reemerge with new and resurgent diseases cited as causes of morbidity and mortality for large populations. since this time, infectious disease rates have continued to rise, new diseases have emerged, and previously eradicated diseases have reemerged. the emergence and reemergence of infectious diseases has mostly been attributed to the unintentional consequences of human behavior, activities, and development. the threat of emerging and infectious diseases (eids) to human health, society, and the global economy is increasing as infectious diseases continue to evolve and spread. to illustrate, in alone over million people died from hiv, and million people contracted malaria. furthermore, the sars outbreak in resulted in death for out of the infected patients within months, with the survivors experiencing ill health and, in some cases, disability. clearly, infectious diseases such as sars place added stress on healthcare systems in affected countries as these systems must mobilize resources to effectively deal with the epidemics. they also create tensions within communities and decrease economic prosperity. for example, travel, tourism, and retail sales dropped dramatically during the sars epidemic, especially in asia where the disease originated and was most prevalent. globally, the economic impact of sars was estimated to be $ - billion usd. emerging and reemerging infectious diseases are largely preventable, and yet with their profound impact and increasing prevalence, they remain a threat to global health, which must be addressed. humans and pathogens have always been in a dynamic state of interaction; however, current trends reveal that this relationship has become unbalanced as human activities are causing pathogens to appear and spread at an increasingly alarming rate. as aforementioned, the st century has seen a rise in infectious disease, largely due to the societal and global trends that are reinforced by an increasingly technological, globalized, and interconnected world. between and , infectious diseases, such as tuberculosis, sars, and h n , emerged or reemerged in both the developed and developing world. these diseases caused the death of an estimated million people per year, with % of these deaths occurring in the concentrated region of south-east asia and the sars outbreak in china affecting other countries. by the end of april , a measles outbreak in the united states affected people, % of whom were unvaccinated. similarly, in the european region, the who reported a total of , measles cases by march , with related deaths in the preceding year. additionally, the world is still battling with diseases such as hiv/aids, malaria, ebola, h n , and dengue fever. each year, approximately million travelers transport pathogens between places and provide a means for infectious disease to spread across borders. the increased globalization of the world has resulted in travel and trade being facilitated on a global scale, which has caused infectious diseases to spread more easily to countries that were previously unaffected. moreover, there is a higher prevalence of infectious diseases in low-income and developing countries due to increased poverty, social inequality, and conflict, leading to increased susceptibility and a decreased capacity to effectively provide sufficient treatment. additionally, environmental variability due to climate change has lengthened the season in which vectors are present and impacted agricultural practices, allowing for increased transmission. furthermore, vectors have also become resistant to numerous pesticides making it harder to control the spread of the diseases, which they carry. another important issue that is feeding the emergence and reemergence of infectious diseases is the issue of drug resistance to treatment. increasingly microorganisms are evolving and adapting which makes it difficult to treat these diseases, thus increasing their rate of transmission. many factors lead to drug-resistant infectious diseases including antibiotic misuse (such as not finishing an entire dose of antibiotics), over-prescription of antibiotics, and the use of antibiotics for agriculture and animal production that can lead to antimicrobial resistance. moreover, vaccinations not being kept up to date or not being administered to the majority of the population to maintain herd immunity has led to drug-resistant diseases, which have evolved and rendered current vaccines ineffective. many infectious diseases are evolving more quickly than effective treatments can be created and tested, as a result, as drug-resistant diseases continue to increase in prevalence, their threat to human health continues to increase. other health trends have also contributed to the infectious disease epidemic. an aging population with a higher life expectancy has resulted in an increased proportion of the population being elderly, thus susceptible to infectious disease, and less likely to have the capacity to combat sickness. much of the world also suffers from multimorbidities, with chronic diseases such as diabetes and heart disease on the rise, once again increasing susceptibility. similarly, the hiv/aids epidemic has resulted in a large immunocompromized population who are vulnerable to contracting multiple infectious diseases. the prevalence of hiv/aids declined globally by % between and . however, as of , . million people were infected globally with hiv/aids, with africa having the highest proportion of hiv/aids cases, a trend which like the general trend of emerging and reemerging infectious disease prevalence is not declining quickly or equitably enough. eids have been posited by researchers to persist as a global health concern for the next couple of decades particularly in poor and under-resourced settings in the global south. however, efforts at the global level yield the potential to address incidences of eids. in line with these objectives, there have been proposed strategies to address current trends of eids. the sustainable development goals (sdgs), created in , identified infectious diseases as a priority area for health policy; sdg . set out to end numerous infectious disease epidemics, such as hiv/aids and malaria by . the sdgs posit that through increased surveillance and allocating more resources and funding to this health issue, diagnostic and treatment programs will be improved, and the epidemic of emerging and reemerging infectious diseases will once again begin to decline. in the meantime, there is urgency in building a global network consisting of a team of specialists in eids. this network will be hinged on improved communication surrounding risks of outbreaks in global hotspots that are difficult to access. this will be foundational to addressing outbreaks, as the who has observed that areas with more frequent outbreaks and endemic cases of reemerging infectious diseases such as hiv, hbv, and ebola tend to be in low-and middle-income countries. budget constraints in allocations for emergencies and laboratory research may therefore be undermining efforts at total eradication of eids in these areas. the lack of coordinated effort in understanding the nature and extent of outbreaks among specialists may be a major contributory factor where isolated cases of eids easily escalate to full-blown epidemics. therefore, a global network of specialist and experts is key in designing future responses to eids. the introduction of vaccines led to the eradication of major infectious disease such as smallpox and measles that plagued earlier centuries as leading causes of death. currently, immunizations alone are estimated to avert two to three million global annual deaths. targeting populations at increased risk, including high-density areas and those in the geographies of elevated risk of zoonosis, with vaccines will lead to a substantial decline in new outbreaks. the trial of ebola vaccines marks a major milestone in reducing mortalities associated with the current perennial outbreak of the ebola virus in west and central africa. as well, after an initial trial of malaria vaccines ended in , the who has recommended an expanded trial in three countries in sub-saharan africa, based on this trial, deployment of this vaccine could be sanctioned across the globe to eradicate malaria. similarly, discussion on the deployment of preexposure prophylaxis (prep) and post exposure prophylaxis (pep) for hiv in both endemic and nonendemic contexts in the global south and north respectively, and among exposed populations, holds the key to achieving sections of the sdgs and the unaids - - . it is worth noting that, an effective surveillance system of people and areas at risk, will lead to early detection of instances of microbial and drug resistance in vaccines and treatment of eids. particularly for eids that are asymptomatic, providing access to testing and screening services is desirable to obtain an early warning and to devise a response to outbreaks. this is largely dependent on available resources at the locale by way of laboratory or clinical technology that will be most effective in the timely detection of new incidences of eids. for instance, only % of people with viral hepatitis have been diagnosed due to limited access to testing services. given that a large percentage of eids are concentrated in countries where governments are under-resourced in responding to outbreaks, well-equipped testing and screening facilities for the public at risk are needed. currently in low-income settings where health infrastructure is under-developed, point-of-care (poc) diagnostics have emerged with the potential for inexpensive, effective, and timely diagnoses of infectious diseases. this also calls for the development and deployment of diagnostic techniques that are suitable for specific regions or geographies. in view of the disproportionate global burden of infectious diseases where some regions are more prone relative to others, a key consideration in eradicating eids may be the reallocation of resources, including expertise and clinical technology to areas that are most impacted. currently, areas least affected by eids receive a higher allocation and concentration of resources to respond to outbreaks. in this regard, in the context of developing countries, hbv, hiv, and ebola have persisted largely due to the absence of funding to secure vaccines and treatment for populations at heightened risk of infection. this is in sharp contrast to the context of the global north where eids are less prevalent but have control of global resources in eradicating infectious diseases. zoonotic pathogens account for . % of all eids, of which an estimated . % are contracted through contact with wildlife. in this context, understanding human contact with wildlife and how to minimize it in hotspots with highly biodiverse fauna can help contain new incidences. moreover, the limited outbreak of eids in areas where conservation efforts have drastically reduced anthropogenic contacts with wildlife means more conservation efforts will lead to a substantial decline of eids. it is also crucial to institute smart surveillance in areas of high population density to avoid new strains of infection from reemerging. see also: epidemiological transition; medical geography. emerging and re-emerging infectious diseases: the third epidemiologic transition emerging infectious diseases: public health issues for the st century global trends in emerging infectious diseases point-of-care testing for infectious diseases: past, present, and future thomas hepatitis b virus epidemiology, disease burden, treatment, arid current and emerging prevention and control measures geography, ecology and emerging infectious diseases emerging infectious diseases: threats to human health and global stability factors in the emergence of infectious diseases the epidemiology of hiv and other sexually transmitted infections in african, caribbean and black men in toronto developments in the diagnostic techniques of infectious diseases: rural and urban prospective shweta sustainable development knowledge platform. www.who.int/whr/ /media_centre/press_release/en/ world health organization www.who.int/news-room/fact-sheets/detail/immunization-coverage world health organization: immunization coverage key: cord- - w wf authors: vignuzzi, marco; lópez, carolina b. title: defective viral genomes are key drivers of the virus–host interaction date: - - journal: nat microbiol doi: . /s - - -y sha: doc_id: cord_uid: w wf viruses survive often harsh host environments, yet we know little about the strategies they utilize to adapt and subsist given their limited genomic resources. we are beginning to appreciate the surprising versatility of viral genomes and how replication-competent and -defective virus variants can provide means for adaptation, immune escape and virus perpetuation. this review summarizes current knowledge of the types of defective viral genomes generated during the replication of rna viruses and the functions that they carry out. we highlight the universality and diversity of defective viral genomes during infections and discuss their predicted role in maintaining a fit virus population, their impact on human and animal health, and their potential to be harnessed as antiviral tools. v iruses are remarkably resilient microorganisms able to adapt and survive in the complex host physiological and immune environment. accumulating evidence demonstrates that viruses can generate modified forms of their genome during replication as tools to adapt to environmental challenges. while the standard genome encodes all the viral proteins required for sustaining viral replication, viral variants contain random mutations that can serve to enhance the ability of the virus to adapt to new conditions . in addition, defective viral genomes (dvgs) and an expanding family of sub-viral particles (box ) that result from either small mutations or drastic truncations and modifications of the viral genome render the virus unable to complete a full replication cycle in the absence of a helper full-length virus to complement the functions lost. dvgs were identified by preben von magnus in the late s as incomplete influenza viruses able to interfere with the replication of the wild-type virus . more than two decades after their discovery, alice huang and david baltimore coined the term defective interfering (di) particles, or dips, to define viral particles that contain normal structural proteins but only a part of the viral genome. in addition, they stipulated that dips can only replicate in the presence of helper virus and that they interfere with the intracellular replication of non-defective homologous virus . huang and baltimore theorized that dips play a critical role in determining viral pathogenesis. follow-up studies using viruses grown to contain either a large content of dips, or depleted of them, revealed that dips reduced virulence in vivo , , induced high levels of interferon (ifn) during infections in vitro [ ] [ ] [ ] , and promoted viral persistence in vitro [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and in vivo , . however, despite their ubiquitous presence and important functions, the lack of appropriate technology to identify dips in infections in vivo led to a widespread belief that dips and their dvgs were largely a product of in vitro virus replication and were not relevant in natural viral infections , . by the late s, the study of dvgs had slowed down drastically and was limited to their use as tools for studying viral replication or as potential antivirals. today, dvgs have been described in most rna viruses (table ) and technological advances have contributed to establishing their role as de facto danger signals for triggering of antiviral immunity in many infections. in addition, we are beginning to appreciate their impact on the clinical outcome of natural infections and on the evolution of viruses. moreover, we are witnessing rapid advancements in the understanding of the molecular mechanisms that regulate the generation of dvgs and those explaining their paradoxical roles in promoting antiviral immunity and viral persistence. here, we review evidence accumulated over more than half a century of observations on dvg generation and activity during rna virus infections. we highlight recent advances that illustrate their critical impact on viral dynamics and evolution during both acute and long-term virus-host interactions. see box for a glossary of relevant terms. different types of dvgs are defined by the type of genomic alterations present. next generation sequencing (ngs) has revealed a large variety of dvg species present in some infections, and recent studies have begun to elucidate the distinct functions of these different dvgs. point mutations, hypermutations and frame shifts. while the first dvgs to be identified and distinguished from the wild-type fulllength viral genome lacked large parts of the genome [ ] [ ] [ ] [ ] [ ] , there are a number of dvg types that do not involve drastic genomic alterations. point mutations in rna viruses can result in detrimental alterations because of the highly constrained nature of their genome organization. indeed, a majority of randomly introduced mutations are either lethal or confer a significant fitness cost, as observed for vesicular stomatitis virus (vsv) , poliovirus and influenza virus . while it is inherently understood that detrimental mutations give rise to defective genomes, such genome types have historically not been considered dvgs. a genome harbouring a detrimental mutation in a structural protein could replicate, but not assemble properly; while a detrimental mutation in the replicase would yield a genome that can produce proper structural and assembly proteins, but could not replicate. either of these genomes could potentially hijack the lacking functions from (and thus, interfere with) a fulllength co-infecting virus. indeed, studies where mutation rates are increased to perturb a viral population from viability to non-viability, revealed that the defective rnas that appear prior to population marco vignuzzi and carolina b. lópez * viruses survive often harsh host environments, yet we know little about the strategies they utilize to adapt and subsist given their limited genomic resources. we are beginning to appreciate the surprising versatility of viral genomes and how replicationcompetent and -defective virus variants can provide means for adaptation, immune escape and virus perpetuation. this review summarizes current knowledge of the types of defective viral genomes generated during the replication of rna viruses and the functions that they carry out. we highlight the universality and diversity of defective viral genomes during infections and discuss their predicted role in maintaining a fit virus population, their impact on human and animal health, and their potential to be harnessed as antiviral tools. dvg diversity during infection. historically, it was thought that only certain viruses generated dvgs and that only one or a few distinct dvgs existed for any given virus. most studies originally relied on high multiplicity of infection (moi) passaging conditions that favoured the emergence of larger deletions (and thus, smaller dvgs) that could rapidly outcompete the longer full-length genomes owing to reduced replication times. furthermore, these works relied on methods with low sensitivity of detection (such as visualization and purification on agarose gels) that would only isolate the most abundant dvgs. however, evidence for populations of distinct species of dvgs in a single infection has been reported since the s , . we now know the diversity of dvgs to be much larger than initially appreciated and that certain dvgs are better than others at reaching high abundance, likely by retaining certain properties that confer replication advantage such as packaging signals and replication elements, or by acquiring the ability to interfere with the replication of other variants. ngs has revealed that dvgs are present in virtually any and every virus population, and that the diversity of dvgs is immense; however, the relative abundance of any given deletion or rearrangement varies, likely indicating that a variety of factors drive dvg generation and accumulation. the use of single-cell sequencing technology will allow precise quantification of the frequency and diversity of dvgs in an infected cell and will provide data on the distribution of dvg variants among a cell population. since most ngs alignment tools filter out reads with more than two mismatches, reads corresponding to deletion breakpoints or rearrangement junctions are rejected from a typical virus genome alignment. a re-analysis of rejected reads would identify these dvg hallmarks. with growing interest in this neglected part of the viral population, informatics tools such as virema , di-tector and vodka are emerging. these tools re-examine reads that potentially harbour jumbled or rearranged viral sequences and have enabled a broader appreciation of dvg diversity. a current challenge with ngs data is to determine how best to differentiate between true dvgs and background error of ngs, how to quantify the relative abundance of any given dvg with respect to full-length in addition to dvgs, a growing number of viral particle variants and sub-viral agents have been discovered in viruses of plants, arthropods and mammals. these include viroids, satellite viruses, virophages and viral-like extracellular vesicles (vlvs). the definition of these entities seems, at times, blurry, due to their largely intersecting properties. in general, sub-viral agents, similar to dvgs, depend on complementation with the standard virus to replicate and spread. however, differences in their requirements for complementation, target virus and sequence identity with their helper virus are used to sub-classify them. here, we provide current definitions of these sub-viral entities and highlight those aspects that differentiate them from dips and their dvgs. satellite viruses were originally described in plant viruses as linear or circular rnas ( - , nt long) that require a helper virus to propagate but are unrelated in sequence to the helper virus. satellite viruses are generally dispensable for the replication of the helper virus, with some exceptions , . satellite viruses differ from satellite rnas in that they encode a protein that packages the satellite rna into virions. satellite rnas can interfere with the replication of its helper virus and either attenuate or exacerbate disease. an example of a satellite virus is the satellite tobacco necrosis virus . this positive-sense, singlestranded rna satellite virus suppresses the replication of its helper virus and ameliorates tobacco necrosis virus symptoms . viroids differ from satellite viruses in that they do not encode any protein, do not require helper virus for replication, and are not encapsidated. viroids have a circular rna genome ( - nt long) that is highly complementary and structured, and are adapted to carry out their complete life cycle as a result from interactions with the host cell machinery , . virophages are - kbp long dsdna viruses that normally produce icosahedral particles and parasitize from giant dsdna viruses (mimiviruses and others) for propagation. virophages infect the giant virus factory, thereby harming the giant virus . the first virophage discovered, sputnik, was found together with acanthamoeba castellanii mamavirus and interferes with its propagation . several other virophages, as well as a large number of virophage candidates, have been identified since then , , . recent studies show that virophages could resemble bona fide dna viruses and their reclassification as their own viral family has been proposed . vlvs are produced during viral infection and, in contrast to other extracellular vesicles, contain viral proteins and nucleic acids but lack capsid protein or viral genomes and, therefore, are not infectious. vlvs have been described in both rna and dna viruses, including herpes simplex virus- , hepatitis c virus and kaposi's sarcoma-associated herpes virus. vlvs play functional roles during viral infections by facilitating communication among cells and enhancing viral infection [ ] [ ] [ ] . continued virus, and how best to normalize between samples and between sequencing runs-problems that are similar to transcriptome analysis of genetic isoforms. furthermore, with increasing data sets and samples, it is becoming evident that dvg species are not necessarily the same if generated in different host and cell types, and the factors dictating these differences remain to be uncovered. dvgs have been long considered the result of stochastic mistakes introduced by the viral rna polymerase that lacks proofreading activity. however, new evidence suggests that additional factors control dvg generation, opening the possibility of manipulating their generation for therapeutic purposes. random products or encoded in the viral genome? while dvgs are generated by many viruses, the molecular mechanisms that govern their generation are poorly understood. a predominant theory is that dvgs arise from random errors that occur during viral replication at high viral titers due to the combination of a lack of proofreading activity of the viral polymerase and the presence of lower fidelity variants that favour the generation of deletions. in support, analyses using deep sequencing approaches revealed that multiple species of dvgs are generated during infection. for example, in infections with flock house virus, a positive-sense (ps)rna virus, clickseq and nanopore sequencing identified a large and seemingly random population of deletion dvgs early after infection . in addition, sequencing analysis of nasopharyngeal samples from influenza virus-infected humans or infections in vitro with human metapneumovirus or measles virus (mev) revealed multiple dvg species in these infections [ ] [ ] [ ] . however, different from deletion and point mutation dvgs, copy-back dvgs are frequently found in discrete dominant populations in an infected cell or tissue, and the same copy-back dvg seems to arise in independent infections with the same parental virus , or during infections with different virus strains . the demonstration of hotspots for the generation of copyback dvgs from respiratory syncytial virus (rsv) and the identification of specific nucleotides that determine where copy-back dvgs rejoin further demonstrate that the generation of copy-back dvgs is not completely random, but instead that specific sequences encoded in the viral genome direct or facilitate their formation in some infections, dvg generation is not a completely stochastic process and, instead, virus-encoded sequences favour the production and/or amplification of predominant dvgs. it remains to be determined whether conservation is a property of certain dvg types and which specific sequences and/or rna structures lead to dvg generation in these conditions. a number of viral proteins are implicated in the generation of dvgs, the best studied being the viral rdrp. engineered viral polymerases with a decreased fidelity and an increased mutation rate produce highly attenuated viruses , which, in many instances, correlate with enhanced production of dvgs - (fig. a) . although the mechanism mediating dvg generation by mutant rdrp remains speculative, a few models are emerging. for example, in viruses with low-fidelity rdrp, such as sindbis virus or tombusvirus, an increased production of dvgs correlates with an enhanced rate of viral rna recombination , . in addition, during influenza virus infection, variations in the elongation capacity of the polymerase associate with differential generation of dvgs . a role for the polymerase multimerization activity was also recently proposed as a driver of dvg generation during influenza virus infection . viral proteins implicated in the regulation of viral transcription and replication are also associated with the generation of dvgs. mutations in the influenza virus nuclear export protein (nep, also known as ns ), which regulates the synthesis of complementary rna, result in enhanced dvg production . similarly, deletion or mutations of the paramyxovirus c proteins that regulate template switching from antigenomic to genomic replication result in enhanced copy-back dvg production , (fig. a) . curiously, in infections with influenza virus lacking nep or paramyxovirus lacking c, dvg production is observed in conditions that normally restrict the generation of dvgs, such as infections at low moi. it is possible that the enhanced production of copy-back dvgs in these situations is related to higher template availability or to an indirect effect of viral polymerase activity, but the precise mechanism remains to be established. in addition, a single amino acid mutation in the sendai virus (sev) nucleoprotein that reduced the density of the ribonucleprotein associates with dvg generation (fig. b) . lastly, in lymphocytic choriomeningitis mammarenavirus (lcmv) infection, the ppxy domain encoded within the matrix protein drives the production of viral particles containing defective genomes, but not those containing standard genomes (fig. c) . replication-driven template-switching. rna recombination is a major driver of deletion dvg formation. a predominant model proposes that sequences at the break point or structural signals in the template rna promote the replicase to switch to the acceptor rna and resume synthesis (fig. d) . replicase-driven recombination was proven in biochemical assays using rdrps of a large number of rna viruses [ ] [ ] [ ] . variations of this model include the forced template switch mechanism in which the replicase switches template after encountering the ′ end of the template generating headto-tail rna dimers. if the templates are dvgs, these would lead to head-to-tail dvg dimers. the ′ end could also be modified by endo-or exo-nucleases, leading to new versions of these genomes . rna editing as a driver of dvg diversity. editing of viral rna leads to hypermutations and can result in the generation of dvgs, as reported in persistent measles infections . in addition, a high rate of viral adenine-to-guanine (or uracil-to-cytosine) rna editing by adenosine deaminase acting on rna occurs in dvgs from vsv, human metapneumovirus and mev , , . in some cases, rna editing regulates the immunostimulatory potential of dvgs . however, the impact of dvg editing on viral replication seems to be virus-specific . whether dvgs have a higher rate of editing than the standard viral genome, as well as the impact of editing generated diversity in dvg evolution and selection, remains to be determined. dvgs have three well-described functions that relate to their role in pathogenesis: interference with standard viral replication, immunostimulation and establishment of viral persistence (fig. a) . interference with viral replication and viral production. dvgs were discovered during the search for factors responsible for reduced infectivity of influenza virus after passages at high titers . dvgs with the ability to interfere with the replication of their parental virus both in vitro and in vivo are found in most positive and negative-sense (ns)rna viruses , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . dvgs accumulate at higher rates than full-length viral genomes in co-infected cells due to their shortened length and, in the case of copy-back species, their highly efficient flanking trailer promoters , . a predominant theory for how dvgs interfere with the replication of standard virus is based on the observed competition between defective and full-length viral genomes for viral components needed for replication (fig. b) . as dvgs accumulate to high levels, it is predicted that they can directly interfere with helper virus replication by monopolizing the viral polymerase and/or competing for structural proteins , . it should be noted that while most evidence supports the notion that the shortest dvgs with largest deletions are best able to outcompete full-length virus because their much smaller size can be more rapidly replicated, most of these studies consist of high moi cell culture conditions which favour competition dynamics based on replication kinetics. the question arises as to whether a longer deletion dvg that retains more coding sequence, yet contains mutated proteins (presumably replicating faster than full-length genomes, yet slower than the shortest dvgs), could be a better competitor of wild-type virus in certain conditions because it additionally expresses defective proteins that, in turn, interfere with wild-type proteins (for example, in multi-component structures such as capsids or replicases). furthermore, recent studies have demonstrated that dvgs and full-length viral genomes dominate in different cells during infection, dips. defective interfering particles. viral particles containing a fraction of the viral genome able to replicate only in the presence of helper virus and interfere with the intracellular replication of non-defective homologous virus. dvgs. defective viral genomes. viral genomes with defective ability to replicate in the absence of a co-infecting standard virus. viral genomes can become defective due to mutations, deletions or a variety of gene rearrangements. rdrp. rna-dependent rna polymerase. viral enzyme that copies the viral rna during viral replication. tips. therapeutic interfering particles. synthetic dvgs with strong interfering activity proposed as therapeutics to outcompete standard viruses. conferring distinct functions to different infected cells , . while cells dominated with full-length viral genomes are the predominant producers of viral particles containing either full-length or defective genomes, cells enriched in dvgs do not produce many particles of any species . these data highlight the need to consider the single cell versus population level impacts of dvgs during interference. triggers of antiviral immunity. dvgs, especially those of the copy-back type, strongly induce the expression of type i and iii ifns, tumour necrosis factor (tnf), interleukin (il)- , il- β and other pro-inflammatory cytokines, and are the primary stimuli of antiviral immunity in many infections [ ] [ ] [ ] , , , [ ] [ ] [ ] (fig. c) . in addition, dvg stimulation optimizes the antigen presentation capacity of antigen-presenting cells , . accumulating evidence indicates that the immunostimulatory activity of dvgs is maintained in vivo and during natural infections in humans. increased survival of infected mice in infections containing dvgs have been reported for multiple viruses , , . in mice infected with the respiratory viruses sev, influenza or rsv, ifns and pro-inflammatory cytokines are strongly induced only after dvgs have accumulated to detectable levels , . detection of dvgs in respiratory secretions of children infected with rsv correlates with expression of antiviral genes , and highly pathogenic influenza virus isolates that fail to induce potent antiviral responses in humans have an impaired ability to generate dvgs . immunostimulatory dvgs can be recognized by pattern recognition receptors (prrs), including toll-like receptors (tlrs) and rig-i-like receptors (rlrs). while tlr signalling is not essential for production of type i ifns in vitro in response to dvgs, rlr signalling is required , , [ ] [ ] [ ] [ ] [ ] . sev copy-back dvgs are stronger immunostimulators than deletion dvgs and are among the strongest known inducers of the antiviral response. therefore, much of what we know of dvgs immunostimulatory activity is based on the study of sev copy-back dvgs. copy-back dvg rna binds rig-i , , and dvgs from sev, mev and rsv viruses strongly stimulate rig-i-dependent signalling , , . rig-i is triggered by binding of ′ di-or triphosphates present on uncapped rna or short regions of double-stranded (ds)rna. phosphatase treatment suppresses the ability of in vitro-transcribed copy-back dvgs to trigger ifn production following transfection supporting the role of rig-i in dvg sensing , , . sev dvgs can also associate with melanoma differentiation-associated protein (mda ) , , but the role for mda in sensing other dvgs is less clear , . other cellular proteins can also associate with dvgs. for example, mev dvgs associate with the dsrna binding protein named protein activator of the interferon-induced protein kinase to optimally activate rig-i . dvg-induced rlr signalling is not simply a result of higher viral rna content in the infected cells, as increasing the amount of dvg-deficient virus does not increase the ifn response , , . importantly, efficient sensing of copy-back dvgs occurs even in the presence of virus-encoded antagonists of the cellular sensing pathways , . these observations suggest that unique features of dvgs favour their detection during infection. the predicted long dsrna stretch formed by the reverse complementary ends of copyback dvgs was thought to be a critical factor in their immunostimulatory activity , , . however, recent evidence demonstrates the existence of additional features in dvgs that have a larger impact on their immunostimulatory potential. structural modelling identified a nucleotide (nt)-long stem-loop motif (dvg ) in a sev dvg- , a well-characterized and potent immunostimulatory copy-back dvg, which was absent in the sev genome and spans the unique junction formed between the genomic break and rejoin points that form this copy-back dvg. deletion of dvg reduces the dvg immunostimulatory activity, while introduction of the motif into an immunologically inert rna improves its ability to induce the expression of type i ifns and ifn-stimulated genes . dvg acts in concert with the ′-triphosphate motif to activate rig-i and allows for enhanced rlr polymerization, a marker of activation . dvg lose immunostimulatory potential . failure to detect dsrna through immunostaining during sev infection and normal immunostimulatory activity following disruption of the complementarity between the ′ and ′ ends of copy-back sev dvgs further support the notion that the predicted long complementary ends of copy-back dvgs have a minor role in the onset of anti-viral immunity. it remains to be determined when and how dvg - is exposed during infection, and whether similar motifs are present in other highly immunostimulatory dvgs. dvg immunostimulation is also an important factor in the modulation of infections in insects. similar to the rig-i and mda prrs in mammals, insects sense viral rna through the protein dicer- . this protein processes the viral rna into small interfering rnas that protect insects from re-infection with the same virus . dvgs are the primary targets for dicer- (ref. ). these observations indicate that the immunostimulatory activity of dvgs is widespread and may have a significant impact on the spread of pathogens within and across species. , , as well as one study reporting dvgs in the brain of human patients who had died due to post-mev subacute sclerosing panencephalitis . dips and full-length viruses cycle asynchronously in many persistent infections in vitro , and in vivo , . cycling occurs in a predictable pattern and has been mathematically modelled using variations of the predator-prey model . a theory to explain the asynchronous cycling of full-length viral genomes and dvgs during persistency was put forward in (ref. ). this theory, which is based on the interference effect of dvgs on the replication of the full-length genome, proposed the following: dips accumulate during virus replication until they reach high concentrations and become predominant. in this condition, dvgs interfere with the replication of the full-length viral genome by competing for essential replication machinery, driving a reduction in the full-length virus. during this process, some previously uninfected cells are infected by standard virus and re-initiate the cycle. interestingly, in some persistent infections the amount of dips appears constant . what drives these cyclic patterns in some viruses but not others, and whether host factors such as the infected cell type influences the cycling pattern, remain unknown. recent evidence indicates that the mechanisms involved in the establishment of persistence are more complex than simple intracellular competition for the replication machinery among different types of viral genomes . using rna in situ fluorescent hybridization, xu et al. showed that during infection with sev or rsv containing dips, there is heterogeneity in the content of viral genomes in the infected population . while some cells are enriched in dvgs, others are enriched in standard full-length viral genomes. the mechanisms for this heterogeneity are currently unknown, however, striking functional differences among these cell populations are beginning to emerge , . cells enriched in dvgs engage the rlr sensing pathway and produce ifns and other pro-inflammatory molecules, including tnf. in addition, these cells induce a pro-survival program, also dependent on signalling through the rlr pathway. these programs protect dvg-high cells from tnfmediated death, while cells lacking dvgs die during infection. surviving dvg-high cells can be propagated for months as a persistent infection. this mechanism provides an explanation for the paradoxical stimulation of both antiviral immunity and establishment of persistence by dvgs. it remains unclear how the enhanced survival of dvg-high cells leads to persistence, and how this survival fits into the cycling of dvg and standard virus observed in many infections. cycling may occur at the intracellular level (where each infected cell goes through cycles of standard viral genome or dvg enrichment driven by competition and interference with the viral replication machinery) and/or at the population level, where the strong interfering and immunostimulatory activities of dvgs make them attractive candidates for vaccine adjuvants and antivirals , . the ability of dips to interfere with the replication of standard viruses and diminish virus-associated disease has been extensively demonstrated in mice during infection with virus stocks containing dips , , , , , even when the vaccine contained standard virus inactivated by ultraviolet treatment . a similar protection has been reported in ferrets vaccinated with an influenza vaccine containing only dips . synthetically engineered dips with strong interfering potential, or 'therapeutic interfering particles (tips)' , have been more recently proposed as a possible strategy to control viral infections. tips would theoretically replicate faster than the wild-type virus and therefore outcompete the virus hindering spread and transmission. tips would have the advantage of being active only in organisms already infected with the wild-type virus due to their dependence on a helper virus to replicate , . although still in the exploratory phase, it remains to be determined how tips would impact virus persistence, the generation of adaptive mutations and the generation of new infectious viruses by complementation. whether protection elicited by natural or synthetic dips is due to direct interference with the standard virus replication or through strong immunostimulatory ability of dvgs is unclear. sev copyback dvgs augment the antigen presentation capacity of mouse and human dendritic cells, resulting in enhanced activation of t cells . in addition, experimental vaccines against influenza virus and rsv adjuvanted with in vitro-transcribed sev dvgs delivered subcutaneously, intramuscularly or intranasally show improved antibody production and increased protection from virus challenge , . sev dvg-derived oligonucleotides (ddos) containing the immunostimulatory motif dvg are effective adjuvants able to bias the humoral and cellular responses against inactivated virus and protein vaccines towards type i immune responses including antibodies of the igg a/c isotypes, th cd + t cells and cytotoxic cd + t cells in mice , . ddos also synergize with the emulsion antibody addavax and enhance its type i immunity-driving potential by mechanisms that depends on type i ifns . notably, a dip influenza vaccine conferred protection to an unrelated virus through stimulation of type i ifn production , suggesting that they can be used as prophylactic or therapeutic antivirals. dvgs are present in live attenuated vaccines against polio, measles and influenza viruses , [ ] [ ] [ ] [ ] . however, their impact in the development of protective immunity and vaccine efficacy has not been formally assessed. based on their interfering and immunostimulatory ability, it is speculated that dvgs may enhance the efficiency of the vaccine while enhancing safety of the virus by reducing its replication and spread. if correct, it would be important to carefully regulate the amount of dvgs in vaccine preparations to avoid complete interference and drastic reduction of the virus to a point of ineffectiveness. while recent ngs data reveal that hundreds of dvgs can arise within a single viral infection, the fact that a smaller subset of dominant dvgs are repeatedly detected in different samples indicates that complex dynamics are at play within the viral population. these complexities include competition (and possibly compensation or cooperation) between different dvgs and positive selection of the best competitors that implicates their relative fitness in relation to the wild-type parental virus and other dvgs. parameters such as replication fitness, packaging, immunoregulation and other traits determine the virus dynamics. it is important to stress that most events leading to dvg formation, including mutations, deletions, recombination and translocations, are either non-viable or deleterious to the virus. in addition, although hundreds or even thousands of different dvgs are generated during a virus infection, the vast majority of these will be lost during the population bottlenecks that occur in vivo, for example when crossing anatomical barriers or during transmission from host to host . however, there are instances where these genomes could make it through bottlenecks, such as during infections of hosts that are immunosuppressed or have comorbidities where founding populations are increased. in addition, infections may occur with virions that have co-packaged wild-type genomes and dvgs or virions may aggregate during infection enhancing co-transmission, as seen for vsv mathematical models have generally only considered one dominant dvg , ; further development is required to incorporate the potential cooperation and competition among others. while historically dvgs have been considered replication waste, an artifact of cell culture passaging conditions or simply a nuisance to laboratory experimentation, a renewed interest in this part of the viral population may reveal a more functional or biological relevance to their existence. do dvgs exist for a reason? why keep waste lying around? given the notoriously high recombination and reassortment rates of some viruses, it is possible that for viruses that undergo recombination, dvgs can provide a repertoire of mutations that could feed back into the viable virus population to promote adaptation. it remains to be seen whether dvgs can provide a selective advantage to the virus, and whether the high moi or localized co-infection conditions are biologically relevant outside of cell culture. recent work in insects suggests that dvgs, which are a preferred template for the generation of the viral dna form of rna viruses, provide additional substrate to help boost the rna interference response that is responsible for viral persistence in insects . indeed, it was shown that a change in the amount of viral dna generated during rna virus infection in drosophila, altered the persistence and kinetics of a wild-type rna virus infection. the authors suggested that evolution has perhaps fine-tuned the production of dvgs to balance wild-type infection and promote persistence (and ultimately, transmission of viruses, including arboviruses in mosquitoes). additionally, a closer examination of dvg dynamics within viral populations can help better understand the biology of standard viruses. indeed, in addition to distinguishing between the dispensable and indispensable nucleotide sequences, proteins and rna structures, we can identify what viral components can operate in cis or in trans. a recent study of hcv dvgs, for example, identified novel cis-acting rna structures that were required for replication and packaging . emerging technologies that allow the 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novel proteincoding rna species in hiv-infected patients on combination antiretroviral therapy replication-competent noninduced proviruses in the latent reservoir increase barrier to hiv- cure defective interfering particles of poliovirus: mapping of the deletion and evidence that the deletions in the genomes of di internal genome deletions in two distinct classes of defective interfering particles of vesicular stomatitis virus influenza defective interfering viral rna is formed by internal deletion of genomic rna vesicular stomatitis virus defective interfering particles can contain extensive genomic sequence rearrangements and base substitutions a defective interfering rna that contains a mosaic of a plant virus genome isolation and characterization of an arterivirus defective interfering rna genome isolation and characterization of sendai virus di-rnas the origins of defective interfering particles of the negative-strand rna viruses structure and origin of a novel class of defective 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induction measles virus defective interfering rnas are generated frequently and early in the absence of c protein and pan be destabilized by adenosine deaminase acting on rna- -like hypermutations defective viral genomes arising in vivo provide critical danger signals for the triggering of lung antiviral immunity immunostimulatory defective viral genomes from respiratory syncytial virus promote a strong innate antiviral response during infection in mice and humans loss of sendai virus c protein leads to accumulation of rig-i immunostimulatory defective interfering rna deep sequencing analysis of defective genomes of parainfluenza virus and their role in interferon induction quantitative characterization of defective virus emergence by deep sequencing does the higher order structure of the influenza virus ribonucleoprotein guide sequence rearrangements in influenza viral rna? sequence-specific fidelity alterations associated with west nile virus attenuation in mosquitoes alphavirus mutator variants present host-specific defects and attenuation in mammalian and insect models reduced accumulation of defective viral genomes contributes to severe outcome in influenza virus infected patients low-fidelity polymerases of alphaviruses recombine at higher rates to overproduce defective interfering particles a single amino acid mutation in the pa subunit of the influenza virus rna polymerase promotes the generation of defective interfering rnas mutations in the rna-binding domains of tombusvirus replicase proteins affect rna recombination in vivo mutation in ns , a nonstructural protein of influenza a virus, extragenically causes aberrant replication and expression of the pa gene and leads to generation of defective interfering particles a single amino acid substitution within the paramyxovirus sendai virus nucleoprotein is a critical determinant for production of interferon-beta-inducing copyback-type defective interfering genomes the lymphocytic choriomeningitis virus 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virus replication and pathogenesis in vitro and in vivo defective interfering particles of respiratory syncytial virus induction and biological properties of defective interfering particles of rabies virus defective t particles of vesicular stomatitis virus. ii. biologic role in homologous interference viral interference by defective particles of vesicular stomatitis virus measured in individual cells analysis of extracellular west nile virus particles produced by cell cultures from genetically resistant and susceptible mice indicates enhanced amplification of defective interfering particles by resistant cultures replication signals in the genome of vesicular stoma titis virus and its defective interfering particles: identification of a sequence element that enhances di rna replication functional characterisation of the genomic and antigenomic promoters of sendai virus homologous interference by incomplete sendai virus particles: changes in virus-specific ribonucleic acid synthesis defective viral genomes alter how sendai virus interacts with cellular trafficking machinery leading to heterogeneity in the production of viral particles among infected cells replication defective viral genomes exploit a cellular pro-survival mechanism to establish paramyxovirus persistence mda participates in the detection of paramyxovirus infection and is essential for the early activation of dendritic cells in response to sendai virus defective interfering particles sendai virus defective-interfering genomes and the activation of interferon-beta induction of dendritic cell production of type i and type iii interferons by wild-type and vaccine strains of measles virus: role of defective interfering rnas highly immunostimulatory rna derived from a sendai virus defective viral genome a novel role for viral-defective interfering particles in enhancing dendritic cell maturation modulation of a systemic semliki forest virus infection in mice by defective interfering virus identification of a natural viral rna motif that optimizes sensing of viral rna by rig.-i. mbio activation of the beta interferon promoter by unnatural sendai virus infection requires rig-i and is inhibited by viral c proteins pact-and rig-i-dependent activation of type i interferon production by a defective interfering rna derived from measles virus vaccine preference of rig-i for short viral rna molecules in infected cells revealed by next-generation sequencing tlr-independent induction of dendritic cell maturation and adaptive immunity by negative-strand rna viruses in vivo ligands of mda and rig-i in measles virusinfected cells nonencapsidated ′ copy-back defective interfering genomes produced by recombinant measles viruses are recognized by rig-i and lgp but not mda atpase-driven oligomerization of rig-i on rna allows optimal activation of type-i interferon double-stranded rna is produced by positive-strand rna viruses and dna viruses but not in detectable amounts by negative-strand rna viruses 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associated with persistent infection in cell culture defective interfering influenza virus rnas: time to reevaluate their clinical potential as broad-spectrum antivirals? targeting pattern recognition receptors (prr) for vaccine adjuvantation: from synthetic prr agonists to the potential of defective interfering particles of viruses accumulation of defective interfering viral particles in only a few passages in vero cells attenuates mumps virus neurovirulence in vivo antiviral activity: defective interfering virus protects better against virulent influenza a virus than avirulent virus interfering vaccine (defective interfering influenza a virus) protects ferrets from influenza, and allows them to develop solid immunity to reinfection the case for transmissible antivirals to control population-wide infectious disease conflicting selection pressures will constrain viral escape from interfering particles: principles for designing resistance-proof antivirals a sendai virus-derived rna 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enveloped virus plaques formed by mutagenized viral populations have elevated coinfection frequencies modeling the intracellular replication of influenza a virus in the presence of defective interfering rnas design requirements for interfering particles to maintain coadaptive stability with hiv- genetic analysis of serum-derived defective hepatitis c virus genomes revealed novel viral cis elements for virus replication and assembly identification of new defective interfering rna species associated with porcine reproductive and respiratory syndrome virus infection multiple species of defective rnas in plants infected with citrus tristeza virus a novel class of large and infectious defective rnas of citrus tristeza virus characterization of defective interfering rnas of berne virus bovine coronavirus mrna replication continues throughout persistent infection in cell culture replication and packaging of coronavirus infectious bronchitis virus defective rnas lacking a long open reading frame analysis of efficiently packaged defective interfering rnas of murine coronavirus: localization of a possible rna-packaging signal a domain at the ′ end of the polymerase gene is essential for encapsidation of coronavirus defective interfering rnas molecular characterization of transmissible gastroenteritis coronavirus defective interfering genomes: packaging and heterogeneity long-term transmission of defective rna viruses in humans and aedes mosquitoes detection and sequencing of defective viral genomes in c / cells persistently infected with dengue virus characterization of homologous defective interfering rna during persistent infection of vero cells with japanese encephalitis virus characterization of hepatitis c virus deletion mutants circulating in chronically infected patients naturally occurring hepatitis c virus subgenomic deletion mutants replicate efficiently in huh- cells and are trans-packaged in vitro to generate infectious defective particles characterization of defective viral rna produced during persistent infection of vero cells with murray valley encephalitis virus spontaneous and engineered deletions in the ′ noncoding region of tick-borne encephalitis virus: construction of highly attenuated mutants of a flavivirus characterization of west nile virus persistent infections in genetically resistant and susceptible mouse cells. i. generation of defective nonplaquing virus particles defective rna particles derived from tomato black ring virus genome interfere with the replication of parental virus defective interfering particles of encephalomyocarditis virus a segmented form of foot-and-mouth disease virus interferes with standard virus: a link between interference and competitive fitness generation of defective interfering particles in picornaviruses characterization of a new isolate of poliovirus defective interfering particles characterization of defective-interfering rnas of rubella virus generated during serial undiluted passage prevention of death in semliki forest virus-infected mice by administration of defective-interfering semliki forest virus properties of host and virus which influence defective interfering virus mediated-protection of mice against semliki forest virus lethal encephalitis sindbis virus: defective-interfering particles temperature-sensitive for interferon induction sequence and structure of defective interfering rnas associated with cucumber necrosis virus infections turnip crinkle virus defective interfering rnas intensify viral symptoms and are generated de novo properties of defective lymphocytic choriomeningitis virus a novel type of defective viral genome suggests a unique strategy to establish and maintain persistent lymphocytic choriomeningitis virus infections interference between inactive and active influenza viruses in the chick embryo. ii. the site of interference influenza virus nucleic acid: relationship between biological characteristics of the virus particle and properties of the nucleic acid in vitro transcription of defective interfering particles of influenza virus produces polyadenylic acid-containing complementary rnas characterization of virulent and avirulent a/chicken/pennsylvania/ influenza a viruses: potential role of defective interfering rnas in nature defective interfering virus associated with a/chicken/pennsylvania/ influenza virus defective interfering influenza virus inhibits immunopathological effects of infectious virus in the mouse defective interfering influenza virus confers only short-lived protection against influenza virus disease: evidence for a role for adaptive immunity in di virus-mediated protection in vivo defective interfering particles of human parainfluenza virus defective measles virus in human subacute sclerosing panencephalitis brain generation of defective interfering particles by two vaccine strains of measles virus mumps virus-persistently infected cell cultures release defective interfering virus particles identification of transcriptive and replicative intermediates in sendai virus-infected cells molecular cloning of natural paramyxovirus copy-back defective interfering rnas and their expression from dna characterization of bunyamwera virus defective interfering particles characterization of toscana virus-defective interfering particles generated in vivo characteristics of a respiratory syncytial virus persistently infected macrophage-like culture a transmissible interfering component of vesicular stomatitis virus preparations cyclic production of vesicular stomatitis virus caused by defective interfering particles the complete sequence of a unique rna species synthesized by a di particle of vsv interference among defective interfering particles of vesicular stomatitis virus very rapid generation/amplification of defective interfering particles by vesicular stomatitis virus variants isolated from persistent infection interferon induction by viruses. xix. vesicular stomatitis virus-new jersey: high multiplicity passages generate interferon-inducing, defective-interfering particles generation of envelope and defective interfering rna mutants of tomato spotted wilt virus by mechanical passage a novel betapartitivirus rnpv from rosellinia necatrix tolerates host rna silencing but is interfered by its defective rnas defective virions of reovirus segment-specific inverted repeats found adjacent to conserved terminal sequences in wound tumor virus genome and defective interfering rnas defective proviruses rapidly accumulate during acute hiv- infection accumulation of defective viral genomes in peripheral blood mononuclear cells of human immunodeficiency virus type -infected individuals satellite rnas and satellite viruses plant virus satellite and defective interfering rnas: new paradigms for a new century properties of satellite tobacco mosaic virus phenotypes expressed in the presence and absence of helper virus a classification system for virophages and satellite viruses virus-associated small satellite rnas and viroids display similarities in their replication strategies what has been happening with viroids? the expanding family of virophages the virophage as a unique parasite of the giant mimivirus ecogenomics of virophages and their giant virus hosts assessed through time series metagenomics capsid protein structure, self-assembly, and processing reveal morphogenesis of the marine virophage mavirus virus-like vesicles of kaposi's sarcoma-associated herpesvirus activate lytic replication by triggering differentiation signaling ultrastructural and biophysical characterization of hepatitis c virus particles produced in cell culture extracellular vesicles released by herpes simplex virus infected cells block virus replication in recipient cells in a sting-dependent manner this work was supported by the us national institutes of health national institute of allergy and infectious diseases (grants nos. nih ai , ai and ai to c.b.l.) and the darpa intercept program (to m.v.) managed by j. gimlett and administered though darpa cooperative agreement (grant no. hr - - - ). in addition, this work was supported by a fulbright us scholar award to c.b.l. the views expressed in this article do not necessarily represent the position or the policy of the us government the authors declare no competing interests. reprints and permissions information is available at www.nature.com/reprints.correspondence should be addressed to c.b.l.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -oyhzn kc authors: li, chenxi; di, di; wang, xin; xia, qiqi; wahaab, abdul; anwar, muhammad naveed; li, zongjie; liu, ke; shao, donghua; qiu, yafeng; wei, jianchao; li, beibei; ma, zhiyong title: duck karyopherin α (dukpna ) is involved in type i interferon expression and the antiviral response against japanese encephalitis virus date: - - journal: dev comp immunol doi: . /j.dci. . sha: doc_id: cord_uid: oyhzn kc karyopherin α (kpna ) is an adaptor molecule that mediates type i interferon (ifn) production by facilitating the nuclear translocation of ifn transcription factors. here, we cloned the duck kpna (dukpna ) gene and analyzed its involvement in type i ifn expression as well as antiviral response against japanese encephalitis virus (jev). the full-length dukpna gene encoded a -amino acid protein that shared . – . % sequence similarity with its orthologues in chickens, humans and mice. the dukpna was extensively expressed in various duck tissues at the mrna level. analysis of the subcellular localization of dukpna by immunofluorescence assays indicated that the dukpna was primarily distributed in both the cytoplasm and nucleus in primary duck embryonic fibroblasts (defs). however, it translocated from the cytoplasm to the nucleus in response to poly(i:c) stimulation or jev infection. the dukpna interacted with duck ifn regulatory factor and facilitated its nuclear translocation, thereby up-regulating the expression of ifn-α and ifn-β in defs in the presence of poly(i:c) stimulation. exogenous expression of dukpna significantly elevated the expression of ifn-α and ifn-β induced by jev infection and inhibited jev replication in defs. these data demonstrate the importance of dukpna in type i ifn signaling as well as the antiviral response against jev replication. karyopherin (kpna), also known as importin α, acts as an adaptor molecule between nuclear-localization-sequence (nls)-bearing cargo proteins and importin β (goldfarb et al., ) , and plays an essential role in the active transport of proteins from the cytoplasm to the nucleus (smith et al., ) . in eukaryotic cells, macromolecules (> nm in diameter or > kda) bearing an nls can be translocated from the cytoplasm into the nucleus by importin α/β heterodimers (fagerlund et al., ) . in eukaryotes, multiple subtypes of kpna have been identified. mice and humans have six (kpna , , , , and ) and seven (kpna , , , , , and ) subtypes, respectively, whereas budding yeast contains only a single kpna (srp ) (oka and yoneda, ) . on the basis of homology, kpna subtypes are classified into three subfamilies: α (kpna , kpna and kpna ), α (kpna and kpna ) and α (kpna and kpna ) (pumroy and cingolani, ; smith et al., ) . the three subfamilies of kpna show distinct nls-binding specificity (nadler et al., ) and exhibit multiple biological functions including cell development and differentiation, regulation of the immune response and control of tumorigenesis. among the identified kpnas, kpna in the α subfamily is involved in the innate immune response against viral infection ye et al., ) . several kpna orthologues from different species including humans (köhler et al., ) , mice (mihalas et al., ) , pigs (chen et al., ) and chickens (caldwell et al., ) have been identified. in mammals, human kpna , a specific adaptor molecule, mediates the nuclear import of interferon (ifn) transcriptional regulators, such as nf-κb p /p heterodimers and ifn regulatory factor (irf) , thereby regulating type i ifn-mediated immune responses (canton et al., ; shaw et al., ) . recently, pig kpna has been demonstrated to bind pig irf and initiate ifn-β production during porcine circovirus infection . however, the role of avian kpna , especially duck kpna (dukpna ), in type i ifn expression was unknown. the irf gene is absent in ducks, and the functions of duck irf (duirf ) complement most of irf 's functions in regulating type i ifn expression (magor et al., ) . therefore, we sought to explore whether dukpna interacts with duirf and induces type i li, et al. developmental and comparative immunology ( ) expression. domestic duck farming is a popular business in china. in addition, ducks, as an amplification host, are susceptible to japanese encephalitis virus (jev) infection (xiao et al., ) . jev is a single-stranded positive-sense rna virus of the flavivirus genus that is transmitted by mosquitoes and vertebrate-amplifying hosts including birds (dhanda et al., ) . jev infection causes stunted growth and death in ducklings (xiao et al., ) . in addition, jev ns protein interacts with human kpna and inhibits the nuclear translocation of irf and nf-κb in human cells (ye et al., ) . we therefore cloned the dukpna gene and analyzed its role in type i ifn expression as well as the antiviral response against jev in primary duck embryo fibroblasts (defs). specific pathogen free (spf) ducks, clinically healthy ducks and spf duck embryos were purchased from the harbin veterinary research institute of the chinese academy of agricultural sciences, china. all animal experiments were approved by the institutional animal care and use committee of the shanghai veterinary research institute (iacuc no: shvri-po- ) and were performed in compliance with the guidelines on the humane treatment of laboratory animals (ministry of science and technology of the people's republic of china, policy no. ) . defs were prepared from -to -day-old spf duck embryos and were grown in dulbecco's modified eagle's medium (gibco, grand island, ny, usa) supplemented with % fetal bovine serum (gibco). the jev sh strain (ncbi accession no. mh ) was maintained in our laboratory. the open reading frame (orf) encoding full-length dukpna was amplified by polymerase chain reaction (pcr) from the spleens of spf ducks. the forward primer dukpna -f and reverse primer dukpna -r (supplementary table ) for amplification of dukpna were designed according to the predicted sequence of dukpna (ncbi accession xm_ . ). the amplified sequence was confirmed by dna sequencing and cloned into the pcdna . vector to express dukpna tagged with ha (dukpna -ha). the multiple sequence alignment of dukpna with chicken (nm_ . ), helmeted guineafowl one hundred cells expressing dukpna -ha were randomly selected from different fields of microscope to calculate the cells with nuclear distribution of dukpna -ha. the percentages of cells with nuclear distribution of dukpna -ha in the presence and absence of poly(i:c) treatment were plotted. results are presented as the mean ± standard error from three independent experiments. **, p < . tested by student's t-test. (xm_ . ), human (nm_ . ) and mouse (nm_ . ) kpna was performed with geneious software. the functional domains of dukpna were analyzed with the smart website (http://smart. embl-heidelberg.de/). the phylogenetic tree of kpna was constructed by neighbor-joining in mega version . . samples of issues including the spleen, liver, ileum, jejunum, proventriculus, lung, kidney, gizzard, duodenum, brain, bursa, heart, appendix and blood were collected from three -day-old spf ducks and three -day-old clinically healthy ducks. total rnas from different tissues were extracted with trizol reagent according to the manufacturer's instructions (takara, dalian, china) and subsequently reverse transcribed to cdna with a primescript™ rt reagent kit with gdna eraser (takara). the relative expression levels of dukpna in different tissues were determined by quantitative real-time rt-pcr (qrt-pcr) with tb green™ premix ex taq™ ii (takara) and primers qdukpna -f and qdukpna -r (supplementary table ). the glyceraldehyde- phosphate dehydrogenase (gapdh) gene was used as an internal control. the subcellular localization of dukpna in defs was determined by immunofluorescence assays (ifas). defs were pre-cultured overnight to approximately % confluence and transfected with recombinant plasmids engineered to express dukpna -ha or empty vector, by using lipofectamine (invitrogen, carlsbad, ca, usa). after h of incubation, the transfectants were stimulated with poly(i:c) (invitrogen) fig. . up-regulation of expression of type i ifn by dukpna . defs were transfected with plasmid for expression of dukpna -ha or empty vector in the indicated amounts ( , and ng) for h and stimulated with poly(i:c) treatment (+poly(i:c)) or left untreated (-poly(i:c)) for an additional h. (a) the expression of dukpna -ha in the transfectants was confirmed by western blotting with anti-ha antibody. (b) the expression of ifn-α and ifn-β at the mrna level was detected by qrt-pcr and normalized to the expression of the gapdh gene. (c) the concentrations of ifn-α and ifn-β proteins in the supernatants were detected by elisa. (d, e and f) defs were transfected with three sirnas targeting different regions of dukpna mrna (sidukpna - , sidukpna - and sidukpna - ) or control sirna (sinegative) for silencing dukpna expression. the transfectants were incubated for h and subsequently stimulated with poly(i:c) treatment or left unstimulated for an additional h. the relative expression of dukpna mrna levels detected by qrt-pcr was normalized to gapdh mrna and is presented relative to the level in control sirna (sinegative) cells (set as ) (d). the relative expression of ifn-α and ifn-β mrna levels was normalized to gapdh mrna levels and plotted (e). the concentrations of ifn-α and ifn-β proteins in the supernatants were detected by elisa (f). results are presented as the mean ± standard error from three independent experiments. **, p < . ; *, p < . tested by student's t-test. c. li, et al. developmental and comparative immunology ( ) at a concentration of μg/ml for h. the defs were fixed in % paraformaldehyde for h, permeabilized with . % triton x- for min and blocked with % bovine serum albumin for h. subsequently, the cells were incubated with mouse anti-ha antibody (sigma, st louis, mo, usa) for h and treated with donkey anti-mouse igg(h + l) antibody conjugated with alexa fluor (invitrogen) for h. the nuclei were stained with ′, -diamidino- -phenylindole (dapi) (sigma). fluorescence images were taken with a fluorescence microscope (carl zeiss, zena, germany). defs at - % confluence were transfected with plasmids for expression of dukpna -ha or with empty vector for h and were stimulated with poly(i:c) ( μg/ml) for an additional h. the supernatants were collected to measure the protein levels of ifn-α and ifn-β with an elisa kit (lengton, shanghai, china), and the cells were used to extract total rnas for detection of the mrna expression of ifn-α and ifn-β by qrt-pcr. the primers used are listed in supplementary table . to further analyze the effects of dukpna on type i ifn expression, we synthesized three small interfering rnas (sirnas) targeting different regions of dukpna mrna (sidukpna - , sidukpna - , and sidukpna - ) in vitro to knock down the endogenous dukpna expression (supplementary table ). defs were transfected with the synthesized sirna or negative sirna with lipofectamine rnai max (invitrogen) and incubated for h. the transfectants were stimulated with poly(i:c) for an additional h and harvested for detection of the expression of ifn-α and ifn-β at both the mrna and protein levels, as described above. the orf of duirf was amplified from duck spleen by rt-pcr according to the sequence of duirf (genbank accession no. mg . ). the amplified sequence was confirmed by dna sequencing and cloned into the pcdna . vector to express duirf tagged with myc (duirf -myc) for co-immunoprecipitation assays. defs were co-transfected with recombinant plasmids for expression of dukpna -ha and duirf -myc and incubated for h. the transfectants were stimulated with poly(i:c) ( μg/ml) for h and lysed in icecold ripa lysis buffer (millipore, billerica, ma, usa) containing mm phenylmethyl sulfonylfluoride for min at °c. the supernatants of cell lysates were collected and incubated with anti-ha protein-g-sepharose beads for h at °c. the beads were washed three times with phosphate buffered saline with . % tween- , resuspended in × sds-page sample buffer and subjected to sds-page. the immunoprecipitated protein complexes were detected with western blot analysis with anti-myc antibody (millipore) and mouse anti-ha antibody (sigma), as described previously (zhao et al., ) . the protein levels of dukpna -ha and duirf -myc in the nuclear extracts were detected by western blotting. lamin b was detected as the nuclear marker (c). intensities of protein bands were determined by densitometric analysis. relative protein levels of dukpna -ha and duirf -myc in the nuclear extracts were normalized to those in the wcl and plotted (d). the significant difference was determined using one-way anova followed by tukey's multiple comparisons test. ***, p < . ; **, p < . ; *, p < . . c. li, et al. developmental and comparative immunology ( ) . . analysis of the effects of dukpna on the nuclear translocation of duirf defs were co-transfected with recombinant plasmids for expression of dukpna -ha and duirf -myc and incubated for h. the transfectants were stimulated with poly(i:c) ( μg/ml) for h and lysed with ne-per nuclear and cytoplasmic extraction reagents (invitrogen) to obtain cytoplasmic and nuclear extracts. the protein levels of duirf -myc in the nuclear extract were determined by western blot analysis. lamin b was detected as a nuclear marker with mouse anti-laminb antibody (cell signaling, danvers, ma, usa). defs were transfected with plasmid for expression of dukpna -ha and incubated at °c for h. the transfectants were infected with the jev sh strain at a multiplicity of infection (moi) of . and incubated at °c for the indicated times. the jev-infected cells were collected at , -and -h post-infection (hpi) for analysis of jev replication. the jev titers in the supernatants were determined with % tissue culture infective dose (tcid ) assays. the protein levels of jev ns in the cells were detected by western blotting with polyclonal antibody specific to jev ns (deng et al., ) . the relative number of rna copies of jev e in the cells was measured by qrt-pcr. the βactin gene was used as the internal control. the primers used are listed in supplementary table . the orf of the dukpna gene was cloned from duck spleen, which was bp in length and encoded a -amino acid protein (fig. a) . the nucleotide sequence of dukpna has been deposited in genbank under accession number mn . analysis of the deduced amino acid sequence revealed that dukpna had . %, . %, . % and . . % sequence similarity to the chicken, helmeted guineafowl, human and mouse kpna sequences, respectively (fig. a) , and showed a high degree of sequence similarity to orthologues in other species. structurally, dukpna contained three domains that were conserved in all orthologues of other species, including an n-terminal importin β-binding (ibb) domain, a series of armadillo (arm) repeats and a conserved short acidic cluster (fig. a) . the arm repeats are essential for nls-binding and are composed of eight regions (arm , , , , , , and ), which shared nearly % sequence identity with sequences from other species (fig. a) . phylogenetic analysis indicated that dukpna clustered together with chicken and helmeted guineafowl kpna in the bird group and was closest to chicken kpna (fig. b) . to examine the tissue distribution of dukpna expression, we collected different tissues from clinically healthy ducks and spf ducks and examined dukpna mrna expression by qrt-pcr. dukpna was ubiquitously expressed at varying levels in various tissues, including the spleen, liver, ileum, jejunum, proventriculus, lung, kidney, gizzard, duodenum, brain, bursa, heart, appendix and blood; the expression profiles were similar between clinically healthy ducks ( fig. a) and spf ducks (fig. b) . among the examined tissues, the blood showed the highest level of expression in both spf and clinically healthy ducks, followed by the kidney, lung, gizzard, spleen, and duodenum, whereas low levels of expression were observed in the ileum and jejunum (fig. ) . the subcellular location of dukpna was examined by ifa. because of a lack of a commercial antibody specific to dukpna , we exogenously expressed dukpna -ha in defs and detected the subcellular location of the expressed dukpna with anti-ha antibody. the dukpna -ha was distributed in both the cytoplasm and nucleus, with predominant expression in the cytoplasm (fig. a) . however, in response to poly(i:c) stimulation, dukpna -ha translocated from the cytoplasm to the nucleus, showing a predominant accumulation in the nucleus (fig. a-b) . these data indicated that poly(i:c) stimulation promoted the translocation of dukpna from the cytoplasm to the nucleus, thus implying a possible role of dukpna in type i ifn expression in ducks. to analyze whether dukpna might be involved in type i ifn expression, we transfected defs with plasmid for expression of dukpna -ha at different doses in the presence and absence of poly(i:c) stimulation, and detected the expression of interferon-α (ifn-α) and interferon-β (ifn-β) at the mrna level by qrt-pcr and at the protein level by elisa. the expression of dukpna -ha was confirmed by western blotting with anti-ha antibody (fig. a) . overexpression of dukpna -ha significantly promoted the expression of ifn-α and ifn-β at the mrna level in a dose-dependent manner in the presence of poly(i:c) stimulation, as compared with the expression in the control vector groups (fig. b) . the promotion of ifn-α and ifn-β expression by dukpna -ha was further confirmed at the protein level. the concentrations of ifn-α and ifn-β protein in the supernatants of dukpna -ha transfected cells were clearly higher than those in the control vector groups (fig. c) . to further confirm the involvement of dukpna in type i ifn expression, we knocked down the expression of endogenous dukpna in defs by rna interference with sirnas (sidukpna - , sidukpna - and sidukpna - ) targeting three different regions of dukpna mrna (supplementary table ) and analyzed the effects on the expression of ifn-α and ifn-β at the mrna and protein levels in the presence or absence of poly(i:c) stimulation. the down regulation of dukpna mrna expression in the sirna-treated cells was confirmed at the mrna level by qrt-pcr (fig. d) , because of a lack of a commercial antibody specific to dukpna . in response to poly(i:c) stimulation, the expression of ifn-α and ifn-β was significantly elevated at both the mrna and protein levels, as compared with the levels in cells without poly(i:c) stimulation. however, the elevated expression of ifn-α and ifn-β was significantly reduced at both the mrna and protein levels in cells treated with sirnas (sidukpna - , sidukpna - , and ) . results are presented as the mean ± standard error from three independent experiments. **, p < . ; *, p < . tested by student's t-test. c. li, et al. developmental and comparative immunology ( ) sidukpna - ) (fig. e-f) , thus suggesting that the silencing of endogenous dukpna expression eliminated the production of ifn-α and ifn-β in defs after poly(i:c) stimulation. together, these data indicated that dukpna is involved in type i ifn expression. irf and irf play critical roles in type i ifn-mediated innate immunity (takeuchi and akira, ). in mammalian cells, kpna interacts with irf and subsequently promotes its translocation from the cytoplasm to the nucleus, thus triggering the expression of type i ifn . the irf gene is absent in the duck genome; therefore duirf substitutes for most of irf 's functions in regulating type i ifn expression (chen et al., ) . given that dukpna upregulated type i ifn expression, we investigated whether dukpna might interact with duirf and promote the translocation of duirf from the cytoplasm to the nucleus. defs were co-transfected with plasmids for expression of dukpna -ha and duirf -myc, and their co-localization was determined by ifas in the presence or absence of poly(i:c) stimulation. the expressed dukpna -ha and duirf -myc mainly co-localized in the cytoplasm in the absence of poly(i:c) stimulation (fig. a) . however, in response to poly(i:c) stimulation, both the expressed dukpna -ha and duirf -myc translocated from the cytoplasm to the nucleus and co-localized in the nucleus (fig. a) , thus suggesting a possible interaction between dukpna and duirf . to confirm this interaction, we performed co-immunoprecipitation in defs co-transfected with plasmids for expression of dukpna -ha and duirf -myc, by using anti-ha antibody for immunoprecipitation and anti-myc antibody for detection of the immunoprecipitated proteins. the dukpna -ha co-immunoprecipitated with duirf -myc (fig. b) , thus revealing that dukpna interacted with duirf in defs. to further analyze whether dukpna promoted the nuclear translocation of duirf , we extracted the nuclear fractions from defs co-transfected with plasmids for expression of dukpna -ha and duirf -myc, in the presence of poly(i:c) stimulation, and performed western blotting analysis to detect the protein levels of dukpna -ha and duirf -myc. the expression of dukpna -ha and duirf -myc in the whole cell lysates (wcl) of defs was confirmed by western blotting (fig. c ). analysis of changes in the protein levels of duirf -myc in the nuclear fractions revealed that the nuclear accumulation of duirf -myc increased, an effect correlated with the increased nuclear accumulation of dukpna -ha ( fig. c and d) , thus indicating that dukpna facilitated the nuclear translocation of duirf , thereby upregulating type i ifn expression. given that jev infection induces type i ifn expression and that dukpna was involved in type i ifn expression in the presence of poly(i:c) stimulation (fig. ) , we investigated the involvement of dukpna in type i ifn expression during viral infection by using jev as a model. defs were infected with jev at different moi ( . , and ), and total rna was extracted from jev-infected cells to detect the endogenous mrna expression levels of dukpna . the qrt-pcr analysis indicated that jev infection did not significantly affect the mrna expression of endogenous dukpna in defs (fig. a) . however, the subcellular location of dukpna -ha shifted from the cytoplasm to the nucleus in defs in response to jev infection (fig. b) , similarly to the translocation induced by poly(i:c) stimulation (fig. ) . analysis of type i ifn expression in jev-infected defs with or without overexpression of dukpna -ha revealed that exogenous expression of dukpna -ha significantly elevated the mrna levels of ifn-α and ifnβ induced by jev infection, as compared with the levels in jev-infected defs without dukpna -ha expression (vector control) (fig. c) . the expression of dukpna -ha in jev-infected defs was confirmed by western blotting (fig. d) . together, these data indicated that dukpna resulted in nuclear translocation of duirf and facilitated type i ifn expression during jev infection, thereby implying its involvement in the type i ifn-mediated antiviral response. on the basis of the observation that dukpna facilitated type i ifn expression during jev infection (fig. ) , we investigated the effect of dukpna on jev replication. defs were transfected with plasmid expressing dukpna -ha or empty vector and subsequently infected with jev. jev replication was monitored by tcid assays and qrt-pcr. as shown in fig. a , the jev titers in defs transfected with plasmid expressing dukpna -ha were significantly lower than those in defs transfected with empty vector at , and hpi. the relative number of rna copies of jev e (fig. b ) and the protein levels of jev ns ( fig. c and d) in defs transfected with plasmid for expression of dukpna -ha were also significantly lower than those in defs transfected with empty vector, thus confirming the inhibitory effect of dukpna -ha on jev replication. mammalian kpna plays a role in regulating the nuclear transportation of type i ifn transcription factors, thereby triggering the expression of type i ifn and the subsequent immune response against virus infection (zhu et al., ; chen et al., ; li et al., ) . however, the role of avian kpna in type i ifn expression was unknown. the current study aimed to clone duck kpna and analyze its role in type i ifn expression as well as the antiviral response. the cloned dukpna comprised amino acid and shared . - . % sequence similarity with its orthologues from chicken, helmeted guineafowl, human and mouse, thus suggesting that kpna has been highly conserved through evolutionary history (goldfarb et al., ; miyamoto et al., ) . the ibb domain involved in binding importin β (kobe, ) , the arm repeats functioning as internal cargo nls-binding sites mediating the nuclear translocation of cargo protein (miyamoto et al., ) , and the short acidic cluster that acts as a binding region for the nuclear exporter of importin α (goldfarb et al., ) were highly conserved in dukpna . these observations suggested that dukpna might have biological roles similar to those of its orthologues from mammals. indeed, in response to poly(i:c) stimulation, dukpna facilitated the expression of type i ifn, showing a conserved role in type i ifn expression similar to that of human, mouse and pig kpna xinghui et al., ) . mechanistically, mammalian kpna acts as a specific adaptor molecule that mediates the nuclear translocation of nf-κb p /p heterodimers and irf , thereby inducing type i ifn expression (fagerlund et al., ; li et al., ) . knockdown of human kpna in mcf- cells significantly inhibits nf-κb activity and impairs ifn-α/β expression, thus leading to immunological dysfunction (zhu et al., ) , whereas up-regulation of kpna enhances nf-κb activation (chen et al., ) . porcine kpna has been found to be involved in the irf mediated type i ifn signaling pathway during porcine circovirus type infection . in ducks, the toll-like receptor signaling pathway and the retinoic-acid-inducible gene i signaling pathway largely resemble the human pathways (evseev and magor, ) and play essential roles in the regulation of type i ifn expression and host antiviral response (yu and levine, ) . notably, irf , as a downstream molecule in the toll-like receptor signaling pathway and the retinoicacid-inducible gene i signaling pathway, is absent in ducks, and duirf instead performs most functions of irf in inducing type i ifn expression (santhakumar et al., ) . in the current study, dukpna interacted with duirf and facilitated the nuclear translocation of duirf . mechanistically, dukpna promotes type i ifn expression, probably via the duirf -mediated type i ifn signaling pathway, in contrast to mammalian kpna . human kpna is detected in nearly all tissues at both the mrna and protein levels (nachury et al., ) . in this study, we detected dukpna expression only at the mrna level because of a lack of a commercial antibody specific to dukpna . the expression of dukpna was detectable in all most tissues tested, showing a ubiquitous expression pattern similar to that of human kpna (goldfarb et al., ; miyamoto et al., ) , thus suggesting the multiple biological functions of dukpna , such as involvement in antiviral immune response. indeed, in response to jev infection, dukpna translocated from the cytoplasm to the nucleus and elevated the type i ifn expression induced by jev infection. furthermore, exogenous expression of dukpna significantly inhibited jev replication in defs, thereby suggesting the involvement of dukpna in the host immune response against viral infection in ducks. this observation was consistent with a previous observation in humans, in which human kpna has been found to have a role in the inhibition of jev replication (ye et al., ) . given the highly conserved amino acid sequence similarity and the conserved role in type i ifn expression, kpna may be an important molecule in type i ifn signaling as well as type i ifn-mediated antiviral response, and may be targeted by viruses to antagonize the type i ifnmediated antiviral response. indeed, the b protein of middle east respiratory syndrome coronavirus binds kpna and impairs the interaction between kpna and the nf-κb-p subunit, thus interfering with the host innate immune response (canton et al., ) . jev ns protein competitively binds kpna and inhibits the nuclear translocation of ifn transcription factors, thus suppressing ifn expression (ye et al., ) . these previous observations together with our results emphasize the importance of kpna in the type i ifn-mediated antiviral response. however, these roles must be further mechanistically explored to gain more insight into the full biological functions of kpna . in summary, we cloned the dukpna gene, which encoded a amino acid protein with . - . % sequence similarity to its orthologues from other species. dukpna was extensively expressed in various tissues in ducks and was found to be involved in type i ifn expression in response to poly(i:c) stimulation. dukpna was found to interact with duirf and promote the nuclear translocation of duirf , thereby triggering type i ifn expression. in response to jev infection, dukpna translocated from the cytoplasm into the nucleus and elevated the expression of type i ifn expression. exogenous expression of dukpna significantly inhibited jev replication. together, these data suggested that dukpna is involved in type i ifn expression as well as the type i ifn-mediated antiviral response. full-length cdnas from chicken bursal lymphocytes to facilitate gene function analysis mers-cov b protein interferes with the nf-κb-dependent innate immune response during infection vitamin d deficiency 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virus ns inhibits type i interferon (ifn) production by blocking the nuclear translocation of ifn regulatory factor and nf-κb toll-like receptor , rig-i-like receptors and the nlrp inflammasome: key modulators of innate immune responses to double-stranded rna viruses differential antiviral immunity to japanese encephalitis virus in developing cortical organoids nucleocapsid protein of porcine reproductive and respiratory syndrome virus antagonizes the antiviral activity of trim by interfering with trim -mediated rig-i ubiquitination tdp- inhibits nf-κb activity by blocking p nuclear translocation supplementary data to this article can be found online at https:// doi.org/ . /j.dci. . . key: cord- - qj d f authors: gerace, elisabetta; lo presti, vincenzo di marco; biondo, carmelo title: cryptosporidium infection: epidemiology, pathogenesis, and differential diagnosis date: - - journal: eur j microbiol immunol (bp) doi: . / . . sha: doc_id: cord_uid: qj d f cryptosporidium is a protozoan that infects a wide variety of vertebrates, including humans, causing acute gastroenteritis. the disease manifests with abdominal pain and diarrhea similar to that of choleric infection. in the immunocompromised hosts, the parasite causes prolonged infections that can also be fatal. for this reason, cryptosporidiosis is considered one of riskiest opportunistic infections for patients with acquired immunodeficiency syndrome. the best way to control the infection in these patients is setting up sensitive and specific diagnostic tests for epidemiological surveillance and morbidity reduction. here, we summarized the general aspects of cryptosporidium infection focusing on available diagnostic tools used for the diagnosis of cryptosporidiosis. molecular methods currently available for its detection and progress in the development of new diagnostics for cryptosporidiosis are also discussed. cryptosporidiosis is a worldwide infection caused by the protozoan cryptosporidium, a parasite that infects many species of vertebrates, including humans, causing acute gastroenteritis, abdominal pain, and diarrhea [ ] . cryptosporidiosis is transmitted primarily through the fecal-oral route, i.e., by ingesting viable oocysts of animal and/or human origin, emitted with feces that contaminated food or water [ , ] . although the waterborne transmission of infectious pathogen is well documented, neither the natural reservoir nor the exact infection route of cryptosporidia is well-known [ ] . cryptosporidium was first discovered by tyzzer in [ ] , but for a longtime, it was thought to be a non-pathogenic parasite. only since , it was recognized as an opportunistic pathogenic parasite, when human cases of cryptosporidiosis were reported to be associated with diarrhea [ , ] . however, cryptosporidium remained unknown as a significant human pathogen of acute diarrhea disease until , when it was recognized as a causative agent of self-limiting diarrhea for the general population and a life-threatening disease for immunocompromised persons such as those receiving immunosuppressive agents and acquired immunodeficiency syndrome (aids) patients [ , , ] . the parasite completes its life cycle within a single host (monoxen cycle), alternating asexual and sexual reproduction [ , ] . although more than species have been included in the genus cryptosporidium, only species, namely, cryptosporidium parvum and cryptosporidium hominis, commonly infect humans [ ] [ ] [ ] . c. parvum is responsible for most cattle infections, and consequently, it is considered to be responsible for most zoonotic infections in humans [ ] . cryptosporidium spp. exhibit little host specificity, and different members of this genus have been reported to infect multiple hosts, such as mammals, marsupials, birds, reptiles, and fish [ , ] . the intracellular protozoan parasite cryptosporidium is a human and veterinary pathogena member of the phylum apicomplexa that include other pathogens such as plasmodium spp., eimeria spp., neospora, babesia, and theileria [ ] . however, in contrast to other parasitic protozoa of this phylum, such as toxoplasma gondii or plasmodium falciparum, the species of the genus cryptosporidium cannot be cultivated in vitro [ ] . since there is no vaccine commercially available to prevent cryptosporidium infection, and these parasites have certain characteristics that make them highly contagious (i.e., survival in the environment for a long time and resistance to chlorine-based disinfectants), the only way to avoid the spreading of the parasite to other people is the introduction of preventive measures to control the transmission of the germs that are shed in feces [ ] . these precautions are especially important for people with weakened immune systems [ ] . infection is initiated by ingestion of oocysts, each of which contains sporozoites that hatch at the intestinal level, releasing infectious sporozoites [ , ] . after excystation, these sporozoites are ingested into a modified host membrane separated from the cytoplasm by a dense layer; then, the location of parasites within the host is not intracellular but extracytoplasmic [ ] . within the parasitophorous vacuole, the parasite undergoes asexual or schizogony reproduction, leading to the production of merozoites within a type i meront [ ] . the merozoites can invade the neighboring epithelial cells and propagate the infection to other sites of the intestines. during this stage, the merozoites can undergo distinct replicative cycles: an asexual stage characterized by multiplication of merozoites (type i meront) and production of thin-walled oocysts that autoinfect the host and/or a sexual stage with formation of type ii meront, which, after differentiation in microgametocytes and macrogametocytes, will unite to form the zygote [ ] . the diploid zygote, through a process called sporogony, will form sporozoites within thick or thin-walled oocysts. the thick-walled oocyst, protected by a resistant wall, after releasing in the feces is shed into the environment, ready to infect a new individual ( figure ) [ , ] . although the number of worldwide reported cases of cryptosporidiosis in the last years is increased with a number of cases per , population, numerous indicators (i.e., clinical symptoms) indicate that the frequency of infection is likely to be -fold higher than the number of reported cases [ ] . the prevalence of cryptosporidium infection is significantly lower in industrialized countries compared to developing countries since, in the latter, there are many people that still lack a basic level of drinking water and sanitation [ , ] . in developing countries, cryptosporidiosis is rarely reported in immunocompetent persons, while this infection causes approximately - % of cases of severe diarrheal illness mostly in malnourished children less than five years old [ , ] . cryptosporidium spp. have been isolated worldwide, and outbreaks of cryptosporidiosis associated with swimming pool or contaminated drinking water have been reported in several countries [ , ] . although there are more than species of cryptosporidium, only few species like c. hominis, c. parvum, c. meleagridis, c. felis, and c. canis are commonly found in people [ , , ] . among these species, c. hominis and c. parvum are most frequently found in intestinal infections in humans, although they differ in host range, with the former that infects exclusively humans, while the latter has an infectious cycle involving humans and ruminants, and thereby, usually c. parvum infects people who have close contact with large numbers of animals [ ] [ ] [ ] . although the transmission from animals is possible, it is very uncommon, and cryptosporidium infection transmitted from sheep, horses, goats, and rodents has been rarely reported [ , , ] . c. canis and c. felis, the dog-and the cat-adapted species, respectively, have been reported to cause infection in individuals without being sick at all [ , ] . however, human infections with cryptosporidium species from pets are very rare [ , ] . in immunocompetent persons, cryptosporidium infection usually produces a bout of watery diarrhea, although the infection in some persons may not lead to the symptoms [ , , ] . the disease is likely underestimated, since the diarrhea usually resolves without any treatment [ ] . although even people who do not have direct contact with animals may be infected, those who have direct contact with infected animals (particularly calves) or swallow pool water or drink untreated water are at a higher risk of contracting cryptosporidiosis [ , , ] . cryptosporidium infections are also more common in individuals who are in poor health or who have weakened immune systems (e.g., human immunodeficiency virus (hiv)/ aids, cancer, and transplant patients) [ , [ ] [ ] [ ] . between the parasites that caused about million deaths every year, cryptosporidiosis resulted in over , deaths [ , ] . moreover, cryptosporidium is one of the most important protozoan pathogen that cause waterborne outbreaks worldwide [ , , ] . cryptosporidium lives in the intestines of the infected individuals and animals in the form of oocysts, which will be released in the feces [ ] . after infection, the parasite have been released into the feces from an infected human or animal. in the gastrointestinal tract, the oocysts are broken releasing sporozoites (a) that invade the mucosa to establish a cycle of repeated endogenous reinfection (endogenous autoinfection). the trophozoites remain in the apical portion of the cell (b) and undergo asexual division (merogony) to form merozoites (c). the merozoites released from type i meronts enter adjacent host cells to form type ii meronts. type ii meronts do not recycle but enter host cells to form the sexual stages (d). the type ii meronts form merozoites that differentiate in sexual reproductive microgamonts (male) and macrogamonts (female) (e). after fertilization, the zygote develops into resistant thick-walled oocysts (f) that are excreted from the host in the fecal material. however, some oocysts are thin-walled (g) and excyst within the same host in a process of autoinfection (h) alters the function of the intestinal barrier, increasing its permeability, absorption, and secretion of fluid and electrolytes, and thereby, the severity, persistence, and outcome of the infection depend on the degree of the immunocompromised status [ , ] . the oocysts are very resistant to chlorine, chloramines, and chlorine dioxide, which are commonly used in methods of water system disinfection, and remain vital for infection in the environment for a long time [ ] . humans become infected with cryptosporidium by touching anything that has come in contact with contaminated feces, although the most common mode of transmission is represented by ingestion of oocysts in contaminated food and water or air [ , ] . recent studies indicate that cryptosporidiosis may be transmitted by inhalation of aerosolized droplets via respiratory secretions or by coughing, in addition to the well-documented fecal-oral transmission [ ] . pulmonary infections also have been reported [ , ] . immunocompromised hosts are more susceptible to infection than people with a healthy immune system, and in the subjects with hiv/aids, the parasite often causes a chronic, prolonged form of a disease, which is difficult to treat and can even result in death [ ] . in these patients, fever and malabsorption are common, and the parasite can cause inflammatory disease of the biliary tree leading to biliary tract obstruction, sclerosing cholangitis, papillary stenosis, and pancreatitis [ , ] . for this reason, cryptosporidiosis is considered one of the riskiest opportunistic infections for patients with acquired immune deficiency syndrome [ ] . the diagnosis of cryptosporidiosis is usually made by microscopic detection of the parasite oocysts, oocyst antigens, or oocyst dna in stool samples. since the most common symptom of cryptosporidiosis is a watery diarrhea, the differential diagnosis for cryptosporidium includes bacterial, viral, and parasitic enteric pathogens associated with acute diarrhea such as rotaviruses, coronaviruses, escherichia coli, and salmonella spp. [ , ] . however, gastrointestinal disorders may also have noninfectious causes, such as inflammatory bowel disease in humans [ ] . diagnosis of cryptosporidiosis is usually made by microscopically identifying the presence of oocysts of to μm in diameter in the stool of the infected subjects [ , ] . however, since the detection of cryptosporidium oocysts can be difficult, three fecal samples collected on separate days should be microscopically examined for detection of oocysts prior to exclude a cryptosporidium infection in subjects with severe diarrhea [ , ] . in addition, for detection of oocysts in stool, sample must be concentrated using the formalin-ether sedimentation method prior to microscopic examination [ ] . the oocysts of cryptosporidium can be also observed by acid-fast (modified ziehl-neelsen method) or phenol-auramine staining on unconcentrated fecal smears, where the oocysts stain red and bright yellow, respectively [ , ] . however, much attention should be given to this staining since the oocysts may also appear as "ghost" cells [ ] . in addition, although the oocysts of cryptosporidium are half the size of those of cyclospora cayetanensis (about - μm in diameter vs. - μm in diameter), another coccidian protozoan parasite that infects the intestine of humans causing acute diarrhea, much attention should be given when evaluating stool samples since the oocysts of both parasites are autofluorescent and acid-fast ( figure ) [ , ] . in addition, although c. cayetanensis has a life cycle similar to cryptosporidium, its oocysts are unsporulated and not infective when shed in the feces, and thereby, direct fecal-oral transmission cannot occur [ ] . although routine diagnosis of cryptosporidiosis is generally made by the microscopic identification of oocysts in fecal smears, this method, despite being easy to use and low cost, unfortunately has a low sensitivity (≤ %). moreover, accurate diagnosis of cryptosporidiosis using this technique is largely dependent on the experience of the microscopist. sensitivity can be improved by performing modified acidfast stain, a staining generally performed if there are structures suspicious for cryptosporidium, which has been reported to be associated with a sensitivity of %. however, these methods cannot distinguish between cryptosporidium species [ , ] . in addition to the above described methods, watery or mushy stools can be examined for the laboratory diagnosis of cryptosporidiosis using different techniques such as the enzyme-linked immunosorbent assay (elisa) and immunochromatographic test, which have good sensitivity and specificity for detection of cryptosporidium antigens [ ] [ ] [ ] . although the commercial kits have a range of sensitivities and specificities higher than that of the microscopic methods (ranging from to %), previous studies have shown that these antigen/antibody-based detection methods are also ineffective if the burden of this parasite in the patients is below the minimum threshold [ ] . in addition, these methods are more expensive than polymerase chain reaction (pcr), which is now accepted in most laboratories as the gold standard for the detection of this parasite in the stool. previous studies have shown that compared to pcr, microscopy, elisa, and immunochromatographic test (ict) are less convenient in terms of cost, sensitivity, and specificity and also are more time consuming [ ] [ ] [ ] . although there have been important advances in diagnostic tools (i.e., the availability of multiplex pcr assays for the detection of intestinal protozoa), the accessibility to this molecular technique is limited in some labs and totally absent in others. in addition, the expense and requirement for technical expertise have limited their use particularly in high-prevalence regions such as developing countries. despite the global prevalence and the high impact of cryptosporidiosis, principally in immunocompromised patients, major deficiencies exist in the current control programs, especially in terms of available diagnostic tools. in addition, most common diagnostic tests tend to misdiagnose the disease in endemic areas. in particular, microscopic techniques are the most widely used method for detection of cryptosporidia in stool samples, and the diagnostic accuracy of these methods is largely dependent on the experience of the microscopist. since early diagnosis is the best way to fight the infection, there is a need to develop molecular techniques that are sensitive, specific, easy to perform, cost-effective, and highthroughput. this work was supported by fondo per il finanziamento delle attività base di 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vectors parasitic infection among hiv/aids patients at bela-bela clinic, limpopo province, south africa with special reference to cryptosporidium cryptosporidium infection in solid organ transplantation burden of disease from cryptosporidiosis widespread occurrence of cryptosporidium infections in patients with hiv/aids: epidemiology, clinical feature, diagnosis, and therapy post-infection symptoms following two large waterborne outbreaks of cryptosporidium hominis in northern sweden cryptosporidium parvum disrupts intestinal epithelial barrier function via altering expression of key tight junction and adherens junction proteins intestinal and pulmonary infection by cryptosporidium parvum in two patients with hiv laboratory diagnosis of cryptosporidiosis laboratory diagnosis of cryptosporidiosis comparative diagnostic techniques for cryptosporidium infection comparison of current methods used to detect cryptosporidium oocysts in stools differences in the detection of cryptosporidium and isospora (cystoisospora) oocysts according to the fecal concentration or staining method used in a clinical laboratory detection of intestinal protozoa in the clinical laboratory evaluation of four commercial rapid immunochromatographic assays for detection of cryptosporidium antigens in stool samples: a blind multicenter trial multisite performance evaluation of an enzyme-linked immunosorbent assay for detection of giardia, cryptosporidium, and entamoeba histolytica antigens in human stool evaluation of an immunoassay-based algorithm for screening and identification of giardia and cryptosporidium antigens in human faecal specimens from saudi arabia evaluation of the roche lightmix gastro parasites multiplex pcr assay detecting giardia duodenalis, entamoeba histolytica, cryptosporidia, dientamoeba fragilis, and blastocystis hominis is real-time pcr-based diagnosis similar in performance to routine parasitological examination for the identification of giardia intestinalis, cryptosporidium parvum/cryptosporidium hominis and entamoeba histolytica from stool samples? evaluation of a new commercial multiplex pcr assay and literature review comparison of three commercial multiplex pcr assays for the diagnosis of intestinal protozoa all authors conceived the original idea, cb wrote the manuscript with support from eg and vml. the authors declare no conflict of interest. key: cord- -lvm o r authors: woo, bean; baek, kwang-hyun title: regulatory interplay between deubiquitinating enzymes and cytokines date: - - journal: cytokine growth factor rev doi: . /j.cytogfr. . . sha: doc_id: cord_uid: lvm o r deubiquitinating enzymes (dubs) are cysteine protease proteins that reverse the ubiquitination by removing ubiquitins from the target protein. with over dubs identified and categorized into at least families, many dubs interact with one or more cytokines, influencing cellular processes, such as antiviral responses, inflammatory responses, apoptosis, etc. while some dubs influence cytokine pathway or production, some dubs are cytokine-inducible. in this article, we summarize a list of dubs, their interaction with cytokines, target proteins and mechanisms of action. ubiquitination and deubiquitination are post-translational modifications for numerous proteins, which in turn affect many physiological processes. ubiquitination is defined as an attachment of one or more ubiquitin (ub) molecules onto the target protein through the function of a series of proteins: e , e and e ( fig. ) [ ] . seven lysine residues have been identified (k , k , k , k , k , k and k ) on the ubiquitin molecule [ ] . also, additional ub molecules can be attached onto one of the seven lysine residues or the n-terminal methionine to form polyubiquitin chains (polyub) [ , ] . perhaps, ub is most well-known as the crucial marker of the ubiquitin-proteasome system (ups), in which ubiquitinated proteins enter proteasomal degradation via s proteasome ( fig. ) [ ] . however, ub also affects many other aspects of tagged proteins, such as localization, protein interaction, function, etc. [ ] . on the other hand, deubiquitination refers to the process that reverses ubiquitination via deubiquitinating enzymes (dubs) (fig. ). little less than human dubs have been identified so far [ ] . dubs were categorized into five different families in the past [ ] , but at least seven different families are identified as of now, which include the ubiquitin-specific protease (usp), ubiquitin carboxyl-terminal hydrolase (uch), machado-josephin disease protein (mjd), ovarian tumor (otu), jab /mpn/mov (jamm), permutated papain fold peptidases of dsrna viruses and eukaryotes (pppde) and miu-containing novel dub family (mindy) [ , ] . cytokines are groups of small proteins that play a role in cell signaling and immune system by binding to their respective receptors. since dubs regulate diverse physiological processes, it was to be expected that dubs and cytokines affect one another. as anticipated, the more studies were performed regarding the functions of dubs, the more interaction between dubs and cytokines were revealed. recently, several reviews dealing with how dubs affect pathway of a specific cytokine were published [ ] [ ] [ ] , but none has yet introduced as a whole the interaction between dubs and cytokines. in this review, we wish to provide a brief overview of the dubs discovered to regulate cytokine signaling pathways and cytokine-inducible dubs. we will discuss the dubs that influence the pathways of interferons (ifn), tumor necrosis factors (tnf), tnf-related apoptosis-inducing ligand (trail), interleukins (il) and chemokines. ifn-α and ifn-β cytokines belong to type i ifn family that are essential for antiviral responses, cancer, inflammation, etc. [ ] . when a cell recognizes a viral infection through detecting ifn-stimulating signaling molecules or foreign double stranded dna in the cytosol, retinoic acid-inducible gene-i (rig-i) is activated, triggering the cascade of the second messenger system to activate and translate ifn-α and ifnβ signaling pathways (fig. ) [ ] . dubs interact with some of the key molecules in the ifn signaling pathway, which include, but are not limited to, rig-i, stimulator of interferon genes (sting), tumor necrosis factor receptor-associated factors (trafs), interferon regulatory factor are summarized in table . we will discuss the dubs in the order of ifn signaling pathway shown in fig. . the first group of dubs are those that deubiquitinate rig-i to inhibit the production of ifn. rig-i is a cytosolic protein that plays a significant role in ifn signaling by detecting viral dna and rna [ , ] , which is then ubiquitinated by tripartite motif (trim) to activate the signaling cascade to synthesize ifn [ ] . orf , usp , usp , usp , usp , cylindromatosis (cyld), porcine epidemic diarrhea virus papain-like protease (pedv plp ) and transmissible gastroenteritis virus papain-like protease (tgev pl ) are the dubs found to deubiquitinate rig-i. we will discuss them one by one. in a study using hek t cells and sendai virus (sev), knockdown of the gene transcribing usp resulted in upregulation of type i ifn, while overexpression of usp decreased type i interferon as usp showed dose-dependent inhibition of ifn-β [ ] . further experiment supported the idea that usp deubiquitinates k -polyub from rig-i [ ] . however, when the effects of usp with a mutated catalytic site and wild type usp were compared, the results were surprisingly similar, indicating that usp 's catalytic activity is not necessary for it to inhibit ifn synthesis [ ] . orf is a dub activity containing tegument protein, found within kaposi's sarcoma-associated herpesvirus (kshv) and murine gamma herpesvirus (mhv ) [ , ] . the expression of kshv orf in hek t cells led to suppression of both rig-i-induced and sev infection-induced ifn-β promoter activation [ ] . on the contrary, kshv orf -c g mutant, with defective deubiquitinating activity, resulted in lesser to no suppression, confirming the influence of orf on the ifn synthesis [ ] . also, the overexpression of trim , but not the mutant trim , reversed the orf 's effect on ifn production and reverted the ubiquitination of rig-i, further confirming the result [ ] . it is also noteworthy that orf was not capable of suppressing mavs-induced activation of ifn-β production [ ] . when mhv infected bone marrow derived dendritic cells (bmdc) were induced by mcmv and hsv- for type i ifn induction, no ifn was detected, but tnf-α, il- and il- β were expressed upon high dose stimulation [ ] . this provided evidence that mhv induced innate immunity of the host to a lesser extent [ ] . on the other hand, orf mutant mhv stimulated innate immune response [ ] . by utilizing dub activity of orf , mhv blocked viral dna induced, sting-mediated ifn production [ ] . in a study performed by zhong et el, usp , too, was found to deubiquitinate rig-i and reduce sev-induced ifn-β production in hek t cell line [ ] . knockdown of usp gene also led to augmentation of isre promoter upon sev induction [ ] . mutating the catalytic residue of usp was sufficient to block usp 's effect on ifnβ induction, supporting that ifn-β suppression via usp is dub activity dependent [ ] . usp targeted not only rig-i for deubiquitination, but also extended to traf [ ] , traf [ , ] and traf [ ] and to affect ifn signaling. usp also deubiquitinated traf and traf to regulate in il- signaling [ ] . however, some studies have given different results regarding usp 's ability to deubiquitinate traf . lin et al. stimulated usp knockout bmdc with sev or hsv- infection and added wild type (wt) usp or mutant usp , but k -ub of traf did not differ from one another [ ] . however, zhong et al.'s study using hek t cells supported deubiquitination of traf by usp [ ] . this variance in the result may be caused by the difference of the cell line used for the studies. furthermore, usp showed its ability to suppress phosphorylation of interferon regulatory factor (irf ) and p , also contributing to inhibition of ifn promoter activation [ ] . fan et al., knowing that usp inhibits rig-i-induced ifn-β production, searched for its mechanism [ ] . they unveiled that usp fig. . mechanism of action of ubiquitin proteasome system and deubiquitinating enzymes. ub attaches to the target protein by going through a series of reaction with e (ubiquitin activating), e (ubiquitin conjugating) and e (ubiquitin ligating) enzymes. a target protein could be ubiquitinated once or multiple times on lysine residues. s proteasome identifies target proteins with polyub chain and degrades them into amino acid segments and reusable ub. ubiquitinated proteins could also be deubiquitinated by dubs, resulting in a different fate. inhibited isre reporter activity induced by sev and rig-i-card, but not by tank-binding kinase (tbk ) in mouse embryonic fibroblasts (mef) cells [ ] . usp deubiquitinated rig-i in hek t cells [ ] . also, they found that usp 's function regarding antiviral response is compatible in mef and hek t cell lines by introducing each cell line's usp to the other cell line and observing the effect [ ] . usp 's specificity to rig-i was also confirmed in hela cells through coimmunoprecipitation (co-ip) of usp with rig-i, using rabbit polyclonal antibodies against usp [ ] . usp also deubiquitinated mda to inhibit antiviral response [ ] . usp is also a dub that deubiquitinates k -polyub chain of both rig-i and mda and suppresses ifn-β activation [ ] . usp 's effect was found viable in t, thp- , human peripheral blood mononuclear cells (pbmcs) and raw . cells, supporting that usp 's activity is viable in both human and murine cells [ ] . usp did not inhibit mavs, sting, tbk , irf and tirf, as demonstrated by isre-luc activity induction test [ ] . also co-ip demonstrated the interaction between usp and stimulated rig-i or mda , but not the unstimulated ones, supporting that ligand stimulation is required for usp to interact with rig-i or mda [ ] . more specifically, poly(i:c) (lmw) stimulation leads usp to have a strong interaction with rig-i, but a weak one with mda , while poly(i:c) (hmw) stimulation leads usp to have a strong interaction with mda , but a weak one with rig-i [ ] . cyld is another dub that removes k -ub chain from rig-i to decrease the ifn production [ , ] , but tbk and ikkε were also identified as the target of the deubiquitination of cyld in ebna cells [ ] , resulting in the same effect. cyld also interacted with ips- to negatively regulate it, but did not deubiquitinate it [ ] . schmid et al. found that in brain and peripheral blood of c bl/ , the mrna level of ifn-γ gene decreased with the knockdown of cyld, while the serum concentration of ifn-γ increased [ ] . a study conducted using human kidney mesangial cells (mc) showed slightly different results: silencing cyld in mc cells and stimulating them with poly ic increased the toll-like receptor (tlr )-induced activation of rig-i and mda [ ] ; however, the level of mrna of rig-i and mda actually decreased [ ] . the authors speculated this difference to be caused by the change in cell line used [ ] , but further study is necessary to determine the cause. cyld also decreased ifn promotor activation by deubiquitinating traf and traf in hek t cells, respectively [ , ] . cyld in u os/nod cells were found to deubiquitinate k -ub of ripk proteins, especially ripk , to suppress nod -induced nf-κb activation [ ] . when cyld was suppressed, ubiquitinated receptor interacting protein kinase (ripk ), also called rip , and ripk proteins accumulated within cells [ ] . plps, first discovered in coronavirus in [ ] , are multifunctional proteins with dub activity that are synthesized by many families of viruses that regulate ifn signaling pathway by interacting with rig-i [ ] [ ] [ ] [ ] . recently, the mechanism by which pedv plp suppresses ifn production in the host cell was identified. in hek t cells, pedv plp was found to deubiquitinate rig-i and sting, thereby affecting its downstream pathway, resulting in suppression of ifn production [ ] . tgev pl also was revealed to bind and deubiquitinate both rig-i and sting in hek t cells [ ] . studies on middle east respiratory syndrome coronavirus encoded papain-like protease (mers-cov pl pro ) showed that it also has a dub function [ ] . this was supported by a study by bailey-elkin et al., in which they obtained the crystal structure of pl pro -ub complex and showed that wt mers-cov pl pro , but not the dub mutant pl pro , suppresses ifn-β promotor activity [ ] . the targets of mers-cov pl pro were identified as rig-i, mda and mavs [ , ] . mers-cov pl pro and severe acute respiratory syndrome coronavirus (sars)-cov pl pro inhibited the proinflammatory signaling in hek t cells upon mda stimulation, which included decreased expression of ccl and ifn-β and decreased level of cxcl mrna [ ] . another study on sars-cov pl pro found that irf is ubiquitinated and that deubiquitinating activity of sars-cov pl pro was required for it to affect irf [ ] . sars-cov pl pro did not affect irf in other means, such as dimerization or nuclear translocation [ ] . dub domain mutated plp of equine arteritis virus (eav) also increased in expression of ifn-β and il- in equine long fibroblasts (elf) [ ] . plp domain was also found in nsp protein, which will be discussed in a later section. sting is a transmembrane protein found in mitochondria and endoplasmic reticulum that regulates ifn-promotor activation at the downstream of rig-i [ ] . zhang et al. studied the effect of usp (also known as ubp ) on sting and revealed that usp interacts with sting to affect ifn-promotor activity [ ] . however, when usp -/-mef cells with either wt usp or dub activity-mutated usp were induced with hsv- , hcmv or cytosolic dna, ifnb, ifna , tnf, il- or cxcl genes increased in expression, indicating that the deubiquitinating activity of usp is not responsible for this phenomenon [ ] . subsequently, they searched for dubs that interact with usp and found that knockdown of usp inhibited usp -induced deubiquitination of sting and knockdown of usp inhibited usp -induced deubiquitination of sting [ ] . immunoprecipitation revealed that sting, usp and usp are arranged as usp -usp -sting, but both usp and usp were associated with the n-terminus of sting [ ] . usp deubiquitinated k -or k -linked ubiquitin of sting [ ] . these results together supported that although usp does not deubiquitinate sting itself, usp recruits usp to deubiquitinate sting to suppress ifn synthesis [ ] . another way that usp inhibited nf-κb activation is by deubiquitinating k -ub of tak and nemo [ ] . usp strongly interacted with tak -tab and dub activity dependently deubiquitinated k -ub of tak in t cells [ ] and in th cells [ ] . usp also fig. . tlrs, ifnari and ifnarii induced ifn production pathway. pamps, ifn-α and ifn-β stimulate tlr and ifnar i & ii receptors respectively to induce ifn production as well as nf-κb activation. dubs that play a role in these pathways are indicated in the figure to show the mechanism of their action. decreased k -ub of nemo [ ] . in a study by malakhova et al., usp inhibited ifn-induced gene activation by affecting jak-stat signaling pathway in t cells [ ] . their study showed that usp does not interact with ifnar , but with box -box region of ifnar to disrupt its interaction with jak to inhibit jak's tyrosine kinase activity in a dub activity independent manner [ ] . consistent with this, usp knockout murine cells displayed hyperactivity towards type i ifn signaling, resulting in the increase of the level of phosphorylation of stat and stat [ ] . usp 's interaction with ifnar also interfered with ifnar 's ability to recruit ifnar , hindering ifn i signaling [ ] . herpes simplex virus (hsv- ) invades a host and escapes its ifn-mediated innate immunity by encoding a large tegument protein, ul , which has a motif with dub activity, named ul ubiquitin-specific protease (ul usp) [ ] . when hek t cells were transfected with markers of co-ip and ul usp or the c a (a dub motif mutant) and then infected with sev, the result showed reduction of ubiquitination of traf in cells with wt ul usp, while c a has no reduction of ul usp's ubiquitination [ ] . the result supported that ul usp deubiquitinates traf molecules to inhibit ifn-promotor activation [ ] . in a different study regarding the function of ul usp, it was found that ul usp inhibits cgas and sting dependent ifn-β production [ ] . nf-κb activation from overexpressing sting, tbk , ikkα and ikkβ was also inhibited, but not from overexpressing p [ ] . in this study, human foreskin fibroblast (hff) cells were infected with either hsv- or hsv- c a mutant and stimulated with ifn stimulatory dna [ ] . as a result, the level of endogenous iκbα in hff cells with mutant hsv- significantly decreased compared to hff cells with wt hsv- , supporting that ul usp decreases the degradation of iκbα in a dub activity-dependent manner [ ] . additionally, co-ip study in hek t cells showed decrease in ubiquitination of iκbα in those transfected with wt ul usp, but not in those transfected with c a mutant [ ] . taken together, ul usp deubiquitinates iκbα to inhibit its degradation, suppressing nf-κb activity [ ] . a study by lin et al. revealed that usp is required for both dna and rna virus-induced signaling [ ] . supporting this claim, silencing usp in mefs or mouse lymphatic fibroblasts (mlf) led to inhibition of expression of ifna , tnf, and il- upon triggering them with sev, vesicular stomatitis virus (vsv) or poly(i:c) [ ] . also, the level of ifnα and il- was reduced in mlfs, bmdcs or flt lpdc cells with usp knockdown [ ] . usp 's dub activity was also found to be necessary for virus-induced signaling, as usp knockdown mefs with wt usp reconstitution allowed expression of ifnb, ifna and il- upon sev or hsv- induction, while those with dub activity mutant usp did not [ ] . overexpressing usp in hek t cells resulted in reduction of irf phosphorylation when stimulated with sev, leading to inhibition of nf-κb activity [ ] . isre reporter activity was also inhibited by usp in a dose-dependent manner [ ] . taking it one step further, lin et al. uncovered that usp stabilizes traf in a dub activity dependent manner by deubiquitinating k -ub of traf in bone marrow-derived macrophages (bmdm) cells, inhibiting tlr signalinginduced innate immune responses [ ] . the nonstructural protein (nsp ) is a viral protein with deubiquitinating activity in its plp domain, which was found in scov and in mouse hepatitis virus a (mhv-a ) [ , ] . infecting mef cells with mhv-a did not result in detectable ifn-β induction, while infecting them with sev (the control) did result in ifn-β responses [ ] . when cells were given variants of nsp , the ifn-β induction only took place in cells that lacked wt plp domain [ ] . when the plp domain was present, ifn-β induction was suppressed upon viral infection [ ] . moreover, polyub of irf , which is necessary for ifn-β induction, was deubiquitinated in the presence of plp , which was further confirmed by co-ip indicated formation of a complex of plp and irf [ ] . this deubiquitination inhibited nuclear translocation of irf [ ] . in hek t cells and mef cells, k -polyub was also deubiquitinated by the plp domain of nsp , inactivating tbk -irf complex in the cytoplasm [ ] . monocyte chemotactic protein-inducing protein (mcpip ) is a protein, common to human and mouse, with dub activity toward traf , traf and traf , thereby inhibiting jnk and nf-κb signaling [ ] . a more recent study has uncovered through co-ip in sev infected hek t and hela cells that mcpip interacts with irf and through confocal microscopy that transfection with mcpip inhibited nuclear translocation of irf [ ] . additionally, the presence of mcpip inhibited traf and tbk activated ifn-β expression [ ] . co-ip also revealed possibility of mcpip to interact with ips- and ikkε as well [ ] . a (also known as tnfaip ) inhibited lps-induced nf-κb activity in mef cells in a study by boone et al. [ ] . upon further testing in hek t, they found that wt a , but not dub activity domain mutant a removed k -ub from traf [ ] . in raji cells, the n-terminal dub domain of a interacted with and deubiquitinated irf [ ] . however, in vitro study showed no interaction between a and irf , which is likely due to requirement of other intracellular proteins [ ] . irf has been known to be activated by epstein-barr virus (ebv)'s oncoprotein called latent membrane protein (lmp ) [ ] . a has been known for a long time as a negative regulator of nf-κb pathway mediated by rig-i. a does interact with rig-i, and suppresses rig-i-mediated nf-κb pathway [ ] , but whether a deubiquitinates rig-i or not still requires confirmation. similar to orf in mhv [ ] , bplf is an ebv encoded large tegument protein with dub activity that opposes tlr signaling in the host [ ] . gent et al.'s research revealed that bplf deubiquitinates traf and nemo in t cells [ ] . immunoprecipitation in t cells revealed that k -ub of traf and nemo was reduced when wt bplf was expressed, while mutant bplf did not [ ] . k -ub of iκbα was also identified as a target of bplf 's dub activity [ ] . the brcc isopeptidase complex (brisc) is a nuclear dub complex, composed of abraxas, brcc , brcc and merit , capable of deubiquitinating k -ub [ ] . in a study by zheng et al., serine hydroxymethyltransferase (shmt) formed a complex with brisc to form brisc-shmt complex [ ] . shmt allowed interaction of brisc with ifnar to deubiquitinate k -ub of ifnar , reducing ifnar 's internalization and degradation by lysosome [ ] . taken together, brisc is the first dub complex we discussed that works to actually increase responses to ifn. tnf is a cytokine that plays a significant role in inflammation and regulation of immune cells. since tnf shares some of its pathway with ifn, studies that focused on tnf rather than ifn are included in this section for the purpose of this review (fig. ) (table ) . usp was identified by studies to negatively regulate both tnf-αand il- β-induced nf-κb activation [ ] [ ] [ ] . jiang et al. observed that introducing small interfering usp (siusp ) to decrease usp level in microglia from the spinal cord of sprague-dawley rats led to an increase in p-p and traf expression as well as secretion of tnf-α and il- β, all of which decreased upon introduction of ha-usp plasmid [ ] . xiao et al.'s study added on to this by demonstrating usp 's interaction and deubiquitination of traf and traf , but not traf , both in vivo, in hek t cells, and in vitro [ ] . as a result, usp negatively influenced tnf-α-induced-nf-κb activation-mediated cytokine induction, including il- and il- in a and h cells [ ] . usp also deubiquitinates tak in hek t cells [ ] . usp also protected iκbα from degradation, [ ] , a necessary step for tnf-α-induced nf-κb activation [ ] . this was further supported by knockout of usp aiding iκbα degradation [ ] . l. infantum otubain (otuli) has been shown to induce inflammatory responses in peritoneal macrophages from c bl/ , shown by production of tnf-α and il- as well as lipid droplet synthesis [ ] . also otuli demonstrated strong dub activity on k -ub and weak activity on k -ub in vitro at ph . [ ] . we mentioned in a previous section that usp deubiquitinates traf and interacts with traf , increasing expression of tnf in hek t cells [ , ] , while in mef cells, usp negatively affects tnf-α-induced nf-κb activation [ ] . a also affects tumor necrosis factor receptor (tnfr ) signaling pathway. in bmdms and bmdc, a worked together with tax bp to interact with ubc , an e enzyme, resulting in the inhibition of e ligase activities of traf , traf and ciap [ ] . futhermore, a and tax bp participated in degrading ubc upon il- and tnf-α stimulation in mef cells [ ] . a 's zf motif was found to recruit a dimers to bind with ripk in the tnfr signaling complexes and inhibit ubiquitination of k -ub and k -ub chains of ripk , hindering tnf signaling [ ] . in intestinal epithelial cells (iec), a dimer interacted with the ripoptosome (also known as complex iia), which allowed ubiquitins on ripk to sustain, increasing caspase- activation to promote tnf-induced apoptosis [ ] . however, this effect was not dub activity dependent [ ] . cezanne is a dub that belongs to the a subgroup of otu family. similar to a , cezanne also was shown to suppress nf-κb signaling by deubiquitinating k -polyub from ripk [ , ] and traf [ ] . also, cezanne was recruited to the activated tnfr prior to deubiquitinating ripk , which was dependent on the ubiquitin-associated (uba) domain of cezanne [ , ] . consistnent with the findings, inhibiting cezanne production via sirna resulted in an increased production of il- upon tnf-α stimulation [ ] . cezanne's dub activity was required for inhibiting phosphorylation and degradation of iκbα [ ] . usp (also known as usp ) interacted with and deubiquitinated traf in beas b cells [ ] . a noteworthy fact is that traf in jnk pathway was targeted by usp , but not traf in nf-κb signaling [ ] . trail is a cytokine that binds to death receptors (dr) and induces apoptosis, especially in tumor cells. its specificity for tumor cells have made trail and its receptor as the targets for anti-cancer therapeutics. trail inducing dubs are also summarized in table . in a study of malignant mesothelioma, a loss of function mutation of brca associated protein (bap ) resulted in increased sensitivity fo trail induction [ ] . when testing for domains that play a role in trail sensitivity in h mm cells, only asxl / binding site-mutated bap and dub domain-mutated bap resulted reduction in rtrail sensitivity, indicating that asxl / binding sites play a role in trail sensitivity [ ] . this was in congruence with the fact that bap binds to asxl to form the polycomb repressive deubiquitinase complex (pr-dub) that deubiquitinates histone h a [ ] . also, flow cytometry analysis confirmed that the mutation of c a, or the deubiquitinating domain, of dub resulted in decreased expression of dr and dr in h cells [ ] . only dr expression increased in h cells upon bap knockout [ ] . on the same line with bap , usp knockout also increased trail sensitivity [ ] . a study by leznicki et al. introduced three isoforms of usp in hek cells: usp iso , usp iso and usp iso , although the focus was on the first two [ ] . usp iso was revealed as an integral membrane protein on endoplasmic reticulum, while usp iso was identified as a cytosolic protein [ ] . moreover, the proteins that they interacted with also varied [ ] . usp iso led to er stress, bap cleavage and activation of caspase- and caspase- , resulting in apoptosis [ ] . usp iso upregulated c/ebp homologous protein (chop) and dr in u osfipin and hela fipin cells [ ] . on the other hand, overexpressing usp iso exerted an opposite effect of delaying caspase- processing in trail-induced apoptosis [ ] . this effect was dependent on dub activity [ ] . b-ap is a small therapeutic molecule that inhibits usp and uchl [ ] . b-ap has been identified as an agent that increases trail receptors on many types of cancer cells, increasing their likelihood to enter apoptosis via nk cells [ ] . introduction of b-ap in a , hct and calu- cells increased the level of dr , but not the other death-inducing signaling complex (disc) components [ ] . the increase in the level of dr protein was due to reduction in the degradation of dr , leading to an increase in trail-induced apoptosis [ ] . in a different study, caspase-denependent apoptosis was increased in mantle cell lymphoma (mcl) cells when exposed to b-ap , which was confirmed with addition of pan-caspase inhibitor zvad-fmk, which resulted in an inhibition of apoptosis [ ] . this study indirectly demonstrated that usp and/or uchl partakes in decreasing dr expression. mcpip is another dub that decreases dr [ ] . exposing mda-mb- cells to doxycycline (dox) led to induction of mcpip , which then led to a decrease in dr [ ] . similarly, when a human lung cancer cells were exposed to mcp , mcpip level increased in a dosedependent manner, also resulting in a decrease in dr [ ] . likewise, dr level increased when mcpip was knocked down via short hairpin rna (sirna) [ ] . mcpip successfully achieved this by deubiquitinating dr , thereby stimulating lysosomal degradation of dr [ ] . also, the increase in dr level following mcpip knockdown catalized the formation of disc during the dr -induced apoptosis [ ] . we will now discuss dubs that induce interleukins, which are listed in table . usp has been shown to deubiquitinate traf and tlr and to interact with traf , increasing expression of il- in mef cells [ , ] . on top of decreasing production of ifn [ , ] , orf in mhv also induced the production of il- β [ ] . il- β production was dependent on nlrp and asc, rather than aim [ ] . usp negatively regulates il- β-induced nf-κb activation [ ] [ ] [ ] . increases (c bl/ lung homogenate) [ ] increase = inc. in production, + = induce positive effect. eeyarestatin i (esi), a small molecule that inhibits deubiquitination, has been found responsible for blocking il- β release [ ] . lopex-castejon et al., who reported this finding speculated uch or usp was responsible for this phenomenon, but futher study showed that they do not regulate il- β secretion individually or cooperatively [ ] . this result left possibility of an uncharacterized dubs or an additional dub(s) partaking in the process. usp knockout murine splenocytes and naïve t cells produced more il- compared to the wt splenocytes [ ] . under th polarizing condition, il- production was significantly higher in the usp knockout naïve cd + t cells compared to the wt naïve cd + t cells [ ] . also usp knockout naïve cd + t cells underwent hyperproliferation under th polarizing condition, which was reversed by adding il- neutralizing antibody [ ] . taken together, usp downregulates il- synthesis and tcr-induced t cell proliferation [ ] . additional mechanism of action has been discussed in the previous seciton. dufner et al. found that usp was essential in t cell maturation and homeostasis, although it was not required for negative selection [ ] . inhibiting usp leads to decrease in il- ra mrna as well as ccr [ ] . also, il- , il- p , ifn-γ and tnf levels were increased in the blood of usp f/f cd -cre mice than usp f/f [ ] . deleting otulin gene in mouse immune cells, t, b, natural killer cells (nk), dendritic cells (dc) and macrophage cells, resulted in production of cytokines specifically responsible for acute systemic inflammation, such as tnf, il- β, il- , mcp- , mip- α and g-csf [ ] . cytokines responsible for adaptive immunity were not affected by the deletion [ ] . it is noteworthy that this study suggests that although many cytokines partake in the inflammatory response in murine cells without otulin, the primarily responsible cytokine is tnf [ ] . unlike in immune cells, deleting otulin in myeloid cells resulted in activation of cytokines responsible for acute and chronic inflammation as well as autoimmunity, showing an increase in the level of out of cytokines tested [ ] . deficiency of otulin in macrophages resulted in nf-κb activation without an induction, which was due to the inability to manage polyub chains synthesized by linear ubiquitin chain assembly complex (lubac) [ ] . another study found that otulin overexpression inhibited tnf-α-induced nuclear translocation of p in hek t cells [ ] . interestingly, both wt and dub domain mutant otulin disabled lubac-induced nf-κb activation, indicating that otulin-mediated met -polyub is not the only factor influencing nf-κb activation [ ] . otulin has also been identified to deubiquitinate met -polyub of ripk and inhibited the binding of nemo and ripk , blocking the tnf-α-induced nf-κb response [ ] . ebv bplf suppresses ifn production, but il- production by cells upon malp- ligand stimulation was also abrogated upon bplf expression [ ] . ebv bplf suppressed production of proinflammatory cytokine il- in -tlr /cd cells [ ] . trabid is a dub from otu family, translated from the gene zranb [ ] . upon zranb knockout in mice, il- , tnf, il- a, il- b and il- a displayed a decrease in expression [ ] . trabid's deubiquitinating activity was necessary for recruiting c-rel and p to il- promoter by influencing histone modifications [ ] . knockout of trabid rendered bmdc incapable of producing il- and il- , leading to a decrease in the number of differentiation of cd + t cells to t h and t h cells, which was reversible with adding il- and il- [ ] . not many studies have focused their study objectives on discovering relationship between chemokines and dubs. some discovered interactions are listed in table . we have discussed how dubs induce cytokine production, signaling and effects. compared to dub's effects on cytokines, cytokine-inducible dubs are far less studied due to the difficult nature of planning such studies. however, this information can be as important as dub-induced cytokines. we will now discuss some known examples of cytokine-inducible dubs, as listed in table . dub- is one of the early identified cytokine-inducible dubs. studying the sequence of dub- gene, unveiled that it contained a il- inducible enhancer in ba/f murine lymphocyte cell line [ ] . the timing of il- induced dub- mrna increase was identified as early g phase [ ] . moreover, when dub- was constitutively expressed, majority of ba/f cells were arrested in g phase of the cell cycle, which was dub activity dependent [ ] . induction of dub- was dependent on viable jak and raf- , but not stat , suggesting that dub- expression is dependent on two pathways: jak and ras/raf- /mapk pathway [ ] . il- and granulocyte-macrophage colony-stimulating factor (gm-csf) also induced dub- transcription, which supported that β common (βc) subunit plays a part [ ] . dub- a decreased in expression when jak was suppressed, also suggesting dub- a to be affected by il- and jak pathway [ ] . dub- is similar to dub- in its sequence of amino acids and is also induced by jak /stat pathway [ ] . however, unlike dub- , dub- was induced only by il- in t cells, but not by il- [ ] . dub- also increase = inc. in production. + = induce positive effect. showed increase in jak/stat signaling pathway products by decreasing il- induced dephosphorylation of stat [ ] . also, dub- decreased apoptosis in ba/f cells upon withdrawal of cytokines [ ] . the mechanism by which dub- achieves these effects needs to be further studied. in myeloid d cells, dub- stabilized csf r and increased its signaling activity by decreasing lysosomal degradation of csf r by deubiquitinating it, leading to prolongation of stat phosphorylation in csf signaling pathway [ ] . on the other hand, dub- a is a dub expressed in hematopoietic cells, such as b and t cells [ ] . unlike dub- , which was more expressed by il- , dub- a was further expressed upon exposure to il- [ ] . although similar to dub- induction by il- [ ] , dub- a induction by colony-stimulating factor (csf ) in myeloid d cells did not require erk [ ] . dub- (also known as usp ) is a cytokine-inducible human dub that was found to deubiquitinate sds and block proliferation in hela cells [ ] . in mrna level, dub- expression increased in raji cells when treated with il- and in u cells when treated with il- [ ] . dub- also influenced the ras/mek/erk signaling pathway and deubiquitinated ras converting enzyme (rce ), decreasing proliferation of cells [ ] . otud- b, a dub from otu family, was upregulated in a mouse pro-b cells, ba/f cells, upon cytokine stimulation [ ] . stimulation with il- , il- , il- or gm-csf resulted in a dose-dependent increase of otud- b mrna in the first to h of stimulation, but quick decrease was observed from to h [ ] . overexpressing otud- b in ba/f cells resulted in downregulation of proliferation and increased the frequency of apoptosis [ ] . usp has been known to be induced by viral infection, genotoxic stress or interferon [ ] . consistent with this, usp 's mrna level increased in thp- cells and thp- -derived macrophages upon exposure to ifn-β [ ] . furthermore, tlr ligands, lps, pam csk and cl all gave the same result of increased mrna level of usp , supporting that tlr-induced signaling pathway induces usp expression [ ] . exposure to tnfα caused gsk β-mediated phosphorylation of usp , leading to deubiquitination of traf in beas b cells, increasing jnk signaling upon tnf-α-induction [ ] . a was identified since as a tnf-α-induced dub in human umbilical vein endothelial cells (huvec) [ ] . mrna of cezanne quickly increased in hek and huvec cells upon exposure to tnf-α, but not upon shear stress [ ] . we have discussed some known cases of dub-regulated cytokines and cytokine-inducible dubs. cytokines are intertwined in numerous cellular processes and dubs are closely related to cytokines. this review dealt with many dubs, but less than half of all known dubs are discussed. moreover, we cannot say for certain that all the functions of the dubs discussed here are discovered. this grants us to further investigate the molecular mechanism and their effects. studying dubs and their effects could enlighten us with a novel therapeutic approach to various diseases, including but not limited to immunological diseases and cancer. structural and functional insights to ubiquitin-like protein conjugation ubiquitin modifications the ubiquitin code in the ubiquitin-proteasome system and autophagy a genomic and functional inventory of deubiquitinating enzymes regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes an atlas of altered expression of deubiquitinating enzymes in human cancer mindy- is a member of an evolutionarily conserved and structurally distinct new family of deubiquitinating enzymes broad and diverse mechanisms used by deubiquitinase family members in regulating the type i interferon signaling pathway during antiviral responses the roles of ubiquitin modifying enzymes in neoplastic disease viral deubiquitinases: role in evasion of anti-viral innate immunity type i interferon in chronic virus infection and cancer triggering antiviral response by rig-i-related rna helicases double-stranded dna and doublestranded rna induce a common antiviral signaling pathway in human cells rna recognition and signal transduction by rig-i-like receptors trim ring-finger e ubiquitin ligase is essential for rig-i-mediated antiviral activity ubiquitin-specific protease negatively regulates virus-induced type i interferon signaling via catalytically-dependent and -independent mechanisms a functional ubiquitin-specific protease embedded in the large tegument protein (orf ) of murine gammaherpesvirus is active during the course of infection inhibition of rig-i-mediated signaling by kaposi's sarcoma-associated herpesvirusencoded deubiquitinase orf evasion of innate cytosolic dna sensing by a gammaherpesvirus facilitates establishment of latent infection ubiquitin-specific proteases negatively regulates virus-induced type i interferon signaling induction of usp by viral infection promotes innate antiviral responses by mediating the stabilization of traf and traf ubiquitin-specific protease regulates tlr -dependent innate immune responses through deubiquitination of the adaptor protein traf negative regulation of il- -mediated signaling and inflammation by the ubiquitinspecific protease usp usp negatively regulates antiviral response by acting as a rig-i deubiquitinase usp inhibits type i interferon signaling by deubiquitinating rig-i-like receptors cylindromatosis (cyld), a deubiquitinase, attenuates inflammatory signaling pathways by activating toll-like receptor in human mesangial cells the tumour suppressor cyld is a negative regulator of rig-i-mediated antiviral response the deubiquitinating enzyme cylindromatosis dampens cd (+) t cell responses and is a critical factor for experimental cerebral malaria and blood-brain barrier damage loss of the cylindromatosis tumour suppressor inhibits apoptosis by activating nf-kappab the ubiquitinmodifying enzyme a is required for termination of toll-like receptor responses cyld limits lys -and met -linked ubiquitin at receptor complexes to regulate innate immune signaling deubiquitination, a new function of the severe acute respiratory syndrome coronavirus papain-like protease? crystal structure of the middle east respiratory syndrome coronavirus (mers-cov) papain-like protease bound to ubiquitin facilitates targeted disruption of deubiquitinating activity to demonstrate its role in innate immune suppression mers-cov papain-like protease has deisgylating and deubiquitinating activities the papain-like protease of porcine epidemic diarrhea virus negatively regulates type i interferon pathway by acting as a viral deubiquitinase transmissible gastroenteritis virus papain-like protease antagonizes production of interferon-beta through its deubiquitinase activity proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease the sars coronavirus papain like protease can inhibit irf at a post activation step that requires deubiquitination activity deubiquitinase function of arterivirus papain-like protease suppresses the innate immune response in infected host cells modulation of stimulator of interferon genes (sting) expression by interferongamma in human keratinocytes usp recruits usp to promote innate antiviral response through deubiquitinating sting/mita usp negatively regulates nf-kappab signaling by targeting tak and nemo for deubiquitination through distinct mechanisms usp inhibits nf-kappab and nfat activation during th differentiation by deubiquitinating the tak -tab complex ubp is a novel regulator of interferon signaling independent of its isg isopeptidase activity microarray analysis reveals that type i interferon strongly increases the expression of immuneresponse related genes in ubp (usp ) deficient macrophages receptor dimerization dynamics as a regulatory valve for plasticity of type i interferon signaling a deubiquitinating activity is conserved in the large tegument protein of the herpesviridae herpes simplex virus ubiquitin-specific protease ul inhibits beta interferon production by deubiquitinating traf herpes simplex virus ubiquitin-specific protease ul abrogates nf-kappab activation in dna sensing signal pathway the papainlike protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity plp , a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production plp of mouse hepatitis virus a (mhv-a ) targets tbk to negatively regulate cellular type i interferon signaling pathway mcpinduced protein deubiquitinates traf proteins and negatively regulates jnk and nf-kappab signaling mcpip negatively regulate cellular antiviral innate immune responses through dub and disruption of traf -tbk -ikkepsilon complex the a deubiquitinase activity negatively regulates lmp activation of irf intracellular signaling molecules activated by epstein-barr virus for induction of interferon regulatory factor negative regulation of the retinoic acid-inducible gene i-induced antiviral state by the ubiquitin-editing protein a epstein-barr virus large tegument protein bplf contributes to innate immune evasion through interference with toll-like receptor signaling a brisc-shmt complex deubiquitinates ifnar and regulates interferon responses downregulation of usp promotes activation of microglia and subsequent neuronal inflammation in rat spinal cord after injury ubiquitin-specific protease (usp ) targets traf and traf for deubiquitination and inhibits tnfalpha-induced cancer cell migration usp targets tak to downregulate tnfalpha-induced nf-kappab activation ubiquitin signalling in the nf-kappab pathway revealing a novel otubain-like enzyme from leishmania infantum with deubiquitinating activity toward k -linked substrate inhibition of nf-kappab signaling by a through disruption of ubiquitin enzyme complexes dimerization and ubiquitin mediated recruitment of a , a complex deubiquitinating enzyme elevated a promotes tnf-induced and ripk -dependent intestinal epithelial cell death nf-kappab suppression by the deubiquitinating enzyme cezanne: a novel negative feedback loop in pro-inflammatory signaling the n-terminal ubiquitin-associated domain of cezanne is crucial for its function to suppress nf-kappab pathway cezanne regulates inflammatory responses to hypoxia in endothelial cells by targeting traf for deubiquitination the deubiquitinating enzyme usp stabilizes traf and reduces e-cadherin-mediated adherens junctions loss of functional bap augments sensitivity to trail in cancer cells histone h a deubiquitinase activity of the polycomb repressive complex pr-dub expansion of dub functionality generated by alternative isoforms -usp , a case study proteasome deubiquitinases as novel targets for cancer therapy a novel inhibitor of proteasome deubiquitinating activity renders tumor cells sensitive to trail-mediated apoptosis by natural killer cells and t cells the proteasome deubiquitinase inhibitor b-ap enhances dr activation-induced apoptosis through stabilizing dr the novel deubiquitinase inhibitor b-ap induces direct and nk cell-mediated antitumor effects in human mantle cell lymphoma monocyte chemotactic protein-induced protein- enhances dr degradation and negatively regulates dr activation-induced apoptosis through its deubiquitinase function deubiquitinases regulate the activity of caspase- and interleukin- beta secretion via assembly of the inflammasome the ubiquitin-specific protease usp is critical for the development and homeostasis of t cells the deubiquitinase otulin is an essential negative regulator of inflammation and autoimmunity otulin antagonizes lubac signaling by specifically hydrolyzing met -linked polyubiquitin epigenetic regulation of the expression of il and il and autoimmune inflammation by the deubiquitinase trabid dub- , a deubiquitinating enzyme with growth-suppressing activity jak is required for induction of the murine dub- gene the murine dub- gene is specifically induced by the betac subunit of interleukin- receptor dub- a, a novel deubiquitinating enzyme subfamily member, is polyubiquitinated and cytokine-inducible in blymphocytes the deubiquitinating enzyme dub- prolongs cytokine-induced signal transducers and activators of transcription activation and suppresses apoptosis following cytokine withdrawal dub- is a member of a novel family of cytokine-inducible deubiquitinating enzymes the deubiquitinating enzyme dub a enhances csf signalling by attenuating lysosomal routing of the csf receptor dub- a, a new member of the dub subfamily of hematopoietic deubiquitinating enzymes lys- -specific deubiquitination of sds by usp regulates hdac activity dub- , a cytokine-inducible deubiquitinating enzyme that blocks proliferation usp regulates ras activation and cell proliferation by blocking rce activity evidence for otud- b participation in b lymphocytes cell cycle after cytokine stimulation ubp (usp ) specifically removes isg from conjugated proteins the a cdna induced by tumor necrosis factor alpha encodes a novel type of zinc finger protein we would like to thank members of baek's laboratory for their critical comments this study was funded by the korea ministry of environment (moe) as 'the environmental health action program ( )'. he has been serving as an editorial board member of more than a dozen of international journals. current research and clinical interests are in the molecular genetics of ubiquitination and deubiquitination systems relevant to various cancers. he first coined terms ubiquitomics and deubiquitomics in the field of the ubiquitin-proteasome system. in addition, he is also interested in genomics and proteomics during stem cell proliferation, differentiation, and reprogramming. key: cord- -eydkfqi authors: feng, mingxiang; shaw, shih-lung; fang, zhixiang; cheng, hao title: relative space-based gis data model to analyze the group dynamics of moving objects date: - - journal: isprs j photogramm remote sens doi: . /j.isprsjprs. . . sha: doc_id: cord_uid: eydkfqi the relative motion of moving objects is an essential research topic in geographical information science (giscience), which supports the innovation of geodatabases, spatial indexing, and geospatial services. this analysis is very popular in the domains of urban governance, transportation engineering, logistics and geospatial information services for individuals or industrials. importantly, data models of moving objects are one of the most crucial approaches to support the analysis for dynamic relative motion between moving objects, even in the age of big data and cloud computing. traditional geographic information systems (gis) usually organize moving objects as point objects in absolute coordinated space. the derivation of relative motions among moving objects is not efficient because of the additional geo-computation of transformation between absolute space and relative space. therefore, current giss require an innovative approach to directly store, analyze and interpret the relative relationships of moving objects to support their efficient analysis. this paper proposes a relative space-based gis data model of moving objects (rsmo) to construct, operate and analyze moving objects’ relationships and introduces two algorithms (relationship querying and relative relationship dynamic pattern matching) to derive and analyze the dynamic relationships of moving objects. three scenarios (epidemic spreading, tracker finding, and motion-trend derivation of nearby crowds) are implemented to demonstrate the feasibility of the proposed model. the experimental results indicates the execution times of the proposed model are approximately – % those of the absolute gis method for the same function of these three scenarios. it’s better computational performance of the proposed model when analyzing the relative relationships of moving objects than the absolute methods in a famous commercial gis software based on this experimental results. the proposed approach fills the gap of traditional gis and shows promise for relative space-based geo-computation, analysis and service. moving objects are the most common and important component in a diverse range of phenomena, such as human mobility (fang et al., ; jiang et al., ; almuhisen et al., ) , urban transportation , tang et al., tu et al., ) , ship logistics in the ocean (yu et al., b; fang et al., ) and even animal migrations (bastille-rousseau et al., ) . many research projects have been driven and improved by moving-object data analysis, such as individual/group behavior analysis, path discovery and behavior prediction. because of the large amount of moving objects in real applications, these analyses require a powerful gis data model to store, analyze and interpret physical and contextual information (e.g., absolute topology and relative motion) of moving objects. current gis data models usually record essential information for moving objects within an absolute space. in absolute space, geocoded locations are bound to previously existing geometry and topology relationships among the corresponding points in the space (meentemeyer, ; couclelis, ) . therefore, moving objects are always represented as a series of observations that consist of an id, location and time (hornsby and egenhofer, ; spaccapietra et al., ) , alongside additional information such as activities (wang and cheng, ; shaw and yu, ) and semantics vandecasteele et al., ) . furthermore, these models describe their movement processes and interactions alongside basic geographical information (such as land use, poi, and events). in fact, these models require a large https://doi.org/ . /j.isprsjprs. . . received november ; received in revised form april ; accepted may amount of computation to support analysis from the perspectives of individual or groups of moving objects (which could be called relative space). moreover, several analysis, such as the surrounding dynamics and motion trends of nearby crowds, are critical to moving objects in highly complex and dynamic environments according to the personal requirements of decision-making (e.g., on relax life style, feeling of safety). current gis data models must be improved in terms of the geocomputation of these analyses. actually, relative space is an instinctual approach to moving objects and is a powerful theoretic framework to represent the surrounding dynamics and motion trends of moving objects in nearby crowds. traditional relative space is often studied in the research communities of mathematical theories (einstein, ) , physics (veltman, ; ruggiero, ) , astronomy (rots et al., ) and aerospace science (sinclair et al., ) . currently, very few gis data models have been built for relative space, which requires additional space-transformation computations to implement this instinctual approach in applications. in relative space, relative dynamic relationships between moving objects are easy to build independently of whether they can be geocoded by coordinates. importantly, the analysis of moving objects in relative space could easily follow instinctual requirements. therefore, the motivation of this paper is to create a relative space-based gis data model of moving objects and propose some basic gis operators for analyzing moving objects, which changes the analysis of current absolute space-based gis models and facilitates the efficient computation of real-time relative relationship dynamics, such as the surrounding dynamics and motion trends of crowds near moving objects. the contributions from this paper are summarized as follows: • a relative space-based gis data model of moving objects (rsmo) is introduced to construct, operate and analyze moving objects' relative relationships. • a relationship-querying algorithm and relative dynamic patternmatching algorithm are introduced to analyze the dynamic relationships of moving objects. • three scenarios (epidemic spreading, tracker finding, and derivation of the motion trends of nearby crowds) are implemented to demonstrate the feasibility of the proposed model, which also shows better performance compared to absolute methods. this paper is structured as follows. related work is discussed in section . section proposes the modeling process, structure and operation of this model. section illustrates the implementation of rsmo and the two algorithms. experiments with three case studies and performance testing are reported in section . section discusses the proposed model. finally, conclusions are presented in section . moving objects are a very popular representation requirement in current gis applications because of the large volume of their tracked trajectories, e.g., cars in cities, vessels at sea, and even people around the world. therefore, many research projects have been conducted to store, manage and query moving-object data in the communities of computer science and geographical information science. in computer science, several data structures have been defined to support the storage and querying of moving objects. the first structure is the storage unit in dbmss (database management systems). güting and his group abstracted moving objects into moving points and moving regions and developed a discrete model in a dbms to store, manage and query moving objects' data (forlizzi et al., ; güting et al., güting et al., , güting and schneider, ) . based on abstracted data types, moving objects could be managed and queried by using sql. these works provided a solid foundation to support research on moving objects. the second is tree structures. moving objects were usually abstracted as points (ranu et al., ) , single continuous polynomials (ni and ravishankar, ) , or non-regulated sequences of roads in transportation networks (sandu popa et al., ) . then, these objects were indexed by the tree's derivatives, for example, b + -trees (sandu popa et al., ) , r-trees (yue et al., ) , d r-trees (xu et al., ) , fn (fixed network) r-trees , pa-trees (ni and ravishankar, ) , and grid trees (yan et al., ) . these tree-based indices could facilitate efficient queries on moving objects. the third structure is moving object-oriented databases. to satisfy the demand of faster processing with big data, postgreesql , mon-godb , monetdb (boncz et al., ) , and cassandra (hospital et al., ) were developed to store and analyze large volumes of moving objects' information. in the community of geographical information science, traditional gis data models, such as field-based models (e.g., raster-based models) (cova and goodchild, ) and feature models (e.g., object-and vector-based models) (tang et al., ) , usually depended on absolute coordinates in their reference frames to describe spatial object's motion or relationships. these models were often used to represent some key concepts that were related to moving objects, i.e., the place, activity and semantics. the concept of "place" was used to indicate the location of activity, which usually complied with a certain upper-bound distance and lower-bound duration (kang et al., ) . human daily activities were always represented as a sequence of place visits (do and gatica-perez, ) . additionally, space time path was a key concept in classical time geography framework that was used to represent and analyze activities and interactions in a hybrid physical-virtual space (shaw and yu, ) and social space (yin and shaw, ) . several basic semantics of moving objects were derived from the trajectories of moving objects, for example, episodes, stops, and moves (ilarri et al., ) . based on these basic semantics, researchers (parent et al., ; bogorny et al., ; jiang et al., ; wan et al., ) built semantic trajectories from semantic point sequences. semantic trajectories were easy for urban planners and migration or transportation agencies to use to mine spatial-temporal mobility patterns. moving object-oriented operators (i.e., range queries, nearestneighbor queries) were derived to support the analysis of moving objects' relationships. the first common operator is range queries, which specify a value of moving objects to fall within a lower and upper boundary zhan et al., ; xu et al., ; yue et al., ) , e.g., finding all the objects of a specific traveler between am and am. filtering and refining are two important steps of range queries. filtering determines candidate locations (such as a node in a tree structure) that contain specified attribute values and overlap the query area in space and time, while refining retrieves well-matched objects under the querying conditions through some advanced filtering techniques, such as statistics-based approaches (zhan et al., ) and time-based partitioning techniques (yue et al., ) . the second common operation is nearest-neighbor (nn) queries, which find the nearest neighbors to a given object in a database for a specific time interval. two similar phases of nn queries include searching for candidate units based on the idea of node pruning with large coverage and determining the well-matched results according to various conditions, such as time limits (güting et al., ) , obstacles (gao et al., ) , spatial-network structures (cheema et al., ) , reverse nearest neighbors (cheema et al., ) and movement uncertainties (niedermayer et al., ) . these operators are usually applied by calculating the distance between the coordinates of moving objects to derive relative metrics such as the distance, direction and time. this situation could produce repeated computations and misrepresent the relative relationships of moving objects without a geocoding technique. a wide spectrum of applications can be conducted for individuals, groups or the public. the first application is the movement-behavior analysis of moving objects, for example, individuals (gonzález et al., ; ando and suzuki, ; gao et al., ; renso et al., ; song et al., ) and groups (zheng et al., ; li et al., ; gupta et al., ; mcguire et al., ; liu et al., ) . the second application is path recommendation from historical trajectories (luo et al., ; dai et al., ; yang et al., ; zheng et al., ) . the third application is location prediction for tourists (lee et al., ; yu et al., a) , navigators (li et al., ; besse et al., ) , and driverless vehicles . these applications require that the dynamic relationships of moving objects in space over time are inferred. however, very few models can directly organize the dynamic relationships of moving objects. these applications greatly depend on computationally intensive infrastructures. the studies about relative space mainly appear in the fields of vessels in maritime and airplane in aviation. in maritime, the moving object data model also can be applied for assessing collision risk (bye and aalberg, ; fang et al., ) , predicting vessel behavior (zissis et al., ; xiao et al., ) and planning path (cummings et al., ; hornauer et al., ) . in aviation field, motion guidance and control (yu et al., ; sun et al., ; li and zhu, ; zhu et al., ) for spacecraft rendezvous, position and attitude estimation (philip and ananthasayanam, ; qiao et al., ) between the chaser and the target satellites were also referred to the applications of moving object data model. these studies aim to control the moving object in real time to avoid risk and complete the established movement in the ocean or aerospace, where no appropriate global reference for them. those studies focus on the real-time motion control to respond to surrounding moving objects in a high dynamic scenario. these missions need an efficient data model to handle relationships of moving objects as well. in short, current research on moving objects rarely organizes their dynamic relationships directly because of a lack of relative space-based data models and analytic frameworks in the communities of computer science and geographical information science. this paper attempts to fill this gap by introducing a relative space-based gis data model to analyze the group dynamics of moving objects, which should reduce the intensive computation that occurs when deriving the relationships of moving objects and provide better analytic performance than traditional absolute coordinate-based gis data models. this section introduces a relative space-based gis data model of moving objects (rsmo). rsmo is extended from the space-time cube (stc) structure in arcgis. an stc is a three-dimensional cube that includes space-time bins in the x and y dimensions and time in the t dimension and represents limited space coverage over a fixed period. the basic idea of rsmo is to directly record moving objects' relationships and relative dynamics with a three-dimensional space-time cube to facilitate transfer to absolute space by maintaining the current locations of moving objects via additional space-time bins. this data model reduces the calculation of moving objects' relationships and relative dynamics in traditional gis data models to provide efficient relative space-based queries between moving objects for real applications with cars, vessels, people, etc. rsmo is designed to support some basic functions such as moving objects' motion storage and organization, queries based on relative relationships, relative motion-pattern analysis and mining, and transformation between absolute space and relative space. the following section will first introduce rsmo and then the basic functions that depend on rsmo. before introducing the proposed rsmo, this section defines some basic concepts at first as follows: definition : space-time cube (stc) is a three-dimensional ( d) euclidean space containing of a d geographical space (x and y) plus time (t) for visualization and analysis of objects' behavior and interactions across space and time (bach et al., ) . definition : space-time bin (stb) is a basic unit of space-time cube, which lies in the fixed position based on their x and y dimensions in d geographical space and t dimension in time to represent a limited location with a certain time. definition : relative relationship bin (rrb) is a derived stb with a substituted reference framework of reference object, target object and time (fig. ). it contains a quadruple < t, ref_obj, tar_obj, relationship > , which indicates the relationships of corresponding reference object (ref_obj) and target object (tar_obj) during a specific time t. definition : relative relationship matrix is a matrix of all moving objects' rrbs in the time t and within a specific environment. definition : relative relationship cube is a time series of relative relationship matrices, which is organized in three-dimensional space (two dimensions in space and one dimension in time) according to time. the proposed rsmo model extends the structural organization of space-time bins in arcgis to model the relative relationships among moving objects and facilitate their relative space-based analysis. here, the motion in the stc is represented by the relationships of moving objects, which is a natural approach to represent the actual cognitive processes of moving objects in an environment. fig. illustrates the extended-entity-relationship (eer) diagram of the rsmo model, which shows the elements (entities, relationships and attributes) and the hierarchical relationships among these elements. this model defines six basic entity types, i.e., "object", "relative location", "relationship", "relative relationship bin", "relative relationship matrix" and "relative relationship cube". here, object represents the moving object in a real scenario, such as the people in fig. , vehicles/ unmanned ground vehicles, unmanned aerial vehicles, vessels, planes, and so on. relative location is the position of any object relative to other object (fig. ) , which can be represented by the relative distance and angle. relationship indicates the mutual spatial or social relationships among objects, for example, closeness, friendship, colleague does not require much additional computation to build relationships among objects from the coordinates in traditional stcs and provides a natural approach to analyze a moving object's behavior. fig. provides an example of rsmo's organization. fig. (a) illustrates the spatial trajectories of five moving objects (objects labelled as a, b, c, d and e). fig. (b) illustrates a right-handed coordinate system for detecting objects' relative location from each object's view. fig. (c) shows a geometric transformation method from relative locations to ) of moving objects are organized akin to x and y coordinates. in this matrix, rows represent "reference" objects and columns represent their detected "target" objects. fig. (e) shows a relative relationship cube that includes all relative relationship matrices sorted by time. the proposed model replaces the orthogonal coordinate axes (x, y) in the stc with two object axes that represent reference and target objects to store their relationships. recording the changing relationships between moving objects is insufficient because this process only allows relationship comparison between two different moving objects and cannot support transformation with absolute space to match the computational tasks in current gis tools. to solve these problems, an additional object r (called the initial reference object), which is a fixed coordinate in absolute space, was added to rsmo (the object r in fig. (b) ). this object represents a fixed location acting as a local reference system. by comparing the location change in this local reference system, each object's motion relative to itself can be derived. after determining the coordinates in absolute space, this object also facilitates transformation between relative space and absolute space. the details of this procedure will be introduced in . . . this relative relationship cube forms the basic structure of rsmo. this section introduces a set of basic operators for the implemented gis functions. those operators are organized into six classes according to the type of relationship outcome. ( ) query processing is an operator to get the direct relative relationship in terms of relative distance and angle between objects and time. ( ) group relative dynamic calculation is an operator to derive the relative dynamic characteristics of the group. ( ) transformation between absolute space and relative space is an operator to transform the data between absolute space (e.g. coordinates) and relative space (e.g. relative distance and angle). ( ) initial reference object transformation is an operator to change the reference object of relative relationship cube, and update all relative relationships of all objects. ( ) attribute transformation is an operator to derive the relationship attributes based on relative relationship cube, such as, closeness. ( ) relative relationship dynamic pattern transformation is an operation to derive the relative dynamic pattern between object, such as moving trends of them. these operators are expanded and developed from stcs to adapt the features of a moving object's motion in relative space. the following subsections will describe these classes in detail. query processing searches for the relative mutual relationships of moving objects and their changes. the six operators of query processing are explained as follows: ( ) point extraction retrieves the relationships between designated objects at specific times. this operator is a basic function for querying and analysis. ( ) time drilling retrieves the dynamic processes of the relationships between designated objects. ( ) target drilling extracts the specific reference object's relationships with other target objects at a designated time. ( ) relationship curvilinear drilling retrieves the target objects that present a specific relationship with the designated reference object. this operator can be used to extract the reference object's behavior relative to other objects, such as the interactions between two moving objects if "distance < m". the reference object's interactions can be expressed as a planar d curve that consists of target objects for each time. ( ) time cutting retrieves all the objects' mutual relationships at any designated time. ( ) reference cutting retrieves the movement of all objects relative to the reference object. detailed descriptions of these query-processing operators are listed . obtain relative relationship bins with different references and target objects by time drilling; . obtain the results from fun(), whose input is relative space-time lines from the previous step; . fill the results in the corresponding position of the two-dimensional matrix built by the reference and target axes. . obtain relative relationship bins with different references and target objects by target drilling; . obtain the results from fun(), whose input is relative space-time lines from the previous step; . fill the results in the corresponding position of the two-dimensional matrix built by the reference and time axes. note: in group relative dynamic calculations, min in example is used to find the minimum in the dataset. process details the procedures in the illustration and provides text to help readers understand this operator. flattening(axis, fun()) is used to express a uniform form. two parameters are required: the axis indicates the input of the operator and fun() is a function to process the values along the axis. additionally, fun() can be set by the user for different purposes. in table . group relative dynamic calculation computes the group relative dynamic characteristics (i.e., average distance, interaction frequency in social media, and the closeness of social relationships) based on the relationship changes of all objects. the two operators time flattening and target flattening are explained as follows: ( ) time flattening computes the relative dynamic statistical characteristics between each pair of objects during the entire period and retrieves a matrix that contains the results. for instance, the average distance is calculated through a statistics function, and the unit in the resulting matrix shows the average distance between each pair of objects, which infers the closeness of their relationship during the entire period. ( ) target flattening computes the relative dynamic statistical characteristics of all the target objects relative to each reference object at each time stamp and retrieves a matrix that contains the results. for example, if we want to calculate the interaction frequency, we can obtain a result matrix that indicates the interaction changes of each reference object relative to other objects over time. detailed descriptions of these group relative dynamic calculations are listed in table . the purpose of this operator is to build a transformation method between absolute space and relative space. this operator is used to adapt current gis modules because most gis software analyzes moving objects only in absolute space. if we know the coordinates ( x y ( ) the increment in the x and y directions ( x, y) in absolute space can be calculated with equation ( ) by distance decomposition: ( ) based on ( x, y), the coordinates of a in absolute space can be expressed with eq. ( ): initial reference transformation transforms the relationship of any moving object with another object to that of another object. this process changes the analysis view between objects, which could support the relative space-based analysis of any individual object in parallel. a detailed description of initial reference transformation is listed in table . below, we present an example to explain this process. an example is introduced to explain the transformation process in fig. (a) . a is the initial reference object of object a at t . if we want to shift the initial reference object from a to b (object b at t ), where b is the new initial reference object, we compute all the objects' unknown relationships with b . for example, fig. (a) shows the unknown mutual relationships b -a and b -c in the new cube's matrix. the mutual relationship between b and c ( fig. (b) ) can be derived by the following steps. first, the coordinates in absolute space are calculated with the transformation operator between absolute space and relative space from section . . . second, the distance between b and c is calculated by the law of cosines. the distance between a b and a c can be derived by computing the difference between ang a c , and ang a b , . then, the distance between b and c (dis finally, the angle between b and c is calculated by the following rotation method to ensure uniqueness. ( ) the rotations of b and c relative to object a (yaw a b , and yaw a c , ) are calculated with equation ( ) thus, we set shift(a ,x) as the transform function to convey the initial reference object from a to x , where the purpose of attribution transformation is to label new attributions to each relative relationship bin and filter bins with any specific conditions. the two operators (labeling and filtering) are explained as follows: ( ) labeling adds new attributions to each relative relationship bin note: label(fun()) and filter(fun()) are used to express uniform forms. the parameters of fun() in labeling is a function to acquire new attributes based on the relative relationships of bins. and fun() in filtering is used to select the relative relationship bins based on the new attribute by labeling (e.g. closeness is intimate). additionally, fun() in two operators can be set by user for different purposes. based on the conditions or classifier set from fun(). ( ) filtering is used to remove some bins from the results of the labeling step. ( ) detailed descriptions of these attribution transformations are listed in table . the goal of relative relationship dynamic pattern transformation is to analyze the motion features of moving objects in relative space. two common operators are explained as follows: ( ) trending computes motion features (such as distance changes, angle changes, relative speeds and relative acceleration) by comparing relationship changes between each pair of objects. ( ) matching discovers relative motion patterns with the condition of a movement pattern, such as accompaniments and trackers. the detailed descriptions of these relative relationship dynamic pattern transformations are listed in table . a prototype (fig. ) was developed to implement the proposed rsmo model. all the functions store, manage and analyze relative space data were encoded into a dynamic link library within c++ environment. the visualization of this prototype was developed by the qgis (quantum gis) framework in a python environment. two typical analysis modules were implemented, namely, query processing and relative relationship dynamic pattern transformation. the query processing contained all six operators (point extraction, time drilling, target drilling, time curvilinear drilling, time cutting and target cutting). two relative relationship dynamic pattern matching algorithms (accompaniments and trackers) were implemented in this prototype. a query algorithm called query processing is described here to for retrieving relative relationship in relative space. the main idea of this algorithm are to organize the relative relationship as three dimensions, namely, reference object, target object and time, which is based on model's structure, and then to index relative relationship by corresponding objects and time. by using a composite index consisting of reference object, target object and time, this algorithm can quickly find out the relative relationship completely covered by the given restricted objects and time. in the query, this algorithm retrieves relationships by accessing the relative relationship according to the corresponding composite index of given object and time, instead of extracting each relative relationship bins of reference or target object one by one in the cube. therefore, this algorithm can be integrated with all the operators shown in section . . and constrained by six parameters, namely, cube, query_type, c_ref, c_ tar, c_t, and rel, which are explained in algorithm . cube is a structure of map, whose key consists of a reference and target object, while value is also a map whose key is the time and value is the relationship. the detail of the query processing algorithm is described in algorithm . when comparing this algorithm with traditional gis querying in absolute space, this algorithm can avoid many redundant computational address coordinates, which can improve the efficiency of the relative relationship querying. we will test this approach in section . . algorithm , which is called pattern matching, illustrates the process of searching a specific relative pattern in relative space. dynamic pattern is represented as a space-time trajectory (bao et al., ) , which is consist of specific locations in absolute space (shown in fig. ) . given a specific pattern (pattern_s), each object's distance to the pattern_s needs to be calculated to measure its similarity to pattern_s. finally, the trajectory, whose distances are all in matching threshold (in fig. ) , would be got as the outcome of pattern matching in absolute space. in relative space, dynamic pattern is represented as a relative relationship bin series, which is composed of bin with specific relative distance. in this algorithm, the bins with each object's relative distance to pattern_s can be directly got by reference cutting at first. then, they are labeled according to whether their relative distance is in matching threshold. finally, the relative relationship bin series are obtained, which match the pattern_s in all time. this algorithm provides a more efficient way for relative relationship dynamic pattern matching. it simplifies the spatial computation to measure the similarity on basis of reference cutting and labeling in the proposed model. the details of the pattern matching algorithm are described in algorithm . algorithm : pattern matching (cube, c_ref, pattern_s) input: cube -the relationships between all objects in a relative space-time cube form; c_ref -the reference object, an index on ref of a relative relationship bin in cube; pattern_s -a specific relative relationship pattern to be found, which is expressed as a time series that consists of a triple of time, rel. output: the object in cube, which produces a trend of pattern_s relative to c_ref. let q be a sequence of bins in the cube. q contains pairs of the form < objects_pair, relptr > , where objects_pair is the index on attribute ref and tar in a pair form and relptr is a map structure whose time is the key and relationship is the value; let objectslist be the set of objects that contains the codes of all the objects in the scenario; let timestamps be the total number of time stamps in the data; let * be any value; let labeling be the function that get the relative relationship bins, which match the pattern_s; this algorithm allows users to search objects with specific motion patterns relative to dynamic reference objects. this advantage can help users discover several meaningful behaviors of objects that are hidden in a moving group, for example, being followed. the proposed rsmo model was tested with three application scenarios, such as epidemic spreading, tracker finding, and the motiontrend derivation of nearby crowds. each case study was used to demonstrate the feasibility and advantages of the proposed model. this paper collected pedestrians' (experimental subjects) walking trajectories on the street in the three experimental scenarios. a lenovo phab pro phablet was used to record their trajectories in both absolute and relative space. this device is openly available and equipped with sensors for motion tracking. this phablet can record the position and rotation angle relative to the starting pose of the device. the gps receiver recorded the motion in absolute space and relative space. fig. shows all trajectories of the experimental subjects walking along a road in wuhan city, china. the detailed information includes the following fields in table : • "user id" is the unique identifier for each object. • "time stamp" is the time when the location was recorded. • "longitude" and "latitude" were used to record the pedestrians' location in absolute space with gps receiver. • "x increment" and "y increment" show the relative locations in the reference frame that was built by the user's starting pose. • "rotation angle" records the change in angle relative to the initial pose in a counterclockwise direction. • "initial azimuth" is the angle of the user's face relative to the north. the collected data needed to be pre-processed before saved into the proposed model as the following steps. ( ) derive relative distance and angle between all objects. first, the relative distance could be computed by the operator of transformation between absolute space and relative space with the coordinates in the fields of longitude and latitude in collected data. then, the relative angle was computed based on the movementdirection azimuth ( absolute ), which was derived from rotation angle ( ) and initial azimuth ( ) according to the equation ( ). ( ) set the initial reference object for the model. initial reference object was set as object at t in this experiment by computing all objects' relationships to object at t . the operator of transformation between absolute space and relative space was implemented for this task under the condition that each objects' coordinates (longitude and latitude), rotation angle and initial azimuth are known in collected data. the first scenario involved finding the person who contacted with an epidemic carrier. in the research community of epidemiology, the spread of diseases is closely related to the spatial and social activities of patients, and the propagation of diseases greatly depends on the trajectory of a patient's social activities. thus, tracing the back-propagation path of a virus and finding close contacts are meaningful to prevent the spread of diseases. this experiment assumed a severe acute respiratory syndrome (sars) carrier's walking path to find its close contacts. sars is a viral respiratory disease of zoonotic origin that is caused by the sars coronavirus (sars-cov). the primary route of transmission for sars is the contact of mucous membranes with respiratory droplets or fomites (world health organization, ) . the research about respiratory droplets transmission shows that the largest droplets will totally evaporate before falling m away (xie et al., ) . therefore, a person will be identified as a close contact if the distance to the carrier (whose id is in fig. ) is less than m. therefore, this paper uses m as the parameter for querying close contacts. this query was implemented by combining the reference cutting and labeling operators (shown in fig. (a) and (b) ). first, the relative relationships (distance) of all the experimental subjects relative to the carrier were derived by reference cutting. here, a matrix (shown in fig. ) was used to show the change in distance to the carrier for each experimental subject; blue means a closer distance to the carrier, and red means a farther distance to the carrier. then, the relative relationship bin indicating a close contact (in green) was determined by labeling for distances below m. thus, the subjects ( and ) were found as close contacts. reference cutting (a) and labeling (b) are used to search the relative relationship bins containing close contacts in the data model, which are labeled in red. the close contacts' relative locations to the carrier are marked in (c). and their location in absolute space are shown in (d) by the operator of transformation between relative space and absolute space. by using the transformation between absolute space and relative space , this study could also locate the exact locations of close contacts. fig. (c) and (d) show the two locations of the subjects and in relative space and absolute space. one was much close to the carrier, the first being at . m at : : and the other at . m at : : . according to these querying results, public health agencies should take actions to control these two people. thus, this query is very helpful for public health agencies to control epidemic spreading if embedded into any real-time gis analytical systems. the second scenario was to find any tracker of a subject in a crowd. in the research community of crime, tracking is a basic behavior for further crime (beauregard et al., ) . finding trackers are helpful to take caution actions to prevent crime. usually, the tracker's behavior is similar to other normal pedestrians in the crowd. therefore, victims and public security organizations may experience difficulty discovering a tracker in absolute space. in this experiment, we assumed that the subject carried large amounts of cash, and he had to be aware of potential trackers. to remain unnoticed, trackers usually stay within a certain range from the target to neither be found nor lose the target. this distance range is a critical parameter to build specific patterns for trackers. according to the sociological theory (moore et al., ) , trackers are easily found if their distance to the victim is less than m. therefore, this study set the parameter of m as the lower distance limit for this query. on the other hand, the upper distance limit must guarantee that the target is within the tracker's sight. this study set the upper distance limit as m to ensure that the tracker did not lose the target. this parameter was calculated according to the waiting time of intersections in this study area ( s). therefore, s × . m/s = m, where . m/s is the normal average walking speed (fitzpatrick et al., ) . sometimes, the distance is probably larger than m, which may cause the tracker to lose the target; thus, the tracker will speed up to follow the target. in this case, the tracker must find the target within s because of the constraints of traffic lights. the conditions of tracker finding can be expressed as follows: ( ) the distance between the tracker and target is always [ m, m); ( ) the time that the distance between the tracker and target is not [ m, m) is below s, which occurs only once while speeding up because of the traffic-light locations in the study area. three main operators (reference cutting, labeling, matching) were used to find the tracker by meeting the above conditions. similar to the close-contact querying of epidemic spreading, this study used the reference cutting operator to find a matrix with all subjects' relationships relative to . then, this process used the labeling operator to label the relative distance to all relative relationship bins. fig. shows the labeled results. in this figure, bins that were labeled in red and yellow had a relative distance less than m, while bins that were labelled in blue had a relative distance larger than m. fig. illustrates the change in the relative distances between all the subjects and the target. two related patterns could be derived through matching: ( ) the subject was close to the target most of the time (i.e., , and ), or ( ) the tracker lost the target after a very short time (i.e., ). fig. shows the speed pattern of this subject. obvious acceleration was observed between t and t , meeting the second condition of identifying the tracker. based on the above results, the four subjects were viewed as trackers. in order to demonstrate the advantages of the proposed approach, this study also shows the trajectories of and . by the operator of transformation between absolute space and relative space, the trajectories of these individuals and the target did not present obvious abnormal features compared to other trajectories in absolute space ( fig. (a) ). however, their relative trajectories relative to the target presented an obvious accompaniment feature in relative space ( fig. (b) and (c)). therefore, this proposed approach can mine hidden patterns that are ignored by traditional gis data models. the third scenario was to derive the motion trends of nearby crowds. the motion trend of a nearby crowd is an important feature of surrounding dynamics and has a large influence on decision-making processes (fang et al., ) . this concept is necessary for individuals to understand nearby motion trends to avoid heavy congestion or stampedes. this experiment assumed the subject as a walker who fig. . distances of all the subjects relative to . fig. . acceleration of , which was positive when the target was lost. m. feng, et al. isprs journal of photogrammetry and remote sensing ( ) - hoped to be aware his surroundings, for example, how many people moved closer to or farther from him and whether they were accelerating towards him. five main operators (reference cutting, trending, labeling, filtering and time flattening) were used to implement this application. first, the relative relationships of all the experimental subjects relative to the feng, et al. isprs journal of photogrammetry and remote sensing ( ) - walker were derived by using reference cutting. then, a matrix that contained the motion trend (relative distance change and relative acceleration) was calculated through the trending operator. this matrix was labeled with a legend of colors in fig. through the labeling operator, which illustrates changes in the relative trend motion of the other subjects compared to the walker. in fig. , the red bins in the matrix mean that these subjects are accelerating towards the walker, orange bins mean that the subjects are decelerating towards the walker, gray bins mean that the subjects are accelerating away from the walker, blue bins mean that the subjects are decelerating away from the walker and yellow bins mean no change in distance and speed. the gray, blue and yellow relative relationship bins were removed with the filtering operator. fig. (a) shows the results after this operation. then, the number of bins where the subjects moved towards the walker was counted by time flattening, which counted any red or orange bins. the updated result is shown in fig. (b) , indicating that more than half of the individuals were moving closer to the walker from t to t ( : : - : : ) . similarly, fig. (c) shows the number of subjects that were accelerating towards the walker at each time, indicating that more than half of the subjects were accelerating towards the walker at eight times (t , t , t -t , t , t , t , t -t and t -t ) . this study transformed these bins from relative space to absolute space by the operator of transformation between absolute space and relative space, and the locations of surrounding people are shown in fig. (a) and (b) at : : and : : , respectively. by combining all the locations of surrounding people close to the walker, we could find the concentrated area of surrounding people, which is labeled in red in fig. (c) . in this case, the walker may have felt uncomfortable and potentially at risk in the crowd road. setting walker as initial reference object, this model derived the crowd's motion trends for all subjects by the operator of initial reference transformation. fig. showed the results of initial reference transformation from object at t to object at t . those who were moving closer to subject in fig. (a) didn't show clear motion trend for the new walker (subject at t ) in fig. (b) . this method is helpful for people-oriented routing services, such as pedestrian navigation or tourism. this study tested the performance of the proposed model in two aspects. one was the time complexity of querying functions; the other was time complexity between the proposed model and the absolute method in arcgis geodatabase. this study used a dataset (gramaglia et al., ) of vehicle trajectories on two highways around madrid, spain. the detailed information in these trajectories contained five fields (time, id, x position (m), y position (m) and speed (m/s)). before the comparison, this study chose datasets for this open-access dataset that contained m subjects for n times, where m = , , , , , and n = , or . table lists these datasets and the sizes of their storage spaces. all the computational tasks were singlethreaded implemented on a dell desktop (four intel(r) xeon(r) processors with cpu e - v @ . ghz, g of ram and a -bit operating system). six main functions (point extraction, time drilling, reference cutting, querying close contacts, finding trackers and deriving the motion trends of nearby crowds (deriving trend)) were selected to test the time table lists the execution times for the datasets and fig. illustrates the computation time of each function among these datasets. both tables show that the computation time of all the selected functions was less than s if the data volume of the datasets was smaller than gb. the execution times significantly increased when the dataset contained more than . * records. among the six functions, point extraction and finding track spent significantly less time than the other four functions when the records increased to * in the dataset. this result shows that the proposed model could work effectively with more than gb of data and meet behavior-analysis requirements in scenarios with subjects. the second comparison examined the time efficiency between the proposed model and the absolute model (geodatabase in arcgis). the proposed model presents an obvious advantage in terms of conducting three functions (point extraction, time drilling, reference cutting) because these functions only require a simple query, while the absolute model requires additional relative relationship transformation. the other three functions (querying close contacts, finding tracker and deriving the motion trend of crowd nearby) are meaningful to real situations and require a relatively complicated implementation. therefore, this study compared the second set of three functions that were implemented by the proposed model and absolute model. in arcgis, "buffer" and "overlay analysis" were used to implement querying close contacts. the point features of the reference subject's trajectory were the input features of buffer analysis in arctoolbox. the outputs of this operation were face features that represented the close-contact area. then, the intersect tool in overlay analysis could be used to check the status of the subjects' trajectories inside or outside the close contact area. however, no appropriate tools exist in arcgis to directly implement finding tracker or deriving the motion trend of crowd nearby. in terms of finding tracker, we computed each subject's distance relative to the reference subject at each time via coordinates in geodatabase and filtered the subjects according to the conditions in section . . in terms of deriving the motion trend of crowd nearby, we calculated all the subjects' relative speeds and relative accelerations relative to the reference subject and statistically counted five types of motion trends, as described in section . . table lists the execution times of the three selected functions for the proposed model and absolute gis method with of the datasets; these last two datasets were omitted because the absolute gis method would stop responding if the volume the of dataset increased to . gb. fig. illustrates the computation time of each function among these datasets. the execution times of the proposed model were approximately - % those of the absolute gis method for the same function. the computation time of querying close contacts for the proposed model was approximately % that for the absolute gis method, the most evident improvement from the proposed model. this improvement considerably increased with increasing dataset volume ( fig. (a) ), and the execution-time difference was s when the volume was . gb. the second function was deriving the motion trend of the crowd nearby, whose time-efficiency improvement was similar to that of querying close contacts. the execution time of the proposed model was approximately % that of the absolute gis method. additionally, the improvement from the proposed model became significant if the dataset's records were larger than * (fig. (c) ). the execution-time difference was s when the volume was . gb. the execution time of the third function (finding tracker) with the proposed model was only %- % that of the absolute gis method; this improvement was not as evident as that for the previous two functions. in fig. (b) , the execution time from the proposed model and absolute gis method steadily increased as the number of dataset records increased. however, the execution-time difference only reached . s when the dataset's volume was . gb, an insignificant increase compared to the previous two functions. this result indicates that the proposed model exhibits better efficiency when implementing these functions compared to the absolute gis method. in this study, the execution times of the three selected functions (querying close contacts, finding tracker and deriving the motion trend of crowd nearby) for the proposed model and absolute gis method with of the datasets are used to indicate the efficiency improvement of the proposed model. thus, this section discusses the improvement in respect of model's structure and operator for the previous three case studies. ( ) in the case of epidemic spread and close-contact querying, this paper analyzed the processes of the proposed model and absolute gis method for querying close contacts of the carrier. in absolute gis, this task could be achieved by buffer analysis in arcgis software. corresponding buffer areas related to a carrier had to be built while it was moving all the time in absolute space. however, in the proposed model, there is no need to build many buffer areas to recognize relationships in all time. it only executed once reference cutting to get all object's relationships to the carrier, no matter how many objects in the scenario. this is fundamental reason for the better performance of our model in this case study. table and fig. (a) show that more relationships had to be recognized in absolute gis with the increasing number of objects in the scenario, which leads to the execution time increase more quickly compared with the proposed model. ( ) in the case of tracker finding, the pattern matching approach in absolute space needs to compute the relative relationships of moving objects first, then uses data structure to store the time series of relative relationships between target objects with others, and finally filters the time series of relative relationships with distance constraints to match the pattern of the tracker. it is a complicated computation process in absolute gis method. the proposed model directly extracted each object's relationship referred to the target in relative space by reference cutting. then, the operators of trending and matching were used to find tracker. these computation processes are simple matrix-based computation. therefore, it could have a better computation performance than the pattern matching approach in absolute space. results in table and fig. (b) demonstrate the execution times by the proposed model is much shorter than that in absolute gis method. ( ) in the case of deriving the motion trends of nearby crowds, motion trends of crowd nearby in absolute gis method need to computing each object's distance to the walker from coordinates in each time first, then compares the time series of relative distance to find the moving trend of close or far away, and finally to plot it as a distribution map in absolute space, which supports the choosing of walk routes. this computation task is also an implicated computation process. the proposed model could analyze their distance change characteristic to the walker by the operators of trending and matching, which are also matrix-based computations suitable to computer environment. table and fig. (c) shows that the proposed model saved approximately % of the execution time compared to absolute space-based gis method.. in short, the proposed model outperforms the absolute space-based approach in relative motion analysis by simplifying the process of relationships computation and querying.. by extending stc in arcgis, this paper presents a novel model for storing, managing and analyzing moving objects based on their relationships in relative space. in this model, the x and y in stc are substituted as the reference and target objects, and the stb in stc are extended to store the data structure related to relative relationship among moving objects. to support the analysis of moving objects, this paper introduces six classes of operators according to the type of relationship outcome, namely, query processing, group relative dynamic calculation, transformation between absolute space and relative space, initial reference object transformation, attribute transformation, relative relationship dynamic pattern transformation. then, two common used algorithms, relative relationship querying and relative relationship dynamic pattern matching, are introduced on the base of these defined operators. the former algorithm organizes the relative relationship as three dimensions (reference object, target object and time) based on the model's structure, and queries relative relationship by corresponding objects and time. the later algorithm implements reference cutting and labeling to match each object's relative motion with specific pattern, which simplifies similarity measure in absolute space. this study collected the trajectories of walking pedestrian and used these data to demonstrate the feasibility of the proposed model. finally, the proposed model was successfully tested in three real-life scenarios to analyze dynamic and complex relationships between moving objects. the results validated the capabilities of these designed functions and indicated that the proposed model saved up to %- % execution time compared with traditional absolute gis methods. and the proposed model could be widely applied in the domains of public health, security, individual-based services. therefore, the contributions of this paper could be summarized as follows: ( ) a relative space-based gis data model of moving objects (rsmo) is proposed to construct, operate and analyze moving objects' relative relationships in relative space. ( ) relative space-based relationship-querying algorithm and relative fig. . curves of the execution times for "querying close contacts" (a), "finding tracker" (b) and "deriving the motion trends of nearby crowds" (c) with the proposed model (blue line) and geodatabase (red line). (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) dynamic pattern-matching algorithm are proposed on the base of the proposed model. ( ) three real-life scenarios are implemented to demonstrate the feasibility and usefulness of this proposed model in discovering interesting behavior hidden in crowd (e.g. querying close contact, finding tracker and deriving the crowd's motion trends). moreover, the computational time of these real-life scenario analysis could save up to - % while compared with absolute space based methods. the proposed model still has a big disadvantage. the data volume of the proposed data model was much larger than that of the traditional absolute space-based data organization. the compression of these relative data is a challenge. future research need study efficient compressing algorithms to simplify the data pre-process and reduce the volume of relative data. the handling of multiple types of relationships could also be examined in this model, e.g., social and mental relationships. powerful operators (i.e., closeness analysis in groups) in the social domain could be integrated into the model to 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real-time vessel behavior prediction the research was supported in part by the national natural science foundation of china (grants , ), and the fundamental research funds for the central university. key: cord- - n f wrz authors: węglarz-tomczak, ewelina; talma, michał; giurg, mirosław; westerhoff, hans v.; janowski, robert; mucha, artur title: neutral metalloaminopeptidases apn and metap as newly discovered anticancer molecular targets of actinomycin d and its simple analogs date: - - journal: oncotarget doi: . /oncotarget. sha: doc_id: cord_uid: n f wrz the potent transcription inhibitor actinomycin d is used with several cancers. here, we report the discovery that this naturally occurring antibiotic inhibits two human neutral aminopeptidases, the cell-surface alanine aminopeptidase and intracellular methionine aminopeptidase type . these metallo-containing exopeptidases participate in tumor cell expansion and motility and are targets for anticancer therapies. we show that the peptide portions of actinomycin d and actinomycin x( ) are not required for effective inhibition, but the loss of these regions changes the mechanism of interaction. two structurally less complex actinomycin d analogs containing the phenoxazone chromophores, questiomycin a and actinocin, appear to be competitive inhibitors of both aminopeptidases, with potencies similar to the non-competitive macrocyclic parent compound (k(i) in the micromolar range). the mode of action for all four compounds and both enzymes was demonstrated by molecular modeling and docking in the corresponding active sites. this knowledge gives new perspectives to actinomycin d's action on tumors and suggests new avenues and molecules for medical applications. natural products are excellent leads for drug development. particularly in the areas of antibiotic and anticancer therapies, natural products and their derivatives comprise a significant percentage of clinically used drugs [ ] . actinomycin d (actd), also known as dactinomycin, a small molecule naturally produced by streptomyces bacteria [ ] , was the first antibiotic shown to have anticancer activity. approved for medical use in [ ] , dactinomycin has been mostly used as a remedy for a variety of mainly pediatric tumors, such as wilms' tumor, rhabdomyosarcoma and ewing's sarcoma [ ] [ ] [ ] [ ] [ ] [ ] . actd is a neutral molecule consisting of a planar phenoxazone ring substituted with two carboxylatelinked cyclic pentapeptides ( figure ). its anticancer role has been associated with dna functionality, leading to rna, and consequently, protein synthesis inhibition [ ] . actd binds to double and single-stranded dna (but not to rna) with its phenoxazone ring intercalating with high specificity between gpc base pairs. moreover, it stabilizes cleavable topoisomerase i and ii complexes with dna, in which the polypeptide lactone rings occupy a position in the minor groove of the dna helix or the drug penetrates the topoisomerase-dna binding area [ , ] . after years of actinomycin d usage, its action was also correlated with apoptosis; at low doses, the drug is a potent inducer of apoptosis [ , ] , triggering this process in normal and tumor cells, as well as in cells that rarely undergo apoptosis [ ] . it was shown that the molecular mechanism of actinomycin d-induced apoptosis was associated with activation of the apoptosis fas surface protein, which is also known as cd (cluster of differentiation ) [ ] , the non-mitochondrial stress-activated protein/junminoterminal kinases apoptotic pathway, and with increased expression of the apoptosis regulator bax [ ] . cells inducing apoptosis in the immune response pathway via actinomycin d may also be associated with the expression of the cd surface antigen [ ] . additionally, the presence of intracellular atp is necessary for the induction of apoptosis by actinomycin d [ , ] . in recent years, intensive research in laboratories and clinics has focused on the correlation between actinomycin d activity and cellular tumor antigen p functionality and expression [ , ] . actd induces p expression [ , ] . apoptosis in patients with mutated or deleted p suggests a p -independent cell death mechanism [ ] . however, the function of p was restored after low doses of actinomycin d in various tp wildtype tumor cell lines [ , ] . -amino- h-phenoxazin- -ones missing the cyclic pentapeptide fragments, such as actinocin and questiomycin a (figure ), are also naturally produced by streptomyces [ ] . they occur in nature as pigments, fungal metabolites and allelo-chemicals [ ] [ ] [ ] . compounds composing the -aminophenoxazin- -one skeleton exhibit antitumor, antimicrobial, and antiviral activities in vitro and in vivo [ ] [ ] [ ] [ ] [ ] . soon after the discovery of actinomycin d and its properties, the anticancer actions of questiomycin a were also described [ ] , but the success of actinomycin d turned attention away from its simpler analogues. human alanine aminopeptidase (hsapn) and methionine aminopeptidase type (hsmetap ) are representative metalloproteases that have been targeted as a therapeutic intervention against pathological disorders. among others, they participate in proteolytic pathways associated with angiogenesis, apoptosis and proliferation, which makes them interesting objects of oncological research [ ] [ ] [ ] . metap is the best recognized member of the three known intracellular methionine aminopeptidases that catalyze the removal of the n-terminal methionine residue from a number of nascent proteins. this step is required before folding into their functional forms [ , ] . the enzyme binds two cobalt [ ] or manganese ions [ ] in its active site. as higher metap expression was observed in tumor (mesothelioma, [ ] neuroblastoma, [ ] and colorectal carcinoma [ ] ) cells compared with normal cells, the enzyme serves as a target for anti-angiogenic compounds of natural origin, such as fumagillin and ovalicin [ ] . selective inhibition of metap stops vascularization and tumor growth in animal models [ , ] . aminopeptidase n is a membrane-bound, zincdependent metalloproteinase also known as cd (cluster of differentiation ) [ ] . apn specificity is directed towards the cleavage of a neutral or basic amino acid from the n-terminal position of peptides and proteins [ ] . in contrast to metap , its active site is accessible to the exterior environment of the cell and the cell membrane anchoring its n-terminus [ ] . aminopeptidase n is described as a moonlighting protease with multiple functions, including antigen presentation. it is also a receptor for some human viruses, e.g., coronaviruses. these functions are connected not only to peptide cleavage but also to endocytosis, signaling and ligation or the inhibition of its enzymatic activity, which might result in complex and systemic effects [ ] . apn overexpression has been observed in tumor cells from melanoma [ ] , renal [ ] , colon [ ] , gastric [ , ] , pancreatic [ ] , and thyroid [ ] cancers and on leukemic blasts in acute myeloid leukemia [ ] . compelling studies have implicated apn protease activity in tumor-associated processes, particularly angiogenesis, apoptosis and metastasis [ ] [ ] [ ] . here, we show that actinomycin d and its analogues inhibit human metap and apn. developments in synthetic ligands of metalloaminopeptidases, substrates and inhibitors, indicated that not only amino acid and peptide mimetics but also heterocyclic compounds may effectively interact with these enzymes [ , [ ] [ ] [ ] [ ] . inspired by these accounts we have suggested the -amino- h-phenoxazin- -one scaffold as capable of binding to the aminopeptidase active sites. indeed, the well-known antibiotic and anticancer drug actinomycin d inhibits the two aminopeptidases hsapn and hsmetap with inhibition constants approximately μm (table and figure a ). kinetic studies allowed us to determine the mechanism www.oncotarget.com of inhibition of actinomycin d as non-competitive, with steady-state binding being achieved immediately ( figure b ). comparable results (within the experimental error) were obtained for actinomycin x , a structure closely related to actd. a minor variation in the amino acid composition of the α peptide side chain ( figure ) did not influence their binding affinities. despite structural similarity to actd medicinal properties of actinomycin x have been scarcely investigated. for example, actinomycin x was reported to show higher cytotoxicity of the human leukemia cell line hl- (lc = nm, lc is the lethal concentration for a half of population) than actinomycin d (lc = nm) [ ] . further studies proposed actinomycin x as an inducer of apoptosis of human prostate cancer cells through the mtor pathway compounded by mirna [ ] . the analogs that do not possess the macrocyclic polypeptide fragments, actinocin ( figure c and d) and questiomycin a, exhibited competitive inhibition mechanisms for each aminopeptidase. regarding their potency, they differed significantly from one another. actinocin was twice as effective as actinomycin d/x in the case of hsapn and four times as effective in the case of hsmetap . in contrast, questiomycin was times less effective; the unsubstituted -aminophenoxazone ring gave rise to a five-fold decrease in activity for hsapn and hsmetap compared to the lead compound. cytotoxicity of actinomycin d and questiomycin a reported in the literature is visibly different to each other and to some extend corresponds with these results. -aminophenoxazine- -ones induced apoptosis in many types of cancer cells [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , however, in comparable cases of human acute b-and t-lymphoblastic leukemia, cervix carcinoma and larynx carcinoma, the cytotoxicity of questiomycin a is up to three orders of magnitude lower (ic = - μm) than actinomycin d (ic = - nm) [ ] . the mechanisms of binding for each inhibitor must be significantly different at the molecular level due to the the inhibitors were screened in tris buffer. the release of the fluorophore was monitored continuously. the linear portion of the progress curve was used to calculate the velocity. each experiment was repeated at least three times and the results are presented as the average with standard deviation. for more details, please see the materials and methods section. * calculated based on the cheng-prusoff equations for non-competitive and competitive inhibitors [ ] . fundamental variations in their structures. this issue was studied in additional detail by molecular modeling. the modeled binding of actd/actinomycin x with hsapn ( figure ) and hsmetap ( figure ) showed complex sets of contacts based on typical peptide-protein interactions at different conformations. in the case of apn [ ] , both drugs most typically penetrated the interior of the enzyme and filled the characteristic central cavity, which was apt to accept large peptide substrates ( figure a and b). the modeled assembly was generally stabilized by a hydrogen bonding network ( figure c and d), particularly welldefined for actinomycin x . arg of hsapn has been identified as a residue highly involved in binding. for actinomycin d its terminal guanidine nitrogen atom forms a convenient hydrogen bond bridge between both c=o groups of the heteroaromatic ring-substituting carboxyamides ( . Å and . Å, respectively, figure c ). in contrast, for the most favorable actinomycin x conformation ( figure d) , the corresponding n atom of arg is engaged in interactions with the α cyclic peptide via two tight contacts with c=o of d-val ( . Å) and thr ( . Å). the extra oxygen atom of oxopro is pointed towards the α-nitrogen of the same enzyme residue ( . Å). the β peptidyl ring of the inhibitor is also favorably bound by the guanidine residue of arg , with distances . Å and . Å to c=o of thr and n-meval, respectively. the thr residue is in additional contacts with the hydroxyl group of ser ( . Å, via c=o) and the carboxylate of asp ( . Å, via n-h). finally, the amino group of phenoxazinone interacts with -oh of tyr , a group which is involved in the transition state stabilization during the natural substrate cleavage. for metap [ ] , protein-actinomycin d/x interactions mainly involved the surface of the enzyme ( figure a and b). the inhibitors are favorably located at the entrance of the active site, thereby blocking substrate inclusion and processing. in the lowest energy conformation, moieties of the cyclic peptide α of actinomycin d are partially inserted into the active site cavity and surrounded by the hydrophobic enzyme residues: his , leu , his , ile , pro and leu ( figure c ). the heterocyclic system is exposed to the outside and hardly contributes to this lipophilic binding. nevertheless, the functional groups of -amino- hphenoxazin- -one form a set of hydrogen bond contacts. the -amino group is bridged between the carboxylate of asp ( . Å) and the hydroxyl o atom of tyr ( . Å). the latter is also in a favorable position to n-h of d-val of the cyclic peptide α ( . Å). in addition, the -oxo group of the inhibitor forms a contact with the n-h of phe ( . Å). the β peptidyl ring of actinomycin d interacts less tightly with the enzyme and only two specific hydrogen bonds are recognized. they involve the thr residue, the c=o group binds with the side chain -nh of asn , and n-h with the hydroxyl o atom of thr . for actinomycin x the overall ligand-enzyme architecture is somewhat different as the β ring and the heterocyclic system are mostly involved in hydrophobic interactions with phe , his , ile , his , phe , his , met , ala and leu of the protein surface ( figure d ). this fit is emphasized by hydrogen bond contacts of the hydroxyl group of tyr bridged between n-h and c=o of thr ( . Å and . Å, respectively), and the guanidino group of arg with c=o of d-val ( . Å). the α peptidyl ring of the ligand forms two specific interactions which involve asn and asn (the side chain -nh ) of the protein, and oxopro and n-meval (oxo/c=o, . Å and . Å, respectively) of actinomycin x . elimination of the cyclic peptide fragments from the structure of actinomycin d/x allowed the resulting actinocin to penetrate much further into the active sites of the studied metallopeptidases and to act as a classical competitive ligand by interacting with the metal ions (figures and ). for human apn, the heteroaromatic ring is conveniently positioned in the hydrophobic environment while a functional group oxygen atom is directed towards the zn ion. interestingly, flipping the molecule does not seem to disturb the preferential binding. the highest rating docking score was shown for the conformation where the carbonyl oxygen atom is involved in an interaction with the metal cation ( . Å, figure a ). the oxygen was also in a tight hydrogen bond proximity to the tyr -oh group ( . Å). these two contacts and the stabilization of the neighboring amino group by glu ( . Å) and gln ( . Å), similar to the peptide n-termini, closely resembles the substrate-enzyme complex, including the carbonyl positioning for the nucleophilic water attack. the zinc bidentate complexation with the -carboxylate group ( . Å and . Å) was an alternative thermodynamically favored conformation ( figure b ). in this case, the -oxo group was in close hydrogen bond proximity to the arg guanidine nitrogen atoms ( . Å and . Å, respectively). the -carboxylate also formed a potential o-h … o=c contact with the glu side chain group ( . Å). similarly, the main driving force for the binding of actinocin to hsmetap was a tight bidentate complexation between one of the cobalt ions and -carboxylate ( . Å and . Å, figure ). this interaction was further strengthened by the close three-dimensional fit of the molecule in the active site provided by hydrophobic contacts with phe , ala , ile and his and hydrogen bonds. in particular, the latter involved interactions between the -amino group and the ala ( . Å) and lys ( . Å) oxygen atoms, as well as the -oxo group and the ser hydroxyl ( . Å). this set of favorable interactions gave rise to the highest theoretical docking score for actinocin to metap and was consistent with the lowest obtained inhibition constant values. lacking both carboxylate groups, questiomycin a loses the affinity of actinocin to both apn and metap. the decrease in inhibitory activity is not dramatic, approximately one order of magnitude, but demonstrates that the metal-binding moieties are important but not critical for the structure and potency of a good ligand. indeed, according to the molecular modeling results, the general fit of the heteroaromatic system and specific hydrogen bonds are reproduced for questiomycin a in the active sites of the human metallopeptidases ( figure a and b). for hsapn ( figure a ) the -oxo group of questiomycin a is firmly bridged between arg guanidine nitrogen atoms ( . Å and . Å, respectively) while the heteroaromatic ligand core is nested in the hydrophobic environment of ala , ala , val and his . similarly, the structurally simplest inhibitor reproduces interactions of the functional groups with metap. -amino group is hydrogen-bonded with c=o of ala ( . Å), lys ( . Å) and gln ( . Å), while the -oxo group with the ser hydroxyl ( . Å). the lipophilic contacts involve ala , his and ile . taking in account these close similarities of questiomycin a versus actinocin binding, it can concluded that the difference in their activities are predominantly caused by the absence of metal coordination by carboxylates. actinomycin d is a long-known drug that was developed as an anticancer agent years before apoptosis and other cell death mechanisms and cancer progression were elucidated. it is still present in the world health organization's list of essential medicines [ ] , which contains the most effective and safest medicines required by any proper health system. while actd is widely used in the treatment of cancer, intensive research on its mechanism of action, as well as on clinical trials with actinomycin d in combination with other drugs, are progressing. at low concentrations, actinomycin d inhibits proliferation and induces cell death in neuroblastoma cell lines, as well as tumor regression in xenograft tumor models. cortes et al. proved experimentally that apoptosis is the major cell death mechanism caused by actinomycin d treatment in p wt cells, though less so in p -deficient cell lines [ ] . the combination of actinomycin with drugs as nutlin- [ ] and the immunotoxin rg [ ] have improved the anticancer activities of the latter by activating apoptosis and p . questiomycin a was demonstrated as costive agent of apoptotic cell death in the gastric and colon cancer cell line by dysregulating the function of mitochondria, and activating the caspase signaling [ , , ] . questinomycin a also induced cellular apoptosis by causing rapid intracellular acidification in several cancer cells [ , , , ] , generating reactive oxygen species in human lung adenocarcinoma cells and human glioblastoma cell line [ , ] and by dna laddering and upregulated fas expression in mouse melanoma b cells [ ] . direct connection of questiomycin with p still remains an issue to be elucidated. there are deep premises that it exists. the cell-surface receptor fas, a member of the tnf-r family of receptors, is a key component of the extrinsic death pathway [ ] . it has been reported that p can activate the extrinsic apoptotic pathway through the induction of genes encoding transmembrane protein fas [ ] that could be unregulated by questiomycin a [ ] . in contrast, aminopeptidases have also been subject to intensive research in oncological studies. accordingly, in recent years, methionine aminopeptidase type , as well as alanyl aminopeptidase, have become extremely specialized targets for the development of new inhibitors [ , - , , , , ] . most known metap inhibitors are irreversible binding synthetic analogues of fumagillin [ , ] , two of which, tnp- and beloranib, have advanced to clinical trials. tnp- has been shown to inhibit angiogenesis in vitro and in vivo. it entered clinical development for cancer as an anti-angiogenic agent and has achieved phase i/ii trials for kaposi's sarcoma, renal cell carcinoma, brain cancer, breast cancer, cervical cancer and prostate cancer. however, the trials have not led to its approval for clinical applications. beloranib was originally designed as an angiogenesis inhibitor similar to tnp- for the treatment of cancer. once the potential anti-obesity effects of metap inhibition became apparent during trials, clinical development began to focus on these effects, and beloranib has shown positive results inhibitor and enzyme amino acid residues are shown as sticks. the catalytic zinc ion is shown as a dark blue sphere. hydrogen bonds and hydrophobic contacts are shown as green lines. www.oncotarget.com in preliminary clinical trials for this indication [ ] . moreover, there are tens of reversible inhibitors of both natural and synthetic origin. they include anthranilic acid sulfonamides [ ] , bengamides [ ] , benzoselnazalones [ ] and compounds based on bestatin [ ] , , , -triazole [ ] , and pyrazolo [ , -b] indole [ ] . research on new, reversible agents that are safe for organisms is ongoing. the most known and widely studied reversible apn inhibitors, bestatin and chr- , also known under the commercial names ubenimex and tosedostat, respectively, have been investigated clinically for their anticancer effects. ubenimex is approved for the treatment of lung cancer and nasopharyngeal cancer, and tosedostat showed significant anti-leukemic activities in phase i/ii clinical trials. currently, phase i/ii studies of tosedostat in combination with other chemotherapeutic agents are in progress for aml and metastatic pancreatic adenocarcinoma [ ] . compounds with apn inhibitory activities improved the anti-metastasis and anti-angiogenesis effects in vivo [ , ] . additionally, intensive studies on enzyme structure specificity led to the development of highly active competitive inhibitors of apn, with inhibition constants in the nanomolar range. phosphonic and phosphinic analogues of amioacid and dipeptides [ , ] , benzosuberone [ ] , naturally occurring curcumin [ ] and even more recently, a cyclic peptide inhibitor [ ] can be counted in this group of compounds. the recent research on actinomycin d highlights its huge therapeutic potential and suggests that low doses of this drug could be used in combination with other agents to take advantage of its unique activity and avoid non-specific effects. nevertheless, there is no correlation between these properties and the inhibition of the neutral aminopeptidases metap and apn that is highly important in cancer progression. blocking the activity of metap and apn with actinomycin d or its analogs seems to be promising for the development of new generations of potent anticancer agents that would be implicated in different mechanisms of action and directed against multiple molecular targets. in view of the network nature of cancer [ ] , the use of combinations of inhibitors appears to be indicated, as well as alternative drugs with multiple specificities. actinomycin d may prove to be such a drug for targeting a network rather than a single molecule. the discovery reported here may contribute to the development of better therapies. actinomycin d was purchased as a lyophilized powder from sigma-aldrich (poznan, poland), actinomycin x was purchased form adipogen (liestal, switzerland). actinocine and questiomycin a were synthesized according to a previously described procedure [ ] . recombinant human apn and metap were purchased as a lyophilized powder from r&d systems (minneapolis, usa). fluorogenic substrates ala-amc (l-alanine- -methylcoumaryl- -amide) and met-amc (l-methionine- -methylcoumaryl- -amide) were purchased from peptanova (sandhausen, germany). spectrofluorometry was performed on a spectra max gemini em fluorimeter (molecular devices, sunnyvale, usa) in a -well plate format (excitation = nm, emission = nm). calculations of the kinetic parameters were performed using the softmax pro, graphpad prism and microsoft excel programs. recombinant human apn enzyme was dissolved in mm tris-hcl buffer (ph = . ) and used directly in the kinetic experiments. recombinant human metap was obtained as a lyophilized powder from r&d systems. the enzyme was dissolved in mm tris-maleate buffer containing . mm cocl , mm nacl and . % v/v albumin at ph = . and incubated for minutes. after this activation, the enzyme was used directly in the kinetic experiments. the inhibitors were screened against human apn and human metap at °c in the assay buffer as described above. for the steady-state measurements, the enzymes were preincubated for - min at °c with an inhibitor before adding the substrate (ala-amc, μm for human apn or met-amc and μm for metap ) to the wells. for the initial state measurements, the enzymes were preincubated for - min at °c before adding the substrate and inhibitor to the wells. the following conditions were used: ( ) total volume μl, ( ) eight different inhibitor concentrations, and ( ) enzyme concentration of . nm for human apn and nm for metap . the release of the amc fluorophore was monitored. the linear portion of the progress curve was used to calculate the velocity. hphepch phe [ , ] for apn and ebselen [ ] for metap were used as a positive control. the inhibition mechanism was determined from the lineweaver-burk and dixon plot ( figure b and d) . the ic values were calculated from the reaction velocity to inhibitor concentration plot fitted to the equation log[i] vs. response. the k i values were equal to the ic for non-competitive inhibitors [ ] and were calculated for competitive inhibitors using the following equation: k i = (ic -[e]/ )/ ( + ([s]/km)) [ ] , where the ic is the concentration of the inhibitor that effected % inhibition, [s] and k m are the substrate concentration and michaelis constant in the absence of inhibitor, respectively, and [e] is the concentration of the enzyme. www.oncotarget.com molecular modeling studies were performed using the biovia discovery studio program package (san diego, ca, usa). the crystal structures of human aminopeptidase n (cd ) in complex with amastatin (pdb id. fyt) [ ] and methionine aminopeptidase type (pdb id. d e) [ ] were used as the starting points for the calculations of the enzymes complexes with actinomycin d, actinomycin x , actinocin and questiomycin a. the enzyme models were protonated at ph . and minimized with the charmm force field. the water molecules and former ligands were removed. new ligands were protonated at the same ph and the partial charges of all the atoms were computed using the momany-rone algorithm. minimizations used the charmm force field with the smart minimizer algorithm up to an rms gradient of . Å. actinomycin d, actinomycin x , actinocin and questiomycin a were docked into the enzyme active center using the libdock docking algorithm. the number of hotspots was changed from to (standard method). the 'best' conformation method was selected and the minimization of the results was achieved using the charmm force field. natural products: a continuing source of novel drug leads bacteriostatic and bacteriocidal substances produced by soil actinomycetes dactinomycin basis of actinomycin action. i. dna binding and inhibition of rna-polymerase synthetic reactions by actinomycin the treatment of wilms' tumor: results of the second national wilms' tumor study results with ema/co (etoposide, methotrexate, actinomycin d, cyclophosphamide, vincristine) chemotherapy in gestational trophoblastic neoplasia and national wilms tumor study group. treatment of wilms tumor relapsing after initial treatment with vincristine, actinomycin d, and doxorubicin. a report from the national wilms tumor 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sensitization of pancreatic cancer cells to cd mediated apoptosis induction of surface antigen cd expression in t-lymphocytes following exposure to actinomycin d intracellular atp is required for actinomycin d-induced apoptotic cell death in hela cells actinomycin d and its mechanisms of action akt mediates actinomycin d-induced p expression methylated actinomycin d, a novel actinomycin d analog induces apoptosis in hepg cells through fasand mitochondria-mediated pathways specific activation of the p pathway by low dose actinomycin d: a new route to p based cyclotherapy actinomycin d induces p -independent cell death and prolongs survival in high-risk chronic lymphocytic leukemia activation of p in cervical carcinoma cells by small molecules phenoxazinone synthase: mechanism for the formation of the phenoxazinone chromophore of actinomycin contributions of phenoxazone-based pigments to the structure and function of nanostructured granules in squid chromatophores plectosphaeroic acids 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drug targets: broad applications or therapeutic niche? human melanoma invasion and metastasis enhancement by high expression of aminopeptidase n/cd urinary excretion of glycine.prolile dipeptidile aminopeptidase, n-acetyl-beta-d-glucosaminidase, alanine aminopeptidase and low molecular protein in patients with renal cell carcinoma aminopeptidase n is involved in cell motility and angiogenesis: its clinical significance in human colon cancer aminopeptidase n (apn/cd ) as a target molecule for scirrhous gastric cancer serum apn/cd as a novel diagnostic and prognostic biomarker of pancreatic cancer biological significance of aminopeptidase n/cd in thyroid carcinomas hematopoietic stem cells express multiple myeloid markers: implications for the origin and targeted therapy of acute myeloid leukemia role of aminopeptidase n (cd ) in tumor-cell invasion and extracellular matrix degradation aminopeptidase-n/ cd is a potential proapoptotic target in human myeloid tumor cells aminopeptidase n (cd ) as a target for cancer chemotherapy s pocket fingerprints of human and bacterial methionine aminopeptidases determined using fluorogenic libraries of substrates and phosphorus based inhibitors exploring s plasticity and probing s ′ subsite of mammalian aminopeptidase n/ cd with highly potent and selective aminobenzosuberone inhibitors metalloaminopeptidase inhibitors identification of methionine aminopeptidase as a molecular target of the organoselenium drug ebselen and its derivatives/analogues: synthesis, inhibitory activity and www.oncotarget.com molecular modeling study characterization of streptomyces mitkk- apoptosis of human prostate cancer cells induced by marine actinomycin x through the mtor pathway compounded by mirna ic -to-ki: a web-based tool for converting ic to ki values for inhibitors of enzyme activity and ligand binding synthesis, biological evaluation, and molecular modeling of h-pyrido[ , -a]phenoxazin- -one, a compound with potent antiproliferative activity anticancer activity of phenoxazines produced by bovine erythrocytes on colon cancer cells prevention of carcinogenesis and development of gastric and colon cancers by -aminophenoxazine- -one (phx- ): direct and indirect anti-cancer activity of phx- suppression of the proliferation of cancer cell lines, kb- - and k cells preceded by a decrease in intracellular ph caused by phenoxazine derivatives -aminophenoxazine- -one-induced apoptosis via generation of reactive oxygen species followed by c-jun n-terminal kinase activation in the human glioblastoma cell line ln -aminophenoxazine- -one and -amino- , α-dihydro- α, -dimethyl- h-phenoxazine- -one cause cellular apoptosis by reducing higher intracellular ph in cancer cells apoptosis induction preceded by mitochondrial depolarization in multiple myeloma cell line u by -aminophenoxazine- -one rapid decrease of intracellular ph associated with inhibition of na+/h+ exchanger precedes apoptotic events in the mnk and mnk gastric cancer cell lines treated with -aminophenoxazine- -one -aminophenoxazine- -one prevents pulmonary metastasis of mouse b melanoma cells in mice the x-ray crystal structure of human aminopeptidase n reveals a novel dimer and the basis for peptide processing spiroepoxytriazoles are fumagillin-like irreversible inhibitors of metap with potent cellular activity effect of low doses of actinomycin d on neuroblastoma cell lines low-dose actinomycin-d treatment re-establishes the tumoursuppressive function of p in rela-positive ependymoma actinomycin d enhances killing of cancer cells by immunotoxin rg through activation of the extrinsic pathway of apoptosis the fas death factor apoptosis -the p network the rational design of therapeutic peptides for aminopeptidase n using a substrate-based approach allosteric inhibition of aminopeptidase n functions related to tumor growth and virus infection phase i trial of the angiogenesis www.oncotarget.com inhibitor tnp- for progressive androgen-independent prostate cancer methionine aminopeptidase type- inhibitors targeting angiogenesis development of sulfonamide compounds as potent methionine aminopeptidase type ii inhibitors with antiproliferative properties the bengamides: a mini-review of natural sources, analogues, biological properties, biosynthetic origins, and future prospects -amino- -hydroxyamides and related compounds as inhibitors of methionine aminopeptidase- novel reversible methionine aminopeptidase- (metap- ) inhibitors based on purine and related bicyclic templates discovery of potent, reversible metap inhibitors via fragment based drug discovery and structure based drug design-part phase i/ii clinical study of tosedostat, an inhibitor of aminopeptidases, in patients with acute myeloid leukemia and myelodysplasia design, synthesis and biological evaluation of novel amino acid ureido derivatives as aminopeptidase n/cd inhibitors structureguided, single-point modifications in the phosphinic dipeptide structure yield highly potent and selective inhibitors of neutral aminopeptidases a structural insight into the p s binding mode of diaminoethylphosphonic and phosphinic acids, selective inhibitors of alanine aminopeptidases irreversible inhibition of cd /aminopeptidase n by the antiangiogenic agent curcumin cancer: a systems biology disease catalytic oxidative cyclocondensation of o-aminophenols to -amino- h-phenoxazin- -ones the authors declare no conflicts of interest. key: cord- -ltx w zh authors: zhu, liqian; jiang, xinyi; fu, xiaotian; qi, yanhua; zhu, guoqiang title: the involvement of histone h acetylation in bovine herpesvirus replication in mdbk cells date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: ltx w zh during bovine herpesvirus (bohv- ) productive infection in cell cultures, partial of intranuclear viral dna is present in nucleosomes, and viral protein vp associates with histones and decreases histone h acetylation, indicating the involvement of histone h acetylation in virus replication. in this study, we demonstrated that bohv- infection at the late stage (at h after infection) dramatically decreased histone h acetylation [at residues k (h k ac) and k (h k ac)], which was supported by the pronounced depletion of histone acetyltransferases (hats) including cbp/p (creb binding protein and p ), gcn l (general control of amino acid synthesis yeast homolog like ) and pcaf (p /cbp-associated factor). the depletion of gcn l promoted by virus infection was partially mediated by ubiquitin-proteasome pathway. interestingly, the viral replication was enhanced by hat (histone acetyltransferase) activator ctpb [n-( -chloro- -trifluoromethylphenyl)- -ethoxy- -pentadecylbenzamide], and vice versa, inhibited by hat inhibitor anacardic acid (aa), suggesting that bohv- may take advantage of histone acetylation for efficient replication. taken together, we proposed that the hat-dependent histone h acetylation plays an important role in bohv- replication in mdbk (madin-darby bovine kidney) cells. bovine herpesvirus (bohv- ) is an important pathogen that causes pneumonia, conjunctivitis, genital disorders, and abortions in cattle [ ] . bohv- infection induces severe inflammatory response though diverse mechanisms, such as by overexpression of pro-inflammatory cytokines and reactive oxidative species [ ] [ ] [ ] . the suppression of host immune response by virus infection may render secondary infection by diverse pathogens, such as bovine viral diarrhea viruses (bvdv), bovine respiratory syncytial virus (brsv), parainfluenza- virus (pi v), bovine coronaviruses, mannheimia haemolytica, pasteurella multocida, histophilus somni and mycoplasma spp. [ , ] , and consequently lead to a life-threatening pneumonia known as bovine respiratory disease complex (brdc), one of the costliest ailments in cattle feeding [ , ] . in eukaryotes, dna is packaged into a protein-dna complex called chromatin, with nucleosome as monomeric subunit containing a core of histone proteins (h a, h b, h , and h ) surrounding by~ bp of genomic dna [ ] . the chromatin is dynamically organized into regions of either loosely packaged actively transcribed chromatin (euchromatin) or highly condensed cbp/p rabbit mab (monoclonal antibody) (cat# , : ), pcaf rabbit mab (cat# , : ), gcn l rabbit mab (cat# , : ), histone h rabbit mab (cat# , : ), acetyl-histone h (lys ) rabbit mab (cat# , : ), acetyl-histone h (lys ) rabbit mab (cat# , : ), ubiquitin mouse mab(cat# , : ), hdac (histone deacetylas) mouse mab (cat# , : ), hdac mouse mab (cat# , : ), hdac mouse mab (cat # , : ), hdac rabbit mab (cat # , : ), β-actin rabbit mab(cat# , : ), hrp (horseradish peroxidase) labeled anti-mouse igg (cat# , : ) and hrp labeled anti-rabbit igg (cat# , : ), were purchased from cell signaling technology (beverly, ma, usa). bohv- vp antibody ( : ) is kindly provided by prof. vikram misra at the university of saskatchewan [ ] . anacardic acid (aa) (cat#a ), trichostatin a (tsa) (# ). mg (cat# - ), ammonium chloride (nh cl) (cat# ), were ordered from sigma-aldrich (st. louis, mo, usa). bortezomib (#s ) was obtained from selleckchem.com (houston, tx, usa). n-( -chloro- -trifluoromethyl-phenyl)- -ethoxy- -pentadecyl-benzamide (ctpb) (cat# - - ) was provided by santa cruz biotechnology (dallas, tx, usa). cytotoxicity of indicated chemicals in mdbk cells was assessed by trypan-blue exclusion test, as described by fiorito et al. [ , ] , with modification. in brief, mdbk cells in -well plates were treated with or without chemicals at indicated concentrations for h. then the cells were collected by trypsinization, and an aliquot of the cell suspension was mixed with an equal volume of . % trypan-blue ( . %) (bio-rad, hercules, ca, usa, # ). after incubation for min, cells were counted using a burker chamber under a light microscope. the percentage of cell viability in the chemical treatment groups was calculated by normalization of the number of live cells to that in the control samples. the value of cell viability in the control was arbitrarily set to %. confluent mdbk cells in mm dishes were infected with bohv- (moi = ) for , and h. cell lysates were prepared using lysis buffer ( % triton x- , mm sodium chloride, mm edta, mm egta, mm sodium fluoride, mm sodium pyrophosphate, mm phenylmethylsulfonyl fluoride, . g/ml leupeptin, mm benzamidine, and mm sodium orthovanadate in mm tris-hcl, ph . ). to test the effects of certain inhibitors on the designated signaling, mdbk cells were infected for h along with treatment with indicated chemicals at the designated concentrations. cell lysates were prepared using lysis buffer as described above. cell lysates were separated on or % sds-polyacrylamide gels, and proteins were transferred to a polyvinylidene difluoride (pvdf) membrane (bio-rad, hercules, ca, usa). targeted proteins were detected using respective antibodies. the intensity of immune reactive bands was analyzed with free software image j (https://imagej.nih.gov/ij/download.html). to calculate the relative protein expression levels, the band intensity of target proteins was firstly normalized to β-actin, then normalized to the control lane. for ip studies, mdbk cells in -mm dishes were infected with bohv- at an moi of . at h after infection, cells were lysed with ml of ripa buffer ( × pbs, % np- , . % sodium deoxycholate, . % sds) supplemented with protease inhibitor as described above in western blots analysis. cell lysates were clarified by centrifugation at , rpm for min, and incubated with dynabeads ® (life technologies, carlsbad, ca, usa, cat. no. d), which have been incubated with µl of acetyl-histone h (lys ) rabbit mab (cell signaling technology, cat# ) or gcn l rabbit mab (cell signaling technology, cat# ) for h at room temperature with rotation. after overnight incubation at • c with rotation, the beads were collected with the help of a magnet (dynamag™) (life technologies, cat. no. d). after three washing with pbs, beads were boiled in sds loading buffer and western blots were performed to detect the designated proteins. mdbk cell in -well plates were infected with bohv- (moi of ) along with the treatment of indicated chemicals(paa, anacardic acid, tsa, and ctpb) at the designated concentration for h at • c, after three washing with pbs, fresh medium with designated chemicals was added to each well. at h after infection, viral yields were titrated in mdbk cells. the cell cultures treated with dmso was used as a control. the results are expressed as tcid /ml calculated using the reed-muench formula. confluent mdbk cells in mm dishes were infected with bohv- using an moi of . at , and h post infection(hpi) total rna was purified with trizol ls reagent (ambion, thermo fisher scientific, waltham, ma, usa, cat# ) following the manufacturers' instructions. freshly prepared rna ( µg) was used as a template for the synthesis of the first-strand cdna with commercial random hexamer primers for viral mrna detection using thermoscript™ rt-pcr system kit (invitrogen, carlsbad, ca, usa, cat# - ). the cdna products were used as templates for relative qrt-pcr to measure levels of viral mrna of bicp , and bicp as well as cellular gene glyceraldehyde- -phosphate dehydrogenase (gapdh) with specific primers as previously described in the reference [ ] . analysis of gapdh mrna was used as an internal control. relative qrt-pcr was carried out using the abi fast real-time system (applied biosystems, foster city, ca, usa). separate gapdh amplification was used to normalize gene expression. the data were analyzed using the equation −∆∆ct method. acetylation of histone h is involved in transcription activation, and h k ac is an epigenetic marker for histone acetylation. to test whether bohv- infection alters histone h acetylation, we evaluated the expression levels of h k ac, h k ac as well as h in virus infected mdbk cells at , , and hpi. as a result, virus infection consistently decreased the expression levels of h k ac, h k ac and h , and peaked at hpi ( figure a ). quantitative analysis indicated that the levels of h k ac, h k ac and h were decreased to . %, . % and . % relative to the control, respectively( figure b ), suggesting that bohv- infection reduced histone h acetylation (h k ac and h k ac). histone acetylation and deacetylation are reversible processes regulated enzymatically by hats and histone deacetylases (hdacs). hats such as cbp/p , gcn l , and pcaf, are enzymes that ultraviolet (uv) light-inactivated viruses are replication deficient because it could bind to the receptors and enter the cells, but are unable to express viral genes [ , ] . to further understand whether complete viral replication cycle was required to affect the histone h acetylation, uv-inactivated viral particles were employed for further investigation. as shown in figure c ,d, uv-inactivated virus had no effects on histone h acetylation. thus, these results suggested that de novo viral protein production and/or dna replication seems to be associated with the decreased acetylation of histone h . histone acetylation and deacetylation are reversible processes regulated enzymatically by hats and histone deacetylases (hdacs). hats such as cbp/p , gcn l , and pcaf, are enzymes that acetylate conserved lysine residues on histones. to understand the mechanisms underlying the decreased histone h acetylation by virus infection, we initially detected the protein levels of cbp/p , pcaf, and gcn l following bohv- infection at , , and hpi. virus infection altered the expression of cbp/p , pcaf, and gcn l , only at hpi, all of them were robustly decreased in comparison to the mock infected control (figure a ). the protein levels of cbp/p , pcaf, and gcn l were reduced to approximately . %, . %, and . % relative to the control, respectively ( figure b ). the global decease of hats expression at h after infection may reflect their reduced capability for histone acetylation, which is in agreement with the decreased histone h acetylation. hdacs are a family of enzymes controlling deacetylation of histones. the family of mammalian hdacs is comprised of at least members which are classified into four classes: class i (hdac , , and ), class ii (hdac , , , , and ), class iii (sirt to ) and class iv (hdac ) [ ] . in this study, the expression of hdac - in response to virus infection was detected using western blots. as can be seen in figure c ,d, virus infection altered the expression of both hdac and hdac with distinct manners, while neither hdac nor hdac were apparently affected. at hpi, the expression levels of hdac and hdac were decreased to approximately . % and . % relative to the control, respectively. the unexpected decreased expression levels of both hdac and hdac may undermine the finding that virus infection decreased the acetylation of histone h . taken together, virus infection altered the expression of hats and hdacs with distinct manners. relative to hdacs, the prominently decreased expression of hats strongly supported the reduced acetylation of histone h at hpi. hdacs are a family of enzymes controlling deacetylation of histones. the family of mammalian hdacs is comprised of at least members which are classified into four classes: class i (hdac , , and ), class ii (hdac , , , , and ), class iii (sirt to ) and class iv (hdac ) [ ] . in this study, the expression of hdac - in response to virus infection was detected using western blots. as can be seen in figure c ,d, virus infection altered the expression of both hdac and hdac with distinct manners, while neither hdac nor hdac were apparently affected. at hpi, the expression levels of hdac and hdac were decreased to approximately . % and . % relative to the control, respectively. the unexpected decreased expression levels of both hdac and hdac may undermine the finding that virus infection decreased the acetylation of histone h . taken together, virus infection altered the expression of hats and hdacs with distinct manners. relative to hdacs, the prominently decreased expression of hats strongly supported the reduced acetylation of histone h at hpi. our foregoing results demonstrated that virus infection differentially altered hats and hdacs expression, particularly the depletion of hats correlated with the reduced histone h acetylation. therefore, the role of hats and hdacs in bohv- productive infection was independently investigated using hat inhibitor anacardic acid (aa) and hdac inhibitor trichostatin a (tsa), respectively. aa specifically inhibits the enzymatic activity of hats, such as cbp/p and pcaf, and thereby affects hat-dependent gene transcription [ ] . also, aa has been reported to have multiple other biological effects such as antitumor activity and antioxidant activity [ ] . in this study, we found that the treatment of virus-infected cells with hat inhibitor aa at a concentration of µm and µm resulted in a . -and . -log reduction of the virus titer comparing to that in the mock-treated control, respectively ( figure a) . indeed, µm of aa treatment could inhibit histone h acetylation as demonstrated by the reduced levels of h k ac relative to the control, but aa increased the levels of h k ac in the context of virus infection in comparison to the mock treated but infected cells ( figure e ,f). maybe the significantly decreased progeny virus by aa treatment led to rescuing the depletion of h k ac attributed to virus infection. tsa is an hdac-specific inhibitor that can selectively inhibit the enzymatic activities of class i and ii hdacs, but not class iii hdacs [ ] . interestingly, it was reported that influenza virus infection decreased hdac expression in a cells, and the treatment of infected cells with and µm of tsa resulted in . -fold and . -fold increase of progeny virus relative to the control, respectively [ ] . in this study, we used much fewer concentrations of tsa to investigate its role in bohv- replication. we found that the treatment with nm of tsa could restore histone h acetylation ( figure g ,h), but had no impact on the viral replication because the virus titer was only increased~ . log (equal to -fold), with a difference not statistically significant (p > . ) ( figure b ). of note, all the concentrations used for indicated inhibitors had no cytotoxicity to mdbk cells ( figure e ). but we noticed that in the context of virus infection the cell viability was reduced to approximately . % by tsa ( nm) treatment ( figure i ), which is a possible reason for why tsa treatment could not evidently booster viral replication. these results suggested that the maintenance of hat activities was essential for virus productive infection. ctpb is a potent activator of cbp/p , but not of pcaf activities. to further confirm the role of hat played in the virus infection, ctpb, was employed for further investigation. ctpb at a concentration of µm showing no cytotoxicity to mdbk cells can significantly promote virus productive infection ( figure c ,e). the virus titer was increased~ . log by the treatment with µm of ctpb in comparison with the mock-treated control ( figure c ). this observation further suggested that the histone acetylation by hat played an essential role in bohv- productive infection. considering that histone acetylation regulated by hat is an important factor controlling gene expression, we further investigated the effects of chemical inhibition of hats on viral gene expression. for this purpose, the virus-infected cells were treated with µm of aa or dmso as a control, and the mrna levels of immediate early (ie) genes including bicp and bicp were detected with relative qrt-pcr. in aa treated virus-infected cell cultures, the mrna levels of bicp considering that histone acetylation regulated by hat is an important factor controlling gene expression, we further investigated the effects of chemical inhibition of hats on viral gene expression. for this purpose, the virus-infected cells were treated with µm of aa or dmso as a control, and the mrna levels of immediate early (ie) genes including bicp and bicp were detected with relative qrt-pcr. in aa treated virus-infected cell cultures, the mrna levels of bicp decreased to . % and . % relative to the mock-treated control, at and hpi, respectively, but at hpi the chemical treatment showed minor effects ( figure a ). bicp mrna levels were reduced to . %, . % and . % by aa treatment relative to the control samples, at , and hpi, respectively ( figure b ). though these ie proteins were not detected due to the unavailability of given antibodies, these results of qrt-pcr indicated that the hat inhibitor aa affected the transcription of these ie genes. an additional study was performed to examine the effects of aa on the expression of viral tegument protein vp by western blots using an antibody against vp . as demonstrated in figure c , at hpi vp protein expression levels decreased approximately . -fold by the treatment with aa relative to that in the mock-treated control, indicating that aa affected vp expression. in summary, these findings further suggested that this hat inhibitor affected bohv- productive infection in mdbk cells, and therefore the maintaining of hat activity is essential for virus efficient replication. viruses , , x for peer review of decreased to . % and . % relative to the mock-treated control, at and hpi, respectively, but at hpi the chemical treatment showed minor effects ( figure a ). bicp mrna levels were reduced to . %, . % and . % by aa treatment relative to the control samples, at , and hpi, respectively ( figure b ). though these ie proteins were not detected due to the unavailability of given antibodies, these results of qrt-pcr indicated that the hat inhibitor aa affected the transcription of these ie genes. an additional study was performed to examine the effects of aa on the expression of viral tegument protein vp by western blots using an antibody against vp . as demonstrated in figure c , at hpi vp protein expression levels decreased approximately . -fold by the treatment with aa relative to that in the mock-treated control, indicating that aa affected vp expression. in summary, these findings further suggested that this hat inhibitor affected bohv- productive infection in mdbk cells, and therefore the maintaining of hat activity is essential for virus efficient replication. the virus infected cells were treated with dmso or aa ( µm). total rna was prepared at indicated time points, and qrt-pcr was performed to determine the mrna levels of bicp (a) and bicp (b). (c) mdbk cells in mm dishes were infected by bohv- at an moi of with the treatment of dmso or aa ( µm), at hpi, cell lysate was prepared and subjected to western blots to detect vp protein. the band intensity was analyzed with software image j. and analysis was compared with that of untreated but infected control. data represent three independent experiments. significance was assessed with the student t test (* p < . , ** p < . ). ns: not significant. generally, there are two potential pathways to control protein degradation in eukaryotic cells, one mediated by ubiquitin-proteasome and other mediated by lysosome [ ] , which can be efficiently inhibited by chemical inhibitors, such as mg and nh cl, respectively. to identify whether the ubiquitin-proteasome and/or lysosome pathways are involved in the reduced acetylation of histone h at h after infection, the virus-infected mdbk cells were treated with either proteasome inhibitor mg or lysosome inhibitor nh cl throughout infection as determined elsewhere [ , ] . the treatment of mg ( µm) reversed the depletion of both h k ac and h k ac attributed to virus infection, and rescued their expression to a level higher than that in the uninfected control, whereas nh cl treatment did not show any effect ( figure a,b) . the increased levels of ubiquitinated proteins . total rna was prepared at indicated time points, and qrt-pcr was performed to determine the mrna levels of bicp (a) and bicp (b). (c) mdbk cells in mm dishes were infected by bohv- at an moi of with the treatment of dmso or aa ( µm), at hpi, cell lysate was prepared and subjected to western blots to detect vp protein. the band intensity was analyzed with software image j. and analysis was compared with that of untreated but infected control. data represent three independent experiments. significance was assessed with the student t test (* p < . , ** p < . ). ns: not significant. generally, there are two potential pathways to control protein degradation in eukaryotic cells, one mediated by ubiquitin-proteasome and other mediated by lysosome [ ] , which can be efficiently inhibited by chemical inhibitors, such as mg and nh cl, respectively. to identify whether the ubiquitin-proteasome and/or lysosome pathways are involved in the reduced acetylation of histone h at h after infection, the virus-infected mdbk cells were treated with either proteasome inhibitor mg or lysosome inhibitor nh cl throughout infection as determined elsewhere [ , ] . the treatment of mg ( µm) reversed the depletion of both h k ac and h k ac attributed to virus infection, and rescued their expression to a level higher than that in the uninfected control, whereas nh cl treatment did not show any effect ( figure a,b) . the increased levels of ubiquitinated proteins in mg -treated cells but not in nh cl-treated cells confirmed the efficiency of mg as an inhibitor for the proteasome pathway in mdbk cells ( figure c ). though the concentration of mg used in this study did not show obvious cytotoxicity to mdbk cells ( figure f ), the chemical may have off-target effects. so, another proteasome inhibitor bortezomib was employed to validate the rescued effects of mg . as expected, bortezomib at a concentration of nm showing no cytotoxicity to mdbk cells could reverse bohv- -reduced expression of histone acetylation marker h k ac to a level a little bit higher than that in the uninfected control ( figure d-f) , though bortezomib showed relative lower capability than mg . these results suggested that the ubiquitin-proteasome pathway may contribute to the decreased acetylation of histone h in the virus infected cells. viruses , , x for peer review of in mg -treated cells but not in nh cl-treated cells confirmed the efficiency of mg as an inhibitor for the proteasome pathway in mdbk cells ( figure c ). though the concentration of mg used in this study did not show obvious cytotoxicity to mdbk cells ( figure f ), the chemical may have off-target effects. so, another proteasome inhibitor bortezomib was employed to validate the rescued effects of mg . as expected, bortezomib at a concentration of nm showing no cytotoxicity to mdbk cells could reverse bohv- -reduced expression of histone acetylation marker h k ac to a level a little bit higher than that in the uninfected control ( figure d-f) , though bortezomib showed relative lower capability than mg . these results suggested that the ubiquitinproteasome pathway may contribute to the decreased acetylation of histone h in the virus infected cells. in view that the proteasome inhibitors of both mg and bortezomib could rescue the depletion of h k ac (a marker for histone h acetylation) attributed to virus infection, we investigated whether h k ac was ubiquitinated in the cells with or without infection by ip assay. unexpectedly, when we performed ip with the h k ac specific monoclonal antibody, the ubiquitinated protein bands could not be detected in the cells with or without infection using ubiquitin specific antibody ( figure a ), indicating that h k ac is not ubiquitinated in mdbk cells. so it was highly possible that the depletion of acetylated histone h due to virus infection was not caused by the proteasomemediated h k ac degradation. in view that the proteasome inhibitors of both mg and bortezomib could rescue the depletion of h k ac (a marker for histone h acetylation) attributed to virus infection, we investigated whether h k ac was ubiquitinated in the cells with or without infection by ip assay. unexpectedly, when we performed ip with the h k ac specific monoclonal antibody, the ubiquitinated protein bands could not be detected in the cells with or without infection using ubiquitin specific antibody ( figure a ), indicating that h k ac is not ubiquitinated in mdbk cells. so it was highly possible that the depletion of acetylated histone h due to virus infection was not caused by the proteasome-mediated h k ac degradation. our foregoing results indicated that both pcaf and gcn l were significantly decreased by the virus infection ( figure a) . interestingly, the treatment of virus-infected cells with µm of mg could significantly reverse the depletion of gcn l but not pcaf ( figure b,c) . we speculated that virus infection may promote gcn l degradation via proteasome pathway. therefore, ip was performed with gcn l specific monoclonal antibody, and ubiquitin specific antibody was used to detect the ubiquitination of gcn l in the cell cultures. as a result, the ubiquitinated gcn l could be detected from the ip sample by the immunoblot using ubiquitin specific antibody ( figure d ). this result indicated that virus infection might target gcn l for ubiquitin-mediated degradation, which would partially account for the depletion of acetylated histone h and correlated with the reversed depletion of h k ac by the treatment of mg in the virus-infected cells. our foregoing results indicated that both pcaf and gcn l were significantly decreased by the virus infection ( figure a) . interestingly, the treatment of virus-infected cells with µm of mg could significantly reverse the depletion of gcn l but not pcaf ( figure b ,c). we speculated that virus infection may promote gcn l degradation via proteasome pathway. therefore, ip was performed with gcn l specific monoclonal antibody, and ubiquitin specific antibody was used to detect the ubiquitination of gcn l in the cell cultures. as a result, the ubiquitinated gcn l could be detected from the ip sample by the immunoblot using ubiquitin specific antibody ( figure d ). this result indicated that virus infection might target gcn l for ubiquitin-mediated degradation, which would partially account for the depletion of acetylated histone h and correlated with the reversed depletion of h k ac by the treatment of mg in the virus-infected cells. acetylation is one of the best-characterized covalent modifications of histones. hyperacetylation of histones is associated with an "open chromatin" conformation and transcriptional activation, whilst hypoacetylation of histones is associated with condensed chromatin and gene silencing [ , ] . for some dna viruses, such as hsv- and canine parvovirus, much is known about the effects of histone modification on virus replication. during hsv- productive infection, the viral genomes are associated with histones immediately after injection into the nucleus, and viral proteins icp and vp are required to enhance histone acetylation on the viral genome to enable efficient viral gene expression [ , ] . during canine parvovirus infection, cellular histones are associated with viral dna, and histone acetylation on parvoviral dna is essential for viral gene expression [ ] . bohv- tegument protein vp associates with histones and thereby decreased histone h acetylation in infected cells or vp transfected cells [ ] . in this study, for the first time we demonstrated that histone h acetylation (h k ac and h k ac) was significantly decreased during bohv- infection in mdbk cells, and peaked at h after infection (figure ) , which suggested the involvement of histone h acetylation in bohv- infection. to further elucidate the potential mechanisms for the decreased histone h acetylation in response to the virus infection, the steady state expression of certain hats and hdacs were investigated because they regulate enzymatically histone acetylation with opposite effects. we found that the virus infection led to a global decrease of all the detected hats, including cbp/p , gcn l and pcaf (figure a) , which was clearly in favor of the finding that virus infection decreased histone h acetylation (figure ). however, it seems that downregulation of both hdac and hdac by virus infection was not correlated with the decreased levels of histone h acetylation, which emphasized the complexity of the mechanisms for the regulation of histone acetylation following virus infection. it has been reported that hdac regulates influenza a virus (iav) replication independent of its deacetylation activity [ ] . hdac stimulates host type i interferon antiviral response, therefore iav infection decreases hdac expression for efficient replication [ ] . the treatment with tsa, an hdac inhibitor, increases iav infection via the inhibition of signal transducer and activator of transcription i (stat ) pathway, and the depression of interferon-stimulated genes including ifitm , isg , and viperin in iav-infected cells [ ] . hdac is required for inflammatory gene expression in response to lps stimulation [ ] . so, hdac and hdac can stimulate host antiviral response or inflammatory response, which tend to be depressed by virus infection. moreover, hdac inhibitors promote hsv- productive infection in neural cells [ , ] . likewise, we found that tsa enhanced bohv- virus yield approximately -fold, but the difference was not statistically significant ( figure ). therefore, we assumed that the downregulation of hdac and hadc would be beneficial for bohv- efficient replication independent of their deacetylation activity, which needs further extensive studies, in the future. it has been reported that the ubiquitin-proteasome pathway, generally known to mediate protein degradation, is involved in bohv- productive infection and immune invasion [ , ] . here, we found that the ubiquitin-proteasome inhibitors could reverse the depletion of acetylated h in bohv- infected cells (figure ) . mechanistically, the virus infection targeted gcn l but not acetylated histone h for proteasome-mediated degradation (figure ), which may consequently reduce the acetylation of histone h . previous studies have reported that some bohv- -encoded viral proteins promote the proteasome-mediated degradation of certain cellular proteins. for example, both interferon response factor (irf ) and promyelocytic leukemia (pml) are targeted by viral proteins bicp for proteasome-dependent degradation [ , ] . viral ul . protein is involved in the proteasome-mediated degradation of the transporter associated with antigen presentation [ ] . the bohv- host shutoff protein ul destabilizes the expression of immune responses related genes by ubiquitin-proteasome pathway [ ] . taken together, the above evidence indicates that virus-encoded proteins may target specific cellular proteins for proteasome-dependent degradation. whether a specific viral protein(s) is involved in the depletion of gcn l through proteasome pathway is an interesting subject which is remained to be determined in the future. in this study, we found that bohv- replication was positively correlated with hat activity because the treatment with hat activator (ctbp) increased the virus yield, and vice versa, it was significantly decreased by hat inhibitor (aa) partially through affecting virus gene expression (figures and ) . this finding is supported by a previous report that cbp/p , an hat, enhances bohv- productive infection and transactivation of late viral protein gc promoter [ ] . taken together, these findings suggested that bohv- infection may take advantage of histone acetylation for efficient replication. we speculated that hat-dependent histone h acetylation plays an important role in bohv- replication in mdbk cells. in this study, the expression status of acetylated h including h k ac and h k ac was investigated in the context of virus infection. we demonstrated that histone acetylation played an important role in bohv- replication, while the virus infection decreased histone h acetylation by differentially altered expression of hats and hdacs. in addition, virus infection targeted gcn l for degradation via the proteasome pathway, which correlated well with the reversed depletion of h k ac by the treatment of mg in the virus-infected cells. we suggested that the hat-dependent histone (h ) acetylation plays an important role in bohv- replication in mdbk cells in summary, we provided evidence that bohv- infection decreased histone h acetylation in mdbk cells, while histone acetylation played an important role in bohv- replication. we suggested that hat-dependent acetylation of histone h plays a vital role in bohv- replication. this finding might add our knowledge on understanding the mechanism for the viral pathogenesis. bovine herpesvirus (bhv- ): biology, pathogenesis, and control active genes are tri-methylated at k of histone h response of proinflammatory and anti-inflammatory cytokines in calves with subclinical bovine viral diarrhea challenged with bovine herpesvirus- bhv- induced oxidative stress contributes to mitochondrial dysfunction in mdbk cells regulation of innate immune responses by bovine herpesvirus and infected cell protein (bicp ). viruses a review of the biology of bovine herpesvirus type (bhv- ), its role as a cofactor in the bovine respiratory disease complex and development of improved vaccines susceptibility loci revealed for bovine respiratory disease complex in pre-weaned holstein calves detection and characterization of viruses as field and vaccine strains in feedlot cattle with bovine respiratory disease chromatin structure: a repeating unit of histones and dna histone exchange, chromatin structure and the regulation of transcription regulation of histone modifying enzymes by the ubiquitin-proteasome system histone h a variants in nucleosomes and chromatin: more or less stable? regulation of chromatin by histone modifications linking 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bovine herpesvirus- dna in nucleosomes and chromatin of bovine herpesvirus- -infected cells: identification of a virion-associated protein in chromatin of infected cells protein composition of the bovine herpesvirus . virion the role of phospholipase c signaling in bovine herpesvirus infection protein and dna elements involved in transactivation of the promoter of the bovine herpesvirus (bhv) ie- transcription unit by the bhv alpha gene trans-inducing factor tetrachlorodibenzo-p-dioxin regulates bovine herpesvirus type induced apoptosis by modulating bcl- family members mg- reduces virus release in bovine herpesvirus- infection the high mobility group at-hook protein stimulates bovine herpesvirus productive infection the cellular protein p ipk regulates influenza virus mrna translation and replication through a pkr-mediated mechanism histone deacetylase is a component of influenza a virus-induced host antiviral response -alkylsalicylates are selective tip inhibitors and target the acetyl-coa binding site inhibition of histone acetyltransferase activity by anacardic acid sensitizes tumor cells to ionizing radiation trichostatin a and trapoxin: novel chemical probes for the role of histone acetylation in chromatin structure and function influenza a virus dysregulates host histone deacetylase that inhibits viral infection in lung epithelial cells the ubiquitin-proteasome pathway and proteasome inhibitors influenza a virus-induced degradation of eukaryotic translation initiation factor b contributes to viral replication by suppressing ifitm protein expression histone acetylation increases chromatin accessibility histone modification patterns during gene activation herpes simplex virus vp , but not icp , is required to reduce histone occupancy and enhance histone acetylation on viral genomes in u os osteosarcoma cells histone deacetylase plays an acetylation-independent role in influenza a virus replication requirement for the histone deacetylase hdac for the inflammatory gene expression program in macrophages histone deacetylase inhibition enhances oncolytic viral replication in glioma histone deacetylase inhibitors induce reactivation of herpes simplex virus type in a latency-associated transcript-independent manner in neuronal cells the infected cell protein encoded by bovine herpesvirus (bicp ) induces degradation of interferon response factor and, consequently, inhibits beta interferon promoter activity regulation of promyelocytic leukemia (pml) protein levels and cell morphology by bovine herpesvirus infected cell protein (bicp ) and mutant bicp proteins that do not localize to the nucleus varicelloviruses avoid t cell recognition by ul . -mediated inactivation of the transporter associated with antigen processing the ul -encoded virion host shutoff (vhs) protein and vhs-independent mechanisms are responsible for down-regulation of mhc class i molecules by bovine herpesvirus bovine herpesvirus immediate-early protein (bicp ) interacts with the histone acetyltransferase p , which stimulates productive infection and gc promoter activity key: cord- -aek mvdw authors: ishiguro, takashi; kobayashi, yasuhito; takano, kenji; ozawa, ryota; shimizu, yoshihiko; takayanagi, noboru title: two cases of primary human parainfluenza virus pneumonia in which bronchoalveolar lavage fluid yielded human parainfluenza virus date: - - journal: intern med doi: . /internalmedicine. - sha: doc_id: cord_uid: aek mvdw two patients, a -year-old woman and -year-old woman, presented to our hospital with symptoms of lower respiratory tract infection. both patients showed chest imaging findings of bilateral ground-glass opacities and consolidations. we initially suspected these patients of having influenza-associated pneumonia and cryptogenic organizing pneumonia, respectively, and performed bronchoalveolar lavage, but only human parainfluenza virus- infection was detected by multiplex polymerase chain reaction testing. these findings suggest that pneumonia due to human parainfluenza virus- should be included in the differential diagnosis of such cases. human parainfluenza viruses (hpivs) are single-stranded, enveloped rna viruses belonging to the paramyxoviridae family. compared with findings in studies of hpiv infection in children, less is known about these infections in adults. in previous studies, . to . % of hospitalized patients with pneumonia were found to have hpiv infection ( , ) ; however, the characteristics of primary viral pneumonia due to hpiv are not well known. we recently experienced two cases of pneumonia in which hpiv- was isolated from bronchoalveolar lavage (bal) fluid and confirmed by a multiplex polymerase chain reaction (pcr) test (fast track diagnostics resp kit, silema, malta), which detects the following respiratory pathogens: influenza a and b viruses; coronaviruses nl , e, oc , and hku ; human parainfluenza viruses , , , and ; human metapneumovirus a/b; rhinovirus; respiratory syncytial virus a/b; adenovirus; enterovirus; human parechovirus; bocavirus; and mycoplasma pneumoniae. we herein report these cases and review the clinical and radiological features. a -year-old woman presented to our hospital with appetite loss and fever in march. on the first day of illness, she initially developed appetite loss, and on the second day, she developed a fever of . . on the fourth day, she developed dyspnea, sore throat, and cough, and on the ffth day, she presented to a local physician who found abnormal shadows on chest x-ray and referred her to our hospital. she had no noteworthy past history. she was a never-smoker and was never exposed to dust. on admission, her vital signs included a body temperature of . , respiratory rate of /min, systolic blood pressure of mmhg, and heart rate of beats per min. chest auscultation showed bilateral coarse crackles. an arterial blood gas analysis under o of l/min by nasal canula showed a ph of . , partial pressure of carbon dioxide in arterial blood (paco ) (fig. a) . chest computed tomography showed ggos and consolidations in both lung fields ( fig. b and c) . pleural effusion or lymphadenopathy were not found. we performed bronchoscopy and the bal procedure from the internal segment of the right middle lobe. bal fluid ( of ml recovered) showed . × cells/ ml (neutrophils, . %; lymphocytes, . %; macrophages, . %; and eosinophils, . %) but did not show any microorganisms by gram staining or yield significant pathogens including m. pneumoniae or legionella spp. cytology of the bal fluid showed no significant findings. a transbronchial lung biopsy specimen showed alveolitis, fibrin exudation, and intraluminal organization (fig. ). blood culture was negative. we suspected her of having influenza-associated pneumonia and started ampicillin/sulbactam and peramivir. her chest shadows gradually improved, and her fever abated on hospital day (hd) . oxygen by nasal canula was stopped on hd , and she was discharged on hd . specific antibody titers against m. pneumoniae, c. pneumoniae, c. psittaci, legionella spp., influenza virus, adenovirus, and respiratory syncytial virus in paired sera did not increase, but antibodies against hpiv were not measured. we analyzed the bal fluid with multiplex pcr, which showed positive results only for hpiv- . two years after discharge, she has not relapsed. a -year-old woman presented to our hospital with cough, sputum, and sore throat of weeks duration in may. she initially visited a local physician who detected abnormal shadows on her chest x-ray and then referred her to our hospital. she had not received any antibiotics before presenting to our hospital. she had a history of gastric carcinoma when she was years old and had undergone gastrectomy. her cancer had not recurred. she was an ex-smoker with brinkman index of , but she had never been exposed to dust. on admission, her vital signs included a body temperature of . , respiratory rate of /min, systolic blood pressure of mmhg, and heart rate of beats per min. chest auscultation showed bilateral coarse crackles. an arterial blood gas analysis under ambient air showed a ph of . , paco of . torr, pao of . torr, and hco − of . mmol/l. laboratory data showed a white blood cell count of , /mm (neutrophils, . %; lymphocytes, . %; eosinophils, . %; basophils, . %; and monocytes, . %), hemoglobin of . g/dl, platelets of . × /mm , ast of iu/l, ldh of iu/l, creatinine of . mg/ dl, crp of . mg/dl, and kl- of u/ml. autoantibodies were negative, as was an anti-hiv antibody test. rapid nasopharyngeal or oropharyngeal diagnostic tests for influenza virus and mycoplasma pneumoniae and urinary antigen tests for s. pneumoniae and legionella spp. were all negative. chest x-ray showed bilateral consolidation (fig. a) , and chest computed tomography showed nonsegmental and subpleural consolidation bilaterally in the lower lobes (fig. b) . no centrilobular pulmonary nodules, pleural effusion, or lymphadenopathy was detected. we performed bronchoscopy and bal from the lateral segment of the right lower lobe. bal fluid ( of ml recovered) showed . × cells/ml (neutrophils, . %; lymphocytes, . %; macrophages, . %; and eosinophils, . %) but did not show any microorganisms by gram staining or yield significant pathogens including m. pneumoniae. cytology of the bal fluid also showed no significant findings. we could not obtain adequate specimens for evaluation via transbronchial lung biopsy. we initially suspected her of having cryptogenic organizing pneumonia (cop) and administered prednisolone mg daily. her chest shadows gradually improved, and she was discharged on hd . we analyzed the bal fluid with multiplex pcr, which showed positive results only for hpiv- . specific antibody titers against m. pneumoniae, c. pneumoniae, c. psittaci, legionella spp., influenza virus, adenovirus, and respiratory syncytial virus in paired sera did not increase, but antibodies against hpiv were not measured. we then gradually tapered the prednisolone and stopped it two months after hospital discharge. at four years since her treatment, she has not developed a relapse. we herein report two immunocompetent patients with primary hpiv- pneumonia. one patient required oxygen supplementation on admission, and both patients recovered without the administration of antiviral agents. most hpiv infections in adults cause mild upper respiratory tract symptoms, but the elderly or those with a compromised immune system are associated with progressive disease ( ) ( ) ( ) . hpiv is responsible for to % of acute respiratory illnesses in adults. in other studies, . to . % of hospitalized patients with pneumonia were found to have hpiv infection ( , ) . furthermore, hpiv is present in . % of severe pneumonias, which indicates that hpiv is a frequent pathogen of both non-severe and severe pneumonia ( ) . however, previous reports that investigated virus infections in patients with pneumonia used nasopharyngeal or oropharyngeal swabs to detect viruses, which raises the possibility of upper respiratory tract infection by hpiv. furthermore, these studies include mixed viral and bacterial infections, and the clinical characteristics of the immunocompetent patients with primary hpiv pneumonia are not fully known. in our two cases, hpiv was detected in bal fluid, which indicated the hpiv in our cases to be a pathogen of not the upper airways but the lower respiratory tract and of pneumonia. in addition, bal fluid did not yield other significant pathogens, nor did specific antibody titers against other pathogens of pneumonia in paired sera increase, which support the diagnosis of primary hpiv infection in our patients. four subtypes of hpiv (hpiv- , , , and ) have been identified, and hpiv- is the most prevalent serotype of infections. among adults hospitalized with pneumonia, hpiv- was found in . % of patients ( , ) . the causative virus in our patients was hpiv- , a subtype found in . % of adult pneumonias ( , ) . although our patients recovered successfully, tamaki et al. reported a fatal case of viremia due to hpiv- in a patient with adult t-cell leukemialymphoma treated with mogamulizumab ( ), which indicates that hpiv- can also be a fatal pathogen in immunocompromised patients. diseases mainly suspected in our patients before identification of hpiv- via multiplex pcr testing were primary influenza virus pneumonia and cop, respectively. a previous study reported that viral pneumonia is frequently misdiagnosed as cop ( ) . in the differentiation of these diseases, sore throat is a more frequent symptom of viral pneumonia than of acute interstitial lung diseases including cop ( ) , and our patients both had sore throats. however, typical symptoms in adult patients with hpiv infection include fever, rhinorrhea, cough, and a sore throat, which do not clinically distinguish it from other respiratory viruses ( ). cunha et al. reported that the clinical presentation of hpiv pneumonia closely mimics h n pneumonia ( ) . chest ct findings in our patients included bronchial wall thickening, bilateral consolidations, and ggos, which were compatible with typical ct findings of hpiv pneumonia, e. g., multifocal patchy consolidation with ggos, and centrilobular nodules with bronchial wall thickening ( ); however, these findings seem nonspecific for hpiv pneumonia. pokharel et al. reported multinucleated giant cells with organizing pneumonia as findings suggestive of hpiv infection ( ) , but the sensitivity and specificity of the pathologic findings were not known and these findings were not present in our patients. thus, it is important to include viral infections in the differential diagnosis and investigate respiratory specimens with viral pcr tests. hpiv causes epidemics during the spring season or a small secondary period of in- creased activity in the fall, and thus seasonality and local epidemiology may be clues suggestive of hpiv pneumonia. currently, no treatments with proven efficacy are approved for the treatment of hpiv infections. corticosteroids, generally dexamethasone and budesonide, are associated with fewer clinic visits and admissions for croup and the reduced use of nebulized epinephrine ( ) . we initially misdiagnosed our patient in case as having cop and administered corticosteroids. the efficacy of corticosteroid administration in immunocompetent adult patients with hpiv pneumonia is not known. some data suggest favorable effects on varicella-zoster virus (in combination with acyclovir) and hantavirus ( ) and in influenza-associated pneumonia in some clinical settings ( , ) . among previously reported patients with influenza-associated pneumonia, corticosteroids were administered without neuraminidase inhibitors in and were effective ( ) . among the patients with viral pneumonia due to non-influenza viruses, corticosteroids were administered to , of whom did not survive, but were effective in the other patients ( . %) ( ) . however, the mainstay of therapy in patients with hpiv infection is considered to be the reduction of immune suppression, which includes corticosteroids use ( ) . further studies are needed to clarify the significance of corticosteroids as a treatment option for primary hpiv pneumonia in immunocompetent patients. however, much of the available data for the use of antiviral agents in the treatment of hpiv comes from immunocompromised patients. the efficacy of antiviral drugs in immunocompetent adults with hpiv pneumonia has not been fully evaluated, and thus further studies are needed. one limitation of this report is that the possibility of detecting viruses from the upper respiratory tracts when using the bal technique cannot be denied. to avoid this concern and ensure that samples are obtained only from the lower respiratory tract, intubation or use of a protected specimen brush is required. in conclusion, we experienced two immunocompetent pa-tients with primary hpiv- pneumonia. primary hpiv pneumonia can resemble influenza-associated pneumonia and cop, and hpiv pneumonia should be included in the differential diagnosis of such cases. parainfluenza virus in the hospitalized adults infections with viruses in patients hospitalized with acute respiratory illness epidemiology and clinical impact of parainfluenza virus infections in otherwise healthy infants and young children < years old respiratory syncytial virus and parainfluenza virus respiratory syncytial viral infections in transplant recipients viral infections in patients with severe pneumonia requiring intensive care unit admission viral pneumonia in older adults in: mandell, douglas, and bennett's principles and practice of infectious diseases respiratory syncytial viral infections in transplant recipients fatal pneumonia and viremia due to human parainfluenza virus type in a patient with adult tcell leukemia-lymphoma treated with mogamulizumab viral pneumonia requiring differentiation from acute and progressive diffuse interstitial lung diseases human parainfluenza virus type (hpiv ) viral community-acquired pneumonia (cap) mimicking swine influenza (h n ) during the swine flu pandemic radiographic and ct features of viral pneumonia parainfluenza virus- -induced cytopathic effects on lung tissue and bronchoalveolar lavage fluid in a bone marrow transplant recipient: a case report glucocorticoids for croup. cochrane database syst rev cd medical treatment of viral pneumonia including sars in immunocompetent adult the role of corticosteroids in severe communityacquired pneumonia: a systematic review effect of low-to-moderatedose corticosteroids on mortality of hospitalized adolescents and adults with influenza a(h n )pdm viral pneumonia the internal medicine is an open access journal distributed under the creative commons attribution-noncommercial-noderivatives . international license. to view the details of this license a part of this study was supported by a research fund from saitama cardiovascular and respiratory center under grant no. es. key: cord- -seonrtn authors: alraddadi, basem m.; qushmaq, ismael; al‐hameed, fahad m.; mandourah, yasser; almekhlafi, ghaleb a.; jose, jesna; al‐omari, awad; kharaba, ayman; almotairi, abdullah; al khatib, kasim; shalhoub, sarah; abdulmomen, ahmed; mady, ahmed; solaiman, othman; al‐aithan, abdulsalam m.; al‐raddadi, rajaa; ragab, ahmed; balkhy, hanan h.; al harthy, abdulrahman; sadat, musharaf; tlayjeh, haytham; merson, laura; hayden, frederick g.; fowler, robert a.; arabi, yaseen m. title: noninvasive ventilation in critically ill patients with the middle east respiratory syndrome date: - - journal: influenza other respir viruses doi: . /irv. sha: doc_id: cord_uid: seonrtn background: noninvasive ventilation (niv) has been used in patients with the middle east respiratory syndrome (mers) with acute hypoxemic respiratory failure, but the effectiveness of this approach has not been studied. methods: patients with mers from saudi arabian centers were included in this analysis. patients who were initially managed with niv were compared to patients who were managed only with invasive mechanical ventilation (invasive mv). results: of mers critically ill patients, niv was used initially in ( %) patients, whereas ( %) patients were only managed with invasive mv. patients who were managed with niv initially had lower baseline sofa score and less extensive infiltrates on chest radiograph compared with patients managed with invasive mv. the vast majority ( . %) of patients who were managed initially with niv required intubation and invasive mechanical ventilation, and were more likely to require inhaled nitric oxide compared to those who were managed initially with invasive mv. icu and hospital length of stay were similar between niv patients and invasive mv patients. the use of niv was not independently associated with ‐day mortality (propensity score‐adjusted odds ratio . , % ci [ . , . ] p = . ). conclusions: in patients with mers and acute hypoxemic respiratory failure, niv failure was very high. the use of niv was not associated with improved outcomes. middle east respiratory syndrome (mers) has emerged as a cause of severe respiratory illness in humans. , as of march , , cases of mers have been reported including deaths. the disease presentation ranges from asymptomatic infection to severe respiratory illness, multiorgan failure, and death. [ ] [ ] [ ] acute hypoxemic respiratory failure (ahrf) develops in up to % of hospitalized patients with mers and is associated with high mortality. , to date, there is no specific antiviral therapy for mers of proven effectiveness; supportive therapy remains the cornerstone of management. noninvasive ventilation has been increasingly used in the management of ahrf with variable success. [ ] [ ] [ ] [ ] while niv may initially avoid the need for intubation and invasive mechanical ventilation (mv) , several studies have reported high failure rates and the need for invasive ventilation among patients with severe acute respiratory distress syndrome (ards) and an association with increased mortality. in a recent analysis from the lung safe study on unselected patients with ards, niv was associated with higher intensive care unit (icu) mortality in patients with the ratio of partial pressure of oxygen to the fraction of inspired oxygen (pao /fio ) lower than mm hg. the role of niv in ahrf secondary to viral respiratory infections is unclear. although some uncontrolled studies suggested that niv was effective and safe in management of patients with severe acute respiratory syndrome (sars), [ ] [ ] [ ] others have highlighted concern of increased transmission risk to healthcare workers when patients with sars are treated with niv. use of niv in ahrf caused by pandemic h n virus (pdmh n ) infection has been reported from several countries, [ ] [ ] [ ] with reported niv failure reaching up to %. all studies were limited by their retrospective nature and, often, small sample size. noninvasive ventilation has been used in patients with mers, , but its value in preventing intubation and impact on clinical outcomes has not been studied. the objective of this study was to assess the success of niv in mers patients with ahrf in avoiding intubation and its association with mortality and icu and hospital length of stay. our secondary objective was to identify factors associated with niv failure in mers patients. we conducted this analysis on a multicenter retrospective cohort of critically ill mers patients from in this study, we included all patients with ahrf who required mechanical ventilation support in the icu, whether invasively or noninvasively. all patients who were managed initially with niv were compared to those who were managed with invasive mv without niv. for this analysis, we extracted baseline data including demographics, comorbidities, duration from onset of symptoms to emergency room admission, icu admission, and intubation. arterial blood gases, continuous variables were described as medians and interquartile ranges (q , q ) or means and standard deviations and were tested we performed a secondary comparison of patients who had failed niv to those who had been successfully treated with niv. all statistical tests were two-sided with significance set at α < . . analyses were conducted using sas version . (sas institute, cary, nc). in patients who were managed initially with niv, niv was used for a median duration of ( , ) day and patients ( . %) eventually required intubation and invasive mv ( [ , ] , p = . ). there was no significant difference in the duration of invasive mv and total duration of niv and invasive mv between the two groups, although invasive mv-free days and total niv and invasive mv-free days were significantly longer among niv patients compared to invasive mv patients (table , figure s ). overall, only / ( . %) of the niv patients avoided subsequent intubation (table s ). these patients were significantly younger than tables s and s ). crude -day mortality was significantly higher in patients who failed niv compared with patients successfully treated only with niv (table s and figure s ). we have shown that among patients with mers-related ahrf, niv was commonly used, but nearly always resulted in subsequent transition to invasive ventilation. our results suggest that while the initial niv use in mers patients was not associated with reduction of mortality or length of icu or hospital stay, these patients had greater requirement for subsequent inhaled nitric oxide. a minority of patients were successfully managed with niv-those who were young and had less severe disease. these findings have important implications for early management of patients infected with mers, specifically, that there is little advantage to initial niv treatment for most patients with mers-related ahrf and that niv may be associated with greater subsequent need for oxygenation rescue therapy such as inhaled nitric oxide. noninvasive ventilation has been proven to be useful as a means to avoid intubation and improve clinical outcomes in certain conditions, generally, with the possibility for rather rapid reversal of respiratory failure-for example, pulmonary edema due to congestive heart failure, and respiratory failure due to copd exacerbations. , for conditions that typically worsen or do not improve in the range of many hours (eg, most causes of pneumonia), there appears to be little advantage in using niv as a means to avoid intubation. , , in choosing to use niv for as initial treatment for patients with hypoxemic respiratory failure, there is a practical risk of patients worsening on niv and requiring intubation at a time when they already have more advanced organ failure. few studies have assessed the effectiveness of niv in patients with ahrf secondary to ards and acute lung injury. the overall effectiveness of niv in reducing intubation rate or improving clinical outcome in these patients remains controversial. [ ] [ ] [ ] post hoc analysis of the lung safe study found that niv was used in % of patients with ards and was associated with higher icu mortality in subset of patients with severe ards. a randomized controlled trial of patients niv is generally not recommended for patients with hypoxia secondary to respiratory infections due to lack of efficacy and the potential for pathogen transmission. , , it is also considered one of the aerosol-generating procedures that may increase risk of transmission to healthcare workers. broad dispersion of exhaled air during niv via a face mask has previously been shown using a simulated patient encounter. in conclusion, we report the results of niv use in mers patients from a large cohort of critically ill patients. we observed that there is little advantage to initial niv treatment for most patients with mers-related ahrf and that niv may be associated with greater subsequent need for oxygen rescue therapy. we would like to thank the international 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group noninvasive ventilation in critically ill patients with the middle east respiratory syndrome key: cord- -skmxrkhf authors: park, inchae; yoon, byungun title: technological opportunity discovery for technological convergence based on the prediction of technology knowledge flow in a citation network date: - - journal: j informetr doi: . /j.joi. . . sha: doc_id: cord_uid: skmxrkhf as technological convergence has recently become a mainstream innovation trend, technological opportunities need to be explored in heterogeneous technology fields. most of the previous convergence studies have taken a retrospective view in measuring the degree of convergence and monitoring the converging trends. this paper proposes a quantitative future-oriented approach to technological opportunity discovery for convergence using patent information. in a future-oriented approach, technological opportunities for convergence are suggested by predicting potential technological knowledge flows (tkfs) between heterogeneous fields. the potential tkfs are predicted by a link prediction method in a directed network, which is suggested in this paper to represent the direction of the predicted tkfs by adapting the concept of bibliographic coupling and edge-betweenness centrality. converging technological opportunities are proposed as incremental and radical technological opportunities by extracting the potential increased knowledge flow links and emerging knowledge flow links. moreover, the direction and themes of the predicted potential tkfs are provided as technological opportunities for convergence. as an illustration of the proposed method, the technological opportunities between biotechnology (bt) and information technology (it) are explored. firms and researchers can use the proposed method to seek out new technological opportunities from various technologies so that r&d policymakers can plan new r&d projects on technological convergence. it is critical to monitor technological trends and anticipate the direction of technological change under rapidly changing technological circumstances. many attempts have been made for technological opportunity discovery (tod) to identify technological opportunities and threats that could affect future growth and survival because they are considered some of the most crucial issues for companies (cozzens et al., ) . the studies on tod have largely been conducted to anticipate promising technologies that have not yet been developed and to analyze potential new markets that can be created by utilizing existing technologies (cho, yoon, coh, & lee, ) . expert-based qualitative tod approaches such as delphi, questionnaire survey analysis, and triz (teoriya resheniya izobretatelskih zadach) have the advantage of easy validation and the disadvantage of expensive costs and of being timeconsuming. quantitative approaches such as computer-based methods and bibliometric analysis can play a complementary role to the disadvantage of the qualitative approaches and propose objective results based on a large volume of data. as massive technological data have been accumulated and analytic techniques have advanced, it is possible to identify technological opportunities using a quantitative approach. patent analysis has been employed as a representative quantitative approach for tod, and additional quantitative analytic techniques such as morphology analysis, text mining, network analysis, and novelty detection have been combined with patent analysis for tod (geum, jeon, & seol, ; lee, kang, & shin, ) . the tod studies based on patent analysis in the initial stage have focused on anticipating newly emerging technological opportunities. recently, a few attempts have been made to identify technological opportunities via technological convergence (an, kim, mortara, & lee, ) . thus, tod research has been extended from technological opportunities in a specific technology area to those merged with heterogeneous technology fields, which are different from the existing technology domain. technological convergence is also a critical issue because it can be a technological breakthrough. many quantitative approaches have been taken to tackle technology convergence using patent analysis. from the perspective of combining the research themes of tod and technological convergence, the previous quantitative technological convergence studies have some limitations. first, most of the previous studies on technological convergence using patent analysis have analyzed patent data with a retrospective view by measuring the degree of convergence (geum, kim, lee, & kim, ; takano, mejia, & kajikawa, ) . however, it is necessary to identify technological opportunities for convergence in terms of technology planning for the future. this paper suggests a prospective tod approach for technological convergence to overcome the limitation. second, many convergence studies investigate industry-and macro-level technology fusion (curran & leker, ) . it is insufficient to utilize the results at the stage of converging technology development because those studies analyze technological convergence at a high level. third, from a methodological point of view, patent co-classification has been largely utilized to measure the degree of technological or industrial convergence in the previous convergence studies because patent classification is suitable to represent technologies as a proxy and to measure the overlap between technologies (bongioanni, daraio, & ruocco, ) . although patent classification enables the monitoring status of convergence, it is difficult to suggest practical information in terms of a future-oriented perspective for converging technology development. to overcome the aforementioned limitations, a concept of technological knowledge flows (tkfs) can be utilized because technological opportunities can be diversified by using the tkfs from the heterogeneous technology fields to detect signals of technological convergence for the future (caviggioli, ) . patent co-classification analysis has been utilized in most of the previous convergence studies, whereas patent citation analysis can be utilized because the citation information on patents or scientific papers has been considered a reliable proxy for tkf (jaffe, trajtenberg, & henderson, ) . in addition, to emphasize the future-oriented view based on the tkf approach, link prediction, which is a technique used to detect missing links in the current networks and to predict new links in future networks, is utilized to identify technological opportunities for convergence. this research contributes to existing literature in several ways by filling the research gap and overcoming previous studies' limitations. first, a tod framework for converging technology opportunities is suggested as an extension of existing tod studies. second, a future-oriented tod approach is proposed by overcoming the retrospective view. third, technological opportunities are suggested at a micro level by subgrouping potential tkfs and suggesting promising technological themes. finally, a link prediction method is utilized to identify technological opportunities to overcome the methodological limitation. this paper aims to identify technological opportunities for convergence by predicting potential tkfs between heterogeneous fields using quantitative data analysis. three research questions are suggested in the present study. first, what are the potential technological opportunities for convergence? second, what are the characteristics of the predicted technological convergence areas? third, what are the possible technological themes for technological convergence? to solve the research questions, a new approach to exploring the tod for technological convergence is proposed by means of link prediction in patent-citation networks and tkf networks, as well as by investigation of knowledge flow properties and technological knowledge themes. in addition, technological opportunities for convergence are discussed with the direction and strength of the predicted potential tkfs. technological opportunity is defined as the potential or possibility for technological progress in a general or particular field (yang, huang, & su, ) . similar terms, such as technology intelligence (ti) and tod, have been used to identify technological opportunities. cho et al. ( ) elucidated the concept of tod with ti, which was suggested by kerr, mortara, phaal, and probert ( ) , in two perspectives: objects and activity. although ti focuses on opportunities and threats in the perspective of objects and is interested in information management and an analysis process in the view of activity, tod concentrates on opportunities and the strategic use of information in terms of objects and activity. in this paper, we used the concept of tod to identify technological opportunities because we deal with future-oriented opportunities and the strategic use of technological information. although technological opportunity is generally discovered through qualitative approaches such as delphi and scouting (rohrbeck, heuer, & arnold, ) , porter and detampel ( ) suggested technology opportunities analysis (toa) as a start toward the quantitative tod approach that identifies technological trends through bibliometric analysis and measurement of technological literature such as patents and publications. as data analytic techniques have advanced, technology opportunities have been identified mainly using patent information through many quantitative techniques such as keyword-based patent maps (lee, yoon, & park, ; yoon, yoon, & park, ) , keyword-based morphology analysis (yoon & park, ) , and novelty detection technique (geum et al., ) . the tod studies initially focused on identifying the technological opportunity itself. in previous tod studies using patentometric analysis, technological opportunities are proposed as the patent vacancy in the patent map, the outlier patent, and the recombination of technological functions. first, the vacant area was suggested as technological opportunities by developing the patent map based on textual information using data mining techniques such as the self-organizing feature map (sofm) (yoon et al., ) , principle component analysis (pca) (lee, yoon et al., ) , and generative topology mapping (gtm) (son, suh, jeon, & park, ) . the vacant area in the patent map was proposed as technological opportunities because it indicated the area that is not yet developed when the patent information is visualized as a map. second, outlier patents that were distinctive from existing technology were proposed using the outlier detection technique (geum et al., ; lee, kang et al., ; yoon et al., ) , which can suggest more explicit potential technological opportunities by identifying a novel patent that is new and distinct from the existing technology rather than identifying a patent vacancy representing a set of keywords. third, the recombination of technological functions was proposed as a technological opportunity by using keyword-based morphology analysis (yoon & park, ; yoon, park, & coh, ) and the function-based tod approach using the subject-action-object (sao) structure (yoon et al., ) . this approach is based on the assumption that new knowledge or invention is the result of the recombination of the known components of knowledge (aharonson & schilling, ; fleming, ; funk & owen-smith, ; notten, mairesse, & verspagen, ) . as tod-relevant studies have progressed, the coverage of research on tod has been extended by identifying potential opportunities from the convergence of various technological areas. although existing studies have proposed the tod methods for convergence based on patent citation analysis, they offered insufficient implications because they suggested patent pairs as potential technological opportunities. the coverage of recent tod studies tends to be extended by identifying technological opportunities from external areas. this paper concentrates on the new subject by meeting the recently required tod research trend, in that it proposes a tod approach for technological convergence on the basis of tkf from the future-oriented perspective. the concept of technological convergence was first introduced by rosenberg ( ) as a process commonly used by unrelated industry sectors and in various stages of tool production. kodama ( ) used the term "technology fusion" to represent a type of breakthrough innovation created by merging more than two existing technologies as a similar concept. although they have been used interchangeably in some cases to refer to a blurring of boundaries between more than two different areas, curran and leker ( ) differentiated between them in terms of loci where convergence and fusion take place. in this research, we conducted a literature survey on both as technological convergence research. the evolutionary stages of the convergence phenomenon are defined as knowledge (or scientific disciplines), technological, applicational (or market), and industrial convergence (curran, bröring, & leker, ) . research on the convergence of scientific disciplines has investigated the discovery of new knowledge through the exchange of knowledge. in this process, bibliometric analysis, such as co-word, co-citation, co-authorship, and journal subject category co-classification analysis, has been widely conducted using scientific article data (leydesdorff, ; porter & rafols, ; porter, cohen, roessner, & perreault, ; schummer, ; sun, ding, & lin, ; zhang, zhang, zhu, & lu, ; zitt, ramanana-rahary, & bassecoulard, ). research on market-level convergence has analyzed product specifications using press release data (lee, lee, & cho, ; han, chung, & sohn, ) . in this research, we focused on technological and industrial convergence using patent data. research on the convergence of technology at the industry level used patent data because patents are considered an indicator of technological innovation (curran & leker, ; lee, ) . in particular, the research on industry convergence analyzes industry-patent concordance information, such as standard industry classification (sic) and international patent classification (ipc) concordance (curran et al., ) , to link technology information to industry information. most of the previous studies on technological convergence using patent data have concentrated on measuring the degree of convergence or monitoring technological change. from a patent indicator perspective, most of the studies developed patent indicators based on a patent classification system to measure the degree of convergence and to monitor the phenomenon of technological convergence. trajectory patterns of technology fusion were identified by measuring fusion degree indicators based on the citing-cited relationship between patent classification codes (no & park, ) . a fusion index (fi), which represented the degree of fusion, was identified to predict promising fusion technologies (lee & yoo, ) . furthermore, indicators for identifying technological convergence were suggested based on the ipc-level network using the concept of entropy and gravity (han & sohn, ) . from the patent-citation analysis perspective, a few studies measured technological convergence and monitored the trends of convergence from knowledge flow based on patent citation with patent classification. geum et al. ( ) measured convergence intensity and coverage by means of patent citation and co-classification analysis. ko, yoon, and seo ( ) analyzed the trends of industry-wide technology fusion by constructing a knowledge-flow matrix using the citation analysis between ipcs. moreover, as a semantic analysis perspective, moehrle and passing ( ) suggested the possibility of convergence by applying an anchor-based patent mapping approach based on the semantic similarity. most of the previous research on technological convergence has utilized patent classification because it is appropriate to represent the technological area and to measure the overlap between technologies. although many previous studies on technological convergence have focused on analyzing the phenomena using a retrospective perspective, some researchers have attempted to identify technological convergence using a future-oriented perspective (kim & lee, ; lee, han, & sohn, ; song, elvers, & leker, ) . lee, han et al. ( ) predicted the pattern of technological convergence by applying both an association rule and link prediction based on ipc co-occurrence. song et al. ( ) and kim and lee ( ) anticipated technological convergence using the concept of tkf based on patent classification and patent citation, respectively. patent citation analysis has been utilized in many studies on tkf diffusion and transfer (a.j. r.r. nelson, ; park & suh, ) , technological trajectories and technological frontiers (mina, ramlogan, tampubolon, & metcalfe, ; wong & wang, ) , technological knowledge domain (weng & daim, ) , and so on. link prediction is utilized to identify a potential link in a tkf network based on patent citation information because this research aims to anticipate the future converging technological opportunity. link prediction is a technique to detect missing links in current networks and to predict new links in future networks. many algorithms are introduced to solve the link prediction problems from diverse types of networks (heterogeneous and bipartite) and links (multi-relational, active/inactive, and appearing/disappearing). it is categorized into ( ) node-based metrics, ( ) topology-based metrics, ( ) social-theorybased metrics, and ( ) learning-based methods (wang, xu, wu, & zhou, ) . below is a summary of a comprehensive review of the state-of-the-art link prediction in social networks by wang et al. ( ) . first, node-based link prediction metrics mainly calculate the similarities between node pairs by using attributes and actions that reflect node attributes and actions. second, topology-based link prediction metrics are based on the topological information. many topology-based metrics were proposed after liben-nowell and kleinberg ( ) discussed several metrics based on structural graph features. the topology-based metrics can be divided into neighbor-, path-, and random-based metrics. third, social theory-based metrics employ classical social theories, such as community, triadic closure, strong and weak ties, homophily, and structural balance. finally, learning-based methods, as proposed in recent works, differ from the previous approaches by using link prediction metrics, internal attributes, and external information. these are divided into feature-based classification, probabilistic graph models, and matrix factorization. for more information on link prediction, several excellent survey papers used diverse perspectives (liben-nowell & kleinberg, ; wang et al., ) . link prediction has been used for various applications within business areas, including recommendation systems to search for potential collaborators (mori, kajikawa, kashima, & sakata, ) , patent partners in enterprise social networks (wu, sun, & tang, ) , and interesting items in online shopping (akcora, carminati, & ferrari, ) . in academic social networks, a co-author relationship prediction is proposed in the heterogeneous bibliographic network in sun, barber, gupta, aggarwal, and han ( ) . in terms of technology forecasting, lee, han et al. ( ) proposed technological convergence pattern prediction using ipc. in this research, link prediction is used to predict tkfs between heterogeneous technology fields. the predicted tkfs between the fields can suggest the potential of technological convergence with strength and direction. this research proposes a systematic method to investigate the current tkf between heterogeneous fields by using patent analysis and to identify technological opportunities for convergence by predicting the potential tkf. to this end, first, patent-level networks based on patent-citation relationships are generated by network analysis, which is widely used to comprehend knowledge flow. two types of patent citation networks, patent citation networks, and potential patent citation networks are constructed. the patent citation network represents the current citation relationship, and the potential patentcitation network expresses the potential link between patents that do not have any citation relationship. the potential patent-citation network is generated by a link-prediction technique that predicts new links in future networks. in this paper, a link-prediction method based on edge-betweenness centrality is proposed to reflect the context of convergence and tkf between heterogeneous fields. second, technology-level networks, tkf networks, and potential tkf networks are visualized by merging patents in the same technology group in the patent citation network. the types of change in tkf are identified based on the comparison of tkf networks and potential tkf networks. third, a tkf property map is generated through the use of patent indicators that measure the property of tkf. the tkf property map represents the position of technology based on the property of knowledge flow and the direction of the predicted knowledge flow. technological opportunities for convergence are identified by a comprehensive approach based on the types of change in tkf and the trends of the predicted tkf. additionally, the themes of technological opportunities are suggested as a micro-level approach. as shown in fig. , our proposed approach consists of four steps: data collection and preprocessing, construction of tkf network, construction of potential tkf network, and tod for convergence. the first step is to collect patent data and preprocess the data for analysis. the second step is to generate a patent-citation network, which is then merged into the tkf network. the third step is to predict the potential patent citation. in this step, the proposed edge-betweenness centralitybased link-prediction method is used. thus, the potential patent-citation network and potential tkf network are generated by merging information on patent-level citations and the results of link prediction, and then the type of change in tkf is identified. finally, in the fourth step, technological opportunities for convergence are identified by reflecting the knowledgeflow property of technology and the change in knowledge flow and by suggesting themes of technological opportunities for convergence. in the following section, the detailed procedures of step and step are explained as a proposed approach for the present research. the potential tkf is predicted by link prediction from tkf based on the current citation relationships in the patentcitation network in step of fig. . in other words, the link prediction detects missing links that are likely to have citation relationships between patents even though patent citation actually does not occur. in the present study, a link-prediction method to anticipate knowledge flow between target technologies and heterogeneous technologies, which are different from the target technology, is proposed considering the context of technological convergence. most of the studies on link prediction methods have been focused on undirected networks (yu & wang, ) . to solve this problem, schall ( ) suggested a link-prediction method in a directed graph based on the probability that a given type of triad pattern will be closed. jawed, kaya, and alhajj ( ) also proposed a time frame-based link prediction using the triad pattern. the link-prediction approach using a triad pattern is not appropriate for patent-citation networks, in that a citation cannot occur mutually between patents. that is, the patent citation network is a directed network because of the acyclic characteristic of patent citation (a patent never cites later patents). yu and wang ( ) proposed a different approach for link prediction in directed networks by identifying three types of similar and candidate nodes for target nodes. in this research, the link-prediction technique suggested by yu and wang ( ) is adopted and revised to reflect the context of technological convergence opportunities. although the strength of the link is important in the patent citation network in terms of tkf, many previous link prediction approaches have not focused on the strength of links but on the node-based metrics. in this research, the proposed method is to predict potential links using edge-betweenness centrality, as suggested by girvan and newman ( ) . the concept of edge-betweenness centrality is adopted from the vertex-betweenness centrality proposed by freeman ( ) . the vertexbetweenness centrality of node i, which is the number of shortest paths between pairs of other vertices that run through node i, measures the influence of a node over the flow of information between other nodes. the edge-betweenness centrality of an edge is defined as the number of shortest paths between pairs of vertices that run alongside it. the edge with a high betweenness centrality value can be interpreted as a bridge connecting different communities. although liu, hu, haddadi, and tian ( ) proposed a link-prediction model based on node centrality, it gave weight based on the node centrality of common neighbors. the proposed link-prediction method gives weight based on edge-betweenness centrality because the links with a high edge-betweenness centrality value can be considered important links connecting heterogeneous fields in terms of tkf. the concept of the proposed link-prediction method is shown in fig. . the concept of similar and candidate nodes for target nodes from yu and wang ( ) is adopted, but with a difference in the method for selecting nodes. a directly linked node (called a -hop-out neighborhood) and an indirectly linked node (called a -hop-out neighborhood), among three types of similar nodes, as suggested by yu and wang ( ) , are not considered similar nodes in this study because technological knowledge is transferred and accumulated by patent citation. in this research, bibliographic-coupled patents in heterogeneous technologies are considered similar nodes for target nodes in ways that yu and wang ( ) considered to be sharing nodes as a third type of similar nodes. after searching for similar nodes in different technology fields for a target node, the list of candidate nodes is identified based on the concept of edge-betweenness centrality in networks. the proposed method consists of three steps: ( ) locating similar nodes of a target node, ( ) identifying candidate nodes, and ( ) ranking candidate nodes. first of all, the nodes similar to target nodes are defined as bibliographic-coupled nodes, which are greater than a cut-off value, in a technology that is different from the target node's. a directed link, <u, v> ε, e exists between nodes u and v if u links to v. according to eq. ( ), s(u) is a similar node to the target node u. b(u,v) is a normalized bibliographic-coupling strength. b cut-offvalue (u,v) is the cut-off value for the selection of a similar node. tech (u) or tech (v) is the technology field to which patent node u or v belongs. the bibliographic-coupling strength is defined as the number of common references, and the normalized bibliographic-coupling strength, which is shown by glänzel and czerwon ( ) , is defined as the following: where nbc uv is the normalized bibliographic-coupling strength between patent nodes u and v, r uv is the number of common references between patents u and v, and n u or n v is the number of references in the reference list of patent u or v. second, after selecting the list of similar nodes s(u) for target nodes u, a list of candidate nodes c(u) is identified. it is assumed that a target node may link to nodes that its similar nodes also link to. ( ) according to eq. ( ), the set of out neighbors of node u is out is the set of out neighbors of node v. third, candidate nodes are ranked by scores composed of weight w(u,c,s) and edge-betweenness centrality eb(e sc ), which is the concept suggested by girvan and newman ( ) : according to eq. ( ), score(u, c) is the score that links target node u to candidate node c reflecting weight w(u, c, s) and edge-betweenness centrality eb(e sc ), w(u, c, s) are the weight scores that link target node u to candidate node c through similar node s, and eb(e sc ) is an edge-betweenness centrality that links similar node s to candidate node c. as shown in eq. ( ), weight score w(u, c, s) is calculated by the score of a similar node s ∈ s(u) for each candidate c ∈ c(u), if s follows c: according to eq. ( ), v i v j is the number of shortest paths between vertices v i and v j , and v i v j (e sc ) is the number of shortest paths between vertices v i and v j that run through the edge between vertices s and c. for example, patent u in fig. , which belongs to technology b, is selected as a similar node to target patent u , which belongs to technology a because both patents are bibliographically coupled and have a normalized bibliographic-coupling strength of / when the cut-off value is / and belong to heterogeneous technology fields. the candidate nodes are identified as u , u , and u because the similar node, u , is connected to those nodes in the patent-citation network. finally, the candidate nodes u and u are predicted to link to target node u even though u belongs to technology a, whereas u and u belong to technologies b and c, respectively, after the candidate nodes are ranked using a score based on edge-betweenness centrality when using an appropriate cut-off value. based on the patent-citation relationship, a potential patent-citation link is predicted by the proposed link-prediction method, which is described above in detail. potential patent-citation links are derived as a potential patent-citation network through link prediction based on the bibliographic coupling relationship and edge-betweenness centrality. then the potential tkf networks are visualized by integrating the patent nodes by technology level. the predicted link indicates the direction of the potential patent citation and the linked patent information that is positioned in a different technology field. thus, the purpose of step is to recommend the patents that might be cited and that are positioned in heterogeneous technology fields by using a systematic link prediction method. the change in direction and strength of the predicted links is categorized in table through comparison of the tkf network and potential tkf network. the tkfs increase when the predicted links are added from the tkf network to the potential tkf network; otherwise those newly predicted links emerge from the tkf network. two types of links can exist as unidirectional and bidirectional links; otherwise, the link does not exist in the tkf network. in terms of increased change, first, tkf can increase in an identical-directional way when unidirectional links exist between technologies in the tkf network and when the identical-directional links are predicted in the potential tkf network. for example, patent-citation links are predicted from technology a to b in the potential tkf network when patent citation occurred from technology a to b, and then the knowledge flow increased identical-directionally. second, the tkf can increase unidirectionally when bidirectional links exist between technologies in the tkf network and the links are only predicted to be one-way in the potential tkf network. for example, patent-citation links are predicted only from technology a to b in the potential tkf network when patent citation occurred from technology a to b and vice versa; then the knowledge flow unidirectionally increased. third, tkf can become bidirectional when bidirectional links exist between technologies in the tkf network and the links are predicted to be two-way in the potential tkf network. for example, patent-citation links are predicted from technology a to b and vice versa in the potential tkf network when patent citation occurred from technology a to b and vice versa; then the knowledge flow became bidirectional. from the perspective of emerging change, first, tkf can emerge as different-directional when unidirectional links exist between technologies in the tkf network and the different-unidirectional links are predicted in the potential tkf network. for example, patent-citation links are predicted from technology b to a in the potential tkf network when patent citation occurred from technology a to b; then the knowledge flow emerges different-directionally. second, tkf can emerge as unidirectional when the link does not exist between technologies in the tkf network and the unidirectional links are predicted in the potential tkf network. for example, patent-citation links are predicted from technology a to b in the potential tkf network when patent citation did not occur between technologies a and b; then the knowledge flow emerges unidirectionally. third, tkf can emerge as bidirectional when the link does not exist between technologies in the tkf network and the bidirectional links are predicted in the potential tkf network. for example, patent-citation links are predicted from technology a to b and vice versa in the potential tkf network when patent citation did not occur between technologies a and b; then the knowledge flow emerged bidirectionally. the tkf property is determined whether the technology is mainly absorptive or diffusive between heterogeneous technologies by using a patent classification code, which is assigned to patents in focal technologies. degree centrality and the ratio of patent classification in technology are used to measure the tkf property as eqs. ( ) and ( ). to this end, a citation network whose node is a patent classification is generated when the patents whose citations occur between heterogeneous fields are used. the inflow and outflow properties are defined as the following: where p if,t and p of,t are the inflow and outflow properties for technology t, respectively; if i and of i are the inflow and outflow properties for patent classification i by using in-degree centrality and out-degree centrality, respectively; and r i,t is the ratio for patent classification i in technology t. the in-degree and out-degree centrality are measured as follows (freeman, ) : where if i and of i are the in-degree and out-degree centrality for patent classification i respectively; g ij represents the relation from patent class i to j in the network. the technologies are mapped in the tkf property map with the x-axis representing the inflow property and the y-axis the outflow property, as shown in fig. . the tkf property is categorized into the technology knowledge absorption and diffusion active group (a&da), tkf absorption-oriented group (ao), tkf diffusion-oriented group (do), tkf absorption and diffusion neutral group (a&dn), and tkf absorption and diffusion passive group (a&dp), using proper cut-off values. the tkf property map shows the position of technology property and the direction and type of change (increased or emerging) for predicted knowledge flows. the position can indicate which technologies are active, passive, or biased between the heterogeneous fields in terms of tkf absorption and diffusion. in the context of convergence, the tkf property (absorption or diffusion) represents which technologies already technologically converged between two fields in the status quo, in that technology convergence may come from technological interaction. technologies in the tkf a&da (or a&dp) group have actively (or passively) interacted with each other between the heterogeneous fields, because the technologies in the a&da (or a&dp) group have both high (or low) in-and out-flow property values. technologies in tkf ao (or do) that have a high (or low) inflow but low (or high) outflow property value serve as knowledge absorbers (or diffusers), largely in terms of the tkf bias between the fields in the current situation. technologies in tkf a&dn that have a middle inflow and outflow property value or have a middle inflow (or outflow) but a low outflow (or inflow) property value interacted neutrally with each other between the fields. the predicted tkfs that are represented as dashed and solid lines show the directions of the potential tkfs in the future. the increased knowledge flows are considered incremental converging technology opportunities, and the emerging knowledge flows are considered radical converging technology opportunities. in the context of convergence, the predicted tkf is able to suggest which technologies have the potential to converge with each other with in terms of direction, strength, and type. technological opportunities for convergence are identified by suggesting technological themes for predicted knowledge flows. many studies have conducted exemplary case studies on the convergence between bt and it because it provides tools and platforms for the investigation and transformation of biological systems (geum et al., ) . to illustrate the proposed approach, we also conducted a case study of bt-it convergence and adopted the technology field classification of bt and it proposed by geum et al. ( ) , which measured the convergence of bt and it at the technology level because the classification was the accumulated results based on the technology classification of research in it (lee, kim, & park, ) and in bt (no & park, ) , and it was derived systematically using information such as the us patent classification (uspc) and uspc to ipc concordance. the technology fields bt and it were split, as shown in table , into bt-related technologies and it-related technologies. . . step : data collection and preprocessing the granted patent data that were registered between january and december were collected from the uspto online database, as shown in table , using searching queries combining relevant uspcs by technology field, as proposed by geum et al. ( ) , and keywords in abstracts that represent the technology's title. the collected granted patent data from to were used for the proposed approach, and the data registered between and were used to validate the proposed method. the , collected patents, registered between and , among , patents registered between and , were preprocessed by removing noisy and duplicated data in multiple technological fields. the data were transformed to the cited-citing format, with the patent number, assigned focal technology, registration year, and patent classification code for further steps. the patent-citation network was generated based on the , , cited-citing pairs, as shown (a) in fig. , using nodexl, a free and open-source network analysis and visualization software package. the tkf network was developed, as shown (b) in fig. , by merging patents in the same technology group in (a) in fig. . the tkf network still shows a complex relationship between technologies, and the widths of the links in the network indicate the number of citations between technologies. the tkf between technologies in homogeneous technology sectors (i.e., it to it or bt to bt) account for approximately % ( , patent-citation pairs), and the tkf between technologies in heterogeneous technology sectors (i.e., it to bt or bt to it) account for approximately % ( , patent-citation pairs). in this research, we focused on the tkf between bt and it because the purpose of the research is to identify technological opportunities for convergence. step : construction of potential knowledge flow network the potential patent-citation network was developed through link prediction, and the potential tkf networks were visualized by merging the patent nodes by technology level, as shown in fig. , after conducting link prediction and selecting cut-off value for the tkf network. the black solid line and gray dashed line in the potential tkf network in fig. denote emerging links and increased links in comparison with the tkf network. to conduct the proposed link prediction, cutoff values for bibliographic-coupling strength and edge-betweenness centrality should be selected. the distribution of the respective value on normalized bibliographic coupling strength and the edge-betweenness centrality for the method is shown in appendixes a and b. the x-axis represents the intervals of continuous numerical value, the bar represents the rate of each interval, and the curve represents the cumulative rate as the y-axis. both values follow long-tail distributions that have few very large events and many very small events. most of the links have low normalized bibliographic-coupling strength values as shown in appendix a because few sharing references exist between the patents in heterogeneous fields. moreover, many zero values are found in the edge-betweenness centrality in the patent citation graph as shown in appendix b. it is assumed that the distributions roughly follow the pareto principle (also known as the / rule), in that % of the effects come from % of the causes. most of the normalized bibliographic coupling strengths had a low value because we searched for similar patents in different technological fields, and most of the edge-betweenness centrality had zero value in the patent citation network. we focused on the long tail in the distribution because the link prediction for converging tod is intended to detect weak signals from the knowledge flow between heterogeneous fields. in other words, we chose the threshold value to catch weak signals by removing % of the general results. thus, link prediction was conducted with the cut-off value for the normalized bibliographic-coupling strength as . , which is in the top %, and for the edgebetweenness centrality as , , which is also in the top %. the bibliographic-coupling value is calculated from the data assigned in heterogeneous technology sectors (i.e., the patents between bt and it). the edge-betweenness centrality value was calculated for the patent-citation graph using the "igraph" package in r. the appropriate cut-off value should be selected to categorize the change of tkf to remove noise because patent citations occurred in pairs among possible pairs between bt and it. the number of patent citations follows a long-tail distribution, as shown in appendix c. the x-axis of the graph represents tkf combinations as discrete categories that are sorted in descending order of patent-citation frequency, the bar represents the rate of each category, and the curve represents the cumulative rate as the y-axis. for instance, the pair with the most patent citations among the pairs is the pair from sgy (surgery) to sig (signal processing), labeled as "tech tech " in appendix c. we selected the top pairs, those whose frequency of citation between technologies was larger than , to include around the top % ( . %) of all pairs based on the statistical significance level of . , and we removed the rest of the pairs (around . %) in the technology knowledge flow. the change of tkf resulting from the link prediction with the cutoff value mentioned in step , is shown in appendix d and is compared with the original citation links. among the potential tkfs, increased from the tkf network, and had newly emerged. most of the increased potential links were predicted to increase tkf bidirectionally; however, three tkf pairs (bmd→net, sgy→net, sgy→rob) were predicted to be unidirectional although the tkfs were bidirectional in their network. the six unidirectional tkfs in the tkf network increased identical-directionally, and the five pairs (except bip→net) newly emerged in a different unidirectional way. the tkfs that had not existed in the network emerged unidirectionally for pairs and bidirectionally for pairs. the property of tkfs between heterogeneous fields was analyzed using ipc codes assigned in patents and in eqs. ( ) and ( ) in section . . as suggested in fig. , the technologies were categorized into the four types shown in appendix e, with the inflow and outflow property values using cut-off points that divide the maximum values of inflow and outflow into three equal parts. a tkf property map was developed in fig. . the width of a link represents the number of predicted patents between technologies as knowledge flow, and the black solid line and gray dashed line denote emerging links and increased links, respectively, in the map. the degree centralities for ipcs were calculated, and the percentage of ipc in the respective technologies was calculated to measure the technology inflow and outflow. the top potential tkfs were proposed as technological opportunities for convergence between bt and it fields among potential tkfs in the increased links and emerging links groups. appendix f shows the technological opportunities for convergence between bt and it with type, the predicted tkf's direction, and the tkf property type. to identify the technological opportunities in the respective potential tkfs, patents were grouped according to the girvan-newman clustering method (girvan & newman, ) . the number of clusters was selected based on the results of modularity value, which is often used to detect an optimal number of clusters. the number of clusters in appendix g was chosen when the network had a high modularity value through repeated calculations because networks with high modularity have dense connections between the nodes within clusters but sparse connections between nodes in different clusters. after the clustering method was conducted, technological themes of the respective groups were proposed, as shown in appendix g, by reviewing patents' titles and abstracts in groups. several groups in the potential tkfs presented incremental technological opportunities for convergence, but only one group presented radical technological opportunities for convergence because many predicted patent-citation links for increased links, and only a few patent citation links in the emerging link group existed. to sum up the data between and , tkfs appeared among possible tkfs ( bt and it fields in a direct network). in order to delete noise, we adopted pairs among tkfs, which account for approximately % in terms of patent citation distribution as shown in appendix c. after performing link prediction with data between and , pairs are predicted. to validate the results, , patents registered between and were used as a future point data. in the data between and , tkfs appeared among possible tkfs. under the situation, we calculated precision and recall indices to validate the proposed method. first, the precision is the fraction of predicted pairs that are correctly predicted. it is calculated by dividing the number of correct results by the number of all predicted pairs. the pairs in future point data occurred among the predicted tkfs. thus, the value of precision is / = . . second, the recall is the fraction of correctly predicted pairs to actual pairs that should be correctly predicted. it can be calculated by dividing the number of correct predicted results by the number of actual tkfs in future point. to calculate this, we took pairs among tkfs as the actual results in future point data by adopting the same cut-off rule selecting % pairs among all pairs in terms of patent citation distribution. the pairs in future point data occurred among predicted tkfs. thus, the recall value is / = . . under the link prediction power, the additional validation was conducted specifically by conducting correlation analysis between the predicted results and the future actual data with the correctly predicted tkfs. we performed the correlation analysis using spearman's rank correlation coefficient on the rate of knowledge flow that each pair has in the predicted and actual data. it was conducted from two perspectives, knowledge flow prediction and degree of convergence, because tkfs were predicted to detect the signals of technological convergence. therefore, we investigated whether the knowledge flows and whether two technological fields derived from the predicted knowledge flows could converge in the future. more specifically, the first investigation determined whether the distribution of correctly predicted knowledge flows was similar to future distribution of actual knowledge flows. the second investigation determined whether the distribution of correctly predicted knowledge flows was similar to the future distribution of the degree of convergence. first, the rate of the correctly predicted tkfs based on the patent data registered between and was compared to the rate of the correctly predicted tkfs based on the patent data registered between and using correlation analysis. we suggested a correlation between predicted results of knowledge flows based on the data at the present point ( ) ( ) ( ) ( ) ( ) ( ) ( ) and the future real knowledge flows ( ) ( ) ( ) . the spearman's rank correlation coefficient was . . second, the degree of convergence in the future real knowledge flows was measured by using co-classification analysis based on a -digit ipc, which represented a subclass, as co-classification analysis was widely used to calculate the degree of convergence. the rate of the correctly predicted tkfs based on the patent data registered between and was compared to the degree of convergence in the correctly predicted tkfs based on the patent data registered between and using correlation analysis. the spearman's rank correlation coefficient was . . it has strong correlation in both perspectives when we measure spearman's rho, which was calculated to reflect the difference between the present and future in terms of components of knowledge flow and the difference between the rate of knowledge flow and degree of convergence. therefore, the technological opportunities for convergence can be discussed based on the results of proposed tkf prediction because the proposed link prediction power is high and the correctly predicted tkfs have roughly strong positive correlation with future point data. overall, all the technologies in bt and it were balanced in terms of tkf without bias toward absorption or diffusion, as shown in fig. , against the expectation that some technologies play an absorption-oriented or diffusion-oriented role under the circumstance of bt it convergence. that is, no technologies took the definite absorptive-or diffusive-oriented roles in circumstances of tkf between bt and it. consequently, technological knowledge had been exchanged between bt and it fields through equal reciprocal cooperation, and technological convergence between bt and it has been in progress. these results are similar to those suggested by hur ( ) showing that technological sectors coevolve with reciprocal knowledge exchange with each other. the properties of tkf between bt and it could be divided into active, neutral, and passive properties, as suggested in appendix e. the it fields tended to actively absorb and diffuse technological knowledge compared to bt because three technologies ( (sig), (rob), and (sol)) in it fields were located only in tkf absorption and diffusion active groups (a&da), but almost all technologies in the bt fields (except for two technologies, (sgy) and (bmd)) were located in the tkf absorption and diffusion passive group (a&dp). although some technologies such as (bmd), (elc), and (soc) belonged to the tkf absorption and diffusion neutral group (a&dn) and (sgy) belonged to the tkf diffusion oriented group (do), six bts and four its were assigned to tkf a&dp in the current situation. one can interpret that some tkfs between bt and it have been exchanged, but the tkfs rarely interacted with each other in over half of the technological areas. in the context of the convergence status quo, technological convergence has proceeded between bt and it through reciprocal tkfs. however, several it-relevant technologies tend to technologically converge more with some bt technologies because the tkf property (absorption or diffusion) represents the technologies that have already technologically converged between two fields in the status quo. appendix d shows that tkfs were predicted equally because the tkfs from bt to it and from it to bt were predicted among potential knowledge flows. there were knowledge flows from bt to it, which accounted for approximately % in terms of increased link prediction, and there were knowledge flows from it to bt, which accounted for approximately % in terms of emerging link prediction. in appendix f, knowledge flows from it to bt and knowledge flows from it to bt were in the top predicted increased knowledge flows. although tkfs were predicted to be at a similar level from bt to it and vice versa in all potential technological flows, technological flows from it to bt accounted for to percent of the increased links and emerging links in terms of the top tkfs, which we considered incremental or radical technological opportunities for convergence between bt and it. therefore, it can play more of a leading role in terms of technological convergence between bt and it because it provides technological tools and platforms for analysis to bt (geum et al., ) . the tkfs from it to bt tend to be predicted for the future in the context of direction and strength for technological convergence. in this research, biotechnology (bt) and information technology (it) were illustrated for application of the proposed method. it supports other technologies as an enabling technology. if the proposed approach applied to other technologies, they could have various characteristics. however, the proposed method can be generalized because it is a prediction methodology based on tkf in patent citation networks between various technological areas if knowledge flows occur between distinct technology fields. fortunately, we could define technologies in bt and it using predefined technological classification based on ipc and uspc. an issue of reliability may arise for the classification defining technology with combinations of patent classification. if this method was applied to other technology fields, defining technology using patents could be one of the tricky issues. ipc at a high level can be used as a proxy for technology if a proper way to define technologies is not found. although an argument can be made for using ipc to represent technologies, patent classifications such as ipc and uspc are utilized as proxies for technology in some research because the ipc-classification system's granularity varies greatly between various technology areas. in the proposed method, cut-off selection steps arose in several phases. these cut-off values should be carefully chosen because these are a sensitive factor. to choose the cut-off value in respective phase, first of all, data distribution should be observed. then, the cut-off value should be selected by considering the objective of cut-off value with sensitive analysis. we mentioned three types of cutoff values in terms of link prediction, tkf network, and tkf property maps. first, the goal of using cutoff values in the link prediction is the detection of weak signals between two distinct heterogeneous technology fields. in our case, we used the pareto rule and focused on the long tail in the distribution. we selected the threshold with top % to catch weak signals by removing % of general results in the distribution. the cutoff value in the link prediction process can be chosen with high values of bibliographic coupling and edge-betweenness centrality between two heterogeneous fields using sensitive analysis. this criterion can be utilized when applying this method between other distinct technology fields. it is expected that most of the bibliographic coupling strength and edge-betweenness centrality may have low value or zero value because this approach searches for similar patents between two distinct technological fields. second, the cut-off value in the tkf network was selected to remove patent citation links, which can be considered noise because a technology-level network is merged with a patent network. in our case, we took . % of patent citations and removed the rest based on the statistical significance level of . because the significant level indicates a % of risk of conclusion statistically. this cut-off value that selects a strategy using significance level can be applied to other cases, however, the distribution should be firstly checked and an analyst's review was also necessary with sensitive analysis because the distribution of patent citation between technology fields can be dependent on the technology fields. finally, the cutoff point in the tkf property map was selected by dividing the inflow and outflow maximum values evenly to define the tkf property type in order to identify types of tkf property. it can be generally used when applying this method to other technological fields. when the proposed approach is applied to other cases, the cut-off value should be selected by considering the objective in each step. qualitative approaches using sensitive analysis as well as quantitative criterion are necessary to determine a cut-off value because two types of cut-off value can be sensitively affected by the criterion. we adopted the concept of link prediction in a direct network, which is suggested by yu and wang ( ) . in the previous research, a unified weighting approach was taken with three voting strategies, which is termed as v , v ra , and v sim , to compute w(u, c, s). v is the weight strategy as a binary value that we are taking in this research, v ra strategy is the way weighting the similar nodes by applying the inverse of its out-degree, and v sim strategy is the way weighting a candidate by measuring pearson's correlation coefficients between a target node and similar nodes according to overlap of their out neighbors. these three voting strategies can be weighted by using unified ranking algorithm with ˛, ˇ, and . it is suggested that the ˛, ˇ, and can be determined in order to obtain practical outcome in the research. the current study adopts only v strategy by simplifying the model because the parameters (˛, ˇ, and ) vary with settings and objectives and moreover, this research concentrates on predicting knowledge flow by merging predicted links between patents. more information on retrieving crucial links can be offered by applying other weighting schemes in further research. this paper addresses three research questions on identifying opportunities for technological convergence as presented in the introduction. appendix f shows the results of the first and second research questions, determining potential technological opportunities and their characteristics. in the context of potential types of technological convergence, the type of technological opportunity is suggested as incremental or radical technological opportunities for convergence. in terms of increased link prediction, half of the knowledge flows go from the a&dn group to the a&dp group; for the emerging link predictions, half of the knowledge flows go from the a&dp group to the a&dp group, and two knowledge flows go from the a&da group to the a&dp group, as shown in appendix f. the potential increased tkfs can be considered incremental technological opportunities for convergence because many tkfs already existed between bt and it. furthermore, many links were predicted to increase the existing tkfs. however, the potential emerging tkfs can be considered radical technological opportunities for convergence because the knowledge flows had not existed in the tkf network, and the tkfs that had passive properties in terms of knowledge flow between bt and it accounted for half of all emerging tkfs. it can be interpreted that the potential tkfs that have both passive properties signify a more radical change than tkfs that have the active property because they are predicted to be technological opportunities for convergence although the property of technologies is presently passive. moreover, bidirectional emerging tkfs tended to be more active than the unidirectional emerging tkfs because they included the a&da, a&dn, and do groups. unidirectional emerging tkfs can be considered an incremental change among emerging tkfs. therefore, the emerging tkfs as radical technological opportunities for convergence can be weak signals for technological convergence because all emerging tkfs had fewer predicted citation links than the increased tkfs, and the flows' properties tended to be more passive than the increased flows. the information on potential emerging tkfs can be riskier than the information on potential increased tkfs. the third research question in this paper is what technological themes on technological convergence exist at the micro level. appendix g presents the list of potential technological themes as the results of the third research question by dividing them into two groups (incremental and radical opportunities). the tkfs for incremental technological opportunities were all predicted as bidirectional increased links. only two bidirectional tkfs ( (sig) → (sgy) and (sgy) → (sig)) were included in the top potential tkfs for incremental technological opportunities. the two technological flows have some difference in technological themes. the potential technological flow from signal processing (sig) to surgery (sgy) showed knowledge flows ( ) from touch detection to blood glucose level control, analysis of eating habits, and gastric stimulation; ( ) from display for medical procedures to surgical navigation, brain stimulation, and neuron signal analysis; ( ) from imaging an interior surface of a cavity to endoscopy and visualization systems; and ( ) from x-ray images and methods for bone diseases to endoscopy and -d bone organic matrix density. however, the potential technological flow from surgery (sgy) to signal processing (sig) showed knowledge flows ( ) from data processing for use in tissue characterization to image processing and pattern recognition of images, ( ) from cochlear implant sound processors to audio signal processing, ( ) from surgical instruments to user interfaces and -d pointing devices, ( ) from illustration methods to handheld devices as visual indicators and portable media devices, ( ) from hearing aid appliances to optical waveguide vibration sensors and noise cancellation systems, ( ) from biopsy devices to touch screens, and ( ) from analytic sensors to video data recording. although two tkfs are predicted as bidirectional increased knowledge flows and as incremental technological opportunities, their themes are rather different. the technological contents on sig, which are mainly about image processing, flow to technological contents on sgy, which are related to surgical systems. in terms of the flows from sgy to sig, not only image technological themes but also audio signal processing and biopsy-relevant technology flows to technological themes on user interfaces, such as -d pointers and touch screens. the potential emerging tkfs can also provide weak signals for radical convergence although the number of patents in the potential knowledge flows is small compared to those in the potential increased knowledge flows. therefore, the directions of potential knowledge flows and technological themes can provide useful micro-level information when one builds a plan for technological convergence and conducts technology cooperation between heterogeneous fields. it can identify target technology fields for technological convergence, micro-level contents for technological convergence, and the role of knowledge diffuser or receiver on a cooperative r&d for convergence. for example, when one conducts technology cooperation for technological convergence between the technology fields of sig and sgy, image processing techniques for medical procedures in sig should be primarily used in collaboration with the techniques for surgical navigation and neuron signal analysis in sgy, and technology on hearing aid appliances in sgy should take the leadership with the noise cancellation system in sig. the proposed method adds to the existing knowledge base in three ways. first, the predicted results are derived through a future-oriented approach based on link prediction that overcomes the retrospective perspective of previous convergence research. second, patent citation information that traces tkf direction is utilized to overcome the prior approach to measuring the degree of convergence using patent classification overlap that does not include the direction of information flow. third, technological themes are suggested with direction information by clusters in the predicted tkf, which can be considered micro-level technological convergence as the way of overcoming the macro-level approach. this paper suggested technological opportunities for convergence by predicting tkfs based on patent citations between bt and it. several findings emerge from this research. first of all, tkfs between bt and it were balanced based on patent data registered from to in terms of absorption and diffusion of knowledge flow without bias; consequently, technological convergence had already been in progress across many technologies in bt and it. therefore, many tkfs were predicted to increase, and some tkfs had recently emerged. second, tkfs from bt to it and from it to bt were equally predicted in general. in terms of increased knowledge flow prediction, more knowledge flows occurred from bt to it than from it to bt, and more knowledge flows occurred from it to bt than from bt to it in terms of emerging knowledge flow prediction. many tkfs moved from it to bt when we extracted the top potential tkfs as technological opportunities for convergence. therefore, tkfs from it to bt were mainly predicted because it was a knowledge diffuser in knowledge flow between them although several knowledge flows that had already proceeded at a higher level than a certain standard moved from bt to it. third, the emerging tkfs can be considered radical opportunities for convergence because they were predicted as technological opportunities although they presently exhibited passivity. this study contributes to several perspectives. first, from an academic perspective, three research questions from the introduction are answered. to answer the first research question, the top ten potential tkfs are suggested as incremental and radical technological opportunities for convergence by the proposed methodology. to answer the second research question, the characteristics of the predicted technological convergence are proposed by analysis of the tkf's property and by prediction of the tkf's direction and strength between the bt and it fields with a future-oriented approach. to answer the third research question, technological themes are suggested at the micro level by grouping potential tkfs together. second, from a research methodological perspective, a novel link-prediction approach is proposed by applying the concept of edge-betweenness centrality to a link-prediction method for a directed network to reflect the importance of links between heterogeneous fields in terms of tkf. third, from the perspective of research policy, this research can offer improved processes and systems related to technological collaboration between heterogeneous fields for technological convergence. the proposed method and results can be used in various ways. first, it helps firms seek technological themes for convergence, considering their own technological capability, which enables them to prepare strategic technology development for the future based on the results of quantitative analysis. second, r&d policymakers can plan new research projects on technological convergence that act as a bridge in terms of technology collaboration for convergence. third, the results help research institutions and universities improve interior systems to develop converging technology and professional technology manpower training systems across disciplines effectively. this research has several limitations. first, we suggested the results of correlation with perspectives of knowledge flow and degree of convergence because the causality between the results of prediction and the future cannot be analyzed. it can, however, be validated by applying the proposed predicting methodology with patent data and comparing the actual cases of technological convergence quantitatively. in addition, the proposed method could be more reliable if the qualitative evaluation was conducted by domain experts. second, non-patent references were not considered for analysis because we focused on technological convergence. the addition of non-patent data could provide many more implications in terms of scientific discipline convergence and evolution between science and technology. third, analyst's subjectivity and a sensitive analysis are required when one selects cut-off values in several steps of the proposed method. finally, we focused on technological opportunities for convergence between only bt and it. in future research, to surmount the above limitations, technological opportunities for convergence based on data in multiple technologies can be identified with improved massive data processing techniques. inchae park: collected the data; contributed data or analysis tools; performedthe analysis; wrote the paper. byungun yoon: conceived and designed the analysis; performedthe analysis; wrote the paper. see fig. b . see table d . 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opportunity discovery (tod) from existing technologies and products: a function-based tod framework scientific evolutionary pathways: identifying and visualizing relationships for scientific topics relativity of citation performance and excellence measures: from cross-field to cross-scale effects of field-normalisation this work was supported by the national research foundation of korea grant funded by the korean government (nrf- r d a a ) and dongguk university (s- -g - ). key: cord- - e zjaz authors: park, ji-eun; jung, soyoung; kim, aeran; park, ji-eun title: mers transmission and risk factors: a systematic review date: - - journal: bmc public health doi: . /s - - - sha: doc_id: cord_uid: e zjaz background: since middle east respiratory syndrome (mers) infection was first reported in , many studies have analysed its transmissibility and severity. however, the methodology and results of these studies have varied, and there has been no systematic review of mers. this study reviews the characteristics and associated risk factors of mers. method: we searched international (pubmed, sciencedirect, cochrane) and korean databases (dbpia, kiss) for english- or korean-language articles using the terms “mers” and “middle east respiratory syndrome”. only human studies with > participants were analysed to exclude studies with low representation. epidemiologic studies with information on transmissibility and severity of mers as well as studies containing mers risk factors were included. result: a total of studies were included. most studies from saudi arabia reported higher mortality ( – . %) than those from south korea ( . %). while the r( ) value in saudi arabia was < in all but one study, in south korea, the r( ) value was . – . in the early stage and decreased to < in the later stage. the incubation period was . – . days in saudi arabia and – . days in south korea. duration from onset was – days to confirmation, . – . days to hospitalization, – days to death, and – days to discharge. older age and concomitant disease were the most common factors related to mers infection, severity, and mortality. conclusion: the transmissibility and severity of mers differed by outbreak region and patient characteristics. further studies assessing the risk of mers should consider these factors. middle east respiratory syndrome (mers) was first reported in in saudi arabia [ ] . although most patients are linked to the arabian peninsula geographically, mers has been detected in many other parts of the world [ ] . a large mers cluster was also observed in in south korea [ ] . mers causes sporadic infection and intrafamilial and healthcare-associated infection. its symptoms can vary from asymptomatic infection to death. despite the infection's association with high mortality, specified antiviral therapy is lacking, especially for patients with concomitant diseases [ ] . many previous studies have assessed the risks of mers, such as factors dictating severity or an infection risk, yet the indices they present vary. for example, the case fatality rate was found to be . % in the middle east area, but . % in south korea [ ] . the incubation period was reported to be . - days in south korea [ , ] , but . in a study using data from multiple areas [ ] and . in saudi arabia [ ] . accurate assessment of the risk of mers is essential for predicting and preventing infection. a systematic review of the risk of mers, as covered in previous studies, is potentially helpful for predicting this spread, and its future impact. this study aimed at reviewing the risk of mers, focusing on indices related to infectivity and severity. we searched international (pubmed, sciencedirect, cochrane) and korean databases (dbpia, kiss) using the term "mers" or "middle east respiratory syndrome", encompassing articles published after . the search process was conducted in october . we also manually searched the reference lists of the included studies. human studies were included, while animal studies and reviews were excluded. only articles in english or korean were included. even if a study collected data on humans, such as collecting specimens from religious pilgrims, it was excluded if there were no mers patients in the study sample. additionally, case studies including fewer than mers patients were excluded as they were considered as having insufficient mers patient numbers and representative information. the included studies were classified as epidemiologic studies and those covering risk factors of mers. in the epidemiologic category, indices related to the risk of mers were divided into two categories; related to infectivity and related to severity. the index related to infectivity included the reproduction number (r), attack rate, incubation period, serial interval, and days from onset to confirmation. the index related to severity included the case fatality rate (cfr), days from onset to hospitalization, days from onset to discharge, days from onset to death, and days from hospitalization to death. in the risk factor category, factors related to infection, transmission, severity, and mortality of mers were analysed. even if the included studies investigated factors that were related to mortality, when they did not analyse risk factors of severity or mortality using appropriate statistical methods (e.g., regression analysis, cox proportional hazards model) or only compared prevalence factors, we excluded them from the risk factor category. in all categories, we extracted the study period, number of participants, and geographical region where the data were collected using a data extraction form confirmed after pilot assessment. a total of studies were searched, and were reviewed, excluding duplicate studies. after the title and abstract review, a further and were excluded, respectively. another four studies were included via a manual search, which left a total of studies for analysis ( fig. ). the of total included studies were classified as epidemiologic studies (table ) . r value, representing the reproduction number, indicates the average number of secondary cases generated by infectious individuals. thirteen studies reported r value of mers. four studies that used data from multiple areas had r < . [ , [ ] [ ] [ ] . studies using saudi arabia or middle east area data reported r < , at . - . [ ] [ ] [ ] [ ] , though one reported . - . [ ] . studies using south korea data showed higher values, at . - . [ ] [ ] [ ] [ ] , in the early stage, and < in the later period [ ] or with control intervention [ ] . a total of eight studies reported the attack rate. four reported the overall or secondary attack rate, and the other four reported the attack rate of specific participant groups. two studies conducted in saudi arabia showed . % [ ] and % [ ] secondary attack rates. studies in south korea showed secondary attack rates of . % in one study [ ] and . - . % in another [ ] . two studies reported the attack rate among healthcare workers (hcws). one study in south korea reported a mers incidence of . % among hcws [ ] , and another study using multiple area data reported a . - . % infection rate among hcws [ ] . the attack rate among hospital patients was % in one study [ ] and % in the early and % in the later period in another [ ] . the incubation period is the period between infection and appearance of signs of a disease. a total of studies reported the incubation period of mers. nine used data from south korea and showed a - . day incubation period [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . one study using data from saudi arabia reported a . day incubation period [ ] , and another using data from multiple areas reported a . day incubation period [ ] . sha et al. compared the incubation periods between the middle east area and south korea and reported . - and days, respectively [ ] . the serial interval of an infectious disease represents the duration between symptom onset of a primary case and of its secondary cases. two studies used south korea data, reporting serial intervals of mers of . and . days, respectively [ , ] . among five studies reporting days from onset to confirmation, three studies used data from south korea. one study analysing all south korea cases reported days from onset to confirmation [ ] . park et al. reported . days for all cases, for second generation and for third generation [ ] . one study from taiwan reported days for hcws and for non-hcws [ ] . a study from saudi arabia reported days from onset to confirmation [ ] . sha et al. compared the data from middle east and south korea areas and reported - and - days, respectively [ ] . two studies from saudi arabia reported days from onset to hospitalization. one reported . - days [ ] , and the other reported . days [ ] . twenty-six studies reported on mers-related mortality. ten reported the mortality rate in south korea as . - . % [ , , - , , , , ] ; one of which, including all mers patients in south korea, reported a mortality rate of . % [ ] . ten studies analysing data from saudi arabia reported higher mortality rates, of - . % [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] , although others reported mortality rates % [ ] and . % [ ] . a taiwanese study reported a mortality rate of . % [ ] . studies using data from multiple areas reported mortality rates ranging from . % [ ] to . % [ , ] . three studies reported days from mers onset to discharge. sha et al. reported days in the middle east area and in south korea [ ] . one study from saudi arabia reported days [ ] , and another in south korea reported [ ] . two korean studies reported similar periods of - days from onset to death: - . in park et al. [ ] and in ki et al. [ ] . although one study from saudi arabia reported longer than days from onset to death [ ] , sha et al., comparing data between the middle east and south korea, reported similar periods of . and days, respectively [ ] . one taiwanese study also reported a similar period of - days [ ] . two studies reported a similar length of hospitalization: [ ] and . days [ ] . of the studies included in the risk factor category, four were duplicates of studies in the epidemiologic category as they had information regarding the epidemiologic index and risk factors ( table ) . two studies reported on the risk factors of mers infection. alraddadi et al. [ ] analysed the effect of nonhuman contact, including travel history, animal-related exposure, food exposure, health condition, and behaviour and reported direct dromedary exposure, diabetes or heart disease, and smoking as risk factors of mers infection. another study reported older age, outbreak week, and nationality as risk factors [ ] . three studies analysed factors associated with spreaders. non-isolated in-hospital days, hospitalization or emergency room visits before isolation, deceased patients, and clinical symptoms, including fever, chest x-ray abnormality in more than three lung zones, and the cycle threshold value, were related to spreaders [ , , ] . four studies reported risk factors of mers severity. the included studies showed that the prnt and cd t cell response [ ] as well as a high mers virus load [ ] were associated with the severity of mers. additionally, male sex; older age; concomitant disease, including hypertension; and symptoms, including fever, thrombocytopenia, lymphopenia, and low albumin concentration, were related to mers severity or secondary disease [ ] [ ] [ ] . fifteen studies reported risk factors of mortality in mers patients. older age [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] and comorbidity [ , [ ] [ ] [ ] ] , including diabetes [ , ] , chronic kidney disease [ ] , respiratory disease [ , ] , pneumonia [ ] , cardiac disease, and cancer [ ] , were the most prevalent in the included studies. male sex was reported as a risk factor in one study [ ] . smoking [ , ] and location of acquisition [ , ] were also reported. while one study noted that hcw, as a profession, was associated with mortality [ ] , non-hcws were reported to be related to mortality in two other studies [ , ] . additionally, a shorter incubation period [ , ] , longer duration of symptoms [ ] , more days from onset to confirmation [ ] , later epidemic period [ ] , and longer hospitalized days [ ] were reported as mortality risk factors. symptoms at diagnosis, including abnormal renal function [ ] , respiratory symptoms [ ] , gastrointestinal symptoms [ ] , lower blood pressure [ , ] , and leucocytosis [ , ] , were also found to be associated with mortality in mers patients. severity of illness, [ , ] such as need for vasopressors [ ] , chest radiographic score [ ] , health condition [ ] , use of mechanical ventilation [ ] , and occurrence of dyspnoea [ ] were also found to increase the mortality risk. the characteristics of mers differ between south korea and the middle east area. the r value of mers was reported to be below in the middle east area, except in one study [ ] , but was from . - . in south korea [ ] [ ] [ ] [ ] [ ] . although studies using data from the middle east area reported . - % secondary attack rates, studies in south korea reported - % secondary attack rates for patients or hospital visitors [ ] , and . - . % for the overall attack rate [ , ] . the mers incubation period was reported to be . - . days in the middle east area [ , ] , but this period was found to be slightly longer in south korea [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the severity of mers also differed between the middle east area and south korea. mortality of mers patients was found to be . % in south korea based on a report including all cases [ ] , but most studies from saudi arabia reported higher rates, from to . % [ , , , [ ] [ ] [ ] . days from onset to confirmation were similar, - days in the middle east area [ , ] and - . days in south korea [ , , ] . days from onset to discharge were slightly longer in south korea, - days in the middle east area [ , ] and - days in south korea [ , ] (table ). the transmissibility and severity of mers were different by outbreak countries, especially between the middle east area and south korea. the virus, host, and environmental factors may be the causes of the mers outbreakrelated differences between the two regions. from the standpoint of viral factors, there was a mutation of the mers coronavirus (mers-cov) in the south korea outbreak. kim et al. [ ] reported a point mutation in the receptor-binding domain of the viral spike protein in mers-cov, and another study showed that mers-cov in south korea had higher genetic variability and mutation rates [ ] . individual characteristics can also affect mers transmission. as previous studies showed, there is an association between older age and mers infection [ ] , severity [ ] , and mortality [ , ] , and the population structure may be related to transmission and severity. additionally, individuals aware of mers were found to be more likely to practice preventive behaviour [ ] , which differed by demographic characteristics [ , ] . the transmission environment may also contribute to the difference. while many mers cases were contracted through exposure to camels in saudi arabia [ ] , the south korea outbreak involved multiple generations of secondary infections caused by intra-hospital and hospital-tohospital transmission [ , ] . strategies considering various factors are therefore needed to assess the impact of mers and to better control its spread. although several studies have reported the overall r value [ , , , ] , others have shown that this value this can be variable based on the generation or a control intervention [ , , ] . especially in the south korea epidemic, the r value was particularly high in the early stage or first generation, at . - . , though it later decreased to . - . [ , ] . further studies should consider and analyse the variation of the r value depending on the period or control intervention. while earlier studies on infectious diseases assumed a homogeneous infection ability of a population, recent studies have shown the existence of so-called super spreaders, individuals with a high potential to infect others in many infectious diseases, including ebola and severe acute respiratory syndrome (sars) [ ] . the role of the super spreader is also important in the spread of mers. in south korea, . % of mers patients were associated with five super-spreading events [ ] . stein et al. [ ] asserted that super spreaders were related with the host, pathogen, and environmental factors, and wong et al. [ ] reported that individual behaviours could also contribute to disease spread. there are variations in the mortality and attack rates among studies using south korea data. for example, park et al. [ ] reported a . % mers mortality, while reports from the korean ministry of health and welfare showed . % mers mortality. this disparity may, in part, be due to small sample sizes. park et al. [ ] included only patients because the study was conducted in an early phase of a mers outbreak. we excluded studies that included cases with < subjects, which were mostly case series, to reduce those types of biases. the present review found that older age and concomitant disease were risk factors of mers infection and mortality. these results are consistent with a recent systematic review that reported older age, male, and an underlying medical condition as predictors of death related to mers [ ] ; therefore, these factors should be prioritized in protection and treatment procedures. one limitation of this study was the possibility of subject duplication. especially in south korea, the korean government publishes mers reports that include all patients. the epidemiologic index in other studies might be biased since they included partial korean patients and were analysed in the middle of an outbreak. however, we included those studies because they showed the characteristics of mers in different situations and different stages. we did not conduct a meta-analysis because of the small number of studies for each index, which might be another limitation of this study. although this study reviewed the risk factors of mers and their impact, assessing the effect size of each risk factor is important. more studies investigating the effect of risk factors on mers need to be constantly conducted. most studies on the transmissibility and severity of mers have originated from saudi arabia and south korea. even though the r value in south korea was higher than that in saudi arabia, mortality was higher in saudi arabia. the most common factors behind mers infection and mortality were older age and concomitant disease. future studies should consider the risk of mers based on the outbreak region and patient characteristics. the results of the present study are valuable for informing further studies and health policy in preparation for mers outbreaks. isolation of a novel coronavirus 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respiratory syndrome coronavirus and the multiple generations of secondary infection in south korea the role of super-spreaders in infectious disease super-spreaders in infectious diseases clinical determinants of the severity of middle east respiratory syndrome (mers): a systematic review and meta-analysis authors' contributions jep (corresponding author) designed the study, and conducted the data search and the analysis with jep ( st author). syj and ark participated in the data review. jep (corresponding) drafted the manuscript, and jep ( st), syj, and ark revised it. all authors read and approved the final manuscript.ethics approval and consent to participate not applicable. the authors declare that they have no competing interests. key: cord- - laifjgz authors: anuj, samir a.; gajera, harsukh p.; hirpara, darshna g.; golakiya, baljibhai a. title: bactericidal assessment of nano-silver on emerging and re-emerging human pathogens date: - - journal: j trace elem med biol doi: . /j.jtemb. . . sha: doc_id: cord_uid: laifjgz with the threat of the growing number of bacteria resistant to antibiotics, the re-emergence of previously deadly infections and the emergence of new infections, there is an urgent need for novel therapeutic agent. silver in the nano form, which is being used increasingly as antibacterial agents, may extend its antibacterial application to emerging and re-emerging multidrug-resistant pathogens, the main cause of nosocomial diseases worldwide. in the present study, a completely bottom up method to prepare green nano-silver was used. to explore the action of nano-silver on emerging bacillus megaterium mtcc and re-emerging pseudomonas aeruginosa mtcc pathogenic bacteria, the study includes an analysis of the bacterial membrane damage through scanning electron microscope (sem) as well as alternation of zeta potential and intracellular leakages. in this work, we observed genuine bactericidal property of nano-silver as compare to broad spectrum antibiotics against emerging and re-emerging mode. after being exposed to nano-silver, the membrane becomes scattered from their original ordered arrangement based on sem observation. moreover, our results also suggested that alternation of zeta potential enhanced membrane permeability, and beyond a critical point, it leads to cell death. the leakages of intracellular constituents were confirmed by gas chromatography-mass spectrometry (gc–ms). in conclusion, the combine results suggested that at a specific dose, nano-silver may destroy the structure of bacterial membrane and depress its activity, which causes bacteria to die eventually. antibiotics deserve much of the credit for the dramatic increase in life expectancy around the world in the th century. a famous physician expert once noted that the discovery of penicillin in the early s showed more curative power to a lone provider than the collective talent of all the physicians in new york at that time [ ] . unfortunately, it is inevitable that, after sometime, bacteria develop resistance to existing antibiotics, making infectious disease more difficult to treat [ ] . the resistance profile has probably been smouldering issue for years, but nowadays it's almost like a switch got triggered. infectious disease experts are alarmed by the prospect that effective antibiotics may not be available to control the re-emergence of previously deadly infections and the emergence of new infections caused by drugresistant bacteria in near future. recent outbreaks of pneumonia (pseudomonas aeruginosa) and tuberculosis (mycobacterium tuberculosis), for example, both of which have re-emerged within the region itself, have also focused regional, national and international attention on the threat developed by re-emerging infectious diseases and in particular on southeast asia as the epicentre of these types of diseases. indeed, tuberculosis and pneumonia are re-emerging in a drug-resistant form [ , ] . southeast asia is also a hotspot for emerging infectious disease in particular, zoonotic diseases as a result of many factors including population growth, mobility and environmental changes. severe acute respiratory syndrome (sars), nipah virus and the most recent pandemic influenza a h n are only a few of many examples of emerging infectious diseases in the southeast asia; each of these diseases has caused global societal impact related to unexpected illnesses [ ] . other emerging infectious diseases caused by bacteria are less terrible than these examples; however, they nonetheless may take a significant increase in morbidity as well as cost related to prolong treatments. in recent years, the infection caused by bacteria in man to have probably emerged in china was brain abscess during infection with bacillus megaterium [ ] . to the best of our knowledge, infections caused by these bacteria are rare and have not been reported as the cause of any important clinical diseases [ , ] . while it is not known which new infectious diseases will emerge tomorrow, populations have to be protected by staying one step ahead of the microbes by searching a new antimicrobial drugs. moreover, researchers also discovered some bacterial species that are resistant to different classes of antibiotics, including new synthetic drugs, even though these bacteria have never exposed to antibiotics [ , ] . these studies support a hypothesis that antibiotic resistance is genetically rich natural phenomenon, deeply embedded in the bacterial pan-genome in which bacteria evolve; it can be slowed but not stopped. if these types of non-pathogenic bacteria create any health problems, then it's very difficult to control them by conventional antibiotics. hence, we need new antibiotics to control resistant bacteria, which must act at several cellular and molecular levels. the mode of action of nano-silver is not known for specific at single sight but to influence bacterial membrane permeability and many metabolic pathways at same time. the present study aimed to understand the antibacterial activity and acting mechanism of nano-silver, the mechanism of inhibition against emerging b. megaterium mtcc and re-emerging p. aeruginosa mtcc through alternation of overall surface charges and destroying membranous structure with consequent leakages of important intracellular constituents such as amino acids, fatty acids, organic acids and sugar acids. the study represented a potential template for the design of novel antibacterial agents to overcome the issue of emerging and re-emerging infectious diseases. the medicinal plant, tinospora cordifolia was selected from gir forest region of gujarat, india on the basis of its medicinal property [ ] . silver nitrate, ar grade was procured from thermo fisher scientific, vadodara, india. the two bacterial strains one gram positive (bacillus megaterium mtcc ) and one gram negative (pseudomonas aeruginosa mtcc ) are clinical isolates that were obtained from the microbial type culture collection (mtcc), institute of microbial technology, chandigarh, india. bacterial strains were cultivated on nutrient broth medium (ph . ± . ; himedia, vadodara, india) at °c with shaking at rpm. bacterial cell suspensions were diluted with a sterile milli-q water (ph ) to obtain a final concentration of cfu/ ml. four broad spectrum antibiotics, namely, amoxicillin (sun pharmaceutical, ankleshwar, india), chloramphenicol (alps pharmaceuticals, uttarakhand, india), trimethoprim-sulfamethoxazole (abbott healthcare, mumbai, maharashtra), and linezolid (glenmark pharmaceutical, ankleshwar, india) were used for bactericidal assay. all other chemicals used in this study were analytical grade (sigma aldrich, ahmedabad, india). for the preparation of aqueous extract, g of dry stem powder was boiled in a conical flask with ml of deionised water for min at °c on magnetic stirrer at rpm, then the mixture was filtered through whatmanno. filter paper, and the stem extract was collected and stored at °c for further use in synthesis of nano-silver. aqueous solution ( − m) of silver nitrate (agno ) was prepared and stem extract of t. cordifolia was added ( : ratio) in dark condition for the reduction of silver nitrate at room temperature [ ] . the bioreduction of silver nitrate was confirmed by uv-vis spectrophotometric (agilent technologies, cary ) analysis. dynamic light scattering (dls) technique was used to determine the size and the potential stability of the nano-silver in the suspension using nanotrac wave (s ). the surface morphology of nano-silver along with elemental analysis was determined by scanning electron microscope (zeiss, evo- ) coupled with an energy dispersive x-ray analysis (edx) facility by applying an acceleration voltage of kv. the comparative antibacterial activities of nano-silver and broad spectrum antibiotics was effectively accessed against emerging pathogens bacillus megaterium mtcc and re-emerging pathogens pseudomonas aeruginosa mtcc using agar well diffusion assay method [ ] . the drug of choice for each tested strain was used to prove the unique bactericidal action of nano-silver compared to conventional broad spectrum antibiotics, namely, amoxicillin (inhibits bacterial cell wall synthesis), trimethoprim-sulfamethoxazol (inhibits synthesis of proteins and nucleic acids), chloramphenicol (inhibits bacterial protein synthesis by preventing elongation process) and linezolid (inhibits the initiation process in bacterial protein synthesis at a very earlier stage) [ ] . the concentration of nano-silver and antibiotics was adjusted to μg/ml, and the concentration of bacterial cells was cfu/ml. after incubation at °c for h the zones of inhibition was measured [ ] . the assays were performed with three replications and standard deviation between replication was calculated. samples from bacterial cultures (bacillus megaterium mtcc and pseudomonas aeruginosa mtcc ), mixed with μg/ml of nano-silver, were collected at h and pre-fixed with . % glutaraldehyde for min; these were then washed two times in the same buffer and post-fixed for h in % osmium tetroxide. after washing with buffer, dehydration process was conducted with , , , , and % of ethanol. prior to analysis by sem (zeiss, model evo- ), the samples were dried at °c and subjected to analysis with kv accelerating voltage. control experiment was conducted in absence of nano-silver [ ] . bacterial surfaces are characterised by their surface electric charge, which allows the measurement of zeta potential through nanotrac wave (model s ). the bacterial cultures were harvested by centrifugation at , rpm for min, and the cell pellets washed three times with . mm potassium phosphate buffer solution (ph . ) and finally re-suspended in the same buffer to the final concentration of cfu/ml. the washed bacterial cell suspensions were incubated with μg/ml of nano-silver for h [ ] . zeta potential was also carried out for normal and autoclaved (at °c, psi for min) bacterial cells [ ] . the average bacterial size of the sample was also measured in nanotrac wave (model s ) for autoclaved, non-treated and treated bacterial cells at ambient temperature ( °c). gc-ms profile was carried out to detect the leakages of intracellular constituents from non-treated and nano-silver treated bacterial cells (same as zeta potential). the known volume of bacterial supernatant (water v/v) were dried at °c in nitrogen turbo evaporator (biotage, turbovap®lv); followed by derivatized with μl of a mg/ ml methoxyamine hydrochloride solution in ml of pyridine for min at °c on shaker. subsequently, the samples were silylated for min at °c with μl n,o-bis (trimethylsily) trifluoroacetamide (bstfa) on shaker. the derivatized bacterial supernatants were analyzed with gas chromatograph-mass spectrometer (gcms-qp plus, shimadzu, japan) coupled with shimadzu gcms-qp se mass selective detector. about μl aliquots of the supernatants were injected into a db- ms capillary column ( m × . mm i.d., . μm film thickness) using split less injection ( °c, . min). helium gas was used as a carrier gas at a flow rate of ml/min. leakages of intracellular compounds were identified by mass spectra analysis ( - m/z range) through the national institute standard and technology (nist) library and gcms solution software to calculate the similarity index and above % match was considered acceptable [ ] . the mass spectrum of identified intracellular compounds of treated bacteria was compared with the spectrum of the non-treated bacteria (control) and identified leakage constituents due to nano-silver treatment on bacterial surface. the gc-ms profile of aqueous stem extract of t. cordifolia was also carried out. the common compounds between t. cordifolia aqueous extract and nano-silver treatment of bacterial culture as well as between bacterial suspension without silver and nano-silver treatment were identified and subtracted to identify unique compounds (linkages) release due to nano-silver treatment on bacterial surface. the nano-silver was successfully synthesized from aqueous stem extract of t. cordifolia by mixing with mm silver nitrate solution. additions of plant extract into the silver nitrate solution resulted in the gradual change of the colour of silver nitrate solution from light yellow to dark brown, indicating the formation of nano-silver (fig. a) . similar colour changes were reported in previous studies and hence confirmed the completion of reaction between silver nitrate and stem extract [ ] . the synthesis of nano-silver was further confirmed by uv-vis spectrophotometer. a strong specific peak for the synthesized nano-silver was obtained at nm (fig. a) . dynamic light scattering data showed that the synthesized nano-silver were in the range of . nm + . (fig. b) , which is well in agreement with sem result (fig. c) . the surface zeta potential value of nano-silver was measured to be slightly positive and was found to be . mv (data not shown in fig. ) . the positive charge of nano-silver prevents them from aggregation and increases their stability, as well as help to enhance their antibacterial activity by interacting with negatively charged biomolecules of bacterial membrane [ ] . the sem image of the sample showed the formation of spherical nano-silver in aggregated form with average . nm size, which was confirmed to be silver by edx analysis (fig. c and d) . the edx analysis showed a strong peak at kev, which is typical for the absorption of metallic nano-crystallites silver due to unique surface plasmon resonance (spr), thereby confirming the formation of nano-silver [ ] . the presence of additional peak for oxygen indicates that some nano-silver got oxidized by forming silver oxide [ ] . the comparative antibacterial activities of the nano-silver and broad spectrum antibiotics were assessed against emerging b. megaterium (mtcc ) and re-emerging p. aeruginosa (mtcc ) bacteria. a direct comparison of the nano-silver and the broad spectrum antibiotics under the same dose range ( μg/ μl for cfu/ml inoculum) showed that the tested microorganisms developed resistance to antibiotics belonging to different classes (fig. ) . the mean of three replicates of the diameter of inhibition zone around each well of nanosilver and broad spectrum antibiotics is represented in table . the inhibition zones of nano-silver obtained in this study indicated that a nano-silver has potential to control emerging and re-emerging multidrug-resistant pathogens compared to tested antibiotics. these findings further agree with previous results by other researchers, where it was proven that nano-silver exert the same result on multidrug-resistant bacteria [ ] . our findings showed that b. megaterium mtcc developed resistance to all tested antibiotics, except linezolid ( fig. a and b) . linezolid is a new class of antibiotics called the oxazolidinones that are useful to control the emergence of drug resistance gram-positive organisms [ ] . unfortunately, b. megaterium mtcc developed resistance to trimethoprim-sulfamethoxazol, which has been valuable combinatorial therapy for treating a variety of infections. this emerging resistance to trimethoprim-sulfamethoxazol is disturbing because it is a combination of two broad spectrum antibiotics that act synergistically against a wide variety of urinary and respiratory tract infections [ ] . there are limited data available to the infections caused by b. megaterium, there have been no previous data on resistant to antibiotics, except cloxacillin and streptomycin and thus, careful investigation is required [ , ] . the increasing bacterial resistance among gram-positive species is concerning because they are responsible for / of nosocomial infectious diseases [ ] . on the other hand, p. aeruginosa mtcc developed resistance to all tested broad spectrum antibiotics ( fig. c and d ). our findings of bactericidal activity of nano-silver against multidrug-resistant p. aeruginosa are exactly in accordance with findings shown by lara et al. [ ] . in recent years, p. aeruginosa bacteria that are resistant to different classes of antibacterial agents is one of the most feared bacteria that causes pneumonia [ , ] . the high antibiotic prescribing rate is one of the leading causes for the development of antibiotic resistant microbes in case of re-emergence of infectious diseases. however, the speed of the resistance development varies depending on the antibiotics and microbes. overall, our findings suggests that there was significant difference between the bactericidal efficacy of nano-silver and antibiotics on emerging b. megaterium mtcc and re-emerging p. aeruginosa mtcc , demonstrating that the bactericidal mechanism of nano-silver was not affected by those known resistance mechanisms that differentiate these strains from susceptible strains. the data presented in the study are novel as nano-silver exhibit excellent bactericidal effect towards emerging and re-emerging bacterial infections regardless of their drug resistant mechanisms. to observe the membrane deformities upon the nano-silver treatment, we scanned the nano-silver treated and non-treated bacterial cells using sem. the images indicate clumping of nano-silver on bacterial membrane in treated cells than non-treated cells (fig. ) . upon interaction with nano-silver, the surface potential of bacterial membrane is neutralized, resulting into increased in surface tension. after h of treatment, the interactions result in surface potential changes which lead into the membrane depolarization at the point of clumping. as a result, bacterial membrane become scattered from their original ordered arrangement. the scattered membrane containing cells no longer remain intact, often found in aggregates (fig. b, d) . surface charge neutralizations of the bacterial cell membrane are important target site of the certain membrane acting compounds particularly with antimicrobial properties, which acts on bacterial cell surface [ ] . according to our observations, nano-silver produced alternation of zeta potential in both gram-negative and gram-positive bacteria. as shown in fig. , the initial interaction of nano-silver with a cell surface of b. megaterium mtcc and p. aeruginosa mtcc cells display zeta potential of − . and − . , respectively. however, dead (autoclaved) bacterial cells of b. megaterium mtcc and p. aeruginosa mtcc had a lower zeta potential of . and − . mv compared to normal bacterial cells (data not shown in fig. ) . in our study, it was found that, the zeta potential value moved towards neutral with treatment of nano-silver. the calculated zeta potential values of b. megaterium mtcc and p. aeruginosa mtcc at different time intervals are shown in fig. . there were significant differences in the zeta potential of b. megaterium mtcc compared to p. aeruginosa mtcc , when they were normal or exposed to nano-silver or autoclaved. moreover, it was also found that the magnitude of decrease in the negativity of the zeta potential of bacterial cell surface was found to be greater in b. megaterium mtcc and such alternation was found to be time dependent (studied till h) as well. these low values of the zeta potential indicated the interaction between cationic nano-silver and the negative surface potential of bacterial surface. interestingly, such change in zeta potential through nano-silver can be correlated with the increased bacterial membrane permeability as it was evident from gc-ms analysis (fig. supplementary material). the present findings are in conformity with earlier reports, where it has been stated that surface neutralization of the bacterial membrane leads to increase in cell permeability [ ] . gc-ms based secretome analysis has important applications in discovering the mode of action of nano-silver and helps unravel the effect of nano-silver on membrane permeability of bacteria. in this study, the leakages of the intracellular constituents from bacterial cells were identified from their mass spectra and retention times, as indicated by the chromatogram of the extra cellular components of the bacterial cells after treatment of nano-silver ( fig. ; supplementary material). in case of treated bacterial cells, some constituents were from plant extract, by which we synthesized nano-silver. in general, the total constituents present in extracellular materials in treated cells, nontreated cells as well as plant extract detected in gc-ms were shown in table . an increase in some unique intracellular components in treated cells was observed, compared with those in the plant extract and nontreated cells. the common and unique compounds identified from bacillus megaterium mtcc and pseudomonas aeruginosa mtcc , before and after treatment with nano-silver along with plant extract are depicted in fig. . total compounds were identified common to bm_t, pa_t and pl_e followed by unique compounds in bm_t and in pa_t. moreover, compounds found to be common for pa_t and pl_e. the nature of compounds identified after treatments were sugars, organic acids, amino acids and fatty acids, which showed significant differences with non-treated cells and plant extracts (table ; supplementary material). this may indicate that nano-silver were found to interact with bacterial cell surfaces and results in membrane damage. nano-silver is considered a novel antibacterial agent, which offers several advantages such as broad spectrum antibacterial activity and lower tendency to induce resistance. the present study demonstrating the interaction of green synthesized nano-silver exerted a slight stress on the bacterial cell membrane through alternation of zeta potential. the strong antibacterial activity of nano-silver possesses the potential to serve as a platform for developing nano-silver into a novel antibacterial drug to overcome the problem of emerging b. megaterium mtcc and re-emerging p. aeruginosa mtcc . the antibacterial efficacy of nano-silver against multidrug-resistance indicated that the mode of action of nano-silver is not the same as the mode of action exerted by the antibiotics. hence, with the threat of multidrug-resistant strains of bacteria, nano-silver could be a good alternative to control emerging and re-emerging infectious diseases. the authors declare that they alone are responsible for the writing and content of this paper. the authors report no conflicts of interest in this research work. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. authors do not declare any conflict of interest. as antibiotic discovery stagnates, a public. health crisis brews accelerating resistance, inadequate antibacterial drug pipelines and international responses emerging and re-emerging infections overview of nosocomial infections caused by gram-negative bacilli brain abscess caused by bacillus megaterium in an adult patient bacillus megaterium delayed onset lamellar keratitis after lasik primary cutaneous infection with bacillus megaterium mimicking cutaneous anthrax antibiotic resitance is ancient an antibiotic-resistance enzyme from a deep-sea bacterium tinospora cordifolia; a pharmacological update plant mediated synthesis of nano-silver by using dried stem powder of tinospora cordifolia, its antibacterial activity and comparison with antibiotics preparation of agar wells for antibiotic assay antibiotics: classification and mechanisms of action with emphasis on molecular perspectives biosynthesis of nano-silver from staphylococcus aureus and its antimicrobial activity against mrsa and mrse antibacterial activity and mechanism of nano-silver on escherichia coli alteration of zeta potential and membrane permeability in bacteria: a study with cationic agents surface charge and zeta potential of metabolically active and dead cyanobacteria gc-msstudy of nutritious leaves of ehretia laevis synthesis of nano-silver using azadirachta indica (neem) extract and usage in water purification the effects of interfacial potential on antimicrobial propensity of zno nanoparticle synthesis and effect of nano-silver on the antibacterial activity of different antibiotics against staphylococcus aureus and escherichia coli nanomed synthesis of silver nanoparticles by silver salt reduction and its characterization production of nano-silver with strong and stable antimicrobial activity against highly pathogenic and multidrug resistant bacteria linezolid: it's role in the treatment of grampositive, drug-resistant bacterial infections increases in rates of resistance to trimethoprim bacillus megaterium resistance to cloxacillin accompanied by a compensatory change in penicillin binding proteins streptomycin resistance in bacillus megaterium antibiotic resistance. nosocomial gram-positive infection ixtepan turrent, cristina rodrıguez padilla, bactericidal effect of silver nanoparticles against multidrug-resistant bacteria unique level of multidrug resistance (mdr) in pseudomonas aeruginosa strain deb high prevalence of multidrug resistant pseudomonas aeruginosa recovered from infected burn wounds in children the application of biophysical techniques to study antimicrobial peptides, spectrosc escherichia coli cell surface perturbation and disruption induced by antimicrobial peptides bp and pepr the authors convey their thanks to department of biotechnology, junagadh agricultural university, junagadh, gujarat, india, for providing laboratory facilities to conduct experiment and analysis with sophisticated instruments. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.jtemb. . . . key: cord- -x zp authors: esposito, susanna; mencacci, antonella; cenci, elio; camilloni, barbara; silvestri, ettore; principi, nicola title: multiplex platforms for the identification of respiratory pathogens: are they useful in pediatric clinical practice? date: - - journal: front cell infect microbiol doi: . /fcimb. . sha: doc_id: cord_uid: x zp respiratory tract infections (rtis) are extremely common especially in the first year of life. knowledge of the etiology of a rti is essential to facilitate the appropriate management and the implementation of the most effective control measures. this perspective explains why laboratory methods that can identify pathogens in respiratory secretions have been developed over the course of many years. high-complexity multiplex panel assays that can simultaneously detect up to viruses and up to four bacteria within a few hours have been marketed. however, are these platforms actually useful in pediatric clinical practice? in this manuscript, we showed that these platforms appear to be particularly important for epidemiological studies and clinical research. on the contrary, their routine use in pediatric clinical practice remains debatable. they can be used only in the hospital as they require specific equipment and laboratory technicians with considerable knowledge, training, and experience. moreover, despite more sensitive and specific than other tests routinely used for respiratory pathogen identification, they do not offer significantly advantage for detection of the true etiology of a respiratory disease. furthermore, knowledge of which virus is the cause of a respiratory disease is not useful from a therapeutic point of view unless influenza virus or respiratory syncytial virus are the infecting agents as effective drugs are available only for these pathogens. on the other hand, multiplex platforms can be justified in the presence of severe clinical manifestations, and in immunocompromised patients for whom specific treatment option can be available, particularly when they can be used simultaneously with platforms that allow identification of antimicrobial resistance to commonly used drugs. it is highly likely that these platforms, particularly those with high sensitivity and specificity and with low turnaround time, will become essential when new drugs effective and safe against most of the respiratory viruses will be available. further studies on how to differentiate carriers from patients with true disease, as well as studies on the implications of coinfections and identification of antimicrobial resistance, are warranted. respiratory tract infections (rtis) are extremely common especially in the first year of life (everard, ) . most of these infections are due to one of the many respiratory viruses, mainly respiratory syncytial virus (rsv) (griffiths et al., ) , influenza virus (iv) (antonova et al., ) , and rhinovirus (rv) . however, parainfluenza virus (piv) (branche and falsey, ) , adenovirus (adv) (esposito et al., b) , human metapneumovirus (hmpv) , bocavirus (bv) (principi et al., a) , and enterovirus (ev) (hellferscee et al., ) can also play a relevant role, particularly during epidemics. when bacteria are the cause, streptococcus pyogenes is a common cause of pharyngitis (paradise, ) , and streptococcus pneumoniae is a typical cause of lower rtis (esposito and principi, ) . in some cases, coinfections with two or more viruses (scotta et al., ) or with viruses and bacteria (brealey et al., ) can occur. for many years, it was thought that knowledge of the etiology of a respiratory infection was essential to facilitate the appropriate management and the implementation of the most effective control measures. it was presupposed that evidence showing that a given viral pathogen was the cause of a respiratory infection could reduce the prescription of further diagnostic tests and the use of antibiotics. theoretically, clinician uncertainty and the anxiety of patients and their family members could also be reduced (gill et al., ) . this perspective explains why laboratory methods that can identify pathogens in respiratory secretions have been developed over the course of many years. initially, cell cultures, immunofluorescence assays, and rapid antigen direct tests were used. these tests were mainly used for virus identification due to the higher frequency of viral rtis. however, none of these tests were considered completely satisfactory for clinical use. although cell cultures exhibited a high specificity and good sensitivity, they were very expensive and had a long turnaround time (leland and ginocchio, ; gharabaghi et al., ) . immunofluorescence assays achieved moderate sensitivity, identified no more than eight viruses, and sometimes required a long turnaround time (ginocchio and mcadam, ) . finally, rapid antigen direct tests, although able to provide results in few minutes with high specificity, were available only for rsv, iv, and adenovirus and have low sensitivity (gharabaghi et al., ; ginocchio and mcadam, ) . these problems have been overcome, at least in part, in recent years, when methods based on nucleic acid amplification became available. such methods exhibit enhanced sensitivity and specificity, and they can detect a broad range of pathogens within an acceptable turnaround time. single polymerase chain reactions (pcrs) for all the known respiratory viruses and several multiplex platforms using pcr and methods for nucleic acid amplification for the simultaneous detection of two or more viruses have been developed (hanson and couturier, ) . high-complexity multiplex panel assays that can simultaneously detect up to viruses (mahony et al., ) , viruses and two or three atypical bacteria (gonsalves et al., ) , viruses and four bacteria (beckmann and hirsch, ) and a total of pathogens including bacteria (fast track diagnostic, ) within a few hours have been developed. however, not all the bacteria that play a relevant role in the determination of respiratory infections are systematically included. some of these multiplex platforms have been marketed and largely evaluated in clinical practice in patients admitted to the emergency departments, hospital wards and intensive care units. despite these evaluations, the actual role of these diagnostic measures is not precisely defined. in particular, it has not yet been established whether the availability of a laboratory measure able to identify several potential etiologic agents of a respiratory infection offers real advantages in term of diagnostic accuracy, choice of appropriate therapy and reduction of the social and economic problems strictly associated with pediatric respiratory diseases. moreover, in the majority of the cases they don't allow the identification of antimicrobial resistance to commonly used drugs. in this paper, what can be derived from the presently available studies in this regard is discussed. multiplex platforms based on molecular methods can be used only in the hospital, as they require specific equipment and laboratory technicians with considerable knowledge, training, and experience (beckmann and hirsch, ; esposito et al., a; biomerieux, ; fast track diagnostic, ) . moreover, these platforms have a turnaround time that is significantly shorter than that of culture but generally much longer than that of rapid tests as they take some hours to give reliable results. this can be a limitation in the emergency department or in the intensive care unit, where many patients require immediate diagnostic and therapeutic decisions (beckmann and hirsch, ; esposito et al., a; biomerieux, ; fast track diagnostic, ) . only the most recently developed platforms, such as the biofire r filmarray r respiratory panel , have an acceptable turnaround time of about h, not much longer than a rapid test (biofire, ) . finally, the number of samples that can be processed per run can significantly vary from assay to assay. in some cases, such in the case of the already cited biofire r assay, only one sample could be processed per run, while most platforms have higher sample throughput (up to samples) (chan et al., ) . this can be a problem during epidemics when several patients have to be tested simultaneously. multiplex assays are significantly more sensitive and specific compared with rapid immunochromatographic tests and immunofluorescence assays; however, as multiplex assays detect both viable and non-viable viruses and bacteria, they can lead to debatable results (beckmann and hirsch, ; esposito et al., a; biomerieux, ; fast track diagnostic, ) . generally, the presently developed and marketed multiplex assays exhibit comparable performance with regards to sensitivities and specificities, and detection of coinfections. a previous comparison of the (luminex, ) nx tag rpp assay, biofire film array respiratory panel (fa-rp) (biomerieux, ) , respifinder (rf ) (beckmann and hirsch, ) , and the (luminex, ) rvp fast assay v (esposito et al., a) in terms of the ability to detect common pathogens revealed that the discordance was lower than %, although the turnaround time, workflow simplicity and risk of contamination were lower for the (luminex, ) nx tag rpp (chen et al., ; tang et al., ) . a systematic review and meta-analysis (huang et al., ) of studies on the accuracy of fa-rp, nanosphere verigene rv+ test (hologic, ; luminex, ) gen-probe prodesse assays (hologic, ) in the detection of iv a, iv b virus, rsv, hmpv, and av showed that all of these assays had high diagnostic accuracy, with an area under the receiver operating characteristic curve (auroc) equal to or > . for all tested viruses. the only exception was adenovirus, for which the auroc was . . finally, a similarly high accuracy was demonstrated for anyplex ii rv , advansure rv, and real-q rv (yun et al., ) . the allplex respiratory panels (allplex; seegene, republic of korea), a recently released one-step real-time reverse transcription-pcr method for the simultaneous detection of multiple pathogens, has been demonstrated to be a rapid and accurate method for detecting respiratory viruses, particularly in case of multiple viral infections (lee and lee, ) . a study comparing allplex rp assay with prodesseproflu+ and profast+ (hologic, madison, wi, usa) , and genexpert flu/rsv xc (cepheid, usa) for iv and rsv detection, found accuracy of , , and % and sensitivity of , , and %, respectively. the three assays showed a % specificity and positive predictive value (ppv), while the negative predictive values (npv) were , , and % for allplex rp , prodesse and genexpert, respectively (gimferrer et al., ) . in general, the accuracy of multiplex platforms in identifying respiratory pathogens is similar to those of single pcr, although in some cases, a slightly lower efficiency has been shown. for example, when the luminex ( ) nx tag rpp was compared with single pcr, it was shown that the multiplex assay had a lower sensitivity against cv hku and cv o and was oversensitive for several other viruses, leading to several false-positive results. the worst accuracy was found for hmpv, for which the ppv was < % (chen et al., ) . similarly, compared with standard pcr assays, the sensitivity of taqman array card was , , and % for adv, piv- and− , respectively, and - % for the other tested viruses. assay specificity was %, and coefficients of variation for virus controls ranged from . to . % (weinberg et al., ) . however, as previously reported, not all the bacterial pathogens that cause respiratory infections are systematically included in the available multiplex platforms. this is a relevant limitation in clinical practice for most of these assays as it is the lack of information regarding antimicrobial resistance to the most commonly used anti-infective drugs. evidence showing that a virus is present in the respiratory secretions of a patient with an rti does not necessarily mean that that particular infectious agent is the cause of the disease. a virus can be the etiologic agent, but viruses can also be asymptomatically carried or shed for several weeks after an infection that has been cured. therefore, viruses can be identified in the asymptomatic incubation period without having an actual role in the disease. contrary to what was thought some years ago, viruses can asymptomatically colonize the airways. both children admitted to the hospital for nonrespiratory diseases and healthy children attending day care were shown to be carriers of at least one respiratory virus in ∼ % of cases, although the viral load was generally lower in carriers than in symptomatic subjects (jansen et al., ; moe et al., ) . notably, the rate of asymptomatic colonization varies significantly from virus to virus. the detection of iv, rsv, hmpv, and piv is generally indicative of disease, as the frequency of asymptomatic carriage of these agents is relatively uncommon. in contrast, the detection of certain other viruses raises many doubts due to the high frequency with which these viruses can be identified in asymptomatic children (rhedin et al., ) . despite the capacity to cause upper and lower rtis and to trigger asthma and chronic obstructive pulmonary disease exacerbations (esposito et al., b; parker et al., ; , rv is the virus that is most frequently found in asymptomatic patients. principi et al. ( b) studied healthy children < years old who were followed with weekly nasopharyngeal samples during the months of the winter season of - . a total of , nasopharyngeal samples were obtained, and samples tested positive for rv ( . %). of these, ( . %) were not associated with respiratory symptoms, suggesting asymptomatic colonization. carriage is common also for bv (chow and esper, ), which can be detected in the respiratory secretions of healthy children with the same frequency as that observed in patients with rti (von linstow et al., ) . similar findings have been reported for adv, which has been detected in the nasopharynx of healthy children in up to % of cases (colvin et al., ; rhedin et al., ) . pathogens that are asymptomatically carried are also the pathogens that can be most commonly detected with the most recent multiplex platforms. the carriage of mycoplasma pneumoniae (mp) has been identified in many asymptomatic children, although the prevalence varied according to the site of the study [ % in denmark (spuesens et al., ) , and % in the usa (wood et al., ) ] and the period of respiratory sample collection [ % in the spring, and % in the summer (colvin et al., ) ]. similarly, a certain percentage of healthy children ( - %) has been shown to test positive for chlamydophila pneumoniae (cp) (emre et al., ; block et al., ; falck et al., ) . long-term viral shedding after a previous infection can further render the results of multiplex platforms poorly effective in identifying the etiology of a disease. however, there are significant differences in long-term shedding among the various infectious agents. generally, those agents that are uncommonly carried are the same agents that are shed only for a few days, and the opposite trend applies for those agents that are frequently asymptomatically carried. although certain influenza patients can shed the virus up to days, shedding typically peaks on day after the onset of symptoms (ip et al., ) . although there are exceptions (munywoki et al., ) , rsv shedding has been estimated to last between . and . days (hall et al., ; okiro et al., ) . in contrast, rv and bc are generally shed for a longer time. in a study involving children aged - months with bocavirus infection, it was shown that in % of cases, the virus persisted in the respiratory secretions for more than days, despite the rapid disappearance of clinical manifestations (wagner et al., ) . the same prolonged shedding has been reported for rv (loeffelholz et al., ) , although there are differences according to the type of the infecting strain (daleno et al., ) . long-lasting persistence in the respiratory secretions after acute infection has been demonstrated for adv (kalu et al., ) and atypical bacteria. the persistence of mp dna in the throat is common, with a median carriage time of weeks after the disease onset (range days− months) (nilsson et al., ) . cp can be detected for months, as this pathogen temporarily interrupts its replication cycle when exposed to antimicrobials but later resumes replication, leading to the generation of infectious particles (panzetta et al., ) . finally, viruses can be detected during the incubation period without playing any role in the determination of the actual disease. the incubation period for viral respiratory infections is generally short. it has been calculated (lessler et al., ) that this period lasts . days ( % confidence interval [ci], . - . ) for adv, . days ( % ci, . - . ) for human coronavirus, . days ( % ci, . - . ) for iv a, . days ( % ci, . - . ) for iv b, . days ( % ci, . - . ) for piv, . days ( % ci, . - . ) for rsv, and . days ( % ci, . - . ) for rv. however, during this period, these viruses are detectable in the respiratory secretions and can lead to diagnostic mistakes. defining the etiology of disease is further complicated by the detection of coinfections, as it is practically impossible to establish which agent is the true causative agent. unfortunately, coinfections are common. in a study involving children with radiographically confirmed cap, viral coinfections were demonstrated in cases ( . % of the enrolled patients and . % of those with viral infections). similar findings were reported when viral-bacterial coinfections were studied (nolan et al., ) . these findings are not surprising, as a previous or concurrent viral rti can favor the development of a secondary bacterial coinfection throughout the airway. augmented bacterial adherence and colonization, dysregulation of the innate and adaptive immune response, immunosuppression, the release of bacteria from biofilms, and alteration of the microbiome are mechanisms through which viruses can favor bacterial superinfection (bakaletz, ) . in conclusion, multiplex platforms, despite significantly increasing the possibility to detect which pathogens are present in the respiratory secretions of a child with a respiratory infection, do not offer any advantage in comparison to tradition diagnostic tests regarding the identification of the true etiologic agent of the disease. when multiplex assays are used, the benefits of determining which infectious agent(s) is (are) potentially responsible for an rti are strongly limited by the low number of drugs that are active against the respiratory targets that are currently available on these diagnostic platforms. presently, only drugs against influenza virus, rsv and atypical bacteria are licensed. furthermore, it is debated that these drugs should be used in all the subjects suffering from infections due to sensitive agents. the systematic use of neuraminidase inhibitors (european center for disease prevention and control, ) and baloxavir marboxil (hayden et al., ) is not recommended for all the cases of influenza because influenza is frequently a mild disease, and the advantage of drug administration is limited to a marginal reduction in the disease duration. consequently, the use of these drugs is reserved only for extremely severe cases, although their true efficacy in these cases has not been definitively demonstrated (lessler et al., ; european center for disease prevention and control, ) . rsv infection can be treated with ribavirin, which is the only licensed drug for the treatment of this virus. however, ribavirin is difficult to use, costly, and teratogenic, and there is weak evidence for its efficacy. ribavirin is typically used in severe cases that occur in immunocompromised subjects (brendish and clark, ) . although several new drugs against rsv are in development, and it is likely that in next few years some of them will be licensed for universal use, the present treatment of rsv infection remains based on supporting measures, such as hydration and o administration (xing and proesmans, ) . antibiotics that are effective in vitro against atypical bacteria are generally recommended and largely used when these pathogens are the suspected or demonstrated cause of an rti (esposito et al., (esposito et al., , a kohlhoff and hammerschlag, ) . however, the actual relevance of these drugs in clinical practice is debated, and there is evidence that seems to suggest that they are only slightly effective or not effective (spuesens et al., ; gardiner et al., ) . in children, only macrolides can be used against mp and cp, as other drugs that are effective against atypical bacteria cannot be prescribed to these patients, particularly the youngest. the administration of tetracyclines and chloramphenicol can lead to severe adverse events; ketolides and streptogramins have limited use in pediatrics; and fluoroquinolones are not licensed for use in subjects < years of age (principi and esposito, ) . however, many studies that have compared the clinical course of mp and cp infections in children treated with macrolides or other drugs that are ineffective against these pathogens have reported no difference between the two groups, suggesting that macrolides are useless (principi and esposito, ) . in addition, macrolides should be used only when their use is presumed to be effective in reducing the abuse of antibiotics and the emergence of resistant strains . finally, despite its high risk of nephrotoxicity, cidofovir, which is a drug licensed for the treatment of cytomegalovirus (cmv) retinitis in hiv-infected patients, has been used to treat adv infections in immunocompromised subjects (ganapathi et al., ) . a retrospective evaluation including children showed that in / cases, viral clearance and clinical response were achieved. however, four patients expired despite the viral clearance, and one of these deaths was directly ascribed to the adv infection. however, cidofovir is not licensed for the treatment of adv; this drug is reserved only for very severe cases occurring in immunocompromised children and cannot be considered as a potential solution to treat mild-to-moderate cases of respiratory disease in otherwise healthy children (ganapathi et al., ) . a recent prospective study including children and adults with rti, aimed to determine antibiotic misuse, showed that viral infection was more common in children than in adults, while antibiotic overuse occurred both in children ( %) and, at a significantly higher level, in adults ( %). the study highlights the needing for effective interventions to decrease antibiotic overuse in rti patients of all ages (van houten et al., ) . it has been proposed that the use of tests that can identify viruses reduce the use of antibiotics, the prescription of other diagnostic measures, the risk of hospital acquisition among other patients and the length of stay in the pediatric clinical setting. the advantages of these tests from a medical, social, and economic point of view should therefore be enormous. however, the results of studies on multiplex assays are few and conflicting. when these tests were used in pediatric patients admitted to the intensive care unit, no advantage in antibiotic prescriptions was demonstrated (byington et al., ) . in contrast, a retrospective evaluation of the use of these tests in pediatric inpatients showed that a positive test result was associated with a decreased length of stay in the hospital and a shorter duration of antibiotics, at least in patients with common respiratory diagnoses (schulert et al., ) . however, in wishaupt et al. ( ) the availability of results within - h did not lead to any advantage in terms of hospital admissions, length of hospital stay or the duration of antibiotic use when antibiotic treatment had been initiated. a more recent retrospective study in which a multiplex assay was tested in children admitted to the emergency department prior to admission or within the first days of hospitalization revealed that patients for whom the results of the test were available were less likely to receive antibiotics for ≥ days; moreover, these patients were more likely to be in isolation for ≥ days compared with the controls (subramony et al., ) . finally, a recent study conducted in special high-risk settings such as hematology and oncology units, suggests that the diagnoses of asymptomatic virus infections, such as rsv and influenza, can be useful to lower the risk of hospital acquisition. the screening program proved useful for identifying asymptomatically infected patients with viral shedding, thus reducing the risk of transmission and potential nosocomial clusters of rsv and influenza virus on hemato-oncological wards (baier et al., ) . on the other hand, less than satisfactory results were also reported in studies that evaluated the impact of rapid tests or immunofluorescence assays, although marketed preparations have been shown to exhibit sensitivity and specific higher than % (vos et al., ) . a cochrane review (doan et al., ) that analyzed four studies regarding the impact of rapid viral diagnosis in children admitted to the emergency department reported that the only advantage of the rapid diagnosis was a lower rate of chest radiography (relative risk [rr], . ; % ci, . - . ). no effect on the length of the visit or blood or urine testing was demonstrated. moreover, a trend toward decreased antibiotic use was demonstrated, but the difference between the tested and untested children was not significant. the knowledge that a bronchiolitis case was associated with the detection of rsv in the nasopharyngeal secretions did not lead to any advantage. children with positive results from the rapid test received similar blood tests and radiological examinations. moreover, although the children who tested positive had a more severe disease, virologic testing for rsv did not identify children at risk. globally, the rapid test for rsv is considered useless, and this attitude explains why the test is not recommended by several experts (ralston et al., ) . the only rapid test for a single virus that seems to positively impact physician attitudes and have a satisfactory effect on antibiotic consumption the clinical course of disease is the rapid test for influenza. several years ago, esposito et al. ( ) showed that children with a positive rapid influenza test were significantly less likely than those with a negative test or no test to undergo routine blood examinations ( . % vs. . % and . %; p = . and p = . ) or receive antibiotics ( . % vs. . % and . %; p < . and p = . ). children with positive tests also tended to have a lower incidence (although not significant) of chest radiographs ( . % vs. . % and . %) and a lower likelihood of admission ( % vs. . % and . %). similar results were reported more recently by cantais et al. ( ) , who showed that the diagnosis of influenza was followed by a . % reduction in blood puncture, a . % reduction in chest x-rays, a . % reduction in lumbar puncture, a . % reduction in urine culture, a . % reduction in antibiotic treatments, and a . % reduction in hospital stay; altogether, these reductions resulted in a reduction of medical costs estimated to be >€ , per season. finally, no studies have evaluated the impact of multiplex platforms on the expectations of patient and parents. generally, laboratory tests are requested because they are considered to be effective measures to improve the judgement of clinicians. however, the diagnosis of a viral disease by a laboratory test can be interpreted as a simplification of a more complex clinical problem. bronchiolitis due to rsv can be very severe, and its inclusion among a viral diagnosis may lead to parental dissatisfaction, especially given that no specific therapies are available for rsv, and only supportive measures are prescribed (cabral et al., ) . moreover, no definitive information is available on the impact of multiplex assays on clinician anxiety. all these data are needed for a more complete evaluation of the impact of multiplex assays in clinical practice. multiplex platforms for the identification of respiratory viruses and atypical bacteria allow for the identification of most of the infectious agents that cause respiratory infections in infants and children. it is highly likely that these platforms can be particularly important for studies specifically planned to evaluate epidemiology of respiratory pathogens and clinical research. on the contrary, their routine use in pediatric clinical practice remains debatable. they cannot be used in the community where most of the pediatric respiratory diseases are diagnosed. moreover, they cannot allow to overcome the limitation of the traditional diagnostic tests for respiratory pathogens as they do not differentiate carriage from infection, do not seem to influence therapy as effective drugs are available only for iv and rsv, and do not seem to significantly impact of the socioeconomic problems strictly related to pediatric respiratory infections. they seem, however, justified in the presence of severe clinical manifestations, and in immunocompromised patients for whom specific treatment option can be available, particularly when they can be used simultaneously with platforms that allow identification of antimicrobial resistance to commonly used drugs. it is highly likely that these platforms, particularly those with high sensitivity and specificity and with low turnaround time, will become essential when new drugs effective and safe against most of the respiratory viruses will be available. se wrote the first draft of the manuscript. am, ec, and bc gave a substantial scientific contribution. es performed the literature review. np co-wrote the first draft of the manuscript and supervised the project. all authors approved the 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mycobacterium bovis is that the true incidence of this type of tb in humans is not known and is likely to be underestimated. to effectively address challenges posed by ztb, an integrated one health approach is needed. in this manuscript, we describe the rationale, major steps, timeline, stakeholders, and important events that led to the assembling of a true integrated multi-institutional and interdisciplinary team that accomplished the ambitious goal of developing a ztb roadmap, published in october, . it outlines key activities to address the global challenges regarding the prevention, surveillance, diagnosis, and treatment of ztb. we discuss and emphasize the importance of integrated approaches to be able to accomplish the short (year ) and medium term (year ) goals outlined in the ztb roadmap. worldwide, there is consensus that solutions to complex issues need the participation and involvement of different stakeholders. recent outbreaks of emerging and re-emerging zoonoses [i.e., zika, ebola, middle east respiratory syndrome (mers), avian and swine influenza] ( ) have heightened the public's awareness about the close and complex interrelationship between the health of humans, wildlife species, and domestic animals. tuberculosis (tb), is one such disease that continues to have devastating consequences for not only humans, but also cattle and several wildlife species ( ) ( ) ( ) ( ) . caused by bacteria belonging to the mycobacterium tuberculosis complex (mtbc), tb presents additional challenges to human and veterinary health authorities given the zoonotic nature of the pathogens responsible for the disease and the ability of mtbc agents to be shared across species ( , ) . tb in humans is caused primarily by mycobacterium tuberculosis (m. tb); worldwide, it is the leading cause of death in humans by an infectious disease ( ) . bovine tb caused by mycobacterium bovis (m. bovis) is widely distributed around the world ( , ) and continues to cause considerable economic losses to farmers and countries due to the reduced production of affected animals, culling of animals from herds (or entire herd depopulation in some cases), and the elimination of affected (or all) parts of animal carcasses at slaughter ( ) . m. bovis can also infect and cause tb in humans (zoonotic tuberculosis (ztb) ( ) ( ) ( ) ( ) . the world health organization (who) estimated that "in there were , new cases of ztb and , deaths due to this type of tb" ( ) . furthermore, m. bovis has the ability to cause tb and cause death in several wildlife species ( , ) . one of the main public health challenges regarding ztb is that its true incidence in humans is not known and is likely to be underestimated due to the lack of systematic surveillance for m. bovis as a causal agent of tb in people in all low-income, high tb burden countries where bovine tb is endemic, and the inability of laboratory procedures most commonly used to diagnose human tb to identify and differentiate m. bovis from m. tb ( ) . to effectively address challenges posed by ztb (and other diseases at the "animal-human" interface), a crosssectorial and multidisciplinary one health approach linking animal, human, and environmental health is required. in this manuscript, we describe the rationale, major steps, timeline, stakeholders, and important events that lead to the assembling of a true integrated multi-institutional and interdisciplinary team that worked toward and accomplished the ambitious goal of developing a ztb roadmap that was published in english, spanish, and french ( ) ( ) ( ) to address the global challenges regarding the prevention, surveillance, diagnosis, and treatment of zoonotic tb (ztb), globally. we discuss and emphasize the importance of integrated approaches to be able to accomplish the short (year ) and medium term (year ) goals outlined in the ztb roadmap. the international union against tuberculosis and lung disease (the union) is an international scientific organization that works with partners, including governments, academia, and civil society, to fight tb, tobacco use and other lung diseases in low-and middle-income countries, through technical assistance, training, and research. through its volunteer members, it houses scientific sections that promote areas of specific interest. the union's ztb sub-section is a global network of physicians, veterinarians, researchers, economists and social anthropologists that works to understand the dynamics of ztb, create global awareness, and facilitate multi-institutional collaboration to address the challenges posed by it. efforts conducted by the ztb sub-section has led to a continuous and stable increase in the number of activities, attention, and attendees to ztb-related activities at the annual union world conference on lung health. for example, at the conference in berlin, germany, there was only one ztb session (symposium) attended by less than people. over the last seven years, the ztb activities at the annual conference have increased to two scientific symposia, one poster session, one meet the expert session, press releases, and a keynote talk on ztb at the plenary session in south africa during the conference. during the last conference in guadalajara, mexico in october , an audience of approximately professionals from different disciplines attended each of the two ztb scientific symposia. prior to the union's initial activities to create global awareness of ztb, in , the who, oie and fao pioneered one health approaches under a tripartite partnership, which shares responsibilities and jointly develop and implement integrated strategies for addressing health risks at the human animal-ecosystem interface ( ) . the combined involvement and commitment of these three institutions has been crucial to successfully develop a ztb roadmap since these institutions jointly: ( ) provide global leadership for tb prevention, care and control (who); ( ) is responsible for improving animal health and welfare (oie); and ( ) work toward improving food security, nutrition and agricultural productivity and reduce rural poverty (fao). in march , the ztb sub-section created a working group to raise awareness of the public health risk posed by ztb. this working group included participants from key parties including the: one of the accomplishments of this working group was the publication of a manuscript ( ) calling for a call to action in the lancet infectious disease journal. a constellation of events has occurred to bring ztb to the awareness of scientists, policymakers, government officials and the general public. in may , the world health assembly, who's yearly gathering of the world's ministers of health, approved the new post- global tb strategy. the strategy aims to end, rather than merely control, the global tb epidemic, with targets to reduce tb deaths by % and to reduce new cases by % between and , and to ensure that no family is burdened with catastrophic expenses due to tb. it set interim milestones for , , and ( ) . thus, finding and treating every case of tb, whether caused by m. tb or m. bovis, will count toward the achievement of this ambitious goal. for this reason, as countries move toward detecting the million tb cases estimated to be missed annually, and in light of the endorsed who "end tb" strategy, the tripartite, the union and the key organizations concerned with human and animal health, agriculture and tb joined forces to develop a zoonotic tb road map outlining medium-and long-term milestones to globally address the prevention, surveillance, diagnostic, and treatment challenges faced by persons with ztb. in september , the united nations declared the end of the millennium development goals and used them as the foundation for the sustainable development goals (sdgs) ( ) for - , encompassing broad and comprehensive topic areas, ranging from elimination of poverty and hunger, improved education and gender equality, to clean water and energy, action on climate and improvement of life under water and on land. the third sdg addresses global health, with tb highlighted as one of the priorities, thus presenting a key opportunity to improve the health of communities affected by ztb. the stop tb partnership, the global advocacy organization for tb, published the th edition of its global plan to end tb, - entitled "the paradigm shift" ( ) . this is a costed plan that includes actions needed to decrease tb, and is in full support of who's end tb strategy. the global plan has set its -( )- targets, which are to: identify % of all tb cases; concentrate on identifying % of those in key populations; and ensure % obtain appropriate treatment until cure. for the first time in this document, communities and people at risk of contracting ztb were included as a key population. a meeting co-organized by who and the union, with contributions from leading international organizations for human and animal health, academic institutions, and nongovernmental organizations took place at who in geneva. there, the first steps toward formally conceptualizing a roadmap for ztb began, in which ten priorities were identified to be presented to who's global tb programme's strategic technical advisory group (stag) for tb in june . in addition to the institutions initially working on increasing global awareness of ztb, the following institutions joined the efforts at this specific meeting: -swiss tropical and public health institute, switzerland -animal and plant health agency (apha), united kingdom -university of ibadan, nigeria -the global research alliance for bovine tuberculosis (grabtb) the involvement and participation of grabtb was an important addition and played a strategic role in partnering with colleagues focusing in improve the understanding and control of bovine tb and developing novel and improved tools to control the disease at its bovine source ( ) . grabtb was established in and as part of its strategic goals, the alliance seeks not only to enhance collaboration within its members and institutions, but also with the broader human and animal tb research community. the priorities proposed for the ztb roadmap were endorsed by the stag, and a working group was created and tasked to produce and publish a ztb road map during . at the stag meeting, a former ztb survivor, from the masaai community in kenya shared with the scientific community the challenges she faced while suffering from this disease, which included: initial misdiagnosis, development of extrapulmonary (abdominal) tb, antimicrobial resistance to anti-tb drugs additional to the inherent resistance m. bovis has against pyrazinamide, and the need for longer ( months) antimicrobial treatment, compared to the standard of months. during the th union world conference on lung health in liverpool, england, a british veterinarian and former ztb patient/survivor also shared his experience and challenges of initial misdiagnosis, extrapulmonary (pleural) tb, drug resistance to isoniazid in addition to pyrazinamide, and the longer treatment required while battling ztb in the year . these patients' testimonies further emphasized that ztb is not a disease from the past, and highlighted the challenges faced by certain communities at higher risk of contracting ztb. both patients emphasized the need of more awareness among the medical community to better diagnose and treat ztb patients, and thus prevent the additional complications they had to endure. the heads of state comprising the g forum declared that "shaping an interconnected world, calls for a one health approach to tackling the spread of antimicrobial resistance and highlighted the need to foster research and development for tb" ( ) . the roadmap for ztb was published and launched at the union world conference for lung health in guadalajara, mexico. the roadmap is the product of efforts of the tripartite partnership on zoonotic diseases comprising who, oie, and fao and the union, and is available in english, french, and spanish ( ) . the role of media over the past years, all efforts, activities, and events related to ztb were highlighted by a considerable number of local, regional, national, and global media sources including central news network (cnn), the british broadcasting corporation (bbc), and le monde newspaper in france, to name a few. the zoonotic tb roadmap ( ) outlines priorities to address the existing challenges posed by ztb, divided into three major core themes: ( ) improve the scientific evidence, ( ) reduce transmission at the animal-human interface, and ( ) strengthen intersectoral and collaborative approaches. "identify opportunities for community-tailored interventions that jointly address human and animal health interventions that jointly address human and animal health can increase health and economic benefits for communities. sharing of human resources, equipment and transport across sectors can reduce operational costs. this increased cost-effectiveness is especially relevant given the public funding constraints that often exist in settings where people are most at risk of zoonotic tb. for example, outreach childhood immunization campaigns or other existing livestock vaccination or testing programmes conducted in rural communities could be used to concurrently deliver educational and behavior change messages about food safety, to test livestock for bovine tb or, potentially in the future, to implement livestock vaccination campaigns against bovine tb. interventions must be tailored to the cultural and socioeconomic characteristics of each setting. community-driven participatory initiatives are key to achieving sustainability." table | timeline for action and milestones to be achieved in the short ( ) and medium term ( ) outlines in the ztb roadmap ( ) . reduce transmission at the animal-human interface although the publication of the ztb roadmap represents an unprecedented and historical accomplishment in the fight against global tb ( ) , there is still much work to be conducted in order to implement the actions needed to improve the prevention, diagnosis, control and treatment of ztb. table is an excerpt from the ztb roadmap in which the short (year ) and medium term (year ) milestones to be accomplished are outlined under the three core themes of the roadmap. one of the key elements toward accomplishing these goals is that the unique cultural and socioeconomic factors that shape the relationship between people, livestock, and wildlife species in different ecosystems must be taken into account, while including the at-risk communities in future efforts to reduce the risk of zoonotic transmission of m. bovis across species. these efforts not only need to focus on preventing transmission from livestock (mostly cattle) to humans, but also, and in parallel, to reduce the prevalence of the disease in both domestic and wildlife species. the availability of improved diagnostic tools for ztb in different species, as well as the implementation of disease monitoring, surveillance, and prevention strategies in livestock and wildlife species will be a crucial and much needed component to be able to implement comprehensive programs that will account for the complexities ztb poses due to its zoonotic nature. finally, including vaccination of wildlife (where feasible), not only has the potential for reducing the burden of disease, but also could play an important role in conservation efforts, especially among endangered and protected species. implementing an integrated approach to develop the ztb road map did not come without the inherent challenges of multidisciplinary and multi-institutional projects. that said, the accomplishment of this milestone in the fight against tb was the vision of a world free of tb, no matter what its source, the strong support of who and the stop tb partnership, a motivated core group that drove the process by providing clear goals and timelines, being inclusive by inviting all interested parties, and fostering a strong commitment to work together from colleagues and institutions both from the human and animal sectors that previously were working in isolation. this collaboration has opened new doors and opportunities as the world fights to end tb. all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. the national institute for occupational safety and health (niosh): emerging infectious diseases world health organization report of the meeting of the oie ad hoc group on tuberculosis, annex of the oie scientific commission for animal diseases report -september one health in the shrinking world: experiences with tuberculosis at the human-livestock-wildlife interface evidence of increasing intra and inter-species transmission of mycobacterium bovis in south africa: are we losing the battle? available online at mycobacterium bovis infection and control in domestic livestock zoonotic tuberculosis due to mycobacterium bovis in developing countries zoonotic mycobacterium bovis-induced tuberculosis in humans current knowledge and pending challenges in zoonosis caused by mycobacterium bovis: a review zoonotic tuberculosis in human beings caused by mycobacterium bovis -a call for action why has zoonotic tuberculosis not received much attention? world health organization, world organisation for animal health, food and agricultural organization of the united nations world health organization, world organisation for animal health, food and agricultural organization of the united nations, the international union against tb and lung disease. hoja de ruta contra la tuberculosis zoonotica world health organization, world organisation for animal health, food and agricultural organization of the united nations, the international union against tb and lung disease available online at united national sustainable development goals (sdgs) global plan to end tb: the paradigm shift g leaders declaration: shaping an interconnected world a roadmap for zoonotic tuberculosis: a one health approach to ending tuberculosis in addition to the institutions listed on the manuscript the authors wish to acknowledge the work of additional institutions that contributed to the development of the ztb roadmap: servicio key: cord- -d r nji authors: harrath, rafik; abu duhier, faisel m. title: sero‐prevalence of middle east respiratory syndrome coronavirus (mers‐cov) specific antibodies in dromedary camels in tabuk, saudi arabia date: - - journal: j med virol doi: . /jmv. sha: doc_id: cord_uid: d r nji the middle east respiratory syndrome coronavirus (mers‐cov) is a novel coronavirus which was responsible of the first case of human acute respiratory syndrome in the kingdom of saudi arabia (ksa), . dromedary camels are considered as potential reservoirs for the virus and seem to be the only animal host which may transmit the infection to human. further studies are required to better understand the animal sources of zoonotic transmission route and the risks of this infection. a primary sero‐prevalence study of mers‐cov preexisting neutralizing antibodies in dromedary camel serum was conducted in tabuk, western north region of ksa, in order to assess the seopositivity of these animals and to explain their possible role in the transmission of the infection to human. one hundred seventy one ( ) serum samples were collected from healthy dromedary camels with different ages and genders in tabuk city and tested for specific serum igg by elisa using the receptor‐binding s subunits of spike proteins of mers‐cov. ( , %) of the total camel sera shown the presence of protein‐specific antibodies against mers‐cov. these results may provide evidence that mers‐cov has previously infected dromedary camels in tabuk and may support the possible role of camels in the human infection. blood sera were separated, diluted at : and analyzed for mers-cov specific antibodies using the anti-mers-cov elisa camel (igg) kit manufactured by euroimmun ag (lübeck, germany). this test is based on the recombinant mers-cov spike protein subunit- and has successfully been used by other authors evaluating mers-cov in camels. optical density (od) was measured at nm using a mindray mr- elisa reader. statistical analysis was performed on spss v. . software (spss inc., chicago, il). data were expressed as percentage for continuous variables, which were normally distributed, or as percentages of total for categorical variables. pearson χ test was used to assess intergroup significance. this research was ethically approved by the research ethic committee at the university of tabuk. one hundred seventy one serum samples were collected from three areas in tabuk a primary study was conducted in tabuk instructions. this test has been successfully used and evaluated by many authors for mers-cov detection in camels. , , results have shown that a high number ( %) of dromedary camels from the different farms of tabuk riyadh and screened by elisa test showed that % of the animals were found to have antibodies to mers-cov. in the same study, archived serum samples collected from dromedary camels from to in riyadh and kharj were also analyzed by elisa and showed a high seroprevalence ( %) of mers-cov neutralizing antibodies. our data agree also with previous studies reporting wide antibody prevalence in camels in many countries, including egypt and oman. , in another study conducted in oman, all serum samples from dromedary camels were positive for mers-cov specific antibodies. similar results were also reached from a larger study conducted in united arab emirates (uae), where dromedary camels' sera screened in revealed % seropositivity. in africa, an outbreak investigating serum samples for mers-cov assessment in camels has also shown similar results in nigeria ( %), tunisia ( . %), and ethiopia ( . %). likewise, in a study conducted on archived camel sera samples collected in from egypt and between and from sudan and somalia, % were found to have neutralizing antibodies to mers-cov. similar results were also obtained in a study from kenya. while camels in the middle east and africa were highly exposed to mers-cov infection, camels from europe and australia which are geographically isolated were not exposed to the virus. however, serum samples collected from dromedary camels living in the archipelago of canary islands, between and showed that % have antibodies against mers-cov. in another study carried out in the same islands, dromedary camel sera were analyzed and only . % were seropositive for mers-cov. data showed that all the seropositive dromedary camels for mers-cov were all imported from africa. in addition, dromedary camels' sera collected from different regions between and in the islands were all tested negative for specific mers-cov antibodies. all these findings indicate that active mers-cov infection did not occur in the canary islands. the same author explained the increase of the seropositivity in adult camels by the age range of the animals which were exposed for long time to the virus infection. for young camels, specific mers-cov antibodies could be acquired from their mothers as it was reported by kamber who suggests that maternal igg antibodies in camels are acquired through the colostrum intake during the first h post-parturition and not via the trans-placental route. after h, antibody levels in the dam's milk decrease rapidly, and igg levels in serum cease to rise. we also suggest that maternal antibodies might not have been sufficient to mediate protective immunity for them. animals would then be exposed to new mers-cov infection and specific antibodies would consequently increase in - years after, which can explain the higher seroprevalence in juveniles then in adults. the predominance of viral antibodies in young camels could also be explained by animal exposure to the virus few times postparturition and not by mother immunity, which will stimulate antibody secretion in the blood. in the two last possibilities, young camels could contract viral infection after , period of epidemic mers circulation in ksa, and production of specific antibodies would consequently increase in some months after birth. respiratory specimens, as nasal swabs, and rectal swabs will be required for analysis to know whether anti-mers antibodies are issued from recent viral infection. in relation to sex, our results have shown a high seropositivity in both males ( , %) and females ( . %) without a significant difference between the two genders. this information was not reported in previous studies and indicates that the sex cannot be a limiting factor in camel's infection by the virus. in conclusion, our findings in dromedary camel's sera, even if limited to serology, constitute a strong argument suggesting that mers-cov has previously infected dromedary camels in tabuk region. we speculate that dromedary camels may support the role of these animals as potential reservoirs for human infection but we cannot confirm this relationship from the current data alone. further studies including more animals and respiratory samples are needed for a larger evaluation of camel population in this region. in addition, a whole genome sequencing of camel mers-cov is required to confirm its incrimination in human infection. the work was financially supported by the deanship of scientific research (dsr) at the university of tabuk. http://orcid.org/ - - - middle east respiratory syndrome coronavirus (mers-cov) update severe respiratory illness caused by a novel coronavirus middle east respiratory syndrome coronavirus (mers-cov) summary and literature update human betacoronavirus c emc/ -related viruses in bats, ghana and europe close relative of human middle east respiratory syndrome coronavirus in bat middle east respiratory syndrome coronavirus in bats, saudi arabia middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation human infection with mers coronavirus after exposure to infected camels, saudi arabia middle east respiratory syndrome coronavirus neutralizing serum antibodies in dromedary camels: a comparative serological study disappearance of antibodies to sars-associated coronavirus after recovery assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections specific serology for emerging human coronaviruses by protein microarray antibodies against mers coronavirus in dromedary camels antibodies against mers coronavirus in dromedary camels seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt geographic distribution of mers coronavirus among dromedary camels absence of mers-cov antibodies in feral camels in australia: implications for the pathogen's origin and spread presence of antibodies but no evidence for circulation of mers-cov in dromedaries on the canary islands time course of mers-cov infection and immunity in dromedary camels middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia studies on the supply of immunoglobulin g to newborn camel calves (camelus dromedarius) how to cite this article: harrath r, abu duhier fm. seroprevalence of middle east respiratory syndrome coronavirus (mers-cov) specific antibodies in dromedary camels in key: cord- - qx v w authors: brown, paul a.; courtillon, céline; weerts, erik a. w. s.; andraud, mathieu; allée, chantal; vendembeuche, anthony; amelot, michel; rose, nicolas; verheije, monique h.; eterradossi, nicolas title: transmission kinetics and histopathology induced by european turkey coronavirus during experimental infection of specific pathogen free turkeys date: - - journal: transbound emerg dis doi: . /tbed. sha: doc_id: cord_uid: qx v w numerous viruses, mostly in mixed infections, have been associated worldwide with poult enteritis complex (pec). in a coronavirus (fr‐tcov d) was isolated in france from turkey poults exhibiting clinical signs compatible with this syndrome. in the present study, the median infectious dose (id ( ))(,) transmission kinetics and pathogenicity of fr‐tcov were investigated in ‐day‐old spf turkeys. results revealed a titre of ( . ) id ( )/ml with id ( )/ml being beyond the limit of genome detection using a well‐characterized qrt‐pcr for avian coronaviruses. horizontal transmission of the virus via the airborne route was not observed however, via the oro‐faecal route this proved to be extremely rapid (one infectious individual infecting another every . hr) and infectious virus was excreted for at least weeks in several birds. histological examination of different zones of the intestinal tract of the fr‐tcov‐infected turkeys showed that the virus had a preference for the lower part of the intestinal tract with an abundance of viral antigen being present in epithelial cells of the ileum, caecum and bursa of fabricius. viral antigen was also detected in dendritic cells, monocytes and macrophages in these areas, which may indicate a potential for fr‐tcov to replicate in antigen‐presenting cells. together these results highlight the importance of good sanitary practices in turkey farms to avoid introducing minute amounts of virus that could suffice to initiate an outbreak, and the need to consider that infected individuals may still be infectious long after a clinical episode, to avoid virus dissemination through the movements of apparently recovered birds. infectious bronchitis virus is a highly contagious virus transmitted very quickly among naive birds in the field. it is responsible worldwide for respiratory diseases, egg drop with poor eggshell quality, reduced hatchability, nephritis and sometimes, in early infection of future breeders, genital atrophy responsible for the syndrome of "false laying" in chicken breeders or layers (jackwood, & wit, ) . turkey coronavirus, originally identified in the usa in the s as one of the agents responsible for an acute enteritis named bluecomb (panigrahy, naqi, & hall, ; ritchie, deshmukh, larsen, & pomeroy, ) and since with a multifactorial disease known as poult enteritis complex of turkeys (pec) , has now been detected in most areas where turkeys are farmed cavanagh et al., ; dea & tijssen, ; domańska-blicharz, seroka, lisowska, tomczyk, & minta, ; martin, vinco, cordioli, & lavazza, ; maurel et al., ; teixeira et al., ) , although tcovs isolated in europe have been shown to have a different genetic lineage to those isolated in the usa (brown et al., ; maurel et al., ) . pec includes several intestinal disorders that occur in turkeys mostly within the first three weeks of life (guy, ) and its clinical signs often include diarrhea, stunting, anorexia, dehydration, weight loss, and immune dysfunction (atrophy of the thymus and the bursa of fabricius) that promotes secondary infections. the wide distribution of both ibv and tcov and their highly contagious nature have considerable economic repercussions. the contagious nature of a disease can be measured by the "reproduction number" (r ) defined as "the expected number of secondary cases produced by a single (typical) infection in a totally susceptible population" (masters & perlman, ) . the parameters necessary to calculate r are (a) the speed of transmission and (b) the shedding duration of the infectious viruses. generally, a virus with an r less than will disappear quickly because an infected individual will have a low ability to infect another. a virus with an r greater than one will spread in the susceptible population. for ibv, an r of . has been estimated (de wit, de jong, pijpers, & verheijden, ) , which is a figure comparable to the r of highly contagious human viruses such as measles virus (r - ) (masters & perlman, ) . for tcov, r has not yet been fully calculated; however, a study with an american tcov isolate demonstrated that infectious virus particles can be shed up to six weeks post-infection in experimentally infected turkeys . the current study focused on strain fr-tcov d that was detected in france in in turkeys with clinical signs compatible with pec. fr-tcov is the only european tcov strain isolated to date, although coronaviruses have been detected in turkeys in poland, great britain and italy (cavanagh, ; domańska-blicharz et al., ; martin et al., ) . the aim of this study was to determine the transmission properties of the virus by evaluating its id and reproduction number (r ) under experimental conditions in day-old spf turkeys, in order to better understand the diffusion of the disease. histopathological examination and in-situ detection of tcov antigen at the sites of replication in the intestinal tract were also performed. three animal experiments (exp , and ) were performed in agreement with the national regulations of the french ministry for higher education and research on animal welfare and after approval from the french agency for food, environmental and occupational health & safety's (anses) ethical committee. virus fr-tcov d isolated from duodenal contents of -dayold turkeys affected by pec in november was propagated by inoculating embryonated spf turkey eggs (anses, ploufragan, france) via the intra-amniotic route, as previously described (guionie et al., ) . because fr-tcov d does not induce clinical lesions in the embryo, the intestines of inoculated embryos were screened days post-inoculation by qrt-pcr (maurel et al., ) , and the intestines of positive embryos were collected and pooled to prepare a virus stock ( ). five-fold serial dilutions of this stock were inoculated into seven eggs per dilution, and a titre of . eid /ml was calculated according to reed & muench ( ) . one hundred microliters of intestinal or cloacal swab material was lysed with μl of buffer rlt (qiagen, france) by mixing and incubating at room temperature for min. rna was extracted using magattract rna tissue mini m kit or magattract virus mini m kit for biorobot m (qiagen, france) and eluted in μl of buffer ave following the manufacturer's instructions. the presence of tcov genome was detected using a qrt-pcr specific for avian coronaviruses (maurel et al., ) . the limit of detection (lod) and the linear phase of this qrt-pcr were described as log and from to log copies per microliter of extracted rna, respectively. in this study, samples were considered positive with a result higher than log copies per microliter of extracted rna. all results are given as copy number (cp)/μl of extracted rna expressed in log together with the sd . | exp . titration of fr-tcov in -day-old spf thirty -day-old spf turkeys were separated in groups of birds, and housed for days in negative pressure isolators allowing ad lib feeding and drinking. each isolator had a cardboard floor with a metal grid platform underneath and a surface area of . m . groups , , and were inoculated via the oral route with . ml of brown et al. strain fr-tcov d diluted to − . , − . , − . and − . respectively in mem hepes (gibco, france) supplemented with penicillin ( μ/ml final concentration) and streptomycin ( . mg/ml final concentration). control group was inoculated with memh plus antibiotics alone via the same route. at -day post-inoculation (dpi), two spf turkey contacts were introduced into groups - as sentinels to demonstrate horizontal transmission of infectious virus. from to dpi, cloacal swabs were collected from all subjects, sampling the contacts first, followed by those that had been inoculated. rna was extracted from these samples for molecular analysis as described above. the % endpoint was calculated using the method of reed and muench (reed & muench, ) . thirty-two -day-old spf turkeys were separated into groups, one containing subjects and a second containing . each group was housed in a separate negative pressure room at a density of seven birds per m² and floors were covered with wood chippings (reproducing common commercial rearing conditions in france). the group of three subjects was inoculated with . ml of strain fr-tcov d diluted at − . in the same media as used in exp. , via the oral route. at dpi, cloacal swabs were collected to confirm their fr-tcov d positive status by qrt-pcr. at dpi, one positive subject was placed as a seeder infected bird among the group of spf subjects (contacts). cloacal swabs were collected from all subjects every hr until hr post-contact (hpc), at hpc and days post-contact (dpc) then weekly until dpc. during the -hr-sampling regime, the order in which the subjects were taken was respected throughout. this ensured that each subject was sampled precisely every two hours. sampling staff wore a new pair of sterile gloves for each sampled bird, so as not to transfer the virus through bird-handling. rna was extracted from these samples to perform qrt-pcr, to determine infection and the excretion period for each subject. one representative positive sample selected at dpc of exp . (codified t ) was diluted (same media as exp. ) so as to inoculate via the oral route . rna copies in three -day-old spf turkeys. they were housed in a negative pressure room, under the same rearing conditions as in exp , with three -day-old spf turkeys introduced as contact-birds at dpi to demonstrate horizontal transmission. cloacal swabs were collected daily for qrt-pcr analyses from all birds until dpi, when the birds were humanely euthanized and duodenum, jejunum, ileocaecal junction and bursa of fabricius were collected. these samples were fixed for hr in % formaldehyde then transferred to % ethanol and finally embedded in paraffin wax for histopathology and anti-tcov immunohistochemistry (see section histopathology). this process was repeated using one representative positive sample from , , , and dpc of exp . (codified t , t , t , t and t , respectively) to make a total of six experiments. airborne transmission was evaluated in each of these experiments by using six -day-old spf turkeys housed in a park in the same containment cell but separated from the other animals, at a distance of meters. the sampling programme was as described above. housing, circulation of personal, change of boots, clothes and gloves was organized to minimize physical contamination. fr-tcov was detected with qrt-pcr at dpi in all six inoculated subjects of group (dilution − . , mean ± sd . ± . log cp/μl), in out of subjects of group ( − , . ± . log cp/ μl) and in out of subjects in group ( − . , . ± . log cp/μl). at and dpi, all subjects of these groups, including contactbirds, were positive, demonstrating horizontal transmission. no viral rna was detected throughout the experiment in groups ( − ) and (memh). the result obtained at dpi (before horizontal transmission) gave a virus titre of , id /ml. the following data are shown graphically in figure . an inoculated subject with a viral rna load of . log cp/μl at dpi that had been placed among contacts, transmitted the virus to one contact between and hpc, though the level of viral rna detected at hpc in this newly infected bird ( . log cp/μl) was almost at the lod. however, between and hpc the level of viral rna detected in the same bird increased to . log cp/μl and a second contact was positive at . log cp/μl. the data are shown graphically in figure . in three out of six exp . samples (t , t and t ), the number of positive inoculated birds and the level of viral rna detection increased over time during the sampling period, culminating at dpi with rna detected in all birds including contacts (mean ± sd = . ± . , . ± . and . ± . cp/μl, respectively). no viral rna was detected throughout the period, neither in inoculated or contact subjects exposed to t , t and t , nor in subjects assigned to the assessment of airborne transmission. intestinal samples taken from infected subjects at dpi from exp. showed well-preserved characteristic architectural features. except for some very mild hyperemia and rare epithelial desquamation, no clear histopathological changes were seen in any of the samples (figure a) . immunohistochemical staining showed an abundance of viral protein expressed in the ileum, caeca and bursa of all inoculated or contact subjects exposed to t , t and t (expression in the caecum and bursa is shown for t in figure ). as shown in figure histograms, antigen detection in the other regions of the intestine (duodenum or jejunum) was inconsistent in both the inoculated and contact-birds exposed to the same samples, as illustrated by the fact that no viral protein was detected in the duodenum of any contact subject exposed to t , t and t . no viral protein expression was seen in any of the intestines taken from the inoculated and contact subjects exposed to t , t and t . to be taken into consideration. similarly, at one id /ml, viral rna levels were also beyond the limit of detection of a well characterized qrt-pcr (maurel et al., ) , so that -day-old turkeys might be also more sensitive than qrt-pcr to detect infectious tcov. such a high susceptibility of young hosts was also reported in a recent paper where an enteric coronavirus of pigs (porcine epidemic diarrhea virus, pedv) was shown to infect more efficiently -day-old piglets than tissue culture (minimal infectious dose = . tcid ) (thomas et al., ) . rna levels at the pedv mid have also been reported to be beyond the limit of detection by qrt-pcr (goyal, ) . the effects of turkey age on the id of fr-tcovwere not investigated in the current study however, the fact that tcov associated enteric disorders such as pec or poult enteritis mortality syndrome (pems) are predominantly diseases of younger subjects lends support to more resistance in older birds. was also difficult to justify on an ethical level in respect to the principals of the rs (russell & burch, ) . although the "duration of excretion" for every individual could not be obtained, the experiments performed in the current study (in which one sample from each date was re-inoculated) revealed that some subjects continued to shed infectious virus for at least six weeks when others ceased at two. at this time the authors have no data on why some subjects stopped excreting infectious virus four weeks in advance of others or if, in fact, excretion detected at six weeks was representative of subjects with intermittent excretion profiles as has been observed in cats experimentally infected with fecv (kipar, meli, baptiste, bowker, & lutz, ) . in cats, this intermittent excretion has been suggested to be linked with persistent infection in the colon (lower intestine) from which viruses then have the potential to re-infect the small intestine at any time. the presence of virus in both regions of the gut can then result in renewed excretion (kipar et al., ) . concerning fr-tcov these questions should be assigned to specifically designed trials and histopathological examination, however, the present study seems to indicate a clear tropism of fr-tcov for lower intestine, as described for fecv (kipar et al., ) . indeed, in the current study, fr-tcov's ability to infect the turkey intestinal tract was successfully demon- (reddy et al., ) . however, although the stellate morphology and the localization of the depicted cell (see inset figure c ) suggest that it likely belongs to one of the mentioned apc families, additional assays characterizing these cell types (for example double immunohistochemical staining for both viral protein expression and cell-characterizing protein epitopes) are needed to confirm a apc tropism for tcov. fr-tcov viral protein expression was not correlated with histopathologic changes in the sampled tissues collected here, contrary to previous observations following inoculation with a tcov of us lineage that did induce lesions, albeit without associated clinical signs . this discrepancy could be explained by the f i g u r e detection of viral antigen in intestinal tissues. immunohistochemistry of: intestinal tissues d = duodenum, j = jejunum, i = ileum, c = caeca b = bursa of fabricius, taken dpi from subjects inoculated with samples t , t , t , t , t ort of exp (a) and from their corresponding contacts (b) difference in age of the birds inoculated as in the us tcov study, birds had been inoculated at days of age; alternatively, like the distribution pattern discussed above, this discrepancy might be a time-dependent feature, with dpi in our study being too early for morphologic changesresulting from epithelial damage, local tissue reactions and influx of immune cells to manifest. furthermore the specific date postinoculation when microscopic lesions were observed in the us tcov study was not given . it is equally possible that under experimental conditions the european lineage of tcov simply has a different pathogenic profile to those of the us lineage. infection studies for eu and us tcovs in turkeys of the same age and under the same controlled conditions are required for comparative analysis into the pathological profiles of these different lineages. in conclusion an extremely low dose of european isolate fr-tcov strain d is required for infection of -day-old turkey poults under experimental conditions. the virus spreads very quickly via the oro-faecal route among susceptible subjects (a new subject at least every . hr) and infectious virus may continue to be excreted for at least six weeks after the initial infection which may be linked to a preferential tropism for the lower intestines. these results stress the importance of good sanitary practices at the entrance to livestock buildings and the need to consider that infected individuals may still be infectious long after the clinical episode. novel receptor specificity of avian gammacoronaviruses that cause enteritis poult enteritis 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replication of nephropathogenic infectious bronchitis virus in peripheral blood monocytic cells, a strategy for viral dissemination and kidney infection in chickens a simple method of estimating fifty percent end points electron microscopy of coronavirus-like particles characteristic of turkey bluecomb disease the principles of humane experimental technique detection of turkey coronavirus in commercial turkey poults in brazil effect of porcine epidemic diarrhea virus infectious doses on infection outcomes in naive conventional neonatal and weaned pigs design and analysis of an actinobacillus pleuropneumoniae transmission experiment transmission of infectious bronchitis virus within vaccinated and unvaccinated groups of chickens transmission kinetics and histopathology induced by european turkey coronavirus during experimental infection of specific pathogen free turkeys the authors wish to thank the département des côtes d'armor/conseil régional de bretagne/conseil régional des pays de loire/france agrimer/office de l'élevage/comité interprofessionnel de la dinde française for their financial support. this research was part of the epicorem anr programme: eco-epidemiology of coronaviruses:from wildlife to human & emergence threat assessment. the authors declare that they have no conflict of interest. http://orcid.org/ - - - key: cord- -piust s authors: oh, hyang soon title: knowledge, perceptions, and self-reported performance of hand hygiene among registered nurses at community-based hospitals in the republic of korea: a cross-sectional multi-center study date: - - journal: j prev med public health doi: . /jpmph. . sha: doc_id: cord_uid: piust s objectives: to assess the nurses’ hand hygiene (hh) knowledge, perception, attitude, and self-reported performance in small- and medium-sized hospitals after middle east respiratory syndrome outbreak. methods: the structured questionnaire was adapted from the world health organization’s survey. data were collected between june and july , . results: nurses showed scores on knowledge ( . ± . ), perception ( . ± . ), self-reported hh performance of non-self ( . ± . ), self-reported performance of self ( . ± . ), and attitude ( . ± . ). hh performance rate of non-self was y( )= . + . x (hh performance rate of self) (adjusted r( )= . , p< . ). the regression model for performance was y( )= . + . x( ) (peception)+ . x( ) (attitude)+ . x( ) (role model); coefficients were significant statistically except attitude, and this model significant statistically (adjusted r( )= . , p< . ). conclusions: advanced hh education program would be developed and operated continuously. perception, attitude, role model was found to be a significant predictors of hh performance of self. so these findings could be used in future hh promotion strategies for nurses. health care-associated infections (hais) critically impact patient outcomes, increase hospital costs, and extend hospital pissn - eissn - stays [ ] . at any given time, about in inpatients has an hai. this leads to the loss of tens of thousands of lives and costs the us health care system billions of dollars annually [ ] . the primary transmission route of pathogens between patients in hais is via health care workers (hcws)' hands [ ] . thus, hand hygiene (hh) is the single most important factor for preventing hais [ ] . proper hh among hcws is one of the foremost techniques for reducing hais [ , ] . however, the reported rates of hh performance among hcws are low, ranging from . to . %, which is insufficient to satisfactorily prevent hais [ , ] . during the middle east respiratory syndrome (mers) outbreak from may to july , in the republic of korea (hereafter korea), there were confirmed mers cases nationwide [ , ] . we learned several important lessons from the mers outbreak, including that hh is the key to infection control and prevention. thereafter, various types of educational initiatives, campaigns, and training about hh have been implemented nationally, ranging from large cities to small-tomedium cities. in korea, infection control programs have been developed and implemented at university-affiliated hospitals in large cities, and these programs have spread to hospitals in small-tomedium cities [ ] . studies of hh performance and knowledge among hcws have been conducted in larger hospitals with good resources for hh [ ] [ ] [ ] . however, few studies have investigated knowledge and perceptions of hh among hcws in small-to-medium hospitals with relatively limited resources. because of their frequent contact with patients, nurses' proper execution of hh plays an especially important role in the prevention of hais, and nurses should therefore be provided with essential and up-to-date hh information. it has also been established that nurses' knowledge, perceptions, and attitudes about hh influence their hh performance [ ] [ ] [ ] . as such, this study was conducted to assess the status of knowledge, perceptions, and performance of hh among nurses in community-based hospitals in a small-to-medium-sized city in an urban region in korea after the mers outbreak, to identify the factors that influenced their knowledge, perceptions, and performance, and to identify relationships among their knowledge, perceptions, and performance. a cross-sectional design was used to administer a self-reported questionnaire, which took approximately minutes to complete. before the start of the study, the author contacted the hospital directors to explain the purpose of the study and to obtain permission for recruitment. the enrolment criteria were hospitals that were affiliated with teaching hospitals and permitted nurses to participate voluntarily in this study. finally, community-based hospitals affiliated with teaching hospitals located in a small-to-medium-sized city in the south jeolla province of korea were enrolled in this study. we performed a power analysis using g*power version . . . (franz faul, universitat kiel, germany) to determine that a sample size of would be required to achieve a power of . with an effect size of . (a medium effect size for multiple correlations) with an alpha of . . a convenience sample of registered nurses (rns) was recruited from the hospital study sites. the hospitals fully understood the purposes of the study and permitted voluntary recruitment; subjects' participation was voluntary and anonymous. questionnaires were delivered directly to, and later collected from, each hospital. data were collected from june to july , . a total of questionnaires were distributed and were returned (response rate, . %). after excluding incomplete questionnaires, questionnaires were used for the analysis. the questionnaire included domains: (a) hh knowledge, (b) hh perceptions, (c) hh attitudes and role models, and (d) participant demographics and hospital characteristics. the knowledge domain (a) was adapted from the revision of the world health organization (who) hand hygiene knowledge questionnaires for health-care workers, which are composed of questions about the main route of transmission of germs, sources of hais, the timing of hh to prevent transmission of germs to patients or to other healthcare workers, knowledge about handwashing and alcohol-based hand rubs, knowledge about hh methods for clinical situations, and practices for increasing the prevalence of hh [ ] . the items included multiple-choice, true/false, and yes/no (coded as right answer= , wrong answer= ) items, with a total score range of - points (table s ). the perceptions domain (b) was also adapted from the who questionnaire to identify perceptions and performance [ ] . three questions (b , b , and b in table s ) were excluded from the total score because they reduced the reliability of the questionnaire (cronbach alpha, . with all questions; . after exclusion). eleven of the items were on a -to -point scale from 'not effective' to 'very effective', or 'very low' to 'very high', with a total score range of - points. question b was used to assess self-reported hai rates. two other questions (b and b ) were analysed separately as indicators of the self-reported hh performance of non-self and self. the attitudes and role models domain (c) was adapted from hand hygiene knowledge and performance a previous study [ ] . it was a self-report questionnaire consisting of items on a -to -point scale from 'not effective' to 'very effective' [ ] . the total scores ranged from to . hh role models were assessed with items on a -to -point scale from 'do not agree' to 'strongly agree', with a total score range of - points. a higher score in each domain indicated greater knowledge, more positive perceptions, more frequent performance, better attitudes, and higher scores for role models, respectively. the who-based questions were translated into korean and a pilot study was conducted from june to , , during which the translated items were reviewed by nursing professors and rns to assess the content validity and to refine a checklist. pilot study participants were asked to comment on whether the questionnaire items adequately sampled each domain; the questions were accurate, clear, and easy to understand; the instructions were clear and complete; and any of the questions or statements might lead to discord. the suitability of the questionnaire for use was confirmed by the pilot study. participants' time to complete the questionnaire was recorded and within-domain reliability was calculated. demographic variables included participants' age, sex, religion, marital status, education level, clinical work experience (years), department, and position. hospital characteristics included the type of hospital, number of beds, hh guidelines, presence of an infection control department (icd), presence of an infection control nurse (icn), number of hh sinks, number of alcohol-based hand rubs, experiences of hh education within the last year, hh campaigns, hh monitoring and feedback, and mass media information. yes/no answers were coded as 'yes'= , 'no'= . data were analysed using spss version . (ibm corp., armonk, ny, usa), and alpha values < . were considered to indicate statistical significance. the cronbach alpha was calculated to determine reliability. descriptive statistics were calculated. descriptive data for knowledge, perceptions, attitudes, role models, and self-reported hh performance are presented as mean±standard deviation (sd), minimum, maximum, and median. the per-centage of correct answers to each question in the knowledge domain was scaled as high ( % and over), medium ( - %), and low ( % and below). this scale was established after the proportion of correct answers of each question was analysed (table s ) ; the mean and sd were calculated as . and . %, respectively. thus, % was assigned as a medium score, and a high score would be recommended to correspond to the mean or sd; however in this study, that figure would be over %, so % was assigned as the cutoff for a high score. the data were found not to be distributed normally based on the kolmogorov-smirnov test (p< . ). non-parametric univariate statistical analyses were conducted using the mann-whitney u and kruskal-wallis tests. simple linear regression analysis was conducted to confirm the relationship between the self-reported hh performance of self and that of non-self. pearson correlation analysis was conducted to identify associations among knowledge, perceptions, attitudes, and self-reported hh performance of self. multivariate analysis with multiple linear regression with stepwise variable selection was conducted using variables that were confirmed to be statistically significant in the univariate analysis and correlation analysis. the cronbach alpha values were . (domain a, knowledge), . (domain b, perceptions), . (domain c, attitudes) and . (domain c, role models) in this study. these values were . , . , . , and . , respectively, in the pilot study. the study was approved by the institutional review board of sunchon national university ( - -hr- - , - -hr- - ). prior to participating, written informed consent was obtained from each participant; participants were also informed that their consent could be withdrawn at any time during the study. in the hospitals included in this study, there were . ± . beds (mean±sd) and . ± . (mean±sd) hcws, had an icd and had an icn ( had a part-time icn, had full-time icns), and were general hospitals. the demographics and general characteristics of the participants are presented in table . participants' mean±sd score of knowledge was . ± . ; the proportion of high and medium levels of correct answers for knowledge was . % (table ) . some questions showed a low proportion of correct answers, as follows (table s ): "a : what is the most frequent source of germs responsible for health care-associated infections?" ( . %); "a - : hand rubbing causes skin dryness more than hand washing" ( . %); "a - : after exposure to the immediate surroundings of a patient" ( . %); "a - : after exposure to the immediate surroundings of a patient" ( . %); "a - : regular use of a hand cream" ( . %). participants' mean±sd perception score was . ± . (table s ). the following questions about perceptions showed low mean±sd scores: "b . : hh posters are displayed at points of care as reminders" ( . ± . ), "b : what importance does the head of your department attach to the fact that you perform optimal hh?" ( . ± . ), "b : what importance do your colleagues attach to the fact that you perform optimal hh?" ( . ± . ), and "b : what importance do patients attach to the fact that you perform optimal hh?" ( . ± . ). the self-reported hai rate (%) was identified as . ± . (mean±sd). the self-reported hh performance of non-self (other hcws) was . ± . (mean±sd). the self-reported hh performance of self was . ± . (mean±sd); this was highest after body fluid exposure/risk ( . ± . ) and lowest before touching a patient ( . ± . ). participants' mean±sd scores in the attitudes and role models of c domains were . ± . and . ± . (table s ) . some questions on attitudes showed extraordinarily low mean±sd scores. in particular, the scores of "c : hh is convenient." and "c : hh is protective" were . ± . , and . ± . , respectively. some questions on role models showed low mean±sd scores, as follows (table s ) : "cr : i think that the charge nurse is performing according to the hospital's regulations" and "cr : i think that my colleague nurses are performing hh according to the hospital's regulations" both received a score of . ± . . the mean knowledge scores were significantly higher among participants who had received hh education within the past year; those who worked at a hospital with an icd, icn, or hh campaign, or where hh performance was monitored; those who worked in general hospitals; and those whose hospitals employed a full-time icn (compared with those with no icn or a part-time icn) ( table ) . the mean perceptions scores were significantly higher among participants whose hh performance was monitored; those who monitored their colleagues' hh performance; those who had experienced hh campaigns; those who were married; those who had higher education levels; and those who had higher positions ( table ). the mean scores for self-reported hh performance of self were significantly higher among hcws who had received hh education within the past year; those whose hh performance was monitored; those who monitored their colleagues' hh performance; and those who had higher positions (table ) . no independent variables were associated with significant differences in the scores for attitudes or role models in the univariate analyses. the correlation analysis among knowledge, perceptions, attitudes, and self-reported hh performance of self identified significant positive correlations among all categories except knowledge (table ). the model for the self-reported hh performance rates of self and non-self was follows. the hh performance rate of non-self was calculated as y = . + . x (hh performance rate of self), and a significant linear relationship was found (adjusted r = . , p< . ) ( table ). the regression model for knowledge was calculated as y = . + . x (receiving education within the past year)+ . x (icd)+ . x (icn) - . x (type of hospital). the coefficient of each predictor was not statistically significant, but the model as a whole did show statistical significance (adjusted r = . , p< . ) ( table ). the regression model for perceptions was calculated as y = . + . x (hh performance was monitored)+ . x (monitoring colleagues' hh performance)+ . x (hh campaign)+ . x (marital status)+ . x (education level)+ . x (position). the coefficients were not statistically significant, but the model as a whole did show statistical significance (adjusted r = . , p< . ) ( table ). the regression model for self-reported hh performance of self was not calculated by multiple linear regression using the variables found to be significant in the univariate analysis. the regression model for performance was calculated as y = . + . x (perceptions)+ . x (attitudes)+ . x (role model); the coefficients were statistically signifi- in terms of infection control infrastructure [ ] , icds and icns were not fully allocated across the hospitals analysed in this study. the values for numbers of sinks and the placement of alcohol-based hand rub stations in this study were no worse than has been reported in previous studies (in ) of large korean hospitals [ ] . however, as resources for hh, the placement of sinks in every room and alcohol-based hand rub stations by every bed, as well as supplying alcohol-based hand rub to every hcw should be improved continuously. in terms of hh activities, most participants ( . %) had received hh education; however, the scores for other activities such as hh campaign experience and hh monitoring activities were low. these issues can be easily resolved with icd and icn placement [ , ] . such improvements should be made continuously until the korean health care quality standards are satisfied [ ] . the mean score of knowledge among our participants ( . ± . ) was higher than was reported in previous studies ( . ± . [ ] , . ± . [ ] , and . ± . [ ] ) conducted by the same method (who questionnaire). however, the proportion of medium and high levels of correct answers was . %. moreover, serious weaknesses in knowledge were found in response to the following questions: "what is the most frequent source of germs responsible for health care-associated infections?", "hand rubbing causes skin dryness more than hand washing", "a - : after exposure to the immediate surroundings of a patient", "a - : after exposure to the immediate surroundings of a patient", and "regular use of a hand cream". therefore, hh education programs should be promptly reviewed, and systemic and advanced hh education and training programs must be developed and implemented to enhance hh knowledge broadly, not just focusing on these specific knowledge questions. the perceptions in this study ( . ± . ; total score, ) were somewhat higher than observed in a previous study ( . ± . ; total score, ) [ ] . however, some perceptions-related items that received low scores should be improved, because perceptions have been shown to be significant predictors of nurses' hh intentions and adherence [ ] , and an important predictor of hh performance [ , ] . self-reported hh performance of self was highest after body fluid exposure/risk and lowest before touching a patient. this finding corresponds to those of previous observational studies [ , ] and is consistent with a previous self-reported performance study [ ] . interestingly, the relationship between the hh performance of self and that of non-self was positive and linear. this finding is also consistent with a previous report [ ] . participants evaluated the hh performance of non-self at . times the hh performance of self. attitudes about hh were relatively poor in responses to both "hh is convenient" and "hh is protective". these findings may represent barriers to maintaining good hh, and also demonstrate the need for strategies to promote the perceived convenience and protectiveness of hh [ ] . in the role model domain, "it is important for my colleagues to perform hh according to the hospital's regulations" showed high endorsement, and nursing colleagues were identified as the most important hh role models. these results are consistent with previous studies [ ] [ ] [ ] , ] . moreover, the perception of being a role model for one's colleagues can be used to improve hh compliance [ , ] . according to the multiple linear regression analysis, receiving education within the past year (yes), having an icd (yes), and having an icn (yes) positively affected knowledge, while type of hospitals negatively affected knowledge. hh performance being monitored (yes), monitoring colleagues' hh performance (yes), and the presence of an hh campaign (yes) positively affected perceptions. therefore, these findings can be used to improve knowledge and perceptions. as this study was not an observational study of hh performance, this study has some limitations in terms of the self-reporting of hh performance. the regression model of self-reported hh performance of self showed increases with . (perceptions)+ . (attitudes)+ . (role models). knowledge was excluded from this model. consistently with previous studies [ , , , ] , our participants' self-reported hh performance rate of self was positively correlated with their scores for perceptions, attitudes, and role models. an explanation for this is that general perceptions of hh [ , , ] and the perception of being a role model for one's colleagues [ , ] are very important for improving hh compliance among hcws [ ] . as such, these findings could be used in future hh promotion strategies for nurses. in this study, the hh knowledge, perceptions, attitudes, and role models of rns in community-based hospitals in a small-to-medium urban area were characterized. the presence of some items with relatively low scores revealed some room for improvements in knowledge. receiving education within the past year, having an icd, and having an icn were found to be related to knowledge. in addition, hh campaigns and monitoring were associated with perceptions. the self-reported hh performance rate of self was associated with perceptions, attitudes, and role models. world health organization. who guidelines on hand hygiene in health care: first global patient safety challenge clean care is safer care office of disease prevention and health promotion evidence-based model for hand transmission during patient care and the role of improved practices point of care hand hygiene-where's the rub? a survey of us and canadian health care workers' knowledge, attitudes, and practices mers outbreak in korea: hospital-to-hospital transmission middle east respiratory syndrome coronavirus (mers-cov) outbreak in south korea, : epidemiology, characteristics and public health implications national survey of the status of infection surveillance and control programs in acute care hospitals with more than beds in the republic of korea knowledge and beliefs about hand hygiene among hospital nurses hand hygiene compliance of healthcare workers in a children's hospital p : knowledge and perception toward hand hygiene among health-care workers in teaching hospital perceptions, attitudes, and behavior towards patient hand hygiene key beliefs of hospital nurses' hand-hygiene behaviour: protecting your peers and needing effective reminders improved hand hygiene compliance is associated with the change of perception toward hand hygiene among medical personnel world health organization. hand hygiene knowledge questionnaire for health-care workers world health organization. perception survey for health-care workers study on the efficacy of nosocomial infection control (senic project): results and implications for the future the status of healthcare-associated infection control among healthcare facilities in korea development and application of evaluation indices for hospital infection surveillance and control programs in the republic of korea reference material knowledge, attitudes, and practices study on hand hygiene among imam hossein hospital's residents in understanding the determinants of australian hospital nurses' hand hygiene decisions following the implementation of a national hand hygiene initiative nurses and physicians' perceptions of the importance and impact of healthcare-associated infections and hand hygiene: a multi-center exploratory study in hong kong analysis of hand hygiene practices of health care personnels understanding healthcare workers self-reported practices, knowledge and attitude about hand hygiene in a medical setting in rural india importance of structured training programs and good role models in hand hygiene in developing countries nurses' knowledge regarding hand hygiene and its individual and organizational predictors this research was supported by sunchon national university funds ( ). the author has no conflicts of interest associated with the material presented in this paper. key: cord- - ubzr f authors: grasso, daniel; renna, felipe javier; vaccaro, maria ines title: initial steps in mammalian autophagosome biogenesis date: - - journal: front cell dev biol doi: . /fcell. . sha: doc_id: cord_uid: ubzr f during the last decade, autophagy has been pointed out as a central process in cellular homeostasis with the consequent implication in most cellular settings and human diseases pathology. at present, there is significant data available about molecular mechanisms that regulate autophagy. nevertheless, autophagy pathway itself and its importance in different cellular aspects are still not completely clear. in this article, we are focused in four main aspects: (a) induction of autophagy: autophagy is an evolutionarily conserved mechanism induced by nutrient starvation or lack of growth factors. in higher eukaryotes, autophagy is a cell response to stress which starts as a consequence of organelle damage, such as oxidative species and other stress conditions. (b) initiation of autophagy; the two major actors in this signaling process are mtor and ampk. these multitasking protein complexes are capable to summarize the whole environmental, nutritional, and energetic status of the cell and promote the autophagy induction by means of the ulk -complex, that is the first member in the autophagy initiation. (c) ulk -complex: this is a highly regulated complex responsible for the initiation of autophagosome formation. we review the post-transductional modifications of this complex, considering the targets of ulk . (d)the mechanisms involved in autophagosome formation. in this section we discuss the main events that lead to the initial structures in autophagy. the becn -complex with pi k activity and the proper recognition of pi p are one of these. also, the transmembrane proteins, such as vmp and atg , are critically involved. the membrane origin and the cellular localization of autophagosome biogenesis will be also considered. hence, in this article we present an overview of the current knowledge of the molecular mechanisms involved in the initial steps of mammalian cell autophagosome biogenesis. during the last decade, autophagy has been pointed out as a central process in cellular homeostasis with the consequent implication in most cellular settings and human diseases pathology. at present, there is significant data available about molecular mechanisms that regulate autophagy. nevertheless, autophagy pathway itself and its importance in different cellular aspects are still not completely clear. in this article, we are focused in four main aspects: (a) induction of autophagy: autophagy is an evolutionarily conserved mechanism induced by nutrient starvation or lack of growth factors. in higher eukaryotes, autophagy is a cell response to stress which starts as a consequence of organelle damage, such as oxidative species and other stress conditions. (b) initiation of autophagy; the two major actors in this signaling process are mtor and ampk. these multitasking protein complexes are capable to summarize the whole environmental, nutritional, and energetic status of the cell and promote the autophagy induction by means of the ulk -complex, that is the first member in the autophagy initiation. (c) ulk -complex: this is a highly regulated complex responsible for the initiation of autophagosome formation. we review the post-transductional modifications of this complex, considering the targets of ulk . (d)the mechanisms involved in autophagosome formation. in this section we discuss the main events that lead to the initial structures in autophagy. the becn -complex with pi k activity and the proper recognition of pi p are one of these. also, the transmembrane proteins, such as vmp and atg , are critically involved. the membrane origin and the cellular localization of autophagosome biogenesis will be also considered. hence, in this article we present an overview of the current knowledge of the molecular mechanisms involved in the initial steps of mammalian cell autophagosome biogenesis. keywords: autophagy regulation, mtor, ampk, ulk , vmp there are three types of autophagy, processes where cytoplasmic components are delivered to lysosomes for degradation: microautophagy/endosomal microautophagy galluzzi et al., ) , chaperone-mediated autophagy (cma) (cuervo and wong, ; kaushik and cuervo, ) and macroautophagy (hereafter mentioned as autophagy). this is the engulfment of cytoplasmic contents by a double membrane vesicle, named autophagosome. the outer membrane of the autophagosome eventually fuses with the lysosome, where the inner vesicle is delivered (figure ) . here we present a brief overview of the mechanisms involved in the initial steps of mammalian cell autophagosome biogenesis. the main task of autophagy is to deal against poor nutrient environments. in superior eukaryote cells, mtor, which is a serine/threonine kinase, checks the presence of growth factors and nutrients. in presence of amino acids (mainly leucine, glutamine and arginine), mtorc maintains the autophagy inhibition. when nutrients are no longer available, the inhibition of mtorc releases the 'brake' and autophagy is eventually induced (carroll et al., ) . growth factors negatively regulate the autophagy by activation of mtor. activation of the insulin receptor induces the phosphorylation of tsc , avoiding the tsc / complex formation and the mtorc inhibition (haeusler et al., ) . other growth factors induce the ras pathway, which activates the erk / dimer that inhibits the tsc / complex and phosphorylates raptor activating mtorc and suppressing autophagy (carriere et al., ) . ampk is a key serine/threonine kinase that is activated in low energy conditions (egan et al., ) . then, ampk activates the autophagosome formation by mean of direct and indirect ways. furthermore, ampk can be activated by camkkb in the eroverloaded response (hoyer-hansen et al., ) . the unfolded protein response, by mean of ire α, perk and atf , is also an autophagy triggering event, enhancing lc conjugation (ding et al., ; kouroku et al., ) . during quick and intense oxygen fluctuations, autophagy is induced by mtorc -dependent pathways and/or by er stress. abbreviations: ambra , activating molecule in becn -regulated autophagy protein ; ampk, amp-activated kinase; ap- , adaptor protein ; atf , activating transcription factor ; atg, autophagy related gen or protein; bcl- , b-cell lymphoma ; becn , coiled-coil moesin-like bcl -interacting protein; bh , bcl- homology ; bip, binding immunoglobulin protein; bnip , bcl interacting protein ; camkkb, calcium/calmodulin-dependent protein kinase kinase ; copii, coat complex protein ii; ctages , cutaneous t-cell linphomaassociated antigen ; cul , cullin- ; dapk, death-associated protein kinase; deptor, dep domain containing mtor-interacting protein; dfcp , double fyve containing protein ; ep , histone acetyltransferase p ; erk / , mitogen-activated protein kinase; esyt, extended synaptotagmin; fip , fak family-interacting protein of kda (also known as rb cc ); foxo , forkhead box protein o ; gsk , glycogen synthase kinase ; hif α, hypoxia-inducible factor alpha; horma, hop/rev /mad domain; idr, intrinsically disordered region; ire α, inositol-requiring enzyme alpha; jnk, c-jun n-terminal kinases; kap , e sumo-protein ligase trim ; khlh , kelch-like protein ; lc , microtubule-associated proteins a/ b light chain b (also known as map lc b); lir, lc -interacting region; lkb , serine/threonine-protein kinase stkb ; mit, microtubule interacting and trafficking domain; mlst , mammalian letal with sec protein ; nedd , neural precursor cell expressed developmentally down-regulated protein ; nedd l, neural precursor cell expressed developmentally downregulated gene -like; nrf , nuclear factor erythroid -related factor ; pdk , -phosphoinositide-dependent protein kinase ; perk, proline-rich receptor-like protein kinase; pi k, phosphatidylinositol -kinase; pi p, phosphatidylinositol -phosphate; pkcδ, protein kinase c delta type; pras , proline-rich akt substrate of kda; proppin, β-propeller that bind polyphosphoinositides; rab, ras-related protein; raptor, regulatoryassociated protein of mtor; rheb, ras homolog enriched in brain; ros, reactive oxygen species; serca, sarco/endoplasmic reticulum ca +-atpase; sirt , sirtuin ; sqstm , sequestosome (also known as p ); stx , sintaxin ; sumo, small ubiquitin-like modifier; tfeb, transcription factor eb; tip , kda tat-interactive protein; mtor, mammalian target of rapamycin; traf , tumor necrosis factor receptor (tnfr)-associated factor ; trappiii, transport protein particle (trapp) iii complex; tsc / , tuberous sclerosis / ; ulk , unc- -like kinase ; uvrag, uv radiation resistance associated protein; vmp , vacuole membrane protein ; vps, vacuolar protein sorting; wipi, wd repeat domain phosphoinositide-interacting protein. (papandreou et al., ; rouschop et al., ) . in moderate but chronic hypoxia, autophagy is triggered mainly by hif α and pkcδ-jnk pathways (mazure and pouyssegur, ) . hif α is the major transcription factor involved in cell response to hypoxia (brocato et al., ) . among the genes transcribed by hif α is bnip which disrupts the bcl -becn interaction releasing becn to be part of the autophagy process (zhang et al., ) , and vmp , which interacts with becn and is required for autophagosome formation (ropolo et al., ; rodriguez et al., ) . regarding to the pkcδ pathway, this kinase activates jnk that in turn phosphorylates bcl to release it from becn (pattingre et al., ) . oxidative stress induces autophagy in order to recycle damaged mitochondria (and other damaged organelles), and eliminate proteins aggregates (ureshino et al., ) . nrf is bound to antioxidant response elements promoting the transcription of p , a cargo receptor for autophagy (puissant et al., ) . foxo induces the expression of lc (an atg protein that is described below) and bnip (mahalingaiah and singh, ) . finally, ros inhibit atg -mediated lc delipidation, that takes place immediately after formation of the autolysosome, conferring stability to lc and favoring its recruitment to the autophagosome (scherz-shouval et al., ) . independently of the induction agent, in canonical autophagy, the initiation of autophagosome biogenesis is managed by the kinases mtor and ampk. in fact, through the association with raptor, deptor, pras and mlst , mtor constitutes the complex [mtorc ]. at basal conditions, mtorc is stimulated by the small gtpase rheb. in turn, mtor triggers cell growth and diverse anabolic processes such as lipids, proteins and nucleotides synthesis (lamb et al., ; klionsky and schulman, ) . on the other hand, active mtorc abolishes most of catabolic processes including the autophagy (lamb et al., ; klionsky and schulman, ; figure b) . therefore, mtor inhibits autophagy, by several phosphorylations on the first complex of the pathway (see further), when optimal nutrients concentration is available. during starvation, rheb is inhibited by the tsc / heterodimer removing the activation stimulus on mtor (huang and manning, ) . this inhibition of mtorc decreases its influence on autophagy and as a consequence, the mechanism of autophagosome biogenesis is triggered (carroll et al., ; figure e ). moreover, the inactivation of mtorc allows that the dephosphorylated tfeb translocates to the nucleus (puertollano et al., ) where it induces the transcription of atg genes, such as uvrag, wipi, maplc b, sqstm , vps , vps , and atg b. tfeb also promotes the lysosomal function in the cell (settembre et al., ) . ampk is a heterotrimeric complex composed by a catalytic α subunit and two regulatory subunits, β and γ (egan et al., ) . since ampk is activated in low energy conditions, this kinase inhibits anabolic processes, and induces catabolic pathways, such figure | (a) schematic overview of autophagy. ukl activation leads to autophagosome biogenesis. on the er surface, the transmembrane protein vmp recruits a pi k complex. the consequent pi p subdomain is recognized by dfcp on the omegasome structure. then, in the isolation membrane, wipi proteins recruit the atg -atg -atg complex which in turn make possible the lipidation of lc on the membrane. the formation of autophagosome, a double membrane vesicle, allows the carrying of cargo to lysosome. eventually, cargo is degraded in the resulted autophagolysosome. er, endoplasmic reticulum; pi k, phosphatidylinositol -kinase; pi p, phosphatidylinositol ( , , ) triphosphate (pi p). as autophagy (egan et al., ; zhang et al., ; figure b) . amp binding allows lkb to phosphorylate ampk (thr ) (xiao et al., ; zhang et al., ) , which in turn directly and indirectly activates the autophagosome formation as is explained in the next sections. ulk is so far the first complex in the core molecular machinery involved in the biogenesis of autophagosomes. this complex is composed by the serin/threonin protein kinase ulk , atg , fip , and atg . activated ulk is capable of triggering series of phosphorylations that enable the nucleation process and autophagosome biogenesis. at n-terminal ulk is the kinase domain followed by a disordered region that is postulated as highly regulated. on the opposite side, there are two mit domains in tandem that compose a globular structure (noda and fujioka, ) . ulk structure was characterized in complex with atg . on the c-terminal of atg there are two mit-interacting motifs in a helical region for recognitioninteraction with the ulk mit domains (noda and fujioka, ; qi et al., ) . additionally, both proteins, ulk and atg , have a lir domain for interaction with lc family members. atg , the smallest member of the complex, is essential for autophagy (mercer et al., ) . atg is almost fully composed by a horma domain with direct interaction with the horma domain at the n-terminus of atg . atg stabilizes atg and ulk (mercer et al., ; suzuki et al., ) and seems to recruit downstream molecules through its wf finger motif (suzuki et al., ) . the last member of ulk -complex is fip , that is the largest molecule involved in this complex (hara et al., ; figures c,d) . ulk complex is regulated by the two major key proteins related to nutritional and energetic sensing, mtor and ampk (he and klionsky, ) . under growth factors stimulation and nutrient availability, the activated mtorc interacts with ulk through raptor and phosphorylates several sites of ulk (ser / in mouse, ser in human) (alers et al., ) and atg (ser in mouse) subunits (kim et al., ; puente et al., ) . then, ulk complex remains inactivated and autophagy repressed. ampk induces ulk -mediated autophagy by three strategies: -ampk phosphorylates tsc at ser enhancing the activity of this mtorc inhibitor (inoki et al., ) . -ampk is able to inhibit mtorc activity directly by phosphorylation of raptor in ser / (gwinn et al., ; egan et al., ) . -ampk interacts with and phosphorylates ulk in ser / for its activation (kim et al., ; figure e ). another pathway for ulk autophagy activation has been proposed: ambra may act as a bridge between ulk and the ubiquitin ligase e traf (nazio et al., ; grumati and dikic, ) . traf -mediated poly ubiquitination, k type branched ubiquitin, potentiates autophagy activation by promoting stabilization and self-association of ulk . this event initiates a positive loop, where ulk phosphorylates ambra enhancing traf -mediated ulk ubiquitination (nazio et al., ; grumati and dikic, ) . further, growth factors withdrawal might induce the activation of tip by gsk -mediated phosphorylation at ser . tip is an acetyltransferase that induces the activation of ulk by acetylation of lys / enhancing the triggering of autophagy (lin et al., ) . once activated, ulk is able to phosphorylate several substrates. among them, there are two initial complexes, the ulk complex itself and the pi kc complex (pi kc -c ). in the first complex, ulk phosphorylates to itself (thr / , ser ) (bach et al., ) , and the other members of the complex, atg (ser / ), fip (ser / / ) and atg (ser / ) (lin and hurley, ; orhon and reggiori, ; figure e ). in the second complex, ulk potentiates the pi k activity of the catalytic subunit vps , by the phosphorylation of two members of the complex, becn (ser ) and atg l (ser ), resulting in the increment of pi p production (russell et al., ) . following to ulk complex activation, the transmembrane protein vmp interacts with the bh domain of becn through its atg domain, recruiting the pi kc -c to the autophagosomal membrane (molejon et al., ) . there are two main pi kc complexes in autophagosome biogenesis. the complex is composed by becn , atg l, vps and vps , which is a key component in autophagosome initiation. the other complex, pi kc -c , is related to autophagosome maturation and endosomal trafficking and is composed by the same members except for the regulatory protein atg l which is replaced by uvrag. structurally, the pi kc -c is stabilized in pairs, becn /atg l and vps /vps (stjepanovic et al., ) . upon autophagy induction, becn recruitment induces the complex assembly, through the adaptor atg l, where the wd domain of vps organizes the proteins into the complex allowing the activity of vps (stjepanovic et al., ) . moreover, the kap -mediated sumoylation of vps enhances the interaction of this protein with the rest of the complex (yang et al., ) . as it was commented before, ulk -mediated phosphorylation of becn , atg l and vps potentiates pi k activity in this complex. the tumor suppressor dapk, a calcium/calmodulin serine/threonine kinase, also contributes to the pi kc -c recruitment to the autophagosome membrane. this kinase phosphorylates becn on its bh domain interfering with the becn -bcl-xl association and releasing becn (zalckvar et al., ). this effect is reaffirmed by traf which ubiquitinates becn on the same region (shi and kehrl, ) . recently, it has been proposed that vps activity may be switched on/off by an ep -dependent acetylation/deacetylation on k , as another regulation of the pi kc -c (su and liu, ; . the cascade of subsequent activations of ulk and pi kc -c complex members is limited by a series of degradative processes. the deubiquitinase a (dub a ) controls becn participation on autophagosome formation by elimination of poly ubiquitin chain in the bh domain placed by atf e ligase. beyond that regulation, the e ligases nedd and nedd l induce degradation of key members in ulk , and vps complexes respectively (platta et al., ; nazio et al., ) . becn is poly ubiquitinated with k -linked ubiquitin chain by nedd to be eliminated in the proteasome. similar activity is carried out by nedd l on ulk targeting this protein with k -and k -linked ubiquitin chains. in both cases, the proteasome-mediated elimination of those proteins causes the destabilization of its respective complexes. in a redundant way of labeling for degradation, the poly ubiquitination with k -linked ubiquitin chains on ulk , becn , and vps is catalyzed by the complex cul -khlh (liu and chen, ) . the local enrichment of pi p in er-subdomains acts as the signal for the nucleation of several autophagy-related proteins in a structure named omegasome that resembles the greek letter omega (ktistakis and tooze, ) . the first protein which recognizes the pi p is dfcp . dfcp possesses a diffuse pattern over the er, mitochondria and golgi but it is rapidly mobilized to the pi p spots by the recognition of this phospholipid with the two fyve motifs of its structure. although it is a marker of omegasome, little is known about its role during the initial steps of autophagosome biogenesis. additionally, the dfcp depletion does not seem to interfere with the progression of autophagy. the rising omegasome leads to extension of a sack-like structure named isolation membrane or phagophore. wipi b, a member of the proppin family, recognizes the local pi p by the frrg motif of its wd -repeat β-propeller on the isolation membrane (nascimbeni et al., a) . the process continues with two ubiquitin like systems: atg and lc . cytoplasmic atg is covalently attached to a c-terminal glycine of atg . this catalytic reaction resembles the ubiquitination process where atg and atg are subrogated to e and e enzymes, respectively (klionsky and schulman, ) . atg -atg complex is highly important, since it functions as e enzyme for lc conjugation to phosphatidylethanolamine (pe) on the autophagosomal membrane. this process seems to be mediated by atg l, which is composed by a wd -repeat β-propeller domain localized in the c-terminal sequence. at n-terminal sequences, atg l possesses a binding domain that allows the interaction with atg to eventually form the atg -atg -atg l complex (wilson et al., ) . the middle sequence of atg l expands a coil-coil (cc) dimerization domain that induces the formation of atg l dimers (wilson et al., ) . then, wipi b is recognized by a region of atg l, between the cc-dimerization domain and the wd -repeated β-propeller domain. consequently, the atg -atg -atg l complex is recruited to the isolation membrane. lc plays a central role in autophagy being involved in vesicle elongation, maturation, fusion of autophagosome-lysosome and even as an adaptor to cargo recognition (nakatogawa et al., ; lee and lee, ) . lc shows a diffuse pattern distributed over the cytoplasm and into the nucleus (known as lc -i) in basal conditions. upon autophagy triggering, lc is deacetylated in the nucleus by sirt (huang et al., ) and is cleaved in cytoplasm by atg b, which eliminates the c-terminal arginine residue to expose a glycine (satoo et al., ; maruyama and noda, ) . in an ubiquitin-like reaction, the exposed glycine is combined to form a thioester bound, first with atg (e -like enzyme) and then with atg (the e -like enzyme) (satoo et al., ; maruyama and noda, ) . atg is recognized by atg of the atg -atg -atg l complex which has been already recruited to isolation membrane through wipi b. the atg -atg -atg l complex functions as the e enzyme leading the formation of an amide bound with the amine headgroup of pe otomo et al., ; dooley et al., ) . the lipidated lc (lc -ii) is present at the isolation membrane and on the autophagosome, in both sides of the membrane. the arrival of autophagosome to the lysosome is a fusion dependent mechanism of the hops complex, through stx (jiang et al., ) , and rab (gutierrez et al., ) . since lc is present in both membranes of autophagosome, once exposed to lysosomal hydrolases, there is a pool of lc that is degraded with cargo. however, the lc localized in the external membrane is cleaved from the pe, by atg b, and then recycled. otomo et al., ; dooley et al., ) . furthermore, of which is explained above, autophagy is able to follow unconventional pathways. er-stress or glucose influx after starvation in nih t , can induce autophagy independent of mtor inhibition and where ampk activation is not essential (corona velazquez and jackson, ) . moreover, the glucose influx in mouse embryonic fibroblast can trigger autophagy independent of ulk / . starved chicken dt cells show an autophagy dependent of atg -fip interaction but independent of ulk . similar behavior is observed in some viral infection, such as coronaviruses, hbv or poliovirus, which induce a non-degradative ulk -independent form of autophagy. even more interesting is that the oleate fatty acid can induce an autophagy mechanism that lacks of pi p synthesis, since it cannot be inhibited by knocking-down of becn , vps , or atg . these examples suggest that autophagy is flexible and the pathways in autophagosome biogenesis may adapt to different situations depending on the inductor and the biological context (corona velazquez and jackson, ). it is accepted that the initial structure related to autophagy is located on the er. the data suggest that ulk complex translocates to phosphatidylinositol-enriched er-subdomains and then, the membrane structure is fed by atg a-containing vesicles (nishimura et al., ) . then, autophagosomes are formed in highly active er-subdomains where lipidic interchange between er and other cytoplasmic organelles occurs. two sites of autophagosome biogenesis have been recently demonstrated: the er-plasma membrane contact site (er-pm) and the er-mitochondria contact site (hamasaki et al., ; nascimbeni et al., b) . vmp is a key player in the biogenesis of autophagosomes that remains in the autophagosomal membrane (grasso et al., ) . vmp -becn interaction allows the recruitment of pi kc -c to the er-pm contact site by the interaction with the proteins esyt , , and (nascimbeni et al., b) . moreover, vmp was suggested to also regulate the er-mitochondria contact site during autophagy and to be involved in the release of the initial autophagosome vesicle by activation of serca pump (tabara and escalante, ; zhao et al., ) . the transmembrane protein atg a is in golgi and endosomal system, in early and late endosomes with a minimal percentage of recycling ones (feng and klionsky, ) . in starvation, the trappiii complex, related to er-golgi vesicular trafficking, mobilizes atg a vesicles to the sites of nascent autophagosomes (shirahama- noda et al., ) . the adaptor protein ap- is required for this event, since it mediates the trafficking of atg a from trans-golgi network to the site of autophagosomes maturation (mattera et al., ) . this event would potentiate the expansion of the isolation membrane. nevertheless, the contribution of this membrane by the atg a vesicles is not enough to explain the growth of the membrane itself. moreover, atg seems to take a distinctive role in different systems. in contrast to mammals, yeast atg has a fundamental role at very early steps in the pre-autophagosomal structure. on the other hand, in plants, the depletions of arabidopsis atg still allows formation of autophagosomal structures supplemented with atg (lc ortholog) suggesting divergent regulation and mechanisms of this types of vesicles (zhuang et al., ) . ribosomes-free regions specialized in er-golgi communication are present in the rough er. vesicles arise targeted to the golgi from these areas, described as er-exit sites (eres). these vesicles are supplemented by the proteins sar , sec , sec , sec and sec , that constitute the copii coat (zahoor and farhan, ) . before reaching golgi, the copiicoated vesicles go through an intermediated structure named er-golgi intermediate compartment (ergic) (ben-tekaya et al., ) . the function of these structures is not completely understood, but they might participate in the autophagosome biogenesis. an impairment of these compartments causes an autophagy downregulation (karanasios et al., ; zahoor and farhan, ) . data suggest that the bulk contribution for the growth of the autophagosome membrane comes from the er-golgi vesicular trafficking. during starvation, the fip -ctages interaction induces the remodeling and enlargement of eres positives for sec (ge et al., ) . this allows the production of copiicoated vesicles that are released to contribute to autophagosome formation. moreover, ulk phosphorylates sec a, a member of the copii multiprotein complex. this event is related to morphological variations on eres during starvation and might turn the secretory machinery from anabolic to catabolic state. a recent work shows a previously unexpected key role of rab a-positive membranes in autophagosome biogenesis (puri et al., ) . they demonstrated that wipi relies, beyond the recognition of pi p, in the interaction with rab a for recruitment of atg l. also, the authors suggest a model where isolation membrane is represented by rab apositive membrane, likely to be recycling endosomes. in this context, rab a-positive membranes constitute the platform for autophagosome formation initial steps. the initial molecular steps in autophagosome biogenesis are determined by three mains complexes: ulk complex; pi kc -c ; and atg l -atg -atg which eventually favors lc lipidation in the growing isolation membrane. lc family seems to play a relevant role in cargo recognition, autophagosome closure and fusion with lysosomes. however, while the initial molecular steps seem to be essential and well-known in canonical autophagy, the subsequent events in mammalian autophagosome biogenesis are less characterized. moreover, the wide spectrum of autophagy-related events and the number of molecules involved (table ) leads to the concept that different pathways might account for diverse types of autophagy and may reveal different functions of autophagy in physiological and pathological cellular processes. furthermore, the meaning of different origins and composition of the autophagosomal membrane, such as those supplied by atg a and cop-ii vesicles (feng and klionsky, ) , are still not fully understood. moreover, autophagosome biogenesis is regulated by a variety of signaling pathways through posttranslational modification, such as phosphorylations, ubiquitinations, sumoylations and acetylation, that may account for diverse conditions, functions or selectivity. furthermore, this molecular regulation, that are eminently druggable, may be relevant in the development of therapeutic strategies of autophagy modulation for complex pathologies such as cancer (galluzzi et al., ) or neurodegenerative diseases (zare-shahabadi et al., ) . although there are many aspects still unclear on mammalian autophagosome biogenesis, future findings that shed light on this sophisticated intracellular process can be taken for granted. role of ampk-mtor-ulk / in the regulation of autophagy: cross talk, shortcuts, and feedbacks the serine/threonine kinase ulk is a target of multiple phosphorylation events live imaging of bidirectional traffic from the ergic molecular responses to hypoxiainducible factor alpha and beyond erk / phosphorylate raptor to promote ras-dependent activation of mtor complex (mtorc ) control of tsc -rheb signaling axis by arginine regulates mtorc activity so many roads: the multifaceted regulation of autophagy induction chaperone-mediated autophagy: roles in disease and aging differential effects of endoplasmic reticulum stress-induced autophagy on cell survival wipi b links ptdins p to lc lipidation through binding atg l phosphorylation of ulk (hatg ) by amp-activated protein kinase connects energy sensing to mitophagy autophagic membrane delivery through atg molecular definitions of autophagy and related processes autophagy in malignant transformation and cancer progression remodeling of er-exit sites initiates a membrane supply pathway for autophagosome biogenesis zymophagy, a novel selective autophagy pathway mediated by vmp -usp x-p , prevents pancreatic cell death ubiquitin signaling and autophagy rab is required for the normal progression of the autophagic pathway in mammalian cells ampk phosphorylation of raptor mediates a metabolic checkpoint biochemical and cellular properties of insulin receptor signalling autophagosomes form at er-mitochondria contact sites fip , a ulk-interacting protein, is required for autophagosome formation in mammalian cells regulation mechanisms and signaling pathways of autophagy control of macroautophagy by calcium, calmodulindependent kinase kinase-beta, and bcl- the tsc -tsc complex: a molecular switchboard controlling cell growth deacetylation of nuclear lc drives autophagy initiation under starvation tsc mediates cellular energy response to control cell growth and survival the hops complex mediates autophagosome-lysosome fusion through interaction with syntaxin autophagy initiation by ulk complex assembly on er tubulovesicular regions marked by atg vesicles the coming of age of chaperone-mediated autophagy ampk and mtor regulate autophagy through direct phosphorylation of ulk dynamic regulation of macroautophagy by distinctive ubiquitin-like proteins er stress (perk/eif alpha phosphorylation) mediates the polyglutamine-induced lc conversion, an essential step for autophagy formation digesting the expanding mechanisms of autophagy the autophagosome: origins unknown, biogenesis complex role of the mammalian atg /lc family in autophagy: differential and compensatory roles in the spatiotemporal regulation of autophagy microautophagy: lesser-known self-eating structure and function of the ulk complex in autophagy gsk -tip -ulk signaling pathway links growth factor deprivation to autophagy klhl links the ubiquitin-proteasome system to autophagy termination chronic oxidative stress increases growth and tumorigenic potential of mcf- breast cancer cells autophagy-regulating protease atg : structure, function, regulation and inhibition ap- mediates export of atg a from the trans-golgi network to promote autophagosome formation hypoxia-induced autophagy: cell death or cell survival? a novel, human atg binding protein, atg , interacts with ulk and is essential for macroautophagy the vmp -beclin interaction regulates autophagy induction atg , a ubiquitin-like protein required for autophagosome formation, mediates membrane tethering and hemifusion local detection of ptdins p at autophagosome biogenesis membrane platforms er-plasma membrane contact sites contribute to autophagosome biogenesis by regulation of local pi p synthesis fine-tuning of ulk mrna and protein levels is required for autophagy oscillation mtor inhibits autophagy by controlling ulk ubiquitylation, selfassociation and function through ambra and traf autophagosome formation is initiated at phosphatidylinositol synthase-enriched er subdomains atg family kinases in autophagy initiation structure of the atg -atg conjugate reveals a platform for stimulating atg -pe conjugation assays to monitor autophagy progression in cell cultures structure of the human atg ∼atg conjugate required for lc lipidation in autophagy hypoxia signals autophagy in tumor cells via ampk activity, independent of hif- , bnip , and bnip l role of jnk -dependent bcl- phosphorylation in ceramide-induced macroautophagy nedd -dependent lysine- -linked polyubiquitination of the tumour suppressor beclin nutrient-regulated phosphorylation of atg inhibits starvation-induced autophagy the complex relationship between tfeb transcription factor phosphorylation and subcellular localization when autophagy meets cancer through p /sqstm the rab a-positive compartment is a primary platform for autophagosome assembly mediated by wipi recognition of structure of the human atg -atg horma heterodimer: an interaction hub within the ulk complex a novel hif- alpha/vmp -autophagic pathway induces resistance to photodynamic therapy in colon cancer cells the pancreatitis-induced vacuole membrane protein triggers autophagy in mammalian cells the unfolded protein response protects human tumor cells during hypoxia through regulation of the autophagy genes map lc b and atg ulk induces autophagy by phosphorylating beclin- and activating vps lipid kinase the structure of atg b-lc complex reveals the mechanism of lc processing and delipidation during autophagy reactive oxygen species are essential for autophagy and specifically regulate the activity of atg tfeb links autophagy to lysosomal biogenesis traf and a regulate lysine -linked ubiquitination of beclin- to control tlr -induced autophagy trappiii is responsible for vesicular transport from early endosomes to golgi, facilitating atg cycling in autophagy unveiling the role of vps kinase domain dynamics in regulation of the autophagic pi k complex pik c /vps control by acetylation vps acetylation controls its lipid kinase activity and the initiation of canonical and non-canonical autophagy structure of the atg -atg complex reveals essential roles of atg in autophagy initiation vmp establishes er-microdomains that regulate membrane contact sites and autophagy calcium signaling alterations, oxidative stress, and autophagy in aging wipi b and atg l : setting the stage for autophagosome formation structural basis for amp binding to mammalian amp-activated protein kinase acetylated hsp and kap -mediated vps sumoylation is required for autophagosome creation in autophagy crosstalk of autophagy and the secretory pathway and its role in diseases dap-kinase-mediated phosphorylation on the bh domain of beclin promotes dissociation of beclin from bcl-xl and induction of autophagy autophagy in alzheimer's disease mitochondrial autophagy is an hif- -dependent adaptive metabolic response to hypoxia amp as a low-energy charge signal autonomously initiates assembly of axin-ampk-lkb complex for ampk activation the erlocalized transmembrane protein epg- /vmp regulates serca activity to control er-isolation membrane contacts for autophagosome formation atg regulates autophagosome progression from the endoplasmic reticulum in arabidopsis the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © grasso, renna and vaccaro. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - w bxqx authors: casadei, elisa; salinas, irene title: comparative models for human nasal infections and immunity date: - - journal: dev comp immunol doi: . /j.dci. . . sha: doc_id: cord_uid: w bxqx the human olfactory system is a mucosal surface and a major portal of entry for respiratory and neurotropic pathogens into the body. understanding how the human nasopharynx-associated lymphoid tissue (nalt) halts the progression of pathogens into the lower respiratory tract or the central nervous system is key for developing effective cures. although traditionally mice have been used as the gold-standard model for the study of human nasal diseases, mouse models present important caveats due to major anatomical and functional differences of the human and murine olfactory system and nalt. we summarize the nalt anatomy of different animal groups that have thus far been used to study host-pathogen interactions at the olfactory mucosa and to test nasal vaccines. the goal of this review is to highlight the strengths and limitations of each animal model of nasal immunity and to identify the areas of research that require further investigation to advance human health. olfaction is one of the most ancient and conserved senses among animals (ache and young, ) and it is fundamental for animals to perceive and react to their world (ache and young, ; leinwand and chalasani, ) . the main function of the olfactory system is the detection of chemical stimuli present in the host environment and the transduction of these stimuli into electrical signals that reach the higher regions of the brain. one of the unique features of the olfactory system is that the sensory neurons that make up the olfactory neuroepithelium are constantly exposed to pathogens and symbionts, establishing the most intimate interactions between neurons and microorganisms found in the human body. the anatomical organization of the human olfactory system makes it an attractive site for pathogens to gain entry into the host. first, the olfactory system is directly connected to the central nervous system (cns) via the olfactory bulb and therefore numerous neurotropic agents including parasites, bacteria and viruses can reach the cns via transport along the olfactory nerve. examples of neurotropic human pathogens that exploit the nasal route of infection include viruses such as herpes simplex type (hsv- ), coronavirus (hcov), vesicular stomatitis virus (vsv) and rabies virus; bacteria such as streptococcus pneumoniae, burkholderia pseudomallei. listeria monocytogenes; and the protozoan naegleria fowleri (dando et al., ) (table ) . second, the olfactory system is part of the upper respiratory tract in mammals and therefore, pathogens can reach other parts of the respiratory system once they successfully invade the olfactory mucosa. examples of respiratory pathogens that are known to infect the human olfactory organ include influenza virus, respiratory syncytial virus (rsv), rhinovirus, staphylococcus aureus, s. pneumoniae. third, the upper respiratory system is also connected to the gastrointestinal tract via the esophagus and therefore it is possible for pathogens that cause gastric infection to cause nasal diseases. although this route is less well studied, some examples may include human bocavirus (hbov), human rotavirus (hrv), epstein-barr virus (ebv) and salmonella enterica ( table ) . the human nasopharyngeal cavity has a nasopharynx-associated lymphoid tissue (nalt) that consists of both organized lymphoid structures (o-nalt) and diffuse nalt (d-nalt) . d-nalt is found in all vertebrates whereas o-nalt may have first appeared in sarcopterygian fish (tacchi et al., ; sepahi et al., ) . human o-nalt is formed by a series of tonsils (palatine, nasopharyngeal and lingual) arranged in a circular fashion in the oropharyngeal cavity that form the waldeyer's ring ( fig. a ) (zaman et al., ) . the human waldeyer's ring is an induction site for mucosal immune responses (zaman et al., ) . as we will discuss later, o-nalt anatomy differs among different vertebrate groups and a waldeyer's ring similar to that of humans is not always present. in order to establish models for the study of human nasal pathogens, it is important to understand the parallelisms and differences that exist among the olfactory systems and nalt of different species at the cellular, molecular and functional level. in this review, we summarize the main differences in the anatomical organization of nalt and the nasal immune responses in different vertebrate groups as a way to identify species that are more or less suitable for the investigation of specific finally, several human nasal diseases appear to originate from members of the nasal microbiome, which harbors numerous pathobionts including s. aureus. although the study of host-microbiota interactions in the olfactory organ is still at its infancy, we will review established and emerging models that could help advance this field. invertebrates do not have an anatomical structure that forms an olfactory organ. instead, a network of sensory neurons is found in their body. for example, the c. elegans nervous system is composed of neurons which allow the animal to respond adequately to a variety of environmental stimuli and/or stressors such as chemotaxis, thermotaxis, mechanosensation, navigation, learning and olfactory sensation (bargmann et al., ; ha et al., ; iino and yoshida, ; kato et al., ; troemel et al., ) . together with awb, two other pairs of neurons ash and adl are responsible for the avoidance behavior. ash neurons mainly respond to high osmolarity, heavy metals, bitter alkaloids, detergents, and light touch to the tip of the nose (active et al., ; hilliard et al., ; kaplan and horvitz, ) . adl neurons, in turn, respond to social feeding behavior (bono et al., ) . therefore, c. elegans has four neuronal cells ( awa and awc) to sense and discriminate a variety of volatile attractants. as in humans and other animals, c. elegans is also able to respond differently to the same odorants at different concentrations. whereas high concentrations of an odorant result in repulsive behavior, low concentrations of the same odorant trigger attraction behavior (yoshida et al., ) . the first olfactory system may be found in the protochordate amphioxus, where only a rudimental olfactory bulb was identified by the presence of dopaminergic neurons in the anterior nerve cord (lacalli, ; satoh, ) . both cartilaginous and bony fish have an olfactory system which largely resembles the overall organization of its mammalian counterpart. in elasmobranchs, the olfactory organ is characterized by a wide epithelial surface giving sharks the name of "swimming noses" (yopak et al., ) . interestingly, it appears that these large surfaces do not provide cartilaginous fish with more sensitivity or lower threshold odor discrimination compared to teleost fish (meredith and kajiura, ) . the olfactory organ in teleost fish is located in the nasal cavity on the dorsal surface of the head to the front and medial position of the eyes (kasumyan, ) and is composed of two paired olfactory rosettes containing neurons that express vomeronasal receptors (vnr) as well as olfactory receptors (ors) and therefore it may be considered as both the main and accessory olfactory systems found in tetrapods. during evolution, lungfish appear to be the most ancient species in which compartmentalization between the main olfactory epithelium (moe) and vomeronasal organ (vno) occurs (nakamuta et al., ; schnell et al., ) melioidosis bacteria (dando et al., ; hsueh et al., ) listeriosis, sepsis, meningitis, encephalitis, spontaneous abort bacteria (farber and peterkin, ; inoue et al., ; koopmans et al., ) pneumonia, meningitis, sepsis, osteomyelitis, endocarditis bacteria (bogaert et al., ; varon, northcutt and rink, ) . in amphibians, reptiles and mammals the olfactory organ presents a much more complex structure with a clear separation between the moe and the vno. although found in two distinct anatomical regions, recent findings have highlighted the cooperative role of moe and vno in the olfactory perception and the regulation of social communications in mammals (ache and young, ; baum and kelliher, ; kelliher, ) . importantly, the vno of humans is not functional in adulthood. in the human embryo, the vno is similar to that of others species, with bipolar neurons similar to vomeronasal sensory neurons. during development, the vno is reduced to a more simplified structure until it disappears at weeks (meredith, ) . similarly, other tetrapod species have also lost functional vnos including cetaceans, some bats, crocodiles and primates (mucignat-caretta et al., ) . this is not the case of mice, in which the vno is functional throughout life and it plays a vital role in the induction of neuro-hormonal changes due to direct contact with pheromonal stimuli (mucignat-caretta et al., ) . . evolution of olfactory systems: cellular and molecular organization one of the major differences between the invertebrate and vertebrate sensory neurons is that most vertebrate olfactory sensory neurons (osns) express only one functional receptor gene for each neuron. c. elegans neurons, in turn, express several functional receptor genes at the same time (axel, ) . due to the limited number of neurons in the c. elegans body, odorant recognition power is limited (active et al., ) . the fact that each neuron expresses several g-protein-coupled receptors (gpcrs) allows the worm to respond to multiple stimuli using only few neuronal cells . in the majority of vertebrates, the olfactory epithelium is composed by three major cell types: the osns, responsible for odor recognition, the sustentacular cells, that provide osns with support and other utilities, and the basal cells, which are the stem cells of the olfactory epithelium. as mentioned earlier, one of the most prominent characteristics of osns is their ability to express only one type of or. additionally, all osns expressing the same receptor converge in the same glomerular area in the olfactory bulb (gao et al., ) . several molecular features of sensory systems are not only conserved within vertebrates but are also found in invertebrates. among these commonalities is the presence of gpcrs, expressed by the osns, as the main odorant receptors in worms, flies, several fishes (i.e. trout and zebrafish), mouse and human (mori and sakano, ) . in mouse, for example there are more than different ors and each osn express only one or gene. neuropeptides are also conserved in many species including nematodes, flies and mammals and are important modulators of odorant responses in the osns. in fact, neuropeptides may facilitate or reduce the activity of osns and this regulation helps the animal to localize odors and food sources with great accuracy (leinwand and chalasani, ; zhang et al., ) . perhaps the best described invertebrate model system in neuroimmunology is c. elegans. c. elegans feed on bacteria and therefore are attracted to bacterial-derived food odorants (stensmyr et al., ) . additionally, several laboratories have identified pathogenic bacteria that infect c. elegans including pseudomonas aeruginosa and serratia marcensens (pradel et al., ) . pathogenic bacteria and some of their metabolites induce aversive behaviors in c. elegans and this behavior is mediated by chemosensory neurons (beale et al., ; schulenburg and ewbank, ) . moreover, elegant studies have demonstrated the key role that sensory neurons play in the regulation of the inflammatory response of worms against pathogens. the c. elegans model has therefore demonstrated that animals have the ability to discern between pathogenic and non-pathogenic bacteria using sensory neurons (pradel et al., ) . additionally, microsporidia, natural viruses and nonnatural viruses including vsv (balla and troemel, ) , can infect c. elegans and these models are readily available for the study of neuroimmune responses during infection. thus, the detailed map of the c. elegans nervous system and the conserved nature of the chemosensory transduction machinery between c. elegans and vertebrates, makes the worm a suitable laboratory model for the understanding of how animals respond to microorganisms. additional strengths of this model include: high throughput screening protocols, established behavioral assays and availability of bacterial and viral infection models. however, c. elegans lacks a complex neuroepithelium similar to the vertebrate olfactory epithelium as well as a cns. thus, using this model, the immunological contributions of different cell types to pathogens as well as the crosstalk between the peripheral and cns during the course of an infection cannot be investigated (table ) . . bony fish: an emerging model for the study of nasal immunity the teleost olfactory organ is not connected with the oral cavity or respiratory tract, offering a unique and elegant model for the study of human nasal pathologies that have a cns component. teleost fish have also recently emerged as a great model to investigate nasal immunity. the discovery of teleost nalt a few years ago (salinas, ; tacchi et al., ) revealed the conserved nature of this lymphoid tissue in vertebrates. since teleost nalt only contains d-nalt (tacchi et al., ) , teleost fish can now be used to investigate d-nalt responses to pathogens without the use of transgenic animals or surgical procedures. teleost nalt is composed of b cells (igm + and igt + b cells) that are scattered throughout the olfactory epithelium and are mostly located intraepithelially (tacchi et al., ) . additionally, two different cd α + t cell populations have been described in trout nalt, one in the lateral sensory epithelium and one in the apical mucosal epithelium (sepahi et al., ) . cd + t cells have not been investigated in teleost nalt thus far. finally, abundant mhc-ii + antigen presenting cells are found in both the sensory and mucosal epithelium in the trout olfactory organ, but those located in the mucosal tips are found more apically and therefore closer to luminal antigens (sepahi et al., ) . nasal immune responses of rainbow trout against neurotropic viruses such as rhabdoviruses (tacchi et al., ; larragoite et al., ) and protozoan parasites such as ichthyophthirius multifiliis (yu et al., ) have been described. these studies indicate that nalt mounts strong innate and adaptive immune responses upon vaccination or infection in bony fish. importantly, nasal delivery of a live attenuated viral vaccine not only causes local nasal immune responses and systemic immune responses (tacchi et al., ) but also immune responses in the cns (larragoite et al., ) . interestingly, these responses are recorded even in the absence of virus in cns tissues (larragoite et al., ; sepahi et al., ) . with regards to specific antibody responses, it appears that igt is the main antibody isotype involved in specific immune responses against protozoan parasites causing chronic infections in trout nalt (yu et al., ) . these findings confirmed that igt is the functional equivalent to mammalian iga in nasal secretions and that nasal igt responses are likely the goldstandard to evaluate specific immune responses, at least in chronic infection models. thus, rainbow trout has recently emerged as a model to understand neuroimmune interaction from the olfactory periphery to the cns. although to date only the effects of fish pathogens on nasal immune responses have been investigated, we anticipate that bony fish models will be soon adapted to the study of human nasal pathologies. finally, since the presence of nalt in teleosts meant the development of nasal vaccines for use in aquaculture, we can now exploit this model to better understand the mechanisms of protection induced by nasal vaccines sepahi et al., sepahi et al., , . to date, the zebrafish (danio rerio) model has been widely used for the study of many human pathogens (antoine et al., ; harris and harris, ; medina and luis, ; neely et al., ; santoriello and zon, ; swaim et al., ) . zebrafish is largely used due to the small body size, rapid life cycle, transparency at the early stages of life and the large numbers of eggs available every week. moreover, zebrafish resilience and amenability to genetic manipulation techniques have allowed the generation of a broad number of transgenic lines used for studying hematopoiesis, aging, development or cancer. complex hostmicrobe interactions can be investigated in real time using life imaging and fluorescently labeled pathogens. finally, germ-free (gf) zebrafish provide a platform for the identification of microbial contributions to disease onset and progression (kostic et al., ; melancon et al., ; rawls et al., ) . despite the fact that these infection models have been optimized, to date there are no studies evaluating the effects of any pathogen on the olfactory organ of zebrafish. this may be partly due to the fact that nalt has not been characterized in zebrafish yet, although e. casadei, i. salinas developmental and comparative immunology ( ) - comparative histological studies across teleost suggest that nalt is present in most if not all bony fish (tacchi et al., ) . with regards to human nasal pathogens, zebrafish has been used to study the neurotropic mammalian vsv and embryos showed % mortality (guerra-varela et al., ) . additionally, the zebrafish model has been widely used to study influenza virus a, gabor et al., ) , herpes simplex virus (antoine et al., ) and l. monocytogenes (levraud et al., ) . as summarized in table , zebrafish offers many advantages for the study of human pathogen-host interactions but there are some caveats to this model too. one of the limiting factors of the zebrafish model is the difference in temperature between zebrafish and the majority of the human virulent pathogens. in fact, zebrafish requires a temperature of °c, while human pathogens require a temperature of °c at which the development as well as the stress and immune response of zebrafish are significantly altered from those at °c (duggan and mostowy, ; long et al., ) . aves have both o-nalt and d-nalt (fig. d) . o-nalt consists of lymphoid nodules at the root of the nasal septum and the dorsal side of the choanal cleft and does not form a waldeyer's ring (kang et al., ) . these lymphoid nodules are well-organized with defined b and t cell areas and support the formation of germinal centers. developmentally, o-nalt in the chicken begins to form days post-hatch and is fully formed by day . chicken nalt and its capacity to uptake different classes of antigen was thoroughly studied by kang et al. ( ) . with regards to b cell composition, both in chicken o-nalt and d-nalt, igy b cells are the most abundant whereas igm and iga b cells are less frequent (ohshima and hiramatsu, ) . in ducks, similar to chicken, both o-nalt and d-nalt are found. duck o-nalt has been described as several lymphoid aggregates found in the ventral wall of the nasal cavity near the choanal cleft and on both sides of the nasal septum (kang et al., ) . the study of nasal immune responses in birds is extensive, not as models of human disease, but rather due to the important threatening pathogens that infect the respiratory tract of birds including avian influenza virus, infectious bronchitis virus (ibv) and newcastle disease virus (ndv) (de geus et al., ) . as a consequence, oculo-nasal vaccination is one of the most common routes of vaccination in poultry farms (nochi et al., ) . for instance, msd manufactures oculo-nasal spray vaccines for ibv and ndv. intranasal vaccination induces specific iga responses locally and in serum in chickens and ducks (kang et al., ) , making the induction of specific iga titers a common readout following nasal vaccination both in humans and birds (table ) . comparisons between the nasal immune response of chickens and ducks to influenza virus are interesting because ducks are asymptomatic and chickens are highly susceptible accidental hosts (chaise et al., ) . pigs have a respiratory system that largely resembles the human respiratory system (meurens et al., ) . pigs, like other farmed animals, present a waldeyer's ring of lymphoid aggregates. in pigs, the palatine tonsil is not present (casteleyn et al., ; horter et al., ; liu et al., ). it appears that porcine tonsils are richer in b cells than in t cells (yang and parkhouse, ) . importantly, nasal vaccination of pigs results in local and systemic iga and igg specific antibodies (li et al., ; dhakal et al., ) , highlighting the value of this vaccination route and the commonalities between the antibody responses of pigs and other species such as chickens, mice and human. pigs have been proposed as potential models for the study of human influenza h n virus . in support of this model, avian and mammalian influenza viruses infect pig epithelial cells in the respiratory tract, h n swine influenza virus can be transmitted to humans and the cytokine responses in bronchoalveolar lavage (bal) fluid from infected pigs are identical to those observed for nasal lavage of infected humans (khatri et al., ; hayden et al., ) . pigs can be intranasally infected with influenza viruses and therefore they represent a well-established model of influenza nasal immunity (table ) . although the delivery of nasal probiotics and other immunostimulants has rarely been investigated, pigs have been used for testing nasal immunostimulants such as bacillus subtilis (yang et al., ) . horses have five tonsils that form a waldeyer's ring similar to that of humans (liebler-tenorio and pabst, ) . the nasal immune responses of horses to bacteria and viruses have been well studied. some pathogens such as streptococcus equi, cause severe upper respiratory infections in horses since they attach to the equine tonsils (sweeney et al., ) . nasal secretions of horses infected with s. equi or intranasally vaccinated with the usda-approved s. equi live attenuated vaccine had specific iga and igg antibodies as well as systemic iga titers (galan and timoney, ; delph et al., ) . horses are however not considered model organisms of human disease due to the large size, maintenance costs and need for large enclosures where pathogen containment is hard to achieve (table ) . of all model organisms, ferret (mustela putorius furo) is the elective model to study multiple human respiratory infections including avian influenza virus, morbillivirus, coronavirus and others (belser et al., ) . apart from the small size, human and ferrets have similar lung physiological characteristics, and when intranasally inoculated with influenza virus, ferrets showed human clinical symptoms such as fever, nasal secretion, coughing and sneezing; which are not present in the classical mouse model (belser et al., ; zitzow et al., ) (table ) . importantly, ferrets have pharyngeal tonsils which are often analyzed in experimental infection models (van den brand et al., ; lipatov et al., ) . moreover, an aged ferret model has been developed to better investigate the effects of aging on the immune responses to influenza a h n strain (paquette et al., ) . the high susceptibility to influenza along with the ability to infect healthy ferrets, make these animals a suitable model to follow host-virus interactions (belser et al., ) . along with the influenza infection model, ferrets have been used to evaluate the effectiveness of novel vaccine candidates including flumist and m sr. m sr evokes a wide antibody immune response in ferrets both locally and systemically. moreover, this vaccine has a protective effect against the drifted viruses h n and h n in both ferrets with and without pre-existing immunity. thus, m rs have been considered as a suitable candidate for a large spectrum human vaccine suitable for all ages (hatta et al., ) . rabbits only have palatine tonsils (fig. c) and therefore a full waldeyer's ring similar to that of humans is not present (casteleyn et al., ) . rabbits have been used as a model for paranasal sinuses fungal infections such as aspergillosis (chakrabarti et al., ) as well as a model for rhinosinusitis (al-sayed et al., ; dao-yu et al., ) . strengths of the rabbit model include the anatomical similarities of the rabbit sinus with that of humans, the easy access to this area in rabbits and the fact that immune responses are largely conserved between rabbits and humans (table ) . domestic rabbits are commonly infected by pasteurella multocida and bordetella bronchiseptica. at weaning, about % of rabbits were e. casadei, i. salinas developmental and comparative immunology ( ) - found to have nasal infections with p. multocida and % had infections with b. bronchiseptica (deeb et al., ) . intranasal immunization of rabbits with p. multocida toxin induces specific iga responses in the nasal and bronchoalveolar lavage (jarvinen et al., ) indicating again that, similar to human, iga responses are the gold-standard for evaluation of nasal vaccine efficacy in several animal models including rabbits. rats do not have any tonsils and therefore lack the waldeyer's ring (casteleyn et al., ) . rats, however, have o-nalt structures that resemble those found in mice. the cotton rat (sigmodon hispidus) model has traditionally been used to understand many viral human respiratory infections including influenza. both innate and adaptive immune responses and disease pathogenesis were characterized in cotton rats intranasally infected with influenza virus a and b (eichelberger, ) . in particular, cotton rats were infected with several influenza virus a strains including h n , h n and influenza virus b to determine the infectivity as well as the pathogenesis. interestingly, all the viruses were able to replicate in the nose and lung. moreover, concomitant to the lung infection with influenza virus a, a strong immune response characterized by ifnα, ifnγ, tnfα, il- α and il- was detected (eichelberger, ) . however, similar to mice, cotton rats are macrosmotic animals and do not have a sneezing impulse. the latter is a limiting factor in the study of disease propagation. in contrast, cotton rats offer many advantages to the study of rsv pathology because they are more permissive to rsv infection than mice and they show human-like disease symptoms upon infection (boukhvalova et al., ) (table ) . thus, cotton rats offer a valid model to study the pathogenesis of rsv and to develop and test vaccines and therapies against this virus (citron et al., ; sami et al., ) . as an example, intramuscular immunization with virus-like particles containing the pre-fusion protein f of rsv (pre-f/f vlps), induced high titers of neutralizing antibodies and protected the cotton rat lung and nasal cavity from viral replication. interestingly, animals previously infected and then challenged with rsv had undetectable viral loads in the nose. hence, intranasal immunization with vlps may lead to better stimulation of mucosal immune responses and greater protection, compared to other routes of immunization (cullen et al., ) . finally, the cotton rat model is of particular interest to understand the biology and pathogenesis of s. aureus. nasal carriage of s. aureus is an important risk factor for s. aureus infections in both adults and children (kluytmans and verbrugh, ; mulcahy and mcloughlin, ) . cotton rats can successfully be infected with s. aureus by nasal instillation of live bacteria and therefore they have been used to assess the efficacy of therapies to control s. aureus infections in humans (kokai-kun, ) . gene expression profile was analyzed during the initial phases of the nasal colonization with a human isolate of s. aureus (burian et al., ) . within the genes identified, s. aureus adhesin molecules (tago, tark, clfb and isda) and virulence factors (walkr, sak, sced) were fundamental for the nasal colonization of s. aureus (burian et al., ). based on the higher level of igg against four s. aureus antigens, two iron-responsive surface determinants isda and isdh were selected as vaccine candidates against s. aureus. in fact, s. aureus isda adhesin molecule is responsible for the binding to human squamous nasal epithelial cells and clumping factor b (clfb), playing a complementary role in such binding. when tested in cotton rats, both antibodies raised against isda antigen conferred protection against nasal carriage (clarke et al., ) . although the full protective mechanism is still not totally understood, this vaccination strategy may be a promising prophylactic tool in humans (clarke et al., ) . a caveat to this model, however, is that the nasal microbial community of the cotton rat is uniquely composed of several bacterial species that do not necessarily reflect the microbial complex interactions that occur in human anterior nares (chaves-moreno et al., ) (table ) . this is important since previous studies have shown that the nasal microbiota regulates nasal immune responses following intranasal vaccination (henriksson et al., ) . thus, careful evaluation of nasal microbial communities may be critical when performing nasal vaccination trials. guinea pigs (cavea porcellus) were one of the first animal models used to study human infectious diseases, such as diphtheria and tuberculosis (padilla-carlin et al., ) . guinea pigs o-nalt consists of - lymphoid follicles at the junction of the nasal cavity and the nasopharyngeal duct (okada et al., ) . interestingly, guinea pigs are more susceptible than mice to l. pneumophila (mcdade et al., ) . thus, this model organism was the first to be used to isolate l. pneumophila and to determine its virulence factors. moreover, guinea pigs present all the symptoms present in humans infected with l. pneumophila. guinea pigs can be intranasally infected with l. pneumophila (padilla-carlin et al., ) but studies pertaining nasal immune responses to legionella in guinea pigs are missing. susceptibility to viral nasal infection, especially for human influenza viruses a and b (palese et al., ) , and ebola (wong et al., ) was investigated in guinea pigs. in the case of h n avian influenza viruses, the guinea pig was also used as a model to investigate the origin of viral resistance in this species . interesting, at day post infection the viral titer in the nasal wash and lung were already very low and proteomic profiling of the lung showed that more than ifn-stimulated genes (isgs) were activated during the infection. thus, unique and potent nalt and lung responses against h n may confer protection against this virus in guinea pigs. guinea pigs have also been intranasally infected with zika virus (zikav). nasal infection with zikav results in infection of the brain, parotid glands, tears, saliva and sera but nalt immune responses were not evaluated (deng et al., ) . of interest, nasal vaccination of guinea pigs with a genital herpesvirus hsv vaccine results in protection to genital herpes and elicits systemic specific igg but not iga titers (persson et al., ) . overall, guinea pigs continue to be used as models for the study of nasal pathogens and can illuminate mechanisms by which nasal vaccines confer protection at distant mucosal sites such as the vaginal tract. however, more detailed characterization of the local nasal immune responses against relevant human nasal pathogens in this species is still needed. mice are one of the most widely used models to study human infections. this organism offers many advantages including relative low cost, easy access to reagents and transgenic mice with specific gene manipulation including over-expression or gene disruptions. however, human nasal pathogens do not always replicate efficiently in mice. apart from the abovementioned examples of rsv and l. pneumophila, human influenza virus does not replicate efficiently in mice. mice are also known to be resistant to many upper respiratory infections and they do not exhibit sneezing reflex, which is an important factor in the study of the viral transmission (grieves et al., ) . a final consideration is that mice, like dogs, have a very sensitive sense of smell and therefore they are considered macrosmatic. primates, on the other hand, are generally considered microsmatic, although this classification may not always be the most adequate depending on the primate species (smith and bhatnagar, ) (table ) . thus, evolutionary speaking, microsmatic and microsmatic mammals have likely co-opted different strategies to detect and defend their olfactory organs against pathogen infection. unlike humans, rodents do not have the ring of tonsils that forms the waldeyer's ring. rodents have o-nalt structures described as bellshaped paired lymphoid aggregates in the floor of the nasal cavity at the entrance of the pharyngeal duct (fig. b) (van der ven and sminia, e. casadei, i. salinas developmental and comparative immunology ( ) - ) . murine o-nalt is considered the functional equivalent to the human waldeyer's ring. murine nalt is well organized with b and t cell areas and a modified epithelium covering the lymphocytes (asanuma et al., ) . apart from o-malt, d-nalt is also present in rodents. detection of specific antibodies in nasal washes, nalt explant culture and phenotyping of nalt leucocytes are all standard techniques in the evaluation of nasal immune responses to vaccination in mice (cisney et al., ) . surgical ablation of o-nalt structures can be performed in mice allowing the investigation of the contributions of d-nalt to vaccination responses (cisney et al., ) . nasal neuroepithelial responses following multiple poly i:c instillations were studied in mice. poly i:c administration resulted in infiltration of neutrophils, macrophages and t cells into the olfactory mucosa and presence of caspase- positive apoptotic osns (kanaya et al., ) . the same study observed tlr expression in the apical part of the supporting cells and in the cytoplasm of acinar cells of bowman's glands (kanaya et al., ) suggesting that collaborations between multiple cell types of the olfactory epithelium likely take place upon nasal viral infections. as an example of this cooperation, several studies have shown the innate immune responses of olfactory ensheathing cells (oecs), which are unique glial cells of the moe, against pathogenic bacteria. in this rat model, the olfactory mucosa is first damaged by irrigation with zinc sulfate or triton x- and then bacteria are administered. oecs are robust inos producers (vincent et al., ; harris et al., ) . finally, oecs appear to respond to several pamps and express tlr (vincent et al., ) . intranasal administration of lps in mice induces persistent rhinitis (hasegawa-ishii et al., ) with a pathology that is characterized by leukocyte infiltration as well as the production of the pro-inflammatory cytokine il- β. interestingly, loss of osns in the main olfactory mucosa as well as synaptic loss in the olfactory bulb have been reported in this model (hasegawa-ishii et al., ) . mice have also been extensively used to study cns neuroinflammation in different disease models (manglani and mcgavern, ) . vsv, which is not lethal to mice, can be nasally delivered as a model to track cns immune responses. recent studies have shown the role of t cells in this response and the presence of local myeloid cells that cross-present antigen in the cns (moseman et al., ) . thus, similar to fish, the nasal vsv mouse model has evolved as a platform to study neuroimmune interactions. finally, mice have been used for the evaluation of several human nasal vaccine formulations including vaccines against influenza viruses, s. aureus or zikav (brown et al., ; schaffer et al., ; sumathy et al., ) . similar to birds and other mammals, induction of specific local iga titers and systemic igg titers are usually detectable in mice following intranasal vaccination depending on the antigenic model (kurono et al., ; hirano et al., ) . s. aureus nasal colonization in mice was reduced by the immunization with killed s. aureus and/or the recombinant clumping factor b (rclfb) (schaffer et al., ) . clfb is known to bind mouse and human cytokeratin and human desquamated nasal epithelial cells allowing s. aureus to colonize the nasal mucosa. moreover, clfb is also conserved among s. aureus strains, hence is a suitable candidate for a multi-strain vaccine. both intranasal immunizations showed protection against s. aureus along with a reduction of nasal colonization (schaffer et al., ) . the immunocompromised mouse strain a that lacks interferonα/β receptor , was intranasally inoculated with zikav and all mice showed a prolonged viremia. after days post inoculation, igm and iga antibodies were detected, followed by igg. thus, a strong infection in the nasal mucosa was established in a mice (deng et al., ) . a vaccine against the envelope protein e of zikav was tested in this mouse strain and production of neutralizing antibodies was able to confer protection against zikav and other flaviviridae (sumathy et al., ) . overall, mice continue to be the model of choice for many nasal vaccine trials despite the drawbacks summarized in table . . primates as models for the study of human nasal diseases and immunity due to anatomical and immunological similarities, the use of nonhuman primate models confers great advantages in the evaluation of therapeutic drugs, treatments and vaccines. of particular interest for the study of nasal immune responses and human nasal diseases, primates have tonsils arranged in a waldeyer's ring similar to humans (ludlow et al., ) . macaques are often used because they are easy to breed compared to other primates. several macaque species have been used in preclinical evaluations of infectious agents and in vaccine development studies including streptococcus a (gas), influenza a virus, measles virus, hiv, ebola, variola major, hrsv and zikav (brown et al., ; rivera-hernandez et al., ; lucas-hourani et al., ; sumathy et al., ; ludlow et al., ) . in the case of gas, an intranasal infection model in monkeys was used to evaluate the transcriptomic gas profile during different phases of infection and the grade of inflammation caused to the animals (virtaneva et al., ) . primates, like the rest of the endotherms discussed in this review, mount specific iga antibody responses in the nasal mucosa following intranasal infection or vaccination (komada et al., ; mizuno et al., ) . cynomolgus monkeys, also known as crab-eating macaques (m. fascicularis), have been used to evaluate the effects of age on immune responses following intranasal infections or intranasal vaccination (mizuno et al., ; lucas-hourani et al., ) . these types of studies are very important when making decisions about vaccination programs in children and adults. even if non-human primates are the closest model species to human and maybe the most reliable to study host-pathogen interactions, the similarities between humans and monkeys have often raised ethical issues and public outrage against scientists. other caveats of this model include the difficult genetic manipulation, the intense husbandry demand and the high costs to maintain animals physically and mentally healthy (table ) . for all these reasons, the use of non-human primates in research is highly regulated and often used as a "last resource" when any other model cannot be applied to the particular research question. in this final section, we want to introduce the concept of organoids as a model to study human nasal diseases. one of the bottlenecks for the investigation of host-microbe interactions is the complexity of the microbial communities and the lack of in vitro models to study one-to-one cause-effect mechanisms for each microbe. in general, organoids are considered miniaturized and simplified organs. although organoids are very similar to the real organs, due to the lack of vascularization, structure and sometimes mature function, organoids remain an imperfect model and they are still under development. therefore, organoids are used along with more classical approaches such as in vivo studies. the first organoid was created years ago from a single intestinal crypt cell; where even in the absence of a non-epithelial cellular niche, a single cell was able to self-renew and differentiate, generating a villus-like epithelial structure in which all the different cell types present in the real organ were found (sato et al., ) . since then several "mini-tissue" have been reproduced successfully in many animals including human pancreas, kidney, thyroid, liver, inner ear, retina, skin tissue with follicular hair and brain (barkauskas et al., ; lee et al., ; koehler et al., ; orsini et al., ; ramachandran et al., ; turksen, ; ueda et al., ) . nasal organoids were also created from nasal human turbinate epithelia (mocs) almost years ago (kleinsasser et al., ) . although nasal organoids have been used for genotoxicity studies, their full potential has not been exploited to study human nasal diseases and host-microbe interactions at the human nasal mucosa. due to the impervious and sometimes inaccessible airway anatomy, it is often difficult to determine the contribution of nalt in the cascade of events that e. casadei, i. salinas developmental and comparative immunology ( ) - lead to respiratory pathologies. here, we propose the use of nasal organoids as a model for human nasal infections and other diseases, such as nasal polyps, rhinosinusitis, anosmia, allergy or aspergillosis. organoids could also constitute a suitable model to study nasal-specific microbial interactions helping to identify virulence factors required for invasion and infection. additionally, nasal organoids could shed light on potentially favorable competition between commensals and specific pathogens involved, for example, s. pneumoniae rhinosinusitis. many animal models have helped unveil host-pathogen interactions at the nasal mucosa but many questions remain to be answered. overall, nasal mucosal immune responses are not well understood compared to other mucosal immune responses. this is despite the large variety of pathogens that exploit the nasal route to gain entry to the host. understanding nasal immunity in different animal models is vital for establishing models of human disease, but our current view of the diversity of nasal immune systems is patchy and reflects specific research efforts rather than unbiased comparisons. although specific antibody responses following intranasal vaccination are a common readout in most animal experimental models as well as humans, very little is known about the early immune responses that halt pathogen entry or the contributions and cross-talk between d-nalt and o-nalt. additionally, new insights in teleost fish suggest that nasal immunization triggers distant immune responses in the cns suggesting tight neuroimmune regulation in the olfactory-cns axis. vertebrates have a wide diversity of olfactory systems and the anatomical differences found in nalt across species makes the translation of animal studies to human difficult. anatomically, the o-nalt of primates and farmed animals is the closest to human o-nalt, however these models also present their own limitations. some of most refined animal models, such as the pig nasal influenza model, illustrate the importance of validating human disease symptoms, disease progression and immune responses in the laboratory setting. further efforts are necessary to exploit the best attributes of each animal model and to translate 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for attractive odorants in caenorhabditis elegans the signaling pathway of caenorhabditis elegans mediates chemotaxis response to the attractant -heptanone in a trojan horse-like pathogenesis the innate immunity of guinea pigs against highly pathogenic avian influenza virus infection iga response and protection following nasal vaccination of chickens with newcastle disease virus dna vaccine nanoencapsulated with ag@sio hollow nanoparticles pathogenesis of avian influenza a ( h n ) viruses in ferrets this work was funded by national science foundation (nsf) ios award # and us department of agriculture (usda) afri award # - - to is. key: cord- -r hgfsxz authors: chakraborty, supriyo; barman, antara; deb, bornali title: japanese encephalitis virus: a multi-epitope loaded peptide vaccine formulation using reverse vaccinology approach date: - - journal: infect genet evol doi: . /j.meegid. . sha: doc_id: cord_uid: r hgfsxz japanese encephalitis (je) is a serious leading health complication emerging expansively that has severely affected the survival rate of human beings. this fatal disease is caused by je virus (jev). the current study was carried out for designing a multi-epitope loaded peptide vaccine to prevent jev. based on reverse vaccinology and in silico approaches, octapeptide b-cell and hexapeptide t-cell epitopes belonging to five proteins, viz. e, prm, ns , ns and ns of jev were determined. hydrophilicity, antigenicity, immunogenicity and aliphatic amino acids of the epitopes were estimated. further, the epitopes were analyzed for different physicochemical parameters, e.g. total net charges, amino acid composition and boman index. out of all the epitopes, a total of four t-cell epitopes namely kradss, krsrrs, skrsrr and kecpde and one b-cell epitope i.e. pkpcskgd were found to have potential for raising immunity in human against the pathogen. taking into account the outcome of this study, the pharmaceutical industries could initiate efforts to combine the identified epitopes together with adjuvant or carrier protein to develop a multi-epitope-loaded peptide vaccine against jev. the peptide vaccine, being cost effective, could be administered as a prophylactic measure and in jev infected individuals to combat the spread of this virus in human population. however, prior to administration into human beings, the vaccine must pass through several clinical trials. japanese encephalitis (je) is a major emerging, dreadful infection worldwide as this fatal disease has affected the lives of many individuals resulting into , deaths and , cases of infection per year (solomon, ; tsai, ) . the causal agent of the disease is je virus (jev). jev is transmitted by mosquitoes and is categorized into genus flavivirus and family flaviviridae (westaway et al., ) . jev severely affects the central nervous system of human and results into infectious disease. the transmission cycle of jev occurs between mosquitoes and birds or swine. however, the transmission of the virus to humans usually takes place through infected mosquitoes of the species, culex tritaeniorrhynchus (porterfield, ) . jev is found to prevail in many asian nations namely india, nepal, sri lanka, china, japan, korea, vietnam, thailand, myanmar, taiwan, siberia, cambodia, bhutan, bangladesh, malaysia and indonesia. the je epidemic has spread from eastern asia to southeast and southern asia (burke and leake, b; oya, ; vaughn and hoke jr, ; endy and nisalak, ; mackenzie et al., ) . apart from asia, je has affected many geographic regions of other continents as well, namely northern australia and western pacific (paul et al., ; hanna et al., ; hanna et al., ) . the outbreak of je was first observed in japan during s and the first isolate of jev was obtained by culturing the brain cells of an infected individual in (solomon et al., ) . although children are the primary targets of je infection, it also causes dreadful infection in adolescents and adults. in temperate regions of asia je outbreaks mainly occur in summer; while outbreaks in torrid zone and subtropics of asia prevail throughout the year and the occurrence of je infections rapidly increases during rainy days (burke and leake, b; halstead and jacobson, ; fischer et al., ) . symptoms of jev infection include fever, meningoencephalopmyelitis, aseptic meningitis, seizures or poliomyelitis-like paralysis (solomon et al., ; solomon and vaughn, ) . death occurs in about - % of je infected cases and about - % of surviving people usually encounter constant abnormalities associated with nervous system, e.g. mental disorientation, mental retardation and hemiparesis (solomon et al., ; fischer et al., ; ooi et al., ) . there are five different genotypes of jev (uchil and satchidanandam, ; solomon et al., ) and all the strains belong to only one serotype (tsarev et al., ; erra and kantele, ) . jev genome consists of a positive-sense single stranded rna molecule of size kb (westaway et al., ) . the viral rna synthesizes one polyprotein, which undergoes proteolytic cleavages within the jevinfected cells and produces proteins, viz. envelope (e), capsid (c), membrane (m or precursor membrane i.e. prm), ns , ns a, ns b, ns , ns a, ns b and ns proteins (ns-non-structural) (chambers et al., ; marin et al., ; rice, ; zanotto et al., ) . the envelope of jev, made up of glycoprotein, has a size of nm. this envelope encircles the nucleocapsid formed by the joining of capsid and rna. the e protein helps the virus adhere and penetrate into the host cell; in addition to helping at the time of membrane fusion (allison et al., ; kuhn et al., ) . the prm protein is secreted only during immature stage of the virion. at the later stage of viral infection, prm protein is broken down into m protein with the help of proteases. as a result, the virion develops into a mature virion. sometimes, prm protein fails to break down into m protein (bray and lai, ) . in jev-affected host cells, the virus produces ns protein externally and it acquires importance in virion maturation (fan and mason, ; rice, ) . the two other proteins namely ns and ns help jev undergo replication process (rice et al., ; bartholomeusz and wright, ) . various vaccines are formulated time to time for preventing the spread of je. the vaccines include inactivated cells acquired from mouse brain or vero cell culture, live-attenuated vaccines developed from jev or other viruses resulting into the formation of chimera (halstead and thomas, ; baig et al., ; hegde and gore, ) . all these vaccines have shown effective results in infected individuals. the who suggests to utilize these vaccines in those regions of the world where jev has caused acute infection (organization, ) . novel vaccines based on replicon (kofler et al., ) , subviral particles (konishi et al., ; konishi et al., ) , and jev-pulsed dendritic cells (li et al., ) have been under development and tested only in animal models. reverse vaccinology approach is based on the genomics of a pathogen and elucidates the antigenic determinants in the proteins of the pathogen (rinaudo et al., ; tantray et al., ) . it, in fact, follows an in silico approach to screen the entire genome of the infectious pathogens to determine the epitopes of proteins that could be used as potential molecules for peptide vaccine formulation and development. in comparison to the traditional technique of vaccine design that requires laborious as well as expensive wet lab experiments, the reverse vaccinology approach relies mostly on genomic information and involves much lesser cost and time. the technique of reverse vaccinology provides enormous advantage in designing peptide vaccines against highly infectious pathogens, including those which cannot be cultured in lab due to high risk imposed on researchers and health professionals. antigenic determinants or epitopes are the regions of the proteins that interact with the receptors on the t-cells or with antibodies produced by the b-cells and generate significant immune response in host. for this purpose, various bioinformatic tools have been developed to identify the potential epitopes for peptide vaccine formulation (weiss and littman, ) . peptide vaccines are not only safer, less expensive and easily manufacturable but also require less time for manufacture as compared to the traditional vaccines (von hoff, ; tang et al., ) . unlike traditional vaccines, peptide vaccines do not cause autoimmune disorders or skin irritations (skwarczynski and toth, ) . moreover, the newly discovered epitopes need to be well characterized for biological functions. development of immunity in a host against a pathogen or antigen stems from the presence of t-and b-lymphocytes. t-lymphocytes recognize the antigen only when it is displayed by a class of proteins, called major histocompatibility complex (mhc) molecules, as exterior part to the antigen presenting cells (apcs). the t-cell receptor binds with the antigen and causes its destruction (lafuente and reche, ). on the other hand, the destruction of antigen by b-cells takes place via the secretion of antibodies that interact with the antigen. b-cells are also capable of providing immunity during future exposure to the antigen as some b-cells differentiate into memory cells in the host. the current study was performed for predicting the potential t-cell and b-cell epitopes of five proteins in jev namely e, prm, ns , ns and ns of jev for designing a multi-epitope loaded peptide vaccine. this study allows the identification of unique peptides as vaccine candidates that might be much cheaper than the existing vaccine formulation against jev. our analysis suggested that e, prm and ns proteins possess capability for generating immunological response in the host body (monath et al., ) . another study reported that ns and ns proteins can induce immune reaction in the host body (turtle et al., ) . the predicted epitopes identified in five proteins selected in this study could be promising for formulating a peptide vaccine against jev and hence, could prevent the spread of jev in affected individuals. complete sequences of the proteins, viz. e, prm, ns , ns and ns of jev were collected from the protein database of national center for biotechnology information (ncbi) (http://www.ncbi.nlm.nih.gov). all the proteins were identified to possess their respective accession numbers: np_ . , np_ . , np_ . , np_ . and np_ . . using the algorithm based on wet lab experiments and established by hopp and woods, the linear hexamer t-cell epitopes in five selected proteins of jev were identified (hopp and woods, ) . further, the set of linear octamer b-cell epitopes in five selected jev proteins was determined by using the algorithm outlined by kolaskar and tongaonkar (kolaskar and tongaonkar, ). hydrophilicity score is a measure that describes the hydrophilicity of an epitope or a protein. it is the mean of the hydrophilicity values of all the amino acids contained in an epitope or a protein. it is useful in determining the structure of the protein (hopp and woods, ) . hydropathy index, a measure of hydrophobicity of a given epitope or a protein, was determined as the average of hydrophobicity values from all amino acids present in an epitope or a protein (kyte and doolittle, ) . estimation of hydropathy index values of all epitopes was achieved by applying the abdesigner algorithm (pisitkun et al., ) . beta-turn regions within a protein have significant role in generating immunogenicity. chou-fasman algorithm was used to determine the properties of secondary structure of peptide (chou and fasman, ) . immunogenicity score reports the immunogenic nature of a given epitope or protein. the immunogenicity scores of all the epitopes were estimated on the basis of hydropathy index and chou-fasman conformation parameters of each epitope. further, hydrophilicity, antigenicity and immunogenicity scores as well as aliphatic amino acids in each epitope were estimated using a computer program written in perl language by sc (corresponding author). the physico-chemical parameters of epitopes such as net charge and amino acid composition were determined in protparam tool of expasy (https://web.expasy.org/protparam/). further, the cleavage sites in epitopes were identified using the online tool peptide cutter (https:// web.expasy.org/peptide_cutter/) (shown in s and s ). boman index i.e. the protein binding potential of each epitope was determined by using the online tool apd i.e. (http://aps.unmc.edu/ap/prediction/ prediction_main.php). the epitopes that acquire boman index values higher than . kcal/mol are considered being capable of attaching to the mhc molecules or antibodies. with the help of bioinformatic tools, five hexapeptide t-cells epitopes and five octapeptide b-cell epitopes were identified in five proteins namely e, prm, ns , ns and ns of jev. in order to combat the spread of jev, hexapeptide t-cell epitopes would possess the ability to raise high immune response in the host (hopp and woods, ) . the epitope positions, hydrophilicity scores and total net charges of all t-cell epitopes were determined (table a) . out of five epitopes in e protein, the epitope ekrads (epitope position - ) was recorded with the greatest hydrophilicity score ( . ) but without any net charge. two epitopes namely krsrrs (epitope position - ) and skrsrr (epitope position - ) located within prm protein exhibited the greatest hydrophilicity value ( . ) with positive net charge (+ ). similarly, within ns protein, two epitopes with maximum hydrophilicity score of . were identified as eestde ( - ) and reestd ( - ) carrying net charges of − and − , respectively. the epitope rgeekk ( - ) of ns protein was estimated with the highest hydrophilicity value ( ) along with a positive net charge (+ ). in ns protein, the epitope deeren ( - ) was recorded with high hydrophilicity value ( . ) associated with a negative net charge (− ). high hydrophilicity scores of t-cell epitopes along with their positions usually represent the hydrophilic sections in proteins and these regions tend to remain exposed on the surface of the proteins. these regions can bind easily with the mhc molecules and the complexes so formed would be exhibited by the apcs on their surfaces. the t-cells can easily recognize these cells and destroy them. the epitope positions, hydrophilicity scores and total net charge of each octapeptide b-cell epitope were evaluated (table b) . in e protein, the epitope sgsdgpck ( - ) occupied maximal hydrophilicity value ( . ) but without any net charge. in the case of prm protein, two epitopes namely skrsrrsv ( - ) and krsrrsvs ( - ) were identified with the highest hydrophilicity score of . with net charge of + each. in ns protein, the epitope tkecpdeh ( - ) was recorded with the highest hydrophilicity value ( . ) and a negative net charge (− ). in ns protein, the epitope pkpcskgd ( - ) was found to possess high hydrophilicity value ( . ) with a positive net charge (+ ). in ns protein, the epitope pyvgkred ( - ) was found to possess the highest hydrophilicity score of . without any net charge. high hydrophilicity scores of b-cell epitopes imply that these epitopes lie in the hydrophilic regions of the respective proteins and can interact with the antibodies produced by b-cells. this interaction might lead to the generation of immunological response in host against jev. all t-cell epitopes obtained from five proteins were examined and evaluated for antigenicity (fig. a) . antigenicity values of the t-cell epitopes were found to range from . (deeren) to . (kecpde). likewise, total b-cell epitopes were also analyzed for antigenicity score (fig. b) and the antigenicity scores of b-cell epitopes varied from . (skrsrrsv, krsrrsvs, rctrtrhs and cgrggwsy) to . (pspkpcsk). the epitopes possessing high antigenicity values normally represent highly antigenic nature of the epitopes. notably, high antigenicity score of an epitope is treated as a salient feature for the formulation of a peptide vaccine. like antigenicity analysis, all the hexapeptide t-cell epitopes were evaluated for their immunogenicity scores (table a) . in e protein, the epitope nardrs was found to possess the highest immunogenicity score ( . ) whereas, in prm protein the epitope gndped recorded table a t-cell epitopes from five proteins of jev representing their hydrophilicity scores and total net charges (hopp & woods approach, maximum immunogenicity score ( . ). in ns protein the epitope khnrre was identified with the highest immunogenicity score ( . ) but in ns protein the epitope drqeep was found to possess the maximum immunogenicity score of . . in ns protein two epitopes, viz. deeren and rekrpr each possessed the highest immunogenicity score ( . ). aliphatic amino acid plays an important role in binding affinity. it is believed that the epitopes comprising of aliphatic amino acids viz. ala, gly, ile, lys, leu, met and val can interact easily with either of the two lymphocytes. in the present study, the t-cell epitopes were examined to calculate their aliphatic amino acid number (table a) . one epitope, gkrekk of ns protein consisted of aliphatic amino acids. four epitopes, namely grgdkq of e protein, rgeekk and geekkn of ns protein, and krekkp of ns protein had aliphatic amino acids each. six epitopes were identified to consist of aliphatic amino acids namely ekrads, nekrad and kradss of e protein; skgenr of prm protein; edcgkr of ns protein; and gperek of ns protein. ten epitopes, i.e. nardrs of e protein; krsrrs, skrsrr, dpedvd and gndped of prm protein; khnrre and kecpde of ns protein; gdrqee of ns protein; and rekrpr and rrarre of ns protein contained a single aliphatic amino acid. however, four epitopes were identified with no aliphatic amino acid and these were eestde and reestd of ns protein, drqeep of ns protein, and deeren of ns protein. in the case of all octapeptide b-cell epitopes, the immunogenicity score as well as the number of aliphatic amino acids contained in each b-cell epitope were determined (table b ). in e protein the epitope ysgsdgpc was identified to possess the highest immunogenicity score of . whereas, in prm protein the epitope ndpedvdc was recorded with maximal immunogenicity score ( . ). in ns protein the b-cell epitope tkecpdeh was estimated with maximum immunogenicity score of . . further, in ns protein the epitope ypkckngd recorded the highest immunogenicity score ( . ) whereas, in ns protein the epitope hkdpehpy recorded the maximum immunogenicity score of . . nine epitopes namely sgsdgpck and lsysgsdg of e protein; kpvgryrs of ns protein; ypkckngd, pkpcskgd and eypkckng of ns protein; gkenyvdy, pyvgkred and cgrggwsy of ns protein were identified to contain aliphatic amino acids. seven epitopes were found to possess aliphatic amino acids i.e. ysgsdgpc and elsysgsd of e protein, skrsrrsv and krsrrsvs of prm protein, drykylpe and kylpetpr of ns protein and pspkpcsk of ns protein. seven epitopes, i.e. eppfgdsy of e protein, ndpedvdc of prm protein, tkec-pdeh and ecpdehra of ns protein, dtpspkpc of ns protein, and hkdpehpy and fykpseps of ns protein contained a single aliphatic amino acid. moreover, two epitopes namely dcwcdnqe and rctrt-rhs of prm protein contained no aliphatic amino acid. it is believed that the epitopes that possess high immunogenicity score are highly immunogenic and the immunogenicity score of a peptide epitope is a determining factor in choosing epitopes for peptide vaccine formulation. boman index values of all hexapeptide t-cell epitopes present in five jev proteins were estimated (fig. a) . we used boman value . kcal/ mol as the minimum cut-off value. notably, all the t-cell epitopes in our study possessed boman value > . kcal/mol and were considered to have potential for binding successfully to mhc molecules. from these results we concluded that the complexes would be presented to tcells by apcs and would elicit immune response in the host. further, we noted that the boman index of t-cell epitopes ranged from . (kecpde) to . (rrarre). similarly, we estimated boman index of all the b-cell epitopes (fig. b) . eight b-cell epitopes, viz. sgsdgpck, ysgsdgpc, eppfgdsy and lsysgsdg of e protein; pspkpcsk and dtpspkpc of ns protein; and fykpseps and cgrggwsy of ns protein were found to have boman index < . kcal/mol which indicated they may not attach to immunoglobulins secreted by b-cells. the remaining epitopes possessed boman index > . kcal/mol and hence, these epitopes could bind to immunoglobulins effectively. the t-cell epitopes of jev proteins were analyzed for their amino acid arrangement (fig. a) . different amino acids were found in epitopes. interestingly, some epitopes of e, prm, ns and ns proteins comprised of amino acids like lys, asp, gly, ser, glu, arg constituting nearly . % of the total composition. on the other hand, some (kolaskar & tongaonkar approach, epitopes of prm, ns and ns proteins contained arg, asp, glu and lys to the extent of . %. only one epitope rrarre of ns protein was found to contain arg constituting . % of the total epitope composition. likewise, the b-cell epitopes of jev proteins were analyzed for amino acid composition (fig. b) . all the epitopes contained varying amino acid composition. some b-cell epitopes contained amino acids namely tyr, his, asp, ser, gly, lys, thr, cys, glu, pro and arg constituting nearly . % of the total composition. further, some other epitopes of e, prm, ns and ns proteins were found to possess ser, gly, arg, pro and asp constituting . % of the total epitope composition. further, the t-cell and b-cell epitopes against jev are amenable to cleavage by various enzymes and chemicals and the results of cleavage are presented in supplementary tables (s , s ). in this study, the potential epitopes for peptide vaccine formulation were identified in five proteins of jev namely e, prm, ns , ns and ns . all the epitopes were analyzed for various parameters using different bioinformatic tools. only a few epitopes were found to possess the properties essential for generating immune response in the host against jev and the selected epitopes might be used in formulating an effective peptide vaccine to combat the menace of jev. earlier several studies were carried out by other researchers for designing peptide vaccine based on genome derived or reverse vaccinology approach. in a study conducted by wei et al. ( ) , the epitopes existing in e protein of jev were identified that possessed the ability for developing peptide vaccine. two t-cell epitopes were determined positioning at amino acid sequences - and - , while six b-cell epitopes positioning at amino acid sequences - , - , - , - , - , and - were identified (wei et al., ) . chikungunya infection, a viral disease spread by aedes mosquitoes, is caused by chikungunya virus (chikv). kori et al. ( ) identified the epitopes from the proteins of three chikv strains having potential for peptide vaccine formulation. the t-cell epitopes identified by them included daekeaeeereaelt, aeeereael and kkkpgrrermcmkie whereas, the b-cell epitopes were found to be qvlkaknigl and sskydlecaq (kori et al., ) . zika virus (zikv) that causes deadly infectious disease results in malformed babies and the decline of survival rate in human. yadav et al. ( ) identified epitopes in the envelope glycoprotein sequence of zikv that could be employed for creating a peptide vaccine to prevent zikv. the t-cell epitope yrimlsvhg possessed the competence for developing immunity (yadav et al., ) . nipah virus (niv) causes respiratory infection and encephalitis in human and the transmission usually occurs through pigs or bats. in order to design peptide vaccines for treatment of niv diseases, two potential epitopes from niv proteins i.e. glycoprotein (g) and fusion (f) protein were detected with the help of immunoinformatics approach. it was reported that either the epitope ewisivpnfilvrnt from g-protein or the epitope gpkvslidtsstiti from f-protein could be used for vaccine development (sakib et al., ) . high-risk human papillomavirus (hrhpvs) is known to cause viral infections as well as cervical cancers in human. in a study, the probable epitopes were identified from e protein of hrhpvs and these epitopes were reported to possess competence in preparing successful peptide vaccine against hrhpvs. based on in silico approach, it was suggested table a immunogenicity (ig) and number of aliphatic amino acids in t-cell epitopes of jev (hopp & woods approach, immunogenicity (ig) and number of aliphatic amino acids in b-cell epitopes of jev (kolaskar & tongaonkar approach, that the t-cell epitopes fafrdlcivyr and rrevydfaf or their mixture could be considered for vaccine formulation and development. moreover, another potential t-cell epitope positioning at - amino acids was also identified as a potential peptide vaccine candidate (khan et al., ) . human coronavirus (hcov) has been reported to cause pneumonia as well as some diseases of the respiratory and the gastrointestinal tracts in human. for developing peptide vaccines against this virus, oany et al. ( ) identified the epitopes in spike proteins that was suggested for boosting immunological response within human body against hcov. from the study, it was evident that the b-cell epitope positioned at - amino acids and the t-cell epitope ksstgfvyf had the ability for designing a peptide vaccine (oany et al., ) . ebola virus disease (evd) is considered to be one of the deadly viral diseases affecting human beings. epitope-based peptide vaccine formulation against evd using reverse vaccinology approach was reported, wherein the potential t-cell epitopes viz., rrtrre, rrkrrd, ktgkkg and dedded and the potential b-cell epitopes viz., hlglddq, pdyddch, qpkcnpn, dqekkil, shyeppn, dyddchs, ptsppqd and eytypds were identified from the viral surface proteins (chakraborty, ) . the present study suggested that a multi-epitope-based peptide vaccine against jev could be developed by combining the promising bcell and t-cell epitopes found in e, prm, ns , ns and ns proteins. in order to design a peptide vaccine, the first criterion is to select the epitopes possessing high antigenicity scores. out of t-cell epitopes examined in five proteins of jev, eight epitopes were identified to possess high antigenicity scores. these epitopes were ekrads, krsrrs, skrsrr, dpedvd, edcgkr, kecpde and krekkp. the epitope ekrads possessed low immunogenicity score and so, it was excluded from further analysis. the remaining seven epitopes possessed high immunogenicity and hydrophilicity values in addition to the desired boman index value. on the basis of total net charges, three epitopes, i.e. dpedvd, edcgkr and kecpde were excluded from the study as these might not bind with the virus. we concluded that four t- among octapeptide b-cell epitopes, seven epitopes namely drykylpe, kpvgryrs, pkpcskgd, pspkpcsk, dtpspkpc, hkdpe-hpy and fykpseps were estimated to possess high antigenicity scores. only one epitope pkpcskgd was identified with excessive immunogenicity, hydrophilicity as well as boman index value. this b-cell epitope could be used in formulating the peptide vaccine. pharmaceutical companies could initiate efforts to synthesize a multi-epitope-loaded peptide vaccine by combining the promising four t-cell epitopes and one b-cell epitope in varying proportions with a strong adjuvant or carrier protein. the peptide vaccine could be administered in jev affected persons following proper clinical trials to ensure its safety and efficacy and might prevent the spread of jev by raising immunity in human. in view of the endless struggle between human and jev in evolutionary context, it is advisable to construct different sets of peptide vaccines comprising the epitopes in varied combinations. to build up a continuing resistance against jev through vaccination, the temporal and spatial deployment strategy (over time and space) of vaccine administration could be followed using different peptide vaccine sets in the countries frequently affected by jev. supplementary data to this 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encephalitis origin and evolution of japanese encephalitis virus in southeast asia the epitopes of foot and mouth disease a new vaccine candidate using reverse vaccinology for vibrio cholera in cholera disease factors in the changing epidemiology of japanese encephalitis and west nile fever. fac. emerg. arbovirus dis phylogenetic analysis suggests only one serotype of japanese encephalitis virus human t cell responses to japanese encephalitis virus in health and disease phylogenetic analysis of japanese encephalitis virus: envelope gene based analysis reveals a fifth genotype, geographic clustering, and multiple introductions of the virus into the indian subcontinent the epidemiology of japanese encephalitis: prospects for prevention design and evaluation of a multi-epitope peptide against japanese encephalitis virus infection in balb/c mice signal transduction by lymphocyte antigen receptors flaviviridae intervirology. computational modeling and analysis of prominent t-cell epitopes for assisting in designing vaccine of zika virus population dynamics of flaviviruses revealed by molecular phylogenies the authors are grateful to assam university, silchar, assam, india for providing necessary facilities to carry out this research work. further, the research work is dedicated to those great souls who died of jev due to lack of proper medication and affordable medical facility. key: cord- - vlxa i authors: williamson, e. d.; westlake, g. e. title: vaccines for emerging pathogens: prospects for licensure date: - - journal: clin exp immunol doi: . /cei. sha: doc_id: cord_uid: vlxa i globally, there are a number of emerging pathogens. for most, there are no licensed vaccines available for human use, although there is ongoing research and development. however, given the extensive and increasing list of emerging pathogens and the investment required to bring vaccines into clinical use, the task is huge. overlaid on this task is the risk of anti‐microbial resistance (amr) acquisition by micro‐organisms which can endow a relatively harmless organism with pathogenic potential. furthermore, climate change also introduces a challenge by causing some of the insect vectors and environmental conditions prevalent in tropical regions to begin to spread out from these traditional areas, thus increasing the risk of migration of zoonotic disease. vaccination provides a defence against these emerging pathogens. however, vaccines for pathogens which cause severe, but occasional, disease outbreaks in endemic pockets have suffered from a lack of commercial incentive for development to a clinical standard, encompassing phase iii clinical trials for efficacy. an alternative is to develop such vaccines to request us emergency use authorization (eua), or equivalent status in the united states, canada and the european union, making use of a considerable number of regulatory mechanisms that are available prior to licensing. this review covers the status of vaccine development for some of the emerging pathogens, the hurdles that need to be overcome to achieve eua or an equivalent regional or national status and how these considerations may impact vaccine development for the future, such that a more comprehensive stockpile of promising vaccines can be achieved. globally, there are a number of emerging and re-emerging pathogens. some of these cause endemic disease in regions of the globe, where they are maintained in zoonotic reservoirs and transmitted to man either by direct or indirect contact. for most of the emerging pathogens there are no licensed vaccines available for human use, although there is ongoing research and development. however, given the extensive and increasing list of emerging pathogens and the time and investment required to bring vaccines into clinical use, the task is huge. overlaid on this task is the risk of anti-microbial resistance (amr) acquisition by micro-organisms which can endow a relatively harmless organism with pathogenic potential. furthermore, climate change also introduces a challenge by causing some of the insect vectors and environmental conditions prevalent in tropical regions to begin to spread out from these traditional areas, thus increasing the risk of migration of zoonotic disease. vaccination provides a defence against these emerging pathogens. however, to date, vaccines for pathogens which cause severe, but occasional, disease outbreaks in endemic pockets have suffered from a lack of commercial incentive for development to a clinical standard. while approval of vaccines for diseases caused by such pathogens would clinical and experimental immunology review article series editor: e diane williamson make a significant impact on disease outbreaks, taking niche vaccines into clinical development, including phase iii clinical trials for efficacy, requires a large investment in time and money. an alternative is to develop such vaccines to request us emergency use authorization (eua), or an alternative status in the united states, canada and european union (eu) making use of a considerable number of alternative regulatory mechanisms that are available prior to licensing, so that the products are deployable at the first indications of a disease outbreak. this review covers the status of vaccine development for some of the emerging pathogens, the hurdles that need to be overcome to achieve eua or an equivalent regional or national status and how these considerations may impact vaccine development for the future, such that a more comprehensive stockpile of promising vaccines can be achieved. pathogens which are classed as emerging or re-emerging are identified through global surveillance programmes and organizations such as the world health organization (who). although labelled 'emerging', most of these pathogens are not new and have either been quiescent in the environment until the conditions are opportune to emerge or have evolved from a parent organism to adapt to the prevailing conditions. thus, there is an intricate relationship between the environment, the climate, wildlife and human existence and lifestyle. the coronaviruses exemplify this point: the ancestral virus possibly existed approximately years ago [ ] . coronaviruses have a wide species range infecting birds, bats, chickens, pigs, dogs, cats and rodents [ ] . however, the first human coronavirus was described only in the s [ , ] , and the coronavirus causing severe acute respiratory syndrome (sars) was discovered only in [ ] [ ] [ ] , while that causing middle east respiratory syndrome (mers) first emerged in [ ] . it is likely that warm-blooded flying birds and bats have co-evolved with the coronaviruses to aid dissemination [ ] . for example, sars is thought to have first infected old world bats, then spreading to horseshoe bats [ ] , civets and finally to man [ ] . in the outbreak of sars in china and adjacent countries, phylogenetic analysis suggested that the virus spread from bats to humans, possibly through the intermediary civet species. neither bats or civets showed any clinical signs of infection, and it is thought that bats are the main zoonotic reservoirs for the virus [ ] . organizations such as the who, national institutes for allergy and infectious diseases (niaid) and the us centers for disease control (cdc) publish lists of emerging pathogens which may be viral, bacterial or rickettsial in nature. the who priority list contains viruses which have been prioritized as the most likely to cause epidemics and for which the who will establish a blueprint programme for accelerated research and development (r&d) [ ] . the list published in february is shown in table . niaid [ ] and cdc [ ] also publish lists of pathogens of priority which comprise bacteria and viruses, but categorize these into three groups depending on pathogenicity, accessibility and the availability of vaccines and therapies. all the viruses listed by the who also occur on these lists, alongside bacterial pathogens of concern. one of these is yersinia pestis, causative of bubonic and pneumonic plague, which is recognized by all three bodies (who, cdc, niaid) as a current priority following the exceptionally large and serious outbreak between september to april in madagascar [ ] , where the disease is endemic. in addition, niaid recognizes the added threat to human health posed by the acquisition of amr by pathogens and who has also published a priority list [ ] of bacterial species for which r&d is required to develop new antibiotics (table ) . globally, there are many regions of endemic disease which are maintained by reservoirs of zoonotic pathogens in the local wild animal species. these pathogens comprise viral, bacterial and rickettsial species and some have complex lifecycles, infecting an environmental (e.g. standing water) or animal reservoir and then being transmitted either by direct contact with man or indirectly, via an insect (e.g. mosquito) or mammalian (e.g. bat, civet, camel) vector, to man, from which they may be spread by human-tohuman transmission. (fig. ). an example of a serious and widespread bacterial infection is mosquito-transmitted plasmodium falciparum, causing malaria in many tropical regions. malaria causes significant morbidity and co-morbidity in these regions in which it is endemic. due to the complex life cycle of the causative bacterium, it has been challenging to achieve a vaccine for malaria. the most advanced candidate is the rts,s/as vaccine [ ] , which is undergoing a pilot implementation in three countries in sub-saharan africa, with a view to determining its impact on disease prior to a wider implementation [ ] . despite the availability of approved vaccines [ , ] , typhoid fever and cholera remain significantly debilitating enteric diseases in regions of the world where hygienic living conditions are poor and there is little access to health care. both infections are caused by bacteria (salmonella typhi and vibrio cholera, respectively) which exist in contaminated water and food. tuberculosis (tb) is a respiratory infection which is widespread globally. caused by mycobacterium tuberculosis, and transmitted to man from zoonotic reservoirs (badgers, cattle) with a high potential for subsequent human-to-human transmission, tb is prevalent in susceptible individuals living in overcrowded conditions [ ] . although the bcg vaccine has been in routine use for many years, variable efficacy has been reported, depending on region of use [ , ] with - % reported in the united kingdom but lower levels in equatorial countries. the emergence of multi-drugresistant tb (mdr tb) in recent years raises the bar for treatment of this disease and makes a high level of vaccine efficacy even more important [ ] . examples of zoonotic reservoirs which maintain endemic viral diseases are numerous and are summarized in table . in particular, viral endemic disease is caused by the coronaviruses (mers and sars) in saudi arabia and the eastern mediterranean countries; and by the lassa arenavirus, which is endemic in west african countries and caused a serious outbreak of lassa fever in nigeria in [ ] ; other viruses which are endemic and cause viral haemorrhagic fever include dengue, which is widespread in tropical and subtropical regions worldwide [ ] , the filoviruses (ebola and marburg) which have a zoonotic reservoir in bats in sub-saharan africa [ ] ; and yellow fever virus, which is prevalent in africa, central and south america and the caribbean [ ] . interestingly, the same mosquito (aedes aegypti) which spreads yellow fever virus also transmits the dengue, chikungunya and zika viruses [ ] . chikungunya virus has a widespread distribution in africa, asia, india and south america and with occasional cases in europe, and causes a debilitating, but rarely fatal, disease [ ] . zika virus emerged in brazil in [ ] , although it was first detected in monkeys in uganda as early as [ ] , and the first documented human case occurred in nigeria in [ ] . the large brazilian outbreak of zika viral disease culminated in , with thousands of cases reported [ ] . rift valley fever virus (rvfv) is another zoonotic virus which is endemic in sub-saharan africa and primarily infects cattle, sheep and goats, but can be transmitted to man by mosquito bite to cause an acute fever [ ] . an example of a bacterium which has been classed as a re-emerging pathogen and which is endemic in global regions is y. pestis, causative of plague. bubonic [ ] [ ] [ ] . over thousands of years, y. pestis has evolved away from the enteric yersinia species to become a lethal flea-transmitted bacterium [ ] . it is transmitted to man, typically from a zoonotic reservoir in infected rats or other rodents (e.g. ground squirrels or prairie dogs), by flea-bite to cause bubonic plague [ ] (fig. ). if not detected and treated, this can develop into either septicemic plague or the most serious form of all, a secondary pneumonic plague. in turn, individuals with pneumonic plague can transmit this by aerosol droplet to others, to establish a primary pneumonic plague infection. each year, a few cases of plague are also reported in the southwestern united states, where the disease has been endemic in the rodent population since the late s [ ] . as well as infecting the rodent population, infected fleas can spread y. pestis to other wildlife species (e.g. rabbits/hares or, rarely, domesticated animals [ ] ) which, in turn, raises the potential of transmission to man by inhalation to cause a primary pneumonic plague. in endemic areas, outbreaks of plague are often associated with seasonal environmental changes, causing rodents to stray closer to human habitation. the plague outbreak in madagascar during / was particularly serious, with an estimated cases and deaths ( · % fatality rate) [ ] . this outbreak was approximately sixfold greater than usual, with an unusual predominance of pneumonic, rather than bubonic plague. although y. pestis is susceptible to antibiotics, such as the aminoglycosides (gentamycin, streptomycin), the fluoroquinolones (e.g. ciprofloxacin) or tretracyclines (e.g. doxycycline) [ ] , these need to be administered very early to a suspected plague case, and ideally before symptoms emerge. additionally, there have been reported instances of antibiotic resistance including to multiple antibiotics [ , ] . hence, there is a clear and increasingly urgent need for an efficacious vaccine. other bacterial endemic diseases include melioidosis and glanders which, although not caused by zoonotic pathogens, are endemic in southeast asia where the bacteria reside in soil and are transmitted to humans through occupational exposure, e.g. working in paddy fields [ ] . another bacterial disease which is endemic in the northern hemisphere is tularaemia, caused by the bacterium francisella tularensis, which has zoonotic reservoirs in the rabbit, hare and rodent populations in the south, central and western united states and is transmitted to man by ticks and biting flies [ ] . rickettsial species, such as coxiella burnetii, causative of q-fever, comprise bacteria which exist within another cell, and as such q-fever is contracted when humans are exposed to aerosolized droplets from the urine, milk, faeces or birth detritus from infected sheep, goats and cattle [ ] . for all these pathogens, there is a requirement for efficacious approved vaccines to curtail or prevent regular disease outbreaks. for some of these pathogens (e.g. rvfv, cchf) there are vaccines [ ] , but these are not widely available [ ] or have been used and withdrawn for safety or regulatory reasons (e.g. the live vaccine strain for tularaemia) [ ] or they are not universally suitable, requiring a screening test prior to administration due to the potential for a hypersensitivity response (as for the vaccine for q-fever) [ ] . there is no readily available licensed plague vaccine, although a series of killed whole cell vaccines (kwcv) has been produced and used in the past, mainly in the biodefence context (reviewed in [ ] ). additionally, live attenuated plague vaccines, derived from an attenuated mutant strain as the ev series, have been used in the former ussr (fussr) and are still used in asia, notably in russia and china [ , ] . ev , the most commonly cited of these, provides protective efficacy against plague, but is licensed for human use only in countries of the fussr and is documented to cause serious adverse effects in non-human primates and malaise and adverse effects in human vaccinees [ ] . whatever the context, all these diseases would be positively impacted by the availability of efficacious and approved vaccines. however, to a greater or lesser extent they are all niche diseases with no major commercial incentives to drive vaccine development programmes. this is a space that non-governmental organizations (ngos) such as the coalition of epidemic preparedness innovations (cepi) [ ] and global vaccine alliance (gavi) [ ] have entered and they are supporting vaccine efforts for some of the diseases listed above. additionally, philanthropic funders such as the gates foundation are supporting vaccine r&d efforts [ ] . in the united kingdom, and subsequent to the ebola outbreak in west africa, vaccine networks for human and veterinary vaccines have formed to prioritize vaccine efforts in these respective contexts [ ], while the department of health, together with innovate uk, has supported r&d of vaccine candidates for the prioritized pathogens [ ] . as a result of these global initiatives, a number of candidate vaccines are being developed for emerging bacterial and viral pathogens, examples of which, although by no means exhaustive, are cited here [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . who reports ongoing global vaccine r&d efforts [ ] and also tracks the progress of clinical trials for emerging pathogens [ ] . here it may be worth drawing a distinction between prophylactic vaccination, i.e. general use prophylaxis (gup), given routinely and not necessarily in the face of specific, predicted outbreaks, in contrast to post-exposure prophylactic (pep) vaccination, to be given after a suspected exposure to a pathogen, or to ring-fence an outbreak or, indeed, post-exposure therapeutic (pet) vaccination, to be given after an actual exposure. in the pep and pet contexts, the benefit of vaccination vastly outweighs the risk of disease (i.e. the risk : benefit ratio is low), while in the prophylactic context the risk : benefit ratio may be greater than, or equal to, · ). for infections with short incubation times, e.g. less than h, as for pneumonic plague, pep or pet vaccination may only be useful if administered under antibiotic cover. in the united states, once a vaccine candidate has been thoroughly tested for safety in non-clinical models, and in an escalating-dose, statistically powered, phase i design in the clinic, from then on it may be possible to pursue approval for an eua, rather than pursue the full-length pathway to biological licensing authorization (bla). this can enable the earlier availability of vaccines for use in endemic regions. however, it should be noted that eua is only available through the food and drug agency (fda) in the united states. some alternative regulatory mechanisms that may be considered prior to licensing are outlined below. from the discovery phase to the clinic, vaccine development requires the completion of a series of steps represented as a generic outline in fig. . the regulatory agencies lay down specific guidance for these steps [ , ] and this review presumes no authority in this regard, but seeks to give a generalized overview of a generic vaccine development process. exit from the discovery and preclinical phases requires substantive data demonstrating immunogenicity and efficacy in at least one suitable animal model(s). where possible, efficacy in the animal model should be demonstrated by direct exposure to the pathogen concerned. technology transfer for manufacture under good manufacturing practice (gmp) will require a demonstration of known provenance of all essential materials required in the manufacture of the vaccine candidate. this includes seed stocks of cell lines from which recombinant proteins may be expressed, e.g. escherichia coli, human embryo kidney cells, baculovirus, tobacco mosaic virus; or seed stocks of attenuated vaccine vectors, e.g. viral vectors such as adenovirus, vesicular stomatitis virus, modified vaccinia ankara; or bacterial vaccine vectors, e.g. salmonella, listeria; the genetic constructs cloned into the cell line in question; all culture media and components and all formulations and excipients. transfer of the manufacturing process to gmp may include scale-up and conversion to, for example, fermentation conditions, or to plant-based, mammalian cell line or insect cell line expression on an expanded scale. this transfer will probably require the demonstration of consistency between consecutive batches at gmp, which will also enable the development of scaled-up downstream processing methodology. vaccine components (the drug substance) from these batches may be formulated (the drug product) as required and used in safety/toxicology studies and for immunogenicity/efficacy in an appropriate second animal model, which will be as representative as possible of the human response. the second animal model is often, but not necessarily, a non-human primate, and the selection of this second model will depend entirely on the vaccine indication. there will also be a requirement to generate sufficient stability data on both the drug substance and the drug product and to demonstrate that the drug product is stable for at least the duration of the intended phase i clinical trial under prevalent conditions in that location. clearly, extended stability testing under a range of conditions, including accelerated conditions (high ambient temperature and relative humidity), will also be required to progress the vaccine through development. as well as determining its stability, both the drug substance and product will require characterization for properties such as identity, purity, isoelectric point, osmolality, endotoxin levels and potency, to demonstrate consistency between batches and to allow for release of these for clinical trials. it is essential to ensure safety of the drug product before entering a clinical trial, and the drug product may be tested in suitable small animal models for the absence of adverse effects under conditions of repeated dosing at the anticipated human dose level, as well as in the intended human schedule, but at multiples of the anticipated human dose-level. the use of rodent models (mouse or rat) for this testing allows for sufficient statistical powering of such safety/toxicological testing. if the clinical trial is to be conducted in women of child-bearing age, it may be necessary to carry out reproductive toxicology testing of the drug product; in this case it may be necessary also to use a sensitive rabbit model and to administer the vaccine to pregnant rabbits to screen for adverse effects in the mother and potential teratogenic effects in the f generation. the national regulatory authority will expect to review the existing safety data and outlines of further protocols to be used. a review of all the data pertaining to the candidate vaccine will be required by the regulators in order to proceed to a clinical trial. depending on the specific requirements of the regulatory authorities in the country of origin/manufacture of the vaccine and also the intended location of the clinical trial, this may take the form of an investigator's brochure, protocol and summaries of manufacturing, non-clinical and clinical data in a clinical trial application (for the european medicines agency, ema) or an investigational new drug (ind) application (e.g. us fda). other international regulators (e.g. in canada, japan and china) will have variations on the documentation required. in the united states, the alternative approaches to full marketing approval that are available for vaccines under development and which may be considered through the fda are to either request an eua or to request expanded access (ea), sometimes called compassionate use. the eua is issued in support of potential and actual public health, military and domestic emergencies involving chemical, biological, radiological and nuclear agents (cbrn), including emerging infectious diseases, e.g. pandemic influenza. such fda-approved medical products which may be stockpiled for use in emergencies are referred to as medical countermeasures (mcm) and include biological products, e.g. vaccines, drugs and devices. specifically, the eua authority is separate and distinct from use of an investigational medical product held under an ind and must be able to treat serious or life-threatening diseases or conditions. in contrast, ea submissions are for products used under an ind or a protocol (treatment plan), or submitted as a protocol amendment to an existing or new ind. the ea categories cover use under either an individual patient ind or protocol, individual emergency ea use, or ea for an intermediate-size population or for widespread use (large populations). other us regulatory programmes for biologicals and drugs to treat serious or life-threatening conditions include fast-track designation, breakthrough therapy designation, accelerated approval pathway and priority review designation. in canada, access to drugs through special access programmes (saps) exists for serious and life-threatening conditions, when marketed alternative products are not available or unsuitable and evidence supports the intended use. priority review of marketing submissions and 'notice of compliance with conditions' for such products may also be considered and granted by health canada. in the european union, the ema supports early patient access to new medicines and which are eligible for a marketing authorization application under the centralized procedure and either target unmet medical needs or those which have a major public health interest. the ema has launched a priority medicines (prime) scheme to facilitate early dialogue and support the development of medicines that target unmet medical needs and which promotes an accelerated regulatory assessment. in addition, the prime scheme is intended for seriously debilitating or life-threatening diseases, compassionate use of unauthorized medicines for patients with an unmet medical need (when no satisfactory treatment is available in the european union) and also provides conditional marketing authorization. the regulation and rules of access to compassionate use programmes varies between national government authorities across eu member states. in the united kingdom, the early access to medicines scheme (eams) gives patients with life-threatening or debilitating conditions access to innovative medicines without marketing authorization, but for which there is a clear medical need. a two-stage evaluation process initially considers promising innovative medicine (pim) designation before an eams scientific opinion. also in the united kingdom, the potential supply of unlicensed medicinal products (specials) may be considered by the medicines and healthcare products regulatory agency (mhra) for the importation of medicines that have marketing authorization status from countries outside the united kingdom and european union. where there is an unmet need of priority to the who for a new vaccine, the who may establish a blueprint programme [ ] . as vaccine candidates for the specific blueprint programme advance through the development process, and particularly when they have attached clinical data, the who may run a prequalification check to determine whether the candidate meets their requirements on behalf of un agencies that will ultimately purchase vaccines [ ] . to do this, who will engage with subject matter experts to draft and publish a target product profile (tpp) for a vaccine requirement. who will use its scientific advisory group of experts (sage) to review vaccine candidate performance against the tpp and to prequalify a vaccine candidate(s) which fulfils the tpp. assuming that the preclinical safety and toxicology testing has been satisfactory, a protocol for phase i testing of the vaccine candidate may be submitted to the regulatory authorities for approval. the primary objective of the phase i clinical trial is to test the vaccine candidate for safety in informed and consenting adult volunteers who will be selected according to pre-agreed inclusion/exclusion criteria. as this is the first time the candidate is being used in man, the phase i trial is typically small and cautious. if a range of dose-levels is being tested, it may be necessary to dose a sentinel group of single subjects from each arm of the study to assess safety through week of immunization and starting at the lowest level before proceeding to the next level. the data from the sentinel group would be reviewed before proceeding with the main study. then the first cohort would be dosed at the lowest level before proceeding to the next level, and so on. volunteers will be closely monitored for adverse effects in the clinic at each dosing time-point and will typically maintain a diary at home to record any symptoms arising between time-points. an independent safety monitoring panel, including medically qualified personnel, will be required to monitor the reporting of adverse effects. volunteers may also be blood-sampled to assess vaccine immunogenicity from baseline and serial samples, and depending on vaccine type may be required to supply additional samples which can be collected non-invasively, e.g. saliva/stool samples. all volunteers may be followed up for a pre-agreed period on completion of the study, to check for latent adverse events and to monitor, e.g. for maintenance of circulating antibody titres or memory response to the vaccine. the second phase of clinical trials typically allows for an enlarged study to assess vaccine safety and to monitor immunogenicity. during this phase, significantly expanded cohorts of volunteers may be dosed with vaccine at doselevel(s) selected as optimum from the phase i trial and can be monitored for safety and a more detailed immunogenicity assessment, which may include assays of both serological and cellular memory responses. once again, volunteers may be blood-sampled for baseline and serial serum/plasma samples, and depending on vaccine type may be required to supply additional samples which can be collected non-invasively, e.g. whole blood/saliva/stool. in the event that a phase iii trial for efficacy is not feasible, for ethical or practical reasons, it may be necessary to plan a blood-sampling regimen in the volunteers which will enable sufficient sample volumes to be available for animal rule studies (discussed below). if sufficient safety data are gained from a phase ii trial, the regulatory authorities may be able to consider the vaccine for eua. the third phase of clinical trials is typically designed to assess efficacy. however, where this is not feasible for either practical/logistical or ethical reasons, phase iii could be regarded as a further, significantly enlarged trial for vaccine safety with adequate follow-up of vaccinated volunteers. a phase iii field trial of vaccine efficacy may be feasible in the event of an anticipated seasonal outbreak, in which case vaccination could be given prophylactically and well in advance. as long as effective biosurveillance programmes are in place to instigate a rapid response to new infections in an unvaccinated placebo cohort, the latter group may be included in a prophylactic efficacy trial. conversely, reactive mode vaccination in the context of an actual outbreak is likely to be more complicated, as it would be unethical to omit anyone at risk of infection from the vaccination programme. hence, sufficient safety data on the candidate vaccine would be required in case of need to administer it to pregnant/nursing mothers, children and elderly people as well as to the general adult population. additionally, it may be necessary to administer an adjunct to the vaccine, such as an appropriate antibiotic, to both cohorts (vaccinated and placebo). whatever the context, careful consideration of the trial design needs to be made and approved by the regulators to achieve adequate statistical powering and to determine the need to provide supplementary antimicrobial therapy, and also the need for a placebo cohort. after the anthrax letters incident in the united states in in which anthrax was released through the postal system, resulting in five fatalities and widespread anxiety [ ] , the fda issued the animal rule (fda cfr) [ ] to expedite the development of vaccines and therapies for biothreat agents for which it is neither feasible nor ethical to carry out phase iii efficacy studies in man. in this situation, the animal rule makes provision for the substitution of animal efficacy data for human efficacy data. the animal efficacy data should demonstrate a reasonable likelihood that the candidate vaccine or therapy would be efficacious in man [ ] . the animal efficacy data should be provided ideally from more than one animal model, or from one model only if that model is well-characterized and authentically represents the human disease syndrome [ ] . in addition, the fda may require supporting data to include pharmacokinetic/pharmacodynamic (pk/pd data) and a determination of the pathophysiological mechanism of action of the biothreat agent and a 'reasonable understanding' of the protective mechanism of the proposed vaccine or therapy. thus, if the candidate is a vaccine, a reasonable understanding of the protective mechanism(s) being invoked by it requires an identification of the immune correlates of protection. immune parameters which correlate with protection in the selected animal models may include the total titre of circulating antibody induced to the vaccine and the determination of a minimum cut-off titre required to confer protection. alternatively, the titre of functional or neutralizing antibody within that total may provide the correlate, particularly where the neutralization of specific virulence factors produced by the biothreat agent is identified and quantifiable [ ] . for some vaccines, the induction of a cellular memory response instead of, or in addition to, circulating antibody will provide the correlate. this is measurable by the ex-vivo recall response of peripheral blood mononuclear cells (pbmcs) on stimulation with the vaccine antigen(s) [ ] . in the absence of direct efficacy testing in the human vaccinee, the immune correlate identified above provides a surrogate marker of efficacy. if this is a functional antibody, the titre induced in man needs to be compared with the titre required in the animal model(s) to provide protection. this can be achieved, for example, by the in-vitro neutralization of a specific virulence factor, or by a competitive enzyme-linked immunosorbent assay (elisa), where the test antibody is competed with a known neutralizing antibody for binding to the target antigen or by the passive transfer of antibody from a human vaccinee into a naive animal, followed by pathogen challenge [ ] . whatever the immune correlate may be, its effect would be expected to follow the pattern shown in fig. , where as the value increases, the likelihood of death in the vaccinee decreases [ ] . thus, vaccine efficacy = relative reduction in risk of death = − (% of vaccinated subjects at < protective level) (% of unvaccinated subjects at< protective level) . although a number of immunotherapies and pretreatments have been licensed under the animal rule, the first vaccine to be licensed in these circumstances is biothrax for therapeutic vaccination in suspected exposure to anthrax [ ] . a case for the ancient origin of coronaviruses identification of alpha and beta coronaviruses in wildlife species in france: bats, rodents, rabbits and hedgehogs cultivation of novel type of commoncold virus in organ cultures a new virus isolated from the human respiratory tract identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome the aetiology of sars: koch's postulates fulfilled the emergence of the middle east respiratory syndrome coronavirus discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats bats, civets and the emergence of sars review of bats and sars naiad/nih priority list of pathogens. available at cdc priority list of pathogens. available at the plague outbreak in madagascar: data descriptions and epidemic modelling who priority list of bacteria rts,s malaria vaccine efficacy and immunogenicity during plasmodium falciparum challenge is associated with hla genotype malaria vaccine implementation programme (mvip) -programme advisory group vaccines for preventing typhoid fever global economic evaluation of oral cholera vaccine: a systematic review diagnosis and treatment of tuberculosis: latest developments and future priorities efficacy of bcg vaccine in the prevention of tuberculosis variation in protection by bcg: implications of and for heterologous immunity drug-resistant tb: deadly, costly and in need of a vaccine genomic analysis offers insight into nigeria lassa fever outbreak ecological niche modeling for filoviruses: a risk map for ebola and marburg virus disease outbreaks in uganda sep yellow fever in africa and the americas: a historical and epidemiological perspective zika, chikungunya and dengue: the causes and threats of new and re-emerging arboviral diseases chikungunya: bending over the americas and the rest of the world the zika virus epidemic in brazil: from discovery to future implications zika: the origin and spread of a mosquito-borne virus zika virus outbreak of zika virus infections the pathogenesis of rift valley fever ecologic features of plague outbreak areas, democratic republic of the congo mechanism study on a plague outbreak driven by the construction of a large reservoir in southwest china (surveillance from - ) the natural history and incidence of yersinia pestis and prospects for vaccination yersinia pestis, the cause of plague, is a recently emerged clone of yersinia pseudotuberculosis plague into the st century yersinia pestis: examining wildlife plague surveillance in china and usa cat-transmitted fatal pneumonic plague in a person who travelled from colorado to arizona cdc plague treatment transferable plasmid-mediated resistance to streptomycin in a clinical isolate of yersinia pestis resistance of yersinia pestis to antimicrobial agents melioidosis in thailand: present and future tularemia transmission to humans: a multifaceted surveillance approach airborne geographical dispersal of q fever from livestock holdings to human communities: a systematic review and critical appraisal of evidence current status of rift valley fever vaccine development the use of veterinary vaccines for prevention and control of rift valley fever: memorandum from who/fao meeting protection induced by a francisella tularensis subunit vaccine delivered by glucan particles standardized guinea pig model for q fever vaccine reactogenicity plague vaccine development: current research and future trends developing live vaccines against plague russian vaccines against especially dangerous bacterial pathogens cepi new vaccines for a safer world about gavi, the vaccine alliance vaccine delivery:bill and melinda gates foundation dual route vaccination for plague with emergency use applications immunogenicity and safety of subunit plague vaccine: a randomized phase a clinical trial rift valley fever mp- vaccine phase clinical trial: safety, immunogenicity and genetic characterisation of virus isolates development of vaccines against cchf virus a monovalent chimpanzee adenovirus ebola vaccine boosted with mva effect of dengue serostatus on dengue vaccine safety and efficacy non-neutralising antibodies elicited by recombinant lassa-rabies vaccine are critical for protection against lassa fever chadox and mva based vaccine candidates against mers-cov elicit neutralising antibodies and cellular immune responses in mice vaccine and therapeutic options to control chikungunya virus chikungunya vaccines in the pipeline a bacteriophage t nanoparticle based dual vaccine against anthrax and plague who: health products in the pipeline for infectious diseases world health organization. who vaccine pipeline tracker yjurcmg xwo kvuyedybmzdcxqbyjgdczm/pubhtml# fda guidances for vaccine development vaccines for emerging pathogens: from research to the clinic. part regulatoryinformation/guidances/vaccines/default.htm ema guideline for vaccine development who guidance for clinical evaluation of vaccines who vaccine standards: vaccine prequalification. available at the anthrax vaccine and research: reactions from postal workers and public health professionals code of federal regulations (cfr) cfr . - . evidence needed to demonstrate effectiveness of new drugs when human efficacy studies are not ethical or feasible predictive models and correlates of protection for testing biodefence vaccines first vaccine approval under the fda animal rule the authors declare no competing interests. key: cord- -qnijw y authors: morgene, m. fedy; botelho-nevers, elisabeth; grattard, florence; pillet, sylvie; berthelot, philippe; pozzetto, bruno; verhoeven, paul o. title: staphylococcus aureus colonization and non-influenza respiratory viruses: interactions and synergism mechanisms date: - - journal: virulence doi: . / . . sha: doc_id: cord_uid: qnijw y viral infections of the respiratory tract can be complicated by bacterial superinfection, resulting in a significantly longer duration of illness and even a fatal outcome. in this review, we focused on interactions between s. aureus and non-influenza viruses. clinical data evidenced that rhinovirus infection may increase the s. aureus carriage load in humans and its spread. in children, respiratory syncytial virus infection is associated with s. aureus carriage. the mechanisms by which some non-influenza respiratory viruses predispose host cells to s. aureus superinfection can be summarized in three categories: i) modifying expression levels of cellular patterns involved in s. aureus adhesion and/or internalization, ii) inducing s. aureus invasion of epithelial cells due to the disruption of tight junctions, and iii) decreasing s. aureus clearance by altering the immune response. the comprehension of pathways involved in s. aureus-respiratory virus interactions may help developing new strategies of preventive and curative therapy. the development of upper and lower respiratory tract infections is determined by the interaction between one micro-organism and the host immune response. more recently, the interaction between the resident microbiota and incoming pathogens may also participate in the development of respiratory tract infections. it has been postulated for a long time that viral infection of the respiratory tract indirectly predispose to bacterial superinfection by disruption of the respiratory mucosal epithelium [ ], or passively through anatomical and mechanical changes like eustachian tube dysfunction [ ], ostiomeatal obstruction and reduced mucocilliary clearance [ ] . over the last decades, there was an increasing interest to investigate the contribution of bacterial colonization in the outcome of viral respiratory tract infections. the human respiratory tract is known to be the reservoir of diverse commensals and potential pathogens including mainly staphylococcus aureus, streptococcus pneumoniae and haemophilus influenzae that compose a significant part of the respiratory tract microbiota [ ] . the role of interactions between viruses and bacteria in the pathogenesis of respiratory infections have been extensively studied in the literature and notably those between influenza viruses and s. aureus or s. pneumoniae [ ] [ ] [ ] [ ] [ ] [ ] . however, interactions between s. aureus and non-influenza respiratory viruses were not reviewed recently. the aim of this report is to describe the current knowledge on possible interactions between s. aureus and non-influenza viral pathogens in the respiratory tract, with a focus on the mechanisms by which these interactions are potentially mediated. impact of viral infections in the respiratory tract on staphylococcus aureus colonization s. aureus is a commensal bacterium of the skin and mucosa, colonizing to % of the whole population [ , ] . the main reservoir of s. aureus carriage in humans remains the nose [ ] , but other sites of carriage have been reported, such as the skin [ ] , pharynx [ , ] , vagina [ ] , and rectum [ ] . s. aureus is however a janus-faced bacterium and beyond its commensal status, it is also a life-threatening pathogen. it is in fact considered to be one of the leading causes of nosocomial and community-acquired bacterial infections [ ] . in addition to be the most common cause of bacteremia, with a % mortality rate despite appropriate treatment [ ] , it is also known as an etiological agent of other deep-seated infections including osteomyelitis, septic arthritis, endocarditis and device-related infections [ , ] . s. aureus is also an important pathogen in lung infection, mostly implicated in hospital-acquired pneumonia [ ] . s. aureus expresses a wide repertoire of surface proteins that recognize cellular adhesive molecules and it is therefore able to adhere to and internalize into lung epithelial cells, which protects the bacteria from the host immune system and facilitate chronic infection [ ] . in vitro experiments have demonstrated that the activation of nf-κb (nuclear factor kappa-light-chain-enhancer of activated b cells) signaling pathway in infected pulmonary epithelial cells results in inflammation enhancement via il- expression; furthermore, intracellular s. aureus can lead, after an initial lag period, to the apoptosis of these cells. [ , ] in necrotizing pneumonia, the key virulence factors of s. aureus associated with the apoptosis of lung cells were shown to be pore-forming toxins, namely panton-valentine leukocidin (pvl) and alpha-hemolysin [ , ] . besides, s. aureus is frequently involved in secondary bacterial pneumonia occurring during seasonal influenza outbreaks [ ] . during the a/h n influenza pandemic, bacterial co-infections complicated up to one-third of influenza cases in the united states in which s. aureus was the most common pathogen, accounting for % of the cases in both critically ill children and adults [ , ] . s. aureus co-infection was associated with significantly higher morbidity and mortality [ ] . to date, the clinical association between the healthy carriage of s. aureus and the secondary staphylococcal pneumonia is still unclear. however, recent evidence from in vitro and in vivo tests showed that host physiologic changes induced by influenza virus can lead to the transition from asymptomatic colonization to invasive disease [ ] . in addition to influenza viruses, s. aureus nasopharyngeal carriage has been found to be associated with some other respiratory viruses like rhinovirus and respiratory syncytial virus (rsv). rhinovirus is the second respiratory virus that has been most frequently reported to interact with s. aureus. several studies showed that natural or experimental rhinovirus infection in s. aureus nasal carriers leads to increased s. aureus airborne dispersal, especially when sneezing is a part of the syndrome [ ] [ ] [ ] [ ] [ ] [ ] . these studies emphasized that rhinovirus infection may facilitate the spreading of s. aureus from staphylococcal carriers to their environment and the transmission of the bacterium between humans. a recent study showed that rhinovirus infection is associated with changes in the upper respiratory tract microbiota [ ] . in this study, healthy adults who were experimentally infected by rhinovirus showed increase in the relative abundance of h. parainfluenzae, neisseria subflava and s. aureus, and returned to their baseline level after the infection was cleared [ ] . it has been also shown that experimental rhinovirus infection significantly increases s. aureus nasal load by % compared to baseline bacterial load [ ] . these findings suggest that changes in the composition of respiratory microbiota following rhinovirus infection may play a role in the development of bacterial superinfection. however, the role of rhinovirus infection on the onset or increase of staphylococcal carriage in human remains poorly studied. a prospective microbiological analysis showed that % of children with severe rsv bronchiolitis had a bacterial co-infection in their lower airways and were at increased risk for bacterial pneumonia [ ] . bacterial co-infection in children with rsv bronchiolitis seems to increase inflammatory markers, abnormal radiologic patterns and hospital stay [ , ] . in young infants with rsv bronchiolitis, s. aureus, h. influenzae, m. catarrhalis and s. pneumoniae are the most common pathogens colonizing the nasopharynx and the lower airways [ , ] . a recent prospective study investigated the differences in the nasopharyngeal microbiome during acute respiratory tract infections due to human rhinovirus or rsv in infants aged less than months [ ] . by contrast to previous studies, s. aureus was not found among the most abundant bacteria. however, the difference of s. aureus abundance was significantly higher in rsv than in rhinovirus-infected infants [ ] . another prospective study investigated the nasopharyngeal microbiota in young infants with rsv infections by s-rna sequencing; the authors found that rsv infection was positively associated with h. influenzae and s. pneumoniae, but negatively associated with s. aureus nasopharyngeal colonization [ ] . in another study concerning nasopharyngeal aspirates from children with rsv infection aged between months and years, s. aureus was shown to colonize % of rsvinfected patients with a positive association between rsv and s. aureus nasopharyngeal carriage [ ] . the conflicting data from the two latter studies may be explained by differences in target populations, sampling procedures and/or microbiological methods. to date, most of the studies dedicated to the impact of rsv infection on the bacterial colonization of the upper respiratory airways identified a synergistic interaction between rsv and s. pneumoniae [ , ] . nevertheless, nasopharyngeal colonization with s. aureus is clearly more than a passive phenomenon during rsv infection and further studies are needed to elucidate the interactions between these pathogens. there is no doubt that the impact of infection with respiratory non-influenza viruses on s. aureus colonization is important and should not be neglected as this may worsen the disease outcome and even be fatal in some cases [ ] . the recent technical breakthrough of molecular diagnostic will be of precious help to investigate the changes in nasopharyngeal microbiota composition and notably in staphylococcal carriage during non-influenza viral infections of the respiratory tract. the high incidence of staphylococcal superinfection during pandemic and seasonal influenza and the important mortality risk associated to this condition promoted the in-depth investigation of the molecular and immunologic mechanisms that are involved in the bidirectional synergism between both pathogens. influenza virus is known to promote staphylococcal superinfection by alteration of the host immune system via increased production of pro-inflammatory cytokines and interferons (ifns), impairment of phagocytic cell functions and suppression of type immunity [ , ] . in addition, influenza virus has been shown to promote s. aureus adhesion and internalization within non-professional phagocytic cells through at least two distinct mechanisms: binding of bacteria to the membrane-associated hemagglutinin of influenza-infected cells, and binding of bacteria to free virions, followed by internalization of virus-coated bacteria into non-infected cells [ ] . besides, s. aureus coinfection promotes influenza virus replication and pathogenicity; indeed, extracellular bacteria secrete staphylokinase that facilitates the binding of influenza virus to the host cells, whereas intracellular s. aureus inhibits influenza virus-induced type i ifn signaling through impaired signal transducers and activators of transcription (stat and stat ) dimerization [ , ] . while the mechanisms of interaction between s. aureus and influenza virus seem to be deeply understood, the interactions between s. aureus and other respiratory viruses were less investigated. table summarizes the main mechanisms and pathways potentially involved in the interactions between s. aureus and non-influenza respiratory viruses. rhinovirus, the most common cause of upper respiratory tract infections (urti), primarily targets the nasal and nasopharyngeal epithelial cells [ ] . the innate immune system recognizes rhinovirus by different pattern recognition receptors (prrs) including membrane and endosomal toll-like receptors (tlrs) and cytoplasmic inducible rna helicases like retinoic acid-inducible gene- (rig-i) or melanoma differentiation-associated protein (mda ) [ ] . rhinovirus infection promotes proinflammatory cytokines and ifn production mainly through the activation of nfκb [ , ] . several potential mechanisms through which rhinovirus increases susceptibility to bacterial infection have been demonstrated in vitro in epithelial cells of the upper and lower airways. in , selinger, reed and mclaren developed an in vitro model for studying bacterial adherence to virus-infected epithelial cells [ ] . they reported that the adherence of s. aureus was significantly higher in rhinovirusinfected cells compared to uninfected cells. only recently, various in vitro studies have shown that inflammation due to rhinovirus infection increased cellular patterns that facilitate the adhesion and internalization of s. aureus within host cells [ ] [ ] [ ] [ ] [ ] . in primary human nasal epithelial cells, it has been demonstrated that rhinovirus infection up-regulates the expression of cellular fibronectin [ , ] . the upregulation of fibronectin was observed at both transcriptional and translational levels and seems to be related to the rhinovirus-induced nfκb activation the intercellular adhesion molecule (icam- ), which is the receptor of the major group of rhinoviruses [ ], have been found to be overexpressed during rhinovirus infection [ , ] . adhesion and internalization of s. aureus to epithelial cells is also mediated by ] . both immortalized pneumocytes (a cell line) and primary human nasal epithelial cells have been found to release higher levels of il- and il- when infected by rhinovirus and subsequently overexpressed icam- . the pro-inflammatory effect of il- and il- also induces an overexpression of icam- in the surrounding uninfected cells, which increases s. aureus uptake by the epithelial cells [ ] . the up-regulation of icam- and pro-inflammatory cytokines in rhinovirus-infected epithelial cells seems to follow the nfκb pathway [ ] [ ] [ ] . interestingly, the extracellular adherence protein (eap), an adhesin naturally secreted by s. aureus, was found to bind icam- [ ]. eap belongs to secreted expanded repertoire adhesive molecules (serams) and is involved in bacterial aggregation [ ], adhesion and invasion of epithelial and fibroblastic cells [ ] [ ] [ ] , and in preventing neutrophil recruitment [ , [ ] [ ] [ ] [ ] . in addition to icam- , eap can also bind a large variety of extracellular matrix and plasma proteins including collagen, fibrinogen, fibronectin, vitronectin, laminin, thrombospondin and prothrombin [ , , [ ] [ ] [ ] . taken together, it can be hypothesized that the ligation of s. aureus to icam- , whose expression is enhanced by rhinovirus infection, is mediated by eap. the mucosal barrier of the nasal cavity is the first site of exposure to inhaled respiratory pathogens and plays an important role in host defenses in terms of innate immunity. its integrity is regulated in large part by tight junctions of epithelial cells [ ] , which is a complex of several internal and membrane proteins including occludin and zona occludens (zo) proteins [ ] . rhinovirus infection disrupts the barrier function of the airway epithelium through the dissociation of zo- and occludin from the tight junction complex [ ] . rhinovirus infection has been shown to induce oxidative stress in respiratory epithelial cells by generating reactive oxygen species (ros) [ ] . ros generation is induced by double-strands of viral rna (dsrna) that appear transiently during rhinovirus replication, and is produced by nadph oxidase [ ] . in nonphagocytic cells, ros act as a molecular switch to stimulate pro-inflammatory responses [ ] . nevertheless, ros generation disrupts the barrier function of rhinovirusinfected cells [ ] . thus, a perturbation of the tight junction barrier function increases paracellular permeability, facilitates translocation of pathogens and their soluble products, and exposes basolateral receptors. it has been found that the infection of primary human airway epithelial cells with both major and minor groups of rhinovirus promoted the paracellular migration of s. aureus and its epithelium invasion [ ] . the rhinovirus infection facilitates also the transmigration of other bacterial species like h. influenzae and pseudomonas aeruginosa [ ] . this provides insights into another mechanism by which rhinovirus can predispose the host to secondary bacterial infections. interactions between rhinovirus and s. aureus have been shown to be bi-directional: whereas rhinovirus promotes s. aureus adhesion and internalization within host cells, s. aureus enhances the rhinovirus replication [ ]. s. aureus secretes several toxins including pore-forming toxins, toxic shock syndrome toxin and enterotoxins [ ] . in vitro, staphylococcal enterotoxins a and b (sea and seb) were shown to enhance the rhinovirus replication in a epithelial cells in a dose-dependent manner; however, they were found able to enhance neither icam- expression nor il- β, il- and il- secretion [ ] . in contrast, studies using other cellular models (normal human keratinocytes, coculture of human peripheral blood mononuclear cells and a cells, or primary nasal epithelial cell cultures) showed that these enterotoxins induced icam- expression or the secretion of pro-inflammatory cytokines [ ] [ ] [ ] . therefore, the mechanisms involved in the promotion of rhinovirus replication via staphylococcal enterotoxins need to be studied more in-depth. all these experimental data provide evidence of bidirectional synergism between rhinovirus and s. aureus. the different mechanisms described above are summarized in figure . nevertheless, the molecular pathways of these mechanisms remain poorly understood and deserve further investigation. rsv is a worldwide seasonal virus that affects mainly young children. rsv disease manifestations can vary from mild urti to severe pneumonia or bronchiolitis, which can lead to hospitalization and serious complications like respiratory failure [ ] . despite the high incidence of bacterial and rsv co-infection in both children and adults, only few studies aimed to investigate the possible interactions between s. aureus and this virus [ ] [ ] [ ] [ ] . rsv is supposed to exacerbate s. aureus co-infection via at least two mechanisms: enhancing the adhesion of s. aureus to the rsv-infected cells [ , ] , and decreasing the bacterial clearance by altering the immune response to s. aureus [ , ] . in vitro studies had shown that rsv-infected epithelial cells bind more s. aureus than uninfected cells [ , ] . study of nasal washings in patients with respiratory virus diseases has shown that secretion of lewis blood group antigens is associated with rsv infection [ ] . in addition, epithelial cells (hep ) expressing high concentrations of antigens of the lewis blood group bound significantly more s. aureus than cells expressing low concentrations of these antigens [ , ] . while rsv infects about % of infants by the first year of life, lewis antigen is expressed in secretions of nearly % of month-aged infants [ , ] . this may explain the high susceptibility of rsv-infected infants to secondary staphylococcal infection, sometimes resulting in lethal issue [ ] . nevertheless, the molecular patterns involved in rsv-related increase of s. aureus adhesion to host cells remain unknown. pro-inflammatory cytokines and type i ifns are known to be secreted by rsv-infected epithelial cells after viral sensing by different prrs, mainly through nfκb and interferon regulation factors (irfs) pathways [ ] . in addition, ifn-γ (type ii ifn) seems to play an important role in the immune response to rsv as it activates the t cell response [ ] . it has been demonstrated that rsv infection of human adult or neonatal mononuclear leukocytes results in significant inhibition of the lymphoproliferative response to heat-killed s. aureus [ ] . this inhibitory effect on the development of cell-mediated immune response could be due in part to the increased secretion of ifn-γ [ ]. ifn-γ was shown to reduce rsv replication in epithelial cells and also to inhibit b cell responses, which may alter the humoral response to s. aureus infection and decrease the bacterial clearance [ , ] . murine models had been used to demonstrate that, despite the increased number of inflammatory cells, rsv decreases the clearance of s. aureus and other pathogenic bacteria like s. pneumoniae and p. aeruginosa from the lungs of mice following secondary bacterial infection [ ] . it was suggested that rsv alters neutrophil function via changes in inflammatory response and cytokine secretion in the lung. further studies are needed to delineate the different immunological pathways that sustain the decrease of bacterial clearance and the increase of susceptibility to secondary infections by s. aureus following rsv infection. s. aureus interactions with rhinovirus or rsv have been actively, yet insufficiently, investigated. however, bacterial interactions with other respiratory viruses are poorly studied and only partial studies are available. for example, parainfluenza virus has been shown to enhance the ability of non-typeable h. influenzae and s. pneumoniae to adhere to human respiratory epithelial cells [ ] . however, there is no information available about the interaction of these agents with s. aureus in humans. only one in vitro study conducted on bovine embryonic lung cells investigated the effect of bovine parainfluenza virus infection on adherence of several bacterial agents, including s. aureus, and found no virus-specific effect on any of the tested bacteria [ ] . besides, normal human bronchial epithelial cells that were pre-incubated with s. pneumoniae resulted in an increased susceptibility to infection with human metapneumovirus. nevertheless, this was not the case for cells pre-incubated with h. influenzae, m. catarrhalis or s. aureus [ ] . on the contrary, synergistic effect between s. aureus and coronavirus has been demonstrated in vivo in a swine model. lipoteichoic acid from s. aureus increased the susceptibility to coronavirus infection in pigs via increased secretion of pro-inflammatory cytokines il- , il- , . to date, the lack of clinical and experimental data about the relationship between s. aureus colonization and other respiratory viruses complicates the understanding of potential interactions between these pathogens. while clinical and mechanistic aspects of the synergism between s. aureus and influenza virus have been deeply studied, the interactions between this bacterium and other common respiratory viruses were only partially investigated [ ] . literature still lacks more data about the relations between s. aureus carriage and non-influenza respiratory virus infections, as well as deeper insights into mechanisms of interactions between these different pathogens. the evaluation of the concrete risk of switch from commensal s. aureus colonization to invasive disease during viral respiratory tract infections may help us to propose efficient strategies of decolonization when needed. furthermore, the comprehension of the involved molecular pathways may help to develop new strategies of preventive and curative treatments. factor kappa-light-chain-enhancer of activated b cells; nkg d: natural killer group d receptor; nod : nucleotide-binding oligomerization domain-containing protein ; nox: membrane-bound nadph oxidase; rac : ras-related c botulinum toxin substrate ; rantes: regulated on activation, normal t cell expressed and secreted chemokine; rig-i: retinoic acid inducible gene- ; ros: reactive oxygen species; s. aureus: staphylococcus aureus; sea: staphylococcal enterotoxin a; seb: staphylococcal enterotoxin b; ssrna: single-stranded rna; tlr: toll-like receptor; zo- : zona occludens . no potential conflict of interest was reported by the authors. viral potentiation of bacterial superinfection of the respiratory tract alterations of the eustachian tube, middle ear, and nose in rhinovirus infection acute community-acquired sinusitis colonization and infection of the respiratory tract: what do we know? paediatr child health insights into the interaction between influenza virus and pneumococcus axis of coinfection evil immune dysfunction and bacterial coinfections following influenza severe influenza and s.aureus pneumonia the immunology of influenza virus-associated bacterial pneumonia influenza and bacterial superinfection: illuminating the immunologic mechanisms of disease the role of nasal carriage in staphylococcus aureus infections detection and clinical relevance of staphylococcus 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exposure is associated with human metapneumovirus seroconversion and increased susceptibility to in vitro hmpv infection lipoteichoic acid from staphylococcus aureus exacerbates respiratory disease in porcine respiratory coronavirus-infected pigs natural killer cells are involved in acute lung immune injury caused by respiratory syncytial virus infection key: cord- -purplsjn authors: fernández-ponce, cecilia; durán-ruiz, maria c.; narbona-sánchez, isaac; muñoz-miranda, juan p.; arbulo-echevarria, mikel m.; serna-sanz, antonio; baumann, christian; litrán, rocío; aguado, enrique; bloch, wilhelm; garcía-cozar, francisco title: ultrastructural localization and molecular associations of hcv capsid protein in jurkat t cells date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: purplsjn hepatitis c virus core protein is a highly basic viral protein that multimerizes with itself to form the viral capsid. when expressed in cd (+) t lymphocytes, it can induce modifications in several essential cellular and biological networks. to shed light on the mechanisms underlying the alterations caused by the viral protein, we have analyzed hcv-core subcellular localization and its associations with host proteins in jurkat t cells. in order to investigate the intracellular localization of hepatitis c virus core protein, we have used a lentiviral system to transduce jurkat t cells and subsequently localize the protein using immunoelectron microscopy techniques. we found that in jurkat t cells, hepatitis c virus core protein mostly localizes in the nucleus and specifically in the nucleolus. in addition, we performed pull-down assays combined with mass spectrometry analysis, to identify proteins that associate with hepatitis c virus core in jurkat t cells. we found proteins such as nolc , pp γ, ilf , and c qbp implicated in localization and/or traffic to the nucleolus. hcv-core associated proteins are implicated in rna processing and rna virus infection as well as in functions previously shown to be altered in hepatitis c virus core expressing cd (+) t cells, such as cell cycle delay, decreased proliferation, and induction of a regulatory phenotype. thus, in the current work, we show the ultrastructural localization of hepatitis c virus core and the first profile of hcv core associated proteins in t cells, and we discuss the functions and interconnections of these proteins in molecular networks where relevant biological modifications have been described upon the expression of hepatitis c virus core protein. thereby, the current work constitutes a necessary step toward understanding the mechanisms underlying hcv core mediated alterations that had been described in relevant biological processes in cd (+) t cells. hepatitis c virus core protein is a highly basic viral protein that multimerizes with itself to form the viral capsid. when expressed in cd + t lymphocytes, it can induce modifications in several essential cellular and biological networks. to shed light on the mechanisms underlying the alterations caused by the viral protein, we have analyzed hcv-core subcellular localization and its associations with host proteins in jurkat t cells. in order to investigate the intracellular localization of hepatitis c virus core protein, we have used a lentiviral system to transduce jurkat t cells and subsequently localize the protein using immunoelectron microscopy techniques. we found that in jurkat t cells, hepatitis c virus core protein mostly localizes in the nucleus and specifically in the nucleolus. in addition, we performed pull-down assays combined with mass spectrometry analysis, to identify proteins that associate with hepatitis c virus core in jurkat t cells. we found proteins such as nolc , pp γ, ilf , and c qbp implicated in localization and/or traffic to the nucleolus. hcv-core associated proteins are implicated in rna processing and rna virus infection as well as in functions previously shown to be altered in hepatitis c virus core expressing cd + t cells, such as cell cycle delay, decreased proliferation, and induction of a regulatory phenotype. thus, in the current work, we show the ultrastructural localization of hepatitis c virus core and the first profile of hcv core associated proteins in t cells, and we discuss the functions and interconnections of these proteins in molecular networks where relevant biological modifications have been described upon the expression of hepatitis c virus core protein. thereby, the current work constitutes a necessary step toward understanding the mechanisms underlying hcv core mediated alterations that had been described in relevant biological processes in cd + t cells. keywords: hepatitis c virus, immune evasion, proteomics, interactome, ultrastructure, regulatory t cells, immune tolerance introduction hepatitis c virus (hcv) infection is an important cause for chronic viral liver disease and one of the main indications for liver transplantation (anzola, ; dustin and rice, ) . hcv affects million people worldwide and in more than % of the patients leads to chronicity (gower et al., ) . the high level of chronicity and the absence of a protective vaccine, makes hcv infection a significant public health problem (anzola, ; dustin and rice, ; gower et al., ) . the molecular mechanisms harnessed by hcv to establish a chronic infection and their implications in the innate and adaptive immune systems have not been fully elucidated. in this regard, several hcv viral proteins have been described as modulators of immunological phenomena (yao et al., ; krishnadas et al., ; tu et al., ; chen et al., ) . among them, hcv core protein has been widely associated with pathogenicity, virulence, immune evasion and immune regulation (dominguez-villar et al., , a waggoner et al., ; tu et al., ; doumba et al., ; fernandez-ponce et al., , . however, the underlying molecular processes, as well as the behavior of hcv core protein or its interactions with host cell components, remain unclear. hcv core is a highly basic protein, which binds and protects the viral rna, multimerizing with itself to form the viral capsid (santolini et al., ) . in mammalian infected cells, hcv core interacts with endoplasmic reticulum membranes, lipid droplets and other cellular and viral proteins to promote assembly of new virions. however, several inhibitory molecules and the lack of some host factors can hamper viral production by disrupting the core multimerization process and the coordinated interactions with other viral proteins (mousseau et al., ; gawlik and gallay, ) . in this way, non-enveloped hcv core proteins can be directed to alternative subcellular locations and also be released into the extracellular space (maillard et al., ; polyak et al., ; tan et al., ) . according to these findings, in hcv chronically infected patients, hcv core protein, has been detected as non-enveloped isolated nucleocapsids, not only in hepatocytes (falcon et al., ) , but also in serum (maillard et al., ) , peripheral blood cd + t cells (fernandez-ponce et al., ) and other nonparenchymal liver cells, such as, lymphocyte, pit, endothelial, stellate, kupffer and polymorphonuclear cells (falcon et al., ) . in t cell lines, non-enveloped isolated nucleocapsids binding and internalization has also been described in vitro (doumba et al., ) . these data further support the presence of hcv core protein inside immune cells, including lymphocytes, during hcv chronic infection. interestingly, hcv core protein intracellular expression in cd + t lymphocytes has been shown to induce modifications in cell proliferation, cell cycle progression, expression of anergy genes, transcription of genes involved in cytoskeleton reorganization, vesicle trafficking, endocytosis, transcription and translation, cytokine production, cell death and generation of a t cell regulatory phenotype with exhausted features (bergqvist and rice, ; bergqvist et al., ; dominguez-villar et al., , a doumba et al., ; fernandez-ponce et al., ) , characterized by an increased expression of foxp (forkhead box p ) and ctla- (cytotoxic t-lymphocyte antigen- ) (dominguez-villar et al., a) , high levels of il- secretion, and decreased il- and ifn-γ production (doumba et al., ; fernandez-ponce et al., ) . it has been described for several viruses that the specific subcellular localization of viral proteins and their interactions with host molecules can alter the spatial distribution and organization of cellular proteins, and in this way, induce diverse molecular and cellular effects (chen et al., ; yoo et al., ; ning and shih, ; bertrand and pearson, ; ponti et al., ; hiscox et al., ; zhu et al., ; raval et al., ) . as studies using the whole virus do not allow for the elucidation of the specific molecular mechanisms in which each protein is implicated, in this work, we focused on a single viral protein, showing that in cd + t cells, hcv core protein mostly localizes in the nucleus and specifically in the nucleolus where it is greatly enriched. in addition, we performed pull down assays, combined with mass spectrometry analysis, in order to identify host proteins associated with hcv core, which could be implicated in the functional effect previously observed to be induced by the presence of hcv-core in t cells. we found several proteins implicated in important functions that are associated with hcv core protein. thereby, our results shed light on the molecular mechanisms underlying the alterations in biological cell processes and the generation of adaptive regulatory-like cd + t cells in the periphery by the intracellular presence of a single hcv viral protein. human embryonic kidney (hek) lenti-x tm t cell line (clontech) and jurkat cell line (american type culture collection, manassas, va, usa) were maintained in dulbecco's modified eagle's medium (dmem tm ) supplemented with % (v/v) heat inactivated fetal bovine serum (fbs), mm l-glutamine, mm hepes, % (v/v) sodium pyruvate, µm -mercaptoethanol, u/ml penicillin and µg/ml streptomycin at • c, % co . human peripheral blood samples were obtained from healthy donors upon signature of an informed consent and following approval by the ethic sub-commission of the puerta del mar university hospital (dependent from the central quality commission), in accordance to spanish and european union regulations. peripheral blood mononuclear cells (pbmcs) were isolated by density gradient centrifugation using lymphocyte separation medium (eurobiotm, montpellier, france). cells were washed three times with pbs, subsequently stimulated with mg/ml phytohemagglutinin-p (pha) (sigmatm, saint louis, missouri, usa) and cultured in dmem supplemented with % (v/v) sodium pyruvate, non-essential aminoacids, vitamins, larginin, l-asparragin, folic acid, mm hepes, mm -mercaptoethanol, mg/ml streptomycin, u/ml penicillin (life technologies, carlsbad, ca, usa) and % heat-inactivated fbs (gibco) at • c, in a % co atmosphere. u/ml il- was added to the cultures every h, for a total of days to obtain blasts. hek lenti-x tm t cells (clontech) were used as packaging cell lines to produce lentiviral supernatant by co-transfecting plasmids pcmvdr . , coding for hiv- gag and pol proteins and pmd .g for the vesicular stomatitis virus g protein (vsvg) together with the transfer vector phrsincppt-sewhcv-core-gfp (hcv-core) coding for the first amino acids of the hcv polyprotein (serotype a) corresponding to hcv-core protein. transfer vector phrsincppt-sewgfp expressing only gfp (green fluorescent protein) was used as a control (dominguez-villar et al., ) . cells were transfected in optimem tm medium using cms diameter cell-culture dishes coated with collagen (collagen i, rat tail gibco r invitrogen cell culture), and polyethylenimine (pei), branched (sigma-aldrich) as cationic polymer. transfection efficiency was evaluated by facs analysis. supernatants were collected at and h, centrifuged to remove cells and debris and concentrated using lenti-x tm concentrator (clontech r ) to obtain a high-titer virus-containing pellet. pellets were frozen at − • c and stored until use. viral titer in supernatants was determined evaluating their efficiency in infecting jurkat cells. jurkat cells were transduced using hcvcore-gfp and gfp (as control) lentiviral supernatants, previously prepared, at a multiplicity of infection (moi) of . aliquots from the cultures were collected after h to determine the transduction efficiency. gfp expression was monitored by facs (cyanadp-mle tm ; dakocytomation tm ) presenting a transduction range > % in all experiments. untransduced, hcvcore-gfp and gfp transduced jurkat cells were collected h post-transduction, counted, washed with phosphate buffer saline (pbs) and fixed for h using % paraformaldehyde (pfa) in pbs, ph . . subsequently, cell pellets were pre-embedded in agarose x, taken out of the tube with a needle and sliced into approximately mm pieces. pre-embedding immunogold was performed. agarose pieces were washed in pbs, permeabilized using . % triton x− in pbs and incubated in aurion blocking solution containing normal goat serum (aurion r . ), for min at • c. sections were stained immunochemically with anti-gfp antibody (abcam ab ) diluted : , in pbs containing . % bovine serum albumin (bsa) at • c, overnight, and subsequently washed with the same buffer. for gold particle staining, sections were incubated with nm gold-labeled anti-rabbit igg (sigma-aldrich) diluted : in . % bsa/pbs at • c, overnight. agarose pieces were washed in . m caco-buffer ph . at room temperature. sections were post-fixed in . % osmium tetroxide and . m caco-buffer at room temperature, for h and washed with . m caco-buffer. fixed sections were dehydrated by sequential incubation in ethanol - % and propylene oxide, embedded in epoxy resin and polymerized at • c for h. semi ( nm) and ultra-thin ( - nm) sections, were obtained using a leica em uc ultramicrotome. uultra-thin sections were collected onto plastic-coated nickel grids and immunochemically contraststained with uranium salts (uranyl acetate) and lead citrate to reveal cell ultrastructure. finally, samples were analyzed using a transmission electron microscope em a fa.zeiss with the item soft imaging system software (olympus). untransduced jurkat cells and pbmcs cultured as was previously described, were collected, counted, washed with pbs and cells were incubated min with µl lysis buffer ( mm tris-hcl ph . , mm nacl, % nonidetp- , mm edta, mm phenylmethylsulfonyl fluoride, mm na vo , mm naf, on ice. cell debris was discarded by centrifugation at , g for min, • c, and soluble proteins were stored at − • c before further analysis. protein levels were measured using bradford protein assay kit (pierce), according to manufactures recommendations. two hundred microliter magnetic beads (dynabeads r myone tm streptavidin t invitrogen) were washed following manufacturer's recommendations and conjugated with hcv core genotype- b biotin-prospec (hcv- ) by incubating µl ( mg/ml) dynabeads with µl ( pmoles) hcv core protein in pbs, for h at • c, with gentle rotation. hcv core protein-coated beads were washed three times by resuspension in pbs containing . % twin- and decanting the supernatant while placed on a magnet. for mass spectrometry analysis, coated dynabeads (or uncoated as control for unspecific binding) were incubated overnight, at • c, with jurkat cells protein lysates, with gentle rotation, in the following ratio: µl of coated beads with µg of total protein lysate. for western blot experiments, coated dynabeads were incubated overnight, at • c, with protein lysates from pbmc blasts, with gentle rotation, in the following ratio: µl coated beads with mg protein sample. µl uncoated beads were incubated with mg protein lysate, in the same conditions, as a control of unspecific binding. samples were washed twice with pbs and once with ultrapure water and incubated with µl of mm dithiothreitol (dtt) in mm ammonium bicarbonate for min at • c. eluated proteins were quantified and µg were precipitated with acetone during h at − • c and centrifuged min at , g. subsequently, proteins were resuspended in urea m, reduced with mm dtt in mm ammonium bicarbonate at room temperature for min and alquilated with mm iodacetamide min, in the dark, subsequently trypsin digestion was performed with sequencing grade trypsin (promega, madison, wi, usa) at • c overnight (enzyme/substrate ratio : ). finally, the digestion was quenched adding % formic acid. the resulting peptides were transferred to a clean tube, dried in speed-vac and stored at − • c prior to analysis by mass spectrometry (ms). peptides were resolved by reverse phase chromatography using an eksigent ultra pump fitted with a µm id column cm length (acclaim pepmap c µm a). samples were initially loaded for desalting into a cm length µm id precolumn packed with the same chemistry as the resolving column prior to analytical chromatography. mobile phases used were % water . % formic acid (buffer a), % acetonitrile . % formic acid (buffer b). gradient was developed during min up to % b. column was equilibrated in % b for min and % b for min, using a constant flow of nl/min. peptides eluted from the column were analyzed using a sciex tripletof tm system. data dependent acquisition was collected upon a survey scan performed in a mass range from to , m/z in a scan time of ms. the top most intense precursors were selected for fragmentation with a transmission window width of . da. minimum accumulation time for ms/ms was set to ms giving a total cycle time of , ms. fragment ions were collected in a mass range from to , m/z in order to have a single q transmission window. precursors were excluded from further fragmentation during s. raw data files were processed using proteinpilot tm . software from sciex and mgf files were loaded onto proteinscape tm . software (bruker) for data grouping and protein identification via mascot program version . (matrix science ltd, london, uk). search parameters were: homo sapiens taxonomy. trypsin specificity was used and only one missed cleavage allowed, cysteine carbamidomethylation as fixed and oxidized methionine was used as variable modification. mass tolerance of precursor ion and fragment ions were ppm and . da, respectively. peptides charges of +, +, and + were selected and minimum peptide length was set at five residues. peptides and proteins were considered positively identified if their mascot score was higher than and , respectively. proteins were identified with the minimum of one peptide and a false discovery rate < %. proteins identified with one single peptide were only included if peptide scores were significant, and peptide spectra contained most "y" and/or "b" representative ions for the corresponding peptide sequence. examples are included in supplementary table . subsequently, we compared the identified proteins that bound to the dynabeads vs. the ones identified binding to hcv core protein using the "compare results" option from proteinscape software . . thus, proteins were found to specifically associate with hcv core protein. proteome profiling data has been deposited in the peptideatlas repository, identified as pass . samples obtained from the pull down assay were washed twice with pbs using the magnet, and incubated with x laemmli buffer at • c for min. subsequently, beads were removed from the proteins using a magnet and proteins resolved by sds-page and transferred to pvdf membranes (immobilon-fl). membranes were incubated with the corresponding primary antibody, followed by the appropriated secondary antibody, conjugated to hrp. primary antibodies used were pp gamma antibody (pa - ) from thermo fisher scientific r and mypt antibody (# ) from cell signaling technology r . reactive proteins were visualized using an ecl system and images were acquired in a chemidoc touch imaging system (bio-rad r laboratories, hercules, ca, usa). localization and network analysis of identified candidate proteins associated with hcv core protein string database version (search tool for the retrieval of interacting genes/proteins) (http://string-db.org) (szklarczyk et al., ) was applied to visualize detailed information about the subcellular localization and the main interactions of the identified proteins. ingenuity pathways analysis (ipa; ingenuity systems, redwood city, calif.) software was used for further protein characterization according to known and predicted associations into function canonical pathways and networks recorded in the ipa library. nuclear localization sequence (nolss) in hcv core protein prediction was obtained by entering a protein sequence in fasta format on the nod web server http://www.compbio.dundee.ac. uk/www-nod/ (scott et al., ) . nolss were predicted if the average score output by the artificial neural network ann of consecutive windows was at least . (scott et al., (scott et al., , . to analyze the localization of hcv core protein in t cells and to circumvent the lack of optimal anti-hcv core antibodies, we took advantage of a gfp-hcv core fusion construct that has been extensively used in our laboratory (dominguez-villar et al., , a fernandez-ponce et al., ) and an unfused gfp expressing construct as a control. jurkat cells were efficiently transduced with lentiviral vectors expressing hcvcore-gfp or gfp as a control. percentage of transduced cells analyzed by flow cytometry was > % in all cases (data not shown). jurkat cells transduced with gfp or hcv core gfp-expressing lentiviral construct or left untransduced were subsequently immunostained with anti-gfp antibody and a nm gold-labeled anti-rabbit igg and analyzed by transmission electron microscopy. as shown in figure , hcv core was mostly localized in the nucleus (figures b,d,e) and specifically in the nucleolus where it was greatly enriched (figures b-f) , although some immunostaining was observed in cytoplasm ( figure a) . hcv-core-gfp was detected in the nucleus in % out of hcv core gfp expressing jurkat cells analyzed, % showed the presence of gfp inside both nucleus and cytoplasm, % of the cells only in cytoplasm (figure a) , while % showed gfp immunolabelling in nucleus with enrichment in the nucleolus (figures b-f) . % of the cells were not stained. in hcv core expressing jurkat cells which showed core protein nucleolar localization, the total raw number of gold particles counted inside the nucleoli was , with an average of gold particles per cell and a standard deviation of . . regarding gfp transduced control cells, gfp was localized in the nucleus of % from gfp-expressing jurkat cells analyzed and in both nucleus and cytoplasm in %. there was no recognizable co-localizing with any organelle. nucleolus localization was not visible in any cell (figures g,h) . no immunogold staining was observed in untransduced jurkat cells (data not shown). thus, the study revealed specific immunolabeling of hcv core protein in the nucleolus of jurkat cells. based on the findings obtained with the electron microscopy assay, we decided to identify hcv core associated proteins that could mechanistically be correlated with hcv core protein nucleolar localization. thus, we performed pull down experiments using biotinilated hcv core protein as bait. further analysis with the string platform allowed us to classify the identified proteins depending on their localization (figure ) . thus, from the associated proteins identified (supplementary table ), have been previously described to localize in the nucleus (figure a) and in the nucleolus (figure b) , while most of the proteins described in the cytoplasm (figure c ) have also been identified in the nucleus and/or nucleolus. string also allowed us to determine potential protein functionalities according to previously published reports. interestingly, most of the identified hcv core associated proteins were found to participate in binding processes, as indicated in figure . regarding protein function, we mainly focused on the functional classification given by the ipa software, which, based on biomedical literature and integrated databases, allows to determine the most probable pathways and/or functions in which identified proteins are involved. thus, ipa core analysis of our dataset (figure ) revealed that the identified hcv core associated proteins were mainly described to participate in splicing and processing of rna, cell cycle progression, cell proliferation, apoptosis and rna virus infection. our findings support the concept that the subcellular localization of hcv core protein might have an impact on cd + t cells functions. in order to confirm, by an alternative method, some of the associations identified by mass spectrometry analysis, and most importantly to evaluate whether such associations were also present in primary t cells (pbmc), thus enhancing the relevance of our findings. postnuclear lysates from human pbmcs blasts (see methods), were subjected to hcv core protein pull down, using a biotinilated hcv core protein ( pmoles) bound to magnetic beads as a bait. uncoated magnetic beads were used as a control. experiments were first carried out in jurkat cells and inputs from jurkat and pbmc were loaded in parallel ( . % from the amount used per pull down), to evaluate the relative amount of each target protein (data not shown). serine/threonine-protein phosphatase was selected due to its known localization (it has a dynamic and predominantly nucleolar distribution) and to its function (it has been implicated in the regulation of several biological pathways previously described in t cells transduced with hcv core protein) (aggen et al., ; ceulemans and bollen, ; nie et al., ) . it was also selected based on the fact that both its catalytic subunit (pp γ) and its regulatory subunit a (mypt ), were identified by mass spectrometry, showing a despairing mascot score of . and . respectively. as shown in figure , western blot analysis of (pp γ) and (mypt ), confirmed the presence of both frontiers in microbiology | www.frontiersin.org figure | "actions" view, according to the "go cellular components" distribution option, in order to identify their subcellular localization. string analysis reported that proteins were nuclear (a) ( from ), nucleolar (b) ( from ), and cytoplasmic (c) ( from ). colored lines between proteins indicate the type of evidence for each interaction, with a minimum required confidence score of . . proteins in pbmcs lysates obtained from the pull down of hcv core protein coated magnetic beads (figure ) . in order to identify potential nucleolar localization sequences in hcv core protein, we used the nod web server (http://www. compbio.dundee.ac.uk/www-nod/), a program that predicts the presence of nolss in eukaryotic and viral proteins, based on a database of statistically analyzed human nucleolar localization sequences (scott et al., ) . two nolss were identified in hcv core protein ( several biological and immunological consequences of hcv core intracellular expression in cd + t cells have previously been described by us and others, (bergqvist and rice, ; bergqvist et al., ; dominguez-villar et al., , a doumba et al., ; fernandez-ponce et al., ) . these findings suggest an important role for the intracellular presence of hcv core in hcv pathogenesis and chronification. in this study, we have analyzed the ultrastructural localization of hcv core protein in cd + t cells. according to studies using cell lines unrelated to the immune system, hcv core protein has been shown to localize in the endoplasmic reticulum, mitochondrial outer membrane and nucleus of human embryonic kidney t cells (suzuki et al., ) ; associated to lipid droplets in cho, hepg and huh cells lines (barba et al., ; boulant et al., ; qiang and jhaveri, ) , in the cytoplasm, endoplasmic reticulum, in the proximity of the nuclear membrane, in the nucleus and nucleoli of hepatocytes isolated from chronically hcv infected patients (falcon et al., ) ; and in the cytoplasm and nucleus of non-parenchymal liver cells such as, lymphocyte-like cells, kupffer-like cells, polymorphonuclear-like cells, pit, endothelial, stellate, and fibroblast-like cells isolated from livers of chronically hcv infected patients (falcon et al., ) . in the present work, we found that in cd + t lymphocytes, hcv core protein mostly localizes in the nucleus and specifically in the nucleolus where it is greatly enriched and mainly organized in clusters (figure ) . regarding these findings, nuclear localization signals (nlss) described previously in hcv core protein, could be responsible for nuclear localization (chang et al., ; suzuki et al., suzuki et al., , , while hcv core protein traffic and residence in the nucleolus, could be explained by the presence of two nucleolar localization sequences (nolss), identified using the bioinformatic nucleolar localization sequence detector web server for eukaryotic and viral proteins (scott et al., ) . nolss are showed in figure . the statistical variation in core protein nucleolar localization shown by hcv-core expressing cells, could be due to differences in cellular cycle stage, as it has been described for other nucleolar resident proteins (chen and huang, ; stoldt et al., ; pirlot et al., ) . presence of nolss in cellular or viral proteins is a key factor in their dynamic traffic and residence within the nucleolus. presumably, nolss interact with nucleolar proteins and/or rna to mediate nucleolar targeting and retention. thus, in chimeric viral proteins with mutated nolss, trafficking to the nucleolus is abrogated, and heterologous nolss insertion restores their trafficking pattern (boyne and whitehouse, ; emmott et al., ) . interestingly, the function of hcv core protein inside the virus, can partly explain its subcellular localization as hcv core protein mainly interacts with hcv genomic rna, multimerizing around it and forming the capsid shell. while multimerizing inside the virus, we have not seen any multimerization in our experiments, which is in agreement with several studies showing that mammalian cell lines fail to produce capsid assembly (bukh et al., ; pietschmann et al., ; polyak et al., ; rouille et al., ; hourioux et al., ) , due to the lack of host cells factors that are essential for hcv core multimerization and assembly, or to the presence of inhibitory factors that induce the majority of hcv-core to be targeted away from the er (hope and mclauchlan, ; mclauchlan et al., ; polyak et al., ) . thus, un-multimerized hcv core protein traffics to alternate subcellular compartments. hcv core has been shown to bind rnas in addition to hcv genomic rna (kunkel et al., ; cristofari et al., ) , including ribosomal rna (santolini et al., ) and trna (kunkel et al., ) . since the nucleolus contains several copies of rrna genes and it could be a recruiting site for trnas (carmo-fonseca et al., ) it is likely that the nucleolar rna constitutes another important molecular target structure for hcv core protein. in agreement with our findings, several dna viruses, retroviruses and rna viruses, as well as viral proteins, have been described to traffic to the nucleolus and be associated with nucleolar proteins wurm et al., ; chen et al., ; dove et al., ; michienzi et al., ; cawood et al., ; hiscox, ; emmott et al., ; lam et al., ; jarboui et al., ) . the nucleolus is a dynamic nuclear organelle whose proteome is continuously changing. it seems to be a temporary storage or a sequestration site for a multiplicity of proteins (emmott and hiscox, ; hiscox et al., ) . functionally, there is extensive evidence that the nucleolus is implicated in ribosome biogenesis (stoykova et al., ; warner, ; scheer et al., ) , cell cycle regulation, cell growth, senescence, stress response signaling (andersen et al., ; emmott and hiscox, ; tsai and pederson, ; lam and trinkle-mulcahy, ) and the pathogenesis of several diseases such as cancer (james et al., ; orsolic et al., ; yang et al., ) , cardiovascular disease (hariharan and sussman, ) and neurodegenerative disorders (payao et al., ; lu et al., ; rieker et al., ; tsoi and chan, ; lee et al., a; parlato and liss, ; hernandez-ortega et al., ) . viral protein trafficking and localization into the nucleolus has shown implications in both viral life cycle and in host cell physiology and it has been narrowly related with the loss of essential nucleolar functions (hiscox, ) . in addition, it has been shown that accumulation of viral proteins in the nucleolus can cause volume exclusion and crowding effects, disrupting the nucleolar architecture (hancock, ; hiscox, ) . virus infection and some viral proteins from poliovirus, avian infectious bronchitis virus (ibv), coronavirus and human immunodeficiency virus- (hiv- ) induce disruption of the nucleolar architecture and changes on the subcellular distribution of nucleolar proteins or proteins that traffic to the nucleolus, such as nucleolin, p , b . (waggoner and sarnow, ; hiscox, ; dove et al., ) . these findings are closely related to the presence of perturbations in cell cycle, cytokinesis and apoptosis in the host cells (miyazaki et al., ; chen et al., ; galati et al., ; hiscox, ) . semliki forest virus nucleocapsid migrates to the nucleolus (jakob, ) and porcine reproductive and respiratory syndrome virus nucleocapsid specifically interacts with the small nucleolar rna (snorna)-associated protein, fibrillarin in virus infected cells (rowland et al., ; yoo et al., ) , while hepatitis b virus (hbv) core protein usually co-localizes with the nucleolar proteins, nucleolin and b (ning and shih, ) . in addition, it has been shown that coronavirus nucleocapsid protein localization in the nucleolus of infected cells and its association to the nucleolar protein b . is related to cell cycle stage and could be involved in cell cycle delay or arrest to promote virus replication wurm et al., ; cawood et al., ) . thus, trafficking to the nucleolus and nucleolar residency time of hcv core protein in cd + t cells could be narrowly related with previous findings from us and others showing that the intracellular presence of hcv core in cd + t cells induces decreasing cell proliferation, delay in cell cycle progression and a differential expression pattern of genes with relevant function, including anergy-associated genes, genes involved in cytoskeleton reorganization, vesicle trafficking, endocytosis, cytokines production, cell death, transcription, and translation (dominguez-villar et al., ) . in agreement with the described localization, we found hcv core protein association with several nuclear, nucleolar proteins or proteins described to traffic to the nucleolus, which showed mainly binding connections (figure ) . the wide range of proteins associated with hcv core, correlate with the findings obtained by dolan et al. who identified two computationally predicted molecular recognition features within the n-terminal intrinsically disordered region (idr) in the hcv core sequence. the identified molecular recognition features, mediate hcv core protein binding to hcv rna and to multiple host proteins, suggesting that hcv core protein exhibits hub protein properties (dolan et al., ) . interestingly, pathway and network-based analysis of proteins identified to be associated with hcv core protein, indicate that a wide range of these proteins are involved in several biological signaling pathways, such as, rna processing and splicing (figure a) , cell cycle progression, cell proliferation, apoptosis ( figure b ) and infection and replication of rna viruses, including hcv ( figure c) . with the integrated analysis of the present data, we confer a better description of the hcv core -human t lymphocyte relationship. concerning ribosomal biogenesis, cellular proliferation, cell cycle and apoptosis; several large (rpl) and small (rps) ribosomal proteins, and proteins involved in rna processing and splicing as dead-box rna helicases, precipitated with figure | predicted nucleolar localization sequences in hcv core protein. graph obtained from the nucleolar localization sequence detector web server (nod), displaying nols prediction score for each residue of hcv core protein. pink shaded regions represent the range of scores within which a -residues segment is predicted to be a nols. thus, pink shaded regions represent the nols candidate segment, which highlights scores above . . hcv core (figures a,b) (rocak and linder, ; xu et al., ) . interactions between viral and ribosomal proteins, splicing factors and dead-box rna helicases including ddx and ddx , have been previously described and suggested as a key mechanism for viral replication and production as well as for life cycle progression and survival (bortz et al., ; naji et al., ; yasuda-inoue et al., ; cervantes-salazar et al., ; klymenko et al., ; li et al., ) . implication of other identified proteins, such as protein red (ik) and filamin a (flna) (figure b ) in proliferation and cell cycle progression, have also been widely demonstrated (lee et al., b; sun et al., ) . thus, associations of hcv core protein in t cells could alter the function of the associated proteins, explaining some of the effects shown for hcv-core expression, including cell cycle delay and inhibition of cell proliferation (dominguez-villar et al., , a fernandez-ponce et al., ) . in addition, we found that many host proteins associated with hcv core, are involved in replication and infection of rna viruses including hcv ( figure c) . proteins as dexd/hbox helicases have been extensively studied in virus infection and have been described as proteins hijacked by viruses for their benefit. specifically, dhx is involved in virus replication, innate immunity response to viral dsrna and participation in the expression of ifn-stimulated genes (fullam and schroder, ) . rna binding proteins (rbps) such as the host poly(rc)-binding protein (pcbp) and different heterogeneous nuclear ribonucleoproteins (hnrps), are also interesting as they stabilize viral rnas, co-localize with the viral replicase complex and facilitate viral rna template selection (li and nagy, ) . in addition, interleukin enhancer-binding factor (ilf ) has been shown to be recruited to hcv replication complexes (li et al., ) and in other infections by rna viruses, ilf interaction with viral proteins has been described to affect virus replication among other virus life cycle stages (patino et al., ) . furthermore, the subcellular localization of hcv core and its association with nuclear and nucleolar proteins found in the present study, can aid in explaining the cd + t cell regulatory/exhausted phenotype described by us and others, in cd + t cells expressing hcv core protein (dominguez-villar et al., a; doumba et al., ; fernandez-ponce et al., ) . some such associations are with serine/threonine-protein phosphatase catalytic subunit (pp γ), protein phosphatase regulatory subunit a (mypt ) (nie et al., ) , interleukin enhancer-binding factor (ilf ) (shi et al., a,b) , complement component c q binding protein (c qbp) (kittlesen et al., ) and runt-related transcription factor (runx ) (klunker et al., ) . in conclusion, analysis of the association of hcv core with host proteins in cd + t cells and the study of its ultrastructural localization, open an extensive field of study poised to understand the mechanisms underlying functional findings previously described in cd + t cells expressing hcv core protein, that have demonstrated to be relevant for hcv immune system evasion and thus hcv chronification. conception, design and or interpretation of the work: fg-c, ea, and rl. wb (for localization studies). cb and md-r (for association studies). performed association studies: cf-p, md-r, and as-s. performed localization studies: cf-p, jm-m, and ma-e. run statistical analyses: rl and cf-p. wrote the paper: fg-c, cf-p, ea, rl, and md-r. performed confirmation co-ip experimentos: in-s and cf-p. all authors participated in critical revision and subsequently approved the manuscript. all authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. for ea. the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. regulation of protein phosphatase- nucleolar proteome dynamics hepatocellular carcinoma: role of hepatitis b and hepatitis c viruses proteins in hepatocarcinogenesis hepatitis c virus core protein shows a cytoplasmic localization and associates to cellular lipid storage droplets transcriptional activation of the interleukin- promoter by hepatitis c virus core protein the hepatitis c virus core protein modulates t cell responses by inducing spontaneous and altering t-cell receptor-triggered ca + oscillations the conserved 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cell division the role of ribosomal proteins in the regulation of cell proliferation, tumorigenesis, and genomic integrity nucleolar repression facilitates initiation and maintenance of senescence direct binding of hepatitis c virus core to gc qr on cd + and cd + t cells leads to impaired activation of lck and akt distinct ddx deadbox rna helicases cooperate to modulate the hiv- rev function colocalization and interaction of the porcine arterivirus nucleocapsid protein with the small nucleolar rna-associated protein fibrillarin interaction of avian influenza virus ns protein and nucleolar and coiledbody phosphoprotein we would like to thank christian hoffmann, evelyn janssen, beatrix martiny, jessicahausmann, mojgan ghilav and consuelo rivera for their excellent technical support, the plataforma andaluza de bioinformática (centro de supercomputación y bioinformática, university of málaga) for the use of the ingenuity pathways analysis (ipa) software and the core biomedical research facility of the university of cadiz for the use of core infrastructure. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmicb. . /full#supplementary-material key: cord- -t jm n authors: severance, emily g.; dickerson, faith b.; yolken, robert h. title: autoimmune phenotypes in schizophrenia reveal novel treatment targets date: - - journal: pharmacology & therapeutics doi: . /j.pharmthera. . . sha: doc_id: cord_uid: t jm n abstract typical and atypical antipsychotics are the first-line treatments for schizophrenia, but these classes of drugs are not universally effective, and they can have serious side effects that impact compliance. antipsychotic drugs generally target the dopamine pathways with some variation. as research of schizophrenia pathophysiology has shifted away from a strictly dopamine-centric focus, the development of new pharmacotherapies has waned. a field of inquiry with centuries-old roots is gaining traction in psychiatric research circles and may represent a new frontier for drug discovery in schizophrenia. at the forefront of this investigative effort is the immune system and its many components, pathways and phenotypes, which are now known to actively engage the brain. studies in schizophrenia reveal an intricate association of environmentally-driven immune activation in concert with a disrupted genetic template. a consistent conduit through this gene-environmental milieu is the gut-brain axis, which when dysregulated can generate pathological autoimmunity. in this review, we present epidemiological and biochemical evidence in support of an autoimmune component in schizophrenia and depict gut processes and a dysbiotic microbiome as a source and perpetuator of autoimmune dysfunction in the brain. within this framework, we review the role of infectious agents, inflammation, gut dysbioses and autoantibody propagation on cns pathologies such as neurotransmitter receptor hypofunction and complement pathway-mediated synaptic pruning. we then review the new pharmacotherapeutic horizon and novel agents directed to impact these pathological conditions. at the core of this discourse is the understanding that schizophrenia is etiologically and pathophysiologically heterogeneous and thus its treatment requires individualized attention with disease state variants diagnosed with objective biomarkers. schizophrenia is a polygenic psychiatric disorder defined by a complex of positive, negative and cognitive symptoms. positive symptoms include delusions and hallucinations, whereas negative symptoms reflect the absence of an expected response such as blunted affect, anhedonia or loss of social drive. individuals with schizophrenia also often suffer from cognitive difficulties that impact organized thought processes, concentration, task completion and memory (apa, ) . the causes of schizophrenia are not known, but the disorder is thought to be a product of gene and environmental factors interacting during critical neurodevelopmental time points (demjaha, maccabe, & murray, ; european network of national networks studying gene-environment interactions in et al., ; kavanagh, tansey, o'donovan, & owen, ; modinos et al., ; nimgaonkar, prasad, chowdari, severance, & yolken, ; schizophrenia working group of the psychiatric genomics, ; tsuang, ) . over the years, research has concentrated on the dopamine receptor, but pharmacological modulation of subtypes of this receptor is not an effective treatment for all cases of schizophrenia. more specifically, although dopamine receptor modifying drugs can be effective for positive symptoms in many, these agents are rarely effective for negative and cognitive symptoms, which account for much of the chronic morbidity of the condition. this research focus beyond the dopamine receptors has broadened to include other neurotransmitter receptor types including serotonergic, cholinergic, glutamatergic, and gabaergic pathway targets, yet the discovery of definitive causative mechanisms for this disease that might effectively guide treatment strategies has remained elusive (erhart, marder, & carpenter, ; marder, ) . schizophrenia is currently treated with typical and atypical antipsychotics. the older, typical, antipsychotics produce a sedative effect and closely target dopamine receptors. atypical antipsychotics bind dopamine receptors but with less affinity and with additional blockade of the serotonin -ht a receptors (seeman, ) . the secondgeneration atypical agents generally overcome some of the extrapyramidal motor side effects associated with the first-generation typical antipsychotics, but themselves can produce metabolic side effects including diabetes mellitus, weight gain and hyperlipidemia (crossley, constante, mcguire, & power, ; ucok & gaebel, ) . with much inter-agent and inter-patient variability, both generations of treatments can be effective in reducing positive psychiatric symptoms such as delusions and hallucinations, but there remains a significant proportion of patients who are partially or fully resistant to treatment or who discontinue the medications (leucht et al., ; muller, ) . furthermore, as mentioned, many of the currently available antipsychotics fail to adequately elicit an improvement in negative symptoms or cognition (meyer, schwarz, & muller, ; muller, ) . the differences in effectiveness and side effect profiles from a meta-analysis as well as from the clinical antipsychotic trials of intervention effectiveness (catie) and cost utility of the latest antipsychotic drugs in schizophrenia study (cutlass) trials are well discussed by leucht et al. ( ) . in addition, pharmaceutical companies are downsizing psychiatric research budgets, as the currently applied drugs, though flawed, are still the most effective medications that are available. these agents are also becoming accessible generically (bartfai & lees, ) . technical advances fueled by the deep sequencing revolution and genome wide association studies (gwas) have not brought about the identification of new genetic loci to direct treatment research efforts or shed light on new mechanisms guiding schizophrenia disease pathophysiology, with one exception that is the focus of our review, the immune system. immune system dysregulation in schizophrenia is a consistently replicated physiological target that encompasses gene by environmental interactions (international schizophrenia et al., ; jones, mowry, pender, & greer, ; kirch, ; knuesel et al., ; meyer, ; muller, ; rothermundt, arolt, & bayer, ; severance, yolken, & eaton, ; shi et al., ; stefansson et al., ; torrey & peterson, ; . gwas studies identified the p - p chromosomal portion that houses the major histocompatibility complex/human leukocyte antigen mhc/hla and complement c loci as one of the most important genetic susceptibility regions for schizophrenia (corvin & morris, ; international schizophrenia et al., ; sekar et al., ; shi et al., ; stefansson et al., ). the mhc/hla gene family encodes cell surface proteins that bind and present antigens as either self or non-self to t-cells; thus, any dysfunction thereof predisposes a susceptibility to infectious disease, graft rejection, cancer and autoimmunity. the complement c association is interesting because its variation in gene copy numbers may cause abnormal synaptic pruning in schizophrenia, presumably as a consequence of perturbation during normal neurodevelopment and not necessarily as any intrinsic abnormality in immunity (sekar et al., ) . other genetic risk alleles found by gwas outside of this chromosomal region also are involved in immune pathways (network and pathway analysis subgroup of psychiatric genomics, ) . this genetic information, coupled with decades of evidence for environmentally triggered immune risk factors and newfound functions in the brain by classic peripheral immune pathways, all point to an integral immune system role in schizophrenia. among the dysregulated immune conditions associated with schizophrenia are those with distinct autoimmune components. here we review the autoimmune component of schizophrenia and mechanisms by which this comorbidity could impact central nervous system (cns) function via the gut-brain axis. an overview of our topic is modelled in fig. . we will first present the basic tenets of autoimmunity in a context that is relevant to our subsequent evaluation of autoimmunity in schizophrenia. we will then discuss the autoimmune phenotype of schizophrenia in terms of risk factors for disease including the role of infection, a peripheral and central inflammatory state, gut dysbioses and the presence of autoantibodies. we will integrate our discussion with mechanisms that contribute to relevant cns pathologies, including behavioral indices such as severity of psychiatric symptoms and cognitive deficits and biochemical indices such as complement pathway dysfunction and autoimmune-related neurotransmitter receptor hypofunction. in so doing, we bring to light new therapeutic targets and review some of the clinical trials that have already been implemented to test the efficacy of treatment of these immune pathways in schizophrenia. the maintenance of good health relies on an immune system that can recognize and destroy non-self-entities including bacteria, viruses and other foreign antigens, as well as remove injured or mutated selfentities such as apoptotic cells and environmentally-or geneticallymodified native proteins. autoimmunity is a complex condition that reflects the inability of a host to differentiate self from non-self. in affected individuals, autoimmunity generally indicates the insufficient establishment of or loss of immune tolerance. tolerance is a highly regulated process that is dually created centrally and peripherally. central tolerance is established through primary clonal selection aimed at eliminating immature self-reactive t and b lymphocytes from the thymus and bone marrow, respectively, before mature cells are distributed systemically. self-reactive lymphocytes can escape into the systemic circulation, and peripheral tolerance refers to the inactivation of these systemic mature auto-reactive t and b cells that are typically concentrated in the body's lymph nodes or non-lymphoid tissues. methods to control these wayward lymphocytes include their placement into an anergic state or their removal by regulatory t cells (tregs) (murphy, travers, walport, & janeway, ; theofilopoulos, kono, & baccala, ) . when immune tolerance is disrupted, autoimmune diseases ensue. these conditions are characterized by chronic inflammation, autoantibodies, and tissue injury. the autoimmune pathophysiology can occur systemically (systemic lupus erythematosus) or in organ-specific patterns (hashimoto's thyroiditisthyroid; type diabetes mellituspancreas; rheumatoid arthritisconnective tissue). an imbalanced immune system where over-activated pro-inflammatory effector t cells outnumber the immunosuppressive tregs can result in autoimmune pathologies, and there are a variety of mechanisms that can bring about this imbalance. one suspected trigger of autoimmunity is infection in genetically susceptible people (murphy et al., ; theofilopoulos et al., ) . in a recent study of the danish civil registration system, it was found that hospital admission for infection was a risk factor for autoimmune disorders (nielsen, kragstrup, deleuran, & benros, ) . infection-mediated loss of tolerance can involve the polyclonal activation of autoreactive t cells, molecular mimicry, uncovering of cryptic antigens and the creation of novel self-reactive antigens. for example, in molecular mimicry, antibodies directed against the infectious agent or corresponding t cells become cross reactive to self-antigens. new antigenic epitopes can be generated when pathogen-derived proteins bind to a host's cell or tissue or when self-entities undergo post-translational or pathogen-elicited modifications. infection can also break down the sequestering of tissue-specific antigens that were previously quiescent in areas known as immunologically privileged. these generated autoantibodies initiate the cellular immune machinery to clear perceived invading antigens and thus lead to inflammation, tissue damage, and hypersensitivity that typifies an autoimmune condition (murphy et al., ; theofilopoulos et al., ) . autoantibodies can be pathogenic through binding and inactivation of cell surface receptors and extracellular molecules and the formation of immune complexes that collect and cause tissue damage. autoantibodies generated by the mother also have the potential of affecting fetal development as part of antibody transfers across the placenta (lleo, invernizzi, gao, podda, & gershwin, ) . it is possible that these autoantibodies and their potential to bind to important brain proteins may underlie some neuropsychiatric symptoms. . . the gut as a source of autoimmunity as described later, low grade central and peripheral inflammation is an emerging risk factor for the development of schizophrenia (bechter, ; bechter et al., ) , but the source of this inflammation is not known and likely has multiple origins in different people. the largest immune organ in the body is the gastrointestinal (gi) tract which serves as a critical hub regulating self and non-self interactions via the gutassociated lymphoid tissue (galt). as such, the galt functions to convey immune tolerance to a complex community of commensal microbes, externally-derived dietary products, and the host's own cellular machinery, while simultaneously protecting the body from destructive pathogens and other dangerous antigens. (chistiakov, bobryshev, kozarov, sobenin, & orekhov, ; dinan & cryan, ; sandhya, danda, sharma, & scaria, ; wekerle, ) . members of the gut microbiome include bacteria, viruses, archaea and fungi, which comprise a functional system whose goal it is to extract, metabolize and absorb nutrients to fuel and maintain a healthy body. these microbes and downstream products, however, also have important immunemodulatory roles. when functioning correctly, they dynamically control the immune response and inflammatory status of the gi tract. the breakdown of tryptophan, for example, leads to a broad range of kynureninerelated metabolites that function as both pro-and anti-inflammatory triggers and are involved with excitatory neurotransmission (cervenka, agudelo, & ruas, ) . bacterial metabolites and structures such as short chain fatty acids (scfas) and polysaccharide moieties function diversely to maintain an immune homeostasis with pro-inflammatory th effects that are dampened by anti-inflammatory treg cellmediated immunosuppression (wekerle, ) . importantly, many of these microbial metabolites are neuromodulatory and either are themselves, or else trigger the production of, neurotransmitters and neuropeptides such as dopamine, acetylcholine, gamma-aminobutyric acid, serotonin and brain-derived neurotrophic factor (cryan & dinan, ; rieder, wisniewski, alderman, & campbell, ) . a homeostatic microbiome can become imbalanced, or dysbiotic, by many intrinsic or extrinsic factors including diet, pharmaceuticals, toxic chemicals, infectious agents and even by the genetics of the host (abegunde, muhammad, bhatti, & ali, ; sandhya et al., ; wu et al., ) . during this dysbiosis, microbial control of inflammation shifts toward a pro-inflammatory th , th and th dominance and suppression of anti-inflammatory treg cells (chistiakov et al., ; hornig, ; wekerle, ) . in response, there is an increase in the expression of antimicrobial proteins, fig. . model of autoimmune phenotypes operating along the gut-brain axis in schizophrenia. as described in the main text, infection, inflammation, gut dysbioses and autoantibodies are all related to measurable autoimmune features in schizophrenia. these features contribute to permeabilization of the blood-gut barrier, which allows the translocation of gut-based products into circulation. interactions of these gut products with neurotransmitter receptors located in the enteric nervous system may be one source of autoantibody propagation especially in the presence of inflammation. systemically, a low-grade inflammatory state is perpetuated as the immune system is activated, including complement and cytokine production pathways. in turn, this inflammation may contribute to a compromised bbb and allow gut-derived products, peripheral immune factors and autoantibodies access to the brain. immune dysfunction, susceptibility to infections and barrier stabilities in schizophrenia may be under genetic control. in the diagram, immune cells in peyer's patches and in systemic circulation include macrophages, dendritic cells, b lymphocytes and t lymphocytes. yy refers to the immunoglobulins iga, igg and igm. downregulation of inflammatory pathways, and an increase in the production of mucus to protect epithelial surfaces and repair damaged intestinal tissue and cellular barriers (ismail & hooper, ; round, o'connell, & mazmanian, ; smith & garrett, ; sommer & backhed, ) . when commensal bacteria (and invading pathogens) do breach the gi mucosa and gut-vasculature barrier, the setting is prime for the generation of an autoimmune state with the creation of an inflammatory environment that both develops from and fosters the genesis of autoantibodies. microbial dysbiosis has a hypothesized role in the etiology of autoimmune-based human inflammatory bowel disorders including crohn's disease and ulcerative colitis (powell, walker, & talley, ; theofilopoulos et al., ) . similarly, the gut is in constant contact with food-derived antigens that, in a setting when tolerance has not been sufficiently established, can also trigger autoimmunity. tolerance to the food load also involves microbial maintenance of epithelial cellular barriers, efficient digestion of food proteins and immune mediation by regulatory t and dendritic cells (steele, mayer, & berin, ) . a gi disorder with an autoimmune mechanism directed against food antigens is celiac disease. celiac disease is characterized by a hypersensitivity autoimmune reaction directed against a wheat gluten antigen combined to an enzyme necessary for its metabolism, tissue transglutaminase, in individuals with a genetic predisposition as evidenced by specific hla-dq types. increasingly, a psychiatric comorbidity is being recognized in individuals with autoimmune gi disorders (fadgyas-stanculete, buga, popa-wagner, & dumitrascu, ; filipovic & filipovic, ; gupta, masand, kaplan, bhandary, & hendricks, ; vaknin, eliakim, ackerman, & steiner, ; vu, kushnir, cassell, gyawali, & sayuk, ) . conversely, there is a long history of data demonstrating gi inflammation in individuals with psychiatric disorders (buscaino, ; hemmings, ; severance, prandovszky, castiglione, & yolken, ) . autoimmune conditions of the brain can arise via such varied means as injury-induced cytokine release, pathogen-and tumor-directed antibodies that are cross-reactive to brain proteins, t-cells that are exposed to previously inaccessible brain antigens, and genes that confer a susceptibility to immune dysfunction (pilli, zou, tea, dale, & brilot, ; wekerle, ) . cns-related autoimmune disorders include multiple sclerosis, amyotrophic lateral sclerosis (als), limbic encephalitis, seizures, rasmussen's encephalitis, pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (pandas) and anti-n-methyl-d-aspartate (nmda) receptor encephalitis (dalmau, geis, & graus, ; diamond, honig, mader, brimberg, & volpe, ; pilli et al., ) . in spite of a varied etiology, many autoimmune encephalopathies are similarly characterized by an inflammatory state and the presence of antibodies directed against such brain structures as neuronal surface receptors and synaptic proteins (dalmau et al., ; diamond et al., ; newman et al., ) . as research in this field progresses, the intricate and extensive role that peripheral immunity has within the cns is becoming better understood and appreciated. the cns has long been considered an immune-privileged organ and thus impervious to processes involving peripheral immune cells. structural protection comes from the blood brain barrier (bbb), an endothelial cell-vascular interface reinforced with tight junction proteins and astrocyte processes, which has been thought to prevent peripheral immune factors and cells from entering the brain. recent data bring to question a more contentious issue regarding the requirement that the integrity of the bbb be compromised in order for autoimmune features to affect the cns. bbb permeability would presumably be required for autoantibodies generated peripherally to be active in the brain, as well as for the cns entry of inflammatory molecules or other immune entities (pollak et al., ) . it is becoming evident that subsets of lymphocytes are able to gain access to and take up residence in the cns in spite of an intact bbb, and further, these b and t cells may become active in the presence of microbial infection (wekerle, ) . the perceived impenetrability of this structural and immune barrier in the brain was further disputed with the discovery of lymphatic vessels lining the dural sinuses and connecting the csf with deep cervical lymph nodes; in these vessels were t cells suggesting that this conduit between the cns and lymph nodes provides evidence that the cns is a site accessible to immunosurveillance (louveau et al., ) . nevertheless, inflammation renders the bbb more permeable and therefore, there are likely numerous autoimmune conditions that are accelerated when a bbb has been inappropriately breached (hammer et al., ) , such as during pathogen infection. interestingly, in germfree experimental mouse studies, the absence of gut microbes was associated with a dysfunctional bbb, altered myelination of cortical neurons, cognitive deficits and behavioral changes (blander, longman, iliev, sonnenberg, & artis, ; sharon, sampson, geschwind, & mazmanian, ) . the wide range of types of antigens that bring about similar immune effects suggests it is the immune system response or dysregulation thereof rather than a specific pathogen or food exposure that contributes to neuropsychiatric disease. the immune system response can take the form of adaptive immunity, where an antigen-specific response elicits the machinery to generate immunological memory through clonal selection of lymphocytes. the immune response can also be innate, a form characterized by less specificity and immediate cellular defense to clear invading pathogens, or antigens identified as foreign (murphy et al., ) . the complement pathway bridges innate and adaptive immunity and when initially activated forms immune complexes to aid clearing of foreign antigens, damaged cells, and autoantigens from circulation. defects of the complement pathway lead to increased susceptibility to infection and to autoimmune disorders. complement deficits result in the accumulation of apoptotic debris, immune complex deposits, inflammation and tissue damage. lingering apoptotic waste is a source for autoantigens especially during inflammation in genetically susceptible individuals, and subsequent t cell activation can lead to autoreactive b cells and autoantibodies (walport, a (walport, , b . systemic lupus erythematosus (sle), for example, is an autoimmune disease characterized by systemic autoimmune symptoms that result from tissue injury in multiple organs due to high levels of immune complex deposits. sle is associated with alterations in the levels of multiple complement proteins including c q and c . c q is a first-line antigen recognition protein that forms immune complexes with antigens bound to antibodies; c is found further downstream from c q in the classic pathway and also as part of the pamp-or damp-initiated lectin pathway, in both cases involved in enzymatic reactions to assemble the c -convertase and eventual activation of the membrane attack complex (ballanti et al., ; martin & blom, ; walport, a walport, , b . intriguingly in recent discoveries over the last decade, this peripheral complement pathway was found to be active in the brain. as described in rodent models, complement proteins functioned in synapse formation and elimination during neuronal development as well as in response to the presence of brain pathogens in adults (bialas & stevens, ; boulanger, ; fourgeaud & boulanger, ; hong et al., ; lui et al., ; presumey, bialas, & carroll, ; ransohoff & stevens, ; schafer et al., ; stephan et al., ; stevens et al., ; vasek et al., ) . the subsequent identification of c -related polymorphisms as genetic risk factors for schizophrenia (sekar et al., ) has potentially important implications for autoimmune studies of schizophrenia and may represent a nexus molecular pathway by which genetic and environmental etiologies for the disorder can be reconciled (nimgaonkar et al., ) . with respect to schizophrenia, the timing of exposure to an infectious disease with maternal generation of antibodies during the prenatal or early postnatal period is a topic that has been the focus of numerous investigations, the results of which generally support neurodevelopmental hypotheses for schizophrenia etiology (allswede, buka, yolken, torrey, & cannon, ; blomstrom et al., ; brown, cohen, greenwald, & susser, ; buka et al., ; ellman, yolken, buka, torrey, & cannon, ; karlsson et al., ; mortensen et al., ; pedersen, stevens, pedersen, norgaard-pedersen, & mortensen, ; severance, gressitt, buka, cannon, & yolken, ; xiao et al., ). the precise activation of complement in response to a maternal antibody load implicates a mechanism of inappropriate fetal activation of synaptic pruning as an etiological pathway to schizophrenia. as described to this point, autoimmunity can materialize through multiple mechanisms that reflect the body's failure to accurately recognize its own cellular make-up. emerging data indicate that these pathologies may very well impact the brain. a similar dysregulation of adaptive and cellular immunity is increasingly demonstrated in schizophrenia, suggesting that this disorder, although not a classic autoimmune disease, may share some common, potentially treatable, physiological features. early records examining the associations between autoimmune disorders and schizophrenia document the discovery of an inverse correlation in prevalence between rheumatoid arthritis and schizophrenia, and this relationship was also more recently replicated (chen et al., ; eaton, hayward, & ram, ; gorwood et al., ; sellgren, frisell, lichtenstein, landen, & askling, ; trevathan & tatum, ) . other investigations, including some recent large-scale birth registry studies, found a positive association between an array of autoimmune disorders (including multiple sclerosis, systemic lupus erythematosus, autoimmune thyrotoxicosis, autoimmune hepatitis and psoriasis) and a diagnosis of schizophrenia or psychosis chen et al., ; eaton et al., ; gilvarry et al., ; wright et al., ) . in addition, it has also been found that a history of infection further contributed to elevate the risk of autoimmune disorders on the development of schizophrenia (benros et al., ) . similarly, having a history of an infection or having a family member with schizophrenia also significantly elevated the risk for developing an autoimmune disease . very recently, an analysis of this registry data indicated that the combination of a history of infections and exposure to anti-infective agents such as antibiotics further augmented the risk for developing schizophrenia (kohler et al., ) . it is likely that in certain cases, autoimmune disorders and schizophrenia may be the result of a genetically-encoded insufficient or over-active immune response triggered by exposure to an environmental factor such as infection. data from studies of the microbiome also suggest that the genesis of autoimmunity can occur at least in part when the gut is dysbiotic (de oliveira, leite, higuchi, gonzaga, & mariano, ; fung, olson, & hsiao, ; thaiss, zmora, levy, & elinav, ). an intestinal origin for autoimmunity also reconciles numerous risk factors for the development of schizophrenia, which in turn supports the existence of a gut-brain connection. as such, similarly faulty genetic templates perhaps involving variations of mutations in mhc, hla or complement loci in autoimmune disorders and schizophrenia may result in a dysregulated immune response to not only pathogens but to a wide array of environmentally-and endogenously-derived antigens. the reaction against these antigens provokes inflammation, a hallmark characteristic of the autoimmune state and often observed in people with schizophrenia (bechter, ; dickerson et al., ; fillman et al., ; miller, buckley, seabolt, mellor, & kirkpatrick, ; . again, of interest with respect to brain disorders are the many components of the complement pathway which are not only part of inflammation-generating trajectories but also, as mentioned, have newly-discovered functions in synapse formation and pruning. autoantibodies also prime and perpetuate the autoimmune inflammatory state. if these autoantibodies are directed against important brain proteins such as neurotransmitter receptors, neuronal function can be greatly compromised. in this section, we review the evidence in support of pathogen exposures, inflammation, the gut-brain axis and autoantibodies as autoimmune phenotypes in schizophrenia. there is a long history of research and support for infection as a process leading to autoimmune disorders (ercolini & miller, ; nielsen et al., ) ; therefore, it is not surprising that exposure to pathogens has been posited as an autoimmune risk factor for the development of schizophrenia (hornig, ; torrey & peterson, ; . among the documented infectious disease agents and types of infections that have been associated with schizophrenia are the protozoan toxoplasma gondii, influenza viruses, cytomegalovirus and other human herpesviruses, borna disease virus, endogenous retroviruses, coronaviruses, urinary tract infections, chlamydophillas, and candida yeast infections (arias et al., ; hornig, ; karlsson et al., ; karlsson, schroder, bachmann, bottmer, & yolken, ; leweke et al., ; miller et al., ; severance et al., ; torrey et al., ; torrey, bartko, lun, & yolken, ; torrey, bartko, & yolken, ; . in some studies, pathogen exposures were associated with a decrease in cognitive function and progressive gray matter loss prasad et al., ; prasad, watson, dickerson, yolken, & nimgaonkar, ; schretlen et al., ; severance, gressitt, et al., ; shirts et al., ; watson et al., ; yolken, torrey, lieberman, yang, & dickerson, ) . the focus of this review is on identifying autoimmune conditions in people with schizophrenia with a direction toward identification of new treatments; however, it should be noted that there is also a large literature base about prenatal exposures to specific microbes and to the infectious disease process as a risk factor for the future development of schizophrenia brown et al., ; brown, begg, et al., ; brown & derkits, ; brown, hooton, et al., ; buka et al., ; ellman et al., ; estes & mcallister, ; mortensen et al., ; pedersen et al., ; xiao et al., ) . perhaps the most consistently replicated finding of a pathogen exposure associated with a diagnosis of schizophrenia involves the parasitic pathogen, t. gondii (arias et al., ; monroe, buckley, & miller, ; torrey et al., ; torrey et al., ) . only in recent years has this connection been regarded in the context of t. gondii as a gut pathogen showing significant associations with gut-based antigens and inflammatory processes in people with schizophrenia severance, yolken, & eaton, ) . indeed, t. gondii is used routinely in experimental rodents to model inflammatory bowel diseases (craven et al., ; grainger et al., ; hand et al., ; heimesaat et al., ) . thus, as an inflammation-generating, neurotropic parasite able to permeabilize endothelial cell barriers, t. gondii is uniquely equipped to pathologically impact the brain directly resulting in glial cell activation or indirectly via the facilitated entry of systemic immune and gut-derived factors to the cns. in a mouse model, we demonstrated extensive t. gondii-generated complement activation in brains in the vicinity of the parasite and in proximity to synapses (xiao et al., ) . in a recent translational study, we found parallel increases in mice and humans of autoantibodies directed against the nmda receptor in those who had been exposed to t. gondii, with corresponding elevations of surrogate markers of compromised blood-gut-barrier and bbb (kannan et al., ) . results from this and other studies also further documented the effects of parasite exposure on clinical endpoints such as decreased cognition, suicidal behavior and severity of psychotic symptoms esshili et al., ; hamdani et al., ; kannan et al., ; lindgren et al., ) . just as schizophrenia is not a classic autoimmune disorder, it is also not a classic inflammatory disorder. in schizophrenia, the detectable inflammation has been ascribed to be of a low-grade nature with peripheral and central inflammatory states measured using a variety of biomarkers in different research labs (bechter, ; catts, wong, fillman, fung, & shannon weickert, ; dickerson et al., ; fillman et al., ; fillman et al., ; fillman, sinclair, fung, webster, & shannon weickert, ; kirkpatrick & miller, ; miller et al., ; muller, ; severance et al., ; . in most cases, the source of this inflammation in these studies is not known, but it is evident in the form of pro-and anti-inflammatory cytokine dysregulation, disrupted tryptophan and kynurenine metabolism, exposure to microbial pathogens and antigens, food hypersensitivities, complement activation, post-mortem pathologies and brain imaging. a large meta-analysis of cytokines in schizophrenia provided strong evidence for upregulation of proinflammatory cytokines associated with schizophrenia and particularly of il- , ifn-gamma, and tnf-alpha compared to controls (miller et al., ) . interestingly, those patients receiving antipsychotic medication showed significantly decreased levels of il- and ifn gamma suggesting an immunosuppressive effect of these drugs; such an effect was previously demonstrated by other investigators for haloperidol and clozapine (leykin, mayer, & shinitzky, ; muller, ) . simultaneous increases in il and il- receptors in schizophrenia, however, suggest the cytokine network is dysregulated and not necessarily uniformly pro-inflammatory in direction. as noted earlier, complement activation is part of the body's inflammatory defense response culminating in the launching of the membrane attack complex. altered peripheral complement components in schizophrenia include c , c , and c , although not all of the studies reported consistent findings (arakelyan et al., ; boyajyan, khoyetsyan, tsakanova, & sim, ; kucharska-mazur et al., ; mayilyan, ; mayilyan, weinberger, & sim, ; santos soria, moura gubert, cereser, gama, & kapczinski, ; . c is also upregulated centrally with postmortem human brains samples showing increased rna expression in schizophrenia compared to controls (sekar et al., ) . as noted, c polymorphisms have been identified as conferring a genetic susceptibility for schizophrenia with copy number variations impacting levels of gene expression (mayilyan, ; mayilyan, dodds, boyajyan, soghoyan, & sim, ; sekar et al., ) . preliminary evidence in a study of twins with schizophrenia implicated peripheral expression of complement genes c and serping with cortical thinning (allswede et al., ) . while centrally-derived complement activity has implications for brain biochemistry and synaptic pruning, chronic peripheral inflammation also has brain effects and was associated with cognitive impairment in schizophrenia (bulzacka et al., ) . as we posit in the next section, a logical possible source of this inflammation derives from a gut microbiome that is imbalanced; this imbalance may cause the translocation of microbes, microbial products, antigenic food products across the gut vasculature, and instigate a low level of inflammation and autoimmune-generating potential (severance, yolken, & eaton, ) . as discussed earlier, an imbalanced gi environment is a prime venue for the production of autoantigens. a leading autoimmune disorder with often replicated ties to schizophrenia is, in fact, celiac disease (baldwin, ; bender, ; dohan, dohan, , dohan, , eaton et al., ; graff & handford, ) . as we discussed earlier, people with celiac disease have an intolerance to wheat gluten which when ingested causes the body to launch an autoimmune attack against what it perceives to be a novel antigenic complex of gluten bound to cell-derived tissue transglutaminase in the small intestine (alaedini & green, ; green et al., ; guandalini & assiri, ) . gluten antibodies have also been shown to bind synapse-related brain proteins (alaedini et al., ; briani et al., ; hadjivassiliou et al., ) . this connection between gluten exposure and psychiatric disorders was first hypothesized by fc dohan who observed that psychiatric hospitalization rates in europe strongly correlated with the amount of wheat consumption, which varied considerably during and immediately after world war ii (dohan, a (dohan, , b . since then, numerous studies and meta-analyses have examined the association of food antigens with schizophrenia and with varied results (cascella et al., ; dickerson et al., ; jin et al., ; mclean, wilson, st clair, mustard, & wei, ; reichelt & landmark, ; reichelt & stensrud, ; samaroo et al., ; severance et al., ) . in our studies, these gluten antibody profiles correlated with measures of gi inflammation (severance et al., ; . interestingly, in a recent clinical study, antigliadin antibodies were found to predict brain inflammation as measured via proton magnetic resonance spectroscopy (rowland et al., ) . celiac disease is marked by strong hla associations, pathogenic cd + and autoantibody generation. as such, celiac disease represents the classic interaction of genes, the environment and immunology (hardy & tye-din, ) . similar operative mechanisms may be present in certain people with schizophrenia who suffer from gluten sensitivities. inflammatory bowel diseases are among the autoimmune disorders that are found with increased frequency in schizophrenia (gupta et al., ; severance, prandovszky, et al., ) . likewise as we have indicated, there are emerging data that people diagnosed with these gi disorders have increased rates of psychiatric symptoms (fadgyas-stanculete et al., ; filipovic & filipovic, ; gupta et al., ; vaknin et al., ; vu et al., ) . gi disturbances related to mental illnesses are recorded as early as the first and second centuries when hippocrates and later galen advocated the practice of treating the gut in order to improve brain function (severance, prandovszky, et al., ) . since then, the search for a physiological basis for the gut-brain connection has resurfaced periodically, particularly starting in the th century, and interest in this topic is currently at an all-time high. the historical precedence of gi studies of mental illness is important, because of a present misbelief that the gi comorbidities are primarily the result of medications. while intestinal motility is impaired by the anticholinergic side effects of antipsychotics, gi dysfunction not attributable to medications has been documented in numerous studies, including those that predate the introduction of antipsychotic medications which occurred in the s and those that examine various girelated biomarkers in antipsychotic-naïve patients (severance, prandovszky, et al., ) . a pre-antipsychotic era study of post mortem tissue reported - % of the schizophrenia group had extensive bowel inflammation (buscaino, ; hemmings, ) . in our studies, food antigen exposures, yeast exposures and bacterial dysbiosis are all entities that contribute to gi-related inflammation in individuals with schizophrenia (severance et al., (severance et al., , severance, gressitt, et al., ) . parallel studies in antipsychotic naïve individuals support that the biomarker patterns are specific to the disease state and not the result of immune effects of the drug. these biomarker studies also document the translocation of gut-based products such as food peptides and microbial products into systemic circulation at increased rates in people with schizophrenia compared to controls. as discussed earlier, it is known that gi inflammation produces an environment that has deleterious effects on the gut vasculature barrier. we found evidence for compromised endothelial barrier pathologies in the gut and cns, which were associated with schizophrenia. in people with intact endothelial barriers, gut-derived food antigen antibodies found peripherally in serum should not be associated with those found centrally in the csf. in people with schizophrenia, we found strong correlations between serum and csf antibodies, which were not present in control individuals without a psychiatric disorder . gut-derived inflammation can be the product or source of a gut microbiome that is out of balance. the gut microbiome itself has not been yet extensively studied in schizophrenia, but initial deep sequencing studies of microbial taxa suggest altered patterns of diversity associated with disease compared to controls (castro-nallar et al., ; schwarz et al., ; yolken et al., ) . in the oropharyngeal microbiome, species of lactic acid bacteria, lactobacilli and bifidobacterium, known to regulate chronic inflammation, were more abundant in schizophrenia compared to controls (castro-nallar et al., ) . interestingly, levels of the viral phage, lactobacillus phage phiadh, were also altered in schizophrenia and correlated with levels of its corresponding bacterial host, lactobacillus gasseri . a study of the fecal microbiome also pointed toward elevations of lactobacillus bacteria in individuals with first episode psychosis compared to controls and importantly indicated that these levels were related to the severity of psychotic symptoms and response to treatment (schwarz et al., ) . these few studies of the microbiome as well as those documenting microbial translocation collectively indicate that gut dysbioses are putatively prevalent in schizophrenia (dickerson, severance, & yolken, ) . further studies aimed to elucidate the functional consequences of this microbial dysbiosis on cns endpoints such as psychiatric symptoms, cognition and treatment resistance are desperately needed. thus, inflammation in the intestinal tract and associated compromise of the gut-blood cytological barrier has varied implications for people with psychiatric disorders who may have co-existing autoimmune conditions. microbial and related products in the bloodstream lead to systemic immune activation, a potentially pathological state that may be already aggravated in individuals who have genetically-encoded immune dysfunctions. endothelial barrier permeability not only impacts the gi-vasculature interface but provides a means by which gut-derived products might penetrate the blood-brain barrier, a cytological architecture that is structurally similar. these faulty barriers may be particularly important if autoantibodies generated in the inflammatory gut environment were able to cross a compromised bbb. an intestinal system in flux may predispose to autoimmunity by means of a wide array of neurotransmitter targets found in the enteric nervous system that are identical to those found in the brain. understanding the role of autoantibodies that are reactive against brain proteins is a longstanding subject of studies that examine autoimmunity in psychiatric disorders (boehme, cottrell, dohan, & hillegass, ; durell & archer, ; fessel, a fessel, , b glebov, ; gurevich, ; heath, ; heath & krupp, ; heath, krupp, byers, & liljekvist, a; heath, krupp, byers, & liljekvist, b; jones et al., ; kirch, ; lehmann-facius, ; mellsop, whittingham, & ungar, ; stamboliev, ; stoimenov, ) . a recent meta-analysis recorded a significant elevation of different autoantibodies in persons with schizophrenia compared with controls (ezeoke, mellor, buckley, & miller, ) . in schizophrenia, autoantibodies directed to a number of brain proteins have been examined, and particularly but not exclusively, the target has been the neurotransmitter receptors: cholinergic muscarinic receptors, nicotinic acetylcholine receptors, dopamine d receptors, mu-opioid receptors, serotonin receptors, ampa receptors, glutamate receptors, gamma-aminobutyric acid (gaba) receptors, glutamic acid decarboxylase (gad), and potassium channel receptors (deakin, lennox, & zandi, ; ezeoke et al., ; jones et al., ; masdeu et al., ) . these studies also highlight other brain-related entities that may be regulated by autoantibodies including neuregulin- , human endogenous retroviruses (hervs), cardiolipin, dna, histones, and mitochondria. the rationale for studying neurotransmitter receptors is based on the idea that these autoantibodies bind and suppress the activity or otherwise block and contribute to receptor hypofunction. of note, cell surface targets such as neurotransmitter receptors are presumably more accessible than intracellular targets such as dna and mitochondria. nmda receptor hypofunction has been a longheld hypothesis for schizophrenia etiology with evidence that its blockage in healthy people precipitates some of the symptoms of schizophrenia (gilmour et al., ) . positive associations of nmda receptor autoantibodies and schizophrenia have not, however, been uniformly replicated suggesting the possibility that autoantibody presence may not be solely disease associated (ando et al., ; castillo-gomez et al., ; dahm et al., ; de witte et al., ; deakin et al., ; dickerson et al., ; haussleiter et al., ; masdeu et al., ; masopust et al., ; pearlman & najjar, ; pollak, mccormack, peakman, nicholson, & david, ; rhoads, guirgis, mcknight, & duchemin, ; steiner et al., ; steiner et al., ; timucin, ozdemir, & parlak, ; zandi et al., ) . for example, these putatively pathogenic anti-nmda receptor antibodies and other brain-directed antibodies were found to be present in multiple non-psychiatric control groups (castillo-gomez et al., ; glass et al., ) . as the autoantibody presence in controls may also be significant, it is possible that these autoantibodies may serve some regulatory function. it is additionally a possibility that a two-hit hypothesis is in effect; for these autoantibodies to be pathogenic, access through the bbb is required. as discussed with respect to the parasite, t. gondii, infection may be a potent agent of both barrier permeabilization and autoantibody generation (kannan et al., ) . in this review, we have focused on t. gondii as the perpetrator of autoimmunity, but other infectious disease agents may also produce autoantibodies, including hiv (arboleya et al., ) . the alpha nicotinic acetylcholine receptor represents an interesting autoimmune target because it introduces the putative contribution of the enterically-located vagus nerve and thus another gut-brain connection. the vagus nerve carries information between the enteric nervous system and the cns. it is hypothesized that in a certain subset of patients, the vagus nerve when stimulated can control peripheral and central inflammation through modulation of nicotinic acetylcholine receptor subtypes (corsi-zuelli et al., ) . thus, if autoantibodies are present against this receptor, as several studies of schizophrenia have found (chandley, miller, kwasigroch, wilson, & miller, ; mukherjee et al., ) , suppressed inflammation or dysregulated anti-inflammatory modulation may ensue. this set of studies further implicates the enteric nervous system as a rich autoantibody source for relevant brain targets, as the enteric nervous system is equipped with much the same extensive array of neurotransmitter receptors that are found in the brain. finally, with respect to autoantibodies, we address the timing of exposure to these potentially pathological agents, particularly for the fetus. a large repertoire of work in rodents documents the role of maternal immune activation as detrimental to fetal brain biochemistry (brown & derkits, ; estes & mcallister, ) . among the pathways studied, complement is found to be elevated peripherally as well as centrally following infection of the mother (han, zhang, & hashimoto, ; . results from human studies also support a neurodevelopmental role for maternal immune activation in conferring risk to the development of schizophrenia or psychosis in adult offspring (allswede et al., ; brown & derkits, ; estes & mcallister, ) . a large high profile study of the swedish birth registry demonstrated elevated gluten antibodies in mothers whose children went on to develop psychosis as adults . because we had previously found that c q bound and formed immune complexes with gluten at increased rates in schizophrenia compared to controls, we examined maternal c q levels in a birth cohort from the national collaborative perinatal project and also found elevated levels of c q in mothers whose children went on to develop psychosis (severance et al., ) . in this study, maternal c q levels were significantly correlated with gluten, hsv- and adenovirus. in a rodent model of maternal immune activation, c q was elevated in the prefrontal cortex of adult offspring from mothers treated with poly(i:c) during pregnancy . placental transfer of maternal antibodies is a primary means of conferring offspring immunity. indeed, in recent work, autoantibodies to the contactin-associated protein-like (caspr ), a protein important in brain development, have been isolated from mothers of children with neurodevelopmental disorders, with some variation (brimberg et al., ; . modelling of antibody capping of caspr in mice during pregnancy resulted in offspring with significant microglial activation, synaptic loss, structural insufficiencies of the cortex and hippocampus and behavioral abnormalities (brimberg et al., ; . thus, the presence maternally of autoantibodies to important brain proteins including complement and neurotransmitter receptors may be a very tangible threat to the developing fetus. by studying the comorbid physiological conditions present in individuals with schizophrenia, we are offered clues to the cellular and pathway deficiencies that could be evaluated for treatment. as described in this review, the immune system has many complexities that when dysregulated can result in an autoimmune state. as such, this immune network represents a novel group of therapeutic targets that may be relevant to the study of brain disorders. the intricacies of the immune system and the poly-genic and poly-environmental heterogeneity of the schizophrenia disorder, itself, however, reinforces the likelihood that any one treatment approach will not be relevant for every patient. it will be critical to pre-identify those subsets of patients who are experiencing specific autoimmune characteristics and to diagnose these comorbidities with appropriate biomarkers so that an effective individualized treatment approach can be designed and implemented. it will be additionally important to develop biomarkers of treatment response and side effects . at the present time, the treatments described below are best considered as potential adjuncts to currently employed antipsychotics. in this section, we review some of the evidence in support of varied therapeutic approaches to treat autoimmune-derived abnormalities in schizophrenia. given the overlapping mechanisms of some of the treated pathological conditions, certain therapeutic approaches may be appropriate for managing multiple autoimmune conditions. minocycline is a tetracycline antibiotic that has been evaluated in several clinical trials as an adjunctive treatment to antipsychotics and was associated with improvement of certain psychiatric symptoms and cognition (chaudhry et al., ; kelly et al., ; levkovitz et al., ; liu et al., ; miyaoka et al., ) , with several meta-analyses supporting its safety and efficacy (solmi et al., ; xiang et al., ) . interestingly, the effects of minocycline in schizophrenia may not be solely a result of its direct anti-microbial activity. minocycline may also inhibit some of the detrimental effects of microglial activation including free radical generation and impaired adult neurogenesis (mattei et al., ) . other antibiotics such as azithromycin and d-cycloserine have been tested in small studies with mixed results and remain candidates for follow-up studies when biomarkers are available to identify the most appropriate patients goff, ; schade & paulus, ) . the mechanism of action of d-cycloserine may be especially informative for schizophrenia hypotheses linked to dysfunction of the nmda receptor, as d-cycloserine is a known partial agonist at the glycine modulatory site of this receptor (goff, ; mcbain, kleckner, wyrick, & dingledine, ) . more experimental data are also needed to determine the role of antibiotics in beneficially or detrimentally modulating gut microbiota and thus the gut-brain axis. several antiviral agents have been tested as adjunctive therapies including valacyclovir in various patient populations pre-screened for seropositivity to certain viruses, with mixed findings. in the first study, a significant improvement in psychiatric symptoms by valacyclovir was evident in individuals who were seropositive for cytomegalovirus but not for those who were seropositive for other herpesviruses (dickerson, boronow, stallings, origoni, & yolken, ) . in a placebo-controlled follow-up study, no differences in psychiatric symptoms were evident between the two treatment groups . no clinical trials to date have demonstrated a change in psychiatric symptoms or cognition with therapy aimed to treat t. gondii infections (chorlton, ) . although anti-malarial compounds involving artemisinin derivatives did not show a significant effect on psychiatric endpoints in two studies wang et al., ) , a reduction of antibodies directed to the food antigen, gluten, was found in the treatment group compared to placebo . because inflammation has been documented in many individuals with schizophrenia and because it can create a physiological state that predisposes susceptible individuals to autoimmunity, it is possible that anti-inflammatory pharmacotherapy may be beneficial (meyer et al., ) . agents of therapeutic potential include cox- & inhibitors, n-acetylcysteine, statins, or use of monoclonal antibodies as cytokine blockers (miller & buckley, ; muller, ) . acetylsalicylic acid (aspirin) is a cox- and - inhibitor shown to significantly lower positive psychiatric scores in smaller studies as well as meta-analyses (laan et al., ; nitta et al., ; sommer et al., ) . the cox- inhibitor celecoxib administered as an adjunct to antipsychotic agents also resulted in improvement of symptoms/cognition compared to risperidone alone in several study trials, although most benefit was observed in earlier disease stages (akhondzadeh et al., ; marini et al., ; muller, ; muller et al., ; muller et al., ) . however, this benefit by celecoxib was not confirmed in two meta-analyses of anti-inflammatory agents (nitta et al., ; sommer et al., ) , but appeared efficacious in improving psychotic symptoms in first-episode schizophrenia in a third meta-analysis . oxidative stress, as evidenced by a shift in balance in favor of proversus anti-oxidants, results in disrupted redox signaling, which is a phenotype increasingly associated with schizophrenia (do, cuenod, & hensch, ) . biomarkers of oxidative stress such as glutathione, glutathione peroxidase, and superoxide dismutase are thought to reflect this disruption, and alterations of these biomarkers have been recorded in schizophrenia and in psychosis (coughlin et al., ; coughlin et al., ; langbein et al., ; nucifora et al., ; zeni-graiff et al., ; zhang, catts, & shannon weickert, ) . n-acetylcysteine, a precursor of glutathione which has potent anti-oxidant as well as antiinflammatory properties, was found with some variation to significantly improve psychiatric symptom severity and cognition in a variety of psychiatric disorders including schizophrenia (berk et al., ; chen, chibnall, & nasrallah, ; conus et al., ; farokhnia et al., ; minarini et al., ; nitta et al., ; sommer et al., ; zheng et al., ) . n-acetylcysteine is interesting because of its multiplicity of effects on cytokine generation and removal and its role in glutamatergic and dopaminergic neurotransmission (bumb, enning, & leweke, ; sommer et al., ) . adjunctive pravastatin, a cholesterol lowering medication, was found to significantly lower positive psychiatric scores from baseline to weeks, although the benefit was not maintained through weeks (vincenzi et al., ) . a trend toward some benefit in symptom reduction was also observed for simvastatin and the serotonin ( -ht ) receptor antagonist, ondansestron (chaudhry et al., ) . adjunctive treatment with another statin, lovastatin, was not associated with improvement in psychiatric symptoms (ghanizadeh, rezaee, dehbozorgi, berk, & akhondzadeh, ) . a meta-analysis of adjunctive statin therapy in schizophrenia indicated a favorable improvement of psychiatric symptoms associated with treatment (shen et al., ) . based on results from several small studies, potential cytokine targets that might be promising in schizophrenia include monoclonal antibodies directed against il and its receptor (tocilizumab) and against tumor necrosis factor (infliximab) (miller & buckley, ) . in people with inflammatory bowel disease, for example, the antitumor necrosis factor-α aided in the reduction of mucosal inflammation (pache, rogler, & felley, ) . several trials of monoclonal antibodies for the treatment of schizophrenia are underway. inflammation in schizophrenia may be related to dysregulation of the complement system that, when coupled with complement function in the brain, make this pathway a prime candidate to harness for adjunctive treatments in schizophrenia. therapeutic strategies have focused on inhibiting complement activation which has been primarily successful with targets in the terminal pathway (barnum, a (barnum, , b . barnum ( a barnum ( , b provides comprehensive reviews on the progress and history of targeting the complement pathway using small molecules, sirna inhibitors and antibodies. the anti-c antibody, eculizumab, for example, has favorable safety and effectiveness results for managing other indications (hillmen et al., ) . the end result is blocked formation of the membrane attack complex, so complement pathways upstream from c would remain intact. targeted development of complement inhibitors for autoimmunity are in progress (morgan & harris, ) . for schizophrenia, it will be important to understand precisely how and which complement components are dysfunctional peripherally and in the brain in order for an effective complement-based treatment to be designed. the operative rationale for correcting microbial imbalances is that decreasing gut inflammation and resolving a source autoimmunity will eliminate a sequence of pathologies such as compromised barrier permeability and inappropriate immune activation. in this way, progress may be made toward therapeutics that will improve psychiatric symptoms and cognition. several approaches thus fall into this category including use of prebiotics, short chain fatty acids (scfas), probiotics, fecal transplants and various combinations thereof. most studies with this objective have been performed in experimental rodent models, but results from a number of human clinical studies also show promise. prebiotics are specialized plant products used to stimulate the colonization of the intestinal tract with beneficial bacteria but that are not metabolized by the host. scfas are metabolites generated from the fermentation of these fibers by gut microbes. scfas modulate inflammatory and metabolic pathways and are likely able to cross the bbb (joseph, depp, shih, cadenhead, & schmid-schonbein, ) . joseph et al. ( ) proposed that a diet enriched with fiber in conjunction with scfas, such as the mediterranean diet, might improve immune outcomes that are dysregulated in schizophrenia. intestinal permeability may also be improved with the scfas propionate and butyrate, and the polyunsaturated fatty acids, omega- fatty acids (michielan & d'inca, ) . satogami, takahashi, yamada, ukai, and shinosaki ( ) showed that reduced omega- levels in the blood were associated with significant cognitive impairment in individuals with schizophrenia, but a number of trials of omega- supplementation in schizophrenia have been negative (chen, chibnall, & nasrallah, ; fenton, dickerson, boronow, hibbeln, & knable, ; qiao et al., ; satogami et al., ) . results from a rodent study showed that a diet enriched for the microbial metabolites acetate and butyrate increased regulatory t cell densities, improved gut barrier cohesion and reduced diabetogenic cytokines such as il (marino et al., ) . probiotics are supplements containing live beneficial gut bacteria and sometimes yeast, administered with the rationale that these microorganisms will themselves re-establish a balanced microbiome. probiotics have been evaluated in schizophrenia in three analyses of a placebo-controlled study. it was found that probiotics (lactobacillus rhammosus strain gg and bifidobacterium animals subsp. lactis strain bb ) given once per day improved gi symptoms, altered il- mediated immune and intestinal epithelial pathways and was associated with decreased antibody levels against the opportunistic pathogen candida albicans (dickerson et al., ; severance, gressitt, et al., ; tomasik, yolken, bahn, & dickerson, ) . in these analyses, probiotics did not deliver a benefit for improvement of residual psychotic symptoms. interestingly an impact on psychiatric symptoms by probiotics was suggested only in those who did not have evidence of a c. albicans infection. fecal transplantation also involves the re-priming of a dysfunctional gut microbiome with live bacteria from a healthy donor, usually performed for the purposes of treating clostridium difficile infections. it is also experimentally used in autoimmune diseases and has shown promise in inflammatory bowel diseases (millan, laffin, & madsen, ; weingarden & vaughn, ) . case reports also describe its benefit for treating autism, parkinson's disease, multiple sclerosis and chronic fatigue syndrome (evrensel & ceylan, ) . dietary modification to avoid a high animal protein and high carbohydrate content is essential for improving the microbiome composition toward a healthy balance statovci, aguilera, macsharry, & melgar, ) . the dietary avoidance of gluten is the standard treatment of celiac disease and gluten intolerance. gluten-free diets in people with schizophrenia in fact were found to be associated with a decreased prevalence of thyroid autoantibodies (toscano et al., ; ventura et al., ) . pharmacological agents in development to treat celiac disease include gluten degrading enzymes, (alv and an-pep), which are in clinical trials and show promise in reducing symptoms and gluten t-cell responses (mitea et al., ; siegel et al., ; tye-din et al., ) . zonulin inhibitors such as larazotide are also in development to block gluten transit across the intestinal barrier and have demonstrated improved symptoms, fewer autoantibodies and reduced proinflammatory state patients with celiac disease (parzanese et al., ) . in mice, an endogenous serine protease inhibitor, elafin, has been shown to normalize inflammation and restore intestinal barrier function (galipeau et al., ) , while the compound, sevelamer, available to treat symptoms of chronic kidney disease, binds bacterial proteins and prevents their translocation from the gut into circulation (kristoff et al., ) . some of these compounds are in the beginning stages of preclinical development and thus will require extensive safety, efficacy and other regulatory testing before clinical trials are considered. therapeutic strategies to diminish the effects of autoantibodies involve plasmapheresis to remove the antibodies from systemic circulation or immunosuppression to offset the actual production of antibodies. plasmapheresis in schizophrenia with the objective to eliminate autoantibodies has been tested with limited success in small studies with very select patients and in case reports (kramina et al., ; schulz et al., ) . directed pharmacotherapy to rejuvenate the receptor can be successful if the autoantibody target, such as the identity of a specific neurotransmitter receptor, is known. autoantibodies directed against the glutamate system in schizophrenia are best exemplified by the multitude of studies that examine nmda receptors, as described earlier. hypofunction of this receptor is a hypothesized mechanism underlying schizophrenia symptoms with the presence of autoantibodies that will bind and internalize the receptor lending support to this rationale. this target is attractive because of the numerous agents currently available that are known to modulate this receptor. some promising research has been implicated by agents that enhance nmda receptor function via stimulation of the glycine modulatory site (d-serine, d-cycloserine and bitopertin), although with some mixed results in terms of the treatment of schizophrenia (alberati et al., ; bugarski-kirola et al., ; bugarski-kirola et al., ; heresco-levy et al., ; kantrowitz et al., ; umbricht et al., ) . the use of immunotherapy including steroids, intravenous immunoglobulin, and plasma exchange demonstrated promising improvements in symptoms and reductions in autoantibodies in a case series of individuals with nmda receptor autoantibody-related psychosis, a finding that will be further investigated in randomized placebocontrolled trials . pharmacological strategies to increase nmda receptor function in schizophrenia are well-reviewed elsewhere (balu, ) . here we presented evidence in support of an autoimmune component of schizophrenia in the context of mechanistic or impacted pathways in order to better understand how to formulate novel treatment strategies. currently, we cannot advocate or suggest any compound to replace antipsychotics. new clinical trials that examine the safety and efficacy of adjunctive therapies in carefully identified individuals who demonstrate specific comorbidities or evidence of inflammation are needed. as we have discussed in the context of the autoimmune phenotype of schizophrenia, a number of moieties potentially treatable by immunemodulatory agents emerge including pathogens, inflammation, gut dysbioses, and effects from autoantibodies. it is likely that a combination of approaches may offer the most effective evaluation of adjunctive therapies. for example, in patients presenting with gi symptoms, the genetic screening for hla subtypes coupled with antibody studies might identify a subset of patients who would benefit from an approach to treat celiac disease and gluten intolerance. microbiome screening of fecal samples coupled with blood tests using biomarkers of microbial dysbioses could identify those who might benefit from probiotics and other gut-related treatments. genetic screening for complement deficits could identify people with c gene susceptibilities and who might be candidates for complement activation or inhibition-based treatment. for a long time, treating schizophrenia has been focused on dopamine since this pathway is most likely central to the physiological and therapeutic puzzle of schizophrenia. very recently, it has been discovered that dopamine participates in signaling between immune cells, in a similar manner to synapse communications in the brain. papa et al. 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authors: goggin, rachel k.; bennett, catherine a.; bassiouni, ahmed; bialasiewicz, seweryn; vreugde, sarah; wormald, peter-john; psaltis, alkis j. title: comparative viral sampling in the sinonasal passages; different viruses at different sites date: - - journal: front cell infect microbiol doi: . /fcimb. . sha: doc_id: cord_uid: awng h background: with the emergence of the microbiome as an important factor in health and disease in the respiratory tract standardised, validated techniques are required for its accurate characterisation. no standardised technique has been reported specifically for viral sampling in the sinonasal passages. aim: to optimise viral sampling techniques from the sinonasal cavity. methods: sterile cytology brushes were used under endoscopic guidance to sample the sinonasal mucosa at time of endoscopic sinus surgery at both the middle and inferior meatuses (mm and im). dna and rna were extracted from the samples and underwent pcr or rt-pcr testing, respectively, for a panel of common upper respiratory tract viruses. results: twenty-four adult patients were recruited for this study. / ( %) patients were positive for virus in at least one site, while / ( %) were positive for virus at both sites. the mean number of viruses identified at the two sites were similar ( . ± . at the mm vs. . ± . at the im). / ( %) of patients showed no virus at either site, while / ( . %) demonstrated the same viral species at both sites. conclusion: although the number of viruses present at different sites with the nasal cavity are similar, discord exists in the viral species between sites. it is therefore recommended that both sites are sampled in the clinical and research setting better to characterise the viral species within the nasal cavity. the role of the healthy human microbiome in prevention and eradication of disease is an area of burgeoning interest in recent years. the interplay between various colonising organisms, their relative abundance, and the importance of a fine microbial balance has been shown to be essential for normal functioning of multiple organ systems, not least respiratory (lloyd-price et al., ; mitchell and glanville, ) . conversely, disruption of this balance between viruses, bacteria, and single-celled eukaryotes has been implicated in numerous disease processes, including acute infective processes as well as many chronic inflammatory diseases (lloyd-price et al., ) . microbial dysbiosis (specifically bacterial) has been implicated in several respiratory diseases, including asthma (fazlollahi et al., ) and chronic rhinosinusitis (crs) (cleland et al., ) . persistent nasal and paranasal sinus inflammation characteristic of crs affects up to % of the western population (fokkens et al., ) and manifests as nasal congestion, facial pain or pressure, anterior or post-nasal drainage, and reduction or loss of smell (benninger et al., ) . although the exact aetiopathogenesis of this condition remains elusive, it is considered multifactorial in origin. current theories include the fungal hypothesis, the bacterial hypothesis (implicating dysbiosis with staphylococcus aureus overgrowth, superantigen production, and biofilm formation), and an overactive immune response (resulting in chronic inflammation and defective mechanical and innate immune barriers to infection in the crs population) (lam et al., ) . an area that is anecdotally suggested to play a role in crs pathogenesis is a viral dysbiosis (jang et al., ; cho et al., ). this is due to self-reports by many crs patients that their symptoms almost invariably developed after an initial viral upper respiratory tract infection (urti) . research into the ideal method to sample the sinonasal bacterial microbiome is ongoing (copeland et al., ) , however similar efforts to investigate and standardise sampling of the virome have not been made. studies attempting to investigate the upper respiratory virome are limited. the lack of standardisation in sampling has led to conflicting results regarding the presence of virus and the composition of the virome. collection techniques employed thus far include nasal washes, aspirates, brushings, and traditional viral swabs, with viral analysis performed by pcr (cheung et al., ; tao et al., tao et al., , ramadan et al., ; jang et al., ; zaravinos et al., ; wood et al., ; cho et al., ; costa et al., ; liao et al., ; abshirini et al., ; lima et al., ; rowan et al., ) . few studies have compared sampling methods; heikkinen et al. found no difference in the detection of childhood influenza comparing nasal swabs and aspirates (heikkinen et al., ) . spyridaki et al. found a higher detection of rhinovirus (rv) in nasal lavages compared with nasal brushings, but found no difference in any other viruses tested when comparing these to nasal aspirates and swabs (spyridaki et al., ) . to date there have been no studies that have compared different sites within the sinuses and nasopharynx in terms of viral detection. the aim of the study here presented was to establish differences in viral detection and species sampled from different sinonasal sites, in an effort to validate and standardise viral collection techniques, and facilitate further investigation of the sinonasal virome. patients for this study were recruited from the tertiary rhinologic practices of the two senior authors (pjw and ajp). this study was carried out in accordance with the recommendations of the central adelaide local health network ethics committee (hrec/ /tqeh/ ). the protocol was approved by the same. all subjects gave written informed consent in accordance with the declaration of helsinki. patients were included in this study if they were older than years of age and were undergoing endoscopic surgery. control patients consisted of patients with an absence of clinical or radiologic evidence of crs. crs patients fulfilled the diagnostic criteria for crs as outlined in the american guidelines (rosenfeld et al., ) . the radiological severity of disease was scored for all patients using a lund-mackay score (lms) (lund and mackay, ) . using an aseptic technique, endoscan cytology brushes (mcfarlane medical, melbourne, australia) were used to sample the sinonasal mucosa (figure ) of the left and right middle meatuses (mm) and inferior meatuses (im) of each patient. this was done under endoscopic visualisation with caution to avoid cross-contamination from neighbouring tissue. the samples were then placed in a viral transport medium [ % roswell park memorial institute medium supplemented with % foetal bovine serum, % amphotericin b, and % penicillin streptomycin (all gibco by thermofisher, waltham, usa)] and immediately transported on ice to the laboratory for processing. sample material was removed from the brushes and centrifuged at • c and , rpm for min in order to isolate cellular material. the supernatant was discarded, after which samples were stabilised with µl rpe buffer (qiagen, hilden, germany) and . µl beta-mercaptoethanol (gibco by thermofisher, waltham, usa) and stored at − • c. samples were thawed in batches to undergo rna and dna extraction using an allprep dna/rna mini kit (qiagen, hilden, germany). this yielded dna samples of µl (average concentration . ng/µl, range . - . ng/µl) and rna samples of µl (average concentration . ng/µl, range - . ng/µl). extracted dna and rna were stored at − • c until batch testing for a range of upper respiratory tract viruses using realtime pcr. the panel included rv, influenza a-c, parainfluenza (piv) - , respiratory syncytial virus (rsv) a and b, coronavirus (cov) hku- , oc , nl , and e, enterovirus (env), metapneumovirus (hmpv), adenovirus (adv), bocavirus (bov), polyomaviruses wupyv and kipyv, epstein-barr virus (ebv), cytomegalovirus (cmv), herpes virus (hhv ), herpes simplex virus (hsv) and , and varicella zoster virus (vzv). all dna extracts first underwent an endogenous retrovirus (erv ) assay (present as two copies per human diploid cell) in order to confirm respiratory sample collection quality. briefly, dna extracts were screened for erv , adv, bov, wupyv, kipyv, cmv, ebv, vzv, hsv and , and hhv using an identical set of conditions previously optimised so as not to compromise sensitivity ( table ) . said conditions were pmol of each primer, . pmol of the respective probe(s), and µl of template, made up to a final reaction volume of µl using the bioline sensi mix ii probe pcr mix kit (bioline australia). details of the target genes, primer, and probe sequences are summarised in tables , . samples then underwent the following cycling conditions: • c for min, followed by cycles of • c for s and • c for s. the rna extracts were tested for rv, influenza a-c, piv - , rsv a and b, covs hku- , nl , oc , and e, env, and hmpv (table ) using identical quantities of primer, probe, and template to the dna reactions but with the bioline sensifast probe one-step rt-pcr kit (bioline, sydney, australia). there were two exceptions to these quantities; the iv a/b duplex where asymmetric probe amounts were used ( . and . pmol, respectively) and the rv assay where pmol of probe was used. samples then underwent the following "+" indicates a locked nucleic acid (e.g., +a is a locked nucleic adenine analogue). frontiers in cellular and infection microbiology | www.frontiersin.org cycling conditions: • c for min, and cycles of • c for s and • c for s. all samples were run with both positive and negative controls; the positive controls were either previously established clinically positive samples, or synthetic controls specific for each assay. all cycling was conducted on viia instruments (thermofisher, scoresby, australia). viral detection was defined as a cycle threshold (ct) of forty or less. statistics were performed using software from scientific python, namely scipy and pandas through the jupyter notebook interface (oliphant, twenty-four patients were recruited at time of endoscopic surgery; this included men and women, with an age range of - years, and a mean age of years. seven patients had crs without polyps (crssnp), eight had crs with polyps (crswnp), and nine were controls. demographics and patient characteristics are summarised in table . all patients in the crs groups underwent functional endoscopic sinus surgery (fess), while those in the control group underwent trans-sphenoidal resections of pituitary masses. erv was detected in all patient samples, with a median ct of . (range . - . ), showing adequate cellular material was captured throughout the collection and dna extraction phases. eighteen patients were positive for at least one virus in at least one site ( / , %), while six ( / , %) were negative for any of the viruses for which the samples were screened ( a standardised, validated technique for viral sampling in the sinonasal passages has not yet been described. this study shows a significant discrepancy in viral presence and species between just two of the sites commonly sampled, highlighting the need for such a standardisation. this indicates that viral sampling needs to be conducted with a cytobrush in both the im and mm. collection variability has the potential to impact respiratory viral detection significantly. the sample volume and location, as well as the documented uneven distribution of viruses within the nasal cavity, can all contribute to false negatives (van wesenbeeck et al., ) . given that clinically relevant, actively replicating viruses of the upper respiratory tract are intracellular and largely reside in the upper epithelial layers of the mucosa (vareille et al., ) , adequate cell sampling is an important consideration when searching for viruses. traditional viral sampling brushes have the advantages of causing less trauma to the delicate mucosa and thus less discomfort to a conscious patient, but risk sampling largely secreted elements rather than the cells themselves (spyridaki et al., ) . viruses do certainly reside in these secretions, but this may not necessarily represent actively replicating virus causing disease. for these reasons we elected to use cytology brushes for this study. cytology brushes are designed specifically for cell sampling due to their larger and more rigid design than traditional viral sampling brushes. although this may potentially increase the risk of trauma or discomfort to the awake patient, when used in the anaesthetised patient, as was the case in this study, they have the significant advantage of increased cellular sampling yield (stokes et al., ) . as mentioned viral yields are also difficult to compare in respiratory samples, as sample volume can vary widely. the samples here averaged a dna concentration of . ng/µl and an rna concentration of . ng/µl, but with ranges of . - . and - . ng/µl, respectively. to minimise the impact of this variability on results all samples underwent an erv assay prior to pcr. this has been identified previously as a positive indicator of respiratory sample quality, and all samples were wellwithin previously published target ranges (alsaleh et al., ; sarna et al., ) . viral sampling is traditionally performed from the inferior nasal septum and anterior nasal floor as they are easily accessible and cause minimal patient discomfort. the posterior nasopharyngeal wall is also traditionally endorsed, but confirmation of access to this site is difficult without endoscopic equipment. there is no evidence however that these three sites are any more or less appropriate. these areas may indeed be less than ideal due to their relative proximity to airborne pathogens (and therefore risk of contamination), their distance from areas of particular interest (such as the paranasal sinuses), and the tendency for pooling of potentially contaminating secretions in these areas. the mm (sampled in our study) remains relatively simple to access but is further away from potential sources of contamination, and receives drainage from a much wider area including the maxillary, frontal, and anterior ethmoid sinuses. there are indeed a number of other sites in the nasopharynx not investigated here, for example the superior meatus, the sphenoethmoidal recess, and the post-nasal space, however these are difficult to reach without endoscopic equipment that is not readily available in the primary care setting, and can be subject to contamination from other more anterior sites during insertion and removal of sampling instruments. should these areas demonstrate greater viral presence than the mm and im the specialist input required to access the sinuses themselves would likely delay or miss altogether the diagnosis and window for anti-viral treatment. large-scale economic viability of the collection method here proposed also warrants mention; pooling of viral samples from the same patient prior to analysis and limitation of viral testing to a smaller panel of more prevalent, clinically relevant pathogens would be prudent, however selection of such a panel requires further investigation. common, clinically relevant upper respiratory viruses are largely of the rna subtype, and include rv, influenza, rsv, and hmpv, and to a lesser extent cov, piv, and env. of the dna viruses here investigated adv is certainly a notable urt pathogen. bov has been linked largely with lower respiratory illness (gottlieb, ) . the other dna viruses here investigated were chosen not primarily for their clinical relevance in viral respiratory disease, but instead for either their near-ubiquity, their ability to remain latent in the respiratory tract, or both. ebv and hhv have here shown themselves to be particularly useful in testing viral sampling methods as they are almost omnipresent in the adult sinonasal passages, and are rarely entirely cleared after first infection. effort was made in this study to identify any correlation between control/crssnp/crswnp status and viral presence. patient reports of recent viral infection, sinonasal outcome test (snot- ) scores, lund-mackay computed tomography scores, lund-kennedy endoscopic scores, and rt-pcr cycle threshold values were collected for all patient and samples, but the sample size here was too small to demonstrate any significant differences. the inclusion of the extremely common herpesviruses (seen, as expected, in many of our controls) also skewed any such results. this is an area that requires significant further investigation. neither of the sites from whence samples were taken were more or less likely to be positive than the other. our observation that the mm and im only completely agree in terms of viral presence or absence as well as viral species in % of cases indicates a significant proportion of viruses present would not be identified were only one site to be sampled. our findings suggest viral sampling from the sinonasal passages should be taken from both sites in both nasal cavities. the sampling method here described has significant implications for further research into a field of emerging importance in both rhinologic and also respiratory disease on a larger scale. the raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher. rg contributed to study design, sample collection and processing, data analysis, and writing of the manuscript. cb contributed to sample collection. ab contributed to statistical analysis. sb contributed to sample processing 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prevalence of human papilloma virus and human herpes virus types - in human nasal polyposis the authors thank prof. eric gowans for his assistance in establishing the methodology and editing this work. the authors also acknowledge the contributions of dr. mian ooi and mr. aden lau in the collection of samples. key: cord- - p scli authors: majzoub, karim; wrensch, florian; baumert, thomas f. title: the innate antiviral response in animals: an evolutionary perspective from flagellates to humans date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: p scli animal cells have evolved dedicated molecular systems for sensing and delivering a coordinated response to viral threats. our understanding of these pathways is almost entirely defined by studies in humans or model organisms like mice, fruit flies and worms. however, new genomic and functional data from organisms such as sponges, anemones and mollusks are helping redefine our understanding of these immune systems and their evolution. in this review, we will discuss our current knowledge of the innate immune pathways involved in sensing, signaling and inducing genes to counter viral infections in vertebrate animals. we will then focus on some central conserved players of this response including toll-like receptors (tlrs), rig-i-like receptors (rlrs) and cgas-sting, attempting to put their evolution into perspective. to conclude, we will reflect on the arms race that exists between viruses and their animal hosts, illustrated by the dynamic evolution and diversification of innate immune pathways. these concepts are not only important to understand virus-host interactions in general but may also be relevant for the development of novel curative approaches against human disease. the animal kingdom, including humans, has evolved while facing constant threats from viral elements. viruses can be, in some cases, beneficial for a given animal species and drive its evolution [ ] . however, their uncontrolled replication may cause disease and prove fatal to their hosts. consequently, animal cells have evolved devoted pathways which ( ) sense and recognize pathogen-associated molecular patterns (pamps) and, more particularly, virus-associated molecular signatures; ( ) initiate signaling cascades stemming from the site of detection, translocating the information to the nucleus; and ( ) induce a transcriptional program that confers an antiviral state to the host ( figure ). interestingly, a closer examination of individual factors constituting these pathways shows a different conservation status between different animal species. while genes encoding sensors and signaling platforms are generally well conserved amongst animals, virus-stimulated genes (vsgs) are clearly less so and are subject to faster evolution [ , ] . in vertebrates, one such vsg is the secreted interferon (ifn) cytokine, that signals in an autocrine and paracrine fashion. secreted ifn molecules bind to cell-surface receptors and initiate signal transduction involving the janus kinase/signal transducer and activator of transcription (jak-stat) pathway. this pathway induces the transcription of a major antiviral program composed of hundreds of so-called ifn-stimulated genes (isgs) that comprise effectors of the cell-autonomous antiviral defense [ ] . a lot of our understanding of the innate antiviral immune system in animals is a result of studies conducted in vertebrates and more particularly in mammalian species. therefore, the ifn system has been heavily studied over the last years. however, during the last two decades, we came to appreciate that the ifn system, as we know it, is a vertebrate particularity. indeed, while some animal species like insects or nematodes are devoid of ifns and rely on rna interference (rnai) as the major antiviral pathway, some others, like mollusks, have conserved all the components that lead to ifn production but have no obvious homologs of type i ifn cytokines. nevertheless, these are predicted to use an ifn-like antiviral cytokine [ ] . in fact, the ifn cytokine itself seems to be an evolutionary novelty, however, the pathways dictating its production existed early in metazoan evolution [ , ] (figure ). interestingly, ifn is not the only vsg induced upon viral detection in mammals. certain isgs that directly interfere with the viral life cycle like viperin are also immediately induced after viral infection in an ifn-independent fashion [ ] [ ] [ ] . in this review, we will focus on the conserved pathways that are responsible for sensing viral pamps, signaling and inducing antiviral genes upon infection in animals. we will start by describing our current view on the immediate activation of the ifn and nf-kb pathways in vertebrate species. we will then zoom in on two important viral nucleic acid receptor families, toll-like receptors (tlrs) and rig-i-like receptors (rlrs), describe their function in viral rna detection and their conservation across animal species. next, we will focus on a central hub in the signaling pathways induced by dna viruses, the stimulator of ifn genes (sting). we will then examine how the evolutionary conflicts between viruses and host immune factors are shaping antiviral immunity in animals. viruses and transposable elements are very powerful drivers of evolution [ , ] . however, their uncontrolled replication and spread can be catastrophic to host cells. it is therefore not surprising that every known living species on the planet has evolved measures to recognize and counteract parasitic genetic elements. it is suggested that anti-sense mediated targeting of viral nucleic acids was the most primordial strategy protocells used to fend off viral threats [ ] . this is illustrated by argonaute and crispr-based defenses in bacteria, archaea and rnai systems in plants, all of which rely on anti-sense nucleic acids that program a nuclease to target and degrade the complementary invading viral genome [ ] [ ] [ ] . because many of the core rnai machinery components can be found in all eukaryotic superkingdoms, it is thought that this antiviral defense mechanism predated the emergence of pattern-recognition receptor (prr)-based immunity ( figure ) [ ] . interestingly, antiviral defense in eukaryotes has diversified greatly during evolution, with some species maintaining rnai-based defenses [ ] and others innovating and adopting completely novel antiviral strategies. in chordates and more particularly in vertebrate animals, the emergence of ifn-i and a recombinational adaptive immune system seems to coincide with the loss of rnai as the main antiviral mechanism in somatic cells. there is strong evidence of an intrinsic incompatibility between an antiviral rnai and the prr-ifn system. for instance, while long dsrnas can produce functional small interfering rnas (sirnas) in stem cells in the absence of ifn, the same dsrna molecule is not processed onto sirna and is sensed as a pamp in vertebrate somatic cells [ , ] . interestingly, experimental evidence of antiviral rnai in mammals seems to be limited to specialized pluripotent cells in which the prr-ifn system is not fully deployed yet. the emergence of both the ifn-i (innate) and somatic dna recombination systems (adaptive) in vertebrates constituted a major evolutionary event that dispensed them from using rnai for antiviral purposes. interestingly, retrotransposition and selfish transposable elements were determinants in the acquisition of these two systems [ , [ ] [ ] [ ] . the emergence of somatic dna recombination in vertebrate animals was considered an "immunological big bang" [ , ] . indeed, somatic dna recombination in specialized b and t cell lineages provided jawed vertebrates with large repertoires of major histocompatibility complexes (mhc), t-cell receptors (tcrs) and immunoglobulins (igs). until relatively recently, adaptive immunity was believed to be exclusive to gnathostomes (jawed vertebrates). we now know that agnathans (jawless vertebrates), including lampreys and hagfish, have also evolved an equivalent adaptive immune system with specialized lymphocytes termed, vlra, vlrb and vlrc cells, in which specific variable lymphocyte receptors (vlrs) are produced through somatic leucine-rich repeat (lrr) rearrangements [ ] . in both gnathostomes and agnathans, somatic recombination events in specialized cells permitted a pathogen-tailored response and endowed vertebrate species with an immune memory. until the end of the last century, most vertebrate immunologists concentrated their efforts on studying the adaptive arm of the immune system. nearly years ago, charles janeway predicted the presence of an evolutionary ancient immune system that detects conserved microbial and danger signals, termed the pathogen-associated molecular patterns (pamps) and danger-associated molecular patterns (damps), respectively. janeway predicted that this innate immune system precedes and instructs the adaptive system [ , ] and posited that pamps and damps must be sensed by germ-line encoded prrs. at that time, the innate immune components were severely understudied and tlrs, rlrs and sting's respective functions in immunity were completely unknown. this illustrates the immense leap forward the innate immunity field has experienced in the last three decades. today, we know that vertebrates share with other invertebrate animals specialized phagocytic cells that are able to discriminate between self and non-self. by recognizing general pathogen molecular patterns (pamps, e.g., viral double-stranded rna) and danger signals (damps), these cells establish an immediate and general inflammatory response translated into an antimicrobial and/or antiviral state. although mainly studied in vertebrates, the machineries responsible for these responses transcend this group and are fairly conserved in all animals. we will describe a particular arm of the innate immune pathways, the innate antiviral system that is best studied in mammals. unlike bacteria, viruses represent a unique challenge for prrs because they possess few unique signatures that could serve as pamps [ ] . however, viral nucleic acids (dna or rna) could have peculiar biochemical features that differentiate them from endogenous host rna [ ] . in rna molecules, for example, the lack of a -methylguanosine cap structure, double strandedness or the trior bi-phosphorylation at their ends are often used by prrs for self/non-self-discrimination. prrs that detect viral infection can be classified into four families: tlrs, rlrs, aim -like receptors (alrs) and the cgas-sting sensors [ ] . after viral detection, prr-mediated signaling directly or indirectly induces transcription factors, including ifn-regulatory factors (irfs) and nuclear factor k-b (nf-kb) to upregulate expression of vsgs including pro-inflammatory cytokines. another class of prrs such as double-stranded rna (dsrna) activated protein kinase r (pkr; also known as eif ak ), adenosine deaminase acting on rna (adar ) and - -oligoadenylate synthetase (oas ) also contribute to innate immunity [ ] . these also recognize viral signatures; however, their main function is not necessarily to induce a transcriptional immune response, but rather to directly attack viral rna by degrading it or inhibiting its translation. for this reason, these are not usually considered receptors. viruses are obligatory intracellular parasites, therefore their detection by prrs most often occurs in the intracellular milieu. endosomal transmembrane tlrs, including tlr , tlr and tlr recognize dsrna in the endosome lumen [ ] [ ] [ ] . rlrs including rig-i [ ] , melanoma differentiation associated gene (mda ) [ ] , and laboratory of genetics and physiology (lgp ) [ , ] detect viral rnas in the cytosol, whereas cytosolic viral dna is mainly recognized by cyclic-gmp-amp (cgamp) synthase (cgas) [ ] . therefore, the nature of the viral particle (e.g., enveloped vs. non-enveloped) and the viral genome (e.g., dna vs. rna) dictates which of these receptors recognizes the infection first ( figure single-stranded rna (ssrna) is a potent tlr and tlr ligand, while tlr is specific for dsrna. tlr , for example, recognizes dsrna viruses from reoviruses [ ] , but can probably also recognize dsrna intermediates from (+) strand rna viruses like coxsackievirus and west nile virus (wnv) and (-) strand rna viruses like the human respiratory syncytial virus (hrsv) [ ] . indeed, all rna viruses are thought to produce dsrna intermediates as part of their replication cycle, so both ssrna and dsrna viruses have the potential to be sensed by tlr . tlr and , on the other hand, have been shown to prefer ssrna ligands from (-) strand rna viruses such as vesicular stomatitis virus (vsv) and influenza a virus (iav) [ , ] . when it comes to rlrs, most rna viruses have been shown to be detected by rig-i or mda , as these receptors have a high affinity to dsrnas. while rig-i prefers viral rnas bearing di-or tri-phosphate groups at their while the cytosolic recognition of viral rna is almost exclusively mediated by rlrs, several proteins have been proposed to play a role in dna sensing and triggering innate immune responses, such as the dna-dependent activator of ifn-regulatory factors (dai), ddx , rna polymerase iii, ifi and dna-pk [ ] [ ] [ ] [ ] [ ] [ ] . however, among all the proposed sensors, only cgas knock-outs can completely shut down ifn production in response to cytosolic dna [ ] . the cgas protein is now thought to be the major viral dna sensor and has been shown to detect adenovirus, human papillomavirus (hpv), herpes simplex virus- (hsv- ) and cytomegalovirus (cmv) [ , [ ] [ ] [ ] . aim has also been shown to activate the inflammasome upon dna stimulation [ ] but will not be discussed in this review. rna ligands cause the endosomal transmembrane tlr , and to dimerize and then to oligomerize through their cytoplasmic tir (toll/il- receptor) domains. this allows tlrs to recruit signaling adaptors via tir-tir interactions [ ] . tlr recruits the adaptor protein trif (tir-domain-containing adapter-inducing ifn-β) [ , ] . trif is able to play a dual role by inducing the ifn or the nf-kb pathways. when it comes to ifn, after activation, trif recruits the ubiquitin ligase traf (tumor necrosis factor receptor-associated factor ) through an ubiquitination mechanism which in turn recruits tank-binding kinase (tbk ) [ , ] . the trif/tbk complex is then able to phosphorylate the transcription factor irf , triggering its dimerization and nuclear translocation. phosphorylated irf dimers specifically bind to ifn-stimulated response elements (isres) present in the ifn-β gene promoter which leads to the transcription of this cytokine [ ] . trif can also recruit ripk (receptor-interacting serine/threonine-protein kinase ) that leads to the activation of the ikk complex, releasing the nf-kb transcription factor from its ikb inhibitory subunit and resulting in its translocation to the nucleus to induce the transcription of pro-inflammatory cytokines [ ] (figure ). unlike tlr , the activation of tlr and tlr recruits the adaptor protein myd (myeloid differentiation primary response ) through tir-tir domain interaction. myd death domains oligomerize which triggers the formation of the myddosome signaling complex consisting of myd and the irak family of kinases (il- receptor-associated kinases), irak , and . through a series of phosphorylations and the help of the e ubiquitin ligase traf , the myddosome is able to recruit and activate the transcription factors irf , irf and nf-kb that translocate to the nucleus to induce the transcription of ifn-α genes and other proinflammatory cytokines [ ] [ ] [ ] [ ] [ ] [ ] [ ] (figure ). rlrs (rig-i, mda and lgp ) are characterized by a central dead-box helicase/atpase domain and a c-terminal regulatory domain (ctd) essential for rna recognition and autorepression in the absence of rna ligands. with the exception of lgp , rlrs also possess two n-terminal caspase activation and recruitment domains (cards). upon rna binding rig-i is remodeled into an active conformation in which the ctd and helicase domains organize into a ring around the rna ligand and the card domains are exposed [ , ] which facilitate their interactions with other card domains resulting in rig-i tetramers. although rig-i and mda share similar domain architectures, mda seems to prefer longer dsrna, assembling along these molecules to form helical, filamentous oligomers [ , ] . a poly-ubiquitination reaction by ubiquitin ligases like riplet and trim (tripartite motif-containing ), is thought to enhance rig-i and mda oligomerization and activation [ ] [ ] [ ] [ ] . rig-i and mda oligomers then serve as a scaffold for binding to the adaptor protein mavs (mitochondrial antiviral signaling protein, also known as ips- , visa, and cardif) [ ] [ ] [ ] . mavs has been shown to be critical for mounting an efficient immune response to infection by several rna viruses [ ] . its c-terminal transmembrane domain is inserted into the outer mitochondrial membrane [ ] , whereas its n-terminal card domain mediates its aggregation on the mitochondrial surface by interacting with the tandem cards of rig-i or mda oligomers [ , ] . mavs aggregates then recruit several e ubiquitin ligases including traf , traf and traf . although traf-mediated ubiquitination is essential to activate mavs downstream signaling, the ubiquitination targets of traf remain unknown [ ] . subsequently, the ubiquitin sensor nemo (nf-κb essential modulator, also known as ikkγ) [ , ] is then recruited to the mavs/trafs complex, which in turn recruits ikk and tbk to the mavs complex leading to activation of nf-kb and irf and their translocation to the nucleus to induce the transcription of antiviral genes [ ] [ ] [ ] [ ] (figure ). after trif and mavs were discovered, sting was identified as a third adaptor protein that is also able to activate irf and ifn production [ , ] . sting is an endoplasmic reticulum (er) resident membrane protein with cytoplasmic c-and n-termini. sting has been shown to be essential for dna-mediated ifn production in different tissues, for example, it is crucial for host defense against the dna virus hsv- [ ] . sting has also been shown to sense cyclic dinucleotides (cdns), which are the second messengers known to be produced by bacteria such as listeria monocytogenes [ ] [ ] [ ] [ ] . although it can bind bacterial cdns, sting is unable to bind dna and relies on an upstream sensor, cgas [ ] . cgas is an enzyme that contains a nucleotidyltransferase (ntase) domain and can synthesize the second messenger -cyclic gmp-amp (cgamp) from atp and gtp upon dna recognition ( figure ). loss of cgas in various cell lines and also in vivo results in a complete loss of type i ifn induction upon dna delivery or viral infections [ , ] . cgas preferentially binds longer dna (> bp) as a dimer to form stable protein-dna ladder networks responsible for strong cgamp production [ , ] . a unique cgamp isomer termed -cgamp with particular phosphodiester linkages is produced by cgas [ , ] . -cgamp is a potent sting ligand and has a higher affinity to this protein than other cgamp molecules containing different phosphodiester linkages such as -cgamp, -cgamp or bacterial cdns [ , ] . apart from activating sting in the cell where cgas initially detects viral dna, cgamp second messengers can also travel to neighboring cells, through gap-junctions [ ] or after being packaged in newly formed virions [ , ] . this intercellular transfer of free or packaged cgamp permits uninfected cells to mount a preventive ifn response, protecting them from infection or providing a faster response to dna viruses that encode cgas antagonists. upon cgamp binding, sting undergoes a conformational change that results in the release of its c-terminal tail (ctt) from its autoinhibitory state and in the formation of sting homodimers that translocate to perinuclear regions to colocalize with tbk [ , , ] . tbk recruitment results in the phosphorylation of sting and the phosphorylated site serves as a platform for irf dimerization and activation which ultimately results in ifn-β induction [ ] (figure ). sting has also been shown to induce nf-kb, map kinase and stat activation, as well as the stimulation of lc puncta formation, a hallmark associated with autophagosome formation [ , [ ] [ ] [ ] . however, the molecular mechanisms by which sting induces these non-ifn responses remain poorly understood. tlrs comprise an ancient family of membrane-spanning receptors that recognize ligands through their extracellular domains and initiate an intracellular response upon stimulation (see above). the toll gene was first identified as a developmentally important gene in drosophila in [ ] . in the mid- s the discovery that this gene also plays an essential role in the ability of drosophila to resist fungal infections connected for the first time toll receptors to innate immunity [ , ] . although in flies toll functions as a cytokine receptor, a human toll receptor (tlr ) was rapidly identified [ , ] and shown to induce an immune response in mice after induction by lps [ ] . we now know that there are ten tlrs in humans that can respond to many bacterial and viral pamps [ ] . prototypical tlrs contain three structural elements, a hydrophobic ectodomain containing a variable number of lrrs, a transmembrane domain and a tir domain, which mediates downstream signaling through adaptor proteins [ ] . tlrs are likely very ancient immune sentinels since two of their characteristic building blocks (lrr and tir domains) are observed in placozoans (e.g., trichoplax animals) [ ] and porifera (e.g., sponges) [ ] . full tlrs were detected in cnidarian species, like the starlet sea anemone (nematostella vectensis; one single tlr) [ , ] and the acroporid corals (acropora digitifera; four tlrs) [ ] (figure ) . interestingly, both developmental and immunological roles of tlrs have been described in cnidarians. tlrs from both the sea anemone (nematostella vectensis) and the mountainous star coral (orbicella faveolata) have been shown to signal via myd leading to nf-kb activation [ , ] . in the bilateria phylum, tlrs can be found in most studied species, however, their numbers vary greatly among species, ranging from a single tlr in nematodes like caenorhabditis elegans, to over two hundred in echinoderms like the pacific purple sea urchin strongylocentrotus purpuratus (figure ). the expansion of the tlr repertoire in some animals like the sea urchin, reflects the adaptation of their immune arsenal to rapidly changing environmental stressors [ ] . amongst a multitude of other innate immune factors in this species, such as nacht domain-lrrs and scavenger receptors, sea urchin genomes encode for tlrs. among those, tlrs belong to a greatly expanded set of genes with vertebrate like features, many of which seem to have duplicated recently. the high prevalence of pseudogenes ( % to %) among those might reflect a history of strong positive selective pressures. another phylum where tlrs have undergone a significant expansion is in mollusca [ ] , like the pacific oyster crassotrea gigas [ ] (figure ). the pacific oyster encodes for tlrs in total, potentially reflecting a highly specialized response to environmental challenges and response to pathogens. the spread of pathogens in c. gigas natural habitats occurs very quickly, which is highlighted by the mass mortality events the ostreid herpesvirus (oshv ) has caused in many oyster nurseries. tlr sensing of oshv results in the differential regulation of more than a thousand genes, many of which are related to viral infection (e.g., cytosolic dna sensing and dna replication) [ , ] . in contrast to the very diverse set of tlr repertoires found in other bilateria species (e.g., nematodes, sea urchins and oysters), chordates and more particularly vertebrates contain roughly equal numbers of tlrs, reflecting the reduced need for highly diversified pattern recognition due to the acquisition of adaptive immune components (figure ). in general, vertebrate tlrs can be grouped into six major families [ ] . the families responsible for sensing of viral pamps are the tlr family, which recognizes dsrna, the tlr family (including tlrs , and ) which recognizes nucleic acid motifs and the large tlr family (tlr , , , , , , , and ) . the reduced number of tlrs in vertebrates does not necessarily mean that the tlr-response in those species cannot be tailored to a particular environment. a peculiar example is tlr , one of two virus sensing tlrs present in the pufferfish takifugu rubripes. tlr is widely conserved among teleosts and amphibians but does not seem to be present in avian or mammalian animals, which indicates that tlr might be required only in vertebrates living in water [ ] . in mammals, one last case of tlr adaptation and rapid evolution that is worth mentioning comes from bat species. analyses of tlr evolution in bats reveal adaptations acquired by tlrs , , and , with unique mutations fixed in ligand-binding sites [ , ] . these adaptations are thought to stem from the unique lifestyle of bat species, that are the only known flying mammals, and that represent important viral reservoirs [ ] . evolutionary studies paint a complex and dynamic picture of the emergence and functional diversification of rlrs across the animal kingdom. initially, several studies proposed that rig-i and mda /lgp evolved in animals independently through gene fusion and domain grafting events [ , ] . for instance, it has been proposed that the two card domains have been acquired by rig-i and mda in two separate events: the first domain being gained by the ancestor of rig-i and mda before their duplication and the second acquired after their divergence [ ] . these studies suggested that full-length rlrs are a vertebrate-specific evolutionary novelty, although their building blocks may have been present in closely related invertebrate animals [ , ] . a more recent study challenges this view and finds that the rlr-based immunity is not vertebrate-specific but originated in the earliest multicellular animals [ ] (figure ). in this study, the authors show that rlrs functionally diversified through a series of gene duplication events, followed by protein-coding changes that modulated their rna-binding properties. using homology-based gene prediction based on confirmed human rlrs the authors were able to identify full-length rlrs in early-branching animal genomes, including porifera (e.g., sponges) and cnidaria (e.g., jellyfish). however, they were unable to identify rlrs in non-metazoan eukaryotes, including fungi and choanoflagellates [ ] (figure ). it is therefore proposed that the ancestral rlr (rig-i/mda /lgp anc) duplicated in bilateria to give rise to rig-i and mda /lgp lineages, followed by a more recent duplication of the mda /lgp ancestor, giving rise to mda and lgp lineages in jawed vertebrates after their split from jawless vertebrates [ ] . the emergence of rlrs early in animal evolution is a very plausible scenario, since other components of the signaling pathways downstream of rlrs, like the irf genes, are also found in early metazoans [ ] ( figure ). another recent evidence suggesting that rlrs predated vertebrate evolution comes from studies performed in mollusks (pacific oyster; c. gigas). the invertebrate c. gigas not only encodes up to rlrs, but also mavs, traf , tbk and irf family proteins, which have been shown to have functional antiviral roles [ , , ] (figure ) . even though there is no consensus on the exact evolutionary history of rlrs, it is clear that these receptors (and/or their building blocks) existed very early in metazoan evolution and most importantly, they are subject to a very dynamic evolution. this is illustrated by the lineage-specific loss of rlr genes in many species. for example, although mda and lgp homologs were found in many teleost fish, rig-i homologs have only been identified in some fish species like salmon and carp [ ] . rig-i is absent in the chicken genome although mda and lgp are both present [ , ] . interestingly, chickens suffer severely from avian influenza virus (aiv) infection compared to ducks (that do possess the rig-i gene) which could be due to the loss of rig-i affecting their first line of defense in epithelial cells [ ] . most studied mammals possess rig-i, however, it has been lost in at least one mammalian species; the chinese tree shrew [ ] . interestingly, with the loss of rig-i, both mda and lgp have undergone strong positive selection in chinese tree shrews, and positively selected sites in mda endowed the substitute function for the lost rig-i [ ] . another eloquent example illustrating the dynamic evolution of these receptors is the loss of all rlr genes (rig-i, mda and lgp ) in insects (figure ). in drosophila, for example, although the nf-kb and jak/stat pathways are present and contribute to antiviral defenses [ , ] , all components of the rlr-mavs-irf-axis have been lost. instead, drosophila like other insects and relies on the rnai mechanism as the major antiviral system protecting it from viral infections [ , , ] . interestingly the rnase iii dicer- , a central player in insect antiviral immunity, responsible for generating small interfering rnas (sirnas), also contains an n-terminal dexd/h-box helicase domain that is highly homologous to the helicase domains of vertebrate rlrs [ , ] . moreover, dicer- has been shown to be responsible for the transcriptional upregulation of an antiviral gene (vago) that could function as a cytokine by activating the jak/stat pathway and triggering systemic antiviral immunity in various mosquito tissues [ , ] . although the pathway leading to the transcriptional activation of vago is still poorly understood in insects, these studies established that dexd/h-box helicase containing proteins, like dicer and rlrs, may represent an evolutionarily conserved set of viral nucleic acid sensors that direct antiviral responses in animals [ ] . one last observation exemplifying the dynamic and rapid evolution of these receptors comes from mammalian species. indeed, rlrs seem to be experiencing very recent adaptive changes in some mammals. for example, rig-i seems to have accumulated adaptive changes altering its rna-binding properties throughout mammalian evolution [ ] . moreover, in humans, for example, a number of protein-coding polymorphisms have been identified in rig-i which may contribute to differences in viral susceptibility and risk of autoimmune diseases [ , , ] . sting presence in animal genomes is probably more ancient than that of rlrs, since sting homologs can be found in most animal phyla including unicellular choanoflagellates ( figure ) [ , ] . furthermore, the ability of sting to bind cdns seems to be an ancient property. in an elegant study, kranzusch and colleagues show that a sting homolog in the starlet sea anemone n. vectensis (nvsting) is not only structurally very similar to that of human sting but is also able to bind cgamp with very high affinity [ ] . however, sting's ctt domain, which is crucial for tbk recruitment and downstream ifn induction, appeared only in vertebrate species [ ] . consequently, nvsting lacking the ctt is unable to induce ifn-β production in response to cdns when transfected in mammalian cells [ ] . the lack of a ctt domain in invertebrates does not mean that sting could not have an immune function in these animals. a first indication comes from invertebrate species like the lophotrochozoa phylum that includes the pacific oyster c. gigas and the annelid worm capitella teleta. in these animals, an unusual sting architecture can be found, where a sting domain is fused to a tir domain, known to be involved in innate immune signaling [ , ] . the second indication that sting lacking a ctt could function in immunity comes from arthropods. recent studies in drosophila, that lack an ifn system, clearly show that sting is important for antimicrobial and antiviral nf-kb activation in this model [ , ] (figure ) . interestingly, the emergence of the ctt domain of sting in vertebrate species seems to coincide with the development of the ifn system. nevertheless, sting ctt domain function, which dictates downstream signaling, seems to be plastic amongst vertebrate species. in a recent study, authors show that sting ctt-dependent activation of irf and nf-kb varies between vertebrate species [ ] . while sting ctt from mammalian species is able to induce a strong ifn-β and a weaker nf-kb response, an extension of this domain in ray-finned fish species elicits a dramatic enhancement of nf-kb activation and weaker irf -ifn signaling [ ] . another indication of sting ctt structure-function plasticity comes from bat species. a highly conserved and functionally important serine residue (s ) in sting's ctt domain is lost in bats [ ] . the replacement of this critical residue in this mammalian species significantly dampens sting-dependent ifn activation. the authors of this study suggest that the lifestyle of bat species (e.g., flight induced cytosolic dna, high viral titers) imposes a strong selective pressure on sting. this results in functionally dampened sensing and signaling mechanisms to avoid ifn overactivation and to cope with high cytosolic dna content. taken together, present studies suggest an evolutionarily ancient role of sting in antiviral immunity and modulation of its structure and function to accommodate species-specific pathogen burdens. the picture is less clear for the cgas enzyme when it comes to antiviral immunity. although cgas homologs have been identified in a variety of ancient metazoan lineages [ , ] , it is believed that the ability of cgas to bind and detect dsdna emerged in vertebrates. indeed, cgas' zinc-ribbon domain, required for dna binding and cgamp synthesis in response to dna in the cytosol, seems to be a vertebrate innovation [ ] [ ] [ ] . interestingly, primate cgas seems to have undergone rapid evolution in this lineage, as observed by the positive selection at its nucleic acid binding interfaces [ ] . these studies argue that although the cgas enzyme existed early in metazoans, its function has been repurposed for dna sensing only recently in vertebrates. clearly, cgas and sting seem to have acquired novel features throughout evolution. specifically in vertebrates cgas evolved the zinc ribbon motif to detect dna and sting evolved the ctt domain that expanded its signaling potential. as obligate intracellular parasites, viruses have evolved an array of evasion mechanisms to escape their elimination by the host's immune system. interestingly, viral antagonism is a general strategy and is not a peculiarity of animal viruses. many bacteriophages, for instance, encode crispr-cas inhibitors, termed anti-crisprs, to counter prokaryotic antiviral systems [ ] . plant viruses also encode viral suppressors of rnai (vsrs) the main antiviral system in plant cells [ ] . likewise, several evasion strategies and immune antagonisms by animal viruses have been described [ ] [ ] [ ] [ ] [ ] . these include hiding the viral genome from immune detection, shutting off host translation or transcription machineries, inhibiting host rna processing and trafficking and interfering directly with either proteins that sense viral presence, or factors that signal the information to the nucleus. since interfering with the innate immune system is less damaging for the host than targeting vital cellular machineries (e.g., translation), many studied viruses seem to have opted for this strategy. several studies describe viral evasion mechanisms at both the recognition and sensing step (tlrs, rlrs and cgas-sting) or at the downstream signaling steps through the targeting of proteins such as mavs, tbk , irf , irf and nf-κb. evasion strategies and immune antagonisms by animal viruses are a very active area of research, that have yielded a rich literature in the past few years. we will here just give some select examples of viral strategies that curb sensing and signaling by tlrs, rlrs and cgas-sting in animals, with an obvious bias towards viruses infecting humans. for a more complete picture on the subject, readers can refer to excellent reviews, published recently, describing those strategies [ ] [ ] [ ] [ ] [ ] . tlr signaling has been shown to be inhibited by the vaccinia virus (vacv) protein a r, that targets specific tir-domain-containing adaptor proteins. a r itself contains a tir domain which allows it to competitively interact with tir-domain-containing complexes such as myd , trif or tram, thereby inhibiting the activation of both nfkb and irfs [ , ] . human t-cell leukemia virus type- (htlv- ) is also able to interfere with tlr -dependent signaling. the htlv- encoded viral protein p binds and disables a transcription factor, pu. , required for tlr surface expression [ ] . trif, an important player in the tlr signaling cascade, is a target of choice of many viruses. the ns / a protease of hcv and the c proteases of several picornaviruses such as coxsackievirus b, ev and hepatitis a virus (hav), can all recognize and proteolytically cleave trif, producing trif fragments that are unable to signal [ ] [ ] [ ] [ ] [ ] . when it comes to rlrs, one basic strategy used by cytosolic viruses to escape surveillance is to simply prevent these receptors from accessing viral genomes. denv, for example, replicates in convoluted membranes of the er concealing its dsrna intermediates from the cytosol and thereby prevents the activation of rlrs [ ] . other viruses like ebola virus (ebov) and marburg viruses encode viral protein (vp ) that tightly binds and 'shields' the viral genome from detection by rig-i [ , ] . another strategy used by viruses to 'hide' from rlrs consists of modifying the very molecular features these receptors rely on to recognize viral genomes. for example, both, hantaan viruses from the bunyaviridae family and borna disease virus (bdv) from the bornaviridae family, encode phosphatases that process the triphosphate group at their genome termini, to a -monophosphate to escape rig-i surveillance [ , ] . lassa virus (lasv) from the arenaviridae family evolved a unique strategy in which its nucleoprotein (np) acquired a - exonuclease activity, that enables it to digest free dsrna, preventing the activation of rig-i [ ] . however, the most direct way of interfering with rlr function and their signaling partners is to either directly target them for cleavage and degradation or to manipulate their phosphorylation and ubiquitination statuses, which are crucial for their activation. indeed, many viruses encode proteases that directly cleave rlrs. while the cpro proteases of both poliovirus and ev cleave rig-i, the apro of ev cleaves mda [ , ] . mavs, a crucial hub for both rig-i and mda -mediated signaling is also frequently targeted and cleaved by numerous viral proteases, such as cpro from hav, apro from ev , ns -ns a from hcv, apro and cpro from rhinovirus and cpro from coxsackievirus b (cvb ) [ , , [ ] [ ] [ ] . mavs can also be indirectly degraded by particular viruses. for instance, measles virus (mev) can trigger a selective form of autophagy, called mitophagy, responsible for the degradation of mitochondria, which leads to a decrease of mavs abundance [ ] . another example of indirect mavs degradation comes from studies with severe acute respiratory syndrome (sars)-associated coronavirus (sars-cov). this virus has evolved a strategy in which its b protein localizes to mitochondria and subverts the cellular e ubiquitin ligase atrophin- -interacting protein (aip ) to degrade mavs [ ] . post-translational modifications of both mavs and rlrs have also been shown to be subverted by viruses to inhibit their downstream signaling. ns proteins from many influenza a virus strains (iav) interact with the host ubiquitin ligase trim and inhibit its oligomerization, a crucial step for its enzymatic activity of attaching lys -linked polyubiquitin to the card domains of rig-i [ , ] . other viruses encode deubiquitinating enzymes (dubs) to remove the lys -linked ubiquitination off rig-i. orf from kaposi's sarcoma herpesvirus (kshv), papain-like protease (plp) from sars-cov, leader proteinase (lpro) from foot-and-mouth disease virus (fmdv) and the ovarian tumor (otu)-type proteins of arteriviruses and nairoviruses have all been shown to possess a deubiquitination activity and interfere with rig-i mediated signaling [ , [ ] [ ] [ ] . rig-i and mda phosphorylation status can also be subverted by viruses. in normal conditions, phosphorylation of serine or threonine residues keeps rig-i and mda in an inactive state. upon viral infection, pp phosphatases are recruited to dephosphorylate specific marks on those receptors and activate them. v proteins from measles and nipah viruses (mev and niv) act as decoys and have been shown to bind pp -α and pp -γ, sequestering them away from mda and rig-i [ , ] . similar to evasion strategies that counter the rna sensing machinery described earlier, dna viruses use numerous strategies to escape cgas-sting-dependent detection and signaling. they could either hide their viral genomes or cleave, degrade, post-translationally modify or even relocalize dna sensing and signaling factors [ ] . hepatitis b virus (hbv), that causes chronic hepatitis and increases the risk of developing liver cirrhosis and hepatocellular carcinoma, has developed an array of mechanisms to inhibit the host's immune systems (reviewed in [ ] ). notably, the hbv polymerase can bind to sting to block its lys -linked ubiquitination, inhibiting the production of ifn-β [ ] . moreover, even though cgas is expressed in human hepatocytes and is able to sense and signal upon transfection of naked relaxed-circular hbv dna; during a natural infection, hbv dna seems to escape cgas detection, likely due to packaging of the genome into the viral capsid [ ] . kshv, another dna virus has been shown to act on both cgas and sting. several kshv proteins (e.g., orf and lana) can either sequestrate stimulatory dna or directly bind to cgas inhibiting its enzymatic activity [ , ] . kshv has been also shown to encode a viral interferon regulatory factor (virf ) that interacts with sting thereby preventing tbk binding and sting activation by tbk -dependent phosphorylation [ ] . the ns protease of denv, together with its ns b co-factor, has been shown to target the residues - (lrrg) of human sting, leading to its cleavage and degradation [ , ] . interestingly, mouse sting lacks these lrrg residues, and ns b/ns of denv is neither able to cleave the murine sting, nor to block murine ifn-β production. therefore, it has been proposed that the inability of denv to cleave mouse sting might explain its host tropism, as murine cells are not very susceptible to denv infection [ , ] . in animals, prrs and their associated signaling pathways are early and potent cellular sensors of viral elements, that mobilize the organism's defenses by inducing an antiviral state. major advances have been made in the last two decades in the understanding of their function in mammalian immunity. new genomics data and gene editing tools can now let us interrogate prr-like pathways in poorly studied animal species and define their evolutionary trajectories. studying the evolution of immune components and their interplay with viral pathogens is extremely important since our immune responses to contemporary viruses have been shaped by our evolutionary responses to previous infections. the modern innate immune system is generally not yet optimized against modern viruses but rather was selected for by previous rounds of co-evolution with ancient viruses [ ] . studying the biological arms race between host and virus, referred to as the "red queen hypothesis" [ ] , in which each entity maintains a relatively constant fitness cost, will be instrumental in the fight against future infections. such studies will help us understand many aspects of viral infections including viral zoonoses, tropism, global epidemics and disease progression. furthermore, exploring these pathways and mechanisms for therapeutic purposes may offer novel strategies to cure human disease. indeed, modulating the action of the aforementioned immune sensors is proving to be an effective strategy to develop vaccines and vaccine adjuvants [ ] [ ] [ ] [ ] [ ] or to treat viral infections [ ] [ ] [ ] [ ] [ ] [ ] . finally, the use of tlr, rlr and sting modulators, to treat inflammation, auto-immune disease [ , ] and also in cancer immunotherapy [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] provides an eloquent incentive to continue studying these pathways and to look ahead with great 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system primordial emergence of the recombination activating gene (rag ): sequence of the complete shark gene indicates homology to microbial integrases evolution of immune systems from viruses and transposable elements re-evaluation of the immunological big bang pattern recognition receptors and control of adaptive immunity approaching the asymptote? evolution and revolution in immunology crosstalk between cytoplasmic rig-i and sting sensing pathways discriminating self from non-self in nucleic acid sensing innate immune pattern recognition: a cell biological perspective recognition of double-stranded rna and activation of nf-kappab by toll-like receptor recognition of single-stranded rna viruses by toll-like receptor human tlr- -, - -, and - -mediated induction of ifn-alpha/beta and -lambda is irak- dependent and redundant for protective immunity to viruses the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses inhibition of the rna polymerase iii-mediated dsdna-sensing pathway of innate immunity by vaccinia virus protein e rna polymerase iii detects cytosolic dna and induces type i interferons through the rig-i pathway rig-i-dependent sensing of poly(da:dt) through the induction of an rna polymerase iii-transcribed rna intermediate dna-pk is a dna sensor for irf- -dependent innate immunity dlm- /zbp ) is a cytosolic dna sensor and an activator of innate immune response the interferon response to intracellular dna: why so many receptors? immunobiology ifi is an innate immune sensor for intracellular dna the helicase ddx senses intracellular dna mediated by the adaptor sting in dendritic cells adenovirus detection by the cgas/sting/tbk dna sensing cascade pan-viral specificity of ifn-induced genes reveals new roles for cgas in innate immunity cyclic gmp-amp is an endogenous second messenger in innate immune signaling by cytosolic dna aim recognizes cytosolic dsdna and forms a caspase- -activating inflammasome with asc ticam- , an adaptor molecule that participates in toll-like receptor -mediated interferon-beta induction role of adaptor trif in the myd -independent toll-like receptor signaling pathway specificity in toll-like receptor signalling through distinct effector functions of traf and traf critical role of traf in the toll-like receptor-dependent and -independent antiviral response type i interferon [corrected] gene induction by the interferon regulatory factor family of transcription factors cutting edge: tnfr-associated factor (traf) is essential for myd -dependent pathway but not toll/il- receptor domain-containing adaptor-inducing ifn-beta (trif)-dependent pathway in tlr signaling interferon-alpha induction through toll-like receptors involves a direct interaction of irf with myd and traf helical assembly in the myd -irak -irak complex in tlr/il- r signalling protein kinase ikkbeta-catalyzed phosphorylation of irf at ser induces its dimerization and nuclear translocation in myeloid cells an oligomeric signaling platform formed by the toll-like receptor signal transducers myd and irak- ikkbeta is an irf kinase that instigates inflammation interleukin- receptor-associated kinase- plays an essential role for toll-like receptor (tlr) -and tlr -mediated interferon-{alpha} induction structural basis of rna recognition and activation by innate immune receptor rig-i structural basis for the activation of innate immune pattern-recognition receptor rig-i by viral rna mda assembles into a polar helical filament on dsrna cooperative assembly and dynamic disassembly of mda filaments for viral dsrna recognition trim ring-finger e ubiquitin ligase is essential for rig-i-mediated antiviral activity ubiquitin-induced oligomerization of the rna sensors rig-i and mda activates antiviral innate immune response riplet/rnf , a ring finger protein, ubiquitinates rig-i to promote interferon-beta induction during the early phase of viral infection the ubiquitin ligase riplet is essential for rig-i-dependent innate immune responses to rna virus infection ips- , an adaptor triggering rig-i-and mda -mediated type i interferon induction cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf essential role of ips- in innate immune responses against rna viruses an autoinhibitory mechanism modulates mavs activity in antiviral innate immune response mavs forms functional prion-like aggregates to activate and propagate antiviral innate immune response structural basis for the prion-like mavs filaments in antiviral innate immunity mavs recruits multiple ubiquitin e ligases to activate antiviral signaling cascades activation of ikk by tnfalpha requires site-specific ubiquitination of rip and polyubiquitin binding by nemo sensing of lys -linked polyubiquitination by nemo is a key event in nf-kappab activation key role of ubc and lysine- polyubiquitination in viral activation of irf sting is an endoplasmic reticulum adaptor that facilitates innate immune signalling the adaptor protein mita links virus-sensing receptors to irf transcription factor activation sting regulates intracellular dna-mediated, type i interferon-dependent innate immunity sting is a direct innate immune sensor of cyclic di-gmp coordinated regulation of accessory genetic elements produces cyclic di-nucleotides for v. cholerae virulence mpys is required for ifn response factor activation and type i ifn production in the response of cultured phagocytes to bacterial second messengers cyclic-di-amp and cyclic-di-gmp the n-ethyl-n-nitrosourea-induced goldenticket mouse mutant reveals an essential function of sting in the in vivo interferon response to listeria monocytogenes and cyclic dinucleotides cyclic gmp-amp synthase is an innate immune sensor of hiv and other retroviruses pivotal roles of cgas-cgamp signaling in antiviral defense and immune adjuvant effects cgas senses long and hmgb/tfam-bound u-turn dna by forming protein-dna ladders cyclic gmp-amp synthase is activated by double-stranded dna-induced oligomerization cgas produces a - -linked cyclic dinucleotide second messenger that activates sting cyclic gmp-amp containing mixed phosphodiester linkages is an endogenous high-affinity ligand for sting viruses transfer the antiviral second messenger cgamp between cells transmission of innate immune signaling by packaging of cgamp in viral particles structure-function analysis of sting activation by c[g( , )pa( , )p] and targeting by antiviral dmxaa atg a controls dsdna-driven dynamic translocation of sting and the innate immune response phosphorylation of innate immune adaptor proteins mavs, sting, and trif induces irf activation activation of stat by sting is critical for antiviral innate immunity cytosolic-dna-mediated, sting-dependent proinflammatory gene induction necessitates canonical nf-kappab activation through tbk activation of autophagy by alpha-herpesviruses in myeloid cells is mediated by cytoplasmic viral dna through a mechanism dependent on stimulator of ifn genes establishment of dorsal-ventral polarity in the drosophila embryo: the induction of polarity by the toll gene product signals from the il- receptor homolog, toll, can activate an immune response in a drosophila hemocyte cell line the dorsoventral regulatory gene cassette spatzle/toll/cactus controls the potent antifungal response in drosophila adults chromosomal localization of til, a gene encoding a protein related to the drosophila transmembrane receptor toll, to human chromosome p prediction of the coding sequences of unidentified human genes. i. the coding sequences of new genes (kiaa -kiaa ) deduced by analysis of randomly sampled cdna clones from human immature myeloid cell line kg- defective lps signaling in c h/hej and c bl/ sccr mice: mutations in tlr gene the role of pattern-recognition receptors in innate immunity: update on toll-like receptors evolutionary origins of toll-like receptor signaling innate immunity in the simplest animals-placozoans sea anemone model has a single toll-like receptor that can function in pathogen detection, nf-kappab signal transduction, and development the innate immune repertoire in cnidaria-ancestral complexity and stochastic gene loss differential and convergent utilization of autophagy components by positive-strand rna viruses a conserved toll-like receptor-to-nf-kappab signaling pathway in the endangered coral orbicella faveolata genomic insights into the immune system of the sea urchin massively parallel rna sequencing identifies a complex immune gene repertoire in the lophotrochozoan mytilus edulis massive expansion and functional divergence of innate immune genes in a protostome teleost tlr recognizes rna duplex to induce ifn and protect cells from birnaviruses adaptive evolution of virus-sensing toll-like receptor in bats the evolution of bat nucleic acid-sensing toll-like receptors immune system modulation and viral persistence in bats: understanding viral spillover origin and evolution of the rig-i like rna helicase gene family characterization of the mollusc rig-i/mavs pathway reveals an archaic antiviral signalling framework in invertebrates retinoic acid-inducible gene i (rig-i)-like receptors (rlrs) in fish: current knowledge and future perspectives chicken cells sense influenza a virus infection through mda and cardif signaling involving lgp association of rig-i with innate immunity of ducks to influenza genome of the chinese tree shrew loss of rig-i leads to a functional replacement with mda in the chinese tree shrew the kinase ikkbeta regulates a sting-and nf-kappab-dependent antiviral response pathway in drosophila the jak-stat signaling pathway is required but not sufficient for the antiviral response of drosophila the rna silencing endonuclease argonaute mediates specific antiviral immunity in drosophila melanogaster essential function in vivo for dicer- in host defense against rna viruses in drosophila sensing viral rnas by dicer/rig-i like atpases across species the dexd/h-box helicase dicer- mediates the induction of antiviral activity in drosophila secreted vago restricts west nile virus infection in culex mosquito cells by activating the jak-stat pathway dicer- -dependent activation of culex vago occurs via the traf-rel signaling pathway nucleic acid sensing in invertebrate antiviral immunity the rig-i atpase core has evolved a functional requirement for allosteric stabilization by the pincer domain the selective footprints of viral pressures at the human rig-i-like receptor family evolution and functional impact of rare coding variation from deep sequencing of human exomes cyclic di-nucleotide signaling enters the eukaryote domain evolutionary origins of cgas-sting signaling toll signaling: the tireless quest for specificity analysis of drosophila sting reveals an evolutionarily conserved antimicrobial function modular architecture of the sting c-terminal tail allows interferon and nf-kappab signaling adaptation dampened sting-dependent interferon activation in bats structure of human cgas reveals a conserved family of second-messenger enzymes in innate immunity ] is the metazoan second messenger produced by dna-activated cyclic gmp-amp synthase structural mechanism of cytosolic dna sensing by cgas overlapping patterns of rapid evolution in the nucleic acid sensors cgas and oas suggest a common mechanism of pathogen antagonism and escape the discovery, mechanisms, and evolutionary impact of anti-crisprs decoding type i and iii interferon signalling during viral infection viral evasion of dna-stimulated innate immune responses ten strategies of interferon evasion by viruses viral evasion of intracellular dna and rna sensing viral evasion and subversion of pattern-recognition receptor signalling a r and a r from vaccinia virus are antagonists of host il- and toll-like receptor signaling vaccinia virus protein a r targets multiple toll-like-interleukin- receptor adaptors and contributes to virulence the htlv-i p interferes with tlr signaling and modulates the release of pro-and anti-inflammatory cytokines from human macrophages innate immunity evasion by enteroviruses: insights into virus-host interaction toll-like receptors in antiviral innate immunity hepatitis c virus protease ns / a cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity the coxsackievirus b c protease cleaves mavs and trif to attenuate host type i interferon and apoptotic signaling disruption of tlr signaling due to cleavage of trif by the hepatitis a virus protease-polymerase processing intermediate the dengue virus conceals double-stranded rna in the intracellular membrane to escape from an interferon response ebola virus vp protein binds double-stranded rna and inhibits alpha/beta interferon production induced by rig-i signaling structural basis for marburg virus vp -mediated immune evasion mechanisms sequestration by ifit impairs translation of o-unmethylated capped rna old world hantaviruses do not produce detectable amounts of dsrna in infected cells and the termini of their genomic rnas are monophosphorylated structure of the lassa virus nucleoprotein reveals a dsrna-specific to exonuclease activity essential for immune suppression rig-i is cleaved during picornavirus infection enterovirus apro targets mda and mavs in infected cells cleavage of ips- in cells infected with human rhinovirus disruption of innate immunity due to mitochondrial targeting of a picornaviral protease precursor mitophagy enhances oncolytic measles virus replication by mitigating ddx /rig-i-like receptor signaling sars-coronavirus open reading frame- b suppresses innate immunity by targeting mitochondria and the mavs/traf /traf signalosome influenza a virus ns targets the ubiquitin ligase trim to evade recognition by the host viral rna sensor rig-i deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases inhibition of rig-i-mediated signaling by kaposi's sarcoma-associated herpesvirus-encoded deubiquitinase orf deubiquitinase function of arterivirus papain-like protease suppresses the innate immune response in infected host cells antagonism of the phosphatase pp by the measles virus v protein is required for innate immune escape of mda measles virus suppresses rig-i-like receptor activation in dendritic cells via dc-sign-mediated inhibition of pp phosphatases the cgas-sting defense pathway and its counteraction by viruses immune evasion strategies during chronic hepatitis b and c virus infection. vaccines (basel) , , hepatitis b virus polymerase disrupts k -linked ubiquitination of sting to block innate cytosolic dna-sensing pathways hepatitis b virus evasion from cyclic guanosine monophosphate-adenosine monophosphate synthase sensing in human hepatocytes inhibition of cgas dna sensing by a herpesvirus virion protein cytoplasmic isoforms of kaposi sarcoma herpesvirus lana recruit and antagonize the innate immune dna sensor cgas modulation of the cgas-sting dna sensing pathway by gammaherpesviruses denv inhibits type i ifn production in infected cells by cleaving human sting dengue virus targets the adaptor protein mita to subvert host innate immunity evolutionary conflicts between viruses and restriction factors shape immunity the evolutionary conundrum of pathogen mimicry enhanced influenza virus-like particle vaccination with a structurally optimized rig-i agonist as adjuvant pika as an adjuvant enhances specific humoral and cellular immune responses following the vaccination of mice with hbsag plus pika a tlr ligand that exhibits potent inhibition of influenza virus replication and has strong adjuvant activity has the potential for dual applications in an influenza pandemic as , an aluminum salt-and tlr agonist-based adjuvant system, induces a transient localized innate immune response leading to enhanced adaptive immunity the tlr agonist, monophosphoryl lipid a, attenuates the cytokine storm associated with respiratory syncytial virus vaccine-enhanced disease sting agonists enable antiviral cross-talk between human cells and confer protection against genital herpes in mice the tlr antagonist eritoran protects mice from lethal influenza infection targeting innate immunity for antiviral therapy through small molecule agonists of the rlr pathway direct antiviral properties of tlr ligands against hbv replication in immune-competent hepatocytes safety, efficacy and pharmacodynamics of vesatolimod (gs- ) in virally suppressed patients with chronic hepatitis b antibody and tlr agonist delay viral rebound in shiv-infected monkeys therapeutic effects of the artemisinin analog sm on lupus-prone mrl/lpr mice via inhibition of tlr-triggered b-cell activation and plasma cell formation tak- (resatorvid), a small-molecule inhibitor of toll-like receptor (tlr) signaling, binds selectively to tlr and interferes with interactions between tlr and its adaptor molecules essential for the antitumor effect of immune checkpoint blockade sa- - bbl and monophosphoryl lipid a constitute an efficacious combination adjuvant for cancer vaccines magnitude of therapeutic sting activation determines cd (+) t cell-mediated anti-tumor immunity immunogene therapy using immunomodulating hvj-e vector augments anti-tumor effects in murine malignant glioma promising targets for cancer immunotherapy: tlrs, rlrs, and sting-mediated innate immune pathways immunogenic cell death of human ovarian cancer cells induced by cytosolic poly(i:c) leads to myeloid cell maturation and activates nk cells sting-mediated dna sensing promotes antitumor and autoimmune responses to dying cells sting agonist formulated cancer vaccines can cure established tumors resistant to pd- blockade this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license funding: this work has been supported by the h marie-curie actions msca-if- -hipshot (km). this work was also supported in part by the national institutes of health grants u -ai (tfb) by arc, paris and institut hospitalo-universitaire, strasbourg (therahcc and therahcc . ihuarc ihu and ihuarc to t. acknowledgments: the authors would like to thank jean-luc imler and joao t. marques for their critical reading of the manuscript. the authors apologize to colleagues whose work could not be cited due to space limitations. the authors declare no conflict of interest. key: cord- -yr zfxz authors: le devendec, laetitia; jouy, eric; paboeuf, frederic; de boisséson, claire; lucas, pierrick; drider, djamel; kempf, isabelle title: development of a pig infection model with colistin-resistant escherichia coli date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: yr zfxz colistin-resistant escherichia coli are isolated from pigs suffering from post-weaning diarrhea (pwd). this study was designed to develop an experimental model of pwd using mcr- -carrying shiga toxin-producing e. coli (stec) or enterotoxigenic e. coli (etec), for the future evaluation of control measures. three groups of eight piglets, kept in high biosecurity units, were orally inoculated with mcr- -positive stec or etec, and one unchallenged group was used as a control. clinical signs were recorded. regularly-collected fecal samples and samples obtained from the digestive tract of animals sacrificed one month after inoculation were cultured in selective media and isolates were characterized. blood samples were used to genotype the polymorphisms of the pigs’ intestinal receptors for f and f e. coli adhesins. diarrhea was more frequent and more fecal samples contained the inoculated strain in the group inoculated with the o -f etec strain than with the o -f or o -f stec strains. however, fewer positive samples were obtained from the two pigs with the f resistant genotype. the three inoculated strains could be re-isolated up to the end of the experiment. excretion peaked on the first week after inoculation with the o -f etec strain, and later for the other two. an mcr- gene transfer to other commensal isolates was observed only for o -f stec, while the loss of mcr- from the inoculated strain occurred in all groups. the o -f etec challenge may be used to evaluate alternative solutions to combat pwd caused by colistin-resistant e. coli in pigs. post-weaning diarrhea (pwd) and edema disease are common pathological conditions affecting piglets (rhouma et al., a) . various escherichia coli strains cause these diseases. indeed, different serogroups (o , o , o , o , o …), adhesive factors (f (k ), f , aida, eae…), and other virulence factors (enterotoxins lt, sta, stb, east…) have been reported in these strains (fairbrother and gyles, ) . the control of these infections must be based primarily on optimizing breeding management of the weaning piglets so as to avoid, insofar as possible, stressful conditions as they are separated from the sow, transported, mixed, placed in a new environment and provided with new feed. prophylactic measures may also include vaccination of the sow (rhouma et al., a) . however, these measures may be ineffective in preventing digestive troubles, and in the past, orally administered antimicrobials were almost systematically used to control colibacillosis. as a result, e. coli strains have developed antimicrobial resistance, particularly to the most frequently administered molecules. of note, resistance to colistin (cst) had been detected at relatively low levels for years in commensal isolates, but prevalence was sometimes high for pathogenic strains (kempf et al., ) , and after a publication on the emerging mcr- gene in china (liu et al., ) , it soon appeared that this gene was also present in many other countries, particularly in pig e. coli strains (kempf et al., ) . as these strains are frequently also resistant to many other antimicrobials (delannoy et al., ) , it became necessary to develop and evaluate novel control products or measures. to our knowledge, experimental models of colibacillosis in piglets have been developed with inoculation of cst-susceptible e. coli (madec et al., ; rhouma et al., rhouma et al., , b but not with cstresistant strains. however we believe that such models are needed to evaluate the impact or the efficacy of antimicrobials, including colistin, and alternative strategies on infections caused by strains with resistance or reduced susceptibilities to a number of molecules. furthermore, it has been shown that acquisition and expression of the mcr- gene in a e. coli cell may result in gross morphological changes, including cell membrane impairment, and reduced fitness and attenuated virulence in a galleria mellonella model. moreover the mcr- mediated lps modification reduces the stimulation of macrophages, and a lower production of il- and tnf was induced with lps extracted from mcr- strains compared to control lps (yang et al., ) . thus because of these potential differences of susceptibility and virulence of mcr- strains, we decided to develop an experimental model of colibacillosis in weaned piglets using multi-drug-resistant e. coli strains isolated from clinical cases and carrying the mcr- gene. . . preparation of strains, media and inocula and whole genome sequencing three e. coli strains ( - , - and - ) from our previously characterized collection of mcr- -positive e. coli isolates from diseased pigs were selected on the basis of their resistances, serogroups and virulence genes as detected by high-throughput real-time pcr microarray technology (delannoy et al., ) . the intent was to choose strains representative of cst-resistant isolates obtained from piglets with pwd. all three strains were hemolytic. strains - , - , and - belonged to phylogenetic groups (pgg) a, a, and e, and had markers o -f , o -f , and o -f respectively. all three strains were resistant to colistin, sulfamethoxazole, trimethoprim, tetracycline, and ampicillin. strains - and - were also resistant to gentamicin and chloramphenicol. a rifampicin-resistant mutant ( - m, - m, and - m) was prepared from each strain by culturing a large amount of inoculum on rifampicin-supplemented media ( mg/l). dna from the three mutants was prepared using qiamp dna mini kits (qiagen, courtaboeuf, france) according to the manufacturer's instructions. whole genome sequencing was performed with the ion proton system (ion torrent™, thermofisher, villebon sur yvette, france). we cleaned reads with trimmomatic (bolger et al., ) , then used bwa-mem (li, ) to align them with the e. coli e strain (ncbi reference sequence nz_jend . ) to obtain an estimated coverage depth of . we next performed spades assemblies on cleaned reads (bankevich et al., ) and mira assemblies (chevreux et al., ) on related raw reads. contigs were identified using megablast on a local nt database (chen et al., ) , and those corresponding to "uncultured soil fungus" were deleted. sequences were analyzed using a web tool (https://cge.cbs.dtu.dk/ services) to detect antimicrobial resistance genes and verotoxin virulence genes, and to determine the st of each strain (thomsen et al., ) . other virulence genes, including paa, sfpc, cnf , aida-i, ecf - , stce, and eibg (tseng et al., ), sita-d, iuca-d, iuta, hlyf, ompt, etsa-c, iss, irob-e, iron, cvaa-c, cvi, tsh, and eita-d (touzain et al., ) , tere, ured, and esc (ae . ), hlya (fm . ), orfa and orfb (gu ), were sought using blast (https://blast.ncbi.nlm. nih.gov/blast.cgi). to prepare the inocula, each mutant was cultured in mueller hinton (mh) broth containing colistin ( mg/l) for h at °c, under gentle agitation. each bacterial pellet obtained after centrifugation ( g, min) was re-suspended in peptone buffered solution in order to obtain a titer of colony-forming units (cfu)/ml. the experiment was performed in accordance with french animal welfare regulations and the protocol was approved by the anses/ enva/upec ethical committee (cometh authorization - (apafis no. )). the experiment was conducted in the anses ploufragan animal facilities. strict biosecurity measures were implemented to avoid contamination of the pigs, including the use of an air filtration system and airlocks for each unit, unit-specific clothes, and compulsory showering after visiting the pigs. the trials were conducted with specific-pathogen-free (spf) large white piglets. the spf piglets are naturally born from hysterectomy-derived sows controlled for presence of classical and african swine fever, aujesky's disease, footand-mouth disease, h n /h n swine influenza, transmissible gastroenteritis/porcine respiratory coronavirus, porcine parvovirus, reproductive and respiratory syndrome virus, porcine circovirus type , border disease, mycoplasma hyopneumoniae, pasteurella multocida, bordetella bronchiseptica, actinobacillus pleuropneumoniae, haemophilus parasuis, streptococcus suis type , salmonella, lawsonia intracellularis, brachyspira hyodysenteriae, yersinia, campylobacter, listeria monocytogenes, balantidium coli and trichomonas. the piglets are left with the sow until weaning and bred in a pathogen-free environment. the six-week-old piglets were randomized before the experiment. the animals did not receive any antibiotic treatment prior to the trial and were given the same non-supplemented feed. each room contained two pens (table ) . on day , five piglets in animal room e (group , five pigs) were orally inoculated with ml of sterile broth. on the same day, eight piglets per room (four piglets per pen) in animal rooms e , e , and e , (respectively named groups g , g , and g , eight pigs each) were orally inoculated with ml of the inocula prepared with strains - m, - m, and - m respectively. the weight of each animal was recorded once a week. during the week, daily clinical examinations consisted in looking for general clinical signs and taking rectal temperatures. individual fecal samples were collected from pigs on , , , , , , , , , , , , and (groups g , g , and g ) , and on , , and (group g ) . the pigs were sacrificed on day (group g ), day (group g ) or day (group g ). three pigs from the g group were also sacrificed on day , the others being kept as controls for another assay and necropsied on day following euthanasia. the sacrificed animals were necropsied and samples of the duodenum, jejunum, ileum, cecum, colon, and rectum were collected. the samples collected post-mortem contained both fecal material and scraped mucosa. for culture analysis, all the samples were diluted / in peptone buffered solution containing % glycerol. the / diluted aliquots were stored at - °c. before freezing (fecal samples) or after thawing (organ samples), decimal dilutions were prepared. one hundred microliters of decimal dilutions were inoculated using easyspiral® dilute (interscience, saint-nom-la-bretèche, france) onto macconkey media supplemented with rifampicin ( mg/l) and incubated overnight at °c. moreover, μl of the / thawed dilutions was enriched overnight in . ml of rifampicin-supplemented lb broth ( mg/l) before plating on rifampicin-supplemented macconkey plates. the limit of detection was cfu without enrichment and cfu/g after enrichment. whenever possible, two colonies (from samples positive only after enrichment) or three colonies (from samples positive without enrichment) per sample were stored. pcr was used to confirm that they belonged to the e. coli species (bej et al., ) , to detect the presence table arrangement of pigs inoculated with the different studied strains. groups all pigs were susceptible to e. coli f . a number of pigs resistant to e. coli f . of the mcr- gene (liu et al., ) , and to determine their phylogenetic group (clermont et al., ) . for isolates which did not exhibit the expected results (presence of mcr- and phylogenetic group of the inoculated strain), eric-pcr (rivera et al., ) and pfge profiles (ribot et al., ) after digestion with xbai were studied. the susceptibility of some of these isolates was compared to the susceptibility of the inoculated strains by determining the minimum inhibitory concentrations (mic) using a broth microdilution method on euvsec plates (sensititre, thermofisher scientific, dardilly, france). in addition, fecal samples collected on days , and were inoculated, after thawing, onto the chromagar orientation ™ agar medium (i a, montpellier, france) supplemented with colistin ( mg/l) and vancomycin ( mg/l) (cao-cv) as previously described (mourand et al., ) to detect colistin-resistant rifampicin-susceptible e. coli resulting from transfer of the mcr- gene to commensal e. coli. when e. coli colonies were observed, two colonies per sample were stored and further analyzed. their identity and the presence of the mcr- gene were checked by pcr, and their resistance to rifampicin was tested by culture on rifampicin-supplemented mh media ( mg/l). moreover, μl of the / dilutions was enriched overnight in . ml of colistin-supplemented lb broth ( mg/l) before plating onto rifampicinsupplemented macconkey plates (limit of detection: cfu/g). the colonies were identified and analyzed for the presence of the mcr- gene as previously described. for molecular analysis, thawed fecal and organ samples were tested with a slightly modified version of the protocol described by dona et al. (dona et al., ) . briefly, μl of the / diluted thawed fecal suspension was used to inoculate . ml of luria bertani (lb) broth supplemented with rifampicin ( mg/l). after overnight culture at °c under agitation, the broths were centrifuged at g for ten minutes and a cellular lysate was prepared from the pellet. e. coli dna and the mcr- gene were then detected by pcr in the lysate. pcr was performed on lysates obtained from enrichment in the colistin-supplemented broths ( mg/l), like for samples collected on days , , and . blood was obtained from each pig just before euthanasia. these samples were used to genotype the pigs for the different polymorphisms linked to the intestinal receptors for f and f e. coli adhesins. the porcine mucin (muc ) gene has been mapped on the q region of the pig chromosome , significantly associated with in vitro e. coli f ac adhesion to enterocytes, and muc is the most likely responsible gene governing susceptibility towards enterotoxigenic e. coli (etec) f ac diarrhea (ren et al., ) . the polymorphism of muc was analyzed based on the test described by rasschaert et al. (rasschaert et al., ) to determine the f ac/ab receptor status. briefly, a fragment of the mucin gene was amplified using hemo klen taq polymerase (neb, evry, france) and digested with xbai. the bp amplified muc pcr fragment of the resistant allele is indigestible, unlike the susceptible allele. muc was genotyped by pcr to detect the presence of amplicons of bp (muc a allele) and pb (muc b allele) as described by ren et al (ren et al., ) . susceptible animals usually carry at least one muc b allele. the e. coli f receptor locus is closely linked to the fucosyltransferase gene, and we used the test developed by meijerink et al. (meijerink et al., ) in which amplification followed by restriction with cfoi enables discrimination between the different alleles, based on the number of amplified fragments: two fragments for homozygous a/ a, three for homozygous g/g and four for heterozygous. the resistance is associated with allele a (meijerink et al., ) . the kruskall-wallis non-parametric test was used to compare weight gain. the distributions of pigs or isolates were analyzed using the chi -test or the fisher exact test. this whole genome shotgun project has been deposited at ddbj/ ena/genbank under the accession qzvz , qzwa and qzvy (respectively - m, - m and - m). sequencing enabled us to detect the resistance genes present in the isolates. thus, beside the previously described mcr- gene and the mutations in genes possibly involved in resistance to polymyxins (delannoy et al., ) , the strains harbored the genes coding for their other observed resistances: sulfonamides (sul , sul or sul ), trimethoprim (dfra ), tetracycline (tet(a)), chloramphenicol (cata , cmla ), ampicillin (bla tem a or bla tem b ) or aminoglycosides (aac( )-iia, aac ( )-iva, aada , aada , aada , aph( ′)-ia, aph( ′)- c, aph( )-ia, stra or strb). the virulence genes are listed in table . the genes previously detected by microarray (delannoy et al., ) were confirmed to be present. the two f stx -positive strains belonged to st and , whereas the o f strain containing genes coding for the lt, sta and stb toxins belonged to st- . the inoculum titers were . × cfu/ml, . × cfu/ml, and . × cfu/ml for - m, - m and - m respectively. only pigs could be tested as no blood was obtained from the two control pigs sacrificed on day . as expected, muc and muc gene results were in agreement, and only five animals were found to be of the genotype resistant to e. coli f ac/ab: one in group g , two in g , and two in g (table ) . for the f receptor, ten pigs were heterozygous ag and the other were homozygous gg. thus all pigs were susceptible to e. coli f . all pigs of group g had normal rectal temperature during the assay. moderate pyrexia ( . °c- . °c) was observed during the three days after inoculation in , and pigs in groups g to g respectively. no clinical signs were observed in group g . in groups g to g , moist, cow-dung appearance feces were observed in all pigs during the third week after inoculation. moreover, in group g , two pigs had diarrhea during the four days following inoculation. the mean weight gains of groups g to g from day to day were respectively . ± . , . ± . , . ± . , and . ± . kg (p = . ). the day -day weight gain of group g , but not those of groups g and g , was significantly different from the weight gain of the control group (p = . ). the weight gains from day to day of groups g to g were respectively . ± . , . ± . , . ± . , and . ± . kg and did not differ significantly (p > . ). no lesions could be observed post-mortem. the results are presented in tables and . all cultures from noninoculated pigs or from samples collected before inoculation were negative on rifampicin-supplemented media. some of these samples yielded e. coli colonies after enrichment, but none of the isolates tested had the mcr- gene. for cultures on rifampicin-supplemented media, with or without enrichment, and after mcr- -pcr control of isolates, all inoculated pigs were observed to be positive at least once. in groups g , g , and g , out of sampling days after inoculation, the pigs were positive on to (mean . ), to (mean . ) and to (mean . ) different days respectively. the total positive samples were out of , out of , and out of tested samples in groups g to g respectively, the proportion in g being significantly higher than the other two groups (chi- test, p < . ). in all groups, at least one pig was positive on the last sampling day, four weeks after inoculation. the maximum mean titers were observed on day ( . ) in group g , on days and ( . on each day) in group g , and on day ( . ) for group g . most isolates obtained directly on rifampicin-supplemented media ( / , %) or after enrichment in rifampicin-supplemented media ( / , %) harbored the mcr- gene and showed the expected characteristics of the inoculated strains. considering only isolates obtained without enrichment, for group g , out of isolates shared the characteristics of inoculated strain - m, and one isolate obtained on day , without enrichment, belonged to pgg a, had the eric and pfge profiles and the antimicrobial susceptibility of - m, including resistance to colistin, although it was negative for the mcr- gene. in group g , out of isolates obtained without enrichment shared the characteristics of inoculated strain - m, but nine isolates lacked the mcr- gene. six were fully analyzed and were found to belong to pgg a, and have the eric and pfge profiles of the - m isolate. they were resistant to tetracycline and gentamicin but, contrary to - m, susceptible to sulfamethoxazole, trimethoprim, colistin and ampicillin. the mcr- -negative isolates had been obtained on day , , , and from three pigs bred in the two pens of the animal room. in group g , out of isolates obtained without enrichment had the expected characteristics (presence of the mcr- gene and phylogenetic group e). six had the mcr- gene but belonged to pgg a, and their eric and pfge profiles were identical to each other but different to that of - m. they were obtained on day and day from three pigs placed in the two pens of the animal room. one isolate obtained on day was mcr- -negative. its pfge profile was identical to the inoculated (delannoy et al., ) . a: sample containing rifampicin-resistant, mcr- -positive e. coli of the expected phylogenetic group, obtained directly. b: sample containing rifampicin-resistant, mcr- -positive e. coli of the expected phylogenetic group, obtained after enrichment. c: sample containing rifampicin-resistant, mcr- -positive e. coli of an unexpected phylogenetic group and pfge profile, obtained directly. d: sample containing rifampicin-resistant, mcr- -positive e. coli of an unexpected phylogenetic group and pfge profile, obtained after enrichment (possibly transconjugant). e: sample containing rifampicin-resistant, mcr- -negative e. coli of the expected phylogenetic group and pfge profile, obtained directly (possibly inoculated strain having lost the mcr- gene). f: sample containing rifampicin-resistant, mcr- -positive e. coli detected on cao-cv medium. ns: no sample. grey cells: clinical signs (diarrhea or temperature > . °c). * : pig resistant to e. coli f . ; and for g , three pigs were positive on day . positive pigs were detected in the two pens for groups g and g (table ). there was no difference between the proportion of positive samples in the inoculated groups (fisher's exact test, p > . ). all the samples collected before inoculation were negative. for group g , nine out of samples were positive; they were obtained on days , , , and from seven different pigs. for group g , samples out of obtained on days , , , , , and were positive; seven pigs were positive. for the e group, out of samples were positive; they were obtained on days , , , , and from six different pigs. there was no difference between the proportion of positive samples in the inoculated groups (chi test, p > . ). only samples collected on days , , and were analyzed. all the results were negative except three fecal samples collected on day from two pigs in group g and one in g . for the same days , and , culture on rifampicin media detected positive samples out of samples from inoculated groups and was thus significantly more sensitive (p < . ). the results obtained on rifampicin-supplemented macconkey plates after enrichment in rifampicin-supplemented broth, are given in table . all the samples collected from the non-inoculated pigs and all the duodenum samples were negative. the proportion of positive samples in group g ( / ) was significantly lower than the proportion in g ( / ) and g ( / ) (fisher exact test, p < . ). besides the isolates with the characteristics of the inoculated strains, a few rifampicin-resistant mcr- -negative isolates belonging to the phylogenetic group of the respective inoculated strains were obtained in the jejunum or cecum from three different pigs in group g ; in the ileum or cecum from three different pigs in group g ; and in the ileum, colon, rectum or cecum from three different pigs in group g . the three strains proposed as an experimental model are relevant because they have been reported in literature and seem to be frequently involved in pig diarrhea. indeed sequencing confirmed that they were shiga toxin-producing (stec) ( - m and - m) or etec ( - m). o :f - m is of type st- , which has already been isolated from many different sources, such as humans, animals (including swine in italy and china), food (including pork in the usa) and the environment (water in france) (http://enterobase.warwick.ac. uk/species/ecoli/search_strains). an st- mcr- and bla ndm- e. coli of human origin was also characterized by zhang et al. (zhang et al., ) and several mcr- st- e. coli of healthy swine origin have also been reported in china . the o :f - m e. coli belongs to st- , like several pig or bovine pathogenic isolates of serogroups o :h :f ac reported in different european, american or asian countries, or an o poultry apec isolated in germany. the o :f - m strain belongs to st- , like o :f isolates obtained from pig diarrhea in hungary in (http://enterobase. warwick.ac.uk/species/ecoli/search_strains?query=st_search). an analysis of the different contigs carrying mcr- did not reveal other resistance or virulence genes or notifiable genes. symptoms, including hyperthermia and diarrhea, were more severe in group g . this group also had the highest excretion titers in the first week, and more fecal samples were found positive. however, at necropsy-more than one month after inoculation-the o :f + inoculated strain was less frequently isolated than the other two inoculated strains, both of type f + fimbriae. this may be related to the fact that susceptibility to f + or f + e. coli may vary with the pig's age. verdonck et al. (verdonck et al., ) previously observed a similar delay, with f + etec peak excretion days post-infection (pi), but no excretion days pi, in contrast to the peak excretion of f + vtec infection observed between and days pi, and excretion persisting up to days pi. interestingly, in group g , the only two pigs found to be genetically resistant to e. coli f had no diarrhea and gave only a total of eight positive fecal samples out of , whereas out of samples were positive for the six pigs which were genetically susceptible to e. coli f (p = . ); their mean log titer was . compared to a mean of . (p = . ) for the other pigs in group g . although all the pigs were genetically susceptible to e. coli f , the symptoms remained relatively moderate in groups g and g . the experimental model could be improved by using higher inocula doses, intra-gastric inoculation, withdrawing feed before inoculation, cold stress, pretreatment with antibiotics, co-inoculation of viruses, or inoculating piglets when younger (madec et al., ) , or using pigs known to be genetically susceptible to the inoculated strains. it is acknowledged that post-weaning digestive disorders are generally encountered in inadequate breeding conditions, contrary to the highstandard environment offered to our weaning piglets. using rifampicin-supplemented media, we could evaluate the recovery of the inoculated strain, and search for the possible loss of the mcr- gene from the inoculated strains. the use of colistin-supplemented media was used to try to detect the transfer of the mcr- gene from the inoculated strains to commensal ones. detection of the mcr- gene in colistin-enrichment broths was also performed but the sensitivity of this method was poor as previously observed in experiments with pigs inoculated with a commensal mcr- -positive e. coli strain (mourand et al., ) , and needs further improvements. the three inoculated strains could be recovered from fecal and post-mortem samples up to four and five weeks respectively after inoculation. for group g only, a few isolates carrying the mcr- gene were also obtained, but their other characteristics (phylogenetic group, pfge or eric-pcr profiles, and antimicrobial susceptibility) were different from those of the inoculated strain. as no mcr- -containing e. coli was present in the non-inoculated animals, the uncharacteristic isolates are very probably commensal isolates having acquired the mcr- gene or plasmid from e. coli - m. other analyses are necessary to determine the precise mechanisms of this transfer, which can occur through either conjugation or transposon mobilization. interestingly, in vitro, the mcr- gene was shown to be on a conjugative plasmid in e. coli - , but not in e. coli - or e. coli - . the transconjugant obtained in vitro from e. coli - acquired resistance to colistin, tetracycline, sulfamethoxazole and trimethoprim. different studies have shown that the in vitro acquisition of an mcr- -containing plasmid does not lead to a considerable fitness cost in e. coli as measured by growth kinetics, cytotoxicity, and virulence in a galleria mellonella model (tietgen et al., ; zhang et al., ) , but a high level of mcr- expression compromises growth rate, fitness, and the membrane's structural integrity while increasing cellular death (yang et al., ) . in our experiment, most isolates from samples collected from the e. coli - m-inoculated pigs shared the characteristics of this strain, suggesting that the commensal strains which had acquired the mcr- gene were perhaps not as competitive as e. coli - m. in the three inoculated groups, few of the isolates obtained were resistant to rifampicin, shared the phylogenetic group and pfge profile of the inoculated strain but lacked the mcr- gene. wu et al. ( ) studied the stability of various mcr- -harboring plasmids and showed that the inchi , inci , incx and incy plasmids were maintained after days of passage in a colistin-free medium, but the incfii plasmid was lost from the e. coli dh alpha transformant after day . the in vivo loss of the mcr- gene has already been detected for other strains (mourand et al., ) . interestingly, the loss of the mcr- gene was not accompanied by a loss of resistance to colistin for - m, suggesting that the previously described deletion in the pmrb protein, or other unnoticed mutations, probably played a role in colistin resistance. for e. coli - m, the isolates lacking mcr- were no longer resistant to colistin, trimethoprim, sulfamethoxazole, and ampicillin, suggesting that these resistance genes may be present on the same genetic element, but further analysis is needed to confirm this hypothesis, as the genes were present on contigs different from the mcr- contig. the loss of the colistin resistance 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e. coli strains through resapath network. the authors indicate that there are no conflicts of interest. the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. key: cord- -l zqci authors: holschbach, chelsea l.; peek, simon f. title: salmonella in dairy cattle date: - - journal: veterinary clinics of north america: food animal practice doi: . /j.cvfa. . . sha: doc_id: cord_uid: l zqci as an infectious, contagious pathogen, salmonella is probably rivaled by only bovine viral diarrhea virus in its ability to cause clinical disease, such as enteritis, septicemia, pneumonia, and reproductive losses. the increasing prevalence of salmonella, particularly salmonella dublin, on dairies presents new challenges to producers and veterinarians. no current discussion of bovine salmonellosis is complete without acknowledging the increasing public health concern. increasing antimicrobial resistance among enteric pathogens brings the use of antimicrobials by veterinarians and producers under ever stricter scrutiny. this article provides a comprehensive review of salmonella etiology, prevalence, pathogenesis, diagnostics, treatment, and control. facet to salmonellosis on many modern dairies. the ability to establish lifelong infection, characterized by an asymptomatic carrier status, with intermittent periods of bacteremia and intermittent shedding, challenges control of this serotype. enteric infection with other non-host-adapted serotypes, particularly in calves, can also be associated with true bacteremia, sepsis, and high mortality rates. no current discussion of bovine salmonellosis could be complete without acknowledging the increasing public health concern regarding its relevance as an important zoonosis, the risk that contaminated dairy and dairy beef products can pose to human health, and, just as important, the reality that increasing antimicrobial resistance among zoonotic enteric pathogens such as salmonella brings the use of antimicrobials by veterinarians and producers under ever stricter scrutiny. salmonella is a genus of gram-negative, facultative anaerobic bacteria that belong to the family of enterobacteriaceae. there are recognized species within the genus: s enterica and salmonella bongori. s enterica can be further divided into subspecies, s enterica subspecies enterica being the most relevant in dairy cattle. more than serovars (serotypes), differentiated by their antigenic composition, have been identified. serovars are based on the somatic (o), flagellar (h), and capsular (vi) antigens. most human and veterinary diagnostic laboratories have phenotypically divided salmonella isolates into serogroups based on detection of the o lipopolysaccharide and h flagellar antigens, historically by agglutination methods. , although these traditional serotyping techniques have formed the basis of human and veterinary diagnostic practice for salmonellosis for several decades, they are labor intensive and time consuming, typically taking at least hours. with the advent of more advanced molecular diagnostic methods, genetic approaches to serotyping are beginning to supercede traditional tests. in general, these methods use of types of targets for serotype determination, the first are indirect targets, which use random surrogate genomic markers known to be associated with certain serotypes, and the second method uses direct targets requiring the use of highly specific genetic determinants of a particular serotype. the latter typically involve the rfb gene cluster responsible for o somatic group antigen synthesis and the flic and flib genes encoding the flagellar antigens of salmonella. genomic sequencing is becoming increasingly common for the identification and serotyping of salmonella isolates. , the hope is that, with diminishing costs and continued refinement, more rapid, accurate genoserotyping will improve diagnostic and surveillance efforts for both public health and veterinary purposes. most commonly, clinical bovine isolates have been divided by their o antigens, and serovars are further grouped into serogroups assigned to an early letter of the alphabet (eg, a, b, c, d, and e). by current convention, salmonella isolates are referred to by their serovar/serogroup classification (eg, s enterica subspecies enterica serovar typhimurium, is abbreviated to salmonella typhimurium). despite the diversity of serovars, relatively few are of clinical importance among cattle. the majority of cattle isolates are salmonella of types b, c, and e, which are non-host specific, or salmonella dublin (type d), which is the host-adapted serovar in cattle. the isolation of salmonella from the feces of dairy cows or calves as well as the environment on dairy farms is increasingly common. as part of the united states department of agriculture's national animal health monitoring system (nahms) dairy holschbach & peek study, fecal samples were collected from approximately healthy cows on each of dairy operations across states. forty percent of the dairy operations had at least cow that was salmonella positive on fecal culture. of the roughly healthy cows sampled, % were fecal culture positive. compared with the dairy nahms study, the percentage of salmonella-positive operations had doubled and the percentage of positive cows had more than doubled. for the study, when environmental sampling was performed in conjunction with individual cow sampling, the number of dairies with a positive salmonella culture increased to nearly %. within the study, the most frequently isolated salmonella serotypes included salmonella cerro, salmonella kentucky, salmonella montevideo, and salmonella muenster. these serotypes fall within groups k, c , c , and e, respectively. in a comprehensive study of more than dairy herds in the northeastern united states in , fecal samples were collected from female dairy cattle for salmonella culture based on a suspicion of clinical disease. salmonella was found in % of the dairy herds monitored for approximately year over the course of the study. the herd-level incidence rate was approximately positive herds per herdyears; however, just % of the positive study herds accounted for more than % of the clinical salmonella cases. the predominate serotype identified was salmonella newport, accounting for % of the cases, followed by salmonella typhimurium, accounting for nearly % of cases. clustering of disease among herds was consistent with another us prevalence study that found that % of the enrolled dairy farms accounted for more than % of the salmonella-positive fecal and environmental samples. in this study, sampling of conventional and organic herds on occasions over a period of year resulted in detection at least salmonella-positive fecal sample on more than % of farms ( / ). serogroup e was the most commonly identified serogroup in fecal samples, although serogroup b was the most common isolate across farms, with % of fecal-positive farms having at least serogroup b isolate. data from a more recent study demonstrated that of the nearly salmonella isolates identified at the national veterinary services laboratory from clinical and nonclinical case submissions, the most common serotype was salmonella dublin ( %) followed by salmonella cerro ( %) and salmonella typhimurium ( %). a retrospective study of s enterica isolates submitted to the wisconsin veterinary diagnostic laboratory from to parallels the findings from the national veterinary services laboratory. of the nearly isolates identified, salmonella dublin was the most prevalent serotype identified, accounting for a total of isolates ( % of total). along with dublin, salmonella cerro ( %), newport ( %), montevideo ( %), kentucky ( %), and typhimurium ( %) comprised the top most commonly isolated sertotypes. the emergence of salmonella dublin as one of the most commonly isolated serotypes is of major concern for the dairy industry. as the host-adapted strain of salmonella in cattle, animals infected with salmonella dublin can become chronic, subclinical carriers that have the potential to shed large numbers of organisms into the environment. these carriers also play an important role in maintaining infection within a herd by shedding not only in feces, but also in milk and colostrum. salmonella infections are well-known for their association with clinical signs of enterocolitis, septicemia, and abortion in dairy cattle. pneumonia is an increasingly common manifestation of salmonella dublin infection in calves , and worth bearing in mind when dealing with mild, moderate, or severe respiratory disease on heifer rearing facilities. whether or not this merely represents hematogenous localization of the salmonella in dairy cattle organism to the lungs in much the same way that is seen with septic arthritis, for example, or a more specific organ tropism for the lungs by this serovar is uncertain. however, personal observations by one of the authors and many others suggest that this particular clinical manifestation of salmonella dublin infection is increasingly common during the late nursing and postweaning period. salmonella infection is most commonly transmitted by fecal-oral contamination from other livestock, rodents, birds, or by feeding contaminated protein source animal byproducts. given the increased frequency with which the organism can be isolated on dairy farms, from both symptomatic and asymptomatic cattle, it is reasonable to assume that fecal-oral spread from other cattle is the most common means of spread on modern dairies. older literature establishing that aerosol transmission was possible in closely confined, penned calves would also seem to be currently relevant with respect to the spread of certain salmonella serotypes, especially salmonella dublin, on endemic heifer rearing facilities. , in both calves and adults, those factors that determine pathogenicity and whether or not clinical disease is seen include virulence of the serotype, dose of inoculum, degree of immunity (passive or adaptive) or previous exposure of host to the serotype, and other stressors currently affecting the host. the organism will less frequently penetrate ocular or nasal mucous membranes. the most detailed studies of the pathogenesis of bovine salmonellosis infection come from the literature describing enteric infection via the oral route, mainly in calves. [ ] [ ] [ ] once ingested, salmonella attaches to mucosal cells and is capable of destroying enterocytes. attachment is increased if gastrointestinal stasis is present or the normal flora has been disturbed or is not yet established, as is the case in neonates. the organism penetrates through the enterocytes to the lamina propria of the distal small intestine and colon, where they stimulate an inflammatory response or are engulfed by macrophages and neutrophils. once salmonellae have gained entry to mononuclear phagocytes, they can be rapidly disseminated throughout the body. salmonellae have a predilection for lymphoid tissues, invading through m-cells, and are found in the highest numbers in the peyer patches and mesenteric lymph nodes. from here, the organism often enters the lymphatics and may eventually lead to bacteremia. , experimental studies have also shown that oral exposure can lead to infection and systemic dissemination via pharyngeal lymphoid tissue (tonsils) without the need for true enteric infection. salmonellae are capable of surviving and multiplying in numerous host tissues, often as facultative intracellular bacteria in macrophages and reticuloendothelial cells. these characteristics guard them against the hosts' normal defense mechanisms and potentially facilitate true bacteremia. the virulence mechanisms of salmonellae are, therefore, composed of their ability to invade the intestinal mucosa, locate to and multiply within the lymphoid tissues, and to evade host defense mechanisms. enterocolitis caused by salmonella spp. is due to inflammation with subsequent maldigestion and malabsorption, and to a lesser extent from secretory mechanisms. , inflammation in the colon leads to the commonly observed fresh blood in the feces of both adults and calves. the diarrhea caused by salmonella spp. is principally mediated by the host inflammatory reaction to the infection. to establish infection, enteropathogens such as salmonella must first be able to overcome those host factors that resist colonization of the gut, principle among these being a fairly dense gut microbiota, which secrete a variety of bacteriocins, antibiotics, and colicins that hinder enteropathogen growth. there is increasing evidence that many enteropathogens, including salmonellae, are not able to colonize the gut in the face of a normal microbiota and hence factors that negatively influence this key component of resistance are important in the predisposition to enteric disease. once salmonella density reaches a critical threshold (about colony-forming units per gram in the case of salmonella typhimurium in mice), then a sufficient number of organisms can invade the gut epithelium by first docking with and then invading the epithelial cells. at a molecular level, salmonella typhimurium does this by specific bacterial adhesins for attachment and then a secretion system that injects a cocktail of bacterial toxins (the type iii secretion system) that enables the bacterium to reach the lamina propria. damaged gut cells are expelled into the lumen, as part of the host defense system, giving rise to some of the clinical signs of salmonellosis and a profound inflammatory response is initiated via interleukin- within to hours after infection. there are molecular reasons that underscore the clinical observation that differences in pathogenicity between serotypes exist. some strains of salmonella dublin and salmonella typhimurium, for example, have a virulence plasmid (carrying the spv gene) that facilitates survival of the organism within phagocytes, partly perhaps explaining the increased association of these serotypes with clinical disease in calves and adults. the ease with which genes can be transferred between salmonella and other members of the enterobacteriaceae also provides a rational explanation for the transfer of antimicrobial resistance. precise and eloquent experimental data on the mechanisms by which salmonella infection can lead to reproductive loss and abortion are hard to find. clinically, abortions are most common when serotypes b, c, or d are involved and it makes intuitive sense that abortion in cattle infected with salmonella spp. could arise through several different mechanisms. septicemia could lead to seeding of the fetus and uterus, causing fetal infection and death. , the fact that diagnostic post mortem investigations of aborted fetuses can often recover the organism from fetal samples supports this possibility. endotoxemia leading to inflammatory mediator release might also cause luteolysis secondary to prostaglandin release. high fevers or hyperthermia could also play a role in prostaglandin release or cause abortion through more direct fetal injury. cows may abort at any stage of gestation, but expulsion of the fetus is most common at to months of gestation. , a definitive diagnosis of salmonella infection in the live animal involves detection of the organism, most commonly by aerobic culture. although a clinical history of febrile illness accompanied by hemorrhagic enteritis and anorexia may be suggestive in either calves or adults, there is a sufficient differential diagnosis list in both age groups that diagnostic sampling must be performed. when reproductive losses are encountered in pregnant cattle, unless there are concurrent cases of bloody diarrhea, the clinical signs are even less definitive for salmonellosis and the differential list even longer. for hemorrhagic enteritis in adults, the differential list principally includes winter dysentery and bovine viral diarrhea virus infection; in calves, depending on age, such a presentation merits consideration of several viral (rotavirus, coronavirus), protozoal (cryptosporidium, eimeria), and bacterial causes (escherichia coli, clostridium perfringens). however, one should not rely on the presence of blood in the stool; many cases of enteric salmonellosis present without this clinical finding. remarkable variation in clinical severity will occur based on serovar virulence, host immunologic status, and inoculating dose. in calves, death may occur owing to septicemia before diarrhea salmonella in dairy cattle becomes obvious or a significant clinical abnormality. in large free stall dairies, it is increasingly common to encounter salmonella infection as an endemic challenge with clinical presentations that are highly variable, ranging from the classic textbook description of reproductive losses and enteric disease in adult cattle through to lower impact problems with fevers of unknown origin, little to no diarrhea, and only modest consequences in terms of appetite and milk yield reduction. because salmonella organisms are easily and rapidly out competed by other fecal gram negatives, the majority of diagnostic laboratories use enrichment media such as tetrathionate or selenite broth to improve the chances of salmonella growth and then plate these enriched samples onto selective media such as brilliant green or xylose lysine deso-oxycholate agar. veterinarians working in the field are advised to contact their local diagnostic laboratory for assistance with sample handling, processing, and submission before investigating either individual or group problems with enteric disease suspicious for salmonella infection. it is frequently worthwhile to place samples directly into enrichment media before submission to improve the chances of positive culture and to keep samples chilled until they arrive at the diagnostic laboratory. disadvantages of fecal culture include the fact that shedding can be sporadic, even in true infections (certainly when one considers the sensitivity of bacterial culture) and that, in the face of an ongoing outbreak, one can occasionally encounter clinically normal calves and adults who shed the organism but never develop any clinical signs. the latter situation may still provide useful information, however, both from the perspective of deciding which animals merit treatment but also from the broader standpoint of identifying an enteric pathogen that should never be trivialized or considered a commensal. however, the general pattern is that subclinically or persistently infected cattle shed low numbers of organisms, whereas clinically ill or acutely infected animals may excrete higher numbers in feces. when the clinical suspicion of salmonella is high, a single negative culture is not sufficient to rule out infection. as mentioned, fecal samples should be submitted to qualified diagnostic laboratories that are equipped to culture enteric pathogens and with careful attention to sample handling. although culturing of individual cow fecal samples is the most common method used to assess individual and herd salmonella status, it can be expensive and time consuming, especially in larger herds. in a study comparing individual, pooled, and composite fecal samples, it was found that composite fecal sampling was more sensitive at the sample level than the other methods, primarily because of the increased number of cattle sampled indirectly through this method. hence, if one is merely trying to obtain a yes or no answer or identify and track specific serovars, or antimicrobial susceptibility patterns over time, composite fecal samples are typically collected from areas on dairy operations where manure accumulates from a majority of adult animals, such as holding pens, alleyways, and lagoons. newer techniques for diagnosing salmonella are based on detection of genetic material from the bacteria, that is, polymerase chain reaction (pcr) techniques. , these techniques are generally thought to be more sensitive than culture, but have the disadvantage that subsequent serotyping is not always possible. both species-specific (s enterica) and individual serotype pcr tests are available at some, but not all, veterinary diagnostic laboratories within the united states. there are main pcr methods: the traditional pcr and the real-time pcr. in the traditional pcr method, the test result is qualitative (yes or no). in real-time pcr, a threshold cycle (ct-value) gives a quantitative value of dna in the sample; the ct-value is inversely correlated with the starting concentration of the target dna; hence, the lower the ct number the more salmonella dna there will be in the sample. at the current point in time, only a few veterinary diagnostic laboratories offer both species-specific and serotype (usually salmonella dublin) assays for use with biological samples such as feces, milk, or tracheal and bronchoalveolar lavage fluid. the advantage is a quicker turnaround time and the potential for greater sensitivity, although parallel cultures are still necessary for in vitro antibiograms to be performed to aid treatment decisions (dr keith poulsen, wisconsin state veterinary diagnostic laboratory, personal communication, ). it is possible that, in the near future, pcr assays may become used for environmental samples although these can contain so many potential pcr inhibitors and out-competing organisms that sensitivity and specificity may be lost. the use of pcr methodology to investigate contamination of milk is also of increasing relevance, potentially for veterinarians, but also from the public health perspective. certain serovars, notoriously salmonella dublin, but also to include salmonella typhimurium and newport, can be found in the milk or colostrum of infected lactating animals. although conventional pasteurization should kill the organism, there is an understandable desire for food safety reasons to use highly sensitive methods to detect the organism after harvest. although fecal culture remains the gold standard at most laboratories, blood culture, a culture of transtracheal wash or bronchoalveolar lavage fluid, and joint fluid may all be useful choices for individuals experiencing bacteremic salmonellosis. the propensity for bacteremia in neonatal calves with salmonellosis makes aseptically obtained aerobic blood cultures a particularly useful diagnostic sample to consider in valuable animals. , culture of these nonfecal samples is far less likely to be diagnostically valuable in adults, although pcr methods on such samples may potentially improve sensitivity in the future. although gross post mortem findings of severe, diffuse, fibrinonecrotic ileotyphlocolitis with watery, often bloody content are highly suggestive, they are neither consistent enough or definitive for enteric salmonella infection in calves or adults. however, in both calves and adults, necropsy material can provide excellent diagnostic material for the definitive diagnosis of salmonella infection. in all age groups, it is advised to obtain numerous samples from the gastrointestinal tract (ileum, cecum, colon), mesenteric lymph node, and gall bladder (bile is a particularly useful sample), as well as lung tissue, especially when consideration of salmonella dublin is warranted, as increasingly is the case. because veterinarians are rarely only interested in the diagnosis of salmonella infection during a field necropsy, one may need to take multiple samples from such sites and handle the samples specifically as described to enhance the chances of a positive salmonella culture. culture remains the most common method used by most diagnostic laboratories to confirm salmonella infection in post mortem samples. samples from abortion cases that may have been caused by salmonella, should include fluid or tissue from both the dam and the fetus. most salmonella-associated abortions are in the last trimester so there will be a fetus to work with, preferably relatively fresh depending on the delay before the fetus is discovered. samples from the dam might include milk or colostrum, serum, and feces. feces and milk can be screened via culture or pcr, whereas the serum sample can be used for salmonella dublin serology (described elsewhere in this article). providing the fetus is not severely autolyzed, heart blood, abomasal contents, and intestinal or biliary samples might be useful but diagnostically veterinarians are all too commonly challenged by the "freshness" of an abortus. as is true of many enteritis investigations, with abortion cases veterinarians are typically attempting to submit samples that might reveal one of many possible infectious etiologies and it may be simpler to submit the entire fetus if this can be done in a timely manner. environmental sampling on dairy farms and heifer rearing facilities has largely been a research tool rather than a clinically applicable procedure. however, quite a lot of information has been learned regarding areas of large free stall facilities where positive salmonella cultures can often be repeatedly obtained either in herds with or without known clinical disease. , not surprisingly, areas of high traffic use and density and where sick cows and cows soon to calve are located are frequently discovered to yield positive cultures. just as was discussed under individual cow fecal sampling, veterinarians are advised to seek the input of the laboratory to which they are going to submit samples before obtaining on-farm environmental specimens. the use of buffered peptone water or more specific enrichment broths before submission may improve chances of salmonella being isolated from heavily contaminated samples. drag swabs, milk filters, and even absorbent socks worn over shoes, as have been used for environmental sampling in poultry houses, can be used. proof of current infection with salmonella dublin can be achieved via conventional culture with serotyping or pcr methodologies if available. , in addition, both in the united states and several countries in europe it is also currently possible to use an enzymelinked immunosorbent assay (elisa) to measure the level of antibodies directed against o-antigens from salmonella dublin in blood and milk. in this way, one can measure the humoral immune response as an indicator of current or previous infection. , some laboratories report the elisa result as a semiquantitative percentage value, giving an optical density reading referable to a standard set of controls. in addition, elisa tests can also be used for individual or bulk tank milk sample screening, and have come to be used quite extensively in countries such as denmark, where active surveillance programs for this serovar are in effect. , sensitivity for the serum elisa is considerably higher than fecal culture for the identification of salmonella dublin infected cattle, and as a diagnostic test the serum elisa is reported to perform best when used in animals between and months of age (box ). treatment fluid therapy is the mainstay of treatment for cattle with enteric salmonellosis. the type of fluid and route of administration is based on the severity of clinical signs box salmonella diagnostic testing options individual animal fecal culture using enrichment and selective media. composite fecal sampling. salmonella polymerase chain reaction (feces, milk, tracheal or bronchoalveolar lavage fluid). blood, transtracheal wash, bronchoalveolar lavage, or joint fluid culture when bacteremia is suspected in calves. culture of post mortem samples: gastrointestinal tract, mesenteric lymph node, bile, and lung. salmonella dublin enzyme-linked immunosorbent assay: serum or milk. and the economic value of the animal. in calves with acute, severe diarrhea showing signs of hypovolemic shock, intravenous fluid therapy using a balanced electrolyte solution, such as lactated ringers, is necessary. , in severely depressed or comatose animals, resuscitative fluids, such as hypertonic saline, are indicated. if administered, hypertonic saline, dosed at - ml/kg, should always be followed with isotonic crystalloids or water to replace the "borrowed" water from the intracellular space. dextrose supplementation can be a critical part of the intravenous fluid therapy plan for calves with salmonellosis, not only because of poor feed intake, but because of the increased risk of hypoglycemia that may accompany septicemia. calves that are ambulatory, have a suckle, and are only moderately dehydrated can often be managed with oral fluids. calves and even adult cattle can develop severe metabolic acidosis with peracute salmonella infections and intravenous bicarbonate-rich fluids should be considered when profound depression or shocklike signs accompany diarrhea. oral electrolyte solutions have proven to be helpful in correcting mild to moderate dehydration; however, depending on the degree of bowel inflammation, fluid absorption and digestion may be altered. fluid therapy for adult cattle in the field setting can prove to be more challenging owing to the sheer volume of fluid needed in cases of severe dehydration. hypertonic saline followed by at least gallons of oral electrolytes or water, either consumed voluntarily or given by orogastric tube, is a highly efficient method of fluid resuscitation in adult cattle. in valuable calves or adults, colloids (plasma or hetastarch) are often indicated as a result of hypoproteinemia secondary to albumin loss from the gastrointestinal tract. synthetic colloids, such as hetastarch, are a more reasonably priced option, but only augment colloidal pressure. plasma has the added benefit of immunoglobulins and acute phase proteins, which provide therapeutic benefits in septic or inflammatory conditions. antimicrobial therapy for the treatment of salmonellosis was, is, and probably always will be, controversial. of utmost concern is the potential for the creation of antibiotic-resistant strains of salmonella that may present a risk to humans or animals in the future. although antimicrobial therapy may aid in clinical recovery, it has also been criticized as failing to limit fecal shedding or to impart a positive effect on the duration of fecal shedding. in truth, this criticism is largely extrapolated from research in other species. in cattle, the effect of prior antibiotic use on fecal shedding may be age variable, with research identifying that the risk of fecal shedding after antibiotic treatment is greater for adults and heifers than in calves. however, the risk of true bacteremia in calves with enteric salmonellosis is substantial, justifying the use of antimicrobials in patients of this age. bacteremic spread of the organism can result in concurrent disease in multiple organs, such as pneumonia, arthritis, and meningitis. the presence of these clinical infections should always merit antimicrobial administration. the comparative risks for such systemic complications in adults are less than in calves, making the routine use of antimicrobials in mature animals less justifiable. if possible, antimicrobial selection should be based on culture and susceptibility of the salmonella isolate. the dilemma faced by practitioners is frequently that real-time decisions regarding antimicrobial use and selection have to be made in advance of any definitive microbiologic data. some guidelines regarding salmonella susceptibility can be provided, however. according to the nahms study, isolates were found to be most resistant to tetracycline, streptomycin, ampicillin, and ceftiofur, but were frequently sensitive to aminoglycosides, fluoroquinolones, and trimethoprim-sulfas. to the us readership, these lists will not provide much comfort because of restrictions on antimicrobial use under the current animal medicinal drug use clarification act. fluoroquinolones and certain sulfonamides may not be used extra-label in the united states. additionally, there is a voluntary ban on the use of aminoglycosides, such as gentamicin and amikacin, in food-producing animals because of long-term tissue residues. as of , the extra-label use of ceftiofur in regard to dose, route, and frequency of administration is also prohibited. owing to the facultative intracellular nature of the organism, it is also worth bearing in mind that antimicrobial penetration into the cell can be limited, even for antimicrobials that show in vitro efficacy. when chosen, antibiotic therapy should be continued for at least to days in cases of acute or peracute salmonellosis. appropriate withdrawal times should be observed for all antimicrobial usage and animal medicinal drug use clarification act guidelines followed at all times. for questions regarding extended withdrawal times and extralabel use of antimicrobials, us readers are advised to contact the food animal residue avoidance database. in addition to crystalloid fluid therapy, colloid administration when indicated by hypoproteinemia, and responsible, legal, and signalment appropriate selection of antibiotics, the third and final component of therapy for salmonellosis is antiinflammatory use. the inflammatory cascade triggered by local or systemic infection with salmonella is a critical component of the pathogenesis of this organism and culminates in many of the clinical signs observed. direct endotoxin-mediated effects alongside the host systemic inflammatory response are major components of many calf and adult salmonella infections that can be mitigated, at least in part, by the use of nonsteroidal antiinflammatory drugs. cattle may be dosed with flunixin meglumine at . mg/kg of body weight intravenously every hours and then tapered to . mg/ kg every hours, or the medication discontinued after the patient stabilizes. label use of flunixin meglumine includes dosages of up to . mg/kg in the united states. prolonged administration of nonsteroidal antiinflammatory drugs, particularly at the higher dose or in the face of dehydration, can lead to abomasal ulceration and renal papillary necrosis. , , in rare circumstances, some clinicians elect to administer "shock" doses of corticosteroids, but this measure would be uncommon in either general or referral practice. soluble prednisolone sodium succinate would be the preferred agent in such circumstances. from both the literature and personal experience, it seems that not only are herd epidemics becoming more common, but perhaps more worryingly the disease has become endemic on an increasing number of facilities. endemicity is obviously problematic with any serovar, but is inevitable when the herd prevalence of salmonella dublin infection increases. frequently, the disease becomes a cyclical problem responsible for a spectrum of illness that varies from the more classic presentations described through to milder illness perhaps characterized by fever, looser than normal stool, and mild production loss. depending on the interaction of general cow health, other concurrent stressors, climatologic stress, and the level of fecal-oral challenge at any one time, adult cows may or may not become clinically ill. transition cow management becomes an important factor in whether or not new infections are acquired and subsequently result in clinical illness in the late dry and early lactation period, a time when cattle may be at their most susceptible to infectious disease. as with any fecally-orally spread organism, control strategies are broadly speaking simple to describe, but not necessarily so easy to put into place for many dairies. larger herd size, crowded husbandry, and free stall housing all contribute to an increased propensity for exposure to contaminated manure, and although purchased feedstuffs are still occasionally incriminated as a means by which new salmonella infections are introduced onto farms, as are rodent and bird populations, the major source of infection are other cattle shedding the organism in their feces. the high likelihood of feces being contaminated with salmonella organisms on many diaries should mitigate against the spreading of manure on fields that are to be used for forages, or common use equipment for manure handling and feed distribution. evidence suggests that heating of manure to greater than c for more than days, alongside aeration of composted manure using straw, markedly and significantly reduces the number of salmonella organisms, although it is uncertain how practical this information is to larger dairies with modern large-volume manure handling systems. peculiarly, and perhaps rather worryingly, study looking at risk factors for increased antimicrobial resistance among salmonella isolates on dairy farms identified the use of composted manure for bedding as a significant problem. the most directly applicable research regarding modern manure handling systems and survival of salmonella organisms under natural rather than laboratory conditions demonstrated that a multiple-drug-resistant strain of salmonella newport survived for less than hours in a compost pile at c, but would survive for more than months and more than months in an effluent lagoon and field soil, respectively. once salmonellosis has been confirmed in adult cattle, there are a number of further investigative and control measures that may be implemented. these measures do not differ according to serotype, but there are some specific challenges concerning the host adapted serovar salmonella dublin that will be discussed in a later section. it is prudent to consider the possible source(s) of the infection. although commodities, especially protein feed sources, and wild bird and rodent populations have been incriminated in many texts over the years, it seems quite uncommon these days for a single point source event to have introduced the infection onto a dairy de novo. environmental sampling of feed, water, and storage facilities can be helpful in identifying contamination in this regard, but if, as is commonly the case on larger dairies, management continues to purchase replacement animals or expand from other herds, it seems inevitable from prevalence data that the infection will be introduced via infected cattle and their feces. in all probability, many "new" outbreaks are likely surges in clinical disease and new infections in a herd where the infection already existed but hitherto had remained subclinical. factors in transition cow management that reduce immunologic competence or increase exposure risk, are likely to contribute to the onset of clinical disease in such circumstances. the isolation of affected animals and strict attention to hygiene are pieces of advice routinely given but difficult to implement on large dairies. the numbers of affected animals can be overwhelming and lactating cows have to be milked at least twice a day, requiring them to walk and congregate in frequently trafficked areas and holding pens for the parlor into which they release enormous numbers of organism whenever they defecate. avoidance of common use equipment for manure handling and feed distribution have already been mentioned, but should be in place on well-managed dairies anyway. sick, transition, and maternity animals should never be housed together, but unfortunately are for convenience on many occasions; this condition merely ensures exposure of the most susceptible animals to those most likely to be contagious. cleaning and disinfection of the environment are also important, but again somewhat intimidating in the context of a larger dairy. proper cleaning and disinfection of the environment and equipment after a salmonella outbreak can, however, be critically important in decreasing the risk of disease transmission to both cattle and humans. cleaning is defined as the removal of all visible debris and is arguably the most important step in decontamination of animal environments. even the best disinfectants will be minimally effective when used in the presence of organic matter, such as feces and bedding material. not only does cleaning remove the physical barrier between disinfectants and the organism, but it also removes a majority of the organisms so that fewer need to be killed by the disinfectants. this is especially helpful with fecally-orally spread infections like salmonella. where the infectious dose is relatively high (often in the order of - organisms , ). livestock trailers, maternity and calf pens, feeding equipment, and other areas suspect of being contaminated with salmonella should be the main focus for cleaning and disinfection. although high-power washing can be quite helpful in removing organic debris, its use is not recommended because of the risk of cross-contamination of the environment, and splashing and aerosolization of contaminated material, which can lead to human and animal infection. , power washing also fails to remove biofilm, which is an essential and vital component to proper cleaning. in place of power washing, hand-held foamers can be used to apply alkaline detergent and acid rinses for cleaning. the wisconsin veterinary diagnostic laboratory has formulated a cleaning and disinfecting protocol specifically for premises with confirmed salmonella, which can be found at www.wvdl.wisc.edu. a recent paper examining disinfection efficacy against several common bacterial pathogens in a large animal hospital environment showed an approximately % reduction in colony-forming units per milliliter of s enterica when either an accelerated hydrogen peroxide or peroxy monosulfate disinfectant product was used via a mist application technique, provided adequate cleaning was performed first. as with antimicrobial drugs, disinfectants have a spectrum of activity that can be highly variable between disinfectant classes. examples of disinfectants commonly used in veterinary medicine include bleach (sodium hypochlorite), quaternary ammonium, phenols, and peroxides. bleach is rapidly inactivated by organic debris, but has a broad spectrum of activity. quaternary ammonium has moderate activity in organic debris and is effective against gram-negative bacteria, such as salmonella. the principle advantage of phenols is better activity in organic debris. peroxides are increasingly used for environmental disinfection, footbaths, and environmental misting and fogging, , and are perceived as being more environmentally friendly than chemicals such as phenols and bleach. chlorine dioxide is a powerful oxidant as well as disinfectant, and it can be used to remove and prevent biofilm formation. its use in the dairy industry is becoming more common. current recommendations from the wisconsin state veterinary diagnostic laboratory are for its use in solution at ppm. although rarely done on farm, the effectiveness of environmental cleaning and subsequent disinfection for salmonella control can be assessed by postdisinfection sampling. ongoing efforts at animal isolation and environmental hygiene will be important because shedding of salmonella will continue for many weeks after the initial cases have seemingly resolved. with respect to control, shedding continues periodically for the life of the animal in the case of salmonella dublin. once salmonella has been identified on a farm, veterinarians and management should increase awareness of the public health risk among workers and revisit personal hygiene, protective clothing, and appropriate disinfectant footbath use for employees. if time and labor resources are limited, then concentrating cleaning and disinfection efforts toward highrisk groups (transition cows, maternity pen) and high use traffic areas may be a reasonable compromise. inevitably, the identification of salmonella infection in adult cows or calves will lead to a conversation about vaccine use as a preventative strategy. many farms have at one time or another tried a commercially available or autogenous salmonella vaccine as an adjunct component of control. the safety and efficacy of autogenous products are questioned by many academicians, but individual experiences are sometimes compelling, at least in the short term in the face of an outbreak. as with other infectious contagious diseases such as infectious bovine keratoconjunctivitis, when any vaccine product is used during an outbreak it is impossible to know whether improvement was associated with vaccine use or natural immunologic exposure and protective antibody responses. the most commonly used product in the united states currently for the control of salmonellosis in adults is a siderophore receptor/porin vaccine derived from salmonella newport (salmonella newport bacterial extract, zoetis animal health, parsippany, nj). it is administered to dry cows as an initial injection series and boostered annually. it can, however, be given at any stage of lactation or to heifers. it will not prevent infection, but has been associated with an amelioration in disease severity. it does result in demonstrable antibody levels in colostrum when administered twice during the dry period, although the protective effect of these antibodies against challenge postnatally in calves at this time is unknown. the efficacy of other gram-negative core vaccines to prevent or decrease salmonella disease, such as the j product (enviracor, zoetis animal health) or endovac-bovi (immvac, columbia, mo), which are specifically marketed for protection against coliform mastitis, is highly debatable. the maintenance of good general health, excellent hygiene, and particular attention to the well-being of late gestation and early lactation animals are all critical components of salmonella control. a closed herd is ideal, but rarely achieved, making exposure to the organism inevitable on most dairies. prompt diagnosis, treatment, and isolation are important during an outbreak in adult cattle and environmental sampling to include bulk tank milk and high-risk housing areas should now be considered a routine part of disease prevention and surveillance. many of the important components of adult cow control programs mentioned in the previous section overlap with specific measures recommended for calves. an article in a previous volume of this journal provided an excellent review of control measures specific to calves. as in adult herds, endemic disease is increasingly common among calves. commercial heifer rearing facilities that manage preweaned calves from as young as a few hours of age onward, sourced and transported from multiple farms of origin, create a high-risk environment for the acquisition and spread of neonatal salmonellosis. adequate passive transfer, although imperative for rearing healthy calves, is not an absolute guarantee for protection from salmonella infection. fecal-oral transmission is a prime means of spread for enteric and septicemic salmonella infection in calves, but one must be mindful of the risk posed by other secretions such as colostrum, unpasteurized milk, and respiratory secretions, especially in the case of salmonella dublin. hygiene, isolation, and treatment principles for calves, calf housing, and personnel working with calves are very similar to those discussed in the adult section. special consideration should be given to fecal contamination of milk, milk replacer, colostrum, feeding equipment, and starter rations as a means of cross-infection. periodic environmental sampling of equipment such as nipple feeders, buckets, and housing can be valuable tools to trouble shoot outbreaks and improve quality control and prevention efforts. milk and colostrum are effective enrichment media for salmonella, so sampling these sources should be done "as fed" rather than as initially mixed or prepared. the increased availability of colostrum pasteurizers has added a very helpful tool to control not only salmonella dublin, but also other serotypes that can also be found in colostrum. maternity area hygiene and management are extremely important salmonella in dairy cattle in the control of neonatal salmonellosis. decreasing the postpartum exposure to the dam reduces the chances of immediate infection. a rather alarming recent publication has identified that true vertical transmission in newborn calves is documented with several serovars common to cattle in the united states. if further studies confirm this finding, it would add yet another serious challenge to the control of salmonellosis in calves. because exposure of calves to salmonella is very likely in the commercial dairy environment, management efforts should be directed toward limiting dose and maximizing health and disease resistance in the young replacement animal population. there are no revelations within this advice, but just as occurs with adult cattle, the degree to which farms are able to dedicate personnel and time may only be prioritized in the midst of, or immediately after, an outbreak of clinical disease. prompt diagnosis, separation, and treatment are important, but group housing of calves can quickly create a "perfect storm" for contagious disease spread. as with adults, vaccination and immunization with modified live or killed (autogenous or commercially available) products is often part of the control and prevention measures instituted. there is very little evidence to support effective control of salmonella infection in calves via passive transfer from immunized dams with any type of vaccine although the siderophore/porin product mentioned in the previous section in adults will stimulate colostral antibody. salmonella is predominantly cleared by cellular immune responses and humoral antibody alone may not provide satisfactory protection. vaccine use in calves is best considered when management efforts at control and prevention have already been put in place, or if these have been implemented but found to make little difference in the pattern or severity of disease. autogenous products derived from a specific serovar isolated from clinical cases must be used very carefully owing to the risk of anaphylactic reactions, and only from reputable biologic manufacturers. similarly, caution is advised regarding modified live vaccine use in calves owing to the potential for adverse reactions. killed vaccines have performed inconsistently in the small number of trials carried out in the past in calves. , comments regarding salmonella dublin control the increasing prevalence of salmonella dublin infection in the us dairy industry , and its unique status as the host adapted serovar of s enterica subspecies enterica in cattle merit some more specific attention. for readers who wish more, and a greater in-depth discussion of this serovar, we refer you to the excellent primary sources and review paper authored by dr liza nielsen from denmark who, together with her international collaborators, has published a great deal of excellent work, particularly as it applies to disease impact as well as control and surveillance strategies. , , , , [ ] [ ] [ ] within the european community, especially within the scandinavian countries, there are currently several active surveillance and certification programs that are designed to control, and potentially eradicate salmonella dublin infection in cattle herds. it is doubtful whether the immediate future holds much promise for such coordinated efforts within the us dairy industry, but there are undoubtedly useful lessons to be learned from experiences in other countries. all of the control measures described in this article for adults and calves can be applied to salmonella dublin infection, just as they can to other serovars. however, the serologic response to salmonella dublin, and the ability to measure that as a potential surrogate marker of the carrier status, opens up possibilities for identification and control. currently within the united states, the serologic test for salmonella dublin is available commercially through the animal health diagnostic center at cornell university and can be applied to either blood (serum) or bulk tank milk samples. it is important to recognize that a single time point positive test result does not confirm the carrier status, but indicates an antibody response owing to previous exposure, current infection, or passively derived antibody in a calf less than months of age. repeated individual animal sampling at specified intervals can be used during surveillance programs to identify animals that are likely to be carriers based on the persistence of an elisa positive result with a high optical density reading. , , using the data generated by nielsen as a guide, the animal health diagnostic center at cornell university categorizes a carrier as any animal that has strong positive serum elisa results over an -month period (dr belinda thompson, personal communication). from the currently available literature it does not seem to be possible to predict or estimate what percentage of infected calves or adults will go on to become true carriers, although the number is probably quite low. in herds classified as being endemic for salmonella dublin in denmark, the seroprevalence is highly variable but may only be at % of the whole herd, with a higher proportion of infection in young stock compared with adults. reinfection of previously infected and seemingly recovered animals also seems to be possible when individuals are followed over long periods of time. some of these subsequent infections may also result in the development of carrier status (dr belinda thompson, personal communication). bulk tank samples can be used for periodic milking herd surveillance, or, if applied to selected milking groups, to identify whether salmonella dublin has been introduced into a herd or is present in a particular population of cattle within the herd. from epidemiologic data, it seems that the risk of becoming a carrier after infection is greater for calves and for adults infected around the time of calving. another study shows that salmonella dublin infection in endemic herds can be reduced when an individual employee was dedicated to colostrum administration to newborn calves and calving cows were moved into a specific maternity pen before calving. a number of epidemiologic investigations in endemic salmonella dublin herds in scandinavia have identified risk factors and important control points for eradication of infection. , [ ] [ ] [ ] [ ] many of the risk factors and management tools demonstrated to improve control of salmonella dublin infection are intuitively sensible and relevant to other salmonella serovars. improving the likelihood of control is associated with avoiding cattle purchases from other farms and ensuring good calving area management and individual calf-rearing practices with solid, not permeable, barriers between calves. aggressive culling programs are not practical in situations where prevalence is high and may only become reasonable once new calf infections are serologically proven to decline to very low, or absent, levels. , it may be difficult for some producers and heifer rearers to instigate all of the management changes and practices that have been successful in european countries, but readers are directed to information available through the animal health diagnostic center at cornell university website for very helpful guidelines concerning control of salmonella dublin. in the united states, there is a commercial live salmonella dublin vaccine (entervene d, boehringer ingelheim vetmedica, st. joseph, mo) that is being used as a component of salmonella dublin control on many farms. the product is administered parenterally to newborn calves to stimulate an immune response before initial exposure to the pathogen. the goal is to prevent the serious health consequences of natural infection as well as the development of the carrier status in what is the most susceptible population of animals within endemic herds. however, when given according to label instructions the product will interfere with serologic testing, giving a false-positive result at up to months of life. furthermore, the product can be associated with fatal anaphylactic reactions in some recipient calves. these reactions seem to be more common in endemic herds than in naïve ones. this product can stimulate colostral antibody production when given to dry cows and was not associated with any adverse reactions when given to late pregnant animals. the vaccinated cohort in this study were from a farm with no clinical history of salmonellosis in recent years. whether this colostral antibody might provide protection against neonatal infection is currently unknown. herd biosecurity? maintain a closed herd. if purchasing cattle, ensure a negative serologic test from individual animals or a negative bulk tank milk test from the herd of origin within the last months. maintain separate maternity and sick cow pens. have separate equipment for feed and manure handling. dedicate personnel to solely work with high-risk or sick cattle versus neonates. salmonellosis not only can cause severe disease in cattle, but also poses a significant zoonotic risk. farm workers, calf handlers, and their families are clearly at risk of becoming infected by salmonella spp. during outbreaks of clinical illness, but the risk of exposure goes far beyond farm workers or veterinarians with direct animal contact during outbreaks of disease. asymptomatic shedding of salmonella, a characteristic of salmonella dublin infection, but also an issue with many other common bovine serovars such as newport and typhimurium, creates risk for people in direct contact with the animal, its feces, or milk. , , however, the majority of human salmonellosis cases do not derive from direct animal contact, but are instead acquired through foodborne exposure. so-called nontyphoidal salmonellosis is one of the leading causes of acute bacterial gastroenteritis in humans in the united states, responsible for an estimated . million cases of illness annually. the predominant risk for zoonotic salmonellosis from cattle lies in exposure to contaminated meat from beef, which would include dairy beef and cull dairy cows, typically via fecal contamination of the carcass at the time of slaughter. [ ] [ ] [ ] although salmonella mastitis is extremely uncommon, shedding of the organism in milk is not, and its presence has been documented in bulk tank milk in several studies. - a positive bulk tank or milk filter sample may represent fecal contamination, true lactational shedding, or a combination of both. conventional pasteurization should kill the organism, provided effective temperature and duration are reached. it is important to consider the diagnostic procedure performed to identify the salmonella in bulk tank or milk filter samples when interpreting these studies. studies using pcr , , rather than culture will detect a greater prevalence of salmonella-contaminated samples because of genomic material from both live and dead organisms in the sample. side-by-side comparisons of conventional culture and pcr using the same samples have been performed and show that approximately one-quarter ( . % vs . %) of those bulk tank samples that are pcr positive for s enterica will be positive by culture. true "dairy" products actually account for only a small percentage of human salmonellosis in the united states, and many of these outbreaks are due to the consumption of raw milk and raw milk products. , bacterial antimicrobial resistance represents an important current and future problem in infectious disease public health. concerns regarding zoonotic salmonella infections have been amplified in recent years by the emergence of multiple drug-resistant strains of several s enterica serovars associated with cattle. [ ] [ ] [ ] [ ] it is generally accepted that antimicrobial-resistant bacteria are produced, maintained, and disseminated as a result of selection pressure introduced by the use of antimicrobial drugs. suspected principal foci of selection pressure include use of antimicrobials for the treatment of humans and in food-producing animals for treatment or prevention of disease and growth promotion. , modern molecular methods combined with other conventional techniques such as pulse field gel electrophoresis can be used to investigate the origins of foodborne human enteric disease and the role of antimicrobial use in cattle with the occurrence of multiple drug-resistant salmonella infection in humans. at this point in time, there are few published studies establishing such links from "farm to fork." a recent extensive systematic literature review of publications on the effect of antimicrobial use in agricultural animals on drug-resistant foodborne salmonellosis in humans from to concluded that, although antibiotic use in cattle increased the likelihood of colonization in the host, there were no studies that traced antimicrobial-resistant salmonella in humans back to the farm. the antimicrobials of choice for treating bacterial gastroenteritis in humans are generally the fluoroquinolone, ciprofloxacin, for adults and the cephalosporin, ceftriaxone, for children. , at issue today is whether the veterinary analogs of these drugs may be responsible for the emergence of antimicrobial resistance in foodborne pathogens like salmonella. the mechanism by which s enterica typically acquires antimicrobial resistance to fluoroquinolones differs quite markedly and, importantly, from that by which resistance to cephalosporins develops. specifically, fluoroquinolone resistance is usually acquired through clonal dissemination of salmonella isolates with mutations in chromosomally encoded resistance genes. cephalosporin resistance usually is obtained via independent acquisition of mobile genetic elements via plasmids and transposons. further work is needed in this area to determine whether there is a connection between veterinary use of ceftiofur and the emergence of ceftriaxone resistance in salmonella spp. although ceftriaxone-resistant salmonella typhimurium has been documented in cattle, other larger studies have demonstrated little to no resistance to this particular third-generation cephalosporin in cattle sourced serovars despite more common resistance to other cephalosporins. , although it is now years old, interested readers are directed to the excellent review of antimicrobial resistant salmonella in dairy cattle by alexander and colleagues. in a more recent publication, a significant decrease was observed in antimicrobial resistance among dairy cattle salmonella isolates in the northeastern united states. many practitioners and diagnostic laboratories will be very familiar with the wide variety of antimicrobial sensitivity patterns demonstrated by different s enterica serovars obtained from individual animal and environmental samples. certain serovars seem to be more commonly associated with greater in vitro resistance than others. the paper by cummings and colleagues demonstrated a decrease in resistance trends between and . it was postulated that this might have been related to an increase in the prevalence of the serovar cerro in fecal samples from their study population. the biggest concern arises with serovars that have historically been more common in dairy cattle and that are associated with human disease outbreaks, such as newport and typhimurium. in particular, several human foodborne outbreaks caused by salmonella typhimurium dt of dairy or beef origin that are characteristically resistant to the antibiotics ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline have been reported. , an interesting and rigorously investigated example of zoonotic multiple drug resistant salmonella from cattle is provided by the wisconsin experience with salmonella heidelberg over the last years. since , the wisconsin state veterinary diagnostic laboratory (wvdl), in conjunction with human and veterinary health organizations throughout wisconsin, have been tracking a multidrug-resistant strain of salmonella heidelberg, a group b serovar (dr keith poulsen, wvdl personal communication). as of november , there were confirmed human infections from different wisconsin counties. upon questioning, more than % of the infected individuals reported purchasing holstein bull calves from livestock dealers or sale barns. during and , the wvdl also isolated several multidrug-resistant salmonella heidelberg isolates from calves located mostly in wisconsin. pulse-field gel electrophoresis and whole genome sequencing of isolates indicated that the human and bovine isolates were very closely related. this strain of salmonella heidelberg is highly pathogenic and multidrug resistant. only antimicrobial drug is an effective treatment option for human cases and no effective, legal (united states) options exist for cattle (dr keith poulsen, wvdl, personal communication). as the application of modern molecular techniques becomes more commonplace, it is probable that diagnostic and surveillance efforts will place food animal species and production methods under greater scrutiny with respect to zoonotic enteric diseases. increased awareness, rigor, and possibly limitations regarding antimicrobial use in food animals should not be surprising outcomes. large animal internal medicine escherichia, shigella, and salmonella methodologies for salmonella enterica subsp. enterica subtyping, gold standards and new methodologies the validation and implications of using whole genome sequencing as a replacement for traditional serotyping for a national salmonella reference laboratory salmonella serotype determination utilizing high throughput genome sequencing data biosynthesis of o-antigens: genes and pathways involved in nucleotide sugar precursor synthesis and o-antigen assembly sequence invariance of the antigen-coding central region of the phase i flagellar filament (flic) gene among strains of salmonella typhimurium the salmonella in silico typing resource (sistr): an open web-accessible tool for rapidly typing and subtyping draft salmonella genome assemblies infectious diseases of the gastrointestinal tract united states department of agriculture (usda). salmonella, listeria and campylobacter on us dairy operations united states department of agriculture (usda). e.coli o :h and salmonella status on dairy farms. fort collins (co): usda-aphis-vs-ceah the incidence of salmonellosis among dairy herds in the northeastern united states prevalence of salmonella spp on conventional and organic dairy farms salmonella serotypes isolated from animals in the united states antimicrobial resistance patterns of bovine salmonella enterica isolates submitted to the wisconsin veterinary diagnostic laboratory: - histopathology case definition of naturally acquired salmonella enterica serovar dublin infection in young holstein cattle in the northeastern united states review of pathogenesis and diagnostic methods of immediate relevance for epidemiology and control of salmonella dublin in cattle transmission of salmonellae among calves penned individually aerosol infection of calves and mice with salmonella typhimurium salmonella in calves cross protective immunity conferred by a dna adenine methylase deficient salmonella eneterica serovar typhimurium vaccine in calves challenged with salmonella serovar newport contribution of salmonella typhimurium virulence factors to diarrheal disease in calves salmonella infections in cattle salmonellosis in calves -the effect of dose rate and other factors on the transmission salmonella typhimurium diarrhea reveals basic principles of enteropathogen infection and disease-promoted dna exchange the roles of inflammation, nutrient availability and the commensal microbiota in enteric pathogen infection an nk cell perforin response elicited via il- controls mucosal inflammation kinetics during salmonella gut infection methods for the cultural isolation of salmonella comparison of individual, pooled, and composite fecal sampling methods for detection of salmonella on u.s. dairy operations development and evaluation of a real time multiplex polymerase chain reaction procedure to clinically type prevalent salmonella enterica serovars validation of a h real time pcr method for screening of salmonella in poultry fecal samples real time pcr method for detection of salmonella spp. in environmental samples recombinant plasmid based quantitative real time pcr analysis of salmonella enterica serotypes and its application to milk samples isolation of salmonella spp. from the environment of dairies without any clinical history of salmonellosis cattle and environmental sample level factors associated with the presence of salmonella in a multi-state study of conventional and organic dairy farms age stratified validation of an indirect salmonella dublin serum elisa for individual diagnosis in cattle humoral antibody responses to experimental and spontaneous salmonella infections in cattle measured by elisa factors associated with variation in bulk tank milk salmonella dublin elisa odc% in dairy herds simulation model estimates of test accuracy and predictive values for the danish salmonella surveillance program in dairy herds disease management of dairy calves and heifers effect of previous antimicrobial treatment on fecal shedding of salmonella enterica subsp. enterica serogroup b in new york dairy herds with recent salmonellosis pathogen reduction in minimally managed composting of bovine manure survival characteristics of salmonella enterica serovar newport in the dairy farm environment farm level associations with the shedding of salmonella and antimicrobial resistant salmonella in us dairy cattle salmonella transmission through splash exposure during a bovine necropsy comparison of disinfectant efficacy when using high-volume directed mist application of accelerated hydrogen peroxide and peroxymonosulfate disinfectants in a large animal hospital passive immunity stimulated by vaccination of dry cows with a salmonella bacterial extract evidence supporting vertical transmission of salmonella in dairy cattle salmonella typhimurium infection in calves: protection and survival of virulent challenge bacteria after immunization with live or inactivated vaccines immunization of calves against salmonellosis effect of prevention of salmonella dublin exposure of calves during a one-year control programme in danish dairy herds dynamic changes in antibody levels as an early warning of salmonella dublin in bovine dairy herds gross margin losses due to salmonella dublin infection in danish dairy cattle herds estimated by simulation modelling salmonella dublin infection in dairy cattle: risk factors for becoming a carrier a questionnaire study of associations between potential risk factors and salmonella status in swedish dairy herds culling decisions of dairy farmers during a -year salmonella control study salmonella dublin infection in young dairy calves: transmission parameters estimated from field data and an sir model nyschap recommendations for the control of salmonella dublin in dairy calf and heifer raising operations characterization of the serologic response induced by vaccination of late gestation cows with a salmonella dublin vaccine the effect of clinical outbreaks of salmonellosis on the prevalence of fecal shedding among dairy cattle in new york human multi-drug resistant salmonella newport infections food related illness and death in the united states animal sources of salmonellosis in humans outbreak of multi-drug-resistant salmonella enterica serotype typhimurium definitive phage type infection linked to commercial ground beef, northeastern united states highly resistant salmonella newport-mdrampc transmitted through the domestic us food supply: a foodnet case -control study of sporadic salmonella newport infections antimicrobial resistance of salmonella enterica isolates from bulk tank milk and milk filters in the united states prevalence of salmonella enterica, listeria monocytogenes and escherichia coli virulence factors in bulk tank milk and in line filters from us dairies prevalence of salmonella enterica in bulk tank milk from us dairies as determined by polymerase chain reaction factors associated with salmonella presence in environmental samples and bulk tank milk from us dairies a survey of foodborne pathogens in bulk tank milk and raw milk consumption among farm families in pennsylvania antimicrobial resistance trends among salmonella isolates obtained from dairy cattle in the northeastern united states prevalence of antimicrobial resistance among salmonella on midwest and northeast dairy farms ceftriaxone-resistant salmonella infection acquired by a child from cattle antimicrobial resistance and serotype prevalence of salmonella isolated from dairy cattle in the southwestern united states antimicrobial resistance of commensal escherichia coli from dairy cattle associated with recent multiresistant salmonellosis outbreaks effects of antimicrobial use in agricultural animals on drug resistant foodborne salmonellosis in humans. a systematic literature review nontyphoidal salmonellosis ceftiofur resistant salmonella strains isolated from dairy farms represent multiple widely distributed subtypes that evolved by independent horizontal gene transfer antimicrobial resistant salmonella in dairy cattle in the united states analysis of antimicrobial resistance genes detected in multi-drug resistant salmonella enterica serovar typhimurium isolated from food animals key: cord- -gh m gi authors: dziąbowska, karolina; czaczyk, elżbieta; nidzworski, dawid title: detection methods of human and animal influenza virus—current trends date: - - journal: biosensors (basel) doi: . /bios sha: doc_id: cord_uid: gh m gi the basic affairs connected to the influenza virus were reviewed in the article, highlighting the newest trends in its diagnostic methods. awareness of the threat of influenza arises from its ability to spread and cause a pandemic. the undiagnosed and untreated viral infection can have a fatal effect on humans. thus, the early detection seems pivotal for an accurate treatment, when vaccines and other contemporary prevention methods are not faultless. public health is being attacked with influenza containing new genes from a genetic assortment between animals and humankind. unfortunately, the population does not have immunity for mutant genes and is attacked in every viral outbreak season. for these reasons, fast and accurate devices are in high demand. as currently used methods like rapid influenza diagnostic tests lack specificity, time and cost-savings, new methods are being developed. in the article, various novel detection methods, such as electrical and optical were compared. different viral elements used as detection targets and analysis parameters, such as sensitivity and specificity, were presented and discussed. from humanity's common illnesses, the most frequent are acute respiratory infections (aris). influenza plays a role of the most serious virus causing aris and is the most often detected in lung infections. other causatives of aris can be rhinovirus, parainfluenza, adenovirus, enterovirus, respiratory syncytial virus (rsv) and others [ , ] . influenza wears the name of 'the mother of all pandemics'. in particular, influenza a type has the ability to cause worldwide epidemics. in the last hundred years, virus attacks were documented four times: the spanish flu (h n ) in , asian flu (h n ) in , hong kong influenza (h n ) in and swine influenza (h n ) in . it seems obvious to expect an influenza pandemic to return [ , ] . the illness affects groups of all ages, and this pattern is not common for most viruses. there is a high possibility for every human to suffer from influenza. groups infected are mostly schoolchildren, the elderly and patients with serious medical conditions. infections of respiratory viruses, mainly influenza, and rsv, occur seasonally in winter months (from december to march) in europe, what is less common in hot climates where rhinovirus is seen in fall and spring, while adenovirus infects all year [ ] . influenza spreads very easily among the population and shows high attack rates. in the us, it is the th highest cause of death, infects around . to . billion of children and . to . billion adults worldwide [ ] , kills . to million people annually with numbers still increasing [ , ] . the influenza virus construction is comparatively simple. it mainly contains -segmented rna and surface proteins with highly immunogenic properties. distinguishable are three types-influenza a, b, and c, all belonging to the orthomyxovirus family. the a-type is mostly responsible for pandemics in the th- st century. two glycoproteins cover the viral surface: haemagglutinin (ha) and neuraminidase (na) in a ratio of four to one [ ] . based on surface composition, distinguishable are h (h -h ) and n (n -n ) subtypes forming potentially combinations [ ] . influenza a infects the human population, birds, pigs, dogs, horses and more [ ] . genetic recombination is possible through the segmented genome. reassortment of genes is highly important in the epidemics. human population does not have the immunity against mutants with new ha and na antigens on the virion surface. there is a possibility of interspecies transmission without genetic reassortment, like in the case of h n virus between swine and humans (and conversely) or h n from poultry to humans. in other cases, rna segment reassortment occurs if at least two influenza viruses infect a single hosts cell [ , ] . animal influenza viruses which occasionally infecting humans are called zoonotic influenza viruses (in direct and indirect contact) [ ] . this high possibility of genetic variation can have subsequently pandemic effects. most of the new influenza viruses are mutants forming from antigenic drift [ ] . the b-type influenza virus has similar biological properties to the a-type one. however, through electron microscopy, they are indistinguishable in size and shape. influenza b infects mainly humans and rarely other species. the antigenic drift occurs less often than in the a-type virus [ , ] . the c-type influenza virus naturally infects humans but is less frequently detected, causes mild pediatric infections and sometimes affects adults [ ] . it differs from a and b types through a shorter genome ( segment less), and its major surface glycoprotein is hemagglutinin-esterase-fusion (hef), functioning as h and n together [ ] . additional minor protein m is categorized as a single-pass integral membrane protein. it plays the role of a proton-selective ion channel, ph sensitive [ ] . recently, new influenza virus genus was isolated from pigs and cattle and specified as d virus. it shows many similarities to c type virus. however, its structural differences make it a danger to public health due to the ability of binding human tracheal epithelia [ ] . some studies have shown that - % of workers exposed to cattle breeding have specific antibodies against influenza d, what means a risk of zoonotic infections. real-time polymerase chain reaction (rt-pcr) assay is believed to be adequate for influenza d virus infection diagnosis [ ] . the influenza virus has a diameter of around nm [ ] . influenza a virus proteins (ha, na, and m ) are localized externally on the surface, more specifically they protrude above the lipid membrane. the infection starts with virus linking to the host respiratory epithelial cells. it recognizes and binds to sialic acid receptors via h proteins. sialic acids are nine-carbon acidic monosaccharides mainly found at the end of many glycoconjugates. the terminal carbon- can bind to carbon- or carbon- of galactose, forming different α-linkages and steric configurations. in human population dominate α- , bonds, while α- , are also common; however, the latter are more common in other species (ducks, birds). so there is a possibility of human infection by avian influenza, but less efficiently [ , ] . the next step is neuraminidase activity. sialic acid is rifted from the cell's surface, what enables the influenza virus release and distribution in the respiratory tract. the na protein plays a role in replication of a and b influenza types. the m protein is essential after cell entry through uncoating of influenza a virus [ ] . influenza incubation lasts to days, then the virus sheds and symptoms appear. viruses circulate in a host for to days but decrease - days after the first symptoms [ ] . viral rna genome is segmented thus recombination between different strains is possible. this process is called antigenic shift; however, it is sporadic and occurs less than once per decade [ ] . as a result, surface glycoproteins undergo some variations (minor changes in amino acid sequence like point mutations in genes). the ability of influenza virus to progressive antigenic change forces updates of vaccines composition [ ] . influenza glycoproteins are an excellent target for virus detection due to many copies of ha (around ) and na (around ) on one viral particle [ , ] . the 'gold star' are commercial ridts showing results in around min. ridts are immunoassays that can identify influenza viral nucleoprotein antigens in respiratory specimens. monoclonal antibodies target viral nucleoprotein using immunochromatographic or immunoassay techniques. observed results are color changes or some other optical signals [ ] . for example 'alere and influenza a&b' test uses an isothermal nucleic acid amplification, and 'bd veritor' uses chromatographic techniques as influenza detection methods. the mode of action of ridt is presented in figure . the 'gold star' are commercial ridts showing results in around min. ridts are immunoassays that can identify influenza viral nucleoprotein antigens in respiratory specimens. monoclonal antibodies target viral nucleoprotein using immunochromatographic or immunoassay techniques. observed results are color changes or some other optical signals [ ] . for example 'alere and influenza a&b' test uses an isothermal nucleic acid amplification, and 'bd veritor' uses chromatographic techniques as influenza detection methods. the mode of action of ridt is presented in figure . the main advantage is the possibility of providing the test in a physician's office in an effortless way with approximately moderate sensitivity ( - %) [ ] and high specificity ( %), but only in a qualitative way (positive/negative). thus, false positive and false negative results should be considered, especially during high influenza activity season [ ] . false-negatives occur more often than false-positives. negative results cannot exclude virus infection [ ] . undeniably, this kind of tests contributes to shortening the time of infection diagnosis, especially when the algorithm of influenza detection is long and laboratory procedures and requirements are high [ , ] . ridts performance is better in children than adults (around % greater) due to higher viral loads and longer viral shedding [ ] . tests differ in the virus type detected and with distinguishing between types, it means detection of only a type, a and b types with or without distinguishing between them [ ] . sensitivity for b-type virus detection is lower than a-type. none of ridts can distinguish influenza a subtypes. in some tests, the results standardization is possible due to analyzing devices. they are available in cassette, card and dipstick formats, with a visual inspection or automated readers [ ] . ridts belong to ii class of devices which need general and special controls. they were the main advantage is the possibility of providing the test in a physician's office in an effortless way with approximately moderate sensitivity ( - %) [ ] and high specificity ( %), but only in a qualitative way (positive/negative). thus, false positive and false negative results should be considered, especially during high influenza activity season [ ] . false-negatives occur more often than false-positives. negative results cannot exclude virus infection [ ] . undeniably, this kind of tests contributes to shortening the time of infection diagnosis, especially when the algorithm of influenza detection is long and laboratory procedures and requirements are high [ , ] . ridts performance is better in children than adults (around % greater) due to higher viral loads and longer viral shedding [ ] . tests differ in the virus type detected and with distinguishing between types, it means detection of only a type, a and b types with or without distinguishing between them [ ] . sensitivity for b-type virus detection is lower than a-type. none of ridts can distinguish influenza a subtypes. in some tests, the results standardization is possible due to analyzing devices. they are available in cassette, card and dipstick formats, with a visual inspection or automated readers [ ] . ridts belong to ii class of devices which need general and special controls. they were reclassified by food and drug administration (fda) from i class with low risk due to many failures in the h n pandemic in . also, fda evaluated some criteria for ridts that must be adhered [ ] . as the market is enriched in commercial ridts, many authors have compared their sensitivities and specificities in influenza a/b detection. ryu et al. [ ] have compared three digital ridts 'sofia influenza a + b fluorescence immunoassay', 'bd veritor system flu a + b assay' and 'buddi influenza a and b test' with conventional 'sd bioline'. results have shown for influenza a: buddi, sofia, veritor, and bioline sensitivity on level . %, . %, . %, and . %, respectively and specificity: %, . %, . %, and %, respectively, which are only partially accurate with producers assurance. for influenza b, sensitivities and specificities were on similar levels. none of the tests showed cross-reactivity with other respiratory viruses. the sofia test with the fluorescence reader had the best sensitivities. ridts with digital readout systems showed many similarities to conventional assays like small sample volume (less than µl) and short analysis time (around min) but exhibited much better sensitivities, even one order of magnitude lower limits of detection (lods). 'gold sign flu' and 'quick navi-flu' two ridts basing on immunochromatography were compared by akaishi et al. [ ] they were applied for nasopharyngeal swabs as appropriate for the influenza a and b antigens detection. quick navi-flu demonstrated better sensitivities and specificities. advanced were short detection time (less than min for most probes) compared to fluorescence-based tests. another type of point-of-care (poc) test 'the cobas ® liat ® influenza a/b' was evaluated by melchers et al. [ ] the molecular assay based on rt-pcr showed results in min. detection time of immunofluorescent pads was shorter, but sensitivities varied from % to % and often had to be confirmed by pcr methods. the cobas ® liat ® longer analysis time was balanced with an automated system, higher sensitivities ( %) and specificities ( %) compared to another test (diagenode). this assay was faster than others fda-approved rt-pcr-based tests with analysis time ≥ h. two authors evaluated and compared different ridts on a wide range of patients for influenza and rsv detection and distinction. gómez et al. [ ] tested breath samples (nasopharyngeal swabs or aspirates) from both adults and children and moesker et al. [ ] used > swabs only from pediatric patients (aged - years). fluorescence immunoassays (fias) with europium dye 'sofia ® influenza a + b' and 'sofia ® rsv' were compared to reference methods, rt-pcr, and cell culture. automatic analyzers showed higher sensitivities and specificities than other market ridts [ ] . comparison of 'influenza ab ® ' and 'binaxnow rsv ® ' tests with a traditional rt-pcr confirmed the lower accuracy of ridts than in virus isolation and rt-pcr methods [ ] . pediatric specimens were showing higher sensitivities as children have higher viral titers thus better ridts responses. in the literature, ridts evaluation or comparison to each other or the reference points are trending in the last few years. besides the examples described above, available are also 'alere i influenza a&b' nucleic acid amplification versus 'xpert flu/rsv' [ ] , 'the bd veritor™ system flu a + b' versus the 'sd bioline assay' [ ] , maripoc ® test [ ] , becton dickinson test [ ] , 'flu a + b' vs real-time-pcr [ ] , and many more [ ] [ ] [ ] [ ] [ ] [ ] [ ] , focusing on saliva specimens or nasopharyngeal swabs [ ] . immunofluorescence method is an antigen detection with fluorescent microscope usage. the results are received in - h with high specificity and moderate sensitivity. distinguishable are direct (dfa) and indirect (ifa) fluorescent antibody staining assays for influenza a and b detection. subtyping of influenza a virus is not possible [ ] . dfa assays are believed to have an easy procedure and short response time, so they are popularly used for influenza diagnosis. in the test, respiratory epithelial cells from nasopharyngeal swabs are directly stained with fluorescent-labeled antibodies and examined under fluorescent microscope. sensitivities are around - % levels. on the usa market, only two approved tests by fda can be found, which are the d fastpoint l-dfa and bartels viral respiratory screening and identification kit [ ] . dfa tests do not win in the influenza diagnosis with molecular assays which are giving higher sensitivities. from serological tests, the most commonly used are hemagglutination inhibition assay (hai), microneutralization/virus neutralization assay (vn), single radial hemolysis (srh), complement fixation assay, enzyme-linked immunosorbent assay (elisa) and western blotting [ ] . these kinds of tests are generally not recommended because of paired serum samples necessity. the first swab must be collected as soon as possible at the beginning of an infection and the second about - weeks after. there is also a problem with the test availability. the results from a single serum specimen are not interpretable. although the assay is cheap and simple, the sensitivity is unsatisfactory [ , ] . hai is generally used for influenza antibodies detection, which inhibits the interaction between h glycoprotein and red blood cells receptors. the test can be performed on inactivated viruses and is positive when four-fold or more rise of specific antibody titer is observed. the rise is between acute and convalescent serum samples and is measured by hemagglutination inhibition [ ] . in the srh method, the formed complex of antigen-antibody induces the measurement of hemolysis areas which are proportional to antibodies quantity. there is no pretreatment of the serum needed. the technique is commonly used in natural infections and vaccinations [ ] . the srh gives higher sensitivities than the hai assay. the vn measures virus-specific antibodies induction and their ability for virus neutralization. as a result, viral cells infection is prevented. this method is also routinely used in natural infections and vaccinations in influenza seasons. the vn assay gives higher sensitivities than the hai assay, but there are some restrictions in the diagnostic application (certified laboratories necessity). this assay requires infectious, active viruses [ ] . elisa tests [ ] are performed since the s with high sensitivity and specificity. they are available in two forms: paper strips and microtiter plates. despite the big popularity of the test, the major disadvantage is still the lower sensitivity compared to tests based on nucleic acid-techniques. in a conventional test, the influenza virus is detected through specific antigen-antibody interaction and immunocomplex-enzyme linkage, resulting in color change [ ] . some research groups are working on enhancing the sensitivity of these tests by using gold and europium nanoparticles with positive results. the europium nanoparticle-based immunoassay (enia) detects strains of influenza a virus and some b virus subtypes with -times higher sensitivity than commercial elisa assay [ ] . the viral culture method was introduced in the s and is believed to be the most traditional and the gold standard for influenza diagnosis [ ] . influenza viruses are recovered in clinical samples through propagation in mammalian cells or embryonated eggs. the principle is inoculation of permissive cell lines or embryonated eggs with infectious samples, propagation for a week (up to days), observation of the cytopathic effect, and checking virus infection by various methods: immunofluorescence microscopy, antibody staining or erythrocytes hemadsorption [ ] . these methods base on pcr and detection of specific dna/rna sequences of the virus. nats offer higher sensitivity than antigen-based tests and in much shorter time. currently available are reverse transcriptase-pcr (rt-pcr), sequencing-based tests like next-generation sequencing (ngs), ligase chain reaction, dna microarray tests, simple amplification-based assay (samba), nucleic acid sequencing-based amplification (nasba), loop-mediated isothermal amplification-based assay (lamp) and more. general majority of nats is performed within - h with influenza a subtypes information. commercially available are fda licensed nats for influenza virus detection [ ] . rt-pcr allows identification of influenza viral rna in respiratory specimens and is believed to be the most powerful influenza identification assay all over the world. it uses nested primers to detect and subtype influenza viruses [ ] . results of the analysis offer very high specificity and the sensitivity is believed to be the highest of all conventional detecting methods [ ] . the test procedure contains viral rna extraction from a specimen, rna reverse transcription to single-stranded complementary dna (sscdna) by reverse transcriptase enzyme and product amplification with fluorescent detection. some molecular assays using rt-pcr technique cannot only distinguish a and b types of influenza viruses but even identify specific seasonal influenza a subtypes, like h n or h n [ ] . rt-pcr method compared to cell culture and elisa shows -and -times higher sensitivity, respectively. additionally, this sensitivity is not dependent on the patients' age. the procedure of combining multiple primers sets in 'multiplex rt-pcr' method enables detection of several respiratory viruses in one reaction. the main disadvantage is the - h long reaction time and diagnosis costs, as rt-pcr is the most expensive test kind [ , ] . except conventional rt-quantitative pcr (rt-qpcr), specialists are working on speeding up the analysis, like one-step high-speed droplet-rt-pcr, getting results within min [ ] . samba method involves isothermal nucleic acid amplification with the three-step procedure: viral rna extraction, dna amplification by isothermal dna polymerase and dipstick system detection. results are obtained in around h. this test is appropriate for avian and human seasonal influenza and gives high sensitivity ( % and . % for influenza a and b) [ ] . nasba is an isothermal amplification assay, pcr-independent. in one reaction it uses three enzymes: rnase h, t rna polymerase, avian myeloblastosis virus reverse transcriptase (amv-rt). single-stranded dna probes are used to capture viral rna sequences, then separated on a microfluidic chip. the test is suitable for seasonal influenza, in outbreaks of aris. moore et al. [ ] showed % sensitivity on clinical samples. this method is applicable for detecting other viruses, like hiv, rsv, sars [ ] . lamp approach is used for the detection of many viruses like sars, adenovirus, rhinovirus, influenza virus and others. it uses dna polymerase or rna reverse transcriptase and two sets of primers which can recognize six distinct regions in viral complementary dna (cdna) [ ] . the target gene is amplificated and determined by photometrical methods (color change when sybr green added). the sensitivity is similar to rt-pcr assay [ ] . rt-lamp variety proposed by parida et al. [ ] showed a ten times higher sensitivity compared to an rt-pcr method, what is . tcid /ml (tissue culture infection dose at % end point). ngs is one of the most influencing techniques in genetic and medicine fields. ngs compared to the original sanger sequencing extremely speeds up the analysis due to automation possibilities. the next advantage is decreasing the costs from $ million for human genome sequencing in , by sanger method, to $ nowadays. some other technique, illumina platform, offers one million bases sequencing for the cost from $ . to $ . [ , ] . in general, illumina platform is basing on the amplification of nucleic acid fragments on solid substrate or bridge amplification. whitehead et al. [ ] applied illumina ngs platform to create sequence-function map to optimize influenza-binding proteins. this technique has big chances to develop new effective influenza inhibitors. another platform, roche life sciences, developed ngs platforms based on pyrosequencing. it has found application in influenza a case for molecular markers identification, gene coding m protein mutation identification or single nucleotide polymorphism detection in the gene coding hemagglutinin [ ] . except listed above, there are more ngs platforms, like pacific bioscience, ion proton, complete genomics and more, which use different sequencing techniques and have both advantages and limitations, vary on costs, read length, analysis time and error rate. for example, the chembio portable lateral flow platform can be performed outside the laboratory giving results in min. the luminex analysis requires fewer reagents, offers fewer errors and a wide linear range [ ] . among methods mentioned, general diagnostic tests for influenza base on virus culture (conventional and shellvial), detection of viral nucleic acid (pcr) or antigens (by neuraminidase enzymatic activity, fluorescent antibody or enzyme/optical immunoassay) and serologic tests. from them, culture methods can be excluded due to specialized laboratory requirement. also, serologic tests are impractical, need two adequate specimens and are time-consuming. rapid results can only give detection of nucleic acids or specific viral antigens and promote practical and useful diagnosis [ ] . however, rt-pcr is still considered as time-consuming and expensive, and the elisa test does not offer high sensitivity [ , ] . due to various limitations in the conventional detection methods, new diagnostic approaches are being developed. main trends for influenza virus detection are: (i) modifications of traditional 'gold star' methods like pcr, ridts, elisa what results in analysis time shortening, costs lowering, lod and limit of quantification (loq) improvement, (ii) conjugating of traditional methods and creating new platforms, micro-biochips and others, (iii) introducing known solutions to new ones, like smartphone-based analysis control with results data insertion into google maps, (iv) reuse of the functions of known devices, like glucometer, smartphone cameras, (v) the most common used detection methods: spectral/optical, electrical, (vi) and entirely new approaches. some of the approaches for influenza virus are presented in figure . detection limits were shown in plaque forming units (pfu), g/l, viral copies, m, hemagglutinating units (hau), and tcid units. they were difficult to compare as relied on different quantification methods. moreover, influenza have many components acting as target analytes, which in general are in different quantities/ratios in one viral particle or between influenza types/subtypes. exemplary are dna/rna (quantified in copies/ml or m), ha (quantified in hau) or whole viral particles (quantified in pfu). comparison of most lods between newly developed biosensors and different conventional methods were given. give detection of nucleic acids or specific viral antigens and promote practical and useful diagnosis [ ] . however, rt-pcr is still considered as time-consuming and expensive, and the elisa test does not offer high sensitivity [ , ] . due small, micro-size devices are trending in point of care (poc) tests. electromechanical systems on micro-or nano-scale have chemical, biological and medical applications. this kind of assays gives high efficiencies, small amounts of used materials and low waste production. they also speed up the analysis time and makes influenza diagnosis laboratory-independent [ ] . another critical feature is the devices' portability. for example, using microfluidic rt-pcr with a continuous-flow and disposable electrical printed (dep) chips an influenza virus of swine-origin can be detected in a min analysis [ ] . combimatrix corporation developed influenza a microarray detecting all known so-far virus subtypes in less than h [ ] . biochips are common. not only for influenza disease but, i.e., for tobacco mosaic virus (tmv), human rhinovirus serotype (hrv ) and others [ ] . small, micro-size devices are trending in point of care (poc) tests. electromechanical systems on micro-or nano-scale have chemical, biological and medical applications. this kind of assays gives high efficiencies, small amounts of used materials and low waste production. they also speed up the analysis time and makes influenza diagnosis laboratory-independent [ ] . another critical feature is the devices' portability. for example, using microfluidic rt-pcr with a continuous-flow and disposable electrical printed (dep) chips an influenza virus of swine-origin can be detected in a min analysis [ ] . combimatrix corporation developed influenza a microarray detecting all known so-far virus subtypes in less than h [ ] . biochips are common. not only for influenza disease but, i.e., for tobacco mosaic virus (tmv), human rhinovirus serotype (hrv ) and others [ ] . the invention of user-friendly techniques brought measurements to smartphone systems. the study of yeo et al. [ ] showed the performance of a smartphone-based rapid fluorescent diagnostic system (srfds) created for the h n virus diagnosis in chickens. the authors used oropharyngeal (op) and cloacal (cl) samples and compared their method with real-time rt-pcr. the limit of detection of srfds was . pfu/ml, what is -fold higher than in conventional colloidal-gold-based avian influenza rapid diagnostic test. the specificity was % and the sensitivity . % for op and . % for cl specimens, making this test comparable to rrt-pcr. pretreatment swab was transferred to the sample pad, after min reaction time the smartphone camera was used as a detector, by using filtration of the excitation light by the emission filter in the light-emitting diode (led) module. the results were displayed on the smartphone screen, as a ratio of a control and test lines on a strip and coordinated with the location on google maps, to check for non-/infected areas. another group, wu et al. [ ] , also have used smartphone assistance for influenza a detection. an automated and portable paper-based microfluidic system was developed. the chip consisted of two modules: the storage module with reagent chambers and the reaction module with the absorbent pad and nitrocellulose membrane functionalized with specific monoclonal antibodies. the smartphone was used for image capturing from the membrane by camera and for processing the image with an algorithm to the application developed with java. the smartphone was used as a guide to the microcontroller, connected via bluetooth. it enhanced multiple reaction steps performance, collected the results and sent it to medical agencies if necessary. another idea was to use already existing techniques and standardized analyzers. zhang et al. [ ] designed an electrochemical assay which used a glucometer. glucose-containing substrate (sg ) exposed to influenza virus (or neuraminidase) released glucose which is determined amperometrically. samples were analyzed directly, without further preparation. the result was the detection of strains of influenza viruses (h n and h n ) in h. this method offers user-friendly, fast and inexpensive detection. when classifying influenza diagnosis methods by used measurement techniques, dominating are optical [ ] and electrical [ ] . they are believed to be fast, easy, adequate (providing qualitative and quantitative analysis) and relatively inexpensive. the influenza virus gold electrode electrochemical sensor was proposed by horiguchi et al. [ ] immobilization of ha specific receptor ( -sialyllactose) onto gold allowed label-free h n detection. quartz crystal microbalance (qcm) technique and electrical detection were developed for biosensor measurements. qcm gave − hau sensitivity with min detection time and electrical detection gave sensitivity of − hau and min analysis time. the sensitivity was higher than conventional immunochromatographic technique (ict). different type of bioreceptor, anti-m antibodies was developed for influenza a detection. nidzworski et al. [ ] modified boron-doped diamond (bdd) electrodes with -aminobenzoic acid and anti-m antibodies in self-assembled monolayer (sam) approach. by electrochemical impedance spectroscopy (eis) measurements authors achieved the lowest limit of detection ( fg/ml) compared to previously reported methods. the assay offered easy sample pretreatment, short incubation time (< min) and non-interference of bacteria and yeast which might be present in patients swab. another antibodies immobilization method was presented by cheng et al. [ ] simple technique of ac electric field application on electrodes induced positive dielectrophoresis. subsequently, viral particles were attracted to the immunosensor. commercially available surface acoustic wave (saw) electrode chip of mm length size was used. the detection limit was . pg/ml with very fast response time s. the authors have achieved the sensitivity of % and specificity of %. apart from electrodes mentioned above, there is a wide range of electroactive materials for biomolecule recognition. available are carbon-based materials like carbon paste, glassy carbon [ , ] , graphene-oxide [ ] , reduced graphene oxide [ ] , boron-doped diamond (presented in figure ) [ ] . there are also noble metal-based materials like silver, gold [ , ] , platinum, zinc, cadmium, and others [ ] . moreover, every listed material can undergo multiple methods of surface modification, not only with nanoparticles [ ] [ ] [ ] [ ] [ ] [ ] [ ] , but hybrids and biological materials, like aptamers [ ] [ ] [ ] , sialic acid [ ] or its derivatives [ ] , composites [ , , ] , nanohybrids [ ] and dyes [ ] . for influenza biosensing the most often used measurement techniques are differential pulse voltammetry [ , , ] , cyclic voltammetry [ ] , electrochemical impedance spectroscopy [ , ] , and amperometry [ , ] . interesting research of mubarok et al. [ ] showed whole blood analysis for neuraminidase activity label-free detection. the cleavage of glycosidic linkage of self-synthetized n-acetyl- -o-( aminophenyl)-α-neuraminic acid (ap-neu ac) in the presence of na released p-aminophenol molecules. as p-aminophenol showed electroactivity, the electrical signal onto a bare glassy carbon (gc) electrode was recorded. lod of na activity was . ng/ml. the sensor was applicable also for urine, saliva and nasal swabs. currently, many authors construct glycan-based biosensors [ , , ] as they are natural viral receptors with selectivity for pathogenic subtypes. they form a compact layer on the measurement surface, so-called glycocalyx, reaching even mm concentration. simple electrode modification might exclude time-consuming and expensive antibodies assay. for example, lod for h n was achieved on viral particles per µ l level [ ] . for glycan-derivatives sams the most often used electrochemical techniques are impedance spectroscopy and amperometry. except influenza proteins mentioned in ' . influenza pathogenesis' section, some non-structural proteins pa-x, pb -f , ns , ns and others were recently found [ ] they were also identified as pathogenic for the host organism [ ] [ ] [ ] . electrochemical eis biosensor for pb -f was developed by miodek et al. [ ] the authors used antibody-antigen approach and modified gold electrode with a specific anti-pb -f antibody with three steps. firstly, pyrrole and ferrocene derivatives were electrochemically polymerized onto the au surface. the next step was biotin/streptavidin linkage and the closing step was biotinylated antibodies immobilization. for confirmation of proper sensor modification the surface plasmon resonance (spr), atomic force microscopy (afm) and cyclic voltammetry (cv) techniques were used. by differential pulse voltammetry (dpv) two linear ranges were observed, - nm and . - . mm of pb -f what was probably caused by two specific antibody sites. lod was on . nm level. from optical/spectral methods the most common are fluorescence, ultraviolet/visible (uv/vis) spectroscopy, surface enhanced raman spectroscopy (sers) [ , ] and others [ ] . recently developed influenza sensor by liu et al. [ ] used fluorescence for neuraminidase detection. the macrocyclic dye substrate (squaraine-derived core blocked on ends with sialic acid) reacted with viral na releasing the blockages. subsequently, the free core was encapsulated with there are also noble metal-based materials like silver, gold [ , ] , platinum, zinc, cadmium, and others [ ] . moreover, every listed material can undergo multiple methods of surface modification, not only with nanoparticles [ ] [ ] [ ] [ ] [ ] [ ] [ ] , but hybrids and biological materials, like aptamers [ ] [ ] [ ] , sialic acid [ ] or its derivatives [ ] , composites [ , , ] , nanohybrids [ ] and dyes [ ] . for influenza biosensing the most often used measurement techniques are differential pulse voltammetry [ , , ] , cyclic voltammetry [ ] , electrochemical impedance spectroscopy [ , ] , and amperometry [ , ] . interesting research of mubarok et al. [ ] showed whole blood analysis for neuraminidase activity label-free detection. the cleavage of glycosidic linkage of self-synthetized n-acetyl- -o-( aminophenyl)-α-neuraminic acid (ap-neu ac) in the presence of na released p-aminophenol molecules. as p-aminophenol showed electroactivity, the electrical signal onto a bare glassy carbon (gc) electrode was recorded. lod of na activity was . ng/ml. the sensor was applicable also for urine, saliva and nasal swabs. currently, many authors construct glycan-based biosensors [ , , ] as they are natural viral receptors with selectivity for pathogenic subtypes. they form a compact layer on the measurement surface, so-called glycocalyx, reaching even mm concentration. simple electrode modification might exclude time-consuming and expensive antibodies assay. for example, lod for h n was achieved on viral particles per µl level [ ] . for glycan-derivatives sams the most often used electrochemical techniques are impedance spectroscopy and amperometry. except influenza proteins mentioned in ' . influenza pathogenesis' section, some non-structural proteins pa-x, pb -f , ns , ns and others were recently found [ ] they were also identified as pathogenic for the host organism [ ] [ ] [ ] . electrochemical eis biosensor for pb -f was developed by miodek et al. [ ] the authors used antibody-antigen approach and modified gold electrode with a specific anti-pb -f antibody with three steps. firstly, pyrrole and ferrocene derivatives were electrochemically polymerized onto the au surface. the next step was biotin/streptavidin linkage and the closing step was biotinylated antibodies immobilization. for confirmation of proper sensor modification the surface plasmon resonance (spr), atomic force microscopy (afm) and cyclic voltammetry (cv) techniques were used. by differential pulse voltammetry (dpv) two linear ranges were observed, - nm and . - . mm of pb -f what was probably caused by two specific antibody sites. lod was on . nm level. from optical/spectral methods the most common are fluorescence, ultraviolet/visible (uv/vis) spectroscopy, surface enhanced raman spectroscopy (sers) [ , ] and others [ ] . recently developed influenza sensor by liu et al. [ ] used fluorescence for neuraminidase detection. the macrocyclic dye substrate (squaraine-derived core blocked on ends with sialic acid) reacted with viral na releasing the blockages. subsequently, the free core was encapsulated with macrocyclic tetralactam what caused spectral changes. red-shift in absorbance and fluorescence emission of squaraine were observed in the na presence. results were visible with naked eye. with some optimization, this portable sensor might be used in influenza diagnosis in poc or for effective determination of antiviral inhibitor drugs. the authors claimed possibilities of differentiation for classes of mammalian, bacterial and viral neuraminidase and suitability for quantitative analysis. another technique, sers, was used in magnetic immunosensor for avian influenza detection [ ] . sam approach of -mercaptobenzoic acid ( -mba) molecules chemisorption on the gold nanoparticles (aunps) was used for further influenza a igg antibodies immobilization. the sandwich-type biosensor structure enabled qualitative and quantitative analysis. h n was detected at lod tcid /ml with linearity range - × tcid /ml. the assay had a potential for poc use due to time efficiency, sensitivity, and portability. a similar study by park et al. [ ] has shown sers-based assay in a lateral flow strip. the principle was exchanging aunps from the commercial kit with sers-active nanotags. this innovation enhanced precision and sensitivity of the raman signal. the au-nanotags-influenza-antibodies complex was captured onto an antibodies-modified strip with positive probes giving two red strains (control line and sers-test tine). negative probes gave only one response on the control line. lod was calculated as . × pfu/ml and this value was one order of magnitude higher than standard colorimetric kits. a different detection-type, immunochromatographic test (fict), for avian influenza a was proposed by yeo et al. [ ] the authors have chosen fluorescent red dye which intensity increased intensity by additional fluorescent phosphor groups linkage. the assay was to europium-based fict and standard ridt. influenza a virus from nasopharyngeal swabs was detected by a portable fluorescent strip reader. analysis time was only min, the lod was estimated at hau/ml, and the linearity range was - hau/ml. fict was -times more sensitive than europium-based fict and -times more sensitive than the rapid diagnostic test. another use of dye, , , , -tetramethylbenzidine (tmb), was in colorimetric immunosensor assays. lin et al. [ ] similarly used antibodies in complex with streptavidin/biotin linker, liposome and antigen. innovative was horseradish peroxidase (hrp) encapsulation in liposome. after the addition of hydrogen peroxide (h o ) and tmb to the complex, lysis of liposomes occurred. hrp catalyzed the decomposition of h o generating · oh radicals which oxidized tmb and gave a color change to the solution. the detection was possible by the naked eye and spectrophotometric technique. lod of h n was . ng/ml with linearity from . to . ng/ml. compared to standard elisa where very small absorbance and visual no color change was observed with the concentration below . ng/ml, the authors approach showed much more sensitivity. the modification of tmb-based assay was proposed by ahmed et al. [ ] for spectrophotometric h n virus detection authors have used gold nanoparticles-carbon nanotubes (aunps-cnts) hybrids, which showed the high catalytic activity of tmb oxidation. the complex of aunps-cnts-tmb-h o in the influenza presence showed a change in color. blue tone intensity varied depending on the virus concentration. the complex absorbed the light in λ max = nm which was measured using a microplate reader. this method had a limit of detection times lower ( . pfu/ml) than conventional elisa. ahmed et al. [ ] similarly have used (+)aunps in tmb-based method achieving h n virus detection at even lower levels of . pg/ml and for h n of . pfu/ml improving sensitivity to -times higher than elisa. next optical detection technique upconversion luminescence resonance energy transfer (lret) was used by ye et al. [ ] . the authors detected avian influenza h virus subtypes. the biosensor contained donor fluorophores (bagdf :yb/er upconversion nanoparticles, ucnps) and acceptor fluorophores (aunps). lret measurement was activated by hybridization between complimentary oligonucleotides which were linked to nps. the target h gene was conjugated to acceptor nps and complimentary genes to donor nps. hybridization process decreased the fluorescence of ucnps and enhanced light absorption of aunps. upconversion spectra were registered after h probes incubation. lod was pm of the hemagglutinin gene and linear response from pm to nm. glycan-based methods mentioned in the electrical assays also have found application in optical biosensors. zheng et al. [ ] used glycan-functionalized gold nanoparticles (ggnps) that bounded and aggregated on the viral surface. the authors have differentiated fourteen influenza strains and distinguished them from a human respiratory syncytial virus. the principle was different ha-binding preferences depending on the configuration of c-c bond in sialic acid receptors (described in section . 'influenza pathogenesis'). the one-step procedure, mixing virus with ggnps and min incubation resulted in a color change from red to purple and was measured spectrophotometrically. except for natural glycans, he et al. [ ] have synthesized influenza virus na resistant sialosides, with c-, s-and triazole linkage ends and printed onto a glass surface. these molecules could capture eight virus strains at very low concentration. caught viruses gave fluorescence intensity rise. lod was on ceid (chicken embryo infectious dose). adegoke et al. [ ] as first evaluated the synthesis of cdznsetes quantum dots (qd) with the one-pot hot-injection method. they were used for near-infrared-emitting viral rna detection. the fluorescence signal was measured after min incubation of qd and h n probe. low lod level ( copy/ml) confirmed much better sensitivity than ridts. the standard influenza virus detection method (rt-pcr) was modified by hmila et al. group [ ] . the novel assay development was motivated by new mutations in ha and na of tunisian poultry. the risk of economic losses and human infections significantly increased. the authors have used an aptamer-real time-pcr. advanced was using one step selex procedure for h n specific ssdna aptamers selection and later use as ligands for virus capture. the assay was adequate for direct swabs with no need for sample pretreatment and showed rapid, label-free results and high sensitivity. conventional elisa showed lod on . × tcid /ml and this assay . × tcid /ml. another group [ ] has performed a new platform in china where standard pcr was conjugated with mass spectrometry-electrospray ionization (esi-ms) for identification of respiratory viruses. the authors have used nasopharyngeal aspirates for the analysis and have compared their method with dfa and later confirmed by rt-pcr plus sequencing. pcr-esi-ms showed higher sensitivities because detected more viruses in patients' specimens and was adequate for co-infections determination. the method could detect many virus families like coronaviruses, adenoviruses, alphaviruses and others. pcr-esi-ms was believed as faster and more automatic than conventional fda but required expensive equipment and had no practical usage in poc devices. for rapid and accurate influenza detection with high sensitivity and short time, eboigbodin et al. [ ] have proposed the assay combining two techniques. reverse transcription (rt) of cdna to rna in single-step reaction and strand invasion-based amplification (siba ® ) in isothermal conditions allowed to detect both influenza a and b in only min where traditional rt-pcr required more than min. beneficial was rna usage for detection instead of dna as it is major influenza genetic material. copies of h n rna were detected with -times higher sensitivity than rt-pcr. modification of lamp method was proposed by ge et al. [ ] lamp is believed to be the most willingly used nucleic acid-based isothermal amplification assay. however, differentiation of products containing multiple targets is still challenging as currently existing methods (e.g., electrophoresis) require specialized equipment. simple colorimetric method with invasive gold nanoparticles reaction was used. nps aggregation and dispersion resulted in solution color change. the sensor could subtype h an h with lod of rna copies and influenza b with lod rna copies. coordination of two trending viral detection methods, electrical and optical was proposed by sepunaru et al. [ ] virus tracking was achieved by uv-vis spectroscopy. influenza particles were able to absorb silver nps and showed maximum absorbance at nm. moreover, the tagged virus could be absorbed on the gc electrode and gave an electrical response. chronoamperometric signal of silver nps oxidation process was proportional to virus quantity. this assay enabled viral and bacterial infection distinction. sakurai et al. [ ] have improved the common antigen-detection rapid influenza test known as immunochromatography (ic). ic could be accomplished in less than min, however, had low sensitivity (around %) and lod on pfu which is lower than pcr-based methods. the improvement was based on antibodies conjugation with fluorescent beads what enhanced the sensitivity -times. moreover, the assay was more accurate for early infections detection than ic. gouma et al. [ ] have recently invented the novel isoprene sensor for an influenza virus ( figure ). the authors have claimed that infected patients generate more volatile products compared to healthy ones. volatile products come from the alveolar and airway epithelium as well as leukocytes infiltrating the lungs, like volatile organic compounds (vocs) and nitric oxide (no). they were used as biomarkers to detect the disease. the constructed device was a portable -sensor array microsystem offering rapid non-invasive screening. the measurement needed to be conducted as fast as the disease was (potentially) present to observe biomarkers changes in time. the sensor was believed to give satisfying specificity and sensitivity. more precisely, the sensor could detect three gases: isoprene, ammonia, no in the temperature control conditions. it measured resistance changes of h-wo material with exposure to no, no , methanol, and isoprene at • c. improvement was based on antibodies conjugation with fluorescent beads what enhanced the sensitivity -times. moreover, the assay was more accurate for early infections detection than ic. gouma et al. [ ] have recently invented the novel isoprene sensor for an influenza virus ( figure ). the authors have claimed that infected patients generate more volatile products compared to healthy ones. volatile products come from the alveolar and airway epithelium as well as leukocytes infiltrating the lungs, like volatile organic compounds (vocs) and nitric oxide (no). they were used as biomarkers to detect the disease. the constructed device was a portable -sensor array microsystem offering rapid non-invasive screening. the measurement needed to be conducted as fast as the disease was (potentially) present to observe biomarkers changes in time. the sensor was believed to give satisfying specificity and sensitivity. more precisely, the sensor could detect three gases: isoprene, ammonia, no in the temperature control conditions. it measured resistance changes of h-wo material with exposure to no, no , methanol, and isoprene at °c. jiang et al. [ ] have invented an influenza sensor using a saw platform. the piezoelectric linbo wafers were coated with sio . in this technique, the propagation of acoustic waves was changing at the measured material, depending on analytes located on the surface. love waves detected antigens through specific antigen-antibody interaction. the authors have used two reagents that were effective for immobilizing ha antibodies on the measurement surface. they were triethoxysilybutyladehyde (altes) and triethoxysilylundecanal ethylene glycol acetal (actes), which were end-functionalized with carboxylic acid and aldehyde. lod for h n ha antigen was ng/ml. oyama et al. [ ] have developed their point-of-care testing chip. it was based on antigenantibody interactions with fluorescence detection. the authors applied absorbing polymer providing continuous sample flow and separation of bounded and free antibody residues. a glass fiber sheet was chosen as a flow medium. antibodies were fluorescently labeled by dylight and dylight . the achieved sensitivity was more than thousand times better compared to immunochromatographic jiang et al. [ ] have invented an influenza sensor using a saw platform. the piezoelectric linbo wafers were coated with sio . in this technique, the propagation of acoustic waves was changing at the measured material, depending on analytes located on the surface. love waves detected antigens through specific antigen-antibody interaction. the authors have used two reagents that were effective for immobilizing ha antibodies on the measurement surface. they were triethoxysilybutyladehyde (altes) and triethoxysilylundecanal ethylene glycol acetal (actes), which were end-functionalized with carboxylic acid and aldehyde. lod for h n ha antigen was ng/ml. oyama et al. [ ] have developed their point-of-care testing chip. it was based on antigen-antibody interactions with fluorescence detection. the authors applied absorbing polymer providing continuous sample flow and separation of bounded and free antibody residues. a glass fiber sheet was chosen as a flow medium. antibodies were fluorescently labeled by dylight and dylight . the achieved sensitivity was more than thousand times better compared to immunochromatographic commercial assays (lod above ng/ml). krishna et al. [ ] have developed giant magnetoresistance (gmr) sensor, which adapted monoclonal antibodies to influenza h h v nucleoprotein with magnetic nanoparticles (mnps). in the virus presence, mnps were bonded to the sensor, and the resistance change was measured. lod was . × tcid /ml. this assay was applicable for nasal swabs. except for the methods mentioned above, many authors have proposed novel ideas. for example, shelby et al. [ ] invented the magnetic relaxation nanosensor (mrns) which was selectively binding to hemagglutinins. the influenza virus variants detection was at . nm concentration level. kirkegaard et al. [ ] invented screen-printed aptasensor (pedot) for impedance-based influenza a detection. ozcelik et al. [ ] evaluated the optofluidic wavelength division multiplexing method for single-virus detection. the authors could distinguish three subtypes of influenza a using two approaches. first was virus labeling with different color labels, and second was the combination of two different colors for every viral strain. lee et al. [ ] presented quantitative h n virus screening without dna amplification, basing on single-particle dual-mode total internal reflection scattering (sd-tirs) with transmission grating (tg). chan et al. [ ] claimed that the flow graphene transistor-based dna sensors have not been explored yet, so they proposed microfluidic integrated reduced graphene oxide (rgo) transistor for the h n influenza virus gene detection. zhang et al. [ ] elaborated a label-free optical sensor for influenza serotyping. the authors have used the pattern recognition method based on arrayed imaging reflectometry (air) platform. when antireflective chip condition was perturbed due to binding to, e.g., an antibody, the reflected light changes were quantitatively detected. katayama et al. [ ] created an influenza virus biosensor based on two methods: electrochemiluminescence (ecl) of modified au electrode combined with immunoliposome encapsulating ru (ii) complex. the authors have achieved sensitivity higher than elisa; the detection range was from . × to . × pfu/ml. li et al. [ ] combined complement fixation and luminol chemiluminescence for rh n detection, which is a recombinant avian influenza virus protein. this assay could be completed in . h with a linear detection from . fg/ml to ng/ml. tran et al. [ ] detected influenza a viruses using carbon nanotubes field effect transistor (cntfet) dna sensor. the response time was less than min, and the detection range was linear from pm (lod) to nm. the sensor signal recovery was % after -month storage in ph-controlled conditions. the most interesting biosensors were compared in table . rt-siba rna copies -< min [ ] mrt-lamp-cirn rna / copies - rna copies/µl~ min [ ] other novel ideas vp-viral particles, nir-near-infrared fluorescence, ha-p-hemagglutinin peptide, rp-recombinant protein, lsv linear sweep voltammetry, sers + lfa-surface-enhanced raman scattering-based lateral flow assay, rt-siba-reverse transcription strand invasion-based amplification, mrt-lamp-cirn-reverse transcription loop-mediated isothermal amplification-based assay coupled with cascade invasive reaction, cft + cl-complement fixation test+ luminol chemiluminescence. the scope of this article was presenting progress in the influenza sensing area, discussing the chosen materials and recognition elements, measurement techniques, sensitivities, specificities and further applications in poc devices. from all the presented influenza virus diagnosis methods, the most used in laboratories are 'gold standard' conventional methods. however, the majority of these require long time analysis ( - h for rt-pcr) [ ] but compensating it with high sensitivities and the most reliable results. that is why the need for improvement for 'gold standards' increase significantly. despite the fact of low and variable sensitivity, ridts are the main improvement in influenza diagnostics, especially in pandemic seasons. they are pivotal in the emergency rooms by decreasing numbers of unnecessary antibiotics dosages. performing ridts at physicians' offices relieves laboratories work and enables the focus on specimens designed for culture or other time-consuming methods. moreover, the increased interest in specific laboratory diagnosis is due to minimizing the antibiotics resistance of patients and improves the influenza recognition system. as influenza is a public-health threat and influenza a is pandemic, portable, fast and accurate tools are in high demand in the medical industry to control virus outbreaks and spreads. also, this kind of devices needs constant updates due to genetic assortment of human, swine and avian influenza. new h/n genes are produced, against which human population lacks immunity. generally understood the 'biosensors' field has attracted many scientists. from a wide range of proposed assays, it is possible to choose these with reasonable cost, good selectivity and sensitivity, and practical application in poc. for authors of this article, the electrochemical sensors win the competition. it seems that they meet the need for rapid and accurate influenza diagnosis. they offer a vast number of electrode materials and target detection molecules with practically endless modification methods. furthermore, these sensors successfully can be expanded for other pathogen detection by changing the kind of probe immobilized on the electrode surface. funding: this research was funded by the ncbr, techmatstrateg / / /ncbr/ ; ventures program of the foundation for polish science, co-financed by the european union, regional development fund; the project "research and development work on an innovative, ultra-sensitive, fast and cheap micro-test for detecting the influenza virus-flusensor", rpma. . . - - / as part of action . "enterprise r&d activity" of the regional operational programme of mazowieckie voivodeship for - , co-financed by the european regional development fund, priority axis i "use of r&d activity in economy"; the project "research and development works to develop a multisensor prototype-an innovative micro-sensor for identifying bacterial or viral causes of upper respiratory tract infections" as part of action . "enterprise r&d projects" sub-action . . "industrial research and development activities carried out by enterprises" of the smart growth operational programme - , co-financed by the funds from the european regional development fund. the authors declare no conflict of interest. -viral diseases chapter -viral upper respiratory infection past pandemics | pandemic influenza (flu) | cdc. available online highly specific and rapid glycan based amperometric detection of influenza viruses rapid diagnostic assay for intact influenza virus using a high affinity hemagglutinin binding protein influenza (seasonal) the biology of influenza viruses an open receptor-binding cavity of hemagglutinin-esterase-fusion glycoprotein from newly-identified influenza d virus: basis for its broad cell tropism structure and function of the influenza virus genome rates of spontaneous mutation among rna viruses review of rapid diagnostic tests for influenza sense rna viruses-negative sense rna viruses influenza a virus m protein: roles from ingress to 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combining electrochemiluminescence on binary sams modified au electrode with an immunoliposome encapsulating ru (ii) complex combining complement fixation and luminol chemiluminescence for ultrasensitive detection of avian influenza a rh n detection of influenza a virus using carbon nanotubes field effect transistor based dna sensor key: cord- -yr uxok authors: guo, zijing; he, qifu; tang, cheng; zhang, bin; yue, hua title: identification and genomic characterization of a novel cress dna virus from a calf with severe hemorrhagic enteritis in china date: - - journal: virus res doi: . /j.virusres. . . sha: doc_id: cord_uid: yr uxok in this study, a novel circular replication-associated protein (rep)-encoding single stranded (cress) dna virus was discovered in diarrheic sample of a calf with severe hemorrhagic enteritis. the virus, named bo-circo-like virus ch, has a circular genome with nucleotides (nt). six putative open reading frames (orfs) were identified, including rep, capsid (cap) and four proteins of unknown function. both the genome size and the number as well as the organization of encoded orfs, bo-circo-like virus ch is most closely related to po-circo-like virus detected in pig faeces. a preliminary survey using specific primers for the rep region showed that . % ( / ) of diarrheic samples were positive for bo-circo-like virus, and all healthy samples were negative. in conclusion, our results indicate that bo-circo-like virus ch may represent a new virus in bovine. further investigation is needed to determine the relationship between the virus infection and diarrhea. viruses with a circular ssdna genome are found in archaea, bacteria, and eukaryotic organisms (shulman and davidson, ) . until recently, the cress dna viruses have been classified into six families by the international committee on taxonomy of viruses (ictv), namely geminiviridae, nanoviridae, circoviridae, genomoviridae, bacilladnaviridae and smacoviridae. viruses of families geminiviridae nanoviridae and bacilladnaviridae were known to infect plants (ramesh et al., ; rybicki and martin, ; kazlauskas et al., ) . viruses of the new families smacoviridae and genomoviridae had been identified in faecal samples of various vertebrates, including humans and bovine krupovic et al., ; steel et al., ; kim et al., ) . the family circoviridae includes the genera circovirus and cyclovirus, which widely infected humans and certain animals (fu et al., ; hattermann et al., ; li et al., ; lorincz et al., ; phan et al., ) . the genomes of circoviruses and cycloviruses range in size from . to . kb and contain two major orfs, which encode rep and cap proteins (breitbart et al., ) . the taxonomic classification for the family circoviridae suggested that rep identities and genome organization should be used to identify a genome as belonging to either the circovirus or cyclovirus genus (rosario et al., ) . furthermore, a new species demarcation threshold of % genome-wide pairwise identity for members of the family circoviridae (breitbart et al., ; rosario et al., ) . in addition to six recognized cress dna viral families, a large number of novel cress dna viruses, waiting to be classified by ictv, have been rapidly discovered in faeces of humans and animals through viral metagenomics in recent years. the hosts include chimpanzees (blinkova et al., ) , pigs zhang et al., ) , rodents (phan et al., ) , bats (ge et al., ) , humans (castrignano et al., ) , dromedaries (woo et al., ) , duck (steel et al., ) , deer (steel et al., ) , llama (steel et al., ) , chamois (steel et al., ) , bovine (steel et al., ) , flying fox (male et al., ) , chickens (lima et al., ) and macaques (kapusinszky et al., ) . among these novel cress dna viruses, a group of viruses with large genomes have been proposed family kirkoviridae according to genomic and rep-phylogenetic characteristics (li et al., ; zhao et al., ) . until now, seven reported cress-dna viruses genomes can be classified as the proposed family kirkoviridae (li et al., ; shan et al., ; zhao et al., ) , including four genomes determined in the feces of healthy and diarrhoeic pigs , one genome determined in the liver and spleen of a horse with fatal idiopathic hepatopathy (li et al., ) and two genomes determined in patients feces with type diabetes (zhao et al., ) , successively. a significant characteristic of these viruses in the proposed family kirkoviridae shows large genomes ranging in size from to nt with three to six orfs. additional, all the rep proteins of these viruses show significant genetic distance with that of the viruses within family circoviridae. although one member named kirkovirus equ in the proposed family kirkoviridae was suggested that it may be potentially related to disease (li et al., ) , the pathogenesis of these viruses is still unknown. in this study, a novel bovine-derived cress dna virus was found in diarrheic faecal sample through viral metagenomics. the fecal sample was collected from a -month-old simmental cross calf with severe hemorrhagic enteritis accompanied by dyspnea and fever in october in sichuan province, china. the sample was suspended in phosphate-buffered saline (pbs) to create a % suspension and centrifuged at g for min at °c. the supernatant was then filtered through a . mm filter (millipore) to remove intact bacteria and other large cellular debris. the filtrates was treated with u dnase and . mg rnase enzymes (takara) at °c for min to remove unprotected nucleic acids. total nucleic acid from the sample was extracted using a qiaamp viral rna mini kit (qiagen) following the manufacturer's instructions. reverse transcription was performed using superscript iii reverse transcriptase (invitrogen) and random hexamers (invitrogen). the quantity of cdna was determined by a qubit fluorometer (life technologies). the sample of ng cdna was used to construct a library according to the manufacturer's instructions (truseq rna sample preparation kit) and sequenced on hiseq (illumina), as described in the previous studies (chen et al., ) . raw sequence reads were trimmed to remove the reads of adaptors, duplicate reads and bovine genomic sequences, and a minimum length of bp was selected. the reads that passed the data processing were considered the useful sequences. in parallel, all useful reads were subjected to de novo contig assembly using the idba_ud (peng et al., ) , and evaluate assembly results using soap software (http:// soap.genomics.org.cn/). the assembly data were aligned with sequences in the ncbi nonredundant nucleic database (nt) and the nonredundant protein database (nr) using blastn and blastx, respectively. the taxonomies of the aligned read with the best blast value (e value < − ) were selected and used for further grouping analysis. the abundance of viruses was scanned by soap aligner software to further analyse the diversity of the species identified from the calf diarrheic sample. a total of bases were generated from the illumina sequencing run, and approximately . % of the sequence data matched those of mammalian viruses. the raw sequence reads from the viral metagenomic libraries were deposited in genbank database under the accession number srp . seven konwn mammalian viruses consist of bovine enterovirus ( reads), bovine kobuvirus ( reads), bovine herpesvirus ( reads), nebovirus ( reads), bovine coronavirus ( reads), bovine adenovirus type ( reads) and bocaparvovirus ( reads) were found, and the length of each read was bp. notably, reads reveal significant sequence similarity with po-circo-like viruses (genbank accession no. nc_ . and jf . ), and the reads were assembly into two large contigs. one contig consists of nt (position: - - - nt) and the other contig consists of nt (position: - - - nt), referencing the genome ( nt) of strain po-circo-like virus (genbank accession no. nc_ . ). to acquire the full-length genome of the cress-dna virus and further verify the sequences of viral metagenomic, four set of primers were designed based on the viral metagenomic sequencing data (genbank accession no. srp ). the primers sequences were shown in table . pcr was performed with premix taq™ (takara, china), following the manufacturer's protocol. the pcr amplification products were gel-purified using a gel extraction kit (omega, usa) and then cloned into the pmd -t vector and transformed into escherichia coli dh αcompetent cells (fig. ) . the recombinant plasmids were extracted using a plasmid extraction kit (omega, usa) and then sequencing was performed by sangon biotech (china, chengdu). the sequences were assembled using seqman software (version . ; dnastar, usa). putative orfs in the circular genomes were identified using the orf finder tool (http://www.ncbi.nlm.nih.gov/gorf/gorf. html). the hairpin and stem-loop structures were identified in mfold webserver (zuker, ) . nucleotide and deduced amino acid sequences were analyzed using the megalign program of dnastar . software (dnastar inc., wi, usa) to determine sequence homology. mega . was used to perform the multiple sequence alignment and subsequently to build a maximum likelihood phylogenetic tree using the lg model with bootstrap support (kumar et al., ) . the novel circular dna virus has a complete genome of nt with g+c content of . % (genbank accession no. mh ). six putative orfs (> aa) were identified, including rep protein of aa, cap protein of aa and four proteins of unknown function of , , and aa. a stem-loop structure was predicted to be located close to the putative rep gene stop codon in the intergenic region (fig. ) . both the genome size and the number as well as the organization of encoded orfs of the virus were similar to those of the viruses of recently proposed kirkoviridae (li et al., ; shan et al., ; zhao et al., ) . (li et al., ; zhao et al., ; wang et al., ) . due to the remarkably diversity in the genome size, number of orfs and genome sequences of these cress dna viruses, it is only possible to construct a phylogenetic tree for them based on the rep gene, which is the most conserved gene in the genomes of these novel viruses (castrignano et al., ; varsani and krupovic, ) . both the genome size and the number as well as the organization of encoded orfs of strain bo-circo-like virus ch indicated that it showed closest genetic relationship with strain po-circo-like virus . however, two orfs ( residues and residues) of the virus did not have significant similarity to any orfs in genbank, and a recombination event seems occurred in a orf ( residues). generally, recombination and horizontal acquisition of non-homologous genes were significant features in ssdna virus (krupovic and koonin, ; lefeuvre et al., ; martin et al., ) . this phenomenon was also observed in the cress dna virus (kazlauskas et al., (kazlauskas et al., , lefeuvre and moriones, ) , which may contribute to evolution of the cress dna virus. the putative rep protein ( residues) of bo-circo-like virus ch shares . % aa identity and . % aa identity with that of po-circolike virus and po-circo-like virus , respectively, but shares . - . % aa identity with that of other five reported members of the proposed family kirkoviridae. the rep-representative members of the families circoviridae, nanoviridae, geminiviridae genomoviridae, bacilladnaviridae and smacoviridae, and still unassigned novel ssdna viruses available in genbank database were used constructed a maximum-likelihood phylogenetic tree. the result showed that bo-circo-like virus ch is clustered into a independent branch with seven reported strains of proposed family kirkoviridae and eight cress-dna virus strains recently submitted to genbank database; bo-circo-like virus ch is more closely related to po-circo-like virus and shows significant genetic differences with viruses in the families circoviridae, nanoviridae, geminiviridae genomoviridae, bacilladnaviridae and smacoviridae (fig. ) . further analysis of strains in the new clade suggested that these strains display large genomes ranging in size from to nt with three to six orfs. the hosts includs human, macaca, bovine, porcine, horse, dromedary and rodent. although the classification criteria of the proposed family kirkoviridae is not yet clear (li et al., ; zhao et al., ) , it seems that these viruses may be classified into the family proposed kirkoviridae. the third-largest orf ( residues) of bo-circo-like virus ch was predicted to encode cap protein since high percentage ( / ) of the basic amino acids arginine and lysine near the n-terminus of the orf was observed, which is characteristic of circovirus cap proteins (stewart et al., ) . additional, a blastp search using the orf showed that it hits the putative cap protein of macaca mulatta cg (genbank accession no. ku . ; qc = %, i = %, ev = e- ). the putative cap protein of bo-circo-like virus ch also matches hypothetical protein of six strains of the proposed family kirkoviridae except for kirkovirus equ . the putative cap protein of bo-circo-like virus ch shares . % aa identity and . % aa identity with that of po-circo-like virus and po-circo-like virus , respectively, and shares . - . % aa identity with that of other four members of the proposed family kirkoviridae. four orfs of unknown function were also identified in bo-circo-like virus ch. among these orfs, two orfs ( residues and residues) did not have significant similarity to any orfs in genbank database, including orfs of the viruses belong to family kirkoviridae. one orf ( residues) of other two orfs showed that it only hits a putative protein of po-circo-like virus (qc = %, i = %, ev = e- ) using a blastp search. interestingly, for another orf ( residues), partial aa fragment ( - aa) in the n-terminus showed % aa identity with the strain po-circo-like virus , but the region was deleted in strain po-circo-like virus , and the second region ( - aa) only showed . % aa identity with two-component system sensor histidine kinase bass of pectobacterium atrosepticum (genbank accession no. wp_ . ), which seems a recombination event in the orf. due to the lack of the parental nt sequences in genbank database, recombination analysis could not be further performed. in fact, it is not surpring that high rates of recombination in cress dna genomes had been reported (kazlauskas et al., ; lefeuvre and moriones, ) . to investigate the presence of this newly identified virus in other cattles, diarrheic and healthy faecal samples were collected from the provinces of sichuan (two farms), henan (three farms), liaoning (three farms) and shandong (three farms), china. a primer set was designed based on conserved rep sequences. the primer sequences were as follows; f: ′-aagaacacctggatgaaggaacgc- ′; r: ′-gccagtcatcaatcacaaccctct- ′, the amplified fragment was bp. viral dna was extracted from individual calf fecal samples using phenol-chloroform. the pcr amplification products were gelpurified using a gel extraction kit (omega, usa) and then cloned into the pmd -t vector and transformed into escherichia coli dh αcompetent cells. the recombinant plasmids were extracted using a plasmid extraction kit (omega, usa) and then sequencing was performed by sangon biotech (china, chengdu). in the case of bovine fecal samples, approximately . % ( / ) of diarrheic faecal samples were positive for bo-circo-like virus, and all healthy samples were negative by pcr assays. four positive samples were detected from two farms (detection rate: / and / , fig. . predicted genome organization of strain bo-circo-like virus ch. six orfs (> aa) were predicted in strain bo-circo-like virus ch, including cap, rep and four proteins of unknown function. the potential stem-loop was predicted to be located close to the putative rep gene stop codon in the intergenic region; detailed nucleotides sequences are also presented. respectively), and the geographical distance between two farms is more than km. the bp of the four bo-circo-like virus sequences shared . %- . % nt identity with bo-circo-like virus ch, and shares . %- . % nt identity with strains po-cicro-like virus . phylogenetic analysis of these partial rep nt sequences showed that all bo-circo-like virus were clustered into a dependent branch and were more related to po-cicro-like virus (fig. ) . the pcr screening sequence fragments were deposited in genbank under the following fig. . phylogenetic analysis was performed based on the amino acid sequence of orf presumable encodes rep protein. the sequence alignments included strain bo-circo-like virus ch in this study, representative members of the circoviridae, geminiviridae, nanoviridae, genomoviridae, bacilladnaviridae and smacoviridae families, the proposed new genera of krikoviruses, and still unassigned novel cress-dna viruses with the best blastp matchs in genbank database. the tree was constructed by the maximum-likelihood method with lg model and bootstrap replicates in mega . software. only bootstrap values > % are shown. the strain in this study is marked with red box, and seven reported strains of the proposed family kirkoviridae are marked with black boxes. (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article). accession numbers mh -mh . although the number of fecal samples was limited, all bo-circo-like virus in this study were detected from diarrheic faecal samples. recently, one member named kirkovirus equ in the proposed family kirkoviridae was suggested that it may be potentially related to disease, and the virus was found in the liver and spleen of a horse with fatal idiopathic hepatopathy (li et al., ) . thus, further investigation is needed to determine the pathogenesis of the bo-circo-like virus. in conclusion, a novel bovine-derived cress dna virus, named bo-circo-like virus ch, was identified from a calf with fatal hemorrhagic enteritis in china. it shows a circular genome of nt with six putative orfs, including rep protein, cap protein and four proteins of unknown function. based genome organization and phylogenetic analysis, the bo-circo-like virus ch may represent a new virus in the proposed family kirkoviridae. further investigation is needed to determine the pathogenesis of the virus. the raw sequence reads from the viral metagenomic libraries have been deposited into as bioproject, biosample and sra at ncbi, the accession number were prjna , samn and srp , respectively. the complete genome sequence of bo-circolike virus ch has been deposited in genbank under accession no. mh ; the partial rep sequences of bo-circo-like virus have been deposited in genbank under accession nos. mh -mf . this work was funded by sichuan province applied foundation project (grant number jy ), the innovation team for animal epidemic diseases prevention and control on qinghai-tibet plateau, state ethnic affairs commission (grant number td ) and the innovative research project of graduate students in southwest university for nationalities (grant number cx sz ). the authors declare that they have no conflicts of interest. the study does not contain any studies with human participants or animals performed by any of the authors, and all experiments in this study are in compliance with ethical standards for research. fig. . phylogenetic analysis based on partial orf nucleotides sequences ( -nt) of presumable encodes rep gene. the sequence alignments included five bo-circo-like virus strains detected in this study and seven reported strains of the proposed family kirkoviridae. the tree was constructed by the maximum likelihood method with hasegawa-kishino-yano model and bootstrap replicates in mega . software. only bootstrap values > % are shown. the strains in this study are marked with red boxes. (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article). novel circular dna viruses in stool samples of wild-living chimpanzees ictv report consortium, . ictv virus taxonomy profile: circoviridae two novel circo-like viruses detected in human feces: complete genome sequencing and electron microscopy analysis identification of circo-like virus-brazil genomic sequences in raw sewage from the metropolitan area of são paulo: evidence of circulation two and three years after the first detection a novel astrovirus species in the gut of yaks with, diarrhoea in the qinghai-tibetan plateau insights into the epidemic characteristics and evolutionary history of the novel porcine circovirus type in southern china genetic diversity of novel circular ssdna viruses in bats in china cloning and sequencing of duck circovirus (ducv) case control comparison of enteric viromes in captive rhesus macaques with acute or idiopathic chronic diarrhea evolutionary history of ssdna bacilladnaviruses features horizontal acquisition of the capsid gene from ssrna nodaviruses pervasive chimerism in the replicationassociated proteins of uncultured single-stranded dna viruses identification of a novel single-stranded, circular dna virus from bovine stool evolution of eukaryotic single-stranded dna viruses of the bidnaviridae family from genes of four other groups of widely different viruses genomoviridae : a new family of widespread single-stranded dna viruses mega : molecular evolutionary genetics analysis version . for bigger datasets mycovirus-like dna virus sequences from cattle serum and human brain and serum samples from multiple sclerosis patients recombination as a motor of host switches and virus emergence: geminiviruses as case studies widely conserved recombination patterns among single-stranded dna viruses possible cross-species transmission of circoviruses and cycloviruses among farm animals exploring the virome of diseased horses fecal virome of healthy chickens reveals a large diversity of the eukaryote viral community, including novel circular single-stranded dna viruses first detection and analysis of a fish circovirus cycloviruses, gemycircularviruses and other novel replication-associated protein encoding circular viruses in pacific flying fox (pteropus tonganus) faeces recombination in eukaryotic single stranded dna viruses idba-ud: a de novo assembler for single-cell and metagenomic sequencing data with highly uneven depth the fecal viral flora of wild rodents cyclovirus in nasopharyngeal aspirates of chilean children with respiratory infections geminiviruses and plant hosts: a closer examination of the molecular arms race revisiting the txonomy of the family circoviridae: establishment of the genus cyclovirus and removal of the genus gyrovirus virus-derived ssdna vectors for the expression of foreign proteins in plants the fecal virome of pigs on a high-density farm viruses with circular single-stranded dna genomes are everywhere! circular replication-associated protein encoding dna viruses identified in the faecal matter of various animals in new zealand identification of a novel circovirus in australian ravens (corvus coronoides) with feather disease sequence-based taxonomic framework for the classification of uncultured single-stranded dna viruses of the family genomoviridae smacoviridae: a new family of animal-associated singlestranded dna viruses plasma virome of cattle from forest region revealed diverse small circular ssdna viral genomes metagenomic analysis of viromes of dromedary camel fecal samples reveals large number and high diversity of circoviruses and picobirnaviruses viral metagenomics analysis demonstrates the diversity of viral flora in piglet diarrhoeic faeces in china intestinal virome changes precede autoimmunity in type i diabetes-susceptible children mfold web server for nucleic acid folding and hybridization prediction key: cord- -dbrp vxc authors: gibbs, shawn g.; herstein, jocelyn j.; le, aurora b.; beam, elizabeth l.; cieslak, theodore j.; lawler, james v.; santarpia, joshua l.; stentz, terry l.; kopocis-herstein, kelli r.; achutan, chandran; carter, gary w.; lowe, john j. title: review of literature for air medical evacuation high-level containment transport date: - - journal: air medical journal doi: . /j.amj. . . sha: doc_id: cord_uid: dbrp vxc abstract introduction aeromedical evacuation (ae) is a challenging process, further complicated when a patient has a highly hazardous communicable disease (hhcd). we conducted a review of the literature to evaluate the processes and procedures utilized for safe ae high-level containment transport (ae-hlct) of patients with hhcds. methods a literature search was performed in pubmed/medline (from through january ). authors screened abstracts for inclusion criteria and full articles were reviewed if the abstract was deemed to contain information related to the aim. results our search criteria yielded publications and were separated based upon publication dates, with the natural break point being the beginning of the - ebola virus disease epidemic. best practices and recommendations from identified articles are subdivided into pre-flight preparations, inflight operations, and post-flight procedures. conclusions limited peer-reviewed literature exists on ae-hlct, including important aspects related to healthcare worker fatigue, alertness, shift scheduling, and clinical care performance. this hinders the sharing of best practices to inform evacuations and equip teams for future outbreaks. despite the successful use of different aircraft and technologies, the unique nature of the mission opens the opportunity for greater coordination and development of consensus standards for ae-hlct operations. air medical evacuation (ae) is a challenging process, further complicated when a patient has a highly hazardous communicable disease (hhcd). the ease of air travel, tourism, and expansion of international commerce exposes all regions of the world to these diseases. the preference is to treat patients with hhcds on-site, rather than transport from the outbreak area ; however, high-level containment transport (hlct) evacuations may be preferred when ) there is an incapacity of the local infrastructure to provide care, ) there is a potential detrimental effect to local health care workers (hcws) (ie, the patient is a colleague), ) the outbreak is in an active war or conflict zone, ) it is a policy decision (to increase volunteerism), or ) there are local or national political concerns. regardless, successful ae hclts of patients with hhcds requires a discussion on risks, benefits, planning, training, and resources. the to ebola virus disease (evd) epidemic prompted multiple ae hlcts; at least nations conducted ae hlcts for at least patients with evd within the country and internationally. [ ] [ ] [ ] [ ] the ae-hlcts were conducted by single-patient isolation transports. since that epidemic, multiple groups have developed ae hlct systems enabling simultaneous isolation and care of multiple patients; these include the us department of state containerized bio-containment system and the us department of defense (dod) transport isolation system. the us centers for disease control and prevention issued ae guidance for evd in . although portions of the guidance were broadly applicable, it was evd specific, lacked discussion of logistical challenges, and did not include experiences from recently conducted ae hlcts. no ae hlcts during the epidemic had secondary transmissions although they were conducted differently by each organization. some evacuation procedures were preestablished and drilled, whereas others were based on situational needs. ae hlct has increased since it was introduced in the s, but no literature review comparing approaches has been published. a literature review queried the intersection of key words "biological warfare" and "aeromedical evacuation" or "transportation of patients" and yielded a single citation; today, that same search yields results. [ ] [ ] [ ] [ ] this study's purpose is to provide a more comprehensive evaluation of the processes and procedures used for safe ae hlcts of patients with hhcds in preflight, in-flight, and postflight environments. a literature search was performed in pubmed/medline (from through january ) with the following terms: ) "aeromedical isolation," ) "aeromedical evacuation" or "transportation of patients" or "air ambulance" or "hems" or "helicopter" and "ebola" or "lassa" or "viral hemorrhagic" or "highly infectious" or "highly hazardous" or "contagious" or "communicable" or "middle east respiratory syndrome (mers)" or "sars" or "smallpox", and ) "mobile" or "transport" and "high-level isolation" or "high containment". authors screened abstracts for the following inclusion criteria: peer-reviewed literature, written in english, and described ae hlct of persons with an hhcd. diseases considered highly hazardous were identified based on the following definition by the european network for highly infectious diseases: "an infection that is easily transmissible from person to person; life-threatening; presents a serious hazard in the health-care setting and the community; and requires specific control measures (e.g., high-level isolation)." this definition is understood to include various viral hemorrhagic fevers, severe acute respiratory syndrome (sars), and other easily transmissible emerging infectious diseases. articles were reviewed if the abstract contained information related to the aim, with those focused exclusively on ground transport or ae of non-hhcd patients excluded. the search terms yielded publications; met the inclusion criteria and were included in the study (tables and ). the articles were separated based on publication dates, with the natural break point being the to evd epidemic. thoms et al discussed drawing on the operational experience from phoenix air corporation, a private organization that began ae hlcts in when it developed the aeromedical biological containment system. the us department of state, united nations, and other governments used that single-patient transport system for ae hlcts of patients with evd in to . although phoenix air corporation's ae hlct experience is widely known, details of their procedures and policies were not published in peer-reviewed literature and were not available. planning for and executing ae hlcts must account for multiple variables; our review is organized around "preflight, in-flight, and postflight" environments. a broad spectrum of diseases was covered in the reviewed articles, including airborne diseases, biological warfare agents, , , and viral hemorrhagic fevers. articles published before targeted many diseases (table ) , whereas articles published after (table ) focused almost exclusively on evd. pre- articles included considerations for airborne isolation, whereas post- articles stressed contact isolation associated with evd. the reviewed articles understated the considerable collaborations involved in ae hlct decision making because most only vaguely mentioned frequent discussions and multiagency requests must occur before transport. [ ] [ ] [ ] nicol et al did indicate that the decision to evacuate patients is a "complex process that considers the clinical, public health, and political contexts." although no article identified a decision-making rubric for deploying ae hlct assets, several discussed factors involved in the decision-making process (eg, recommendations by domestic and international agencies). lotz and raffin indicated their transport met recommendations set by the world health organization for medical evacuation of patients with high infectious risk ( - hours). thoms et al noted that ". . . u.s. military policy is to treat highly infectious patients 'in place', and avoid unnecessary evacuation to the u.s." but acknowledged instances in which transport would occur, such as index cases or for political considerations. given the current emphasis on military participation in nation-building efforts, it is unlikely that adequate resources for "treatment in place" will be present during future outbreaks. as such, the military may become increasingly reliant on ae hlcts. patient stability and survivability were noted as principal factors in the decision to conduct an ae hlct; a patient moved before the onset of severe disease manifestations is preferable and, at times, a requirement for transport because of limited isolation units. , , , [ ] [ ] [ ] ae hlct places additional stressors associated with altitude on the patient that impact their physical condition (eg, hypoxia and claustrophobia). , , , , articles identified a lack of local facilities with resources and capabilities as a reason for domestic or international evacuation. , , volunteers supporting humanitarian endeavors overseas are often assured that they will be repatriated should they become ill, as was the case during the to evd epidemic when at least evd-infected hcws/volunteers were evacuated to their home countries. no reviewed articles detailed the types, duration, requirements, or frequency of training. biselli et al noted training includes personal protective equipment (ppe), patient management on ground and inflight, and equipment decontamination, whereas christopher and eitzen, which detailed a royal air force mission, remarked on the benefit of in-flight, just-in-time training that occurred on the flight to the patient, while also stating that the mission resulted in routine air transport isolator exercises. as with many fields, it is difficult to determine applicable training and exercise needs for ae hlct. organizations work internally (considering equipment, mission, and personnel) to determine the appropriate training and delivery to maintain competency. the regulations and legal limitations associated with the ae hlct were not fully explored in any reviewed article. two articles mentioned the need to adhere to organizational policies, , noted a requirement to obtain consent from governments for transports and one stated ae hlct teams routinely seek diplomatic clearance when flying over other nations, but none discussed applicable federal or international regulations. withers and christopher , discussed the need for regulations to address the unpredictable reaction of the international community in a hhcd event but primarily focused on the biological weapons and toxin convention protocol. schilling et al discussed the need for flight certification to ensure materials are deemed safe to fly. withers and adams et al detailed preference for long-range capable aircrafts to limit refueling stops. all us military aircraft used in ae hlct missions are capable of midair refueling and are able to eliminate stops for fuel and/or extend flights to avoid the airspace of hostile or reluctant nations. four studies mentioned the importance of effective communication and coordination among partners, but none discussed details of communication plans. , , , seven articles did identify organizations that would be contacted to initiate a transport, albeit at a high level. , , , , , , thoms et al detailed crew predeparture briefings. the article by christopher and eitzen was the only one to detail communication plans with the patient in-flight, namely, -way radios between team members and patients. ewington et al and thoms et al detailed the space layout within the c aircraft that both evacuations used, including placement and securing of the isolation unit. thoms et al detailed "aircraft containment zones" for patient areas within the aircraft where hcws and crew could move within the aircraft and procedures and level of ppe that hcws and crew would need for each zone. nicol et al also demarcated clean and dirty zones for confirmed patients and established a corridor for access to toilets and eating spaces for exposed, asymptomatic cases. zone designation lends the ability to transport multiple patients at different stages of disease progression for the same disease or, more likely, to transport both suspected and confirmed patients. in the air medical transportation of a lassa fever patient from nigeria to germany, the following zones were established: a containment zone where the patient was located, a crew zone where ppe was not worn, and a neutral zone between the that was also available for plane-related emergency procedures. withers and christopher , stated that military "flight nurses know that cabin airflow is 'top to bottom, front to back' on the c- a nightingale; therefore, contagious patients are placed as far aft and as low as possible." withers and christopher , also noted particular considerations (eg, high-efficiency particulate air systems, air exchanges/hour, and negative pressure zones) are made on the ventilation systems within each aircraft for potential dispersion of aerosolized microbes from a contagious patient that is either uncontained in an isolation unit or may have been unknowingly contagious. the professional training level of ae hlct personnel varied ( table ). the articles by thoms et al and nicol et al were the only ones that explicitly noted the care team could be augmented with additional support to ensure adequate staff levels for the full flight dependent on the number of patients transported and, in the case of thoms et al, for flight duration; however, no details were provided on the targeted staffing-to-patient ratio or the flight duration that would demand augmented staff. although a critical issue, the time personnel spent in ppe is not extensively discussed. schilling et al noted a portable anteroom is used for ppe donning and doffing when flights exceeded hours. lamb noted the ae hlct team worked shifts consisting of nurse and paramedic, enabling the rest of the team to eat, sleep, and rest. other articles lacked analysis and recommendations for hcw fatigue x indicates the subject was included in the article. and shift rotation during longer transports. ppe can be cumbersome and trigger hcw physiological and psychological distress-even in environmentally controlled biocontainment facilities -and may be exacerbated at altitude. appropriate work-rest cycles; considerations to time in ppe; and fatigue, alertness, and clinical performance monitoring are important during ae hlct. the objective analysis of these factors is necessary to maximize performance and safety. every article mentioned the importance of proper ppe use, but few detailed ppe ensembles, and none described donning and doffing procedures. ewington et al noted that decontamination procedures were overseen by a designated and trained ppe monitor but lacked details on the ppe level or type. dindart et al stated their personnel used "full ppe" with no details provided; however, based on article images, it appears they used the world health organization−recommended ppe (goggles, procedure masks, fluid-resistant hood, fluidresistant coveralls, gloves, and boots). thoms et al described their use of "coveralls, multiple pairs of surgical gloves, rubber outer boots and a powered air purifying respirator (papr) system to prevent skin exposure"; christopher and eitzen and withers and christopher described similar configurations. schilling et al discussed the physical stress of working in a respirator but did not specify type; however, images indicate a ppe configuration similar to thoms et al. nicol et al repeatedly noted that once sealed, patient care during transport with the trexlar air transport isolator (t-ati) does not require staff to wear ppe. although lamb did not specify in-flight use, ppe similar to that described in the article by dindart et al was used for personnel that helped transport the patient onto the aircraft. most articles indicated that a portable isolation unit, such as the air transport isolators used by the italian air force and british military (previously used by usamriid), the t-ati currently used by the british military, the vickers aircraft transport isolator (previously used by usamriid), or the human stretcher transit isolator-total containment (oxford) limited (hsti-tcol) used domestically in guinea, were operated in-flight. , , , , , , the hsti-tcol was described in detail with significant limitations, including the inability to restrain the patient during turbulence or place items (eg, medicine, devices) into the unit once the patient is enclosed. although these portable units were described in varying levels of detail, each offered complete enclosure for a single patient, barrier protection for the hcws, and high-efficiency particulate air−filtered negative pressure air. , , most depended on batteries with a hour life, whereas others had the ability to use the aircraft's electrical system. , experiments showed that portable isolation chambers may leak or rupture when exposed to an explosive decompression ; therefore, contingency procedures should be in place. sweden and italy use a combined ground and air transport whereby a specially designed and equipped ambulance is driven inside of a c- . the patient remains in the ambulance in-flight; essentially, the ambulance becomes an isolation unit. this combination reduces loading time and the likelihood of aircraft contamination. the british military uses a dedicated road transport vehicle for the t-ati positioned at the receiving air base for seamless transport to the destination facility. a major limitation of transport systems was the inability to house multiple patients. newer systems currently in validation seek to alleviate this limitation. the transport isolation system is a dod containment modality designed and approved for loading onto c- and c- military aircraft; each system (aluminum frame with clear plastic liner that maintains a negative pressure isolation environment) is capable of moving multiple patients simultaneously, and such systems can be accommodated on the larger c- platform. the containerized bio-containment system is a us state department−sponsored platform similar to a hard-sided shipping container with viewing ports and a negative-pressure isolation environment. it has the capability to transport patients simultaneously with space for multiple caregivers and is designed to be loaded onto the c- (not yet approved by the us air force) or the boeing airframe. , procedures/capabilities in-flight care provided during ae hlcts will not be equivalent to that available at a dedicated health care facility. however, several articles detailed the ability to provide a wide range of medical procedures in-flight (eg, endotracheal incubation and defibrillation) , , , ; other articles implied in-flight procedures were limited to monitoring. the type of isolation unit limits capabilities in-flight; for example, the hsti-tcol detailed in the study by dindart et al is a sealed pod and enables only limited interventions (eg, intravenous rehydration and antiemetics). in reviewing articles and operational experiences for evd, we found a lack of consideration and planning for liquid and solid waste. there is a general underestimation of the volumes of both produced in-flight and an unclear understanding of the rules and regulations that govern waste during each transport phase. lotz and raffin and ewington et al indicated waste generated by the patient in-flight were kept within the isolation unit but segregated from the patient. upon transport completion, the isolation unit was enveloped, and all associated waste destroyed , ; however, methods for packaging, transporting, and subsequent waste destruction were not described. thoms et al stated a transportable lavatory would be included on the aircraft and used to capture liquid waste but did not discuss the handling and storage of solid wastes generated in-flight (eg, ppe). nicol et al noted waste can be minimized in-flight by using containers with absorbent powder or solidifying agents but did not detail the process. lamb discussed the process of double bagging ppe used for patient receival with the intention to dispose with waste generated in-flight but did not elaborate. dindart et al indicated that waste generated in-flight would be collected and incinerated postflight; however, no details were provided. withers and christopher , discussed criteria for a decontaminating compound (eg, effective within a short time, in low concentrations with low human toxicity, stable shelf life, and compatible with aircraft materials). this stressed the importance of the compound compatibility with aircraft materials. only nicol et al mentioned the existence of a mortuary protocol if the patient were to pass away in-flight, stating only that a death inflight is "managed with standard procedures, which vary depending on the jurisdiction of the flight." in the case of a death in-flight, a decision would have to be made to either continue to the destination or return to the departure origin based on factors such as distance traveled, available fuel, political considerations, and other patients awaiting transport. although such a decision would be made in communication with decision makers on the ground, preliminary discussions of this contingency would be beneficial. several potential in-flight emergency scenarios were discussed. ewington et al acknowledged the potential of an isolation unit breach and noted the medical engineer would conduct repairs immediately. in discussing emergency evacuation procedures, thoms et al noted crew would don patients in ppe to reduce exposure and minimize contact with rescuers or nonmission personnel. postflight details were limited in most reviewed articles. dindart et al stated "the plane is decontaminated using a chlorine solution at every point of contact between the pod and the plane, which take about min." thoms et al indicated that a dilution of disinfectant solution calla (zip-chem products, morgan hill, ca) and sani-wipes (disposables international, incorporated, orangeburg, ny) were available during the transport. it also stated that postflight "medical crewmembers and/or equipment will be decontaminated per current policy"; however, there were no policy details. lotz and raffin indicated that "disinfection of the cabin of the aircraft and medical equipment with nocolyse (oxy'pharm; champigny-sur-marne, france) spray (hydrogen peroxide, catalyst, biosurfactant)" is conducted after mission conclusion. schilling et al detailed the use of formaldehyde fumigation as the final decontamination posttransport and indicated that sweden used a nonflammable peracetic acid for decontamination of the staff. nicol et al stated the t-ati system was fumigated with vaporized hydrogen peroxide and the frame decontaminated and returned for reuse after days. tsai et al detailed the use of bleach solution spray on the isolation unit and ppe before air transport of patients with sars, and the use of water spray and desiccation on the isolation unit upon transport completion. wilson and driscoll also reported the use of bleach for surface decontamination before boarding the aircraft. posttransport decontamination of aircraft differed. efficacy is the primary intention; however, decontamination agents must also comply with aircraft material compatibility. the viability and stability of pathogens differ; therefore, decontamination methods may be adapted based on the hhcd. lufthansa technik, a german laboratory, found disinfectant components effective against hhcds while also aircraft compatible (alcohol, formaldehyde, and hydrogen peroxide) and detailed standard operating procedures for aircraft disinfection. more research and information on regulations are needed to support safe aircraft decontamination. waste disposal details were lacking. two articles indicated waste was incinerated , but did not specify how it was packaged or transported before incineration. likewise, nicol et al noted the isolator envelope is autoclaved on-site and disposed of as regulated clinical waste after decontamination but did not provide additional details. in the united states, the terminal disposal of category a waste (of which evd and many other hhcds are classified) is costly and requires specific packaging and a vendor with a department of transportation special permit to move and process the waste. all transporting organizations should have written protocols and procedures for the terminal disposal of category a waste and, when necessary, preidentify a certified vendor if the waste is not able to be autoclaved, incinerated, or deactivated on-site to downgrade the hazardous materials classification. thoms et al mentioned only that an infectious disease physician might screen medical personnel postflight. tsai et al indicated that personnel performed twice daily temperature monitoring for days after a sars transport. lamb noted that personnel were monitored for only hours after returning to the united kingdom because the transported patient later tested negative for lassa fever and positive for malaria. as with monitoring of hcws providing care in high-level containment facilities, postmission monitoring of crew and hcws should be included in written protocols to minimize the opportunity for further transmission. there are limitations to this review. ae of trauma patients and cases of other communicable diseases that are not highly hazardous may offer important considerations for operating procedures that were not included in this review. there also exists an inherent bias in the exclusion of non−english language documents, as well as the lack of access to publicly available non−peer-reviewed resources produced by various organizations. additionally, our review was conducted specifically searching for "highly hazardous" and "highly infectious" diseases. other terms are also used, but these were not included in the literature because we were aware that european high-level containment facilities and the majority of federal documents used the terms "highly infectious" or "highly hazardous communicable" diseases before and during the evd epidemic. moreover, this review focuses specifically on ae of patients with hhcds; clearly, the ground transportation facet is a critical component of the safe movement of such patients and has its own challenges and risks. since the evd epidemic, the us state department and dod have developed systems for ae hlcts of multiple patients of varying levels of hhcd acuity during the same operation. although ae hclt during the evd epidemic was managed within phoenix air corporation's capabilities, a larger global epidemic may demand scalability. ae hlct systems advancement with increased space and ability to perform care within the unit enables more advanced patient care procedures than available in single-patient isolation. however, with the improved capability for in-flight care, discussions are needed on what medical procedures should be conducted in-flight, focusing on minimizing aerosol generation. additionally, post- reviewed articles (table ) reflect the increasing staffing demands for patients with evd; the transport of multiple patients with hhcds will only enhance the resource-intensive nature of these missions. ae hlct poses significant risks to crews. high hhcd mortality rates and the unstable environment inherent in aes require policies and procedures to decrease transmission risks and maximize patient management. the designation of high-level isolation facilities in the united states and europe narrows the list of potential receiving facilities; procedures should be well discussed and thoroughly exercised between transporting organizations and their respective receiving facilities. a future outbreak of a hhcd is likely; advancing the field of ae hlct is critical. there is limited peer-reviewed literature available on ae hlct, including important aspects related to hcw fatigue, alertness, shift scheduling, and clinical care performance. few experienced teams have published details on their processes, experience, and operations, and this limited breadth of literature hinders the sharing of best practices to inform evacuations and equip teams for future outbreaks. despite the successful use of different aircraft and technologies, the unique nature of the mission opens the opportunity for greater coordination and the development of consensus standards for ae hlct operations. supplementary material associated with this article can be found, in the online version, at doi: . /j.amj. . . . mobile high-containment isolation: a unique patient care modality long-range transportation of ebolaexposed patients: an evidence-based protocol the immediate psychological and occupational impact of the sars outbreak in a teaching hospital pre-hospital transportation in western countries for ebola patients, comparison of guidelines aerial medical evacuation of health workers with suspected ebola virus disease in guinea conakry-interest of a negative pressure isolation pod-a case series transferring patients with ebola by land and air: the british military experience clinical management of ebola virus disease in the united states and europe guidance on air medical transport (amt) for patients with ebola virus disease (evd) aeromedical evacuation: management of acute and stabilized patient aeromedical evacuation of biological warfare casualties: a treatise on infectious diseases on aircraft new scenarios in major accidents−use and adaption of current concepts to ward off damage jubail−an aeromedical staging facility during the gulf conflict: discussion paper association for professionals in infection control and epidemiology inc. and centers for disease control and prevention framework for the design and operation of high-level isolation units: consensus of the european network of infectious diseases how phoenix air entered the ebola business transporting patient with suspected sars air evacuation under high-level biosafety containment: the aeromedical isolation team transporting patients with lethal contagious infections aeromedical transfer of patients with viral hemorrhagic fever aeromedical evacuation using an aircraft transit isolator of a patient with lassa fever european concepts for the domestic transport of highly infectious patients the added value of preparedness for aeromedical evacuation of a patient with ebola containment aircraft transit isolator evaluation of infection control practices during an ae neuilly sur seine, france: advisory group for aerospace research and development ebola virus disease: preparedness and infection control lessons learned from two biocontainment units world health organization. personal protective equipment in the context of filovrius disease outbreak response airplane transport isolators may lose leak tightness after rapid cabin decompression ready for the challenge: dobbins selected as home for new biocontainment system mil/desktopmodules/articlecs/print.aspx?portalid= &moduleid= &ar-ticle= nebraska biocontainment unit perspective on disposal of ebola medical waste disinfection of aircraft: appropriate disinfectants and standard operating procedures for highly infectious diseases need for aeromedical evacuation high-level containment transport guidelines key: cord- -k g r lw authors: maykowski, philip; smithgall, marie; zachariah, philip; oberhardt, matthew; vargas, celibell; reed, carrie; demmer, ryan t.; stockwell, melissa s.; saiman, lisa title: seasonality and clinical impact of human parainfluenza viruses date: - - journal: influenza other respir viruses doi: . /irv. sha: doc_id: cord_uid: k g r lw background: widespread availability of rapid diagnostic testing for respiratory viruses allows more in‐depth studies of human parainfluenza viruses (hpiv). objectives: this study aimed to assess seasonality of hpiv types ‐ , clinical outcomes by hpiv type, and risk factors for illness severity. patients/methods: this retrospective study was performed from january to december in children and adults with hpiv, detected by multiplex reverse transcription polymerase chain reaction, participating in a community surveillance study of acute respiratory infections (aris) in new york city and patients admitted to a tertiary care center in the same neighborhood. seasonality trends by hpiv type were compared between the community and hospital groups. the associations between hpiv type, demographics, clinical characteristics, and illness severity were assessed. results: hpiv was detected in ( %) of community surveillance participants (median age . years) and hospitalized patients (median age . years). seasonality for hpiv types ‐ agreed with previously described patterns; hpiv‐ occurred annually in late summer and fall. in the community cohort, ( %) participants sought medical care, ( %) reported antibiotic use, and ( %) reported ≥ day of missed work or school. among hospitalized patients, % had ≥ chronic conditions. multivariable ordinal logistic regression demonstrated that increased severity of illness was significantly associated with hpiv‐ and chronic cardiovascular and respiratory conditions in children and with age ≥ years and chronic respiratory conditions in adults. conclusions: hpiv‐ presented late summer and early fall annually and was associated with increased severity of illness in hospitalized children. human parainfluenza viruses (hpiv) types - are common respiratory pathogens that cause both upper and lower respiratory tract illnesses, especially in young children. hpiv- and hpiv- are more common in children and more often associated with croup. hpiv- is more frequently associated with bronchiolitis, bronchitis, and pneumonia. hpiv- , while least well-characterized, has been described as associated with both mild illness in children and lower respiratory tract disease. hpiv types have varying seasonality, prevalence, and clinical manifestations. in the united states, hpiv- usually occurs in the fall of odd numbered years, hpiv- occurs every fall, and hpiv- occurs in spring and summer. , the seasonality of hpiv- has not been as well defined, often due to its low prevalence. a recent study conducted in colorado found a year-round prevalence of hpiv- with peaks in the fall of odd numbered years. however, estimates of hpiv- prevalence are increasing, potentially due to improved and increased diagnostic testing, but few recent studies have assessed this. , widespread availability of multiplex reverse transcription polymerase chain reaction (rt-pcr) assays allows for more in-depth studies of the epidemiology and impact of hpiv types. the population of this study included both a community-based cohort and hospitalized children and adults with laboratory-detected hpiv. the inclusion of the community cohort allowed for a broader evaluation of the impact of hpiv than has been previously assessed. the study's objectives were to (a) assess the seasonality of hpiv types - , (b) determine clinical outcomes by hpiv type, and (c) identify risk factors for increased severity of illness. a retrospective study of participants in a community surveillance cohort and hospitalized patients with hpiv types - detected from january , to december , was performed. the community cohort was derived from the mobile surveillance for acute respiratory infections (aris) and influenza-like illness (ili) in the community (mosaic) study, a -year community-based surveillance ordinal logistic regression demonstrated that increased severity of illness was significantly associated with hpiv- and chronic cardiovascular and respiratory conditions in children and with age ≥ years and chronic respiratory conditions in adults. conclusions: hpiv- presented late summer and early fall annually and was associated with increased severity of illness in hospitalized children. epidemiology, parainfluenza, respiratory, seasonality, viruses f i g u r e flowcharts depicting the overall number of respiratory viral panel (rvp) tests ordered which yielded the final number of human parainfluenza virus (hpiv) types in the community cohort ( a) and in hospitalized patients ( b) study in new york city (nyc) that includes households annually. , households were identified by contacting a random sample of participants who had taken part in a large population-based survey of an urban, primarily immigrant latino community. for this study, eligible households had or more members with at least member under years of age, were spanish-or english-speaking, and had a cellular telephone with text messaging. text messages were sent twice weekly inquiring about possible respiratory illness among household members. nasal swabs were collected from ill participants by research staff if at least two of the following were reported to be present: fever/feverishness, runny nose/congestion, sore throat, cough, and/or myalgia. for infants, either runny nose or congestion prompted collection of a swab. hospitalized adults and children with laboratory-detected hpiv admitted to a university-affiliated medical center in nyc located in the same neighborhood as the community cohort were included. testing was requested by treating clinicians, and as per the standard of care, is generally recommended for all hospitalizations to evaluate acute respiratory symptoms and guide transmission precautions. patients hospitalized within calendar-days of hpiv detection were included. those with hpiv detected > days before admission, > days after admission and those with a second positive test for the same hpiv type within weeks of the first positive test were excluded. nasopharyngeal swabs for both the community cohort and the hospitalized patients were tested for respiratory pathogens using multiplex rt-pcr (biofire diagnostics, inc. salt lake city, utah) which has been reported to have %- % sensitivity and %- % , swabs from the community were tested in a research laboratory at columbia university medical center (cumc). swabs from hospitalized patients were tested in the clinical microbiology laboratory at newyork-presbyterian hospital at cumc. blood, urine, and respiratory tract cultures for bacteria were sent by treating clinicians as per the standard of care for suspected infections and also processed by the clinical microbiology laboratory. for the community cohort, days of missed school or work; medical care in primary care clinics, urgent care, or emergency departments; hospitalizations; and use of antibiotics were collected by follow-up calls at least days after the resolution of respiratory illness. for hospitalized patients, demographic and clinical characteristics were obtained from electronic medical records (emr). primary icd- diagnoses were categorized as respiratory or nonrespiratory. patients' primary and secondary diagnoses were categorized into groups of chronic conditions previously described as risk factors for severe respiratory disease in adults and children with respiratory viruses. [ ] [ ] [ ] conditions were excluded if they could not be classified within a chronic condition category, had < occurrences across all patients, or have not been associated with severe respiratory disease, for example bipolar disorder. manual chart review of readmitted patients was performed to determine whether readmissions within weeks were due to respiratory illness. hospital course and healthcare utilization data were obtained from the emr including radiographs within days of hpiv detection; all antibiotics within days prior to or days after hpiv detection; icu admission and -day mortality. respiratory support including use of continuous positive airway pressure (cpap), mechanical ventilation, or extracorporeal membrane oxygenation (ecmo) was determined by billed procedure codes. use of bilevel positive airway pressure (bipap) or oxygen supplementation was not assessed, as accurate usage data were unavailable in structured electronic sources. ta b l e comparison of demographic characteristics, symptoms, and outcomes of participants in a community cohort, by human parainfluenza viruses (hpiv) type to assess seasonality, epidemiologic curves that modeled the detection of each hpiv type in the community cohort versus hospitalized patients were created. demographic and clinical characteristics of the two groups were analyzed as proportions, means, and/or medians; associations between hpiv type and clinical course were examined using chi-square, fisher's exact, and anova tests, as appropriate. ta b l e comparison of demographic and clinical characteristics of hospitalized patients with different human parainfluenza viruses (hpiv) in the community cohort, swabs were obtained, of which ( %) of participants were positive for at least one hpiv type ( figure a ). the hpiv-positive swabs were from households; hospital community at least one hpiv type ( figure b) the mean length of hospitalization was . (sd ± . ) days and varied by hpiv type ( were associated with higher odds of increased severity of illness. among the adults, ( %) had severe, ( %) had moderate, and ( %) had mild illness (table ). in the final adjusted model for adults, age ≥ years and respiratory conditions were associated with higher odds of increased severity of illness. we had a unique opportunity to compare the epidemiology and clinical impact of hpiv types among individuals in a communitybased surveillance study and among hospitalized patients during the same time and in the same geographic area. the seasonality of hpiv types - was consistent with published trends, , but we found that hpiv- occurred annually in late summer and fall, suggestive of a local epidemiologic trend that has not been previously described. we found that hpiv- was most common among hospitalized patients ( %) while hpiv- was most common in the community cohort ( % we modified a previously validated severity of illness score to further explore the predictors of illness severity. in children, increased severity of illness was associated with respiratory conditions, cardiovascular conditions and hpiv- while in adults, increased severity of illness was associated with age ≥ years and cardiovascular conditions. the association of comorbid conditions and severe illness in those with hpiv is consistent with other respiratory viruses, most notably rsv and influenza, which both can exacerbate underlying cardiac and pulmonary conditions. [ ] [ ] [ ] , this may also explain why % of hospitalized patients had a nonrespiratory primary diagnosis. the rate of co-detection was similar across hpiv strains and as others have shown, viral co-detections did not impact severity of disease. however, due to limited sample size, we could not assess the impact of specific co-infections. while only % of hpiv-positive patients had a positive bacterial culture, % received at least one dose of an antibiotic between days before and days after hpiv detection. thus, most antibiotic usage represented empiric therapy, suggesting an opportunity for antibiotic stewardship. this study had limitations. the community cohort was % hispanic/latino, reducing the study's generalizability, and small, limiting our ability to find differences in severity of illness or symptomatology associated with hpiv types. our hospital is a referral center and cares for many patients with underlying conditions. furthermore, its referral status could impact accurate interpretation of "local" epidemiology as only % of the hospitalized patients lived in the same zip codes as the mosaic study participants (m. stockwell, personal communication). testing of hospitalized patients relied on clinicians' judgement. we did not review radiographic findings or capture bipap use which may have biased assessment of severity of illness. the severity of illness score was developed for children and not previously tested in adults. use of administrative data restricted our ability to assess patients' symptomatology. although most patients had a respiratory diagnosis, we may have misclassified hpiv infections that actually represented prolonged hpiv shedding from previous illnesses. we did not classify positive bacterial cultures as consistent with infection vs. colonization vs. contamination. in this study, each hpiv type demonstrated seasonality, but each was similar in the community and among hospitalized patients. we found that hpiv- had distinct epidemiology, not previously described, and was associated with increased severity of illness in hospitalized children. as demonstrated for other respiratory viruses, older age and underlying conditions, particularly respiratory and cardiac conditions, were associated with increased severity of illness. additional studies exploring hpiv incidence and severity in both children and adults could help reinforce the need for vaccine development. the authors thank alexandra hill-ricciuti for analytic assistance. seasonal trends of human parainfluenza viral infections: united states estimates of parainfluenza virus-associated hospitalizations and cost among children aged less than years in the united states epidemiology and clinical presentation of parainfluenza type in children: a -year comparative study to parainfluenza types - epidemiology of parainfluenza infection in england and wales, - : any evidence of change? human parainfluenza type infections mosaic: mobile surveillance for acute respiratory infections and influenza-like illness in the community community -and hospital laboratory-based surveillance for respiratory viruses. influenza other respir viruses filmarray respiratory panel information sheet multicenter evaluation of the eplex respiratory pathogen panel for the detection of viral and bacterial respiratory tract pathogens in nasopharyngeal swabs respiratory syncytial virus infection in elderly and high-risk adults children with complex chronic conditions in inpatient hospital settings in the united states pediatric deaths attributable to complex chronic conditions: a population-based study of washington state predicting severe pneumonia outcomes in children human parainfluenza virus types - in hospitalized children with acute lower respiratory infections in china a prospective study of parainfluenza virus type infections in children attending daycare severe pneumonia after cardiac surgery as a result of infection with parainfluenza virus type human parainfluenza virus outbreak and the role of diagnostic tests defining the risk and associated morbidity and mortality of severe respiratory syncytial virus infection among infants with chronic lung disease influenza virus infections among patients attending emergency department according to main reason to presenting to ed: a -year prospective observational study during seasonal epidemic periods single-and multiple viral respiratory infections in children: disease and management cannot be related to a specific pathogen seasonality and clinical impact of human parainfluenza viruses key: cord- -eumuid r authors: widagdo, w.; okba, nisreen m. a.; richard, mathilde; de meulder, dennis; bestebroer, theo m.; lexmond, pascal; farag, elmoubasher a. b. a.; al-hajri, mohammed; stittelaar, koert j.; de waal, leon; van amerongen, geert; van den brand, judith m. a.; haagmans, bart l.; herfst, sander title: lack of middle east respiratory syndrome coronavirus transmission in rabbits date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: eumuid r middle east respiratory syndrome coronavirus (mers-cov) transmission from dromedaries to humans has resulted in major outbreaks in the middle east. although some other livestock animal species have been shown to be susceptible to mers-cov, it is not fully understood why the spread of the virus in these animal species has not been observed in the field. in this study, we used rabbits to further characterize the transmission potential of mers-cov. in line with the presence of mers-cov receptor in the rabbit nasal epithelium, high levels of viral rna were shed from the nose following virus inoculation. however, unlike mers-cov-infected dromedaries, these rabbits did not develop clinical manifestations including nasal discharge and did shed only limited amounts of infectious virus from the nose. consistently, no transmission by contact or airborne routes was observed in rabbits. our data indicate that despite relatively high viral rna levels produced, low levels of infectious virus are excreted in the upper respiratory tract of rabbits as compared to dromedary camels, thus resulting in a lack of viral transmission. middle east respiratory syndrome coronavirus (mers-cov) is a novel pathogen that is known to infect dromedary camels and humans [ , ] . seroepidemiological studies indicate that this virus has been circulating in dromedary camels in the arabian peninsula and africa for decades [ ] [ ] [ ] . mers-cov sequences obtained from these camels are largely similar to those obtained from human mers cases in corresponding regions, thus providing evidence that dromedary camels act as the zoonotic source of this virus [ , ] . however, many primary human mers cases do not have a history of direct contact with these animals [ ] . this suggests the presence of unidentified routes of human-to-human transmission or the involvement of other animal species in spreading the virus to humans. besides dromedary camels, other animal species, i.e. llamas, alpacas, and pigs have been shown to be susceptible and develop upper respiratory tract infection upon experimental intranasal mers-cov inoculation [ ] [ ] [ ] . this is in line with the expression of the mers-cov receptor, dipeptidyl peptidase- (dpp ), in their nasal epithelium [ ] . mers-cov-seropositive llamas and alpacas have been reported in the field, and mers-cov-experimentally-inoculated alpacas have also been shown to transmit the virus via contact [ ] [ ] [ ] . to further understand the zoonotic potential of mers-cov, it is crucial to delineate the factors involved in the spread of the virus among dromedaries as well as other animal species. in order to gain insight into these factors, we performed mers-cov transmission experiments in rabbits. we have previously shown that rabbits are susceptible to mers-cov and develop both upper and lower respiratory tract infection upon virus inoculation [ ] . naïve recipient rabbits were housed with mers-cov-inoculated donor rabbits either in the same or in adjacent cages to determine whether mers-cov can be transmitted via contact or airborne routes, respectively [ ] . donor rabbits were found to shed high levels of viral rna but a limited amount of infectious virus thus potentially restricting mers-cov transmission in these animals. in vivo experiments in this study were performed using passage human isolate mers-cov emc strain (hcov-emc/ ) and passage isolate mers-cov (qatar / ; genbank acc. no. mk ) that were propagated in vero cells as described earlier [ ] . qatar was isolated from a years old qatari man that developed severe pneumonia and was pcr confirmed to have a mers-cov infection [ ] . animal experiments were approved and performed according to the guidelines from the institutional animal welfare committee (no. approved on july , - - approved on september , and avd -wp approved on november ). the studies were performed under biosafety level (bsl ) conditions. to compare whether different routes of mers-cov inoculation generate similar clinical outcomes, twelve -month-old new zealand rabbits (oryctolagus cuniculus), specific pathogen free, and seronegative for mers-cov were divided into four groups. animals were inoculated under ketamine-medetomidine anesthesia either (a) intranasally with µl of × tcid /ml mers-cov; (b) intranasally with ml × tcid /ml mers-cov; (c) intranasally with µl of × tcid /ml mers-cov and intratracheally with ml × tcid /ml mers-cov; or (d) intranasally with ml pbs. the intranasal inoculums were divided equally over both nostrils. nasal and throat swabs were obtained daily from day up to day post inoculation. these animals were then sacrificed on day and respiratory tract tissues were collected. to compare the clinical outcomes of the mers-cov emc strain and the qatar strain, ten new zealand rabbits were divided into two groups and inoculated with ml of × tcid /ml of each mers-cov strain intranasally. nasal and throat swabs were obtained from day up to day post inoculation and these animals were sacrificed on day . to study mers-cov transmission, a modified version of the previously described influenza a virus ferret transmission set-up was used. this set-up consists of two clear polymethyl methacrylate cages of different sizes. donor rabbits and direct contact recipients were housed in a cage of cm × cm × cm (w × h × l), whereas airborne recipients were housed in a cage of cm × cm × cm (w × h × l). these cages are separated by two stainless steel grids cm apart to prevent direct contact but still allow airflow from the donor rabbit to the airborne recipient rabbit. these transmission cages allow the experiment to be conducted in negatively pressured isolators in the bsl facility, with hepa-filtered airflow < . m/s [ ] . since these cages were too small for new zealand rabbits, we chose a smaller-sized breed, the netherland dwarf rabbits (oryctolagus cuniculus), for the mers-cov transmission experiment. we used both male and female rabbits with an age viruses , , of range of months- years in these experiments. first, three mers-cov seronegative netherland dwarf rabbits were inoculated intranasally with ml of × tcid /ml mers-cov qatar strain ( µl per nostril) to show that they are equally susceptible to mers-cov as the new zealand rabbits. nasal and throat swabs were obtained daily up to days post inoculation. these animals were then sacrificed on day and their respiratory tract tissues were collected. for the virus transmission experiment, twelve netherland dwarf rabbits were randomly distributed into four individually housed groups. one naïve rabbit from each group was inoculated intranasally with ml of × tcid /ml mers-cov ( µl per nostril), thus acting as donor rabbits. the other two rabbits were used as direct contact and airborne recipients, respectively. donor and direct contact animals were of the same sex. all animals were sacrificed at days post exposure and blood was collected to assess seroconversion. nasal swabs, throat swabs, and respiratory tract tissue samples were evaluated for the presence of infectious virus by virus titration, and for viral rna by rt-qpcr against the upe gene as previously described [ ] . samples with a cycle threshold less than forty were considered as positive for mers-cov rna. viral rna was quantified as genome equivalents (ge) using mers-cov strain emc (containing tcid /ml) as a calibrator. virus titration was performed in serial -fold dilutions on vero cells. cells were monitored under a light microscope at day for cytopathic effect. the amount of infectious virus in swab samples was calculated by determining the tcid . statistical analysis was performed using the graphpad prism program (la jolla, ca, usa). kruskal-wallis test was applied due to the non-normal distribution of our data as priorly determined by shapiro-wilk test. the significant difference between groups was determined at a p-value < . . respiratory tract tissue samples were collected in formalin and embedded in paraffin for pathological analysis. hematoxylin-eosin staining was performed for histopathological analysis. the presence of mers-cov nucleoprotein and mers-cov rna was detected by immunohistochemistry and in-situ hybridization, respectively, using previously published protocols [ ] . the localization of dpp in the respiratory tract of non-infected new zealand rabbits was analyzed using an optimized immunohistochemical assay [ , ] . collected serum samples were tested for mers-cov neutralizing antibodies using a virus neutralization assay and for mers-cov s -specific antibodies using mers-cov s elisa according to the previously published protocols [ ] . goat anti-rabbit igg conjugated with hrp ( : , dako, glostrup, denmark) was used as a secondary antibody in the elisa. rabbits are the smallest animal species that can be naturally infected by mers-cov. we previously reported that they develop both upper and lower respiratory tract infection upon mers-cov inoculation [ ] , suggesting the expression of the viral receptor at these locations. using immunohistochemistry, we analyzed the dpp expression in rabbit respiratory tract tissues. in the upper respiratory tract, dpp is strongly expressed at the apical surface of both nasal respiratory and olfactory epithelium ( figure ). in the lower respiratory tract, dpp is present in both bronchiolar and alveolar epithelial cells, although some variation in dpp expression was observed throughout the lungs. dpp is either absent, limitedly expressed on alveolar type ii cells, or expressed on both alveolar type i and ii cells ( figure ). thus, these data highlight a broad dpp expression in the respiratory tract different human mers-cov strains have been isolated since the emc/ strain was first characterized [ , ] . however, studies that evaluate phenotypic differences between these strains in animals are currently lacking. we investigated whether a more recent mers-cov strain (qatar / ) replicates differently in rabbits in comparison to the emc strain. we found that rabbits inoculated with the mers-cov emc strain and those with the qatar strain developed an equally mild infection and shed similar levels of viral rna in their nasal and throat swabs ( figure ). following this result, the mers-cov transmission experiment was performed using the qatar strain, the more recent strain of these two. type ii cells are indicated with arrows, and type i cells with an arrowhead. nasal epithelium and bronchiolar epithelium pictures were taken at a × magnification, and alveolar epithelium at ×. the isotype control showed no background signal in our assay. in our previous study, we inoculated rabbits both intranasally and intratracheally [ ] . intratracheal inoculation is quite invasive, and thus requires a skillful operator to minimize procedure-related damage in the respiratory tract. in contrast, intranasal inoculation is less invasive and had been used in other studies to infect rabbits with mers-cov [ , ] . here we investigated whether intranasal mers-cov inoculation is sufficient to induce both upper and lower respiratory tract infection in rabbits, in comparison to combined intranasal and intratracheal inoculation. three new zealand rabbits (oryctolagus cuniculus) were inoculated with mers-cov emc strain either intranasally with µl of × tcid /ml (group a); intranasally with ml of × tcid /ml (group b); intranasally with µl of × tcid /ml and intratracheally with ml of × tcid /ml (group c); or intranasally with ml of pbs as a negative control (group d). all three groups of mers-cov inoculated rabbits developed minimal clinical manifestations and histopathological lesions. the amount of viral rna shed in the nasal and throat swabs did not vary among the inoculated groups (figure a,b) . however, in the lungs of the rabbits, the amount of viral rna was significantly lower in group a than in groups b and c ( figure c ). in line with these observations, fewer mers-cov-infected cells were observed in the lungs of group a animals compared to groups b and c ( figure d ). based on these results, we decided to use intranasal inoculation with ml of × tcid /ml mers-cov for our subsequent experiments. pictures were taken at a × magnification, and alveolar epithelium at ×. the isotype control showed no background signal in our assay. different human mers-cov strains have been isolated since the emc/ strain was first characterized [ , ] . however, studies that evaluate phenotypic differences between these strains in animals are currently lacking. we investigated whether a more recent mers-cov strain (qatar / ) replicates differently in rabbits in comparison to the emc strain. we found that rabbits inoculated with the mers-cov emc strain and those with the qatar strain developed an equally mild infection and shed similar levels of viral rna in their nasal and throat swabs (figure ). following this result, to study mers-cov transmission, an experimental set up previously used to investigate influenza a virus transmission between ferrets was used. this set up consists of two polymethyl methacrylate cages separated with two steel grids, cm apart [ ] . because this set-up was too small to house new zealand rabbits, we used a smaller-sized breed, the netherland dwarf rabbits. prior to the virus transmission experiment, netherland dwarf rabbits were inoculated with mers-cov qatar strain to determine their susceptibility to the virus. similar to the new zealand rabbits [ ] , netherland dwarf rabbits shed viral rna in the nasal and throat swabs as well as in the respiratory tract tissues upon intranasal inoculation ( figure a,b) . identical to the new zealand rabbits [ ] , the mers-cov-inoculated netherland dwarf rabbits did not develop any clinical signs, including nasal discharge, and showed minimal histopathological lesions and immune cell infiltration in the respiratory tract. to study mers-cov transmission, four netherland dwarf rabbits were intranasally inoculated with mers-cov. six-hours later, each of them was co-housed with one naïve rabbit in the same cage, and h later another one was co-housed in an adjacent cage to determine whether mers-cov could be transmitted through contact and/or airborne routes. nasal and throat swabs were collected every other day up to day or post inoculation/exposure for the donor and direct contact rabbits, respectively, and day post exposure for the airborne recipient ones. both viral rna and infectious virus were quantified in these samples. we found that all donor rabbits shed high loads of viral rna in both the nasal swabs (~ - tcid ge/ml) and the throat swabs (~ - tcid ge/ml). the nasal and throat swabs were obtained from day (before inoculation) until day post inoculation. the amount of viral rna is displayed in genome equivalents per ml (ge/ml). dashed lines depict the detection limit of the assay. all error bars represent standard deviations. to study mers-cov transmission, an experimental set up previously used to investigate influenza a virus transmission between ferrets was used. this set up consists of two polymethyl methacrylate cages separated with two steel grids, cm apart [ ] . because this set-up was too small to house new zealand rabbits, we used a smaller-sized breed, the netherland dwarf rabbits. prior to the virus transmission experiment, netherland dwarf rabbits were inoculated with mers-cov qatar strain to determine their susceptibility to the virus. similar to the new zealand rabbits [ ] , netherland dwarf rabbits shed viral rna in the nasal and throat swabs as well as in the respiratory tract tissues upon intranasal inoculation ( figure a,b) . identical to the new zealand rabbits [ ] , the mers-cov-inoculated netherland dwarf rabbits did not develop any clinical signs, including nasal discharge, and showed minimal histopathological lesions and immune cell infiltration in the respiratory tract. donor rabbits at day post inoculation, and in one of the donors up to day . in the throat swabs, infectious virus was only detected in two donors on day , up to day in one of them. in contrast, none of the swabs from recipient rabbits was positive for virus titration (figure c,d) . serological analysis of samples collected days after exposure showed that only the donor rabbits seroconverted and developed neutralizing antibodies ( figure a,b) . the antibody response of these directly inoculated rabbits was relatively low, confirming the results of previous studies [ , ] . this indicates that these rabbits developed mers-cov infection while the contact and airborne-exposed rabbits did not, supporting the results of the virus titration. to study mers-cov transmission, four netherland dwarf rabbits were intranasally inoculated with mers-cov. six-hours later, each of them was co-housed with one naïve rabbit in the same cage, and h later another one was co-housed in an adjacent cage to determine whether mers-cov could be transmitted through contact and/or airborne routes. nasal and throat swabs were collected every other day up to day or post inoculation/exposure for the donor and direct contact rabbits, respectively, and day post exposure for the airborne recipient ones. both viral rna and infectious virus were quantified in these samples. we found that all donor rabbits shed high loads of viral rna in both the nasal swabs (~ - tcid ge/ml) and the throat swabs (~ - tcid ge/ml). the amount of viral rna shed by the inoculated rabbits remained high until day post inoculation. on the other hand, recipient rabbits housed in the same cage (direct contact recipients), or adjacent cage (airborne recipients), shed limited amounts of viral rna (~ tcid ge/ml) in both nasal and throat swabs. among four animals in each group, only two direct contact recipient and two airborne recipient rabbits had detectable viral rna up to day post inoculation in the nasal swabs, while in the throat swabs, viral rna was only detected in one direct contact recipient and one airborne recipient rabbit at day post inoculation ( figure a,b) . infectious virus was detected at low level (~ tcid /ml) both in the nasal and throat swabs of the donor rabbits; in the nasal swabs of all donor rabbits at day post inoculation, and in one of the donors up to day . in the throat swabs, infectious virus was only detected in two donors on day , up to day in one of them. in contrast, none of the swabs from recipient rabbits was positive for virus titration ( figure c,d) . serological analysis of samples collected days after exposure showed that only the donor rabbits seroconverted and developed neutralizing antibodies ( figure a,b) . the antibody response of these directly inoculated rabbits was relatively low, confirming the results of previous studies [ , ] . this indicates that these rabbits developed mers-cov infection while the contact and airborne-exposed rabbits did not, supporting the results of the virus titration. current data indicate that mers-cov is highly endemic in dromedary camels in the arabian peninsula and africa and has been circulating in these animals for decades [ ] [ ] [ ] ] . this suggests that this virus is easily transmitted between dromedary camels. from an epidemiological point of current data indicate that mers-cov is highly endemic in dromedary camels in the arabian peninsula and africa and has been circulating in these animals for decades [ ] [ ] [ ] ] . this suggests that this virus is easily transmitted between dromedary camels. from an epidemiological point of view, it is important to know whether other animal species in the region may also spread the virus to humans or other animal species. in vitro, mers-cov has been found to infect cells from a broad range of animal species including old and new world camelids as well as primates, bats, cows, sheep, goats, pigs, horses, and rabbits [ , , ] . the dpp viral receptor of these species, especially rabbits, has high similarity to that of humans and dromedary camels, especially in the region that interacts with the spike protein, and thus can facilitate mers-cov infection [ ] [ ] [ ] . the new world camelids, i.e. llamas and alpacas, have been shown to seroconvert to mers-cov when present in regions where mers-cov is circulating and may transmit the virus [ ] [ ] [ ] . it is currently unclear why, besides camelids, other livestock animals do not seem to transmit the virus to humans [ , [ ] [ ] [ ] . to further understand the transmission potential of mers-cov, we performed virus transmission experiments using rabbits as animal model. to perform the virus transmission experiments, we housed mers-cov-inoculated rabbits together with naïve contact rabbits either in the same or adjacent cages. rabbits developed both upper and lower respiratory tract infection upon mers-cov inoculation [ ] , either via intranasal or combined intranasal and intratracheal routes, in line with the localization of dpp in their respiratory tract epithelium. the amount of viral rna being shed by the inoculated rabbits during the first three days post inoculation is almost as high as that of the mers-cov-inoculated dromedary camels [ , ] . however, none of the direct contact and airborne-exposed rabbits developed any clinical signs, shed significant levels of viral rna, shed infectious virus, nor did they seroconvert. one possible reason for this lack of transmission is the limited amount of infectious virus being shed by the inoculated rabbits [ ] . previous studies have shown that in the nasal and lung tissues of experimentally infected rabbits, infectious virus was generally detected in a limited amount despite the abundant presence of viral rna and virus nucleoprotein [ , ] . alternatively, low levels of infectious virus transmitted to recipient animals may be unable to initiate a productive infection due to the presence of host proteins that restrict replication. comparable to rabbits, mers-cov-infected pigs and goats develop minimal clinical signs, hardly shed infectious virus, and barely spread the virus to naïve animals [ , ] . in contrast, mers-cov-infected dromedary camels develop nasal discharge and shed a high amount infectious virus ( - tcid /ml), almost equal to the amount of viral rna being shed ( - ge/ml), during the first days post inoculation [ , ] . these interspecies differences may indicate presence of nasal discharge and infectious virus shedding as critical factors in mers-cov transmission. in humans, levels of infectious virus shed by mers-cov patients have rarely been reported. however, mers-cov patients that transmit the virus have been shown to shed a higher amount of viral rna in their swabs compared to those that do not, supporting the quantity of virus shed as an important factor in the transmission of mers-cov between humans [ ] . for influenza a viruses, infectious virus shedding has been documented as one of the main determinants of airborne virus transmission. using ferrets as an animal model, it has been reported that a reduction in infectious virus shedding in the nasal swabs can subsequently limit virus transmission [ ] [ ] [ ] . our results show that despite the presence of dpp in the upper respiratory tract, accompanied by mers-cov replication at this site, a limited amount of infectious virus was shed. similarly, titration of rabbit lung homogenates that show high levels of viral rna and presence of nucleoprotein ( figure c ,d) resulted in only low levels of infectious virus, in line with earlier observations [ , ] . at this stage, it is not clear which host mediated mechanisms limit the production of infectious virus while allowing viral rna to still be shed at high levels. since restriction of infectious virus shedding in the rabbits already occurred one day post inoculation, activation of host innate immune responses, including type i interferon induction, may be relevant. in vitro studies have shown that mers-cov is relatively sensitive to type i interferon-mediated antiviral activities [ , ] . in human plasmacytoid dendritic cells, mers-cov inoculation leads to secretion of large amount of type i and iii interferons and production of viral rna, but hardly any infectious virus is being produced [ ] . it is also possible that most infectious virus shed by these rabbits is defective, lacking the capacity to efficiently infect target cells. further studies are needed to elucidate the mechanisms that restrict mers-cov replication in rabbits compared to dromedary camels. potentially, some of the mers-cov accessory proteins shown to antagonize immune responses including production of interferon, may not work efficiently in some mers-cov susceptible species, including rabbits. it is intriguing to investigate whether a similar phenomenon occurs in some human mers-cov infections and whether this is linked to the development of asymptomatic to mild clinical manifestations [ ] . this might partly explain why mers-cov transmission in humans is rather inefficient in comparison to dromedary camels [ , ] , and why camelids that secrete high levels of infectious virus are the only known zoonotic source of mers-cov [ , , , ] . deciphering these mechanisms could potentially offer insight into understanding mers-cov transmission as well as developing novel treatments to tackle the ongoing outbreaks. isolation of a novel coronavirus from a man with pneumonia in saudi arabia isolation of mers coronavirus from a dromedary camel middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia geographic distribution of mers coronavirus among dromedary camels zoonotic origin and transmission of middle east respiratory syndrome coronavirus in the uae middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation reported direct and indirect contact with dromedary camels among laboratory-confirmed mers-cov cases 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mammals in egypt an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels middle east respiratory syndrome coronavirus experimental transmission using a pig model risk factors for transmission of middle east respiratory syndrome coronavirus infection during the outbreak in south korea impact of prior seasonal h n influenza vaccination or infection on protection and transmission of emerging variants of influenza a(h n )v virus in ferrets efficacy of seasonal live attenuated influenza vaccine against virus replication and transmission of a pandemic h n virus in ferrets predicting "airborne" influenza viruses: (trans-) mission impossible? mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-alpha treatment inhibition of novel beta coronavirus replication by a combination of interferon-alpha b and ribavirin high secretion of interferons by human plasmacytoid dendritic cells upon recognition of middle east respiratory syndrome coronavirus world health organization mers situation update transmission of mers-coronavirus in household contacts mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -kv ekg authors: takano, tomomi; yamada, shinji; doki, tomoyoshi; hohdatsu, tsutomu title: pathogenesis of oral type i feline infectious peritonitis virus (fipv) infection: antibody-dependent enhancement infection of cats with type i fipv via the oral route date: - - journal: j vet med sci doi: . /jvms. - sha: doc_id: cord_uid: kv ekg feline infectious peritonitis virus (fipv) causes a severe, immune-mediated disease called fip in domestic and wild cats. it is unclear whether fip transmits from cat to cat through the oral route of fipv infection, and the reason for this includes that fip is caused by oral inoculation with some fipv strains (e.g., type ii fipv wsu - ), but is not caused by other fipv (e.g., type i fipv ku- strain: fipv-i ku- ). in this study, when cats passively immunized with anti-fipv-i ku- antibodies were orally inoculated with fipv-i ku- , fip was caused at a % probability, i.e., fipv not causing fip through oral infection caused fip by inducing antibody-dependent enhancement. many strains of type i fipv do not cause fip by inoculation through the oral route in cats. based on the findings of this study, type i fipv which orally infected cats may cause fip depending on the condition. feline coronavirus (fcov) belongs to the genus alphacoronavirus, subfamily coronavirinae, family coronaviridae [ ] . fcov is classified into two serotypes (i and ii), based on differences in the amino acid (aa) sequence of spike (s) protein and antibody neutralization. furthermore, fcov exists as two different biotypes: feline enteric coronavirus (fecv: avirulent fcov) and feline infectious peritonitis virus (fipv: virulent fcov) [ ] . antibodies against virus enhance viral load and disease severity in some viral infections including fip. this phenomenon is known as antibody-dependent enhancement (ade) of viral infection [ ] . ade of fipv infection can be induced by the presence of sub-neutralizing levels of anti-fipv s antibodies [ ] . unlike dengue virus infection, ade was induced by re-infection with the identical serotype virus in fipv infection [ ] . fecv is spread predominantly through the oral route in cats [ ] . on the contrary, it is unclear fipv transmits from cat to cat through the oral route of fipv infection. it is suggested that the incidence of fip in cats is dependent on the route of fipv infection. the incidences of fip in cats intraperitoneally and oronasally inoculated with the type i fipv ucd strain were . and %, respectively [ ] . in a similar fashion, a previous study described that oral inoculation with type i fipv ku- strain (fipv-i ku- ) cannot lead to fip in cats, but subcutaneous and intraperitoneal inoculation with fipv-i ku- can lead to fip [ , ] . in this study, we investigated whether oral inoculation with fipv-i ku- causes fip in cats passively immunized with anti-fipv-i ku- antibodies. in addition, we investigated whether ade of type i fipv infection can be promoted in vitro using feline macrophages. anti-fcov antibody-negative seven specific pathogen free (spf) cats aged - months were used. the cats were maintained in a temperature-controlled isolated facility. all experiments were approved by the president of kitasato university through the judgment of the institutional animal care and use committee of kitasato university ( , and - ), and performed in accordance with the guidelines for animal experiments of kitasato university. sample sizes were determined based on the previous study, and the minimum number of cats was used. passive immunization with anti-fipv-i ku- antibodies was performed as described before [ ] . briefly, three cats (ab , ab , and ab ) were subcutaneously administrated with serum from fipv-i ku- -infected healthy cats, and one cat (ab ) was administrated with igg purified from fipv-i ku- -infected cats-derived ascites by ammonium sulfate precipitation followed affinity purification on a protein a column. the neutralizing antibody titer against fipv-i ku- was : in both the serum-and purified igg. the cats were orally inoculated with fipv-i ku- ( . tcid /head) days after passive immunization. as a control, three cats (c , c , and c ) without passive immunization were inoculated with the virus (fig. a) . cats were euthanized when reaching the humane endpoint or days after inoculation with fipv. the test sera were serially -fold diluted in medium and mixed with an equal volume of a virus suspension containing approximately tcid / µl and the mixtures were incubated at °c for min. each mixture was then inoculated into the felis catus whole fetus- cells (kindly supplied by dr. m. c. horzinek of utrecht university) in -well flat-bottomed plates, and incubation was made in an atmosphere of % co in air at °c for days. for each serum dilution, tests were duplicated. the neutralizing antibody titer (nt) was expressed as a reciprocal of the highest dilution of the test sera that inhibiting cytopathic effect completely. the elisa for anti-fcov antibodies was performed as described by takano et al [ ] . briefly, detergent-disrupted, purified fipv virions were diluted appropriately with carbonate buffer ( . m, ph . ). a total of µl of the dilution was pipetted into each well of a -well flat-bottomed plate. the plates were allowed to stand overnight at °c, washed with pbs containing . % tween- , and µl of the test serum sample was then added to each well. horseradish peroxidase-conjugated goat anti-cat igg (icn pharmaceuticals inc., costa mesa, ca, u.s.a.) was diluted to optimal concentration with pbs containing % fcs and . % tween- , and µl of dilution was added to each well of plates. after incubation at °c for min, µl of the substrate solution was added to each well and plates were incubated at °c for min in a dark room. the substrate solution was prepared by dissolving o-phenylenediamine dihydrochloride at a concentration of . mg/ml in . m citric acid and . m na hpo buffer (ph . ), and . µl/ml of % h o was then added. the reaction was stopped with n h so solution and the optical density (o.d.) at nm was determined. rna was isolated from rectal swab samples by a method reported previously [ ] . to synthesize cdna from fipv genomic rna, µl rna extract and . mol sense primer for the fipv nucleocapsid (n) gene (positions - , ′-caactggggagatgaacctt- ′) were added to ready-to-go rt-pcr beads (ge healthcare life sciences) and the volume was adjusted to µl with water. the resulting solution was incubated at °c for hr to synthesize cdna. cdna was amplified by pcr using primers specific for the fcov n gene (sense primer, positions - , ′-caactggggagatgaacctt- ′; antisense primer, positions - , ′-ggtagcatttggcagcgtta- ′). pcr was performed as reported previously [ ] . for the macrophages, feline alveolar macrophages, which are used for analysis of ade of type ii fipv infection [ ] , were selected. feline alveolar macrophages were obtained from spf cats by broncho-alveolar lavage with hank's balanced salt solution (hbss) as described previously by hohdatsu et al. [ ] . feline alveolar macrophages were maintained in rpmi growth medium supplemented with % fcs, u of penicillin per ml, µg of streptomycin per ml, and µm -mercaptoethanol. viral suspension (fipv-i ku- , . tcid / ml) and igg purified from fipv-i ku- -infected cats-derived ascites (subneutralizing titer: final concentration of : ) were mixed in an equal volume ratio and allowed to react at °c for hr, and . ml of this reaction solution was used to inoculate feline alveolar macrophages ( cells) cultured in each well of -well lab-tek chamber slide (thermo fisher scientific, waltham, ma, u.s.a.). as controls, igg alone and virus suspension alone were added to feline alveolar macrophages. after virus adsorption at °c for hr, the cells were washed with hbss and then added ml of growth medium. after hr, n protein levels were determined by immunofluorescence assay (ifa), as described previously [ ] . for recognizing fipv-i ku- n protein, mab yn (mouse igg b) prepared by our laboratory [ ] was used. fipv-infected cells were analyzed using a leica dm microscope and las x integrated imaging system (leica microsystems, wetzlar, germany). two cats (ab and ab ) were euthanized at and day of post fipv infection (dpi) with fipv-i ku- , respectively, when they reached the humane endpoint. these animals showed febrile (> . °c), lethargy, anorexia, and jaundice. upon necropsy, ascites was noted in two cats with fip and pyogranulomatous lesions were present in the intestine and spleen. pleural effusion and inflammatory lesions in the lung were noted in ab . in passively immunized cats without clinical symptoms after fipv infection, several - -mm nodules were observed in the intestine, but there was no other lesion. cats inoculated orally with fipv-i ku- without passive immunization did not develop clinical symptoms. no fip-related lesion was noted on necropsy at dpi with fipv-i ku- . figure b shows the survival rate of cats infected with fipv-i ku- . the survival rate of cats with passive immunization was lower than that of the cats without passive immunization, and the average survival time after infection with fipv-i ku- was also shorter. we tested for the presence of neutralizing antibodies against fipv-i ku- in cats with passive immunization. in cats with passive immunization, nt was increased to - fold on the day of virus inoculation (day post passive immunization) ( fig. a) . nt was maintained at a constant level in cats excluding cats (ab and ab ) which developed fip after viral challenge. in cats without passive immunization, nt started to increase on dpi with fipv-i ku- , and reached - fold on dpi (fig. b) . time-course changes in the serum anti-fipv antibody level were investigated by elisa using purified soluble fipv antigen. in cats passive immunization, the elisa od value was increased to . - . on the day of virus inoculation (day post passive immunization). as with the nt titer, the elisa od value was continuously increased in cats excluding cats developed fip (ab and ab ; fig. c ). in cats without passive immunization, the elisa od value continuously increased - dpi after inoculation with fipv-i ku- , and reached . - . on - dpi (fig. d ). rectal swab samples from cats were subjected to rt-pcr targeting fcov n gene. in cats with passive immunization, fcov n gene was detected day after virus inoculation in ab . however, fcov n gene was not detected in any samples of the other cats (table ) . based on the findings described above, fipv-i ku- was suggested to promote ade through not only subcutaneous but also oral infection. however, the mechanism of ade induced by fipv-i ku- infection is unclear. to elucidate this mechanism, it is necessary to promote fipv-i ku- -induced ade in vitro. therefore, the ade infection with fipv-i ku- was performed in feline macrophage. no fcov n protein was detected in macrophages treated with only igg purified from ascites of fipv-i ku- -infected cats (fig. a) . in macrophages treated with only fipv-i ku- , fcov n protein was detected in . ± . % (mean ± s.d.) of cells (fig. b) . in macrophages treated with both purified igg and fipv-i ku- , fcov n protein was detected in . ± . % (mean ± s.d.) of cells (fig. c) , showing that the fipv-i ku- infection rate in macrophages increased in the presence of the antibody. cats orally infected with fipv-i ku- do not develop fip. on the basis of this fact, the biotype (phenotype) of the orally inoculated fipv-i ku- is classified as "fecv". however, fipv-i ku- has the genetic characteristics of "fipv", i.e., the sequence of the s /s site of fipv-i ku- is rsrss (p r→s) [ ] , and aa at position has been changed from methionine to leucine [ ] . in addition, aa is deleted from c protein in fipv-i ku- [ , ] . furthermore, no amino acid deletion was noted in b protein of fipv-i ku- [ , ] . it is unclear which of these regions is involved in the pathogenicity of fipv-i ku- . it is now possible to prepare recombinant type i fcov by reverse genetics [ , ] . it is desired to mutate the regions associated with pathogenicity in fipv-i ku- , inoculate cats with these mutants through various routes, and confirm whether the mutant causes fip. fcov n genes were hardly detectable from rectal swab samples of cats infected with fipv-i ku- . the reason for this is unclear. we previously confirmed that fcov n genes were detected from rectal swab samples after fipv-i ku- subcutaneous -i ku- ab ------ab - ------+: fcov n gene positive; -: fcov n gene negative. on the basis of these facts, we suggested that inoculation routes result in differential patterns of virus shedding in cats infected with fipv. generally, when a cat developed fip in multi-cat environments, cats living together also develop fip at a high probability [ ] . fipv excreted from cats with fip may infect other cats through the oral route. however, orally inoculated type i fipv mostly does not cause fip in cats [ ] . it has been difficult to explain these contradictory facts. it was clarified that even fipv not causing fip through oral infection may cause fip in anti-type i fcov-seropositive cats. however, not all anti-fipv seropositive cats develop fip. for example, ade is not promoted and the virus is neutralized in cats with a high anti-fipv neutralizing antibody level [ ] . fip also does not develop when cellular immunity is strongly induced after fipv infection [ ] . based on these findings, to elucidate the clinical state of fip, it is necessary to analyze the status of immunity in cats after viral infection. however, many recent studies on fip do not focus on the host but focus on the virus. we suggest that fip is a "multi-causal disease" involving various risk factors (virulence of fcov, the status of immunity in host, and the route of virus infection etc.). we confirmed that fip was caused in % when cats passively immunized with anti-fipv-i ku- antibodies were inoculated orally with fipv-i ku- , i.e., fipv not causing fip through oral infection caused fip by inducing ade. moreover, we were able to demonstrate that infection of fipv-i ku- to feline macrophages was enhanced by anti-fipv-i ku- igg. this study may provide a platform for understanding the mechanism of ade induced by oral viral infection. acknowledgment. this work was in part supported by mext/jsps kakenhi grant number jp k . feline infectious peritonitis. abcd guidelines on prevention and management spike protein fusion peptide and feline coronavirus virulence feline infectious peritonitis: insights into feline coronavirus pathobiogenesis and epidemiology based on genetic analysis of the viral c gene reverse genetics for type i feline coronavirus field isolate to study the molecular pathogenesis of feline infectious peritonitis the molecular genetics of feline coronaviruses: comparative sequence analysis of the orf a/ b transcription unit of different biotypes a study on the mechanism of antibody-dependent enhancement of feline infectious peritonitis 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upon re-infection with the identical serotype virus in feline infectious peritonitis virus infection tnf-alpha, produced by feline infectious peritonitis virus (fipv)-infected macrophages, upregulates expression of type ii fipv receptor feline aminopeptidase n in feline macrophages mutation of neutralizing/antibody-dependent enhancing epitope on spike protein and b gene of feline infectious peritonitis virus: influences of viral replication in monocytes/macrophages and virulence in cats genome organization and reverse genetic analysis of a type i feline coronavirus feline coronaviruses: pathogenesis of feline infectious peritonitis feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses key: cord- -cbtf y f authors: giuliano, a.; watson, p.; owen, l.; skelly, b.; davison, l.; dobson, j.; costantino‐casas, f. title: idiopathic sterile pyogranuloma in three domestic cats date: - - journal: j small anim pract doi: . /jsap. sha: doc_id: cord_uid: cbtf y f pyogranulomatous inflammation has been extensively described in cats, in particular in cases of feline infectious peritonitis and also associated with mycobacteria, actinomyces, nocardia, rhodococcus and fungal infections. idiopathic sterile pyogranulomatous dermatitis has also been described. in this case series we describe the clinical presentation, histopathology and outcome of three cases of feline idiopathic sterile steroid‐responsive pyogranuloma with different presentation and different locations of the lesion, but with the common feature of having a mass with no superficial skin involvement. pyogranulomatous inflammation (pi) is a chronic inflammatory lesion characterised by a predominance of macrophages and neutrophils, often in combination with plasma cells and giant cells (raskin ) . generalised pi has been extensively described in the veterinary literature, particularly in cases of feline infectious peritonitis (fip) (weiss & scott , kipar et al. . cutaneous, subcutaneous and, more rarely, systemic pi in cats is mainly associated with mycobacteria and fungal infections (malik et al. , , brömel & sykes , baral et al. with sporadic implication of actinomyces, nocardia, rhodococcus, streptomyces, francisella, bartonella and leishmania (patel ; valentine et al. ; malik et al. ; farias et al., ; santero et al. ; sharman et al. ; varanat et al. ; traslavina et al. ) . idiopathic sterile pyogranulomatous dermatitis has also been described in cats (scott et al. ). we describe the clinical presentation and outcome of three cases of feline idiopathic sterile pyogranuloma with different presentation and locations, but with the common picture of a mass with no superficial skin involvement. to our knowledge sterile pyogranuloma with mass appearance and absence of skin involvement have not previously been described in cats. a -year-old male neutered domestic short hair (dsh) cat was presented for investigation of a right sided naso-maxillary mass. generalised gingival inflammation, more severe over the right canine tooth, was noticed at a routine medical check. a dental polish, descale and a right canine root extraction was performed by the referring veterinarian. the facial swelling appeared weeks following the dental procedure and the cat was treated with weeks of amoxicillin-clavulanic acid without improvement. the owner did not report any clinical signs apart from occasional sneezing during the previous few weeks. on physical examination the patient was bright and alert. there was a firm, poorly defined mass extending from the right proximal maxilla to the nasal region causing facial deformation. no ulceration or skin lesions were present; oral examination was unremarkable except for a swelling on the right side of the proximal maxilla, close to the area of previously removed canine tooth. complete blood count (cbc) and biochemistry were unremarkable, felv antigen and fiv antibody tests were negative. oral radiography ruled out a remaining canine tooth root. due to the high suspicion of neoplasia a ct scan of head, neck and thorax was performed. the ct scan confirmed the soft tissue mass on the nasal dorsum extending laterally to the missing canine tooth and associated with lysis of the incisive and nasal bones. the mass extended into the right nasal cavity with destruction of the nasal turbinates and mild deviation of the nasal septum (fig ) . surgical wedge biopsies were taken after incision and blunt dissection of the skin and subcutis. the biopsies were taken from two different sites within the mass to increase the chances of collecting a representative sample and were immediately submitted for histopathology. due to the high suspicion of neoplasia, tissue samples for culture were not taken and no antibiotics or other types of treatment were started. microscopic examination exhibited severe pyogranulomatous and lymphocytic cellulitis and mild pyogranulomatous and lymphocytic myositis (fig. a, b) . there was no evidence of neoplasia in any of the samples examined. no infectious etiological agents could be detected following gram, ziehl nielsen (zn) and periodic acid schiff (pas) staining. immunohistochemistry for coronavirus fipv - antifeline coronavirus antibodies (tammer et al. ) was nega-tive. the sample was sent for pan-fungal (its -its region of ribosomal dna) as previously published (lau et al. ) and mycobacteria pcr assay (mycobacterial its -its regions) on the paraffin block sample as previously published (hughes et al. , fyfe et al. ) both of which were negative. bacterial s rna probe fish analysis for eubacteria was negative (maunder et al. ). on clinical examination the mass remained of similar size for the following months but the sneezing became more frequent. prednisolone treatment was started at mg/kg daily orally and the mass reduced in size and the sneezing markedly improved, the same dose of prednisolone was continued for months until last follow-up. the cat was still alive at the time of writing months after presentation and the facial swelling and the sneezing resolved almost completely. a -year-old female neutered outdoor dsh was presented for further investigation of an abdominal mass. the referring veterinary surgeon detected the mass as an incidental finding when the cat presented for annual booster vaccination. the patient did not have any history of previous illness and the owner did not report any current problems or clinical signs; the cat was otherwise fit and well. on abdominal palpation a non-painful, firm, partially mobile mass was present in the mid-caudal abdomen. at this stage, considering the size of the mass in the abdomen and the clinical presentation, the most likely diagnosis was neoplasia although fip, fungal and mycobacterial granuloma were also considered. cbc and biochemistry were unremarkable apart from mild hyperglobulinaemia g/l (reference interval to ). fiv antibody and felv antigen tests were negative. abdominal ultrasound showed a heterogeneous mass with mixed echogenicity and no other abnormalities. the mass measured approximately × cm, and was thought to originate from the mesenteric lymph node. cytology of fine needle aspirates (fnas) of liver and spleen taken to rule out the possibility of related disease in those organs was unremarkable. an fna of the mass revealed pi with mixed, mainly neutrophils and macrophages, infiltrates. an ultrasound-guided tru-cut biopsy of the mass was obtained under general anaesthesia, which confirmed the lesion to be pyogranulomatous. in view of the continued high suspicion of neoplasia, an exploratory laparotomy was performed. the mass was confirmed to originate from the mesenteric lymph node and could not be resected without damage to the intestinal blood supply. multiple wedge biopsies were obtained to submit for culture and histopathology. histopathological examination showed a pyogranulomatous and necrotising lymphadenitis, with fibrosis. no aetiological agents were visible on gram, zn and pas staining. immunohistochemistry for coronavirus (fipv - anti-feline coronavirus antibodies) was also negative. the sample was sent for pan-fungal pcr (its -its region of ribosomal dna) on paraffin tissue block and mycobacteria pcr (mycobacterial its region) on frozen sample that were both negative. bacterial s rna probe fish analysis for eubacteria was negative. due to the incidental finding and its asymptomatic nature it was decided to monitor the mass and no treatment was given. the mass remained of similar size and no clinical signs related to it were ever reported. the cat was still alive and well almost years after diagnosis when she died in a road traffic accident. a -year-old male neutered dsh was presented for further investigation of a to cm right submandibular subcutaneous mass. the mass was first noticed by the owner months previously when it was approximately cm in diameter; the mass was not painful and there were no associated clinical signs. the patient was treated by the referring veterinarian with a -day course of meloxicam and amoxicillin-clavulanic acid with no improvement. on presentation the cat was still clinically well and the owner did not report any problems or clinical signs except for the mass. it was suspected to be neoplastic and thoracic radiography and a wedge biopsy sample were taken under general anaesthesia. thoracic radiography showed focal mineralisation of the left caudal lung lobe -considered an incidental finding -but was otherwise unremarkable. the histopathological diagnosis was marked pyogranulomatous and lymphocytic cellulitis with mild myositis and no evidence of neoplasia. the mass continued to grow and, neoplasia remained high on the differential list, so the mass was surgically resected. the second histopathology report confirmed the previous diagnosis of a pyogranuloma, and no aetiological agents could be found with pas, gram and zn staining. pcr and culture for mycobacteria were performed on frozen (stored at − °c) and fresh tissue, respectively, and were both negative. a pan-fungal pcr (its -its region of ribosomal dna) on paraffin block tissue was also negative except for isolation of "candida albicans." although this was most likely a contaminant, the patient was prescribed a -month course of itraconazole. one month later new numerous nodules non-associated with the previous removed mass were found in the lymphatic chain of the neck, together with an enlarged prescapular lymph node, but there was no regrowth of the previous removed granuloma. mri confirmed the presence of the nodules in the neck and an enlarged right prescapular lymph node; no other abnormalities were found. radiography of the thorax and abdominal ultrasound were unremarkable. the prescapular lymph node and three nodules in the neck were removed for diagnostic purposes. histopathology confirmed the presence of pyogranulomatous cellulitis and myositis, with mild dermal fibrosis and pyogranulomatous lymphadenitis. no aetiological agents could be found despite repeated examination using pas, gram and zn stains. tissue culture, pan-fungal and mycobacteria pcr were again negative. immunohistochemistry for coronavirus antigen (fipv - antifeline coronavirus antibodies) and s rna probe fish analysis for eubacteria were performed and were both negative. the lesions remained unchanged for the following weeks and a dose of prednisolone mg/kg daily for weeks was prescribed but produced no improvement. the dose was increased to mg/kg for the following weeks, reducing to mg/kg for the following month. despite the initial lack of response, the lesions markedly improved and completely disappeared over the following months. the prednisolone was gradually reduced and stopped. at the time of writing the cat was still asymptomatic and no recurrence of the lesions has been documented, more than year after initial presentation. to our knowledge this is the first report of feline idiopathic sterile pyogranuloma without skin involvement. the pyogranulomas here described affected young to middle-aged dsh cats, they had varied locations but similar histopathological features, with no aetiological agents found despite extensive investigations. the s rrna pcr for mycobacteria species in fresh, frozen or even in paraffin-embedded tissue sample is considered a very sensitive and specific test for diagnosis of lepromatosis/mycobacteriosis in cats compared to histopathology and zn staining, so the possibility of a false negative is unlikely (hughes et al. ) . the pan-fungal pcr amplification of the its - · s-its region of ribosomal dna is considered a sensitive test for detection of fungi and, in combination with conventional laboratory tests, it improves the accuracy of fungal detection in tissue specimens (lau et al. ) , so a fungal infection was considered extremely unlikely. all the formalin-fixed and paraffin-embedded tissue samples for pcr were fixed for a maximum of hours and analysed within days from sample collection, limiting false-negative results as previously published (hughes et al. , reppas et al. ). on the s rna probe fish analysis no bacteria could be found. this test is considered more sensitive than culture in the detection of bacteria, ruling out the possibility of a bacterial involvement. bartonella is often asymptomatic in cats, although recently bartonella has been isolated in pyogranulomatous myocarditis in two cats (varanat et al. the manifestation of any concurrent disease can be difficult to prove (barnes et al. ) . in our case we did not test for bartonella, so although unlikely we could not completely rule out this possibility. furthermore, the lack of significant progression or development of systemic lesions in particular after treatment with prednisolone in cases and was more consistent with a non-infectious process. the clinical response to prednisolone in cases and may, in fact, indicate a possible immune-mediated/ inflammatory aetiology, similar to that reported in dogs (fraga-manteiga et al. ) and currently still under investigation (bexfield et al. ) . in conclusion we have reported three cases of idiopathic sterile granuloma with mass appearance in cats. the granulomas had an overall slow growth and benign progression, despite the aggressive clinical appearance and the remarkable size of the masses in all cases. in case , surgical resection originally controlled the disease, but due to the development of other lesions month later, the benefit of surgery, except for diagnostic purposes, remains uncertain. immunosuppressive treatment with prednisolone could be beneficial but more studies are required to support this notion. none of the authors of this article has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. disseminated mycobacterium avium infection in young cats: overrepresentation of abyssinian cats evidence of bartonella henselae infection in cats and dogs in the united kingdom study into sterile neutrophilic-macrophagic lymphadenitis in the english springer spaniel epidemiology, diagnosis, and treatment of blastomycosis in dogs and cats cutaneous pyogranuloma in a cat caused by virulent rhodococcus equi containing an kb type i plasmid idiopathic sterile pyogranulomatous lymphadenitis in a nine-month-old springer spaniel molecular characterization of a novel fastidious mycobacterium causing lepromatous lesions of the skin, subcutis, cornea, and conjunctiva of cats living in victoria determination of the aetiology of presumptive feline leprosy by s rrna gene analysis pcr studies of feline leprosy cases morphologic features and development of granulomatous vasculitis in feline infectious peritonitis development and clinical application of a panfungal pcr assay to detect and identify fungal dna in tissue specimens cryptococcosis in cats: clinical and mycological assessment of cases and evaluation of treatment using orally administered fluconazole diagnosis and treatment of pyogranulomatous panniculitis due to mycobacterium smegmatis in cats infection of the subcutis and skin of cats with rapidly growing mycobacteria: a review of microbiological and clinical findings nocardia infections in cats: a retrospective multi-institutional study of cases campylobacter species and neutrophilic inflammatory bowel disease in cats pyogranulomatous skin disease and cellulitis in a cat caused by rhodococcus equi general categories of cytologic interpretation detection and identification of mycobacteria in fixed stained smears and formalin-fixed paraffin-embedded tissues using pcr cutaneous sterile granulomas/pyogranulomas, leishmaniasis and mycobacterial infections idiopathic sterile granulomatous and pyogranulomatous dermatitis in cats intra-abdominal actinomycetoma in a cat immunohistochemical demonstration of feline infectious peritonitis virus antigen in paraffin-embedded tissues using feline ascites or murine monoclonal antibodies laser capture microdissection of feline streptomyces spp pyogranulomatous dermatitis and cellulitis localized cutaneous infection with francisella tularensis resembling ulceroglandular tularemia in a cat identification of bartonella henselae in cats with pyogranulomatous myocarditis and diaphragmatic myositis pathogenesis of feline infetious peritonitis: pathologic changes and immunofluorescence key: cord- -qtlevnbt authors: al hosani, farida ismail; kim, lindsay; khudhair, ahmed; pham, huong; al mulla, mariam; al bandar, zyad; pradeep, krishna; elkheir, kheir abou; weber, stefan; khoury, mary; donnelly, george; younis, naima; el saleh, feda; abdalla, muna; imambaccus, hala; haynes, lia m; thornburg, natalie j; harcourt, jennifer l; miao, congrong; tamin, azaibi; hall, aron j; russell, elizabeth s; harris, aaron m; kiebler, craig; mir, roger a; pringle, kimberly; alami, negar n; abedi, glen r; gerber, susan i title: serologic follow-up of middle east respiratory syndrome coronavirus cases and contacts—abu dhabi, united arab emirates date: - - journal: clin infect dis doi: . /cid/ciy sha: doc_id: cord_uid: qtlevnbt background: although there is evidence of person-to-person transmission of middle east respiratory syndrome coronavirus (mers-cov) in household and healthcare settings, more data are needed to describe and better understand the risk factors and transmission routes in both settings, as well as the extent to which disease severity affects transmission. methods: a seroepidemiological investigation was conducted among mers-cov case patients (cases) and their household contacts to investigate transmission risk in abu dhabi, united arab emirates. cases diagnosed between january and may and their household contacts were approached for enrollment. demographic, clinical, and exposure history data were collected. sera were screened by mers-cov nucleocapsid protein enzyme-linked immunosorbent assay and indirect immunofluorescence, with results confirmed by microneutralization assay. results: thirty-one of ( %) case patients were asymptomatic or mildly symptomatic and did not require oxygen during hospitalization. mers-cov antibodies were detected in of ( %) case patients with available sera, including severely symptomatic, mildly symptomatic, and asymptomatic case patients. no serologic evidence of mers-cov transmission was found among household contacts with available sera. conclusions: transmission of mers-cov was not documented in this investigation of mostly asymptomatic and mildly symptomatic cases and their household contacts. these results have implications for clinical management of cases and formulation of isolation policies to reduce the risk of transmission. although there is evidence of person-to-person transmission in household and healthcare settings [ ] [ ] [ ] [ ] [ ] , more data are needed to describe and better understand the risk factors and transmission routes in both settings, as well as the extent to which disease severity affects transmission. these data would be of importance to the public health response given that approximately % of confirmed mers-cov cases reported to the world health organization have been described as mildly symptomatic or asymptomatic [ ] . during january - may , the department of health-abu dhabi (doh) investigated laboratory-confirmed cases and conducted extensive contact investigations in both household and healthcare settings [ ] . through these investigations, % of the laboratory-confirmed cases reported no symptoms or mild illness [ ] . contacts of case patients were tested by diagnostic pcr assays; however, results could include false negatives due to the -day incubation period. in this investigation, we use serological detection of mers-cov antibodies to evaluate if asymptomatic or mildly ill case patients had detectable mers-cov antibodies, estimate transmission rates from known cases to their household contacts, and identify potential risk factors. this investigation occurred in the emirate of abu dhabi, which occupies > % of the uae's total area [ ] and is comprised of regions: abu dhabi (capital city), al ain region, and al dhafra. the emirate of abu dhabi has a population of . million ( estimate) [ ] . the al ain region borders oman and saudi arabia and houses the second largest city in the emirate, al ain city. while al ain city is an oasis, the rest of the region primarily consists of desert and mountains. the al dhafra region is mainly desert and rural with approximately residents and a population density of residents/km [ ] . all laboratory-confirmed mers-cov cases (n = ) in the emirate of abu dhabi diagnosed between january and may and their household contacts (n = ) were eligible for the investigation. these cases were a convenience sample during the ongoing mers-cov outbreak. two of the ( . %) household contacts tested for mers-cov during initial contact investigations were pcr positive and eligible to be enrolled as cases for our investigation (figure ). the enrolled case was a healthcare worker who might have been exposed by another coworker, who also lived in the case's household; therefore, the enrolled case was a result of either household or healthcare transmission prior to this investigation's initiation. the case not enrolled in this investigation was exposed in the household. household contacts were defined as any person who stayed at least night at the same location as the case patient during the days prior to the case patient's symptom onset or the date of first positive specimen if the case patient was asymptomatic. excluded cases included palace workers and other high-level officials; their associated household contacts were also excluded. for each mers-cov case identified in the investigation, clinical information, including symptoms, was collected using the international severe acute respiratory and emerging infection consortium form, which was filled out in real time by healthcare providers and subsequently verified by retrospective chart review. in abu dhabi during this time period, all individuals who tested positive for mers-cov were admitted to a healthcare facility for observation and infection control regardless of symptom status. the same definitions for case severity were used as in al hosani et al [ ] including the following: asymptomatic cases reported no symptoms at the time of a positive test as recorded by a healthcare provider in the medical chart; mildly symptomatic cases reported symptoms, such as pharyngitis, rhinorrhea, or cough, and did not require oxygen during their hospitalization; and severely symptomatic cases required supplemental oxygenation during their hospitalization, ranging from nasal cannula to mechanical ventilation. using data collected from doh's surveillance of mers-cov cases, households with mers-cov case patients were approached. household contacts who were eligible for the investigation included those that had been identified through contact investigations associated with the case patient performed by doh officials within hours' notification. three attempts were made to contact each household. if no response was received after attempts, the household was not enrolled. households that agreed to be enrolled were given an appointment at the local disease prevention and screening center for questionnaire administration and serum collection. questionnaires were administered in english, arabic, or, if an interpreter was available, the participant's native language. data collected included demographics; residence/household description; exposure history to other mers-cov cases, healthcare settings, and animals; travel history; and medical history, including any long-term effects reported by case patients. for deceased case patients, a proxy completed the case patient questionnaire using recall. the real-time reverse-transcription pcr (rrt-pcr) results were obtained from the doh surveillance data. upper (ie, nasopharyngeal, oropharyngeal) and lower respiratory tract specimens (ie, sputum, bronchoalveolar lavage fluid, tracheal aspirates) were analyzed using rrt-pcr in the sheikh khalifa medical center laboratory. additional laboratory result verifications were performed in a random sample of specimens using nucleocapsid-based rrt-pcr [ ] . serum samples were inactivated using × rads gamma irradiation and stored at ≤ - °c until use. screening of serum specimens by mers-cov nucleocapsid enzyme-linked immunosorbent assay (elisa) was performed at the sheik khalifa medical city in abu dhabi, uae and the centers for disease control and prevention (cdc), atlanta, georgia. titers of ≥ : were reported as positive. recombinant full length mers-cov nucleocapsid protein indirect elisa was used to screen serum specimens as described by al-abdallat et al [ ] . serum samples were tested for the presence of neutralizing antibodies to mers-cov using a microneutralization assay (mnt) [ ] . the neutralization titer was measured as the reciprocal of the highest serum dilution that completely inhibited vero cell lysis in at least of the triplicate wells. positive and negative controls were included for each mnt performed and included back-titration and mock-infected cells. titers of ≥ : were reported as positive. all work with live mers-cov was done in biosafety level containment at the cdc. immunofluorescence assays (ifas) were performed by screening sera at a dilution of : and : on paraformaldehyde-fixed, acetone-methanol permeabilized mers-cov (strain mers-cov hu/england-n / ) infected or uninfected control vero cells. antihuman immunoglobulin g, m, and a fluorescein isothiocyanate conjugate was used to detect anti-mers-cov antibodies in human serum, and nuclei were counterstained with ′, -diamidino- -phenylindole to allow identification of individual mers-cov-infected cells. fluorescence was detected using a zeiss axioimager fluorescence microscope. the positive control for the assay is a serum sample from a patient infected with mers-cov hu/ england-n / . a positive result was scored when these conditions were met: cells were evenly stained (instead of punctate staining); fluorescence intensity was higher than that of the negative controls; and signal intensity declined with serial dilution. a minimum of negative controls were included with each ifa. approximately % of specimens negative by nucleocapsid elisa were screened by both ifa and mnt to confirm the negative result. mers-cov antibody positivity was defined as one of the following: ( ) of tests (ie, mers-cov nucleocapsid elisa, mers-cov mnt, and ifa) were positive; or ( ) mers co-v mnt was the only positive test. household survey data were entered into electronic forms in epi info version . (cdc). quality control and assurance were performed through epi info intelligent codes programmed into the forms. household survey data were merged with the laboratory results, and descriptive analysis was completed. differences in proportions were compared using the mantel-haenszel χ test, while differences in continuous variables were compared using the student t test. p < . was considered statistically significant. data analysis of the merged dataset was conducted with sas version . software (sas institute, cary, north carolina). following local customs, informed consent was obtained from the head of the household, who provided consent for all members of a household; however, each individual was still able to decline participation. this investigation was determined by doh and cdc to be part of a public health response, not research, and therefore not subject to institutional review board review. thirty-four case patients' households were included (supplementary table ). household residences ranged in size from m to m (interquartile range [iqr], - m ). a median of individuals (range, - ) lived in the households days prior to the diagnosis of a mers-cov household case patient. more than half of mers-cov case patients shared a bathroom with others in the household. all households reported having air conditioning. thirty-four cases of ( %) and household contacts of ( %) participated (table ) . females comprised a higher proportion of case patients compared with household contacts ( . % vs . %), and case patients were older compared with household contacts (median, years vs years). most case patients and contacts were from the al ain region of the abu dhabi emirate. seventy-one percent (n = ) of case patients reported working in a healthcare setting days prior to diagnosis, with nurses being most represented ( %, n = ) ( table ) ; only % (n = ) of household contacts worked in a healthcare setting days prior to a case patient's diagnosis. compared with household contacts, case patients less frequently reported visiting or owning a farm ( % vs %), but reported camel exposure more frequently ( % vs %). household contacts reported the following frequent exposures to mers-cov case patients: hugging ( %, n = ), using the same bathroom ( %, n = ), sharing meals ( %, n = ), and kissing or nose-kissing (ie, rubbing tips of noses against one another) ( %, n = ). case patients reported a higher proportion of underlying medical conditions than household contacts, including diabetes ( % vs %, p = . ), hypertension ( % vs %, p < . ), kidney failure ( % vs %, p = . ), and heart failure ( % vs %, p < . ) ( table ). case patients also reported taking medications for any illness more frequently than contacts ( % vs %). three case patients ( %) reported limitation to activities due to mers-cov with a median duration of days (iqr, - days) ( table ) . normal activities were resumed at a median of days (iqr, - days). of case patients, ( %) reported being asymptomatic, ( %) reported being mildly symptomatic, and ( %) were severely symptomatic. age and proportion having underlying medical conditions increased with symptom severity ( table ) . symptom duration did not have any noticeable trend with symptom severity (data not shown). all severe case patients were treated in the intensive care unit, as well as asymptomatic case, who had underlying diabetes, hypertension, and kidney disease. the median days hospitalized increased with symptom severity ( table ) . sera were obtained from of ( %) case patients and of ( %) household contacts. among the case patients with available sera (table ) among the case patients with detectable antibodies against mers-cov, all of them were aged < years, with a median age of years, compared to a median of years for case patients without detectable antibodies (table , p = . ). number of days of pcr positivity was notably higher among those who had detectable antibodies compared to those who did not (median, days vs days, p = . ). we describe the results of follow-up of mers-cov case patients and of their household contacts from the emirate of abu dhabi during - . notably, serologic testing did not find any evidence of mers-cov transmission in the households of mers-cov case patients in our investigation, suggesting that viral transmission from asymptomatic or mildly symptomatic individuals to household contacts does not readily occur. sera were tested with a combination of different laboratory assays (nucleocapsid elisa, ifa, and mnt); we feel confident that individuals identified as "negative" did not seroconvert. although there was clear evidence of household transmission in household not enrolled in this investigation, our investigation's results did not show evidence of additional household transmission. overall, our findings support current recommendations that home isolation may be appropriate for asymptomatic cases and close contacts who are ill and do not require hospitalization in consultation with local public health departments [ , ] . because this investigation occurred during may-june , many case patients were recruited from the april healthcare-associated outbreak at an al ain region hospital [ ] . a kingdom of saudi arabia study found that while healthcare personnel were at high risk for infection, most illness was relatively mild and could be unrecognized, highlighting potential undetected transmission of the virus to others [ ] . in our investigation, case patients tended to be younger ( - years) , and most reported working in a healthcare setting days prior to their diagnosis where they were exposed to a similar to previous studies, case patients with severe disease had higher frequency of comorbid conditions and required intensive care, including intubation [ ] [ ] [ ] . in a recent investigation from south korea, patients with a higher host infectivity, which included evaluation of pcr cycle threshold values, along with higher numbers of contacts, were more likely to transmit mers-cov [ ] . it is likely that most of the primary case patients in this investigation had lower host infectivity. while our investigation found that some asymptomatic or mildly symptomatic case patients had detectable antibodies, we did not find any detectable antibodies in asymptomatic and mildly symptomatic case patients. other studies also did not find detectable antibodies in some asymptomatic and mildly ill cases [ , ] . if seroconversion is to occur in case patients, studies have demonstrated that this usually occurs within the first month of illness [ ] [ ] [ ] . for the majority of case patients with detectable antibodies, we found persistence of antibody response for several months after the initial diagnosis, even close to a year. additionally, these case patients had a longer duration of mers-cov pcr positivity than those who did not have detectable antibodies, indicating a potential relationship between longer viral shedding and seroconversion. previous studies have demonstrated that asymptomatic and mildly symptomatic case patients can test pcr positive > weeks from lower respiratory tract specimens [ , ] . our investigation's serology results do not provide additional evidence of transmission to household contacts, though there is evidence from other settings to suggest limited household transmission [ ] . also, very low rates of household transmission have been reported during hospital-based outbreaks [ , ] . more robust transmission studies involving larger numbers of case patients representing a range of clinical and demographic characteristics and their contacts are needed to further investigate risk exposures. there are several limitations to this investigation. first, serum samples were collected at varying intervals after illness onset for each case patient, potentially affecting serology results. the duration of antibody response is unknown. second, recall bias might have led to the misclassification of symptom severity among household contacts; however, for case patients, to minimize this bias, we relied upon retrospective medical chart review, though this also might not be as complete since it depended on the initial healthcare provider's history and physical. third, these case patients were immediately isolated in hospitals after pcrpositive results were discovered. the removal from the household setting might have reduced exposure to household contacts although the case patients were residing with household contacts at the time of the contact investigations. last, because our investigation did not detect household transmission, we cannot comment on any behaviors or exposures that would increase risk among household contacts of case patients. in summary, we did not document additional household transmission in this investigation that included a preponderance of asymptomatic and mildly symptomatic confirmed mers-cov case patients. our investigation findings support the recommendation to consider home isolation for asymptomatic and mildly ill cases that do not require hospitalization while using proper precautions, including face masks, frequent hand washing, and minimizing exposure to the case patient in the household [ , , ] . while no vaccines or antivirals against mers-cov are currently available, reducing transmission through effective infection control management remains a major priority. understanding transmission risk for different mers-cov-infected patients who live in different settings will be important data that must be factored into prevention strategies. further studies on human-to-human transmission in different settings should be conducted to inform mers-cov prevention and control guidelines. supplementary materials are available at clinical infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or 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respiratory syndrome coronavirus infection clinical aspects and outcomes of patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia middle east respiratory syndrome risk factors for transmission of middle east respiratory syndrome coronavirus infection during the outbreak in south korea serologic responses of mers-coronavirusinfected patients according to the disease severity viral shedding and antibody response in patients with middle east respiratory syndrome coronavirus infection kinetics of serologic responses to mers coronavirus infection in humans a case of long-term excretion and subclinical infection with middle east respiratory syndrome coronavirus in a healthcare worker health care worker contact with mers patient, saudi arabia interim guidance for healthcare professionals acknowledgments. the authors gratefully thank the participants; the teams from the disease prevention and screening center-al ain, the disease prevention and screening center-abu dhabi, ghayathi hospital, and silla hospital that supported this investigation; and suvang trivedi and seyhan boyoglu-barnum at the centers for disease control and prevention (cdc) for technical support.disclaimer. the conclusions, findings, and opinions expressed herein are those of the authors and do not necessarily reflect the official position of the us department of health and human services, the us public health service, the cdc, or the authors' affiliated institutions.financial support. this work was supported by the cdc. potential conflicts of interest. all authors: no reported conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord- -sqetz h authors: hou, yixuan; meulia, tea; gao, xiang; saif, linda j.; wang, qiuhong title: deletion of both the tyrosine-based endocytosis signal and the endoplasmic reticulum retrieval signal in the cytoplasmic tail of spike protein attenuates porcine epidemic diarrhea virus in pigs date: - - journal: journal of virology doi: . /jvi. - sha: doc_id: cord_uid: sqetz h porcine epidemic diarrhea virus (pedv) causes high mortality in neonatal piglets. the pedv spike (s) protein contains two intracellular sorting motifs, yxxΦ (tyrosine-based motif yevf or yeaf) and kvhvq at the cytoplasmic tail, yet their functions have not been fully elucidated. some vero cell-adapted and/or attenuated pedv variants contain ablations in these two motifs. we hypothesized that these motifs contribute to viral pathogenicity. by transiently expressing pedv s proteins with mutations in the motifs, we confirmed that the motif kvhvq is involved in retention of the s proteins in the endoplasmic reticulum (er)-golgi intermediate compartment (ergic). in addition, we showed that the yxxΦ motif triggers endocytosis of s proteins. these two motifs synergistically regulate the level of s expressed on the cell surface. to investigate their role in viral pathogenicity, we generated three recombinant pedvs by introducing deletions or a mutation in the two motifs of the infectious clone of pedv pc a strain (icpc a): (i) icΔ aa (ΔyxxΦekvhvq), (ii) icΔ aa (Δkvhvq), and (iii) icya (y a, to an inactivated motif, aevf). infection of vero cells with icΔ aa resulted in larger syncytia and more virions, with reduced numbers of s protein projections on the surface compared with icpc a. furthermore, we orally inoculated five groups of -day-old gnotobiotic piglets with the three mutants, icpc a, or a mock treatment. mutant icΔ aa caused less severe diarrhea rate and significantly milder intestinal lesions than icpc a, icΔ aa, and icya. these data suggest that the deletion of both motifs can reduce the virulence of pedv in piglets. importance many coronaviruses (covs) possess conserved motifs yxxΦ and/or kxhxx/kkxx in the cytoplasmic tail of the s protein. the kxhxx/kkxx motif has been identified as the er retrieval signal, but the function of the yxxΦ motif in the intracellular sorting of cov s proteins remains controversial. in this study, we showed that the yxxΦ of pedv s protein is an endocytosis signal. furthermore, using reverse genetics technology, we evaluated its role in pedv pathogenicity in neonatal piglets. our results explain one attenuation mechanism of vero cell-adapted pedv variants lacking functional yxxΦ and kvhvq motifs. knowledge from this study may aid in the design of efficacious live attenuated vaccines against pedv, as well as other covs bearing the same motif in their s protein. taxonomically, pedv is a member of the alphacoronavirus genus within the coronaviridae family. the mature pedv virion consists of four structural proteins: spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins. as the major glycoprotein on the pedv envelope, s proteins form trimers, which appear as projections on the surface of a virion using an electron microscope, and bind to cellular receptors and mediate virus-host membrane fusion. proteolytic cleavage of s proteins expressed on the cell surface triggers syncytium formation ( , ) . like those of other coronaviruses (covs), pedv virions assemble at the endoplasmic reticulum (er)-golgi intermediate compartments (ergic) ( ) ( ) ( ) . the amounts of pedv s proteins in the ergic, in other organelles, or on the cell surface are likely regulated by two nearby motifs in its cytoplasmic tail (ct): a tyrosine-based motif, yxx⌽ (x is any residue and ⌽ is a bulky hydrophobic residue: f, m, i, l, or v), and an er retrieval signal (errs), kvhvq ( ) ( ) ( ) ( ) , as well as other viral and cellular proteins. the cov errs, either in the dilysine or the dibasic form (kxkxx, kkxx, or kxhxx), is a weak ergic retention signal ( , ) . it interacts with coatomer complex i (copi), a cellular protein involved in cargo transportation from the golgi to er, and prevents large amounts of the s proteins from being transported to the cell surface through the canonical secretory pathway ( , ) . in addition, the errs in the s protein of severe acute respiratory syndrome cov (sars-cov) promotes the interaction between s and m proteins in the golgi region ( ) . inactivation of the errs in the sars-cov s protein impaired its incorporation into virus-like particles when coexpressed with the m in the cells ( ) . for pedv, the amino acid sequence of the errs is kvhvq, which is highly conserved among different genotypes. one study demonstrated that a single amino acid substitution in this motif (kvhvq to kvrvq) weakens the intracellular retention function of the s proteins of the th passage of a murine-adapted pedv variant, mk-p ( ) , resulting in enhanced syncytium formation in vero cells. however, this impaired kvrvq motif does not alter the incorporation of s into the mk-p virions ( ) . although the yxx⌽ motif is a well-studied, clathrin-dependent endocytosis signal among numerous viral and host cellular transmembrane proteins ( ) ( ) ( ) ( ) ( ) ( ) ( ) , its function in cov s proteins has not been fully understood. most s proteins of alphacoronaviruses, such as transmissible gastroenteritis virus (tgev), and gammacoronaviruses, such as infectious bronchitis virus (ibv), contain this motif in their cts (fig. a) . a previous study demonstrated that the yxx⌽ motif is responsible for intracellular retention but not endocytosis of tgev s proteins into cells ( ) . interestingly, this retention signal could be transformed into a strong endocytosis signal when the first lysine residue of the tgev kxhxx motif was replaced by methionine (yepiekvhvh to yepiemvhvh; change is in bold type and yxx⌽ and kxhxx motifs are underlined) ( ) . for ibv, the role of the yxx⌽ motif in s protein intracellular transport is controversial. one group reported that this motif is not an endocytosis signal ( ) . the other group demonstrated that the yxx⌽ motif functioned as an endocytosis signal in a chimeric protein composed of the vesicular stomatitis virus (vsv) g protein ectodomain and the ct of the ibv s protein ( ) . it has also been demonstrated that a recombinant ibv harboring a mutated yxx⌽ (yttf to ytta) in the ct of the s protein could not be recovered in cell culture, suggesting the essential role of this motif in the ibv life cycle. moreover, since the =end of the ibv s gene overlaps with the transcription regulatory sequence (trs) of accessory gene , mutagenesis of the ibv errs (kksv to aasv) impaired the synthesis of subgenomic rna (sgrna ), resulting in decreased expression of e proteins in the ibv-infected cells ( ) . previously, we generated an attenuated pedv strain by serially passaging a highly virulent strain, pc a, in vero cells. at the th (p ) and later passages, we identified a premature stop codon in the =end of the s gene ( ) . this stop codon truncates the s protein by amino acids (aa) (evfekvhvq) . interestingly, at least five pedv variants (fl , sm- , chm , avct , and knu- ) containing a premature terminated s gene have been reported (table ) ( ) ( ) ( ) ( ) . all these pedv variants are either cell culture attenuated or clinically mild in pigs, except for avct , whose pathogenicity is unknown. these variants share the same s truncation pattern as pc a-p : partially ablated yxx⌽ and completely deleted kvhvq. it is possible that the loss of kvhvq may enhance the surface s protein level and increase its ability to induce syncytia in cell culture. however, the function of the incomplete yxx⌽ motif in viral replication and pathogenicity is unknown. notably, two types of yxx⌽ motif exist in the cts of s proteins among pedv genotypes: classical strains (g a) and some highly virulent asian strains (g a) contain a yeaf motif, whereas highly virulent north american strains (g b), s insertion and deletion (indel) strains (g b), and some g a strains harbor a yevf motif. we hypothesized that the yxx⌽ motif of pedv, regardless of its amino acid sequence, is an endocytosis signal and determines viral virulence in pigs. in this study, we evaluated the phenotypes of transiently expressed s mutants containing mutations or deletions in these two motifs in mammalian cells and the virulence of three representative recombinant pedvs in gnotobiotic piglets. motifs yxx⌽ and kvhvq regulate the levels of pedv s proteins on the cell surface. to determine the roles of the two motifs in the intracellular sorting of s proteins, we constructed plasmids bearing a wild-type s gene (of pc a) or an s gene with different mutations in the cts ( fig. b and c) . also, the dispensable -aa region (residues to ) in domain of each s protein was replaced by fluorescent protein mcherry ( aa), as we demonstrated previously that this -aa region does not interfere with the formation of s protein trimers ( ) . wt and wt contain the yevf and yeaf motifs, respectively. using these two wild-type constructs as templates, we generated additional mcherry-tagged s mutants, including mutants bearing point mutations and the ct-truncated variants ( fig. ; table ). the Δ aa mutant lacks both motifs. the Δ aa and hd mutants contain the cts of pedv strains pc a-p and knu- , respectively. the Δ aa mutant bears the same ct as four s-truncated pedv variants, fl , sm- , avct , and chm . mk-p contains the identical ct to that of the pedv strain mk-p . to evaluate the difference in functions between yevf and yeaf, three pairs of mutants (ya and ya-a, Δ aa and Δ aa-a, and mk-p and mk-p -v) were generated. to confirm the role of the kvhvq motif in ergic retention, transiently transfected vero cells were fixed with methanol at h posttransfection (hpt) and stained with an antibody against ergic marker ergic- ( fig. a) . we observed distinct sorting patterns among the recombinant s proteins. quantification of the colocalization between the s protein and ergic signals was indicated as pearson's correlation coefficient (pcc) value ( , ) , by measuring to individual cells from each sample (fig. b) . the higher pcc values indicate the higher levels of colocalization between the two fluorescent signals. for the four s proteins (wt , wt , ya, and ya-a) with an intact kvhvq motif in the ct, most intracellular s proteins were concentrated at the ergic region, with significantly higher pcc values than for the other s mutants (p Ͻ . ). interestingly, only the six s proteins with an intact yxx⌽ motif in their cts (wt , wt , Δ aa, Δ aa-a, mk-p , and mk-p -v) formed clear puncta in the cells. to determine the steady-state levels of these s proteins, we quantified the fluorescent intensities of the mcherry signals in individual vero cells of each sample at hpt (fig. b) . no statistically significant difference (p Ͼ . ) was observed among samples, indicating that all the recombinant s proteins were expressed at similar levels. next, we measured the levels of s proteins on the cell surface using digital quantitative method and syncytium induction assay. the transfected cells were fixed with % formaldehyde without permeabilization, and the surface s proteins were stained with green fluorescence (fig. a) . total (both surface and intracellular) s proteins were observed for the red fluorescence. since each s protein produces equal intensity of red fluorescent signal, we quantified the levels of surface s proteins by measuring the ratios of gray values of the green fluorescent signal to mcherry signal and designated it the surface/total ratio. fifty individual cells in each sample were measured and are plotted in fig. c . wt did not differ statistically from wt . eight mutants (Δ aa, ya, ya-a, Δ aa, Δ aa, hd, mk-p , and mk-p -v) expressed higher levels of s proteins than the two wild-type s proteins on the cell surface, while Δ aa and Δ aa-a expressed significantly lower levels (p Ͻ . ). because proteolytic cleavage of surface s proteins triggers cell-cell fusion, a syncytium induction assay was performed by culturing the transfected cells in medium containing trypsin. the syncytia induced by the eight mutants (Δ aa, ya, ya-a, Δ aa, Δ aa, hd, mk-p , and mk-p -v) contained significantly more nuclei than those induced by the two wild types (wt and wt ), while almost no syncytia were induced by the Δ aa and Δ aa-a mutants ( fig. d and e). collectively, these data suggest that an intact kvhvq motif (wt , wt , ya, and ya-a) retains s proteins in the ergic, and the two motifs synergistically regulate the level of pedv s protein expressed on the cell surface via different mechanisms. the yxx⌽ motif is an endocytosis signal and regulates surface s protein internalization to early endosomes. to determine whether the yxx⌽ motif functions as an endocytosis signal in pedv s protein, we performed an antibody-antigen (gp -s protein) uptake assay. we evaluated the portions of the gp -bound surface s proteins (green) being endocytosed after incubation at °c. as controls, one set of cells were incubated at °c, whereby the cell endocytosis activities ceased. therefore, both the gp -bound s proteins (green) and the surface s proteins (red), which were labeled after incubation and fixation without permeabilization, were colocalized on the cell surface ( fig. ) . on the other hand, for the cells being incubated at °c, a large portion of the gp -bound s proteins (green) of wt , wt , mk-p , mk-p -v, Δ aa and Δ aa-a formed puncta in the cells and were not colocalized with the surface s proteins (red). this was further confirmed when we compared the pcc values between the gp -bound s protein signal and total surface s protein signal of each s protein at °c and °c (fig. a ). we found that the pcc values of the six s proteins (wt , wt , mk-p , mk-p -v, Δ aa, and Δ aa-a) at °c were significantly lower than those at °c, suggesting that partial gp -bound s proteins of those six samples were internalized from the cell surface after incubation at °c. additionally, we probed the total gp signals on the cell surface without permeabilization in the gp -s uptake assay (fig. b ). we measured the ratios of surface s (gp ) signal to total s (mcherry) signal in the transfected cells incubated at °c and °c. wt , wt , and the mk-p , mk-p -v, Δ aa, and Δ aa-a mutants had significantly lower ratios at °c than at °c. in contrast, the other six mutants (Δ aa, ya, ya-a, Δ aa, Δ aa, and hd) had similar pcc values (fig. a ) and surface/total ratios (fig. b ) at °c and °c. these results indicate that the s mutants without an intact yxx⌽ motif were defective in endocytosis. endocytotic vesicles deliver cell surface proteins to early endosomes in the cytoplasm ( ) . to determine whether the internalized s puncta of wt , wt , mk-p , mk-p -v, Δ aa, and Δ aa-a ( fig. a and fig. ) were located in early endosomes, we performed the antibody-antigen (gp -s protein) uptake assay (fig. ). after performing fixation and permeabilization of cells, we immunostained the gp -bound s proteins (green) and the early endosome marker rab proteins (red). the gp -bound s protein signals in the cells incubated at °c showed a significantly higher percentage ). collectively, these data suggest that an intact yxx⌽ motif in the ct is necessary for endocytosis of the s protein from the cell surface to early endosome. pig anti-s antiserum gp at °c for min. after washing with pbs three times, one set of cells was cultured at °c for min to allow endocytosis. another set of cells was kept at °c for min as the control group. cells were fixed with % formaldehyde without permeabilization. surface s proteins were stained with mouse anti-s monoclonal antibody sd - and goat anti-mouse af -conjugated secondary antibodies (red). then the cells were permeabilized with triton x- and stained with goat anti-guinea pig af -conjugated secondary antibodies (green). nuclei were stained with dapi (blue). cells were randomly selected, and images were taken by using a leica tcs sp confocal microscope. scale bar: m. virulence of the recombinant pedv lacking both the yxx⌽ and kvhvq motifs was reduced in neonatal gnotobiotic pigs. to investigate the function of the two motifs in pedv replication and pathogenesis, we generated three recombinant pedvs by introducing the Δ aa, Δ aa, and ya mutations into the ct of the pedv infectious cdna clone of strain pc a (icpc a) ( ) . we inoculated -day-old gnotobiotic piglets with the three mutant pedvs, icpc a, and phosphate-buffered saline (pbs; mock treatment) to determine the role of the two motifs in viral pathogenicity. clinical signs fig. . to stain the gp bound on the cell surface, cells were fixed with % formaldehyde without permeabilization. gp was probed using af -conjugated secondary antibodies (green). the fluorescent intensities of surface (af ) to total s (mcherry) ratios were measured in to individual cells. values are plotted in a bar chart, and shown as means Ϯ sds. values of the same sample cultured at °c and °c were analyzed by student's t test. ns, p Ͼ . ; **, p Ͻ . ; ***, p Ͻ . ; ****, p Ͻ . . deletion of yxx and kvhvq from s attenuates pedv journal of virology internalized s proteins colocalized with early endosomes. antibody-s protein uptake assays were performed at hpt for the two wild-type s proteins (wt and wt ) and the four mutants, Δ aa, Δ aa-a, mk-p , and mk-p -v, that formed puncta as shown in fig. . cells were incubated with guinea pig anti-s antiserum gp at °c for min. after washing with pbs three times, one set of cells were cultured at °c for min to allow endocytosis and another set of cells were incubated at °c for min. after fixation with methanol, early endosome marker rab proteins were stained with rabbit anti-rab mab (c b ) followed by af -conjugated goat anti-rabbit antibody (red). gp -bound s proteins were stained with goat anti-guinea pig af -conjugated secondary antibodies (green a ). the villous atrophy in the duodenum, jejunum, and ileum was quantified by measuring villus height to crypt depth (vh/cd) ratios of each piglet (fig. d to f). a lower vh/cd value indicates more severe atrophy in the intestines. compared with the virulent icpc a group, the lesions of icΔ aa-infected pigs were the least severe in all three small intestinal regions (duodenum, jejunum, and ileum). although there were no statistically significant differences in duodenum and ileum, the lesions in the jejunum of icya-infected piglets were significantly milder than those of the icpc a-and icΔ aa-infected pigs. we did not observe significant differences in clinical signs, fecal viral shedding titers, or vh/cd ratios between the icΔ aa and icpc a groups. we concluded that the virulence of icΔ aa was milder than that of icpc a, and the icΔ aa maintained virulence similar to that of icpc a. these data suggest that deletion of both the yxx⌽ and kvhvq motifs from the s protein significantly impaired the pathogenicity of the highly virulent pedv icpc a. the yxx⌽ and kvhvq motifs are dispensable for pedv replication, but the lack of either impairs viral replication efficiency in vitro. we measured the one-step (with a multiplicity of infection [moi] of ) growth kinetics of the viruses in vero cells (fig. a) . the three pedv mutants had significantly lower peak titers than icpc a at hpi. subcellular structures of the four recombinant pedv-infected vero cells at hpi were visualized by transmission electronic microscopy (tem) of cellular sections. like other covs, replication of pedv induces dramatic membrane rearrangements ( , ). we did not observe differences in the rearranged membrane structures among cells infected with the four recombinant pedvs. however, in the icpc a-infected cells, golgi vacuoles contained more mature virions, which are smaller particles with denser cores than immature virions ( ), than those in the icΔ aa-and icya-infected cells (fig. f and g) . the icΔ aa-infected cells contained intermediate amounts of mature virions in the vacuoles. trans-golgi vacuoles containing large numbers of mature virions were observed exclusively in icpc a-infected cells. this single-membrane structure contains viral particles prior to their egress. collectively, these data suggest that neither the yxx⌽ nor kvhvq motif is essential for pedv replication, but the absence of either motif impairs the generation of infectious pedv viral particles. recombinant pedvs lacking an intact yxx⌽ motif in the ct of the s protein formed larger syncytia than the wild-type virus. we measured multistep growth kinetics (moi ϭ . ) of the four recombinant pedv in vero cells (fig. b) . we found that the speed of viral replication varied among the four recombinant viruses and the infectious titers gradually decreased after the majority of cells were lysed due to the formation of syncytia. in the growth curve, the peak titers of icΔ aa and icya occurred earlier but were lower than those of icpc a and icΔ aa. replication of icΔ aa in vero cells showed a peak titer similar to that of icpc a, but with a delay. we performed plaque assays and measured the kinetics of the plaque formation of the four viruses ( fig. c and d) . at hpi, the icΔ aa virus had induced the largest plaques, followed by the icya and then by the parental icpc a, whereas the icΔ aa had formed the smallest plaques. we stained the surface s proteins of infected cells at an early time point ( hpi) (fig. e) . the fluorescent signals represented s proteins anchoring either on cell membrane or on the egressing virions on the surface. the s proteins of icpc a-infected cells clustered mainly in a small area on the cell surface, presumably virions clustering at the egress sites, in contrast to the evenly distributed surface s proteins of icΔ aa and icya on the entire cytoplasmic membrane, presumably s proteins anchoring directly on the cell membrane. the surface s proteins of icΔ aainfected cells were distributed in a pattern similar to that of icpc a but showed less intense staining. these data suggest that the yxx⌽ motif decreases the amount of s proteins on the cell surface in pedv-infected cells. to determine whether the attenuation of icΔ aa in vivo was due to the amount of s proteins incorporated into virions, we visualized the s proteins on the purified virion surface using tem. the culture and purification of the four recombinant pedvs were performed simultaneously to minimize the variations due to sample preparation. we observed virions with or without s protein projections on virions in all four samples (fig. a) . the virions without s projections could be those that lack enough s proteins on the surface or whose s projections were lost during sample preparation for tem. the icΔ aa pedv contained the lowest percentage of virions with s protein projections, followed by icya and then by icpc a and icΔ aa. next, we quantified the number of s projections on the individual virions of each pedv sample. among the four mutants, the icΔ aa pedv contained significantly fewer s projections than icpc a, icΔ aa, and icya (p Ͻ . ). these data suggest that the deletion of both the yxx⌽ and kvhvq motifs from the s protein impairs the incorporation of s proteins into pedv virions. previous studies showed that the m protein retained the s protein of sars-cov in the golgi regions when they were coexpressed in the same cell ( , ) . to characterize whether the s proteins of the four recombinant pedvs can still interact with m, we cloned the pedv m gene into the vector puc -sgrna. the s and m proteins were coexpressed in vero cells (fig. a ) or the s protein alone was expressed. when the s and m proteins were coexpressed in cells, we observed that they colocalized, with equal pcc values ( fig. b and c) . we stained the surface s proteins of the cells cotransfected with the s and m plasmids or transfected with the s plasmid alone and measured the ratios of fluorescent intensities of surface s to total s. we found that the expression of m significantly decreased the surface s protein levels in the same cell (fig. c) , as evident by the significantly lower surface s to total s ratios in the m and s plasmidcotransfected cells compared with those in the cells transfected with only s plasmid. in this study, we determined that the yxx⌽ and kvhvq motifs at the ct of pedv s proteins are dispensable for pedv replication. however, the deletion of both motifs reduced the viral virulence in vivo and the incorporation of s proteins into virus journal of virology particles in vitro. the yxx⌽ motif and the kvhvq motif function as an endocytosis signal and an er retrieval signal, respectively. yxx⌽ and kvhvq together regulate surface s protein levels and s proteininduced syncytium formation. the number of nuclei in a pedv s protein-induced syncytium, or the size of a syncytium, correlates with the amount of s proteins on the cell surface ( ) . collectively, our data support the hypothesis that both motifs are involved in the regulation of pedv s proteins on the cell surface: the kvhvq motif retains the s proteins in the ergic, limiting its transport to the cell surface, whereas the yxx⌽ motif decreases the amount of surface s proteins via endocytosis. the mutants Δ aa, Δ aa, Δ aa, and hd, which lack the complete kvhvq motif and complete/partial yxx⌽ motif, had the highest level of surface s proteins ( fig. a and c) , probably due to the lack of both er retrieval and endocytosis. the mutants ya, ya-a, mk-p , and mk-p -v had the second highest level of cell surface s proteins. for the ya and ya-a, this is probably due to the inactivation of the endocytosis signal, while their errs remained intact. for mk-p and mk-p -v, it may be due to the impaired errs (kvrvq; r is the mutation that leads to the impaired errs) while their yxx⌽ motif was intact. surprisingly, the mutants Δ aa and Δ aa-a had significantly lower levels of surface s proteins than wt and wt . the lack of the entire kvhvq motif resulted in significantly lower levels of ergic colocalization of Δ aa and Δ aa-a than of wt and wt (fig. ) , confirming that the kvhvq motif functions as an errs in pedv as described previously ( , ) . why, then, did we not observe an increased level of surface s proteins for these two mutants? we hypothesized that the loss of the entire kvhvq motif in the ct of the s protein altered the intracellular sorting of s proteins. one possibility could be that the potency of their yxx⌽ motifs, as the endocytosis signal, was dramatically enhanced due to the lack of the entire errs motif. a similar endocytosis enhancement effect was reported for the yxx⌽ motif of tgev when its kxhxx motif's conformation was changed due to the k-to-m mutation ( ) . another possibility could be that the deletion of the entire kvhvq motif may cause other deficiencies in s protein sorting, such as impaired export from the er or altered transportation to other organelles. detailed mechanistic studies regarding the sorting of Δ aa and Δ aa-a s proteins are necessary to answer this question. the endocytosis role of the yxx⌽ motif in pedv s intracellular sorting. the conserved location of the yxx⌽ and kxhxx/kkxx motifs in the ct implies that the s protein sorting mechanisms may be similar among different covs (fig. a) . however, previous studies on the yxx⌽ motif of tgev and ibv s proteins did not provide consistent conclusions ( , , , ) . one reason could be that the expression efficiencies of vectors varied in those studies. due to the small amount of s proteins expressed on the cell surface, the endocytosed s proteins might be too subtle to be detected in the antibody-antigen uptake assay. in our study, the plasmid puc -sgrna overexpressed pedv s proteins in vero cells compared to some commercially available vectors (data not shown), probably due to the inclusion of the =leader sequence of subgenomic rna (sgrna) and pedv =untranslated region (utr) that enhanced the translation. although we determined that both yevf and yeaf are endocytosis signals of pedv s proteins, it is possible that their efficiencies may vary. in the future, more advanced quantitative approaches, such as mechanically induced trapping of molecular interactions (mitomi) ( ) , could be used to determine their affinities for binding to cellular factors. additionally, one limitation of this study is that we did not applied approaches to monitor the kinetic trafficking of the endocytosis of s proteins from the cell surface. future studies to address these points are necessary. in this study, we did not detect the colocalization of endocytosed s protein puncta and lysosomal marker lamp in wt -, wt -, Δ aa-, Δ aa-a, mk-p -and mk-p -v-puc -sgrna transfected cells (data not shown). also, the total s protein levels (mcherry signals) of those mutants did not decrease during up to days' culture. we hypothesize that the internalized pedv s proteins are transported back to the virion assembly sites rather than being degraded in the lysosome. for pedv, this endocytosis may be a strategy to efficiently use the expressed s proteins for the generation of infectious viral particles. this hypothesis may be supported by the quantitative data on s proteins incorporated into virions (fig. ) , because the icΔ aa pedv with an intact yxx⌽ motif had a number of s projections similar to that of icpc a. this endocytosis signal yxx⌽ motif may also help pedv evade host immune responses by constantly decreasing the amounts of viral s proteins on the cell surface. previous studies reported that inactivation of the yxx⌽ motif in the envelope proteins of simian immunodeficiency virus (siv) and human immunodeficiency virus (hiv) attenuated the corresponding virus and enhanced the t cell responses in vivo ( , ) . moreover, host antibodies that have already bound to cell surface s proteins would be endocytosed into the cytoplasm, to evade antibody-dependent cell lysis ( , ) . another possible function of this yxx⌽ motif in pedv s protein is that it could downregulate the levels of viral receptors on the cell surface, which may be involved in superinfection exclusion. the role of the yxx⌽ and kvhvq motifs in pedv pathogenicity. in the gnotobiotic piglet study, we observed that the virulence of icΔ aa was significantly reduced compared with that of the parental virus, icpc a. in contrast, no statistically significant differences were observed in the virulence between icpc a and the icΔ aa mutant, which lacks the kvhvq motif. mutant icya caused significantly milder villous atrophy in the jejunum than icpc a. although not significant, it caused a delayed onset of diarrhea and lower peak fecal shedding titers than did icpc a. therefore, icya pedv with an inactivated yxx⌽ motif was slightly attenuated. we investigated why the deletion of both motifs from the s protein (icΔ aa), but not the deletion of errs alone (icΔ aa), significantly attenuates a virulent pedv. tem images showed that icΔ aa-infected vero cells contained significantly higher numbers of mature virions in the golgi vacuoles than those infected with icΔ aa ( fig. f and g) . particularly, icΔ aa had a significantly lower percentage of purified virions with s projections on the surface and lower numbers of s projections on individual virions with surface s projections than the other three viruses (fig. ) . therefore, icΔ aa may generate the highest level of defective virions (without enough s protein projections on the viral surface) among the four pedvs, causing attenuation in vivo. the difference in s incorporation into viral particles among the pedv mutants may be due to three reasons. first, the lack of both sorting signals renders more icΔ aa s proteins to be transported onto the cell surface, decreasing its level in the virus assembly site, ergic. second, the intense and rapid cell-cell fusion induced by surface s proteins in the infected cells causes significant intracellular membrane rearrangements ( ) , which may interfere with the icΔ aa budding and egress process. in contrast, icΔ aa does not cause significant syncytium formation. the third reason may be the altered interaction between s and m proteins in the virion assembly site. although some previous studies revealed that mutation of the errs (klhyt) of sars-cov s protein altered the s-m interaction and s incorporation into virions ( , ) , another study suggested that inactivation of this motif in the s of ibv did not affect the s-m interaction ( ) . for pedv, a previous study of mk-p also showed that the impaired kvrvq motif did not alter the s incorporation ( ) , which is similar to our observation for icΔ aa (fig. ). in our s and m coexpression assay, we found that the four s proteins colocalized with the m proteins in the cytoplasm at the same level, and the coexpression of m protein significantly decreased the transportation of the four s proteins onto the cell surface, implying that those four s mutants may still interact properly with m proteins, although more experiments to evaluate s-m physical interactions are needed. our current data support the first and second possibilities. another reason for the attenuation of icΔ aa pedv in pigs is that mutation of pedv yxx⌽ may also interfere with the synthesis of orf sgrna, since the predicted trs of open reading frame (orf ) sgrna overlaps with the nucleotide sequences encoding the tyrosine of the yxx⌽ motif ( ) . it was reported that a recombinant ibv bearing mutations in its dilysine motif (kkxx) expressed less e protein, due to the decrease of sgrna synthesis ( ) . in this study, the sgrna levels in the icΔ aa-and icya-infected cells were about log and log lower than that in the icpc a-infected cells, as determined by sgrna -specific taqman real-time reverse transcription-qpcr (rt-qpcr) (data not shown). sgrna expresses the accessory protein orf . we do not know whether the e gene in the sgrna can be translated during pedv infection. therefore, it is highly possible that the orf protein level was decreased in the icΔ aa-and icya-infected cells. however, due to the lack of effective antibodies against pedv orf and e proteins, we could not confirm these results at the protein level. the decrease of orf expression may also contribute to the significant attenuation of icΔ aa and the slight attenuation of icya in piglets because we determined previously that a pedv lacking an intact orf gene is partially attenuated in piglets ( ) . the role of s ct truncation in vero cell-adapted pedvs and pedv vaccine development. in our study, the deletion of both the yxx⌽ and kvhvq motifs from the s protein could not completely attenuate a highly virulent pedv strain in piglets, as icΔ aa still caused moderate diarrhea in all the piglets (n ϭ ). therefore, the ablation of the two motifs was probably not the only determinant of the reduced virulence for those attenuated/mild pedv variants (fl , sm- , chm , pc a-p , and knu- ). the attenuation of these strains must be the combined consequence of all the mutations in their genomes. we investigated why some vero cell-adapted pedv variants (pc a-p , sm- , and knu- ) lost function of the yxx⌽ and kvhvq motifs during continuous passaging. we found that the corresponding mcherry-tagged s proteins (Δ aa, Δ aa, and hd) caused larger syncytia than the wild-type s proteins. the loss of the two motifs may facilitate the rapid spread of those pedv variants between cells through enhanced syncytium formation. this idea was supported by a study of the mutant avct . jengarn et al. ( ) demonstrated that recombinant avct was rescued in vero cells only if the infectious cdna clone contained the Δ aa mutation in the s ct. we conclude that inactivation of both the yxx⌽ and kvhvq motifs of the s protein could be applied in the rational design of a pedv vaccine since it results in more s proteins being transported onto cell surface in the infected enterocytes, facilitating antigenic recognition by the host immune system, and may not decrease protective immunogenicity because no neutralizing epitope has been identified in the s ct of pedv. we aim to test this hypothesis in future studies. to achieve complete attenuation and to increase the safety of an attenuated vaccine, the inactivation of the two motifs should be combined with mutations in other key genes in the pedv genome. in conclusion, we showed that the yxx⌽ and kvhvq motifs in the ct of pedv s protein regulate the surface s protein level. the deletion of both motifs significantly reduced the virulence of pedv in pigs. our discovery can aid in the rational design of attenuated vaccines for pedv and other covs containing similar motifs in their s proteins. cells, reagents, and antibodies. vero cells (atcc ccl ) were cultured in dulbecco modified eagle medium (dmem; gibco, carlsbad, ca) supplemented with % fetal bovine serum (fbs) (atlanta biologicals, flowery branch, ga). the guinea pig polyclonal antibody (pab) gp against pedv s was generated in our laboratory as described below. pedv s subunit (aa to ) gene of vero cell-adapted pedv pc strain at passage level (genbank accession no. km ) was cloned into pfuse-higg -fc vector (invivogen, san diego, ca), expressed in hek- t cells, and purified using a protein a affinity column (pierce, waltham, ma). guinea pigs were first immunized with the purified s subunit mixed with freund's complete adjuvant via subcutaneous injection, followed by two booster injections of the same dose in freund's incomplete adjuvant with a -to -week interval between doses. the total igg antibodies in the hyperimmune serum were purified. the production of guinea pig serum was approved by the institutional animal care and use committee (iacuc) of the ohio state university. mouse monoclonal antibodies (mabs) sd - against pedv s (residues to ) ( ) and sd - against pedv n proteins were kindly provided by diego diel and eric nelson at south dakota state university, respectively. rabbit hyperimmune serum against pedv m proteins was kindly provided by ying fang at kansas state university. mouse mab against the ergic- (oti a ) was purchased from enzo life science (farmingdale, ny). rabbit mab against rab (c b ) were purchased from cell signaling technology (danvers, ma). alexa fluor (af )-or af -conjugated secondary goat antibodies against the igg antibodies of different species were all purchased from life technologies (gibco, carlsbad, ca). icΔ aa (n ϭ ), icya (n ϭ ), or pbs (mock treatment; n ϭ ). postinoculation, we observed clinical signs and collected rectal swabs every h. all the piglets were euthanized at hpi to evaluate histopathological lesions at the acute phase of infection. taqman rt-qpcr for the detection of fecal pedv n rna, plaque assay for the detection of fecal infectious pedv titers, and immunohistochemistry staining of pedv antigens using mouse mab against pedv n protein (sd - ) were performed as described previously ( , ) . this animal study was approved by the iacuc of the ohio state university. growth curves of infectious pedvs. the growth curves of pedv replication in vero cells were determined by inoculation of monolayers of cells with each of the four recombinant pedvs with an moi of . or . after h absorption, the inocula were removed and the cell monolayers were washed with pbs three times. maintenance medium containing g/ml of trypsin was added to the cell culture. the total viruses (intracellular and extracellular) in each sample were collected by freezing and thawing the culture at appropriate time points. the infectious titers of viral samples were titrated by plaque assays. plaque assays. plaque assays were performed as described previously ( ) . briefly, monolayers of vero cells were inoculated with appropriate dilutions of recombinant pedvs for h. then the inocula were removed and the cells were washed with pbs three times. the cells were overlaid with an agarose-mem (gibco, carlsbad, ca) mixture containing g/ml of trypsin. the diameters of individual plaques were measured by using an ix- fluorescence microscope (olympus, tokyo, japan) and its software. tem. transmission electron microscopy (tem) of pedv-infected cell sections was performed as described previously ( ) . to visualize the subcellular structure in tem, recombinant pedv-infected vero cells (moi ϭ ) were cultured in trypsin-free medium for h and fixed with fixative ( % glutaraldehyde, % paraformaldehyde in . m potassium phosphate buffer [ph . ]) for h. samples were embedded in agarose, dehydrated, and re-embedded in em bed resin (electron microscopy science, hartfield, pa). ultrathin sections of each sample were prepared and stained. to visualize s protein projections on the virions, vero cells were inoculated with the recombinant pedvs (moi ϭ ) in the presence of trypsin. without freezing and thawing, virions in -ml culture supernatants of each recombinant pedv were purified by using peg- (sigma-aldrich, st. louis, mo) precipitation, followed by % sucrose-cushion ultracentrifugation ( , ϫ g for h), according to a previous protocol ( ) . to maintain consistency, the four pedvs were cultured and prepared simultaneously. samples of subcellular section and purified virions were stained with % uranyl acetate prior to tem imaging. images were visualized using an h- tem (hitachi, japan). statistical analysis. the statistical analyses were performed using graphpad prism . . the data in fig. b , b to e, d to f, g, and b are shown in box-whisker plots. the outliers were the values / greater than the upper quartile or / smaller than the lower quartile ( ) . comparisons of means of groups in these figures and fig. a, d , b, and c were analyzed by one-way analysis of variance (anova) followed by tukey's multiple-comparison test. in the fig. a , b, b, and c, the two groups were analyzed by student's t test. a p value between each two groups less than . was considered significantly different. porcine epidemic diarrhea in china updated estimated economic welfare impacts of porcine epidemic diarrhea virus (pedv) assessment of the economic impacts of porcine epidemic diarrhea virus in the united states emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences proteolytic activation of the porcine epidemic diarrhea coronavirus spike fusion protein by trypsin in cell culture mutation in the cytoplasmic retrieval signal of porcine epidemic diarrhea virus spike (s) protein is responsible for enhanced fusion activity coronavirus m 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complement-mediated lysis syncytia formation induced by coronavirus infection is associated with fragmentation and rearrangement of the golgi apparatus pedv leader sequence and junction sites characterization of a pathogenic full-length cdna clone and transmission model for porcine epidemic diarrhea virus strain pc a the s glycoprotein subunit of porcine epidemic diarrhea virus contains immunodominant neutralizing epitopes engineering the largest rna virus genome as an infectious bacterial artificial chromosome measurement of colocalization of objects in dual-colour confocal images the gnotobiotic piglet as a model for studies of disease pathogenesis and immunity to human rotaviruses experimental infection of a us spike-insertion deletion porcine epidemic diarrhea virus in conventional nursing piglets and cross-protection to the original us pedv infection pathology of us porcine epidemic diarrhea virus strain pc a in gnotobiotic pigs cell culture isolation and sequence analysis of genetically diverse us porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene purification and electron cryomicroscopy of coronavirus particles some implementations of the boxplot complete genome sequence of novel porcine epidemic diarrhea virus strain gd- in china sequence of the spike protein of the porcine epidemic diarrhoea virus phylogenetic analysis of porcine epidemic diarrhea virus field strains prevailing recently in china this study was funded by the national institute of food and agriculture, u.s. department of agriculture, under award number - - (q.w., principal investigator [pi]; l.j.s., co-pi).we thank andrea kaszas and xiaohong wang for technical assistance and juliette hanson, ronna wood, megan strother, dennis hartzler, sara tallmadge, and jeff ogg for animal care assistance. we thank chang-won lee and scott kenney for their critical reviews of the manuscript. we are grateful to diego diel and eric nelson at south dakota state university for providing us the mouse mabs against pedv s and n proteins and to ying fang at kansas state university for providing us the rabbit pab against pedv m proteins. key: cord- -a ba dz authors: chen, qi; wang, leyi; yang, chenghuai; zheng, ying; gauger, phillip c.; anderson, tavis; harmon, karen m.; zhang, jianqiang; yoon, kyoung-jin; main, rodger g.; li, ganwu title: the emergence of novel sparrow deltacoronaviruses in the united states more closely related to porcine deltacoronaviruses than sparrow deltacoronavirus hku date: - - journal: emerg microbes infect doi: . /s - - -z sha: doc_id: cord_uid: a ba dz nan porcine deltacoronavirus (pdcov), has been identified as one of the major enteric pathogens causing diarrhea in pigs since , . both clinical and experimental evidence demonstrated that pdcov is pathogenic to swine and causes similar lesions in the pig intestine as porcine epidemic diarrhea virus (pedv) , . pdcov strains reported from the united states and other countries had . - . % nucleotide identity at the whole-genome level , suggesting that a single genotype is circulating worldwide. regarding sparrow deltacoronavirus (spdcov), one sparrow cov strain, hku - , has been reported thus far . the genomic diversity of sparrow deltacoronaviruses remains unclear. we report the detection and genetic characterization of novel spdcov in fecal samples collected from sparrows found on swine farms in the midwestern united states. in january , multiple batches of porcine oral fluid (pen-based) and floor-picked fecal samples were submitted from a grow-finish stage pig farm in illinois for routine health monitoring. unexpectedly, multiple oral fluid samples and one fecal sample were tested positive for pdcov (the ct value ranged between and ) by duplex pedv and pdcov real-time rt-pcr (see technical appendix). however, no clinical diarrhea or other signs were observed among pigs on the index farm, which prompted us to further investigate the source of positive pcr results. since the pigs were housed in hoop-style buildings with uncontrolled access for wild bird entry and congregation, fecal samples were also collected from sparrows found inside the pig barn (isu - to − ) and were submitted to our laboratory for testing in january , along with feed samples (isu - to − ). surprisingly, / fecal samples were positive using pcr targeting the pdcov n gene, with a ct of . - . , while all feed samples were negative. four additional sparrow fecal samples (isu - to − ) collected in june from the same farm were tested, and one of the samples (isu - ) was positive by pdcov n gene-based pcr, with a ct of . . similarly, in an unrelated grow-finish swine farm in minnesota, two oral fluid samples collected from clinically healthy pigs at the end of october were tested positive (ct of . and . ), and one sparrow fecal sample (isu - ) collected in early november from that barn were tested positive (ct of . ) using a pdcov n gene-based pcr. the pdcov pcr-positive sparrow samples then underwent next-generation sequencing (ngs) using an illumina miseq platform (see supplementary methods). after using an in-house bioinformatics analysis pipeline (supplementary methods), four nearly complete genomic sequences of spdcov (isu - , isu - , isu , and isu ; genbank accession numbers mg , mg , mg , and mg , respectively) were obtained. the genome organizations of all four spdcov strains are similar to those of sparrow cov hku and pdcov with the characteristic gene order ′replicase orf ab, spike (s), envelope (e), membrane (m), and nucleocapsid (n)- ′ (fig. a) . similar to hku , the four spdcov strains contain three orfs downstream of n encoding three nonstructural proteins (ns a, ns b, and ns c), as illustrated in fig. a . whole-genome nucleotide sequence comparison showed that spdcov isu shares a higher identity with isu ( . %) and isu - and − ( . %) than with pdcov ( . %) and hku ( . %), and it has an even lower identity with other avian deltacoronaviruses, such as hku ( . %), hku ( . %), and hku ( . %) (fig. b, supplementary table s ). spdcov isu - and isu - share . % wholegenome nucleotide identity with each other, whereas both strains share . %, . %, . %, and . % identity with isu , isu , pdcov, and hku , respectively, and they share an even lower identity with other avian deltacoronaviruses, such as hku ( . %), hku ( . %), and hku ( . %). these data suggest that the newly identified spdcov isu - /isu - , isu , and isu represent novel spdcov strains. to exclude the possible issue of contamination of sparrow fecal samples with porcine samples, all fecal samples directly collected from swine were tested and were negative for both sdcov and spdcov. in addition, sanger sequencing of four rt-pcr products of the orf ab, m, and n genes of spdcov showed that the sequences had % identity to those from ngs. the four spdcov strains identified in this study share a slightly higher identity ( . - . % and . - . %) to a sparrow cov strain hku - than to pdcov strains ( . - . % and . - . %) in their orf ab and n proteins, respectively ( fig. b and supplementary table s ). in contrast, the four spdcov strains share a significantly lower identity to sparrow cov hku ( . - . %) than to three pdcov strains ( . - . %) in their s protein (fig. b and supplementary table s ). in addition, they share a relatively lower identity ( . - . % and . - . %) to sparrow cov hku than the pdcov strains ( . - . % and . - . %) in their e and m proteins, respectively (supplementary table s ). these findings were also confirmed by phylogenetic tree analysis of amino-acid sequences in which the four spdcov strains clustered together and were closely related to pdcov and sparrow cov hku strains in orf ab, e, m, and n trees ( fig. c and supplementary figure s ). however, the four spdcov strains were close to pdcov but distant from sparrow cov hku in the s tree (fig. d) . in the case of non-structural proteins (ns , ns a, and ns b), the spdcov strains share a high amino-acid identity with each other. however, spdcov isu - / has . % and % identity to isu and isu , respectively, in ns c, which is much lower than the . % identity shared between isu and isu (supplementary table s ). an -nt deletion in the ns c gene of isu - and - resulted in a protein that was amino acids shorter than that of isu and isu in length (supplementary figure s ) . overall, these data further support that the sparrow covs identified in our study are novel spdcov. recombination analysis revealed that pdcov and sparrow cov hku are highly variable from spdcovs in two regions. the most prominent variable region is located in the s-e-m gene region, and another region is located within the orf ab gene (~ nt to nt) (supplementary figure s ) . however, there is no obvious pattern showing that the emergence of spdcov strains identified in the study was through recombination between pdcov and sparrow cov hku . comparison of pdcov pcr primer and probe sequences (targeting the n gene) with spdcov sequences revealed that the probe sequence was exactly identical to the sequence observed in spdcov, and only and / nucleotide mismatches were observed in the forward and reverse primers, respectively (supplementary figure s ) . therefore, pdcov real-time rt-pcr can cross-react with spdcov, which complicates the molecular detection of pdcov. it is plausible that contamination with sparrow feces containing spdcovs caused non-specific positive results for pdcov when the oral fluid and fecal samples collected from clinical healthy pigs were tested using pedv/pdcov duplex rt-pcr. this also raises a concern regarding the specificity of detecting pdcov from environmental and environmentally contaminated samples. to avoid the cross-reaction issue, further modification of the primers and probe for pdcov real-time rt-pcr is needed. pdcov, as a causative agent for pig diarrhea, has been reported in many countries, including the united states, canada, japan, south korea, thailand, vietnam, mainland china, and laos, indicating that pdcov is widespread in pig populations. however, all known pdcovs to date have been relatively conserved and shared . - . % nucleotide identity at the whole genome level. in contrast, the spdcov strains identified in the present study (isu - / , isu , and isu ) as well as one previously identified sparrow cov strain, hku - , exhibited higher genetic diversity ( . - . % nucleotide identity at the whole-genome level). further studies to understand the genetic diversity of spdcov by sequencing more spdcov strains are warranted. another important finding is that, due to the relative conservation between pdcov and spdcov n gene sequences in some regions, the pdcov n gene-based pcr used in this study can cross-react with spdcov. thus, pdcov pcr results should be interpreted carefully when pig samples that can be contaminated by bird feces or other excretions are tested. the spdcov sequence data reported in this study may help determine the evolutionary relationship of various dcovs and help to understand the interspecies transmission of dcovs in future studies. virus taxonomy, classification and nomenclature of viruses coronavirus diversity, phylogeny and interspecies jumping discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus full-length genome sequence of porcine deltacoronavirus strain usa/ia/ / detection and genetic characterization of deltacoronavirus in pigs porcine deltacoronavirus: histological lesions and genetic characterization pathogenicity and pathogenesis of a united states porcine deltacoronavirus cell culture isolate in -day-old neonatal piglets analysis was completed using cgview comparison tool software. the corresponding strains/samples for rings are detailed on the bottom. orf open reading frame, s spike, e envelope, m membrane, n nucleocapsid, ns , ns a, ns b, and ns c represent nonstructural proteins , a, b, and c, respectively. c phylogeny of orf ab amino-acid sequences. d phylogeny of spike protein sequences. the sparrow deltacoronavirus strains identified in this study are indicated with red squares, hku - is marked with a blue square, and three hku strains are marked with a pink triangle. the phylogeny was inferred using the neighbor-joining method in mega version . . statistical support was obtained using bootstrap resampling ( replications) and is drawn on the inferred tree. reference sequences representing coronavirus diversity were obtained from ncbi genbank (pedv, porcine epidemic diarrhea virus, nc_ ; sc-batcov- , scotophilus bat coronavirus , nc_ , tgev transmissible gastroenteritis virus, aj , fipv feline infectious peritonitis virus, nc_ , prcv porcine respiratory coronavirus, dq , phev porcine hemagglutinating encephalomyelitis virus, dq , bcov bovine coronavirus, nc_ , ibv infectious bronchitis virus, nc_ , ibv-partridge partridge coronavirus, ay , tcov turkey coronavirus, nc_ ; bulbul, cov strain hku - , fj ; bulbul cov strain hku - , fj ; thrush cov strain hku - , nc_ ; munia cov strain hku - , nc_ supplementary information accompanies this paper at https://doi.org/ . /s - - -z. key: cord- -txaoz oh authors: jordan, paul c; stevens, sarah k; deval, jerome title: nucleosides for the treatment of respiratory rna virus infections date: - - journal: antivir chem chemother doi: . / sha: doc_id: cord_uid: txaoz oh influenza virus, respiratory syncytial virus, human metapneumovirus, parainfluenza virus, coronaviruses, and rhinoviruses are among the most common viruses causing mild seasonal colds. these rna viruses can also cause lower respiratory tract infections leading to bronchiolitis and pneumonia. young children, the elderly, and patients with compromised cardiac, pulmonary, or immune systems are at greatest risk for serious disease associated with these rna virus respiratory infections. in addition, swine and avian influenza viruses, together with severe acute respiratory syndrome-associated and middle eastern respiratory syndrome coronaviruses, represent significant pandemic threats to the general population. in this review, we describe the current medical need resulting from respiratory infections caused by rna viruses, which justifies drug discovery efforts to identify new therapeutic agents. the rna polymerase of respiratory viruses represents an attractive target for nucleoside and nucleotide analogs acting as inhibitors of rna chain synthesis. here, we present the molecular, biochemical, and structural fundamentals of the polymerase of the four major families of rna respiratory viruses: orthomyxoviridae, pneumoviridae/paramyxoviridae, coronaviridae, and picornaviridae. we summarize past and current efforts to develop nucleoside and nucleotide analogs as antiviral agents against respiratory virus infections. this includes molecules with very broad antiviral spectrum such as ribavirin and t- (favipiravir), and others targeting more specifically one or a few virus families. recent advances in our understanding of the structure(s) and function(s) of respiratory virus polymerases will likely support the discovery and development of novel nucleoside analogs. respiratory viral infections are a global health concern caused by dozens of different types of viruses. the respiratory diseases resulting from these viral infections represent one of the main causes of death in developing countries. a more thorough understanding of respiratory viruses, their epidemiology, as well as medical impact on the communities they affect will delineate the path toward eventual treatments and future abatement of the illnesses. while symptoms of many respiratory viruses are similar, the viruses themselves are characteristically unique. categorically, viruses are grouped based on similarities such as the nature of their nucleic acid genome, envelope presence, overall size, and even capsid uniformity. this review focuses on the following families of rna viruses: orthomyxoviridae, paramyxoviridae and pneumoviridae, picornaviridae, and coronaviridae. orthomyxoviridae comprise negative (-) sense singlestranded (ss) rna viruses that are segmented, enveloped, and includes the influenza viruses (see table ). paramyxoviridae and pneumoviridae are also (-)ssrna viruses, but are non-segmented and enveloped, and include parainfluenza virus (piv), human respiratory syncytial virus (rsv), and human metapneumovirus (hmpv). the picornaviridae family, which contains positive (þ)ssrna viruses are non-enveloped; the key members include the rhinoviruses and enteroviruses. lastly, the coronaviridae are (þ)ssrna enveloped viruses, which include, chiefly, human coronavirus (hcov), and severe acute respiratory syndrome (sars)-associated and middle eastern respiratory syndrome (mers) cov. young children, the elderly, and patients with compromised cardiac, pulmonary, or immune systems are at greatest risk for serious disease associated with these rna virus respiratory infections. in a -year study, over % of acute respiratory viral infections in critically ill children admitted to a pediatric intensive care unit were caused by either a picornavirus, rsv, piv, or hmpv (see figure ). other dna viruses such as adenovirus can be the source of respiratory infections but will not be discussed here. in addition to their wide variation in viral characteristics, respiratory rna viruses are also remarkable in their epidemiological variety. they differ in ( ) their outbreak calendar, where some are seasonal and others are year-round, ( ) their patient profile, whether infant, geriatric, or otherwise healthy adults, and ( ) the morbidity and/or mortality associated with infection. influenza virus is a (-)ssrna virus and a member of the orthomyxoviridae family. there are four influenza genera within this family, called a, b, c, or d. influenza a and b contain hemagglutinin and neuraminidase envelope glycoproteins. influenza c and d have a single surface glycoprotein called the hemagglutinin-esterase fusion protein. , antigenic variation in these glycoproteins results in limited vaccine protection. influenza, or the flu, presents with symptoms such as headache, cough, fever, sore throat, malaise, and chills. generally, the flu lasts from days to weeks and the severity of infection is determined by the host. the highest incidence of influenza infection occurs in younger patients (< years old) where a shorter infection is typical, while those at risk for longer and more severe illness and complications associated with infection are the pediatric (< years old) and geriatric populations (> years old), pregnant women, and immunocompromised individuals. , it is estimated that - million cases of the flu occur annually around the globe, with a quarter to half million deaths resulting from these illnesses. piv (paramyxoviridae family), rsv, and hmpv (pneumoviridae family) until recently, piv, hmpv, and rsv were all categorized in the paramyxoviridae family due to their phylogenetic proximity in the order mononegavirales, the non-segmented negative-strand rna viruses. more recently, rsv and hmpv have been assigned as members of the newly formed pneumoviridae family. while influenza outbreaks are most prevalent in the winter, some viruses such as piv persist year-round. human piv has four types ( to ) and was known historically to induce respiratory complications mainly in children and the immunocompromised; however, more recently, it has been identified as a concern in the adult population as well. symptoms of piv include upper and lower respiratory tract infection, middle ear inflammation, bronchitis, pneumonia, and croup, the last of which results in the most hospitalizations in the pediatric patients infected by this virus. , up to one-third of the nearly million annual cases of lower respiratory tract infection in children is at least partially due to the presence of pivs. rsv and piv infections are among the most common reason for hospitalization of young children. , the two strains of rsv, a and b, are distinguished by genetic variations in the g surface glycoprotein. dissimilar to piv, rsv occurs mostly in the winter months in its target pediatric population. symptoms include runny nose, nasal inflammation, cough, sore throat, low-grade fever, wheezing, bronchiolitis, and pneumonia. current estimates in developing and industrialized countries suggest as many as million cases of rsv worldwide in the pediatric population less than years old, % of which require hospitalization, and % to % of hospitalized cases result in mortality. this amounts to between , and , deaths in young children annually. , hmpv, like rsv and influenza, tends to have greatest prevalence in the winter and studies have shown that by the age of years, nearly all children have been infected with this virus. the clinical manifestations of infection with this virus are upper and lower respiratory tract infections, bronchiolitis, middle ear inflammation, fever, chills, pneumonitis, and wheezing. of note, hmpv tends to occur in populations with seasonal inconsistency as studies done on italian populations shortly after its discovery from - , showed a range of infection from % to % depending on the year. patterns of seasonal irregularity like this have been noted with other respiratory viruses, particularly rsv and influenza. rhinoviruses are thought to be the cause of up to twothirds of what is termed the common cold, worldwide. children tend to experience up to of these infections, or colds, per year, while this incidence drops in adults to just - per year. there are three distinct species of rhinovirus, rv-a, rv-b, and rv-c, each of which infects humans at different periods throughout the year. symptoms include cough, fever, sneezing, nasal congestion, sore throat, fever, and headache and usually last - days after an initial -h viral incubation. in addition to the three rhinoviruses, four enterovirus species result in disease in humans, ev-a, ev-b, ev-c, and ev-d, while ev-e through ev-h, and ev-j affect non-human hosts. , enteroviruses differ from rhinoviruses in that while rhinoviruses are limited to the respiratory airways, enteroviruses infect a wide range of cell types. they result in a large array of complications associated with the respiratory, gastrointestinal, and central nervous systems. manifestations of enterovirus infection range from a febrile cold to encephalitis, pneumonia, viral meningitis, and even death. although ev-c and ev-d are the principal enteroviruses that cause respiratory illness, ev-a also includes ev . ev causes hand-foot-and-mouth disease, a highly contagious pathogen in children that mainly results in a maculopapular rash, blisters on the limbs, and ulcers in the mouth. ev is most prevalent in the summer months and tends to be more ubiquitous in tropical zones of the globe. in rare cases of severe ev infection, respiratory illness can lead to pulmonary edema, hemorrhage, and lung failure. presently, six hcov are recognized: hcov- e, hcov-nl , hcov-oc , hcov-hku , and the well-known sars and mers cov. these cov can be further characterized based on genera of alpha, beta, gamma, or delta; the alpha and beta cov comprise the six viruses mentioned above, and are those that infect humans. cov e and oc are both pathogens associated with the common cold, but can cause pneumonia as well. hcov-nl and hcov-hku infection show similar clinical features to those in patients with e and oc , but clinicians have also reported bronchiolitis, croup, and pneumonia in infected individuals. , the first cov recognized as pandemic threat is sars-cov. sars was discovered in - after a perplexing epidemic of pneumonia among hospital workers in china. by the end of its global epidemic, sars disseminated to countries, infecting over individuals, and killing roughly % of those infected. roughly a decade later, a similar pattern occurred with mers, which began in in jordan with an outbreak of a respiratory illness among hospital workers, one of whom died of the infection. later that year, a man with pneumonia and multiple organ failure in saudi arabia was found to have the mers pathogen. adults are the target population for both sars and mers with a median age range of - years; mers occurs predominantly in men whereas sars does not. the clinical features of both sars and mers range from mild to severe respiratory illness, fever, chills, cough, shortness of breath, vomiting, and diarrhea, with the latter displaying a more lethal pneumonia and renal failure. , even worse than sars mortality, retrospective analysis has shown that of the confirmed cases of mers, % were fatal. viral polymerase: an important molecular target for antiviral therapy nucleoside analogs represent one of the dominant classes of antiviral agents due to their widespread use against the common chronic infections caused by human immunodeficiency virus (hiv), hepatitis b virus, and herpesviruses. in the past years, multiple nucleoside and nucleotide analogs have been developed as direct-acting agents against rna virus infections such as hepatitis c virus (hcv), but have not yet been successfully applied to acute infections caused by respiratory viruses. only a handful of nonnucleoside drugs have been developed for the treatment and prevention of these viruses. such drugs include the fda approved oseltamivir, zanamivir, and peramivir for influenza virus infection, palivizumab for rsv prevention, as well as the two discontinued clinical candidates targeting rhinovirus, pleconaril and rupintrivir. these molecules possess limitations preventing their widespread use, such as short therapeutic window and risk of resistance selection for the neuraminidase inhibitors, and only partial protection associated with prophylactic use for palivizumab. this has provided the impetus for the approval of new drugs with a broader therapeutic use. the recognized advantages of direct-acting agent nucleosides over other classes of antiviral agents are ( ) their propensity to cover a broad-spectrum of virus strains and sometimes species and ( ) their high barrier to antiviral resistance. both properties are best explained by the mechanism of action common to most antiviral nucleosides: targeting viral polymerases. after being metabolized by host kinases to their triphosphate form, antiviral nucleotides compete with natural nucleoside triphosphates (ntps) to bind to the active site of viral polymerases and alter dna or rna synthesis. the nucleotide binding site of these proteins is usually well conserved among virus families and any changes in neighboring amino acids often comes at a cost for the enzyme and the virus. the functional and structural features of rna polymerases of respiratory viruses targeted by antiviral nucleosides are described in the following paragraphs. influenza virus is a (-)ssrna virus and a member of the orthomyxoviridae family. the viral genome has eight segments in influenza a and b and seven segments in influenza c and d. in influenza a, these encode for or proteins. these are non-structural protein (ns ), non-structural protein (ns ), matrix protein m and ion channel protein m , polymerase acidic (pa) protein, polymerase basic protein (pb ), polymerase basic protein -f , polymerase basic protein (pb ), nucleoprotein (np), hemagglutinin (ha), and neuraminidase (na). some viruses express the pb -n protein. all four species of influenza adopt similar arrangements with the viral genomic segments forming viral ribonucleoprotein complexes associated with one heterotrimeric polymerase. influenza a polymerase is composed of three subunits that yield a kda heterotrimeric complex. the longest viral rna segments encode for the pa protein, pb , and pb , which assemble to form the influenza polymerase complex (see figure ). the three subunits interact noncovalently to exert their polymerase activity. the polymerase transcribes viral rna into messenger rna (mrna) and then replicates it using a complementary rna intermediate. the process of transcription includes cap snatching, where short-capped cellular rna are bound by the pb subunit, cleaved from the pa endonuclease domain, and then utilized for priming mrna synthesis by the pb domain. [ ] [ ] [ ] [ ] [ ] [ ] the pb subunit has an n-terminal domain (pb -n) from residues to and a c-terminal domain (pb -c) from residues to . pb -n, including a lid domain, interacts with the c-terminal extension and thumb of pb . the pb -c includes several notable structural features and subdomains such as the c-terminal nuclear localization signal, the pb -domain, the pb cap- linker, the mid domain, and a cap binding domain. based on structural biology, the pb domain has a key exterior, positively charged residue at the position within a flexible loop that partially wraps around an alpha helix to form what is known as a phi-loop. importantly, this residue is in the middle of a set of highly conserved, basic residues forming a net positive charge. a signature structural element is the conserved p[f/p]aaapp motif on the n-terminal side of the residue that is part of the alpha helix previously described. mutation of the p or f residue significantly decreased the ability of the virus to replicate, presumably by causing a slight kink in the alpha helix that alters the polymerase function. the exact role of the pb domain remains unclear, but recent evidence suggests it is not necessary for in vitro binding and transcription of viral rna; this has not been proven true in a cell-based format. the pb domain is at the center of the polymerase complex and within its center is a classic right-handed shape with the signature fingers, palm, and thumb subdomains (see figure ). these subdomains are described as conserved rna-dependent rna polymerase (rdrp) motifs pre-a/f and a-e. the pre-a/f motif describes the fingertips and a small loop, which spreads over to the thumb domain; the tip of this loop is stabilized by an alpha helix within the pa domain. residues within the pre-a/f may guide and bind ntps and the incoming template. in addition to these subdomains, n-terminal and c-terminal extensions interact with pa and pb domains. the fingers and palm are covered by a linker connecting the two subdomains of pa. the pb possesses a b-hairpin loop within motif e from residues to in the thumb subdomain of influenza a. in de novo initiation, it has been shown with other related polymerases that this priming loop may serve as a platform for the initial ntp on the end of the template and ensure against double-stranded rna. biochemical investigations have shown that the proline found within the loop tip motif of -ala-his-gly-pro is necessary for in vitro and cell-based rna synthesis. this loop may also be necessary during replication mode for terminal de novo initiation but unnecessary for internal initiation and transcription. the pa endonuclease domain or the pa subunit, as it will be named here, has little homology to other proteins and its exact enzymatic function was discovered only recently. the subunit was expressed in insect cells to reveal an n-terminal third (pa-nter) and a c-terminal two-thirds (pa-cter) subdomains. they have molecular weights of and kda, respectively. these two subdomains are connected by a flexible linker. the endonuclease activity was originally thought to occur through the pb or pb domains but the structure of the pa-nter was solved by two groups to reveal that the catalytic residues for endonuclease activity reside in the pa domain, not in the pb subunit as originally thought. , the pa endonuclease domain contains a signature (p)dxn(d/e)xk motif that is conserved among influenza viruses and coordinate divalent cations such as magnesium or manganese. although the exact quantity and identity of ions present in the native influenza enzyme are unclear, the endonuclease activity is strongly activated by metal ion binding through hydrolysis of ssdna and ssrna substrates. human metapneumovirus and human rsv are nonsegmented, negative strand rna viruses from the pneumoviridae family of the order mononegavirales. the polymerases from this class of viruses are multifaceted with multiple enzymatic functions contained within a single protein. it exists as part of a ribonucleoprotein complex, or replicase, composed of l, n, p, and m - proteins in complex with rna. these include rna synthesis activities carried out by an rdrp domain but also include capping and methylation functions. the rdrp carries out transcription and replication of its genome in response to cis-acting elements within the genome. the genome of rsv is approximately kilobases long and is transcribed into capped and poly-adenylated mrnas. the hmpv genome is kilobases long. current understanding of hmpv transcription is based on knowledge gained from the more extensive characterization of rsv transcription, which is the focus here. at the beginning and end of each viral gene lies a conserved region (cr) of - nucleotides and - nucleotides, respectively. these are termed the gene start (gs) and gene end (ge) signals with an intergenic, non-transcribed region between these genes. at the side of the genome, prior to the first gene, is the leader (le) extragenic region and at the end is the trailer (tr) extragenic region. the lengths of these extragenic regions can vary based on the virus but in rsv the le region is nucleotides long. to transcribe its rna, the polymerase initiates at the third nucleotide to transcribe a short uncapped transcript of about nucleotides. this rna is released but the polymerase remains affixed to the template where it proceeds along until it encounters the gs signal for the first gene and subsequently begins rna synthesis. these mrnas are modified with a methyl cap and when the ge signal is reached, a polyadenylated tail is added, and the mrna is released. the polymerase then scans for the next gs region. the genome is replicated starting at the leader promoter in a processive manner to yield a positive sense antigenome rna. the and ends of the antigenome contain the trailer and leader complement. the trailer complement ultimately directs genome rna synthesis. the core rsv polymerase consists of a kda large (l) protein of approximately amino acids and a kda phosphoprotein (p) that synthesizes an rna product upon the addition of an rna template. [ ] [ ] [ ] [ ] the p protein is thought to act as a chaperone to aid in the stability and expression of the polymerase. during rna synthesis, the p protein anchors the l protein to the n protein and also binds to the m - transcription antitermination factor. [ ] [ ] [ ] [ ] this matrix protein, m - , serves as an elongation factor and is necessary for the polymerase to be fully processive in producing long mrna products. no structure is available for any l protein from the paramyxoviruses and pneumoviruses, largely due to the size, solubility, and complexities with yielding enough highly pure protein. however, the cryo-electron microscopic (em) structure of the l-p complex from a highly similar virus, vesicular stomatitis virus (vsv), has recently been solved. vsv is a non-segmented, negative strand virus from the rhabdoviridae family and given the high sequence conservation of the l protein, the structure of vsv l has provided important structural insights. the l proteins of rsv, vsv, and other related negative sense rna polymerases can be divided into six cr. the first three regions (cri-iii) of vsv generate a doughnut-like structure in negative stain em and cryo-em analysis. , the remaining crs appear as globular appendages on this doughnut. the doughnut structure adopts a classic right-handed configuration in the cryo-em reconstruction composed of fingers, palm, and thumb domains, like other polymerases (see figure ). the criv and crv contain conserved residues and catalytic motifs necessary for enzymatic function. among these is the gxxt[n]hr motif, a highly conserved set of residues necessary for ntp binding and the hr motif, which forms covalent histidine rna intermediate. within crvi there is a motif with sequence similarity to the -o-ribose methyltransferase (mtase) domain, which has been characterized in the vsv l system. while it is unknown exactly how similar the capping mechanism of vsv is to rsv and hmpv, the detailed mechanism of the vsv capping biochemistry provides insights. the capping of vsv is unique in that it is accomplished through an unconventional rna:gdp polyribonucleotidyltransferase (prntase) rather than a guanyltransferase. [ ] [ ] [ ] the vsv capping occurs in two parts starting with conversion of gtp to gdp through a gtpase followed by the covalent attachment of a histidine to prna to form a phosphamide bond. this gdp then serves as a nucleophile to attack the prna and results in the release of the gppprna. the second part of the reaction consists of the mtase reaction, which uses s-adenosylmethionine to methylate nitrogen and the -oxygen of the cap. a recent crystal structure of the mtase domain from hmpv has provided additional clues into this reaction (see figure ). a -residue fragment was expressed, consisting of the cr-iv containing the putative mtase with an additional c-terminal k-k-g motif (termed cr-viþ). with the exception of the k-k-g motif, the fold of the hmpv mtase domain indicates a conserved fold compared to vsv. while the cr-viþ was active, the reaction rate was very slow and structural and biochemical results did not clearly identify active site residues. this suggests that mtase requires other co-factors or additional parts of the l protein to be catalytic. human rhinovirus (hrv), enterovirus (ev ), and poliovirus (pv) are nonenveloped, positive strand rna viruses and are all members of the picornaviridae family. picornaviruses replicate their genome using an rdrp, called d pol . replication takes place in one of two forms: primer-dependent format or de novo rna synthesis. de novo rna synthesis, which uses a single initiation nucleotide, gives the -hydroxyl group for adding the sequential nucleotide whereas a primer-dependent format uses a protein-based primer or an oligonucleotide for the hydroxyl group donor. the polymerases from the picornavirus family only use a protein-primed mechanism of initiation. , the d pol uses a small viral peptide (vpg) to initiate both plus and minus rna synthesis in vivo, making picornaviridae unique in their initiation mechanism. rna synthesis is initiated using a highly conserved tyrosine residue within vpg using cis-acting replication element as a template whose position varies depending on the genus. , the d pol aids in the binding of two ump molecules to the tyrosine hydroxyl group of vpg. [ ] [ ] [ ] the product of this reaction, vpg-pu is then extended by an additional uridine to form vpg-pupu. the d pol is located at the c-terminal end of a longer viral polyprotein of approximately kda and the structures of d pol have been solved for ev , hrv , and pv, among many others. [ ] [ ] [ ] the structures of these polymerases are largely similar and have sequence domains a-g indicating this conservation. the -residue polymerase domain adopts a right-handed configuration with fingers, palm, and thumb subdomains providing an optimal arrangement for substrate and metal cation access during the catalytic cycle. the palm, fingers, and thumb subdomains contain these sequence motifs (motifs a-e in the palm subdomain shown in figure ) . , these motifs have specialized roles in catalysis including nucleotide binding and overall structural integrity of the active site. the palm contains the active site of all the rdrps and its structure consists of an antiparallel beta-sheet surrounded by three alpha helices. additional substructures within the fingers domain are referred to as the index, middle, ring, and pinky fingers. , all but the pinky fingers build an extended beta-sheet that seems to be conserved. the index finger within the fingers subdomain makes an important contact with the thumb subdomain and pushes the ring finger down to trap it. this conformation results in the ring finger being the roof for ntp entry and making important interactions with the triphosphate. an additional structural feature of motif f is a positively charged tunnel that modulates the interactions of ntp. this tunnel aides in the diffusion of nucleotides and is conserved among this family of viruses. proteins and enzymes rarely exist in a monomeric state in nature but are energetically driven to higher order, oligomeric states through polar and hydrophobic interactions and disulfide bond formation. the development of increasingly sophisticated structural biology techniques, including high-resolution x-ray crystal structures and cryo-em, has provided a snapshot into how polymerases may adopt such oligomeric states. the understanding and characterization of the oligomeric states place these multifunctional enzymes in a greater biological context. an example of such oligomeric states and how this impacts the catalytic function is the polymerase from pv. pv rdrp is described as having macromolecular contacts at two polymerase interfaces (interface i and interface ii). interface i is defined by the interaction of the thumb domain from one polymerase with the palm of another polymerase. interface ii describes interactions of the n-terminus of one polymerase with the thumb of a neighboring polymerase within the crystal lattice. additionally, pv can form tube-like structures suggesting it is a highly dynamic structure able to undergo and adopt multiple conformational arrangements. the oligomeric state of pv polymerase is required for membrane-associated rna replication in infected cells, as demonstrated by mutating residues involved in protein-protein interactions. , the polymerase of cov the cov are part of the larger nidovirus family and have exceptionally large genomes of up to kilobases in length. cov are positive sense rna viruses, with a notable example being sars as one of the most pathogenic member of this viral family. the cov genome has a -cap, is polyadenylated on the end, and generates a total of non-structural proteins (nsp to nsp ). the two-thirds of its genome encodes for non-structural proteins that combine to form a replication and transcription complex that completes viral rna synthesis. the nsp protein is the rdrp and is typically composed of a n-terminal domain composed of a nidovirusspecific rdrp-associated nucleotidyltransferase (niran) and a c-terminal containing the rdrp domain, which contains a set of six conserved motifs (motifs a-f) responsible for recognizing substrates and template. , the niran domain has only been identified in nidoviruses and is approximately residues long in cov. in sars-cov, a reverse genetic system identified this motif as a requirement for replication of its viral genome. while niran activity has not been directly observed outside of a reverse genetic system for cov, based on the nucleotidylation activity of eav nsp , the niran domain is hypothesized to play a role in protein priming, capping, or as a potential universal ligation mechanism. the active site of the polymerase is located within motif c and is composed of conserved (within the nidovirus family) ser-asp-asp residues. importantly, conserved aspartates found in motif a of sars-cov, which combined with those found in motif c, contribute to the polymerase and rna synthesis activities. this is different from other positive strand rna viruses which contain a gdd active site. motif a along with motif c aid in coordinating the metal ions necessary for catalysis. the sars-cov harbors a signature sequence in motif g necessary for primer-dependent rna synthesis. , due to difficulties in obtaining large enough amounts of highly pure protein, the structures of cov nsp have not been solved either by x-ray crystallography or cryo-em. therefore, the structural information currently available is based solely on structural models obtained via sequence alignment and homology modeling techniques. these models indicate a right-handed fold composed of fingers, palm, and thumb subdomains with clearly defined entry and exit channels, consistent with rdrp domains for other structurally characterized positive sense rna polymerases (for example, foot-and-mouth disease virus) but clearly distinct from the known molecular topology of negative-stranded rdrps. no structural models predict the presence of a priming loop. these data combined with biochemical data indicating no de novo initiation of rna synthesis may account for the functionality of motif g. a complete characterization of the in vitro nsp rdrp activity has demonstrated overall weak activity. initial evidence suggested that a previously uncharacterized n-terminal domain may have been required for polymerase activity. however, the addition of this domain using a c-terminally tagged protein still yielded protein with poor activity and processivity. based on these results and to increase the in vitro activity of the nsp , two other non-structural proteins, nsp and nsp , were added to the nsp protein in a primer extension mode. the addition of nsp and nsp to nsp appear to activate and increase the processivity of the polymerase allowing it to produce an rna synthesis product of nucleotides in the presence of mg þ . linking the nsp and nsp subunits together also increased the polymerase reaction efficiency suggesting that nsp -nsp complex formation may influence the reaction rate. importantly, nsp can interact with an nsp -nsp -nsp complex without influencing rna synthesis activities. nsp contains an exoribonuclease domain that has been shown to decrease nucleotide mismatch, in many ways similar to the proofreading exoribonuclease activities correlated with a polymerase. in this section, we aim to answer the following questions that are important to medicinal chemists, biologists, and drug developers working in the field of virology. are there clinically relevant nucleoside analogs that are potent against one or multiple respiratory viruses? how were these molecules first identified, and which ones have been approved for commercial use? why aren't molecules like ribavirin and its analogs more widely used to treat respiratory viral infections? what are the current approaches to develop new nucleoside analogs against respiratory rna viruses? the broad-spectrum antiviral effect of ribavirin, a guanosine analog, was first reported in the s. , it was found at the time that ribavirin inhibits dna and rna viruses, including herpesviruses, vaccinia, vsv, as well as respiratory infections caused by influenza a and b viruses and parainfluenza virus. ribavirin is currently approved for the treatment of chronic hcv infection in combination therapy and against severe rsv infection in monotherapy. in the case of rsv infection, ribavirin is administered as a small-particle aerosol that requires use of a mask and a tent. ribavirin was originally developed against influenza based on its efficacy in a mouse model of influenza, , but its effect in human clinical trials was less clear, so it was not approved for the treatment of influenza. its clinical use for the treatment of rsv infection via aerosol delivery remains limited due the inconvenient route of administration, lack of clear evidence for efficacy, and safety concerns associated with anemia and risk of teratogenicity. studies evaluating the mechanism of action of ribavirin have produced contradictory results. it is usually acknowledged that ribavirin exerts its main effect through its monophosphate metabolite by inhibiting the host inosine monophosphate dehydrogenase (impdh) enzyme, leading to depletion of intracellular gtp pools, which results in indirect inhibition of rna synthesis during viral replication (for review ). the nucleoside form of ribavirin is also believed to enhance t-cell-mediated immune response through increased expression of interferon-gamma and tumor necrosis factor-alpha. in addition, it has been proposed that prolonged replication of pv in the presence of ribavirin increases the viral mutation frequency and decreases infectivity. one hypothesis is that the mutagenic effect of ribavirin is caused by its triphosphate form that is recognized by the viral rna polymerase. once incorporated into the viral genome, ribavirin monophosphate could equally base pair with cytidine and uridine, therefore causing random mutations throughout the viral genome. ribo-cytidine and adenosine analogs containing a methyl group at the -position on the ribose are known inhibitors of hcv and other related members of the flaviviridae family. [ ] [ ] [ ] [ ] [ ] [ ] [ ] one of the most potent molecules of this series, -deaza- -c-methyladenosine ( dma, mk- ), was once a development candidate for the treatment of hcv infection ( figure ) . , the adenosine analog dma also inhibits hrv type a infection in vitro, with ec values ranging from to mm. a subgenomic replicon assay was used in transient transfection experiments to demonstrate that dma is equipotent against multiple strains of hrv type c. this important proof-of-concept experiment demonstrated that -methyl nucleosides prevent picornavirus replication, most likely by inhibiting the viral rna polymerase function. this class effect was confirmed with nitd , another adenosine analog containing a -c-ethynyl on its -ribose ( figure ). just like dma, nitd was previously known to inhibit flaviviruses, and was once a development candidate for the treatment of dengue infection. nitd blocks the replication of dengue virus in cell culture and in mice by inhibiting the rdrp activity of the ns protein. , therefore, the possibility that nitd would inhibit other (þ)ssrna viruses is not unexpected. indeed, nitd blocks the replication of ev , another enterovirus-related to rhinovirus. this in vitro antiviral effect was confirmed in a separate study that also demonstrated in vivo efficacy. investigators in the latter study infected -week-old ag immunocompromised mice with ev by intraperitoneal inoculation. treatment of the infected animals with nitd given orally at mg/kg twice a day for days resulted in % survival at the end of the study, compared to % survival for the vehicle control group. nitd cannot be developed in the clinic due to severe toxicity seen in -day studies in rats and dogs. however, the results summarized here indicate that nucleoside analogs targeting the viral rna polymerase of rhinovirus, ev , and other enteroviruses have the potential to be efficacious in preclinical animal models, providing a rationale to conduct human studies with safer molecules sharing the same mode of action. -deoxy- -fluoro nucleosides for influenza fluorinated nucleosides are well known for their antiviral and anticancer properties (for review ). in particular, -deoxy- -fluoro guanosine ( fdg) was at one time considered a potential candidate for influenza treatment ( figure ). in vitro, fdg inhibits influenza a virus replication with an ec of about mm, without causing apparent cell toxicity. in ferrets, treatment with fdg at mg/kg starting h postinfection significantly reduced h n influenza a virus titers in nasal washes, associated with reduction in fever and inflammation. although time-of-addition experiments suggested that the molecule inhibits an early step of virus replication, more direct evidence for the mechanism of action came from enzyme inhibition studies. in cell-free transcription experiments, fdg triphosphate inhibited influenza a virus rna polymerase activity by competing with natural gtp. the inhibition of the enzyme was caused by the incorporation of fdg monophosphate into the viral rna. more recently, the related nucleoside analog -deoxy- -fluoro cytidine ( fdc) was evaluated against the highly pathogenic h n and the pandemic h n strains. when administered intraperitoneally, fdc significantly enhanced survival of balb/c mice infected with a lethal dose of either h n or h n viruses. although these studies show compelling evidence of in vivo efficacy in preclinical species, fdg and fdc are not suitable candidates for clinical development. one of the main limitations of these molecules is their lack of specificity for influenza virus polymerase. the ability of a nucleotide to inhibit distant molecular targets is not detrimental per se. as such, -deoxy- -fluoro nucleotides and their derivatives interact with the rna polymerase of hcv. [ ] [ ] [ ] [ ] but the substitution of the -hydroxy by a fluoro group also makes the resulting nucleotides broad substrates for viral and human dna polymerases. in the latter study, the authors have shown that the monophosphate form of both fdc and fdg can be incorporated into dna by human dna polymerase alpha and gamma. this might explain the changes in cell cycle distribution and cytostatic effect caused by prolonged in vitro incubation with fdc. paradoxically, the same molecule was well tolerated when administered intravenously to rats and woodchucks for up to days. one hypothesis for this discrepancy is that a low organ exposure of the phosphorylated metabolite(s) of fdc could limit the toxic effect on dividing cells in these animals. the antiviral -fluoro- -hydroxy- -pyrazinecarboxamide (t- , favipiravir, avigan) has been approved in japan for the treatment of influenza infection since . t- is a nucleoside precursor inhibiting influenza virus with broad-strain coverage , ( figure ). it is often proposed that t- exerts its antiviral activity through its ntp form (t- rtp) by directly inhibiting the rdrp activity of influenza a virus polymerase, but the exact mode of action and precise molecular interaction between the nucleotide and the viral polymerase has been elusive. in vitro, t- is efficiently converted to its ribofuranosyl -triphosphate (t- rtp) form by cellular enzymes. treatment of influenza a virus-infected cells with t- results in a significant increase of lethal mutations within the viral genome, a phenomenon also described as error catastrophe. the lethal mutagenesis hypothesis is supported by enzymatic assays showing that t- rtp is efficiently recognized by influenza a virus polymerase both as a guanosine and an adenosine analog. in addition, single events of t- rmp incorporation into rna by influenza a virus polymerase delayed but did not block the extension of the rna primer strand. the antiviral potency of t- covers other virus families well beyond orthomyxoviruses. t- has been shown to inhibit a number of diverse rna viruses unrelated to influenza, including representatives of noroviruses, bunyaviruses, arenaviruses, flaviviruses, and filoviruses. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] it is interesting to point out that the mutagenic effect of t- has also been documented for hcv. at the biochemical level, we showed that t- is recognized as substrate for rna synthesis not only by viral polymerases, but also by human mitochondrial rna polymerase. , this host-based interaction did not result in any measurable in vitro mitochondrial toxicity, but it raised more questions about the mechanism of action of the compound. recently, the possibility that t- exerts its main antiviral effect without converting to its triphosphate form came from the observation that t- ribonucleoside is chemically unstable under biological conditions. even though t- does not seem to potently inhibit the human impdh enzyme, its very broad antiviral spectrum and its capacity to induce lethal mutagenesis are somewhat reminiscent of ribavirin, another nucleoside that inhibits hcv replication through host-based mechanisms. therefore, the possibility that t- exerts its inhibition through interactions with host proteins cannot be ruled out and remains to be further explored. considering its similarities with ribavirin in terms of antiviral spectrum and mode of action, it will be interesting to see if t- becomes more widely used in patients suffering from respiratory viral infections, or if it will remain limited to stockpiling for potential influenza pandemic in japan. the discovery of als- , the parent molecule of the prodrug als- (lumicitabine), was the result of a screening campaign using a focused library of structurally diverse nucleoside and nucleotide analogs tested against rsv in an in vitro infectious assay. the main scaffold identified from this screen was difluoro- azido-cytidine. further modifications at the -and -positions to improve anti-rsv potency and selectivity, led to the identification of als- ( fluoro- chloromethyl-cytidine) ( figure ). in vitro, als- inhibits a broad panel of rsv a and b subtypes, as well as related pneumo-, paramyxo-, and rhabdoviruses. in particular, we recently reported that als- inhibits rsv and hmpv with similar in vitro potency. the molecular target of als- was determined by two independent methods. the polymerization function of the rsv l protein was identified as the target of als- inhibition, first, by selecting and characterizing drug resistanceassociated mutations located in the l gene. when introduced into a wild-type rsv genome, four amino acid mutations (m l, a v, l i, and i v) were phenotypically associated with resistance to als- . enzymatic assays using purified recombinant rsv polymerase were critical to validate the mode of action of als- . in these assays, the -triphosphate form of als- (als- -tp) caused immediate chain termination of rna synthesis and inhibition of the viral polymerization activity. this inhibitory effect was specific to rsv polymerase, since als- -tp did not inhibit polymerases from host or viruses unrelated to rsv such as hcv. the lack of inhibition against hcv was rationalized by molecular modeling, predicting steric clashing of als- -tp inside the active site of hcv polymerase. because of the low oral bioavailability of als- , a series of , -diester prodrugs was evaluated for improved pharmacokinetic properties. one prodrug, als- , formed high levels of monophosphate and triphosphate in the lungs when administered orally to nonhuman primates. because of its high oral bioavailability, als- was evaluated for in vivo efficacy in african green monkeys infected with rsv. at the end of treatment, rsv rna was undetectable in bronchoalveolar lavage samples from all four als- -treated animals. subsequently, a randomized, double-blind, clinical trial evaluated als- given for days to healthy adults inoculated with rsv. the reduction in viral load in nasal washes associated with als- treatment varied from % to % depending on the dose regimen. rsv rna was undetectable . to . days after the start of als- treatment compared with . days for placebo. assessment of symptom scores and quantity of mucus produced also showed a clear effect on rsv-induced disease. this important result represents the first proofof-concept validation that an rsv replication inhibitor can be efficacious in humans. als- is currently in clinical development for the treatment of rsv infection in hospitalized infants and adults (clinicaltrials.gov identifier: nct , nct ). the recent ebola virus outbreak of - in west africa triggered increased efforts to identify new antivirals targeting filoviruses. as a result, the development of a new series of c-linked nucleoside analogs with anti-ebola properties was soon reported. in a cellbased infectious assay, the -cyano c-linked adenosine derivative (gs- , or compound ) was moderately active against ebola replication with ec values around . mm, whereas the -methyl and -ethynyl counterparts were completely inactive. gs- is also a broad-spectrum inhibitor of a variety of rna viruses from four families (filoviridae, flaviviridae, paramyxoviridae, and pneumoviridae), including hcv and rsv. , however, the addition of a -c-methyl group, as in the case of the gs- , , significantly reduces the antiviral spectrum to hcv only. the relatively weak antiviral activity of gs- across all viruses ( . - mm ec ) was attributed to its inefficient intracellular phosphorylation, which could be improved by adding a monophosphate prodrug to the parent nucleoside. the resulting compound, gs- ( figure ) inhibits the zaire and sudan species of ebola virus and marburg virus with ec values ranging from . to . mm, and exhibits moderate cytotoxicity (cc ¼ to > mm) in multiple human cell types. gs- exhibits the same broad antiviral spectrum as its parent molecule. the triphosphate form of gs- is recognized as substrate by rsv polymerase, but its incorporation into rna does not lead to immediate chain termination. the favorable in vitro data led to further evaluation of gs- in a macaque lethal model of ebola virus disease. complete protection was achieved when gs- was administered at a daily intravenous dose of mg/kg, beginning on day post-infection. following phase i safety testing in healthy human volunteers, gs- was first given as a -day course for compassionate use to an ebola-infected nurse who had survived the disease and developed a recurrence in the central nervous system. soon after, a neonate who had congenital ebola virus infection received three different experimental therapies, including a -day treatment with gs- . in both cases, patients cleared the virus and survived the infection. the characterization of the broad antiviral spectrum of gs- was further expanded to another (þ) ssrna virus family: coronaviridae. it was shown that gs- inhibits sars-cov and mers-cov replication in multiple in vitro systems, including primary human airway epithelial cell cultures with sub-micromolar ec values. gs- was also effective against other human and bat cov. in a mouse model of sars-cov infection, prophylactic and early therapeutic administration of gs- reduced lung viral load and improved clinical signs of disease as well as respiratory function. although there is limited data to confirm the proposed mechanism of action of gs- against each virus, it is generally assumed that the molecule targets the rdrp function of the viral polymerase. in the case of cov, this is supported by the identification of two mutations (f l and v l) within the predicted fingers subdomain of the rdrp protein nsp from murine hepatitis virus. these mutations emerged over passages and confer -to -fold resistance to gs- , combined with overall reduced replication fitness. at this point, the precise mechanism of action of gs- against cov remains elusive. it is possible that gs- triphosphate is not excised by the proofreading activity of nsp because of lack of immediate chain termination, as observed for rsv polymerase. in this case, could the resistance mutations identified in nsp alter the chain termination profile of gs- , and make it more susceptible to excision? such studies are needed, not only to understand how gs- works but also to design new molecules against cov polymerases. in this review, we summarized the exciting advances in discovery and development of novel nucleoside analogs as potential new treatments for respiratory rna virus infections. the medical need is high because very few drugs have been approved for the treatment of respiratory viral infections despite worldwide health impacts attributed to them. the approved drugs include zanamivir, oseltamivir, peramivir, and favipiravir (japan only) for influenza virus and palivizumab for rsv, all of which have limitations that prevent their widespread use in a therapeutic setting. drug candidates intended for use against rhinovirus infections, such as the capsid inhibitor pleconaril and the protease inhibitor rupintrivir, have been tested in the clinic without success. the first nucleoside analog developed for respiratory viral infection was ribavirin, but despite its approval for use in rsv, its utility for treating severe viral infections remains low. therefore, the concept of nucleoside analogs against respiratory viruses remains relatively new and needs to be further explored. what are the molecular determinants of polymerase selectivity against nucleotide analogs? we currently do not understand well how specific changes made in nucleotide analogs alter their recognition as substrates for rna synthesis, and how substrate selectivity differs among positive and negative strand rna virus polymerases. for example, many -modified nucleotide analogs are known to inhibit hcv polymerase, often with an antiviral spectrum extended to flaviviruses and picornaviruses. however, there is no clear mechanistic basis to explain why none of these compounds inhibit (-)ssrna viruses, or even other (þ)ssrna viruses such as cov. could the exonuclease/proofreading activity of cov polymerases excise chain terminators and resume rna synthesis? are there specific amino acid within the active site of (-)ssrna virus polymerases responsible for the discrimination of -c-methyl nucleotides? these hypotheses have not been tested, in part, due to the difficulty to conduct biochemical and structural studies on viral polymerases from respiratory viruses. until recently, the production of soluble, pure viral protein targets has been limiting, especially in the case of large protein complexes. as mentioned earlier in this review, the development of robust expression systems for influenza polymerase trimer, as an example, have made it possible to use x-ray crystallography and potentially cryo-electron microscopy to provide molecular visualization of binding pockets for small molecule inhibitors, entry and exit channels for substrate(s), and potential new ways to disrupt domain interactions. these structural insights will tremendously aid the development of new drugs as well as to further elucidate the mechanisms of action and binding of existing drugs to their protein targets. molecular modeling is also a useful approach that we and others have used to rationalize the differences in selectivity of lumicitabine against rsv and hcv polymerase. more studies such as these ones will be needed to rationally design new nucleotide analogs targeting respiratory virus polymerases. in the past, many nucleoside analogs failed during development for safety/toxicity reasons, especially molecules with suboptimal specificity for their viral polymerase target and those used for chronic treatment of infections such as hiv and hcv. in the context of acute respiratory infections, evaluation of safety must be based on both the intended duration of treatment and the targeted patients, which sometimes include vulnerable populations such as children and the elderly. other considerations to ensure successful future development of nucleoside analogs directed against respiratory infections will be to 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culture and in a hamster model a -deazaadenosine analog is a potent and selective inhibitor of hepatitis c virus replication with excellent pharmacokinetic properties a dengue fever viremia model in mice shows reduction in viral replication and suppression of the inflammatory response after treatment with antiviral drugs synthesis and pharmacokinetics of valopicitabine (nm ), an efficient prodrug of the potent anti-hcv agent -c-methylcytidine robust antiviral efficacy upon administration of a nucleoside analog to hepatitis c virus-infected chimpanzees multiple classes of antiviral agents exhibit in vitro activity against human rhinovirus type c an adenosine nucleoside inhibitor of dengue virus biochemical characterization of the inhibition of the dengue virus rna polymerase by beta-d- '-ethynyl- -deazaadenosine triphosphate an adenosine nucleoside analogue nitd inhibits ev proliferation inhibition of enterovirus by adenosine analog nitd fluorinated nucleosides as antiviral and 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in a model of arenaviral hemorrhagic fever: t- efficacy and reduced toxicity suggests an alternative to ribavirin in vitro and in vivo activities of t- against arenavirus and bunyavirus infections efficacy of favipiravir (t- ) and t- pyrazine derivatives in phlebovirus disease models activity of t- in a hamster model of yellow fever virus infection in comparison with that of a chemically related compound, t- effect of t- treatment on western equine encephalitis in a mouse model t- (favipiravir) inhibition of arenavirus replication in cell culture t- ) inhibits in vitro norovirus replication lethal mutagenesis of hepatitis c virus induced by favipiravir structure-activity relationship analysis of mitochondrial toxicity caused by antiviral ribonucleoside analogs biochemical evaluation of the inhibition properties of favipiravir and '-c-methylcytidine triphosphates against human and mouse norovirus rna polymerases synthesis of t- -ribonucleoside and t- -ribonucleotide and studies of chemical stability distinct effects of t- (favipiravir) and ribavirin on influenza virus replication and viral rna synthesis discovery of '-chloromethyl- '-deoxy- ', '-di-o-isobutyryl- '-fluorocytidine (als- ), a first-in-class rsv polymerase inhibitor for treatment of human respiratory syncytial virus infection molecular basis for the selective inhibition of respiratory syncytial virus rna polymerase by '-fluoro- '-chloromethyl-cytidine triphosphate development of als- /als- as an effective replication inhibitor of human metapneumovirus activity of oral als- in a respiratory syncytial virus challenge study therapeutic efficacy of the small molecule gs- against ebola virus in rhesus monkeys gs- and its parent nucleoside analog inhibit filo-, pneumo-, and paramyxoviruses discovery and synthesis of a phosphoramidate prodrug of a pyrrolo [ , -f][triazin- -amino] adenine c-nucleoside (gs- ) for the treatment of ebola and emerging viruses inhibition of hepatitis c virus replication by gs- , a potent c-nucleoside monophosphate prodrug discovery of the first c-nucleoside hcv polymerase inhibitor (gs- ) with demonstrated antiviral response in hcv infected patients late ebola virus relapse causing meningoencephalitis: a case report first newborn baby to receive experimental therapies survives ebola virus disease broad-spectrum antiviral gs- inhibits both epidemic and zoonotic coronaviruses the nucleoside prodrug gs- inhibits multiple coronaviruses and selects for resistance mutations in the rna-dependent rna polymerase that are associated with a decrease in viral replication fitness we wish to thank leo beigelman, julian symons, andreas jekle, and peggy korn for their review of the manuscript. the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. janssen research & development provided support in the form of salaries for all authors but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. key: cord- -rrx r n authors: fan, wensheng; tang, ning; dong, zhihua; chen, jiming; zhang, wen; zhao, changrun; he, yining; li, meng; wu, cuilan; wei, tianchao; huang, teng; mo, meilan; wei, ping title: genetic analysis of avian coronavirus infectious bronchitis virus in yellow chickens in southern china over the past decade: revealing the changes of genetic diversity, dominant genotypes, and selection pressure date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: rrx r n the high mutation rates of infectious bronchitis virus (ibv) pose economic threats to the poultry industry. in order to track the genetic evolutionary of ibv isolates circulating in yellow chickens, we continued to conduct the genetic analyses of the structural genes s , e, m, and n from ibv isolates in southern china during – . the results showed that the dominant genotypes based on the four genes had changed when compared with those during – . based on the s gene phylogenetic tree, lx -type (gi- ) was the most dominant genotype, which was different from that during – . the second most dominant genotype was ldt -a-type, but this genotype disappeared after . new-type (gvi- ) isolates showed increasing tendency and there were four aa (qkep) located in the hypervariable region (hvr) iii and one aa (s) insertion in all the new-type isolates. both the analyses of amino acid entropy and molecular evolutionary rate revealed that the variations from large to small were s , e, m, and n. purifying selection was detected in the s , e, m, and n gene proteins, which was different from the positive selection during – . six isolates were confirmed to be recombinants, possibly generated from a vaccine virus of the / -type or ldt -a-type and a circulating virus. the estimated times for the most recent common ancestors based on the s , e, m, and n genes were the years of , , , and , respectively. bayesian skyline analysis revealed a sharp decrease in genetic diversity of all the four structural genes after and since late , the viral population rapidly rose. in conclusion, the ibvs circulating in southern china over the past decade have experienced a remarkable change in genetic diversity, dominant genotypes, and selection pressure, indicating the importance of permanent monitoring of circulating strains and the urgency for developing new vaccines to counteract the emerging lx -type and new-type ibvs. infectious bronchitis (ib) is one of the major viral diseases affecting the poultry industry globally. the causative agent avian infectious bronchitis virus (ibv) is a member of the genus gammacoronaviruses, subfamily coronavirinae, family coronaviridae and is prone to mutate. there are multiple genotypes and serotypes of ibv isolates identified worldwide and limited cross-protection confers between serotypes of ibvs [ ] [ ] [ ] [ ] [ ] [ ] , which poses great challenge to the control of ib by vaccination. the ibv has a single-stranded rna genome of approximately . kb in length [ ] and encodes four structural proteins: the spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins [ , ] . the s protein is cleaved into subunits s and s by proteases [ , ] . the function of the four structural proteins has been extensively reviewed by others [ ] [ ] [ ] . the genetic analysis based on the s gene has become the primary method of classifying ibv strains because of variability and functional importance [ ] . however, those previous ibv molecular characterizations that were merely focused on the analysis of the s gene or partial s gene sequence could not explain the changes in the serotypes and pathotypes of ibv variants [ ] . sometimes, single gene analysis even is misleading. therefore, it is necessary to analyze all the structural protein-coding genes s , e, m, and n simultaneously in order to obtain comprehensive genetic information and molecular mechanism of variation of circulating ibv isolates. a variety of ibv genotypes and variants are distributed globally. so far, a total of seven genotypes comprising distinct viral lineages have been defined worldwide based on the complete s gene sequences [ ] . ibv strains within a certain country or region are unique even though many countries share some common antigenic types. for example, two distinct lineages that fall in two different genotypes-gi- and gii- -were identified as unique to europe. gi- , gi- , and giv- -genotypes have been implicated in widespread disease disseminations and persistent virus infections in north american [ , , ] . gi- , gi- , and gi- are currently circulating in south american flocks [ ] . lx -type (qx-type or which was firstly isolated in china in spread westward invading russia, middle east, and europe, and then becoming the most prevalent genotype in many countries, such as korea, russia, iran, italy, uk, malaysia, sudan, and so on [ , , [ ] [ ] [ ] [ ] [ ] . nowadays, lx -type and ck/ch/lsc/ i-type (gi- ) appear to be the dominant viruses based on the s gene in china [ ] . because of differences in breeding variabilities and feeding patterns of chickens in china, the characteristics of circulating ibvs at different times and in different regions are variable. hence, it is necessary to conduct long-term tracking of the ibv circulating isolates in specific geographic regions or countries for the effective control of ib [ , , ] . the appearance of ibv variants was related to high mutation and recombination rates, which result in the generation of genetic diversity and phenotypic heterogeneity. however, ibv evolution is not driven by genetic drift alone. evolution involves two fundamental steps-i.e., generation of genetic diversity and selection [ ] . the selection process was affected by multiple factors, such as immune responses, the microenvironment of infected hosts, physical and biosafety conditions [ , ] . vaccines not only give rise to new variants through recombination, but also impose selection pressure on the evolution of field strains [ ] [ ] [ ] . it is essential for appropriately controlling and prevention of the disease to understand the evolution of ibv [ ] . southern china is the major production region of yellow chickens ( . billion in and made a proportion of % of the total chickens) in the country. guangxi province, located in southwest of china, has the biggest production of local breeds of chickens ( . billion birds in ) [ ] , and most of the birds are free-range and raised for a longer time (about -day-old) at a rather high density, and the chicks are raised in relatively closed environment and lack of ventilation during brooding [ , ] . also, flocks of various companies, with different chicken breeds and differing ages and vaccination programs located in the same areas are common [ ] . these situations surely increase the odds of multiple-infection of several ibv strains including the vaccine strains or/and field strains in flocks of birds [ ] . despite the widespread use of mass-type (massachusetts genotype) (h , h , ma , m , and w ), / , ltd -a live vaccines and inactivated vaccines, ib has been a continuing problem in vaccinated flocks in these regions [ ] . our previous studies found the serotype and genotype diversity of guangxi ibvs from to and the important role of vaccine strains in the emerging of new ibv strains via recombination [ , , ] . however, the comprehensive genetic information of circulating ibv strains in this region was unavailable over the past decade. hence, we continued to carry out the genetic analysis of ibv isolates during - . the aim was to track the genetic evolutionary trends of ibv field strains circulating over the past decade and their possible causes through relatively new and comprehensive analyses of virus genes, and then provide valuable reference and countermeasure against ibv field breakouts in southern china. sixty-four ibv strains isolated from flocks of yellow chickens during - were analyzed in the present study (table ). all ibv field isolates were obtained from the birds of previously vaccinated flocks with h , ldt -a and/or / vaccines and experienced clinical signs of the ibv infection. all ibvs were isolated and propagated as previously described [ ] . nanning jx jx jx kj gx-gl guilin jx jx jx kj gx-gl guilin jx jx jx kj gx-gl n/a guilin jx jx jx kj gx-nn- nanning jx jx jx kj gx-nn- nanning jx jx jx kj gx-nn- nanning jx jx jx kj gx-nn- nanning jx jx jx kj gx-nn- nanning jx jx jx kj gx-nn- nanning jx jx jx kj gx-nn- nanning jx jx jx kj gx-nn- nanning jx jx jx kj gx-nn- nanning jx jx jx kj gx-nn nanning jx jx jx kj gx-yl yulin jx jx jx kj gx-yl yulin jx jx jx kj gx-nn nanning jx jx jx kj gx-nn table . cont. days of age locations a genbank accession numbers s n m e gx-nn nanning mk mk mk mk gx-qz qinzhou mk mk mk mk gx-yl yulin mk mk mk mk gx-lz liuzhou mk mk mk mk gx-yl yulin mk mk mk mk gx-lz liuzhou mk mk mk mk gx-yl yulin mk mk mk mk gx-yl yulin mk mk mk the entire s , e, m, and n genes were amplified for each ibv strain and the primers used were designed as previously described [ , ] . the anticipated amplification segments for the s , e, m, and n genes are bp, bp, bp, and bp in lengths, respectively. . . rna extraction and amplification of s , e, m, and n genes viral rna was extracted and the first cdna strand was synthesized as previously described [ ] . the pcr conditions for the s , m, and n gene amplification were the same as previously described [ ] . the pcr conditions for the e gene amplification were • c for min, cycles of • c for s, • c for s, and • c for s, followed by • c for min. the pcr products were analyzed on . % agarose-gel electrophoresis. the pcr products of s , e, m, and n genes were sequenced by beijing genomics institute (bgi) (shenzhen, china) after cloning. the open reading frames of ibvs were determined and their nucleotide sequences were submitted to genbank database and assigned accession numbers ( table ) . sequences of reference ibv strains (with the exception of strains for e gene) retrieved from the genbank database were used (supplementary table s ). the ibvs isolated during - in guangxi [ ] were also analyzed together in order to get a general profile of ibv evolution. the nucleotide and deduced amino acid (aa) sequences of the s , e, m, and n genes obtained from the ibv isolates were aligned using the editseq program in the lasergene package (dnastar inc., madison, wi, usa) and compared to those of ibv reference strains representing the main well-established lineages and genotypes using the megalign program in the same package. to ensure the scientificity and reliability of the results, we extracted the s , e, m, and n genes from the complete genome sequences of reference strains. phylogenetic trees based on the aa sequences of s , e, m, and n genes were constructed using mega version . according to previous description with nodal support values obtained by posterior probabilities and bootstrap replicates [ ] . aligned nucleotide sequences of s , e, m, and n genes were subjected to the recombination detection program (rdp version . ) to detect potential recombination events by seven algorithms (rdp, geneconv, bootscan, maxchi, chimera, siscan and seq) in rdp . [ ] . the detection of recombination breakpoints by at least four of these methods were considered as confirmation of any putative recombination event. the potential recombination events and breakpoints were further verified by similarity plots (simplots) analysis in simplot version . . [ , ] . entropy of amino acid sequences within the s , e, m, and n proteins of ibv isolates was calculated by bioedit version . . . in order to understand the variation degree of these four structural protein genes [ ] . positive selection and positively selected sites within the s , e, m, and n proteins were analyzed by the single-likelihood ancestor counting (slac), fixed effects likelihood (fel), and internal fixed effects likelihood (ifel) methods of datamonkey version (http://www.datamonkey.org/ / / ) [ ] to detect whether these proteins have undergone positive selection. the recombinants were excluded in order to reduce a false detection of positive selection. the potential n-glycosylation sites were predicted within the s , e, m, and n proteins. the analysis was performed using netnglyc server . software available at http://www.cbs.dtu.dk/services/ netnglyc [ ] . the results of aligned sequences were computed by multiple alignment with fast fourier transformation (mafft) [ ] . the nucleotide substitution process was modelled independently for each partition with gtr (general time reversible) + g (gamma distribution with a discrete) + i (proportion of invariant sites) based on aic by the jmodel test . . . bayesian tree reconstructions were performed in beast version . . . a bayesian skyline coalescent model and strict molecular clock were selected. the bayesian markov chain monte carlo (mcmc) chains of the s , e, m, and n genes were run for million, million, million, and million generations, respectively. results were analyzed using tracer version . and confirmed convergence of mcmc chains with % of each chain discarded as burn-in and sampled every , steps [ ] . statistical uncertainty (reflected calculating % high probability density (hpd) values) and convergence (reflected calculating effective sample size) in parameter estimates were evaluated in tracer version . program. the posterior sets of trees were summarized as a maximum clade credibility (mcc) tree using tree annotator version . . with % burn-in and then displayed the created mcc tree using figtree version l. . [ ] . the mutation rates and the most recent common ancestor (tmrca) of the aligned sequences were estimated. the change of effective population size over time was inferred by bayesian skyline plots (bsp). the nucleotide and deduced aa sequence similarities of the s , e, m, and n genes among the isolates during - were . - % and . - %, . - % and . - %, . - % and . - %, and . - % and . - %, respectively. compared with h , the isolates gx-qz , gx-lz , and gx-nn have higher amino acid sequence similarities of . - . %, . - %, . - %, and . - % in the s , e, m, and n genes, respectively. within the s gene, there were different nucleotide lengths (from to bp), and the most common lengths were bp ( / isolates, . %) and bp ( / isolates, . %). there were eight types of s protein cleavage site motifs found: rrfrr ( / ), hrrrr ( / ), rrlrr ( / ), rrsrr ( / ), hrrkr ( / ), hrikr ( / ), hrskr ( / ), and rkrkr ( / ) among the isolates. there were four aa (qkep) (located in the hypervariable region (hvr) iii) and one aa (s) insertion found in the s genes of six isolates (gx-nn , gx-nn , gx-nn , gx-nn , gx-qz , and gx-qz ), respectively (supplementary figure s ). the s gene phylogenetic tree showed that the ibv isolates during - were divided into eight distinct groups (figure a ). counts of , , , , and out of ibv isolates during - belonged to lx -type (qx or gi- ), ldt -a-type (gi- ), / -type (gi- ), mass-type (gi- ), and ck/ch/lsc/ i-type (gi- ), respectively. five isolates (gx-nn , gx-nn , gx-nn , gx-nn , and gx-nn ) in recent years were grouped with taiwan reference strains tw / as taiwan-i-type (gi- ). six isolates (gx-nn , gx-nn , gx-nn , gx-nn , gx-qz , and gx-qz ) and two isolates (gx-nn and gx-yl ) showed considerable low similarities ( . - . %, . - . %) with the above genotypes and belonged to two separate groups new-type (gvi- ) and new-type (gvii- ). viruses , , x for peer review of ( / ), hrrkr ( / ), hrikr ( / ), hrskr ( / ), and rkrkr ( / ) among the isolates. there were four aa (qkep) (located in the hypervariable region (hvr) iii) and one aa (s) insertion found in the s genes of six isolates (gx-nn , gx-nn , gx-nn , gx-nn , gx-qz , and gx-qz ), respectively (supplementary figure s ). the s gene phylogenetic tree showed that the ibv isolates during - were divided into eight distinct groups (figure a ). counts of , , , , and out of ibv isolates during - belonged to lx -type (qx or gi- ), ldt -a-type (gi- ), / -type (gi- ), mass-type (gi- ), and ck/ch/lsc/ i-type (gi- ), respectively. five isolates (gx-nn , gx-nn , gx-nn , gx-nn , and gx-nn ) in recent years were grouped with taiwan reference strains tw / as taiwan-i-type (gi- ). six isolates (gx-nn , gx-nn , gx-nn , gx-nn , gx-qz , and gx-qz ) and two isolates (gx-nn and gx-yl ) showed considerable low similarities ( . - . %, . - . %) with the above genotypes and belonged to two separate groups new-type (gvi- ) and new-type (gvii- ). the phylogenetic trees of e, m, and n genes of the isolates were segregated into six, four, and six unique groups, respectively (figure b-d). and their phylogenetic trees exhibited considerably different topology compared with that of the s gene. no obvious geographic differences were found among the isolates, while there was a high degree of sequence identity among the isolates in the same period of time (supplementary tables s -s ) . the percentages of different genotypes based on s , e, m, and n genes of ibv isolates in different years were summarized in figure . based on the s gene, the ck/ch/lsc/ i-type was the predominant genotype during - , but the lx -type was the predominant genotype circulating in the field during - . the ldt -a-type was the second most dominant genotype, but this genotype disappeared after . based on the e and m genes, the ck/ch/lsc/ i-type was the predominant genotype during - , while the ck/ch/lsc/ i-type and lx -type were the predominant genotypes during - . based on the n gene, the ck/ch/lsc/ i-type and the lx -type were the predominant genotypes during - , but only the lx -type was the predominant genotype during - . therefore, our results demonstrated that the ck/ch/lsc/ i-type isolates were the predominant ibvs according to the phylogenetic study of the s , e, m, and n genes from to . thereafter, the proportion of lx -type, ldt -a-type, and the phylogenetic trees of e, m, and n genes of the isolates were segregated into six, four, and six unique groups, respectively (figure b-d) . and their phylogenetic trees exhibited considerably different topology compared with that of the s gene. no obvious geographic differences were found among the isolates, while there was a high degree of sequence identity among the isolates in the same period of time (supplementary tables s -s ) . the percentages of different genotypes based on s , e, m, and n genes of ibv isolates in different years were summarized in figure . based on the s gene, the ck/ch/lsc/ i-type was the predominant genotype during - , but the lx -type was the predominant genotype circulating in the field during - . the ldt -a-type was the second most dominant genotype, but this genotype disappeared after . based on the e and m genes, the ck/ch/lsc/ i-type was the predominant genotype during - , while the ck/ch/lsc/ i-type and lx -type were the predominant genotypes during - . based on the n gene, the ck/ch/lsc/ i-type and the lx -type were the predominant genotypes during - , but only the lx -type was the predominant genotype during - . therefore, our results demonstrated that the ck/ch/lsc/ i-type isolates were the predominant ibvs according to the phylogenetic study of the s , e, m, and n genes from to . thereafter, the proportion of lx -type, ldt -a-type, and new-type strains increased over time. recombination events of the s , e, m, and n genes of isolates were examined using the rdp in the study. the results showed that recombinant events were found in the s gene of six isolates ( figure and table ). gx-nn was derived from recombination between the two lx -type strains gx-nn (major parent) and gx-nn- (minor parent). gx-nn was derived from recombination between lx -type strain qx (major parent) and ck/ch/lsc/ i-type strain saibk (minor parent). gx-nn was derived from recombination between lx -type strain gx-hc (major parent) and / strain (minor parent). gx-nn was found to be a recombinant between the new-type isolate gx-nn (major parent) and the vaccine strain ldt -a (minor parent). gx-nn was found to be a recombinant isolate formed by a major parent isolate gx-yl (ldt -a-type) and a minor parent isolate gx-yl (ck/ch/lsc/ i-type). the potential parents of gx-yl were proved to be a major parent isolate gx-nn (tw / -type) and a minor parent isolate gx-nn ( / -type). in addition, it was found that a-t rich hotspot sequence atttt (t/a) was near the breakpoint site of the s subunit gene in all of recombinant isolates except gx-yl (supplementary figure s ) . in order to confirm the results of the rdp analysis, genomic sequences analyses of the six ibv isolates were carried out by the simplot software, and the results were consistent with those of the rdp analysis ( figure ) . therefore, the dominant recombinants were the lx -type isolates from to . recombination events of the s , e, m, and n genes of isolates were examined using the rdp in the study. the results showed that recombinant events were found in the s gene of six isolates ( figure and table ). gx-nn was derived from recombination between the two lx -type strains gx-nn (major parent) and gx-nn- (minor parent). gx-nn was derived from recombination between lx -type strain qx (major parent) and ck/ch/lsc/ i-type strain saibk (minor parent). gx-nn was derived from recombination between lx -type strain gx-hc (major parent) and / strain (minor parent). gx-nn was found to be a recombinant between the new-type isolate gx-nn (major parent) and the vaccine strain ldt -a (minor parent). gx-nn was found to be a recombinant isolate formed by a major parent isolate gx-yl (ldt -a-type) and a minor parent isolate gx-yl (ck/ch/lsc/ i-type). the potential parents of gx-yl were proved to be a major parent isolate gx-nn (tw / -type) and a minor parent isolate gx-nn ( / -type). in addition, it was found that a-t rich hotspot sequence atttt (t/a) was near the breakpoint site of the s subunit gene in all of recombinant isolates except gx-yl (supplementary figure s ) . in order to confirm the results of the rdp analysis, genomic sequences analyses of the six ibv isolates were carried out by the simplot software, and the results were consistent with those of the rdp analysis ( figure ) . therefore, the dominant recombinants were the lx -type isolates from to . the entropy of the amino acid sequences of s , e, m, and n genes showed that there were many high entropy amino acid sites within the s gene, but a few high entropy amino acid sites were scattered within the e, m, and n genes (supplementary figure s ) . the average entropy values sorted from large to small as follows: s ( . ), e ( . ), m ( . ), and n ( . ). the percentages of entropy value which was bigger than . in s , e, m, and n genes were . % ( / ), . % ( / ), . % ( / ), and . % ( / ), respectively. therefore, the largest variation was observed in the s gene, and the e gene was also more variable than the m and n genes. the selection profile of s , e, m, and n proteins of totally (with deleted recombinant strains) ibvs were showed in table . the dn/ds ratio of s , e, m, and n proteins of eighty isolates were . , . , . , and . , respectively, indicating that the s , e, m, and n proteins of these ibv isolates had evolved under purifying selection (table ) . however, a few positively selected sites were detected although most sites were under neutral selection and purifying selection (table and supplementary table s ). aa residues , , , , , and of the s protein were consistently highlighted by positive selection models (slac, fel, and ifel) as positive selection sites. similarly, residue of the e protein, residues , , , , and of the m protein, residues , , , , and of the n protein were identified as positively selected sites. in addition, all of the positively selected sites within s , e, m, and n genes had high entropy values of larger than . (supplementary table s ). the results showed that there were - , - , - , - potential glycosylation sites within the s , e, m, and n proteins (except that gx-nn strain did not have a glycosylation site in the n protein). the comparison of the estimated n-glycosylation sites of s protein between the isolates and the reference strains showed that all the strains presented one n-glycosylation site at the residue / (nfsd) (except ck/ch/lsc/ i-type (gi- ) strains). and at the residue / (nitl), all the strains presented one n-glycosylation site (except taiwan-type (gi- ) and new-type (gvi- )). similarly, all the strains presented one n-glycosylation site at e protein residue / (ngsf) and residue / (nktl) (except ck/ch/lsc/ i-type and conn-type), all the strains presented one n-glycosylation site at m protein residue / / (nctl) and at n protein residue (nasw) (except gx-nn strain) (table and supplementary figure s -s ). the mean substitution rates for the s , e, m, and n genes of the epidemic isolates during - were calculated to be . × − , . × − , . × − , and . × − substitutions/site/year (s/s/y), respectively ( table ), indicating that s gene is the most easily mutated and n gene is the most stable among the four structural genes. the estimated times for most recent common ancestor (tmrca) of s , e, m, and n genes were before . , . , . , and . , respectively ( figure ). based on s , genotypes of ck/ch/lsc/ i (gi- ), new (gvi- ), mass (gi- ), lx (gi- ), / (gi- ), taiwan (gi- ), ldt -a (gi- ), and new (gvii- ) were dated back to . , . , . , . , . , hpd, highest probability density. viruses , , x for peer review of the reconstruction of population history was assessed using a bayesian skyline plot coalescent model. results showed that the effective population size of s , e, m, and n genes of ibvs was featured by a continuous and slow reduction in viral population size between s and s. a more obvious decrease was observed between s and s base on the s gene ( figure a ). remarkably, a sudden and sharp decline in the effective population size was observed after s base on the s , e, m, and n genes. however, since late , the viral population rapidly rose ( figure ). the reconstruction of population history was assessed using a bayesian skyline plot coalescent model. results showed that the effective population size of s , e, m, and n genes of ibvs was featured by a continuous and slow reduction in viral population size between s and s. a more obvious decrease was observed between s and s base on the s gene ( figure a ). remarkably, a sudden and sharp decline in the effective population size was observed after s base on the s , e, m, and n genes. however, since late , the viral population rapidly rose ( figure ). the reconstruction of population history was assessed using a bayesian skyline plot coalescent model. results showed that the effective population size of s , e, m, and n genes of ibvs was featured by a continuous and slow reduction in viral population size between s and s. a more obvious decrease was observed between s and s base on the s gene ( figure a ). remarkably, a sudden and sharp decline in the effective population size was observed after s base on the s , e, m, and n genes. however, since late , the viral population rapidly rose ( figure ). southern china is the largest region of yellow chickens produced in china. despite extensive vaccination, ib continues to be a serious problem. in the present study, strains of ibv were isolated from diseased chicken flocks in southern china during - and the genetic properties of the entire s , e, m, and n genes analyzed. to increase our insight into the comprehensive epidemiological situation and evolutionary trend of ibv in southern china, ibv isolates from to in southern china were also used to analyze together. the representative reference strains included the live vaccine strains commonly used and the prevalent strains in china and other countries worldwide, which represented the main well-established lineages and genotypes in recent years in china [ ] . our results indicated that there was a remarkable change in genetic diversity, dominant genotypes, and selection pressure of ibv strains in southern china over the past decade compared with the previous period of - . the co-circulation of multiple ibv genotypes and the increasing of ibv variants have resulted in great challenges for the controlling ib through vaccination. in our study, multiple ibv genotypes were also identified in guangxi over the past decade. a total of eight, six, four, and six genotypes were identified based on the s , e, m, and n genes, respectively. therefore, there is still ongoing genetic diversity of ibvs in southern china, which is the same as previous epidemics in this region and other parts of china [ , , ] . interestingly, the predominant genotype changed from ck/ch/lsc/ i-type lx -type was the most dominant genotype in our study, which agreed with many previous descriptions [ , , , ] . surprisingly, the second most dominant genotype of ibv circulating in southern china was ldt -a-type, which was a pandemic type and frequently isolated from chicken flocks in china [ , , ] . ldt -a commercial live vaccine has been issued by the official authority in china since [ ] . the re-isolation of vaccine strain is possible when ldt -a live vaccine strain is extensively used. we noticed that among the ldt -a type isolates during - , four, eight and two strains were isolated in , , and respectively. however, only one ldt -a type strain was isolated during - . we are not sure whether or not the strains of ldt -a type isolated between and are re-isolation of vaccine strains, but it seems that the ldt -a type vaccine could provide sufficient protection against the circulating homologous field strains in southern china because none of ldt -a genotype strains were isolated after . at the same time, we found the e genes of most ldt -a type isolates belonged to the ck/ch/lsc/ i-type, and the m and n genes of most ldt -a type isolates belonged to lx (qx)-type. these results implied that ldt -a-type might isolate the recombinants from ldt -a vaccine strain and other circulating ibv strains. considering that the ldt -a types were occasionally isolated in other regions of china recently [ ] , the ldt -a type isolates in southern china still need to be further monitored. the taiwan-type ibvs were firstly isolated in taiwan in s and have been divided into the taiwan-i and taiwan-ii subgroups [ , ] . more studies reported that an increasing number of taiwan-type strains have been isolated in southern china in recent years [ , , , ] . taiwan-ii-type strains (gx-xd and gx-g) were isolated in and no taiwan-ii-type strain was isolated in guangxi after that [ ] , but five taiwan-i-type strains were isolated during - . in our study, . % ( / ), . % ( / ), and % ( / ) of taiwan-i-type strains were isolated in , , and , respectively. it seemed that the taiwan-i-type strains were increasing in recent years. evidences proved that the taiwan-i-type strains were widely spread from taiwan to guangdong, guangxi, fujian, sichuan hunan, zhejiang, yunnan province and so on [ , , [ ] [ ] [ ] , and the increasing isolates demonstrated that the currently used vaccines were lacking protective efficacy against the taiwan-i-type strains. the recombinants between taiwan-i-type and lx (qx) causing severe economic losses have been reported [ ] . how the taiwan-type strains spread from taiwan to mainland china remains unknown. recently, wild birds have been of concern as natural ibv carriers, since infected birds have been showed to carry the viruses over long distances [ , , , ] . therefore, monitoring of wild birds should be needed in further. of course, ibv live vaccines and poultry products will also need to be followed. the earliest strain of new-type (gvi- ) was isolated in [ ] and has in recent years spread to many chicken flocks [ , , , , ] . surprisingly, majorities of the new-type strains from other studies were isolated from south china [ , , , ] . recently, another novel genotype vii (gvii- ) was reported [ ] . both these two new-type viruses were identified in our study and the total number of their strains showed new-type was the third most dominant genotype. we found that there was a four aa (qkep) insertion located in the hvr iii and one aa (s) insertion in all the new-type isolates. interestingly, the four aa (qkep) were predicted b cell epitopes by the bepipred /iedb tool and half of the new-type viruses' serotype were different from that of / or other vaccine strains [ ] . the aa mutation in the hvr of the s gene maybe lead to the occurrence of new serotype and immunity escape. the origin of new-type viruses in china remains unknown. interestingly, the "novel" isolates were shown to undergo recombination in our study. the new-type isolate gx-nn was a major parent to the recombinant ldt -a-type isolate gx-nn . to the best of our knowledge, this first report of a recombination event needs to be further investigated. analyzing the complete-genome and identifying the antigenicity and pathogenicity of new-type isolates will be further studied. it is uncertain whether the new-type would become the predominant type in china in further, but it continues circulating in the field suggests that the new-type ibvs are still endemic in china and more attention should be paid to them. high rates of recombination result in ibv variability. many recombinants were confirmed. some recombination occurred between field and vaccine viruses [ , , , ] , some occurred between field isolates [ , , ] . six recombinants were identified in this study and three had a major parent of the lx -type, and two with minor parents of / -type genotype. our previous studies confirmed five recombinants, which were between the vaccine strain / and the ck/ch/lsc/ i-type field strain gx-yl during - [ ] . therefore, we have identified recombinants so far and recombinants were derived from the / -type strain during - . high frequencies of recombination between vaccine and field strains have been reported frequently worldwide [ , , , , , ] . the / vaccine strain has been commonly used in china for a long time, so it is not surprising that / -type recombinants were found in the field. however, there were more recombinants involved in the / -type vaccine strain when compared to the mass-type vaccine strains' recombinants according to our and other previous reports [ , , , , ] , although both mass-type and / -type vaccine strains were widely used in china. the phenomenon may have the following three explanations. firstly, incomplete protection of / -type vaccine strain against heterologous strains might result in co-infection of vaccine and heterologous strains in the same birds and eventually led to recombination. secondly, the / -type vaccine strain recombinants may be more likely to escape vaccine immunization than mass-type vaccine strain recombinants. thirdly, the / -type vaccine strain persisted longer in the immunized birds than the mass-type vaccine strain, increasing the likelihood of co-infection with the latter infected strain/strains, as a previous report showed that / vaccine could persist in birds for days [ ] . the complete genome, pathogenicity, and immunogenicity of / -derived recombinants should be assessed in further studies. the isolation ratio of / (gi- ) genotype strains was relatively stable recently. therefore, in order to reduce the recombination occurred between field and vaccine viruses, the multi-valent live vaccine combined with / strain or other new vaccines (such as ldt -a strain, lx strain, and so on) should be used with caution and thoughtful consideration. in order to understand the variation degree of the s , e, m, and n genes and the correlation between gene variation and selection pressure, the analyses of entropy of amino acid sequences, molecular evolutionary rate, and positive selection were carried out in our study. the results showed that mean substitution rates were between . × − and . × − substitutions per site per year in different genes, which is comparable with previous description [ ] . both the average entropy and mean substitution rates sorted from large to small as follows: s , e, m, and n genes, and the dn/ds ratios of s , e, m, and n genes were . , . , . , and . , respectively. the dn/ds ratios of s , e, m, and n genes were less than , meaning that the analyzed region were under purifying selection. therefore, our results indicated that the degree of variation sorted from large to small as follows: s , e, m, and n genes and there was a positive correlation between selection pressure and gene variation. it is known that s genes had the largest variation, but the mutation of e gene has not been paid enough attention. a recent study reported that the e gene evolved at the fastest rate among four structural protein-coding genes [ ] . the e gene mutation needs to be focused on in the future. to our knowledge, it is the first report to analyze systematically the variation degree of the four structural genes of ibvs and correlation between gene variation and selection pressure. as mentioned above, the s , e, m, and n genes of guangxi ibvs during - were under purifying selection. despite that the four structural genes underwent purifying selection, there were six, one, five, and five positively selected sites within the s , e, m, and n genes respectively. the dn/ds value and positively selected sites of guangxi isolates were different from those of isolates from other countries and regions [ , , ] . in addition, all of the positively selected sites in s , e, m, and n genes had high entropy values of bigger than . . the accumulation of amino acid variation will have an important effect on the gene characteristics and evolutionary direction of viruses [ ] . therefore, more attention should be paid to those positively selected sites with high entropy values. in the present study, positive selection was not detected in the s , e, m, and n proteins of ibv isolates in southern china during - although there were high number of mutations. zhao et al. also found that purifying selection was the main evolutionary pressure in the protein-coding regions in china over the past two decades [ ] . however, positive selection was detected in the s , m, and n proteins of ibv isolates in southern china during - according to our previous investigation [ ] . therefore, positive selection in the s , m, and n proteins in different periods in southern china was changeable. this phenomenon was also seen in some other previous reports [ ] [ ] [ ] . the change in selection pressures may be due to immune responses induced by multiple types of vaccines, the microenvironment of infected hosts, or physical and biosafety conditions [ ] . molecular evolutionary rate analysis can accurately estimate the molecular dating and it was used to determine successfully the timing of ancestors of classical swine fever virus (csfv) [ ] , sars virus [ ] , and ibv [ , ] . according our previous investigation [ ] , the first ibv strain was isolated in , demonstrating that the ibv was already present at that time. this study further backdated the ibvs emergence of more than years (during - ) and indicated that the most recent ancestor of guangxi ibv isolates based on the genes s , e, m, and n existed in the early , , , and , respectively. different structural protein genes of the ibvs had different time of the most recent ancestor. this phenomenon was also reported in other viruses [ , , ] . the data of tmrca estimated from our study suggested ibv has been already circulating in field for a period before its first outbreak. interestingly, the evolution rates sorted from large to small as follows: s , e, m, and n genes; the similarity plot data showed high genetic divergence and recombination was found in the s gene of ibv isolates, which agreed with the dates of most recent ancestor. although ibv was discovered in s, it is thought that it may have a long history as a species. the similar results were reported in other viruses [ , ] . in our early study, maybe unavailability of advanced diagnostic and sequencing technologies, the backyard poultry farming, inefficient surveillance systems, and limited financial resources resulted in the non-detection of ibv isolates for such a long time in guangxi. bayesian skyline analysis of s , e, m, and n genes of ibvs revealed that a persistent and slow decrease in relative genetic diversity between s and s, mirroring the implementation of mass-or / -based vaccine was effective in controlling the virus in field conditions to some extent, although not being able to eradicate the pathogen. analysis of s showed a more obvious decrease between s and s. the live vaccine h has been used in china for many years and the / -like live vaccine was also commonly used in china since s even without official authorization. therefore, the / -like vaccine was effective between s and s. surprisingly, a sudden and sharp decrease in genetic diversity base on the s , e, m, and n genes was observed after , mirroring the implementation with a newly registered ldt -a-based vaccine. in our study, the ldt -a-type disappeared after . ldt -a-like strains were also rarely isolated in other recent studies [ , ] . it was reported that the change of viral population size had a strong association with the vaccine administration/withdrawal [ , ] . therefore, it can be speculated that the ldt -a vaccine could provide a better protection against the circulating strains in china. however, since late , a sharp increase was observed in viral population size, which may suggest the implementation of current vaccines could not provide higher protection against the circulating strains. the lx -type isolates had become the predominant ibvs since in our study. the proportion of lx -like genotype strains increased over time in china [ ] . increasing of lx genotype viruses over time agreed with the rapid rise in the viral population since late . our previous study showed that the h , / , or ldt -a vaccines could not provide complete protection against the prevalent local strains of ibv (such as lx genotype isolates) in southern china [ ] . therefore, it is predicted that the epidemic trend of lx type strain will not decline in the next few years and will become more and more intense. therefore, new lx type vaccine was urgently needed. lx live vaccine was officially authorized in china last year, but has not yet come into the market. lx commercial vaccine urgently needs to go to the market in china. taken together, our results of genetic analyses of the four structural protein genes s , e, m, and n clearly demonstrated that there has been a remarkable genetic change of ibv in southern china over the past decade. the coexistence of multiple genotypes of ibv, the changes of dominant genotypes in different periods, the emergence of new genotypes, the recombination of vaccine strains with the field strains, the rapid evolving rate of ibv, the ever-changing of viral population size, and the presence of multiple positively selected sites in the structural proteins suggest a complexity of ibv epidemic strains in southern china. this epidemiological complexity may be caused by multiple factors: simultaneous vaccination of multiple live vaccines, illegal use of live vaccines, large number of avian varieties, raising of chickens at a rather high densities and the patterns of raising (free-range style and lack of biosecurity), etc. the complexity also suggests the importance of molecular epidemiological investigation and permanent monitoring of circulating strains as well as more serious challenges for ib prevention and control. based on the present results, it is predicted that lx -type will remain to be dominant genotype in near future and new-type ibv isolates will be more and more prevalent in the future. therefore, it is necessary to choose and/or develop new vaccines to counteract the ibvs of these two genotypes. in addition, multivalent new vaccines should be developed and rational modified vaccination strategies and the strict biosafety should be observed. to our knowledge, this is the first report showing the changes of genetic diversity, dominant genotypes, and selection pressure of ibv strains. the present study extends our knowledge about past and present ibv variability in southern china, providing valuable reference and countermeasures against ibv field breakouts in china, also emphasizing the importance of constant dynamic surveillance of the circulating isolates. in conclusion, our results indicate that the ibv strains in southern china have experienced a remarkable change in genetic diversity, dominant genotypes, and selection pressure, indicating the importance of permanent monitoring of circulating strains and the urgency for developing new vaccines which counteract the emerging lx -type and new-type ibvs and should be resistant to recombination and mutation. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , table s : ibv reference strains used in this study; table s : estimates of evolutionary distances over sequence pairs between groups of s gene; table s : estimates of evolutionary distances over sequence pairs between groups of e gene; table s : estimates of evolutionary distances over sequence pairs between groups of m gene; table s : estimates of evolutionary distances over sequence pairs between groups of n gene; table s : the selection profile and entropy values of s , e, m, and n proteins of guangxi ibv isolates; figure s : alignment of the deduced amino acid sequences of the s subunit of the spike protein of ibv isolates (new-type or gvi- ) and other strains. the insertion of critical amino acids , , , , and are highlighted in red; figure s : the sequences were aligned using the clustal w method in the bioedit version . . . package. the hotspot sequence atttt(t/a) (green underline) was found near the breakpoint site (red box) of the s subunit gene in all of recombinant isolates except gx-yl . the gx-yl isolate hotspot sequence and breakpoint site were marked in blue box; figure s : entropy plot of amino acid of s (a), e (b), m (c), and n (d) protein genes of ibvs. x-axis shows the amino acid sites of s , e, m, and n genes; y-axis shows the entropy of each amino acid site; figure s : the location of positive selection sites and putative glycosylation sites of the s subunit of ibv isolates. the positively selected sites and n-glycosylation site are highlighted in red and blue separately; figure s : the location of positive selection sites and putative glycosylation sites of the e subunit of ibv isolates. the positively selected sites and n-glycosylation site are highlighted in red and blue separately; figure s : the location of positive selection sites and putative glycosylation sites of the m subunit of ibv isolates. the positively selected sites and n-glycosylation site are highlighted in red and blue separately; figure s : the location of positive selection sites and putative glycosylation sites of the n subunit of ibv isolates. the positively selected sites and n-glycosylation site are highlighted in red and blue separately. serotype and genotype diversity of infectious bronchitis viruses isolated during - in guangxi continuous evolution of avian infectious bronchitis virus resulting in different variants co-circulating in southern china complete genome sequences of two chinese virulent avian coronavirus infectious bronchitis virus variants molecular characterization of major structural protein genes of avian coronavirus infectious bronchitis virus isolates in southern china analysis of s gene of avian infectious bronchitis virus isolated in southern china during - immune protection conferred 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of the creative commons attribution (cc by) license the authors declare no conflicts of interest. key: cord- - e qah d authors: zhai, shao-lun; lu, shou-sheng; wei, wen-kang; lv, dian-hong; wen, xiao-hui; zhai, qi; chen, qin-ling; sun, yan-wei; xi, yun title: reservoirs of porcine circoviruses: a mini review date: - - journal: front vet sci doi: . /fvets. . sha: doc_id: cord_uid: e qah d porcine circovirus (pcv) is one of the smallest known dna viruses in mammals. at present, pcvs are divided into three species, pcv , pcv , and pcv . pcv and pcv were found in the s and the s, respectively, whereas pcv was discovered recently in . pcv does not cause diseases in pigs. however, pcv , similar to pcv , is reported to be associated with several swine diseases, including porcine dermatitis and nephropathy syndrome (pdns) and reproductive failure. pcvs are very common in domestic pigs as well as wild boars. however, pcvs have been occasionally isolated from non-porcine animals, including ruminants (such as cattle, goats, wild chamois, and roe deers), rodents (such as nmri mice, balb/c mice, black c mice, icr mice, mus musculus, and rattus rattus), canines (such as dogs, minks, foxes, and raccoon dogs), insects (such as flies, mosquitoes, and ticks), and shellfish. moreover, pcvs are frequently reported in biological products, including human vaccines, animal vaccines, porcine-derived commercial pepsin products, and many cell lines. pcvs are also abundant in the environment, including water samples and air samples. interestingly, pcv and/or pcv antibody or antigen has also been detected in sera, stool samples and respiratory swab samples of human, revealing zoonotic potential of pcvs. thus, pcvs inhabit many types of reservoirs. in this review, we summarize the reservoirs of pcvs, and this information would be helpful in understanding the natural circulating status and possible cross-species transmission of pcvs. porcine circoviruses (pcvs) are members of the circovirus genus of the circoviridae family. currently, there are three species in the genus, pcv type (pcv ), pcv type (pcv ), and pcv type (pcv ), respectively. in , pcv was discovered as a contaminant in porcine kidney cell lines ( ) . subsequent studies have confirmed that pcv is apathogenic in pigs ( ) . in late s, a new porcine disease, called post-weaning multisystemic wasting syndrome (pmws), emerged in north america and europe ( ) ( ) ( ) ( ) , and pcv was confirmed as the causal pathogen ( , ) . pcv has a global distribution and diverse genotypes ( , ) . in , the third pcv, named pcv , was discovered using high-throughput sequencing technology in u.s. swine herds suffering from porcine dermatitis and nephropathy syndrome (pdns), reproductive failure and other syndromes ( , ) . a recent study suggested that pcv fulfilled koch's postulates and could cause pdns in piglets ( ) . pcvs, especially pcv and pcv , are very common in pigs, and can cause diverse clinical presentations including pmws, pdns, reproductive failure, interstitial pneumonia, and so on. both, pcv and pcv have garnered immense interest in the world swine industry. hitherto, most studies found in literature have focused on pcvs derived from swine sources. occasionally, pcvs have also been isolated from non-porcine animals, biological products, and environmental samples. in , for the first time, tischer et al. confirmed the presence of pcv antibodies in cattle in germany using indirect immunofluorescence assay (ifa) and enzyme-linked immunosorbent assay (elisa) ( ) . later, a pcv nucleotide ( , bp) was identified in cattle with respiratory diseases and aborted bovine fetuses in canada ( ) . in , a new disease called hemorrhagic diathesis broke out in calves in germany, and pcv b ( , bp) was suggested as a potential causal pathogen ( , ) . in the united states and china, pcv was frequently detected using metagenomic sequencing method and special pcr method in beef from supermarkets, beef stalls, and goat samples ( ) ( ) ( ) ( ) ( ) . interestingly, one previous study confirmed that calves were susceptible to pcv and presented lymph node swelling, reddening of oral and ocular mucosa, and diarrhea post inoculation ( ) . in , a circovirus-like virus sequence (porknw , genbank accession number hq ) was discovered in both pork and beef samples from stores in san francisco ( ) . in , two similar sequences were re-detected in beef samples from the sunset district of san francisco ( ) . in fact, these circovirus-like virus sequences were significantly close to the aforementioned pcv in pigs ( , ) . based on these research reports, it is speculated that pcv is also widespread in cattle. a recent epidemiological investigation from china confirmed our speculation. their results showed that out of ( . %) clinically healthy bovine samples from eight regions in the shandong province were positive for pcv , and that the bovine-origin pcv genome sequences had a close relationship with porcine-origin pcv genome sequences ( ) . surprisingly, pcv was also reported in the samples of wild chamois and roe deers ( ) . the data suggest that ruminants, especially cattle, are noteworthy reservoirs of pcvs. these epidemiological data bring to light two important scientific issues that warrant further research: ( ) to determine whether pcv has a multihost receptor and ( ) to identify whether some cross-species transmission routes (such as direct contact, insect biting) might contribute to the circulation of pcv in pigs and ruminants. seroconversion of pcv antibody was detected in several murine species including nmri, balb/c, and black c ( ) . based on evidence from numerous studies, mice are often considered important animal models of pcv infection ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . however, only a few studies have conformed pcv infection in field rodent samples. since , several research teams from hungary, brazil, korea and china have provided molecular evidence of pcv in rodents circulating on swine farms ( ) ( ) ( ) ( ) . in the study from brazil, pcv antigen was identified in the spleen, lungs and kidneys of rodent species mus musculus and rattus rattus using immunohistochemistry assay ( ) . in addition, rodent-origin pcv genome sequences had remarkable similarity with gene sequences of pcv isolated from pigs ( , ) . it is noteworthy that one recent study identified pcv in commercially sourced laboratory mice including balb/c and icr mice ( ) . this is in contrast with from the hungarian study, which claims that rodents need certain contact with pigs, without which pcv is negative in rodents ( ) . owing to limited information, we are currently unable to ascertain the origin of murine pcv in laboratory mice. without significant clinical manifestation, rodents are more of a carrying reservoir and an animal model of pcvs. on farms, unconstrained rodents may promote the spread of pcvs. fur animals have significant economic value. in recent years, cases of infections due to porcine pathogens (such as hepatitis e virus, pseudorabies virus) have progressively in minks and foxes ( ) ( ) ( ) ( ) . pcv was also reported in minks, foxes, and raccoon dogs with diarrhea or reproductive failure in china ( ) ( ) ( ) . interestingly, the samples were tested negative for common pathogens (such as mink enteritis virus, canine distemper virus, coronavirus, rotavirus, or astrovirus) of minks and foxes, indicating that pcv was also an important pathogen contributing to the clinical diseases of minks and foxes. infection of pcv in fur animals may be caused by consumption of food containing porcine-origin products and possibly by cross-species transmission. these mechanisms could also explain pcv and pcv infection in dogs ( , ) . generally speaking, pcv is not considered as an arbovirus. however, pcv has been often detected in m. domestica flies and culex mosquitoes in pig farms ( , ) . surprisingly, pcv was first identified in ticks (ixodes ricinus) collected from wild roe deers that were negative for pcv , making the source of pcv ambiguous ( ) . pcv infection is postulated to occur when hosts get bitten by carrier flies, mosquitoes, and ticks. bivalve shellfishes (such as oysters, clams, and mussels) intake nutrition by filtering up to . l/h of surrounding water and simultaneously concentrate microorganisms that are present ( ) ( ) ( ) . animal pathogens can contaminate the beds via runoff from fields fertilized using animal waste. one previous study reported high detection rate for pcv and e. coli ( %, / and %, / ) in blue mussels (mytilus edulis) from danish commercial harvesting areas, while common food-borne pathogens (such as hepatitis e virus, rotavirus, salmonella) were absent. the geographic distribution of the pcv -positive shellfish samples revealed that positive samples were localized to limfjord in the northern part of jutland and a bay area in the south-western part of jutland, denmark ( ) . this suggested that these bay areas were contaminated by porcine waste, and pcv may be a specific indicator of porcine waste in shellfish. there has always been controversy regarding pcvs infecting human. at the outset, tischer et al. confirmed the presence of pcv antibodies in human sera, and the ifa results showed a significantly higher number of positive sera ( %) in non-hospitalized "healthy" persons from the former german democratic republic than that ( . %) in blood donors from berlin-west ( ) . interestingly, scientists from northern ireland, the united states, and germany could not detect antibodies or antigens of pcv in human samples ( ) ( ) ( ) ( ) . however, in other studies, antibody of pcv or antigen of pcv was detected in human sera, digestive tract samples and respiratory tract samples ( ) ( ) ( ) ( ) ( ) ( ) . although there is serological and molecular proof of pcv presence in humans, the detection rate is very low. thus, the clinical significance of pcvs in human beings remains largely unknown. pcv was first confirmed as a contaminant in porcine kidney cell lines ( ), following which, non-infectious pcv and pcv were detected in porcine-derived commercial pepsin products used for humans ( ) . owing to its porcine origin, the detection of pcvs in pepsin and other porcine biological products is perfectly plausible. pcv was also reported in commercial veterinary vaccines against classic swine fever virus (csfv), porcine parvovirus (ppv), and pseudorabies virus (prv) ( , ) . however, in , pcv was reported in human rotavirus vaccine ( ) , which caused a significant stir because of unknown clinical significance of pcvs in humans. since that incident, the food and drug administration (fda) and several research institutions began investigating the source of the vaccine contamination and assessing the safety of the vaccine. their results indicated that several cell lines, virus seeds and vaccines were contaminated by pcv and/or pcv ( ) ( ) ( ) ( ) ( ) ( ) . porcinederived commercial pepsin product used for cell culture and vaccine production was considered as the main source of vaccine contamination ( ) . although the contaminated vaccines caused no clinical diseases in people, humans can be considered likely reservoirs for pcvs ( , ) . thus, via human fecal matter, pcvs might have been introduced into environmental water or other places ( ). as we know, many viruses can spread through aerosols. whether pcvs can spread via aerosol is little known. however, canadian researchers found that high viral loads (up to genomes per cubic meter of air) of pcv existed in swine confinement buildings ( ) . another study also identified the existence of pcv in bioaerosol samples from pig farms and abattoirs. in addition, pcv was detected in nasal washes of workers ( / ) from pig farms ( ) . this revealed that pcv was a potential airborne virus. intriguingly, pcv can be detected in various types of water samples from brazil, including (a) water used for swine consumption, subjected to conventional treatment followed by chlorination; (b) tap water meant for human consumption, subjected to conventional treatment followed by chlorination; (c) surface water without treatment, used for swine consumption; (d) water from the pinhal river, which crosses; (e) groundwater collected from a tap located at a school; and (f) water from the jacutinga river, which is redirected and treated for domestic supply ( ) . this indicated that pcv was widely prevalent in the environment. in summary, from the current knowledge, we infer that pcvs have multiple reservoirs; they are not limited to the swine family, but have broad distributed in ruminants, rodents, canines, insects, and other species. consequently, we deduce the occurrence of possible cross-species transmission (such as pigs to rodents, pigs to cattle, pigs to fur animals) of pcvs. in most cases of detection or infection in non-porcine animals, the main reason has been ingestion of porcine products or 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credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - lfpy d authors: niu, ting-jiang; yi, shuai-shu; wang, xin; wang, lei-hua; guo, bing-yan; zhao, li-yan; zhang, shuang; dong, hao; wang, kai; hu, xue-gui title: detection and genetic characterization of kobuvirus in cats: the first molecular evidence from northeast china date: - - journal: infect genet evol doi: . /j.meegid. . . sha: doc_id: cord_uid: lfpy d feline kobuvirus (fekov), a novel picornavirus of the genus kobuvirus, was initially identified in the feces of cats with diarrhea in south korea in . to date, there is only one report of the circulation of kobuvirus in cats in southern china. to investigate the presence and genetic variability of fekov in northeast china, fecal samples were collected from cats with obvious diarrhea and asymptomatic cats in shenyang, jinzhou, changchun, jilin and harbin regions, northeast china, and viruses were detected by rt-pcr with universal primers targeting all kobuviruses. kobuvirus was identified in fecal samples with an overall prevalence of . % ( / ) of which samples were co-infected with feline parvovirus (fpv) and/or feline bocavirus (fbov). diarrhoeic cats had a higher kobuvirus prevalence ( . %, / ) than asymptomatic cats ( . %, / ). by genetic analysis based on partial d gene, all kobuvirus-positive samples were more closely related to previous fekov strains with high identities of . %– . % and . %– % at the nucleotide and amino acid levels. additionally, phylogenetic analysis based on the complete vp gene indicated that all fekov strains identified in this study were placed into a cluster, which separated from other reference strains previously reported, and three identical amino acid substitutions were present at the c-terminal of the vp protein for these fekov strains. furthermore, two complete fekov polyprotein genomes were successfully obtained from two positive samples and designated jz and cc , respectively. the two strains shared . %– . % nucleotide identities and . %– . % amino acid identities to fekov prototype strains. phylogenetic analysis indicated that fekovs were clustered according to their geographical regions, albeit with limited sequences support. this study provides the first molecular evidence that fekov circulates in cats in northeast china, and these fekovs exhibit genetic diversity and unique evolutionary trend. kobuvirus (kov), which belongs to a recently classified genus (kobuvirus) of the family picornaviridae, is a small, non-enveloped, spherical virus approximately - nm in diameter (zell ) . kobuvirus has a single-stranded, positive-sense rna genome of . - . kb consisting of ′ untranslated region (utr), one single open reading frame (orf), ′utr and poly a tail (han et al. ) . this orf encodes a large polyprotein which is cleaved to yield a non-structural protein l, three structural proteins (vp , vp and vp ) and seven other nonstructural proteins ( a- c and a- d) (reuter et al. ) . of these, vp is the most important viral capsid protein determining the antigenicity and pathogenicity for kobuvirus. d gene encodes the rnadependent rna polymerase (rdrp), which plays a critical role in viral replication (lescar and canard ) . based on the function of encoded proteins, the kobuvirus genome is generally divided into three functional regions: p (encoding structural protein vp , vp and vp ), p and p (encoding non-structural protein a- c and a- d, respectively) (han et al. ; reuter et al. ) . since human aichi virus (aiv) was first recognized in as the cause of oyster-associated nonbacterial gastroenteritis in humans in aichi prefecture, japan (yamashita et al. ) , the novel kobuviruses have been identified in many mammalian animals. in , bovine kobuvirus (bkv) was first identified from a contaminant of hela cells in japan (yamashita et al. ). subsequently, porcine kobuvirus (pkov) was found in the stools of domestic pigs in hungary in (reuter et al. ) . canine kobuvirus (cakov) that was genetically related to human aiv was first identified in a domestic dog with acute gastroenteritis in usa in (kapoor et al. ) , and was subsequently found in healthy domestic dogs (oem et al. a ) and wild carnivores, including wolves (melegari et al. ) , red foxes (di martino et al. ) , golden jackals, side-striped jackal and spotted hyena (olarte-castillo et al. ) . to date, kobuviruses have been reported in human (yamashita et al. ) , cattle (yamashita et al. ) , sheep (reuter et al. ) , pig (reuter et al. ) , rodents (phan et al. ) , goat (oem et al. b) , wild boars (reuter et al. ) , roe deer (di martino et al. a) , rabbits (pankovics et al. ) , bats (wu et al. ) , ferrets (smits et al. ) , domestic and wild carnivores (olarte-castillo et al. ) and cats (chung et al. ) . according to the recent report of international committee on taxonomy of viruses (ictv) in (https://talk.ictvonline.org/taxonomy/), the genus kobuvirus was classified into six officially recognized species, namely aichivirus a (formerly aichi virus), aichivirus b (formerly bovine kobuvirus), aichivirus c (porcine kobuvirus), aichivirus d (kagovirus ), aichivirus e (rabbit kobuvirus) and aichivirus f (bat kobuvirus), respectively (adams et al. ; adams et al. ) . aichivirus a includes six types: human aiv, cakov, murine kobuvirus, roller kobuvirus, kathmandu sewage kobuvirus and feline kobuvirus (fekov). feline kobuvirus (fekov), a member of the species aichivirus a, was first identified in feces of cats with diarrhea in south korea in (chung et al. ) . the genetic analysis based on the partial rdrp gene indicated that fekov strains shared higher nucleotide ( . %- . %) and amino acid identities ( . %- . %) with cakov strains previously reported (kapoor et al. ) . in a study by cho, et al., it was demonstrated that kobuvirus widely circulated in domestic cats and was associated with viral diarrhea (cho et al. ) . recently, fekov infection in cats was reported in italy (di martino et al. b ). in , lu et al. first reported the circulation of fekov in diarrhoeic cats in southern china. fekov rna was found in domestic cats with diarrhea, but was undetected in healthy cats (lu et al. ) . however, only four complete genomes of fekov strains, including fk- (cho et al. ) , d (choi et al. ) , te/ /it/ (di martino et al. b ) and whj- (lu et al. ) , have been sequenced until now. furthermore, there is no data of kobuvirus infections in cats in northeast china. in this study, we provide the first molecular evidence for the circulation of fekov in northeast china, and investigate the prevalent levels, as well as genetic characteristics. in total, fresh fecal samples were collected from cats with diarrhea and asymptomatic cats from five different regions in northeast china, including shenyang, jinzhou, changchun, jilin and harbin, during january to november . individual fresh feces were immediately placed in rnase-free tubes and were stored at − °c until further use. fecal samples were suspended in phosphate-buffered saline (pbs, ph = . ) at a concentration of approximately . g/ml, and then the suspension was centrifuged at ×g for min at °c to collect the supernatant. total rna was extracted from μl of supernatant using axyprep body viral rna miniprep kit (corning, china), and reverse transcribed to synthesize cdna using the revertaid first strand cdna synthesis kit (invitrogen, usa) according to the manufacturer's instructions. viral dna of fecal samples was extracted using viral dna extraction kit i (omega, china) according to the manufacturer's instruction. the detection of kobuvirus was performed by rt-pcr using a pair of universal primer previously described (the primer sequences are shown in table ), univ-kobu-f/univ-kobu-r targeting -bp of partial d gene for all kobuviruses (reuter et al. ). these samples were also examined for other feline enteric viruses, including feline parvovirus (fpv) and feline bocavirus (fbov), using pcr assays previously described (takano et al. ; yang et al. ). the amplified products were separated after electrophoresis on . % agarose gels at v for t.-j. niu et al. infection, genetics and evolution ( ) - min, and were visualized using a gel documentation system (wealtec, usa). to further investigate the genetic diversity of fekov detected in the present study, two pairs of primers for the amplification of partial d gene and the complete vp gene were designed. the primer sequences are shown in table . the pcr conditions were as follows: initial denaturation at °c for min, followed by cycles of °c for min, °c for s and °c for min, and a final extension at °c for min. all pcr products were purified using axyprep dna gel extraction kit (corning, china), and then were cloned into pmd- t vector (takara, china). plasmid dna was extracted using axyprep plasmid miniprep kit (corning, china), and positive dh- α clones (three clones per sample) were sent to sangon biotech (shanghai, china) for sanger sequencing. two fekov-positive samples were randomly selected to amplify the complete polyprotein gene sequences using six primer sets designed in the present study (primer sequences are shown in table ). the reaction conditions were described as follows: pre-denaturation at °c for min followed by cycles of denaturation at °c for min, annealing at °c for min, extension at °c for s, and a final extension at °c for min. purification of pcr products, clone of purified fragment, plasmid extraction and sequencing were performed with the same methods as previously described. the nucleotide sequences were assembled using seqman program, and the complete fekov polyprotein gene sequences were deposited in genbank under accession numbers mh ( nt) and mh ( nt). pairwise alignments among different kobuviruses based on the partial d gene and the complete vp gene were performed using online blast (https://blast.ncbi.nlm.nih.gov/blast.cgi). the nucleotide and amino acid identities among all sequences were calculated using bioedit. for the obtained genome sequences, the orf was predicted using orf finder (https://www.ncbi.nlm.nih.gov/orffinder/), and the potential cleavage sites of polyprotein were predicted using netpicorna . and were further verified via the nucleotide and amino acid alignments with reference kobuviruses. similarity plot analysis of the complete polyprotein gene was performed using simplot . software. phylogenetic tree was constructed using the neighbour-joining method with bootstrap replicates in mega . software. we tested a total of fecal samples from shenyang (n = ), jinzhou (n = ), changchun (n = ), jilin (n = ) and harbin (n = ) in northeast china. background data was available for all cats of which were diarrhoeic and were healthy; were collected from private veterinary clinics and were collected from animal shelter centers. the screening results showed that of the samples ( . %) were positive for kobuvirus. out of these kobuvirus-positive samples, were positive for fpv, were positive for fbov and were co-infected with fpv and fbov. detailed information about kobuviruspositive samples was shown in table . the diarrhoeic cats had a higher kobuvirus prevalence ( . %, / ) than asymptomatic cats ( . %, / ) and the positive rate of cats from animal shelter centers ( . %, / ) were also higher than that of cats from private veterinary clinics ( . %, / ). moreover, there was no significant difference in the prevalence of samples from different regions (from . % to . %). partial d genes of kobuvirus-positive samples were sequenced in this study, and the nucleotide sequences were deposited in genbank under accession numbers mh -mh . the sequences shared . %- % nucleotide identities and . %- % amino acid identities with each other. these sequences had the highest nucleotide ( . %- . %) and amino acid identities ( . %- %) with fekov reference sequences deposited in genbank, suggesting that all twentyeight fecal samples were fekov-positive. furthermore, the sequences were . %- . %, . %- . % and . %- . % similar to cakovs, human aivs and murine kobuvirus at the nucleotide level, respectively. phylogenetic analysis based on the partial d gene showed that the sequences were more closely related to fekovs, clustering in the aichivirus a, which included also human aivs, cakovs and murine kobuvirus. the fekov sequences were divided into two major groups: sequences clustered with the chinese fekov strain, whj- (lu et al. ) , and formed a major group, while the other sequences and fekov reference strains identified in south korea and italy formed another group. interestingly, only one sequence identified in this study appeared more closely related to the korean fekov strain, d (choi et al. ) , while other sequences were separated from these reference sequences (fig. ) . eight samples were randomly selected from fekov-positive samples, and their complete vp gene sequences were obtained in the present study (genbank accession numbers mh -mh ). the eight sequences shared nucleotide and deduced amino acid identities of . %- % and . %- % with each other and the highest nucleotide identities of . %- . % with the chinese fekov strain, whj- (lu et al. ) , when compared with fekov reference strains. the neighbour-joining tree based on the vp nucleotide sequences showed that our sequences were more closely related to fekov strain whj- and clustered within a major group, while other fekov reference strains identified in south korea and italy formed another group (fig. a) . interestingly, the eight sequences and fekov strain whj- placed in different branches in the phylogenetic tree based on the deduced amino acid sequences (fig. b) . then, we analyzed the amino acid mutation sites in the vp gene between the eight sequences and other fekov sequences previously described, and discovered that three identical amino acid substitutions at amino acid positions (t → a), (p → s) and (s → t) were exhibited in all sequences identified in this study (table ) . two complete fekov polyprotein genomes were successfully sequenced from two kobuvirus-positive samples in the present study using six pairs of primers, and designated jz and cc , respectively. the obtained genome sequence of jz was nt in length and contained one orf ( nt) encoding the polyprotein of aa, while the cc was nt long. the orf of cc was nt in length with one-amino-acid deletion in vp gene. moreover, we predicted and verified the genomic organization and potential cleavage sites for the complete polyprotein gene of jz and cc , which were identical to other fekov strains previously described (fig. a) . the jz shared . % nucleotide identity and . % amino acid identity with cc . compared with other fekov reference strains, jz and cc had . %- . % nucleotide identities and . %- . % amino acid identities with the korean strains, fk- and d , and the italian strain, te/ /it/ , and shared a higher sequences homologies with the chinese fekov strain, whj- , at the nucleotide ( . %- . %) and deduced amino acid ( . %- . %) levels. we next compared the similarities of each functional region among jz , cc and other kobuviruses. for jz , the highest nucleotide and amino acid identities were found in the whj- p region, with values of . % and . %, while the highest nucleotide and amino acid divergences were found in the d l region, with values of . % and . %. cc shared amino acid identities of . %- %, . %- . %, . %- . % and . %- . % with fekov reference strains at the l, p , p and p regions, respectively. furthermore, the nucleotide and amino acid homologies between cc and whj- were higher than that between jz and whj- at each region of the polyprotein gene (table ) . in order to further analyze the genetic characteristics of the complete polyprotein gene, the similarity plot analysis which compared polyprotein nucleotide sequences of jz , cc and one cakov sequence (used as a out-group sequence) to fekov reference strain d /kj (used as a query sequence) was performed in this study. the analytical results showed that jz and cc shared similar similarities with reference strain d in the vp , vp , vp , a and from a to c regions, but different similarities in the l, b, c and d regions. in the l, a and b regions, cc was more similar to reference strain than jz , while cc presented considerably lower similarities in other regions of polyprotein gene than jz . moreover, higher range of genetic variability was observed in p and p regions of polyprotein gene (fig. b) . phylogenetic analysis based on the complete polyprotein nucleotide sequences indicated that jz and cc were more closely related to fekov reference strains than other kobuvirus strains and these fekov strains formed a group distinct from cakovs, human aivs and murine kobuvirus, within the aichivirus a. in the group of fekov, jz and cc clustered with the chinese fekov strain, whj- , and formed a tight branch, while other fekov strains also formed different branches according to their geographical regions (fig. ) . these results suggested that genetic diversity of fekov was presented in different geographical regions, albeit with limited sequences support. in the past few years, the circulations of kobuvirus in cats had been reported in south korea, italy and southern china (chung et al. ; di martino et al. b; lu et al. ). however, the related data of fekov in other countries and regions is lacking. this study presents the first identification and genetic characterization of kobuvirus in cats in northeast china. we investigated fecal samples of which ( . %) were positive for kobuvirus. the prevalence rate is similar to previous reports in south korea ( . %, / ), italy ( . %, / ) and southern china ( . %, / ) (chung et al. ; di martino et al. b; lu et al. ) , suggesting that kobuvirus widely circulates in domestic cats in northeast china. the prevalence of kobuvirus in diarrhoeic cats ( . %, / ) is significantly higher than that in healthy cats ( . %, / ), similar to a previous study by cho, et al. (cho et al. ) . moreover, we also tested other enteric viruses (fpv and fbov) for kobuvirus-positive samples of which samples were positive for fpv and/or fbov. in previous investigations, the co-infection of fekov, fpv and feline enteric coronavirus (fecv) in diarrheal cats with a higher prevalence had been reported (di martino et al. b) , and it was also determined that human aivs and other animal kobuvirus were associated with gastroenteritis (yang et al. ; zhai et al. ). these reveal that fekov, as a potential enteric virus, may be associated with viral diarrhea in cats. phylogenetic analysis based on partial d gene indicates that the fekov sequences identified in this study clustered into two large groups, majority had a higher nucleotide identity with the chinese fekov strain, whj- (lu et al. ) , and formed a novel group, while only sequences are divided into another group which formed with other fekov strains identified in south korea and italy (fig. ) . moreover, two unique amino acid replacements (amino acid positions and in the complete d gene) were present in most sequences (excluding jz , jz , sy , sy , sy and (caption on next page) t.-j. niu et al. infection, genetics and evolution ( ) - sy ) identified in the present study and whj- . these results suggest that a unique evolutionary trend is present in fekov strains circulating in china, when compared with fekov strains in other countries. the vp protein of picornaviruses is not only an important capsid protein determining the antigenicity and pathogenicity for kobuvirus, but also is the most variable structural protein in the kobuvirus (reuter et al. ) . phylogenetic analyses based on the complete vp sequences indicated that fekov strains identified in this study clustered together and formed a separate cluster compared to other fekov strains, and the identical amino acid mutations were present in the cterminal of vp protein. additionally, the different amino acid substitutions of the vp protein were observed in fekov strains identified in different regions (table ). these results suggest that the vp protein may be used as a considerable indication for the geographical distribution of fekovs. furthermore, a recent study indicated that a polyproline helix structure, as integrin binding motifs, is present at the c-terminal of vp protein on the outer surface of human aiv, and predicted this polyproline motif may be associated with signal transduction, antigen recognition, and viral infectivity and pathogenicity ). subsequently, the identical proline-rich motif was also reported in cakovs . a polyproline fragment is present in amino acid positions - of the vp protein for all fekov strains, similar to human aivs and cakovs. interestingly, one amino acid substitution is observed in this polyproline motif for fekov strains identified in this study (one substitution from proline to serine at position ) and that identified in italy (one substitution from proline to alanine at position ). the impact on viral infectivity for this amino acid mutation needs to be further investigated via structure prediction and viral isolation. fig. . phylogenetic analysis based on the nucleotide sequences ( nt) of partial d genes for kobuviruses. the phylogenetic tree was conducted using the neighbour-joining method with , bootstrap replicates using mega . software. black triangles indicate sequences identified in the present study, and black diamond indicates the chinese fekov strain, whj- . the silhouettes of hosts for different kobuviruses were observed on the branches. aiv, aichi virus; bkv, bovine kobuvirus; cakov, canine kobuvirus; fekov, feline kobuvirus; mokov, murine kobuvirus; pkov, porcine kobuvirus. br, brazil; chn, china; de, germany; egy, egypt; hun, hungary; it, italy; jp, japan; nl, nederland; kor, south korea; uk, the united kingdom; usa, the united states. the amino acid positions are referred to the complete vp gene. bold text indicates the identical amino acid substitutions for fekov sequences that identified in the present study. the complete polyprotein gene sequences of jz ( nt) and cc ( nt) were successfully obtained in this study. genomic analysis showed that the polyprotein of the two strains were all cleaved into viral proteins, l, vp , vp , vp , a, b, c, a, b, c and d. the predicted cleavage sites were q/g, q/h, q/a and q/s, in accordance with the korean fekov strains, d and fk- , and the chinese strain, whj- (cho et al. ; choi et al. ; lu et al. ). one of the important finding is that one-amino-acid deletion in vp gene of cc . in a previous study, thirty-amino-acid deletion was presented in b gene of pkov from healthy piglets, and these deletions were possibly related to the pathogenicity of pkov (jin et al. ) . however, in this study, jz and cc were all identified from diarrhoeic cats, the pathogenicity of fekov seemed unaffected by this amino-acid deletion. consequently, more complete polyprotein gene sequencing of chinese fekov strains is needed to determine whether this amino-acid deletion is widely existent in fekov table nucleotide and deduced amino acid sequence identities of the complete polyprotein gene and l, p -p regions between two chinese fekov strains, jz and cc , and other kobuviruses. t.-j. niu et al. infection, genetics and evolution ( ) - strains circulating in china, and further targated research is also needed to demonstrate the effect of this deletion in fekov. phylogenetic analysis based on the complete polyprotein sequences showed that jz and cc shared higher nucleotide and amino acid identities with the chinese strain, whj- , compared to other fekov strains, and all fekov strains were clustered according to their geographical regions, albeit with limited sequences support (fig. ) . moreover, jz and cc shared high nucleotide and amino acid identities to each other in the vp , a and c regions, but low identities to fekov reference strains. recent research indicates that the a protein plays a vital role in hijacking host acyl-coa-binding domaincontaining protein- (acbd ), which provides a site for the replication of kobuviruses (klima et al. ). c protein of most picornaviruses is also important for viral replication (fujita et al. ). therefore, these mutations in vp , a and b regions of jz and cc may affect viral replication and antigenicity, more targeted researches are needed to further determine. taken toghter, the considerable genetic diversity is existent in chinese fekov strains. furthermore, these fekov strains shared high amino acid identity with cakovs and human aivs in the complete polyprotein gene and different functional regions, especially in p region. considering the close genetic relationship of these kobuviruses, and frequent contact among their own host, the cross-species transmission of kobuviruses is worth investigating. in previous studies, several findings had provided considerable evidences for the potential risks of cross-species transmission for fekov, including frequent genetic variation in vp gene of fekov with . × − substitutions/site/year substitution rates (cho et al. ) , high nucleotide and amino acid identities between fekovs and cakovs (chung et al. ; di martino et al. b; lu et al. ) , and the detection of igg antibodies specific for aiv in cats (carmona-vicente et al. ) . moreover, it is also important to mention about conducting serological studies to investigate kobuvirus pathogenicity and whether the genetic diversity in fekov affects pathogenicity. thus, periodic genetic and serological investigations of fekovs will be helpful for the assessment of cross-species transmission and pathogenicity for fekovs. in conclusion, we provide the first molecular evidence for the circulation of feline kobuvirus in cats in northeast china. our findings indicate that the circulation of fekov in domestic cats with diarrhea was more prevalent, suggesting fekov infections may be related to viral diarrhea in cats. phylogenetic analyses based on partial d gene and complete vp gene indicate that the considerable genetic diversity is exhibited in chinese fekov strains, and novel fekov strains with unique evolutionary trends are circulating in china. moreover, the complete polyprotein genes of fekov strains jz and cc are successfully sequenced in this study. these findings will help us to understand the epidemics and genetics of kobuvirus in cats in china. further epidemiological and molecular investigations are also required to demonstrate the distribution, genetic diversity and potential risk of cross-species transmission of feline kobuvirus. ratification vote on taxonomic proposals to the international committee on taxonomy of viruses changes to taxonomy and the international code of virus classification and nomenclature ratified by the international committee on phylogeny and prevalence of kobuviruses in dogs and cats in the uk molecular characterization of the full kobuvirus genome in a cat genetic characteristics of the complete feline kobuvirus genome detection and genetic characterization of feline kobuviruses molecular evidence of kobuviruses in free-ranging red foxes (vulpes vulpes) molecular detection of kobuviruses in european roe deer (capreolus capreolus) in italy detection of feline kobuviruses in diarrhoeic cats membrane topography of the hydrophobic anchor sequence of poliovirus a and ab proteins and the functional effect of a/ ab membrane association upon rna replication sequence analysis reveals mosaic genome of aichi virus genetic characterization of porcine kobuvirus variants identified from healthy piglets in china characterization of a canine homolog of human aichivirus kobuviral non-structural a proteins act as molecular harnesses to hijack the host acbd protein rna-dependent rna polymerases from flaviviruses and picornaviridae prevalence and genomic characteristics of canine kobuvirus in southwest china first report and genetic characterization of feline 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prevalence of newly isolated, cytopathic small round virus (aichi strain) in japan isolation and characterization of a new species of kobuvirus associated with cattle aichi virus strains in children with gastroenteritis isolation and characterization of feline panleukopenia virus from a diarrheic monkey picornaviridae-the ever-growing virus family a novel porcine kobuvirus emerged in piglets with severe diarrhoea in china structure of human aichi virus and implications for receptor binding this study was supported by the research project of the national key research and development plan of china (grant no. yfd ). this study was also funded by national key r&d program of china (grant no. yfd ). jtn, ssy and hlw conceived and designed the experiments; jtn, ssy, hlw and ybg performed the experiments; jtn, ssy, ylz and hd analyzed the data; ybg, kw, sz and gxh contributed reagents/materials/analysis tools; jtn, ssy, hd and gxh drafted the manuscript; jtn, ssy, hd, xw and ylz revised the manuscript; hd and gxh supervised and approved the message for publication. all authors declare that they have no competing interests. key: cord- -u yrcto authors: jung, kyung sook; jang, yu mi; hwang, ji hye; park, gi jun; son, tae jong title: risk of water and food-borne communicable diseases in travelers entering korea date: - - journal: osong public health res perspect doi: . /j.phrp. . . . sha: doc_id: cord_uid: u yrcto objectives: it was supposed to analyze status and affecting factors in water and food-borne communicable disease by screening entrants with diarrhea symptom at the point of entry in korea methods: symptomatic travelers with water and food-borne communicable diseases who entered korea were diagnosed by a health declaration and detection of causative agents in water and food using laboratory tests. among those entered in , the affecting factors in the incidence of communicable diseases among those who had diarrhea at the entry into korea, were analyzed, with frequency and chi-square test. results: the number of travel entrants with gastrointestinal communicable diseases increased by . % from to . the percentage of causative agents of water and food-borne communicable diseases was the highest at . % from july to september. the rate of detection of causative agents of communicable disease pathogens in travelers from southeast asia entering korea was . %, which was higher than people arriving from east asia and central asia ( . %; p < . ). conclusion: the positive ratio of causative agents of water and food-borne communicable diseases was high among travelers that had entered korea from july to september, with a high number among entrants from southeast asia. based on the positive detection of causative agents, the entry period and countries visited were statistically significant affecting factors (p < . ). the major communicable diseases that have been reported to the korean centers for disease control and prevention (kcdc) are dengue fever, malaria, hepatitis a, shigellosis and zika virus [ ] . in recent times, the number of cases of shigellosis (a category in communicable diseases prevention act), is increasing. the incidence rate of shigellosis is high among travelers from southeast asia such as vietnam, philippines, and india [ ] . among cases of shigellosis reported to the kcdc in , cases ( . %) had been infected from abroad. this means that there has been a . -fold increase in cases from the cases of shigellosis reported in [ ] . the number of water and food-borne (wfb) communicable diseases (such as shigellosis, cholera, typhoid, paratyphoid, enterohemorrhagic e. there were cases reported, which was . -times higher than the number of cases reported in the previous year [ ] . using data from the quarantine information system in kcdc, this study surveyed the health status of travelers entered korea who were quarantined during to because they had symptoms of communicable disease. the incidence status and its related factors of overseas wfb communicable diseases were analyzed. in this study, cases of fever, diarrhea and respiratory symptoms among people quarantined within the period - were confirmed. causative agents of wfb communicable disease were determined using laboratory tests at the point of entry and related factors affecting gastrointestinal wfb communicable diseases were analyzed. table ). countries [ , ] . travelers' diarrhea is a symptom of eating infected food and to a lesser extent drinking unclean water [ , ] . globally, - % of travelers experience diarrhea during travel, and usually have bacterial pathogens such as e. coli, campylobacter jejuni, shigella spp., and salmonella spp. these are major causes of diarrhea symptoms in travelers [ , ] . the authors have no conflicts of interest to declare. korea immigration service statistics study on analysis of quarantine legislations and systems in australia for building an integrative quarantine system korea centers for disease control & prevention. typhoid fever, shigellosis during traveling southeast asia global epidemiology of diseases and travel medicine korea centers for disease control & prevention. standard operating procedure for overseas infectious disease in quarantine korea centers for disease control & prevention korea centers for disease control & prevention. yearbook of infectious disease surveillance korea centers for disease control & prevention. shigellosis during traveling to the philippines travelers' diarrhea: clinical manifestations, diagnosis, and treatment epidemiology of travelers' diarrhea: details of a global survey management of travellers' diarrhea traveler's diarrhea in foreign travelers in southeast asia: a cross-sectional survey study in traveler's diarrhea foreign travel-associated illness-a focus on travelers' diarrhea. london (uk): health protection agency detection of the causative agents of traveler's diarrhea using a real-time pcr screening method traveler's diarrhea clinical characteristics and etiology of traveler's diarrhea among korean travelers visiting south-east asia an epidemiological investigation on an outbreak of shigellosis during travelling cambodia and vietnam among korean rural people emerging infectious disease in southeast asia: regional challenges to control overview of travel clinic: focus on travel-related infectious disease key: cord- - hf qw authors: romette, j. l.; prat, c. m.; gould, e. a.; de lamballerie, x.; charrel, r.; coutard, b.; fooks, a. r.; bardsley, m.; carroll, m.; drosten, c.; drexler, j. f.; günther, s.; klempa, b.; pinschewer, d.; klimkait, t.; avsic-zupanc, t.; capobianchi, m. r.; dicaro, a.; ippolito, g.; nitsche, a.; koopmans, m.; reusken, c.; gorbalenya, a.; raoul, h.; bourhy, h.; mettenleiter, t.; reiche, s.; batten, c.; sabeta, c.; paweska, j. t.; eropkin, m.; zverev, v.; hu, z.; mac cullough, s.; mirazimi, a.; pradel, f.; lieutaud, p. title: the european virus archive goes global: a growing resource for research date: - - journal: antiviral research doi: . /j.antiviral. . . sha: doc_id: cord_uid: hf qw abstract the european virus archive (eva) was created in with funding from the fp -eu infrastructure programme, in response to the need for a coordinated and readily accessible collection of viruses that could be made available to academia, public health organisations and industry. within three years, it developed from a consortium of nine european laboratories to encompass associated partners in africa, russia, china, turkey, germany and italy. in , the h research and innovation framework programme (infras projects) provided support for the transformation of the eva from a european to a global organization (evag). the evag now operates as a non-profit consortium, with partners and associated partners from eu and non-eu countries. in this paper, we outline the structure, management and goals of the evag, to bring to the attention of researchers the wealth of products it can provide and to illustrate how end-users can gain access to these resources. organisations or individuals who would like to be considered as contributors are invited to contact the evag coordinator, jean-louis romette, at jean-louis.romette@univmed.fr. the european virus archive (eva) was created in with funding from the fp -eu infrastructure programme, in response to the need for a coordinated and readily accessible collection of viruses that could be made available to academia, public health organisations and industry (gould et al., ) . within three years, it developed from a consortium of nine european laboratories to encompass associated partners in africa, russia, china, turkey, germany and italy. following the h , call for research and innovation framework programme (infras projects) the concept of developing the eva into a global organization was a natural progression and the project was funded for a four-year period. in april , the evag started to operate as a non-profit consortium including partners and associated partners from eu and non-eu countries. based on excellent progress during the first two operational years, we have every reason to believe that the consortium will continue to expand and will ultimately constitute the world's largest virus collection. in this paper, we outline the structure, management and goals of the evag, to bring to the attention of researchers the wealth of products it can provide and to illustrate how end-users can gain access to these resources. laboratory isolation and characterisation of viral pathogens, such as vaccinia virus, poliovirus, foot and mouth disease virus, yellow fever virus and others, began at the turn of the twentieth century. this represented a pioneering era in the development of medical and veterinary virology. over the years, diagnostic and research centres were established worldwide to investigate viruses causing a wide range of diseases including those that had hitherto escaped all attempts to be identified and cultured under laboratory conditions. in the st century, ever-growing numbers of mammalian viruses continue to be isolated. they may represent only the tip of the iceberg, as more viruses are being discovered and reported using rapidly developing molecular technologies. as a result of these advances, the isolation, identification, characterisation and preservation of growing virus collections has inevitably generated new challenges. many of the known viruses are in collections that may be lost or discarded when the virologist in charge retires or when a laboratory closes or is reorganized. other collections may not be available to the wider virology community, because scientists are not always willing or in a position to share their viral isolates. they may lack the infrastructure, time (manpower), facilities and experience to characterize and present their collection in an accessible catalogue and further be able to ship their collected strains to foreign countries due to the restraints imposed by increasingly complex transportation regulations. furthermore, many governments have introduced additional security measures that specifically regulate the transfer and/or exchange, between countries, of high-risk-group pathogens that may attract the interest of terrorist organisations. additionally, laboratories in russia, china, india, southeast asia and south america hold their own virus collections, many of which, to date, have been relatively inaccessible to european laboratories. viruses from many taxonomic families have also been identified on the african continent and some well-developed laboratories in africa have made their collections readily accessible to researchers in europe and the americas. currently, however, except for a few countries, including senegal, south africa and kenya, authenticated virus collections from other regions of africa are virtually nonexistent. one objective of the evag therefore has been to establish trust among these widely disparate institutes and to associate the scientists in these countries with partners in the consortium under carefully monitored conditions that protect these developing laboratories, to the mutual benefit of everyone. the current project aims at the development of virus collections in low-income countries where, in some cases, there has been an understandable reluctance to exchange viruses for fear of losing their resource to institutes from more highly developed countries. the evag infrastructure provides assured access to modern capacities, thereby assisting these institutes to train their staff in developing their own resources and expanding and maintaining these capacities locally. end-user requests for viruses and reagents from these developing laboratories will be entirely managed from within, but supported by the quality management and distribution systems of the consortium. in common with all other members of the consortium, these developing laboratories will automatically retain full ownership of their collections. elsewhere, research laboratories often accumulate collections of viruses that are primarily dependent on the speciality of the laboratory scientists. for example, the american type culture collection (atcc) and the us centres for disease control and prevention (cdc) maintain large collections of mammalian viral pathogens, including risk group (rg ) agents. laboratories in russia, china, india, southeast asia, south africa, australia and new zealand hold collections that have been isolated specifically in their countries. relatively large collections of arboviruses are currently held in the usa (galveston national laboratory, texas), france, the czech republic, slovakia and also in some countries of scandinavia. european collections of specified animal pathogens, particularly lyssaviruses, pestiviruses, arboviruses and influenza viruses are maintained at the animal and plant health agency and at the pirbright institute in the united kingdom; in italy at the istituto zooprofilattico sperimentale delle venezie (izsve) hosting the world organization for animal health/food and agriculture organization (oie/fao) and eu reference laboratory for avian influenza; and in france at the institut pasteur. rg viruses such as ebola and nipah are maintained in specialised facilities in france, germany russia, the usa, china, south africa and the uk. diverse viruses of medical or veterinary importance (coronaviruses, herpesviruses, retroviruses, adenoviruses, enteroviruses, etc.) are held in laboratories around the world. as the evag continues to develop and to expand its boundaries, access to a greater proportion of these viruses will be enabled where possible. recently, in meeting the challenges of emerging viruses, the consortium has voluntarily oriented a part of its activities to the support of the world health organization (who) during emerging health crises by dedicating a new work-package entitled "response to emergence". moreover, the evag is currently an associated partner of the global outbreak alert and response network (goarn). current processes of coordinating virus standardisation, characterisation, preservation and distribution are inevitably relatively arbitrary, and are largely dependent on the speciality of each laboratory. in fact, besides the evag, we are unaware of any non-profit organization that is concerned with facilitating reliable access globally to viruses and associated reagents from individual virus collections for research and/or diagnostic laboratories, teaching centres or industries involved in the production of diagnostic reagents, pharmaceuticals and vaccines solely for the benefit of science, in a safe and carefully regulated manner. it would be virtually impossible to establish a single laboratory to maintain supplies of all recognised pathogenic mammalian viruses. based on the knowledge and experience gained from the former eva project, we have addressed these problems by using web-based tools to centralise access to quality-controlled, standardised preparations of viruses held in laboratories worldwide. the underlying concept is simple, but because virologists are naturally protective of their collections, its success has required a paradigm shift in the mind-set of the partners or associated partners in the individual laboratories. the h -infraia- - framework programme of the european union (eu) placed a call for funding applications under the heading "research and innovation action," with sub-headings, one of which was "research infrastructures." accordingly, the concept of the evag was created and a funding proposal was submitted. in preparing the proposal, it was emphasised that it would be virtually impossible to generate a wide-ranging virus archive in a single laboratory complex. thus, although many high-calibre virology laboratories operate in europe, it was decided to retain the "virology club tradition" by developing the principle of an integrated international infrastructure via consortia, operating through validated networks. as far as we are aware, this concept had not previously been encompassed by virologists in the context of producing and providing access to viruses. the evag was therefore conceived to fill the gaps in accessing viruses through identification of qualified international laboratories that could become partners of the consortium. it was agreed that by integrating their collections or resources and devising appropriate validated protocols and effective dissemination procedures, the partner laboratories would be able to achieve common high standards in producing and supplying authenticated viruses to the scientific community, both within and outside europe. the evag is unique. it was conceived to move beyond the current state of the art and provide access to the largest collection of mammalian viruses and associated reagents in the world to the global scientific community, to government health and environmental departments, to higher education institutes and to industry, through appropriate information systems. the new approach was, in the first instance, to integrate non-eu partners as beneficiaries of the project. this was possible because the rule of reciprocal benefit was agreed: the non-eu partner provision activity of material to an eu entity (research institute, health organization, animal health-care structure, etc.) was eligible for financial compensation from the consortium eu budget. consequently, highcalibre european and non-european partner laboratories agreed to become the primary members of the evag consortium. these are listed in table partner laboratories that form the core of the evag, listed in chronological order of their integration into the project. both medical and veterinarian institutes are included to cover viral zoonotic diseases. it also illustrates the wide geographic coverage already attained and demonstrates that bsl laboratories are major contributors to this consortium. the choice of institutes also reflects the specific available skills and know-how of scientists in institutes closely associated with the most well-known contemporary emerging viruses. we have also begun to integrate network organisations such as the global approach to biological research, infectious diseases and epidemics in low-income countries (gabriel) network, of the fondation merieux and the international pasteur institutes. future plans include expansion of partnerships to countries not currently represented, such as malaysia, vietnam, cambodia, indonesia, south america, and additional african countries. in addition, associate partners who share the same interest in the dissemination of viruses and reagents, but are not beneficiaries of the eu grant, have now been included (see table ). this association was formalized by the signature of a memorandum of understanding. the archive is founded on the principle that members and associate members of the consortium retain ownership of the viruses that they disseminate via the centralised distribution centre. the virologist or institution that supplies the viruses to the customer retains the ownership and enters into agreements with the customer via material transfer agreements (mtas). all orders for viruses and associated reagents (cell cultures, recombinant proteins, rna, dna, recombinant viruses, plasmids, monoclonal antibodies), are placed via the website https://www. european-virus-archive.com/which is managed centrally in marseille by the management team (partner# , aix-marseille university). during the early stages of the existence of the original eva, emphasis was placed on establishing the collections of viruses and associated reagents and ensuring that quality control was managed centrally. this ensured that all laboratories complied with identical standards. this principle has been further developed in the project. a manager was appointed to head up a management quality committee with the responsibility of establishing and maintaining appropriate standards through close monitoring of procedures, protocols and record maintenance. to ensure uniformity throughout the consortium, the quality managers representing each partner form the body of the committee. previously, non-eu participants were given the status of associate partners, i.e. they were associated with the consortium, but were not full members. the objective was to evaluate the feasibility of this approach concerning both the collaborative exchanges related to development of a common infrastructure and the operational effectiveness of resource distribution. from the point of view of scientific collaboration, the process was established rapidly and functioned efficiently. however, standardisation of the procedure for the distribution of materials to end-users proved to be challenging, and it is still being developed and improved within the current consortium. the reason for this slow progress is almost entirely due to the complexity of national regulations. whilst the european regulations retain the principle of being national and are enforced primarily to control the circulation of pathogens, they have been developed to maintain uniformity throughout the eu countries. thus, the administrative pathway to obtaining authorisation for an exchange of resources between, for example, france and germany or the netherlands and italy are very similar and are principally based on agreements provided by the national biosafety agencies. in contrast, the process of exchanging resources between the eu and russia or between the eu and the usa involves many more constraints: the intervention of different administrations in processing authorisation for delivery of hazardous goods including, biosafety agencies, customs administration, commerce and trade administration, ministry of health, etc. (also, the law in russia does not permit the import or export of pathogens.) nevertheless, our expertise in overcoming these hurdles is exemplified by the report of studies on the rg virus crimean-congo haemorrhagic fever virus in which the virus was supplied by a european partner to an end-user in the usa (haddock et al., ) . not surprisingly, when zika virus emerged in south america and was found to be responsible for microcephaly, we received numerous requests for zika virus, and many papers have been published with recognition of the evag as the source of virus. five examples encompassing, i) simultaneous transmission with chikungunya virus, ii) diagnosis, iii) signal processing pathways, iv) pathogenesis in relation to foetal infections and v) virus structure, are cited here (goertz et al., ; chan et al., ; zhang et al., ; miner et al., ; sirohi et al., ) . these anticipated problems of transporting pathogenic viruses between countries, particularly outside europe, were considered during the preparation of the project. each potential barrier to efficient exchanges between different countries was extensively discussed and product dissemination objectives for each non-eu partner were defined depending on their specific status. once established and operating satisfactorily, the management team continued the process of contacting high-calibre laboratories outside europe, organising meetings and conferences with these laboratories and subsequently inviting them to become associate partners. currently associate partnerships have been negotiated and agreed with laboratories in africa, russia, china, turkey, australia, japan, germany, italy, hungary, south korea, south east asia and south america ( table ). the initiation and early progress of the evag are already tremendous achievements, and subject to its continued success and expansion, it is anticipated that it will become the largest global network of laboratories contributing to the sharing of resources and promoting advances in virology. currently agreed associate partners in the evag. the integration of institutes that share the same interest in research and in the provision of high quality products to the scientific community is made through an association formalized by the signature of a memorandum of understanding. the choice of the associate partners has been established to fill gaps or to extend the list of viruses currently offered in the catalogue, and to have evag present in geographical areas concerned by emerging virus outbreaks. dissemination of resources including know-how and material to the scientific community is one of the major deliverable of the project. it has been addressed by the creation of web-based catalogue accessible on the evag portal. quality of the offer is a key factor to differentiate evag from the other infrastructures proposing similar products to the end-user. the gold standard products listed on the catalogue have been clearly at the origin of the fast growing dissemination activity of the consortium and of its robust reputation as reliable provider of key components for the detection of emerging viruses acting under the umbrella of international health organisations. the evag infrastructure provides wide-ranging and efficient access to virus collections held in laboratories worldwide, with the potential for access to recently isolated viruses from clinical, veterinary and field samples. the collection will continually expand, as the number of contributing laboratories increases and will provide participants with access to materials and capacities, including: • freeze-dried viruses for long-term storage; • viruses validated by sequencing, including the "rescue" and characterisation of archived material; • reagents and kits, including primer sequences and appropriate protocols for virus identification; • standardised diagnostic kits and recombinant proteins representing genes of the viruses, or hyperimmune antisera with defined specificities; • custom-prepared kits, proteins and antibodies; • high-quality associated research laboratories accessible to perform experiments and theoretical and practical training. relevant information can be obtained by placing an enquiry on the website (www.european-virus-archive.com/evag-portal). at the time of writing the catalogue shows more than products consisting of viruses and associated reagents. the list which is constantly increasing includes human, animal, insect, fish and plant viruses of medical, veterinary, academic, industrial and economic importance in addition, a range of arthropod tissue culture cell lines, monoclonal antibodies, recombinant protein expression systems, purified viral rna, specialised kits for diagnosis and custom-prepared proteins, can also be obtained through the website. the approach to quality management is directed by the project's own quality standard, based upon organization for economic cooperation and development (oecd) guidelines, and critically focusses on the acquisition, characterisation and storage of virus products. each partner institute seeks to comply with this way of working to harmonise laboratory processes across the consortium. the quality manager conducts "best practice" audits to quantify improvement progress. all facilities are inspected and regulated by their own national bio safety authorities, and employ staff trained in procedures compliant with the requirements of the international air transport association (iata) for the safe dispatch of potentially hazardous materials. trans-national access (tna) ensures free of charge access to european research infrastructures and the costs of the research, products and associated activities. this opportunity is open to all european researchers and to some extent to researchers from non-eu countries. tna dispatches include regulatory documentation such as import/export permits, and a certificate of analysis (coa) provided by the supplying partner. the evag project has established a grading system that defines virus product quality ranked from to , in which grade is the basic level, described as partially sequenced to confirm identity; not checked for mycoplasma contamination; unknown infectivity; and stored frozen. the "gold standard" is a fully sequenced virus, mycoplasma-free, with defined infectivity and lyophilised for long-term storage. derived products such as proteins, nucleic acids or antigens are also prepared using standardised protocols and checked for quality before storage or dispatch. security at the consortium institutes is maintained using cctv, pincoded passes and at remote sites, -h manned security/vehicle identification. entry into bio containment laboratories is strictly controlled. supply of products to the applicants operates via the tna approach defined above, whereby research groups apply through the web portal by submission of a completed request form. a successful application must be justified by an explanation of scientific objectives, techniques to be utilised/developed, list of named researchers, list of relevant publications and confirmation that appropriate expertise and facilities to handle the virus are in place. a review of these criteria is carried out by the project quality manager and a selection panel that includes external experts. a team of scientists and administrators is dedicated to the management of the evag, to ensure continuity and to maximize efficiency. whilst the individual laboratories operate through the web-based catalogue, they supply to the end-user only the viruses and products that are held in their collections. the website is the main communication tool with which to inform the end-user and to disseminate the products through the dedicated portal. the portal is conceived as a user-friendly marketplace that lists the viruses and associated reagents of each partner. this web-based catalogue constitutes a single entry point to the archived viruses and reagents. it provides access for end-users to place any enquiry about products of interest through a commonly used e-shop format. any end-user could be entitled to free access to the virus or associated reagent(s). the provider of the requested item(s) receives financial compensation from the eu budget. eu support for free-ofcharge access provided to the end-user has as its main objective to facilitate access of the scientific community to high quality resources. however, this is conditional on the excellence of the project which the end-user aims to develop using the resource. an international panel, consisting of independent experts, reviews the request for free access and subject to approval the customer will receive the item without charge, other than covering the cost of transportation. in the case of a charge being required to supply the virus or reagent, the evag only recovers the cost of production and transportation to the customer. free-of-charge access to end-users employed outside eu-member or euassociate states is limited to % of the total accesses provided in the grant. in addition to core partners, the project also integrates associate partners as an efficient means of enlarging the collection of viruses and reagents in the web-catalogue. easy and efficient access to the viruses and reagents and to the service offered to the scientific community is attracting increasing numbers of requests for the products in the catalogue. in addition, the quality of the products distributed and the assistance provided by the management team to answer specific questions from scientists are establishing the evag as a key biological resource centre worldwide. it is now a recognised entity acting under the umbrella of medical and veterinarian international health organisations which include the who, goarn, oie and the major centres for disease control and prevention around the world which underpin the control of emerging viral diseases. to place an enquiry and receive a quotation for a product, open a web browser and type in "evag." then click on login/register to create an account. the online catalogue can then be accessed via the portal link on the home page. products in the catalogue can be found by typing in a keyword/and/or the appropriate taxonomic name. the filters on the right hand side of the portal can also be used to browse through the products by category. the product(s) of interest should then simply be added to an enquiry cart, and the enquiry form should be completed. a material transfer agreement (mta) and a quotation for the products and shipping costs will then be sent to the end-user. the enquiry then becomes an order when the quote is accepted and the mta is signed. all enquiries are evaluated in terms of biosafety, and an official (signed and stamped) end-user qualification form of the laboratory to receive and handle the products of interest is requested. the emergence of sars at the end of has clearly demonstrated the lack of preparedness of countries to face emerging virus outbreaks. when the concept of eva was discussed in , this aspect of the consortium activity was clearly stated in the project submitted to the european commission. this was reinforced in the second project: evag, submitted in , with a contingency fund dedicated to support this activity. the emergence of the novel mers-cov in humans in saudi arabia in provided an opportunity for consortium partners and members of a european epidemic response network to develop a precise and rapid mers cov diagnostic tool, suitable for field diagnosis under emergency conditions (corman et al., ) . during december , the who recommended pcr assays as the method to detect the virus in blood samples, and identified the evag as a reliable source for the delivery of positive control reagents (who, ) . to date more than kits have been distributed worldwide, to laboratories in countries. this achievement demonstrated the capacity of the consortium to mobilize high-calibre scientists capable of rapidly developing and supplying a technical solution to a high-level emergency situation involving human pathogenic agents. another key feature of this achievement was the efficiency of our logistics platform to distribute the material worldwide under the demanding conditions prevailing at the time. zika virus emerged in french polynesia and spread rapidly to south america and the caribbean, causing at least , clinical cases, within the first year according to paho/who estimates. on the st of february , who declared a public health emergency of international concern (pheic) (see ref ). on the th of february, the us centre for disease control (uscdc) elevated its response to "level activation" i.e. the highest level. prior to may , there were two strains of zika virus in the evag online catalogue. by the first trimester of , at the peak of international concern, partners had added eight more products of direct relevance to the emergency situation. since november, , the evag has received enquiries for more than products to be distributed in countries. a total of products were provided in february, alone, with the highest demands in the usa, singapore, netherlands, germany, france and china. in its interim guidance for zika virus laboratory testing (march , ), who identified the evag as the provider for pcr quality-control material (corman et al., ) . this emergency situation and the resulting need for a coordinated distribution logistic was very challenging. consequently, the management team introduced procedures to alleviate the burden on partners and to improve product access. this was achieved by establishing additional distribution hubs for zika virus products obtainable from: the centre for molecular diagnostics and therapy (crie), moscow, russia; wuhan institute of virology (wiv), wuhan, southern china; national institute of infectious diseases (niid), tokyo, japan. the management team also negotiated a common material transfer agreement (mta), with industry on behalf of all consortium members and a centralised exchange logistics platform was established. this experience underlines the reality that anticipation and preparedness are key components for an effective response to public health emergencies. from december, through may, brazil reported its largest yellow fever (yf) outbreak in decades, with a total of suspected cases and deaths (fischer et al., ) , prompting widespread yellow fever virus (yfv) vaccination campaigns and the need to distinguish between vaccine-and wild-type yfv-associated disease. novel multiplex real-time reverse transcription pcrs that differentiate between vaccine and american wild-type yfv were developed by consortium partners in response to this outbreak and validated under field conditions (fischer et al., ) . centres for disease control (cdc) in countries play an important role for the control of virus outbreaks by the collecting data and samples during the identification phase of the pathogens. however the academic research combined with material collections appears to be an unavoidable actor for the preparedness and the control of an emerging virus disease, each time a variant form or a new form of a pathogen is responsible of it. combining, in the same organization, cdc and high calibre research laboratories was foreseen as the best solution to reach both an efficient control of the virus propagation and a preparedness for the next outbreak. at the same time, to cover the all range of pathogens from rg to rg was only made possible by the constitution of a group of high containment facilities within the organization. sixteen registered biosafety level- (bsl- ) laboratories, with the resources to handle the highest risk hg pathogens and five cdcs are partners or associate partners in the consortium. each cdc contributes directly to the management and control of emerging virus diseases, under the umbrella of the who. the evag is also associated with the global outbreak alert and response network (goarn) and is recognised by goarn as a supplier of high quality viruses and virusrelated reagents and resources. during the - ebola virus epidemics in west africa, all of the bsl- institutes were directly involved in helping local authorities to contain the epidemic. they all gained important experience in the management of such health crises, collecting field samples and isolated relevant virus strains many of which are now characterised and stored in the facilities of the evag bsl- partner at the bernard nocht institute in hamburg, germany. in addition, they addressed specific issues involving management of the exchange of high risk pathogens and activation of platforms dedicated to offering services to appropriate endusers. the consortium is now in its third year of existence under the h framework programme. its ultimate objective is to become a permanent archive that provides access to a very wide range of viruses and reagents globally. this will be achieved firstly, through extension of the funding arrangements, secondly through further increasing the range of contributors to the collection, and thirdly by bridging large european or international infrastructures having activities related to those of evag, including: it would be naive to suggest that the long-term survival of the consortium will be straightforward. for example, its perpetuation will require a continuous and increasingly large funding stream to meet the almost restrictive costs of research, laboratory development and upkeep in the face of newly emerging highly pathogenic viruses. sustainability is a key issue for such an infrastructure and the consortium plan will be to remain a non-profit organization. the obvious consequence of this fundamental choice is to have access to diversified sources of funding. the nations to which partners belong will have to support the collection activities including infrastructure management, as well as research concerning the development of new tools necessary to keep the archiving process up to date. the financial support for all the actions involved in preparedness for emerging viral diseases e.g., distribution of bio-resources to supply reagents to detect infected patients at local level will have to come from government health departments but at the international level, from the european commission, charitable international health funding agencies such as the wellcome trust foundation, the bill and melinda gates foundation, the who, the oie. future financial planning also includes the creation of a specific interface between the industrial sector and the consortium. in the first instance, this interface will explore bi-directional opportunities to organize scientific exchanges, priority access to bio-resources including reference material development of diagnostic services, validation of diagnostic assays and commercialization by industrial partners to the benefit of both parties. signed contracts of collaboration will be facilitated, and procedures harmonized. as noted above, the concept of the evag is unique. as far as we are aware, there is no equivalent viral archive, nor does any other collection provide the accessibility, reagent backup, sequence data, provenance, quality control, and capacity to advertise, inform, negotiate and conduct the entire transaction to the end-user via the web and internationally registered secure transportation companies the reputations, high quality, experience and knowledge of the consortium partners, combined with the integration of the european commission infrastructure, will provide endusers with opportunities to approach new fields of research in structural viral genomics, evolutionary biology, control of infectious diseases, antiviral drug design, fundamental research, public and environmental health, pathogenesis, immunology and a wide variety of associated disciplines. subsequently, subject to continuation of funding and the appropriate justification, the evag aims to extend the diversity of its disciplines to encompass fish, plant, bacterial, fungal, protist and other currently unrecognised viruses. this will be achieved first through expansion within the european community, followed by integration of specialist laboratories in the americas, russia, asia, southeast asia, australia and africa. the recently created african cdc will provide a unique opportunity to establish strong links with public health institutes in africa. the substance of this relationship will be based on trust and mutual benefit. for instance, training workshops and staff exchanges will be organized with the objective of developing appropriate infrastructures that can be directly integrated into the evag format and the levels of scientific expertise and quality and range of products that will justify their integration into evag as full partners within the consortium. the evag provides a range of viruses and relevant reagents to endusers engaged in a wide variety of established infrastructures. for instance, the newly created eu infrastructure, infravec (european infrastructures for insect disease vector research and control -https:// infravec .eu/) supplies infectious vectors (e.g. mosquitoes and ticks) to their end-users. the evag is closely associated with this activity, via the provision of viruses that can infect the vectors. erinha (http://www. erinha.eu/), a european strategy forum on research infrastructures (esfri www.esfri.eu/), offering access to high containment facilities to industrial end-users for their preclinical trials including bsl pathogens, recommends evag as the provider of the viruses necessary for their trials. bbmri, another esfri, plans to create a service unit dedicated to the provision of viruses and derived material from viruses. evag is identified as the recommended supplier. the evag contributes to the functioning of other eu infrastructures involved in related research topics including zoonotic diseases, drug discovery, vaccine development, viral epidemiology and emerging virus diseases. these eu-funded projects include prepare (platform for european preparedness against (re-) emerging epidemics -https://cordis. europa.eu/project/rcn/ _en.html), compare (collaborative management platform for detection and analyses of (re-) emerging and foodborne outbreaks in europe -http://www.compare-europe.eu/about), emerge (efficient response to highly dangerous and emerging pathogens at eu level -https://www.emerge.rki.eu/), zikalliance (a multinational and multidisciplinary research consortium coordinated by insermhttps://zikalliance.tghn.org/about/). as a future objective, one can imagine the generation of links among all of these organisations to constitute a "life science infrastructure cooperative" dedicated to virology-related topics, in which the evag could play a central role. for the long-term future, we are planning to establish closer collaborative links with other compatible scientific infrastructures by initiating teaching and training programmes amongst younger scientists to ensure that people of the right calibre will be able to undertake the roles currently occupied by experienced but "maturing" scientists. indeed, one could envisage e-learning programmes being built into diploma courses in universities. as we continue our ambitious mission to build a scientific infrastructure that can benefit scientists operating in all areas of the viral world, we recognise that other agencies and research teams can provide valuable datasets, and analytical expertise from which disease intervention strategies might be developed. indeed, other compatible agencies are approaching the consortium to share their resources and to promote their activities through the consortium website. this is yet another avenue that is being explored, based on the complementarities of these parties with the existing group of partners. at the time of writing, we are witnessing the massive impact of nextgeneration sequencing technology that will undoubtedly continue to evolve and provide even more discoveries, any one of which might then be exploitable by a future larger and more comprehensive form of agency. since the first recognition of viruses and our obsession with trying to understand and control them, it has been an exciting time for virologists. long may the evag and its successors ensure that this is always the case! this publication was supported by the european virus archive global (evag) project that has received funding from the european union's horizon research and innovation programme under grant agreement no . improved detection of zika virus rna in human and animal specimens by a novel, highly sensitive and specific real-time rt-pcr assay targeting the '-untranslated region of zika virus detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction assay optimization for molecular detection of zika virus lineage-specific real-time rt-pcr for yellow fever virus outbreak surveillance mosquito co-infection with zika and chikungunya virus allows simultaneous transmission without affecting vector competence of aedes aegypti the european virus archive: a new resource for virology research a cynomolgus macaque model for crimean-congo haemorrhagic fever zika virus infection during pregnancy in mice causes placental damage and fetal demise the . a resolution cryo-em structure of zika virus laboratory testing for middle east respiratory syncytial coronavirus: interim guidance a crispr screen defines a signal peptide processing pathway required by flaviviruses we wish to thank the following scientists for their expertise and contributions during the successful establishment and development of supplementary data related to this article can be found at https:// doi.org/ . /j.antiviral. . . . key: cord- -v uiuhih authors: lemmin, thomas; soto, cinque title: glycosylator: a python framework for the rapid modeling of glycans date: - - journal: bmc bioinformatics doi: . /s - - - sha: doc_id: cord_uid: v uiuhih background: carbohydrates are a class of large and diverse biomolecules, ranging from a simple monosaccharide to large multi-branching glycan structures. the covalent linkage of a carbohydrate to the nitrogen atom of an asparagine, a process referred to as n-linked glycosylation, plays an important role in the physiology of many living organisms. most software for glycan modeling on a personal desktop computer requires knowledge of molecular dynamics to interface with specialized programs such as charmm or amber. there are a number of popular web-based tools that are available for modeling glycans (e.g., glycam-web (http://https://dev.glycam.org/gp/) or glycosciences.db (http://www.glycosciences.de/)). however, these web-based tools are generally limited to a few canonical glycan conformations and do not allow the user to incorporate glycan modeling into their protein structure modeling workflow. results: here, we present glycosylator, a python framework for the identification, modeling and modification of glycans in protein structure that can be used directly in a python script through its application programming interface (api) or through its graphical user interface (gui). the gui provides a straightforward two-dimensional ( d) rendering of a glycoprotein that allows for a quick visual inspection of the glycosylation state of all the sequons on a protein structure. modeled glycans can be further refined by a genetic algorithm for removing clashes and sampling alternative conformations. glycosylator can also identify specific three-dimensional ( d) glycans on a protein structure using a library of predefined templates. conclusions: glycosylator was used to generate models of glycosylated protein without steric clashes. since the molecular topology is based on the charmm force field, new complex sugar moieties can be generated without modifying the internals of the code. glycosylator provides more functionality for analyzing and modeling glycans than any other available software or webserver at present. glycosylator will be a valuable tool for the glycoinformatics and biomolecular modeling communities. glycosylation is an important post-translational modification of proteins, where a carbohydrate is covalently attached by an enzyme to specific amino acids motifs known as sequons space [ ] [ ] [ ] [ ] . glycosylation has several principal structural and functional roles in biology, that include protein folding [ ] , tissue repair [ ] , and cell migration [ ] . in eukaryotes, nearly % of the proteome is believed to be glycosylated [ ] . more recently, glycosylation has been observed in bacteria where it has been associated with their virulence and the formation of biofilms [ ] . for viruses, such as hiv and influenza, glycosylation allows for evasion of the host's immune system [ , ] . thus, determining the role of glycan structure in biology is essential in order to understand pathogenesis. the diverse and dynamic nature of glycan structures makes it difficult to resolve their structure experimentally through traditional approaches (e.g., x-ray crystallography, cryogenic electron microscopy (cryo-em) or nuclear magnetic resonance (nmr)). computational methods, such as molecular dynamics (md) can help resolve glycan dynamics but this method is computationally intensive and cannot be used for the rapid modeling of glycan structure. complementary techniques that are more rapid and available through a graphical user interface (gui) should allow users to gain new insights into glycanprotein structure. in silico modeling of glycoprotein is a tedious and time consuming process and tools, such as carb-builder [ ] , polys [ ] , doglycans [ ] , sweet-ii [ ] , glycam-web [ ] , glycan reader [ , ] and charmm-gui glycan modeler [ ] were developed to facilitate the modeling of glycans. carbbuilder, polys and doglycans are open source programs that allow building glycan structures from their primary sequence of monosaccharide units. sweet-ii is part of the website glycosciences.db [ ] and can be used to build d structures of glycans. furthermore, the website provides a number of tools for manipulating and analyzing glycans. glycam-web offers several options that simplify the building and set-up of molecular dynamics simulations of glycoproteins. it uses the glycam force field [ ] that is compatible with the amber force field. finally, glycan reader recognizes most types of glycans and their chemical modifications found in the protein data bank (pdb), which are all available in the charmm force field [ ] . it also provides the option for editing their threedimensional structure. glycan modeler generates complex glycans and glycoconjugates by searching templates from a fragment database. glycan reader and modeler have both been integrated into charmm-gui [ ] , a powerful website widely employed for setting up molecular dynamics simulation. in addition, charmm-gui provides the functionality for modelling glycolipids and lipopolysaccharides (lps) and to combine them with complex biological membrane simulations [ ] . while many of these tools are available as webservers making them ideal for their ease of use and distribution, this limits their ability to be customized for the specific needs of some users; for example, for tasks that require batch modeling of several glycoforms for a given protein or adding non-canonical saccharides to a protein structure. we describe here glycosylator, a python framework designed for the rapid modeling of glycoprotein. it can be used directly in a python terminal or script to identify, manipulate, and build glycans. in addition, the gui allows for the quick visualization and modification of glycosylated proteins (such as one downloaded directly from the pdb). the molecular description of glycans is based on the charmm force field [ ] . new saccharides appearing in updated versions of the force field or defined by the user can easily be added. modeled glycans can be further refined by removing clashes and sampling alternate conformations. since glycosylator is distributed as a python package, users can easily adapt the code to meet their specific needs. the glycosylator framework is composed of classes, several of which can be employed as standalone instances for other applications in molecular modeling (additional file : figure s in the supporting information (si) section). at the core of glycosylator is the molecule class. a molecule is defined as a single covalently linked set of atoms and is implemented around the prody [ ] and networkx [ ] packages. prody is widely used for studying biomolecules and offers several functions for storing and manipulating structures. the functions and classes provided are used in the molecule class for saving and quickly accessing the structural data of a molecule. the topological properties of a molecule are represented here as a graph using the net-workx package. a molecule can be instantiated directly with a d structure (pdb) or using a moleculebuilder instance and the topology information provided for the charmm force field [ ] . when loading a glycoprotein, glycosylator will identify all oand n-linked sequons and their glycans. the structure and topology of each of the glycans can then be modified. clashes and alternative conformations for glycans can be optimized with the sampler class. finally, the graphical representation of glycans provided by the drawer class makes use of matplotlib [ ] , a python package used for plotting. taken together, glycosylator provides more functionality for analyzing and modeling glycans than many popular software packages and webservers ( table ). the main functions used for glycosylating a protein can be conveniently accessed through glycosylator's gui (additional file : figure s ). below, we briefly describe each class. detailed examples for the usage of each class are provided in the supporting information (additional file : example s ) section and on the github repository. charmm force field topology and parameter files are parsed using the charmmtopology and charmm-parameters classes, respectively. the data is stored in a dictionary for a quick and easy access. the charmm-topology class creates and stores an additional dictionary for looking up patches. the patches are used to define the glycosidic bonds between saccharide units and are required for modification (e.g., deleting atoms). the molecule class is used for storing the coordinates (prody's atomgroup) and connectivity (networkx graph) of a molecule. the bonds, angles and dihedrals are assigned either by the user or automatically based on the distances between atoms. the molecule's connectivity is saved as a directed graph. the user can provide the root atom to define the direction of the connectivity graph; by default, the first atom of the molecule is chosen. ring structures are automatically detected identifying all rotatable torsional angles that are not part of a cycle. these torsional angles can be measured, set to a specific value or rotated by a given amount. an interresidue graph is also built in order to quickly parse through a molecule composed of several residues. the moleculebuilder class is employed for building and editing molecules. information about the connectivity and atoms of a molecule are extracted from a charmmtopology instance. this class allows for the initialization of a prody residue (atomgroup). applying a patch (charmm) will modify one or several residues. for glycans, patches are typically used to define the glycosidic linkage. moleculebuilder interfaces directly with the prody atomgroup and returns all the information required for creating a molecule instance. glycosylator class was designed to deal specifically with glycans/glycoprotein. it can import a pdb file and automatically extract all the oand n-linked sequons and associated atoms. each glycan is saved as a molecule instance in a dictionary. the key of the dictionary is the residue number and chain of the sequon. glycosylator uses an internal text representation for storing a topology tree for each glycan structure. these trees describe the connectivity and saccharide units that compose a polysaccharide. a library of these structures can be imported into a glycosylator instance or saved as a simple text file or a sql database. glycosylator can then compare the extracted connectivity tree to the internal dataset of known glycans to identify them based on the glycosidic linkage and residue type. we do note that chemical post-modifications of glycans are not supported in the current version. glycans can be extended, trimmed or modeled ab initio. this can be achieved by providing the identification of a known oligosaccharide (in the library) or with a topology tree describing the connectivity and glycan units of the desired oligosaccharide. the topology tree is a string representation of a glycan. sampler class implements a genetic algorithm for removing clashes between molecules and their environment (e.g., protein). the charmm force field energy function for the torsional angles will be used for biasing table list of functionalities offered by the available software and webservers for modeling glycans. charmm-gui includes glycan reader and modelers, as well as the glycolipid and lps modelers the random number generator and to sample more energetically favorable torsional angles [ ] . the generation of the initial population can be skewed towards the common co-dependence of angles. the fast clash detection algorithm is based on k-d trees for intra-and interclashes of glycans. standard grid mapping is used for the detection of clashes between glycans and their environment. to reduce the search space, the genetic algorithm iteratively optimizes subsets of glycans with the highest number of steric clashes. drawer class is used for generating d symbolic representations of glycans according to the iupac standard. the inter-residue connectivity graph stored in a molecule is used for drawing the connectivity of a glycan. the protein is represented as a ribbon, each sequon is highlighted and the linked glycans are shown as a tree topology. the graphical representation is produced with matplotlib and can be further modified by the users (e.g., add text, rescale) and exported in various image formats. we compared the performance of glycosylator and doglycans, another python framework for modeling glycans using three representative viral envelope glycoproteins, each containing different numbers of glycosylation sites and overall glycan density. the glycans on the surface of these proteins create a shield that helps them to evade the host's immune system [ ] . for the benchmark, a mannose was modeled at each sequon, mimicking the glycosylation state before exiting the endoplasmic reticulum [ ] . the topology of the glycosylated structure was generated with the autopsf plug-in of vmd [ ] . each glycoprotein was then minimized with steps of conjugate gradient optimization in namd [ ] . the resulting energy-minimized model was then submitted for a sanity check to pdb-care (http:// www.glycosciences.de/tools/pdb-care/), a powerful tool that checks the connectivity and nomenclature in glycoproteins [ ] . we observed that all glycoproteins modeled with glycosylator had a lower potential energy and were devoid of any steric clashes and topological errors ( table ) . for structures with a low density of sequons, such as influenza's hemagglutinin, glycosylator and doglycans performed similarly. however, a simple minimization was insufficient for removing steric clashes from the hiv- envelope trimer and delta coronavirus spike protein structures using doglycans. the density of sequons at the surface of these glycoproteins is high, requiring a more effective strategy for removing clashes, such as provided by glycosylator's sampler class. the steric clashes present in the structures produced with doglycans lead topological errors, such as ring puckering after minimizations. in order to solve this issue, the torsional angles would have to be manually adjusted by the user. as an additional test case, we modeled the glycan shield of the hiv- env trimer using glycosylator. the hiv- env trimer consists of - sequons making it one of the most highly glycosylated proteins currently known. we chose the bg -sosip structure with pdb: id fyl, [ ] ) as the starting structure. first, all crystallographically-determined glycans were identified and hydrogenated (fig. , upper left triangle). the ribbon representation allowed for a quick visual inspection of the identified n-linked sequons and linked glycans. a combination of mannose , mannose and complex glycans was then modeled ab initio or by extending existing glycans to produce a more biologically relevant glycoform of the hiv- env trimer (fig. , lower right triangle) . the sampler function in glycosylator was then used to remove all major clashes, such that the table benchmark comparing glycosylator and doglycans. the average minimum distance between sequons was computed between the closest pairs of asparagine cα atoms. the number of issues accounts for errors in glycan connectivity and nomenclature due to steric clashes. the potential energy was calculated after steps of conjugate gradient energy minimization better performance are highlighted in bold topology of the full glycoprotein could be generated directly with the autopsf plug-in of vmd [ ] . the remaining clashes were quickly removed with steps of conjugate gradient energy minimization in namd [ ] . the resulting model was then submitted to the pdb-care server [ ] for a sanity check and we found no discrepancies in connectivity. the python script used for this example is available in the github repository. two additional examples for building and identifying glycans can be found in the supporting information section (additional file : examples s and s ). glycosylator is a versatile python framework for manipulating glycans and glycoproteins that facilitates the structural study of glycans. it will significantly improve the ability of the glycobiology community to model glycan structure without requiring advanced expertise in protein modeling or molecular dynamics. glycosylator has already been successfully used for several studies investigating the dynamics of glycans over long timescales ( ns to μs) [ ] [ ] [ ] . glycosylator is a valuable asset for glycoinformatics and biomolecular modeling communities. furthermore, it should be noted that glycosylator can also be used to model other polymers (d _polymer in github). project name: glycosylator. project home page: https://github.com/tlemmin/ glycosylator operating system(s): platform independent. programming language: python. license: mit. supplementary information accompanies this paper at https://doi.org/ . /s - - - . additional file : figure s . architecture of glycosylator, a python framework for the rapid modeling of glycans. each class is represented fig. identification, visualization and modeling of n-linked glycans onto the hiv- env trimer. protein surface representation of the highmannose glycoform of the hiv- env trimer (pdb id: fyl). crystallographically-determined glycans are shown in licorice representation. each subunit (gp and gp ) is represented as a ribbon with sequons indicated with gray squares and the n-linked glycans shown above or below the sequon (upper left triangular panel). glycosylator was used to produce a complex glycoform variant of the hiv- env trimer by modeling glycans ab initio or extending existing glycans (lower right triangular panel) by a hexagon. full circles connecting classes indicate a class that contains an instance of the previous one as an attribute, e.g. instances of molecule and moleculebuilder are attributes of glycosylator. several attributes from glycosylator can be directly shared with drawer and sampler (white squares). glycosylator can parse a pdb file of a glycoprotein and identify all the sequons (orange rhombus). the glycans (blue squares and green circles) will be extracted and saved as molecule instances. glycans at each sequon can then be built, modified or identified. figure s . glycosylator graphical user interface. a) the main window is used to import a pdb file of a glycoprotein. glycosylator will produce a symbolic representation (orange dashed line rectangle). a specific sequon can be selected in the right panel (purple dashed line rectangle). the glycan can be modified by clicking on the symbolic representation. b) the user can select a glycan from the common library or a library that they created. the selected glycan is highlighted with a red square. example s . building a glycan. the structure of an n-acetyl-d-glucosamine will be imported as a molecule instance. all missing atoms will be added according to the charmm force field. a second n-acetyl-d-glucosamine will then be linked through a - glycosidic bond. finally, an alpha-d-added according to the charmm force field. a second n-acetyl-d-glucosamine will then be linked through a - glycosidic bond. finally, an alpha-d-mannosewill be built ab initio and saved to a pdb file. example s . importing, identifying and editing of a glycan. the structure of a mannose will be imported as a molecule instance. a glycosylator instance will then identify it against a database of known structures. finally, the molecule will be trimmed down to a mannose . on the frequency of protein glycosylation, as deduced from analysis of the swiss-prot database unraveling the mechanism of protein n-glycosylation protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptides bonds sequence differences between glycosylated and non-glycosylated asn-x-thr/ser acceptor sites: implications for protein engineering glycosylation-directed quality control of protein folding glycosylation as a main regulator of growth and death factor receptors signaling cell migration-the role of integrin glycosylation similarities and differences in the glycosylation mechanisms in prokaryotes and eukaryotes insights into bacterial protein glycosylation in human microbiota playing hide and seek: how glycosylation of the influenza virus hemagglutinin can modulate the immune response to infection virus glycosylation: role in virulence and immune interactions implementation of glycanbuilder to draw a wide variety of ambiguous glycans a molecular builder for carbohydrates: application to polysaccharides and complex carbohydrates doglycans-tools for preparing carbohydrate structures for atomistic simulations of glycoproteins, glycolipids, and carbohydrate polymers for gromacs sweet -www-based rapid d construction of oligo-and polysaccharides glycam glycan reader: automated sugar identification and simulation preparation for carbohydrates and glycoproteins glycan reader is improved to recognize most sugar types and chemical modifications in the protein data bank charmm-gui glycan modeler for modeling and simulation of carbohydrates and glycoconjugates db: an annotated data collection linking glycomics and proteomics data glycam : a generalizable biomolecular force field. carbohydrates charmm additive all-atom force field for carbohydrate derivatives and its utility in polysaccharide and carbohydrate-protein modeling charmm-gui: a web-based graphical user interface for charmm charmm-gui membrane builder for complex biological membrane simulations with glycolipids and lipoglycans prody: protein dynamics inferred from theory and experiments exploring network structure, dynamics, and function using networkx matplotlib: a d graphics environment a bioinformatics view of glycan-virus interactions comprehensive glycoscience (from chemistry to systems biology) vmd: visual molecular dynamics scalable molecular dynamics with namd pdb-care (pdb carbohydrate residue check): a program to support annotation of complex carbohydrate structures in pdb files trimeric hiv- -env structures define glycan shields from clades a, b, and g insights from nmr spectroscopy into the conformational properties of man- and its recognition by two hiv binding proteins microsecond dynamics and network analysis of the hiv- sosip env trimer reveal collective behavior and conserved microdomains of the glycan shield publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank dr. james e. crowe for his support and comments on the manuscript, and dr. peter d. kwong and dr. gwo-yu chuang for their constructive feedback. authors' contributions tl and cs conceived of the idea; tl designed and wrote the glycosylator package; tl and cs wrote the paper. all authors read and approved the final manuscript. this work was supported by a grant from the human vaccines project. tl would like to acknowledge the support of the swiss national science foundation (grant p p pa_ ). the funding bodies human vaccines project and swiss national science foundation had no role in the design of the study; collection, analysis, and interpretation of data; or in writing the manuscript. glycosylator is available from the following github repository: https://github. com/tlemmin/glycosylator ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests. key: cord- -amg uz authors: keske, Şiran; gümüş, terman; köymen, tamer; sandıkçı, sunay; tabak, levet; ergönül, Önder title: human metapneumovirus infection: diagnostic impact of radiologic imaging date: - - journal: j med virol doi: . /jmv. sha: doc_id: cord_uid: amg uz background: human metapneumovirus (hmpv) is a recently detected virus, which can cause mild to severe respiratory tract infections. through this study, we aimed to detail the outcomes of hmpv infections. materials/methods: between january and november , patients who had hmpv detected in nasopharyngeal or bronchoalveolar lavage by molecular respiratory pathogen tests were evaluated. the food and drug administration cleared multiplexed‐polymerase chain reaction system (idaho technology, salt lake city, ut) was used for diagnosis. chest radiography (cr) and computed tomography (ct) were evaluated by an expert radiologist. results: in total patients were included, the mean age was . ( ‐ ) years, and % were male. the hospitalization rate was %. lower respiratory system infection (lrti) was diagnosed in patients with clinical findings, and in patients out of the radiological findings supported the diagnosis. the lrti rate was significantly higher in adults than children ( . %‐ . %; p = . ). in cr, peribronchovascular infiltration (pi) was the most common feature seen in out of patients and was generally bilateral ( out of patients). in ct imaging, ground‐glass opacity was the most common finding seen in out of patients and nodular consolidation in five patients. ribavirin was given to four patients, three of whom were severe and required respiratory support. none of the patients died of hmpv infection. conclusions: the ground‐glass opacity in ct was similar to other respiratory virus infections, and pi in cr was very common and typical; however, nodular consolidation that may mimic bacterial infection was seen in one‐fourth of ct. human metapneumovirus (hmpv) was defined in . it causes mild to severe respiratory system infections. [ ] [ ] [ ] due to the increasing availability of molecular based tests, reporting of hmpv infection is increasing all over the world. [ ] [ ] [ ] the hmpv has been reported to be one of the most commonly detected viruses that cause influenza-like illness and community-acquired pneumonia in both adults and children. radiologic findings of hmpv infection have not been reported to be different from other viral pathogens in studies with high sample sizes; however, it has been reported that studies including radiologic findings of hmpv infections were commonly done among immunocompromised hosts. hmpv infection may cause fatality in patients with hematologic malignancy. , the infection presents with a wide variation of clinical conditions from asymptomatic to fatal. we aimed to describe the clinical course of the disease by detailing the clinical and radiological findings and the outcome. we included patients from inpatient and outpatient departments who had hmpv in nasopharyngeal or bronchoalveolar lavage, diagnosed using the molecular respiratory pathogen test. the study was conducted in a -bed private hospital in istanbul between january and november . this was a retrospective study performed by reviewing the patient charts. the clinical features of the patients, demographic characteristics, chronic diseases including malignant disorders, diabetes mellitus, chronic kidney and liver diseases, and detailed information about antibiotic and antiviral drugs were collected by chart review. complete blood count, c-reactive protein, and liver and kidney function tests were studied among all patients. bacterial cultures (throat, sputum, blood, and urine) were obtained, and procalcitonin (pct) was studied in patients with suspicion of bacterial infection and/or in critical patients. chlamydia pneumoniae and mycoplasma pneumoniae were included in multiplex polymerase chain reaction (pcr) tests, and urine legionella antigen test was performed in case of suspicion. the food and drug administration cleared multiplexed-respiratory pcr system biofirefilmarray (idaho technology), which detects viral pathogens including hmpv, and three bacterial species were used for the molecular detection of hmpv. chest radiography (cr) and computed tomography (ct) were done if lower respiratory system infection (lrsi) was suspected. the radiological imaging of patients was evaluated by a single dedicated radiologist. the upper respiratory system infection (ursi) was defined based on the modified centor score that was developed for acute tonsillitis/ pharyngitis and deduced from the recommendation of the centers for disease control and prevention and infectious diseases society of america. , the lrsi is defined as infectious inflammation of the lower respiratory tract and refers to acute pneumonia, acute bronchitis, and acute bronchiolitis. clinical lrsi is a syndrome characterized by symptoms consistent with respiratory tract infection (such as fever, cough, sputum, and dyspnea) and lung auscultation findings (crackles, rhonchus, and decreased lung sounds). radiological lrsi is defined as radiological findings of the lung, including consolidation, cavitation, peribronchial ground-glass opacity (ggo), airspace consolidation, and small nodules. in the statistical analysis, the t test for continuous variables and the χ test for the comparison of categorical variables were used. in the analysis, stata (statacorp, college station, tx) was used, and p < . was set as significant. the institutional review board of koç university approved the study. our study included patients ( figure ). the mean age of the patients was . ( - ) years, and % of them were male (table ) . two-third ( %) of the patients were under years old. in patients, more than one virus was detected. concomitant agents were as follows: seven rhino/enterovirus, five influenza, two coronavirus, and one respiratory syncytial virus. the cases were most commonly seen between november and june, and % of the cases were seen in december ( the antibiotics were given in % of the patients, despite detection of virus. the rate of antibiotic prescription was higher among lrti when compared with ursi ( %- %; p = . ). the third generation cephalosporins, β-lactam/β-lactamase inhibitor combination, and quinolones were the most commonly used antibiotics ( %, %, and %, respectively). finally, ribavirin was given to four patients, three of whom were severe and required respiratory support. none of the patients died of hmpv infection. our study demonstrates the radiologic findings of hmpv infections in patients with lrsi (figures and ) . one of the most interesting radiologic findings was the presence of nodular consolidation in ct, which is generally expected to be seen in bacterial infection. the pi in cr and ggo in ct are the most common radiologic findings, which are frequently seen in viral pneumonia. in a recent review for radiologic imaging of viral agents that may cause pneumonia, the general radiologic findings of hmpv infections were followed as bilateral centrilobular nodules, ggo, and multilobar infiltrations; however, there was no information about nodular consolidation. in addition, they noted that the radiologic findings of hmpv infections were most commonly reported in patients with hematologic malignancy, but there were limited data on immunocompetent patients. the time from onset of symptoms to hospital admission may affect radiologic findings. in our study, among patients with normal cr, which were lrsi-based on their clinical findings, time from onset of symptoms to hospital admission was lower ( . to . days; p = . antibiotic prescription rate was still high, which was % among patients with lrsi. there is no confirmed antiviral treatment in hmpv infection. ribavirin use in hmpv infections is limited to case reports in immunocompromised patients. in a recent study, ribavirin use was not associated with a better clinical outcome. they reported that the duration of viral shedding was shorter in patients using ribavirin. a recent review also concluded that there is no evidence to recommend ribavirin in immunocompromised patients. the fourth european conference on infections in leukemia (ecil- ) guideline also commented that general recommendations for treatment of hmpv infection could not be made with current evidence. in our study, ribavirin was used in four cases, three of whom were severe and required respiratory support and none of them died. in conclusion, ggo in ct was similar to other respiratory virus infections and the pi in cr was very common and typical; 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sun, gengxin; chen, chih-cheng title: spread of infectious disease modeling and analysis of different factors on spread of infectious disease based on cellular automata date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: x qf yu infectious diseases are an important cause of human death. the study of the pathogenesis, spread regularity, and development trend of infectious diseases not only provides a theoretical basis for future research on infectious diseases, but also has practical guiding significance for the prevention and control of their spread. in this paper, a controlled differential equation and an objective function of infectious diseases were established by mathematical modeling. based on cellular automata theory and a compartmental model, the slirds (susceptible-latent-infected-recovered-dead-susceptible) model was constructed, a model which can better reflect the actual infectious process of infectious diseases. considering the spread of disease in different populations, the model combines population density, sex ratio, and age structure to set the evolution rules of the model. finally, on the basis of the slirds model, the complex spread process of pandemic influenza a (h n ) was simulated. the simulation results are similar to the macroscopic characteristics of pandemic influenza a (h n ) in real life, thus the accuracy and rationality of the slirds model are confirmed. infectious diseases are diseases that can be transmitted from person to person, from person to animal, or from animal to animal after proto-microorganisms and parasites infect human beings or animals [ ] [ ] [ ] . infectivity, epidemic, and uncertainty are the three main characteristics of infectious diseases. a thorough study of the spread causes, spread routes, spread processes, and epidemic laws of infectious diseases is the main method for effective prevention, control, and elimination of infectious diseases. at present, the mathematical study of infectious diseases is mainly based on the theory and method of infectious disease dynamics [ ] [ ] [ ] . the essence of infectious disease dynamics is to establish a mathematical model that can reflect the spread process, spread law, and spread trend of infectious diseases. its advantage is that, according to the characteristics of infectious diseases, the model of infectious diseases is reasonably assumed, the appropriate parameters are set, and the appropriate variables are selected. then, the dynamic characteristics of infectious diseases can be clearly revealed. it has laid a solid foundation for further analysis of the causes and key factors of the spread of infectious diseases, and for seeking the optimal strategies for the prevention and control of such diseases. the main method to study and forecast the spread mechanism of infectious diseases is to establish mathematical models. some of these are used to study the general laws of infectious diseases, while others are used to study specific infectious diseases, such as hfmd (hand foot and mouth disease), tuberculosis, aids, and so on. in , bernoulli [ ] began using mathematical models to study the spread of smallpox by vaccination. in , hamer [ ] constructed and analyzed a discrete time model for the study of recurrent measles epidemics. in , kermack and mckendrick [ ] proposed the sir (susceptible-infected-recovered) compartmental model for the first time in order to study the epidemic law of the black death prevailing in europe at that time. on the basis of the sir model's analysis, the "threshold theory" was proposed to distinguish the spread or regression of the disease. the validity of the sir model has been proven by the data regarding large-scale infectious diseases in history, thus the deterministic model [ ] based on a differential equation has been widely accepted. with the deepening of research, the factors involved in establishing the mathematical model are increasing, and the dimension of the model is also increasing. based on the classical sir model, aron and schwartz [ ] proposed the seir(susceptible-exposed-infective-recovered) model in . that model considers that the latency of infectious diseases also has an impact on infectious diseases, so the model is more realistic. focusing on severe acute respiratory syndrome (sars) infectious disease spread in recent years, safi and gumel [ ] constructed the seqijr(susceptible-exposed-quarantine-infective-isolation-recovered) model according to the characteristics of sars. small and chi [ ] studied the effects of vaccination and isolation on the sars epidemic and constructed the seirp (susceptible-exposed-infective-recovered-persevered) model. in addition, some researchers start with the population structure of infectious disease spread and study the spread model of infectious disease. according to the influence of age on the spread of infectious diseases, boklund et al. [ ] proposed a model to better characterize the effect of age heterogeneity on the spread of infectious diseases. according to the difference in population, meng et al. [ ] divided the population into different groups, and they proved the global stability of disease-free equilibrium and endemic equilibrium. with the development of artificial intelligence, the network dynamics model has gradually become a new research method of infectious disease model. the most common network dynamics models include the ordinary differential equation model, the discrete differential equation model, the impulsive differential equation model, and the differential equation model with time delay. the main methods are finite equation theory, matrix theory, bifurcation theory, k-order monotone system theory, central manifold theory, lasalle invariant principle, etc. however, these research methods are all theoretical studies on infectious diseases, but it is difficult to apply them to practical problems. the concept of cellular automata was proposed in the s. cellular automata can be described as a dynamic system consisting of a transformation function in cellular space, which is discrete in time and space. the cellular automata model can be formally expressed as ca = (l c , s, m, f ), where ca represents the cellular automaton system, l c represents the mesh space which is divided according to given rules, a mesh corresponding to a cell of cellular automata, s represents the set of cell states, m = (c , c , . . . , c n ) represents the set of current cell adjacent to cells, and f represents the transformation function which can transform c n to c, the function that can calculate the state of cell c at t + time according to the state of adjacent cells of cell c at t time. adjacent cells are moore neighbors with radius = . as shown in figure , cells can move in eight directions. misra et al. [ ] gave a comprehensive introduction to the theory and application of cellular automata. since the s, epidemic spread models based on cellular automata [ ] [ ] [ ] [ ] have been extensively studied. according to the characteristics and mechanism of aids, pan et al. [ ] proposed a spread model of aids based on cellular automata. lópez et al. [ ] proposed an epidemic spread misra et al. [ ] gave a comprehensive introduction to the theory and application of cellular automata. since the s, epidemic spread models based on cellular automata [ ] [ ] [ ] [ ] have been extensively studied. according to the characteristics and mechanism of aids, pan et al. [ ] proposed a spread model of aids based on cellular automata. lópez et al. [ ] proposed an epidemic spread model based on cellular automata that considers individual heterogeneity, population mobility ratio, and individual maximum moving distance. because cellular automata can perform some experiments that cannot be done in real life by modeling, we can analyze the actual situation and obtain the results, in order to solve the complex problems that cannot be dealt with in the deterministic model. it is thus becoming a typical representative of the network dynamics model. based on the ability of cellular automata to model complex problems, this paper considered that, in real society, population mobility is caused by economic development, living environment, education level, and other factors, and that population density, sex ratio, and age structure of area also have some influence on the spread of infectious diseases. an epidemic spread model susceptible-latent-infected-recovered-dead-susceptible (slirds) based on cellular automata was therefore established. the contributions of our research are as follows: • a more realistic epidemic spread model based on cellular automata was established and achieved good results in simulation experiments. the effects of population density, sex ratio, and age structure on the spread of infectious diseases were discussed, and the simulation results were analyzed to observe the effects of the above three factors on the spread process of infectious diseases. the suggestions given in this paper based on the three influencing factors provided strong support for researchers to study the spread process of infectious diseases in different environments. the rest of this paper is organized as follows. the methodology of our research is introduced in section . simulation results and analysis are given in section . discussions are presented in section . finally, conclusions are given in section . on the basis of the sir and sis (susceptible-infected-susceptible) models, the state of the population was divided into susceptible, latent, infected, recovered, and dead. the total number of members of the population is denoted as n(t). s(t) represents susceptible population, meaning the number of members of the population who are not infected but are susceptible to infection at time t. latent population is denoted as l(t), meaning the number of members of the population infected at time t but not yet affected, and at this time the individual is not infectious. infected population is denoted as i(t), meaning the number of members of the population who are infected and have infectivity at time t. recovered population is denoted as r(t), meaning the number of members of the population who are immune at t time and will not be infected for a certain period of time. dead population is denoted as d(t), meaning the number of members of the population who died of infectious diseases at time t, and individuals are not infectious at the moment. the slirds model can be described through the following differential equation models: where δ represents the proportion of the population who lost immunity to the infectious disease, β is the ratio coefficient of the infection rate, ω is the latency ratio coefficient of the infectious disease, γ is the ratio coefficient of the recovered infected population, and i o and s o represent the ratio of infected and susceptible individuals in the initial population, respectively. the transition relationships of states in the slirds model are shown in figure . where represents the proportion of the population who lost immunity to the infectious disease, is the ratio coefficient of the infection rate, ω is the latency ratio coefficient of the infectious disease, is the ratio coefficient of the recovered infected population, and and represent the ratio of infected and susceptible individuals in the initial population, respectively. the transition relationships of states in the slirds model are shown in figure . in order to simulate the phenomenon of crowd movement in the real world, this paper introduced the idea of random walk cellular automata to simulate individual movement in the crowd. considering the limitation of individual movement, the maximum step length l is set for individual movement. at the same time, considering the individual activity m, all individuals are scanned randomly in each time step, and the individuals whose proportion is m are selected. and ( | |, ≤ ) are chosen randomly for each selected individual ( , ) , and then ( , ) and ( , ) are exchanged to complete the individual movement. in this paper, the slirds epidemic model based on cellular automata is proposed. assuming that the environment of the crowd is a regular = × mesh space, a sparse matrix whose density is = ⁄ ( is the set of individuals) is generated randomly. each non-zero element of the matrix represents an effective individual. represents the neighbor set of cellular nodes, and it uses a moore neighbor with radius = . ( , ) ( ) = , , , , is used to represent the cell state in the i-th row and the j-th column at time t. different values represent different states as follows: in order to simulate the phenomenon of crowd movement in the real world, this paper introduced the idea of random walk cellular automata to simulate individual movement in the crowd. considering the limitation of individual movement, the maximum step length l is set for individual movement. at the same time, considering the individual activity m, all individuals are scanned randomly in each time step, and the individuals whose proportion is m are selected. d i and d j (|d i |, d j ≤ l) are chosen randomly for each selected individual c (i,j) , and then c (i,j) and c (i+d i ,j+d j ) are exchanged to complete the individual movement. in this paper, the slirds epidemic model based on cellular automata is proposed. assuming that the environment of the crowd is a regular n = n × n mesh space, a sparse matrix whose density is ρ = c n /n (c n is the set of individuals) is generated randomly. each non-zero element of the matrix represents an effective individual. m represents the neighbor set of cellular nodes, and it uses a moore neighbor with radius = . s (i,j) (t) = { , , , , } is used to represent the cell state in the i-th row and the j-th column at time t. different values represent different states as follows: s (i,j) (t) = represents susceptible state, meaning that individuals are not infected and they are immune to this infectious disease; s (i,j) (t) = represents latent state, meaning that individuals have been infected, but they do not have infectivity; s (i,j) (t) = represents infected state, meaning that individuals are infected and infectious; s (i,j) (t) = represents recovered state, meaning that individuals have recovered and acquired immunity within a certain period of time; s (i,j) (t) = represents dead state, meaning that individuals are dead and they do not have infectivity. because cellular automata cannot reflect every individual's and their neighbors' randomness, unified parameters t , t , and t are introduced, t representing the maximum peak of latency time for each individual, t representing the maximum peak of illness time for each individual, and t representing the maximum peak of immunization time for each individual. t s (i,j) (t) represents latency time of the individual, t s (i,j) (t) represents illness time of the individual, and t s (i,j) (t) represents immunization time of the individual. because of heterogeneity among individuals, each individual shows different resistance, infectivity, and infectious range to disease. this paper considers the effects of population density, sex ratio, and age structure on infectious disease spread in the population, and discusses the influence of different factors on infectious disease spread. in real life, because there are differences in climate, economy, education, and medical treatment, the population is not divided by rules like cellular automata. for example, in china, the population density in the southeast coastal areas is greater than that in the northwest. in addition, because of the different distribution of business districts, schools, and hospitals, the distribution of population in the same city is not uniform. in areas with a large population density, the distance between individuals is shorter and the spread range of individuals is wider. individuals in the population have higher contact frequency and more neighbors around them, so their infectivity and the probability of being infected also increase. in order to study and analyze the influence of population density on infectious disease spread, each individual is mapped into a cell in the cellular automata model. when there is no individual and the individual is in dead state in a cell, they are not infectious. in order to simulate the difference in population density, the population density can be simulated by setting the value of d(t) in the initial state. at this time, d(t) does not represent the number of dead individuals, but represents that there is no individual in the cell. in this paper, a sparse matrix was used to simulate the random distribution of population and the infectious disease spread, and then the trend of infectious disease spread under different population densities as well as the influence of different population densities on infectious disease spread were analyzed. due to the influence of economic development and other factors, the population ratio and age structure in different regions are also different. for example, young and middle-aged people in remote mountainous areas go to work in big cities, resulting in a large number of old and young people in the original area. in areas where labor is scarce, such as coal mines and crude oil mining areas, there is an imbalance in the proportion of men to women. therefore, it is of great practical significance to study the influence of sex ratio and age structure on infectious disease spread. in real life, because of different living environments, living habits, resistance levels to viruses, infectious abilities to diseases, levels of drug resistance, and spread ranges, and in order to simulate the spread mechanism of infectious diseases more accurately, it is particularly important to consider individual heterogeneity, establish an infectious disease spread model, and further analyze and predict the spread mechanism of the epidemic situation. in this paper, the probability of infection p (i,j) (t) was used to describe individual heterogeneity. individual heterogeneity is determined by the individual's resistance to disease and the infectivity of neighbor cells. the state of neighbor cells of cell c (i,j) at (i, j) can be expressed by an adjacency matrix as follows: after three times spread, its adjacency matrix is defined as follows: the infection rate of cell c (i,j) at time t is defined as follows: because the probability of infection is inversely proportional to one's own resistance, it is proportional to the infectivity of one's neighbors. thus, it can be expressed as follows: where f c (i,j) ,c(k,l) represents the infectivity of cell c (k,l) to cell c (i,j) (because of the difference in the constitution of different individuals, they have different infectivity and resistance) and f c (i,j) ,c(k,l) obeys ( , ) uniform distribution [ ] . r c (i,j) represents the infectious disease resistance of cell c (i,j) . some diseases have different influences on different sex and age groups, that is, individual sex and age differences are also important factors affecting an individual's resistance to disease. thus, it can be expressed as follows: where g m and g f represent the proportion of males and females in the population, f m and f f represent the influence coefficient of infectious diseases on males and females, y , . . . , y n are the proportion of various age groups in the population, f , . . . , f n are the influence coefficient of infectious diseases on n groups of populations, and t c (i,j) obeys ( , ) uniform distribution [ ] . the infection probability of each individual is determined by its own resistance to infectious diseases and the infectivity of its neighbors. at each time step, the individual state is updated synchronously. according to a given population density, the sparse matrix is generated to simulate the distribution of population, and then through the age structure and sex ratio, each individual sets their attribute values. the initial state of all individuals is set to s = , the state of the infected individuals is set to s = . individuals in cells are updated according to the following rules: ( ) when s (i,j) (t) = , individual infection probability p (i,j) (t) is calculated, and then whether the individual will be transformed into s (i,j) (t) = is determined. otherwise, s (i,j) (t) = . meanwhile, individual latency time t s (i,j) (t) = t s (i,j) (t) + . ( ) when s (i,j) (t) = , when t s (i,j) (t) < t , s (i,j) (t + ) = . otherwise, s (i,j) (t + ) = . meanwhile, individual illness time t s (i,j) (t) = t s (i,j) (t) + . ( ) when s (i,j) (t) = , when t s (i,j) (t) < t , s (i,j) (t + ) = . otherwise, the individual enters into dead state with probability λ, and s (i,j) (t + ) = ; the rest of individuals have recovered and acquired immunity, and s (i,j) (t + ) = . meanwhile, individual immunization time t s (i,j) (t) = t s (i,j) (t) + . ( ) when s (i,j) (t) = , when t s (i,j) (t) ≥ t , individual immunity to the infectious disease disappears with probability δ. the individual then turns into susceptible state, s (i,j) (t) = . ( ) at each time step, all individuals move. without considering other factors, this paper focused on the influence of three factors, namely, population density, individual heterogeneity, and mobility on infectious disease spread, and the slirds model based on cellular automata was constructed. in order to verify the validity of the model, this paper took pandemic influenza a (h n ) as an example to simulate the spread process of pandemic influenza a (h n ). in this paper, we used matlab simulation software (r b, mathworks, natick, ma, usa) to carry out simulation experiments; the simulated curves are realizations of the average from all simulations. according to the latent and infectious characteristics of pandemic influenza a (h n ), the time step of simulation is in days, and the total time is set to t = . the simulated initial number of members of the infected population was consistent with the actual number of members of the infected population, and we assumed that the proportion of the initial latent population was . %. first, the number of members of the infected population in the slirds model simulation experiments was compared with the actual data of pandemic influenza a (h n ) in beijing in mainland china (june-july ) [ ] . the comparison results are shown in figure . ( ) when ( , ) ( ) = , when ( ( , ) ( )) < , ( , ) ( + ) = . otherwise, ( , ) ( + ) = . meanwhile, individual illness time ( ( , ) ( )) = ( ( , ) ( )) + . ( ) when ( , ) ( ) = , when ( ( , ) ( )) < , ( , ) ( + ) = . otherwise, the individual enters into dead state with probability , and ( , ) ( + ) = ; the rest of individuals have recovered and acquired immunity, and ( , ) ( + ) = . meanwhile, individual immunization time ( ( , ) ( )) = ( ( , ) ( )) + . ( ) when ( , ) ( ) = , when ( ( , ) ( )) ≥ , individual immunity to the infectious disease disappears with probability . the individual then turns into susceptible state, ( , ) ( ) = . ( ) at each time step, all individuals move. without considering other factors, this paper focused on the influence of three factors, namely, population density, individual heterogeneity, and mobility on infectious disease spread, and the slirds model based on cellular automata was constructed. in order to verify the validity of the model, this paper took pandemic influenza a (h n ) as an example to simulate the spread process of pandemic influenza a (h n ). in this paper, we used matlab simulation software (r b, mathworks, natick, ma, usa) to carry out simulation experiments; the simulated curves are realizations of the average from all simulations. according to the latent and infectious characteristics of pandemic influenza a (h n ), the time step of simulation is in days, and the total time is set to t = . the simulated initial number of members of the infected population was consistent with the actual number of members of the infected population, and we assumed that the proportion of the initial latent population was . %. first, the number of members of the infected population in the slirds model simulation experiments was compared with the actual data of pandemic influenza a (h n ) in beijing in mainland china (june-july ) [ ] . the comparison results are shown in figure . in figure , the abscissa is the time step of simulation and the ordinate is the number of infected individuals. the correlation coefficient of the two sets of data is . by t-test. it shows that the simulation results are close to the actual data and that the model is reasonable and effective. in figure , the abscissa is the time step of simulation and the ordinate is the number of infected individuals. the correlation coefficient of the two sets of data is . by t-test. it shows that the simulation results are close to the actual data and that the model is reasonable and effective. all things being equal, the parameters of two simulations for the slirds model were set as follows: in figure , considering the difference in population base, the number of members of the population that died, were susceptible, were infected, and were immunized was replaced by death, susceptibility, infection and immunization rates to describe the changes in population in different states. from the death rate curve in figure a , it can be seen that the death rate increases with the increase in figure , considering the difference in population base, the number of members of the population that died, were susceptible, were infected, and were immunized was replaced by death, susceptibility, infection and immunization rates to describe the changes in population in different states. from the death rate curve in figure a , it can be seen that the death rate increases with the increase of population density, but the overall trend is rising and tending to be stable. from the susceptibility rate curve in figure b , it can be seen that the change in population density has little influence on the susceptible population, and the susceptibility will first decrease and then reach a stable value when the population density is large. from the infection rate curve in figure c , it can be seen that the change in population density has little influence on the infected population. when the population density is large, the number of members of the infected population is greater, but the general trend is rising first, then falling, and finally tends to be stable. from the immunization rate curve in figure d , it can be seen that the change in population density has little influence on the immune population. when the population density is large, the immunity first rises and then reaches a stable value. according to the above analysis, it is known that when the population density is large, the spread rate of infectious diseases is faster. all things being equal, the ratio of males to females was : . the parameters of two simulations for the slirds model were set as follows: ( ) the influence coefficients of infectious disease on males and females were . and . , respectively. ( ) the influence coefficients of infectious disease on males and females were . and . , respectively. two simulation results are shown in figure . as shown in figure a , we can see that when infectious diseases have a greater influence on males, the number of deaths is higher, but the overall trend is rising and gradually stable. as shown in figure b , we can see that infectious diseases have less influence on susceptible population under different influence coefficients; when infectious diseases have a greater influence on females, the number of members of the susceptible population first decreases and then reaches a stable value. as shown in figure c , we can see that infectious diseases have less influence on infected population under different influence coefficients; when infectious diseases have a greater influence on males, the number of members of the susceptible population first decreases and then reaches a stable value. however, the overall trend is first rising and then falling, and finally tends to be stable. as shown in figure d , we can see that infectious diseases have less influence on recovered population under different influence coefficients; when infectious diseases have greater influence on males, the number of members of the susceptible population increases first, and then reaches a stable value. according to the above analysis, it is known that in cities with more males than females, when the infectious disease has a great influence on males, infectious diseases have a greater influence on the population because of the large population base of males. similarly, there are corresponding phenomena in cities with more females than males. ( ) the influence coefficients of infectious disease on males and females were . and . , respectively. ( ) the influence coefficients of infectious disease on males and females were . and . , respectively. two simulation results are shown in figure . as shown in figure a , we can see that when infectious diseases have a greater influence on males, the number of deaths is higher, but the overall trend is rising and gradually stable. as shown in figure b , we can see that infectious diseases have less influence on susceptible population under different influence coefficients; when infectious diseases have a greater influence on females, the number of members of the susceptible population first decreases and then reaches a stable according to related materials [ ] , the influence coefficient of pandemic influenza a (h n ) on males and females is very different. in simulation experiments, the influence coefficients of pandemic influenza a (h n ) on males and females were set to g m = . , g f = . , respectively. the parameters of two simulations for the slirds model were set as follows: ( ) the ratio of males to females is : . ( ) the ratio of males to females is : . two simulation results are shown in figure . from the death curve in figure a , we can see that when the number of males is large, the number of deaths is higher, but the overall trend is rising and gradually stable. from the susceptibility curve in figure b , we can see that under different sex ratios, infectious diseases have little influence on susceptible population. when the number of females is large, the number of members of the susceptible population first decreases and then reaches a stable value. from the infection curve in figure c , we can see that under different sex ratios, infectious diseases have little influence on the infected population. when the number of males is large, the number of members of the infected population decreases first and then reaches a stable value. from the immunization curve in figure d , we can see that under different sex ratios, infectious diseases have little influence on the immunization population. when the number of males is large, the number of members of the immunization population increases first, and then reaches a stable value. according to related materials [ ] , the influence coefficient of pandemic influenza a (h n ) on males and females is very different. in simulation experiments, the influence coefficients of pandemic influenza a (h n ) on males and females were set to = . , = . , respectively. the parameters of two simulations for the slirds model were set as follows: ( ) the ratio of males to females is : . ( ) the ratio of males to females is : . two simulation results are shown in figure . from the death curve in figure a , we can see that when the number of males is large, the number of deaths is higher, but the overall trend is rising and gradually stable. from the susceptibility curve in figure b , we can see that under different sex ratios, infectious diseases have little influence on susceptible population. when the number of females is large, the number of members of the susceptible population first decreases and then reaches a stable value. from the infection curve in figure c , we can see that under different sex ratios, infectious diseases have little influence on the infected population. when the number of males is large, the number of members of the infected population decreases first and then reaches a stable value. from the immunization curve in figure d , we can see that under different sex ratios, infectious diseases have little influence on the immunization population. when the number of males is large, the number of members of the immunization population increases first, and then reaches a stable value. according to the above analysis, it is known that when the number of males is larger in the cities where infectious diseases affect men more, infectious diseases have a greater influence on the population. due to factors such as mobility and spatial environment, age structure of the population presents different distributions. the age structure of a city can be divided into three types: young, adult, and aged according to the proportion of children, adolescents, youth, middle-aged people, and elderly people. according to the above analysis, it is known that when the number of males is larger in the cities where infectious diseases affect men more, infectious diseases have a greater influence on the population. due to factors such as mobility and spatial environment, age structure of the population presents different distributions. the age structure of a city can be divided into three types: young, adult, and aged according to the proportion of children, adolescents, youth, middle-aged people, and elderly people. according to related materials [ ] , the influence coefficient of pandemic influenza a (h n ) on children, adolescents, youth, middle-aged people, and elderly people is very different. in simulation experiments, the influence coefficients of pandemic influenza a (h n ) on children, adolescents, youth, middle-aged people, and elderly people were set to f = . , f = . , f = . , f = . , f = . , respectively. all things being equal, the parameters of three simulations for the slirds model were set as follows: figure . the death, susceptibility, infection, and immunization curves are shown in figure a -d, respectively. it can be seen that the number of deaths in the aged cities is the largest. the number of young urban deaths is only inferior to that of the aged cities, whereas the number of deaths in the adult cities is the least. however, the overall trend of change is gradually stable after rising for all types of cities. the difference in age structure of the population has little influence on the susceptible population, and the number of members of the susceptible population in adult cities decreases first and then reaches a stable value. the difference in age structure of the population has little influence on the infected population. the number of members of the infected population in the aged cities is the highest, but the general trend is rising first and then decreasing for all types of cities. the difference in age structure of the population has little influence on the immunization population, and the number of immune individuals in the adult city rises first to then reach a stable level. according to the above analysis, it is known that infectious diseases spread more slowly in adult cities than in aged and young cities, but the resistance of young cities to infectious diseases is slightly greater than that of aged cities. according to related materials [ ] , the influence coefficient of pandemic influenza a (h n ) on children, adolescents, youth, middle-aged people, and elderly people is very different. in simulation experiments, the influence coefficients of pandemic influenza a (h n ) on children, adolescents, youth, middle-aged people, and elderly people were set to = . , = . , = . , = . , = . , respectively. all things being equal, the parameters of three simulations for the slirds model were set as follows: ( figure . the death, susceptibility, infection, and immunization curves are shown in figure a -d, respectively. it can be seen that the number of deaths in the aged cities is the largest. the number of young urban deaths is only inferior to that of the aged cities, whereas the number of deaths in the adult cities is the least. however, the overall trend of change is gradually stable after rising for all types of cities. the difference in age structure of the population has little influence on the susceptible population, and the number of members of the susceptible population in adult cities decreases first and then reaches a stable value. the difference in age structure of the population has little influence on the infected population. the number of members of the infected population in the aged cities is the highest, but the general trend is rising first and then decreasing for all types of cities. the difference in age structure of the population has little influence on the immunization population, and the number of immune individuals in the adult city rises first to then reach a stable level. according to the above analysis, it is known that infectious diseases spread more slowly in adult cities than in aged and young cities, but the resistance of young cities to infectious diseases is slightly greater than that of aged cities. in this paper, we used the idea of a sparse matrix to add population density, sex ratio, and age structure factors into the slirds model. population density was set to and . , respectively. all things being equal, with the increase of population density, infectious diseases spread faster, and infectious diseases have a greater influence on the population. when analyzing the influence of sex ratio on the spread of infectious diseases, we considered two factors, namely, different influence coefficient and different sex ratio. first, the ratio of males to females was set to : . because of the large population base of males, infectious diseases have a greater influence on the population when the infection coefficient is greater. second, the influence coefficients of infectious diseases on males and females were . and . , respectively. because infectious diseases have a greater influence on males, when the number of males is larger, the influence of infectious diseases on the population is greater. when analyzing the influence of age structure on the spread of infectious diseases, we simulated three types of population distribution structure, namely, young, adult, and aged, according to the age structure distribution ratio. the number of members of the infected population and deaths in the aged cities were the largest, and the susceptibility of adult cities to infectious diseases was stronger. that is, the uniform distribution of age plays a more active role in the spread of infectious diseases. in order to effectively prevent the spread of infectious diseases in the population, we offer three suggestions according to the three influencing factors. ( ) population density: the regional economy should be balanced, the large-scale turnover of personnel should be reduced, the density of urban population should be controlled, the population in densely populated areas such as schools should be evacuated during the epidemic period of infectious diseases. ( ) sex ratio: when infectious disease has a greater influence on a certain sex, or if the sex ratio is larger in the population, attention should be paid to prevention and treatment with respect to that sex. ( ) age structure: the age structure should be optimized and the age structure of the city should be stabilized. on this basis, we should pay attention to prevention and treatment with respect to disadvantaged groups (such as the elderly and children) in the spread of infectious diseases. many factors affect the spread of infectious diseases. this paper only studied the influence of the above three factors on the spread of infectious diseases. the many factors that must be further explored in the future include the following: first, the influence of population activity on the spread of infectious diseases; second, the influence of population size on the spread of infectious diseases; and third, in view of the analysis of the influence factors, how to implement effective prevention and control measures against the spread of infectious diseases in specific cities. in order to study the main factors that affect the spread process of infectious diseases, the slirds model was proposed in this paper. combined with cellular automata, an epidemic model based on cellular automata was established. in the simulation experiment, the influence of population density, sex ratio, and age structure on infectious disease spread was analyzed by comparing the results with those from the actual spread process of pandemic influenza a (h n ), and the accuracy of the slirds model was confirmed. with research on the spread of infectious diseases, the advantage of using cellular automata to model complex problems can be used to optimize epidemic models. the system can better analyze the factors affecting the spread of infectious diseases, and provide better theoretical support for the prevention and control of infectious diseases. because cellular automata cannot reflect every individual's and their neighbors' randomness, there was a lack of individual randomness in the slirds model for the maximum peak of each state for the different durations. this will be the direction that we take in the future to focus on improvement. the mathematical theory of infectious diseases and its applications the mathematics of infectious diseases modelling the influence of human behaviour on the spread of infectious diseases: a review modeling infectious disease dynamics in the complex landscape of global health mathematical modeling of infectious disease dynamics effects of limited medical resource on a filippov infectious disease model induced by selection pressure une nouvelle analyse de la mortalite cause par la petite werole et desavantages de i' inoculation pour al prevenir epidemic disease in england contributions to the mathematical theory of epidemics router-level internet topology evolution model based on multi-subnet composited complex network model seasonality and period-doubling bifurcations in an epidemic model mathematical analysis of a disease transmission model with quarantine, isolation and an imperfect vaccine small world and scale free model of transmission of sars comparing the epidemiological and economic effects of control strategies against classical swine fever in denmark asymptotic stability of a two-group stochastic seir model with infinite delays cellular automata-theory and applications a simple model of recurrent epidemics the impact of the wavelet propagation distribution on seirs modeling with delay optimizing content dissemination in vehicular networks with radio heterogeneity a cellular automaton model for the transmission of chagas disease in heterogeneous landscape and host community a model research on aids diffusion based on cellular automaton addressing population heterogeneity and distribution in epidemics models using a cellular automata approach a heterogeneous cellular automata model for sars transmission the national legal report of infectious diseases from the year this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license author contributions: all authors contributed to the work in this paper. s.b., g.s., and c.-c.c. designed the research and wrote the paper. g.s. participated in the creation of the graphics.funding: this research was funded by the shandong provincial natural science foundation, china, grant number zr mg . the authors declare no conflict of interest. key: cord- -p b b t authors: wolf, yuri i.; kazlauskas, darius; iranzo, jaime; lucía-sanz, adriana; kuhn, jens h.; krupovic, mart; dolja, valerian v.; koonin, eugene v. title: origins and evolution of the global rna virome date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: p b b t viruses with rna genomes dominate the eukaryotic virome, reaching enormous diversity in animals and plants. the recent advances of metaviromics prompted us to perform a detailed phylogenomic reconstruction of the evolution of the dramatically expanded global rna virome. the only universal gene among rna viruses is the gene encoding the rna-dependent rna polymerase (rdrp). we developed an iterative computational procedure that alternates the rdrp phylogenetic tree construction with refinement of the underlying multiple-sequence alignments. the resulting tree encompasses , rna virus rdrps and consists of major branches; of the branches include positive-sense rna viruses, is a mix of positive-sense (+) rna and double-stranded rna (dsrna) viruses, and consist of dsrna and negative-sense (−) rna viruses, respectively. this tree topology implies that dsrna viruses evolved from +rna viruses on at least two independent occasions, whereas −rna viruses evolved from dsrna viruses. reconstruction of rna virus evolution using the rdrp tree as the scaffold suggests that the last common ancestors of the major branches of +rna viruses encoded only the rdrp and a single jelly-roll capsid protein. subsequent evolution involved independent capture of additional genes, in particular, those encoding distinct rna helicases, enabling replication of larger rna genomes and facilitating virus genome expression and virus-host interactions. phylogenomic analysis reveals extensive gene module exchange among diverse viruses and horizontal virus transfer between distantly related hosts. although the network of evolutionary relationships within the rna virome is bound to further expand, the present results call for a thorough reevaluation of the rna virus taxonomy. required for polymerase activity ( ) ( ) ( ) . rdrps belong to the expansive class of polymerases containing so-called palm catalytic domains along with the accessory fingers and thumb domains ( , ) . in addition to viral rdrps, palm domain polymerases include reverse transcriptases (rts) of retroelements and reverse-transcribing viruses and the dna polymerases that are responsible for genome replication in cellular organisms and diverse dna viruses. within the palm domain class of proteins, rt and the ϩrna virus rdrps are significantly similar in sequence and structure and appear to comprise a monophyletic group ( , ( ) ( ) ( ) . more specifically, the highest similarity is observed between the ϩrna virus rdrps and the rts of group ii introns. these introns are widespread retrotransposons in prokaryotes that are thought to be ancestral to the rts of all other retrotransposons as well as retroviruses and pararetroviruses (recently jointly classified as the order ortervirales) of eukaryotes ( ) ( ) ( ) ( ) . a phylogenetic analysis of the ϩrna virus rdrps revealed only a distant relationship between the leviviruses and the bulk of the eukaryotic ϩrna viruses, leaving the ancestral relationships uncertain ( ) . the origin of eukaryotic ϩrna viruses from their prokaryotic counterparts is an obvious possibility. however, given the dramatically greater prevalence of ϩrna viruses among eukaryotes compared to the narrow spread of leviviruses and "levi-like viruses" in bacteria, an alternative scenario has been proposed. in this scenario, rdrps of the prokaryotic and eukaryotic ϩrna viruses independently descended from distinct rts ( , ) . among eukaryotic ϩrna viruses, phylogenetic analysis of the rdrps strongly supports the existence of picornavirus and alphavirus "supergroups," which are further validated by additional signature genes ( , ) . however, both the exact compositions of these supergroups and the evolutionary relationships among many additional groups of viruses remain uncertain. some rdrp phylogenies suggest a third supergroup combining animal "flavi-like viruses" and plant tombusviruses, but this unification is not supported by additional shared genes and thus remains tenuous ( , , ) . the similarity of rdrps among dsrna viruses is limited, but these rdrps are similar to various degrees to ϩrna virus rdrps. therefore, different groups of dsrna viruses might have evolved from different ϩrna viruses independently, on multiple occasions ( , ) . although it is not entirely clear how prokaryotic dsrna viruses fit into this concept, evidence of an evolutionary affinity between cystoviruses and reoviruses has been presented ( , ) . for a long time, the evolutionary provenance of Ϫrna virus rdrps remained uncertain due to their low sequence similarity to other rdrps and rts ( ) . however, recent protein structure comparisons point to a striking similarity between the rdrps of Ϫrna orthomyxoviruses and those of ϩrna flaviviruses and dsrna cystoviruses ( ) . all these findings notwithstanding, the overall evolutionary relationships among the rdrps of ϩrna, Ϫrna, and dsrna viruses and rts remain unresolved. in particular, whether the rdrps of ϩrna and Ϫrna viruses are mono-or polyphyletic is unclear. many deep evolutionary connections between rna virus groups that were originally thought to be unrelated have been delineated using the results of pre-metagenomicera evolutionary studies. these discoveries culminated in the establishment of rna virus supergroups ( , , ) . however, the evolutionary provenance of many other rna virus groups remained unclear, as did the relationships between the rna viruses of the baltimore classes and retroelements and their ultimate origins. the prospects of substantial progress appeared dim because of the extreme sequence divergence among rna viruses, which could amount to irrevocable loss of evolutionary information. recent revolutionary developments in virus metagenomics (metaviromics) dramatically expanded knowledge of the diversity of rna viruses and provided an unprecedented amount of sequence data for informed investigation into rna virus evolution ( , , ) . the foremost development was the massive expansion of the known invertebrate virome, which was achieved primarily through meta-transcriptome sequencing of various holobionts. the subsequent phylogenetic analysis revealed previously unknown lineages of ϩrna and Ϫrna viruses and prompted reconsideration of high-rank virus unifications, such as ϩrna virus supergroups ( , ( ) ( ) ( ) ( ) . the rna viromes of fungi and prokaryotes also underwent notable expansion, albeit it was not as extensive as that of invertebrates ( , , ) . here we reexamine the evolutionary relationships among and within the baltimore classes of rna viruses through a comprehensive analysis of the available genomic and metagenomic sequences. in particular, to build a phylogenetic tree of thousands of viral rdrps, we designed an iterative computational procedure that alternates phylogeny construction with refinement of the underlying multiple alignments. although rna viruses have relatively short genomes (ϳ to kb), the combined gene repertoire (pangenome) of these viruses includes numerous genes that are shared, to various degrees, by related subsets of rna viruses. to obtain further insight into virus evolution, we therefore attempted to reconstruct the history of gain and loss of conserved proteins and domains in different virus lineages. we also investigated evolution of the single jelly-roll capsid protein (sjr-cp), the dominant type of capsid protein among ϩrna viruses. our analysis revealed patterns that are generally congruent with the rdrp phylogeny and provide further insights into the evolution of different branches of rna viruses. finally, we analyzed a bipartite network in which rna virus genomes are connected via nodes representing virus genes ( , ) to identify distinct modules in the rna virosphere. the results shed light on the evolution of rna viruses, revealing, in particular, the monophyly of Ϫrna viruses and their apparent origin from dsrna viruses, which seem to have evolved from distinct branches of ϩrna viruses on at least two independent occasions. comprehensive phylogeny of rna virus rna-dependent rna polymerases: overall structure of the tree and the major branches. amino acid sequences of rdrps and rts were collected from the nonredundant national center for biotechnology information (ncbi) database and analyzed using an iterative clustering-alignmentphylogeny procedure (see fig. s in the supplemental material) (see materials and methods for details). this procedure ultimately yielded a single multiple alignment of the complete set of , virus rdrp sequences (see data set s in the supplemental material) and , rt sequences organized in ϩ clusters ( clusters of rdrps and clusters of rts; see materials and methods for details). this sequence set did not include rdrps of members of the families birnaviridae and permutatetraviridae, distinct groups of rna viruses that encompass a circular permutation within the rdrp palm domain ( ) and therefore could not be confidently included in the alignment over their entire lengths. the phylogenetic tree of rdrps and rts ( fig. ; see also data set s ) was assembled from a set of trees that represent three hierarchical levels of relationships. at the lowest level, full complements of sequences from each cluster were used to construct clusterspecific trees. at the intermediate level, up to representatives from each cluster were selected to elucidate supergroup-level phylogeny. at the highest level, up to representatives from each cluster were taken to resolve global relationships (data set s a and s ). the final tree (data set s ) was assembled by replacing the cluster representatives with the trees from the previous steps. the large number and immense diversity of the viruses included in our analysis create serious challenges for a systematic, phylogeny-based nomenclature of the identified evolutionary lineages of rna viruses. many such lineages consist of viruses newly discovered by metaviromics and are not yet formally classified by the international committee on taxonomy of viruses (ictv) and therefore cannot be assigned formal names. for the purpose of the present work, we adopted a semiarbitrary naming scheme using the following approach. (i) we use taxon names that had been fully accepted by the ictv as of march ( ) whenever possible. these names are recognizable through their capitalization and italicization and rank-specific suffixes (e.g., -virales for orders and -viridae for families). as is the common practice in virus taxonomy, the officially classified members of each ictv-approved taxon are referred to via vernacular designations (recognizable through their lack of capitalization and italicization). for instance, the members of the ictv-approved order bunyavirales are called bunyaviruses, whereas those of the family tombusviridae are called tombusviruses. however, in this work, both taxon and vernacular terms are to be understood sensu lato: if our analysis indicates certain viruses to be members of or very closely related to an ictv-established taxon, we consider them members of that taxon despite the lack of current ictv recognition. as a result, the order bunyavirales has more members in our analysis than in the official taxonomy. (ii) we use vernacular names in quotation marks for viruses/lineages that are clearly distinct from those covered by the official ictv framework. whenever possible, we use names that circulate in the literature (e.g., "hepeliviruses," "statoviruses"). in the absence of such unofficial names, we name the lineage reminiscent of the next most closely related lineage (e.g., "levi-like viruses" are a clearly distinct sister group to leviviridae/leviviruses). (iii) monophyletic clusters that transcend the currently highest ictv-accepted rank (i.e., order) are labeled according to terms circulating in the literature (i.e., "alphavirus supergroup," "flavivirus supergroup," and "picornavirus supergroup"). (iv) lineages represented by a single virus are labeled with the respective virus name. rooting the phylogenetic tree, generated using phyml ( ) , between the rts and rdrps resulted in a well-resolved topology of rna viruses in which the tree splits into major branches, each including a substantial diversity of viruses (fig. ) . branch consists of leviviruses and their eukaryotic relatives, namely, "mitoviruses," "narnaviruses," and "ourmiaviruses" (the latter three terms are placed in quotation marks as our analysis contradicts the current ictv framework, which classifies mitoviruses and narnaviruses as members of one family, narnaviridae, and ourmiaviruses as members of a free-floating genus, ourmiavirus). branch ("picornavirus supergroup") consists of a large assemblage of ϩrna viruses of eukaryotes, in particular, those of the orders picornavirales and nidovirales; the families caliciviridae, potyviridae, astroviridae, and solemoviridae, a lineage of dsrna viruses that includes partitiviruses and picobirnaviruses; and several other, smaller groups of ϩrna and dsrna viruses. branch consists of a distinct subset of ϩrna viruses, including the "alphavirus supergroup" along with the "flavivirus supergroup," nodaviruses, and tombusviruses; the "statovirus," "wèivirus," "yànvirus," and "zhàovirus" groups; and several additional, smaller groups. branch consists of dsrna viruses, including cystoviruses, reoviruses, and totiviruses and several additional families. branch consists of Ϫrna viruses. each of these major branches of the tree is strongly supported by bootstrap replications (fig. ). assuming the rt rooting of the tree, branch , which consists of leviviruses and their relatives infecting eukaryotes, is a sister group to the rest of rna viruses; this position is highly robust with respect to the choice of the phylogenetic method and parameters. this tree topology is compatible with the monophyly of the rdrps and, by inference, of rna viruses and the origin of eukaryotic rna viruses from a prokaryotic rna virus ancestor shared with leviviruses. the deeper history remains murky. we have no information on the nature of the common ancestor of retroelements and rna viruses, let alone on whether the ancestor was an rna virus or a retroelement. however, parsimony considerations suggest that a retroelement ancestor is more likely given that capsids first appeared in the virus part of the tree rather than having been lost in retroelements. the next split in the tree occurs between branch and the short stem that formally joins branches , , and . however, the unification of branch with branches and is weakly supported and might not reflect actual common ancestry. arguably, the most striking feature of the rna virus tree topology is the paraphyly of ϩrna viruses relative to dsrna and Ϫrna viruses. indeed, according to this phylogeny, Ϫrna viruses evolved from within dsrna viruses, whereas dsrna viruses are polyphyletic (fig. ). one major group of dsrna viruses that includes partitiviruses and picobirnaviruses is firmly embedded within ϩrna virus branch , whereas another, the members of a larger dsrna virus group that includes cystoviruses, reoviruses, totiviruses, and viruses from several other families comprise a distinct branch, branch , which might be related to ϩrna virus branch (fig. ). this placement of the two branches of dsrna viruses is conceptually compatible with the previous evolutionary scenarios of independent origins from ϩrna viruses. however, the presence of a strongly supported branch combining lineages of dsrna viruses that infect both prokaryotes and eukaryotes suggests a lesser extent of polyphyly in the evolution of dsrna viruses than originally proposed ( , ) . an alternative phylogenetic analysis of the same rdrp alignment using raxml yielded the same main branches, albeit some with weak support (data set s b). furthermore, although the dsrna viruses are split the same way using raxml as in the phyml tree, the nested tree topology, in which branch (the bulk of dsrna viruses) is lodged deep within ϩrna viruses and branch (Ϫrna viruses) is located inside branch , is not reproduced (data set s b). instead, branches and are separate and positioned deep in the tree, immediately above the split between branch and the rest of the rdrps. given the poor resolution of the raxml tree and a strong biological argument, namely, the absence of identified Ϫrna viruses in prokaryotes or protists (with the exception of the "leishbuviruses" infecting kinetoplastids [ , ] ; see also discussion below), we believe that the tree topology presented in fig. carries more credence than that shown in data set s b. nevertheless, these discrepancies emphasize that utmost caution is due when biological interpretation of deep branching in trees of highly divergent proteins is attempted. the rdrp is the only universal protein of rna viruses. accordingly, other viral genes must have been gained and/or lost at different stages of evolution. thus, after performing the phylogenetic analysis of rdrps, we assigned the proteins and domains shared by diverse viruses to the branches of the rdrp tree. multiple alignments and hidden markov model (hmm) profiles were constructed for , proteins and domains encoded by rna viruses, and a computational pipeline was developed to map these domains on the viral genomes (data set s ). the resulting patterns of domain presence/absence in the branches of the rdrp tree were used to reconstruct the history of the gains and losses of rna virus genes (or proteins and domains, for simplicity), both formally, using the ml-based gloome method ( ) , and informally, from parsimony considerations. these reconstructions reveal a high level of branch specificity in the evolution of the gene repertoire of rna viruses. the only protein that is likely to have been gained at the base of the eukaryotic virus subtree (branches to ) (which also includes the bacterial cystoviruses) is the single jelly-roll capsid protein (sjr-cp) (fig. s a ). retroelements lack capsid proteins, and therefore, there is no indication that sjr-cp was present in the hypothetical element that encoded the common ancestor of rdrps and rts. furthermore, reconstruction of the evolution of branch (leviviruses and their relatives) argues against the ancestral status of sjr-cp in this branch. (ii) branch : leviviruses and their descendants. the current rdrp tree topology combined with gene gain-loss reconstruction suggests the following evolutionary scenario for branch ( fig. a) : a levivirus-like ancestor that, like the extant members of the leviviridae, possessed a capsid protein unrelated to sjr-cp ( , ) gave rise to naked eukaryotic rna replicons known as "mitoviruses" and "narnaviruses." these replicons consist of a single rdrp gene (fig. b ) and replicate in mitochondria and in the cytosol of the host cells of fungal and invertebrate hosts, respectively (the latter hosts were identified in metaviromic holobiont analyses) ( , ) . recently, the existence of plant "mitoviruses" has been reported although it is not known whether these viruses reproduce in the mitochondria ( ) . the "narnavirus" rdrp is also the ancestor of the rdrp of the expanding group of "ourmiaviruses" (fig. a) . "ourmiaviruses" were originally identified in a narrow range of plants, and genomic analysis revealed the chimeric nature of these viruses, with a "narnavirus"-like rdrp but sjr-cps and movement proteins (mps) which were apparently acquired from "picorna-like" and "tombuslike" viruses, respectively ( ) . use of metaviromics has led to the identification of numerous related viruses associated with invertebrates, many of which encode distinct sjr-cp variants and some of which acquired an rna helicase ( fig. b and s ) ( ) . thus, the evolution of this branch apparently involved the loss of the structural module of leviviruses, which yielded naked rna replicons that reproduced in the mitochondria of early eukaryotes. a group of these replicons subsequently escaped to the cytosol, which was followed by the reacquisition of unrelated structural modules from distinct lineages of eukaryotic viruses inhabiting the same environment (fig. b ). this complex evolutionary scenario emphasizes the key role of modular gene exchange in the evolution of rna viruses. (iii) branch : picornavirus supergroup. expansive branch generally corresponds to the previously described "picornavirus supergroup" (fig. and ) ( , ) . some of the virus groups that were previously considered peripheral members of this supergroup, such as totiviruses and nodaviruses, were relocated to different branches in the present tree (branches and , respectively), whereas the viruses of the order nidovirales were moved inside branch from an uncertain position in the tree. nevertheless, the core of the supergroup remains coherent, suggestive of common ancestry. within branch , major clades are strongly supported (fig. a) ; however, many of the internal branches are less reliable, so that the relative positions of partitivirus-picobirnavirus, potyvirusastrovirus, and nidovirus clades within branch remain uncertain. the largest and most coherent of the branch clades includes the cornerstone of the picornavirus supergroup, the ϳ viruses-strong order picornavirales ( ), expanded with caliciviruses, solinviviruses, and a multitude of unclassified viruses infecting invertebrates, vertebrates, fungi, protists, and undefined hosts (for viruses discovered by metaviromics) (fig. ) ( , , , ( ) ( ) ( ) . the second largest, deep-branching clade consists of two lineages that include, respectively, ϩrna and dsrna viruses (fig. ). the ϩrna virus lineage combines astroviruses and potyviruses, the evolutionary affinity of which is well recognized ( , ) . the dsrna lineage includes the members of the families amalgaviridae, hypoviridae, partitiviridae, and picobirnaviridae, with each of these families greatly expanded by unclassified affiliates. finally, the "middle" clade is smaller and less diverse; it encompasses nidoviruses, including the longest of all ϩrna virus genomes ( ) , and solemoviruses with much shorter genomes (fig. ) . notably, some members of the family luteoviridae and heterocapsa circularisquama rna virus, the only known alvernavirus ( ) , are nested within the solemovirus clade. given the lack of support beyond the phylogenetic affinity of the rdrps and the dramatic differences in the genomic architectures of nidoviruses and solemoviruses, the possibility that this unification was caused by a tree construction artifact is difficult to rule out (the branch support notwithstanding). hypoviruses, representing a group of fungal capsidless rna replicons, have been traditionally viewed as dsrna viruses. however, comparisons of genome architectures and phylogenetic analyses suggested that hypoviruses are derivatives of potyviruses (continued on next page) global rna virome ® that have lost the capsid protein ( , ) . in the current rdrp tree, hypoviruses cluster with the dsrna viruses of the partitivirus-picobirnavirus clade rather than with potyviruses (fig. ). whether this position is an artifact of tree construction or whether hypoviruses actually share the rdrps with dsrna viruses is unclear. the partitivirus-picobirnavirus clade within branch represents a transition to the bona fide dsrna baltimore class (fig. ) . typical partitiviruses and picobirnaviruses have minimalist genomes that consist of two dsrna segments encapsidated separately into distinct -subunit tϭ capsids ( ) ( ) ( ) ( ) . these genome segments encode, respectively, rdrps and cps that are clearly homologous between the two families. the cps of the partitivirus-picobirnavirus clade have been suggested to be distantly related to those of other dsrna viruses that belong to branch ( , ) . notably, this clade also includes some naked rna replicons that reproduce in algal mitochondria or chloroplasts, use a mitochondrial genetic code, and, in terms of lifestyle, resemble "mitoviruses" ( , , ) . by analogy, the origin of the partitivirus-picobirnavirus group in an as-yet-undiscovered lineage of prokaryotic rna viruses seems likely. more specifically, this group of dsrna viruses could have evolved through reassortment of genomic segments encoding, respectively, a ϩrna virus rdrp of branch (possibly a naked rna replicon) and a dsrna virus capsid protein related to those of branch viruses. the most recently evolved branch of partitiviruses is characterized by larger, -to -partite genomes, in contrast to the mono-or bipartite genomes in the deeper branches ( ) . this observation emphasizes a major tendency in virus evolution: increases in genome complexity via gradual acquisition of accessory genes ( ) . apart from the sjr-cp, an apparently ancestral protein that is likely to represent a shared derived character (synapomorphy) of branch is a serine protease that is present in members of the order picornavirales (with the diagnostic substitution of cysteine for the catalytic serine), members of the potyvirus-astrovirus clade, solemoviruses, alvernavirus, and nidoviruses ( fig. b ; see also fig. s b ). as demonstrated previously, this viral protease derives from a distinct bacterial protease, probably of mitochondrial origin, which is compatible with an early origin of branch in eukaryotic evolution ( ) . the reconstruction of protein gain and loss, together with the comparison of genome architectures in this branch, revealed extensive rearrangements as well as gene and module displacement ( fig. b ; see also fig. s b). branch includes viruses with relatively long genomes and complex gene repertoires (nidoviruses, potyviruses, and many members of picornavirales) along with viruses with much shorter genomes and minimal sets of genes (astroviruses and solemoviruses). clearly, evolution of branch viruses involved multiple gene gains. of special note is the gain of distinct helicases in clades within this branch: superfamily helicases (s h) in members of picornavirales, superfamily helicases (s h) in potyviruses, and superfamily helicases (s h) in nidoviruses ( fig. b ; see also fig. s b ). this independent, convergent gain of distinct helicases reflects the trend noticed early in the study of rna virus evolution, namely, that most viruses with genomes longer than ϳ kb encode helicases, whereas smaller ones do not. this difference conceivably exists because helicase activity is required for the replication of longer rna genomes ( ) . another notable feature is the change of virion morphologies among potyviruses (replacement of ancestral sjr-cp by an unrelated cp forming filamentous virions) and nidoviruses (displacement by a distinct nucleocapsid protein). the dramatic change in virion morphology and mode of ge- where a conserved domain comprises only a part of the larger protein, the rest of this protein is shown in light gray. the locations of such domains are approximated (indicated by fuzzy boundaries). c pro , c chymotrypsin-like protease; cp, capsid protein; e, envelope protein; en, nidoviral uridylate-specific endoribonuclease (nendou); exo, =-to- = exoribonuclease domain; fcp, capsid protein forming filamentous virions; m, membrane protein; md, macrodomain; mp, movement protein; mt, ribose- -o-methyltransferase domain; n, nucleocapsid protein; n , guanine-n -methyltransferase; ppro, papain-like protease; sjr and sjr , single jelly-roll capsid proteins of type and type ; spike, spike protein; s h, superfamily helicase; s h, superfamily helicase; s h, superfamily helicase; vp , virion protein ; z, zn-finger domain; spro, serine protease; p , protein . distinct hues of same color (e.g., green for mps) are used to indicate cases where proteins that share analogous function are not homologous. wolf et al. ) that most likely were acquired independently of the capping enzymes of other rna viruses. nidoviruses also gained ribonucleases and other accessory proteins that are involved in genome replication, virulence, and other aspects of the infection cycle of these largest known rna viruses ( , ( ) ( ) ( ) . (iv) branch : "alphavirus supergroup" and "flavivirus supergroup" and the extensive diversity of "tombus-like viruses." branch is the part of the ϩrna virus rdrp tree that underwent the most dramatic rearrangements compared to previous versions. this branch consists of two strongly supported clades of ϩrna viruses: (i) the assemblage that originally was defined as the "alphavirus supergroup" ( , ) joined by several additional groups of viruses and (ii) flaviviruses and related viruses ("flavivirus supergroup"; fig. and ). in the former clade, the alphavirus supergroup encompasses an enormous diversity of plant, fungal, and animal ϩrna viruses and consists of well-supported lineages, namely, tymoviruses, virgaviruses/alphaviruses/endornaviruses, and hepeviruses/benyviruses, each accompanied by related viruses that often form as-yet-unclassified lineages (fig. a ). within the "alphavirus supergroup" alone, the genome lengths range from ϳ to ϳ kb. despite this length variation, all supergroup members harbor a conserved rna replication gene module encoding a cape, s h, and rdrp; the conservation of this module attests to the monophyly of the supergroup (fig. b) . in contrast, virion architectures differ dramatically even within each of the three lineages of the "alphavirus supergroup." the major structural themes include variants of icosahedral capsids formed by sjr-cp (e.g., bromoviruses and tymoviruses); unrelated icosahedral capsids enveloped in a lipoprotein bilayer (togaviruses); flexuous filamentous capsids formed by a distinct type of cp (alphaflexiviruses, betaflexiviruses, gammaflexiviruses, and closteroviruses); and rigid rod-shaped capsids assembled from another distinct cp (benyiviruses and virgaviruses). it was traditionally thought that the latter capsid type is specific to viruses of flowering plants ( ) . however, the recent discovery of a virgavirus-like cp in invertebrate viruses (e.g., bě ihǎi charybdis crab virus [ fig. b ]) ( ) suggests that the emergence of this unique cp fold antedates land colonization by plants at ϳ million years ago (mya). yet another "structural" theme is offered by endornaviruses, naked rna replicons which, similarly to the hypoviruses in branch (see above), originally were classified as dsrna viruses. however, endornaviruses possess all the hallmarks of the alphavirus supergroup and are clearly derived from ϩrna viruses of this group. they seem to have been mislabeled dsrna viruses due to the accumulation of dsrna replication intermediates in infected cells ( , ) . a parallel loss of the cp genes apparently occurred in the deltaflexiviruses, which, in rdrp phylogenies, form a sister group to the flexible filamentous gammaflexiviruses ( ) , and in the umbraviruses that are included in the family tombusviridae based on the rdrp phylogeny. notably, unlike most other capsidless viruses that are vertically inherited, umbraviruses can hijack capsids of coinfecting luteoviruses for aphid transmission ( ) . within branch , the phylogenetically compact alphavirus supergroup is embedded within the radiation of diverse virus groups, including the well-known tombusviruses and nodaviruses, along with several newcomers discovered via metaviromics, such as the "statovirus," "wèivirus," "yànvirus," and "zhàovirus" groups ( , , ) (fig. a ). our rdrp analysis revealed remarkable phylogenetic heterogeneity within and among these groups and split the "tombus-like viruses" into lineages with distinct evolutionary affinities (groups "uncl. [unclassified] inv. [invertebrates]" and subsets of "tombus-like viruses" and "nodaviruses" in fig. a ). this subdivision is also supported by the analysis of the cps of these viruses (see section on sjr-cp evolution) (fig. ) . therefore, in contrast to the alphavirus supergroup, nodaviruses, and the flavivirus supergroup, the term "tombus-like" loses its evolutionary and taxonomic coherence. accordingly, we use the term "tombusviruses" (without quotation marks) only for one lineage that includes the members of the current family tombusviridae along with a broad variety of related plant and invertebrate holobiont viruses ( ) . the previously suggested, tenuous flaviviridae-tombusviridae affinity is gone in the present tree, although members of both families belong to the same major branch, branch . plant tombusviruses (and members of closely related plant virus genera), representing the only group of "tombus-like viruses" that was available at the time of previous analyses ( , ) , now form but a small twig deep within the large assemblage that we refer to as tombusviruses. tombusviruses are affiliated with "statoviruses" ( ) and with a subset of unclassified viruses from invertebrate holobionts rather than with flaviviruses (fig. a) . flaviviruses now form a separate clade within branch , the flavivirus supergroup that includes members of four recognized flaviviral genera (pegivirus, hepacivirus, flavivirus, and pestivirus), the newly discovered "jı ngménviruses" with segmented genomes ( ) , and a variety of unclassified, extremely divergent "flavi-like viruses" of animals and plants. this clade is split into two well-supported lineages; one includes pegiviruses and hepaciviruses, and the other consists of the rest of flaviviruses (fig. a ). flaviviral virions are enveloped, with the envelope proteins forming an external icosahedral shell, whereas the core nucleocapsid is apparently disordered; the evolutionary provenance of the core protein, with its distinct fold, is unclear ( , , ) . notably, flaviviral envelope proteins are class ii fusion proteins that are closely related to alphavirus envelope e proteins ( ) . the theme of gene swapping between these distantly related virus groups of branch is further emphasized by the homology between the alphavirus cps that form icosahedral capsids under the lipid envelopes and the flavivirus nonstructural ns proteases that share a chymotrypsin-like fold ( ) . because the rdrp tree topology implies that the alphavirus ancestor is more recent than the ancestor of flaviviruses (fig. ) , such adoption of the ns protease for a structural role is suggestive of emerging alphaviruses borrowing their structural module from preexisting flaviviruses ( ) . the hallmark of branch is the capping enzyme (cape), which is present in the entire "alphavirus supergroup" and in flaviviruses ( fig. ; see also fig. s b). a highly divergent version of cape has been identified in nodaviruses ( ) and, in our present analysis, in the additional subset of viruses that grouped with nodaviruses, as well as in a few viruses scattered throughout the clade. formally, cape is inferred to be ancestral in the entire branch . however, capes of "alphavirus supergroup" members, nodaviruses, and flaviviruses are related only distantly to one another, and at least the latter have closer eukaryotic homologs, namely, the ftsj family methyltransferases ( , ) . furthermore, tombusviruses, statoviruses, yànviruses, zhàoviruses, wèiviruses, and members of the pegivirus-hepacivirus lineage of flaviviruses lack cape, putting into question its presence in the ancestor of this branch ( to an even greater extent than in branch , the apparent routes of virus evolution in branch involve lineage-specific gene capture that resulted in evolution of complex cp-spro, capsid protein-serine protease; e, envelope protein; fcp, divergent copies of the capsid protein forming filamentous virions; hsp h, hsp homolog; mp, movement protein; ns, nonstructural protein; nsp to nsp , nonstructural proteins; ppro, papain-like protease; prm, precursor of membrane protein; rcp, capsid protein forming rod-shaped virions; ris, rna interference suppressor; s h, superfamily helicase; s h, superfamily helicase; sjr , single jelly-roll capsid proteins of type ; spro, serine protease; votu, virus otu-like protease; ns, nonstructural protein. distinct hues of same color (e.g., green for mps) are used to indicate the cases when proteins that share analogous function are not homologous. global rna virome ® genome architectures (fig. b) . the most notable cases are the closteroviruses and divergent flaviviruses that have genomes of up to to kb, rivalling coronaviruses in terms of genome length and the complexity of the gene repertoire ( , ( ) ( ) ( ) . the lack of genes assigned to the common ancestor of branch (with the obvious exception of the rdrp) prevents development of a coherent evolutionary scenario for the entire branch. in the case of the clade encompassing the "alphavirus supergroup" and related viruses, a potential common ancestor could be a simple virus that encoded only an rdrp and a sjr-cp, a cp fold most broadly represented in this clade, including diverse tombusviruses, nodaviruses and members of bromoviridae, and tymoviridae within the "alphavirus supergroup." proposal of such an ancestor for the flavivirus clade is challenged by the lack of viruses with short and simple genomes among flaviviruses. indeed, the lengths of the genomes in this clade range from ϳ kb to ϳ kb, with even the shortest ones encoding at least three of the flavivirus signature genes (serine protease [spro], s h, and rdrp). one potential clue, however, is provided by "jı ngménviruses," with tetrapartite genomes in which the protease-helicase modules and the rdrps are encoded by separate genome segments; two other segments apparently encode structural proteins of unclear provenance ( ) . this genome architecture could hint at an ancestral flavivirus genome that was assembled from genes borrowed from preexisting viruses, one of which possessed a divergent "tombus-like virus" rdrp. although the origins of branch are murky, major trends in its subsequent evolution clearly included lineage-specific gene capture, starting with helicases and capes in the ancestors of the major lineages and followed by diverse genes in smaller groups (fig. b) . (v) branch : dsrna viruses. branch , which joins branch with weak support, includes the bulk of the dsrna viruses ( fig. and ). all dsrna viruses in this branch share a unique virion organization and encode homologous cps. in particular, the specialized icosahedral capsids of these viruses, involved in transcription and replication of the dsrna genome inside cells, are constructed from homo-or heterodimers of cp subunits organized on an unusual tϭ (also known as pseudo-tϭ ) lattice ( , ) . the only exceptions are the chrysoviruses, which encode large cps corresponding to the cp dimers of other dsrna viruses and form genuine tϭ capsids ( ) . the icosahedral capsids of partitiviruses and picobirnaviruses, which encode rdrps belonging to branch , are also constructed from homodimers ( , , ) and have been suggested to be evolutionarily related to those of the dsrna viruses from rdrp branch ( ) despite little structural similarity between the corresponding cps. totiviruses, many of which have "minimal" genomes encoding only rdrps and cps, comprise one of the two major clades in branch , whereas cystoviruses, the only known prokaryotic dsrna viruses, together with members of the vast family reoviridae, which consists of multisegmented dsrna viruses infecting diverse eukaryotes, comprise the second clade (fig. ) . the concept of the closer phylogenetic affinity between cystoviruses and reoviruses appears to be corroborated by the fact that the inner tϭ icosahedral capsid is uniquely encased by the outer icosahedral shell constructed on a tϭ lattice in both families ( ) . both cystoviruses and reoviruses appear to have gained many cladespecific genes, in particular, reca-like packaging atpases of the former ( ) and the capes of the latter that are only distantly related to capes of other rna viruses and likely were acquired independently ( , ) ( (vi) branch : ؊rna viruses. branch , the % supported lineage combining all Ϫrna viruses, is lodged within branch as the sister group of reoviruses, and this position is upheld by two strongly supported internal branches in the rdrp tree ( fig. and ). the Ϫrna branch splits into strongly supported clades. the first clade encompasses the viruses-strong membership of the order mononegavirales ( ), along with the members of the distantly related family aspiviridae ( ), groups of Ϫrna viruses discovered through metaviromics ("chǔ viruses," "qínviruses," and "yuèviruses") ( , ) , and a group of unclassified fungal viruses (fig. a) ( , ) . in contrast to the members of the mononegavirales, most of which possess unsegmented genomes, the remainder of this clade is characterized by bi-, tri-, or even tetraseg-mented genomes (fig. b) . the second clade combines the family orthomyxoviridae, the genus tilapinevirus ( ), and the large order bunyavirales ( viruses) ( ) . the latter order consists of two branches, one of which is the sister group to the orthomyxovirus/tilapinevirus clade (albeit with weak support). the numbers of negativesense or ambisense genome segments in this clade range from to in most of bunyaviruses to in viruses of the genus emaravirus and the orthomyxovirus/tilapinevirus group ( , , , ) . a notable acquisition in the first clade is a cape, whereas members of the second clade share "cap-snatching" endonucleases (en) ( ) . patterns of the single jelly-roll capsid protein evolution. the sjr-cp is the dominant type of cp among ϩrna viruses and is also found in members of one family of dsrna viruses (birnaviridae). structural comparisons indicate that sjr-cps of rna viruses form a monophyletic group and likely were recruited from cellular sjr proteins on a single occasion during the evolution of rna viruses ( ) . the short length and high divergence of sjr-cps preclude adequate resolution in phylogenetic analysis; thus, we performed profile-profile sequence comparisons and clustering of all viral sjr-cp sequences in our data set (see materials and methods for details). analysis of the resulting network reveals patterns that are generally congruent with the rdrp phylogeny and provides further insights into the evolution of different branches of rna viruses. at conservative p value thresholds (p Ͻ e Ϫ ), the majority of sjr-cps segregated into two large clusters, both of which contained representatives from rdrp branch . cluster included the members of picornavirales and caliciviridae and diverse "picornalike viruses" of invertebrates, whereas cluster consisted of the members of the families astroviridae, luteoviridae, and solemoviridae and "sobemo-like viruses" (fig. s ). in addition, cluster contained members of several families from rdrp branch , namely, tombusviridae (and diverse "tombus-like viruses"), hepeviridae, a subgroup of nodaviridae, and "statoviruses." at less-restrictive p value thresholds (p Ͻ e Ϫ ), all sjr-cps were interconnected, largely through making contacts to the core of cluster . only "ourmiaviruses" had stronger affinity to picornaviruses in cluster (fig. ) . this pattern of connectivity is consistent with the radiation of sjr-cps from a common ancestor, likely resembling sequences from cluster of branch . this analysis also reveals high cp sequence divergence among members of some families (e.g., bromoviridae) and numerous cases of apparent cp gene replacement. for instance, the cps of nodaviruses fall into two groups; one is related to the turreted cps of tetraviruses, and the other is similar to cps of tombusviruses, mirroring the rdrp phylogeny (fig. ) . at a greater phylogenetic distance, cps of astroviruses and hepeviruses are closely related despite their affiliation with branches and , respectively, suggesting cp gene replacement in the ancestor of one of the two families. given that the cps of hepeviruses connect to sjr-cps of other viruses through astroviruses, cp gene replacement most likely occurred in the ancestor of hepeviruses (fig. s ) . notably, the cps of "zhàoviruses," "wèiviruses," and "tombus-like" and "sobemo-like" viruses (representing a diverse virus assemblage within the solemovirus branch, to the exclusion of bona fide solemoviridae) did not form discrete clusters but rather were affiliated with diverse virus groups, suggesting extensive recombination in these viruses, with multiple cp gene exchanges (fig. ) . in the case of unclassified "narnaviruses" and "ourmiaviruses," the cp genes apparently were acquired on more than independent occasions from different groups of viruses, emphasizing the impact of recombination and gene shuffling in the evolution of rna viruses. previously, a similar extent of chimerism was also observed among singlestranded dna (ssdna) viruses ( , ) , highlighting the evolutionary and functional plasticity of short viral genomes. the modular gene-sharing network of rna viruses: gene transfer and module shuffling. the pronounced structural and functional modularity of virus proteomes and the pervasive shuffling of the genomic regions encoding distinct protein modules are key features of virus evolution ( , , ) . therefore, a productive approach to the study of the virosphere that complements phylogenetics is the construction and analysis of networks of gene sharing. bipartite networks, in which one type of node corresponds to genes and another to genomes, have been employed to investigate the double-stranded dna (dsdna) domain of the virosphere ( ) . this analysis revealed hierarchical modular organization of the network, with several modules that included global rna virome ® nonobvious connections between disparate groups of viruses ( , ) . although this type of analysis is less informative for rna viruses due to the small number of proteins encoded in each viral genome, the "pan-proteome" of rna viruses is large (data set s ), prompting us to experiment with bipartite gene sharing networks for rna viruses. the initial search for statistically significant modularity identified distinct modules, most of which included a single virus family (fig. a and b) . remarkably, the family reoviridae has been split into modules, highlighting the vast diversity of this family, comparable to that seen in order-level taxa. among the exceptions, the most expansive module included the viruses of the order picornavirales (module ), together with those of the family caliciviridae (module ), which are linked through the conserved suite of genes that includes those encoding sjr-cp, chymotrypsin-like protease, s h, and the rdrp (fig. a and c) . viruses of the order bunyavirales were also recovered in a single module characterized by the presence of a conserved nucleocapsid (with the exception of the families nairoviridae and arenaviridae) and the cap-snatching endonuclease (module ; fig. a and c) . the next stage of the network analysis aimed at detecting the supermodules that are formed from the primary modules via connecting genes. the results of the analyses of the supermodules of rna viruses failed to attain statistical significance due to the small number of shared genes; nevertheless, some notable connections were revealed by this analysis. specifically, overlapping supermodules were identified (fig. c) . the largest and, arguably, most remarkable is a supermodule that combines ϩrna, dsrna, and Ϫrna viruses that share the capping enzymes, s h (with the exception of reoviridae and mononegavirales), and additional connector genes (e.g., the otu family protease) that link some of the constituent modules. the second largest supermodule combines large subsets of viruses from rdrp branches and that are connected through the sjr-cp and the chymotrypsin-like protease. another supermodule encompasses enveloped ϩrna viruses of the families flaviviridae and togaviridae and Ϫrna viruses of the order bunyavirales (except for arenaviridae) that share homologous envelope glycoproteins (except for flaviviruses) and class ii fusion proteins. information from gene sharing is inherently limited for rna viruses due to the small number of genes in each genome. nevertheless, the bipartite network analysis revealed prominent "horizontal" connections that are underlain either by actual gene exchange or by parallel acquisition of homologous genes by distinct rna viruses. host ranges of rna viruses: evolutionary implications and horizontal virus transfer (hvt). rna viruses have been identified in representatives of all major divisions of eukaryotes, whereas in prokaryotes, members of two families of rna viruses are known to infect only a limited range of hosts ( , , , ) . for branch in our phylogenetic tree of rdrps, the route of evolution from leviviruses infecting prokaryotes to eukaryotic "ourmiaviruses" of plants and invertebrates is readily traceable and involves a merger between a levivirus-derived naked rna replicon that eukaryotes most likely inherited from the mitochondrial endosymbiont with the sjr-cp of a eukaryotic "picorna-like virus." notably, such a merger seems to have occurred on at least three other independent occasions in branch because several groups of invertebrate holobiont "narnaviruses" and some "ourmiaviruses" encode distantly related sjr-cps that apparently were acquired from different groups of plant and animal viruses (fig. ) . the case of cystoviruses is less clear given that this clade is sandwiched between eukaryotic viruses in branch and therefore does not seem to be a good candidate for classification as the ancestor of this branch. it appears more likely that the ancestor was a toti-like virus, whereas cystoviruses were derived forms, which would imply virus transfer from eukaryotes to prokaryotes. however, an alternative scenario might be considered. no known prokaryotic viruses are classified in branch , but it has been proposed that the picobirnaviruses, for which no hosts have been reliably identified, actually are prokaryotic viruses. this proposal is based on the conspicuous conservation of functional, bacterium-type, ribosome-binding sites (shine-dalgarno sequences) in picobirnavirus genomes ( , ) . should that be the case, viruses of prokaryotes might be lurking among totiviruses as well. then, branch would stem from a prokaryotic ancestor, obviating the need to invoke virus transfer from eukaryotes to prokaryotes to explain the origin of the cystoviruses. we made an attempt to quantify the potential horizontal virus transfer (hvt) events in the rna viruses that represent the major branches of the rdrp tree. the leaves of the tree were labeled with the known hosts, the entropy of the host ranges for each subtree was calculated, and the resulting values were plotted against the distance from the root (fig. ). by design, for all branches, entropy (host diversity) drops from the maximum values at the root to zero at the leaves. all branches show substantial host range diversity such that, for example, at the half-distance from the root to the leaves, all branches, except for branch , retain at least half of the diversity (fig. ). furthermore, the differences between the branches are substantial, with the highest entropy observed in branch (dsrna viruses) and branch (Ϫrna viruses). with all the caveats due to potential errors and ambiguities in host assignment, this analysis strongly suggests that hvt played an important role in the evolution of all major groups of rna viruses. rna virus evolution coming into focus. this work was prompted by the advances of metaviromics, which have dramatically increased the known diversity of rna viruses ( , , , , ) . we reasoned that this expansion of the rna virosphere could provide an improved understanding of virus evolution. although further progress of metaviromics and enhanced phylogenomic methods will undoubtedly change current ideas, we believe that some key aspects of rna virus evolution are indeed coming into focus. the expanded diversity of rna viruses, combined with the iterative procedure for phylogenetic analysis, allowed us to obtain a tree of all rdrps and the most closely related rts in which the main branches are strongly supported and thus appear to be reliable (fig. ) . to our knowledge, the picture of rna virus evolution emerging from the tree has not been presented previously. the tree seems to clarify the relationships between the baltimore classes of rna viruses by revealing the nested tree structure in which dsrna viruses evolved, on at least two occasions, from ϩrna viruses, whereas Ϫrna viruses evolved from a distinct group of dsrna viruses. the derivation of Ϫrna viruses from dsrna viruses is, arguably, the most unexpected outcome of the present analysis, considering the lack of genes (other than the rdrp gene) shared by these virus classes. clearly, given that the primary evidence behind the derivation of Ϫrna viruses from within dsrna viruses comes from deep phylogeny, extreme caution is due in the interpretation of this observation. however, the pronounced similarity between the three-dimensional ( d) structures of the rdrps of Ϫrna influenza virus a and bacteriophage dsrna cystovirus ( ) is compatible with our findings. further, because virtually no Ϫrna viruses are known in prokaryotes or unicellular eukaryotes (with the single exception of the "leischbuviruses" in parasitic trypanosomatids [ ] , which were likely acquired from the animal hosts of these protists), their later origin from a preexisting group of ϩrna or dsrna viruses appears most likely. the ϩrna to dsrna to Ϫrna scenario of rna virus genome evolution also makes sense in terms of the molecular logic of genome replication-expression strategies. indeed, ϩrna viruses use the simplest genomic strategy and, in all likelihood, represent the primary pool of rna viruses. the dsrna viruses conceivably evolved when a ϩrna virus switched to encapsidating a replicative intermediate (dsrna) together with the rdrp. naked replicons similar to those of "mitoviruses," hypoviruses, and endornaviruses might have been evolutionary intermediates in this process. this switch does not seem to be as "easy" and common as previously suspected ( , ) but, nevertheless, appears to have occurred at least twice during the evolution of rna viruses. the global rna virome ® origin of Ϫrna viruses is the next step during which the plus strand is discarded from the virions, perhaps simplifying the processes of transcription and replication. conceivably, the evolution of dsrna and Ϫrna viruses, in which transcription and replication of the viral genomes were confined to the interior of virions or nucleocapsid transcription/replication complexes and no dsrna accumulated in the infected cells, was driven by the advantage of escaping some of the host defense mechanisms, in particular, rna interference (rnai) ( , ) . the membrane-associated replication complexes of ϩrna viruses could represent an initial step in this direction ( ) . obviously, evolution of the rdrp does not equal evolution of viruses; other genes, in particular, those encoding capsid and other structural proteins, are crucial for virus reproduction, and these genes often have different histories. the reconstruction of gene gain and loss sheds some light on these aspects of rna virus evolution. the ancestors of each of the major branches of rna viruses (except for branch ) appear to have been simply organized ϩrna viruses resembling tombusviruses (fig. ) . thus, these types of viruses encoding rdrps and sjr-cps might have been ancestral to the bulk of eukaryotic rna viruses (apart from those in branch that derive directly from prokaryotic leviviruses). the subsequent parallel capture of different helicases enabled evolution of increasingly complex genomes via accumulation of additional genes (fig. ) . notably, similar levels of complexity, with a complete coding capacity of to kb, were reached independently in branches of the rdrp tree, namely, branch (coronaviruses), branch (closteroviruses and flaviviruses), branch (reoviruses), and branch (filoviruses and paramyxoviruses) (fig. and ) . gene exchange and shuffling of gene modules are important factors of rna virus evolution. for example, it appears that all dsrna viruses in the two major clades within fig. legend. only the genes corresponding to rdrp, cp, and helicases (s h, s h, and s h for the helicases of superfamilies , , and , respectively) are shown systematically. the helicases appear to have been captured independently and in parallel in branches of ϩrna viruses, facilitating the evolution of larger, more complex genomes. additional genes, namely, the endo and exo genes (for endonuclease and exonuclease, respectively) and the hsp h gene (heat shock protein homolog), are shown selectively, to emphasize the increased genome complexity, respectively, in nidovirales and in closteroviridae. the virion architecture is shown schematically for each included group of viruses. icosahedral capsids composed of unelated cps are indicated by different colors (see the text for details). the question mark at the hypothetical ancestral eukaryotic rna virus indicates the uncertainty with regard to the nature of the host (prokaryotic or eukaryotic) of this ancestral form. the block arrow at the bottom indicates the time flow and the complexification trend in rna virus evolution. wolf et al. branches and share homologous structural modules that combine with distinct rdrps. at least in the case of partiti-picobirnaviruses in branch , the dsrna virus ("toti-like virus") cp apparently displaced the ancestral sjr-cp. however, this particular protein structure does not seem to be essential to encapsidate a dsrna genome; birnaviruses, whose provenance is uncertain due to the permutation in their rdrps, retain sjr-cp, which is most closely related to sjr-cp of nodaviruses and tetraviruses ( ) . an even more striking example of module shuffling is presented by amalgaviruses, dsrna viruses that group with partitiviruses in the rdrp tree but encode a distant homolog of the nucleocapsid protein of Ϫrna bunyaviruses ( ) ( ) ( ) ( ) . more generally, structural and replication modules have been repeatedly shuffled during the evolution of ϩrna viruses. examples include displacement of the ancestral sjr-cp by a filamentous cp in potyviruses and by a helical nucleocapsid protein in nidoviruses and multiple cases of displacement with rod-shaped-like cp and unique nucleocapsid proteins in branch . thus, exchange of genes and gene modules among rna viruses is pervasive and can cross the boundaries of baltimore classes. another recurring trend in rna virus evolution is the loss of the structural module, resulting in the emergence of naked rna replicons such as "narnaviruses" and "mitoviruses" in branch , hypoviruses in branch , and endornaviruses, umbraviruses, and deltaflexiviruses in branch ( ) . on some occasions, broad horizontal spread of a gene led to a major shift in the lifestyle of viruses, such as adaptation of viruses to new types of hosts. the primary cases in point are the movement proteins (mps) of plant viruses that are represented in all branches of the rdrp tree and, outside the rna part of the virosphere, in plant caulimoviruses and badnaviruses and in ssdna viruses ( ) . the prevalence of hvt and the overall course of rna virus evolution. arguably, the most striking realization brought about by metaviromics analyses concerns the diverse host range of numerous groups of viruses, even tight ones that occupy positions near the tips of the rdrp tree. extending early observations on the highly unexpected similarities between viruses of animals and plants, recent metaviromic analyses revealed numerous clusters of indisputably related viruses infecting animals and plants or plants and fungi or, in some cases, animals or plants and protists. the results of the analyses of evolutionary relationships between viruses with distinct host ranges are supported not only by the phylogeny of the rdrp but also by the fact that these viruses share additional conserved domains, such as, for example, sjr , s h, and c-pro in the case of picornavirales members infecting protists, plants, fungi, invertebrates, and vertebrates (fig. ) . invertebrates are particularly promiscuous hosts for viruses, often sharing the same virus group with distantly related organisms. certainly, much caution is due in the interpretation of host range assignments from metaviromics, especially those resulting from holobiont studies. viruses identified in holobiont samples of (for example) invertebrates could actually infect protists associated with these animals ( ) or could represent contamination from fungal or plant or even prokaryotic sources. these uncertainties notwithstanding, the extensive diversity of hosts even within small branches of the rdrp tree is undeniable. a coevolution scenario in which the ancestors of all these viruses originated from the common ancestor of the respective groups of eukaryotes and coevolved with the hosts implies an enormous diversity of rna viruses in early eukaryotes. this scenario appears to be highly unlikely given the apparent paucity of rna viruses in the extant protists (although new metaviromic studies might substantially expand the range of protist viruses). the pervasive hvt alternative seems much more plausible, especially given that arthropods, nematodes, and other invertebrates are well known as virus vectors and thus fit the role of rna virus reservoirs ( ) . in addition to invertebrates, which appear to be the dominant hvt agents, fungi could also play an important role in hvt within the global rna virome. indeed, fungi that are tightly associated with plants and insects often share closely related viruses with these organisms ( ) ( ) ( ) . furthermore, an indisputable case of cross-kingdom transfer of an insect iflavirus to an entomopathogenic fungus has been recently described ( ) . these findings appear to be most extensively compatible with a grand evolutionary scenario (fig. ) in which the ancestor of the eukaryotic rna virome was a levi/ narnavirus-like naked rna replicon that originally reproduced in mitochondria and then combined with a host carbohydrate-binding sjr protein ( ) or a preexisting sjr-cp from a dna virus to form a simple ancestral virus. given that viruses of branches , , and are present in modern protists, it appears likely that these branches emerged in early eukaryotes. however, because of the apparent dominance of the viruses of rdrp branch (members of the "picornavirus supergroup" in general and of the "aquatic picorna-like viruses" clade in particular) in protists, this branch likely diversified first, whereas the diversification of branches and occurred later, after ancestral protist viruses were transferred to marine invertebrates during the cambrian explosion. results of the recent analysis of the viromes of ctenophores, sponges, and cnidarians suggest that substantial diversification of rna viruses had already occurred in these deeply branching metazoa ( ) . invertebrates brought their already highly diverse rna virome to land at the time of terrestrialization and subsequently inoculated land plants. in land plants, rna viruses, particularly those of branch , dramatically expanded, perhaps in part because of the exclusion of competing large dna viruses. finally, it seems plausible that, given the high prevalence of Ϫrna viruses in metazoa and their virtual absence in protists (with the exception of the recently discovered "leishbuviruses" that likely invaded their parasitic protist hosts via hvt from an animal host [ , ] ), the viruses that comprise rdrp branch evolved in animals via mixing and matching genes from reovirus-like and flavivirus-like ancestors. the impending overhaul of rna virus taxonomy. the expansion of the global rna virome thanks to the advances of metaviromics, combined with the phylogenomics results, seems to call for an overhaul of the current virus taxonomy on multiple levels. most importantly, creation of a coherent, hierarchical system with multiple taxonomic ranks seems to be imminent. this process has already started with the proposal of a phylum rank for Ϫrna viruses, for which monophyly is unequivocally supported by the present analysis ( fig. and ). this phylum could consist of two subphyla with multiple classes and orders. at least additional phyla of rna viruses can be confidently predicted to emerge, including the dsrna viruses of branch and the ϩrna viruses of branches , , and . each of these phyla will undoubtedly have a rich internal structure. in addition, some of the current families do not seem to be compatible with the expansive rdrp trees presented here and in a previous analysis ( ) . for instance, the families coronaviridae, togaviridae, and rhabdoviridae are likely to be split into two families each. while the present study was in preparation, a major attempt at a comprehensive virus classification was published ( ) . this work analyzed a dendrogram that was produced from matrices of distances between viruses derived from sequence similarity scores combined with measures of gene composition similarity. unlike our present analysis, this approach presupposes monophyly of each of the baltimore classes. furthermore, given that their analysis was based on measures of similarity rather than on phylogenetic analysis proper, this approach is best regarded as producing a phenetic classification of viruses rather that an evolutionary reconstruction as such. some of the groups delineated by this method, particularly those among ϩrna viruses, are closely similar to those reported here. others, however, are extensively different; for instance, the order mononegavirales is not classified as monophyletic in their dendrograms. we did not attempt a complete comparison; such an exercise could be useful in the future for better understanding the routes of rna virus evolution. concluding remarks. through metaviromics, many aspects of the global rna virome evolution can be clarified. certainly, reconstruction of the deepest events in this evolutionary history is bound to remain tentative, especially because the rdrp gene is the only universal gene of the rna viruses and is hence the only one that can serve as the template for evolutionary reconstructions. at the depth of divergence characteristic of rdrps, the relationship between the major branches in the tree cannot be established with confidence. nevertheless, monophyly of several expansive groups, in particular, the main branches in the rdrp tree, is strongly supported. because of the stability of these branches, biologically plausible scenarios of evolution emerge under which dsrna viruses evolved from different groups of ϩrna viruses whereas Ϫrna viruses evolved from dsrna viruses. evolutionary reconstructions suggest that the last common ancestors of each major lineage of eukaryotic ϩrna viruses were simple viruses that encoded only the rdrp and a cp, most likely of the sjr fold. subsequent evolution involved independent capture of distinct helicases which apparently facilitate replication of larger, more complex ϩrna genomes. the helicase-assisted replication of ϩrna genomes created the opportunities for parallel acquisition of additional genes encoding proteins involved in polyprotein processing and virus genome expression, such as proteases and capping enzymes, respectively, and proteins involved in virus-host interactions, such as mps or rnai suppressors of plant viruses. in addition to these processes of vertical evolution of rna viruses, the results from phylogenomic analyses reveal multiple cases of gene module exchange among diverse viruses and pervasive hvt, often between distantly related hosts, such as animals and plants. together, these processes have shaped a complex network of evolutionary relationships among rna viruses. the much-anticipated comprehensive exploration of the rna viromes of prokaryotes and unicellular eukaryotes, such as free-living excavates, chromalveolates, rhizaria, amoebozoa, and choanoflagellates, as well as deeply rooted metazoa, will undoubtedly help in developing better-supported evolutionary scenarios for each of the major branches of the rna virus tree. nevertheless, it is already clear that the current taxonomy of rna viruses is due for a complete overhaul. phylogeny of rna-dependent rna polymerases. protein sequences belonging to rna viruses (excluding retroviruses) and to unclassified viruses were downloaded from the ncbi genbank database in april ( ) . initial screening for rdrp domains was performed using psi-blast ( ) (e value of . , effective database size of ϫ sequences) with position-specific scoring matrices (pssms) produced from the available rdrp alignments. the sources included group-specific alignments for ϩrna viruses and dsrna viruses ( , ) and the pfam alignments for the Ϫrna viruses (pfam , pfam , pfam , and pfam ) from the ncbi conserved domain database (cdd) ( ) . additionally, a set of rts from group ii introns and non-long-terminal-repeat (non-ltr) retrotransposons was extracted from genbank as an outgroup. extracted rdrp footprints were filtered for the fraction of unresolved amino acids (at most %) and clustered using uclust ( ) with a similarity threshold of . . one representative from each cluster was selected for further analysis. the resulting set contained , virus rdrps. this set went through several rounds of semimanual curation whereby sequences were clustered using uclust, aligned using muscle ( ) , and cross-searched against each other and their parent sequences (often representing complete viral polyproteins) using psi-blast and hhsearch ( ) . upon obtaining the results of these searches, the boundaries of the rdrp domain were expanded or trimmed to improve their compatibility with each other. the rdrp and rt sequences were subjected to an iterative clustering and aligning procedure, organized as follows. initially, sequences were clustered using uclust with a similarity threshold of . . the clustered sequences were aligned using muscle, and singletons were converted to pseudoalignments consisting of just one sequence. sites containing more than % gaps were temporarily removed from alignments, and pairwise similarity scores were obtained for clusters by the use of hhsearch. scores for a pair of clusters were converted to distances as follows: the d a,b ϭ Ϫlog[s a,b /min(s a,a ,s b,b ) formula, in which d a,b is the distance between cluster a and cluster b and s a,b is the hhsearch score for the comparison of these clusters, was used to convert scores s to distances d. the matrix of pairwise distances was used to calculate the mean by the unweighted pair group method using average linkages (upgma) ( ) . shallow tips of the tree were used as the guide tree for a progressive pairwise alignment of the clusters at the tree leaves using hhalign ( ) , resulting in larger clusters. this procedure was reiterative, ultimately resulting in the single alignment of the whole set of , virus rdrp sequences and , rt sequences. during the clustering procedure, virus rdrp clusters, consisting of to sequences, were defined. these clusters represented either well-established groups of related viruses (roughly comparable to the ictv family rank) or, in case of poorly characterized and unclassified viruses, groups of well-aligned rdrps that were clearly distinct from others. in all cases, uncertainties were treated conservatively, i.e., favoring placing sequences with questionable relatedness into separate clusters. additionally, rt sequences were placed into two clusters consisting of group ii intron rts and non-ltr retrotransposon rts. for each cluster consisting of three or more sequences, an approximate maximum likelihood (ml) phylogenetic tree was constructed from the cluster-specific alignment using the fasttree program ( ) (whelan and goldman [wag] evolutionary model with gamma-distributed site rates) with sites that contained more than % gaps removed from the alignment. trees were rooted using a variant of the midpoint rooting procedure such that the differences between the (weighted) average root-to-tip distances in the subtrees on the opposite sides of the root were minimized. to resolve the structure of the global relationships, up to five representatives from each cluster were selected using the within-cluster trees to ensure the diversity of the selected sequences. this procedure resulted in a set of virus rdrps and rts. the alignments of the selected sequences were extracted from the master alignment and filtered for sites containing more than % gaps. a ml phylogenetic tree was reconstructed for the resulting alignment using phyml ( ) (le gascuel [lg] evolutionary model with gamma-distributed site rates and empirical amino acid frequencies; support values were calculated using abayes method implemented in phyml). another form of branch support, i.e., bootstrap support provided by the transfer (booster) phylogenetic bootstrap method ( ) , was also used to assess the reliability of the major tree divisions. alternatively, the same rdrp alignment was used as the input for ml phylogenetic analysis using raxml (lg evolutionary model with gamma-distributed site rates and empirical amino acid frequencies). rdrps in the global tree were divided into major branches (supergroups). up to representatives from each cluster were selected to form supergroup-level alignments. the respective trees were reconstructed from these alignments using the same procedure (phyml tree with lg evolutionary model with gamma-distributed site rates and empirical amino acid frequencies). the overall tree was assembled manually by first replacing the supergroup representatives in the global tree with the supergroup trees and then replacing the cluster representatives with the cluster trees. the lower-level trees were rooted according to the arrangement of the representatives in the upper-level tree. identification of protein domains. protein domains were identified using a representative set of rna virus genomes, including representative members of ictv-approved virus families and unclassified virus groups. this set was annotated manually using sensitive profile-profile comparisons with the hhsuite package ( ) , and hmm profiles for annotated proteins or their domains were generated by running one iteration of hhblits against the latest (october ) uniclust database ( ) . each annotated profile was assigned to a functional category (e.g., Љcapsid protein_jelly-roll,Љ Љchymotrypsinlike proteaseЉ). these profiles were used to annotate the genomes of all of the viruses that were included in the rdrp phylogenetic analysis and for which complete (or near-complete) genome sequences were available. profiles for the latter proteins were generated by running one iteration of jackhmmer ( ) against the uniref database. protein regions that did not have significant hits were extracted and clustered with cluster analysis of sequences (clans) ( ) , and groups containing at least three members were identified, annotated (if possible), and added to the manually annotated profile database. the last step of the domain identification procedure was then repeated using the updated rna virus profile database. highly similar viruses (with identical domain organizations and Ͼ % identical rdrps) were removed from the data set. in addition, only one representative genome was retained for some members of overrepresented species (e.g., hepacivirus c). many genomes (e.g., alphaviruses, tombusviruses, and nidoviruses) encoded readthrough proteins, resulting in domains from the shorter protein contained within the longer readthrough protein. such redundancies were removed by filtering out the shorter of the two proteins sharing Ͼ % identity. the final set of annotated genomes included , viruses. reconstruction of the history of gene gain and loss. the protein domains identified in the , virus genomes were mapped onto the composite tree of virus rdrps. a set of representatives was chosen among the (mostly complete) genomes. the domain complement data and the respective tree were analyzed using the gloome program ( ) . domain gains and losses were inferred at each tree branch using the difference of posterior probabilities for the domain occurrence in the nodes at the proximal and the distal ends of the branch (differences with values above . imply gain; differences with values below Ϫ . imply loss). clustering analysis. the sjr cp network was generated by performing all-against-all comparisons of the sjr cp profiles. to generate hmm profiles, sjr cp sequences were extracted from the total proteome of rna viruses and two iterations of hhblits were performed against the uniprot _ _ database. the resulting profiles were compared to each other with hhsearch ( ) . their similarity p values were extracted from the result files and used as an input for clans program ( ) . clusters were identified using a network-based algorithm implemented in clans. resulting clusters were manually inspected and refined. analysis of bipartite gene-genome networks. a bipartite network was built to study the patterns of gene sharing among viral genomes ( , ) . after removal of genomes with fewer than domains and of domains that appeared in less than genomes, the network consisted of , nodes, of which , corresponded to genomes and to domains. genome and domain nodes were connected by links wherever a domain was present in a genome. a consensus community detection approach was used to identify the modules of the network ( , ) . first, we ran replicas with infomap ( ) (bipartite setting, using domains as factors) and built a similarity matrix by assigning to each pair of nodes a similarity value equal to the fraction of replicas in which both nodes were placed in the same module. then, hierarchical clustering was performed on the similarity matrix, setting the number of clusters equal to the median number of modules obtained in the replicas. order statistics local optimization method (oslom) software ( ) was subsequently used to filter significant modules with a p value threshold equal to . . to detect higher-order (super)modules, we first identified connector domains as those present in at least modules with prevalence greater than . . the second-order network, composed of modules and connector domains, was searched for second-order modules with infomap ( replicas, bipartite setting with connector domains as factors). due to the small size of the second-order network, consensus community detection did not qualitatively improve the results of the search, and therefore we took the replica with the best infomap score and skipped hierarchical clustering for this and subsequent steps of the higher-order module search. after assessing statistical significance with oslom, second-order modules were recovered, encompassing of the original modules associated with closely related virus families. a third-order network was built by pooling the second-order modules with the modules that remained unmerged. the third-order network was analyzed in the same manner to obtain the supermodules. none of these supermodules was assessed by oslom as significant. quantification of virus host range diversity. known hosts or, in case of viruses isolated from holobionts, virus sources were identified for , viruses. the host taxonomy (analyzed to the phylum level) was mapped to the leaves of the combined tree. the tree was ultrametrized, and the weights were computed for all leaves as described in reference . each internal node of the tree therefore defines a set of leaves with assigned hosts; the distribution of (weighted) relative frequencies of hosts can be characterized by its shannon entropy. each leaf has one host defined such that the entropy of the host range distribution is whereas the host 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networks reveal community structure finding statistically significant communities in networks extreme deviations from expected evolutionary rates in archaeal protein families key: cord- -ur g vp authors: miłek, justyna; blicharz-domańska, katarzyna title: coronaviruses in avian species – review with focus on epidemiology and diagnosis in wild birds date: - - journal: j vet res doi: . /jvetres- - sha: doc_id: cord_uid: ur g vp coronaviruses (covs) are a large group of enveloped viruses with a single-strand rna genome, which continuously circulate in mammals and birds and pose a threat to livestock, companion animals, and humans. covs harboured by avian species are classified to the genera gamma- and deltacoronaviruses. within the gamma-covs the main representative is avian coronavirus, a taxonomic name which includes the highly contagious infectious bronchitis viruses (ibvs) in chickens and similar viruses infecting other domestic birds such as turkeys, guinea fowls, or quails. additionally, ibvs have been detected in healthy wild birds, demonstrating that they may act as the vector between domestic and free-living birds. moreover, covs other than ibvs, are identified in wild birds, which suggests that wild birds play a key role in the epidemiology of other gammacovs and deltacovs. development of molecular techniques has significantly improved knowledge of the prevalence of covs in avian species. the methods adopted in monitoring studies of covs in different avian species are mainly based on detection of conservative regions within the viral replicase, nucleocapsid genes, and ’utr or ’utr. the purpose of this review is to summarise recent discoveries in the areas of epidemiology and diagnosis of covs in avian species and to understand the role of wild birds in the virus distribution. among the most abundant viruses infecting a wide variety of animals, including birds and humans are representatives of the large coronaviridae family. their virions contain the largest single-stranded positive sense rna (ssrna) genome, the feature which distinguishes them from other known viral rna genomes ( ) . similarly to other rna viruses, coronaviruses (covs) are characterised by high genetic diversity driven by mutation and recombination, which can lead to the emergence of new viruses. such new pathogens can have new features which even enable them to switch to new hosts ( ) . these newly created viruses can acquire zoonotic potential, as witnessed by the severe acute respiratory syndrome (sars), the epidemic from southern china in caused by sars-covs. this disease, termed "atypical pneumonia", was diagnosed in humans in countries and had a nearly % mortality rate. in , there emerged a subsequent disease caused by a novel coronavirus, the so-called middle east respiratory syndrome (mers) with even higher mortality rates. both sars-and mers-covs crossed the species barrier from bats to humans through civet cats and camels as intermediate organisms ( ) . wild bird species serve as a natural reservoir of many emerging zoonotic pathogens and thus have a significant impact on public health. they are also the source of pathogens dangerous to domestic animals, and such infections could have socio-economic consequences. this is the reason why over the years wild birds have been under epidemiological surveillance. among the viruses transmitted by wild birds, the most well-known are influenza a viruses ( ) . wild birds are also implicated in the spread of the west nile virus, borrelia burgdorferi, and other bacterial infections such as salmonella or campylobacter, and with them also resistance genes to antibiotics ( ) . studies from the last years have also shown the presence of covs in wild birds ( , , , ) . among factors which make birds an excellent reservoir of various pathogens and also a bioreactor contributing to their variability, there are the high biodiversity of bird species, their ecological traits such gathering/grouping during feeding and roosting, but most importantly their capability to fly long distances ( ) . in this paper, we will focus more on different aspects related to covs identified in wild birds. however, acknowledging the gaps in understanding of the biology of these viruses, we will also refer to the most attention-worthy representatives of avian coronavirus species, i.e. infectious bronchitis viruses (ibv). taxonomic classification. covs belong to the family coronaviridae, subfamily coronavirinae, and order nidovirales. initially, classification of members of this subfamily was based on their serological relationships as opposed to the new taxonomic revision based on a threshold level of sequence identity of a few replicase regions (the pp ab polyprotein and the orf ab gene). according to these criteria, coronavirinae are divided into four genera: alpha-, beta-, gamma-, and deltacoronavirus, replacing the traditional division into antigenic groups , , and ( ) . the final list of species proposed by the international committee on taxonomy of viruses (ictv) including the genus identified in birds, is presented in table . generally, alpha-and betacovs infect humans and domestic animals, while gamma-and deltacovs are largely associated with avian hosts although they were also detected in marine mammal species as well as in some asian carnivores ( , ) . the main representative of the gammacoronavirus genus is avian coronavirus. this taxonomic name includes ibv which causes a highly contagious disease of chickens, and genetically similar viruses isolated from other domestic galliformes: turkey coronavirus (tcov), responsible for turkey enteritis, and the more recently detected guinea fowl coronavirus (gfcov), the aetiological factor of fulminating disease in this species ( , , ) . analogous viruses were also detected in pheasants, peafowl, and quails, but also in nongalliformes, namely columbiformes, pelecaniformes, ciconiiformes, psittaciformes, and anseriformes ( , , , , ) . there are plenty of ibv variants with divergent molecular and biological properties. due to the lack of a clear method for ibv classification, new rules based on the spike gene fragment (s ) sequence were recently proposed. it distinguished and named lineages, categorised into six genotypes (gi to gvi) ( ) . the difference between other members of the avian coronavirus family, namely tcov and gfcov, is also in the s gene structure. interestingly, all other regions of the genome were found to be similar to ibv, suggesting that they come from the same ancestor ( , ) . in , surprising information about a new gammacov species from the white beluga whale appeared, which challenged the prevailing opinion of the specificity of gammacovs only to birds ( ) . further studies identified another coronavirus in ducks in whose genome fulfilled the official ictv criteria required to distinguish a new species in the gammacoronavirus genus. ictv approval is still awaited for this new species designation ( ) . coronaviruses identified in in birds of the passeriformes order, namely munia, bulbul, and thrush, appeared to be similar to each, but distinct from known coronaviruses, and they formed a unique cluster in the phylogenetic tree which was the basis for generation of a novel genus: deltacoronavirus ( ). interestingly, deltacovs were also identified in pigs in hong kong and the united states ( ) . additionally, these viruses clustered with previously unclassified coronaviruses detected in the asian carnivores, the asian leopard cat, and ferret badger ( ) . currently, the deltacoronavirus genus comprises eight species, including seven avian and one swine coronaviruses. it is suspected that covs appeared over million years ago, corresponding in time to the coevolution and codivergence of bat and avian species ( ) . their subsequent diversification was a product of differences in alimentation, reproduction, and roosting ecology. genomic organisation of avian coronaviruses. the coronavirinae subfamily is characterised by an exceptionally large rna genome ( ) . the genome size among viruses, included under the taxonomic species name avian coronavirus, is about , nucleotides (nt), the smallest being the , nt of / ibv strain (genbank no kx ), and the largest the , nt of ck/ch/lgd/ (kc ). duck cov (genbank no. km ), a candidate for a separate species within the gammacoronavirus genus, has a similar size of , nt. the complete genomic sequences of deltacov strains, identified in wild birds available in genbank, are about , nucleotides shorter, their sizes ranging from , nt in white-eye cov hku (nc ) to , nt in magpie-robin cov hku (nc ) strains. the genomes of all coronaviruses have similar structures and organisation, but also display unique groups or even strain-specific genomic structures, including accessory genes. generally, the genome is contained between the 'capped end and poly(a) tail at the '-end, comprising short untranslated regions (utrs). about two-thirds of the genome is occupied by two overlapping large open reading frames (orfs), encoding replicase (rna-dependent rna polymerase (rdrp)) polyproteins a and ab. these two large polyproteins are formed by a ribosomal frameshift mechanism and subsequently cleaved by viral proteases into the nonstructural proteins (nsp) ( ) . the number of nsps of viral replicase in the most complex of the family of positive-strand rna viruses and in alphaand betacovs is , whereas gamma-and deltacovs have nsps because they lack nsp . part of these replicase domains, the seven most conserved, which are nsp , nsp , and nsp - , serve as species demarcation among coronaviruses ( ) . the other third of the genome includes four structural protein genes organised in the following, canonical way: s, e, m, and n ( ) . among these four genes, there are a set of lowmolecular accessory proteins the presence of which could be strain-dependent, and these are a, b, b, c, a, b, and b, this being the most recently identified in some ibv and tcov strains and only orf downstream of n protein, - b ( , ) . it is believed that accessory proteins are not essential for virus replication, but could play a role in virus virulence as b protein does. in addition to these four canonical structural proteins s, e, m, and n, deltacovs seem to have a smaller number of nonstructural accessory proteins. they contain nonstructural protein ns and a number of proteins located downstream of the n protein, designated ns a, b, and sometimes also c and d ( ) . the functions of nonstructural proteins of gamma-and deltacovs are largely unknown. viral proteins. the name "coronavirus" aptly represents their appearance of solar corona in negatively stained electron micrographs, which displays characteristic club-shaped spike (s) proteins on the envelope of the virion. in addition to protruding s proteins, the envelope is formed by the most abundant membrane (m) proteins and non-glycosylated envelope proteins (e) present in lower quantities. such an envelope surrounds the viral genomic rna which forms the complex with a few copies of the nucleocapsid (n) protein ( ). information about the role of virus proteins in the course of infection comes from studies on ibv and tcov. the crucial stage in the virus life cycle relies on interaction of a viral attachment protein with a particular host cell receptor and then release of the genome inside the cell through the fusion with this cell membrane. the key player in both stages is the s protein, which is therefore recognised as a determinant of tissue and cell tropism and pathogenesis. this protein consists of two subunits: the n-terminal s subunit which forms a globular head structure and the c-terminal s subunit which is a transmembrane stalk. the s subunit is responsible for recognising and binding the receptor cell to the host, and the s domain specialises in the fusion process ( ) . coronaviruses identify their various specific receptors and co-receptors, which may be proteins and sugars. the ibv has a primary affinity for the respiratory systems of chickens, but its variants could have tropism also to other organs such as kidneys, oviduct, testes, bursa of fabricius, caecal tonsils, or the alimentary system ( ). the main attachment factor for respiratory ibv is α , -linked sialic acid glycan, widely distributed on host tissue, and this explains why such strains can also have affinity to other organs. additionally, the diversity of the s domain sequence as high as %- % among different ibv variants could further contribute to the binding capacity of these viruses. it is suspected that nephropathogenic ibv strains could use a different or additional receptor for tissue binding. studies on enteric coronaviruses (tcov and gfcov) also revealed their different receptor specificity, as binding of their s protein to the host glycan receptor is independent of the presence of sialic acid residues and recognises poly-lacnacon complextype n-glycans. the difference in receptor binding observed between respiratory ibvs and enteric gammacovs could be concluded from the difference between their s coding regions which is as high as % ( ) . the function of the s protein in avian deltacov seems to be analogous to ibv or tcov, but nothing is known about their receptor specificity. diagnosis. information about the range of avian species infected with covs and the prevalence of these infections has been accumulated from the increasing number of studies based on the use of virus-specific molecular tests. however, such detection methods depend on the specificity of the primers used. the method which was applied for the first time in a systemic monitoring of coronaviruses in wild birds amplified bp of the replicase gene of all coronaviruses known at that time ( ) . this allowed the identification of novel coronaviruses infecting graylag geese, feral pigeons, and mallards ( ) . next, the methods targeting the conserved regions of ibv viruses, i.e. 'utr, 'utr, or even s gene fragments ( , , ) were used for monitoring purposes. additionally, in a molecular survey of avcov in ducks the viral nucleocapsid (n) gene was amplified in a reverse-transcription polymerase chain reaction (rt-pcr) ( ) . the viruses identified in such way were designated ibv-like. the summary of monitoring studies of coronaviruses in wild birds is given in table . however, this approach changed after a few findings; firstly, it turned out that other gammacovs such as tcov or gfcov have genomes closely related to ibv, and the only diverged gene between them is the s gene; secondly, recombination events were discerned to be a quite common phenomenon in these groups of viruses; and the most important discovery was that of deltacov and determination of the criteria which enable discrimination between gamma-and deltacov ( , , ) . more recently, primers specific to the appropriate conserved site of the replicase gene have commonly been applied ( , , ) . all these primers are within a region of about nt occupied by the nsp of orf ab which is instrumental in coronavirus species differentiation and contains different numbers of degenerative nucleotides to detect the broader spectrum of coronavirus strains. the position of the used primers, aimed at the replicase gene, is shown in table . epidemiology. it is well known that ibv is ubiquitous in most parts of the world in regions with intensive poultry production, where it causes huge economic losses. ibv strains are responsible for diseases of the respiratory, urogenital, and digestive tracts of domestic fowl (gallus gallus). however, there are many reports of ibv presence in other bird species, which indicate that the virus can cross the species barrier. recently, ibvs homologous to vaccine h and field ibv strains were found in healthy domestic teal (anas) and peafowl (pavo cristatus), respectively. this indicates that the virus can replicate in these bird species without clinical signs ( ) . moreover, a virus similar to field ibv inoculated into specific-pathogen-free chickens caused nephritis and high mortality, in contrast to h -like ibv which revealed itself to be apathogenic. these findings indicate the possible role of these birds as asymptomatic carriers of ibv and the possibility of virus transmission to susceptible chicken populations. similar conclusions were drawn from chinese and brazilian studies which showed the presence of mass-like ibv strains in wild peafowl and pigeons ( , ) . the detection of virus closely related to the h ibv vaccine strain in faeces of free living ducks and whooper swans also suggested cross-species infection from a poultry population to synanthropic birds ( ) . the presence of gammacov was also found in wild birds sampled in poland. ibv-like strains were identified in anseriformes, charadriiformes, and galliformes, and the detected gene fragments were highly similar to the most frequently detected lineages of ibv in this geographical region, i.e. mass, /b and qx ( ) . similarly, ibv strains were identified in studied samples from wild birds of the corvidae, ardeidae and anatidae families in egypt, and some of them had s gene fragments highly homologous to the ma vaccine strain. such findings suggest the possibility for vaccine strains to spillover to wildlife which may serve as the asymptomatic host, enabling these strains to undergo some genetic changes. such modified virus could then spillover in the reverse direction back to a poultry population, and the possibility of its higher pathogenicity could not be ruled out ( ) . recently, interesting results for the presence of both gamma-and deltacoronaviruses in quails were reported ( ) ( ) ( ) . the identified gammacovs revealed a similarity to ibv strains unique to south america ( ) . furthermore, in some cases, both genera of the viruses were identified simultaneously ( ) . the summary of monitoring studies on the occurrence of coronaviruses in wild birds is presented in table . the rate of positives of gamma-and deltacovs differs greatly from . % to %, depending on temporal/seasonal and spatial features and depending on applied detection methods. however, even using the same method, the rate of positives varies from . % to %. among factors influencing the prevalence of cov in wild birds, there could also be the age of the birds sampled, bird order/species, and their behaviour (migratory versus resident, water birds or land-dwelling birds). very interesting is the identification of betacovs in south american wild birds, these being viruses which were previously detected only in mammals ( ) . it seems, however, that these studies may suffer from methodological deficiencies and require a thorough re-analysis. there is much evidence that cov could be transmitted from poultry to wild birds and from wild birds to poultry. in this way, the virus could spread over long distances. wild birds are suspected of spreading different ibv variants into new geographical regions such as variant qx (gi- lineage) from china to europe or var (gi- lineage) from the middle east to poland ( , ) . if the hosted wild bird acquires infections of various coronaviruses, it could be an excellent environment for recombination events, which may lead to the emergence of a new disease dangerous to humans as evidenced by sars-or mers-covs. that is the reason why wild birds have to be continuously studied for the presence of various coronaviruses. the authors declare that there is no conflict of interests regarding the publication of this article. financial disclosure statement: this publication was possible due to the polish ministry of science and higher education (grant no. know /cb/pro / within the scientific consortium "healthy animal -safe food"). animal rights statement: none required. phylogenetic analysis of partial s and n gene sequences of infectious bronchitis virus isolates from italy revealed genetic 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betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus genomic analysis and surveillance of the coronavirus dominant in ducks in china key: cord- -jhm u ka authors: wang, dang; chen, jiyao; yu, chaoliang; zhu, xinyu; xu, shangen; fang, liurong; xiao, shaobo title: porcine reproductive and respiratory syndrome virus nsp antagonizes type i interferon signaling by targeting irf date: - - journal: journal of virology doi: . /jvi. - sha: doc_id: cord_uid: jhm u ka porcine reproductive and respiratory syndrome virus (prrsv) is an arterivirus from the nidovirales order that causes reproductive failure and respiratory disease in pigs and poses a constant threat to the global pig industry. the prrsv-encoded nonstructural protein (nsp ) is a nidovirus-specific endoribonuclease (nendou) that is conserved throughout the arteriviridae and coronaviridae families. previously, our research and that of others demonstrated that prrsv nsp inhibits type i interferon (ifn) production through nendou activity-dependent mechanisms. here, we found that prrsv nsp also inhibited ifn-stimulated response element (isre) promoter activity and subsequent transcription of ifn-stimulated genes (isgs). detailed analysis showed that nsp targeted interferon regulatory factor (irf ), but not transducer and activator of transcription (stat ) or stat , key molecules in the type i ifn signaling pathway. furthermore, the nsp -irf interaction impaired the formation and nuclear translocation of the transcription factor complex ifn-stimulated gene factor (isgf ) in both nsp -overexpressed and prrsv-infected cells. importantly, nsp mutations (h a, h a, and k a) that ablate nendou activity or its cell cytotoxicity also interacted with irf and retained the ability to block ifn signaling, indicating that the nsp -irf interaction is independent of nendou activity or cell cytotoxicity of nsp . taking the results together, our study demonstrated that prrsv nsp antagonizes type i ifn signaling by targeting irf via a nendou activity-independent mechanism, and this report describes a novel strategy evolved by prrsv to counteract host innate antiviral responses, revealing a potential new function for prrsv nsp in type i ifn signaling. importance the nidovirus-specific endoribonuclease (nendou) encoded by prrsv nonstructural protein (nsp ) is a unique nendou of nidoviruses that infect vertebrates; thus, it is an attractive target for the development of antinidovirus drugs. previous studies have revealed that the nendou of nidoviruses, including porcine reproductive and respiratory syndrome virus (prrsv) and human coronavirus e (hcov- e), acts as a type i interferon (ifn) antagonist. here, for the first time, we demonstrated that overexpression of prrsv nsp also inhibits ifn signaling by targeting the c-terminal interferon regulatory factor (irf) association domain of irf . this interaction impaired the ability of irf to form the transcription factor complex ifn-stimulated gene factor (isgf ) and to act as a signaling protein of ifn signaling. collectively, our data identify irf as a natural target of prrsv nendou and reveal a novel mechanism evolved by an arterivirus to counteract innate immune signaling. agent, prrs virus (prrsv) (family arteriviridae, order nidovirales), has a positive-polarity single-stranded rna genome, approximately kb in length, encoding two nonstructural polyproteins (orf ab and orf b) and eight structural proteins (gp , e, gp , gp , gp , orf a, m, and n) ( , ) . after being cleaved by four virus-encoded proteases, nonstructural protein ␣ (nsp ␣), nsp ␤, nsp , and nsp , the two polyproteins are further cleaved into individual nonstructural proteins that perform different functions during the viral life cycle ( ) . to reproduce rapidly and establish a persistent infection in pigs, prrsv has developed the ability to resist host interferon (ifn) signaling, which is the key signaling pathway for innate immune responses against viral invasion or replication ( , , ) . innate immune responses are activated by host pattern recognition receptors (prrs), which recognize molecular structures called pathogen-associated molecular patterns (pamps) that are structurally conserved within a large number of pathogenic organisms ( ) . upon recognition of pamps, prrs initiate signaling pathways that ultimately trigger the production of type i ifns. subsequently, the janus kinase/signal transduction and transcription activator (jak/stat) pathway is activated by type i ifns ( , ) . briefly, type i ifns bind to their surface receptors (ifnar and ifnar ) and then activate and phosphorylate the two intracellular tyrosine kinases janus kinase (jak ) and tyrosine kinase (tyk ). these activated tyrosine kinases mainly phosphorylate two transcription factors, namely, signal transducer and activator of transcription (stat ) and stat , which interact with ifn regulatory factor (irf ) to form ifnstimulated gene factor (isgf ) ( ) . the transcription factor complex isgf (stat / stat /irf ) is transported to the nucleus, where it recognizes ifn-stimulated response elements (isres), leading to the induction of hundreds of ifn-stimulating genes (isgs) ( ) . many isgs function as potent antiviral effectors, directly preventing viral infections by targeting viral processes such as viral entry, viral genome replication, viral protein synthesis, or viral body release ( ) . during coevolution with their hosts, many viruses have developed elaborate strategies to circumvent the jak/stat pathway in ifn signaling ( ) . among several known viral evasion strategies, targeting the transcription factor complex isgf is a particularly powerful means of inactivating ifn signaling, because this strategy effectively inhibits common downstream isg expression. for example, our recent study showed that porcine deltacoronavirus blocks the jak/stat pathway by c-like protease-mediated cleavage of stat ( ) . the nonstructural protein (nsp ) of rotavirus mediates the degradation of irf by targeting the c-proximal irf-binding domain and inhibits ifn-mediated phosphorylation of stat ( , ) . sendai virus c protein binds stat to block the formation of stat /stat heterodimers or stat /stat homodimers, which inhibit ifn signaling ( ) . previous studies have shown that prrsv nsp inhibits type i ifn production through nidovirus-specific endoribonuclease (nendou) activitydependent mechanisms ( ) ( ) ( ) . however, the nendou activity of overexpressed nsp unexpectedly exhibited extensive substrate specificity in vitro and is extremely toxic to prokaryotic and eukaryotic cells, indicating that the inhibition of ifn production by wild-type (wt) prrsv nsp may be due to its cytotoxicity ( ) . here, we found that prrsv nsp also inhibits isre promoter activity and the transcription of isgs, thereby interfering with the type i ifn signaling pathway. importantly, mutations that eliminate nendou activity and its cytotoxicity in nsp retain the ability to block ifn signaling. detailed analysis showed that nsp inhibited type i ifn signaling by targeting irf , a key molecule in the isgf complex, revealing a potential novel function of prrsv nsp in type i ifn signaling. identification of prrsv nsp as an antagonist of type i ifn signaling. type i ifn signaling induces a potent antiviral response in cells by inducing the expression of hundreds of isgs, which is vital for the control of viral infections ( ) . to assess the potential role of prrsv nsp in type i ifn signaling, the mrna levels of ifn-stimulated gene (isg ), isg , isg , and '- '-oligoadenylate synthetase (oas ) were analyzed in human embryonic kidney cells (hek- t) overexpressing hemagglutinin (ha)-tagged prrsv nsp . as shown in fig. a , prrsv nsp significantly inhibited the transcription of isgs induced by ifn-␣ compared with the control group results. because of the presence of isre in the isg promoter regions, various concentrations of prrsv nsp expression plasmid and isre-luciferase reporter plasmid were cotransfected into hek- t cells or porcine kidney cells (pk- ). the results showed that nsp strongly inhibited ifn-␣-induced isre promoter activity in a dose-dependent manner in hek- t cells (fig. b) and pk- cells (fig. c) . these results confirm the antagonistic nature of prrsv nsp in type i ifn signaling. prrsv nsp inhibits type i ifn signaling in an endoribonuclease activityindependent manner. in arterivirus, the nsp endoribonuclease is important for viral replication ( , , ( ) ( ) ( ) ( ) . several previous studies have shown that prrsv nsp inhibits type i ifn production in a nendou activity-dependent manner ( ) ( ) ( ) . we considered such a possibility for prrsv nsp -mediated inhibition of type i ifn signaling to be associated also with its nendou activity. on the basis of their chemical properties and known residue requirements, the three catalytic residues of endoribonucleases (his , his , and lys [numbering based on prrsv nsp ]) were shown to be directly involved in catalysis ( , , ) . thus, three endoribonuclease catalytic residue mutations, his ala (h a), his ala (h a), and lys ala (k a), were introduced into prrsv nsp and the corresponding eukaryotic expression plasmids expressing the n-terminally ha-tagged nsp endoribonuclease inactive mutants were constructed. to test whether the ha-tagged nsp mutants lose endoribonuclease activity, ha-tagged wt nsp and three mutants were expressed in escherichia coli. the recombinant proteins were purified ( fig. a) , and fluorescence resonance energy transfer (fret) assays were performed to detect the endoribonuclease activity. as shown in fig. b , the levels of endoribonuclease activity of the three ha-tagged nsp mutants were significantly decreased compared with the wt nsp (fig. b) . however, none of the endoribonuclease inactive mutants (h a, h a, or k a) showed a loss of the ability of nsp to inhibit isre promoter activity in cells overexpressing ha-tagged nsp mutants (fig. c) . we also used prrsv nsp as a negative control and porcine epidemic diarrhea virus (pedv) nsp as a positive control. in agreement with a previous study ( ) , pedv wt nsp suppressed ifn-␣-induced isre promoter activity whereas such an inhibitory effect was not observed in cells expressing a pedv nsp endoribonuclease inactive mutant (h a) or prrsv nsp (fig. c) . these data suggest that although the nendou activity of pedv nsp is important for inhibiting ifn signaling, prrsv nsp does not depend on the nendou activity. we further tested the ability of nsp mutants to inhibit ifn-␣-induced isg expression. as shown in fig. d , each endoribonuclease inactive mutant (h a, h a, or k a) of nsp also inhibited the ifn-␣-induced transcription of isgs. since these three mutants are also devoid of cell cytotoxicity ( ), our results indicated that the inhibition of ifn signaling observed in nsp -expressing cells is independent of its endoribonuclease activity and cell cytotoxicity. prrsv nsp disrupts isgf -mediated activation of the isre promoter. isgs are antiviral effectors induced by ifns through the formation of a tripartite transcription factor, isgf , which is composed of stat , stat , and irf ( ) . given the pivotal role of the transcription factor complex isgf in type i ifn signaling, we further investigated whether overexpression of nsp inhibits isgf -mediated signaling. as shown in fig. , coexpression of the components of transcription factor complex isgf (stat , stat , and irf ) significantly activated the isre promoter compared with the results seen with the empty plasmid control. however, activation of the isre promoter by isgf was significantly inhibited in the presence of prrsv nsp (fig. ) . similarly to the results seen with wt nsp , each endoribonuclease inactive mutant (h a, h a, or k a) was also able to suppress the isgf -mediated ires promoter (fig. ) , suggesting that the endoribonuclease activity of nsp does not govern the ability of nsp to block the activity of the isgf -induced ires promoter. consequently, we speculated that prrsv nsp might target the isgf complex to inhibit type i ifn signaling. prrsv nsp does not degrade or block the phosphorylation of stat and stat . since downregulation of molecules responsible for ifn-activated signal transduction is a mechanism commonly employed by viruses ( , ( ) ( ) ( ) , we investigated whether nsp impairs the endogenous protein levels of stat , stat , and irf . as shown in fig. , no reduction was observed in the protein levels of stat , stat , and irf as a consequence of the presence of wt nsp or expression of its endoribonuclease inactive mutants (h a, h a, and k a [compare lanes and to lane ]), suggesting that nsp does not induce downregulation of the isgf complex. the type i ifn-activated isgf transcription complex containing tyrosine-phosphorylated stat and stat associated with irf is rapidly translocated to the nucleus fig prrsv nsp inhibits isgf -induced isre promoter activity. hek- t cells were cotransfected with prrsv nsp or its endoribonuclease activity-defective mutants ( . g/well), along with porcine isgf complex (stat /stat /irf ; . g/well), isre-luc plasmid ( . g/well), and prl-tk plasmid ( . g/well). after h, cells were harvested for luciferase assays. *, p Ͻ . . after ifn treatment, and phosphorylation-dependent activation of stat and stat is critical to mediate ifn-inducible antiviral responses ( , ) . to investigate whether prrsv nsp alters the stat and stat phosphorylation status after ifn-␣ stimulation, lysates from nsp -expressing cells were subjected to western blotting using phospho-stat (stat -y ) and phospho-stat (stat -y ) antibodies (abs), respectively. the levels of phosphorylated stat and stat were greatly increased after ifn-␣ treatment, and no difference in the levels was found between nsp -expressing cells and nsp -nonexpressing cells (fig. , lanes and ) . similarly, nsp mutants lacking endoribonuclease activity (h a, h a, and k a) had no effect on the phosphorylation of stat and stat (fig. [compare lane to lanes to ]). these results indicated that prrsv nsp and its endoribonuclease inactive mutants do not target the isgf complex for degradation or prevent type i ifn-induced stat proteinactivating tyrosine phosphorylation. prrsv nsp interacts with the irf-association domain (iad) of irf . previous studies have shown that many viral proteins can interact with components of the isgf complex to inhibit type i ifn signaling ( , , ) . thus, we next investigated whether nsp functions by interacting with the isgf component. to this end, hek- t cells were transfected with expression constructs encoding ha-tagged prrsv nsp protein and flag-tagged porcine stat , stat , and irf . coimmunoprecipitation (co-ip) and immunoblotting analyses showed that nsp interacted with porcine irf , but not stat or stat ( fig. a to c). to further confirm the interaction of nsp with endogenous irf , coprecipitated endogenous irf and prrsv nsp were analyzed by immunoblotting with an anti-irf antibody and an anti-ha antibody, respectively. as shown in fig. d , reciprocal pulldown of both nsp and endogenous irf further confirmed the interaction between nsp and irf . previous studies demonstrated that irf contains an n-terminal dna-binding domain (dbd; amino acids [aa] to ) and a c-terminal iad (aa to ) connected by a flexible linker (fl) ( ) ( ) ( ) . to investigate which domain of porcine irf is involved in nsp protein binding, four mutants with deletions of different domains of irf , including irf (aa to ; dbd), irf (aa to ; dbd-fl), irf (aa to ; iad), and irf (aa to ; fl-iad), were constructed by mutagenesis (fig. e ). hek- t cells were cotransfected with various combinations of flag-tagged full-length or deleted versions of irf and the ha-tagged prrsv nsp protein. as shown in fig. f , prrsv nsp -irf interaction impairs the ifn-induced formation and nuclear accumulation of isgf . since the function of isgf relies on the selective interaction between phosphorylated stat and the irf-association domain of irf ( , ) , the observed interaction between nsp and irf -iad led us to speculate that this interaction may impair the recruitment of phosphorylated stat by irf and the subsequent nuclear accumulation of isgf . to test this hypothesis, hek- t cells were transfected with the indicated nsp expression plasmids and then treated with ifn-␣. the prrsv nsp protein and phosphorylated stat /stat (stat -y and stat -y ) were immunoprecipitated with an anti-irf antibody. as shown in fig. a , ifn-␣ treatment significantly induced the phosphorylation of stat and stat , and irf efficiently pulled down phosphorylated stat /stat after ifn-␣ treatment (compare lanes and ). however, the amount of phosphorylated stat /stat that bound to irf remarkably decreased in the presence of prrsv nsp and its mutants (h a, h a, and k a) (fig. a, lanes and to ) . to further test whether prrsv nsp prevents the ifn-induced nuclear accumulation of isgf , we performed nuclear and cytoplasmic fractionation of the cells following ifn-␣ treatment. antibodies against stat -y and stat -y were used to detect the presence of the phosphorylated proteins in the two fractions. as expected, higher levels of stat -y and stat -y were found in the nuclear fraction than in the cytoplasmic fraction after ifn-␣ treatment of mock-transfected cells (fig. b , lanes and ). in contrast, in nsp -transfected cells after ifn-␣ stimulation, more isgf complex components (stat -y , stat -y , and irf ) were detected in the cytoplasmic fraction than in the nuclear fraction (fig. b, lanes and ) . taken together, these findings indicate that prrsv nsp impairs the formation and nuclear translocation of isgf . because the endoribonuclease inactive mutants of nsp retained the ability to block type i ifn signaling, these mutants in particular were still capable of interacting with irf . next, we investigated whether these mutants, lacking endoribonuclease activity, still impaired the formation and nuclear accumulation of isgf . indeed, our data revealed that the prrsv nsp mutants (h a, h a, and k a) still significantly inhibited the ifn-induced formation and nuclear accumulation of isgf and did so to similar extents (fig. b [compare lanes to and lanes to ] ). this provided further support for the notion that the ability of prrsv nsp to block type i ifn signaling is independent of its endoribonuclease activity and cell cytotoxicity, because these three mutants had similar effects with respect to inhibiting the formation and nuclear translocation of isgf compared with wt nsp . prrsv infection inhibits the formation of isgf through the interaction of nsp with irf . to exclude the possibility that the observed prrsv nsp -irf interaction was an artifact of plasmid overexpression in cell culture, we analyzed this interaction in the context of prrsv infection. african green monkey kidney cells (marc- ) were infected with prrsv for h and then collected at h after ifn-␣ treatment for immunoprecipitation with anti-nsp or anti-irf antibodies to detect the interaction between nsp and endogenous irf . as shown in fig. a , nsp was coimmunoprecipitated with endogenous irf in prrsv-infected cells. furthermore, phosphorylated stat /stat was also coimmunoprecipitated with endogenous irf upon ifn-␣ stimulation. however, the formation of stat /stat /irf heterotrimers was significantly inhibited following prrsv infection (fig. a [compare lanes and ] ). hek- t cells were transfected with expression vectors encoding ha-nsp and its mutants. endogenous irf was precipitated and immunoblotting analysis was performed with an anti-pstat antibody (the top panel), anti-pstat antibody (the second panel), or anti-ha antibody (the fourth panel) to detect the interaction with endogenous irf . (b) phosphorylated stat and stat in nuclear and cytoplasmic fractions. subcellular fractionation of hek- t cells, h after ifn-␣ treatment, was performed for nuclear and cytoplasmic fractions, followed by western blotting using stat -y antibody, stat -y antibody, anti-hsp antibody as a cytoplasmic marker, and anti-parp antibody as a nuclear protein marker, as indicated. ha antibody was used to detect the expression of prrsv nsp and its mutants (ha-nsp ). to detect the interaction with endogenous irf . (b) phosphorylated stat and stat in the nuclear and cytoplasmic fractions. an experiment parallel to that outlined for panel a was performed. subcellular fractionation of marc- cells was performed to obtain nuclear and cytoplasmic fractions, followed by western blotting using anti-stat -y , anti-stat -y , anti-irf , and anti-nsp antibodies, along with anti-hsp antibody as a cytoplasmic marker and anti-parp antibody as a nuclear protein marker, as indicated. (c to e) marc- cells were infected with prrsv (moi ϭ . ) or uninfected. at h postinfection, the cells were mock-treated or treated with ifn-␣ ( iu/ml) for h. after the cells were fixed and permeabilized, p-stat (c), p-stat (d), and irf (e) was visualized by immunofluorescence staining with rabbit anti-pstat , rabbit anti-pstat , or rabbit anti-irf antibody. the nsp protein was detected by the use of a nsp -specific mab. to further address whether the ifn-induced nuclear accumulation of isgf was also affected by prrsv infection, nuclear and cytoplasmic fractionation was performed. compared with uninfected cells, the levels of stat -y , stat -y , and irf proteins were reduced in the nuclear fraction of prrsv-infected cells after ifn-␣ stimulation (fig. b [compare lanes to and lanes to ]) . immunofluorescence experiments showed that prrsv infection inhibited ifn-induced nuclear accumulation of stat -y (fig. c) , stat -y (fig. d) , and irf proteins (fig. e ) compared to mock-infected cells. in prrsv-infected cells with ifn stimulation, nsp and irf were primarily colocated in the cytoplasm (fig. e) . together, these data indicated that prrsv infection inhibits the formation and nuclear accumulation of isgf through the nsp -irf interaction, which was consistent with the results of gene transfection experiments in cells expressing prrsv nsp . during coevolution with their hosts, many viruses have acquired mechanisms to circumvent host innate immune responses. of note, prrsv infection has been found to produce abnormally low levels of type i ifns and to inhibit the ability of type i ifns to induce antiviral responses ( , ) . the endoribonuclease encoded within the prrsv nsp sequence, which has the uridylate-preferred cleavage site for rna that is necessary for virus replication, has been found to be a multifunctional protein ( , ) . previously, it has been demonstrated that prrsv nsp is involved in the inhibition of ifn production through multiple distinct mechanisms, as follows. (i) nsp represses the transcription of type i ifns by inhibiting the activation of transcription factors irf and nuclear factor kappa b (nf-b), which directly activate the promoters of type i ifns ( , ) . (ii) nsp reduces the levels of transcripts and proteins of mitochondrial antiviral signaling protein (mavs) and retinoic acid-inducible gene i (rig-i), two critical factors in the ifn induction pathway ( ) . (iii) nsp removes the ubiquitin chains from ib␣ (inhibitor of nf-b alpha), thereby preventing the proteasomal degradation of ib␣ and subsequent liberation of nf-b ( ). (iv) nsp recruits the otu deubiquitinase with linear linkage specificity (otulin) to enhance its ability to remove the cellular protein ubiquitin associated with innate immunity, resulting in the additive effect of suppressing type i ifn production ( ) . however, whether nsp also regulates ifn signaling remains unclear. in this report, we present evidence that prrsv nsp suppressed type i ifn signaling by targeting irf , a key molecule in the jak/stat pathway, revealing a potential new function for the nidovirus endoribonuclease in type i ifn signaling. prrsv nsp , a conserved nendou within the arteriviridae and coronaviridae families, belongs to the xenopus laevis poly(u)-specific endoribonuclease (xendou) superfamily and plays an important role in nidovirus replication and pathogenesis ( ) . the structures of the arterivirus nsp , coronavirus (cov) nsp , and xendou catalytic domains, essential for endoribonuclease activity, and particularly the active site residues (his , his , and lys ; numbering based on prrsv nsp ), were found to be highly conserved. besides prrsv nsp , several other studies have reported that cov nsp inhibits ifn production in ectopic expression experiments. in support of this, infection with nendou activity-deficient covs, such as pedv, murine hepatitis virus, and human cov e (hcov- e), produced a remarkably high level of type i ifn in primary cells compared with wt infection, which effectively demonstrated the ifnantagonistic properties of nsp of covs ( , , ) . these data appear to indicate that endoribonuclease activity is sufficient for nendou-mediated ifn transcriptional repression; however, the nendou activity of overexpressed nsp /nsp may unexpectedly mediate nonspecific cleavage, thereby inducing cytotoxicity ( , ) . unfortunately, it was impossible to determine whether the cell cytotoxicity also contributed to the inhibition of ifn induction by nendou, as mutations disrupting the ability of nendou to block ifn production abrogated not only endoribonuclease activity but also its cell cytotoxicity ( ) . thus, the observation of ifn transcriptional suppression in previous studies could have been the result of cytotoxicity as a consequence of ectopic over-expression of nendou. previous studies have shown that prrsv wt nsp is toxic to e. coli and that the expression levels of wt nsp are extremely low and that one of the nendou mutants (nsp k a) can be expressed at high levels under identical conditions ( , ) . similar phenomena were observed in our experiments designed to express and purify nsp . indeed, in our previous study ( ) , we found that prrsv wt nsp exhibited cytotoxicity to eukaryotic cells whereas no cytotoxicity associated with the nendou mutants of nsp was detected. similarly, recombinant nsp of equine arteritis virus (eav) has been reported to be extremely toxic to a variety of hosts ( ) . thus, the lower level of expression of prrsv wt nsp may have been due to its cytotoxicity (fig. ) . interestingly, our results clearly showed that the prrsv nsp mutants (h a, h a, and k a) devoid of both nendou activity and cell cytotoxicity retained the ability to block type i ifn signaling by targeting irf , as did wt nsp , indicating that nsp has evolved a new mechanism for the impairment of ifn signaling that operates in an endoribonuclease activity-independent and cell cytotoxicity-independent manner, distinct from the inhibition of ifn induction. as a potent antiviral response, type i ifn signaling can control viral infections by activating the transcription factor complex isgf (stat /stat /irf ), resulting in increased transcription of hundreds of isgs and contributing to the development of an antiviral state ( , ) . it is becoming increasingly apparent that irf is a central factor not only for mediation of but also for regulation and direction of type i ifn responses ( ) . while many ifn effects, in particular, those associated with type i ifns, require all three canonical signaling molecules, studies of irf deficiency revealed unique roles for irf that were distinct from those of stat and stat ( , ) . recently, evidence has emerged that irf is the main viral target of the host's innate immune response. for example, our previous study ( ) revealed that porcine bocavirus nonstructural protein (ns ) inhibits the dna-binding activity of isgf by interacting with irf . another study showed that the e oncoprotein of papillomavirus binds to irf to block the formation of the isgf complex ( ) . moreover, many virus-encoded proteins, such as varicella-zoster virus orf , adenovirus early region a protein (e a), and rotavirus nsp , mediate irf degradation ( , , ) . human cytomegalovirus also reduces the protein levels of irf in human embryonic lung fibroblasts ( ) . to survive in the host, prrsv has also evolved strategies to block ifn signaling. previous studies have revealed that prrsv nsp ␤, a papain-like proteinase, blocks the nuclear translocation of isgf by inducing degradation of karyopherin-␣ ( , ) . here, we showed that nsp is another prrsvencoded antagonist of ifn signaling. prrsv nsp adopts a mechanism distinct from that of nsp ␤, i.e., interaction with irf , an essential component in the formation of isgf . our data not only highlight the multifaceted control of type i ifn signaling by prrsv but also uncover a novel mechanism by which prrsv antagonizes innate immune signaling. it is well established that irf exhibits several functionally conserved regions among the members of the irf family, such as an n-terminal dna-binding domain that recognizes and binds to isre motifs in the promoter region of most isgs and a c-terminal irf-association domain that is responsible for selective interaction with the coiled-coil domain (ccd) of stat ( , ) . recently, rengachari and colleagues reported the crystal structures of irf -iad alone and in a complex with stat -ccd ( ) . the structure of the irf -iad/stat -ccd complex revealed that surface features had deviated from their respective paralogs to enable a specific interaction between irf -iad and stat -ccd required for isgf function in cells ( ) . in this study, we found that prrsv nsp specifically interacted with irf -iad. thus, it may be the case that the targeting of irf -iad by nsp sequesters the interaction between irf and stat . this may help to explain why the formation and nuclear translocation of isgf were severely impaired in prrsv nsp -transfected cells and prrsv-infected cells. interestingly, prrsv nsp mutants (h a, h a, and k a) also impaired the formation and nuclear translocation of isgf by interacting with irf . furthermore, the three-dimensional ( d) structures of nendou activity-defective arterivirus nsp (k a in prrsv nsp ; h a and k a in eav nsp ) are remarkably similar to those of wt prrsv nsp ( , ) , with an overall root mean square deviation (rmsd) range of . to . Å (data not shown), suggesting that the ala substitution in his , his , and lys of prrsv nsp in this study might not prevent the core conformations of nsp protein from folding correctly. on the basis of data presented here and those reported by others mentioned above, we speculate that the ability to interact with irf correlated with the correct folding of nsp but not with nendou activity or cell cytotoxicity. future investigations will be required to identify the structural basis of the nsp -irf interaction. although our data clearly demonstrated that prrsv nsp could be coimmunoprecipitated with endogenous irf in prrsv-infected cells, thereby inhibiting the formation and nuclear translocation of isgf (fig. ) , whether prrsv nsp also functions as an antagonist of ifn signaling during prrsv infection has not been fully understood. one of the obstacles to studying arterivirus with nendou activitydeficient nsp mutants in cell culture is that catalytic residue mutations have been reported to nearly completely abolish viral replication even in ifn-deficient cells ( , ) . future investigations designed to identify other nonactive site residues of nsp that are involved in the nsp -irf interaction but that do not affect prrsv replication will be required to fully elucidate the function of nsp in ifn signaling. in summary, our data identify prrsv nsp as a newly recognized antagonist in ifn signaling and show that the nsp -irf interaction is involved in inhibition of the formation and nuclear translocation of isgf . this novel function of prrsv nsp offers new insight into the interaction between a viral endou and the ifn signaling pathway, potentially aiding the development of novel therapeutic targets and more-effective vaccines against prrsv. cells and viruses. hek- t, marc- , and pk- cells, obtained from the china center for type culture collection, were cultured at °c and % co in dulbecco's modified eagle's medium (invitrogen, usa) supplemented with % fetal bovine serum. prrsv strain wuh (genbank accession number hm . ), isolated from the brain of a pig suffering from "high fever" syndrome in china at the end of , was amplified and titrated as described previously ( ) . plasmids. the luciferase reporter plasmid isre-luc and the cdna expression constructs encoding porcine stat , stat , and irf with an n-terminal flag tag have been described previously ( ) . the luciferase reporter plasmid isre-luc contained ifn-stimulated response elements in the promoter of most of the isgs cloned upstream of the firefly luciferase reporter gene. prl-tk plasmid (promega) was used as an internal control for normalization of the transfection efficiency. the nsp gene of prrsv strain wuh was amplified and cloned into pcaggs-ha with an n-terminal ha tag. prrsv nsp mutants (h a, h a, and k a) were constructed by overlap extension pcr using specific mutagenic primers (available upon request) in a pcaggs-ha background. prrsv nsp and its mutants with an n-terminal ha tag were cloned into pgex- p- with a glutathione s-transferase (gst) tag in the n terminus. irf deletion mutants were also amplified by pcr and constructed in a pcaggs-flag background. pedv nsp was amplified from pedv strain aj ( ) and cloned into vector pcaggs-ha with a ha tag. pedv nsp mutants (h a) were constructed using pedv nsp as the template. all constructed plasmids were confirmed by sequencing. luciferase reporter gene assay. hek- t or pk- cells grown in -well plates were cotransfected with reporter plasmid isre-luc ( . g/well), prl-tk ( . g/well), and the indicated expression plasmids or an empty control plasmid. where indicated, cells were further treated with ifn-␣ (pbl assay science) ( , u/ml) h after the initial cotransfection. cells were lysed h later, and firefly luciferase and renilla luciferase activities were measured using a dual-luciferase reporter assay system (promega) according to the manufacturer's protocol. the relative levels of firefly luciferase activities were standardized as prl-tk activities. data are presented as means and standard deviations (sd). p values of Ͻ . were considered statistically significant, and p values of Ͻ . were considered highly statistically significant. rna extraction and quantitative real-time pcr. in brief, total rna was extracted from cells using trizol reagent (invitrogen), and rna ( g) was reverse transcribed into cdna using avian myeloblastosis virus reverse transcriptase (takara, japan). the cdna ( l of l) was then used in a sybr green real-time pcr assay (applied biosystems). the abundance of individual mrna transcripts in each sample was determined three times and normalized to that of glyceraldehyde- -phosphate dehydrogenase (gapdh) mrna. all quantitative pcr (qpcr) primers used in this study have been described previously ( ) . protein expression and purification. for protein expression, the recombinant prokaryotic expression plasmids encoding the n-terminally gst-ha-tagged wt nsp and mutant (h a, h a, k a) proteins were transformed into e. coli strain trans bl (de ) and cultured at °c in lb medium containing g/ml ampicillin. when the culture optical density at nm (od ) reached . to . , the cells were induced by the use of . mm iptg (isopropyl-d- -thiogalactopyranoside) for h at °c. for protein purification, the cultured cells were collected by centrifugation at , rpm for min and resuspended in phosphate-buffered saline (pbs) followed by disruption with ats ah- . the supernatant was filtered by the use of a filter with a . -mm-pore-size membrane and was then loaded onto a glutathione-sepharose b column (ge healthcare). to gain gst-free proteins, the harvested fusion proteins with an n-terminally gst tag were incubated with gst- c rhinovirus protease at a ratio of : for h at °c, and the cleavage products in the cleavage buffer were further separated by reloading onto glutathione-sepharose b column. finally, the target proteins were eluted by the use of ml cleavage buffer. the purified proteins were stored at Ϫ °c for detection of enzyme activity. enzyme activity assay. the rna substrate ( =- -carboxyfluorescein-da-ru-da-da- -carboxy-n,n,n,n-tetramethylrhodamine- =) was purchased from nanjing genscript company. the n-terminally ha-tagged prrsv nsp /h a/h a/k a proteins and the rna substrate were placed in a reaction buffer containing mm hepes (ph . ), mm kcl, and mm dithiothreitol (dtt) diluted with . % diethyl pyrocarbonate-treated water. the concentration of proteins used in the assay was m, and the substrate concentration was m. the endoribonuclease activity was monitored every min for min in a fluorescence plate reader, with excitation at a wavelength of nm and emission at a wavelength of nm. the results were analyzed by the use of graphpad prism software . . western blot analyses and subcellular fractionation. cells were grown in -mm-diameter dishes and harvested with lysis buffer (beyotime, china) plus nm phenylmethylsulfonyl fluoride (pmsf) and phosstop phosphatase inhibitor (sigma, usa). samples were then separated by sds-page and transferred to polyvinylidene difluoride membranes (millipore, usa) to determine the protein expression levels. overexpression of stat , stat , irf , and irf deletion mutants was evaluated using mouse monoclonal anti-flag antibody (macgene, china). the expression of prrsv nsp and its mutants was analyzed using mouse monoclonal anti-ha antibody (mbl, japan). rabbit polyclonal antibodies against irf (santa cruz, usa), stat (santa cruz, usa), stat (abclonal, china), phospho-stat (stat -y , cell signaling technology, usa), and phospho-stat (stat -y , abclonal, china) were used to detect the protein levels and phosphorylated forms of each of the respective endogenous proteins. monoclonal antibody (mab) against prrsv nsp was produced from hybridoma cells derived from sp / myeloma cells and spleen cells of balb/c mice immunized with recombinant nsp protein from prrsv strain wuh . the isotype of nsp -specific mab is mouse igg . the specificity of mab against prrsv nsp was confirmed by specific reactions performed with pcaggs-ha-nsp -transfected cells and prrsv-infected cells. to analyze the nuclear translocation of isgf , nuclear fractions were extracted from cells using an ne-per nuclear and cytoplasmic extraction reagents kit (thermo fisher scientific, usa) according to the manufacturer's protocol. the cytoplasmic and nuclear fractions were subjected to western blotting. successful isolation was assessed using rabbit polyclonal antibodies against heat shock protein (hsp ; proteintech, china) and parp (proteintech, china) with cytoplasmic and nuclear protein markers, respectively. coimmunoprecipitation and immunoblotting analyses. to test the interactions between proteins, hek- t cells or marc- cells from each -mm-diameter dish were lysed in radioimmunoprecipitation assay (ripa) lysis buffer containing mm tris-hcl (ph . ), mm nacl, % triton x- , % sodium deoxycholate, . % sds, nm pmsf, and phosstop phosphatase inhibitor (sigma, usa), and the protein concentration was measured and adjusted. for each immunoprecipitation, g of cell lysate protein was incubated with . g of the indicated antibody and l of protein aϩg agarose (beyotime, china) overnight at °c. the agarose beads were then washed three times with ml of lysis buffer. the precipitates were subjected to % sds-page and subsequent immunoblot analysis using the indicated antibodies. in some immunoblotting analyses, the horseradish peroxidase-conjugated goat anti-rabbit igg light chain (ipkine, usa) were used to eliminate the interference of the rabbit igg heavy chain. indirect immunofluorescence assay. marc- cells cultured on coverslips in -well plates were infected with prrsv at a multiplicity of infection (moi) of . . at h postinfection, the infected cells were mock treated or treated with recombinant human ifn-␣ (pbl assay science) ( , u/ml) for h. the cells were then fixed with % paraformaldehyde for min and immediately permeabilized using methanol (precooled at Ϫ °c) for min at room temperature (rt). after three washes with pbs, cells were incubated with % bovine serum albumin (bsa)-pbs overnight at °c and incubated with the primary antibody for h. the antibodies used were as follows: anti-phospho-stat (cell signaling technology, usa), anti-phospho-stat (cell signaling technology, usa), anti-irf (abclonal, china), and anti-nsp mab. anti-mouse igg (hϩl) antibody conjugated to alexa fluor or anti-rabbit igg (hϩl) antibody conjugated to alexa fluor was diluted to : for use as the secondary antibody, after which the cells were stained with ', -diamidino- -phenylindole (dapi; beyotime, nantong, china)-pbs for min. fluorescent images were acquired with a confocal laser scanning microscope (olympus fluoview ver. . , japan). porcine reproductive and respiratory syndrome virus (prrsv): pathogenesis and interaction with the immune system the ever-expanding diversity of porcine reproductive and respiratory syndrome virus the prrsv replicase: exploring the multifunctionality of an intriguing set of nonstructural proteins prrsv structure, replication and recombination: origin of phenotype and genotype diversity the structural biology of prrsv antiviral strategies against prrsv infection porcine reproductive and respiratory syndrome (prrs): an immune dysregulatory pandemic interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures jak-stat pathways and transcriptional activation in response to ifns and other extracellular signaling proteins the janus protein tyrosine kinase family and its role in cytokine signaling tyrosinephosphorylated stat and stat plus a -kda protein all contact dna in forming interferon-stimulated-gene factor how cells respond to interferons interferon-stimulated genes and their antiviral effector functions interplay between janus kinase/signal transducer and activator of transcription signaling activated by type i interferons and viral antagonism porcine deltacoronavirus nsp antagonizes type i interferon signaling by cleaving stat rotavirus nsp mediates degradation of interferon regulatory factors through targeting of the dimerization domain rotavirus nsp protein inhibits interferon-mediated stat activation structural basis of the inhibition of stat activity by sendai virus c protein nonstructural protein of porcine reproductive and respiratory syndrome virus suppresses both mavs and rig-i expression as one of the mechanisms to antagonize type i interferon production endoribonuclease activities of porcine reproductive and respiratory syndrome virus nsp was essential for nsp to inhibit ifn-beta induction a dimerization-dependent mechanism drives the endoribonuclease function of porcine reproductive and respiratory syndrome virus nsp interferon signalling network in innate defence structural biology of the arterivirus nsp endoribonucleases nonstructural protein (nsp ) of porcine reproductive and respiratory syndrome virus (prrsv) promotes prrsv infection in marc- cells biochemical characterization of arterivirus nonstructural protein reveals the nidovirus-wide conservation of a replicative endoribonuclease site-directed mutagenesis of the nidovirus replicative endoribonuclease nendou exerts pleiotropic effects on the arterivirus life cycle coronavirus endoribonuclease activity in porcine epidemic diarrhea virus suppresses type i and type iii interferon responses porcine epidemic diarrhea virus infection inhibits interferon signaling by targeted degradation of stat ns of dengue virus mediates stat binding and degradation zika virus targets human stat to inhibit type i interferon signaling porcine bocavirus np negatively regulates interferon signaling pathway by targeting the dna-binding domain of irf the ebola virus interferon antagonist vp directly binds stat and has a novel, pyramidal fold the irf family transcription factors in immunity and oncogenesis stat nuclear trafficking mapping of nine porcine interferon regulatory factor genes distinct stat structure promotes interaction of stat with the p subunit of the interferon-alpha-stimulated transcription factor isgf two domains of isgf gamma that mediate protein-dna and protein-protein interactions during transcription factor assembly contribute to dna-binding specificity the viral innate immune antagonism and an alternative vaccine design for prrs virus the nonstructural protein of porcine reproductive and respiratory syndrome virus inhibits nf-kappab signaling by means of its deubiquitinating activity the superimposed deubiquitination effect of otulin and prrsv nsp promoted the multiplication of prrsv an "old" protein with a new story: coronavirus endoribonuclease is important for evading host antiviral defenses coronavirus nonstructural protein mediates evasion of dsrna sensors and limits apoptosis in macrophages early endonuclease-mediated evasion of rna sensing ensures efficient coronavirus replication the emerging role of interferon regulatory factor in the antiviral host response and beyond the type i interferon-alpha mediates a more severe neurological disease in the absence of the canonical signaling molecule interferon regulatory factor mice deficient in stat but not stat or irf develop a lethal cd ϩ t-cellmediated disease following infection with lymphocytic choriomeningitis virus the human papillomavirus e oncoprotein abrogates signaling mediated by interferon-alpha varicella viruses inhibit interferon-stimulated jak-stat signaling through multiple mechanisms effects of adenovirus e a protein on interferonsignaling human cytomegalovirus inhibits ifn-alpha-stimulated antiviral and immunoregulatory responses by blocking multiple levels of ifn-alpha signal transduction porcine reproductive and respiratory syndrome virus nsp beta inhibits interferon-activated jak/stat signal transduction by inducing karyopherin-alpha degradation porcine reproductive and respiratory syndrome virus inhibits type i interferon signaling by blocking stat /stat nuclear translocation structural basis of stat recognition by irf reveals molecular insights into isgf function mir- b reduces porcine reproductive and respiratory syndrome virus replication by negatively regulating the nf-kappab pathway complete genome sequence of porcine epidemic diarrhea virus strain aj isolated from a suckling piglet with acute diarrhea in china this work was supported by the major project of the national natural science foundation of china (grant ), the national basic research program ( ) of china (grant cb ), the national natural science foundation of china (grants key: cord- - vql u authors: belova, natalya v.; girichev, georgiy v.; kotova, vitaliya e.; korolkova, kseniya a.; trang, nguyen hoang title: the molecular structure of -methylpyridine-n-oxide: gas-phase electron diffraction and quantum chemical calculations date: - - journal: journal of molecular structure doi: . /j.molstruc. . . sha: doc_id: cord_uid: vql u abstract the molecular structure of -methylpiridine-n-oxide, -mepyo, has been studied by gas-phase electron diffraction monitored by mass spectrometry (ged/ms) and quantum chemical (dft) calculations. both, quantum chemistry and ged analyses resulted in c s molecular symmetry with the planar pyridine ring. obtained molecular parameters confirm the hyperconjugation in the pyridine ring and the sp hybridization concept of the nitrogen and carbon atoms in the ring. the experimental geometric parameters are in a good agreement with the parameters for non-substituted n-oxide and reproduced very closely by dft calculations. the presence of the electron-donating ch substituent in -mepyo leads to a decrease of the ipso-angle and to an increase of r(n→o) in comparison with the non-substituted pyo. electron density distribution analysis has been performed in terms of natural bond orbitals (nbo) scheme. the nature of the semipolar n→o bond is discussed. the considerable interest to the heterocyclic compounds containing n-oxide group is caused by their pronounced biological activity [ ] . among the representatives of this class, there are compounds with bactericidal, analgesic, anticonvulsant activity, as well as ones with apoptotic, antifungal and antimicrobial activity [ , ] . these properties allow to use n-oxides as inhibitor of hiv- reverse transcriptase [ , ] , as antiviral agent against various sars coronavirus strains [ ] , and as antiadhesive and quorum-sensitive inhibitor [ ] . in the literature one can find the evidence, that some complexes with pyridine-n-oxides are used in agriculture to regulate plant growth rates [ ] . it was shown that pyridine-n-oxide derivatives are genotoxic [ ] . the biochemical activity of n-oxides is usually considered to be the result of complexation with the metalloporphyrins in living organisms [ e ] . the reactivity of noxides may vary due to different substituents in the ring [ ] . the variation of the substituents gives wide possibilities to chemical modifications of n-oxides and allows to influence their complexing properties, and, as the result, their biological activity. to understand the complexation ability of n-oxides with different substituents the detailed information about the properties of free molecules is needed. the available literature data about molecular structure of noxides can not be considered comprehensive. thus, only pyridine-n-oxide and three of its para-substituted compounds have been studied by gas-electron diffraction (ged) [ , ] . analyzing the average values of bond lengths in the pyridine ring the authors of [ ] have concluded that the aromaticity increases in the series: nitropyridine-n-oxide ( -no pyo), -chloropyridine-n-oxide ( -clpyo), pyridine-n-oxide (pyo), -methylpyridine-n-oxide( -mepyo). the crystal structures of pyo, -no pyo and -mepyo have been studied by x-ray crystallography [ , ] . the quantum chemical study of the substituent effect on the properties of pyridine-n-oxides has been performed for ten compounds with different substituents [ ] . the comparative analysis of structural data obtained by different methods suggests that the molecular parameters of n-oxides obtained by ged [ ] look rather strange. thus, the nec bond lengths in -no pyo, -mepyo, -clpyo recommended in ref. [ ] are even longer than r(cec) in benzene ring, and seems to be overestimated compare to the calculated values and x-ray data. moreover, the bond distance r(n/o) in -mepyo molecule recommended by authors [ ] appears to be significantly overestimated and conflicting with the calculated and x-ray values. furthermore, the accuracy of ged data obtained in refs. [ , ] cannot be considered today as satisfactory. the discrepancies in molecular structure of substituted n-oxides according to ged and other methods motivated us to perform a new gas-electron diffraction study of -methylpyridine-n-oxide. this molecule has been chosen because of the largest structural contradictions. now more sophisticated methods for the analysis of ged data are available, including the possibility of quantum chemical results to be used in the interpretation of experimental data. this allows us to determine the structural parameters more precisely compared to the studies performed in . the electron diffraction patterns and the mass spectra were recorded simultaneously using the techniques described previously [ , ] . two series of ged/ms experiments at two different nozzleto-plate distances were performed. the conditions of ged/ms experiments and the relative abundance of the characteristic ions of -mepyo are shown in tables and , respectively. the temperature of stainless steel effusion cell was measured by a w/re- / thermocouple, which was standardised using the melting points of sn and al. polycrystalline zno was used to establish the wavelength of the fast electrons. microphotometric measurements were carried out by means of automatic techniques [ ] . the molecular intensities were obtained in the ranges . ÷ . Å À (short camera) and . ÷ . Å À (long camera). the molecular intensities and the radial distributing curves are shown in figs. and respectively. quantum chemical calculations were carried out with gaussian program set [ ] . a hybrid dft computational methods, namely b lyp [ e ] and pbe [ ] , as well as mp method, were used. structure optimizations were followed by calculations of the vibrational frequencies in order to ensure that a minimum on the potential energy hyper-surface had been reached. analysis of the potential functions of methyl group internal rotation shows that there is only one stable geometric configuration with h c c c about (see fig. for atom numbering). although it should be noted that the internal rotation barrier is negligibly small, . kcal/mol (b lyp/cc-pvtz) or . kcal/mol (mp /cc-pvtz), thus the rotation of ch group in -mepyo can be considered as free. the geometrical parameters of the calculated equilibrium structure are listed in table . vibrational amplitudes and corrections, dr ¼ r h À r a , were derived from theoretical force fields (b lyp/cc-pvtz) by sipachev's method (approximation with taking into account the nonlinear kinematic effects at the level of the first order perturbation theory for the transformation of cartesian coordinates into internal coordinates), using the program shrink [ e ] . relevant values are listed in table (excluding nonbonded distances involving hydrogen). the nbo g program [ ] , implemented for natural orbital analysis in pc gamess [ ] , was used to obtain the net atomic charges, and to study the effect of hyperconjugation on the structure. b lyp/aug-cc-pvtz wave functions were used in the nbo analyses. relevant values of net atomic charges, wiberg bond indexes, and second order interaction energies (e ( ) ) between donoracceptor orbitals are collected in tables and . visualization of the natural orbitals performed by the chemcraft program [ ] . the heaviest ion in the mass spectrum observed during the ged/ ms experiment was [me-pyo] þ (table ) . no ions were detected, which could arise from impurities. this proves the monomeric vapour composition under the conditions of the ged experiment. the theoretical sm(s) functions were calculated with the following assumptions. independent r h parameters were used to describe the molecular structure. starting parameters from b lyp/ cc-pvtz calculations were refined. the differences between all cec, as well as between all ceh bond lengths were constrained to calculated values (b lyp/cc-pvtz). in compliance with the quantum chemical calculation results, a planar skeleton with c v symmetry of the pyridine ring was assumed. the methyl group was considered in the framework of c v symmetry. vibrational amplitudes were refined in groups with fixed differences. with the abovementioned assumptions, four bond distances and four bond angles (table ) were refined simultaneously with six groups of vibrational amplitudes. vibrational amplitudes were refined in groups with fixed differences (table ). only two correlation coefficients had absolute values larger than . : (r(nec ))/ (r(c ec )) ¼ À . , and (:nc c )/(:c nc ) ¼ - . . the best agreement factor is r f ¼ . %. final results of the least squares analysis are given in table (geometric parameters) and table (vibrational amplitudes). table summarizes the structural parameters of -me-pyo. we can note a perfect agreement between calculated and experimental molecular structure parameters. the molecule possesses almost c s symmetry (the torsional angle h c c c is close to ) with the planar pyridine ring. table compares gas-phase structures of pyo and -me-pyo obtained in refs. [ , ] and in this work. the parameters obtained in our research is in a good agreement with the theoretical results (therein and [ ] ) and, also, with the parameters for non-substituted n-oxide in contrast to the values recommended by chiang j. f. et al. [ ] . furthermore, the parameters of free molecules of pyo [ ] and -me-pyo obtained in this work are in a good agreement with the crystal structures [ , ] . apparently, some increase of the semipolar n/o bond lengths in the crystal phase compared to free molecules is due to intermolecular hydrogen bonding. all parameters presented in table confirm the hyperconjugation in the pyridine ring and the sp hybridization concept of the nitrogen and carbon atoms in the ring. the exceptions are only the data by chiang j. f. et al. [ ] . which look rather strange. unfortunately, the correct comparison of ged parameters of -mepyo and non-substituted pyridine-n-oxide looks to be impossible because of large uncertainties in ged values obtained for pyo [ ] . according to quantum chemical calculations the insertion of the methyl group in para e position to the pyo has no significant effect on the structural parameters of the pyridine ring, excluding only the ipso-angle : c c c , which changes from . in pyo to . in -mepyo (b lyp/cc-pvtz). it is interesting to note, that the substituent effects on the geometry of heterocyclic ring are rather similar to the tendencies for the benzene ring [ , ] . thus, the presence of the electron-donating ch substituent leads to the decrease of the ipso-angle. furthermore, according to the calculations the r(n/o) bond lengths appear to be also sensitive to a fig. . b uncertainties in r h s¼(s sc þ( . s ls ) ) / (s sc ¼ , r, s ls estandard deviation in least-squares refinement), for angles s ¼ s ls . c p i e parameter refined independently. table . the cec and cen bond orders in the ring for both molecules, as well as the values of the distances from tables and show that these bonds do not correspond to either single nor double bonds. this confirms the existence of p-conjugation in the pyridine rings. the c ec bond order for -mepyo corresponds to the single cec bond. it is interesting to note that q(neo) in both molecules can not be interpreted as exact single or double bond. moreover, the introduction of an electron-donating substituent increases the bond length n/o, slightly reduces the corresponding bond order and significantly increases the electron density on the oxygen. one can conclude that in this case the nucleophilic properties of the molecule increase. furthermore, as it has been mentioned previously [ ] the introduction of electron-donating substituents to the heterocyclic ring of n-oxides results in increasing ability for complex formation. our experimental and calculated data (tables , and ) reveal the incorrectness of the authors of [ ] in their conclusion about more single neo bond character in -mepyo. the delocalization of electron density between occupied lewis type (bonds or lone pairs) nbo orbitals and formally unoccupied (antibonding) non-lewis nbo orbitals corresponds to a stabilizing donor-acceptor interaction. the energy of these interactions can be estimated by the second order perturbation theory. table collects the largest values of calculated second order interaction energies (e ) between donor-acceptor orbitals in pyo and -mepyo. in both cases several values correspond to the interactions related to the resonance in the pyridine ring, i.e. the interactions between p(cec) and p*(cec) and between p(cec) and p*(cen). the nature of semipolar neo bond was explained in detail [ ] . the largest second order interaction energies (e ) in both molecules corre- to study the substituent effect on the aromaticity the nuclear independent chemical shifts nics( ) have been calculated by using b lyp/aug-cc-pvtz approximation. nics( ) values correspond to the negative isotropic shielding (in ppm) at Å above the ring plane. the calculated values of nics( ): À . (pyo) and À . ( -me-pyo) are very close to each other. thus, from the nics( ) values one can conclude that the introduction of ch group does not noticeably affect the aromaticity of the pyridine-n-oxide. furthermore, the data of table do not confirm the conclusion of the authors of [ ] about changing the aromaticity with substitution, which was made by analyzing the average values of bond length in the pyridine ring. ab initio scf and ci study of the electronic spectrum of pyridine noxide -and -nitro- -azabenzo[a]pyrens and their n-oxides: hifly mutagenic nitrated azaarenes pridine oxide derivatives: structureactivity relationship for inhibition of human immunodeficiency virus and cytomegalovirus replication in cell culture pyridine n-oxide derivatives are inhibitory to the human sars and feline infectious peritonitis coronavirus in cell culture aromatic amine oxides genotoxic impurities,strategies for identification and control purification and properties of trimethylamine n-oxide reductase from aerobic photosynthetic bacterium roseobacter denitrificans non-enzymatic reduction of aliphatic tertiary amine n-oxides mediated by the haem moiety of cytochrome p nonenzymatic reduction of brucine n-oxide by the heme group of ctocrome p electronic factors effects on the reactivity of heteroaromatic noxides molecular structure of pyridine n oxide molecular structures of -nitro-, -methyl-and -chloropyridine-n-oxides substituent effect on the properties of pyridine-n-oxides upgrading the emr- electrondiffraction camera for use with gases apparatus for study of molecular structure of valence-unsaturated compounds, instruments and experimental techniques cr revisited density-functional thermochemistry. iii. the role of exact exchange density-functional exchange-energy approximation with correct asymptotic behavior development of the colle-salvetti correlation-energy formula into a functional of the electron density accurate spin-dependent electron liquid correlation energies for local spin density calculations: a critical analysis toward reliable density functional methods without adjustable parameters: the pbe model local centrifugal distortions caused by internal motions of molecules calculation of shrikage corrections in harmonic aproximation advances in molecular structure research chemcraft version . (build ) crystal structure of -methylpyridine noxide isothermal and isochoric crystallization of highly hygroscopic pyridine n-oxide of aqueous solution molecular geometry of substituted benzene derivatives. i. on the nature of the ring deformations indused by sublimations group electronegativities from benzene ring deformations: a quantum chemical study key: cord- -nuhrd z authors: hung, kevin k. c.; mark, carman k. m.; yeung, may p. s.; chan, emily y. y.; graham, colin a. title: the role of the hotel industry in the response to emerging epidemics: a case study of sars in and h n swine flu in in hong kong date: - - journal: global health doi: . /s - - - sha: doc_id: cord_uid: nuhrd z background: the global travel and tourism industry has been rapidly expanding in the past decades. the traditional focus on border screening, and by airline and cruise industries may be inadequate due to the incubation period of an infectious disease. this case study highlights the potential role of the hotel industry in epidemic preparedness and response. methods: this case study focuses on the epidemic outbreaks of sars in and h n swine flu in in hong kong, and the subsequent guidelines published by the health authority in relation to the hotel industry in hong kong which provide the backbone for discussion. results: the metropole hotel hastened the international spread of the sars outbreak by the index case infecting visitors from singapore, vietnam, canada as well as local people via close contact with the index case and the environmental contamination. the one-week quarantine of more than guests and staff at the metropark hotel during the h n swine flu exposed gaps in the partnership with the hotel industry. the subsequent guidelines for the hotel industry from the centre of health protection focused largely on the maintenance of hygiene within the hotel premises. conclusion: positive collaborations may bring about effective preparedness across the health and the tourism sectors for future epidemics. regular hygiene surveillance at hotel facilities, and developing coordination mechanism for impending epidemics on the use of screening, swift reporting and isolation of infected persons may help mitigate the impact of future events. preparedness and contingency plans for infectious disease control for the hotel industry requires continuous engagement and dialogue. the global travel and tourism industry has expanded rapidly in recent years. the global number of international tourist arrivals increased from approximately million in to million in [ ] . the ever greater numbers present enormous challenges to the entire global community for epidemic preparedness and control. the increasing complexity of frequent international travel opens an ideal route for local outbreaks of infectious disease to becoming global pandemics. the health and wellbeing of travellers warrants appropriate consultation and treatment in its own right, but in the case of infectious diseases of major public health concern, it is important to address the public health aspects of their illness as patients are also disease carriers promoting the spread of infectious disease on a potentially global scale. the international health regulations (ihr) ( ) form one of the few legally binding instruments of the world health organisation (who). the purpose and the scope of ihr ( ) are "to prevent, protect against, control and provide a public health response to the international spread of disease in ways that are commensurate with and restricted to public health risks, and which avoid unnecessary interference with international traffic and trade" [ ] . most of the existing research in travel medicine and the current guidelines for international health authorities emphasise the role of the air-travel industry in tracking and thus containing the potential international spread of infectious disease. for instance, the who guideline on international travel and health highlights the role of airlines as well as shipping companies, together with that of tour operators and travel agents in limiting the spread of infectious disease across borders [ ] . similarly, the centres for disease control and prevention (cdc) guidelines focus on the airline and cruise industries [ ] . the role of the other important sector in tourism, namely the hotel industry, have been less clearly defined and discussed. this case study will use the example of the metropole hotel in hong kong in the international spread of severe acute respiratory syndrome (sars) in , and the effect of the government mandated quarantine of the metropark hotel during the swine flu in hong kong. sars was caused by the sars-associated coronavirus, with the primary mode of transmission being direct mucous membrane contact with infectious respiratory droplets [ , ] . it had a basic reproduction number of approximately , and the spread was mainly to those with close contact in health care and household settings. the first cases were identified in guangdong province of china in november , and a number of natural reservoirs have been found including civet cats and bats. incubation period ranged from to days [ ] . the overall case fatality ratio was . % worldwide, with deaths resulting from pneumonia and subsequent respiratory failure. the travel industry contributed to the speed of the spread of this unknown disease at that time, particularly within the metropole hotel as it was the first site for global dissemination of the virus [ ] . sars subsequently caused deaths in countries, with the disease spreading to cover five continents [ ] . this case vividly illustrates how a local outbreak can rapidly evolve into a global pandemic. the h n swine flu pandemic in was caused by the a(h n ) pdm influenza virus [ ] . this strain containing genes from pig, bird and human influenza viruses has never been reported before , and the outbreak was first identified in march in mexico and the united states. it was estimated that between , and , deaths resulted globally, with % in people younger than years old from respiratory and cardiovascular influenza related complications. the h n virus infection mortality was estimated to be . - . % of the world's population, much lower than the previous . % during the pandemic and the - % of the pandemic [ ] . the first imported case in hong kong was tested positive for swine influenza on the may [ ] . this has led to the subsequent quarantine of all guests and staff at the metropark hotel for week. hotels can be a critical component in the evolution of a local outbreak into a global pandemic, and an initial contact point of the import of an impending global pandemic. the aim of this paper is to highlight the role of hotel in the spread of epidemic, and discuss control measures that can be implemented. this case study focuses on the epidemic outbreaks of sars in and h n swine flu in in hong kong. secondary information was extracted from the literature search and the grey literature looking for published official reports, statements, policy papers, field reports and guidelines for further discussion on the role of the hotel industry on the epidemics. public health principles of disaster response were used to provide the backbone for discussion. medline was used to identify research articles published between january and december in the english language, to answer these specific questions: . what factors in the hotel setting contributed to international spread of sars; . the decision making and implementation of hotel quarantine during swine flu, and the impact of the hotel quarantine. any study design regarding aspects on timelines of sequence of events, environmental sampling and contamination, evaluation of hotel related interventions, modelling of interventions were included. studies not meeting the above inclusion criteria or answering the research questions was excluded. the the search identified records from medline of which five were relevant (references [ , [ ] [ ] [ ] [ ] ), and an additional records were identified through other sources and the grey literature. a medical professor from guangzhou in china arrived in hong kong on february and checked into a room on the ninth floor of the metropole hotel in kowloon [ ] . during his stay, he infected at least seven other guests and visitors staying on the ninth floor of the hotel including three visitors from singapore, one visitor from vietnam, two visitors from canada and a local individual [ ] . on march , the singapore ministry of health reported to the hong kong department of health that three patients who presented with pneumonia were admitted to hospital after returning to singapore from hong kong. they had all stayed in the metropole hotel. during the conversation, laboratory investigations were pending and there was not sufficient evidence to suggest that their illnesses had been related to the metropole hotel [ ] . on march , the who issued a global alert about unusual cases of an acute respiratory syndrome. on march , the index case for the significant outbreak at the prince of wales hospital was identified. it was not until march , after multiple enquiries, the patient revealed that he had visited the metropole hotel around that period as a visitor but not a guest [ ] . on the same day, the hong kong department of health reported the chain of transmission of the outbreak at the metropole hotel [ ] . the metropole hotel exemplified the potential international spread of infectious diseases. the index cases in the hong kong, toronto, singapore and hanoi outbreaks were all associated with the hotel. sars patients in ireland and united states had also visited the metropole hotel around the same time when there were other sick guests present in the hotel [ ] [ ] [ ] ] . subsequently there were hospital outbreaks when these index cases returned and were treated at their home countries. table showed the timeline of the sars action of the department of health. little was known about the new disease sars when the outbreak began at hotel and hospitals in february and early march. the who did not issue its first emergency travel advisory naming the illness as sars until march [ ] . there was local media coverage of an outbreak of atypical pneumonia in guangzhou on february , and the health authority in hong kong had made contact with the guangzhou and guangdong authorities. however, accurate information about the atypical pneumonia outbreak in guangdong province was not available to hong kong or the international community at the time. case investigation and contact tracing conducted by the department of health on february on the index case revealed that the guangzhou professor and his wife had stayed at the metropole hotel. however, no contact tracing was conducted in the hotel at that stage since the department of health had not received any other reports of severe community-acquired pneumonia related to the hotel [ ] . there were no environmental factors or triggers identified that warranted further action. however, environmental sampling on the carpet outside the room in which the index case resided, and elevator area showed a hot zone which tested positive for the sars virus by polymerase chain reaction (pcr) months after the index case stayed at the hotel [ ] . another study investigating german guests staying at the hotel also suggested the possibility of environmental contamination as a source of infection [ ] . it is not known how long the infectious virus persists in the surroundings of a sars patient. the established practice was for contact tracing note: materials extracted from references [ , ] to be conducted on close contacts (friends or family), but not on the basis of a shared location [ ] . a number of researchers estimated the basic reproduction number of sars by fitting models to the initial growth of the epidemics in a number of countries [ ] . modelling of sars epidemiology in hong kong and china showed rapid public health measures such as contact tracing for confirmed and suspected cases, and quarantining, monitoring, and restricting the travel of contacts had an effective reduction in reproduction number [ ] [ ] [ ] [ ] . a -year-old male from mexico arrived in hong kong on april , and stayed at the metropark hotel. he attended hospital on the same evening where he was admitted to an isolation ward. he subsequently developed a fever and was confirmed to have swine flu on may [ ] . the hong kong special administrative region (hksar) government raised the response level to 'emergency' on the same day under the emergency preparedness plan for influenza pandemic. an 'emergency response level steering committee on human swine influenza (flu a h n ) pandemic' was also established on the st may to formulate the overall disease control strategy [ ] . under the prevention and control of disease ordinance, the director of health ordered that all guests and staff at the metropark hotel should be quarantined on the evening of may [ ] . the quarantine was led by the department of health in collaboration with other government departments. quarantined persons were provided with oseltamivir (tamiflu) and other medical treatment. the social welfare department provided daily necessities and emotional support to the quarantined. a help desk was set up at the hotel involving the department of health, the home affairs department, the social welfare department, the immigration department, the civil aid service, the auxiliary medical service and the police. the quarantine ended week later, which covered the incubation period of influenza of to days. for those persons who had completed the quarantine period without showing symptoms of being infected, they were issued with certificates of conclusion of quarantine. at the same time, the centre for health protection (chp), other government departments and relevant agencies conducted contact tracing starting on the st may . close and selected social contacts were prescribed chemoprophylaxis and put under medical surveillance. the hotel and nearby streets, as well as other public places, were cleansed and disinfected. hygiene guidelines had been issued to all licensed hotels/ guesthouses and rented rooms to encourage enhanced cleansing and improvement of hygiene. all industrial associations had been informed of the situation and reminded the need to take precautionary measures. the occupational safety and health council had organised public seminars to raise public awareness of preparedness for influenza in the workplace [ ] . table showed the timeline of the swine flu action of the department of health. both the metropole hotel at kowloon (now renamed metropark hotel kowloon) and the metropark hotel at wan chai (now renamed kew green hotel) were four stars hotels situated at busy part of the city. both hotels were managed by the same management groupthe china travel service (holdings) hong kong limited. the two hotels were no different in terms of access to public health facilities and general standard of care. the difference in the timing of public health actions by the health authority was likely contributed by experience of sars preceding swine flu. after the sars outbreak in hong kong the health authority established the guidelines for hotels in preventing severe acute respiratory syndrome (sars) [ ] hygiene guidelines have been issued to all owners' corporations, owners' committee, mutual assistance committees and the hong kong association of property management companies, licensed hotels/guesthouses and bed space apartments to encourage enhanced cleansing and improvement of hygiene. note: materials taken from references [ , ] and guidelines on infection control & prevention in the hotel industry [ ] . the guidelines provided practical information for hotel staff members on how measures to prevent communicable diseases should be done. it offered comprehensive information on ways to implement infection control measures, in particular the maintenance of good hygiene on hotel premises [ ] . the chp organised infection control seminars for the hotel industry on a regular basis. for instance, in response to the ebola virus outbreak in west africa in - , the chp provided advice for the local hotel industry on receiving guests with a travel history or residence in an ebola virus disease affected area. the guideline stressed the importance of enquiring about the travel history of guests and outlined procedures on handling these guests who may feel unwell. the guideline reiterated the need to keep a record of staff and residents who had stayed in the hotel, with their personal and contact details, for possible future public health actions and contact tracing [ ] . ideally, hotels should be setting their own standards of hygiene measures and providing training to staff before an outbreak occurs. further roles and responsibilities included contingency arrangement, plan of acquisition of protective equipment, disease reporting and surveillance mechanism during outbreak period [ ] . from our online and database internet search, however, there is little mention of collaboration between the government and the hotel industry. no documentation was found on setting up of task forces or committees, or of invitation to hotel representatives to the working group advising on infection control guidelines in hong kong. sars served as the classic example of how tourism and international travel can present challenges to the global health system. the spread of the illness within a single hotel and the subsequent international air travel of the victims contributed to and accelerated the speed of the spread of sars across the globe. the experience from sars in hong kong had a profound impact on the public health reform especially on the infectious disease surveillance and epidemic response [ ] . these included strengthening the surveillance and the isolation and treatment of individuals with the disease according to case definitions, the establishment of communication channels between hotel and the government system, and the development of guidelines and response plans that allows the implementation of stringent infection control measures when necessary. the establishment of contingency plans and command structures including the 'emergency response level steering committee on influenza pandemic' allowed a clear structural framework and key lines of responsibility [ ] . however, collaboration with the private sector and the hotel industry were found to be limited and focused around infection control measures. according to subsequently published literature, application of appropriate measures had likely reduced the number of people who were infected, requiring medical care and died during the influenza pandemic [ ] . it has been shown that case isolation or household quarantine could have a significant impact at reducing attack rates in the community, and chemoprophylaxis can greatly reduce disease transmission during the pandemic [ , ] . however, the quarantine of guests at the metropark hotel in inevitably stirs up much discussion and controversy among the media and the public health community on the balance between the need to protect the public health and the need to safeguard civil liberties. the decision of quarantine created enormous tension between the government, guests and the hotel management. the decision of the need for quarantine and the scale of the quarantine needs to be scientifically justified. the negative effects overall of such a policy on the tourism attractiveness of a destination cannot be neglected. the quarantine at the metropark hotel during swine flu also highlighted the extensive assistance needed for the quarantined persons, and the cooperation necessary in the possible future need for a hotel quarantine. pre-established partnerships and coordination between the government and the hotel industry is key in epidemic preparedness and response. studies have shown that the psychological impacts of sars and the government restrictions on travel, had a great impact on the travel industry far beyond the region of sars hit areas [ ] . for the hotel industry in hong kong, the number of hotel guests dropped dramatically to a level that was never experienced before [ ] . in order for hotels to sustain their business, the hong kong hotel industry adopted an industry-wide recovery effort and empathized on mutual support [ ] . previous papers called for a better preparedness of the hotel industry for future crises and epidemics [ , ] . collaborations with the hotel industry to mitigate the impact of epidemics hotels are often the first point of contact for tourists arriving at a host country. hotels could provide an additional line of defence beyond entry border screening, and they could offer another layer of protection against illnesses that border screening processes may have missed, for example in the situation where travel occurs during the incubation period of an infectious disease. in view of this, the capacity of hotels in the detection of potential illness and the launching of an initial response should be fully recognised and utilised. the who pandemic influenza risk management recommended involving civil society and the private business sector in pandemic preparedness planning and national committees [ ] . the case of hotel industry collaboration with the health sector in hong kong has the potential to provide a positive example of effective disaster risk reduction coordination. the epidemic preparedness and infection control measures mounted against sars and h n swine flu demonstrated a role that needed to be filled by the hotel industry. during sars, late recognition of the environmental contamination of hotel facilities and the failure of timely intervention on the hotel guests with close contact contributed to the spread of the disease internationally. while the appropriateness and best method of quarantine in future pandemic influenza warrants further research, the swine flu hotel quarantine exposed gaps in the partnership with hotel industry. health authorities in hong kong had since provided guidelines mostly in the area of disinfection and hygiene, and focused on educating hotel workers on basic hygiene to prevent the spread of infectious diseases. the potential to establish traveller screening, timely reporting and isolation for the infected guests during epidemics could be explored. the capacity of the hotel industry in controlling infections should be recognised not only in hong kong but also in other parts of the world. the world bank. international tourism, number of arrivals world health organization. international health regulations european centre for disease prevention and control. facts about severe acute respiratory syndrome (sars) sars: clinical presentation, 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hong kong special administrative region european centre for disease prevention and control. guide to public health measures to reduce the impact of influenza pandemics in europe strategies for mitigating an influenza pandemic public health interventions and sars spread the severe acute respiratory syndrome: impact on travel and tourism the survival of hotels during disaster: a case study of hong kong in pandemic influenza risk management: who interim guidance. geneva: world health organization chronology of major events of the sars incident in hong kong available at legislative council panel on health services not applicable. no funding available for this study.availability of data and materials not applicable. ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -wels wt authors: gottlieb, jens title: community-acquired respiratory viruses date: - - journal: semin respir crit care med doi: . /s- - sha: doc_id: cord_uid: wels wt the incidence of community-acquired respiratory viruses (carvs) is ∼ cases per patient-years after lung transplantation (ltx). paramyxoviruses account for almost % of the cases of carv infection in ltx. most patients will be symptomatic with a mean decline of to % in forced expiratory volume in second. the attributable death rate is low in recent years to % carv infected ltx patients will develop chronic lung allograft dysfunction within a year after carv infection. this risk seems to be increased in comparison to the noninfected ltx recipient. detection rate of carv dependent on clinical awareness, sampling, and diagnostic method with nucleic acid testing by polymerase chain reaction in bronchoalveolar lavage is the gold standard after ltx. there is no approved treatment for paramyxoviruses, most centers use ribavirin by various routes. toxicity of systemic ribavirin is of concern and some patients will have contraindication to this treatment modality. treatment may reduce the risk to develop chronic lung allograft dysfunction and respiratory failure. agents under development are inhibiting viral attachment and use silencing mechanisms of viral replication. the respiratory tract is a major entry through which viruses initiate infection. the respiratory tract can be divided anatomically into the upper respiratory tract and the lower respiratory tract divided by the lymphoid tissue of waldeyer's ring. community-acquired respiratory viruses (carvs) are highly developed and ubiquitous pathogens which can cause infection of both the upper and lower respiratory tract. infection with these viruses usually results in a mild, self-limited disease in the nonimmunocompromised adult. some carv may result in respiratory failure even with fatal outcome (e.g., middle east respiratory syndrome coronavirus, corona virus associated severe acute respiratory syndrome). carvs have been increasingly recognized as common pathogens after lung transplantation (ltx). carv are a diverse group of viruses including the paramyxoviridae: respiratory syncytial virus (rsv), parainfluenza virus (pv), human metapneumovirus (hmpv); the orthomyxoviridae: influenza a and b (flu); the picornaviridae: rhinovirus (rv) and enterovirus; the coronaviridae: coronavirus (cov); and the adenoviridae: adenovirus (av) (see ►table ). most viruses will cause local infection in the respiratory tract first with secondary dissemination to other sites in the body, while other viruses typically remain limited to the respiratory tract and induce tissue injury locally. various defense mechanisms have evolved in the respiratory tract to prevent and control infection. advances in the pathomechanisms involved in carv infection has been made recently. excessive inflammation associated with severe infection can be controlled by blocking costimulatory signals, without altering immune-mediated virus clearance. recent studies suggest that the activation of effector t cells in virus-infected lungs is generated by inflammatory cells locally. this modification of the immune response in the infected epithelium may lead to virus clearance and will regulate acute inflammation, and possibly immunological memory. resolution of respiratory virus infection requires not only the elimination of the keywords ► lung transplantation ► community-acquired respiratory viruses ► ribavirin ► bronchiolitis obliterns syndrome the incidence of community-acquired respiratory viruses (carvs) is $ cases per patient-years after lung transplantation (ltx). paramyxoviruses account for almost % of the cases of carv infection in ltx. most patients will be symptomatic with a mean decline of to % in forced expiratory volume in second. the attributable death rate is low in recent years to % carv infected ltx patients will develop chronic lung allograft dysfunction within a year after carv infection. this risk seems to be increased in comparison to the noninfected ltx recipient. detection rate of carv dependent on clinical awareness, sampling, and diagnostic method with nucleic acid testing by polymerase chain reaction in bronchoalveolar lavage is the gold standard after ltx. there is no approved treatment for paramyxoviruses, most centers use ribavirin by various routes. toxicity of systemic ribavirin is of concern and some patients will have contraindication to this treatment modality. treatment may reduce the risk to develop chronic lung allograft dysfunction and respiratory failure. agents under development are inhibiting viral attachment and use silencing mechanisms of viral replication. virus but also the repair and regeneration of normal lung structures and restoration of normal pulmonary function. cells and molecules regulate processes such as epithelial cell to mesenchymal cell transitions, as well as the transformation of fibroblasts and fibrocytes into myofibroblasts. dysregulation of these processes in response to lung inflammation is associated with progressive pulmonary injury and lung fibrosis. in ltx recipients, carv infection may cause inflammatory processes mediated by both innate and adaptive immune responses that result in injury to airway epithelial cells and subepithelial structures leading to obliteration of small airways. apart from direct sequelae, carv may promote immunologically mediated lung injury resulting in the development of acute rejection (ar). a clinical term for chronic lung allograft dysfunction (clad) is bronchiolitis obliterans syndrome (bos). the clinical picture is an irreversible decline in forced expiratory volume in second (fev ). histopathologically obliteration of terminal bronchioli by fibromyxomatous tissue is recognized. bos is the most important prognosis limiting factor following ltx, and remains the major impediment to long-term graft and patient survival after ltx. on average, it affects every second recipient after years. the annual incidence of bos after ltx is $ %. bos is a progressive disease and % of bos patients die from respiratory failure with a -year survival in affected patients of %. in ltx recipients, symptoms of carv infections may resemble those of other clinical problems like other causes of infection (bacterial or fungal), acute rejection, and drug toxicities. bacteria (mycoplasma pneumoniae, chlamydia pneumoniae, and legionella pneumophila) also cause community outbreaks, share similar clinical features, and may present diagnostic difficulties. in contrast to the nonimmunosuppressed host, carv infection usually leads to more severe illness in the lung transplanted recipient with a higher incidence of respiratory failure. in a recent clinical trial with rsv-infected ltx patients, median decline in fev compared with the preinfection level was %. seventy-four percent of these patients had symptoms of a lower respiratory tract infection and % a significant infiltrate on chest x-ray. in a retrospective singlecenter study over a -year time span, % of ltx patients presenting with carv infection ( % coxsackie and rv, % pv, % cov, % rsv, % hmpv, % flu) demonstrated a pulmonary infiltrate and % were asymptomatic. acute consequences of rsv infection include bronchiolitis, pneumonia, and respiratory failure. permanent long-term effects following ltx include the development of new or progressive chronic allograft dysfunction that manifest clinically as bos. , rhinoviral infection can be persistent in lung transplant recipients with graft dysfunction. no temporal association was observed between carv infection and ar. , cumulative infection rates of carv in lung transplant recipients in earlier retrospective single-center studies of to years ranged from to %. [ ] [ ] [ ] [ ] retrospective studies did not test for viruses newly recognized as important pathogens for bos, especially hmpv. therefore, apart from methodological issues, bos incidence may not be exactly estimated from these studies. there are several published prospective studies using polymerase chain reaction (pcr) techniques of carv in ltx involving between and patients during surveillance periods of to months. , - symptoms of respiratory tract infection (rti) were frequent to % in screened patients but incidence of carv was variable between . and %. the different incidences can be explained by different methodology and inclusion criteria. in the swiss study, only patients who underwent bronchoscopy were studied, making it difficult to calculate the true incidence of respiratory viral infections and comparing it with uninfected patients. in a canadian study, only stable patients were included and screened for carv and matched with uninfected patients, which excluded an estimation of the true incidence of carv during the observation period. in a u.s. study, ltx recipients were contacted weekly by telephone and screened for symptoms. symptoms of rti were, therefore, more frequent. serology identified most of the carv in this study and hmpv was not investigated. pvs were the dominant pathogens in a german surveillance study using pcr testing in upper respiratory samples and antigen testing in sample from the lower respiratory tract followed by rsv ( - % in other prospective studies). in the german surveillance study, an annual incidence of % in carv-positive recipients was described, which is higher than to % in other prospective studies using pcr techniques. , in summary, prospective studies have described an incidence of to cases per patientyears (►table ). infected ltx recipients in early studies presented with respiratory failure. interestingly, in retrospective studies , , the bos incidence in carv-positive lung transplant recipients was much more common after year than in the prospective studies ( - % versus - %). , , several surveillance and retrospective studies confirmed an association of carv infection (as well other carv) with the onset of bos. these studies are heterogeneous and have limitations in design, case selection, and diagnostic procedures. this association was identified mainly for paramyxoviruses, and the association with influenza and av is less well documented. according to the published literature, the incidence of bos was to % in ltx recipients infected with carv during the following months. , , , , patients may present with onset of clad immediately with carv infection or later after an initial recovery of fev after the viral infection. there is a lack of information on the potential role of viruses that are difficult to grow, such as rv, human cov, enteroviruses, and hmpv. some of them have recently been recognized as important pathogens in ltx. , , virus isolation is still the gold standard for the laboratory detection of respiratory viruses. however, virus isolation in cell cultures is slow and not always technically successful. therefore, this method is no longer used in the transplant setting where rapid workup is crucial. rapid antigen detection tests or antibodies for immunofluorescence testing (ift) are not available for all carv. the detection of viral antigens is reported to be less sensitive and less specific than cell cultures but allows rapid detection. nucleic acid amplification tests for carv by pcr are rapid and sensitive. they are commercially available in single or multiplex format. the significance of virus detection by pcrs in asymptomatic patients is unknown and virus detection by pcr may not reflect active infection. serology plays no role in detecting carv infection after ltx. upper respiratory sampling by naso-oropharyngeal swab (nos) are useful in combination with pcr techniques avoiding the need for bronchoscopy. special swabs samples should be used. mouth rinses are an acceptable alternative. in ltx recipients, bronchoalveolar lavage (bal) is a widespread diagnostic tool. given the broad spectrum of other possible diagnosis in a ltx patient with symptoms of upper respiratory tract infection (urti) or lower respiratory tract infection (lrti), bronchoscopy with bal is advised in most circumstances. bal was most sensitive do detect carv in surveillance studies. in a recent randomized controlled trial (rct), % of all rsv infections were detected by pcr. the potential incidence of bos after untreated carv infections and the threat of respiratory failure in infected ltx recipients illustrate the need for effective treatment of carv infections. unfortunately, respiratory viruses are a hard target for various reasons. most of the time the virus spends in the host and will be inside the host cell. the virus is protected from the host immune system as well as from available circulating enzymes. there are a limited number of potential drug targets since viruses use the host biochemical mechanisms to multiply. viruses multiply very quickly, so antiviral drugs will often have little effect by the time symptoms appear. in addition, resistance to commonly used antivirals develops early. theoretically, viruses can be targeted by inhibiting the stage of viral attachment, internalization, fusion and uncoating, replication, assembly, and release. unfortunately, except the neuraminidase inhibitors for influenza, no registered drugs are available for most the treatment of carv infections in adults. treatment strategy may be symptomatic in most cases. it usually consists of steroids, oxygen, and antibiotics in case of concomitant bacterial infection. ribavirin is known since and is registered for use in chronic hepatitis c virus infection. ribavirin, a purine community-acquired respiratory viruses gottlieb nucleoside analogue with efficacy against many rna viruses, including rsv, hmpv, and pv, represents a viable treatment option for pv infection. ribavirin has been shown to have in vitro activity against rsv and the aerosolized form has been approved for the treatment of lower respiratory tract disease due to rsv in certain at-risk populations. data for intravenous use of ribavirin remain limited in ltx recipients. drawbacks with inhaled ribavirin include difficulties in administration, requiring continuous inhalation, along with associated risks of bronchoconstriction and respiratory distress, which may necessitate discontinuation. in addition, aerosolized ribavirin is considered to be potentially hazardous and teratogenic to the environment including health care workers as well as being excessively expensive. considering these limitations, widespread acceptance of the nebulized administration is missing. several centers use oral ribavirin as an off-label alternative treatment in paramyxovirus infections in ltx. a retrospective study demon-strates no significant differences in -month outcomes between oral and inhaled ribavirin therapy for rsv infection after ltx. in a recent multicenter trial involving rsv-infected patients from sites in australia, austria, germany, france, canada, and the united states, inhaled ribavirin was used in % infected individuals, oral ribavirin in %, intravenous ribavirin in %, intravenous immunoglobulin (ivig) in %, pulsed steroids in %, and palivizumab in %. the optimal duration of ribavirin therapy is unknown, although treatment orally for days led to virus elimination in % of patients in a retrospective study. of particular note, ribavirin was contraindicated in % of pv-infected recipients in this study and was terminated early in another quarter of cases due to adverse effects. twice-weekly monitoring of blood cell counts (risk of hemolytic anemia) and renal function is mandatory while under ribavirin therapy. compared with other patient populations treated with ribavirin, more frequent discontinuation due to toxicity was necessary in the ltx population. the most likely explanation for this remains combined toxicity of ribavirin and that inherent to the maintenance medication required after ltx. distinction must be made between the short-and longterm effects of treatment with ribavirin. prior studies have focused on short-term effects of ribavirin treatment, such as survival of the acute phase or shorter hospital length of stay, and found no beneficial effect. retrospective single-center studies suggest that ltx patients treated with ribavirin have a better pulmonary function months after paramyxovirus infection and have found that oral ribavirin reduces the number of complicated courses of paramyxovirus infection and reduces the long-term risk of bos %. recovery of pulmonary function postinfection was significantly better in ribavirin-treated patients (n ¼ ) than in patients treated with supportive care (n ¼ ) in a retrospective study. patients treated with oral ribavirin had a lower incidence of bos ( % of the ribavirin group versus % of the nonribavirin group [p ¼ . ]) within months. the latter subgroup was treated with supportive care in this study and had contraindications for ribavirin (e.g., advanced kidney disease, anemia). all ribavirin studies in ltx lack a randomized placebocontrolled design, which makes them unsuitable to make evidence-based recommendations. there is clearly the need for a rct to determine the efficacy of oral ribavirin. since unnecessary treatment with drugs should always be avoided and ribavirin has some unfavorable side effects, a firm conclusion is needed about the clinical benefits of this treatment. oral ribavirin seems to be a promising and inexpensive therapy which may have a significant impact on long-term morbidity and mortality of lung transplant recipients. anecdotal cases have reported the off-label use of pavilizumab and ivig in rsv-infected ltx recipients. several agents have been studied against influenza including das (fludase) and nitazoxanide, but no data are published for ltx patients. rna interference is a natural biological process whereby small interfering rnas (sirnas) can direct sequence-specific degradation of mrna, leading to reduced expression of the corresponding protein. aln-rsv is a sirna targeting the rsv nucleocapsid messenger rna, preventing the formation of the nucleocapsid protein and thereby reducing viral replication. intranasal aln-rsv administration significantly inhibited the rate of rsv infection in a phase experimental infection study in healthy adults. in a pivotal study in rsv-infected ltx patients, nebulized aln-rsv treatment proved to be safe and well-tolerated and incidence of new or progressive bos was decreased at day in patients treated with aln-rsv compared with placebo. the efficacy of aln-rsv administration in addition to standard of care on preventing new or progressive bos in rsvinfected ltx patients was evaluated in this large randomized, double-blind, placebo-controlled trial. in this phase b, trial subjects were randomized to receive aerosolized aln-rsv or placebo daily for days. aln-rsv was found to be safe and well tolerated. at day in aln-rsv -treated patients (n ¼ ) compared with placebo (n ¼ ), there was a trend toward a decrease in new or progressive bos ( . % vs. . %, p ¼ . ), which was significant in the per-protocol cohort (p ¼ . ). treatment effect was enhanced when aln-rsv was started < days from symptom onset, and the effect was independent from ribavirin treatment. presatovir (gs- ) is an oral rsv fusion inhibitor with potent and selective anti-rsv activity in vitro. a phase a, randomized, double-blind, placebo-controlled study was conducted to evaluate the safety, tolerability, and efficacy of presatovir in healthy adult volunteers infected with an rsv challenge virus. treatment with presatovir resulted in lower mean area under the curve (auc) viral load from initial dose through end of quarantine. viral load was assessed twice daily using nasal washes and in total symptom score during the entire quarantine period. results from a phase b, rct evaluating the effect of gs- in ltx recipients with rsv infection (nct ) are expected soon. there are several unmet needs in the development of effective therapies for carv. one is a wider treatment window. most agents are reported to be less effective if the patient presents more than hours of symptom onset. therapies should be cost-effective and reduce the threat of resistance by continuous antigenic drifting and antigenic shifting of viruses. alternative formulations (e.g., intravenous drugs) for hospitalized patients should be available as well as drugs for severely ill, hospitalized patients, and for pediatric patients. prevention of carv infection is of utmost importance in ltx. annual influenza vaccination is strongly recommended for all lung transplant recipients including all their household members. wearing face masks, avoiding contact with infected persons, and skin disinfection are usually recommended as preventive measures in ltx recipients, although they were not systematically evaluated in this high-risk cohort. viral pneumonia community-acquired respiratory viral infections in lung transplant recipients regulating the adaptive immune response to respiratory virus infection an international ishlt/ats/ers clinical practice guideline: diagnosis and management of bronchiolitis obliterans syndrome post-transplant bronchiolitis obliterans international society for heart and lung transplantation. the registry of the international society for heart and lung transplantation: thirty-third adult lung and heart-lung transplant report- ; focus theme: primary diagnostic indications for transplant survival after bronchiolitis obliterans syndrome among seminars in respiratory community-acquired respiratory viruses gottlieb unauthorized distribution is strictly prohibited. bilateral lung transplant recipients chlamydia pneumoniae infection after lung transplantation aln-rsv for prevention of 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respiratory viruses in lung transplant recipients a single-season prospective study of respiratory viral infections in lung transplant recipients the value of polymerase chain reaction for the diagnosis of viral respiratory tract infections in lung transplant recipients community-acquired respiratory viral infections in lung transplant recipients: a single season cohort study respiratory viruses and severe lower respiratory tract complications in hospitalized patients incidence and morbidity of human metapneumovirus and other communityacquired respiratory viruses in lung transplant recipients human metapneumovirus in lung transplant recipients and comparison to respiratory syncytial virus intravenous ribavirin is a safe and cost-effective treatment for respiratory syncytial virus infection after lung transplantation oral ribavirin for the treatment of noninfluenza respiratory viral infections: a systematic review oral versus inhaled ribavirin therapy for respiratory syncytial virus infection after lung transplantation single-centre experience with oral ribavirin in lung transplant recipients with paramyxovirus infections rna interference therapy in lung transplant patients infected with respiratory syncytial virus oral gs- activity in a respiratory syncytial virus challenge study lower respiratory viral illnesses: improved diagnosis by molecular methods and clinical impact the impact of viral respiratory tract infections on long-term morbidity and mortality following lung transplantation: a retrospective cohort study using a multiplex pcr panel key: cord- -i tzr z authors: cockrell, adam s.; leist, sarah r.; douglas, madeline g.; baric, ralph s. title: modeling pathogenesis of emergent and pre-emergent human coronaviruses in mice date: - - journal: mamm genome doi: . /s - - - sha: doc_id: cord_uid: i tzr z the emergence of highly pathogenic human coronaviruses (hcovs) in the last two decades has illuminated their potential to cause high morbidity and mortality in human populations and disrupt global economies. global pandemic concerns stem from their high mortality rates, capacity for human-to-human spread by respiratory transmission, and complete lack of approved therapeutic countermeasures. limiting disease may require the development of virus-directed and host-directed therapeutic strategies due to the acute etiology of hcov infections. therefore, understanding how hcov–host interactions cause pathogenic outcomes relies upon mammalian models that closely recapitulate the pathogenesis of hcovs in humans. pragmatism has largely been the driving force underpinning mice as highly effective mammalian models for elucidating hcov–host interactions that govern pathogenesis. notably, tractable mouse genetics combined with hcov reverse genetic systems has afforded the concomitant manipulation of virus and host genetics to evaluate virus–host interaction networks in disease. in addition to assessing etiologies of known hcovs, mouse models have clinically predictive value as tools to appraise potential disease phenotypes associated with pre-emergent covs. knowledge of cov pathogenic potential before it crosses the species barrier into the human population provides a highly desirable preclinical platform for addressing global pathogen preparedness, an overarching directive of the world health organization. although we recognize that results obtained in robust mouse models require evaluation in non-human primates, we focus this review on the current state of hcov mouse models, their use as tractable complex genetic organisms for untangling complex hcov–host interactions, and as pathogenesis models for preclinical evaluation of novel therapeutic interventions. the etiologies of coronaviruses (covs) comprise an extensive host range that includes reptiles, birds, pigs, dogs, cats, cattle, rodents, bats, camels, and humans. the extensive nonhuman host range may serve as reservoirs for pre-emergent coronaviruses poised for zoonotic transmission into human and animal populations (forni et al. ; zhou et al. ) . there are six known human coronaviruses (oc , e, nl- , hku , sars-cov, and mers-cov), four of which hku , were identified in the last years. considering that bats may be the pinnacle reservoir for the origin of most hcovs (sars-cov, mers-cov, e, and nl ), evidence for sars-cov and mers-cov indicates that intermediate zoonotic hosts (civet cat and camel, respectively) may be as important for the evolution of a pathogenic cov to emerge into the human population (forni et al. ) . the emergence of novel pathogenic covs into the human population can result in highly lethal respiratory hcovs with global pandemic potential through human-to-human spread, which is underscored by the global spread of sars-cov (~ % mortality) in - and the ongoing re-introduction of mers-cov (~ % mortality) into humans on the arabian peninsula. human-to-human transmission of mers-cov was most apparent in the south korean outbreak in , established by a single individual returning from a visit to the arabian peninsula, and resulting in individuals infected with an ~ % mortality rate (lee ) . the high mortality rate in south korea also established a society-wide panic that led to a severe economic toll, anticipated to cut . % from the gdp rate in (lee ) . once an hcov emerges in humans new tools and robust mouse models are needed to decipher pathogenic mechanisms. ultimately, understanding the hcov-host molecular interaction networks that regulate pathogenesis can guide the engineering of therapeutic countermeasures. an ideal strategy for development of models that recapitulate human respiratory covs clearly includes the development of a genetically tractable mammalian model that effectively recapitulates human pathogenic phenotypes, and an efficient reverse genetics system for seamless manipulation of the hcov genome. genetic control of both host and hcov genomes facilitates the ability to draw causeand-effect relationships between virus/host genetics and pathogenic outcomes. reverse systems genetic tools were developed to manipulate the large hcov genomes (~ kb) that encode non-structural proteins; structural proteins including spike (s), envelope (e), membrane (m), and nucleocapsid (n); and a number of accessory proteins that vary in sequence and function ( fig. ) [reviewed in (cockrell et al. ) ]. a number of in vitro experiments (biochemical and tissue culture based) demonstrated that hcov proteins interact with host cell proteins to ensure fidelity during virus replication or facilitate evasion of host cell immune responses. the most prominent hcov-host cell interaction is between the major determinant of viral tropism, the spike protein, and a host cell-specific receptor. the spike protein forms a trimer on the surface of the virus with each monomer harboring a receptor binding domain (rbd) that interacts with its cognate receptor on the host cell (ace for sars-cov and dpp for mers-cov) (fig. ) . the spike protein, especially the rbd, is a major target for development of antibody and vaccine therapeutics. other highly conserved structural and non-structural proteins, chiefly enzymatic proteins, may serve as targets for broadly effective anti-hcov therapeutic targets. the accessory proteins are not conserved between hcovs, and numerous studies revealed that these gene sets encode critical determinants of species-specific pathogenesis by modulating host immune responses (fig. ) . in vitro studies have shown a number of accessory proteins to be important for evasion of host innate immune responses through interactions with host proteins [reviewed in (de wit et al. ; totura and baric ) ]. although in vitro studies are imperative for understanding molecular and biochemical mechanisms, only in vivo studies can decipher how complex hcov-host interactions relate to pathogenic phenotypes following a respiratory infection. discerning the fine balance between pathogenesis and protection, that is governed by the relationship between host innate and adaptive immune responses, requires the development of mammalian models that effectively recapitulate the pathogenesis of respiratory hcovs. the challenges associated with modeling pathogenesis of emergent and pre-emergent hcovs are borne out of models developed for sars-cov and mers-cov over the last years (table ; fig. ). mice, ferrets, and non-human primates (nhps) are traditionally used for the studies involving respiratory pathogens and are among the species initially investigated for sars-cov and mers-cov replication and pathogenesis in the lungs. taking into account a number of practical considerations when comparing models for both sars-cov and mers-cov, mouse models appear to be the most pragmatic (table ) . although we emphasize the practicality of hcov mouse models for the purposes of this review and the absolute critical need for robust primate models for downstream drug and vaccine testing, it is important to note that research (e.g., efficacy of therapeutic countermeasures) established in a robust mouse model should effectively translate to large animal models, such as nhps, that more closely reflect human physiology. in general, mouse models for both sars-cov and mers-cov are affordable, readily available, require fig. emergent and pre-emergent coronavirus genome organization. the orf a and orf b genes (green) encode non-structural proteins (nsp -nsp ) that are highly conserved throughout coronaviruses. the structural genes (red) encode the structural proteins spike (s), envelope (e), membrane (m), and nucleocapsid (n), which are common to all coronaviruses. the accessory genes (dark shade) are unique to different coronaviruses with regard to number, genomic organization, sequence, and function nominal training for handling, amenable to manipulation under bsl conditions, amenable to analysis with commercially available molecular reagents, amenable to large numbers for purposes of experimental reproducibility, and are genetically tractable (table ) . despite these commonalities, establishing a mouse model for mers-cov presented challenges not encountered with sars-cov (table ) . even now, years after the first case of mers-cov, one of the biggest challenges remains a very limited understanding of the pathology of mers-cov in humans. only two studies have described the pathology of mers-cov in two fatal human cases (alsaad et al. ; ng et al. ). the two fatal human mers-cov cases were similar to those observed in cases of sars-cov, wherein patients exhibited severe acute respiratory distress with diffuse alveolar damage and formation of hyaline membranes, alveolar fibrin deposits, edema, hemorrhaging, cellular debris, alveolar and interstitial inflammation throughout lungs, and extensive pulmonary tissue necrosis (alsaad et al. ; ng et al. ) . fatal cases of sars-cov and mers-cov were commonly associated with a gradual loss of respiratory function with available intervention restricted to mechanical respiratory support and oxygen supplementation [reviewed in ]. therefore, effective mouse models for sars-cov and mers-cov should minimally be able to recapitulate fatal respiratory disease having pathology similar to that observed in humans. as described below, a number of mouse models exhibiting fatal respiratory disease were developed for sars-cov and mers-cov; however, a single impediment was realized early in model development for mers-cov that was not confronted for sars-cov. the mouse orthologue of the human receptor for mers-cov, dipeptidyl peptidase (dpp ), did not support interaction with the mers-cov spike glycoprotein rbd (cockrell et al. ) . therefore, unlike sars-cov, commercially available mice were not susceptible to mers-cov (towler et al. ) ]. in order to establish lethal mouse models, the spike protein, and other genomic determinants, have been modified through adaptive evolution in mouse lungs (day et al. ; frieman et al. ; roberts et al. a ). thereby, any mouse subspecies that exhibits an unaltered mouse ace can be infected with the mouse-adapted sars-cov. wild-type mers-cov column (green). mers-cov infects humans via an interaction of the spike protein [rcsb pdb id: x f (yuan et al. ) ] with its cognate receptor, dpp [rcsb pdb id: onc (feng et al. ) ]. the mers-cov spike protein is not able to interact with the mouse orthologue of human dpp (cockrell et al. ; coleman et al. ) . therefore, the mouse dpp gene had to be genetically modified in order to allow for infection with mers-cov (cockrell et al. ; li et al. ; pascal et al. ) . pre-emergent covs column (grey). pre-emergent covs can use either known human receptors for covs (ace or dpp ) (ge et al. ; menachery et al. ; wang et al. ; yang et al. yang et al. , , or novel, unknown receptors to infect their host. the genetically highly diverse panel of mouse strains from the collaborative cross has the potential to provide mouse models for studies of newly emerging covs due to the high genetic variability present in this resource. for all virus images: yellow represents the envelope protein; light/dark blue represents the membrane protein; and red represents the nucleocapsid protein infection and replication (coleman et al. ). susceptibility and mers-cov pathogenesis was achieved using unique approaches to genetically engineer mice, as described in detail below. establishing mouse models of fatal disease for sars-cov and mers-cov has afforded the necessary knowledge to institute a preclinical in vivo platform that can be used to evaluate pre-emergent hcov respiratory pathogens with unknown disease outcomes (fig. ) . the strategy is widely portable to other important human pathogens that replicate poorly or not at all in the mouse. the need for sars-cov mouse models was recognized shortly after the sars-cov outbreak that spread globally in - . three different strategies were employed for development of sars-cov mouse models: (i) different mouse species (or subspecies) were challenged with wildtype human sars-cov isolates in order to find a model that allows for replication and reflects severe respiratory disease symptoms observed in infected human patients; (ii) mice were genetically engineered to modify the host receptor, which facilitated productive sars-cov replication and pathogenesis; and (iii) adaptive evolution of wild-type sars-cov to a chosen mouse species was done to enhance pathogenesis, and associated clinical phenotypes in vivo. initial studies to develop a mouse model for sars-cov evaluated the susceptibility of various mouse subspecies to clinical isolates of sars-cov. in early , the first study using the mouse as a model organism for sars-cov was reported ). - weeks old female balb/c mice were intra-nasally infected with a clinical isolate of sars-cov (urbani) and efficient viral replication was observed from the upper and lower respiratory tract with peak titers on day and clearance by day , whereas viral replication was not detected in other organs. however, mice continued to gain weight and did not show other signs of clinical disease. passive transfer of immune sera to naïve mice was sufficient to prevent viral replication in the respiratory tract, indicating that wild-type sars-cov could elicit an effective humoral immune response. nonetheless, in the absence of pathogenesis with no clinical signs of disease, it is difficult to properly evaluate the efficacy of therapeutic countermeasures. in a second study, glass et al. used c bl/ mice which were known to exhibit a th -biased immune response in contrast to balb/c mice table practical considerations for establishing an effective hcov animal model a yes-required virus adaptation b yes-after genetically engineering a mouse c affordability-more cost effective relative to ferret and nhp models d availability-developed models that can be readily acquired and studied by the broader scientific community e ease of handling-mice require least amount of specialized training compared to ferrets and nhps f amenable to absl conditions-mice are most amenable to absl conditions due to space limitations, specialized handling requirements, and personnel limitations g maybe-dependent on species and techniques h n/d-not determined that were considered to be th -biased (glass et al. ) , which may account for differences in pathogenic outcomes. like the balb/c study, - weeks old c bl/ mice were infected with the clinical sars-cov urbani isolate. infected c bl/ mice exhibited slower weight gain than mock-infected mice and sars-cov was found not only in the upper and lower respiratory tract but also in the brain, which was not associated with clinical disease in humans. developing a sars-cov model in c bl/ mice had the added benefit of expanding the applicability of the model to genetically modified knock-out (ko) mice that are predominantly generated on a c bl/ background, thereby facilitating the investigation of host genetics on sars-cov pathogenesis (glass et al. ) . utilizing cd- −/− and rag −/− knock-out mice, glass et al. demonstrated that nk cells, nk-t cells, or t and b lymphocytes are not required for clearance of sars-cov in the mouse (glass et al. ). these initial studies to evaluate different murine subspecies as models for sars-cov resulted in replication in the respiratory tract with no indication of the clinical pathogenesis often observed in human cases experiencing severe respiratory distress. knock-out mice deficient in the innate immune response were the first to exhibit clinical alterations that included weight loss and progressively worsening pulmonary disease (hogan et al. ). svev and stat −/− mice on a svev background infected with a sars-cov clinical isolate (toronto- ) showed that stat was required for the resolution of sars-cov infection. clinical signs of disease were only observed in ko mice, whereas efficient viral replication in the lower respiratory tract caused no clinical symptoms in svev wild-type mice. in contrast, stat −/− mice supported viral replication with peak titers on day and persistence throughout day (hogan et al. ). most features of pathology that were found in stat −/− mice were different from those found in humans. however, some pathological features common to humans, such as bronchiolar injury with focal respiratory epithelial cell necrosis, supported the idea that the mouse species could be a good model for sars-cov-associated clinical findings observed in the respiratory tract of infected human patients. increased fatality as consequence of acute respiratory distress syndrome (ards) from sars-cov infection was often associated with advanced age (> years old) in humans (booth et al. ; donnelly et al. ; tsui et al. ) , which was exploited to improve mouse models of sars-cov pathogenesis. to replicate advanced age in humans, - months old balb/c (roberts et al. ) , c bl/ , and s (roberts et al. ) mice were intra-nasally infected with a clinical isolate of sars-cov (urbani). balb/c and c bl/ mice showed comparable levels and kinetics of viral replication while aged s mice cleared sars-cov by day (roberts et al. ). the predominant clinical feature regarding mouse studies is weight loss, a consequence of appetite loss and dehydration. all three lines of aged mice showed transient signs of clinical disease including weight loss, ruffled fur, hunching, and dehydration that resolved by day . moreover, all three strains exhibited histopathological findings such as perivascular and peribronchiolar infiltrates, necrotic debris in bronchioles, and interstitial pneumonitis. importantly, extensive alveolar damage in aged balb/c mice persisted through day post-infection (p.i.), findings that closely resembled pathology observed in humans. these are some of the first controlled studies to demonstrate that differences in host genetics can significantly influence sars-cov pathogenesis, a topic that is revisited below with regard to using a tractable population of genetically distinct mice referred to as the collaborative cross. concurrent with these studies, rockx et al. demonstrated that pathogenesis related to sars-cov clinical isolates was also dependent upon the genetics of the sars-cov strain evaluated in vivo (rockx et al. ). using reverse genetics rockx et al. modified the spike protein from the sars-cov urbani strain to encode the spike from middle (cuhk-w ) and early (gz ) phase clinical isolates or a civet strain spike gene from hc/ sz/ / (rockx et al. ). an infectious clone harboring the clinical isolate gz or civet hc/sz/ / spikes exhibited severe weight loss and death, as well as pathology consistent with acute respiratory distress syndrome in aged, but not young mice (rockx et al. ). despite these early achievements with aged mice for sars-cov mouse model development, it is impractical to maintain large colonies of aged mice; and immune senescence in aged mice may complicate studies involving pathogenesis and effective evaluation of immune responses to therapeutic countermeasures. in addition to these limitations, full-length clinical isolates of sars-cov were not shown to be lethal in aged mice, a clinical outcome that impacted nearly % of all human cases. lethality was not achievable using clinical sars-cov isolates in various young wild-type or immune-incompetent mouse strains. with this in mind, attention turned to genetic modification of mice to acquire a lethal model for sars-cov pathogenesis. in order to continue working with unaltered human clinical isolates of sars-cov, mouse strains constitutively expressing the human receptor for sars-cov, human angiotensin converting enzyme (hace ), were generated. the logic followed that human clinical isolates of sars-cov are evolved to use the hace receptor more effectively than the innate mouse ace (mace ) receptor; therefore, may elicit pathology consistent with a lethal respiratory infection. different constitutive promotors were used to express hace in mice: cytokeratin promotor (mccray et al. ; netland et al. ) , chicken beta-actin promotor with a cytomegalovirus ie enhancer (tseng et al. ) , and the mouse ace promotor (yang et al. ). expression levels of human ace correlated with disease severity in all transgenic mouse models. human ace overexpression mice generated with the cytokeratin promoter and the mace promoter were attempts to limit expression to respiratory cells and cells that exhibited innate mace receptor expression, respectively (mccray et al. ; netland et al. ; yang et al. ). even though these transgenic mice showed infection of airway epithelium, all of them exhibited high levels of hace expression in the brains of transgenic mice which supported increased viral load in brain tissue (mccray et al. ; netland et al. ; tseng et al. ; yang et al. ). the increased viral loads in the brains of infected animals ultimately led to mortality caused by extensive dissemination and encephalitis in the brain (mccray et al. ; netland et al. ; tseng et al. ; yang et al. ) . accordingly, despite the successful generation of lethal hace overexpression mouse models for sars-cov pathogenesis, neurological-related mortality confounded their value as models that could effectively mimic lethal respiratory disease often observed in infected humans. adaptive experimental evolution has been a staple of virology for > years (kalter ) , providing critical insights into pathogenic mechanisms while bestowing robust, lethal mouse models to interrogate the efficacy of vaccines and therapeutics (bolles et al. ; cockrell et al. ; rasmussen et al. ; roberts et al. a ). adaptive experimental evolution is performed by serial in vivo passages in the tissue of interest to adapt the virus to the host innate and adaptive immune responses. this is similar to the ongoing battle between a virus and the host immune responses occurring in nature, but on an expedited time-scale and in a tissue-specific manner. tissue-specific adaptive evolution places the virus under selective pressure for mutations that allow for more efficient replication. to adapt sars-cov to cause severe acute respiratory disease in mouse lungs, -week-old female balb/c mice were intra-nasally infected with the clinical urbani isolate (roberts et al. a ). this process was repeated ( rounds) until signs of clinical disease were observed, predominantly weight loss that reached clinical endpoints for humane euthanasia (considered humane lethality) (roberts et al. a ). the resulting mouse-adapted sars-cov was designated ma . rapid weight loss was accompanied by high viral titer in the lungs, viremia and dissemination to extrapulmonary sites, lymphopenia, neutrophilia, and histopathological changes in the lung commonly associated with pneumonitis. accordingly, mortality was a consequence of extensive viral replication which led to virus-associated destruction of pneumocytes and epithelial cells. only six coding mutations were sufficient to render the sars-cov urbani human clinical isolate % lethal in weeks old, - weeks old, and aged balb/c mice (roberts et al. a ). the ma sars-cov has since been used in a multitude of studies and proven to not only cause lethal disease in balb/c mice but also in other mouse strains (deng et al. ; frieman et al. ; gralinski et al. gralinski et al. , totura et al. ; zhao et al. ) . additional passaging of ma resulted in the generation of other mouse-adapted sars-cov strains including ma ( passages) (frieman et al. ) , while an independent research group acquired a lethal sars-cov model by passaging the human urbani isolate rounds through the lungs of balb/c mice, resulting in v (day et al. ). in addition to acquiring lethal sars-cov models of disease, comparing various passages of mouse-adapted sars-cov provides the unique advantage of being able to identify which sars-cov proteins acquire specific mutations that can elicit severe respiratory phenotypes. identification of adaptive mutations acquired in specific sars-cov proteins provided insight into virus-host interaction networks that may be used for the development of virusdirected therapeutic countermeasures. combining mouseadapted sars-cov strains with mouse models that support interrogation of the host genome for molecules that influence sars-cov pathogenesis can reveal import interaction nodes amenable to host-directed therapeutic intervention. as mentioned above, a number of early sars-cov mouse model studies showed that disease progression and outcome are mouse strain dependent, indicating that host genetics have a considerable influence on sars-cov pathogenesis. this is not surprising since human gene association studies have indicated that differences in an individual's genetics may govern susceptibility and clinical outcomes of respiratory viral pathogenesis (everitt et al. ; forton et al. ; kenney et al. ; mills et al. ; pasanen et al. ; patarcic et al. ; zhou et al. ), including retrospective studies of sars-cov-infected patients (chan et al. , ching et al. ; wang et al. ; zhu et al. ). however, human genetic associations do not indicate cause-and-effect, which require the capacity to model the impact of host genetics on respiratory viral pathogenesis, in this case sars-cov pathogenesis. a recently developed, innovative resource for genetic mapping, called the collaborative cross (cc), comprises a panel of recombinant inbred mouse strains containing tractable genetic diversity that approaches the genetic diversity in the human population (churchill et al. ; threadgill et al. ) . using an octo-parental breeding scheme that includes classical laboratory stains (a/j, c bl/ j, and /svimj), mouse models for human diseases (nod/shiltj for diabetes; nzo/hlltj for obesity), and wild-derived mouse strains (cast/eij, pwk/phj, and wsb/eij), the cc captures % of the genetic variation present in the three major mouse subspecies (mus musculus musculus, mus musculus domesticus, mus musculus castaneus) (roberts et al. b) . virus infection studies in cc mouse lines, including sars-cov, have led to mapping of high and low host response alleles as they relate to development of clinical signs of disease following viral pathogenesis (bottomly et al. ; brinkmeyer-langford et al. ; ferris et al. ; gralinski et al. gralinski et al. , green et al. ; rasmussen et al. ) . rapid genetic mapping is most successful through comparative analysis of cc strains that show extreme phenotypes such as high versus low weight loss, or high versus low lung titers. extreme phenotypes in select cc strains may reflect clinical outcomes observed in human disease, as observed in testing mouse-adapted ebola virus in different cc strains . applying the cc technology to sars-cov pathogenesis identified two cc strains (cc / unc and cc /unc) with opposing susceptibility profiles . genetic mapping revealed an adaptor protein (ticam ) in the toll-like receptor pathway as a strong candidate driving severe respiratory disease phenotypes . based on these observations, the cc mouse platform can be used to identify novel mouse models that recapitulate human clinical outcomes resulting from pathogenic viruses. the cc mouse strains are an extraordinarily powerful platform to establish novel mouse models for emergent and pre-emergent isolates of respiratory coronaviruses. harnessing the power of cc host genetic mapping will support the identification of novel hcov-host molecular interaction networks that can be subsequently validated in genetically engineered mice. innovations in gene editing technologies such as crispr/cas , talens, and synthetic zinc fingers have augmented the efficiency of genetic engineering in mice, making genomic modifications (i.e., allele-specific mutations, allele swaps, knock-outs, knock-ins) feasible in multiple mouse species, on a large-scale (doench ; kim and kim ) . coincidentally, advances in the crispr/ cas technology overlapped with the outbreak of mers-cov in , which was fortuitous for the development of lethal mers-cov mouse models. building on expertise from the sars-cov outbreak, coronavirus researchers immediately recognized the overwhelming need for an effective mers-cov mouse model following the emergence of mers-cov in . however, researchers were perplexed to find that mouse lines conventionally susceptible to sars-cov infection/replication were completely resistant to infection with clinical mers-cov isolates (coleman et al. ) . initial studies demonstrated that non-human primates were susceptible to clinical isolates of mers-cov (de wit et al. b; falzarano et al. ; johnson et al. johnson et al. , munster et al. ) ; therefore, the inability to infect mice with mers-cov was likely not due to restriction by the host immune responses. this was validated in immune-incompetent mouse lines that also lacked the capacity for infection/replication of mers-cov (coleman et al. ) . concurrent with some of the initial mers-cov studies in mice, raj et al. identified a novel receptor for mers-cov, human dipeptidyl peptidase (hdpp ) (raj et al. ) . unlike sars-cov which could infect mice through interaction of the rbd in the spike protein with the mace receptor, mouse dpp (mdpp ) did not support an interaction with the mers-cov spike protein (cockrell et al. ) . crystal structures of the mers-cov spike-hdpp interface revealed a number of specific amino acids necessary for an interaction between the rbd in spike and two specific domains, referred to as blades iv and v on the β-propeller structure, of the hdpp structure (lu et al. ; wang et al. ) . ectopic expression studies with the mouse dpp receptor revealed that altering a minimum of two amino acids on the mdpp (positions a and t ) conferred susceptibility of the human fibroblast cell line, t, to mers-cov infection (cockrell et al. ; peck et al. ) . importantly, in mice t is an n-linked glycosylation site that may sterically hinder mers-cov infection, and is not present in dpp orthologues from susceptible species including human, nhps, bats, and camels (peck et al. ) . apparently, ferret and hamster dpp may also have similar glycosylation's, which may explain the inability of mers-cov to also infect/replicate in these conventional small animal models (de wit et al. a; peck et al. ; raj et al. ; van doremalen et al. ). species-specific challenges utilizing the dpp receptor not only limited development of mouse models, but stymied development of all small animal models required for evaluating therapeutic countermeasures. accordingly, the initial development of mouse models for mers-cov required employing a number of methods that focused on engineering mice to express/overexpress the human dpp receptor (agrawal et al. ; li et al. ; zhao et al. zhao et al. , (fig. ) . the first mouse model, developed by zhao et al., established transient expression of human dpp in the airway of balb/c mice, c bl/ mice, and several knock-out mice using an adenoviral vector to transduce an hdpp overexpression cassette (ad-hdpp ) to the lungs of mice (zhao et al. ) . transient hdpp expression rendered mice susceptible to clinical isolates of mers-cov infection [ × plaque forming units (pfu) dose], supporting viral replication in the lungs and producing transient weight loss with mild pneumonia, particularly in older and immunodeficient mice (zhao et al. ) . no mortality or signs of advanced clinical respiratory disease were observed (zhao et al. ) . as a platform strategy that could be applied to novel emerging viruses, the ad-hdpp model provided a means to rapidly evaluate mers-cov-directed therapeutic countermeasures with regard to interfering with replication in the lungs of infected mice. nevertheless, the ad-hdpp model was not an effective means of understanding how therapeutic countermeasures could prevent development of severe respiratory disease that resulted in ~ % mortality in infected human cases. to develop more clinically relevant mouse models, a number of groups turned to more conventional hdpp knock-in approaches, resulting in ubiquitous, constitutive overexpression of hdpp throughout the mouse (fig. ) . in , agrawal et al. reported development of a robust mers-cov mouse model by placing expression of hdpp under the cag promoter (agrawal et al. ) . infection with clinical isolates of mers-cov resulted in high viral loads in the lungs that produced severe respiratory disease and was fatal by day post-infection (agrawal et al. ) . however, disease was confounded by high viral loads in extrapulmonary tissues including the brain, heart, spleen, kidney, and intestines (agrawal et al. ) . in an independent study, zhao et al. demonstrated multi-organ damage in an hdpp overexpression mouse model employing the same promoter (zhao et al. ) . the mers-cov viral loads were nearly two logs higher in the brains of infected mice than in the lungs and corresponded to viral encephalitis observed in the brain by day , with mice exhibiting signs of paralysis (zhao et al. ) . li et al. went a step further by generating hdpp overexpression models with the goal of restricting expression to epithelial cells . two different models were generated using either the cytokeratin (k ) or surfactant c protein (spc) promoter to drive expression of hdpp . mouse lines derived from the spc-hdpp cassette did not exhibit mortality or signs of respiratory disease. in contrast, infection of k -hdpp mice with × pfu of a mers-cov clinical isolate (emc_ ) produced weight loss, lung hemorrhaging and mortality by - days p.i., and lung pathology consistent with severe respiratory disease . unfortunately, the k promoter did not limit mers-cov infection to the lungs, instead high viral loads in the brains of infected mice caused neurological disease that was consistent with the observed mortality . in a novel genetic engineering approach, pascal et al. employed regeneron's velocigene technology to precisely replace the entire mdpp coding region (~ kb) with the hdpp gene, which included ~ kb of sequence encoding both introns and exons (pascal et al. ) (fig. ) . despite removal of nearly the entire mdpp gene, the ′ and ′ untranslated regions of mdpp were maintained; thereby, retaining the endogenous mdpp promoter and rna termination regions conferred expression levels and tissue distribution for hdpp similar to that obtained for mdpp . infection with × pfu of a mers-cov clinical isolate resulted in viral replication in the lungs with mild clinical disease and pathology indicative of inflammation, with no weight loss or mortality through day p.i. (pascal et al. ) . subsequent studies with the velocigene mice comprised a more detailed evaluation of the model, wherein mice infected with . × pfu achieved dramatic weight loss and mortality (using % cut-off) by days p.i. . c dpp sequence used in velocigene hdpp knock-in mouse model (pascal et al. ) . mouse genomic sequence, from exon through the stop codon in exon , was deleted and replaced with exon through exon and a portion of ′ untranslated sequence of human genomic sequence. d dpp sequence used in hdpp knock-in model (li et al. ) . mouse genomic sequence from codon i in exon to codon v in exon was replaced with the human equivalent. e dpp sequence used in - +/+ mouse model (cockrell et al. ) . crispr/cas technology used to make a l substitution in exon and t r substitution in exon of mouse dpp . f dpp construct used in hdpp overexpression mouse models. constructs included cdna from human dpp flanked by constitutive promoter, polyadenylation signals, and other regulatory elements (agrawal et al. ) ; or by ′ and ′ genomic regions of human cytokeratin or human surfactant protein c ) (coleman et al. ) . lung pathology indicated that mice developed moderate signs of respiratory infection with little indication of severe respiratory disease. these studies were also the first indication that cd + t cells and macrophages play a significant role in mers-cov pathogenesis (coleman et al. ) . importantly, mers-cov infection/ replication was primarily in the lungs, with little involvement of extrapulmonary tissues (coleman et al. ; pascal et al. ) . the lack of brain lesions in this model was a significant advancement over transgenic models that relied on global/ubiquitous hdpp expression. nonetheless, this model is not readily available to the scientific community by regeneron, and if the model is obtained it comes with a number of commercial restrictions from regeneron that limit its utility when considering development of therapeutic countermeasures (declaration by the authors of this manuscript). another limitation often overlooked with hdpp expression models is the innate functions of dpp that have evolved in a species-specific manner to maintain various physiological processes inherent to the host. altered dpp activity and/or expression is associated with many pathological conditions that include psycho-neuroendocrine disorders, solid tumor cancers, hematological malignancies, infectious disease, and autoimmune/inflammatory diseases (klemann et al. ) . the enormous biological breadth of dpp is predominantly through its enzymatic activity wherein it cleaves off the amino terminal dipeptides of various biological substrates having l-alanine or l-proline at the penultimate position (klemann et al. ) . one of the more important physiological processes of dpp , also known as cd , is the modification of numerous cytokine and chemokines involved in the maintenance of immunological homeostasis (klemann et al. ; ohnuma et al. ) . as a cell surface molecule on cd + and cd + t cells, dpp influences numerous immunological processes including t cell activation and proliferation, cytokine production, differentiation to immunoglobulin producing plasma cells, and transendothelial migration (ohnuma et al. ) . therefore, overexpressing full-length hdpp or replacing mdpp with full-length hdpp in the knock-in mouse models may significantly alter the inherent biological and immunological processes that mdpp has evolved to execute in a species-specific manner, in the mouse. ultimately, if hdpp expression results in a modified immune response in mice, these could artificially influence the pathogenic outcomes of mers-cov infections and immunological responses that are central to evaluating the efficacy of therapeutic countermeasures. the goal of the most recent generation of mouse models was to genetically modify the mdpp receptor so that it would be amenable to interaction with the mers-cov spike rbd and subsequent infection, thereby avoiding the need to introduce the hdpp receptor. achieving a mers-cov susceptible mdpp with minimal modifications would be central to limiting any functional alteration to mdpp that could interfere with its broad influence over multiple host physiological processes. in , cockrell et al. utilized the crispr/cas technology to make two amino acid substitutions in the mdpp gene (a l and t r) of c bl/ j mice (cockrell et al. ) (fig. ) . as discussed above, in vitro overexpression of the modified mdpp was previously found to alter the susceptibility of cell lines to mers-cov infection (cockrell et al. ; peck et al. ) . mice with the modified dpp (referred to as - +/+ mice) encode both amino acid substitutions on both chromosomes. characterization of this model demonstrated that innate mdpp expression profiles and physiological processes influenced by dpp (glucose metabolism and t cell activation) were not altered by the two amino acid modifications of mdpp . the - +/+ mice infected with various isolates of mers-cov (clinical isolates, a camel isolate, and molecular clones) supported high viral replication in the lungs, but failed to exhibit clinical signs of disease (cockrell et al. ) . adaptive evolution through rounds of serial passage in - +/− mice yielded a mouse-adapted mers-cov capable of achieving lethality, decreased respiratory function, and lung pathology indicative of severe acute respiratory distress syndrome at viral doses of × pfu (cockrell et al. ) . mers-cov infected - +/+ mice that exhibited severe respiratory disease were absent of any detectable virus in the brain. the development of severe respiratory disease and mortality could be prevented using a mers-cov spike-directed vaccine, and through prophylactic administration of an antibody therapeutic (cockrell et al. ) . although the - +/+ model is highly effective for studying mers-cov pathogenesis and evaluating therapeutic countermeasures, the high infectious dose ( × pfu) required to achieve severe respiratory disease and mortality left room for improvement. an additional rounds of passaging resulted in a mouse-adapted virus capable of achieving similar severe respiratory disease and mortality, but at substantially lower doses ( - pfu) (douglas et al. ) . overall, limiting changes in the mdpp receptor to two amino acids, researchers were able to minimize disruption of the natural expression levels, distribution, and biological functions of mdpp . the - +/+ mers-cov mouse model continues to have a central role in studies investigating mers-cov pathogenesis and therapeutic countermeasures (menachery et al. b, c) . the most recent mouse model consists of a genetically modified mdpp receptor through replacement of exons - (including introns) of the mdpp locus with the hdpp equivalent, resulting in a minimal knock-in mouse model referred to as hdpp -ki (li et al. ) (fig. ) . modification of mdpp in the hdpp -ki model is much smaller than the regeneron full-length hdpp knock-in model, but larger than the two amino acid modifications to mdpp in the - +/+ model, placing it somewhere between the two types of models. regarding biological and immunological function, it is not clear if the modified mdpp in the hdpp -ki model has altered expression, biodistribution, or functional profiles compared to wild-type mdpp receptor. nevertheless, the hdpp -ki mice were susceptible to infection with clinical isolates of mers-cov, resulting in high levels of viral replication in the lungs but no signs of respiratory disease. a mouse-adapted mers-cov was achieved through serial passages of mers-cov, inducing severe respiratory disease accompanied by high mortality at infectious doses of - pfu (li et al. ) . similar to the - +/+ model, the hdpp -ki model exhibited little involvement of extrapulmonary tissues in mers-cov pathogenesis (li et al. ). the mouse-adapted viruses acquired by li et al. revealed unique mutations in specific viral proteins that may be critical for achieving and evading host immune responses. comparison of these viruses to those obtained by douglas et al. demonstrated that changes at amino acid in the mers-cov spike protein may be critical to achieve lethal infection at low infectious doses (douglas et al. ; li et al. ). importantly, this mutation was not present in earlier passages by cockrell et al. in the - +/+ model, which required high infectious doses to achieve severe respiratory disease and lethality (cockrell et al. ) . mouse-adapted viruses, such as those used by cockrell et al., douglas et al. and li et al. can help provide insight into adaptive evolution and identify specific mutations or regions of the virus that are important for enhancing virus fidelity in the host and/or evading the host immune responses. adaptations in mers-cov most notably occur in the spike region, which is an important determinant of host tropism. adapted mers-cov strains with mutations in this region may not provide an accurate representation of the observed viral pathogenesis in humans. currently, achieving lethal disease in mice with clinical isolates of mers-cov require expression of full-length hdpp , as demonstrated for the regeneron hdpp knock-in mice. however, altering dpp significantly through overexpression or replacement of mdpp can disrupt the immunological homeostasis of the animal, making it difficult to analyze disease etiology and immune responses to therapeutic countermeasures. a mers-cov mouse model would ideally involve infection with clinical mers-cov isolates in an unmodified mouse. one possibility is utilizing cc mice, as described above for sars-cov, which offer a wider range of genetic variation than the genetically engineered c bl/ mice used in currently established mouse models. however, unlike sars-cov, the cc mice are not susceptible to mers-cov. crossing various cc lines with mice that have minimal alterations to mdpp (e.g., - +/+ mice), or direct genetic engineering of cc lines using crispr/cas technologies, can produce mouse lines that maintain native mouse dpp expression, distribution, and functional profiles, but can be screened for differential susceptibility to clinical isolates of mers-cov. the capacity to genetically engineer receptors to alter mouse susceptibility to hcov infection, combined with adaptive hcov evolution and genetically diverse cc mouse strains, establishes a platform that can be used to evaluate the pathogenesis of pre-emergent hcovs with unknown etiologies. the mouse tools founded from studies of sars-cov and mers-cov pathogenesis will aid in understanding if specific pre-emergent covs have pathogenic potential in mammalian models of severe respiratory disease. most hcovs (sars, mers, e, and nl ) are considered to have their origin in bats [reviewed in (forni et al. ; menachery et al. a) ]. metagenomics analysis of various bat species has identified a diverse repertoire of sarslike (sl-bcovs) and mers-like (ml-bcovs) bat coronaviruses (ge et al. (ge et al. , he et al. ; lau et al. lau et al. , lau et al. , wu et al. ; yang et al. ) . those with pre-emergent potential include sl-covs wiv and wiv , shown to utilize the ace receptor (ge et al. ; menachery et al. ; yang et al. ) , and an ml-cov hku , demonstrated to utilize the dpp receptor for infection yang et al. ) . notably, wiv and wiv are the only pre-emergent covs directly isolated from bat feces on vero e cells and shown to replicate in various cell lines expressing the human, or nhp, ace receptor (ge et al. ; yang et al. ) . in contrast to wiv and wiv , isolated hku was not demonstrated to infect cells, but rather lentiviral particles pseudotyped with hku spike protein supported efficient binding and infection of cells expressing hdpp yang et al. ) . the structure of cov spike proteins in the context of pseudotyped lentiviral particles may differ to that in cov particles, and may account for the difficulty of hku infecting and replicating on cells expressing hdpp . the spike proteins of many pre-emergent bat covs (bcovs) have amino acid differences that preclude efficient interaction with known human host receptors, such as hace or mace . transmission to intermediate hosts (civets or camels) and humans may require additional adaptations in the spike protein [reviewed in (graham and baric ; menachery et al. a) ]. therefore, to evaluate pathogenesis of pre-emergent bcovs in mouse models, the spike proteins were replaced by generating infectious molecular clones with known spike proteins from clinical isolates of sars or mouse-adapted sars-cov becker et al. ; menachery et al. menachery et al. , . the first of these studies derived an infectious molecular clone sl-bcov from sequences of four previously identified sl-bcovs (hku - , hku - , hku - , and rp ) (becker et al. ). the chimeric virus was able to replicate in balb/c mouse lungs, but did not elicit signs of respiratory disease commonly associated with sars-cov (becker et al. ) . similarly, a chimeric wiv (wiv -ma ) replicated in balb/c mouse lungs, but was attenuated with regard to causing respiratory disease (menachery et al. ) . attenuation of wiv -ma was partially overcome by infecting hfh mice that overexpress the hace receptor (menachery et al. ) , indicating that mouse adaptation of the wiv -ma to mace could enhance disease in wild-type balb/c mice. indeed, agnihothram et al. demonstrated that mouse adaptation of an infectious molecular clone of an hku -se chimeric resulted in dramatic respiratory disease in aged balb/c mice . hku is closely related to mers-cov, but since there was no mouse model available for mers-cov at the time, it was more practical to replace the hku spike with the mouse-adapted sars-cov spike. these studies demonstrated that adaptive mutations outside of the spike are necessary to elicit respiratory disease in different mammalian species. however, this was not the case for the shc bcov chimeric virus (shc -ma ), which caused significant weight loss in balb/c mice and replicated to titers comparable to sars-cov ma in the lungs . moreover, wild-type shc spike protein supported infection/replication in the lungs of balb/c mice, but did not result in disease. applying the classical approach of adapting shc to mice by serial passaging may be beneficial, but can be time-consuming. screening shc , or other pre-emergent bcovs, in cc mouse strains has the potential to rapidly lead to mouse models capturing different, or even all, aspects of clinical disease in human patients. the ultimate goal for mouse model development is capturing disease etiologies that closely reflect what is observed during human cov infection, so the efficacy of various therapeutic countermeasures can be evaluated for effective resolution of pathogenic outcomes. a number of hcov-directed drug, antibody, and vaccine therapeutics were evaluated for efficacy against sars-cov until when the focus of therapeutic development shifted to mers-cov [reviewed in (dyall et al. ; zumla et al. ) ]. currently, there are no fda-approved therapeutics for the specific treatment of sars-cov or mers-cov. various combinations/types of broad spectrum antivirals (e.g., ribavirin) combined with interferon therapies (interferon α or β), and/ or immune modulators (e.g., corticosteroids) were ineffective for sars-cov and mers-cov, and in some cases appeared to worsen disease [reviewed in (dyall et al. ; zumla et al. )] . a recent clinical trial seeks to interfere with mers-cov replication using a combination therapy that includes fda-approved protease inhibitors (lopinavir/ ritonavir) (https ://clini caltr ials.gov/ct /show/study /nct ). repurposing fda-approved drugs has been the interest of numerous therapeutic studies, but little is known regarding the efficacy of drug therapeutics against sars-cov or mers-cov outside of cell-based in vitro studies [reviewed in (dyall et al. ) ]. mouse models confer distinct advantages for therapeutic examination including cost savings through small scale production for testing, and experimental reproducibility. the recent development of a novel nucleotide prodrug, gs- , demonstrated efficacy against ebola virus in nhps (warren et al. ) , and has since moved into phase clinical trials for ebola [https ://clini caltr ials.gov/ct /show/ nct ?cond=preva il+iv&rank= ; (dornemann et al. )] . a recent study demonstrated broad spectrum efficacy of gs- against multiple hcovs (sars-cov, mers-cov, and nl ) and pre-emergent bcovs (hku , hku , sch , and wiv ) on primary human airway epithelial cells (sheahan et al. ) . importantly, gs- exhibited in vivo efficacy against sars-cov-induced immunopathology if the drug was administered prophylactically, or a therapeutic dose -h post-infection (sheahan et al. ) . delaying treatment until -h post-infection did not ameliorate disease symptoms. although the kinetics of sars-cov pathogenesis will differ between mouse and human, these results indicate that the molecular programs leading to respiratory immunopathology are established shortly after infection, thereby limiting effective therapeutic treatment to a short window, post-infection. the study by sheahan et al. also demonstrates the importance of having mouse models that recapitulate severe respiratory disease often seen in humans (sheahan et al. ). an effective therapeutic should not only prevent viral replication, but more importantly should effectively restrict respiratory pathogenesis. achieving both may require therapeutic intervention with a combination of an hcov antiviral and a hostdirected therapy that curtails immunopathology associated with acute respiratory distress syndrome. only in the last years have mers-cov mouse models, described above, become available to assess therapeutics; therefore, future studies will include evaluating in vivo efficacy of gs- against mers-cov in the - +/+ model described above. as an ongoing threat to worldwide public health, a plethora of therapeutic research focused on development of mers-cov-specific antibodies and vaccines, both of which have been extensively reviewed [reviewed in dyall et al. ; okba et al. )] . importantly, few studies have evaluated the therapeutic efficacy of mers-cov antibodies and vaccines in mammalian challenge models that elicit severe respiratory disease, often resulting in death (cockrell et al. ; munster et al. ; tai et al. ; van doremalen et al. ; wang et al. ). rather, many preclinical studies exhibited efficacy in the mouse or nhp challenge models with protection defined as reduced viral loads in the lungs by plaque assay or rt-pcr. preclinical results have expedited the process of moving some antibody and vaccine therapeutics into early clinical trials [reviewed in dyall et al. ; okba et al. ) ]. based on results obtained in studies with gs- , it will be critical to determine efficacy of antibody and vaccine therapeutics in lethal respiratory models of mers-cov infection. vaccine countermeasures are not only being considered as intervention strategies for staving off disease in humans, but have also exhibited efficacy in dromedary camels, the mers-cov zoonotic host (haagmans et al. ; muthumani et al. ) . targeting zoonotic hosts, such as dromedary camels or bat populations, may be an effective means of curtailing transmission into the human population. thinking beyond vaccine countermeasures, we now have the ability to harness genome editing technologies that could allow us to intervene with the process of zoonotic transmission. c bl/ j mice were made susceptible to mers-cov by using crispr/cas technologies to modify two amino acids at positions and on mouse dpp , to the orthologous amino acids encoded by human dpp (cockrell et al. ) . genetic modification of the human dpp orthologues in camel and bat to look like mouse dpp at these positions may result in camels and bats that are now resistant to mers-cov infection. although introduction of mers-cov-resistant bats into the environment may take a long time, camels are considered farm animals in many parts of northern africa and the middle east with controlled breeding practices; therefore, it may be feasible to establish resistant herds that are unable to transmit mers-cov to humans. genome editing of farm animals is clearly achievable as recently demonstrated in pigs modified with crispr/cas for genome-wide inactivation of porcine endogenous retroviruses (pervs), thereby eliminating the potential of cross-species transmission of pervs during xenotransplantation of pig organs into humans, with the intention of easing the shortage of available human organs (niu et al. ; yang et al. ) . the combined power of genetic engineering of mice, reverse genetic systems for covs, mouse-adaptation, and genetically diverse mouse populations afford the tools to develop models that reproducibly recapitulate severe respiratory disease that can be caused by hcovs in humans. establishing mouse models for sars-cov and mers-cov have largely depended on one or more of these tools. regardless of how effective models for sars-cov and mers-cov are for studying pathogenesis and therapeutic interventions, there are drawbacks related to mouse-adapted and transgenic mouse models that alter susceptibility and pathogenesis to hcovs. acquired mutations associated with severe respiratory disease in mouse-adapted hcovs may have no bearing on human pathogenesis or have altered pathogenic outcomes in humans. genetically engineering a specific host species to enhance susceptibility restricts applications of the mouse model, requiring cross-breeding to introduce novel host mutations from knock-out mouse lines and cc mouse strains. this process is labor intensive and requires an extensive timeline. alternatively, one could introduce mutations directly into genetically diverse lines; however, this is not cost effective. additionally, it is difficult to replicate and study co-morbidities in mice that are associated with lethal respiratory disease seen in humans. pre-existing conditions such as advanced age, diabetes, chronic lung diseases, heart disease, and kidney disease have been reported in many of the severe/lethal mers-cov human cases [reviewed in ]. one way to address many of these concerns could be to screen genetically diverse populations of mice (e.g., collaborative cross) with clinical isolates or infectious molecular clones of wild-type strains from emergent, or pre-emergent, hcovs. screening in genetically diverse mouse populations may yield mouse models with a range of clinically relevant phenotypes that more closely reflect the plethora of respiratory disease outcomes observed in the human population. experiences from sars-cov and mers-cov have taught researchers that each hcov may require unique approaches for mouse model development. the mouse tools instituted for sars-cov and mers-cov constitute a versatile preclinical platform for addressing global pathogen preparedness, a directive 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generation of a mouse model for middle east respiratory syndrome multi-organ damage in human dipeptidyl peptidase transgenic mice infected with middle east respiratory syndrome-coronavirus a functional variation in cd increases the severity of pandemic h n influenza a virus infection fatal swine acute diarrhoea syndrome caused by an hku -related coronavirus of bat origin genetic variation of the human alpha- -heremans-schmid glycoprotein (ahsg) gene associated with the risk of sars-cov infection coronaviruses-drug discovery and therapeutic options key: cord- -t yioyl authors: rohr, jason r.; barrett, christopher b.; civitello, david j.; craft, meggan e.; delius, bryan; deleo, giulio a.; hudson, peter j.; jouanard, nicolas; nguyen, karena h.; ostfeld, richard s.; remais, justin v.; riveau, gilles; sokolow, susanne h.; tilman, david title: emerging human infectious diseases and the links to global food production date: - - journal: nat sustain doi: . /s - - - sha: doc_id: cord_uid: t yioyl infectious diseases are emerging globally at an unprecedented rate while global food demand is projected to increase sharply by . here, we synthesize the pathways by which projected agricultural expansion and intensification will influence human infectious diseases and how human infectious diseases might likewise affect food production and distribution. feeding billion people will require substantial increases in crop and animal production that will expand agricultural use of antibiotics, water, pesticides and fertilizer, and contact rates between humans and both wild and domestic animals, all with consequences for the emergence and spread of infectious agents. indeed, our synthesis of the literature suggests that, since , agricultural drivers were associated with > % of all — and > % of zoonotic — infectious diseases that emerged in humans, proportions that will likely increase as agriculture expands and intensifies. we identify agricultural and disease management and policy actions, and additional research, needed to address the public health challenge posed by feeding billion people. i nfectious diseases are emerging at an unprecedented rate with significant impacts on global economies and public health . the social and environmental conditions that give rise to disease emergence are thus of particular interest, as are management approaches that might reduce the risk of emergence or re-emergence . at the same time, undernutrition -the insufficient intake of one or more nutrients -remains a major source of global ill health [ ] [ ] [ ] [ ] . together, the unprecedented rate of infectious disease emergence and the need to sustainably feed the global population represent two of the most formidable ecological and public health challenges of the twentyfirst century [ ] [ ] [ ] [ ] [ ] (fig. ) , and they interact in complex ways (fig. ) . although modern agricultural technologies have reduced hunger, improved nutrition and spared some natural ecosystems from conversion to agriculture, current global agricultural production and distribution infrastructure still leaves billions of people's diets deficient in one or more crucial nutrients, with major consequences for global morbidity and mortality , and these deficiencies are expected to worsen with climate change [ ] [ ] [ ] [ ] . credible estimates are available for specific nutrient shortfalls -for example, . billion people suffering iron-or vitamin b -deficiency anemia , . billion people with insufficient dietary energy (that is, calorie) intake , % of pre-school age children and % of pregnant women at risk of vitamin a deficiency -but the world still lacks a rigorous, recent assessment of the population suffering shortfalls of any one or more nutrients, although the number is surely in the billions. by , the united nations projects that the global population will grow by nearly billion, to exceed billion people . meeting the united nations' sustainable development goal, to "eradicate hunger" (https://sustainabledevelopment.un.org/) for this expanding human population will necessitate a large increase in food supplies, with major changes to agricultural production and distribution systems, infrastructure and social protection programmes (fig. ) . despite large and, in many cases, growing inequalities, the global population is on average expected to become richer, and historically, with greater affluence has come greater consumption of food products in general and more animal-sourced foods in particular, both of which further increase food demand and thus the requirement for agricultural expansion and/or intensification . agriculture already occupies about half of the world's land and uses more than two-thirds of the world's fresh water , and recent studies have suggested that agricultural production might need to double or triple by to keep pace with projected population growth and food demand [ ] [ ] [ ] (fig. ) . meeting this demand using present agricultural production systems could require replacing > hectares of natural ecosystems with agricultural production, approximately % of the global land area, which is larger than the continental united states, although continuous efficiency improvements in agriculture will compel re-evaluation of these estimates. in turn, this agricultural expansion could result in an estimated ~ -fold increase in irrigation, ~ . -fold increase in fertilizer , , and -fold increase in pesticide use (fig. ) . the challenges of feeding > billion people and managing infectious diseases intersect in several ways ( figs. and ) . first, the expansion and intensification of agriculture are disproportionately occurring in tropical, developing countries , , , where % of deaths are attributable to infectious diseases , where the risk of disease emergence might be greatest, and where disease surveillance and access to health care, particularly for those infections that accompany extreme poverty, are most limited , . second, agricultural expansion and intensification have historically come with massive habitat conversion, contamination with animal waste and increasing use of agricultural inputs, such as pesticides and antibiotic growth promoters. beyond their direct adverse effects on human health , , agricultural biochemical inputs are known to have direct effects on emerging human infectious diseases, and can also serve as indirect drivers by contributing to the emergence of wildlife diseases that constitute important sources of emerging infections in humans , . however, the strengthening of agricultural production systems can also improve nutrition, which has pronounced benefits for combating many infectious diseases at the individual and population levels , . although concerns have been raised regarding the environmental impact of agricultural expansion and intensification , - , , , their effects on infectious disease risk have not been synthesized. here, we review both the beneficial and adverse effects of agricultural expansion and intensification on the transmission of human infectious diseases. we synthesize the pathways through which agricultural practices influence human infectious diseases, and vice versa, and identify opportunities to minimize the adverse consequences of agricultural growth while maximizing the human health benefits of agricultural development (fig. ). agricultural development can yield direct improvements to nutrition, and through several mechanisms, nutrition can be a critical determinant of infectious disease susceptibility and progression , (figs. and ). for example, immune responses are energetically costly and thus undernutrition often reduces the development and effectiveness of immune responses that can limit or clear infections. the relationship between infectious disease and nutritional agricultural production can improve human health by reducing food prices and enhancing nutrition, which can increase resistance to infectious diseases. however, freshwater habitats established for irrigation, as well as other agricultural inputs, often increase the risk of vector-borne diseases, such as malaria and schistosomiasis. in general, rural residents are most vulnerable to these increases in infectious disease, whereas consumers some distance away derive most of the benefits from increased food production. to maximize human health given the impending billion humans projected on the planet by , society must minimize the adverse consequences of agricultural growth while maximizing the health benefits. images by kate marx. status can function in reverse as well, because many parasitic infections place direct demands on host nutrition, causing undernutrition when food is limited . in fact, some parasites, such as helminths, can even cause eating disorders, such as geophagy (desire to eat soil), bulimia and anorexia . enduring infections often require rapid and effective repair of tissue damage caused by parasites, which is also costly. hence, certain infections can cause undernutrition, which itself can compromise both resistance and tolerance to infections , . by improving nutrition, agricultural development should facilitate combating many infectious diseases. for example, death rates from acute respiratory infections, diarrhoea, malaria and measles, diseases that on average kill more than a child every seconds ( million per year) , are much higher in children who suffer undernutrition than in those that do not , . in addition, poor maternal nutrition and associated impaired fetal growth are strongly associated with neonatal death from sepsis, pneumonia and diarrhoea ; undernourishment is a well-understood risk factor for . although the traditional green revolution approach to food production has succeeded in reducing undernourishment arising from insufficient calorie and protein intake, it has been very slow in reducing micronutrient deficiencies , which can be have significant effects on defence against disease. to make matters worse, where violence, unrest and terrorism impede access to food, such as in parts of africa and central asia, morbidity and mortality attributed to communicable diseases can be further exacerbated . although research suggests that improving nutrition will generally improve responses to infectious diseases, there may be important exceptions. for certain diseases, such as schistosomiasis and many respiratory infections, pathogenesis is a product of host immunity, often from hyperinflammation , . thus, undernutrition can actually decrease symptoms by reducing the strength and pathogenicity of immune responses . in addition, as nutrition improves, some parasites might proliferate faster than immunity can increase , resulting in more, rather than less, morbidity, with the classic example that dietary intake of iron can increase malariainduced mortality . in this section, we review the links between rural economy, infrastructure and infectious disease. , phosphorous (c), fertilizer and pesticide use (d), cropland area (e), and irrigated land area (f) and associated % prediction bands (light blue bands). 'nitrogen' refers to the normalized estimated global amount of nitrogen nutrients in fertilizers produced (originally reported in thousands of tonnes, now in metric tonnes). 'phosphorous' refers to the normalized estimated global amount of p o nutrients in fertilizers produced (originally reported in thousands of tonnes, now in metric tonnes). for a, data were collected from the world bank. for b-f, data were collected from the food and agriculture organization of the united nations and statistical models were developed based on the relationships between these variables and global human population density. after the best model was identified, year was substituted for human population density based on the fit in a. these projections should be evaluated with considerable caution because the models assume that human population size is the only factor that affects fertilizer and pesticide use and the amount of arable and irrigated cropland, when in reality, many factors affect these responses (for example, climate, diets, type of crops grown and so on). they merely illustrate that even an extremely parsimonious model seems to track past patterns tolerably well and thus could serve as a basis for coarse projections. see supplementary methods for details of the statistical models used to generate these figures and supplementary table for coefficients and statistics for the best-fitting models. economic development. economic development, especially agricultural development, has historically driven reductions in both infectious diseases and poverty across many settings. in fact, the poor financially benefit more from economic growth in the agricultural sector than in industrial or service sectors , . the early stages of economic development often involve the construction of infrastructure to facilitate food production and distribution, including roads, dams and irrigation networks , . more recently, the early stages of rural development often include rapid expansion of telecommunication, and to a lesser degree, electrification, both of which are promising but underutilized resources for disease monitoring and control in the developing world . other rural infrastructure that is more crucial to infectious disease prevention, such as safe water, sanitation and energy supplies, often follows or is developed concurrently , . in developing countries, rural infrastructure that provided access to sanitation and safe water explained % and % of the difference in the prevalence of malnutrition and child mortality rates between the poorest and richest quintiles, respectively . moreover, clean water, sanitation and electricity can also facilitate the construction of schools and health clinics that can help to further reduce disease through education, prevention and treatment. hence, if the history of the developed world repeats itself in the developing world, then it seems plausible that agricultural development necessary to feed billion people might help to reduce infectious diseases by promoting economic development and rural infrastructure. there are several important assumptions to this hypothesis, however, such as: ( ) it is possible to feed billion people; ( ) the rate of economic development will match or exceed the pace of human population growth; and ( ) developing countries are not in an infectious disease-driven 'poverty trap' , which is the notion that poverty increases the chances of acquiring and succumbing to disease, and chronic disease traps humans in poverty [ ] [ ] [ ] [ ] [ ] . if agricultural development lags behind population growth or if clean water supplies, sanitation and electricity do not immediately follow road, dam and irrigation construction, then the prevalence of many infectious diseases could remain unchanged or even increase as human populations grow . agricultural irrigation and freshwater redistribution. one major change in rural infrastructure that often accompanies agricultural development is the redistribution of fresh water, which has well-known consequences for the transmission of infectious diseases. for example, because % of crop production comes from only the % of agricultural land that is irrigated, and irrigated lands accounted for much of the increased yields experienced during the green revolution , the potential for increased irrigation infrastructure to exacerbate infectious diseases is a critical concern (fig. ) . importantly, agricultural development has caused both declines of certain types of fresh water, such as wetlands, and increases of others, such as dams, reservoirs and irrigation schemes. one of the largest drivers of global wetland losses has been conversion to agriculture , which has led to declines in diseases that rely on wetland habitats. for example, japan's successful eradication of schistosomiasis during the mid-twentieth century relied in part on conversion of wetlands to orchards in schistosomiasis-endemic areas , although replacing oxen with horses, which are less susceptible to schistosoma japonicum, as draft animals is also thought to have contributed substantially . similarly, some of the earliest successes in malaria control at the turn of the twentieth century occurred in the southern united states, where wetland drainageincluding for agricultural conversion -was one of a suite of antitransmission strategies used by the rockefeller foundation in its landmark malaria control efforts . although wetlands often decline with agriculture, dams, reservoirs and irrigation networks often increase, and this redistribution of fresh water has been widely associated with increases in some vectors and hosts of human pathogens . for example, construction of the aswan high dam in egypt and the accompanying irrigation network was associated with a rise in mosquito vectors and the disfiguring mosquito-borne disease lymphatic filariasis, commonly known as human elephantiasis . likewise, dam and irrigation construction resulted in increases in malaria in sri lanka and india , , and a sevenfold increase in malaria in ethiopia . a meta-analysis of studies revealed that humans living near irrigation schemes or in close proximity to large dam reservoirs had significantly higher risk of schistosome infections than humans that did not live near these water resources , at least partly because of increased freshwater habitat for intermediate snail hosts. similarly, a recent analysis of schistosomiasis case data from the past years across sub-saharan africa showed far-reaching effects of dams and irrigation schemes, with increases in schistosomiasis risk extending up to hundreds of kilometres upstream from the dams themselves . hence, dams, reservoirs and irrigation networks related to agricultural expansion are likely to increase vector-borne and waterborne diseases unless water resources are developed in deliberate and coordinated ways to mitigate these risks . urbanization, globalization and the movement of agricultural products. the world's population became majority urban in and urbanization continues to outpace population growth, especially in developing countries. urbanization necessarily extends food supply chains, as consumers reside further from farms and fisheries. although only % of the food produced globally for human consumption is traded across international borders , globalization is likewise elongating food supply chains. furthermore, as the costs of international travel have declined over time, the movement of people across borders has likewise increased. the expanded spatial scope and increased frequency, speed and volume of people and agricultural products moving within and among countries necessarily facilitates the spread of pathogens. for example, in ecuador, there is evidence that the construction of new roads has affected the epidemiology of diarrheal diseases by changing contact rates among people as well as between people and contaminated water sources . each year in the united states, there are already an estimated million people with illnesses, , hospitalizations and , deaths from food-borne infections , and imported foods -especially from developing countries with poor sanitation infrastructure and weak food safety enforcement -have been associated with a rise in food-borne illness . in addition, globalization is already thought to be a factor in the spread of human influenza viruses that spillover from poultry and swine , . although modernization of food supply chains, especially those linking farms in developing countries to high-income consumers in cities and high-income countries, has typically accelerated the diffusion of stricter food safety and quality standards , , as urbanization and globalization further extend food supply chains, enhanced monitoring for the spread of pathogens across transportation networks and strengthened food safety regulations will be needed. in this section, we review the effects of agricultural industrialization. anti-parasitic and antibiotic drug use. as the global human population increases, there will almost certainly be an increase in high density, industrialized livestock and aquaculture operations. currently, such livestock operations are vulnerable to devastating losses of animals to disease. for instance, in just the last years, an influenza a virus (h n ) and a foot-and-mouth outbreak led to the destruction of more than . million chickens and million livestock in china and great britain , respectively, and a 'mad cow disease' epizootic led to the slaughter of million cattle worldwide . similar scenarios have occurred in aquaculture. in the past three decades, there has been more than fourfold growth in industrial aquaculture worldwide , which should continue to increase as food demand grows and wild fish and shellfish captures push the limits of renewable production . bacterial outbreaks in aquaculture are common, especially in developing countries where there are sanitary shortcomings . in an effort to prevent these catastrophic disease-associated losses and improve animal growth, the agricultural industry uses a larger proportion of global antibiotic and anthelmintic production than human medicine, and most of the antibiotics are provided at non-therapeutic doses in the absence of any known disease , . in fact, although estimates are lacking for most countries in the world, in the united states, nearly nine times more antibiotics are given to animals than humans and, of the antibiotics given to animals, more than times as many are used nontherapeutically as therapeutically . this widespread use of antibiotics and anti-parasitic drugs (for example, anthelmintics) in industrialized agriculture and aquaculture could have important implications for human infectious diseases because it seems to be driving microbial resistance to these drugs, some of which are also used in human medicine , , . for example, livestock are a primary source of antibiotic-resistant salmonella, campylobacter and escherichia coli strains that are pathogenic to humans . there is evidence that antibiotic-resistance genes acquired from aquaculture are being transferred to human systems and these pathogens have subsequently caused outbreaks . anthelmintic resistance is also rife among parasitic worms of livestock and is strongly implied for parasitic worms of humans . as livestock and aquaculture production expand to address growing food demands, it is likely that current antibiotics and anthelmintics will become less effective because of evolved resistance, and thus infectious diseases of domesticated animals and humans will be more difficult to treat . pesticides, fertilizers and disease. pesticides, particularly insecticides, have shared value for suppression of agricultural pest populations and disease-carrying insect vectors, such as mosquitoes and other flies. because of the anticipated sharp increase in pesticide use by (fig. ) , insecticide resistance is also expected to increase, with important implications for the control of diseases carried by insect vectors. insecticides with widespread shared use for both crop protection and vector control include pyrethroid, organophosphate and organochlorine insecticides . several mosquito vectors of human and livestock pathogens have already evolved some resistance to these compounds . if agricultural expansion and intensification is accompanied by an increased use of insecticides, vector resistance may become more common and the control of vectorborne diseases more challenging. in addition to potentially driving vector resistance, increased pesticide use is likely to cause numerous other effects on host-parasite interactions. pesticides can alter disease risk by modifying host susceptibility to parasites [ ] [ ] [ ] . for example, many pesticides are immunomodulators that can increase infectious diseases of wildlife and humans , , or are endocrine disruptors of humans with potential downstream effects on immunity . even if pesticides do not directly affect immunity, detoxification of pesticides is energetically 'expensive' for the host, and thus pesticide exposure can reduce available energy resources for humans and zoonotic hosts to invest into parasite defences , . pesticides can also affect human disease risk by altering the densities of hosts or parasites or their natural enemies or mutualists , . several pesticides can alter host behaviours or be directly toxic to hosts and parasites, which in turn can modify contact rates between parasites and human hosts. in addition, pesticides can alter community composition, which can indirectly affect behaviours or densities of intermediate and zoonotic hosts and parasites. although chemical contaminants can be deadly to many free-living stages of parasites, both empirical trends and theoretical models suggest that stress associated with pesticide exposure can increase non-specific or generalist pathogens , , . in addition to the impacts that pesticides can have on infectious diseases, they can also contribute to non-infectious diseases, such as cancers, birth defects, miscarriages and impaired childhood development , which can further strengthen the poverty-infectious disease trap. indeed, among african agricultural households, there is evidence of an association between increased pesticide use and increased time lost from work due to sickness . in addition to an increase in pesticides, nitrogen-and phosphorous-based fertilizers are expected to increase threefold by to boost food production, and most of this increase will occur in tropical regions already rich with pathogens , , . although the effects of environmental nutrient enrichment on disease are indirect and complex, with some infections increasing and others decreasing, two reviews on the subject suggest that elevated nutrient levels more often than not exacerbate the impact of infectious disease , . for example, phosphorous enrichment can benefit mosquitoes that transmit malaria and west nile virus , . in addition, nitrogen-and phosphorous-based fertilizer use can increase the number of snails that transmit flatworms that cause human schistosomiasis , . conversion of natural habitat to agriculture can increase the abundance of ecotones (boundaries between ecological systems), change species composition and reduce native biodiversity , . ecotones play an important role in a number of important emerging infectious diseases , , and reduced biodiversity that accompanies agricultural intensification can increase zoonotic disease emergence and can worsen already endemic diseases [ ] [ ] [ ] . a recent global meta-analysis suggests that, based on the available literature, such biodiversity losses generally increase infections of wildlife and zoonotic infections of humans . however, studies of the relationship between biodiversity change and infectious disease risk tend to focus on a single parasite or disease, often of non-human animals, which limits the ability to determine more broadly the effects of agricultural intensification on the overall burden of infectious disease in human populations . recent studies have found that the local loss of dense forests, largely from agricultural expansion, affected diarrheal diseases, acute respiratory infections and general fever in cambodian children and infectious disease incidence in nigerian children over a decade , . proximate causes probably included reduced regulation of microbial contaminants in surface and ground waters, increased smoke from biomass burning, shifts in the ranges of insect vectors and decreased access to forest ecosystem services. a major concern is the potential for positive feedbacks between poverty, biodiversity loss, soil degradation and infectious disease , . several mechanisms can underpin these reinforcing relationships. the first arises as a result of poor rural peoples' reliance on limited biophysical assets for their livelihoods. as households clear forests, deplete soils or overharvest biota to meet near-term consumption needs, this resource degradation can compromise future economic productivity and increase disease risk. in addition, poverty, environmental degradation, biodiversity loss and disease can quickly become mutually reinforcing responses to natural shocks, such as droughts or floods, or to protracted conflict in a region. there are also more direct examples. for instance, global net losses of tropical forests remained unchanged during the s and s (at approximately million ha yr − or . % annually ; also see https://glad.umd.edu/projects/global-forest-watch and https:// www.globalforestwatch.org) and forest clearing can lead to transmission of zoonotic disease by increasing contact with wild animals. conversely, encroachment of people and domesticated animals into natural areas can introduce diseases to wildlife that can devastate wild populations and create reservoirs for the disease to be transmitted back to domesticated animals (see next section for further discussion and citations). especially as the agricultural frontier expands, considerable attention must be paid to monitoring and managing these biodiversity-poverty-disease feedbacks . a central tenet of epidemiology is that the incidence of many infectious diseases should increase proportionally with host density because of increased contact rates and thus transmission among hosts . hence, increasing human and livestock densities could cause increases in infectious diseases unless investments in disease prevention are sufficient to prevent these increases. given that most of the increase in human and livestock densities are expected to occur in developing countries where disease surveillance, pest control, sanitation, and medical and veterinary care are limited, there is little reason to expect that control efforts will keep up with the expected increases in infectious diseases associated with increasing densities of these hosts. as host densities and thus transmission increase, theory suggests that parasite virulence should also increase under some circumstances . when virulence of a pathogen is tied to its propagule generation within the body (such as occurs with many viral infections), the intermediate virulence hypothesis posits that an intermediate level of virulence maximizes parasite transmission because it balances producing many parasite offspring (increasing parasite fitness) with detriments to host survival due to pathology (decreasing parasite fitness). the balance in this trade-off determines parasite persistence. hence, as host densities increase and transmission becomes more frequent, the cost of increased virulence declines, shifting the optimum towards higher virulence . consequently, for pathogens that experience this virulence trade-off, increases in human, crop and livestock densities have the potential to augment both the incidence and severity of infectious diseases. feeding billion people -and the associated increase of land converted to agricultural production and livestock grazing -is expected to cause a surge in human-livestock, human-wild animal and livestock-wild animal contact rates, increasing the likelihood of 'spillover' events, which are defined as pathogen transmission from a reservoir host population to a novel host population , . as natural ecosystems are converted to crop land or range land, interactions among humans, and domesticated and wild animals, could increase . furthermore, if developing countries follow a trajectory similar to developed countries, then their demand for meat will increase, further increasing human-livestock, human-wild animal and livestock-wild animal contact rates . these interactions are crucial because % of livestock pathogens are capable of infecting multiple host species, including wildlife and humans , and based on published estimates from the s, over half of all recognized human pathogens are currently or originally zoonotic , , , as are - % of recent emerging infectious disease events , , (fig. ) . examples of recent zoonotic disease emergences with enormous impacts on either livestock, humans or both, many of which might have agricultural drivers, include avian influenza, salmonellosis (poultry and humans), newcastle disease (poultry), swine flu, nipah virus (pigs and humans), middle east respiratory syndrome (camels and humans), bovine tuberculosis, brucellosis (mostly cattle and humans), rabies (dogs and humans), west nile virus, severe acute respiratory syndrome (sars) and ebola (humans) , . to quantify the relationship between agricultural factors and disease emergence in humans through time, we used the human disease emergence database of jones et al. . we classified land-use change, food industry and agricultural industry as agricultural drivers of human disease emergence (see supplementary methods) . these analyses revealed that agricultural drivers were associated with % of all diseases and nearly % of zoonotic diseases that emerged in humans since (fig. ) . these values are even higher if we include the use of antimicrobial agents as an agricultural driver of human disease emergence, given that agricultural uses of antibiotics outpace medical uses in the developed world nearly nine to one , . several factors have materialized that facilitate spillover events associated with disease emergence . spillover appears to be a function of the frequency, duration and intimacy of interactions between a reservoir and novel host population . for example, influenza is believed to have jumped from horses to humans soon after domesticating horses and then made additional jumps to humans from other domesticated animals, such as poultry and swine . similarly, when free-range turkeys were prevented from interacting with wild birds and when interactions between domesticated pigs and wild fruit bats were reduced, influenza and nipah virus incidence , human pathogens that are currently or originally zoonotic , , and recent emerging pathogens that are zoonotic , . dropped, respectively, suggesting wild-animal sources of these infections , . in sub-saharan africa, the high frequency and duration of environmental interactions between different species of schistosoma worms infecting humans and cattle has undoubtedly facilitated their hybridization, and these hybrid schistosomes are more virulent to humans than their non-hybrid counterparts . these factors associated with spillover and disease emergence could be targeted to reduce transmission potential as human populations and agricultural productivity increase. given that most human population growth is expected to occur in developing, tropical countries, hunting, fishing and gathering pressures will almost certainly rise in subsistence economies , . the consequences of these pressures might initially increase contact rates between humans and wildlife. this could increase disease risk as bushmeat hunting is already thought to be responsible for several emerging human infectious diseases . to make matters worse, bushmeat hunting is the second biggest threat to biodiversity behind habitat loss, and biodiversity losses can contribute to disease emergence . however, if humans overexploit these natural resources, then human-wildlife interactions could eventually decline as species go extinct, which could reduce spillover events. changes in the composition of biodiversity associated with overhunting and overfishing can also have complex indirect effects on disease risk mediated by species interactions. for example, empirical evidence supports the notion that defaunation of large vertebrates in africa should increase zoonotic disease risk by reducing the predators and competitors (large herbivores) of rodents, a common reservoir of human pathogens . similarly, overfishing of snail-eating cichlids in lake malawi in africa seems to have been a causal factor in snail population and human schistosomiasis increases there . investigations of the effects of overexploitation on infectious diseases remain in their infancy, but the consequences for human populations could be profound. up to this point, we have focused on the effects of feeding billion people on infectious diseases, but the relationship is bidirectional. that is, human infectious diseases can also impact the agricultural and economic development necessary to feed the growing human population (fig. ) . for example, the overuse of antibiotics, anthelmintics and pesticides to prevent diseases is driving resistance to these chemicals that will compromise future crop, livestock and aquaculture production. in sub-saharan africa, areas with higher historical tsetse-fly abundance, the vector of the parasite (trypanosoma brucei) that causes african sleeping sickness in humans and cattle, experienced greater lags in the adoption of animal husbandry practices (for fertilizer and labour in agricultural enterprise) that hindered agricultural development and prosperity in africa long before and after europeans colonized . in rural subsistence communities, any source of ill health can significantly impact people's productivity, yields and agricultural output [ ] [ ] [ ] . for example, human immunodeficiency virus/aids has reduced average life expectancy in sub-saharan africa by years since , and a kenyan study found that crop production by rural subsistence-farming families dropped % after the death of a male head of household . many neglected tropical diseases (ntds) impose devastating productivity losses for affected people that can impede agricultural development from local to national to regional scales . some low-income communities appear mired in this poverty-disease trap, and thus might require substantial investment in health systems to promote the necessary agricultural and economic growth to pull them out of the povertydisease cycle [ ] [ ] [ ] [ ] [ ] . the goal of identifying potential changes to infectious disease risk associated with feeding the growing human population is not only to draw attention to this important current and future problem but to also encourage preparation for these changes by stimulating agricultural-and disease-related research, management and policy that could maximize the human health benefits of agricultural development . one urgent challenge is the problem of antibiotic, anthelmintic and pesticide resistance. analyses demonstrate that, in some cases, improvements in growth and feed consumption can be achieved by improved hygiene instead of antibiotics . indeed, all antibiotics as growth promoters were banned in the european union in . to curb antimicrobial consumption in food animal production, van boeckel et al. suggest: ( ) enforcing global regulations to cap antimicrobial use, ( ) adhering to nutritional guidelines leading to reduced meat consumption, and ( ) imposing a global user fee on veterinary antimicrobial use. in addition, as recommended by the world health organization, international organization for epizootics, and food and agriculture organization, national and international policies based on best management practices should be developed and implemented that document when and how antibiotics should be used, and agencies should be established to monitor their use, mandate reporting and enforce these policies . finally, given that host genetic variability can reduce disease risk , large-scale industrial livestock operations could add genetic variability into their artificially selected food animals in an effort to reduce epidemics and epizootics. these changes promise to secure the long-term viability of antibiotics and anthelmintics for curing diseases of humans and non-human animals. another challenge that seems surmountable is to enhance education and health literacy. not surprisingly, education has been documented as a major contributing factor to reducing infectious diseases, especially ntds, and reducing ntds can have reinforcing positive effects on the ability of humans to fight more deadly diseases, such as aids, malaria and tuberculosis . limiting ntds could also have knock-on effects for education and health literacy because ntds impede cognition, learning and school attendance . indeed, an investment of just us$ . per child for ntd control can result in the equivalent of an extra school year of education . this is likely an underestimate because of unaccounted for indirect effects of deworming on learning. a recent study revealed large cognitive gains among children who were not dewormed but had older siblings who were . thus, enhanced education and ntd control have the potential to synergistically fuel agricultural and economic development and facilitate escape from the poverty-disease trap. national and international shifts in investments could also potentially pay large dividends for nutrition, infectious disease control and poverty reduction. there is considerable evidence that the developing world will struggle to feed its growing human population because of the poverty trap of infectious disease . however, evidence also suggests that this trap might be broken through investments in health infrastructure and preventive chemotherapy [ ] [ ] [ ] . curing worm diseases has the potential added benefit of reducing nutritional needs of cured individuals by ceasing the feeding of their parasites. by reducing food demand, these drugs could also protect the environment by reducing the conversion rate of natural areas to agriculture. this, in turn, might help to curb climate change, which is one of the greatest threats to global food security and might also increase disease risk , . understanding the economic relationships among infectious disease treatment and prevention, food production, poverty and climate change in the developing world are important areas of future research. as human populations increase, there might be more pathogen spillover events that could result in new emerging human infectious diseases. historically, humans have combated emerging diseases through early detection followed by coordinated quarantine, as demonstrated by the sars outbreak in and the ebola outbreak in . we recommend continued and improved coordinated international disease surveillance, but this approach reacts to rather than prevents disease. we recommend a shift in both research and disease management efforts towards proactive management approaches. one proactive approach that has promise for preventing zoonotic disease emergence is biodiversity conservation , , but more research is needed to evaluate its effectiveness across various types of zoonotic diseases and its costs and benefits relative to more proven reactive public health interventions. ultimately, science needs a better understanding of pathogen spillover, the origins of disease emergence and post-spillover evolution so that human disease emergence can be better predicted and prevented , , . improved and diversified plant and animal genetic material could also help to simultaneously reduce hunger and disease. many efforts are already well underway; for example, introducing c traits into rice to enhance higher photosynthetic capacity in a warming world, and breeding drought-and flood-tolerant cereals, pulses and vegetables better suited to increased frequency of extreme weather events that can help sustainably enhance productivity. crops and livestock genetically adapted to resist more pests can reduce pesticide and antibiotic use. in addition, given that micronutrient deficiencies are now the most widespread source of undernutrition globally, commodity research must diversify beyond merely boosting productivity of staple cereals. far greater attention is needed on approaches that add mineral and vitamin content to foods . other, perhaps more challenging, interventions also merit attention. closing 'yield gaps' on underperforming lands, increasing cropping efficiency, eating less meat and excess per capita food consumption -which also have adverse health impacts on noninfectious diseases, such as high blood pressure, diabetes and heart disease -and reducing food waste and loss have the potential of doubling global food supplies , , , , while simultaneously allowing financial savings to be redirected towards health infrastructure to control disease. in addition, family planning, promoting female education and job market prospects, and enhancing early childhood survival have proved very effective at reducing human population growth . finally, producing food in more urban and suburban environments through vertical farming (the practice of growing produce in vertically stacked layers) also has potential to enhance food production locally, and might have reduced agrochemical and transportation costs and non-target effects relative to more traditional farming . investments into predictive models could also pay dividends. agricultural environments are complicated, multi-species systems that exhibit interactions at many levels. the complexity further increases from the tensions that arise in balancing expanding agricultural systems, social benefits and infectious disease risk. all of this complexity can overwhelm many analytical tools and experiments, which makes advances in mathematical modelling especially crucial to addressing these issues. box describes several examples of advances in mathematical modelling that illustrate their promise for analysing the links between agricultural practices and the dynamics of infectious diseases, projecting risks to future decades, influencing risks through either intentional (policies and interventions) or unintentional (continued habitat box | modelling tools for quantitative analysis of agriculture-infectious disease systems mathematical models facilitate the investigation of a complex web of interactions between agricultural systems, social dynamics and infectious diseases in the presence of the substantial nonlinearities inherent in these disparate processes. a notable example is a body of theory and analyses that have emerged to examine the links between agricultural systems and the dynamics of infectious diseases in the context of global development . the approach combines ecological and economic theory to model population subsistence and health, with the goal of examining the conditions under which subsistence and health needs of populations are met. the fundamental consumer-resource relationships that underlie the biological generation of capital via agricultural production can be formalized in ordinary differential equation models, which can be coupled with similarly structured models that estimate the gains and losses in human capital associated with population health and the increasing risk of acquiring and succumbing to infectious diseases associated with poverty. key insights emerge from such integrated models. high prevalence of infectious diseases among populations that rely heavily on subsistence, labour-intensive agriculture can reduce agricultural productivity, degrade human capital, undermine economic growth and generate conditions that systematically reinforce poverty . poverty, in turn, increases disease transmission as the lack of access to clean water and preventive care often associated with the lifestyle of the poor make them continuously exposed to parasites and pathogens embedded in the environment . malnutrition, a common consequence of poverty, may weaken immune response and increase susceptibility to disease. poor education and lack of resources lead to further reduction of health-seeking behaviour. modelling shows that the reinforcing feedback between poor health, labour productivity and capital formation triggers a cycle of increasing poverty and disease, known as disease-driven 'poverty trap' . it also shows that poverty traps are reinforced by the asymmetry between the slow pace of investment in disease protection associated with increasing capital due to agricultural development, and the relatively faster ecological changes in transformed landscape that foster disease transmission . modelling can also reveal the impact of temporary versus lasting structural interventions in changing the development trajectories of poor populations. these intrinsically quantitative and mechanistic investigations can indicate specific conditions for economic growth or resilience that can inform development strategies, via, for instance, targeted investments in agricultural development, human health or ecological conservation that may reinforce globally stable development equilibria . a final major contribution of such models is to encourage primary data collection in support of deeper understanding of system dynamics, and greater inference of underlying determinants of health and wealth. because there are strong traditions of primary data collection in population biology, epidemiology and other disciplines wherein mathematical modelling is being advanced, the expansion of model-based research on coupled agricultural-health systems is likely to yield new field data collection efforts and direct the design of experiments, which, in turn, will yield greater opportunities for model parameterization and validation. these new empirical field data are what are desperately needed to explore whether the predictions of highly stylized models are supported in settings far more complex than modelled. simulation modelling, in turn, can also permit exploration of the uncertainty intrinsic to field experiments that generate data in a specific place and time, advancing understanding of system performance under a range of feasible weather, market and other conditions not realized during the period of data collection. conversion and antibiotic use) actions, and incorporating economic and social costs of various public health or agricultural interventions. these models should help direct field experiments, which are desperately needed to parameterize and validate these models and to quantify key sources of uncertainty. ultimately, we believe 'win-win' scenarios for controlling infectious diseases, increasing agricultural productivity and improving nutrition are attainable. although this sort of integrative thinking has been historically rare because agriculture and public health have been perceived as disparate disciplines, it is beginning to slowly penetrate these distinct fields of study. for example, several agrochemicals seem to increase the risk of human schistosomiasis and agriculturally derived zoonotic pathogens, and thus researchers are actively attempting to identify agrochemicals that might 'kill two birds with one stone' , reducing crop pests and thus increasing food production while not increasing or even decreasing human pathogens [ ] [ ] [ ] . similarly, researchers are beginning to consider the introduction of prawns as biological control agents of schistosomiasis, which could simultaneously decrease disease and increase nutrition -because restored prawns can be fished, harvested and consumed without compromising their diseasecontrolling benefits , . we believe 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institutional affiliations. key: cord- -xfze ah authors: monto, arnold s; malosh, ryan e; evans, richard; lauring, adam s; gordon, aubree; thompson, mark g; fry, alicia m; flannery, brendan; ohmit, suzanne e; petrie, joshua g; martin, emily t title: data resource profile: household influenza vaccine evaluation (hive) study date: - - journal: int j epidemiol doi: . /ije/dyz sha: doc_id: cord_uid: xfze ah nan acute respiratory infections (ari) are a major cause of morbidity worldwide. some of the earliest studies to describe the epidemiology of these infections were household cohort studies. [ ] [ ] [ ] [ ] [ ] [ ] the largest of these were conducted in seattle, washington and tecumseh, michigan in the s and s , and they provided information on the relative frequency, seasonality and symptomatic characteristics of ari shortly after many respiratory viruses were first identified. given that the role of household structure in seasonal incidence and transmission of respiratory viruses was the primary objective, an individual's longitudinal history of infection was not a major focus. indeed, during the first phase of the tecumseh study of respiratory illness, households were maintained on report for only year, and then gradually replaced so that the entire community could be represented over time. these historical studies relied on cell culture for virus identification, a method that requires specimens to be processed quickly and is considerably less sensitive than current molecular methods. for this reason, the tecumseh study and others relied extensively on serodiagnosis of influenza infection using twice yearly blood specimens. it was not possible to time infections in those who only were serologically positive, limiting the ability to do robust analyses of transmission patterns. with current molecular techniques, it has become much easier to identify a broad range of agents of respiratory infection. this allows documentation of infection, co-infection and subsequent re-infection. collecting specimens within a short time from the onset of symptoms still maximizes the likelihood of accurate and timely identification of viruses associated with a respiratory illness for studies of transmission and vaccine effectiveness. collection of regular blood specimens continues to be valuable as well, for both virus identification and for analysis of serologic correlates of protection. the household influenza vaccine evaluation (hive) study is an ongoing prospective household cohort study that began in . the hive study was based on the original tecumseh study of respiratory illness with several key modifications to illness surveillance and to laboratory methods for identification of respiratory viruses. while respiratory virus infections in general could be studied, the primary objective was to estimate the effectiveness of influenza vaccines using a cohort design for comparison with studies using the testnegative design (tnd). under the tnd, specimens are collected from participants who meet a respiratory illness case definition, and vaccination frequency is compared between those who test positive for influenza and those who test negative. the tnd is focused on those individuals with illnesses severe enough to seek care, which reduces bias due to differential care-seeking behaviour but misses milder presentations of respiratory virus illnesses. [ ] [ ] [ ] the use of a prospective cohort design has allowed inclusion of these mild illnesses in evaluations of vaccine effectiveness. here we report on the first phase of the hive study, conducted from through . who is included? from - through - , eligible households were those who lived within miles of the study clinic in ann arbor mi, and who had at least four individuals who received primary care from the university of michigan health system (umhs) and at least two children < years old; in - the eligibility criteria were changed to allow three-person households. we recruited or re-recruited households each summer or autumn. previously participating households were invited to re-enroll provided that they continued to meet the eligibility criteria. we supplemented this recruitment with direct mail or email invitations to households in the source population. in total, we have collected data and specimens with active ari surveillance for individuals from households ( table ). the majority of these individuals were children < years of age, % were white (non-hispanic), and % were female. the study cohort size has ranged from to participants in each year ( to households), and ( %) individuals have been followed longitudinally over multiple years. how often have they been followed up? active surveillance for ari meeting a case definition was conducted seasonally (october-may) from - . beginning in october , year round surveillance was initiated. hive study participants were contacted weekly by email or phone to ascertain new ari meeting the study case definition. for participants years of age, the case definition consisted of two or more of the following symptoms: cough, fever/feverishness, nasal congestion, sore throat, body aches, chills, headache. for participants < years of age, the case definition consisted of two or more of the following symptoms: cough, fever/feverishness, nasal congestion/runny nose, trouble breathing, fussiness/ irritability, decreased appetite. each household received a calendar at enrollment to assist with recording illness onset dates. when illnesses were reported, the household was contacted by study staff to schedule a clinic visit for sample collection. at illness visits for ari meeting the case definition, trained research staff collected nasal and throat swabs (or nasal swabs only for participants < years of age) for detection of influenza and other respiratory viruses. since october , an additional, self-collected or parentcollected specimen was also collected in the home on the day of symptom onset. beginning in december , all household members years of age were asked to contribute a blood specimen at the initial enrollment visit and at scheduled visits twice annually thereafter. each year, autumn blood specimens (pre-season) were collected after the majority of study participants had received influenza vaccine and prior to the start of the influenza season (beginning in november). spring (post-season) specimens were collected after local influenza circulation ended each year (may-june). at the annual enrollment visits, individual participants were queried about household and demographic characteristics and advisory committee on immunization practices (acip)-defined high-risk conditions. a detailed clinical history for these participants is extracted from the umhs electronic medical record (emr), or from their regular primary care provider if they are not part of the health system. influenza vaccination information, including date, lot number, vaccine type, dose and manufacturer, was confirmed using multiple sources. information from the michigan care improvement registry (a state-maintained repository of vaccination records that requires reporting of all childhood vaccines in michigan) was requested for all study participants. provider-based vaccine records were obtained directly from the umhs emr or through medical record requests for individuals with outside-system care providers. these records were supplemented by vaccine diary cards that were filled out by participants at the time of vaccination. there has historically been a high level of vaccine uptake in the hive study population, ranging from - % (table ) . respiratory specimens collected at illness visits were tested by real-time reverse transcriptase polymerase chain reaction (rt-pcr) for laboratory confirmation of influenza, using primers and probes from the centers for disease control and prevention. influenza subtype was determined for influenza a specimens and lineage was determined for influenza b specimens. from through , a total of influenza infections were identified. specimens are also tested by pcr for non-influenza respiratory viruses including respiratory syncytial virus, human metapneumovirus, parainfluenza, coronavirus and rhinovirus. symptoms present since illness onset were recorded and participants completed a follow-up survey reporting illness outcomes (e.g. duration of symptoms, care seeking behaviour). blood specimens were centrifuged, and the serum was separated and stored in ml aliquots at - c until processing in serologic assays. collected sera were tested in parallel using hemagglutinin inhibition (hai) assays. hai assays use inactivated influenza vaccine subunit material (sanofi-pasteur) from relevant a/h n , a/h n and b vaccine strains. anti-neuraminidase antibody (nai) was measured by an enzyme-linked lectin assay (ella) using inactivated, reassortant viral targets containing contemporary neuraminidase segments and a mismatched h hemagglutinin ha. antibody titres measured from blood specimens collected in the autumn have been analysed as both pre-season titres for the coming influenza season as well as post-vaccination titres for those individuals who were vaccinated prior to collection. similarly, antibody titres measured from blood specimens collected in the spring have served as post-season titres as well as prevaccination titres in analyses for the following study year. a full description of data collected throughout study follow-up can be found in table and the timeline for key study activities can be found in figure . the original aims of this study were to estimate influenza vaccine effectiveness annually and to compare those estimates with those from tnd studies conducted in outpatient clinics during the same seasons. with additional funding, we have expanded on these original aims by collecting blood specimens for studies of antibody-mediated households participants influenza-positive individuals influenza-positive specimens strain a c h n pdm h n immunity, extending ari surveillance year-round, and incorporating laboratory testing for other respiratory viruses. statistical analyses to estimate vaccine effectiveness were performed annually. adjusted hazard ratios (ahr) were estimated by cox proportional hazard models with robust variance using a sandwich estimator to account for household clustering. adjusted models included age, high-risk health status and vaccination status. vaccination status was modelled as a time-varying covariate, with subjects considered vaccinated days after vaccine receipt. vaccine effectiveness was calculated as x . vaccine effectiveness (ve) has varied markedly by year and age group. in - and - , for example, we observed ve against any influenza infection of approximately % in two of three age groups. ve was lowest among adults ( years) in - , however, young children ( months to years) had the lowest ve in - . , in - , when influenza a/h n predominated, we observed ve against community acquired influenza a/h n infection of % ( % confidence interval - to ). during the - season, we observed no effect of vaccine on the risk of infection with antigenically drifted influenza a/h n , but point estimates against b yamagata were near %. the estimated ve against b yamagata appeared to be primarily driven by high effectiveness in young children. in general our annual estimates of influenza ve have been consistent with those from the us flu ve network. [ ] [ ] [ ] [ ] [ ] [ ] during the - influenza season, we found that those who had been vaccinated in the previous and current year were less protected than those who received the vaccine in the current year only. this was the first time in the recent era that such a reduction was documented, thus continuing historical debates on whether repeat vaccination resulted in reduced protection. [ ] [ ] [ ] [ ] these original findings of a repeat vaccination effect prompted a furthering of the hypothesis that antibody response to vaccination was decreased after multiple annual administration of vaccines of similar antigenic make-up. in order to evaluate this hypothesis, we began collecting blood specimens in december . in the - season, the predominant circulating influenza virus was a(h n ). relative to those unvaccinated in both years, ve was higher in those vaccinated in the current year, lower in those vaccinated in the current and previous year, and lowest in those vaccinated in the previous year only. with the addition of blood collection in the cohort, we found that hai titres correlated with the observed ve for the type a viruses. lower post-vaccination titres were found among those vaccinated in both years relative to those vaccinated only in the current season. a similar difference in post-vaccination titres was not observed for influenza b in - . importantly, the effects of prior season vaccination on ve and serologic susceptibility to infection have not been observed in all years. during the - season, a year in which influenza a(h n )pdm predominated, we found similar levels of effectiveness among those vaccinated in both the current and previous seasons compared with those vaccinated only in the current season. consistent with this finding, hai and nai titres against influenza a(h n )pdm were similar comparing those vaccinated in both the current and prior season to those vaccinated only in the current season. antibody titres measured in twice annual blood specimens have enabled additional evaluations of the relationship between vaccination and antibody-mediated protection. the fact that participants were observed over time allowed us to demonstrate that in this highly vaccinated cohort, prevaccination antibody titres were generally high and a fold rise in titre following vaccination was less frequent than expected. , finally, measuring antibody titres both against viruses similar to those that circulated locally and against those in the vaccine, we observed increased susceptibility among those with high vaccine-strain-specific antibody titres but low circulating-strain-specific antibody titres. interrupting transmission is a major goal of influenza prevention strategies and could be key to controlling the burden of disease during a pandemic. the household may be an effective place to accomplish this goal due to the high proportion of transmission estimated to occur in this setting. we have used standard epidemiologic methods to estimate serial intervals by virus type and to identify household and individual level characteristics associated with secondary infection risk. these methods remain susceptible to misclassification of a transmission event, as infections are generally linked retrospectively based on the time between onset of each illness and viral type and subtype despite continued risk of infection from the community. we have attempted to address this potential for misclassification in two ways. first, it is now possible using next-generation sequencing to differentiate the two types of transmission on the basis of genetic similarity as we have demonstrated with influenza a viruses. second, we have adapted individual-based transmission models to account for risk of infection from both the community and the household and to allow for chains of transmission. the hive study from the start has identified viruses other than influenza, and, for one season, bacterial agents associated with respiratory illnesses. surveillance initially extended through much of the respiratory season and has been conducted year-round since october ; thus it has been possible to determine seasonality of these respiratory viruses. in addition, the detection of multiple respiratory viruses has allowed us to describe the frequency of coinfection in different age groups. the hive study has recently received continued core funding and is now recruiting existing and new households into a second phase of the cohort. a commitment to years of follow-up with year-round surveillance for ari is now required of those entering the study. blood specimen collection, which in the past was limited to those years of age, is now extended to younger children and includes collection of peripheral blood mononuclear cells (pbmcs) for enrollment / re-engagement blood collecƟon ari surveillance figure . timeline of study enrollment, acute respiratory illness (ari) surveillance and blood collection activities. blood collection for antibody studies began in the autumn of . ari surveillance has been carried out continuously on a year-round basis since the autumn of ; ari surveillance was limited to the typical influenza season prior to that. studies of cell-mediated immunity. in addition, targeted recruitment strategies are now focused on children < months old and their households in an effort to study infections and immune response in early life. the main strength of the hive study is the longitudinal, prospective study design with intensive data collection. active surveillance for symptomatic illness with collection of respiratory specimens for laboratory-confirmation of influenza and other viruses allows for calculations of the incidence and determinants of infection over time, and builds on previous cohorts in the region that defined illness based on serology alone. using multiple sources, including participant medical records, to document influenza vaccination status and regular serologic sampling are major strengths of the cohort. the number of participants that have been prospectively followed for multiple years has enabled multi-year studies of immune response to infection and vaccination, studies that are increasingly important for understanding how individual influenza immunity evolves over time. the illness sampling strategy requires participant symptom reporting. it is therefore important to note that subclinical infections are not captured by illness sampling, as this would require routine swabs in symptom-free individuals. however, the availability of end-of-season serologic specimens allows for analysis for uncaptured influenza virus infections in unvaccinated individuals. the intensive data collection increases the burden on study participants. it is therefore necessary to build relationships with the community to ensure enrollment and long-term participation. as a result the study is resource intensive and the study population is limited in terms of both sample size and generalizability. in particular the source population from which participants is drawn is limited geographically to households who are able to travel to the university of michigan study site for enrollment visits, blood draws and illness specimen collection. the hive study population consists of suburban residents and largely reflects the high education level and vaccination uptake in our region. one approach to increase sample size and generalizability would be to establish multi-site cohorts, presenting logistical considerations that might be challenging to unify under a common protocol. indeed, other longitudinal cohorts of respiratory illness have used varied recruitment strategies or follow-up methods tailored to their unique communities. , our history of involvement in this community, stretching back to the s, has given us the opportunity to optimize the cohort for longterm success. the investigators regularly collaborate with others to address key questions in respiratory virus epidemiology and immune correlates of protection. proposals for future collaborations using hive study specimens and data can be submitted to the study investigators for consideration. the hive study was supported by the centers for disease control and prevention (u ip , u ip ) and the national institute of allergy and infectious diseases (r ai , r ai ). conflict of interest: e.t.m has received grant support from merck and pfizer for work unrelated to this report. a.s.m. has received consultancy fees from sanofi, seqirus and novavax for work unrelated to this report. a.s.l. has received consultancy fees from sanofi for work unrelated to this report. all other authors declare no conflict of interest. • we collected longitudinal data on demographics, health history, influenza vaccination status and ari incidence. we collected specimens to detect occurrence by rt-pcr of influenza and other respiratory viruses and serum to determine antibody titres to hemagglutinin and neuraminidase. specimens are archived. • investigators interested in learning more about the hive study as well as data and specimen availability are welcome to email the study investigators. estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower respiratory tract infections in countries: a systematic analysis for the global burden of disease study acute minor 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year period variable efficacy of repeated annual influenza vaccination antibodies against the current influenza a(h n ) vaccine strain do not protect some individuals from infection with contemporary circulating influenza a(h n ) virus strains influenza transmission in a cohort of households with children stochastic processes constrain the within and between host evolution of influenza virus application of an individual-based transmission hazard model for estimation of influenza vaccine effectiveness in a household cohort co-colonization by streptococcus pneumoniae and staphylococcus aureus in the throat during acute respiratory illnesses frequency of acute respiratory illnesses and circulation of respiratory viruses in households with children over surveillance seasons a universal influenza vaccine: the strategic plan for the national institute of allergy and infectious diseases mosaic: mobile surveillance for acute respiratory infections and influenzalike illness in the community community surveillance of respiratory viruses among families in the utah better identification of germs-longitudinal viral epidemiology (big-love) study the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention. hive study research staff: barbara aaron; amy p. callear; rachel truscon; emileigh johnson; caroline k. cheng; anne kaniclides; natalie williams; casey martens. key: cord- - zf qtlh authors: farag, elmoubasher; sikkema, reina s.; vinks, tinka; islam, md mazharul; nour, mohamed; al-romaihi, hamad; al thani, mohammed; atta, muzzamil; alhajri, farhoud h.; al-marri, salih; alhajri, mohd; reusken, chantal; koopmans, marion title: drivers of mers-cov emergence in qatar date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: zf qtlh mers-cov (middle east respiratory syndrome corona virus) antibodies were detected in camels since , but the first human case was only detected in . this study sought to identify and quantify possible drivers for the mers-cov emergence and spillover to humans. a list of potential human, animal and environmental drivers for disease emergence were identified from literature. trends in possible drivers were analyzed from national and international databases, and through structured interviews with experts in qatar. the discovery and exploitation of oil and gas led to a -fold increase in qatar gdp coupled with a -fold population growth in the past years. the lifestyle gradually transformed from bedouin life to urban sedentary life, along with a sharp increase in obesity and other comorbidities. owing to substantial governmental support, camel husbandry and competitions flourished, exacerbating the already rapidly occurring desertification that forced banning of free grazing in . consequently, camels were housed in compact barns alongside their workers. the transition in husbandry leading to high density camel farming along with increased exposure to humans, combined with the increase of camel movement for the racing and breeding industry, have led to a convergence of factors driving spillover of mers-cov from camels to humans. emerging infectious diseases are a cause for increasing global concern, because of their impact on global health and economics [ ] . the ebola outbreak in west africa during - showed that pathogens which previously caused small and easy to control outbreaks had the potential to infect thousands of people under the right circumstances [ ] . this is also a concern for the middle east respiratory syndrome coronavirus (mers-cov), which until now has been the cause of sporadic cases and hospital outbreaks [ ] . to date, there have been confirmed laboratory cases worldwide, with deaths [ ] . all mers index cases are linked to the arabian peninsula. dromedary camels have been identified as a reservoir of mers-cov with occasional zoonotic transmission to humans [ , ] . human-to-human transmission is also common, with around % of the mers cases reported to who being health care associated [ , ] . however, the source of infection of many index cases remains unclear [ , ] . studies have shown that mers-cov, or related viruses have been circulating among camels at least since [ ] . since that period, massive changes have occurred in people's lives and in animal husbandry across the arabian peninsula. understanding these changes may help to reconstruct the events that led to the emergence of mers-cov as a human disease. past research identified several drivers of emerging zoonoses, such as urbanisation, population growth and demography, and environmental and agricultural changes [ ] [ ] [ ] . the drivers which could have potentially influenced the mers-cov emergence in humans have only sporadically been investigated [ , ] . by reviewing changes involving humans and camels over the past years in qatar, this study sought to identify the key drivers of the emergence and spread of mers-cov. potential drivers for disease emergence were identified from literature and from discussions with national and international experts in mers-cov. the final list had the following categories: economic development; human demography and behavior; international travel, commerce, sports and leisure; political environment; agriculture and food industry change, including camel demography, husbandry and movement; changes in climate and land use. data from onwards were collected from national and international databases. if multiple data sources were available, data from both sources were collected. all data were entered in an excel datasheet and reviewed and discussed with the project team (supplementary ). qualitative information and remaining data gaps were addressed by interviews with a group of experts and stakeholders from qatar. criteria to select experts included years or more experience in a camel-related business (farming, trading and racing) or professional services related to camels and being familiar with cultural aspects of the qatari community. using a structured interview guide (supplementary ) and a moderator, a series of interviews were conducted in arabic, each lasting approximately for hours. the main themes that were covered during the interviews included: (changes in) people's living conditions; customs and purposes of camel ownership; cultural habits related to camels; educational level and personal behaviors of camel owners and workers; camel movement; demographic distribution of camels in qatar; camel farming practices: feeding, grazing, and slaughter. a detailed transcript was shared with the experts for authentication. a literature search was done to complement findings from the quantitative and qualitative study, using pubmed, google scholar and the local sources of information including the ministry of public health (moph), ministry of municipality and environment (mme), ministry of development and planning statistics (mdps), and qatar statistical authority (qsa). the funder had no role in study design, data analysis, data interpretation, or writing of the review. historically, qatari inhabitants were mostly bedouins along with a few settled people [ , ] . the bedouins owned limited numbers of camels, sheep, and goats [ ] . camels were used as a source of food (milk and meat) and means for transportation. in , oil and natural gas resources were discovered. however, large-scale exploitation started in the s [ ] . from the s onwards, qatar's economy has been steadily growing. however, the year marked a significant turning point as qatar's gdp almost increased by more than -fold during the period - ( figure a ) [ , ] . qatar is currently considered to be one of the wealthiest countries in the world [ ] . the thriving economy was paralleled by major demographic and life style changes. in the late s, around , people lived in qatar [ ] . in response to demands for a larger workforce after the exploitation of oil and gas began, foreign laborers started to migrate to qatar from countries in the region, like palestine, oman, iran, and the kingdom of saudi arabia (ksa). later, immigrants from pakistan, india, nepal, sri lanka, bangladesh, the philippines, and indonesia joined the older migrant populations, increasing the number of inhabitants to , by and recently to , , ( figure a ) [ ] . in , non-qatari males made up % of the residents of working age ( - ) and non-qatari made up more than % of the total number of qatar inhabitants older than years of age ( figure b ,c) [ ] . most recent estimations of the origins of the non-qatari population are that % is indian, % bangladeshi, % nepali, % filipinos, % egyptian, % pakistani, and % iranian [ ] . the total number of males in qatar increased from . % of the total population in to . % in ( figure b ). in , almost % of residents were between and years old, and this has risen to more than % in ( figure a ). detailed accounts on age distribution were not available before [ ] . most people in qatar live in urban areas. the percentage of residents living in cities increased from . % in , to . % in , and . % in [ ] . doha, the capital and the biggest city of qatar, hosts the greatest number of people. however, there has also been a large increase in number of people living in the al-rayan area, where most of the camel farms are located. the number of tourists visiting qatar also increased, especially since . most tourists came from other gcc countries, but the number of visitors from europe and america were also increasing ( figure d ) [ , ] . according to experts, the economic development and population increase coincided with major changes in life style. the bedouin nomadic lifestyle gradually decreased as most of the qatari tribes shifted to an urban, settled lifestyle; cars and planes rapidly replaced camels as transportation means. this transformation to a more sedentary lifestyle is reflected in the profile of comorbidities. more than % of adults are overweight and almost half of them obese [ ] . male obesity increased from % in to % in , which is extremely high compared to the current % prevalence in men worldwide [ ] . in , % of residents above years were hypertensive, rising to % in , and % in [ , ] . prevalence of high blood sugar among adults in was %, compared to a worldwide prevalence of % [ ] . the qatar stepwise report reported in that % of adults were daily smokers. yet, qatar has a low death rate: . / , compared to the worldwide death rate of . / , and its healthcare system has developed rapidly over the past twenty years [ , , ] . the increase in the number of dromedary camels reflects the increasing popularity of camels as sports animals ( figure b ). with the changing life style and increasing wealth, the purchase and breeding of (expensive) racing camels came within reach of an increasingly large segment of the qatari national population. according to experts, although camel racing has traditionally been part of the bedouin culture, the organized racing business went through major changes over the past decades. this was partly due to financial and regulatory support from the qatari government. this support increased the social and economic value of camels in qatar, further stimulating their popularity. the al-shehaniya camel-racing track, one of the biggest tracks in the gulf, was opened in [ ] . the camel farms that are located near the al-shehaniya camel racing area are mostly used for racing camels. there are about racing camel holdings at the al-shehaniya camel racing area. some of the camels in qatar are used to compete in camel beauty contests that are organized around the arabian peninsula. according to the fao, in there were about , camels in qatar. this rose to over , in , , in , and more than , in ( figure b ). more than % of the animals are currently kept for racing [ ] . across the gulf region, qatar has the highest camel density, with . units/km , compared to . units/km in united arab emirates (uae) and . units/km in the ksa [ ] . in , the total number of camel farms was and by it had increased to [ ] . as a result of the loss of traditional methods of rangeland management, the vegetation coverage decreased from % to only % of total land cover. overgrazing of the green areas due to the increased population of camels and other livestock accelerated the desertification of qatar [ ] . therefore, the government decided to assign natural protected areas in [ , ] , and started to sanction the free grazing of livestock since [ ] . by , open grazing was completely banned [ ] . according to the experts' opinions, this led to changes in farming practices, as herds were then moved outside of qatar to areas where free grazing remained possible. moreover, in qatar, camels are now raised in closed systems and within of designated farming areas (camel complexes) in the residential districts. camel workers also live on the premises of the camel complexes. typically, a camel complex has a reception room (majlis) for social activities of the camel owners. the al-rayyan municipality, where the al-shehaniya camel racing area is also located, currently holds about % of the total camel population and % of camel holdingss (figure ) [ , ] . according to the experts, this newly adopted closed farming system led to the increase of disease incidence, especially of parasitic diseases. however, we did not find any disease statistics to substantiate these findings. the increasing focus on camel race competitions caused big changes in camel farming practices. previously, the calves were weaned when the next calf was born. currently, weaning occurs at around months of age. after being weaned, young camels are directly taken for acclimatization (during the period mid-july through mid-august) from the general livestock farms (located across the region) to the racing farms, mostly located within the al-shehaniya area. this involves drastic changes in feeding systems, intense training for races, and mock races alongside camels from other farms and older training camels. the off-season for camel racing is during summer (mid-april to august) ( figure ). during this time, most of the owners travel abroad, the frequency of visits to the farms substantially decreases, and workers are permitted to take annual vacations. from september onward, training intensifies, in preparation of the racing season, which lasts from mid-september through mid-april. during that time, , registered camels from different origins, ages, gender, nationalities, and breeds compete together at the al-shehaniya camel-racing track. during the racing season, up to rounds take place, approximately five days per week. an unprecedented, increasingly intensified mobility of camels inside and outside qatar has been seen over the recent decades. the domestic and cross border mobility does not only involve camels, but also people who look after the camels to provide care along the journey. import and export of camels have especially increased since the year ( figure c ). the imported camels mainly come from the uae and ksa ( figure d) . the dynamics and travel patterns of qatari camels are complex (figure ). camels are transported to and from different locations, for a variety of purposes, and with a noticeable seasonal pattern. mobility gets more intensive during the racing and trading season (september to april). experts believe that the ban of open grazing in qatar played a key role in the intensity and frequency of camel movements. they mention that there has been a remarkable increase after in numbers of camel workers and owners who cross the borders to and from ksa along with their animals, although this recently stopped with the ksa-qatar political situation. the ban of open grazing stimulated camel owners to establish farms in ksa and uae where open grazing is still permitted. therefore, camels are moved through gulf countries, particularly during the winter season. camel races and beauty contests that are routinely organized in nearly all gulf countries are another factor that boost the national and international movement of camels. compared to other types of camels, racing camels dominate in terms of numbers and frequency of mobility both across borders and domestically, particularly between september and april. as per the records of the camel racing committee, in the racing competitions, , camels from qatar and camels from the other gcc countries contested [ ] . however, owing to the lack of standardized identification system, it was difficult to determine the exact figures and the extent of these movements. camels are also being mobilized for reproduction purposes (figure ). mating season (also known as camels' honeymoon) starts in the middle of august and continues through february of the next year, with the high season in the september-october period. female camels are usually taken from their own location to other farms where selected males are kept particularly for reproduction purposes. about , female camels are annually being moved for mating. they spend around week at a breeding farm with male camels before they are taken back to their original farms. programmed mating is exclusively being practiced for race and show camels. the mating season is another seasonal activity that entails intensive movements of camels, camel owners, workers, car drivers and veterinarians. the doha wholesale market constitutes the primary hub for camel trading. in parallel with the increased number of camel races, al-shehaniya city also grew as a market and has become a hub for trade of racing and beauty show camels in qatar. the wholesale market in doha hosts camels and other types of livestock from countries all over the gulf region. the camels typically stay at the market until they are sold. camel workers live at the market premises. camels that are being sold (calves in particular) serve a variety of purposes. they are sold to be slaughtered at the doha wholesale market abattoir, for breeding purposes, to be trained as racing camel, or to be prepared for camel show competitions. in recent years, the doha wholesale market has been surrounded by rapidly growing residential areas. animals in the market are now in close proximity to the residents. as of , slaughter practices were banned inside residential premises, and can only be performed in official slaughterhouses and exclusively by licensed persons. camel meat and milk are no longer part of the daily diet of most qatar inhabitants. nonetheless, camel meat is a fundamental ingredient of qatari social events and family celebrations. production of camel meat and milk has remained stable in the past years. camel milk is generally kept for personal use, particularly for the perceived therapeutic merits of raw camel milk, as well as camel urine. experts state that there is an unshakable belief that the regular consumption of camel milk helps to prevent and control diabetes. it is also widely believed in the qatari community that camel urine and milk can heal skin lesions and other diseases. camel urine is also regularly used to whiten the skin and face and lighten the hair. the majority of camel owners offer camel milk and urine for free, as a practice of generosity. the role of camels in the transmission of mers-cov is well documented [ ] . despite the fact that mers antibodies have already been detected in camels since [ ] and human contact with animals is not new, human mers cases were only detected in [ ] . based on institutional and literature data and in-depth interviews with key professionals, this study sought to examine the changes involving the human, animal, and environmental drivers that may have contributed to the spread and virus spillover to humans. our reconstruction of events over the past decades, based on available literature, statistics, and expert opinions, lead to the conclusion that the discovery of oil and natural gas resources has been the starting point of a chain of events that ultimately led to conditions favoring the emergence of mers-cov ( figure ). this discovery led to massive economic growth. owning a camel represents the wealth and status of its owner in arabic culture. governmental sponsorship of camel ownership and camel racing further stimulated the camel industry, especially the camel-racing sector. this in turn lead to an accelerating increase of the camel population, a change in camel farming, and a concomitant increase in the number of camel workers [ ] . the human population of qatar has increased by -fold over the last decades [ ] . this is unlike other high-income countries, that have a yearly overall population growth of only . % [ ] . population growth and high population density have been shown previously to be important risk factors for disease emergence [ ] . moreover, consistent with the disease profile of wealthy countries where sedentary lifestyle prevails, the prevalence of chronic diseases increased in qatar in accordance with the increasing gpd, ultimately rendering the qatar population not only vulnerable to virus transmission, but also to its deadly complications [ ] . the intimate nature and number of interactions between camels and humans has also increased significantly in the past years, increasing the risk of any zoonotic spillover. at camel complexes, workers intimately reside, sleep, and eat with their camels. camel owners, on the other hand, pay regular visits to their barns and stay there for considerable hours every day (even longer during weekends, holidays, and winter season) in the majlis built at the corner of their barns. owners, who are often of advanced age with multiple comorbidities, enjoy drinking fresh camel milk and entertaining guests. those who suffer certain diseases tend to visit the camel barns to use camel urine or drink fresh camel milk for its perceived curative properties. among the variety of changes that involved camel husbandry in qatar, the shift from open grazing to close housing systems seems to be most significant. opportunities for camel-to-camel and camel-to-human spread have greatly increased since then. it is possible that housing camels in barns, with poor biosecurity and hygienic standards, turned these barns into 'melting pots' for the virus that ultimately acquired the ability to cross the human-animal barrier. the increase of cross-border movement of camels increased chances and frequency of (international) virus spread. camels are transported freely across borders for a variety of purposes through multiple routes and means of transportation. when camels and the humans that accompany them, arrive at the site of a race or beauty event in qatar, they are housed with the local camels. owners are welcomed in the majlis at the camel complexes. the mixing of camel and human of different origins further increase chances of virus transmission. viruses , , x; doi: for peer review www.mdpi.com/journal/viruses species variation in dpp [ ] . as such, the increasing human-animal interface that is described in this paper may have facilitated the adaptation of the spike protein to human ddp . however, much remains unknown, also in view of the findings that mers-cov from east africa were not phenotypically different from the viruses from the middle east, while human mers patients have not been reported from the african continent [ ] . finally, the changes in animal husbandry practices, earlier weaning, frequent grouping and transportation of animals, and the introduction of an entirely new feeding system, may induce stress in the camels. these changes and movements often involve young weaned animals, at the same time as maternal antibodies are waning, which are linked to the shedding of the virus [ , ] . most of the limitations of this study were related to the availability of data. firstly, statistics on animals, import and export, animal workers, and land use were only found since onwards, limiting the chance although much effort was made to study mers-cov viral sequences and mers-cov transmission between dromedary camels and humans, it is still unknown which genetic mechanisms have caused the viral spillover of dromedary camels to humans. however, the most important determinant of host specificity seems to be the spike s protein, that recognizes and binds to host-cell receptor dpp [ ] . recently it has been shown that the mers-cov spike can rapidly adapt to species variation in dpp [ ] . as such, the increasing human-animal interface that is described in this paper may have facilitated the adaptation of the spike protein to human ddp . however, much remains unknown, also in view of the findings that mers-cov from east africa were not phenotypically different from the viruses from the middle east, while human mers patients have not been reported from the african continent [ ] . finally, the changes in animal husbandry practices, earlier weaning, frequent grouping and transportation of animals, and the introduction of an entirely new feeding system, may induce stress in the camels. these changes and movements often involve young weaned animals, at the same time as maternal antibodies are waning, which are linked to the shedding of the virus [ , ] . most of the limitations of this study were related to the availability of data. firstly, statistics on animals, import and export, animal workers, and land use were only found since onwards, limiting the chance to study the trends and changes prior to that year. secondly, even the available national data on the animals, humans, and environment were found to be sometimes inconsistent, limiting the possibility global trends in emerging infectious diseases the ebola outbreak, - : old lessions for new epidemics middle east respiratory syndrome middle east respiratory syndrome coronavirus cross host transmission in the emergency of mers coronavirus evidence for camel-to-human transmission of mers coronavirus middle east respiratory syndrome coronavirus transmission coronavirus transmission in extended family mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia the clinical and virological features of the first imported case causing mers-cov outbreak in south korea urbanization and disease emergence: dynamics at the wildlife-livestock-human interface zoonosis emergence linked to agricultural intensification and environmental change reasons for the increase in emerging and re-emerging viral infectious diseases. microbes infect dromedary camels and the transmission of middle east respiratory syndrome coronavirus (mers-cov) unraveling the drivers of mers-cov transmission divers are a pearl's best friend: pearl diving in the gulf s- s. qatar digital library national identity formation in modern qatar: new perspective oil and politics in the gulf qatar country indicators ministry of development planning and statistics. quarterly bulletin for population and social statistics-third quarter population of qatar by nationality- report ministry of development planning and statistics. population and social statistics annual tourism performance report chronic disease risk factor surveillance world health organization. global health observatory (gho) data: overweight and obesity noncommunicable diseases and their risk factors; stepwise approach to surveillance (steps) death rate, crude death the reports of the camel racing organizing committee; ministry of culture and sport department of animal resources animal population; world animal health information database (wahis interface)-version ministry of development planning and statics supreme council for environment and natural reserves opportunities for the sustainable use of the camel in qatar isolation of a novel coronavirus from a man with pneumonia in saudi arabia mers-cov in upper respiratory tract and lungs of dromedary camels prevalence of comorbidities in the middle east respiratory syndrome coronavirus (mers-cov): a systematic review and meta-analysis bat-to-human: spike features determining 'host jump' of coronaviruses sars-cov, mers-cov, and beyond adaptive evolution of mers-cov to species variation in dpp replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels mers coronaviruses from camels in africa exhibit region-dependent genetic diversity key: cord- -gt ey wz authors: wang, weiwen; ng, edward title: air ventilation assessment under unstable atmospheric stratification — a comparative study for hong kong date: - - journal: build environ doi: . /j.buildenv. . . sha: doc_id: cord_uid: gt ey wz in most current air ventilation assessment (ava) studies, a simple neutral assumption that does not consider thermal effects is adopted, particularly for numerical simulation practices. with statistics of daytime observations during summer in hong kong as an example, this study demonstrates that neutral atmospheric boundary conditions occur with a very low probability, which implies that current practices are indeed far away from reality. this study is devoted to addressing this knowledge gap by cross-comparisons of field measurements, wind tunnel tests, and large-eddy simulations (les) under neutral and unstable conditions. it is found that les-computed velocity ratios under unstable conditions are in line with field measurements, while results of simulations under neutral conditions are close to those of wind tunnel tests. enhanced vertical mixing due to surface heating produces improved ventilation performance in the unstable case. the neutral assumption tends to underestimate pedestrian-level velocity ratios compared to a diabatic condition; hence it is deemed conservative when it is adopted in ava practices. moreover, stronger wind direction variance under unstable conditions results in weaker correlation between velocity ratios and frontal area indices than neutral conditions, which implies that street orientations become less important in ventilation under unstable conditions. wind comfort and wind safety for pedestrians are important requirements for city design and urban planning [ ] . for subtropical high-density cities such as hong kong, in order to mitigate the negative effects of the urban heat island, good ventilation is required for healthy living and comfortable thermal sensations [ , ] . after hong kong was hit by the severe acute respiratory syndrome (sars), from which many people died in , the hong kong government started discussions among various government departments and consulted relevant professional institutes and stakeholders regarding the standards, scope, and mechanism for application of an air ventilation assessment (ava) system to improve the urban wind environment [ ] . afterwards, a set of planning guidelines was promoted based on a series of studies. the housing, planning and lands bureau and the environment, transport and works bureau jointly issued the air ventilation assessment technical guidelines (ava-tc - ) in . meanwhile, a new chapter on ventilation assessment was also incorporated into the hong kong planning standards and guidelines (hkpsg). these two documents, setting out the framework and requiring all major government departments to include ava as one of their planning and design considerations, were the first to address weak wind problems [ ] . unlike the guidelines of many countries for dealing with wind gust problems, the hong kong ava is specifically designed to deal with weak wind conditions in congested urban areas under a hot and humid subtropical climate [ ] . the world's first weak-wind urban ava system has been applied in hong kong in the city's design and planning practices for more than a decade. however, the current ava methodology is known to have limitations in that it cannot correctly predict urban air ventilation performance in some areas of hong kong where convective motion is prominent due to inhomogeneous surface heating in weak wind conditions [ ] . one possible cause is that the current ava methodology adopts the neutral assumption for the atmospheric boundary condition, which is justified only when mechanical turbulence has a greater influence on ventilation than buoyancy-induced turbulence under high background wind speed. however, the neutral assumption does not adequately represent the actual situation, as the daytime atmospheric boundary is likely to be unstably stratified, particularly under clear sky and low wind conditions [ ] . more importantly, the most crucial situations for urban ventilation are those in which only a low-speed background wind is present. the neutral assumption that supposes that buoyancy-induced turbulence has only a minor influence on ventilation is not true in such a condition. the atmospheric stability of the urban boundary layer is well known [ ] . meanwhile, many urban ventilation studies have used wind tunnel testing and computational fluid dynamics (cfd) models with a neutral assumption [ ] [ ] [ ] [ ] [ ] [ ] [ ] . but very few studies have addressed the effects of diabatic conditions on urban ventilation. a study of thermal effects on turbulence coherent structures found that when thermal effects are included by surface heating in large-eddy simulation (les), the spanwise flow is stronger within an idealized building array compared to the neutral case [ ] . another les study suggested that roof heating, in combination with building walls and ground heating, is important in the strength and location of the canyon vortex [ ] . both of these les studies used idealized urban geometries (cubical building arrays). using reynolds-averaged navier-stokes (rans) simulations of two simplified hong kong city models, yang and li pointed out that airflow in street canyons is dependent on thermal stratification when wind speed is small relative to the buoyancy force [ ] . however, how pedestrianlevel ventilation under thermal stratification differs from neutral conditions in a realistic urban complex is indistinct. the neutral assumption is widely adopted in ava practices, mainly because of its low computational cost and the ease with which it can be realized in numerical simulations and wind tunnel tests. when thermal conditions, specifically unstable stratification, are considered in ventilation, there will be additional challenges: first, a larger model domain is required to catch the larger turbulent structures in unstable simulations than in the neutral condition, while the grid size has to be kept small to sufficiently resolve the street-level air flows [ ] . second, special attention should be paid to the lateral boundary conditions to ensure that they will not artificially modify the wind environment within the assessment region. for instance, the radiative outflow condition in the non-cyclic boundary requires a positive outflow at all times [ , ] . therefore, while a weak background wind is provided for generating buoyancy-driven turbulence, extra model domain is needed on the leeward side of the city. this also requires extra computational cost. third, more uncertainties will be incorporated in the estimation of initial thermal conditions. heat fluxes are more difficult to monitor than winds. in such cases, validation of model results can be another obstacle. the objective of this study is to demonstrate the knowledge gap between current practices and reality by comparing wind tunnel test results, field measurements, and a pair of les experiments in hong kong, and to propose possible adaptations for future ava practices based on the comparative results and knowledge of atmospheric boundaries under various conditions. in ava studies, we are especially interested in pedestrian-level wind velocity. the wind velocity ratio (vr) is used as an indicator, which is calculated by vr = v p /v ∞ , where v p is the wind velocity at the pedestrian level ( m above the ground), and v ∞ is the wind velocity at the boundary layer not affected by ground roughness. apart from the meteorological data used in estimating the atmospheric stability class, this study includes some wind tunnel test results and field measurements from previous ava studies, and a pair of les experiments are undertaken for cross-comparison. utilizing observations from two urban weather stations in hong kong during - , we estimate the probability of stability classes according to pasquill's atmospheric stability classification scheme [ ] . hourly cloud cover data are taken from hong kong observatory headquarters, while wind speed and global solar radiation data are taken from king's park. the locations of these two weather stations are indicated in fig. . we consider daytime hours ( a.m.- p.m.) during all summer (june-august) days in - . as listed in table , atmospheric turbulences are categorized into six stability classes, namely a, b, c, d, e, and f, with class a being the most unstable, class f the most stable, and class d corresponding to neutral conditions. thresholds for wind speed and cloud cover are quantitatively specified, while the threshold for daytime insolation is given only qualitatively, in terms of "strong," "moderate," and "slight," which has to be transferred as specific values. based on hourly observations of global solar radiation from a.m. to p.m. on summer days, the th, th, and th percentiles are computed as . , . , and . mj m − , respectively. according to table , daytime stability is classified. for example, when wind speed is lower than , classifications a and b are divided by the th percentile of solar radiation; when wind speed is between and , classifications a and b are divided by the th percentile of solar radiation, classifications b and c are divided by the th percentile, and so forth. the probability of various classes of stability is calculated using all summer data during - . the estimated result suggests that neutral conditions occur with a very low probability of about only %; even the weak unstable class c occurs less than % of the time. during most of the daytime (about %), atmospheric stratification in urban hong kong can be classified as moderate and strongly unstable (classes b and a). however, the estimation may not be very accurate due to some deficiencies of this methodology. there are three limitations to the use of the pasquill stability classification scheme: first, the pasquill scheme is widely used mainly because it requires only routine meteorological data, but it was originally developed in the united kingdom at a location some miles from the coast, and should be restricted to the insolation values and climate conditions there. it has been found that various stability classification methods result in large discrepancies [ ] . second is the restriction of local observations to hong kong. hko is one of the two sites that have cloud cover observations (the other site is hong kong international airport). it is located in a small park that is surrounded by a high-density urban area in tsim sha tsui (fig. ) , with an altitude of m above mean sea level. kp is surrounded by a relatively larger green space on a small hill with an altitude of m on the kowloon peninsula. the uncertainties introduced to the classification of stability by such environmental conditions are difficult to control. finally, there is no information available as to how the parameters of strong, moderate, and slight should be chosen when the pasquill scheme is used locally in hong kong. due to the abovementioned limitations of stability classification using the pasquill scheme and ordinary meteorological observations, we use some samples of radiosonde observations to further demonstrate the probability of various types of atmospheric conditions in hong kong in summer. the radiosonde data are observed at king's park (fig. generally, sounding data are available only at : and : greenwich mean time (gmt), that is, : and : hong kong local time. in this study, sounding data recorded at : local time are used. we demonstrate the results from september (fig. ), when the street-level field measurements (introduced in section . ) are conducted. meanwhile, data from july , representing midsummer of the same year, are shown in fig. as well. fig. a boundary conditions should increase during summer afternoon hours compared to this time. in september, when the weather is cooler, the probability of unstable conditions decreases while the probability of stable and near-neutral conditions increases compared to july (fig. b ). in general, using both the pasquill stability classification scheme and the vertical gradient of θ e from radiosonde observations suggests that the probability of a neutrally stratified boundary layer in urban hong kong in summertime is not high; specifically, it is lower than the possibility of a thermally stratified boundary layer. the assumption of a neutral atmospheric boundary condition in air ventilation studies of hong kong is not representative. wind tunnel data are taken from an earlier ava study [ ] . the study was conducted by local researchers under the instruction of the hong kong planning department (hkpd). final reports can be downloaded from the hkpd web page [ ] . the wind tunnel tests were undertaken in accordance with international practice requirements and conducted at the clp power wind/wave tunnel facility (wwtf) of the hong kong university of science and technology [ ] . the clp power wwtf has a state-of-the-art subsonic boundary-layer wind tunnel. the overall size is . ground measurement data are adopted from the field measurements in "urban climatic map and standards for wind environment -feasibility study" [ ] . spot measurements were conducted using handheld equipment along preselected paths in tsim sha tsui. measurements were made along the paths at fixed points (fig. ) . the equipment used in the measurement included a -function sensor probe from testo [ ] for the measurement of air temperature and wind speed, and a testo data logger for instant processing of the measured data. the data logger was set with a sampling time of s and an averaging time of min. all the equipment was iso certified and was cross-calibrated in the laboratory before actual measurements were taken. field measurements carried out on september from : to : local time are utilized. it was a sunny and hot summer day. the air temperature observed at hko during the field measurements was - °c, and the relative humidity was %- %. the field measurements covered measurement points and reference point at the seafront in tsim sha tsui (fig. ) . twelve trained researchers were employed and grouped into teams to conduct the measurements. each group started the measurements at the first point (close to the seashore) of their corresponding path at the same time; that is, group started at g - , while group started at g - , etc. (fig. ) . during the measurements, measuring sensors were located m above the ground and logged the data. when the measurement time was up, the researchers recorded the data and moved on to the next point at the designated time. both temperature and wind speed were measured, but only wind data are used in this study. the values are listed in table . the rightmost column of table gives the time of measurement. mapping of the spatial distribution requires relatively stable weather conditions during the entire measurement period; that is, temporal variation of the weather conditions should be insignificant [ ] . this requirement has been fulfilled for the field measurements used in this study. however, as all test points cannot be measured at the same time, uncertainties that could affect the results of the comparison between the measurements and numerical simulations are unavoidable. moreover, the limited sampling time ( min) for each point could import another uncertainty, as the les represents a more stable situation with an hourly average. the realistic environment is difficult to fully consider in numerical modeling-for instance, the effects of greenery and traffic. we employ an les model to carry out cfd simulations in this study. les overcomes the deficiencies of the rans model by explicitly resolving large, energy-containing turbulent eddies and parameterizing only subgrid-scale (sgs) turbulence [ , ] . les provides not only mean flow fields but also instantaneous turbulences, which are especially important for human comfort at the pedestrian level in the urban canopy layer. the les model used in this study is the parallelized les model (palm), which was developed in , when it was one of the first parallelized les models for atmospheric research [ ] . the les model has six prognostic quantities by default: the velocity components u v w , , on a cartesian grid, the potential temperature θ, specific humidity q v or a passive scalar s, and the sgs turbulent kinetic energy e. the governing equations are based on the non-hydrostatic, filtered, incompressible navier-stokes equations with boussinesq approximation and are filtered implicitly using the volume-balance approach of schumann [ ] . the governing equations of palm are given in maronga et al. [ ] . the modified version [ , ] of the . -order deardorff scheme [ ] is used for turbulence closure. the temperton algorithm [ ] for the fast fourier transform is used to solve the poisson equation for the perturbation pressure. for the time integration, a third-order runge-kutta scheme [ ] is used. the advection scheme used is the secondorder scheme of piacsek and williams [ ] . alternatively, a fifth-order scheme developed by wicker and skamarock [ ] can be utilized. the monin-obukhov similarity theory (most) is applied between the surface and the first grid level. following the most, a constant flux layer is assumed as the boundary condition between the surface and the first grid level where scalars and horizontal velocities are defined [ ] . a prandtl layer is assumed at each surface. if non-cyclic horizontal boundary conditions are used, palm provides the possibility of generating time-dependent turbulent inflow by using a turbulence-recycling method, which follows the one described by lund et al. [ ] with modifications introduced by kataoka and mizuno [ ] . in front of the simulation domain, a recycling area is attached. the outflow boundary of this recycling area is the recycling plane, from where the turbulence signal ′ φ y z t ( , , ) is recycled: where x recycle is the distance from the inflow boundary to the recycling plane, that is added to the mean inflow profile after each time step. the recycling area length x recycle should be much larger than the integral length scale of the respective turbulent flow to avoid the same turbulence structures being recycled repeatedly. hence a precursor run, which can have a comparatively small domain horizontally for generating the initial turbulence field of the main run, is promoted [ ] . the computational domain is depicted in fig. . the urban area is taken from the black box in fig. , which encloses an area of km by km on the southeastern edge of the kowloon peninsula. in front of the city, a turbulence recycling area is added to the domain, where the turbulence-recycling method is applied to create a turbulent inflow for the simulation. the size of the recycling area is m by km, and it is m away from the city area. the m × km buffer zone between x recycle and the city helps prevent the blocking effects of the buildings from reaching the recycling area, while the km × km buffer zone on the leeward side of the city helps ensure a positive outflow. as introduced, the radiative outflow condition in this non-cyclic boundary always requires a positive outflow. the topography was rotated °( north is downward) due to the requirement of the turbulence-recycling method that inflow must come from the left. horizontal grid spacing is equidistantly m. grid-sensitive tests using palm . for urban ventilation simulations have been conducted in recent studies and suggest that a grid size of m is sufficient for such a task [ , ] . vertical grid spacing is m below m and is stretched with a stretch factor of . above. with vertical levels, the top level is at about m. in the les model, scalar variables are defined at the grid centers, while velocity components are shifted by half of the grid spacing. therefore, horizontal wind velocity output from the m and m levels is linearly interpolated (averaged) to obtain v p at m above the ground. the no-slip bottom boundary condition and the freeslip top boundary condition are applied to the horizontal velocity components. a cyclic (periodic) boundary condition setup in the spanwise direction is implemented. a low-velocity wind of . ms- is prescribed. as introduced, we focus on the crucial situation for urban ventilation in which only a lowspeed background wind is present. under such low wind conditions, the influence of buoyancy-induced turbulence becomes important, and ventilation will depend on the balance between thermal forcing and wind forcing. in addition, if high wind speed is used, more computational time will be needed because the time step has to be shorter. but a low background wind speed also requires a large buffer zone behind the city to ensure a positive outflow (fig. ) . the time step lengths are optimized in the les model. in the les experiments described below, the mean time step lengths are between . s (for the unstable cases) and . s (for the neutral case). a h precursor run, with a horizontal domain of m × m and the same height as the main run, is promoted for generating initial turbulent inflow profiles. the total simulation time for the main run is h. the first hour is excluded in the analysis of the results, as the turbulences need this time to spin-up. the simulated results of the second hour are averaged for analysis. simulations are performed with different atmospheric stratification. the first simulation features a neutral stratification in which the influence of temperature on the flow field is excluded; that is, the calculation of the temperature equation is switched off. in this case, the major output elements are velocity components. other simulations feature an unstable stratification realized by a prescribed homogeneous potential temperature of k below m and a capping inversion layer above with a potential temperature gradient of k per m, which is a general temperature setup for simulations of a convective boundary layer. more importantly, prescribed heat fluxes are generated from building rooftops and side walls. as in a realistic urban area with rather complex urban form, the thermal conditions and their balance with the velocity field are difficult to quantitatively decide by heat flux table for details. generation in the numerical simulation. therefore, we carry out a set of test runs, namely t , t , t , and t , with various prescribed rooftop and side wall heat flux settings, as listed in table . we deem that the approaching winds are always under neutral stratification due to general high wind speeds and very small surface roughness over open sea waters. therefore, no surface heat fluxes are prescribed in the turbulence recycling area and the buffer zones. the bulk richardson number (r b ) is estimated to examine the unstable stratified boundary layers of the simulations: where = − g m s . is the acceleration of gravity, z ct is the height of the canopy top, estimated at m in this study (refer to fig. ), θ is the potential temperature at the bottom (the lowest model level), and θ ct and u ct are the potential temperature and wind speed at the canopy top ( m), respectively. the parameters in table are averaged from the km × km city area. herein, r b for diabatic test runs is estimated, ranging from − . to − . in table . the more heat fluxes that are given in the les, the larger the absolute r b value that will be obtained. fig. demonstrates the potential temperature profiles averaged from y-z sections that cross the city (at x = m, m, and m in fig. ) from diabatic les experiments. the les model has been validated for simulating flows and turbulence characteristics at the street-canyon and neighbourhood scale [ ] and has been widely used in studies of urban street-canyon flows [ ] [ ] [ ] [ ] , including high-density urban areas in hong kong [ ] and macau [ ] . the code used in this study (palm version . ) has been validated by a cfd guideline [ ] for pedestrian-level ventilation under neutral atmospheric conditions in recent studies [ , ] . for simulation of the thermally stratified urban boundary layer, the model has been validated by uehara's wind tunnel data [ ] for thermally stratified street canyons [ ] . this study focuses on pedestrian-level ventilation. simulation outputs from the les model are cross-compared with wind tunnel tests (under neutral conditions) and field measurements (under unstable conditions), which will be presented in the following section. wind tunnel test data, field measurements, and les outputs are cross-compared to quantify their similarities and differences. we attempt to discover what causes deviations in pedestrian-level ventilation in different atmospheric conditions. in addition to wind speed, wind direction will be investigated as well. but first of all, we have to determine which diabatic simulation (t , t , t , or t ) best represents the typical actual boundary conditions. the atmospheric stability of the urban boundary during the field measurements is estimated by meteorological observations. it is found that the hourly-averaged wind speed and global solar radiation observed at king's park (fig. ) at p.m. on september are . ms - and . mj m − , respectively. according to pasquill's classification scheme (table ) and the thresholds introduced above (here we simply apply the summer thresholds to the field measurement days in september), it can be deduced that there were unstable (class b) atmospheric conditions at the time the field measurements were taken. there are very few data regarding r b in urban areas, and we are short of quantitative measurements for estimating r b in the urban boundary in hong kong. nakamura and oke measured the temperature and wind distribution in a real street canyon and provided an r b range from − . to − . on a clear midsummer afternoon [ ] . the estimated values of r b for the diabatic runs in the present study, ranging from − . to − . (table ) , are close to the field-measured values of nakamura and oke [ ] . the stratified conditions of the les experiments can reproduce those observed in realistic urban areas. meanwhile, taking the comparison of the observed r b in nakamura and oke [ ] into consideration, wind tunnel experiments with thermal stratification by uehara et al. [ ] suggest a set of tested values of − . , − . , and − . for a weakly to moderately unstable stratified urban boundary layer. evaluating our les results in table by this set of tested values in wind tunnel experiments of unstable stratification, t , with an r b of − . , is deemed to be the best at reproducing a typical unstable stratified urban boundary layer. it is noteworthy that the wind tunnel speed v ∞ of uehara et al. [ ] is . ms - as well, the same as the setting in our les experiments. therefore, t , with a mean kinematic heat flux of . km s- (about . w m - ) prescribed to all building rooftops, and a mean kinematic heat flux of . kms - prescribed to all building side walls, is chosen as the final diabatic simulation for the following comparative study. it is difficult to estimate v ∞ in calculating vr for field measurements. the reference point at the seafront (fig. ) is deemed unsuitable, as it measures near-surface ( m above the ground) wind speed in front of the city. we propose the wind observed at waglan island as v ∞ . waglan island is located approximately km southeast of hong kong island and has uninterrupted exposure to winds (fig. ) . with a very small surface roughness over open sea waters [ ] and a relatively high anemometer elevation ( m above mean sea level) [ ] , data collected at waglan island are considered to be representative of the boundary-layer wind approaching hong kong. hourly wind speed observed at waglan island at p.m. on september is . ms - . the wind speeds in table are divided by this value to gain the field-measured vr. wind tunnel tests described in section . are conducted under the neutral assumption. among wind tunnel test points and field measurement points, there are overlapping points (excluding the reference point at the seafront). the ids of these points in both the field measurements and wind tunnel tests are listed in table , together with their corresponding vr values. the mean values in table suggest that the wind tunnel experiments may have underestimated the mean vr compared to field measurements. les-computed vr under neutral and unstable atmospheric conditions in tsim sha tsui are shown in fig. . it is obvious that the overall ventilation inside the city in the unstable experiment is better (with larger vr) than that in the neutral experiment. the overlapping test points of the wind tunnel and field measurements are located on the vr map of les. corresponding values are listed in table to enable crosscomparison. the mean values given in table show that the les experiment under neutral conditions is more comparable to the wind tunnel tests, while the les experiment under unstable conditions is more comparable to the field measurements. this is further demonstrated in the scatter plots and linear regressions in fig. . fig. shows that the wind tunnel predicts a relatively lower vr compared to field measurements, with a regression coefficient of . in fig. a . also, given an r-squared of . , the regression between wind tunnel tests and field measurements is not very convincing. the [ ] . v bl is the boundary-layer velocity. building and environment ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] regression between the wind tunnel and les neutral is better, with a regression coefficient closer to ( . ) and a larger r-squared of . (fig. b) . the linear relation between field measurements and the diabatic les experiment is like that of fig. c , giving a regression coefficient of . and an r-squared of . (fig. c) . moreover, geometrical features of pedestrian-level vr obtained from these methods are shown in fig. . the best-ventilated areas are located around the main chatham road south (see fig. ) for all cases in fig. . in the narrow streets west of chatham road south, vr is around . for field measurements and the les unstable run, but decreases to about . in the les neutral run and even below . in some areas in the wind tunnel tests. according to our knowledge of boundary-layer meteorology, nearsurface velocity over boundary-layer velocity is larger in an unstable atmospheric boundary layer than in a neutral one (fig. ) . therefore, the wind tunnel tests predict a lower vr for the overlapping test points compared to the field measurements, which should be caused by the difference in atmospheric conditions; that is, neutral in the wind tunnel but unstable in the field measurements. wind profiles under different types of atmospheric boundary layers in fig. also explain why ventilation in the diabatic les experiment is overall better than that under neutral conditions. fig. depicts some les-simulated zonal wind (u-wind) profiles, both in front of the city and in the middle of the city. these profiles demonstrate that the diabatic les experiment produces greater near-surface velocity than the neutral case, especially inside the city where surface (building) heating is provided. the higher wind speed in the diabatic case is related to the additional convective motion caused by heating from the buildings, which increases vertical mixing throughout the boundary layer in and above the city and leads to higher pedestrian-level ventilation compared to the neutral case. the vertical velocity sections in fig. show that in front of the city, the vertical velocity fluxes are generated by the turbulence recycling method, without the surface heating difference, and the diabatic case (fig. b) is similar to the neutral case (fig. a) in the lower levels. the differences in the top levels are produced by temperature gradients in the capping inversion. in the middle of the city, much stronger vertical motion is found in and above the street canyons in the unstable case (fig. d ) than in the neutral case (fig. c) . the previous section illustrates that better ventilation under unstable conditions is caused by surface heating and enhanced vertical mixing. in this section, the differences between the les experiments under neutral and unstable conditions will be quantitatively compared. this is shown in fig. , which compares vrs taken from random test points in the city of the two les experiments. in this procedure, all street (unbuilt) grid points in each les experiment are stored in a one-dimensional array, and , test points (about % of the total unbuilt grid points) are randomly taken from each array. the random function calculates the interval between test points using a normal distribution with a mean of the array size divided by the number of test points ( , ) and a standard deviation of % of the mean. the test points are randomly spread throughout the entire assessment area. sensitivity tests were conducted regarding the number of test points, and no significant differences were found when the number of test points was larger than , , which means that , test points are sufficient to produce the graphs in fig. . both the probability and the boxplot are produced by these , data points. fig. a shows that the diabatic les experiment improves ventilation performance by increasing (decreasing) the probability of vr above (below) . compared to the neutral case. in each box of building and environment ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] fig. b, the central mark is the median, the edges of the box are the th and th percentiles, and the whiskers extend to the most extreme data points [ ] . it is obvious that all statistics except the minimum value in the diabatic les experiment are significantly larger than in the neutral case. the median of vr in the unstable case is . , twice that in the neutral case ( . ). the maximum vr in the unstable and neutral cases is . and . , respectively. additional convective motion caused by heating in the unstable experiment increases not only wind speed, but also fluctuations in wind direction. we select two points, one located in a narrow street parallel to the input wind direction (point as denoted in fig. ), the other located in a wide street perpendicular to the inflow wind direction (point as denoted in fig. ). the surrounding building shapes of these two points vary largely, but we focus only on the wind variation under the two atmospheric conditions, rather than the surrounding urban wind direction in the parallel street canyon is more homogeneous (fig. a ) compared to that in the perpendicular location (fig. b) , where intense rotations of wind occur in the lee of the buildings. but in both locations, we experience larger variance of wind direction in the unstable case than in the neutral case. additional convective motion and enlarged eddy size due to surface heating should be major contributors to this difference in wind direction variance between the neutral and unstable cases. stronger variation in wind direction under unstable conditions implies that street orientation may be less important in urban ventilation compared to neutral conditions. this is verified by fig. a . the frontal area index (λ f ), which is defined as the frontal area of buildings in a certain wind direction over a site area [ ] , is calculated from building data used in the simulations. this index has been widely used in urban wind environment investigations and is found to be an important morphological factor that influences urban ventilation [ , ] . we compute only the frontal area index, rather than frontal area density, as we have only one inflow wind direction. frontal area density is the accumulation of frontal area indices in all wind directions, weighted by the observed occurrences of different directions. frontal area indices are calculated from m × m averaging areas inside the km × km city domain. in order to obtain more data samples, the averaging areas overlap. this is also a way to guarantee that the results are not influenced by random positioning of the averaging areas. in this procedure, the first averaging area covers a m × m region in one corner, say, the left-bottom corner of the city domain. the next averaging area moves m ( model grids) forward in one (x or y) direction, and so forth. in this way, a total of data samples are obtained in the km city domain. the vrs are also averaged in such a way in both the neutral and unstable cases. fig. a demonstrates that pedestrian-level vr under neutral conditions gives a correlation of − . with the λ f , which is significantly larger than − . in the unstable case. hence, street orientation seems to be less important for urban ventilation under unstable conditions due to larger wind direction variance compared to neutral conditions. the limitation of this is that the relationship between ventilation and λ f is hard to justify given that only one wind angle is tested. fig. b further demonstrates a scatter plot and regression between vr and the ground coverage ratio (λ p ), which is defined as the ratio of ground area that is covered by the building to the site area. this parameter is computed using the same averaging areas and moving-window algorithm that were used in the calculation of λ f . more importantly, λ p is independent of wind direction. fig. suggests that the correlation between vr and λ p is higher than that between vr and λ f in both the neutral and unstable cases. the vr under neutral conditions gives a correlation of − . with λ p , which is still larger than − . in the unstable case (fig. b) . one possible cause is that stronger convective motion (fig. ) and greater wind direction variance (fig. ) in the unstable case reduce the effects of urban form and urban density on ventilation. nevertheless, these results are still restricted by the limited domain and the complexity of the realistic urban morphology tested in the simulations. future studies using parametric urban forms and urban densities while considering more input wind directions should be conducted to expand the preliminary result of this study. in general, the neutral assumption, which does not consider thermal stratification in air dynamics, is a simple but widely accepted assumption for urban wind studies. but the fact that neutral conditions occur with a relatively low probability in summertime, as shown in both the pasquill stability classification scheme and the vertical gradient θ e from radiosonde observations in section . , invokes the consideration of thermal stratification in studies of urban ventilation. in this case study, we cross-compare pedestrian-level vr taken from field measurements, wind tunnel tests, and a pair of les experiments in a high-density area of hong kong. the wind tunnel test under the neutral assumption is found to underestimate the ventilation as measured by pedestrian-level vr compared to the field measurements, which were taken during unstable atmospheric conditions (fig. a) . at the same time, the results of the les experiment under the neutral assumption are consistent with those of the wind tunnel tests (fig. b) , while the unstable les run captures the reality (field measurements) well (fig. c) . such findings theoretically agree with our knowledge that near-surface wind speed over boundary-layer velocity should be significantly larger in unstable stratification than in neutral stratification (fig. ) . the major cause is additional convective motion caused by surface heating, which increases vertical mixing throughout the boundary layer in and above the city. these thermodynamical features are well captured by the les experiments (figs. and ). the quantitative difference in the les results under neutral and unstable conditions are analyzed by vrs taken from random test points in the city (fig. ) . impacts of different types of atmospheric conditions on wind direction variance in simulations of ventilation are evaluated. an unstable boundary induces stronger wind direction variance compared to a neutral boundary in the urban areas (fig. ) . therefore, the effects of street orientation on urban ventilation may be less important in an unstable boundary layer than in a neutral boundary layer. this is demonstrated by the regressions of the frontal area index and pedestrianlevel vr under different types of atmospheric conditions (fig. ) . according to the findings in sections . - . , we can deduce that current ava practices under the neutral assumption are conservative if the actual atmospheric boundary is unstably stratified. but it is also noteworthy that unstable stratification generally occurs under weak wind conditions ( table ) . as the background wind (the boundary-layer velocity in fig. ) is weak, though the pedestrian-level vr under unstable conditions is significantly larger than that under neutral conditions, the actual wind environment, that is, the absolute wind speed at the pedestrian level, will not improve as much as the vr. we considered only neutral and unstable stratifications in this study and did not look at stable stratification. urban ventilation studies under stable atmospheric conditions are difficult due to a lack of in situ observations, and modeling (with a wind tunnel or cfd) of this type of thermal stratification is challenging. but the effects of a stably stratified boundary on urban ventilation should be the subject of a future study. according to fig. , stable boundary conditions may be the worst situation for urban ventilation, as they produce the smallest near-surface velocity over boundary-layer velocity compared to the other two types of conditions, and the background wind is generally not very strong (table ) . to conclude, wind tunnel tests under the neutral assumption underestimate the overall ventilation as measured by pedestrian-level vr compared to field measurements, which were conducted during an unstably stratified atmospheric boundary. vrs in the unstable les case are in line with field measurements, while those in the neutral case are close to the wind tunnel tests. overall ventilation under unstable conditions is found to be better than that under neutral conditions. current ava practices under the neutral assumption are conservative if the actual situation includes an unstably stratified boundary layer. enhanced convective motion due to surface heating in unstable conditions is the main reason the neutral assumption can be conservative. moreover, stronger wind direction variance under unstable conditions results in weaker correlation between vrs and frontal area indices than under neutral conditions. this implies that street orientation is less important in street-level ventilation under unstable conditions than in neutral conditions, due to the importance of surface heating effects in unstable conditions. cfd simulation for pedestrian wind comfort and wind safety in urban areas: general decision framework and case study for the eindhoven university campus urban heat islands in hong kong: statistical modeling and trend detection urban human thermal comfort in hot and humid hong kong, energy build policies and technical guidelines for urban planning of high-density citiesair ventilation assessment (ava) of hong kong urban climate challenges in the tropics: rethinking planning and design opportunities determining site wind availability for air ventilation assessment in hong kong's coastal and highly complex topographical conditions parametric studies of urban morphologies of high density cities and their air ventilation performance under neutral and unstable atmospheric conditions using advanced large-eddy simulations progress in observing and modelling the urban boundary layer 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simulation study of thermal effects on turbulent flow and dispersion in and above a street canyon wind, temperature and stability condition in an east-west oriented urban canyon determining the power-law wind-profile exponent under near-neutral stability conditions at sea quantifying urban heat island an introduction to boundary layer meteorology variations of boxplots urban morphology detection and computation for urban climate research improving the wind environment in high-density cities by understanding urban morphology and surface roughness: a study in hong kong quantitative ventilation assessments of idealized urban canopy layers with various urban layouts and the same building packing density key: cord- -mcfxye f authors: chung, woo-chang; hwang, kwang yeon; kang, suk-jo; kim, jae-ouk; song, moon jung title: development of a neutralization assay based on the pseudotyped chikungunya virus of a korean isolate date: - - journal: j microbiol doi: . /s - - - sha: doc_id: cord_uid: mcfxye f the chikungunya virus (chikv) belongs to the alphavirus genus of togaviridae family and contains a positive-sense single stranded rna genome. infection by this virus mainly causes sudden high fever, rashes, headache, and severe joint pain that can last for several months or years. chikv, a mosquito-borne arbovirus, is considered a re-emerging pathogen that has become one of the most pressing global health concerns due to a rapid increase in epidemics. because handling of chikv is restricted to biosafety level (bsl- ) facilities, the evaluation of prophylactic vaccines or antivirals has been substantially hampered. in this study, we first iden-tified the whole structural polyprotein sequence of a chikv strain isolated in south korea (knih/ / ). phylogenetic analysis showed that this sequence clustered within the east/ central/south african chikv genotype. using this sequence information, we constructed a chikv-pseudotyped lenti-virus expressing the structural polyprotein of the korean chikv isolate (chikvpseudo) and dual reporter genes of green fluorescence protein and luciferase. we then developed a pseudovirus-based neutralization assay (pbna) using chikvpseudo. results from this assay compared to those from the conventional plaque reduction neutralization test showed that our pbna was a reliable and rapid method to evaluate the efficacy of neutralizing antibodies. more importantly, the neutralizing activities of human sera from chikv-infected individuals were quantitated by pbna using chikvpseudo. taken together, these results suggest that our pbna for chikv may serve as a useful and safe method for testing the neutralizing activity of antibodies against chikv in bsl- facilities. chikungunya virus (chikv) belongs to the alphavirus genus of the togaviridae family and is an arbovirus that can be transmitted by mosquitoes such as aedes aegypti and aedes albopictus. its infection causes chikungunya fever, characterized by a high fever, rashes, headaches, and severe joint pain that can last for several months or years. chikv is considered a re-emerging human pathogen and has caused millions of cases of infection around the world (schwartz and albert, ; thiboutot et al., ) . as a tropical disease, chikv epidemic has mostly been reported in regions near and including the indian ocean, pacific islands, and the americas (morrison, ) . however, global warming and international transportation have increased the risk of spreading chikv infections to non-endemic regions, including south korea, where travel-associated or imported cases of chikv infection have been reported (cha et al., ; hwang and lee, ; yeom, ) . in addition, there may be a potential risk of local transmission of chikv, because the habitat of aedes albopictus has been increased significantly in south korea (chang el al., ) . the complete genome of chikv is a linear, positive-sense, single-stranded rna with , nucleotides that encodes two polyproteins (schwartz and albert, ; morrison, ) . one polyprotein consists of the nonstructural proteins nsp , nsp , nsp , and nsp that are responsible for viral genome replication and viral gene transcription in infected cells. the other polyprotein consists of the capsid, e , e , k, and e structural proteins that are responsible for virion formation. the translated structural polyprotein is autocatalytically processed into capsid protein c, e , k, and p . the capsid protein c encapsidates viral genomic rna, while the glycoproteins e and p interact to form heterodimers that subsequently trimerize into a viral spike in the endoplasmic reticulum (jose et al., ; li et al., ) . the glycoprotein p is then cleaved into e and e by cellular furin. glycoprotein e is important for receptor binding during viral entry, while glycoprotein e is responsible for membrane fusion during viral infection (bréhin et al., ; kuo et al., ) . although chikv glycoprotein e is released after budding and not associated with the mature virion, glycoprotein e facilitates e -p heterodimerization and prevents the exposure of the e fusion loops from premature fusogenic activity (mulvey and brown, ; carleton et al., ; solignat et al., ; voss et al., ; snyder and muk- yap et al., ) . in addition, the e protein has been shown to be a major epitope for neutralizing antibodies (voss et al., ) . despite the increasing global concerns about the worldwide reemergence of chikv, there is no approved antiviral treatment for chikv infection or vaccine against chikv (jain et al., ; tharmarajah et al., ) . multiple technology platforms have been applied to develop chikv vaccines using live attenuated viruses, inactivated viruses, recombinant viruses, chimeric alphaviruses, or virus-like particles (vlp), although such vaccines are still in various stages of the clinical or pre-clinical processes (weaver et al., ; powers, ) . in addition, vaccine efficacy is often evaluated using the conventional plaque reduction neutralization test (prnt) (azami et al., ) . prnt determines the neutralizing antibody titer of a test sample by counting the number of plaques formed by cytopathic effects of viral infection. however, it is difficult to handle live, biohazardous chikv because its use requires a biosafety level- (bsl- ) facility, therefore an alternative assay to evaluate vaccine efficacy may facilitate vaccine development. in this study, we aimed to develop a pseudovirus-based neutralization assay (pbna) using knih/ / , a korean isolate of chikv (cha et al., ) . a chikv-pseudotyped virus (chikvpseudo) expressing the structural polyprotein of chikv, was constructed using a lentiviral vector, which expressed both luciferase and green fluorescence protein (gfp) reporters. validation and comparisons of pbna results with those from conventional prnt indicate that the pbna using chikvpseudo may be a safe, rapid, and reliable neutralization assay for evaluating the neutralizing activity of anti-sera against chikv. vero, hela, bhk- , and hek t cells were cultured in complete dulbecco's modified eagle's medium (hyclone) containing % fetal bovine serum (fbs; hyclone) and supplemented with u/ml penicillin and μg/ml streptomycin (hyclone). knih/ / , the korean isolated strain of chikv, was provided by the national culture collection for pathogens, korea. a neutralizing antibody against chikv e protein (anti-e ; chk- from absolute antibody ltd.) was used for plaque reduction neutralization tests (prnts) and pseudovirus-based neutralization assays (pbnas) (pal et al., ) , while a neutralizing antibody against the spike protein of the middle east respiratory syndrome coronavirus (mers-cov) (sino biological inc., cat: -r ) was used as a control for pbna specificity. human sera were obtained from a study titled "burden of dengue infection in children and adults of ouagadougon, burkina faso" with informed consent of the subjects. the subjects authorized researchers for the utilization of the left-over blood samples to identify other pathogens to study disease transmission in this area. this additional study was approved by the institutional review board of international vaccine institute (ivi irb protocol number: - ). knih/ / infected bhk- cells were harvested with tri reagent (mrc). rna was extracted using a chloroform extraction method. the cdnas were synthesized using the revertaid first-strand cdna synthesis kit (invitrogen) with oligo dt primers. the synthesized cdna was used as template dna for pcr amplification of the structural polyprotein coding region, which was then cloned into the pcmv -flag vector (pcmv -flag-chikvst) using the following primers: f: -cttgcggccgcgatggagttcatc ccaacccaaact- and r: -tatattggatccttag tgcctgctgaacgacacgc- . underlined letters indicate the noti and bamhi restriction enzyme sites used for cloning. to sequence the cloned structural polyprotein, additional primers targeting the capsid gene ( -gccatccc agttatgtgcctgttgg- ) and the e gene ( -cag tataactccccctctggtcacgcgt- ) were used and the sequence submission can be found in genbank. the nucleotide sequences of structural polyprotein coding genes from chikv isolates were selected for phylogenetic analysis (table ) . amino acid sequences were aligned by the clustal omega multiple sequence alignment program. the neighbor-joining algorithm and the maximum composite likelihood model were used for tree construction by mega software (tamura et al., ) . the reliability of the analysis was evaluated by a bootstrap test with , replicates. the expression of structural proteins of chikv from pcmv -flag-chikvst was tested. the plasmid was transfected into hek t cells using polyethyleneimine (fukumoto et al., ) . at h post-transfection, cells were harvested and analyzed by western blot as described below. to produce chikvpseudo, hek t cells were transfected with pcmv -flag-chikvst, pspax (a gift from dr. seungmin hwang, the university of chicago, il, usa), and luc p plvx-ires-zsgreen , which is able to express two reporter genes, luciferase and gfp (a gift from dr. nam-hyuk cho, seoul na- tional university, south korea). pmd .g encoding vsv-g (a gift from dr. seungmin hwang, the university of chicago, il, usa) was used for control pseudotyped virus production. cell media was changed around h post-transfection. the supernatant containing cultured viruses was harvested at days after the media change, then filtered using a . μm syringe filter. the virus stock was aliquoted in small volumes and stored at - °c until use. for titration of the pseudotyped chikv, hela cells were infected with chikvpseudo by incubation with virus-containing media for days. the number of gfp expressing cells were counted using facs (facscalibur, bd biosciences) to calculate the titer of infectious chikvpseudo. cells were then harvested and analyzed by western blotting to evaluate structural protein expression. sds-page resolved lysates were transferred to a polyvinylidene fluoride (pvdf) membrane and probed with primary antibodies against flag-m ( : , ; sigma), e ( : ; absolute antibody ltd.), or α-tubulin ( : , ; sigma). antimouse immunoglobulin g antibody conjugated with horseradish peroxide ( : , santa cruz biotechnology) was used as a secondary antibody. detection was performed using ecl plus western blotting detection reagents (elpis) and an las- chemiluminescent image analyzer (fujifilm). the ability of an antibody to neutralize chikv was determined using the prnt with vero cells. we used a neutralizing antibody against chikv e protein (anti-e ; chk- from absolute antibody ltd.) as a reference antibody (pal et al., ) and experiments were done in triplicate. threefold serial dilutions of stock antibody were made with dmem containing % heat-inactivated fbs-containing, then mixed with a fixed amount of chikv (knih/ / , pfu/well in well plates) and incubated at °c for h. after incubation, a vero cell monolayer was infected with the virus/ antibody mixtures and incubated at °c for min to adsorb the virus. next, after removing the inoculum, the overlay media containing % methylcellulose was added to the infected vero cells. after days of incubation, cells were fixed and stained with % crystal violet in % ethanol. plaques were counted to calculate the inhibitory concentration of the antibody against chikv. the neutralization curve and the effective concentration of % neutralization (logic ) were calculated using graphpad prism (graphpad). pbna experiments were done in triplicate. for the pbna, three-fold serial dilutions of the anti-e antibody were mixed with chikvpseudo solution at an estimated titer of gfp unit/well and then incubated at °c for h. all antibody dilution was performed with dmem containing % heat-inactivated fbs. then, a hela cell monolayer was infected with the chikvpseudo/antibody mixtures and incubated at °c for h. infected cells were cultured in complete media for additional days. cells were then harvested and analyzed by a luciferase activity using the luciferase reporter assay system (promega) following manufacturer's instructions. the neutralization curve and the effective concentration for % neutralization (logic ) were calculated using graphpad prism. although not frequent, travel-associated cases of chikv infection have recently been increased and detected in % of patients with dengue-like symptoms (cha et al., ; hwang and lee, ; yeom, ) . knih/ / was isolated from such a patient and deposited to the national culture collection for pathogens (cha et al., ) . to further perform a genetic analysis of knih/ / , we amplified the structural polyprotein coding region from cdna of knih/ / using a reverse transcription system. the amplified structural polyprotein dna of . kb in length was cloned into an expression vector system (pcmv -flag-chikvst) and primers used to specifically amplify portions of the capsid (c) and e genes. an ncbi-blast search showed that the sequence of the structural polyprotein has % sequence similarity with other chikv isolates belonging to the east/central/south african (ecsa) genotype. the structural polyprotein sequence of knih/ / was then aligned with sequences from asian, west african, or ecsa genotypes using clustal w alignment and the phylogenetic position of the knih/ / isolate identified using mega software. the knih/ / isolate clustered with ecsa genotypes and with isolates from singapore and malaysia (table and fig. ). we have established a reliable plaque reduction neutraliza-tion test (prnt) using knih/ / . however, because of the potential limitations of handling chikv in laboratories owing to its biohazard risk level, we set out to develop an alternative pseudovirus-based neutralization assay (pbna) that may replace the conventional prnt. a pseudotyped virus of knih/ / (chikvpseudo) was designed to express the full-length structural polyprotein rather than individual structural proteins to enhance its infectivity (hu et al., ) . a schematic diagram of the structural polyprotein expression plasmid of knih/ / (pcmv -flag-chikvst) is shown in fig. a . to produce chikvpesudo, pcmv -flag-chikvst was transfected into hek t cells with a lentivirus-based dual reporter vector (luc p-plvx-ires-zsgreen ) and a packaging vector of lentivirus (pspax ) (fig. b) . flag-tagged capsid protein was detected in hek- t cells infected with chikvpseudo using anti-flag antibody (fig. c) . the expression of glycoprotein e was also observed in chikvpseudo-transduced cells, as in knih/ / -infected cells using anti-e antibody. chikvpseudotransduced cells also showed expression of both the gfp and firefly luciferase dual reporter genes, allowing us to quantify the infectivity of chikvpseudo ( fig. d and e) . although both hek t cells and hela cells resulted in high efficiency in transduction of chikvpseudo, we set to use hela cells for pbna, because hela cells were more durable for the process of pbna, providing more reliable values (data not shown). to check whether chikvpseudo can be utilized for a neutralization assay, we assessed the neutralizing activity of an antibody against the structural protein e (anti-e ), ranging the concentrations from μg/ml to . ng/ml, as produced from chikvpseudo infection. first, the concentration of antibody required to inhibit chikvpseudo infection by % (ic ) was calculated. chikvpseudo infection levels were decreased by the anti-e in a dose-dependent manner and the logic of anti-e was estimated to be . (pg/ml) (fig. a) . we next compared the results of the neutralization assay using chikvpseudo with the conventional prnt for knih/ / to validate our results. the neutralizing activity of the same anti-e antibody was tested with prnt for infection with knih/ / and each calculated value was obtained from independent experiments. pbnas using anti-e and chikvpseudo elicited an average logic value of . ± . pg/ml while a prnt with knih/ / yielded a logic of . ± . pg/ml. we then used the estimated values of logic from those independent experiments to determine assay precision by calculating the coefficient of variation (cv) for each assay. neutralization assays with chikvpseudo showed a cv value of . %, which is similar to the prnt cv of . %, indicating that pbna is a sufficiently precise neutralization assay. when we used serial three-fold dilutions of the anti-e antibody, starting from the concentrations of μg/ml in our pbna, the linear regression analyses of the neutralizing activity showed linearity in the range of % to % infectivity reduction (fig. ) . pbnas with chikvpseudo yielded an average regression coefficient (r ) value of . from independent pbnas (fig. a) . similarly, conventional prnts yielded an average r value of . (fig. b) . these results thus indicate that for both the neutralization assay with chikvpseudo and the prnt, there is a significant correlation between the level of anti-e and the inhibition of chikvpseudo infectivity. to evaluate the specificity of pbnas for anti-e targeting of chikv, we compared the neutralizing ability of anti-e and an antibody against the spike protein of mers-cov (antispike), an unrelated protein. serial three-fold dilutions of the antibody solution, starting from the concentrations of μg/ml were incubated with gfp unit of chikvpseudo per dilution and added to the cells for infection. results from these infections indicated that the anti-e antibody inhibits chikvpseudo in a dose-dependent manner. however, the anti-spike antibody did not show dose-dependent inhibition (fig. ) , indicating that chikvpseudo infection was specifically inhibited by the antibody against the chikv protein e . as previously reported, pseudotyped virus can be applied in detection of neutralizing antibodies in infected patients (hu et al., ; noranate et al., ) . for this purpose, we tested the neutralization of chikvpseudo with serum samples from chikv-positive individuals. these sera were only positive for chikv, but negative for all other mosquitoborne viruses including dengue virus (denv), zika virus, yellow fever virus, west nile virus, and japanese encephalitis virus, as confirmed in elisa (data not shown). serum samples that were negative for all abovementioned mosquitoborne viruses were also used as negative controls (chikvnegative). four out of five serum samples from chikvpositive individuals neutralized chikvpseudo in a dilutiondependent manner to various degrees, while none of serum samples from chikv-negative controls did (fig. ) . this result indicates that chikvpseudo can be applied to measure the neutralizing activity of clinical samples. chikv, a member of the alphavirus genus of the togaviridae family, is a tropical disease-causing virus that is transmitted by mosquitoes. global warming and frequent international travel have increased the risk of mosquito-borne diseases globally, and chikv is no exception. over millions of chikv infection cases have been reported in more than countries, suggesting that chikv has re-emerged as a global pathogen (morrison, ) . there are three strains of chikv based on their partial e sequences: east/central/south african (ecsa), west african, and asian (powers et al., ; rezza et al., ; volk et al., ) . each strain has been associated with regional epidemics, but the ecsa strain caused an extensive nationwide outbreak in ectopic areas such as malaysia in (sam et al., ; morrison, ) . recently in south korea, the cases of various mosquito-borne viral infections have increased, and chikv infections have been detected in % of patients with dengue-like symptoms, suggesting that surveillance and prevention programs for chikv are required (cha et al., ; hwang and lee, ; yeom, ) . in this study, we identified the whole sequence of the structural polyprotein of a chikv isolated in korea, knih/ / , using reverse transcription. in addition, similar to a previous report analyzing the e sequence, we submitted our structural polyprotein sequence to an ncbi-blast search and phylogenetic analysis and confirmed that the knih/ / isolate is clustered within ecsa genotype of chikv (cha et al., ) . to evaluate the titer of neutralizing antibodies in a tested serum, the prnt has been conventionally used in laboratories. although it is considered a gold standard for neutralization assays, the use of hazardous live chikv and the requirement of a bsl- facility have impeded research for treatment of diseases caused by this virus. in addition, it takes a relatively long time (about - days) to visualize and count the plaques for the prnt. to overcome these limitations, alternative neutralization assays using non-infectious virus replicon particles (vrps) or pseudotyped viruses have been developed (gläsker et al., ; kishishita et al., ; hu et al., ; lee et al., ) . here, we adopted the strategy of expressing the entire structural polyprotein of chikv to generate the pseudotyped virus form of knih/ / (chikvpseudo) for the enhanced infectivity compared to a virion expressing either an individual e or e protein (hu et al., ) . in addition to increased safety, the pbna using chikvpseudo takes only days to evaluate the neutralizing activity of a tested antibody and makes it easy to quantify the infectivity level using the luciferase activity of infected cells. furthermore, chikvpseudo has additional advantages when compared to other pseudotyped virus systems. first, chikvpseudo expresses a flag-tagged capsid protein that can be easily detected by a commercially available antibody against flag, so that one does not need to generate or use a specific antibody against chikv proteins to check expression. second, we used a dual reporter gene construct that expresses both gfp and luciferase in an infected cell. many studies using pseudotyped viruses or vlps often titrate viral infectivity using time-and labor-intensive methods, like measuring the viral genome copy number or virion protein concentration, as is the case for the hiv- protein p (salvador et al., ; gläsker et al., ; kishishita et al., ; wichit et al., ) . compared to these cases, chikvpseudo can be more easily titrated by measuring the gfp signal or luciferase activity. since gfp is expressed only in infected cells, gfp-positive foci can serve as infectious centers similar to the plaques formed by infectious chikv. in addition, we validated the pbna using chikvpseudo in terms of precision, linearity, and specifi-city by comparison with the conventional prnt. the calculated ic and validation values were comparable in both assays despite differences in cell types and incubation times. these results indicate that pbna with chikvpseudo can be successfully employed in a rapid neutralization assay with improved safety compared to prnts. furthermore, the pbna results with clinical samples from chikv-infected patients indicate that our chikvpseudo system can be applied to evaluate the neutralizing activities of human sera. in agreement of our results, a recent study reported an application of pseudotyped chikv to assess chikv dna-vaccine candidates in their ability to raise neutralizing antibodies (wu et al., ) . likewise, chikvpseudo can be used for assessment of vaccine efficacy. taken together, we have identified the whole structural polyprotein sequence of a korean isolated chikv and its phylogenetic position. using this sequence, we constructed chikvpseudo containing the entire structural polyprotein and developed a pseudovirus-based neutralization assay for chikv with improved safety and speed. validation reports of our pbna also support its reliability as a neutralization test compared to conventional prnts. clinical samples can be also used for our pbna to measure the neutralizing activity against chikv. these results indicate a potential use of chikvpseudo to detect neutralizing antibodies for vaccine efficacy tests as well as during clinical diagnosis. neutralization assay for chikungunya virus infection: plaque reduction neutralization test production and characterization of mouse monoclonal antibodies reactive to chikungunya envelope e glycoprotein role of glycoprotein pe in formation and maturation of the sindbis virus spike travel-associated chikungunya cases in south development of pseudovirus-based neutralization assay for chikungunya virus korea during monitoring and control of aedes albopictus, a vector of zika virus, near residences of imported zika virus patients during in south korea cost-effective gene transfection by dna compaction at ph . using acidified, long shelf-life polyethylenimine virus replicon particle based chikungunya virus neutralization assay using gaussia luciferase as readout chikungunya virus glycoproteins pseudotype with lentiviral vectors and reveal a broad spectrum of cellular tropism the first imported case infected with chikungunya virus in korea chikungunya: a review a structural and functional perspective of alphavirus replication and assembly development of a pseudotyped lentiviral vector-based neutralization assay for chikungunya virus infection cell-based analysis of chikungunya virus e protein in membrane fusion comparison of jev neutralization assay using pseudotyped jev with the conventional plaquereduction neutralization test structural changes of envelope proteins during alphavirus fusion re-emergence of chikungunya virus involvement of the molecular chaperone bip in maturation of sindbis virus envelope glycoproteins development of a highly protective combination monoclonal antibody therapy against chikungunya virus reemergence of chikungunya and o'nyong-nyong viruses: evidence for distinct geographical lineages and distant evolutionary relationships infection with chikungunya virus in italy: an outbreak in a temperate region characterization of chikungunya pseudotyped viruses: identification of refractory cell lines and demonstration of cellular tropism differences mediated by mutations in e glycoprotein genotypic and phenotypic characterization of chikungunya virus of different genotypes from malaysia biology and pathogenesis of chikungunya virus the alphavirus e glycoprotein functions in a clade-specific manner replication cycle of chikungunya: a re-emerging arbovirus mega : molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods chikungunya: vaccines and therapeutics. f res chikungunya: a potentially emerging epidemic? genome-scale phylogenetic analyses of chikungunya virus reveal independent emergences of recent epidemics and various evolutionary rates glycoprotein organization of chikungunya virus particles revealed by x-ray crystallography chikungunya virus and prospects for a vaccine imipramine inhibits chikungunya virus replication in human skin fibroblasts through interference with intracellular cholesterol trafficking development and application of a bioluminescent imaging mouse model for chikungunya virus based on pseudovirus system structural studies of chikungunya virus maturation current status and outlook of mosquito-borne diseases in this research was supported by a grant ( mfds ) from the ministry of food and drug safety in - , the national research foundation of korea (nrf) funded by the ministry of education ( r a b ), and a grant from korea ministry of health and welfare (hi c ). the chikv strain isolated in korea (knih/ / ) was obtained from the national culture collection for pathogens, republic of korea. the luc p-plvx-ires-zsgreen plasmid for chikvpseudo was a kind gift from dr. nam-hyuk cho at seoul national university, republic of korea. human serum samples used in this paper were originally collected as part of the dengue vaccine initiative (dvi) study conducted in ouagadougou, burkina faso, by centre muraz and agir. key: cord- - l rsa authors: choi, juwhan; oh, jee youn; lee, young seok; hur, gyu young; lee, sung yong; shim, jae jeong; kang, kyung ho; min, kyung hoon title: the association between blood eosinophil percent and bacterial infection in acute exacerbation of chronic obstructive pulmonary disease date: - - journal: int j chron obstruct pulmon dis doi: . /copd.s sha: doc_id: cord_uid: l rsa introduction: the use of antibiotics is based on the clinician’s experience and judgment, and antibiotics may often be overused in the treatment of acute exacerbations of chronic obstructive pulmonary disease (aecopd). eosinophils have been studied as biomarkers of bacterial infection and prognostic factors in chronic obstructive pulmonary disease and aecopd. thus, the purpose of this study was to determine whether eosinophils could be used to determine bacterial infection in aecopd events. methods: we retrospectively analyzed the medical records of patients admitted to korea university guro hospital for aecopd between january and may . data pertaining to baseline characteristics, results of previous pulmonary function tests, treatment information during the admission period, and history of pulmonary treatment were collected before admission. results: a total of aecopd events were eligible for inclusion and were divided into two groups based on the eosinophil count: those involving eosinophil counts of less than % ( events) and those involving counts of % or more ( events). in univariate analysis, the only bacterial pathogen identification events and bacterial-viral pathogen co-identification events were significantly more frequent in the group with eosinophil counts of less than % (p= . and p= . , respectively). in logistic regression analysis, the rates of only bacterial pathogen identification [odds ratios = . ; % confidence interval, . – . ; p= . ] and bacterial-viral pathogen co-identification [odds ratios= . ; % confidence interval, . – . ; p= . ] were higher in the group with eosinophil count less than %. conclusion: in conclusion, eosinophil counts of less than % are potential indicators of a bacterial infection in aecopd events. eosinophils could thus serve as a reference for the use of antibiotics in aecopd treatment. chronic obstructive pulmonary disease (copd) is an airway and lung disease that impairs the immune lung defense system making it susceptible to bacterial infections. , copd patients may experience acute exacerbations due to these bacterial infections. the global initiatives for chronic obstructive lung disease (gold) guideline recommend the use of antibiotics when a bacterial infection is suspected in events of acute exacerbations of chronic obstructive pulmonary disease (aecopd). the gold guideline also suggests that symptoms such as increase in dyspnea, sputum volume, and sputum purulence are the criteria for antibiotic usage. since these symptoms are not presented as absolute numerical values, the use of antibiotics is based on clinical experience and judgment. hence, antibiotics are often overused and stray from the gold guidelines, as reported by a study conducted in europe. to solve this problem, various biomarkers that distinguish bacterial infections have been studied, which include c-reactive protein (crp) and procalcitonin. however, crp commonly elevates in viral infections and hence cannot specifically distinguish bacterial infections. , procalcitonin is useful for distinguishing bacterial infections but is expensive and less accessible. therefore, we need a biomarker that can specifically distinguish bacterial infections, while being cost-effective and user-friendly. it is known that eosinophils are lowered in pneumonia and other infectious diseases caused by bacteria. [ ] [ ] [ ] eosinophils are a simple test that can be easily measured and are inexpensive to use in the clinical field. thus, the purpose of this study was to determine whether eosinophils could be used to determine bacterial infection in aecopd events. we retrospectively reviewed and analyzed the medical records of aecopd events in patients admitted in the korea university guro hospital from january to may . we searched our electronic medical records database using keywords such as "copd" and "acute exacerbation". this study was approved by our institutional review board (kugh - ). this study was a retrospective study, so patient consent was not necessary, and we maintained patient confidentiality with the declaration of helsinki. copd was diagnosed based on the gold guidelines, where a ratio of forced expiratory volume in the first second (fev ) to forced vital capacity was less than % in post-bronchodilator spirometry prior to admission, and aecopd was defined as "an acute event characterized by a worsening of the patient's respiratory symptoms that is beyond normal day-to-day variation and leads to a change in medication". , all the patients were older than years. patients were excluded ) if the cause of admission was not aecopd, for example, acute heart failure, acute pulmonary edema, acute pulmonary embolism, pneumothorax, or arrhythmia; or ) if they had a co-morbidity that could affect the eosinophilic count, for example, cancer, allergic disorder, autoimmune disease, or hematologic disease; or ) if they had no clinical data, such as pulmonary function test (pft), laboratory test, and culture test results. medical records were reviewed and analyzed for the following data: age, gender, smoking history, comorbidities, treatment information during admission period, laboratory test, culture test, polymerase-chain-reaction (pcr) assay, previous pft, and pulmonary-related treatment before admission. laboratory test, culture test, and pcr assay were conducted within hrs after admission. blood, sputum, and urine culture were conducted for identification of the bacterial infection. sputum pcr assay was conducted for identification of viral infection. sputum pcr assays were performed using nasopharyngeal aspiration by a trained doctor. and sputum pcr assay can detect influenza virus, respiratory syncytial virus, parainfluenza virus, coronavirus, rhinovirus, enterovirus, adenovirus, bocavirus, and metapneumovirus. we defined "maintenance oral steroid" as using steroid for more than three weeks and "short-term oral steroid use" as using steroid for three weeks or less. data were analyzed by spss software (spss for windows, spss inc., chicago, il, usa). data were reported as mean ± standard deviation or number and percent of each group. we divided the two groups with respect to eosinophil count of %. continuous variables were compared using the mann-whitney test or independent t-test, while categorical variables were compared using the chi-square test or fisher`s exact test. variables with p-value < . on univariate analysis were tested in logistic regression analysis. when analyzing logistic regression analysis, white blood cell (wbc) and crp were converted into binary variables based on the optimal cut-off values, and these optimal cutoff values were calculated using receiver operating characteristic. the cut-off values of wbc and crp were , . cells/mm and . mg/l, respectively. the logistic regression analysis was assessed by the hosmer-lemeshow test, with p-value < . considered statistically significant. multivariate analysis was performed in three different models. model- analyzed only bacterial pathogen identification, bacterial-viral pathogen co-identification, and laboratory findings (wbc, crp). model- analyzed all other factors that were statistically significant in the univariate analysis except for the nopathogen identification factor. model- analyzed the age and gender along with the factors of model- . with respect to the exclusion criteria, only aecopd events were eligible. out of these, events with eosinophil counts less than % and events with eosinophil counts of % or more were analyzed. table shows baseline characteristics of the total events and the two groups. there were statistically significant differences between the two groups: length of hospital stay (p< . ), experience of intensive unit care (p= . ), wbc (p< . ), eosinophil percent (p< . ), eosinophil count (p< . ), crp (p< . ), and fev (liters) (p= . ). there were no statistically significant differences in the other factors (p-value for all other variables > . ) ( table ) . we analyzed any past treatment associated with pulmonary conditions before admission. there was a statistically significant difference between the two groups for maintenance oral steroid (p= . ), but no statistically significant differences in the other factors were noted (p-value for all other variables > . ) ( table ) . we classified all events into four groups according to bacteria or virus identification: only bacterial pathogen identification, only viral pathogen identification, bacterial-viral pathogen co-identification, and no-pathogen identification. the only bacterial pathogen identification events and bacterial-viral pathogen co-identification events were significantly more frequent in the group with eosinophil counts of less than % (p= . and p= . , respectively). the no-pathogen identification events were significantly more frequent in the group with eosinophil counts of % or more (p< . ). the frequency of only viral pathogen identification events did not show any statistically significant difference between the two groups (p= . ; table ). in all multivariate models, the only bacterial pathogen identification events and bacterial-viral co-identification events showed statistically significant differences between the two groups (table ). table shows odds ratio, % confidence interval, and p-value by logistic regression analysis. this study was the first to analyze whether bacterial infections can be differentiated based on the eosinophil percent of % in aecopd patients in korea. our results showed that only bacterial pathogen identification events and bacterial-viral pathogen coidentification events are significantly more frequent in groups with eosinophil counts of less than %. since some studies used eosinophil percent of % as a cut-off value to predict the prognosis and treatment response in copd, we also used the same cut-off value. for example, some studies analyzed the prognosis of steroid inhaler response and treatment in copd based on the % value, , while some analyzed the pneumonia risk and stability in stable copd based on the % value. , eosinophils, which make up - % of the total wbcs, have several receptors on their cell surface and secrete various cytokines and mediators. research in eosinophils was initially focused on helminth infection and allergic disorders. as research progressed, it was found that eosinophils performed various functions in the human body such as regulation of innate and adaptive immunity and responses to infection and inflammation. , since animal studies showed that eosinophil count decreased with acute bacterial infection, eosinophils have been studied as a marker of bacterial infections. , the reason for the decrease in peripheral eosinophils count in acute bacterial infection was the accumulation of eosinophils at the inflammatory site and inhibition of egress from the bone marrow. additionally, bacterial infection in lungs affects the cytokine and chemokine release from the airway smooth muscle cells (asmc). bacterial infections activate human asmc to release c-x-c motif chemokine (cxcl)- that increases neutrophil recruitment. on the other hand, bacterial infections inhibit the release of eotaxin- that is proven to induce blood eosinophilia in studies on mice. many studies have been conducted on the relationship between eosinophil and copd and aecopd. in copd analysis, it was thought that sputum eosinophilia could be indirectly predicted by the peripheral blood eosinophils, and blood or sputum eosinophil count were inversely related to the bacterial load. in aecopd analysis, eosinopenia is predicted to have a poor prognosis. eosinopenia group based on eosinophil count of /mm is associated with long hospital day and high mortality. our results suggested that the eosinopenia group showed an increase in the mortality rate and duration of hospitalization because the rate of bacterial infection was higher than the eosinophilia group. studies on the relationship between eosinophils and viral infection have shown heterogeneous results. there have been studies of the relationship between viral infections and eosinophil in children and infants, but not with copd. although these studies were not conducted for copd patients, it was reported that respiratory viral infections showed various cytokines and eosinophil activation depending on the type of virus. , furthermore, in the murine asthma model study, airway eosinophil infiltration increased following a rhinovirus infection unlike that in the control mice. in conclusion, eosinophil is expected to show various patterns depending on the virus type and airway reactivity. however, additional studies are needed because of the lack of studies in copd patients. biomarkers should be such that they are quick and easy to perform, and cost-effective despite medical and economic levels. however, crp and procalcitonin are difficult to perform in all hospitals, including primary care hospitals. on the other hand, eosinophil test, belonging to complete blood count test, is easily accessible anywhere and is inexpensive. a disadvantage of using eosinophil as a biomarker is that there are many factors that affect eosinophils, such as hematologic disorders, cancer, allergy diseases, medications, and steroids. in this study, patients with diseases that could affect eosinophil were initially excluded and the use of inhaled corticosteroids or oral steroid was analyzed using univariate and multivariate analysis. in pulmonary-related treatment before admission, the use rate of β oral agonist was high. korean copd patients prefer oral medicines over inhalers. also, previous epidemiological studies on the use of medications in copd patients in korea show a low use rate of inhalers. because there was no difference in the use rate of β oral agonist between the two groups, we think that it will not affect the results of the study. this study had several limitations. first, it had a retrospective design and was a single center study. we wanted to analyze other biomarkers like procalcitonin, but only half the patients were tested for procalcitonin and hence, could not evaluate the procalcitonin together. second, colonization and contamination could not be distinguished in the analysis of isolation. to compensate for this as much as possible, we also analyzed the condition bronchiectasis that was known to be highly colonized, and the difference between the two groups was not apparent. additionally, culture test and pcr assay were performed by trained doctors. third, we could not analyze the effect of the steroid dose and type. in the inhaled corticosteroids group, different doses and different kinds of inhalers were used, and the dose varied between and mg in the oral steroid use group. fourth, eosinophil may be affected by various conditions and diseases. although eosinophil has the advantage of being cheap and easy to use in practice, it is dangerous to decide alone by eosinophil percent whether to use antibiotics in aecopd. it would be useful to use it as a reference. although this study was a retrospective and singlecenter study, we analyzed various factors in a largescale group, and multiple factors that affected eosinophil count were analyzed. however, an additional large-scale multicenter, randomized control study will be required to demonstrate our results better. eosinophil count of % may be an indicator to distinguish various bacterial infections in aecopd patients. furthermore, by predicting the presence or absence of bacterial infection, a more reliable antibiotic treatment can be determined. copd, chronic obstructive pulmonary disease; aecopd, acute exacerbation of chronic obstructive pulmonary disease. the international journal of copd is an international, peer-reviewed journal of therapeutics and pharmacology focusing on concise rapid reporting of clinical studies and reviews in copd. special focus is given to the pathophysiological processes underlying the disease, intervention programs, patient focused education, and self management protocols. this journal is indexed on pubmed central, medline and cas. the manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. visit http://www.dovepress.com/testimonials.php to read real quotes from published authors. submit your manuscript here: https://www.dovepress.com/international-journal-of-chronic-obstructive-pulmonary-disease-journal defective phagocytosis in airways disease oxidative stress-mediated inkt-cell activation is involved in copd pathogenesis copd exacerbations: causes, prevention, and treatment global strategy for the diagnosis, management and prevention of chronic obstructive lung disease report: gold executive summary antibiotic therapy in exacerbations of chronic obstructive pulmonary disease antibiotic prescription for copd exacerbations admitted to hospital: european copd audit c-reactive protein level and microbial aetiology in patients hospitalised with acute exacerbation of copd c-reactive protein levels predict bacterial exacerbation in patients with chronic obstructive pulmonary disease effect of procalcitonin-guided treatment on antibiotic use and outcome in lower respiratory tract infections: cluster-randomised, single-blinded intervention trial blood and 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eosinophil leukocytes in acute inflammation. ii. eosinophil dynamics during acute inflammation differential regulation of ccl- /eotaxin- and cxcl- /il- by gram-positive and gram-negative bacteria in human airway smooth muscle cells neutrophil activation induced by artinm: release of inflammatory mediators and enhancement of effector functions relationship between interleukin- and eotaxin in regulating blood and tissue eosinophilia in mice peripheral blood eosinophils: a surrogate marker for airway eosinophilia in stable copd eosinopenia as a marker of mortality and length of stay in patients admitted with exacerbations of chronic obstructive pulmonary disease different cytokine profile and eosinophil activation are involved in rhinovirus-and rs virus-induced acute exacerbation of childhood wheezing a comparison of cytokine responses in respiratory syncytial virus and influenza a infections in infants the effect of rhinovirus on airway inflammation in a murine asthma model possible health effects of noise induced cortisol increase this study was supported by a korea university guro hospital grant (o ). all authors contributed to data analysis, drafting and revising the article, gave final approval of the version to be published, and agree to be accountable for all aspects of the work. the authors report no conflicts of interest in this work. key: cord- -pgxxkfc authors: wang, cong; hua, chen; xia, shuai; li, weihua; lu, lu; jiang, shibo title: combining a fusion inhibitory peptide targeting the mers-cov s protein hr domain and a neutralizing antibody specific for the s protein receptor-binding domain (rbd) showed potent synergism against pseudotyped mers-cov with or without mutations in rbd date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: pgxxkfc middle east respiratory syndrome coronavirus (mers-cov) has continuously posed a threat to public health worldwide, yet no therapeutics or vaccines are currently available to prevent or treat mers-cov infection. we previously identified a fusion inhibitory peptide (hr p-m ) targeting the mers-cov s protein hr domain and a highly potent neutralizing monoclonal antibody (m ) specific to the s spike protein receptor-binding domain (rbd). however, m was found to have reduced efficacy against mers-cov strains with mutations in rbd, and hr p-m showed low potency, thus limiting the clinical application of each when administered separately. however, we herein report that the combination of m and hr p-m exhibited potent synergism in inhibiting mers-cov s protein-mediated cell–cell fusion and infection by mers-cov pseudoviruses with or without mutations in the rbd, resulting in the enhancement of antiviral activity in contrast to either one administered alone. thus, this combinatorial strategy could be used in clinics for the urgent treatment of mers-cov-infected patients. middle east respiratory syndrome (mers) coronavirus (mers-cov), a lineage c beta-coronavirus, was reported to cause severe respiratory tract infection [ , ] . to date, laboratory-confirmed cases of infection with mers-cov, including mers-cov associated deaths, have been reported to the world health organization (who) from countries. currently, no effective therapeutics or vaccines are available to treat or prevent mers-cov infection. the spike (s) protein of mers-cov plays important roles in virus attachment, fusion, and entry into the target cell [ ] [ ] [ ] . similar to other coronaviruses, the s protein of mers-cov consists of s and s subunits. the s subunit is responsible for the binding of the virion by its receptor binding domain (rbd) to the cellular receptor, dipeptidyl peptidase- (dpp ), while the s subunit mediates the fusion the t cell line was obtained from atcc (manassas, va, usa), and the huh- cell line was from the cell bank of the chinese academy of sciences (shanghai, china). these two cell lines were propagated in dulbecco's modified eagle's medium (dmem) supplemented with % fetal bovine serum (fbs). peptide hr p-m was synthesized by solid phase peptide synthesis at syn inc. (shanghai, china), and human mab m was provided by prof. tianlei ying at fudan university, shanghai, china. recombinant plasmids encoding the mers-cov s protein with d g, d g, q h, or i t mutations were kindly provided by dr. lanying du at the new york blood center, ny, usa. mers-cov pseudoviruses were constructed as described previously [ , ] . briefly, t cells were plated in a t tissue culture flask and incubated at • c for h. cells were cotransfected with plasmids pnl - .luc.re encoding env-defective, luciferase-expressing hiv- and pcdna . -mers-cov-s encoding s protein with or without mutation in rbd at mass ratio of : using vigofect (vigorous biotechnology, beijing, china), according to the manufacturer's recommendation. the supernatant was replaced with fresh dmem at - h post-transfection and harvested after incubation for an additional h. in order to remove cell debris, the supernatant was centrifuged at viruses , , of rpm for min, followed by filtration through a . µm filter. mers-cov pseudovirus in the supernatant was quantified by testing p content in the product of mers-cov pseudovirus. a mers-cov pseudovirus inhibition assay was performed as previously described [ , , ] . briefly, huh- cells were seeded ( cells/well) into a -well plate and incubated overnight at • c. mers-cov pseudovirus was incubated with a serially diluted inhibitor for min at • c, followed by the addition of huh- cells. the cells were incubated with or without pseudovirus as virus control and cell control, respectively. the culture was replaced with fresh medium h post-infection and incubated for an additional h. cells were lysed using lysis reagent (promega, madison, wi, usa), and cell lysates were transferred to a -well costar flat-bottom luminometer plate (corning costar, new york, ny, usa), followed by the addition of luciferase substrate (promega) to measure luminescence using an infinite m pro (tecan, grödig, austria). mers-cov s protein-mediated cell-cell fusion was performed as previously described [ ] . briefly, plasmid paav-ires-mers-egfp encoding the mers-cov s protein was transfected into t cells ( t/mers/egfp) using the transfection reagent, vigofect (vigorous biotechnology, beijing, china). the target huh- cells expressing dpp were incubated at × cells/well in wells of a -well plate for h. the effector t/mers/egfp cells that express mers-cov s protein and egfp or the control t/egfp cells that express egfp only were preincubated at cells/well with an inhibitor at the indicated concentration or phosphate buffered saline (pbs) as control at • c for min. the mixture of t/mers/egfp cells and an inhibitor or pbs were added to huh- cells in the wells, followed by a co-culture at • c for h. the t/mers/egfp cells fused or unfused with huh- cells were fixed with % pfa and counted under an inverted fluorescence microscope (nikon, tokyo, japan). the fused cell showed much larger size and weaker fluorescence intensity than the unfused cell because of the diffusion of egfp from one cell to more cells (figure ). almost no fused cells could be observed in the groups of negative control (pbs+ t/egfp+huh- ) or peptide treatment (hr p-m + t/mers/egfp+huh- ) (figure ). the concentration for % inhibition (ic ) was calculated using calcusyn software kindly provided by dr. t.c. chou [ ] . viruses , , x for peer review of was centrifuged at rpm for min, followed by filtration through a . µm filter. mers-cov pseudovirus in the supernatant was quantified by testing p content in the product of mers-cov pseudovirus. a mers-cov pseudovirus inhibition assay was performed as previously described [ , , ] . briefly, huh- cells were seeded ( cells/well) into a -well plate and incubated overnight at °c. mers-cov pseudovirus was incubated with a serially diluted inhibitor for min at °c, followed by the addition of huh- cells. the cells were incubated with or without pseudovirus as virus control and cell control, respectively. the culture was replaced with fresh medium h post-infection and incubated for an additional h. cells were lysed using lysis reagent (promega, madison, wi, usa), and cell lysates were transferred to a -well costar flat-bottom luminometer plate (corning costar, new york, ny, usa), followed by the addition of luciferase substrate (promega) to measure luminescence using an infinite m pro (tecan, grödig, austria). mers-cov s protein-mediated cell-cell fusion was performed as previously described [ ] . briefly, plasmid paav-ires-mers-egfp encoding the mers-cov s protein was transfected into t cells ( t/mers/egfp) using the transfection reagent, vigofect (vigorous biotechnology, beijing, china). the target huh- cells expressing dpp were incubated at × cells/well in wells of a -well plate for h. the effector t/mers/egfp cells that express mers-cov s protein and egfp or the control t/egfp cells that express egfp only were preincubated at cells/well with an inhibitor at the indicated concentration or phosphate buffered saline (pbs) as control at °c for min. the mixture of t/mers/egfp cells and an inhibitor or pbs were added to huh- cells in the wells, followed by a co-culture at °c for h. the t/mers/egfp cells fused or unfused with huh- cells were fixed with % pfa and counted under an inverted fluorescence microscope (nikon, tokyo, japan). the fused cell showed much larger size and weaker fluorescence intensity than the unfused cell because of the diffusion of egfp from one cell to more cells ( figure ). almost no fused cells could be observed in the groups of negative control (pbs+ t/egfp+huh- ) or peptide treatment (hr p-m + t/mers/egfp+huh- ) (figure ). the concentration for % inhibition (ic ) was calculated using calcusyn software kindly provided by dr. t.c. chou [ ] . six-week-old female specific-pathogen-free (spf) balb/c mice (bodyweight about g) were divided into groups of mice each. mice in group , , and were intraperitoneally (i.p.) injected with m ( . mg in µl pbs) alone, hr p-m ( mg in µl pbs) alone, and the combination of m ( . mg in µl pbs) and hr p-m ( mg in µl pbs), respectively. mice were sedated with nembutal ( mg/kg body weight) before and h after injection of the inhibitors, respectively, and bled retro-orbitally. the blood was centrifuged at rpm for min after standing at room temperature for h. the sera were collected and heat-inactivated at • c for min. inhibitory activity of the inhibitors on mers-cov pseudovirus was evaluated in serum as described above. to assess the potential synergistic effect, hr p-m and m were mixed at the indicated molar concentration ratio, while hr p-m alone and m alone were included as controls. the mixtures were serially diluted and tested for their inhibitory activity on mers-cov pseudovirus infection as described above. each sample was tested in triplicate, and data were analyzed for synergistic effect by calculating the combination index (ci), using the calcusyn program. ci values of < and > indicate synergy and antagonism, respectively, and synergy was divided into different strengths, according to ci values, as follows: < . indicates very strong synergism; . - . indicates strong synergism; . - . indicates synergism; . - . indicates moderate synergism; and . - . indicates slight synergism [ , ] . fold of potency enhancement was calculated with the ratio of concentrations of inhibitor testing alone and in combination. to determine the significance of difference in sensitivity between wild-type and mutant viruses to inhibitors and the inhibitory activity detected in sera from balb/c mice treated with inhibitors alone or combination, statistical analyses were performed using a two-tailed unpaired student's t-test, using graphpad prism, version . . values with p < . and p < . were considered statistically significant and very significant, respectively. we first investigated the potential cooperative effects of combining hr p-m with m on mers-cov pseudovirus infection. in our preliminary study, we found that ic values of hr p-m and m for inhibiting mers-cov pseudovirus infection were about nm and . nm, respectively. therefore, we tested the inhibitory activity of hr p-m alone, m alone, and hr p-m /m in combination at a molar concentration ratio of , : , respectively. as shown in figure and table , combining hr p-m and m resulted in strong synergistic inhibitory activity against mers-cov pseudovirus infection with ci values of . - . for - % inhibition, including potency enhancement of . -to . -fold for m and . -to . -fold for hr p-m . this result suggested that the mers-cov fusion inhibitory peptide hr p-m and the mers-cov neutralizing mab m could be used in combination to enhance anti-mers-cov activity. note: each sample was tested in triplicate, and the mean values are presented. ratio of molar concentration of hr p-m and m in combination is , : . next, we tested the potential synergistic activity of the hr p-m /m combination on mers-cov s protein-mediated cell-cell fusion. we adjusted the molar concentration ratio of hr p-m and m in the combination to : , since the ic values of hr p-m and m for inhibiting mers-cov s protein-mediated cell-cell fusion in our preliminary studies were about nm and . nm, respectively. as shown in figure and table , the combination also exhibited strong synergism against mers-cov s protein-mediated cell-cell fusion (ci = . ) with enhancement of -fold for m and -fold for hr p-m . this result confirms that combining hr p-m , a mers-cov fusion inhibitor, with m , a human neutralizing mab, results in strong synergism on s protein-mediated membrane fusion because they target the different stages of mers-cov fusion and entry processes. the blue curves represent inhibitors used alone, and the red curves represent each inhibitor used in combination. the width between two curves represents the fold of enhancement between an inhibitor used alone and in combination. note: each sample was tested in triplicate, and the mean values are presented. ratio of molar concentration of hr p-m and m in combination is , : . next, we tested the potential synergistic activity of the hr p-m /m combination on mers-cov s protein-mediated cell-cell fusion. we adjusted the molar concentration ratio of hr p-m and m in the combination to : , since the ic values of hr p-m and m for inhibiting mers-cov s protein-mediated cell-cell fusion in our preliminary studies were about nm and . nm, respectively. as shown in figure and table , the combination also exhibited strong synergism against mers-cov s protein-mediated cell-cell fusion (ci = . ) with enhancement of -fold for m and -fold for hr p-m . this result confirms that combining hr p-m , a mers-cov fusion inhibitor, with m , a human neutralizing mab, results in strong synergism on s protein-mediated membrane fusion because they target the different stages of mers-cov fusion and entry processes. note: each sample was tested in triplicate, and the mean values are presented. the molar concentration ratio of hr p-m and m in combination is : . du et al. have previously shown that mers-cov pseudoviruses with mutations in rbd, such as d g and d g detected in some mers-cov strains isolated from different regions and at different times [ , ] , are resistant to the neutralizing activity of an rbd-specific mouse mab mersmab [ ] . in the present study, the sensitivity of pseudotyped mers-cov strains with key mutations in rbd, as identified in some mers-cov mutants isolated during the - outbreaks [ ] , including d g, d g, q h, and i t, along with wild-type mers-cov, was compared between the inhibitory activity of hr p-m peptide alone and m neutralizing mab alone. as shown in table , the resistance of mers-cov mutants to the neutralizing activity of m is about -to -fold, whereas the pseudoviruses with or without mutations were equally sensitive to fusion inhibitory activity of hr p-m . this result suggested that use of mab m alone is unable to control the infection by mers-cov strains with mutations in rbd. table . sensitivity of mers-cov pseudoviruses with or without mutations in the receptor-binding domain (rbd) to the inhibitory activity of m (nm) and hr p-m (µm) separately. [ , ] , are resistant to the neutralizing activity of an rbd-specific mouse mab mersmab [ ] . in the present study, the sensitivity of pseudotyped mers-cov strains with key mutations in rbd, as identified in some mers-cov mutants isolated during the - outbreaks [ ] , including d g, d g, q h, and i t, along with wild-type mers-cov, was compared between the inhibitory activity of hr p-m peptide alone and m neutralizing mab alone. as shown in table , the resistance of mers-cov mutants to the neutralizing activity of m is about -to -fold, whereas the pseudoviruses with or without mutations were equally sensitive to fusion inhibitory activity of hr p-m . this result suggested that use of mab m alone is unable to control the infection by mers-cov strains with mutations in rbd. table . sensitivity of mers-cov pseudoviruses with or without mutations in the receptor-binding domain (rbd) to the inhibitory activity of m (nm) and hr p-m (µm) separately. to determine whether the combination of hr p-m and m also exhibited synergistic antiviral activity against infection of mers-cov strains with mutations in rbd or in the hr domain, we constructed pseudoviruses bearing mers-cov s protein with mutations in rbd, including d g, d g, q h, or i t, and those in the hr domain, including q h and q r [ , ] . we then tested their sensitivity to the inhibition of hr p-m alone, m alone, and the hr p-m /m combination. as shown in table , combining m with hr p-m exhibited strong synergism against infection by pseudotyped mers-cov strains with or without mutations in the rbd or hr domain with ci value less than . and potency enhancement in the range of -to -fold, suggesting that this combinational therapy has potential to be further developed for treatment of patients infected by different mers-cov strains, including those with resistance to rbd-specific neutralizing antibodies. table . combination index and fold of enhancement for inhibiting mers-cov pseudoviruses with or without mutations in rbd in s subunit and hr in s subunit of mers-cov s protein by hr p-m and m . to determine whether the hr p-m /m combination could sustain its efficacy in vivo compared to hr p-m or m alone, we tested the anti-mers-cov pseudovirus activity of the inhibitors in sera of mice treated with i.p. injection of hr p-m , m , and the hr p-m /m combination, respectively. as shown in figure , the inhibitory activity detected in sera from mice treated with hr p-m or m alone was significantly higher than that detected in sera from mice before inhibition of any inhibitor. on the other hand, the anti-mers-cov activity detected in sera from mice treated with the hr p-m /m combination was significantly more potent than that detected in sera of mice administered with hr p-m or m alone. this result confirms that combining hr p-m with m affords synergism against mers-cov s infection, both in vitro and in vivo. note: the molar concentration ratio of hr p-m and m in combination against wildtype virus, viruses with mutations in rbd, and those in hr is , : , : , and , : , respectively. to determine whether the hr p-m /m combination could sustain its efficacy in vivo compared to hr p-m or m alone, we tested the anti-mers-cov pseudovirus activity of the inhibitors in sera of mice treated with i.p. injection of hr p-m , m , and the hr p-m /m combination, respectively. as shown in figure , the inhibitory activity detected in sera from mice treated with hr p-m or m alone was significantly higher than that detected in sera from mice before inhibition of any inhibitor. on the other hand, the anti-mers-cov activity detected in sera from mice treated with the hr p-m /m combination was significantly more potent than that detected in sera of mice administered with hr p-m or m alone. this result confirms that combining hr p-m with m affords synergism against mers-cov s infection, both in vitro and in vivo. the high mortality of mers-cov-infected patients [ ] [ ] [ ] calls for the development of highly effective anti-mers-cov therapeutics. although we and others have previously identified a mers-cov fusion inhibitory peptide (hr p-m ) targeting the mers-cov s protein hr domain and a highly potent human neutralizing mab (m ) targeting the mers-cov s protein rbd data are presented as means ± sd. **, and *** represent p < . , and p < . , respectively. the high mortality of mers-cov-infected patients [ ] [ ] [ ] calls for the development of highly effective anti-mers-cov therapeutics. although we and others have previously identified a mers-cov fusion inhibitory peptide (hr p-m ) targeting the mers-cov s protein hr domain and a highly potent human neutralizing mab (m ) targeting the mers-cov s protein rbd [ , , ] , their further development is limited by low potency in the case of hr p-m and low efficacy to neutralize mers-cov strains with rbd mutations in the case of m [ , [ ] [ ] [ ] . the combinatorial use of drugs with different mechanisms of action, i.e., cocktail regimen, has been widely applied in clinics [ ] . for example, the combinatorial use of hiv reverse transcriptase (rt) inhibitors and protease inhibitors, known as highly active anti-retrovirus therapy (haart), has shown significant synergism in inhibiting hiv- infection, reducing adverse effects and delaying the emergence of drug resistance, thus extending the lifespan of millions of hiv/aids patients [ ] [ ] [ ] . moreover, we previously showed that combining hiv- attachment inhibitors with rt inhibitors, or combining the st, nd, and/or rd generation hiv fusion inhibitors that target different sites in the hiv- gp hr domain, exhibited synergistic and complementary effect against infection by a broad spectrum of hiv- strains, including those resistant to hiv attachment inhibitors, fusion inhibitors, and rt inhibitors [ ] [ ] [ ] . in this study, we compared the anti-mers-cov activity of hr p-m alone and m alone with that of hr p-m /m in combination and found that the inhibitory activity of the hr p-m /m combination was significantly more potent than either one administered alone against mers-cov s protein-mediated cell-cell fusion and mers-cov pseudovirus infection, suggesting synergistic activity based on the dual mechanisms of action whereby hr p-m targets the s subunit hr domain for inhibiting s -mediated virus-cell or cell-cell fusion [ ] and m targets the s subunit rbd for inhibiting virus-cell binding or virus attachment [ ] . it has been well known that drug synergism can be expected when drugs that act by different mechanisms of action are mixed together [ ] . while mers-cov pseudoviruses with mutations in rbd were resistant to the rbd-specific mab m , they were equally sensitive to hr -targeting peptide hr p-m . notably, however, the hr p-m /m combination exhibited strong synergistic antiviral activity against all pseudotyped mers-cov strains, including those with mutations in rbd of s protein, which are even resistant to an rbd-specific mouse mab mersmab [ ] . we also demonstrated that sera from mice treated with the hr p-m /m combination revealed significant efficacy in inhibiting mers-cov pseudovirus infection compared to hr p-m or m alone. collectively, these results suggest that the combinatorial strategy overcomes the weaknesses of hr p-m peptide and m mab, while, at the same time, takes advantage of the unique mechanism of action of each to provide, by the sum of both, much more effective inhibitory activity against mers-cov infection than either peptide or mab used alone. the strong synergy of the combination is expected to reduce the dosage of the individual inhibitor in such combinational therapy, resulting in decreased cost and toxicity, thus making the final product more affordable and safer. therefore, this combinational therapy shows promise for further clinical development. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus 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for hiv infection combination of candidate microbicides cellulose acetate , -benzenedicarboxylate and uc has synergistic and complementary effects against human immunodeficiency virus type infection combinations of the first and next generations of human immunodeficiency virus (hiv) fusion inhibitors exhibit a highly potent synergistic effect against enfuvirtidesensitive and -resistant hiv type strains synergistic efficacy of combination of enfuvirtide and sifuvirtide, the firstand next-generation hiv-fusion inhibitors we thank lanying du at the new york blood center for providing the plasmid encoding mers-cov s protein with mutation in rbd and tianlei ying at fudan university for providing mab m . the authors declare no competing financial interests.viruses , , key: cord- - ie c f authors: heimer, carol a. title: the uses of disorder in negotiated information orders: information leveraging and changing norms in global public health governance date: - - journal: br j sociol doi: . / - . sha: doc_id: cord_uid: ie c f the sars epidemic that broke out in late in china’s guangdong province highlighted the difficulties of reliance on state‐provided information when states have incentives to conceal discrediting information about public health threats. using sars and the international health regulations (ihr) as a starting point, this article examines negotiated information orders in global public health governance and the irregularities in the supply of data that underlie them. negotiated information orders within and among the organizations in a field (here, e.g., the world health organization, member states, government agencies, and international non‐governmental organizations) spell out relationships among different categories of knowledge and non‐knowledge – what is known, acknowledged to be known, and available for use in decision making versus what might be known but cannot be acknowledged or officially used. through information leveraging, technically sufficient information then becomes socially sufficient information. thus it is especially information initially categorized as non‐knowledge – including suppressed data, rumour, unverified evidence, and unofficial information – that creates pressure for the renegotiation of information orders. the argument and evidence of the article also address broader issues about how international law and global norms are realigned, how global norms change, and how social groups manage risk. to mean well-defined ignorance (gross and mcgoey : ) or a type of knowledge about the unknown (gross : ) . to be sure, influential research has considered such important matters as the distinctions between levels and kinds of ignorance, uncertainty, and risk, strategies for handling asymmetric information, and procedures for coping with dishonesty and duplicity (see, e.g., ackerlof ; arrow ; cook ; ericson and doyle ; goffman ; granovetter ; heimer b; knight knight [ ; shapiro ; williamson ) . what is missing, though, is a thorough incorporation of various forms of ignorance, such as non-knowledge, into existing theories of how people, groups, and organizations seek, assign meaning to, and use information (gross and mcgoey ; heimer ; mcgoey ) . researchers need to consider how exactly non-knowledge fits into the negotiated information orders that anchor organizational and interorganizational action. using sars as an example, this article examines negotiated information orders in global public health governance and the irregularities in the supply of the data that underlie them. information may be in short supply because it is suppressed, and it may also be of uncertain quality because it is incomplete or purposefully misleading. in effect, the sars case suggests, whether information is acquired from legitimate sources shapes not only the nature and quality of the information itself but also the uses to which it can be put. in addition to seeking information, then, actors strategically seek information from particular sources and deploy the information they have in hand to pressure others to augment or confirm existing information. through information leveraging, technically sufficient information becomes socially sufficient information. in this way, the article shows what a negotiated information order might look like when we more fully incorporate the social uses of non-knowledge and other forms of ignorance into our analysis. in particular, the article suggests that it is especially information categorized initially as non-knowledge -including suppressed data, rumour, unverified evidence, and unofficial information -that creates pressure for the renegotiation of information orders. although it is a truism that information is needed before rational decisions can be made, the importance of information for organizational decision making is often overestimated. since the pioneering work of herbert simon (march and simon ; simon ) , organization theorists have understood that the model of rational decision making was a poor description of reality and did not capture how information is actually used by organizations. because of limited cognitive and computational capacities, theorists suggest, organizations are only boundedly rational, accepting satisfactory solutions rather than continuing decision-making processes until they find optimal ones. besides using less information than might be expected, organizations also use it on a different timetable and for different purposes. for instance, information intensive solutions often are produced somewhat independently of the problems with which they are eventually matched (feldman ) . employing the metaphor of a garbage can, other scholars suggest that organizational decision making is not linear, but instead depends on how the semi-autonomous streams of choice points, problems, solutions, and participants come together (cohen et al. ; heimer and stinchcombe ) . moreover, information has symbolic as well as instrumental uses, often serving to legitimate decisions even when it plays little role in identifying problems and selecting or crafting solutions (feldman and march ) . decision making is a quintessentially social matter. decisions may depend less on whether decision makers have enough high-quality information than on whether they agree that the available information meets a variety of normatively or even legally established criteria. that is, whether or not information is technically sufficient, it must also be socially sufficient to be usable in decision making (heimer a) . information is technically sufficient if it can be used to answer key questions confronting an organization and if it can be used, perhaps with some modification, in an organization's decision-making algorithms. if decision makers cannot cite data of the sort conventionally used or recognized by their organizational field as sufficient for decision making, their decisions may be subject to challenge. a negotiated information order emerges when consensus is reached within or between organizations in a field regarding the criteria for socially sufficient information -about the type of information usable in decision making, the priority given to different types of information, and allocation of responsibility for gathering and interpreting that information (heimer a: ) . as these conceptual distinctions suggest, the symbolic nature of information penetrates even more deeply into organizational decision making than previous research might lead us to expect (feldman and march ; meyer and rowan ) . in particular, such symbolic considerations shape assessments of both decision-making processes and the information on which they are based. to work its symbolic magic, information must be seen as legitimate, and organizational actors will spar over whose data passes that test. but, crucially, such tests are layered. socially sufficient information is thus information that is widely agreed to be adequate to its intended purposes. technically sufficient information is more contested, with some actors touting its virtues and others casting doubt. technical sufficiency can therefore be a way-station along the path to social sufficiency or an intermediate category that permits some uses of information while prohibiting others. although participants experience these discussions as realist, social scientists would be quick to point out the deeply constructionist character of claims about the quality and veracity of information. the lines dividing categories of information are necessarily fluid, with discoveries shoring up some claims while undermining others and regularly adding to the stores of both knowledge and ignorance. as we will see, it is especially the boundary between knowledge and non-knowledge where contests are focused, because crossing that boundary makes otherwise prohibited actions possible. transposed into an organizational register, the dividing line between knowledge and non-knowledge takes the form of a distinction between technically and socially sufficient information. to say that the acceptability of information depends on a negotiated information order says only that the meaning of information is not given a priori but must be worked out collectively. norms about the sufficiency of information may be grounded in rules or laws. or they may reflect a broad, but informal consensus. what consensus is ultimately reached will depend on such factors as power differences, inter-organizational dependencies, and pre-existing loyalties. the preferences of powerful actors who have a vested interest in perpetuating practices associated with traditional types of information may have an outsized influence on the norms that emerge. previous agreements about the acceptability of various kinds and quantities of information provide important starting points, but will be less influential when decision makers face situations that seem unprecedented. thus negotiated information orders can be destabilized by modifications in technology, by the arrival of new problems or opportunities, or by changes in relationships among parties. three examples illustrate the importance of negotiated information orders in assigning meaning and determining how information is interpreted and used. clarke's mission improbable ( ) shows how information orders negotiated by powerful actors can exclude other voices that might challenge the meaning assigned to information. analysing organizations' plans to avert, control or cope with disasters, clarke considered the 'fantasy plans' created to clean up oil spills in open waters, evacuate long island in the event of a nuclear power plant accident, and protect the population during and after a nuclear war. in each case, rather than frankly acknowledging the impossibility of averting, controlling or mitigating disaster, key actors developed elaborate analogies and conducted careful simulations to convince themselves and others of the truth of essentially untenable propositions. the problem, of course, is that such analogies and simulations rarely worknuclear meltdown is not much like an ice storm, a major oil spill in open waters cannot be simulated by scooping up oranges from calm seas, and the evacuation of long island because of a nuclear accident cannot in good conscience be equated with the flow of people during rush hour. but when discussion is confined to a circle of experts, others may be unable to point out the obvious. here, the negotiated information order precluded consideration of information that other parties could have introduced by dismissing suppressed perspectives as unusable non-knowledge. with sars, as we will see, the first impulse of health workers, scientists, and policy makers was also to assume that they were seeing a variant of something they had encountered in the past -an atypical pneumonia or a disease caused by chlamydia (normile ; who a) . in sars, as in clarke's cases, suppressing information allowed actors at least temporarily to move forward with existing routines. even when divergent views are not completely suppressed, the context in which information is considered can shape conclusions. in last best gifts, healy ( ) asks how the major organizations supplying blood and blood products to american patients responded to early evidence that hiv could be transmitted through their products. in the american blood industry, a nonprofit whole-blood sector (the blood banks), reliant on donors, coexists with a for-profit plasma industry (the plasma fractionators) that purchases plasma from suppliers. between and , when no one was sure whether hiv could be transmitted through tainted blood and blood products, the us centers for disease control (cdc) presented their accumulating evidence and made recommendations about how to keep blood supplies safe (healy : - ) . representatives of blood banks and plasma fractionators received identical information, often at the same meetings. interestingly, though, these two sectors interpreted the information differently and adopted divergent strategies. blood banks, dependent on donors, saw blood borne transmission as 'still unproven' (healy : ) and were unwilling to ask intrusive questions about donor lifestyles and sexual practices. in contrast, plasma fractionators, working in a competitive market that made them more dependent on consumers than suppliers, adopted a policy of questioning potential donors and excluding putatively high-risk groups from their supplier pools. in short, the negotiated information order of the plasma fractionators led them to see the early information about hiv transmission as knowledge to be acted on, while blood banks' information order constructed the same information as non-knowledge to be ignored. with sars, people in different social contexts not only interpreted the data differently but also concluded that the data implied different things about their obligations under the ostensibly clear rules of the ihr. the final example shows how attempts to solve a new problem, not easily managed within the constraints imposed by an existing information order, can lead to modifications in that information order and changes in power relations in the field. contrasting the insurance of mobile rigs used for exploration and drilling with the insurance of fixed platforms used later for the production of oil in the norwegian north sea, heimer ( a) shows how norwegian insurers gradually altered the negotiated information order dominated by powerful british marine insurers. during the crucial early period of the exploration and development of the oil fields, insurers lacked the experience-based information needed for rating and underwriting, making them dependent on the reinsurance offered by british insurers. because multiple companies had to cooperate to assemble these insurance contracts with their uncertain risks and astronomical face values, insurers had to agree about what information was acceptable for rating and underwriting. british insurers stubbornly insisted on using conventional types of information. some types of information that, from a norwegian perspective, addressed key uncertainties thus could not be used simply because they had not been used in the past; social sufficiency dominated (alleged) technical sufficiency because of the requirement for consensus. because the total insurance capacity was insufficient to adequately insure the north sea oil fields, norwegian insurers were strongly motivated to create new routines for collecting and analysing data. they worked around and then modified conventions about what information could be used for ratemaking and underwriting. and gradually the situation changed. for mobile rigs, experiencebased data slowly became available and pooling risks over time and over similar units became increasingly feasible. this in turn further decreased dependence on the british reinsurance market and enabled norwegian insurers to be more flexible about what data to use and how to construct the policies. in contrast, little changed in the insurance arrangements for fixed installations, which were more expensive, less uniform, less numerous, and introduced later in the development of the oil fields. in the sars case as well, as we will see, pressure to change the rules became acute when new sources of helpful information became available but could not be efficiently exploited unless rules and norms were modified. as these examples demonstrate, organizations' information use is strongly shaped by social conventions. negotiated information orders spell out the relationships among different categories of knowledge and non-knowledge -what is known, acknowledged to be known, supplied by official sources, categorized as socially sufficient and therefore available for use versus what might be known, with varying degrees of uncertainty, but cannot be acknowledged or officially used. non-knowledge, which often comes from unofficial, back-channel sources, may be disregarded because it seems dangerous, threatening, harmful, or simply uncertain; ignored because decision makers are reluctant to bear the costs of retooling to collect, evaluate, and use new forms of information; or discarded because of the symbolic importance attached to information from official sources and the rights accorded to those who possess such high-value information. negotiated information orders thus introduce a modicum of stability in information use for some period of time until a new opportunity or danger arises. when that occurs, key actors pointing to the strategic value of certain information may successfully advocate for the reclassification of some non-knowledge as usable and technically sufficient and for the renegotiation of the information order. the arrival of a new disease -hiv, for the negotiated information order of the american blood industry; sars, for the ihr, the corresponding information order of global public health governance -can bring into sharp relief the irrationalities of established understandings about the reliability of information and the appropriate ways of using it. this article draws on a case study of sars to demonstrate how the deficiencies of the existing information order, institutionalized in the ihr, became painfully apparent in the wake of the epidemic. recognizing the substantial contributions that unofficial, previously illegitimate sources of knowledge could make in the fight against deadly infectious disease in turn helped to solidify the consensus around ihr reform efforts already underway in - when the sars epidemic occurred. in preparing this article, i have drawn especially on primary who documentation about the ihr and sars epidemic, supplemented by reports and commentaries from governmental bodies (e.g., the cdc and us congress) and non-governmental organizations and policy institutes (e.g., the national academy of medicine and chatham house). i have also drawn extensively on existing journalistic and scholarly accounts chronicling and analysing various features of the epidemic and scholarly articles and books investigating the epidemic's legal ramifications and the revision of the ihr. these documents were drawn from a larger body of primary and secondary materials collected primarily in - for a larger project examining the relationship between law and globalization in healthcare more generally. the article contends that in this case, a strong argument about technical sufficiency ultimately led to a new rule system that recategorized such non-knowledge as socially sufficient, legitimate, usable knowledge. international disease surveillance and global health governance have a long history before the sars epidemic. this history includes a century of international sanitary conferences to standardize quarantine regulations to prevent the spread of cholera, yellow fever and plague (and, previously, relapsing fever, typhus and smallpox); the crafting and revision of the international sanitary regulations (first written in ); and the formation of a series of international organizations to oversee disease surveillance and international public health, culminating with the creation of the world health organization, whose member states formally adopted the international sanitary regulations in . revised and renamed the international health regulations (ihr) in , these rules were in turn replaced by the revision, which went into effect in (fidler ; fidler and gostin ; gostin : - ; scales : - ) . a key issue in these agreements has been the collection and publication of information about disease outbreaks, with careful rules about who has to report and to whom, what they must report about, and what information they must transmit -in short, a negotiated information order that became more fully institutionalized over time. only with transparency, the argument went, was there any hope of protecting public health and curbing the spread of disease. yet, as the history of disease surveillance makes clear, because nations also worry about threats to trade, tourism and national reputation, they often strategize about what to reveal and on what timetable, hoping that diseases can be brought under control before discrediting information damages the economy or spoils the national reputation. the objective of the international conferences, conventions, and the ihr has been to induce more timely and more complete sharing of information, previously narrowly focused on reporting on a few infectious diseases and now more expansively redefined to include both infectious diseases and a wide variety of other threats to public health, by recognizing and working with this tradeoff. under the ihr (who ) , security against the spread of disease was to be achieved by requiring member states to notify the who of disease outbreaks within their borders (part ii, notifications and epidemiological information, articles - ) and maintain public health capabilities at ports and airports to monitor and reduce cross-border transmission of disease (part iii, health organization, articles - ). minimization of interference with trade and travel was to be achieved by specifying the range of responses states would be permitted or required to take in response to public health threats (part iv, health measures and procedures, articles - ). in effect, commitments to report outbreaks were traded for promises that responses to such information would be moderate, reasonable and scientifically grounded. even with this exchange in place, though, the record of compliance has been poor, and poor on both counts (carvalho and zacher ; fidler : ; kamradt-scott : - ; scales ; woodall ) . countries frequently failed to report disease outbreaks, but they also imposed overly restrictive protective measures, including quarantines and outdated vaccination requirements, that violated the spirit and the letter of the ihr rules on trade and travel. diseases continued to spread across borders whether or not travellers and goods were impounded, quarantined or otherwise delayed. although neither the who nor the member states seemed very committed to it, as the governing information order, the ihr continued to be consequential in shaping the circulation of information, categorizing information as actionable or not, and providing an excuse for states to shirk or evade pressures to report even as new transparency norms were emerging. the ihr's history suggests that this information order is primarily organized around concerns with trade and travel and has favoured the interests of rich countries (chorev b; fidler ; kamradt-scott ) . similar patterns of favoring the interests of rich countries in global health governance have been noted by other scholars (king ; erikson ; but see wenham on recent changes in emphasis). 'the rising commercial costs imposed by a system of uncoordinated, unregulated national quarantine practices meant that trade rather than health drove the development of international governance on infectious diseases', concludes fidler ( : ) . quite emblematically, the treaty and its predecessors focused only on diseases that seemed likely to be spread by trade and travel, and particularly those that might move from poor to rich countries. infectious diseases that plagued only poor countries, such as polio, were not listed, and south-south contagion was a secondary concern. adjustments were unidirectional: diseases were removed from the list, but re-emerging or new diseases were not added; no adjustments were made to take account of changes in modes and speed of transportation. the official rules of the ihr in some senses imagined a static information order in which states interacted with the who -what fidler ( ) describes as a westphalian system. yet the information order has evolved over time in important ways, with the official information order often out of step with informal practices. two key drivers of change have been innovations in information technologies, which vastly increased the amount of information available while simultaneously reducing state control of information, and the creation of new types of actors in the loosely organized global public health system. as reporting rules were first being developed, it was diplomats who certified that a ship's last port of call was disease free, allowing ships to avoid quarantine as they entered ports to offload cargo and passengers (fidler : ) . although diplomats no longer verify bills of health, the treaty's reliance on national reporters remains a core element of the reporting framework even though the new categories of actors (ngos, ingos, international health workers, laboratory workers, scientists, etc.) have access to much relevant information. thus an evolving information order peopled with these new actors co-existed with a static legal framework that only recently acknowledged and incorporated them. this meant that the who was unable to act even when it possessed information that it believed to be technically sufficient. because it was bound by a strictly formalized set of rules (few things are more rigid than a treaty with a long list of signatories), it could not adjust to evolving communication patterns. although analysts have often described disease surveillance as a collective action problem in which the global interest in transparency is pitted against national interests in episodic strategic concealment, characterizing the problem this way vastly understates the complexity of the interactions among actors. in particular, although it is nation states that have ihr treaty obligations, information about disease may be generated and controlled not only by non-state actors (as mentioned above) but also below the level of the nation-state, by agencies of the state, provincial health departments, individual public or private hospitals, and doctors and other medical personnel. as we will see, the norms and rules about how these lower level actors fit into the ihr negotiated information order have not always been entirely clear. until recently, the ihr made the who exceedingly dependent on official country reports by prohibiting the use of other sources of information. although promed-mail became an important unofficial source of information about threats to public health after its founding in , for many years the who was constrained from officially using it (woodall ). over time, the who's stance on these alternative sources of information evolved. a world epidemiological record piece suggested that 'public health authorities should give more attention to information from sources other than the public health sector, including ngos and the media. the capacity of public health authorities to rapidly respond to outbreak-related information from any source is essential for the efficiency and credibility of the entire surveillance effort' (who : ) . as the volume of information available from electronic sources and from health experts dispersed around the world increased, the pressure to use such information also increased. with increasingly sophisticated tracking systems, for instance, it became possible to demonstrate that deaths (even of particular named individuals) could have been prevented by earlier issuance of travelers' advisories (woodall ) . often, though, sub rosa information was less useful for issuing official warnings than for pressuring countries to report or for asking pointed questions about the adequacy or accuracy of reported information. 'they have accused us of spreading unfounded rumors and posting reports that have had no peer review. but we're just reporting what is being said or published. we tell health officials, you might as well report this, because you'll be reading it on promed tomorrow', commented charles calisher, an early moderator of promed (miller ) . some kinds of action required only that information be seen as technically sufficient (adequate in volume and coverage), but other kinds of action required that information also be socially sufficient (supplied by legitimate sources and arriving through specified routes). were the ihr ever an effective information order? undoubtedly the treaty was an improvement over earlier agreements, both in clarifying expectations and obligations and in institutionalizing a set of practices for reporting on disease outbreaks and keeping protective reactions in bounds. although it was an admirable attempt to create a worldwide consensus that balanced health interests against economic ones, it also had several clear deficiencies. an especially important deficiency was the limited coverage of the ihr, which cast doubt on the legitimacy of the treaty. beyond this severely limited coverage, the ihr were also compromised as an information order by a naïve conception of states as unitary actors and by rules that allowed the who to use only limited kinds of information supplied by specified, state-based actors. over time, informal norms supported fuller reporting on a broader range of threats and exploitation of information from unofficial as well as official sources. but in the medium term, although some nation-states adhered to the new norms, others hid behind the inadequate formal rules of the ihr, and still others continued to ignore even the limited formal requirements of the ihr. how well did this imperfect, outdated information order function when the ihr encountered sars, a new, deadly infectious disease that seemed poised to spread rapidly around the world? did the deficiencies of the information order in fact prevent the who from acting quickly and appropriately to contain the disease? many accounts of the - sars episode describe the chinese as concealing information or misrepresenting the situation in the first months, often suggesting that the country acted illicitly or illegitimately in doing so (altman ; the guardian ). yet closer examination of the record (see especially huang ) suggests that something considerably more complex occurred -there were multiple legitimate reasons for china to conceal early evidence of the outbreak. to begin, during the first days, there was nothing to report because no one understood that this was a new viral disease. because most apparently new diseases are in fact not new, physicians are reminded to think of horses not zebras when they hear hoof beats. as perhaps happened with sars, this advice sometimes leads people astray. with the benefit of hindsight, it is easy to conclude that chinese health workers should have been more diligent in forwarding reports about early cases of 'atypical pneumonia'. but we must be careful not to interpret actions taken in the confusion of the earliest days with knowledge acquired only later. still, local hospitals did call on provincial authorities for help. provincial authorities contacted the national ministry of health. a group of experts conducted an investigation. a report was prepared and circulated to all of the hospitals in the province. but here chinese law altered the disease's trajectory because the report became a state secret that could be shared only with specified people (such as the heads of hospitals). and then the trajectory was modified serendipitously when the report arrived in hospitals during the chinese new year celebrations. because no one read or acted on the report for a three-day period, precautionary measures were not implemented, creating an opportunity for the disease to spread. as noted, several months passed between the first appearance of sars and the first reports to the who. had the first suspicious cases been reported promptly, the disease likely would not have spread beyond guangdong province and hundreds of deaths could have been prevented. reports on the case suggest that in the earliest period, people 'knew' but 'didn't know' about the epidemic. and although healthcare workers, officials and other actors suspected a problem, at least in some instances they were either forbidden to share information or prohibited from acting on the information they received. in this case, the complex interplay of international, national and local rules and norms seems to have done as much to delay as to accelerate the spread of information about threats to public health. information about sars gradually leaked out, though, with a report from the chinese ministry of health finally reaching the who on february (who d). accounts of this period mention 'medical whistle-blowers' (see, e.g., eckholm ), promed-mail, the global health intelligence network (gphin, the 'rumor list'), the global outbreak alert and response network (goarn), and the move of the disease across borders into hong kong and then vietnam. although who personnel were investigating cases of what turned out to be sars in china as early as late february (enserink a (enserink : , the who issued its first alert about a severe form of atypical pneumonia only on march . according to david heymann (then executive director of who's communicable diseases cluster), vietnam was 'the trigger' for this announcement (enserink a (enserink : . a march report from carlo urbani, a who parasitologist consulting on a case in the french hospital in hanoi, provided the first indication that the new disease had spread beyond guangdong and hong kong. (urbani himself subsequently died from sars.) with the second who announcement, the world was informed that the atypical pneumonia, now named sars, was a new and very serious communicable disease. the secret was out. during this period, it could be hard to discern the signal in the noise. many things contributed to the noise -the irreducible uncertainties of the early days of a new disease, fear, mistakes, lack of preparation, incompetence, reputational concerns, and of course deliberate obfuscation. to be sure, there was ample evidence of outright concealment, foot-dragging, and obfuscation. the guangdong provincial government 'initially banned the press from writing about the disease and downplayed its significance' (enserink b (enserink : . although the who diplomatically reported cooperative efforts (see, e.g., who b), it carefully avoided comment on chinese silence or obfuscation between november and february, and even later. in fact, although chinese officials agreed to share information, their first promises were followed by more deceptions (fidler ; huang ; knobler et al. ). when the chinese government began to share information, who officials were still unable to get meetings with chinese health officials and were refused permission for travel to guangdong (enserink b (enserink : . when the early undercount of sars patients was attributed to the inadvertent exclusion of patients in military facilities, eckholm pointed out that the high proportion of beijing sars patients in military hospitals 'could [instead] indicate that patients were placed there to avoid their inclusion in civilian disease reports ' ( ) . what the nation-state does not know, it cannot report. but the ihr was little help in dampening the noise or strengthening the signal. although the ihr treaty was officially the governing document when the - sars outbreak occurred, it was an imperfect information order that did not authoritatively mandate a clear course of action. '[n]othing compelled china, or any other country, to tell the rest of the world what was happening within its borders early in ' (enserink b (enserink : . indeed, the shocking weakness of the international health governance system was surely a factor in china's failure to report the outbreak quickly. under the ihr, most disease outbreaks, including those of previously unknown diseases, were domestic business. but if china had no formal obligation to report, why was it so soundly condemned for its delay in transmitting information to the who? although the treaty -international law -did not require reporting, emerging norms around the management of global public health governance diverged from formal law. under these emerging norms, a failure to report a new disease, an environmental disaster, or some other occurrence that might affect global public health was a serious infraction (heymann ) . indeed, it was the conflict between these emerging norms and the existing treaty provisions, along with the emergence of new infectious diseases like ebola, that helped spur the ihr revision, first called for in a resolution, well ahead of the sars outbreak. an important difference between formal treaty obligations and norms, though, is that the first applies uniformly and the second does not. being a signatory to a treaty is a bright line. membership in a moral community is more ambiguous, with some treaty signatories more fully incorporated and others more peripheral. thus although the long silence of the chinese government was not technically a violation of the ihr, it nevertheless appeared dishonest and inappropriate to the international community, undermining rather than supporting emerging cooperative norms and in fact harming global public health by allowing the new disease to spread beyond china's borders. the institutional incoherence around global public health governance was in fact deeper than this; the treaty provisions were inconsistent with domestic law as well as with emerging norms. until treaty provision and domestic law are harmonized, health workers can be caught between local and global legal obligations, two distinct sets of rules laying out inconsistent requirements for partially overlapping groups of actors. although only state representatives were responsible for reporting to the who, domestic law compelled medical workers to preserve state secrets about the very matters that international norms -but not ihr treaty provisions -compelled them (or others in their chain of command) to report. many chinese actors were in a terrible bind, legally required to protect state secrets but morally obligated to share information so fellow citizens could protect themselves from a virulent emerging disease and so international bodies could study the disease and develop methods to combat it. individual and global interests both demanded transmission of information, yet the chinese state initially mandated secrecy instead. moreover, the ihr specified roles and obligations for only a few actors, thus offering no guidance about appropriate courses of action for many other actors who possessed relevant information. beyond legalistic matters about obligations to report or to conceal, the evidence from sars also suggests that fears about economic consequences of adverse publicity associated with disease outbreaks strongly shaped the thinking of chinese authorities (huang : ) . these economic concerns were in fact justified, though overstated, in hindsight. the economic effects of sars include much more than the cost of providing medical care for those affected, as analysts acknowledge. lee and mckibben ( ) estimated the short-term impact of sars to be about $ billion for alone if people expected the epidemic to be a one-time event and considerably higher if they behaved as if they anticipated recurrences. subsequent research suggests that the economic impacts were considerably smaller than anticipated and that recovery occurred quickly (keogh-brown and smith ) . although the economic impact was widely dispersed, the losses were greater in asian countries than in the rest of the world, with strong shocks to mainland china, which experienced a decline in foreign investment, and especially to hong kong whose service economy depends on travel and tourism. for government officials responsible for the overall welfare of a society, including both physical and economic health, worries about commercial impacts cannot be dismissed. as a negotiated information order, the ihr was thus ineffective, unstable, and ripe for change for a host of reasons. first, legal obligations were out of sync with the higher expectations of an evolving normative system. second, international law and domestic law often had not been harmonized and disagreed about whether threats to public health should be reported or kept secret, creating a serious conundrum for health workers. third, the ihr failed to take account of the social complexity of a system in which information was produced and controlled by a wide variety of actors, including not just official national representatives (e.g., ministries of health) and provincial or other substate actors (e.g., provincial departments of health), but also actors who were not state representatives but nevertheless had relevant roles and expertise (e.g., heads of hospitals, whether private, public or military), journalists, and private citizens all with varying relationships to the international treaty, emerging norms, and domestic law. fourth, although the ihr did not envision that the who would act on the basis of information other than that provided officially by nation-states, pressure to use such 'non-knowledge' had increased over time as information sources multiplied, tools to parse such information were created, and threats to public health came to seem increasingly urgent. one important effect of sars was to shift the boundary between official and unofficial knowledge, ultimately modifying the information order so that unofficial information of questionable quality could be used as leverage, forcing states to reveal what they might have preferred to conceal. the revision of the ihr was adopted by the world health assembly (wha), the governing body of the who, in and put into force in . just as revisions to the ihr were being crafted, the deficiencies of the existing legal framework were made glaringly apparent by the rapid spread of sars and the numerous -and avoidable -deaths it caused. although china had not in fact violated the existing treaty, it clearly violated emerging norms on the reporting of infectious diseases. the objective of the new treaty provisions was to induce earlier and fuller reporting by acknowledging the importance of non-state actors as suppliers of information and recrafting the information order so that previously unusable kinds of information -information that might have been seen as technically sufficient but was not socially sufficient -could now be used. the revision brought important changes in what has to be reportedany 'public health emergency of international concern'. along with this broader range of reportable threats, the ihr introduced a decision tool to replace the short, simple list and guide reporting; offered considerable guidance about who should report and how (e.g., mandates for designated reporters, now called 'national focal points'); and created tool kits for implementation including for harmonizing the ihr with domestic law (who ). in effect, these changes move the ihr from the realm of 'soft law' further into the domain of 'hard law' (abbott and snidal ) by making the rules more specific and more obligatory, by adding processes for interpretation of law and for dispute settlement, and by inserting rudimentary enforcement mechanisms. some of the work of hardening the ihr is delegated to individual member states as they bring domestic law into harmony with the ihr. as treaty provisions and domestic law are harmonized and gaps bridged, excuses for non-compliance are eliminated and domestic supports for compliance are added (see, e.g., the agreement between the australian federal government and its states and territories to ensure timely reporting [commonwealth of australia ; scales : ]). fidler ( ) argues that sars exposed the conflict between an outdated, unworkable, westphalian system of international governance and a world in which global diseases required a global governance system. states have lost their primacy, he suggests, in a world in which they can control neither the movement of disease nor the movement of information. believing it had the right to suppress information, the chinese government attempted to treat information about infectious disease as it always had: as a matter of state secrets. but in a world of cell phones and internet, text messages and email allowed both patients and physicians to circumvent the state. prohibiting news media from reporting the outbreak of the deadly disease did not keep individuals from communicating with one another inside china and sending information and questions to contacts outside the country. with the growth of new information technologies, state monopolies on information have decayed and the balance between socially and technically sufficient information has shifted. as the volume of information considered technically sufficient has increased and the who has developed more sophisticated techniques for extracting high-quality information, its capacity to pressure states to meet their treaty obligations has increased. something like an enforcement capacity, albeit one not formally recognized in the ihr, grew up in the midst of all this complexity. with the vote of the world health assembly (wha) and the subsequent revision of the ihr, this enforcement capacity has been recognized, endorsed and formalized, first with the wha's blessing of the who's use of unofficial information and then with the incorporation of this information use into the procedures outlined in the revised ihr. in this case, changes in practice preceded changes in the legal infrastructure as the who increasingly drew on information that did not come directly from the official reporters of member states. but in a pattern of 'punctuated globalization' (heimer ) , the legal framework seems now to have reclaimed the lead in moving forward global coordination around public health surveillance. as countries and agencies adjust to the ihr, we can expect the development of a host of new strategies for exploiting the opportunities created by this new framework. the revisions have required many countries to invest heavily in improving their systems for tracking and reporting threats to public health. this, in turn, has created an opening for many joint activities between rich and poor countries, including construction of new cdc facilities around the world (gootnick ) . do these changes then signal the end of the gap between actionable, socially sufficient information and technically sufficient information in global health governance? rather than an end to the gap, we should expect a shift of the gap's location. gaps arise because parties with imperfectly aligned interests have some incentive to game systems. such discrepancies between global, collective interests and regional, state or local interests will continue to exist and some evidence suggests both continued and fresh strategies for gaming and non-compliance (scales : - ) . the exact configuration of the gaps will change, of course, as the nature of the key actors changes (less emphasis on states, perhaps) and as technologies change (easier transmission of information by both official and lay actors). the gap itself will not vanish. states will remain relevant actorsindeed world politics suggests that national borders are as often reinforced as demolished and that states continue to have responsibilities and interests that might motivate them to conceal information. moreover, a clarification of treaty obligations and the introduction of a new lever for the who will not entirely resolve the problem. in the past, with no uncertainty about obligations to report, countries nevertheless failed to report outbreaks (carvalho and zacher ; fidler : ; kamradt-scott : - ; scales ) . although the who can more nimbly alert the world about an outbreak, it can do little beyond that: no sanctions, no fines, no cancellation of membership. and new incentives for non-compliance will continue to arise. until samples were used to create flu vaccines, countries had little reason to withhold samples of new influenza strains. but under a regime that protects intellectual property and gives those supplying samples no share of the income from the sale of resulting vaccines, countries now have an incentive not to offer their samples for the common good. when indonesia, responding to this incentive structure, began withholding flu samples, a new who working group developed a non-binding framework to encourage both virus and benefit sharing (fidler ; fidler and gostin ; scales ; smith ) . in the argument of this article, sars plays a central (albeit non-determinative) role. but is sars simply a useful case on which to hang the argument? or could the argument have been built around hiv/aids, h n , ebola, zika, or some other infectious disease? in fact, other diseases and sars are not interchangeable in this argument; sars is not 'merely' an example. because of historical timing, sars was the epidemic that brought the previously recognized failings of existing disease surveillance systems into the spotlight and stiffened the spines of those pushing for change. the features and timing of sars helped to bring the shortcomings of the ihr into sharp relief, undermining their legitimacy and making it essentially impossible for the who and public health specialists to continue working under the old rules. the legitimacy of the who increasingly depended on denying the legitimacy of the ihr. by the time sars appeared, the deficiencies of the ihr had become so glaringly apparent that the wha had endorsed the who's use of unofficial information even before the rules changed. but particular features of the disease, namely its brief incubation period and moderate transmissibility, meant that the adage that microbes do not respect national borders was all too applicable. local outbreaks of sars had global relevance in a way that local outbreaks of hiv/aids, with its long period of dormancy, did not. sars quickly became a global threat. but it also mattered that the disease arose in a country that wished to suppress information about the outbreak. in the age of the internet and cell phones, information, like microbes, neither respects borders nor governmental edicts on secrecy. thus sars brought to a head a long-standing clash between national governments' desires to keep secrets and new capacities to transmit information with or without governments' blessing. in fidler's view, 'china's behavior [at the start of the sars epidemic] put the final nail in the coffin of basing global surveillance for infectious diseases only on government information ' ( : ) as the rules required. sars was a 'historic moment in public health governance' (fidler : ) , the tipping point for new governance strategies (fidler : ) . in a limited sense, then, sars was a boon to the who because it provided an added inducement for the wha and member states to modify the rules in ways that benefited the entire group and gave the who and ihr new relevance. although the ihr's limitations had long been apparent, by making it impossible to deny that the treaty provisions were outmoded sars accelerated the process of reaching consensus on proposed changes. the revisions of the ihr attempted to deal with two kinds of ignorance: ignorance about outbreaks of known diseases and ignorance about newly emerging diseases and other threats to public health. before revision, the ihr had focused only on outbreaks of known diseases and therefore on ignorance that could in principle be reduced or even eliminated by full and honest disclosure. as it became clear that infectious diseases were not going to be eradicated, as new diseases continued to emerge, and as natural disasters, industrial accidents, air and water pollution, and so forth came to be understood as threats to public health, the ihr's focus shifted to these less tractable forms of ignorance and thinking changed about what should be reportable under the ihr. this expanded understanding of threats to public health brought both expanded obligations for states and expanded obligations for the who. the who's remit now included not just spreading the word and issuing advisories about a larger package of threats to public health, but also overseeing and orchestrating the scientific work of untangling the etiology, symptom patterns, modes of detection, and effective remedies for these threats. into this changed environment, the reworked information order introduced a more sophisticated understanding of the relationship between what was or could be known and what was unknown and perhaps even unknowable. the modified procedures of the ihr in some senses acknowledged the difference between technically sufficient information that was also socially sufficient -because it had been supplied by mandated state reporters -and technically sufficient information that was not socially sufficient because it travelled to the who by unconventional or even clandestine routes. but the loosening of constraints on the sourcing of information did more than simply make information usable by recategorizing previously unofficial, socially insufficient information. the modified procedures also opened the door to using information as leverage, with information of inferior quality or illegitimate provenance being used to pry loose information of better quality or from official sources. moreover, in casting a wider net and exhibiting its willingness to draw on an expanded network of informants and more variable kinds of information, the ihr seem to acknowledge the essential irreducibility of ignorance. when uncertainty cannot be eliminated, and when the transmission and withholding of information is at least in part a strategic game, an entity such as the who is in no position to sharply limit the information it will consider. the ihr, a renegotiated global public health information order, thus incorporate into their structure an acknowledgement of the complex relationship between knowledge and ignorance, socially sufficient information and technically sufficient information, and the socially constructed nature of these distinctions. although this article focuses on negotiated information orders in global public health governance, its argument and evidence address broader issues about how global norms change and how social groups manage risk. the story of the - sars epidemic, the core empirical component of the article, is about the possibility that a virulent new disease would become a devastating pandemic and about an emerging (but not yet formalized) obligation to inform the who about serious threats to public health. the comparison points -the threat of aids contamination in banked blood (healy ) ; threats from oil spills, nuclear power accidents, and nuclear war (clarke ) ; and threats from accidents on north sea oil rigs and platforms (heimer a ) -are also about how key actors assessed novel risks. in all of these cases, the assessment of the core risk was implicitly balanced against other risks -risks to trade and tourism for sars; risks to relationships with important constituencies for the blood banks (healy ) ; risks to desired investments in business and government enterprises (clarke ) ; and risks to vested interests in the insurance business (heimer a) . generally speaking, though, as discussions unfolded, only some of the risks were fully on the table, perhaps because people were not wholly aware of how other considerations were shaping their thinking, perhaps because of the questionable legitimacy of balancing other risks (trade and tourism, in the sars case) against threats to life and health. the result is often a pattern of minimizing assessments of danger and normalizing those (implicit) assessments. as noted earlier in the article, many disease outbreaks, even of the three reportable diseases, had not been reported to the who. somewhat like the normalization of deviance that diane vaughan ( ) so carefully describes in the challenger launch decision, the deviant non-reporting of disease outbreaks had been normalized. some countriesespecially poorer ones -were learning from one another that they would suffer no consequences from ignoring ihr treaty obligations. although the ihr were described as regulations to protect health in all countries, in fact they focused on stemming the spread of disease from poor countries to richer ones. as chorev ( a) suggests, international obligations perceived as coercive are more likely to be reinterpreted locally and perhaps ultimately transformed through processes of reactive diffusion. in the case of the ihr, reactive diffusion essentially made the already unenforceable ihr progressively less useful. but in the pre-sars period, the evidence in fact suggests a more complex process of normative change. two rather different norms were being institutionalized simultaneously in global public health governance. at the same time that ignoring ihr treaty obligations was becoming the norm in some circles, a different norm was spreading in other circles. some countries -especially the richer ones -were adopting a more cooperative stance, sharing information not only on ihr reportable diseases but also on other infectious diseases and threats to public health. it was this cooperative norm, not the norm of non-reporting, that ultimately diffused and, coupled with the sars epidemic, led to a reinvention of the ihr as a treaty with a few more teeth. how did this happen? here a comparison with the space shuttle launch decision is instructive. although nasa carried out rigorous, carefully scripted pre-launch reviews, contextual pressures to launch could subtly shift thinking about which risks could be dismissed and which warning signs ignored. over time, these modified assessments were institutionalized and the insularity of the process made it hard for alternative viewpoints to force a recalibration. the conflict between protecting against rare events and attending to business is utterly mundane (vaughan ) , so mundane that insurers have institutionalized methods for protecting key risk management tasks from production pressures (heimer b) . the job of the ihr, arguably, is to rebalance risk assessments so global public health interests are not regularly sacrificed when discrediting information about health threats is concealed to protect a country's trade and tourism. yet the ihr treaty gave the who few levers to induce such a rebalancing. unlike space shuttle launch decisions, though, global public health governance does not take place behind a single set of closed doors. thus, although a practice of non-reporting -normalized deviance -seemed to be developing in some sectors, changes in information technologies and communication patterns made secret keeping more difficult and shifted the balance in favour of the more cooperative norm. even with china's strict control over the internet and the press, text messages and emails spread news about 'atypical pneumonia', forcing public officials to acknowledge the outbreak. although any single medium might fail to pick up the news, the proliferation of methods for detecting signals makes suppression of information more difficult. a news blackout might make gphin, which scrapes information from news outlets, less effective, but have less effect on promed-mail, which relies on medical workers' postings. working together over some considerable period of time and in a series of discrete steps, the new information technologies and the emerging norm of information sharing reconfigured the rules about global public health governance and reshaped understandings about what information could be used and who could supply it. information technologies first reshaped some practices of the who. as the who began to use the unofficial information supplied by entities like gphin, it also initiated the process of redefining non-knowledge as technically sufficient, at least for some purposes. as the who rebuilt its routines to use unofficial information alongside official country reports, new relationships and resources (e.g., goarn) were created around those new information sources. both the suppliers of information and the who increasingly treated this new information as technically sufficient. with the endorsement of the wha, these new practices and new definitions of the adequacy of unofficial information were further institutionalized, moving one step further to a formal change in the treaty itself. with the adoption of the ihr, the process was complete -what had previously been categorized as unusable non-knowledge was first reconceptualized as technically sufficient, and ultimately accepted as socially sufficient for use in an expanded menu of actions. nevertheless, information categorized as unusable non-knowledge will always exist and will continue to be important precisely because it comes from different social locations than those tapped by official information. as mary douglas would remind us, we need the sentinels on society's margin to warn us of unexpected dangers every bit as much as we need people working in core institutions to protect us from more routine risks (douglas and wildavsky ) . although admittedly the uses of non-knowledge or clandestine knowledge are typically different than the uses of official knowledge, that should not lead us to underestimate either the vital strategic value of non-knowledge or the importance of using it efficiently in a smoothly functioning, adaptable information order. just ask kim philby or david john moor cornwell, aka john le carré. gphin in (who c , notes that '[m]ore than % of the initial outbreak reports come from unofficial informal sources, including sources other than the electronic media, which require verification' (who n.d.) . gphin is often credited with picking up news of a disease outbreak in china in late november (heymann and rodier : ) . set up in by the who and formally launched in , goarn is a collaboration of other networks, linking a wide variety of experts and combining both surveillance and response (fidler ; heymann ; heymann et al. ; who c: ) . as of , goarn includes as members over technical institutions and networks concerned in one way or another with public health (https://extranet.who.int/goarn/; last viewed march ). fidler ( ) , especially, credits goarn with a major role in containing sars. according to virologist malik peiris, 'if something untoward was happening across the border, it would come to hong kong pretty quickly' (enserink a (enserink : . vietnam was a reluctant trigger, though. as hospital staff fell ill, the vietnamese government had to be persuaded that this was not simply a 'private problem in a private hospital' but might instead be 'very important' (enserink a : . according to the who, the resurgence of cholera in south america and plague in india, as well as the emergence of new infectious agents such as the ebola virus, 'resulted in a resolution at the th world health assembly in calling for the revision of the regulations' (https://www.who.int/ihr/ about/faq/en/; last viewed march ). some observers (e.g., katz and fischer ; wenham ) contend that states have not lost their primacy. for similar assessments of the role of 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'epidemic intelligence -systematic event detection international health regulations ( key: cord- -te fv gq authors: mergeay, matthias; coeckelbergh, evelien; de cauwer, harald; viaene, mineke; van der mieren, gerry title: an adult case of metapneumovirus-induced acute encephalitis date: - - journal: acta neurol belg doi: . /s - - - sha: doc_id: cord_uid: te fv gq nan because a (meningo-)encephalitis was suspected a spinal tap was performed and acyclovir, amoxicilline and ceftriaxone were initiated. csf results, indicative for viral encephalitis, are shown in table . due to impaired consciousness and the need of vital parameter monitoring, the patient was transferred to the intensive care unit (icu). during the first hours on the icu the patient presented with two episodes of tonic-clonic seizures, successfully treated with intravenous benzodiazepines. after the second episode of seizures, levetiracetam g dd was added to the therapeutic regimen. the next day the patïent was fully awake, he had a glasgow coma scale of / , with a normal neurological examination. pcr revealed to be negative for enterovirus, cytomegalovirus, varicella zoster virus, herpes simplex, cryptococcus neoformans, listeria monocytogenes, haemophilus influenza, neisseria meningitides, str. pneumoniae in csf and negative for influenza a/b, parainfluenzavirus, rhinovirus, bocavirus, adenovirus and coronavirus in serum. mycoplasma pneumoniae, chlamydophila pneumoniae, bordetella pertussis and bordetella parapertussis also were excluded. nasopharyngeal aspirate was negative for coronavirus, but showed rna strands of human metapneumovirus (hmpv) with a low viral load suggestive for a recent infection. control eeg the same day showed a normalization of the basal rhythm to hz. viral conjunctivitis was conf ir med by the ophthalmologist. cultures of blood and csf remained sterile. because hmpv encephalitis was diagnosed, antibiotics were discontinued. mri of the brain did not reveal cerebral infectious disease nor recent ischemia. levetiracetam was discontinued after months because of favourable outcome with complete remission. in adult hmpv encephalitis cases influenza-like symptoms or respiratory infection (bronchiolitis, bronchitis or interstitial pneumonia) were reported in all, though in our and tan's case the patient was admitted because of other complaints, and respiratory tract infection was only revealed by chest radiography [ , , ] . neurological features of hmpv encephalitis include coma, delirious behavior, impaired consciousness, seizures, and refractory status epilepticus [ ] . in the previously reported adult cases two presented with altered mental status, one with seizures. in all cases encephalitis was diagnosed on clinical grounds despite lacking laboratory support e.g. despite normal csf analysis in tan's and fok's cases. in jeannet's and our case, clinical presentation, csf pleocytosis and elevated protein levels were indicative for viral encephalitis (table ) [ , , ] . antiviral drugs e.g. acyclovir or ribavirin were used in three patients, in two antibiotic regimen was given because of the associated interstitial pneumonia. in our patient acyclovir and antibiotics were initiated and administered until serological testing, pcr and cultures were available [ , , ] . extensive testing for viral and bacterial pathogens can help the clinician in getting a much faster diagnosis, initiating proper treatment, and predicting outcome. treatment for hmpv essentially remains supportive, although ribavirin was shown to be active against rsv and hmpv [ ] . in fok's case the patient was treated with a days course of methylprednisolone because of no clinical improvement was noticed and autoimmune encephalitis/cerebral vasculitis was suspected [ ] . in some patients mr imaging does suggest autoimmune pathogenesis [ , ] . in hmpv-induced encephalitis scattered cortical and subcortical t w/flair hyper intensities have been described. this is in contrast with the mr findings in herpes encephalitis or influenza-associated encephalopathy (iae) [ ] . however, it is unclear whether direct viral cerebral invasion, or nonspecific inflammation/vasculitis, or excessive extracellular release of neurotransmitters is the responsible pathogenic factor [ , , ] . in our patient mri was within normal limits: this might be due to the uncomplicated clinical course or because of the time lapse between mri and the clinical symptoms. in iae rapid recovery both clinically and radiographically has been reported [ ] . in patients with suspected viral encephalitis, hmpv may be considered as the causative agent, and testing for hmpv in nasopharyngeal aspirate and csf is then required. conflict of interest all the authors report no disclosure nor conflict of interest relevant to the manuscript. all authors report no financial disclosure. ethical approval this manuscript does not contain any studies with human participants or animals performed by any of the authors. informed consent informed consent was obtained from all individual participants included in the study. additional informed consent was obtained from all individual participants for whom identifying information is included in this article. adult human metapneumovirus encephalitis: a case report highlighting challenges in clinical management and functional outcome human metapneumovirus in the cerebrospinal fluid of a patient with acute encephalitis encephalitis-associated human metapneumovirus pneumonia in adult cerebrospinal fluid findings in an adult with human metapneumovirus-associated encephalitis influenza-associated encephalopathy with extensive reversible restricted diffusion within the white matter key: cord- - um jmhk authors: imperiale, michael j.; casadevall, arturo title: a new approach to evaluating the risk–benefit equation for dual-use and gain-of-function research of concern date: - - journal: front bioeng biotechnol doi: . /fbioe. . sha: doc_id: cord_uid: um jmhk in the twenty-first century, biology faces a problem that has previously vexed other disciplines such as physics, namely the prospect that its knowledge domain could be used to generate biological agents with altered properties that enhanced their weapon potential. biological weapons bring the additional dimension that these could be self-replicating, easy to manufacture and synthesized with commonly available expertise. this resulted in increasing concern about the type of research done and communicated, despite the fact that such research often has direct societal benefits, bringing the dual-use dilemma to biology. the conundrum of dual use research of concern was crystallized by the so-called “gain-of-function” type of experiments in which avian influenza viruses were endowed with new properties in the laboratory such as increased virulence and the capacity for mammalian transmission. after more than a decade of intensive discussion and controversy involving biological experiments with dual-use potential, there is no consensus on the issue except for the need to carry out such experiments in the safest conditions possible. in this essay, we review the topic with the hindsight of several years and suggest that instead of prescribing prohibitions and experimental limitations the focus should be on the importance of scientific questions at hand. we posit that the importance of a scientific question for medical and scientific progress provides a benchmark to determine the acceptable level of risk in biological experimentation. revolution in the late twentieth century that the potential of biology, and in particular microbiology, in biological warfare and terrorism came into focus. these fears came true with a single act of terrorism involving the mailing of anthrax spores in the u.s. in , which caused several deaths and considerable disruption to government facilities such as congressional offices and the postal system (bush and perez, ) . this act of bioterrorism catalyzed a series of events that led to greater awareness of the potential of using biological knowledge in nefarious ways, new regulations in biological research in the u.s. such as the select agents and toxins list, and heighted concerns about some types of microbiology research. one of the immediate effects of the post- environment was increased scrutiny of published findings, and several papers were particularly noteworthy for eliciting discussion and concern. in , researchers in australia showed that insertion of the gene for il- into the ectromelia virus genome defeated vaccine immunity (jackson et al., ) , a report that drew immediate concerns because of its possible implication for similar effects with variola major virus. similarly, the finding that complement inhibition potentiated variola virus virulence suggested a mechanism for enhancing its pathogenicity (rosengard et al., ) . an analysis of the u.s. milk distribution system revealed vulnerable nodes where the introduction of botulinum toxin could lead to mass casualties (wein and liu, ) , leading to an outcry about journals publishing research that could be exploited by terrorists. advances in molecular biology led to the chemical synthesis of poliovirus (cello et al., ) and the reconstruction of the pandemic influenza virus (tumpey et al., ) , which greatly heightened concern about the re-introduction of extinct, controlled, and new viruses to naïve human populations. in this environment, the u.s. government created the national science advisory board for biosecurity (nsabb) in to provide guidance on issues relating to biological risk and security. both authors of this article were inaugural members of the nsabb and served on that board for years. when the nsabb first began their deliberations, one of the major early topics was that of dual-use research in the biological sciences. this issue had previously vexed the physics community in the twentieth century as advances in physics and associated technologies had led to radar, nuclear weapons, ballistic missiles, and other military hardware. however, there was little or no precedent for similar concerns in the biological research community, especially since biological weapons were outlawed by the biological weapons convention in . moreover, the term dual use, which had meant civilian and military use in the physics arena, was redefined in its application to biology by an nrc report in to mean technologies and information that had both beneficial and maleficent use (nr council, ) . nevertheless, among the early accomplishments of the nsabb was formulating a definition for dual use research of concern (durc), which limited the scope of worrisome work to a small subset of biological research. the nsabb also generated a set of recommendations for communicating durc in publications and other venues. the work of the nsabb proceeded quietly and in relative obscurity until when the u.s. government learned about two submitted manuscripts reporting that highly pathogenic avian influenza virus (hpaiv) could be made mammalian transmissible by laboratory passage in ferrets, and sought an opinion from the board as to whether publication should be permitted. before considering the gof controversy involving influenza virus, it is worthwhile reviewing what is meant by "gof" and the limitations of the lexicon in the controversy. at the most basic level, a gof experiment, as the name implies, is one that gives a biological entity any new property. gof experiments can be highly beneficial and can generate pest resistant crops, microbes expressing proteins for medical therapy such as recombinant human insulin, and new cancer therapies by enhancing lymphocyte function. the controversial aspects of gof experiments in microbiology arise when microbes are modified to have new properties that can enhance their virulence and transmissibility because such experiments raise worries about new diseases, outbreaks, pandemics, biosafety, and biosecurity. in the experience of the authors, the nsabb probably did not anticipate what a problematic dual-use paper would look like until they had the opportunity to consider the two papers that reported the gain of mammalian transmissibility for hpaiv (herfst et al., ; imai et al., ) . what made the two papers highly controversial was that they reported that only a few amino acid changes were sufficient to confer mammalian transmissibility to the h n hpaiv, and there was concern that this information could be used to generate a virus capable of pandemic potential with high mortality by anyone with basic molecular biology and virology skills. the nsabb initially recommended redacting the sequence information, but this was considered not feasible given the legal framework in the u.s., and the two papers (herfst et al., ; imai et al., ) were eventually published in after some modifications to the text. the arguments, counterarguments, and events surrounding that episode are described in various publications (berns et al., a,b; fouchier et al., a) , often by the participants themselves, and will not be repeated here. the major outcome of the great gof controversy of is that it defined and crystallized some of the issues of dual-use research in biology by providing clear examples of experiments that were of great scientific value while also raising biosecurity and biosafety concerns. although both papers were eventually published with full information, the debate and controversy did not solve the central questions of what work should be performed moving forward and how it should be reported. in fact, the controversy erupted again in the summer of following the publication of additional papers describing other gof experiments (e.g., watanabe et al., ) as well as a series of biosafety lapses at u.s. government laboratories, which led the government to impose a pause on these types of experiments with certain viruses until the nsabb could formulate new recommendations and guidelines. the pause was lifted in mid-december, , alongside policy guidance about how gof funding decisions should be made going forward. one of the most vexing questions of the problem of dual-use research is publication of scientific findings. in the u.s., the research community is guided by national security decision directive (nsdd) , formulated in by the reagan admini stration, which states that all fundamental research results could be published freely. in other words, there was either open publication or classification, and nothing in between. viewed from the context of nsdd- the papers involved in the gof controversy were either to be published in their entirety or classified, with the latter alternative being impractical and unfeasible since the work had been carried out in an unclassified environment. with this binary policy, redacting papers because they might contain information useful for nefarious purposes is not an option unless export control regulations are invoked [for a full discussion of the legalities involved, see nas em durc report (national academies of sciences engineering and medicine, )]. consequently, when faced with gof papers containing information that could conceivably be used to enhance the pathogenicity or transmissibility of a virus, editors and journals have almost always opted for full publication, usually requiring more details from the authors about biosafety and biosecurity methods, and often publishing an accompanying editorial emphasizing the scientifically useful aspects of the research [for examples, see dermody et al. ( dermody et al. ( , a ]. such decisions have sometimes elicited strong criticism from members of the virology community (dermody et al., b; wain-hobson, a) . a more recent situation illustrates the difficult issues faced by authors, editors, and journals when publishing papers that contain durc. in , the journal of infectious disease faced the quandary of receiving a paper reporting a new botulinum toxin that was resistant to neutralization by the then-available antisera. in response, the journal took the remarkable decision of publishing the paper without the toxin sequence and vowed to keep the data confidential until an antidote was available (casadevall et al., ; dover et al., ; relman, ; hooper and hirsch, ) . a major factor in withholding the sequence data was the realization that this toxin could be easily produced using standard techniques if the toxin sequence was available and that the availability of such a toxin might pose a major public health risk. this decision was highly controversial since without the sequence information other investigators could not verify the findings reported in the publication or begin to develop medical countermeasures. in fact, subsequent work revealed that the new toxin was indeed neutralized by available sera, thus diminishing the initial concerns (enserink, ) . on one hand, withholding the sequence information from public scrutiny was the responsible action given the potential threat, while on the other hand, such an action delayed verification of the findings and the generation of countermeasures had the threat been real. clearly, manuscripts containing durc pose vexing problems for journals that often must make such decisions alone. at american society for microbiology journals, there are protocols in place for reviewing such manuscripts and the journals have available many individuals with microbiological and safety expertise who can provide input (casadevall et al., ) . other situations are likely to pose different challenges, but it is clear that journals are often poorly equipped to handle the difficult problems involved in publishing durc content. in this regard, we have argued for the need of a national board that can help journals make publication decisions (casadevall et al., ) , although to date no such body exists. another major challenge to the control of durc information is the emergence of preprint servers in biology that allow the posting of research findings before peer review and the proliferation of predatory open access journals that will publish essentially any paper for a fee. consequently, there now exist alternative systems for publication even if standard journals decline to publish a particular article over durc concerns. bypassing traditional publication methods could also allow authors to avoid government scrutiny. it is not an exaggeration that the situation today regarding durc and gof experiments remains highly unsatisfactory. experimental work involving gof experiments on pathogens with pandemic potential is highly regulated with proponents of such work arguing that restrictions on experimental design are unwarranted (fouchier et al., b) while opponents of such experiments argue that such work cannot be justified (wain-hobson, a,b). proponents of gof experiments have noted that data generated in such experiments is useful in epidemiological surveillance (schultz-cherry et al., ), whereas opponents have argued that such work cannot be morally justified because of its inherent risk (lipsitch and galvani, ) . in the u.s., government-mandated pauses on certain types of experiments remain in effect for federally funded research but such prohibitions do not extend to other countries, or to work funded by other sources. although the nsabb continues to analyze and debate the issues involved with durc and gof experiments in the u.s., there is no comparable body on the international scene, where much of this type of research is done. there is no consensus in the scientific community on whether the value of some experiments justifies the risks involved. the central problem in finding a consensus is that the value of the research cannot be measured in real time, while assessments of risk involve assumptions that can lead to widely divergent estimates. for example, some risk-benefit calculations have suggested that a high consequence accident is likely to occur in the near future (lipsitch and bloom, ; lipsitch and inglesby, ) , while others have estimated such probabilities to be near zero (fouchier, ) . however, there is some evidence that the incessant debate, which shows no sign of resolution, is taking its toll on the virology community as shown by surveys suggesting that scientists in training may avoid these areas of research (pfeiffer, ) . this is of particular concern given that recent years have seen new viral threats in the forms of the west africa ebola outbreak, the emergence of zika virus in the americas, and middle east respiratory syndrome coronavirus in the arabian peninsula, highlighting the importance of virology in human preparedness against pandemic threats casadevall, a, ) . one of the most important developments in the past few decades has been a change in the zeitgeist of the field that concerns itself with durc research and gof experiments. in the early years of the twenty-first century, as the u.s. reeled from the / terrorist attacks, which were shortly followed by the anthrax spore attacks, the focus was primarily on biosecurity. at that time, the concern was that terrorists and state actors would use the tools of the molecular biology revolution to create new and more devastating biological weapons. however, as the years passed and no new attacks developed, combined with the attribution of the anthrax spores in mail attacks to a lone insider in a u.s. government laboratory (bhattacharjee and enserink, ) , this security threat appeared to recede. at the same time, a series of unfortunate biosafety lapses in government laboratories heightened concerns about biosafety. hence, today people (at least those outside the security community) appear to be more worried about an unintended release of a pathogen with pandemic potential than a deliberate biological attack. this change in emphasis from biosecurity to biosafety has developed slowly and could easily change if there is another deliberate biological attack or if becomes apparent that adversaries are developing biological weapons. a major problem contributing to the unsatisfactory nature of the current situation is that the nsabb definition of durc does not work well in day-to-day practice. the nsabb defined durc as "life sciences research that, based on current understanding, can be reasonably anticipated to provide knowledge, information, products, or technologies that could be directly misapplied to pose a significant threat with broad potential consequences to public health and safety, agricultural crops and other plants, animals, the environment, materiel, or national security. " although the crafting of this definition was a major achievement at the time in being able to limit the scope of durc to a relatively small portion of biomedical research, thus leaving most biological research to proceed unfettered, subsequent experience showed that the clause "reasonably anticipated" is so subjective in nature that it requires a risk assessment beyond the capabilities of most scientists, reviewers, and editors (casadevall et al., ) . in essence, the definition was helpful in keeping whole swaths of biological research outside the durc category, such as cancer and immunology research, but it difficult to apply in determining what is included in the durc definition. also contributing to an unsatisfactory situation are concerns whether such regulations as the select agents and toxins list help or hinder societal security. on one hand, placing great restrictions on the accessibility to a number of agents and toxins does increase security by making them more difficult to obtain, with the caveat that these are naturally occurring agents that could be obtained from nature by a determined actor. on the other hand, there is the concern that focusing on lists and a relatively short list of agents means that the overwhelming majority of microbial threats are not on the durc radar screen. for example, zika virus emerged as a pathogen of pandemic potential with little or no anticipation from experts. hence, making lists and focusing attention on those agents in the list has the paradoxical potential to reduce biosecurity since regulations are tightened for listed agents, possibly hindering research, while other dangerous agents are neglected (casadevall and relman, ) . a similar argument applies to the current durc oversight policy of the u.s. government, which is focused on a specific list of agents. this process, which is akin to searching under the lamppost, carries great danger because it ignores what could be significant threats. in this regard, it is noteworthy that fungal pathogens are seldom considered as biological threats despite the fact that members of this kingdom have significant weapon potential (casadevall and pirofski, ; casadevall, ) . against this backdrop of dissatisfaction is the fact that science continues to progress very rapidly, introducing new technologies such crispr/cas , gene drives, and more efficient synthetic biology, each of which brings with it new possibilities for research that improves the human condition as well as new tools for nefarious purposes. furthermore, as the technologies improve they are increasingly accessible to more individuals and countries for whom this type of research was previously beyond reach. hence, the problem of durc is likely to become significantly more urgent in the near future. in a world where new infectious agents that can rapidly spread among vulnerable populations are described on a regular basis, humanity needs a healthy research establishment focused on microbial threats. it is estimated that there are a minimum of , mammalian viruses (anthony et al., ) : some fraction of these are probably zoonotic events waiting to happen. as ian malcolm, the fictional character created by michael crichton in the novel jurassic park, stated, "life finds a way. " information and knowledge are the best defenses against these threats. in addition to terror from nature, a new crisis will almost certainly occur in the future arising from a deliberate attack, a new provocative paper, or another biosafety lapse. gof experiments and durc research are essential for preparedness and the question is not whether this research should be pursued but rather how to do it safely and mitigate risk. to date, each of the controversies has been reactive, with proponents and opponents of such experiments responding to a specific finding or study. after more than a decade of discussion on what constitutes durc, benefits and risks of gof experiments, regulations, pauses, and moratoriums, it is increasingly apparent that current approaches are inadequate for the challenges at hand. given these limitations, we have proposed a new framework for durc research that focuses on answering specific questions (imperiale and casadevall, b) . hence, instead of prohibiting certain types of experimentation, we suggest that the way forward is to focus on specific scientific questions and the problems that need answers. for example, if there is a need to determine whether a particular feral virus pathogen has the capacity for mammalian infection and transmission, then gof experiments performed in safe and controlled conditions can be justified. on the other hand, endowing hiv with new transmission properties is not a medically important gof experiment (national academies of sciences engineering and medicine, ). all human activities that involve probing the unknown, ranging from space exploration to tissue culture procedures, carry some degree of risk, and it is nature of humanity to accept risk to attain progress. opponents and proponents of this type of experimentation need to maintain open channels for continued discussion because the dialectic of ideas is likely to result in better decisions. institutional bodies such as the nsabb need to be supported for they constitute important venues for such discussion and recommendations that mitigate risk. in fact, it is important to create similar institutions that can work at the international level since u.s.-based research is a small portion of all microbiological work done worldwide. most importantly, we should remain optimistic that the research community can do the research necessary to obtain information critical to protect our species while minimizing the risks of such work. a strategy to estimate unknown viral diversity in mammals public health and biosecurity. adaptations of avian flu virus are a cause for concern policy: adaptations of avian flu virus are a cause for concern anthrax investigation. fbi discusses microbial forensics -but key questions remain unanswered the anthrax attacks years later don't forget the fungi when considering global catastrophic biorisks on the need for a national board to assess dual use research of concern dual-use research of concern (durc) review at american society for microbiology journals redaction of sensitive data in the publication of dual use research of concern the weapon potential of human pathogenic fungi microbial threat lists: obstacles in the quest for biosecurity? chemical synthesis of poliovirus cdna: generation of infectious virus in the absence of natural template a new determinant of h n influenza virus pathogenesis in mammals sequence changes associated with respiratory transmission of h n influenza virus in mammals the decision to publish an avian h n influenza virus gain-offunction experiment molecular characterization of a novel botulinum neurotoxin type h gene smallpox and the indians in the american colonies biosecurity. as new botulism threat implodes, more questions studies on influenza virus transmission between ferrets: the public health risks revisited pause on avian flu transmission research the pause on avian h n influenza virus transmission research should be ended airborne transmission of influenza a/h n virus between ferrets novel clostridium botulinum toxin and dual use research of concern issues experimental adaptation of an influenza h ha confers respiratory droplet transmission to a reassortant h ha/h n virus in ferrets the importance of virology at a time of great need and great jeopardy a new synthesis for dual use research of concern zika virus focuses the gain-of-function debate expression of mouse interleukin- by a recombinant ectromelia virus suppresses cytolytic lymphocyte responses and overcomes genetic resistance to mousepox rethinking biosafety in research on potential pandemic pathogens ethical alternatives to experiments with novel potential pandemic pathogens moratorium on research intended to create novel potential pandemic pathogens dual use research of concern in the life sciences: current issues and controversies biotechnology research in an age of terrorism is the debate and "pause" on experiments that alter pathogens with pandemic potential influencing future plans of graduate students and postdoctoral fellows? inconvenient truths" in the pursuit of scientific knowledge and public health variola virus immune evasion design: expression of a highly efficient inhibitor of human complement influenza gain-of-function experiments: their role in vaccine virus recommendation and pandemic preparedness characterization of the reconstructed spanish influenza pandemic virus an avian h n gain-of-function experiment of great concern the irrationality of gof avian influenza virus research circulating avian influenza viruses closely related to the virus have pandemic potential analyzing a bioterror attack on the food supply: the case of botulinum toxin in milk biological warfare at the siege of caffa both authors contributed to the writing of this manuscript. references conflict of interest statement: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -x hq b authors: versluys, anne birgitta; boelens, jaap jan title: morbidity and mortality associated with respiratory virus infections in allogeneic hematopoietic cell transplant: too little defense or harmful immunity? date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: x hq b the impact on morbidity and mortality of community acquired respiratory virus (carv) infections in patients undergoing allogeneic hematopoietic cell transplant (hct) is widely studied. here we give an overview of the current literature on the incidence and chance of progression to severe disease in this highly immune compromised population. we discuss the issue whether it is predominantly direct viral damage that causes clinical deterioration, or that it is in fact the allogeneic immuneresponse to the virus that is most important. this is an important question as it will guide therapeutic decision making. it asks for further collaborative studies focusing on sensitive surveillance with pcr techniques and relating clinical data with parameters of immune reconstitution. community acquired respiratory virus infections (carv) include a variety of viruses such as rhinovirus, coronavirus, respiratory syncytial virus (rsv), influenza virus, para influenza virus, and metapneumo virus. carv infections range from asymptomatic carriership to significant respiratory disease. prevalence of carv largely depends on season, detection mode, age of patient and immune status (shah et al., ; hirsch et al., ; green, ) . influenza and rsv have significant seasonal variation, whereas para influenza or rhinovirus cause disease year round (green, ) . highly sensitive diagnostic techniques like polymerase chain reaction (pcr) for the detection of viral dna and rna reveal a high prevalence of carv in the normal population. in healthy children attending day care prevalence is as high as % (moe et al., ) . in children admitted to hospital for respiratory disease - % are tested pcr positive, almost twice as high as in adults admitted to hospital for respiratory disease (ching et al., ) . in the immune compromised pediatric population the prevalence of carv is around - % (fazekas et al., ) with mostly mild symptoms at time of detection. there are many reports on carv prior to, or early after, hematopoietic cell transplantation (hct). recently a large multicenter retrospective analysis in , pediatric hct recipients showed an incidence of . % symptomatic carv infections within year after transplant (fisher et al., ) . surveillance studies in the same population on nasopharyngeal aspirates (npa) routinely performed prior to transplant showed an incidence of % (versluys et al., ) . most studies discuss the risk of progression to viral pneumonia, for various types of carv. only few groups looked at long term outcome. results are conflicting, reported risk of progression is - % (bredius et al., ; hirsch et al., ; chemaly et al., b; fisher et al., ; green, ) . hct is a curative treatment for several malignant and non-malignant childhood diseases. its success is limited by infections, alloimmunity and toxic events. respiratory viruses contribute to post-transplantation morbidity and mortality in different ways. here we provide an overview of recent literature on carv in the hct setting, focusing on the risk of progressive viral lung disease, the role of viruses in the lung microbiome and the potential viral trigger for allo-immunity after hct. reported incidence of rsv in hct-recipients varies between and % (shah et al., ; robinson et al., ; fisher et al., ) , depending largely on season, patients' age and detection methods. progression to lrti occurs in - % (shah et al., ; kim et al., ; fisher et al., ) , with risk factors for progression related to age, donor source, use of steroids, immune status, and concomitant infections (renaud et al., ; chemaly et al., b; kim et al., ; shah et al., ) . mortality rates are around % (chemaly et al., a; robinson et al., ; fisher et al., ) it is important to notice the in most pediatric studies, rsv-positive patients were treated with the antiviral drug ribavirin (molinos-quintana et al., ) , with anti-rsv monoclonal antibodies (palivizumab) or with nonspecific intravenous immunoglobulins (ivig) (el-bietar et al., ) , or with a combination (chávez-bueno et al., ) . aerosolized ribavirin and ivig is recommended for adult hct recipients with rsv lrti (dignan et al., ; waghmare et al., ) . para influenza virus (piv) in hct recipients is systematically reviewed by shah et al. ( a) . the incidence of piv in hct recipients is % (range . - %), % progressing to lrti. significant predictors of lrti progression were infection within days after hct, lymphocytopenia/neutropenia at the onset of infection, use of corticosteroids, younger age, and respiratory co-infections. reported overall mortality is % ( - %), in piv-lrti %. there is currently no licensed therapy for piv pneumonia. influenza is diagnosed in approximately - % of hct recipients, and in up to % of patients with respiratory symptoms in the flu-season. progression to viral pneumonia occurs in - % of patients, and is associated with mortality in - % (fisher et al., ; green, ) . these numbers are strongly influenced by seasonal outbreaks and the subtype of the influenza virus. risk factors for progressive influenza disease are lymphocytpenia/neutropenia and steroid use (kmeid et al., ) . in contrast to many other respiratory viruses, influenza can be treated with neuraminidase inhibitors (waghmare et al., ; green, ) . human rhino virus (hrv)is the most common cause of respiratory virus infections in both immunocompetent and immunocompromised individuals. reported incidence in hct recipients is - % (versluys et al., ; shah et al., ; campbell et al., ; fisher et al., ) . for a long time there was uncertainty about the ability of hrv to cause lower respiratory tract disease. recent studies however, suggest that hrv may be a clinically significant pathogen with the potential to cause serious pulmonary disease in hct recipients (campbell et al., ; seo et al., seo et al., , versluys et al., ) with risk of progression to lrti of - % (shah et al., ; campbell et al., ; fisher et al., ) and hrv related mortality of - % (shah et al., ; campbell et al., ; fisher et al., ) . there is increasing interest in the pathogenecity of human metapneumo virus (hmpv). in a systematic review (shah et al., b) shah et al. summarized all published data on hmpv in patients with hematologic malignancies or undergoing hct. about one third of described cases were children. they report an overall incidence of % (range - %), with a risk for progression to lrti of % (range - %) and a mortality rate of % ( - %) in the total hmpv positive group, and % in the hmpv-lrti group. other respiratory viruses like bocavirus (bov) and coronavirus (cov) in hct setting, are scarcely studied. prevalence of either bov or cov is % in the adult hct population with respiratory symptoms. a significant proportion of the cov infected patients required hospitalization, and some progressed to lrti. in contrast, bov detection was rare and almost always related to co-pathogens (pinana et al., ) . a surveillance study on bov in children found % of children with rti to be pcr positive, as well as % of the healthy controls; thus showing a high prevalence of the virus without necessarily causing disease. progression to lower infection however was associated with higher bov load and viremia, suggesting a pathogenic role in a subgroup of patients (christensen et al., ) . figure shows the incidence of carv in mainly adult hct population, and the impact of carv on lrti and mortality. profound immunosuppression in patients undergoing hct obviously leads to a greater risk of infection, with prolonged shedding of the virus, a higher chance of transmission of disease and a greater risk of progression to severe lower respiratory tract disease. as antiviral therapy is only effective in the minority of the respiratory viruses, and vaccines are not widely available, prevention is very important. the working group of the fourth european conference on infections in leukemia (ecil- ) reviewed the literature on carv in leukemic patients and patients after hct (hirsch et al., ) . for prevention they recommend infection control measures like good personal hygiene, avoiding contact with individuals with a respiratory tract infection and restricting young children from visiting patients. administration if ivig preparations in patients with hypogammaglobulinemia (igg < g/l), and the use of intravenous monoclonal antibody specific for the rsf-f protein (palivizumab) during rsv outbreaks may be considered. they do not advocate routine screening for carv. deferral of chemotherapy or conditioning should be considered, figure | incidence of common respiratory viral infections (crv) and associated progression to lower respiratory tract infections (lrti) and mortality in hct recipients. updated from shah et al. ( ) , based on (versluys et al., ; shah et al., ; chemaly et al., b; campbell et al., ; robinson et al., ; fisher et al., ; green, ) . as well as treatment for rsv and hpiv only in the hct setting. in a joint working group in the uk (dignan et al., ) has reviewed the available literature and made recommendations for the diagnosis and management of respiratory viral infections in patients with hematological malignancies or those undergoing hematopoietic stem cell transplantation. as far as prevention is concerned they come to the same conclusions as ecil- , and add the recommendation for influenza vaccinations in household contacts and medical staff, and post-exposure prophylaxis with oseltamivir in hct patients who have been in contact with influenza. in a recent large prospective study, including adults and children undergoing allogeneic hct, clinical outcomes associated with respiratory viruses (rv) detected prior to hct were analyzed (campbell et al., ) . multiplex pcr testing for rv was done on nasal washes or nasopharyngeal swabs. in % of patients a rv was detected, % of them being asymptomatic. in the pediatric subgroup, defined as aged < years, the prevalence of rv was higher ( %), with a larger proportion being without symptoms ( %). the rv positive patients were significantly younger and had higher risk underlying disease with lower lymphocyte count. overall mortality at day was significantly higher in rv-patients than in non-rv patients ( . vs. . %; hr . ; % ci . - . ; p = . ). in % of the deceased patients the cause of death was thought to be directly related to the pre-hct rv, no data are given about the cause of death in the other patients. patients with rhinovirus performed worse compared to the other rv. this may be partly explained by the fact that most patients with rsv or influenza where treated with antiviral therapy or had their transplant delayed. data on longer follow up are lacking (campbell et al., ) . hutspardol et al. retrospectively studied treatment related mortality (trm) and long-term pulmonary complications in children who had respiratory symptoms and a rv detected within days after allogeneic hct. the overall frequency of documented rv infections was . %, half of the patients presented with signs of a lrti and mortality rate at day was %. cause of death was pneumonitis/ards in all, with symptoms occurring on day - after hct. during follow up ( . years, range . - . ) no chronic pulmonary complications nor allo-immune lung syndrome was observed (hutspardol et al., ) . with regard to long term pulmonary function chien et al. studied , adult hct recipients by performing routine pulmonary function tests years after hct. airflow obstruction, defined as an annualized decline in fev of more than %, occurred in % of patients and had impact on overall mortality. higher age at transplant, gvhd category, pulmonary function pre-transplant and the occurrence of a respiratory virus infection within the first days after hct were significant risk factors for airflow obstruction (chien et al., ) . erard et al. further studied the association of rv and airflow decline, and found that this was particularly true in patients after lrti caused by parainfluenza virus or respiratory syncytial virus (erard et al., ) . in a retrospective study among , pediatric hct recipients in us centers . % acquired symptomatic rv within the first year after hct (fisher et al., ) . in line with others, rhinovirus was the most common virus, followed by rsv and piv. rv was detected after a median of ( - ) days after hct. most children had urti only, in patients with hmpv there was significantly more lrti. during months follow up % required mechanical ventilation and % had significant pulmonary sequelae like bronchiolitis obliterans, subacute pulmonary problems and other not specified pulmonary complications. all cause mortality among rv positive patients was %, compared to % in the non-carv group. recent steroid exposure and rv detection within days after hct were poor prognostic factors for morbidity and death. at least % of death were not attributable to carv infection. the timing of the events is also remarkable, as % of deaths occurred more than days after diagnosing carv infection, which is at least months after hct for most. the widespread use of pcr diagnostics has led to an increase in the detection of carv in patients undergoing hct. many of these patients become symptomatic and a significant proportion develops lrti. there is a clear increased risk for mortality in carv positive patients. hence, prevention and development of anti-viral drugs are of great importance. however, one could debate about the reason for severe morbidity and mortality in carv positive patients. how do you diagnose progressive viral infection? the carv will not be cleared for months because of the immunocompromised state of the host after hct, so finding positive pcrs is not convincing enough. timing of (progression of) symptoms in relation to immune reconstitution might be helpful in answering the question if it is progression of viral damage or if the donor derived immunity actually is targeting the lung. in last decade more and more evidence has emerged that "triggered" alloreactivity may play a crucial role in toxicity and mortality. this holds true for hct, but is also recognized in solid organ transplantation. in the context of lung transplantation several studies have examined the role of rv in the development of chronic lung allograft dysfunction (clad), a form of chronic rejection of the lung (kumar et al., (kumar et al., , fisher et al., ) . many, but not all, reported an association between rv and clad. pooled analyses of studies on rv and clad (vu et al., ) did not confirm the association, mainly due to the heterogeneity of studies and limitations in design, diagnostic techniques and definitions. fisher et al. tried to overcome these limitations by studying a more homogenous cohort of lung transplant recipients, using modern molecular assays to detect rv and applying consensus definitions of clad (fisher et al., ) . in patients, ( %) developed clad at a median of weeks (interquartile range (iqr): - weeks). in patients ( . %) a respiratory viral episode was seen, after a median of weeks post lung transplantation (iqr: - weeks). in multivariate analysis rv was associated with clad (hr . , % ci . - . ; p = . ). this association was stronger the more proximate the rv occurred after lung transplantation. our group studied the role of respiratory viruses (rv) in immune mediated lung disease after hct, analogous to this phenomenon as described after lung transplantation (versluys et al., (versluys et al., , . the host-vs.-graft chronic allograft rejection in lung transplantation is in many ways comparable to the graft-vs.-host inflammation in hematopoietic cell transplantation. in a cohort of children undergoing allogeneic hct routine npa and bal sampling for the presence of rv was done prior to transplant. rv was found in % ( % in bal/npa, % in npa-only). rhinovirus was the most frequently detected rv ( %). allo-immune lung syndrome (allo-ls), defined as bronchiolitis obliterans syndrome (bos) or idiopathic pneumonia syndrome (ips), occurred in %, after a median of . weeks ( - weeks). rv-positivity in bal was a predictor for allo-ls (hr . , % ci . - . ; p = . ). no other predictors were found. the hypothesis is that rv causes epithelial damage and triggers an allogeneic immune response leading to severe lung disease. so the lung disease does not occur primarily from progressive viral infection during the period of low immunity, but from allo-immune mediated damage weeks after hct. this inflammatory aspect of disease might explain the reported benefit of steroid use on the risk of mechanical ventilation among hct recipients with influenza (choi et al., ) , and the controversy on risks of steroid use in case of carv after hct (waghmare et al., ) . more and more is known about the role of microbiota in human health. so far research has largely focused on the gut microbiome, also in the context of gvhd. but the microbial ecosystem at other body sites, including the respiratory tract is attracting growing attention. host and environmental factors influencing the respiratory microbiota include genetics, microbial exposure (birth mode, feeding type, day care), vaccination, infections and antibiotics (man et al., ) . viral infection interacts with the microbiome by disrupting the airway epithelial barrier facilitating bacterial adhesion, liberating host derived nutrients and decreasing muco-ciliary clearance. in addition respiratory viruses can modulate innate and adaptive immune responses promoting bacterial colonization (man et al., ) . moreover, it is becoming clear that the virome should be seen as a part of the microbiome, that affects the function of the host immune system (cadwell, ) . the role of a disturbed respiratory microbiome/virome in lung disease is postulated for asthma and chronic obstructive pulmonary disease (copd) (zou et al., ) . impact of carv on outcomes after hct is an intriguing topic where pathogenesis is not completely understood. is the poor immune system associated with progressive infection? does alloimmunity play a crucial role in lung toxicity? most studies describe data on symptomatic patients where carv is detected at time of symptoms. only few studies report on pre-hct sampling, although we know a large proportion of our patients is carv positive with only mild symptoms. time of (worsening) of symptoms, warranting viral diagnostics and thus detecting the carv, is often weeks after hct. are these nosocomial acquired viruses, or were these virus already present and giving symptoms after a certain period of time? are the viruses acquired after discharge? but then immunity is usually restored to a certain degree. from various infectious diseases, like rsv bronchiolitis (fonseca et al., ) , immune reconstitution inflammatory syndrome (iris) in hiv patients (with cryptococcal meningitis, cmv retinitis or bcg-itis) (walker et al., ) or iris in non-hiv immunesuppressed patients with immune recovery (followed by worsening of treated tuberculosis, idiopatic pneumonia or hepatitis) (sueki et al., ) , we know the harmful effect of immune response on the patient. the debate about inappropriate immune response on infectious triggers is especially intriguing in the allogeneic setting. the definition criteria for alloimmune mediated lung syndromes (panoskaltsis-mortari et al., ; jagasia et al., ) describe the clinical, radiologic and functional aspects of lung pathology, with exclusion of other evident causes of this phenotype, like heart failure and infection, including respiratory virus infection. one can argue if this holds true for respiratory viruses detected by pcr. the detection modes have become much more sensitive over time, so the impact of positive findings on the disease criteria should be reevaluated. in the hct population with its high prevalence of rv, these viruses will have a long persistence making them detectable for weeks after initial infection, with uncertain meaning for their role in pathology. an interesting paper in this matter was recently published by seo et al. ( ) . in patients with ips, they went back to bal samples at time of diagnosis, and applied more sensitive diagnostics for microbial pathogens. in % of patients an occult pathogen was found, % being a respiratory virus. all patients were treated with steroids because of ips. overall mortality was higher in the group of patients with an occult pathogen, than in the group without. the authors conclude that these patients had had to be excluded as ips patients, that they had infectious pneumonia and that steroid treatment had adversely influenced their outcome. however, as we are not informed about rv status pre-hct, this could also be persisting rv after hct, triggering immune-mediated lung disease (ips). in that situation steroids are beneficial in the treatment at the moment of clinical deterioration. in conclusion, despite the growing awareness of carv infections in hct patients, well-designed studies are lacking that systematically evaluate diagnostic and therapeutic strategies of 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patients research on the human virome: where are we and what is next all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. the reviewer pv declared a past co-authorship with one of the authors jb to the handling editor.the remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © versluys and boelens. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -ava u y authors: rady, hanaa i.; kholy, amani el title: prevalence of human rhinovirus infection in young children with acute wheezing() date: - - journal: gaz egypt paediatr assoc doi: . /j.epag. . . sha: doc_id: cord_uid: ava u y introduction: recurrent wheezing is one of the leading causes of chronic illness in childhood. we aimed to evaluate the prevalence of human rhinovirus (hrv) infection in the acute attack of wheezy chest which began after a respiratory illness. methodology: the study was conducted on children aged months to years presenting to the emergency department with an acute wheezy episode either for the first time or recurrent wheeze defined as > reports of wheezing in the first years of life. all subjects were subjected to a complete history and clinical examination. chest x-ray was done to all subjects. nasopharyngeal and oropharyngeal swabs were obtained from all subjects and the presence of hrv was determined by pcr examination. results: by pcr method, patients ( . %) were positive for viral infection. due to viral co-infection, . % ( cases) were +ve for respiratory syncytial virus followed by hrv . % ( cases). conclusion: hrv was the second common viral infection in children with wheezes. its prevalence was more in winter with higher incidence of recurrence. compared to the other respiratory viruses, it had the higher mortality . %. asthma is a heterogeneous and multi-factorial disease that manifests as episodes of coughing, wheezing, and shortness of breath mainly at night. the major pathophysiology of asthma is bronchial inflammation with airway hyper-responsiveness, which results in reversible airway obstruction. among the various factors that have been involved in the pathogenesis of asthma, viral infections are the most prominent. viral infection affects wheezing and asthma in children and adults of all ages. wheezing illnesses are usually viral in origin, and children with more severe wheezing episodes are more likely to develop asthma later on in their life. human rhinoviruses (hrv) are not only the main pathogens responsible for the common cold, but are also now recognized to have a major impact on asthma pathogenesis. children who experience repeated rhinovirus-induced wheezing episodes in infancy have a significantly increased risk of developing asthma, even when compared to children who experience wheezing induced by respiratory syncytial virus (rsv). the aim of this study was to determine the prevalence of human rhinovirus as a cause of acute wheezing in egyptian children after an acute respiratory illness. a prospective study including children aged months to years presenting to the emergency department (ed) of cairo university children hospitals, with an acute wheezy episode (signs of respiratory distress and expiratory wheezes on auscultation and/or hyperinflation of the chest on chest radiograph) either for the first time or recurrent wheeze defined as > reports of wheezing in the first years of life. we excluded children with underlying cardiac or chronic pulmonary disease (other than asthma), the presence of stridor or egyptian pediatric association gazette j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / e p a g daily treatment with oral corticosteroids for > days prior to presentation. all included cases had complete physical examination including grades of respiratory distress chest x-ray and laboratory investigations in the form of oxygen saturation, blood gases, and complete blood count. a nasopharyngeal and oropharyngeal swabs were taken and reverse transcription pcr was used to screen the samples for hrv. oropharyngeal swabbing: a dry sterile tip flocked with nylon fiber swab applicator was used to swab both the tonsils and the posterior pharynx. nasopharyngeal swabbing: a flexible sterile nylon fiber swab applicator was inserted into the nostril and back to the nasopharynx. it was then slowly withdrawn with a rotating motion. sample processing: the swabs were placed in a ml centrifuge tube labeled with the patient unique id and containing ml viral transport media (vtm: consisting of a sterile solution of bovine albumin fraction v, hepes buffer, penicillin, and streptomycin in hank's balanced salt solution). the received swabs inside the ml tube were agitated vigorously for s using a vortex mixer to free cells from the swab tip, and then both swabs were removed from the tube and discarded using a forceps. the sample was kept in a À c deep freezer until processed. nucleic acid extraction: automated extraction was performed using the qiacube machine with qiaamp Ò viral mini kit cat# , (qiagen) using the manual lysis protocol which consists of purification from manually lysed cell-free body fluids. multiplex real-time pcr was performed using the anyplex tm ii rv detection cat# rv g y from seegene inc, compatible with cfx tm real-time pcr bio-rad, the interpretation was done using the see gene viewer program. management of patients was followed depending on their condition whether received ambulatory therapy, required hospital admission or required intensive care unit admission. we recorded the length of hospital stay and patient's outcome: discharged, transferred or died. the study was explained for each parent before inclusion and an informed written consent was obtained from parents before enrollment. statistical package for social science (spss) version . was used for analysis of data. data were summarized as mean, sd and percentages. non parametric (mann-whitney u) test was used for analysis of quantitative data, as data were not symmetrically distributed. while chi square test was used for detection of risk factor for rhinovirus infection. p value was considered significant if < . . we studied children that presented to children hospital of cairo university ed with acute wheezy chest. the patients' condition varies from requiring nebulizer at the ed, hospital admission and oxygen supplementation to pediatric intensive care admission for infusion therapies or mechanical ventilation. the studied patients were male and female aged months to years. by pcr method, patients ( . %) were positive for viral infection and ( . %) patients were negative. from the viral infected patients patients ( . %) had single infection, while patients ( . %) had co-infection with more than one virus. rsv affected . % ( cases), followed by hrv . % ( cases) ( table ) . all the demographic and clinical data of cases were demonstrated in table . the median age of patients who were positive for rhinovirus was younger than rhinovirus negative patients, and this was statistically significant. regarding clinical data cough, grunting, fever and vomiting were more prevalent among hrv +ve patients p = . ; . ; . and . ; respectively, while cyanosis and diarrhea were more prevalent among hrv -ve patients p = . and . ; respectively. patients with history of recurrent wheezes ( . %) were more common than patients with first-time wheeze ( . %), p = . as shown in table . recurrent attacks of wheezes were more common among hrv +ve cases ( . %) compared to other viruses ( . %), a difference which was statistically significant p = . . there was a statistically significant difference between the frequency of rhinovirus positive and negative patients throughout the studied period. rhinovirus was most frequently detected throughout the winter months from december to february, as shown in fig. . table comparing the fate between patients positive for rhinovirus and negative for rhinovirus among virus positive patients: there was no significant difference regarding the illness severity between patients infected with hrv compared to other viruses: all patients who tested positive for rhinovirus infection by pcr were admitted to the pediatric intensive care unit (picu) with . % needing mechanical ventilation while . % of patients who tested negative for rhinovirus by pcr required icu admission with % needing mechanical ventilation, p = . . as regards outcome there was statistically significant difference in outcome between rhinovirus positive and negative cases. mortality rate was higher among hrv positive cases ( . %) vs ( . %), p = . . among children that presented to the ed of cairo university children hospital with wheezes, atopic wheezes were only in ( . %) while the majority was viral induced ( . %). the predominance of hrv infection among younger age was in accordance with guittet and colleagues, who studied children, median age was . ( . - . ) months, and where more than half, ( . %) were under months. jartti and colleagues, studied children hospitalized with acute expiratory wheezing and the presence of rhinovirus, rsv, coronavirus, metapneumovirus and enterovirus rnas were detected in the nasal secretions. in our study, we had almost the same viruses isolated from wheezy infants and children but rsv was most common, followed by hrv then adenovirus. another study done on hospitalized children admitted with acute expiratory wheezes, viruses were detected in the nasopharyngeal aspirates by pcr. a viral pathogen was identified in ( %). rsv was the most frequently detected ( %), followed by rhinovirus ( %). in our study, hrv +ve cases represented with recurrent attacks of wheezes rather than single attack. hrvs have been recognized as an extremely common cause of recurrent wheezing in early childhood by message and his colleagues, . hrv infection was associated with a -fold risk of developing asthma. among rhinovirus positive patients, co-infection with more than one virus was ( . %, cases) than infection with single virus ( . %, cases). fujitsuks and his colleagues , had co-infection detected in ( . %) patients while, smuts and his colleagues , it was %. , interestingly, our study comes in agreement with a study done by feng and his colleagues, which was performed on nasopharyngeal swabs obtained from children hospitalized for acute lower respiratory tract infection. approximately % were confirmed as hrv-positive cases with % confirmed as rsvpositive cases. this study shows also that hrv infection occurs sporadically throughout the year with an hrv-positive rate higher in winter and autumn. all our hrv +ve cases were admitted to picu and this could be explained by the presence of bacterial super-infection. this comes in agreement with a study done by guittet and colleagues . the results showed that . % of patients with rhinovirus showed pneumonia and % of them showed abnormal chest x-ray in the form of increased bronchovascular markings, consolidation, pneumothorax and pleural effusion. conclusion hrv was the second common viral infection in children with wheezes. its prevalence was more in winter with higher incidence of recurrence. compared to the other respiratory viruses, it had the higher mortality . %. no conflict of interest. no funding from any source. variants of dennd b associated with asthma in children gina guidelines on asthma and beyond rhinoviruses, allergic inflammation, and asthma role of rhinovirus infections in asthma rhinovirus and acute respiratory infections in hospitalized children. retrospective study persistance of rhinovirus and enteroviruses rna after acute respiratory illness in children role of emerging respiratory viruses in children with severe acute wheezing rhinovirus-induced lower respiratory illness is increased in asthma and related to virus load and th / cytokine and il- production the role of rhinovirus infections in the development of early childhood asthma a molecular epidemiological study of respiratory viruses detected in japanese children with acute wheezing illness human rhinovirus infection in young african children with acute wheezing detection and clinical features of human rhinovirus in hospitalized children with acute respiratory tract infection in eastern areas of guangdong province i would like to thank all the icu team, the laboratory team and all my patients for their cooperation and sincerity during the study period. key: cord- -w m ci i authors: yamin, mohammad title: it applications in healthcare management: a survey date: - - journal: int j inf technol doi: . /s - - - sha: doc_id: cord_uid: w m ci i healthcare management is currently undergoing substantial changes, and reshaping our perception of the medical field. one spectrum is that of the considerable changes that we see in surgical machines and equipment, and the way the procedures are performed. computing power, internet and associated technologies are transforming surgical operations into model based procedures. the other spectrum is the management side of healthcare, which is equally critical to the medical profession. in particular, recent advances in the field of information technology (it) is assisting in better management of health appointments and record management. with the proliferation of it and management, data is now playing a vital role in diagnostics, drug administration and management of healthcare services. with the advancement in data processing, large amounts of medical data collected by medical centres and providers, can now be mined and analysed to assist in planning and making appropriate decisions. in this article, we shall provide an overview of the role of it that have been reshaping the healthcare management, hospital, health profession and industry. internet and web . have been instrumental in evolving most of the information technology (it) applications that we are now accustomed to. barely years ago, we had not imagined the technological advancements, which are now implemented into our lives. technological advancement, riding on the wave of disruptive technologies of the last quarter of a century, is reshaping and redefining the world's social order and norms. the medical field is part and parcel of our lives and is perhaps one that we care the most about. in the recent times, this field has undergone vast changes. the medical profession today has evolved greatly since the beginning of this century. not only the medical procedures, the medical data flow and management has also undergone considerable improvement. advance data transfer and management techniques have made improvements in disease diagnostic and have been a critical role in national health planning and efficient record keeping. in particular, the medical profession has undergone substantial changes through the capabilities of database management, which has given rise to the healthcare information systems (his). medical data collected from different institutions can now be mined and analysed in time using big data analytics tools. this article shall provide an overview of the involvement, importance and benefits of it and how data management is contributing to the medical science field. in particular, the following will be discussed: . patient and data management . big medical data analytics . privacy and security of healthcare data artificial intelligence and robotics in healthcare artificial intelligence (ai) is a field of computer science which has its roots in logic (mathematics), psychology, philosophy, linguistics, arts, science and management among others. many ai-dominant tools and applications can be found in games, auto spare parts, heavy machinery and various medical instrumentations. according to [ ] , many programs are developed with the help of ai to perform specific tasks which make use of many activities including medical diagnostic, time sharing, interactive interpreters, graphical user interfaces and the computer mouse, rapid development environments, the linked listdata structure, automatic storage management, symbolic, functional, dynamic, and object-oriented programming. here are some other examples. pharmaceutical developments involve extensive clinical trials in real environment and may take many years, sometimes more than a decade. this process requires a considerable amount of resources and can cost billions of dollars. in order to save lives and money, it is desirable to expedite this time consuming process to make healthcare cheaper. an urgency of this nature was witnessed during the ebola crisis in some west african nations [ ] . an urgency of this nature was witnessed during the ebola crisis in some west african nations [ ] , where a program powered by ai was used to scan existing medicines that could be redesigned to fight the disease. nowadays medical consultation, with the help of an online illness database, can take place without the direct interaction of a medical practitioner. an example of such a practice can be found in [ ] . advantages of such consultations lies in the fact that the symptoms presented online by the patients are matched with those of about ten thousand known diseases [ ] , whereas doctors can only match the symptoms against a fraction of the known diseases which they are familiar without the intervention of an it database. likewise virtual nursing to support patients online is also available from [ ] . yet another app [ ] developed by boston children hospital provides basic health information and advice for parents of ill children. since the introduction in s, robots have been used in many scientific and social fields. their use in fields like nuclear arsenal, production of automobiles, and manipulations in space has been widespread. in comparison, the medical science field has somewhat been gradual to take advantage of this technology. nevertheless, robots are now days being utilised to assist in or perform many complex medical procedures, which has given rise to the term robotic surgery. a comprehensive discussion of robotic surgery is available in [ ] . indeed there are many advantages of using robots in healthcare management. for example, developing pharmaceuticals through clinical trials can take many years and cost may rise to billions of dollars. making this process faster and more affordable could change the world. robots can play a critical role in such trials. robots are also very effective in medico training where simulations are achieved. use of robots in abdominal surgery [ ] is innovative in comparison to the conventional laparoscopy procedures, and has the potential to eliminate the existing shortcomings and drawbacks. according to [ ] , the most promising procedures are those in which the robot enables a laparoscopic approach where open surgery is usually required. likewise robotic surgery for gynaecology related ailments [ ] can also be very effective. the conventional laparoscopy, the surgeon would have limited degree of freedom with a d vision, whereas the robotic system would provide a d view with intuitive motion and enable additional degrees of freedom. prostatic surgery, being complex in the way of the degree of freedom, is another area where robots are very effective [ ] . nosocomial infections pose a major problem hospitals and clinics around the world. this becomes worst in case of large and intense gatherings of people like in hajj [ ] . to keep the environment free of these viruses and bacteria, the environments needs to be cleaned efficiently and regularly. however some viruses like ebola [ ] and mers coronavirus [ ] are highly contagious and cannot be cleaned by humans without having protective gear, which can be costly and may or may not be effective. cleaning the hospital with chlorine is an effective way. however, there are many drawbacks of this method including a high risk for cleaners to be infected themselves. a sophisticated approach was implemented in us hospitals by using robots [ ] to purify the space. however, some of the robots used are not motorized and may require a human to place them in the infected room or area, posing the risk of infection to humans. it is not known as to which substance or matter can effectively eliminate deadly viruses like ebola and mers coronavirus. however, according to [ ] , beta-coronaviruses, which is one of the four strands of mers coronavirus, can be effectively eliminated by uv light. for such a deadly and highly contagious virus clearing, a robot that can be remote controlled is needed, which can move forward, backward and sideways, and is able to clean itself. concept and related technology of virtual reality (vr), augmented reality (ar) and mixed reality (mr), which originate from image processing and make extensive use of ai, have been in use for decades in games and movies. for some time now, the vr, ar and mr tools and technology are assisting the medical field in a significant way. virtual reality has been used in robotic surgery to develop simulators to train surgeons in a stress free environment using realistic models and surgical simulation, without adversely affecting operating time or patient safety [ ] . an account of ar and vr technologies, including the advantages and disadvantages can be found in [ ] . the ar and vr technology can be very useful in medical and healthcare education, an account of which can be found in [ ] . applications of vr, ar and mr can also be very helpful in urology [ ] . healthcare management information systems (hmis) is the generic name for many kinds of information systems used in healthcare industry. the use of hmis is widespread in various units of hospitals, clinics, and insurance companies. a doctor records patient data (habits, complaints, prescriptions, vital signs, etc.) in one such system they use. a number of hospital/clinic units like radiology, pathology, and pharmacy store patient data in these systems for later retrieval and usage by different stakeholders as shown in fig. . appointment system itself can also be part of a hmis or a standalone system. modern healthcare without hmis cannot be comprehended. an information system or electronic system dealing includes a database system, sometimes known as the backend of the main system. a database is a collection of data, tailored to the information needs of an organisation for a specific purpose. it can grow bigger or shrink to a smaller size depending on the needs of different times. a database in itself would be of no use unless it was coupled with a software system known as the database management system (dbms), which facilitates various functions and processes like data entry and storage, retrieval, and update, data redundancy, data sharing, data consistency, data integrity, concurrency, and data privacy & security. more information about database systems can be found in [ ] . data in an information system can be organised in several different ways. classical representation of data [ ] is tabular. most tables can be regarded as mathematical relations, for this reason, a dbms with tabular data organisation is known as relational. relational databases have by far been dominant in the business organisations until recently. in the eighties and nineties of the last century, object-oriented and semantic data models were also introduced. in particular, object-oriented databases [ ] became very popular for managing audio-visual data. semantic database technology [ ] , although very efficient for search queries, did not reach its expected peak, perhaps because of its complex data model dealing with ontologies. a comparative study of relational, object oriented and semantic database systems is carried out in [ ] . although the evolution of information technologies began much earlier, however, its implementation by businesses and corporations occurred in the second half of the last century. the healthcare industry a bit slow to take the advantage of the technology. earlier, medical records and systems were paper based. thus the first phase of the usage of information technology and systems in hospital and healthcare management was to transform paper based records to database systems. not only did it change paper based systems to electronic ones but also allowed data manipulation. as a result, more data could be processed in a very short time, which was critical in making timely decisions. a discussion of early stages of this transformation can be found in [ ] . consolidation of this technology resulted in healthcare management information systems (hmis). timely decision is critical in all walks of life but it assumes a much greater urgency in some healthcare cases. hmis is capable to transferring data from one location or application to another within split of a second, as opposed to days without the help of electronic systems. this allows users to access, use and manipulate data instantly and concurrently. as the technology continues to evolve and refine, the performance of information systems operations, in particular those of hmis, is bound to improve. ubiquitous health system is meant to provide healthcare services remotely. according to [ ] , ubiquitous healthcare system means the environment that users can receive the medical treatment regardless of the location and time. essentially, a ubiquitous healthcare system would monitor daily or periodic activities of patients and alert the patients or health workers of problems, if any. a discussion of national healthcare systems in korea, including the concept of u-hospitals, is discussed in [ ] . a rich picture of a ubiquitous healthcare system is shown in fig. . internet of things (iot) is an emerging technology. data in organisations, including hospitals, clinics, and insurance companies, is increasing every day. to process this large data, advanced data processing techniques are combined into what is now known as the big data analytics. internet of things (iot) is a general paradigm. when dealing with medical environment, it is known as internet of medical of things (iomt), which is same as the medical internet of things. a discussion of iomt can be found in [ ] . the fundamental objective of iot is to provide ubiquitous access to numerous devices and machines on service providers (sps) [ ] , covering many areas such as location based services (lbs), smart home, smart city, e-health [ , ] . these tools, devices and machines cover service providers in many areas of location based services. a few other could be smart home, street or city, elements of ubiquitous health systems, tools for electronic learning and electronic business, the number which is increasing day by day and it may reach fifty billions in [ , ] . we have conceptualised this in fig. . the iot applications invariably use cloud storage coupled with fog computing [ ] . data in organisations is growing at a rapid rate. some organisations have collected huge pile from their operations. organisational data needs to be mined and analysed on a regular basis to uncover hidden patterns, unknown correlations, market trends, customers, patients and treatment records to help organizations to plan and make more informed decisions. mining and analysis of big data captures the immense wealth of intelligence hidden in organisational data, makes information transparent and usable at much higher frequency, and enhance performance. all of these contribute to providing higher value in efficient decision making. when data grows, it cannot be handled manually, often big data organisations use robotic arm to perform data loading and unloading operations. medical data is also growing at a rapid rate in many hospitals, clinics and insurance companies. mining and analysis of healthcare data is even more critical as it relates to the wellbeing of the society. data centric research is highly desirable to find answers to questions on many chronical diseases, and to plan and manage national and international health programs. potential befits of big data analytics in healthcare are discussed in [ ] . in particular, authors have provided five strategies for success with big data. these are ( ) implementing big data governance, ( ) developing an information sharing culture, ( ) training key personnel to use big data analytics, ( ) incorporating cloud computing into the organization's big data analytics, and ( ) generating new business ideas from big data analytics. a survey of big data analytics in healthcare and government is conducted in [ ] , where the need for the analytics is linked to the provision and improvement of patient centric services, detection of diseases before they spread, monitoring the quality of services in hospitals, improving methods of treatment, and provision of quality medical education. authors in [ ] identify medical signals as a major source of big data, discuss analytical techniques of such data. some of the well known tools used for big data analytics are apache tm hadoop Ò is highly scalable storage platform. it provides cost-effective storage solution for large data volumes. it doesn't require any particular format. apache spark is an open source, in-memory processing machine. its performance is much faster than that of hadoop. mapreduce is used for iterative algorithms or interactive data mining. there are many other big data analytics tools, a list of them can be found at http://bigdata-madesimple.com/top- -big-data-tools-data-analysis/. there are always security and privacy concerns associated with data. protection of privacy and security is a responsibility of data holding organisations. with medical data these concerns assumes higher pronounce and priority. medical data can be very sensitive, and may be linked to life and death of the patients. often socio political issues are linked with medical data. it is well known that india was divided into two countries giving rise to pakistan in . mohammad ali jinnah, very clever and shrewd, was the leader of pakistan campaign, larry collins and dominique lapierre [ ] argue that the partition of india could have been avoided if ''the most closely guarded secret in india'' had become known. jinnah was suffering from tuberculosis which was slowly but surely killing him. in [ , , ] , privacy issues of data are discussed. healthcare procedures and management are now greatly relying on it applications, which are providing realtime access to data and utilities. without these applications, healthcare will be limited, compromised and prone to major problems. many of the medical equipments, which are used in procedures, are highly dependent on technology. ai, robots, vr, ar, mr, iomt, ubiquitous medical services, and big data analytics are all directly or indirectly related to it. hmis is critical for efficient management of records, appointment systems, diagnostics 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journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: up q k q porcine epidemic diarrhea virus (pedv) is the highly contagious, causative agent of an economically important acute enteric disease in pigs of all ages. the disease is characterized by diarrhea and dehydration causing mortality and growth retardation. in the last few decades, only classical pedv was reported sporadically in europe, but in outbreaks of pedv were described in germany. phylogenetic analysis showed a very high nucleotide similarity with a variant of pedv that was isolated in the us in january . the epidemiological situation of pedv infections in the netherlands in was unknown and a seroprevalence study in swine was performed. in total, blood samples from sows from farms and samples from wild boars were collected from may till november and tested for antibodies against pedv by elisa. the apparent herd prevalence of . % suggests that pedv was not circulating on a large scale in the netherlands at this time. however, in november a clinical outbreak of pedv was diagnosed in a fattener farm by pcr testing. this was the first confirmed pedv outbreak since the early nineties. sequence analyses showed that the viruses isolated in and in the netherlands cluster with recently found european g b strains. this suggests a one event introduction of pedv g b strains in europe in , which made the netherlands and other european countries endemic for this type of strains since then. porcine epidemic diarrhea (ped) is an economically important acute enteric disease in pigs of all ages. the disease is characterized by diarrhea and dehydration causing mortality -particularly in neonatal pigletsand growth retardation. the causative agent is porcine epidemic diarrhea virus (pedv), which is an enveloped, positive singlestranded rna virus, belonging to the family of coronaviridae (pensaert and de bouck, ) . the genome of pedv is approximately kb long and about two-third encodes for non-structural proteins and one-third of structural proteins (kocherhans et al., ) . among these proteins, the main research interest is focused on the spike (s) gene and its glycoprotein product s, mediating receptor binding and membrane fusion (li et al., ) . although only one serotype has been described, phylogenetic studies of the s gene showed that pedv can be genetically separated into two groups: genogroup (g ) and genogroup (g ). each genogroup can be further divided into subgroups a and b, and a and b, respectively (lee, ) . classical ped, now grouped g a, was first recognized as a severe swine enteric disease separate from transmissible gastro enteritis (tge) in the united kingdom in and first described in belgium in (pensaert and de bouck, ) . in the eighties and nineties, the virus was detected in many countries in europe including the netherlands and from europe pedv spread to asia, where it caused large outbreaks with considerable losses in the pig industry (song and park, ) . until , north america was considered to be free of pedv infections (cima, ) , but in that same year highly virulent strains of pedv emerged in the united states of america (us), causing diarrhea, vomiting and loss of appetite in pigs of all age groups and up to % of mortality in suckling piglets (chen et al., ; huang et al., ; stevenson et al., ) . this strain, typed as g b, rapidly spread across the us, canada, mexico and several countries in south america . in the last few decades, only classical pedv (or g a) was reported sporadically in europe (alborali et al., ; martelli et al., ) . in , outbreaks of pedv were described in germany and phylogenetic t analysis showed a very high nucleotide similarity with a variant of pedv (oh ) containing nucleotide insertions and deletions in the s gene (s-indel) that was isolated in the us in january stadler et al., ) . this variant, typed as g b, caused mild clinical signs and lower mortality rates in suckling piglets compared to other circulating pedv g b strains in the us . since the reports of outbreaks in germany, more reports about outbreaks of this particular s-indel virus in several european countries have been published, among which france, belgium, spain, portugal and austria (efsa, ; grasland et al., ; mesquita et al., ; steinrigl et al., ) . this suggests that this mild pedv variant is circulating in europe since the beginning of . the aim of this study was to determine the status of pedv in the netherlands with a serological survey and to investigate the first pedv outbreak in the netherlands since the early nineties. the number of required blood samples from animals and farms to estimate the seroprevalence of pedv in dutch sow herds was calculated based on the following assumptions: pedv is highly contagious and no vaccination against this virus was carried out in the netherlands. as a result, it was expected that, if pedv was present in a sow herd, the within herd prevalence would be very high (bertasio et al., ; goede et al., ) . the required number of blood samples per herd was calculated using winepiscope . (thrusfield et al., ) . to detect infection in a herd with % probability, an estimated within herd prevalence of %, and an average herd size of sows per farm (wur, ), three blood samples per farm were required. in , there were approximately sow farms in the netherlands (wur, ) . in order to show with a high probability ( %), that less than % of the dutch farms (n = ) were infected, farms would need to be tested (thrusfield et al., ) . herds were randomly selected stratified by pig density per province to represent the total dutch sow herd population ( fig. a and b). statistical analyses were performed using stata/se version . software (stata corporation, ). for the serological survey, blood samples were selected from samples collected for the obligatory monitoring of aujeszky's disease (pseudorabies), swine vesicular disease (svd) and classical swine fever (csfv). additionally, blood samples were collected from sows at slaughter (vion, groenlo, the netherlands). herds were identified based on their unique herd number (ubn). samples were randomly selected given the availability of enough serum to perform all assays. samples were collected from may till up to and including november . commissioned by the dutch government, gd animal health was also monitoring wild boars for aujeszky's disease, svd, foot and mouth disease, csfv, trichinellosis and african swine fever. because it is suggested that wild boars may play an important role as a pedv reservoir (lee et al., ) , blood samples of wild boar collected as part of this monitor were also tested. these blood samples were collected from regions close to the borders with germany and belgium, from april till august . blood samples collected were stored at − °c, before dispatched to the virology division of the faculty of veterinary medicine, at utrecht university (uu). for the detection of pedv antibodies in serum samples an in-house indirect elisa based on the viral spike (s) protein s -part of the g b strain gdu (non-s-indel, genbank ku . ) was used, similar as the elisa previously described (gerber et al., ) . the s antigen used in this study is produced in hek t cells, a mammalian expression system. to facilitate the purification from the cell culture supernatant, the protein is associated with the fc part of murine igg. per well ng of protein was used to coat the plates. the in-house elisa was optimized by uu with sera from pedvinfected (g b) pigs from the us and with hyperimmune sera of animals vaccinated with the pedv s protein. sera were tested in duplicate at a : dilution, the absorbance was measured with an elisa plate reader at nm. the mean od value of pedv-negative sera was . . sera with od values of > . ( × od value pedv negative sera) were considered positive. the aforementioned pedv-positive sera from infected animals were high positive (> . od) in this elisa ( supplementary fig. ). the calculated sensitivity and specificity of the elisa was % (ci %: - %; n = ) and % (ci %: - %; n = ), respectively. for the detection of virus-specific antibodies in serum samples the virus neutralization test (vnt) was used as an alternative sero-diagnostic assay. a previously described vnt was used to test all samples positive by elisa. the validation of this test is shown in fecal swabs or eswabs (copan diagnostics incorporated) of individual animals were taken by the local practitioner and sent in the same day for pcr testing. also three pigs of the index farm with severe diarrhea were euthanized and submitted for post mortem examination. rna was isolated from fecal samples and the detection pcr was performed as previously described (kim et al., ; lowe et al., ) . in order to type the viruses the pedv s gene was sequenced with primers previously described (chen et al., ; oka et al., ) and designed in this study (table ) , and compared with sequences available in genbank. a phylogenetic tree was constructed using mega software by neighbor-joining method maximum likelihood method. branch lengths represent the predicted number of substitutions and are proportional to the differences between the isolates (tamura et al., ) . after confirmation of the first g b outbreak in the netherlands it was unquestionable to try to prevent the further spread of the outbreak. therefore, it was decided to monitoring and follow up of specific management advices to the farmers. five fattening herds, five sow herds and one nursery herd were selected after pedv infection was confirmed by pcr on feces. for control and eradication of pedv, a tailor made advice per farm, mainly based on biosecurity measures, was given. advise was mainly focused on the separation between pedvinfected and non-infected healthy animals. this separation was applied in stables, animal categories and compartments. it was made using the following measures: dress code (change between compartments), additional disinfection cleaning and disinfection of compartments and corridors and improving pest control. furthermore, all professional herd visitors were informed and required to take measures, in particular those aimed at cleaning and disinfection, which would prevent the transfer of the disease to another pig farm. regular testing of pooled fecal samples was done to monitor the effect of interventions. in sow herds, nursery piglets and replacement gilts were sampled; in fattening herds a random sampling in all age groups was performed. after introduction of pedv, the virus could be detected in a compartment for - weeks. at farm level the virus was detectable much longer due to transmission to new susceptible animals. farms were presumed negative for pedv if three sampling rounds with at least -weeks interval, of thirty randomly taken individual fecal samples each, proved to be pcr negative. in total, sera from sows originating from farms, and sera from wild boar were collected. the blood samples came from all provinces in the netherlands and are fairly representative for the distribution of farms across provinces (fig. b ). all sera were tested in the indirect pedv s -based elisa (group , table ). nine samples, originating from nine different farms located in four different provinces, tested seropositive. the od values were low in all seropositive samples (od: . - . ) relative to positive control sera (convalescent or hyperimmune sera) with values od: . - . . two of the nine elisa-positive sera from the serological study also tested positive in the vnt. with two confirmed positive samples of the samples the animal prevalence was . % (ci %: - . %). the herd prevalence with out of farms was . % (ci %: - . %). for all herds, three samples were tested. the additional samples of seven herds with a seropositive sample of which one vnt positive, were examined. these samples, in total, were all negative in the pedv elisa. the proportion of seropositive samples ( / ) falls within the expected proportion of false positives given the specificity of the elisa which is estimated at > %. so we either detected pedv outbreaks that had not spread yet or the seven original samples were false positives. for two herds no additional samples from the monitoring programs were available (including one sow farm with a vnt positive sample). all tested sera obtained from wild boar were negative for pedv antibodies in the used indirect elisa (group , table ). in the first week of november , gd animal health received a report of pigs showing lethargy and anorexia for up to h after which profuse diarrhea occurred in almost all pigs in nine compartments within the fattening barn. diarrhea was watery, light greyish, sometimes yellow or green colored. body temperature in the clinical phase reached . °c. after the first days of disease lethargy subsided and lack of appetite diminished, a profuse diarrhea became more prominent. in later stages of the infection the consistency of the diarrhea changed to slightly more solid. pigs did not seem to suffer much and no animals died, although some pigs did not grow for a week and within the group body weights started to differ. after an extra week of feeding, pigs did recover and had a normal weight at slaughter. the fattening barn, located in a pig dense area of the netherlands, consisted of compartments with pigs each, divided over pens. first symptoms occurred on october th in one compartment followed by symptoms in consecutive compartments in the following days. initial diagnostic tests for the presence of e. coli, salmonella, lawsonia intracellularis and brachyspira pilosicoli were, except for low numbers of pathogenic e. coli, negative. based on the low numbers of pathogenic e. coli and the age of the pigs involved, e. coli was ruled out as causative agent in this case. subsequently, it was decided to test for porcine deltacoronavirus (pdcov) and pedv. six fecal samples were found to be negative for pdcov, but positive for pedv rna on november th . on the same day, three pigs with severe diarrhea were euthanized and were submitted for post mortem examination. pathological examination showed severe villus atrophy in the small intestine, and pcr tested positive for pedv. during the outbreak, in total % of the animals ( compartments) were affected by pedv as confirmed by pcr on fecal samples. based on the results of the serological survey, over % of the dutch farms were pedv negative in ; it was decided to try to prevent the further spread of the outbreak. to control and eradicate pedv the biosecurity measures taken, as described in the materials and methods section, seemed to be of great importance. also pet control was applied since some farms suffered from mice and rats. furthermore, it became clear that in compartments in which the infection was present, after thorough cleaning and disinfection, pedv free piglets could be introduced and that those stayed free from pedv infection. three fattening and three sow herds were presumed free of pedv within months after the diagnosis of ped was confirmed. the nursery herd was depopulated, and, after double cleaning and disinfection of all compartments, repopulated. despite the immediate action of all parties in the dutch pig production industry to optimize their hygiene measures to ensure that infection by pedv would be avoided as much as possible, it could not be prevented that pedv spread to other farms. most infected farms were located on the east side of the netherlands at the border of germany (fig. c) . at the end of , in total farms were confirmed pedv positive by gd animal health. in fig. the number of new pedv pcr positive farms per month since the beginning of the outbreak is shown. in order to characterize the virus originating from outbreaks in and , the s gene of three isolates of different farms was sequenced and the sequences ned/gd / , ned/gd / and ned/ gd / were deposited in the genbank database and received accession numbers kr (index farm), kr and mf , respectively. together with sequences available in genbank, the isolates were compared in a phylogenetic tree (fig. ) . sequence analyses showed that the isolates had a . % homology with the usa/oh strain and a . % homology with the german g b strains. furthermore, the european strains available in genbank ( - ) cluster together with this oh strain, or the so called s-indel strain (fig. ) . this study showed that most likely pedv and particularly the genogroup b (s-indel) strains did not circulate in the netherlands on a large scale till the end of . only a very small part of the dutch sow farms tested positive on antibodies against pedv ( . % (ci %: - . %)) and no pedv antibodies were detected in wild boars. the used elisa based on the s protein of a g b strain showed a high sensitivity and specificity against antibodies raised against g a, g b en g b strains. however, the elisa was validated with a limited amount of samples ( supplementary fig. ) and field samples may react differently, just like in a similar elisa recently described (gerber et al., ) . the viral spike (s) protein is prominent on the virus surface and is very immunogenic. all animals that have been infected with pedv have antibodies against the s protein and particularly against the s part. the s -part of the spike protein is the most variable part between related coronaviruses and there is no cross reactivity with other coronaviruses such as transmissible gastroenteritis coronavirus (tgev), porcine respiratory coronavirus (prcov) and porcine delta coronavirus (pdcov) as previously described (gimenez-lirola et al., ; lin et al., b) . because results of immunoassays based on the s protein correlate well with viral neutralization (paudel et al., ) , as shown in supplementary table , we decided to use the vnt as an alternative sero-diagnostic assay. based on case reports from germany stadler et al., ) , austria (steinrigl et al., ) and the netherlands (gd animal health) a pedv infection with the european circulating strain (g b) will spread very rapidly within a herd and a single pedv positive animal on a farm is not likely to occur. the number of elisa positive animals ( %, table , group ) on the index farm after the clinical signs started, seemed to justify the assumption of the high within herd prevalence of % for sample size calculation for the serological survey. eventually, there were fewer herds sampled than planned ( instead of ) since it was decided to stop the serological survey, because the first case of pedv was diagnosed in the netherlands on november th . sequence analyses of the viruses isolated in - showed a % homology with oh , a less virulent pedv strain found in the us and in germany in . although the g b virus strain present in europe is less virulent (efsa, ; grasland et al., ; hanke et al., ; mesquita et al., ; steinrigl et al., ) compared to the strain that caused the us outbreaks in , this european strain showed to be very contagious and still could cause severe economic damage in pedv naive herds. after infection on sow farms loss off piglets could range up to % in the sucking piglets ( (lin et al., a) and individual case reports, gd animal health). in the foreseeable future, vaccination will not be possible. therefore, the authorities in the netherlands advised all parties in the pig production industry to optimize their hygiene measures to ensure that infection by this virus would be avoided as much as possible. the strict preventive biosecurity measures taken in these herds demonstrated that most herds that were monitored could prevent the transfer to pedv naive compartments within the herd and some were able to obtain a presumed pedv free status for the whole farm. additionally, a higher biosecurity level helps preventing the introduction of other pathogens and controls the spread of infection within that herd (fao, ) . however, an increasing number of herds became infected (fig. ) and pedv spread across the country after the initial outbreak (fig. c) . the transport trucks with positive animals between herds seemed to have the highest transmission risk (data not shown). the number of new pedv pcr positive farms per month since the beginning of the outbreak is most likely an underestimation (fig. ) . since ped is not a notifiable disease, nor have all veterinarians performed diagnostic testing in all cases, as the clinical signs of an outbreak are very typical. it seems that pedv g b became endemic in the netherlands since its initial outbreak in november , just like in most countries in europe. that g b viruses isolated in europe in - phylogenetically cluster together with a high similarity (fig. ) suggests a onetime introduction event. however, pedv is endemic and many other coronaviruses are circulating that may result in coronavirus variants through mutation or recombination (fehr and perlman, ) . therefore, it is of upmost importance that the presence of circulating pedv is being monitored and genetically analyzed, in order to update diagnostic tools where necessary. we declare no conflict of interest. surveillance and control of ped coronavirus in pigs in italy porcine epidemic diarrhea virus shedding and antibody response in swine farms: a longitudinal study isolation and characterization of porcine epidemic diarrhea viruses associated with the disease outbreak among swine in the united states viral disease affects u.s. pigs: porcine epidemic diarrhea found in at least states updated epidemiological data on ped good practices for biosecurity in the pig sector -issues and options in developing and transition countries coronaviruses: an overview of their replication and pathogenesis detection of antibodies against porcine epidemic diarrhea virus in serum and colostrum by indirect elisa reactivity of porcine epidemic diarrhea virus structural proteins to antibodies against porcine enteric coronaviruses: diagnostic implications previous infection of sows with a "mild" strain of porcine epidemic diarrhea virus confers protection against infection with a "severe complete genome sequence of a porcine epidemic diarrhea s gene indel strain isolated in france comparison of porcine epidemic diarrhea viruses from germany and the united states origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states multiplex realtime rt-pcr for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus completion of the porcine epidemic diarrhoea coronavirus (pedv) genome sequence porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus wild boars harboring porcine epidemic diarrhea virus (pedv) may play an important role as a pedv reservoir manipulation of the porcine epidemic diarrhea virus genome using targeted rna recombination cellular entry of the porcine epidemic diarrhea virus experimental infection of a us spike-insertion deletion porcine epidemic diarrhea virus in conventional nursing piglets and cross-protection to the original us pedv infection antigenic relationships among porcine epidemic diarrhea virus and transmissible gastroenteritis virus strains role of transportation in spread of porcine epidemic diarrhea virus infection epidemic of diarrhoea caused by porcine epidemic diarrhoea virus in italy outbreak of porcine epidemic diarrhea virus in portugal cell culture isolation and sequence analysis of genetically diverse us porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene comparison of serum neutralization and enzyme-linked immunosorbent assay on sera from porcine epidemic diarrhea virus vaccinated pigs a new coronavirus-like particle associated with diarrhea in swine porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines emergence of porcine epidemic diarrhea virus in southern germany first detection, clinical presentation and phylogenetic characterization of porcine epidemic diarrhea virus in austria emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences mega : molecular evolutionary genetics analysis version . win episcope . : improved epidemiological software for veterinary medicine distinct characteristics and complex evolution of pedv strains new variant of porcine epidemic diarrhea virus pig farming the authors would like to thank d. oorburg en c. sima of vion, groenlo, the netherlands, for providing blood samples from slaughter sows, g. spierts and laboratory staff for collecting and archiving blood samples and n. schuurman and j. de jong for performing the elisa. furthermore, r. dijkman for implementing the pcr at the gd animal health lab, a. van lenthe for advice during the survey, m. meijerink for assisting in sample collection during herd visits and a. veldhuis and h. brouwer-middelesch for making the maps of the netherlands (fig. ) . the authors thank the owner of the index farm and the practitioner involved in this farm for their information and their cooperation in collecting the samples.the authors would like to thank the dutch ministry of economic affairs and the product board for livestock and meat for funding the monitoring system for pig health in the netherlands. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.vetmic. . . . key: cord- - lzjhn j authors: tian, bin; zhou, ming; yang, yu; yu, lan; luo, zhaochen; tian, dayong; wang, ke; cui, min; chen, huanchun; fu, zhen f.; zhao, ling title: lab-attenuated rabies virus causes abortive infection and induces cytokine expression in astrocytes by activating mitochondrial antiviral-signaling protein signaling pathway date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: lzjhn j rabies is an ancient disease but remains endemic in most parts of the world and causes approximately , deaths annually. the mechanism through which the causative agent, rabies virus (rabv), evades the host immune response and infects the host central nervous system (cns) has not been completely elucidated thus far. our previous studies have shown that lab-attenuated, but not wild-type (wt), rabv activates the innate immune response in the mouse and dog models. in this present study, we demonstrate that lab-attenuated rabv causes abortive infection in astrocytes, the most abundant glial cells in the cns. furthermore, we found that lab-attenuated rabv produces more double-stranded rna (dsrna) than wt rabv, which is recognized by retinoic acid-inducible gene i (rig-i) or melanoma differentiation-associated protein (mda ). activation of mitochondrial antiviral-signaling protein (mavs), the common adaptor molecule for rig-i and mda , results in the production of type i interferon (ifn) and the expression of hundreds of ifn-stimulated genes, which suppress rabv replication and spread in astrocytes. notably, lab-attenuated rabv replicates in a manner identical to that of wt rabv in mavs−/− astrocytes. it was also found that lab-attenuated, but not wt, rabv induces the expression of inflammatory cytokines via the mavs- p /nf-κb signaling pathway. these inflammatory cytokines increase the blood–brain barrier permeability and thus enable immune cells and antibodies infiltrate the cns parenchyma, resulting in rabv control and elimination. in contrast, wt rabv restricts dsrna production and thus evades innate recognition by rig-i/mda in astrocytes, which could be one of the mechanisms by which wt rabv evades the host immune response in resident cns cells. our findings suggest that astrocytes play a critical role in limiting the replication of lab-attenuated rabv in the cns. rabies is an ancient disease but remains endemic in most parts of the world and causes approximately , deaths annually. the mechanism through which the causative agent, rabies virus (rabv), evades the host immune response and infects the host central nervous system (cns) has not been completely elucidated thus far. our previous studies have shown that lab-attenuated, but not wild-type (wt), rabv activates the innate immune response in the mouse and dog models. in this present study, we demonstrate that lab-attenuated rabv causes abortive infection in astrocytes, the most abundant glial cells in the cns. furthermore, we found that lab-attenuated rabv produces more double-stranded rna (dsrna) than wt rabv, which is recognized by retinoic acidinducible gene i (rig-i) or melanoma differentiation-associated protein (mda ). activation of mitochondrial antiviral-signaling protein (mavs), the common adaptor molecule for rig-i and mda , results in the production of type i interferon (ifn) and the expression of hundreds of ifn-stimulated genes, which suppress rabv replication and spread in astrocytes. notably, lab-attenuated rabv replicates in a manner identical to that of wt rabv in mavs−/− astrocytes. it was also found that lab-attenuated, but not wt, rabv induces the expression of inflammatory cytokines via the mavs-p /nf-κb signaling pathway. these inflammatory cytokines increase the blood-brain barrier permeability and thus enable immune cells and antibodies infiltrate the cns parenchyma, resulting in rabv control and elimination. in contrast, wt rabv restricts dsrna production and thus evades innate recognition by rig-i/mda in astrocytes, which could be one of the mechanisms by which wt rabv evades the host immune response in resident cns cells. our findings suggest that astrocytes play a critical role in limiting the replication of lab-attenuated rabv in the cns. introduction rabies is an acute encephalomyelitis. the hallmark of rabies is that the disease is almost always fatal once clinical signs develop ( , ) . the causative agent, rabies virus (rabv), is a negative-strand rna virus belonging to the genus lyssavirus in the family rhabdoviridae ( ) . rabv enters neurons from the neuromuscular junction closest to the site of infection. after a short incubation, rabv travels to the central nervous system (cns) through sensory or motor neurons. only slight tissue damage and neuroinflammation can be observed in the brains of rabid patients ( ) . in contrast, lab-attenuated rabv induces extensive inflammation and apoptosis, as well as increases in the expression levels of innate immunity-related genes in the cns of infected mice ( ) ( ) ( ) ( ) ( ) ( ) . these findings suggest that wt, but not lab-attenuated, rabv evades the host immune responses. the innate immune system is the first line of defense against viral invasion. viruses are usually confronted by various pattern recognition receptors (prrs), including toll-like receptors (tlrs) and retinoic acid-inducible gene i (rig-i) like helicases (rlrs) ( ) . tlr family members, such as tlr and tlr , are generally involved in recognizing negative-strand rna viruses ( ) . tlr binds double-stranded rna (dsrna), whereas tlr recognizes single-strand rna. the main sources of dsrna in infections with single-strand rna viruses are the replicative intermediates generated by viral rna-dependent rna polymerase ( ) . tlr and tlr initiate signaling though the adaptor molecules trif and myd , respectively. both signaling cascades triggered by these proteins lead to irf phosphorylation in the c terminal region at serine , which is critical for irf activation by two iκb kinases (tbk- and ikkε) ( , ) . activated irf homo-or hetero-dimerizes with irf and then translocates into the nucleus, to interacts with the creb binding protein cbp/p and stimulates the transcription of interferon (ifn)-β, as well as some ifn-stimulated genes (isgs) ( , ) . alternatively, rna viruses can be recognized by two rlrs, rig-i, and melanoma differentiation-associated protein (mda ), located in the cytoplasm ( , ) . rig-i solely senses short and blunt dsrna of negative-strand rna viruses containing ′ triphosphate rna in the panhandle region of their single-stranded genome ( ) . unlike rig-i, mda preferentially binds blunt-ended dsrna, with or without ′ triphosphate ( ) . rig-i and mda signaling is mediated through mitochondrial antiviral-signaling protein (mavs), which is also known as ips , visa or cardif ( ) . similar to tlr signaling, rlr signaling results in irf activation and nuclear translocation ( ) . several groups have attempted to identify the prrs that recognize rabv. prehaud et al. found that tlr mrna expression is upregulated following rabv infection in postmitotic human neurons ( ) . furthermore, enhanced tlr expression has also been observed in the cerebellar cortical tissues of rabies patients ( ) . the observation that tlr is upregulated following infection suggests that tlr plays a role in the innate recognition of rabv. faul et al. found that both rig-i and mda were responsible for inducing dendritic cell (dc) activation and type i ifn production upon rabv infection ( ) . mavs, the adaptor protein for both rig-i and mda , is essential for inducing innate immune responses in dcs. in another study, li et al. found that mice lacking tlr exhibited a phenotype associated with intermediate mortality rates between those of myd −/− and control mice, indicating that tlr may play an important role in controlling rabv infections. however, the role of tlr in rabv infection is not entirely understood ( ) . astrocytes are the most abundant glial cells in the cns and constitute the blood-brain barrier (bbb) along with endothelial cells and pericytes ( ) . astrocytes are generally involved in regulating the cns microenvironment and also play roles in neuronal metabolic support, synaptic transmission, and neurotropism. moreover, astrocytes participate in developing and maintaining the bbb and guiding neuronal migration during development. the roles of astrocytes in innate immunity and inflammation have been reported recently ( ) . astrocytes are the reservoir for many neuroinvasive viruses, such as human immunodeficiency virus, theiler's murine encephalomyelitis virus (tmev), john cunningham virus, and herpes simplex virus (hsv) ( ) ( ) ( ) ( ) . astrocytes have also been shown to play multiple roles in viral infections. specifically, they can increase bbb permeability ( ) by producing cytokines and degrading tight junction proteins ( ) . moreover, astrocytes have been shown to induce the innate immune response to produce ifn ( ) . an early report showed that both wt and lab-attenuated rabv could successfully infect primary astrocytes in the early stages of rabv infection ( ) . a recent study demonstrated that a rabv vaccine strain sad-l caused an abortive infection in astrocytes, but the detailed mechanism was not revealed ( ) . in this study, it was found that lab-attenuated rabv produces higher level of dsrna than wt rabv, which is recognized by rig-i/mda and results in the activation of mavs signaling pathway. following mavs activation, the production of isg and inflammatory cytokines helps to clear the lab-attenuated rabv from the cns. in contrast, wt rabv can maintain a persistent infection in astrocytes by evading the innate recognition. mouse bend. cells and vero cells were obtained from the american type culture collection (atcc; manassas, va, usa) and were maintained in dulbecco's modified eagle medium (dmem) supplemented with fetal bovine serum (fbs; gibco, carlsbad, ca, usa). mouse neuroblastoma (na) cells were maintained in rpmi- medium (thermo-fisher, usa) supplemented with % fbs. the drv-ah (drv) was isolated from a rabid dog in anhui province, china ( , ) . cvs-b c (b c), originated from cvs- virus by passage in bhk- cells ( ), has been used as a lab-attenuated rabv ( ) ( ) ( ) . both drv and cvs-b c were propagated in suckling icr mouse brains. all the viruses were manipulated under the standard biosecurity procedures made by the ministry of agriculture of china. a rabbit anti-ubiquitin protein c (ubc) polyconal antibody was purchased from abclonal technology (woburn, ma, usa). a rabbit anti-rig-i monoclonal antibody was purchased from enzo life technology (farmingdale, ny, usa), and rabbit polyclonal antibodies against irf , stat and occludin were obtained from santa cruz biotechnology (santa cruz, ca, usa). a rabbit antiphospho-irf monoclonal antibody was purchased from cell signaling technology (washington, dc, usa), and rabbit anti-ifit and irf monoclonal antibodies were purchased from abcam (cambridge, ma, usa). a rabbit polyclonal anti-claudin- antibody, a biotinylated goat anti-rabbit or mouse antibody, and an alexa fluor -conjugated goat antimouse or rabbit antibody were purchased from invitrogen (grand island, ny, usa). mouse monoclonal anti-zoluna occludens- (anti-zo- ) antibodies were obtained from sigma (st. louis, mo, usa), and mouse monoclonal anti-gapdh antibody was purchased from proteintech (wuhan, china). the mouse anti-rabv n and p monoclonal antibodies were prepared in our laboratory, and the fluorescein isothiocyanate (fitc)-conjugated anti-rabv nucleoprotein antibody used herein was obtained from female wt or mavs-knockout (mavs−/−) c bl/ mice ( - weeks old) were infected intracerebally (i.c.) with µl of drv-ah ( ffu), b c ( ffu), or mock infected with the same volume of dmem. at days postinfection (d.p.i.), the mice were euthanized with co when moribund, and their brains were collected for immunohistochemistry analysis. blood-brain barrier permeability was determined by measuring sodium fluorescein (naf) uptake as described previously with minor modifications ( ) . briefly, µl of naf ( mg/ml) was injected intraperitoneally (i.p.) into each mouse. after anesthetization, peripheral blood and brains of each mouse were collected. the fluorescence in serum and brain homogenate samples was analyzed by a spectrophotometer (biotek instruments, vt, usa) with excitation at nm and emission at nm. standards ( - , g/ml) were prepared to calculate the naf content. naf uptake into tissue is calculated as (μg of fluorescence spinal cord/mg of tissue)/(μg of fluorescence sera/ml of blood) to normalize values for blood levels of the dye at the time of tissue collection. to detect cd + cell in the brain, infected mice were anesthetized with ketamine-xylazine ( . ml/ g body weight), perfused with ml pbs, and then the brains were transferred into % neutral buffered paraformaldehyde (pfa) for at least h ( ) . briefly, the brain sections were antigenic recovered and blocked with donkey serum, incubated with primary antibodies overnight at °c, and then secondary antibodies was applied. pbs was treated as a negative control by replacing primary antibodies. sections were photographed and analyzed using an olympus bx microscope (tokyo, japan). primary mixed glial cell cultures were established as described previously ( ) . briefly, the brain cells of -to -day-old neonatal c bl/ mice were dissociated by repeated pipetting and then passed through a -nm nylon mesh (corning, ny, usa). the cells were subsequently washed once in cold pbs and cultured in dmem (with high glucose) supplemented with % fbs and % penicillin-streptomycin. the medium was changed on days , , and for the astrocytes and on day only for the microglia. on day , the flasks were shaken at rpm for h to remove any non-adherent cells (mainly microglia). the remaining adherent astrocytes were detached with trypsin-edta and then plated again for further experiments. the purity of the astrocyte cultures was greater than %. mouse neurons were obtained from embryonic mouse brains as previously described ( ) and then dissociated by repeated pipetting (approximately times) before being passed through a -nm nylon mesh. the cells were then washed once in cold pbs and cultured in dmem (with high glucose) supplemented with % fbs and % penicillin-streptomycin for h, after which the medium was replaced with serum-free neural-basal medium (invitrogen, carlsbad, ca, usa) supplemented with % b- (invitrogen, carlsbad, ca, usa). viral titers were determined by direct fluorescent antibody assay ( ) . na cells cultured in -well plates were inoculated with viruses diluted serial -fold and then incubated at °c for h. then the culture supernatant was subsequently removed, and the cells were fixed and stained with fitc-conjugated anti-rabv n antibodies. the antigen-positive foci were counted under a fluorescence microscope (zeiss, germany), and the viral titers were calculated as ffu per milliliter. all titrations were conducted in quadruplicate. confocal microscopy table | primers used for quantification of viral mrna, ifn-stimulated genes, chemokines, and cytokines. retinoic acid-inducible gene i (rig-i) f gcgtctcagtgcagcacatcatt qrt-pcr antibodies against dsrna, rabv n, rabv p, claudin- , occludin, zo- , or dapi. infected wt or mavs−/− mice were anesthetized with ketamine-xylazine ( . ml/ g body weight) and then perfused with pbs followed by % neutral buffered formalin, as described previously ( ) . three independent mouse brain samples were collected from each group and embedded in paraffin for coronal sectioning. the sections were subsequently stained with antibodies against gfap, map , rabv p, rabv n, or dapi. after being washed, the cells or sections were incubated with an alexa fluor -conjugated goat antirabbit or mouse secondary antibodies or an alexa fluor -conjugated goat antirabbit or mouse secondary antibodies for h at room temperature. staining was visualized with a nikon a confocal laser microscope system equipped with nis-elements imaging software (nikon, melville, ny, usa) and was quantified using fiji, an imagej distribution package manufactured by nih (http://imagej.net/introduction). mean fluorescence intensity (mfi) was quantified using the region of interest, which encompassed the entire cell to include the membrane, and background staining was quantified using three negatively stained regions per cell. these regions were subtracted from the total mfi. for the protein synthesis inhibition tests, primary astrocytes were pretreated with cycloheximide (chx) (invivogen, san diego, ca, usa) for h at a dose of µg/ml, infected with drv or b c at an moi of . and then continued to be incubated with chx for another h. for the tbk activation blockage assays, bx (invivogen, san diego, ca, usa) at a dose of µm was used to treat primary astrocytes. for the inflammatory pathway blockage assays, the primary astrocytes were pretreated with a p inhibitor (skepinone-l; sellek, houston, tx, usa), a jnk inhibitor (jnk inhibitor ix; sellek, houston, tx, usa), an nf-κb inhibitor (sc ; sellek, houston, tx, usa), or dmso as control at a dose of µg/ml for h. then the cells were infected with drv or b c at moi and incubated with the above inhibitors for h. the concentrations of tnf-α and il- in the supernatant of astrocytes were measured by the commercial elisa kits according to the manufacturer's instruction (abclonal technology, woburn, ma, usa). rna was isolated with trizol ® reagent (invitrogen), according to the manufacturer's instructions, and qrt-pcr was performed as described previously ( ) . briefly, ng of total rna (from either cells or tissue) was transcribed into cdna in a reaction mixture with a total of volume of µl using a superscript iii reverse transcription kit (toyobo). the reaction mixture comprised μl of cdna combined with µl of iq sybr green mix (biorad, hercules, ca, usa), µl of diethyl pyrocarbonatetreated water, and . µl of primer mix (the concentration of each primer was mm). the cdna was amplified using an iq icycler (bio-rad), and the cycle threshold (ct) values were recorded. the ct value was inversely correlated with the mrna concentration, and each ct unit represented a twofold change in the mrna concentration. basal mrna expression levels were expressed as Δ ct values and were normalized to β-actin mrna expression levels [ Δ ct ct (isg)/ Δ ct (β-actin)]. induced mrna expression levels were expressed as fold changes relative to mock-infection levels using the ΔΔct method. all the primer sequences are listed in (table ) . to quantify cellular rabv n rna levels, we transcribed the total rna using avian myeloblastosis virus reverse transcriptase xl (takara, kusatsu, japan) and a primer specific for the rabv n genomic sequence. a standard curve was generated from serially diluted plasmids carrying a rabv n gene and the copy numbers of n mrna were normalized to mg of total rna. the cells were lysed with ripa buffer containing protease inhibitors (roche), and the protein concentrations were measured using a dc protein assay kit (bio-rad). equal quantities of protein were resolved by or % sds-page and then transferred to polyvinylidene difluoride membranes (bio-rad), which were blocked with % nonfat milk before being incubated with primary antibodies against rig-i, phosphorylated irf (p-irf ), stat , ifit , rabv n, claudin- , occludin, zo- , or gapdh and then probed with the appropriate secondary antibodies. the blots were then visualized using ecl reagent (ge, pittsburgh, pa, usa) and detected under an intelligent dark box ii (ge, pittsburgh, pa, usa). the -gain pmt scan generated the optimal standard curve, and the results of this scan were analyzed using q-analyzer software for qam-cyt- (raybiotech). transendothelial permeability assay was performed according to the methods described previously ( ), with some modifications. b.end cells were grown on -μm-pore transwell filter inserts until they reached % confluency. the medium was then treated with uv-inactivated cell culture supernatants collected from rabv-infected astrocytes. after the cells had incubated for h, they were treated apically with dextran-fitc at a dose of . µg/ml for min. the samples were then removed from the lower chamber and subjected to fluorescence measurement, which were performed using a fluorimeter (biotek, winooski, vt, usa; the excitation wavelength was nm, and the emission wavelength was nm). the fluorescence values for the experimental cells were subsequently compared to corresponding values for a control cell monolayer. data are expressed as the mean and standard error of the mean (sem), and the significance of the differences between groups was evaluated by student's t test or one-way analysis of variance followed by tukey's post hoc test. the survival ratios were analyzed by log-rank (mantel-cox) test. the asterisks indicated statistical significance (*, p < . ; **, p < . ; ***, p < . ). graphs were plotted and analyzed using graphpad prism software, version . (graphpad software, la jolla, ca, usa). results comparison of the pathogenicity of drv-ah and cvs-b c in mice first, the pathogenicity of the wt rabv strain drv-ah (drv) and lab-attenuated strain cvs-b c (b c) used in this study were compared in a mouse model. c /bl mice were i.c. inoculated with ffu b c or drv and the development of rabies was observed. as expected, drv-infected mice displayed development of the diseases at d.p.i. and all moribund at d.p.i., earlier than b c-infected mice which all succumb to rabies at d.p.i. (figure a) . we found that b c replicated faster than drv at the early stage of the infection in the cns. to ensure that the viral load in the brains is similar, c /bl mice were i.c. inoculated with ffu b c or ffu drv. then the viral load in the mice brain were determined at , , , , , and d.p.i. the results showed that the genomic rna of drv was lower than that of b c at and d.p.i., but reached the same level as that of b c at and d.p.i. (figure b) . previous studies have shown that the lab-attenuated rabv infection enhances the bbb permeability and induces inflammation in the brain ( , ) . thus, the na-fluorescence (na-f) uptake of both strains at d.p.i. was measured, and the data showed that na-f uptake in b c-infected brain was significantly higher than that of drv or mock-infected brains ( figure c) . consistently, there were more cd + lymphocytes in b c-infected brains than drv-infected mouse brains (figures d,e) . all these results are consistent with the previous findings related to the cns inflammation and bbb permeability change during comparison of the pathogenicity between wt and lab-attenuated rabv ( , , ) . astrocytes play an important role in the induction of innate immunity in the cns. to investigate the role of astrocytes in the pathogenesis of rabv, we isolated primary astrocytes and neurons from suckling mice and infected them with drv or b c. the growth kinetics of both viruses in astrocytes was assessed by virus titration and immunofluorescence assay (ifa). the viral loads in the cell culture supernatants infected with b c quickly reached peak at d.p.i., and then gradually decreased until d.p.i. in contrast, the virus titer of drv was initially relatively low but subsequently increased steadily until the endpoint (figure a) . to be noted, the viral titer of drv in the cell supernatant maintained at a relatively low level than that of b c in astrocyte at and d.p.i., but n mrna transcription levels of drv was significantly higher than that of b c at and d.p.i. (figure c) . the ifa results showed that drv persistently replicated in astrocytes and large immunofluorescence foci could be observed at d.p.i., while no obvious immunofluorescence foci could be found in b c-infected cells ( figure d) . as a control, virus replication kinetics in primary neurons was also compared. it was found that viral titers of b c were always higher than those of wt rabv at the indicated time points (figure b) . the ifa results also showed that both drv and b c could efficiently replicate in neuron ( figure d) . to confirm these observations in vivo, c bl/ mice were i.c. infected with ffu b c or ffu drv. infected mice were euthanized when moribund and the brains were harvested for fluorescence ihc analysis. gfap was a well-known surface marker for astrocytes ( ) , and the gfap staining demonstrated drv could effectively infect astrocytes in the brains ( figure e) . however, few infected astrocytes could be observed in b cinfected mouse brains (figure e ). in contrast, neuron was intensively infected by both drv and b c ( figure f) . together, these results show that the b c causes abortive infection in astrocytes both in vitro and vivo. previous studies have demonstrated that rabv can be recognized by rig-i and mda , which share a common adaptor mavs in dcs ( ) . to assess innate immune responses in astrocytes, cells were infected with drv or b c at an moi of . and the expression of several proteins involved in the mavs signaling pathway, namely, rig-i, p-irf , stat and ifit (isg ), was measured by western blot. the ubiquitin ligase trim mediates lysine -linked ubiquitination of rig-i's n-terminal card domains is indispensable to induce type i ifn production and antiviral immunity ( ) . thus, the immunoprecipitation of rig-i was carried out and then resolved by western blot by using an anti-ubiquitin antibody. as expected, rig-i was much more robustly ubiquitinated in astrocytes infected with b c than that in astrocytes infected with drv. consistently, the expression levels of rig-i, p-irf , stat , and ifit in b c-infected astrocytes were higher than those in drv-infected cells ( figure a) . next, we attempted to determine which viral product (rna or protein) activates mavs signaling pathway in astrocytes. primary astrocytes were treated with chx, a protein synthesis inhibitor, to inhibit viral protein synthesis. it was found that b c and drv n transcription levels were similar between chxtreated and mock-treated astrocytes at h p.i. (figure b) . however, the expression levels of the genes involved in the mavs signaling pathway, namely, rig-i, mda , mavs, irf , ifn-β, stat , and ifit , were significantly upregulated by b c compared with drv in both chx-treated and mock-treated astrocytes, indicating that viral rna rather than proteins activates ifn pathway depending on mavs (figures c-i) . taken together, these data demonstrate that the lab-attenuated rabv, but not wt rabv, activates the mavs signaling pathway by viral rna, resulting in the production of ifn as well as isgs in astrocytes. retinoic acid-inducible gene i and mda activation is induced mostly by dsrna, which is produced during viral replication ( ) . to compare the amount of dsrna produced by wt and labattenuated rabv, primary astrocytes were infected with drv or b c at an moi of . . at and d.p.i., dsrna was stained with specific antibody and observed by a confocal fluorescence microscope. the results demonstrate that more dsrna is synthesized in b c-infected astrocytes than those in drv-infected cells ( figure a) . the dsrna intensity per cell in b c-infected cell was significantly higher than that in drv-infected cells (figure b) , considering the similar rabv intensity per cell (figure c) . the ratio of dsrna intensity to rabv intensity in b c-infected astrocyte was significantly higher than that in drv-infected cells ( figure d) . these results suggest that lab-attenuated rabv produces more viral dsrna than wt rabv during viral replication, ( ffu) . (e) at d.p.i., mice were euthanized, perfused with pbs and then fixed with % pfa. the brains were subsequently coated with paraffin, and the brain sections were stained with antibodies against gfap (red), rabv p protein (green), or dapi (blue). the white arrows indicate rabv-infected astrocytes. the scale bars represent µm. (f) the same brain sections as (e) were stained with antibodies against map (green), rabv p protein (red), or dapi (blue), then visualized under a confocal microscope. the scale bars represent µm. notably, b c titers were significantly increased in mavs−/− astrocytes compared with wt astrocytes (figures a,b) . moreover, the cell numbers of immunofluorescence plaques in mavs−/− astrocytes caused by b c infection were significantly more than those in wt astrocytes (figures e,f) . in contrast, mavs deficiency had no significant influence on drv replication and spread in astrocytes (figures a,e) . tbk is an iκb kinase downstream of the mavs signaling pathway and is critical for irf phosphorylation. treatment with the tbk specific inhibitor bx significantly increased b c titers in astrocytes (figures c,d) . to verify these observations in vivo, mavs−/− mice were i.c. infected with drv or b c, and virus infection of astrocytes was determined by ifa. we found that both drv and b c could efficiently infected mavs−/− astrocytes (figures g,h) . taken together, these findings suggest that mavs signaling significantly restricts the replication and spread of lab-attenuated but not wt rabv in astrocytes. recent studies have shown that tlr may be another innate immune molecule that recognizes rabv ( ) . thus, the role of tlr in rabv replication was investigated in astrocytes. astrocytes from tlr −/− and control mice were isolated and then infected with drv or b c at an moi of . . at different time points p.i., viral titers (figures a,b ) and the formation of viral immunofluorescence foci was analyzed (figures e,f) . the results demonstrated that tlr deficiency did not significantly affect the replication and spread of either drv or b c in astrocytes, suggesting that tlr does not play a role in containing rabv replication and spread in astrocytes. astrocytes are one of the major sources of inflammatory cytokines in the cns post viral infection, and these cytokines play an important role in regulating bbb permeability. to investigate rabv-induced cytokine production, primary astrocytes from wt and mavs−/− mice were prepared and infected with drv or b c at an moi of . . at indicated time points, the cell culture supernatants were harvested and analyzed with a cytokine array kit. the results showed that b c induced significantly higher levels of cytokine expression, namely, tnf-α, il- , il- β, ifn-γ, il- , and vegf expression, than drv in wt and mavs−/− astrocytes. the levels of cytokine expression in mavs−/− astrocytes were significantly lower than those in wt astrocytes, indicating that rabv-induced inflammatory cytokine production in astrocytes is dependent on the mavs signaling pathway (figures a-f) . the specific pathway through which rabv induces cytokine production was subsequently identified in astrocytes. a previous study demonstrated that rabv induces cytokine production in macrophages mainly through p , jnk, and nf-κb pathways ( , ) . thus, primary astrocytes were treated with p , jnk, and nf-κb pathway inhibitors skepinone-l, jnk ix, and sc , respectively, and the concentrations of tnf-α and il- in the cell supernatant were measured by elisa. the results showed that skepinone-l and sc caused greater reductions in tnf-α ( figure g ) and il- ( figure h ) protein levels than jnk ix in b c-infected astrocytes. none of these inhibitors significantly altered the expression levels of tnf-α and il- in drv-infected astrocytes. these findings suggest that lab-attenuated rabv induces cytokine expression in astrocytes mainly through the p and nf-κb pathways and that wt rabv suppresses cytokine production in astrocytes. our previous studies demonstrated that the chemokines/ cytokines induced by rabv infection are responsible for reducing tj protein expression and enhancing bbb permeability ( ) . to investigate the effect of these cytokines on bbb permeability, a mouse brain microvascular endothelial cell line b.end , was cocultured with uv-inactivated supernatants infected with drv or b c and collected them at different time points after infection. treatment with the supernatants from b c-infected astrocytes for h (figure a ) induced significant increase in dextran-fitc infiltration from to h p.i. notably, treatment with the supernatants from b c-infected mavs−/− astrocytes elicited significant increases in dextran-fitc permeability at h p.i. (figure b) . no significant increases in dextran-fitc permeability were observed after treatment with the supernatants from wt or mavs−/− astrocytes infected with drv. to gain an insight into the mechanisms by which labattenuated rabv enhances bbb permeability, the expression levels of the indicated tj proteins (claudin- , occludin, and zo- ) were assessed in astrocytes by western blot. it was found that the expression levels of claudin- and occludin were unchanged, while zo- was significantly reduced after the cells were treated with the supernatants from b c-infected wt astrocyte ( figure c) . however, this reduction was attenuated in cells treated with the supernatants from mavs−/− astrocytes infected with b c. no significant changes in zo- expression were observed in b.end cells treated with supernatants from wt or mavs−/− astrocyte infected with drv ( figure d) . similarly, ifa results showed that zo- expression was significantly decreased in b.end cells cocultured with the supernatants from b c-infected astrocytes collected at h p.i. (figure e) . zo- fluorescence intensity was only slightly decreased in b.end cells cocultured with the supernatants from b c-infected mavs−/− astrocytes collected at h p.i., indicating that cell-to-cell contact was not significantly decreased in these cells. moreover, no zo- degradation was observed in b.end cells cocultured with the supernatants from wt or mavs−/− astrocytes infected with drv. to be noted, no significant changes in claudin- and occludin expression were detected in either wt or mavs−/− astrocytes upon rabv infection (figures c,e) . together, these results illustrate that lab-attenuated, but not wt, rabv upregulates the production of inflammatory cytokines in astrocytes, resulting in zo- degradation in bmecs and subsequent increases in bbb permeability. furthermore, these results indicate that cytokine production is dependent on mavs signaling pathway. astrocytes play critical roles in host defense during viral infections of the cns ( ) . prr activation in astrocytes results in the expression of many immune mediators, including type i ifns and inflammatory cytokines ( , ) . during infections caused by pathogens for which glia are not susceptible targets, activation of the innate immune system caused by pathogen recognition in astrocytes may promote antiviral immune responses in susceptible neurons, as well as cns leukocyte trafficking ( , , ) . in an early report, primary murine, feline, and human astrocytes were infected with wt (srv) and lab-attenuated rabv (era) ( ), after which viral loads and replication were assessed by infectivity assay and immunofluorescence. the results showed that astrocytes can be infected by rabv, suggesting that astrocytes may play a role in viral spread and persistence and/or neuronal dysfunction ( ) . however, in that study, viral loads and replication were evaluated only at the early time point after infection ( d.p.i.) . in this present study, the growth of drv with that of b c, which were used as a pair of wt and lab-attenuated rabv in previous studies ( ) ( ) ( ) , was compared in a long-term experiment. surprisingly, we found that lab-attenuated rabv, but not wt rabv, caused abortive replication in astrocytes, a feature that may be associated with the ability of the virus to evade the innate immune response. productive infection of the astrocyte is critical for neurotropic pathogens to induce encephalitis. astrocytes sensed viral entry into the cns and mounted a type i ifn response, which rapidly restricts the virus after neuronal transport into the cns. previous studies demonstrated that tlr −/− astrocytes were more permissive to hsv infection and caused severe symptom of encephalitis and tissue damage, which was due to impaired type i ifn production in the absence of tlr ( ) . a recently work found that abortively infected astrocytes are the major producers of ifn-β after infection of the brain with diverse neurotropic viruses, including tmev, rabv, and vesicular stomatitis virus (vsv) ( ) . consistent with these studies, we also found that the abortive infection of lab-attenuated rabv in astrocytes was related to its ability to activate ifn signaling pathway. basal isg expression levels are an important determinant of susceptibility to viral infection. we have found that astrocytes have higher basal expression levels of the mrnas encoding isg proteins, such mda and stat , and other molecules crucial for recognizing viral invasion and creating an more antiviral environment than neurons ( ) , which may explain why lab-attenuated rabv activates the innate immune response to a degree sufficient to restrict viral replication in astrocytes but not in neurons. double-stranded rna is a viral product that plays an essential role in inducing innate immunity, which leads to the production of type i ifns and the activation of hundreds of isgs. early biochemical studies of viral replication suggested that most viruses produce dsrnas ( ) . however, in , weber et al. reported that dsrna could be detected by ifa in cells infected with positive-stranded rna viruses, but not with negative-stranded rna viruses ( ) . this notion was challenged by two other studies on dsrna production in cells infected with negative-stranded rna viruses ( , ) . moreover, a recent study demonstrated the dsrna formation in cells infected with several negative-stranded rna viruses, such as vsv, measles virus (mev), and influenza a virus, although the intensity of the staining of dsrna tended to be weaker in cells infected with negative-stranded rna viruses when compared with those infected with positive-stranded rna viruses ( ) . consistent with this finding, the production of dsrna was detected in both lab-attenuated and wt rabv-infected astrocytes, although lab-attenuated rabv produced significantly more dsrna in the cytoplasm than wt rabv. we attempted to investigate why lab-attenuated rabv produces more dsrna than wt rabv. pfaller et al. found that dsrna expression was much lower in cells infected with wt mev than in cells infected with a mutant mev whose c protein was knocked out which is known to control genome replication and transcription ( ) . similarly, takeuchi et al. observed dsrna formation in cells infected with sendai virus ( ) with c protein knocked out but not in cells infected with wt sev, indicating that the c protein limits or masks dsrna production ( ) . both mev and sev are within the family paramyxoviridae, thus it is possible that their c protein possess the similar function to subvert the production of dsrna. ebola virus protein vp adopts a unique strategy to mask key cellular recognition sites on dsrna ( ) . a recently study showed that the coronavirus endonuclease (endou) activity is the key to prevent early induction of dsrna. replication of endou-deficient coronaviruses is greatly attenuated in vivo and severely restricted in primary cells even during the early phase of virus infection ( ) . in this present study, by exchanging viral genes between wt and lab-attenuated rabv, we found that single n, p, and g of wt rabv could not suppress dsrna formation (data not shown). however, multiple viral proteins of rabv may work together to limit the production of dsrna. further studies are needed to elucidate the mechanism through which wt rabv restricts dsrna formation and thus evades recognition by the innate immune system in infected cells. the bbb, which is composed of specialized bmecs joined by tjs and ensheathed by astrocytes and pericytes, plays an important role in protecting the cns. our previous studies have shown that rabv does not infect bmecs, nor does it modulate tj protein expression in bmecs ( ) . however, brain extracts prepared from mice infected with lab-attenuated rabv but not wt rabv reduced tj protein expression in bmecs, indicating that the above enhancements of bbb permeability and reductions in tj protein expression are not caused by rabv infection. rather, they are caused by virus-induced inflammatory chemokines/ cytokines. the innate immune mechanisms that regulate bbb function in the setting of infectious diseases have been appreciated only recently. multiple inflammatory cytokines, including tnf-α, il- , il- β, ifn-γ, il- , and vegf, disrupt bbb and tj integrity in bmecs ( , , ( ) ( ) ( ) , and inflammatory cytokine signaling at the bbb during infection facilitates leukocyte trafficking into the cns, which is essential for the clearance of many pathogens ( , ) . our present study demonstrates that lab-attenuated rabv induces production of several inflammatory cytokines in astrocytes, especially tnf-α, il- , il- β, ifn-γ, il- , and vegf, which cause disruption of the bbb and tj integrity. our findings suggest that astrocytes play a critical role in regulating bbb permeability as a major source of cytokines during viral infection. furthermore, we found that the production of inflammatory cytokines in astrocytes by lab-attenuated rabv was dependent on mavs signaling pathway, underscoring the critical role of mavs signaling in defensing against rabv infection in cns. | the proposed model for the mechanism through which wild-type (wt) and lab-attenuated rabies virus (rabv) infect astrocytes. during infection, lab-attenuated rabv produces double-stranded rna (dsrna), which is recognized by retinoic acid-inducible gene i (rig-i)/melanoma differentiation-associated protein (mda ). activation of mitochondrial antiviral-signaling protein (mavs), the common adaptor protein for rig-i and mda , leads to enhanced production of interferon, as well as some ifn-stimulated genes, which limit rabv replication and spread. mavs activation stimulates the p and nf-κb signaling pathways and induces cytokine production in astrocytes. inflammatory cytokines promote blood-brain barrier (bbb) permeability, enabling peripheral inflammatory cells and antibodies to infiltrate into the central nervous system, thereby facilitating rabv clearance. in contrast, wt rabv prevents activation of the mavs signaling pathway by restricting dsrna production. conclusion based on the results, we propose the following model: labattenuated rabv produces dsrna recognized by rig-i, mda , or both, resulting in the activation of the mavs signaling pathway in astrocytes. ifn expression induces the transcription of hundreds of isgs to inhibit rabv replication in astrocytes and causes the abortive infection by lab-attenuated rabv. the inflammatory cytokines induced by lab-attenuated rabv enhance bbb permeability, enabling immune cells and antibodies to infiltrate the cns and facilitate rabv clearance. conversely, wt rabv restricts dsrna production and then evades recognition by the innate immune system, resulting in persistent viral replication in astrocytes (figure ). current status of rabies and prospects for elimination the cell biology of rabies virus: using stealth to reach the brain rhabdoviruses: rabies virus role of apoptosis in rabies viral encephalitis: a comparative 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abortively infected astrocytes appear to represent the main source of interferon beta in the virus-infected brain complete genome sequence of a street rabies virus isolated from a rabid dog in china rabies viruses leader rna interacts with host hsc and inhibits virus replication role of chemokines in the enhancement of bbb permeability and inflammatory infiltration after rabies virus infection enhancement of blood-brain barrier permeability and reduction of tight junction protein expression are modulated by chemokines/cytokines induced by rabies virus infection the inability of wild-type rabies virus to activate dendritic cells is dependent on the glycoprotein and correlates with its low level of the de novo-synthesized leader rna expression of neuronal cxcl induced by rabies virus infection initiates infiltration of inflammatory cells, production of chemokines and cytokines, and enhancement of blood-brain barrier permeability cell-type-specific type i interferon antagonism influences organ tropism of murine coronavirus infection of pericytes in vitro by japanese encephalitis virus disrupts the integrity of the endothelial barrier regional astrocyte ifn signaling restricts pathogenesis during neurotropic viral infection influenza a virus ns targets the ubiquitin ligase trim to evade recognition by the host viral rna sensor rig-i overexpression of tumor necrosis factor alpha by a recombinant rabies virus attenuates replication in neurons and prevents lethal infection in mice rabies virus-induced activation of mitogen-activated protein kinase and nf-kappab signaling pathways regulates expression of cxc and cc chemokine ligands in microglia doublestranded rna is produced by positive-strand rna viruses and dna viruses but not in detectable amounts by negative-strand rna viruses measles virus c protein impairs production of defective copyback double-stranded viral rna and activation of protein kinase r production of il- , il- , ifn-gamma and ip- in human astrocytes correlates with alphavirus attenuation sendai virus c protein plays a role in restricting pkr activation by limiting the generation of intracellular double-stranded rna ebolavirus vp coats the backbone of double-stranded rna for interferon antagonism early endonuclease-mediated evasion of rna sensing ensures efficient coronavirus replication astrocytes produce and release interleukin- , interleukin- , tumor necrosis factor alpha and interferon-gamma following traumatic and metabolic injury cellular mechanisms of il- -induced blood-brain barrier disruption expression of interferon gamma by a recombinant rabies virus strongly attenuates the pathogenicity of the virus via induction of type i interferon the authors wish to thank the staff members of the animal facility at huazhong agricultural university for caring for the mice. this work was partially supported by the national natural science foundation of china ( and to zf and and to lz); the national program on key research project of china ( yfd to lz). key: cord- -bqtflk r authors: leblanc, j. j.; elsherif, m.; mulpuru, s.; warhuus, m.; ambrose, a.; andrew, m.; boivin, g.; bowie, w.; chit, a.; dos santos, g.; green, k.; halperin, s. a.; hatchette, t. f.; ibarguchi, b.; johnstone, j.; katz, k.; langley, j. m.; lagacé-wiens, p.; loeb, m.; lund, a.; mackinnon-cameron, d.; mccarthy, a.; mcelhaney, j. e.; mcgeer, a.; poirier, a.; powis, j.; richardson, d.; semret, m.; shinde, v.; smyth, d.; trottier, s.; valiquette, l.; webster, d.; ye, l.; mcneil, s. a. title: validation of the seegene rv multiplex pcr for the detection of influenza a subtypes and influenza b lineages during national influenza surveillance in hospitalized adults date: - - journal: j med microbiol doi: . /jmm. . sha: doc_id: cord_uid: bqtflk r background: the serious outcomes surveillance network of the canadian immunization research network (cirn sos) has been performing active influenza surveillance since (clinicaltrials.gov identifier: nct ). influenza a and b viruses are identified and characterized using real-time reverse-transcriptase polymerase chain reaction (rt-pcr), and multiplex testing has been performed on a subset of patients to identify other respiratory virus aetiologies. since both methods can identify influenza a and b, a direct comparison was performed. methods: validated real-time rt-pcrs from the world health organization (who) to identify influenza a and b viruses, characterize influenza a viruses into the h n or h n subtypes and describe influenza b viruses belonging to the yamagata or victoria lineages. in a subset of patients, the seeplex rv one-step ace detection assay (rv ) kit was also used for the detection of other respiratory viruses. results: in total, nasopharyngeal swabs were tested by rv and real-time rt-pcrs for influenza a and b identification and characterization. for influenza a, rv showed . % sensitivity, % specificity and . % accuracy. the performance characteristics of rv were similar for influenza a subtypes h n and h n . for influenza b, rv had . % sensitivity, % specificity and . % accuracy, with similar assay performance being shown for both the yamagata and victoria lineages. conclusions: overall, the detection of circulating subtypes of influenza a and lineages of influenza b by rv was similar to detection by real-time rt-pcr. multiplex testing with rv allows for a more comprehensive respiratory virus surveillance in hospitalized adults, without significantly compromising the reliability of influenza a or b virus detection. influenza virus infection is a leading infectious cause of morbidity and mortality in developed countries, and is of considerable public health concern [ ] [ ] [ ] [ ] [ ] [ ] [ ] . many vaccine formulations have been developed to reduce the burden of influenza illness, but the continued evolution of the viruses through antigenic drift requires that the vaccines be reformulated each year [ ] [ ] [ ] . monitoring the epidemiology and burden associated with circulating influenza viruses is important to make informed recommendations on vaccine use [ ] [ ] [ ] , and the currently circulating strains include influenza a virus subtypes h n and h n and the yamagata and victoria lineages of influenza b [ ] . since , the serious outcomes surveillance network of the canadian immunization research network (cirn sos) has been conducting active surveillance for acute respiratory illness in hospitalized adults to monitor the burden of influenza illness and assess the effectiveness of seasonal influenza vaccines against laboratory-confirmed influenza [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the cirn sos network comprises to acute care (depending on the year) hospitals across canada, and influenza testing is performed using real-time reverse-transcriptase polymerase chain reaction (rt-pcr) methods derived from the world health organization (who) [ ] [ ] [ ] . these methods allow the identification of influenza a and b, the discrimination of influenza a viruses into h n and h n subtypes, and the characterization of influenza b viruses into yamagata or victoria lineages [ ] [ ] [ ] . these viruses were all circulating in canada at the time of this study. while who-based real-time rt-pcrs methods are often viewed as the reference standard for influenza virus detection, diagnostic laboratories and surveillance studies often test for other viral aetiologies of respiratory illness [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . to avoid the high cost and labour associated with individual virus detection, multiplex rt-pcr technologies have been developed and are now commercially available [ ] . multiplex rt-pcr can detect influenza a and b, as well as non-influenza respiratory viruses (nirvs), such as respiratory syncytial virus (rsv), coronaviruses, rhinoviruses, human metapneumovirus (hmpv), human parainfluenza viruses, adenovirus and enteroviruses [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . while the focus often lies on influenza, nirvs can also be a significant causes of morbidity and mortality [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . nirvs can also co-circulate with influenza [ , ] , and can mirror the clinical presentations of influenza, or other viral or bacterial respiratory tract infections [ ] [ ] [ ] [ ] [ ] [ ] [ ] . from a diagnostic perspective, rapid identification of viral respiratory viruses has potential benefits, such as a decrease in antibiotic prescriptions, a decrease in laboratory investigations and more judicious use of oseltamivir, and allows for the implementation of infection control practices, such as patient cohorting [ , , ] . from a surveillance perspective, understanding the epidemiology of nirvs can inform guidelines for patient management [ ] [ ] [ ] ] and help guide the development of new vaccines or therapeutics [ ] . this study focuses on molecular detection of influenza viruses for the purpose of surveillance. the cirn sos network has performed testing for nirvs on a subset of nasopharyngeal swabs collected for influenza surveillance. nirv testing was performed using a health canada approved test, the seeplex rv one-step ace detection assay (seegene, inc., seoul, republic of korea) (rv ). this conventional multiplex rt-pcr uses dual-priming oligonucleotide (dpo) technology [ , ] for the detection of influenza a and b, as well as other respiratory viruses. the performance of rv has previously been compared against cell culture, direct immunofluorescence, real-time rt-pcr and other multiplex rt-pcrs for respiratory viruses [ - , , ] ; however, to the best of our knowledge, the performance of rv for the detection of influenza a and b has yet to be compared against that of the who real-time rt-pcr reference methods. given that rv showed variable performance for other viruses compared to other molecular methods [ , , , ] , an assessment of rv against the who reference methods for influenza a and b is well justified. both rv and the who real-time rt-pcrs were used by the cirn sos network, allowing a direct comparison of these methods. (toronto, on), le comité d'éthique de la recherche de trois-rivières-centre hospitalier affilié universitaire regional (trois-rivières, qc), the university of british columbia clinical research ethics board (vancouver, bc) and the university of manitoba health research ethics board (winnipeg, mb). active surveillance for influenza was performed across up to hospitals in canadian provinces over consecutive influenza seasons starting in , but the specimens from this study were collected between november and may . on a daily basis, dedicated sos network surveillance monitors reviewed all adult admissions (aged ≥ years) to identify patients with an acute respiratory illness, and patient demographics and outcomes were collected. patient demographics and outcomes have been the subject of several cirn sos network publications [ ] [ ] [ ] [ ] [ ] [ ] [ ] . within days of onset of illness, consenting patients were enrolled and tested for influenza viruses a and b from np swabs collected in universal transport media (utm) (copan diagnostics). all swabs were divided into aliquots and archived at − °c for batch shipment on dry ice to the cirn sos reference laboratory at the canadian center for vaccinology (ccfv) (halifax, ns). the cirn sos reference laboratory retested all specimens from each cirn site using validated real-time rt-pcr methods [ ] [ ] [ ] . total nucleic acids (tnas) were extracted from µl of np swab material using a magnapure lc . instrument (roche diagnostics). the tnas were eluted into µl, and µl served as a template for each µl real-time rt-pcr reaction consisting of . µl superscript iii rt/platinum taq mix pcr enzyme mix (invitrogen, carlsbad, ca, usa), ×pcr master mix, . µm primers and . µm probes (table s , available in the online version of this article). initially, influenza strains were identified as a or b with a duplex real-time rt-pcr using primers targeting the matrix genes for each influenza type (table s ). subsequently, tna that was positive for influenza a was subjected to a real-time rt-pcr subtyping assay targeting the haemagglutinin (ha) genes specific to either h or h . tna that was positive for influenza b was subjected to realtime rt-pcr characterization into victoria or yamagata lineages. amplifications of all real-time rt-pcr assays were performed on an applied biosystems fast instrument (life technologies), under the following conditions: reverse transcription at °c for min; activation of the taq dna polymerase at °c for min; and cycles of °c for s (denaturation) and °c for s (combined annealing and extension). threshold cycle (c t ) values were provided by the manufacturer's software, and the c t cutoff for positivity was determined using previously validated values at defined thresholds (table s ) . following the same tna extraction method as described for real-time rt-pcr analyses, tna was extracted from a separate aliquot of np swab material and µl of tna was used as a template for rv reactions, as recommended in the manufacturer's instructions. amplification was performed in a -well plate in a c touch thermocycler (biorad laboratories ltd, mississauga, on, canada). amplicons were resolved using . % (w/v) agarose gel electrophoresis with staining using . µg ml − ethidium bromide (final concentration), and visualized on a geldoc xr+instrument with imagelab software (version . ) (biorad laboratories). influenza a and b results from the rv or real-time rt-pcr assays were classified as positive or negative, and compared to a composite reference standard where concordant results between two of three methods were considered a true positive or negative result. discrepant analyses were performed using real-time rt-pcr and/or sequencing by the national microbiology laboratory (winnipeg, mb, canada). statistical analysis software (sas) version . (sas institute, cary, nc, usa) was used to assess significant differences between methods using × contingency tables and mcnemar's chi square test. a p value ≤ . was considered statistically significant. sensitivity, specificity, accuracy, misclassification rates and kappa statistics were reported with % confidence intervals (cis). viral loads in np swabs were assessed based on standard curves generated by amplification of the matrix gene targets for influenza a and b using conventional rt-pcr, cloning of each target into plasmids and performance of real-time rt-pcr on serial dilutions of the quantified plasmids. to directly compare the limits of detection (lod) of rv and the who real-time rt-pcrs, parallel testing was performed using -fold serially diluted viruses. the viruses used were reference strains of the cirn sos network triplicates values from three independent experiments were analysed, and the c t values obtained from real-time rt-pcr for influenza a and b were used to estimate viral concentrations (relative to the standard curves generated using plasmid controls). the lod was estimated at a probability of % by probit analysis [ , ] using statplus professional version . . . laboratory data were available on adults hospitalized with acute respiratory illness, who had all molecular tests of interest performed: rv , influenza a/b duplex real-time rt-pcr for influenza a or b screening; and h /h real-time rt-pcr subtyping for positive influenza a specimens or realtime rt-pcr yamagata/victoria lineage discrimination for positive influenza b. of the patients, were positive for influenza a ( h n and h n ) and were positive for influenza b ( yamagata lineage and victoria lineage). most of the results overlapped between testing methods, but some discrepant results were observed (figs and ). compared to the composite reference standard, rv showed a sensitivity for influenza a of . % ( / ; % ci: . - . %) and a specificity of . % ( / ; % ci: . - . %). discordant results were seen in three influenza a positive results (table ; fig. ). the accuracy of rv for influenza a was . % ( / ; % ci: . - . %). similar performance characteristics were noted for influenza a h n and h n ( in this study, the estimated lod of rv for each influenza a subtype and influenza b lineage was higher than that of the real-time rt-pcr for influenza a/b (table ), but below the detection limits of the real-time rt-pcrs for influenza a subtyping or b lineage characterization (tables and s ). this is consistent with previously validated cutoffs for positivity for the real-time rt-pcr methods (table s ) . at viral concentrations that were below the detectable limit of rv but detected using influenza a/b real-time rt-pcr, while this was not statistically significant, rv was unable to detect influenza a or b in a small subset of specimens in which the viral loads were low. conventional multiplex pcrs such as rv are often less sensitive than real-time rt-pcr, and this can be explained in part by the assay principle. realtime rt-pcr detects fluorescence signals captured during pcr amplification cycles, and results are defined objectively using validated cutoffs for positivity. conventional rt-pcr is an end-point detection of amplicons following electrophoresis and staining; a process where visualization of amplicons can be difficult and subjective when working with specimens with low concentrations of target [ - , - , , ] . whether conventional or real-time rt-pcr is used, the performance of these molecular methods can also vary, depending on factors such as genetic mismatches in the pcr target region, which could arise in influenza over time via antigenic drift. this emphasizes the need to verify the performance for characterized and circulating subtypes and lineages of influenza. however, for rv , only a limited number of studies have assessed its ability to detect influenza a and b viruses, and none have specifically looked at its performance for the detection of influenza a virus subtypes or influenza b lineages [ , , , , ] . cho et al. [ ] compared rv to a composite reference standard that included culture and a commercial real-time rt-pcr and demonstrated that rv *the concentration that could reproducibly (at % confidence) be detected by the test method is described as the lod. the lod was determined by probit analysis by testing replicate aliquots of virus dilutions (see table s ). for each virus aliquot, the c t values obtained by the who real-time rt-pcr were used to infer viral load. concentrations of virus dilutions were estimated using standard curves generated with plasmids pflua and pflub from this study (see supplementary material) [ ] assessed the performance of rv against direct fluorescent antibody testing, virus culture and isolation, and three additional multiplex pcr methods. the specificities for influenza a and b were . and %, and the sensitivities were . % ( % ci: . - . %) and % ( . - %), respectively. in the present study, the performance of rv was verified for the detection of recent influenza a subtypes (h n and h n ) and influenza b lineages (yamagata and victoria), and it showed similar performance characteristics to the who real-time rt-pcr. since this study used specimen collected prospectively from to , it could be argued that the performance of the molecular methods assessed could vary with more recent circulating influenza strains, if primer or probe mismatches occur in the assay gene targets. however, the cirn sos laboratory participates in yearly quality assurance programmes proficiency testing for influenza a and b detection and characterization using all the assays from this study (all real-time rt-pcrs and the rv assay), and no differences in assay performance have been observed over time (data not shown). in this study, only a small subset of results in specimens with low viral loads were not detected by rv , with this representing . and . % of the total tests for influenza a and b, respectively. while the lower sensitivity of multiplex pcrs such as rv was expected, the possibility of target gene sequence mismatches cannot be excluded. it should be noted that viral loads were estimated using quantification relative to a standard curve generated using plasmid dna controls. with influenza virus being an rna virus, comparison to plasmid dna does not account for the reverse transcription step. regardless, all viruses were subjected to direct method comparison in the analytical analyses, which includes the reverse transcription step. subsequently, the resulting c t values were subjected to comparison against the standard curve derived from plasmid dna results, as plasmid dna was more readily quantifiable. overall, with this approach it remains valid to infer that the specimens not detected by rv had low viral loads, but the absolute quantity of virus should be considered to be an estimate (tables , s and s ) . overall, this would have little impact on the conclusions from this study. this study's strengths include prospectively collected specimens from a defined patient population (adults hospitalized with acute respiratory illness), comparison of results against reference methods for influenza a and b detection, and analyses performed on influenza viruses characterized by subtyping or lineage determination. the main limitation of the study was that it was focused solely on influenza virus. however, this is justified because influenza viruses are the only respiratory viruses for which vaccines are currently available, and the performance for the detection of other viruses has been assessed previously [ , [ ] [ ] [ ] [ ] [ ] . while assessing the performance of rv against validated real-time rt-pcrs for influenza is well justified, this study focused on the use of rv for population-based surveillance in hospitalized adults. these findings should not be extrapolated to other patient populations or for applications in clinical diagnostic testing where individual-level results are prioritized. the patients tested in this study were hospitalized adults, often presenting with co-morbidities and severe outcomes [ ] [ ] [ ] [ ] [ ] [ ] [ ] . further, respiratory virus testing is often performed using testing algorithms following initial screening methods for influenza a and b. as such, nucleic acids extracted from clinical specimens are sometimes inadvertently subjected to a freeze/thaw cycle prior to testing. the impact of freeze/ thaw was not assessed in this study, as testing was performed following independent nucleic acid extraction on a different specimen aliquot. however, given that rv failed to detect a small subset of influenza a and b specimens at low viral loads, additional freeze/thaw cycles may further compromise influenza virus detection, and should thus be avoided. finally, the workflow is relatively simple with rv for small numbers of specimens, but additional benefits could be afforded by using imaging software enabling automated amplicon detection if high-throughput specimen processing is required [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . such automated analyses of rv amplicons could also reduce reduce result subjectivity compared to interpretations made from visual assessment of amplicons [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . automated analyses were not evaluated in this study, but the technical staff performing rv testing were blinded to the real-time rt-pcr method results to avoid bias, and the rv results were remained unchanged with subsequent independent review by other blinded staff members. overall, the performance of rv was comparable to the who real-time rt-pcr standards for the detection of recently circulating subtypes of influenza a and lineages of influenza b, and it only missed a very small subset of influenza a and b results at low viral loads. given that the performance characteristics of the rv multiplex pcr are not provided in the manufacturer kit insert, these data are of value for its users, which include several acute care hospitals and provincial public health laboratories in canada [ , ] . this study shows that the rv conventional multiplex pcr can be used for surveillance studies for respiratory viruses without significantly compromising detection of influenza a and b. the use of multiplex technologies such as rv can help better define the epidemiology of influenza and nirvs, and these data are important for the development of novel therapeutics and vaccines. influenza virus surveillance was funded by the public health agency of canada (phac) and the canadian institutes of health research (cihr), and through a collaborative research agreement between glaxosmith-kline biologicals sa and the cirn sos network. cirn and sm provided funding for rv testing. the authors received no financial support or other form of compensation related to the development of the manuscript, and were solely responsible for final manuscript content and data interpretation. the many dedicated cirn sos network surveillance monitors, the hospital staff and the ccfv staff, who were instrumental in the recruitment, collection, processing and archiving of specimens, and data collection. interim estimates of / influenza clinical severity and vaccine effectiveness in the prevention of laboratory-confirmed influenzarelated hospitalisation interim estimates of / influenza vaccine effectiveness in preventing laboratory-confirmed influenza-related hospitalisation from the serious outcomes surveillance network of the canadian immunization research network influenza vaccine effectiveness against influenza-related hospitalization during a season with mixed outbreaks of four influenza viruses: a test-negative case-control study in adults in canada the importance of frailty in the assessment of influenza vaccine effectiveness against influenza-related hospitalization in elderly people influenza vaccine effectiveness to prevent influenza-related hospitalizations and serious outcomes in canadian adults over the / through / influenza seasons: a pooled analysis from the canadian immunization research network (cirn) serious outcomes surveillance (sos network) effectiveness of influenza vaccination on hospitalizations and risk factors for severe outcomes in hospitalized patients with copd the impact of prior season vaccination on subsequent influenza vaccine effectiveness to prevent influenza-related hospitalizations over influenza seasons in canada the rationale for quadrivalent influenza vaccines national advisory committee on immunization (naci) who recommendations on the composition of influenza virus vaccines respiratory virus detections in canada cdc protocol of real-time rt-pcr for influenza a(h n ) who information for molecular diagnosis of influenza virus in humans -update differentiation of influenza b virus lineages yamagata and victoria by real-time pcr evaluation of allplex respiratory panel / / multiplex real-time pcr assays for the detection of respiratory viruses with influenza a virus subtyping evaluation of anyplex™ ii rv and 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seeplex rv for simultaneous detection of respiratory viruses use of the seeplex rv detection kit for surveillance of respiratory viral outbreaks in the clinical significance of filmarray respiratory panel in diagnosing community-acquired pneumonia clinical evaluation of the luminex nxtag respiratory pathogen panel comparison of the biofire filmarray rp, genmark esensor rvp, luminex xtag rvpv , and luminex xtag rvp fast multiplex assays for detection of respiratory viruses cost analysis of multiplex pcr testing for diagnosing respiratory virus infections co-infections with influenza and other respiratory viruses influenza-like illness incidence is not reduced by influenza vaccination in a cohort of older adults, despite effectively reducing laboratory-confirmed influenza virus infections global epidemiology of non-influenza rna respiratory viruses: data gaps and a growing need for surveillance severe morbidity and mortality associated with respiratory syncytial virus versus influenza infection in hospitalized older adults respiratory syncytial virus and other noninfluenza respiratory viruses in older adults viral respiratory infections of adults in the intensive care unit unexpectedly higher morbidity and mortality of hospitalized elderly patients associated with rhinovirus compared with influenza virus respiratory tract infection detection of enterovirus d in canadian laboratories practical guidance for clinical microbiology laboratories: viruses causing acute respiratory tract infections respiratory outbreak investigations: how many specimens should be tested? rapid detection and identification of respiratory viruses using a dual priming oligonucleotide systembased multiplex pcr assay a multiplex pcr assay for the detection of five influenza viruses using a dual priming oligonucleotide system detection of influenza h n virus: all molecular tests are not equal this study was approved by the research ethics boards (rebs) at each participating hospital ( clinicaltrials. gov identifier: nct the authors would like to thank the patients and families whose participation made this study possible. the authors would also like to thank author contributions all authors participated design and implementation or implementation or analysis, interpretation of the study, and the development of the manuscript. all authors had full access to the data and gave final approval before submission.conflicts of interest j. l. received research grants from merck for work outside the study, but no personal payments. t. f. h. and s. a. m. report payments to their institution from the gsk group of companies for the conduct of the study, and payments from pfizer, merck, novartis and sanofi-pasteur outside the submitted work. m. k. a. reports grants from the gsk group of companies, pfizer and sanofi, but no personal payments. j. m. reports payments to her institution from the gsk group of companies and sanofi for her participation in advisory boards. a. p. reports payments from actelion, sanofi-pasteur and genentech. j. p. reports payments from the gsk group of companies, merck, roche and synthetic biologics outside the submitted work. l. v. received research grants from the gsk group of companies, pfizer, optimer, cubist and merck, and personal fees from merck, optimer and cubist. g. d. s. reports he was external consultant at business and decision life sciences (on behalf of gsk) at the time of the study, and is currently employed by the gsk group of companies and holds shares in the gsk group of companies. no other conflicts were declared. five reasons to publish your next article with a microbiology society journal . the microbiology society is a not-for-profit organization. . we offer fast and rigorous peer review -average time to first decision is - weeks. . our journals have a global readership with subscriptions held in research institutions around the world. . % of our authors rate our submission process as 'excellent' or 'very good'. . your article will be published on an interactive journal platform with advanced metrics.find out more and submit your article at microbiologyresearch.org. key: cord- - tss ydx authors: chen, jiao; shang, jiayu; wang, jianrong; sun, yanni title: a binning tool to reconstruct viral haplotypes from assembled contigs date: - - journal: bmc bioinformatics doi: . /s - - - sha: doc_id: cord_uid: tss ydx background: infections by rna viruses such as influenza, hiv still pose a serious threat to human health despite extensive research on viral diseases. one challenge for producing effective prevention and treatment strategies is high intra-species genetic diversity. as different strains may have different biological properties, characterizing the genetic diversity is thus important to vaccine and drug design. next-generation sequencing technology enables comprehensive characterization of both known and novel strains and has been widely adopted for sequencing viral populations. however, genome-scale reconstruction of haplotypes is still a challenging problem. in particular, haplotype assembly programs often produce contigs rather than full genomes. as a mutation in one gene can mask the phenotypic effects of a mutation at another locus, clustering these contigs into genome-scale haplotypes is still needed. results: we developed a contig binning tool, virbin, which clusters contigs into different groups so that each group represents a haplotype. commonly used features based on sequence composition and contig coverage cannot effectively distinguish viral haplotypes because of their high sequence similarity and heterogeneous sequencing coverage for rna viruses. virbin applied prototype-based clustering to cluster regions that are more likely to contain mutations specific to a haplotype. the tool was tested on multiple simulated sequencing data with different haplotype abundance distributions and contig sizes, and also on mock quasispecies sequencing data. the benchmark results with other contig binning tools demonstrated the superior sensitivity and precision of virbin in contig binning for viral haplotype reconstruction. conclusions: in this work, we presented virbin, a new contig binning tool for distinguishing contigs from different viral haplotypes with high sequence similarity. it competes favorably with other tools on viral contig binning. the source codes are available at: https://github.com/chjiao/virbin. high genetic diversity within viral populations has been observed in patients with chronic infection with rna viruses such as human immunodeficiency virus (hiv), hepatitis c virus (hcv), etc [ ] [ ] [ ] [ ] [ ] . the genetic diversity could be caused by multiple infections of different strains or by mutations during the virus replication inside the host. in the latter case, the high replication rate, coupled with the low fidelity of the viral polymerase in most rna viruses, results in a group of different but related strains infecting the same host, which is often termed as *correspondence: yannisun@cityu.edu.hk electrical engineering, city university of hong kong, hong kong, china full list of author information is available at the end of the article "quasispecies" [ ] . previous studies have revealed that patients with chronic virus infections, such as acquired immune deficiency syndrome (aids), are often the reservoir of new viral variants, which are likely produced during the replication process [ ] . because different strains could have very different biological properties such as virulence, transmissibility, antiviral drug resistance etc, characterizing the genetic diversity within viral populations is very important for developing effective prevention and treatment strategies. for example, if some strains have developed antiviral drug resistance, they may become the dominant strains and lead to treatment failure. thus, characterization of the strain-level diversity of viral populations is indispensable for understanding the viruses and is of great clinical importance. sequencing viral quasispecies for genetic diversity analysis was one of the first applications of nextgeneration sequencing (ngs) technologies [ ] . applying whole genome shot-gun sequencing to viral quasispecies does not require cultivation and can sequence divergent strains from known virus families. it thus has become a favored choice for characterizing the diversity of quasispecies. given the sequenced viral quasispecies, different types of analysis can be conducted to probe the genetic diversity. a relatively straightforward analysis is to understand the local diversity of known viruses by performing read mapping against reference genomes. while this type of analysis can produce a collection of local changes (mutations, insertions, or deletions) of the strains inside the quasispecies, it is not sufficient to infer the biological properties of the strains, which are more likely to be determined by multiple genes. in particular, epistatic interactions are abundant in rna viruses, where the mutation of one gene masks the phenotypic effects of a mutation at another locus. thus genome-scale reconstruction of the strains is essential for phenotype prediction of viruses [ ] . reconstructing the genome-scale strain sequences inside quasispecies is often referred to as genome-scale haplotype reconstruction, where the genomes of strains are called haplotypes. the goal is to assemble short reads from sequenced viral populations into correct haplotype sequences. when the reference genome is available, read mapping can be conducted first to identify local mutations and then cluster the local mutations (or short contigs) into genome-scale haplotypes. when quality reference genomes are not available, which is often the case for emerging viruses such as severe acute respiratory syndrome (sars) coronavirus, read mapping is not a very effective strategy to identify all mutations. thus, de novo assembly is needed to stitch the reads into haplotypes. with or without reference genomes, genome-scale haplotype reconstruction in quasispecies remains a computationally challenging problem. high similarity between haplotypes in the same quasispecies and the heterogeneous sequencing depth along the viral genomes present barriers to adoption of existing assembly programs. a recently published comparison showed that none of the tested haplotype reconstruction tools were able to successfully reconstruct the five known strains for a mock hiv quasispecies [ ] . we had the same observations when comparing several popular metagenomic assembly tools and haplotype assembly tools such as idba-ud [ ] , iva [ ] , savage [ ] , mlehaplo [ ] on the same data set [ ] . many methods output a set of contigs with various sizes that are much shorter than the genomes. with these outputted contigs from assembly programs, it still remains to infer the number of haplotypes and to match the contigs to their originating haplotypes. thus, there is a need to cluster the contigs into different groups so that each group represents a haplotype. this step is called contig scaffolding or binning and has been applied for bacterial strain characterization. contig binning for viral quasispecies has its unique challenges. first, the goal of binning is to distinguish contigs from different viral strains rather than species. thus, composition-based features such as tetranucleotide frequencies or gc contents are not informative enough to separate contigs from different haplotypes, which usually share high sequence similarity (over %). tools that heavily rely on sequence composition-based features will not be able to estimate the number of haplotypes correctly. second, rna virus sequencing tends to be compounded by gene expression and fast degradation and thus the observed sequencing coverage along each haplotype, or even a contig, can be more heterogeneous than expected. in addition, if a contig contains a region that is common to multiple haplotypes, that region tends to have higher coverage than a haplotype-specific segment. all these challenges require carefully designed methods to use the coverage information for contig binning. although a number of contig binning algorithms have been developed [ ] [ ] [ ] [ ] [ ] [ ] [ ] , they all possess limitations in distinguishing contigs from different viral strains of the same species. most of the existing contig binning tools for microbiome sequencing data are designed for bacteria. these methods usually estimate the bin number by aligning metagenomic data to a pre-established marker gene database, and then assign assembled contigs to different bins using sequence composition information and read coverage levels. for example, maxbin [ ] uses both tetranucleotide frequencies and contig coverage levels to assign assembled contigs into different bins. some binning tools [ ] leverage co-abundance of genes across multiple metagenomic samples. the rationale is that if two contigs are from the same bin, their coverage profiles across multiple samples should be highly correlated. recently, there are a couple of newly developed tools for strain level analysis from metagenomic data, such as constrain [ ] and strainphlan [ ] . both rely on species identification using clade-specific genes, then zoom in to identify the strains. however, both tools were mainly tested on bacteria. our method is designed to cluster contigs produced by existing assembly tools. there are another group of methods conducting haplotype reconstruction via read clustering [ , ] , which groups variant sites obtained by read mapping against reference genomes. these tools don't usually output contigs and thus do not use contig binning. here we present virbin, a method designed specifically for binning contigs derived from viral quasispecies data. the input to virbin is a set of contigs derived from assembly tools. the output includes the estimated number of haplotypes, the grouped contigs for each haplotype, and the corresponding relative abundances. unlike many bacterial contig binning tools that require multiple samples, our method works on a single sample. we evaluate the haplotype number estimation and clustering performance of virbin on both simulated and mock hiv quasispecies sequencing data. the simulated data provide us with known ground-truth for accurate evaluation of the clustering performance. to evaluate how the number of haplotypes affects the haplotype inference performance, we produced simulated quasispecies sequencing data consisting of haplotypes and haplotypes, respectively. for each experiment, we evaluate the performance of virbin from three aspects: haplotype number estimation, clustering performance, and the computed haplotype abundance. when the originating haplotypes of the input contigs are known, we can evaluate both the recall and precision for the clustering step. first, we map the clusters to haplotypes based on the consensus haplotype label of the component contigs. if there is no consensus haplotype membership (e.g. a tie), we map the clusters to haplotypes based on the ranking of the abundance. let a cluster be b and its paired haplotype be h. as the input to our program is a set of contigs, let the contig set originating from h be c h . define b ∩ c h as the common regions between the two contig sets. following other contig binning tools [ , ] , the base-level recall for h is thus |b∩c h | |c h | , which quantified how many of the bases in c h are correctly clustered in b. the base-level precision is defined as |b∩c h | |b| , which quantifies how many of the bases in cluster b are from contig set c h . similar metrics can be defined for contig-level, which can be found in the additional file . first, we constructed haplotypes with average sequence similarity around %. second, in order to simulate haplotypes of different relative abundances, we generated sets of reads following different abundance distributions. third, we generated contigs using two different methods. in method , we simulated sets of error-free contigs of different sizes directly from the reference genomes. in method , we applied available assembly tools to generate contigs from the reads. the simulated contigs are not dependent on any assembly tool and thus are ideal for evaluating the binning method. for simulated contigs, we have (sets of reads) × (sets of simulated contigs), i.e., sets of input to our program. for assembled contigs, we have (sets of reads) × (sets of assembled contigs) as input to virbin because we applied two assembly tools. additional file : figure s sketches the process of input data generation. the data simulation details can be found below. hiv haplotype construction: there are many sequenced hiv strains in the hiv sequence database [ ] . however, many of the strains do not possess sufficiently high similarity to be included in simulated quasispecies. thus, we use both real and simulated strain sequences to simulate haplotypes of high similarity. simulated strains were produced by mutating, deleting, or inserting bases at random positions from a real strain in the hiv database. as a result, the five-haplotype dataset contains a hiv- strain fj from hiv sequence database, simulated haplotypes from fj , and another hiv- strain fj . the sequence similarity between the simulated haplotypes and its originating sequence is %. the average sequence similarity between all the five haplotypes is around %, which is comparable to the sequence similarity between haplotypes in a mock hiv quasispecies dataset [ ] . reads simulation using different haplotype abundance distributions: with available hiv haplotypes, simulated reads were generated from them by art-illumina [ ] as error-containing miseq paired-end reads, with read length of bp, average insert size of bp, and standard deviation of bp. with the total coverage of -x, three sets of reads are produced using different abundance distributions. the first one is based on the power law equation [ ] . the second and the third sets of reads represent challenging cases where different haplotypes have similar abundances, which create difficulties for abundance-based binning algorithms. the abundance differences in the second and third data set are . and . , respectively. in total, there are , simulated reads for hiv haplotypes. the relative abundance for five haplotypes in each read set can be found in table . "power" is the read sets generated based on the power law equation. "equal ( %)" is the abundance distribution with equal difference of . . "equal ( %)" is the abundance distribution with equal difference of . as the total coverage is -x, the sequencing coverage of each haplotype is the product of the total coverage and the relative abundance. contig simulation: for each reference genome (denote its length as l), we randomly generated a list of location pairs (p , p ), where ≤ p < p ≤ l. each location pair represents a candidate contig's starting and ending position. then, in the simulated contigs, we only keep the ones above bp (i.e. p − p + ≥ ). in addition, we would like to simulate the hard case where the contigs cannot be extended any more using large overlaps. thus, we sort all the remaining contigs by p and remove the ones that have overlaps of size above bp with previous contigs in the sorted list. the five sets of simulated contigs have different n values and are referred to as " " to " ", indicating the upper bound of the contig length in each set. additional file : table s shows the detailed properties of the five sets of contigs. all the simulated data sets can be downloaded from virbin's github repository. according to our methods, the haplotype number estimation only depends on the alignment results of contigs. for all five sets of simulated contigs with different average lengths, the consensus window depth of the longest windows is for all. the histogram of window depth for simulated contig sets is shown in fig. . it is clear that window depth dominates longest windows. thus, the estimated number of haplotypes is , reflecting the truth for our data sets. in general, the percentage of windows with depth increases with increasing contig lengths. we applied virbin to cluster contigs into groups. since the ground truth about the haplotype membership of each contig is known, we were able to evaluate the clustering results by calculating the precision and recall at the base level. the evaluation results are shown in fig. . the performance of clustering is worst for shortest contig set (denoted as along the y-axis). with increasing contig lengths, the clustering performance becomes better for all three different abundance distributions. when the contigs are long, the clustering performance for haplotypes with different abundance distributions is comparable. the results were compared with maxbin, which is a binning tool for metagenomic contigs based on tetranucleotide frequencies and reads coverage levels. maxbin requires marker genes to identify seed contigs for binning. we were able to run the core clustering program of maxbin by inputting both the number of haplotypes (i.e. ) and the seed contigs manually. we randomly chose one contig from each haplotype as the seed contig and calculated the contigs' abundances by mapping reads to them using bowtie . although the haplotype number was explicitly provided to maxbin, empty clusters can be produced by maxbin. the results from maxbin are shown in fig. . for the shortest contig set, maxbin only reported two clusters with one contig for each cluster, leaving ( . %) contigs unclassified. for contigs sets from - , maxbin was able to generate five clusters, but with~ % contigs unclassified. the results of maxbin usually have lower precision or recall values than virbinİn addition, fig. the histogram of window depth for the longest windows. x-axis: the observed window depth, from to . y-axis: the number of windows with the corresponding window depth on x-axis. " " to " " represent the five sets of contigs for contig sets from - , there are haplotypes without correctly assigned contigs. the lower sensitivity of maxbin could be caused by its dependency on both sequence composition and contig coverage for clustering. due to high sequence similarities between viral haplotypes, sequence composition is not informative enough in differentiating contigs from different viral strains. instead, maxbin could mistakenly cluster contigs from the homogeneous regions of the viral genome, leading to more chimeric clusters. strainphlan [ ] is also able to to characterize the genetic structure of viral strains in metagenomes. it takes the raw sequencing reads and metaphlan [ ] database of species-specific reference sequences as input and aims to output the most abundant strain for each sample. however, it failed to detect any viral species at the first step running metaphlan . constrains [ ] is another tool designed to identify strain structures from metagenomic data. it uses bowtie to map reads to a set of universal genes and infers the within-species strains abundances by fig. the recall and precision of contig binning results by maxbin. x-axis represents each haplotype, in decreasing order of relative abundance. y-axis is the index of the data sets utilizing single-nucleotide polymorphism (snp) patterns. this tool again did not get enough mapped reads to report any strain abundance. and it takes considerable efforts for us to modify their codes for our inputs. thus, we cannot report the results from strainphlan or constrains. relative abundance computation: once the iterative clustering algorithm converges, the abundance of each cluster can be computed as the weighted average abundances for all contigs from this cluster. fig. compares the known haplotype abundance profiles with our computed ones. there are three read sets with different abundance distributions (table ) . for each distribution, there are five sets of contigs (additional file : table s ). thus, three plots of five curves are presented to compare the ground truth and the computed abundance. in addition, we applied χ -test to compare the ground-truth distribution and each computed abundance distribution. the p-values from all the tests are larger than . , indicating that the distributions are not statistically different. as maxbin only correctly clustered several contigs, we did not include the abundance comparison. fig. compare the known haplotype abundance distributions with computed ones by virbin. x-axis represents each haplotype. y-axis is the ground truth or predicted abundance for each haplotype in addition to simulated contigs, we also tested virbin on assembled contigs by two de novo assembly tools: a generic assembly tool sga [ ] and a viral haplotype reconstruction tool pehaplo. the assembled contigs were evaluated by metaquast [ ] and the results are listed in additional file : table s . both pehaplo and sga produced enough contigs to cover almost all the five haplotypes. contigs produced by pehaplo have larger n values than contigs by sga. while both tools apply overlap graph for assembly, pehaplo utilizes a pairedend information guided method for path finding, which can potentially connect some of the nodes together. therefore, it produced longer contigs than sga. the contigs are paired with haplotypes based on the highest sequence similarity. all of the contigs and their originating haplotypes have similarity of at least %. for all three sets of contigs assembled by pehaplo and sga on three sets of reads, the consensus window depth of the longest windows is , revealing the actual haplotype number. figure a presents the clustering results on contigs generated by pehaplo and sga. it shows that virbin achieved good clustering results on contigs assembled by both assembly tools. the clustering results on sga's contigs are similar to pehaplo's contigs, with both high precision and recall. this observation is consistent with the results on simulated contigs that when the contigs are long enough, virbin can produce good results. again we compared our results with maxbin. the clustering results of maxbin on assembled contigs are shown in fig. b . for contigs assembled by pehaplo, maxbin correctly clustered all corresponding contigs to the strain fj -h as the recall is . . however, this cluster also the comparison between the predicted abundance by virbin and the ground-truth on two sets of assembled contigs is presented in additional file : figure s . we also simulated reads from haplotypes and tested virbin on this data set. the data generation and also the detailed results, clustering results is presented in additional file : table s and the relative abundances for each haplotype are shown in additional file : figure s , can be found in the section of additional file . in this experiment, we applied virbin to a mock hiv quasispecies data set (srr ), sequenced from the mix of five hiv- strains ( . , hxb , jrcsf, nl , yu ) with illumina miseq sequencing technology [ ] . this data set contains , , ( bp) of reads that cover the five strains to , x. the raw data set was preprocessed with faqcs/ . [ ] and trimmomatic [ ] to trim and filter low-quality reads or adapters. the remaining reads were then error-corrected with karect [ ] . after pre-processing, , reads were left. by mapping pre-processed reads to the available reference genomes by bowtie , we were able to estimate the abundance for each haplotype as shown in fig. . we use the contigs assembled by pehaplo as input for virbin. pehaplo produced contigs from the real miseq hiv data set that can cover about % of the five hiv- strains. these contigs have a n value of bp and the longest contig is bp. virbin was applied to the aligned contigs for haplotype number estimation. all the windows were sorted in descending order of window length. out of the top windows, contain contigs, contain contigs, and contain contigs. out of the top windows, contain contigs, contain contigs, and contains contigs. similar to the simulated data, using the consensus window depth (i.e. ) correctly predicted the haplotype number. clustering results: the clustering algorithm was applied to cluster the contigs into groups. for each contig, its originating haplotype is determined by comparing the contig with all reference genomes. the haplotype with the highest similarity and above % is assigned. the outputs of virbin and maxbin are shown in table . strainphlan and constrains were applied on this real hiv data set. strainphlan was able to identify the hiv species, but could not report any strain information. constrains could not align enough reads to marker genes for further strain-level analysis. compared to the simulated contigs or assembled contigs using simulated reads, the results of virbin on the real sequencing data have generally lower sensitivity and precision. there are two major reasons. first, the assembled contigs for real reads are more likely to contain errors. second, this data set has several haplotypes with very similar average abundances. referring to fig. , the abundance difference between the least abundant haplotypes is < %. thus, the clustering algorithm could mix contigs from these haplotypes. for the mock data experiment, we also present the recall and precision at contig level in additional file : table s . we again compared the predicted abundance profile with the known one in fig. . the χ -test output p-value . and . for virbin and maxbin, respectively, indicating that the predicted abundance distributions by virbin and maxbin are not statistically different from the ground truth. overall, virbin can cluster more contigs into their originating haplotypes than maxbin. while virbin focuses on sub-contigs that are more likely unique to one haplotype, maxbin clusters whole contigs, which could contain regions common to multiple haplotypes and makes read coverage more heterogeneous. in addition, sequence composition-based features such as tetranucleotide frequencies are not effective in distinguishing highly similar viral strains. our experimental results show that virbin works better for longer contigs that can cover bigger regions of the underlying genomes. when the genome coverage by the contigs is below %, the performance of virbin deteriorates because it becomes harder to estimate the correct number of haplotypes. in addition, the empirical experience shows that it is difficult to classify two viral strains when the abundance difference between them is below %. thus, although we have demonstrated much better contig binning performance for distinguishing viral in this work, we presented virbin, a new contig binning tool for distinguishing contigs from different viral haplotypes with high sequence similarity. by conducting experiments on multiple simulated data sets of different haplotype abundance distribution, different number of haplotypes, and different sets of simulated or assembled contigs, we demonstrated that our tool can produce more accurate clustering results than popular contig binning tools for viral haplotype reconstruction. the overall pipeline of our method is shown in fig. . there are mainly two steps: ( ) estimate the number of haplotypes by aligning contigs and identifying windows; ( ) calculate relative abundances in each window and apply a clustering algorithm to group clusters of the same haplotype. the underlying algorithm of grouping contigs into haplotypes is prototype-based clustering [ ] . features such as the overlaps and paired-end connections have limited usage in grouping distant contigs from the same haplotype. the clustering will mainly use the features based on the abundance distributions. although abundance-based clustering has been used for contig binning from multiple samples [ , ] , existing tools are not designed fig. the pipeline of virbin to tackle key challenges of distinguishing contigs of different haplotypes. first, the observed coverage of each contig not only depends on the abundance of the underlying haplotype, but also depends on whether it is a unique or shared region by two or more haplotypes. second, heterogeneous coverage of each haplotype in an rna viral quasispecies is common, which is caused by sequencing-related biases and compounded by gene expression. thus, directly applying existing prototypebased clustering models such as gaussian-mixture model to contigs is not expected to produce accurate clustering. our solution to this problem is to cut the contigs into "windows" and to apply the clustering on sub-contigs that are more likely to represent one haplotype. in addition, instead of assuming any parametric distribution, which is usually not the case for haplotype contigs, we will use a non-parametric distribution. although the high similarity between haplotypes presents a barrier to adoption of kmer-based features for distinguishing contigs from different haplotypes, it brings opportunities for haplotype number estimation. with stringent alignment threshold, contigs that can be aligned with each other usually come from the same region of the virus and thus the number of aligned contigs can be carefully used for haplotype number estimation. we progressively build multiple sequence alignments using contigs' pairwise alignments. in this step, base-level accuracy of the alignment is not a major concern and thus progressive construction of the alignment between contigs can serve the purpose well. we first sort the contigs by their lengths in descending order. the longest contig will be used as the first reference. all the other contigs will be aligned to the reference using blast+ [ ] to generate an alignment profile similar to multiple sequence alignment. two types of alignments are kept from the output of blast+. one is the alignment that is local to the reference but global to the shorter contigs. the other is overlap alignment, which is the alignment between the suffix/prefix strings of the contigs. if not all the shorter contigs can be aligned to the reference contig, this process will continue by using the second longest contig as the reference until all the contigs are used. figure c shows the alignment between contigs from three haplotypes in fig. a using the longest contig as the reference, which is usually produced for the most abundant haplotype. each multiple alignment can be divided into many windows, which are formed whenever there is a change of the sequences in the alignment. we define the number of contigs inside each window as the window depth, d. based on these definitions, we have the following observations. when each position of the underlying haplotypes can be covered by at least one contig, d is equal to or larger than the number of haplotypes. note that the common regions between different haplotypes are regarded as different positions and thus should be covered by different contigs. this conclusion can be proved by contradiction easily. fig. b shows the contigs satisfying the conditions in the ideal case. there are three haplotypes with different abundances. they only contain mutations at three positions that are far away from each other. because of the long common regions among them, assembly programs usually won't be able to recover all the three genomes. instead, they can generate short but correct contigs. in fig. b , each position in the three haplotypes is covered by at least one contig. in this case, all the windows have depth of at least . if every position of a haplotype is only covered by one contig, the windows will have depth n, which is the number of haplotypes. as some positions can be covered by multiple contigs, the overlaps between contigs contribute to window depth larger than n. for example, in fig. b , the middle window contains the overlaps between fig. window construction from aligned contigs. a three haplotypes with mutations at three locations. line weights represent the haplotype abundance. s and s are two mutation-free regions common to three haplotypes. s and s are at least k bp. b the alignment of contigs that satisfy the ideal condition. the grey-scale intensity represents the coverage of a contig. three windows are produced. c the contigs that cannot cover all the three haplotypes. there are six windows. their depth values are denoted below each window. for contig marked with "a s ", its sequencing coverage is plotted above the contig two contigs from each haplotype and thus has depth . in this ideal case, we can choose the smallest window depth as the number of haplotypes in a sample. in practice though, the assumptions about the contigs' properties are not always true. thus, in our implementation, we will use the consensus window depth as the number of haplotypes, by assuming that most windows cover all haplotypes and contain haplotype-specific mutations. for the contigs shown in fig. c, window depth is the most frequent one. in the implementation, we first sort all the windows in descending order of window length. then we choose the most frequent window depth of the top x windows as the number of haplotypes. the default value of x is in our implementation. we will present the results of haplotype number estimation using consensus window depth for both simulated and real quasispecies data. step : contig clustering based on relative abundance distribution let the number of haplotypes estimated by step be n. the final goal of the pipeline is to divide the contigs into n groups so that each group contains contigs originating from the same haplotype. in this step, we conduct clustering on subcontigs from windows with depth n. unlike many existing contig clustering tools, our clustering is not applied to a complete contig. because each contig can contain both haplotype-specific region and shared regions among different haplotypes, using the read coverage profile of the whole contig will confuse the clustering algorithm and makes the convergence slow or leads to wrong assignment of the objects. for example, in fig. c , the contig "a s " contains mutation a from one haplotype and also a shared region s . thus, significantly more reads will be mapped to the shared region and make the coverage for this contig highly heterogeneous. thus, the objects as input to the clustering algorithm are "subcontigs" in windows of depth n, where the sub-contigs are substrings of the contigs in these windows. they are more likely to represent the relative abundance of one haplotype. after clustering on sub-contigs, a post-processing step will be applied to get the cluster membership for each contig based on its sub-contigs' memberships. the clustering algorithm we adopt is prototype-based clustering and is essentially an augmented k-means algorithm. in a standard k-means algorithm, the centroid of the objects in a cluster is the prototype of the cluster. in our algorithm, the prototype is a distribution that is derived from the sub-contigs and empirically describes the relative abundance distribution. the clustering algorithm will assign each sub-contig to one cluster based on the posterior probability of the abundance distribution. although different clustering methods such as gaussian mixture model can be applied to cluster the sub-contigs, the augmented k-means as shown below has the fastest convergence with better clustering accuracy according to our tests. before we describe the main components, we first introduce the notations. the average relative abundance (denote asc) for a sub-contig c i in a window of depth n is calculated as: where s(c i ) is the total number of reads covering subcontig c i . similarly, we can calculate the position-specific relative abundance vector c for a sub-contig c i as where c i [k] represents the reads coverage at position k of sub-contig c i . |c i | is the number of bases in the sub-contig. n is the number of bins or haplotypes estimated by step . virbin utilizes the position-specific relative abundances of sub-contigs in windows with depth n to estimate the probability that a sub-contig belongs to a bin. let the n bins be h , h , . . . , h n . letẼ i (x) be the probability density function of relative abundance for sub-contigs from bin i, where x is an observed abundance. the iterative clustering algorithm contains four steps as shown below: initialization: initialize n groups by randomly assign sub-contigs to them. updating the empirical abundance distributionẼ i : for each bin i, the component sub-contigs' relative abundance profiles cs are aggregated to calculate the empirical probability density functionẼ i . the aggregation is performed by calculating the normalized histograms for these relative abundance profiles, so that the summation of histogram values will be . the number of bars of the histogram is a parameter that can be set by users. the default number is . given an abundance value x, it is normalized to its closest bar value to get the corresponding probabilityẼ i (x). re-assignment of the sub-contigs: onceẼ i is derived, the relative likelihood of c j being produced from the ith prototype distribution can be calculated asẼ i (c j ). the prior probability of each bin (or haplotype) is a weighted sum of the likelihoods of all the component sub-contigs. the weights are determined by the total bases in the sub-contigs. this weighted sum enables us to incorporate both the total sub-contig bases and also the associated abundance generation likelihood for estimating the prior probability of a particular haplotype. the prior probability pr(h i ) for bin i is: with both likelihood and prior from iteration t, the expected probability that c j belongs to haplotype h i at iteration t + can be calculated as likelihood * prior, that is with the posterior probabilities calculated for each group distribution, we can reassign the sub-contig c i to the haplotype with the maximum posterior probability. the same reassigning procedures are applied for all the subcontigs. with the assignment results, the distribution e i and prior probability p(h i ) can be updated. iteration: iterate step and until the clustering results do not change or the maximum number of runs have been achieved. the default maximum number of runs is . step : post-processing the output of the augmented k-means is the clustered sub-contigs. for each cluster, its average abundance is calculated as the weighted average of the abundances of all sub-contigs in the cluster and the weight is determined by the length of a sub-contig. the haplotypes' abundances are the average abundances of the clusters. as each contig can contain multiple sub-contigs, which could have different membership, the contig's membership is determined by the dominant membership of its sub-contigs. for example, if a sub-contig is not in the window of depth n, it is not an input to the clustering step and will not be clustered. this could happen when a region of a contig is common to multiple haplotypes. it is also possible that the sub-contigs of a contig are assigned to different clusters, which could be caused by assembly errors. hepatitis c virus dynamics during natural infection are associated with long-term histological outcome of chronic hepatitis c disease hiv treatment failure: testing for hiv resistance in clinical practice deep sequencing of evolving pathogen populations: applications, errors, and bioinformatic solutions contribution of intra-and interhost dynamics to norovirus evolution deep sequencing reveals mixed infection with pandemic influenza a (h n ) virus strains and the emergence of oseltamivir resistance evolutionary dynamics: exploring the equations of life chapter -pathogenesis of viral infections and diseases viral quasispecies assembly via maximal clique enumeration viquas: an improved reconstruction pipeline for viral quasispecies spectra generated by next-generation sequencing idba-ud: a de novo assembler for single-cell and metagenomic sequencing data with highly uneven depth iva: accurate de novo assembly of rna virus genomes de novo assembly of viral quasispecies using overlap graphs maximum likelihood de novo reconstruction of viral populations using paired end sequencing data de novo haplotype reconstruction in viral quasispecies using paired-end read guided path finding maxbin: an automated binning method to recover individual genomes from metagenomes using an expectation-maximization algorithm binning metagenomic contigs by coverage and composition metabat, an efficient tool for accurately reconstructing single genomes from complex microbial communities cocacola: binning metagenomic contigs using sequence composition, read coverage, co-alignment and paired-end read linkage desman: a new tool for de novo extraction of strains from metagenomes constrains identifies microbial strains in metagenomic datasets microbial strain-level population structure and genetic diversity from metagenomes viral quasispecies reconstruction via tensor factorization with successive read removal qsdpr: viral quasispecies reconstruction via correlation clustering art: a next-generation sequencing read simulator quasispecies dynamics with network constraints metaphlan for enhanced metagenomic taxonomic profiling efficient de novo assembly of large genomes using compressed data structures metaquast: evaluation of metagenome assemblies full-length haplotype reconstruction to infer the structure of heterogeneous virus populations rapid evaluation and quality control of next generation sequencing data with faqcs trimmomatic: a flexible trimmer for illumina sequence data karect: accurate correction of substitution, insertion and deletion errors for next-generation sequencing data introduction to data mining blast+: architecture and applications publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. supplementary information accompanies this paper at https://doi.org/ . /s - - - . this work was supported by michigan state university (east lansing, usa) and city university of hong kong (hong kong, china sar) project . the funding did not play any role in design/conclusion. virbin can be download from https://github.com/chjiao/virbin. data are available upon request.ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests. key: cord- -lw xd m authors: ha, kyoo-man title: integrating the resources of korean disaster management research via the johari window date: - - journal: eval program plann doi: . /j.evalprogplan. . sha: doc_id: cord_uid: lw xd m it is not widely known that quite a few researchers are faced with difficulties in using various resources of disaster management research in korea. the article aims to assess how rigorously the korean field of disaster management research resources has been managed or how it can be improved for the ultimate goal of disaster management. descriptive content analysis has been used as the major methodology by referring to the johari window. in doing so, electronic research resources have been systematically compared with integrated research resources via the perspective of korean-speaking researchers and that of english-speaking researchers. the conclusion is that two researchers have to be integrated with all four research resources (open, blind, hidden, and unknown resources) by implementing assigned responsibilities as well as freely asking questions. ultimately, this will be conducive to reducing down the impacts of disaster in korea. south korea (hereinafter korea) has continually been hit not only by natural disasters but also by man-made emergencies (on that point, this article covers all kinds of disaster). sample disasters include the collapse of sampoong department store in , typhoon maemi in , the sinking of ferry sewol in , the outbreak of middle-east respiratory syndrome (mers) in , gyeongju earthquakes in , pohang earthquakes in , miryang fires in , the chronological outbreak of avian influenza and foot-and-mouth disease, and others (ministry of interior & safety (mois), ). due to the many occurrences of disasters, the people asked the government to take progressive actions, but its policy was incomplete because of many factors, including the lack of disaster awareness, poor level of scientific research, lack of appropriate education, and lack of efficient government efforts. as a policy tool, impact assessment deals with evaluating or assessing the consequences of various environmental or disaster programs (durning, ; morgan, ) . with impact assessment, there is a need to identify the critical impacts of certain programs and then provide alternatives to reduce these impacts. in the field of disaster management research, impact assessment may improve the effectiveness of disaster management. disasters cause not only human loss and economic damages but also psychological impacts to human society. many researchers have also studied such impacts. however, with much more complex issues of disasters or disaster management, several complicated questions remain unanswered (benight & mcfarlane, ) . indeed, more sophisticated and in-depth researches on disaster management are urgently needed. to illustrate, there are some researchers who concentrate on a single resource in a particular topic. other researchers also fail to access research resources in a timely manner. in the case of disaster management, availability of integrated resources and timely access are of the same importance, given the need for organized action and quick response when it comes to curtailing the impact of disasters (alamdar, kalantari, & rajabifard, ; srinivasan et al., ) . despite the availability of diverse research resources in the internet, many researchers continue to encounter difficulties in using and making them more useful in their works (scott & few, ) . in many cases, this is also the experience in the field of korean disaster management. this raises the question: how can the integration of research resources on disaster management be improved to benefit not only korea but also the international community? this article aims to assess the state of integration of korean disaster management research resources. descriptive content analysis was used to compare resources with the viewpoints of korean-speaking researchers and english-speaking researchers using the johari window. electronic research resources refer to materials available in computers or via the internet that may be structured, but not necessarily coherent. integrated research resources are not only available electronically; they are also structured, organized, and maintained in a system that allows them to be searched and used significantly. the key is the need for transformation to be in an integrated setup or platform for both korean-speaking researchers and english-speaking researchers. in , researchers joseph luft and harrington ingham developed the johari window (named after them) (clayton, ) . it is a tool used to help assess self-awareness and understanding, while observing multiple relationships between the self and others. many internationally known researchers have discussed the issue of research in the field of disaster management. at the same time, some researchers have applied the concept of the johari window to their disaster management research (lander & atkinson-grosjean, ) . however, based on the literature review, not many international researchers have made the same efforts to systematically assess the korean disaster management research or the johari window in the korean context. the johari window has four behavioral areas, specifically, open, blind, hidden, and unknown. the use of this technique plays a role in setting up goals, improving critical thinking, studying prejudices, identifying all elements or personal boundaries, and developing relationships. by analyzing the four areas, concerned individuals can improve their knowledge and information through an appropriate assessment (south, ) . research, to include not only research itself but also its development, has played many roles in supporting disaster management. throughout the life cycle of disaster management, namely, disaster prevention/mitigation, preparedness, response, and recovery, research has contributed in mitigating the loss of human lives, economic damages, and psychological impact in the international community. although invention or use of cutting-edge technologies or new theories continues, research has changed how the field of disaster management operates (king, edwards, watling, & hair, ) . surely, research has many resources, depending on an individual's perspective. as an example, disaster management research may have two resources, namely, primary resources and secondary resources (university of maryland university college (umuc), ). primary resources as original materials include raw data, survey and interview results, official documents, and other firsthand accounts. on the other hand, secondary resources as secondary materials include assessments, syntheses, discussions, and others. although disaster management research consists of many sub-areas, each sub-area has its own characteristics (xu, wang, shen, quyang, & tu, ) . for example, research on natural disaster as a sub-area has not adequately predicted disaster victims' psychological change, as the field has not provided enough amount of pre-disaster data or related statistical analysis (green, lowe, & rhodes, ) . although this may depend on the type of natural disaster, research on natural disaster has relied so much on victims' post-disaster data. in general, disaster management research has more noteworthy characteristics, as compared with other academic researches. similar to the thought that a disaster may be efficiently managed by multiple professionals or complicated knowledge, related research is also a sort of multidisciplinary one. also, the contents of disaster management research are specific and thus may be applicable to concrete cases. by using common and basic terminologies, disaster management research allows ordinary citizens to understand the research results. disaster management needs organized and quick action, especially in the case of researching and developing vaccines, such as with mers, ebola, and h ni virus (callaghan, ) . without timely resolution, the adverse impacts of pandemic outbreaks may be serious and damaging. in disaster management, integration does not only concern uniting data resources but also stakeholders, methodologies, disciplines, etc. (ye, browne, grdisa, beyene, & thabane, ) . integration is also about highly coordinated and collaborative networks available to extend services toward effective disaster management. integrated disaster management research was strongly vindicated for the past or years in the international community, mainly because disaster has a complex structure, wherein one or two experts alone may not be enough to easily solve or handle disaster management. however, the majority of integrated research was carried out by european researchers or north american researchers concerning their own topics or fields of interest. surprisingly, even though they maintained their achievement on their own integrated research, some critics raised questions on whether related research was indeed performed or not (gall, nguyen, & cutter, ) . like disaster management research in many countries, the korean field of disaster management research has a wide scope. it is surrounded by unique management styles, cultures, or other environments. furthermore, the korean field includes many international principles on disaster management, considering that it has started to associate itself with the international community. if the korean research field fails to assess or use the networks of all disaster management research resources, it may not smoothly suggest appropriate alternatives not only for decision-makers but also for disaster victims. alternatively, without the help of self-assessment via the johari perspective, the adverse impacts of disasters would not be mitigated in korea. integration of all research resources relative to disaster management is imperative. doing so may help lessen or address the potential ripple effects of disasters. using the johari window as an assessment tool will aid in achieving this integration goal (hills, ). an increasing number of korean researchers have examined disaster management in korea. however, not many korean researchers have studied the issue of disaster management research. furthermore, no rigorous study has ever been attempted to assess or evaluate disaster management research or its resources in terms of the johari window (national disaster management institute (ndmi), ). therefore, the goal of this article has potential value to the study. descriptive content analysis was the methodology used for this study, as it has been considered as one of the most effective tools in analyzing the important features of korean disaster management research resources and evaluating not only the tangible effects of research resources but also their intangible effects (fenriam, ; vo, ) . the procedure of descriptive content analysis includes four steps: defining a research question, identifying information and data, reviewing collected information and data, and documenting a descriptive summary (bengtsson, ) . in short, descriptive content analysis plays a role in assessing the significant characteristics of disaster management research resources by describing the outcomes of disaster management research resources. meanwhile, relevant subjects and texts were identified, collected, and then interpreted for the research. for information and data identification, some terms (e.g., johari window, principles of disaster management, science and technology policy, korean disaster management, etc.) were searched in widely used search engines, such as sciencedirect, ebsco, google.com, oxford university press, korean search engines, etc. moreover, the author performed subjective assessment to qualify the identified materials and resources. primarily, as the johari window was proposed about years ago, not many researchers have discussed related issues in the st century. thus, the author identified or included as many articles or documents as possible on the johari window into the references or resources for analyzed texts. in general, the majority of identified or collected references were labeled as favorites as shown in the results of the article. using impact assessment, this article assesses (or evaluates) how the korean field of disaster management research has been doing with its resources and what the field should do to improve the current situation and establish appropriate alternatives. in the process, the article analyzes all the resources of korean disaster management research using the johari perspective, with the goal of naming a current model and an alternative model on disaster management research resources. similarly, this research has shown a comparative perspective between korean-speaking researchers and english-speaking researchers regarding disaster management research resources. the comparative study leads to identifying many factors that are unique to national disaster management system when reflecting on traditional approaches, such as a single-case study, being too parochial, rigid, or even narrow in their theoretical process (jreisat, ; luft & fakhouri, ) . additionally, traditional approaches have not been based on cross-cultural situation. therefore, this comparative study plays a role in fostering the development of effective planning and programs in the field, particularly by referring to the cross-language perspective. as can be seen in table , the two major stakeholders identified are korean-speaking researchers and english-speaking researchers. these two groups have been directly or indirectly involved in all major resources of korean disaster management research. research resources written in other languages are excluded from the scope of this framework. by cross-checking the two stakeholders, four research resources, including open research resources, blind research resources, hidden research resources, and unknown research resources, are analyzed. descriptive content analysis has been well known for its systematic review of the subject (kim & lennon, ) . similarly, the johari window has thoroughly pursued the systematic aspect of disaster management research resources by referring to open/free resources, blind resources, hidden resources, and unknown resources in that order (newstrom & rubenfeld, ) . the johari window comprehensively includes not only the bright sides of research resources but also their dark sides. considering these aspects, the johari window technique is quite relevant for the methodology used in this article. open research resources include four major english resources, such as international journals, the united nations' official documents, googlescholar.com, and yahoo.com, which are some of the many open research resources. it is worth noting that those four major english resources have been widely used by both english-speaking researchers and korean-speaking researchers. to elaborate, all english-speaking researchers may access such resources and generally understand related contents. also, although only a few korean-speaking researchers can understand the contents from such resources, all korean-speaking researchers have equal access to them. many publications have held a monopoly of their research results. without the internet, it would be more difficult for researchers to communicate or share their research results with their colleagues. through the use of internet, many researchers have overcome these difficulties. with the support of public funds, many researchers have been able to read, copy, print, download, and distribute research results without much restrictions. nevertheless, open research resources are, at times, said to be useless resources to many korean-speaking researchers, mainly because they do not understand the english language very well. furthermore, even though those open resources are electronically suitable to all researchers, even english-speaking researchers still face difficulties in freely accessing such resources because of strict copyright laws (sharma, ) . a good example of blind research resource is the emergency management institute (emi)'s independent study program (isp). various english-speaking researchers have recognized important courses in the program. at the same time, they have used it as their research resource (emergency management institute (emi), ). however, almost all korean-speaking researchers were not able to use this program as their research resource. the emi's program includes many disaster management principles, such as comprehensive emergency management, integrated emergency management system, incident command system, whole community approach, and many others. the international association of emergency managers (iaem) has contributed to spreading the emi's program to many isolated areas while encouraging local emergency managers to take appropriate courses and become certified emergency managers. blind research resources have been frequently ascertained as a sort of "liability" in the field of disaster management research (katsikopoulos & gigerenzer, ) . a liability is something that causes negative problems and, thus, should be efficiently solved. the emi's program has been electronically usable to korean-speaking researchers; however, they have not recognized its existence. some have surely recognized it, but they have not fully understood it due to language barrier. thus, at present, the emi's program is considered a challenge for korean-speaking researchers. hidden research resources include local definitions on disaster management, local community data, and knowledge of indigenous resources in korea. although all korean-speaking researchers have comparatively understood them, many english-speaking researchers are not aware of them. a good example is that "banjae" to korean-speaking researchers means "to manage typhoon accompanied by flood," but it has frequently been translated as "disaster prevention" in english. language does matter in the field of disaster management research (kim, ferrini-mundy, & sfard, ) . when english-speaking researchers work on their mathematical research, they predominantly explain the issue in procedural terms. on the other hand, koreanspeaking researchers respond to the issue via structural ways. when researchers work on the issue either in the english language or in the korean language, they are less prolific than when bilingual researchers resolve the same issue. electronically, hidden research resources have been certainly available to both korean language researchers and english-speaking researchers. however, many english-speaking researchers have not been able to use those resources for their research because of the language barrier or the lack of appropriate understanding of the local culture. in terms of understanding relevant resources written in english, table analytical framework via the johari window. sources: (chapman, ; luft & ingham, korean-speaking researchers are at a disadvantage. on the contrary, english-speaking researchers are at an advantage because they do not face any challenge in understanding the language. unknown resources, as the name suggests, are unfamiliar to many. they remain to be determined or discovered for meaningful use. one example would be the precise differences between korean language research and english language research regarding disaster management. further examples are the applications coded in programming languages and experiments with results that are not yet available to the public and are known only to some scientists. a few researchers have pointed out the existence of unknown research resources in the field of disaster management (ramasesh & browning, ) , such that they have willingly or unwillingly faced unknown research resources while attempting to develop new disaster management products or implement new disaster management processes. despite these unknown research resources, they have paradoxically made their new theories. a superficial form of integration is based on the fact that albeit far too many resources are available in the internet, they remain unknown to many. exactly evaluating the gap between and among researchers and precisely assessing the gap between and among resources are quite a challenge. with the above-mentioned analysis of the four research resources, it is summarized that the current korean model should be named as electronic research resources. although research resources have been located and then directly or indirectly managed under their own circumstance, they have all been just electronically suitable for not only korean-speaking researchers but also english-speaking researchers via the internet. as long as only electronic research resources are used in korea, the field of research will surely lose important knowledge and information on disaster management. in other words, disaster management researchers may not efficiently use knowledge and information from electronic research resources due to the associated challenges. without the appropriate research results as their source, most researchers will not be able to produce better alternatives. therefore, electronic research resources must be changed into integrated research resources. integrated research resources mean that all research resources are not only electronically usable but also substantially used by various researchers without much difficulty. both korean-speaking researchers and english-speaking researchers have to re-evaluate the way they use integrated research resources. it has been known that any change in one research resource may influence the other research resources in many cases (luft, ) . thus, all researchers should initiate their drive within appropriate resources to start realizing appropriate changes. based on the initial change, those researchers need to work together to reach the apex of the targeted model. while thinking that disasters cause not only physical impact but also social impact to the human society, this article has examined critical problems and appropriate alternatives on disaster management in the viewpoint of impact assessment. to illustrate, this article has made efforts to provide theories on research resources, while also trying to mitigate or assess the physical impact and social impact of disasters. in summary, both electronic research resources and integrated research resources have been engaged under the context of impact assessment. fundamentally, both korean-speaking researchers and englishspeaking researchers should be aware of the negative aspects of electronic research resources or the necessity of integrated research resources. up to present, some researchers are not aware how the four resources are being used. in other words, they are not interested in examining the issue and, thus, work for their own research under a limited scope. specifically, without related awareness, they would not be willing to work for change. based on the high level of related awareness, all researchers need to plan the integration of disaster management research resources in advance (chang, wilkinson, brunsdon, seville, & potangaroa, ; jugend, araujo, pimenta, gobbo, & hilletofth, ) . by systematically planning how to overcome the status of superficially integrated resources or how to implement related countermeasures, they may flexibly tackle diverse research issues, as shown in table . while changing to integrated research resources, it is inevitable for the research field to coordinate thorny research issues. without voluntarily coordinating the stark differences among various researchers, the research field will be in a rut and unable to advance. also, it is quite necessary not only for english-speaking researchers but also for korean language researchers to give up their vested interests or privileges, if any, for the ultimate goal of disaster management. considering that disaster management is a public service, the korean governments, such as the ministry of the interior and safety (mois), should play an appropriate role in supporting the integration of research resources (hakaloba, mumba, dambe, & michelo, ) . by officially addressing the changing process via legalization, budget allocation, tax reliefs, international relations, and others, the mois may contribute to the sustainability of resource integration. furthermore, the mois may develop its research management section as a formal international organization and then facilitate it to work for integrated research resources with the full participation of both korean-speaking researchers and english-speaking researchers. in doing so, the mois may create a series of working committees between and among countries. as they approach integrated research resources, all korean-speaking researchers and english-speaking researchers should be allowed to ask questions at anytime (armstrong, ) . many english-speaking researchers have more frequently asked questions on their researches under the western culture than what korean-speaking researchers have performed under the confucian culture. with barriers in freely asking questions, the research field would not be able to sufficiently advance. alternatives toward integrated research resources. sources: (lee & kim, ; westerwick, kleinman, & knobloch-westerwick, ; yoon, ) . appropriate alternatives ① open research resources -all kinds of open research resources should be freely accessible to korean-speaking researchers as well as english-speaking researchers without any restriction, for the goal of distributing research results. ② blind research resources -with the support of iaem, korean-speaking researchers must understand the status of emi's isp as a fundamental research resource. also, the emi (or english-speaking researchers) may add the characteristics on korean disaster management or its research resources into isp. ③ hidden research resources -to reduce the extent of language barrier between korean-speaking researchers and english-speaking researchers, the field of research must operate on diverse networks in multi-languages. ④ unknown research resources -bilingual (english language and korean language) researchers need to work on comparative perspectives between korean language research and english language research or joint international research. k. -m. ha evaluation and program planning ( ) accordingly, all answers need to be composed of appropriate knowledge and information. if those answers do not provide related information or data to those who ask questions or they just keep silent due to political reasons, those questions will just be meaningless. therefore, when there is a question from any researcher, there should be an answer for the goal of integrated research resources. without an answer, the embodiment of integrated research resources would not succeed. although a huge amount of information has been advanced with high speed, almost no one has ever been completely competent in understanding or interpreting them. diverse kinds of information have been produced in incremental pieces. hence, the free flow of information exchange has to be facilitated in the research field before jumping into integrated research resources. basing on the concept of open communication in the research field, various researchers must freely exchange appropriate research knowledge and information with others (little, ) . for the ultimate goal of disaster management, all researchers need to utilize interpersonal communication as well as twoway communication. the use of johari window has been an excellent technique, which makes individuals and groups better grasp their relationship with not only themselves but also others. on the effect of the johari window, it has provided a fair opportunity to see how the field of disaster management research resources has been operated. by assessing how the self and others are doing, the johari window has played a role in positively supporting the field of disaster management research. also, the johari perspective may be considered as a cornerstone in evaluating the level of korean disaster management research, particularly by equally investigating all important areas/aspects. similarly, as the realization of disaster management research is much related to either the success or failure of disaster management, the johari window is regarded as a supervision tool, as well as a selfassessment tool for disaster management research resources (halpern, ) . while comprehensively overseeing the big picture of scientific research, the field of disaster management can efficiently use the johari window in supervising research processes. it is known that the johari perspective has provided new research opportunities for many past researchers (giarratano, savage, barcelona-demendoza, & harville, ) . on this point, appropriate researchers may meet with new research agenda on disaster management research, with the support of this article. in other words, when considering that the johari window has shown both the resilience and vulnerability of disaster management research in korea, related researchers may figure out much more research agenda. ultimately, the general direction of korean disaster management research will be able to free itself from fragmentation. if research resources will remain as only electronically or superficially available, related researches will just be fragmented. however, when the integrated research resources are addressed, related researches will be carried out in a more holistic way (waring, ) . as a result, researches in the future will have a more comprehensive quality. on top of doing impactful assessment, the purpose of this article is to assess how efficiently the korean field of disaster management research resources should be improved to contribute to the ultimate goal of disaster management. considering that both electronic research resources and integrated research resources have been systematically drawn via the johari perspective, this article has been successful in achieving its original goal. the biggest finding is that not only korean-speaking researchers but also english-speaking researchers must exert efforts to change electronic research resources into integrated research resources. for its implementation, all four research resources, namely, open, blind, hidden, and unknown researches, have to carry out their own assignment, such as distributing open research data, approaching the emi's isp as a basic research resource, enhancing diverse networks in multi-languages, and encouraging comparative perspectives and joint international researches. while reflecting on the fact that no rigorous study has ever been attempted yet to analyze the korean field of disaster management research resources via the johari perspective, this article has many advantages as a pioneer study. at the same time, this study has illustrated that both korean-and english-speaking researchers can benefit from using the johari perspective. moreover, it has shown the problems and the alternatives surrounding research resources in the field of disaster management in korea. this article has directly indicated the need for diverse studies in the future. for example, it will be necessary for korean-speaking researchers and english-speaking researchers to further delve into their assigned responsibilities regarding open, blind, hidden, and unknown research resources. in addition, researchers are encouraged to apply the johari perspective to their own case studies. then, they may eventually compare or contrast their results with a korean case or among themselves. the author declares that he has no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. towards multi-agency sensor information integration for disaster management. computers, environment and urban systems revisiting the johari window: improving communications through self-disclosure and feedback how to plan and perform a qualitative using content analysis challenges for disaster research: recommendations for planning and implementing disaster mental health studies disaster management, crowdsourced r&d and probabilistic innovation theory: toward real time disaster response capability an integrated approach: managing resources for post-disaster reconstruction johari window: a model for 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integrated service network a case study of fluent korean-english bilingual speakers: group membership and code choices he is working for the we company as a researcher as well as pusan national university (in korea) as an adjunct professor. also, he, as a certified emergency manager, is serving as the korean representative for the international association of emergency managers. his research interests include emergency management and public policy key: cord- - p i authors: wang, shiu‐mei; huang, kuo‐jung; wang, chin‐tien title: severe acute respiratory syndrome coronavirus spike protein counteracts bst ‐mediated restriction of virus‐like particle release date: - - journal: j med virol doi: . /jmv. sha: doc_id: cord_uid: p i bst /tetherin, an interferon‐inducible antiviral factor, can block the cellular release of various enveloped viruses. we previously reported that human coronavirus e (hcov‐ e) infection can alleviate the bst tethering of hiv‐ virions by downregulating cell surface bst , suggesting that coronaviruses are capable of encoding anti‐bst factors. here we report our new finding that severe acute respiratory syndrome coronavirus (sars‐cov) spike (s) glycoprotein, similar to vpu, is capable of antagonizing the bst tethering of sars‐cov, hcov‐ e, and hiv‐ virus‐like particles via bst downregulation. however, unlike vpu (which downmodulates bst by means of proteasomal and lysosomal degradation pathways), bst downregulation is apparently mediated by sars‐cov s through the lysosomal degradation pathway only. we found that sars‐cov s colocalized with both bst and reduced cell surface bst , suggesting an association between sars‐cov s and bst that targets the lysosomal degradation pathway. according to one recent report, sars‐cov orf a antagonizes bst by interfering with bst glycosylation( ). our data provide support for the proposal that sars‐cov and other enveloped viruses are capable of evolving supplementary anti‐bst factors in a manner that requires virus replication. further experiments are required to determine whether the bst ‐mediated restriction of authentic sars‐cov virions is alleviated by the sars‐cov spike protein. bone marrow stromal antigen (bst , also designated as cd or tetherin) is a type ii integral membrane protein containing a cytoplasmic n-terminal region followed by a spanning transmembrane domain and a carboxy-terminal glycosyl-phosphatidylinositol (gpi) anchor. bst is an interferon-inducible gene that functions as an innate defense system against virus infections. it has been described as a host restriction factor capable of impeding the release of several types of enveloped viruses, including retroviruses, - filoviruses, [ ] [ ] [ ] arenaviruses, influenza, and the sendai virus. one research team has proposed that bst inhibits virus release by tethering nascent virions to cell surfaces via the n-terminal transmembrane domain and c-terminal gpi anchor. most bst -restricted enveloped viruses bud directly from cell surfaces, but a small number of enveloped viruses (eg, herpesviruses) are subject to bst -related restrictions even though their final envelopment entails membranes from tgn and/or endosomal compartments and egression via exocytosis. , in a previous study we reported that the human coronavirus e (hcov- e), whose assembly and budding occurs at the er-golgi intermediate compartment and whose virions are released via vesicle exocytosis, [ ] [ ] [ ] is also subject to bst inhibition. results from electron microscopy analyses indicate the presence of hcov- e virions on cell surfaces or on the membranes of intracellular vesicles that tend to cluster with bst . this suggests the bst -triggered tethering of budding virions to vesicle membranes that remain on cell surfaces at the plasma membrane after exocytosis. bst has been described as moderately restricting the release of the hepatitis c virus, whose assembly takes place in the er and whose release from cells via secretory pathways occurs in a manner similar to that of coronaviruses. , combined, these data support the assumption that enveloped virus budding and release occurring at the plasma membrane or in an intracellular compartment is subject to bst blocking. bst is a component of innate immune response in the form of restricted enveloped virion release, and many viruses have evolved specific antagonists to counteract bst antiviral activity: hiv- vpu, hiv- env, simian immunodeficiency virus (siv) nef and env, ebola and sendai virus gp, kaposi's sarcoma-associated herpesvirus k , and influenza virus neuraminidase are all capable of antagonizing bst . [ ] [ ] [ ] [ ] , , , , since some of these anti-bst viral factors are viral envelope glycoproteins, there is speculation that sars-cov spike glycoprotein may possess the property to counteract the bst blocking of virus release. our work is built in part on an earlier finding by another research team that the orf a accessory protein (encoded by sars-cov) inhibits the bst tethering of virions. we also found that the sars-cov spike (s) protein is capable of downmodulating bst , thus mitigating the bst -mediated restriction of virus-like particle (vlp) release, and suggesting that sars-cov and other enveloped viruses are capable of evolving additional anti-bst factors. mammalian expression vectors encoding sars-cov m, n, s, and e were provided by g. j. nabel. bst dimerization-defective mutant (bst / c a) was a gift from klaus strebel. plasmid ptre-hn, kindly provided by volker thiel, served as a template to generate pcr product containing hcov- e nucldocapsid coding sequence, using a forward primer ′-cgcaatcgattcatgaaggcagttgct- ′ and a reverse supplemented with % fetal calf serum (invitrogen). confluent cells were trypsinized and seeded onto cm dish plates hours before transfection. for each construct, cells were transfected with μg of plasmid dna by the calcium phosphate precipitation method; μm chloroquine was added to enhance transfection efficiency. unless otherwise indicated, μg of each plasmid was used for cotransfection. for hela transfection, plasmid dna was mixed with gencarrier (epoch biolabs) at a ratio of μg to μl; the transfection procedure was performed according to the manufacturer's protocols. human coronavirus e (hcov- e) were propagated in hela or a cells as described previously. detection, a mouse monoclonal antibody was used. , bst was probed with a human bst mouse antiserum (ab , abcam) or a rabbit antiserum. vpu was detected with a rabbit antiserum. the secondary antibody was a sheep antimouse or donkey antirabbit horseradish peroxidase-(hrp) conjugated antibody (invitrogen). we also used hela cells (which constitutively express bst ) to assess the impact of endogenous bst on vlp yields and found that bst forms stable cysteine-linked dimers. blocking bst dimerization by replacing cysteines c , c , and c with alanine in the bst ectodomain, in turn, blocks the bst inhibition of hiv- release, suggesting that such dimerization is required for virion release blocking. to test whether bst dimerization is also required for n (panel b) , or nl . delvpu (panel c) plus a wild-type or mutant bst expression vector (bst /c a) containing alanine substitutions for three cysteine residues in the bst ectodomain. cells and supernatants were harvested and subjected to western immunoblotting at to hours posttransfection. bst , bone marrow stromal antigen ; vlp, virus-like particle further suggesting that bst dimerization is required to inhibit coronavirus vlp release. as stated above, several viral membrane glycoproteins such as hiv- and siv env, as well as ebola and sendai virus gp proteins, exert counteractive effects on bst . we, therefore, attempted to identify anti-bst activity associated with the sars-cov spike (s) protein. since bst is known to restrict hiv- release in the absence of vpu, we performed tests to determine whether sars-cov s counteracts bst and therefore supports hiv- release. as shown in the upper panel of figure a (lane vs. lanes and ) , the inhibitory effect of bst on nl . delvpu virion release decreased in the presence of either sars-cov s or vpu in step with reduced bst expression ( figure a, middle panel, lanes - ) , suggesting that sars-cov s is capable of promoting the release of hiv- virus particles from cells via bst downregulation. additional experiments confirmed that sars-cov s, like vpu, is capable of reducing bst expression in a dose-dependent manner (figures b and c) . combined, the data suggest that sars-cov s may counteract the bst -mediated restriction of vlp release. after binding to bst , vpu moves bst toward lysosomal-and proteasomal-degradation pathways. , - our next task was to examine whether sars-cov s mediates bst degradation via the same or similar pathway. transfectants were treated with either mg (a proteasome inhibitor) or ammonium chloride (nh cl, a lysosome inhibitor). we found that bst downregulation mediated by sars-cov s was not significantly affected when the proteasome function was inhibited, but was noticeably reduced following lysosome function inhibition ( figure , lanes - ) . consistent with previous reports, we observed that proteasome or lysosome function inhibition resulted in markedly reduced vpu-mediated bst downregulation ( figure , lanes - ) , suggesting that bst downregulation as mediated by sars-cov s largely occurs via the lysosomal degradation pathway. since bst largely localizes at cell surfaces (where they tether virions to prevent their release), we tested whether sars-cov s antagonizes bst via surface bst downregulation. cells were cotransfected with bst and sars-cov s expression vectors. we observed that bst and s colocalized in perinuclear areas, but bst signals were barely detectable on cell surfaces. instead, bst largely localized in perinuclear areas regardless of whether or not it was coexpressed with s ( figure a ). this is consistent with a previous report that unlike hela-endogenous bst (which localizes on cell surfaces as well as in the perinuclear area), exogenous bst predominantly localizes in perinuclear areas, with little distribution on cell surfaces. nevertheless, flow cytometry quantification suggests that bst cell surface expression is noticeably reduced in t cells following sars-cov s coexpression ( figure c ). in the case of hela cells (which constitutively express bst ), we did found sars-cov s colocalized with bst in the plasma membrane ( figure b ). bst green fluorescence intensity on hela cell surfaces decreased slightly following sars-cov s coexpression, suggesting that the capacity of sars-cov s to counteract the bst -associated inhibition of virion release was due in part to cell surface bst downmodulation. bst is capable of inhibiting sars-cov and hcov- e vlp release ( figure ). since the bst inhibition of hiv- release via virion tethering at cell surfaces is well documented, we used a vpudeficient hiv- virus-producing vector (nl . delvpu) as a control in our experiments. as shown in figure , coronavirus vlps (similar to those of hiv- ) were tethered to cell surfaces by bst , and bst inhibited sars-cov and hcov- e vlp release in a bst dimerization-dependent manner, similar to hiv- ( figure ). further, in the same manner, as vpu, sars-cov s facilitated hiv- release via bst downregulation (figure ) . we determined that vpu downmodulated bst via proteasomal and lysosomal degradation pathways and that the predominant lysosomal pathway was mediated by sars-cov s ( figure ). our confocal microscopy observations suggest that sars-cov s colocalized with bst at hela cell surfaces ( figure ). sars-cov s likely binds to bst , after which it serves as a target for lysosomal degradation. vpu is capable of trapping bst intracellularly and preventing its recycling back into the plasma membrane. , , , , whether sars-cov s similarly counteracts bst requires further investigation. the transmembrane protein sars-cov orf a has been shown to counteract bst tethering by interfering with bst glycosylation. , in addition to the likelihood that bst -associated restriction of f i g u r e sars-cov glycoprotein s colocalizes with bst . cells (panel a) were cotransfected with bst and a sras-cov expression vector. hela cells (panel b) were transfected with the sars-cov s expression vector. at to hours posttransfection, cells were probed with an anti-bst antibody before cell membrane permeabilization. sars-cov s was probed with an anti-s polyclonal antiserum. a rhodamineconjugated or fitc-conjugated antirabbit or antimouse antibody served as a secondary antibody. c, sars-cov s coexpression reduces bst cell surface expression. t cells were transfected with bst alone (middle panel) or together with a sars-cov s expression vector (bottom panel). at to hours posttransfection, cells were fixed and probed with a rabbit anti-bst antibody before the permeabilization of cell membranes, followed by a secondary fitc-conjugated antirabbit antibody. cells then were analyzed by flow cytometry. bst , bone marrow stromal antigen ; sars-cov, severe acute respiratory syndrome coronavirus sars-cov virion release is mitigated by sars-cov s, it is possible that a number of enveloped viruses have developed supplementary anti-bst factors over time-note that in addition to vpu, hiv- nef is capable of overcoming bst restrictions on virus release under certain conditions. siv nef and env are capable of antagonizing bst , and influenza neuraminidase and m proteins both possess anti-bst capabilities. some researchers have suggested that influenza and/or ebola vlp release, but not virion release, is inhibited by bst . , due to biosafety requirements, we are currently unable to perform tests to determine whether sars-cov s is capable of overcoming bst restrictions on sars-cov virion release. bst- /hm . is a raft-associated apical membrane protein with an unusual topology simian immunodeficiency virus envelope glycoprotein counteracts tetherin/bst- /cd by intracellular sequestration species-specific activity of siv nef and hiv- vpu in overcoming restriction by tetherin/ bst antagonism to and intracellular sequestration of human tetherin by the human immunodeficiency virus type envelope glycoprotein tetherin inhibits retrovirus release and is antagonized by hiv- vpu the interferon-induced protein bst- restricts hiv- release and is downregulated from the cell surface by the viral vpu protein nef proteins from simian immunodeficiency viruses are tetherin antagonists broad-spectrum inhibition of retroviral and filoviral particle release by tetherin tetherinmediated restriction of filovirus budding is antagonized by the ebola glycoprotein dimerization of tetherin is not essential for its antiviral activity against lassa and marburg viruses infectious lassa virus, but not filoviruses, is restricted by bst- /tetherin influenza virus partially counteracts restriction imposed by tetherin/bst- antagonism to human bst- /tetherin by sendai virus glycoproteins tetherin inhibits hiv- release by directly tethering virions to cells molecular mechanism of bst /tetherin downregulation by k /mir of kaposi's sarcomaassociated herpesvirus herpesvirus assembly and egress bst /cd counteracts human coronavirus e productive infection by tethering virions at the cell surface the molecular biology of coronaviruses virus assembly hepatitis c virus: assembly and release of virus particles modulation of hepatitis c virus release by the interferoninduced protein bst- /tetherin the ring-ch ligase k antagonizes restriction of kshv and hiv- particle release by mediating ubiquitin-dependent endosomal degradation of tetherin severe acute respiratory syndrome coronavirus orf a inhibits bone marrow stromal antigen virion tethering through a novel mechanism of glycosylation interference generation of synthetic severe acute respiratory syndrome coronavirus pseudoparticles: implications for assembly and vaccine production the formation of cysteinelinked dimers of bst- /tetherin is important for inhibition of hiv- virus release but not for sensitivity to vpu selective replication of coronavirus genomes that express nucleocapsid protein identifying epitopes responsible for neutralizing antibody and dc-sign binding on the spike glycoprotein of the severe acute respiratory syndrome coronavirus apobec g cytidine deaminase association with coronavirus nucleocapsid protein vpu enhances hiv- virus release in the absence of bst- cell surface down-modulation and intracellular depletion human immunodeficiency virus type vpu protein is an oligomeric type i integral membrane protein self-assembly of severe acute respiratory syndrome coronavirus membrane protein sars-cov envelope protein palmitoylation or nucleocapid association is not required for promoting virus-like particle production an interferon-αinduced tethering mechanism inhibits hiv- and ebola virus particle release but is counteracted by the hiv- vpu protein. cell host microbe vpu directs the degradation of the human immunodeficiency virus restriction factor bst- /tetherin via a {beta}trcp-dependent mechanism vpu antagonizes bst- -mediated restriction of hiv- release via beta-trcp and endolysosomal trafficking hiv- antagonism of cd is species specific and involves vpu-mediated proteasomal degradation of the restriction factor antagonism of tetherin restriction of hiv- release by vpu involves binding and sequestration of the restriction factor in a perinuclear compartment hiv- vpu neutralizes the antiviral factor tetherin/bst- by binding it and directing its beta-trcp -dependent degradation differential inhibition of calpain and proteasome activities by peptidyl aldehydes of di-leucine and tri-leucine ammonium chloride, an inhibitor of phagosomelysosome fusion in macrophages, concurrently induces phagosomeendosome fusion, and opens a novel pathway: studies of a pathogenic mycobacterium and a nonpathogenic yeast the tetherin/bst- coiled-coil ectodomain mediates plasma membrane microdomain localization and restriction of particle release membrane anchoring by a c-terminal tryptophan enables hiv- vpu to displace bone marrow stromal antigen (bst ) from sites of viral assembly hiv- vpu blocks recycling and biosynthetic transport of the intrinsic immunity factor cd /tetherin to overcome the virion release restriction structure and intracellular targeting of the sars-coronavirus orf a accessory protein tetherin antagonism by hiv- group m nef proteins bst- restricts iav release and is countered by the viral m protein influenza virus is not restricted by tetherin whereas influenza vlp production is restricted by tetherin severe acute respiratory syndrome coronavirus spike protein counteracts bst -mediated restriction of virus-like particle release key: cord- - ymjmbz authors: naug, dhruba; choe, jae c. title: disease transmission and networks date: - - journal: encyclopedia of animal behavior doi: . /b - - - - . - sha: doc_id: cord_uid: ymjmbz while epidemiological models have traditionally assumed that diseases spread by the mass action principle, actual contact networks within social groups do not meet this assumption. theoretical models have shown that disease dynamics could vary considerably under different types of contact networks, but these models face challenges in terms of their evaluation due to the difficulty of collecting empirical data. the honeybee colony with its elaborate social organization and large repertoire of diseases provides an ideal setting to explore how the structure of the contact network contributes to the transmission of a disease. animals living in large groups are particularly vulnerable to infectious diseases. the close proximity of individuals offers excellent transmission opportunities to a pathogen that is spread by direct contact between hosts. many studies show a positive relationship between group size and parasitism in terms of prevalence (proportion of infected individuals in a group) and intensity (number of pathogens per individual) (strassmann, ; côté and poulinb, ; rifkin et al., ) . if the host population is homogeneous in exposure and susceptibility to a pathogen, the birth and death rates of the host and the contact rate between susceptible and infected individuals are sufficient to predict the infection dynamics. however, groups are rarely homogeneous and individuals differ among themselves in various respects such as age, sex, physiological state, behavior, and spatial location. this causes individuals to differ in their probability of becoming infected and transmitting the infection, making it more difficult to predict the trajectory of an infection. the rate at which an infection spreads and whether it persists in the population depend on the magnitude of the key epidemiological parameter, r , or the mean number of infections caused by a single infected individual. in order to stop an epidemic outbreak, r must be maintained below . according to the mass-action sir (susceptible-infected-recovered) model, the most basic model of epidemic spread, r ¼bts, where b is the transmission coefficient that incorporates both infectiousness and contact rate of the infected individuals, t is the duration of infectiousness, and s is the available number of susceptible individuals (bjørnstad et al., ; volz and meyers, ) . the simple sir model has provided many important insights into the epidemiology of a wide range of pathogens but its fundamental assumption of homogeneous mixing among individuals is clearly unrealistic. population-level estimates of r can obscure the considerable variation in contact rate and infectiousness among individuals. several studies have shown that typically, % of the transmission events are contributed by % of the host population: a trend that is referred to as the / rule (perkins et al., ; paull et al., ) . this was highlighted during the recent global epidemic of severe acute respiratory syndrome (sars) when a few infected individuals were responsible for giving rise to an unusually large number of secondary cases (galvani and may, ) . whether or not infected individuals have contact rates that are disproportionately higher than the population average has important implications because public-health programs generally rely on the immunization of only a fraction of the hosts to protect the entire population. the effects of host heterogeneity on the spread of infectious disease can be most simply modeled by dividing a population into subpopulations with different within-group and between-group transmission rates. a more explicit approach is to use models that incorporate the structure of the actual contact network in the population (scott, ) . unlike the continually changing set of contacts in random mixing models, each individual is assigned a finite set of contacts to who they can transmit infection and from who they can be infected. predictions from network models can be considerably different from those that use mean-based approaches. although individuals may have the same number of contacts per unit time in both network and mass action models, the fixed contact structure in networks can lead to rapid, localized spread of an infection followed by a slowing down of the process as the number of susceptible individuals depletes locally. this makes disease extinctions more likely than outbreaks though the latter are more explosive if they occur. the use of network models also has bearing on the evolution of the pathogens themselves. given their high reproductive rates, pathogens are likely to undergo rapid selection to adapt to the available transmission routes between infected and susceptible individuals (bishop et al., ) . both theoretical and experimental results show that high transmission rates are selected in localized networks where there is intense competition for susceptible hosts while networks that are more global in their connectivity select for lower transmission rates due to lack of such competition. localized contact structure also selects for a higher diversity in the pathogen population in contrast to a randomly mixed host population where cross-immunity to similar strains structures the pathogen population into discrete, nonoverlapping strains. a transmission network is generally defined by a matrix x that describes the connections among all the individuals within a group. in its simplest form, the matrix is unweighted, with x ij ¼ , if there is one or more interactions that can transmit an infection and x ij ¼ if there is none. the matrix is also generally undirected, meaning that infection can pass either way across an interaction or x ij ¼x ji . more detailed models can be constructed using weighted, directed networks (opsahl et al., ) . the structure of the transmission network can be characterized by a number of parameters that can be quantified from these matrices. the most commonly used ones are ( ) degree, the number of connections an individual has; ( ) density, the proportion of existing connections out of all possible ones; ( ) path length, the average number of links that connect any two individuals; and ( ) clustering, the density of the local neighborhood or cliquishness (boccaletti et al., ) . focal measures such as degree can identify high-risk individuals in the population and can be used to inform surveillance and infection control strategies. network level measures such as average path length and clustering coefficient can make predictions about the spread of the infection in the population. critical points that reflect order of magnitude shifts in network properties and the consequent propagation of an epidemic can be identified from phase transitions in network parameters. network models are difficult and time consuming to build because they require information about the connectivity between every pair of individuals in a group. in this effort, researchers have mainly relied on infection tracing that describes the actual connections through which the infection spreads or contact tracing that looks at all the potential connections from a source individual (keeling and eames, ) . network models are also complex in terms of their statistical evaluation unlike differential equations based mass-mixing models. moreover, as different diseases are transmitted via different transmission pathways, network models are disease specific and cannot be easily generalized. in the face of these difficulties, simulating networks with different structures ( fig. ) and studying the parameters that influence transmission dynamics has been an important and influential research paradigm. in these types of networks, each individual has a fixed number of random connections, resulting in a network with no clustering and short path lengths (barabási and albert, ) . the early growth rate of an infectious process and the final epidemic size are lower in these networks compared with the mass-action model, largely because of the quick depletion of the local environment of susceptible individuals around an infected individual. in these networks, individuals are connected only to their adjacent neighbors, leading to a homogeneous network with high clustering and long path lengths (olfati-saber and shamma, ) . this leads to an even stronger depletion of the local environment and thus the growth rate of the infection. the transmission properties of small-world networks have generated a lot of interest and are important to understand because many biological networks including human social networks show small-world properties lago-fernández et al., ) . they lie somewhere between regular and random networks, displaying high clustering but small path lengths due to the existence of a few long-range connections. even though the transmission process is still largely localized, the few longrange links allow the infection to spread relatively quickly and more synchronously over the entire network. small-world networks may or may not have a scale-free structure. these networks are characterized by an extreme heterogeneity in connectivity, the number of contacts per individual being described by a power law distribution (barabási and bonabeau, ) . a few highly connected individuals, called superspreaders in the epidemic context, have a disproportionately high influence on the transmission process (zhou et al., ) . networks of human sexual contacts have been shown to follow such a distribution and the transmission and maintenance of sexually transmitted diseases thus depends mainly on a few promiscuous individuals (liljeros et al., ) . in such networks, control measures directed at random individuals are quite ineffective while targeted interventions work really well. by immunizing the superspreaders, the contact network becomes sparser by orders of magnitude and brings about a drastic reduction in the number of transmission events. complex network models being hard to parameterize can lead to predictions that are no more reliable or maybe even worse than those from simpler frameworks. however, with the relative dearth of suitable experimental systems with sufficient social complexity, opportunistically obtained data about the course of natural epidemics in humans have been the only major recourse for testing network models. in this context, the honeybee colony can prove to be an ideal model system. honeybees not only provide the setting of a crowded social group that is susceptible to a vast array of infectious diseases but they are also extremely amenable to a variety of experimental paradigms at both the individual and the social level. the long association of honeybees and their pathogens over evolutionary time provides a backdrop to test how the network of social interactions in the colony could serve as the central arena for host-pathogen dynamics (nazzi et al., ) . the pathogens can exploit the network to rapidly spread across the colony, while the host can use its structural properties as a mechanism to resist the spread. the recent finding that honeybees possess only one-third as many genes for immunity as other insects strongly suggests that the structure of social organization is an important mechanism that compensates for a lower physiological immunocompetence (evans et al., ) . interaction networks in a social insect colony could be organized according to one of the following designs: ( ) work-chains with each individual performing all the required parts of a given task, ( ) work-chains with each individual performing one and only one part of a task with one or more other individuals completing the rest, and ( ) work-chains with each individual performing only one part of a task at any given time but performing all the parts equally frequently. the efficiency and the reliability of material and information flow are substantially incremented in each successive type of network, which is adaptive for ergonomic purposes. it is however less recognized that the same design features will also promote the transmission efficiency of pathogens, increasing the vulnerability of the colony to an infectious disease. food, information, as well as pathogens primarily enter a honeybee colony from the environment through the foragers. nearestneighbor based interactions drive the subsequent transfer process, spreading these across the colony. with the individuals spatially distributed within the colony according to their ages, this results in a centripetal flow from the oldest individuals at the outer edge of the colony to the youngest ones residing at the center (tilman, ) . this flow pattern imparts some amount of protection to the most valuable youngest members from invading pathogens: a phenomenon that can be termed 'organizational immunity'. (ping and yihua, ) . this social contact network in the colony is therefore highly structured and nonrandom, leading to a pool of individuals that is heterogeneous with respect to its probability of contacting, manifesting, and transmitting an infection, presenting an invading pathogen with the challenge of negotiating this complex landscape (fig. ) . superimposed on this general age-based interaction pattern, one also sees that only a minority of the individuals in the colony are the primary drivers of the majority of the transfer process. this gives the interaction network an appearance of a scale-free structure. in contrast to such heterogeneous connectivity observed in the large honeybee colonies, individuals are more uniformly connected to each other in social insect species with smaller colony sizes. within-species comparisons suggest that colony size is a primary driver of network structure and complete mixing becomes more and more improbable with increasing number of individuals. there is considerable variation in network structure even among colonies of similar sizes and it has been shown that the density of contact network in the colony determines the spread of a contagious pathogen within it. small perturbations in the structure of social organization can bring about large changes in transmission dynamics. many honeybee diseases, which remain in the background at a low level in the colony, can rapidly turn lethal and erupt into an epidemic under certain conditions generally referred to as 'stress'. investigation of these so-called 'stress' conditions suggest that they translate into disruption of the normal social organization in the colony in the face of contingencies such as a nectar flow in the environment, high demand for a certain task, or a rapid increase in colony population size (martin, ; guzmán-novoa et al., ) . the resulting higher activity level and more generalization of labor profiles can lead to higher contact rates or other changes in social network structure. a disease can also bring about some restructuring of the social organization in the colony. disease at an individual level is defined as a disruption of homeostatic mechanisms, leading to an alteration in the normal set point of an organism and its symptoms are the physiological mechanisms that restore it. this definition can be extended to an epidemic being a process that disrupts the social organization critical to the functioning of a group and its symptoms are mechanisms that, via collective action of its members, attempt to restore the social structure. it has been speculated that a disease symptom such as bees starting to forage at a younger age is an adaptive response on the part of the host that serves to reduce within-colony transmission of the disease by keeping infected bees outside . however, it is equally plausible that such a response can in fact increase transmission rates by contaminating the food they collect. behavioral fever in response to an infection, which can inhibit the development of a pathogen, requires bees to cluster more tightly that can in turn increase the contact rate among them (mayack and naug, ) . bees infected with a pathogen have also been shown to incur an energetic stress that increases their hunger level, leading them to be more eager solicitors but more reluctant donors of food. this could lead uninfected and infected bees to occupy different positions in the contact network in terms of sources and sinks in the transmission chain. it is important to note here that the structure of the social network in the colony is an emergent property that arises from individual behavior, which can be altered by simple pathophysiological mechanisms arising from a disease. for disease ecologists interested in using network theory, the development of network statistics remains a major research focus. a second area of rapidly developing interest is dynamic networks which account for the possibility that the structure of the contact networks might not remain constant over time, maybe partly as a consequence of the disease outbreak itself. more importantly, empirical research has lagged behind the pace of theoretical work made possible by increased computational power. matching efforts to develop laboratory experimental systems are urgently needed to explore the interaction between network structure and disease dynamics. integration of behavioral biology and physiology to the already existing framework of ecology, evolution, and mathematical modeling would also be critical to our understanding of the structural and functional properties of biological networks. research on the proximate basis underlying the behavioral interactions among individuals will give insights into the role of demographic and environmental factors on disease dynamics via their effects on social structure. it will also help answer the important questions of how the pathophysiology of a disease can alter the structure of the contact network and whether such symptomatic restructuring benefits the host or the pathogen. in social insect groups where the colony social network is considered to be primarily a product of ergonomic considerations, it is important to explore whether pathogens have played any selective role in its design. this addresses the broad issue of how any group of interconnected units, whether a bee colony or a computer cluster, deals with the challenge of shielding its network from attacks without seriously compromising its performance. it is being increasingly recognized that excessive use of antimicrobials to treat diseases selects for resistant strains of pathogens that can no longer be eliminated by the same drugs. intervention measures that have short-term epidemiological benefits but long-term evolutionary repercussions have led to the recent resurgence of many diseases and the heightened virulence of pathogen populations. this has led to the suggestion that understanding the natural dynamics of a disease from an evolutionary, ecological, and behavioral perspective might provide pointers to preventive and curative methods that are more sustainable. there are plenty of accounts concerning behavior and customs in humans that affect the transmission of infectious diseases. agricultural practices such as the clearing of land and irrigation have brought increased contact between human populations and animal reservoirs of diseases such as schistosomiasis and malaria (steinmann et al., ) . urbanization has brought about increased transmission of lyme disease, cholera, dengue, and leishmaniasis (kendall et al., ; desjeux, ; bradley and altizer, ; chowdhury et al., ) . changes in sexual behavior have had a large influence on the spread of human immunodeficiency virus (hiv), human papillomavirus (hpv), chlamydia, gonorrhea, and other sexually transmitted diseases (bunnell et al., ; satterwhite et al., ) . with the current threat of these numerous emerging diseases, it has become extremely important to understand the dynamics of infectious processes in the context of crowded living conditions that characterize many animal groups and humans. an understanding of the behavioral processes that define the structure of a social group will help identify the transmission pathways used by pathogens to spread and suggest possible ways to manage the social structure as a counteractive measure to both prevent and control the spread of a likely epidemic. emergence of scaling in random networks scale-free networks rapid evolution in plant chitinases: molecular targets of selection in plant-pathogen coevolution dynamics of measles epidemics: estimating scaling of transmission rates using a time series sir model complex networks: structure and dynamics urbanization and the ecology of wildlife diseases changes in sexual behavior and risk of hiv transmission after antiretroviral therapy and prevention interventions in rural uganda parasitism and 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mediated by host immunity can drive the collapse of honeybee colonies consensus filters for sensor networks and distributed sensor fusion node centrality in weighted networks: generalizing degree and shortest paths from superspreaders to disease hotspots: linking transmission across hosts and space empirical evidence for key hosts in persistence of a tick-borne disease the study on organizational immunity-based enterprise adaptation do animals living in larger groups experience greater parasitism? a meta-analysis changes in sexual behavior and std prevalence among heterosexual std clinic attendees social network analysis schistosomiasis and water resources development: systematic review, meta-analysis, and estimates of people at risk. the lancet infectious diseases parasitoids, predators, and group size in the paper wasp, polistes exclamans resource competition and community structure susceptible-infected-recovered epidemics in dynamic contact networks collective dynamics of 'small-world' networks behaviors of susceptible-infected epidemics on scale-free networks with identical infectivity structure of the social network and its influence on transmission dynamics in a honeybee colony the role of colony organization on pathogen transmission in social insects the structure and function of complex networks collective dynamics of 'small-world' networks key: cord- -xhfmio k authors: fagre, anna c.; kading, rebekah c. title: can bats serve as reservoirs for arboviruses? date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: xhfmio k bats are known to harbor and transmit many emerging and re-emerging viruses, many of which are extremely pathogenic in humans but do not cause overt pathology in their bat reservoir hosts: henipaviruses (nipah and hendra), filoviruses (ebola and marburg), and coronaviruses (sars-cov and mers-cov). direct transmission cycles are often implicated in these outbreaks, with virus shed in bat feces, urine, and saliva. an additional mode of virus transmission between bats and humans requiring further exploration is the spread of disease via arthropod vectors. despite the shared ecological niches that bats fill with many hematophagous arthropods (e.g., mosquitoes, ticks, biting midges, etc.) known to play a role in the transmission of medically important arboviruses, knowledge surrounding the potential for bats to act as reservoirs for arboviruses is limited. to this end, a comprehensive literature review was undertaken examining the current understanding and potential for bats to act as reservoirs for viruses transmitted by blood-feeding arthropods. serosurveillance and viral isolation from either free-ranging or captive bats are described in relation to four arboviral groups (bunyavirales, flaviviridae, reoviridae, togaviridae). further, ecological associations between bats and hematophagous viral vectors are characterized (e.g., bat bloodmeals in mosquitoes, ingestion of mosquitoes by bats, etc). lastly, knowledge gaps related to hematophagous ectoparasites (bat bugs and bed bugs (cimicidae) and bat flies (nycteribiidae and streblidae)), in addition to future directions for characterization of bat-vector-virus relationships are described. bats and the viruses they harbor have been of interest to the scientific community due to the unique association with some high consequence human pathogens in the absence of overt pathology. virologic and serologic reports in the literature demonstrate the exposure of bats worldwide to arboviruses (arthropod-borne viruses) of medical and veterinary importance [ ] . however, the epidemiological significance of these observations is unclear as to whether or not bats are contributing to the circulation of arboviruses. historically, a zoonotic virus reservoir has been considered a vertebrate species which develops a persistent infection in the absence of pathology or loss of function, while maintaining the ability to shed the virus (e.g., urine, feces, saliva) [ ] [ ] [ ] . haydon et al. extended this definition of a reservoir to include epidemiologically-connected populations or environments in which the pathogen can be permanently maintained and from which infection is transmitted to the defined target population. the significance of the relative pathogenicity of the infectious agent to the purported reservoir host has been debated [ ] . in the case of bats as a reservoir species, rigorous field and experimental evidence now exist to solidify the role of the egyptian rousette bat (rousettus aegyptiacus) as the reservoir for marburg virus [ ] [ ] [ ] . considering arboviruses, additional criteria must be met in order to consider a particular vertebrate species a reservoir. reviewed by kuno et al., these criteria include the periodic isolation of the infectious agent from the vertebrate species in the absence of seasonal vector activity, and the coincidence of transmission with vector activity [ ] . further, the vertebrate reservoir must also develop viremia sufficient to allow the hematophagous arthropod to acquire an infectious bloodmeal [ ] in order for vector-borne transmission to occur. bats have long been suspected as reservoirs for arboviruses [ ] , but experimental data that would support a role of bats as reservoir hosts for certain arboviruses remain difficult to collect. here we synthesize what information is currently known regarding the exposure history and permissiveness of bats to arbovirus infections, and identify knowledge gaps regarding their designation as arbovirus reservoirs. the order bunyavirales is divided into eight families, four of which pose threats to public health and veterinary medicine-families nairoviridae, peribunyaviridae, phenuiviridae, and hantaviridae [ ] . while bats have been demonstrated to host hantaviruses, these viruses do not rely on an arthropod in their transmission cycle and thus will not be discussed [ ] . viruses in order bunyavirales that have been experimentally examined in bats or described in field studies are descried in table . members of the genus orthonairovirus of medical and veterinary significance include crimean congo hemorrhagic fever virus (cchfv) and nairobi sheep disease virus (nsdv) [ ] . cchfv is transmitted by ticks in genera rhipicephalus and hyalomma [ ] . while neither live virus nor nucleic acid of cchfv has been detected from bats, serologic evidence suggests past infection of populations of bats across a diverse geographic range [ ] [ ] [ ] . further, bats are often parasitized by both soft and hard ticks, which occupy a diverse range of ecological niches in endemic countries [ ] [ ] [ ] . a seroprevalance study by müller and colleagues examining african bat species (n = , ) found that the prevalence of antibodies against cchfv was much higher in cave-dwelling bats ( . %- . %, depending on species) than foliage-living bats ( . %- . %) [ ] . they also screened , serum samples by rt-pcr, but all were negative for cchfv nucleic acid [ ] . experimental studies to assess the ability of bats to support replication of cchfv have not been published. members of the genus orthobunyavirus include many viruses of importance to human and veterinary medicine, including bunyamwera virus, california encephalitis virus, jamestown canyon virus, kaeng khoi virus, and la crosse encephalitis virus [ ] , but limited evidence exists regarding the exposure or potential involvement of bats in the circulation of viruses in this family. kaeng khoi virus (kkv) has been isolated from cimicid bugs (order: hemiptera, family: cimicidae) (stricticimex parvus and cimex insuetus) and from suckling wrinkle-lipped bats (tadarida plicata ) in caves in thailand, but was not isolated from soft ticks tested in the same area (ornithodorus hermsi) [ ] . additionally, kkv has been implicated in the case of several mine workers who reported illness and were discovered to have seroconverted [ ] , demonstrating spillover of this virus to humans in association with the cave environment, and suggesting that cimicids may play a role in vectoring virus between bat and human hosts. to date, no experimental data have been generated to address this hypothesis. spence and colleagues attempted to experimentally infect jamaican fruit bats (artibeus jamaicensis) via intramuscular injection with nepuyo virus (group c serogroup), yet no infectious virus was subsequently recovered from the bats [ ] . this is interesting considering two strains of nepuyo virus were isolated from jamaican fruit bats (artibeus jamaicensis) and great fruit-eating bats (artibeus literatus) in honduras, and protective sera were found in jamaican fruit bats in trinidad. [ , ] . bats of undetermined species were involved in a large serosurvey in brazil that examined antibodies in wildlife against the gamboa serogroup orthobunyaviruses, though none were found to be positive [ ] . seven and twelve species of trinidadian bats were examined for antibodies by hi against caraparu (group c serogroup) and maguari (bunyamwera serogroup) viruses, respectively, and were all found to be negative [ ] . viruses in the genus phlebovirus (family phenuiviridae) of importance to human and animal health include rift valley fever virus (rvfv) and severe fever with thrombocytopenia syndrome virus (sftsv) [ ] . bats of the species miniopterus schreibersii (n = ) and eptesicus capensis (n = ) were experimentally infected with rvfv and the m. schreibersii bat's urine and liver tested positive for antigen [ ] . a recent study by balkema-buschmann and colleagues experimentally infected egyptian rousette bats (rousettus aegyptiacus) with vaccine strain mp- and recovered infectious virus from spleen and liver of some animals [ ] . oelofsen & van der ryst ( ) examined samples from field-caught bats in africa, yet none were positive for antigen by use of elisa [ ] . kading et al ( ) detected neutralizing antibodies against rvfv in egyptian rousette bats and little epauletted fruit bats (epomophorus labiatus) in uganda, a country that has recently experienced human cases of rvfv [ , ] . whether or not bats serve as a reservoir of rvfv during interepidemic periods remains to be determined. bangui virus (bgiv) is an unclassified bunyavirus and was isolated from an unidentified bat in the central african republic (car) [ ] . mojuí dos campos virus (mdcv) is another ungrouped bunyavirus isolated from an unidentified bat species [ , ] . the family flaviviridae includes many high-consequence emerging arboviruses, including zika virus (zikav), yellow fever virus (yfv), and dengue virus (denv). flaviviruses associated with bats that do not appear to utilize an arthropod vector ("no-known vector flaviviruses") have been reviewed elsewhere [ ] . viruses in family flaviviridae that have been experimentally examined in bats or described in field studies are descried in table . interestingly, despite denv isolations from artibeus spp. bats in the wild, experimental infections of great fruit-eating bats (a. intermedius) with denv- and jamaican fruit bats with denv serotypes and resulted in low levels of viremia, low rates of seroconversion, and lack of detection of viral rna in the organs via rt-pcr, indicating that bats may not act as a suitable reservoir host [ ] [ ] [ ] . experimental infection of the indian flying fox (pteropus giganteus) resulted in no viremia or clinical signs, but intracerebral inoculation of little brown bats (myotis lucifugus) resulted in irritability, paralysis, and death [ , ] . denv nucleic acid and anti-denv antibodies have been detected in mexican bats on the gulf and pacific coast, and nucleic acid has been detected in the liver and/or sera of wild-caught bats in french guiana [ , ] . anti-denv antibodies have been detected in multiple bat species in uganda [ ] . however, a survey in central and southern mexico analyzing individuals representing bat species by rt-pcr resulted in no detection of viral nucleic acid [ ] . a study by vicente-santos and colleagues examined bat species from costa rica and found a cumulative seroprevalence of . % ( / ) by prnt and a prevalence of . % ( / ) in organs tested by rt-pcr [ ] . no infectious virus was isolated and viral loads were considered too low for the bats to function as amplifying hosts. rather, vicente-santos and colleagues surmised a spillover event from humans to bats, with bats functioning as a dead-end host [ ] . the serum of jamaican fruit bats (artibeus jamaicensis) and great fruit-eating bats (a. literatus) from grenada (n = ) were also tested for antibodies against denv , , , and , and none were seropositive [ ] . while field evidence supports the exposure of bats to denv in multiple geographic areas, experimental infections conducted to date are consistent in that bats are not likely to support denv replication and circulation to levels high enough to infect blood-feeding mosquitoes. multiple studies conducted experimental infections of insectivorous bats with japanese encephalitis virus (jbev) and found that bats were susceptible to infection with this virus. three species of bats (big brown bats (eptesicus fuscus), little brown bats (myotis lucifigus) and eastern pipistrelles (pipistrellus subflavus)) were inoculated with jbev in the laboratory and maintained infection while held under simulated hibernation conditions. bats infected prior to hibernation were viremic upon arousing from hibernation, with circulating virus detectable as long as days after the initial infection [ ] . big brown bats also demonstrated recurrent viremia in the absence of clinical signs in a subsequent study [ ] . importantly, researchers demonstrated a mosquito-bat-mosquito transmission cycle and postulated this may be an overwintering mechanism for jbev since mosquitoes did successfully transmit jbev to bats at low temperatures [ ] . eastern pipistrelles also became infected with jbev after consumption of infected mosquitoes, demonstrating that bats could be infected orally as well as through a mosquito bite [ ] . no demonstrable pathologic effects noted during infection of three bat species [big brown bats (eptesicus fuscus), little brown bats (myotis lucifigus) and mexican free-tailed bats (tadarida brasiliensie mexicana) with various strains of jbev or st. louis encephalitis virus (slev) [ ] . no pathology nor viremia was appreciated when pipistrelles (pipistrellus abramus) were infected with jbev [ ] . while experimental data demonstrated that some bat species can sustain jbev infections and support mosquito-borne transmission of this virus, the epidemiological significance of these observations in the field remains unclear. jbev has been isolated from wild-caught bats in taiwan (miniopterus fuliginosus and hipposideros armiger terasensis [ , ] , china (rousettus leschenaultia and murina aurata [ , ] , japan (miniopterus schreibersi fuliginosus and rhinolophus cornutus cornutus [ ] . antibodies against jbev have been detected in wild-caught bats in indonesia (unspecified species) [ ] , china (rousettus leschenaultia, cynopterus sphinx, taphozous melanopogon, miniopterus schreibersii, pipistrellus abramus, rhinolophus macrotis and miniopterus fuliginosus [ , ] , australia (pteropus scapulatus and pteropus gouldi) [ ] , taiwan (unspecified species) [ ] , india (pteropus giganteus, hipposideros pomona, hipposideros speoris, hipposideros bicolor, hipposideros cineraceus, megaderma lyra, cynopterus sphynx, and rhinolophus rouxi) [ ] [ ] [ ] , and japan (miniopterus schreibersi fuliginosus, rhinolophus ferrum equinum nippon, vespertilio superans, myotis macrodactylus, rhinolophus cornutus cornutus, pipistrellus abramus, myotis mystacinus, plecotus auritus sacrimontis, and murina leucogaster hilgendorfi) [ ] . multiple isolations of jbev from locations where the virus is endemic, in addition to the fact that genetic characterization of isolates has supported their similarity to strains identified from human and mosquito isolates, support the role of bats in ongoing circulation of jbev [ ] . another medically-important flavivirus with both field-obtained information and in vivo experimental inoculation is slev. a study by herbold and colleagues demonstrated that % of wild-caught eptesicus fuscus and myotis lucifugus (n = ) in ohio possessed neutralizing antibodies to slev [ ] . other serosurveillance efforts in north america and grenada focused on detection of slev in free-ranging bat populations have resulted in largely negative findings [ , ] . following experimental infection, viremia and transplacental transmission (albeit infrequent) was appreciated in mexican free-tailed bats (tadarida brasiliensis) [ , ] . the viremia in these bats reached log units, likely too low a titer to facilitate transmission to a blood-feeding mosquito [ ] . upon inoculation, little brown bats (myotis lucifugus) appear to be resistant or only slightly susceptible to slev [ ] . herbold and colleagues ( ) demonstrated that inoculation of eptesicus fuscus with slev results in infection and virus was maintained throughout hibernation ( days), with viremia developing four days following arousal ( days post-infection) [ ] . low levels of viremia upon experimental inoculation in conjunction with low seroprevalence data indicate this virus likely does not utilize bats as a reservoir host in nature. to date, biosurveillance testing of bats in central america for wnv have turned up negative results. grenadian artibeus jamaicensis and artibeus literatus (n = ) bats were negative for wnv neutralizing antibodies by prnt [ ] , trinidadian bat species (n = ) were negative by elisa for wnv antibodies [ ] , and mexican bat species (n = ) tested for wnv rna by rt-pcr were negative [ ] . in north america, results have been negative or indicative of low levels of circulation in bat populations tested. tissues from field-collected bats representing seven species in illinois tested by rt-pcr were all negative for wnv, and the same study reported one big brown bat (eptesicus fuscus) with neutralizing antibodies (n = ) [ ] . a field survey taking place in new jersey and new york reported one big brown bat and one northern long-eared bat (myotis septentrionalis) with neutralizing antibodies to wnv (n = ) [ ] . in another field study, only two of free-tailed bats (tadarida brasiliensis) possessed neutralizing antibodies against wnv [ ] . in uganda, kading et al. ( ) detected neutralizing antibodies to wnv in / african straw-colored flying foxes (eidolon helvum), and / little epauletted fruit bats (epomophorus labiatus) [ ] . simpson and o'sullivan ( ) demonstrated experimental inoculation of african straw-colored flying foxes did not result in viremia though two of three bats developed neutralizing antibody. in the same study, two of three egyptian rousette bats were infected but only trace viremia was detected and seroconversion was not appreciated [ ] . experimental inoculation of free-tailed bats (tadarida brasiliensis) did not result in viremia, and infection of big brown bats resulted in low titers ( - pfu/ml) [ ] , not capable of supporting transmission to feeding mosquitoes [ ] . attempts to experimentally infect vampire bats (desmodus rotundus) and black mastiff bats (molossus rufus) by mosquito bite (aedes aegypti) were unsuccessful [ ] . experimental inoculation of multiple bat species (eumops perotis, carollia perspicillata, phyllostomus hastatus and bats in the genus mollosus) were similarly unsuccessful [ ] . still, kading et al. detected a significant neutralizing antibody titer against yfv in one egyptian rousette bat in uganda in , indicating bats are exposed to this virus in nature [ ] . uganda has experienced outbreaks of yfv in recent years [ ] . while multiple african bat species (eidolon helvum, rousettus aegyptiacus, and rousettus angolensis) demonstrated viremia following inoculation with zikav, mops condylurus did not become viremic, although did contain low virus titers in the kidney [ , ] . experimentally-infected little brown bats were susceptible to the zikav by the intraperitoneal, intradermal, intracerebral and intrarectal routes of exposure, but not susceptible intranasally [ ] . however, it is unclear how zikav could circulate in bat populations. kading et al. ( ) did not detect neutralizing antibodies to zikav among ugandan bats screened. flavivirus infections of bats with an emphasis on the potential role in zika virus ecology has been reviewed elsewhere [ ] . flavivirus serology has been historically challenging due to the cross-reactivity of viral epitopes to circulating antibodies [ ] . therefore, the results of serologic surveillance studies must be interpreted cautiously [ , ] . further, multiple methods exist for antibody detection (e.g., hi, prnt, elisa), and the biological significance of neutralizing vs. non-neutralizing antibodies must be taken into account. in , the serum of mexican bats from three species (glossophaga soricina, artibeus jamaicensis, and artibeus literatus) was assayed by prnt using wnv, slev, and denv - , and were positive for flavivirus-specific antibodies ( %). none of the titers exceeded , and all samples were also negative when tested for flavivirus nucleic acid by rt-pcr [ ] . in a serosurvey, eight bats ( . %) displayed non-specific hemagglutination-inhibition (hi) results indicating cross-reactivity or antibodies against an undetermined flavivirus [ ] . kading and colleagues performed a serosurveillance study in ugandan bats and identified . % ( / ) had non-specific flavivirus antibodies by plaque reduction neutralization assay (chaerephon pumilus, hipposideros ruber, mops condylurus, nycteris macrotus, eidolon helvum, epomophorus minor, and rousettus aegyptiacus) [ ] . still, results generally supported the widespread exposure of bats in uganda to flaviviruses [ ] . in , sotomayor-bonilla and colleagues reported that liver and spleen samples from mexican bat species tested negative using pan-flavivirus ns primers [ ] . a recent study in brazil suggested a lack of arboviral circulation in bat populations, as individuals from species were tested for molecular and serologic evidence of alphavirus and flavivirus infection and all were negative [ ] . results of experimental infection of egyptian rousette bats with wnv and of angolan free-tailed bats (mops condylurus) with ntaya virus resulted in very low levels of viremia, while infection of african straw-colored fruit bats with ntaya virus resulted in neither pathology nor detectable viremia [ ] . few studies have examined the presence of viruses in genus coltivirus in bat populations, and to date, a single isolation has been made (table ) [ ] . a study by chastel and colleagues failed to detect antibodies to eyach virus (reoviridae, colorado tick fever group) in the serum of two field-caught bats [ ] . to date, five orbiviruses have been isolated from wild-caught bats and serologic evidence exists for exposure of australian and south american bats to orbiviruses (table ) . while no evidence of human exposure exists for these bat-associated orbiviruses, bukakata (bukv) and fomede (fomv) appear to be strains of the chobar gorge species [ ] . cgv was isolated from ornithodoros species ticks in nepal, and serum from nearby humans and ruminants possessed anti-cgv antibodies, indicating past exposure [ ] . further investigation is warranted to determine the true vector-host association of these viruses and their zoonotic potential. viruses in family reoviridae that have been experimentally examined in bats or described in field studies are descried in table . viruses in genus alphavirus (family togaviridae) that have been experimentally examined in bats or described in field studies are descried in table . enzootic circulation of chikv is understood to occur among non-human primates and forest-dwelling mosquitoes [ ] , but other vertebrates including rodents, bats, reptiles and amphibians have been shown to support chikv replication [ , ] . the range of peak viremia developed by big brown bats was relatively low, but within the range of infectivity to blood feeding mosquitoes [ , ] . when indian flying foxes (pteropus giganteus) and big brown bats were experimentally infected with chikv, bats developed viremia but no clinical signs of disease, indicating they could play a role in the natural transmission of this virus [ , ] . experimental infection of african straw-colored flying foxes did not result in viremia or seroconversion to chikv, supporting a separate study which reported lack of viremia in experimentally infected egyptian rousette bats and african straw-colored flying foxes [ , ] . in , the serum of wild-caught grenadian bats (genus artibeus) were subjected to prnt and ( %) were found to possess neutralizing antibody to chikv [ ] . chikv has been circulating in central and south america since [ ] . whether or not bats are contributing to the natural circulation of chikv in endemic areas or areas of introduction remains to be determined. serological evidence exists supporting exposure of bats to encephalitic alphaviruses in the field, and experimental data demonstrate the susceptibility of bats to infection with alphaviruses including veev. four mexican bat species were examined for molecular evidence of infection with venezuelan equine encephalitis virus (veev), western equine encephalitis virus (weev), and eastern equine encephalitis virus (eeev). no individual bats were positive for weev or eeev, but % ( / ) representing all four species were positive for veev [ ] . field-caught jamaican fruit bats (artibeus jamaicensis) and great fruit-eating bats (artibeus literatus) were negative by prnt for eeev and weev antibodies, but . % ( / ) had neutralizing antibodies to veev [ ] . similarly, the serum of bats representing species was subjected to elisa, and . % ( / ) contained veev-specific antibodies. elisa and hi assays for eeev and weev antibodies, respectively, were all negative [ ] . four species of wild-caught bats from the northeastern united states were tested for neutralizing antibody against eeev and weev. samples were negative for antibodies against weev, but . % of the bats tested did possess eeev-neutralizing antibody [ ] . bats of the genera myotis and eptesicus were experimentally infected with eeev, and developed viremia but failed to develop neutralizing antibodies. infection of big brown bats by bite of culiseta melanura and aedes aegypti mosquitoes was successful. more non-hibernating than hibernating bats were seropositive for eeev [ ] . in a recent serosurveillance study, / bats were seropositive by plaque reduction neutralization assay to babanki virus (bbkv) and / egyptian rousette bats had non-specific alphavirus antibodies (table ) [ ] . multiple isolates of bbkv were obtained from cx. perfuscus mosquitoes collected from multiple locations in uganda during this same sampling period as when bats were sampled [ ] . mosquito blood meals from bats comprised . % of the total blood meals identified from the species cx. perfuscus [ ] . it is unclear whether bats contribute to the transmission cycle of bbkv or are merely incidentally exposed through mosquito bites ten pteropus poliocephalus bats were experimentally infected with ross river virus, and five developed low (log . tcid / µl) detectable and short-lived ( days) viremia. still, % of the colonized mosquitoes (aedes vigilax) that fed on the bats between days - post-infection became infected [ ] . kading et al. ( ) modeled that for viremias <log . /ml, the probability of a mosquito becoming infected was around . or less given the low circulating titer and the volumetric constraints of a small mosquito blood meal; therefore, at least mosquitoes would need to feed on an animal with a low viremia in order for one mosquito to ingest virus [ ] . in the case of rrv, published data demonstrate that infection of mosquitoes fed on rrv-viremic bats is still possible despite a viremia and low titer [ ] . therefore, if bat and mosquito populations are in high numbers, % of bats develop a detectable viremia, and % of mosquitoes become infected, mosquito-borne transmission could take place even though experimentally-determined efficiencies are low [ ] . antibodies against mayaro virus were detected by hi in trinidadian bat species tested [ ] . a number of hematophagous arthropods feed on bats, including bat flies (genera nycteribiidae and streblidae), bat bugs and bed bugs (family cimicidae), and ticks (families argasidae and ixodidae) [ , , , [ ] [ ] [ ] [ ] [ ] [ ] . viruses of medical and veterinary significance have also been isolated from these arthropods [ , [ ] [ ] [ ] . however, the contribution that these ectoparasites play in the circulation of medically important viruses among bats and other hosts is unclear and necessitates further investigation. kading and schountz ( ) reviewed instances in the literature where mosquito blood meals have been identified as originating from bats [ ] . information on primary mosquito vectors feeding on bats is very limited. tiawsirisup et al. collected mosquitoes from five genera inside a bat cave in thailand to investigate sylvatic circulation of jbev. while these collections included arbovirus vectors cx. quinquefasciatus and cx. tritaenhiorhynchus, the only blood-fed mosquitoes collected from the cave were cx. quinquefasciatus, at least of which had fed on leschnault's rousette (rousettus leschenaulti) bats [ ] . culiseta morsitans theobald mosquitoes (vector of eastern equine encephalitis virus) were found to have fed on eastern pipistrelle bats (pipistrellus subflavus), but these blood meals comprised only % of the total blood meals identified from this mosquito species [ ] . no information was found on any blood meals from bats being detected in aedes (stegomyia) species, but it is unclear how much investigation has been done in this area. sixteen of field-collected ae. funereus mosquitoes (vector of rrv) had fed on pteropus alecto bats [ ] . in africa, mosquito species in which bat blood meals have been identified and are known to be associated with a number of medically-important arboviruses include: coquillettidia (cq.) fuscopennata (theobald) (yfv, sindbis, chikungunya viruses), culex (cx.) perfuscus edwards (wnv, oropouche, sindbis, wesselsbron, usutu, babanki viruses), cx. (cx.) neavei theobald (wnv, babanki, spondweni, sindbis, koutango viruses), and cx. (cx.) decens group (wnv, chikungunya, babanki viruses [ , ] . while mosquitoes in the subgenus cx. (cx.) are recognized as primary vectors of wnv, sindbis, babanki, and usutu viruses, only for babanki virus has additional field data been collected so far that support a potential role for bats in virus circulation (discussed above) [ ] . bat flies (order: diptera; families: nycteribiidae, streblidae) are highly host-specific obligate hematophagus ectoparasites of bats [ , ] . a novel fusogenic orthoreovirus, tentatively named mahlapitsi virus (mahlv), was discovered in bat flies (eucampsipoda africana) associated with egyptian rousette bats [ ] . a novel orthobunyavirus, tentatively named wolkberg virus (wbv), was isolated from the same species of bat flies in a similar geographic region [ ] . dengue virus rna was detected by rt-pcr in bat flies (strebla wiedemanni, trichobius parasiticus) associated with common vampire bats (desmodus rotundus) [ ] . bartonella spp. bacteria have also been found infecting both fruit bats and the bat flies parasitizing them in madagascar, providing a documented example of a pathogen-vector-bat association involving bat flies [ ] . more research in this area is needed to elucidate the role of bat ectoparasites in spreading pathogens among individual bats in a roost. in addition to parasitizing humans by infesting their dwellings, bed bugs and other arthropods in family cimicidae are found in close association with bat populations [ ] . some cimicids are known to play a role in alphavirus transmission. cliff swallow bugs (oeciacus vicarious) transmit fort morgan virus and buggy creek virus [ , , , ] among passerine birds. tonate virus, which is closely related to veev, and also causes fatal encephalitis in man, has also been isolated from swallow bugs in the 's [ ] . as previously described, bat bugs (cimex insuetus and strcticimex parvus) host the orthobunyavirus kkv which likely causes disease in humans [ , ] , but this also remains a largely understudied research area. ticks and mites are known to parasitize bats and they both fill similar ecological niches. many studies have identified viruses in bats that cluster phylogenetically with other arboviruses transmitted by ticks. other isolations are indicative of bats playing a role in viruses isolated from either hard-bodied or soft-bodied ticks. for example, estero real virus was first isolated from ticks (ornithodoros tadaridae) obtained from a palm tree colonized by cuban bats [ ] . due to the close ecological association between egyptian rousette bats (r. aegyptiacus) and soft-bodied ticks in caves, the argasid tick ornithodoros faini was screened for marburgvirus rna but all pools were found to be negative [ ] . to date, evidence does not support the role of arthropods in the transmission of filoviruses in nature. bats are also known to host mite species in families macronyssidae, dermanyssidae, and spinturnicidae, though the significance of these arthropod's role in virus transmission between bats and other vertebrates is largely unknown [ ] [ ] [ ] . species in macronyssidae and dermanyssidae have also been demonstrated to efficiently transmit arboviruses (further reviewed in [ ] ). a recent study explored viruses of potential arthropod-origin in the fruit bat hypsignathus monstrosus and identified five viruses: one dicistrovirus (family dicistroviridae, order picornavirales), one nodavirus (family nodaviridae), and two tombus-like viruses. they also detected a related tombus-like virus isolated from fig wasps and primitive crane flies co-habitating with bats, suggesting the ability of arthropods to host "bat-associated" viruses [ ] . tombus viruses typically infect plants, but tombus-like viruses infect a wide range of hosts, including arthropods and marine invertebrates [ ] [ ] [ ] . ingestion of arthropods by fruit bats has been documented [ , ] , and could be a potential mechanism explaining the association between viromes of arthropods and frugivorous bat species. with regard to experimental infections, one must take into account the method of inoculation (e.g., intracerebral, subcutaneous, intraperitoneal, intramuscular), the inoculating titer, as well as whether or not vector bites were associated with the infection. in analyzing serologic results, one must take into account whether the assay is detecting neutralizing or non-neutralizing antibodies, and consider the dynamics of antibody decay which may impact detection sensitivity [ ] . arthropod saliva has been shown to potentiate arbovirus infection, affecting not only the magnitude of peak viremia but also the viral loads in certain organs [ ] . the inoculating viral strain used is also important, as it should align geographically with where the species under study resides to truly characterize their potential as a reservoir species. komar et al. ( ) presented detailed methods for estimating the reservoir competence of vertebrate species for mosquito-borne viruses [ ] , including calculating a reservoir competence index based on the susceptibility to infection, mean daily infectiousness, and the duration of infectiousness. this reservoir competence index is interpreted as the relative number of infectious vectors that derive from a particular vertebrate species [ ] , and would be easily transferrable to evaluating the reservoir competence of bats. further, bats are very long-lived (up to years) compared to other small mammals, potentially increasing the chances of exposure and subsequent transmission to other vertebrates or invertebrate vectors [ ] . cell culture also offers a viable starting point for initial assessments of viral replication in a particular host species, particularly when in vivo studies are not feasible. to date, many cell lines derived from the organs of bats have been established [ ] . importantly, the intent of this review is not to vilify bats, but to analyze the existing literature and its support for bats as arboviral reservoirs, in addition to identifying areas for future study. bats provide vital ecosystem services, such as arthropod suppression, pollination, and seed dispersal [ ] . past depopulation efforts in response to viral outbreaks resulted in increased viral spillover, and are not a viable means of disease control and have even resulted in higher virus infection rates in the bat species when colonies repopulated from neighboring roosts [ ] . further work should encompass directed field surveillance complemented by both in vitro and in vivo approaches, with field surveillance efforts focused on optimization of non-lethal and non-invasive sampling [ ] [ ] [ ] [ ] . when possible, longitudinal sampling of identifiable animals through use of marking or tagging allows for monitoring of seroconversion and viremia persistence, providing a chance to better characterize transmission periodicity and seasonal changes in viremia profiles. to truly elucidate the role of bats as reservoirs for arboviruses, field surveillance studies documenting natural infection and transmission dynamics among vector and vertebrate species must be supplemented with experimental infections to characterize viremia profiles and infectiousness to vectors, virus-induced pathology, and immune kinetics following infection. with bats, these tasks are not trivial, and carry significant challenges in both the field and the lab. these challenges are evidenced by the few arboviruses for which there are substantial field and laboratory data involving the infection of bats. while many studies have presented serologic data indicative of past exposure of free-ranging bats to arboviruses of medical and veterinary importance, these studies should be followed up with laboratory assessments of reservoir host competence to shed light on the true epidemiological significance of the field data. further, the detection of viral nucleic acid in free-ranging bats does not necessarily implicate the species as an arboviral reservoir. rather, recovery and isolation of live virus at biologically relevant titers, and demonstrating the persistence of the pathogen in nature among connected populations of a potential reservoir species is more definitive. unfortunately, few established bat colonies exist for use in vivo viral pathogenesis studies, limiting the achievability of these studies. to fulfill the vertebrate reservoir host paradigm, in vivo infections must support findings in the field. the isolation of marburg virus from egyptian rousette bats in uganda in addition to experimental infections demonstrating viremia and shedding in the absence of overt pathology support the role of this bat species as the reservoir for marburg virus [ , , ] . for arboviruses, the combination of field work and in vivo pathogenesis studies are lacking. still, several examples have emerged from this review that point to bats as potentially competent amplifying hosts for arboviruses. kaeng khoi virus was isolated from both bats and cimicid bugs in thailand, and was also shown to be the causative agent behind sick mine workers [ ] . some bat species did develop a viremia at a level that would be infectious to mosquitoes when inoculated experimentally with chikv, and bats have been found exposed to chikv during field investigations [ , ] . experimental evidence supporting transmission of rrv from pteropus poliocephalus to recipient mosquitoes in addition to identification of rrv-seropositive bats trapped in australia and indonesia highlights the need for further investigation into the role of bats in the ecology of this disease [ , , ] . a mosquito-bat-mosquito transmission cycle was established in the lab for jbev [ ] . serological evidence from the field documenting bat exposure to jbev, the sustained viremia in bats during hibernation, and demonstration of mosquito transmission 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optimizing non-invasive sampling of an infectious bat virus experimental inoculation of egyptian rousette bats (rousettus aegyptiacus) with viruses of the ebolavirus and marburgvirus genera key: cord- -uceukiie authors: jones, arwel wyn; davison, glen title: chapter exercise, immunity, and illness date: - - journal: muscle and exercise physiology doi: . /b - - - - . - sha: doc_id: cord_uid: uceukiie abstract it is generally accepted that moderate amounts of exercise improve immune system functions and hence reduce the risk of infection whereas athletes engaged in regular prolonged and/or intensive training have a higher than “normal” incidence of minor infections, especially of the upper respiratory tract (urt, e.g., common cold and influenza). this is likely related to regular acute (and possibly chronic) periods of exercise-induced changes in immune function. urt infections can compromise performance directly if suffered shortly before or during competition or indirectly if suffered at other times via effects on training and/or physiological adaptations. this chapter covers the effects of exercise (acute and chronic), both positive and negative, on immune function and consequent infection risk, and considers the current state-of-the-art for monitoring and assessing this in athletes. it is generally accepted that moderate amounts of exercise improve immune system functions and hence reduce the risk of infection. however, there is strong evidence that athletes engaged in regular prolonged and/or intensive training have a higher than "normal" incidence of minor infections, especially of the upper respiratory tract (urt, e.g., common cold and influenza) (gleeson and walsh, ) . this is particularly apparent in endurance athletes such as cyclists, runners, swimmers, and triathletes, but any athletes with a high training load and/or suboptimal recovery may be at increased risk. such infections can compromise training and/or competition performance . upper respiratory tract infections (urti) are among the most frequent presentations to general practitioners (hasham and hall, ) . these do not usually require hospital admission, but such illnesses have a significant economic and social impact-e.g., absence from work, healthcare costs, increased morbidity, reduced feelings of well-being, health and quality of life, and reduced social interaction. broadly, moderate amounts of exercise are associated with enhanced immunity and resistance to such infections whereas high amounts or prolonged and/or vigorous training may increase the risk. indeed, urti are also suggested to be the most common type of infection in the athletic population (roberts, ; cannon, ; peters, ; gleeson and walsh, ) . in fact they have shown to be a highly prevalent medical condition in athletes at clinics in both the summer and winter olympic games (e.g., robinson and milne; engebretsen et al., engebretsen et al., , . observational and experimental studies have investigated the proposed greater resistance to pathogens with moderately active lifestyles. animal investigations have demonstrated that brief bouts of moderate physical activity ( À min treadmill running) compared to inactivity prior to or immediately following inoculation with pathogens leads to decreased mortality and morbidity from infection (davis et al., ; lowder et al., ) . early exercise training studies of older and obese humans also demonstrated that À weeks of moderate exercise [ min walking at %À % of maximal oxygen uptake ( _ vo max )] resulted in lower incidence or duration of selfreported urti compared to sedentary individuals (nieman et al., b (nieman et al., , . these effects have been supported by several longitudinal studies of the wider general population (ages À years) where maintenance of a moderately active lifestyle leads to lower self-reported or laboratory confirmed uri/urti episodes (kostka et al., ; matthews et al., ; kohut and senchina, ; ciloglu, ; kostka and praczko ; kostka et al., ; nieman, ) ; (spence et al., ; barrett et al., ) . although patterns vary between sports, a review by walsh et al. ( b) suggested that athletes tend to report urti either during the high-intensity and tapering period prior to competition (e.g., swimming, team sports) or in the period following competition (e.g., long distance running). it has long been hypothesized that a j-shaped relationship exists between exercise workload and susceptibility to urti (nieman, ) . this model suggests that an individual involved in regular moderate exercise is less likely to contract urti compared to a sedentary individual but prolonged high-intensity exercise or periods of strenuous exercise training are associated with an above-average risk of urti (see fig. . ). indeed, the j-shaped model was initially based on findings of increased self-reporting of urti in the À week period following participation in competitive endurance races (e.g., nieman et al., a) . further support for an adverse effect of prolonged/ strenuous exercise on susceptibility to urti has come from animal studies (davis et al., ; gross et al., ; folsom et al., ; lowder et al., ; murphy et al., ) . prolonged exercise (treadmill running for . h) has been shown to increase morbidity and mortality of mice inoculated with respiratory viruses (e.g., herpes simplex type virus, influenza) prior to (davis et al., ; murphy et al., ) or following (lowder et al., ) this type of exertion compared to resting and/or moderately exercised mice. in addition to acute exertion, equine studies have also demonstrated that intensified periods ( À days) of exercise training prior to or following inoculation with influenza leads to greater severity of infection in vaccinated (folsom et al., ) and nonvaccinated horses (gross et al., ) . generalizing these results to the human in vivo environment is questionable (albers et al., (albers et al., , bermon et al., ) and such approaches (i.e., pathogen challenge) with human volunteers do have considerable ethical constraints (hope and mcmillan, ) . although not yet investigated in an exercise context, pathogen challenge studies in humans have demonstrated lower resistance to urti due to other life stressors (e.g., psychological stress, sleep disturbance) (cohen et al., (cohen et al., , prather et al., ) . in addition to the potential exposure to these stressors in a training and competition environment (coutts et al., ; hausswirth et al., ) , the relevance of these findings to athletes is emphasized by the impact of transient modulation in host resistance to urti by a stressor. exercise immunology research (epidemiological and experimental) has grown substantially over the last few decades to investigate the relationship between exercise and urti in humans (shepard, ) . in contrast to animal research, human studies (attempting to discern the effects of prolonged exercise/intense training on urti) have mainly involved monitoring athletes following heavy exertion (i.e., relied on natural exposure to pathogens) but only a limited number of these have verified that symptoms are due to infectious agents (pathogens) (spence et al., ; schwellnus et al., ; hanstock et al., ) . this has raised concerns regarding the validity of urti episodes (i.e., self-reported) in athletes that occur in and around competition or heavy periods of training (bermon, ; walsh et al., b) . discrepancies between physician and laboratory diagnosed urti has also highlighted the limitations with evaluation of urti episodes (cox et al., ) . upon presentation of symptoms at a sports medicine clinic, the study of cox et al. ( ) observed that only % of cases were found to be indicative of infection with laboratory methods (e.g., identified pathogen) while % were diagnosed as urti by physicians. in a surveillance study of a range of athletes (recreational and elite) and sedentary controls, spence et al. ( ) demonstrated that the first two days of symptoms with infectious or noninfectious (see later) cases were similar but duration and severity of symptoms on subsequent days were greater with infectious cases. on the other hand, the common symptom scoring methods applied in self-report questionnaire studies usually require symptoms to be present for two or more days in order to be counted as an "episode" (e.g., fricker et al., ; gleeson et al., ) , which may offer some protection against such limitations. it is also worthy of note that a subsequent uk-based study observed an % agreement rate between self-report and laboratory-confirmed pathogen detection (hanstock et al., ) . the difference to earlier studies could be due to time of year, location, or perhaps the sensitivity of detection methods has improved since the earlier studies (e.g., spence et al., ) . nevertheless, for clarity the term upper respiratory tract symptoms or upper respiratory illness (uri) are generally accepted in this area unless infection has been clinically confirmed (when urti can be used). although there were numerous early anecdotal reports and retrospective survey data to support the proposed jshaped relationship (simon, ; shepard et al., ; nieman, ) , such observations alone cannot validate the influence of exercise on uri. however, further support to the j-shaped model (i.e., heavy exercise workload) was provided by a number of prospective and retrospective studies which suggested that marathon or ultramarathon runners suffer from an increased risk of uri (e.g., À weeks following competitions) (nieman et al., a; peters et al., ) . the study of peters and bateman ( ) was a seminal study in highlighting this by randomly recruiting a sample of runners who competed in the two oceans marathon in cape town. in the days following the km event, % of runners reported uri compared to % of age-matched controls that did not participate in the marathon but shared living space with runners (i.e., to control for exposure to pathogens and other environmental factors). nieman et al ( a) went on to show that, compared to equally experienced runners who did not compete, there was a sixfold increase of uri in runners during the seven days following the los angeles marathon. taking into account other factors influencing risk of uri (age, stress levels, and illness at home), the likelihood of uri was doubled in those who ran . km compared to those who ran , km as part of their weekly training programmes leading up to the event. heath et al. ( ) also highlighted running mileage as a significant risk factor for incidence of uri in a cohort of runners followed for a period of months. more recently, matthews et al. ( ) have also suggested that runners with higher training loads tend to be more prone to uri and that endurance athletes in particular suffer from longer episodes of uri than their recreational counterparts. however, such findings have not been demonstrated consistently as shorter observational studies have failed to observe any associations of uri with differences in training mileage, intensity, and load . despite much interest, there remains more uncertainties than evidence based facts regarding the notion that high volumes of training are associated with an increase in the incidence of uri (walsh et al., b) . one suggestion is that such inconsistent findings may be related to whether participants within studies are considered "elite" or "highly trained." malm ( ) suggests that a prerequisite to achieving elite athlete status is an immune system which can withstand the strenuous nature of training and competition as susceptibility to infections is incompatible with elite performance. for this reason, malm ( ) proposed an s-shaped rather than a j-shaped curve to include elite training which is associated with a lower risk of infection compared to high exercise workload (see fig. . ). however, it is highly likely that elite/ professions athletes will have considerable support (financial, medical, sports science, and nutrition, etc.) and their support team may implement preventive and treatment strategies to reduce the risk and limit the effects of uri and this may also contribute to better management of other stressors. it could be, therefore, that rather than being naturally able to withstand strenuous training and infections, it is simply that they are better supported (compared to counterparts who lack such support mechanisms) to reduce controllable risk factors. nieman ( ) suggested most athletes may not report uri or suffer from an increased risk if they avoid periods of overreaching or overtraining (nieman et al., ) . it seems that an increase in uri may only be attributed to participation in acute prolonged exertion (e.g., marathon, ultra-marathon) or more importantly when athletes are exposed to a greater strain of training through exceeding individual training thresholds coupled with inadequate recovery or other life stressors (e.g., sleep disturbance) (foster, ; pyne and gleeson, ; hausswirth et al., ) . in other words, training load or volume alone does not give full information on the level of stress that an athlete (and their immune system) is under. indeed, the way training in distributed or periodized is of key important also. in support of this, svendsen et al. ( ) have shown that rapid changes in training load (i.e., increasing too quickly) are better predictors of uri risk than total load alone, which goes some way to explaining the above mentioned discrepancies where only total load or volumes were considered. in line with the general population, when pathogen identification has been attempted, bacteria are rare causes of urti in athletes, with viruses being responsible in most cases (in particular rhinovirus, adenovirus, and parainfluenza) (roberts, ; mäkelä et al., ) . urti of viral origin last approximately À days but clearance of the particular virus from the system may take longer (winther et al., ; heikkinen and järvinen, ) . the route of entry into the body by most viruses is the respiratory tract where symptoms reflect the perturbations in function of infected cells and the attempts of the immune system to contain the infection (roberts, ) . as the nature of symptoms of some (i.e., infectious) uri may be similar to noninfectious inflammatory factors and/or presentation of allergic conditions, identification of antibody titers, and isolation of specific pathogens from body fluids of athletes have been recommended to clarify causes of symptoms and provide the most appropriate treatment or management strategies (gleeson, ; cox et al., ) . it was generally believed that upper respiratory symptoms in athletes were due to an infective cause, however, it is only more recently that other causes ("noninfectious hypothesis") have been proposed during training and competition (bermon, ; schwellnus et al., ) . robson-ansley et al. ( ) reported that a higher incidence of uri in runners following a marathon ( %) compared to nonrunners ( %) was significantly associated with positive responses in the allergy questionnaire for athletes. as the prevalence of inhalant allergy may be as common as %À % of highly trained athletes, it may partly provide an explanation to incidence of uri (langdeau et al., ; lumme et al., ; schwellnus et al., ) . environmental influences have been considered relevant to certain groups of competitive athletes, in particular swimmers who are exposed to chlorine derivatives from swimming pool disinfectants while inhaling large amounts of air above the water surface (piacentini et al., ; bougault et al., ). in addition to inhaled irritants, the combination of high ventilation rate in heavy training and surrounding cold, dry air is another possible source of noninfectious, nonallergenic inflammatory stimuli to uri in some athletes (e.g., runners, cyclists) (bermon, ; cox et al., ) . there is also potential for direct damage in epithelial tissue of the urt and/or fibers of the contracting skeletal muscle following strenuous exercise to contribute to the noninfectious, local, and systemic inflammatory origin of uri (peters, ) . schwellnus et al. ( ) reported that the use of an antiinflammatory agent reduced uri in participants following an ultra-marathon event, but as this nasal, buccopharyngeal spray also contained antimicrobial properties it does not provide any conclusive evidence to support a purely noninfectious inflammatory cause for uri. in contrast, administration of an antiinflammatory throat spray in the period leading up to and following a half-marathon event had no influence on the incidence of uri in runners but did reduce the severity of recorded symptoms (cox et al., ) . although conflicting evidence here may purely reflect site-specific differences between agents, together these studies do provide evidence that noninfectious inflammation may not be the underlying cause of all uri, but may interact with infectious causes to potentiate the symptoms that occur as a result of responses to pathogen challenge. in their surveillance study, spence et al. ( ) demonstrated that the distribution of uri closely followed the j-shaped curve in terms of training status (i.e., both the control and elite athlete group suffered from greater days of illness than their recreational counterparts), but only % of reported illnesses were confirmed by identification of common respiratory pathogens. it is worthy to note that only specific pathogens were tested in spence et al. ( ) , thus uri were possibly caused by known pathogens not tested for, unknown pathogens and/or new strains of viruses which were yet to be identified (bermon, ) . despite the low number of identified pathogens highlighted with laboratory evaluation of uri in some studies (i.e., spence et al., ; cox et al., ) , it must be emphasized that this does not rule out infectious causes for these cases as such diagnostics procedures do have inherent limitations in identifying causative agents from an evolving diverse pool of pathogens (e.g., b common cold viruses) (heikkinen and järvinen, ; eccles, ) . furthermore, a more recent (albeit smaller) study based in the united kingdom during the winter months (i.e., typical uri season) observed that a much higher proportion ( %) of reported illnesses were confirmed by identification of urti-causing pathogens (hanstock et al., ) . in this study, recreational-level athletes completed the study, which included a -week monitoring period during which subjects reported uri using the daily jackson common cold questionnaire. of these subjects, urti-causing pathogens were detected in (all were positive for rhinovirus and one was concurrently positive for coronavirus). not all cases of uri can present as a typical response to a primary viral infection of initial upper respiratory symptoms followed by local and systemic inflammation (e.g., fever, aches) (gleeson et al., ) . occasionally symptoms may be minor and/or short-lasting ( À days) resulting in training being unaffected or be a reflection of persistent fatigue and recurrent infections as a result of chronic heavy exertion (reid et al., ) . in these cases it has been suggested that, in addition to exercise-induced inflammation, uri following exercise may be related to reactivation of latent viruses within the upper airways rather than the incidence of primary infections in the recovery period (gleeson et al., ) or prior urt infections that have not been fully eliminated even after symptoms have subsided. ekblom et al. ( ) found in a group of recreational runners that prerace uri was significantly associated with uri incidence following the marathon. this was supported by a longitudinal observational field study, where it was suggested that reactivation of prerace viruses and exercise-induced inflammatory responses were the primary causes of elevated uri incidence prior to and following an . km marathon . the % of runners, who recorded incidence of uri during the À days following the race, also recorded symptoms in the time leading up to the race. these findings may support the aforementioned animal studies whereby participation in prolonged exercise may worsen symptom severity induced by existing infection (malm, ) . it has also been suggested that the reactivation of latent viruses (e.g., epsteinÀbarr virus, ebv) could be implicated in the etiology of urt infections (gleeson et al., ) . ebv is a herpes virus that typically infects b %À % of the world's adult population (gleeson et al., ; staras et al., ; bate et al., ) . after initial primary infection, these viruses lay dormant within the cells of the immune system and immunocompetent individuals are generally asymptomatic (kano and shiohara, ; crawford, ) . however, under significant physical and/or psychological stress the immune system's ability to control these infections (keeping latent) may be lost and such latent viruses may become reactivated (tingate et al., ; glaser et al., ; mehta et al., b; stowe et al., ) . in an exercising population, ebv has received attention due to its ability to replicate continuously or intermittently from the oropharynx (faulkner et al., ; nadal et al., ) . gleeson et al. ( ) found a significant relationship between previous ebv infection and uri in elite swimmers: while all seronegative swimmers remained unaffected (no reported uri) during a day period of intensive training out of the seropositive swimmers had ebv dna detected in saliva during the study with of these going on to develop uri which appeared À days following first detection of ebv dna. it was postulated that ebv was implicated in the symptoms. however, cox et al. ( ) treated a group of endurance runners with a herpes-virus-specific antiviral treatment and although this was able to significantly reduce ebv expression, this had no effect on uri. it is possible, therefore, that ebv is not directly involved/the cause of uri per se, but rather an in vivo indication of immunodepression/compromised immunity and, therefore, increased susceptibility to other uri-causing pathogens. indeed, for the reactivation of ebv to occur, there must be a disturbance within certain parameters of the immune system which usually keep the virus tightly-regulated and latent. this reflection of immune perturbations within the host (mehta et al., a) occur in line with the most researched hypothesis ("open window") behind an increased risk of primary infections in athletes who participate in prolonged exercise. this theory is still it its infancy however and requires further study, although it does seem to be supported by studies showing athletes to have greater levels of detectable ebv dna compared to controls (hoffmann et al., ) . in a clinical investigation of elite athletes suffering recurrent episodes of uri, ebv viral shedding was detected in % of the cohort (reid et al., ) . yamauchi et al. ( ) also found in an intensive training period with rugby players that salivary expression of ebv dna was . times greater in participants with uri compared to those without uri. the "open window" hypothesis suggests that prolonged/heavy exercise causes depression of the immune system which leaves the body less resistant to viruses and bacteria and thus increases the risk of subclinical and clinical infection for between and h (pedersen and ullum, ; nieman, ) , which reflects that the increased risk of uri with heavy exertion was due to a decrease in immunosurveillance (and vice versa, the decreased risk with moderate exercise was due to an increase in immunosurveillance). the immunological response to acute exercise is deemed a subset of stress immunology (hoffman-goetz and , where responses have been likened to those caused by infection, sepsis, burns, or trauma (pedersen and hoffman-goetz, ) . although exercise does share some similarities to the hormonal and immunological responses of these clinical physical stressors, there are also important distinct differences in the magnitude and temporal responses (shepard, (shepard, , . strenuous exercise induces an ordered sequence of modest changes in pro-inflammatory signaling followed predominantly by antiinflammatory responses which down-regulates immune function whereas some of the clinical stressors listed above (e.g., sepsis) trigger an excessive and overwhelming elevation in systemic pro-inflammatory responses (shepard, ) . exercise immunologists have controlled and adapted the duration and intensity of the stress model of exercise to gain a deeper understanding into how alterations in immune function following acute exercise and training may lead to changes in susceptibility to pathogens. the immune system has evolved to protect the human body from pathogens (viruses, bacteria, and parasites). it encompasses the ability to maintain homeostasis even when exposed to a wide range of foreign and self molecules (i.e., antigens). the components of the nonspecific innate system and the specific acquired system overlap to ensure that a state of immunity against infection is established. one of the main mechanisms responsible for changes in host defense with moderate activity seems to be a greater immunosurveillance associated with moderate activity. regular bouts of moderate intensity activity generally induce transient improvements in the immune system . there are various factors which mediate these relationships, one of which is the fitness status of participants, although many studies assessing the immune response to moderate exercise have focused on interventions for previously sedentary individuals. many components of the immune system are temporarily reduced (exercise-induced immunodepression) after strenuous and/or prolonged bouts of exercise (gleeson and walsh, ) . this may persist for as little as a few hours or a long as a few days (depending on the nature of the exercise). in particular, if subsequent bouts are commenced too soon, before the immune system has fully recovered, then a progressive accumulation of immunodepression may ensue. periods of depressed immunity, whether small acute periods or more chronic periods, are termed "open windows" (as discussed earlier) and this is a likely mechanism explaining increased susceptible. however, it is important to point out that this does not necessarily mean that changes in isolated immune markers alone can predict illness risk. there is considerable redundancy in the immune system, so it is important to note that changes in isolated in vitro and ex vivo markers may not give a good representation of the ability of the whole immune system to mount an effective response. even if it was possible to measure every component of the immune system (in vitro) concurrently, it would still be virtually impossible to calculate how such results could be combined to predict the whole integrated (in vivo) immune response. for this reason in vivo measures or markers that represent the ability of the whole immune system to mount a coordinated, integrated response are the most useful and clinically relevant markers (see albers et al., , and albers et al., for detailed reviews). it remains important to study such in vitro and ex vivo markers nonetheless as they provide mechanistic information and insight on the effects of exercise on immunity, but for most of these measures researchers (and practitioners) must exercise caution in their interpretation of such data, which should be used to supplement more clinically relevant markers and provide additional mechanistic insight. the number of circulating immune cells (leukocytes) is profoundly influenced by acute exercise with reports from over a century ago highlighting the exercise-induced mobilization following the boston marathon (larrabbe, ) . circulating leukocytes consist of the granulocytes (neutrophils, eosinophils, and basophils; %À % of total), monocytes ( %À %), dendritic cells (less than %), and the lymphocytes ( %À %) which can be divided into innate [natural killer (nk) cells] and acquired (t helper, t cytotoxic, and b cells) cells. it is now clear that leukocytosis, which is an increase in the total number of circulating leukocytes (mainly neutrophils and lymphocytes) occurs (up to %) during and immediately post exercise (simpson, ) . the observed changes are dependent on the exercise intensity and duration (gleeson, ) with prolonged endurance exercise (. . h) causing a greater leukocytosis (three-to fourfold increase) than brief ( À min) high-intensity exercise (robson et al., b) . at rest, it is estimated that an equal amount of leukocytes are circulating within the blood and located within marginated pools, adhered to blood vessel walls of the circulatory system (athens et al., ; berkow and dodson, ) . foster et al. ( ) suggested the increased cardiac output during exercise and the subsequent shear stress (increased blood flow) within blood vessels induces leukocytes to enter circulation in a process known as demargination. however, it is anticipated that marginal pools within the liver, lung, spleen and other vital organs (e.g., bone marrow, intestines) also contribute to the large leukocytosis following exercise as pools in the lung alone possess lymphocytes which are present in times larger amounts than the circulatory pool (hogg and doerschuck, ; simpson, ) . activation of the sympathetic nervous system (elevated concentrations of plasma catecholamines) and the hypotha-lamicÀpituitaryÀadrenal (hpa) axis (cortisol release) during prolonged exercise also play an integral role in exercise-induced leukocytosis (nieman, ; atanackovic et al., ; anane et al., ). the immediate leukocytosis, particularly neutrophilia (increased neutrophil count) upon onset of exercise is suggested to be due to the haemodynamic and catecholamine induced demargination of vascular and pulmonary pools but this is later followed by a cortisol-induced release of neutrophils from the bone marrow otherwise known as delayed leukocytosis (allsop et al., ) . this effect is seen simultaneously with the immediate leukocytosis during prolonged exercise of . h as cortisol level is sufficiently elevated and its action is prolonged, explaining the previously mentioned greater leukocytosis than following shorter, higher intensity exercise (robson et al., b) . neutrophils released from the bone marrow following stimulation by cortisol are suggested to include a greater proportion of immature cells (e.g., band cells) compared to neutrophils that demarginate from the endothelial walls upon onset of exercise which have similar maturity levels to those already in circulation (hetherington and quie, ; mccarthy et al., ) . although effects on dendritic cells remain unclear, the bone marrow is also a source of maturation for both monocytes and b cells where monocytes also increase proportionately with exercise duration and b cells are associated with limited redistribution (pedersen et al., ; shek et al., ; nieman et al., ; lancaster et al., a; okutsu et al., ) . nk and t cells (mostly cytotoxic) also increase proportionately with exercise intensity and duration unlike neutrophils where increases are predominantly influenced by duration (mccarthy and dale, ; gabriel et al., ; shek et al., ; campbell et al., ) . even though lymphocytes are present at numerous sites within the body, evidence suggests that mobilization of these cells during exercise mainly occurs from secondary lymphoid organs, for example, spleen, intestinal peyer's patches (pp) rather than primary lymphoid organs such as the thymus or bone marrow (simpson et al., (simpson et al., , campbell et al., ). the spleen is considered to be an abundant source of the lymphocytes deployed during exercise (baum et al., ; nielsen et al., ) , with shear stress and catecholamines being important release mechanisms (kappel et al., ; benschop et al., ; shepard, ; timmons and cieslak, ; dimitrov et al., ) . there is a clear consensus developing that exercise triggers a redistribution of t cells with a longer history of antigen exposure rather than naive t cells (campbell et al., ; simpson, ) . following the lymphocytosis during and upon completion of prolonged exercise, the high concentration of adrenaline and cortisol often cause lymphocytopenia (lymphocyte count below resting levels) (nieman et al., ; nieman, ) during the recovery. this biphasic response (lymphocytosis during exercise and lymphocytopenia during recovery) has been found with various protocols requiring heavy/prolonged exertion or exhaustive exercise (e.g., fry et al., ; shek et al., ) , with lymphocyte count up to % below resting levels reported post exercise (simpson, ) . lymphocytopenia is due to the selective extravasation of lymphocyte subsets, nk and t cytotoxic cells, from blood to the surrounding tissues (gabriel et al., ; simpson et al., ; kruger et al., ) . the return of total leukocyte count to resting levels generally begins immediately post exercise with diminishing activation of the sympathetic nervous system and hpa axis, but in the case of very intense exercise, leukocyte (primarily neutrophils) count may continue to increase as described above (mccarthy and dale, ; allsop et al., ) . given these leukocyte perturbations, an increase in neutrophil:lymphocyte ratio is suggested to be an indicator of the overall magnitude of the stress response induced by exercise . the immune system consists of many physical barriers (e.g., skin, mucus, cilia) which act to prevent entry of pathogens into the human body. if such attempts fail, infectious agents will be detected by receptors present on the cellular components of the innate immune system such as the granulocytes (mostly neutrophils, but include basophils and eosinophils), nk lymphocytes, monocytes (which mature into macrophages within tissue), and dendritic cells. the recognition of foreign material is one way in which the innate and acquired immune systems differ from one another. unlike acquired immunity, the innate system does not exhibit memory of previous encounters with antigens (i.e., foreign molecule), therefore a similar response takes place during any future exposure. present on the cell surface of innate cells are pattern recognition receptors (prr) that distinguish self from nonself material by recognizing molecules on microbes which have been conserved through evolution due to their essential function i.e., bacterial dna, lipopolysaccharides (lps), and other bacterial cell wall components (kimbrell and beutler, ; beutler and rietschel, ) . the majority of these structures comprise of the toll-like receptors (tlr) which are crucial components of the antigen presentation cells (apc) (discussed later in this section) of innate immunity (monocytes/macrophages). neutrophils also known as polymorphonuclear (pmn) cells (due to their multilobed nucleus) are the most abundant circulating leukocyte population of the immune system. these leukocytes are considered specialised short-lived cells with deficits in numbers and/or function of this subset being associated with increased risk of potentially fatal bacterial infections (smith et al., ; boxer, ; viscoli et al., ) . once released from the bone marrow, neutrophils migrate out of the circulation within À h to marginated and tissue pools where they reside for a further À days (smith, ; summers et al., ) . neutrophils respond to chemotactic stimuli (e.g., formylated peptides such as formyl methionyl leucyl phenylalanine, fmlp) from fragments of invading microorganisms and tissue damage at sites of infection and inflammation respectively where they engage in numerous intrinsic conformation adaptations during and following their migration (chemotaxis) (smith and pyne, ) . similar to other phagocytes (e.g., apc, discussed later in this section), the detection of pathogens or tissue damage through prr (e.g., fpr , a fmlp receptor) on the neutrophil surface leads to internalization (phagocytosis) and holding of these fragments within the cytoplasm (via a membrane bound vesicle known as phagosome) thereby activating a series of effector functions in the cell (nathan, ; borregaard, ) . the mechanistic detail of engulfment by neutrophils may depend on the involvement of the specific ligand (i.e., antigen) attached to the receptors or whether fragments have been opsonised by soluble immune factors, but the process will ultimately involve internalization into a phagosome (amulic et al., ) . soluble components which act as opsonins (promote attachment of antigen to phagocyte) include acute phase proteins, antibodies (see section . . ) and complement proteins (e.g., c ) (thiel et al., ; gordon, ; brekke et al., ) . it is worthy to note that elevations in circulating concentrations of acute phase proteins (c-reactive protein, crp) in response (i.e., acute phase response) to perturbations in homeostasis (e.g., trauma, tissue damage) are considered one example of why the strenuous nature of prolonged exercise is likened to medical conditions (e.g., sepsis, burns) (kushner and rzewnicki, ; pedersen and hoffman-goetz, ; fallon, ) . neutrophils are also known as granulocytes due to the characteristic release (degranulation) of primary azurophil [defensins, elastase, myeloperoxidase (mpo)] and secondary specific (lactoferrin, lysozyme) granule contents into the cytoplasm to fuse with the phagosome or the plasma membrane to create an antimicrobial milieu inside and outside of the cell. these granules do show varying readiness to mobilize in response to inflammatory signaling, with the azurophilic subset being the most difficult to mobilize (amulic et al., ) . the degradation of particles within the phagosome or extracellular space of infected or damaged tissue is aided further by the process of neutrophil oxidative burst involving the formation of reactive oxygen species (ros) through the nadph oxidase system (weiss, ; babior, ) . depending on the stimulus (see section . . . for relevant stimuli of this thesis), the nadph oxidase system may assemble on the membrane of the phagosome and/or the cell where the main ros produced are the superoxide anions which react to form other intermediates including hydrogen peroxide, hypochlorous acid, hydroxyl radical, and singlet oxygen (peake, ) . it has been suggested that components of azurophilic (e.g., mpo) and specific (e.g., flavocytochrome b ) granules may regulate the activity of the nadph oxidase (tal et al., ; amulic et al., ) , highlighting that intracellular signaling cascades triggered by attachments of ligands to surface receptors, may lead to coordinated degranulation and oxidative burst responses to invading pathogens (daniels et al., ; pyne, ) . it has also been demonstrated that neutrophils also possess antimicrobial capacity independent of phagocytosis known as neutrophil extracellular traps (nets) (mantovani et al., ) . although mechanisms of nets are not completely understood, it has been established that classical effector functions of neutrophils (degranulation and oxidative burst) play important roles in mediating the response (fuchs et al., ; patel et al., ; metzler et al., ; amulic et al., ) . nets are suggested to form upon an active form of cell death which results in the release of a network of nuclear filaments (dna and histone) into the extracellular space from the degrading neutrophil (brinkmann et al., ; fuchs et al., ) . on the basis of the exercise-induced changes in neutrophil counts it is not surprising that exercise also affects functional responses, but due to the variation in study design the early evidence was conflicting (peake, ) . nevertheless, it can be argued that when responses are controlled for on a per cell basis the effects of exercise duration (i.e., prolonged exercise) are clearer. neutrophils may present on a continuum of state of activation from dormant to primed through to being fully activated (smith, ) . during exercise, there is a release of agents into the circulation which may prime (e.g., induce assembly of nadph on membrane) or desensitize (internalization of receptors) the capacity of neutrophils for enhanced responsiveness to later stimulation or inhibit such functional responses respectively (pyne, ; peake, ; amulic et al., ) . the number of neutrophils engaging in phagocytic activity is increased following prolonged exercise but the phagocytic capacity of each neutrophil is decreased within the circulation and the nasal cavity (gabriel et al., ; müns, ; blannin et al. a; nieman et al., ; chinda et al., ) . albers et al. ( ) suggested that neutrophil phagocytosis may have low suitability as a marker of the immunomodulatory effects of exercise and assessing the killing capacity (degranulation and oxidative burst) may be more sensitive to reflect a susceptibility to infection. simultaneous with neutrophilia following prolonged exercise there is an increase in unstimulated degranulation and oxidative burst responses (e.g., measured by total plasma elastase, spontaneous ros production) (blannin et al., b; suzuki et al., ; bishop et al., ) . these findings provide support that prolonged exercise induces neutrophil activation (possibly due to muscle damage) that may result in the cells entering a "refractory period" in the recovery from prolonged exercise whereby there is a transient inability to respond to subsequent stimulation (e.g., in vitro) (peake, ) . numerous studies have found significant decreases in neutrophil degranulation and/or oxidative burst responses to in vitro stimulation by bacterial peptides (fmlp and lps) and/or synthetic stimuli (phorbol- -myristate- acetate, pma) during the recovery from prolonged ( . . h) exercise (chinda et al., ; suzuki et al., ; davison and gleeson, laing et al., ; davison and diment, ) . this decline in killing capacity has not been coupled to changes in cell surface receptors, suggesting that the modulatory effects of exercise on in vitro stimulants occur downstream in intracellular signal transduction pathways of neutrophils such as phosphorylation cascades or secondary messengers (e.g., cyclic adenosine monophosphate, calcium) (mooren et al., ; peake, ) . the magnitude of the effects of prolonged exercise on neutrophil effector functions will depend on the balance of immunosuppressive (e.g., "refractory period") and immunostimulating factors (e.g., priming agents) which largely depend on the extent of neutrophilia (peake, ) . as previously mentioned, prolonged exercise (i.e., elevations in stress hormones) induces a mobilization of immature neutrophils into the circulation which have been shown to exhibit reduced nadph oxidase activity and granular content (hetherington and quie, ; berkow and dodson, ) . the immuodepressive effects of cortisol and the subsequent release of subpopulations from the bone marrow was supported by robson et al. ( b) who found that the degree of exercise-induced decrease in stimulated degranulation per neutrophil and neutrophilia was greater following prolonged exercise (b h) compared to short, intense exercise (b min). although considered to be a major factor, activation of the hpa axis alone cannot wholly account for changes in the neutrophil function (laing et al., ) , as some of the noted inhibitory effects of cortisol on receptor mediated responses (e.g., fmlp) would not explain decreased responses to stimulants that activate neutrophils independent of surface receptors (e.g., pma) (o'flaherty et al., ; tomchek et al., ; peake, ) . thus other mechanisms have also been suggested to be involved in neutrophil dysfunction, where elevations in catecholamines, cyclic adenosine monophosphate, complement proteins (c a), direct cellular oxidative damage and growth hormone may occur with the inflammatory response to prolonged exercise and have been shown to interfere with calcium signaling or other intermediates of intracellular pathways (henson et al., ; hack et al., ; suzuki et al., ; thibault et al., ; tintinger et al., ; robson et al., ; laing et al., ) . the susceptibility of phagocytes to modulation by strenuous exertion are also demonstrated by the decreased expression of tlr on the cell surface of monocytes immediately and up to h following prolonged exercise (lancaster et al., b; oliveria and gleeson, ) which are not explained by exercise-induced changes in monocyte numbers alone (simpson et al., ) . the phagocytic function of monocytes has been shown to increase following prolonged exercise which may be related to the increased pro-inflammatory phenotype observed with exercise in this leukocyte (steppich et al., ; hong and mills, ) . once monocytes reach the tissues they will form mature macrophages, therefore the biological significance of changes in monocytes is unclear as it may not reflect exercise-induced changes in immunosurveillance within tissue (i.e., at sites of inflammation or infection) (simpson et al., ; walsh et al., b) . these macrophages along with other phagocytes (dendritic cells) are present in majority of body tissues where unlike the direct killing capacity of neutrophils, macrophages and dendritic cells mostly act as professional apc (beutler, ; iwazaki and medzhitov, ) . investigations within exercise stress models thus far have been limited to animal studies which are difficult to generalize to the human response, but do suggest dysfunction in the capacity of these cells to present antigens following prolonged exercise (davis et al., ; woods et al., ; ceddia and woods, ; ceddia et al., ; woods et al., ; murphy et al., ; liao et al., ; chiang et al., ) . nk cells are large granular cells that can sense structures of high-molecular weight glycoproteins expressed on virus-infected cells via prr on their cell surface. they form up to % of the lymphocytes within the body and are vital in defense against viral infection. however, compared to other peripheral lymphocytes (b and t cells, see section . . ), prior sensitisation is not required (cerwenka and lanier, ) . thus upon activation nk cells trigger apoptosis or lysis of a virusinfected cell by releasing granule contents such as the pore forming proteins perforin and cytolysin. the initial investigations into nk cell activity (nkca) showed that exercise-induced responses were largely mediated by the duration and intensity of the bout (gannon et al., ) . the nkca was demonstrated to mirror the increases in nk cells following moderate or exhaustive exercise (gannon et al., ; woods et al., ) . however, when the exercise bout is intense and prolonged, ncka on a per cell basis has been shown to be reduced for several hours (kappel et al., ; nieman et al., ; mcfarlin et al., ) . binding of microorganisms to prr (e.g., tlr) of innate immune cells not only triggers activation of innate parameters but also induces the generation of protein messengers (e.g., cytokines) and other signaling components to stimulate acquired immunity (takeda and akira, ) . however, compared to innate immunity, the acquired immune response to a pathogen is delayed due to a lag period for recognition by the vast range of antigen receptors within b and t lymphocyte populations, clonal selection/expansion of the pertinent lymphocytes and clonal elimination for tolerance of self to occur (kimbrell and beutler, ; medzhitov, ) . t and b cells form %À % and %À % of the circulating pool of lymphocytes respectively, where the t cell population mature in the thymus gland and the b cells in the bone marrow. t lymphocytes are subdivided further into cytotoxic (tc, cd ), helper (th, cd ), and regulatory (treg) where the treg are formed from a naive cd cell and modulate immune responses compared to the primary involvement in removal of pathogens by tc and th. the induction of a primary immune response to pathogens involves presentation of antigens to the cell surface receptor of t lymphocytes (t cell receptor) by dendritic cells (lanzavecchia and sallusto, ; mellman and steinman, ) , while all other apc (e.g., monocytes, macrophages) are involved in the initiation of secondary immune responses via memory t cells which have encountered the antigen previously (gallucci and matzinger, ) . t cells are able to recognize antigens via major histocompatibilty complex (mhc) class i and ii molecules on the cell surface of an apc (banchereau and steinman, ) . the number of antigens encountered by an individual will determine the proportion of naive (unactivated) or memory (activated) t cells circulating within the body. although structurally similar to the t cell receptor, the surface receptor of the b lymphocyte is a membraneanchored immunoglobulin (ig) (also termed antibody). upon interaction of antigen and receptor, the b cell undergoes clonal expansion to form both short-lived plasma effector cells and long-lived memory cells or internalize the bound antigen and act as an apc to t cells (lebien and tedder, ) . the short-lived plasma cells secrete igs into the blood to aid destruction of the pathogen while the memory cells with their greater affinity to the antigens can remain in the circulation throughout life to rapidly differentiate into plasma effector cells if the same antigen is encountered again (walsh et al., b) . the circulating igs fall into five major classes, igm, igg, iga, igd, and ige where each of these classes can be divided further into subclasses. this diverse repertoire of igs possess functional regions capable of binding to a vast range of antigen sites (epitopes) to form immune (antigenÀantibody) complexes that neutralize the toxicity of certain antigens or common ig regions which can activate phagocyte ingestion and soluble innate factors (e.g., complement) . following a lag period for accumulation of igs within the blood, the igm class predominates during any primary response to an antigen whereas igg predominates under normal resting conditions as well as in the rapid ig response to secondary antigen exposure (mckune et al., ) . therefore the acquired immune system consists of a cellular (i.e., t cells) and humoral component (i.e., ig). determining which component of the acquired immune system predominates, are the subpopulations of th cells (th and th ) and the cytokine profile they produce and release (walsh et al., b) . cytokines act as protein messengers between one immune cell and other, where the cell receiving the signal may proliferate, secrete additional cytokines, migrate to the area of origin of the signal, differentiate into another type of cell or die (undergo apoptosis) (curfs et al., ) . the main cytokine group responsible for leukocyte communication is the interleukin (il) family but other major cytokine groups are the colony stimulating factors (e.g., granulocyte macrophage colony stimulating factor, granulocyte-colony stimulating factor), tumor necrosis factors, and interferons (ifn) where their main roles are stimulation of cell growth, tumor cytoxicity, and inhibition of viral replication respectively (curfs et al., ) . the stimulation of th cells and production of cytokines such as il- , and ifn γ promotes cell-mediated immunity (tc responses) for defense against intracellular pathogens while activation of th cells (il- , il- , il- , and il- ) coordinates humoral immunity (b cells) and release of igs for defense against extracellular pathogens (seder, ) . it has been suggested that intense/prolonged exercise can modulate this balance by decreasing the proportion of th cells in circulation whereas th cells remain unaffected (steensberg et al., ; lancaster et al., lancaster et al., , a . this shift in immunity has been proposed to provide an explanation for potential increases in the susceptibility to uri following prolonged exercise given the importance of type responses towards viral infection (steensberg et al., ; fabbri et al., ) . such effects of exercise are suggested to be mediated via the suppression of type cytokine production by elevations in stress in hormones (adrenaline and cortisol) and the release of cytokines from contracting skeletal muscle (primarily il- ) that favor production of type cytokines (gleeson, ) . supporting evidence for cytokine modulations following intense exercise has been provided by decreases in il- and ifn and no changes in il- shown with in vitro mitogen-stimulated isolated cells and whole blood culture (tvede et al., ; moyna et al., ; smits et al., ; starkie et al., ) . these changes in cytokine production overlap with influences on other stages of t cell activation where decreases in mitogeninduced t cell proliferation have been observed following intense/prolonged exercise (fry et al., ; nieman et al., nieman et al., , henson et al., ; bishop et al., ) . despite these observations, the evidence suggesting that t cell activation is down-regulated by prolonged exercise is confounded by numerous methodological limitations (walsh et al., b) . t cell proliferation assays generally use a fixed amount of total lymphocytes or whole blood so changes following exercise may only reflect changes in the proportion of lymphocyte subsets due to the greater increase in nk cells post exercise which do not respond to mitogen (green and rowbottom, ) . depletion of nk cells from cell culture has been found to remove the significance of changes in mitogeninduced proliferation following exercise (green et al., ) . nieman et al. ( ) found that proliferative responses were substantially different based on adjustments for t cell populations, but there was still a significant fall in proliferation following intense exercise compared to preexercise. nevertheless, the validity and sensitivity of mitogen-induced culture to assess t cell function have been questioned due to their nonspecific effects (activating other cell types, e.g., b cells) and lack of ability to detect subtle changes in individual t cell populations (bishop et al., ) . demonstrated that ex vivo migration of cd and cd cells to a human-rhinovirus infected bronchial (lung) epithelial cell line was decreased following a prolonged exercise bout ( h of running). it is also difficult to extrapolate such observations on isolated cells from a circulating pool that is considerably lower than total lymphocyte mass within the complex in vivo environments at the skin, mucosa, and lymph nodes (gleeson, ) . although investigations on whole blood maintains the proximity of leukocytes and the extracellular milieu of leukocytes compared to leukocyte isolation, the use of in vivo measures to assess response to antigenic challenge may be more clinically relevant (albers et al., ; albers et al., ; walsh et al., b; bermon et al., ) . both cell-mediated and adaptive parameters contribute to the largest component of the immune system, mucosal immunity (total surface area of m ) (brandtzaeg et al., ) . the importance in host defense against pathogens becomes apparent when recognizing that the mucosal surfaces of the upper and lower respiratory tract account for b %À % of total immune protection by the body and the small intestine along with the colon are responsible for % of all igs produced (kudsk, ) . increases in illness and morbidity have been attributed to impairment in mucosal immunity (daele and zicot, ) , highlighting the importance that immune competence at mucosal surfaces has in the health and well-being of athletes (west et al., ) . although not functioning independently of the systemic immune system, mucosal immunity is also considered a distinct entity due to its autonomously regulated, localized defense mechanisms (toy and mayer, ) . the gut-associated lymphoid tissue, urogenital tracts, lacrimal glands, lactating mammary glands, and respiratory tracts which include the bronchus-associated lymphoid tissue (balt), salivary glands and nasalassociated lymphoid tissue are all mucosal surfaces which fall under the network of immune structures known as the common mucosal immune system (cmis) (gleeson and pyne, ) . the immunological protection provided by this network may be via organized tissue with wellformed follicles (muscosa-associated lymphoid tissue) such as pp of the small intestine or as a diffuse accumulation of leukocytes (lymphocytes, plasma cells, and phagocytes) as found in the lung and the lamina propria (connective tissue) of the small intestine (kyd and cripps, ) . these immune structures are pivotal for the monolayered epithelial layer of mucosal surfaces which is continually exposed to a wide array of antigens or allergens including pathogenic bacteria or viruses, gut microflora and ingested food (johansen et al., ) . indeed, the mucosae are considered to be the first line of defense as they are the sites where most pathogens enter the body (macpherson et al., ) . the cmis are differentiated into inductive and effector sites, where the induction sites (primarily pp) involve the sensitization of immune response following antigen presentation while the effector sites consist of interconnected distal sites (respiratory tract, lamina propria) where the array of activated b cells and plasma cells home and migrate to provide local protection (kyd and cripps, ; kudsk, ) . at least % of the body's plasma cells (activated b cells) reside in the mucosal effector tissues, with local production of igs representing a major immunological barrier at all mucosal surfaces and iga being the predominant antibody (brandtzaeg et al., ; . within the bloodstream, iga under most circumstances is found as a monomeric peptide (yel, ) . however, in all mucosal secretions iga exists as a dimeric protein covalently linked by a j chain containing another peptide termed the secretory component (gleeson and pyne, ) . the secretory component is the cleaved segment of the polymeric ig receptor (pigr) that is produced by the mucosal and glandular epithelial cells and expressed on the basolateral membrane (teeuw et al., ; ). the proteolytic cleavage of pigr occurs following its binding and induction of the active transport (exocytosis) of dimeric iga through epithelial cells on to the mucosal surface (strugnell and wijburg, ) . the remaining secretory component wrapped around the j chain-linked dimeric iga forms secretory iga (siga) which is considered to be resistant to proteases secreted at mucosal sites (e.g., intestinal mucosa) (underdown and dorrington, ; lindh, ; johansen et al., ; strugnell and wijburg, ) . it is this local production of siga that forms the major effector function of mucosal immunity . only in the neonate or situations of iga deficiency does igm represent a significant defense at mucosal surfaces with igg also being found in low quantities at mucosae (brandtzaeg et al., ; gleeson and pyne, ) . iga can be further divided into subclasses, where iga is the most abundant in the distal gastrointestinal tract ( %), whereas iga predominates in the salivary glands ( %À %) and nasal lymphoid tissue ( . %) (gleeson, ) . protection of mucosal surfaces via siga occurs through multiple mechanisms. one such mechanism known as immune exclusion involves the binding of antigens to regulate the commensal microorganisms (microbiota) and prevent the attachment and invasion of mucosal surfaces by pathogens (strugnell and wijburg, ; sutherland and fagarsan, ) . other mechanisms include the binding of antigens which have already crossed the mucosal barrier and actively transporting them back across the epithelial layer into the lumen or intracellular neutralisation of viruses when bound to pigr within the mucosal epithelia (lamm, ) . the presence of siga (formed by b cells adjacent to salivary ducts and glands) in saliva has tended to be the mucosal immune marker of choice due to the ease of collection (korsrud and brandtzaeg, ; gleeson, ; sari-sarraf et al., ; ). most of the salivary fluid itself is formed by three pairs of major salivary glands (parotid, submandibular, sublingual) but production is supplemented by a vast amount of small submucosal glands that lie on and around the tissue (e.g., palate, tongue) within the oral cavity (proctor and carpenter, ) . although saliva drains from the acini (cluster of cells) of each of these glands into the mouth via striated and excretory ducts, the nature of the secretion differs whereby a serous (watery) fluid is produced by the parotid, mucous fluid by the submandibular and sero-mucous mixture by the sublingual (aps and martens, ) . in unstimulated saliva secretion, the proportion of the fluid provided by parotid, submandibular, sublingual and the remaining submucosal glands are suggested to be %, %, %, and % respectively on average (dawes, ) . in addition to the aforementioned transcytosis of siga into the mucosal secretion, saliva benefits from the import of several antimicrobial peptides (amps) that contribute to the first line of defense by providing innate defences compared to the specific nature of iga (bals, ) . amps are categorized as being small cationic peptides (, amino acids) that represent inducible, constituent factors of mucosal secretions (west et al., ) . although numerous amps require some form of enzymatic modification prior to their functional configuration, they act in synergy with other components of the innate immune system to prevent and aid clearance of infections (wakabayashi et al., ; bowdish et al., ; de smet and contreras, ; ibrahim et al., ; radek and gallo, ) . several amps can modulate other immune processes such as leukocyte cytokine secretion, chemotaxis and remodeling of injured epithelia (ganz, ; bowdish et al., ; tjabringa et al., ) . the most abundant amps in the secretions of the urt are lysozyme and lactoferrin (singh et al., ) . lysozyme is a small protein ( kda) released into saliva by neutrophils, macrophages, and the submucosal glands that possesses bactericidal capacity through hydrolyzing the polysaccharide of bacterial cell walls (jolles and jolles, ; travis et al., ; bosch et al., ; west et al., ; fabian et al., ) . the antimicrobial properties of the smaller molecule lactoferrin ( kda) from neutrophils and submucosal glands are due to its ability to bind free iron, depriving bacteria of this nutrient that is essential for growth and multiplication (legrand et al., ; bowdish et al., ; ward et al., ) . although not an inhibitor of the main causative agent of urti (rhinovirus), lactoferrin is considered to be effective against other common respiratory viruses [adenovirus, respiratory syncytial virus (west et al., ) ]. there is a wide variety of other amps, most of which have been grouped into three main families; cathelicidins (e.g., ll ), defensins (α and β subfamilies), and histatins (bals, ) . these are primarily released into the oral cavity by the epithelial cells, salivary glands, and/or neutrophils (de smet and contreras, ; fabian et al., ) . the amps work synergistically in low concentrations to destabilize cell walls of microorganisms and provide a broad spectrum of activity against gram-positive and gram-negative bacteria (bals, ) . other salivary proteins that contribute an important line of defense via the inhibition of adherence and growth of specific bacteria (e.g., streptococcus) at the oral mucosa include α-amylase (scannapieco et al., ) . in addition to influx of invading microorganisms from the external environment, the human mouth has a constant microbial presence that needs to be regulated (bender et al., ) . surfaces of the oral cavity are bathed with saliva, whereby the fluid is recognized to provide a "fingerprint" of the vast range of the resident microorganisms (li and gleeson, ; boutaga et al., ; fabian et al., ; dewhirst et al., ) . reduced salivary flow rate can directly impact on the oral microbiome by inducing a shift towards colonization via pathogenic microorganisms (meurman, ) . indeed, the entire surface of the respiratory tract is a source of commensal microorganisms which, similar to exogenous antigens, possess the ability to become pathogenic in the host (watson et al., ; bosch et al., ) . amps not only play a crucial role in the protection against foreign pathogens, but also act as a synergic arsenal of molecules that regulate the response to commensal bacteria (boman, ; davison et al., ; jones et al., ) . therefore the importance of amps is twofold; preventing disruption of the epithelial layer by acting as a critical first line of defense (biofilm) against pathogens, and maintaining homeostasis of the commensal community that act to outcompete invading microorganisms (blaser and falkow, ; murphy et al., ; . the overlapping nature of these defenses is highlighted when reduction in expressions of amps in in vitro models lead to changes in bacterial colonization (bals et al., ; liu and modlin, ) . subsequently, perturbations in the balance of the microbiota on the mucosal surface can lead to overgrowth and further amplification of microbes which may have direct effects locally (e.g., oral infection, urti) or have indirect effects through predisposing to respiratory illness with the many established interactions between microorganisms (bacteriaÀbacteria, viralÀbacteria) (slots and genco, ; blaser and falkow, ; murphy et al., ; meurman, ; bosch et al., ) . such debilitating effects are likely to occur if the imbalance in microbiota occurred due to a lack of immune competence (e.g., decreased amps) (bosch et al., ) . the resistance of the microbiota within the urt to the immune perturbations associated with prolonged exercise is currently unclear. the most popular parameter of mucosal immunity that has been investigated in an acute exercise setting has been salivary siga due to the notion that individuals who suffer from iga deficiency contract urti regularly (gleeson and pyne, ) . physiological changes (e.g., nervous stimulation, dehydration) during exercise can influence the secretion of saliva and its protein components (walsh et al., ; ). the salivary glands are innervated by both the parasympathetic and sympathetic nervous system, with changes in stimulation of either of these having an influence on the volume, viscosity, protein and mucin concentration (aps and martens, ) . parasympathetic nervous stimulation via vasodilation of the salivary glands is believed to trigger a high volume of watery saliva, low in protein concentration ). on the other hand, sympathetic stimulation produces salivary secretions which are low in volume but high in protein that is primarily due to enhanced active transport of proteins from salivary cells (proctor and carpenter, ) . thus similar to the other discussed immune parameters, salivary siga concentration is susceptible to effects (sympathetic and parasympathetic responses) of exercise intensity and duration (walsh et al., b) . the following discussion will consider those studies which have used the most reproducible collection method, unstimulated whole saliva collection (also termed passive drool), due to the potential for stimulated saliva flow (e.g., chewing) and other collection methods (e.g., swabs) to preferentially induce secretion from certain glands and/ or influence saliva composition once secreted (navazesh and christensen, ; navazesh, ; proctor and carpenter, ; harmon et al., ; beltzer et al., ; granger et al., ; allgrove et al., ) . although there is inconsistency in study design within the literature to draw a definitive conclusion, walsh et al. ( b) suggested that siga concentration in saliva generally decreases (e.g., tomasi, et al., ; nieman et al., nieman et al., , palmer et al., ) or remains unchanged (e.g., mackinnon and hooper, ; sari-sarraf et al., ) following prolonged exercise ($ . h at %À % maximum oxygen uptake, _ vo max ). it seems that the combination of high-intensity and exercise of a long duration has the most significant impact (i.e., depressive) on salivary siga concentration (mackinnon, ; nieman et al., ) . the discrepancies in the literature are also believed to be partly due to the way in which salivary siga concentration is expressed relative to the exerciseinduced changes in physiological responses (walsh et al., ) . in an attempt to account for such changes, salivary siga concentration has been expressed as a secretion rate or relative to total salivary protein/albumin/osmolality (gleeson, ) , but this makes the comparison between studies difficult ). the secretion rate or expression relative to saliva osmolality are preferred over measures of siga as ratio to total protein (blannin et al., ) . one reason for this is that other salivary proteins (e.g., amylase) are known to increase with exercise without a change in siga (walsh et al., ) . furthermore, expression as a secretion rate may better reflect the amount of available siga on the mucosal surface (mackinnon et al., ) . in contrast, it has been argued that salivary siga concentration is of greater significance as secretion rate may only provide an explanation to how salivary flow rate has changed . having said that, salivary siga when expressed as a secretion rate has been found to be the best predictor of uri incidence in athletes following a km race or during intense training and competitive phases (fahlman and engels, ) . additionally, given that the majority of saliva is water, expression relative to secretion rate also accounts for the concentrating effect of other salivary components following any dehydration (bishop et al., ; oliver et al., ) . as saliva osmolality reflects the inorganic electrolyte concentration (rather than protein content) and hence falls in proportion with decreases in flow rate, siga:osmolality provides an alternative method (blannin et al., ; . although iga is the dominant ig in mucosal secretions, some have also attempted to identify changes in salivary igg and igm concentrations following exercise. limited findings suggest igg remains unchanged but igm parallels decreases in salivary siga, highlighting the potential effects that acute strenuous exercise has on salivary immune parameters (gleeson and pyne, ; ). another aspect of mucosal immunity which has received little attention to date is the responses of amps to exercise (walsh et al., b) . despite the interest of the dentistry field in the role of amps in oral health and infection, (putsep et al., ; tanida et al., ; tao et al., ; dale et al., ) , the relationship of amps with exercise-induced immune dysfunction is yet to be explored conclusively (davison et al., ) . to date, the few investigations that have been conducted suggest in accordance with other immunological measures, responses of amps may be dependent on the intensity and duration of the exercise bout (hoffman-goetz and . a h cycling bout at b % _ vo max resulted in a significant decrease in salivary lysozyme (slys) concentration, slys secretion rate and slys:osmolality which recovered after h of rest (davison and diment, ) . in contrast, expressions of other amps (ll and defensins: human neutrophil peptide À ) following similar stressors ( . h at b % _ vo max ) have been shown to significantly increased immediately post exercise. in the recovery period ( À . h post) following a km mountain trail race (mean running time b h) there was a significant decrease in salivary lactoferrin (slac) concentration and nonsignificant decreases in slac and slys secretion rate (gillum et al., ) . the mechanism behind the effects of prolonged exercise on salivary parameters (amps, siga) remains unclear (walsh et al., b) . the flow rate of saliva is considered to be the major source of variation in concentration of mucosal parameters (gleeson and pyne, ) . in general, saliva flow rate decreases in response to a prolonged exercise bout (walsh et al., ; bishop et al., ) . decreases have been attributed to a withdrawal of parasympathetic stimulation rather than sympatheticinduced vasoconstriction of salivary glands . parasympathetic withdrawal associated with sensations of a dry mouth in response to other acute stressors (e.g., psychological) supports such proposals as does the lack of effect on saliva flow rate with interventions aimed to increase sympathetic stimulation (bosch et al., ; bishop et al., ) . a crucial determinant of siga release into saliva is the presence of pigr to permit transport across the epithelial layer (bosch et al., ) . evidence from animal studies suggests that increased mobilization of pigr occurs only above a certain threshold of increased sympathetic stimulation (proctor et al., ) . this may explain why a brief bout of high-intensity exercise leads to increases in salivary siga (e.g., davison, ) . however, this does not explain the decrease found with prolonged exercise bouts ). it has been speculated that this nervous stimulation over a longer period (i.e., prolonged exercise) may deplete the available iga (proctor et al., ; allgrove et al., ) or there may be a further threshold or interaction with duration where pigr mobilization is down-regulated (walsh et al., b) . as brief bouts of maximal effort exercise result in increased slac and slys, it is reasonable to suggest that mobilization of amps during exercise is also influenced by sympathetic stimulation in relation to certain thresholds (allgrove et al., ; west et al., ; usui et al., ) . however, the reductions in slys following prolonged exercise may rather be related to an increased stimulation of the hpa axis and hence increases in salivary cortisol concentration, which have been associated (note, not cause, and effect) with reduced concentration of slys (perera et al., ; west et al., ) . additionally, it is worthy to note that the source of slac and slys (phagocytes, epithelial cells) is different to siga. therefore it is possible that increases in amps during high-intensity exercise may be as a result of the effects of hyperventilation (e.g., drying of the mucosal surfaces) given that some of these changes are lost or reduced when expressed relative to osmolality or as a secretion rate (davison, ) . additionally, the exercise-induced damage to the epithelial layer could induce an inflammatory response whereby epithelial cells increase release of amps and other sources of amps are recruited (e.g., neutrophils) (west et al., ) . the relative contribution of amps from each of these sources to saliva levels at both rest and following exercise is unclear. however, it is reasonable to suggest that an airway inflammatory response partly accounts for the increases in amps identified immediately following prolonged exercise (davison et al., ) . certain amps (α defensins) have been suggested to make up % of the protein found in neutrophil azurophilic granules (radek and gallo, ) . müns ( ) demonstrated a significant (twofold) increase in the neutrophil count of mucosal secretions (nasal lavage fluid) following a prolonged exercise bout. although neutrophils continually migrate into saliva from the circulation via gingival crevices (lukac et al., ; bender et al., ) , the extent of neutrophila that occurs in the circulation following exercise may lead to the presence of neutrophils and their contents in saliva (e.g., amps) being substantially increased (davison et al., ) . increased levels of amps (e.g., α defensins) have been observed in other body fluids (e.g., plasma) following a circulating neutrophila (shiomi et al., ) . the variation in the responses of different amps to prolonged exercise (davison et al., ; davison and diment, ) , therefore, may be due to the changes in the maturity of circulating neutrophils (as expressions of amps can vary throughout the maturation of the neutrophil in the bone marrow) or decreases in some amps may merely reflect neutrophils undergoing a refractory period post exercise (cowland et al., ; borregard and cowland, ; gullberg et al., ; nagaoka et al., ; sorensen et al., ; nagaoka et al., nagaoka et al., , peake, ) . based on the evidence presented to date regarding acute prolonged exercise, it may be expected that the highly trained athletes demonstrate lower immune function at rest compared to nonexercising controls. however, numerous cross-sectional studies have attempted to discern such differences and found that when measures of immune function are gathered in a "resting state" (at least h following the previous bout) there seems to be very little difference between athletes and controls (gleeson, ) . a review of early investigations in the area (nieman, ) suggested even when significant immune perturbations had been observed in athletes participating in strenuous exercise, investigators had limited success in identifying such measures that influence alteration in rates of uri (nieman et al., b; mackinnon et al., ; nieman et al., nieman et al., , . this actually adds further support to the open window theory, whereby these periods of depressed immunity are transient. however, for athletes who train many times per week, they will experience multiple open windows and the total time of increased susceptibility will be greater, meaning a higher overall risk of contracting an illness. despite the large inter-individual variation in concentration of salivary siga, (walsh et al., b) some of the early work (salivary siga responses to training) reported lower concentrations in endurance athletes compared to sedentary counterparts (tomasi et al., ) . in contrast, most of the subsequent investigations support evidence of other parameters by showing salivary siga to be broadly similar in the two populations (gleeson and pyne, ; ). conversely, findings in elite rowers had %À % lower slac concentration than controls at the start and mid-way through a month training period (west et al., ) , suggesting investigations of the mucosal immune compartment in a "resting state" between these populations requires further attention. inverse relationships between salivary siga concentration/secretion and uri incidence in some studies of athletes undergoing periods of intensive training (i.e., athletes not necessarily in "resting state") (fahlman and engels, ; neville et al., ) further supports the need for longitudinal research on mucosal immunity with large athletic populations (nieman, ) . the common observations of this mucosal parameter within intensive training studies represents a narrow range that have established a link between an immune measure and uri in athletes (gleeson, ; walsh et al., b) . given the complexity of the immune system, it is unlikely that salivary siga alone would explain uri risk for all athletes, thus other factors in combination with the immune measure need to be determined (nieman and bishop, ) . investigators have begun attempts to address this by examining differences between illness prone and healthy athletes with early evidence suggesting that the blood obtained from the illness prone athletes has greater ex vivo production of antiinflammatory cytokines (e.g., il- and il- ) in response to multiantigen challenge, indicative of a downregulation of cell-mediated immunity gleeson and walsh ; gleeson and bishop, ) . multifactorial mechanisms beyond salivary siga alone in uri incidence become more apparent when athletes intensify their training over a short period of time (gleeson, ) . indeed, À weeks of intensified training has been shown to induce marked reductions in neutrophil and monocyte function, lymphocyte proliferation and the circulating number of t cells (verde et al., ; robson et al., a; lancaster et al., lancaster et al., , . in addition to intensive experimental protocols, longitudinal monitoring over a competitive seasons showed that team sports (rugby) and endurance athletes (cyclists) are sensitive to the intensive training periods with decreases in t cell counts, slys, neutrophil oxidative burst, and il- production (baj et al., ; cunniffe et al., ) . indeed, rapid changes (increases) in training load/stimulus may be the key risk factor (more so than total training load) for uri occurrence in athletes (e.g., svendsen et al., ) . these findings suggest that there is a cumulative effect of repeated bouts of strenuous exercise due to an inadequate recovery time for the immune system (papacosta and gleeson, ) . furthermore, it may be that responses to acute prolonged exercise are more relevant than resting immunity in athletes where the immediate recovery period represents the "open window" when athletes are most vulnerable to infection (nieman et al., b (nieman et al., , pedersen and bruunsgaard, ; abbasi et al., ) , since the chronic effects represent the accumulation of each acute response. despite the lack of clear difference in measures at rest, as mentioned earlier, as athletes are exposed to more frequent acute bouts ("open windows") of immunodepression, the overall risk may be considered greater than nonexercising controls and each acute response dictates the size and duration of each individual "open window." the presence of any other risk factors (e.g., dietary deficiency, sleep disturbance, psychological stress) will have additive effects on the transient immune responses and increase the risk of illness following prolonged exercise as seen in many studies (gleeson, ) . although underlying causes of uri remain uncertain, decrements in performance as a result of uri have been reported (reid et al., ; pyne et al., ) . the monitoring of the risk status of an athlete is paramount in order to determine the appropriate preventive or therapeutic interventions required during stages of strenuous training/ competition. regular moderate activity in both sedentary and active populations is considered to promote an antiinflammatory environment. this is an important underlying mechanism in the protection against chronic inflammatory conditions gained from physical activity in older populations (e.g., cardiovascular disease, type diabetes, obesity) (walsh et al., a) . despite moderate activity induced decreases in resting concentrations of inflammatory mediators (e.g., acute phase reactants, crp) (mcfarlin et al., ) , evidence of an influence of regular moderate activity on resting leukocyte function remains equivocal however. it is likely that additional mechanisms are also responsible for the benefits of physical activity on overall immunity and infection risk. moderate activity (cycling) of h duration has been shown to increase the capacity of blood neutrophils to respond to both receptor independent and dependent in vitro stimulation (smith et al., ) . this exercise-induced priming of neutrophil microbiocidal capacity has been suggested to be due to an increased presence of immunostimulating factors in the circulation and a greater proportion of responsive cells. dhabar ( ) suggests that the immunopotentiation from moderate exercise is due to the bidirectional effect of stress hormones on immunity where subtle elevations are beneficial whereas significant and sustained elevations (as seen with prolonged and/or intensive exertion) are detrimental to the host. it is these short-lived changes in cell-mediated immunity that occur during and shortly after each acute moderate exercise bout itself that are proposed to contribute to the lower risk of urti (nieman et al., ) . to further investigate the effect of moderate activity on cell-mediated immunity, animal and human experimental studies have monitored the production of cytokines by t lymphocytes. it is well established that intracellular pathogens trigger a (type ) cell-mediated immune response resulting in the differentiation of naive cd and cd t cells into t helper and t cytotoxic type lymphocytes (th /tc ) characterized by a phenotype of interferon (ifn)-γ and il- production (seder, ) . in contrast, extracellular pathogens, induce a humoral (type ) immune response resulting in the differentiation of naive cd and cd t cells into th / tc lymphocytes characterized by a phenotype of il- , il- , il- , and il- production (seder, ) . type and type phenotypes are mutually inhibitory, whereby up-regulation or downregulation of either leads to an imbalance in immune responses (zhao et al., ) . for example, it has been suggested that moderate activity can induce heightened type responses and, given the importance of type responses towards viral infection (steensberg et al., ; fabbri et al., ) , contribute to decreased urti risk. baum et al. ( ) showed that an acute bout of moderate cycling caused increases in ifn-γ production by peripheral blood lymphocytes of young adults where concentrations remained elevated compared to a control group h post exercise. longitudinal studies ( . À years) of moderate activity (cycling or walking for min each day) have to date only been investigated in the older population where an age-associated decline in type lymphocytes and cytokines have been reversed (ogawa et al., ; shimizu et al., ) . although a type response is critical for immmunosurveillance and early viral clearance, a sustained or excessive shift towards this phenotype has been implicated in tissue damage within lung pathology (van reeth, ) . martin et al. ( ) have hypothesized, albeit from animal models, that moderate activity also potentiates immunoregulatory mechanisms to skew towards a th phenotype and antiinflammatory environment during responses to infection. further research is warranted to determine the role of type /type balance and cell-derived cytokines towards infection risk. longitudinal interventions (b weeks) also suggest that aerobic activities of a moderate intensity mediate the resting levels of other immune parameters in the circulatory and mucosal (saliva) compartments. this may include increasing ("restoring") plasma ig or preventing further age-related decline in salivary concentrations (secretory iga, siga) in the elderly or increasing levels (e.g., siga secretion rate) in the younger adult population klentrou et al., ; akimoto et al., ; martins et al., ; sloan et al, ) . siga of the mucosal immune system expressed as a saliva secretion rate has previously been found to be the best predictor of urti incidence (risk) during exposure to intense, prolonged exertion . as a reduction in incidence of urti symptoms during weeks of moderate activity has been associated with an increase in siga (klentrou et al., ) , it is likely that this parameter plays an important role in the differential doseÀresponse relationship of physical activity and infection risk. the underlying mechanisms of these changes are unclear but evidence from animal studies suggest that moderate activity may mediate an up-regulation of siga production by stimulating the expression of cytokines involved in the synthesis of the ig (drago-serano et al., ) . based on the available evidence, regular aerobic moderate activity should be promoted as a preventive strategy against urti due to a combination of short-term acute changes after each bout and a summation effect over a longer period, with the exact temporal pattern being dependent on the investigated immune parameter. resistance exercise has been studied less comprehensively in the context of its effects on immunity. the majority or research in this area has focused on the postexercise changes in leukocyte counts (freidenrick and volek, ) . following an acute bout of resistance exercise, nk cells, monocytes and neutrophils increase in the circulation whereby the number of some of these subsets (monocytes, neutrophils) remain elevated for up to h post exercise (ramel et al., ) . the duration and magnitude of the postexercise leukocytosis is diminished in the older population as a consequence of age-related decline in leukocytes (freidenrick and volek, ) . differentiation of monocytes into macrophages and their infiltration into muscle tissue with neutrophils is essential for the regeneration and repair of muscle tissue after resistance exercise induced damage (arnold et al., ; tidball et al., ) . this leukocyte redistribution has been shown to be dependent on individual components of the resistance exercise (e.g., rest length, training load) (mayhew et al., ; carlson et al., ) . the extent of this damage depends largely on the eccentric component of the exercise bout (sorichtere et al., ) . the infiltration of immune cells and release of chemokines and other inflammatory mediators associated with this muscle damage creates a localized pro-inflammatory environment (edwards et al., ) . although focus of investigations with resistance exercise has not been on changes in infection risk per se, this heightened inflammatory environment has been proposed to be an effective adjuvant that may enhance responses to vaccination as discussed in the vaccination section below. as discussed earlier, in vitro and ex vivo markers can help understanding of how the immune system responds to exercise but the most valuable (clinically relevant) measures are those which assess the whole integrated immune response (albers et al., (albers et al., , . in the exercise immunology literature, such measures include challenge-type tests [such as delayed type hypersensitivity (dth) or contact hypersensitivity (chs) tests], response to vaccination, and possibly the reactivation of latent viruses (as discussed earlier). dth is a complex local immunological reaction to an intracutaneous antigen (following previous encounter/sensitisation) that is primarily t-cell-mediated but involves interaction of various cell types and chemical mediators of the immune system to induce a cell-mediated immune response (albers et al., ) . although the antigen presentation to t cells within a dth reaction is similar to the response to intracellular pathogens (kaufmann, ) , it can be considered an amplified reflection of the stages that occur with a "conventional" antigen which allows for the cell-mediated immune response to be noninvasively quantified (kobayashi et al., ) . the dth reaction is characterized by an infiltration of predominantly peripheral blood mononuclear cells [t cells (antigen-specific) and monocytes/macrophages] which release cytokines and other inflammatory mediators that present as eczematous plaques at the site (i.e., skin) (bruunsgaard et al., ; schwarz, ; kaplan et al., ; malaijan and belsito, ) . such epidermal indurations (edema and erythema) that peak in the À h following antigen reexposure can then provide the means by which cell-mediated immune response (the outcome) can be measured (albers et al., ) . bruunsgaard et al. ( ) showed that triathletes who completed prolonged exercise prior to intradermal injections of seven previously encountered antigens had significantly reduced skin responses compared to resting controls, indicating immunodepression. this method, however, is limited to the recall (elicitation) of preexisting immunological memory and does not provide any evidence on the effects of prolonged exercise on the primary immune (induction) response to a novel antigen (harper-smith et al., ) . contact sensitisation which involves topical skin exposure to novel synthetic chemicals provides an experimental model where both the elicitation and induction of t-cell-mediated responses can be measured (palmer and friedmann, ; albers et al., ; friedmann, ) . harper -smith et al. ( ) found that participating in prolonged exercise ( h of running) compared with seated rest prior to cutaneous sensitisation with a novel antigen (diphenylcyclopropenone, dpcp) reduced the recall (elicitation) of the immune response (skin erythema and edema) to that same antigen four weeks later. when participants completed the same prolonged exercise immediately prior to the elicitation phase (recall at four weeks), there was also a reduction in skin responses to dpcp compared to resting controls. however, the magnitude of reduction in response to dpcp at this phase (elicitation, %) was considerably lower than the reduction observed when prolonged exercise was completed prior to dpcp sensitisation (induction, %). this suggests that the primary immune response to antigens (apc and induction of t cell specific memory) is more susceptible to the perturbations induced by prolonged exercise than responses to recall (previously exposed) antigens, which may have relevance regarding the susceptibility of athletes to new/ novel compared to nonnovel pathogens. a number of follow-up studies using the chs model have since demonstrated that this is a controllable, reproducible, sensitive, and valid in vivo marker of exercise-induced immunodepression (diment et al., ; davison et al., ; jones et al., ) . the study by diment et al. ( ) also demonstrated no effect of the same exercise ( h of treadmill running) on the skin's response to the irritant croton oil, which provided strong evidence that the observed decrease in in vivo immunity assessed by this chs method of immune induction to dpcp is an antigen-specific, t-cell-mediated, response. simultaneously to the aforementioned assessment of cell-mediated immunity, bruunsgaard et al. ( ) also vaccinated all groups with previously encountered antigens that would stimulate b cell function. there were no differences between the prolonged exercise and control group in the ability of b cells to generate antibody responses to these recall antigens in the days following vaccination. this may provide further supporting evidence to proposals that primary immune responses are more susceptible to the effects of exercise or it may suggest that perturbations are more pronounced in the immediate period following prolonged exercise ( À h, i.e., proposed open window). the response of circulating igs to prolonged exercise has not been extensively researched with the studies that have investigated the predominant igs in blood (a, g, m) reporting conflicted findings (decreases, increases, and no change) peters et al., ; walsh et al., b; bishop, ) . this may not be surprising given the small proportions of b cells ( %À %) in the circulating lymphocyte pool and little redistribution of this subset following exercise. for the investigation of activated b cells and ig responses to exercise, studies have focused primarily on other immune compartments which may have greater relevance to urti (i.e., mucosa). the role of moderate exercise on in vivo immune markers has received relatively little attention in the literature. the majority of relevant research has studied the response to vaccination. however, most of these studies were designed to assess the utility of exercise as a vaccine adjuvant (e.g., in individuals who are less responsive to vaccination such as the elderly or those with certain chronic diseases) rather than exploring the effects of exercise on in vivo immunity per se. such studies can provide some insight into the benefits of exercise to immunity but the extent to which this can be translated to other populations requires much further study. long-term physical activity in such populations (pre and post vaccination) has been shown to improve antibody responses and immunogenicity to influenza, pneumococcus and meningococus vaccination (kohut et al., keylock et al., ; yang et al., ; grant et al., ; woods et al., ; bachi et al, ) , which support the beneficial effects of moderate exercise on in vivo immunity. however, there are a number of potential confounding factors (such as social interaction and support, psychological factors etc, from the exercise interventions) that may contribute to the benefit but are difficult to control in such studies. the acute effects of various types of exercise has also been investigated in a number of studies (edwards et al., (edwards et al., , campbell et al., ; edwards et al., edwards et al., , long et al., ; ranadive et al., ) . generally, these studies have been conducted with younger rather than older subjects and in most of these cases no benefits are observed. this could be because younger populations generally mount a strong (optimal) response to vaccination anyway, leaving little scope for benefit. as such, in this context, response to vaccination may not be the best model of in vivo immune response since it does not completely mimic a real infective challenge (i.e., as the virus is usually not live, or is attenuated). nevertheless, more work is needed in this area and other in vivo markers are desirable. athletes and individuals involved in heavy training programmes and/or prolonged bouts of exercise appear to have an increased risk of contracting uri. this is likely related to regular acute (and possibly chronic) periods of exercise-induced immunodepression (open windows). regular moderate exercise, on the other hand, appears to have the opposite effect and reduces infection risk. although in vitro and ex vivo measures and markers can contribute mechanistic information to the understanding of these relationships, they should not be considered as a substitute for clinically relevant/in vivo immune markers as most in vitro/ex vivo markers do not predict infection risk 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on innate mucosal immunity sites of rhinovirus recovery after point inoculation of the upper airway effects of exercise on the macrophage mhc ii response to inflammation effects of maximal exercise on natural killer nk cell cytotoxicity and responsiveness to interferon-alpha in the young and old exercise and cellular innate immune function special feature for the olympics: effects of exercise on the immune system: exerciseinduced modulation of macrophage function cardiovascular exercise training extends influenza vaccine seroprotection in sedentary older adults: the immune function intervention trial virus activation and immune function during intense training in rugby football players effects of a taiji and qigong intervention on the antibody response to influenza vaccine in older adults effects of moderate and high intensity exercise on t /t balance key: cord- -u ohm p authors: liu, xiaonan; zhang, chao; zhao, mengye; liu, kewu; li, hang; li, ningning; gao, linlin; yang, xuemin; ma, ting; zhu, juanli; hui, wenli; hua, kai; cui, yali title: a direct isothermal amplification system adapted for rapid snp genotyping of multifarious sample types date: - - journal: biosens bioelectron doi: . /j.bios. . . sha: doc_id: cord_uid: u ohm p genotyping of single nucleotide polymorphisms (snps) in point-of-care (poc) settings could be further improved through simplifying the treatment of samples. in this study, we devised an accurate, rapid and easy-to-use snp detection system based on direct loop-mediated isothermal amplification (lamp) without dna extraction, known as direct-lamp. samples from various sources (including whole blood, dried blood spot, buccal swab and saliva), treated with naoh, can be used directly in amplification. the turnaround time was about min from sample collection to provision of results. the accuracy was evaluated by assessing the polymorphisms of methylenetetrahydrofolate reductase (mthfr) c t and aldehyde dehydrogenase- (aldh ) glu lys, which are better known for their critical role in folate and ethanol metabolism, respectively. completely consistent genotyping results reveal that direct-lamp is generally concordant with sequencing. this system can serve as a very promising platform in the fields of disease predisposition, drug metabolism and personalized medicine. the most abundant source of genetic variation (approximately %) in the human genome is represented by single nucleotide polymorphisms (snps), which can account for heritable inter-individual differences in complex phenotypes (mhlanga and malmberg, ; yu et al., ) . hence, snp detection has great potential for direct clinical application by providing highly accurate diagnostic information for facilitating early diagnosis, prevention, and treatment of genetic diseases (everitt et al., ; moore et al., ) . because of its accuracy, dna sequencing is considered to be the gold standard for snp detection analysis. however, given the subsequent massive data analysis required by the method, sequencing is more suitable for discovery of novel snps rather than detection of specific known snps (grant et al., ; hendre et al., ; muhammad et al., ) . other technologies rely on sample amplification, including dna microarrays michal et al., ; zhang et al., ) , real-time polymerase chain reaction (rt-pcr) (martinez-serra et al., ; psifidi et al., ) , selfsustained sequence replication ( sr) (ren et al., ) , strand displacement amplification (sda) (toley et al., ) , high resolution melting (hrm) (norambuena and copeland, ) , mass array (kriegsmann et al., ; suthandiram et al., ) , and multiplex pcr-rflp method (loo et al., ) also offer sensitive approaches for snp detection. however, the tremendous potential of snp detection in clinical practice has so far often been limited by the lack of efficient screening methods which cannot meet the requirements of point-of-care (poc) testing strategies due to their complex experimental procedures and long testing times (tost and gut, ) . based on the status quo, development of a new detection platform that shortens assay times and simplifies procedures would be highly desirable. saving time from dna purification, a conventional step in snp detection that not only complicates the procedure, but also enhances the risk of invalid results and cross-contamination, is a promising approach. it has been reported that new snp detection methods have been developed based on pcr that do not require dna extraction from whole blood, hair root and saliva (cascella et al., ; hallie and reena, ; hayashida et al., ) . by treating them with specific chemicals, samples can be used directly in pcr without an initial dna purification step, which would save approximately min compared with conventional snp detection systems. however, pcr-based detection methods still require long processing times due to thermal cycling. in order to shorten the testing time further, it would be interested to adopt another simple and fast detection method, instead of pcr. loop-mediated isothermal amplification (lamp) is a highly specific, sensitive, and rapid gene amplification method that was established by notomi et al. (notomi et al., ) . this method amplifies nucleic acids under isothermal conditions and employs self-recurring strand-displacement synthesis primed by a specially designed set of target-specific primers (duan et al., ; tomita et al., ) . lamp has been used for detection of pathogens via the amplification of gene segments, including japanese encephalitis virus infection (mori et al., ) , rapid genotyping of carcinogenic human papillomavirus and herpesvirus (mori et al., ) , and the detection of middle east respiratory syndrome coronavirus (bhadra et al., ; mori et al., ; notomi et al., ) , demonstrating the method's great potential in molecular diagnostics. based on the advantages of this technology, lamp has been used for genotyping with buccal swab samples, combining gold nanoparticles functionalized with ssdna and colorimetric turbidimetry detection (carlos et al., ) . although it no longer requires dna purification, this method still takes h to perform due to its complex experimental procedure. methylenetetrahydrofolate reductase (mthfr) is the key enzyme in folate metabolism. mutations in the mthfr gene would influence the activity of this enzyme and increase the risk of various diseases, such as cancer, neurological disorders, cardiovascular diseases, and pregnancy complications (nazik et al., ) . aldehyde dehydrogenase- (aldh ) is responsible for the oxidation of aldehydes in the liver. differences in aldh expression contribute to a wide variety of human diseases, including cardiovascular disease, diabetes, and cancer. in addition, genetic polymorphisms of aldh alter susceptibility to ethanol intake, as well as the risk of alcoholism and alcoholic complications, and aldh may possess important therapeutic potential against alcoholism and other forms of myocardial damage (chen et al., ) . in this study, we devised a direct-lamp procedure, amplifying nucleic acids with various samples (including whole blood, dried blood spot, buccal swab and saliva) without dna purification, which is essential for conventional nucleic acid detection methods. the turnaround time was about min from sample collection to provision of results. mthfr c t, and aldh glu lys were utilized as models for snp detection with the direct-lamp method. accuracy was evaluated against clinical samples, and complete consistency between genotyping results reveals that this direct lamp procedure generally concords with sequencing. the proof of concept scheme described in this paper uses direct-lamp for snp detection. as exhibited in fig. a , samples are treated with naoh solution, followed by amplification under isothermal conditions using specifically designed primers. for convenience, we refer to the wild type as "wt" and the mutation type as "m". for each sample to be detected, two separate reactions (m tube and wt tube) are run simultaneously using the same sample. two sets of allele-specific primers (wt sequence and m sequence) are added to two different tubes for amplification, respectively. the result can be obtained automatically through a real-time fluorometer. the operating procedures of the direct-lamp only take min from sample collection to result. based on point mutations in the mthfr c t and aldh glu lys genes, four specific primers were designed to discriminate each snp. as shown in fig. b , the direct-lamp procedure relies on the specific primers, with forward inner primer (fip) and backward inner primer (bip) that are designed to contain a snp nucleotide at the ′ terminus. each reaction includes two common primers (f and b ) and two special primers (fip-wt and bip-wt for wild type allele or fip-m and bip-m for mutation type allele). the fluorescence signal coming along with target fragment amplification would be read in real-time only when the ′-end of the specific primer is complementary with the template. the structures of the specific primers used in this study are presented in fig. b . the target snp was characterized by using four different primer regions specifically designed to recognize six distinct regions on the target gene, which were selected to ensure that the primers would specifically amplify the c t and glu lys substitutions. the result depicted in fig. c illustrates that the direct-lamp could accurately detect all possible homozygotes and heterozygotes of mthfr c t with whole blood sample. for mthfr c t and aldh glu lys polymorphism genotyping, respectively, two sets of primers for direct-lamp were designed using the primer . software program (primer-e ltd., plymouth, uk), including two outer primers (f and b ) and four inner primers (fip-wt, fip-m, bip-wt and bip-m) that recognize six distinct regions of each target gene. two primers were also designed for sequencing. the sequences of the primers are shown in supplementary table s . all primers were synthesized by invitrogen biotechnology ltd. (shanghai, china). matched fresh human whole blood and dried blood spot samples were collected from unrelated chinese volunteers at the shaanxi provincial people's hospital (xi'an, china) with informed consent. whole blood sample was collected using edta-coated tubes. dried blood spot sample was prepared by dropping the peripheral blood on the filter paper and drying in the air. matched saliva and buccal swab samples were obtained from chinese volunteers at northwest university (xi'an, china) with informed consent. saliva samples were collected in eppendorf tubes. buccal swab sample was collected by swabbing the insides of cheeks times. the study was approved by the ethics committee of the college of life sciences, northwest university (xi'an, china). peripheral blood and saliva samples were collected in edta-coated tubes and eppendorf tubes, respectively, followed by mixing of the samples with mm naoh in a : ratio, with a final volume of μl, and incubation at room temperature for min. in total, μl of the mixture was taken for subsequent amplification. the buccal swab head was cut off (about mm under the head) and placed into μl of mm naoh, followed by incubation at room temperature for min, and μl of the mixture was taken for subsequent amplification. dried blood spot of mm diameter was put into an eppendorf tube and mixed with μl of mm naoh, followed by incubation at room temperature for min, and μl of the mixture was taken for subsequent amplification. the mixture must be used immediately after preparation. samples treated with naoh were observed using an optical microscope (ix , olympus optical co., ltd., tokyo, japan) with w halogen light source (u-lh l- , olympus); cells to be observed were stained with rapid wright-giemsa staining solution kit (sangon biotech co., ltd., shanghai, china). the initial conditions of the direct-lamp procedure were adopted as described by zhang et al. ( ) . the direct-lamp was performed with different combinations of primer concentration ( . μm fip/bip with . μm f /b , . μm fip/bip with . μm f /b , . μm fip/ bip with . μm f /b and . μm fip/bip with . μm f /b ) to determine the best primer concentration. the best reaction temperature was also investigated by running the direct-lamp at , , , , or °c for min. the four sample types were used to establish the direct-lamp, and the genotypes of the used samples had been sequenced. the reaction mixture contained μl of isothermal master mix (optigene ltd., uk) (including geobacillus dna polymerase, thermostable inorganic pyrophosphatase, optimized buffer including mgcl , dntps and ds-dna dye), μl of primer mix consisting of four primers each for f and b primers at . μm, fip and bip primers at . μm, μl sample treated solution, and ddh o up to μl. the reaction was performed in -well . -ml tubes, with incubation at °c for min. the fluorescence intensity of the ds-dna dye was simultaneously monitored in a realtime fluorometer (genie ii from optigene ltd. uk). the detection limit of optimized direct-lamp was evaluated by a serial dilution of the target concentrations ( %, %, %, . %, . %, . %, . %) of whole blood sample and saliva sample with two different genotypes (wild type and homozygous mutation, confirmed by sequencing) of mthfr c t and aldh glu lys. the four sample types with two different genotypes (wild type and homozygous mutation which confirmed by sequencing) of mthfr c t and aldh glu lys, respectively, were used to evaluate the specificity of the detection system. the accuracy of the optimized direct-lamp was further verified using clinical samples, which had been obtained with informed consent. the study was approved by the ethics committee of the college of life sciences, northwest university (xi'an, china). the genotype of each sample was analyzed by direct-lamp and compared with the results obtained via sequencing by bgi (beijing genomic institute, beijing, china). based on the statistical data, the coincidence rate and total agreements of three genotypes of mthfr c t and aldh glu gly were calculated to evaluate the accuracy of our method. in the current study, samples were treated with naoh to induce cells lysis and dna release, saving time ordinarily spent on extraction of dna from sample. to evaluate the effect of naoh on the performance of direct-lamp, the morphologies of leukocytes and oral epithelial cells in whole blood and saliva were observed, and the ph value at each step of direct-lamp process was assessed. in fig. a , a great number of cellular debris were found with the whole blood treated with naoh, while integrated cells were observed when blood was incubated with physiological saline. similar results were found for saliva treated with naoh and physiological saline, respectively. the results indicated that naoh treatment lead to cell lysis, followed by release of dna for use in subsequent amplification. in fig. b , it is shown that the ph value of naoh solution was . , and by adding the naoh solution to blood sample and saliva sample, the ph value of the mixture was . - . . however, the ph value drops dramatically to . - . by mixing the naoh treated sample with reaction buffer, and no significant difference in ph value was observed between the reaction buffer with and without naoh treated sample. the reason can be explained as the buffering effect of tris-hcl buffer, which provided a benign working environment for dna polymerase, according to verma et al. ( ) . to determine the best working condition, the performance of the direct-lamp was evaluated under different concentrations of primers and reaction temperatures by using whole blood sample with two different genotypes of mthfr c t (wild type and homozygous mutation, which were confirmed by sequencing). firstly, the best combination of primer concentrations was found to be . μm of the fip/bip and . μm of the f /b (fig. a) . the reaction temperature of direct-lamp was also optimized, as shown in fig. b ; the best amplification efficiency and specificity was obtained when the reaction temperature was °c. to evaluate the performance of the direct-lamp, a serial dilution of the target concentrations of whole blood sample and saliva sample with two different genotypes (wild type and homozygous mutation which confirmed by sequencing) of mthfr c t and aldh glu lys, respectively, were used to determine the detection limit. the x. liu et al. biosensors and bioelectronics ( ) - amplification efficiency reflected by the threshold time is inversely proportional to the dilution ratio of biological sample (fig. ) . accurate genotyping results were still obtained with the direct-lamp when biological sample was diluted to times. the specificity of the detection system for every target and sample type was evaluated, accurate genotyping results were still obtained under interference of background in all cases (supplementary fig. s and fig. s ), demonstrating great potential for direct clinical application. the accuracy of the direct-lamp was further verified with clinical samples (matched whole blood and dried blood spot samples, matched buccal swab and saliva samples) with informed consent. the study was approved by the ethics committee of the college of life sciences, northwest university (xi'an, china). mthfr c t of whole blood samples and aldh glu lys of saliva sample were also sequenced by bgi. in fig. , the threshold time of wild type were defined as a negative value, the threshold time of mutation type were defined as a positive value. for mthfr c t genotyping, samples of homozygous mutation, samples of wild type and samples of heterozygous mutation were observed with whole blood in fig. a , which was same as those found with dried blood spot in fig. b . for aldh glu lys genotyping, sample of homozygous mutation, samples of wild type and samples of heterozygous mutation were observed with saliva in fig. c , which was same as those found with buccal swab in fig. d . the genotyping results provided by the direct-lamp showed no discrepancies with those found with sequencing. as shown in supplementary table s , the derived allele frequencies of mthfr c t based on the direct-lamp detection were % and % for c and t, respectively (calculated from: c: f +f / , t: f / +f ), which is in agreement with those reported by hui et al. . as shown in supplementary table s , the observed allele frequencies of aldh glu lys were % and % for g and a respectively, which is also in agreement with those reported by eng et al. ( ) in the chinese population. loop-mediated isothermal amplification (lamp) was established by notomi et al. and this method has been used for detection of pathogens via the amplification of gene segments, demonstrating the method's great potential in molecular diagnostics. based on the advantages of this technology, some researchers are working on this technology can be applied for snps detection. a few reports indicated that this method has been applied for snps detection successfully (carlos et al., ; iwasaki et al., ; kwong et al., kwong et al., , nakamura et al., ; nakamura and ito, ; zhang et al., ) , which shorten the testing time further than pcr. however, dna purification or complex clinical sample pre-treatment are required for snps detection in these methods. the sample pre-treatment procedures including repeating incubation, elution and centrifugation consume a lot of time and energy during the detection process. although commercial kits simplify the procedure of dna purification and clinical sample pre-treatment, there are restrictions on the clinical sample types, such as colorless clinical samples are required (fta™ indicated micro card, whatman, uk) . the need for rapid snps detection to provide highly accurate diagnostic information is realized by all major health organizations, including the centers for disease control and prevention and the world health organization. however, no method combining lamp exists for snps detection that can be performed within a single patient visit ( min) directly from various clinical samples. here, we have established a rapid, easy-to-use and accurate snp detection platform using direct-lamp, which enables us to use whole blood, dried blood spot, buccal swab or saliva as samples for genotyping without dna purification. the whole testing procedure required only min from sample collection to result, which is much faster than with conventional snp detection systems. this is the first report to introduce the pretreatment of various kinds of samples with a simple naoh solution before lamp for genotyping. designed as a universal genotyping platform, this direct-lamp protocol has been successfully applied not only to the four sample types previously mentioned, but also verified with clinical sample, indicating that this genotyping platform could be applied to more sample types in further study. the treatment of sample with naoh plays an important role in this direct-lamp, making the whole testing procedure convenience and useable. first of all, compared with other snp detection methods, the dna purification step is eliminated in our assay with the help of naoh treatment, reducing the sample preparation time. secondly, using naoh-treated sample simplifies the complex procedures of conventional genotyping methods and eliminates the problem of cross-contamination, which happened in traditional dna purification processes. furthermore, various natural dna polymerase inhibitors in whole blood, such as hemoglobin, igg, lactoferrin, and proteases (adams, ; connelly et al., ; monroe et al., ) , can be inactivated by treatment with naoh solution. by using direct-lamp, qualitative analysis can be carried out by reading the fluorescence signal automatically in real time. the major advantage of this assay system is rapid qualitative answer in ''yes'' or ''no'' according to the fluorescence signal. compared with conventional snp detection methods based on pcr or dna sequencing, direct-lamp shows significant advantages (jung et al., ; ngo et al., ) . for conventional genotyping methods that require purified dna as template, such as pcr-microarray, qpcr and dna sequencing, the process of dna purification required a fair amount of time during the whole testing, whereas the present assay system requires less than min for sample preparation. traditional genotyping techniques based on pcr always require more than . h for dna amplification, whereas the present methods based on lamp shorten the testing time further. the process of nucleic acid amplification under isothermal conditions, employing self-recurring strand-displacement synthesis primed by a specially designed set of target-specific primers, enable this special identification system, which is faster than pcr-based methods, which use only two primers to recognize two regions (duan et al., ; tomita et al., ) . thus, compared with conventional snp detection techniques usually requiring a complex operation procedure that limits their application, the present methods provide an easy-to-operate onsite technique for genotyping with high efficiency. therefore, the direct-lamp will greatly benefit molecular diagnostics by dramatically reducing turnaround time, which makes this snp detection method very promising for poc testing in clinical diagnostics. with further development for additional snps and sample types, direct-lamp could enable rapid clinical decision-making, improve management of disease prevention and facilitate personalized medicine in each level of medical institutions. in this study, we presented a direct-lamp for snp detection by using whole blood, dried blood spot, buccal swab or saliva as samples without dna purification. this is the first report that a snps detection platform combined the direct amplification and lamp for genotyping with various clinical sample validation. our data have demonstrated that this system is highly applicable for detection of snps in clinical samples due to its unparalleled advantages, such as short processing time, simple procedure and accuracy. since detection of snps has significant clinical value, in our laboratory this method has already been extended to detection of snps in other genes related to folic acid metabolism. it is envisaged that our newly established direct-lamp could be quickly adapted for the detection of other snps of genes that are associated with disease risk, drug metabolism, or drug reaction. analytical sciences the international plos one , e this study was supported by the national natural science foundation of china ( ), national natural science foundation of china ( ), shaanxi provincial nano-biomedical detection innovation team founds ( kct- ) and northwest university graduate innovation and creativity funds (yzz ). supplementary data associated with this article can be found in the online version at http://dx.doi.org/ . /j.bios. . . . key: cord- -q neat x authors: zhang, haoqing; xu, ying; fohlerova, zdenka; chang, honglong; iliescu, ciprian; neuzil, pavel title: lamp-on-a-chip: revising microfluidic platforms for loop-mediated dna amplification date: - - journal: trends analyt chem doi: . /j.trac. . . sha: doc_id: cord_uid: q neat x nucleic acid amplification for the detection of infectious diseases, food pathogens, or assessment of genetic disorders require a laboratory setting with specialized equipment and technical expertise. isothermal deoxyribonucleic acid amplification methods, such as loop-mediated isothermal amplification (lamp), exhibit characteristics ideal for point-of-care (poc) applications, since their instrumentation is simpler in comparison with the standard method of polymerase chain reaction. other key advantages of lamp are robustness and the production of pyrophosphate in the presence of the target gene, enabling to detect the reaction products using the naked eye. polymerase inhibitors, presented in clinical samples, do not affect the amplification process, making lamp suitable for a simple sample-to-answer diagnostic systems with simplified sample preparation. in this review, we discuss the trends in miniaturized lamp techniques, such as microfluidic, paper-based, and digital with their advantages and disadvantages, especially for poc applications alongside our opinion of the future development of miniaturized lamp. polymerase chain reaction (pcr) [ ] is currently the most widely used nucleic acid amplification test (naat) method. the integration of heating, and, especially cooling control modules used for thermal cycling to perform pcr increases the challenges and cost of the point-of-care (poc) system's design and operation. this demands careful design for the pcr system to optimize thermal properties, which directly influences its performance, as extensive transition times between temperature segments of each cycle could result in non-specific amplification results. with an advent of microfluidics methods over the past years, the pcr technique was one of the first utilizing this technology [ ] . since then, the microfluidics were extensively exploited for pcr applications in their conventional [ ] , quantitative [ ] , and digital form [ , ] , including different poc assays [ , ] . integration of a conventional pcr with pre-or post-processing samples is difficult, as it requires specially developed hybridization probes, which can pass the nucleic acid (na) denaturation steps with a temperature of z c [ ] . isothermal amplification (ia) methods became promising alternatives [ ] to pcr, especially for poc applications [ , ] . ia conducts deoxyribonucleic acid (dna) amplification at elevated constant temperatures, simplifying the system design and operation. the temperature range required to perform ia is typically from z c to z c, lower than the one used for pcr denaturation, making the ia system simpler in comparison with pcr and suitable for integration with sample preparation [ e ] . the most widely used ia method is loop-mediated isothermal amplification (lamp) as it provides excellent specificity in comparison with other ia methods [ ] . lamp utilizes six specific oligonucleotides sequences in the initial (material-producing) step and then four sequences during amplification, elongation, and recycling steps. the process is based on primer annealing followed by auto-cycling strand displacement. it was originally performed at elevated temperatures of z c [ e ] (fig. ) . the lamp is a rapid, sensitive technique for na amplification, with applications in clinical diagnostics and pathogen detection [ e ] , food safety testing [ ] , and others. here, we present the trends in the lamp-on-a-chip systems for naat including its microfluidic, paper-based and digital variants. for each specific system, we reviewed their impacts and potentials. we also briefly described the detection methods used for lamp techniques as well as lamp applications. the potential of lamp-on-a-chip technology relies on simplified hardware, easy options to integrate it with sample preparation steps, and lamp detection capability. the insensitivity of the lamp technique to the inhibitors presented in the clinical sample makes lamp an exciting method for the detection of infectious diseases or genetic maladies. nucleic acids amplification methods are primarily required to be performed, as the original number of either dna or ribonucleic acid (rna) copies in the clinical sample is insufficient for their direct detection. they can be categorized according to the operating procedures, as either temperature cycling or isothermal. polymerase chain reaction is the most common technique used for na amplification [ , ] . the reagents are dna polymerase, two sets of primers (oligonucleotides), and deoxynucleoside triphosphates. the pcr protocol requires a temperature-cycling process in three sub-steps: denaturation of a double-stranded dna (dsdna) molecule to two single-stranded dna (ssdna) molecules at z c, annealing of the target sequence of specific primers to ssdnas at z c, and enzymatic elongation to finish the dsdna at z c. the cycling is typically repeated e times to achieve a detectable number of dna copies. the speed of the pcr process is defined mainly by the activity of dna polymerase and the heat transfer rate defined by the hardware setup. the microfluidic pcr systems have rapid heat transfer due to a small sample volume and high surface-to-volume ratio. they can be divided into two major groups according to the temperature cycling method: -stationary chamber pcr [ ] is also called "time domain pcr" [ ] . a defined volume of sample positioned in a chamber [ ] or sealed in a droplet [ ] is heated and cooled, either actively using an external element, or passively by simply switching the heater off with a stationary sample. different heating methods are developed for a fast process, including infrared-mediated [ ] , plasmonassisted heating [ ] , and ultrafast with active heating and passive cooling, achieving cycles pcr in < min [ ] . -continuous flow pcr method can be called, more generally, the "space domain pcr". the sample moves between heaters with fig. . schematic of lamp. reproduced with permission from ref. [ ] , open access. different temperatures. the chip in early reported work [ ] had a serpentine channel build over three heating blocks. overall, the continuous flow pcr system does not need the temperature cycling from the heaters but does need an external pumping unit and an accurate flow control. recently, a pcr system with an ultimate reaction time, was demonstrated achieving a cycle time of between z . s and z . s, resulting in the total time required for cycles of pcr within an incredible range of z s to z s [ ] . the pcr system's utilization for poc is limited by the system hardware and the sample-to-answer protocol complexity. numerous steps are required to remove polymerase inhibitors from clinical samples. isothermal amplification techniques require simple hardware and they are rather insensitive to polymerase inhibitors, making the amplification process robust. they typically require a processing time within a range from z min to z min, significantly longer than microfluidic-based pcr running time. the most promising ia techniques suitable for conduction in microfluidic devices are nucleic acid sequence-based amplification (nasba) [ ] , recombinase polymerase amplification (rpa) [ ] , helicase dependent amplification (hda) [ ] , and lamp [ ] . nasba [ ] is an ia method operating at a temperature of z c, designed to amplify target rna. nasba can achieve up to z fold target amplification in z min [ ] . this technique requires the initial removal of secondary structures from target rnas to achieve specific amplification products conducted at z c. this method, integrated with a sample preparation, was demonstrated to detect the norovirus in oysters [ ] . rpa [ ] is an ia operating entirely at a relatively low temperature range from z c to z c. it can amplify a target sequence using a recombinase, single-strand binding proteins, and a stranddisplacement polymerase. rpa has the advantage of a fast reaction with relaxed requirement for temperature control. this method has been demonstrated by the lab-on-a-foil system, based on a centrifugal microfluidic cartridge. the system was capable of detecting < dna copies in z min [ ] . another rpa-performing device, constructed of a substrate comprising of a paper made from a combination of glass fiber and cellulose could detect human immune-deficiency virus (hiv). results were detected by lateral flow strips achieving a limit of detection (lod) of copies in z min [ ] . hda [ ] is another type of ia working in a temperature range from z c to z c using a dna helicase for denaturation of dsdna into ssdna. unfortunately, its speed of long-target sequence amplification is a limiting factor. the strength of this method is its combination with paper-based microfluidics, as demonstrated by the detection of mycobacterium tuberculosis [ ] . lamp [ ] is one of the ia methods for dna amplification, operating at temperatures of z c. its low running cost and the simplicity of the hardware makes lamp one of the most promising techniques for genetic analysis in low-resource settings. the development of lamp-on-a-chip techniques will be further elaborated in the next sections. microfluidics-based lamp chips with different structures were proposed for poc diagnostics. here, we summarized the development of those chips based on single gene detection, multi-gene detection, and reverse transcription lamp (rt-lamp) (fig. ) . a magnetic bead-based lamp system, integrating na extraction and temperature control, was proposed for the rapid detection of methicillin-resistant staphylococcus aureus (mrsa) ( fig. a ) [ ] . the clinical samples, along with the specific nucleotide-coated magnetic beads, the washing buffer, and the reaction mixture were first loaded into the chambers of three different chips. the dna was then extracted from the bacteria (cell lysis), bound to magnetic particles, and concentrated, using a magnetic field followed by lamp. the fluids transportation and their mixing were assisted by a vacuum pump and a micro valve. the temperatures for cell lysis and lamp were controlled by a built-in micro temperature control module. the results were analyzed using a spectrophotometer and the lod was z fg ml À . this system was significantly faster in comparison with standard mrsa detection, as timeconsuming cell culturing was eliminated. also, the risk of contamination during cell culture was greatly suppressed as the system keeps the bacteria enclosed. nevertheless, the solution movement in the chip and the optical detection were achieved by external components, such as the pump and spectrophotometer. a microfluidic disk-based lamp chip, integrating sample preparation and detection, was developed [ ] (fig. b ). the disk consisted of a pressure-sensitive adhesive material film sandwiched between two poly(methyl methacrylate) (pmma) layers. the lamp reagents and the solution containing the dna template were preloaded into respective chambers and sealed within the chip, prior to the experiment. the solution mixing was achieved by modulating the rotation rate of the disk, alternating the centrifugal force. the chip was kept at an elevated temperature using a hot air gun with a chip temperature monitored by an infrared camera. the results were optically detected as fluorescence change using sybr green i intercalating dye with achieved lod of z pg ml À . the large size of the hardware, due to bulky temperature and disk rotation control systems, made it challenging for poc applications. single gene detection using a compact integrated microfluidic lamp chip is rather costly. therefore, chip structures were modified to meet the requirements of multiplexed gene detection with high throughput. an octopus-like multi-channel chip was fabricated using the soft lithography method (fig. c ) [ ] . the microchannels were connected to the related wells via gradient bridges. the designed microchannels present a low-mass-transfer coefficient to avoid the "cross-talk". the different sample solutions were loaded into the chamber by capillary force and sealed. then the lamp was performed at z c. the results were detected using an optical sensor, obtaining lod < copies$ml À . nevertheless, the sample pre-treatment, heating, and optical detection was performed off chip. compared with the previous design, the lamp disk system was proposed, integrated with dna extraction and amplicon detection using a colorimetric lateral flow strip (fig. d ) [ ] . it consisted of three major microfluidic layers from top to bottom for solution loading and transportation, dna extraction and amplification, and lateral flow strip detection, respectively. the reagents for lamp were loaded and sealed in the chamber. the chip was mounted on a centrifugal system, and the solution mixing and transportation via microchannels were controlled by operating different revolutions per min. three identical units on the chip allowed simultaneous multi-gene detection with lod of colony-forming units. nevertheless, the off-chip temperature control module limited its application for poc diagnostics. an rt-lamp system (fig. e ) [ ] , made from polydimethylsiloxane (pdms), was designed to integrate rna extraction and one-step rt-lamp with an external temperature control. the sample was transported by operating the integrated pneumatic pump and valve. the rna was extracted using specific probeconjugated magnetic beads after sample loading. the rt and lamp steps were performed using integrated micro-heater systems. this research presented a hands-off integrated system for virus detection from the sample containing rna using the one-step rt-lamp process. however, off-chip electrophoresis was performed for amplicon detection. table . an integrated rt-lamp system for rapid detection of the zika virus ( fig. f ) was proposed [ ] . the disposable chip consisted of four independent multifunctional reactors. the rna was extracted and transferred into the amplification chamber. the chip was then placed inside a thermally insulated portable chamber, heated by an exothermic chemical reaction to perform lamp. the results were recorded using a mobile phone camera, but it was also directly visible using the naked eye. this low-cost, simple, integrated sample-to-answer-based rt-lamp system underlines the potential of this technology for poc applications. a combination of multiplexing and rt-lamp has also been demonstrated detecting two rnas, both from hiv, thus increasing the specificity of its diagnostics [ ] . classical microfluidic lamp devices require pumping systems and valves for fluid manipulation, increasing the cost and operational complexity of the system. the researchers explored the potential of much simpler paper-based devices, well-known for their robustness, cost-effectiveness, and user-friendliness [ , ] . in this section, we summarize their characteristics, showing their potential applications (fig. ) . a critical aspect for paper-based na amplification is the paper's material selection. the nonspecific binding of dna molecules to the paper's fibers as well as paper selffluorescence, negatively influence the noise level and thus the lod. the most commonly used paper types are either nitrocellulosebased fta [ e ] or glass-based [ ] . a disposable cassette for the detection of hiv was proposed [ ] (fig. a) . the cassette, made from pmma, consisted of a single amplification chamber connected to the inlet and outlet ports. the fta membrane was incorporated into the chamber for rna isolation, concentration, and purification. as a result, the captured rna was directly used as a template for rt-lamp, without the elution or removal of the amplification inhibitors. a thin film heater, together with a conventional thermocouple, was mounted to the cassette holder to provide key components of a closed-feedback loopthermal control system. the fluorescence of the sample was monitored in real-time using a portable and compact optical detector, achieving an lod of < hiv virions. the system could be improved to detect nucleic acids related to other pathogens in saliva and urine, as well as in water. a magnetic-based sliding-strip device [ ] was fabricated for gene detection (fig. b) . a magnetic strip with an fta-based disc was sandwiched between two magnetic layers, containing serial ports for sampling, washing, amplification, and detection. the linear motion of the sliding strip acted as a valve to control all processing steps. the probe's fluorescence was detected using a hand-held ultraviolet source, achieving an lod of z cells. roll-to-roll thermal imprinting (molding) technology was proposed for the fabrication of integrated pdmsepaper microfluidic device for molecular diagnostics [ ] . this technology enabled the scaling up of production to thousands of devices in an hour (fig. c) . the mold was made by photolithography on a nickel plate. the pdms coating and pattern replication were performed on alcoated paper. the metal coating effectively suppressed the paper's auto-fluorescence and enhanced fluorescence intensity, due to its high reflectivity. a rolling cylinder was used to transfer microfluidic structures in pdms by thermal imprinting. the performance of this integrated device was validated by the detection of viral rna by lamp. the method created a bridge between academic research and industrialization in poc diagnostics, upscaling fabrication processes. an integrated sample-to-answer microcapillary-based lamp system [ ] was presented for single nucleotide polymorphism genotyping (fig. d) . the fta was placed inside this capillary as well as all reagents for dna extraction, purification, and amplification. the sample flow was controlled by capillary forces as well as an external pressure. once the sample pretreatment was completed, the glass capillary was kept at a temperature of z c for an hour, using an external heating source, to complete the lamp and the results were subsequently observed by the naked eye. a pdms-paper hybrid sample-to-answer device incorporating lamp and lfa was proposed (fig. e) to enhance lamp sensitivity [ ] using the shunt and pdms barrier. it consisted of following four layers from top to bottom: a polyvinyl chloride for later flow, a glass fiber for performing lamp, an fta for sample loading and extraction, and a cellulose for waste absorption. the reagents for lamp were pipetted onto the strip and then heated up using a hand-held heating system [ ] . this device was capable of performing a simple colorimetric readout, achieving a lod of z pm, -fold signal enhancement compared with the lfa strip without the shunt and pdms barrier. this chip-based system is a good candidate for poc, due to its portability and simplicity. these molecular diagnostic platforms generated a wide range of applications in medicine, healthcare, agriculture, environment, and water monitoring due to their low cost, rapidity, and accuracy. digital pcr (dpcr) was reported almost two decades ago [ ] and sparked the interest of numerous applications primarily for early cancer detection and non-invasive, prenatal diagnostics. naturally, the dpcr system has lamp alternatives (fig. ) . based on the sample compartmentalization methods, digital lamp (dlamp) systems can be grouped into two categories: droplet-based and well-plate-based systems. the droplet-based dlamp devices utilizes the sample droplets suspended in oil, thus preventing dropletto-droplet cross-contamination. the chip-based dlamp devices are of two types, both containing micro-reaction chambers. the sample is loaded into wells, either via a microfluidic network and then sealed by oil, or by pipetting it into wells and sealed by a lid. results from both the droplet-based and chip-based dlamp devices are analyzed in the same way as dpcr results, using either flow cytometry or digital image processing. the first chip-based dlamp system was proposed in [ ] . this self-digitalized chip was fabricated based on the pdms having microwells. the sample and the oil were sequentially loaded using force originated by air pressure (fig. a ). the chip with the loaded sample was placed into a water reservoir, keeping the temperature at z c to perform the lamp process. during the same year, a self-priming system for chip-based dlamp was presented [ ] . its major advantage was the absence of external pumping or valves for the manipulation of the sample as table characteristics of lamp-on-a-chip. fta stands for flinders technology associates, producing the fta membranes, and lfa stands for lateral flow assays. well as the oil. the pdms chip was kept in a vacuum environment to remove air from the pdms. the sample was loaded into the chip and pulled into the chambers due to the force caused by pressure difference between the external ambient and low internal pressure with degaussed pdms working as a sorption pump (fig. b) . however, the system independence on fluidic elements, such as pumps and valves, was compromised by a low "digitalization" level having only wells. a droplet-based dlamp device was presented in [ ] (fig. c) . the droplets were generated using a classical cross between two microfluidic channels and then transferred into a heated region to perform amplification. the droplet volume was z pl with a processing time of z min required for a volume of z ml. that is equivalent to amplifying dna in z droplets in h. the elevated temperature of z c was achieved by placing the dlamp device on a thermoelectric element. an optical setup was used for fluorescence detection, using a dual excitation wavelength and a dual detection band. results monitoring was performed by two methods: dna binding to evagreen and calcein dye-based indicators. another method was introduced using disposable polymeric dropchip, generating the droplets by centrifugal force [ ] . once the droplet generation was completed, the chip was transferred to a thermal cycler to perform lamp in z h and the amplification results were monitored by a microarray scanner. the chip consisted of two loosely connected plates, enabling them to slip over each other, thus named "slipchip" technology [ ] (fig. d) . the sample droplets were generated and then mixed by layer slipping. the slipchip contained wells and was used to test antimicrobial susceptibility, measuring the phenotypic response of escherichia coli (e. coli) present in a clinical sample of urine. the results of slipchip technology showed a fast processing time of < min. monitoring methods used for lamp are commonly adopted procedures developed earlier for pcr and other bio-molecular techniques. their utilization for lamp has been extensively studied and was summarized [ ] . mainstream detection methods of lamp results often rely on naked-eye monitoring to observe precipitates [ ] , dna-binding dyes [ ] , and colorimetric indicators [ ] . off-chip end-point detections are commonly conducted by performing gel electrophoresis [ ] . there are also real-time monitoring optical methods, such as turbidity measurement monitoring optical attenuation of the fluid [ ] and fluorescence with a suitable dye [ ] . electrochemical methods can also be either real-time or endpoint detected, and they are also commonly used for lamp monitoring, either in form of a sensor [ ] or biosensor [ ] . finally, there are antibody-based methods such as immunochromatic technique (lateral flow dipstick) [ ] and the gold standard for protein detection such as antibody enzyme-linked immunosorbent assay [ ] . besides these major techniques, researchers have also tested numerous other methods based on a nano-au probe [ ] , field effect transistors [ ] , surface plasmon resonance [ ] , bioluminescence [ , ] , or the giant magnetoresistive effect [ ] . nevertheless, compared with mainstream detection methods, these techniques are not commonly used as they require chemical preparation or additional instruments to perform product detection. the lamp method is an innovative technique for gene amplification, due to the simplicity of its hardware [ ] . it has been utilized in a wide range of applications, including infectious disease diagnostics [ , ] and food safety tests [ ] . in particular, lamp is an effective gene amplification method for poc devices [ ] . with the progression of globalization, the spread of infectious diseases is threatening people's health and lives around the world, regardless of regions' wealth or development status. it is essential to diagnose and cure infected patients to prevent the spread of diseases. rapid, accurate, and sensitive diagnostic tools to identify pathogens, ideally in poc format, are vital to tackle the spread of infectious diseases [ ] . lamp was first recognized as a potent alternative to pcr in , when it was shown to detect the rna of a severe acute respiratory syndrome virus [ ] . since then, it has attracted a lot of attention and has become an increasingly popular method for clinical diagnosis. so far, lamp has been employed for rapid detection of both dna and rna possessing human-and nonhuman-carried viruses, such as mrsa [ ] , pseudorabies [ ] , tuberculosis [ ] , influenza a [ ] , zika [ ] , nervous necrosis [ ] , and aqua pathogens in fish [ ] . food safety and food quality control are, inevitably, an important part of the public-health system. lamp has been used in food safety detection [ , e ] including staphylococcus aureus [ ] , salmonella [ e ], e. coli o [ ] , and vibrio parahaemolyticus [ ] , as well as food allergens [ ] . lamp is an isothermal amplification technique that can detect target sequences with high specificity and sensitivity. its technology has been further developed recently, having advantages over classic pcr techniques, such as isothermal operation. the specificity of the pcr achieved by annealing at an optimized temperature is substituted by lamp technology with more complex primer designs. however, a processing time of a single sample lamp cannot compete with ultimate pcr systems, capable of performing the amplification in < min [ ] . the dlamp technique seems different, as its conduction was reported in < min, a difficult time to achieve for dpcr [ ] . therefore, lamp has its merits, as the heating system is simpler, and there is no temperature cycling and cooling as required for pcr. also, the sample pretreatment module is easier to incorporate due to a more robust lamp process compared to pcr. the results analysis of lamp is also much easier, as the resulting fluorescence after amplification can be observed by the naked eye. a lower lod can be better achieved by lamp in food-borne pathogen detection than pcr [ ] . moreover, the simple structure of lamp chips provides opportunities for mass production and industrialization. as a result, lamp-based integrated systems [ ] are more suitable for application in poc diagnostics [ ] than pcrs. lamp-based diagnostics are not likely to replace conventional techniques such as pcr; they will coexist in parallel, as each one has its strengths and weaknesses. since its discovery, lamp has been further developed, often involving the combination of this technique with other molecular approaches, such as reverse transcription and multiplex amplification. lamp is easy to handle, and is compatible with multiple detection methods, including turbidity detection, real-time fluorescence detection, and end-point visualization. in previous sections, we discussed lamp's potential based on its specificity, efficiency, speed (more related to the dlamp), and practical implementations. the utilization of lamp was demonstrated for the diagnosis of viruses, bacteria, and allergens in food. nevertheless, there is still much room for improvement in detection methods for the integration of lamp in poc applications. ideally, the detection modules have to be miniaturized and integrated with the on-chip lamp system. thus, developing a mobile biosensor with a lamp technology application may result in significant advancement towards the detection of pathogens directly from clinical samples, which is especially important in cashstrapped countries or when working with highly contagious clinical samples. due to its high specificity, lamp can also be used for the early detection of genetic disorders, an important aspect of prevention diseases associated with them and their treatment. in summary, lamp could be the ideal method for diagnostics in an everyday poc setting. specific enzymatic amplification of dna in vitro: the polymerase chain reaction, cold spring harbor symp chemical amplification: continuous-flow pcr on a chip pcr microfluidic devices for dna amplification polymerase chain reaction in microfluidic devices digital polymerase chain reaction technology e recent advances and future perspectives advances in microfluidic pcr for point-of-care infectious disease diagnostics point-of-care diagnostics for global 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a spiral chip for absolute quantification of nucleic acids this work was supported by the "foreign experts program" of p.r. china (w ). it was also partially supported by the grant agency of the czech republic under the contract ga - s and from the ministry of education, youth and sports of the czech republic under the project op vvv ceitec nanoþ (cz. . . / . / . / _ / ). key: cord- - rsfs d authors: yan, nan; tang, cheng; kan, ruici; feng, fan; yue, hua. title: genome analysis of a g p[ ] group a rotavirus isolated from a dog with diarrhea in china date: - - journal: infect genet evol doi: . /j.meegid. . . sha: doc_id: cord_uid: rsfs d genotype g is an emerging genotype among species a rotavirus (rva) circulating in humans and pigs worldwide. in this study, an rva strain designated rva/dog-tc/chn/sccd-a/ /g p[ ] was isolated in cell culture from a pet dog stool sample with acute diarrhea, and its whole genome was sequenced. the genotype constellation of sccd-a was g -p[ ]-i -r -c -m -a -n -t -e -h . all genome segments except the vp gene were closely related to the genes from porcine rva strains or porcine-like human rva strains. on the other hand, the vp gene clustered in a distinct lineage only with that of a g p[ ] porcine-like human rva, preventing the identification of the exact host species origin, but very unlikely to be originated from human rva. in addition, phylogenetic analysis showed that the g vp gene of sccd-a clustered into a novel sublineage within the lineage iii of g . this first isolation of a g p[ ] rva from a pet dog may justify the exploration of the role dogs play in the interaction of rva circulating in pigs and humans. rotavirus a (rva), family reoviridae, are the major pathogens causing diarrhea in animals and children worldwide. the rva virion encapsidates a genome of dsrna segments, encoding six structural viral proteins (vp -vp , vp and vp ) and five or six nonstructural proteins (nsp -nsp / ) (vlasova et al., ) . rva has two outer capsid proteins, vp and vp , which define the g and p genotypes, respectively. to date, at least g and p genotypes have been identified by the rotavirus classification working group (rcwg) (https://rega.kuleuven.be/cev/viralmetagenomics/virus-classification/ rcwg). for highly genetically diverse rva strains, the dual (g/p) typing system was extended to a full-genome sequence classification system, with the notations gx-p[x]-ix-rx-cx-mx-ax-nx-tx-ex-hx used for the genes encoding vp -vp -vp -vp -vp -vp -nsp -nsp -nsp -nsp -nsp / , respectively (matthijnssens et al., ) . in dogs, rva is usually associated with mild diarrhea in puppies (greene, ) . previously, only the g p[ ] genotype had been described in dogs, until a bovine g p[ ] genotype was isolated from a young dog in (luchs et al., ; sieg et al., ) . the isolation and characterization of g p [ ] have been extensively reported in dogs, but information about the prevalence of rva in dogs is still limited (otto et al., ; ortega et al., ; alves et al., ) . as canine rvas have zoonotic potential between humans and dogs, further investigation of the molecular prevalence of rva in dogs is needed (wu et al., ; luchs et al., ; papp et al., ) . g rotavirus is an emerging genotype spreading worldwide in pigs and humans (wu et al., ) . in mainland china, the first g strain was detected in , and g strains were uncommon before (li et al., ) . recently, however, g rva has emerged as the predominant genotype in china (dian et al., ; yu et al., ) . the rva g p[ ] genotype has been detected in thailand, the united states, japan, south korea, italy, belgium, brazil, mainland china and taiwan . recently, a g p[ ] strain was isolated from a child with severe diarrhea in thailand, showing that porcine g p[ ] can infect humans directly (komoto et al., ) . in this study, an rva positive sample, detected by rt-pcr as described by ortega et al. ( ) , was used to isolate the virus. moreover, this fecal sample was detected as negative for canine parvovirus type , canine coronavirus and canine distemper virus by pcr assay (decaro et al., ; decaro et al., ; elia et al., ) . the sample was collected from a three-month-old labrador with acute diarrhea in october at the animal hospital of southwest university for nationalities in sichuan province, china. rva isolation was conducted on embryonic rhesus monkey kidney tissue cells line (ma- cells, atcc crl- . ) as previously described . characterized by cell shrinking, cell layer splitting, lysis, detachment, and shedding, was observed at h post-infection (hpi). after four continuous passages, virus cytopathic effect (cpe) was more visible, and the time of occurrence of cpe was stable. the fourth-generation cultures were used to plaque purification the virus. in passages - , the time of occurrence of cpe was stable at hpi. the vp , vp and vp genes of the purified virus were sequenced at passages , , and , and they shared % nucleotide sequence identity between each passage, which indicated that the virus stock contained only a single virus strain. this rva strain was designated rva/dog-tc/chn/sccd-a/ /g p [ ] . at passage , virus titration was performed in -well plates with tenfold serial dilutions with eight replicates per dilution. the virus titer was determined by the reed-muench method, and endpoints were expressed as % tissue culture infective dose (tcid )/ml, the virus titer was . tcid / ml. to determine the rva genome sequence, pairs of primers were designed (table s , available in the online supplementary material). viral rna was extracted from culture-adapted virus using rnaiso plus® (takara bio inc., japan) and then reverse transcribed into cdna using the primescript™ rt reagent kit (takara bio inc., japan) according to the manufacturer's instructions. the resulting cdna was stored at − °c for pcr amplification. the pcr products were purified and cloned into the pmd -t simple vector prior to sequencing, and the sequences were assembled using seqman software (version . ; dnastar). the open reading frame (orf) was identified by orf finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). genotype assignments were carried out using the rotac v . online tool according to the genotyping recommendations of the rcwg (maes et al., ) . sequence identity analyses were performed with aligned nucleotide and amino acid sequences by the clustalw method using the megalign . program (dnastar). phylogenetic trees were constructed using the maximum likelihood method with the jukes-cantor model, bootstrap replicates and default parameters in mega . recombination events were assessed using simplot software (version . . ) and rdp . with rdp, geneconv, chimaera, maxchi, bootscan, siscan, seq methods and lard. the genome of rva/dog-tc/chn/sccd-a/ /g p[ ] was successfully determined, and the constellation of this strain was g -p [ ]-i -r -c -m -a -n -t -e -h . to exclude the possibility of virus reassortment between rva strains that might have been presented in the clinical sample during the cell culture adaptation, we amplified fragments of all genome segments from the original fecal sample. we confirmed that the nucleotide sequences were % identical between the virus genome present in the original fecal sample and the cultureadapted strain. the accession numbers for the segments for vp -vp , vp , vp and nsp -nsp were deposited in genbank (mh -mh ). according to nucleotide identity of the n. yan, et al. infection, genetics and evolution ( ) - coding region sequences, the nsp , nsp , nsp , vp , vp , vp , vp and vp genes were closely related to cognate genes of porcine rotavirus strains. the sequence closest to the nsp gene of rva/dog-tc/ chn/sccd-a/ /g p [ ] was that of human strain r which was previously shown to be of porcine rotavirus origin (wang et al., ) . similarly, the sequence closest to the nsp gene of sccd-a was that of human strain nt which was previously shown to be of porcine rotavirus origin (do et al., ) . on the other hand, the sequence closest to the vp gene of sccd-a was that of strain ll which was reported to be the result of a porcine rotavirus having transmitted to a human, but the phylogenetic tree for the vp genes showed that strains sccd-a and ll clustered together in an independent group that was distinct from any of the previously established lineages (fig. ) . as the exact host species origin of the vp gene of strain ll was reported to be indeterminable (li et al., ) , so was the origin of the vp gene of sccd-a. the nucleotide and amino acid identity of genes in strain sccd-a with the closest sequences was shown in table , the phylogenetic tree of vp was shown in fig. . the phylogenetic tree of vp and vp genes were shown in fig. s and fig. s (phan et al., ; shi et al., ; esona et al., ) . phylogenetic tree based on complete vp gene coding region nucleotide sequence bp, sequence alignment and clustering were performed by clustalw using mega . software. the tree was constructed by the maximum likelihood method with bootstrap values calculated for replicates. ▲ marks the strain in this study. prevalence of the g p[ ] strain in dogs is required. due to rotavirus is transmitted via fecal-oral transmission, whether the infectious source of this virus in the dog was from dietary contamination needed to be further investigated. rva vp defines the g genotype and induces neutralizing antibodies (aoki et al., ) . to accurately understand the antigenicity of g rva, it was proposed that g can be divided into six distinct lineages (i-vi) based on the vp nucleotide sequence (phan et al., ) . lineages i, ii, iv and v are found only in humans, while lineages iii (with sublineages a-d) and vi (with sublineages a-g) are found in both humans and pigs (shi et al., ; esona et al., ) . according to previous reports about the division of g lineages (phan et al., ; shi et al., ; esona et al., ) , all strains in the three papers which represented every lineages and sublineages in g and g sequences which had the highest similarity with strain sccd-a in the genbank were used for phylogenetic analysis. vp of strain rva/dog-tc/chn/ sccd-a/ /g p[ ] clustered into lineage iii, but was located at a unique sub-branch with six g strains (kc . , ky . , kt . , kt . , kj . and kc . ), which were distinct from the other known sublineages in lineage iii, indicating that these seven strains may represent a novel sublineage of lineage iii (fig. ) . only representative strains and strains of potential novel sublineage were retained in fig. . the table of amino acids comparison between the potential novel lineage and other lineages was shown in table s (available in the online supplementary material). no recombination event was identified in these strains, g lineage iii is an emerging genotype, and both porcine and human strains of lineage iii might have a common progenitor (phan et al., ) . in conclusion, a g p[ ] rva strain, named rva/dog-tc/chn/ sccd-a/ /g p[ ], was isolated from a young pet dog with acute diarrhea, and the genome constellation of this strain was g -p[ ]-i -r -c -m -a -n -t -e -h . this strain was considered as a porcine origin of rva strain, which indicated that dogs may play a role in rva g p[ ] circulating in pigs and humans. the potential public health significance this poses, because of close contact between dogs and humans, highlights the necessity of further surveillance for this virus in dogs. to our knowledge, this is the first isolation of the g p[ ] genotype from dogs. the full-length genome of rva/dog-tc/chn/sccd-a/ /g p [ ] has been deposited in genbank under accession no. mh -mh . identification of enteric viruses circulating in a dog population with low vaccine coverage structure of rotavirus outer-layer protein vp bound with a neutralizing fab quantitation of canine coronavirus rna in the faeces of dogs by taqman rt-pcr a realtime pcr assay for rapid detection and quantitation of canine parvovirus type in the feces of dogs completely genomic and evolutionary characteristics of human-dominant g p group a rotavirus strains in yunnan molecular epidemiology of rotavirus a, causing acute gastroenteritis hospitalizations among children in nha trang detection of canine distemper virus in dogs by real-time rt-pcr genetic relationships with other g strains and detection of a new g subtype infectious diseases of the dog and cat identification and characterization of a human g p[ ] rotavirus strain from a child with diarrhoea in thailand: evidence for porcine-to-human interspecies transmission molecular characterization of unusual human g p[ ] rotaviruses identified in china molecular epidemiology of g rotavirus strains in children with diarrhoea hospitalized in mainland china from rare g p[ ] rotavirus strain detected in brazil: possible human-canine interspecies transmission rotac: a web-based tool for the complete genome classification of group a rotaviruses full genome-based classification of rotaviruses reveals a common origin between human wa-like and porcine rotavirus strains and human ds- -like and bovine rotavirus strains identification of co-infection by rotavirus and parvovirus in dogs with gastroenteritis in mexico detection of rotavirus species a, b and c in domestic mammalian animals with diarrhoea and genotyping of bovine species a rotavirus strains full-genome sequencing of a hungarian canine g p[ ] rotavirus a strain reveals high genetic relatedness with a historic italian human strain genetic heterogeneity, evolution and recombination in emerging g rotaviruses molecular characterization of a rare g p[ ] porcine rotavirus isolate from china a bovine g p[ ] group a rotavirus isolated from an asymptomatically infected dog porcine rotaviruses: epidemiology, immune responses and control strategies full genomic analysis of a porcine-bovine reassortant g p[ ] rotavirus strain r isolated from an infant in china the dynamics of a chinese porcine g p[ ] rotavirus production in ma- cells and intestines of -day-old piglets putative canine origin of rotavirus strain detected in a child with diarrhea novel g rotavirus strains co-circulate in children and pigs prevalence of rotavirus and rapid changes in circulating rotavirus strains among children with acute diarrhea in china the authors declare that they have no conflicts of interest. this study did not involve animal experiments besides the fecal sampling of diarrhea dogs that visited animal hospitals for clinical treatment. supplementary data to this article can be found online at https:// doi.org/ . /j.meegid. . . . key: cord- -mw ujjrk authors: loré, nicola ivan; cigana, cristina; sipione, barbara; bragonzi, alessandra title: the impact of host genetic background in the pseudomonas aeruginosa respiratory infections date: - - journal: mamm genome doi: . /s - - - sha: doc_id: cord_uid: mw ujjrk understanding the significance of human genetic diversity in modulating host susceptibility to opportunistic infections is an emerging challenge in the field of respiratory illnesses. while it is recognized that diverse bacterial strains account for differential disease manifestations, emerging data indicate that host genetic diversity is an important determinant factor that influences the severity of opportunistic infections. with particular regard to respiratory illnesses mediated by the gram-negative bacterium pseudomonas aeruginosa, diverse genetic background is also emerging as a key contributor. human-genome-wide association studies are a common approach for determining the inter-individual genetic variation associated with variability of the pulmonary infections. historically, diverse murine inbred mouse strains and ex-vivo cellular models were considered complementary to human studies for establishing the contribution of genetic background to p. aeruginosa respiratory infections. more recently, the development of a new mouse model of infection, mirroring human airway diseases, combined with innovative murine resource populations, modelling human genetic variation, provides additional insights into the mechanisms of genetic susceptibility. in this review, we cover the recent state of the art of human and animal studies and we discuss future potential challenges in the field of p. aeruginosa respiratory infections. genetic diversity is a key determinant in modulating human susceptibility to several diseases, including the respiratory infections (chapman and hill ) . the relevance of host genetic background in driving disease phenotypic diversity is also emerging in the context of monogenic diseases characterized by airway infection (cutting ) . based on recent findings, we can now assume that pathogens are not the unique factors determining the wide spectrum of disease outcomes, as there is a considerable contribution from the host genetic background. the relevance of individual genetic variations to pseudomonas aeruginosa infection is emerging in several respiratory illnesses of different etiologies including cystic fibrosis (cf), chronic obstructive pulmonary disease (copd), or bronchiectasis. p. aeruginosa is one of the most common hospital-acquired opportunistic pathogens and mediates extremely variable outcomes of respiratory illnesses (gellatly and hancock ) . so far, human studies have identified candidate genetic loci or modifier genes associated with the disease diversity during p. aeruginosa respiratory infection (weiler and drumm ) . in parallel, several studies using animal and ex-vivo models have been carried over to achieve deep understanding of the biological relevance and significance of the host genetic profile in modulating the disease outcome of p. aeruginosa respiratory infection. recently, the use of classical murine inbred strains and new murine resource populations has supported several progressive steps forward in this field, generating new knowledge with a strong translational significance. in this review, we discuss the latest state of the art that mainly covers human and murine studies to define the role of host genetic background and host genetic traits during respiratory infection by p. aeruginosa. outcomes of p. aeruginosa infections in patients with respiratory illnesses are difficult to predict and show a high degree of variability in terms of disease severity, complications and survival (chapman and hill ; cutting ) . the association between genotype and phenotype is difficult to determine in humans due to the small size of cohorts with detailed clinical outcomes that are often influenced by non-genetic contributors, such as environmental and stochastic factors. the most characterized population, affected by respiratory infections, is that suffering from cf genetic disease and followed from birth; their clinical data, including their microbiological status (e.g., p. aeruginosa), are periodically recorded. in cf, it has been shown that host genetic factors (e.g., multiple genetic loci or polymorphic variations) influence the progression of p. aeruginosa lung infections and age at establishment, regardless of the cystic fibrosis transmembrane conductance regulator (cftr)-genotype. two main genetic approaches have been exploited in humans to identify genetic modifiers that directly influence the phenotype of cf disease: (i) an "a priori" approach, i.e., the candidate gene approach, where the genes selected for investigation are identified from the current understanding of cf lung disease pathophysiology, and (ii) an "unbiased" approach, analyzing the whole genome or exome sequencing by linkage and genome-wide association studies (gwas). from the candidate gene studies, several genes have been proposed as potential cf pulmonary disease phenotype modifiers (weiler and drumm ) . these could be classified into different categories including inflammatory response, infection response, and tissue damage and repair. genetic association has been found for several phenotypes spanning from the age of first infection, establishment of chronic p. aeruginosa infection or particular p. aeruginosa phenotypes (e.g., mucoid). first acquisition of airway p. aeruginosa has been associated with variants in the mannose-binding lectin (mbl ), in the na + /h + exchanger (scl a ), in the amino acid transporter (slc a ) (dorfman et al. (dorfman et al. , pereira et al. ) and in the tumor necrosis factor-α (tnf-α) (coutinho et al. ) genes. significant associations between age-of-onset of chronic p. aeruginosa infection have been reported for ficolin (fnc , fnc ), mbl-associated serine protease (masp ), caveolin- (cav ), and transmembrane-like channel (tmc ) genes (dorfman et al. ; emond et al. ; haerynck et al. ). time to first p. aeruginosa airway infection, chronic p. aeruginosa infection, and mucoid p. aeruginosa have been associated with genetic variants in dynactin protein (dctn ) (emond et al. ) . p. aeruginosa mucoidy has been associated with pro-inflammatory cytokines interleukin (il- ) gene variants (furlan et al. ). however, even collectively, these human studies only partially explain the contribution of genetic variance to the risk of p. aeruginosa airway infection and should be validated in additional cohorts. similar approaches should also be exploited in other populations affected by p. aeruginosa respiratory infection (e.g., copd). different mouse models of acute respiratory infection by p. aeruginosa have been used to dissect the role of genetic backgrounds in determining lung disease severity (table ) . these models exploited and compared different inbred murine strains during acute pneumonia lasting for hours or at most a few days. in this regard, a comprehensive classification of mouse inbred strains into categories according to their resistance or susceptibility is difficult to achieve. the differences in the disease outcome may be affected by several factors including routes of administration (aerosol vs intratracheal), size of the bacterial load and different p. aeruginosa strains used (laboratory vs clinical), length of infection, as well as age, gender and the specific murine substrains. despite these issues, a wide range of disease severity after p. aeruginosa infection with some common trends were observed indicating a critical role of the host genetic background in modulating the outcome of the pulmonary disease. here, we focus mainly on the murine inbred strains most commonly tested in models of p. aeruginosa respiratory infection, namely dba/ , a/j, c h/he, balb/c, and c bl/ mice (table ) . susceptibility and resistance to p. aeruginosa infection were initially based on survival rates and bacterial loads in the lungs followed by additional characterization of immunological phenotypes. in an early extensive study, inbred mouse strains were compared using aerosol models of p. aeruginosa infection (wilson et al. ). dba/ j mice were the most susceptible, while a/j and c h/hen were the most resistant when monitored for mortality. in more details, the ability of the dba/ j strain to contain bacterial growth in the lungs was limited and this supported the role of host genetic makeup in resistance to infections. wilson et al. affirmed that this severe disease phenotypic trait was due to a limited bactericidal activity when compared with c bl/ macrophage cells (wilson et al. ) . of note, these immune-mediated mechanistic conclusions were different to a previous study that focused on the macrophage phagocytic ability in different genetic backgrounds (morissette et al. ) , although both studies identified dba/ j mice as susceptible murine strains. (morissette et al. ) ≈ bactericidal activities of lung macrophages (morissette et al. ) c bl/ balb/c ↑ splenocyte proliferation (barclay et al. ) ↓ tnf-α production by alveolar macrophages (sapru et al. ) ↓ proliferation of lung t cells (stevenson et al. ) ↓ il- and il- production from lung t cells (kondratieva et al. ) ↑ ifn-γ production from lung t cells (kondratieva et al. ) c h balb/c ↑ ifn-γ production by splenocytes (moser et al. ) ↓ il- production by splenocytes (moser et al. ) in a recent extensive study, we compared nine inbred mouse strains, including murine sub-strains, in a pneumonia model system induced by intratracheal delivery of the p. aeruginosa clinical strain to define deviant survival phenotypes. the highest susceptibility was observed in a/j and dba/ j, while balb/canncrl and c h/heouj were the most resistant to p. aeruginosa infection (de simone et al. ) . when distinct sub-strains of balb/c from jackson (balb/cj and balb/cbyj) or charles river (balb/canncrl) were compared, significant differences between balb/canncrl and balb/cbyj were found supporting marked genetic and behavioral differences. a more detailed characterization of immunological phenotype was carried out on a/j and c h/heouj mice, as the most deviant murine strains (de simone et al. ) . when bacterial loads and neutrophils recovered in the lungs were correlated, a/j mice showed a delayed neutrophil recruitment and a higher bacterial burden in the lungs, suggesting their inefficiency to promptly keep the bacterial infection in check compared to c h/heouj mice. we speculated that a/j mice do not mount a proper, early immune defense leading to a permissive environment for bacterial replication and bacteremia, ultimately leading to rapid disease progression. furthermore, a/j mice showed an excessive release of pro-inflammatory cytokines that did not correlate with the cellular response, suggesting a defect in leukocyte recruitment. as stated above, variability in the methods of infection (aerosol vs intratracheal) and measurable outcomes (body weight vs mortality vs bacterial load vs inflammation) are two key factors affecting the interpretation of the results. therefore, a direct comparison among studies should be carefully weighted. based on the definition of host resistance to infection (schneider and ayres ) , an accurate evaluation of bacterial loads at different post-infection timepoints may represent the most unbiased biological parameter for comparing results across different studies. mouse strains presenting deviant disease phenotypes can be further exploited to identify genetic loci underlying host susceptibility to infections of different origins (gruenheid and gros ; vidal et al. ) . while the forward genetic approach has been widely implemented for several pathogens, only one study has applied it to p. aeruginosa infection. in this regard, we mated p. aeruginosa-resistant c h/heouj, and susceptible a/j mice and the deriving f intercross population was phenotyped and used for quantitative trait locus (qtl) mapping . a significant qtl associated with the survival rate was mapped on chromosome , and named p. aeruginosa infection resistance locus (pairl ). through gene prioritization analysis within the pairl genomic interval (~ mbps), a set of promising candidate disease modifier genes was proposed that mediated host-pathogen sensing, neutrophils and macrophages recruitment, and inflammatory processes. it is becoming evident that complex combinations and variations of genetic locus/genes affect the outcome of p. aeruginosa respiratory infection. although mouse inbred strains are the starting point for the exploration of causal genotype-phenotype relationships and gene mapping, they do not offer a wide genetic variability for the identification and study of the polymorphic variations that are hallmarks of the human population (peters et al. ). this limits genetic and phenotypic diversity and the value of qtl detection. the new collaborative cross (cc) mice population may overcome the limitation of previous genetic mapping approach (churchill et al. ) , such as the low mapping resolution based on f intercross population. the cc is a murine inbred reference population with high genetic diversity designed by randomly outcrossing eight founder inbred strains, followed by inbreeding processes (https :// csbio .unc.edu/ccsta tus/index .py). it is worth noting that the cc murine population has been used to model infectious diseases caused by a number of different virus and bacteria including ebola virus (rasmussen et al. ) , mycobacterium tuberculosis (smith et al. ), west nile virus (graham et al. ) , severe acute respiratory coronavirus (sars-cov) (gralinski et al. ) , influenza a virus (ferris et al. ) , and non-typhoidal salmonella (zhang et al. ) . in these studies, the power of the cc resource led to the identification of the host genes controlling susceptibility to infections. when we challenged a cohort of different cc murine strains with p. aeruginosa into the lung, they displayed a widely marked phenotypic disease variation in terms of mortality (survival rate) and morbidity (changes in body weight). when broad-sense heritability of these phenotypic traits was evaluated, we confirmed the influence of the genetic profile, rather than environmental factors, among cc lines during p. aeruginosa infection (lore et al. ) . this study confirmed the ability the cc murine population has to mirror human disease diversity in the context of acute lung infection by p. aeruginosa. it is evident that the cc population may be exploited to identify genetic loci associated with acute lung infection by p. aeruginosa, with a higher mapping resolution in comparison to previous studies. studies with different inbred mouse strains support the contribution of the host genetic background to predispose the animals to chronic infection and different degrees of disease severity due to a different immunoreactivity to pathogens (table ). in this regard, several mouse models of chronic infection have been established by including p. aeruginosa clinical isolates in different types of immobilizing agents such as agar, agarose, or seaweed alginate to prevent clearance by the host (bragonzi ) . recently, we exploited the agar-beads mouse model of long-term chronic lung infection, refined and characterized in c bl/ ncrl , in five phylogenetically different inbred strains of mice to identify deviant disease phenotypes and to dissect the cell-mediated immunity . in this study, we classified a/j and dba/ j as the most susceptible to p. aeruginosa chronic infection, while c h/ heouj strains were classified as the most resistant. several clinical phenotypes including survival rates, weight losses, bacterial loads and capacity to establish long-term chronic infection were considered in this ranking. other works that focused on chronic infection mediated by bacteria embedded in agar-beads are generally in agreement with this classification and in addition include balb/c as a resistant murine strain (morissette et al. ; tam et al. ) . moreover, a different classification for c bl/ n susceptibility to chronic infection was reported, probably due to the use of different bacterial strains embedded in agar-beads. to define possible causes for predisposition, immune-related events possibly linked to diverse p. aeruginosa susceptibility and resistance have been investigated. morissette et al. reported that in a model of p. aeruginosa chronic infection, leukocyte recruitment in the early phases of infection was higher in resistant hosts compared to susceptible ones. this could account for a differential predisposition to the development of chronic infection (morissette et al. ; spagnuolo et al. ) . in line with this work, we found in the early phase of chronic infection that neutrophils were similar in the lungs of susceptible a/j and resistant c h/heouj strains, despite higher bacterial loads in susceptible mice; in addition, resistant mice showed higher alveolar macrophage numbers. immunity mediated by t-helper (th) cell subsets, such as cd + t cells, may play a critical role in host protection vs immunopathology of infectious diseases. early studies in the seaweed alginate beads model had defined th (ifnγ -producing cd + t) response of resistant c h/hen mice as being associated with a better disease outcome when compared to the th (il -producing cd + t) response of susceptible balb/c mice (moser et al. (moser et al. , . while the th /th paradigm dominated the field of p. aeruginosaassociated immunological responses, the discovery of a third subset of il- -producing effector th cells has fostered new hypotheses. indeed, we have recently demonstrated that the il- -mediated immune response may be beneficial in the early phase chronic infection by contrasting p. aeruginosa, while its overload can be detrimental in the advanced phase by exacerbating immunopathology (lore et al. a, b) . in line with this report, by comparing different murine inbred strains, we showed that the high clearance of p. aeruginosa and the progressive resolution of inflammation in resistant c h/heouj mice was associated with early superior lung infiltration by an il -dominated t-cell response . instead, il -producing cd + t cells were negligible in both resistant c h/heouj and susceptible a/j mice, while levels of ifnγ -producing cd + t cells were similar in the early stage chronic infection. in the late phase, susceptible a/j mice showed a massive infiltration of the lung by cd + il + cells compared to p. aeruginosaresistant strain associated with an exacerbated immunopathology. thus, host genetic backgrounds predisposing to the modulation of il -mediated immunity can determine the outcomes of early and advanced chronic p. aeruginosa respiratory infection. these results open up new avenues for translational studies aimed at testing new therapeutic approaches. as in the context of acute respiratory infection, mouse strains presenting deviant disease and immunological phenotypes can be further exploited to identify genetic loci underlying host susceptibility to chronic respiratory infection. at the time of writing, human and murine studies addressing the role of genetic traits associated with chronic respiratory infection are limited. thus, future efforts exploiting new murine population (e.g., cc mice) may contribute to new knowledge in this field. the use of ex-vivo models has been exploited but not widely implemented to investigate the cellular/molecular mechanisms underlying the impact of host genetics on the susceptibility to p. aeruginosa infection (table ) . innate immune recognition and clearance mechanisms play a critical role in controlling p. aeruginosa. the host genetic background could influence the neutrophil and macrophage capacity to phagocytize and kill p. aeruginosa. despite an initial increase in phagocytosis, bone-marrow-derived macrophages of susceptible dba/ displayed a severely reduced bactericidal capacity compared to those of c bl/ macrophages resistant to p. aeruginosa infection (wilson et al. ). differently, the in vitro bactericidal activity of resident and inflammatory alveolar and interstitial macrophages recruited did not differ between susceptible dba/ and balb/c mice resistant to p. aeruginosa infection (morissette et al. ) . the different origins of macrophages (bone marrow vs lung) could explain the different results of these reports. however, in our recent report, we did not find differences either in phagocytizing or bactericidal activities of bone-marrow-derived neutrophils and peritoneal macrophages from susceptible a/j and resistant c h/heouj mice, suggesting that the different outcomes of diverse genetic backgrounds to p. aeruginosa infection cannot be assigned to the bactericidal activity of phagocytosing cells . it is worth noting that the tissue origin may account for a different stage of neutrophil differentiation and function. thus, future studies focusing on neutrophil cells isolated from murine lungs or airways during an inflammatory response should light on the role of the host genetic background in this context. immune cells derived from susceptible and resistant mouse strains could produce different amounts of cytokines/ chemokines thus impacting on leukocyte recruitment. it has been demonstrated that alveolar macrophages from resistant balb/c mice produce significantly higher levels of tnf-α following stimulation with heat-killed p. aeruginosa compared to those from susceptible c bl/ mice (sapru et al. ) . bone-marrow-derived macrophages from a/j mice secrete less il- β protein than those from c bl/ and balb/c mice when stimulated in vitro by p. aeruginosa (miao et al. ) . the genetic background may also influence t-cell responses. splenocytes from a/j and c bl/ mice were found to be high responders to proliferation stimulated in vitro by p. aeruginosa exoenzyme s, while balb/c mice were low responders (barclay et al. ) . taking into consideration that the authors identified a/j and c bl/ mice as susceptible to p. aeruginosa infection and balb/c mice as resistant, they speculated that lymphocyte proliferation could impair the host response to p. aeruginosa. differently, it has been shown that heat-killed p. aeruginosa induced significantly higher in vitro proliferative responses of lung t cells from resistant balb/c than those from susceptible c bl/ mice (stevenson et al. ) . however, the differences between the last two reports could result from the origin of t cells (spleen vs lung) as well as the use of different p. aeruginosa factors. afterwards, the latter group showed that t-cell clones derived from lymph nodes and lungs of different mouse strains produced different cytokines following treatment with heat-killed p. aeruginosa. t-cell clones from resistant balb/c mice produced more il- and il- and were annotated as th -like cells, while those from susceptible c bl/ mice produced more ifn-γ and were considered th -like cells (kondratieva et al. ) . other studies on ex-vivo polarization of th cells from different mouse strains showed divergent results. splenic cells from resistant c h/hen mice produced more ifn-γ following stimulation with p. aeruginosa outer membrane protein, while those from susceptible balb/c mice produced more il- (moser et al. ) . the th polarization in resistant mouse strains is in line with the observation that this type of response is commonly associated with p. aeruginosa clearance or with a milder course of the infection (yonker et al. ) . it is clear from these ex-vivo studies that several contrasting results have been reported probably reflecting the complexity of the host-p. aeruginosa interplay. differences among studies may reflect experimental models of infection, the inbred mouse sub-strains, the use of different p. aeruginosa strains or its products, the type of cells stimulated, their physical origin, the timing and other unconsidered confounders. the relevance of the inter-individual genetic variation, intended as the diversity of host genetic backgrounds or host genetic modifier locus/i, has emerged in the context of respiratory infection by p. aeruginosa. several proofs have been produced thanks to the complementary findings of both human and animal studies. however, additional efforts are required to clearly identify the candidate host genetic modifier locus/i with translational potential. as discussed in this review, genetics-based variability in the disease outcomes evidenced by animal models, and in particular different inbred murine strains, represents important progress in the field, at least in the context of p. aeruginosa infection. overall, these studies highlight how mouse models can be exploited to mirror host genetic diversity and the associated host defense variations. future studies may exploit cc mice and mouse models of p. aeruginosa infection and will reveal genetic loci associated with the severity of acute or chronic lung infection as previously demonstrated for other respiratory pathogens (ferris et al. ; gralinski et al. ) . further efforts should aim at confirming the potential biological and translational relevance in humans in the context of respiratory infection induced by p. aeruginosa. regarding rare monogenic diseases characterized by airway infection (e.g., cf), new mouse models mirroring inter-individual genetic variation are essential to advance or complement human studies, currently limited by the small size of cohorts and phenotypic data. once more, the cc or the diversity outbred (do) mice represent strong tools for the future to model genetic diversity of monogenic diseases, providing new knowledge in this research area. moreover, refined mouse models of infection may prove to be essential to determine the relevance of genetic diversity in modulating differential activation of the immune system during p. aeruginosa respiratory infection. as we discussed in this review, previous studies had highlighted the potential role of mouse inbred strains in this field, although they do not completely reflect the host genetic variability in humans. in this regard, to our knowledge, no data from ex-vivo studies on cc-or do-derived cells stimulated by p. aeruginosa have been published. however, the availability of powerful tools, as cc and do mice, may be essential to associate in vivo phenotypic descriptive investigations to in vitro mechanistic studies in these populations to identify pathways involved in the host susceptibility to p. aeruginosa lung infection. in conclusion, further efforts are required to correlate human and animal data. the technological advancement of dna sequencing and analysis both in humans and animals has given a significant boost to studies in this field. this will allow the scientific community to identify novel candidate modifiers of genetic loci or genes with a translational clinical relevance in the context of p. aeruginosa respiratory infection. pseudomonas aeruginosa exoenzyme s stimulates murine lymphocyte proliferation in vitro murine models of acute and chronic lung infection with cystic fibrosis pathogens human genetic susceptibility to infectious influences the response to the opportunistic pseudomonas aeruginosa infection altering cell-mediated immunity and bacterial replication mapping genetic determinants of host susceptibility to pseudomonas aeruginosa lung infection in mice complex two-gene modulation of lung disease severity in children with cystic fibrosis modulatory effect of the slc a gene on susceptibility to infections and pulmonary function in children with cystic fibrosis exome sequencing of extreme phenotypes identifies dctn as a modifier of chronic pseudomonas aeruginosa infection in cystic fibrosis exome sequencing of phenotypic extremes identifies cav and tmc as interacting modifiers of chronic pseudomonas aeruginosa infection in cystic fibrosis modeling host genetic regulation of influenza pathogenesis in the collaborative cross il gene as modifier of cystic fibrosis: unraveling the factors which influence clinical variability pseudomonas aeruginosa: new insights into pathogenesis and host defenses genetic diversity in the collaborative cross model recapitulates human west nile virus disease outcomes genome wide identification of sars-cov susceptibility loci using the collaborative cross forward genetic dissection of innate response to infection in inbred mouse strains: selected success stories polymorphisms in the lectin pathway genes as a possible cause of early chronic pseudomonas aeruginosa colonization in cystic fibrosis patients characterization of t cell clones derived from lymph nodes and lungs of pseudomonas aeruginosa-susceptible and resistant mice following immunization with heat-killed bacteria host genetic diversity influences the severity of pseudomonas aeruginosa pneumonia in the collaborative cross mice the il- a/il- ra axis in pulmonary defence and immunopathology il- a impairs host tolerance during airway chronic infection by pseudomonas aeruginosa pseudomonas aeruginosa activates caspase through ipaf endobronchial inflammation following pseudomonas aeruginosa infection in resistant and susceptible strains of mice lung phagocyte bactericidal function in strains of mice resistant and susceptible to pseudomonas aeruginosa chronic pseudomonas aeruginosa lung infection is more severe in th responding balb/c mice compared to th responding c h/hen mice early immune response in susceptible and resistant mice strains with chronic pseudomonas aeruginosa lung infection determines the type of t-helper cell response association of clinical severity of cystic fibrosis with variants in the slc gene family (slc a , slc a , slc a and slc a ) the mouse as a model for human biology: a resource guide for complex trait analysis host genetic diversity enables ebola hemorrhagic fever pathogenesis and resistance quantitative and qualitative differences in bronchoalveolar inflammatory cells in pseudomonas aeruginosa-resistant and -susceptible mice two ways to survive infection: what resistance and tolerance can teach us about treating infectious diseases tuberculosis susceptibility and vaccine protection are independently controlled by host genotype the host genetic background defines diverse immune-reactivity and susceptibility to chronic pseudomonas aeruginosa respiratory infection in vitro and in vivo t cell responses in mice during bronchopulmonary infection with mucoid pseudomonas aeruginosa characterization of chronic bronchopulmonary pseudomonas aeruginosa infection in resistant and susceptible inbred mouse strains forward genetic dissection of immunity to infection in the mouse genetic influences on cystic fibrosis lung disease severity defect in early lung defence against pseudomonas aeruginosa in dba/ mice is associated with acute inflammatory lung injury and reduced bactericidal activity in naive macrophages host-pathogen interplay in the respiratory environment of cystic fibrosis identification of new loci involved in the host susceptibility to salmonella typhimurium in collaborative cross mice key: cord- - t o u authors: borkenhagen, laura k; wang, guo-lin; simmons, ryan a; bi, zhen-qiang; lu, bing; wang, xian-jun; wang, chuang-xin; chen, shan-hui; song, shao-xia; li, min; zhao, teng; wu, meng-na; park, lawrence p; cao, wu-chun; ma, mai-juan; gray, gregory c title: high risk of influenza virus infection among swine workers: examining a dynamic cohort in china date: - - journal: clin infect dis doi: . /cid/ciz sha: doc_id: cord_uid: t o u background: china is thought to be a hotspot for zoonotic influenza virus emergence, yet there have been few prospective studies examining the occupational risks of such infections. methods: we present the first years of data collected from a -year, prospective, cohort study of swine-exposed and -unexposed participants at swine farms in china. we conducted serological and virological surveillance to examine evidence for swine influenza a virus infection in humans. results: of the participants ( swine-exposed and swine-unexposed), ( . %) seroconverted against at least swine influenza virus subtype (swine h n or h n ). swine-exposed participants’ microneutralization titers, especially those enrolled at confined animal feeding operations (cafos), were higher against the swine h n virus than were other participants at and months. despite elevated titers, among the study subjects for whom we had complete follow-up, participants working at swine cafos had significantly greater odds of seroconverting against both the swine h n (odds ratio [or] . , % confidence interval [ci] . – . ) and swine h n (or . , % ci . – . ) viruses, compared to unexposed and non-cafo swine workers with less intense swine exposure. conclusions: while some of the observed increased risk against swine viruses may have been explained by exposure to human influenza strains, study data suggest that even with elevated preexisting antibodies, swine-exposed workers were at high risk of infection with enzootic swine influenza a viruses. since the s, china has led the world in swine farming. currently, china produces nearly half of the world's pork supply (approximately million head in ) [ , ] and the number of pigs continues to grow as china moves from small-scale swine farms to industrial farming practices [ ] . these large, industrial swine farms, commonly known as confined animal feeding operations (cafos), are thought to be sites for novel pathogen emergence and transmission. in recent years, swine pathogen epizootics in china have included porcine reproductive and respiratory syndrome virus [ ] , porcine epidemic diarrhea virus [ ] , swine acute diarrhea syndrome coronavirus [ ] , and, most recently, a - massive outbreak of african swine fever [ ] . beyond these swine-only pathogens, swine zoonotic pathogens threaten to harm not only the animals, but also the people exposed to them. in swine cafos, the continual introduction of naive animals, through onsite farrowing or the frequent introduction of new pigs, sustains the pathogen prevalences within a large farm by providing opportunities for pathogens to move from barn to another. in particular, this is true for influenza a viruses (iavs). the sustained circulation of iavs and often poor biosecurity provides great opportunity for swine iavs to reassort or recombine with avian or human iavs, which can result in novel progeny-virus zoonotic transmission events [ , ] . previous such events have underlined the importance of pigs in the generation of novel iavs, including the pandemic outbreak in [ ] . still, there are relatively few robust, prospective studies examining the risk of zoonotic transmission of swine iavs to swine workers [ ] . even fewer studies have captured the incidences of asymptomatic swine influenza virus (siv) infections among humans through routine surveillance [ , ] . in this -year ( - ) prospective cohort study of swine workers in china, we are employing virological and immunological surveillance to identify patterns of iav emergence and transmission. the primary aim of our study is to identify the demographic and occupational risk factors for incident siv infections in humans through influenza-like illness (ili) surveillance and serological evidence of exposure to sivs. secondarily, we seek to examine the use of serological and mucosal immunities as biomarkers for protection against siv infections in humans. results from participant enrollment and year of ili surveillance have been previously reported [ , ] . here, we augment these results with data from the subsequent months of follow-up. as previously reported [ , ] , swine-exposed participants ("exposed") were enrolled at the beginning in march of at swine cafos; small, house-holding swine farms; abattoirs, veterinary station; and animal market in shandong and jiangsu provinces in china. we also enrolled nonswine-exposed participants ("unexposed") in these regions in . participants were asked to complete an enrollment survey with questions pertaining to demographics, health status, type of work performed, animal exposure, and personal protective equipment use. participants were also asked to permit the collection of a ml blood draw. annual follow-up visits took place in march of ( months) and march of ( months) to collect survey data and blood samples. replacements for participants lost to follow-up were enrolled as necessary to maintain a similar sample size. throughout the -month study period, participants were asked to contact research staff within hours of developing an ili event (acute onset of a respiratory illness with a measured oral temperature ≥ . °c and a sore throat or cough for > hours) and were also contacted weekly by study staff to monitor for such events. if a participant reported an ili event, they were asked to permit the collection of a nasal swab and nasal wash, as well as a ml blood draw, within days of the report (acute). a second blood draw was collected more than days after the illness (convalescent). nasal washes were performed by irrigating nostril with saline while the participant's head was tilted back to a ° angle; they were then coached to tilt forward as the return fluid was collected, and the procedure was repeated with the second nostril. we employed influenza a virus molecular detection, microneutralization (mn), and immunoglobin a (iga) assays, as described in our previous study [ ] . to assess the possibility of neutralizing-antibody cross-reactivity against circulating human influenza strains, the same mn assay procedure was performed using sera drawn at and months from a random subset of participants ( exposed and unexposed). a detailed description is in the supplementary information. we used standard descriptive, bivariate, and multivariable risk factor analyses to compare various subgroups of participants. the primary outcomes were seroconversion against the swine h n and h n viruses. our primary modeling framework was a complete case analysis, based on all individuals with full follow-ups through months, using a multivariable logistic regression model to estimate the association between various demographic and occupational risk factors and seroconversion against each virus. generalized estimating equations adjusted for the correlation of participants within a swine facility and within participants over time. inverse probability weighting (ipw) was used to adjust the complete case analysis for possible biases arising from losses to follow-up. a variable selection procedure was applied to find a parsimonious model. see the supplementary information for additional details. the industrialization of pork production in china has led to significant consolidation and the shut down of millions of farms since [ ] ; in , of the farms enrolled in this study were closed, and have since been replaced ( figure ). replacements for participants lost to follow-up were enrolled to maintain a similar sample size. of the participants enrolled at any time point ( exposed and unexposed), ( . %) were followed for months ( exposed and unexposed), ( . %) were followed for months ( exposed and unexposed), and ( . %) were observed at a single time point ( exposed and unexposed; figure ). the complete case analysis was based on the participants ( exposed and unexposed) with follow-ups through months. detailed descriptions of the cohort characteristics and changes over time can be found in the supplementary information. considering sera collected at , , and months, ( . %) of the enrolled participants (open cohort) seroconverted against at least iav subtype, with a total of seroconversion events ( table ) . of these events, ( . %) occurred in individuals enrolled from cafos, ( . %) in individuals enrolled from non-cafo swine exposure sites (ie, small, house-holding swine farms; abattoirs; a veterinary station; and an animal market), and ( . %) occurred in swineunexposed individuals. there were seroconversion events ( . %) against swine h n , ( . %) against swine h n , ( . %) against avian h n , ( . %) against avian h n , and ( . %) against avian h n . there were no observed seroconversions against avian h n . the multivariable model results for h n and h n seroconversion in the complete case cohort are shown in table . variable selection revealed several baseline characteristics significantly associated with seroconversion; see the supplementary information for a full list of risk factors considered. participants with elevated (≥ ) baseline h n mn titers had lower odds of seroconversion (odds ratio [or] . , % confidence interval [ci] . -. ), as did participants who had taken medication in the prior days (or . , % ci . -. ) or who reported a respiratory infection in their household in the prior months (or . , % ci . -. ; table ). there was an association between higher h n -specific iga titers at baseline and future seroconversion (or . for an increase of standard deviation in h n specific iga, % ci . - . ). similar to as in h n titers, individuals with elevated baseline h n mn titers had lower odds of seroconverting against h n (or . , % ci . -. ). seroconversion against h n was also found to be positively associated with a participant's report of an outbreak of illness among animals at work in the prior days (or . , % ci . - . ; table ). after including ipw, only the enrollment site type and baseline mn titer maintained statistically significant associations with seroconversion against h n , though the point estimates for all risk factors remained similar. for h n , ipw did not significantly alter the point estimates or cis for the included risk factors. detailed multivariable modeling results from the open cohort are available in the supplementary information. in the random sample of participants additionally tested with mn assays against seasonal human h n and h n viruses, ( . %) seroconverted against swine h n , ( . %) seroconverted against human h n , ( . %) seroconverted against swine h n , and ( . %) seroconverted against human h n . examining possible cross-reactivity, the odds of participants who seroconverted against human h n also seroconverting against swine h n were . ( % ci . - . ). similarly, the odds of participants who seroconverted against human h n also seroconverting against swine h n were . ( % ci . - . ). in the open cohort, the swine-exposed participants were less likely than unexposed participants to seroconvert against sivs; adjusted ors for seroconversion against swine h n and swine h n were . ( % ci . -. ) and . ( % ci . -. ), respectively ( table ). this effect was also observed for seroconversion against either or both sivs (or . , % ci . -. ), after adjusting for differences in follow-ups using the number of follow-up visits as a proxy for person-time at risk (or . , % ci . -. ). stratification of the exposed group into those exposed and not exposed to a cafo revealed no significant differences in seroconversion against the swine h n virus for the exposure groups. however, the geometric mean of mn titers against swine h n at and months were the same or higher among participants enrolled at cafo facilities, compared both to those enrolled at non-cafo swine facilities and to unexposed participants (table ) . conversely, higher odds of seroconversion against swine h n virus were observed among the unexposed (or . , % ci . - . ) and cafo-exposed (or . , % ci . - . ), when compared to the non-cafo swine workers; the unexposed participants had higher geometric mean mn titers, compared to the other groups, for all time points (table ) . there was no correlation between years of swine exposure and seroconversion against swine h n at enrollment, and no differences in this relationship by enrollment site type. a negative correlation was observed between years of swine exposure and seroconversion against swine h n after enrollment (spearman ρ = − . , % ci −. to −. ). among cafo swine workers, the spearman correlation for seroconversion against swine h n was − . ( % ci −. to . ); however, among non-cafo swine workers, the spearman correlation for the same outcome was − . ( % ci −. to −. ). follow-up n= figure . diagram of enrollments, replacements, and losses to follow-up for this longitudinal study. participants exposed and unexposed (unexp.) to swine were followed for influenza-like illness and sampled every months for months. in the complete case cohort, cafo swine workers had significantly greater odds of seroconverting against swine h n , compared to either non-cafo swine workers (or . , % ci . - . ) or unexposed participants (or . , % ci . - . ; table ). cafo swine workers had higher odds (though not statistically significant) of seroconverting against h n than unexposed participants (or . , % ci . - . ) and significantly higher odds of seroconverting against h n than non-cafo swine workers (or . , % ci . - . ; table ). further, cafo swine workers had higher odds of seroconverting against h n (or . , % ci . - . ) and h n (or . , % ci . - . ) when compared to both unexposed participants and non-cafo swine workers with less intense swine exposure. between and months of follow-up, ili events were identified among exposed and unexposed participants out of the open cohort, for incidence proportions of . and . , respectively (table ) . of these participants, were enrolled at baseline and followed through months, was lost to follow-up before their -month visit, and was enrolled in and thus only had months of available follow-up. among the exposed participants, worked at a swine cafo, worked at house-holding swine farms, and worked at abattoirs. the average number of pigs housed per day among the enrollment sites where these participants were enrolled was , compared to a lower average of pigs among the enrollment sites of the exposed participants who did not have recorded ilis in this time period. all but of these events occurred between december and january . molecular evidence of iav was detected from either a nasal wash or swab specimen in participants ( unexposed, exposed). there was exposed participant who seroconverted against swine h n , ( exposed and unexposed) who seroconverted against swine h n , and exposed participants who seroconverted against both swine h n and h n viruses. in this report, we present the first months of data from a -year prospective, cohort study of iav among participants exposed and unexposed to swine in china. while we were able to replace the participants lost to follow-up for a consistent sample size for the first months, by months, we were unable to maintain the original target sample size. in general, older and less educated participants were more often lost to follow-up. the characteristics of replacement participants were different for exposed and unexposed groups over time. in both follow-up visits at and months, new, unexposed participants were more likely to be female and more likely to have reported at least respiratory tract infection in the past months. this influx of participants with a history of respiratory tract infection and possible exposure to iav, as well as disproportionate follow-ups between the exposed and unexposed groups, may be partially responsible for the elevated titers and higher number of seroconversions observed in the unexposed group. thus, we considered the open cohort in several ways to explore relationships between the follow-up time and the history and extent of exposure. in addition to comparing the exposed and unexposed groups, an adjustment for person-time showed little change in the results; we suspect there were too few observation times (up to ) for a meaningful effect to be observed with this adjustment. though the number of reported ili events (n = ) was too low to perform statistical analyses, more ili events were observed among the swine-exposed than -unexposed participants during the study period. these exposed participants worked at enrollment sites with higher average numbers of pigs housed per day, compared to exposed participants who did not experience an ili. along this line, we further stratified the exposed group into cafo and non-cafo workers, assuming that individuals ( ) swine-exposed and enrolled at cafo (exposed to cafo swine); ( ) swine-exposed and enrolled at sites other than cafo (not exposed to cafo); and ( ) not swine-exposed (unexposed). non-cafo sites included house-holding swine farms, abattoirs, a veterinary station, and an animal market. samples were collected at (baseline), , and months, with additional acute and convalescent sera samples collected at individually reported influenza-like illness events between follow-up visits. seroconversion against each virus was defined as a -fold rise in titer, relative to any previously gathered sample, and titer value ≥ . predicted probabilities and ors were calculated from a univariate logistic regression model using empirical (sandwich) covariance estimates to adjust for repeated measurements within individuals over time. abbreviations: σ, standard deviation; cafo, confined animal feeding operations; ci, confidence interval; h n or h n , swine influenza virus subtype; or, odds ratio; ref., referent group. data are stratified by swine-exposed (exposed) and non-swine exposed (unexposed) participants. sera samples were collected from exposed (n = ) and working at cafos have more intense exposure to large populations of pigs. here, we observed conflicting results, with no significant association with seroconversion against swine h n by enrollment site type and increased odds of seroconversion against swine h n among unexposed participants. we hypothesized that some of this effect for swine h n might have been due to cross-reactivity with this human subtype, and this was supported by the subset of sera samples we tested that demonstrated higher odds of seroconversion against human h n if the participant had also seroconverted against swine h n , a trend that was not observed for h n . finally, the complete case analysis we conducted among participants followed through months demonstrated results more consistent with previous literature [ , [ ] [ ] [ ] [ ] . the results obtained from the complete case analysis aligned with those from the first year of this study [ ] , as well as results of a similar cross-sectional study of swine workers in southern china [ ] that demonstrated elevated antibody titers for sivs among swine-exposed participants. despite these elevated titers, the longitudinal data still showed that cafo swine workers had a significantly higher risk of seroconverting against swine h n , compared to non-cafo swine workers (or . , % ci . - . ) and unexposed participants (or . , % ci . - . ). this is similar to results from a longitudinal study of swine workers in iowa, which found that swine-exposed individuals (or . , % ci . - . )-and even their nonexposed spouses (or . , % ci . - . )had a higher risk of seroconverting against swine h n virus than other, nonexposed individuals [ ] . while exposed participants were not found to be at a higher risk of seroconversion against swine h n virus, the ors trended in that direction, and we present evidence that suggests some of this effect may be due to cross-reactivity with the seasonal h n virus in humans. although complete case analyses are known to be biased when the data are not missing completely at random, there are several reasons our analysis is justified here. first, our primary outcome measure, by definition, required a minimum of follow-up measurements; therefore, participants observed only at enrollment could not contribute to an analysis of seroconversion. participants observed at only time points are similarly sera samples were collected from participants who were exposed to swine and who were not exposed to swine (unexposed) in the shandong and jiangsu provinces of china between march and december . swine-exposed participants were either enrolled at cafo facilities (exposed to cafo swine) or at non-cafo facilities (not exposed to cafo swine), including house-holding swine farms, abattoirs, a veterinary station, and an animal market. samples were collected at each of time points ( , , and months), with additional acute and convalescent sera samples collected at individually reported influenza-like illness events between follow-up visits. seroconversion against each virus was defined as a -fold rise in titer, relative to any previously gathered sample, and titer value ≥ . a multivariable logistic regression using empirical (sandwich) covariance estimates was used to adjust for repeated measures over time. abbreviations: cafo, confined animal feeding operations; ci, confidence interval; h n or h n , swine influenza virus subtype; iga, immunoglobin a; mn, microneutralization; or, odds ratio; ref., referent group; sd, standard deviation. less likely to have a seroconversion event, since individuals with time points have more opportunities to seroconvert. further, we observed systematic differences in demographicand exposure-related characteristics between participants who dropped out, participants who remained in the study, and participants recruited as replacement subjects at subsequent time points. overcoming these issues would require strict assumptions about the missing data mechanisms (eg, multiple imputation), which we did not feel were tenable. thus, we opted for a pragmatic approach, reporting the complete case analyses as our primary models, with sensitivity analyses using ipw. even with considerable losses to follow up in the original cohort, compelling analyses were obtained from the study subjects with complete data. among these participants, the most intensively swine-exposed participants (cafo swine workers) had higher titers against swine h n virus and yet higher odds of seroconversion during follow-up to both swine h n and the swine h n viruses. while some of this risk of seroconversion against swine h n could likely be explained by cross-reactivity against human h n iav, these data support the premise that persons with intense occupational exposure to pigs are at high risk (ors ranging from - , depending upon the occupational comparison group) of infection with enzootic swine viruses. it seems logical, then, that more efforts should be made to conduct surveillance for novel iavs, which may first emerge at cafo human-swine interfaces. supplementary materials are available at clinical infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. china's pork imports rise along with production costs united states department of agriculture foreign agricultural services united states department of agriculture foreign agricultural services. peoples republic of china livestock and products annual: multiple outbreaks of african swine fever create uncertainty for the world's largest pork producer origin of highly pathogenic 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increased risk of infection with zoonotic influenza virus? serologic evidence of h swine influenza virus infection in swine farm residents and employees serological evidence and risk factors for swine influenza infections among chinese swine workers in guangdong province acknowledgments. the authors thank the professionals from the licheng district center for disease control and prevention (cdc), jinan, china; the shandong provincial cdc, junan, china; and the wuxi cdc, wuxi, china, who greatly helped in engaging the swine farms and in the collection of samples. they thank the farm managers and the study participants for their much-appreciated cooperation.disclaimer. the contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of the funding sources listed. potential conflicts of interest. all authors report no potential conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord- -zrdsr nh authors: beigel, john h; voell, jocelyn; kumar, parag; raviprakash, kanakatte; wu, hua; jiao, jin-an; sullivan, eddie; luke, thomas; davey, richard t title: safety and tolerability of a novel, polyclonal human anti-mers coronavirus antibody produced from transchromosomic cattle: a phase randomised, double-blind, single-dose-escalation study date: - - journal: the lancet infectious diseases doi: . /s - ( ) - sha: doc_id: cord_uid: zrdsr nh summary background middle east respiratory syndrome (mers) is a severe respiratory illness with an overall mortality of %. there is no licensed or proven treatment. passive immunotherapy approaches are being developed to prevent and treat several human medical conditions where alternative therapeutic options are absent. we report the safety of a fully human polyclonal igg antibody (sab- ) produced from the hyperimmune plasma of transchromosomic cattle immunised with a mers coronavirus vaccine. methods we did a phase double-blind, placebo-controlled, single-dose escalation trial at the national institutes of health clinical center. we recruited healthy participants aged – years who had normal laboratory parameters at enrolment, a body-mass index of – kg/m , and a creatinine clearance of ml/min or more, and who did not have any chronic medical problems that required daily oral medications, a positive rheumatoid factor (≥ iu/ml), iga deficiency (< mg/dl), or history of allergy to intravenous immunoglobulin or human blood products. participants were randomly assigned by a computer-generated table, made by a masked pharmacist, to one of six cohorts (containing between three and ten participants each). cohorts and had three participants, randomly assigned : to receive active drug sab- versus normal saline placebo; cohorts and had six participants randomised : ; and cohorts and had ten participants, randomised : . participants received mg/kg, · mg/kg, mg/kg, mg/kg, mg/kg, or mg/kg of sab- , or equivalent volume placebo (saline control), on day , and were followed up by clinical, laboratory, and pharmacokinetic assessments on days , , , , , and . the primary outcome was safety, and immunogenicity was a secondary outcome. we analysed the intention-to-treat population. this trial is registered with clinicaltrials.gov, number nct . findings between june , , and jan , , we screened participants, of whom were eligible and randomly assigned to receive sab- (n= ) or placebo (n= ). adverse events were reported: adverse events occurred in ( %) of participants receiving sab- (mean · adverse events per participant). adverse events occurred in all ten participants receiving placebo (mean · adverse events per participant). the most common adverse events were headache (n= [ %] in participants who received sab- and n= [ %] in those receiving placebo), albuminuria (n= [ %] vs n= [ %]), myalgia (n= [ %] vs n= [ %]), increased creatine kinase (n= [ %] vs [ %]), and common cold (n= [ %] vs n= [ %]). there was one serious adverse event (hospital admission for suicide attempt) in one participant who received mg/kg of sab- . the area under the concentration–time curve (auc) in the mg/kg dose ( μg × days per ml) is comparable to the auc that was associated with efficacy in a preclinical model. interpretation single infusions of sab- up to mg/kg appear to be safe and well tolerated in healthy participants. human immunoglobulin derived from transchromosomic cattle could offer a new platform technology to produce fully human polyclonal igg antibodies for other medical conditions. funding national institute of allergy and infectious diseases, national institutes of health, and biomedical advanced research and development authority. middle east respiratory syndrome (mers) is a severe respiratory illness caused by a novel coronavirus (cov). the spectrum of clinical illness ranges from asymptomatic illness to a severe acute respiratory disease requiring intensive care and mechanical ventilation. mers has an overall mortality of % and there is no licensed or proven treatment. the use of immune anti-mers coronavirus (mers-cov) plasma has been suggested as one potential therapeutic approach. however, it is difficult to collect sufficient anti-mers-cov plasma from infected patients to implement this strategy. the use of passive immune therapy, either in the form of direct plasma infusion or intravenous immunoglobulin, is often invoked both for emerging infectious diseases, such as ebola virus disease, as well as more common diseases with high morbidity (despite the availability of antiviral therapeutics), such as severe influenza. however, the use of immune plasma in all of these conditions has similar constraints. consistent production of large quantities of anti-pathogen-neutralising human plasma or immunoglobulin requires collection of plasma from convalescent or vaccinated human volunteers. the limited plasma supply restricts wide implementation of these therapeutics. one novel alternative method of manufacturing neutralising intravenous antibodies of consistently high affinity and avidity is to use the hyperimmune plasma of transchromosomic cattle, which produce highly potent and antigen-specific, fully human polyclonal igg de novo, and which mount a robust antibody immune response after vaccination. to develop the transchromosomic cattle, a human artificial chromosome was constructed that comprised the entire human immunoglobulin gene repertoire (human immunoglobulin heavy chain [igh] and human κ light chain [igk] ), which resides on two different chromosomes in human beings, specifically the igh locus from chromosome and the igk locus from chromosome . the immunoglobulin gene repertoires from the human artificial chromosome provide the large diversity of human polyclonal antibodies. to avoid possible human-bovine interspecies incompatibility between the human immunoglobulin-μ-chain protein (higm) and bovine transmembrane α and β immunoglobulins (bigα and bigβ) in the pre-b-cell-receptor complex, the higm constant domain was partly replaced with the counterpart of bovine igm (bigm). furthermore, the dna regulatory elements iγ and sγ were bovinised on the human artificial chromosome to facilitate dna-protein interactions in the bovine b cell. this human artificial chromosome is designated as iskchac∆, and is transferred to bovine fibroblasts that are homozygous triple knockouts of the two bovine immunoglobulin-μ heavy-chain loci (bighm and bighml ) and the bovine λ light-chain locus (bigl). the triple-knockout bovine fibroblasts carrying the human artificial chromosome were used as nuclear donors to clone transchromosomic cattle. the mers-cov spike protein attaches to the host-cell receptor cd (also known as dipeptidyl peptidase or dpp ), and mediates subsequent cell fusion. vaccination of mice with the plasmid vaccines expressing the spike glycoprotein showed the generation of neutralising antibodies through blocking both the receptor binding and non-receptor-binding domains. seroconversion to the spike protein (measured by igg) was associated with resolution of clinical illness in patients with mers-cov infection, and the levels of anti-spike igg were inversely correlated with lower respiratory tract viral loads. for these reasons, purified al-hasa strain mers-cov spike protein nanoparticles (a clade b strain, manufactured by novavax inc, gaithersburg, md, usa) were chosen as the immunogen to generate an anti-mers-cov intravenous immunoglobulin in this transchromosomic bovine system (thereafter known as sab- ). the purpose of this phase study was to evaluate the safety, tolerability, and pharmacokinetics of sab- after a single infusion of sab- in healthy adults. this trial represents the first-in-human clinical study using a fully human antibody derived from transchromosomic cattle. we did this double-blind, placebo-controlled, single-dose escalation, phase randomised controlled trial at the national institutes of health (nih) clinical center (bethesda, md). the study was conducted in accordance with the applicable regulatory and international conference on harmonization-good clinical practice requirements. the study protocol was approved by the national institute of allergy and infectious diseases institutional review board (protocol -i- ). evidence before this study we searched pubmed on oct , , for studies using these search terms in the title or abstract: ("middle east respiratory syndrome" or "mers") and ("antivirals" or "treatment"). we restricted the article type to clinical trials and the species to human, and there were no language restrictions. we found no articles. after removing the search restriction of title or abstract for these terms, removing the species restrictions to human beings, and excluding studies that did not assess mers therapeutics (rather they mentioned it in the background or discussion), we found no previous publications of mers therapeutics in human beings. additionally, we searched for studies using the search terms ("intravenous immunoglobulin" or "igiv"), and ("bovine" or "cow"), and restricted the article type to clinical trials and the species to human. after excluding studies that did not assess bovine intravenous immunoglobulin, we identified no previous publications of mers therapeutics in humans. to our knowledge, this is the first study to show the safety, tolerability, and pharmacokinetics of intravenous immunoglobulin derived from transchromosomic cattle, and the first study to describe the safety of a putative therapeutic specifically for mers. the evidence from this study shows that single infusions of sab- up to mg/kg appear to be safe and well tolerated in healthy participants, and should be considered for further investigation in the treatment of mers. eligible participants were healthy adult men or women aged - years, with a body-mass index of - kg/m and a creatinine clearance of ml/min or more (calculated using the chronic kidney disease epidemiology collaboration formula). all participants were required to have normal laboratory parameters at enrolment, and could not have any chronic medical problem that required daily oral medications, a positive rheumatoid factor (defined as a rheumatoid factor ≥ iu/ml), or iga deficiency (defined as iga < mg/dl), nor any history of an allergy to intravenous immunoglobulin or human blood products. all participants were required to use two effective forms of contraception, at least one of which had to be a barrier method. a full list of exclusion criteria is included in the appendix. participants provided written informed consent before screening. an unmasked pharmacist at the nih clinical center pharmacy generated a blinded randomisation scheme before study initiation. after screening, study participants were enrolled sequentially in one of six cohorts. on the day of study drug dosing, the unmasked pharmacist determined treatment allocation by referring to the next position on the randomisation scheme. cohorts and had three participants, randomly assigned : to receive active drug sab- versus normal saline placebo; cohorts and had six participants randomly assigned : ; and cohorts and had ten participants, randomly assigned : . study participants and study team members remained masked throughout the entire study. the study treatment was provided in an infusion bag, the contents of which were obscured by an opaque covering over the bag and tubing. sab- was manufactured by sab biotherapeutics inc (sioux falls, sd, usa). two transchromosomic bovines were hyperimmunised with purified al-hasa strain mers-cov s protein nanoparticles (novavax inc; - mg/kg) formulated with sab's proprietary adjuvant formulation (sab-adj- ). the transchromosomic cattle were vaccinated five times at - -week intervals. hyperimmune plasma (up to · % of bodyweight) was collected from each transchromosomic cow on days , , and days after the third, fourth, and fifth vaccination. plasma was stored at - °c until use. additional sab- manufacturing methods are detailed in the appendix. sab- was supplied in vials containing mg per ml. the lot of sab- we used had an elisa binding titre of u/mg. we assayed sab- for its ability to neutralise mers-cov in vitro using a fluorescencereduction neutralising assay in vero e cells. the concentration of sab- at which % inhibition of relative fluorescence intensity was observed was · µg/ml. the doses used in the six cohorts were: mg/kg (cohort ; prepared at a concentration of mg/ml in normal saline), · mg/kg (cohort ; prepared at mg/ml), mg/kg (cohort ; prepared at mg/ml), mg/kg (cohort ; prepared at mg/ml), mg/kg (cohort ; prepared at mg/ml), and mg/kg (cohort ; prepared at mg/ml). participants randomly assigned to placebo received a normal saline control at a volume equivalent to receiving active drug above. the infusions were started at a rate of · ml/kg per h, escalating by · ml/kg per h increments every min to a maximum rate of - ml/kg per h, depending on cohort. in the highest dose cohort at the maximum infusion rate of mg/kg per h, this infusion was less than a tenth of the maximum rate of standard intravenous immunoglobulin as per administration guidelines at the nih clinical center (maximum mg/kg per h). the starting dose of mg/kg was -times lower than the maximum preclinical animal toxicity study dose of mg/kg. the target dose of mg/kg exceeds the effective doses in preclinical models, and is · -times lower than the maximum nonclinical dose (sab biotherapeutics inc, unpublished). participants were screened up to weeks before dosing. vital signs were obtained before the start of infusion, and then approximately and min after the start of the infusion, every min thereafter until the end of the infusion, and then every h for h after infusion. participants were seen on days , , , , , and . blood samples for the pharmacokinetic analysis were obtained at baseline, h after the end of infusion, h after the end of infusion, and on days , , , , , and . at each study visit, participants were questioned about new symptoms, their medical condition, and new medications. all new symptoms or abnormal laboratory results were captured as adverse events. we tested serum for sab- pharmacokinetics using an elisa assay and functional inhibition (pharmacodynamics) using a mers micro-neutralisation assay. we assessed immunogenicity by testing for anti-igg antibodies (using rheumatoid factor), sab- antidrug antibodies, anti-bovine κ antibodies, and anti-camelid antibodies (an antibody used in the manufacturing process; appendix). the primary outcome was safety-ie, the type and frequency of adverse events experienced by participants receiving sab- compared with placebo. secondary outcomes were the pharmacokinetic profile, mers virus neutralisation assay over time (pharmacodynamics), and immunogenicity. we determined the study sample size to detect an adverse event with a true rate of % and a probability of · in the cohorts with larger doses (cohort and ), whereas see online for appendix rarer events with a true rate of % could be detected with a probability of · . we did the analysis in the intention-to-treat population. we did not impute missing data. we analysed pharmacokinetic data using phoenix winnonlin software (version . ; cetara, st louis, mo) for both non-compartmental analysis and compartmental modelling. for the drug in the serum, key non-compartmental analysis pharmacokinetic parameters of interest included the maximum observed concentration (c max ); apparent elimination rate constant (λ z , determined by calculating the absolute value of the slope of the log-linear regression plasma concentration-time plot, with a minimum of three concentrations along the terminal phase); the elimination half-life (t ½ , calculated as · /λ z ); the area under the concentration-time curve (auc), from h to the last pharmacokinetic sample post-dose (auc last ) and to infinity (auc -∞ ), calculated with the linear-up, log-down trapezoidal rule; clearance calculated as dose/auc -∞ ; and volume of distribution, calculated as dose/(auc last × λ z ). we calculated geometric mean ratios and % cis to compare pharmacokinetic parameters between cohorts for assessment of dose linearity and proportionality. we tested structural models of one, two, and three compartments. to determine goodness of fit of the compartmental models, we visually inspected plots of observed and predicted concentrations versus time, and weighted residuals versus predicted concentrations, pharmacokinetic parameter coefficient of variation, and akaike information criteria. the two-compartment model that we selected was parameterised in terms of central compartment clearance, central compartment volume of distribution, inter-compartmental clearance, peripheral volume of distribution, and secondary parameters of distribution elimination rate and distributional half-life. we plotted the reciprocal of the highest % neutralisation titres over time and used them to calculate the auc last . we assessed the correlation of neutralisation titre auc last with sab- auc last using linear regression. this trial is registered with clinicaltrials.gov, number nct . employees of the sponsor of the study were involved with study design, data collection, data analysis, data interpretation, and writing of the report. the corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. between june , , and jan , , we screened participants for this study, of whom were sequentially randomly assigned to six cohorts (figure ). the study ended after being fully enrolled. after allocation to study groups, all participants completed the planned follow-up. baseline characteristics of the randomly assigned participants are shown in table . adverse events were reported: occurred in ( %) of participants receiving sab- (mean · adverse events per participant), and adverse events occurred in all ten participants receiving placebo (mean · adverse events per participant; table ). there was one grade adverse event of depression, and one grade adverse event of suicide attempt, both occurring in the same participant. there was one serious adverse event of hospital admission for a suicide attempt by the same participant who had received mg/kg of sab- , which, as the other two adverse events in this participant, was judged to be unrelated to study intervention. this participant had a history of depression for several years that was not disclosed at the time of screening, and the participant was not receiving medical care or treatment at the time of enrolment. the most common adverse events were headache, albuminuria, increased creatine kinase, common cold, myalgia, and low serum bicarbonate. most adverse events were noted in similar proportions in those receiving sab- and placebo (table ). there were two hypotensive events that occurred during study drug administration, both in cohort ( mg/kg of sab- ): one in a participant who received sab- and the other in one who received placebo. regarding the hypotensive event in the participant receiving sab- , min after the start of the infusion, the participant complained of feeling warm and slightly lethargic. blood pressure at the time was / (about mm hg lower than pre-infusion), and there was no increase in heart rate. the infusion was stopped for min with no other intervention, and the hypotension resolved. the infusion was restarted with no further episodes of hypotension. low serum bicarbonate was seen only in participants receiving sab- (three [ %] of ), and were only noted in cohorts ( mg/kg of sab- ) and ( mg/kg of sab- ). two vasovagal reactions occurred in participants assigned to receive sab- , dose-proportional increases in the parameters of c max and auc -∞ over the -fold range of doses from · mg/kg to mg/kg (table ) . we did not use pharmacokinetic data from the mg/kg cohort because a terminal phase could not be identified because of multiple concentrations that were below the limit of assay detection ( · µg/ml). mean concentration versus time profiles of the sab- cohorts at each dose are shown in figure a . sab- concentrations after infusion appeared to follow a bi-exponential decline and best fit a two-compartmental pharmacokinetic model. results for key pharmacokinetic parameters from non-compartmental analysis are shown in table and parameters obtained from the two-compartmental model are presented in the appendix. the average terminal elimination half-life (t / ) was within the range of a typical antibody in human beings (approximately days) across all dose cohorts, excluding the mg/kg cohort. both uncorrected and baselineadjusted serum immunoglobulin concentrations over time did not appear to correlate with dose of sab- administered or sab- pharmacokinetic concentrations (data not shown). we determined anti-mers neutralising antibody titres using the jordan-n / strain of mers-cov (a clade a strain). microneutralisation titres correlated with sab- concentrations in serum ( figure b) . the functional neutralisation of the mers virus seems to follow the pharmacokinetic exposure of sab- described previously (r²= · , p< · ). pharmacodynamic analysis using the microneutralisation titres showed similar results, with geometric mean titres in the mg/kg cohort of / at days and / at days (appendix). we investigated the immunogenicity of sab- in several ways. we assessed the presence of anti-igg antibodies using rheumatoid factor. one participant in cohort ( mg/kg of sab- ) became rheumatoidfactor positive on day , with a maximum rheumatoid factor of iu/ml. this positive rhematoid factor persisted throughout the study, and there were no associated symptoms with this finding (the only adverse event reported in this participant was albuminuria). we also assessed anti-drug antibody (anti-sab- ). three participants had anti-drug antibody present at baseline (before study drug administration), which persisted and did not change throughout the study (one participant in the mg/kg of sab- cohort and two in the mg/kg of sab- cohort). the pharmacokinetic parameters for these three participants were compared with the other participants in their respective cohorts, and there was no significant difference in t ½ , c max , or auc. all participants with preexisting anti-sab- had higher results for t ½ and auc than the cohort geometric mean. no new anti-drug antibodies were developed after administration of sab- . we assessed the presence of anti-bovine κ light chain, representing residual bovine proteins that might be present in the final intravenous immunoglobulin product. seven participants had antibodies to the bovine κ light chain detected at baseline. there was no development of new anti-bovine κ-light-chain antibodies after administration of sab- . we also assessed whether anti-camelid antibody was present, representing immunogenicity towards a llama anti-human κ-light-chain antibody used in the manufacturing process to purify sab- . no participants had anti-camelid antibody at baseline, or developed these antibodies during the study. sab- is a novel anti-mers-cov intravenous immunoglobulin manufactured from the hyperimmune plasma of transchromosomic cattle that produce fully human polyclonal igg. the use of passive immunotherapy, either as immune plasma or intravenous immunoglobulin, has been recommended for multiple severe and emerging infectious diseases such as severe seasonal influenza, pandemic influenza, severe acute respiratory syndrome, mers, and ebola. there are often limitations in collecting sufficient human plasma for production. , albeit novel, the transchromosomic bovine production system is, more importantly, rapid and scalable. in transchromosomic cattle, new antigen-specific and high-titre human intravenous immunoglobulin can be generated within months of a vaccine being available. therefore, our study is noteworthy not only by advancing a potential therapeutic for mers, but also by showing the potential safety of a novel production platform that can quickly generate a new putative drug candidate to an emerging infectious disease. the results of this first-in-human phase study suggest that sab- appears to be safe and well tolerated at single doses up to mg/kg. the adverse events seen in participants given sab- were largely comparable to those given placebo. in events occurring in more than one participant given sab- , low bicarbonate, fatigue, loose stools, sore throat, vasovagal reaction, and aspartate aminotransferase increase did not have any corresponding events in the placebo group. both vasovagal reactions occurred before study drug administration. only low blood bicarbonate and sore throat seemed to occur more commonly in the higher dose cohort. with a small number of participants (as is typical in a phase study), it is difficult to discern with certainty the attribution of adverse events to the study drug. these potential events will need to be investigated closely in any future studies. the pharmacokinetic profile of sab- is similar to what would be expected for human polyclonal or monoclonal antibodies with no human targets. in the critically ill population, the clinical illness of mers (defined by duration of hospital stay) is a median days (iqr - ), and mers-cov shedding (as detected by pcr) is median days ( % ci - ) in survivors and can exceed days in those that die from mers. the pharmacodynamic profile of sab- , with a neutralisation titre of / at days and / at days for mg/kg, suggests good viral neutralisation activity for a period that exceeds the clinical illness and viral shedding after administration of a single dose. the maximum concentration occurred in the initial sample ( h after the end of infusion), and sab- concentrations remained detectable at the end of days following single mg/kg, mg/kg, and mg/kg doses. of course, these might be different in an infected patient, in light of antibody-virus binding and subsequent alteration in pharmacokinetics or pharmacodynamics. the efficacy of sab- has previously been shown in an adenovirus human dipeptidyl peptidase (ad -hdpp )-transduced balb/c mouse model. ad -hdpp transduced balb/c mice were intranasally infected with mers-cov and given sab- at or h after infection. for mice given μg ( mg/kg, assuming g body weight for mice) of sab- at h post infection, mers-cov titres were below the level of detection at day (p< · ). when sab- was administered h after infection, viral titres were detectable but reduced by about -fold by day compared with the untreated control (p< · ). given that animal models of mers-cov challenge do not replicate human disease effectively, the relevance of this model to extrapolate human efficacy is not known. the mouse dose of μg (equivalent to mg/kg) equates to a human dose of approximately mg/kg based on body surface area conversion. in view of the high mortality rate in people with mers-cov infection and the difficulties extrapolating preclinical models to human disease, we suggest the highest tolerated dose in this phase trial ( mg/kg) should be used initially in human efficacy trials. the vaccine used for immunising transchromosomic cattle to generate sab- is the spike protein nanoparticle vaccine of the al-hasa strain, which belongs to a clade b of mers-cov, and the pharmacokinetic data show high antibody titres towards this clade b strain. sab- was previously shown to neutralise the jordan (jordan-n / ) and erasmus medical center (emc/ ) strains of mers-cov, both of which are clade a. therefore sab- is anticipated to have naturalising capacity to current clade a and b strains of mer-cov. to our knowledge, this is the first study to show the safety, tolerability, and pharmacokinetics of a novel therapeutic for mers, as well as for this new source of fully human igg produced in transchromosomic cattle. the data attained in this study suggest that sab- is safe and well tolerated at potentially therapeutic exposures. further clinical investigation of sab- for the treatment of mers is warranted. contributors jhb, jv, es, tl, and rtd were responsible for initial study design. jhb, jv, and rtd were responsible for study implementation and enrolment of participants. kr was responsible for the micro-neutralisation assay. hw, j-aj, and es were responsible for the pharmacokinetic assay and the immunogenicity assays. jhb, jv, pk, and rtd analysed and interpreted the data and wrote the first draft of the report. all authors had opportunity to review the data and edited the final report. hw, j-aj, and es have financial interests in sab biotherapeutics inc. all other authors declare no competing interests. who. middle east respiratory syndrome coronavirus middle east respiratory syndrome feasibility of using convalescent plasma immunotherapy for mers-covinfection, saudi arabia the use of convalescent plasma to treat emerging infectious diseases: focus on ebola virus disease immune plasma for the treatment of severe influenza: an open-label, multicentre, phase randomised study species-specific chromosome engineering greatly improves fully human polyclonal antibody production profile in cattle molecular basis of binding between novel human coronavirus mers-cov and its receptor cd evaluation of candidate vaccine approaches for mers-cov viral shedding and antibody response in patients with middle east respiratory syndrome coronavirus infection purified coronavirus spike protein nanoparticles induce coronavirus neutralizing antibodies in mice human polyclonal immunoglobulin g from transchromosomic bovines inhibits mers-cov in vivo meta-analysis: convalescent blood products for spanish influenza pneumonia: a future h n treatment? experience of using convalescent plasma for severe acute respiratory syndrome among healthcare workers in a taiwan hospital critically ill patients with the middle east respiratory syndrome: a multicenter retrospective cohort study the authors thank the volunteers who participated in this research study, without whom the study would not have been possible. we also thank the physicians, nurses, and other health-care personnel at the national institutes of health who provided invaluable services and support for this study. this project was funded in part with federal funds from intramural research programmes of the national institute of key: cord- -yd idp authors: zhang, chengfei; yan, yan; he, hongwang; wang, li; zhang, na; zhang, jie; huang, hongjun; wu, nannan; ren, hua; qian, min; liu, mingyao; du, bing title: ifn-stimulated p y( ) protects mice from viral infection by suppressing the camp/epac signaling pathway date: - - journal: j mol cell biol doi: . /jmcb/mjy sha: doc_id: cord_uid: yd idp among the most important sensors of extracellular danger signals, purinergic receptors have been demonstrated to play crucial roles in host defense against infection. however, the function of p receptors in viral infection has been little explored. here we demonstrated that p y( ) and its ligand adp play an important role in protecting hosts from viral infections. first, we demonstrate that p y( ), as a typical interferon-stimulated gene, is induced together with extracellular adp during viral infection. most importantly, extracellular adp restricts the replication of different kinds of viruses, including vesicular stomatitis virus, newcastle disease virus, herpes simplex virus , and murine leukemia virus. this kind of protection is dependent on p y( ) but not p y( ) or p y( ), which are also considered as receptors for adp. furthermore, cyclic adenosine monophosphate and epac are downregulated by extracellular adp through the p y( )-coupled gi alpha subunit. accordingly, inhibition or deletion of epac significantly eliminates adp/p y( )-mediated antiviral activities. taken together, our results show that p y( ) and adp play pivotal roles in the clearance of invaded virus and have the potential as antiviral targets. upon viral infection, the host can sense the virus through the recognition of pathogen-associated molecular patterns by pattern recognition receptors, especially toll-like receptors (tlrs) and rig-i-like receptors (rlrs) (kawai and akira, ; chen et al., ) . when activated by viral nucleic acids, tlrs and rlrs will activate the tbk -irf axis, leading to type i interferon (ifn) expression and secretion (garcia-sastre and biron, ; he et al., ) . then, the released ifns induce hundreds of interferon-stimulated genes (isgs) via the janus kinase-signal transducer and activator of transcription (jak-stat) signaling pathway to clear the invaded virus (platanias, ; garcia-sastre and biron, ; shu et al., ) . among them, the double-stranded rna-dependent protein kinase pkr, the myxovirus resistance gene mx, the ′- ′ oligoadenylate synthetases, and interferon induced transmembrane protein (ifitm ) have been shown to disrupt viral infection in various manners rafique et al., ; liu et al., ) . more than genes have been characterized as isgs. however, their roles and mechanisms in fighting against multiple distinct classes of viruses remain largely unexplored. purinergic signaling and purinergic receptors are important in both innate and adaptive immune responses. purinergic signaling was first reported in and, after several decades of study, many of the biologic effects of atp, utp, adp, udp, and adenosine signaling were discovered (eltzschig et al., ; idzko et al., ) . in tissue injury or pathogen infection, nucleotides can be released from stressed cells as a danger signal, alerting the cells by interaction with purinergic receptors in an autocrine or paracrine manner . nineteen different purinergic receptor subtypes have been identified, which can be classified into p and p receptors. four p receptor subtypes can be activated by adenosine. p receptors can be further divided into p y receptors (p yrs) and p x receptors (p xrs) based on their structures and distinct signaltransduction mechanisms. p yrs are g-protein-coupled receptors for atp, adp, utp, and udp, while p xrs are nucleotide-gated ion channels and can be activated by atp (ralevic and burnstock, ; junger, ; idzko et al., ) . many studies revealed crucial roles for p receptors during inflammatory and infectious diseases. for example, atp-triggered p x activation is crucial for the activation of nlrp inflammasome and the subsequent release of interleukin- β (di virgilio et al., ) . atp/utp receptor p y signaling has been identified as a 'find-me' signal to recruit motile phagocytes, thereby promoting clearance of apoptotic cells or bacteria (elliott et al., ; chen et al., ) . our previous study demonstrated that the adp-mediated p y /p y signal pathway protects the host defense against bacterial infection via erk signaling (zhang et al., ) . however, whether adp-(or its receptors-) mediated immune regulation is also involved in viral infection remains unclear. p y , p y , and p y are all receptors for adp. p y binds to gtp-binding protein subunit alpha q (gq), which can evoke calcium signaling. while p y and p y interact with gtpbinding protein subunit alpha i (gi), which can suppress the activity of adenylate cyclase (adcy) and the production of cyclic adenosine monophosphate (camp) (zhang et al., ; shinozaki et al., ) . in this study, we demonstrate that p y , as an isg, and its ligand, adp, is released from infected cells as a danger signal during viral infection, which restricts the replication of both dna and rna viruses. adp/p y -mediated protection against viral infection operates by suppressing the expression of exchange protein activated by camp (epac ), which is an alternative key intracellular sensor for camp. therefore, we demonstrated that adp/p y restricts viral infection through suppressing the camp/epac signaling pathway, which has potential therapeutic significance in the prevention and control of viral diseases. the expression of p y is increased by ifn through jak/stat pathway to identify p y family members potentially involved in antiviral immune responses, we treated mouse bone marrowderived macrophages (bmdms) with ifn-α, ifn-β, and ifn-γ to appraise the rna expression of all p y family members by quantitative real-time pcr (q-pcr). as shown in figure a , the rna expression of p y was upregulated obviously by all three ifns. the protein level of p y also upregulated by ifn-β ( figure b) . besides, when blocked ifn receptor (ifnar) with the anti-ifnar antibody, the upregulated expression of p y was decreased ( figure c ), suggesting that p y upregulation is dependent on signals downstream of ifn. the jak/stat pathway is crucial for ifn-induced cellular antiviral signaling. to determine whether ifn-induced p y upregulation via the jak/stat pathway, we pretreated mouse peritoneal macrophages (pems) with ruxolitinib (a jak inhibitor) and fludarabine (a stat inhibitor) and found that ifnβ-induced p y expression was blocked by both inhibitors ( figure d and e). inhibition of stat also reduced vsv-induced p y expression ( figure f ). in addition, knockout of tlr to block tlr -mediated activation of ifn also suppressed poly(i:c)-induced p y expression ( figure g ). taken together, these data demonstrated that p y has the typical characteristics of isgs. as an endogenous ligand to purinergic receptors, adp can be released from injured cells to activate p y , p y , and p y receptors. to investigate the potential role of adp as a danger signal in viral infection, the release of endogenous adp in viralinfected cells was measured. as shown in figure a and b, extracellular adp in vsv-infected cell supernatant was clearly increased in a time-dependent manner. besides, ndv-infected bmdms and hsv- -infected hek t cells also release large amounts of adp ( figure c and supplementary figure s a ), suggesting adp can be released as a danger signal during viral infection. to investigate the biological significance of adp, we pretreated the cells with adp before vsv infection. to our surprise, the rna replication of vsv in adp-treated raw . cells was reduced significantly in a time-( figure d ) and concentration-( figure e ) dependent manner. accordingly, the viability of vsv-infected raw . cells was increased dramatically by adp in a concentration-dependent manner while have little influence on uninfected cells ( figure f and supplementary figure s b ), indicating that extracellular adp protects cells from vsv infection. to confirm whether adp has broad-spectrum antiviral activities, raw . cells and bmdms were pretreated with adp before vsv, ndv, and hsv- infection. as shown in figure g -j and supplementary figure s c , the rna expression of vsv, ndv, hsv- and the titers of vsv were all strikingly decreased by adp. similar results were observed in hek t cells, where cell cytopathic effects were induced by vsv and hsv- , as determined using a crystal violet staining assay (supplementary figure s d and e). we further infected hek t and hela cells with vsv-gfp and mlv-gfp and found that the number of gfppositive cells was decreased in adp-treated cells, indicating that vsv-gfp and mlv-gfp were restricted by adp (supplementary figure s f-i). to rule out the possibility that adp degradation products mediate the suppression of viral replication, we treated cells with non-hydrolyzable adp analog adp-β-s. as shown in supplementary figure s j , adp-β-s has a similar role as adp in restricting vsv replication. taken together, our data suggested that extracellular adp has broad-spectrum antiviral activities, which have great potential in protecting hosts from a viral infection. to explore the key receptors involved in adp-mediated antiviral activities, we detected the expression of p y , p y , and p y after vsv infection. as shown in figure a , only the expression of p y was increased significantly, whereas the expression of p y and p y was little changed in vsv-infected raw . cells. similar data were observed in poly(i:c)-treated bmdms ( figure b ). we then used mrs and mrs to inhibit p y and p y , respectively, to verify whether p y and p y contributed to adp-mediated antiviral activities. we found that adp-mediated antiviral activities were little influenced by mrs but significantly decreased by mrs as shown in figure c -f, and both of mrs and mrs have little influence on cell viability (supplementary figure s a and b). consistently, when infected wild-type, p y -, p y -, and p y deficient bmdms with vsv, the vsv rna replication was increased only in p y -deficient bmdms, not in p y -or p y -knockout bmdms ( figure g ). meanwhile, adp-reduced vsv rna replication was compensated in p y -deficient pems ( figure h) . furthermore, the protein expression of vesicular stomatitis virus glycoprotein (vsv-g) was significantly increased in p y -deficient pems and overexpression of p y in hela cells significantly repressed vsv replication ( figure i ; supplementary figure ifn signal pathway increases p y expression. (a) mouse bmdms were treated with ifn-α, ifn-β, or ifn-γ ( ng/ml) for h, and mrna expression of p y family was detected by q-pcr. (b) mouse pems were treated with ifn-β ( ng/ml) for or h, and the expression of p y protein was detected by western blotting. (c) mouse bmdms were pretreated or not with anti-ifn-α/β receptor igg ( μg/ml) for h before being treated with ifn-β ( ng/ml) for h, and the expression of p y mrna was detected by q-pcr. (d) mouse pems were pretreated or not with ruxolitinib ( μg/ml) for h and then stimulated with ifn-β ( ng/ml) for h. the expression of isg and p y mrna was detected by q-pcr. (e) mouse pems were pretreated with or without fludarabine ( μg/ml) for min and then stimulated with ifn-β ( ng/ml) for h. the expression of isg and p y mrna was detected by q-pcr. (f) mouse pems were pretreated or not with fludarabine ( μg/ml) for min and then infected with vsv (moi = ) for h. the expression of p y mrna was detected by q-pcr. (g) tlr +/+ and tlr −/− mouse pems were treated with poly(i:c) ( μg/ml) for h. the expression of ifn-β, isg , and p y mrna was detected by q-pcr. data are shown as mean ± sd. **p < . ; ***p < . . figure s c and d). taken together, our data suggested that p y deficiency facilitates vsv infection and adp-mediated antiviral activities primarily through the p y receptor. to further confirm the in vivo antiviral activities of adp/p y , we challenged wild-type and p y -deficient mice with a lethal and then infected with vsv (moi = ) for h. vsv titers in supernatants were measured by plaque assay. (i and j) mouse bmdms were treated with adp ( μm) for h and then infected with ndv (moi = . ) and hsv- (moi = . ) for h virus rna replicates were detected by q-pcr. data are shown as mean ± sd. *p < . ; **p < . ; ***p < . . dose of vsv. as shown in figure a , the survival of adp-treated wild-type mice was increased obviously, but there was little change in that of p y -deficient mice. p y -deficient mice were more susceptible to vsv infection. on the other hand, if we intraperitoneally injected mice with adp to activate p y , vsv distributions in the liver, spleen, and lung were all significantly decreased ( figure b ). similar data were observed during hsv- infection ( figure c ). besides, more vsv rna replicates were found in p y -deficient mice liver ( figure d ). as a neurotropic virus, vsv can invade the nervous system and is transmitted directly via the transcutaneous or transmucosal route. thus, to mimic the natural infection of vsv, we treated mice with adp through nasal dripping and then intranasally infected with vsv. as shown in figure e and f, vsv rna replication and vsv-g in olfactory bulbs were both significantly reduced by adp. taken together, these data demonstrated that adp/p y plays an important role in protecting mice from vsv infection, suggesting that adp/p y has potential as a novel antiviral drug target. adp/p y restricts viral replication by inhibiting camp signaling type i ifn plays pivotal roles in fighting against the invaded virus, so we tested whether it was involved in adp/p y mediated antiviral activities. however, when we treated bmdms with adp, the rna expression of ifn-α and ifn-β was little changed (supplementary figure s a and b) . p y signaling can suppress the activity of adcy and the production of camp. to confirm that, raw . cells were pretreated or not with mrs before being treated with adp. as shown in figure a , adp significantly suppressed the production of camp, while p y antagonist mrs rescued this phenomenon. adp ( mg/kg) for h and then intraperitoneally injected with vsv ( × pfu/g). mice were executed for days and mouse survival was recorded for days. (b) six-week-old mice were intraperitoneally treated with adp ( mg/kg) for h and then intraperitoneally injected with vsv ( × pfu/g) for h. vsv rna replicates in organs were detected by q-pcr. (c) six-week-old mice were intraperitoneally treated with adp ( mg/kg) for h and then intraperitoneally injected with hsv- ( × pfu/g) for h. hsv- rna replicates in livers were detected by q-pcr. (d) eight-week-old p y +/+ or p y −/− mice were intraperitoneally injected with vsv ( × pfu/g) for h and vsv rna replicates in livers were detected by q-pcr. (e) six-week-old mice were treated with adp ( mg/kg) through nasal dropping for h and then intranasally infected with vsv ( × pfu/g) for h. vsv rna replicates in olfactory tissues were detected by q-pcr. (f) six-week-old mice were treated as in e, and vsv invaded in olfactory tissues were detected by immunofluorescence. scale bar, μm. data are shown as mean ± sd. **p < . ; ***p < . . however, to our surprise, vsv infection caused an obvious increase in cellular camp levels ( figure b) . besides, p y -deficient pems showed higher cellular camp levels than wild-type pems during vsv infection ( figure c ). so, we examined the role of camp in adp/p y -mediated antiviral activities. we first found that adcy activator forskolin blocked adp-mediated the cells were then infected with vsv (moi = . ) for h. vsv rna replicates were detected by q-pcr and vsv titers in supernatants were measured by plaque assay. (i) raw . cells were pretreated or not with kh ( μm) for h before being treated with adp ( μm) for h. the cells were then infected with hsv- (moi = . ) for h, and hsv- rna replicates were detected by q-pcr. data are shown as mean ± sd. *p < . ; **p < . ; ***p < . ; ns, not significant. antiviral activity completely ( figure d and e). furthermore, camp treatment promoted viral replication obviously and also rescued the adp-reduced cell cytopathic effects and vsv replication ( figure f and g; supplementary figure s c and d). consistent with this, kh (a selective inhibitor of adcy) treatment significantly inhibited cell cytopathic effects and vsv replication (supplementary figure s e and f). adp-mediated antiviral activities were also significantly blocked by kh ( figure h and i). the cell viability assays revealed that these phenomena were not caused by the toxicity of forskolin, camp, and kh (supplementary figure s g-i) . taken together, these results suggested that camp plays a key role in adp/p y -mediated antiviral activities. adp/p y restricts viral replication in an epac -dependent manner when intracellular camp is increased by upstream signaling, epac and protein kinase a (pka) could be activated as classical sensors for camp. thus, we treated hela cells with esi- (epac / inhibitor), hjc (epac selective inhibitor), and h (pka inhibitor) at a safety concentration (supplementary figure s a -c). as shown in supplementary figure s d , the replication of vsv is dramatically suppressed by both esi- and h , but not hjc , suggesting that epac and pka may be involved in adp/p y -mediated restriction to vsv. however, when we inhibited pka with h , the replication of vsv ( figure a ) and hsv- ( figure b ) in hela cells are still suppressed by adp, whereas adp-reduced vsv, hsv- , and ndv replication was significantly blocked by esi- , but not hjc ( figure c -f and supplementary figure s e) . besides, the adpreduced vsv-g expression was also blocked by esi- ( figure g) . these data show that epac is crucial in adp/p y mediated antiviral activities. in addition, we generated an epac knockout cell line using the crispr/cas system ( figure h ). as shown in figure i and j, when epac is deficient, adp-mediated suppression of vsv rna replication and vsv-g expression were eliminated. taken together, these results demonstrated that adp/p y -mediated antiviral activities are dependent on epac . inhibiting epac with esi- in raw . cells significantly suppressed vsv and hsv- replication, and epac knockout also inhibited hsv- replication, demonstrating that either inhibition or deletion of epac has broad-spectrum antiviral activities (supplementary figure s f-h) . to further confirm the key role of epac in viral infection, we detected the expression of epac in viral-infected cells. as shown in figure a , when infected raw . cells with vsv, ndv, and hsv- , rna expression of epac was increased significantly. deficiency in p y maintained higher expression of epac in pems after infection with vsv ( figure b ). in addition, the expression of epac protein also upregulated after virus infection as shown in figure c and supplementary figure s a , b. however, to our surprise, the increased expression of epac induced by vsv was reduced by adp in a dose-dependent manner (supplementary figure s c and d) . to exclude that the downregulation of epac may be due to the attenuation of vsv replication by adp, cell lines were only treated with adp. as shown in figure d -f and supplementary figure s e , the endogenous rna expression of epac was directly inhibited by adp. the protein expression of epac was also inhibited by adp ( figure g and h). taken together, these data demonstrated that the virusincreased epac expression is suppressed by adp, which restricts the viral infection in host cells. p y and p y , as membrane receptors for adp, are both gi-coupled receptors that inhibit the activity of adcy and reduce the production of camp (vitiello et al., ) . p y is highly expressed in platelets and plays a central role in platelet activation, which has become a drug target for atherothrombotic diseases, whereas p y is mainly expressed in the bone marrow, spleen, liver, and brain (savi et al., ; vitiello et al., ) . previous studies have shown that p y and p y are both involved in the development of pain behavior during nerve injury and the expression of pro-inflammatory cytokines (kobayashi et al., ; liu et al., ) . however, the role of p y and p y receptors in viral infection remains unclear. here we demonstrate that p y is upregulated by type i ifn (p y is little changed) and its endogenous ligand, adp, is released as danger signals from virusinfected cells. consequently, extracellular adp-activated p y protects the host against viral infection in a camp-dependent manner. thus, our study reveals an important role of adp/p y in fighting against viral infection, which demonstrates the potential of purinergic receptors as novel antiviral targets. upon viral infection, the host initiates a series of signaling cascades to induce the production of type i ifn, which results in the induction of isgs to exclude invaded virus (ooi et al., ; zhan et al., ) . previous studies reported that isgs could further promote the production of type i ifn to eliminate the virus, such as isg and ifit (lu et al., ; liu et al., ) . therefore, we tested whether adp/p y influences the expression of type i ifn. our results show that adp enhanced ifn expression by only a little. because adp clearly inhibited virus replication, we hypothesized that there must be another way for adp/p y to restrict viral infection. thus, the classic gi-coupled signaling pathway came to mind. interestingly, we found that adp-inhibited vsv replication and cell cytopathic effects were rescued by both adcy activator forskolin and camp. moreover, similar to adp, the adcy inhibitor kh also inhibited vsv replication, suggesting that camp plays a key role in adp/p y mediated antiviral activities. as the first identified second messenger, camp plays a crucial role in microbial pathogenesis (mcdonough and rodriguez, ) . pka and epac are the two classical sensors for intracellular camp, which are essential for many biological processes (gloerich and bos, ) . pka plays important roles in metabolism, cell cycle, cell migration, differentiation, and apoptosis (yan et al., ) . previous studies have reported that pka can inhibit ifn induction of the jak/stat pathway (david et al., ) . recently, a new study reported that pka catalytic (pkac) figure adp-mediated antiviral activities are dependent on epac . (a) hela cells were treated or not with h ( μm) for h before being treated with adp ( μm) for h and then infected with vsv (moi = . ) for h. vsv rna replicates were detected by q-pcr. (b) hela cells were treated or not with h ( μm) for h before being treated with adp ( μm) for h and then infected with hsv- (moi = . ) for h. hsv- rna replicates were detected by q-pcr. (c) hela cells were pretreated or not with esi- ( μm) for h before being exposed to adp ( μm) for h. the cells were then infected with vsv (moi = . ) for h and vsv rna replicates were detected by q-pcr. (d and e) hela cells were pretreated or not with esi- ( μm) for h before being exposed to adp ( μm) for h. the cells were then infected with hsv- (moi = . ) or ndv (moi = . ) for h and viral rna replicates were detected by q-pcr. (f) raw . cells were treated or not with esi- ( μm) for h before being exposed to adp ( μm) for h. the cells were then infected with hsv- (moi = . ) for h, and hsv- rna replicates were detected by q-pcr. (g) hela cells were pretreated or not with esi- ( μm) for h before being exposed to adp ( μm) for h. the cells were then infected with vsv (moi = . ) for h and vsv-g protein levels were detected by western blotting. (h) epac +/+ and epac −/− hela cells were exposed to vsv (moi = . ) for h, and the expression of epac was detected by western blotting. (i) epac +/+ and epac −/− hela cells were treated with adp ( μm) for h and then infected with vsv (moi = . ) for h. vsv rna replicates were detected by q-pcr. (j) epac +/+ and epac −/− hela cells were treated with adp ( μm) for h and infected with vsv (moi = . ) for h. vsv-g protein levels were detected by western blotting. data are shown as mean ± sd. *p < . ; **p < . ; ***p < . ; ns, not significant. subunits α and β could negatively regulate rna virus-triggered signaling and ultimately attenuate the innate antiviral response (yan et al., ) . epac has two isoforms, epac and epac . the camp/epac signal pathway is important for controlling various cellular functions, including cell adhesion, exocytosis, and microtubule dynamics. previous studies have reported that epac is a potential target for the treatment of fatal rickettsioses and that the silencing of epac gene expression renders cells resistant to mers-cov infection (gong et al., ; tao et al., ) . in our study, we found that both pka and epac inhibitors suppress viral replication. however, only epac inhibition could block adp/p y -mediated antiviral activities. also, we found that the epac inhibitor could not block adp/p y -mediated antiviral activities. these findings suggest that adp/p y -mediated antiviral activities are mainly dependent on epac . knockout of epac in hela cells showed similar results and further proved this point. taken together, our results showed that, upon viral infection, the virus can trigger an upregulation of intracellular camp levels to activate epac and thereby facilitating virus replication. however, the virus also triggers adp released from these cells, which will activate p y and suppress the camp/ epac signal pathway to restrain virus replication. this phenomenon revealed a negative feedback regulation in animals during viral infection, and that activation of p y and inhibition of the camp/epac signal pathway are potential antiviral strategies. p y , p y , and p y knockout mice were described previously (zhang et al., ) . tlr knockout mice were a gift from professor yuping lai (east china normal university) and were described previously (wu et al., ) . c bl/ wild-type mice were purchased from the shanghai laboratory animal company. all of the mice were maintained in specific pathogen-free (spf) facilities at the animal center of east china normal university. all animal experiments were done following institutional guidelines. reagents adp, adp-β-s, mrs , forskolin, camp, kh , esi- , h , anti-β-actin antibody, and anti-vsv-g antibody were purchased from sigma-aldrich. ruxolitinib was from targetmol. fludarabine was obtained from calbiochem. polyinosinic-polycytidylic acid [poly(i:c)] was purchased from invivogen. adp-glo™ kinase assay kit was purchased from promega. mrs and anti-gpr (p y ) antibodies were purchased from abcam. hjc was purchased from selleck. the anti-epac antibody was from bioss. primescript rt master mix and sybr premix ex taq were from takara. ifn-α, ifn-β, and ifn-γ were purchased from sino biological inc. the camp elisa kit, mouse ifn-α/β receptor block/neutralize polyclonal goat igg (af ), and normal goat igg control were from r&d systems. p y plasmid was obtained from biogot technology, co., ltd. the bmdms were prepared as follows: bone marrow cells were collected from tibias and femurs by flushing with dmem, and the cell suspension was filtered through a -μm cell strainer to remove any cell clumps. bone marrow cells were then cultured in dmem containing % fbs, % penicillin/streptomycin, and % l culture supernatants. the pems were prepared as follows: mice were intraperitoneally injected with ml of % sterile thioglycollate medium and, days later, mice were sacrificed and the peritoneal cavities washed with ml pbs. the cell suspension was filtered through a -μm cell strainer to remove any cell clumps. pems were then cultured in dmem containing % fbs and % penicillin/streptomycin. raw . , hek t, and hela cells were obtained from the american type culture collection and cultured in dmem containing % fbs and % penicillin/streptomycin. after stimulation, total rna was extracted with rnaiso plus (takara) from the cells. cdna was synthesized from extracted total rna using the primescript rt master mix perfect real time kit (takara) according to the manufacturer's protocol. quantitative real-time pcr was performed using a sybr-green premix (takara). the sequence-specific primers are listed in supplementary table s . adp release assay raw . cells, mouse bmdms, and hek t cells were plated in -well plates ( × cells per well) overnight and then infected with viruses for various periods. adp release into the cell culture supernatants was detected using adp-glo™ kinase assay kit according to the manufacturer's protocol. raw . cells were plated in -well plates ( × cells per well) overnight. the cells were treated with adp for h and then infected with vsv for h. mts ( μl, promega) was added to each well. after incubated for h at °c, the absorption was measured at nm. cell viability of the untreated group was normalized to %. hela and hek t cells were seeded into -well plates ( . × cells per well) overnight. the cells were treated with adp for h and then infected with vsv-gfp or mlv-gfp for h. cells containing virus were determined after gating on gfp-positive cells. hek t cells were plated in -well plates ( × cells per well) overnight. twelve hours after vsv (moi = . ) or hsv- (moi = . ) infection, cells were fixed with % paraformaldehyde in pbs for min at room temperature. after incubation with crystal violet for min, the cells were then washed with pbs five times before the photographs were taken. cells were grown in -well plates. after stimulation, cells were lysed with radio immunoprecipitation assay (ripa) buffer supplemented with protease and phosphatase inhibitors. whole-cell lysates were separated by % sds-page, transferred to nitrocellulose membranes, and blocked with % bsa. immunoblots were hybridized with the respective primary antibodies. protein bands were visualized with the appropriate fluorescent secondary antibodies and the odyssey laser digital imaging system (gene company). cells were infected with viruses for the indicated time and moi. for mouse survival assay, -week-old p y +/+ or p y −/− mice were intraperitoneally treated with adp ( mg/kg) or not treated for h and then intraperitoneally injected with vsv ( × pfu/g). executed these for days and mouse survival was recorded for days. for in vivo studies of viral replication, to -week-old mice were intraperitoneally treated with adp ( mg/kg) or not treated for h and then intraperitoneally injected with vsv ( × pfu/g) or hsv- ( × pfu/g) for the indicated time and viral rna replicates in organs were detected by q-pcr. vero cells were plated in -well plates ( × cells per well) overnight. the supernatants from vsv-infected cells were serially diluted and infected vero cells for h. then the supernatant was removed and the cells were covered with dmem containing % low-melting point agarose. plaques were counted after h. for immunofluorescence assay, -week-old mice were anesthetized with ketamine/rompun and treated with mg/kg adp through nasal dropping for h before being intranasally infected with vsv ( × pfu/g). after h of infection, mice were sacrificed, and the olfactories were fixed with % paraformaldehyde in pbs overnight. slices ( -μm) from olfactories were permeabilized with . % triton x- and blocked with % bsa. tissues were then incubated with anti-vsv-g antibody overnight and alexa-fluor- -conjugated secondary antibody for h. finally, the tissues were stained with dapi and visualized with a fluorescence microscope. for camp detection, raw . cells or pems were plated in -well plates ( × or × cells per well, respectively) overnight. after stimulation, the cells were lysed with μl cell lysis buffer and the amounts of camp were measured by elisa (r&d systems) according to the manufacturer's instructions. to generate an epac -knockout cell line, a crispr/cas system was used. the epac -knockout target sequence used was ′-aaatgcactccggaccacg- ′. a lenticrisprv plasmid containing the epac -knockout target sequence was transduced into hela cells. three days later, puromycin-resistant single clones were selected, and the epac -knockout clones were identified by western blotting with the anti-epac antibody. all data were analyzed using the graphpad prism software and shown as mean ± sd. the student t-test (two-tailed) was used to analyze the significance of data and p-value < . was considered statistically significant. supplementary material is available at journal of molecular cell biology online. induction of siglec-g by rna viruses inhibits the innate immune response by promoting rig-i degradation purinergic signaling: a fundamental mechanism in neutrophil activation purinergic p y receptor modulates stress-induced hematopoietic stem/progenitor cell senescence activation of protein kinase a inhibits interferon induction of the jak/stat pathway in u cells the p x receptor in infection and inflammation nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance purinergic signaling during inflammation impact of protein kinase pkr in cell biology: from antiviral to antiproliferative action. microbiol type interferons and the virushost relationship: a lesson in detente epac: defining a new mechanism for camp action exchange protein directly activated by camp plays a critical role in bacterial invasion during fatal rickettsioses errα negatively regulates type i interferon induction by inhibiting tbk -irf interaction nucleotide signalling during inflammation immune cell regulation by autocrine purinergic signalling the role of pattern-recognition receptors in innate immunity: update on toll-like receptors multiple p y subtypes in spinal microglia are involved in neuropathic pain after peripheral nerve injury interferon-inducible cholesterol- -hydroxylase broadly inhibits viral entry by production of -hydroxycholesterol ifn-induced tpr protein ifit potentiates antiviral signaling by bridging mavs and tbk p y and p y receptors involved in adpβs induced the release of il- β, il- and tnf-α from cultured dorsal horn microglia isg enhances the innate antiviral response by inhibition of irf- degradation the myriad roles of cyclic amp in microbial pathogens: from signal to sword novel antiviral host factor, tnk , regulates ifn signaling through serine phosphorylation of stat mechanisms of type-i-and type-ii-interferon-mediated signalling positional effect of phosphorylation sites and in the cytoplasmic domain of the e protein of hepatitis c virus a genotype: interferon resistance analysis via sequence alignment receptors for purines and pyrimidines the active metabolite of clopidogrel disrupts p y receptor oligomers and partitions them out of lipid rafts transformation of astrocytes to a neuroprotective phenotype by microglia via p y receptor downregulation adap is an interferon stimulated gene that restricts rna virus entry blocking of exchange proteins directly activated by camp leads to reduced replication of middle east respiratory syndrome coronavirus immunoregulation through extracellular nucleotides hyperglycaemia inhibits reg a expression to exacerbate tlr -mediated skin inflammation in diabetes pkacs attenuate innate antiviral response by phosphorylating visa and priming it for march -mediated degradation phosphatase pp negatively regulates type i ifn production and antiviral innate immunity by dephosphorylating and deactivating tbk two disparate ligand-binding sites in the human p y receptor extracellular adp facilitates monocyte recruitment in bacterial infection via erk signaling this work was supported by the national key r&d program of china ( yfa to b.d.), the national natural science conflict of interest: none declared. key: cord- - xi lqf authors: albarrak, ali; alotaibi, badriah; yassin, yara; mushi, abdulaziz; maashi, fuad; seedahmed, yassein; alshaer, mohamed; altaweel, abdulaziz; elshiekh, husameddin; turkistani, abdulhafiz; petigara, tanaz; grabenstein, john; yezli, saber title: proportion of adult community-acquired pneumonia cases attributable to streptococcus pneumoniae among hajj pilgrims in date: - - journal: int j infect dis doi: . /j.ijid. . . sha: doc_id: cord_uid: xi lqf background: the hajj mass gathering is a risk for pneumococcal disease. this study was performed to evaluate the proportion of adult community-acquired pneumonia (cap) cases attributable to streptococcus pneumoniae among hajj pilgrims in . to add sensitivity to etiological attribution, a urine antigen test was used in addition to culture-based methods. methods: adult subjects hospitalized with x-ray-confirmed cap were enrolled prospectively from all general hospitals designated to treat hajj pilgrims in the holy cities of mecca and medina. patients were treated according to local standard of care and administered the binaxnow s. pneumoniae urine antigen test. results: from august to september , , a total of patients with cap were enrolled in the study, . % of whom were admitted to hospitals in mecca; % of the cases were admitted after the peak of hajj. patients originated from countries. their mean age was . years and the male to female ratio was : . just over % of the cases had diabetes, % declared that they were smokers, and . % of cases were treated in the intensive care unit (icu). the overall case-fatality rate was . %, but was higher among those treated in the icu and in those with invasive disease. the proportion of cap cases positive for s. pneumoniae, based on culture or urine antigen test, was . % ( % confidence interval . – . %). conclusions: cap during hajj has an important clinical impact. a proportion of cap cases among hajj pilgrims were attributable to s. pneumoniae, a pathogen for which vaccines are available. additional studies to determine the serotypes causing pneumococcal disease could further inform vaccine policy for hajj pilgrims. the hajj religious mass gathering hosted by the kingdom of saudi arabia (ksa) is attended by millions of muslims annually from all over the globe (yezli et al., ) . the event can facilitate the acquisition and transmission of infectious agents, including those responsible for respiratory tract infection, and has been linked to both local and international outbreaks of diseases (ahmed et al., ; memish et al., a,b; yezli et al., a) . examples include meningococcal disease and influenza (salmon-rousseau et al., ; yezli et al., a) . experience from hajj shows that the implementation of appropriate prevention measures such as vaccination can significantly reduce the incidence of disease and outbreaks associated with this mass gathering. of note is the prevention of meningococcal disease outbreaks since , after the introduction of compulsory vaccination with the quadrivalent meningococcal vaccine and targeted chemoprophylaxis (yezli et al., b) . streptococcus pneumoniae is a common cause of pneumonia and an important cause of morbidity and mortality worldwide (feldman and anderson, ; varon et al., ) . hajj presents many risk factors for pneumococcal disease acquisition and transmission. many pilgrims are elderly with pre-existing underlying health conditions and worship under crowded conditions that promote respiratory disease transmission and infection . crowding in particular has been associated with pneumococcal disease outbreaks (banerjee et al., ; mercat et al., ) . the acquisition and transmission of s. pneumoniae is well documented during hajj, independent of clinical status (memish et al., , a , and the organism is a leading cause of pneumonia-related hospitalizations and intensive care unit (icu) admissions during the event (al-tawfiq and memish et al., ) . vaccines against pneumococcal disease are available and are recommended for those at risk (such as the elderly and those with underlying health conditions) in many countries, including countries in the gulf states such as bahrain, kuwait, oman, qatar, and the united arab emirates (feldman et al., ; tomczyk et al., ) . the saudi thoracic society has also recently published guidelines on pneumococcal vaccination for hajj pilgrims (alharbi et al., ) . however, there is no official ksa recommendation for vaccination for hajj pilgrims (saudi ministry of health, ) . appropriate evidence-based policies regarding vaccination for pilgrims require a better understanding of the clinical burden of the disease associated with the event (al-tawfiq and . the evidence currently available for the burden of hajjassociated pneumococcal disease is suggestive, but limited by the insensitivity of bacterial cultures as a means of diagnosing the full burden of invasive or non-invasive pneumococcal pneumonia (bartlett, ) . the addition of urine antigen testing for adult pneumonia patients is expected to add sensitivity to the etiological attribution, without inappropriately minimizing specificity (mandell et al., ) . the sensitivity and specificity of this test in the diagnosis of community-acquired pneumonia (cap) due to s. pneumoniae have been reported to be in the range of %- % and %- %, respectively (gutierrez et al., ; klugman et al., ; molinos et al., ; song et al., ) . the aim of this study was to evaluate the proportion of hospitalized, x-ray-confirmed cap attributable to s. pneumoniae among adult hajj pilgrims in , using the urine antigen test as well as standard culture-based tests, in order to determine the clinical burden of disease associated with hajj and inform vaccination policy-making. this was a prospective case-series study conducted in hospitals in the holy cities of mecca and medina, ksa. the study was conducted over a -month period from august to september , ( dull qida to dull hija h in the islamic calendar) around the date of the hajj peak of september . the study was therefore able to capture three time periods: pre-hajj (august to september ), hajj (september to september ), and post-hajj (september to september ). patients were enrolled from all general hospitals (excluding specialty hospitals such as obstetrics and gynecology hospitals and pediatric hospitals) designated to treat hajj pilgrims. these included four general hospitals and seven temporary (holy sites) hospitals in mecca and four general hospitals in medina. the study population comprised adult pilgrim patients aged years old diagnosed with x-ray-confirmed cap. for this protocol, cap was defined in accordance with the us food and drug administration (fda) (us food and drug administration, ) as an acute infection of the pulmonary parenchyma associated with symptoms such as fever or hypothermia, chills, rigors, cough, chest pain, or dyspnea, accompanied by the presence of a new lobar or multilobar infiltrate on a chest radiograph within h of hospital admission. patients with known or suspected active tuberculosis (tb; defined as smear-positive after three acidfast bacilli tests), those < years old, non-hajj pilgrims, and patients admitted to the study hospitals with suspected cap patients excluded (not pilgrims or under years of age) patients excluded (did not fit the definition of cap, confirmed tb cases, refused to participate) patients excluded (missing crfs, no urine test performed (missed or patient unable to provide sample)), and no culture based tests performed x-ray confirmed cap cases fulfilling the inclusion criteria with crfs and urine and/or culture tests performed patients who did not consent to participate were excluded from the study. based on the above criteria, patients were enrolled in the study. following informed consent, x-ray-confirmed cap patients were treated according to local standard of care and administered a urine antigen test for s. pneumoniae (alere binaxnow s. pneumoniae urine antigen test; alere, waltham, ma, usa). a case report form (crf) containing patient demographic and clinical characteristics was filled out by trained investigators (to ensure consistency across sites) for each patient using the patient's medical chart and information from the patient or a family member. a case of x-ray-confirmed cap was recorded as positive or negative for s. pneumoniae on the crf based on findings from any one of the following microbiological tests: alere binaxnow s. pneumoniae urine antigen test, culture from a normally sterile site if conducted during routine investigation (i.e., blood, bone, cerebrospinal fluid, joint fluid, pericardial fluid), culture from a respiratory specimen if conducted during routine investigation and obtained by any of the following means (us food and drug administration, ): ( ) endotracheal aspiration in intubated patients; ( ) bronchoscopy with bronchoalveolar lavage or table demographic and other characteristics of the pilgrim population enrolled. number ( protected-brush sampling; ( ) sputum obtained by deep expectoration. the urine antigen test was performed at the patient's bedside as soon as possible after enrolment using the alere binaxnow s. pneumoniae test as per the manufacturer's recommendation. other samples were handled and processed in the same hospital as per the hospital's standard procedures. the results of microbiological investigations were collected and recorded on the crfs once available. characteristics of the study population were summarized as frequencies and percentages for categorical variables and as means with the range for quantitative variables. the association between explanatory variables and outcomes was evaluated by chi-square test or fisher's exact test, as appropriate. in addition, odds ratios (or) and their % confidence intervals (ci) were calculated in multivariate analyses. all tests for significance were two-sided, and a p-value of < . was considered statistically significant. all analyses were performed using ibm spss statistics version . (ibm corp., armonk, ny, usa). over the study period, patients with suspected cap were admitted to the hospitals included in this study. of these patients, had x-ray-confirmed cap and were enrolled in the study (figure ). demographic and other characteristics of the enrolled study population are shown in table . patients originated from countries, with the most represented being indonesia (n = , . %), egypt (n = , . %), and india (n = , . %). all but one case entered ksa between august and september , . most patients were elderly males (mean age . years, range - years; male to female ratio : ). the majority of cases (n = , . %) were admitted to hospitals in the city of mecca, the main site of the hajj pilgrimage, including cases admitted to the four temporary hospitals at the mecca holy sites. the pattern of admission shows that the number of cases admitted to hospitals increased over the study period and that most cases of cap occurred post hajj (figure , table ). cough and difficulty breathing were the most common symptoms and were present in % (n = ) and . % (n = ) of the cases, respectively. similarly, fever and tachypnea were the most common vital sign abnormalities, recorded in . % (n = ) and . % (n = ) of the cases, respectively. at least one clinical or laboratory abnormality was recorded for % of cases. only . % (n = ) of cases were initially admitted to the icu upon arrival at the hospital, but . % (n = ) of cases were treated in the icu during their hospital stay. diabetes mellitus was reported by . % (n = ) of the study population, and % (n = ) declared that they were cigarette smokers. only . % (n = ) of the cases acknowledged having used antibiotics in the days prior to their admission, although antibiotic use was unknown in a further . % (n = ) of the cases. all but one of the cases who had used antibiotics prior to hospital admission had been on a single antibiotic. one person had taken both ceftriaxone and clarithromycin prior to hospital admission. the most common antibiotics used prior to hospital admission were cephalosporins (mainly third-generation) and penicillins. culture-based methods (sterile sites or respiratory specimens) were performed in . % (n = ) of the cases, with the etiology determined in . % ( / ) of these cases. s. pneumoniae was identified in % (n = ) of the samples and in % (n = ) of samples from normally sterile sites. other pathogens were identified in six samples, including staphylococci (n = ) and klebsiella pneumoniae (n = ). urine antigen tests to detect s. pneumoniae infection were performed in . % (n = ) of cap cases; . % (n = ) were positive. the overall proportion of cap cases with a positive result for s. pneumoniae (based on either culture-based tests or the urine antigen test) was % (n = ). valid test results for both culture-based methods and the urine antigen test were available for cases. overall, agreement in results (both negative and positive) between the two methods was figure . general pattern of community-acquired pneumonia (cap) case admissions to hospitals during the study period. found in cases ( . %). based on these results, the sensitivity and specificity of the urine antigen test compared to the culturebased methods were calculated to be . % and . %, respectively. hospital location and treatment in the icu were significantly associated with s. pneumoniae cap cases (p = . ). cap patients admitted to medina hospitals were less likely to have s. pneumoniae-attributable cap than those admitted to mecca hospitals (or . , % ci . - . ). cap patients treated in the icu were . times more likely to be s. pneumoniae cap cases than those not treated in the icu (or . , % ci . - . ). no significant association was observed between s. pneumoniae-attributable cap and age, gender, pilgrim's country of origin, antibiotic use in the days prior to hospital admission, smoking, or diabetes status (table ) . disposition at discharge was recorded for cap cases. twenty-four patients died, resulting in an overall case-fatality rate of . %. the case-fatality rate among cap patients treated in the icu was nearly nine times that of non-icu patients ( . % vs. . %). the case-fatality rate among all s. pneumoniae-positive cases was . %, among s. pneumoniae-positive urine antigen test cases was . %, and among blood culture-positive s. pneumoniae cases was %. only admission to the icu on arrival and treatment in the icu were significantly associated with mortality in cap cases. patients with cap treated in the icu were over times more likely to die than those not treated in the icu (or . , % ci . - . ). this study is the first to systematically enroll cases of x-rayconfirmed cap among pilgrims during the whole hajj season and from hospitals in the two holy cities of mecca and medina, giving the best estimate of the burden of cap associated with hajj. as there was active triaging of all cases of suspected cap admitted to hospitals during hajj (due to middle east respiratory syndrome coronavirus (mers-cov) screening), the study is likely to have captured almost all cap cases admitted to hospitals. cap patients originated from a wide variety of countries with a sizable proportion being older males, which is reflective of the general population of hajj. a number of other studies have reported that pneumonia is a leading cause of hospital admission (accounting for - % of hospital admissions) during the pilgrimage (al-ghamdi et al., ; khan et al., ; madani et al., ; shirah et al., ) . however, most of these studies were limited to mecca city alone, specific hospital(s), or to the hajj rituals days only or a few days around that period. hence, previous studies have likely underestimated the true burden of hajj-associated pneumonia. it was found that % of hospitalized x-ray-confirmed cap cases among adult hajj pilgrims in were attributable to s. pneumoniae. the organism is commonly isolated from hajj pilgrims treated in clinics or hospitals during their pilgrimage. several studies have found s. pneumoniae to be the cause of pneumonia in up to % of cases during hajj (alzeer et al., ; asghar et al., ; mandourah et al., ; shirah et al., ) , while one study found that among patients treated for severe cap at facilities during the hajj, s. pneumoniae was found in %, using the randox respiratory multiplex array (memish et al., ) . other important causative pathogens reported in these studies have included other bacteria such as staphylococcus aureus, k. pneumoniae, haemophilus influenzae, and pseudomonas aeruginosa and viruses including human rhinovirus, influenza a virus, and human coronaviruses, as well as the fungus candida albicans. the results of this study are likely to be a more accurate reflection of the actual proportion of cap caused by s. pneumoniae during hajj, as the urine antigen test was used in addition to the standard culture-based methods. the test adds sensitivity to the etiological attribution without minimizing specificity (mandell et al., ) and overcomes many of the limitations and difficulties in culturing s. pneumoniae from clinical samples (bartlett, ) . although the test was highly specific in this study, it had lower sensitivity than that reported in other studies (mandell et al., ) . nearly half of the cap cases seen in this study were treated in the icu, reflecting the severity of the disease during hajj. admission to an icu was based on patient clinical assessment and the need for respiratory or hemodynamic support (arabi and alhamid, ) . pneumonia is a leading cause of icu admission during hajj, accounting for - % of icu admissions, and is a major cause of severe sepsis and septic shock in icus during the event (madani et al., ; mandourah et al., ; shirah et al., ) . the case-fatality rate for cap patients in this study was . %, which is within the range of rates reported for cap internationally (drijkoningen and rohde, ; vila-corcoles et al., ) . also, case fatality was higher among those admitted/treated in an icu and among those with invasive disease. this is also in accordance with other reports, including those among hajj pilgrims (drijkoningen and rohde, ; mandourah et al., ; shirah et al., ) , and is likely because patients treated in the icu or those with invasive disease have a more severe illness and are at a higher risk of death. most cap cases in this study were admitted to mecca hospitals, and the number of cases increased over the study period, with the highest admission rate being after the hajj rituals days. this pattern is in accordance with the hajj journey and its characteristics. most pilgrims arrive in ksa a few days (or weeks) before the hajj ritual dates and spend time in the holy cities of mecca and/or medina. during the hajj dates, all pilgrims return to mecca to perform the hajj rituals, most of which take place at the mecca holy sites. hence, the pre-hajj period is characterized by a smaller number of pilgrims, living in less crowded environments, spread across both mecca and medina, and relatively free of stressors associated with performing the hajj rituals. during the hajj dates, the maximum numbers of pilgrims are located in a small area of mecca performing physically challenging hajj rituals in crowded conditions, under both physical and environmental stressors. these conditions facilitate disease transmission and render pilgrims more prone to infection. it is likely that many cap cases admitted after hajj were infected during the hajj dates. this may explain the increase in the number of cap cases post-hajj, while the number of pilgrims in mecca and medina was decreasing. most cap cases were elderly males and many had diabetes or were smokers. although no significant association was found between these factors and pneumococcal pneumonia in this study, age, co-morbidities, and smoking are established risk factors for cap, including pneumococcal pneumonia (almirall et al., ; lynch and zhanel, ). the latter is a vaccine-preventable disease and the above risk factors are indications for pneumococcal vaccination in adults (tomczyk et al., ) . the finding that a proportion of cap during hajj was caused by s. pneumoniae, and that most of it was among individuals at risk of the disease, is significant. currently, pneumococcal vaccination is not one of the officially recommended/compulsory vaccinations in the hajj health requirements set by the saudi authorities (saudi ministry of health, ). although some countries have recommended pneumococcal vaccination for their hajj pilgrims (feldman et al., ; mathai et al., ; rashid et al., ) , an evidence-based policy requires a better understanding of the clinical and economic burden of the disease associated with hajj. this study is a first step in providing such data, by defining the burden of the disease in the hajj season using a more reliable diagnostic test for pneumococcal pneumonia. however, further studies are warranted, including accurate estimations of the incidence of the disease during the mass gathering and determining the serotypes causing the illness. this study has some limitations. it was aimed to systematically enroll all hospitalized x-ray-confirmed cap cases among hajj pilgrims during the study period. however, very early pneumonia may not be apparent on chest radiographs and may have led to the exclusion of some cases from the study. not all cap cases were investigated using culture-based methods, which are not routinely conducted by hospitals during hajj due to feasibility. some of the information collected from pilgrims was self-reported (e.g., smoking status) and hence may be subject to underreporting. also, information on the pneumococcal disease vaccination status of the cases was not collected, so it was not possible to investigate the effect of vaccination. in addition, as no accurate data on the adult population at risk during the study period were available, it was not possible to accurately estimate the incidence of cap during hajj. in conclusion, s. pneumoniae-attributable cap during hajj has an important clinical burden. further studies, including investigations of the incidence of the disease and s. pneumoniae serotypes involved in the disease, as well as the identification of the population at risk, are warranted to provide a comprehensive evidence base for appropriate policy-making regarding vaccination of hajj pilgrims. this study was funded by merck & co., inc. the study was approved by the king fahad medical city ethics committee and the institutional review board. all participants gave verbal consent before enrolment and the study was conducted in accordance with the guidelines of the ethics committee. tanaz petigara and john grabenstein are full-time employees of merck & co., inc. the other authors have no conflicts of interest to declare. health risks at the hajj pattern of admission to hospitals during muslim pilgrimage (hajj) prevention of pneumococcal infections during mass gathering the saudi thoracic society pneumococcal vaccination guidelines- risk factors for community-acquired pneumonia in adults: a systematic review of observational studies tuberculosis is the commonest cause of pneumonia requiring hospitalization during hajj (pilgrimage to makkah) emergency room to the intensive care unit in hajj. the chain of life profile of bacterial pneumonia during hajj outbreak of pneumococcal pneumonia among military recruits diagnostic tests for agents of community-acquired pneumonia pneumococcal infection in adults: burden of disease pneumococcal disease in the arabian gulf: recognizing the challenge and moving toward a solution epidemiology, virulence factors and management of the pneumococcus evaluation of the immunochromatographic binax now assay for detection of streptococcus pneumoniae urinary antigen in a prospective study of community-acquired pneumonia in spain pattern of medical diseases and determinants of prognosis of hospitalization during muslim pilgrimage hajj in a tertiary care hospital. a prospective cohort study novel approaches to the identification of streptococcus pneumoniae as the cause of community-acquired pneumonia streptococcus pneumoniae: epidemiology, risk factors, and strategies for prevention causes of hospitalization of pilgrims in the hajj season of the islamic year causes of admission to intensive care units in the hajj period of the islamic year update of practice guidelines for the management of community-acquired pneumonia in immunocompetent adults clinical and temporal patterns of severe pneumonia causing critical illness during hajj consensus recommendation for india and bangladesh for the use of pneumococcal vaccine in mass gatherings with special reference to hajj pilgrims a cohort study of the impact and acquisition of naspharyngeal carriage of streptococcus pneumoniae during the hajj etiology of severe community-acquired pneumonia during the hajj-part of the mers-cov surveillance program impact of the hajj on pneumococcal transmission mass gathering and globalization of respiratory pathogens during the hajj an outbreak of pneumococcal pneumonia in two men's shelters sensitivity, specificity, and positivity predictors of the pneumococcal urinary antigen test in community-acquired pneumonia the potential for pneumococcal vaccination in hajj pilgrims: expert opinion hajj-associated infections saudi ministry of health. health requirements for travellers to saudi arabia for pilgrimage to makkah mass gathering medicine (hajj pilgrimage in saudi arabia): the clinical pattern of pneumonia among pilgrims during hajj diagnosis of pneumococcal pneumonia: current pitfalls and the way forward use of -valent pneumococcal conjugate vaccine and -valent pneumococcal polysaccharide vaccine among adults aged >/= years: recommendations of the advisory committee on immunization practices (acip) guidance for industry: community-acquired bacterial pneumonia: developing drugs for treatment streptococcus pneumoniae: still a major pathogen pneumococcal pneumonia in adults years or older: incidence, mortality and prevention meningococcal disease during the hajj and umrah mass gatherings prevention of meningococcal disease during the hajj and umrah mass gatherings: past and current measures and future prospects an opportunity for mass gatherings health research we thank the mecca and medina regional general directorate of health affairs for their contribution to the study. key: cord- -io fvy authors: lorusso, eleonora; mari, viviana; losurdo, michele; lanave, gianvito; trotta, adriana; dowgier, giulia; colaianni, maria loredana; zatelli, andrea; elia, gabriella; buonavoglia, domenico; decaro, nicola title: discrepancies between feline coronavirus antibody and nucleic acid detection in effusions of cats with suspected feline infectious peritonitis date: - - journal: research in veterinary science doi: . /j.rvsc. . . sha: doc_id: cord_uid: io fvy abstract intra-vitam diagnosis of feline infectious peritonitis (fip) is a challenge for veterinary diagnosticians, since there are no highly specific and sensitive assays currently available. with the aim to contribute to fill this diagnostic gap, a total of effusions from cats with suspected effusive fip were collected intra-vitam for detection of feline coronavirus (fcov) antibodies and rna by means of indirect immunofluorescence (iif) assay and real-time rt-pcr (qrt-pcr), respectively. in effusions there was no evidence for either fcov rna or antibodies, and specimens tested positive by iif and qrt-pcr, respectively, although antibody titres≥ : , which are considered highly suggestive of fip, were detected only in effusions. three samples with high antibody levels tested negative by qrt-pcr, whereas qrt-pcr positive effusions contained no or low-titre antibodies. qrt-pcr positive samples with low antibody titres mostly contained low fcov rna loads, although the highest antibody titres were detected in effusions with c t values> . in conclusion, combining the two methods, i.e., antibody and rna detection would help improving the intra-vitam diagnosis of effusive fip. feline infectious peritonitis (fip) is a lethal disease of cats caused by a hypervirulent variant of feline coronavirus (fcov), an alphacoronavirus that usually causes self-limiting infections of the intestinal epithelium, leading to mild or no gastroenteric signs (addie et al., ) . two different fcov genotypes are currently known, fcov type i (fcov-i) and type ii (fcov-ii), both involved in the occurrence of mild gastroenteritis or fatal fip (decaro and buonavoglia, ) . fip is a perivascular pyogranulomatosis that may occur in two clinical forms, effusive and non-effusive fip, which are characterized by prevalence of effusions in the body cavities and of pyogranulomatous lesion in organs, respectively. fip diagnosis is challenging since the 'gold standard' is the post-mortem demonstration of fcov antigens in tissues by immunohistochemistry. therefore, alternative tools are commonly used for the intra-vitam diagnosis. haematological and biochemical analyses can support a presumptive diagnosis of fip, but they usually require further investigations, such as assessment of the fcov antibody titres and molecular detection of fcov rna in the effusions (effusive form) or bioptic samples (non-effusive fip) from ill cats. unfortunately, both methods lack specificity and sensitivity, thus often leading to an inconclusive diagnosis (addie et al., ) . recently, a comparison between the intra-vitam detection of fcov antibodies and that of fcov rna in the effusions of cats with confirmed fip has been carried out, showing a trend toward negative or low antibody levels in cats with high viral rna titres (meli et al., ) . however, these findings have not been confirmed by other studies. in the present paper, a total of effusions from cats with confirmed fip have been screened for fcov antibodies and rna, suggesting that intra-vitam diagnosis of effusive fip needs to be assessed by means of combined antibody-and virus-detection methods. t reported (meli et al., ) . all samples were sent to our lab for fip confirmation by diagnostic labs that had carried out some preliminary analyses on the effusions, including rivalta's test, total proteins, albumin/globulin ratio, total leukocyte counts and identity of cells (table ) . collected samples included ascitic fluids and pleuric effusions. for fcov antibody detection and titration, an indirect immunofluorescent (iif) assay was used (campolo et al., ) , with minor modifications. briefly, fcov-ii strain / (buonavoglia et al., ) was cultivated on crandell feline kidney (crfk) cells grown on coverslips. infected cells were fixed in acetone % and twofold dilutions of the effusion (starting from dilution : to : , ) were tested. goat anti-cat igg conjugated with fluorescein isothiocyanate was used as secondary antibody solution (sigma aldrich srl). the assay was proven to detect both fcov-i and fcov-ii antibodies (addie and jarrett, ; campolo et al., ) . effusion with qrt-pcr positive and iif-negative results were treated with ammonium thiocyanate to dissociate immune complexes, as previously described (pullen et al., ; macdonald et al., ) . for fcov rna detection, μl of the effusions were used for rna extraction by means of qiaamp® viral rna mini kit (qiagen s.p.a., milan, italy), following the manufacturer's protocol and the rna templates were stored at − °c until their use. fcov reverse-transcriptase quantitative pcr (fcov qrt-pcr) was performed as previously described (gut et al., ) , with minor modifications. in brief, a one-step method was adopted using platinum® quantitative pcr supermix-udg (invitrogen srl, milan, italy) and the following -μl mixture: μl of master mix, nm of primers fcov f (gatttgatttggcaatg-ctagattt) and fcov r (aacaatcactagatccagacgttagct), nm of probe fcov p (fam-tccattgttggctcgtcatagcg-ga-bhq ) and μl of template rna. the employed oligonucleotides bind to the ′ untranslated region (gut et al., ) . the thermal profile consisted of incubation with udg at °c for min and activation of platinum taq dna polymerase at °c for min, followed by cycles of denaturation at °c for s, annealing at °c for s and extension at °c for s. threshold cycle (c t ) number was used as the measure of viral load. the lower the c t , the more virus present in the sample. spearman's rank correlation coefficient was calculated to evaluate the possible correlation between viral rna loads and antibody titres by the use of the online tool social science statistics (http://www. socscistatistics.com/tests/spearman). fifty-one ( ascitic and pleuric fluids) of the tested samples had fcov antibody (table and fig. ), although only positive effusions contained antibody levels ≥ : , which are considered highly suggestive of fip diagnosis (hartmann et al., ) . additional samples presented fcov antibody titres between : and : , which are quite high for an enteric infection but cannot be considered enough high for a systemic infection. only one effusion had an antibody titre of : and two samples displayed an antibody titre of : , . by means of qrt-pcr, fcov rna was detected in a total of samples ( ascitic and pleuric fluids). c t values were generally above (mean c t value of . ), accounting for low viral titres, with higher viral rna loads (c t values < ) being detected in only effusions. by comparing the results qrt-pcr with those of iif assay using an antibody titre ≥ : as cut-off (fig. b) , samples tested negative by both assays (no viral rna and no fcov antibodies), possibly accounting for diseases other than fip, and samples tested negative only by qrt-pcr, although they contained fcov antibody titres between : and : , , which were highly suggestive of fip. eighteen effusions were found to contain fcov rna in the absence of specific antibodies (or at least in the presence of antibody titres < : ); of these qrt-pcr positive specimens had no fcov antibodies (or at least antibody titres < : ), while additional effusions contained antibody titres ranging from : to : , which are not considered as suggestive of fip. therefore, based only on antibody detection, a total of cats whose effusions contained viral rna were predicted not to be affected by fip, while taking advantage on molecular detection of fcov rna, animals with high antibody titres would have been considered fip negative. unfortunately, samples with fcov rna tested negative by iif even after treatment with the chaotropic thiocyanate ion, which had been proven to dissociate immune complexes pullen et al., . most effusions displaying the highest viral loads (c t values < ) contained antibody titres ≥ : ; only one sample with a low c t value displayed an antibody titre ( : ) not suggestive of fip (table ) . therefore, qrt-pcr positive samples with low antibody titres mostly contained low fcov rna loads, although the highest antibody titres were detected in effusions with c t values > . overall, no statistically significant correlation (r = . ; twotailed p-value = . ) was found between viral rna loads and antibody titres. intra-vitam fip diagnosis still represents a challenge for veterinarians and diagnosticians, since there is no available tool to unambiguously diagnose the disease. fip cannot be differentiated from an fcov enteric infection based on serology because the antibodies are directed against the same pathogen and there are no relevant antigenic differences between the enteric and hypervirulent strains. it is recognised that fip-ill cats have very high antibody titres in their serum and effusions due to the systemic spreading of the virus through the infected monocytes/macrophages (addie et al., ). however, detection of high antibody titres alone is not a confirmatory test. in addition, the absence of specific antibodies or the presence of very low antibody titres has been recently demonstrated in the effusions of cats with confirmed fip, likely due to antibody sequestration by the high number of viral particles in the same sample of some cats (meli et al., ) . hartmann et al. ( ) demonstrated that about % of cats with fip tested seronegative for fcov. however, in that study a transmissible gastroenteritis virus strain was used as antigen, which could affect the sensitivity of fcov-antibody testing (giori et al., ) . accordingly, fcov antibody titres were found to dramatically drop in terminal cases of fip (pedersen, ) . this phenomenon is not restricted to fip, but it has been also demonstrated for other viral infections characterized by high-level virus replication (quirós-roldán et al., ; guihot et al., ) . overall, detection of fcov antibodies in the effusions is affected by poor specificity and sensitivity. molecular methods have been used for detection of fcov rna in the effusions of cats with suspected fip (gut et al., ; simons et al., ; hornyák et al., ; soma et al., ; doenges et al., ; felten et al., ; longstaff et al., ) . however, these methods display similar issues related to the diagnostic performances (lack of sensitivity and specificity). in fact, they are not able to distinguish between enteric and virulent fcovs, since no specific genetic markers have been identified for the latter strains. in addition, the enteric fcovs have been proven to cause transient viremia and even have a low replication in the blood (can-sahna et al., ; kipar et al., ; fish et al., ) , thus potentially being able to passively spread to the effusions associated to other diseases. a recent paper (meli et al., ) has investigated the agreement between fcov antibody titres and rna detection in the effusions of cats with confirmed fip, showing a correlation between high amounts of virus and lower signals in iif assay, likely due to the fact that antibodies bound to viral antigens of the effusions are not able to bind to the antigens of the fcov-infected cells used in serological tests. here, we have analysed by the same methods the effusions of cats with suspected fip, thus including also potential samples from animals with non-fip related diseases. accordingly, using an iif antibody titre of : as a cut-off, samples tested negative by both iif and qrt-pcr assays, possibly accounting for diseases other than fip, while effusions gave contrasting results (low-titre or no antibodies in the presence of fcov rna or viceversa). these samples with conflicting results are likely to be true positive since an iif-negative result could be related to antibody sequestration by high viral loads (meli et al., ) . in addition, addie et al. ( ) demonstrated that up to % antibodypositive effusions from fip cases were negative for fcov rna, likely as a consequence of pcr inhibition by interfering substances or rna degradation during sample transportation and storage. however, in the absence of alternative diagnosis, even those cats with neither fcov antibodies nor rna in their effusions could not be definitively considered as non-fip animals (addie et al., ) . unfortunately, clinical cases were mostly untraceable and confirmatory necropsy was not done in any case, so that the lack of confirmatory testing represents the main limitation of the present study. in contrast with what observed by meli et al. ( ) , there was no statistically significant correlation between high viral loads and lowtitre or negative antibody results. in fact, most effusions with low or no fcov antibody titres displayed low amounts of virus, although samples with very high levels of fcov rna contained slightly lower antibody titres (generally < : ) in comparison with effusions with the lowest amounts of virus, which reached iif antibody titres of : , - : , ( table ) . the present study confirms that, when performed singularly, neither the detection of fcov nucleic acid nor that of specific antibodies in the effusions of cats with suspected fip is able to warrant an affordable diagnosis of the disease. therefore, in order to increase the diagnostic performances, we suggest combining the two methods (antibody and rna detection) for an intra-vitam diagnosis of effusive fip. using this diagnostic approach, only out of cats whose effusions were analysed would be considered fip negative, even if also in these cases fip could not be completely ruled out (meli et al., ) . thus, the combined serological and molecular protocol should improve the ability of fig. . comparison between indirect immunofluorescence (iif) assay and real-time rt-pcr (qrt-pcr) carried out on effusions from cats with suspected feline infectious peritonitis (fip) . numbers indicate the samples positive (+) or negative (−) for fcov antibodies or rna. results according to both techniques are shown in bold. for iif assay, the cut-off was set to : (a) or : (b), the latter being considered highly suggestive of fip. laboratories to diagnose effusive fip, especially if the test results are supported by clinical and haematological findings. however, intravitam diagnosis of non-effusive fip still remains highly inconclusive, even if recent studies tried to address this issue (doenges et al., ) . therefore, future studies are needed to develop and validate tools for the intra-vitam diagnosis of non-effusive fip, which still represents a 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measurement of relative antibody affinity by elisa using thiocyanate elution samples with high virus load cause a trend toward lower signal in feline coronavirus antibody tests the history and interpretation of feline coronavirus serology antibody avidity determination by elisa using thiocyanate elution anti-hepatitic c virus antibodies hidden in circulating antibody/antigen aggregates in hcv-rna positive patients a mrna pcr for the diagnosis of feline infectious peritonitis detection of ascitic feline coronavirus rna from cats with clinically suspected feline infectious peritonitis this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. key: cord- -xeq yix authors: nurtay, anel; hennessy, matthew g.; sardanyés, josep; alsedà, lluís; elena, santiago f. title: theoretical conditions for the coexistence of viral strains with differences in phenotypic traits: a bifurcation analysis date: - - journal: r soc open sci doi: . /rsos. sha: doc_id: cord_uid: xeq yix we investigate the dynamics of a wild-type viral strain which generates mutant strains differing in phenotypic properties for infectivity, virulence and mutation rates. we study, by means of a mathematical model and bifurcation analysis, conditions under which the wild-type and mutant viruses, which compete for the same host cells, can coexist. the coexistence conditions are formulated in terms of the basic reproductive numbers of the strains, a maximum value of the mutation rate and the virulence of the pathogens. the analysis reveals that parameter space can be divided into five regions, each with distinct dynamics, that are organized around degenerate bogdanov–takens and zero-hopf bifurcations, the latter of which gives rise to a curve of transcritical bifurcations of periodic orbits. these results provide new insights into the conditions by which viral populations may contain multiple coexisting strains in a stable manner. the combination of very large population sizes, very short generation times, and lack of proof-reading mechanisms during genome replication confer viral populations with an extremely high evolutionary plasticity that allow them to quickly adapt to system. nearly all mathematical models in epidemiology detecting various dynamical behaviours with multi-strain infections illustrate the necessity of numerical approaches and the dependency of such models on a large number of parameters [ ] [ ] [ ] [ ] [ ] . a classical approach used in epidemiology [ ] to circumvent this difficulty is to introduce dimensionless parameter groups, such as the basic reproduction number r , as in [ ] . however, the number of dimensionless groups can still become large as additional complexity is introduced into the model, as is the case here. thus, we perform a bifurcation analysis to systematically track how the dynamics of the system change as multiple parameters are varied. in general, the study of parameters in terms of their effect on the stability of certain states of a model is a highly effective way to gain important insights into the investigated system [ ] [ ] [ ] . the paper is organized as follows. we first introduce a detailed description of the model in § . then, in § , we provide the equilibrium points of the system, study their stability and investigate, in detail, the effect of phenotypic differences in infection rate, virulence and mutation rate. the biological interpretations of the results are present throughout the work, however, concrete statements are found in § . some technical details of the bifurcation analyses are discussed in appendix a. here we introduce the mathematical model describing the infection dynamics of wt and mutant strains. the model is based on a nonlinear system of five ordinary differential equations. the state variables of the model are: uninfected susceptible cells, x; two different virus strains, given by the wt (z w ) and the mutant (z m ) species; and two types of infected cells, one infected by the wt strain, y w , and another one infected by the mutant strains, y m (figure ). the time evolution of the interacting populations is described with the following model: . schematic diagram of the processes included in the mathematical model for the main system investigated, which does not consider backward mutations. the system is composed of two pathogenic strains: wild-type (variable z w ) and mutant (variable z m ) species, that compete for the infection of healthy cells (variable x, green cells). infection of healthy cells gives rise to two different populations of infected cells with wt (variable y w ) and mutant (variable y m ) strains, displayed by red and blue cells, respectively. viral strains are assumed to grow and mutate (dashed lines) within the cells, being released again to the system for further infection after cell lysis. the terms in model ( . ) are labelled with the biological processes. the meaning of these processes as well as of the parameters is discussed in the following lines. the uninfected host cells, which are limited by the carrying capacity of their environment, k, proliferate and die proportionally to parameters b and d, respectively. as previously mentioned, the mutant and the wt strains infect the host cells at rates a m and a w , respectively. mathematical and statistical models of multiple infections, coinfection and superinfection have been studied in detail [ ] [ ] [ ] [ ] and are not considered here. acknowledging the importance of differences between lytic and lysogenic infection cycles in the production of virions, yet unlike [ ] [ ] [ ] , we consider only lytic infections, i.e. the populations of infected cells do not grow as uninfected cells do [ ] . in fact, the model is based on the same assumptions of the classic lotka-volterra equations, which were adapted to virus dynamics by nowak & may [ ] and many others [ ] [ ] [ ] [ ] [ ] . in the study of coexistence of viral populations, the main role of infected cells is viewed in the scope of the processes of the lytic cycle, i.e. a direct impact in change and production of free virions. viruses replicate and produce their own type of virions via infected cells. strains can change and mutations occur only during the process of viral replication inside an infected cell (see dashed arrow in figure ). therefore, cells from population y w infected by the wt strain can mutate at rate m into cells of population y m . that would increase the size of the latter population at the same rate m. we note that our analyses are mainly developed considering mutations from the wt to the mutant strains. this is a standard strategy to keep the model as simple as possible, assuming that the probability of backward mutations is extremely small due to the enormous size of the sequence space. however, some results considering backward mutations will also be presented. while considering mutation in infected cells, we implicitly study the mutation of the viral genomes. it is clear that infection affects the life-span of infected cells in a nontrivial way [ ] . in this study, the virulence of the strains is depicted by including new decay mechanisms for the uninfected cells. however, for simplicity, we consider death rates of infected cells to be g m and g w , and as opposed to d þ dg m and d þ dg w . the absence of a mechanism for virus replication makes the multiplication of viral strains entirely dependent on the machinery of the infected cells. therefore, the overall number of virions produced in one lytic cycle must be proportional to the virulence and the number of virions produced by a single-infected cell, the latter of which we refer to as the burst size. we average the burst sizes of viral strains and consider them as constants k m and k w for mutant-and wt-infected cells, respectively. au contraire, the infection from the perspective of the virus is associated with an average number of virions spent to ensure a successful infection process. owing to the absence of co-infection by different strains in our model, this number can be considered as the multiplicity of infection of the viral strain. we take the multiplicity of infections to be constants n m and n w for mutant and wt strains, respectively. in this model, we assume that populations are not being harvested, but do consider a decay of all the populations in the system. in the case of the virus populations, this decrease is described as an outflow or 'death' of free virus particles from the system. the overall 'death' rates of the virions in the system are z w and z m for wt and mutant strains, respectively. initial conditions for system ( . ) are non-negative values: the model ( . ) can be simplified by introducing dimensionless variables that are based on characteristic timescales and population sizes. the quantity b d describes the effective growth rate of uninfected cells and its inverse, (b d) , is used to define the characteristic time scale of the system. in a virus-free environment, the maximum size of the uninfected cell population isx max ¼ ( À d=b)k, which is used to define the characteristic population size for both the uninfected and infected cells. the characteristic population size of the viral strains is chosen to be the product of the mutant burst size k m and the characteristic size of the infected cell population,x max . the former is required due to differences in sizes and measurement units of the viral loads and the cell populations in the system. we therefore non-dimensionalize the variables according to where bars are used to denote dimensionless quantities. we also introduce dimensionless parameters defined by where p i (recall that i ¼ m, w) stands for other 'rate' parameters, namely: g m , g w , z m and z w . notice that all non-dimensional 'rate' parameters, including mutation m, are relative rates, i.e. the rate relative to the effective growth rate of uninfected cells. meanwhile, non-dimensional parameters related to the virus populations are inevitably linked to the burst size due to the choice of z i . the non-dimensional burst size k is simply the ratio of the wt and mutant burst sizes. both multiplicity of infections are scaled with respect to the burst size of the mutant strain. the dependence of the dimensionless infection rates a i on the burst size of the mutant-type-infected cells, the carrying capacity, growth rate of uninfected cells and dimensional infection rate shows how each of these quantities affects the overall rate of infection. understandably, the non-dimensionalization places a restriction on the parameter values and requires b . d. however, this restriction is biologically justified. taking b . d, as shown later, forces the trivial equilibrium to be unstable, thus avoiding scenarios where all populations become extinct. upon ignoring bars for clarity purposes, we obtain the non-dimensionalized system and _ z w ¼ kg w y w À n w a w z w x À z w z w : ( : e) the dimensionless model ( . ) is now analysed to understand how the dynamics change under parameter variation. we calculate the equilibria of ( . ), conduct a linear stability analysis, and identify analytical conditions that lead to transcritical and hopf bifurcations. we find that curves of these bifurcations can intersect at specific points in parameter space, giving rise to degenerate bogdanov-takens (dbt) and zero-hopf (dzh) bifurcations. the role of dbt and dzh bifurcations is to organize the stability (or phase) diagram into regions with distinct dynamics. the numerical continuation package matcont [ ] is used to track how the equilibria and bifurcations evolve as the infection rate, virulence, and mutation rate are varied. the matcont source code and data are available online [ ] . the non-dimensional model given by equations ( . ) has four equilibria. for mathematical convenience, we define the population vector v(t) ¼ {x(t), y m (t), y w (t), z m (t), z w (t)}. the first equilibrium is the origin if stable, the trivial solution v corresponds to the extinction of all of the populations. the second equilibrium describes, whenever stable, a virus-free state whereby only the uninfected cells persist. owing to our choice of non-dimensionalization, the maximum population of uninfected cells is equal to one. the third equilibrium is given by and, if stable, corresponds to the persistence of uninfected cells and the mutant strain of the virus. there is no wt strain of the virus and the viral population is only composed of mutant genotypes. in order for the wt-free state v to be biologically meaningful, the condition n m , must hold. this condition corresponds to the burst size of the mutant virus being greater than its multiplicity of infection. throughout the remainder of the paper, it will be assumed that n m , . finally, the fourth equilibrium point is given by where a ¼ g w k À (m þ g w )n w , b ¼ À((n w À k)a w þ z w )g w À m(a w n w þ z w ) and c ¼ a m z w (g w ( À n m ) À mn m ) À a w z m (g w (k À n w ) À mn w ): this equilibrium point involves, whenever stable, a state of coexistence for the wt and mutant strains. similar to the wt-free state v , the coexistence state v can only be biologically meaningful if the burst size of wt strain satisfies k . n w ( þ m/g w ). this inequality is a generalization of that derived for the mutant virus (n m , ) which accounts for mutation. the singularity that occurs in the coexistence state v when c ¼ leads to difficulties when using numerical methods to track how this equilibrium evolves under parameter variation. the assumption of uni-directional mutation prevents the existence of an equilibrium point that is analogous to v whereby only the wt virus exists. a linear stability analysis is carried out to determine the behaviour of the system close to the equilibrium points. the jacobian matrix for equations ( . ) is given by straightforward calculations for the trivial solution v yield eigenvalues of the jacobian given by thus, for all parameter values, the trivial solution v , as mentioned before, is always unstable, i.e. it is a saddle point with a one-dimensional unstable manifold. although it is possible to find analytical expressions for the eigenvalues of the jacobian evaluated at v , they are sufficiently complicated that little insight is gained from analysing them directly. in order to conduct a stability analysis for v , it is more useful to consider the characteristic polynomial for the eigenvalues l. the polynomial det (j(v ) À li) ¼ can be regrouped into the form of three factors and the first factor, p , provides a constant eigenvalue, l ¼ , which reserves the possibility for stability of v . although the next two factors p and p do not provide obvious eigenvalues, they enable the identification of critical parameter groups for which v undergoes a bifurcation. based on our knowledge of the bifurcations that can occur in models similar to ( . ), we may expect to find transcritical and hopf bifurcations. for a varying parameter (or set of parameters), the onset of a hopf bifurcation leads to the creation of periodic orbits (pos) after the change of stability of the equilibrium point. furthermore, hopf bifurcations are characterized by the jacobian matrix having a single pair of complex conjugate eigenvalues with zero real part. an analysis of the factors p and p given by ( . b,c) reveals that v cannot undergo a hopf bifurcation. this is because the coefficients of the linear terms are strictly positive, thus preventing the eigenvalues from ever being purely imaginary. transcritical bifurcations occur when two equilibria collide non-destructively, exchanging their stability and resulting in the jacobian matrix having a single eigenvalue that is equal to zero. since v exists for all parameter combinations, transcritical bifurcations can be straightforwardly detected by forcing l to be zero in p and p . by solving it will be shown below that r w and r m are the basic reproductive numbers for the wt and mutant strains, respectively. when r w ¼ , the virus-free state v and the coexistence state v intersect. by expanding v around r w ¼ , we find that it has negative components when r w , and thus lies outside of the biologically meaningful phase space. however, all of the components of v become positive when r w . . likewise, expanding the equilibrium v around r m ¼ shows that this condition corresponds to points where the virus-free state v and wt-free state v intersect. for r m , , some components of v are negative; for r m . , all components are positive. in the case when both r w ¼ r m ¼ hold, there is a triple intersection of v , v and v . furthermore, the jacobian has a double zero eigenvalue at this point, indicating the onset of a non-degenerate or degenerate bogdanov-takens bifurcation. as will be shown in § . , the bogdanov-takens bifurcations in this model are of degenerate type. more generally, we find that v and v intersect when thus, there are three curves of transcritical bifurcations defined by the dynamics of the system near the dbt bifurcation can be determined by expanding the equilibria and their eigenvalues around r w ¼ and r m ¼ . the eigenvalues of all of the equilibria are real, ruling out the possibility of a branch of hopf bifurcations emanating from the dbt point, which is a generic feature of non-degenerate bogdanov-takens bifurcations [ ] . when r w , and r m , , the virusfree state v is locally asymptotically stable and the wt-free state v and the coexistence state v are unstable, meaning that the virus-free state is achieved. for r m . and r w , , the virus-free and wtfree state exchange stability, with v becoming unstable and v locally asymptotically stable, with v remaining unstable. similarly, for r w . and r m , , the virus-free and coexistence state exchange stability: v becomes unstable, v becomes locally asymptotically stable, and v remains unstable. the stability in the region r m . and r w . is governed by the transcritical bifurcation occurring along royalsocietypublishing.org/journal/rsos r. soc. open sci. : the curve r m ¼ r w , which leads to an exchange of stability between wt-free and coexistence states, v and v . the transcritical bifurcations that occur for with r w , do not result in an exchange of stability. thus, the local stability diagram near the dbt bifurcation can be drawn as in figure . there are three distinct regions, denoted by i, ii and iii, where v , v and v are local attractors, respectively. based on these results, we can interpret the quantities r m and r w as basic reproduction numbers for the mutant and wt viral strains, respectively. in general, for a strain to persist, its basic reproduction number has to be strictly greater than one. besides, the value of a basic reproduction number is proportional to the number of new infections arising in a following generation of virions from an infected cell. identifying basic reproduction numbers enables different models of population dynamics to be compared in a consistent manner. however, as it is not possible to write the dimensionless model ( . ) solely in terms of the basic reproduction numbers r m and r w , we will consider how the dynamics change under the variation of specific individual parameters that are of biological interest. to examine the stability of the wt-free state v , we obtain the eigenvalues from the characteristic polynomial computed from det (j(v )Àli) ¼ . this equation can be factorized and rewritten as q (l) Á q (l) ¼ , where q and q are quadratic and cubic polynomials in l, respectively. the exact form of q is not required here. we write following the analysis scheme discussed earlier, to obtain conditions for hopf bifurcations of v , we search for a purely imaginary pair of eigenvalues. setting q (l ¼ +iv)¼ , we obtain the critical condition for a hopf bifurcation, being the angular frequency of the emerging pos. interestingly, for this hopf bifurcation, there is no dependence on parameters associated with the wt strain of virus. for all biologically meaningful solutions of ad ¼ bc, the mutant strain of virus has the potential to gain periodic behaviour through the creation of a stable po. the other factor, q (l), provides no possibility for a hopf bifurcation due to a strictly positive linear coefficient in the quadratic polynomial. transcritical bifurcations of v may occur if which is equivalent to r m ¼ and corresponds to an intersection of v and v . second, from q (l ¼ ) ¼ , we obtain an expression which matches with ( . ), that is, the parameter combination yielding an intersection of v and v . as will be shown in § . , it is possible to simultaneously satisfy q (l ¼ +iv)¼ and q (l ¼ ) ¼ , implying that the jacobian matrix at v ¼ v has a pair of purely imaginary complex conjugate eigenvalues and a zero eigenvalue. this corresponds to the onset of a degenerate zero-hopf (dzh) bifurcation. like the dbt bifurcation, the dzh bifurcation will be shown to have an unusual structure that does not coincide with any of the standard normal forms (this point is further discussed in appendix a). in particular, our analysis reveals that a curve of global transcritical bifurcations of pos (tpo bifurcation) emanates from the dzh point rather than a curve of torus bifurcations [ ] . a tpo bifurcation occurs when an unstable po and a stable po collide with each other in a non-destructive way (differently from what would happen in a saddle-node bifurcation of pos, with destruction of pos), and subsequently exchange stability. the detailed stability and bifurcation analysis of v , v , as well as the dzh point, will be performed numerically. in particular, we will construct one-and two-dimensional bifurcation diagrams using specific pairs of parameters. we first examine the influence of the infection rates a m and a w on the dynamics. the values of the other parameters are set to we begin our investigation by constructing one-dimensional bifurcation diagrams using a m as the bifurcation parameter and fixing a w . we first consider two values of the wt strain infections rate given by a w ¼ . and a w ¼ , corresponding to basic reproduction numbers r w ¼ : and r w ¼ : , respectively. these values of a w therefore lie on opposite sides of the curve of transcritical bifurcations involving v and v defined by r w ¼ . the resulting bifurcation diagrams are shown in figure . solid and dashed lines represent stable and unstable equilibria, respectively. solid circles represent maxima and minima of stable pos. blue and orange lines denote equilibria that exist in biologically meaningful and non-meaningful phase space, respectively. the parametric dependence of the population of uninfected cell population x is used as this is the only component that differs between all four equilibria. figure a shows, for the case a w ¼ . when the infection rate of wt virus is increased to a w ¼ , the virus-free and coexistence states undergo a transcritical bifurcation. consequently, the bifurcation diagram in figure b shows that for mutant-type infection rates given by a m , . , the coexistence state v is stable whereas the virus-free state v is unstable. this implies that region i (v stable) is replaced by region iii (v stable). although v and v undergo a transcritical bifurcation at a m ¼ . , there is no exchange of stability. instead, it is v and v which exchange stability through a transcritical bifurcation at a m ¼ . , corresponding to the case of equal basic reproduction numbers, r m ¼ r w ¼ : . both regions ii and iv persist for a w ¼ , with the supercritical hopf bifurcation occurring at a m ¼ . , the same location as in figure a . the one-dimensional bifurcation diagrams shown in figure can be rebuilt using the infection rate of the wt virus a w as the bifurcation parameter for fixed values of a m that lie on opposite sides of r m ¼ . however, the resulting diagrams are qualitatively similar to those in figure , with the exception that the wt-free state v is switched with the coexistence state v and vice versa. the supercritical hopf bifurcation now occurs from v and thus gives rise to region v, characterized by the periodic coexistence of the viral populations. the five regions identified from the bifurcation analysis can be conveniently visualized by constructing a two-dimensional bifurcation diagram where both infection rates a m and a w are continuously varied. in this diagram, displayed in figure a, the locations of the bifurcations that separate the five regions are traced out as the infection rates vary. not all of the bifurcations shown in figure a lead to a biologically meaningful change in the dynamics, i.e. they do not represent a boundary between two distinct regions. thus, in figure b we represent the two-dimensional diagram with only the biologically meaningful bifurcations shown. figure a reveals a complicated bifurcation scenario that is largely centred about dbt and dzh bifurcations occurring at a w ¼ . , a m ¼ . and a w ¼ . , a m ¼ . , respectively. the dbt bifurcation lies at the intersection of three curves of transcritical bifurcations satisfying r w ¼ , r m ¼ and r m ¼ r w , which may be written, respectively, in terms of the infection rates as ( : ) the bifurcation structure around the dbt point is identical to that predicted from the local stability analysis. thus, we can eliminate from figure a the biologically irrelevant curves of transcritical bifurcations emerging from the dbt point in order to obtain the boundaries between regions i, ii and iii shown in figure b. the dzh bifurcation occurs at the simultaneous intersection of two curves of hopf bifurcations associated with v and v and the curve of transcritical bifurcations involving v and v . as predicted from linear stability analysis, the hopf bifurcation curve associated with the wt-free state v does not depend on the infection rate of the wt virus a w and thus appears as a straight line given by a m ¼ . . emanating from the dzh point is a curve of tpo bifurcations. to better understand the dynamics that occur near the dzh point, one-dimensional bifurcation diagrams are obtained by setting a w ¼ and treating a m as a bifurcation parameter and then setting a m ¼ and treating a w as the bifurcation parameter. the resulting diagrams are shown in figure , with open circles denoting unstable pos. figure a shows that as a m is increased from zero when a w ¼ , the wt-free state v first undergoes a transcritical bifurcation with the virus-free state v , then a subcritical hopf bifurcation, and finally a transcritical bifurcation with the coexistent state v . none of these bifurcations change the stability of the equilibria. thus, the associated curves of transcritical and hopf bifurcations in figure a do not represent boundaries between the five characteristic regions and are not shown in figure b. for these infection rates, all of the equilibria are unstable and the dynamics are therefore determined by the stability of pos. for sufficiently small values of a m , the system is in region v, and there is a stable po around the coexistence state v . this po is created by the hopf bifurcation that defines the boundary between regions iii and v shown in figure b. as a m is increased, the unstable pos around the wt-free state v that emerge from the subcritical hopf bifurcation grow in size. eventually, this unstable po collides and exchanges stability with the stable po around v through a tpo bifurcation at a m ¼ . . during the tpo bifurcation, the stable and biologically meaningful po around v becomes unstable and some components enter negative phase space. afterwards, the po around the wt-free state is stable and the system is in region iv. the one-dimensional bifurcation diagram created with a m ¼ and shown in figure b shares the same qualitative features as figure a, with v and its pos exchanging roles with v and its pos. none of the bifurcation of equilibria have biological significance. the main difference in this case is that the subcritical hopf bifurcation and the pos it creates exist outside the biologically meaningful phase space. as these pos are involved in a biologically meaningful tpo bifurcation, this figure demonstrates how it can still be useful to understand and resolve features that lie outside of the biologically meaningful space. upon removing the biologically redundant bifurcation curves from figure a, the stability diagram in figure b is obtained. the transcritical bifurcations at the boundaries between regions i and ii, and i and iii, capture the transition from virus-free to virus-persistent states that tend to a stable equilibrium. supercritical hopf bifurcations separate regions ii from iv and iii from v, and mark the transition between stationary and periodic population dynamics. the curve of tpo bifurcations separates regions iv and v, both of which are characterized by periodic viral populations, in the same way that a curve of transcritical bifurcations separates regions ii and iii, which describe stationary viral populations. thus, despite the system exhibiting a range of complex dynamics, they can be elegantly classified and organized in terms of the regions shown in figure b . overall, in figure b , we notice quite a large area of coexistence, regions iii and v. in population genetics, this corresponds to the most simple case of the emergence and persistence of a polymorphism in a population and maintenance of biodiversity. an example of an experiment that illustrates a simplified competition was conducted for escherichia coli in [ ] . these experiments focused on studying the behaviour of a newly emerging mutant in a population of a few strains of bacteria that compete with each other in a spatially homogeneous environment for the same type of nutrients. the results of the competition experiments demonstrate that in the vast majority of cases, the competitors stably coexist in steady or periodic states, which align with the outcome of our theoretical model. when even a slight change is possible in the genome, new mutations will appear. however, if all the other characteristics of an initial and a mutant population are the same, then, for both populations to persist, the initial population must have larger fitness than a mutant population. otherwise, the initial population will be out-competed because of the constant 'leak' into the mutant population. a more detailed analysis of the impact of mutation rates in the dynamics can be found in § . . we now study the effect of the strains' virulence ( parametrized by g w and g m ) on the dynamics of the system by building bifurcation diagrams. the infection rates are chosen to be from the different regions of the stability diagram shown in figure b , which are based on the reference values g m ¼ g w ¼ . . the other parameters are fixed to we focus on regions i, iii and iv of figure b as a starting point, covering all the other regions from there. we begin with the case where the infection rates are chosen to coincide with region i at the virulence reference values. these infection rates therefore correspond to basic reproduction numbers that are less than one. from the definition of r m given by ( . ) , we see that the basic reproduction number of the mutant is independent of the virulence. thus, changes in g m or g w cannot increase r m beyond one. therefore, regions ii and iv, where the mutant-type virus persists at the expense of the wt virus becoming extinct, cannot be entered. the basic reproduction number for the wt virus given by ( . ) is an increasing function of the virulence of the wt virus. in the limit of very large virulence, g w ! , we find that r w ! a w (k À n w )=z w . thus, if the infection rate a w is so small that the limit of r w is less than one, then it will not be possible to leave region i and both strains of the virus will always become extinct. however, if a w is sufficiently large and the basic reproduction number increases beyond one, then a change in dynamics will be observed as g w is increased. by solving r w ¼ , a critical value of the virulence of the wt virus is obtained the above equation is the condition for a transcritical bifurcation between the virus-free state v and the coexistence state v and defines the boundary between regions i and iii. thus, only for values of g w . g crit w will the virus persist in the system for this choice of infection rates. the stability diagram for a w ¼ . and a m ¼ . has been numerically computed and is shown in figure a . these values of the infection rate correspond to region i at the reference values of the virulence (figure b). as predicted, regions ii and iv are absent from the stability diagram. however, region v is also missing. thus, for this choice of infection rates, only regions i and iii can be entered by changing the values of the virulence. regions i and iii are separated by a straight dash line given by the critical condition ( . ) . we now consider infection rates given by a w ¼ , a m ¼ , which correspond to region iii at the reference virulence (figure b). the resulting stability diagram in terms of the virulence is shown in figure b . the choice of a m ¼ along with the values of the parameters in ( . ) leads to r m ¼ : . thus, variations in the virulence cannot bring the system to region i and at least one type of virus will always persist. a transition between regions ii and iii can occur via the transcritical bifurcation between the wt-free and coexistence states v and v . the critical condition for this transcritical bifurcation is given by equation ( . ) , and depends only on the virulence of the wt viral strain. thus, the transition between regions ii and iii appears as the vertical dashed line in figure b . interestingly, this figure shows that as the virulence of the wt virus is increased, the system transitions from region iii to region v and then back to region iii. these transitions occur via supercritical hopf bifurcations, denoted by solid lines. this scenario corresponds to the so-called bubble bifurcation (found in other epidemiological systems, e.g. [ ] ), in which the system is in a stationary state, then enters into an oscillating one, and finally goes back to a stationary state as the control parameter is changed. to understand why region iv does not appear in the stability diagrams on figure a ,b, we first recall that region iv is separated from region ii by a hopf bifurcation curve of the wt-free state v . this hopf curve has been calculated analytically from the equality ad ¼ bc, where a, b, c and d are given in ( . ) , and is a quadratic expression for g m independent of g w and a w . solving ad ¼ bc to find g crit m (not shown due to complexity of the expression), at the values of infection rates chosen for figure a,b, we notice they are outside of the positive parameters space. in fact, only for values of the mutant-type infection rate that satisfy equivalent to a m . . for the chosen parameters, does the hopf curve of v appear on a g w versus g m stability diagram ( figure c) . greater values of a m lead to increases in the area of region iv under region ii. the stability diagrams of figure indicate the impact of virulence on the complexity of the dynamics. clearly, the virulence of the mutant strain does not affect the survival of the wt strain. that is, increases in g m do not lead to an appearance or disappearance of the wt strain. however, the survival of the mutant strain does not appear to depend on g m either. importantly, the virulence of the wt strain, g w , strongly controls the dynamics and the persistence of the wt strain. as can be seen for small values of g w , this strain can become extinct as shown in figure b. in other words, even for a superior infection rate of the wt viral strain, there is a threshold of g w that must be surpassed in order for this strain to exist. for inferior values that are below this threshold, the survival of the wt strain is impossible because the rate of wt virion production, i.e. the death of rate infected cells, is insufficient. the qualitative behaviour of solutions, i.e. whether the populations of wt strains undergo oscillations or stabilize to a certain value, depends on the virulence in a non-trivial way. as shown in figure b, there is an interval of values for g w for which the system has a stable po governing the coexistence of strains, marked as region v. outside of this interval, the populations reach stable stationary states. in the previous analyses, the mutation rate has been fixed to m ¼ . . we now consider the effect of m on the dynamics. this is a key parameter that has been largely investigated within the framework of the error threshold [ , ] and lethal mutagenesis [ ] in quasi-species theory. in general, the coexistence of two strains requires the basic reproduction number of the wt virus to be greater than one, r w . , which can be interpreted as a condition on the mutation rate: m , g w k z w a w À þ n w À : thus, for two strains to coexist, the mutation rate must be sufficiently small. this result supports a conjecture that excess mutation exhausts the population of the wt strain, thereby leading to a process similar to the well-known error catastrophe [ ] . we would expect that a gradual increase of the mutation rate contributes to a better success of the mutant strain as the frequency of mutants generated de novo increases. the right-hand side of ( . ) is an increasing function of a w , reflecting the fact that a greater rate of infection by the wt virus will offset a greater mutation rate. a stability diagram in terms of the mutation rate m and the wt infection rate a w is shown in figure a for the figure . two-dimensional bifurcation diagrams for g w versus g m at different sets of a i , with i ¼ m, w. parameter values are given by ( . ) and (a) ( . ) . the dashed line marking the boundary between regions i and iii, and also defining the region of coexisting populations, has been obtained by replacing the inequality with equality in ( . ) . as the infection rate a w increases, the boundary between regions i and iii reaches a horizontal asymptote given by for mutation rates that satisfy m , m c , regions iii and v can be entered from region i by increases in the infection rate, promoting coexistence. however, for m . m c , only the wt-free state can occur. hence, equation ( . ) defines a critical, finite mutation rate at which coexistence no longer becomes possible due to extinction of the wt virus due to the outcompetition by the mutant strains. to explore this in more detail, we have repeated the stability diagram shown in figure b using a value of m ¼ . . m c . the stability diagram changes drastically, becoming that shown in figure b , and contains only three regions: i, ii and iv. the stability diagrams in figure b and b are linked through the fact that as the mutation rate increases, the dbt bifurcation shifts to the right, eventually tending to infinity as the critical value is approached. finally, figure c,d illustrates how the equilibrium populations of infected cells and viral strains change with increasing mutation rate m. specifically, we have used a w ¼ . and a m ¼ . , along with the parameters in ( . ), in both panels. as m increases beyond the point where ( . ) is satisfied, the population of mutants (virions and infected cells) outcompetes the wt populations ( figure c,d) . this phenomenon is similar to the error threshold defined in quasi-species theory. here we show that mutation is not only involved in this shift, but also depends (at the infection-cell level) on virulence, burst size and multiplicity of infection. throughout the previous analyses, we have assumed that the wt strain of the virus mutates and the new strain has no chance of mutating exactly into the original strain. although it is highly unlikely, the full picture of the proposed model would require a consideration of a possibility for backwards mutation, especially when modelling phenotypic traits. we therefore replace ( . b,c) with we begin our investigation of the role of backwards mutation by re-building the two-dimensional bifurcation diagram and stability diagrams shown in figure using the parameters in ( . ) . the mutation rate of the wt virus is set to m w ¼ . and we consider two values of the mutation rate of the mutant virus. first, we take m m ¼ ( m w , so that the new form of mutation can be considered as a small perturbation to the original system of equations ( . ). then, we equate the mutation rates of both strains and set m m ¼ . . the two-dimensional bifurcation diagrams in figure a ,b reveal that backwards mutation results in several important changes to the dynamics. importantly, the behaviour of the system can be described using three main states: the virus-free state (analogous to region i and displayed in white in figure ), stationary coexistence all populations ( polka dot pattern in figure , analogous to region iii), and oscillatory coexistence of all the populations (checkerboard pattern in figure , analogous to region v). furthermore, the dbt and the dzh bifurcations have vanished, the latter of which implies that the curve of tpo bifurcations no longer exists either. the vertical and horizontal lines of transcritical bifurcations defined by r w ¼ and r m ¼ , which intersected at right angles in figure a , have now merged into two separate branches that do not intersect. the curves of hopf bifurcations, which also intersected in the case of uni-directional mutation, have also merged into two distinct non-intersecting branches. finally, the transcritical bifurcation between the wt-free and coexistent states v and v has vanished as well. the resulting stability diagram, shown in figure by patterns, is now considerably simpler and involves only analogues of regions i, iii and v. thus, the only virus-persistent states are those in which both virus strains coexist. the wt-free state can no longer occur due to the creation of the wt virus through backwards mutation. in this paper, we studied a mathematical model of population dynamics of two viral strains infecting a population of the same cell type. unlike previous models of parasite-host-like interaction, we consider the emergence of a second strain by mutation from a single strain introduced into the environment. the mechanism of virus replication forces the consideration of two types of infected cells, one for each viral strain, in the model. we analysed the system of differential equations and its solutions. by studying parameters space, we identified five different regions, each characterized by distinct dynamics: (region i) the virus-free state which maximizes the population of host cells, (region ii) the stationary existence of mutant virus with nonzero cell populations supporting it, (region iii) the stationary persistence of both viral strains with nonzero cell populations, (region iv) the oscillating existence of the mutant virus and corresponding cell populations but extinct wt virus population, and finally, (region v) the oscillating coexistence of all populations. the population of the mutant-type virus exists as long as the mutation rate of the wt strain is positive. the broken symmetry between the wt and mutant strains is clear from the stability diagrams based on infection rates. from our results, we observe that survival of the wt virus is essential for coexistence. however, the growth of the wt virus population jeopardizes its persistence in the system by creating its own competitor: the mutant-type virus. moreover, the larger the mutation rate, the greater the infection rate of the original wt strain should be in order to remain in the system. interestingly, we have found a maximum-critical value-of the mutation rate in the context of the model which allows for the persistence of the wt strain and hence coexistence. values of the mutation rate that are higher than the critical value make coexistence impossible even for the most infectious wt strain. furthermore, our model shows that the concept of the error threshold may not be considered as a one-parameter-driven effect, i.e. based solely on the mutation rate [ , ] . the critical value of mutation is shown here to be proportional to the virulence and burst size of the wt virus and proportionally inverse to its multiplicity of infection. hence, our results extend the phenomenon of the error threshold at the infected cell population level. data accessibility. matcont source files, output data and matlab files used to create the figures in the manuscript are available from the dryad digital repository: https://doi.org/ . /dryad. g v [ ] . and _ j ¼ b þ b j þ a j þ b j j , ( a b) provided that the coefficients a and b are not equal to zero, a b = . the quantities b and b in equations (a ) play the role of bifurcation parameters, with b ¼ b ¼ corresponding to the bt point. the bifurcation diagram of system (a ) consists of curves of saddle-node and hopf bifurcations, neither of which were detected in the local analysis of the model analysed in this article ( . ) near the bt point, as well as curves of global bifurcations involving homoclinic orbits. the procedure outlined by kuzetnsov [ ] enables the normal form coefficients to be related to the model studied here. remarkably, the coefficient a can be calculated analytically and is found to be equal to zero for all parameter combinations. thus, the bt bifurcation occurring in our model is always degenerate and the local dynamics will be different from those of equation (a ). the vanishing of a can be rationalized in terms of the number of equilibria at the degenerate bogdanov-takens (dbt) point. an equilibrium analysis shows that (a ) will only have two equilibria at the dbt point b ¼ b ¼ if a = ; however, the model under study has three. thus, a ¼ is needed to capture the triple equilibrium at the dbt point in our model. this also suggests that higher-order terms must be included in (a ) in order for it to capture the dbt bifurcation in the model investigated here. while an extended system of normal form equations for degenerate bt bifurcations is proposed in kuznetsov [ ] , it cannot capture the transcritical bifurcations found in the original model, suggesting that an alternative form is required. however, due to the simple nature of the local dynamics near the degenerate bt point shown in figure , which can be obtained analytically from the full model ( . ), we do not pursue this point further. the poincaré normal form of a zh bifurcation can be written as [ , § . ] is a vector of bifurcation parameters. analyses of the normal form equations for the zh bifurcation show that local dynamics depend on the values of the normal form coefficients h ijk and g ijk . a common feature between the various cases is that the zh bifurcation occurs at the tangential intersection of curves of saddle-node and hopf bifurcations. however, figure a shows that the zero-hopf bifurcation in our model lies at a transversal intersection of curves of transcritical and hopf bifurcations. furthermore, none of the normal forms predict that a curve of tpo bifurcations should emanate from a zh point. the calculation of the three normal form coefficients for the dzh bifurcation is rather involved and must be performed numerically. we find that the normal form coefficient g ( ) ¼ across a range of parameter values, indicating the zh bifurcation is degenerate. the normal form equations for degenerate zh bifurcations are not well known and previous studies have focused on different cases [ , ] . tigan et al. [ ] showed that a degenerate zh bifurcation with g ( ) ¼ occurs in a rö ssler-type system and used averaging theory to detect periodic orbits, leaving the structure of global bifurcations unresolved. we believe, as far as we know, that this is the first time a curve of tpo bifurcations has been observed to emanate from a degenerate zh bifurcation of this type. we leave determining the corresponding normal form as an interesting area of future work. viral quasispecies. virology - frequency of hepatitis b virus e-minus mutants varies among patients from different areas of china coexistence of hepatitis b surface antigen (hbs ag) and anti-hbs antibodies in chronic hepatitis b virus carriers: influence of "a" determinant variants evolution and emergence of plant viruses emerging pathogens: the epidemiology and evolution of species jump ultra-deep sequencing for the analysis of viral populations deep sequencing analysis of viral infection and evolution allows rapid and 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bifurcations in a rössler-type system acknowledgements. we thank antoni guillamon for interesting discussions about the zero-hopf bifurcation. m.h., j.s. and l.a. acknowledge the hospitality of the instituto de biología molecular y celular de plantas which led to fruitful meetings. competing interests. we declare we have no competing interests. funding. the research leading to these results has received funding from 'la caixa' foundation. this work has been also appendix a. further study of degenerate bogdanov -takens and zero-hopf bifurcationsnormal form theory provides a means of predicting the local dynamics that occur near a given bifurcation. this is possible because the local structure of a bifurcation is model independent and thus can be immediately determined from pre-existing analyses of simpler systems of equations. the simplest system of equations that completely captures the dynamics of a particular bifurcation is called the normal form. the conducted numerical studies on the system ( . ) revealed that the bogdanov-takens and zero-hopf bifurcations are non-standard in the sense that the local dynamics do not agree with those predicted by their normal forms. such a mismatch can occur when one of the terms in the normal form equations vanishes due to its coefficient being equal to zero, implying that an extended system of equations needs to be considered. we now examine the normal forms of the bogdanov-takens (bt) and zero-hopf (zh) bifurcations in order to rationalize the discrepancies between the two-dimensional bifurcation diagram shown in figure and those predicted from normal form theory.the local bifurcation diagram near a bt point can be determined from the normal form equations given by (e.g. kuznetsov [ , § . ]) key: cord- -c lszcu authors: miao, faming; zhang, jingyuan; li, nan; chen, teng; wang, lidong; zhang, fei; mi, lijuan; zhang, jinxia; wang, shuchao; wang, ying; zhou, xintao; zhang, yanyan; li, min; zhang, shoufeng; hu, rongliang title: rapid and sensitive recombinase polymerase amplification combined with lateral flow strip for detecting african swine fever virus date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: c lszcu african swine fever virus (asfv), the etiological agent of african swine fever (asf), a hemorrhagic fever of domestic pigs, has devastating consequences for the pig farming industry. more than , , pigs have been slaughtered since august in china. however, vaccines or drugs for asf have yet to be developed. as such, a rapid test that can accurately detect asfv on-site is important to the timely implementation of control measures. in this study, we developed a rapid test that combines recombinase polymerase amplification (rpa) of the asfv p gene with lateral flow detection (lfd). results showed that the sensitivity of recombinase polymerase amplification with lateral flow dipstick (rpa-lfd) for asfv was copies per reaction within min at °c. the assay was highly specific to asfv and had no cross-reactions with other porcine viruses, including classical swine fever virus (csfv). a total of field samples were examined using our method, and the agreement of the positive rate between rpa-lfd ( / ) and real-time pcr ( / ) was %. overall, rpa-lfd provides a novel alternative for the simple, sensitive, and specific identification of asfv and showed potential for on-site asfv detection. african swine fever (asf) is a severe viral disease that manifests clinical symptoms of hemorrhagic fever caused by african swine fever virus (asfv) and can result in case fatality rates of up to % in domestic pigs, depending on the virus strain (galindo and alonso, ) . asf, which was first identified by montgomery ( ) in kenya in the s, made its first incursion into europe via two successive entries into portugal (in and again in ) , spreading rapidly throughout western europe and then to south america and the caribbean (michaud et al., ) . it was eventually eradicated by the mid- s, with the exception of sardinia (mur et al., ) . however, since the second major incursion of the disease, initially into georgia in , asf has spread to eastern europe and russia (rowlands et al., ; revilla et al., ) . the virus has continued to spread worldwide, including china. since the first asf case emerged in china in august , more than cases have been recorded in provinces (zhou et al., ) . african swine fever normally presents with non-specific symptoms, including fever, anorexia, vomiting, and diffused hemorrhage in superficial skin. post-mortem examination shows pericardial effusion, kidney enlargement, lymphadenectasis, and darkened and enlarged spleen. all these features are indistinguishable from those seen in classical swine fever virus (csfv) infection (tauscher et al., ; li and tian, ) . at present, no effective treatment or vaccine for asfv is available, and disease control is based mainly on animal slaughtering and strict sanitary measures (cisek et al., ; rock, ) . rapid laboratory diagnosis is important for timely triage and confirmation to control and preventing this disease because of its rapid progression to death and spread. molecular tools based on the detection of the genetic information of asfv have become more widely accepted for asf diagnosis (oura et al., ) . polymerase chain reaction (pcr) and real-time pcr techniques have provided a supportive method to post-mortem asf diagnosis (aguero et al., ; zsak et al., ; fernandez-pinero et al., ) ; however, they cannot be used for field (on-site) detection in pig farms. recently, liu et al. ( ) reported that the improved realtime pcr assay using a universal probe library (upl) probe could be applied to asfv molecular diagnosis under field conditions. due to limitations of the battery-powered realtime pcr instrument, it can process only a moderate number of samples. isothermal recombinase polymerase amplification (rpa) has been successfully used to detect multiple viral pathogens, including infectious bovine rhinotracheitis virus (hou et al., ) , bovine coronavirus (amer et al., ) , ebola virus (yang et al., ) , bovine viral diarrhea virus, or foot-and-mouth disease virus (wang et al., ) . as reports of asfv detection by rpa are limited, we aimed to develop and evaluate a rapid detection tool that combines immunochromatographic strip tests, more commonly referred to as lateral flow devices (lfds), with rpa targeting the conserved asfv p gene. this study was conducted as part of the surveillance of the asf outbreak in china. samples were collected for asf testing and surveillance under the agreement between the ministry of agriculture and rural affairs of the chinese government and farm owners. sample collecting treatment was conducted in accordance with the protocols for viral hemorrhagic fever under the urgent interim guidance for case management established by the world organization for animal health. the protocol for this study was approved by the ethics committee of the military veterinary research, academy of military medical sciences. samples were collected by animal centers for disease control and prevention of jilin province. the viruses were inactivated in the bsl- lab, and the inactivated samples were transferred to the bsl- lab for genomic dna extraction and detection. for standard plasmids, a plasmid extraction kit was used in accordance with the manufacturer's instructions (axygen, united states). for field samples, tissue homogenates were prepared in formaldehyde, and the inactivated virus homogenate was subjected to dna extraction by using a nanomagnetic bead adsorption kit (bailing biotechnology, beijing co., ltd., china). for the whole extraction procedure, lysis, adsorption, and washing were performed, and elution with µl of rnase-free water was conducted to dissolve the dna. the extracted dna was then stored at − • c. the p gene was first amplified in the rpa reaction system comprising the designed primers (labeled biotin) and a -carboxyfluorescein (fitc) labeled-probe, and the expected amplicons were labeled with -fitc and -biotin at the ends. then, the amplified products were recognized by anti-biotin and anti-fitc monoclonal antibodies on the test line, where gold nanoparticles were prefixed (figure ) . the whole genome sequence of a p genotype ii virus-asfv-sy , which was sequenced in our lab (genbank accession number mh )-was used as the reference sequence for rpa primer selection. rpa sense primer ( -aagaagaaagttaatagcagatgccgataccac- ), rpa anti-sense primer ( -biotin-gctcttacatacccttccacta cggaggcaatg- ), and rpa probe primer ( -fam-tgg gttggtattcctcccgtggcttcaaagcaaag[thf]taa tcatcatcgcac-p- ) were designed by using twistdx nfo rpa kits (cambridge, united kingdom) in accordance with the manufacturer's guidelines. taqman pcr was performed to detect asfv p in an mx p multiplex quantitative pcr system (stratagene) in accordance with the manufacturer's instructions. real-time pcr primers for p were used as described before (king et al., ) . the sequences spanning the rpa amplification region were obtained through pcr and cloned into the pmd -t plasmid to generate figure | schematic of rpa-lfd assay. frontiers in microbiology | www.frontiersin.org pmd -p . taqman runs of the experimental samples involved at least three replicates with no-template or no-primer control and the combination of the primers. the pcr conditions were • c for min and cycles of • c for s and • c for s. a standard curve was generated from serially diluted p recombinant plasmids of known copy numbers. a pair of primers and rpa probe primer was designed to specifically detect asfv circulating strains in china. the rpa assay was conducted in a µl system by using a twistamp nfo kit (twistdx tm , cambridge, united kingdom). all the reagents ( nm rpa primers, nm rpa probe, × rehydration buffer, and purified h o) except the template or the sample dna and magnesium acetate were prepared in a master mix, which was aliquoted into each tube of a . ml tube strip containing a dried enzyme pellet. magnesium acetate ( . µl) was pipetted slowly into the tube lids, and subsequently, µl of the sample dna was added to the tubes, the lids were closed, and magnesium acetate was centrifuged into the tubes by using a mini-spin centrifuge. the tubes were immediately used for isothermal amplification. asfv-rpa detection was combined with an lfd (yoshida, co., ltd., china). then, µl of the amplified product was added to the sample pad, and the dipstick was inserted into µl of the sample buffer for - min. a dilution range of to copies per reaction of pmd -p recombinant plasmid was used to evaluate the sensitivity of recombinase polymerase amplification with lateral flow dipstick (rpa-lfd), and the amplicons were evaluated through agarose gel electrophoresis. the reactions were evaluated with prrsv/ch r, csfv/shimen, prv/jl , jev/sa - - , pedv/cv , and pcv b-sd to examine the specificity of the developed rpa-lfd assay. the thermo-reaction procedures were optimized after different primers, probe combinations, and thermo-cycles were run in the real-time pcr system to evaluate the sensitivity of taqman asfv real-time pcr assay. the real-time pcr conditions that yielded the highest amplification efficiency were • c for min and cycles of • c for s and • c for s by using the primer combination reported by king et al. ( ) . the realtime pcr assay was sufficiently sensitive to detect copies per reaction (figure ) . no cross-reactions with prrsv/ch r, csfv/shimen, prv/jl , jev/sa - - , pedv/cv , and pcv b-sd were observed, and a positive signal was detected in asfv-sy and asfv-jl . in this study, copies of the asfv-positive pmd -p plasmid were used as a template to determine the optimal rpa reaction temperature. the reaction mixture was incubated at eight different temperatures ( • c, • c, • c, • c, • c, • c, • c, and • c) for min. the detection result showed that the test line was short at • c and • c, and the test line density did not enhance significantly from • c to • c ( figure a) . in this study, copies of the asfv-positive plasmid were used as a template to determine the optimal rpa reaction time. in figure b , the rpa assay tested positive for asfv in min. the measured dna band density revealed that the dna yield doubled after a reaction time of min and increased slightly after min. the intensity of the dna bands in agarose gel did not significantly change at reaction times of , , , and min. this finding indicated that the rpa reactions during the remaining parts of this study were completed after min. however, the false positive test lines appeared when the reaction time was more than min (invalid result). therefore, min was considered the optimum reaction time for the rpa-lfd assay, and the assay worked from • c to • c. in terms of convenience and safety, • c was chosen as the optimum reaction temperature. the sensitivity results showed that the detection limit of the asfv rpa-lfd assay was copies per reaction of the recombinant plasmid pmd -p . the amplicon in the asfv rpa-lfd assay ( bp) was also tested through subsequent visualization with % agarose gel-electrophoresis after purification, and the sensitivity of rpa based on gelelectrophoresis visualization was copies per reaction (figure ) . in terms of the specificity of asfv rpa-lfd assay, no cross-reactions with other important viruses of pigs were observed (figure ) , and this observation was confirmed by agarose gel electrophoresis. the samples included spleen samples and blood samples of animals (jilin cdc). of these samples, spleen tissues and blood samples were positive for asfv nucleic acid as detected by rpa-lfd. taqman real-time pcr showed that samples were positive. the rpa-lfd detection results were consistent with those of real-time pcr. rpa-lfd revealed that the samples showed a light test line and high cq values. of these samples, one tested sample, which showed with a short test line or no test line, was rotten, and the cq value in real-time pcr was , indicating weakly positive result. although the detection results between the two methods were consistent (table ) , the rpa-lfd assay was more rapid than real-time pcr. in particular, the whole rpa-lfd assay yielded results in min, whereas real-time pcr generated results approximately h later. the average time from symptom onset to outcome in asf is - days; however, the pig farms were usually in rural areas or even in a mountainous area far away from the city laboratory, which required more time for sampling and transportation. once diagnosis and timely treatment of suspected asf pigs is delayed, the risk of asf exposure to other pig farms was increased. in the current outbreak, laboratory testing with taqman real-time pcr is being widely used in affected areas. however, requirements-including a sophisticated thermocycler, complex sample treatment procedures, and expensive reaction instruments-have limited its applications in point-of-care testing (king et al., ) . different isothermal molecule amplification assays, including loop-mediated isothermal amplification assay (lamp) (james et al., ) , polymerase cross-linking spiral reaction (wozniakowski et al., ) , cross-priming amplification method (fraczyk et al., ) , chimeric dna/lna-based biosensor (biagetti et al., ) , and droplet digital pcr (wu et al., ) , have been developed as a rapid, simple, and cost-effective alternative to pcr-based molecule assay. rpa has several advantages. for example, initial heating for dna denaturation is not required, and test conditions ( • c within min) are easily implemented. lamp assay for asfv detection requires a long time ( min) and high temperature ( • c− • c) (james et al., ) . by comparison, rpa assay takes less than min at • c to complete. as such, rpa assay is simpler and more easily used than lamp assay. wang et al. ( ) reported the field validation of real-time rpa for asfv, although this technique can specifically detect asfv plasmid with a detection limit of dna copies per reaction. however, this detection assay is based on an expensive scanner device. detection methods for actual clinical samples have yet to be designed, so the application of this test to clinical samples is limited. gao et al. ( ) developed crosspriming amplification combined with immune-chromatographic strips for the rapid on-site detection of asfv, but its reaction conditions include min at • c and six primers for a reaction system, thereby causing non-specific amplification. the use of lateral flow assays combined with a monoclonal antibody against p protein of asfv can be used to detect p viral and recombinant protein or inactivated culture virus (sastre et al., ) ; however, the sensitivity of the assay is -fold lower than genomic amplification. recombinase polymerase amplification with lateral flow dipstick overcomes the technical difficulties posed by current amplification methods; for example, it operates at a low and constant temperature, does not require the thermal denaturation of templates, and does not rely on an expensive thermocycler (aguero et al., ) . in combination with a commercially magnetic nanobead-based dna extraction kit, this approach can be applied to on-site testing or rapid diagnosis under poorly equipped conditions. rpa is highly resistant to crude samples, suggesting that it can be used for on-the-spot field testing with crude nucleic acid extraction. in an rpa-nfo reaction system, nfo cleaves the primer of the probe at the thf position and effectively deblocks the probe, thereby generating a new hydroxyl group that can act as a primer for polymerase extension; thus, the probe is transformed into an extension primer with an increased specificity of amplification (james and macdonald, ) . the potential defect of rpa-lfd assay for asfv is that it may carry over contaminants in fields because its tube must be opened after amplification is completed. therefore, precautions should be taken. for example, reaction tubes should be carefully opened and closed, gloves should frequently be changed, the progress of pre-and post-rpa amplification should be separated, and reaction time should be shortened if possible. the replacement of dttp with dutp may help prevent such carryover contamination as demonstrated in other nucleic amplified system assays. in summary, we evaluated a rapid rpa-lfd test for the rapid diagnosis of asfv. the superior performance of rpa-lfd for asfv and its consistent detection results with those of real-time pcr indicated its appropriateness for laboratory diagnosis and that it has great potential for on-site testing of asfv circulating in china. the raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher. this study was conducted as part of the surveillance of the asf outbreak in china. samples were collected for asf testing and surveillance under the agreement between the ministry of agriculture and rural affairs of the chinese government and farm owners. sample collecting treatment was conducted in accordance with the protocols for viral hemorrhagic fever under the urgent interim guidance for case management established by the world organization for animal health. the protocol for this study was approved by the ethics committee of the military veterinary research institute, academy of military medical sciences. samples were collected by animals centers for disease control and prevention of jilin province. the viruses were inactivated in the bsl- lab, and the inactivated samples were transferred to the bsl- lab for genomic dna extraction and detection. highly sensitive pcr assay for routine diagnosis of african swine fever virus in clinical samples a new approach for diagnosis of bovine coronavirus using a reverse transcription recombinase polymerase amplification assay chimeric dna/lna-based biosensor for the rapid detection of african swine fever virus african swine fever virus: a new old enemy of europe molecular diagnosis of african swine fever by a new realtime pcr using universal probe library development of cross-priming amplification for direct detection of the african swine fever virus, in pig and wild boar blood and sera samples african swine fever virus: a review cross-priming amplification combined with immunochromatographic strip for rapid on-site detection of african swine fever virus rapid detection of infectious bovine rhinotracheitis virus using recombinase polymerase amplification assays recombinase polymerase amplification: emergence as a critical molecular technology for rapid, low-resource diagnostics detection of african swine fever virus by loop-mediated isothermal amplification development of a taqman pcr assay with internal amplification control for the detection of african swine fever virus african swine fever in china overcoming the challenges of pen-side molecular diagnosis of african swine fever to support outbreak investigations under field conditions comprehensive phylogenetic reconstructions of african swine fever virus: proposal for a new classification and molecular dating of the virus on a form of swine fever occurring in british east africa (kenya colony) thirty-five-year presence of african swine fever in sardinia: history, evolution and risk factors for disease maintenance virological diagnosis of african swine fever-comparative study of available tests african swine fever virus biology and vaccine approaches challenges for african swine fever vaccine developmentperhaps the end of the beginning african swine fever virus isolate, georgia development of a novel lateral flow assay for detection of african swine fever in blood on the situation of african swine fever and the biological characterization of recent virus isolates rapid detection of foot-and-mouth disease virus using reverse transcription recombinase polymerase amplification combined with a lateral flow dipstick a recombinase polymerase amplification-based assay for rapid detection of african swine fever virus polymerase cross-linking spiral reaction (pclsr) for detection of african swine fever virus (asfv) in pigs and wild boars development and application of a droplet digital polymerase chain reaction (ddpcr) for detection and investigation of african swine fever virus development and evaluation of a rapid and sensitive ebov-rpa test for rapid diagnosis of ebola virus disease emergence of african swine fever in china preclinical diagnosis of african swine fever in contact-exposed swine by a real-time pcr assay key: cord- -e ylkonb authors: mo, xiao-dong; zhang, xiao-hui; xu, lan-ping; wang, yu; yan, chen-hua; chen, huan; chen, yu-hong; han, wei; wang, feng-rong; wang, jing-zhi; liu, kai-yan; huang, xiao-jun title: treatment of late-onset hemorrhagic cystitis after allogeneic hematopoietic stem cell transplantation: the role of corticosteroids date: - - journal: ann hematol doi: . /s - - - sha: doc_id: cord_uid: e ylkonb we aimed to evaluate the treatments, particularly the role of corticosteroids, in patients with late-onset hemorrhagic cystitis (lohc) after allogeneic hematopoietic stem cell transplantation (allo-hsct). one hundred and sixty-three consecutive patients who underwent non-t-cell-depleted allo-hsct and met the criterion of lohc after allo-hsct were enrolled in this study. the median time from allo-hsct to the occurrence of lohc was (range, – ) days. pathogens identified in blood and/or urine samples from patients were mostly viruses. all of the patients with lohc received intravenous fluid hydration, alkalization, and forced diuresis, of which patients achieved complete remission (cr) after these treatments. the remaining patients received anti-infection therapies and achieved cr after the therapies. corticosteroids were additionally applied to out of patients who did not achieve cr after anti-infection therapies, and . % (n = ) of them showed a grade to lohc at the beginning of corticosteroid therapy. thirty-five patients showed an immediate response (cr or downgraded at least one grade) within week after the beginning of the corticosteroid therapy. sixty-four patients ( . %) achieved cr after corticosteroid therapy, and the median period from the beginning of corticosteroid therapy to cr was days. thus, we observed that viruses were the most common pathogens in lohc after allo-hsct and that anti-infection therapies were critical. for patients not showing a satisfactory response to anti-infection therapies, additional corticosteroid therapy may help to achieve cr. allogeneic hematopoietic stem cell transplantation (allo-hsct) is an effective treatment for hematological malignancies. although transplantation techniques have progressed significantly, late-onset hemorrhagic cystitis (lohc) is still one of the major complications, with an incidence rate varying from . to % [ , ] . it can cause mortality and lead to a low survival rate [ , ] . there are several therapeutic methods for lohc, including ensuring appropriate hydration, hematological homeostasis (maintaining high platelet counts, appropriate red cell counts, and levels of clotting factors), pain relief, catheterization for cystoscopic clot extraction, continuous bladder irrigation with normal saline for prevention of clots and bladder tamponade, anti-infection (particularly antiviral), hyperbaric oxygen, estrogen, clotting factors, and keratinocyte growth factor therapies [ ] . although lohc tends to be selflimiting after or weeks, some patients, particularly the patients who received alternative donor allo-hsct, can show refractory lohc which can persist for several months [ ] . refractory lohc may lead to poor quality of life, even mortality, after allo-hsct. given the assumption that alloimmune injury might also be involved in the pathogenesis of lohc [ ] , huang et al. [ ] used corticosteroids to treat patients with refractory lohc, and all of them achieved complete remission (cr). however, the small sample size of the patients (n = ) in this pilot study limited the ability to further identify the efficacy of corticosteroids in the treatment of lohc. thus, in the present retrospective study, we aimed to further evaluate the treatments, particularly the use of corticosteroids, in patients with lohc after allo-hsct. patients a total of consecutive patients underwent non-t-celldepleted allo-hsct at the peking university institute of hematology (puih) from january , , to december , : , , and had undergone human leukocyte antigen (hla)-unrelated donor (urd) hsct, hla-identical sibling donor (isd) hsct, and hla-haploidentical related donor (haplo-rd) hsct, respectively. one hundred and sixty-three patients who met the criterion of lohc after allo-hsct were enrolled in this study : , , and had undergone urd hsct, isd hsct, and haplo-rd hsct, respectively ( table ). the final follow-up visits for the endpoint analysis were conducted on july , . informed consent was obtained from all patients or their guardians, and the study was conducted in accordance with the declaration of helsinki. the study protocol was approved by the ethics committee of peking university people's hospital. preconditioning consisted of cytarabine, busulfan ( . mg kg − day − , administered intravenously on days − to − ; day being the first day of donor cell infusion), cyclophosphamide ( . g m − day − , days − to − ), and simustine ( mg m − , day − ). cytarabine was administered at g m − day − (days − to − ) to the haplo-rd group, at g m − day − (days − to − ) to the urd group, and at g m − day − (day − ) to the isd group. rabbit antithymocyte globulin (thymoglobulin, . mg kg − day − , days − to − ; sanofi, france) was administered to the haplo-rd and urd groups [ , ] . granulocyte colony-stimulating factor (g-csf)-mobilized, fresh, and unmanipulated bone marrow (bm) and peripheral blood harvests were infused into the recipients on the day of collection. in addition, the patients received cyclosporine a (csa), mycophenolate mofetil (mmf), and short-term methotrexate (mtx) as graft-versushost disease (gvhd) prophylaxis [ ] . donor selection, hla typing, and stem cell harvesting were performed as described previously [ , ] . minimal residual disease (mrd)-directed immunotherapy (e.g., donor lymphocyte infusion and interferon-α) was given before hematological relapse, as a preemptive intervention therapy, months post-hsct [ , ] . comorbidities in hsct recipients were assessed by the hematopoietic cell transplantation-specific comorbidity index (hct-ci) [ ] . all of the patients were hospitalized in rooms with highefficiency particulate-arresting (hepa) air filters for - weeks, from day − until the time at which neutrophil recovery was achieved. all of the patients received oral antibiotics (e.g., fluoroquinolone) for gastrointestinal decontamination before and during the period of neutropenia. the patients also received trimethoprim-sulfamethoxazole to prevent pneumocystis carinii infection from days − to + , fluconazole or itraconazole capsules for invasive fungal infection from days − to + , and acyclovir ( - mg) for herpes simplex virus and varicella-zoster virus, administered orally, twice daily, from day + until the time of csa discontinuation. ganciclovir ( mg kg − ) was administered intravenously, twice daily, from days − to − , for prophylaxis against cytomegalovirus (cmv) infection. the infection surveillance and treatment protocols used at our institute have been described in detail elsewhere [ ] . [ ] . for positive cmv and ebv samples, the pathogen load was determined by quantitative pcr. additionally, urine samples were subjected to gram, fungal, and acid-fast bacilli staining, and to bacterial and fungal cultures. these staining and cultures were also performed on blood samples from patients who presented with fever. blood and urine samples were tested repeatedly weekly. diagnosis of hemorrhagic cystitis (hc) required the presence of sustained hematuria along with dysuria and/or lower abdominal pain. diagnosis of lohc required that the symptoms of hc occurred beyond days post-transplantation. the severity of hc was graded according to the published criteria: grade , microscopic hematuria on more than two consecutive days; grade , macroscopic hematuria; grade , macroscopic hematuria with clots; grade , macroscopic hematuria with clots and impaired renal function secondary to urinary tract obstruction [ ] . severe lohc was defined as grade to lohc, and non-severe lohc was defined as grade to lohc. as prophylaxis for hc, all of the patients were given l m − day − of intravenous fluid, from h before to h after the administration of cyclophosphamide. sodium mercaptoethanesulfonate was given intravenously, at a dose of mg kg − , prior to cyclophosphamide administration and every h thereafter, over h, until the last dose of cyclophosphamide [ ] . with the diagnosis of lohc, along with the supportive therapies (ensuring appropriate hydration, alkalization, forced diuresis, hematological homeostasis, and pain relief), the patients without gvhd were subjected to tapering of immunosuppressant. the anti-infection therapies included antiviral and antimicrobial therapies. the patients showing cmvnegative blood and urine samples could receive empirical antiviral therapies with mg kg − day − of intravenous ganciclovir or - mg kg − day − of intravenous foscarnet sodium [ , ] . however, the patients showing cmv-positive blood and/or urine samples should receive anti-cmv therapies, i.e., mg kg − day − of intravenous ganciclovir or mg kg − day − of intravenous foscarnet sodium. the patients with refractory cmv infection, that is, cmv dnaemia lasting for > weeks despite the administration of a full dose of antiviral drug therapy, received the combination of foscarnet and ganciclovir. antiviral therapies were given for - days or until week after the virus tests became negative. the patients could also receive empirical antimicrobial therapies, such as carbapenem, cephalosporins containing the β lactamase inhibitor, fourth-or third-generation cephalosporin, or quinolone, for at least days. for the patients who did not achieve cr after anti-infection therapy, an additional corticosteroid therapy was applied. the dose was prednisone mg kg − day − , if the patient's weight was < kg, or prednisone mg day − for patients whose weight was > kg. prednisone could be changed by equivalent doses of dexamethasone or methylprednisolone. the patients' response was evaluated - days later. for the patients responding to corticosteroids, the dose of corticosteroids was reduced, weekly, to two thirds of the dose of the previous week [ ] . however, for the patients having other transplant-related complications that also needed corticosteroid therapy during lohc (e.g., gvhd), the initial time, dosage, and duration of corticosteroid therapy were applied according to these transplanted complications instead of lohc. how to manage patients with concurrent active gvhd, cmv infection, and lohc was critical in the treatment of refractory lohc in the present study. for the patients having concurrent cmv infection and refractory lohc, we suggested that systemic corticosteroid therapy could only be considered when the virus was cleared but the loch was not cr. however, for the patients having concurrent grade ii to iv acute gvhd and refractory lohc, systemic corticosteroid therapy should be added immediately with the use of empirical antiviral therapies or anti-cmv therapy (fig. ) . the criteria of therapeutic efficiency for lohc were as follows: ( ) cr, hc symptoms were relieved and microscopic hematuria disappeared, and there was no recurrence in the week after achieving cr; ( ) partial remission (pr), hc symptoms had significantly improved (such as > % reduction in the frequency of urination or relief from dysuria and burning) and/or gross hematuria was downgraded at least one grade, but cr was not achieved; and ( ) non-remission (nr), hc symptoms and hematuria did not improve or showed significant deterioration (e.g., gross hematuria upgraded at least one grade). the patients were classified as bhigh risk^if they ( ) had acute leukemia in its third complete remission (cr ) or greater; ( ) had acute leukemia in pr, in nr, or in a state of relapse before hsct; and ( ) had chronic myeloid leukemia after the first chronic phase. all the other patients were stratified into standard-risk categories. neutrophil engraftment was defined as the first day when the absolute neutrophil count was ≥ . × l − for three consecutive days, and platelet engraftment was defined as the first day when the platelet count was ≥ × l − for seven consecutive days without transfusion. gvhd was diagnosed in accordance with the accepted international criteria [ , ] . relapse was defined as morphologic evidence of disease in samples from the peripheral blood, bone marrow, or extramedullary sites, or by the recurrence and sustained presence of pre-transplantation chromosomal abnormalities. patients exhibiting mrd were not classified as showing relapse. non-relapse mortality (nrm) was defined as death by any cause in the first days post-hsct or death without evidence of disease recurrence beyond day . overall survival (os) events were defined as death from any cause. disease-free survival (dfs) was defined as the survival period with continuous cr. data were censored at the time of death, relapse, or the last available follow-up. continuous variables were compared using the mann-whitney u test; categorical variables were compared using the χ test and fisher's exact test. the kaplan-meier method was used to estimate the probability of survival. competing risk analyses were used to calculate the cumulative incidence of lohc, using gray's test to evaluate differences between the groups [ ] . nrm and relapse were the competing events. the level of significance was set at p < . . all reported p values were based on two-sided tests. data analyses were primarily conducted with spss software (spss inc., chicago, il, usa), while the r software package (version . . ; http://www.r-project.org) was used for competing risk analysis. table summarizes the characteristics of the patients with lohc in this study. fifteen were children ( . %) and the other were adults ( . %). all the patients achieved neutrophil engraftment within days after hsct, with a median time to neutrophil engraftment of (range, - ) days. during the follow-up period, patients exhibited platelet engraftment, with a median time to platelet engraftment of (range, - ) days. six patients showed relapse and died of nrm after hsct. the -year probability of dfs and os after hsct was . and . %, respectively. one hundred fig. the management of concurrent active gvhd, cmv infection, and lohc. for the patients with refractory lohc only, the dose of systemic corticosteroid therapy was prednisone mg kg − day − , for patients whose weight was < kg, or prednisone mg day − for patients whose weight was > kg. for the patients with grade ii to iv acute gvhd, the dose of systemic corticosteroid therapy was methylprednisolone to mg kg − day − and four patients ( . %) showed acute gvhd, and patients ( . %) showed grade iii to iv acute gvhd. the median time from transplantation to the occurrence of lohc was ( - ) days. all the patients exhibited frequent and urgent urination and odynuria. thirty-four patients exhibited a fever, and the median temperature at lohc diagnosis was . ( . - . )°c. the median count of white blood cells, hemoglobin, and platelets on lohc diagnosis was . (range, . - . ) × cells l − , (range, - ) g l − , and (range, - ) × cells l − , respectively. sixteen ( . %), ( . %), and ( . %) patients showed grade , , and lohc, respectively, at diagnosis. ninety-six patients showed progression from the initial grade to a more advanced grade, and ( . %), ( . %), ( . %), and ( . %) patients showed grade , , , and lohc, respectively, at peak. the cumulative incidence of lohc at days after hsct was . % for the total population and . , . , and . % for isd hsct, haplo-hsct, and urd hsct recipients (p < . ; fig. a ). the cumulative incidence of grade to lohc at days after hsct was . % for the total population and . , . , and . % for isd hsct, haplo-hsct, and urd hsct recipients, respectively (p = . ; fig. b ). pathogens were identified in blood and/or urine samples from patients: had positive blood samples, had positive urine samples, and had simultaneous positive blood and urine samples. forty-three and patients had infections caused by single and multiple viruses (≥ types of viruses), respectively. in blood samples, cmv was the most common virus ( . %), followed by bk virus ( . %), ebv ( . %), hsv ( . %), jc virus ( . %), hhv- ( . %), adv ( . %), and piv ( . %). in urine samples, the most common virus was bk virus ( . %), followed by jc virus ( . %), cmv ( . %), and adv ( . %). the median plasma and urine viral loads of cmv were . × /copies ( . × /copies- . × /copies) and . × /copies ( . × /copies- . × /copies), respectively, and it was identified simultaneously in plasma and urine samples from patients. the median plasma loads of ebv were . × /copies ( . × /copies- . × /copies). bk virus was identified simultaneously in plasma and urine samples from patients, and jc virus was identified simultaneously in plasma and urine samples from patients. the most common bacterium identified in urine samples was escherichia coli (n = ; . %), followed by enterococcus faecalis (n = ; . %), proteus mirabilis (n = ; . %), and acinetobacter junii (n = ; . %). all patients with lohc received supportive therapies, and achieved cr after these treatments (grade : n = ; grade : n = ; no pathogens were detected in blood and urine samples). the remaining patients received anti-infection therapies. all of them received antiviral therapy ( . % of patients received anti-cmv therapies, and the other . % of patients received empirical antiviral therapies). a total of patients received antibacterial therapies. seventy-one out of patients ( . %) achieved cr after anti-infection therapies, and the period from the beginning of anti-infection therapies to cr was (range, - ) days. the remaining patients ( . %) did not achieve cr (nr: n = ; pr: n = ). the rate of cr after anti-infection therapies was fig. cumulative incidence of late-onset hemorrhagic cystitis. the cumulative incidence of total (a) and grade to (b) late-onset hemorrhagic cystitis according to different donors significantly lower in the severe lohc group compared to that of the non-severe loch group ( . vs. . %, p < . ). for the patients that did not achieve cr after anti-infection therapies, of them were not subjected to corticosteroid therapy (severe lohc: n = ; non-severe lohc: n = ), and none of them achieved cr until the last available follow-up. the other patients received the additional corticosteroid therapy, and . % (n = ) of them showed a severe lohc at the beginning of corticosteroid therapy. nine patients showed virus positivity when corticosteroids were administered (cmv: n = ; ebv: n = ; bk virus: n = ). thirty-three of the patients received corticosteroid therapy because of other transplant-related complications (group a), including gvhd (n = ), engraftment syndrome (n = ), poor graft function (n = ), and diffuse alveolar hemorrhage (n = ). the other patients that received corticosteroid therapy did not have additional transplant-related complications (group b). the median period from the beginning of anti-infection therapies to the beginning of corticosteroid therapy was (range, - ) days, which was significantly shorter in group a than that in group b ( vs. days, p < . ). sixty-nine out of patients ( . %) responded to corticosteroid therapy (cr: n = ; downgraded at least one grade, although not achieving cr: n = ). thirty-five out of ( . %) patients showed an immediate response within week after the beginning of the therapy; particularly, achieved cr within week. for the patients that downgraded at least one grade after therapy, finally achieved cr. thus, a total of patients ( . %) achieved cr after corticosteroid therapy. the median period from the beginning of corticosteroid therapy to cr was (range, - ) days, which was comparable between groups a and b ( . vs. days, p = . ). in addition, among the other five patients who downgraded at least one grade after corticosteroid therapy, all the symptoms of lohc were relieved persistently; nevertheless, continuous microscopic hematuria was observed until the last available follow-up. among the patients that did not achieve cr after antiinfection therapies, the cumulative incidence of cr for lohc was significantly higher in the corticosteroid-treated group than that in the corticosteroid non-treated group ( . vs. . %, p = . ; fig. ). for the patients that did not show any response to additional corticosteroid therapy, relapsed and did not receive further therapies for lohc, and died of nrm (infection: n = ; gvhd: n = ; thrombotic microangiopathy: n = ). for the other patients, were subjected to electrocoagulation hemostasis with cystoscope, and all of them achieved cr after cystoscopy. for the remaining patients, corticosteroid therapy was discontinued, and the patients were subjected to intravenous fluid hydration, alkalization, forced diuresis, continuous bladder irrigation with normal saline, and antiinfection therapies. cr was achieved at the last available follow-up. the treatments of lohc are summarized in fig. . although transplantation techniques have progressed significantly, lohc after allo-hsct continues to seriously influence patients' quality of life. in the present study, we observed that viruses were the most common pathogens in lohc after allo-hsct. for patients not showing a satisfactory response to supportive and anti-infection therapies, additional corticosteroid therapy may help to achieve cr. the present study was the largest study identifying the efficacy of corticosteroid therapy in the treatment of lohc after allo-hsct. in the present study, cmv was the most common virus in patients with lohc. cmv-associated lohc has been described, as case reports, in some centers [ , ] . xu et al. [ ] showed that cmv viremia is a risk factor for lohc (rr = . ; % ci, . - . ; p = . ). han et al. [ ] showed that the cumulative incidence of lohc at day in patients with and without cmv viremia (prior to or at the onset of lohc) was . and . % (p = . ), respectively, and that cmv viremia (hr = . ; % ci, . - . ; p = . ) was an independent risk factor for the development of lohc. thus, decreasing the occurrence of cmv viremia is important to decrease the risk of lohc after allo-hsct. however, we observed that cmv was identified in urine in only patients, and cmv loads were lower in urine than in plasma. thus, the role of cmv in lohc pathogenesis should be further elucidated. bk virus was another important virus for lohc, which was the second most common virus in blood samples and the most common virus in urine samples in the present study. several studies showed that lohc is strongly associated with the presence of bk virus, although the role of bk virus in lohc pathogenesis has not yet been fully elucidated [ , ] . however, we did not detect copies of bk virus, and we could not further identify the association between the burden of bk virus and lohc. we observed that pathogens were identified in most patients ( / ) and viruses were the most common pathogens. in addition, many patients ( / ) showed infection with multiple viruses. previous studies also reported that virus infection was the most important pathogenesis for lohc [ , , , , ] . thus, we hypothesized that anti-infection therapies should be the basis for the treatment of lohc after allo-hsct. in the present study, more than % of the patients with grade to lohc achieved cr after anti-infection therapies. although infection may be the most important cause of lohc in allo-hsct recipients [ ] , some patients showed unsatisfactory response to anti-infection treatments, particularly for those with severe lohc. in the present study, only . % of patients with grade to lohc could achieve cr after anti-infection therapies alone. thus, other pathogeneses, such as immune injury, may also contribute to the occurrence of lohc after allo-hsct. ost et al. [ ] suggested that post-engraftment lohc represent uroepithelial gvhd. several studies also reported the association between acute gvhd and lohc after allo-hsct [ , ] . in the present study, we observed that more than % of the patients showed acute gvhd. thus, the association of lohc with gvhd regarding timing, incidence, and severity suggests that its pathogenesis may involve a local inflammatory environment, cellular immune responses, effector mechanisms, and hla as well as non-hla genetics [ ] . in addition, several infectious agents can trigger autoimmunity via different mechanisms [ ] , and several studies have observed that infections, particularly viral infections, can trigger a graft-versus-host reaction [ ] [ ] [ ] . in the model of leung et al. [ ] for lohc, infected uroepithelial cells are attacked by donor lymphoid cells, leading to tissue destruction. thus, the role of additional corticosteroid therapy after anti-infection and supportive therapies is worth considering in the treatment of patients with lohc, particularly those showing unsatisfactory responses to anti-infection therapies. huang et al. [ ] reported that patients with refractory lohc received a low dose of corticosteroids, and all of them achieved cr. in the present study, although . % of the patients who showed an unsatisfactory response to supportive and anti-infection therapies and received additional corticosteroid therapies had severe lohc, more than half of them showed an immediate response to corticosteroid and . % finally achieved cr. thus, the additional corticosteroid therapies may be able to significantly shorten and change the course of lohc and it may be a potential therapy for lohc patients showing unsatisfactory response to antiinfection therapies. the present study had several limitations. first, this was a retrospective study, which might have influenced the accuracy fig. treatments for patients with late-onset hemorrhagic cystitis. cr, complete remission; nr, non-remission; nrm, nonrelapse mortality; pr, partial remission of our findings. second, we might have underestimated the occurrence of virus-associated lohc in this study, because the number of viruses that could be tested was relatively small. third, the several, varied treatments for lohc introduced heterogeneity into our study results and reduced the reliability of our conclusion about the advantage of corticosteroid therapy for clinical outcomes in patients with lohc. future prospective and multicenter studies will provide more information about pathogens and treatments of lohc after allo-hsct. in summary, we observed that viruses were the most common pathogens in lohc allo-hsct and that anti-infection therapies were critical for these patients. for patients showing unsatisfactory response to anti-infection therapies, additional corticosteroid therapy may help to achieve cr, particularly for those with severe lohc. hemorrhagic cystitis following high-dose chemotherapy and bone marrow transplantation in children with malignancies: incidence, clinical course, and outcome haemorrhagic cystitis in paediatric bone marrow transplant patients: an association with infective agents, gvhd and prior cyclophosphamide mesna compared with continuous bladder irrigation as uroprotection during highdosechemotherapy and transplantation: a randomized trial a prospective study of bk-virus-associated haemorrhagic cystitis in paediatric patients undergoing allogeneic haematopoietic stem cell transplantation prevention and management of bk-virus associated haemorrhagic cystitis in children following haematopoietic stem cell transplantation-a systematic review and evidence-based guidance for clinical management comparison of clinical features of hemorrhagic cystitis after haploidentical and matched sibling donor allogeneic hematopoietic stem cell transplantation ciprofloxacin decreased polyoma bk virus load in patients who underwent allogeneic hematopoietic stem cell transplantation immunerelated late-onset hemorrhagic cystitis post allogeneic hematopoietic stem cell transplantation partially matched related donor transplantation can achieve outcomes comparable with unrelated donor transplantation for patients with hematologic malignancies haploidentical vs identical-sibling transplant for aml in remission: a multicenter, prospective study haploidentical hematopoietic stem cell transplantation without in vitro t cell depletion for the treatment of hematological malignancies treatment of acute leukemia with unmanipulated hla-mismatched/haploidentical blood and bone marrow transplantation how do we choose the best donor for t-cell-replete, hla-haploidentical transplantation? 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polyoma bk virus and haemorrhagic cystitis in haematopoietic stem cell transplantation: a changing paradigm the risk of polyomavirus bk-associated hemorrhagic cystitis after allogeneic hematopoietic sct is associated with myeloablative conditioning, cmv viremia and severe acute gvhd incidence, clinical outcome, and management of virus-induced hemorrhagic cystitis in children and adolescents after allogeneic hematopoietic cell transplantation hemorrhagic cystitis-a manifestation of graft versus host disease? infection and autoimmune disease viral infection as a trigger in flares of acute graft-versus-host disease chronic graft-versus-host disease following varicella-zoster virus infection in allogeneic stem cell transplant recipients the complex relationship between human herpesvirus and acute graft-versus-host disease acknowledgments the authors thank editage for their assistance in editing this manuscript and dr. yu-jia chi, wei guo, and li-jia ma for their assistance in collecting the data. informed consent was obtained from all patients or their guardians, and the study was conducted in accordance with the declaration of helsinki. the study protocol was approved by the ethics committee of peking university people's hospital. the authors declare that they have no conflict of interest. key: cord- -tvguutak authors: praznik, ajda; vinšek, neža; prodan, ana; erčulj, vanja; pokorn, marko; mrvič, tatjana; paro, darja; krivec, uroš; strle, franc; petrovec, miroslav; Žnidaršič eržen, marta; grosek, Štefan title: risk factors for bronchiolitis severity: a retrospective review of patients admitted to the university hospital from central region of slovenia date: - - journal: influenza other respir viruses doi: . /irv. sha: doc_id: cord_uid: tvguutak aim: study's objective was to identify risk factors associated with bronchiolitis severity. methods: a retrospective chart review of all children < years old diagnosed with bronchiolitis at the university medical centre ljubljana between may and april , who were treated as outpatients (paediatric emergency department, ped group) or as inpatients in the standard hospital setting (ward group) or in the paediatric intensive care unit (picu group). detection of respiratory viruses in nasopharyngeal swab was accomplished by rt‐pcr. severity was assessed by wang respiratory score and hospitalization longer than hours. results: the study included children. the three most frequently detected viruses were respiratory syncytial virus (rsv), human rhinovirus (hrv) and human bocavirus (hbov) ( . %, / ; . %, / ; . %, / ). patient groups differed in wang respiratory score for the severity of bronchiolitis (p < . ). no differences regarding the causative viruses were found. there was a lower proportion of children with the presence of more than one virus in picu group compared to other two groups (p = . ). the three groups significantly differed in age, birthweight, comorbidities, bronchodilator treatment and antibiotic usage. however, multiple regression analysis revealed that younger age and the use of antibiotics were associated with bronchiolitis severity defined as hospitalization for > hours. conclusions: respiratory syncytial virus, hrv and hbov were the most frequently detected viruses. the majority of patients admitted to the picu had only one virus detected. younger age and the use of antibiotics were associated with bronchiolitis severity. bronchiolitis is a potentially life-threatening viral respiratory infection that affects children under years of age. it is common and occurs worldwide. the bronchiolitis mortality rate is approximately per infants and is higher in developing than in developed countries. the most commonly identified causative agent is respiratory syncytial virus (rsv). other viruses implicated in the aetiology of bronchiolitis include human metapneumovirus (hmpv), parainfluenza virus (piv), influenza virus a and b virus (infv), human rhinovirus (hrv), human coronavirus (hcov), adenovirus (adv), enterovirus (ev) and human bocavirus (hbov). the majority of children with bronchiolitis can be managed as outpatients; approximately %- % of patients younger than year are hospitalized, usually during the seasonal rsv epidemics that occur in cold months. treatment of bronchiolitis is symptomatic and focuses on the maintenance of hydration, oxygenation and antipyresis. in severe cases, intensive care management may be needed with continuous positive airway pressure (cpap), intubation and mechanical ventilation. some infants with secondary bacterial infection need antibiotic treatment. [ ] [ ] [ ] in infants with pre-existing medical conditions and immunodeficiency, complications such as acute respiratory distress syndrome (ards), bronchiolitis obliterans, congestive heart failure and secondary bacterial infection may develop. [ ] [ ] [ ] [ ] [ ] although there is no universally accepted measure for the assessment of bronchiolitis severity, the wang respiratory score, using clinical observation (general appearance, respiratory rate, presence of wheezing and retractions), is widely used. children with bronchiolitis and wang respiratory score or less can be managed on an outpatient basis, while children with scores - usually require hospitalization and those with even higher scores are admitted to the picu. the objective of our study was to ascertain demographic characteristics, clinical findings and presumptive aetiologic agents (respiratory viruses demonstrated in nasopharyngeal swab) associated with bronchiolitis severity defined as length of hospitalization for > hours. the study was approved by the national medical ethics committee of the republic of slovenia (kme / / ) (nmec rs). the study was conducted at the university medical centre ljubljana (umcl), a university hospital serving the central region of slovenia with a population of around inhabitants (approximately one-third of all slovenian population). we performed a retrospective chart review of all patients with bronchiolitis < years of age, referred to the three paediatric departments, that is umcl children's hospital, department of infectious diseases and paediatric intensive care unit (picu) between may and april . we excluded patients that had been previously diagnosed with asthma, and all previous episodes if a patient had more than one episode of bronchiolitis in studied time span. patients were identified through a query of the electronic medical record based on a bronchiolitis diagnosis (icd- code j . - ). electronic medical records of patients included in the study were reviewed, and statistical analysis was performed for the following clinical and laboratory data: gender, chronological age at admission, prematurity (defined as birth before weeks of gestation), birthweight, history of allergies, number of previous bronchiolitis episodes, clinical manifestations of bronchiolitis using the wang respiratory score, comorbidities (chronic lung disease, congenital heart disease, immune deficiency or neuromuscular diseases), body temperature at admission, treatment with bronchodilators, antibiotics or supplemental oxygen and respiratory virus detected in the nasopharyngeal swab. only the first swab taken from individual patient was included. swabs were routinely taken from patients in two departments, while in another one sampling was sparse. high season and low season of incidence were also included as variables. using rrt-pcr, the sensitivity of the nasopharyngeal swab is above % compared to a composite gold standard. , for patients, no swab analysis was performed. bronchiolitis severity was assessed by wang respiratory score and as a duration of hospitalization for > hours. numerical data were presented as median (range) and categorical as frequencies (percentages). the differences in categorical variables among the three groups were tested using chi-square test or likelihood ratio test; kruskal-wallis test was performed for numerical variables. the clinical and laboratory findings were compared between the three groups of patients, defined according to the site of management: patients treated as outpatients (paediatric emergency department, ped group), patients treated in the standard hospital setting (ward group) and children admitted to the paediatric intensive care unit (picu group). multiple logistic regression analysis was used to identify key variables associated with severe bronchiolitis, defined with the duration of hospitalization for > hours. a p-value < . was considered statistically significant. all analyses were performed using spss . . the study group comprised children with bronchiolitis. there was a male predominance with ( . %) boys. of children, were treated as outpatients (ped group), were hospitalized at a regular paediatric unit (ward group), and were treated in a paediatric intensive care unit (picu group). nasopharyngeal swabs were taken in / ( %) children. of patients admitted to hospital, ( %) were hospitalized for > hours. the median length of hospital stay was days in picu group and days in ward group (p < . ). the three study groups differed in wang's score for particular clinical sign parameter (p < . ) apart from wheezing, and in the need for supplemental oxygen treatment (p < . )-only a few patients required oxygen treatment in the outpatient ped group, while almost all patients needed oxygen treatment in picu group. the three study groups differed in wang score (p < . ) ( figure ). they also differed in several clinical and microbiological characteristics ( table ) analysis of the presence of more than one virus in nasopharyngeal swab revealed that rsv and hbov were the two most frequently simultaneously detected viruses and that the proportion of patients with more than one of detected viruses in individual swab was statistically significantly lower in picu than in ped or ward (p = . ) ( table ) . multiple logistic regression analysis showed that out of several factors only younger chronological age (p < . ) and treatment with antibiotics (p = . ) were associated with severe bronchiolitis defined as hospitalization > hours. (table ). in the present study, we compared characteristics of children with bronchiolitis treated in the outpatient clinic, admitted to the ward or admitted to the paediatric intensive care unit. age, prematurity, comorbidity and treatment with bronchodilators and antibiotics, differed in the three groups suggesting these factors relate to disease severity. the type of virus did not correlate with treatment group (table ). further analyses revealed that chronological younger age and treatment with antibiotics independently were associated with hospitalization longer than hours (table ) . chronological age is known as the most important predictor of severe bronchiolitis. similar to study, several others have found a significant association between the age of less than months and a higher risk of hospitalization and severe bronchiolitis. [ ] [ ] [ ] [ ] however, grimwood et al did not find a significant correlation between children aged less than months and bronchiolitis severity in a multivariate analysis. hospitalization rates that are attributable to rsv bronchiolitis are usually the highest between and days after birth. this age coincides with the declining concentration of transplacentally acquired maternal anti-rsv immunoglobulin, which protects infants against disease. , certain studies have shown that prematurity is independently associated with more severe bronchiolitis, , , while the others have not found significantly higher rates of hospitalization and severe bronchiolitis among premature compared to full-term infants. , , in the present study, the proportion of preterm children was highest in the picu group and lowest in the ped group, which suggested a more severe bronchiolitis. however, multiple logistic regression model did not show a correlation between prematurity and in the present study, antibiotic treatment was significantly associated with severe bronchiolitis, which is in accordance with some previous reports. , although the design of our study did not enable analysis of the appropriateness of antibiotic therapy, the finding that almost all patients in the picu group received antibiotic treatment compared to only . % in the ped group might suggest that bacterial superinfections lead to a more severe course of bronchiolitis. another frequently mentioned risk factor for severe bronchiolitis is comorbidity. , [ ] [ ] [ ] in our study, one-third of children hospitalized in picu had at least one comorbidity, while in the ped group the corresponding proportion was significantly lower. however, again, in the multiple logistic regression analysis comorbidities did not correlate with the severe course of bronchiolitis (table ) as shown also in some other studies. , one should take into account that our study did not analyse the association of comorbidities with specific virus types as done in mentioned studies. , , , another limitation is that we did not analyse specific comorbidities, but only as a group determined by previous studies. , , this is because there were only children with comorbidity and we believe that dividing the comorbidity group into smaller groups would not give reliable information. the present study showed that rsv, hrv and hbov were the most frequently present viruses in nasopharyngeal swab of children with bronchiolitis. the predominance of rsv is in agreement with several previous reports. , , the second most common pathogen is usually hrv, followed by infv or piv , , while in our study hbov ranked the third. some reports indicated that hbov is rarely detected as a single agent in hospitalized children with bronchiolitis, leading to speculation that this virus is more likely to be an innocent bystander than a true pathogen. however, uršič et al have recently reported the first fatal case of an extremely severe bronchiolitis caused by hbov in an immunocompetent child. the absence of association between virus type and characteristics of patients is in agreement with the findings of several previous articles. , , nevertheless, in some reports, differences in the severity of the disease caused by various types of viruses or viral co-infections were established. , , according to our study, multiple viruses detected in the nasopharyngeal swab were moderately associated with a more severe course of the bronchiolitis, although not significant (p = . ). other studies proved that children with viral coinfection were less likely to be admitted to intensive care unit than children with single virus infection. , possible explanation is that as one or more viral respiratory pathogens can be detected in the upper respiratory tract of as many as % of asymptomatic young children, , it is likely that in several patients with bronchiolitis and more than one virus detected in nasopharyngeal swab, one of the viruses is responsible for the acute infection while the others are innocent bystanders, possibly as a result of prolonged shedding after an already resolved infection. it is not unusual that a child has a history of more than one episode of bronchiolitis. studies suggest that repeated infections by rsv can be in part due to variability of the virus strains. acute bronchiolitis can cause (transitory) anatomical and histological changes in lower respiratory tract that may prone to a more severe new episode. however, in accordance with some previous reports in our study, the number of previous episodes of bronchiolitis did not correlate with bronchiolitis severity. our study also showed that the proportion of children who received bronchodilators was statistically different in the ped, ward and picu group (table ) . this is probably due to the common practice in several countries (such as united states, switzerland and belgium) that children exhibiting a moderately severe bronchiolitis are more often given bronchodilators than those with mild or severe disease. [ ] [ ] [ ] the data on the efficiency of bronchodilators in bronchiolitis are conflicting. many studies agree that bronchodilators may improve clinical symptom scores, but they do not affect disease resolution, need for hospitalization, or length of stay. interpretation. in addition, children with performed viral diagnosis were younger and were treated in the high seasonal months. comparison of the three groups regarding virus repartition should be taken with precaution as the sample of children treated in the standard hospital setting with viral data available seems to be biased. data are not missing at random, but are available to higher extent for younger children and for children treated in the high season months. in conclusion, our study revealed that rsv, hrv and hbov were the most frequently detected viruses in children with bronchiolitis. chronological younger age and the use of antibiotics were associated with severe bronchiolitis defined as hospitalization longer than day. further prospective studies are needed to assess the importance of various respiratory viruses and host factors in this common paediatric illness. we thank the institute of microbiology and immunology and the national institute of public health who provided us with data necessary for statistical analysis. the authors have no competing interests. ana prodan http://orcid.org/ - - - the impact of dual viral infection in infants admitted to a pediatric intensive care unit associated with severe bronchiolitis risk factors for bronchiolitis-associated deaths among infants in the united states impact of human rhinovirus types and viral load on the severity of illness in hospitalized children with lower respiratory tract infections ljubljana: združenje za infektologijo, slovensko zdravniško društvo ljubljana viral bronchiolitis in children diagnosis and management of bronchiolitis clinical findings and severity of acute bronchiolitis bronchodilators for treatment of mild bronchiolitis: a 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title: phosphatidylserine’s role in ebola’s inflammatory cytokine storm and hemorrhagic consumptive coagulopathy and the therapeutic potential of annexin v date: - - journal: med hypotheses doi: . /j.mehy. . sha: doc_id: cord_uid: egw bvzf the phosphatidylserine (ps) molecule is present in cell membranes where it is actively kept on their inner leaflets but when cells are damaged it moves to the surface and become a signal for their removal, the platform upon which the coagulation cascade takes place and a ligand that activates a feedback cycle of inflammatory cytokine secretion and initiates the wakeup call for the innate immune response. these are physiologic responses to ps but the ebola virus displays ps molecules on its membrane’s surface and the huge numbers of viruses cause a pathologic inflammatory cytokine storm and a hemorrhagic consumptive coagulopathy. annexin v is an innate molecule that can cloak membrane displayed ps and prevents its th cell’s inflammatory cytokine generation and cascade thrombin generation. the hypothesis presented is that its administration will cloak ps and prevent ebola’s consumptive coagulopathy and its cytokine storm. the phosphatidylserine (ps) molecule is present in cell membranes where it is actively kept on their inner leaflets but when cells are damaged it moves to the surface and become a signal for their removal, the platform upon which the coagulation cascade takes place and a ligand that activates a feedback cycle of inflammatory cytokine secretion and initiates the wakeup call for the innate immune response. these are physiologic responses to ps but the ebola virus displays ps molecules on its membrane's surface and the huge numbers of viruses cause a pathologic inflammatory cytokine storm and a hemorrhagic consumptive coagulopathy. annexin v is an innate molecule that can cloak membrane displayed ps and prevents its th cell's inflammatory cytokine generation and cascade thrombin generation. the hypothesis presented is that its administration will cloak ps and prevent ebola's consumptive coagulopathy and its cytokine storm. phosphatidylserine (ps) molecules are present in cell membranes where they are actively kept on their inner leaflets. in senescent and otherwise damaged cells ps moves to their surface where it becomes a signal for their phagocytic removal [ ] , the platform upon which the coagulation cascade takes place [ ] , a ligand for the tim- receptor on t helper one (th ) cells that activates its inflammatory cytokine secretion [ ] and a ligand that binds to tim receptors on mononuclear immune cells to provide the wakeup call for the innate immune response [ ] . billions of cells become apoptotic daily and display ps on their surface but they are rapidly removed by phagocytes that recognize it [ ] and the th inflammation [ ] , cascade generated blood coagulation [ ] and the innate immune activation do not take place [ ] . in ebola ps on the surface of its virus activates th inflammatory cytokine secretion and the ps exposure is so great that a cytokine storm is produced [ ] and all the thrombin generated on the cascade's ps platforms generates enough thrombin to consume coagulation components and produce a hemorrhagic consumptive coagulopathy [ ] . macrophages and dendritic cells are phagocytes that secrete inflammatory cytokines when tlrs on their surface recognize foreign or damage associated molecular patterns but they also display tim- receptors and in ebola those receptors bind to and phagocytize the ps+ viruses and they become infected [ ] . when infected these cells are prime viral replicators that generate viruses to infect other cells. in addition, the infected dendritic cells may no longer be able to mhc present antigenic pathogen peptides to immune cells in the adaptive immune response producing immunologic depression. in it was discovered that ps on the ebola virus could bind to the tim- receptor on th cells and independently produce a lethal cytokine storm in mice [ ] . this was proven when an ebola infection in knockout tim- −/− negative did not produce a cytokine storm and the mice survived even though the viral load was only minimally affected [ ] . the hypothesis presented here is that annexin v's therapeutic administration in ebola can prevent its th cell generated inflammatory cytokine storm, stop the cascade generated hemorrhagic consumptive coagulopathy and prevent macrophage and dendritic cell infection. unfortunately when annexin v cloaks ps the cascade can't function and hemostasis will not be possible so let us very briefly examine the components of inflammation, blood coagulation and annexin v with regard to the possibility that its administration can prevent ebola's cytokine storm and coagulopathy without causing bleeding. t continued in a late mediated inflammatory response when some of the cells damaged by those cytokines become necrotic causing high mobility group box one (hmgb ) molecules release by nuclear disruption. the hmgb molecules then bind to the tlr receptors on dendritic cells and cause their inflammatory cytokine secretion [ ] . the ps exposed on the surface of cells damaged by the innate and late mediated inflammatory responses binds to the tim- receptor on th cells and they begin a feedback cycle of inflammatory cytokine secretion that expose more ps and amplifies the inflammation [ ] . this amplified inflammation damages and kills both pathogens and healthy cells and the ps displayed on the latter activate the innate immune response when an infection is present and make hemostasis possible when a vascular wall is breached. this physiologic ps feedback generation contrast with the pathologic cytokine storm and hemorrhagic consumptive coagulopathy in ebola where membrane displayed ps is on every virus. for blood coagulation to begin thrombin must be present and its generation begins when cells are damaged and the procoagulant molecules tissue factor (tf) and ps are exposed. in ebola the inflammatory storm damages many cells and the large numbers of tf and ps molecules exposed cause intravascular blood coagulation that consumes coagulation components and leads to a hemorrhagic consumptive coagulopathy. tf initiates blood coagulation but ps is the platform upon which the coagulation cascade's thrombin generation produces the consumptive coagulopathy. in ebola ps is not only exposed by inflammatory cell damage it is also on every virus. tf begins blood coagulation by activating factors x and ix. activated factor xa changes prothrombin to thrombin and factor ixa is an essential component of the coagulation cascade. tf generated thrombin directly or indirectly activates all coagulation components including the coagulation cascade. once activated the cascade generates thrombin in a feedback cycle of factor x activation in its tenase complex and by a catalytic generation of thrombin in its prothrombinase complex [ ] . in the tenase complex activated factor viiia and ixa bind to membrane displayed ps and are joined there by factor x to be activated [ ] . the activated factor xa then joins factor va on ps in the prothrombinase complex and the factor xa, factor va and ps amalgam catalytically begins changing prothrombin to thrombin and exponentially increases its generation making hemostasis possible [ ] . in hemophilia factors viii and/or ix are not available and so the cascade can't function and hemostasis is not possible even though tf thrombin generation is not affected [ ] . factor ixa is required for cascade thrombin generation but tf can't supply enough of it for cascade function so cascade generated thrombin activates factor xi and it activates factor ix. this increases thrombin generation but not exponentially. this is a good thing because though fibrin generation is desirable during cellulitis intravascular blood coagulation is not. for the cascade's exponential thrombin generation to take place factor xii must activate factor xi so it can activate factor ix and to do this factor xii must be activated by binding to sulfatide on activated platelets [ ] . this is not possible intravascularly because activated platelets secrete a factor xii activation inhibitor [ ] . it is possible when a vascular breach is present and collagen is exposed because platelets displaying sulfatide and ps on their surface bind to the collagen and the blood flow washes away the inhibitor. in a vascular breach tf and ps are only exposed at the breach site and factor ix is activated there by factor xiia making the cascade's exponential increase in thrombin generation possible. the same thing happens when an atherosclerotic plaque ruptures and collagen is exposed. the physiologic feedback cycles of ps exposure that cause inflammation and thrombin generation are needed to assure a rapid innate immune response to an infection and to rapidly control bleeding when a vascular wall is breached but such cycles, like atomic chain reactions, are dangerous and require controls and it is proposed that annexin v, like factor ixa is one of them. the following study by park supports the hypothesis that annexin v can block cytokine storms and consumptive coagulopathies [ ] . in this study gram negative septicemia was induced in mice where the lipopolysaccharide (lps) molecule on those bacteria generated a lethal cytokine storm and generated excess fibrin (not a coagulopathy). lps generates thrombin by binding to tlr on dendritic cells. it was found that a single injection of annexin v stopped the cytokine storm, reduced fibrin generation and enabled the mice survival [ ] . the annexin v did this by cloaking ps on damaged cells and by blocking the binding of lps to tlr . let us now take a brief look at annexin v. annexin v is an innate molecule found in cells and in nano molecular amounts in plasma where its levels increase during inflammation and blood coagulation [ ] . what it does in cells and how it gets from there to plasma is uncertain [ ] . it is a small protein molecule that is rapidly removed by kidneys causing it to have a half-life of minutes in plasma [ ] . it has a great affinity for ps and because of this its labeled molecules are used in the lab and clinically for its identification but it has no therapeutic application. when annexin v binds to ps on a cell's membrane it crystalizes and this forms a two dimensional ps cloaking shield that can internalize it into the cells cytoplasm [ ] . when annexin v cloaks ps the th cell's inflammatory cytokine secretion that causes ebola's cytokine storm [ ] and the cascade thrombin generation that produces its hemorrhagic consumptive coagulopathy [ ] can't take place. this will also prevent hemostasis. will this prevent its therapeutic use? using annexin v to treat the pathologic aspects of inflammation and blood coagulation will only be possible if its therapeutic benefits are rapid and long lasting, its pathologic ones transitory and the physiologic recovery from its ps cloaking is rapid. all seem possible. when annexin v cloaks exposed ps this will instantly and permanently prevent those ps's ability to activate the th cell's inflammatory cytokine secretion [ ] and the blood coagulation that ps makes possible [ , ] . annexin v's half-life of minutes [ ] means it will be rapidly eliminated so that ps exposed by a subsequent vascular breach will be met by breach exposed tf generating thrombin and by ps and sulfatide on activated platelets adhering to the collagen. regarding annexin v's blocking of the innate immune cell activation [ ] , annexin v will only be given during an infection so that the innate immune response will already have been initiated. the ability to monitor the effect of annexin v administration would be desirable and since radio and florescent labeled annexin v molecules are used in the lab and clinically to detect ps+ cells it is possible that they can be used to quantify the ps+ cells that produce the inflammation and blood coagulation. could conditions other than ebola be considered for annexin v treatment? the same inflammatory and coagulation pathology is present in severe septicemia as is present in ebola and as in ebola it has little effective treatment, annexin v may be effective there. coronavirus sars and severe influenza infections are other lethal conditions to consider for annexin v therapy. in them their lethal respiratory failure may be the result of the inflammation and blood coagulation caused by the ps exposure on infected alveolar and bronchiolar cells resulting in the consolidation of the air space by the massive infiltration of mixed mononuclear/neutrophilic cells, edema and fibrin deposits [ ] . the administration of annexin v in ebola and in other critically ill patients should probably be by intravenous bolus so that its cloaking of ps will instantly stop further th cell feedback secretion of inflammatory cytokines and coagulation cascade thrombin generation that is causing the inflammatory organ damage and the thrombotic coagulopathy. in ebola its bolus administration may also prevent further macrophage and dendritic cell infection. in non-critical sars and influenza infections monitoring the levels of ps+ cells could be used to determine the necessity for annexin v use and when it is needed the ps + cell count can be used to monitor its continuous intravenous administration. annexin v's ability to cloak ps and block its roles in inflammation and blood coagulation is well documented but the therapeutic possibilities presented are speculative and require animal studies for their verification. at present there is little or no effective treatment for these lethal conditions so those studies would seem to be indicated. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. the role of phosphatidylserine in recognition of apoptotic cells by phagocytes the regulator of phosphatidylserine-catalyzed inflammation and coagulation during apoptosis ebola virus binding to tim- on t lymphocytes induces a cytokine storm tim genes: a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity find-me and eat-me signals in apoptotic cell clearance: progress and conundrums phosphatidylserine receptors: enhancers of enveloped virus entry and infection cell death in the pathogenesis of immune-mediated diseases: the role of hmgb and damp-pamp complexes differential roles of tissue factor and phosphatidylserine in activation of coagulation kinetics of factor x activation by the membrane-bound complex of factor ixa and factor viiia membrane-dependent interaction of factor xa and prothrombin with factor va in the prothrombinase complex ratelimiting roles of the tenase complex of factors viii and ix in platelet procoagulant activity and formation of platelet-fibrin thrombi underflow human annexin v binds to sulfatide: contribution to regulation of blood coagulation platelet surface-associated activation and secretion-mediated inhibition of coagulation factor xii annexin a increases survival in murine sepsis model by inhibiting hmgb -mediated pro-inflammation and coagulation clinical significance of measurement of plasma annexin v concentration of patients in the emergency room extracellular annexin a : functions of phosphatidylserine-binding and two-dimensional crystallization diannexin, an annexin a homodimer, binds phosphatidylserine with high affinity and is a potent inhibitor of platelet-mediated events during thrombus formation animal models of acute lung injury not applicable. none. supplementary data to this article can be found online at https:// doi.org/ . /j.mehy. . . key: cord- - ytt j authors: de vivo, darryl c.; bertini, enrico; swoboda, kathryn j.; hwu, wuh-liang; crawford, thomas o.; finkel, richard s.; kirschner, janbernd; kuntz, nancy l.; parsons, julie a.; ryan, monique m.; butterfield, russell j.; topaloglu, haluk; ben-omran, tawfeg; sansone, valeria a.; jong, yuh-jyh; shu, francy; staropoli, john f.; kerr, douglas; sandrock, alfred w.; stebbins, christopher; petrillo, marco; braley, gabriel; johnson, kristina; foster, richard; gheuens, sarah; bhan, ishir; reyna, sandra p.; fradette, stephanie; farwell, wildon title: nusinersen initiated in infants during the presymptomatic stage of spinal muscular atrophy: interim efficacy and safety results from the phase nurture study date: - - journal: neuromuscul disord doi: . /j.nmd. . . sha: doc_id: cord_uid: ytt j spinal muscular atrophy (sma) is a neurodegenerative disease associated with severe muscle atrophy and weakness in the limbs and trunk. we report interim efficacy and safety outcomes as of march , in children with genetically diagnosed sma who first received nusinersen in infancy while presymptomatic in the ongoing phase , multisite, open-label, single-arm nurture trial. fifteen children have two smn copies and have three smn copies. at last visit, children were median (range) . [ . – . ] months of age and past the expected age of symptom onset for sma types i or ii; all were alive and none required tracheostomy or permanent ventilation. four ( %) participants with two smn copies utilized respiratory support for ≥ h/day for ≥ consecutive days that was initiated during acute, reversible illnesses. all participants achieved the ability to sit without support, / ( %) achieved walking with assistance, and / ( %) achieved walking independently. eight infants had adverse events considered possibly related to nusinersen by the study investigators. these results, representing a median . years of follow up, emphasize the importance of proactive treatment with nusinersen immediately after establishing the genetic diagnosis of sma in presymptomatic infants and emerging newborn screening efforts. spinal muscular atrophy (sma) is an autosomal recessive neurodegenerative disease associated with progressive and often severe muscle weakness and atrophy and is a leading cause of death in infants [ ] [ ] [ ] [ ] . sma is caused by homozygous deletions ( ∼ % of sma patients) or compound heterozygous mutations ( ∼ % of sma patients) in the survival motor neuron ( smn ) gene that prevent production of full-length functional smn protein [ ] . the paralogous gene smn undergoes aberrant splicing and produces mostly truncated, dysfunctional protein ( ∼ %) [ ] . symptom onset most often occurs in infancy and early childhood, although onset can be in adulthood in the mildest form. symptoms are generally more severe with earlier onset [ ] . most individuals who develop sma have a symptom-free period after birth, which differs in duration for every individual. sma is divided into four major subtypes based on age at symptom onset and maximum motor function achieved [ , , ] , but maximum motor function achievement can be influenced by sma treatment. many infants with infantile-onset sma and children with later-onset sma treated with nusinersen in the endear and cherish studies, respectively, achieved motor function incongruent with their expected sma subtypes based on smn copy number and age of symptom onset [ , ] . prior to the onset of symptoms, other markers must be relied upon for diagnosis and classification of predicted sma subtype. smn gene copy number is roughly correlated with disease severity, as an increased number of smn gene copies typically leads to an increased amount of functional smn protein and a milder phenotype [ ] . among individuals who have smn disruptions and are therefore expected to develop sma symptoms, approximately % of individuals with two copies of the smn gene are predicted to develop the type i form of sma, while approximately % of those with three copies of the smn gene are predicted to develop sma type ii [ , ] . additionally, phosphorylated neurofilament heavy chain (pnf-h) levels have recently been shown to be a promising biomarker of disease activity and treatment response in individuals with sma [ , ] . pnf-h is a neuron-specific cytoskeletal structural protein released into the plasma and cerebrospinal fluid (csf) during axonal damage [ , ] . in the endear and cherish studies, concentrations of pnf-h were found to correlate with baseline clinical characteristics indicative of disease severity. furthermore, pnf-h concentrations declined rapidly then stabilized at a lower level following nusinersen treatment in a manner not seen in the sham control group [ ] . nusinersen is an antisense oligonucleotide that alters the splicing of smn pre-mrna to promote expression of fulllength smn protein [ , - ] and is the first disease-modifying treatment approved for sma [ ] . in clinical studies in a range of symptomatic children across several sma populations, nusinersen demonstrated significant and clinically meaningful benefit as assessed by achievement of motor milestones, measures of motor function, and survival in those with infantile-onset sma, along with a favorable safety profile [ , , ] . importantly, subgroup analyses of the shamcontrolled studies in infantile-onset (endear) and lateronset (cherish) sma indicated that greater improvements in motor function following nusinersen treatment were observed in those with relatively shorter disease duration at treatment initiation [ , ] , suggesting that earlier treatment may lead to better clinical outcomes. nurture is an ongoing phase , open-label study aimed to evaluate the safety and efficacy of nusinersen in preventing or profoundly attenuating the severity of sma when initiated prior to the onset of symptoms. infants enrolled in nurture had genetic confirmation of q sma, were ≤ weeks old at first dose, and were considered most likely to develop sma type i or type ii based upon the smn gene copy number and the expected concordance with the phenotype of an affected sibling(s) based on previous studies [ , , ] . in the absence of treatment, many of these infants would not be expected to achieve independent sitting (those likely to develop sma type i), and few if any would be expected to ever walk independently [ ] . most would also be expected to require respiratory intervention and some would not survive beyond their early years [ ] . nurture (nct ) is an ongoing, phase , open-label, single-arm, multinational study to evaluate the long-term safety and efficacy of intrathecal nusinersen in infants who initiate treatment early, prior to the onset of clinical signs of sma. given the expectation that infants with genetically diagnosed sma without a functioning copy of the smn gene and with two or three copies of the smn gene will develop severe or fatal symptoms during the first years of life, an internal control group was considered unnecessary and ethically unjustifiable. nurture consists of a -year treatment period and a post-treatment follow-up evaluation. key eligibility criteria were age ≤ weeks at first dose, genetic documentation of q sma (biallelic deletion or protein disabling mutation of the smn gene), two or three copies of the smn gene, baseline compound muscle action potential (cmap) amplitude ≥ mv (protocol amended on september [version ] to baseline ulnar cmap amplitude ≥ mv), absence of hypoxemia, and no clinical signs or symptoms suggestive of sma. participants received nusinersen mg administered as intrathecal injections by lumbar puncture. the nusinersen treatment regimen consists of four loading doses (administered on days , , , and ), followed by a maintenance dose every days over five years ( fig. ) . intention-to-treat population is all infants who received ≥ dose of study drug ( n = ). a infants who attended or had the opportunity to attend the visit. b infants treated with nusinersen mg; some infants received a -mg scaled equivalent dose before the protocol was revised in march . this interim analysis from the ongoing nurture study reports data from the march , , data cut. at the time of this interim analysis, the nurture infants were . - . (median . ) months of age and were past the expected age of symptom onset for sma types i or ii [ , ] . the nurture trial is taking place at active study sites in seven countries. the study was approved by the local ethics committee at each participating site and is being conducted in accordance with the international council on harmonization (ich) guidelines for good clinical practice and the declaration of helsinki. written informed consent was obtained from the parent(s) or legal guardian(s) of each study participant in accordance with local practice and regulations. the primary endpoint for nurture is time to death or respiratory intervention (invasive or non-invasive for ≥ h per day continuously for ≥ days or tracheostomy). secondary endpoints reported in this interim analysis include ( ) proportion of participants alive; ( ) attainment of motor milestones as assessed by world health organization (who) criteria; ( ) attainment of motor milestones by hammersmith infant neurologic examination, section (hine- ); ( ) change from baseline in the children's hospital of philadelphia infant test of neuromuscular disorders (chop intend) motor function scale; ( ) change from baseline in growth parameters: weight for age/length, head circumference, chest circumference, head-to-chest circumference ratio, and arm circumference; and ( ) proportion of participants developing clinically manifested sma at and months of age as defined by any of the following conditions: (a) age-adjusted weight < th percentile or decrease of ≥ major weight growth curve percentiles ( rd, th, th, th, or th) compared with baseline, or a percutaneous gastric tube placement for nutritional support at or months of age; (b) failure to achieve sitting without support, standing with assistance, and hands-and-knees crawling at age months; or (c) failure to achieve the milestones defined at age months and failure to achieve walking with assistance, standing alone, and walking alone at age months. secondary safety endpoints include ( ) incidences of adverse events (aes) and serious adverse events (saes); ( ) change from baseline in clinical laboratory parameters, electrocardiograms (ecgs), and vital signs; and ( ) neurological examinations (hine- [neurological status] and hine- [behavior] for participants age ≤ months; standard neurological exam for participants age > months). exploratory endpoints, including ( ) change from baseline in cmap amplitude; ( ) time to death or permanent ventilation (defined as ≥ h/day continuously for > days in the absence of an acute reversible event or tracheostomy); ( ) time to death or ventilation for ≥ h/day continuously for ≥ day or tracheostomy; and ( ) plasma and csf pnf-h concentration and change over time were also assessed in this interim analysis. the following clinical assessments were performed to evaluate nusinersen efficacy: survival, respiratory events, who and hine- motor milestone achievement, hine- neurological evaluation, chop intend motor function scale, growth parameters, ulnar and peroneal cmap amplitudes, and plasma/csf pnf-h levels. a listing of study visits at which data was collected for the study assessments is provided in supplementary table . achievement of motor milestones was evaluated using who criteria [ ] and the hine- assessment tool [ ] . who motor milestones are a set of six milestones (sitting without support, standing with assistance, hands-and-knees crawling, walking with assistance, standing alone, and walking alone) expected to be attained by age months in healthy children [ ] . who motor milestones were assessed by physical therapists; age of first achievement of each milestone was recorded by caregivers. hine is a quantifiable, three-part assessment to evaluate neurological status (section ), development of motor function (section ), and behavior (section ) [ ] . neurological items assessed by the hine- include sucking/swallowing ability during assessment of cranial nerve function [ ] . scores range from to , with higher scores indicating better function. hine- measures developmental progress in eight motor milestones: voluntary grasp, ability to kick in supine position, head control, rolling, sitting, crawling, standing, and walking. total scores range from to , with higher scores indicating better motor function [ ] . the hine- score was assessed until day . chop intend is a -item, -point motor assessment developed to evaluate motor skills in infants with sma type i, with higher scores indicating better motor function [ , ] . the chop intend was assessed until a participant reached a maximum score of , after which it was not assessed. motor function assessments were performed by trained clinical evaluators. cmap amplitude is an electrophysiological technique that reflects the mass of excitable muscle tissue depolarized with whole nerve stimulation, and in sma relates to the approximate number of motor neurons innervating a muscle. ulnar cmap amplitude is a well-validated method for tracking disease progression in sma [ , ] . measurement of growth parameters included length, weight-for-age, length-for-age, weight-for-length, head circumference, chest circumference, head-to-chest circumference ratio, and arm circumference. who child growth standards were used to determine percentiles for each parameter [ ] . pnf-h levels recently have been shown to be a potential biomarker of disease severity and response to treatment in individuals with sma. plasma and csf samples were tested for pnf-h. pnf-h levels were summarized as geometric mean for nurture infants by smn copy number and compared with levels reported for infants without sma aged < year. geometric mean was used to appropriately account for the underlying distribution of the data. samples from individuals were donated by boston children's hospital (boston, ma) and confirmed to be from individuals with no known neurological or musculoskeletal disorders or other chronic illness. pnf-h levels were measured using a pnf-h enzyme-linked lectin assay from proteinsimple. safety assessments included treatment-emergent ae monitoring, neurological examinations, physical examinations, vital sign measurements, pulse oximetry, -lead ecgs, and laboratory testing (hematology, blood chemistry, urinalysis, and coagulation) and were performed by trained physicians and ancillary medical professionals. when possible, comparisons in survival and motor milestone achievement were made between nurture participants and non-nusinersen-treated siblings with sma. only non-invasive data were collected from siblings, including smn gene copy number, sibling sma history, and sibling treatment history. time to death or respiratory intervention, proportion of participants alive, proportion of participants achieving who motor milestones, proportion of participants developing clinically manifested sma, and safety analyses were analyzed in all study participants who received ≥ dose of nusinersen (intent-to-treat population). all other efficacy analyses were performed on interim efficacy sets that comprised all dosed participants who attended or had the opportunity to attend the targeted visit of the analysis (efficacy set). all secondary endpoints were assessed at ages and months. proportion of participants alive and proportion who achieved who motor milestones at ages and months were estimated using the kaplan-meier method. chop intend total score, hine- total motor milestone score, change in growth parameters, ulnar and peroneal cmap amplitude, and pnf-h concentrations were summarized using descriptive statistics. the proportion of participants developing clinically manifested sma at day ( -month assessment) or day ( -month assessment) were reported with a corresponding wilson score confidence interval (ci) with continuity correction [ ] . the age at which a who motor milestone was achieved was determined by using the caregiver-reported date if confirmed at the subsequent study visit by the physical therapist. to identify early predictors of motor function, spearman correlation coefficients were calculated. specifically, the relationships between participant characteristics at baseline and at the end of the loading dose period on day (age at first dose of nusinersen, gestational age, plasma pnf-h levels, weight for age, chop intend total score, hine- motor milestone total score, and ulnar cmap amplitude) and future motor function (day total hine- motor milestone score and age of achievement of walking alone) were evaluated. the statistical software sas r version . (cary, nc) or above was used for all summaries and statistical analyses. a total of infants were screened; infants ( with two smn copies and with three smn copies) met eligibility criteria and were enrolled in the study. five infants were ineligible; one did not have a diagnosis of q sma, one did not have two or three copies of the smn gene, and three had an ulnar cmap amplitude equal to or below one mv at screening (two of whom also had clinical signs or symptoms suggestive of sma). the first participant's initial visit was in may and enrollment was complete in february . as of data cutoff, no participants have withdrawn from the study or discontinued treatment. the median (range) age at first dose of nusinersen among enrolled infants was . months. baseline characteristics by smn copy number are shown in table . median (range) chop intend total score and total hine- motor milestone scores were . ( . - . ) and . ( - ), respectively, in two-copy participants ( n = ) and . ( . - . ) and . ( - ), respectively, in three-copy participants ( n = ). twenty-four participants consented to collection of plasma and csf for future investigations of possible biomarkers of sma disease. baseline plasma values were unavailable in two participants, and baseline csf values were unavailable in one participant. geometric mean ( % ci) plasma pnf-h concentrations were , . ( . - , . ) pg/ml in two-copy participants ( n = ), and . ( . - . ) pg/ml in three-copy participants ( n = ). geometric mean ( % ci) csf pnf-h concentrations were , . ( , . - , . ) pg/ml in two-copy participants ( n = ) and . ( . - . ) pg/ml in three-copy participants ( n = ). nineteen nurture participants had ≥ sibling with sma; in total, there were full siblings and two half siblings. of the siblings, eight had two smn copies, eight had three smn copies, and eight had an undocumented number of smn copies. one of the siblings was related to two nurture participants (a set of twins). as of this interim analysis, all nurture participants were alive and none required permanent ventilation. the median time to death or respiratory intervention (invasive or non-invasive ventilation for ≥ h/day continuously for ≥ days or tracheostomy) could not be estimated, as there were too few events. four ( %) infants (all with two smn copies) utilized respiratory intervention for ≥ h per day continuously for ≥ days ( fig. ) , all of whom initiated respiratory intervention during an acute, reversible illness. at the last study day prior to data cutoff, two of these infants no longer utilized respiratory intervention; these infants had previously received respiratory intervention for ≥ h per day for totals of and days during the course of the study. the other two infants continued to receive respiratory intervention for two and h per day, respectively, at the last study day prior to data cutoff; these infants received respiratory intervention for ≥ h per day for totals of and days, respectively, over the course of the study. no other infants received respiratory intervention during the course of the study. achievement of motor milestones. as of data cutoff, all ( / ; %) nurture infants achieved the who motor milestone "sitting without support", while / ( %; / with two smn copies and / with three smn copies) achieved "walking with assistance", and / ( %; / with two smn copies and / with three smn copies) achieved "walking alone". site-or caregiver-reported ages of first achievement for who motor milestones are shown in fig. [ , ] . the median ( % ci) ages for first achievement of sitting without support, walking with assistance, and walking alone in two smn copy participants were . ( . - . ) months, . ( . - . ) months, and . ( . , . ) months, respectively. in those with three smn copies, the median ( % ci) ages for first achievement of sitting without support, walking with assistance, and walking alone were . ( . - . ) months, . ( . - . ) months, and . ( . - . ) months, respectively. hine- motor milestone total scores also increased over time for all participants regardless of smn copy number. mean (range) total scores increased from a baseline of . ( - ) to . ( - ) at the last observed visit, up to and including day , for participants with two smn copies and from . ( - ) to . for those with three smn copies. mean scores for infants with two or three smn copies approached the scale maximum of points; three-copy participants approached the maximum earlier than participants with two smn copies ( fig. ) . motor function. mean chop intend total scores rose steadily from baseline until approximately day and then remained stable over time ( fig. (a) ). at the last visit, mean (range) chop intend total score was . ( - ) in those with two smn copies and . ( - ) in those with three smn copies. at the time of this interim analysis, / ( %) of participants with smn copies and / ( %) of those with smn copies had achieved a maximum score of ( fig. (b) ). beyond day , chop intend total scores appear to be constrained by a plateau ceiling effect of ∼ points and was no longer assessed after participants had achieved the maximum score of ; however, continued improvement was observed in hine- and who milestones outcomes. at the last observed visit up to and including day , all nurture participants had the ability to suck and swallow as measured by the hine- neurological assessment. twentytwo participants ( / with two smn copies, / with three smn copies) achieved the maximum score of (good sucking and swallowing) on the hine- . three infants (all fig. . site-or caregiver-reported a age at first who motor milestone achievement. smn , survival motor neuron ; who, world health organization. a if caregiver-reported, achievement was confirmed by the study site at the next study visit with a yes or no response. b who motor milestone windows of achievement were determined based on the who multicenter growth reference study windows of achievement in healthy children [ ] . with two smn copies) achieved a score of (poor sucking and/or swallowing); each of these three had gastrostomy tubes placed. clinically manifested sma. the proportions of two smn copy participants who had protocol-defined symptoms of sma were . ( % ci . - . ; n = of total) at age months and . ( % ci . - . ; n = of total) at age months. the proportions of three smn copy participants who had protocol-defined symptoms of sma were . ( % ci . - . ; n = of total) at age months and . ( % ci . - . ; n = of total) at age months. the seven infants who developed protocol-defined symptoms of sma by months (all with two smn copies) were all continuing to grow and achieve who motor milestones inconsistent with type i sma and with the milestone attainment of their siblings with sma. all seven were sitting without support, five were walking with or without assistance, and four were walking alone. six of these seven participants had siblings with sma; none of the siblings achieved sitting independently and / required tracheostomy and/or died by months of age. the seven infants with clinically manifested sma at age months included the three participants with a poor suck/swallow reflex who had gastrostomy tubes placed. while these three infants undoubtedly exceeded their expected two smn copy type i predicted phenotype by achieving the ability to sit independently, they achieved motor milestones at a slower pace than other participants with two smn copies in the study. all three participants were able to stand with assistance, two were able to walk with assistance, and one achieved walking alone; two were infants who received respiratory intervention at last visit. baseline pnf-h levels were higher in most presymptomatic infants with sma compared with non-sma infants and were significantly higher in most participants with two smn copies compared with those with three smn copies (plasma p = . ; csf p = . ). the geometric mean plasma pnf-h level in healthy infants < year of age was . pg/ml (median . [range - ] pg/ml; n = ; fig. (a) ). in presymptomatic infants with sma, geometric mean plasma pnf-h levels declined rapidly during the loading phase of nusinersen treatment and then stabilized ( fig. (b) ). a similar pattern of decline followed by stabilization was observed for geometric mean csf pnf-h levels ( supplementary fig. ). ) ulnar nerve cmap amplitudes at baseline were . ( . ) mv and . ( . ) mv in participants with two and three smn copies, respectively ( p = . ), and remained stable over time ( supplementary fig. (a) ). similar results were observed for peroneal nerve to tibialis anterior cmap amplitude (baseline peroneal cmap amplitude in two versus three smn copy participants, p = . ; supplementary fig. correlations were calculated to identify the earliest, strongest predictors of motor function. among the baseline characteristics analyzed, baseline plasma pnf-h level was the strongest predictor of hine- total motor milestone score at day ( r s = − . ; p = . ; n = ) and age of achievement of who walking alone ( r s = . ; p = . ; n = ). in the analysis of participant characteristics at day , plasma pnf-h levels at day were the best predictor of future motor function achievement in the overall population. plasma pnf-h levels at day were significantly correlated with the total hine- motor milestone score at day ( r s = − . ; p = . ; n = ) and with achievement of the who motor milestone walking alone ( r s = . ; p = . ; n = ). lower plasma pnf-h levels at day were associated with earlier achievement of walking alone ( fig. ) . in participants with smn copies, day weight for age ( r s = . ; p = . ; n = ) and day cmap amplitude ( r s = . ; p = . ; n = ) were correlated with total hine- motor milestone achievement at day , and similar results were observed for age at achievement of who walking alone. the associations between day weight for age or day cmap amplitude and achievement of who walking alone are shown in supplementary figs. (a) and (b) . plasma pnf-h levels at day were evaluated in relationship to those from individuals without sma and to motor milestone achievement and other outcomes. plasma samples were analyzed from non-sma individuals who ranged in age from . to . years. these individuals had plasma pnf-h levels ranging from to pg/ml (q , pg/ml; q , pg/ml). nurture day was selected for this analysis based on the age range of non-sma individuals. twenty-two nurture participants had a recorded measurement for day plasma pnf-h level, of whom ( with smn copies; with smn copies) had a result below q ( ≤ pg/ml) for non-sma individuals < year of age. among these nurture participants, all children who initiated ventilation support ( n = ), met the protocol definition of clinically manifested sma at day ( n = ), and/or had delayed (or no) achievement of sitting or walking alone (per who th percentile window) had a day plasma pnf-h level above the q ( > pg/ml) for non-sma individuals < year of age. aes were reported in / ( %) nurture participants ( table ). twenty of ( %) participants had an ae that was mild or moderate in severity. there were no aes considered to be definitely related to study drug by the investigators. all aes considered by the investigator to be possibly related to study drug ( / infants; %) resolved despite continued treatment, with the exception of one case each of proteinuria and clonus, which were ongoing at the time of data cutoff. a total of saes were reported in / ( %) participants. treatment-emergent saes in those participants included tendon disorder and dehydration ( n = ); bronchitis, choking, pneumonia ( n = ); pneumonia ( n = ); mycoplasmal pneumonia ( n = ); viral upper respiratory tract infection ( n = ); abdominal distension, respiratory distress, dehydration, enterovirus infection, corona virus infection, respiratory syncytial virus bronchiolitis, bacterial pneumonia, acute respiratory failure, respiratory failure, tachycardia, viral gastroenteritis, pneumonia ( n = ); respiratory distress, respiratory syncytial virus bronchiolitis, aspiration pneumonia, pneumonia ( n = ); failure to thrive ( n = ); urinary tract infection ( n = ); pyrexia, pneumonia, pneumococcal pneumonia, pseudomonal pneumonia, upper respiratory tract infection ( n = ); respiratory syncytial virus infection ( n = ); upper respiratory tract infection ( n = ). there were no saes related to study drug. the lumbar puncture procedure was generally well tolerated. eight participants had an event determined by investigators to be possibly related or definitely related to the lumbar puncture procedure: traumatic lumbar puncture ( n = ); vomiting ( n = ); subdural hematoma, post-procedure discomfort ( n = ); headache ( n = ); postlumbar puncture procedure syndrome, lower lumbar spinal canal hematoma, hypertension ( n = ); post-procedural swelling ( n = ); extradural hematoma, tachycardia, weightbearing difficulty, muscular weakness ( n = ); and epidural hemorrhage, spinal subarachnoid hemorrhage, tachycardia, hyperreflexia ( n = ). five hemorrhages near the thecal space in four participants occurred in the setting of multiple lumbar puncture attempts; all events occurred when participants were < weeks old, and none were in the context of thrombocytopenia. the only lumbar puncture-related event classified as an sae was one case of post-lumbar puncture syndrome that occurred before the first dose of study drug following a failed dosing attempt. no clinically relevant trends with respect to thrombocytopenia, coagulation abnormalities, abnormal liver and kidney function tests, or proteinuria were observed in the nurture cohort. there were no observed cases of heart disease, liver failure, bacterial meningitis, aseptic meningitis, hypersensitivity, or hydrocephalus. alanine aminotransferase (alt), aspartate aminotransferase (ast), creatine kinase, creatinine, cystatin c, and platelet levels remained stable over time (supplementary figs. - ). to our knowledge, nurture is the first study to investigate a treatment targeting the underlying cause of the sma disease in a presymptomatic period and one of the first two studies to investigate any treatment for presymptomatic sma [ ] . the results of this interim analysis, representing a median . years of follow up, demonstrate substantial clinical benefit in infants with two or three copies of the smn gene (considered most likely to develop type i or ii sma) as a result of early initiation of nusinersen treatment. nurture infants, who at the time of this interim analysis were age ≥ months and past the age of symptom onset [ , ] , were all alive without requirement for permanent ventilation. the contrast to the natural history of untreated sma is dramatic: those with sma type i (the expected phenotype for approximately half our cohort based on smn copy number) die or require permanent ventilation, on average, by . months of age [ ] . that early treatment is key to this rescue arises from comparison of the nurture experience with that of the endear study, in which treatment with nusinersen was initiated in a symptomatic period. by the end of the endear study, / ( %) nusinersen-treated infants with infantile-onset sma died or required permanent ventilation [ ] . all nurture participants are alive without permanent ventilation and able to sit independently; nearly all children ( / ) can walk with assistance or independently, and all continued to gain motor skills throughout the study interval. in both groups of children with two and three smn copies, nusinersen showed rapid onset of improvement and durability of effect on mean chop intend motor function scores. these results are clearly incongruent with the natural history of individuals with two or three copies of smn [ ] . these results demonstrate that treatment with nusinersen of genetically diagnosed infants with sma in the presymptomatic period allows for gains in motor function closer to normal development than that expected in individuals with sma type i or ii. the motor milestone and motor function achievements of many nurture infants are also drastically discordant from their untreated siblings with the same smn copy number. this discordance is notable, given that a previous study found that . % of sibling pairs with sma had a concordant phenotype [ ] , further emphasizing the clinically meaningful benefit of nusinersen treatment in the presymptomatic period. chop intend scores for nurture infants were similar to those observed for healthy infants over the first three study months, the interval for which natural history data on healthy infants are available [ ] . nurture infants' chop intend scores greatly exceed those observed in a natural history cohort of symptomatic infants with sma with two copies of smn over the course of the study, who had a mean (sd) decline of . ( . ) points over - months [ ] . similarly, the mean ulnar cmap amplitude of nurture infants remained stable over time in participants with two and three smn copies in nurture, while amplitude fell rapidly and was never higher than . mv in a natural history cohort of infants with sma aged ≥ months with two smn copies [ ] . overall, the nurture infants who initiated treatment before the appearance of clinical symptoms had earlier and substantially greater improvement in hine- total scores than did infants and children who initiated treatment after symptom onset in the endear and cs a trials [ , ] . nurture infants also had higher baseline chop intend scores and were able to attain maximum or near-maximum chop intend scores more quickly than did infants who initiated treatment after symptom onset [ , ] . while the differences in population ages and study designs between studies must be kept in mind when interpreting these data, the differences in hine- score trajectories and chop intend maximum scores indicate that treatment with nusinersen can have a meaningful impact on clinical outcome beyond that seen when treatment is initiated after symptom onset. the functional outcomes of nurture participants with three smn copies were generally better than those observed in participants with two copies of smn . specifically, all participants with three smn copies have achieved all of the more advanced who motor milestones, including walking independently; whereas three participants with two smn copies have yet to achieve these milestones. participants with three smn copies also achieved higher mean chop intend and hine- total scores by last observed visit than participants with two smn copies. baseline chop intend and hine- scores were also higher in participants with three smn copies versus two smn copies. while slight differences in the outcomes of patients with three smn copies and two smn copies were observed, all far exceed outcomes predicted by sma type i and ii natural history. these findings highlight the substantial benefit of early therapy, and thus point to the value of early diagnosis. population-based newborn screening (nbs) is a method for identifying presymptomatic individuals with treatable diseases that aims to provide the opportunity for early intervention [ ] . in the united states, nbs for homozygous deletion of exon in the smn gene was added to the recommended uniform screening panel (rusp) on july th, . other nbs programs are in development globally [ ] [ ] [ ] [ ] . presymptomatic individuals with sma are now being identified in several us states through nbs programs, and many additional states will begin screening later in . additional nbs efforts for sma are ongoing globally and are supported by the general population in several countries [ - , , ] . a treatment algorithm based on smn copy number for sma positive infants identified though nbs was recently developed by a group of sma experts [ ] . the recommendation was for infants with two or three smn copies to receive immediate treatment following confirmatory testing. an additional recommendation by other sma experts suggests immediate treatment for presymptomatic infants with up to four smn copies [ ] . nurture results support immediate treatment of infants with two or three smn copies and support the notion that earlier treatment across the spectrum improves eventual outcome as was demonstrated by the endear and cherish studies [ , ] . pnf-h levels recently have been shown to be a potential biomarker of disease severity and response to treatment in individuals with sma. concentrations of pnf-h in both the endear and cherish studies were found to correlate with several baseline clinical characteristics indicative of disease severity and decline rapidly after treatment with nusinersen before stabilizing at lower plateau levels [ ] . to our knowledge, nurture is the first study to evaluate pnf-h levels in a presymptomatic sma population. geometric mean plasma and csf levels of pnf-h were substantially higher in nurture participants than in infants without sma and, within nurture participants, plasma and csf pnf-h levels were significantly higher in participants with two versus three smn copies. after nusinersen treatment initiation, pnf-h levels declined rapidly during the loading period before apparently stabilizing at lower levels. the rapid decline of pnf-h levels suggests the value of pnf-h as a potential biomarker of treatment response to smn-enhancing therapies. levels of pnf-h in plasma at the end of the nusinersen loading dose period may predict future motor function such as walking alone by the who th percentile for expected age of achievement. the very high pnf-h levels measured before onset of clinical symptoms suggest that pathophysiologic processes precede the onset of clinical symptoms; whether release of pnf-h into interstitial fluids marks a treatment-reversible pathology, or instead an early degeneration that is not yet of sufficient magnitude to be clinically apparent, is yet unknown. in either case, pre-treatment elevation of pnf-h levels in these presymptomatic infants further emphasizes the value of nbs initiatives worldwide and stress the importance of proactive intervention in the presymptomatic phase of sma, consistent with other chronic progressive neurological diseases [ ] [ ] [ ] [ ] . nusinersen demonstrated a favorable benefit-risk profile consistent with data from previous studies [ , ] and no new safety concerns were identified. the lumbar puncture procedure was generally well tolerated. a small number of hemorrhages near the thecal space occurred in the setting of multiple lp attempts in infants who were < weeks old; none were in the context of thrombocytopenia. careful monitoring of hematology, blood chemistry, urinalysis, coagulation, vital signs, and ecgs demonstrated no clinically relevant trends related to nusinersen treatment. levels of creatine kinase and transaminases (alt and ast) remained stable over time, suggesting no predisposition to muscle injury and no hepatic abnormalities. though the nurture outcomes are dramatic, key limitations of the study should be noted. nurture is an open-label study with a relatively small number of participants. while several participants had untreated siblings with sma that allowed for the comparison of motor function achievement with natural history, formal assessments were not performed in the siblings with sma, and the study has no sham-control group. it should be noted that infants had variable presentation across clinical measures at baseline (notably tendon reflexes, ulnar cmap, and chop-intend). however, although there is no uniformly established definition of sma symptom onset, all infants met the entry criteria and were considered presymptomatic at enrollment according to the investigator. chop intend score was assessed until a participant achieved the maximum score of , after which time it was no longer assessed, but a ceiling effect of the chop intend score was apparent at a score of approximately . hine- score was assessed until day but was not assessed at later study visits. additionally, comparisons of changes in chop intend and hine- scores over time across the nusinersen clinical development program must be made cautiously; differences in study populations and study designs should be taken into consideration when interpreting the data. caregiver-reported versus study visit documentation of achievement of motor milestones could lead to variation in the reported timing of achievements. additionally, time of follow-up varies among participants, and first achievement of motor milestones may occur between study visits where these are assessed and documented. results from the nurture study demonstrate the potential benefits of initiating nusinersen during the presymptomatic period in infants with sma. in this interim analysis, many infants and children treated during the presymptomatic period achieved motor milestones in timelines consistent with normal development. data demonstrate durability of effect over a median of . years of follow up, with children continuing to make progress throughout the study with no evidence of sustained regression. these results not only exceed expectations based on the natural history of sma and the phenotypes of participants' siblings with sma, but also represent treatment benefits exceeding those observed when treatment is initiated in a symptomatic period. additionally, pnf-h data from nurture demonstrate that the underlying sma disease is biologically active in the presymptomatic period and merits treatment. results from nurture strongly emphasize the need for early identification of infants with sma through nbs and support the value of treatment initiation in presymptomatic infants, genetically diagnosed with sma with two or three copies of the smn gene, immediately after sma diagnosis. this conclusion is consistent with current 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muscular atrophy phosphorylated neurofilament heavy chain (pnf-h) levels in infants and children with sma: evaluation of pnf-h as a potential biomarker of sma disease activity association of phosphorylated neurofilament heavy chain (pnf-h) with nusinersen treatment of sma: analyses from the endear and cherish studies neurofilaments and neurofilament proteins in health and disease neurofilament phosphoforms: surrogate markers for axonal injury, degeneration and loss newborn screening: history, current status, and future directions newborn blood spot screening test using multiplexed real-time pcr to simultaneously screen for spinal muscular atrophy and severe combined immunodeficiency presymptomatic diagnosis of spinal muscular atrophy through newborn screening pilot study of population-based newborn screening for spinal muscular atrophy in new york state sma added to list of recommended screenings for disease given to newborns in us antisense correction of smn splicing in the cns rescues necrosis in a type iii sma mouse model antisense oligonucleotides delivered to the mouse cns ameliorate symptoms of severe spinal muscular atrophy results from a phase study of nusinersen (isis-smn rx ) in children with spinal muscular atrophy nusinersen: first global approval treatment of infantile-onset spinal muscular atrophy with nusinersen: a phase , open-label, dose-escalation study sma subtype concordance in siblings: findings from the cure sma cohort observational study of spinal muscular atrophy type i and implications for clinical trials who multicentre growth reference study group. who motor development study: windows of achievement for six gross motor development milestones optimality score for the neurologic examination of the infant at and months of age the children's hospital of philadelphia infant test of neuromuscular disorders (chop intend): test development and reliability validation of the children's hospital of philadelphia infant test of neuromuscular disorders (chop intend) compound muscle action potential and motor function in children with spinal muscular atrophy natural history of denervation in sma: relation to age, smn copy number, and function two-sided confidence intervals for the single proportion: comparison of seven methods by study to evaluate sodium phenylbutyrate in presymptomatic infants with spinal muscular atrophy (stopsma) natural history of infantile-onset spinal muscular atrophy newborn genetic screening for spinal muscular atrophy in the uk: the views of the general population willingness to pay for a newborn screening test for spinal muscular atrophy treatment algorithm for infants diagnosed with spinal muscular atrophy through newborn screening precious sma natural history data: a benchmark to measure future treatment successes presymptomatic studies in als: rationale, challenges, and approach amyotrophic lateral sclerosis: a long preclinical period? detection of huntington's disease decades before diagnosis: the predict-hd study toward defining the preclinical stages of alzheimer's disease: recommendations from the national institute on aging-alzheimer's association workgroups on diagnostic guidelines for alzheimer's disease the authors thank the patients who are participating in this study and their parents/guardians and family members, without whom this effort cannot succeed. the authors also thank the people who are contributing to this study, including the study site principal investigators, clinical monitors, study coordinators, physical therapists, and laboratory technicians. biogen provided funding for medical writing support in the development of this manuscript; susan chow, phd, and allison green, phd, from excel scientific solutions wrote the first draft of the manuscript based on input from authors, and nathaniel hoover from excel scientific solutions copyedited and styled the manuscript per journal requirements. the authors had full editorial control of the manuscript and provided their final approval of all content. requests for data supporting this manuscript should be submitted to the biogen clinical data request portal ( www. biogenclinicaldatarequest.com). supplementary material associated with this article can be found, in the online version, at doi: . /j.nmd. . . . key: cord- -ji emajn authors: zhou, jie‐ying; peng, ying; peng, xiao‐you; gao, han‐chun; sun, ya‐ping; xie, le‐yun; zhong, li‐li; duan, zhao‐jun; xie, zhi‐ping; cao, you‐de title: human bocavirus and human metapneumovirus in hospitalized children with lower respiratory tract illness in changsha, china date: - - journal: influenza other respir viruses doi: . /irv. sha: doc_id: cord_uid: ji emajn background: lower respiratory tract illness is a major cause of morbidity and mortality in children worldwide, however, information about the epidemiological and clinical characteristics of lrtis caused by hmpv and hbov in china is limited. objectives: human bocavirus (hbov) and human metapneumovirus (hmpv) are two important viruses for children with lower respiratory tract infections (lrti). we aimed to assay the correlation between viral load and clinical characteristics of hbov and hmpv with lrti in changsha, china. methods: nasopharyngeal aspirates (npas) from children with lrti were collected. real‐time pcr was used to screen hbov and hmpv. analyses were performed using spss . software. results: pneumonia was the most frequent diagnosis. there was no significant difference between hbov‐ and hmpv‐positive patients in age (p = . ) or hospitalization duration (p = . ); . % and . % were positive for hbov and hmpv. hbov infections peaked in summer ( . %), and hmpv infections peaked in winter ( . %). the hbov‐positive patients had a shorter hospitalization duration than the hbov‐negative patients (p = . ), and the hmpv‐positive patients had a higher prevalence of fever than the hmpv‐negative patients (p = . ). the hbov viral load was significantly higher among patients aged < year (p = . ). the mean hbov and hmpv viral loads were not significantly different between patients with single infections and coinfections. patients infected with hbov only were older than those coinfected with hbov and other respiratory viruses (p = . ). no significant difference was found in the clinical characteristics of patients infected with hmpv only and those coinfected with hmpv and other respiratory viruses. conclusion: pneumonia was the most frequent diagnosis caused by hbov and hmpv. neither hbov nor hmpv viral load was correlated with disease severity. hmpv, which was originally isolated in the netherlands and is closely related to avian metapneumovirus, was the first human disease-causing pathogen identified in the genus metapneumovirus. hmpv causes various clinical symptoms, such as cough, wheezing, and fever. young children and the elderly are more susceptible to hmpv infection. , indeed, hmpv mainly infects children under years of age and causes upper respiratory tract and severe lower respiratory tract infections. [ ] [ ] [ ] [ ] [ ] hbov was discovered in sweden and classified in the family parvoviridae, and causes various clinical symptoms, including cough, wheezing, and fever. hbov infections are largely confined to infants and young children (< months). globally, the average prevalence of hbov in respiratory tract samples ranges from . (ci: . - . ) to . % (ci: . - . ). however, information about the epidemiological and clinical char- (table ) . to standardize quantification of hbov and hmpv, known numbers of dna or rna transcripts containing the primer targets were used in -fold serial dilutions ( to copies/μl). any amplification detected before cycles was considered positive. quantification of > copies/μl ( copies/ml) was considered a positive result. an internal positive control gene (gapdh), positive control, and negative (water) control were included in all reactions. viral loads were expressed as initial copy numbers per real-time pcr reaction, and quantitative results were transformed as the log number of viral copies/μl. continuous variables are expressed as means ± standard deviation (sd) and were compared by independent samples t test. categorical variables are expressed as frequencies or percentages. comparisons were performed by chi-squared or fisher's exact test. analyses were performed using spss . software. two-sided p-values <. were considered to indicate statistical significance. in total, we analyzed samples collected from lrti patients patients were divided into two groups: those with and without hbov or hmpv infections (table ) (table ). according to the british thoracic society guidelines for the management of community-acquired pneumonia in children and the clinical diagnosis, lrtis were classified as mild to moderate or severe. the mean hbov and hmpv viral loads did not differ significantly between the two groups (p = . and p = . ) (figure ). in this study, we used real-time pcr to explore the etiology and ex- with lrtis. this is higher than previous study ( . %) in changsha that used traditional pcr, and lower than that a study in lanzhou that used real-time pcr. on the one hand, the high detection rate in our study could be caused by the greater sensitivity of real-time pcr compared with traditional pcr. on the other hand, it is possible that the detection rate differs geographically. rsv was the most frequently virus detected in patients with respiratory infections, followed by piv , hbov, adv, and hmpv. our data support previous reports of rsv, piv , hbov, adv, and hmpv as the major agents associated with lrtis among children in a hospital setting. , [ ] [ ] [ ] [ ] the hbov infection rate ( . %) in the present study is consistent with earlier reports ( . %- . % , , , [ ] [ ] [ ] [ ] , and the incidence of hmpv ( . %) in patients with respiratory tract infections is similar to that in other regions ( . %- % , , , , [ ] [ ] [ ] . therefore, hmpv and hbov are major causes of lrtis worldwide. the seasonal peaks of hbov and hmpv infections vary among countries because of differences in climatic and geographic factors. in this study, hbov activity peaked in summer, in agreement with the report by jiang. in contrast, detection of hbov in lanzhou peaked in december and april, possibly due to the dry, cold weather in lanzhou and the warm, humid weather in changsha. hmpv detection peaked in winter, which is in line with previous studies. , , , in contrast, in hong kong, hmpv detection peaks in spring/summer. the seasonality of hbov differed geographically, possibly due to climatic factors. the most frequent symptoms of hbov-and hmpv-positive patients were cough and fever, in accordance with previous reports. , , however, deng reported that in chongqing, wheezing was the most frequent symptom exhibited by hbov-positive patients with severe lrtis. there was no difference between the hbov-and hmpv-positive patients in the incidence of fever, tachypnea, cyanosis, o therapy, or wbc count. the hbov-positive patients had a shorter hospitalization duration than hbov-negative patients (p = . ). in contrast, deng reported that hbov-positive patients had a longer hospitalization duration. the longer hospitalization duration of hbov-negative patients in our study may have been caused by the presence of other viruses (such as rsv and piv ) in many of them. also, hospitalization duration was significantly associated with age (≤ months), maternal smoking during pregnancy, and a family history of asthma. a high hmpv viral load contributes to development of fever. , indeed, hmpv-positive patients had a higher incidence of fever than hmpv-negative patients (p = . ) in our study. we assessed the relationship between viral load and clinical features (age, gender, respiratory rate, temperature, cyanosis, hospitalization duration, and wbc count). the only significant association of hbov-or hpmv-positive patients was a higher viral load in < -year-old hbov-positive patients. this is in agreement with jiang's report that patients with a high viral load were significantly younger. in contrast, the duration of wheezing and hospitalization was longer in children with a high than a low hbov viral load in the study by deng, possibly due to inclusion of only patients with severe lrtis. hmpv and hbov coinfections with other viruses are common in this study has the following strengths: a considerable duration, large number of patients, and comparison of the most common viruses. its limitation includes the lack of a control group without clinical evidence of illness. further studies should compare a symptomatic group with a control group and a symptomatic period with an asymptomatic period. in summary, we present a prospective study of lrtis caused by hbov and hmpv. a further comprehensive and in-depth study of the role of hbov and hmpv in lrtis in china is warranted. a newly discovered human pneumovirus isolated from young children with respiratory tract disease human metapneumovirus: insights from a ten-year molecular and epidemiological analysis in germany outbreak of human metapneumovirus infection in a severe motor-and-intellectual disabilities ward in japan viral load and acute otitis media development after human metapneumovirus upper respiratory tract infection clinical disease and viral load in children infected with 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in otherwise healthy children who attended an emergency department real-time quantitative pcr detection of four human bocaviruses human bocavirus and human metapneumovirus in hospitalized children with lower respiratory tract illness in changsha, china we thank li-li li, na-liu, jie-mei yu, li-li pang, and other staff members in department of viral diarrhea (national institute for viral disease control and prevention) for detection of virus pathogens. the authors have no competing interests to report. the process obtained families' informed consent, and the study protocol was approved by the first affiliated hospital of hunan normal university, changsha, china. http://orcid.org/ - - - key: cord- -bcaerchf authors: van der heden van noort, gerbrand j.; ovaa, huib title: how to target viral and bacterial effector proteins interfering with ubiquitin signaling date: - - journal: activity-based protein profiling doi: . / _ _ sha: doc_id: cord_uid: bcaerchf ubiquitination is a frequently occurring and very diverse posttranslational modification influencing a wide scope of cellular processes. ubiquitin (ub) has the unique ability to form eight different lysine-linked polymeric chains, mixed chains and engages with ubiquitin-like (ubl) molecules. the distinct signals evoked by specific enzymes play a crucial role in, for instance, proteasome-mediated protein degradation, cell cycle regulation, and dna damage responses. due to the large variety of cellular functions that this posttranslational modification influences, the enzymes that construct such ub modifications, and subsequently controle and degrade these signals, is enormous. in this chapter, we will discuss the current state-of-the-art of activity-based probes, reporter substrates, and other relevant tools based on ub as recognition element, to study the enzymes involved in the complex system of ubiquitination. state-of-the-art of activity-based probes, reporter substrates, and other relevant tools based on ub as recognition element, to study the enzymes involved in the complex system of ubiquitination. protein activity is regulated by the attachment and removal of posttranslational modifications (ptms). nucleophilic side chain functionalities in the protein can be decorated with such ptms aided by a large class of enzymes that can monitor the substrate and the need to activate or deactivate its activity. some of those ptms are intensively studied such as, for instance, phosphorylation, but other ptms are less well understood due to their high complexity, such as glycosylation and ubiquitination. in the latter case, a amino acid protein ubiquitin (ub) is transferred mostly to the e-amine of a lysine residue in a target protein. ubiquitination is involved in almost all aspects of eukaryotic biology including the regulation of immune responses, cell cycle progression, and protein degradation. ub is a stable protein with a denaturation temperature of over °c that is highly conserved from yeast to man with only three respective changes. another interesting feature of ub is its ability to form polymeric ub chains in which the e-amine functionality of any of the seven internal lysine residues or the n-terminal amine of ub can be linked to the c-terminal carboxylic acid of a subsequent ub. eight different topologies can thus be formed all exerting different signaling outcomes. lys- -linked poly-ub, for instance, is involved in proteasome-mediated protein degradation, whereas the lys- -linked poly-ub is implicated to play a role in dna damage repair. most linkage types have been connected to one or more specific functions but new discoveries keep expanding the repertoire of involvement of ub in cell signaling. on top of this already complex system with eight different homotypic poly-ub chains, the possibility to form mixed heterotypic chains and branched chains complicates the ub system even further. posttranslational modifications such as phosphorylation, acetylation and newly discovered adp-ribosylation of ub and hybrid chains with ubiquitin-like modifiers such as sumo and nedd are also found to play distinct roles. the numerical amount of combinations one could think of our vast, but are all biologically relevant? the general mechanism of ub attachment to a target protein or a predecessor ub (forming a poly-ub chain) depends on a series of so-called e -, e -, and e -enzymes that work together to activate and ligate ub to its target using atp as energy source. this process, known as the canonical ub cascade, can undergo repetitive cycles and lead to poly-ubiquitinated substrates. one of two e activating enzymes, known to date, adenylates the c-terminal carboxylic acid of ub and subsequently forms a reactive thioester intermediate. one of * e conjugating enzymes takes over the ub cargo by means of a trans-thiolation reaction (stewart et al. ) . one of over e -enzymes encoded in the human genome either mediates the transfer of ub from the e to the substrate or takes over the ub cargo itself prior to transferring it to the target, depending on whether the e belongs to the ring, hect or rbr class of conjugating enzymes (buetow and huang ) . the specific combination of e /e not only ensures the correct target proteins are ubiquitinated but also dictates the type of poly-ub linkage that is installed; actively controlling the signaling outcome of the ubiquitination event. counteracting the buildup of (poly-)ubiquitinated proteins is a group of deubiquitinating proteases (dubs) that break down the ubiquitin modification, liberating the substrate protein, recycling ub, and ending the ub invoked signaling. nearly a hundred of such deubiquitinating proteases have been identified in human cells that can be classified into seven distinct families. the subfamilies of ubiquitin-specific proteases (usps), ubiquitin c-terminal hydrolases (uchs), ovarian tumor domain proteases (otus), machado-joseph disease proteases (mjd), motif interacting with ub-containing novel dub family (mindys), and zinc finger with ufm -specific peptidase domain protein (zufsps) are cysteine proteases, whereas jab /mpn/mov proteases (jamms) are zinc-dependent metallo-proteases. to understand the complex signaling network brought about by the writers, readers, and erasers of the ubiquitin code, tools to study them in detail and on molecular level are of great interest. below we describe the current state of the molecular toolbox available and three examples of their use in unraveling some of the secrets in viral and bacterial interference with ubiquitin biology. a diversity of tools has been developed to study the activities of enzymes involved in constructing and deconstructing ubiquitin chains and ubiquitinated proteins. the probes based on the ub core as recognition element will be discussed below classified on their ability to target different enzymatic functions. both chemical and semi-synthetic techniques are used to construct such probes, each having distinct advantages and drawbacks that, however, will not be discussed in great detail in this chapter. the first activity-based probe based on ub forming a covalent complex between probe and target protease was based on replacing gly -gly with -aminobutyraldehyde (pickart and rose ) . the ubiquitin aldehyde (ubal) reagent was found to form a complex and inhibit the activity of the uch protease studied. modifications on this reactive c-terminal element with, for instance, a nitrile moiety (lam et al. ) and glycine vinyl sulfone (vs) or glycine vinyl methyl ester amine (vme) (borodovsky et al. (borodovsky et al. , led to the development of a bigger panel of activity-based probes able to capture the active site cysteines of deubiquitinating enzymes. these electron poor vinyl motifs act as michael acceptor element and allow the sulfur nucleophile of the active site cysteine to be trapped by forming a covalent intermediate in an irreversible reaction. later on, the total chemical synthesis of ub (mutants) using solid phase peptide chemistry (el oualid et al. ; kumar et al. ; pasunooti et al. ) opened the way to prepare ub probes carrying fluorescent labels or affinity handles on large scale (de jong et al. ) . exploiting a similar mode of action as ub-vme is ub-propargyl (prg), where the terminal alkyne of the propargyl moiety unexpectedly is able to react with the active site cysteine leading to a covalent adduct in the form of a vinyl thioether linkage (see fig. a ) (ekkebus et al. ) . major advantage of this last variant, ub-prg, is its unreactiveness toward other cysteine proteases, making it really a dub-specific probe. in general, specificity of such probes for dubs is based on the ub element of the probe to be recognized by the protease on a binding interface or so-called s -pocket preceding the active site placing the reactive fig. activity-based probes to target dub activity. a reaction details of cysteine protease reaction with ub-prg, b different probes targeting s , s -s ′, or s -s interactions, c misplacement of probes on dubs allowing the study of different binding pockets element directly over the active site cysteine. in this situation, the two reacting partners are optimally aligned and lead to the formation of a covalent adduct (see fig. a ). some dubs have been shown to have a preference to specifically cleave certain poly-ub chains. in order to investigate such possible preferences, in a classical experiment all native diub molecules are incubated with a purified recombinant dub and cleavage of the diub is monitored over time. biggest drawback of this method is that it is not compatible with complex biological settings (such as cell lysate), and it is limited to isolated dubs. to overcome such issues, a second generation of probes to investigate linkage-specific proteolysis of dubs has emerged more recently. these probes generally consist of two ubiquitin moieties carrying a michael acceptor element in the isopeptide linkage region in-between the two ub moieties. initial reports show the two ub regions to be linked together using non-native connections such as a triazole (mcgouran joanna et al. ) and thiol ether linkage (li et al. ) . two type of probes presenting either a dehydroalanine (dha) (haj-yahya et al. ) or vme-like electrophilic trap (mulder et al. ) mimic the native lysine-glycine linkage the closest using amide linkages. a panel of all seven isopeptide-linked diub probes can be constructed and used to covalently capture the active site cysteine of the dub showing its reactivity and preference toward certain linkage types. using such probes the n-terminal or distal ub molecule will be positioned before the active site in the so-called s -pocket, and the c-terminal or proximal ub molecule will be positioned after the active site cysteine in the so-called s ′-pocket. due to the geometrical differences between al lys-linked diub probes, the dub will only be able to position the probes mimicking its natural substrates in such a way that the active site cysteine is able to react with the reactive element (see fig. b ). some dubs are able to recognize ub chain topologies using other binding surfaces further away from the active site, such as, for instance, an s -site preceding the s -site. a third generation of probes targeting such s binding sites has been developed where a diub molecule is equipped with a reactive element at the proximal c-terminus (flierman et al. ). these probes are only able to react with dubs that contain a s -site that plays a determining role in positioning the diub molecule in the s -and s -sites thereby placing the alkyne directly over the active site cysteine (see fig. b ). noteworthy is that the isopeptide linkage between proximal and distal ub has been replaced by a protease stable triazole linkage, prohibiting the protease of interest to degrade the probe during assays. if a dub recognizes such a third generation diub probe using its s -and s ′sites, the alkyne will not be in the vicinity of the active site cysteine and no covalent adduct will be formed. conversely, if a second generation probe will be reacted with a dub recognizing the diub moiety using it's s -and s -sites, no reaction will occur since the reactive element will not be aligned with the reactive cysteine (see fig. c ). having access to both second and third generation probes offers an exciting combination to investigate the binding interfaces that play a role in determining binding preferences of dubs and cast a light on their molecular mechanism of action. more recently a new probe able to reversibly capture cysteine dubs followed by the release of the still active proteases has been published, making use of subtle chemistry to form, and disrupt disulfide bridges between the active site cysteine and ub-based thiol-containing probe (de jong et al. ) . although only the proof of principal studies has been performed, this novel technology holds great promise for the future capture, release, and follow-up investigations of native active cysteine dubs. not all dubs are cysteine proteases, however, and the metallo-dub family currently is understudied due to the lack of probes. selective probes targeting such proteases are needed and will no doubtably be developed in the future. the covalent capture of active dubs with ub-based activity-based probes allows for purification of the formed complexes and subsequent crystallization efforts to study the interactions between protease and ub in detail. a large number of crystal structures has been solved using such ub-based abps as is reviewed by van tilburg et al. ( ) . synthetic procedures to prepare above-mentioned probes and reagents on large scale are in place and are finding their way into the related fields of ub-like proteins such as, for instance, sumo biology (mulder et al. ). due to the diversity and complexity in the conjugating and ligation machinery, development of probes targeting the constructing of ubiquitinated substrates has only emerged more recently than probes targeting the deconstructing proteases, for the simple reason that targeting a sequential enzymatic cascade is more difficult than targeting a single proteolytic step. the first step in the cascade is activation of the c-terminal carboxylate of ub by and e -activating enzyme. the e firstly adenylates the carboxylic acid group of glycine at the expensive of atp in one region of the e and subsequently the catalytic cysteine of the e in another region of the enzyme takes over the ub cargo by forming a thioester and expelling amp. initial probes based on ub -cgg-vinyl sulfonamides show reactivity toward the cysteine of yeast e uba , although the c-terminal arg-leu-arg-gly-gly region of ub is replaced by the smaller cys-gly-gly vinyl adenyl sulfonamide, rendering a shorter ub mutant with a rl deletion (see fig. a ) (lu et al. ) . these ub probes are based on small molecule adenyl sulfonamide analogues that can also be used to monitor intracellular e activity and specific inhibitory potential toward different e 's (an and statsyuk ; misra et al. ) . other probes based on ub -dehydroalanine adenyl amides also show reactivity toward a. o. uba although these probes are longer than the native ub substrate . combined this data implies that unlike the active sites of dubs the e 's are more tolerant toward their substrates or the flexibility of the c-terminal tail of ub corrects for possible misalignment during the ub transfer from the atp binding domain to the catalytic cysteine domain of the e . these adenylate mimic abps are important to study e 's but in view of the bigger picture are quite restricted. due to the stabilized adenylate (either amide or sulfonamide) linkage, these probes lack the ability to be transferred down the enzymatic cascade, being stuck in the first stage. the second step in the cascade is transfer of the ub cargo from e to e , a processes that can be trapped and studied using a e based-abp (stanley et al. ) . recombinant expression of an e and modification with an tosyl-substituted double-activated ene-reagent (tdae) forms an electron poor activated vinyl sulfide that upon juxta-positioning of the e 's cysteine is able to form a stable bis thioether e -e complex (see fig. b ). in an analogues approach, the third step in the fig. activity-based probes to capture a e -enzymes, b e -e interactions or ub-e -e interactions, c ub-e , ub-e , and ub-e interactions cascade, ub transfer from e to e can be interrogated. a ubiquitin-charged e carrying a tdae element allows for trapping the trans-thiolation event toward an e , forming a stable complex between ub, e , and e (see fig. b ) (pao et al. ) . of note is that in this last tdae derived probe, the c-terminal rgg motif of ub is replaced by the reactive tdae element, which might limit the generality of such probes as it is implicated that r and the digly motif can play an important role of recognition of the appropriate ubl. all approaches mentioned above are useful in specifically studying one step in the cascade of ub-ligation. to amend the need to be able to pass down the probe through the complete cascade and target e 's, e 's, and e 's; simultaneously, a 'cascading'-probe based on a c-terminal dehydroalanine (dha) moiety was developed (mulder et al. ). this reactive element has been employed earlier in diub probes targeting cysteine dubs (haj-yahya et al. ), but was found in other studies to show low reactivity. this low reactivity can be harnessed in trapping the e -e -e enzymatic cascade consecutively (see fig. c ). the ub dha can be activated by an e to form a thioester intermediate and transferred to an e and subsequent rbr-or hect-e via a trans-thiolation reaction. in each step, only a small portion of the active cysteines will be trapped by the dha-moiety, while the rest of the cysteine will continue to function normal in the trans-thiolation step. by doing so, the probe will capture all components in the cascade, which has been shown to indeed be detectable using proteomic approaches in a proof of principle study. the above-mentioned arsenal of probes has emerged very recently, and most likely the near future holds exciting insights in the ubiquitination cascade through implementation of these probes. complicating factor in the investigation of e -ligases is that of the three major classes of e 's only the hect and rbr classes have an active cysteine residue that takes over the ub cargo from an e and transfer it to the final substrate protein. ring e s do not possess such an active site cysteine and merely serve as platforms to bring ub charged e 's and substrates together, thereby making them unsuited for direct probing using abps. other type of probes or assay reagents based on a ub scaffold have also been developed allowing the real-time monitoring of dub or ligase activity. in contrast to the probes described above, these reagents do not have a michael acceptor element and thus do not form a covalent complex with the targeted enzyme, but mostly rely on a fluorescent signal that is altered. advantage of the probes described below is that one enzyme can perform multiple catalytic cycles, and hence, signal amplification occurs allowing a more accurate readout of the enzymes native activity and/or specificity. an important class of ub-based assay reagents is the fluorogenic ub reagents, where a quenched fluorophore is conjugated at the c-terminus of ub. dub activity will cleave the amide bond at position , and the fluorophore will be released from the ub and simultaneously start to fluoresce (see fig. a ). hence, the increase in fluorescence is a direct measure of dub activity. fluorogenic reporters used in such type of reagents are aminomethylcoumarin (amc) (dang et al. ) or substituted rhodamine- (rho) (hassiepen et al. ) scaffolds that show favorable fluorescent properties. in a similar setup, dub mediated aminoluciferin release can be assayed in a bioluminescence approach using a luciferase assay (orcutt et al. ). all of these reporters are conjugated to a mono-ub recognition element, and hence, the preference of a dub to specific poly-ub topologies cannot be assessed. to allow monitoring chain-specific proteolysis mediated by s -s interactions on the dub, diub-amc substrates were generated (flierman et al. ) . in analogy to the diub-prg covalent probes, these diub-amc substrates are linked via a non-hydrolysable triazole linker preventing proteolysis of the diub entity during the assay. one important note is that the fluorogenic substrates do not contain an isopeptide linkage at the side where the dub would perform its proteolytic action, whereas the natural substrates for most dubs would. to mimic this isopeptide link more closely, fluorescent polarization (fp) reagents were developed where ub is conjugated via a native isopeptide linkage to a fluorophore carrying substrate derived peptide (tirat et al. ; geurink et al. ) . rationale behind these reagents is that once the fluorophore-containing peptide is linked to ub, the large construct tumbles slowly in solution and light remains polarized, whereas after proteolysis the small fluorophore-containing peptide tumbles faster and hence the polarization of light decreases (see fig. b ). such tools are not only reported for ub but also for other ubl's such as the three sumo's, nedd , and isg . another type of reagent makes use of the fact that a fluorophore and quenching moiety can be placed in close proximity of each other on a diub or ub-substrate peptide and upon dub action both entities are separated and hence fluorescence is restored (see fig. c ). probes based on such a time-resolved fluorescence resonance energy transfer (fret) principle consisting of ub-peptide (ohayon et al. ) and diub moieties ye et al. ) have been described. all seven lysine-linked diub fret pairs are available which form a platform to measure dub preference and kinetic parameters in real time. using similar technology, the e mediated constructing of poly-ub chains equipped with a fret acceptor onto a fret donor-containing ub results in a fret signal (madiraju et al. ) . in this particular study, inhibitors for the e can be identified when a decrease in fret signal is observed in the assay. although it was long assumed that in order to ubiquitinate the target substrate the complete cascade, so e , e , and e functions are needed, recent reports show that ub-thioesters can be used in in vitro studies to monitor hect-or rbr-e ligase activity. in these assays, the e -and e -functions are bypassed and ub-thioesters [ub-mes (park et al. ) or fluorescent analogue ub-fluor (krist et al. ) ] are directly trans-thiolated by the active cysteines in several e 's. the fluorescent thioester allows real-time quantitative monitoring of e -ligase activity using a fluorescence polarization set up (park et al. ) (see fig. d ). all probes mentioned above are based on mono-ubiquitinated peptides, mono-ub or diub carrying a reactive or fluorescent entity. moving forward in our understanding of the ub system, it would be of great value to have access to well defined larger ub chains and probes made thereof. synthetic, semi-synthetic and biochemical methods are in place to produce poly-ub chains, carrying native isopeptide linkages or artificial (non-hydrolysable) linkages. biochemical methodology to obtain all except k -poly-ub chains, are reported and rely on the use of linkage-specific e /e enzyme combinations as has been reviewed elsewhere (faggiano et al. ) (see fig. a ). although the majority of poly-ub chains can be produced, several drawbacks come with such methods. crucial in biochemical production of ub chains is that in order to have a defined polymer length, the mixture of simultaneously produced poly-ub's needs to be separated, most frequently done using cation exchange chromatography. inherent in producing polymers is that the isolated yield of one specific poly-ub species is low and scalability of biochemical methodology hence can be an issue. some of the e / e combinations are not absolute topology specific and additional treatment using dubs might be necessary to guarantee a homogenous preparation, further complicating procedures. all in all six out of seven lysine-linked poly-ub chains can be prepared biochemically although preparation of high quantities of pure material still is a laborious task. on the other hand, the total chemical synthesis of poly-ub chains has been undertaken. several methods using auxiliaries, different thiol modified amino acids and ligation/desulfurization strategies are in place as reviewed elsewhere , allowing for the sequential construction of ub chains with the impressive highlight of k -linked tetra-ub (kumar et al. ) . although for such time-consuming approaches a high level of expertise is required, it does allow the introduction of fluorophores, affinity handles or conjugation to substrate peptides or proteins. of note is that using an isoub native chemical ligation approach also k tetra-ub chains and mixed chains have been prepared and employed in structural studies (tang et al. ) . in contrast to sequential strategies, chemical polymerization approaches using a bifunctional thiolysine/thioester-ub mutants (van der heden van noort et al. ; moyal et al. ) (see fig. b ) leading to native isopeptide-linked ub chains, bifunctional thiol/ene ub mutants leading to thioether-linked ub chains (trang et al. ) (see fig. c ) or azide/alkyne-ub mutants leading to triazole-linked ub chains (schneider et al. ) (see fig. d ) give rise to larger poly-ub analogues including the biochemical unavailable k -linked ub chains. such stable poly-ub chains can be employed to study the interacting proteins or 'readers' of the ub code by performing pull-down experiments and subsequent proteomic analysis using mass spectrometry approaches (zhao et al. ; zhang et al. ). fig. poly-ub generation using a the native e -e -e cascade, b a native chemical ligation approach, c a thiol-ene radical coupling approach, and d a copper-catalyzed huisgen cycloaddition approach the above-described arsenal of ubiquitin-based probes, substrates, chains, and analogues are each designed to target-specific enzymatic functions or give insight into unanswered question regarding the ubiquitin-proteasome system. apart from the proof of principle studies conducted during the development of these probes, it is important to note that these tools have made the translational step from the drawing board to actual biochemical and structural studies on viral and bacterial enzymes playing a role in the manipulation of (human) host pathways. viruses have defense mechanism in place to either utilize the host's ubiquitin system in their own advantage or to combat the host's immune response by suppressing pro-inflammatory ub signaling. human coronaviruses (hcov), responsible for pandemic outbreaks of severe acute respiratory syndrome (sars) in and middle east respiratory syndrome (mers) in , are two of such viruses that are interfering with the host's ub signaling. one way these coronaviruses act is by dampening the immune response through action of viral proteases that possess deubiquitinase and deisgylase functions. interferon stimulated gene (isg ) is a ubiquitin-like (ubl) modification that has two ub domains in tandem and is upregulated and attached to substrates during an antiviral response. the papain-like proteases (plpro's) in both mers and sars hcov are identified to cleave isg and ub from cellular proteins. in a study comparing mers and sars plpro, it was found that fluorogenic substrates ub-amc and isg -amc were both cleaved to a similar extent by mers plpro, where sars plpro preferred isg -amc (békés et al. ) . covalent capture of these proteases by abp ub-propargyl again showed a higher reactivity for the mers then for the sars protease. cleavage assays using native k -linked tetra-ub also show a distinct pattern. sars plpro cleaves the ub into ub and is hardly able to process ub any further, whereas mers plpro cleaves ub into a mix of ub , ub , and ub. sars plpro seems to have a di-distributive mechanism only making a cut after a ub moiety, whereas mers is mono-distributive cleaving after one ub residue. diub abp's having the reactive element either at the proximal side of the ub or in-between the ub moieties help to explain how these two different mechanism function, as mers plpro is able to react with the in-between probe and not the proximal probe, and sars plpro shows opposite reactivity toward these two probes (bekes et al. ) . hence, activity of sars plpro seems to be largely influenced by an s site, whereas mers plpro does not seem to contain such a site. a crystal structure of the complex between the s -s targeting diub abp and sars plpro allows identification of the crucial contacts in the s -site of the dub and the diub abp. all in all these hcov proteases, although homologous, are shown to have a different mode of action using the three generations of activity-based probes (discussed in sect. . ) and accompanying kinetic parameters could be deduced using fluorogenic mono-and diub-amc substrates (discussed in sect. . ). similar to viral interference with host immune response, bacteria also have sophisticated mechanisms in place to counteract host immune responses and create optimal conditions for the bacterium to survive and promote replication. bacterial effector proteins typically are directly secreted into the host cells to interfere with host kinase activities or ub(l) signaling pathways. interestingly, those bacteria do not have ub or ubl systems themselves and such effector proteins are only in place to promote survival within the host organism. much of these effector proteases fall in the class of ce clan effector enzymes, which in humans consists of sumo-and nedd -specific proteases. bacterial ce clan proteases do not only show activity toward ubl's but some members also show acetyltransferase activity. in a comparative study on such ce clan effectors from intracellular pathogens salmonella typhimurium, chlamydia trachomatis, escherichia coli, yersinia pestis, rickettsia bellii, shigella flexneri, and legionella pneumophila, activity, specificity, and structure were investigated (pruneda jonathan et al. ) . although all share a common fold most but not all of them were able to react with abp ub-prg. using ub-, sumo -, nedd -and isg -fp substrates (discussed in sect. . ), the three effectors unreactive toward ub-prg were also shown to be unreactive toward any of the fp-reagents whereas the others showed reactivity toward both ub and nedd reagents. in contrast to their unreactivity toward ubl's, the three ce effectors did show acyl transferase activity. using diub substrates, the panel of bacterial dubs was all shown to have a strong preference for k -linked chains, followed by k and k at later time points or higher enzyme concentrations. this specificity mostly was shown to be regulated by the s ′-site of these dubs. taken together, this study uses both mutational analyses, crystal structure information, and data obtained by ub(l)-based probes and assay reagents to classify and study the molecular details of a bacterial class of effector proteins in detail. we thought we understood the basis of the ub system, but do we actually? several enzymes have been recently identified to control the ub system by attaching adenosine diphosphate ribose (adpr) onto specific positions in ub, exerting a new layer of control. the intracellular pathogenic bacterium l. pneumophila, the cause of legionnaires disease, releases effector proteins into its host to hijack the host ub pool (bhogaraju et al. ). these multi-domain side family proteins catalyze the ubiquitination of substrates proteins independent of the e -e -e cascade and without the use of energy source atp. instead they use nad + (see fig. structure ii) to attach adpr on the nucleophilic side chain of arginine of ub using their mono adpr transferase (mart)-domain (see fig. -structure i (qiu et al. ). subsequently, the phosphodiesterase (pde) domain within side catalyzes the reaction between a serine-containing substrate (see fig. -structure iv) and the adp-ribosylated ub (see fig. -structure iii), affording a ribosyl phosphate linkage between ub and the target protein (see fig. -structure v) (kotewicz et al. ) . besides a mart and pde domain side family members also contain a n-terminal canonical dub domain that is not necessary for bacterial replication, but is crucial in regulating the extend of ubiquitination at the bacterial vacuolar surface. cleavage assays using diub substrates show a preference of k over k and k , and crystallization efforts of the formed complex with abp ub-vme reveal a different binding modus, not involving the i patch on ub, of the cysteine dub and ub (sheedlo et al. ) . besides the multi-domain multi-function side's, legionella also expresses a deubiquitinase sidj that is able to cleave the formed phosphodiester linkage to ub, liberating the substrate protein and phosphoribose modified ubiquitin (see fig. -structure vi) ). this phosphoribose-ub also interferes with the host ub machinery as it prevents the canonical e to activate and further process the ub molecule . by controlling the construction and deconstruction of modified ub, without competing with the canonical system, the bacterium tries to hijack the host ub system. lots of questions on the mechanism of action, substrate recognition, and scope of this e -e -e independent ubiquitination and attached deubiquitination process call for the development of novel probes specifically targeting such pathways. so far the development of adp-ribosylated ub carrying a protease stable triazole linkage between the ribose and arg of ubiquitin has been shown to reflect the native auto-ubiquitination activity of side family member sdea (liu et al. ) . no doubtably probes targeting this interesting new way of ub conjugation to substrates will appear in the near future. the complexity of the ubiquitin system and its involvement in a wide variety of important biological processes makes it a widely and intensively studied field. the roles ub play in neurodegenerative disease and cancers, for instance, make it a potential target for therapeutic intervention. unraveling the complexity of this, highly sophisticated system is aided greatly by the development of (activity-based) probes reporting on the dynamics and structural mechanisms used by activating enzymes, ligases, and proteases counteracting the buildup of (poly)-ubiquitinated substrates. ranging from probes that covalently trap the active cysteines involved in catalysis to probes that have altered fluorescent properties upon enzyme activity and stabilized substrates or poly-ub chains allowing preference studies and profiling of the ub interactome have been developed over the past decade or so and already have shed their light on some fundamental question in ub biology. the more knowledge is gathered on the functioning of those (de)ubiquitinating proteins the more tailored solutions to interrogate their biology, and hence, specific probes involved in such approaches become. without a doubt will the next generation of ub-based tools help to increase our knowledge on the system and perhaps ultimately lead to diagnostic tools or therapeutics making it to the clinic. development of activity-based probes for ubiquitin and ubiquitin-like protein signaling pathways facile synthesis of covalent probes to capture enzymatic intermediates during e enzyme catalysis sars hcov papain-like protease is a unique lys linkage-specific di-distributive deubiquitinating enzyme recognition of lys -linked di-ubiquitin and deubiquitinating activities of the sars coronavirus papain-like protease phosphoribosylation of ubiquitin promotes serine ubiquitination and impairs conventional ubiquitination a novel active site-directed probe specific for deubiquitylating enzymes reveals proteasome association of usp chemistry-based functional proteomics reveals novel members of the deubiquitinating enzyme family structural insights into the catalysis and regulation of e ubiquitin ligases kinetic and mechanistic studies on the hydrolysis of ubiquitin c-terminal -amido- -methylcoumarin by deubiquitinating enzymes ubiquitin-based probes prepared by total synthesis to profile the activity of deubiquitinating enzymes release of enzymatically active deubiquitinating enzymes upon reversible capture by disulfide ubiquitin reagents on terminal alkynes that can react with active-site cysteine nucleophiles in proteases chemical synthesis of ubiquitin, ubiquitin-based probes, and diubiquitin the missing links to link ubiquitin: methods for the enzymatic production of polyubiquitin chains non-hydrolyzable diubiquitin probes reveal linkage-specific reactivity of deubiquitylating enzymes mediated by s pockets a general chemical ligation approach towards isopeptide-linked ubiquitin and ubiquitin-like assay reagents development of diubiquitin-based fret probes to quantify ubiquitin linkage specificity of deubiquitinating enzymes dehydroalanine-based diubiquitin activity probes a sensitive fluorescence intensity assay for deubiquitinating proteases using ubiquitin-rhodamine -glycine as substrate a single legionella effector catalyzes a multistep ubiquitination pathway to rearrange tubular endoplasmic reticulum for replication ubfluor: a mechanism-based probe for hect e ligases highly efficient and chemoselective peptide ubiquitylation total chemical synthesis of a amino acid k -linked tetraubiquitin protein editing of ubiquitin conjugates by an isopeptidase in the s proteasome activity-based diubiquitin probes for elucidating the linkage specificity of deubiquitinating enzymes a general approach towards triazole-linked adenosine diphosphate ribosylated peptides and proteins designed semisynthetic protein inhibitors of ub/ubl e activating enzymes tr-fret-based high-throughput screening assay for identification of ubc inhibitors deubiquitinating enzyme specificity for ubiquitin chain topology profiled by di-ubiquitin activity probes dissecting the specificity of adenosyl sulfamate inhibitors targeting the ubiquitin-activating enzyme polymerization behavior of a bifunctional ubiquitin monomer as a function of the nucleophile site and folding conditions a native chemical ligation handle that enables the synthesis of advanced activity-based probes: diubiquitin as a case study a cascading activity-based probe sequentially targets e -e -e ubiquitin enzymes total chemical synthesis of sumo and sumo-based probes for profiling the activity of sumo-specific proteases targeting deubiquitinases enabled by chemical synthesis of proteins bioluminescence assay platform for selective and sensitive detection of ub/ubl proteases probes of ubiquitin e ligases enable systematic dissection of parkin activation protein ubiquitination and formation of polyubiquitin chains without atp, e and e enzymes ubmes and ubfluor: novel probes for ring-between-ring (rbr) e ubiquitin ligase parkin synthesis of -mercapto-l-lysine derivatives: potential building blocks for sequential native chemical ligation mechanism of ubiquitin carboxyl-terminal hydrolase. borohydride and hydroxylamine inactivate in the presence of ubiquitin the molecular basis for ubiquitin and ubiquitin-like specificities in bacterial effector proteases ubiquitin chains modified by the bacterial ligase sdea are protected from deubiquitinase hydrolysis ubiquitination independent of e and e enzymes by bacterial effectors a unique deubiquitinase that deconjugates phosphoribosyl-linked protein ubiquitination dissecting ubiquitin signaling with linkage-defined and protease resistant ubiquitin chains structural basis of substrate recognition by a bacterial deubiquitinase important for dynamics of phagosome ubiquitination chemistry and biology of the ubiquitin signal orthogonal thiol functionalization at a single atomic center for profiling transthiolation activity of e activating enzymes e enzymes: more than just middle men practical chemical synthesis of atypical ubiquitin chains by using an isopeptide-linked ub isomer synthesis and characterization of fluorescent ubiquitin derivatives as highly sensitive substrates for the deubiquitinating enzymes uch-l and usp- nonenzymatic polymerization of ubiquitin: single-step synthesis and isolation of discrete ubiquitin oligomers synthesis of poly-ubiquitin chains using a bifunctional ubiquitin monomer synthetic and semi-synthetic strategies to study ubiquitin signaling ubiquitin chain conformation regulates recognition and activity of interacting proteins an interaction landscape of ubiquitin signaling identification of proteins interacting with ubiquitin chains key: cord- -b vg ap authors: bonelli, f.; turini, l.; sarri, g.; serra, a.; buccioni, a.; mele, m. title: oral administration of chestnut tannins to reduce the duration of neonatal calf diarrhea date: - - journal: bmc vet res doi: . /s - - - sha: doc_id: cord_uid: b vg ap background: neonatal calf diarrhea is generally caused by infectious agents and is a very common disease in bovine practice, leading to substantial economic losses. tannins are known for their astringent and anti-inflammatory properties in the gastro-enteric tract. the aim of this study was to evaluate the effect of the oral administration of chestnut tannins (castanea sativa mill.) in order to reduce the duration of calf neonatal diarrhea. twenty-four italian friesian calves affected by neonatal diarrhea were included. the duration of the diarrheic episode (dde) was recorded and the animals were divided into a control group (c), which received effydral® in l of warm water, and a tannin-treated group (t), which received effydral® in l of warm water plus g of extract of chestnut tannins powder. a mann-whitney test was performed to verify differences for the dde values between the two groups. results: the dde was significantly higher in group c than in group t (p = . ), resulting in . ± . and . ± . days, respectively. conclusions: phytotherapic treatments for various diseases have become more common both in human and in veterinary medicine, in order to reduce the presence of antibiotic molecules in the food chain and in the environment. administration of tannins in calves with diarrhea seemed to shorten the dde in t by almost days compared to c, suggesting an effective astringent action of chestnut tannins in the calf, as already reported in humans. the use of chestnut tannins in calves could represent an effective, low-impact treatment for neonatal diarrhea. calf diarrhea is a very common disease in bovine practice, and is generally caused by infectious agents [ ] . the most common pathogens involved are rotavirus, coronavirus, cryptosporidium parvum and escherichia coli, especially in animals less than one month old [ , ] . calf diarrhea causes substantial economic losses in the dairy industry, due treatment costs, a decreased growth rate in the calves, and a higher replacement rate due to culling or death [ ] . in order to reduce the presence of antibiotic molecules in the food chain and in the environment, phytotherapic treatments are now commonly used both in human [ ] and in veterinary medicine. various formulations of polyphenols, such as bacteriostatic, anti-clostridial and astringent, have been tested, especially in monogastric nutrition [ ] [ ] [ ] [ ] . some phytotherapic options have been evaluated for gastrointestinal diseases, such as gastric ulcers and gastric lesions, in rats, piglets, horses, and calves [ ] [ ] [ ] . pomegranate-residue supplements have been used in neonatal calves affected by cryptosporidium parvum, resulting in a reduction in the fecal oocyst count, as well as in intensity and duration of diarrhea [ ] . tannins are a complex group of polyphenolic compounds which are present in several plants as secondary metabolites against pathogens [ ] . in ruminants, in vitro and in vivo trials have demonstrated that tannins can improve animal performance and reduce the impact of gastrointestinal parasitism, nitrogen pollution, and methane emission from rumen fermentation [ , ] . tannins may interfere with digestive processes by binding dietary protein, by modulating the activity of rumen micro-organisms, and by reducing the growth of the bacterial population [ ] [ ] [ ] . in ruminants, tannins tend to decrease the rate of protein degradation in the rumen [ ] . tannins, in fact, inhibit the growth of proteolytic bacteria and form tannin-protein complexes in the rumen. this effect may significantly vary according to the chemical structure of the tannin and to the amount of tannin included in the diet. however, the reduction of protein degradation in the rumen increases the total amount of protein digested in the duodenum, because the tannin-protein complexes are dissociated in the abomasum, releasing protein. as a consequence, the use of moderate doses of tannins in the diet of ruminants usually improves animal performance [ , ] . the astringent and anti-inflammatory effects of tannins on the gastro-enteric tract have been demonstrated in avian [ , ] and swine species [ ] , and the effects of tannins on liver function have been evaluated in newborn calves [ ] . the aim of the present study was to evaluate the effect of oral administration of chestnut tannins (castanea sativa) in the treatment of calf neonatal diarrhea. this in vivo blinded study was approved by the institutional animal care and use committee (oba, pisa, prot. n. , / of . . ) and was carried out at the university of pisa's dairy farm, where nearly animals are maintained in free-stall conditions. during the study period, the population consisted of italian friesian calves aged between and days. all the calves underwent the same management condition. briefly, immediately after birth they were weighed and then housed in a single straw-bedding pen ( . × m) that leads contact between each other. two l of good colostrum (≥ g/l of ig) from their own dam, or from the colostrum bank, were administered as soon as the calf could drink ( min - h). another l were administered within the next - h in order to achieve a good passive transfer immunity [ ] . all the calves received a total of l of colostrum, twice a day, until the third day of life. then, they received l of whole milk at °c, twice a day until the third week of life. all the feeding procedures were conducted by an expert operator and by using a nipple bucket. even when there were cases of diarrhea, there were still no feeding restrictions or changes of feeding regime. from the third day of life, fresh and clean water were provided to each calf ad libitum. free choice-hay was administered after the first week of life. the calves were weighed and moved into a collective pen after the third week of life. the inclusion criteria were calves up to three weeks of life and the manifestation of diarrhea, defined as a fecal score ≥ [ ] . briefly, the fecal score represents the evaluation of feces fluidity as reported in literature: score means "normal", thus firm, but not hard and the original form is distorted slightly after dropping to the floor and settling; score means "soft", thus does not hold its form, but piles and spreads slightly (like soft-serve ice cream); score means "runny", thus spreads readily to about mm depth. (i.e., pancake batter); score means "watery", thus liquid consistency, splatters. (i.e., orange juice) [ ] . since the first day of diarrhea (t ), the fecal score (fs) was recorded daily by the same expert operator until the complete recovery from diarrhea, at the same time, the physical examination has been done. the duration of a diarrheic episode (dde) was defined as the period (in days) between the first diarrhea outbreak (fecal score ≥ ) and the normalizing of the fs (fecal score = ). once the diarrhea had started, a fecal sample was collected from each calf by manual restraint. a gloved, lubricated finger was passed gently through the anus in order to massage the rectal wall and to stimulate rectal evacuation. fresh feces were then collected in two different sterile tubes: one aliquot was immediately tested with a rapid elisa test (test strips for detection of rotavirus, coronavirus, e. coli f and c. parvum in bovine feces, biox diagnostics, belgium), while the second aliquot was stored in a refrigerated bag and evaluated within one hour for gastrointestinal parasites, according to izzo et al. ( ) [ ] . at the inclusion time, calves were randomly assigned to a control group (c) or a tannin-treated group (t), both made up of calves ( males and females in each of the two groups). all the calves enrolled in group c received effydral® (italy zoetis ltd.) (sodium chloride . g, potassium chloride . g, sodium bicarbonate . g, citric acid anhydrous . g, lactose monohydrate . g, glycine . g) in l of warm water q h. the calves assigned to group t received effydral® (italy zoetis ltd.) in l of warm water plus g of chestnut tannins as extract powder ( g/kg of dry matter equivalent of tannic acid; mauro saviola group srl, radicofani, siena, italy) q h. the chemical composition of the powder is described in campo et al. ( ) [ ] . the powder adopted in the present study was produced in a single batch and analyzed by the manufacturer at the beginning of the study. both solutions were administered using a graduated calves bottle in order to ensure the intake of the entire quantity. the bottle was equipped with a flexible rubber nipple ( cm of length) specific for calf feeding. calves in both groups received the solutions until the normalization of the fs. before the administration of the treatments, all the calves were daily submitted to a complete physical examination and fs evaluation until the resolution of diarrhea. physical examination included evaluation from a distance plus hands-on examination, focusing on: physical appearance, body weight, body condition, head, mouth, eyes, ears, neck and back, thorax, abdomen, umbilicus, musculoskeletal system, perianal region, body temperature, feces, urine, and external genitalia [ , ] . dehydration status was assessed with a score system [ ] . milk intake was recorded during the entire diarrheic episode. at the conclusion of the study, the female calves were raised in the farm as rearing cows, while the male calves were sold for fattening as meat animals. data concerning the weight at birth and at the third week of life, the age of diarrhea onset and the t fecal scores (t -fs) recorded for both groups were assessed for normal distribution by the shapiro-wilk normality test and then a mann-whitney test was applied in order to verify differences between the two groups at the inclusion time [ ] . the average daily gain between birth date and third week of life was calculated for all the calves in both groups. data concerning the average daily gain, the dde and fecal scores recorded throughout the dde were analyzed by a linear model including treatments and sex and their interaction as fixed factors. a shapiro-wilk normality test was performed to assess data distribution. a mann-whitney test was carried out to verify differences between the two groups regarding the average daily gain, the dde and the fecal scoring [ ] . values with p < . were considered statistically significant. twenty-four out of the calves, males and females met the inclusion criteria, and were thus included in this study. the average weight at birth was kg (minimum value of . kg and maximum value of kg) and . kg (minimum value of kg and maximum value of . kg) for groups c and t, respectively. the mean age at t was . ± . days for group c, and . ± . days for group t. the mean t -fs were . ± . and . ± . for groups c and t, respectively. no significant differences were found in terms of the weight at birth, the age of diarrhea onset, and the t -fs between the two groups (p > . ). there were no alterations at the physical examination, and the hydration status was normal for all calves during the entire study period. no differences between groups were found for milk intake. thus, no pharmacological treatments or fluid therapy were needed. seven out of twelve ( / ) calves belonging to group c resulted positive for cryptosporidium parvum, / for rotavirus and coronavirus, and / negative. nine/ calves belonging to group t were positive to cryptosporidium parvum, / for rotavirus and coronavirus, and / were negative to the rapid elisa test performed. fecal flotation did not show intestinal parasites. the average weight at the third week of life was kg (minimum value of kg and maximum value of kg) and . kg (minimum value of kg and maximum value of . kg) for groups c and t, respectively. the average daily gain was . kg/day (minimum value of . kg/day and maximum value of . kg/day), and . kg/day (minimum value of . kg/day and maximum value of . kg/day), for groups c and t, respectively. differences between the two groups were not significant. the mean dde in days was . ± . for group c, and . ± . for group t ( table ) . a significant difference between the two groups was found (p = . ). the mean fecal score recorded throughout the dde for group c was . ± . , while for group t the score was . ± . , with a statistically significant difference between the two groups (p = . ). no statistically significant differences were found between male and female calves for the dde and fecal score recorded throughout the dde. no calves refused the tannins solution. due to the high amount of antimicrobials used every year in the livestock industry and possible cross-resistance between human and animal pathogens [ , ] , for many years alternative treatments have been investigated [ ] . chestnut tannins have been proposed to modulate rumen fermentation and the degradability of food proteins in cattle [ , ] . however, there is little information on the effects of chestnut tannins on diarrhea in neonatal calves, especially in relation to different pathogens. the pathogen that was most isolated in our population of diarrheic calves was cryptosporidium parvum, in line with those reported in the literature [ , , ] . the mean age of diarrhea onset for all calves included was also in line with literature data [ , ] . it is well known that natural plant tannins are able to control intestinal parasites in ruminants [ ] and, more table duration of the diarrheic episode (dde), fecal score recorded throughout the dde of control group (c) and tannintreated group (t) expressed as mean (x) ± standard deviation (sd) in general, have antimicrobial properties [ ] . the supplementation of chestnut extract in grazing heifers has been shown to lead to a significant increase in the average daily gain due to a decrease in fecal parasites infections, above all nematodes [ ] . several mechanisms of actions have been proposed for tannins, including enzyme inhibition and substrate or metal ions deprivation, and bacterial cell membrane integrity [ ] . however, most studies have reported the effects of tannins (above all condensed tannins) on bacteria, fungi and nematode, whereas only few have investigated the effect of tannins on protozoa and, in particular, on cryptosporidium parvum [ , ] . the anti-protozoal activity of polyphenols has been reported for eimeria [ ] [ ] [ ] . a suggested mechanism is the ability of tannins to directly decrease the viability of the larval stage and disrupt egg hatching, as previously observed also for nematodes [ ] . however, there have been conflicting results. bhatta et al. ( ) found a decrease in protozoa when hydrolysable tannins from six different plant sources were applied [ ] . carulla et al. ( ) who reported that condensed tannins from leucaena, quebracho and from acacia mearnsii respectively, can decrease protozoal numbers [ ] [ ] [ ] . in contrast, vasta et al. ( ) found that quebracho tannins were able to increase protozoa in rumen liquor [ ] . differences in the concentration of tannins, plant sources, protozoal species and environment considered in such studies may explain the conflicting results obtained, because these parameters play an important role in the antiprotozoal activity of polyphenols. to the best of our knowledge this is the first study that has investigated orally administrating tannins as a treatment for calf diarrhea. tannins solution appeared to be pleasant for all the calves enrolled in the study and was easy to administer. the length of diarrhea was almost four days shorter in the group treated with tannins compared to the control group. chestnut tannins seem to shorten the length of diarrhea in calves, as already reported in humans [ ] . chestnut polyphenols are ellagitannins and those from wood distillation are particularly rich in ellagic acid, such as pomegranate polyphenols [ ] . few studies have reported on the absorption and metabolism of ellagitannins in animal models. studies of rat intestinal content demonstrated that at the caecum level ellagitannins are hydrolyzed to ellagic acid [ ] . other authors have detected free ellagic acid in human plasma after h post ingestion of pomegranate and attributed this to the release after hydrolysis of ellagitannins according to an optimal physiological ph and gut microbiota activity. in addition, cerdà et al. ( ; ; ) suggested a microbial involvement at the colon level in converting ellagic acid into urolithins, which are potent anti-inflammatory metabolites [ ] [ ] [ ] . although the length of diarrhea was significantly shorter in calves from group t, no significant difference was found in terms of average daily gain between control calves and calves that received chestnut tannins. similarly, a study based on tannins from pomegranate reported no effects of tannins on average daily gain in the first days of life in dairy calves [ ] . further studies are needed in order to investigate the possible effect of hydrolysable tannins on specific etiological pathogens, in particular on cryptosporidium parvum, as already reported for the extract of pomegranate polyphenols [ ] . also, increasing the number of animals included might clarify the effect of chestnut tannins on the average daily gain in calves affected by diarrhea. chestnut tannins might represent a low-impact treatment of neonatal diarrhea in calves. however, the effects of chestnut tannins on the onset of diarrhea and on the weight gain of calves need further investigation due to the lack of data on the metabolism of ellagitannins in ruminants. abbreviations c: control group; dde: duration of a diarrheic episode; elisa: enzyme-linked immunosorbent assay; fs: fecal score; t: tannin-treated group neonatal diarrhea an overview of calf diarrhea-infectious etiology, diagnosis, and intervention antibacterial potential of plant volatile oils: a review useful plants for animal therapy efficacy test of a hydrolysable tannin extract against necrotic enteritis in challenged broiler chickens metabolic and microbial modulation of the large intestine ecosystem by non-absorbed diet phenolic compounds: a review use of polyphenol-rich grape byproducts in monogastric nutrition a review antioxidant effect of dry olive (olea europaea l.) leaf extract on ethanol-induced gastric lesions in rats medicinal plants -prophylactic and therapeutic options for gastrointestinal and respiratory diseases in calves and piglets? a systematic review phylogastro in the treatment of equine gastric ulcer lesions effect of pomegrate-residue supplement on cryptosporidium parvum oocystis shedding in neonatal calves review. tannins and ruminant nutrition methane emission by goats consuming different sources of condensed tannins the influence of addition of gallic acid, tannic acid, or quebracho tannins to alfalfa hay on in vitro rumen fermentation and microbial protein synthesis tannins for suppression of internal parasites milk fatty acid composition, rumen microbial population, and animal performances in response to diets rich in linoleic acid supplemented with chestnut or quebracho tannins in dairy ewes milk production, composition, and milk fatty acid profile from grazing sheep fed diets supplemented with chestnut tannin extract and extruded linseed exploitation of dietary tannins to improve rumen metabolism and ruminant 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properties of tannins effects of plant tannin supplementation on animal responses and in vivo ruminal bacterial populations associated with bloat in heifers grazing wheat forage pomegranate (punica granatum) peel is effective in a murine model of experimental cryptosporidium parvum anthelmintic activity of pistacia lentiscus foliage in two middle eastern breeds of goats differing in their propensity to consume tannin-rich browse consumption of pistacia lentiscus foliage alleviates coccidiosis in young goats sericea lespdeza as an aid in the control of emeria spp. in lambs difference in the nature of tannins on in vitro ruminal methane and volatile fatty acid production and on methanogenic archaea and protozoal populations supplementation of acacia mearnsii tannins decreases methanogenesis and urinary nitrogen in foragefed sheep digestion, ruminal fermentation, ciliate protozoal populations, and milk production from dairy cows fed cinnamaldehyde, quebracho condensed tannin, or yucca schidigera saponin extracts effects of condensed tannins from leucaena on methane production, rumen fermentation and populations of methanogens and protozoa in vitro bacterial and protozoal communities and fatty acid profile in the rumen of sheep fed a diet containing added tannins composition of european chestnut (castanea sativa mill.) and association with health effects:fresh and processed products the effects of ph and rat intestinal contents on the liberation of ellagic acid from purified and crude ellagitannins evaluation of the bioavailability and metabolism in the rat of punicalagin and antioxidant polyphenol from pomegranate juice the potent in vitro antioxidant ellagitannins from pomegranate juice are metabolized into bioavailable but poor antioxidant hydroxy- h-dibenzopyran- -one derivatives by the colonic microflora in healthy humans metabolism of antioxidant and chemopreventive ellagitannins from strawberries, raspberries, walnuts, and oak-aged wine in humans: identification of biomarkers and individual variability the authors would like to thank mauro saviola group srl, radicofani, siena, italy for providing the chestnut tannin extract. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. the datasets used and analyzed are available from the corresponding author upon reasonable request. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - vtcochx authors: lee, jeong yoon; lee, ji sun; materne, emma c.; rajala, rahul; ismail, ashrafali m.; seto, donald; dyer, david w.; rajaiya, jaya; chodosh, james title: bacterial reca protein promotes adenoviral recombination during in vitro infection date: - - journal: msphere doi: . /msphere. - sha: doc_id: cord_uid: vtcochx adenovirus infections in humans are common and sometimes lethal. adenovirus-derived vectors are also commonly chosen for gene therapy in human clinical trials. we have shown in previous work that homologous recombination between adenoviral genomes of human adenovirus species d (hadv-d), the largest and fastest growing hadv species, is responsible for the rapid evolution of this species. because adenovirus infection initiates in mucosal epithelia, particularly at the gastrointestinal, respiratory, genitourinary, and ocular surfaces, we sought to determine a possible role for mucosal microbiota in adenovirus genome diversity. by analysis of known recombination hot spots across human adenovirus genomes in species d (hadv-d), we identified nucleotide sequence motifs similar to bacterial chi sequences, which facilitate homologous recombination in the presence of bacterial rec enzymes. these motifs, referred to here as chi(ad), were identified immediately ′ to the sequence encoding penton base hypervariable loop , which expresses the arginine-glycine-aspartate moiety critical to adenoviral cellular entry. coinfection with two hadv-ds in the presence of an escherichia coli lysate increased recombination; this was blocked in a reca mutant strain, e. coli dh α, or upon reca depletion. recombination increased in the presence of e. coli lysate despite a general reduction in viral replication. reca colocalized with viral dna in hadv-d-infected cell nuclei and was shown to bind specifically to chi(ad) sequences. these results indicate that adenoviruses may repurpose bacterial recombination machinery, a sharing of evolutionary mechanisms across a diverse microbiota, and unique example of viral commensalism. importance adenoviruses are common human mucosal pathogens of the gastrointestinal, respiratory, and genitourinary tracts and ocular surface. here, we report finding chi-like sequences in adenovirus recombination hot spots. adenovirus coinfection in the presence of bacterial reca protein facilitated homologous recombination between viruses. genetic recombination led to evolution of an important external feature on the adenoviral capsid, namely, the penton base protein hypervariable loop , which contains the arginine-glycine-aspartic acid motif critical to viral internalization. we speculate that free rec proteins present in gastrointestinal secretions upon bacterial cell death facilitate the evolution of human adenoviruses through homologous recombination, an example of viral commensalism and the complexity of virus-host interactions, including regional microbiota. h uman adenovirus (hadv), now known to be both a common enteric and respiratory pathogen ( , ) , was first identified in from a child's adenoid specimen in organ culture ( , ) . a double-stranded dna virus with a linear genome of~ kb, hadv segregates by phylogenomics to seven species (a to g), comprising distinct hadv genotypes. the largest and most rapidly expanding hadv species is hadv species d (hadv-d) with unique genotypes. numerous hadv-d types were first discovered in the feces of human patients with aids ( ) ( ) ( ) ( ) ( ) ( ) , and simultaneous coinfection with more than one adenovirus type is common ( , ) . single nucleotide polymorphisms in hadv occur uncommonly, even over decades ( ) , and the viruses within hadv-d are highly conserved (around % at the nucleotide level). however, homologous recombination between viruses within hadv-d occurs commonly, particularly at transitions from conserved to hypervariable nucleotide sequence where the relatively high gc content of hadv-d (~ % overall) drops abruptly ( ) . remarkably, every fully sequenced hadv-d shows evidence for at least two prior homologous recombination events among seven, stereotypically hypervariable, gene segments ( ) . taken together, these data suggest that adenoviruses can persistently infect the human intestine, where coinfections set the stage for homologous recombination between highly related genotypes. the nonenveloped adenovirus capsid takes an icosahedral shape with apices, each marked by a trimeric fiber protein with its distal knob and encircled by five, linked penton base proteins. each individual penton base protein contains two hypervariable loops (hvl and hvl ); hvl expresses the canonical arginine-glycine-aspartic acid (rgd) moiety critical to integrin-mediated internalization of the virus ( , ) . structural data ( ) shows that after binding of the fiber knob to one of several possible primary receptors on the target cell, viral internalization is mediated through binding of each rgd in the five-sided penton base capsomer to integrins, inducing in turn their aggregation, conformational change, and autophosphorylation to catalyze downstream intracellular signaling. previous analysis of hypervariable gene segments in hadv-ds showed only distinguishable amino acid patterns (proteotypes) ( ) for penton base hvl and only for hvl ( ) , consistent with homologous recombination at penton base gene segments as a major driver in the ontogeny of new hadv-d types ( ) . because hvl and hvl are separated by only amino acids and yet their coding regions recombine independently of one another ( ) , we predicted the existence of a recombination signal in the intervening, relatively conserved area of the genome between the gene segments for these two hypervariable regions. in bacteria and bacteriophage, a signal for recombination between homologous dna is the crossover hot spot instigator, or chi nucleotide sequence. this was first discovered in bacteriophage lambda and then in bacterial dna and later was shown to mediate recombination between them ( ) . the chi sequence in bacteriophage and in escherichia coli (chi ec ) is =-gctggtgg- = ( , ) , and its presence induces the exonuclease function of the bacterial recbcd enzyme ( ) . the reca protein of e. coli is then loaded onto unwound single-stranded dna (ssdna) by recbcd to create an ssdna-protein filament, which invades homologous double-stranded dna (dsdna), leading to homologous recombination ( ) . a conserved chi sequence in bacterial genera does not exist ( ) ; the repair enzymes that repair dsdna breaks and mediate homologous recombination also differ in genera. however, reca has significant homology to eukaryotic rad and its paralogs ( ) , enzymes that repair dsdna breaks in human cells, and facilitate homologous recombination in the human genome ( ) . also, the adenovirus and bacteriophage prd exhibit striking structural similarities consistent with a common ancestor ( ) , suggesting the possibility that mechanisms of phage evolution have survived in the adenoviruses. these disparate observations led us to consider whether the presence of intestinal bacterial flora during adenovirus coinfection might facilitate homologous recombination and evolution of enteric hadv-ds. evidence for transkingdom interactions between bacteria of the human gut microbiome and enteric viruses has been accumulating recently. in enteric infection with certain rna viruses, virus hijacks surface bacterial glycans of normal bacterial flora to gain entry to intestinal epithelial cells ( ) ( ) ( ) ( ) ( ) . recently, data suggesting that several enteric bacteria can promote recombination during poliovirus infection have appeared ( ) . as another common enteric agent, adenoviruses are regularly shed in feces of humans and nonhuman primates for months to years after infection ( ) ( ) ( ) ( ) , suggesting that adenoviruses might also exploit enteric bacterial flora to its own advantage. in the work described herein, we utilized a previously published database of whole hadv-d genomes ( ) to analyze the gc/at transition zone at the = end of penton base hvl . we show the presence of chi-like sequences at the exact juncture of conserved and hypervariable sequence and further show an increase in homologous recombination in the presence of bacterial reca protein. these data demonstrate a means by which bacterial flora can facilitate genetic exchange between adenoviruses. we began by searching the penton base genes of hadv-d for chi nucleotide sequences that, in bacteria and bacteriophage, act as signals for recombination between homologous dna ( ) . by careful inspection, we identified chi-like (chi ad ) sequences, for example, =-tctcctga- = in hadv-d , in the relatively conserved region immediately = to hvl (fig. ) . we also noted that the nucleotide sequences of putative chi ad were generally conserved within proteotypes but not between them. in other studies, patterned alterations of gc content in multiples of nucleotides were shown to facilitate homologous recombination between adjacent brome mosaic virus ssrnas through the formation of hairpin loops in the rna ( , ) . our computational analysis of similar gc content transitions across whole hadv-d genomes showed comparable patterns of gc/at transition at predicted recombination hot spots around hypervariable gene segments in the three major capsid genes-the same segments that constitute the molecular identity of each virus ( , , ( ) ( ) ( ) -including the region containing chi ad immediately = to penton base hvl (see fig. s and table s in the supplemental material). we applied mfold (http://unafold.rna.albany.edu/ ?qϭmfold/dna-folding-form) to model the secondary structures of ssdna surrounding and including chi ad , within the gc/at transition zone at hvl . the structures were highly similar within proteotypes, less so between proteotypes (fig. s ) , suggesting that patterned alterations in gc content generate secondary structures that facilitate homologous recombination between hadv-d types sharing the same chi ad . to examine the determinant(s) of homologous recombination among hadv-ds and specifically the role of chi ad , we generated constructs with the chi ad -containing gc/at transition zones from the penton base genes of hadv-d and -d , both from the same hvl proteotype ( ) , the former with the green fluorescent protein (gfp) gene without a promoter (d gfp), and the latter with a cmvt (cmv stands for cytomegalovirus) promoter inserted (d cmv) ( fig. a) . in a cells, cotransfection of linearized d cmv and d gfp constructs resulted in gfp expression ( fig. b and c). by pcr with primers specific to the recombinant product (table s ) and subsequent sequencing, we confirmed that the two constructs had indeed recombined at the expected location ( fig. d and e), the same recombination locus often seen in hadv-d recombination in nature ( ) . prior work suggested that a single nucleotide change in chi can reduce homologous recombination events ( ) , with particular emphasis on the importance of the thymidine (t) at the third position ( , ) . we generated targeted mutations in both constructs to test the specificity of chi ad at the single nucleotide level, while maintaining homology between constructs for cotransfections. chi ad nucleotide identities were determined for hvl in each of hadv-ds to determine a consensus sequence (fig. f ). mutants not found in any known virus were generated, and nine were selected for testing, including one with nucleotide changes synonymous to d / (sy), one with reverse sequence (re), one with antisense sequence (as), and six randomly selected. mutating chi ad reduced homologous recombination between vector constructs except for the sy mutant (fig. g) . subsequent mfold analysis showed that the predicted secondary structure for the sy construct and the location of chi ad in that secondary structure were highly similar to those of the parent chi ad (fig. h) . none of the other predicted structures for chi ad mutants were similar. the re mutation, which maintained t at the third position and was expected to undergo homologous recombination, had a predicted secondary structure distinctly different from that of the naturally occurring chi ad . therefore, nucleotide sequence and secondary structure may be considered covariant determinants of homologous recombination. the human gut microbiota includes trillions of bacteria and countless bacteriophage ( ) . their evolution is driven in part by rec protein-mediated homologous recombination, specifically the unwinding of dsdna by the recbcd complex and upon recognition of chi, the loading of reca onto the ssdna ( ) . we quantified homologous recombination between chi ad constructs in the presence of an endotoxin-free lysate of e. coli strain k- in comparison to a lysate from the k- reca mutant strain dh ␣. by trypan blue exclusion, bacterial cell lysates induced no cell toxicity (data not shown). as predicted, the k- lysate promoted more homologous recombination between constructs than the dh ␣ lysate (fig. a) . the presence of reca in lysates from each strain . thirty-eight hadv-d genomes were aligned by maximum likelihood analysis, a tree (left) was built based on the amino acid sequences of penton base hypervariable loop (hvl ), and the hadv-d genomes were divided into proteotypes as previously described ( ) , shown by a horizontal line separating the virus type names. the nucleotides shown include the junction between conserved nucleotide sequence (black) and hvl nucleotide sequence (blue), with nucleotides on either side of the junction. the chi ad motifs (shown on gray background) fall predominantly within the conserved sequence but include one nucleotide within the hypervariable sequence. was confirmed by western blotting. to determine whether the effect was specific to reca, we also tested k- lysates from which we depleted reca protein with immunomagnetic beads and found that reca-depleted k- lysate induced less recombination than lysate with mock depletion (fig. b ). we next examined the effects of lysates from k- , dh ␣, and reca-depleted k- on penton base hvl recombination between two wild-type viruses with conserved chi ad , hadv-d and hadv-d , chosen because of sufficient sequence disparity in hvl and hvl to permit pcr discrimination of recombinants. we coinfected both viruses in the intestinal adenocarcinoma cell line caco- (clone c bbe ) and used conventional and quantitative pcr (qpcr) ( fig. c and d, respectively) to identify recombination of penton base hvl between viruses. e. coli k- lysate promoted recombination relative to phosphate-buffered saline (pbs), and to a greater degree than either e. coli dh ␣ or reca-depleted k- , with the effect statistically significant by days postinfection. the same results were evident in a cells, a lung carcinoma cell line (fig. s ). because an increase in viral replication due to bacterial lysate could have accounted for the apparent increase in recombined viral dna ( ), we also infected each cell type in the presence of k- lysate or pbs control and performed qpcr with primers specific to sequence conserved between the hexon genes of both viruses (table s ). relative to treatment with pbs, k- lysate appeared to reduce but not prevent viral replication through days postinfection in either cell line ( fig. a to d). upon coinfection and subsequent assay by qpcr, the recombinant accounted for less than . % of total viral dna in either c bbe (fig. e ) or a cells (fig. f) . the relative increase in the ratio of recombinant viral dna to total viral dna in the presence of k- lysate was maintained when total viral replication was taken into account. therefore, the absolute number of viral recombinants increased in the presence of bacterial lysates despite a general inhibition of viral replication. adenovirus-infected cells at the time of viral replication have lost the capacity for cellular biosynthesis, and these cells are in the early phases of virus-induced cell death with reduced integrity to their cellular membranes ( ) . in addition, hadv-d was previously shown to release the ectodomain of muc from ocular surface cells, suggesting that adenoviruses may have intrinsic means of reducing overall mucosal barrier function ( ) . we next performed confocal microscopy to determine whether reca would colocalize with viral dna in infection of c bbe cells (fig. a) . coinfection with -ethynyl- =-deoxyuridine (edu)-labeled hadv-d and hadv-d was performed in the presence of e. coli k- lysate, and the cells were imaged at h postinfection. reca protein was identified only in the nuclei of infected cells, where it colocalized with (fig. s ) . healthy, uninfected cells excluded rec proteins. we next performed chromatin immunoprecipitation (chip) days after coinfection of c bbe cells pretreated with k- lysate. binding of the igg control was extremely low (data not shown), so we compared reca binding of hvl chi ad with binding of reca to conserved genomic regions without chi ad : one in protein vi and another at the conserved = end of the penton base gene. reca binding to penton base sequence containing chi ad was~ -fold greater than to control regions of hadv dna (fig. b ). there are noncanonical pathways of reca loading that function in the absence of recbcd in phage ( ) . to determine whether recbcd or other bacterial proteins are required for a putative interaction between reca and chi ad , we repeated chip with recombinant reca instead of e. coli lysate. the ratios of binding to chi ad relative to other regions of the genome were similar (fig. b) . relative to igg, binding with k- lysate was twice that observed with recombinant reca (data not shown). these data show specificity of reca binding to chi ad and suggest that recbcd dispensably improves binding. hadv infects mucosal sites to cause a myriad of human diseases, and it can be lethal in the immunocompromised host ( ) . how adenoviruses evolve is therefore of considerable significance, as new types can be associated with enhanced virulence and pathogenicity. single nucleotide substitutions in hadv genomes are relatively slow to accrue, with nucleotide-specific stability seen over decades ( , ) . in contrast, homologous recombination among circulating hadv is widely recognized as the principal means of their evolution ( , ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , and recombinational evolution of hadv is now codified in genbank/ncbi criteria for typing newly emerging or historically known but previously uncharacterized hadv ( , ) . in hadv-d, specific homologous recombination patterns predominate ( , , ( ) ( ) ( ) ( ) ( ) ( ) ( ) . for example, homologous recombination involving the hypervariable region corresponding to penton base hvl is particularly widespread ( ) , seen in almost every hadv-d analyzed thus far. the emergent hadv-d , now a common cause of severe epidemic keratoconjunctivitis ( ), expresses hexon epitopes of the nonpathogenic hadv-d , but the penton base of hadv-d , a previously characterized and highly virulent eye pathogen ( ) . another eye pathogen, hadv-d , expresses the hexon epitopes of the nonpathogenic hadv-d on a genome "chassis" of hadv-d with its penton base gene contributed by hadv-d ( ). homologous recombination is not restricted to hadv-d. in hadv-b , homologous recombination of the hexon gene hypervariable regions, prime determinants of type-specific humoral immune responses, permitted the escape of a serious respiratory pathogen from immune pressure ( ) ( ) ( ) ( ) ( ) . recombination between adenoviruses requires at least two homologous adenoviral sequences, one an intact dsdna and the other an ssdna, as would be present in cells undergoing viral dna replication during coinfection ( , ) . coinfection with two or more hadv types occurs commonly ( , , ) and is tolerated by the host because host immunity is mostly type specific ( ) . hadv has been shown to cause persistent infections of healthy persons ( ) within secondary lymphoid tissues, including those at waldeyer's ring (tonsils and adenoids), the gastrointestinal tract ( , - ), and even the ocular surface ( ) . viral persistence in infected tissues increases the likelihood of coinfection with two or more adenoviruses. however, host factors that promote recombination between hadv genomes in vivo are unknown. homologous recombination was first identified by lederberg and tatum in ( ) as a means to ensure the viability of phage and bacteria under host and environmental selection pressures and as a repair mechanism for dsdna and ssdna breaks during genome replication ( ) ( ) ( ) . homologous recombination between bacterial genomes is driven by the presence of the ubiquitously expressed, bacterial reca ( -kda) protein, which is loaded onto bacterial and phage dna by the heterotrimer recbcd or alternate rec proteins. reca-mediated strand exchange (branch migration) occurs upon strict base pairing between adjacent dna molecules ( ) , and the recombination event can then extend to include thousands of subsequent base pairs. experimental null mutations in e. coli reca diminished bacterial recombination as much as , -fold, while mutations in the other rec proteins had substantially lower effects ( ), consistent with known redundancy in the reca loading function of recbcd, but not in the recombination function of reca. although we showed a statistically significant increase in viral recombination in the presence of bacterial lysate, with reduced effect upon reca depletion, the impact on generation of new recombinant viral dna was modest in comparison to the known effect of reca on bacteria and phage. if hadv-ds utilize bacterial recombination machinery in vivo, they likely do so with considerably lower efficiency than for bacteria and phage. chi sequences are typically nucleotides long with a thymidine (t) at the third position, but they differ in bacterial genera. chi sequences appear more frequently than would be expected by chance alone, suggesting their positive selection to facilitate dna repair and recombinational evolution ( ) . in bacteria and phage, the specific and highly conserved nucleotide sequence for chi is critical to its function, while in our experiments, chi ad sequence specificity appeared considerably less stringent. strictly speaking, confirmation of a canonical chi-like effect of chi ad will require further experimental study ( ) . however, chi-like sequences have been identified in human immunodeficiency virus ( ) and in human immunoglobulin and abo genes ( , ) and implicated in translocation-associated human malignancies ( ) ( ) ( ) ( ) . we identified chi-like sequences in hadv-d genomes (chi ad ) at a known recombination hot spot, just = to the sequence encoding penton base hvl , the latter critical to viral internalization and a presumed pathogenesis determinant. we showed in dna constructs that chi ad sequence and directionality impact recombination, although not with the same degree of sequence specificity as chi in bacteria. regardless, hadv-d coinfection in the presence of e. coli lysate increased recombination, not seen with a reca mutant strain or with reca depletion, and reca entered hadv-infected cell nuclei where it localized with viral dna. therefore, although reca-mediated adenoviral recombination appears to be less efficient and less sequence specific than in bacteria and phage, it nevertheless appears that adenoviruses can benefit from the recombination machinery of resident bacterial flora. while adenoviruses may exploit the recombination machinery of local bacterial flora, the efficiency of replication by the recombinant virus and subsequent selection pressure by the host would determine whether a new recombinant survives and is transmitted. in other words, postrecombination selection must also play a role in the emergence of any new viruses. regardless, our work is consistent with the idea that human, bacterial, and viral genomes in the gut may utilize common recombination machinery to foster microbial and viral diversity. it is also possible that adenoviral recombination occurs during infection of other mucosal sites. for instance, while the presence of a stable conjunctival microbiome is a matter of some conjecture ( , ) , it is clear that bacterial superinfection coincident to adenovirus infection can occur in epidemic keratoconjunctivitis ( , ) , which could conceivably promote adenoviral recombination at the ocular surface. secondary structure is also known to impact recombination between two homologous nucleic acid molecules. our analysis showed recombination in one construct (sy) not predicted to undergo recombination, but the secondary structure and location of chi ad within that structure were highly similar to those of the parent chi ad . imputed secondary structures were also found to be highly similar within but not between penton base hvl proteotypes. in rna viruses, stem-loop secondary structures were shown to be particularly critical to recombination between two homologous ssrna ( ) ( ) ( ) . adjacent regions that were = gc-rich and = au-rich ( , ) were considerably more likely to recombine in brome mosaic virus ( ) , particularly when the region of relatively greater gc content is followed by a greater au-rich content of equal length with preference for a gc/au transition of , , or nucleotides. the au-rich regions formed hairpin loops thought to induce a pause of the rna replicase, followed by template switching of the replicase ( , ), i.e., polymerase jumping, when loop-to-loop complementarity was also present ( ) . polymerase hesitation due to secondary loop structures was also shown to promote homologous recombination between retroviruses ( , ) , poliovirus ( ) , norovirus ( ) , and coronavirus ( ), suggesting a common mechanism. a relationship between a virus and local bacterial flora could be detrimental to one or both, beneficial to one or both, or even obligate, with the outcome likely to shift over time due to the dynamics of constantly changing host factors and environmental influences. commensalism characterizes a relationship between two disparate organisms when one organism benefits while the other is unaffected. certain rna viruses benefit by hijacking bacterial surface glycans in the gut to achieve host cell entry ( ) . we propose a novel example of viral commensalism in which chi ad sequences in the hadv-d genome are bound within infected cells by bacterial reca, present because of local bacterial flora, to facilitate homologous recombination between viruses. free rec proteins may be present in gastrointestinal secretions because of bacterial senescence and loss of structural competency, competition and killing by other bacterial species vying for the same mucosal niche, bacteriophage-mediated bacterial cell lysis, host inflammatory cells and factors induced by adenovirus infection that can lyse bacterial cells, and/or the presence of endogenous antimicrobial peptides at the mucosal surfaces ( ) ( ) ( ) ( ) ( ) ( ) . however, to characterize the relationship as commensal may be an oversimplification, as the interactions between adenoviruses, bacterial flora, and the host are certain to be multifaceted. for example, ␣-defensins, peptides generated by the host in response to microbes present in the gastrointestinal tract, limited adenovirus uncoating within endosomes ( , ) and potentiated neutralizing antibody responses to infection ( ) , but also enhanced viral entry into host cells ( ) . our work is another example of complexity in the dynamic evolution of virus-host interactions, including the host's microbiota. cells, viruses, and bacteria. the a cell line was obtained from thermo fisher scientific (waltham, ma) (r ), and the a and caco- (c bbe clone) cell lines were obtained from atcc (manassas, va) (atcc ccl- and atcc crl- ). cell lines were tested for and verified as mycoplasma negative. escherichia coli strain k- (atcc ) was purchased from atcc. e. coli strain dh ␣ was a gift from michael gilmore at massachusetts eye and ear infirmary, harvard medical school. hadv-d (atcc vr- ) and hadv-d (atcc vr- ) were purchased from atcc and verified by molecular typing of the major capsid genes. viruses were purified using the cesium chloride gradient method, verified as endotoxin negative, and the titers of the virus were determined by the tissue culture infectious dose method. hadv-d gene segments of interest in viruses of hadv-d were organized by maximum likelihood trees, constructed using mega (http://www .megasoftware.net/), and segregated into proteotypes by % difference in amino acid content and confirmed by simple inspection. crossover hot spot instigator (chi) sequences inside gc/at transition zones were identified by searching for =-nntnntnn- = in which n could be any nucleotide. the same viruses in hadv-d were studied individually with the recombination site pattern finder ( ) . a threshold of % difference in gc content was applied to identify regions of -, -, or -nucleotide transition from gc rich to at rich, with a sliding -nucleotide window. all gc/at transition zones for viruses were combined by matching nucleotide positions. cloning. the pcdna . -hygromycin vector was obtained from thermo fisher scientific, and the hygromycin resistance gene replaced with one for neomycin from pcdna . -myc-his-a(ϩ) (thermo fisher scientific). the cytomegalovirus (cmv) and t promoters (pcvmt ) of both vectors were removed using nrui-hf and nhei-hf, restriction enzymes from new england biolabs (neb, ipswich, ma), and an adaptor for sanger sequencing (see table s in the supplemental material) synthesized at integrated dna technologies (idt) was cloned into the same region using t dna ligase (neb) to generate pcdna . -hygromycine/neomycin-nocmvt . using q hot start high-fidelity dna polymerase (neb), cmvt promoter and green fluorescent protein (gfp) genes without start codon were amplified from pcdna . -hygromycin and pegfp-n (egfp stands for enhanced gfp) (takara, mountain view, ca), respectively. the cmvt promoter was inserted into the penton base gene of hadv-d between nucleotides and , just after the gc/at transition zone marking the transition from conserved sequence to hvl (d cmv). the gfp gene without a start codon was inserted into the penton base gene of hadv-d between nucleotides and (d gfp). d cmv and d gfp were then cloned into pcdna . -hygromycin-nocmvt and pcdna . -neomycin-nocmvt vectors, respectively. chi ad mutants were created on both pcdna . -hygromycin-nocmvt -d cmv and pcdna . neomycin-nocmvt -d gfp vectors using overlap extension pcr. all constructs were verified by sanger sequencing (ocular genomics institute, massachusetts eye and ear, boston, ma). transfection and measurement of gfp signal. a cells were seeded on black -well cell culture plates (greiner bio-one, monroe, nc) in dulbecco's modified eagle medium (dmem), with % fetal bovine serum (fbs) and % penicillin-streptomycin (thermo fisher scientific). after incubation at °c in % co for h, ng of each plasmid per well was transfected using lipofectamine (thermo fisher scientific) according to the manufacturer's instructions. at days posttransfection, gfp expression level in each well was measured on a spectramax m microplate reader (molecular devices, sunnyvale, ca) and visualized on a leica sp confocal system (leica microsystems, buffalo grove, il) with ϫ magnification. for comparison of gfp expression levels in the presence of bacterial lysates, a cells were treated with g/well of either e. coli k- , dh ␣, or reca-depleted (reca-) lysate for h prior to transfection. pcr and quantitative pcr. pcr primers were synthesized by integrated dna technologies (idt) (coralville, ia), and these primers are shown in table s . viral dna was isolated using genejet viral dna/rna purification kit (thermo fisher scientific) per the manufacturer's instructions. dna was quantified and quality checked on a nanodrop c spectrophotometer (thermo fisher scientific). pcr was performed on a ptc- thermal cycler (bio-rad, hercules, ca) with ng of dna, . l of gotaq g hot start green master mix (promega, madison, wi), and ng of forward and reverse primer with each primers in a total volume of l. pcr was performed as follows: °c for min; cycles, with cycle consisting of °c for s, °c for min, °c for s, and °c for min. quantitative pcr (qpcr) was performed on a quantstudio real-time pcr system (thermo fisher scientific) with ng of dna, l of fast sybr green master mix (thermo fisher scientific), and ng of forward and reverse primer with each primer in a total volume of l. qpcr was performed as follows: °c for s; bacteria assist hadv evolution cycles, with cycle consisting of °c for s and °c for s with data collection and melting curve analysis. secondary structure modeling. the mfold web server's dna folding form was used for dna secondary structure analysis and free energy measurements, without application of constraints, and set for a linear dna sequence with a folding temperature of °c. for default values, ionic conditions were specified at m sodium and no magnesium, maximum distance between paired bases as unlimited, percent suboptimality number fixed at , structure rotation angle set at auto, and regularization angle at °. e. coli lysate preparation and treatment. e. coli k- and dh ␣ were each inoculated into luria broth (lb), incubated at °c for h in a shaking incubator at rpm, then collected by centrifugation in -ml conical tubes at , rpm for min, and resuspended in ml of phosphate-buffered saline (pbs), and aliquots of the bacterial solutions were added to lysing matrix b tubes (mp biomedicals, santa ana, ca). lysis was performed using a fastprep- g instrument (mp biomedicals) at , rpm for six -s pulses. endotoxin was extracted from bacterial lysates with pierce high capacity endotoxin removal spin columns (thermo fisher scientific). protein concentrations were measured by pierce bca protein assay kit (thermo fisher scientific). lysate was added to each well of six-well cell cultures at a final concentration of -g bacterial protein/ml of culture medium, i.e., g/tissue culture well. for pbs control, an equivalent volume of pbs was added to tissue culture medium per well. the presence of endotoxin in bacterial lysates was ruled out using the toxinsensor chromogenic lal (limulus amebocyte lysate) endotoxin assay kit (genscript, piscataway, nj); the final endotoxin levels in cell culture medium after the addition of bacterial lysates were below the detection limit of . endotoxin unit (eu)/ml. reca depletion from the e. coli k- strain was performed with magnetic beads. after e. coli was vortexed for min, -l aliquots of dynabeads m- sheep anti-rabbit igg (thermo fisher scientific) were added to . -ml eppendorf tubes and suspended in ml of washing buffer consisting of . % bovine serum albumin (bsa) in magnesium-and calcium-free pbs. the tubes were placed in a magjet magnetic separation rack (thermo fisher scientific) for min, and then the wash buffer was removed. five hundred microliters of fresh wash buffer and g of anti-reca antibody (catalog no. ab ; abcam, cambridge, ma) were added to the beads, and after gentle mixing, the tubes were incubated at °c for h in rotation. control lysates (without reca depletion) were treated with rabbit igg isotype control (catalog no. ab ; abcam). the tubes were then placed in the magnetic separation rack for min, and the supernatant was removed. the beads were washed with ml of washing buffer three times. five hundred micrograms of e. coli k- lysate in l was added and incubated at °c for h in rotation. after the tubes were placed in the magnetic separation rack for min, the supernatant was transferred into new tubes. fifty microliters of elution buffer ( . m citrate [ph . ]) was added to the beads and boiled for min. the tubes were placed in the magnetic separation rack for min, and the buffer was transferred into new tubes. the protein concentration was measured by using the pierce bca protein assay kit as described above, and -g portions were used for western blotting with anti-reca (catalog no. md- - ; mbl international, woburn, ma) and anti-␤-galactosidase (catalog no. ab ; abcam). bacterial lysate toxicity was assessed by trypan blue exclusion; treatment of cell cultures with g bacterial lysate/ml of tissue culture medium for week showed no cellular toxicity. c bbe and a cells were seeded in six-well plates (corning, corning, ny). after -h incubation, g in l for each bacterial lysate was added to ml dmem with % insulin-transferrin-selenium in each well for h, followed by coinfection with hadv-d and hadv-d , each at a multiplicity of infection (moi) of . . one hundred eighty microliters of supernatant was collected from days postinfection (dpi) to dpi. two units of dnase i (neb) was treated with l of ϫ dnase i buffer at °c for h, and then l of . m edta (ph . ) was added and incubated at °c for min to inactivate dnase i, prior to viral dna isolation and pcr. confocal microscopy. c bbe cells were cultured on four-well nunc lab-tek chamber slides (thermo fisher scientific) for h. bacterial lysate was added to each well as described above for another h prior to viral infection. -ethynyl- =-deoxyuridine (edu)-labeled hadv-d and hadv-d were prepared using click-it plus edu alexa fluor imaging kit (thermo fisher scientific) and added to each well at an moi of~ (for each virus) and incubated at °c in an incubator with % co for h. the cells were washed three times with pbs, fixed for min in l of % paraformaldehyde, washed three times in pbs containing % bsa, treated with l of permeabilization buffer ( . % triton x- in washing buffer) for min, and washed again. the click-it plus reaction cocktail was added for min at room temperature, protected from light. after the cells were washed three times, l of pbs containing % bsa was added for min for blocking. primary antibodies were prepared as follows: : , dilution for anti-reca (mbl international) in washing buffer, : , dilution for anti-recb, anti-recc, and anti-recd (gifts of gerry smith at fred hutchinson cancer research center, seattle, wa). three hundred microliters of each primary antibody was incubated with the cells for h at room temperature, protected from light. after the cells were washed three times, l of : , dilution in washing buffer of secondary antibody was added to goat anti-mouse igg-alexa fluor conjugate (thermo fisher scientific) for anti-reca and to goat anti-rabbit igg-alexa fluor conjugate (thermo fisher scientific) for anti-recb, anti-recc, and anti-recd and incubated for min at room temperature. after three washes each in washing buffer and then in pbs, the cells were mounted with vectashield antifade mounting medium containing =, =-diamidino- -phenylindole (dapi) (vector laboratories, burlingame, ca). photomicrographs were obtained on the leica sp confocal system with ϫ magnification. chromatin immunoprecipitation. c bbe cells were seeded on -mm tissue culture dishes (greiner bio-one) for h, and g of e. coli k- lysate was added for an additional h. hadv-d and hadv-d were added to the cells at an moi of . , with bacterial lysate at the same concentration. at dpi, cells were collected by scraping into pbs, centrifuged, and washed twice in pbs. dna was extracted using a pierce magnetic chip (chromatin immunoprecipitation) kit (thermo fisher scientific) per the manufacturer's instructions, sheared with a q sonicator (qsonica, newtown, ct) and then treated with micrococcal nuclease (mnase). the sheared dna was run on an agarose gel to confirm dna fragments ranging between and bp in length. chip assays were performed with anti-reca antibody (abcam), using the same pierce kit with qpcr primers as listed in table s . statistical analysis. all experiments were performed at least three times. data were analyzed by either student's t test for pairwise comparisons or by analysis of variance (anova) with preplanned comparisons, using sas (cary, nc). significance was set a priori at p Ͻ . . supplemental material for this article may be found at https://doi.org/ . / msphere. - . nearly constant shedding of diverse enteric viruses by two healthy 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was designed to describe the molecular epidemiology and seasonality of acute respiratory infection (ari)‐related respiratory viruses in the united arab emirates (uae). methods: both upper and lower respiratory specimens were collected for the analysis from all the patients who visited the sheikh khalifa specialty hospital (sksh) with ari for over years. the multiplex real‐time reverse transcription polymerase chain reaction (rrt‐pcr) test was used to detect respiratory viruses, which include human adenovirus, influenza virus (flu) a and b, respiratory syncytial virus, parainfluenza viruses, human rhinovirus (hrv), human metapneumovirus, human enterovirus, human coronavirus, and human bocavirus. results: a total of , respiratory samples were collected from ( . %) male and ( . %) female patients with ari who visited the sksh between november and february . the rrt‐pcr test revealed an overall positivity rate of . % ( / ). the positive rate increased during winter; it was highest in december and lowest in september. flu was the most frequently detected virus ( / [ . %]), followed by human rhinovirus ( / [ . %]). the flu positivity rate showed two peaks, which occurred in august and december. the peak‐to‐low ratio for flu was . ( % confidence interval: . ‐ . ). conclusions: the pattern of flu in the uae parallels to that of temperate countries. the trend of the small peak of flu in the summer suggests a possibility of semi‐seasonal pattern in the uae. accordingly, understanding the epidemiology of respiratory viruses is important for promoting preparedness to tackle this public health threat. [ ] [ ] [ ] [ ] [ ] [ ] the epidemiology of ari-related respiratory viruses in developed countries with temperate climates has been well studied. , , contrary to the accumulating knowledge of aris in temperate regions, epidemiological research on acute respiratory viral illness in tropical and subtropical areas is limited, although the epidemiological diversity, according to local climate and latitude, has been well studied. , [ ] [ ] [ ] the knowledge of the regional distribution of respiratory viruses is essential not only for local prevention and control of aris but also for global health decision-making. to our knowledge, limited information is available on the epidemiology and clinical characteristics of respiratory viral infections in the united arab emirates (uae). although a few studies in nearby countries with similar meteorology have described the epidemiology of aris, the reports cover a limited population group. our study was designed to describe the molecular epidemiology of ari-related respiratory viruses, including the seasonality of the viruses in the northern uae for over years. we collected both upper and lower respiratory specimens for the analysis from all the patients who visited the sheikh khalifa specialty hospital (sksh) with acute respiratory illness between november and february . physicians were frequently encouraged throughout the year via sms and e-mails to perform viral real-time reverse transcription polymerase chain reaction (rrt-pcr) tests to diagnose suspected acute ari cases. an ari was defined as the simultaneous occurrence of at least one respiratory symptom or sign (cough, purulent sputum, sore throat, nasal congestion, rhinorrhea, dyspnea, wheezing, or injected tonsils) and at least one of the following systemic symptoms: fever, chills, myalgia, or malaise. we used the edwards harmonic technique method to measure the peak-to-low ratio. , the edwards technique is a geometrical model, which is an approach that fits a sine curve to a time series of frequencies by the use of ordinary regression methods. the peakto-low ratio was interpreted as a measure of relative risk that compares the month with the highest incidence (peak) with the month with the lowest incidence (low or trough). the positivity rates for respiratory viruses during the discrete peak and low periods were compared using a direct method (χ -test) to analyze statistical significance. we applied the chi-squared (χ ) test and fisher's exact test to paired nominal data. student t test was applied to analyze the means of continuous data. a two-sided alpha level of . defined statistical significance. all comparative statistical analyses were conducted using spss version . software (spss inc, chicago, il). among the virus-positive cases, the admission rate was significantly higher in the elderly group than in the other two age groups ( table ). the number of flu and hrv cases was large enough to evaluate the associated seasonality. the peak-to-low ratio of flu was . ( % ci: . - . ) and it had significant seasonality (p < . ). hrv was detected year-round and showed no significant seasonality. the peak-to-low ratio of hrv was . ( % ci: . - . ), which was insignificant (p = . ). ari is highly prevalent and is responsible for a high burden of disease in many countries. respiratory viral infection is the most common cause of ari, and predicting the timing of peak respiratory virus activity is important for improving disease control. in this study, the overall positivity rate of respiratory viruses and the incidence of each virus by age group were similar to those reported by previous studies describing the epidemiology of respiratory viruses. , , , several studies have reported that men are more susceptible to viral infection, and have more vigorous immune and behavioral responses. [ ] [ ] [ ] however, there was no difference in the positivity rate or the incidence of ari by sex in our study. the higher positivity rate of respiratory viruses in the pediatric group was compatible with that reported in other studies. , of the patients included in our study, ( . %) with ari were admitted for further management. our data showed a lower admission rate for virus-positive than virus-negative patients ( . % vs . %), implying a better prognosis for virus-related aris than that for aris of other etiologies, as noted in several previous studies. , the higher admission rate in the elderly group is also in agreement with that reported by earlier studies. , , flu was the most common respiratory virus in all age groups, and the positivity rate was . %, which is similar to previous data reported from studies in oman. , a/h n was the most common subtype detected throughout the year. the high incidence of flu and a/h n may be related to the severe symptoms characteristic of these infections, which increase the likelihood of patients visiting the emergency department. many studies have reported that ari symptoms are more likely to be experienced by individuals infected with flu than by those infected with other respiratory viruses. in addition, a/h n infections are more severe than a/h n or b infections. , flu vaccination could have influenced the incidence of flu and its subtypes in the present study. however, regional information on flu vaccination is not available in the uae. in this study, hrv was the second-most commonly detected respiratory in all age groups. this finding is in agreement with other studies. , good accessibility of patients with common cold to emergency department might explain the higher incidence of hrv in the uae. however, the incidence of hrv could have been overestimated. many patients tested positive for hrv at the emergency department, but actually had bacterial coinfections, and their more severe symptoms could have been attributable to the bacterial infections or combined comorbidities like asthma. rsv is known as the leading cause of ari. , , however, the incidence of rsv among positive cases in our study was lower than that reported in other publications. , it is possible that the incidence of rsv was underestimated in the current study because comparatively small numbers of infants and young children were included. the hot and dry climate of the uae could also be a possible explanation for the low incidence of rsv in this region. rsv is more common during the rainy season in tropical and subtropical areas, and is reported to have a low survival rate at high temperatures. , small numbers of hcov, hadv, hmpv, hbov, hev, and hpiv cases were sporadically detected in our study. reports suggest that because of the mildness of symptoms and the self-limiting nature of these infections, patients may not seek medical care, and this may lead to an underestimation of the incidence of infection. , however, our data suggest that in the uae, like in other temperate countries, a diverse set of respiratory viruses contribute to the ari cases that compel patients to visit medical facilities, because of their severity. because we have reported the incidence of such severe infections, we believe that the data in this study will be of value to medical institutions in the uae. among the virus-positive cases in our study, the coinfection rate was . % overall and . % in the pediatric group. these are compatible with the values mentioned in previous reports. however, several similarly designed studies showed higher rates of coinfection (from . %- . %), particularly in young children. , , hrv was the most common virus to be found in cases of coinfection with other viruses in our study; this finding was in agreement with that reported in the literature. , coinfection with flu and hrv was common in our study although other studies have reported a negative association between the two viruses. , flu and hrv may have been detected in coinfected patients more frequently because the incidence of both viruses was higher than that of the other respiratory viruses in this study. a characteristic semi-seasonality pattern on respiratory viral infection rates was observed in our study. flu was the main contributing organism to the seasonal pattern; the subtypes a/h n /pdm and flu b contributed predominantly to the winter peak. low temperatures and low specific humidity during the winter season in the uae may be the cause of the peak in the number of flu cases in the winter. in addition to the major peak in the winter, a small peak in august was also observed for flu (semi-seasonal pattern), which parallels the patterns reported from temperate and tropical countries in the northern hemisphere. , , most of the respiratory viral infections, including flu, are more common in the winter in temperate countries. however, flu and rsv infections tend to show annual peaks in association with the rainy season in the tropical area. , , in subtropical areas, previous studies have observed that respiratory viruses, including flu, peaked in the coldest months in temperate areas. , the uae is located in a subtropical area and does not have a rainy season. thus, we expected a peak of flu in the winter season, as is common in other subtropical areas. however, flu in the uae showed a two-peaks semi-seasonal pattern, which has also been observed in some other regions located at similar latitudes (taiwan and nepal). the majority of the population of the uae is exposed to a dry, air-conditioned environment for most of the day, especially during the summer season, because outside temperatures range from to °c. despite this relatively controlled environment, the summer peak of flu in the uae could be explained by prolonged effective contact rates, because of the increased indoor activity, and lowered relative humidity, which have been reported to be related to the incidence of viral illnesses. , this finding implies the potential necessity for sustained flu vaccination campaigns and repeated vaccinations to reduce the severity of flu infections, especially for high-risk individuals. , nationwide data are needed to confirm the semi-seasonal pattern of flu in the uae. our study was subject to some limitations. first, this was a single center study with relatively small sample size; moreover, the decision of whether to collect a sample from the patient for viral testing was at the physician's discretion. physicians in the emergency department may have preferred to perform a rapid influenza antigen test rather than to conduct a multiplex rrt-pcr test, which requires a longer time to produce results. hence, the incidence of respiratory viruses may have been underestimated because of the low sensitivity of the rapid antigen test, and because viruses, other than the flu, may not have been identified even if present in the samples. , second, our results may not be generalizable to the entire population of the uae because most of the study specimens were taken from patients in the northern emirates. in addition, our study could have underestimated the degree of seasonality in the uae because the seasonality of viral infections tends to be more apparent when analyses include mild cases that require minimal conservative care (for example, outpatient care in the primary clinic). to our knowledge, this is the first study to describe the rrt-pcrbased seasonal pattern of respiratory viruses related to ari in the uae. moreover, the high sensitivity of the detection method enabled the collection of more reliable epidemiologic data. to date, very few studies have provided such data for the middle-eastern region. the seasonality of flu was similar to that observed in temperate countries and was also consistent with a semi-seasonal pattern of incidence. this knowledge can serve as baseline data for more expansive future surveillance studies of respiratory viruses in the uae and in the middle-eastern region. we would like to acknowledge the physicians and employees of the department of pulmonology, intensive care units, and the emergency department of the sheikh khalifa specialty hospital who participated in the collection of study specimens. this project did not receive any financial or other support from any organization. the economic burden of non-influenza-related viral respiratory tract infection in the united states burden of respiratory viruses in patients with acute respiratory failure evaluation of the bioactivity of influenza vaccine strains in vitro suggests that the introduction of new strains in the southern hemisphere trivalent influenza vaccine is associated with adverse events seasonal variations of respiratory viruses and etiology of human rhinovirus infection in children respiratory viral infections during the - winter season in central england, uk: incidence and patterns of multiple virus co-infections nation-wide surveillance of human acute 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http://orcid.org/ - - - key: cord- -u xryoo authors: mingorance, lidia; castro, victoria; Ávila-pérez, ginés; calvo, gema; rodriguez, maría josefa; carrascosa, josé l.; pérez-del-pulgar, sofía; forns, xavier; gastaminza, pablo title: host phosphatidic acid phosphatase lipin is rate limiting for functional hepatitis c virus replicase complex formation date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: u xryoo hepatitis c virus (hcv) infection constitutes a significant health burden worldwide, because it is a major etiologic agent of chronic liver disease, cirrhosis and hepatocellular carcinoma. hcv replication cycle is closely tied to lipid metabolism and infection by this virus causes profound changes in host lipid homeostasis. we focused our attention on a phosphatidate phosphate (pap) enzyme family (the lipin family), which mediate the conversion of phosphatidate to diacylglycerol in the cytoplasm, playing a key role in triglyceride biosynthesis and in phospholipid homeostasis. lipins may also translocate to the nucleus to act as transcriptional regulators of genes involved in lipid metabolism. the best-characterized member of this family is lipin , which cooperates with lipin to maintain glycerophospholipid homeostasis in the liver. lipin -deficient cell lines were generated by rnai to study the role of this protein in different steps of hcv replication cycle. using surrogate models that recapitulate different aspects of hcv infection, we concluded that lipin is rate limiting for the generation of functional replicase complexes, in a step downstream primary translation that leads to early hcv rna replication. infection studies in lipin -deficient cells overexpressing wild type or phosphatase-defective lipin proteins suggest that lipin phosphatase activity is required to support hcv infection. finally, ultrastructural and biochemical analyses in replication-independent models suggest that lipin may facilitate the generation of the membranous compartment that contains functional hcv replicase complexes. a a a a a millions of humans are chronically infected by hepatitis c virus (hcv) worldwide [ ] . chronic hcv infection is a major biomedical problem as it causes liver inflammation and fibrosis, which can lead to severe liver disease, such as cirrhosis and hepatocellular carcinoma [ , ] . there is no vaccine against hcv and, although blood-screening tests and other prophylactic measures have reduced the dissemination of this pathogen, a number of newly acquired infections still occur associated with risk behavior or with unknown origin [ , ] . however, chronic hcv infection can be successfully eradicated from chronically infected individuals through specific direct-acting antiviral (daa) combination therapies, virtually in all treated patients [ ] . since these specific treatments have only been in place recently, there are no sufficient clinical data on the long-term benefit of these treatments in relieving the severity of advanced liver disease [ , ] . hcv is a hepacivirus (flaviviridae) with a positive sense, single-strand rna genome that encodes a single open reading frame (orf) flanked by untranslated regions (utr), which are essential for viral polyprotein translation and viral genome replication. hcv orf is co-and post-translationally processed by cellular and viral proteases to produce ten major proteins. these have been functionally classified in a replication module, that includes the minimal viral components of the rna replicase (ns , ns a, ns b, ns a and ns b) and an assembly module, which comprises the major structural components of enveloped hcv virions, the capsid protein (core) and the envelope glycoprotein complex formed by e and e heterodimers; as well as the polypeptides p and ns , which are not structural components of virions but contribute to infectious particle assembly in a concerted action with the viral replicase [ ] . hcv utilizes key aspects of cellular lipid metabolism for essentially every aspect of the virus replication cycle and strongly interferes with host cell lipid homeostasis [ ] [ ] [ ] . in fact, chronic hcv patients display high rates of liver steatosis, severity of which inversely correlates with serum liver derived-lipoprotein [ ] . thus, although host immune response remains a major component in hcv pathogenesis, direct interference of hcv infection with hepatocyte lipid metabolism may contribute to overall disease progression [ ] . identified a limiting role of lipin pap activity in the generation of hcv replicase complexes. defective replicase assembly leads to strong inhibition of hcv propagation in lipin -deficient cells but not that of another (+) strand rna virus, indicating a specific role for lipin in hcv infection. given the relevance of lipin for lipid metabolism and its steatogenic potential, we set out to independently verify data from published differential transcriptomic profile studies that suggested that hcv infection may alter lpin mrna abundance in cell culture [ , ] . a specific and statistically significant lpin mrna induction (fig a) was observed in huh- cells after single cycle infection experiments [multiplicity of infection (moi) ] with a cell cultureadapted genotype a hcv (d ) variant [ ] at the peak of the infection ( and hours post-infection) and as compared with mock-infected cells (fig b) . lpin mrna induction was prevented when infected cells were treated with μm sofosbuvir (fig c) , an hcv rna polymerase inhibitor [ ] that reduced viral rna accumulation by more than two orders of magnitude (fig d) , indicating that active hcv replication is required to induce lpin mrna accumulation. lpin mrna has been shown to be upregulated by mechanisms that involve induction of reactive oxygen species (ros), as treatment of the cells with ros-scavenger molecule n-acetylcysteine (nac) is capable of preventing lpin mrna induction under glucose deprivation [ ] or during h o treatment [ ] . given that transcriptional activation of a subset of lipogenic genes during hcv infection is also prevented by addition of antioxidants [ ] , we sought to determine if lpin mrna induction by hcv infection is mediated by ros production and therefore dampened by nac treatment. lpin mrna induction was prevented in the presence of the antioxidant (fig e) , despite comparable hcv rna accumulation in mock-treated and nac-treated hcv-infected cells (fig f) , suggesting that virus replicationinduced ros production is required to induce lpin mrna accumulation. western-blot analysis confirmed that the observed transcriptional change leads to a correlative protein accumulation (fig g and h) ; reinforcing the notion that acute hcv infection alters lipin expression. in order to determine if lipin subcellular localization was altered during hcv infection, we performed confocal microscopy studies in control and hcv-infected huh- cells. lipin staining was observed as cytoplasmic punctated structures both in control and hcv-infected cells. to study if lipin signal colocalized with viral antigens, we performed double staining with antibodies against lipin and double-stranded rna (dsrna) or replicase subunits ns and ns a. none of the viral antigens strictly colocalized with lipin (pearson´s< . ) (s a fig). however, mander´s coefficients indicate that the majority of lipin overlapped with a small fraction of ns and ns a (s c fig). in contrast to lipin , lipin signal did not overlap with that of viral proteins ns and ns a (s b fig). our results indicate that no major lipin rearrangements are observed after hcv infection and that only a minor fraction of ns proteins colocalize with lipin . to study if lipin plays any role in hcv infection, lipin -deficient cells were generated by transducing human hepatoma (huh- ) cells with lentiviral vectors expressing shrnas targeting lpin mrna or a control vector expressing an irrelevant shrna. lipin expression silencing was verified by western-blot, typically days post-transduction (fig a) . a partial ( %; shlpin - ) and a more profound (> %; shlpin - ) reduction in lipin accumulation was observed after transduction with specific shrnas as compared with the control (fig b) . . rnas from mock-infected cells and hcv infected. impact of sofosbuvir ( μm; daa) (c, d) or n-acetylcysteine ( mm; nac) treatment (e, f) on hcv rna accumulation and lpin mrna levels. data are shown as average and standard deviation of two independent infection experiments performed in triplicate (n = ). g-protein samples of infected and control cells collected at hours post-infection were subjected to western-blot analysis to determine lipin and ns levels, using beta-actin as loading control h-quantitation of the relative lipin protein levels in mock and hcv-infected cells (n = ). statistical significance was determined using student´s t-test ( Ã p< . ; ÃÃ p< . ). https://doi.org/ . /journal.ppat. .g huh- cells were transduced with lentiviral vectors expressing control or lpin -specific shrnas. seven days after transduction samples of the cells were collected for western-blot analysis using antibodies against lipin and lipin and actin as loading control. parallel cultures were subjected to an mtt assay to determine their viability as described in the methods section. (a) representative western-blot showing cellular lipin and lipin protein expression levels and a loading control (actin). (b) average expression values for lipin and lipin as determined by western-blot and cell viability as determined by an mtt assay. data are shown as average and sd of six independent transduction experiments (n = ). (c) huh- cells were transduced with lentiviral vectors expressing control or lpin -specific shrnas. at day post-transduction, cells were infected at moi . with hcv d virus. samples of cell supernatants were collected at days and post-infection to determine the extracellular infectivity titer. average and sd of the infectivity titers at day and of two independent infections performed in triplicate (n = ). (d) huh- . cells were transduced with lentiviral vectors expressing control or lpin -specific shrnas. silenced cells were infected days after transduction at moi . with genotype a hcv (tncc) in the presence or absence of made ( μm; shcontrol+daa). intracellular hcv rna was determined by rt-qpcr hours post-infection. data are shown as average and sd of three experiments performed in triplicate (n = ). statistical significance was determined using student´s t-test ( Ã p< . ; ÃÃ p< . ). as expected, lipin and lipin expression is inversely correlated in lipin shrna-expressing cells (fig b) . the viability of lipin-deficient cells, as determined by mtt assay [ ] was comparable to that of control cells (fig b) . these results illustrate that lipin shrna-expressing cells respond homeostatically to functionally compensate partial (shlpin - ) and more pronounced (shlpin - ) loss of lipin (fig b) . control and lipin -deficient cells were infected with a genotype a d at moi . to study viral spread by determining extracellular infectivity titers at different times post infection. fig c shows limited propagation of this virus in lipin -deficient cells, with a statistically significant reduction of up to two (shlpin - ) and three orders of magnitude (shlpin - ) in viral titer between the control and lipin -deficient cell lines at day post-infection. these results indicate that lipin is required for efficient hcv propagation and that homeostatic lipin accumulation (fig b) is not sufficient to support efficient hcv infection. since important differences in the interaction of different hcv genotypes with cellular lipid metabolism have previously been described [ ] , we set out to determine if the observations made with a jfh -derived virus (genotype a) were extensive to other hcv genotypes. we performed low multiplicity infections (moi . ) with genotype a tncc virus strain in lipin -deficient huh- . cell lines, which are susceptible to infection by this recombinant virus [ ] . first, we verified that huh- . . also display a significant reduction in genotype a infection efficiency when lipin is silenced (s b fig) . in order to determine tncc infection efficiency, intracellular hcv rna accumulation was determined hours post-inoculation in control and lipin -deficient huh- . cells. viral rna detected under these conditions reflects the ability of genotype a to infect and replicate viral rna in the different cell lines, as treatment of control cells with an hcv polymerase inhibitor ´-c-methyladenosine ( made; μm) [ ] reduced viral rna content by two orders of magnitude ( fig d) . lipin silencing consistently and significantly reduced tncc replication by approximately -fold both in shlpin - and shlpin - cell lines (fig d) , indicating that lipin is also limiting for genotype a hcv infection. to determine the specificity of these observations, identical lipin -deficient and control huh- cultures were inoculated at moi . with a human alpha-coronavirus cov- e bearing a gfp reporter gene (hcov- e-gfp) [ ] . inoculation of control and lipin -deficient cells with this virus resulted in comparable progeny virus production, as determined by infectivity titration in cell supernatants hours post-infection (s fig) . these results suggest that lipin is not rate limiting for cov- e-gfp infection and that lipin expression is particularly limiting for hcv. to determine which aspects of the hcv replication cycle are limited by lipin silencing, single cycle infection experiments were conducted by inoculating control and lipin -deficient cell cultures at moi with genotype a d virus. infection efficiency was measured by titration of progeny virus infectivity present in the supernatant of infected cells and intracellular hcv rna accumulation at and hours post-infection. infection of lipin -deficient cells resulted in a significant reduction of progeny infectious virus production in shlpin - and shlpin - cells as compared with the titers observed in the supernatants of control cells ( fig a) , reinforcing the notion that lipin silencing interferes with hcv infection. reduced virus production is likely due to parallel reduction of intracellular hcv rna levels observed in lipin -deficient cells as compared with the control cell line (fig b) . this reduction was observed at all time points, except for that at hours, indicating that the size of the inoculum and initial virus adsorption is comparable among the different cell lines (fig b) . thus, lipin silencing suppresses hcv infection by interfering with a step of the hcv lifecycle preceding intracellular hcv rna accumulation. next, we set out to determine if lipin silencing has any impact on persistent hcv infections to verify if lipin is also limiting for late aspects of the virus lifecycle. persistently infected cells continuously replicate viral rna, express viral antigens and secrete infectious virions. thus, it is a valuable system to measure steady-state hcv rna replication as well as infectious particle assembly and secretion. persistently infected cultures were transduced with the lentiviral vectors described above to produce persistently infected, lipin -deficient cells (s fig) . shown as genome equivalents per microgram of total rna ge/μg). panels c, d-persistently infected cultures were generated by inoculation with jfh- virus at moi . . once cultures reached > % of hcv-positive cells, they were transduced with lentiviral vectors expressing control, hcv rna-targeting or lpin -specific shrnas. at day post-transduction, cells were split and samples of the cells and supernatants were collected hours later to determine infectious virus production rate by infectivity titration hcv (c) and rna levels by rt-qpcr (d). all data are shown as mean and sd of independent experiments performed in triplicate (n = ). statistical significance was determined using student´s t-test ( Ã p< . ; ÃÃ p< . ). analysis of extracellular infectivity titers revealed that infectivity titers in lipin -deficient and control cells were comparable, with the exception of a marginal reduction in shlpin - expressing cells, indicating that lipin silencing does not strongly interfere with infectious virus production ( fig c) . intracellular hcv rna levels in lipin -deficient cells were comparable to that of the control cells (fig d) , indicating that lipin expression is not rate limiting for hcv rna replication once infection has been established. taken together, the results shown above indicate that lipin is only limiting at early steps of hcv infection leading to viral rna accumulation. as an independent verification of the hypothesis that lipin is limiting for early aspects of hcv infection, we used a single cycle surrogate infection model based on the production of hcv virions bearing a defective reporter genome encapsidated by trans-complementation (hcv tcp ). hcv tcp are capable of producing abortive single-cycle infections, efficiency of which is proportional to the luciferase activity found in the target cells [ ] . as expected from the results shown in fig , infection of lipin -deficient cells with hcv tcp resulted in a % reduction in the reporter luciferase activity in shlpin - cells and % in shlpin - determined at hours post-infection, proportionally to the degree of silencing in these cells ( fig a and b ). these results underscore the role that lipin plays at early steps of hcv infection and indicate that either viral entry or a step leading to efficient hcv rna replication is impaired in these cells. lpin- mrna is alternatively spliced in human liver to produce isoforms α and β, which may differ in catalytic activity, subcellular localization and gene expression regulation [ ] . to determine the relative contribution of these isoforms to hcv infection, isoform-specific shrnas were generated and used to transduce huh- cells, as described above. lpin α-specific shrna (shlpin - ) reduced total lipin protein by %, while lpin β-specific shrna (shlpin - ) reduced total lipin expression by only % (fig a) . infection of control and lipin isoform-deficient cells with hcv tcp revealed that lipin α silencing resulted in a strong ( %) reduction in hcv infection while lipin β silencing resulted in a milder ( %) but significant reduction in hcv infection efficiency ( fig b) . these results indicate that both isoforms alpha and beta are limiting for hcv infection and suggest that the total amount of lipin present in the cell determines hcv infection efficiency. taken together, the results obtained with four different shrnas indicate that total lipin expression levels strongly correlate with hcv tcp infection efficiency (fig c) , underscoring a role for this host protein in early aspects of hcv infection. reduced hcv rna accumulation in a single cycle infection (fig ) as well as reduced hcv tcp infection (fig ) may be due to a defect in entry of incoming virions. hcv e e -pseudotyped retroviral vectors bearing a luciferase gene (hcv pp ) were used to measure viral entry because they constitute a sound model to study viral adsorption, receptor-mediated internalization and e e -mediated fusion in endosomes [ ] . to assess the specificity of these observations, parallel cultures were inoculated with vsv-g pseudotyped retroviral vectors (vsv pp ). control and lipin -deficient cell lines were infected with hcv pp (genotype a; jfh- strain) and vsvpp. as a positive control of inhibition of hcv entry, we used hydroxyzine ( μm), which efficiently blocks hcv infection by interfering with viral entry [ ] . as expected, hydroxyzine selectively inhibited hcv pp infection, as shown by reduced luciferase levels hours postinoculation only in hcv pp -infected cells ( fig a) . interestingly, lipin -deficient cells (shlpin - and shlpin - ) were fully susceptible to hcv pp and vsv pp infection, as comparable luciferase activity levels were found in all cell lines hours post-inoculation ( fig a) . these results indicate that lipin is not rate limiting for receptor binding, particle internalization or e e -mediated endosomal fusion, which are steps recapitulated in this model [ ] . based on the data presented thus far, we hypothesized that lipin is limiting for a step in the hcv lifecycle downstream of hcv entry, leading to hcv rna accumulation. to verify the hypothesis that lipin silencing causes strong reduction in initial hcv rna accumulation by interfering with a step downstream of viral entry, we bypassed this step of the viral replication cycle by transfecting a subgenomic hcv rna replicon bearing a reporter luciferase gene into control and lipin -deficient cells. first, we evaluated hcv-ires driven primary translation of incoming genomes by transfection of a replication-deficient mutant replicon that bears an inactivation mutation in the catalytic site of ns b rna polymerase [ ] . luciferase activity measured at hours post-transfection was not reduced in any of the cell lines, indicating that transfection efficiency and hcv ires-dependent primary translation was not significantly affected by lipin silencing (fig b) . a significant increase in renilla luciferase activity was observed in shlpin - cells both when transfecting a replicon ( fig b) or a plasmid expressing renilla luciferase under a minimal rna polymerase ii promoter (s a fig), suggesting that the increase in luciferase activity observed in these cells is not related with hcv. hcv rna replication was evaluated by measuring accumulation of a reporter luciferase gene at and hours post-transfection of a replication competent hcv subgenomic replicon. under these experimental conditions luciferase accumulation at hours also represents hcv ires-driven primary translation of the input rna, while reporter luciferase activity at hours depends on effective hcv rna replication. in contrast to primary translation, hcv rna replication inferred by luciferase activity at hours was strongly reduced in lipin -deficient cells ( % in shlpin - and % in shlpin - cells) (fig c) , indicating that initiation of hcv replication is dependent on normal lipin expression. this reduction was not due to a non-specific defect in luciferase expression, as co-transfection of a plasmid expressing renilla luciferase lead to comparable luciferase accumulation in all cell lines, discarding the possibility that death of transfected cells or other spurious effects are responsible for the reduced luciferase activity accumulation (s a fig) . similar experiments were conducted in atg b-deficient cells, as this host factor was shown to be limiting for primary hcv translation [ ] . our studies confirmed that, while partial atg b silencing (s b fig overall, these data indicate that hcv rna replication is not initiated efficiently in lipin -deficient cells and that blockade occurs at a step downstream translation of incoming hcv genomes. in order to determine if any of the known functions ascribed to lipin is required for hcv infection, we tested the ability to restore hcv infection susceptibility of silencing-resistant wild-type (wt) or mutant lipin versions bearing a mutation in the catalytic site responsible for its phosphatase activity (dxdxt) and a mutant in the lxxil motif, which is inactive both for transcriptional activation as well as for phosphatase activity [ ] . control and lipin -deficient cells were transfected with wt lipin beta cdna as well as with dxdxt and lxxil mutants. comparable overexpression levels of the wt and mutant proteins was obtained in each cell line, although overexpressed lipin levels were consistently higher in lipin -deficient cells (fig a) . cells were subsequently inoculated with hcv d at moi and relative infection efficiency was calculated by determining extracellular infectivity titers hours post-infection. overexpression of wt lipin did not significantly alter susceptibility to hcv infection in control cells, although we observed a small but consistent reduction in extracellular infectivity titers when overexpressing wt and mutant lipin constructs (s a fig using relative infection efficiency as readout of this set of experiments, we could clearly observe a statistically significant increase ( -fold) in the relative infection susceptibility in cells overexpressing wt lipin cdna as compared with mock-transfected cells or cells expressing similar or higher levels of the mutants (fig a) , suggesting that wt lipin modestly, though significantly, rescues hcv infection while phosphatase (dxdxt) and transcriptional coactivation (lxxil) mutants do not ( fig b) . these results suggest that lipin transcriptional coactivation capacity is not sufficient to support hcv infection while lipin phosphatase activity is essential. however, given that lxxil mutant is deficient both in transcriptional co-activation and pap activity [ ], we cannot determine if transcriptional co-activation by lipin is also required to support hcv infection. the data described above suggest a role for lipin phosphatase activity in a step of hcv replication cycle between translation of the viral genome and formation of functional replicase complexes. data regarding primary translation where inferred from a surrogate model of translation based on a reporter luciferase gene ( fig b) . to address if indeed viral polyprotein is properly processed and inserted into detergent-resistant microdomains to form the characteristic membranous ultrastructures bearing the viral replicase, we used a replication-independent surrogate model of polyprotein expression. this system is based on a vector encoding the portion of the viral polyprotein corresponding to the replicase (ns -ns b) under the transcriptional control of the t polymerase and the translational control of encephalomyocarditis virus (emcv) ires (ptm-ns / b) [ ] . replication-independent polyprotein overexpression control and silenced cells were inoculated with hcv e e (hcv pp ) or vsv-g-pseudotyped retroviral vectors (vsv pp ) days post-transduction. control cells treated with hydroxyzine ( μm) were used as positive inhibition control. luciferase activity was determined in the different cell lines at hours post-inoculation. hdx, hydroxyzine pamoate ( μm). panels b, c-control and lipin -deficient cells were transfected with a replication-deficient mutant (b) or replication competent subgenomic hcv replicon bearing a luciferase gene (c). luciferase activity was determined in the different cell lines at hours post-transfection for both replicons and hours post-transfection for the replication-competent replicon rna. data are expressed as average and sd of three independent experiments performed in triplicate (n = ). statistical significance was determined using student´s t-test ( Ã p< . ; ÃÃ p< . ). https://doi.org/ . /journal.ppat. .g systems enable assessment of polyprotein processing as well as studying the formation of virus-derived membranous structures [ , ] . control and lipin -deficient cells were infected with a recombinant vaccinia virus expressing t rna polymerase (vact ) and subsequently transfected with the plasmid ptm-ns / b to enable viral replicase expression. sixteen hours post-transfection, cells were processed for lipin is required for hcv replicase formation western-blot using anti-ns . accumulation of ns is comparable in control and both lipin deficient cells, underscoring the notion that lipin is not limiting for polyprotein translation and processing (fig a) . similarly, ns and ns a expression and subcellular distribution was similar in all cell lines (fig b) . these results suggest that there are no major differences in accumulation of viral proteins in lipin -deficient cells and that a step downstream is affected in these cells. transmission electron microscopy (tem) of ultrathin cell sections of cells expressing hcv replicase components shows the expected accumulation of a mixture of characteristic doublemembrane vesicles (dmv) as well as multiple membrane vesicles (mmv) (fig d, e and f) that were not found in mock-transfected cells (fig c) , as reported in previous studies using similar systems [ ] . individual vesicle diameter displays heterogeneous size distribution in which the predominant population is distributed between - nm (median nm; average ± sd: ± nm, n = ) with larger vesicles being less predominant (fig g and s fig) . this size distribution is compatible with that observed similar replication-deficient systems and during hcv infection. treatment of these cells with nm daclatasvir (dctv), resulted in a strong reduction in the number of vesicles per section area (s b fig) , without significantly altering the size distribution of the remaining vesicles (fig g) , as reported by berger et al. [ ] . similarly, the diameter distribution of the vesicles found in lipin -deficient cells was comparable to that in control cells ( fig g) . however, hcv-induced structures were significantly less abundant hours post-transfection in lipin -deficient cells than in controls cells (s c fig) . this reduced abundance is illustrated by a significant reduction in the fraction of cells displaying vesicular structures in lipin -deficient cell cultures (fig h) despite comparable transfection efficiency and viral protein expression levels, indicating that lipin may be required in a critical step leading to formation of the hcv-induced vesicular compartment. to validate the tem results independently, we set out to establish a biochemical assay to evaluate replication-independent replicase complex formation. one of the characteristics of the hcv replicase complexes is that they are located in detergent-resistant membranes (drm) [ , ] . in this sense, ns proteins are associated with replicase complexes that co-sediment with drm markers such as caveolin- or sigma- receptor (sigmar ) in low-density fractions in isopycnic gradient ultracentrifugation experiments [ , ] . control and lipin deficient cells were infected with vact and subsequently transfected with limiting doses of the plasmid ptm-ns / b [ ] . parallel samples were treated with dctv ( nm). sixteen hours post-transfection, cell lysates were generated and subjected to equilibrium ultracentrifugation in - % sucrose gradients. gradient fractions were collected and subjected to western-blot analysis to determine the impact of lipin silencing on ns , sigmar and actin sedimentation profiles. fig a shows how, as previously shown for replicon and jfh -infected cells, ns can be detected in drm fractions (fractions - ), as determined by the presence of sigmar in those fractions (fractions - ; s a fig), although in this experimental system, unlike during viral infection [ ] , most ns co-sediments together with solubilized proteins, as shown for actin (fractions - ; s a fig) . drm-associated ns is reduced in dctv-treated and lipin -deficient cells, while total ns expression remains unchanged (fig a) . four independent experiments were performed in which relative-drm associated ns , normalized to that found in solubilized fractions, was calculated for each experimental condition (fig b) . lipin -deficient cells display a consistent and statistically significant reduction in the drmassociated, but not total ns abundance (fig a and b; shlpin - and shlpin - ) , similar to that observed in control cells in the presence of dctv (figs a and b; shcontrol+dctv) , consistent with the tem data (figs and s ) and the notion that ns in drm fractions may reflect the abundance of replicase complexes formed in these cells. this reduction is not due to an overall reduction in cellular drm abundance in lipin -deficient cells, as lipin -deficient cells display similar sigmar distribution pattern as the control cells (s fig). overall, tem data and drm floatation assays strongly suggest that lipin is rate limiting for the generation of replicase complexes from fully processed polyprotein subunits. hepatitis c virus replication cycle is tightly linked to host cell lipid metabolism and interference with cellular lipid homeostasis contributes to viral pathogenesis [ ] . one of the most evident consequences of this interference is the high prevalence of liver steatosis among chronically infected patients [ , ] . this clinical manifestation of the infection has been linked to, among others, chronic er stress, mitochondrial dysfunction and metabolite depletion induced by hcv infection, which result in the activation of persistent homeostatic adaptation of the cellular lipid metabolism to permit cell survival, at the cost of pathogenic metabolic alterations [ , [ ] [ ] [ ] [ ] . among the different regulatory networks that have been shown to be stimulated during hcv infection, pparα [ ] , pgc- α [ ] , hif- [ ] and srebp [ , ] have also been shown to regulate transcription of lpin mrna [ , , , ] . thus, it is likely that stimulation of one or several of these regulatory networks by hcv infection results in the lpin mrna transcriptional activation observed in this ( fig a) and other studies [ , ] . importantly, prevention of lpin mrna accumulation with nac ( fig e) did not significantly interfere with hcv rna replication (fig f) , suggesting that enhanced lpin mrna accumulation is not required for efficient hcv infection. we favor the hypothesis that ros induced by hcv protein accumulation actively participates in lpin induction, as treatment with the antioxidant nac prevented lpin mrna accumulation, similar to what has been shown for other srebp-regulated genes during hcv infection [ ] . accumulation of lpin mrna during hcv infection results in concomitant protein accumulation (fig g and h) . however, post-translational mechanisms such as phosphorylation, acetylation or sumoylation regulate lipin protein stability, membrane association as well as subcellular localization thus influencing the activity of lipin as pa-phosphatase and as transcriptional coactivator [ ] . hence, it is difficult to predict the implications of lipin protein accumulation during hcv infection. thus, future studies on the interference of hcv infection with cellular lipin functions will be required to determine its role in hcv-related pathogenesis, particularly in its contribution to steatosis. lipin is required for hcv replicase formation the data presented in this study provide evidence that lipin is rate limiting for hcv infection at an early step of the infection leading to formation of membranous hcv replicase complexes, downstream of viral polyprotein expression and processing. we provide evidence for reduced accumulation of viral rna during single cycle infection experiments (fig d) , that is reminiscent of a faulty initiation of viral replication, as suggested by reduced replication of a transfected subgenomic replicon in lipin -deficient cells (fig d) . data obtained in replication-independent polyprotein expression models suggest that generation of the membranous compartment that contains functional replicase complexes is severely limited in lipin -deficient cells, as suggested by a significant reduction of the fraction of cells where these structures could be visualized by tem (figs and s ). this hypothesis is further supported by a significant reduction in drm-associated ns proteins in lipin -deficient cells (fig ) , which may reflect limitations in the association of viral replicase subunits with cholesterol and sphingolipid-rich membranes in lipin -deficient cells [ , , ] . four different shrnas targeting lpin mrna decrease susceptibility to hcv infection proportionally to their ability to reduce total lipin protein accumulation (fig ) . these results, together with the cdna rescue experiments (fig ) , strongly reduce the possibility of observing rnai-associated off-target phenomena. interestingly, homeostatic accumulation of lipin protein in lipin -deficient cells (fig a and b) is not sufficient to compensate for lipin loss to support efficient hcv infection. the notion that, despite being capable of mutually compensating basic liver functions [ , ], lipin and lipin play non-redundant functions in the liver has previously been proposed [ , , ] . lipin is tightly regulated at many different levels and its activity accommodates pap activity in response to different physiological situations such as fasting and insulin signaling [ ] . compelling evidence indicates that, while lipin and lipin cooperate to maintain liver lipid homeostasis, the two proteins differ in many aspects. for instance, lipin is transcriptionally induced by pgc- α and it is also an inducible amplifier of this transcriptional network [ ], whereas lipin is not [ ] . lipin is sumoylated and sumoylation regulates its nuclear localization and function, whereas lipin sumoylation could not be demonstrated, despite the presence of a canonical sumoylation motif in its primary sequence [ ] . lipin enzymatic activity is blocked by mammalian target of rapamycin (mtor)-dependent phosphorylation in response to different metabolic stimuli [ , ], whereas lipin is constitutively active even when phosphorylated [ ] . thus, lipin is considered more as a constitutive phosphatidic acid phosphatase with lower specific activity than lipin [ ] . in addition to these differential regulatory networks, it has been shown that in vitro pap activity of purified lipin and lipin is differentially influenced by the composition of the substrates (liposomes and lipid-detergent micelles) as well as the ph at which the assay is performed [ ] . this differential lipid substrate recognition may be reminiscent of the different preferential association with membranes of different subcellular compartments (s fig) [ , ] . these differences suggest that, while lipin and lipin may share some common features, they are not functionally interchangeable, particularly not in the case of hcv infection [ ] . our data support the notion that lipin silencing has a strong impact on hcv infection without affecting basic cellular functions (fig b) or significantly interfering with infection by an unrelated virus (s fig). despite great efforts and different overexpression systems, functional rescue of lipin functions by wt lipin cdna overexpression in lipin -deficient cells only lead to a small but consistent rescue of virus infection efficiency, which was only observed when overexpressing wt lipin (figs and s ) . given the multiple transcriptional, post-transcriptional and post-translational regulation levels existing for lipin expression, it is conceivable that only a fraction of the overexpressed lipin is fully competent to sustain hcv infection. moreover, high overexpression levels could only be achieved in lipin -deficient cells (fig a) , underscoring the notion that intracellular lipin levels are tightly regulated by the host. nevertheless, overexpression of a mutant lipin lacking phosphatase activity (dxdxt) or a mutant inactive as transcriptional coactivator (lxxil) were not capable of enhancing hcv infection in lipin -deficient cells as compared with the wt lipin ( fig b) . these data reveal that lipin phosphatase activity is required for lipin to support hcv infection and suggest that, while transcriptional co-activation by lipin may be important, this function is not sufficient to support hcv replication. lipins are important enzymes in the main pathway for de novo phospholipid biosynthesis by providing dag derived from the glycerol- -phosphate pathway to produce pc and pe through the kennedy pathway [ ] . production of membranous replicase compartments likely requires de novo synthesis of pc and/or pe, which are major components of biological membranes that depend on dag biosynthesis [ ] . in fact, local pc biosynthesis is required for efficient replication of (+) rna viruses and certain pc species accumulate in hcv-infected cells [ , ] . although increased lipin accumulation may be sufficient to compensate lipin silencing at the whole-cell level [ ], it is possible that acute, local demand of de novo synthesized phospholipids is required at defined suborganellar compartments during early steps of hcv infection and that lipin -deficiency shortens or alters the availability of different membrane components, demand that may not be satisfied by lipin , given the differential regulation [ ] and subcellular localization of these two proteins (s fig). remarkably, deletion of the yeast lipin homologs pah /smp (s. cerevisae) or ned (s. pombe) gene, results in deregulated proliferation of the er and nuclear envelope membranes [ , ] , with concomitant enhancement in (+) rna virus replication [ , ] . the membranous alterations and elevation of total phospholipid content observed in pah -deficient yeast have been ascribed to transcriptional activation of pah -independent alternative phospholipid biosynthetic programs due to pa accumulation [ , ] . our data in mammalian cells are more compatible with a shortage of phospholipid production, which may be at the basis of the reduced abundance viral membranous structure (figs and ) . this opposite outcomes of infection may derive from the fact that yeast use mainly pa (lipin pap substrate) as a precursor for pc biosynthesis, while mammals mainly use dag (lipin pap product) as precursor for pc biosynthesis through the kennedy pathway [ , [ ] [ ] [ ] . moreover, in contrast to yeast and lower eukaryotes, which express only one lipin gene, three different lipin genes coordinate glycerolipid homeostasis in mammals [ ] . thus, interfering with expression of one of the members of the family may not be sufficient to observe the same effects observed when deleting pah , as transcriptional and posttranslational homeostatic compensations are in place in mammals, particularly between lipin and lipin in liver tissue [ , ] . in this sense, deletion of either lipin or lipin in mouse models results in a relatively balanced liver phospholipid content while simultaneous deletion of both lipins is embryonically lethal [ ] . accordingly, only minor alterations of er membranes [ ] and no significant alterations in total pc levels [ ] have been reported in lipin -deficient mouse liver. given the fact that hcov- e is fully capable of replicating in these cells (s fig), it is unlikely that a general disruption of de novo phospholipid biosynthesis occurs in lipin -deficient cells, particularly since hcov- e infection also induces profound er membrane rearrangements required for replication [ ] , some of which are structurally similar to dmvs observed during hcv infection [ ] . thus, we favor the hypothesis that a subcellular pool of glycerophospholipids is managed by lipin in huh- cells and that lipin silencing perturbs local levels of pa and dag, limiting local availability of precursors of structural components of virus-induced membranes. alternatively, lipin deficiency may alter local amounts of important signaling molecules, in particular, that of its substrate (pa) or its product (dag). deregulation of the local pa and dag pools may cause important alterations for the host cell, as both metabolites are potent chemical messengers that regulate different aspects of cellular homeostasis [ ] [ ] [ ] . regarding pa conversion into dag by lipins, it has been shown that pah (yeast lipin homolog) phosphatase activity is critical for transforming local pools of pa into dag at the er membrane to facilitate membrane fusion events mediated by snare complexes [ , ] . mammalian lipin phosphatase activity is also critical for transforming local pools of pa that accumulate at the surface of mitochondria to promote mitochondrial fission [ ] or at the surface of endolysosomes to facilitate autophagy [ ] . thus, lipin and probably other members of the lipin family modulate different aspects of intracellular membrane signaling. given that the function of host factors known to be involved in functional hcv replicase biogenesis, like vapa, vapb and osbp [ ] are indirectly regulated by local pa/dag pools [ ] , it is tempting to propose that lipin silencing interferes with the function of one or several of these, or other yet uncharacterized cellular factors. in contrast to what has been reported for other host factors required for hcv replicase complex formation [ ] , we did not find evidence of lipin protein relocalization during hcv infection (s fig). thus, determining the precise mechanism by which lipin regulates hcv replicase formation is challenging, as association of lipin with different cell membranes is transient and highly regulated by posttranslational modifications [ ] . moreover, some of the known lipin cellular functions may be compensated by other lipins, particularly lipin in the liver. nevertheless, our data clearly indicate that lipin participates at early stages of hcv replication and that the aforementioned homeostatic compensations by other lipins in regards to cellular metabolism may constitute an advantage when considering lipin as a host target for anti-hcv therapy. hcv antiviral compounds ´-c-methyladenosine ( made), sofosbuvir and daclatasvir were obtained from boc sciences (ny, usa), selleckchem (texas, usa) and medchem express (new jersey, usa) respectively and dissolved in dmso to obtain mm stock solutions. nacetylcysteine (nac) and puromycin were obtained from sigma-aldrich (missouri, usa), dissolved in water to a final concentration of . m and mg/ml respectively. hydroxyzine pamoate was purchased from sigma-aldrich (missouri, usa) and dissolved in dmso to a final mm concentration. human hepatoma huh- and derived subclones huh- . , huh- . . (clone ) have been described [ ] [ ] [ ] and were kindly provided by dr. chisari (tsri-la jolla, ca). hek- t cells [ ] were kindly provided by dr. ortin (cnb-madrid, spain). cell cultures were maintained subconfluent in dulbecco´s modified eagle´s medium (gibco) supplemented with mm hepes (gibco), u/ml penicillin/streptomycin (gibco), μm non-essential amino acids (gibco) and % fetal bovine serum (sigma-aldrich). genotype a (jfh- strain), d adapted virus (d ) and genotype a (tncc) virus have been described elsewhere [ , , ] . tncc plasmid was kindly provided by dr. jens bukh (huidovre hospital; copenhagen, denmark). recombinant viruses were generated by electroporation of an in vitro transcribed rna as described previously [ ] , using clone cells (jfh- and d ) virus and huh- . cells for tncc. supernatants containing the infectious virus were used to inoculate naive huh- . . clone cells at low multiplicity of infection (moi . ffu/cell) to prepare working virus stocks. for tncc infections, supernatants of electroporated cells were directly used. lentivirus production. lentiviral vectors shrna expression were produced by co-transfection of plasmids pmdl-rre, pmd g and prsv-rev (a gift from didier trono-addgene plasmids # ; # ; # ), together with the genomic plasmid (mission plko-puro; sigma-aldrich) into hek- t cells [ ] . selected shrna target sequences are: cgagagaa agtggttgacata (shlpin - ); (cctcagacagaaatgcagttt) shlpin - ; cact cccagtccttccggttc (shlpin - ); cctgttccatccttcggaaag (shlpin - ). plasmids encoding a atg b shrna (atg b ) [ ] , non-targeting shrna (shcontrol) as well as an shrna targeting hcv ires (shhcv)were previously described [ ] . a lentiviral vector plasmid encoding lipin -alpha cdna was purchased from genecopeia (cat nbr: ex-t -lv ). to generate lipin beta isoform, exon sequence was inserted using neb-q mutagenesis kit (neb). similarly, silent mutations were introduced in shlpin - target sequence to obtain wt lipin beta protein expression from a cdna resistant to silencing. mutations in the conserved motifs dxdxt (didgt>eidgt) and lxxil (lghil>lghff) were also introduced in lipin beta cdna using neb-q kit, based on previously described mutations [ , ] . the sequence of the entire lpin cdnas was determined by sanger sequencing before use, to assess the introduction of only the desired mutations. coronavirus e-gfp. recombinant human coronavirus e-gfp [ ] was kindly provided by dr. volker thiel (university of bern, switzerland). virus was propagated by infection (moi . ) in huh- cells. supernatants were collected at different time points and titrated by end-point dilution and immunofluorescence microscopy. lentiviral vectors expressing control and lpin -specific shrnas were used to inoculate huh- cells. twenty-four hours later, cells were subjected to selection with . μg/ml of puromycin to assess the lowest lentivirus dose capable of conferring puromycin resistance to % of the cell population. selected cell populations were subsequently cultured in the presence of puromycin until lpin silencing was ascertained by western-blot using anti-lipin antibodies, typically at day - post lentiviral transduction, time at which all experiments were performed in the absence of puromycin. before execution of all the experiments shown in this study, lipin expression was assessed by western-blot. cell viability was determined by a thiazolyl blue tetrazolium blue (mtt) formazan formation assay [ ] . control and lipin -deficient cell lines ( . cells/well) were plated onto -well plates and were inoculated with d virus at a moi ffu/cell. samples of the cells and supernatants were collected , and hours post-infection. for multiple cycle infection experiments (moi . ), samples of the supernatants were collected at day , and post-inoculation. cells were split : in the multiple cycle infection experiments at days and to maintain the cultures subconfluent. extracellular infectivity titers were determined by endpoint dilution and infection foci counting as previously described [ ] . intracellular hcv rna was determined by reverse transcription and quantitative pcr (rt-qpcr) as previously described [ ] . total protein samples were prepared in laemmli buffer and separated using polyacrylamide denaturing gel electrophoresis (sds-page). proteins were subsequently transferred onto pvdf membranes and incubated with % milk (lipins) or % bsa in pbs- . % tween for one hour at room temperature (rt). primary antibodies against lipin (clone b- ; santa cruz), lipin (h- ; santa cruz), ns (clone e ; biofront), beta-actin (ab ; abcam) and tubulin (clone aa ; sigma-aldrich) were diluted in pbs- . % tween and incubated for hour (four hours for lipins) at rt. membranes were subsequently washed for minutes with pbs- . % tween three times. horseradish peroxidase-conjugated secondary antibodies were incubated for hour at room temperature in % milk-pbs- . % tween and subsequently washed three times for minutes at room temperature. protein bands were detected using enhanced chemoluminescence reactions and exposure to photographic films. specific bands were quantitated using the imagej software [ ] on non-saturated, scanned films. confocal microscopy was performed with a leica tcs sp laser scanning system (leica microsystems). images of × pixels at eight bit gray scale depth were acquired sequentially every . - . μm through a x/ . n.a. immersion oil lens, employing las af v . . software (leica microsystems). colocalization indexes were calculated using jacop plugin for image j [ ] from a minimum of regions of interest (roi). images were processed using imagej, were medians of pixel were obtained for the different channels, only for illustration, not for analysis. color levels, brightness and contrast were manipulated for illustration using technical and biological controls as reference. total rna extraction was performed using the gtc extraction method [ ] . purified rna ( - ng) was subjected to rt-qpcr using random hexamers and a reverse transcription kit (applied biosystems). quantitative pcr was performed using x reaction buffer from (applied biosystems) and specific oligonucleotides as previously described [ , ] . standard curves were prepared by serial dilution of a known copy number of the corresponding amplicon cloned in a plasmid vector. control and lipin -deficient huh- . cells were inoculated with tncc virus (moi . ). due to the relatively low propagation levels of the tncc virus in this experimental setup, parallel cultures were infected and treated with ´-c-methyladenosine ( made; μm) to determine the levels of non-replicative, background hcv rna. cells were incubated for hours at ˚c, time after which samples of the cells were collected to determine intracellular hcv rna levels by rt-qpcr. to establish persistently infected cell cultures huh- cells were inoculated at moi . with jfh- hcv strain as previously described [ ] . cell cultures were maintained subconfluent for two weeks, time after which infection rates reach nearly % of the cells, as assessed by immunofluorescence microscopy. at this point cells were split and transduced with the corresponding lentiviral vectors in order to generate lipin -deficient cell cultures as well as control cell lines. once silencing had been verified by western-blot, typically at day - post-transduction, cells were split and samples of the cells and supernatants were collected hours later to determine intracellular hcv rna levels by rt-qpcr and extracellular infectivity titers by end-point dilution and immunofluorescence microscopy. infectious, spread-deficient hcv particles produced by trans-complementation (hcv tcp ) have previously been described [ ] . briefly, huh- . . clone cells expressing core-e and e -ns regions from jfh- by lentiviral transduction, were electroporated with a jfh- subgenomic dicistronic replicon bearing a firefly luciferase gene with reagents kindly provided by dr. ralf bartenschlager (u. of heidelberg). supernatants containing hcv tcp were collected , and hours post-electroporation, pooled and assayed for viral infectivity. hcv tcp infection efficiency was determined by inoculating naïve huh- cells with the electroporation supernatants and measuring luciferase activity hours post-infection using a commercially available kit (promega). retroviral particle production pseudotyped with different viral envelopes has previously been described [ , ] with the materials kindly provided by dr. f. l. cosset (inserm, lyon). control and lipin-deficient cell lines were inoculated with hcv pp and vsv pp and incubated for hours, time at which total cell lysates were assayed for luciferase activity using a commercially available kit (promega). a selective hcv entry inhibitor, hydroxyzine pamoate (hdx) from sigma-aldrich (missouri, usa), was used as positive control of inhibition [ ] . a plasmid containing the sequence corresponding to a subgenomic jfh- replicon bearing a firefly luciferase reporter gene was kindly provided by dr. ralf bartenschlager (u. of heidelberg) [ ] . after digestion with the restriction enzyme mlui, the linearized plasmid was transcribed in vitro using a commercial kit (megascript t ; ambion-paisley, uk). the resulting products were digested with dnase and precipitated with licl. pelleted rna was washed with % and % ethanol, and resuspended in nuclease-free water. in vitro transcribed rna was transfected into the different cell lines together with a plasmid expressing renilla luciferase under a minimal promoter (prl-null; clontech-california, usa) using lipofectamine and the manufacturer´s recommendations (life technologies-california, usa). firefly and renilla luciferase activities were measured in the sample using a commercial kit (dual luciferase assay system; promega-wisconsin, usa) at different times post-transfection. lipin -deficient cells were generated by lentiviral transduction of shlpin - shrna. at day post-transduction, control and lipin -deficient cell populations ( x cells/m well) were transfected in suspension using lipofectamine with plasmids ( ng/m well) expressing wt lipin beta isoform shlpin _ -resistant cdna or dxdxt or lxxil motif mutants [ ] . transfected cell cultures were incubated for hours and subsequently inoculated at moi with d virus. infection efficiency was determined by measuring extracellular infectivity titers hours post-infection. parallel cultures were used to determine relative wt and mutant protein expression efficiency by western-blot. infectivity titers were measured as described above. the relative impact of cdna expression was estimated by determining the ratio between the infectivity found in lipin -deficient cells and the control cells transfected with the same plasmid. in order to average experiments with different raw infection efficiency, all the experiments were referenced to the ratio in the mock-transfected cells. control and lipin -deficient huh- cells were inoculated with cov- e (moi . ) for hours at ˚c. cells were washed twice with warm pbs and replenished with dmem- % fcs. extracellular infectivity titers were determined hours post-infection by end-point dilution and fluorescence microscopy in huh- cells. huh- cells were inoculated at moi with a recombinant vaccinia virus expressing the t phage rna polymerase (vact ) [ ] . two hours later, cells were transfected with the plasmid ptm-ns / b [ , ] (kindly provided by dr. lohmann; u. of heidelberg) and lipofectamine (thermofisher-massachussets, usa) following the manufacturer´s recommendations in terms of total dna per well (typically μg per mm dishes with . x cells/well) and % of the recommended lipofectamine:dna ratio. transfected cells were cultured in the presence of the dna replication inhibitor cytosine β-d-arabinofuranoside (arac; sigma-aldrich) for hours to prevent vact replication [ ] . when indicated, media was also supplemented with nm daclatasvir (dctv). total cell extracts were used to determine viral protein accumulation by western-blot using anti-ns antibody (clone e ; biofront) and β-actin (abcam; ab ) as loading control. for ultrastructural electron microscopy studies, control and lipin -deficient cells expressing ns - b polyprotein (see above) were cultured on glass coverslips and fixed in situ after polyprotein expression with a mixture of % paraformaldehyde (taab) and . % glutaraldehyde (taab) ( h at room temperature), post-fixed with % osmium tetroxide in pbs ( min), treated with % aqueous uranyl acetate ( min), dehydrated with increasing quantities of ethanol and embedded in epoxy resin (taab). ultrathin, -nm-thick sections were cut in parallel to the monolayer, transferred to formvar-coated em buttonhole grids and stained with aqueous uranyl acetate ( min) and lead citrate ( min). sections were visualized on a jeol jem exii electron microscope (operating at kv). quantitation of hcv-induced structures was performed as follows. to quantitate the differences in total vesicle abundance, tem sections were visually inspected under the microscope for the presence/absence of vesicular structures. the number of positive cells and total number of cells were inserted in a x contingency table to determine the statistical significance of the differences between control and lipin -deficient cells using two-tailed fisher´s exact test or two-tailed chi square test. in addition, we determined the frequency of hcvinduced vesicles in dctv-treated control cells by dividing the number of structures per inspected area and calculating the average and sd of the frequencies found in the different images. the diameters of individual vesicles were determined manually using size-calibrated images and image j software. lipin -deficient and control cells ( . x cells) were infected with vact virus (moi ) and transfected with limiting doses of ptm-ns / b plasmid (typically ng/ well), as higher plasmid doses may difficult observing the reported differences. cells were lysed by adding μl of tne ( mm tris-hcl ph . , nacl mm and edta mm) buffer containing . % triton x- and protease inhibitors (complete; roche-basel, sw). lysates were incubated for minutes on ice before clearing them by -minute centrifugation at , r.p.m. clear supernatants were mixed : with % sucrose tne solution. this mixture was applied on top of a % sucrose-tne cushion and was overlaid with % and % sucrose-tne until completing the discontinuous gradient. gradients were centrifuged for hours at , x g. fourteen fractions were collected from the top and analyzed by sds-page and western-blot using antibodies against ns (clone e ; biofront), sigmar (s- ; santa-cruz), caveolin- (epitomics; - ) and beta actin as described previously [ ] . ns signal was quantitated using imagej software and the fraction of drm-associated ns was determined as the ratio of ns signal in fractions , and to the total ns signal in the gradient. global epidemiology and burden of hcv 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lipin gene expression in hepatoblastoma cells lipin proteins and glycerolipid metabolism: roles at the er membrane and beyond morphological and biochemical characterization of the membranous hepatitis c virus replication compartment three mammalian lipins act as phosphatidate phosphatases with distinct tissue expression patterns temporal and spatial regulation of the phosphatidate phosphatases lipin and sumoylation regulates nuclear localization of lipin- alpha in neuronal cells phosphorylation of lipin and charge on the phosphatidic acid head group control its phosphatidic acid phosphatase activity and membrane association lipin binds phosphatidic acid by the electrostatic hydrogen bond switch mechanism independent of phosphorylation the yeast lipin smp couples phospholipid biosynthesis to nuclear membrane growth an evolutionarily conserved fission yeast protein, ned , implicated in normal nuclear morphology and chromosome stability, interacts with dis , pim /rcc and an essential nucleoporin inactivation of the host lipin gene accelerates rna virus replication through viral exploitation of the expanded endoplasmic reticulum membrane host pah p phosphatidate phosphatase limits viral replication by regulating phospholipid synthesis interactions among pathways for phosphatidylcholine metabolism, ctp synthesis and secretion through the golgi apparatus phospholipid biosynthesis in eukaryotes the fatty liver dystrophy mutant mouse: microvesicular steatosis associated with altered expression levels of peroxisome proliferator-regulated proteins inhibition of cytosolic phospholipase a alpha impairs an early step of coronavirus replication in cell culture membranous replication factories induced by plus-strand rna viruses shaping up the membrane: diacylglycerol coordinates spatial orientation of signaling diacylglycerol kinases: shaping diacylglycerol and phosphatidic acid gradients to mitochondria: signaling with phosphatidic acid phosphatidic acid sequesters sec p from cis-snare complexes to inhibit priming a common lipid links mfn-mediated mitochondrial fusion and snare-regulated exocytosis pirna-associated germline nuage formation and spermatogenesis require mitopld profusogenic mitochondrial-surface lipid signaling. developmental cell lipin- regulates autophagy clearance and intersects with statin drug effects in skeletal muscle pkd regulates membrane fission to generate tgn to cell surface transport carriers. cold spring harbor perspectives in biology growth of human hepatoma cells lines with differentiated functions in chemically defined medium highly permissive cell lines for subgenomic and genomic hepatitis c virus rna replication interferon modulation of cellular micrornas as an antiviral mechanism production of high-titer helper-free retroviruses by transient transfection the cellular functions of the yeast lipin homolog pah p are dependent on its phosphatidate phosphatase activity robust hepatitis c virus infection in vitro nih image to imagej: years of image analysis a guided tour into subcellular colocalization analysis in light microscopy single-step method of rna isolation by acid guanidinium thiocyanate-phenol-chloroform extraction expression of the splicing factor gene sfrs is reduced in human obesity and contributes to enhanced lipogenesis cytoplasmic expression system based on constitutive synthesis of bacteriophage t rna polymerase in mammalian cells transient expression of the vaccinia virus dna polymerase is an intrinsic feature of the early phase of infection and is unlinked to dna replication and late gene expression we are grateful to drs. bartenschlager key: cord- - c wttxg authors: lin, huixing; zhou, hong; gao, lu; li, bin; he, kongwang; fan, hongjie title: development and application of an indirect elisa for the detection of antibodies to porcine epidemic diarrhea virus based on a recombinant spike protein date: - - journal: bmc vet res doi: . /s - - - sha: doc_id: cord_uid: c wttxg background: as the major causative agent of swine viral diarrhea, porcine epidemic diarrhea virus (pedv) has caused massive losses to the economies of swine raising countries. accordingly, the serological detection of corresponding antibodies would be beneficial to diagnose pedv indirectly to control the disease. in this study, an indirect enzyme-linked immunosorbent assay (elisa) based on the recombinant truncated spike (s) protein of pedv was developed and validated. results: the reaction conditions of the developed indirect elisa were optimized. this indirect elisa was compared to indirect immunoinfluscent assay (ifa), and the overall coincidence rate was . % based on testing clinical serum samples with different pedv antibody levels. no cross-reactivity with other common swine pathogens was detected for the developed s indirect elisa. finally, the s indirect elisa was applied to detect serum antibodies of field samples collected from different pig farms in eastern china, and it presented an overall substantial agreement on the pedv infection status. conclusions: this established s indirect elisa is capable of detecting serum antibodies against pedv, and due to its high sensitivity and specificity, it could be applied for serological evaluation and indirect diagnosis of pedv infection. porcine epidemic diarrhea (ped) is a highly contagious swine enteritis caused by porcine epidemic diarrhea virus (pedv), which belongs to the order nidovirales and family coronaviridae. the typical symptoms of ped are diarrhea, vomiting, and dehydration, which can be especially dangerous to suckling piglets [ , ] . the mortality of neonatal piglets younger than days old can approach % [ ] [ ] [ ] . ped first appeared in britain in , followed by an outbreak of diarrhea in several pig farms in belgium in [ ] . these outbreaks led to identification of a coronavirus-like particle named cv , which is now recognized as the classic pedv strain. in recent years, ped epidemics have become prevalent in swine-raising countries in asia, including south korea, china, japan, and vietnam, and can cause enormous economic loss [ , ] . pedv is a single-stranded rna virus composed primarily of four structural proteins: the spike protein (s, - kda), membrane protein (m, - kda), envelope protein (e, kda) and nucleocapsid protein (n, - kda). s protein is located on the surface of the virus particle. it is categorized as a type i membrane fusion protein and has the significant biological effect of binding to target cell receptors and entering the cell through plasma membrane fusion [ , ] . the s protein has higher antigenicity than any of the other pedv proteins, and anti-s antibodies detected in pedv-infected pigs persist longer than anti-n antibodies [ ] . the s protein can be separated into the s ( - aa) subunit and the s ( - aa) subunit [ ] . the s subunit is the extracellular domain and can recognize and bind to target cell receptors [ ] , and it is closely linked to the formation of neutralizing antibodies. therefore, this study selected a gene fragment within the s subunit as a coating antigen to develop an indirect enzyme-linked immunosorbent assay (elisa) method for the detection of pedv antibodies. the pedv yc strain was isolated on a breeding farm in yancheng city in (genbank: ku . ). the prokaryotic expression vector pet- a(+) was purchased from biovector ntcc inc. (beijing, china). the hrp-goat anti-pig iga, hrp-goat anti-pig igg, and fitc-goat anti-pig iga was purchased from abcam plc. (shanghai, china). the standard pedv negative serum were collected from specific pathogen free (spf) pigs. the standard pedv positive serum were collected from experimentally pedv immunized spf pigs, at , , , , , and day post-inoculation (dpi). these standard serum were identified of pedv-specific antibodies positive by both indirect immunoinfluscent assay (ifa) and seroneutralization assay (sn) as previously described [ , ] . the swine porv antibody elisa kit and the swine tgev elisa kit were obtained from ingenasa (madrid, spain). the gene sequence of truncated spike protein (named s ) was amplified from the genomic rna of pedv yc strain (genbank: ku . ) by reverse-transcriptase (rt)-pcr. the forward primer was ' cgcggat ccgtcactaggtgccagtccactattaa- 'and the reverse primer was '-cccaagctttcaattgtaaa tatccactttaagaaaacaataa- ′. underlined portions represent bamh i and hind iii restriction sites, respectively. the target gene was bp in length and subcloned into the prokaryotic expression vector pet- a(+), then transformed into a strain of competent e. coli cells, dh α. transformed colonies were selected from luria-bertani (lb) agar plates containing kanamycin ( μg/ml) and were identified by pcr. the resulting recombinant expression plasmid was named a-s and was identified by double enzyme digestion and dna sequence analysis. subsequently, the recombinant expression plasmid a-s was transformed into e. coli bl (de ). the positive transformants were cultured in lb medium containing μg/ml kanamycin with vigorous shaking at °c until the nm optical density (od ) of bacteria cultures reached approximately . . then, by adding isopropyl-β-d-thiogalactopyranoside (iptg), the recombinant protein s was induced for - h at °c. the expression of recombinant proteins was analyzed by % (v/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and the gels were stained with coomassie brilliant blue. induced cells were pelleted and washed in phosphate-buffered saline (pbs) three times, then lysed by sonication in an ice-water bath. the lysed cells were centrifuged at , g for min, then the precipitate (inclusion body) was dissolved with binding buffer containing m urea. the supernatant and the precipitate were then subjected to sds-page analysis. the recombinant protein was purified through affinity chromatography using a ni-nta spin column following the manufacturer's recommendations. purified recombinant protein s was subjected to % (v/v) sds-page, and the gel was prepared for western blotting as follows. recombinant proteins separated in the gel were electrically transferred to a nitrocellulose membrane and the membrane was blocked overnight at °c with tris-buffered saline containing tween- (tbst) which contained % (w/v) skimmed milk powder. the composition of tbst is as follows: mm tris-hcl, mm nacl, and . % tween- . the membrane was then incubated with pig anti-pedv polyclonal antibody ( : dilution in blocking buffer) for h at °c on a plate shaker. following this incubation, the membrane was washed three times with tbst buffer and reacted with hrp-goat anti-pig igg ( : dilution in tbst) at °c for min. after three washes, the final color reaction was developed with a solution of , ′-diaminobenzidine (dab). conventional indirect elisa was performed with the following steps. elisa plates with wells (costar, usa) were coated with μl purified recombinant s protein in bicarbonate buffer (ph = . ) for h at °c. then, plates were washed three times with pbst (pbs containing . % tween- ) and blocked with % skimmed milk in pbs for h at °c. after plates were washed, μl porcine serum samples diluted in pbs containing % (w/v) skimmed milk was added and incubated for min at °c. plates were washed four times and reacted with μl diluted secondary antibody (hrp-goat anti-pig iga or hrp-goat anti-pig igg) for min at °c for the purpose of detecting iga or igg against pedv in serum samples. plates were then washed four times and μl tetramethylbenzidine (tmb) substrate solution was added to each well for a chromogenic reaction at room temperature for min in complete darkness. the color reaction was stopped by the addition μl of m h so to each well. finally, the od was measured and recorded immediately using an infinite pro microplatereader (tecan, männedorf, switzerland). the optimal dilution of recombinant protein s and standard serum was determined by a checkerboard titration based on the method mentioned above. briefly, the concentration of s protein was gradually reduced in the following series: , . , , . , . , and . μg/ml. the standard pedv positive and negative serum were serially diluted in a -fold series from : to : . when the od ratio of positive serum to negative serum was highest, and the od of positive serum was closest to . , the corresponding dilutions of coated antigen and serum sample were considered optimal. in addition to optimal protein dilution, the coating conditions, blocking solution, and reaction time of various materials was explored. furthermore, the optimal concentration of hrp-goat anti-pig iga was tested using the following dilutions: : , : , : , : , and : . two hundred and seventy serum samples were collected for the purpose of determining the positive-negative cut-off value, of which serum samples were collected from spf pigs, serum samples were collected from grow-finish pigs from five farms between and . these five farms were located in areas with no previous history of enteric signs compatible with viral diarrhea, and were pedv rna negative by real-time rt-pcr based on a single collection. these serum samples were confirmed pedv-negative by both ifa and sn assays as previously described [ , ] , and then were used to define the cut-off value in the s indirect elisa. the od value of these pedv-negative serum samples obtained in this s indirect elisa were recorded to calculate the cut-off value. the mean od value of these negative samples (n) + × standard deviations (sd) was defined as the cut-off value. serum samples showing od value greater than or equal to this cut-off were considered pedv-seropositive. to assess the accuracy of this developed s indirect elisa, serum samples from different pig farms were tested using this elisa method. as a comparison, ifa was applied to test these samples and act as a reference method to distinguish positive or negative samples. briefly, vero cells grown on -well plates were infected with the pedv yc strain at multiplicity of infection (m. o. i) of . at h post-infection, cells were washed three times with pbst and fixed with cold methanol for min at − °c. cells were then washed three times with pbst and blocked with % bovine serum albumin (bsa) at °c for h. after been double diluted for six consecutive dilutions in dilution buffer ( % bsa in pbst), the serum samples with varied pedv antibody status were added in the wells of -well plate, and were incubated for h at °c. after three washes with pbst, cells were treated with a fitc-conjugated goat anti-pig iga (thermo scientific) at a : dilution with pbs for min at °c. after a final four washes with pbst, all wells were examined using fluorescence microscopy (axio observer z , zeiss, germany). the pedv antibody titers of the serum samples were expressed as the highest dilution of serum samples producing green fluorescent in the wells of -well plates. the results of this two methods were compared, and the sensitivity and specificity of detection were calculated to evaluate the accuracy of the s indirect elisa. sensitivity was defined as the ratio of positive tests from the developed s indirect elisa to the positive tests from the ifa. specificity was defined as the ratio of negative tests from the developed elisa to the negative tests from the reference ifa. serum cross-reactivity of s indirect elisa to other pathogens to validate the cross-reactivity, this s indirect elisa was utilized to test porcine serum positive for other swine pathogens, namely, porcine transmissible gastroenteritis virus (tgev), swine rotavirus (porv), porcine kobuvirus (pkv), porcine bocavirus (pbov), porcine norovirus (pnov), porcine circovirus type (pcv ), porcine reproductive and respiratory syndrome virus (prrsv), and enterotoxigenic e. coli (etec), jerson prand of the small intestine, clostridium welchii type c. the positive sera were prepared by our lab, by immunizing the specific pathogen free (spf) piglets with purified virus or bacteria. thirty positive serum samples for each virus were tested, and each sample was repeated in triplicate. to test the repeatability of this elisa, serum samples with different pedv antibody levels were chosen. for inter-assay variability, each sample was tested in replicates on plates of different occasions. for intra-assay variability, each sample was tested in replicates on plates within the same occasion. the results were presented as the coefficient of variation (cv), which is the ratio of the standard deviation (sd) to the mean od value of each group of samples (s). a cv value criterion of % was used to meet the repeatability requirement of the test. this s indirect elisa was applied to seroepidemiological analysis of a total of clinical swine serum samples collected from thirty seven farms in eastern china. the pedv infection status of a given farm was determined based on demonstration of pedv rna in fecal samples by real-time rt-pcr and presence of enteric signs [ ] . one thousand one hundred and twenty five serum samples were collected from nursery and grow-finish pigs from ten farms between and at - weeks after the start of pedv outbreaks in these farms. four hundred and eighty two serum samples were collected from nursery and grow-finish pigs from five farms between and . these five farms were located in areas with no previous history of enteric signs compatible with viral diarrhea, were pedv rna negative by real-time rt-pcr based on a single collection, and were considered non-exposed to pedv. three hundred and forty four serum samples from nursery pigs without pedv exposure were collected from six farms between and . these samples were confirmed to be positive for anti-porv antibodies ( farms, n = ) or anti-tgev antibodies ( farms, n = ) by both ifa and commercial elisa kits (obtained from ingenasa). one thousand three hundred and fifty three porcine serum samples with unknown pedv exposure status were randomly selected from sixteen farms from nursery and grow-finish pigs between and . the gene of pedv truncated s fragment ( - nt) was amplified by rt-pcr (fig. a) . a bp pcr product was obtained and subcloned to prokaryotic fig. amplification, sds-page and western blotting analysis of the recombinant protein s . a rt-pcr amplification of the truncated s gene fragment. lane m, dl dna marker. lane and , the truncated s gene fragment. b identifiction of the recombinant expression plasmid a-s by double enzyme digestion. lane m, dl dna marker. lane , the recombinant expression plasmid a-s digested by bamh i/sal i. c sds-page analysis of s protein. lane m, prestained protein molecular weight standard. lane , transformed cells of bl /pet- a(+) after iptg induction for h. lane , transformed cells of bl / a-s after iptg induction for h. lane , purified recombinant protein s by affinity chromatography of ni-nta spin column. d western blotting analysis of s protein. lane m, prestained protein molecular weight standard. lane , e. coli bl with empty vector pet- a(+) reacted with polyclonal mouse anti-pedv antibody. lane , purified s protein reacted with polyclonal mouse anti-pedv antibody. a prominent band with the expected size kda appeared after incubation expression vector pet- a(+), and the inserted gene was sequenced to ensure the correctness of the reading frame. as shown by sds-page (fig. c) , the recombinant protein s was expressed in the form of inclusion body, resulting in a × his-tag fusion protein whose molecular mass was approximately kda. sonicated lysates from recombinant e. coli were harvested, and the precipitate was dissolved in m urea and purified by affinity chromatography of ni + -nta agarose. the immunoreactivity of s protein was examined by western blotting. an obvious band revealed that s protein was specifically bound by pig anti-pedv polyclonal antibody (fig. d) . as expected with checkerboard titration, with concentrations of antigen and serum regularly decreasing, absorbance values of corresponding samples declined. the optimal dilution of coated antigen s protein was measured at . μg/well ( . μg/ml), and optimal serum sample dilution was : (table ) . furthermore, other reaction conditions of the developed elisa were optimized. in brief, the optimum coating condition was h at °c. the best blocking solution was selected as % skimmed milk in pbs. the optimal reaction times for serum, secondary antibodies, and tmb solution were min, min and min, respectively. finally, the best working dilution of the hrp-goat anti-pig iga was : , . to determine the cut-off value of the s indirect elisa, pedv-seronegative samples, verified by both ifa and sn assays, were tested by this elisa method. the average optical density of these negative serum samples (n) was calculated as . , and the standard deviation (sd) of these samples was . . consequently, the cut-off threshold value of s indirect elisa was calculated to be . , indicating that the sample od ≥ . was identified as pedv-seropositive and vice versa. this developed s indirect elisa was applied to serum samples with varied pedv antibody status ( table ). in these samples, the s indirect elisa detected pedv-positive samples, of which tested pedv-positive by ifa. on the other hand, of the remaining samples that tested pedv-seronegative by this s indirect elisa, of them were tested pedv-negative by ifa. hence, the sensitivity of s indirect elisa was . % among pedv-seropositive individuals, and the specificity was . % among pedv-seronegative individuals using ifa as standard evaluation method. in summary, the overall coincidence rate of the s indirect elisa to ifa was . %. to test the cross-reactivity of this s indirect elisa, other viruses known to cause swine diarrhea were examined. the average od of positive serum samples for tgev, porv, pkv, pbov, pnov, pcv , prrsv, etec, jerson prand of the small intestine, and clostridium welchii type c were . , . , . , . , . , . , . , . , . and . , respectively. the results showed that these serum samples were pedv-seronegative and non-cross-reactive with this s indirect elisa, indicating that the established elisa was an effective method for detecting pedv antibodies. the repeatability of s indirect elisa intra-assay variability of the s indirect elisa was assessed by testing swine serum samples, each with replicates. this analysis produced cvs ranging from . - . %, with an average value of . %. inter-assay variability of four batches using identical samples produced cvs ranging from . - . %, with an average value of . %. the results demonstrated that this elisa method yielded low levels of variation, and its repeatability was in the credible range. this s indirect elisa method was used on swine serum samples of different pedv exposure status collected from farms (table this s indirect elisa was applied to test the sera of pedv immunized pigs at , , , , , and day post-inoculation (dpi). the results (fig. ) showed that, both the iga and the igg were positive at dpi. at early infection stage ( dpi), the iga titer was significantly higher than the igg titer; at dpi the iga titer was equivalent to the igg titer; and after dpi, the iga titer was significantly lower than the igg titer. the igg could exist in the sera of infection recovered stage for more time than iga. since december , a large-scale outbreak of diarrhea has been observed in swine farms in china. accumulated evidence indicates that this large-scale outbreak of diarrhea were caused by highly virulent pedv variants [ , ] . serological assays can quickly detect large numbers of samples with both high sensitivity and specificity. several indirect elisa have been developed based on either whole pedv preparations or recombinant viral proteins [ ] [ ] [ ] . the s protein of pedv has numerous epitopes and highly antigenic index regions that induce the production of neutralizing antibodies [ , , ] , and anti-s antibodies detected in pedv-infected pigs persist longer than anti-n antibodies [ ] , thus, we choose s protein as the diagnostic antigen. the recombinant s protein was applied to establish an indirect elisa, and its reaction conditions were optimized. as pedv is an enteric virus, it directly infects and damages enterocytes. mucosal iga, but not systemic igg, plays a crucial role in protection [ , ] . in this research, the titers of iga in the serum were tested for serological evaluation and indirect diagnosis of pedv infection. of the serum samples which were pedv exposed, the overall positive rate of the antibody is . %, which were varied from to % between farms. of the serum samples which were pedv non-exposed, the overall positive rate of the antibody is . %, which were varied from . to . % between farms. the evaluated elisa presented an overall substantial agreement on the pedv infection status of the field swine serum samples. the intra-and inter-assay variability tests proved that this elisa method had good repeatability. when testing other positive serum related to swine viral pathogens, this established elisa demonstrated no cross-reactivity to them. further, the overall rate of coincidence of this elisa was calculated at . % compared with ifa, proving that the diagnostic sensitivity and specificity of this elisa method were favorable. of the numerous pathogens which can cause swine viral diarrhea, pedv, tgev, and porv account for the largest proportion [ ] . in the present study, / anti-porv antibody positive sample and / anti-tgev antibody positive samples were tested pedv antibody positive, which indicated there may be co-infection of pedv and other virus. in conclusion, this established s indirect elisa is capable of detecting serum antibodies against pedv, and due to its high sensitivity and specificity, it could be applied for serological evaluation and indirect diagnosis of pedv infection. fig. determination of iga and igg in the sera of pedv immunized pigs. the sera of pedv immunized pigs were tested by this s indirect elisa at , , , , , and day post-inoculation (dpi). both the iga and the igg were positive at dpi. the igg could exist in the sera for longer time than iga. different letters (a, and b) indicate significant difference between the groups porcine epidemic diarrhea in europe: in-detail analyses of disease dynamics and molecular epidemiology nursery pig growth performance and tissue accretion modulation due to porcine epidemic diarrhea virus or porcine deltacoronavirus challenge in situ 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against porcine epidemic diarrhoea virus (pedv) based on the specific solubility of the viral surface glycoprotein spike protein region (aa ) of porcine epidemic diarrhea virus is essential for induction of neutralizing antibodies identification and comparison of receptor binding characteristics of the spike protein of two porcine epidemic diarrhea virus strains comparison of serum neutralization and enzyme-linked immunosorbent assay on sera from porcine epidemic diarrhea virus vaccinated pigs. the veterinary quarterly epidemic strain yc of porcine epidemic diarrhea virus could provide piglets against homologous challenge multiplex real-time rt-pcr for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus new variants of porcine epidemic diarrhea virus, china sequence and phylogenetic analysis of nucleocapsid genes of porcine epidemic diarrhea virus (pedv) strains in china comparison of an enzyme-linked immunosorbent assay with serum neutralization test for serodiagnosis of porcine epidemic diarrhea virus infection development and application of an elisa for the detection of porcine deltacoronavirus igg antibodies detection of antibodies against porcine epidemic diarrhea virus in serum and colostrum by indirect elisa phage-displayed peptides having antigenic similarities with porcine epidemic diarrhea virus (pedv) neutralizing epitopes identification of two novel b cell epitopes on porcine epidemic diarrhea virus spike protein porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis does circulating antibody play a role in the protection of piglets against porcine epidemic diarrhea virus? antigenic relationships among porcine epidemic diarrhea virus and transmissible gastroenteritis virus strains the data analyzed during the current study are available from the corresponding author on reasonable request. authors' contributions hl, kh and hf designed the study. hl, hz, lg and bl performed and collected data from experiment and analyzed data. hl, hz wrote the manuscript. all authors read and approved the final manuscript. this study was performed in accordance with the recommendations in the guide for the care and use of laboratory animals of the ministry of health, china. all experimental protocols were approved by the institutional animal care and use committee of nanjing agricultural university (no. syxk - ) and performed accordingly. samples were collected only from animals for laboratory analyses, avoiding unnecessary pain and suffering of the animals. the owners gave their written consent for sample collection, and the locations where we sampled are not privately owned or protected in any way. the studies did not involve endangered or protected species. not applicable. the authors declare that they have no competing interests. key: cord- - of ertf authors: lo, catherine yuk-ping title: securitizing hiv/aids: a game changer in state-societal relations in china? date: - - journal: global health doi: . /s - - - sha: doc_id: cord_uid: of ertf background: china has experienced unprecedented economic growth since the s. despite this impressive economic development, this growth exists side by side with the human immunodeficiency virus/acquired immune deficiency syndrome (hiv/aids) and severe acute respiratory syndrome (sars) crises and the persisting deficiencies in public health provision in china. acknowledging the prevailing health problems, the chinese government has encouraged the development of health non-governmental organizations (ngos) to respond to the health challenges and address the gaps in public health provision of the government. hiv/aids-focused ngos have been perceived as the most outstanding civil society group developed in china. considering the low priority of health policies since the economic reform, the limitation of the “third sector” activity permitted in authoritarian china, together with the political sensitivity of the hiv/aids problem in the country, this article aims to explain the proliferation of hiv/aids-focused ngos in china with the usage of the securitization framework in the field of international relations (ir). methods: the research that underpins this article is based on a desk-based literature review as well as in-depth field interviews with individuals working in hiv/aids-focused ngos in china. face-to-face interviews for this research were conducted between january and may in , and between december and january , in china. discourse analysis was in particular employed in the study of the security-threat framing process (securitization) of hiv/aids in china. results: this article argues that the proliferation of hiv/aids-related ngos in china is largely attributed to the normative and technical effects of hiv/aids securitization ushered in by the united nations security council (unsc) and supported by the global fund to fight aids, tuberculosis, and malaria (hereinafter global fund) observed in china. despite depicting a positive scenario, the development of hiv/aids-focused ngos in china generated by the international securitization efforts is largely limited. an internal and external factor was identified to verify the argument, namely ( ) the reduction of international financial commitments, as well as ( ) the fragmentation of hiv/aids-focused ngo community in china. conclusions: this article shows that international securitization weakened with the rise of chinese commitment on hiv/aids interventions. in other words, hiv/aids-related responses delivered by the national government are no longer checked by the global mechanism of hiv/aids; thus it is unclear whether these ngos would remain of interest as partners for the government. the fragmentation of the hiv/aids community would further hinder the development, preventing from ngos with the same interest forming alliances to call for changes in current political environment. such restriction on the concerted efforts of hiv/aids-related ngos in china would make achievement of the sustainable development goals (sdgs) to foster stronger partnerships between the government and civil society difficult, which in turn hindering the realization of ending hiv/aids in the world by . china has experienced remarkable economic growth since the initiation of the economic reform in the s. the average gdp growth in china was . % annually during the first two decades of the economic reform [ ] . the rising nation became the world's leading manufacturing power in , and it surpassed the united states as the world's largest trading country [ ] . jakovljievic further predicted that the economic growth in china would continue at least up to the middle of the st century [ ] . despite such impressive economic performance, the provision of public health and the control of infectious diseases have been marginalized. between the late s and , the chinese national government public health spending as a proportion of total health spending plunged from % to just over % [ ] , despite the fact that china has outperformed other developing countries, such as other brics members, regarding the public health spending in nominal terms [ ] . as argued by yip and mahal, chinese political leaders in the abovementioned period of time simply perceived health as "a consumption activity rather than a productive good and therefore was given lower priority in government funding" [ ] . in this regard, the chinese authorities deliberately curtailed the level of public health expenditures so that economic development could be pursued and the legitimacy of the one-party authoritarian regime bolstered. acknowledging the economic success in the past decades, hiv/aids nevertheless emerged as one of the "negative externalities" of public health threats brought about by the s economic reform. hiv/aids was first discovered in china during the mid- s, the same period when the economic reform was just beginning. the epidemic was exacerbated in china in the s mainly because of government-supported blood plasma selling, which did not follow adequate sterilization procedures. this lack of regulation resulted in the emergence of so-called aids villages (aizibing cun) in henan and its outlying provinces, where a large percentage of rural residents were infected with hiv/aids [ ] . having little benefit from the economic reform in ways that coastal provinces did during the s, many residents in the central plains had little choice but to engage in the blood-plasma industry to improve their standard of living. since , hiv/aids has become a politically sensitive issue in china because of the international condemnation of the henan blood scandal. according to the latest figures released by the joint united nations program on hiv/aids (unaids), , people were estimated to have hiv/aids in china in [ ] . the country has the second largest hiv/aids population in asia after india. the chinese government started addressing the hiv/ aids problem after years of denial and underestimation of the hiv/aids prevalence in the country, especially among the most-at-risk populations (marps) including injecting drug users (idus), female sex workers (fsw), and men who have sex with men (msm) [ ] . however, national hiv/aids policies in the early years were very much driven in a top-down manner with government institutions (i.e. mainly the chinese centers for disease control and prevention [china cdc hereinafter] ) playing a leading role and with limited engagement of the "third sector" [ , ] . given the association of the disease with behaviors that are legally or socially unacceptable in the country (i.e., commercial sex work, injecting drug use, and men having sex with men), hiv/aids high-risk individuals refrain from using the related services provided by the government agencies because they wish to avoid being seized or humiliated by public security officials or by thugs hired by local officials [ ] . in this regard, gåsemyr claimed that the emergence of hiv/aids-focused non-governmental organizations (ngos) in china during the early s could fill the capacity gap of the government by implementing national hiv/aids policies and providing the related services for those marps that are hard to reach by the authorities [ ] . considering the low priority of health policies since the economic reform, the limitation of the "third sector" activity permitted in authoritarian china, together with the political sensitivity of the hiv/aids problem in the country, it is believed that the involvement of hiv/aids-related ngos in china would be largely limited. this is however not the case. over the last years there has been a dramatic increase in the number of hiv/aids-focused ngos operating in china. while gåsemyr argued that the growth of hiv/ aids-related ngos is mainly attributed to the devastating health crisis of sars in china and the new chinese leadership in [ ] , the previous work cannot explain why a high degree of political recognition was observed in solely hiv/aids ngos, instead of other health ngos, such as tb or cancer ngos in china. this article aims to fill this research gap with regard to the proliferation of hiv/aids-focused ngos in china with the use of the securitization framework in the field of ir. the research that underpins this article is based on desk-based literature review as well as in-depth field interviews with individuals working in hiv/aids-focused ngos in china. discourse analysis was in particular employed to study the securitization of hiv/aids in china. potter argued that "discourse analysis has an analytic commitment to studying discourses as texts and talks in social practice…the focus is…on language as… the medium for interaction…one theme that is particularly emphasized here is the rhetorical or augmentative organization of talk and texts" [ ] . silverman pointed out that the intellectual ancestor of discourse analysis is john langshaw austin, the original inventor of the idea of speech acts that later became the core element in securitization theory [ ] . concerning discourse analysis and speech acts share the austinian concern with the rhetorical structure of talk and texts, the founding scholars of securitization theory, buzan, waever and wilde explicitly stated, "the obvious method [to study securitization] is discourse analysis, since we are interested in when and how something is established by whom as a security threat" [ ] . the authors also noted that the technique of employing discourse analysis to securitization studies is to "read[ing], looking for arguments that take the rhetorical and logical form defined here as security" [ ] . the meaning of security is constructed when the political leaders (securitizing actors) frames an issue as an existential threat to the referent objects, claiming the issue as having an absolute priority on the government agenda, thereby invoking a right to handle such an issue with extraordinary/emergency measures to ensure the survival of the referent objects [ ] . in this research, public speeches (in written form) related to hiv/aids problems, which were delivered by the top-level political leaders, were examined. a specific rhetorical structure had to be located in their discourses, consisting of either one or all of the following elements: ( ) the existence of an existential threat; and/or ( ) the perceived threat is challenging the survival of the states or people in the states (referent objects), and/or which it requires to ( ) be urgently dealt with by new political measures of the respective country. an array of documents ranging from official documents, reports, newspaper articles was initially identified via the search engines of google scholar and proquest with the keywords of "hiv/aids", "security threat", and "china". nine key articles were consequently singled out to identify the textual discourses related to hiv/aids threat/ security nexus in china, published or released between and (table ) . a total of semi-structured interviews were conducted along with the discourse analysis. apart from the background information of the respective ngos, the interview questions used in this study are categorized into three main dimensions: ( ) the priority of hiv/aids on the government agenda; ( ) the perceptions on the changing international and national financial commitment on hiv/aids interventions; and ( ) civil society involvement and coordination in national hiv/aids policies. the first dimension aimed to ensure that the ngos respondents were knowledgeable about the changing national government perceptions and policy priority towards hiv/aids in china, therefore, the researcher could rule out those comments given by unknowledgeable respondents so as to enhance the internal validity of the data collected. the second dimension was included to study their perceptions on the level of hiv/aids funding and effect (if any) on the service provision. the third dimension aimed to discover the actual level of involvement of ngos in national hiv/aids interventions as well as the level of coordination and cooperation between different ngos working on hiv/aids. two types of non-probability sampling-purposive sampling, including "theoretical sampling" and snowball sampling-were employed in the selection process of key informants. for purposive sampling method, samples were selected based on the "logic" or "commonsense" of the researcher in terms of the target population, its elements, and the purpose of the study. in this study, the target population consisted of the individuals working in hiv/aids-related ngos at global, national, and grassroots levels. in the course of the data collection, "theoretical sampling" was also applied to understand the subjects from a theoretical perspective. hence, academic scholars who have conducted research on hiv/aids policies in china were also included in the interviewee list. interviewees were also recruited via snowball sampling. the researcher collected data on the few members, and each respondent was asked to suggest people whom the respondent believed to be the most influential for interviewing. interviews for this research were undertaken between january and may in , and between december and january with leaders or senior officers working on hiv/aids in china, including international ngos, nine self-help groups, four gongos, five advocacy groups, two national ngo, one independent consultant, two academia, and two networks (see table ). twenty-six respondents were pinpointed based on the civil society (minjian shehui) was not considered as part of the political system in the pre-modern era; the emergence of a relatively independent civil society is a product of modern china. the changing socio-economic relations brought about by "open door policy" resulted in the recognition and growth of civil society for the first time in the chinese history [ ] . despite the early emergence of civil society, the falung gong incident showcased the destructive power of civil society in the eyes of the chinese government, worrying a robust, well-organized, and out-of-control civil society could overthrow and replace the ccp ruling position. meanwhile, the authorities realized the supplementary role ngos could perform in policy implementation. the chinese government has thus resolved such dilemma by adopting a "state-led" approach to manage civil society in china. based on frolic's concept of "state-led civil society", civil society is created by and belong to the state, thus the independence and autonomy of civil society are at all time bounded by the state [ ] . the state has the role of legitimating social organizations, demanding a disciplined partnership. accordingly, antagonism inherited in the western concept of civil society is not allowed to exist in the state-society relations in chinese authoritarian regime; any alternative force to the state is considered as an attempt to curtail or overturn its political legitimacy and power. accordingly, the state apparatus has managed the numbers and restricted the growth of both international and grassroots ngos via various legislative means, thereby allowing ngos to operate openly but at the same time keep the organization growth in check [ , ] despite these regulations, the chinese authorities had relatively given more autonomy to ingos than grassroots ngos operating in its territory, since the government could access international expertise and tap foreign money to tackle with the emerging social problems in china [ ] . such degree of autonomy is nevertheless problematic after the promulgation of foreign ngos management law (jingwai fei zhengfu zuzhi guanli fa) in april . the law stipulates that ingos operating in china must register with public security officials, and must not engage in political or religious activities that damage "china's national interests" or "ethnic unity". international community and western governments perceived the new law as a reinforcement of restriction of the numbers and scopes of foreign entities operating in the authoritarian regime. grassroots ngos are regulated by the regulation on registration and administration of social organizations (shehui tuanti dengji guanli tiaoli). according to the regulation (amended in february ), a ngo must possess a minimum asset of , yuan and have a "professional management unit" (zhuguan danwei) that acts as a supervisory body to the organization in order to register under the ministry of civic affairs (moca). fulfilling these two requirements is very formidable for grassroots ngos. the financial situation is problematic in many grassroots ngos because of the limited source of funding. on the one hand, most grassroots ngos receive limited financial support from the government, as they usually do not have political ties with the government. grassroots ngos also seldom receive donations from local communities because ( ) newly developed ngos do not have good track records that enable them to win the trust of the locals, and ( ) donors cannot receive tax breaks for their donation to unregistered ngos [ , ] . in addition, most government departments simply reject their applications due to fear of consequences of taking responsibilities for grassroots ngos. many grassroots ngos are hence often unregistered or registered as business entities with the ministry of industry and commerce (moic) [ ] . in addition to the abovementioned restrictions, the regulation also bans "similar organizations" coexisting at the various administrative levels [ ] , facilitating the management and even control the legal status of grassroots ngos in china. having official registration, to certain extent, is sine qua non to the survival of organizations as the legal status entitles ngos as official recognized entities to receive legal and financial supports from the government; unregistered ngos are officially perceived as illegal that would be subjected to prosecution and coercion by the state apparatus. considering a restrictive engagement of grassroots ngos in china, instead of grassroots ngos, early hiv/ aids-related responses at societal level were largely conducted by gongos [ ] , [ ] . considering the low priority of health policies since the economic reform, the limitation of the "third sector" activity permitted in authoritarian china, together with the political sensitivity of the hiv/aids problem in the country, it is intriguing to uncover the reasons for the growth of hiv/aids-focused ngos in china since . gåsemyr argued that the growth of hiv/aids-related ngos is mainly attributed to the devastating health crisis of sars in china and the new chinese leadership in [ ] . having said that the chinese government realized the impacts health problems or infectious diseases could have on its economic and social development, the previous work cannot explain why a high degree of political recognition was observed solely in hiv/ aids ngos, instead of other health ngos, such as tb or cancer ngos in china. considering the regime type of the government, allowing the growth of local ngos would in turn potentially attenuate the supremacy of the chinese communist party (ccp) in ruling the country or erode the autonomy of the state, why the chinese government tolerated, allowed, or even encouraged the growth of ngos in fixing hiv/aids problems, especially the epidemic has been viewed as a politically sensitive issue in china? how can one account for and understand the phenomenon? answering the key question with an ir perspective, this article argues that the rapid development of hiv/ aids-related ngos in china is attributed to the effects of hiv/aids securitization at the international level since . considering one of the most influential theories developed in the field of ir, securitization describes the process of defining an issue as a security threat (speech acts) and the corresponding political responses to block the adverse development of the perceived threat (emergency measures). this article particularly pinpoints the unsc and the global fund, which provide normative and technical supports, to study this endeavor. normative influences in this article refer to the effects of security-threat discourses, primarily resolution of the unsc, on the national government's perception on hiv/aids problems, whereas technical influences refer to the effects of global emergency measures, in particular the global fund, on the funding and management of hiv/aids-related policies at the state level [ ] . this article aims to demonstrate the ways in which international hiv/aids securitization has served as a game changer in altering state-society relations in china. drawing on literature reviews and fieldwork materials, this article further argues that the proliferation of hiv/aids-focused ngos in china generated by the international securitization efforts is nevertheless largely limited owing to the reduction of international financial commitments. the fragmentation of hiv/aids-focused ngo community is another factor hindering the development of a full-fledged third sector in the country. securitization in the ir discipline is a model outlining the process of framing an issue as a security threat and the corresponding political reactions to block the adverse development of the perceived threat. employing the theory, one is required to look for three criteria to determine whether an issue is a threat to the country and its people, including ) discourses uttered by toplevel political leader (securitizing actor) declaring a particular referent objects as an existential threat to security; ) acceptance by the targeted audience convinced of its potential to be an existential threat (audience acceptance); and also ) redirections of existing financial resources, new policies, or practices (emergency measures) are implemented to address the issue following the security-threat claim [ ] . in other words, the resolution of the "existential threat" posed by "x" takes precedence over other problems [ ] . numbers of security studies scholars believed that hiv/aids is the first infectious disease being securitized by the international institutions and national governments as the abovementioned criteria were all fulfilled in the case of hiv/aids [ , [ ] [ ] [ ] . speech acts concerning hiv/aids as a security threat were first identified in a unsc meeting in january . in july of the same year, the unsc passed resolution , acknowledging the urgent need to address hiv/aids, which threatens the stability and security if left unchecked [ ] . it is perceived that the particular security framing of hiv/aids gained acceptance by the national governments (audience of the hiv/aids security-threat claim) since countries unanimously adopted and signed the declaration of commitment on hiv/aids in . the urgency of addressing hiv/aids problems was reinforced in the special united nations general assembly special session (ungass) devoted to hiv/aids that has taken place every years since [ ] [ ] [ ] [ ] . the abovementioned events are significant because they represented the first time that the unsc general assembly devoted an entire session to a single disease, and the first time that the international political authorities framed hiv/ aids as a global health threat to national and international security instead of solely as a developmental or public health problem [ ] . the rhetorical act was backed by operational emergency responses in terms of the augmentation of the global hiv/aids spending since . the amount was exponentially surged from about us$ million in to us$ billion by ; and to an estimated us$ billion in [ ] . several hiv/aids-related funding agencies and health programs formulated after , also referred to global health initiatives (ghis), brought in additional monetary resources for hiv/aids [ ] . perceiving as the most prominent ghis, the global fund pledges to accelerate the end of the three epidemics, [hiv/]aids, tuberculosis, and malaria by providing financial support (us$ billion annually) for low-income countries to respond to the three diseases [ ] . undoubtedly, the introduction of the abovementioned ghis has brought about unprecedented levels of funding for diseases, in particular hiv/aids, in countries that have insufficient resources, whether because of poor socio-economic development or lack of political will, to orchestrate appropriate responses to the intractable disease. acknowledging international securitizing efforts on hiv/aids, this article illustrates the normative effects of resolution of the unsc and the technical influences of the global fund on national-level hiv/aids responses. china is selected as a case study to investigate this endeavor. china's response to the hiv/aids problem changed from an ignorance of "global evidencebased policy recommendations and international advice" into "one of our success stories [in global hiv/aids fight] during this past years," as claimed by michel sidibe, executive director of the unaids [ , ] . the subsequent sections demonstrate the ways in which the securitization effort in the international level acted as a game changer for hiv/aids-focused ngos in participating in national hiv/aids undertakings in china. chinese case study discourse analysis: normative influences and the role of the unsc in china, many of the first cases of hiv/aids since occurred among foreigners and homosexuals. therefore, the official discourses at that time promoted the idea that hiv/aids only affected non-local chinese people and foreigners, asserting "drug taking, alcoholism, robbery, homicide, suicide, divorce, prostitution, homosexuality, syphilis, aids…these kinds of western social ills come from their ideology", and also claiming "china had no sources of hiv/aids" [ , ] . the rhetorical acknowledgement of hiv/aids as a health security threat to the chinese population was largely absent during the early period of time. ramiah also pinpointed that the chinese leadership was extremely reluctant to acknowledge hiv/aids as a problem and securitize hiv/aids in china prior to the s [ ]. one probable reason for the resistance to hiv/aids securitization is economic: the period of early hiv/aids outbreak coincided with the initial years of open door policy and economic reform (gaige kaifang) of china to the outside world. the thirst for economic growth and performance preceded the urgency of disease management: local authorities feared their respected jurisdictions would lose external or foreign investment if the full extent of the problem were known, or that they would be punished by superiors for failing to prevent the hiv/aids spread. the "cover-up" practice also oc- in response to the designated targets, the securitythreat framing of hiv/aids has been fully adopted in the official discourses of chinese leaders and in official documents since . previously viewing hiv/aids as a "western disease" or "non-chinese disease", executive vice minister of health gao qiang stated in the hiv/ aids high-level meeting of the un general assembly that "hiv/aids is a common enemy of the whole mankind as it seriously threatens public health and safety. the chinese government has attached great importance to hiv/aids prevention and treatment and has treated it as a strategic issue for social stability, economic development, national prosperity and security, making it a first priority of the government work" [emphasis added] [ ] . the chinese president hu jintao once claimed in a public speech that "hiv/aids prevention, care and treatment is a major issue pertinent to the quality and prosperity of the chinese nation"; whereas premier wen likewise asserted that "dealing with hiv/aids as an urgent and major issue is related to the fundamental interests of the whole chinese nation" [emphasis added] [ ] . the ideas in the speeches were restated in the official document entitled state council notice on strengthening hiv/aids prevention and control (guowuyuan guanyu qieshi jiangqiang aizibing fangzhi gongzuo de tongzi). this document explicitly demonstrated the determination of the chinese authorities in grappling with the disease and stated that "hiv/aids prevention and control is linked to economic development, social stability, and national security and prosperity. long-term commitment to respond to hiv/aids is hence necessary" [emphasis added] [ ] . china's hiv/ aids prevention and control policies were further strengthened in the state council notice on further strengthening hiv/aids prevention and control (guowuyuan guanyu jinyibu jiangqiang aizibing fangzhi gongzuo de tongzi) in late , claiming that "the prevention and control of hiv/aids is related to the people's physical health and social and economic development, as well as to national security and the rise and fall of the nation. the party central committee and the state council have always attached great importance to the prevention and treatment of hiv/aids" [emphasis added] [ ] . based on the discourse analysis of the official documents and newspaper articles, it is argued that chinese national leaders followed suit the international move (i.e. unsc resolution ) to securitize hiv/aids in the country, framing hiv/ aids as a threat with social, political, economic, and security implications. such an alteration in perception toward the epidemic brought about the political recognition of the hiv/ aids-focused ngos, which has also been highlighted by multiple statements delivered by various top-level government officials since . in , vice premier wu yi stated, "we should mobilize all the partners in society to participate in the fight against hiv/aids. we need to improve our policies and strategies to build a better environment for the society to participate in the response, and try our best to facilitate the involvement of all sectors" [ ] . in , vice minister of health yin li publicly proclaimed "civil societies play an indispensable role in effective hiv/aids interventions led by the government" [ ] . premier li keqiang even promoted tax break for ngos specializing in hiv/aids prevention, claiming the role of ngos in hiv/aids prevention "is an irreplaceable and unique force" [ ] . to a certain extent, these aforementioned statements demonstrate the state's recognition of ngos' works and its willingness to engage with ngos in national hiv/aids interventions, although the political space of involvement is by and large bounded by the state [ ] . the previous section illustrates the change in the perception of the chinese government toward the hiv/ aids problems: from denial to the acceptance of the threats that could be posed by the disease on the country and its people. such threat recognition also brought about the acknowledgement that the government alone could not resolve the hiv/aids problems because of the complicated nature of the epidemic; the involvement of civil society groups was a globally recognized recommendation leading to the successful containment of the epidemic. nevertheless, verbal recognition is not sufficient to enhance the engagement and development of hiv/aids-focused ngos, especially in the preexisting authoritarian government structure; the governance and funding mechanism for hiv/aids responses requires alternation in china. as illustrated in the following section, this was the mechanism of the global fund that enhanced the funding for hiv/aids-focused ngos, facilitating the involvement of civil society groups in translating the rhetorical acts into emergency responses of hiv/aids in china. among the international funding agencies emerging after the hiv/aids securitization launched by the unsc, the global fund was the most renowned major contributor to the hiv/aids interventions in china. the global fund finally accepted the first proposal submitted by the chinese government on hiv/aids interventions in late , after the previous proposals being turned down twice in [ ] . between and , the chinese government has received over us$ billion in grants from the global fund; approximately us$ million were disbursed to hiv/aids intervention programs [ ] . further information regarding the global fund on hiv/aids in china is illustrated in table . intervention of the global fund's funding mechanism was deemed to transform the primary hiv/aids governance in china [ ] . the global fund required the governments to set up a country-level country coordination mechanism (ccm) to construct the grant proposals in line with the national hiv/aids-related strategies, manage the use and distribution of grants, and also oversee the implementation of successful applications. as per the requirement of the global fund, ccm should consist of a broad representation from the government, ngos, multilateral and bilateral agencies, and private sector [ ] . in the existing government structure in china, grassroots ngos have limited channels to participate in policymaking and implementation processes. thus, the multi-sectoral structure of ccm contributed to enhancing the government's commitment to involve ngos in national hiv/aids prevention and control measures [ ] . two among the seats in chinese ccm were reserved for the representatives of hiv/ aids-related grassroots ngos and people living with hiv/aids are a case in point [ ] . considering the existing government structure does not allow the full involvement of ngos in china, the ccm provided valuable opportunities for civil society groups to engage in hiv/aids-related policymaking. the involvement of ngos in national hiv/aids programs was notably highlighted in round of the global fund in china, since the theme of the grant proposal submitted by the ccm was directly linked to the development of civil society groups (refer to table ). in the course of the interview, a director of a hong kongbased ngo commented on the profound influence of the round on the ngo development: "round of the global fund acts as a pushing force for the government to involve more grassroots ngos in hiv/aids interventions" (national ngo- ). in line with the respondent's opinion, kaufman argued the round in particular "was seen by many as a further mechanism to institutionalize hiv/[aids] ngos roles in china's [hiv]/aids response [ ] ." in addition to the increase in participation, financial support for ngos working on hiv/aids-related programs surged through the global fund. as the principal recipient (pr) of the country, the chinese government (i.e., china cdc or the nhfpc) was required to disburse a designated portion of the grants to hiv/aids-related ngos (in the form of sub-recipients) operating in the country [ ] . following the instruction, % of the total grant was allocated to support ngos in round and , whereas % of its grant was distributed to ngos in round of the global fund [ ] . in line with the theme of round entitled "mobilizing civil society to scale up hiv/aids control efforts in china" proposed by the chinese ccm, the proportion granted to hv/aids-related ngos reached % [ ] . owing to the constructive political atmosphere and promising financial resources in the country, a burgeoning number of individual-organized hiv/aids-focused ngos was established in china; over % of the number of grassroots ngos increased in china between and [ ] . the suspension of the global fund in demonstrated that the funding mechanism to some extent held the chinese government accountable for the financial supports of hiv/aids-focused ngos. the global fund once suspended its funding to chinese hiv/aids programs in early because the chinese ccm failed to allocate % of the us$ million hiv/aids grant to grassroots ngos as pledged; less than % was distributed to hiv/aids-focused ngos [ ] . a program officer of a ngo mentioned the failure of ccm to allocate the right portion of the global fund grant to grassroots ngos: "government officials and representatives from gongos dominate the ccm. only a few representatives are from grassroots ngos and civil society" (advocacy group- ). the improper management and use of the global fund was even admitted by a senior government official in the shanghai cdc in times of the interview: first, the global fund required at least percent to percent of the grant should be given to grassroots ngos. however, the government failed to achieve this requirement. second, the management fee in some provinces was too high. third, one of the elements in the rcc program was related to budget aggregation (i.e., monetary contribution by both the national government and the global fund). however, the chinese government did not allocate enough money for the aggregated budget (ccdc/nhfpc- ). the determination of the chinese government to resolve the suspension of the global fund was observed in the course of fieldwork research. during a face-to-face interview inside the office building of the ministry of health (renamed national health and family planning commission in ) in may , the respondent told the investigator "the representatives of the chinese government and the global fund representatives are meeting upstairs to negotiate for the resumption of the grants" (ccdc/nhfpc- ). the dispute was eventually resolved; the chinese government promised to allocate at least % of the global fund grant to ngo work [ ] . in this regard, the global fund mechanism contributed to the political and financial supports of ngos, thereby resulting in the proliferation of hiv/aids-specific grassroots ngos in china, and also the proactive involvement of ngos in national hiv/aids undertakings. in other words, international securitization altered the hiv/aids policy in china, allowing numerous grassroots-level ngos working on hiv/aids to grow in a prevailing political system with "bounded autonomy". an injecting drug use self-help group leader believed the upsurge in the involvement of civil society also resulted in the improvement of state-societal relations, stating, "in the past, the relationship with the government was not good, but it becomes much better now. the government sent officials to visit our center to learn the management strategy of our organization" (self-help group- ). another ngo interviewee in beijing also claimed that "an increase in the acceptance of the government, especially the ministry of health, on grassroots ngos, [is observed in china] . the ministry of health does open the opportunity for ngos to cooperate with the government" (advocacy group- ). having said that the improvement of state-societal relations, it is noted that the chinese state is not a monolithic actor, but a conglomeration of departments and officials with competing and sometimes conflicting agendas in the national and sub-national levels [ ] . a beijing ngo leader pinpointed this phenomenon, stating, "the ministry of health is fine to cooperate with those ngos that can help, regardless of whom. however, state security bureau usually holds a relatively hostile view on the existence of grassroots ngos since the bureau is concerned about social stability" (advocacy group- ). considering hiv/aids as a health threat to the country and its people, the recognition and involvement by the central government and the nhfpc and other related government agencies is of particular vital to the proliferation of hiv/aids-focused ngos in a statedominated society in the current political regime. despite depicting a positive scenario, this article further illustrates that the development of hiv/aids-focused ngos in china generated by the international securitization efforts is largely limited. in other words, the growth of the number of ngos working on hiv/ aids in china would be ceased or even decreased in long term, and also the political space for grassroots to involve in hiv/aids policy making and implementation would be further restricted. an internal and external factor was identified to verify the argument, namely ( ) the reduction of international financial commitment, as well as ( ) the fragmentation of hiv/aids-focused ngo community in china. external factor: the reduction of international financial commitment china resembles some developing countries in that it has faced the problem of decreasing international hiv/ aids financial commitments in supporting its national hiv/aids responses [ ] . given the global financial crisis and the continuous economic boom in china, international and bilateral funding agencies argue that china should be the donor instead of the recipient of international hiv/aids-specific grants and loans [ ] . owing to a lack of international and national hiv/ aids funding, the number of ngos working on hiv/ aids was drastically reduced; many ngos and cbos were simply closed down, only a few of them could barely survive with the support by other funding sources (national ngo- ). the retreat of the global fund has subsequently created a policy vacuum for the chinese government in taking the lead in managing the development of hiv/ aids-related ngos in china. mimicking the funding mechanism of the global fund, the state council launched the china aids fund for non-governmental organizations (cafngo) (shehui zuzhi canyu aizibing fangzhi jijin), providing financial resources for hiv/ aids-related ngos via the submission of grant proposal starting from [ ] . the reduction of the chinese government's reliance on the global fund implies that the hiv/aids-related responses delivered by the national government are no longer checked by the external mechanism. the authoritarian regime is probably the only agency defining/restricting the political space that ngos could work in. explaining the interaction between the chinese authorities and ngos, a university professor in beijing suggested: "in normal circumstances, local cdc staff hand over treatment delivery work to grassroots ngos. nonetheless, cdc officials intend to keep these ngos small, giving them just enough money to run the programs, so that they will not grow too strong to pose potential threats to the government" (academic- ). along with the weakening of international securitization efforts and the rise of chinese government's involvement in managing ngos in the post-global fund era, the continuous proliferation of ngos is further complicated by the fragmented nature of hiv/aids-focused civil society groups in china. it is argued that the existing ngo registration system, as previously mentioned, has brought about a fragmented ngo community in china. apparently, governmentorganized non-government organization (gongo) and registered ngos working on hiv/aids are included in hiv/aids national policy implementation processes, whereas excluding unregistered grassroots ngos that are perceived as illegal or "anti-government" organizations. based on the nature of services that individual hiv/ aids-focused ngos provided, service providers are preferred by the state, whereas ngos serving as advocates of human rights and agencies providing legal services for hiv/aids-infected people would be subjected to prosecution and coercion. a hiv/aids specialist working in an international ngo in beijing stated, "during a hiv/aids ngo meeting, the chinese government clearly claimed that the authorities would like to work with ngos/cbos conducting service delivery, but not with those working on hiv/aids-related human rights or gender issues" (ingo- ). to further illustrate this point, the chinese political leaders publicly praised a guangxi-based ngo named aids care china, recognizing the work the organization had been done in hiv/aids prevention, meanwhile stifling numbers of hiv/aids activists through imprisonment, house arrest, or assault. dr. wang yanhai and dr. gao yaojie were two of the prominent chinese hiv/aids activists that fled to the united states in and , respectively, due to the recurrent pressure and disturbance exerted by chinese authorities owing to their advocacy work. in june , a hiv/aids-related legal aid ngo, beijing yirenping, was raided, and two of its activists were detained. in this regard, the continuous inclusion/exclusion selection process would weaken unregistered or advocacy groups that conceived as threats to the legitimacy of the authoritarian regime [ ] , while preserving ngos that are willing to under control by the state apparatus and prepared to accept the regime's policy measures. for the sake of reaching a modus vivendi, these ngos usually display self-limiting behaviors, avoiding actions that would be interpreted as threats to the regime [ ] , such as having close relationship or cooperation with ngos being "excluded" by the state apparatus. while a formal local network for hiv/aids-related ngos or civil society groups has been established in many countries in asia, such coordination mechanism between non-state actors is largely absent in china. the fragmentation of hiv/aids ngo community is likewise observed in the course of interview sessions. amongst the interviewees, the majority of the ngo respondents believed that cooperation dramatically increased between the government and ngos working on hiv/aids, especially in the areas of treatment, care, and prevention. only a few of them nevertheless stated that communication and coordination occurred amongst ngos in policy implementation. a national-wide ngo respondent claimed that "we seldom cooperate with other ngos or ingos; we have our own teams to do the work" (national ngo- ). some grassroots ngo respondents in shanghai and kunming expressed their unwillingness to cooperate with so-called "unauthentic" ngos or ngos having so-called "government background": few ngos have cooperative relationship. there are lots of "fake" ngos in china. the cdc found some patients to set up grassroots ngos and then through the name of such ngo to apply for the global fund. after they received the money, cdc would become the one who controlled the funding (self-help group- ). eighty-five percent of the hiv/aids-focused ngos in yunnan have government background…for those ngos that are funded by the global fund or china-bill gates projects, these are organizations with the government background…or if you read their introduction saying "with the help and support of government", you will know these are ngos with the government background (self-help group- ). a "we-they" distinction has apparently prevailed amongst the hiv/aids ngos community in china, reinforcing the atomization of hiv/aids-focused ngos, discouraging the proliferation of civil society in china. horizontal relationship amongst ngos working on hiv/aids is relatively weak and ill-organized, thereby avoiding these organizations joining hand to call for favorable political environment or monetary supports to continue their operation in china. owing to the reduction of international financial commitments, as well as the fragmentation of hiv/aids-focused ngo community in china, the proliferation of hiv/aids-focused ngos in china generated by the international securitization efforts is nevertheless largely limited in long run. such restriction on the concerted efforts of hiv/aids-related ngos will inevitably pose challenges to china to fulfill the pledge of the agenda to end hiv/aids as one of the targets of the sustainable development goals (sdgs) [ ] . hiv/aids was officially announced as a security issue in by the unsc resolution , which claimed that the pandemic could pose security threats if left unchecked. the rhetorical security-threat claim resulted in the burgeoning number of ghis organized by the international and bilateral organizations operating at the state level. this article illustrates the ways in which the unsc resolution and the global fund influencing the hiv/aids governance in china. rhetorically acknowledged the hiv/aids problems and the irreplaceable role of hiv/aids-focused ngos in combating the disease by the chinese government, the monetary and technical supports by the global fund enabled hiv/aids-focused ngos to participate in the national policymaking process, facilitating the proliferation and development of civil society in china. this success nevertheless does come with concerns regarding sustainability. this article shows that international securitization weakened with the rise of chinese commitment on hiv/aids interventions. in other words, hiv/aids-related responses delivered by the national government are no longer checked by the global mechanism of hiv/aids; thus it is unclear whether these ngos would remain of interest as partners for the government [ ] . the fragmentation of the hiv/aids community would further hinder the development, preventing from ngos with the same interest forming alliances to call for changes in current political environment. such restriction on the concerted efforts of hiv/ aids-related ngos in china would make achievement of the sdgs to foster stronger partnerships between the government and civil society (goal ) difficult, which in turn hindering the realization of ending hiv/aids in the world by (goal ). this research has several limitations. firstly, there is a lack of available information regarding the operation of local or grassroots hiv/aids-specific ngos that are in particular helping marps in china, owing to the political sensitivity of hiv/aids, the unregistered status of ngos working on hiv/aids, the increasing restrictive environment for ngos operating in china especially under the xi administration, together with the social stigma attached to their service objects as well as the ngos themselves as the service providers. secondly, it is difficult to conduct longitudinal study on the development of hiv/aids-specific ngos in china. the researcher realized that many of the ngos that had been interviewed in were no longer under operation in the follow-up fieldwork in , either because these ngos could not secure sustainable funding, or they were cracked down by local authorities for advocating hiv/aids-related human rights or gender equality, or having very close links to foreign (western) organizations. further research therefore needs to be conducted to remedy these technical weaknesses. it is suggested that comparative research could be conducted on the effects (if any) of the regime types, such as china and thailand, and the level of socio-economic development of the country, such as china and singapore, on the effects of international hiv/aids securitization in the national levels. endnotes here ngos does not include government-organized non-governmental organizations (gongos). referent objects refer to "things that are seen to be existentially threatened and that have a legitimate claim to survival." see buzan b et al., security: a new framework for analysis, p. . "civil society" is often translated as "minjian shehui", "shiming shehui" and "gongming shehui" in chinese. in fact, the three different chinese terms do not have the same meaning. many scholars actually use these different chinese terms of civil society simultaneously. "minjian shehui" is used in this article because the term is widely used in the academic research in china's modern civil organizations. "civil society" in this article refers to a sphere that is conceptually separable from the state and government. at the same time the state council abolished the jijin guanli banfa stipulated in . the chinese and english versions of the revised regulation ( ) can be found in chinalawinfo. "regulation on registration and administration of social organizations ( ) (shehui tuanti dengji guanli tiaoli). nevertheless, grassroots ngos registered under the moic also encounter problems with regard to the amount and source of funding. as registered commercial entities, grassroots ngos are required to pay a % tax on any revenue, even for funding received for non-profit purposes. in recent years, the abovementioned legislative hurdle in ngo registration has been relaxed in some parts of china, first in shenzhen and then in beijing. the local government in these two cities relaxed its ngo registration rule, and allowed ngos in the fields of business, charity, welfare, and social services to register directly with the moca. such a move can also be observed at the national level. in early , the national government started planning to revise the regulations on ngo registration and management. if the revised version is passed by the state council, the individual-organized or grassroots ngos can directly register with the moca without seeking a supervisory body prior to the registration. it is noted that the author is fully aware of the arguments put forward by timothy hildebrandt, stating chinese ngos could be favored or cracked down by the state apparatus irrespective of registration status. the author is argued that unregistered ngos are comparatively easier to be targeted and cracked down by the government officials in the name of "unlawful organizations". for hildebrandt's argument, see hildebrandt, t. the political economy of social organization registration in china. the china quarterly. ; : - . gongos are composed of three types of organizations existed in the s and s: ( ) private organizations from the "old china"; ( ) friendship associations for the promotion of trade, and cultural exchange agencies; and ( ) people's organizations and mass organizations. in the course of the meeting, us vice president al gore claimed that hiv/aids was a security threat because "it threatens not just individual citizens, but the very institutions that define and defend the character of a society. the disease weakens workforces and saps economic strength. hiv/aids strikes at teachers, and denies education to their students. it strikes at the military, and subverts the forces of order and peacekeeping". mcinnes and rushton ( ) argued that the hiv/ aids securitization in was not a successful or complete securitization. however, this article does not intend to examine whether the international hiv/aids securitization has been fully accepted by the target audience. it intends to examine the influences of the hiv/ aids international securitization on the national and sub-national levels in china. in , the bush administration launched the president's plan for emergency aids relief (pepfar), pledging us$ billion from to to "turn the tide against hiv/aids." the who launched the " × " campaign that targets getting million people on antiretroviral treatment by , while the multi-country hiv/aids program of the world bank provided more than us$ . billion for grants and concessional loans to help governments respond to hiv/aids issues. examples included the indian network of people living with hiv/aids, thai network of people living with hiv/aids, asia pacific council of aids service organization in malaysia, and also action for aids singapore, economic reform and growth in china routledge handbook of chinese security. london: routledge bric's growing share of global health spending and their diverging pathways the health care systems of china and india: performance and future challenges evolving health expenditure landscape of the brics nations and projections to rural china, a steep price of poverty: dying of aids: new york times unaids data china's pragmatic approach to aids hiv/aids in china and india: governing health security geopolitics in health: confronting obesity, aids, tuberculosis in the emerging brics economies unleash civil society 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nongovernmental organizations. volunt int j volunt nonprofit org globalization and its methodological discontents: contextualizing globalization through the study of hiv how is health a security issue? politics, responses and issues securitizing infectious disease should hiv/aids be securitized? the ethical dilemmas of linking hiv/ aids and security where are the links? council on foreign relations on the responsibility of the security council in the maintenance of international peace and security: hiv/aids and international peace-keeping operations. united nation security council united nations general assembly united nations general assembly political declaration on hiv/aids: intensifying our efforts to eliminate hiv/ aids. united nations general assembly united nations general assembly aids and security: where are we now? hiv/aids and securitization theory the global fund to fight aids, tuberculosis, and malaria china's evolving aids policy: the influence of global norms and transnational non-governmental organizations china among success stories in global hiv/aids fight: unaids chief. xinhua. peking daily cautions against western threats of aids, drugs: the associated press leexisnexis newspapers . ramiah i. securitizing the aids issue in asia gao qiang, at the hiv/aids high-level meeting of the un general assembly. permanent mission of the people's republic of china to the un state council notice on strengthening hiv/aids prevention and control. ncaids, china cdc 国务院关于进一步加强艾滋病防治工作的通知 [state council notice on further strengthening hiv/aids prevention and control.] 国发 号 [state council document no red ribbon forum redoubles aids fighting bid. china daily keqiang wants tax break for ngos specializing aids/hiv work. south china morning post china after the global fund investment in hiv/ aids programs: does it help strengthen health systems in developing countries? glob health the global fund: managing great expectations china country coordinating mechanism secretariats. the global fund to fight aids, tuberculosis, and malaria people's republic of china: national center for aids/std control and prevention aids funds frozen for china in grant dispute global fund lifts china grant freeze. the body china aids fund for non-governmental organizations making turkey's transformation possible: claiming "securityspeak"-not desecuritization! southeast european and black sea studies estimates of global, regional, and national incidence, prevalence, and mortality of hiv, - : the global burden of disease study the political economy of social organization registration in china this work is supported by a hong kong university research council general research fund grant # . the author would like to thank anonymous reviewers for their useful suggestions. the author would also like to express the appreciation to dr. nicholas thomas for his comments on an earlier version of the manuscript. all errors and omissions are the author's responsibility alone. the author declares that she has no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. the author read and approved the final manuscript. key: cord- -sjab zsk authors: mendez, aaron s; vogt, carolin; bohne, jens; glaunsinger, britt a title: site specific target binding controls rna cleavage efficiency by the kaposi's sarcoma-associated herpesvirus endonuclease sox date: - - journal: nucleic acids res doi: . /nar/gky sha: doc_id: cord_uid: sjab zsk a number of viruses remodel the cellular gene expression landscape by globally accelerating messenger rna (mrna) degradation. unlike the mammalian basal mrna decay enzymes, which largely target mrna from the ′ and ′ end, viruses instead use endonucleases that cleave their targets internally. this is hypothesized to more rapidly inactivate mrna while maintaining selective power, potentially though the use of a targeting motif(s). yet, how mrna endonuclease specificity is achieved in mammalian cells remains largely unresolved. here, we reveal key features underlying the biochemical mechanism of target recognition and cleavage by the sox endonuclease encoded by kaposi's sarcoma-associated herpesvirus (kshv). using purified kshv sox protein, we reconstituted the cleavage reaction in vitro and reveal that sox displays robust, sequence-specific rna binding to residues proximal to the cleavage site, which must be presented in a particular structural context. the strength of sox binding dictates cleavage efficiency, providing an explanation for the breadth of mrna susceptibility observed in cells. importantly, we establish that cleavage site specificity does not require additional cellular cofactors, as had been previously proposed. thus, viral endonucleases may use a combination of rna sequence and structure to capture a broad set of mrna targets while still preserving selectivity. viral infection dramatically reshapes the gene expression landscape of the host cell. by changing overall messenger rna (mrna) abundance or translation, viruses can redirect host machinery towards viral gene expression while si-multaneously dampening immune stimulatory signals ( ) ( ) ( ) . suppression of host gene expression, termed host shutoff, can occur via a variety of mechanisms, but one common strategy is to accelerate degradation of mrna ( ) ( ) ( ) . this occurs during infection with dna viruses such as alphaherpesviruses, gammaherpesvirues, and vaccinia virus, as well as with rna viruses such as influenza a virus and sars and mers coronaviruses ( , , ) . in the majority of these cases, a viral factor promotes endonucleolytic cleavage of target mrnas. this strategy bypasses the normally rate limiting steps of deadenylation and decapping to effect rapid mrna degradation by host exonucleases ( ) . virally encoded host shutoff endonucleases are usually specific for mrna, yet broad-acting in that they target the majority of the mrna population. this is exemplified by herpesviral nucleases, including the sox endonuclease encoded by kaposi's sarcoma-associated herpesvirus (kshv), an oncogenic human gammaherpesvirus that causes kaposi's sarcoma and b cell lymphoproliferative diseases ( , ) . kshv sox is a member of the pd-(d/e)xk type ii restriction endonuclease superfamily that possesses mechanistically distinct dnase and rnase activities ( ) ( ) ( ) . the rnase activity of the gammaherpesvirus sox protein has been shown to play key roles in various aspects of the viral lifecycle, including immune evasion, cell type specific replication, and controlling the gene expression landscape of infected cells ( ) ( ) ( ) ( ) . however, the mechanism by which sox targets mrnas remains largely unknown. sequencing data indicate that within the mrna pool there appears to be a range of sox targeting efficiencies; some transcripts are efficiently cleaved in cells, while others are partially or fully refractory to cleavage ( ) ( ) ( ) ( ) ( ) . additionally, sox has been shown to cut within specific locations of mrnas in cells, further emphasizing that there must be transcript features that confer selectivity ( , ) . indeed, a transcriptome-wide cleavage analysis indicated that sox targeting is directed by a relatively degenerate motif, often containing an unpaired polyadenosine stretch shortly upstream of the cleavage site, which is located in a loop structure ( ) . cleavage within an unpaired loop was confirmed in a recent crystal structure of sox with rna, although additional contacts that could confer sequence specificity were not observed ( ) . thus, a major outstanding question is how rna sequence and/or structure contribute to sox target recognition. in this context, it is unclear how sequence features surrounding the rna cleavage site might impact sox targeting, for example by changing its affinity for a given rna or the efficiency with which cleavage occurs. to address these questions, we sought to reconstitute the sox cleavage reaction in vitro using purified components. using an rna substrate that is efficiently cleaved by sox in cells, we revealed that specific rna sequences within and outside of the cleavage site significantly contribute to sox binding efficiency and target processing. in particular, we found that the polyadenosine stretch adjacent to the cleavage site is critical for sox binding, and we experimentally verified the importance of an open loop structure surrounding the cleavage site. finally, we demonstrated that this in vitro system faithfully recapitulates the initial endonucleolytic cleavage event that is an essential component of mrna target specificity in vivo. collectively, our data reveal that specific sequence features potently impact sox binding, and thus provide key insight into the breadth of sox targeting efficiency observed across the transcriptome. more broadly, this information provides a framework for better understanding the target specificity of endonucleases, which play central roles in mammalian quality control processes and viral infection outcomes. kshv sox was codon optimized for sf expression and synthesized from genewiz. sox was then subcloned using restriction sites bamhi and sali (new england bio-labs) into pfastbac htd. this vector was modified to carry a gst affinity tag and prescission protease cut site as described ( ) . all sox mutants were generated using single primer site-directed mutagenesis ( ) . sequences were validated using standard pgex forward and reverse primers. generation of viral bacmids and transfections were prepared as described in the bac-to-bac ® baculovirus expression system (thermo fisher scientific) manual. after transfection, sf cells (thermo fisher scientific) were grown for h at • c using sf- smf media (gibco) substituted with % fetal bovine serum (fbs) and % antibiotic antimycotic (aa). supernatant was transferred to a six-well tissue culture plate containing ml of × ∧ cells/well. cells were incubated for hr to generate passage (p ). the p supernatant was transferred to a flask containing ml of × cells/ml and incubated for h, a time point sufficient to yield mg of sox per ml of cells. protein expression was confirmed by western blot with an anti-gst antibody (ge health care life sciences). sf cell pellets were suspended in lysis buffer containing mm nacl, % glycerol, . % triton x- , mm dtt, mm hepes ph . with a complete, edta-free protease inhibitor cocktail tablet (roche). cells were sonicated on ice using a macro trip for s bursts with min rests for min at a. cell lysate was cleared using a pre-chilled ( • c) sorvall lynx superspeed centrifuge spun at rpm for min. the cleared lysate was incubated for h at • c with rotation with ml of a gst bead slurry (ge healthcare life sciences) that had been pre-washed × with wash buffer (wb) containing mm nacl, % glycerol, mm dtt, mm hepes ph . . the bead-protein mixture was washed × with ml of wb, then transferred to a ml disposable column (qiagen) and washed with an additional ml of wb followed by ml of low salt buffer (lsb) containing mm nacl, % glycerol, mm dtt, mm hepes ph . with periodic resuspension to prevent compaction. sox was then cleaved on column with prescission protease (ge healthcare life sciences) overnight at • c, and protein eluate was collected for a final volume of ml in lsb. cleaved protein was concentrated to ∼ ml using amicon filter concentrator membrane cut off kda (emd millipore), then loaded onto a hiload superdex s pg gel filtration column (ge healthcare life sciences). protein elutions were concentrated using an amicon concentrator described above to mg/ml and l aliquots were snap frozen in liquid n using nuclease-free . ml microfuge tubes (ambion life technologies) and stored at - • c. all rna substrates (sequences in supplementary table s ) unless stated otherwise were synthesized by dharmacon (ge healthcare) with hplc and page purification. rnas were end labeled with ␥ -[ p]-atp- ci/mmol mci/ml (perkin elmer) using t pnk (new england bi-olabs). rnas were end labeled with -[ p]-pcp ci/mmol mci/ml using t rna ligase (new england biolabs). labeled rna substrates were purified using % urea-page and were isolated from gel slices by incubating overnight at • c in a buffer containing mm tris-hcl, mm edta ph . . eluted rnas were ethanol precipitated and resuspended in rnase-free ddh o. k obs and hill coefficients of sox were determined from the cleavage kinetics of [ p]-labeled rna substrates as previously described ( ) . briefly, l (≤ pm) of [ p]-labeled rna was added to l of premixture containing mm hepes ph . , mm nacl, mm mgcl , mm tcep, % glycerol, and increasing concentrations of purified sox. reactions were performed at room temperature under single turnover conditions, and quenched at the indicated time intervals with l stop solution ( m urea, . % sds, . mm edta, . % xylene cyanol, . % bromophenol blue). samples were resolved by % urea-page, imaged using a typhoon variable mode imager (ge healthcare), and quantified using imagequant and gelquant software packages (molecular devices). the data were plotted and fit to exponential curves using prism software package (graphpad) to determine observed rate constants. a fret probe with excitation at nm and emission at nm (limd flo) was purchased from dharmacon (supplementary table s ). the rna fret probe was added at a final concentration of nm to l of premixture containing mm hepes ph . , mm nacl, mm mgcl , mm tcep, % glycerol with m of sox ( ) . terminator exonuclease (lucigen) was added to reactions using a : dilution of the enzyme. reactions were quenched at indicated time intervals with equal volumes of stop solution containing % formamide and mm edta, then resolved using urea-page and visualized using a typhoon variable mode imager (ge halthcare). the data were plotted using prism software package (graphpad). all experiments were repeated > times and mean values were computed. for assays designed to detect endonucleolytic cleavage intermediates, l of labeled rna substrate was combined with l of reaction solution ( mm hepes ph . , mm nacl, . mm cacl, . mm mgcl , % glycerol, . mm tcep) in the presence or absence of m sox for min at room temperature. rna was then ethanol precipitated, resuspended in % formamide solution containing mm edta, and resolved on a % urea-page analytical grade sequencing gel together with a ss-rna decade ladder (ambion life technologies) for . h at w before imaging as described above. the sequence surrounding the cut site in limd was inserted into a pbssk (-) backbone using the bamhi and xbai restriction sites. mutations were introduced by the quickchange site directed mutagenesis protocol (agilent). the nt sequence surrounding the gfp cut site was inserted using the bamhi and xhoi restriction site. in-line probing was performed as described previously ( ) . briefly, pbssk(-) plasmids containing the indicated sequences (see supplementary table s ) were linearized by digestion with xhoi and scai for gfp or blpi and saci (neb) for limd , gel purified, phenol/chloroform extracted, and ethanol precipitated. the fragments were then used as templates for in vitro transcription with the hiscribe t high yield rna synthesis kit (neb) and afterwards subjected to turbo dnase (ambion by life technologies) treatment. rna was resolved by % urea page, and full length transcripts were excised from the sybr gold stained gel (thermo fisher scientific), eluted overnight in g buffer ( mm tris hcl ph . , mm naoac, mm edta, . % sds), phenol/chloroform extracted, and ethanol precipitated. the rna (∼ pmol) was dephosphorylated using shrimp alkaline phosphatase (rsap, neb), labeled with l [␥ p] atp ( mci/ml) using usb optikinase (affymetrix), then gel purified as described above and dissolved in l of nuclease free water. for the in-line probing reaction, l rna (≥ cpm) was incubated in × reaction buffer ( mm tris-hcl ph . , mm mgcl , mm kcl) at room temperature for or h. the reaction was quenched with × loading buffer ( m urea, . mm edta ph . ). to generate ladders, l of the purified rna was separately subjected to hydrolysis using the next magnesium rna fragmentation module (-oh) or rnase t digestion (t ) (neb). reactions were resolved by % urea-page, exposed on a phoshorimager screen, and scanned using the storm imaging system (ge healthcare). deduced rna structures were drawn using the rna secondary structure visualization tool forna (vienna rna web services). rna probes used in emsa experiments were radiolabeled using the protocol described for ribonuclease activity assays. reactions were incubated at rt for min in buffer containing mm hepes ph . , mm kcl, mm cacl , . % tween- , . tcep, . mg/ml bsa (sigma-aldrich), g/ml of yeast trna (ambion thermo fisher), and the indicated amount of purified sox protein. calcium chloride was used in these binding assays to prevent substrate processing and stabilize rna-protein interactions. reactions volumes were kept at l and stopped with l × emsa loading dye ( mm hepes ph . , mm kcl, % glycerol). reactions were resolved by % native page, and gels were imaged on a typhoon multivariable imager (ge healthcare) and quantified using gelquant software package (molecular dynamics). limd - rna was end labeled with ␥ -[ p]-atp- ci/mmol mci/ml (perkinelmer) using t pnk (new england biolabs). rna was then gel purified as stated previously. emsa gel shifts were first used to determine optimal binding conditions (> % binding, homogeneous complexes of rna-protein). binding buffer contained . % tween (sigma-aldrich), mm cacl , mm kcl, mm nacl, . mm tcep, mm hepes ph . , . mg/ml yeast trna (ambion), . mg/ml nuclease free bovine serum albumin (bsa) (ambion). a dilution series of sox ( - . m) was incubated with l of radiolabeled limd - in the presence of . unit of rnase t (epicentre illumina). reactions were incubated at rt for a total of min before being ethanol precipitated. rna pellets were then resuspended in l of % formamide solution containing mm edta and boiled for min. samples were then loaded onto a % analytical grade urea-page gel and run at w for . h. gels were imaged and analyzed as stated above. in order to produce an rnase t ladder, l of limd - was incubated with . units of rnase t . reactions were incubated at rt for min before being quenched and prepared as stated previously. the limd - hydrolysis ladder was generated as stated in the in-line probing methods. rna probes ( end labeled with biotin) were synthesized from dharmacon (ge healthcare) and hplc and page purified (see supplementary table s ). the octet red e bio-layer interferometry instrument and streptavidin (sa) biosensors were available from fortebio (menlo park, ca, usa). all steps were performed in reaction buffer similar to emsa binding conditions. biosensors were incubated with nm of the biotinylated rna substrate for containing no rna. sox protein was incubated with the rna conjugated biosensors for - s in order to reach saturation. indicated protein concentrations for each bio sensor are located on corresponding binding curves. complexes were dissociated for minimum of min. response curves for each biosensor were normalized against biosensors conjugated to rna in the absence of sox (buffer only control). normalized response curves were processed using octet software version by fitting the group of selected bio sensors to a nonlinear regression model ( ) . dissociation constants (k d ) were determined from k on and k dis values derived from the fitted curves. a complete table of all values is provided in supplementary table s . in cells, the mrna fragments resulting from the primary sox endonucleolytic cleavage are predominantly cleared by the host - exonuclease xrn , while in vitro, rna fragments are rapidly degraded by - exonucleolytic activity intrinsic to purified sox ( ) . thus, it has been challenging to analyze the initial endonucleolytic cleavage event that is an essential component of mrna target specificity in vivo. here, we sought to develop a biochemical system to address these questions. our prior analysis of sox targets in cells identified the human limd mrna, which codes for a protein essential for p body formation and integrity, as being highly susceptible to cleavage by sox ( ) . the minimum sequence required to directly cut the putative cleavage site in limd in cells was mapped to a -nucleotide segment (limd - ), and we therefore chose this as our model substrate to study sox targeting in vitro ( ) . we first expressed and purified kshv sox to greater then % purity from sf insect cells (supplementary figure s a ). using the limd - substrate, we plotted the observed rate constant (k obs ) as a function of sox concentration, yielding a hill coefficient of n = . ( figure a ). thus, in agreement with previous observations ( , ), sox appears to function predominantly as a monomer. under conditions of half maximal activity ( m; figure a ), sox displayed a strong preference for the 'hard' divalent metal mg + and a weaker preference for the 'softer' and larger metals mn + , co + and zn + ( figure b ). this is again consistent with other characterized members of the p/dexk family of enzymes ( , ) . notably, sox activity in the presence of mg + was inhibited in a dose-dependent manner upon competitive addition of ca + ( figure c and supplementary figure s d ). this is likely the result of increased coordination partners engaged by ca + , which decreases the ability of catalytic residues to promote proper base hydrolysis ( ) ( ) ( ) . finally, increasing the nacl concentration above mm led to substantially decreased sox activity ( figure d ), in accordance with the observation that high salt concentrations frequently inhibit nuclease activity by disrupting protein-protein or proteinsubstrate interactions ( ) . given that recombinant sox displays robust - exonuclease activity ( , ), we sought to confirm that limd - was subject to endonucleolytic sox cleavage, as this is the predominant event that directs mrna turnover in sox expressing cells ( , ) . both the and ends limd - were blocked by capping the end with a cy fluorophore and the end with an iowa black quencher (limd - flo). we confirmed this rna was resistant to degradation by the -phosphate dependent exonuclease terminator ( figure e, lane ) . however, in the presence of sox, a cleavage product was observed that correlated with an endonucleolytic cut ( figure e, lane ) . to confirm this processing event was not a result of contamination, we purified a sox mutant containing mutations within two key residues of the sox active site (d n/e q). incubation of this mutant with limd - over the course of . h yielded no rna cleavage (supplementary figure s e ). thus, recombinant sox appears to target limd - for endonucleolytic cleavage in vitro, as has been observed for this substrate in cells. to analyze rna substrate selectivity using our in vitro assay, we first compared sox degradation of limd - to a -nucleotide sequence of the mrna encoding gfp (gfp- ). we have previously shown that gfp mrna is cleaved by sox in cells, and that gfp- is the minimal sequence required to elicit cleavage ( , ) . the cleavage sites for limd - and gfp- are predicted to occur in an open loop region (figure a, red arrow) . upon direct comparison of these two rnas, we observed a ∼ -fold increase in the catalytic efficiency of sox for the limd - substrate compared to gfp- ( figure b ). this difference was not exclusively due to the fact that the gfp substrate was slightly shorter than limd - , as sox also displayed a fold reduction of catalytic efficiency on a longer, nt gfp substrate (gfp- ; figure b ). electrophoretic mobility shift assays (emsa) further revealed a -fold increase in sox binding to limd - compared to gfp- ( figure c ). given that both substrates contain the requisite unpaired bulge at the predicted cleavage site (see figure a and supplementary figure s ), these observations suggest that additional sequence or structural features impact sox targeting efficiency on individual rnas. two sox point mutants, p s and f a, located in an unstructured region of the protein that bridges domains i and ii have been shown to be selectively required for its endonucleolytic processing of rna substrates (supplementary figure s a and s b) ( , ) . structural data indicate that residue f forms a stacking interaction with an adenine base in the rna, likely stabilizing the protein-rna interaction, while p is hypothesized to contribute to structural rearrangements required for f engagement ( ) . we purified both mutants to evaluate their relative rna processing and rna binding activity against the optimal limd - substrate. both mutants displayed purity and elution profiles similar to wild type (wt) sox (see supplementary figure s a-c) . however, the catalytic efficiency of each mutant was > -fold less than wt sox ( figure d ). furthermore, rna binding was severely perturbed; the binding kinetics of wt sox for limd - are in the single digit nanomolar range (k d = nm), while p s and f a display > log defects (k d = nm and nm, respectively) ( figure e and supplementary figure s a c). thus, the large defect in rna binding likely explains the decreased efficiency of rna processing. notably, while there was a dramatic decrease in the relative affinities of the two mutants for limd - , there was not a complete loss of binding or rna processing. this could be a result of secondary nonspecific interactions and/or nonspecific exonucleolytic degradation by sox from the monophosphorylated end of the probe. in silico rna folding predictions of sox targeting motifs, coupled with rna mutagenesis experiments, have indicated that an rna stem loop structure is an important determinant in sox targeting both in vitro and in vivo ( , ) . given the importance of this predicted motif, and in partic- predicted rna fold ular the proposed requirement for unpaired sequence at the cut site, we sought to experimentally determine the structure of limd - using chemical based in-line probing ( figure a ). this showed that the limd - structure contains a largely base paired stem region, followed by a loop at positions - that encompasses the predicted sox cleavage site between nt and , and a short hairpin struc-ture at positions - ( figure b) . notably, some differences exist between the predicted and observed structures of limd - , including a larger loop region and the subsequent short stem-loop (compare figure b to figure a ) however, in both cases the predicted cleavage site of sox resides in a loop region. recently, a high-resolution crystal structure was solved of sox bound to a nt fragment of the kshv pre-microrna k - (k - ). in this structure, the only observed contacts between sox and k - occurred between the four active site residues of sox (y , r , c , f ) and the ugaag motif surrounding the cleavage site of the rna ( ) . it was therefore hypothesized that no other residues beyond this unpaired ugaag motif were involved in transcript recognition ( ) . however, the binding affinity we observed for limd - was -fold stronger that what was previously reported for k - ( ) , suggesting that a more extended interaction surface might distinguish optimal from sub-optimal rna substrates. we therefore used rna footprinting to map the sox binding sites on limd - . indeed, sox protected a region of limd - that included the three adenosine stretch (positions - ) from rnase t digestion in a dose dependent manner ( figure ) . notably, this mapped binding region is the same region predicted from in vivo pare-seq data to be important for sox targeting, although the reason for its importance remained unknown ( ) . we also observed a modest protection of base (g) located directly adjacent to the predicted cleavage site of sox, which represents the region detected in the crystal structure of k - bound to sox. collectively, these findings suggest that while sox may interact with residues directly adjacent to the cut site, a more extensive interaction interface exists for its preferred in vivo targets. to explore the importance of the residues involved in sox binding and cleavage, we engineered mutants of the limd - substrate ( figure a ). first, we preserved the loop structure but replaced the three adenosines bound by sox (residues - ) with guanosines (limd - xa-g). second, we largely abolished the loop structure by providing complementary base pairing (limd - zipper). third, we mutated the residue located at the predicted sox cut site that was also protected in the footprinting assay (limd - a-g). this mutant has been previously identified to block sox cleavage in vivo ( ) . tary figure s ). real-time binding kinetics for sox with wt limd - and each of the three mutant substrates were then measured using bio-layer interferometry (bli). all rna probes were biotinylated and immobilized to a streptavidin-coated bli probe, whereupon the binding and dissociation of sox was measured. to prevent degradation of the probe, excess calcium ion was used in place of magnesium (supplementary figure s e) . sox retained similar binding affinity to the cut site mutant table s ). to rule out the possibility that the effect on binding affinity to the limd - zipper mutant was a result of altered residues within the binding site, we also engineered an additional zipper mutant (limd - zipper ) that did not disrupt the polyadenosine sequence. in agreement with the loop structure playing a critical role in target recognition, this limd - zipper mutant also displayed a substantial defect in binding (k d = . m; supplementary figure s a , b, supplementary table s ). finally, we measured sox binding to the kshv pre-mirna sequence used to obtain the sox-rna cocrystal structure (k - ) ( ) . notably, the affinity of sox for k - was within the range of the limd - structural mutants (k d = . m), suggesting that despite having an ugaag motif upstream of a predicted bulge, this is unlikely to be a sox target ( figure b , supplementary figure s e and supplementary table s ) . we next quantitatively measured the catalytic efficiency of sox towards each of the above rna substrates. despite sox having wt binding affinity for the predicted cleavage site mutant limd - a-g, there was a -fold defect in ' its ability to degrade this mutant ( figure c ). even more marked defects in sox catalytic efficiency were observed for the binding site mutant limd - xa-g, the loop mutants limd - zipper and limd - zipper , and the pre-mirna k - ( figure c and supplementary figure s ). collectively, these data indicate that efficient rna cleavage requires both an appropriate sox binding site and a suitable cut site. in cells, sox cleaves its mrna substrates site-specifically. mutagenesis of residues in mapped cleavage sites generally abolishes sox cleavage at that location ( ) . to determine if our in vitro assay faithfully recapitulated the site specificity of sox endonucleolytic targeting observed in cells, we established reaction conditions that enabled trapping of nucleic acids research, , vol. , no. the early cleavage events. by combining ca + and mg + in our reaction buffer, we were able to sufficiently slow sox processing to visualize cleavage products derived from p labeled substrates. indeed, we observed a predominant nt band, which is the size of the product released upon limd - cleavage at the predicted cut site ( figure a , lane ). additional bands also appeared, likely representing subsequent processing events. importantly, when we incubate sox with the cut site mutant limd - a-g, there is a complete loss of this nt product, as well as the additionally processed intermediates ( figure a, lane ) . production of these cleavage intermediates required sox, as no decay was observed in the rna-only controls ( figure a , lanes - ). finally, we sought to verify that the predominant nt cleavage product we observed was a result of an endonucleolytic cleavage and not end processing. to this end, we generated a limd - substrate containing a p pcp label and a free oh to block end processing. again, in the presence of sox, wt limd - but not the a-g mutant produced a cleavage product whose size corresponded to cleavage at the predicted site ( figure b ). taken together, these data confirm that our in vitro assay faithfully recapitulates sox cleavage site specificity on a true substrate. endonuclease-directed mrna degradation plays key roles in the lifecycle of gammaherpesviruses, yet the fundamental principles governing target specificity by sox and other viral endonucleases are not well understood. here, through the development of the first biochemical system to faithfully recapitulate the internal cleavage specificity observed for sox in cells, we revealed how both rna sequence and structure contribute to targeting. these findings resolve a central feature of the current model of sox activity ( figure ). previous observations established that sequences flanking the cut site were required to direct cleavage by sox ( , ) . however, it was unresolved whether they played a strictly structural role in presenting an exposed loop for cleavage, served as a platform for sox binding, or created a binding site for one or more cellular factors that then indirectly recruited sox to its targets. through a combination of mutational analyses, rna structure probing, and rna footprinting assays, we showed that efficient sox targeting requires both an exposed loop structure and upstream sequences that serve as a sox binding platform. this combination of sequence and structural features within the targeting motif helps explain why some mrnas are efficiently cleaved by sox, whereas others are weaker substrates. a key open question related to sox function is how it can target the majority of mrnas in cells, yet with significant site specificity. our observations suggest that there must be specific mrna features that influence targeting. indeed, pare-seq analyses of cleavage intermediates in sox expressing cells revealed that cleavage sites were associated with a degenerate sequence motif ( ) . sequences proximal to the cleavage site were predicted to be un-base paired and frequently contained a polyadenosine stretch followed by a purine ( ) . the requirement for these sequence features for sox targeting was validated for the limd transcript in cells ( ) . because limd has been established as a particularly robust sox target in cells ( ) , we reasoned that it must contain features optimal for sox processing and therefore would be an ideal substrate to dissect biochemically why these features are important. indeed, sox binding to limd - was -fold better than to the commonly used reporter substrate gfp, and ∼ -fold better than to the k - pre-mirna, which has not been demonstrated to be processed by sox in cells. importantly, these binding differences correlated with the efficiency of sox cleavage in vitro, arguing that the ability to bind the targeting motif is a key step in target recognition. through rna footprinting assays, we were able to show that sox binds to a bulge structure proximal to the cleavage site containing the polyadenosine stretch previously predicted to be important for mrna cleavage by sox in cells ( ) . mutating either just the bulge structure (limd - zipper ) or maintaining the bulge but mutating the polyadenosine stretch (limd - xa-g) resulted in a ∼ -fold reduction in binding affinity, correlating with a dramatic decrease in cleavage efficiency. collectively, these data demonstrate that variability in the efficiency of sox targeting observed in cells is likely due to differences in rna sequences that mediate sox binding. a recent crystal structure of sox bound to the k - pre-mirna captured the importance of the exposed loop region for sox cleavage ( ) . however, the structure did not reveal additional interactions between sox and the rna beyond the three residues surrounding the cut site. our data suggest that this is likely because the k - rna lacks the additional residues necessary for sox binding site found in both limd and gfp. while the k - rna does contain adenosines upstream of the cleavage site, structural predictions indicate these residues are within a stem region ( ) , rather than in an exposed loop as is the case for limd and gfp. together, these observations indicate that while upstream adenosines are important for binding, they must be present in an unpaired state to promote sox binding. it is notable that prior studies reported much weaker interactions between sox and rna (k d = m) compared to its dna substrates (k d = m) ( , , ) . however, in these cases binding assays were conducted with scrambled rna sequences. we found that sox binding affinities to rna substrates vary over several orders of magnitude, in a manner that correlates with cleavage efficiency. interestingly, the crystal structure of sox bound to dna showed more dynamic interactions along the length of the protein (∼ Å interaction surface), when compared to the k - rna bound structure (∼ Å interaction surface). it is therefore possible that more interaction along the length of sox protein might occur with optimal substrates such as limd that are more tightly bound. the fact that purified sox endonucleolytically cleaved limd - at the precise site observed in sox-expressing cells demonstrates that cleavage site selection on an mrna is not mediated by a cellular cofactor. instead, targeting at particular rna motifs is strongly influenced by the strength of sox binding. our observation that the p s and f a sox mutants display significant rna binding defects indicates that their failure to cleave mrnas in cells is due to an inability to efficiently bind the targeting motif. target identification exonucleolytic degradation by xrn / dis l figure . model of mrna targeting by sox. sox is able to distinguish mrna from other types of rna in cells by an as yet unknown mechanism. subsequently, it endonucleolytically cleaves its targets at specific sites, whereupon the fragments are degraded by host exonucleases such as xrn and dis l . here, we revealed that in addition to the requirement for an unpaired loop at the cleavage site, additional upstream rna sequences increase the affinity of sox for individual targets, thereby controlling cleavage efficiency. the mechanism by which sox initially distinguishes rna polymerase ii transcribed mrnas from other types of rna in cells remains an important open question, as this feature of sox selectivity is not preserved in vitro. we hypothesize that cellular co-factors, perhaps though interactions with sox, enable this distinction. more broadly, endonucleases are instrumental in rna processing and degradation. nuclease processing defects lead to several human pathologies ranging from cancer to neurodegeneration ( ) ( ) ( ) ( ) ( ) , and our study provides a framework for better understanding the mechanistic features governing endonuclease targeting. emerging roles for rna degradation in viral replication and antiviral defense modulation of the translational landscape during herpesvirus infection a common strategy for host rna degradation by divergent viruses a two-pronged strategy to suppress host protein synthesis by sars coronavirus nsp protein influenza a virus protein pa-x contributes to viral growth and suppression of the host antiviral and immune responses increasing incidence of cancers associated with the human immunodeficiency virus epidemic human herpesvirus- : kaposi sarcoma, multicentric castleman disease, and primary effusion lymphoma the exonuclease and host shutoff functions of the sox protein of kaposi's sarcoma-associated herpesvirus are genetically separable crystal structure of the shutoff and exonuclease protein from the oncogenic kaposi's sarcoma-associated herpesvirus crystal structure of a kshv-sox-dna complex: insights into the molecular mechanisms underlying dnase activity and host shutoff global mrna degradation during lytic gammaherpesvirus infection contributes to establishment of viral latency gammaherpesviral gene expression and virion composition are broadly controlled by accelerated mrna degradation host shutoff during productive epstein-barr virus infection is mediated by bglf and may contribute to immune evasion aberrant herpesvirus-induced polyadenylation correlates with cellular messenger rna destruction lytic kshv infection inhibits host gene expression by accelerating global mrna turnover coordinated destruction of cellular messages in translation complexes by the gammaherpesvirus host shutoff factor and the mammalian exonuclease xrn deep sequencing reveals direct targets of gammaherpesvirus-induced mrna decay and suggests that multiple mechanisms govern cellular transcript escape an rna element in human interleukin confers escape from degradation by the gammaherpesvirus sox protein nuclease escape elements protect messenger rna against cleavage by multiple viral endonucleases transcriptome-wide cleavage site mapping on cellular mrnas reveals features underlying sequence-specific cleavage by the viral ribonuclease sox kshv sox mediated host shutoff: the molecular mechanism underlying mrna transcript processing t cell costimulatory receptor cd is a primary target for pd- -mediated inhibition the unfolded protein response signals through high-order assembly of ire in-line probing analysis of riboswitches structure of the atp synthase catalytic complex (f( )) from escherichia coli in an autoinhibited conformation identification of new homologs of pd-(d/e)xk nucleases by support vector machines trained on data derived from profile-profile alignments crystal structures of lambda exonuclease in complex with dna suggest an electrostatic ratchet mechanism for processivity why do divalent metal ions either promote or inhibit enzymatic reactions? the case of bamhi restriction endonuclease from combined quantum-classical simulations cofactor-mediated conformational control in the bifunctional kinase/rnase ire the rna exosome and rna exosome-linked disease mutations of exosc /rrp p associated with neurological diseases impact ribosomal rna processing functions of the exosome in s. cerevisiae the rnase ii/rnb family of exoribonucleases: putting the 'dis' in disease nonsense-mediated mrna decay and cancer applying nonsense-mediated mrna decay research to the clinic: progress and challenges mfold web server for nucleic acid folding and hybridization prediction we thank members of the glaunsinger lab for their suggestions and critical reading of the manuscript. we would like to thank the university of california, berkeley tissue culture facility for sf cell maintenance and the university of california san francisco quantitative biosciences institute, antibiome center for use of their octet red e for binding kinetics measurements. nucleic acids research, , vol. , no. key: cord- -jdv u authors: nieto‐rabiela, fabiola; wiratsudakul, anuwat; suzán, gerardo; rico‐chávez, oscar title: viral networks and detection of potential zoonotic viruses in bats and rodents: a worldwide analysis date: - - journal: zoonoses public health doi: . /zph. sha: doc_id: cord_uid: jdv u bats and rodents are recognized to host a great diversity of viruses and several important viral zoonoses, but how this viral diversity is structured and how viruses are connected, shared and distributed among host networks is not well understood. to address this gap in knowledge, we compared the associative capacity of the host–virus networks in rodents and bats with the identification of those viruses with zoonotic potential. a virus database, detected by molecular methods, was constructed in the two taxonomic groups. we compiled , records: in rodents and , in bats. we identified a total of and viruses, of which and are zoonotic viruses, in rodents and bats, respectively. based on a network theory, a non‐directed bipartite host–virus network was built for each group. subsequently, the networks were collapsed to represent the connections among hosts and viruses. we identified both discrete and connected communities. we observed a greater degree of connectivity in bat viruses and more discrete communities in rodents. the coronaviridae recorded in bats have the highest values of degree, betweenness and closeness centralities. in rodents, higher degree positions were distributed homogeneously between viruses and hosts. at least in our database, a higher proportion of rodent viruses were zoonotic. rodents should thus not be underestimated as important reservoirs of zoonotic disease. we found that viruses were more frequently shared among bats than in rodents. network theory can reveal some macroecological patterns and identify risks that were previously unrecognized. for example, we found that parvovirus in megabats and gbagroube virus in rodents may represent a zoonotic risk due to the proximity to humans and other zoonotic viruses. we propose that epidemiological surveillance programmes should consider the connectivity of network actors as a measure of the risks of dispersion and transmission. bats and rodents are hosts of a significant proportion of zoonoses, higher than any other mammalian order. over viruses belonging to viral families have been isolated or detected in bats; however, bat-human transmission has only been observed for viruses, belonging to four different viral families: rhabdoviridae, filoviridae, coronaviridae and paramyxoviridae (allocati et al., ) . some examples of those viruses are as follows: sars-related coronavirus, sosuga rubulavirus, ebola virus and marburg virus, rabies lyssavirus, nipah henipavirus and hendra henipavirus (allocati et al., ; calisher, childs, field, holmes, & schountz, ; hayman, ; o'shea et al., ; plowright et al., ) . rodents have similar zoonotic potential to bats and are associated with a large number of zoonotic viruses, such as sin nombre virus, puumala virus, crimean-congo hemorrhagic fever virus, kyasanur forest virus, tick-borne encephalitis virus, lassa fever virus and venezuelan equine encephalitis virus, among others. all of the aforementioned bat-and rodent-associated viruses have a large impact on public health. however, it is important to take into count that not all of these viruses are obligate pathogens; some are generally commensal. previous studies have explored viral associations on relatively restricted spatial or phylogenetic scales. for example, hayman ( ) propose maps of viral distributions according to the distribution of hosts' families, streicker et al. ( ) explored rabies viruses using a phylogenetic approach, and cui, tachedjian, and wang ( ) compared retrovirus associations between bats and rodents. anthony et al. ( ) explored coronavirus networks at the level of host family, and bordes, caron, blasdell, garine-wichatitsky, and morand ( ) analyse the relationships among zoonotic diseases in southeast asia. luis et al. ( ) analyse viral networks between rodents and bats at global scale identifying several ecology factors to explain virus-host associations. recently, works explored the specificity and frequency of sharing dna and rna viruses among carnivores and bats (wells, morand, wardeh, & baylis, ) and the importance of the phylogeny to explain the viral richness associated with bats and rodents (guy, thiagavel, mideo, & ratcliffe, ) . however, there are currently no studies at a global level that incorporate the human influence in the viral networks. while some authors consider bats and rodents to belong to a similar category of high zoonotic risk potential (han, schmidt, bowden, & drake, ) , other work examines the differences between bats and rodents (luis et al., ) . several different distinctive features of bats have been hypothesized to explain their particularly high viral richness, such as their ability to fly, long migrations, high trophic diversity and social structure (brook & dobson, ; moratelli & calisher, ) . however, currently the viral diversity and connectivity among different species of bats are not well understood (moratelli & calisher, ; o'shea et al., ) (luis et al., ) . rodents, like bats, have been recognized as reservoirs for several zoonotic viruses (han et al., ) , such as virus of hantaviridae (schmaljohn & hjelle, ) and arenaviridae families (charrel & de lamballerie, ) . however, there are differences in rodent-virus associations that impact their zoonotic potential compared with bats. in disease ecology, analytical tools have been used to holistically explain the dynamics of infections and provide novel hypothesis to explain macroecological patterns (johnson, roode, & fenton, ) . one of the theories that helps to predict dynamic changes in host-pathogen systems is graph theory, also known as network theory (bordes et al., ; johnson et al., ) . this approach can be used to gain better understanding of how interactions take place within pathogen communities, how hosts are connected with pathogens, their preferred association and patterns of pathogen transmission (godfrey, ; white, forester, & craft, ). the graphs, better known as networks, focus on the interactions between entities (newman, ) , and they have the potential to infer relationships within a larger framework (hossain & feng, ; luke & stamatakis, ) . a network is capable of emphasizing the preferred union as a process (hartonen & annila, ) and capturing both the individual elements in a system as well as their relevant interconnections (kolaczyk & csardi, ) . in disease ecology, this kind of analysis could be applied to describe viral diversity associated with different hosts and detect hosts and viruses that share associations, and therefore identify groups that share similar characteristics (white et al., ) . in network theory, centrality and dispersion metrics quantify the importance of each component member (martínez-lópez, perez, & sánchez-vizcaíno, ; newman, ; opsahl, agneessens, & skvoretz, ) . the parameter "betweenness" can be used to impacts • the analysis of virus and host networks (rodents and bats) allows us to measure the potential risk of zoonotic diseases. • measuring network connectivity can be a useful tool for identifying hosts and viruses of potential importance in the transmission dynamic of zoonotic diseases. • bats presented twice as many connections between virus and host as rodents, indicating a higher zoonotic potential transmission. recognize dispersing hosts and key viruses in the evolution or viral transmission (opsahl et al., ; white et al., ) , while "closeness" can indicate hosts and viruses that may have little direct connectivity but are surrounded by important highly connected nodes (opsahl et al., ; white et al., ) . in terms of disease ecology, we can employ these and other parameters to explore the host-host, virus-host and virus-virus interactions by collapsing the networks and identifying communities. network analysis thus offers the opportunity to recognize highly diverse viruses and hosts based on a high degree of connectedness. bats and rodents are excellent taxa in which to implement this tool because they harbour a large number of highly adaptable viruses and hosts with high resistance. therefore, in this study we aimed to compare and recognize the differences in the associative capacity of the host-virus networks in rodents and bats worldwide, as well as to identify the viruses that may shift across species, including humans, suggesting zoonotic potential. does not exist in the ictv classification, we assigned all reports from astroviridae family as astrovirus. to increase the certainty of identification of the viruses, only studies that used molecular methods to detect viruses were included in the database we compiled. subsequently, each of the viruses identified in rodents or bats was classified as direct zoonotic or non-zoonotic pathogens (allocati et al., ; calisher et al., ; han et al., ) . an independent undirected bipartite network was built for each of the orders of rodentia and chiroptera. that is to say, each network included two types of nodes-viruses and hosts. viral nodes were connected to host nodes when the virus indicated by the node has been detected in the species indicated by the host node. we included a human host node, which was connected to viruses that have been classified as zoonotic. this helped us to group and identify zoonotic viruses and viruses close to them (which could have zoonotic potential). then, host-to-host and virus-to-virus networks were constructed in order to explore these networks in different dimensions. the "bipartite.projection" function in the igraph package implemented in r software version . . (r core team, ) was used to collapse the bipartite network. basically, in the collapsed networks a host was connected to another host when they shared a common virus and a virus to a virus when they shared a common host ( figure ). in each host-host collapsed network, the host nodes were conserved and the virus nodes were transformed using the weight of corresponding links in order to illustrate the relationships among different hosts (figure a ,b). in each virus-virus network (one for bats and one for rodents), the virus-virus relationship was highlighted by collapsing the host nodes into the weighted links ( figure c ). we measured the networks on two levels: individual node and the entire network. at the node level, we measured different centrality values including: degree (number of links that a node has), betweenness (number of times a node is an intermediary to connect each possible pair of nodes) and closeness (the degrees of average separation in relation to other nodes) (martínez-lópez et al., ; newman, ) . at the network level, we measured density and diameter. network density is the proportion of links that are actually observed in the network divided by those that could possibly occur and network diameter is the length of the longest geodesic distance (newman, ) . network level measurements were useful for summarizing the "big picture" of the network and identify the key nodes that are closely related to humans. network level measurements were generated using the algorithms provided in the packages "igraph" (csárdi table ), and plots were produced using the packages "igraph" and "ggplot " (gómez-rubio, ) in r (r development core team, ). communities were detected using the maximization of modularity method (newman, ) , which recognize nodes with dense and weak connections between groups. we used the function "cluster_ edge_betweenness" (girvan, girvan, newman, & newman, ; newman & girvan, ) in the "igraph" package (csárdi & nepusz, ) to identify the nodes with dense connections with humans, which is based on the following equation: where m denotes the total number of links in the network, a ij refers to the actual number of links between nodes i and node j , γ is a parameter calculated by the algorithm; k, degree of i; δ gigj is a randomized number of links between a pair of nodes. community detection facilitates the recognition of groups of hosts that share viruses and viruses that share hosts, and which therefore may continue to enter in contact with each other because they share similar characteristics. a subnetwork was built by choosing communities with more than four host-virus pairs, which is above the minimum number accepted in statistical normality samples (n = ) (hammer, harper, & ryan, ; royston, ) . these subnetworks were illustrated to gain better community visualization and recognize the most relevant communities for the detection of potentially zoonotic viruses. from the subnetwork, the most important communities were chosen using to the measures of the members (top five nodes) and the number of zoonotic viruses ( %) as selection criteria. then, a sociogram representing the preferential unions and the complex interaction on the largest communities was constructed using the package "visnetwork" (almende, thieurmel, & robert, ) in r. this choice of subnetworks helps us to focus and observe in more detail the interactions within these important communities. the rodent database contained records including rodent species and viruses, of which are zoonotic viruses. the bat database contained , records, consisting of bats species associated with viruses, of which viruses were classified as zoonotic. both databases are detailed in appendix s . table , and centrality values are in appendix s . . % of the nodes had a degree value of or , making them uninformative in terms of epidemiological information, though they may be involved in co-evolutionary processes. thirty-nine different communities were detected. sixteen communities included only two members, while the largest group consisted of members. this particular group included humans, as shown in appendix s . ten communities with at least eight members were selected in the subnetwork (figure b ). we excluded communities and despite fulfilling the inclusion criteria because they are linear, with a single virus that influences the whole. ( ) ta b l e formulas applied to calculate networks parameters degree centrality (d) freeman ( ) closeness centrality (c) this network contained nodes of rodent species with links. the diameter of the network was , and the density was . ( figure a) . the top five nodes in terms of degree, betweenness and closeness are shown in table , and the remaining values are in appendix s . thirty-five different communities were detected, and the largest of which contained members, followed by a group of members (appendix s ). a total of nodes ( bat species, viruses, human node) and links were contained in the bipartite bat network. the network diameter was , and the density was . ( figure a ). three zoonotic viruses had the highest degree and betweenness values; these were bat coronavirus, rabies lyssavirus and bat paramyxovirus (table ). . % of the nodes had degrees of or , so they do not provide much information to the network but they may be involved in co-evolutionary processes. all centrality values are given in appendix s . twenty-nine different communities were detected; four of those had only two members, and the largest community contained members (appendix s ). eleven communities contained eight or more members (figure b ). communities , and were linear and possessed simple edges, with a virus that influenced the whole community. community was a homogeneous community with rich ecological interactions, but which was not highly related to zoonotic viruses. the last community was associated with humans had the highest number of zoonotic viruses involved (figure c ,d, details below). a low number of zoonotic viruses were found in homogeneous bat network communities. we focused only on the community that included the human node. in figure a , three red lines representing the three highly connected viruses in the network followed by a homogeneous community and later in green the human node connections. the community that included humans (figure c ,d) has host nodes (including human) and virus nodes with links. fruit bat parvovirus was the only non-zoonotic virus in that community. miniopterus, mormopterus and saccolaimus were the only bat genera that directly shared viruses with humans. the host-host chiroptera network contained nodes and , links. the diameter and density of the network were and . , respectively ( figure a ). the five nodes with the highest centrality values are shown in table , and detailed results are in appendix s . sixty-seven different communities were detected; the largest had members. the second largest community consisted of members including humans (appendix s ). the virus-virus network contained nodes and , links. the diameter of the network was , and the density was . bats are well-known as excellent reservoirs for zoonotic viruses that usually result in high public health impact (gay et al., ; luis et al., luis et al., , plowright et al., ) . nevertheless, in our database, (tables and ). however, the clusters in the bat network are four times denser compared with rodents; this makes it easier to continue sharing the viruses. in addition, they can act as virus mixers, allowing the viruses to acquire characteristics that allow them to infect other host species, including humans. in addition, the human node is more closely connected to bats. it shows that viruses are shared to a greater degree among bats as discussed earlier (figures a and a) . one plausible explanation is that many species of bats live in high-density populations, with many individuals in close proximity to each other, such as in caves and roosts sites. indeed, there are always a larger number of bat species than rodent species in a given area (kerth, perony, & schweitzer, ) . the difference in connectivity between the bat and rodent hostvirus networks has implications for the zoonotic potential of each taxon. high connectivity facilitates viral transmission within and between species, and so, bats are expected to have higher zoonotic po- for that reason, deeper comprehension is required to unravel this entanglement. one of the objectives covered in this work was to recognize non-zoonotic viruses that may be strongly connected with humans and therefore have zoonotic potential. the bat-virus community that contained humans was composed of bats distributed in africa and australia may be explained by high rates of human-bat contact (allocati et al., ; rupprecht, ). one non-zoonotic virus that was included in the bat-virus community that included humans is fruit bat parvovirus. the parvoviridae family were transmitted from bats to other mammals by a viral ancestor suggesting their zoonotic capacity, and groups of genes in their genome denote this potential (canuti et al., ) . even though the virus currently infects only bats (canuti et al., ) , it is firmly connected with the human node in our network by pteropus poliocephalus, a species endemic to eastern australia (lunney, richards, & dickman, ) . future studies are recommended to elucidate its potential for zoonosis. andes viruses (rodents), cowpox (rodents) and rabies lyssavirus (bats) were defined as main actors (high values of degree, betweenness and closeness) in the bipartite networks. however, their importance disappeared when the network was collapsed to virus-virus interactions, likely because their geographical restriction may limit their viral connectivity. andes virus is only distributed in south america (martinez et al., ) , cowpox in europe (vorou, papavassiliou, & pierroutsakos, ) and rabies lyssavirus in america (moratelli & calisher, ) . therefore, these viruses in the rodent community selected, community has members with predominantly european distribution. further, two non-zoonotic viruses are included among the nine zoonotic viruses, but we do not think they are likely to have zoonotic potential because they do not have direct contact with the human node. therefore, in this community we do not find viruses with zoonotic potential. in the rodent community , m. musculus has high values of connectivity but in the sociogram (figure d) , it is evident that the connectivity is with non-zoonotic viruses. this host species thus is likely less important in public health terms, but highly relevant for disease ecology. in addition, two zoonotic viruses are included in the community , and while they can transfer their zoonotic potential to other non-zoonotic viruses using m. musculus as a virus mixer, we consider this unlikely because the proportion of zoonotic viruses is low, adding the specificity of rodents' viruses and the associative characteristics founding in the rodents. thus, the one-to-one virushost species relationship suggested by the node-to-edge ratio suggests that spillover is unlikely, though not impossible. in the human-rodent community, gbagroube virus is noteworthy because it is the only non-zoonotic virus found in the community. gbagroube virus should be closely monitored along with its host, mus setulosus, found in central africa (granjon, ) . in our study, the human is the most relevant and largest node connected in both groups. the relevance of the human in the network is explained by several factors. first, humans' enormous population and globalization push human populations to nearly everywhere on earth and greatly increases the probability of contact with innumerable organisms, resulting in the emergence of zoonotic diseases (kock, ) . secondly, because zoonotic diseases have clear social implications, once detected in one species, they are much more likely to be tested for, and thus detected, in others (oliver-morales & abarca garcía, ). it is therefore possible that the high apparent importance of humans in the networks is more due to the over-representation of zoonotic viruses in the literature than to humans actually being particularly highly. in the database, we do not have ebola virus reports because in the database the dbatvir database did not identify the host species from which the virus was isolated. similarly, we found that . % of rodent nodes and . % of bats nodes were poorly connected ( - degree). surely, bats have associations that we do not recognize. in addition, there are associations that could occur but do not, but these cannot be identified because cases in which viruses were tested for and not detected are not often reported. we therefore think it is important to report negative samples and the number of animals tested in meticulous reports. a future study may complement and compare our study with models where the influence of humans is omitted. we must take into account that the human node influences the network structure, and the ecological relationships must be analysed without this influence. surely, laxer networks will be observed when the connective force of humans is removed. however, our study does need to include both to identify potentially zoonotic viruses. it is pertinent that, in future investigations, different characteristics of the viruses must be considered simultaneously and not only by their connectivity in the network such as gene sequence, type of transmission and virulence. in the present study, we focus only on viral host capability, not on the symbionts and their associative nature. spatial analysis may help to further explain how our findings apply among different regions of the world. graph theory, beyond allowing the visualization of complex interactions, allows the quantification of many aspects of connectivity and structure. rodents should be taken into account as important reservoirs for zoonotic viruses, since in our database, a greater proportion of the total viruses reported were zoonotic viruses in rodents than in bats. fruit bat parvovirus in bats and gbagroube virus in rodents should be monitored to elucidate their zoonotic potential. in the present study, we only assessed their network proximity to humans and other zoonotic viruses, and molecular genetic approaches may help to confirm our results. counting the number of zoonotic symbionts associated with each order is not a conclusive estimate of their zoonotic potential. our findings reveal that viruses were more frequently shared among bats than rodents. for that reason, bats have more zoonotic potential that the rodents. however, potential emerging zoonotic diseases may arise from both taxonomic groups. we are very grateful to papiit (project ia ), programa castillo for their contribution in the construction of the databases. we declare that we have no conflict of interest. gerardo suzán https://orcid.org/ - - - oscar rico-chávez https://orcid.org/ - - - bat-man disease 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richard n; mulliniks, j travis title: the effect of cow udder score on subsequent calf performance in the nebraska sandhills date: - - journal: transl anim sci doi: . /tas/txy sha: doc_id: cord_uid: ac bhtdj nan selection pressure for increased production has caused producers to remove cows based on factors that include reproductive failure, structural issues, poor health, and disease. producers emphasize improved growth by selecting genetically superior animals through increased milk yield and calf growth. udder conformation and milk yield have been shown to impact calf preweaning adg (neville, ; gleddie and berg, ) . however, beef cows with poor udder conformation may decrease production by decreased calf bw at weaning and increased labor costs, leading producers to cull females with mammary problems. cows with poor udder confirmation at calving are at greater risk of developing mastitis (degroot et al., ) . although mastitis is more prevalent in dairy herds, it does affect % to % of beef females (haggard et al., ; watts et al., ) , which reduces longevity within the cowherd. mastitis-infected females have decreased milk production and altered milk components that effect overall calf growth (crossman et al., ; paape et al., ) . research has shown defects in teat shape and size inhibits the calf's nursing ability thus negatively impacting intake and gain. frisch ( ) reported a correlation between cows with long teats and high calf mortality; however, that same study concluded that a calf would survive if the cow had one functional teat due to dams with poor teat conformation having greater milk yield. in addition, edwards et al. ( ) reported that milk production level did not influence calf bw at weaning and adg preweaning. thus, we hypothesized that cows classified with poor udders would produce calves with similar preweaning and postweaning growth. the objective of this study was to evaluate the effect of beef cow udder score within two calving seasons on preweaning and postweaning progeny performance. cow and calf performance data on cows were collected from through at the gudmundsen sandhills laboratory (whitman, ne). all animal procedures and facilities were approved by the university of nebraska animal care and use committee. cow and subsequent calf performance were obtained from the march (n = ) and may (n = ) calving herds at gudmundsen sandhills laboratory. each year at calving, udder scores were recorded from a to as reported in the intergrade resource management guide (national cattlemen's beef association, ). cows were grouped by udder scores and classified as either bad (udder score or , n = ) or good (udder score or , n = , ). an udder score of was not recorded during the course of the study. the udder score uses a combination of udder conformation and teat score system. calf data was stratified by calving season. calves were vaccinated at mo of age with an infectious bovine rhinotracheitis, parainfluenza- virus, bovine respiratory syncytial virus, and bovine viral diarrhea type i and ii vaccine (bovishield , zoetis, florham park, nj). calves were also weighed, branded, and male calves were castrated. calves were then moved with cows to native upland range pastures. at weaning, calves were weighed and vaccinated against bovine rotavirus-coronavirus clostridium perfringens types c and d and escherichia. after weaning, march-born steer calves were placed in a drylot and consumed ad libitum hay for wk postweaning after which they were transported to the west central research and extension center (wcrec). after weaning, may-born steers grazed subirrigated meadow with . kg supplement or received ad libitum hay with . kg supplement until approximately yr of age then relocated to wcrec. steers were then placed in a growsafe feeding system (growsafe systems ltd., airdrie, alberta, canada) approximately wks after arrival at wcrec. all steer bw was measured on two consecutive days before growsafe entry and again d after growsafe entry to account for the acclimation period to the feeding system. the average of the -d bw after the acclimation period was considered the initial feedlot entry bw and data concerning feedlot performance (bw change, dmi, and adg) was calculated from the average bw. all calves experienced a -d transition period allotted for a common finishing diet of % dry rolled corn, % corn gluten feed, % prairie hay, and % supplement. at feedlot entry, all calves were implanted with mg estradiol benzoate and mg trenbolone acetate (synovex choice, zoetis). approximately d before slaughter, calves were implanted with mg estradiol benzoate and mg trenbolone acetate (synovex plus, zoetis). march-born steer calves were managed similarly during finishing as the may-born calves; however, steer calves were fed as a group in drylot pens. each year, steer calves were sent to a commercial slaughter facility (tyson fresh meats, lexington, ne) when estimated visually to have . cm fat thickness over the th rib. carcass data were collected h post slaughter and final bw was calculated from hcw based on average dressing percentage of %. carcass data included hcw, marbling, yield grade, backfat, and lm area. data were analyzed using the proc mixed and glimmix procedure of sas (sas inst. inc., cary, nc). a mixed-model anova accounted for correlations within udder score and udder score within calving season. models included the effect of treatment, cow age, calving season, and calf sex for all appropriate data. data are presented as lsmeans and p values ≤ . were considered significant and tendencies were considered at a p > . and p ≤ . . calf bw, weaning, and adjusted -d bw is reported in table . influence of sex was not significant in any of the parameters (p ≥ . ), thus, heifer and steer data were pooled together in all preweaning variables. calf bw was similar between udder score groups (p = . ) along with calf weaning bw (p = . ), and adjusted -d bw (p = . ). in agreement, frisch ( ) reported no differences in calf bw at weaning in relation to dam's teat conformation which indicates that udder conformation does not limit calf growth up to weaning through decreased suckling ability or milk yield. however, these results contradict goonewardene et al. ( ) who reported dams with pendulous udders and bottle teats weaned lighter calves compared with well-attached udder and even quarter dams. feedlot performance of steer calves is reported in table . there were no differences between dam udder score group when evaluating postweaning calf feedlot entry bw (p = . ), final feedlot bw (p = . ), dmi (p = . ), adg (p = . ), and g:f (p = . ). cafe et al. ( ) reported cattle that entered the feedlot at similar bw performed with similar growth rates, despite being on a slow or rapid rate of gain from birth to weaning. moreover, hennessy and arthur ( ) evaluated the effect of high and low preweaning growth on calf efficiency in the feedlot, reporting no differences in adg between the two groups. carcass performance is reported in table . calves suckling good udder dams had greater hcw (p = . ) and backfat (p = . ) compared with bad udder counterparts. calves consuming a greater plane of nutrition a few months after birth have increased fat deposition, carcass merit, and heavier carcass weight than those managed on a lower plane of nutrition (stuedemann et al., ; hennessy and morris, ) . although, feedlot entry and final bw were not different between good and bad udder suckling calves, steer feedlot bw were numerically increased, which may have increased hcw. calves suckling dams classified as having bad udders at calving performed similarly during the preweaning period with good udder counterparts, with no differences in overall feedlot performance between udder groups. however, steer calves suckling good udder cows did have heavier carcass weights after the finishing period. further research is required to define the effects of udder score on generational effects of female progeny and how calving season influences the proportion of bad udder cows. a treatments are bad (udder score of or ) and good (udder score of or ). a treatments are bad (udder score of or ) and good (udder score of or ). growth and carcass characteristics of wagyu-sired steers at heavy market weights following slow or rapid growth to weaning the effect of bacterial infection on the milk yield of the individual quarters or the cow's udder genetic parameters and responses of linear type, yield traits, and somatic cell scores to divergent selection for predicted transmitting ability for type in holsteins high milk production decreases cow-calf productivity within a highly available feed resource environment the use of teat-size measurements or calf weaning weight as an aid to selection against teat defects in cattle milk production in range beef cows and its relationship to calf gains. can effect of udder type and calving assistance on weaning traits of beef and dairy × beef calves subclinical mastitis of beef cows the effect of preweaning growth restriction on the feed intake and efficiency of cattle on a grain-based diet before slaughter effect of a preweaning growth restriction on the subsequent growth and meat quality of yearling steers and heifers integrated resource management redbook influence of dam's milk production and other factors on and day weight of hereford calves effects of intramammary infection and parity on calf weaning weight and milk quality in beef cows effect of nutritional level imposed from birth to eight months of age on subsequent growth and development patterns of full-fed beef calves prevalence and effects of intramammary infection in beef cows key: cord- -k ldc m authors: zhang, xuan; ma, shi-lei; liu, zhong-di; he, juan title: correlation analysis of rubella incidence and meteorological variables based on chinese medicine theory of yunqi date: - - journal: chin j integr med doi: . /s - - - sha: doc_id: cord_uid: k ldc m objective: to analyze the correlations between the incidence of rubella and meteorological factors over the same period and previous periods including , , and year ago (defined according to chinese medicine yunqi theory of "pestilence occurring after years") and establish the rubella-meteorological forecast models for beijing area, china. methods: data regarding the incidence of rubella between and from beijing center for disease control and prevention, and the meteorological variables including daily average temperatures, daily average wind speeds, average precipitations, average relative humidity, average vapor pressures and average low cloud covers between and were collected from the beijing meteorological observatory. descriptive statistics and back-propagation artificial neural network for forecast model’s establishment were adopted for data analysis. results: the average temperature and relative humidity have a great contribution ( %) to the rubella morbidity. but the combination of other meteorological factors contributed to improve the accuracy of rubella-meteorological forecast models. the forecast accuracy could be improved by % through utilizing a combination of meteorological variables spanning from years ago to the present rather than utilizing data from a single year or dating back to more earlier time than years. conclusions: there is a close relationship between the incidence of rubella and meteorological variables in current year and previous years. this finding suggests that rubella prediction would benefit from consideration to previous climate changes. doctrine is guided by the overall concept that human and nature correspond and have provided new ideas on infectious diseases research. by the yunqi theory "pestilence occurring after years", the prevalence of infectious disease is not only related to weather factors within the current year but also associated with meteorological factors that occurred years ago. some scholars found that the prevalence of severe acute respiratory syndrome (sars) in in northern china was associated with the climate changes in , which was characteristic of high temperature, less precipitation and a large area of arid climate. ( ) the practical value of "pestilence occurring after years" received increasing more attention in recent years. ( , ) since the meteorological variables may be the early indicators of outbreaks and epidemics, our study focuses on the association of rubella and meteorological variables of current year and particularly previous years. based on the yunqi theory, a forecasting model was developed using the previous data on rubella and meteorological variables. the research involves no direct work with human subjects. the rubella case data were extracted from beijing center for disease control and prevention. these reports were governmental documents that summarized the counts of patients who were diagnosed at health care facilities with a variety of diseases. all individual-level data were anonymous. beijng, the capital city of china, °n and °e, was selected as the study area. many previous studies on yunqi theory have been conducted using climate data of beijing area. ( , ) according to the yunqi theory, the climatic characteristics are distinct during a year, and a cm concept called "six qi" mode describe this seasonal feature using the representative climate factor. ( ) in this study, the meteorological are collected from the beijing meteorological observatory from to , including daily average temperature, daily average wind speed, daily precipitation, daily average relative humidity, daily average vapor pressure and daily average low cloud cover. the rubella data was collected from beijing center for disease control and prevention from to . t h e s t a t i s t i c a l d a t a f o r r u b e l l a a n d t h e meteorological factors are calculated using the concept of 'six qi'. ( ) ( ) ( ) ( ) the incidence of rubella in beijing for each phase of the 'six qi' was calculated to establish a database from to . the data in could not be calculated due to a lack of data about diseases in . similarly, the average values of meteorological variables in beijing for each phase of the 'six qi' from to were also calculated. firstly, a descriptive statistic for meteorological factors and rubella morbidity were made to show the general regularities of weather and rubella prevalence during the past years. secondly, the correlation between meteorological factors and rubella morbidity were analyzed by back-propagation artificial neural networks, a non-linear dynamic systems that belong to the class of feed-forward neural networks. ( ) ( ) ( ) ( ) in this study, the back-propagation artificial neural networks methods were used to assess the importance of various meteorological factors of previous years and the current year on the outbreak of rubella. the meteorological factors with impact degrees (i.e. the standardized significance by model calculations) above % were selected as important variables related to the incidence of rubella. thirdly, the rubellameteorological forecast models were established and assessed. specifi cally, a total of rubella-meteorological forecast models for the prediction of the prevalence of rubella, as presented in table , were established by back-propagation artificial neural networks, and the forecasting accuracy was compared. the models were divided into two groups, including group (e.g. models - ) analyzed rubella morbidity and meteorological factors of a single year; and group (e.g. models - ) analyzed rubella morbidity and combined meteorological factors of previous years. all of the samples were divided into training samples and testing samples to prevent over fitting. an ideal network model could be achieved when the test samples are less than the training samples. consequently, the data for training and forecasting were randomly selected at the default ratio of : that was the most commonly used in the statistical analysis. the criteria of successful model assessment are according to the relative error values of training and testing, which should be less than , and the model will be considered to be successfully created. ( ) indicated in figure , the rubella morbidity showed an obvious seasonality with the highest prevalence in the nd qi, accounting for % of the average annual incidence of rubella. comparatively, rubella was in a state of low prevalence during the th, th, th and st qi, which accounted for . %, . %, . % and . % of the average annual incidence of rubella, respectively. a summary of the meteorological data counted by "six qi" phase from to is presented in table . the peaks of average temperature, average precipitation, average vapor pressure and average low cloud cover appeared in the rd qi (i.e. june and july). in comparison, the low points of these variables appeared in the th qi (i.e., december and january of the next year) and the average low cloud cover has a minimum in the st qi (i.e., february and march). additionally, the peak of average relative humidity appeared in the th qi (i.e., august and september) and low point appeared in the st qi. in contrast, the peak of average wind speed appeared in the nd qi (i.e., april and may) and low point occurred in the th qi. in beijing area, , cases of rubella were reported from january to january . as table , all prediction models (models - ) were successful established, while the prediction accuracy were lower than %. in addition, several meteorological factors from different year periods had significant influences on the incidence of rubella, including average temperature, average relative humidity and average precipitation from the current year, average relative humidity, average temperature and average low cloud cover from year ago; average temperature, average relative humidity, average low cloud cover and average wind speed from years ago; and average precipitation and average low cloud cover from years ago. of them, the largest contribution ( %) factors included average temperature from the current year and years ago, average relative humidity from year ago, and average precipitation from years ago (table ). as presented in table , models to (group ) were successful established and the prediction accuracy of these models were higher than those in models to (group ). the highest forecast accuracy appeared in model ( %), which used the meteorological factors from current year to years ago. however, when data from years ago were added to create model , the prediction accuracy was reduced. thus, there was no need to consider the meteorological factors of earlier periods than years ago. additionally, there were many meteorological factors from different year combinations, which presented infl uences above % on the incidence of rubella. among them, the largest contribution ( %) factors were average temperature and average relative humidity from the current year, and average wind speed from years ago (table ). based on the cm yunqi theory, this retrospective study analyzed the correlations between the rubella morbidity and meteorological factors in beijing area. by using back propagation artificial neural network, several rubella-meteorological forecasting models were established, which were characterized by the time spans ranging from the current year to years ago, and the vital meteorological factors on the incidence of rubella were selected. the study focused on the impact of meteorological variables over the same period and previous periods on the incidence of rubella. the doctrine 'pestilence occurring after years' was found in the huangdi's internal classic which is the source of cm theory. it contributed noteworthy observations about the influence of abrupt climatic changes or unusual weather conditions from to years ago on the present incidence of various infectious diseases. consequently, we found that the prediction accuracies of rubella-meteorological models in group one, which used the data of rubella morbidity and meteorological factors of single year, were undesirable. however, the prediction accuracies of rubellameteorological models in group , which used the data of rubella morbidity and combined meteorological factors of previous years, were obviously improved compared with group . with the addition of more meteorological data coming from previous years, the prediction accuracy showed a rising trend, reaching the top accuracy ( %) when the meteorological data within previous years were combined (current year + year ago + years ago + years ago). then a downward trend appear if more data of previous meteorological data (current year + year ago + years ago + years ago + years ago) were added. thus, there is no need to use the data of earlier periods than years, which prove the doctrine of yunqi theory "pestilence occurring after years". we also found that average temperature and average relative humidity, especially in the current year, were selected as vital meteorological factors because they made the largest contributions to the rubella-meteorological forecasting models, as indicated in the back propagation artificial neural network analysis. this suggests that the conditions of temperature and humidity in beijing area are significantly related to the outbreak of rubella. however, if considering other meteorological factors in - years ago, the prediction results of rubellameteorological forecasting models would be greatly improved. thus, the rubella prevention and control will benefi t from considering the local previous climate changes, which consistent with the findings of other studies in infectious diseases. ( ) ( ) ( ) the innovations of this study are summarized as follows. firstly, based on the cm theory of yunqi, this study divided one year into equal periods ( solar terms per period) that are called the 'six qi' or 'six phases' to describe the seasonal variations in the rubella incidence and meteorological variables. secondly, this study focused on the effects of previous changes of weather that occurred in the past years on the present incidence of rubella, which could improve the timeliness of forecasts based on established early warning models. thirdly, back propagation artificial neural network was introduced to select the most significant weather indicator and establish the rubella-meteorological predictive models. fourthly, this study selected a good model by analyzing and comparing each of the models. this study also has some limitations. although meteorological variables were used to identify possible causal factors for the incidence of rubella in beijing area, the outbreak of rubella is a complex course affected by multiple factors. some other important issues should be considered, such as biological, sociological, and economical factors. t a k e n t o g e t h e r , t h i s s t u d y p r o v i d e s a quantitative evidence that the incidence of rubella in beijing area was associated with meteorological variables, such as temperature and relative humidity, in the corresponding and previous years. moreover, the rubella-meteorological forecast model from the perspective of years ago to the present was successfully established and presented a relatively high accuracy rate, which suggested the use of a combination of meteorological factors spanning from years ago is superior to the use of data from a single. and there is no need to use the data of earlier periods. this information could be helpful in the early prediction of rubella outbreaks and build public health prevention and intervention strategies. therefore, some guiding ideology on yunqi theory regarding "pestilence occurring after years" in cm may be beneficial to provide new ideas for the research of using climate to predict outbreaks of infectious diseases, such as rubella. also, similar studies in other geographical areas and over longer time periods are needed to better understand the impact of weather variables on rubella. the authors declare no competing interests. zhang x analyzed the data and wrote the manuscript. ma sl revised the manuscript. liu zd collected and checked the data. he j conceived and designed the experiments. all authors have read and approved the contents of the fi nal version. rubella and congenital rubella syndrome: global update epidemiological analysis of rubella in beijing seasonal pattern of infectious disease: similarity and differences unusual climatic conditions and infectious diseases: observations made by hippocrates vernacular solution of 'plain questions of the huangdi's canon of medicine'. beijing: people's medical publishing house seasonality of infectious diseases discussion of severe acute respiratory syndromes (sars) from perspective of yunqi theory recognition of generations of doctors on reasons of epidemic disease based on the theory of 'transformation of plague in three years' to explore the correlation between incidence of dysentery and climate changes s mistake in calculation model for annotating yunqi (fi ve circuits and six qi) based on beijing meteorological data for years analysis of climatic characteristic of dominant yun in beijing for years analysis of climate changes in beijing based on the six qi theory of chinese medicine analysis of climate characteristics of solar terms in beijing correlation analysis for the attack of respiratory diseases and meteorological factors correlation between attack of bacillary dysentery and six qi or meteorological factors in beijing coincidence between yun-qi of the heavenly stems and earthly branches and actual climatic changes in beijing for years exploration of the use of mathematical methods in the syndrome analysis research beijing: people's correlation analysis for the attack of bacillary dysentery and meteorological factors based on the chinese medicine theory of yunqi and the medical-meteorological forecast model application of neural network in forecasting tuberculosis caused by meteorological factors case pristine in data analysis and mining of ibm spss relationship between meteorological factors and incidence of dysentery in beijing area based on theory of 'pestilence occurring after three years correlation between typhoid/paratyphoid and meteorological factors and establishment of forecast model based on theory of fi ve circuits and six qi correlation between meteorological factors and mumps incidence in beijing from to we would like to thank the beijing meteorological observatory for collecting the meteorological data and the beijing center for disease control and prevention for the data related to the incidence of rubella. key: cord- -c f lsx authors: taniguchi, akira; tanaka, yasuhito title: an alumina ceramic total talar prosthesis for avascular necrosis of the talus date: - - journal: foot ankle clin doi: . /j.fcl. . . sha: doc_id: cord_uid: c f lsx avascular necrosis tends to occur in the talus because of poor blood supply caused by the extended coverage to the articular cartilage on its surface. treatment is conservative in the earlier stage of this disease; however, surgical treatment is usually indicated in the advanced stage. nonunion, leg length discrepancy, or hindfoot instability may occur in patients treated with ankle or tibio-talo-calcaneal fusion. arthroplasty using a customized total talar prosthesis designed using the computed tomography image of contralateral talus has the potential advantages of weightbearing in the earlier postoperative phase, prevention of lower extremity discrepancy, and maintenance of joint function. techniques and implants. however, the frequency of talar osteonecrosis in patients with talar fracture dislocation is still higher. steroid use increases the blood level of fat, which induces microcontusions, leading to osteonecrosis. delanois and colleagues reported that of subjects with traumatic talar osteonecrosis had a history of steroid therapy. however, no evidence supports the correlation. blair fusion used to be adapted for this condition because it uses the healthy talar neck. , although minimal discrepancy of the lower leg remains after this procedure, hindfoot instability and non union in the fixation site sometimes occurred. in some cases, iliac crest harvest and external fixator may be necessary. the customized alumina ceramic talar prosthesis was developed in to compensate for the disadvantages of fusion treatments. , the first-generation implant was a talar body prosthesis with a bone peg for cement fixation to the talar neck. however, some cases had loosening at the fixation site. in , the second-generation implant was designed as a talar body prosthesis without a bone peg and was expected to perform the bearing function on the talar body. however, talar head rupture occurred in some cases because of higher pressure to the talar head. therefore, total talar prosthesis was developed in and has been used since. in this article, diagnosis and treatment of talar osteonecrosis, as well as surgical technique for artificial talus implantation, are discussed. an etiologic factor of osteonecrosis is that bone ischemia is induced by blood flow disturbance. the cycle of ossification, revascularization, and reabsorption is repeated for the healing system. in early-phase radiography, little difference is seen compared with healthy bone. next, reabsorption revolves around the necrotic bone, which induces bone atrophy followed by decreased bone stock. in the center of osteonecrosis, sclerotic change appears because of blood flow disruption. radiographic staging of osteonecrosis was developed by ficat and arlet and was modified for the ankle by mont and colleagues stage is defined as no radiographic findings, stage is defined as cystic lesion and/or sclerotic change in the talus, stage is defined as crescent sign or collapse in the subchondral bone, and stage is defined as joint space narrowing. in some patients with trauma to the talus, the clear zone can be recognized in the subchondral bone after to weeks of trauma, even in cases with initial sclerotic change, by the restoration of blood flow and that it is free from osteonecrosis (hawkins sign). patients with atraumatic talar osteonecrosis usually consult physicians because of relatively minor ankle pain. sclerotic change in the talar body would be observed even in cases without severe collapse in the talus (fig. ) . the sagittal and coronal planes of a computed tomography (ct) image reveal sclerotic change in the bony structure of the talus (fig. ) . moreover, a -dimensional image makes the evaluation of the an alumina ceramic total talar prosthesis collapse of the talar body and selection of the surgical option possible. in patients treated with artificial talus, ct of the contralateral ankle should be obtained to make a customized implant. mri is useful for the detection of early lesions. in the t -weighted image, the lesion is visualized in the low-density area, reflecting loss of fatty bone marrow. in the t -weighted image, the lesion is visualized in the high-density and low-density areas, reflecting bone marrow edema in the early and advanced stages, respectively. in the fat-suppressed t -weighted image, a mixture of high-intensity and low-intensity areas is seen, reflecting bone marrow edema and necrosis, respectively (fig. ) . bone scintigraphy has potential diagnostic power to detect the early phase. although other modalities evaluate only the local tissue, bone scintigraphy targets the whole body, which is an advantage for the diagnosis of atraumatic necrosis. the lesion has a cold area, reflecting the lack of uptake due to necrosis, and a hot area around the cold area reflecting the higher uptake results from reabsorption (cold in hot) (fig. ) . for patients with stage or without evidence of collapse, dislocation, or degenerative changes in the talus, nonweightbearing using crutches or a knee-bearing orthosis is indicated. partial weightbearing is allowed after bone resorption is recognized because this indicates the restoration of the blood supply, whereas nonweightbearing may continue long-term in cases without findings of bone resorption. surgical treatment is considered in patients not responding to conservative therapy. for patients with stage or , core decompression technique would be used. drills of . mm to . mm in diameter are inserted into the lesion to times from the posterolateral, lateral, or medial sides, or a drill of . mm in diameter is inserted to times to reduce the pressure in the lesion. horst and colleagues reported decreased pain and improved ankle motion in % of subjects after this procedure. for patients with stage and , surgical treatment is usually selected, particularly in patients with talar collapse. tibiocalcaneal fusion connecting the tibia and calcaneus the lesion has a cold area, reflecting the lack of uptake due to necrosis, and a hot area around the cold area, reflecting the higher uptake results from reabsorption (cold in hot). after resecting the collapsed talar body usually results in length discrepancy of the lower legs. blair fusion connecting the tibia and talar neck with the fibula results in less discrepancy; however, long-term mounting of the external fixator is required to address instability in the fixation site. , recently introduced artificial talar prostheses prevent leg length discrepancy, preserve the joint function, and allow early weightbearing. , ct of the healthy side of the talus is obtained to make a customized implant. ct images are reconstructed in the pitch of mm in the coronal and sagittal planes. the talar area is identified in each slice, the reconstructed image of the talus is reversed, and the implant is made using this image (fig. a-f) . the customized ceramic implant is produced in to weeks (fig. g) ; therefore, preventing further collapse of the talus caused by weightbearing is necessary. under spinal or general anesthesia, a -cm skin incision is made in the anterior ankle. the extensor retinaculum is incised, avoiding damage to the superficial peroneal nerve, and the ankle is exposed between the flexor hallucis longus and tibialis anterior tendons. in addition, the talonavicular joint is exposed in the distal side. after the dissection of the joint capsule and ligaments, the talar neck is cut and the talar head is removed. the talar body is cut -cm thick on the coronal plane, dissecting the interosseous talocalcaneal ligament. removal of the entire bone fragment without retaining small fragments in the surgical site is important. after irrigation of the surgical site, the artificial talus is inserted with assistance of foot traction. the ankle, subtalar, and talonavicular joints are ranged to confirm good prosthetic fit. the surgical wound is closed following joint capsule repair (fig. ) . a below-knee walking cast is applied for weeks. weightbearing is avoided in the first week, partial weightbearing is allowed in the second week, and full weightbearing is allowed in the third week (fig. ) . in the authors' experience of ankles in patients with talar osteonecrosis, the japanese society for surgery of the foot ankle-hindfoot scale score improved from . plus or minus . to . plus or minus . . in the subcategory of pain, score improved from . plus or minus . to . plus or minus . , function score improved from . plus or minus . to . plus or minus . , and alignment score improved from . plus or minus . to . plus or minus . . based on the ankle osteoarthritis scale, the score for pain at its worst improved from a mean of . plus or minus . to . plus or minus . . at final follow-up, ankle inversion stress radiography showed a talar tilting angle of . plus or minus . and an anterior drawer distance of . plus or minus . mm. dislocation and migration of the implant were not observed. fractures and fracture-dislocation of the talus the arterial anatomy of the talus talar neck fractures: results and outcomes open reduction and stable fixation of isolated, displaced talar neck and body fractures corticosteroid-induced avascular necrosis of the talus atraumatic aseptic necrosis of the talus aseptic necrosis of the talus and calcaneal insufficiency fractures in a patient with pancreatitis, subcutaneous fat necrosis, and arthritis does avascular necrosis cause collapse of the dome of the talus in severe haemophilia? avascular necrosis of the talus in children with haemophilia postoperative weightbearing radiography. (a) anteroposterior view. (b) lateral view avascular necrosis of bone in severe acute respiratory syndrome osteonecrosis in the foot atraumatic osteonecrosis of the talus comminuted fractures and fracture dislocation of the body of the astragalus the modified blair fusion alumina ceramic talar body prosthesis for idiopathic aseptic necrosis of the talus replacement of the body of the talus with alumina ceramic prosthesis for idiopathic aseptic necrosis ischemia and necrosis of bone avascular necrosis of the talus treated by core decompression avascular necrosis of the talus: a pictorial essay diagnosis of aseptic necrosis of the talus by bone scintigraphy avascular necrosis of the talus: current treatment options an alumina ceramic total talar prosthesis for osteonecrosis of the talus in summary, the talus is surrounded by the tibia, fibula, calcaneus, and navicular, composing some joints in between with these bones. customized alumina ceramic total talar prosthesis is an ideal implant for the treatment of severe talar osteonecrosis. key: cord- -ixho t g authors: guo, hua; cai, chunlin; wang, bo; zhuo, fei; jiang, rendi; wang, ning; li, bei; zhang, wei; zhu, yan; fan, yi; chen, wushen; chen, weihong; yang, xinglou; shi, zhengli title: novel hepacivirus in asian house shrew, china date: - - journal: sci china life sci doi: . /s - - - sha: doc_id: cord_uid: ixho t g nan hepatitis c virus (hcv) is a leading global cause of various liver diseases, including chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. the genome of hcv is monopartite, single-stranded, positive rna, about kb in size. hcv is the prototype species of the hepacivirus genus, which contains species according to the update from the international committee on taxonomy of viruses (smith et al., ) . prior to , humans were thought to be the only host of hcv; however, after that, genetically diverse hepaciviruses were detected or isolated from dogs, cows, horses, primates, bats, and rodents. asian house shrews (suncus murinus, also called asian musk shrews) are small insectivore mammals belonging to the family soricidae, order eulipotyphla. they are widely distributed in southeastern asia, africa, coastal arabia, islands in the indian ocean. many types of viruses have been found in asian house shrews, including coronaviruses, hantaviruses, severe fever with thrombocytopenia syndrome virus, hepatitis e virus, phleboviruses, adenoviruses, and arenaviruses, suggesting that these mammals play an important role as a reservoir for viruses. here, we report a highly diverse group of hepaciviruses discovered in the asian house shrews captured in shenzhen city, china. for virus screening, we captured asian house shrews at districts in shenzhen city, guangdong province, china from to (table s in supporting information). all shrews were humanely sacrificed, and their intestines, lungs, and livers were collected and preserved at - °c. all procedures were carried out with approval from the animal ethics committee of the wuhan institute of virology (approval number: wiva ). rna was extracted from liver tissues and analyzed for the presence of hepacivirus by reverse-transcription nested polymerase chain reaction (rt-pcr) with degenerate primers targeting the ns gene (drexler et al., ) . quantitative rt-pcr (qrt-pcr) and specific pcr were performed with primers designed based on the viral sequences obtained in this study (table s in supporting information). using degenerate primers, hepacivirus sequences were detected in four ( . %) liver samples (table s in supporting information). use gene-specific primers, more liver samples were found positive for hepacivirus (table s in supporting information) (genbank accession nos. mf -mf ). we named these newly discovered viruses as suncus murinus hepacivirus (smhcv). smhcvs were detected out at five sites in shenzhen city, while more than two thirds positive samples came from the bao'an and figure sequence analysis, pathogenesis, and viral rna detection of novel hepaciviruses in asian house shrews. a, phylogenetic tree of smhcvs based on nucleotide sequence from ns to ns regions. neighbor-joining tree was produced using mega software with the p-distance method (https://www. megasoftware.net). bootstrap value (%) was , replicates. the scale bar indicates nucleotide substitutions per site. the sequences marked with the black circle were obtained in this study. b, histopathology and smhcv rna detection in the liver samples of asian house shrew. cryostat sections of liver tissue were stained with hematoxylin-eosin (he) and oil red staining. inflammatory cell infiltration caused by smhcv in szcdc . severe steatosis and piecemeal necrosis caused by smhcv in szcdc . inflammatory cell infiltration caused by smhcv in szcdc . liver sections without virus infection in szcdc . c, dark blue indicates viral rna detected in liver tissue of szcdc by rna probe targeting viral genomic ns gene labelled with digoxigenin (dig). the cryostat sections were scaned on pannoramic midi and pictures were taken by pannoramic viewer . . . scale bars: μm. nanshan districts. more importantly, we found smhcv positive samples collected in three independent years at luohu district, though only small amount of samples were collected in year and . the detected smhcv sequences exhibited . %- % nt identity among themselves and . %- . % nt identity with known rodents hepaciviruses. analysis of the phylogenetic neighbor joining tree based on the bp sequences of the ns region revealed that these sequences formed an independent branch ( figure s a in supporting information). meanwhile, the sequences from the longhua and dapeng were located in separate branch while the sequences from the bao'an, luohu and nanshan crossed together in different branches ( figure s b in supporting information). these results demonstrated that diverse smhcvs were circulated in asian horse shrews in shenzhen city. to further delineate the genetic information of these smhcvs, nearly complete or partial genomic sequences ( , , and bp) were obtained from three samples smszcdc , smszcdc , and smszcdc which had higher viral genome rna copies than others ( figure s in supporting information). pairwise comparison showed that these three strains share %- % identities with each other. the nearly complete genome sequence of smhcv-szcdc shares %- % nt identity with known hepacivirus and its predicted polyprotein shares highest identify of % with rodent hepacivirus g. the phylogenetic tree based on obtained genome sequences (ns to ns b region) showed these hepacivirus strains detected in asian house shrews formed an independent branch ( figure a) . the amino acid p-distances of conserved regions of - and - (relevant to positions - and - of hepacivirus c a, m ) of smhcv-szcdc ranges . - . and . - . with known species in hepacivirus genus, respectively, which meet the demarcation (amino acid p-distances of greater than . in the region - and greater than . in the region - ) for a new species in hepacivirus genus (table s in supporting information) . the viral rna copies in different tissues were quantified by qpcr, with a positive control from the partial genome rna transcript by dna template using a maxiscript kit (applied biosystems, foster city, ca, usa) in vitro. the viral rna load ranged from × to × copies/g liver tissue ( figure s a in supporting information). to further determine the viral tissue tropism, viral rna from three types of tissues of animals was quantified by qpcr. the viral rna was detected in all three tissues of the selected positive samples. moreover, the viral load was higher in the liver than other two tissues and could reach × copies/g tissues ( figure s b in supporting information). interestingly, smhcv rna was also detected in the intestine, which may represent the possibility of fecal spreading. to further evaluate the pathogenesis of these hepaciviruses, liver sections of four shrews were checked by staining with hematoxylin-eosin (he) and oil red staining. these four liver samples were tested negative for hepatovirus, hepadnavirus, and hepevirus based on pcr (table s in supporting information). inflammatory cell infiltration was observed in samples szcdc and szcdc , severe steatosis and piecemeal necrosis were present in sample szcdc ( figure b) . a digoxigenin (dig)-labelled rna probe targeting viral genomic ns gene has succeeded to detect the viral rna in liver tissue of szcdc ( figure c) . in this study, highly diverse hepacivirus (smhcv) sequences were detected in asian house shrews. the viral rna could be detected in samples collected from to , suggesting that these smhcvs had a wide distribution in shenzhen city and had been circulated for a long time in asian house shrews. the obtained genomic sequence of smhcvs share low similarity with known hepaciviruses. analysis of amino acid p-distances of the conserved regions suggests these viruses should be classified as a new species in the hepacivirus genus (smith et al., ) . smhcvs could be detected in the liver, intestine, and lung tissues with high virus concentrations and showed wide tissue tropism. histology analysis demonstrated that smhcvs cause inflammatory cell infiltration, steatosis, and cirrhosis in the target tissues. due to the pathogenesis of smhcv in liver tissue, asian shrews could be a potential animal model for hepacivirus study as well as transgenic mice. recently, several studies have reported hepacivirus-or pegivirus-related sequences in small wild mammals (rodents and bats) and domesticated animals living in close contact with humans (dogs and horses) (drexler et al., , pybus and thézé, . in this study, we found the asian house shrew is another group of animal hosts of hepacivirus. shenzhen is a highly populated and rapid urbanization city with a high chance of close contact with wild animals. with the high diversity of smhcvs presented in this region, there should be a high chance of virus transmission from animals to humans. thus, long-term surveillance should be conducted in the future. as the fourth most commonly reported infectious disease in china, hcv infection maybe get more complicated because of novel hepaciviruses discovery (qin et al., ) . in addition, our investigation was just based on small sample numbers in shenzhen city. considering geographically wide distribution of asian shrews, we believe there should be more hepaciviruses to be discovered in the future. the author(s) declare that they have no conflict of interest. figure s neighbor-joining phylogenetic tree of hepaciviruses. the genome organization of smhcvs obtained in this study. smhcv rna quantification by qpcr in the liver tissues of the asian house shrews. detection of hepacivirus in asian house shrews captured in shenzhen city primers used in this study for viral rna detection and quantification table s the p-distance vaules of pp - and pp - of smhcv-szcdc comparasion with other hepaciviruses the supporting information is available online at http://life.scichina.com and https://link.springer.com. the supporting materials are published as submitted, without typesetting or editing. the responsibility for scientific accuracy and content remains entirely with the authors. evidence for novel hepaciviruses in rodents natural reservoirs for homologs of hepatitis c virus hepacivirus cross-species transmission and the origins of the hepatitis c virus hepatitis c virus infection in china: an emerging public health issue proposed update to the taxonomy of the genera hepacivirus and pegivirus within the flaviviridae family key: cord- -ptkw csu authors: gilbert, gwendolyn l.; kerridge, ian title: the politics and ethics of hospital infection prevention and control: a qualitative case study of senior clinicians’ perceptions of professional and cultural factors that influence doctors’ attitudes and practices in a large australian hospital date: - - journal: bmc health serv res doi: . /s - - -y sha: doc_id: cord_uid: ptkw csu background: hospital infection prevention and control (ipc) programs are designed to minimise rates of preventable healthcare-associated infection (hai) and acquisition of multidrug resistant organisms, which are among the commonest adverse effects of hospitalisation. failures of hospital ipc in recent years have led to nosocomial and community outbreaks of emerging infections, causing preventable deaths and social disruption. therefore, effective ipc programs are essential, but can be difficult to sustain in busy clinical environments. healthcare workers’ adherence to routine ipc practices is often suboptimal, but there is evidence that doctors, as a group, are consistently less compliant than nurses. this is significant because doctors’ behaviours disproportionately influence those of other staff and their peripatetic practice provides more opportunities for pathogen transmission. a better understanding of what drives doctors’ ipc practices will contribute to development of new strategies to improve ipc, overall. methods: this qualitative case study involved in-depth interviews with senior clinicians and clinician-managers/directors ( doctors and nurses) from a broad range of specialties, in a large australian tertiary hospital, to explore their perceptions of professional and cultural factors that influence doctors’ ipc practices, using thematic analysis of data. results: professional/clinical autonomy; leadership and role modelling; uncertainty about the importance of hais and doctors’ responsibilities for preventing them; and lack of clarity about senior consultants’ obligations emerged as major themes. participants described marked variation in practices between individual doctors, influenced by, inter alia, doctors’ own assessment of patients’ infection risk and their beliefs about the efficacy of ipc policies. participants believed that most doctors recognise the significance of hais and choose to [mostly] observe organisational ipc policies, but a minority show apparent contempt for accepted rules, disrespect for colleagues who adhere to, or are expected to enforce, them and indifference to patients whose care is compromised. conclusions: failure of healthcare and professional organisations to address doctors’ poor ipc practices and unprofessional behaviour, more generally, threatens patient safety and staff morale and undermines efforts to minimise the risks of dangerous nosocomial infection. in high income countries, an estimated - % of hospital inpatients develop healthcare-associated infections (hais) [ ] and nosocomial transmission of multidrug resistant organisms (mrdo) is a major contributor to antimicrobial resistance (amr) and its associated healthcare costs [ , ] . it is estimated that - %, or more, of hais are preventable [ , ] , although rates are highly variable, depending on how effectively ipc measures are implemented [ , ] . failures of routine hospital infection prevention and control (ipc) practices, in high income countries during this century, have resulted in devastating nosocomial outbreaks of exotic or emerging infections, such as severe acute respiratory syndrome (sars) in toronto, in [ ] and middle east respiratory syndrome (mers) in seoul in [ ] , causing preventable deaths and massive social and economic disruption. hand hygiene is the most obvious, easily audited and, arguably, the most effective ipc practice [ , ] , its efficacy has been recognised since at least the mid-nineteenth century, with numerous studies showing that significant reductions in pathogen transmission and hai rates are temporally associated with improved hand hygiene compliance [ ] . nevertheless, the use of hand hygiene as a surrogate for ipc quality and the moral status of noncompliance have been questioned, largely as a consequence of ongoing controversy about auditing methods and plausible compliance targets [ ] [ ] [ ] . ethical considerations have particular salience in light of numerous studies reporting lower than average compliance with ipc policies among doctors, compared with other health professionals, albeit with wide variation [ ] [ ] [ ] . doctors' attitudes and behaviours are important, because they disproportionately influence those of other hospital staff and doctors often overestimate their own knowledge and compliance [ , ] . yet their peripatetic clinical practice provides numerous opportunities to transmit pathogens [ ] and to be pathogen "super-spreaders" [ , ] . doctors retain considerable professional autonomy and power, despite repeated challenges from increased regulation, other health professions, evidence-based medicine and consumerism [ ] [ ] [ ] . despite recent attempts to redefine "medical professionalism", a universally agreed definition remains elusive; but all versions include common commitments to e.g. patient welfare; maintenance of knowledge and skills; and securing public trust through professional self-regulation and avoidance of conflicts of interest [ ] [ ] [ ] . how doctors interpret these commitments depends on how they perceive their professional identity [ ] . in practice, their attitudes and practices are complex and sometimes perplexing. assuming that patient welfare is doctors' highest priority [ ] , one may reasonably ask why some would expose their patients to preventable infection risks by failing to observe well-established ipc rules [ , ] ? previous qualitative and mixed methods studies of factors that affect adherence to ipc practices have generally involved mixed groups of healthcare workers and/or focused on particular institutional settings, such as intensive care units. while these studies have identified factors that contribute to ipc practices including: self-protection, role modelling, belief (or not) in the effectiveness of ipc, knowledge, communication and workload [ , , , ] they have not, for the most part, explained why these factors are so influential. the aim of this study was to explore what factors affect doctors' ipc practices and, more specifically, why they are so influential. it took the form of in-depth conversations between researchers and participants, all of whom were senior clinical leaders and or clinician-director/managers with many years' experience. our expectation was that the perspectives of both "insiders" [senior doctors] and more objective "outsiders" [senior nurses] would provide new insights to inform strategies to raise the priority of ipc within the medical community and limit harm from hais and amr more effectively. our research question was: what professional and cultural factors influence doctors' attitudes to and practice of infection prevention and control? both researchers are senior physicians, one female, one male. our special interest in hospital ipc stems from longstanding experience in caring for patients who are at high risk, have suffered and, in some cases, died, from preventable hais and/or have experienced the uncertainty and fear associated with being colonised by a mdro. both of us were employed, for many years before or during this study, at the hospital where it was conducted. most of the participants were known to at least one of us, as colleagues, but with few exceptions, we had not worked closely with them. our aim was to build a rich portrait of doctors' attitudes to and practice of ipc, in one large australian tertiary hospital, by means of in-depth interviews with experienced senior doctors and nurses. together with them, we hoped to formulate new theories to better explain the reasons behind doctors' ipc practices, in order to develop more effective and generalisable strategies to improve them. we used a thematic analysis approach to data analysis [ ] . participants were senior doctors (i.e. medical practitioners) and nurses with varied clinical and/or management responsibilities, who were purposively selected as being likely to have a broad perspective on the attitudes, beliefs and practices of the hospital's medical staff across age-groups and specialties. invitations were sent by email, explaining the purpose of the study. thirty-two potential participants - doctors and nurses -were contacted sequentially. all of the nurses and doctors agreed to participate; two doctors were unavailable, one refused and three did not respond. sixteen participants were facility/divisional directors or unit managers, were specialist consultants; they had had - years' professional experience and most had been employed in the hospital for more than years. doctors were either full-time staff specialists [ss] or visiting medical officers (vmos) contracted on a sessional basis. nurses were all full-time employees. participant characteristics are summarised in table . the setting was a~ -bed university teaching hospital, serving a population of about . million in sydney and providing a broad range of tertiary specialist services to more than , inpatients, annually. the study was conducted at a single site to enable a rich and detailed understanding of the influences on doctors ipc practices and to avoid potential confounding effects of different policies, patient populations or physical environments. semi-structured interviews, lasting - min, were conducted by one researcher. they took the form of frank conversations between colleagues. most were conducted in the participant's -or occasionally the interviewer's -office with no-one else present. interviews were recorded and digitally transcribed by a professional transcription service, with participants' informed consent. examples of questions used to prompt discussion are shown in table . interview transcripts were checked for accuracy by one researcher. both authors reviewed the transcripts, coded them manually and analysed them thematically [ ] , to identify themes and subthemes relevant to the research question. after initial analysis, transcripts and emergent themes/subthemes were reviewed, iteratively, and modified after further discussion. recruitment ceased when data saturation had been reached. participants gave written informed consent to interviews being recorded and transcribed. they were assured that their comments would be confidential and quoted, if at all, only after removal of identifying information. in describing our findings, we identified participants' professions and roles, broadly, to preserve anonymity, as nursing director (nd), manager (nm) or consultant (nc); or medical director (md) or consultant (mc). the relevant local health district human research ethics committee approved the study. four major themes emerged from the data and, within theme , four subthemes were identified. these themes and subthemes are outlined in table . theme is the subject of this paper. other themes/subthemes will be discussed, in detail, elsewhere. you can't tell doctors what to do (medical director [md] ). doctors' highly valued clinical autonomy was seen as one of the most important determinants of ipc practices. participants described doctors, generally, as self-motivated and averse to being told what to do, particularly as they become more senior. this was attributed to: the types of people who become doctors; doctors' perception that others expect them to be confident and decisive; and their tendency to rely on clinical judgment and experience, rather than "rules". however, participants described differences in how doctors enact clinical autonomy in their ipc practices. those of some individual doctors and unitsrepresenting such varied specialties as transplantation surgery, haematology, general surgery and neurosurgerywere described, by participants, as exemplary. a unit director (md ) recounted his own unit's practice, of having a junior doctor audit the team's hand hygiene compliance and present the results at weekly meetings, and his pride in the resulting improvement. at the other extreme, several participantsindependently -cited examples of units in which ipc standards were notoriously poor and hai rates (reputedly) excessive; they also described individual consultants who had to be reminded, repeatedly, about basic ipc precautions and who vehemently demurred, refused or tried to avoid situations where they may be asked, to comply: from a political point of view, we (intensive care unit [icu] clinicians) are in a bind. we encourage outside (surgical) teams to see their high-risk patients daily, (but) they're saying "i don't want to go to icu, they always give me a hard time about washing my hands or taking my jacket off". so, we're trying to make sure that they have easy access to the patients, but in such a way that the whole process is smooth. for some clinicians, hand hygiene presents an objectionable obstacle. (emphasis added) (md ) several (doctor) participants thought their colleagues' objections, to ipc "rules" that they perceived to be inflexible, inappropriately applied or imposed by outsiders, were legitimate. for example, patients being placed in contact isolation, when the risk of cross-infection was minimal, caused increased pressure on already over-burdened junior doctors and potentially endangered other patients, because of misplaced priorities. the infection control nursing staff don't understand the pressures that medical staff, particularly junior medical staff, are under. some of the attitudes to infection control appear to be given in isolation without really an understanding of how they might be integrated into all the various things that have to be done by residents and senior doctors and nurses. a bit of an irritant is if i'm asked to take precautions that i don't think are appropriate (when) nursing staff have instituted a policy (and) gone a bit too far. (for example) a patient who has minor neutropenia and a (mild) fever that you're keeping in just in case.... they're given priority, put in a single room and everybody's in gloves and gowns; and they've got other people out in the ward coughing and hacking, perhaps harbouring something much more significant. (md ) several medical directors attributed doctors' poor ipc practices to ignorance, although on-line ipc training and attendance at education sessions was supposedly mandatory. there's a poor understanding of what's required, even though you're meant to do mandatory training. but apparent adherence to ipc principles sometimes reflected tradition or habituated behaviour, rather than knowledge of organisational policies. a surgeon contrasted his colleagues' almost ritualistic "surgical scrubbing" and donning of theatre attire in the operating theatre, with cursory adherence to standard ipc precautions in the wards: participants emphasised the importance of leadership in moulding the ipc practices of junior doctors, who are likely to emulate those of senior consultants. they characterised good ipc leaders as consistently adhering to good practices, themselves, and being willing to discuss ipc principles and review practices and outcomes with their teams (see md 's anecdote about registrar hand hygiene audits, above, and unit x director's response to his patients' hais below). of course, clinical leadership is about much more than ipc practices: i often talk about (a particular surgical unit) because they are a high performing team and their leadership is very strong across the board in all sorts of areas; they're very solid as a team and the interns know the rules and abide by them even when (the consultants) are not there. (nd ) on the other hand, bad or absent leadership is often also associated with poor ipc practice. if the "boss" ignores or disparages ipc practices, junior doctors would follow suit. it's about leadership and followership. senior staff 's lack of role modelling -automatically the followers see that "it's not a big deal for sir, so it's not a big deal for me". (md ) one participant wondered whether leadership and role modelling might be less critical, now that junior doctors were increasingly aware of the importance of ipc and could influence consultants' ipc behaviours, by reminding them of the policies. the more prevalent view was that the (still) powerful medical hierarchy was a strong deterrent against any attempt to correct the "boss", even implicitly, for fear of retribution. where a junior person speaks to a more senior person (about) a protocol that hasn't been performed correctly, the senior person feels threatened. the normal thing is to stamp your authority and say "well hang on, who are you?". (mc ) most participants felt that any good habits learnt in medical school were likely to be superseded by hidden curricula assimilated during postgraduate training. doctor participants admitted that they would not personally feel comfortable speaking to senior colleagues about poor ipc practices; on the other hand, some nurse participants said they would willingly speak up to a consultant or team with whom they had a good working relationship. participants acknowledged the devastating effects, on patients, of serious hais, but pointed out that most doctors personally manage such complications infrequently and hear about only the worst cases at morbidity and mortality (m&m) meetings. many doctors have little appreciation of the incidence, or impact on patients, of more minor infections. moreover, hais are often regarded as unavoidable; they are rarely attributable to specific actions, omissions or even patterns of behaviour, because of the inevitable delay and multiple factors and people involved. nevertheless, participants described doctors' contrasting reactions to their own patients' hais: one saw it as a disaster, for which he felt personally responsible, another as a driver for change. a nurse manager described starkly contrasting attitudes between two unit directors: (unit x's director says) "what did we do wrong: what are we going to do to make sure this doesn't happen (again)?" he doesn't want to see bad things happen to patients. (unit y's director at the departmental m&m meeting) … . it's: "what's the patient done wrong this time? it's the patient's fault. it's not our fault. the patient's dirty (or) too fat; they should have looked after themselves better". there's no accountability, no insight. (nm ) several participants noted that hais are often overlooked because of inadequate patient follow-up; they were highly critical of some senior consultants' infrequent ward rounds, expectation that trainees would manage and follow-up patients, often with minimal supervision, and apparent lack of interest in patient outcomes. somebody who swans in, looks at something, swans out again and never understands that what he actually did was screwed up the patient's life -that's the problem. interviewer: what can you do about it? participant: throw their noses in the data. (md ) unfortunately, for most departments, very limited data are available. collection of unit-specific hai data was seen as the organisation's, not individual unit's, responsibility. while participants noted that monthly hand hygiene audit data were posted on ward notice boards, they believed that medical teams rarely reviewed them or, if they did, were skeptical about their accuracy and unaware of how they compared with those of other wards. one medical director (md ) acknowledged that ward compliance data showed large discrepancies between wards and the lowest compliance rates amongst doctors, which were masked by the hospitals' satisfactory results, overall. another director (md ) suggested that audit results overestimate actual compliance because auditors were not strict enough, but he rationalised doctors' relatively low rates as being due to the fact that the auditors were nurses. in answer to an open question about general issues of concern, all medical divisional directors mentioned their frustration at some senior consultants' apparent lack of commitment to their public hospital responsibilities, because of the demands or attractions of private practice. [they] ... come in, do an operation, bugger off. they probably won't ever see that [public] patient again; they don't know what the outcome is. they don't have to front these patients in their office. [md ] participants attributed this to better remuneration, working conditions, infrastructure and capital equipment in the private sector. while noting that vmos value the prestige and challenging case-mix of a public hospital appointment, one director (md ) pointed out that their contracts do not stipulate specific obligations (that many would take for granted), such as teaching and supervision of junior staff or accountability for patient outcomes. he suggested that, rather than relying on vmos' "altruism", the public system should provide more attractive conditions. if you want the talent you've got pay for it -an adequate salary, medical indemnity, secretary, reasonable office, parking space. it's a very big package, but that's what you need to do. if you offered them that they would be prepared to take a or per cent cut in income to concentrate their activities in one place and give up all the angst of being an employer. (md ) while this solution seemed unlikely in the current public health system, a senior manager mentioned the hospital executive's plan to revise vmo contracts to define the "rules of engagement", to which applicants must agree, including regular attendance at ward rounds and teaching sessions, trainee supervision and formal delegation of decision-making at weekends. in common with many australian public hospitals, the case hospital's recent devolution of accountability for key performance indicators (kpis), to divisional directors, had made them more aware and less tolerant of consultants, whom they perceived to be failing their implicit obligations, with impunity. in the context of this study, they linked this behaviour to ignorance of ipc policies and poor practice. however, they all agreed that instigating disciplinary action against senior doctors, in these circumstances, would be professionally and legally difficult, divisive and bitterly opposed, on principle, by medical organisations and most doctors, including those whose own behavior was above reproach. none of the divisional directors could offer solutions, other than more explicit employment contracts and/or more pay, but they conceded that it would still be difficult to monitor or enforce compliance. despite their frustration, some directors described recent success, in delegating responsibility for clinical and administrative improvements, including in ipc, to unit directors, and assimilation of autonomous senior consultants into unit teams. while doctors' relatively poor ipc practices, overall, have been well-documented, this qualitative study is one of few which have deeply interrogated why this is so. participants regarded senior doctors' perceived entitlement to professional independence as a major contributor to how they choose to practice ipc and just one expression of how they fulfil implicit obligations to provide evidence-based patient care and clinical leadership. there was consensus that, although doctors are aware of the importance of ipc, for many it is not their highest priority. nevertheless, most observe ipc policiessometimes with modification -and/or do not object to being reminded. uncertainty about the efficacy of some ipc measures and a lack of data about hais are barriers to doctors' becoming more involved in ipc policy development and implementation. however, for most participants, the greatest concern was some senior consultants' hostile disregard for ipc policies and its adverse effects on patients and other staff and their apparent immunity from censure. participants regarded doctors as generally resistant to external rules, which they see as a challenge to their clinical autonomy and self-perception as independent thinkers [ ] . in relation to ipc they may exercise autonomy, either by taking it very seriously (exemplary practice) or by choosing to remain ignorant or dismissive of basic ipc precautions, which participants interpreted as evidence of a perceived entitlement to decide, for themselves, what is important. it has been shown, previously, that compliance with hand hygiene is inversely related to educational level and seniority [ ] . in making sense of this observation, we noted that many senior consultants completed their training and developed habituated behaviours at a time there was little focus on ipc, because of a prevalent belief that infectious diseases had largely disappeared [ ] . while this belief is now known to have been misguided, the fact that it persists could be interpreted as a failure of continuing professional education (cpe). although cpe is a condition of annual medical registration, in australia, it is largely confined to specialty topics. specialist colleges endorse, and expect members to comply with, ipc guidelines and online ipc training is mandatory for all hospital staff, but many senior doctors choose to ignore both, with impunity. college expectations and hospital requirements are futile if professional autonomy is interpreted as meaning that compliance is discretionary [ ] . previous studies have also suggested that doctors' poor compliance with guidelines, in general, reflects ignorance or skepticism about their effectiveness and/or an exaggerated confidence in their own judgment [ , ] . as charani et al suggest: senior doctors consider themselves exempt from following policy and practice, within a culture of perceived autonomous decision-making that relies more on personal knowledge and experience than formal policy [ ] . senior consultants for whom ipc is a high priority may, nevertheless, seek to selectively 'modify' hospital ipc policies. this observation is supported by a recent study [ ] that found that violations of transmission-based precautions were often not due to ignorance, but a clinicians' judgment that the risks did not justify the extra staff time and cost or adverse effects on patients [ ] . participants gave examples of such modifications, which they regarded as sometimes appropriate. however, they are likely to be interpreted as arbitrary and a source of confusion and conflict; they might be avoided if doctors were more willing to be involved in the development and local implementation of ipc policies, as leaders or champions [ , ] . doctors' apparent independence may be partly illusionary, if their clinical practice is guided by habituated behaviours, learnt during their early postgraduate training, by emulating teachers and supervisors, whom they admire or fear [ ] . participants identified leadership, as others have done [ ] , as an important determinant of (good or bad) ipc practice. within a hierarchical hospital system, leadership is usually based on seniority, status and power [ ] . most senior consultants exercise their leadership roles appropriately and model acceptable or exemplary ipc practices but the attitudes of the minority, who repeatedly ignore or dispute basic ipc precautions, will also be transferred to junior doctors. medical students are now taught about the importance of ipc and the risks of hai and amr but, as postgraduate trainees, they absorb the hidden curricula [ ] of the specialties and units in which they train, which may be at odds with what they have been taught. once entrenched, senior doctors' habituated behaviours are difficult to change, since even their peers are deterred, by professional etiquette and their own uncertainties, from challenging colleagues' unsafe practices [ ] . even doctors who are knowledgeable about and aware of the importance of ipc may struggle to understand how hais occur, because the effects of practice breaches are delayed, hidden and uncertain. some regard hais as unavoidable or someone else's fault -"the problem of many hands" [ ] -and many have little concept of patients' fears or the significant personal cost of even minor hais [ ] . these problems are compounded by lack of data. results of mandatory reporting to health authorities or internal incident reports of serious, but rare hai-related events, such as bloodstream infections or unplanned readmissions, have little resonance and there are virtually no data about less serious hais. so it is impossible to monitor trends or reflect on individual consultant's or unit's performances vis-à-vis their peers, despite evidence that surveillance and feedback of results can motivate ipc improvement and reduce hai rates [ , ] . the lack of consistent national hai surveillance, in australia, is a recognised barrier to sustained ipc behavior change, particularly among doctors, and state-or hospitalbased surveillance is highly variable [ ] . the perceived failure of a small proportion of consultants to meet their obligations to the public hospital system was the issue that our participants spoke about most vehemently. our participants felt strongly that senior doctors' infrequent presence in the hospital and poor trainee supervision, (public) patient follow-up and ipc practices were not valid expressions of professional autonomy and agency, but manifestations of disrespect both for patients, who are exposed to unnecessary risk and colleagues who conform with, or are responsible for maintaining, ipc standards. moreover, the hostile reactions of some doctors, to being reminded about basic ipc precautions, were interpreted as a manifestation of the bullying, which is endemic in healthcare settings in many countries, including australia [ , ] , and as 'yet another' failure of professional self-regulation [ ] . in this regard, recent media scrutiny of withdrawal of postgraduate training accreditation from two major sydney public hospitals, largely as a consequence of bullying and harassment by senior medical staff [ , ] , raises the question of whether hospital administrators and specialist colleges will act to address cultural problems in medicine, including bullying, more effectively. where to from here? the vanderbilt university medical centre's system of co-worker observation and reporting is one promising approach to doctors' (and others') unprofessional behaviours. it involves, as a first step, a suitably trained colleague initiating an informal, respectful conversation with a doctor whose behaviour has been the subject of complaint from a patient or co-worker. in most cases this is apparently sufficient but, if a pattern of behaviour emerges, it is followed by staged escalation [ ] . it has proven to be feasible and effective in reducing bullying [ ] , patient complaints [ ] and poor ipc practices [ ] and limits the need for more direct disciplinary action. it has been introduced or considered by several australian hospitals [ ] and the royal australasian college of surgeons [ ] . such an holistic approach to organisationwide culture change, would complement better surveillance and feed-back of hai-related data and innovative strategies, such as the use of video-reflexive methods that have been used successfully to raise clinician awareness and improve ipc practices [ ] . a major strength of this study was the seniority, varied clinical and management experience and broad vision of our participants, who were among the hospital's most senior clinicians and clinician-managers/directors. many of them were responsible for service quality and efficiency across multiple departments and acutely aware of the issues discussed. in addition, we believe that, because the interviewer was an "insider", with comparable seniority and professional background, participants shared their insights and frustrations more candidly than they may have done with an external researcher. as researchers, we were inevitably influenced by our own perspectives and biases, which we sought to mitigate by discussion and self-reflection and by consulting as broad as possible a range of participants. as typical of qualitative research, they were but a small sample of the larger cohort of senior hospital clinicians, but represented a wide range of specialties, seniority, management responsibilities and attitudes to ipc. the fact that participants and researchers were staff of a single hospital is appropriate for a case study but could also be seen as a limitation. however, our observations, experience and published literature suggest that the issues identified are common to most large, tertiary hospitals in australia and countries with similar mixed public/private health systems. moreover, limiting the study to a single site meant that all participants were familiar with the organisation's idiosyncrasies and illustrative examples of doctors' attitudes or practices were corroborated, and given added cogency, by multiple participants. while participants expressed their opinions, frankly, about the attitudes and behaviours of colleagues, they generally did not identify them except by specialty. we did not seek to corroborate opinions about ipc practices with objective evidence of compliance, which was not the focus of this study, and none of the few units mentioned by participants, as having poor ipc practices, was represented amongst participants. this was unintended but may be seen as a limitation. clearly many factors contribute to doctors' ipc practices, other than (and sometimes contrary to) the norms, values and precepts of the medical profession. one of the most salient is how they interpret professional autonomy, which is a strongly defended principle of medical professionalism, but surely not intended to imply that doctors are entitled to ignore the policies of their employer organisations. although participants sometimes defended doctors' objections to, or ignorance of ipc "rules", they clearly regarded doctors who reacted to reminders or requests to comply aggressively or unprofessionally, as an "out-group". in common with other preventive programs, ipc is sometimes a victim of its own (partial) success; there is a widespread but unsubstantiated impression that ipc practices are "good enough" and further improvements not cost-effective. however, the continuing prevalence of preventable hais and nosocomial transmission of mdros and the threat of emerging infections mean that sustained improvement is needed. we argue that this will not be achieved without the full support and participation of all healthcare professions, includingperhaps especiallydoctors. however, our findings suggest that improving doctors' ipc practices will require greater commitment from professional organisations and healthcare administrators to a) more appropriately measure, and inform clinicians about, the adverse effects for patients of non-adherence and b) effectively enforce appropriate policies and practices that prevent these harms. the poor ipc practices of some doctors are just one aspect of the broader issue of unprofessional behaviour and immunity from criticism that must be addressed, if the medical profession, in general, is to be seen to conform with its espoused professional values and those of the 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sustained improvement in hand hygiene adherence: utilizing shared accountability and financial incentives royal melbourne hospital targets bullying with new cognitive institute program: the age building respect and improving patient safety an innovative approach to strengthening health professionals' infection control and limiting hospital-acquired infection: video-reflexive ethnography the authors wish to sincerely thank the senior clinicians who gave so generously of their time to participate in this study and emeritus professor miles little, dr. suyin hor and dr. julie mooney-somers for helpful comments on early versions of this paper. this research received no funding. the datasets generated and/or analysed during the current study are not publicly available because participants did not give consent for interview transcripts to be published but (selected portions) are available from the corresponding author on reasonable request. authors' contributions glg developed the project concept, conducted interviews, reviewed, coded and analysed interview transcripts and wrote the first and subsequent drafts of the manuscript. ik contributed to conception and development of study design, reviewed and coded interview transcripts and contributed to editing of manuscripts drafts. both authors have approved the final version of the article.ethics approval and consent to participate this study was approved by the western sydney local health district human ethics research committee. all participants provided written informed consent to interviews being recorded and transcribed and quotes to be used in publications after removal of identifying information. the information sheet/consent form was approved by the ethics committee. participants were not asked and did not consent for transcripts to be published or for any personal identifying information to be made available other than their professional status (medical/nursing; director/manager/ consultant). quotes were selected to ensure that the source would not be identifiable. the authors declare that they have no competing interests. key: cord- -xty m w authors: marrugal-lorenzo, josé a.; serna-gallego, ana; berastegui-cabrera, judith; pachón, jerónimo; sánchez-céspedes, javier title: repositioning salicylanilide anthelmintic drugs to treat adenovirus infections date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: xty m w the repositioning of drugs already approved by regulatory agencies for other indications is an emerging alternative for the development of new antimicrobial therapies. the repositioning process involves lower risks and costs than the de novo development of novel antimicrobial drugs. currently, infections by adenovirus show a steady increment with a high clinical impact in immunosuppressed and immunocompetent patients. the lack of a safe and efficacious drug to treat these infections supports the search for new antiviral drugs. here we evaluated the anti-adenovirus activity of niclosanide, oxyclozanide, and rafoxanide, three salicylanilide anthelmintic drugs. also, we carried out the cytotoxicity evaluation and partial characterization of the mechanism of action of these drugs. the salicylanilide anthelmintic drugs showed significant anti-adenovirus activity at low micromolar concentrations with little cytotoxicity. moreover, our mechanistic assays suggest differences in the way the drugs exert anti-adenovirus activity. niclosamide and rafoxanide target transport of the hadv particle from the endosome to the nuclear envelope, whilst oxyclozanide specifically targets adenovirus immediately early gene e a transcription. data suggests that the studied salicylanilide anthelmintic drugs could be suitable for further clinical evaluation for the development of new antiviral drugs to treat infections by adenovirus in immunosuppressed patients and in immunocompetent individuals with community-acquired pneumonia. anti-adenovirus activity of salicylanilide anthelmintic drugs. in initial studies, we determined the inhibitory concentrations of the three anthelmintic drugs against hadv in a plaque assay measured as the percentage of plaque formation inhibition compared to a control cells infected in the absence of drugs. the three salicylanilide anthelmintic drugs showed a dose-dependent anti-hadv activity against both hadv and hadv , with % inhibition of plaques formation at . , and . μm for nic, oxy and raf, respectively ( fig. a,b) . the ic values for the three anthelmintic drugs are summarized in table . we also evaluated whether these salicylanilide anthelmintic drugs were able to inhibit virus production using hadv , hadv and hadv yield reduction assays. treatment with nic, oxy and raf was associated with overall reductions in virus yield from to -fold (table ) . in an assay measuring antiviral activity of the three anthelmintic drugs as a function of moi, inhibition was inversely proportional to the number of input particles (fig. ). while they were also inhibitory at high moi, the relationship between the number of infecting hadv particles and the drugs concentration is less marked, as expected. the cellular cytotoxicity of the salicylanilide drugs was also analyzed. the cc values for these molecules were in all cases significantly higher than the ic concentrations required for inhibition in our antiviral activity and mechanistic assays for both β cells (table ) and a cells ( . ± . µm, . ± . µm and . ± . µm for nic, oxy and raf, respectively). step in hadv replicative cycle. as the first step toward identifying the specific step in the hadv replicative cycle that was inhibited by these drugs, we measured the time dependence of the three anthelmintic drugs addition on their ability to block hadv infection. a previous report showed biochemically that hadv viral particles were internalized within min of binding and reach the nuclear pore after min . our results demonstrated that nic, oxy and raf exhibited a time-dependent decrease in their inhibitory activity (fig. a) . all of them showed high inhibition of hadv infection when added at the beginning of the min incubation at °c (− min), and when added immediately prior to warming ( min) or after min. however, when drugs were added at or min, only oxy still showed a significant inhibition higher than % which was lost for nic and raf (fig. a) . to support these findings a western blotting for hadv e a protein was run at h post infection. results obtained showed a significantly lower expression of e a protein in samples treated with nic and raf compared with oxy (fig. b) . impact on hadv genome accessibility to the nucleus. this assay evaluated the impact of the salicylanilide anthelmintic drugs on hadv endosomal escape. after the binding and internalization of the hadv viral particles, the exposure of protein vi inside the endosome triggers endosomolysis and the partially decapsidated hadv escape to the cytosol from where it is transported to the nuclear pore complex at the nuclear membrane by the microtubule network. we used a functional assay comprising hadv-mediated co-delivery of α-sarcin in cells as a marker of the ability of these salicylanilide drugs to alter virus-mediated endosomolysis , . no significant differences in the id ( % inhibitory doses) for hadv-mediated endosome penetration were detected in the presence of any of the salicylanilide drugs ( fig. ) or the dmso negative control. in contrast, the control virus ad ts , an endosome penetration-defective mutant, exhibited a significant increase in the id compared to the dmso control (fig. ) . once viral particles leave the endosome they are transported to the nuclear pore complex where hadv genomes are imported into the nucleus . we hypothesized that if any of the drugs examined was blocking a step in the hadv entry, as expected for nic, the number of genomes that reach the nucleus would be lower. we next evaluated the ability of these salicylanilide drugs to block hadv genome accessibility to the nucleus by the quantification of hadv genomes isolated from the nucleus . as reflected in fig. a , only nic and raf showed a significant block in the accessibility of hadv genomes to the nucleus (fig. a) . the dna copy number of the cellular gene gapdh was also evaluated in both the nucleus and the cytoplasm as a control for the purity of nuclear isolation (data not shown). since our results indicated that nic and raf hindered the accessibility of hadv genome to the nucleus while oxy did not, we resolved to determine if this anthelmintic drug affected later steps in hadv life cycle. impact on hadv replication. the next step was to examine the effect that oxy had upon efficient hadv dna replication using quantitative real-time pcr (qpcr). a cells were infected with hadv and incubated for h at °c. then, the unbound virions were washed-out and cell cultures were incubated for h. hadv dna was extracted at that point to limit the interference caused by subsequent rounds of infection occurring - h post-infection . we used quantitative pcr to quantify in a single round of infection the synthesis of new hadv dna copies as a measure of dna replication efficiency. the presence of oxy ( μm) significantly inhibited hadv dna replication by more than % (p < . , dunnett's multiple comparison test), whilst the quantification of the dna copies of the gapdh gene did not showed a significant effect (fig. b ). this reduction in the hadv dna copy number at the nucleus in the presence of oxy implicated two alternatives for its mechanism of action: i) oxy could inhibit hadv dna replication by interfering with a protein required in this process like hadv dna polymerase, dna binding protein (dbp) or terminal protein (tp) or, alternatively, ii) oxy could impact transcription of the hadv immediate early gene e a, which is a key step before dna replication. to assay the inhibition of hadv mrna transcription, we infected a cells in the presence of oxy ( μm) for h. after the infection, we quantified the rna copy number of the e a gene using quantitative reverse transcription (rt-pcr). as shown in fig. c , oxy exerted a significant decrease in the e a mrnas copy number compared with the control treated with dmso. based on previous works where piperazine derivatives and nucleotide and nucleoside analogues showed antiviral activity against multiple dsdna virus including cytomegalovirus (hcmv) and hadv , , we evaluated the inhibitory activity of nic, oxy and raf on hcmv dna replication. the presence of these anthelmintic drugs generated significant reductions in the quantification of total hcmv dna h after infection of mrc- cells (fig. d ). oxy showed a % decrease whilst nic and raf reached a . % and a . % decrease, respectively of hcmv dna replication, showing no significant differences between samples for the quantification of the gapdh gene (data not shown). salicylanilide anthelmintic drugs combination improves their antiviral activity. since we found different mechanisms of action for these three drugs we hypothesize that their combination should significantly improve their antiviral activity. to evaluate our hypothesis we conducted a combination study based on the chou-talalay method for drug combination using the calcusyn software , . the constant ration for each combination was selected based on the ic values for each drug. the data for all the combinations showed good conformity to the mass action law (r = . - . ) ( table ). all the combinations were classified as synergistic (table ). for the nic:raf (ratio : ) combination the ic and ic levels of inhibition were classified as synergism and the ic level as strong synergism (table ). finally, the combination raf:oxy (ratio : ) was classified as synergism at all the levels of inhibition (table ). the aim of this study was to evaluate the anti-hadv activity of nic, a salicylanilide anthelmintic drug of human use to set the basis for its further experimental and clinical development as a potential new treatment for hadv infections. moreover, we included in our evaluation other two chlorinated salicylanilide anthelmintic derivatives approved for animal use, oxy and raf, because of their closely related structure and mechanism of action with nic. it is well-known that these drugs act as uncouplers of the oxidative phosphorylation, involving dissipation of the membrane potential . in case of nic, jurgeitr et al. previously demonstrated that its antiviral mechanism of action was specifically associated with the neutralization of the endosomal ph, thus showing a broad antiviral activity against ph-dependent viruses. although hadv endosomal escape is not exclusively dependent of ph, a low ph has proven to be associated with the endosomal escape of hadv , . with these premises, we initially assumed a probable common anti-hadv mechanism of action for these three drugs and decided to characterize their anti-hadv activity as a previous step before the evaluation of their safety and efficacy in the syrian hamster model of hadv infection. here, we show that nic, oxy and raf exert significant anti-hadv and anti-hcmv activity at low concentrations. to put this data into perspective, it is noteworthy that the reported ic values for cidofovir, the drug of choice for the treatment of hadv infections, are higher than those shown by these three salicylanilide drugs , . in addition, following our own methodologies, the ic value for cidofovir was . ± . µm, significantly higher than the values obtained for the three salicylanilide drugs. as for their mechanism of action, the data obtained from this work demonstrates that one of these three closely-related drugs did not share the expected mode of action. we have confirmed in our work that nic, and as a novelty raf, acts to decrease the number of hadv genomes associated with the host nucleus, however, as demonstrated by our physiologic assay using the ribotoxin α-sarcin nic and raf may act in a later step after hadv endosomal escape. unlike nic and raf, oxy did not inhibit the access of hadv genomes to the nucleus. this observation was confirmed in their time-course assay where we found that, for nic and raf inhibition occurred at earliest times of the hadv replicative cycle than for oxy as well as in the western-blot assay at hpi that showed higher inhibition of the e a gene expression for nic and raf. as shown by our results, the high inhibition in the hadv dna replication generated by oxy may be the consequence of the previous and significant inhibition of the e a gene transcription, which is supported by our results showing a clear inhibition of the e a transcription at hpi, both by quantitative real-time pcr and by western-blot. it is well known that prior to hadv dna replication, transcription of e a by cellular rna polymerase ii takes place from the e a promoter . then, e a protein promotes its own transcription and is needed for the subsequent expression of the early genes e b, e , e and e from different promoters as well as for dna replication and that is probably why we observed that significant inhibition on hadv dna replication. the existence of different mechanisms for the antiviral activity of these three drugs is supported by the significant combinatory index values obtained using the calcusyn software for all the drug combinations ( table ). the three anthelmintic drugs showed a moderate synergistic to a strong synergistic activity when they were combined in pairs, which was especially strong at all the levels of inhibition evaluated for the nic-oxy combination. likewise, using the combination of the three of them they showed a strong synergistic activity at all the levels of inhibition evaluated. repositioning of drugs for diseases different than those they were approved for is a valuable alternative for drug discovery and development since it reduces significantly the high cost and the time-consumption of developing a new drug [ ] [ ] [ ] [ ] . in case of nic and other anthelmintic drugs, numerous reports support their repurposing as anti-cancer drugs due to their cancer inhibitory properties [ ] [ ] [ ] [ ] . nic is an fda-approved drug that has been used for many years to treat helminthic infections in humans and has previously demonstrated its antiviral potential against different viruses such as japanese encephalitis flavivirus, zika virus, coronavirus, human rhinovirus or influenza virus [ ] [ ] [ ] [ ] . in rats, nic is known to exert low in vivo toxicity, with a median lethal dose % (ld ) of , mg/kg body weight and generate peak serum concentrations between . µm and µm, depending on the dose and the administration route , . in healthy humans orally treated with - , mg nic per person, no signs of intoxication were seen , and the drug remained detectable for - days. peak serum concentrations between . - µm were reported , . as shown in our results, these serum concentrations will be enough to reach the ic for hadv (table ) . oxy and raf are other fda-approved drugs for veterinary use to treat helminthic infections. for oxy, high serum concentrations ranging from . µm to . µm have been reported in sheep and goats after a single oral dose of mg/kg . in the case of raf, an intravenous single dose of mg/kg in goats generated serum concentrations ranging from . µm to . µm registered at . h and h, respectively . after a single oral dose of . mg/kg also in goats, the maximum serum concentrations obtained were . µm and . µm at h and h, respectively . in both cases, oxy and raf serum concentrations are significantly higher than their ic for hadv ( table ) . as for their toxicity, ld of raf has been reported to be higher than , mg/kg of body weight in rats while for oxy, according to the european medicines agency (emea), this ld would be higher than , mg/kg of body weight (also in rats) . our findings show a significant anti-hadv activity for the three salicylanilide drugs targeting different steps on the hadv life cycle. nic and raf would mainly block hadv infection at some point between endosomal escape and dna release into the nucleus, whilst the activity of oxy would be specifically targeting hadv immediately early gene e a transcription. our findings support the evaluation of these three drugs in the syrian hamster model of hadv infection to evaluate their efficacy and safety. the pharmacokinetic profile of these salicylanilide drugs showing low water solubility and oral bioavailability may hamper its further clinical development . in this situation, the generation of derivatives of these salicylanilide drugs based on rational chemical approaches may improve their poor pharmacokinetics, increasing their solubility and bioavailability as potential clinical candidates for antiviral therapy. wild-type hadv (species c), hadv (species b), hadv (species d), and human cytomegalovirus (hcmv) ad were obtained from the atcc. the hadv -gfp and hadv -gfp used in this study are replication-defective viruses with a cmv promoter-driven enhanced green fluorescent protein (egfp) reporter gene cassette in place of the e /e region . hadv were propagated in β cells and isolated from the cellular lysate by cesium chloride (cscl) density gradient combined with ultracentrifugation. virus concentration in mg/ ml was calculated using the bio-rad protein assay (bio-rad laboratories) and converted to virus particles/ml (vp/ml) using × vp/mg. cytotoxicity assay. nic, oxy and raf cytotoxicity was evaluated using the alamar blue cell viability assay (invitrogen) according to the manufacturer's instructions. actively dividing a or β cells were incubated with the salicylanilide anthelmintic drugs for h. after this incubation, the alamar blue reagent was added to the cells ( / th alamar blue reagent in culture medium) for an extra h. their % cytotoxic concentration (cc ) was calculated as previously reported by cheng et al. . the selectivity index (si) was calculated as the ratio of cc to ic , where the ic is defined as the concentration of the anthelmintic drug that inhibits hadv infection by %. hadv plaque assay. anthelmintic drugs were tested in a dose-response assay using an moi of . vp/cell and drug concentrations ranging from to . μm in a plaque assay. briefly, a density of × β cells per well were seeded in -well plates. at - % confluency they were infected with hadv -gfp or hadv -gfp ( . vp/cell) and rocked for h at °c. after this incubation wells were washed-out with pbs. then, cells were carefully overlaid with ml/well of equal parts of . % (water/vol) difco agar noble (becton, dickinson & co., sparks, md) and × emem (minimum essential medium eagle, biowhittaker) supplemented with × penicillin/streptomycin, × l-glutamine, and % fbs. the mixture also contained the drugs in concentrations ranging from to . μm. after incubation for days at °c, virus plaques were scanned with a typhoon imager (ge healthcare life sciences), and quantified with imagej . to determine the time course of the three anthelmintic drugs-mediated inhibition, parallel samples of wild-type hadv were incubated with or without μm of nic and μm of raf or oxy in complete dmem at °c for h. virus ( vp/cell) was then added to a cells ( , cells/well in a -well plate) and incubated at °c. anthelmintic drugs were added at the indicated time points (− , , , , and min) before or during this incubation. after incubation at °c and % co for h dna was purified from the cell lysate using the qiaamp dna mini kit (qiagen, valencia, ca) following the manufacturer's instructions, and the dna was quantified by quantitative pcr following the above described protocol. analysis of anthelmintic drug combinations. the software packet calcusyn (biosoft, ferguson, mo, usa), which compares the drug concentrations required in combination to generate a given effect to the drug concentration that would be needed individually to achieve that same effect was used. for this assay a plaque dose-response assay was carried out using all the possible combination of the three drugs starting from twice the ic obtained previously for each compound and the ratio of those concentrations. calcusyn software interpolates the drugs concentrations needed in combination at the selected ratio to generate effects of %, % and % inhibition and compares these combined drug concentrations with the concentrations from the three drugs' individual dose-response curves required to achieve the same inhibition. the combination effect of the three drugs was reported by the combination index (ci) value, a quantitative estimation of the pharmacological interaction which uses the potency (ic ) and the shape of the dose-response curve of each individual drug and their combinations. the ci value was interpreted in accordance with matthews et al. . nuclear-associated hadv genomes. the nuclear accessibility of hadv genomes was evaluated by real-time pcr following a previously described protocol with a few modifications . briefly, × a cells in -well plates were infected with wild-type hadv (moi , vp/cell) in the presence of μm nic or μm oxy and raf, or the same volume of dmso for negative control. forty-five minutes after infection, a cells were trypsinized and collected, and then washed twice with pbs. then, nuclear and cytoplasmic fractions were separated using a hypotonic buffer solution consisting of mm tris-hcl ph . , mm nacl, and mm mgcl . the cell pellet was resuspended in μl of × hypotonic buffer and incubated for min at °c. then, μl of np- was added and the samples were vortexed. the homogenates were then centrifuged for min at g at °c. hadv dna was isolated from the nuclear (pellet) and the cytoplasmic fractions (supernatant) using the qiaamp dna mini kit (qiagen, valencia, ca). to measure endosome disruption, hadv-mediated ribotoxin (α-sarcin) delivery assays were performed as previously described hadv dna and mrna quantification by real-time pcr. for dna quantification, , a cells/ well in a -well plate were infected with vp/cell (wild-type hadv ) and incubated for h at °c in complete dmem. then, the excess of virus was washed-out, and the medium was replaced with μl of complete dmem containing μm of either anthelmintic drugs or the same volume of dmso (negative control). all samples were done in triplicate. after h of incubation at °c and % co , dna was purified from the cell lysate using the qiaamp dna mini kit (qiagen, valencia, ca) following the manufacturer's instructions. taqman primers, probes and pcr conditions were like those previously reported . for the evaluation of rna expression, same conditions of infection applied for the dna quantification were used. six hours after infection, rna was purified with the mircury rna isolation kit (exiqon inc., ma) following the manufacturer's instructions. quantification of rna copy numbers was performed using primers in conditions previously reported for e a . human glyceraldehyde- -phosphate dehydrogenase (gapdh) gene was used as internal control. primers, probes and conditions applied for gapdh were those previously reported by rivera et al. . for quantification, gene fragments of hexon and gapdh were cloned into the pgem-t easy vector (promega), and known concentrations of those vectors were used to generate a standard curve for each experiment. all assays were performed in a c thermal cycler apparatus (biorad). a burst assay was used to assay the effect of the three anthelmintic drugs on virus production. a cells were infected with wild-type hadv , wild-type hadv or wild-type hadv in the presence or absence of μm nic or μm oxy and raf. after incubation for h, cells were harvested and subjected to three rounds of freeze/thaw. then, serial dilutions of clarified lysates were titrated on a cells, and tcid values were calculated using a previously reported end-point dilution method . to test the anti-hcmv activity of these anthelmintic drugs, mrc- cells were seeded in a -well plate ( . × cells/well), infected with hcmv (moi of . vp/ cell) and incubated in complete dmem in the presence of μm nic or μm in case of oxy and raf or the same volume of dmso in triplicate. then, cells were incubated for h at °c and % co and hcmv dna was purified from the cell lysate using the qiaamp dna mini kit (qiagen, valencia, ca) following the manufacturer's instructions. real-time pcr primers, mixtures and protocols were the same as previously reported . statistical analyses. statistical analyses were carried out using the graphpad prism suite. data are presented as the mean of triplicate samples ± standard deviation (sd), unless otherwise indicated. p < . was considered statistically significant. molecular evolution of human adenoviruses gastroenteritis in childhood: a retrospective study of hospitalized pediatric patients detection of a broad range of human adenoviruses in respiratory tract samples using a sensitive multiplex real-time pcr assay 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single-cell analysis that human {alpha}-defensins block adenovirus uncoating to neutralize infection enhancement of gene transfer to human myeloid cells by adenovirus-fiber complexes antiherpes simplex virus type activity of casuarinin from the bark of terminalia arjuna linn nih image to imagej: years of image analysis inhibition of adenovirus infection by mifepristone mode of transgene expression after fusion to early or late viral genes of a conditionally replicating adenovirus via an optimized internal ribosome entry site in vitro and in vivo a simple method of stimating fifty percent endpoints supported by plan nacional de i + d + i - and instituto de salud carlos iii, ministerio de economía, industria y competitividad, spanish network for research in infectious diseases (reipi rd / / )co-financed by "a way to achieve europe" erdf, the instituto de salud carlos iii, proyectos de investigación en salud (pi / ) and proyectos de desarrollo tecnológico en salud (dts / ), and the spanish adenovirus network (adenonet, bio / -redt). j.s.c. is supported by the "contract to access to the spanish system of research and innovation of the program of r + d + i of the university of seville" (use- -d) grant. j.a.m.l. and a.s.g. have made substantial contributions to acquisition and analysis of data, as well as in the preparation of the manuscript. j.b.c. helped with the acquisition of data. j.p. has made substantial contributions to conception, design and interpretation of data. j.s.c. designed and coordinated the work and the preparation of the manuscript. supplementary information accompanies this paper at https://doi.org/ . /s - - - . the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - pjdutgg authors: awaisu, ahmed; mukhalalati, banan; mohamed ibrahim, mohamed izham title: research designs and methodologies related to pharmacy practice date: - - journal: encyclopedia of pharmacy practice and clinical pharmacy doi: . /b - - - - . - sha: doc_id: cord_uid: pjdutgg abstract the need for evidence to inform policy and practice in pharmacy is becoming increasingly important. in parallel, clinical pharmacy and practice research is evolving. research evidence should be used to identify new areas for improved health service delivery and rigorously evaluate new services in pharmacy. the generation of such evidence through practice-based research should be predicated on appropriate use of robust and rigorous methodologies. in addition to the quantitative and qualitative approaches used in pharmacy practice research, mixed methods and other novel approaches are increasingly being applied in pharmacy practice research. approaches such as discrete choice experiments, delphi techniques, and simulated client technique are now commonly used in pharmacy practice research. therefore, pharmacy practice researchers need to be competent in the selection, application, and interpretation of these methodological and analytical approaches. this chapter focuses on introducing traditional and novel study designs and methodologies that are particularly pertinent to contemporary clinical pharmacy and practice research. this chapter will introduce the fundamentals and structures of these methodologies, but more details regarding the different approaches may be found within the encyclopedia. the mission of pharmacy profession and the role of pharmacists in healthcare have evolved toward patient-centered care in the last few decades. pharmacists with their expertise in drug therapy and accessibility to the public have unprecedented opportunities to assume increasing responsibility for direct patient care (bond, ) . new cognitive pharmaceutical services and new roles for pharmacists continue to emerge. in the era of evidence-based practice and health services, it is not just adequate to propose those new pharmacy services or new roles without evidence of their benefit (awaisu and alsalimy, ; bond, ) . new pharmacy services and new roles must be proven to be feasible, acceptable, cost-effective, and increase health outcomes. pharmacy practice research provides such evidence and can confirm the value of a new service, inform policy, and result in practice changes (bond, ; chen and hughes, ) . research evidence should be used to identify new areas for improved health service delivery and rigorously evaluate new services. the research used to generate such evidence should be grounded in robust and rigorous methodologies (chen and hughes, ) . traditionally, common quantitative and qualitative methods such as randomized controlled trials, cohort study, case control study, questionnaire-based surveys, and phenomenology using qualitative interviews have been used in pharmacy. however, in recent years, novel and more complex methods are being developed and utilized. pharmacy practice researchers need to know how these old and new methodological approaches should be selected, applied, and interpreted in addressing research problems. various study designs, including, but not limited to experimental, quasi-experimental, observational, qualitative, and mixed method designs, have been used in pharmacy practice research. furthermore, different classification systems (e.g., quantitative vs. qualitative, experimental vs. observational, descriptive vs. analytical study designs) have been used in the literature. the choice of a study design to answer a research question in pharmacy practice research is driven by several factors, including the type of the research question or the research hypothesis, expertise of the investigator, availability of data, and funding opportunities. pharmacy practice researchers need to be competent in the selection, design, application, and interpretation of these methodological and analytical approaches. today, many of the research methods used in pharmacy practice research have been adapted from fields such as sociology, anthropology, psychology, economics, and other disciplines. this paradigm shift has led to a greater emphasis on the appropriate choice of a specific research design or method to answer a specific research question (chen and hughes, ) . consequently, pharmacy practice researchers should place an emphasis on the reliability of the methods selected, the correct interpretation of their findings, the testing of a specific hypothesis, and the internal validity of their data, among other considerations. novice and early career researchers should be familiar and have sound foundation in a variety of methods applied in pharmacy practice research, which will be covered in this chapter and other chapters in this encyclopedia. we do believe that more experienced researchers should focus on certain methods in order to advance research in our discipline. traditionally, core quantitative approaches used in pharmacy practice research include nonexperiments, quasi-experimental designs, and true experimental designs such as prospective randomized controlled intervention trials. nonexperiments also include observational study designs that are often described as pharmacoepidemiologic study designs such as case-control study, cohort study, nested case-control study, and cross-sectional study (etminan, ; etminan and samii, ) . in recent years, conventional qualitative approaches and their philosophical paradigms are increasingly been used in pharmacy. these include the five qualitative approaches to inquiry: narrative research, phenomenology, grounded theory, ethnography, and case study. these qualitative methods are often difficult for pharmacy practice researchers to comprehend, and researchers tend to describe the methods of data collection such as individual interviews and focus group discussions as qualitative methods of inquiry. these data collection methods are briefly described later in this chapter, among others. furthermore, there is an increasing importance on the appropriate selection and use of mixed method approach (hadi et al., ; closs, a, b) , which are often designed and applied wrongly. finally, it is worthwhile to be familiar with novel research methodologies such as discrete choice experiments, delphi techniques, simulated client technique, and nominal group techniques, which fall between quantitative and qualitative approaches, often with no clear differentiation on where they belong. although called "novel" in the context of this chapter, these methods are not new in other relevant disciplines, but new and not commonly used in pharmacy practice research. pharmacy practice researchers begin by conception of a research idea or identifying a research question and defining a hypothesis based on the question. the researcher then selects a study design that will be suitable to answer the research question. the study design should be appropriately selected prior to initiation of any research investigation. selecting an inappropriate study design may potentially undermine the validity of a study in its entirety. investigators are encouraged to critically think about the possible study designs to ensure that the research question is adequately addressed and should be able to adequately justify their choice. these study designs have been variously classified and one common classification system is quantitative vs. qualitative study designs. study designs play a major role in determining the scientific value of research studies. inappropriate choice of a study design is impossible to correct after completion of the study. therefore, thorough planning is required to avoid unconvincing results and invalid conclusions. good understanding of basic study design concepts will aid researchers in conducting robust and rigorous practice-based research. this chapter introduces the structure and the fundamentals of common study designs used in pharmacy practice research and discusses the important considerations for conducting pharmacy practice research in terms of study design, data collection, data analyses, and ethical considerations. various classifications for research designs and methods used in pharmacy practice have been used in the literature. the following are some of the approaches for the classification of research designs: . classification based on time orientation: retrospective vs. prospective designs a. retrospective design-a retrospective study design observes what has happened in the past. it begins and ends in the present. this design involves a major limitation as it looks to collect information about events that occurred in the past. an example of this design is retrospective case-control study. case example: investigators were looking for the association between acute myocardial infarction and smoking status, type of tobacco, amount of smoke, etc. (teo et al., ) . another example of a case-control study from published literature is the study investigating the association between the use of phenylpropanolamine and the risk of hemorrhagic stroke (kernan et al., ) . b. prospective design-a prospective study design begins in the present and progresses forward, collecting data from subjects whose outcomes lie in the future. an example of this design is prospective cohort study. case example: investigators were interested to determine the long-term effectiveness of influenza vaccines in elderly people; they recruited cohorts of vaccinated and unvaccinated community-dwelling elderly (nichol et al., ) . . classification based on study purpose: descriptive vs. analytical designs a. descriptive design-a descriptive study describes a population/sample in terms of distribution of the variables, and frequency of outcomes of interest. unlike analytical studies that include control (comparison) group, descriptive studies do not include a comparison group. descriptive studies include case reports, case series reports, cross-sectional studies, surveillance studies, and ecological studies. case example: a case report was written by a physician who contracted severe acute respiratory syndrome (sars) during an outbreak in hong kong (wu and sung, ) . another example is an ecological study examining diet and sunlight as risks for prostate cancer mortality (colli and colli, case example: a group of investigators carried out a study to establish an association between the use of traditional eye medicines (tem) and corneal ulcers. in this case, both case-control and cohort study designs are applicable. in an example of a case control study, archibugi et al. aimed to investigate the association between aspirin and statin exclusive and combined and pancreatic ductal adenocarcinoma occurrence (archibugi et al., ) . another example of a cohort study is a study carried out by wei et al. in which they investigated whether or not acid-suppression medicines increased the risk of bacterial gastroenteritis (wei et al., ) . . classification based on investigator orientation: experimental vs. quasi-experimental vs. observational designs a. experimental design-in experimental design (also known as interventional design), the investigator performs an intervention and evaluates cause and effect relationships. case examples: investigators conducted a study about the newer versus older antihypertensive agents in african hypertensive patients (noaah) trial (nct ) to compare the efficacy of single-pill combinations of newer versus older antihypertensive agents (i.e., a single-pill combination of newer drugs, not involving a diuretic, with a combination of older drugs including a diuretic) (odili et al., ) . in a crossover design, a group of investigators evaluated the effect of spironolactone on nonresolving central serous chorioretinopathy (bousquet et al., ) . b. quasi-experimental design-the quasi-experimental design is very similar to the true experimental design described above and it involves an intervention. the design has been employed when randomization is inappropriate or impossible, especially when implementing complex interventions. case examples: prashanth et al. aimed to understand if (and how) a package of interventions targeting primary health centers and community participation platforms affect utilization and access to generic medicines for people with noncommunicable diseases using quasi-experimental design approach (prashanth et al., (murphy et al., ) . c. mixed method designs-mixed method design brings together qualitative and quantitative methodologies within a single study to answer or understand a research problem (hadi et al., ) . case examples: shiyanbola et al. combined focus group discussion with a survey tool to investigate patients' perceived value and use of quality measures in evaluating and choosing community pharmacies (shiyanbola and mort, ) . below is a brief description of traditional and novel pharmacoepidemiologic study designs. several examples of pharmacoepidemiologic study designs are provided above. some descriptive studies including case reports, case series, and ecological studies will not be described in this chapter. a. case-control studies-in this design, patients (those who develop the disease or outcome of interest) are identified and control patients (those who do not develop the disease or outcome of interest) are sampled at random from the original cohort that gives rise to the cases (etminan and samii, ; newman et al., ) . the distribution of exposure to certain risk factors between the cases and the controls is then explored, and an odds ratio (or) is calculated. b. cohort studies-this can be described as a study in which a group of exposed subjects and a group of unexposed subjects are followed over time and the incidence of the disease or outcome of interest in the exposed group is compared with that in the unexposed group (etminan and samii, ; hulley et al., ) . c. case-crossover studies-the case-crossover may be considered comparable to a crossover randomized controlled trial in which the patients act as their own control (etminan and samii, ) . pattern of exposure among the cases is compared between event time and control time. the between-patient confounding that occurs in a classic case-control study is circumvented in this design. tubiana et al. evaluated the role of antibiotic prophylaxis and assessed the relation between invasive dental procedures and oral streptococcal infective endocarditis, using a nationwide population-based cohort and a case-crossover study design (tubiana et al., ) . d. case-time control studies-this design is an extension of the case-crossover design, but includes a control group (etminan and samii, ) . a group of researchers assessed medication-related hospitalization. they used the case-time control study design to investigate the associations between high risk medication categories (e.g., antidiabetic agents, diuretics, benzodiazepine hypnotics) and unplanned hospitalizations (lin et al., ) . e. nested case-control studies-in this design, a cohort of individuals is followed during certain time periods until a certain outcome is reached and the analysis is conducted as a case-control study in which cases are matched to only a sample of control subjects (etminan, ) . de jong et al. examined the association between interferon-β (ifn-β) and potential adverse events using population-based health administrative data in canada (de jong et al., ) . f. cross-sectional studies-in this type of study, the investigator measures the outcome of interest and the exposures among the study participants at the same time setia, b) . it provides a snapshot of a situation for a particular period. a wide range of quantitative methods are commonly applied in pharmacy practice research. these methods are widely used in published pharmacy practice literature to explore appropriateness of medicines use, appropriateness and quality of prescribing, and medication safety, through analyzing existing datasets, direct observation, or self-report (green and norris, ) . pharmacy practice research questions also seek to determine the knowledge, behaviors, attitudes, and practices of pharmacists, other healthcare providers, patients, policy-makers, regulators, and the general public. quantitative methods are also used in evaluating the effect of new pharmacy services and interventions to improve medicines use. these practice research projects provide valuable insights about how medicines are used, and how to maximize their benefits and minimize their harmful effects. in the context of this chapter, quantitative study designs will be broadly classified into three: ( ) observational, ( ) experimental and quasi experimental, and ( ) other designs. pharmacoepidemiology is a "relatively new science that explores drug efficacy or toxicity using large observational study designs" (etminan, ; etminan and samii, ) . these study designs explore drug use studies that usually cannot be answered using randomized controlled trials or other experimental designs. in several instances, experimental study designs may not be suitable or feasible; in such circumstances, observational study designs are applied . as the name implies, observational studies involve merely observing the subjects in a noncontrolled setting, without investigator intervention or manipulating other aspects of the study. therefore, observational studies are nonexperimental. the observation of the variables of interest can be prospective, retrospective, or current depending on the type of the observational study. in pharmacoepidemiology and other areas of pharmacy practice, researchers are often interested in measuring the relationships between exposure to a drug and its efficacy, toxicity, or other outcomes of interest using observational study designs. it is worthwhile to note that observational study designs investigate association, but, in most cases, not causation. here, we provide descriptions of some commonly used study designs in pharmacoepidemiology and pharmacy practice research in general. case-control study design is used to determine association between risk factors or exposures and outcomes. it is a useful design to study exposures in rare diseases or diseases that take long time to develop . it investigates exposures in individuals with and those without the outcome of interest. nevertheless, case-control studies can help to identify harmful or beneficial exposures. furthermore, the outcome of interest can be undesirable (e.g., mortality) or desirable (e.g., microbiological cure). as the name suggests, in a case-control study design, there are two groups of subjects: ( ) cases (individuals with the outcome of interest) and ( ) controls (individuals without the outcome of interest) . cases are randomly selected based on prespecified eligibility criteria from a population of interest. appropriate representative controls for the cases selected are then identified. the researchers then retrospectively investigate possible exposures to the risk factor. fig. represents a schematic diagram of a case-control study. case-control studies are relatively inexpensive, less time-consuming to conduct, allow investigation of several possible exposures or associations, and are suitable for rare diseases. selection of the control group is a critical component of case-control studies. case-control studies have several drawbacks: confounding must be controlled, subject to recall, observation, and selection biases. or is the measure of association used for the analysis of case-control studies. this is defined as the odds of exposure to a factor in those with a condition or disease compared with those who do not have the condition or disease. similar to case-control studies, cohort studies determine an association between exposures/factors and development of an outcome of interest. as previously described, a cohort study is a study in which a group of exposed subjects and a group of unexposed subjects are followed over time to measure and compare the rate of a disease or an outcome of interest in both groups (etminan and samii, ; hulley et al., ) . a cohort study can be prospective (most common) or retrospective. while a case-control study begins with patients with and those without the outcome of interest (e.g., diseased and nondiseased patients), a cohort study begins with exposed and unexposed patients (e.g., patients with and those without certain risk factor) setia, a) . in a cohort study, both the exposed and the unexposed subjects are members of a larger cohort in which subjects may enter and exit the cohort at different periods in time (etminan and samii, ; hulley et al., ) . typically, a cohort study should have a defined time zero, which is defined as the time of entry into the cohort (etminan and samii, ) . the cohort (a group of exposed and unexposed subjects, who are free of the outcome at time zero) is followed for a certain period until the outcome of interest occurs. in addition, information or data related to all potential confounders or covariates should also be collected as failure to account for these can bias the results and over-or underestimates the risk estimate. there are two types of cohort studies: retrospective cohort and prospective cohort studies. retrospective cohort study, also known as historical cohort study, begins and ends in the present, while looking backward to collect information about exposure that occurred in the past (fig. ) . historical cohort studies are relatively less time-consuming and less expensive than prospective cohort studies (etminan and samii, ; hulley et al., ; setia, a) . in addition, there is no loss to follow-up and researchers can investigate issues not amenable to intervention study designs. however, these studies are only as good as the data available, the investigator has limited control of confounding variables, and it is prone to recall bias. on the other hand, prospective cohort study, also known as longitudinal cohort study, begins in the present and progresses forward, collecting data from enrolled subjects whose outcomes fall in the future (etminan and samii, ; hulley et al., ; [ ( f i g u r e _ ) t d $ f i g ] setia, a) (fig. ) . prospective cohort studies are easier to plan for data collection, have low recall bias, and the researcher has a better control of confounding factors. on the other hand, it is difficult to study rare conditions; they are more prone to selection bias, more time-consuming, expensive, and loss of subjects to follow-up is common. relative risk (rr) is the measure of association used for the analysis of a cohort study. this is defined as the risk of an event or development of an event relative to exposure (i.e., the risk of subjects developing a condition when exposed to a risk factor compared with subjects who have not been exposed to the risk factor). this is a relatively new design in the field of epidemiology in which the patients act as their own controls (maclure, ) . in this design, there is a case and a control element both of which come from the same subject. in other words, each case serves as its own control. it can be considered equivalent to a crossover rct with a washout period (etminan and samii, ) . pattern of exposure to the risk factor is compared between the event time and the control time (etminan and samii, ) . case-crossover study design is useful to investigate triggers within an individual. for instance, it is applicable when studying a transient exposure or risk factor. [ ( f i g u r e _ ) t d $ f i g ] however, determination of the period of the control and case components is a crucial and challenging aspect of a case-crossover study design. since the patients serve as their own controls, the interindividual variability that is inherent in classic case-control studies is eliminated. this is important in studies involving progressive disease states in which disease severity may differ between patients such as multiple sclerosis. or is estimated using techniques such as mantel-haenszel statistics and logistic regression. cross-sectional studies also known as prevalence studies identify the prevalence or characteristics of a condition in a group of individuals. this design provides a snapshot of the prevalence or the characteristics of the study subjects in a single time point. the study investigator measures the outcomes and the exposures in the study subjects simultaneously (etminan and samii, ; hulley et al., ; setia, b) . hence, cross-sectional studies do not follow up patients to observe outcomes or exposures of interest. data are often collected through surveys. cross-sectional design cannot provide cause and effect relationships between certain exposures and outcomes of interest. in a typical experimental study design, the investigator assigns subjects to the intervention and control/comparison groups in an effort to determine the effects of the intervention . since the investigator has the opportunity to control various aspects of the experiment, this allows the researcher to determine the causal link between exposure to the intervention and outcome of interest. the researcher either randomly or conveniently assigns the subjects to an experimental group and a control group. when the investigator performs randomization, the study is considered a true experiment (see fig. ). on the other hand, if subjects are assigned into groups without randomization, the study is considered a quasi-experiment (refer to fig. ). as with experimental designs, quasi-experimental designs also attempt to demonstrate a causal link between the intervention and the outcome of interest. due to the challenges of conducting a true experimental design, the quasi-experimental study designs have been consistently used in pharmacist intervention research. rcts are considered the gold standard of experimental study designs in pharmacy practice and evidence-based research . the investigator randomly assigns a representative sample of the study population into an experimental group and a control group (fig. ) . randomization in rct is to minimize confounding and selection bias; it enables attainment of similar experimental and control groups, thereby isolating the effect of the intervention. the experimental group receives the treatment or intervention (e.g., a new drug or pharmaceutical care for treatment of a certain disease), while the control group receives a placebo treatment, no treatment, or usual care treatment depending on the objective of the study . these groups are then followed prospectively over time to observe the outcomes of interest that are hypothesized to be affected by the treatment or intervention. the result of the study is considered to have high internal validity if significant changes on the outcome variable occur in the experimental group, but not the control group. the investigator can infer that the treatment or intervention is the most probable cause of the changes observed in the intervention group. the unit of randomization in rcts is usually the patient, but can sometimes be clusters to circumvent the drawbacks of contamination. rcts are very challenging to undertake and pharmacy practice researchers should ensure design of robust experiments, while considering all essential elements and adhering to best practices. for instance, to determine the impact of a cognitive pharmaceutical service, the selection of a representative sample of the population is a prime consideration in an rct. moreover, rcts are expensive, labor-intensive, and highly prone to attrition bias or loss to follow-up. in pharmacy practice research, it is often difficult to comply with the stringent requirements of true experimental designs such as rcts, due to logistic reasons and/or ethical considerations krass, ) . whenever true experimental models are not feasible to be applied in pharmacy practice research, the researcher should endeavor to use a more robust quasi-experimental design. for instance, when randomization is not feasible, the researcher can choose from a range of quasi-experimental designs that are non-randomized and often noncontrolled krass, ) . quasi-experimental studies used in pharmacy literature may be classified into five major categories: ( ) quasi-experimental design without control groups (i.e., one group pre-posttest design); ( ) quasi-experimental design that use control groups with no pretest; ( ) quasi-experimental design that use control groups and pretests (i.e., nonequivalent control group design with dependent pretests and posttests) (see fig. ); ( ) interrupted time series and; ( ) stepped wedge designs (brown and lilford, ; grady et al., ; harris et al., ) . the one group pretest posttest design and the nonequivalent control group design (fig. ) are the most commonly applied quasiexperimental designs in practice-based research literature. these designs have been commonly used to evaluate the effect of pharmacist interventions in medications management in general and specific disease states management. the lack of randomization and/or the lack of control group is a major weakness and a threat to internal validity in quasi-experimental designs . the observed changes could be due to some effects other than the treatment. in addition to the common observational, experimental, and quasi-experimental designs described above, there are other designs that are used in pharmacy. these research methods include, but are not limited to, simulated client technique, discrete choice experiments, and delphi techniques. these methods, which are considered relatively new to pharmacy, are now commonly used in pharmacy practice research. in this chapter, we briefly describe these methods and their application in pharmacy. however, a more detailed description of their components and the nitty gritty of their application in pharmacy practice are available elsewhere within this textbook. the use of simulated client or simulated patient (mystery shopper) method to assess practices or behaviors in pharmacy practice has received much attention in recent times (watson et al., (watson et al., , . "a simulated patient is an individual who is trained to visit a pharmacy (or drug store) to enact a scenario that tests a specific behavior of the pharmacist or pharmacy staff" (watson et al., ) . a review by watson et al. demonstrated the versatility and applicability of this method to pharmacy practice research in both developing and developed countries (watson et al., ) . the investigators also identified some important characteristics that should be taken into consideration in designing studies that use this technique. this method can be used to assess wide range of cognitive pharmacy services including counseling and advice provision, treatment of minor ailments, provision of nonprescription medicines, and public health pharmacy, among other things. this method can be a robust and rigorous method of assessing pharmacy practice if used appropriately (watson et al., ; xu et al., ) . more recent developments have documented that the simulated patient methods have been used to provide formative feedback in addition to assessing practice behavior of pharmacists and their staff (xu et al., ) . in a case example, a group of investigators evaluated qatari pharmacists' prescribing, labeling, dispensing, and counseling practices in response to acute community-acquired gastroenteritis (ibrahim et al., ) . in another example, the investigators documented the state of insomnia management at community pharmacies in pakistan (hussain et al., ) . [ ( f i g u r e _ ) t d $ f i g ] figure quasi experimental study design. evidence in healthcare suggests that understanding consumers' preferences can help policy-makers to design services to match their views and preferences (ryan, ) . traditionally, studies to understand patients' and consumers' preferences for pharmaceutical services used opinion or satisfaction survey instruments. nevertheless, such satisfaction surveys lack the ability to identify the drivers of satisfaction or the relative importance of the different characteristics of the service (vass et al., ) . discrete choice experiments are a novel survey-based method in pharmacy that are predicated on economic theories that allow systematic quantification of preferences to help identify which attributes of a good or service consumers like, the relative value of each attribute, and the balance between the different attributes (naik panvelkar et al., ; ryan, ; vass et al., ) . in-depth description of this method and its essential elements are described in another chapter in the encyclopedia. qualitative research methodology is applied to investigate a problem that has unmeasurable variables, to get a comprehensive understanding of the topic, through discussing it with the involved individuals, and to recognize the natural context in which the investigated issue takes place (creswell, ) . the use of qualitative research methodology is becoming increasingly common across diverse health-related disciplines, including pharmacy practice. this is because of its ability to describe social processes and behaviors associated with patients or healthcare professionals, which strengthen the research impact (mclaughlin et al., ) . therefore, pharmacy researchers and practitioners need to be better oriented to qualitative research methods (behar-horenstein et al., ) . in the following section, interpretative frameworks and philosophical orientations, methodologies, data collection and analysis methods, approaches to ensure rigor, and ethical considerations in qualitative research are briefly discussed (cohen et al., ; creswell, ) . interpretative frameworks are the conceptual structures for comprehension, which form researcher's reasoning and views of truth and knowledge (babbie, ) . different scholars have categorized qualitative research paradigms or interpretative frameworks differently. the following are examples of interpretative framework categories that are used in health science research based on the categorization of creswell ( ): ( ) social constructivism (interpretivism) framework; ( ) post-positivism framework; ( ) transformative, feminist, critical frameworks and disabilities theories; ( ) postmodern frameworks; ( ) pragmatism frameworks. philosophical assumptions are theories and perspectives about ontology, epistemology, axiology, and methodology, which underpin the interpretative frameworks selected by qualitative researchers (cohen et al., ) . as with interpretative framework, there are numerous means to categorize the philosophical assumptions that are folded within interpretative framework. the following are explanations of philosophical assumptions based on the categorization of creswell ( ): . ontological assumptions, which define the nature of reality . epistemological assumptions, which clarify means for knowing reality . axiological assumptions, which explain the role and influence of researcher values . methodological assumptions, which identify approaches to inquiry it is important that a qualitative researcher understands how interpretative frameworks (e.g., social constructivism, postpositivism, and pragmatic interpretative frameworks) are differentiated because of their underpinning philosophical assumptions (i.e., ontological, epistemological, axiological, and methodological assumptions). it is important that qualitative researchers understand the differences between the characteristics of the five qualitative approaches to inquiry, in order to select an approach to inquiry and attain methodological congruence (creswell, ) . the five approaches to qualitative research inquiry are: a. narrative research: describes participants' written and spoken stories about their experiences with a phenomenon being investigated, while considering the chronological connection of the phenomenon's series of events (anderson and kirkpatrick, ; creswell, ; czarniawska, ) . b. phenomenological research: describes the essence of participants' common experiences of a phenomenon, so that the description is a general essence rather than an individual experience (creswell, ; giorgi, ; moustakas, ) . c. grounded theory research: aims to generate a theory grounded in participants' data that conceptually explain a social phenomenon, which could involve social processes, or actions or interactions (creswell, ; strauss and corbin, ; woods et al., ) . d. ethnographic research: involves describing the shared patterns of values, behaviors, and beliefs of culture-sharing participants (creswell, ; harris, ; rosenfeld et al., ). e. case study research: provides an in-depth examination of a real-life contemporary phenomenon that researchers cannot change over time, to illustrate the significance of another general topic (baker, ; creswell, ; de leó n-castañ eda et al., ; mukhalalati, ; yin, ) . . data collection methods data collection tools in qualitative research can be categorized into the following fundamental categories (creswell, ): a. observation b. documents c. individual semi-structured interviews d. focus groups (fgs) e. audio-visual materials f. emails chat rooms, weblogs, social media, and instant messaging. a. topic guides: topic guides guide the discussions in focus groups and individual interviews, and contain open-ended questions and probes, to enable the researcher to understand the complete picture, based on participant views and experiences. they are developed based on the literature review, aim and objectives, research questions, and propositions (kleiber, ) . b. audio recording of fgs and interviews: audio recording of discussions that take place in interviews and fgs is essential for managing and analyzing data, and for increasing the accuracy of data collection and analysis, and ultimately enhancing the dependability and credibility of the research (rosenthal, ; tuckett, ) . c. transcription of fgs and interviews recording: verbatim transcription refers to the word-for-word conversion of oral words from an audio-recorded format into a scripted text format. transcribing data is considered as the first data reduction step because it generates texts that can be examined and rechecked (miles et al., ; grossoehme, ) . . data analysis data analysis comprises several fundamental steps, including reading the transcribed text, arranging data, coding data deductively based on prefigured themes or inductively to produce emergent themes, and then summarizing the codes into themes, and finally presenting the analyzed data as results (cohen et al., ; crabtree and miller, ; pope et al., ) . the most commonly used data analysis methods in health science research are: a. thematic analysis thematic analysis is characterized by identifying, analyzing, and reporting themes that are available in the data (braun and clarke, ; castleberry and nolen, ) . b. content analysis content analysis comprises systematic coding followed by quantification of the analyzed data in a logical and unbiased way (berelson, ; vaismoradi et al., ) . c. discourse analysis discourse analysis emphasizes the core format and the structure of texts to examine the assumptions and concealed aspirations behind discourses (brown and yule, ; gee, ) . qualitative research validation involves ensuring the rigor of the utilized data collection, management, and analysis methods, by utilizing approaches to ensure the quality. in pharmacy practice research, closs ( a, b) argued that quality in qualitative research topic has not been discussed widely in the literature, and therefore closs ( a, b) suggested using several trustworthiness criteria to ensure the rigor of qualitative study. the trustworthiness criteria for ensuring quality in qualitative research (lincoln and guba, ) are: . the trustworthiness criteria a. credibility this criterion aims to ensure that the results are true and increases the possibility that the conclusions are credible (cohen and crabtree, ) . this criterion aims to indicate that the research results are repeatable and consistent, in order to support the conclusions of the research (cohen and crabtree, ) . c. confirmability this criterion aims to confirm the neutrality in interpretation by ensuring that the perspectives of participants, not the bias of researchers, influence the results (krefting, ) . d. transferability this criterion involves identifying the contexts to which the study results can be generalized, and indicating if the study conclusions can be applied in similar setting (yin, ) . reflexivity implies revealing and evaluating the effect and biases that researchers can possibly bring to research process, by explaining the researcher's opinion, feelings, and experience with the phenomenon in question, and explaining the influence of this experience on research methods, findings, and write-ups (creswell, ; krefting, ; lincoln and guba, ) . obtaining an ethical approval from the institutional review board (irb) is required before conducting the qualitative research (creswell, ) . the key ethical issues that need to be considered are: a. informed consent and participant information leaflet informed consent refers to the decision taken by a competent individual to voluntarily participate in a research, after adequately understanding the research. participant information leaflet is usually distributed to participants before they consent to participate in the research to clarify them the voluntary nature of research participation, the aim and objectives of the research, the rights of the respondents and the potential risks and harms, the data collection, management and storage conditions, and the right of participants to withdraw from the research (jefford and moore, ) . b. anonymity and confidentiality the anonymity is usually ensured by not disclosing names of participants and by utilizing a code system to identify them during data collection, management, analysis, and in the writing up of the research. the confidentiality of participants and data is ensured by using a code system to identify participants, and by storing all data in a locked cabinet and a password-protected computer for a specified period of time (creswell, ) . c. power relations in qualitative research settings power imbalance is caused by the fact that participants have the experience about the investigated phenomenon, and researchers need to obtain information about these experiences. the power imbalance is usually associated with interaction between the researcher and participants during recruitment stage, and during data collection, analysis, interpretation, and validation stages. hence, researchers should take suitable measures at each stage to decrease the influence of possible power imbalance, and should enhance trust with participants (karnieli-miller et al., ; yardley, ) . research studies in pharmacy practice usually utilize single-method research designs. however, often these report numerous limitations and may not adequately answer the research question. therefore, the combination of more than one research method to answer certain research questions has become increasingly common in pharmacy practice research (ryan et al., ) . mixed methods research design is now a popular and widely used research paradigm in pharmacy practice research fields (hadi et al., (hadi et al., , closs, a, b; ryan et al., ) . mixed methods research allows the expansion of the scope of research to offset the weaknesses of using either quantitative or qualitative approach alone (creswell et al., ; hadi et al., ; closs, a, b; pluye and hong, ) . typically, qualitative and quantitative data are collected concurrently or sequentially in order to increase the validity and the comprehensiveness of the study findings (creswell et al., ; hadi et al., ; closs, a, b; pluye and hong, ; ryan et al., ) . the mixed method approach provides an expanded understanding of phenomenon under investigation through the comparison between qualitative and quantitative data (hadi et al., ; closs, a, b; pluye and hong, ) . this section provides an overview and application of mixed method research in pharmacy practice. however, considerations in selecting, designing, and analyzing mixed methods research studies as well as the various typologies of mixed methods research are discussed elsewhere. johnson et al. ( ) proposed the following definition for mixed methods research: "the type of research in which a researcher or team of researchers combines elements of qualitative and quantitative research approaches (e.g., use of qualitative and quantitative viewpoints, data collection, analysis, inference techniques) for the broad purpose of breadth and depth of understanding and corroboration." mixed methods design allows the viewpoints of participants to be reflected, enables methodological flexibility, and promotes multidisciplinary teamwork (ryan et al., ) . furthermore, the approach allows a more holistic understanding of the research question. however, its major limitations include: need for wide range of research expertise across the research team members, highly labor-intensive, and the complexity of data integration. scholars believe that it is challenging to provide researchers with a step-by-step guide on how to undertake a mixed methods study and that this is driven by the specific research question (ryan et al., ) . nevertheless, the investigator should precisely determine the type of qualitative and quantitative methods to be employed, the order of data collection to be undertaken, the data collection instruments to be used, and the method of data analysis (ryan et al., ) . this approach encompasses a synthesis of findings from both quantitative and qualitative components, which is achieved through integration of the findings from each approach (hadi et al., ; closs, a, b; pluye and hong, ) . different models or typologies for mixed methods research have been described in the literature. the most common typologies used in pharmacy practice and health services research include: concurrent or convergent parallel design, exploratory sequential design, explanatory sequential design, and the embedded design (hadi et al., ; pluye and hong, ) . scholars believe that there are several factors to consider when selecting the typology or model of mixed methods research to use. these factors include: the order of qualitative and quantitative data collection (concurrent vs. sequential); priority of data (i.e., which type of data has priority between quantitative and qualitative data); purpose of integration of the data (e.g., triangulation); and number of data strands (hadi et al., ; pluye and hong, ) . in mixed methods research, integration of qualitative and quantitative findings is critical, and this research approach does not simply involve the collection of these data (ryan et al., ) . • in the era of evidence-based practice, it is not sufficient to propose new pharmacy services or roles without evidence of their benefit. • new pharmacy services and new roles must be proven to be feasible, acceptable, beneficial, and cost-effective. • practice-based research provides such evidence and can inform policy, confirm the value of the new service, and change practice. • various study designs, including, but not limited to experimental, quasi-experimental, observational, qualitative, and mixedmethods designs, have been used in pharmacy practice research. • pharmacy practice researchers need to be competent in the selection, design, application, and interpretation of these methodological and analytical approaches. • the choice of any study design in pharmacy practice research is driven by the expertise of the investigator, type of research question or hypothesis, data availability, time orientation, ethical issues, and availability of funding. there is a great demand for innovation and quality in pharmacy practice. these can be achieved partly through robust and welldesigned pharmacy practice research. pharmacy students, practitioners, educators, and policy-makers are exposed to a variety of research designs and methods. we need to have the best evidence (e.g., in policy, regulation, practice) for making decisions about the optimal research design that ensures delivering an ultimate pharmacy practice and a quality patient care. pharmacy practice research the canadian pharmacists association defines pharmacy practice research as a component of health services research that focuses on the assessment and evaluation of pharmacy practice (koshman and blais, ) . quantitative research is simply defined as "a research in which things are counted." these things might be people, medicines, opinions, behaviors, etc. "while qualitative research is very useful for describing phenomena in depth (particularly motivations, feelings, understandings), quantitative research can tell us how common and widespread the phenomena are" (green and norris, ) . qualitative research qualitative research within the health sciences has developed as a means to gather an in-depth understanding of human behavior, as well as to find the underlying reasons, attitudes, and motivations that govern such behavior (kaae and traulsen, ) . mixed methods research a mixed methods research typically involves both the components of qualitative and quantitative research approaches embedded within a single study or research program for the broad purpose of breadth and depth of understanding and corroboration (creswell et al., ; johnson et al., ; tashakkori and creswell, ) . observational studies observational studies involve merely observing the study subjects in a noncontrolled setting, without investigator intervention or manipulating other aspects of the study in order to determine an association between an exposure and an outcome of interest. experimental studies in experimental design (also known as interventional design), the investigator performs an intervention and evaluates cause and effect relationships between exposure to the intervention and an outcome of interest . subjects are typically randomly assigned into experimental and control groups. quasi-experimental studies quasi-experimental designs are studies that aim to evaluate interventions, but that do not use randomization and at times noncontrolled (harris et al., ; krass, ) . similar to true experimental studies, quasiexperiments aim to determine causality between an intervention and an outcome of interest. narrative interviewing exclusive and combined use of statins and aspirin and the risk of pancreatic cancer: a case-control study pharmacists' involvement in and attitudes toward pharmacy practice research: a systematic review of the literature the contribution of case study research to knowledge of how to improve quality of care perceptions of pharmacy faculty need for development in educational research the need for pharmacy practice research spironolactone for nonresolving central serous chorioretinopathy: a randomized controlled crossover study using thematic analysis in psychology the stepped wedge trial design: a systematic review thematic analysis of qualitative research data: is it as easy as it sounds? why have a special issue on methods used in clinical pharmacy practice research? societal perspective on access to publicly subsidised medicines: a cross sectional survey of adults in australia evaluative criteria for qualitative research in health care: controversies and recommendations research methods in education international comparison of prostate cancer mortality rates with dietary practices and sunlight levels doing qualitative research qualitative inquiry and research design: choosing among five approaches designing a mixed methods study in primary care designing a randomized blinded trial evaluating the safety of β-interferons in ms: a series of nested casecontrol studies healthcare professionals' perceptions related to the provision of clinical pharmacy services in the public health sector of mexico: a case study pharmacoepidemiology i: a review of pharmacoepidemiologic study designs pharmacoepidemiology ii: the nested case-control study-a novel approach in pharmacoepidemiologic research an introduction to discourse analysis: theory and method the theory, practice, and evaluation of the phenomenological method as a qualitative research procedure alternative trial designs and implementation issues quantitative methods in pharmacy practice research overview of qualitative research applications of mixed-methods methodology in clinical pharmacy research ensuring rigour and trustworthiness of qualitative research in clinical pharmacy mixed-methods research in pharmacy practice: basics and beyond (part ) mixed-methods research in pharmacy practice: recommendations for quality reporting (part ) the use and interpretation of quasi-experimental studies in medical informatics emics, etics, and the new ethnography. the rise of anthropological theory: a history of theories of culture. routledge and kegan paul designing cross-sectional and cohort studies assessment of disease management of insomnia at community pharmacies through simulated visits in pakistan evaluating community pharmacy practice in qatar using simulated patient method: acute gastroenteritis management improvement of informed consent and the quality of consent documents toward a definition of mixed methods research qualitative methods in pharmacy practice research power relations in qualitative research phenylpropanolamine and the risk of hemorrhagic stroke focus groups: more than a method of qualitative inquiry what is pharmacy research? can quasi experimental designs in pharmacist intervention research rigor in qualitative research: the assessment of trustworthiness potentially high-risk medication categories and unplanned hospitalizations: a case-time-control study the case-crossover design: a method for studying transient effects on the risk of acute events mixed methods: expanding research methodologies in pharmacy education qualitative data analysis: a method sourcebook examining the disconnect between learning theories and educational practices in the pharmd programme at qatar university: a case study a qualitative study of antipsychotic medication experiences of youth designing case-control studies effectiveness of influenza vaccine in the community-dwelling elderly progress report on the first sub-saharan africa trial of newer versus older antihypertensive drugs in native black patients combining the power of stories and the power of numbers: mixed methods research and mixed studies reviews qualitative research in health care: analysing qualitative data improving access to medicines for non-communicable diseases in rural india: a mixed methods study protocol using quasi-experimental design interdisciplinary medication decision making by pharmacists in pediatric hospital settings: an ethnographic study qualitative research methods: why, when, and how to conduct interviews and focus groups in pharmacy research mixed methods research in pharmacy practice discrete choice experiments in health care methodology series module : cohort studies methodology series module : cross-sectional studies patients' perceived value of pharmacy quality measures: a mixed-methods study tobacco use and risk of myocardial infarction in countries in the interheart study: a case-control study dental procedures, antibiotic prophylaxis, and endocarditis among people with prosthetic heart valves: nationwide population based cohort and a case-crossover study rigour in qualitative research: complexities and solutions: anthony g tuckett outlines the strategies and operational techniques he used to attain rigour in a qualitative research study through relying on guba and lincoln's trustworthiness criterion. research strategies such as use of personal journals, audio recording and transcript auditing, and operational techniques including triangulation strategies and peer review, are examined content analysis and thematic analysis: implications for conducting a qualitative descriptive study discrete choice experiments of pharmacy services: a systematic review simulated patients in the community pharmacy setting -using simulated patients to measure practice in the community pharmacy setting a systematic review of the use of simulated patients and pharmacy practice research acid-suppression medications and bacterial gastroenteritis: a populationbased cohort study communities of practice and the construction of the professional identities of nurse educators: a review of the literature haemorrhagic-fever-like changes and normal chest radiograph in a doctor with sars a systematic review of simulated-patient methods used in community pharmacy to assess the provision of non-prescription medicines dilemmas in qualitative health research qualitative case study methodology: study design and implementation for novice researchers transforming qualitative information: thematic analysis and code development avoiding common pitfalls in qualitative data collection and transcription the enlightened eye: qualitative inquiry and the enhancement of educational practice research methods and methodologies in education postmenopausal hormone use and secondary prevention of coronary events in the nurses' health study. a prospective, observational study is verbatim transcription of interview data always necessary? qualitative research methods for medical educators holding on to the indispensable medication-a grounded theory on medication use from the perspective of persons with medication overuse headache moral dilemmas of community pharmacists: a narrative study a grounded theory of the process of adherence to oral chemotherapy in hispanic and caucasian children and adolescents with acute lymphoblastic leukemia problems of reliability and validity in ethnographic research improving accuracy of transcripts in qualitative research focus groups as qualitative research the contracted relationship: ensuring protection of anonymity and confidentiality conducting small-scale investigations in educational management. nunkoosing, k., . the problems with interviews learning how to lead through engagement with enquiry based learning as a threshold process: a study of how post-graduate certificate in education healthcare professional students learn to lead data analysis in health-related qualitative research essentials of nursing research: appraising evidence for nursing practice determining counselling communication strategies associated with successful quits in the national health service community pharmacy stop smoking programme in east london: a focused ethnography using recorded consultations pharmacists in general practice: a qualitative interview case study of stakeholders' experiences in a west london gp federation quality of qualitative research in the health sciences: analysis of the common criteria present in assessment guidelines by expert users i did not want to take that medicine": african-americans' reasons for diabetes medication nonadherence and perceived solutions for enhancing adherence understanding the meaning of medications for patients: the medication experience doing qualitative research: a practical handbook informed consent: ethical implications for physiotherapy transparency in transcribing: making visible theoretical bases impacting knowledge construction from open-ended interview records i don't think i'd be frightened if the statins went': a phenomenological qualitative study exploring medicines use in palliative care patients, carers and healthcare professionals key: cord- -cynpob b authors: godakova, svetlana a.; noskov, anatoly n.; vinogradova, irina d.; ugriumova, galina a.; solovyev, andrey i.; esmagambetov, ilias b.; tukhvatulin, amir i.; logunov, denis y.; naroditsky, boris s.; shcheblyakov, dmitry v.; gintsburg, aleksandr l. title: camelid vhhs fused to human fc fragments provide long term protection against botulinum neurotoxin a in mice date: - - journal: toxins (basel) doi: . /toxins sha: doc_id: cord_uid: cynpob b the bacterium clostridium botulinum is the causative agent of botulism—a severe intoxication caused by botulinum neurotoxin (bont) and characterized by damage to the nervous system. in an effort to develop novel c. botulinum immunotherapeutics, camelid single-domain antibodies (sdabs, vhhs, or nanobodies) could be used due to their unique structure and characteristics. in this study, vhhs were produced using phage display technology. a total of different monoclonal vhhs were selected based on their comlementarity-determining region (cdr ) sequences. different toxin lethal dose (ld( )) challenges with each selected phage clone were conducted in vivo to check their neutralizing potency. we demonstrated that modification of neutralizing vhhs with a human immunoglobulin g (igg) fc (fragment crystallizable) fragment (fusionbody, vhh-fc) significantly increased the circulation time in the blood (up to days). at the same time, vhh-fc showed the protective activity times higher than monomeric form when challenged with ld( ). moreover, vhh-fcs remained protective even days after antibody administration. these results indicate that this vhh-fc could be used as an effective long term antitoxin protection against botulinum type a. botulinum neurotoxin (bont) is the strongest organic poison for humans and animals. it is produced by the anaerobic, gram-positive, spore-forming, rod-shaped bacterium clostridium botulinum [ , ] . the estimated human lethal dose is about nanograms per kilogram of bodyweight if the toxin is inhaled and one microgram if it is taken orally [ , ] . the most common forms of natural botulism are food-borne, wound, and infant [ ] . food-borne botulism occurs through contaminated food ingestion, and the case fatality rate is now about - % in developed countries (as opposed to - % before ). wound botulism occurs when an open wound is exposed to c. botulinum spores, and the case fatality is approximately - % of patients even with aggressive treatment. infant intestinal botulism occurs with spores ingested and spread over the digestive tract, as infants lack the protective flora of adults, with case fatality rate estimated to be le,ss than - %. inhalation botulism does not occur naturally and may occur in the context of a bioterrorist attack [ , ] . among the four major human pathogenic bonts (bont/a, b, e, and f), bont/a poses the most serious challenge for medical treatment due to its extremely high potency and extraordinary persistence in human patients [ ] . according to the world health organization [ ] , an antitoxin, usually based on equine antitoxin, should be administered as soon as the diagnosis is made. however, side effects, such as allergic reactions, fever serum sickness, and anaphylactic shock, often occur. in the past, at-risk persons and populations have been vaccinated with a chemically inactivated penta-serotype bont/a-e toxoid [ ] ; however, its use has been discontinued due to declining potency [ ] . in other affected individuals, botulism treatment is mainly supportive, with mechanical ventilation being the only effective life-saving treatment [ , ] . a good alternative to antitoxin serum with minimal or no side effects is treatment with monoclonal antibodies (mabs) to bont, which can be prduced in vitro. along with conventional antibodies, single-domain antibodies (sdab), also referred to as vhhs (variable domains of heavy-chain only stibodies) or nanobodies, have been widely used since their discovery in camelids along with classical immunoglobulins (igg) [ ] [ ] [ ] . the absence of light chains in igg and a lack of constant domain (ch ) in the heavy chain are the key characteristics of heavy-chain antibodies (hc-abs). therefore, an antigen-binding site of hc-abs is formed only by a single domain, which is linked directly via a hinge region to the fc (fragment crystallizable) domain. hc-abs recognize the antigen with only one special variable domain, referred to as vhh. the structure of the vhh domain resembles the vh igg domain [ ] . the complementarity-determining region (cdr ) of these hc-abs possesses the extraordinary capacity to form long finger-like extensions, which can extend into cavities on antigens, as the cdr is often much longer than that of conventional vh domains [ , ] . despite their small size (~ kda), sdabs maintain affinities and antigen-binding specificities comparable to those of full-size mabs [ ] . clear advantages include the ability to recognize hidden antigenic sites that are inaccessible for conventional antibodies due to their structure; stability over a wide range of temperatures and ph; high solubility, as well as economical and facile expression and production in large quantities in microorganisms [ , ] . several studies were conducted to test the potency of vhhs as inhibitors of viral infections [ ] and different toxins, produced by such plants and microorganisms as ricinus communis [ ] , mycoplasma hominis [ ] , clostridium tetani [ ] , clostridium difficile [ ] , crotalus durissus terrificus [ ] , and shiga toxigenic escherichia coli (stec) [ ] . it has been demonstrated that the antigen-binding region of vhhs produced by camelids showed strong anti-bont activities in animal models [ , ] . due to their unique features and high efficacy, sdabs are currently in clinical phase trials for the treatment of a wide range of diseases, including cancer, inflammation, hematology, and respiratory diseases [ ] . for instance, cablivi is a nanobody-based medicine, which has recently been approved by the food and drug administration (fda) for acquired thrombotic thrombocytopenic purpura (attp) treatment [ ] . however, the advantages of vhhs may also become their impediments. their small size enables good penetration into tissue and rapid distribution, but their short half-life in the blood circulation limits the time for interaction with hard-to-reach epitopes and for crossing endothelial barriers in sufficient amounts [ , , ] . nevertheless, due to their relatively simple structure, vhhs can be optimized by genetic engineering to obtain desired properties, including extended half-lives [ ] . one approach was to add an albumin-binding peptide to the c-terminus of vhh anti-botulinum, which increases the serum half-life of the vhh to - days [ ] . another approach was to generate virus vectors encoding chimeric proteins with one or more vhhs fused in frame to a cdna encoding the red blood cell membrane proteins glycophorin a or kell. in vitro studies and stem cell transplantation research has demonstrated that the half-life of these vhhs could be extended to several weeks with retained functionality [ ] . the vhh could also be fused to the fragment crystallizable (fc) region of an igg. such modifications, termed fusionbodies, increase the total protein complex size, as well as vhh-fc interaction with neonatal fc-receptor (fcrn), making it impossible to clear by the renal filtration system [ ] . furthermore, the attached fc fragment can promote effector functions, such as phagocytosis and cytotoxicity, thereby enhancing the neutralization of pathogen entry and replication [ ] . in this study, we focused on screening specific bont/a neutralizing alpaca vhhs from a vhh antibody immune library by phage display using bont/a and bont/a treated with dithiothreitol (bont/a-dtt) as antigens. two clones (b and g ) with high neutralizing potency at lethal dose ( ld ) were obtained in phage forms. these clones were produced as vhhs, which were then modified as dimers or fused with human igg fc-fragments (vhh-fc, fusionbody). the vhh-fc modification greatly increased their neutralizing potency and serum half-life. alpaca immunization was performed using five sequential injections with an interval of days between the first and second immunizations and days between all subsequent immunizations to generate an immune library of single-domain antibodies (figure a ). after and days post-immunization, blood was collected, and the bont/a specific antibodies titer was measured by enzyme-linked immunosorbent assay (elisa). the immune serum showed a clear response to bont/a (figure b ). before immunization, all sera showed the background levels of antibody titers. the final titer of toxin-specific antibodies in the alpaca serum was more than / , as the result of the five-fold immunization scheme. to generate a panel of single-domain antibodies to bont/a, we constructed an alpaca immune vhh library for display on the bacteriophage surface by cloning the nucleotide sequences coding for vhh repertoires of the immunized alpaca into an expression phagemid vector. after the transformation of recombinant phagemids into competent e. coli cells, a library, with a size of × phagemids, was obtained. phage display and two rounds of selection were performed for acquiring phage libraries. purified bont/a was used as an antigen for the first round. two independent second rounds were performed separately, with bont/a and bont/a-dtt used as antigens. dtt reduction was used for fragmenting toxins into two constituent parts-the heavy chain (hc) and light chain (lc) [ ] -for more uniform absorption of chains on the immunoplate's surface and to evenly distribute the epitopes on both chains. the polyclonal phage library titer for all rounds was approximately cfu (colony-forming unit)/ml (figure a) . after two rounds of selection, the specificity of each library was determined by elisa ( figure b ). to test the neutralizing potency and specificity of the polyclonal phage libraries obtained in the second round of selection, an in vivo toxin neutralization assay was performed ( figure c ). balb/c female mice were divided into groups and received one intraperitoneal injection with ld or ld bont/a after h incubation with phage libraries. previously obtained phages specific to tetanus neurotoxin (tent) were used for controls as non-specific phages. all mice in the positive control group survived, while all mice in the negative control group and the tent group died. challenging two experimental groups with ld and ld provided full and partial protection, thus allowing further selection of monoclones. a total of clones showing elisa readouts higher than . ( figure a ) and negligible reactivity with bovine serum albumin (bsa) were selected for sequencing. a total of clones with different cdr amino acid sequences were selected for further research. to test the neutralizing potency of the selected clones, an in vivo monoclonal phage neutralization assay was performed in which mice were administered the corresponding phage clone (figure b ). each group received one intraperitoneal injection with ld bont/a previously mixed with the corresponding phage clone at cfu. only the four clones (b , c , b , g ) that fully protected the mice against a ld challenge were chosen to test their protectiveness with a ld challenge. mouse groups, which received clones b and c premixed with bont/a, were partially protected, while two other groups, which received clones b and g premixed with bont/a, showed % protection. thus, clones b and g were chosen for further research as the most protective. it should be noted that the clones obtained by selection on bont/a-dtt showed a greater diversity in the cdr sequence and demonstrated neutralizing activity, unlike the clones, which were selected on bont/a. the most protective clones, b and g , were titrated in series down to cfu and introduced intraperitoneally into mice simultaneously with ld of the toxin. the neutralizing potency of both clones was comparable, with the lower threshold being cfu (figure c ). finally, both clones were tested for cross-reactivity protectiveness with another toxin serotype, bont/b. each group of mice was challenged with ld of the toxin and administered cfu/ml phages. both clones failed to protect the mice, demonstrating that the two protective clones are specific to bont/a. to increase the protectiveness of the selected clones by increasing the antibody circulation time in the blood and/or their additional functionality, two modified constructions were synthesized. one construction was a dimer form of each clone (b -dimer and g -dimer) held by a glycine-serine linker (gly ser) and expressed in the pet plasmid. the second construction was a monomer of each clone linked to a human igg fc fragment (b -fc and g -fc) (figure a ). fc-modifications have been used to increase the antibody circulation time in blood. human fc-fragments cross-react with murine fc-receptors and are capable of binding mouse fcrn [ , ] . human fc was chosen for these constructions to be used in further research and clinics. the dimers were produced and purified from the bacterial periplasmic fraction and verified by sds-page to be~ kda. vhhs fused with fc fragments were produced in chinese hamster ovary (cho-s) cells and verified by sds-page to be~ kda under reducing conditions (figure b ). to determine the polypeptide chain of the toxin that bound specific antibodies, it was denatured by dtt into its hc and lc. sds-page was performed in denaturing conditions followed by western blot. b -fc and g -fc clones were tested, and anti-human igg (fc-specific)-peroxidase antibodies ( : ) were used as detection reagents ( figure c ). these antibodies were associated with the hc ( kda) of the toxin, which corresponds to the receptor domain. an immunoblot with bont/a and bont/a-dtt (under reducing conditions) were performed as control (figure d ). we investigated the affinity of clones b and g via surface plasmon resonance (spr). amine coupling was used to immobilize bont/a and bont/a-dtt on the sensor chip. the kinetic binding on-and off-rates between the antibodies and the toxin were determined from sensorgram analysis and used to calculate the equilibrium dissociation constant (kd) ( table ). table . kinetic parameters of antibody interactions with the toxin obtained by spr (surface plasmon resonance). association (on-rate, k a ), dissociation (off-rate, k d ), maximum analyte binding capacity (r max ), equilibrium association constants (k a ), equilibrium dissociation constants (k d ), and chi for the chosen vhhs (variable domains of heavy-chain only antibodies) binding to botulinum neurotoxin (bont)/a or bont/a-dtt (dithiothreitol). bont/a-dtt k a ( /ms) for the in vivo protection analysis, modified forms of vhhs, different amounts of monomers, dimers, and vhhs fused with fc fragments were premixed with ld bont/a and injected intraperitoneally into mice ( figure a ). monomers had only partial protectiveness even at the highest amount, µg, with protectiveness gradually decreasing and failing at µg. the dimers showed a better result on average, with full protectiveness of the b -dimer at µg and partial protectiveness at lower amounts, failing at µg, as well as partial protectiveness of the g -dimer at - µg amounts, failing at µg. the best results were demonstrated by vhhs fused with fc fragments, with g -fc failing to fully protect the mice at . µg and b -fc protecting the mice at amounts as low as . µg. all forms of vhhs partially protected the mice at high amounts, whereas vhhs fused with fc fragments could protect the mice even at the lowest amounts tested ( . - . µg). to assess the circulation time of various antibody modifications in the blood after a single injection, elisa was used to measure the concentrations of g , b , g -dimer, b -dimer, g -fc, or b -fc in mouse blood taken at different time points after injection. the concentrations of g , b , as well as b -dimer and g -dimer, detected at h post-injection were only about % of the initial level observed at h. however, b -fc and g -fc modification constructs had a relatively long serum half-life. the concentrations of b -fc and g -fc h post-injection were approximately % of the initial level. moreover, two weeks after injection, the concentrations of b -fc and g -fc antibodies were approximately % of the initial level. these data are in agreement with reports on other vhhs with monomeric and fusionbody formats [ ] (figure b ). vhhs fused with fc fragments demonstrated the best protection, and the mice treated with these preparations were alive two weeks after the end of the experiment. based on the analysis of b -fc and g -fc clones' circulation time in the serum (presence of antibodies days after injection), we decided to conduct an experiment on the survival of these mice, which previously received a single injection of the vhhs with the fc fragment, with a repeated administration of only the lethal toxin dose days after the original administration. these mice were challenged with bont/a ld to examine their protection over time (figure c ). all mice that previously received - µg of vhh-fc were still fully protected. the mice which previously received - µg of the vhh-fc were partially protected ( - %) (clone b -fc) or not (clone g -fc). after two weeks, µg of the preparation had no protectiveness. we also tested the prophylactic efficacy and possible treatment of the toxin by administering the two selected fc-fused clones one hour and three hours before and after toxin challenge with ld (figure d ). both clones fully protected the mice before the toxin challenge and one hour after. three hours after toxin administration, the clones lacked protectiveness. overall, we obtained numerous clones after two rounds of biopanning; we selected clones for initial analysis based on their cdr s, chose two clones (b and g ) with the best pre-mixed results in phage form in vivo, produced them in protein form, and modified their structure and characteristics by dimerization via a (gly ser) linker and fusion to a human igg fc fragment to enhance their protective activity. we demonstrated that modification of neutralizing vhhs with an fc fragment (fusionbody) significantly increased the circulation time of antibodies in the blood. at the same time, fc fusion significantly increased the protective activity of vhh clones to more than times compared to monomeric forms. moreover, clone b -fc increased the protective activity at least , times compared to the monomeric form, with % protectiveness even at . µg. the estimated molecular ratio of the ld to . µg pre-mix was : . bont is the most potent and lethal known toxin. therefore, the development of new therapeutic agents for exposure prevention and treatment is essential. therapeutic approaches have been summarized in previous publications [ ] . along with serum therapy, specific antibodies are a promising tool for neutralizing bonts. approaches for the generation, selection, combination, and modification of mabs against bont/a have been described and tested [ ] [ ] [ ] [ ] [ ] . camelid vhhs are a popular tool for constructing recombinant antibodies to detect and neutralize a range of targets [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in particular, it has been shown that hc-only antibodies or vhhs derived from hc-only antibodies, produced by camelids demonstrate strong anti-bont activities in animal models [ , , ] . the main differences of vhhs from conventional antibodies include specific conserved amino acid substitutions in framework region (fr ) that make contact with the lc of classical antibodies, as well as a longer length, more variable amino acid composition in the cdr s, and a folded back loop [ , , ] . specific vhhs can be isolated from vhh libraries. one type of such libraries-immune libraries-can be produced based on peripheral blood lymphocytes isolated from camelids that have been immunized with an antigen of interest in a prime-boost strategy [ ] . in our study, we used a toxoid consisting of bont/a toxin and small amounts of hemagglutinin (ha) as an antigen and screened for vhhs against bont/a and bont/a-dtt using phage display technology. the neutralizing clones in our work were selected on bont/a-dtt. it should be noted that the neutralizing clones were later found in the bont/a library as well. however, there were fewer of them, so a greater number of clones had to be analyzed. overall, approximately clones from bont/a-dtt and more than clones from bont/a libraries were examined. the approach of splitting the toxin into its hc and lc enriches the neutralizing clones in the library. based on their neutralizing activity when pre-mixed with different toxin ld doses, two clones were selected. interestingly, all clones with neutralizing potency did not have the highest elisa signals. therefore, the od (optical density) of the elisa signal does not always mean high neutralizing activity and in vivo protective activity of these vhhs. the next step was to produce and purify vhhs in protein form to test their neutralizing potency in vivo. however, their protection was weak. even when pre-mixing a µg dose of these vhhs with the toxin, they had a - % protection efficiency. administration of lower doses of the preparation was non-protective, with µg lacking effectiveness. therefore, two clones-b and g -were modified to increase their half-life and protection efficacy. increasing the vhh size by oligomerization-the coupling of two or more vhhs via a specific linker-increased their protectiveness; however, even bivalent constructs were rapidly cleared [ ] . another approach was vhh fusion to an fc region of the igg molecule. constructs with fused fc fragments were previously made to extend the antibody half-life to neutralize the middle east respiratory syndrome coronavirus (mers-cov) [ , ] , target the c-x-c chemokine receptor type (cxcr ) to prevent human immunodeficiency virus (hiv- ) strain entry and replication in vitro [ ] , and increase the fc effector functions to neutralize rotavirus [ ] as well as the influenza virus ha [ ] . a recent study of modified vhhs dimers and vhhs fused to fcs targeting unique epitopes on the immunogen, composed of a portion of the central delivery domain and the entire combined repetitive oligopeptides (crops) domain of clostridium difficile type b, showed modest and much greater toxin inhibitions, respectively [ ] . it should be noted that a combined approach for efficient serum clearance of bont/a has been developed before. sepulveda et al. [ ] used single-chain variable fragments (scfvs) as protein binding agents pre-complexed with one or two single anti-tag mabs with an fc domain and tested this construction in vivo for protection and pharmacokinetics. when three or four types of such constructions were given simultaneously, mice were protected at high ld s, and bont/a was rapidly cleared from the sera. protection against bont/a light chain was observed when scfvs fused with fc fragments (scfv-fc) obtained from a macaque immune library were tested ex vivo [ ] . furthermore, the substantial contribution of the homologous fc fragment to the potency of three individual anti-botulinum mabs in antibody preparations has been demonstrated [ ] . in our work, two chosen clones were dimerized via a (gly ser) linker or fusion to human fc fragments to stabilize the molecules and slow down their clearance. these modifications led to improved protectiveness of the preparations. the dimers demonstrated protectiveness at lower doses compared to vhhs, with - % protection efficiency at and µg. clone g -dimer failed to protect the mice at µg, like the vhhs, while clone b -dimer showed partial protectiveness and failed at µg. therefore, the effective dose (ed ) for clone b -dimer was µg, and for the clone g -dimer, was µg. the best results were obtained after the fusion of the vhhs to fc fragments, which is consistent with previous research. both clones fully protected the mice at doses as low as . µg. the protection provided by g -fc was half at . µg, and the clone failed to protect the mice at . µg. b -fc demonstrated full protection down to the lowest tested dose of . µg. the dose at which its protectiveness begins to decrease was not reached. therefore, both clones with fc fragments showed at least a -fold improvement in protectiveness compared to conventional vhhs and dimers. the ed dose for clone b -fc could not be determined, while it was . µg for clone g -fc. besides, the pharmacokinetics analysis of various antibody forms showed that clones b and g containing the igg fc fragment were detected in the mouse serum days after a single injection, which is confirmed by full or partial animal protection from the challenge with the toxin days after administration of various b -fc and g -fc doses. testing the prophylaxis and possible treatment of the toxin by administering b -fc and g -fc one hour and three hours before and after toxin challenge with ld showed that both clones fully protected the mice before the toxin challenge as well as one hour after, but failed to protect the mice three hours after toxin administration, suggesting that these clones could be used for prophylaxis and emergency therapy immediately after toxin administration. this corresponds with the results obtained previously by sepulveda et al. [ ] showing that mice receiving scfvs pre-complexed with anti-tagged mabs with fc fragments were protected only two hours after intoxication, while four hours after, toxin administration lethality was only delayed. the acquisition of antibodies against bont/a and confirmation of their neutralizing potency in vivo in phage forms when pre-mixed with the toxin has provided a new method to screen for neutralizing vhhs before obtaining them in protein form, which can efficiently reduce time and material costs for their production and testing. vhhs, along with their modifications as oligomers and fusions to fc fragments of the igg, increase the range of options for bont/a targeting and neutralization by improving the blood circulation time and therapeutic potential. alpaca immunization and blood collection were performed on one clinically healthy -year-old male alpaca (vicugna pacos) on the "russian alpacas" farm, private land located in pokhodkino, moscow region, russia. this sample collection did not involve endangered or protected species, and no specific permissions were required for these locations/activities. six-week-old female balb/c mice (weighing - g) were purchased from "pushchino breeding facility" (pushchino, moscow region, russia) accredited by association for assessment and accreditation of laboratory animal care (aaalac international) and maintained at the central animal facility at the gamaleya research center of epidemiology and microbiology. mice were kept at a constant temperature ( ± • c) and relative humidity ( %) with h of artificial light per day. they were housed in individual cages ( per cage). mice were fed with dried food and water ad libitum. mice were observed every two hours post-injection except during the night for one week. the animals with characteristic symptoms of botulism, including muscle paralysis and respiratory difficulty, were euthanized by cervical dislocation. bont/a from the c. botulinum strain a was obtained from the gamaleya research center, moscow, russia collection. bont/a is very toxic; therefore, appropriate safety precautions were taken during these experiments. the neurotoxin was handled at a class biosafety cabinet. the antigen was a toxoid consisting of bont/a toxin and small amounts of ha, and . % formaldehyde was used for toxoid preparation. the treatment was carried out for seven days at • c [ ] . mice received intraperitoneal injections to determine residual toxicity, which showed its absence. before use in alpacas, the antigen was purified through a . µm filter. the final concentration was µg/ml. before immunization, the neurotoxin was inactivated by . % formaldehyde at ph = during - days. bont/a preparations were used with freund's adjuvant without pre-adsorption. alpaca immunization was performed using five sequential injections with an interval of days between the first and second immunizations and days between all subsequent immunizations. for the first injection time, µg ( ml) of the antigen and complete freund's adjuvant (sigma, st. louis, mo, usa) at the v/v ratio of : were administered. the four subsequent immunizations were performed with µg ( . ml) of the antigen and incomplete freund's adjuvant (sigma, st. louis, mo, usa) at the v/v ratio of : . small blood samples ( - ml) were collected before immunization, as well as after the third and fifth immunizations as a control. five days after the final injection, ml blood sample was collected and placed into a sterile vacuum collection tube with lithium heparin to prevent blood clotting. mrna isolation, pcr amplification, and library construction were performed, as described elsewhere [ , ] . briefly, camelid vhhs from peripheral blood b-lymphocytes (about cells/ml) of the immunized alpaca were cloned into a phen expression phagemid vector [ ] . the primer set used for pcr amplification of antibody genes appended the sfii (neb) restriction site at the -end and the noti (neb) site at its -end (table ) . recombinant phagemids were introduced into freshly prepared competent suppressor tg e. coli cells (lucigen, middleton, wi, usa). using this method, a library of × individual clones for biopanning and isolation was obtained. the bacteria from the vhh library were added to × yt medium (sigma, st. louis, mo, usa) (with µg/ml ampicillin and % glucose) and incubated at • c in a culture shaker at rpm to an od = . . km helper phages (patrick chames "antibody therapeutics and immunotargeting team" of the cancer research center of marseille) were added to the bacteria (multiplicity of infection, moi = ) and left without shaking at • c for min. the culture was centrifuged at rpm for min at • c, and the cell pellets were resuspended in × yt medium (with µg/ml ampicillin and µg/ml kanamycin) and cultured overnight at • c, in a culture shaker at rpm. the next day, the culture was centrifuged, the supernatant was purified and concentrated by % polyethylene glycol (peg) , . m nacl precipitation, and the pellet was resuspended in phosphate-buffered saline (pbs) with % glycerol. bont/a-dtt was treated with mm dtt for at least min at • c. microtiter plate wells were coated with µg of bont/a for the first round and with bont/a and bont/a-dtt for the second round in . m nahco buffer (ph = . ) at • c overnight. after rinsing three times with pbs with . % tween (tpbs), the plate was blocked with blocking buffer (tpbs with % non-fat dried milk) at • c for h. a total of~ phages were added to each well and incubated at • c for h. unbound phages were removed by washing times with tpbs. the bound phages were eluted by trypsin with a final concentration of mg/ml. tg e. coli cells at od = . were infected with the eluted phages and incubated without shaking at • c for min. after culturing the mixture in × yt agar plates (with µg/ml ampicillin and % glucose) at • c overnight, the cells were scraped. recombinant phages were obtained by packaging with km helper phages, and their titers were determined. a total of two panning rounds were performed. for a polyclonal elisa, an immunoplate (maxisorp, nunc) was coated with ng of bont/a and bont/a-dtt with . m nahco buffer (ph = . ) at • c overnight. the plate was rinsed three times with tpbs, blocked with blocking buffer at • c for h, and a total of~ phages from the starting library or the first or second rounds were added to each well and incubated at • c for h. the unbound phages were removed by washing times with tpbs. the plate wells were detected by horseradish peroxidase (hrp)-conjugated anti-m antibodies ( : ) (abcam, cambridge, uk), followed by the addition of peroxidase substrate , , , -tetramethylbenzidine (tmb) (bio-rad, hercules, ca, usa). the reaction was stopped by m h so , and the absorbance at nm was read with an iems reader mf (thermo labsystems, waltham, ma, usa). a monoclonal elisa was performed following the same protocol, with particular clones bound to bont/a and bont/a-dtt being selected, and the phage plasmids were isolated for sequencing. antibody genes in phagemids were sequenced with a primer set used for pcr according to the protocol of big dye terminator . cycle sequencing kit for the genetic analyzer applied biosystems (waltham, ma, usa). the electrophoretic dna separation was performed in cm capillaries with pop polymer. the selected vhhs expressed in phen plasmid were transformed into e. coli bl cells (neb, ipswich, ma, usa) for expression and purification. an overnight culture was obtained at • c, in a culture shaker at rpm. the next day, the cells were harvested by centrifugation, lysed by bugbuster protein extraction reagent (novagen, madison, wi, usa), and vhhs were purified from the lysate by talon superflow containing co + agarose (ge healthcare bio-sciences ab, uppsala, sweden). the eluted fraction was subjected to dialysis with visking dialysis tubing (mwco - ) (serva, heidelberg, germany). vhhs were separated by sds-page (bio-rad, hercules, ca, usa) under denaturing conditions and had an expected molecular weight of~ kda. antibody affinity was determined by surface plasmon resonance (spr) using a biacore instrument (ge healthcare bio-sciences ab, uppsala, sweden). bont/a and bont/a-dtt were immobilized on the surface of cm sensor chips in the amount of µg each in mm sodium acetate buffer ph = . using the amine coupling kit recommended by the manufacturer (ge healthcare bio-sciences ab, uppsala, sweden). vhhs ( -fold dilutions from µg down to µg) were captured on the sensor chips and submitted at a constant flow rate of µl/min with hbs-ep ( . m hepes ph . , . m nacl, mm edta, . % v/v surfactant p ) as a running buffer at • c with an injection time of min and dissociation time of min. after each injection, the chip surface was regenerated with mm tris-hcl, ph = . for s at a flow rate of µl/min. calculations were performed using biaevaluation software (ge healthcare bio-sciences ab, uppsala, sweden) with reference-subtracted fitting. the selected vhhs were held by a glycine-serine linker (gly ser) and expressed in the pet plasmid. protein expression and purification were performed as for vhhs. nucleotide sequences of vhh genes fused to the human igg fc-fragment were synthesized and cloned into the plasmid pshuttle-cmvfuse (stratagene, la jolla, ca, usa). the cho-s cell culture (thermofisher, waltham, ma, usa, r ) was transiently transfected with pfuse plasmid using the cho gro system (mirus bio, madison, wi, usa), according to the manufacturer's protocol. a plasmid carrying the green fluorescent protein gene in a % amount was used as control. the efficiency of transfection was assessed using axio imager z (carl zeiss, oberkochen, germany) and determined by the number of fluorescence cells compared to their overall amount, which was %. cells were cultured in shake flasks at rpm, % co , % humidity, at • c during transfection and • c h after the transfection for days. starting from day three, cellboosts a ( %), b ( . %) (hyclone, san angeo, tx, usa), and % sigma bioreactor feed (sigma, st. louis, mo, usa) were added each day. after days of cultivation, the culture was clarified by centrifugation at × g and cleared through a . µm filter. the antibodies were purified using protein a affinity chromatography on an akta start chromatography system (ge healthcare bio-sciences ab, uppsala, sweden), with a mabselect sure ml column (ge healthcare bio-sciences ab, uppsala, sweden), according to the manufacturer's protocol. to determine which bont/a polypeptide chain binds vhhs, the toxin was denatured by dtt into its hc and lc. sds-page was performed using a mini-protean tgx stain-free precast gel (bio-rad, hercules, ca, usa) in denaturing conditions. the separated bands were transferred onto a nitrocellulose membrane using a trans-blot turbo system (bio-rad, hercules, ca, usa). the membrane was blocked with a blocking buffer at • c for h. vhhs were diluted in the blocking buffer, added to the membrane, and incubated at • c for h. after rinsing the membrane three times with tpbs, anti-his-tag-hrp-mouse antibodies (genscript, piscataway, nj, usa) diluted : in blocking buffer were added, and the membrane was then incubated at • c for h. the membrane was rinsed three times with tpbs, followed by the addition of clarity western ecl blotting substrates (bio-rad, hercules, ca, usa) for detection. the membrane was visualized on an amersham imager (ge healthcare, buckinghamshire, uk). the neurotoxin as a multi-oligomeric complex toxin with has and ntnha (non-toxic non-hemagglutinin) ( kda) was used for immunization. toxins were prepared and purified as follows. the strain was cultivated under anaerobic conditions for five days. bacterial cells were separated by centrifugation at × g for min at • c. the proteins from the culture filtrate were concentrated by acid precipitation at ph = . for min. the precipitate was separated by centrifugation at , × g for min at • c and dissolved in mm citrate-phosphate buffer with ph = . . gel filtration s and ion-exchange chromatography on akta start chromatography system (ge healthcare bio-sciences ab, uppsala, sweden) on de cellulose (pharmacia, uppsala, sweden) were then carried out. the toxin ( - %, kda) was purified by additional de cellulose chromatography (pharmacia, uppsala, sweden) in borate buffer ph = and eluted by mm nacl. non-sorbed material containing specific antigenic and biological activity was used as bont complex with ha. the specific antigenic activity was a positive reaction with monospecific antibodies to bont/a, ha, and ntnha (gamaleya laboratory of clostridiosis and commercial preparation of scientific centre for expert evaluation of medicinal products russian federation). phages with titers - cfu/ml were prepared, as described above, and mixed with standard saline solution. vhh proteins at different amounts ranging from µg to . µg were prepared, as described above, and mixed with standard saline solution. balb/c - g female mice were divided into groups of four, including the positive and negative control groups. phages or vhh proteins were premixed with the appropriate toxin ld ( ld~ pg bont/a) and incubated for h at • c. all mice received one intraperitoneal µl injection of phages or proteins premixed with various ld s of the toxin and were observed once a day for one week. a specific pathological pattern was observed in sick mice, which is the pathological pattern with dystonia's abdominal muscles (waistline increasing) and death at - pg that is neutralized by monospecific antibodies to bont/a. the positive control group received the rabbit antitoxin igg (gamaleya laboratory of clostridiosis), and the negative control group received a standard saline solution. a group of five six-week-old female balb/c mice was intravenously (i.v.) injected with µg g , b , g -dimer, b -dimer, g -fc, or b -fc into the tail vein. blood was collected from the facial vein at , , , , , , , , h time points. sera were separated and stored at − • c until further use. concentrations of the injected antibody molecules in the above-collected samples were measured by elisa. for elisa, bont/a was coated on microtiter plates (nunc) overnight at • c at ng/well in mm bicarbonate buffer. after washing three times with tpbs, plates were blocked with % dry milk in pbs for one hour at • c. then, × diluted sera were added to the wells, followed by a one-hour incubation. mouse anti-his-tag antibody [hrp] ( : ) (genscript, piscataway, nj, usa) was used to detect monomers and dimers of g and b antibodies in the mouse sera. goat anti-human igg bacterial toxins-a table of lethal amounts clostridial neurotoxins botulinum toxin as a biological weapon-medical and public health management a camelid single-domain antibody neutralizes botulinum neurotoxin a by blocking host receptor binding the effect of formalin on botulinum toxins a, b and c notice of cdc's discontinuation of investigational pentavalent (abcde) botulinum toxoid vaccine for workers at risk for occupational exposure to botulinum toxins clostridium botulinum-international programme on chemical safety-bacteria; world health organization current strategies for designing antidotes against botulinum neurotoxins neutralizing antibodies of botulinum neurotoxin serotype a screened from a fully synthetic human antibody phage display library the european antibotabe framework program and its update: development of innovative botulinum antibodies hammers, r. naturally-occurring antibodies devoid of light-chains properties, production, and applications of camelid single-domain antibody fragments single domain antibodies: promising experimental and therapeutic tools in infection and immunity nanobodies: natural single-domain antibodies single-domain antibodies as therapeutics against human viral diseases development of antiricin single domain antibodies toward detection and therapeutic reagents genetic passive immunization with adenoviral vector expressing chimeric nanobody-fc molecules as therapy for genital infection caused by mycoplasma hominis increasing the potency of neutralizing single-domain antibodies by functionalization with a cd b/cd binding domain selection of nanobodies that block the enzymatic and cytotoxic activities of the binary clostridium difficile toxin camelid single-domain antibodies (vhhs) against crotoxin: a basis for developing modular building blocks for the enhancement of treatment or diagnosis of crotalic envenoming structural basis for the specific neutralization of stx a with a camelid single domain antibody fragment a single-domain llama antibody potently inhibits the enzymatic activity of botulinum neurotoxin by binding to the non-catalytic α-exosite binding region botulinum neurotoxins and botulism: a novel therapeutic approach camelid single-domain antibodies: historical perspective and future outlook current approaches and future perspectives for nanobodies in stroke diagnostic and therapy fusion of higg -fc to in-anti-amyloid single domain antibody fragment vhh-pa h prolongs blood residential time in app/ps mice but does not increase brain uptake prolonged prophylactic protection from botulism with a single adenovirus treatment promoting serum expression of a vhh-based antitoxin protein genetically engineered red cells expressing single domain camelid antibodies confer long-term protection against botulinum neurotoxin beyond binding: antibody effector functions in infectious diseases botulinum neurotoxins: biology, pharmacology, and toxicology the neonatal fc receptor (fcrn) binds independently to both sites of the igg homodimer with identical affinity human igg subclass cross-species reactivity to mouse and cynomolgus monkey fcγ receptors differential tumor-targeting abilities of three single-domain antibody formats characterization of neutralizing antibodies and identification of neutralizing epitope mimics on the clostridium botulinum neurotoxin type a potent neutralization of botulinum neurotoxin by recombinant oligoclonal antibody characterization of botulinum neurotoxin type a neutralizing monoclonal antibodies and influence of their half-lives on therapeutic activity characterization of the monoclonal antibody response to botulinum neurotoxin type a in the complexed and uncomplexed forms development of germline-humanized antibodies neutralizing botulinum neurotoxin a and b llama-derived single-domain antibodies for the detection of botulinum a neurotoxin camelid single domain antibodies (vhhs) as neuronal cell intrabody binding agents and inhibitors of clostridium botulinum neurotoxin (bont) proteases antigen recognition by single-domain antibodies: structural latitudes and constraints single-domain antibodies and their formatting to combat viral infections engineered antibody fragments and the rise of single domains chimeric camel/human heavy-chain antibodies protect against mers-cov infection a novel nanobody targeting middle east respiratory syndrome coronavirus (mers-cov) receptor-binding domain has potent cross-neutralizing activity and protective efficacy against mers-cov nanobody-fc constructs targeting chemokine receptor cxcr potently inhibit signaling and cxcr -mediated hiv-entry and induce antibody effector functions intracellular antibody-bound pathogens stimulate immune signaling via the fc receptor trim universal protection against influenza infection by a multidomain antibody to influenza hemagglutinin neutralization of clostridium difficile toxin b with vhh-fc fusions targeting the delivery and crops domains efficient serum clearance of botulinum neurotoxin achieved using a pool of small antitoxin binding agents development of neutralizing scfv-fc against botulinum neurotoxin a light chain from a macaque immune library role of homologous fc fragment in the potency and efficacy of anti-botulinum antibody preparations monoclonal antibodies to type a, b, e and f botulinum neurotoxins selection and identification of single domain antibody fragments from camel heavy-chain antibodies basics of antibody phage display technology multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (fab) heavy and light chains this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest. key: cord- - xk gln authors: baier, claas; linderkamp, christin; beilken, andreas; thol, felicitas; heuser, michael; ebadi, ella; ganzenmueller, tina; heim, albert; bange, franz-christoph title: influenza and respiratory syncytial virus screening for the detection of asymptomatically infected patients in hematology and oncology date: - - journal: gms hyg infect control doi: . /dgkh sha: doc_id: cord_uid: xk gln introduction: respiratory syncytial virus (rsv) and influenza virus infections are a significant healthcare risk for immunocompromised patients. in addition to community onset, nosocomial acquisition and transmission may also occur. detection of asymptomatic shedders (e.g., patients in the incubation period or immunosuppressed long term shedders) facilitates control of nosocomial transmission. methods: to strengthen the existing infection control concept, a pcr-based screening for rsv and influenza virus was implemented for all patients lacking respiratory symptoms (asymptomatic patients) who were hospitalized on an adult and a pediatric hemato-oncological ward. laboratory results of this screening were analyzed retrospectively. results: respiratory specimens were obtained for screening from patients ( % were years and younger) from december to april . in patients without respiratory symptoms, either influenza virus or rsv infection was found, resulting in a detection rate of about %. in patients, the infection was presumably detected during the incubation period, because an increase of viral load was observed in subsequent specimens. positive screening results facilitated timely implementation of adequate infection control precautions. nosocomial clusters of rsv or influenza were not detected during the screening period on the two wards. conclusion: the seasonal screening program expanded our existing infection control concept in terms of patients lacking respiratory symptoms who shed influenza virus or rsv. it enabled us to identify rsv or influenza infections in patients lacking respiratory symptoms in a -month period and thus to rapidly take isolation precautions. influenza virus (family orthomyxoviridae) and respiratory syncytial virus (rsv; family pneumoviridae) are both single-strand rna viruses and cause upper and lower respiratory tract infections in children and adults [ ] , [ ] , [ ] , [ ] . influenza virus and rsv are spread by droplet and contact transmission. therefore, nosocomial acquisition may occur due to exposure to another infected patient, visitor or healthcare workers (hcws), as well as by contact with contaminated, inanimate surfaces. immunocompromised patients with underlying hemato-oncological disorders or cancer are at risk for increased mortality, especially in the case of lower respiratory tract infection due to these viruses [ ] , [ ] , [ ] . both community and nosocomial acquisition of influenza and rsv are possible in this patient group. nosocomial outbreaks in oncologic care facilities have been described [ ] , [ ] in spite of multiple infection control measures to prevent nosocomial transmission. these measures include stringent hand hygiene, patient isolation, and use of barrier precautions such as surgical masks or vaccination (influenza) [ ] , [ ] , [ ] , [ ] . during a localized nosocomial cluster with rsv at our clinic in the winter season / , we introduced a rt-pcr-based screening of asymptomatic patients for rsv and influenza virus which contributed to the successful control of the rsv cluster [ ] . to strengthen our existing infection control measures in hematology and oncology, we subsequently implemented a systematic influenza and rsv screening of patients lacking respiratory symptoms on a pediatric and an adult hemato-oncological ward as a prophylactic infection control measure in the following winter ( / ). this screening intended to identify patients shedding rsv or influenza virus without signs of disease and -in case of a positive screening test -to establish infection control measures similar to those used for typical symptomatic (e.g. cough, sneezing, fever, respiratory distress) patients with rsv or influenza. in this paper, we describe the laboratory results of this prophylactic screening program for asymptomatic patients (season / ) and discuss its value for infection control in hematology and oncology. the screening program for rsv and influenza was implemented on two wards located in the department of pediatric hematology and oncology and in the department of hematology, hemostasis, oncology and stem cell transplantation at hannover medical school. these departments are tertiary referral centers for children and adults, respectively, with hematologic and solid neoplasia. the pediatric ward contains single (one-bed) and twobed rooms. the adult ward has single (one-bed) rooms, two-bed rooms, and four-bed rooms. all single rooms on the pediatric ward and single rooms on the adult ward have an anteroom and high-efficiency particulate air filtration with the air flow directed to the hallway. single rooms are usually reserved for patients with severe immunosuppression or palliative settings. the wards are serviced by permanent healthcare workers (hcws) and housekeeping staff. parents are allowed to stay overnight with their children on the pediatric ward. during their stay on the ward, parents are permitted to wear street clothes. however, a regular change of their clothing is recommended. the pcr-based seasonal screening for rsv and influenza started in the second half of december and ended at the beginning of april . the start and finish of the screening period were set jointly by the infection control staff, virologists and the clinicians involved based on available epidemiological data. on the adult ward, the screening was performed upon admission (admission screening) and once weekly (prevalence screening). on the pediatric ward, the pcr-based screening was performed once weekly. respiratory material was obtained from all patients lacking respiratory symptoms (asymptomatic patients). prophylactic prevalence screening was continued as long as patients stayed on the wards. patients with any symptoms of respiratory disease, e.g., cough, sneeze, fever or oxygen need (symptomatic patients), received a full diagnostic virology panel including influenza virus and rsv, and other respiratory viruses such as human metapneumovirus, adenovirus, parainfluenza virus, coronavirus and rhino-/enterovirus. in order to guarantee high adherence to the screening program, repeated audits and feedback talks took place. specimens were collected as a combined nasopharyngeal swab or pharyngeal lavage (oral wash) with sterile saline and were processed at the institute of virology. for realtime rt-pcr, rna was extracted using a qiaamp viral rna mini kit in a qiacube according to the manufacturer's instruction (qiagen, hilden, germany). cdna synthesis, amplification and detection of nucleic acid were performed in an applied biosystems ® real-time pcr system (life technologies, carlsbad, california, usa) using a commercially available one-step real-time rt-pcr kit (rsv/hmpv r-gene ® pcr kit, biomérieux, nürtingen, germany) according to the manufacturer's instructions, and an influenza a/b multiplex real time rt-pcr [ ] . the influenza rt-pcr differentiates between types a and b. for semi-quantitative comparison of viral loads with follow-up specimens, the cycle threshold (ct) value of the real time pcr was used. a decrease of the ct by ≥ values (usually equivalent to a more than -fold increase of viral load) was considered as a significant increase of viral load. infection control interventions for asymptomatic patients with rsv or influenza virus, detected by the screening infection control recommendations for asymptomatically infected patients with rsv and influenza virus were the same as for symptomatic patients. they were labeled in an electronic alert system, and it was recommended that the patients each be placed in a single (one-bed) room. each positive pcr test was electronically communicated to the infection control staff. contact and droplet precautions (surgical mask, gown, gloves) were mandatory for hcws and all visitors entering the room of affected patients. the patients were restricted to their single (onebed) room and were trained in hand hygiene. if they had to leave the room, they wore a surgical mask. two negative rt-pcr results at a minimum -day interval were required to stop all isolation precautions. hcws were strongly encouraged to let themselves be vaccinated against influenza before and during the winter season / . the vaccination rate among hcws for influenza was about % on the pediatric ward and about % percent on the adult ward. from december to april , asymptomatic patients were screened. ( %) of these patients were aged or younger and were screened on the pediatric ward. of the remaining adult patients, ( . %) received swabs on the emergency ward, whereas patients were screened exclusively on the adult hematooncological ward. overall, screening samples were obtained, % of which (n= ) were from pediatric patients and % from adults (n= ). the mean screening sample number per patient was . for pediatric patients and . for adults. the majority ( %) of the screening samples were nasopharyngeal swabs (n= ). samples were pharyngeal washes and were exclusively obtained from adult patients. altogether, asymptomatic patients tested positive either for influenza or rsv in a screening sample, i.e., a combined detection rate of %. patients ( adults and one child) tested positive for influenza type a. patients ( adults and children) were tested positive for rsv. co-infections with influenza virus and rsv were not detected. an overview is shown in table . interestingly, in patients ( influenza virus patients and rsv patient), a significant increase of the viral load in samples was observed following the positive screening specimen ( table ) . course of viral load in patients who showed an increase in viral load (presumed detection during the incubation period). the patients showed an increase in viral load during their clinical course and were therefore presumably detected during the incubation period. of the ( %) asymptomatic patients were in twoand four-bed rooms at the time the screening test was reported positive ( positive for rsv, positive for influenza virus). of these patients in shared rooms were immediately moved to separate single rooms, following the infection control recommendations. patients with influenza were kept together, as they occupied the same -bed room when they simultaneously tested positive. patients with rsv and patient with influenza stayed in the shared rooms, and patients had already been discharged when the test result became available. of the asymptomatic patients were already located in single (one-bed) rooms (for reasons other than infection control) and remained in this room after virus detection. for all patients identified (whether located in private or shared rooms after positive test results were available), barrier precautions (droplet and contact) were implemented as described above (see "materials and methods" section). on both wards, all patients presenting with symptoms of acute respiratory infections were routinely tested with a broad panel for respiratory viruses (including rsv and influenza). in these patients with symptoms of acute respiratory infection, we found influenza case and rsv case on the pediatric ward and influenza case and rsv cases on the adult ward from december to april . prevalence of other respiratory viruses was low in this season on these two wards. parainfluenza case, human metapneumovirus cases and rhinovirus cases were found on the pediatric ward. on the adult ward parainfluenza case was detected. clusters of nosocomial transmission of rsv or influenza were not observed during the screening program period. respiratory tract infections caused by rsv and influenza virus are a significant healthcare risk for hospitalized and non-hospitalized patients with underlying hemato-oncological disease. during the winter season, acquisition of these viruses typically occurs in the community, and patients may be admitted with symptoms of infection or infection to a hospital. nosocomial acquisition due to transmission occurs as well [ ] , [ ] , [ ] , [ ] . recom-mendations and suggestions for controlling the spread of influenza and rsv in healthcare settings in general and especially in hemato-oncological patients exist (e.g. [ ] , [ ] ), but should be adapted to each hospital's specific needs. to extend our existing standard infection control measures, we introduced a prophylactic screening program for asymptomatic patients targeting rsv and influenza on an adult and a pediatric hemato-oncological ward. the screening was intended to detect patients that shed the virus without having signs or symptoms of disease. shedding my occur before the onset of disease (incubation period), during an asymptomatic clinical course, or following disease as a result of prolonged viral shedding, particularly in the immunocompromised host [ ] . these groups of asymptomatic patients, who often remain unnoticed, can contribute to nosocomial spread. of the asymptomatic shedders, we detected an increased viral load in patients in follow-up specimens (see table ). it is likely that these patients were in the incubation period. this group of patients is of particular interest for infection control strategies, since with increasing viral load, the risk of transmission also rises. we identified of a total of asymptomatic patients with an rsv or influenza virus infection (detection rate of about %).this means that about asymptomatic patients needed to be screened to detect one shedder of either rsv or influenza virus. other studies found a similar prevalence in asymptomatic individuals for influenza ( . %) and rsv ( . %) using rt-pcr [ ] , [ ] . campbell et al. examined samples from asymptomatic and symptomatic patients prior to hematopoietic stemcell transplantation (hsct) in a study to evaluate the clinical outcomes associated with respiratory virus detection before hsct [ ] . during a -year period, asymptomatic adult and pediatric patients were screened, but only one patient with rsv and none with influenza virus were identified. interestingly, this study found other viruses (e.g., human rhinovirus or coronavirus) in patients. these respiratory viruses were not included in our screening program, as they rarely cause severe lower respiratory tract disease. out of asymptomatic patients who tested positive for rsv or influenza virus in our study occupied shared patient rooms at the time of virus detection. the majority were moved to single rooms (one-bed rooms) or cohorting was used. droplet and contact precautions were used for all affected patients. isolation and barrier precautions regarding virus shedders is important, as transmission to roommates has been described [ ] . the transfer to single (one-bed) rooms was challenging in some cases, due to the lack of single-room availability. nonetheless, because of the high mortality rate of rsv and influenza infection in hemato-oncological patients, isolation and barrier precautions were enforced. with these measures, we aimed to lower the risk of patient-to-patient transmission by reducing the exposure of contact patients. the usage of droplet and contact precautions (such as surgical mask and gown) was also intended to reduce exposure of hcws. all hcws, whether vaccinated against influenza or not, wore masks when they performed care in patients with rsv or influenza virus. a further benefit of early detection of influenza virus is the possibility of early antiviral therapy for infected patients and prophylaxis with antiviral agents for contact patients [ ] . an additional effect of the screening program and the associated audits and feedback talks was the heightened awareness among hcws and visitors for respiratory viral infections in this highly susceptible, immunocompromised patient population. during the screening period, no nosocomial cluster of influenza virus or rsv was observed on the two wards. our investigation has limitations. first, it relied on virological laboratory data analyzed retrospectively. further information, such as the clinical course and outcome of the patients identified by screening, was not accessible for this study. we therefore could not correlate the laboratory results with the clinical course of the patients. viral loads were estimated semi-quantitatively by comparing ct values of real-time pcr between positive screening and follow-up specimens. however, this approach has been established for respiratory specimens by previous studies on influenza and various other pathogens [ ] , [ ] , [ ] , [ ] . multifaceted approaches are necessary to help to control the nosocomial spread of rsv and influenza in healthcare settings. infection control is based on standard precautions (e.g., compliance with hand hygiene, cough etiquette, etc.) together with rsv and influenza pathogenspecific precautionary measures (e.g. droplet precautions, isolation). in special high-risk settings such as hematology and oncology units, additional measures can be useful to lower the risk of hospital acquisition of rsv and influenza. therefore, we implemented a prophylactic rt-pcrbased rsv and influenza screening program targeting asymptomatic patients. this seasonal screening program enabled us to identify patients who lacked symptoms for respiratory disease, but were infected with either rsv or influenza during a period of about months. for these patients the same infection control practices were implemented as for symptomatic patients with rsv and influ-enza infection. in our view, this screening program proved useful for identifying asymptomatically infected patients with viral shedding, thus reducing the risk of transmission and potential nosocomial clusters of rsv and influenza virus on hemato-oncological wards. nevertheless, future studies on larger cohorts, including the analyses of clinical data, are necessary to further validate the use of a seasonal screening program as suggested here. competing interests respiratory syncytial virus -a comprehensive review systematic review of respiratory viral pathogens identified in adults with community-acquired pneumonia in europe community acquired respiratory virus infections in cancer patients -guideline on diagnosis and management by the infectious diseases working party of the german society for haematology and medical oncology respiratory viruses in transplant recipients: more than just a cold. clinical syndromes and infection prevention principles influenza virus infections in patients with malignancies --characteristics and outcome of the season / . a survey conducted by the infectious diseases working party (agiho) of the german society of haematology and medical oncology (dgho) consecutive yearly outbreaks of respiratory syncytial virus in a haemato-oncology ward and efficacy of infection control measures nosocomial outbreak of the pandemic influenza a (h n ) in critical hematologic patients during seasonal influenza - : detection of oseltamivir resistant variant viruses what transmission precautions best control influenza spread in a hospital? fourth european conference on infections in leukaemia (ecil- ): guidelines for diagnosis and treatment of human respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, and coronavirus effect of two-step hygiene management on the prevention of nosocomial influenza in a season with high influenza activity molecular characteristics and successful management of a respiratory syncytial virus outbreak among pediatric patients with hemato-oncological disease diagnostic approach for the differentiation of the pandemic influenza a(h n )v virus from recent human influenza viruses by real-time pcr combining highresolution contact data with virological data to investigate influenza transmission in a tertiary care hospital active surveillance for influenza reduces but does not eliminate hospital exposure to patients with influenza laboratory-based surveillance of hospital-acquired respiratory virus infection in a tertiary care hospital practical prevention of nosocomial influenza transmission, "a hierarchical control" issue the natural history of influenza infection in the severely immunocompromised vs nonimmunocompromised hosts influenza virus prevalence in asymptomatic and symptomatic subjects during pandemic and postpandemic periods respiratory syncytial virus evaluation among asymptomatic and symptomatic subjects in a university hospital in sao paulo, brazil in the period of - . influenza other respi viruses clinical outcomes associated with respiratory virus detection before allogeneic hematopoietic stem cell transplant comparison of simplexa hsv & pcr with culture, immunofluorescence, and laboratorydeveloped taqman pcr for detection of herpes simplex virus in swab specimens a comparison of nasopharyngeal and oropharyngeal swabbing for the detection of influenza virus by real-time pcr assessment of the quantitative ability of advansure tb/ntm real-time pcr in respiratory specimens by comparison with phenotypic methods comparison of real-time pcr assays for diagnosis of pneumocystis jirovecii pneumonia in human immunodeficiency virus (hiv) and non-hiv immunocompromised patients influenza and respiratory syncytial virus screening for the detection of asymptomatically infected patients in hematology and oncology the authors declare that they have no competing interests. we obtained ethical approval for this study from the ethics committee of the hannover medical school (number - ). this research did not receive any specific grant from funding agencies in the public, commercial, or not-forprofit sectors. we acknowledge the technical laboratory staff for the processing of screening specimens. all authors contributed to the manuscript according to the icmje (international committee of medical journal editors) recommendations: ah and cb were responsible for data acquisition. all authors were involved in analysis and interpretation of the data. tg and ah supervised laboratory diagnostics. cb and fcb prepared the manuscript. cb organized the drafting process. cl, ab, mh and ft implemented and supervised the screening program on the wards. all authors critically revised the manuscript and are accountable for accuracy and correctness. all authors have read and agreed to the final draft before submission. key: cord- - xslz bs authors: boroomand, zahra; jafari, ramezan ali; mayahi, mansour title: molecular detection and phylogenetic properties of isolated infectious bronchitis viruses from broilers in ahvaz, southwest iran, based on partial sequences of spike gene date: - - journal: vet res forum doi: . /vrf. . sha: doc_id: cord_uid: xslz bs infectious bronchitis (ib) is a highly contagious disease involving mostly upper respiratory tract in chickens, leading to significant economic losses in the poultry industry worldwide. one of the major concerns regarding to ib is the emergence of new types of infectious bronchitis viruses (ibvs). the purpose of this study was to identify the ibvs isolated from iranian broiler chickens with respiratory symptoms. twenty-five broiler flocks around ahwaz (southwest of iran) were examined for ibv. the specimens including trachea, lung, liver, kidney, and ceacal tonsil, were collected from diseased birds and inoculated into chicken embryonated eggs. harvested allantoic fluids were subjected to reverse transcription polymerase chain reaction (rt-pcr) using primers in order to amplify spike (s ) gene of ibv. the rt-pcr products of four ibv isolates were sequenced. the results showed that from examined flocks with respiratory disease, flocks ( . %) were positive for ibv. in phylogenetic analysis, our isolates were closely related to the qx-like viruses such as pcrlab/ / (iran), qx, hc , hc , ck/ch/gx/nn - , ck/ch/js/yc - , ck/ch/js/ / , ck/ch/js/ / (china), qx/sgk- , qx/sgk- (iraq) with nucleotide homology up to . %. this study indicates the role of ibvs in the respiratory disorders of broiler flocks located in southwest iran, and also the existence of a variant of ibv, which is distinguishable from the other iranian variants. infectious bronchitis (ib) is an acute contagious viral infection with low mortality and a significant reduction in performance in chicken. the causative agent of ib is infectious bronchitis virus (ibv) which is an enveloped, single-stranded, positive sense and rna virus belonging to the family coronaviridae, the genus gamma coronavirus. the virus consists of three important structural proteins: the nucleocapsid (n), the membrane (m), and the spike (s and s ) glycoproteins. the nucleotide sequence of the s gene is highly variable, which makes it prone to mutation and emergence of new variants of the virus. for this reason, the molecular classification of ibvs is based on the investigation of the s gene. vaccination is one of the best ways of immunization of susceptible birds. however, vaccinated flocks may experience disease involvement because there is almost no cross-protection among serotypes. consequently, the viral strains that exist in a different geographical area must first be identified and their pathogenicity should be determined in order to select an appropriate virus strain for vaccination. in iran, aghakhan et al. identified ibv by virus isolation and serological techniques. this isolate belonged to the mass serotype. in previous studies conducted to identify ibv serotypes in iran, the presence of / and massachusetts serotypes have been reported. , recently, a new isolate of ibv (irfibv ) was identified by boroomand et al. which had . % similarity to /b strains. the other ibv serotypes also exist in iran neighboring countries such as dutch strains in pakistan. in ahvaz (southwest iran), ibv vaccines including h and / are used in the vaccination program in broiler chicken flocks. however, ib continues to be responsible for the economic losses of the poultry industry in the region. the purpose of this study was to evaluate the role of ibv in broiler chicken respiratory complexes in ahvaz, and also to study the partial s sequences among field isolates of ibvs. sampling. during january to december , an attempt was made to reveal the role of ibvs in broiler chickens of ahvaz which suffered from severe respiratory distress, including nasal discharge, coughing, wet rale, lacrimation, gasping, and high mortality. twenty five broiler flocks aged - weeks old, some of which had a history of live vaccination against ibv were selected from different geographical parts of ahvaz (north, east, south and west). samples including trachea, lung, kidney, liver and cecal tonsil were obtained from dead birds, transferred in cold chain system to the faculty of veterinary medicine, shahid chamran university of ahvaz and stored at - ˚c until used. the homogeneous tissue samples were centrifuged at ˚c and , rpm for min. the supernatant was taken and antibiotics (penicillin, , iu ml - , streptomycin, μg ml - and gentamycin, μg ml - , all from sigma, st. louis, usa) were added to prevent the growth of bacteria and fungi. an amount of . ml of the suspension was inoculated into the allantoic cavity of -day-old embryonated chicken eggs. the eggs were incubated at ˚c and after hr, the allantoic fluid was harvested and examined for ibv using reverse-transcriptase polymerase chain reaction (rt-pcr). no bacterial and fungal contamination was observed in specific culture media. all experiments were carried out after approval by the animal committee of shahid chamran university of ahvaz, ahvaz, iran. extraction of rna from the allantoic fluid was carried out using rnx-plus solution (cinnagen, tehran, iran) according to manufacturer's instructions. the isolated rnas were stored at - ˚c. the cdna was synthesized using primescript tm rt reagent kit (takara, tokyo, japan) according to manufacturer's instructions. a fragment of the s gene ( bp) was amplified using a pair of specific primers including xce ( '-cactggtaatttttcagatgg- ') and xce ( '-ctctataaacacccttaca- '). the pcr reaction and its thermal condition were set up as previously described. the pcr products were electrophoresed on . % agarose gel. characterization of isolated ibvs. the ibv positive allantoic fluids were investigated for detection of influenza and newcastle disease viruses by rt-pcr using specific primers. out of positive samples, four samples from different flocks were found to be infected with ibv alone. for genotyping, pcr products (s gene) of these four ibv isolates were sequenced in forward direction by bioneer co. (seoul, south korea) and their nucleotide sequences of their partial s gene were compared with each other and with previously reported isolates from iran, neighboring countries, and reference strains of ibv (table ) in genbank by using nblast, and clustalw . the phylogenetic relationship was established by http://www.phylogeny.fr/simple_phylogeny.cgi. the phylogenic tree was constructed using the neighbor joining method and mega software (version . ; biodesign institute, tempe, usa). the topological stability of the tree was evaluated by bootstrap replications. the results showed that flocks ( . %) were positive for ibv (fig. ) . the nucleotide sequences of four isolates were submitted to the genbank sequence database and were given the accession numbers iribvb: kp , iribvc: kp , iribve: kp and iribvf: kp . a phylogenetic tree (fig. ) , based on the hypervariable region of s gene sequences of four ibv isolates from the present study and other strains of ibv retrieved from genbank, was generated. based on the phylogenetic analysis, these four isolates were clustered with qx-like viruses. the results demonstrated the occurrence of qx-like serotype/genotype in ahvaz, iran. the ibv isolates were closely correlated to pcrlab infectious bronchitis virus is one of the main pathogens of commercial and backyard chickens with several serotypes and genotypes circulating in the world. based on our findings, out of examined flocks ( . %) were found to be infected with ibv, which show the prominent role of the virus in respiratory involvement of ahvaz broiler flocks. the ibv isolates in the present study were completely different from ibv vaccine strains (h , ma and / ) used for vaccination in ahvaz, which indicates the incidence of ib infection despite stringent vaccination. the determination of the ibv genotype is necessary not only to understand the evolution of the virus but also for developing vaccines based on circulating ibv strains in the region. one of the significant features of the ib viruses is the emergence of new variants of the virus throughout the world. therefore, new serotypes and new variants of ibvs are still isolated from chickens, even from vaccinated flocks. , the new ibv serotypes should be identified quickly in order to develop an effective vaccination strategy. several years ago, massachusetts serotype was identified in poultry flocks of iran. in recent years, b or / serotype has been identified in iran by several researchers. , , furthermore, the distribution of different ibv genotypes (different from the vaccine strain; massachusetts) was already reported in iran. [ ] [ ] [ ] phylogenetic analysis showed that isolated ibvs in the present study clustered with qx-like viruses. for the first time, in , qx strain of ibv was identified in china, after which the occurrence of the qx-liked virus was informed and it became one of the most prevalent ibv genotypes in various countries. qx-like ibvs were extended from china, to europe and recently to the south of africa. also, in iraq, this genotype was also identified. the pcrlab/ / (jx ) strain of ibv was isolated in iran and classified as qx-like viruses. the findings of the present study revealed that qx strains of ibv may be originated from other countries (probably iraq) which has been transmitted to ahwaz. there is little information about the type of ibv spread between the middle east countries. however, borderline migrations of birds is probably an important factor. taken together, this is the first report indicating the circulation of qx-like viruses in ahvaz broiler chickens with respiratory signs. finally, a comprehensive study on the pathogenesis of these ibv isolates is suggested. infectious bronchitis severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus studies on avian viral infections in iran isolation and molecular characterization of infectious bronchitis virus, isolate shiraz . ibv, by rt-pcr and restriction enzyme analysis isolation and identification of a new isolate of avian infectious bronchitis virus irfibv and study of its pathogenicity detection and seroprevalence of infectious bronchitis virus strains in phylogenetic tree generated based on the hypervariable region of s gene sequences of four ibv isolates from the present study and other strains of ibv retrieved from genbank using the neighbor joining method. commercial poultry in pakistan a laboratory manual for the isolation and identification of avian pathogens a new genotype of nephropathogenic infectious bronchitis virus circulating in vaccinated and non-vaccinated flocks in china molecular analysis of the /b serotype of infectious bronchitis virus in great britain factors influencing the outcome of infectious bronchitis vaccination and challenge experiments molecular epidemiology of infectious bronchitis virus isolates from china and southeast asia a survey of the prevalence of infectious bronchitis virus type / in iran epidemiology of avian infectious bronchitis virus genotypes in iran ( - ) genotyping of avian infectious bronchitis viruses in iran ( - ) reveals domination of is- like virus molecular characterization of infectious bronchitis viruses isolated from broiler chicken farms in iran the pathogenesis of a new variant genotype and qx-like infectious bronchitis virus isolated from chickens in thailand circulation of qxlike infectious bronchitis virus in the middle east genotyping of infectious bronchitis viruses from broiler farms in iraq during detection of the chinese genotype of infectious bronchitis virus (qx-type) in iran isolation and molecular characterization of sul/ / avian infectious bronchitis virus, indicates the emergence of a new genotype in the middle east the authors would like to express their gratitude to the dean for research of shahid chamran university of ahvaz for providing financial support for this work. the author disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: this study was supported by the research grant ( / / / ) provided by shahid chamran university of ahvaz. the authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. key: cord- -nlui d e authors: zahn, matthew; adalja, amesh a; auwaerter, paul g; edelson, paul j; hansen, gail r; hynes, noreen a; jezek, amanda; macarthur, rodger d; manabe, yukari c; mcgoodwin, colin; duchin, jeffrey s title: infectious diseases physicians: improving and protecting the public’s health: why equitable compensation is critical date: - - journal: clin infect dis doi: . /cid/ciy sha: doc_id: cord_uid: nlui d e infectious diseases (id) physicians play a crucial role in public health in a variety of settings. unfortunately, much of this work is undercompensated despite the proven efficacy of public health interventions such as hospital acquired infection prevention, antimicrobial stewardship, disease surveillance, and outbreak response. the lack of compensation makes it difficult to attract the best and the brightest to the field of id, threatening the future of the id workforce. here, we examine compensation data for id physicians compared to their value in population and public health settings and suggest policy recommendations to address the pay disparities that exist between cognitive and procedural specialties that prevent more medical students and residents from entering the field. all id physicians should take an active role in promoting the value of the subspecialty to policymakers and influencers as well as trainees. in recent decades, emerging and reemerging infectious diseases (id) have caused outbreaks with national and international implications and have underscored the critical need for id expertise. id physicians do more than protect the health of their patients. due to the unique communicable nature of id, id physicians provide a population-level service by helping to secure the health of the community. however, as id threats to public health continue to emerge, the number of young physicians applying for id subspecialty training continues to wane. id physicians lead public health responses in their healthcare facilities and communities and at federal and global levels. in the last decades, id specialists have played vital roles in responding to emerging diseases and epidemics including west nile virus ( ), severe acute respiratory syndrome (sars; ), h n pandemic influenza ( ), middle east respiratory syndrome (mers; and ongoing), ebola virus ( - and ongoing), and zika virus ( and ongoing). while these outbreaks garner a great deal of media visibility, id physicians also routinely detect, prevent, or mitigate community outbreaks of vaccine-preventable diseases, foodborne illness, and healthcare-associated infections. the work performed by id doctors in the united states is substantially undercompensated. id physician salaries average around $ a year less than other subspecialties [ , ] . young physicians, who generally complete training with substantial educational debt, can be driven from considering the field [ ] . developing a robust id workforce of the future requires a strategy to attract quality physicians and keep them engaged on the frontline of patient care, research, and public health. despite their documented value, id physicians' services are undervalued by payers compared to primary care and procedure-based specialties. a graduating trainee finishing an internal medicine residency can work immediately as a hospitalist at a higher mean salary than an id physician who will take to additional years of fellowship training. such variances in compensation for direct patient care and insufficient pay for value in other roles such as infection control creates a significant disincentive to pursuing a career in id. the medscape physician compensation report reveals that the median salary of a specialty physician to be approximately $ per year [ ] . the infectious diseases society of america's (idsa) compensation survey showed that the average id physician earns about $ a year. full-time public health physicians averaged $ per year less [ ]. the average medical student leaves school with approximately $ in debt [ ] . this financial disincentive is a significant contributor to the . % decline in the number of applicants to id fellowship training programs over the -year period ending in [ ] . only . % of these programs filled through the match, down % over the same period [ ] . while the - match saw an improvement in the number of positions filled, the match rate was still significantly below that of other specialties, many of which customarily fill at or near % [ ] . it is essential that id physicians promote the value this subspecialty brings to public health in order to affect the policy changes necessary to secure the future of the field. here, we provide concrete examples of id physicians' unique contributions to public health, which can be used to educate policymakers and influencers at the federal, state, and local levels and to advocate for needed investments to sustain the field. id specialty preparation consists of training first in general adult internal medicine or pediatrics followed by to years of training in id. the id subspecialty fellowship includes integrated training to provide patient care and ensure population health. the clinical training includes diagnosis, management, and treatment of patients with id; expertise in techniques for preventing healthcare-associated infections and antimicrobial resistance; prevention strategies; and research approaches to address id-related questions. in , id physicians were practicing in the united states [ ] , often combining clinical care with work as educators, epidemiologists, public health leaders, antimicrobial stewardship or infection prevention and control directors, researchers, administrators, and policymakers. multiple studies have demonstrated the cost-effectiveness and patient benefit of id physician care for hospitalized patients with id [ ] [ ] [ ] [ ] . further, the work of id physicians provides broader public protection against infectious threats through community and healthcare facility-based infection control and prevention activities, surveillance, outbreak response, and other public health activities. the id workforce plays a critical role in managing infections such as human immunodeficiency virus (hiv), tuberculosis, and viral hepatitis, which can cause community outbreaks if not promptly diagnosed and treated. the public health workforce, including id physicians, has been shrinking over the last decade [ , ] . these workforce losses pose a significant barrier to carrying out routine public health activities and responding to public health threats. id physicians employed by federal agencies and state health departments have the community as their patient. along with id physicians in other practice settings, they provide leadership and subject matter expertise to enable a wide variety of community-based interventions. for example, id management and prevention. public health strategies to contain the spread of hiv rely on ensuring that people living with hiv-aids have access to id-driven clinical care. by achieving sustained virologic suppression, people stay healthy and reduce the risk of community transmission [ ] . the hiv and hepatitis c virus (hcv) outbreaks linked to injection drug use in rural scott county, indiana, serve as a stark reminder of the risk for rapid disease transmission in communities that lack robust outbreak prevention resources and activities [ ] . in there was an outbreak of hepatitis a virus in the homeless population of san diego, california, that required significant work from the public health community to resolve [ ] . in march , a national academies of medicine panel declared the elimination of hepatitis b and hcv in the united states by to be a feasible goal. a sufficient workforce with the expertise to treat patients with hcv will be critical to reaching this goal [ ] . as an example of outbreak detection and response to emerging infections, the indiana state health department recognized the first identified case of mers in the united states in a traveler to that state. id specialists at the department helped develop and implement protocols to ensure appropriate care, prevent the spread of infection, and avert an outbreak. id physicians working inside or outside of public health often provide guidance to help allocate community vaccine resources and other preventive services. for example, during measles outbreaks in california in and minnesota in , id physicians within the state and local health departments helped lead vaccination campaigns to halt the spread of the disease [ , ] . even in the absence of a major outbreak, patients in hospitals and healthy individuals in communities are potentially exposed to increasingly drug-resistant and difficult-to-treat pathogens, necessitating id physician leadership to protect the population by limiting the spread of infectious threats [ ] . id public health physicians act as a trusted resource, working through the media, to help educate the public about communicable diseases and prevention strategies. current topics of concern include multidrug-resistant bacteria, zika virus outbreaks, the hepatitis a outbreak in san diego, and mycobacterium chimera infections in patients treated with heart-lung bypass machines. infection prevention and control activities in healthcare settings involve a range of interventions. methods include providing oversight of programs that conduct surveillance and identify risks, providing education regarding the use of appropriate isolation procedures and personal protective equipment, developing policies to respond to novel infections, and ensuring that the healthcare environment and medical devices are properly cleaned and maintained. studies have shown that development and implementation of these activities by id physicians have resulted in improved outcomes and reductions in hospital-acquired infections in a variety of healthcare settings [ ] . prevention of healthcare-associated infections leads to savings of approximately $ to more than $ per patient depending on the specific infection in a variety of healthcare settings [ ] . examples of id physician leadership in infection control and prevention and healthcare epidemiology are described here. during the west africa ebola crisis in and , id physicians served as leaders of special biocontainment units at the national institutes of health, university of nebraska, emory university, and bellevue hospital. these units and their staff developed novel, sophisticated infection prevention strategies to care for patients with potentially deadly infections. they reassured medical providers, political leaders, and our nation's public by providing safe, state-of-the-art care. in seattle, washington, an id physician reported an outbreak of deadly, multidrug-resistant carbapenem-resistant enterobacteriaceae infections were attributed to contaminated, faulty duodenoscopes. this investigation helped lead to changes in guidance on endoscope reprocessing and safety [ , ] . an id physician at vanderbilt university identified contaminated steroids as the source of a fungal meningitis outbreak that ultimately caused more than infections and deaths across states before the cause was established [ , ] . antimicrobial resistance is one of the most urgent public health threats of our time. infections caused by antimicrobial-resistant organisms kill more than people and result in $ billion in unnecessary healthcare costs each year in the united states [ ] . newer antimicrobials will be precious resources, and id physician-driven expertise will be vital to ensure that they are being used appropriately. a recent clinical infectious diseases article highlighted the unique skill set and training id physicians have to lead antimicrobial stewardship programs (asps) in order to ensure that correct antimicrobials are used judiciously [ ] . the centers for disease control and prevention (cdc) have acknowledged the impact and importance of asps in their release of the core elements of hospital antibiotic stewardship programs in [ ] , and the joint commission released their antimicrobial stewardship standard for healthcare settings in . in , legislation was passed in california that mandated that every acute care hospital in the state establish a physician-supervised asp [ ] . adoption of a federal government requirement that other states follow suit would help to ensure that all patients can benefit from the positive impact of asps. asps will become increasingly vital in preventing a post-antibiotic era where common infections become untreatable due to resistant organisms. id physicians need to take an active role in ensuring their facilities have adequate asps that meet the standards laid out by the cdc and joint commission. well-run asps not only help preserve the efficacy of antimicrobials but also improve patient outcomes. in fact, studies have indicated that asps generated several hundred thousand dollars a year in cost savings while also reducing rates of clostridium difficile infections [ , ] . it is vital that the government continue to support the implementation of id-led asps in all healthcare settings to slow the development of resistance and to keep our current antimicrobials useful for as long as possible. in response to the anthrax bioterrorism attacks and the looming threat of pandemic influenza, hospitals began extensive preparations for natural and human-made bioemergencies. these activities substantially increased with the appearance of the sars, mers-coronavirus, and ebola virus epidemics. id physicians provided scientific and clinical expertise that shaped prevention, control, detection, and treatment efforts. natural disasters also carry an increased risk of outbreaks of unusual, serious infections, as seen in connection with the hurricanes and flooding in texas, florida, puerto rico, and the us virgin islands, which led to more than reported cases of leptospirosis and other waterborne and vectorborne diseases [ ] . id physicians also played a leading role in infection prevention at large-scale shelters for people who had been displaced by storms and flooding. hospital and healthcare system emergency preparedness requires an intimate knowledge of hospital infection control procedures and capacities, regional collaboration with other facilities, planning with public health authorities, and development of communication strategies within the hospital community, with public health departments, and with the general public. a review of recent international id outbreaks emphasizes the global dimensions of public health protection-the sars epidemic originated in hong kong, the ebola virus outbreak started in west africa, and the zika virus epidemic spread from brazil throughout south america and the caribbean. a bacterial plasmid that conferred resistance to colistin, often the last antimicrobial line of defense against gram-negative organisms, emerged from bacteria that were colonizing domestic animal populations in china and is now seen in patients on continents. older epidemics, of course, continue; tuberculosis and hiv-aids remain scourges in many countries. emerging and reemerging infections with global consequences have reemphasized efforts to address global health security. the global sars epidemic illustrated how increased international travel and trade introduce new risks for the rapid worldwide spread of new infectious pathogens. the world health organization international health regulations (ihr), issued in , were officially adopted by countries [ ] . id physicians remain crucial to ongoing ihr implementation efforts, including disease outbreak recognition, in-country training, and collaborative partnership in epidemic disease control, diagnostics, and preparedness. with prescient timing, the cdc' s global health security agenda (ghsa) was launched just months before the ebola crisis surfaced in west africa. the ghsa aims to "accelerate progress toward a world safe and secure from infectious disease threats; to promote global health security as an international priority and to spur progress toward full implementation of who ihr " [ ] . the goals fall into major themes: prevention, detection, and response. id physicians provide critical support to all arms. id training and expertise establish the necessary foundations for the prevention goal to address issues such as antimicrobial resistance, biosafety and biosecurity, and immunization needs. the detection goal promotes innovation in the area of accurate, real-time, cost-effective id diagnostics. greater use of diagnostic tests in countries currently lacking such resources is critical for containing emerging and reemerging infections, preventing outbreaks, and halting the development of antimicrobial resistance. the response goal would establish emergency operations centers that link public health with other rapid-response agencies, all of which draw on id physician expertise. multiple studies have demonstrated that consulting an id physician improves treatment outcomes and lowers patient care costs. this benefit has been documented for id consultation in general [ ] , in specific patient populations such as intensive care unit patients [ ] , and for specific illnesses such as staphylococcal bloodstream infections and other multidrug-resistant organism infections [ , ] . id physicians also improve outcomes and reduce costs through antimicrobial stewardship and infection prevention [ , ] . in addition, many complex procedures and treatments (such as bone marrow and solid organ transplantation) could not be safely conducted without input from id specialists. the idsa has implemented several initiatives in an effort to reinvigorate the pipeline of id applicants. these include mentorship programs; scholarships for medical students, residents, and fellows interested in id; research and clinical career meetings; and medical school id interest groups. however, other potential options should be considered in order to properly compensate id physicians for the public health benefit of their work. recommendations for federal, state, local, and institutional policymakers include the following: establish loan repayment opportunities for id physicians who work in public service (eg, local, state, or federal public health departments). significant medical school debt can drive new physicians away from the id specialty and public health. targeted loan repayment opportunities would allow more physicians to pursue these critical career paths. in july , in a move supported by idsa, the us house of representative's energy and commerce committee passed legislation that would authorize the cdc to provide loan repayment for those who serve in the epidemic intelligence services (eis). more than % of eis officers continue serving in public health roles, and this proposal would significantly help make this career path more financially feasible for physicians. such loan forgiveness opportunities could also be broadened to support id physicians who work in public health at the federal, state, or local level and face similar challenges around compensation and medical student debt. establish financial compensation for id physicians who work in public service (eg, local, state, and federal public health agencies) or who do id work that provides broader public health benefits (infection control and antimicrobial stewardship). physicians who perform infection control and antimicrobial stewardship work should be compensated for these activities. as part of hospital accreditation, the centers for medicare and medicaid services (cms) mandates that every participating facility have an active infection control program [ ] . states could attach an accreditation process to such mandates that would formally document id clinicians who oversee hospital infection control and antimicrobial stewardship committees, with loan forgiveness or other financial incentives attached to this accreditation. ensure that id physician compensation reflects public health value added. as a cognitive specialty, many of the clinical services id physicians provide are billed under evaluation and management codes, which have not been reevaluated in more than years. the cms should update these codes to reflect the increasingly complex care provided in inpatient and outpatient settings. public and private payers, healthcare systems, and hospitals should also provide appropriate compensation and protected time for nonclinical services that are crucial to public health, including infection prevention and control, antimicrobial stewardship, and bioemergency preparedness and response. fully fund local, state, federal, and global public health agencies and build a competent workforce. without proper workforce funding, including funding of id physician positions, health departments will not be able to hire and retain the expert workforce necessary to detect, prevent, and respond to public health threats. appropriate compensation for id service in all of these forms will have significant positive effects on individual patient care, nosocomial infections, and community public health, ensuring that the future id workforce has enough of an incentive to pursue this vital field. medscape physician compensation survey results and data, specialties matching service, appointment year. national resident matching program appointment year. national resident matching program association of american medical colleges physician specialty data book employing infectious disease physicians affects clinical and economic outcomes in regional hospitals: evidence from a population-based study the value of an infectious diseases specialist the value of infectious diseases specialists: non-patient care activities infectious diseases consultation reduces -day and -year all-cause mortality for multidrug-resistant organism infections a mismatch between the educational pipeline and public health workforce: can it be reconciled? enumeration of the governmental public health workforce partner study group. sexual activity without condoms and risk of hiv transmission in serodifferent couples when the hivpositive partner is using suppressive antiretroviral therapy hiv infection linked to injection use of oxymorphone in indiana a national strategy for the elimination of hepatitis b and c: phase two report measles outbreak-california antibiotic resistance threats in the united states the value that infectious diseases physicians bring to the healthcare system health care-associated infections: a meta-analysis of costs and financial impact on the us health care system endoscopic retrograde cholangiopancreatography-associated ampc escherichia coli outbreak infections associated with reprocessed duodenoscopes doctor isolates cause in nationwide meningitis outbreak. vanderbilt magazine multistate outbreak of fungal meningitis case count infectious diseases society of america, pediatric infectious diseases society, and the society for healthcare epidemiology of america. infectious diseases physicians: leading the way in antimicrobial stewardship core elements of hospital antibiotic stewardship programs california senate bill no. clinical and economic outcomes of a prospective antimicrobial stewardship program an evaluation of the impact of antibiotic stewardship on reducing the use of high-risk antibiotics and its effect on the incidence of clostridium difficile infection in hospital settings world health organization. international health regulations. available at: www the global health security agenda infectious diseases specialty intervention is associated with decreased mortality and lower healthcare costs impact of regular collaboration between infectious diseases and critical care practitioners on antimicrobial utilization and patient outcome the value of infectious diseases consultation in staphylococcus aureus bacteremia the value that infectious diseases physicians bring to the healthcare system conditions of participation for hospitals potential conflicts of interest. all authors: no reported conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord- -d hsguds authors: coudert, pascal title: les principales maladies du porc date: - - journal: actualités pharmaceutiques doi: . /j.actpha. . . sha: doc_id: cord_uid: d hsguds résumé plusieurs pathologies autrefois mortelles au sein des élevages porcins ont disparu de notre territoire, mais de nombreuses maladies polysystémiques subsistent. si l’antibiothérapie s’avère parfois efficace, la vaccination permet de prévenir un certain nombre de pathologies alors qu’il n’existe aucun traitement préventif ni curatif pour d’autres. par ailleurs, tous les organes du porc sont susceptibles d’être la cible d’agents pathogènes. là encore, des vaccins et des traitements antibiotiques sont disponibles. summary several pathologies which were once mortal within pig farms have disappeared from our territory, numerous polysystemic diseases remain. if antibiotherapy is sometimes effective, vaccination can prevent a certain number of pathologies while there is no preventive of curative treatment for others. moreover, all the pig's organs are susceptible to being targeted by pathogenic agents. here again, vaccines and antibiotic treatments are available. a vec l'amélioration des conditions d'élevage, notamment grâce aux contrôles vétérinaires réguliers, l'état sanitaire des élevages porcins s'est nettement amélioré au fil des années. c'est ainsi qu'en france, certaines affections ont quasiment disparu. À l'inverse, des pathologies, comme la maladie de glässer, sont encore susceptibles d'engendrer d'importantes pertes économiques dans des élevages à haut niveau sanitaire. l'élevage en flux continu sur paille ou en plein air associé à des insuffisances de prophylaxie favorise l'apparition de pathologies comme le rouget, la gale et l'ascaridose. en engraissement, les maladies respiratoires et les entérites sont fréquemment retrouvées. enfin, l'âge des animaux constitue un élément à prendre en compte : la diarrhée colibacillaire peut entraîner une forte mortalité chez le porcelet. en , six maladies et infections à déclaration obligatoire (encéphalite à virus nipah, gastro-entérite transmissible, cysticercose, peste porcine classique et africaine, syndrome dysgénésique et respiratoire) ont été recensées par l'organisation mondiale de la santé animale pour les suidés . un certain nombre de maladies polysystémiques causent encore aujourd'hui des pertes économiques significatives pour les éleveurs [ ] [ ] [ ] . peste porcine ✦ causée par un virus de la famille des flaviridae, la peste porcine classique n'est actuellement plus présente en france tant dans les cheptels porcins domestiques que sauvages. les porcs peuvent se contaminer au contact d'un animal malade, par ingestion de viande de porc contaminée ou de produits dérivés infectés. les sangliers vétérinaire pratique sauvages constituent un réservoir de virus en europe de l'est. ✦ les animaux infectés présentent de la fièvre, une perte d'appétit, une conjonctivite, une constipation suivie de diarrhées et des lésions hémorragiques cutanées, l'ensemble pouvant être fatal dans les formes aiguës. À l'inverse, il existe des formes bénignes sans symptômes apparents. aucun traitement spécifique n'est disponible et la vaccination préventive des cheptels porcins n'est plus autorisée dans l'union européenne depuis suite à la mise en place d'un programme d'éradication de la maladie. ✦ la peste porcine africaine, dont l'agent responsable est un virus à acide désoxyribonucléique (adn) de la famille des asfarviridés, est apparue en europe en mais ne touche actuellement que les pays de l'est (lituanie, pologne, lettonie, estonie, république tchèque) tout en progressant régulièrement vers l'ouest. la symptomatologie de cette maladie est très proche de celle de la peste porcine classique. fièvre aphteuse et maladie vésiculeuse ✦ la fièvre aphteuse est causée par un aphtovirus, virus à arn de la famille de picornaviridae. la symptomatologie de la maladie est caractérisée par de la fièvre à laquelle s'associent des aphtes dans la bouche et des cloques sur les pieds. il n'existe aucun traitement spécifique. si les porcs adultes guérissent généralement spontanément, une mort soudaine suite à des lésions de la musculature cardiaque est possible chez les porcelets. l'europe est considérée indemne mais des épizooties peuvent apparaître suite à l'introduction d'animaux malades, d'objets ou de produits contaminés. ainsi, en , plusieurs foyers ont été recensés en france après utilisation, en angleterre, de déchets de cuisine d'un navire comme aliments pour porcs. ✦ la maladie vésiculeuse, impossible à distinguer d'un point de vue clinique de la fièvre aphteuse, provoque également de la fièvre, des vésicules se transformant en ulcères superficiels sur les pieds, le groin, les mamelles ou dans la bouche. cette affection, pour laquelle il n'existe ni traitement ni vaccin, résulte d'une infection par des entérovirus de la famille des picornaviridae. il est donc nécessaire de procéder à des analyses en laboratoire pour établir un diagnostic de certitude. salmonellose ✦ plusieurs espèces de salmonelles sont à l'origine de la salmonellose porcine, en particulier salmonella derby, s. typhimurium et s. choleraesuis. si l'infection d'un élevage est souvent inapparente, une multiplication importante de ces bactéries au niveau intestinal peut engendrer une diarrhée aqueuse brun jaunâtre avec amaigrissement, pratique vétérinaire des sténoses rectales, une septicémie, voire une toux en cas d'atteinte pulmonaire. ✦ la thérapeutique fait appel à la colistine ou la gentamycine par voie injectable car les animaux malades ne boivent plus et cessent de s'alimenter. néanmoins même à la suite d'un traitement antibiotique, les porcs peuvent rester porteurs sains pendant des semaines, voire des mois. ✦ due à la bactérie haemophilus parasuis, dont il existe sérotypes différents plus ou moins virulents, la maladie de glässer est une affection dont le symptôme principal est une polysérosite fibrineuse. streptococcus suis, mycoplasma hyorhinis peuvent être à l'origine d'un tableau clinique similaire chez le porc. d'autres troubles sont susceptibles d'apparaître tels une polyarthrite, une méningite, une septicémie, une myosite et des difficultés respiratoires. le regroupement d'animaux d'origines différentes et les facteurs de stress (sevrage, transport) favorisent l'appa rition de la maladie au sein de grandes exploitations. ✦ l'antibiothérapie fait appel aux bêtalactamines (amoxicilline, ampicilline, ceftiofur) ou aux associations pénicilline-streptomycine et triméthoprime-sulfamide. toutefois, du fait de l'existence d'un nombre élevé de souches d'h. parasuis multirésistantes, la réalisation d'un antibiogramme est recommandée pour la mise en place d'un traitement. les vaccins disponibles stimulent le développement d'une immunité active uniquement contre les sérotypes les plus prévalents et (suvaxyn ® m. hyo-parasuis) ou seulement (porcilis ® glässer). ✦ l'agent du rouget est une bactérie à gram positif dénommée erysipelothrix rhusiopathiae. les principales voies d'inoculation sont cutanées et intradermiques (écorchures). la maladie peut présenter des évolutions variées : suraiguë ou aiguë, parfois fatales en quelques heures, chronique, septicémique ou localisée. les symptômes sont donc divers : lésions cutanées sous forme de plaques d'urticaire rectangulaires, endocardites, néphrites et arthrites surtout au niveau des jarrets. ✦ les pénicillines a ou g s'avèrent en général efficaces, mais la doxycycline ou les macrolides peuvent être utilisés en cas d'allergie aux bêtalactamines. des vaccins existent en prévention du seul rouget (eryseng ® , ruvax ® ) ou couplés à la parvovirose porcine (eryseng parvo ® , parvoruvax ® , porcilis ® ery + parvo). ces derniers ne protègent pas les porcs contre l'évolution chronique de cette affection et favorisent même parfois l'apparition d'arthrites. ✦ les infections à streptococcus suis font partie des infections majeures dans les élevages porcins. elles affectent surtout les porcelets sevrés et les porcs à l'engraissement. la pathologie peut revêtir plusieurs aspects selon la virulence de la souche et l'organe atteint : hyperthermie, septicémie, méningite, pneumonie, endocardite ou atteinte articulaire. ✦ le traitement fait appel à des antibiotiques : l'ampicilline ou l'amoxicilline. reproduction, mise bas et lactation ✦ un certain nombre d'affections rencontrées au cours de la reproduction est consécutif à des problèmes alimentaires tel le syndrome de la truie maigre qui apparaît en cas de sous-alimentation en gestation, puis en lactation. il est souvent difficile de convaincre les éleveurs que, dans cette situation, un parasitisme externe est la conséquence et non la cause de la maladie. À l'inverse, il existe un syndrome de la truie grasse due à une suralimentation modérée continue, appréciée par des replis graisseux entre les cuisses. ✦ par ailleurs, la présence de problèmes locomoteurs avec une faiblesse des membres en fin de gestation, d'un allongement de la durée moyenne de la mise bas et d'écoulements vulvaires caractérise une truie grasse à la mise bas et maigre au sevrage ("truie accordéon"). il est aujourd'hui admis qu'il est possible d'améliorer le déroulement des mises bas et de la lactation par l'alimentation afin de dynamiser la vitalité des porcelets nouveau-nés. ✦ si les écoulements vulvaires sont physiologiques chez la truie en chaleurs, ils ont pour origine, chez la cochette non saillie, des infections ascendantes provenant de l'environnement et traduisant une immaturité du système immunitaire. chez la truie nullipare, un épisode infectieux peut apparaître à jours après une contamination lors de la saillie. classiquement l'endométrite postpartum se rencontre chez la truie multipare après une colonisation de l'endomètre par des staphylocoques peu fréquente, mais transmissible à l'homme, la brucellose porcine concerne les élevages de plein air contaminés par la faune sauvage. maladies d'élevage des porcs veterinary microbiology and microbial disease merck sharp & dohme corp. msd veterinary manual key: cord- -vfagxsdz authors: althouse, benjamin m; flasche, stefan; minh, le nhat; thiem, vu dinh; hashizume, masahiro; ariyoshi, koya; anh, dang duc; rodgers, gail l.; klugman, keith p.; hu, hao; yoshida, lay-myint title: seasonality of respiratory viruses causing hospitalizations for acute respiratory infections in children in nha trang, vietnam date: - - journal: int j infect dis doi: . /j.ijid. . . sha: doc_id: cord_uid: vfagxsdz background: acute respiratory infections (aris) are the most common causes of death in children under years of age. while the etiology of most pneumonia and ari episodes is undiagnosed, a broad range of ari-causing viruses circulate widely in south east asia. however, the patterns and drivers of the seasonal transmission dynamics are largely unknown. here we identify the seasonal patterns of multiple circulating viruses associated with hospitalizations for aris in nha trang, vietnam. methods: hospital based enhanced surveillance of childhood ari is ongoing at khanh hoa general hospital in nha trang. rt-pcr was performed to detect respiratory viruses in nasopharyngeal samples from enrolled patients. seasonal patterns of childhood ari hospital admissions of various viruses were assessed, as well as their association with rainfall, temperature, and dew point. results: respiratory syncytial virus peaks in the late summer months, and influenza a in april to june. we find significant associations between detection of human parainfluenza and human rhinovirus with the month's mean dew point. using a cross-wavelet transform we find a significant out-of-phase relationship between human parainfluenza and temperature and dew point. conclusions: our results are important for understanding the temporal risk associated with circulating pathogens in southern central vietnam. specifically, our results can inform timing of routing seasonal influenza vaccination and for when observed respiratory illness is likely viral, leading to judicious use of antibiotics in the region. acute respiratory infections (aris) in south east asia cause substantial morbidity and mortality, especially in children under years of age (walker et al., ) . pneumonia continues to be the number one cause of under death despite effective treatments (wardlaw et al., ) . a substantial contributor to this is the largely unknown etiology of most pneumoniae, with both viral and bacterial origin. the patterns of pneumonia and other ari hospitalizations serves as a proxy for determining the transmission dynamics of viruses and bacteria contributing to these hospitalizations, and is of key importance in understanding and limiting burden of this childhood killer. in addition to informing on the relative likelihood of the potential viral etiology of a pneumonia case based on seasonally circulating pathogens, knowledge of seasonal influenza epidemiological dynamics can aid in informing optimal timing for vaccination efforts (saha et al., ; lambach et al., ) and judicious use of antivirals tanaka et al., ) . identification of low incidence seasons would provide a target window for vaccination, with the hope of maximizing population immunity before the onset of the influenza season. similarly, when respiratory syncytial virus (rsv) vaccination becomes available, knowledge of its seasonality will be useful to maximize benefit (drysdale et al., ; anderson et al., ) , and in the potential administration of passive immunoprophylaxis with palivizumab to reduce the number of severe outcomes associated with rsv infection among high-risk infants (committee on infectious diseases, ). finally, knowledge of seasonal patterns of virus circulation can inform the clinical use of antibiotics, again limiting use when viral circulation is traditionally high to minimize antibiotic resistance ( van nguyen et al., ; laxminarayan et al., laxminarayan et al., , van boeckel et al., ) . previous work has identified the potential etiology of pneumonia and other aris in southern central vietnam (do et al., ; yoshida et al., ) . adenovirus (aov), bocavirus (hbov), coronavirus (cov), human metapneumovirus (hmpv), human parainfluenza - viruses (hpiv - ), human rhinovirus (hrv), influenza a and b, and rsv all contribute to the disease burden and circulate widely. information on viral transmission dynamics across seasons in vietnam is relatively under-explored. do et al. found seasonality of rsv infections and slight seasonality of hmpv infections in ho chi minh city, but no seasonality of influenza (do et al., ) . several studies have similarly found high variability in influenza a & b incidence over the year (nguyen et al., ; le et al., ; thai et al., ) . yoshida et al. found rsv occurring in the hot months, influenza a in the cool months, and year-round detection of hrv in nha trang (yoshida et al., ) . few other studies have examined seasonality of these common viruses across south east asia. here we examine the seasonal trends of hospitalizations and circulation of multiple viruses in nha trang, vietnam. using enhanced hospital based surveillance of childhood ari we identify seasonal patterns in hospitalizations as a proxy for transmission and explore the relationship of hospitalizations associated with virus detection with rainfall, temperature, and dew point, to try and identify contributing factors to observed seasonality. the study site is nha trang, central vietnam, where the study population has been described previously (yoshida et al., (yoshida et al., , flasche et al., ) . the hospital based enhanced surveillance of childhood ari is ongoing. we analyze data from january , to april , which is the only tertiary care facility located in khanh hoa province. according to the field site census survey in july , the study catchment area encompassing the non-touristic of the communes in nha trang city, had , residents including , children less than years of age. an ari case was defined as any child presenting to khgh with cough or/and difficulty in breathing. before study enrollment, informed consent was obtained from parents of children who presented with ari and lived in the study catchment area. clinical and demographic information, chest radiographs (cxr), laboratory data, and nasopharyngeal (np) samples were collected from all enrolled patients. khgh is the only hospital in nha trang, khanh hoa province and the only one accessible for residents of the catchment area. hence for incidence calculations we assume that all children with ari are eligible to be hospitalized and enrolled into the study and use the population of the catchment area as denominator. acute respiratory infection patients with normal cxr were categorized as upper respiratory tract infection (urti). patients with abnormal cxr were categorized as lower respiratory tract infection (lrti). np samples were collected at the time of admission and viral nucleic acid was extracted using qia viral rna minikit (qiagen inc., valencia, ca). four multiplex-pcr assays ( : influenza a, influenza b, rsv, hmpv; : piv- , - , - , and - ; : rhinovirus, coronavirus e, coronavirus oc ; : adenovirus and bocavirus) were performed to detect respiratory viruses in each np sample. a second confirmatory-pcr was performed for samples positive on the initial pcr test. samples positive for both pcr assays were defined as positive. reverse transcription-pcr (rt-pcr) assays were performed using one-step rt-pcr kit from qiagen. for the multiplex pcr and hemi-nested pcr assays, taqdna polymerase (promega, san luis obispo, ca) was used as previously described (yoshida et al., ) . positive templates were used in each assay for quality control. three weather variablesrainfall (inches), temperature (f ), and dew point (the temperature to which air must be cooled in order to reach saturation with water)were collected from the nha trang station (id: ) reported by the us national oceanic and atmospheric administration (national oceanic and atmospheric administration, ). we considered monthly averages of all weather variables as well as both the weather variable on the day of admission (t ) as well as averaged over the previous days (t À to t À ) assuming up to a week incubation period for the viral infections (see supplementary material) (lessler et al., ) . the weather in nha trang central vietnam is warm throughout the year (between and c ). in terms of temperature, december to february months are cooler (referred to here as the winter months) while june to august months are hottest months (referred to here as the summer months). september, october, november are the wettest months. for each pcr+ for virus a series of log-link poisson models were fit to assess respective seasonality with calendar month as the main predictor, log-commune population size as an offset term, monthly averaged rainfall, temperature, and dew point, and calendar year as adjusting variables. the outcome was monthly aggregate cases, with resulting coefficients as incidence rate ratios as compared to january. we excluded hospitalizations with more than one virus detected in the nasopharynx to adjust for a potential bias through inclusion of cases who by virtue of being co-infected may otherwise have been asymptomatic (althouse and scarpino, ) . this approach underestimates the true incidence of np carriage among ari cases but allows estimation of seasonal patterns that are not biased by other circulating viruses; although co-circulation of bacteria was not accounted for. we assessed the numbers and variety of viruses in ari hospitalizations using binomial proportion tests for each virus. to examine the relationship between monthly average rain, temperature, and dew point and incidence hospitalized childhood ari infections, we estimated the cross-wavelet transform between the z-standardized time series (we subtracted the mean of the time series and divided by the standard deviation) of weather and viral detections (cazelles et al., ) . the cross-wavelet transform identifies regions of high power in phase-space and identifies the relative phases of each time series, i.e., in-phase or out-of-phase (grinsted et al., ) . the wavelet transform can be thought of a fourier transformation over time that can identify what is the dominant frequency composing a time series as the signal changes in time. the cross-wavelet transform allows us to compare how two time signals co-vary: we can identify if the presence of a particular frequency at a given time in the time series of hospitalizations corresponds to the presence of that same frequency at the same time in a weather covariate (chaves and pascual, ) . additionally, we can identify the magnitude by which weather precedes or follows hospitalizations through the phase angle of the two time series. finally, we can identify the statistical significance of the identified constituent frequencies over time by comparing the observed frequencies to a red-noise process. sensitivity analyses, presented in the supplementary materials, were performed as follows: ) case counts of less than per virus over the whole study period were deemed too low for robust statistical inference; ) alternative poisson regression models where the reference category is july; ) logistic regression models were formulated as an alternative to the poisson regressions above with detection of a virus by pcr (yes/no) as the outcome, with month as the main predictor, adjusted for weather, commune of residence, age, sex, smoking indoors, socioeconomic status (ses), and calendar year, with weather variables on the day of admission (t ) as well as averaged over the previous days (t À to t À ); and ) additional wavelet analyses of viral isolations not presented in the main text. the study enrolled children between to . among those, ( %) had multiple viruses detected in their np swabs, for presence of viruses in the np was not determined, and were excluded from the analyses, thus the total study population was . among all cases with a virus detected, hrv, rsv, and influenza a were the most frequently detected viruses, with ( . % of all viral detections), ( . %), and ( . %) detections, respectively (table and figure ). counts of bocavirus (hbov), coronavirus (cov), and human parainfluenza , , and viruses (hpiv - ) were less than and are reported in the supplementary material. strong seasonality, as defined by at least three consecutive months with a consistently higher or lower incidence than expected (irr or or greater or less than , respectively) and at least one of those statistically significantly different from the baseline, was observed for influenza a, rsv, and the presence of any virus (figures and ) . rsv peaked in july through november, with august seeing a . ( % confidence interval [ci]: . , . ) times higher risk of identifying rsv as the sole viral agent from the nasopharynx of a childhood case as compared to january. influenza a peaked in may with an irr of . ( % ci: . , . ) as compared to january. estimates of odds ratios from the supplemental logistic regression are qualitatively similar to the poisson regression, save for hpiv , which exhibits a consistent yet nonsignificant peak in the cool months in the primary analysis which becomes significant in the supplemental analysis (see supplemental material). weather patterns over the study period were similar to patterns before and after the study period (supplementary material, figure s ). monthly average rainfall (in inches), temperature ( f), and dew point ( f) correlated with the seasonality of some of our endpoints. figure shows the incidence rate ratios for the three weather effects from the seasonally-adjusted models. overall hospitalizations for ari were negatively associated with temperature (irr . per degree increase, % ci: . , . , p = . ) and positively associated with dew point (irr . per degree increase, % ci: . , . , p < . ). of the few other significant effects, influenza a and hrv had a negative associations with temperature (irr . , % ci: . , . , p = . , and irr . , % ci: . , . , p < . , respectively), and hpiv and hrv were positively associated with dew point, with irrs . ( % ci: . , . , p = . ) and . ( % ci: . , . , p = . ), respectively. logistic regression found that rsv was positively associated with the previous week's rain, with an odds ratio of of . ( % ci: . , . , p = . ). previous week's temperature was marginally associated with rsv (or: . ( % ci: . , . , p = . ) (see supplementary material). figure shows the cross-wavelet transform of all hospitalizations and the three weather variables in the month of admission. significant bands of high power around year can be seen for temperature and dew point. this indicates that hospitalizations and temperature and dew point share variability at yearly frequencies over the study period, and the phase relationship indicates that changes in weather slightly precede changes in hospitalizations (arrows point about down). figure shows the cross-wavelet transform of rsv and the three weather variables with similar significance bands around year for temperature and dew point. the phase indicates temperature and dew point lead rsv incidence. while not significant, rsv was found to be leading rainfall by ( months) at the one year period band (see supplementary material). significant bands of power were seen between temperature and dew point with hpiv with an indicated phase relationship of nearly completely out-of-phase (figure ) . similar patterns were seen ( year significant bands between temperature and dew point and virus) for adv, hbov, cov, hpiv , & , hrv, and influenza a, though the phase differences varied across these viruses; hmpv and influenza b had bands lying outside the cone of influence (ie, not statistically significant; see supplementary material). these results in general indicate strong associations between weather covariates and viral hospitalizations at a yearly timescale. here we have identified seasonal trends of several common respiratory viruses in hospitalized children in nha trang, vietnam. by fitting a series of statistical models to the observed data, we allow the data to identify salient features contributing to the seasonality of these viruses. we evaluated seasonal patterns and associations with weather of hospitalizations for several respiratory viruses using three lines of evidence: ) poisson regression examining the relative incidence across months of virus detections adjusted for weather covariates, ) cross-wavelet transforms of hospitalizations with viral detections, and ) a sensitivity analysis with a logistic regression model finding odds ratio of hospitalizations with viral detections and weather variables. any viral detection showed distinct seasonality with peaks in may through september, a negative association with temperature, positive association with dew point, and crosswavelets indicating temperature and dew point leading viral detection. of commonly detected viruses, rsv, influenza a, and hpiv had significant seasonality. rsv peaked in july through december, was positively associated with the week's previous average rainfall. cross-wavelets showed temperature and dew point to lead rsv, rain was found to non-significantly fall behind rsv at year frequencies, and precede rsv at shorter (< day) frequencies. finally, hpiv while not significant, had peaks in january and february, was positively associated with dew point, and was completely out of phase with temperature and dew points in cross-wavelet analyses. these results contribute to the growing body of knowledge on the epidemiology of respiratory pathogens in south east asia and southern central vietnam. using a cross-wavelet transform we evaluated the timedependence of virus hospitalizations with the weather covariates. we found strong yearly associations with rsv over the study period with temperature and dew point in phase with hospitalizations. this seasonality is opposite to observed seasonality in temperate climates, where rsv typically peaks in winter (tang and loh, ; shek and lee, ) . recent reviews of rsv seasonality in tropical regions highlights the uncertainty in the effects of weather on rsv transmission. studies in brazil (straliotto et al., ) , hawaii (reese and marchette, ) , india (agrawal et al., ) , kenya (hazlett et al., ) , and malaysia (chew et al., ; chan et al., ; khor et al., ) , have shown negative associations between temperature and rsv, while studies in hong kong (chan et al., ) , mexico (yusuf et al., ) , singapore (chew et al., ) , and taiwan (huang et al., ) have shown positive associations. we find strongly positive associations between temperature and rsv hospitalizations with a slight lead of temperature on rsv. this is evident both from the cross-wavelet transform and the regression results indicating a positive effect of the previous week's temperature on rsv. there is less uncertainty in the role of rainfall on rsv transmission in tropical areas, where the majority of work indicates that rsv generally occurs during rainy seasons (shek and lee, ) . colombia (bedoya et al., ) , the gambia (weber et al., ) , hong kong (chan et al., ) , kenya (hazlett et al., ) , malaysia (chan et al., ) , and papua new guinea (hierholzer et al., ) all show positive rsv associations with rainfall. omer et al. (omer et al., ) showed significant positive associations between rainfall and temperature in the previous days and rsv incidence in lombok, indonesia. we find similar associations, with the mean rainfall and temperature over the previous days having odds ratios for rsv of . and . , respectively. this association is plausible as the incubation period of rsv is estimated to be between and days (lessler et al., ) . detailed contact tracing studies, coupled with climatological data could refine this association. somewhat surprisingly, the cross-wavelet transform of rsv and rain showed no significant association, though areas of high power were observed in the -year and -month bands, with phase indicating rsv leading weather. however, none of the viruses studied here showed appreciable associations with rain in the cross-wavelet transform, possibly indicating the dominance of other weather effects (temperature and dew point) on virus hospitalizations. as with previous studies examining hpiv incidence, we found a predominance of hpiv ( detections) compared to , , and for hpiv , , and , respectively. typical seasonality for hpiv is the spring and early summer months in the temperate regions (fry et al., ; hall, ) , and has little to no observed seasonality in the tropics and subtropics (chew et al., ; branche and falsey, ) . we find evidence for winter peaks in hpiv hospitalizations when employing both the poisson regression model as well as the logistic regression model to estimate odds ratios, though the peaks were not statistically significant when controlling for weather. the cross-wavelet transforms reveals hpiv to vary significantly at year periodicity with temperature and dew point across the study. it also shows that hpiv hospitalizations are nearly completely out of phase with temperature and dew point throughout the study period. hpiv has been associated with low temperature and low relative humidity (miller and artenstein, ) though there is in general a paucity of data on the transmission routes of hpiv (pica and bouvier, ; meissner et al., ) . future work could explore in more depth the epidemiological relationship between hpiv and weather variables. this study is not without limitations. first, while we have nearly years of data, this is still a relatively short period to assess longterm seasonal trends, or to increase confidence in the estimates of seasonal patterns. however, the length of the analyzed time series is similar to other studies examining seasonal trends in viral respiratory pathogens in the tropics (chew et al., ; hall, ) , and gives indications for areas of future study. second, we excluded individuals with more than one virus detected. examination of changes in the seasonality of other viruses and coinfection over this period is worthy of study and is outside the scope of this paper. third, this study used hospital-based surveillance, necessarily presenting the most ill children. we take as an assumption that hospitalizations are a fraction of all transmission and severity of illness is not related to weather. finally, this study examines the influence of weather on viral hospitalizations and does not address other drivers of seasonal patterns of transmission, such as school closures (fine and clarkson, ) , differences in other social behavior such as contact rates (cook et al., ; dushoff et al., ) , susceptible recruitment through births (metcalf et al., ) , or possible seasonal changes in host immune responses (dowell et al., ) . future work examining these drivers in this setting is necessary. limitations aside, our study adds to the body of literature on seasonality of common respiratory patterns in tropical regions and will be of use when consideration of the epidemiology of these pathogens is necessary. for example the timing of influenza peaks in the mid-spring months (april, may, june) would indicate routine vaccination in the winter months would be of biggest impact. similarly, knowing rsv peaks in the late summer/early fall when rcp is at its lowest may help in limiting unnecessary antibiotic (laxminarayan and malani, ) or antiviral use (tanaka et al., ) . in vietnam and most of asia, most antibiotics are acquired from a pharmacist without a formal prescription ( van nguyen et al., ) . this fact makes results like those presented here of high importance to public health decision-makers to inform pharmacists of seasonality of respiratory infection etiologies and urge judicious prescribing practices. additional future work could include examination of the effects of contact clustering (hébert-dufresne and , coinfection hébert-dufresne et al., ) , and asymptomatic carriers (althouse and scarpino, ) on transmission of the examined viruses all of which may be influenced by weather. comparative evaluation of real-time pcr and conventional rt-pcr during a year surveillance for influenza and respiratory syncytial virus among children with acute respiratory 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to rsv seasonality seasonality of absolute humidity explains seasonality of influenza-like illness in vietnam global antibiotic consumption to : an analysis of national pharmaceutical sales data antibiotic use and resistance in emerging economies: a situation analysis for viet nam global burden of childhood pneumonia and diarrhoea pneumonia: the leading killer of children the clinical spectrum of respiratory syncytial virus disease in the gambia viral pathogens associated with acute respiratory infections in central vietnamese children population based cohort study for pediatric infectious diseases research in vietnam the relationship of meteorological conditions to the epidemic activity of respiratory syncytial virus study design: bma, sf, hh, lmy; data collection: lmy, lnm, vdt; data analysis: bma, sf; writing first draft: bma, writing subsequent drafts: all authors; contributed intellectually: all authors the study was approved by institutional review boards in the national institute of hygiene and epidemiology, vietnam and the institute of tropical medicine, nagasaki university, japan. bma and hh were supported by bill & melinda gates through the global good fund. sf and lmy were partially supported by bill & melinda gates foundation (bmgf opp ). the pediatric ari surveillance study in nha trang, vietnam was supported by the japan initiative for global research network on infectious diseases (j-grid) from ministry of education, culture, sport, science & technology in japan, and japan agency for medical research and development (amed). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. supplementary data associated with this article can be found, in the online version, at https://doi.org/ . /j.ijid. . . . key: cord- -y cp wza authors: negrey, jacob d.; reddy, rachna b.; scully, erik j.; phillips-garcia, sarah; owens, leah a.; langergraber, kevin e.; mitani, john c.; emery thompson, melissa; wrangham, richard w.; muller, martin n.; otali, emily; machanda, zarin; hyeroba, david; grindle, kristine a.; pappas, tressa e.; palmenberg, ann c.; gern, james e.; goldberg, tony l. title: simultaneous outbreaks of respiratory disease in wild chimpanzees caused by distinct viruses of human origin date: - - journal: emerg microbes infect doi: . / . . sha: doc_id: cord_uid: y cp wza respiratory viruses of human origin infect wild apes across africa, sometimes lethally. here we report simultaneous outbreaks of two distinct human respiratory viruses, human metapneumovirus (mpv; pneumoviridae: metapneumovirus) and human respirovirus (hrv ; paramyxoviridae; respirovirus, formerly known as parainfluenza virus ), in two chimpanzee (pan troglodytes schweinfurthii) communities in the same forest in uganda in december and january . the viruses were absent before the outbreaks, but each was present in ill chimpanzees from one community during the outbreak period. clinical signs and gross pathologic changes in affected chimpanzees closely mirrored symptoms and pathology commonly observed in humans for each virus. epidemiologic modelling showed that mpv and hrv were similarly transmissible (r( ) of . and . , respectively), but mpv caused . % mortality mainly in infants and older chimpanzees, whereas hrv caused no direct mortality. these results are consistent with the higher virulence of mpv than hrv in humans, although both mpv and hrv cause a significant global disease burden. both viruses clustered phylogenetically within groups of known human variants, with mpv closely related to a lethal variant from mountain gorillas (gorilla beringei beringei), suggesting two independent and simultaneous reverse zoonotic origins, either directly from humans or via intermediary hosts. these findings expand our knowledge of human origin viruses threatening wild chimpanzees and suggest that such viruses might be differentiated by their comparative epidemiological dynamics and pathogenicity in wild apes. our results also caution against assuming common causation in coincident outbreaks. respiratory viruses of human origin have caused disease in wild apes across sub-saharan africa and pose a significant and growing threat to wild ape health and conservation [ , ] . for example, respiratory disease is the leading cause of morbidity and mortality among chimpanzees (pan troglodytes) in gombe stream national park, tanzania [ , ] and in kibale national park, uganda [ ] , two populations that have been studied continuously for decades. mortality from anthroponotic respiratory pneumoviruses (family pneumoviridae) and paramyxoviruses (family paramyxoviridae) has been documented in western chimpanzees (p. t. verus) in cote d'ivoire [ , ] , eastern chimpanzees (p. t. schweinfurthii) in tanzania [ ] , mountain gorillas (gorilla beringei beringei) in rwanda, lowland gorillas (g. g. gorilla) in central african republic [ ] and bonobos (p. paniscus) in the democratic republic of the congo [ ] . rhinovirus c [ ] and coronavirus oc [ ] of human origin have also caused chimpanzee mortality in uganda and mild respiratory disease in cote d'ivoire, respectively. biological similarities between humans and apes predispose them to cross-species pathogen transmission [ ] , and habitat alterations may exacerbate inter-species contact and anthroponotic transmission risk [ ] . although simultaneous infections of apes and people with the same respiratory virus have rarely been confirmed directly [ , ] , viruses that are relatively benign in humans can cause lethal outbreaks in ape populations, indicating lack of resistance in apes. suitable prevention strategies have included improved hygiene and sanitation [ ] , reduced human visitation [ ] and large-scale vaccination of apes (if effective vaccines someday become available) [ ] . such policies could also benefit human public health by reducing zoonotic transmission risk [ , ] . here, we report simultaneous outbreaks of respiratory disease in two nearby chimpanzee communities in uganda, caused by two distinct negative-sense rna viruses of human origin. the outbreaks occurred from december to february in the ngogo and kanyawara chimpanzee communities in kibale national park [ ] (figure s ). only km apart, the communities are interconnected by contiguous moist evergreen forest, separated by only one intervening chimpanzee community that is not currently studied, and both communities generally experience low mortality rates [ , ] . although outbreaks of respiratory disease in wild chimpanzees are common, their causes often remain undiagnosed [ ] . fresh carcasses are often not recovered, and the remoteness of field sites complicates sample storage and analysis. non-invasive diagnostics (usually from feces) have proven highly informative [ ] , but negative results in such cases are inconclusive [ ] . in the present case, rapid recovery of carcasses, the presence of trained veterinarians and the availability of basic field laboratory capabilities on site provided an opportunity for etiologic diagnoses. furthermore, coordinated, prospective collection of observational data between the two sites offered an unusual chance to compare the clinical and epidemiologic features of the outbreaks directly. from december to february , the ngogo chimpanzee community of kibale national park, uganda ( figure s ), experienced an outbreak of severe respiratory disease. during the same period, the nearby kanyawara community ( figure s ) also experienced a respiratory disease outbreak. at the onset of the epidemics, ngogo community consisted of chimpanzees from < year to years old, and kanyawara community consisted of chimpanzees from < year to years old. epidemic curves ( figure ) show that the ngogo and kanyawara outbreaks each occurred in a single phase, with most cases occurring in january . at ngogo, . % of chimpanzees observed between december and february exhibited respiratory signs. at kanyawara, . % of chimpanzees observed during the same period exhibited respiratory signs. at ngogo, chimpanzees ( . %) died during the outbreak period (figure ). in contrast, no chimpanzees at kanyawara died during the outbreak period, with the exception of a female recovering from disease who died following conspecific aggression (see below). respiratory signs consisted of coughing, sneezing, dyspnea, and nasal exudate; other signs included lethargy, immobility, and dramatic loss of body condition ( figure s ). epidemiologic modelling of the ngogo and kanyawara outbreaks (table and figure s ) yielded daily transmission rate estimates of . and . , and durations of infectivity of . and . days, respectively. these parameters yielded basic reproductive numbers (r ) of . and . for ngogo and kanyawara, respectively. these are similar to values estimated from an outbreak of rhinovirus c in kanyawara in (table ) during which . % of chimpanzees died, and to published values for the human "common cold" [ ] . however, % confidence limits around these estimates were non-overlapping for daily transmission rates of all three outbreaks and duration of infectivity and r in ngogo (both lower than in kanyawara in or / ). risk factor analysis (table s ) showed that age significantly predicted morbidity at both ngogo (χ = . , df = , p = . ) and kanyawara (χ = . , df = , p = . ). in both communities, respiratory signs were least frequently observed among infants and increased through successive age categories. sex did not affect morbidity or mortality at ngogo, but females were significantly more likely to exhibit respiratory signs at kanyawara than were males (χ = . , df = , p = . ). at ngogo, age predicted mortality (χ = . , df = , p < . ), with mortality highest among infants (or . , % ci: . - . ) and individuals ≥ years old (or . , % ci: . - . ), compared to intermediate ages. at ngogo, the carcass of a -year-old female chimpanzee was recovered immediately after the onset of respiratory signs and subsequent death. post-mortem analysis of this individual revealed consolidation of the dependent lobes of both lungs and a serosanguinous pericardial effusion but no other gross pathologic abnormalities ( figure s ). at kanyawara, the carcass of a -year-old female chimpanzee was recovered approximately days after having recovered from respiratory signs (but still remaining weak), immediately after having been attacked by conspecifics (for unclear reasons). post-mortem analysis of this individual showed severe, diffuse pleuropneumonia with fibrinous adhesions to the thoracic wall and severe consolidation of all lobes of both lungs, as well as a serosanguinous pericardial effusion ( figure s ). analysis of paired fecal samples (prior to and during the outbreak period) using a luminex assay that tests for a suite of human respiratory agents revealed different viral etiologies for each community ( table ) . metapneumovirus (mpv, pneumoviridae: metapneumovirus) was detected in of individuals ( . %) from ngogo chimpanzees exhibiting clinical signs during, but not before, the outbreak period (fisher's exact p = . ). human respirovirus (hrv ; paramyxoviridae; respirovirus, formerly known as parainfluenza virus ) was detected in of individuals ( . %) from kanyawara chimpanzees exhibiting clinical signs during, but not before, the outbreak period (fisher's exact p = . ). adenoviruses (adenoviridae) were present in samples from both ngogo ( . %) and kanyawara ( . %) but showed no association with the outbreak period (fisher's exact p = . and . , respectively) and have been previously characterized in this population at comparable frequencies [ ] . enteroviruses (picornaviridae) were also present at low frequency in samples from both communities, similarly showed no association with the outbreak period (fisher's exact p = . in both cases), and have also been previously characterized in this population at comparable frequencies [ ] . metagenomic analysis of respiratory tract swab samples from the chimpanzee examined postmortem at ngogo yielded , , reads after trimming, of which , assembled to yield a coding-complete mpv genome of , bases with average coverage of , consistent with the luminex results described above. this genome (genbank accession number mh ) was most similar ( . %) to a human-derived variant from brazil (genbank accession number mg ). intriguingly, the virus was nearly as similar ( . %) to a variant from a mountain gorilla from rwanda in (genbank accession number hm ) detected during a lethal outbreak [ ] . mpv rna was present in all sections of the respiratory tract, including the lung parenchyma, with the proportion of viral sequence reads declining monotonically from the upper to the lower respiratory tract ( figure s ). sequencing of a nucleotide portion of the viral f gene from fecal samples was successful for three other luminex-positive chimpanzees at ngogo (ab, mi and wi; table ), yielding identical sequences within this variable genomic region (gen-bank accession numbers mh -mh ). by contrast, neither hrv nor any other virus was detected in the respiratory tract of the chimpanzee that died at kanyawara, likely reflecting prior infection and viral clearance. a coding-complete hrv genome ( , bases) was therefore reconstructed from a fecal sample from this same individual collected when she was coughing approximately weeks earlier, using pcr with virus-specific primers and sanger sequencing (table s ). this genome (genbank accession number mh ) was most similar ( . %) to a human-derived variant from the usa (gen-bank accession number ky ). sequencing of a variable and epidemiologically informative nucleotide portion of the viral f gene from fecal samples was successful for three other luminex-positive chimpanzees at kanyawara (al, an and az; table ), yielding identical sequences within this genomic region (genbank accession numbers mh -mh ). re-analysis of respiratory tract metagenomic data from this individual and the individual from ngogo revealed that a small proportion of reads in the upper respiratory tracts of both animals ( . % and . %, respectively) mapped to the reference genome of staphylococcus pneumoniae (genbank accession number nc_ ), which can infect chimpanzees secondarily during viral respiratory disease outbreaks [ , ] , indicating the presence of this or a related bacterium; however no reads mapped to this organism in the lungs of either animal. phylogenetic analysis revealed mpv from the ngogo outbreak to sort within a sub-clade of subtype b [ ] viruses from brazil, peru, rwanda and the usa ( figure ). this sub-clade contains recently collected viruses ( ) ( ) ( ) ( ) ( ) ( ) ( ) , including the mountain gorilla variant from rwanda. the mpv variant from ngogo belongs to a different subtype than a previously reported b virus from a chimpanzee outbreak in tanzania [ ] , and it is less closely related to a previously reported b subtype from a chimpanzee outbreak in cote d'ivoire [ ] than to other variants within its subclade, including the gorilla-derived variant, based on the available nucleotide region of the viral p gene (genbank accession numbers eu -eu ; not shown). phylogenetic analysis revealed hrv from the kanyawara outbreak to sort within a sub-clade of highly similar human-derived viruses from peru and the usa collected between and (no more recently than other sub-clades; figure ). notably, hrv from kanyawara was divergent from viruses from other non-human primates, including viruses from wild zambian baboons (papio cynocephalus) [ ] and simian agent , originally isolated from a blue monkey (cercopithecus mitis) in south africa [ ] . outbreaks of severe and sometimes lethal respiratory disease are common in chimpanzees across sub-saharan africa, but their causes often remain undetermined [ , ] . consequently, and because of differences in field protocols among research sites, it has proven difficult to compare these outbreaks directly. the simultaneity of the two / outbreaks at ngogo and kanyawara, coupled with coordinated data and sample collection efforts between the two sites, allowed a rare direct comparison to be made. analyses demonstrate that the two viruses displayed different pathogenicities and different epidemiological dynamics in the two chimpanzee communities. mpv at ngogo caused substantially higher mortality than hrv at kanyawara, despite causing apparently lower morbidity. however, the estimated daily transmission rate and duration of infectivity of mpv at ngogo differed from corresponding values for hrv at kanyawara (table ), suggesting that, despite comparable r values, the viruses displayed different epidemiologic dynamics. higher mortality due to mpv in infants and older adults compared to intermediate age categories is congruent with data from humans, where infants, young children and older adults are most susceptible to severe clinical manifestations of mpv infection [ , ] , as well as with demographic data from prior mpv outbreaks in chimpanzees [ , ] . neither virus was detected in chimpanzees immediately prior to the outbreaks ( table ) . mpv was detected in . % of affected ngogo chimpanzees sampled during the outbreak, and hrv was detected in . % of affected kanyawara chimpanzees sampled during the outbreak. viral sequences from affected individuals were identical within the variable region sequenced. mpv and hrv were therefore likely single-origin causes of their respective epidemics. adenoviruses and enteroviruses were present both before and during the outbreaks at statistically indistinguishable (adv) and low (ev) frequencies. adenoviruses occur in both healthy and ill wild apes [ ] [ ] [ ] [ ] and in chimpanzees during respiratory outbreaks [ ] , including at kanyawara [ ] , leading to debate about their etiologic role; however, our results support previous conclusions that these viruses are most likely benign. our results support a similar conclusion for enteroviruses, with the notable exceptions of polio virus [ , ] and rhinoviruses [ ] , which can cause deadly disease in chimpanzees. we note that, at the time of the outbreaks, the ngogo and kanyawara epidemics were assumed to have the same cause, with concomitant fears that an infectious agent was spreading rapidly across kibale national park. moreover, avian influenza had recently been reported in waterfowl in lake victoria, on the other side of uganda [ ] . our results highlight how heightened vigilance for emerging infectious diseases can sometimes lead to erroneous inferences. common environmental drivers (e.g. weather, human visitation patterns) may precipitate simultaneous outbreaks of distinct anthroponotic viruses in wild apes, creating the appearance of a single disease with a seasonal pattern. our results also suggest that the transmission of respiratory viruses between chimpanzee communities is probably uncommon, despite high rates of transmission within chimpanzee communities. chimpanzees from neighbouring communities rarely interact [ ] , which may limit opportunities for intercommunity transmission even of highly contagious microbes. mpv and hrv both represent significant burdens to global public health [ ] . mpv infection in humans table s . causes coughing, nasal discharge and weight loss [ ] ; in non-human models signs are often more severe than for other related viruses [ ] . mpv also causes clinical disease in experimentally infected chimpanzees [ ] and severe and sometimes lethal outbreaks of respiratory disease in wild chimpanzees and gorillas, often with similar demographic patterns of infection as we describe herein [ , , , ] , as does a related virus, respiratory syncytial virus (pneumoviridae, orthopneumovirus) [ ] . our results show that this comparatively high virulence of mpv is recapitulated in naturally infected chimpanzees. hrv causes respiratory disease and asthma exacerbation in people worldwide, especially in children and the elderly, but it is less virulent than mpv [ ] . hrv has a wide host range, including primates, ruminants and rodents [ ] . simian agent , first isolated in from an apparently healthy blue monkey [ ] , was later identified as hrv and attributed to anthroponotic transmission [ ] . hrv has been detected in the respiratory tracts of apparently healthy wild yellow baboons in zambia [ ] . experimental infection of marmosets (saguinus mystax) with hrv causes acute and transmissible disease [ ] , but experimental infection of other primates, including rhesus macaques (macaca mulatta) and chimpanzees [ ] , is milder. our results mirror these findings and, more generally, show that some negative-sense rna respiratory viruses of human origin cause only mild and self-limiting infections in wild chimpanzees. we cannot exclude the role of other co-infecting pathogens in modulating the pathogenicity of mpv and hrv in our study populations. for example, hrv can cause upper respiratory disease and predispose chimpanzees to invasive pneumococcal infection [ ] , and the bacterium streptococcus pneumoniae co-occurs with human metapneumoviruses and respiratory syncytial viruses in both wild and in captive apes [ , ] . our finding of a low percentage of metagenomic sequence reads matching s. pneumoniae (or a related bacterium) in the upper respiratory tracts of both necropsied individuals, but not in their lungs, supports the notion that wild chimpanzees host microbes that could easily invade the lower respiratory tract following primary viral infection. population surveys of endemic microbes and serologic assessments of prior exposure to agents that might predispose chimpanzees to infection or enhance pathogenesis could prove informative. clinical signs in ngogo chimpanzees were congruent with those in severely infected humans [ ] . one adult female at ngogo ("kidman") has exhibited persistent wheezing since the outbreak; in human children suffering severe mpv infections, subsequent wheezing episodes are not uncommon [ ] . post-mortem analysis of one individual at ngogo revealed surprisingly little gross pathology of the respiratory tract, consistent with acute infection and rapid death, and mpv was recovered from all sections of that individual's respiratory tract, including the lung parenchyma ( figures s and s ). in contrast, post-mortem analysis of an individual at kanyawara revealed advanced respiratory tract pathology, consistent with longer term infection ( figure s ), but we did not recover hrv from this individual's respiratory tract despite recovering a complete hrv genome from this same individual's feces collected approximately weeks earlier. this individual had likely recovered from the initial viral challenge, cleared the virus, and perhaps acquired a secondary bacterial infection [ , ] . notably, both individuals had pericardial effusions, which in humans are rare but serious sequellae of certain viral infections [ ] . phylogenetic analyses revealed that mpv from ngogo and hrv from kanyawara are each closely related to globally circulating human viruses, indicating independent anthroponotic sources for both viruses. apparent geographic associations in these phylogenies likely represent bias in available sequence data (e.g. high representation of viruses from peru and the usa; table s ). the ngogo mpv variant clusters within a sub-clade of viruses that is more recent ( - ) than other sub-clades, perhaps reflecting antigenic replacement of mpv strains over time. notably, the ngogo variant is very similar to a virus from a fatal infection of a mountain gorilla in rwanda in [ ] . this particular viral lineage may thus have a propensity for infecting apes, or it may simply circulate widely in east africa. the virus is divergent, however, from previously described, lethal viruses from wild chimpanzees in tanzania [ ] and cote d'ivoire [ ] , indicating that multiple mpv variants can infect and kill wild chimpanzees. the pathways by which these viruses entered chimpanzees from humans remain vexingly obscure. people frequent the habitats of both chimpanzee communities for research and associated activities (not solely focused on chimpanzees) and to travel or collect forest products [ ] . tourism is sometimes cited as a risk for anthroponotic transmission of viruses to great apes and helps inform current international union for the conservation of nature (iucn) recommendations for preventing such transmission, such as visitor vaccination requirements, minimum observation distances, alcohol-based sanitizing gel and facemasks [ ] . however, neither ngogo nor kanyawara is a tourism site. ngogo is bordered by a tourism site (kanyanchu) receiving thousands of visitors each year, but there were no reports of a concurrent respiratory disease outbreak in the kanyanchu chimpanzees, although similar events have occurred there at other times. at gombe national park in tanzania, provisioning chimpanzees with bananas (a discontinued practice) was, along with season, the strongest predictor of chimpanzee respiratory signs [ ] , which affected between % and % of chimpanzees per month between and [ ] . however, neither the ngogo nor the kanyawara chimpanzees are provisioned, and biosecurity measures based on iucn recommendations [ ] for visitors to both sites are in place. at kanyawara, data over years show a seasonal pattern of respiratory illness in chimpanzees, typically peaking in march, but no correlation between clinical signs in chimpanzees and seasonal patterns of respiratory illness in humans living nearby [ ] . the role of other species in transmitting these viruses from humans to chimpanzees is also unclear. kibale national park contains a diversity of primate species and social groups, none of which were observed with respiratory signs at the time of the outbreaks, but complex patterns of cross-species transmission from humans to chimpanzees cannot be discounted. such possibilities could be investigated through broad sampling of people (especially research staff) and wildlife during future outbreaks, including serologic assessments of prior exposure. overall, these findings broaden our understanding of human viruses that can cause disease in wild chimpanzees and show that their clinical manifestations can vary markedly. our study also shows that epidemiological analyses, enabled by prospective, coordinated observational data collection, may be able to distinguish among viruses based on early-stage infection patterns. if so, "real-time" epidemiologic analyses would be a valuable complement to molecular diagnostic testing, in that they could help guide response strategies as epidemics progress. epidemiologic analyses could also inform future monitoring efforts to detect and minimize the impact of anthroponotic transmission events on wild ape populations. the study was observational and non-invasive. protocols were approved by institutional animal care and use committees (iacuc) of harvard university (protocol - ) and university of new mexico (protocol - -mcc) and were exempt by boston university's and the university of michigan's iacuc. protocols followed the weatherall report, nih guide for the care and use of laboratory animals, usda animal welfare act, institute for laboratory animal research guide for the care and use of laboratory animals, us public health service and national academies of sciences national research council, and us centers for disease control and prevention. during the outbreaks, trained field assistants collected individual-level observational data on the chimpanzees daily. chimpanzees in both the ngogo and kanyawara communities are individually identifiable, making such data possible to collect. we compiled these data into measures of clinical signs (coughing or sneezing, further classified as mild or severe), and observation time per chimpanzee. we estimated dates of mortality events as the midpoint of the last date a chimpanzee was seen alive and the first date it was absent from a group containing frequent associates, including dependent offspring. to infer epidemiological transmission parameters, we constructed two sir (susceptible-infectious-removed) mathematical modelsone for each communityfollowing althaus [ ] , fitting curves to cumulative incidence data using the nelder and mead optimization algorithm in the optim package in r, version . . [ ] . for both communities, we assumed free admixture and did not incorporate heterogeneity of social interaction to minimize model complexity, as we did previously for a rhinovirus c outbreak in kanyawara [ ] . we analysed risk factors for morbidity and mortality using a generalized linear model (glm) with binomial distribution and log-link function g in r [ ] . age, sex and their interaction were included as predictor variables, and observation hours were included as a control variable. age was modelled as a categorical variable with four levels: infants (< years), juveniles ( - . years), adults ( - . years) and older (≥ years). at ngogo, where mortality occurred, individuals not observed with clinical signs during the outbreak period that disappeared were coded as having died and as positive for respiratory signs. because none of these individuals were adolescent females (the only age-sex category of chimpanzee that can migrate between communities), their sudden disappearance (and the fact that they have never been seen since) confirms that they had, in fact, died. we collected fecal samples from individual chimpanzees in both communities during the outbreak period. two millilitres of feces were placed immediately into rnalater buffer (thermo fisher scientific, waltham, ma, usa) at a : ratio, homogenized and stored at − °c until exported to the united states. paired fecal samples were available from clinically ill chimpanzees ( from ngogo and from kanyawara) during and before (fourth quarter of ) the outbreak period, and we tested these for a suite of respiratory pathogens using the nxtag respiratory pathogen panel (luminex corporation, austin, tx, usa) as previously described [ ] , which tests for influenza virus a (multiple subtypes), human respiratory syncytial viruses a and b, coronaviruses (multiple subtypes), human metapneumovirus, rhinovirus/enterovirus, adenovirus, parainfluenza viruses - , bocavirus, and the bacterial pathogens chlamydophila pneumoniae and mycoplasma pneumoniae. necropsies were performed on two chimpanzees recovered immediately after death (the only two carcasses recovered, despite extensive effort) by trained veterinarians wearing appropriate personal protective equipment. one chimpanzee from ngogo died on january ("stella," a -year-old female) and the other chimpanzee from kanyawara died on january , ("rwanda," a -year-old female). in the latter case, the individual had been coughing days earlier and was attacked repeatedly by conspecifics while still weak; the resulting trauma appeared to be the immediate cause of death. samples (sterile swabs with plastic shafts and dacron tips) were collected from the nose, larynx, trachea, bronchi and lung parenchyma of both individuals and stored in . ml rnalater buffer at − °c. swabs were homogenized and total rna was extracted and converted to cdna libraries in the field, then prepared for sequencing on an illumina miseq instrument (illumina, san diego, ca, usa) using bp paired-end read chemistry as previously described [ ] . polymerase chain reaction (pcr) and sanger sequencing were used to complete viral genomes and to characterize viruses detected by luminex assay in fecal samples (table s ). to infer phylogenetic relationships among the two chimpanzee viruses and published viral sequences, we analysed full genome alignments of all available viruses in genbank (alignment lengths , and , positions, respectively) using phyml . 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phyml . metagenomic assessment of adventitious viruses in commercial bovine sera we thank the uganda wildlife authority, and especially c. tumwesigye, for ongoing help and support. we are also grateful to the uganda national council for science and technology and makerere university biological field station for permission to conduct this research. we thank the field assistants of the ngogo and kibale chimpanzee projects for help in data and sample collection, and k. sabbi, r. donovan and y. bochkov for invaluable assistance, input and discussion. no potential conflict of interest was reported by the authors. key: cord- -i cfmw x authors: peng, ju-yi; horng, yi-bing; wu, ching-ho; chang, chia-yu; chang, yen-chen; tsai, pei-shiue; jeng, chian-ren; cheng, yeong-hsiang; chang, hui-wen title: evaluation of antiviral activity of bacillus licheniformis-fermented products against porcine epidemic diarrhea virus date: - - journal: amb express doi: . /s - - - sha: doc_id: cord_uid: i cfmw x bacillus licheniformis (b. licheniformis) is commonly used as probiotic and its secondary metabolites are attractive anti-microbial candidate. in the present study, we aimed to evaluate the antiviral activity of crude extracts from b. licheniformis against porcine epidemic diarrhea virus (pedv), a highly contagious enveloped porcine virus that has caused great economic loss in pigs. in vivo, pedv-infected piglets supplemented with air-dried solid state fermentative cultivate containing b. licheniformis-fermented products (blfp) showed milder clinical symptoms and decreased viral shedding. importantly, no significant systemic pathological lesions and no reduction in average daily gain were noted in pigs supplemented with the blfp, which suggests that it is safe for use in pigs. in vitro experiments revealed that while b. licheniformis crude extracts exhibited no toxicity in vero cells, co-cultivation of b. licheniformis crude extracts with pedv significantly reduced viral infection and replication. summarized current results suggest that the b. licheniformis-fermented products could be a novel candidate food additive for reducing the impact of ped on the swine industry. beginning in , outbreaks of new variants of porcine epidemic diarrhea virus (pedv) have caused high mortality and morbidity in piglets, leading to great economic losses (lee ) . the virus causes porcine epidemic diarrhea (ped), which is characterized by watery diarrhea, vomiting, and severe dehydration in pigs of all ages, especially suckling piglets (lee ; song and park ) . this highly contagious disease has spread quickly in porcine industries in several countries (jung and saif ; lee ) . furthermore, pedv infection causes severe perturbations of gut microbiota, reducing probiotic bacterial abundance, enriching pathogenic bacteria, and even impairing the growth performance of pedv-surviving pigs (alvarez et al. ; deping song et al. ) . the development of effective protective agents against pedv infection is urgently needed. probiotics is one of the choices to be an alternative to antibiotic growth promoter, agp (abudabos et al. ) . our recent studies reported that lactobacillus species and clostridium butyricum-fermented probiotics product alleviate diarrhea incidence and reduce the gut pathogens in weaning piglets ). on the other hand, bacillus subtilis (b. subtilis) and b. licheniformisfermented products could increase growth performance and mitigate clostridium perfringens-induced necrotic enteritis in broilers (cheng et al. ; lin et al. ) . enhancement of nutrient digestibility and lactobacillus counts of feces by feeding b. subtilis and b. licheniformis has also been reported in pigs (lan and kim ) . it has been demonstrated that b. licheniformis exhibiting antimicrobial activity against pathogens might be due to the production of antibacterial biosurfactants (lin et al. ) . furthermore, our experiment results also demonstrated that b. licheniformis-fermented products exhibit antibacterial activities against clostridium perfringens and staphylococcus aureus in vitro (lin et al. ). in the present study, the antiviral effect of the blfp crude extract against pedv were evaluated in pigs. the surfactin-like peptide in the blfp crude extract was identified from the secondary metabolite of b. licheniformis fermentative cultivates. the in vitro toxicity and antiviral ability of the surfactin-like peptide in the blfp crude extract against pedv were evaluated using the vero cells. vero c cells (american type culture collection (atcc) no. crl- ) were maintained in dmem (gibco, ny, usa) supplemented with % fetal bovine serum (hyclone, utah, usa) and % penicillin-streptomycin-amphotericin b (gibco, ny, usa). the passage pedv-pintung strain (pedvpt-p ) viral stock was used at a titer of × tcid /ml. air-dried solid-state fermentative cultivates of b. licheniformis (weigmann) chester (atcc ® ™ ) were suspended in distilled water and heated at °c for min. supernatants were harvested after centrifugation at , rpm for min. after adjusting the ph value to . with n hcl for protein precipitation, the precipitates were dried in a freeze dryer after washing twice with distilled water. this b. licheniformis-fermented products (blfp) was used in animal study. to characterized the blfp, the surfactin derived from bacillus subtilis (sigma-aldrich, st louis, usa) was used as standard substance. the content of blfp in the fermentative product was determined by high-performance liquid chromatography (hplc) as previously described (schneider and marahiel ) . briefly, after filtration, samples were subjected to analysis via spd- a hplc (shimadzu, japan) with a pre-packed lichrospher rp- column (merck, darmstadt, germany). the mobile phase was a mixture of . mm trifluoroacetic acid and ml ddw with ml % methanol. elution was performed at a flow rate of ml/min and determined with a uv detector ( a vp, shimadzu, tokyo, japan) at nm. the gradient strategy was as follows: - . min, % a to % a; . - min, keeping % a and % b (a, acetonitrile; b, ultrapure water); min pressure bar, max pressure bar, pressure stability bar; injection volume μl; syringe speed μl/s; flush volume μl. liquid chromatography-mass spectrometry (lc-ms) full scan positive mode was performed with m/z ranging from to . fifteen -week-old, pedv-fecal rna and pedv seronegative, large white × duroc crossbred pigs were acquired from a conventional pig farm with no known history of ped. treatments were: ( ) control (n = ); ( ) pedv (n = ); and ( ) pedv + blfp (n = ). these pigs were fed a commercial diet mixed with or without kg/l blfp as feed additives for days prior to the viral challenge, and were challenged with or without × tcid of the virulent pedvpt-p (p ) at weeks of age (table ) . each group was housed in a separate fenced area. clinical symptoms, fecal consistency scoring, and fecal viral shedding were recorded and tested daily. the clinical scores were recorded using a -scale: , normal feces; , pasty; , semi-fluid; , fluid. all piglets were weighed weekly and sacrificed days post-infection (dpi) for safety assessment by pathological examination. the animal experiment was approved by the institutional animal care and use committee of national taiwan university (taipei, taiwan, ntu -el- ). real-time quantitative rt-pcr (qpcr) was performed based on a previously described method with modification (chang et al. ) . the viral rna was extracted from μl of culture supernatants using cador ® pathogen qiacube ® (qiagen, chatsworth, ca, usa) according to the manufacturer's instructions. reverse transcription was performed using the quantinova reverse transcription kit (qiagen) according to the manufacturer's protocol. for qpcr assay, the quantinova probe master mix of fecal swabs were used to determine the genomic equivalents (ge). the detection limit of the real-time rt-pcr was ge of dna using the plasmid encoding the pedv n gene as a standard (data not shown). all pigs were euthanized and necropsy was performed at dpi to evaluate the safety of blfp in pigs. representative tissue samples were collected, fixed in % neutral-buffered formalin, processed routinely, sliced into -μm-thick sections, and stained with hematoxylin and eosin. the histopathological observations were recorded and assessed blindly by one veterinary pathologist. to evaluate the cytotoxicity of blfp crude extract in vitro, vero cells were first grown in a -well microplate (corning life sciences, corning, ny, usa) at a density of , cells per well day prior to the experiment. after removing the culture supernatant, ten-fold serially diluted blfp crude extract in pi medium (dmem supplemented with . % tryptose phosphate broth (sigma-aldrich, mo, usa), . % yeast extract (acumedia, michigan, usa), and μg/ml of trypsin (sigma-aldrich, mo, usa) was added to the cell monolayer. after h, the culture supernatant was removed and μl alamarblue ™ was added (g-biosciences, st. louis, usa; % in pbs). after h of incubation at °c, the plate was read with the excitation wavelength at nm and with the emission wavelength at nm. the od value of each well was calculated to obtain the percentage reduction of alamarblue according to the manufacturer's instructions. additionally, an untreated group g , which consisted of vero cells without any treatment, was included to represent % reduction of alamarblue. all data were further calculated as a percentage of the untreated group g . normalized data were plotted against concentrations of blfp crude extract and fitted to a non-linear regression curve using graph-pad prism (graphpad software, san diego, ca). the % cytotoxicity concentration (cc , the concentration of blfpat which cellular viability was reduced to %) was calculated accordingly. to study the antiviral activity of blfp crude extract against pedv, the biosurfactants were added at different time points during the viral infection. vero cells were seeded in a -well microplate ( , cells/well) day before the experiment. blfp crude extract at ppmin pi medium were added to each well in different orders to treat tcid /ml pedv infections in cells. the experiment included five groups (as illustrated in fig. a to further investigate the antiviral mechanism in the post-drug group (g ), the replication kinetics of pedv in vero cells with or without crude extract of blfp were compared. vero cells were seeded in a -well microplate ( , cells/well) day before the experiment. the crude extract of blfp at ppm in pi medium was added to tcid /ml pedv-infected cells. culture supernatants and cells were collected separately at the , , , , and h post-infection. viral titers in culture supernatants were determined using the reed-müench method (reed ) and are expressed as the % tcid /ml. virus-specific rna in culture supernatants and in cells was quantified by real-time reverse transcription-pcr. virus titration was performed in the culture supernatants. briefly, the first -fold dilution and the subsequent -fold serial dilution of the supernatants were added to , cells/well vero cells on -well microplates. after h of incubation, the inoculants were aspirated, and the cells were washed with pi medium two times and then cultured in pi medium. after h, the plate was subjected to cytopathic effect (cpe) observation and the titer of pedv was determined using the reed-müench method (reed ) and is expressed as the % tcid /ml. to determine the ic , vero cells were seeded in -well microplates ( , cells/well) one day before the experiment. ten-fold serially diluted crude extract of blfp in pi medium was added to vero cells h after % tissue culture infectious dose (tcid ) pedv infection. after h, the percent reduction of alamarbluestaining was examined by the alamarblue ™ assay as described above. all normalized data were plotted and fitted to a nonlinear regression curve in graphpad prism (graphpad software) to generate the ic . to elucidate the direct virucidal activity of crude extract of blfp against pedv, the pedvpt-p viral stock at a titer of × tcid /ml was mixed with or without ppm blfp for h at °c. the mixtures were serially diluted -fold in pi medium and added to vero cells. after h, the viral titers were determined as described above. all data were analyzed and plotted using graphpad prism (graphpad software). all error bars represent standard deviation (sd). the significance of the differences among groups in the cell-based study was determined using student's t test or one-way anova with tukey's multiple comparison. a p-value of < . was interpreted as statistically significant. the results of the average daily gain (adg) and fecal rna shedding in the animal experiments were analyzed and the variables among groups were compared using a non-parametrical kruskal-wallis test, with p < . considered significant. all data were analyzed using graphpad prism software (graphpad prism inc.) as shown in fig. , hplc analysis of the b. licheniformis extract showed a single peak (fig. b) at the retention time of - min, which is identical to the standard substance surfactin derived from bacillus subtilis (fig. a) . (fig. ) . to assess the efficacy of the antiviral ability of blfp against pedv, clinical symptoms after viral challenge were recorded daily (fig. ) . the fecal conditions of pigs in all groups were scored as normal (score = ) before pedv inoculation. in general, pigs supplemented with blfp exhibited milder symptom compared to piglets supplemented with controls. in the control group, all five pigs ( / ) exhibited normal clinical signs (score = ) during the study. in the pedv group in which pigs were supplemented with control food, different severities of pedv-associated clinical signs were first detected days post infection (dpi). two of these five pigs ( / ) exhibited semi-fluid feces (score = ) at dpi, while / pigs showed typical semi-fluid (score = ) to watery diarrhea (score = ) at to dpi, and eventually recovered after dpi. in the pedv + blfp group, most of the pigs exhibited pasty feces (score = ) at - dpi. only one pig exhibited transient typical watery diarrhea (score = ) at dpi. compared to the pedv-infected group with control food, the severity and duration of typical diarrhea (scores - ) were reduced in the pedv + blfp group. to quantify pedv-associated fecal viral shedding to evaluate the antiviral efficacy of blfp against pedvpt-p , rt-qpcr was used to detect fecal viral shedding. in the pedv group (the grey line in fig. ) , fecal viral shedding was first detected ( . ± . log ge) at dpi, found to gradually increase to peak viral load ( . ± . log ge) at dpi, and was continuously detected until dpi. in the pedv + blfp group (the black line in fig. ) , the pattern of viral shedding was similar to but lower than that inthe pedv group during the study, although the difference was not statistically significant. all pigs were weighed weekly in order to evaluate their growth performance with and without biosurfactants as a feed additive. no significant difference in the average daily gain (adg) of pigs was noted among all groups in each week (fig. ) . a b fig. clinical scoring of fecal consistency. compared to pedv-infected groups, the appearance and duration of typical diarrhea (score - ) were reduced in the pedv + blfp group to evaluate the safety of blfp as a feed additive for pigs, necropsy was performed in all piglets weeks postinfection for gross and histopathological examination. for the gross and histopathological examinations, no macroscopic or microscopic lesions were noted in any of the groups. these findings suggest that blfp as a feed additives at kg/ton are safe in conventional pigs up to days of feeding. to determine the median cytotoxic concentration (cc ) of blfp crude extract in vero cells, the blfp crude extract was serially diluted -fold from to . ppm and added to cells. h post-incubation (hpi), no cytotoxicity due to blfp crude extract was observed in vero cells (fig. ) . to determine the maximal inhibitory concentration, the post-drug group g was used to evaluate the maximal inhibitory effect of blfp crude extract by cocultivating fig. fecal shedding of pedvpt-p detected in piglets fed < kg/ton blfp in each group (n = ). the pattern of viral shedding in the pedv + blfp group was similar to but lower than that of the pedv group during the study, with no significant difference detected. changes in the mean values of genomic equivalents (ge)/ml are presented as log values ± sd. the viral rna loads of inoculated groups and the control group were compared using a non-parametrical kruskal-wallis test fig. average daily gain of pigs in each group. all pigs were weighed weekly in order to evaluate their growth performance with or without blfp as feed additives. no statically significant difference in the average daily gain was noted among all groups each week blfp crude extract with pedv-infected cells during the whole study. the results indicated that the antiviral activity of blfp crude extract is dose-dependent, and the inhibition of pedv-induced cpe was calculated with an ic value of . ± . ppm (fig. ) . to elucidate the antiviral activity of blfp crude extract against pedv, ppm of blfp crude extract was added to the culture at different time points during the viral infection. as shown in fig. a , typical pedv-induced cpe was characterized by formation of syncytial cells, cell death, and detachment from the culture plate in pedv-infected cells. the percent reductions of alamar-blue staining in g -g -cells treated with blfp crude extract h before infection, at the same time as infection, and h after viral infection, then replaced with fresh pi medium-were calculated as . ± . %, . ± . %, and . ± . %, respectively, and showed no significant dose-dependent effect of blfp crude extract against pedv. the half-maximal inhibitory concentration (ic ) of blfp crude extract against pedv in vero cells is . ± . ppm. data are presented as the mean ± sd out of three test replicates differences from those in the pedv-infected group g (fig. b) . for the post-drug group g , significant inhibition of pedv-induced cpe, with an . ± . % reduction of alamarblue staining, was observed compared to that in g and other groups (g -g ). similarly, the amount of viral rna in supernatants from g , ranging from . to . log ge, was significantly lower than that of g -g , ranging from . to . log ge (fig. c) . additionally, the viral titer of supernatants in the g group, which was less than tcid /ml, was lower than that of g -g , ranging from × to × tcid /ml. moreover, when evaluating the direct virucidal activity of blfp crude extract against pedv, a reduction of the viral titer of pedv from to tcid /ml was observed. to further investigate the antiviral mechanism in the post-drug group (g ), the replication kinetics of pedv in vero cells in the presence or absence of blfp crude extract were determined. compare to pedv-infected vero cells cultured without blfp crude extract, which showed viral titers from . × at hpi to . × tcid /ml at hpi (fig. a) , cells treated with blfp crude extract had an undetectable extracellular viral titer, which was significantly lower than that of cells without blfp crude extract. similarly, extracellular viral rna levels in pedv-infected cells cultured with biosurfactants were significantly lower than those without blfp crude extract and hpi (fig. b) . however, no significant differences in intracellular viral titers (fig. c ) or intracellular viral rna (fig. d) were noted in pedv-infected cells treated with or without blfp crude extract. since late , new variants of pedv have been identified in several counties and have caused a devastating disease and economic loss. this viral threat has prompted a global effort to develop antivirals against new variant pedvs in vitro (song and choi ; kwon et al. ) and in vivo (cho et al. ; kim et al. ) . herein, we demonstrated that: ( ) blfp crude extract presents no cell toxicity to vero cells, and exhibits significant antiviral ability against pedvpt-p in vero cells when biosurfactants are co-cultivated with pedv; ( ) piglets supplemented with blfp at concentrations of kg/ton or less exhibited milder clinical symptoms and lower viral shedding compared to piglets supplemented with control food, and blfp caused no significant toxicity. therefore, blfp is suggested to be a promising novel feed additive that can act as an additive against pedv in the field. the main antiviral mechanism of blfp crude extract is thought to be related to its unique biochemical structure that increases membrane permeability to disrupt and lyse microbial membranes (huang et al. performing a direct-virucidal study of blfp crude extract against pedv, a ten-fold reduction of the viral titer was able to confirm the directed-antiviral ability of blfp crude extract. according to our hplc analysis, a peak of surfactin-like peptide was identified in the blfp crude extract. several different potential antiviral mechanisms of biosurfactants have been previously reported (vollenbroich et al. ; wang et al. ) . lipopeptide biosurfactants have been previously suggested to possess probiotic attributes for animal and human use (schneider ; sen ) . a previous study demonstrated that the antiviral ability of surfactin from b. subtilis was achieved through competition with the tgev entry receptor . the inhibition of viral enzymes such as proton-atpase that are required for the entry of some viruses into cells by surfactin from b. subtilis (vollenbroich et al. ) has also been reported. it is worth noting that not only did co-cultivation of the surfactin-like peptide in the blfp crude extract with pedv-infected vero cells significantly reduce viral infection and replication in the study, but blfp crude extract-treated pedvinfected cells also exhibited an undetectable extracellular viral titer, suggesting that blfp crude extract may play an important role in reducing the release of virions. these results suggest additional mechanisms of blfp crude extract against pedv in vero cells. although further study of the antiviral mechanisms of blfp crude extract is needed in the future, observation of the diverse antiviral activities of blfp crude extract will lead to further development of this new therapeutic candidate. previous studies demonstrated that lipopeptides produced by various bacillus spp. were safe in mice (youn-hwan hwang et al. ) and weaned piglets (torres et al. ) when administered under a certain concentration. in order to use blfp as a feed additive for its antiviral properties, assessment of its antiviral and safety profile in pigs is essential. our results demonstrated that piglets supplemented with blfp show milder symptoms fig. replication kinetics of pedv in vero cells treated with or without blfp crude extract. a extracellular viral titers in the supernatants of pedv-infected vero cells treated with and without blfp crude extract were determined by viral titration in vero cells using the reed-müench method and expressed as the % tcid /ml. b extracellular viral rna levels in the supernatants of pedv-infected vero cells treated with or without blfp crude extract were determined by real-time reverse transcription (rt)-pcr. c intracellular viral titers in pedv-infected vero cells treated with or without blfp crude extract were determined by viral titration in vero cells. d intracellular viral rna levels in the supernatants of pedv-infected vero cells treated with or without blfp crude extract were determined by real-time rt-pcr. statistical analysis was performed using student's t-test, and statistically significant differences were labeled with *(p < . ) and have reduced viral shedding. importantly, no significant systemic pathological effects and no changes in average daily gain were noted. these results suggest that the biosurfactants at concentrations of kg/ton or less will be very safe in pigs in future applications. in a previous study, it was found that administration of surfactin from b. subtilis at over kg/ton leads to necrosis of hepatocytes in rats (hwang et al. ). in the future, animal experiments using higher dosages of biosurfactants would be necessary to validate its maximal safety dosage. in the present study, blfp was investigated as an antiviral candidate against pedv. in vitro, blfp crude extract exhibited antiviral activities against pedv when incubated long-term with pedv-infected cells. in vivo, we first validated the safety and antiviral ability against of blfp as a feed additive less than kg/ton against pedv after a long period of pedv inoculation. our results suggest that blfp could be a novel candidate feed additive for reducing the titer of pedv in the swine industry. effect of organic acid blend and bacillus subtilis alone or in combination on growth traits, blood biochemical and antioxidant status in broilers exposed to salmonella typhimurium challenge during the starter phase impact of porcine epidemic diarrhea on performance of growing pigs evaluation and comparison of the pathogenicity and host immune 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produced by bacillus subtilis, in rats porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis medicinal herb extracts ameliorate impaired growth performance and intestinal lesion of newborn piglets challenged with the virulent porcine epidemic diarrhea virus in vitro antiviral activity of phlorotannins isolated from ecklonia cava against porcine epidemic diarrhea coronavirus infection and hemagglutination effects of bacillus licheniformis and bacillus subtilis complex on growth performance and faecal noxious gas emissions in growingfinishing pigs porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus optimization of solid-state fermentation conditions of bacillus licheniformis and its effects on clostridium perfringens-induced necrotic enteritis in broilers. r bras zootec :e a simple method of estimating fifty percent endpoints genetic evidence for a role of thioesterase domains, integrated in or associated with peptide synthetases, in non-ribosomal peptide biosynthesis in bacillus subtilis quercetin -rhamnoside reduces porcine epidemic diarrhea virus replication via independent pathway of viral induced reactive oxygen species porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines safety assessment of the antimicrobial lipopeptides synthesized by bacillus subtilis subsp. subtilis cbmdc f mechanism of inactivation of enveloped viruses by the biosurfactant surfactin from bacillus subtilis bacillus subtilis and surfactin inhibit the transmissible gastroenteritis virus from entering the intestinal epithelial cells evaluation of genetic and developmental toxicity of surfactin c from bacillus subtilis bc publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations authors' contributions j-yp, y-bh, and c-yc have drafted, revised the work, and conducted the experiments; y-bh and y-hc provide specimens and substantial contributions to the conception and analysis; h-wc, y-cc, p-st, and c-rj help to interpret data; y-hc and h-wc. design the work and approve the submitted version. all authors read and approved the final manuscript. the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. the animal experiment was approved by the institutional animal care and use committee (iacuc) of national taiwan university (taipei, taiwan, ntu -el- ). not applicable. the authors declare that they have no competing interests. key: cord- -gq lp authors: becker, daniel j.; washburne, alex d.; faust, christina l.; pulliam, juliet r. c.; mordecai, erin a.; lloyd-smith, james o.; plowright, raina k. title: dynamic and integrative approaches to understanding pathogen spillover date: - - journal: philosophical transactions of the royal society b: biological sciences doi: . /rstb. . sha: doc_id: cord_uid: gq lp nan pathogen spillover is the process by which a pathogen is transmitted from a reservoir host species to a recipient host species [ , ] . the term is sometimes used more broadly, particularly in public discourse, blending in elements of onward transmission in the novel host species or even pathogen adaptation to the novel host [ , ] . this theme issue focuses on pathogen spillover sensu stricto, except where explicitly noted. many of the examples considered pertain to zoonotic spillover (i.e. from wildlife or domestic animals to humans), given recent epidemics (e.g. ebola virus [ ] ) and pandemics (e.g. h n influenza virus [ ] ); however, we emphasize the general methods and mechanisms involved in understanding spillover between any two species, such as those that threaten wildlife conservation (e.g. mycoplasma ovipneumoniae from domestic sheep to bighorn sheep [ ] ) and the agricultural sector (e.g. brucella abortus from elk to cattle [ ] ). spillover requires the spatial and temporal alignment of several hierarchical factors that must occur for a pathogen to be transmitted from a reservoir or source host to a recipient host of a different species [ ] . these factors include reservoir host distribution and abundance, pathogen prevalence and shedding from reservoir hosts; pathogen survival in the environment or arthropod vector; recipient host contact with the infectious agent, reservoir host or arthropod vector; and susceptibility of the recipient host. following spillover, another suite of factors determines whether a pathogen is transmitted within the recipient host population (e.g. [ ] ). research on pathogen spillover is often focused on a single component of this process through the lens of a particular discipline. for example, the distribution of reservoir hosts is often studied through ecology, contact between reservoirs and humans is often studied via epidemiology or anthropology, and the pathogenesis of zoonoses in humans is often studied with medical microbiology and immunology. while each factor must be studied and quantified, spillover is the emergent property of these collective processes. studying each factor in isolation fails to account for the hierarchical and often nonlinear dynamics of the spillover system [ ] . pathogen surveillance, epidemic preparedness and management interventions would all benefit from integrative approaches that consider multiple components of pathogen spillover [ ] . this theme issue stemmed from a workshop on cross-species transmission of pathogens, where participants from interlinked fields including ecology, mathematical modelling, epidemiology, virology and immunology discussed how to better understand and predict pathogen spillover. here, we bring together a diverse set of perspectives-including empirical research, theory and synthetic reviews-to highlight cutting-edge research and to provide a roadmap for quantifying and integrating hostpathogen dynamics at each step in the spillover process. manuscripts are organized around three approaches. the first set of manuscripts focuses on integrating data streams to understand spillover dynamics and predict risk. the second set of manuscripts focuses on in-depth analysis of each of the factors affecting cross-species transmission: infection dynamics in reservoir hosts, pathogen survival in the environment, recipient host exposure, dose -response relationships and establishment of infection in recipient hosts. the final set of manuscripts focuses on applied perspectives, with an emphasis on surveillance and interventions. here, we summarize these contributions to highlight key insights, methodologies and future directions to improve our understanding of pathogen spillover. because spillover is the outcome of multiple ecological, epidemiological and immunological factors aligning in space and time [ , ] , predictive frameworks aim to integrate data pertinent to these factors to quantify the relative importance of these processes and to estimate risk. cross et al. [ ] review approaches for estimating spatio-temporal variation in spillover risk, focusing on the wildlife -livestock interface. the authors highlight the challenges inherent in either correlating observed spillover events with relevant covariates or integrating data on host density, distribution and pathogen prevalence using mechanistic models. they highlight that mechanistic approaches may be especially useful in systems where spillovers are infrequent, rarely observed or hard to differentiate from within-species transmission; however, linking datasets on different factors in the spillover pathway requires that such datasets be related to a common spatial and temporal resolution. the authors use case studies of brucellosis in the greater yellowstone ecosystem [ ] and of avian influenza virus in china and north america [ ] to emphasize potential solutions to these challenges for estimating spillover risk. emphasizing that statistical modelling efforts may struggle to detect nonlinear and stochastic relationships inherent in pathogen spillover, childs et al. [ ] provide a strong test of theory governing how hierarchical barriers control crossspecies transmission [ ] . the authors focus their case study on yellow fever, a mosquito-borne viral disease of historical importance in south america that persists in the region largely in sylvatic cycles that occasionally spill over to infect humans [ , ] . specifically, they use mechanistic models that incorporate spatial ecological and immunological data from brazil across years to predict yellow fever spillover in humans. the authors show that a mechanistic model of spillover risk, based on the ecology of mosquito vectors and non-human primate reservoirs, best predicts spillover events compared with models that also include human population size and immunity. this result arises because spillover occurs even in areas with low human population density and high vaccination coverage (e.g. parts of the amazon), so population density and vaccination coverage tend to inflate the predicted risk in locations with low ecological suitability. this integrated approach also highlights a key research gap-cyclical dynamics of susceptible primate populations-that could further improve prediction. this work illustrates that mechanistically modelling the interactions among the environment, viruses, vectors, nonhuman primates and humans can predict rare and seemingly stochastic spillover events with high accuracy. washburne et al. [ ] study the general statistical problems that can arise when aiming to forecast spillover risk. the authors highlight that any such statistical efforts will compile a dataset of explanatory variables expected to relate to pre-spillover processes (e.g., infection prevalence in reservoirs, human vaccination coverage) that are aligned with one of two response variables: the presence and absence of spillover or the number of spillover events at some spatial and temporal resolution (e.g., spatio-temporal counts of yellow fever spillovers [ ] ). the authors show how modelling cross-species transmission as a percolation process, in which pathogens move from infected reservoirs to recipient hosts along a graph representing various spillover pathways [ , ] , reveals first principles for how such datasets will behave and how common statistical tools can produce misleading inferences and poor predictions. for example, percolation theory reveals an inherent nonlinearity in modelling spillover counts, in which statistical inferences are driven by the dominant reservoir sources of infection and the most limiting barriers to cross-species transmission; this nonlinearity can mask the influence of alternative reservoir species or barriers, both of which could be modified through interventions but whose sensitivity as a management tool will appear reduced under linear models. percolation models provide a conceptual framework to connect statistical and mechanistic models with applications to limit risk by illuminating unexpected statistical principles governing pathogen spillover and the nonlinear impacts of management actions. the theme issue's second section uses empirical research, theory and synthetic reviews to understand the processes operating at each stage of pathogen spillover, from infection dynamics within the reservoir hosts to susceptibility and establishment of infection in the recipient host. for the former, the distribution and intensity of infection in reservoir hosts over space and time is the first determinant of spillover risk [ ] . data on these spatio-temporal dynamics help elucidate how pathogens circulate in reservoir hosts and when and where to expect pathogen excretion to be greatest [ ] . however, such field data can be expensive and difficult to collect, and researchers inevitably must tradeoff between the extent and intensity of spatial versus temporal sampling. sampling is thus often opportunistic and fails to adequately describe spillover. plowright et al. [ ] review factors that influence spatial and temporal variation in infection in reservoirs and describe sampling designs that can increase the quality and quantity of this information. although the standard prescription from sampling theory is to sample randomly in space and time [ ] , probabilistic sampling designs are rare in the study of wildlife disease, given logistical challenges and non-random distributions of hosts. the royalsocietypublishing.org/journal/rstb phil. trans. r. soc. b : authors highlight how stratified random sampling designs or adaptive sampling designs can help capture spatio-temporal pulses of infection when researchers have little a priori data on concentrations of infection or spillover events in space and time. these sampling designs can be integrated into modelling approaches and used to better quantify pathogen shedding from reservoirs. accordingly, glennon et al. [ ] present a case study for how to use mechanistic models to differentiate among transmission processes for henipaviruses in straw-coloured fruit bats (eidolon helvum). using this virulent zoonosis as a case study, the authors generalize standard frameworks common in epidemiological modelling [ ] . given that henipavirus infection dynamics in bats are poorly understood, the authors study all possible transitions among infection states in bats to produce potential models. using likelihood-based methods, they fit these models to longitudinal data from captive bats to show strong support for reinfection after virus clearance and cycles of recurrent latent infection: key areas for future empirical work. this inclusive approach to confronting epidemiological models with longitudinal data in poorly understood reservoir host systems holds promise for elucidating spatio-temporal risk of pathogen spillover. following pathogen shedding from reservoir hosts, spillover risk is influenced by the duration of pathogen survival and possible reproduction outside the host in the environment [ ] . for pathogens such as avian influenza virus, persistence in the environment (e.g. ponds) can also facilitate viral reassortment when strains co-occur, promoting co-infections during environmental exposure [ , ] . pepin et al. [ ] review and discuss how genomics, experimental ecology and epidemiological modelling can be leveraged to understand viral reassortment in environmental reservoirs. although no gold standard for capturing, isolating and identifying avian influenza virus diversity from the environment exists, environmental metagenomics and field-based viral diagnostics (e.g. field-based nucleic acid extraction, pcr and sequencing) hold promise for characterizing this context of viral reassortment [ , ] . the authors note how standardizing such field protocols and coupling these data streams with quantitative disease models and natural transmission studies should dramatically improve our understanding of viral co-occurrence and reassortment and thus, this additional process in the pathway to spillover. exposure of recipient hosts to pathogens (e.g. those persisting in the environment) can take a variety of forms; however, in a more general sense, exposure often occurs at elevated rates near boundaries between ecosystems [ ] . borremans et al. [ ] review how ecosystem boundaries can promote spillover by applying ecological theory to understand landscape permeability across ecosystems. the authors highlight that the traits of hosts and pathogens are critical for determining effects of ecosystem boundaries on crossspecies transmission. properties of ecosystem boundaries can also promote or inhibit exposure; for example, edge effects can affect species composition, diversity and population size between ecosystems, as can features of landscape configuration such as patch size and perimeter-to-area ratio [ ] . by considering the analogy between parasite flow and resource flow and by applying concepts from movement ecology, borremans et al. [ ] connect contact rates and spillover risk across ecosystem boundaries to generalize between pathogens and integrate into broader ecological theory. following the complex interactions between reservoir hosts, vectors, pathogens, the environment and recipient hosts, a crucial juncture in any potential spillover event is the point when a recipient host is challenged with a given dose of pathogen (through a particular route and sometimes over a particular duration) and a successful infection does or does not ensue [ ] . lunn et al. [ ] describe how the dose -response relationship, which quantifies the probability of successful infection in the recipient host as a function of challenge dose, can act as a filter on the aforementioned upstream dynamics to shape pathogen spillover risk. the authors integrate recent developments in the dose -response literature, as well as re-analysing data from animal challenge experiments with nipah virus and middle east respiratory syndrome coronavirus [ , ] , to highlight challenges and opportunities arising at the intersection of infectious disease ecology, microbial risk assessment and virology. lunn et al. [ ] call for closer interactions between these fields and for a new generation of pathogen transmission models that link dose -response data to epidemiological dynamics. gostic et al. [ ] next provide an example of the epidemiological insights such an approach can yield. they present a modelling analysis of dose -response experiments for leptospira interrogans, a globally important bacterial zoonosis for which environmental exposure to soil or water contaminated by urine of infected reservoir hosts is the primary transmission route [ ] . by conducting well-designed challenge experiments across a range of exposure routes, and then developing a mechanistic model to identify and quantify the key barriers to infection, gostic et al. [ ] show that intact skin is the crucial defence against leptospiral infection and that skin abrasions or wounds can increase recipient host infection risk by at least a million-fold. this close integration of experimental and modelling approaches isolates a potent and well-defined risk factor for infection with leptospira, opening the door to targeted interventions to reduce spillover risk. once a pathogen has crossed these within-host barriers to replicate and disseminate in the recipient host, the outcome of infection may range from subclinical illness to death and from dead-end spillover to sustained onward transmission [ ] . bonneaud et al. [ ] focus on the conditions favouring pathogen emergence, from the initial jump into the recipient host to adaption in the novel host environment [ ] . the authors highlight that our current understanding of host shifts stems primarily from viral infections, limiting generalizations to other pathogen taxa, given substantial differences in ecology and life history [ ] . they propose several non-mutually exclusive hypotheses to explain why novel bacterial pathogens may be less likely to specialize on their novel hosts and then test these with a mathematical model. the authors demonstrate that high levels of phenotypic plasticity, low rates of evolution and the ability to recombine should reduce propensity to specialize, suggesting that novel bacterial infections may be more likely to result in transient spillovers or increased host ranges than in host shifts. wasik et al. [ ] in turn describe the within-host barriers that pathogens, and viruses in particular, must overcome to replicate and spread in new host populations to cause onward transmission. they present three well-documented examples of viruses that have crossed these barriers to cause epidemics or pandemics in the new host species: influenza a viruses [ ] , human immunodeficiency virus [ ] and royalsocietypublishing.org/journal/rstb phil. trans. r. soc. b : canine parvovirus [ ] . the authors emphasize the role of integrated models that consider all the steps required to go from exposure to spillover to epidemic or pandemic. guth et al. [ ] expand upon these ideas through a comparative study of host and viral traits that predict virulence and the capacity for onward transmission in recipient hosts (i.e. humans). by expanding a previous global dataset of viral zoonoses [ ] , the authors show that increasing reservoir host phylogenetic distance from humans positively correlates with human mortality but negatively correlates with humanto-human transmissibility, suggesting that the virulence induced by reservoirs at high phylogenetic distance may limit viral capacity for onward transmission [ ] . in particular, distantly related reservoirs, such as bats, harbour highly virulent zoonotic viruses with a lower capacity for onward transmission in recipient human hosts, building upon prior work describing bats as special reservoirs [ ] . the theme issue's final section focuses on applied perspectives to detect early spillover events (i.e. surveillance) and the role of interventions focused upstream in the spillover pathway. in particular, early detection is critical for minimizing the spread of zoonotic pathogens following an initial spillover event [ ] . a first series of manuscripts emphasize different approaches to the surveillance of zoonoses. schmidt et al. [ ] use machine learning tools (e.g. boosted regression trees [ ] ) to predict which mammal species are more likely to play roles in ebola virus spillover events. the authors show that large-bodied, frugivorous mammals with slow life histories are likely host species, implicating some insectivorous bats, old world monkeys and forest antelopes as possible ebola virus reservoirs. predictions such as these can help prioritize future wildlife surveillance efforts (e.g. [ ] ). kuisma et al. [ ] in turn describe a community-based surveillance effort focused on wildlife mortality reporting and oriented to early detection of ebola virus disease outbreaks. spanning over a decade and covering km of challenging terrain in the congo basin, this programme has reached hundreds of villages and thousands of hunters and forest gatherers. the programme has educated community members in wildlife carcass reporting and behavioral risk reduction as well as built capacity for safe carcass sampling by trained local responders. this region was not confronted with an ebola virus outbreak during the period described here, and all reported carcasses tested negative. nevertheless, given the well-recognized fact that early intervention can avert massive human and economic costs of widespread epidemics, the low-cost and scalable surveillance programme described by the authors could provide key early detection capability more generally. two other contributions focus on zoonotic pathogen surveillance efforts in domestic animals and human populations. mwangi et al. [ ] present a real-time surveillance system that leverages the existing mobile phone network and shows immense potential to improve adaptive management of spillover. this surveillance system has been implemented in households across rural kenya, where participants are asked to report symptom syndromes in their livestock. zoonotic diseases such as rift valley fever present with severe clinical signs in domestic animal populations, but lack of active surveillance can miss these sentinels [ ] . the authors demonstrate that illnesses were more likely to be reported on mobile phones compared with standard routine household animal surveys. they also show that more severe symptoms are likely to be reported, highlighting the utility of this surveillance method for diseases such as rift valley fever. das et al. [ ] similarly describe the implementation of a surveillance system in hospitals in bangladesh that screens symptomatic patients for potential zoonoses. most patients did not have a laboratory diagnosis for their illness, indicating that unidentified pathogens are likely spilling over in human populations. broad-scale, sustainable human surveillance programmes such as outlined by the authors can play a critical role in early detection of zoonotic spillovers. following these approaches to surveillance, interventions can accordingly focus upstream or downstream in the pathway to spillover, given available data and resources, to limit cross-species transmission. at the wildlife-livestock interface, managing pathogen spillover is a main goal for animal husbandry, conservation and food security [ ] . yet, managers are often forced to make control decisions on the basis of limited evidence about intervention efficacy. manlove et al. [ ] develop a spatially explicit, stochastic model of pathogen transmission within and between wildlife reservoirs and livestock recipient hosts to improve evidence-based decisionmaking. by varying host movement patterns and epidemic growth rates, the authors show that biosecurity, containment and retroactive vaccination of the reservoir are the most effective for limiting the spatial spread and magnitude of spillover risk for fast-moving epidemics in mobile hosts. by contrast, prophylactic vaccination and depopulation of the reservoir host were more successful for fast-moving epidemics with low rates of host movement. this framework provides general intuition for how to manage different pathogens at the wildlife-livestock interface, and a flexible platform for more rigorously investigating disease control strategies. ultimately, one of the primary goals of research focused on pathogen spillover is to design interventions that can reduce or eliminate disease burden in recipient hosts. sokolow et al. [ ] explore how ecological interventions, which target the ecological context in which cross-species transmission occurs, can complement more traditional biomedical and veterinary interventions (e.g. vaccination, culling). the authors provide case studies to illustrate the potential for ecological interventions that target the reservoir host (sometimes indirectly, such as through the restoration of natural enemy populations [ ] ), pathogen survival in the environment, contact between reservoir and recipient hosts, or other aspects of risk in the recipient species. the authors also present a simple mechanistic model, parameterized for two example systems, that shows how nonlinear effects can produce counterintuitive results when comparing potential intervention strategies and highlights the importance of a detailed understanding of underlying ecological dynamics when designing and assessing interventions. lastly, the authors draw attention to the importance of social, economic and political considerations to intervention success, as these can derail even the most efficient or cost-effective intervention. in particular, aligning the benefits of an intervention with the costs incurred is crucial to motivate ecological interventions and may require working across sectors for successful implementation. royalsocietypublishing.org/journal/rstb phil. trans. r. soc. b : pathogen spillover is the result of a complex series of events that result in the successful establishment of infection in a recipient host [ ] . as highlighted in the final paper of this theme issue, developing actionable forecasts of risk is further complicated by the various phylogenetic, spatial and temporal scales over which we study and predict spillover [ ] . the authors here contextualize a diverse range of approaches to pathogen spillover within these scales to illustrate critical areas of pragmatic overlap. by focusing on an ecological perspective, the authors outline a research pipeline that connects pathogen discovery and macroecological analyses with spatio-temporal surveillance in reservoir and recipient hosts. through several case studies (e.g. lyme disease [ ] , hendra virus [ ] , plasmodium knowlesi [ ] ), the authors further demonstrate how ecologically focused research has facilitated predicting spillover of particular pathogens in space and time and facilitated design of intervention strategies. this synthesis shows how greater integration of macroecology, pathogen discovery and surveillance could ultimately generate more actionable predictions and interventions to limit spillover risks. recent epidemics, pandemics and disease emergence events all underscore the need to improve approaches to predict and prevent pathogen spillover. this theme issue highlights a range of methods and their commonalities through diverse host -pathogen systems for which researchers are assessing factors driving spillover risk across varying phylogenetic, spatial and temporal scales. contributing manuscripts further emphasize how developing a mechanistic understanding of the hierarchical factors affecting spillover can facilitate quantifying the drivers of crossspecies transmission, deriving generalizable theory and making robust predictions, even for seemingly rare and idiosyncratic spillover events. importantly, such insights can improve our ability to deploy surveillance efforts, design interventions at early stages of the pathway to spillover and manage disease cases in recipient hosts, thereby limiting or preventing further outbreaks. continued study of pathogen spillover as a repeated and hierarchical phenomenon will only improve our ability to predict, prevent and manage cross-species transmission risks. data accessibility. this manuscript has no additional data. authors' contributions. all authors contributed to the development of ideas and to the writing of this manuscript. competing interests. we declare no competing interests. funding. this work was 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pathogens in novel hosts multiple scales of selection influence the evolutionary emergence of novel pathogens cross-species virus transmission and the emergence of new epidemic diseases. microbiol onward transmission of viruses: how do viruses emerge to cause epidemics after spillover? phil host and viral determinants of influenza a virus species specificity key viral adaptations preceding the aids pandemic the emergence of parvoviruses of carnivores host phylogenetic distance drives trends in virus virulence and transmissibility across the animalhuman interface host and viral traits predict zoonotic spillover from mammals virulence evolution and the trade-off hypothesis: history, current state of affairs and the future bats as 'special' reservoirs for emerging zoonotic pathogens outbreaks in a rapidly changing central africa-lessons from ebola ecological indicators of mammal exposure to ebolavirus a working guide to boosted regression trees prioritizing surveillance of nipah virus in india long-term wildlife mortality surveillance in northern congo: a model for the detection of ebola virus disease epizootics mobile phone-based surveillance for animal disease in rural communities: implications for detection of zoonoses spillover rift valley fever: scientific pathways toward public health prevention and response hospital-based zoonotic disease surveillance in bangladesh: design, field data and royalsocietypublishing.org/journal/rstb phil taming wildlife disease: bridging the gap between science and management epidemic growth rates and host movement patterns shape management performance for pathogen spillover at the wildlife-livestock interface reduced transmission of human schistosomiasis after restoration of a native river prawn that preys on the snail intermediate host the problem of scale in the prediction and management of pathogen spillover climate, deer, rodents, and acorns as determinants of variation in lyme-disease risk hendra virus spillover risk in horses: heightened vigilance and precautions being urged this winter predictive analysis across spatial scales links zoonotic malaria to deforestation ranch (emigrant, montana) for assistance organizing the working group that led to this theme issue. we also thank helen eaton for all her assistance in preparing this theme issue for publication.disclaimer. the views, opinions and/or findings expressed are those of the authors and should not be interpreted as representing the official views or policies of the department of defense or the us government. key: cord- - tqivs f authors: pruvot, mathieu; cappelle, julien; furey, neil; hul, vibol; heng, huy sreang; duong, veasna; dussart, philippe; horwood, paul title: extreme temperature event and mass mortality of insectivorous bats date: - - journal: eur doi: . /s - - - sha: doc_id: cord_uid: tqivs f a mass mortality event involving chaerephon plicatus and taphozous theobaldi bats occurred during a heat wave in april in cambodia. this was investigated to clarify the causes of the die-off and assess the risk to public health. field evidences, clinical signs, and gross pathology findings were consistent with a heat stress hypothesis. however, the detection of a novel bat paramyxovirus raises questions about its role as a contributing factor or a coincidental finding. systematic documentation of bat die-offs related to extreme weather events is necessary to improve understanding of the effect of changing weather patterns on bat populations and the ecosystem services they provide. under a changing global climate, the frequency of extreme weather events is expected to increase, in particular, warm temperature extremes (ipcc ) . although the effects of climate change on wildlife populations is increasingly documented (fao ) , there is still much to learn about the responses of particular species to extreme temperature events, and the potential consequences for their populations and the ecosystem services they support. in - , cambodia experienced the worst drought in years amplified by a strong el niño event in the region (unescap ) , with temperatures soaring to an all-time high of . °c. in april , a mass mortality of bats was reported from the phnom bok temple in the angkor wat complex of siem reap province, cambodia. the die-off was suspected to be related to the concurrent extreme temperatures. however, as bats can host diverse pathogens of public health concern (wang ) , an investigation was undertaken to clarify the causes of the mass mortality and assess the presence of pathogens. the mortality event was reported by local authorities (ministry of environment) to have begun on april . at the time of the site investigation ( - april ), they reported that over bats had died, particularly around - pm. last removal of carcasses had taken place week before the visit, and counts of carcasses during the visit revealed adults and pups. these were mainly females and young of chaerephon plicatus (wrinkle-lipped bats), although the advanced decomposition of most individuals precluded systematic documentation of their sex (fig. ). only two taphozous theobaldi (theobald's tomb bat) individuals were observed in distress but still alive. bat carcasses were only observed at one of the five buildings of the temple, whose roof had collapsed. two other buildings (mostly collapsed) did not have bats. the last two buildings containing live bats were located east of the first three buildings, and had intact roofs covered by large trees (fig. ) . the temperature profiles of the three buildings containing bats, as measured by a digital thermometer (table ) , greatly differed due to variations in sun exposure and vegetation cover. clinical signs reported by observers included panting, dropping on the ground, and convulsions. the two distressed t. theobaldi bats were clinging to the lower part of the temple wall in full daylight (abnormal behavior), with wings slightly open and panting moderately. these were caught with a net, examined, anesthetized, euthanized, and necropsied (sr-b and sr-b in table ). body temperatures were slightly above °c (table ) , compatible with normal body temperature ranges observed in many bat species (o'shea et al. ) . necropsy only revealed mild and localized hemorrhage on the liver, spleen, and lungs of one animal (sr-b ). another recently dead c. plicatus bat was found clinging to another wall of the same building (sr-b ) and sampled. nucleic acids were separately extracted from liver, spleen, kidney, lungs, and oral and rectal swabs samples collected from the three individual bats (table ) using the qiagen rneasy mini kit. extracts were all tested using universal viral family-level rt-pcr assays for filoviruses, lyssaviruses, coronaviruses, paramyxoviruses, influenza viruses, flaviviruses, alphaviruses, and astroviruses (sánchez-seco et al. ; vázquez-morón et al. ; moureau et al. ; zhai et al. ; chu et al. ; tong et al. ; atkins et al. ; quan et al. ; watanabe et al. ; anthony et al. ) . only one kidney sample from a t. theobaldi individual (sr-b ) tested positive using the paramyxovirus universal assay. subsequent phylogenetic analysis of a~ nucleotide region of the rna-dependent rna polymerase (l) gene showed that this was a novel bat paramyxovirus (btpv taphozous-theobaldi sr-b mh ) sharing % nucleotide identity with viruses of the proposed genus jeilongviruses across this segment of the gene (fig. ) . this viral group mostly comprises viruses detected from bats and rodents. the presence of mild hemorrhage in the infected individual was consistent with previous reports of other jeilongvirus infection in rodents (jun et al. ) ; however, these lesions have also been found in bats suffering from heat shock (welbergen et al. ) , and preserved tissues were not available for further histological examination. previous studies have suggested that nutritional and reproductive stressors may lead to increased excretion of the temperature on the building where mortality occurred exceeded °c, often reported as a critical atmospheric temperature threshold for bat thermoregulation (licht and leitner ) . the absence of mortality in the cooler buildings, the timing of death in the hottest part of the day, the behavior and clinical signs observed, the sex and age ratios (female and young primarily affected), gamma distribution and bootstrap. the numbers next to the branches indicate the percentage of bootstrap replicates that support each phylogenetic branch. the tree is drawn to scale, with branch lengths measured in the number of substitutions per site and the non-specific pathology findings were all consistent with previous reports of heat stress events in bats (welbergen et al. ) , although most previous reports involved flying-fox species (pteropus sp.) (welbergen et al. ). other c. plicatus colonies in the battambang province experienced mass mortalities during the same period (furey, n., unpublished data). the absence of data on the initial population size of both species precluded a conclusive assessment of the relative impact on both species. regardless, c. plicatus is a common bat species in cambodia and southeast asia, and provides important ecosystem services (kunz et al. ) . for instance, its services in controlling the rice pest white-backed planthopper (sogatella furcifera) were recently valued at . million usd/year in thailand alone (wanger et al. ). in addition, the bat guano produced by insectivorous bats is widely used as fertilizer (thi et al. ) and represents a profitable market. thus, the inability of c. plicatus to cope with extreme temperature events and the potential consequences on long-term population trends may have major impacts on bat communities, ecosystems, and rural livelihood in the region. our findings imply that maintaining vegetation cover around bat colonies may help them to cope with high temperatures. in the context of rapid land-use change in cambodia (peterson et al. ) , it is necessary to understand how vegetation cover influences coping mechanisms. systematically documenting mass mortality events is important to understand how bat populations could be affected by a changing environment and climate (welbergen et al. ) . it also provides an important opportunity to conduct surveillance of pathogens circulating in bat populations and understand how heatrelated stress may influence the excretion of pathogens hosted by bats. as such, this report contributes to documenting how changes in land-use and weather patterns influence bat population resilience and health. emergence of fatal avian influenza in new england harbor seals characterization of an outbreak of astroviral diarrhea in a group of cheetahs (acinonyx jubatus) novel astroviruses in insectivorous bats amplification of emerging viruses in a bat colony climate change : synthesis report. contribution of working groups i, ii and iii to the fifth assessment report of the intergovernmental panel on climate change a new mouse paramyxovirus (j virus) ecosystem services provided by bats: ecosystem services provided by bats physiological responses to high environmental temperatures in three species of microchiropteran bats a real-time rt-pcr method for the universal detection and identification of flaviviruses bat flight and zoonotic viruses satellites uncover surprising hotspots for tree cover loss | world resources institute identification of a severe acute respiratory syndrome coronavirus-like virus in a leaf-nosed bat in nigeria a generic nested-rt-pcr followed by sequencing for detection and identification of members of the alphavirus genus effect of bat guano on the growth of five economically important plant species sensitive and broadly reactive reverse transcription-pcr assays to detect novel paramyxoviruses niño / : impact outlook and policy implications rt-pcr for detection of all seven genotypes of lyssavirus genus bats and viruses: a new frontier of emerging infectious diseases bat coronaviruses and experimental infection of bats, the philippines climate change and the effects of temperature extremes on australian flying-foxes rapid molecular strategy for filovirus detection and characterization publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments we thank the forestry administration, the ministry of environment, and the angkor center for conservation of biodiversity for facilitating this investigation. all procedures performed in studies involving animals were in accordance with protocol no. : reviewed and approved by wcs's institutional animal care and use committee. access to site and authorization to conduct the study was granted by the ministry of environment of the royal government of cambodia. the authors declare that they have no conflict of interest. key: cord- - fez gu authors: proenca‐modena, josé luiz; de souza cardoso, ricardo; criado, miriã ferreira; milanez, guilherme paier; de souza, william marciel; parise, pierina lorencini; bertol, jéssica wildgrube; de jesus, bruna lais santos; prates, mirela cristina moreira; silva, maria lúcia; buzatto, guilherme pietrucci; demarco, ricardo cassiano; valera, fabiana cardoso pereira; tamashiro, edwin; anselmo‐lima, wilma terezinha; arruda, eurico title: human adenovirus replication and persistence in hypertrophic adenoids and palatine tonsils in children date: - - journal: j med virol doi: . /jmv. sha: doc_id: cord_uid: fez gu the role of human adenovirus (hadv) infection in different acute diseases, such as febrile exudative tonsillitis, conjunctivitis, and pharyngoconjunctival fever is well established. however, the relationships, if any, of hadv persistence and reactivation in the development of the chronic adenotonsillar disease is not fully understood. the present paper reports a ‐year cross‐sectional hospital‐based study aimed at detecting and quantifying hadv dna and mrna of the hadv hexon gene in adenoid and palatine tonsil tissues and nasopharyngeal secretions (nps) from patients with adenotonsillar hypertrophy or recurrent adenotonsillitis. hadv c, b, and e were detectable in nearly % of the patients, with no association with the severity of airway obstruction, nor with the presence of recurrent tonsillitis, sleep apnea or otitis media with effusion (ome). despite the higher rates of respiratory viral coinfections in patients with hadv, the presence of other viruses, including dna and rna viruses, had no association with hadv replication or shedding in secretions. higher hadv loads in adenoids showed a significant positive correlation with the presence of sleep apnea and the absence of ome. although this study indicates that a significant proportion (~ %) of individuals with chronic adenotonsillar diseases have persistent nonproductive hadv infection, including those by hadv c, b, and e, epithelial and subepithelial cells in tonsils seem to be critical for hadv c production and shedding in nps in some patients, since viral antigen was detected in these regions by immunohistochemistry in four patients, all of which were also positive for hadv mrna detection. human adenovirus (hadv) is a nonenveloped icosahedral dna virus that is highly prevalent in human populations. since its discovery in the early s, , more than hadv genotypes, including all the previously characterized serotypes were described. currently, seven hadv species (a-g) have been identified and are classified in the genus mastadenovirus of the family adenoviridae. hadv can infect a large variety of cell types and tissues in humans, leading to a broad array of diseases, including acute respiratory infections (ari), febrile exudative tonsillitis, acute conjunctivitis, cystitis, gastroenteritis, and rare cases of encephalitis, myocarditis, and hepatitis. although hadv infections are generally asymptomatic in immunocompetent individuals, acute hadv diseases have a significant impact on children (especially under years of age), elderly, immunosuppressed individuals, and military recruits. while hadv can replicate in several cells types in vitro and is associated with productive infections in different tissues in humans, several hadv species present varied tissue specificities. for instance, hadv c (serotypes , , , and ) are commonly associated with acute tonsillitis and respiratory diseases, whereas hadv-f (serotypes and ) and hadv d (serotypes , , and ) are typically associated with gastrointestinal infections and a relatively severe and highly contagious form of epidemic keratoconjunctivitis, respectively. following the hadv replication cycle, the viral genome can persist in the nucleus. , such a fact is best exemplified by the persistence of hadv c after primary infections of the respiratory tract, with intermittent viral excretion in nasopharyngeal secretions (nps) and feces. [ ] [ ] [ ] numerous studies have shown that lymphocytes of tonsils and adenoids are essential sites of hadv persistence, namely of the species c. indeed, seminal studies indicated the ability of hadv to persist in tonsils and adenoids, since it was possible to recover hadv from these tissues weeks to months after the establishment of explant cultures. , more recent studies using tissue cell separation and sorting have revealed that hadv dna is present in t lymphocytes of tonsils and adenoids. in addition, several established human lymphocyte cell lines, including a lymphoblastoid cell line derived from a bone marrow transplant recipient with adenovirus pneumonia, may sustain prolonged and noncytopathic adenovirus infection. , although substantial knowledge has been obtained regarding mechanisms associated with viral persistence in human cell lines in vitro, quantification of the hadv genome and the detection of mrna of the hadv hexon gene was performed in human adenoids and tonsils, nps, and peripheral blood (pb) from patients with tonsillar hypertrophy and were compared to those obtained in samples from control patients. the present study was conducted according to the principles expressed in the helsinki declaration and was approved by the local research ethics committee (# / ). all patients and caregivers signed informed consent and voluntarily agreed to participate in the survey. this was a cross-sectional study that evaluated the presence of hadv in different samples of tissues and secretions from the upper respiratory tract of children with obstructive sleep apnea (osa) or recurrent tonsillitis, comparing the results with control patients. fragments of surgically removed adenoids and palatine tonsils (pts), as well as samples of nps and pb, were obtained from patients ( males) aged to years (median . years) who underwent adenotonsillectomy due to osa or recurrent tonsillitis. small punch biopsies from tonsillar tissues, nps, and pb were also obtained from control patients ( males, median . years) undergoing cochlear implantation in the absence of chronic adenotonsillitis, without ari symptoms and with normal nasofibroscopy. all patients enrolled in the study were undergoing treatment at the otorhinolaryngology hadv detection was performed by taqman real-time pcr (real-time pcr) following a previously published protocol. for hours before the pcr to ensure target-specific amplification. as a negative control, the same rna extraction product was used for real-time pcr without previous rt. samples were considered pcrpositive for hadv hexon mrna only when they were also simultaneously negative for the same target using the extracted rna without previous rt. all samples, including all cdnas and the rnas pretreated with dnase, were tested by real-time pcr for β-actin mrna, following the previously described protocol. a molecular typing assay based on conventional nested-pcr amplification and sequencing of a hypervariable region contained in the hexon gene was performed to determine which species of hadv were present in the patients included in this study, following a previously published protocol. briefly, the first pcr reaction was conducted a maximum likelihood (ml) phylogenetic tree was inferred using nucleotide sequences from strains of adenoviruses described in this study and representative members of the adenoviridae family. multiple sequence alignment was generated using mafft v. with manual adjustments. the ml tree was constructed using the iq-tree version . . software with ultrafast bootstraps and the best-fit nucleotides model determined by bayesian information criterion, which considered reversible dna substitution models. , statistical support for individual nodes was estimated using the bootstrap value, and the phylogenetic tree was visualized with the in this study, the association between the replication of hadv and the presence of other respiratory viruses in adenotonsillar tissue were analyzed. all samples were tested for the presence of the following respiratory viruses by real-time pcr, according to previously described procedures the patient groups were compared using the chi-square and fisher's exact tests; viral loads among patient groups were assessed using the mann-whitney or unpaired t test. comparisons between three or more groups were conducted with one-way analysis of variance and the bonferroni test. all assays were carried out using the graphpad prism software version . for mac (graphpad software, san diego, ca), and a p value of less than or equal to . was adopted for significance. (ome), and allergy (tables and ). among all the analyzed parameters, the only fact worth mentioning was that the viral codetections were significantly more frequent (p = . ) in tissues positive for hadv than in hadv-negative ones ( table ). the median hadv load determined by qpcr in adenoids from patients with chronic adenotonsillar disease was . × copies of genome/g (mean . × ± . × copies/g), while in the control patients, the median hadv load was . × copies/g (mean . × ± . × copies/g). in the pts from patients with chronic adenotonsillar disease, the median hadv load was . × copies/g (mean . × ± . × copies/g), whereas, in the control group, the median was . × copies/g (mean . × ± . × copies/g). regarding the nps samples, the median hadv load was . × copies/ml (mean . × ± . × copies/ml) and . × copies/ml (mean . × ± . × copies/ml) in the patients with and without chronic adenotonsillar disease, respectively. the median hadv load was almost three times higher in the adenoids than the other infection sites, although the difference was not significant ( figure a) . however, hadv loads in the adenoids were not uniformly high when compared to the other sampling sites of the same patients ( figure b) . differences in hadv viral loads between patients with chronic adenotonsillar disease and the controls were not significant ( figure c -e). in general, the hadv loads in the adenoids, pts, and nps were not significantly different among the patients with and without any of the several clinical features analyzed in the present study ( figure c -h). of note, the hadv viral loads were significantly higher in patients with sleep apnea ( . × ± . × copies/g) than in those without the condition ( . × ± . × copies/g; p = . ), although lower in patients with ome ( . × ± . × copies/ g) than in those without the disease ( . × ± . × copies/g; p = . ). the hadv load was also significantly lower in pts from patients with ome ( . × ± . × copies/g) than those without the condition ( . × ± . × copies/g; p = . ), suggesting the high frequency ( %) of patients with significant viral loads in the adenotonsillar tissues (> (table ) . remarkably, mrna for the hadv hexon gene was detected in one of the ( . %) hadv-positive adenoid biopsies obtained from control patients without adenotonsillar diseases ( to hadv is among the leading causative agents of ari in humans. in addition to causing ari, a previous study by our group showed that hadv is one of the most frequent respiratory viruses detected in children with chronic adenotonsillar disease in the absence of ari symptoms. the near % detection rate of the hadv genome reported herein confirms previous findings and agrees with adenoids being preferred sites of hadv infection when compared with pts. , hadv has been detected in pb from patients with hadv-related tonsillitis in the presence of interleukin- production by endothelial cells, fibroblasts or activated t lymphocytes, an essential mechanism for the persistence of fever. in the present study of patients without symptoms of ari or acute tonsillitis, hadv was undetectable in pb, suggesting that asymptomatic viremia is not frequent in asymptomatic hadv carriers. as previously published by our group in a small cohort of patients, hadv was detected more frequently in tonsil tissues where codetection of other respiratory viruses was present. however, we demonstrated herein that such codetection is not linked to higher hadv loads (> copies/g), nor with the detection of mrna of the hadv hexon gene. these findings indicate that the although adenovirus dna is frequently found in tonsils, adenoids, and intestinal tissues (varying from %- % of cases), infectious viruses are rarely detected in these tissues, as measured by in situ hybridization or coculture with permissive cells. , corroborating these findings, we were able to detect hadv-specific persistent infection by hadv has been associated with chronic airway obstructive diseases in children, such as asthma. , in those studies, hadv antigen or genome was found in bronchoalveolar lavage from more than % of children with asthma, respectively, by immunohistochemistry or pcr. in the present study, the detection of hadv was not significantly correlated with chronic adenotonsillar disease, respiratory symptoms or ome, nor with any other detectable disease phenotype. some clinical studies have associated the detection of respiratory viruses with ome. viral infections caused by respiratory syncytial virus, influenza virus (types a and b), and adenovirus have been shown to increase the risk of ome, which can be in part attributed to these viral infections facilitating colonization of the nasopharynx by streptococcus pneumoniae, haemophilus influenzae, and m. catarrhalis, , and the adhesion of s. pneumoniae to epithelial cells of the respiratory tract. in contrast, the development of sleep apnea is partially associated with upper airway obstruction due to enlargement of the pts and adenoids, seen significantly more often in obese patients with asymptomatic viral infections, such as those caused by adenoviruses. , proinflammatory cytokines released by visceral adipocytes seem to contribute to tonsillar inflammation and the development of sleep apnea. [ ] [ ] [ ] although no substantial association of hadv and the severity of adenotonsillar enlargement was found in this study, a significant correlation was observed regarding hadv quantities and the presence of sleep apnea or ome. the present study has shown that patients without ome had significantly higher viral loads than individuals with the condition. some viruses, such as human cytomegalovirus, are known to target dendritic cells, subverting and compromising the host's adaptive immunity by interfering with the cellular transport of major histocompatibility complex molecules. , dendritic cells infected with hadv strongly stimulate t cell proliferation, which may result in increased cellular response to other infectious agents, protecting the host from the development of ome. in addition, persistent adenoviral infection, with small hadv loads, could function as a chronic stimulus for the development of ome. in contrast, patients with sleep apnea exhibited significantly higher hadv loads than individuals lacking the condition. cellular and humoral responses are critical for the control of hadv infection. the recruitment of macrophages and natural killer cells leads to the release of a range of proinflammatory cytokines, stimulating both cd + and cd + t cells, and, consequently, b cell proliferation with humoral antibody response. thus, in keeping with this idea, it is reasonable to consider that high levels of hadv may induce the production of proinflammatory and vasoactive cytokines, which increase chances of developing apnea. in conclusion, the present study demonstrated that a high proportion of patients with the chronic adenotonsillar disease had persistent hadv infection in the adenoids and tonsils. however, the presence of productive hadv infection was not associated with the severity of nasal obstruction, recurrent tonsillitis or viral coinfections. the presence of higher hadv loads in patients with apnea, in parallel with a protective effect against 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witnessed apnoea cytomegalovirus us destroys two components of the mhc class ii pathway, preventing recognition by cd + t cells human cytomegalovirus impairs dendritic cell function: a novel mechanism of human cytomegalovirus immune escape dendritic cells are susceptible to infection with wild-type adenovirus, inducing a differentiation arrest in precursor cells and inducing a strong t-cell stimulation principles and practice of clinical virology human adenovirus replication and persistence in hypertrophic adenoids and palatine tonsils in children the authors thank maria cecılia onofre and helder g. key: cord- -jcsfbxgu authors: vogel, hans-arthur title: the nature of airports date: - - journal: foundations of airport economics and finance doi: . /b - - - - . - sha: doc_id: cord_uid: jcsfbxgu this chapter presents the historic development of global air transportation. as it is the main function to accommodate this traffic growth, airports have advanced in response to it. first, their role within the air transport system will be described. a picture of their social, environmental, economic and political impact will be given next. this chapter’s feature is on airport products and services. the nature of airports chapter outline . introduction . the role of airports within the air transport system airports are complex systems, providing infrastructure and services for the operations of aircraft and handling of passengers and cargo. this requires an adequate airfield, including a runway, aircraft parking apron, and terminal facilities for passengers, cargo, general aviation, and aircraft maintenance as well as supporting fixtures for access circulation/car parking, utilities, and other nonaeronautical use facilities. all of them are multifaceted in nature, while the commonality is that they have long lead times to plan and develop and are costly to build and maintain. this very nature of the airport business is a major determinant of the financial performance. another equally important factor is the demand side. airports have developed in response to the overall traffic growth, providing infrastructure and services to their airline customers. the success of aviation and the significant growth rates associated with that is causing an equally significant problem in terms of lacking infrastructure on the ground and in the air. various countries strongly support the industry on the whole, in order to participate in its benefits, for example, economic development and generation of employment. the population in other countries, however, is more critical regarding the environmental effects-especially when living in the vicinity of an airport. these notable differences are partially driven by their individual historical development in parallel to traffic growth and are, to a certain extent, addressing the challenges arising from these, in turn. as evidenced by the partial statistics on commercial aviation's long-term development in table . , it is a continuing success story and has been resilient vis-à-vis oil and/or gulf crises, other acts of war and terrorist attacks, severe acute respiratory syndrome (sars) and the like as well as economic recessions. during the second decade of this century, air transportation is performed by approximately , scheduled airlines with , aircraft in use, air navigation services, and more than , airports (fonseca de almeida, ). according to icao ( ), . billion passengers and . m tons of cargo were handled in . in general, commercial airports have developed in response to the fast growth of the world's airline industry, which in turn is significantly correlated with the annual growth of the gross domestic product (gdp). it is essential to bear this in mind for the rest of the book, since the airport business is fundamentally driven by the numbers or traffic volume. airport operations and management are an important part of the air transport value chain. many other industries would be pleased to cope with the problems associated with the lasting growth (and, in a few regions, maturity) of aviation. some of the ensuing issues, however, are far from being solved and may pose a severe threat. lacking infrastructure on the ground and in the air results in capacity constraints in general and congested hub airports in particular. traffic growth, however, has actually developed significantly differently across the regions. fig. . displays the regional passenger structure based on icao data. a clear trend of airport passengers in relative terms with regard to asia becomes visible, while north america falls behind and europe remains relatively stable. the underlying traffic shift is also reflected by the long-term volumes in passengers, aircraft movements and cargo handled. in spite of worldwide economic uncertainty and political instability in many countries, airports accommodated almost . billion passengers in . as the passengers are being counted at both the departing and arriving airport, the numbers are about double those of icao and the international air transport association (iata). while the total passenger number has almost tripled since , the regional distribution during this period is rather different-with direct implications for the affected airports. the demand for transportation in general, and for air transportation in particular, is primarily dependent on the status and growth of the economy, growth of the population, disposable income per capita and the resulting propensity to travel. the effects of different market maturity and the shift of the center of economic growth from north america (nam) and europe (eur) toward asia-pacific (asp) is illustrated by fig. . . although the middle east (mea) exhibits the highest overall growth rate for the period under scrutiny, it needs to be noted that this is due to the rather low traffic volume in base year . latin america-caribbean (lac) and africa (afr) have grown at par with and slightly higher than global average, respectively. in addition to the long-term development of the passenger segment, fig. . illustrates the development of the freight market during the last decade. after significant growth, volumes appear to stagnate after the effects of the most recent global financial crisis had been made up for in . roughly two-thirds of air cargo are accommodated by the belly hold of passenger aircraft. after inspecting historical data and identifying an overall trend, the future outlook needs to be analyzed in order to fully comprehend the fundamentals driving the airport business. table . displays the expected compound annual growth rate (cagr) or mean annual growth rate of the passenger sector over the specified period. based on rounded billion travelers expected to have flown in , iata projected this figure almost to double to . billion in . this is equivalent to a cagr of . % on average. other traffic projections, for example, airbus ( ) or boeing ( ) foresee healthy growth rates in a corridor between % and %, more toward the higher end regarding the short to medium range and toward the lower end for the longer term. airports council international (aci, ) is forecasting an even higher rate of . % for passengers (and . % for air cargo as well as . % for aircraft movements) in the long term up to . based on the regional trend described above, this scenario results in a tremendous change in passenger market shares, as displayed in fig. . . the uneven regional growth rates (cf. fig. . ) are set to continue, primarily due to market maturity and gdp growth. while this will provide superior growth perspectives to airports in the asia-pacific region, the overall outlook is very positive across the board. regardless of the slightly different projections, volumes will more or less double during the next years. against this background, iata is expecting that almost all of the world's top airports require major infrastructure expansion during this period. moreover, there could be more slot coordinated airports within years' time already. that is in addition to the level and level airports (with some peak congestion) as of november . all regions are concerned by this challenge, although europe is in the limelight of this infrastructure shortage (garcia, ; gittens, ; clark et al., ) . in order to accommodate for this growth scenario, adequate infrastructure is required both in the air and on the ground-substantially beyond current plans to expand capacity. otherwise, eurocontrol ( ) is afraid that according to their most likely scenario, there will be around . million flights more in demand than can be accommodated by , and million passengers will be unable to fly. it also means that the number of airports operating near capacity during most of the day, as it is the case at london heathrow (lhr), for example, will rise from six in summer to then sixteen. . the social, environmental, economic, and political impact of airports although airports are difficult neighbors due to their environmental impact which will be analyzed in section . . , they are an important economic factor for the cities and regions they serve. in addition to providing access to the global air transport system thus supporting connectivity, they also generate direct, indirect, and induced economic activities and employment (mcgraw, ) . the direct effects may be measured in employment and income resulting from the local airport operations, including the operator itself, other service providers, agencies, and the airlines. while indirect impacts refer to employment and economic activities linked to supplying the air transport industry, induced effects comprise employment and activity supported by the spending of air transport employees, for example, suppliers of goods and services purchased by employees. usually, their combined effect is being measured by the economic multiplier. the multiplier concept incorporates the spendings of direct employees as well as employees of supplying companies. finally, airports also support attracting and sustaining business development outside of air transport. these spin-off effects primarily materializing in the tourism and travel and trade industries are termed to be the catalytic impact (aci europe, ; air transport action group, atag, ). in their most recent study on the benefits of aviation, the atag ( ) reports the overall economic impact of aviation to have been around usd . tn in , supporting . % of the global gdp, with . million jobs dependent on aviation and . million thereof directly in aviation on a global scale. furthermore, they estimated direct employment to have been , with airport operators and another . million with a retail and car rental company or government agencies such as customs on-airport. according to aci ( ), the data concerning its airports revealed a ratio of for total employees on the airport site versus employees of the airport operator and a rule-of-thumb industry benchmark of eight airport employees per , passengers on average. significant regional variations need to be noted though for both, since the employment structure is affected by different operational, managerial and ownership models. these differences also apply to the total income of usd . bn generated by aci airports globally. for europe alone, intervistas ( ) has quantified the overall economic impact of airports in terms of , , total jobs and gdp of eur . bn or . % of the regional gdp, including catalytic effects. another local example is the impressive overall total economic impact of aviation on the dubai economy in , which is estimated to be usd . bn, comprising a "core" impact of usd . bn and "tourism" benefits of usd . bn. this is equivalent to . % of dubai's total gdp and was sufficient to support , jobs or % of the dubai's total employment (oxford economics, ) . it needs to be noted, however, that other industries certainly do generate employment, attract additional businesses (including air traffic/airports) and prompt multiplier effects as well. also, impact studies are descriptive in nature and less qualified as a basis for decision-making than costÀbenefit analyses. the latter address the question whether or not a project or sector is beneficial and also account for environmental implications-revealing favorable results for air transport in general (jorge-calderón, ). still, airports are now integrated into their region's socioeconomic engine with considerable economic and social benefits. these have frequently been capitalized on by politics, specifically where airports are in government ownership. quite often, however, the positive effects are overshadowed by the undisputed negative environmental impact, especially on residential areas in the vicinity of airportsÀwhile the value of commercial property in aviation dependent use is enhanced (cohen and brown, ). in recent years, airport planning and development increasingly tend to be a controversial issue. airports have to deal with numerous local issues regarding noise, air quality, waste management, and other environmental concerns, as well as the policies resulting from these matters. eurocontrol ( ) explains the various environmental topics connected to aviation. with air transportation serving the growing needs for the traveling public, aviation, at the same time, encumbers costs or detriments onto society and nature. these main adversities, harming the environment, are identified as aircraft noise, air pollution, climate change, third party risks, and aviation emissions. aircraft noise is a fundamental harm to people living close to the airport, complaining about noise disturbances, especially during nighttime periods. the "balanced approach" presents a means to undertake noise management procedures at airports and implicates the reduction of noise at the source, land-use planning and management measures, noise mitigation procedures, and operational restrictions. the most remarkable aviation emissions responsible for local air pollution are oxides of nitrogen and carbon monoxide. the encroachment of the environment often induces environmentalists and public campaigners to protest against airports, because of possible airport expansion projects or because of the two basic aviation disturbances of noise and gas emissions. these groups have the ability and power to bring about very damaging repercussions to airports, which can lead to night curfews being enforced at airports. one example of the growing public campaigns and citizens' initiatives is the monday demonstrations inside the terminal of the frankfurt airport in against the airport and its generation of noise. being a good neighbor, however, is vital to get planning approval for potential expansion projects. noise insulation of neighboring houses, technological advances such as new aircraft engine technology as well as renewable energies are further mitigations-usually implying financial burdens for the industry. new aircraft engine technology enables the airports' surrounding area to be less harmed by aircraft noise, invoking fewer noise complaints. renewable energies are further technological advances airports can make use of, in order to be part of the move of "going green." due to the large area that airport infrastructure demands, regenerative energy sources, like solar energy devices, can be mounted either to the spacious rooftops or onto the airport's open land masses. additionally, airports can possibly opt for installing wind turbines and use them as regenerative energy sources. such endeavors also would favor the social and environmental influences of airports, as sustainability on the one hand fabricates a positive image of the airport for the public, and simultaneously is welcomed by environmentalists (federal aviation authority, faa, a,b; pagliarello, ) . two initiatives support the efforts of "going green": aci's "carbon accreditation programme" and eurocontrol's "collaborative environmental management specification" (cem). the former was successfully implemented in europe a number of years ago and went global in november . the program certifies airports at four different levels of accreditation covering all stages of carbon management: mapping, reduction, optimization, and neutrality. as of spring , more than airports in more than countries across all regions have actually reached the fourth stage of carbon neutrality. the cem, on the other hand, was only launched in and is limited to europe, but includes aircraft operators, air navigation service providers, and trade associations in addition to airport operators. its objective is to facilitate the already existing activities of these core operational stakeholders by increasing their awareness of the various interdependencies. this, in turn, is to support sustainable airport development as an essential element for improving air transport movements capacity and flight efficiency (bates, ; eurocontrol, ). due to their significant importance for the economic development of a region and/ or an entire country, airports have frequently been subject to political activities. a prime example for the instrumental character of airport development-actually aviation on the whole-is the new dubai world central al maktoum airport, but also the expansion projects of neighboring abu dhabi (and to a certain extent also doha), where these investments are perceived to be a token of preparing the economies for the postÀoil future. similarly, the bric countries have an overriding interest in developing their airports' system, as ground infrastructure may not be comparable to north america or europe. but governments of the developed nations also follow a similar agenda in supporting the competitiveness of their economies. in europe, the european commission (ec)-in its capacity as an economic regulator and competition authority-has come up with several directives and regulations to create a level playing field for airlines and a more efficient use of scarce capacities to the benefit of the traveling public. additional comprehensive measures were adopted to address the capacity shortage at europe's airports and improve the quality of services offered to passengers under the umbrella of the "better airports package" (ec, ) . more recently, the ec ( ) introduced their "new aviation strategy for europe," which is supposed to foster innovation and generate growth for european business, while letting passengers benefit from increasing connectivity and safer, cleaner as well as cheaper flights. (additional aspects of aviation/airport policy will be considered in feature . .) from a commercial perspective, the two main groups of airport users are the airlines, representing the primary customers (b b) and the travelers or consumers using the dedicated facilities (b c). for the airlines, airports are instrumental for offering their product by providing the required infrastructure to operate aircraft and service their customers being passengers and cargo shippers. these genuine airline customers are airport users or consumers at the (continued) same time. this also holds true for meters and greeters as well as employees. the airline business requires adequate airport access as well as facilities and services at competitive fees and charges, plus a growth perspective for future development. in order to address the airlines' demand for adequate capacity and facilities to operate their business, airports provide the required infrastructure and offer a range of services. these represent the main sources of their revenue. at most airports aeronautical charges include the categories summarized by table . . furthermore, a number of airports impose peak charges and/or noise surcharges (or discounts, as applicable), as a constituent of the landing charges. more rarely, additional charges fall due for aircraft emissions and the usage of centralized infrastructure, such as baggage sorters and underground fueling system. ground handling services are not necessarily provided by the airport operator but frequently by third parties. the latter case will then generate commercial income based on a concession fee paid by the handling agent. also fuel charges are usually not collected by the airport but levied by the fuel companies. government taxes bypass the airport operator in general. while chapter , measuring the financial position, will give an example of a table of charges and chapter , regulatory regime, will add pricing aspects, it is worthwhile stating two relevant principles stipulated by icao's ( ) "policies on charges for airports and air navigation services": first, the "user pays principle," which means to say that users should bear the full and fair cost for the provision of required infrastructure; second, airport charges are essentially cost based (leighfisher, ) . the airlines' customers-the passengers consuming at the airport-have a different perspective. today's savvy travelers increasingly expect both air carriers and airports to understand their preferences and provide personalized offers, advice, and guidance for their door-to-door journeys. empowered consumers will be loyal to those addressing their needs for information, control, and individual service. this, for sure, includes easy access to and processing at the airport for boarding their flight (ascend, ) . in order to address this demand, airports provide the required infrastructure and offer a wide range of services and amenities. the latter are frequently provided by third parties instead of the airport operators themselves. for running their businesses on the airport premises, these concessionaires have to pay a percentage fee on their turnover on top of a fixed rental fee. the major services are summarized by table . , representing sources of commercial income for the airport operator generated on the landside, primarily driven by passenger volumes. it needs to be noted that airport employees also make use of several services and contribute to the generation of commercial revenue. the same applies to (continued) airlines (primarily on the airside) generate aeronautical revenue, commercial or nonaeronautical revenue results from activities on the landside of the airport where passengers consume. global market forecast airports council international (aci) the impact of an airport smoothing the airport experience À . boeing, . current market outlook À . boeing commercial airplanes the fight for slots the effect of international airports on commercial property values: case studies of the airport business. routledge specification for collaborative environmental management (cem) environmental issues for aviation challenges of growth-european aviation in . eurocontrol, brussels. european commission (ec), . airport package. com ( ) final. ec, brussels. european commission (ec), . an aviation strategy for europe. com ( ) final. ec environment and energy research & development federal aviation authority (faa) a future perfect? trends and challenges for the aviation industry. th airline marketing workshop forecast reveals air passengers will nearly double to . billion. iata, montreal, press release . international civil aviation organization (icao), . icao database-air transport statistics. icao, montreal. international civil aviation organization (icao), . icao's policies on charges for airports and air navigation services economic impact of european airports-a critical catalyst to economic growth aviation investment-economic appraisal for airports, air traffic management airlines and aeronautics airport design and operation, second ed review of airport charges the heterogeneous impact of airports on population and employment growth in cities the continuing development of airport competition in europe quantifying the economic impact of aviation in dubai the evolving challenge of noise authorities and airlines renting floor space for operations and back offices, parking facilities, as well as associated utilities. major characteristics of concession contracts will be introduced in chapter , measuring the financial position.as with any other business, meeting customer needs and expectations is key to operating an airport successfully. this appears to be mandatory, as copenhagen economics (ce, ) confirmed by oxera ( ) found that: these findings apply primarily to small , million passengers per annum (mppa) and medium sized airports, while larger ones . mppa are affected more by competition for transfer passengers. in general, airports are competing for any additional traffic in terms of passengers and cargo, particularly for: g passengers in a shared local market, g connecting passengers, and g airline services on new and existing routes (oxera, ) .although the focus of this research was on europe, it can be concluded from the wide-spread granting of commercial incentives to airlines across regions that airport competition has become a worldwide phenomenon in the meantime. various enticements have actually developed into a major tool of airport marketing, in addition to putting their products to the shop-window at the different "routes conferences." feature . will discuss the competitive situation in the sector in more detail. this chapter presented the historic development of global air transportation. as it is their main function to accommodate traffic, airports have advanced in response to it. traffic volume and structure, however, are not under full control of airport management but subject to overall economic, political and regional developments. an overview of airports' social, environmental, economic, and political impact was given. environmental concerns as well as changing customer demands and consumer behavior pose new problems in the digital age. addressing these is an ongoing and costly endeavor.this chapter's feature introduced to airport products and services. basically, two main types can be differentiated. while products and services delivered directly to key: cord- -lpd olqq authors: weston, stuart; matthews, krystal l.; lent, rachel; vlk, alexandra; haupt, rob; kingsbury, tami; frieman, matthew b. title: a yeast suppressor screen used to identify mammalian sirt as a proviral factor for middle east respiratory syndrome coronavirus replication date: - - journal: journal of virology doi: . /jvi. - sha: doc_id: cord_uid: lpd olqq viral proteins must intimately interact with the host cell machinery during virus replication. here, we used the yeast saccharomyces cerevisiae as a system to identify novel functional interactions between viral proteins and eukaryotic cells. our work demonstrates that when the middle east respiratory syndrome coronavirus (mers-cov) orf a accessory gene is expressed in yeast it causes a slow-growth phenotype. orf a has been characterized as an interferon antagonist in mammalian cells, and yet yeast lack an interferon system, suggesting further interactions between orf a and eukaryotic cells. using the slow-growth phenotype as a reporter of orf a function, we utilized the yeast knockout library collection to perform a suppressor screen where we identified the ydl c/sir yeast gene as a suppressor of orf a function. the mammalian homologue of sir is sirt , an nad-dependent histone deacetylase. we found that when sirt was inhibited by either chemical or genetic manipulation, there was reduced mers-cov replication, suggesting that sirt is a proviral factor for mers-cov. moreover, orf a inhibited sirt -mediated modulation of nf-κb signaling, demonstrating a functional link between orf a and sirt in mammalian cells. overall, the data presented here demonstrate the utility of yeast studies for identifying genetic interactions between viral proteins and eukaryotic cells. we also demonstrate for the first time that sirt is a proviral factor for mers-cov replication and that orf a has a role in modulating its activity in cells. importance middle east respiratory syndrome coronavirus (mers-cov) initially emerged in and has since been responsible for over , infections, with a case fatality ratio of approximately %. we have used the highly characterized model system of saccharomyces cerevisiae to investigate novel functional interactions between viral proteins and eukaryotic cells that may provide new avenues for antiviral intervention. we identify a functional link between the mers-cov orf a proteins and the ydl c/sir yeast gene. the mammalian homologue of sir is sirt , an nad-dependent histone deacetylase. we demonstrate for the first time that sirt is a proviral factor for mers-cov replication and that orf a has a role in modulating its activity in mammalian cells. camels ( ) ( ) ( ) ( ) . the virus has yet to show sustained transmission between humans, but, with no currently approved antiviral therapeutics or vaccines, mers-cov is considered a "threat to global health" by the who. identifying novel virus-host interactions is essential to guide future intervention strategies. coronaviruses have large (ϳ -kb) positive-sense, single-stranded rna genomes. approximately two-thirds of this rna is dedicated to the production of nonstructural proteins which regulate replication of the viral genome. the remaining third encodes the structural and accessory proteins ( ) .the accessory proteins largely function to promote viral replication through modulating cellular responses to infection. one such protein, produced by mers-cov, is orf a. this accessory protein has been shown in vitro to bind double-stranded rna and to disrupt the innate immune sensing pathways of rig-i and mda to suppress the interferon response ( ) ( ) ( ) . additionally, orf a has been found to inhibit the pkr and stress granule response in cells ( ) ( ) ( ) , further interfering with cellular function to promote viral infection. the yeast saccharomyces cerevisiae have been used extensively throughout the history of eukaryotic cell biology, including its being the first eukaryotic genome to be fully sequenced ( ) . the genome is now highly annotated, due in part to experiments performed with the yeast knockout library collection ( , ) . the yeast knockout collection systematically knocked out each gene in s. cerevisiae with a known dna cassette containing a unique "barcode" region. therefore, by taking any yeast cell from within this library and pcr amplifying and sequencing the barcode region, it is possible to determine which gene has been deleted from that cell. the project managed to knock out ϳ , of the ϳ , yeast genes; deletion of the others resulted in an inability to produce viable yeast, and they were deemed essential for growth in glucose (glu) media. therefore, the yeast knockout collection represents a widely available, semi-genome-wide knockout library of yeast. while extensively used to study eukaryotic cell biology, yeast species have also been used to look for factors involved in replication of rna viruses ( ) ( ) ( ) ( ) ( ) ( ) ( ) . we and others have previously demonstrated that expression of certain proteins from viruses and bacteria in the yeast s. cerevisiae can inhibit growth ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . this growth phenotype can be leveraged to identify compounds with antiviral properties or functionally important residues of the viral proteins. in this study, we utilized the slow-growth phenotype to investigate novel genetic interactions between the viral protein and eukaryotic cells. through screening the mers-cov proteome, we identified various proteins that, when expressed in yeast, caused a slow-growth phenotype, including the protein encoded by the orf a accessory gene. yeast lack an interferon response, and since orf a has largely been studied in experiments designed to disrupt these pathways, we hypothesized that orf a must have further functions in a eukaryotic cell that have yet to be analyzed. to investigate the cellular pathways that orf a interacts with in yeast, we took advantage of the yeast knockout library. we hypothesized that removal of genes involved in the orf a-mediated slow growth would cause a reversion of the slow-growth phenotype, allowing the identification of genetic suppressors. this screening approach identified the ydl c/sir yeast gene as a suppressor of orf a-mediated slow growth, and that gene became the focus of our study. ydl c/sir is a sirtuin protein, first identified through its role in silencing the cryptic mating-type loci hml and hmr ( ) . since that initial identification, sirtuins have been defined as nad-dependent histone deacetylases ( ) involved in regulating a multitude of cellular functions, including metabolism, apoptosis, stress responses, dna repair, and gene expression ( ) ( ) ( ) ( ) . the mammalian homologue of sir , sirt , has been shown to have both proviral and antiviral roles, depending on the virus ( ) ( ) ( ) ( ) ( ) ( ) ( ) , making this an attractive candidate hit from our yeast-based screening for study in mammalian cells. moreover, it has previously been suggested that resveratrol, a compound that activates sirtuins, inhibits mers-cov replication, and yet no mechanism was investigated in that work ( ) . given the combination of sirt being shown to have roles in replication of mammalian viruses and the fact that sir was the most com-monly identified gene in our suppressor screen, this sirtuin became the focus of the study. other genes identified in yeast are yet to be examined in the mammalian context. we found that chemical inhibition of sirt or knockdown with either small interfering rna (sirna) or clustered regularly interspaced short palindromic repeat interference (crispri) inhibited replication of mers-cov, demonstrating that sirt is a proviral factor for viral replication. the sirtuin activator resveratrol was found to inhibit mers-cov replication, but this effect was not dependent on the presence of sirt , suggesting that other sirtuin proteins may have antiviral roles, while sirt appears to have a proviral role. the yeast-based screening system demonstrated a functional link between orf a and sir . we did not observe a direct interaction between orf a and sirt in mammalian cells. however, we do show that orf a can modulate sirt mediated enhancement of nf-b signaling, suggesting a functional link between the two proteins in mammalian cells. our work demonstrates the utility of yeast for identifying novel interactions between viral proteins and eukaryotic cells and defines sirt as a proviral factor for mers-cov replication. mers-cov orf a causes a slow-growth phenotype in yeast, and ydl c/sir is a genetic suppressor. work performed in our laboratory and others previously found that certain proteins from various pathogens can cause a slow-growth phenotype in s. cerevisiae ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . this phenotype can be used to investigate functional aspects of these proteins in eukaryotic cells by the use of suppressor screens. in these screens, yeast genes are knocked out (as in this study) or overexpressed to identify genes that suppress the growth phenotype caused by the expression of the pathogen protein. we took advantage of the yeast model system to investigate cellular interactions of proteins from mers-cov. genes from the mers-cov genome were cloned into a galactose (gal)-inducible (gal ) expression vector, based on pbr ( ) , with a c-terminal green fluorescent protein (gfp) tag, and were transformed into yeast. grown in the presence of % glucose (glu), the expression of viral genes was inhibited and the yeast strain containing this plasmid was able to grow similarly to yeast transformed with a vector control ( fig. b and data not shown). however, grown in the presence of % galactose (gal), viral genes were expressed. we analyzed whether any of the mers-cov-encoded proteins could inhibit yeast growth by performing h growth curves, measuring the optical density at nm (od ) on an automated plate reader as the readout for growth (fig. a) . we found that the following four mers-cov proteins inhibited growth of yeast: nsp (green dotted/dashed line in fig. a ), nsp (maroon line), nsp (orange line), and orf a (gray line). the other mers-cov genes examined had only a modest impact or no impact on growth, suggesting that expression of gfp-tagged proteins alone was not responsible for the slow growth observed. we decided to focus our further study on orf a as this accessory protein has been best described for roles in inhibiting the mammalian interferon (ifn) response, which is not conserved in yeast, therefore suggesting the possibility of hitherto-undescribed functions of orf a in eukaryotic cells. the growth inhibition caused by orf a was further assessed on solid media, where the levels of growth on glu plates (no protein expression) were indistinguishable between vector control and orf a-expressing yeast whereas growth on gal plates displayed growth inhibition of yeast expressing orf a (fig. b) . the results from an initial liquid-culture screening were also further confirmed (fig. c) . we reasoned that the slow-growth phenotype induced by orf a was a result of the viral protein disrupting normal cellular function, allowing us to perform a suppressor screen. we hypothesized that knockout of yeast genes involved in the orf a-mediated slow growth might reduce the level of inhibition and therefore increase the growth rate. to test this hypothesis, the inducible orf a plasmid was transformed into a pooled collection of the yeast knockout library. this library consists of ϳ , nonessential gene knockouts. when this transformed library was plated onto glu plates, the yeast colonies that formed were of similar sizes (fig. d, glu) . however, plated to gal plates to induce orf a expression, a broad range of colony sizes were observed (fig. d , gal). of most interest were the large colonies, as these were thought to have been formed by yeast cells that contain deletions of genes that confer a suppressor phenotype on orf a-mediated slow growth. large colonies were picked from gal plates and were tested for growth in liquid culture and for continued expression of orf a (data not show). suppressors were determined as knockout yeast that expressed orf a and showed enhanced growth compared to orf a-expressing wild-type (wt) yeast. the identity of the knockout was determined by pcr amplification and sequencing of the unique barcode regions that are used in the yeast knockout library. for the screening process, ϳ colonies were assessed for growth phenotypes and were found to have suppressor phenotypes to various degrees (data not shown). these colonies determined as hits were sequenced and found to represent different genes ( table ). the most commonly detected gene knockout was ydl c/ sir (no other sirtuin genes were detected). tested in liquid culture, Δydl c/sir colonies picked from gal plates showed a suppressor phenotype. panel e of fig. shows an example of a single colony that was picked from a galactose screening plate that had a suppressor phenotype. when the growth rate of the initial fast-growing colony was assessed, the identity of the gene knockout was unknown. subsequent sequencing determined it to be Δydl c. this colony represented one of the five Δydl c colonies that were identified as suppressors in the screen. the colony displayed in fig. e still maintained expression of orf a, such that the enhanced growth was not a consequence of a lack of expression of the viral protein (fig. f) . to validate the hypothesis that ydl c/sir is indeed a suppressor of orf a, a confirmed ydl c/sir deletion strain was transformed with the orf a expression plasmid. when the known transformed deletion strain was tested for growth ( fig. g ) and protein expression (fig. h) , the suppressor phenotype was maintained. to further validate identification of ydl c as a specific suppressor of orf a, Δydl c yeast cells were also transformed with an empty vector or a vector expressing sars-cov orf b with a gfp tag (which does not cause a slow-growth phenotype). empty vector and orf b-gfp Δydl c yeast grew at rates similar to those seen with the vector control yeast, suggesting that the Δydl c phenotype did not stimulate enhanced growth and therefore that the suppressor phenotype seen when orf a was present was specific (fig. i ). combined, these data argue that ydl c/sir is a bona fide genetic suppressor of orf a-mediated slow growth. orf a and sirt do not directly interact in mammalian cells. the mammalian homologue of yeast ydl c/sir is the histone deacetylase sirtuin protein sirt ( % similarity at the protein level). we therefore hypothesized that orf a may interact with raffinose to reach saturation. yeast cells were diluted and cultured for h in % gal media at °c on a plate reader. every min, the plate was shaken for s and the od was read. the growth curve has od plotted against time. data are from one representative experiment performed using two separate colonies of yeast. error bars represent deviations between results from the two colonies. (b) yeast cells transformed with a gal-inducible plasmid to express mers-cov orf a or a vector control were cultured for days in media containing % raffinose to reach saturation. the od was measured, yeast cells were diluted to an od of . in % raffinose media, and then a : dilution series was made. this dilution series of the yeast was then replica plated on agar plates containing % glucose (glu) or % gal. (c) as described for panels a and b, vector control or orf a plasmid yeast cells were grown for days in % raffinose to reach saturation. yeast cells were diluted and cultured for h in % gal media at °c on a plate reader. every min, the plate was shaken for s and the od was read. the growth curve has od plotted against time. data are from three independent experiments, with error bars showing standard deviations. (d) the gal-inducible orf a plasmid was transformed into a pool of the yeast knockout (yko) library collection and plated onto agar plates containing glu or gal. a suppressor screen was performed by picking large colonies from the orf a yko library on gal plates and analyzing growth rates for colonies that no longer had the slow-growth phenotype caused by expression of orf a. the most commonly detected gene deletion was ydl c/sir . (e) a representative graph for one of the large colonies picked from a gal plate that was later determined to be Δydl c/sir . (f) western blot to demonstrate that the suppressor phenotype observed in Δydl c/sir colonies was not a result of a lack of orf a expression. (g) a known Δydl c/sir strain was picked from an arrayed collection of the yko library and transformed with gal-inducible orf a plasmid. data show growth curves of these transformed yeast cells from independent experiments (error bars represent standard deviations). (h) western blot to confirm that orf a was still expressed in the transformed Δydl c/sir cells. (i) control experiments testing whether Δydl c/sir yeast cells do or do not grow faster than wild-type yeast. sars-cov-orf b and an empty vector were transformed into yeast that were subsequently grown for h in % raffinose media prior to growth to produce growth curves as described above. sirt in mammalian cells to regulate mers-cov replication. endogenous sirt is localized to the nucleus in huh cells, with areas that appear to have lower-intensity labeling ( fig. a) . transfected into huh cells, a plasmid encoding orf a-gfp showed gfp signal in the cytosol, with additional puncta in the nucleus (fig. b ), in agreement with previously published data ( ) . the colabeling of these transfected cells with antibodies against sirt showed both proteins to be in the nucleus. however, the nuclear puncta of orf a-gfp appeared to be in regions with lower levels of sirt labeling (fig. b) . in cells infected with mers-cov, labeled with antisera to orf a, and colabeled with anti-sirt antibody, orf a was founded in the nucleus and in cytosolic puncta, while sirt was still localized to the nucleus (fig. c) . the more punctate appearance of cytosolic orf a in infected cells was possibly a result of lower expression levels compared to the overexpression construct. overall, it appears that endogenous sirt was localized to the nucleus, while gfp-tagged orf a and orf a expressed during infection were both localized in the nucleus and cytosol. to thoroughly test whether orf a and sirt could interact in huh cells, we acquired both wt and catalytically dead (h y) flag-tagged expression plasmids of sirt (fig. a) , the data in the column labeled "no. of hits" indicate numbers of individual colonies that were found to have the same gene deletion. yko, yeast knockout; orf, open reading frame. reasoning that a higher level of expression of sirt would allow us to detect even low levels of interaction. the overexpression constructs appeared to show a localization pattern similar to that seen with endogenous sirt when expressed individually in cells (data not shown) and when cotransfected with orf a-gfp (fig. b) , suggesting that these are useful for investigating interaction of orf a and sirt proteins. once again, both sirt -flag and orf a-gfp were seen in the nucleus, suggesting that they are in the same compartment of cells and therefore could potentially interact. to investigate this more directly, we used coimmunoprecipitation (co-ip) experiments. cells were transfected with orf a-gfp alone or in combination with wt or h y sirt -flagexpressing plasmids. anti-gfp antibody was used to immunoprecipitate orf a-gfp, which could be detected in the ip fraction in blotting with anti-gfp antibody (fig. c) . in cells cotransfected with orf a-gfp and either of the sirt constructs, while orf a-gfp could be precipitated, sirt was detected only in the nonspecific and flowthrough fractions and never in the ip fraction (fig. c) . these data suggest that orf a and sirt do not directly interact in huh cells. sirt is a proviral factor for mers-cov replication. yeast sir was detected as a genetic suppressor of orf a-mediated slow growth, and yet orf a and sirt were huh cells grown on coverslips were transfected with a plasmid expressing orf a-gfp and were subsequently subjected to immunofluorescence labeling for sirt (visualized with anti-mouse texas red). arrows point toward nuclear and arrowheads point toward cytosolic orf a localizations. (c) huh cells were plated to coverslips day prior to infection with mers-cov at an moi of (uninfected cells were grown as a control). infection was allowed to proceed for h prior to fixation and immunofluorescence labeling for sirt (visualized with anti-mouse texas red) and orf a (visualized with anti-rabbit fitc). arrows point toward nuclear and arrowheads point toward cytosolic orf a localizations. in all experiments, nuclei were labeled with dapi. scale bars ϭ m. found to not directly interact in mammalian cells. the genetic suppression observed in yeast does not necessitate a direct interaction between the two proteins. we therefore hypothesized that orf a may modulate sirt activity indirectly to regulate mers-cov replication. if this were the case, modulation of sirt activity should alter the replication of mers-cov. it has previously been suggested that resveratrol, a chemical compound that broadly activates sirtuin proteins, can inhibit mers-cov replication ( ) . we looked to reproduce these results and indeed found that at higher concentrations, resveratrol was capable of inhibiting mers-cov replication as judged by assessing viral titer released into the supernatant (fig. a ). this inhibition was seen when the drug was added h before, at the same time as, or h after virus was added to the cells, suggesting that resveratrol was not inhibiting entry stages of infection. we additionally tested the impact of ex , a chemical that inhibits sirt . these data showed that sirt inhibition also resulted in inhibition of mers-cov replication (fig. b) . this result was again seen regardless of time of addition. neither of these results appeared to be fully explainable by cell death, as only small differences were observed at the higher concentrations of drug in assessments of cell viability by celltitre-glo assay (fig. c) . in order to investigate the role of sirt more directly, sirna was used to knock down expression of the protein. we used two sirna sequences targeting sirt ( ) . both sirna sequences were capable of knocking down sirt expression (fig. a) , and both resulted in minor drops in cell viability as assessed by celltitre-glo assay (fig. b ). when these samples were infected with mers-cov, significant inhibition of infection was detected (fig. c ). while knockdown of sirt was clearly capable of inhibiting mers-cov replication, overexpression of wt or h y sirt -flag did not have any impact on virus production (fig. d) , suggesting that the endogenous levels of sirt may be sufficient for replication. these data suggest that sirt is a proviral factor for mers-cov infection. however, this result is at odds with the data showing that resveratrol, a sirutin activator, inhibited infection. resveratrol is a broad activator of sirtuin proteins, of which there are in the human genome. we hypothesized that different sirtuin proteins may have different impacts on mers-cov infection. this was tested by transfecting cells with sirt sirna, treating with resveratrol, and infecting with mers-cov. resveratrol was capable of inhibiting mers-cov infection even in the absence of sirt (fig. e) . these data suggest that resveratrol inhibits mers-cov infection independently of sirt . crispri further demonstrates sirt is a proviral factor for mers-cov replication. our sirna-mediated knockdown of sirt in fig. suggests that sirt is a proviral factor for mers-cov replication. in order to further assess the effect of sirt on mers-cov replication, a crispri approach was used ( ) . for this, huh cells that stably express a catalytically dead version of cas (dcas ) were produced through lentiviral transduction. the dcas can be inducibly expressed and binds to dna as directed by the guide rna but does not cleave, instead acting as a steric block to transcription. moreover, a krab element on the dcas further enhances transcriptional repression. these stable huh -dcas cells were further transfected to stably express a short guide rna (sgrna) sequence to target the sirt gene. two lentiviral preparations were used to independently transfect two separate populations of huh cells stably expressing the dcas construct (huh -dcas cells) . the sgrna is constitutively expressed, while the dcas is doxycycline (dox) inducible; therefore, the stable cell populations can be divided into dox-treated and untreated populations for internal controls of target gene knockdown. both huh -dcas -sgrna cell populations demonstrated knockdown of sirt under conditions of treatment with dox for days (fig. a ). when these doxycycline-treated cells were infected with mers-cov, there was a significant inhibition of replication in both populations (fig. b ). knockdown of sirt by both sirna and crispri inhibited mers-cov replication, showing this protein to be a proviral factor for replication. using these crispri cells, we also confirmed that resveratrol inhibited mers-cov replication even in the absence of sirt (fig. c) , validating the sirna results (fig. e ). the level of inhibition of mers-cov replication in control cells appears to have been slightly lower in this experiment; however, under conditions of testing over multiple infections (fig. b) , the inhibition results were statistically significant. overall, it appears that even though the sirtuin activator resveratrol has antiviral activity with respect to mers-cov replication, sirt is dispensable for this effect. orf a modulates sirt activity as assessed by an nf-b pathway readout. sirt appears to function as a proviral factor for mers-cov replication ( fig. ; see also fig. ). our suppressor screen in yeast detected a genetic link between mers-cov orf a and sir (fig. ), but we were unable to detect a direct interaction between orf a and mammalian sirt (fig. ) . we therefore speculated that orf a may have an indirect influence on sirt activity in promoting mers-cov replication. sirt has previously been shown to modulate nf-b signaling through deacetylation of rela/p ( ) . we therefore decided to use nf-b signaling as a readout of sirt activity to determine whether orf a alters this. hek t cells were transfected with combinations of orf a, sirt (wt or h y mutant), and a b-luciferase plasmid (fig. a) . the following day, cells were treated with tumor necrosis factor alpha (tnf-␣) for h and lysed, and the amount of luciferase produced was determined. (results of controls performed for analysis of background luminescence can be seen in fig. d .) at odds with previously published data, we found that transient transfection of sirt in our system significantly enhanced luciferase production following tnf-␣ treatment compared to the results seen with control transfected cells ( fig. ; see also fig. b ). cells transfected with h y sirt also showed an enhanced level of luciferase production (fig. b) . when mers-cov orf a was transfected into cells, inhibition of luciferase production was observed (fig. b) . importantly, when orf a was transfected into cells alongside sirt (wt or h y), the luciferase levels were similar to those seen with orf a alone. these results suggest that sirt no longer enhanced luciferase production in response to tnf-␣ when orf a was present, suggesting that orf a was altering this readout of sirt activity (fig. b) . treatment of cells with resveratrol also gave an enhanced level of nf-b signaling in response to tnf-␣, further suggesting a role for sirtuins in regulating this pathway in hek t cells (fig. c ). however, ex had no effect in our tests (fig. c) . a small but significant increase in nf-b signaling was seen when orf a-transfected cells were treated with resveratrol, but luciferase production was largely blocked compared to the levels seen with cells without transfected orf a (fig. b) . these data further suggest that orf a is capable of modulating sirt -mediated enhancement of nf-b signaling stimulated by tnf-␣ treatment. overall, the results from the sirt transfection and the resveratrol treatment suggest that orf a alters a sirt -mediated response in cells, suggesting a functional link between the two. we have demonstrated that sirt is a proviral cellular protein for mers-cov replication and that yeast cells represent a powerful tool to identify previously unknown virus-host interactions. our initial work started with a suppressor screen in the yeast s. cerevisiae. we found that expression of mers-cov orf a in yeast caused slow growth. orf a functions as a double-stranded rna binding protein that disrupts rig-i, mda , pkr, and stress granule responses in infected cells to promote viral replication ( ) ( ) ( ) ( ) ( ) ( ) . orf a is well characterized as an inhibitor of the interferon response. yeast species do not have an interferon response and yet show growth inhibition by orf a expression, suggesting further functional cellular interactions for the protein. to investigate the ways in which orf a might inhibit yeast growth, a suppressor screen was utilized. by screening the yeast knockout library for suppressors of orf a-induced growth attenuation, the ydl c yeast gene was identified. the ydl c gene encodes the sir protein, and our work validated this as a bona fide suppressor of the the orf a-mediated slow-growth phenotype. the suppressor screen results suggest that there is a functional link between the two proteins but that the link does not rely on a direct physical interaction. while yeast is a powerful starting point for investigating eukaryotic cell biology, in the context of mers-cov infection, it is important to establish relevance in a mammalian system. we did not find a direct interaction between orf a and sirt in huh cells by coimmunoprecipitation. however, when sirt was inhibited either by ex treatment or by knockdown by sirna or crispri, mers-cov replication was inhibited. this establishes sirt is a proviral factor for mers-cov replication. interestingly, our data also suggest that other sirtuin proteins may have antiviral roles in the context of mers-cov replication. resveratrol, a compound derived from various plants, has been shown to activate sirtuin proteins ( ) and was previously shown to inhibit mers-cov replication ( ) , a result that we replicated here. however, we demonstrated that knockdown of sirt by sirna or cripsri had no impact on the resveratrol-mediated inhibition of mers-cov replication, suggesting that other members of the sirtuin family that are activated by the compound may have antiviral properties. alternatively, resveratrol may have other effects on cell biology that inhibit mers-cov replication. since sirt was shown to be a proviral factor by our data and since resveratrol can activate sirt function, it appears that the antiviral properties of resveratrol may outweigh the potential proviral function of sirt or that the proviral role of sirt is not based on its catalytic activity. indeed, our experiments looking at the nf-b pathway (discussed further below) suggest that sirt may have activity in a catalytically dead form, arguing for a role in mers-cov replication that does not require enzymatic function. the role of other sirtuins in mers-cov replication and in how mers-cov modulates these to promote infection is the focus of future studies. the yeast-based screening system identified a genetic interaction between orf a and sir , which led us to hypothesize that orf a may have a functional link with mammalian sirt to regulate its function and promote mers-cov replication. sirt has previously been shown to modulate nf-b signaling in response to tnf-␣ ( ) . this work demonstrated a direct interaction between sirt and p /rela and that sirt inhibited the pathway by deacetylation of this nf-b protein. we therefore reasoned that this modulation of the nf-b pathway could be used as a readout to determine whether orf a expression alters sirt function. interestingly, we found that sirt overexpression in hek t cells actually resulted in an enhanced nf-b response following tnf-␣ treatment and that this enhancement occurred regardless of the level of catalytic activity of sirt . this discrepancy with previously published work could represent a result of the use of different cell lines. our data suggest that sirt promotes nf-b signaling in hek t cells, perhaps by directly binding nf-b and acting as a scaffold protein or by altering steps downstream in the signaling pathway, but that it does so independently of catalytic activity. while we found the effects of sirt on nf-b to be different from those reported from previous studies in different cell lines, sirt still altered the nf-b response, allowing us to investigate the role of orf a in this pathway. our experiments demonstrated that orf a was a potent suppressor of nf-b signaling in hek t cells. when orf a and sirt were coexpressed in cells, there was no longer an enhancement in luciferase production from the b promoter. we cannot rule out the possibility that orf a may influence nf-b signaling independently of sirt , but sirt overexpression was not able to overcome orf a-mediated inhibition. these data, along with the genetic link observed in yeast, suggest that orf a and sirt may have a functional connection without direct binding and that there is an additional node of regulation between the two proteins, which is under investigation. this work adds sirt to the list of host factors that mediate mers-cov replication such as stress granule formation and host innate immune machinery ( ) ( ) ( ) ( ) . the magnitude of inhibition when those factors are affected is ϳ log, demonstrating that, as seen in sirt , host machinery interacts with various virus replication steps to potentially hinder replication. all of these factors demonstrate novel therapeutic targets for future development. overall, we have demonstrated the utility of yeast for investigating the function of viral proteins in a highly tractable heterologous expression platform. we found that studies in yeast are relevant in a mammalian system, as the identification of yeast sir as a suppressor was used to determine that the mammalian deacetylase, sirt , is a proviral factor for mers-cov replication. we currently do not understand how sirt enhances mers-cov replication or the role that orf a plays in this. we hypothesize that acetylation of additional cellular or viral proteins during mers-cov replication is required for efficient replication and that orf a has evolved to modulate this activity. the multifunctional property of orf a has revealed novel sirt functions and pathways that mers-cov and potentially other viruses utilize to impact their replication. was used for all subsequent steps) with % (vol/vol) formaldehyde (thermo) in phosphate-buffered saline (pbs). cells were washed with pbs and excess formaldehyde quenched by min of incubation with mm nh cl-pbs. cells were again washed and lysed in . % (vol/vol) triton x- (sigma) and then incubated for min. samples were blocked for min with . % bovine serum albumin (bsa)-pbs. primary antibodies were then incubated with the sample diluted in . % bsa for h. samples were washed in . % bsa three times for min each time and were then incubated with secondary antibodies in . % bsa for min. finally, coverslips were washed with pbs four times for min each time and mounted to glass slides using prolong gold antifade reagent with dapi ( =, -diamidino- -phenylindole; invitrogen). for the experiment looking at orf a localization with mers-cov infection, huh cells were plated on coverslips day prior to infection with mers-cov. cells were infected for h prior to being fixed with % pfa for h per the standard operating practice (sop) for the biosafety level (bsl ) containment facility. cells were then labeled for immunofluorescence microscopy as described above. the antibodies used were as follows: mouse anti-sirt (clone f ; abcam) ( mg/ml) and anti-mouse texas red and anti-rabbit fluorescein isothiocyanate (fitc; vector labs) ( . mg/ml). all antibodies were diluted : for use. rabbit anti-orf a serum (a kind gift from stanley perlman; used as described previously [ ] ) was diluted : for use. imaging was performed using an echo revolve microscope. sirna knockdown. a double-sirna-transfection approach was used to achieve knockdown. cells were plated in a -well dish the morning of day and allowed to settle on the plates for to h. cells were then transfected with one of two sirt -targeting sirnas (the sequences are described above in the "plasmids and compounds" section and were the same as those reported in reference ) or with scrambled sirna (mission sirna universal negative-control no. [sigma]) using oligofectamine reagent (thermo) and opti-mem (gibco). for a -well dish, l oligofectamine and l opti-mem were mixed and the mixture was incubated for min at rt and mixed with l opti-mem and l m sirna. this transfection mixture was incubated for min at rt. media were removed from cells and replaced with l opti-mem, the transfection mixture was added, and the resulting mixture was incubated at °c/ % co for h. after this incubation period, a : volume of % fbs-dmem was added to the cells. the following day, media were changed for % fbs-dmem. cells were collected on day and replated for a second round of transfection using the same approach. cells were then collected on day and prepared for experimental use. crispri. huh cells were transduced with phage tre dcas -krab lentiviruses, and the transduced cells were selected with geneticin (invitrogen) ( . mg/ml). transduced cells were screened for inducible dcas expression to generate a doxycycline-inducible ( g/ml dox; sigma) gene repression cell line. this cell line was then further transduced with sirt crispri sgrna lentiviruses (sequences for the sgrna are provided above in the "plasmids and compounds" section). sirt crispri sgrnas were designed using the broad institute sgrna design tool. of three sgrnas initially tested, only one resulted in clear and reproducible sirt repression. in each of the tested sgrna sequences, two independent lentivirus preparations (see below) were used for transduction on separate bulk populations of the huh -dcas inducible cell lines. transduction of sirt sgrnas was selected for by the use of puromycin (sigma) ( g/ml). the huh-dcas -sgrna cells were continuously cultured in dmem supplemented with % fbs, % pen/strep, . mg/ml neomycin, and g/ml puromycin. to achieve knockdown of sirt , these cells were cultured in growth medium containing g/ml dox, with this medium being changed every days over a -day period prior to use of the cells for experiments. lentivirus production and transduction. all lentiviruses were produced in hek t cells. lentivectors were cotransfected with vesicular stomatitis virus envelope glycoprotein (vsv-g)-expressing plasmid pmd .g (a gift from didier trono; addgene plasmid no. ) and packaging plasmid pspax (a gift from didier trono; addgene plasmid no. ) and were concentrated using peg-it (system biosciences). for transduction, cells were plated to achieve approximately % confluence in a -well dish. lentivirus preparations were added to cells in media containing g/ml polybrene (americanbio). cells were then subjected to spinoculation at , rpm for min and incubated overnight prior to addition of puromycin selection media. mammalian cell western blot sample preparation. cells for western blotting were plated day prior to sample collection. plates were placed on ice and washed twice with °c pbs. cells were lysed on ice for min by incubation with radioimmunoprecipitation assay (ripa) buffer (sigma) containing ϫ complete protease inhibitor cocktail (complete mini; roche). lysate was collected and centrifuged at , ϫ g for min. supernatant was collected and used for western blotting or stored at Ϫ °c prior to use. the protein content of the sample was quantified using a bicinchoninic acid (bca) system (thermo scientific). a -g volume of whole-cell lysate (wcl) was used in all western blotting experiments. western blotting. samples were separated using % to % mini-protean tgx gels (bio-rad) and transferred to polyvinylidene difluoride (pvdf) membranes (immoblion-fl; millipore) using a trans-blot turbo system (bio-rad) or a wet transfer approach ( h of transfer at ma). membranes were blocked using aquablock (east coast bio) for h at rt and were then incubated with primary antibody (diluted in aquablock) overnight at °c. the following day, membranes were washed with tris-buffered saline with tween (tbst; mm tris, mm nacl, . % tween ) and incubated with secondary antibodies diluted in aquablock for h at rt. membranes were further washed with tbst prior to imaging using a chemidoc imaging system (bio-rad). blot visualization was achieved using either fluorescence or horseradish peroxidase (hrp)-conjugated secondary antibodies, detected by the use of amersham ecl prime reagent (ge healthcare life sciences). the primary antibodies used were as follows: mouse anti-sirt (as described in the "immunofluorescence microscopy" section), mouse anti-flag (clone m ; sigma) ( mg/ml), mouse anti-tubulin (clone dma a; sigma), rabbit anti-gfp (sigma) ( mg/ml), and mouse anti-actin (clone a ; sigma). all primary antibodies were diluted : , for western blotting. the secondary antibodies used were as follows: goat anti-mouse hrp (thermo scientific) ( . mg/ml), goat anti-rabbit hrp (thermo scientific) ( . mg/ ml), goat anti-mouse alexa fluor (life technologies) ( mg/ml), and goat anti-rabbit alexa fluor (life technologies) ( mg/ml). hrp-conjugated secondary antibodies were diluted : , , and fluorescent secondary antibodies were diluted : , . immunoprecipitation. cells were plated day prior to use and lysed in ripa buffer containing ϫ complete protease inhibitor cocktail as described above (similarly to the co-ip protocols previously described in references and , for example). protein content in the whole-cell lysate was determined by bca assay, following the instructions of the manufacturer (thermo scientific). wcl ( g) was used in the ip. nonspecific binding to beads was cleared by incubating the g of wcl for h at °c with protein a magnetic beads (cst). beads were separated from lysate with a magnetic rack, and the supernatant was collected. the nonspecific fraction was prepared by adding ϫ lsb with ␤-mercaptoethanol and heating at °c for min. the collected supernatant was added to fresh magnetic beads and incubated with anti-gfp antibody ( g) overnight at °c. the following day, the beads were separated with a magnetic rack and the supernatant was collected as the flowthrough fraction. beads were washed five times with ripa buffer before addition of ϫ lsb with ␤-mercaptoethanol and heating at °c for min to prepare the immunoprecipitation fraction. viral infection. mers-cov (jordan strain-genbank accession no. kc . ; mers-cov-hu/jordan-n / ) stocks were prepared by infection of vero cells, and titers were determined by plaque assay using these cells (as described previously [ ] ). all experimental infections were performed with huh cells at a multiplicity of infection (moi) of . unless indicated otherwise. for quantification of virus production, medium samples were collected h after infection, centrifuged in a table top centrifuge for min at full speed, and stored at Ϫ °c prior to use. tcid ( % tissue culture infective dose) assays of vero cells were used to quantify the amounts of virus in the collected samples ( ) . luciferase assays. for celltiter-glo (ctg) assays, cells were plated in opaque -well plates day prior to use. for drug treatments, resveratrol or ex was added at the indicated concentrations and incubated for h before ctg assays were performed. for sirna samples, cells were plated and grown overnight and then used for ctg assays. a celltiter-glo luminescent cell viability assay (promega) kit was used per the manufacturer's instructions. luminescence was read using a synergy htx multimode plate reader. reporter assays were used with b luciferase plasmids as previously described ( ) . briefly, hek t cells were plated day prior to transient transfection as described above. these transfections were performed in -well plates using a total of ng of dna per well. at most, three plasmids were required for transfection (sirt /orf a/luciferase for example); in instances of fewer experimental proteins being needed, preceive-gfp was used to balance the amounts of dna. cells were transfected for h before further manipulation. in the cases of drug treatment, cells were pretreated with resveratrol or ex for h and tnf-␣ stimulation was performed for h using ng per 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dynamic imaging of genomic loci in living human cells by an optimized crispr/cas system transformation of yeast by lithium acetate/ single-stranded carrier dna/polyethylene glycol method antagonism of dsrna-induced innate immune pathways by ns a and ns b accessory proteins during mers coronavirus infection posting date. jnk phosphorylates sirt and promotes its enzymatic activity accessory proteins b and ab of severe acute respiratory syndrome coronavirus suppress the interferon signaling pathway by mediating ubiquitindependent rapid degradation of interferon regulatory factor growth and quantification of mers-cov infection, p e. . - e. . we thank kirsten kulcsar and william jackson for valuable comments and critical readings of the manuscript, brendan cormack for the yeast knockout library collection, and stanley perlman for the orf a antisera. we also thank carolyn frenkil for her generous donation that initiated this work. key: cord- -qnbgywgy authors: yilmaz, huseyin; faburay, bonto; turan, nuri; cotton-caballero, maira; cetinkaya, burhan; gurel, aydin; yilmaz, aysun; cizmecigil, utku y.; aydin, ozge; tarakci, eda altan; bayraktar, erhan; richt, juergen a. title: production of recombinant n protein of infectious bronchitis virus using the baculovirus expression system and its assessment as a diagnostic antigen date: - - journal: appl biochem biotechnol doi: . /s - - - sha: doc_id: cord_uid: qnbgywgy the avian coronavirus-infectious bronchitis virus (avcov-ibv) is recognized as an important avian pathogen, and new viral variants are a continuous threat to the poultry industry worldwide. sensitive diagnostics and efficacious vaccines are necessary to combat ibv infections in chickens. the aim of this study was to produce recombinant n protein of ibv in the baculovirus system to use in elisa diagnostic tests in order to enable the assessment of the sero-prevalence and risk of ibv infections in chickens in turkey. for this, the gene encoding the n protein of the beaudette strain of ibv was expressed using a recombinant baculovirus expression system. the recombinant n protein was purified using ni-nta affinity chromatography. an estimated -kda recombinant protein corresponding to the expected molecular weight of ibv n including the xhis tag was detected using an anti-his monoclonal antibody. specific immunoreactivity of the recombinant protein was confirmed by western blot using antiserum obtained from vaccinated and naturally infected chicken from turkey as well as using a monoclonal antibody raised against the n protein of the ibv massachusetts strain. the results obtained with the in-house elisa had high agreement with a commercial elisa. immunoreactivity analysis using antisera in western blotting and the in-house elisa suggests that the recombinant ibv n protein could be broadly cross-reactive with antisera produced against different ibv strains. we conclude that the recombinant baculovirus expressed ibv n protein could serve as a useful diagnostic antigen for detection of ibv infections in chickens by elisa. infectious bronchitis virus (ibv) is the causal agent of infectious bronchitis (ib), an acute and highly contagious disease, threatening the poultry industry worldwide [ , , , , ] . avian coronavirus-infectious bronchitis virus (avcov-ibv) is an enveloped, positive single-stranded rna virus, and belongs to the genus gamma-coronavirus within the family of coronaviridae in the order nidovirales [ ] . the virus is spread mainly by aerosol, consumption of contaminated feed and water, and contact with infected feces or equipment. ib is characterized by various clinical signs in broilers and layer hens: coughing, sneezing, and decreased weight gain [ , ] . specifically, in layers, egg production can drop up to % with eggs that have shells that are wrinkled, thin, and soft. in young chicks, ibv infection can lead to oviduct cysts and reduced laying potential [ , , ] . the to kb genome of ibv encodes nine genes, which includes spike (s), membrane (m), envelope (e), and nucleocapsid (n) [ ] . the spike protein, a viral surface glycoprotein, was shown to induce neutralizing antibody response, and the nucleocpasid protein was shown to elicit strong antibody responses [ , ] . despite the presence and application of ibv vaccines in poultry, there is a high rate of emergence of antigenic variants and recombinant strains, and the lack of cross-protection between different viral genotypes, making disease control difficult and vaccine development rather challenging [ , ] . therefore, genetic characterization of circulating strains of ibv, appropriate vaccination programs, and application of sensitive diagnostic tests to detect and assess disease risk are important regional, national, and international strategies to control ibv infections [ , , , , ] . several elisas have been developed for detection of antibodies to ibv in chickens. recombinant antigens used in these elisas were either based on the s protein [ ] , which is highly variable, or the n protein and often produced in an e. coli expression system [ ] . e. coli is a common flora or pathogen in chickens, thus creating the possibility of serological cross-reactivity and detection of false positives in these diagnostic assays. thus, it is necessary to assess the suitability of other expression platforms for production of diagnostic antigens for use in ibv serology. the objectives of this study were to clone and express ibv n protein using the recombinant baculovirus expression system and assess its use as diagnostic antigen for serological diagnosis of ibv infection in chickens in turkey. the complete coding sequence ( bp) of the ibv n gene of beaudette strain (genbank accession no. m . ) was initially amplified by pcr using primers, jar f: ′-cac cat ggc ttc cgg taa ggc tg- ′ and jar r: ′-cag ctc gtt ctc acc cag agc agc- ′. the pcr product was cloned into pfastbac vector (life technologies), to create a recombinant donor plasmid, pfastbac-n, which was transformed into one shot mach t chemically competent e. coli (life technologies). the donor plasmid was double digested with restriction enzymes bamhi and psti to determine the presence of the correct insert in the right orientation. the accuracy of the sequences was confirmed by dna sequencing. the donor plasmid was transformed into max efficiency dh bac competent e. coli to construct a recombinant bacmid via site-specific transpositioning. to rescue recombinant baculoviruses encoding the ibv n gene, recombinant bacmids were purified using highpure miniprep kit (life technologies) and used to transfect spodoptera frugiperda (sf ) cells grown in sf- ii serum-free medium (sfm) supplemented with % fetal bovine serum and % penicillin-streptomycin as manufacturer's instruction (life technologies). transfection was carried out using cellfectin ii reagent as previously described [ ] and according to the manufacturer's instructions (invitrogen-life technologies). recombinant ibv n protein was expressed using passage or higher passage recombinant baculovirus stocks (> pfu/ml). the protein was expressed with a carboxy-terminal xhis tag, and purification using ni-nta superflow resin (qiagen inc., valencia, ca) was performed as described previously [ ] . concentration of the purified protein was measured by the method of bicinchoninic acid (bca) assay (thermo scientific, rockford, il) at an absorbance of nm, using bovine serum albumin (sigma-aldrich, st. louis, mo) as the protein standard. aliquots of the protein were stored at − c until used. western blot analysis was performed to verify specific protein expression. for this, approximately μg of purified recombinant ibv n protein was resolved in % bis-tris polyacrylamide gel (life technologies) as described previously [ ] . following electroblotting of the proteins onto pvdf membrane, the blots were probed with mouse anti-his (c-terminal)-hrp monoclonal antibodies ( . mg/ml) (life technologies) diluted : to determine expression of the correct molecular size protein. further confirmation of expression was performed using a mouse anti-ibv n (massachusetts strain) monoclonal antibody ( mg/ml) diluted : (cat no. mbs ; mybiosource, san diego, ca) and antiserum (at : dilution) collected from chickens naturally infected with local turkish wild-type ibv strains. the membrane was then incubated with anti-species igg-hrp secondary antibody conjugate (santa cruz biotechnology, dallas, tx) at : dilution, and specific signals were detected using enhanced chemiluminescent (ecl) detection system. to assess the induction of specific ibv n-specific antibodies in the immunized chickens (described below), the antisera were tested in immunoblot assays. approximately, μl of sample containing approximately μg of recombinant ibv n protein was diluted in -μl laemmli sample dilution buffer (santa cruz biotechnology, dallas, tx) and resolved in % bis-tris polyacrylamide gel as described above. following transfer, the blot was probed with ibv n antisera obtained from the chicks immunized with the recombinant ibv n protein at a dilution of : and with antisera from wild-type ibv-infected chicken at : dilution [ ] . ibv negative chicken sera, at a dilution of : , and mouse anti-ibv n monoclonal antibody ( mg/ml) (thermo fisher, cat. no. ) at a dilution of : were used as negative and positive controls, respectively. thereafter, the blots were probed using goat anti-chicken hrp-conjugated secondary antibody ( μg/ . ml) (cat. no. sc ; santa cruz biotechnology, dallas, tx) at a dilution : , and goat anti-mouse hrp conjugated secondary antibody ( μg/ . ml) diluted : (cat. no. sc ; santa cruz biotechnology, dallas, tx). reactivity was detected using ′- ′-diamino benzidine peroxidase substrate system (sigma cat no: d - ) as recommended by the manufacturer. to assess immunogenicity of recombinant baculovirus-expressed ibv n protein, embryonated spf chicken eggs were obtained from the bornova veterinary control institute (izmir, turkey). the eggs were incubated in an incubator for hatching. after hatching, -day-old ibv antibody negative chicks (determined by commercial elisa-biochek, ibv elisa, cat no: ck ibv) were immunized with the recombinant ibv-n protein produced in this study. four chicks were immunized intramuscularly with μg each of the recombinant ibv-n protein mixed with isa vg adjuvant (seppic, france) at a ratio of : (adjuvant/ibv-n solution), and four chicks were given placebo as mock-immunized controls. on day , immunization was repeated using the same amount of adjuvant and antigen. chicks were bled on day (pre-immunization) and then on days , , and to test for presence of antibodies to recombinant ibv-n protein. optimization of elisa an indirect elisa protocol was first optimized by checkerboard titration following the methods as described previously [ , , ] . for this, two-fold dilutions of ibv-n protein were made starting from to ng in a carbonate bi-carbonate coating buffer (sigma, c- ). the negative control serum was from the spf chicks tested antibody negative by a commercial elisa (biochek). positive control was from chickens either vaccinated or had field infection tested ibv-positive by pcr. the ibv positive and negative sera were also diluted two-fold (from : to : ), and the conjugate (hrp-conjugated goat anti-chicken antibody, santa cruz, sc ) was diluted : , : , , : , , and : , , in blocking solution. per the results of the optimization, the optimal amount of the recombinant ibv protein for use in elisa ranged from to ng, whereas the optimal dilution of ibv positive chicken serum ranged from : to : , that of the conjugate at : dilution. following optimization of the elisa protocol, test sera were obtained from broiler chickens exposed to natural wild-type ibv infection, test sera from broiler chickens vaccinated with a live-attenuated commercial ibv vaccine, and sera obtained at different time-points from chicks immunized with recombinant ibv n protein (described above) were analyzed to detect ibv n-specific antibodies. for this, a -well plate (nunc maxisorb, thermo fisher scientific) was coated overnight at °c with ng ibv-n protein/per well in -μl carbonate-bicarbonate coating buffer (sigma, cat. no. c- ). the plate was then blocked for h at room temperature with a blocking buffer (pbs containing % skimmed milk and . % tween ) . a volume of μl of test sera, diluted : in blocking solution, was added and incubated for h at °c. each serum sample was tested in duplicate, and each test plate included duplicate positive and negative control sera. the negative control serum was from the spf chicks tested antibody negative by a commercial elisa (bochek). positive control serum was from chickens either vaccinated or had field infection and tested ibv positive by pcr. goat anti-chicken hrp-conjugated antibody (santa cruz biotechnology, cat. no. sc ), diluted : , was added to the wells and incubated at °c for h. between each step, the plate was washed four times by using the wash buffer (pbs containing . % tween ) . hundred microliters of tmb substrate solution was added to each well and incubated at room temperature for min. the reaction was stopped by adding μl of m h so and absorbance (optical density) was measured at nm using a microplate reader . (slt-spectra, slt lab instruments, germany). the elisa cut-off value was determined by addition of standard deviations to measurements of the mean od value of the negative control sera in each plate. od values above cut-off value were taken as positive. thus, a cut-off od value of . was determined for the in-house indirect elisa. to assess the reliability of the performance of our in-house indirect ibv n elisa, a panel of sera was obtained from chickens naturally infected with local wild-type ibv strains, chickens vaccinated with live-attenuated commercial ibv vaccine, and chickens immunized with recombinant ibv n protein (expressed in a baculovirus expression system). the serum samples were tested simultaneously in the in-house indirect elisa (using the procedure described above) and a commercial elisa (biocheck, san francisco, ca) per manufacturer's instruction. the cut-off value for the commercial elisa was defined according to the manufacturer and determined as samples with a sample to positive ratio (s/p ratio) of . or greater. a bp fragment representing the expected molecular size of the ibv n gene was successfully amplified by high fidelity pcr (fig. ) . the pcr amplicon was successfully cloned into pfastbac/ct plasmid resulting in the creation of a donor plasmid pfastbac-n ( bp), restriction enzyme analysis of the recombinant donor plasmid confirmed the presence of the correct insert in the correct orientation by release of two restriction fragments of the expected size ( and bp) (data not shown). the donor plasmid with the correct sequences was used to create recombinant bacmid for subsequent expression of the target protein. in order to express the recombinant n protein of ibv, sf cells were infected with a recombinant baculovirus encoding the n gene of ibv. at h post-infection, cells were harvested and the recombinant protein was purified via affinity chromatography. an estimated kda recombinant protein was overexpressed, which corresponded to the expected molecular size of ibv n protein as determined by coomassie staining (fig. a) and western blot analysis using anti-his (c-terminal)-hrp monoclonal antibody (fig. b) . to confirm specificity of the target protein, an immunoblot analysis was performed using monoclonal antibodies raised against the n protein of the massachusetts ibv strain (fig. a) and as well as antiserum obtained from local chickens naturally infected with turkish wild-type ibv strain (fig. b) . in both cases, specific reactivity was confirmed by detection of a single band ( kda) of the expected molecular size. to assess the ability of the recombinant ibv n protein to induce host specific antibody responses, four chicks ( -day old) were immunized with the recombinant protein and the sera examined for specific reactivity via immunoblot analysis. specific reactivity was detected with serum samples obtained from of the ibv n-immunized chickens at day post-immunization (example see fig. seen with the other two chicken sera. this result was supported by detection of specific ibv n-reactivity with the ibv-specific monoclonal antibody (fig. , lane ) and with antisera from a chicken exposed to a natural field infection (fig. , lane ) . no specific bands were detected in sera from mock-immunized chickens or sera taken from chicks before immunization (fig. , lanes , , , , , , ) . overall, sera from vaccinated and naturally infected (field-exposed) chickens that tested positive in the immunoblot assay were found to be correspondingly positive in the in-house indirect ibv n elisa (data not shown). to further assess the immunogenicity of the recombinant n protein and the reliability of our results, a panel of antisera from naturally infected ( samples plus negative and positive controls), from chickens vaccinated with live-attenuated ibv ( samples plus negative and positive controls) and from chickens immunized with recombinant ibv n protein ( samples), were tested using both the in-house indirect elisa and a commercial elisa. with the in-house elisa, cut-off value was set at od of . . there was relatively good agreement in performance between the two elisas in detecting ibv n-specific antibodies in the sera from naturally infected chickens (fig. a) and live-attenuated vaccinated chickens (fig. b) . of the serum samples obtained from the naturally infected chickens, % ( / ) tested positive in the in-house indirect elisa, whereas % ( / ) tested positive in the commercial elisa detected (fig. a) . all samples collected from vaccinated chickens tested positive in both the in-house and commercial indirect elisas (fig. b) . of the commercial elisa-positive field serum samples, tested positive in the in-house indirect elisa, indicating a sensitivity of % (using the commercial elisa as reference test). none of the sera obtained from preimmunized or naïve chicks tested positive in either elisas, indicating % specificity (n = ). of the four chicks immunized with the recombinant ibv n protein, two chicks seroconverted at day after immunization. all four chicks seroconverted at days post-immunization in both elisa tests, indicating a % agreement between the two assays. the od values of chicks immunized with recombinant ibv n protein were . , . , . , and . . production of immunogenic recombinant proteins derived from pathogenic organisms represents a good strategy for identification of antigenic proteins that may serve as targets for sero-diagnostic and vaccine development [ , ] . this represents the first study in turkey that expressed recombinant ibv n protein in baculovirus and examined its reactivity against antisera obtained from turkish chickens for potential use as antigen fig. detection of ibv n specific antibodies in sera obtained from naturally infected chicken (a) and vaccinated chickens (b) using an in-house ibv-n elisa and a commercial elisa. the cut-off value for inhouse indirect elisa is set at od . , and for the commercial elisa at s/p (sample to positive) ratio of . in serological investigation of ibv infection in domestic poultry. we have demonstrated that ibv n produced in a recombinant baculovirus expression system is immunogenic in local chickens and could detect ibv n-specific antibodies in sera obtained from chickens exposed to natural infection. the recombinant n protein was reactive in both immunoblot and indirect elisa assays. the performance of the in-house indirect elisa was comparable to the commercial elisa tested in this study, indicating the potential suitability of recombinant baculovirus ibv n as a diagnostic antigen that could be used for serological risk assessment of ibv in domestic chickens in turkey. indeed, the n protein of ibv, in contrast to the s protein, has been associated with high stability and immunogenicity and has been used by others as a diagnostic antigen [ , ] . the recombinant n protein used in the current study was based on nucleotide sequences of the beaudette ibv strain. the reactivity of the recombinant ibv n protein with a monoclonal antibody (b m) produced against the heterologous ibv massachusetts strain, and with sera from local turkish chicken indicate the conserved, cross-reacting nature of the n protein sequence and suggest potential broad cross-reactivity of the target protein among different ibv isolates and its suitability as a diagnostic antigen to detect ibv infection in local turkish poultry. other researchers have utilized recombinant ibv n expressed in various host systems in serological assays. for example, in a study performed by pradhan and others [ ] , ibv-n protein was produced using a prokaryotic expression system. the protein was used for diagnostic purposes and similar to this study, compared the performance with a commercial elisa kit (idexx). the authors reported a . % sensitivity and . % specificity for the in-house elisa based on the recombinant ibv n antigen and they concluded that an indirect elisa based on the recombinant antigen is suitable for use in development of serodiagnostic tools [ ] . in another study, the ibv-n protein was produced in both e. coli and baculovirus expression systems [ ] , and the recombinant antigens were used in elisas to analyze chicken sera collected from farms [ ] . their data from screening of sera for the presence of ibv antibodies indicated that using the recombinant n protein as coating antigen could achieve equivalent performance to an elisa kit based on extracts from infected material as coating antigen [ ] . in a study performed by ding and others [ ] utilizing a multi-fragment antigen composed of s (spike), m (matrix), and n proteins produced in e. coli [ ] , a good reactivity with an anti-ibv chicken serum was demonstrated. an in-house elisa using the multi-fragment protein exhibited a . % concordance with a commercial elisa [ ] . in the present study, recombinant ibv-n protein was produced using a baculovirus expression system and utilized as a diagnostic antigen in an indirect elisa. the chicken sera from farms and the sera from immunized chickens were reactive with the recombinant antigen with comparable performance to a commercial elisa test kit. this study describes the successful cloning and recombinant baculovirus expression of ibv nucleoprotein and its evaluation as a potential serodiagnostic antigen. the recombinant antigen was immunoreactive with hyperimmune sera from local turkish chickens and the performance of an in-house indirect elisa based on the antigen was comparable to a commercial elisa, suggesting the potential utility for serological detection of ibv infections in chickens in turkey. further studies to validate the assay using a larger sample size including comprehensive assessment of assay specificity will be performed. decreased neutralizing antigenicity in ibv s protein expressed from mammalian cells coronavirus avian infectious bronchitis virus evaluation of a nucleoprotein-based enzyme-linked immunosorbent assay for the detection of antibodies against infectious bronchitis virus the long view: years of infectious bronchitis research infectious bronchitis virus variants: a review of the history, current situation and control measures development of an elisa based on a multi-fragment antigen of infectious bronchitis virus for antibodies detection rift valley fever virus structural and nonstructural proteins: recombinant protein expression and immunoreactivity against antisera from sheep. vector borne and zoonotic diseases a glycoprotein subunit vaccine elicits a strong rift valley fever virus neutralizing antibody response in sheep s gene characteristics and efficacy of vaccination against infectious bronchitis virus field isolates from the united states and israel avian infectious bronchitis virus review of infectious bronchitis virus around the world expression and purification of the kda periplasmic protein of brucella abortus: a reagent for the diagnosis of bovine brucellosis cleavage of structural proteins during the assembly of the head of bacteriophage t expression, purification, and improved antigenic specificity of a truncated recombinant bp protein of brucella melitensis m - : a potential antigen for differential serodiagnosis of brucellosis in sheep and goats detection of antibodies to avian infectious bronchitis virus by a recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay immune responses to mucosal vaccination by the recombinant a and n proteins of infectious bronchitis virus molecular epidemiology and evolution of avian infectious bronchitis virus recombinant nucleocapsid protein based single serum dilution elisa for the detection of antibodies to infectious bronchitis virus in poultry s gene-based phylogeny of infectious bronchitis virus: an attempt to harmonize virus classification an elisa for antibodies against infectious bronchitis virus using an s spike polypeptide a reverse transcriptase-polymerase chain reaction survey of infectious bronchitis virus genotypes in western europe from detection of antibodies to equine arteritis virus in horse sera using recombinant chimaeric n/g(l) protein. the veterinary record phylogeny and s gene variation of infectious bronchitis virus detected in broilers and layers in turkey compliance with ethical standards national and international ethical rules were followed during this study. the authors declare that they have no conflicts of interest. key: cord- -y mxcj authors: hoppe, ingrid bortolin affonso lux; medeiros, andréa souza ramos de; arns, clarice weis; samara, samir issa title: bovine respiratory syncytial virus seroprevalence and risk factors in non-vaccinated dairy cattle herds in brazil date: - - journal: bmc vet res doi: . /s - - - sha: doc_id: cord_uid: y mxcj background: the cattle industry is one of the most important brazilian agribusiness sectors and is a strong contributor to the national economy. annually about . million calves are bred, which makes the optimal management of these animals extremely important. several diseases can affect the initial stages of the bovine production chain, being the bovine respiratory syncytial virus (brsv) one of the most relevant pathogens. this study aimed to characterize the epidemiology of brsv infection in dairy cattle herds of são paulo state, brazil, using serological and risk factors analyses. for that, blood samples were collected of animals from farms and a questionnaire about possible risk factors for brsv prevalence was performed. the obtained blood sera were analyzed using virus neutralization test (vnt). results: vnt results showed high brsv prevalence in dairy cattle herds, reaching . % of seropositivity. the brsv seroprevalence among studied farms ranged from to %. the analysis of risk factors indicated that the age group and the occurrence of coinfection with bovine herpesvirus (bohv- ) and bovine viral diarrhea virus (bvdv- ) should be associated with a higher prevalence of brsv, while natural suckling was considered a protective factor. conclusions: the study showed that adult animals over year old are an important risk factor for the high seroprevalence of brsv in herds. the high brsv prevalence associated with bohv- and bvdv- suggests that biosecurity measures should be applied in order to reduce viral dissemination. additionally, the natural suckling may be an important management to protect calves from high brsv seroprevalence. bovine respiratory syncytial virus (brsv) is an economically significant pathogen in cattle production [ ] , as it is one of the most important causes of lower respiratory tract infections in calves [ ] . in dairy cattle, brsv infection usually occurs in young calves aged between weeks and months [ ] . adult animals with subclinical infection are the main source of infection, since reinfections are common in the herds [ , , ] . brsv, bovine herpesvirus (bohv- ), bovine viral diarrhea virus (bvdv) and bovine parainfluenza type- (pi- ) are considered primary agents involved in the bovine respiratory complex. additionally, secondary infection by pasteurella multocida, histophilus somni and mycoplasmas contribute to the aggravation of the disease [ ] . clinical signs are characterized by respiratory symptoms, initially with moderated intensity, such as nasal and ocular discharges which can be aggravated leading to pneumonia. however, mainly in calves, an acute and severe onset is also observed, due to maternal antibodies not effectively protect against brsv infection [ ] . considering the high prevalence of the disease, several studies determined risk factors involved in the epidemiology of brsv. in europe, risk factors were mainly attributed to herd size, herd density, purchasing of new animals, geographic location of the farms, herd type and concomitant bvdv infection [ ] [ ] [ ] [ ] [ ] . similar studies have also been performed in some latin american countries and they showed that most of the animals probably have already been exposed to the virus with consequent high brsv prevalence in cattle herds. in these countries, herd size, age group, presence of bordering farms, herd type and geographic location of the farms were the main risk factors associated with brsv infection [ ] [ ] [ ] [ ] [ ] . in brazil, brsv was first diagnosed in calves in the state of rio grande do sul [ ] and some studies have shown that brsv infection is widespread in southern and southeastern brazil, with high serological prevalence rates [ ] [ ] [ ] . nevertheless, research has not been conducted in order to verify possible risk factors involved in brsv epidemiology. due to this, the current study aimed to determine antibody prevalence against brsv and investigate some risk factors associated with brsv seroprevalence in herds of an important milk producing region in são paulo state, brazil. the study was performed on dairy cattle herds in municipalities in the northern region of the são paulo state, southeastern brazil. this region produces about million liters of milk annually [ ] . the evaluated herds had between and animals and no animals were vaccinated against the pathogens associated with respiratory diseases. the farms were located in a region classified as aw by the climatic classification of koeppen, characterized by dry winter with average temperature higher than °c in the coldest month and precipitation below mm in the driest month. the altitude of these areas was to m above sea level [ ] . sampling of each farm was calculated [ ] with an expected brsv prevalence of % [ ] with acceptable error of % and confidence level of %. after setting the number of samples, bovines of all categories and age group were randomly selected. according to the management adopted in the herds, the age group was defined as ≤ months old "calf" and > months old "adult". in some farms, the number of samples collected was higher than that suggested by the mathematical calculation, once it was also used for serodiagnosis of bohv- and bvdv- . on farms with a small herd size, up to animals, all of them were sampled. the samples from animals were collected from april to june as shown in table . blood samples were collected by jugular or coccygeal vein puncture using disposable needles and vacuum tubes. samples remained at room temperature for h for coagulation and after being transported refrigerated to the laboratory, they were centrifuged at ×g for min to obtain the sera. the sera were aliquoted in . ml identified microtubes that were stored in freezer at − °c until analyses. the presence of antibodies to brsv was tested by virus neutralization test (vnt). serum samples were thawed, inactivated in water bath at °c for min, and diluted in duplicates from : to : in -well microplates with μl of tcid brsv suspended in eagle's minimum essential medium (difco e-mem®). the viral strain was previously titrated [ ] . following during the sample collection, a questionnaire was administered to the owner or manager of each farm with the purpose of identifying potential risk factors related to epidemiology of brsv. thus, the questionnaire was designed according to data of risk factors described in the literature [ , , , ] in addition to the information of the management adopted in dairy farms of são paulo state. the variables explored were: herd size (≤ , > animals), herd density (≤ , > animals/hectare), age group (≤ months old "calf", > months old "adult"), breed (holstein, mixed), type of reproduction (natural mating, artificial insemination), purchase of animals (last months: yes, no), cleaning of facilities (often, rarely), historical of respiratory disease (last months: yes, no), quarantine (yes, no), abortions (yes, no), bohv- infection (serodiagnosis: yes, no), bvdv- infection (serodiagnosis: yes, no), type of calves housing (individual, collective), type of calves feeding (natural suckling, artificial), presence of others domestic animals (yes, no), presence of wild ruminant animals (yes, no). the variables "herd size" and "herd density" were based on the average data; "age group" was defined from the management adopted on the farms; "quarantine" refers to the isolation of purchased animals before adding them to the herd. the serodiagnosis of bohv- and bvdv- was performed at the same time as brsv (unpublished data). the variables "disinfection of the umbilical cord" and "colostrum feeding" were not analyzed because these practices were applied in all dairy farms visited. additionally, the data related to climatic classification and altitude were not included in the analysis because they were similar in all farms. the chi-square tests were employed to compare the brsv, bohv- and bvdv- seropositivity and also with "age group". fisher's exact test was used to compare brsv status according to the expected prevalence of % (≥ %, "high"; < %, "low") and the other variables. only the variables with p < , (two-tailed fisher) were analyzed by a logistic regression model. the analyses were performed using the epi info™ program v. . . serum samples from animals belonging to dairy cattle herds were taken and tested by vnt, in which ( . %) were seropositive to brsv. regarding the age group, % ( / ) of the serum samples were positives for adult animals while the prevalence rate in calves was . % ( / ). antibodies to brsv were detected in all cattle herds, with prevalence rates ranging from to %. the mean antibody titers for adult animals was to , and for calves, to . therefore, prevalence of brsv both in herds and animals was considered high. the chi-square test showed association of brsv seropositivity with "age group" and animals tested seropositives to bohv- and bvdv- (table ). nevertheless, the fisher's exact test only detected statistical difference with the variable "type of calves feeding" (table ). in this case, the relative risk (rr) value was less than one, i.e., the factor "natural suckling" was considered protective. logistic regression (values of p < . , two-tailed fisher) did not show significant results, suggesting that the variables analyzed were not risk factors for the high seroprevalence of brsv in the studied population. this is the first epidemiological study to assess risk factors for brsv seroprevalence carried out in brazil. even though brsv prevalence of . % in the animals sampled was similar to that estimated, the prevalence in adult animals was higher than that expected, reaching % of samples. in calves, the seroprevalence was lower than that found in adult animals ( . %) and could be even lower once vnt does not allow the distinction between antibodies from colostrum and natural infection. thus, this study demonstrated that the prevalence of brsv antibodies was higher in adult animals, as previously reported in other countries [ , ] . adult animals are associated with high seroprevalence of brsv as consequence of a repeated exposure to the virus infection throughout their life and possibility of reinfections. similarly, the highest antibody titers were associated with non-vaccinated adult cattle, probably due to the exposure to successive viral reinfections, which results in a booster effect on antibody titers [ ] . other factor related to high antibody titers is recent brsv infections, which can be confirmed only by paired serology, antibody screening in calves after the period of colostral antibody detection or viral detection by direct methods. as respiratory disease was not reported in half of the herds studied, it is indicative that brsv infection can be subclinical. this is consistent with previous reports [ ] . herds can remain free of clinical brsv infection for many years even in areas of high prevalence of the virus [ ] . the presence of other pathogens is also associated with the prevalence of brsv [ , , , ] . this information explains the association of brsv serological prevalence with the prevalences of bohv- and bvdv- . the infection by these viral agents is also reported in brazilian herds, with high prevalences [ , ] . bvdv infection can cause impairment of the animal's immune function and thereby decrease resistance to other infections [ ] . the synergistic effects of bvdv with other respiratory pathogens have been observed [ , ] . thus, health status of the herds may also be affected indirectly by bvdv control measures [ ] . dairy cattle herds in são paulo state usually have poor biosecurity measures, such as the lack of quarantine of newly purchased animals, lack of diagnosis of respiratory diseases (particularly for brsv) and vaccination is rarely performed against these viruses. therefore, we hypothesized that risk factors for the seroprevalence of bohv- , bvdv- and brsv in the studied population likely to overlap. despite the logistic regression not confirming "type of calves feeding" variable as a risk factor for high prevalence of brsv, the fisher's exact test detected "natural suckling" as a protective factor. "natural suckling" would be important as it may be able to reduce the risk of calves becoming infected by brsv. weaning can be stressful and results in impaired immune function, which may further exacerbate a brsv exposure. suckling reduces the occurrence of diarrhea, prevents the abnormal behavior of cross-suckling of other calves and improves animal health [ , ] . prior to the current study there have been no report about "natural suckling" and its relationship with brsv seroprevalence or its role as a protective factor, therefore, based on the results presented, it has the potential to decrease seroprevalence to brsv. similarities were observed among the results found at the present study and those previously obtained by others conducted in brazil [ ] [ ] [ ] . in latin america countries, equivalents prevalences of brsv have also been reported [ , [ ] [ ] [ ] , as well as difficulties in detecting the risk factors involved in the dissemination of the agent, even using different forms of sampling and analyzing a considerable number of variables. thus, the dynamics of infection may differ even in a particular country or geographic area [ ] . the high serological prevalence of brsv found in this study shows the importance to know more about this infection since it is not considered important in the country, mainly due to the lack of diagnosis. the awareness of the risk factors involved in the brsv dissemination can allow understanding its mechanisms, even though, as in other studies, these factors were not very clear. thereby, further studies as a complement to the current one should be performed until concrete information has been found. adult animals over year old can present high seroprevalences of brsv and are an important risk factor for the virus maintenance in herds. additionally, the concomitant seroprevalence of bohv- and bvdv- leads us to suggest that biosecurity measures to reduce viral dissemination could be applied. non-stressful management, like keeping calves with their mothers promoting natural suckling, may constitute a practical management strategy to reduce the seroprevalence of brsv in brazilian dairy herds. bovine respiratory syncytial virus bovine respiratory syncytial virus (brsv): a review bovine respiratory syncytial virus) infection: its pathogenesis, diagnosis, prevention and treatment seroepizootiologic study of bovine respiratory syncytial virus in a dairy herd respiratory syncytial virus: brief review identificación de agentes virales por inmunohistoquímica en enfermedades respiratórias de bovinos en corral de engorda severe respiratory disease in dairy cows caused by infection with bovine respiratory syncytial virus bovine respiratory syncytial virus seroprevalence and risk factors in endemic dairy cattle herds risk factors for epidemic respiratory disease in norwegian cattle herds risk factors for seropositivity to bovine coronavirus and bovine respiratory syncytial virus in dairy herds seroprevalence of bovine respiratory viruses in north-western turkey bovine respiratory syncytial virus: first serological evidence in uruguay detection on antibodies and risk factors for infection with bovine respiratory syncytial virus and parainfluenza virus in dual purpose farms in colima seroprevalence to bovine virus diarrhoea virus and other viruses of the bovine respiratory complex in venezuela (apure state) prevalence of and risk factors for bovine respiratory syncytial virus (brsv) infection in non-vaccinated dairy and dual-purpose cattle herds in ecuador detection of antibodies and risk factors for infections with bovine respiratory syncytial vírus and parainfluenza virus- in beef cattle of yucatan detection of bovine respiratory syncytial virus in calves of rio grande do sul detection of antibodies against bovine respiratory syncytial virus (brsv) in dairy cattle with different prevalences of bovine herpesvirus type (bhv- ) in são paulo state vírus respiratório sincicial dos bovinos (brsv): situação no brasil serological evidence of bovine respiratory syncytial virus in brazil produção da pecuária municipal campinas: unicamp encuestas por muestreo para estudios epidemiologicos en poblaciones animales simple method of estimating per cent end point dynamics of bovine respiratory syncytial virus infections: a longitudinal epidemiological study in dairy herds a longitudinal study of the dynamics of bovine corona virus and respiratory syncytial virus infections in dairy herds herpesvirus bovino tipo (hvb- ): revisão e situação atual no brasil a infecção pelo vírus da diarreia viral bovina (bvdv) no brasil -histórico, situação atual e perspectivas synergistic effects of bovine respiratory syncytial virus and noncytopathic bovine viral diarrhea virus infection on selected bovine alveolar macrophage functions prevalence, outcome and health consequences associated with persistent infection with bovine viral diarrhoea virus in feedlot cattle the effects of early separation on the dairy cow and calf practical implications of increasing natural living through suckling systems in organic dairy calf rearing the authors thank coordenadoria de assistência técnica integral (cati/ saasp) for providing the dairy cattle herds for the study, dr. estevam g. lux hoppe and andressa de souza pollo for assistance with manuscript revision. this work was supported by fundação de amparo à pesquisa do estado de são paulo, fapesp (financial grant / - and doctoral scholarship / - ). the datasets analyzed within the current study are available from the corresponding author upon request.authors' contributions ibalh and asrm performed the experiment. ibalh analyzed the data and prepared the manuscript. ibalh, cwa and sis designed the study and revised the manuscript. all authors read and approved the final manuscript. the work is in accordance with ethical principles in animal experimentation, adopted by the brazilian code of experimentation (cobea) and approved by the ethics committee on animal use (ceua). protocol / . not applicable. the authors declare that they have no competing interests. key: cord- -usgpe cz authors: zuwala, kaja; riber, camilla f.; løvschall, kaja borup; andersen, anna h.f.; sørensen, lise; gajda, paulina; tolstrup, martin; zelikin, alexander n. title: macromolecular prodrugs of ribavirin: polymer backbone defines blood safety, drug release, and efficacy of anti-inflammatory effects date: - - journal: j control release doi: . /j.jconrel. . . sha: doc_id: cord_uid: usgpe cz macromolecular (pro)drugs hold much promise as broad-spectrum antiviral agents as either microbicides or carriers for intracellular delivery of antiviral drugs. intriguing opportunity exists in combining the two modes of antiviral activity in the same polymer structure such that the same polymer acts as a microbicide and also serves to deliver the conjugated drug (ribavirin) into the cells. we explore this opportunity in detail and focus on the polymer backbone as a decisive constituent of such formulations. fourteen polyanions (polycarboxylates, polyphosphates and polyphosphonates, and polysulfonates) were analyzed for blood pro/anti coagulation effects, albumin binding and albumin aggregation, inhibitory activity on polymerases, cytotoxicity, and anti-inflammatory activity in stimulated macrophages. ribavirin containing monomers were designed to accommodate the synthesis of macromolecular prodrugs with disulfide-exchange triggered drug release. kinetics of drug release was fast in all cases however enhanced hydrophobicity of the polymer significantly slowed release of ribavirin. results of this study present a comprehensive view on polyanions as backbone for macromolecular prodrugs of ribavirin as broad-spectrum antiviral agents. viruses are fascinating products of evolution and life-threatening pathogens at the same time. indeed, these non-cellular assemblies of macromolecules and lipids are simpler than even minimalistic replicating cells but can infect living organisms in both plant and animal kingdoms. throughout history, viral infections repeatedly had a devastating effect on human society and even in modern times, viral outbreaks create an enormous socio-economic burden [ , ] . recent examples include the outbreaks of coronavirus-associated southeast asian and middle eastern respiratory syndromes (sars, mers) as well as the ebola and zika virus outbreaks. to counter this, interdisciplinary efforts have been made over the past decades in the design and development of antiviral drugs. in large part, this was spurred by the spread of the human immunodeficiency virus (hiv) [ ] . currently, there are nearly one hundred antiviral drugs approved by the us fda [ ] . nevertheless, significant challenges remain. it is now understood that a significant limitation of current strategies is that countermeasures to viral pathogens are most typically developed individually and specifically to each emerging pathogen. an aspect that came into the focus of research attention is the development of "broad-spectrum antiviral agents" [ ] . such agents can constitute prophylactic, [ ] preventative, [ , ] or curative [ , ] measures. prophylaxis (vaccination) holds a great appeal but universal antiviral vaccines are yet to be developed [ ] . in fact, the overall majority of pathogenic viruses have no corresponding vaccination product. in turn, curative measures are highly attractive in their own right. however, while replicational cycles of viruses are similar, there is a great variability between viral enzymes and other proteins that could serve as drug targetsmaking the design of universal antiviral agents challenging [ ] . interferon is an endogenous protein that serves as a potent stimulatory signal to human immunity and acts as a non-specific, broadly acting antiviral agent. however, viruses developed multiple interferon inhibitors which compromise induction or activity of interferon [ ] . moreover, due to its side effects, its clinical use is limited [ ] . several small molecule drugs also serve as broadly acting antivirals. of these, ribavirin (rbv) has a long history of medicinal use and appears on the world health organization list of essential medicines for both adults and children [ , ] . however, this drug also has a dose limiting toxicity. several other experimental drug leads are in different stages of (pre)clinical development [ ] [ ] [ ] [ ] . finally, preventative antivirals that act through neutralization of viruses and/or blocking virus cell entry appear to be highly promising as broad-spectrum antiviral agents [ ] [ ] [ ] . a notable class of such agents is polymers. in the early s, it was first observed that viruses interact with polymers [ ] and inhibit virus infectivity [ ] . over subsequent decades, this effect was shown to be affecting viral pathogens across the viral genus and families, and observed for polymers diverse in their structure, natural and synthetic, positively or negatively charged [ ] [ ] [ ] [ ] [ ] [ ] . de clercq et al. proposed that activity of the negatively charged polymers, at least in vivo, is indirect and proceeds via stimulation of interferon production [ ] . however, antiviral activity proceeds potently and efficaciously in vitro, in absence of interferon, and the direct contact between polymers and the virus with ensuing neutralizing activity is now well-documented. inherent skepticism towards polyanions and other polymers as antivirals [ ] originates in the failures of hallmark clinical trials for these agents both when administered systemically or as topical microbicides [ ] . specifically for systemic administration, it has been concluded that preventative antiviral effect would have to rely on a sustained, high concentration of these agents in blood not achievable without toxic effects [ ] . however, other polymers are successfully commercialized as antivirals, specifically as lubricants in condoms acting as microbicides with activity against hiv and herpes simplex viruses [ ] . nucleic acid based polyanions progress through clinical trials as agents against hepatitis b virus with potential of further development as broad-spectrum antiviral activity [ ] . it is important to note that failed clinical trials are indeed discouraging yet important, game-changing developments have occurred in polymer chemistry in recent decades. specifically, novel polymerization techniques are now available to afford polymers with well-controlled molar mass and architecture and a significantly broader available polymer functionality (side chains) [ , ] . another notable advancement includes the development of polymeric virucidal agents that compromise stability of the viral envelope and exert permanent damage to the virion [ , ] . together, the broad spectrum of activity of polymers against viral pathogens and the chemical versatility of these agents renders this class of antiviral agents unique and attractive for deeper investigation. in our past studies, we developed broad-spectrum antiviral agents based on macromolecular prodrugs (mp) of ribavirin. when conjugated to polymers, ribavirin did not accumulate in the red blood cells which in vivo is the main side effect of the drug [ ] [ ] [ ] . mp of ribavirin broadened the therapeutic window of the drug (in vitro) [ ] and were effective against diverse viruses such as hcv (in a replicon cell culture model), measles, and influenza, in the latter case revealing antiviral effects in chicken embryo model [ , ] . we also experimentally confirmed the link between ribavirin and the synthesis of nitric oxide in macrophages, with relevance to the treatment of hepatitis [ ] . key to successful delivery of ribavirin using mp was the development of a disulfide-containing linker that releases the drug from the prodrug upon cell entry [ ] . from a different perspective, we also focused on preventative measures and conducted a systematic study of polymers with anionic charge as inhibitors of virus cell entry [ ] . our broad study considered polymers, infectious zika virus, hiv, and hsv, as well as pseudotyped viral particles for ebola, lassa, lyssa, sars, and other viruses. the main finding of our work was that anionic charge alone (be it carboxylate, phosphate/phosphonate, or sulfonate) did not endow the polymer with broad-spectrum antiviral activity. decisive factor for such activity was the enhanced (but balanced) hydrophobicity of the macromolecule. identified lead candidate polymers exhibited antiviral activity against all the enveloped viral pathogens, including the zika virus. for the latter, we presented evidence of direct contact between the polymer and the viral particle and concurrent inhibition of viral infectivity [ ] . one intriguing possibility lies in the prospect of combining the curative and the preventative antiviral activity within the same macromolecular antiviral agent [ , ] . such agents represent a combination therapy wherein the carrier exerts extracellular preventative effect [ ] whereas the conjugated drug, once released inside the cell, is an intracellularly active therapeutic [ ] . it is also possible that polyanions have an intracellular antiviral effect of their own, that is the inhibition of viral polymerases, [ , , ] presumably through competitive electrostatic interaction with the protein. in this work, we explore the design of such combination therapy in detail. we focus on the choice of the macromolecular backbone as a carrier for the conjugated drug and analyze blood coagulation, binding to albumin, albumin aggregation, inhibitory activity on polymerases, and cytotoxicity for polymers differed by their anionic charge (carboxylates, phosphates and phosphonates, sulfonates). further, we synthesize ribavirin-containing counterparts to the polyanions, investigate kinetics of drug release for the resulting macromolecular prodrugs, and investigate their use for intracellular drug delivery of ribavirin in an anti-inflammatory model in macrophages. as a result, we identify polymers and macromolecular prodrugs that are devoid of blood anti-coagulation activity but are strong as inhibitors of polymerases and efficacious as delivery vehicles for ribavirinthus being attractive for the development of broad-spectrum antiviral agents. we also identified polymers that are benign and devoid of any activity in these tests thus being attractive as "stealth" materials for diverse biomedical applications [ ] . results of this study would be important for the advancement of biomedical engineering, specifically with regards to development of antiviral therapies and drug delivery techniques. homopolymers of different anionic monomers with variations in the anionic functionality (carboxylic acid, sulfonic acid, phosphonic acid and phosphate, fig. ) were obtained through the reversible addition-fragmentation chain transfer (raft) polymerization [ ] . synthetic approach to the synthesis of the corresponding macromolecular prodrugs was chosen to proceed via copolymerization of the drug containing monomer and that corresponding to the carrier polymer. towards this end, rbv containing monomers were designed to be compatible with polymerization of diverse co-monomers, acrylic and methacrylic, hydrophilic and hydrophobic (fig. ). of these, the synthesis of the hydrophobic methacrylate monomer (monomer in fig. ) was reported in our previous publications [ ] . acrylic counterparts were obtained via similar protocols with minor variations. design consideration also concerned polarity of co-monomers, an aspect that exerts limitations to the choice of the solvent for polymerization. polarity of the applied anionic monomers varied from those with a hydrophilic character (phosphates/phosphonates) to those with well pronounced hydrophobic character (ethylacrylic acid, propylacrylic acid). rbv monomers were therefore designed to mimic the polarity of the anionic comonomers through the use of silyl protecting groups to render the monomer more hydrophobic or using deprotected, hydrophilic ′, ′-hydroxyl containing monomers. in total four different rbv prodrug monomers were applied (fig. ) , allowing for the copolymerization to be performed under hydrophobic and hydrophilic conditions with either acrylate/acrylamide or methacrylate/methacrylamide containing monomers. in each case, the monomer structure also comprised a disulfide bond for intracellular degradation, coupled to a self-immolative linker for drug release [ ] . due to the variations in monomer type and monomer polarity, a unique set of reaction conditions was necessary for each copolymerization including the choice of raft agent, solvent (or solvent mixture), reaction time, initiator, and purification procedure (table , for full details see experimental section). synthesized macromolecular (pro)drugs were analyzed by h nmr during synthesis to assess the conversion of the two monomers and in the purified form to confirm the absence of unreacted monomers. polymers were also characterized through size exclusion chromatography (using an -angle static light scattering detector) to determine the molar mass and dispersity of the polymer samples (table ) . we acknowledge that (co)polymers exhibited variability in their degree of polymerization (molar mass). for this reason, analyses presented below do not take into account the likely, non-negligible influence of molar mass on the properties of the polymers. in doing so, we assume that the polymer structure is the factor determining the polymer properties whereas the molar mass is of secondary importance. indeed, our recent publication on the antiviral activity of the polymers revealed clear structure-activity correlations with regards to the polymer functionality whereas no clear correlation with the polymer molar mass was observed [ ] . blood pro/anticoagulation is the most known side effect of polyanions upon their systemic administration. indeed, depending on structure, polyanions may exhibit anticoagulation effect, [ ] as is wellknown for heparin, [ ] a polysaccharide approved by the us fda specifically for the purpose of blood anticoagulation. other polyanions such as polyphosphates (nucleic acids) are reported to be pro-coagulants [ ] . no interference with blood coagulation is a highly desired safety feature of injectable formulations and identification of coagulation-inert polymers was one of the aims of this study. we investigated activated partial thromboplastin time (aptt) in presence of the synthesized polyanions. the platelet-depleted plasma from healthy donors was incubated with polyanions at concentrations of or mg/l and the aptt was measured (fig. ). polysulfonates and pvbza at mg/l enhanced blood coagulation time to a value over s, similarly to heparin, and this indicates that they are strong blood anti-coagulants. this was also observed with pvpa, which doubled the aptt value. in contrast, other polyphosphates and polyphosphonates papa, pmpa, paep and pmep had minor if any influence on blood coagulation time. of the carboxylates, paa and pmaa exhibited a noticeable anti-coagulation effect. in contrast, peaa and ppaa, two polymers with an enhanced hydrophobicity but decreased anionic character compared to paa and pmaa (higher pka) had no effect on blood coagulation time at mg/l. taken together, results in fig. reveal that six out of the tested polymers (polyphosphates, polyphosphonates, peaa and ppaa) appear to have no inhibitory activity with regards to the blood coagulation times. albumin is the most abundant protein in human plasma. it has numerous physiological functions that include the maintenance of table polymerization conditions and macromolecular characteristics of polyanions and macromolecular prodrugs used in this work. for chemical structures and details of synthesis of the rbv containing monomers through , see fig. and experimental section, respectively. chain transfer agents were cyanomethyl dodecyl trithiocarbonate (cdtc), ( -cyano- -[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid) (cdpa), cyanomethyl methyl(phenyl)carbamodithioate (cmpd), -(dodecylthiocarbonothioylthio)- -methylpropionic acid (dcma) or -cyano- -(phenylcarbonothioylthio)pentanoic acid (cppa); polymerization initiators were azobisisobutyronitrile (aibn), , ′-azobis( -methoxy- . -dimethyl valeronitrile) (v- ) or , ′-azobis( -cyanovaleric acid) (acva). m n : number-average molar mass, sec-mals: size exclusion chromatography with multi-angle light scattering detection, dp: degree of polymerization. values of dn/dc were calculated from sec/mals measurements assuming full mass recovery; dp values for ppaa, ppaa-rbv, and pvbza (marked with a * symbol) are calculated from m n (calc). experimental conditions for anionic homopolymers are taken from ref. osmotic pressure and the transport of hydrophobic solutes through the blood. this protein is safe-guarded by the body and physiological mechanisms exist to ensure that albumin is not degraded but retained in the body. as a result, albumin blood residence time reaches a phenomenal value of weeks [ ] . albumin binding is among the most successful methodologies in biomedicine to extend the circulation times of solutes and this has been achieved to a range of drug molecules, from small molecule anticancer drugs [ ] to peptide hormones and proteins [ ] . recently, we observed that synthetic polymers (peaa) can bind albumin and this afforded hepatic deposition of the polymer [ ] . albumin binding for peaa is not altogether surprising in that this protein is well known to bind hydrophobic solutes and anionic solutes, both features being emphasized in the structure of peaa. herein, we aimed to investigate albumin binding for the library of anionic polymers, with and without conjugation of ribavirin. albumin has several binding sites, most notable of which are the so-called sudlow i and sudlow ii sites [ , ] . binding of solutes to these sites can be interrogated using fluorescent probes that exhibit site-specific biding to albumin, specifically dansyl asparagine (sudlow i) and dansyl sarcosine (sudlow ii) [ ] . fluorescence of these probes is significantly decreased upon displacement from the protein globule, and this presents itself as a facile methodology to quantitate binding of albumin with the polymers. polymers were mixed with albumin at μm ( . g/l) concentration of the protein and molar equivalents of the polymer to albumin. the immediate observation from the results of these experiments ( fig. ) is that polymers differed significantly in their capacity to interact with albumin via the two nominated sites. polyphosphonate polymer papa brought about no decrease in the fluorescence of probes indicating no detectable polymer interaction with albumin. papa also exhibited no anticoagulation activity (fig. ) , and together these data suggest that papa is a blood-safe carrier for diverse drug delivery applications. albumin binding for the structurally similar pmpa as well as the phosphate analogues paep and pmep was also not pronounced and at -fold mole excess of the polymer to albumin, probe fluorescence decreased by no more than %. polysulfonates also exhibited minor association with albumin and afforded no change in fluorescence for the sudlow i binding probe and a change in fluorescence for the sudlow ii probe within %. exception to this was the most hydrophobic of these polymers, styrenic psvbs which showed a % probe displacement. in contrast, polycarboxylates exhibited a strong tendency for binding with albumin and exhibited a highly efficacious probe displacement. binding was not site specific and probes were displaced from both sudlow i and ii sites. in the case of paa, displacement of probes for both sudlow i and ii sites was nearcomplete indicating a strong binding. for this polymer, incorporation of rbv abrogated polymer binding to albumin. this illustrates that paa binding to albumin is structure-programmed and change to this macromolecule such as addition of rbv interferes with albumin binding. pmaa, peaa and ppaa also exhibited strong probe displacement capacity albeit not as pronounced as paa. interestingly, unlike paa-rbv, peaa-rbv and ppaa-rbv were strong albumin binders. albumin binding was also analyzed by sec-mals. this experiment confirmed the difference between the polymers in their capacity to binding albumin and highlighted further changes in the result of the polymer-protein association, fig. . elution of albumin was hardly altered upon its incubation with pamps (polysulfonate) taken at an equimolar concentration to albumin and there was minor protein aggregation in the presence of this polymer. this observation was similar for the protein incubation with papa and all polyphosphonates and polyphosphates as well as for pvsa and pspa. in contrast, for the carboxylates paa, pmaa, ppaa, pvbza and the hydrophobic polysulfonate psvbs, polymer-protein interaction afforded strong aggregation and formation of high molar mass products (mda). finally, upon incubation with peaa, a strong sudlow i/ii probe displacing polymer, elution of albumin was shifted to shorter times indicating increased molar mass of the protein, as would be expected for a polymer-protein adduct, with only minor protein aggregation. peaa is a unique polymer and albumin binding without noticeable aggregation was not observed fig. . polyanions and macromolecular prodrugs exhibit structure-dependent binding to albumin via sudlow i/ii sites. interaction with albumin was quantified using fluorescent probe displacement assay using dansyl asparagine and dansyl sarcosine as probes for sudlow i and sudlow ii sites, respectively. fluorescence intensities of the probe in the presence of polyanions were normalized to that in control experiments. % probe fluorescence indicates no probe displacement and % probe fluorescence implies quantitative displacement and indicates strong interaction between the polymer and albumin. results are presented as average of three independent experiments ± sd. statistical significance compared to the control samples was evaluated via one-way anova with dunnett's multicomparison post-hoc analysis. *p ≤ . ; **p ≤ . ; ***p ≤ . . for other polymers studied in this work. next, we examined macromolecular prodrugs with regards to the kinetics of drug release. rbv conjugation was designed such as to be reversed upon the scission of the disulfide linkage, specifically upon cell entry. intracellular environment is characterized by a relatively high concentration of a thiol containing tripeptide, glutathione (gsh), [ ] and this effectively triggers the degradation of disulfide linkages inside the cells [ ] [ ] [ ] . accessibility of the disulfide within the structure of macromolecular prodrugs can vary due to the difference in the size of the side groups functionalities. polymers may thus exhibit significant differences in the drug release kinetics. we have previously investigated the release of rbv and other drugs conjugated to polymers via the same linkage as used in this study (disulfide trigger paired with a self-immolative linker) via hplc [ , , ] . herein, we established an h nmr-based method to quantify the release of rbv as illustrated in fig. on an example of papa-rbv. macromolecular prodrug was incubated in the presence of gsh. disulfide reshuffling initiates the spontaneous cyclization of the self-immolative linker with ensuing release of the pristine drug, ribavirin (fig. , a) . chemical shift of the ribavirin protons exhibits a well-defined migration in the h nmr spectrum and with time, the signal corresponding to the polymer-bound drug decreases whereas that for the free rbv increases (fig. , b) . integration of the corresponding peaks provides a facile means to quantify rbv bound to the polymer and released from the prodrug. release of rbv from the macromolecular prodrug was fast and within h, over % of the drug was released from the macromolecular carrier (fig. ) . this kinetics profile is highly advantageous, significantly faster than that reported for the peptide-based linkers between the drug and the carrier, [ ] and allows for rapid drug release upon prodrug cell entry. we note that data in fig. are rather similar to the drug release data collected in our prior studies via hplc [ , , ] and this serves to validate the established herein nmr-based approach to monitor drug release. h nmr based method to quantify drug release was applied to the macromolecular prodrugs based on other polyanions as well as a nonionic carrier, phpma, fig. . within h of observations, all mp exhibited a pronounced release of their payload. a significant finding from this experiment was that hydrophobicity of the polymer chain has a pronounced effect on the kinetics of drug release. in contrast, no such correlation appears to exist with regards to the charge of the polymer (anionic vs charge neutral) or the nature of the negative charge on the polymer. indeed, structurally similar phpma and pmaa as well as papa and paep released ribavirin quantitatively within h revealing no difference in drug release kinetics. however, relatively small structural change from pmaa to peaa or from peaa to ppaa afforded a significant decrease in the drug release. similarly, a single methyl group variation from papa to pmpa afforded a pronounced drop in the amount of rbv released within h. a likely explanation to this phenomenon is the more compact, less hydrated and thus less accessible polymer coil for increasingly hydrophobic polymers which hinder gsh from initiating drug release. , as well as a significantly slower release from the methacrylamide based phosphonate relative to the acrylamide based phosphonate. all data are displayed as mean ± standard deviation from three independent experiments. for comparison a oneway anova (analysis of variance) was performed, followed by a tukey's post hoc test. ***: p ≤ . . therapeutic and/or side effects elicited by the polyanionic (pro) drugs investigated herein would likely manifest themselves due to the interaction of the polymers with other macromolecules such as proteins. the spectrum of polyanionic endogenous compounds is rather large, and competition [ ] for electrostatic interactions with these polymeric partners is highly likely. in the context of antiviral performance of the polymers, we [ ] [ ] and others [ ] have shown that polyanions can act as efficient inhibitors of polymerases. charge neutral polymers were devoid of such activity [ ] pointing to the anionic charge of the polymer (that is, electrostatic interaction with the target) as the origin of the observed phenomenon. polymers synthesized in this work were analyzed as inhibitors of two types of polymerase enzymes, namely a dna-dependent dna polymerase (replicase) and an rnadependent dna polymerase (reverse transcriptase), fig. . despite being similar in carrying multiple anionic charges, polymers differed markedly in their ability to interfere with the performance of polymerases. polyphosphates and polyphosphonates were nearly devoid of any inhibitory activity in these assays and regardless of the polymer concentration, polymerases remained uninhibited. together with the data presented above, e.g. papa appears to have unique "stealth-like" properties with no-interference in coagulation assays, albumin binding, and competition for binding with polymerases. in contrast, polycarboxylates and polysulfonates exhibited a dose-dependent polymerase inhibition. a rather surprising finding is that high anionic character alone does not make the polymer a strong inhibitor and in the homologous row of carboxylates, it was peaa that exhibited the strongest inhibitory activity (complete inhibition of reverse transcriptase activity at mg/l polymer concentration). this observation echoes our recent findings on the apparent unique pairing of negative character and hydrophobicity of the polymer backbone that renders peaa an efficacious inhibitor of e.g. hepatitis c virus intracellular replication [ ] and a lead polymer with broad-spectrum antiviral activity [ ] . similar observation can be made for the negatively charged pvbza, also characterized by pronounced hydrophobicity of the chain due to the aromatic ring in each repeat unit of the polymer. finally, as a test for activity of the polymers in cell culture, we evaluated polyanions and their counterparts conjugated to rbv as inhibitors of inflammation in stimulated macrophages. our previous findings revealed that paa-rbv inhibited the synthesis of an inflammatory marker nitric oxide (no) in the lipopolysaccharide (lps)stimulated macrophages whereby both the polymer and the released drug elicited their individual anti-inflammatory activity [ ] . we tested the library of polyanions presented above and quantified inflammatory responses in cells incubated with polymers in a range of concentrations from . to mg/l. independently, metabolic activity of the cells (indicative of cell viability) was measured by the presto blue assay. these experiments therefore provide information on both, toxicity of the polymers to macrophages in cell culture and anti-inflammatory activity of the polymers. none of the tested polymers, with or without conjugated ribavirin, exhibited noticeable toxicity at concentrations up to mg/l, fig. . the overall majority of polymers were also devoid of activity with regards to inflammation. however, several polymers comprised notable exceptions and were efficacious inhibitors of inflammation without associated toxicity to the cells. specifically, among the polyanions, pmaa, peaa, pvbza and psvbs afforded significant decrease in the levels of no produced by macrophages. an apparent unifying structural feature of these four polymers is that these are the most hydrophobic of the polyanions used in this work. decreased inflammatory response by pvbza and psvbs was observed already at mg/l making these polymers most potent of the tested polyanions. it is also striking that fig. . activity of the polymerase enzyme in the presence of polyanions ( or mg/l in the reaction mixture) expressed in % of de novo synthesized nucleic acid relative to the un-inhibited polymerase reaction. statistical significance compared to control was evaluated via a one-way anova with dunnett's multicomparison posthoc analysis. *p ≤ . ; **p ≤ . ; ***p ≤ . . these two polymers, as well as peaa, afforded a highly efficacious decrease in the synthesis of no with a near-complete suppression of inflammation without associated toxicity to the cells. this comes in stark contrast with the rbv based treatment which could only afford ca. % reduction in the levels of no at sub-toxic concentrations of the drug [ ] . it is worthy of note that polyanions are typically deemed to have restricted cell entry [ ] . however, confocal laser scanning microscopy evaluation of the polymer uptake by macrophages revealed pronounced polymer-associated fluorescence inside the cells (fig. ) . this was true for all polymers: polycarboxylates, polyphosphates and polyphosphonates, and polysulfonate. contrary to expectations, cell entry does not appear to be decisive in the observed activity of the polymers. macromolecular prodrugs of rbv were synthesized for the (meth) acrylate/(meth)acrylamide type of anionic monomers thus excluding pvbza, pvpa, psvbs, and pvsa. both pmaa and peaa displayed an inherent, structure-driven effect in the anti-inflammatory assay and in these cases it is therefore not possible to discern the effects elicited by the polymer or the intracellularly released rbv. it is likely that both the polymer and the released drug contribute to the overall anti-inflammatory response in macrophages for pmaa-rbv and peaa-rbv. surprising other leads in this assay were macromolecular prodrugs based on pmpa and pamps. in both cases, statistical significance of anti-inflammatory effects was observed at concentrations at and above mg/l. this effect was efficacious and had minor if any associated toxicity. to further probe the mechanism of activity of the polymers in this assay, we investigated potential antagonism of the polyanions with lps. indeed, the latter has anionic component to it and competion with polyanions for interaction with the receptor is not inconceivable. furthermore, prior reports suggested that sulfated polyaromatic suramin can antagonize lps [ ] . to investigate this on the example of peaa, lps stimulation of macrophages was performed using increasing concentration of lps in the presence or absence of a fixed concentration of the polymer (fig. ) . in this assay, antagonism at the receptor in absence of intracellular effects would manifest itself as a shift in the potency of lps without a change in efficacy. in turn, intracellular effects in absence of receptor antagonism would mean a decreased efficacy of the inflammatory response with no change in the potency of lps. experimental data suggest that both effects are likely observed (fig. ) and peaa exerts its activity through both, receptor antagonism and intracellular effects. indeed, the polymer affords statistically significant decrease in the inflammation and also a change, albeit a very minor one, in the ic value for lps with regards to stimulation of macrophages ( . vs . mg/l in the presence of peaa). results of this study provide a comprehensive view on polyanions as the backbone for macromolecular broad-spectrum antiviral therapeutics. together with the results of the screen of these polyanions for inherent antiviral activity, [ ] presented data provide a suggestive view on the utility of polyanionic macromolecular prodrugs of ribavirin as antiviral agents (summarized in table ). first, we wish to evaluate the validity of our assumption that (table ) can mask or skew the observed effects and lead to false negatives or misinterpretation of data. furthermore, terminal groups arising from the different raft agents used in this study to produce the polymers may also considerably change polymer properties [ ] . with regards to the latter point, we note that in our hands, macromolecular prodrugs of rbv prior to and after removal of their cleavable end-groups revealed no difference in the antiinflammatory activity [ ] . in this work, to accommodate the synthetic diversity of polymers, we used five different raft agents (table ) and we see no correlation of polymer activity (table ) and the raft agent used for the polymer synthesis. similarly, experimental results presented in this work appear to show no correlation with the polymer molar mass. blood anti-coagulation effect of the polymers (fig. ) shows strong dependence on the nature of the polymer charged functionality (sulfonates > carboxylates > phosphates and phosphonates) but not with m n (dp). albumin binding results (probe displacement results, fig. ) illustrate that polycarboxylates are strong albumin binders despite the variation of molar mass while phosphate-containing polyanions are weak binders, even the highest molar mass papa. drug release kinetics for macromolecular prodrugs (fig. ) shows a statistically significant correlation with the polymer hydrophobicity but not with polymer dp. polymers most active in rbv delivery were not those characterized by the lowest or the highest dp (in fact, the lowest and the highest dp polymers were not effective in this screen) suggesting that polymer structure, not the number of repeat units in the structure, is decisive in the utility of the polymer as a carrier for the conjugated drug. without doubt, examples of strong, pronounced effects of molar mass on the biomedical properties of polymers are numerous and importance of this factor should not be overlooked. in our own prior work, with regards to the delivery of rbv, we found that high molar mass restricted the polymer cell entry and in doing so limited the observed therapeutic effects [ , ] . not disregarding the importance of these observations, the data of this study strongly suggest chemical structure of the polymer is a dominant factor in its properties (at least in the experiments presented in this study) whereas polymer chain length is of secondary importance. the first important conclusion with regards to the activity of polymers is that the lead polymer structures with inherent broad-spectrum antiviral activity (peaa, pvbza, psvbs) [ ] appear to also be strong anti-coagulants and strong albumin binders (figs. , ) . in turn, weak anti-coagulants and weak albumin binders are also weak inhibitors of virus infectivity. this conclusion may not be altogether surprising in that both anti-coagulation and antiviral effects are likely due to the interaction between the polymers and a proteinaceous target (for antiviral effectsglycoproteins of the virus capsid). as such, this result implies that polyanionic macromolecular (pro)drugs are not well suited for systemic administration to exert antiviral activity in circulation. from a different perspective, the three leads with broad-spectrum antiviral activity [ ] are also shown in this work to be inhibitory to the activity of polymerases and to possess inherent activity within macrophages, in absence of cytotoxicity. this observation is intriguing in that these three polymers may therefore be inhibitory to viruses both extraand intracellularly, in part fulfilling the desired combination of antiviral effects. in our view, these polymers become lead candidates for optimization and design of microbicides for non-systemic, localized antiviral treatments. regretfully, design of ribavirin mp was only considered in this work for (meth)acrylates and (meth)acrylamides; we are currently investigating the synthesis of mp based on pvbza and psvbs as a next step in the optimization these antiviral (pro)drugs. also worthy of note is the discovery of a family of polyanions that are devoid of any inhibitory activity in the screens we have performed, namely the polyphosphates and polyphosphonates. these polymers appear to have only weak and in most cases minimal interaction with the proteinaceous targets in our assays. due to this, as well as their high solubilizing power (as is well used in the design of low molar mass prodrugs [ ] ) and inherent affinity to bone [ ] , these polymers are highly attractive as carriers for diverse drug delivery applications and specifically for bone targeting. the results are an average of three independent experiments n = ± sd. lower standard error bars were omitted for clarity. the statistical difference in inflammation between peaa and non-peaa treated cells was compared using unpaired student's t-test. p < . **. summary of experimental results of this study and the screen for inherent broad-spectrum antiviral activity (**, taken from ref. [ ] ). results are denoted as "+" if the polymer exhibited the nominated property (being increasingly pronounced on a scale from + to +++), "−" if the polymer is devoid of this property or effect, and "n/a" if the polymer was not analyzed in this test. anti-inflammatory activity is denoted '+' if the effect is observed with minimal cytotoxicity of treatment. all chemicals were purchased from sigma aldrich and used without further purification unless stated otherwise. ribavirin was purchased from apichemistry. h nmr and c nmr were recorded with a bruker biospin gmbh mhz nmr spectrometer. multiplicities are indicated by abbreviations. s = singlet, bs = broad singlet, d = doublet, t = triplet, p = pentet, m = multiplet. hrms was recorded on a bruker maxis impact-tof-ms with electrospray ionization (esi+). size-exclusion chromatography (sec) was performed using a system comprising a lc- ad shimadzu hplc pump, a shimadzu rid- a refractive index detector and a wyatt dawn heleos light scattering detector along with a spd-m a pda detector, equipped with either ) a hema-bio linear column with μm particles, a length of mm and an internal diameter of mm from mz-analysentechnik in series with a ohpak sb- hq shodex column with the dimensions . × mm a particle size of μm or b) mz-gel sdplus linear column with μm particles length of mm and an internal diameter of mm from mz-analysentechnik providing an effective molecular weight range of - . . or c) superose increase / gl from ge healthcare with a pore size of um, a particle size of . um, an inner diameter of mm and a length of mm providing an effective molecular weight range of , - , , da with an exclusion limit > , , da. the solvent used was either a) . m pbs filtered through a . μm filter with ppm sodium azide at . ml/ min at °c or b) dmf with mm libr, at . ml/min at °c or c) . m pbs filtered through a . μm filter with ppm sodium azide, at . ml/min at °c. values of dn/dc used for molar mass calculations were established by wyatt mals/astra software assuming full mass recovery. plate reader experiments were performed in black well optiplates on an enspire multilabel reader (perkin elmer®). monomer ( mg, . mmol, equiv.) was dissolved in dry thf ( ml) under a n atmosphere. tea· hf ( . ml, . mmol, equiv.) was added and the reaction was stirred at room temperature for h. the product was purified by flash column chromatography meoh/dcm : to : . residual triethylamine was removed by dissolving the crude mixture in dcm, washing with nh cl twice, once with water, and once with brine. the organic phase was dried over sodium sulfate, filtered, and concentrated in vacuo yielding the pure product. tea ( . g, . mmol, equiv.) was added over a cold ( °c) solution of -hydroxyethyl disulfide ( . g, . mmol, equiv.) in dcm ( ml) under n atmosphere. acryloyl chloride ( . g, . mmol, equiv.) was added dropwise maintaining the temperature at °c. the reaction mixture was heated slowly to room temperature and stirred for h. the reaction was quenched and washed twice with nh cl and brine. the organic phase was dried over sodium sulfate, filtered, and concentrated in vacuo. the product was purified by flash column chromatography pentane/etoac : to : yielding the pure product as a light yellow liquid ( . g, %). a solution of (( r, r, r, r)- , -bis((tert-butyldimethylsilyl)oxy)- -( -carbamoyl- h- , , -triazol- -yl)tetrahydrofuran- -yl)methyl ( nitrophenyl) carbonate ( . g, . mmol, equiv.) in dry dcm ( ml) was added over a cold solution of -(( -hydroxyethyl)disulfaneyl)ethyl acrylate ( . g, . mmol, equiv.), diea ( . g, . mmol, equiv.) and dmap ( . g, . mmol, . equiv.) in dry dcm ( ml) under n atmosphere. the reaction mixture was stirred at room temperature for h resulting in a conversion of %. the reaction was quenched and washed twice with nh cl and brine. the organic phase was dried over magnesium sulfate, filtered, and concentrated in vacuo. the product was purified by flash column chromatography pentane/etaco : to : . residual p-nitrophenol was removed by basic alumina column chromatography etoac/meoh : yielding the pure product as a colorless solid ( . g, %). monomer was synthesized following the protocol described above with an in situ deprotection performed without purification of the monomer . thus, the crude mixture obtained according to the protocol for the synthesis of monomer was dissolved in thf ( ml) under n atmosphere. tea• hf ( . g, . mmol) was added and the reaction was stirred for h. the solvent was removed in vacuo and the product was purified by flash column chromatography dcm/meoh : to : . residual tea was removed by dissolving the product in chloroform and washing twice with nh cl yielding the pure product ( . g, %). experimental details for the syntheses of ethylacrylic acid, propylacrylic acid, -methacrylamidoethyl phosphate, -acrylamidoethyl phosphate, -methacrylamidoethyl phosphonic acid and -acrylamidoethyl phosphonic acid were performed according the protocols published previously [ ] . anionic homopolymers were synthesized as previously described in ref. dcma ( . mg, . mmol, equiv.) and v- ( . mg, . mmol, . equiv.), were dissolved in methanol ( . ml). monomer was dissolved in dmf ( . ml), amps dissolved in water ( . ml) was added. five rounds of freeze-pump-thaw were performed before the ampule was flame sealed and the reaction performed at °c for h. the reaction mixture was diluted with a ph = . nahco / na co buffer and purified by dialysis followed by lyophilisation to obtain the pure product ( mg recovered). . . . poly( -methacrylamidoethyl phosphonic acid-co-rbv) -methacrylamidoethyl phosphonic acid ( . g, . mmol, equiv.) was dissolved in acetate buffer ( . ml). monomer ( . g, . mmol, equiv.) dissolved in meoh ( . ml) was added together with cdpa ( . mmol, equiv.) and acva ( . mmol, . equiv.) stock solutions. methanol ( . ml) was added giving a slightly unclear solution. five rounds of freeze-pumpthaw were performed before the ampule was flame sealed and the reaction performed at °c for h. the reaction mixture was diluted with a ph = . nahco /na co buffer and purified by dialysis followed by lyophilisation to obtain the pure product ( mg). monomer ( . mmol, equiv.), cdpa ( . g, . mmol, equiv.), and v- ( . mmol, . equiv.) dissolved in methanol ( . ml) was added to -acrylamidoethylphosphonic acid ( . g, . mmol, equiv.) dissolved in water ( . ml). six rounds of freeze pump thaw were performed before the ampule was flame sealed and the reaction was performed at °c for h. the reaction mixture was diluted with a ph = . nahco /na co buffer and purified by dialysis, obtaining the pure product upon lyophilisation. cdpa ( . g, . mmol, equiv.), v- ( . g, . mmol, . equiv.), and monomer ( mg, . mmol, equiv.) were dissolved in methanol ( . ml) and added to -methacrylamidoethyl phosphate ( mg, . mmol, equiv.) dissolved in water ( . ml). the mixture was slightly unclear. five rounds of freeze pump thaw were performed before the ampule was flame sealed and the reaction performed at °c for h. a small amount of precipitate was observed upon reaction, the mixture did not change notably in viscosity. the reaction mixture was diluted with a ph = . nahco /na co buffer and purified by dialysis. a second dialysis was performed in : ethanol/water yielding the pure product which was lyophilized. monomer ( . mmol, equiv.), cdpa ( . g, . mmol, equiv.), and v- ( . mmol, . equiv.) dissolved in methanol ( . ml) was added to -acrylamidoethyl phosphate ( . g, . mmol, equiv.) dissolved in water ( . ml). six rounds of freeze pump thaw were performed before the ampule was flame sealed and the reaction performed at °c for h. the reaction mixture was diluted with a ph = . nahco /na co buffer and purified by dialysis. a second dialysis was performed in : ethanol/ water yielding the pure product upon lyophilization. the competition assay was performed in -well plates, and fluorescence analyzed (excitation wavelength nm, emission wavelength nm) on a plate reader. polymers were pre-dissolved in minimal amounts of dmso and then diluted in . m pbs (ph . ). stock solutions of human serum albumin (hsa) and fluorescent probes (dansyl asparagine/dansyl sarcosine) were prepared in . m pbs (ph . ). the components were mixed and pbs added to obtain a total volume of μl in each well, with the following concentrations: hsa μm, dansyl sarcosine μm or dansyl asparagine μm, and polymer at μm, corresponding to equivalents compared to albumin. upon mixing the plates were incubated at °c for h to allow the system to equilibrate. the data represents three independent experiments, which each included three technical replicates. polymers and albumin ( g/l) were dissolved in . m pbs with ppm nan in equimolar amounts. the samples were incubated for h at room temperature and then directly injected into the sec-mals system equipped with a biocolumn and using pbs as an eluent. results were analyzed using the wyatt software astra . a sample of albumin with no polymer added was used as reference in order to compare the albumin peak both with regards to the determined molecular weight and the elution times observed. ribavirin containing polymers were pre-dissolved in minimal amounts of ddmso (if necessary) and diluted in . m pbs (ph . ) prepared from d o. glutathione (gsh) was also dissolved in . m pbs (ph . ) prepared from d o. an aliquot of the polymer solution was mixed with an aliquot of the gsh solution to obtain a . ml sample containing . g/l of polymer and . mm gsh. the sample was incubated for the described time upon which ellmann's reagent was added in equimolar amounts compared to gsh to quench the reaction. the quenched samples were analyzed by h nmr spectroscopy as described in the main text. time point measurements were repeated at least three times for each polymer. raw . macrophage cell line (tested negative for mycoplasma) was used to determine the anti-inflammatory activity of polyanions. the cells were seeded on -well flat-bottomed plate at initial density of · cells per well in μl of media: high glucose dmem, stable lglutamine, without phenol red (gibco, ) supplemented with % fbs and penicillin/streptomycin. three hours after the cells were seeded, the polymers were added at indicated concentrations in μl of the media and incubated with the cells for h. after h, media was removed and the cells were washed with pbs. subsequently, the cells were stimulated with lps (sigma, l ) for h. the level of nitric oxide was quantified via griess assay. briefly, μl of the cell supernatant was mixed with μl of sulfanilic acid ( g/l, % phosphoric acid). after min of incubation, μl of n- -napthylethylenediamine dihydrochloride ( g/l) was added and absorbance ( nm) was measured using a plate reader (bmg labtech, ortenberg, germany). the level of nitrite was quantified against sodium nitrite standard curve and normalized to the negative control which was lpsstimulated cells only. cell viability was measured using prestoblue viability reagent (invitrogen, a ). briefly, the cells were incubated with medium containing % volume of prestoblue reagent. after h of incubation time the fluorescence ( nmexperimental wavelength, nmreference wavelength) was measured on the plate reader (bmg labtech, ortenberg, germany). the cell viability was normalized to the viability of the cells incubated with pure media. the blood obtained from healthy donors was collected into sodium citrate . % tubes. platelet poor plasma (ppp) was obtained by centrifuging the blood at g for min at °c. the polymers were dissolved in pbs buffer containing % dmso. polymers in the solution were mixed with ppp in volume:volume ratio : resulting in the final polymers concentration of and mg/l. prothrombin time was measured using mrx owren's pt reagent (medirox, ghi - ) and activated partial thromboplastin time was measured using dade actin fs (siemens, b - ). all measurements were performed on sysmex cs- i. pbs and pbs with % dmso were used as a negative control. heparin (sigma, h ) was used as a positive control. to analyze inhibitory effect of polymers on dna-dna polymerase activity, quantity pcr (qpcr) reaction was performed using power sybr green pcr master mix (life technologies) according to the protocol provided by the manufacturer. for one sample μl of green pcr master mix were mixed with forward. ′-ggtctctctggttagaccagat- ′ and reverse primer ′-ctgctagagattttccacactg- ′, . ng of phxb -env plasmid (nibsc, programme eva centre for aids reagents, reference number: arp ) and a polymer diluted in pbs to a desired concentration. primers were complementary to ltr upstream element of gag. the program used was °c for min followed by cycles with °c for s, °c for s and during the last cycle, a melting curve was made. a level of inhibition of taq polymerase was calculated by comparing to a positive control without polymer as % of enzyme activity. influence of polymers on activity of reverse transcriptase was determined using enzchek reverse transcriptase assay kit (life technologies). the reaction mixture was prepared according to the protocol provided by the manufacturer. subsequently polymers diluted in pbs were added at the given concentration, and then u of mulv reverse transcriptase (life technologies, cat. nr. n ). the reaction was performed at °c for h. nucleic acids were stained with picogreen dye and fluorescence was measured on a plate reader (bmg labtech, ortenberg, germany). cellular uptake of polyanions in macrophages was evaluated by confocal laser scanning microscopy. coverslips (thermofisher scientific) placed in well plates were coated with . % poly-l-lysine in water (sigma-aldrich) at °c for h, and washed twice with pbs ph . (gibco). raw . cells (mycoplasma negative) were seeded on each coverslip in the -well plate in μl media (high glucose dmem, stable l-glutamine (gibco) supplemented with % fbs and penicillin/streptomycin). h after seeding, media was removed from wells, and μl of mg/l of fluorescently labelled polyanion diluted in media was added to the well. h after addition of polyanions, cells on coverslips were processed for confocal microscopy: coverslips were washed twice with pbs and fixed with % paraformaldehyde in pbs for min at rt, followed by washing twice with pbs. coverslips were stained with μg/ml dapi in pbs for min, and dapi was removed by washing twice with pbs. cell membrane was stained with μg/ml concanavalin a conjugated to alexa fluor® (lifetechnologies) for min at rt. following this staining step, coverslips were washed twice with pbs and transferred, cells facing down, to a microscopy slide (vwr), and then the cells were submerged in a prolong™ diamond antifade mountant (thermofisher scientific). slides were visualised using a zeiss lsm confocal with ×/ . oil dic objective, using excitation line nm for dapi, nm for fitc and nm for alexa fluor® with no spectral overlap in collected emission. emerging viral diseases: confronting threats with new technologies global and regional mortality from causes of death for age groups in and : a systematic analysis for the global burden of disease study the design of drugs for hiv and hcv approved antiviral drugs over the past years combating emerging viral threats advances in antiviral vaccine development topical microbicides to prevent hiv: clinical drug development challenges microbicides and other topical strategies to prevent vaginal transmission of hiv broad-spectrum antiviral agents broad-spectrum agents for flaviviral infections: dengue, zika and beyond the use of interferon-alpha in virus infections is the -year hepatitis c marathon coming to an end to declare victory? ribavirin -current status of a broad 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for the biomedical field we wish to acknowledge financial support from the danish council for independent research, technology and production sciences, denmark (anz; project dff - - ). key: cord- - nwq vxk authors: russo, giuliano; fronteira, inês; jesus, tiago silva; buchan, james title: understanding nurses’ dual practice: a scoping review of what we know and what we still need to ask on nurses holding multiple jobs date: - - journal: hum resour health doi: . /s - - -x sha: doc_id: cord_uid: nwq vxk background: mounting evidence suggests that holding multiple concurrent jobs in public and private (dual practice) is common among health workers in low- as well as high-income countries. nurses are world’s largest health professional workforce and a critical resource for achieving universal health coverage. nonetheless, little is known about nurses’ engagement with dual practice. methods: we conducted a scoping review of the literature on nurses’ dual practice with the objective of generating hypotheses on its nature and consequences, and define a research agenda on the phenomenon. the arksey and o’malley’s methodological steps were followed to develop the research questions, identify relevant studies, include/exclude studies, extract the data, and report the findings. prisma guidelines were additionally used to conduct the review and report on results. results: of the initial records identified, a total of met the inclusion criteria for nurses’ dual practice; the vast majority ( %) were peer-reviewed publications, followed by nursing magazine publications ( %), reports, and doctoral dissertations. twenty publications focused on high-income countries, on low- or middle-income ones, and two had a multi country perspective. although holding multiple jobs not always amounted to dual practice, several ways were found for public-sector nurses to engage concomitantly in public and private employments, in regulated as well as in informal, casual fashions. some of these forms were reported as particularly prevalent, from over % in australia, canada, and the uk, to % in south africa. the opportunity to increase a meagre salary, but also a dissatisfaction with the main job and the flexibility offered by multiple job-holding arrangements, were among the reported reasons for engaging in these practices. discussion and conclusions: limited and mostly circumstantial evidence exists on nurses’ dual practice, with the few existing studies suggesting that the phenomenon is likely to be very common and carry implications for health systems and nurses’ welfare worldwide. we offer an agenda for future research to consolidate the existing evidence and to further explore nurses’ motivation; without a better understanding of nurse dual practice, this will continue to be a largely ‘hidden’ element in nursing workforce policy and practice, with an unclear impact on the delivery of care. electronic supplementary material: the online version of this article ( . /s - - -x) contains supplementary material, which is available to authorized users. health workers' dual practice has been identified as one of the priority research areas in the human resources for health domain [ ] . there is a concern among policymakers and patients alike that simultaneous engagement with public and private sector activities jeopardise the availability of professionals and the quality of services in the public sector and divert patients towards costlier private care, therefore putting at risk the attainment of universal health coverage (uhc) goals [ , ] . 'dual practice' in the health sector has been defined as health workers' concomitant engagement in public and private sector clinical activities, with the public sector job representing the 'primary' one to which the largest proportion of working hours are allocated [ ] . although very common worldwide, the practice has been traditionally treated with suspicion by the public health and health system research literature, amid fears that it may compromise the supply of public services [ ] and encourage absenteeism in public institutions [ ] , as well as the selection and diversion of patients towards private services [ ] . scholars have highlighted the possible potential benefits of the practice, such as the opportunity it offers to provide a wider range of health services to the population and to retain underpaid workers in the public sector [ ] . others have paid attention to the regulatory aspects [ ] , with some focusing on the systems' governance and institutions [ ] and others on the incentives to be offered to achieve the desired level of service provision [ , ] . substantial literature exists on physicians' dual practice [ , ] , most recently building up evidence on its prevalence, forms, and drivers worldwide [ ] [ ] [ ] , as well as on modelling possible regulatory frameworks [ , ] . however, nurses' engagement in multiple job-holding is, in comparison, less explored, despite preliminary evidence of its high prevalence in high-income [ ] as well as in lowincome settings [ ] , and amid concerns of its impact on the nurses' wellbeing [ ] . nurses and midwives are the world's largest group of health professionals, representing % of the global health workforce, and their role is widely considered critical for the delivery of uhc goals in high-as well as low-income countries [ ] . however, the profession has recently come under pressure because of growth of the demand for health services and concomitant scarcity of funds, and the global shifts in the world's health labour market [ ] . as the nursing workforce is predominately female, policy options to address nurses' participation in the public and private labour market will need to take gender into account [ , ] . this scoping review sets out to fill this knowledge gap by systematically searching and reviewing the studies conducted on nurses' simultaneous engagement in public and private clinical activities [ ] . its specific objectives are ( ) to map out the existing literature on the subject, determining its prevalence and distribution across geographies, publication types (e.g. peer-reviewed, grey), and specific topics addressed; ( ) summarise the evidence, perspectives, and specific contents addressed; and ( ) propose an agenda to advance research and development activities to first identify and then mitigate any pervasive effects of nurses' dual practices to uhc, based on the scoping review results. a scoping review was conducted to determine the extent and key themes within the literature on nurses' dual practice, as well as to identify areas for future research on the topic. such knowledge synthesis method is commonly used to address exploratory research questions, to map the existing literature on a field or to preliminarily identify gaps in that literature [ ] [ ] [ ] . we used the five arksey and o'malley's methodological steps to develop the research questions, identify relevant studies, include/ exclude articles, extract the data, and report the findings [ ] . as the methodological guidance for the report of scoping review is still under development, we used the prisma guidelines where appropriate [ ] . in march , we searched medline (through pubmed), the isi web of knowledge, scopus, and the cinhal plus with full texts (through ebsco). we used a set of keywords for the searches and, where appropriate, medical subject headings for nurses combined with keywords and indexed terms related to dual practice (using the bolean operator ' and'). additional file provides full details for the initial search strategy for each of the databases searched. the grey literature also was searched by visiting websites dedicated to nursing and/or health workforce issues. to widen the scope of the review, the searches on databases and grey literature were not filtered for publication date, language, or publication type. human resources for health experts (named in the additional file ) were a priori contacted to provide relevant references on forms of dual practices among nurses. a posteriori (early december ), and based on suggestions coming from the peer-review process, we expand the search terms in the database searches (adding the keywords 'temporary employment' and 'multiple employers') to provide a few additional records which were considered for the review results, as well. iterative rather than strictly streamlined procedures are typical in the process of conducting scoping reviews [ ] . a final search strategy included snowballing searches (reference list scanning, author tracking) performed on the articles preliminarily selected. references from databases or other sources were filtered through the same eligibility criteria. to be included, studies needed to address explicitly both nurses and dual practice issues. the working definition for the 'nurses' category contained explicit reference in the text to the professional label, with midwives included too. the working definition of dual practice, in turn, referred to concomitant practice in two (or more) distinct clinical services, either in the same or in different healthcare institutions. public employment was considered the primary job, whereas the secondary (or subsequent) job(s) was considered the one(s) where fewer working hours were spent, periodically or regularly. alternative labels for dual practice included 'moonlighting' , 'public-private work' , 'multiple profit-generating activities' , 'dual/multiple job-holding' , and 'second jobs'. 'casualization of work' , defined as the process of replacing full-time and regular part-time staff with contract staff employed on an ad hoc basis, is another phenomenon related in many ways to dual practice [ ] . as such, papers addressing this form of employment in relation to dual practice were also included. documents in english, french, portuguese, italian, and spanish were included. with the exception of journal commentaries, editorials, and letters to the editor, we did not exclude references because of the type of article (such as opinion pieces), study output (e.g. final or preliminary results), countries or world regions, publication status (i.e. both peer-reviewed and grey literature), or publication date. titles and abstracts were first screened by one of the authors (tj) and then reviewed in duplicate by the first author (gr), who finally determined the suitability for the full-text review. full-text review was carried out by one of three authors, all with a research track record in nurse workforce and/or dual practice issues (gr, if, jb). any of the authors were able to directly include or exclude papers on the basis of the eligibility criteria; agreement between two or more reviewers was sought for doubtful cases. based on the overarching aim of the paper, the preliminary knowledge of the literature, and a priori consultation with health professionals [ ] , we developed the following set of questions to guide the data extraction for the review: what are the forms in which nurses engage in multiple profit-generating activities? what are the different features of nurses' multiple job-holding? what is the prevalence of this phenomenon in nursing? why do nurses engage in dual practice? what are the enablers and barriers for nurses' dual practice? what are the personal/ professional drivers and consequences? what are the consequences for health systems, specifically for the delivery of quality and safe nursing/health care? what are the consequences for nurses' welfare? what are the consequences for patients? what are health workers', managers', and patients' perceptions around this practice? data extraction tables were then purposively built by the research team to collect data on the specific questions above, either using textual data or synthesis of the articles' findings/conclusions. consistent with the scoping review methodology, the data extraction did not involve quality appraisal or grading of the evidence from the studies. a conventional form of qualitative content analysis, with coding categories derived directly from the text data, was used to analyse data retrieved for each topic [ ] . the first author performed a first synthesis of the extracted material, that was then iteratively edited by two of the other authors (if, jb) following the themes from the data extraction table. from records retrieved, ( %) were excluded after reviewing their titles and abstracts (fig. ). an additional four articles were identified through snowballing search strategies, resulting in a total of full texts assessed for eligibility. of all these, a total of ( %) articles finally met the inclusion criteria for addressing dual practices of nurses: using predominantly quantitative methods and using mostly qualitative designs. the vast majority of the studies were in english, with only four published in portuguese and one in spanish. additional file provides spreadsheets for the (a) list of included articles organised by study-type, (b) the data extraction table, and (c) list of articles excluded with the respective reasons. the vast majority of such documents ( %) were peerreviewed publications, with the remainder being nursing magazine publications ( %), reports, and doctoral dissertations. twenty publications focused on high-income countries (particularly on the usa, uk, canada, and australia), on low-and middle-income ones (south africa, ethiopia, iran, and uganda), and two provided a global view on the phenomenon. many of the documents (n = ) reported information on the prevalence of the phenomenon, and discussed its different forms ( ) . drivers and motivations of nurses' multiple job-holding were the subject of (out of ) of the documents, while individual and institutional consequences of the practice were discussed in and pieces, respectively. only seven of the retrieved documents mentioned policy options associated with nurses' multiple-job holding. below we present the literature retrieved, organised in sections reflecting the emerging themes. from the documents retrieved, it emerged that nurses' engagement in dual practice can take different forms and shapes, with often blurred boundaries. some authors mention 'secondary jobs' and 'moonlighting' practices, where public sector nurses engage with the private sector either individually or through an organised nursing services agency [ ] [ ] [ ] . ribera silva et al. ( ) as well as gupta et al. ( ) refer generally to 'nurses taking up public or private secondary jobs' in brazil, chad, côte d'ivoire, zimbabwe, and mozambique. a similar operational definition is adopted by serra et al. to describe nurses' practicing simultaneously in the national healthcare system and for ngos or private clinics. publications from hics at times use the expression 'casualization of work' to describe job insecurity through a lack of a stable contract of employment, but also the practice of working flexibly for public and private health facilities, often through agencies and banks for outsourced nursing services [ ] [ ] [ ] [ ] . however, a distinction is drawn in the literature between holding multiple jobs concurrently, and dual practice, where the nurse's primary job is in the public sector, and that may be affected in many forms by the simultaneous engagement with other clinical, profitgenerating activities [ ] . three common forms of nurses' dual practice are mentioned, and often used interchangeably, in the literature; primary public sector employment with additional nursing work in the public sector-typically nightly extra shifts in different departments of the same hospital/facility, or other public facilities in the same geographical area [ , ] ; primary public sector employment with additional nursing work in the formal or informal private sector-long-hours shifts, or side jobs during spare time/vacation from main employment [ , , ] ; fixed part-time employment in the public, coupled with multiple flexible contract assignments in public and private sector, often though nurse agencies (referred to as 'casualization') [ , , ] . some authors report that boundaries between public and private sector employment are often blurred, particularly in low-income settings, and that ad hoc classifications of multiple-job holding may be required to capture the essence of the practice for specific countries [ ] . although using different definitions of the practice, a number of studies attempted measuring the prevalence of nurses' engagement in multiple job-holding in highincome as well as low-and middle-income countries (see table below). these are mainly cross-sectional surveys that do not provide data on trends for the phenomenon. for australia, creegan et al. [ ] show that . % of nurses worked part-time in , while in batch and windsor found that the nursing profession had a higher rate of casualization than other professional and highly skilled workforces, and that . % of nurses were employed in non-standard work [ ] . for the uk, tailby reports that % (of ) nurses registered with nhs nurse banks had another nursing job, and % worked occasionally or regularly additional shifts paid at bank or agency rates [ ] . in a survey among nursing magazines' readers in [ ] , % declared taking up extra nursing work, and % another full time job outside nursing; years later, % of the nurses participating in another online magazine readers survey declared engaging in bank and/or agency shifts [ ] . a report from the us bureau of labor statistics [ ] shows that multiple job-holding has grown steadily over the last decades, that . % of nurses had multiple jobs in , and that such prevalence was higher for a small sample of male nurses ( . %). a article from canada [ ] provides evidence that . % of all rural nurses are in casual jobs and that casualization is particularly common among registered nurses and licenced practical nurses ( . %), and more common among those nurses living in the north of the country ( . %). evidence on the phenomenon from lmics is substantial too; gupta et al. report from a multi-country study that nurses dual practice would be more limited than physicians'-the former calculated to be % in chad; % in cote d'ivoire; % in jamaica; % in mozambique; % in sri lanka; and % in zimbabwe [ ] . in a world bank study in ethiopia [ ] , a similar proportion of a cohort of public sector nurses ( %) were found to have secondary jobs years after their initial appointment. several studies by rispel and colleagues from south africa showed the prevalence of different forms of multiple employment to be common (around %) and on the rise among south african nurses [ , ] . and in brazil, portela et al. showed . % of nurses in two public hospitals to be moonlighters [ ] . a few individual and institutional drivers for the practice are recurrent in the literature. at a personal level, the need to increase overall earnings by supplementing income from main salary is by far the most common, such as in northern ireland and elsewhere in the uk-where holding multiple jobs is seen by many nurses as an essential way to increase income [ , ] . however, also for a low-income country like ethiopia where a nurse's salary is typically higher than the country's average gross domestic product (gdp) per capita, serra et al. report that half of the nurses followed in their study took up a second job to support their families [ ] . flexibility of additional part-time employment seems to be another key factor for australian and uk nurses, since nursing is a typically female profession, and some female workers have a strong preference for part-time, flexible jobs in comparison to their male peers [ , ] . in the surveys in south africa [ , ] , the opportunity for learning new nursing skills, the need to introduce diversity in professional routines, a more stimulating working environment, the quality of supervision, and the ability to select their own working hours were the key reported motivating factors for south african nurses. at a more institutional level, also in south africa, the growing demand for nursing services from the private sector is pointed to as the key driver of the phenomenon of casualization of nursing employment. taking a broader organisational perspective, batch and windsor ( ) argue that the 'casualization movement' is really aimed at creating a more flexible, cheaper, and easier to manage the nursing workforce. no specific study appears to have assessed the impact of nurses' dual practice, although many articles offered hypotheses and interpretations in regard. generally, health worker's dual practice is regarded unfavourably in the academic literature. mcpake et al. argue that, depending on its forms and prevalence, it could hamper the attainment of uhc in some countries [ ] . others report that the associated increased tiredness and lack of alertness for casual workers who work long hours in multiple jobs, as well as their difficulty of communication with resident staff, are reported to substantially increase the risk of clinical accidents [ ] . however, a phd dissertation work from the usa shows that, on average, nurses with a secondary job tend to work fewer hours in their primary, public employment than their non-moonlighting colleagues [ ] . studies in south africa suggest that moonlighters are also more likely to take vacation and time out from their main employment to pursue other jobs [ ] , and intentions to leave economic-higher rate for single day fees than in home country of n ireland the public sector and/or migrate have been found to be more frequent among them than in their single-job peers [ ] . baumann et al. argue that employing an unbalanced proportion of full-time and casual nurses reduces flexibility of a hospital management, as these latter would be less available to cover for unforeseen needs [ ] . in the case of ontario, canada's experience with the severe acute respiratory syndrome (sars) epidemic, such factors, together with the increased dependence of many hospitals in high-income countries on agency nurses, have been suggested could compromise the system's 'surge capacity' , that is, its ability to rapidly scale-up services and response in the face of epidemics [ ] . in a qualitative study in rural community hospitals in canada about the changing nature of nursing work, montour et al. argue that employment in multiple organisations contributes to scheduling issues because casual nurses are unavailable to fill vacant shifts [ ] . and finally, according to some studies, some types of health workers' dual practice can critically undermine health service provision and public trust, as it often entails conflict of interest, idleness, and absenteeism [ ] . at a more personal level, portela et al. show that taking extra shifts can seriously affect nurses' general health and exhaustion levels, with night shifts reported to be less disruptive than day ones [ ] . such findings on sleeping patterns are also echoed by ribeiro-silva et al. [ ] for hospital nurses in rio de janeiro, brazil, and by knauth for workers outside the clinical profession [ ] . casualization of work was also identified as a major source of career fatigue and burnout in qualitative interviews with nurses in australia [ ] . marginalisation and exclusion of part-timers by their peers was also reported to be a major source of dissatisfaction and frustration in an ethnographic study on australian nurses [ ] . as a positive individual consequence, nurses were reported to value highly the opportunity dual work offers to complement meagre public salaries in high-income countries [ ] and to support extended families in lowincome ones [ ] ; in the usa in , nurses earned more in their secondary job than in their primary employment [ ] . flexible working hours is another characteristics that nurses would find particularly attractive in secondary, casual jobs in the uk [ ] . in this respect, creegan et al. suggest that flexible working arrangements would be particularly suited for the predominantly female nursing workforce [ ] . only a minority of the studies retrieved in this review (eight) present and discuss possible policy options for managing, regulating, or controlling the practice. mcpake et al. [ ] link the choice of policy measures to the prevalence of the practice and to the country's regulatory capacity. rispel et al. [ ] highlight managing moonlighters as a key human resource for health strategy in south africa; consultation with frontline nurses to counteract the practice's negative impact is suggested as a possible policy option. electronic time recording, cessation of unpaid overtime, and controls over the number of shifts are put forward as alternative measures by other authors [ ] , while developing clinical guidelines for hospitals to ensure safety of services 'in the hands of strangers' has been called for as a possible institutional measure. other scholars have argued for the need for a better understanding of dual practice patterns, in recognition of the fact that more effective planning and management of a flexible workforce could represent a more suitable solution than prohibition [ ] . our review revealed that nurses engage in multiple jobholding activities, with varying forms and prevalence in high-income as well as in low-income countries. the practice appears to be driven by multiple, complex, and varying factors beyond the obvious economic motif, and to have non-trivial consequences, particularly for nurses' welfare, organisation of health services, and health labour market. despite its prevalence and relevance, a surprising paucity of studies was found on nurses' dual practice, and very few policy options have been outlined in the literature to address the phenomenon. although in the nursing profession holding simultaneously multiple jobs cannot be necessarily considered as dual practice, the two areas often overlap, in shapes of poorly demarcated contours. consistently with what is observed for other professions, more than one way seems to exist for nurses to engage with dual practice, both in regulated and informal, casual fashions. this may at least in part explain why the practice has been under-reported and little regulated through the years, with some of its forms driven underground or even considered illegal in some countries [ ] , and other forms-such as the 'casualization' of nursing services-only recently having come to the fore in the context of rapidly evolving health labour markets [ ] . this absence of usable datasets would call for primary research to be conducted to, first, explore through qualitative research the specificities of the phenomenon and, second, to measure them quantitatively. unsurprisingly, our review of the available evidence appears to show that economic considerations are not the sole driver for nurses taking on simultaneous multiple jobs [ , ] . a basic dissatisfaction with the limited range of duties performed in their main job, limited opportunities for development, or availability of time made possible by night shift arrangements, are other important factors that may help explain such a decision. although much effort has been devoted in the past to understanding nurses' burnout [ , ] , surprisingly little attention has been given to the tendency to take on additional work in presence of an already heavy workload. in contrast to the comparatively better understood physician dual practice, the limited evidence reviewed suggests that nurses' dual practice is more likely to be bounded by the very nature of their jobs than it is for physicians, as typically nurses have limited autonomy and tend to work as part of a team, rather than as individual providers. on this basis, a hypothesis could be made that, while nurses are more likely to be part of an established team in their main (public sector) job, second jobs are often taken up as individuals, as agency nurses for one shift, or private home care visits. nurses' personal characteristics also appear to shape forms and extent of the practice in any one country. since taking up additional work in the private sector may be financially rewarding, but will also add to overall workload and may not necessarily increase career prospects, younger and comparatively lower paid nurses seem to be the ones likely to engage more in the practice [ , ] . as a compounding factor, as nurses are predominantly female and often perform a disproportionate share of child-rearing and care for elderly or disabled relatives' duties, we may speculate that having dependents will likely decrease their ability to take up additional hours, unless the additional income generated can compensate for any additional child care costs. the evidence available suggests that the consequences of this phenomenon are not negligible, particularly for the health of those nurses ending up working longer hours and hospital shifts because of their multiple commitments [ , ] , but also for the organisation of public and private health services facing a more 'casual' and less-committed kind of workforce [ ] . interestingly, the most recent literature on nursing and midwifery enterprises [ , ] recognises this limitation and may lay the grounds for a different type of engagement of nursing staff with private sector activities. we also did not find any evidence regarding the importance of economic considerations of nurses' dual practice, or of any difference between higher and lower income countries; as we suspect the implications of such practice may have substantial repercussions on the health labour market, this could represent an area of future research. this paper is based on a scoping exercise and so has limitations. the limited and often incomplete evidence made it difficult to be certain if dual practice is a factor of relevance in all health systems worldwide, if it is a major issue for nurse labour market participation, and its overall impact on the provision of care. with respect to the latter, this may be because some aspects of dual practice are on the margins of 'formal' work and may go unrecognised by formal systems of employment and regulation. all of the above call for a deeper understanding of the phenomenon, with the objective of better harnessing the changing nurses' workforce worldwide. following our review, the core elements of the required research agenda on nurse dual practice appear to be three-fold. first, further research is needed to systematically explore the nature, extent, and impact of nurse dual practice in different systems and countries; this can be achieved through the analysis of employment and professional register/association data sets where these exist, or by adhoc surveys of nurses and/ or workplaces. analysis of specific data sets in some countries (e.g. such as the current population census [ ] and the integrated public use microdata series the united sates; labour force surveys; and professional registries) may provide more evidence on prevalence of dual practice and some of its main forms. secondly, there is a need for developing a more informed picture of the reasons why nurses take on dual practice, their experiences and preferences of dual practice, and the impact on their broader work/life balance. this can be achieved through a qualitative approach, exploring multiple contexts in high-and low-income settings, and different nursing profiles. finally, there is a gap of research that establish the impact of dual practice at the policy level-what is its impact on participation rates, overall nursing hours available in different systems, what are the trends in incidence, what is the impact on nurses, and on the quality of care that is being delivered. measures could be needed to mitigate the effects of nurses' dual practice to protect the provision of free-of-charge public sector for vulnerable populations. this latter area for policy research is the most complex and challenging to interrogate, but also of potentially great significance. without a better understanding of nurse dual practice, it will continue to be a largely 'hidden' element in nursing workforce policy and practice, with an unknown level of significance, and an unclear impact on the delivery of care. priorities for research into human resources for health in low-and middle-income 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on the job partially compensates for sleep loss in night-shift nurses identifying sources and effects of carer fatigue and burnout for mental health nurses: a qualitative approach slaves of the state-medical internship and community service in south africa health worker preferences for job attributes in ethiopia: results from a discrete choice experiment nurses' widespread job dissatisfaction, burnout, and frustration with health benefits signal problems for patient care prevalence of burnout syndrome in clinical nurses at a hospital of excellence current population survey (cps) nurse labor market dynamics are key to global nurse sufficiency the intensive care unit work environment: current challenges and recommendations for the future who's talking? communication and the casual/part-time nurse: a literature review. contemporary nurse relationship between shift work and personality traits of nurses and their coping strategies the health workforce in ethiopia: addressing the remaining challenges advancing the application of systems thinking in health: exploring dual practice and its management in kampala, uganda. health research policy and systems / biomed central the nature and health system consequences of casualisation, agency nursing and moonlighting in south africa stress symptoms in female nurses working in emergency rooms no funding was received for this research. all the data and information included in this review can be found in the annexes. authors' contributions gr elaborated the original idea for the study. gr, if, and jb designed the study. tsj designed the methodology for the review. gr drafted the manuscript. all authors revised, read, and approved the final manuscript.ethics approval and consent to participate n/a. the authors declare that they have no competing interests.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -prgorr d authors: li, zhonghua; zeng, wei; ye, shiyi; lv, jian; nie, axiu; zhang, bingzhou; sun, yumei; han, heyou; he, qigai title: cellular hnrnp a interacts with nucleocapsid protein of porcine epidemic diarrhea virus and impairs viral replication date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: prgorr d the nucleocapsid (n) protein is a major structural component of porcine epidemic diarrhea virus (pedv), which is predicted to be a multifunctional protein in viral replication. heterogeneous nuclear ribonucleoprotein a (hnrnp a ) is a cellular protein participating in the splicing of pre-mrna in the nucleus and translation regulation in the cytoplasm. according to our previous proteomic study about pedv infection in vivo, hnrnp a was thought to be a cellular factor influencing pedv replication. in this report, pedv n protein was discovered to colocalize with cellular hnrnp a in perinuclear region of pedv infected cells. co-immunoprecipitation (co-ip) results clearly demonstrated that pedv n protein could bind to human hnrnp a . replication of pedv was inhibited by silencing the expression of hnrnp a in ccl- cells, suggesting the positive effect of hnrnp a on pedv infection. porcine epidemic diarrhea virus is the most important viral agent which can cause diarrhea in pigs. serious threat has been posed on the world pig industry as a result of the high morbidity and mortality caused by pedv in piglets [ ] [ ] [ ] [ ] [ ] [ ] [ ] . pedv, a single-stranded positive sense rna virus, is a member of coronavirus. four structural proteins, including spike protein (s), membrane protein (m), envelope protein (e) and nucleocapsid protein (n), and the genome constitute the virion. pedv n protein has multiple functions. firstly, as a structural protein, n protein along with the genomic rna forms the nucleocapsid of pedv. furthermore, n protein plays an important role in pedv rna synthesis and enhancing the pedv transcription and virion assembly [ ] [ ] [ ] . however, the effects of n protein on pedv infection usually depend on its interaction with some host factors. for example, pedv n protein binds with phosphoprotein nucleophosmin (npm ) and then protects it from proteolytic degradation by caspase- , enhancing cell survival and pedv growth [ ] . in addition, it has been proved that interaction between pedv n protein and tbk could inhibit irf activation and type i ifn production, further to causing the circumvention of the host's antiviral immunity [ ] . more than heterogeneous nuclear ribonucleoproteins (hnrnps) have been discovered and among them hnrnp a is the best-characterized one [ , ] . hnrnp a is a rna-binding protein which functions as binding to pre-mrna to form hnrnp particles in eukaryotic cells [ ] . this protein contains two rna-binding domains (rbds) and a glycine-rich domain responsible for protein-protein interaction [ , ] . it has been reported that hnrnp a selectively interacts with different rna-binding proteins through its glycine-rich domain [ ] . previous studies have demonstrated hnrnp a could interact with n proteins of sars coronavirus and mouse hepatitis virus (mhv) [ , ] . since pedv is also a member of coronavirus and pedv n protein is a rna binding protein, it is hypothesized that pedv n protein might also be able to interact with hnrnp a during pedv infection. our previous work has proved that hnrnp a underwent different regulations in jejunum tissues of piglets infected with pedv virulent strain and its attenuated strain [ ] . therefore, we assume that hnrnp a may play a role in the life cycle of pedv. in this study, it is found that hnrnp a could interact with pedv n protein and pedv replication could be inhibited by silencing the hnrnp a . the pedv yn (genbank accession no. kt ), yn (genbank accession no. kt ), and cv (genbank accession no. af . ) strain were used throughout this study. yn and yn strains were obtained by passaging the yn strain, a variant strain isolated from the intestine of a piglet with diarrhea, for and generations, respectively. our previous study has demonstrated that yn was a virulent strain and yn was an attenuation strain [ ] . cv strain, a classical pedv strain, was provided by chengdu tecbond biological product co., ltd. (chengdu, china). the ccl- cell line and hek t cell line were purchased from american type culture collection (atcc) and cultured in dulbecco's modified eagle's medium (dmem), supplemented with % fetal bovine serum (invitrogen, carlsbad, ca, usa) at • c with % co . the ccl- cell line was used for virus growth, infection, and cell lysate preparation. hek t cell line was used for co-ip analysis. the rabbit anti-hnrnp a , anti-flag polyclonal antibody (pab) and mouse anti-β-actin, anti-flag mono-antibody (mab) were purchased from abclonal (wuhan, china). mouse mab against pedv n protein was purchased from youlong biotech (shanghai, china). mouse mab against pedv spike protein was established by our laboratory. application of this mab has been described in some previous studies [ , ] . alexa fluor -conjugated anti-rabbit, anti-mouse and alexa fluor -conjugated anti-mouse antibodies were purchased from antgene biological (wuhan, china). the cdna expression construct encoding yn n protein was pcr amplified and cloned into pcaggs-flagc, which encode a c-terminal flag. details of this part have been described in our previous study [ ] . briefly, twelve piglets were randomly divided into three groups of four. the piglets in different groups were orally administrated with . ml yn and . ml yn with the same titer of . median tissue culture infective dose (tcid ) ml − and . ml dmem, respectively. all piglets were euthanized and necropsied when diarrhea was observed in the piglets in yn -infected group. jejunum tissues were separated rapidly, washed with ice-cold pbs buffer, snap-frozen in liquid nitrogen, and kept at − • c for subsequent proteome study. for the proteome study, itraq labeling coupled with lc-ms/ms was chose to analyze whole cell changes of jejunum of piglets, infected with pedv yn strain and yn strain. were precleared with protein a/g agarose (beyotime, shanghai, china) for h then centrifuged at rpm for min at • c. supernatants were incubated with mono-antibody against flag for h, then protein a/g beads were added and incubated at • c for h. the beads were then washed with ip lysis buffer five times and boiled in sample buffer, and the proteins were subjected to sds-page, followed by immunoblotting analysis with anti-flag pab or anti-hnrnp a pab. ccl- cells grown on coverslips were infected with pedv yn strain, yn strain and cv strain, respectively, at a multiplicity of infection (moi) . . at h post infection (hpi), the cells were fixed with % paraformaldehyde for min followed by being treated with methanol. fixed cells were blocked with % bovine serum albumin and incubated with anti-hnrnp a pab and mab against pedv n protein. alexa fluor -conjugated anti-rabbit and alexa fluor -conjugated anti-mouse antibodies were served as the secondary antibody. cell nucleus were stained with , -diamidino- -phenylindole (dapi). the localization of pedv n protein, hnrnp a and cell nucleus were then observed on a zeiss confocal microscope (zeiss, oberkochen, germany). ccl- cells were transfected with sirnas targeting to hnrnp a with lipofectamine reagent (invitrogen, carlsbad, ca, usa) according to the manufacturer's instructions. the sirnas targeting hnrnp a were synthesized by gene pharma (shanghai, china). effect of rna interference was tested by western blot at hpt. ccl- cells seeded in a -well plate were transfected without sirna or with nm nonspecial control sirna or sirna targeting to hnrnp a . at hpt, cells were infected with or without pedv. at hpi (yn ) or hpi (yn and cv ), the cells were fixed with % paraformaldehyde followed by treating with methanol. then the fixed cell was blocked with % bovine serum albumin and then incubated with mab against pedv s protein. alexa fluor -conjugated anti-mouse antibody was applied to detect the primary antibodies. dapi was selected to stain the cell nucleus. rna was extracted by using tripure isolation reagent (roche, in, usa) following the manufacturer's instructions. the cdna was obtained by rt-pcr using the primescript™ rt master mix (takara, tokyo, japan). the real-time rt-pcr assay for quantifying pedv genome used the following primer and probe sequences: pedv forward primer: -cgtacaggtaagtcaattac- , pedv reverse primer: -gatgaagcattgactgaa- , pedv taq-man ® probe: fam-ttcgtcaca gtcgccaagg-tamra. cells were lysed with lysis buffer (beyotime, shanghai, china) containing mm pmsf. these proteins were subjected to sds-page and then the separated protein bands were transferred onto pvdf membrane using a trans-blot (bio-rad, berkeley, ca, usa). the membrane was incubated in blocking buffer (tris-buffered saline (tbs), containing . % tween- (tbst) and % skim milk) for h at room temperature followed by washing three times by pbst. then the membrane was incubated with the corresponding primary antibodies for h at room temperature. after being washed three times by pbst, the membrane was incubated with (hrp)-conjugated goat-anti mouse/rabbit igg at room temperature for . h. finally, the protein bands were visualized using the clarity™ western ecl blotting substrate (bio-rad, hercules, ca, usa). according to the result of lc-ms/ms, hnrnp a was down-regulated in an attenuated pedv strain (yn ) infected jejunum tissues, while showed no apparent change in a virulent pedv strain (yn ) infected jejunum tissues [ ] . western blot assay was applied to confirm the changes of hnrnp a . as shown in figure , regulations of hnrnp a were consistent with the result of lc-ms/ms. hnrnp a has been proved to be involved in the replication process of other coronaviruses [ , ] . therefore, we assumed that hnrnp a may play a role in pedv replication and influence the pathogenicity of pedv in vivo. according to the result of lc-ms/ms, hnrnp a was down-regulated in an attenuated ped rain (yn ) infected jejunum tissues, while showed no apparent change in a virulent pedv stra n ) infected jejunum tissues [ ] . western blot assay was applied to confirm the changes rnp a . as shown in figure , regulations of hnrnp a were consistent with the result of l s/ms. hnrnp a has been proved to be involved in the replication process of other coronavirus , ] . therefore, we assumed that hnrnp a may play a role in pedv replication and influen e pathogenicity of pedv in vivo. previous studies have demonstrated that n protein of some coronaviruses, such as mhv an rs-cov, could interact with hnrnp a . in this study, we determined to investigate whether otein can be colocalized with hnrnp a during pedv infection. ccl- cells were infected wi e yn , yn , and cv strain of pedv, respectively, and the localization of np and hnrnp a as observed by confocal microscopy at hpi. as shown in figure , hnrnp a was main calized in the nucleus without pedv infection. however, some hnrnp a was transported fro e nucleus to the cytoplasm and colocalized with pedv n protein during pedv infectio rthermore, pedv n protein was localized in cytoplasm and no differences were found in t calization of n protein of these pedv strains. the immunofluorescence assay demonstrated th dv n protein and hnrnp a colocalized in cytoplasm. previous studies have demonstrated that n protein of some coronaviruses, such as mhv and sars-cov, could interact with hnrnp a . in this study, we determined to investigate whether n protein can be colocalized with hnrnp a during pedv infection. ccl- cells were infected with the yn , yn , and cv strain of pedv, respectively, and the localization of np and hnrnp a was observed by confocal microscopy at hpi. as shown in figure , hnrnp a was mainly localized in the nucleus without pedv infection. however, some hnrnp a was transported from the nucleus to the cytoplasm and colocalized with pedv n protein during pedv infection. furthermore, pedv n protein was localized in cytoplasm and no differences were found in the localization of n protein of these pedv strains. the immunofluorescence assay demonstrated that pedv n protein and hnrnp a colocalized in cytoplasm. the colocalization of pedv n protein and hnrnp a demonstrated that a certain interaction may exist between these two proteins. thus, co-immunoprecipitation (co-ip) was performed to testify this phenomenon. t cells transfected with plasmids expressing the flag-tagged pedv n protein were subjected to immunoprecipitation using anti-flag antibody. interaction of pedv n protein with host protein hnrnp a was analyzed by immunoblotting with anti-flag antibody and anti-hnrnp a antibody. as shown in figure , cellular hnrnp a protein was only detected in the presence of flag tagged pedv n by co-ip. these results indicate that n protein interacted with hnrnp a . the colocalization of pedv n protein and hnrnp a demonstrated that a certain interaction may exist between these two proteins. thus, co-immunoprecipitation (co-ip) was performed to testify this phenomenon. t cells transfected with plasmids expressing the flag-tagged pedv n protein were subjected to immunoprecipitation using anti-flag antibody. interaction of pedv n protein with host protein hnrnp a was analyzed by immunoblotting with anti-flag antibody and anti-hnrnp a antibody. as shown in figure , cellular hnrnp a protein was only detected in the presence of flag tagged pedv n by co-ip. these results indicate that n protein interacted with hnrnp a . cell lysates were also applied to confirm the expression of proteins (input). in order to investigate whether hnrnp a participates in the replication of pedv, three sirnas targeting to hnrnp a were synthesized to silence the expression of this protein. sequences of the sirnas were shown in table . ccl- cells were transfected with sirnas against hnrnp a expression, cells were collected at hpt to analyze the silencing efficiency of hnrnp a at the protein level. as shown in figure , sirna- showed the best performance for silencing hnrnp a and was chosen for the following research. in order to investigate whether hnrnp a participates in the replication of pedv, three sirnas targeting to hnrnp a were synthesized to silence the expression of this protein. sequences of the sirnas were shown in table . ccl- cells were transfected with sirnas against hnrnp a expression, cells were collected at hpt to analyze the silencing efficiency of hnrnp a at the protein level. as shown in figure , sirna- showed the best performance for silencing hnrnp a and was chosen for the following research. ccl- cells were transfected with sirna- for h followed by infection with pedv yn strain. these cells were subjected to western blot, indirect immunofluorescence assay (ifa) or real-time pcr at hpi figure . according to the results, a conclusion was reached that knockdown of hnrnp a reduced the replication of yn . hek t cells were transfected with a vector expressing pedv n protein with a flag tag (flag-pedv-n) or empty vectors (flag-pcaggs) and the whole-cell lysates obtained at hpt were immunoprecipitated with anti-flag mab. after separation by sds-page, proteins were detected by immunoblotting with the indicated antibodies (ip: flag). cell lysates were also applied to confirm the expression of proteins (input). in order to investigate whether hnrnp a participates in the replication of pedv, three sirnas targeting to hnrnp a were synthesized to silence the expression of this protein. sequences of the sirnas were shown in table . ccl- cells were transfected with sirnas against hnrnp a expression, cells were collected at hpt to analyze the silencing efficiency of hnrnp a at the protein level. as shown in figure , sirna- showed the best performance for silencing hnrnp a and was chosen for the following research. ccl- cells were transfected with sirna- for h followed by infection with pedv yn strain. these cells were subjected to western blot, indirect immunofluorescence assay (ifa) or realtime pcr at hpi figure . according to the results, a conclusion was reached that knockdown of hnrnp a reduced the replication of yn . sequences ( ′- ′) sirna- ggaagaguuguggaaccaatt sirna- ggauuugguaaugauggaatt sirna- gcgguggaggucaauacuutt negative control sirna (nc) uucuccgaacgugucacgutt the pedv virulent strain yn and pedv classical strain cv were further applied to test the positive effects of hnrnp a on pedv replication. as shown in figures and , similar results were obtained. these results demonstrated that hnrnp a is a positive regulator of pedv replication. sirna- ggaagaguuguggaaccaatt sirna- ggauuugguaaugauggaatt sirna- gcgguggaggucaauacuutt negative control sirna (nc) uucuccgaacgugucacgutt the pedv virulent strain yn and pedv classical strain cv were further applied to test the positive effects of hnrnp a on pedv replication. as shown in figures and , similar results were obtained. these results demonstrated that hnrnp a is a positive regulator of pedv replication. figure . knockdown of hnrnp a expression inhibits yn replication. ccl- cells were transfected without sirna (a) or with nc (b) or sirna- (c). at hpt, cells were infected with yn strain at a moi of . or mock infected (d). virus suspensions were harvested at hpi to calculate the virus titer by real-time rt-pcr. the virus copies per ml suspension was calculated (a). cells were harvested at hpi to analyze the expression of pedv n protein by western blot (b) and the numbers of cells infected with pedv by ifa (original magnification ×) (c). pedv n protein levels were quantified by measuring band intensities and normalized with respect to the amount of β-actin. data are shown as means ± the standard errors of the mean (sem) of at least three independent experiments, with the error bars representing the standard deviations. the pedv virulent strain yn and pedv classical strain cv were further applied to test the positive effects of hnrnp a on pedv replication. as shown in figures and , similar results were obtained. these results demonstrated that hnrnp a is a positive regulator of pedv replication. the virus titer by real-time rt-pcr. the virus copies per ml suspension was calculated (a). cells were harvested at hpi to analyze the expression of pedv n protein by western blot (b) and the numbers of cells infected with pedv by ifa (original magnification ×) (c). pedv n protein levels were quantified by measuring band intensities and normalized with respect to the amount of β-actin. data are shown as means ± sem of at least three independent experiments, with the error bars representing the standard deviations. in this study, interaction between hnrnp a and pedv n protein was verified by co-ip and immunofluorescence assay. our results established that n protein interacts with hnrnp a in pedv infected cells and t cells expressing pedv n protein. hnrnp a not only mainly localizes in the cell nucleus, but also shuttles between the nucleus and the cytoplasm [ , ] . however, this protein underwent a relocalization to cytoplasm during pedv infection, suggesting a possible functional link between hnrnp a and pedv infection. this phenomenon is very similar to a previous study about mhv [ ] . furthermore, we found that the pedv n protein and hnrnp a co-localized predominantly in the perinuclear region of pedv infected cells, in which active coronavirus replication/transcription complexes reside. this indicates both pedv n protein and hnrnpa might participate in constituting the pedv replication/transcription complex and their interaction may be involved in regulation of pedv replication. it is well known that hnrnp a is one part of replication/transcription complex of both sars-cov and mhv [ , , , , ] . overexpression of hnrnpa facilitates mhv replication while inhibition of hnrnp a expression results in the reduction of mhv replication [ ] . since pedv is in this study, interaction between hnrnp a and pedv n protein was verified by co-ip and immunofluorescence assay. our results established that n protein interacts with hnrnp a in pedv infected cells and t cells expressing pedv n protein. hnrnp a not only mainly localizes in the cell nucleus, but also shuttles between the nucleus and the cytoplasm [ , ] . however, this protein underwent a relocalization to cytoplasm during pedv infection, suggesting a possible functional link between hnrnp a and pedv infection. this phenomenon is very similar to a previous study about mhv [ ] . furthermore, we found that the pedv n protein and hnrnp a co-localized predominantly in the perinuclear region of pedv infected cells, in which active coronavirus replication/transcription complexes reside. this indicates both pedv n protein and hnrnpa might participate in constituting the pedv replication/transcription complex and their interaction may be involved in regulation of pedv replication. it is well known that hnrnp a is one part of replication/transcription complex of both sars-cov and mhv [ , , , , ] . overexpression of hnrnpa facilitates mhv replication while inhibition of hnrnp a expression results in the reduction of mhv replication [ ] . since pedv is also a member of coronavirus, hnrnp a may be involved in the process of pedv infection. in order to test this hypothesis, we silenced the hnrnp a and then studied its effect on pedv infection. similar to the result of mhv following silencing hnrnp a , replication of three pedv strains were all inhibited, suggesting a positive effect of hnrnp a on pedv infection. replication of coronavirus depend on the generation of nested subgenomic mrnas (sgmrnas) with a common capped leader sequence [ ] . optical transcription of sgmrnas requires the interaction between its leader sequence and the intergenic (ig) sequences of each orf. it has been reported that hnrnp a can bind to both the terminal leader sequences and ig sequences [ ] . so, inhibition of pedv by silencing hnrnp a is likely due to the break of the interaction between terminal leader sequences and ig sequences. however, till now we have not got evidence to support it. in addition, silencing of hnrnp a might also be able to reduce its interaction with pedv n protein. the function of their interaction on pedv infection is under the investigation of our laboratory. our previous study has demonstrated that hnrnp a was downregulated in the jejunum of pedv strain yn infected group, but no apparent change in group infected with yn [ ] . since yn caused diarrhea in piglets while yn could not, therefore, downregulation of hnrnp a was one of the reasons responsible for the lower pathogenicity of yn than yn in vivo. from the field to the lab-an european view on the global spread of pedv porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis new variants of porcine epidemic diarrhea virus, china porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines outbreak-related porcine epidemic diarrhea virus strains similar to us strains distinct characteristics and complex evolution of pedv strains first detection, clinical 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heterogeneous nuclear ribonucleoprotein a controls translation initiation of specific mrnas heterogeneous nuclear ribonucleoprotein a regulates rna synthesis of a cytoplasmic virus multiple type a/b heterogeneous nuclear ribonucleoproteins (hnrnps) can replace hnrnp a in mouse hepatitis virus rna synthesis blocking eif e-eif g interaction as a strategy to impair coronavirus replication nuclear proteins hijacked by mammalian cytoplasmic plus strand rna viruses the authors have declared no conflict of interest. key: cord- - sb moe authors: lagare, adamou; ousmane, sani; dano, ibrahim dan; issaka, bassira; issa, idi; mainassara, halima boubacar; testa, jean; tempia, stefano; mamadou, saidou title: molecular detection of respiratory pathogens among children aged younger than years hospitalized with febrile acute respiratory infections: a prospective hospital‐based observational study in niamey, niger date: - - journal: health sci rep doi: . /hsr . sha: doc_id: cord_uid: sb moe background and aims: in niger, acute respiratory infections (aris) are the second most common cause of death in children aged younger than years. however, the etiology of ari is poorly understood in the country. this study aims to describe viral and bacterial infections among children aged younger than years hospitalized with febrile ari at two hospitals in niamey, niger's capital city, and the reported clinical procedures. methods: we conducted a prospective study among children aged younger than years hospitalized with febrile ari at two national hospitals in niamey between january and december . clinical presentation and procedures during admission were documented using a standardized case investigation form. nasopharyngeal specimens collected from each patient were tested for a panel of respiratory viruses and bacteria using the fast track diagnostic plus kit. results: we enrolled and tested children aged younger than years, of whom ( . %) were aged younger than year, and ( . %) died during the study period. overall, / ( . %) specimens tested positive for at least one respiratory virus or bacterium; of these, ( . %) tested positive for respiratory viruses, ( . %) tested positive for respiratory bacteria, and ( . %) tested positive for both respiratory viruses and bacteria. the predominant viruses detected were respiratory syncytial virus (rsv) ( / ; . %), human parainfluenza virus (hpiv) types to ( / ; . %), human rhinovirus (hrv) ( / ; . %), human adenovirus (hav) ( / ; . %), and influenza virus (inf) ( / ; . %). streptococcus pneumoniae ( / ; . %) was the most frequently detected bacterium, followed by staphylococcus aureus ( / ; . %) and haemophilus influenzae type b ( / ; . %). chest x‐rays were performed at the discretion of the attending physician on ( . %) case patients. of these patients, ( . %) had abnormal radiological findings. a total of / ( . %) and / ( . %) children received antibiotic treatment prior to admission and during admission, respectively. conclusion: a high proportion of respiratory viruses was detected among children aged younger than years with febrile ari, raising concerns about excessive use of antibiotics in niger. acute respiratory infections (aris) are responsible for elevated childhood morbidity and mortality globally, , accounting for approximately four million deaths among children aged younger than years in and resulting in substantial burden of healthcare systems. , in developing countries, aris account for % of all deaths among children aged younger than years and . % of all disabilities and premature deaths. , therefore, data on the epidemiology and seasonality of ari are important to develop control and prevention strategies. in niger, the estimated mortality rate among children aged younger than years was per in (unicef child mortality estimate), and more than % of these deaths were associated with malaria, respiratory infections, and diarrhea. according to the niger health statistics yearbook, the estimated case fatality proportion associated with respiratory infections was . %. in the absence of laboratory diagnosis, it is difficult to clinically differentiate between ari-related causative pathogens, due to similarities of symptoms. , the main causative agents of ari are viruses and bacteria. [ ] [ ] [ ] in niger, there is scarcity of data on aris, although a routine influenza surveillance system exists. inf has been detected in % of samples from children aged younger than years hospitalized with severe acute respiratory illness (sari). this study aims to describe the viral and bacterial infections among children aged younger than years hospitalized with febrile ari at two national hospitals of niamey, the capital city of niger, and the reported clinical procedures. we conducted a prospective study among children aged younger than years hospitalized with febrile ari between january and december . niger has four distinct seasons as categorized by the national directorate of meteorology: the cold season (mid-december to mid-february), the dry season (mid-february to may), the rainy season (june to september), and the hot season (october to mid-december). this study was part of the "total niger infection respiratoire aiguë" (tonira) project, which was funded by the total corporate foundation in order to strengthen medical care of febrile ari among children aged younger than years. the study was conducted at the pediatric wards of two national tertiary hospitals situated in niamey, namely, the hôpital national de niamey (hnn) and the hôpital national lamordé (hnl). these two hospitals provide general health care to the population of niamey estimated at . million in , as well as to patients referred from across the country. laboratory detection of respiratory viruses and bacteria were conducted at the centre de recherche médicale et sanitaire (cermes), niamey, niger (the national reference laboratory for influenza). demographic and clinical data, as well as the use of antibiotics before and during admission, were collected using standard data collection forms. malaria testing was implemented on site using thick blood film microscopy. chest x-rays were performed at the discretion of the attending physician, and typical abnormal radiological findings assessed included pulmonary opacities, acute bronchiolitis, and enlargement of intercostal spaces. malnutrition was determined using the ratio of weight for height, and its level was classified as moderate or severe by the attending physician using national standards based on the technical guidelines for integrated disease surveillance and response in the african region (retrieved from https://www. afro.who.int/sites/default/files/ - /idsr-technical-guidelines_ final_ _ .pdf). febrile ari in conjunction with malaria, malnutrition, and diarrhea were assessed during admission using a standardized case investigation form. in-hospital outcome (ie., discharge, referral, or death) was recorded for all enrolled patients. a febrile ari case was defined as a hospitalized child aged younger than years with onset of fever c or higher and cough within days prior to admission and at least one of the following signs: inability to drink or breastfeed, lethargy, vomiting, convulsions, nasal flaring, chest indrawing, stridor in a calm child, or tachypnea. nasopharyngeal swabs were collected from all enrolled patients, placed in universal transport medium, stored at to c, and trans- amplification. the fluorescence was assessed at the amplification step. the positive and negative virus plasmid controls provided in the kit were included in all runs to monitor assay performance. viral and/or bacterial detection was reported as percentage positive, overall and within selected categories (eg, age groups and seasons). children aged younger than years were stratified into two age groups: infants (aged younger than year) and young children (aged - years), for comparability with other studies from africa. [ ] [ ] [ ] [ ] in addition, the majority of enrolled children were aged younger than year hindering our ability to categorize age in smaller age groups. stata version . (statacorp, college station, texas, usa) was used for the analysis. the protocol was approved by the national ethical consultative com- overall, / ( . %) specimens tested positive for at least one respiratory virus or bacterium (table ) overall, respiratory viruses were detected in / ( . %) speci- figure a ). overall, respiratory bacteria were detected in / ( . %) specimens. s pneumoniae ( / ; . %) was the most frequently detected bacterium, followed by s aureus ( / ; . %) and hib ( / ; . %) ( table ) . m pneumoniae and c pneumoniae were detected individually in less than % of specimens. of the specimens that tested positive for at least one respiratory bacteria, two or more respiratory bacteria were detected in / s pneumoniae and s aureus were mostly detected during the rainy season (june to september) ( table and figure b ). we report the detection of respiratory viruses and bacteria among children aged younger than years hospitalized with febrile ari in t a b l e (continued) , and asia (range %- %). [ ] [ ] [ ] the hpiv were also the most predominant viruses detected in children aged younger than years in a previous study conducted in niger. rsv is considered to be a major cause of ari in children aged younger than years, , [ ] [ ] [ ] and it was the most commonly detected virus also in this study ( / ; . %). among the cases in which hpiv and hcov were detected, hpiv type and hcov type oc were the predominant types, and this is consistent with previous studies. , in this study, infs were detected in . % of the cases, which is in agreement with findings from the national influenza surveillance our study presents some limitations. first, the study spanned over a period of only months, limiting our ability to fully assess the temporal circulation pattern of the investigated agents. due to the small sample size, we may also have been underpowered to detect small variations in the temporal distribution of the investigated agents. second, attribution of causality remains challenging due to the lack of controls in our study. the association of pathogen detection with illness could not be well assessed, although most of the viral and bacterial pathogens identified in this study have been described in previous case control studies as causative agents of ari. , in addition, we did not collect blood samples to assess whether the detection of common bacterial colonizers of the nasopharynx, such as s pneumoniae, were associated with invasive disease. third, we did not record the number of patients with febrile ari that refused enrollment, hindering our ability to estimate underenrollment. last, given that only patients with febrile ari were enrolled, this could have resulted in an underestimation of the rsv burden since a large proportion of rsv-related illness is not associated with fever. in this -year prospective study, both viral and bacterial pathogens were detected in high proportion among hospitalized children aged younger than years with febrile ari in niamey, niger. 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sensitivity profile of streptococcus pneumoniae carried in healthy toddlers before pcv introduction in niamey response to conjugate haemophilus influenzae b vaccine among infants in niamey, niger etiology and factors associated with pneumonia in children under years of age in mali: a prospective case-control study performance of surveillance case definitions in detecting respiratory syncytial virus infection among young children hospitalized with severe respiratory illness-south africa molecular detection of respiratory pathogens among children aged younger than years hospitalized with febrile acute respiratory infections: a prospective hospital-based observational study in niamey we are thankful to the moh, cermes, and clinicians and nurses at the hnn and hnl for their contributions. this work was funded by the total corporate foundation through a partnership with pasteur institute of paris and cermes. the funder was not involved in the study design, collection, analysis, and interpretation of data, writing of the report, and decision to submit the report for publication. adamou lagare had full access to the data and takes responsibility for the integrity of the data and the accuracy of the data analysis. the lead author/manuscript guarantor (adamou lagare) affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained. all authors declare that they have no commercial or other associations that may pose a conflict of interest. https://orcid.org/ - - - stefano tempia https://orcid.org/ - - - x key: cord- -u dhbmht authors: keske, Şiran; ergönül, Önder; tutucu, faik; karaaslan, doruk; palaoğlu, erhan; can, füsun title: the rapid diagnosis of viral respiratory tract infections and its impact on antimicrobial stewardship programs date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: u dhbmht we aimed to describe the potential benefit of new rapid molecular respiratory tests (mrt) in decreasing inappropriate antibiotic use among the inpatients presenting with influenza-like illness (ili). we included patients from inpatient and outpatient departments who had ili and performed mrt between january and december in a -bed private hospital in istanbul. at the end of , we implemented antimicrobial stewardship including systematic use of mrt. then, we compared our observations between the year and the year . we designed the study according to the strengthening the reporting of observational studies in epidemiology (strobe) tool. a u.s. food and drug administration (fda)-cleared multiplexed polymerase chain reaction (pcr) system (biofire filmarray, idaho technology, salt lake city, ut) which detects viruses and three bacteria was used for diagnosis. in total, patients were included; ( %) were inpatients and ( %) were older than years of age. at least one virus was detected in ( %) patients. rhinovirus/enterovirus, influenza virus, and adenovirus were the most commonly detected. among hospitalized patients, in children, a significant decrease in antibiotic use ( . % in and . % in , p = . ) was observed, but in adults, the decrease was not statistically significant ( % in and % in , p = . ). the duration of antibiotic use after the detection of virus was significantly decreased in both children and adults (p < . and p = . , respectively). by using mrt, inappropriate antibiotic use and, also, duration of inappropriate antibiotic use after the detection of virus was significantly decreased. it is time to increase the awareness about the viral etiology in respiratory tract infections (rtis) and implement mrt in clinical practice. respiratory tract infections (rtis) are the most common reasons for admission to healthcare facilities and one of the leading causes of hospitalization [ , ] . the viral pathogens in the etiology of rtis became more detectable along with the improvement in molecular diagnostic methods in recent years. in adults, influenza virus, rhinovirus, adenovirus, respiratory syncytial virus (rsv), human coronavirus, and parainfluenza virus cause infections with considerable morbidity and mortality [ , ] , and in infants, rsv is the most common reason for rtis among hospitalized patients [ ] . the antibiotic prescription rate was reported to be more than % despite the high proportion of viral etiology [ ] [ ] [ ] [ ] . unnecessary antibiotic administration in viral infections and antimicrobial resistance prompted the implementation of antimicrobial stewardship (ams) programs. the bimplementing an antibiotic stewardship program^guideline by the infectious diseases society of america (idsa) suggested rapid viral testing for respiratory pathogens to decrease inappropriate antibiotic use [ ] . in this study, we aimed to describe the viral etiology in influenza-like illness (ili) in children and adults and to show the benefit of new rapid molecular respiratory tests (mrt) in decreasing inappropriate antibiotic use. we designed the study according to the strengthening the reporting of observational studies in epidemiology (strobe) tool [ ] . we included consequent patients from inpatient and outpatient departments, who had moderate to severe ili, and, therefore, needed further diagnostic evaluation between january and december in a -bed private hospital in istanbul. only the patients whom mrt was implemented for diagnosis were included in this study. mrt was introduced in our hospital in , and the demand for mrt increased over the years. at the end of , we enhanced our training on antimicrobial stewardship (ams). the intervention consists of training physicians to increase awareness about mrt, highlighting antimicrobial resistance because of unnecessary antibiotic consumption at ad hoc meetings in each related department. then, we compared our observations between the year and the year . this was a retrospective study performed by reviewing the patient charts. clinical features of the patients, demographic characteristics, chronic diseases including malignant disorders, diabetes mellitus, chronic kidney and liver diseases, and detailed information about antibiotic and antiviral drugs during hospitalization were collected by the chart review. complete blood count, c-reactive protein, and liver and kidney function tests were studied among all patients. bacterial cultures (throat, sputum, blood, and urine) were obtained, and procalcitonin (pct) was studied in all patients with suspicion of bacterial infection and/or in critical patients. chest x-ray and lung computed tomography was done if lower respiratory tract infection (lrti) was suspected. ili was defined according to the world health organization (who) and explained as an acute respiratory infection with fever of ≥ °c and cough and with onset within the last days [ ] . inappropriate antibiotic use was defined as antibiotic use despite detection of virus without documented and/or suspicious bacterial infection. antibiotic use duration was calculated as the duration of antibiotic use after the detection of virus in patients with inappropriate antibiotic use. for molecular detection and identification of respiratory viral pathogens, a u.s. food and drug administration (fda)cleared multiplexed respiratory polymerase chain reaction (pcr) system (mrt), biofire filmarray (idaho technology, salt lake city, ut) which detects viral respiratory pathogens (adenovirus, coronavirus hku , coronavirus nl , coronavirus e, coronavirus oc , hmpv, rhinovirus/enterovirus, influenza a, influenza a/h , influenza a/h - , influenza a/h , influenza b, parainfluenza , parainfluenza , parainfluenza , parainfluenza , and rsv) and three bacteria (bordetella pertussis, chlamydophila pneumoniae, and mycoplasma pneumoniae) was used according to the manufacturer's instructions. mrt yielded results within a few hours and the results were immediately reported to the physician of the patients by laboratory staff. in the statistical analysis, the t-test for continuous variables and the chi-square test for comparison of categorical variables were used. in the analysis, stata (statacorp, college station, tx) was used and a p-value < . was set as being in total, patients whom mrt was implemented were included; ( %) were inpatients and ( %) were older than years of age. at least one virus was detected in ( %) patients and ( %) of them were inpatients (fig. (fig. ) . in patients out of ( . %), multiple viral pathogens were detected. the overall rate of lrtis was % among inpatients. among inpatients infected with rsv, the rate of lrti was % in adults and % in children. similarly, in hmpv infections, the rate of lrti was % in adults and % in children (table ) . in total, among hospitalized patients, antibiotics were continued inappropriately in % ( table ). in children, the antibiotic continuation rate was . % in and decreased to . % in (p = . ); in adults, there was a decrease towards but this was not significant ( table ). the mean duration of inappropriate antibiotic use after the detection of virus decreased in both adults and children in when compared with . the most commonly inappropriately used antibiotics were third-generation cephalosporins, quinolones, and second-generation cephalosporins. in our study, rhinovirus/enterovirus, influenza, and adenovirus were the most commonly detected viruses in the etiology of ili. in a recent study by jain et al. [ ] , a population-based surveillance for community-acquired pneumonia among adults aged years and older was conducted. human rhinovirus, influenza a and b virus, and hmpv were the most commonly detected viruses in etiology. influenza virus and rhinovirus were reported to be the most commonly detected viruses in both adults and children in turkey [ ] . among inpatients older than years of age, % had lrti, and influenza a, rhinovirus, rsv, and hmpv were the most common agents. among children, % had lrti, and rhinovirus, rsv, and hmpv were the most common agents ( fig. ; fig. the most commonly detected viruses among all patients in whom at least one virus was detected. rsv: respiratory syncytial virus; hmpv: human metapneumovirus table ). any effort targeted to reduce the unnecessary use of antibiotics in viral pneumonia could be useful in this era of antibiotic resistance. because of the high rate of inappropriate antibiotic prescription despite the availability of mrt, we decided to improve training sessions about the diagnosis and management of respiratory system infections at the end of . then, we performed an observational comparison between the practice in and . there was a decrease in antibiotic use towards among all inpatients. however when analyzed separately, a significant reduction in children but not in adults was detected ( table ). the significant reduction of antibiotic use in children but not in adults could be explained by differences in physicians' experience in mrt. despite the fact that there was a reduction in antibiotic use in children after the detection of virus, antibiotic prescription rates were still high. in a recent study, the impact of mrt on antibiotic use was investigated and it was seen that antibiotics were still continued in about % of the patients, despite the detection of virus. this finding was explained by being a new test [ ] . in another study, afzal et al. [ ] reported that a positive result for mrt decreased antibiotic use duration and prescription rate but the decrease in antibiotic prescription was not statistically significant. timbrook et al. [ ] studied mrt in combination with pct and concluded that a lower level of pct and detection of virus by mrt were not associated with decreased use of antibiotics in rtis, but in another study, a lower level of pct in combination with detection of virus was found to be associated with shortened duration of antibiotic usage [ ] . in a randomized controlled study, the effect of both mrt and pct on antibiotic use was evaluated and the duration of antibiotic use was found to be decreased in cases of viral infection with lower levels of pct [ ] . one of the strongest parts of our study was having the opportunity of ordering mrt whenever needed in routine clinical practice, so we reached a large sample size. we had the opportunity of getting the results of c-reactive protein, pct, and mrt within a few hours, so we discriminated bacterial or viral etiology quickly. one of the limitations of this study was being conducted in a single center, but our center was unique for implementing this test in clinical practice. we are unique because mrt are still expensive to perform in routine clinical practice in other hospitals. future studies for rapid point-of-care testing for respiratory viruses might improve clinical care by reducing unnecessary antibiotic use [ ] . future cost-effective studies for the implementation of mrt will be useful. by using molecular rapid tests (mrt) in our hospital, inappropriate antibiotic use and also duration of inappropriate antibiotic use after the detection of virus was significantly decreased among inpatients. the treatment paradigm for respiratory tract infections (rtis) is changing, but molecular viral diagnostic tests are in their infancy. it is time to increase the awareness of the viral etiology in rtis and implement mrt in clinical practice. funding no funding of any kind has been received. the data were generated as part of routine work. conflict of interest none to declare. ethical approval koç university irb approved the study. informed consent not applicable. mean duration of inappropriate antibiotic use (days) . (sd . ) . (sd . ) < . . (sd . ) . (sd . ) . rates of hospitalizations for respiratory syncytial virus, human metapneumovirus, and influenza virus in older adults ecil- ): guidelines for diagnosis and treatment of human respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, and coronavirus community-acquired pneumonia requiring hospitalization among u.s. adults role of influenza and other respiratory viruses in admissions of adults to canadian hospitals antibiotic use for viral acute respiratory tract infections remains common ambulatory antibiotic prescribing for acute bronchitis and cough and hospital admissions for respiratory infections: time trends analysis antibiotic prescription rates for acute respiratory tract infections in us ambulatory settings prevalence of inappropriate antibiotic prescriptions among us ambulatory care visits implementing an antibiotic stewardship program: guidelines by the infectious diseases society of america and the society for healthcare epidemiology of america the strengthening the reporting of observational studies in epidemiology (strobe) statement: guidelines for reporting observational studies world health organization (who) ( ) who surveillance case definitions for ili and sari. case definitions for influenza surveillance prevalence and seasonal distribution of respiratory viruses during the - season in istanbul evaluating the impact of the multiplex respiratory virus panel polymerase chain reaction test on the clinical management of suspected respiratory viral infections in adult patients in a hospital setting clinical diagnosis, viral pcr, and antibiotic utilization in community-acquired pneumonia antibiotic discontinuation rates associated with positive respiratory viral panel and low procalcitonin results in proven or suspected respiratory infections the clinical impact of the detection of potential etiologic pathogens of community-acquired pneumonia serum procalcitonin measurement and viral testing to guide antibiotic use for respiratory infections in hospitalized adults: a randomized controlled trial routine molecular point-of-care testing for respiratory viruses in adults presenting to hospital with acute respiratory illness (respoc): a pragmatic, open-label, randomised controlled trial key: cord- -pkovyjo authors: li, yize; dong, beihua; wei, zuzhang; silverman, robert h.; weiss, susan r. title: activation of rnase l in egyptian rousette bat-derived roni/ cells is dependent primarily on oas and independent of mavs signaling date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: pkovyjo bats are reservoirs for many rna viruses that are highly pathogenic in humans yet relatively apathogenic in the natural host. it has been suggested that differences in innate immunity are responsible. the antiviral oas-rnase l pathway is well characterized in humans, but there is little known about its activation and antiviral activity in bats. during infection, oass, upon sensing double-stranded rna (dsrna), produce ′- ′ oligoadenylates ( - a), leading to activation of rnase l which degrades viral and host rna, limiting viral replication. humans encode three active oass (oas to - ). analysis of the egyptian rousette bat genome combined with mrna sequencing from bat roni/ cells revealed three homologous oas proteins. interferon alpha treatment or viral infection induced all three oas mrnas, but rnase l mrna is constitutively expressed. sindbis virus (sinv) or vaccinia virus (vacvΔe l) infection of wild-type (wt) or oas -ko (knockout), oas -ko, or mavs-ko roni/ cells, but not rnase l-ko or oas -ko cells, induces robust rnase l activation. sinv replication is - to -fold higher in the absence of rnase l or oas than in wt cells. however, mavs-ko had no detectable effect on rna degradation or replication. thus, in roni/ bat cells, as in human cells, activation of rnase l during infection and its antiviral activity are dependent primarily on oas while mavs signaling is not required for the activation of rnase l and restriction of infection. our findings indicate that oas proteins serve as pattern recognition receptors (prrs) to recognize viral dsrna and that this pathway is a primary response to virus rather than a secondary effect of interferon signaling. b ats are reservoirs for many rna viruses that are highly pathogenic in humans yet attenuated in their natural host. these include filoviruses (ebola virus and marburg virus) ( ) ( ) ( ) ( ) , coronaviruses (severe acute respiratory syndrome coronavirus [sars-cov] and middle east respiratory syndrome coronavirus [mers-cov]) ( , ) , alphaviruses (venezuelan equine encephalitis virus [veev]) ( ) , and nipah/hendra viruses ( ) . several studies have suggested that bats can tolerate infection due to an enhanced innate immune response that enables early control of infection and prevents systemic dissemination ( ) ( ) ( ) ( ) ( ) ( ) . for example, three alpha interferon (ifn-␣) genes are constitutively expressed in black flying fox, pteropus alecto ( ) . other studies report that bats have a dampened host response, speculated to promote virus-host coexistence ( , ) . for example, the cgas-sting pathway is dampened in some bats species due to a mutation in sting ( ) and the inflammasome dna sensor aim is missing from almost all the known bat species ( ) . thus, there is a need for further investigation into the innate immune response in bats and how it impacts viral infection. double-stranded rna (dsrna)-induced innate immune responses play a critical role in limiting viral infection ( ) . one dsrna-induced and potent antiviral pathway is the oas-rnase l system, which has been well characterized in human cells and murine cells ( ) . the human oas family contains four members, three enzymatic active proteins (oas , oas , and oas ) and one oas-like (oasl) protein, lacking enzymatic activity ( ) . all three enzymatically active oass contain a core unit with dsrna binding and catalytic functions ( , ) . oas and oas duplicate one or two nonenzymatic units which are believed to enhance the binding affinity to dsrna. mice express homologous oas proteins that produce =- = oligoadenylates ( - a), including oas a/g, oas , and oas , as well as oasl and several catalytically inactive oas isoforms, oasl , and additional oas proteins ( , ) . after sensing dsrna, the catalytic domain of oass undergoes a conformational rearrangement to form the catalytic cavity and, from atp, synthesizes - a. - a binds to monomeric rnase l, leading to dimerization and activation to cleave viral and cellular single-stranded rnas, thereby blocking viral replication as well as protein synthesis ( ) . while all three oass (oas to - ) produce - a upon binding dsrna in vitro or when overexpressed ( ) , we showed previously, using a series of cells with oas gene knockouts (kos), that only oas was required for detectable activation of rnase l during infection of three human cell lines with diverse viruses ( ) . there is scant information in the scientific literature about activation or inhibition of the oas-rnase l pathway in bats and its contributions to bat innate immunity, although it was shown that oas-rnase l can be activated by poly(ri)·poly(rc) (pic) in p. alecto bat cells ( ) . we screened a group of bat cells (discussed further below) for activation of rnase l during sindbis virus (sinv) infection and chose to carry out further studies in egyptian rousette (er) bat (egyptian fruit bat)-derived roni/ cells. through bioinformatic analysis of the annotated genomic sequence of egyptian rousette bats from genbank ( ), we identified three oas genes (boas , boas , and boas ) and two oasl genes (boasl and boasl ). (we designate bat genes or proteins as boas to differentiate them from human [h] or mouse [m] genes or proteins.) we utilized crispr-cas gene-editing technology to generate boas-ko egyptian rousette-derived roni/ cells ( ) . we found that, as in human cells, the activation of rnase l is dependent on boas expression during infection with sinv. in addition, ko of boas or rnasel leads to greater than -fold more sinv replication, while ko of either boas or boas promotes more modest increases in replication. as in human cells, activation of rnase l by sinv is independent of mavs expression in bat roni/ cells, suggesting that rnase l may be activated either before or without virus-induced ifn. similar results were obtained with vaccinia virus, vacvΔe l, in bat roni/ cells. finally, our data indicate that the oas-rnase l pathway has a greater antiviral effect than mavs-dependent ifn signaling in roni cells as well as human cells. the bat genome contains three oas genes with interferon-stimulated response elements (isres) in the promoter region, two oasl genes, and one rnase l gene. using the genomic sequences of egyptian rousette bat from genbank, we analyzed the genes and protein sequences of bat oass, oasls, and rnase l ( ) . the genes were annotated as oas (genbank id ), oas -like (genbank id ), oasl (genbank id ), and oasl (genbank id ). no oas was noted. the egyptian rousette (er) boas gene encodes a protein that shares % and % amino acid sequence similarity to human oas and mouse oas , respectively ( table ) . the annotated er oas -like gene encodes a protein of , amino acids. amino acids to share % similarity with human oas ( , amino acids), and amino acids to share % similarity with human oas ( amino acids). these results suggested that the annotated er oas -like gene encoded an oas -oas fusion protein. since in most known species, oas and oas are two separate genes, we could not exclude the possibility that er oas and oas genes were falsely annotated as one gene. to clarify this question, we attempted to clone the mrna encoded by the boas and boas -like genes. we cloned the boas open reading frame (orf) successfully, but we could not clone the mrna predicted to encode a boas -boas fusion protein. instead, we cloned two cdnas which encode boas and boas open reading frames separately. these results suggested that er bats and humans share similar oas gene structures (fig. ). nd nd nd boas yes nd / nd nd nd boas yes nd nd / nd nd boasl no nd nd nd / boasl yes / nd nd / a protein catalytic activity was predicted by the conservation in the active site of the p-loop and aspartic acid triad. b amino acid consensus similarity. genbank protein accession numbers are shown in parentheses as follows: bat proteins, boas (xp_ ), boas (qct . ), boas (qct . ), boasl (xp_ ), and boasl (xp_ ); human proteins, hoas (np_ . ), hoas (np_ . ), hoas (np_ . ), and hoasl (q ); mouse proteins, moas a (p ), moas (e q a ), moas (q vi ), moasl (q vi ), and moasl (q z f ). vector nti was used to do the sequence alignment. c nd, not determined. human and mouse oas genes are induced by interferon (ifn) (i.e., they are interferon-stimulated genes [isgs]) and thus contain interferon-stimulated response elements (isres) in the promoter regions, required for interferon-inducible transcription. to determine whether er oas genes are isgs, we analyzed the genomic sequences for isres (a/gngaaanngaaact or agtttcnntttcnc/t) ( ) . we found that the er oas gene promoter region contains one isre with high sequence identity with that of human oas (fig. ) . upstream of the boas coding region, we found two adjacent isres in the opposite polarity, the first with homology to the one isre found in the human oas gene. the isre of er boas is identical to the first one of two found in the human oas gene. we did not find an isre in the promoter region of the er rnase l gene. boas , boas , and boas mrnas are induced by ifn-␣ or infection with sinv or sev in roni/ cells. roni/ cells were treated with human universal ifn-␣, the relative mrna levels were determined by quantitative reverse transcriptase pcr (qrt-pcr), and the fold increase in expression over mock-treated cells was calculated. induction of bat ifit (isg ) mrna was used as a positive control ( ) and was increased by -fold upon ifn-␣ treatment ( fig. a ). all three boas mrnas were induced by ifn-␣; the boas mrna level increased by -fold, while the mrna level of boas and boas increased by -and -fold, respectively ( fig. a) , consistent with the isres in their promoters. the mrna level of rnase l did not change upon ifn treatment in roni/ cells, indicating that, similarly to the human rnasel gene, bat rnasel is not an isg ( fig. a) , consistent with the lack of an isre in its promoter region (data not shown). to investigate induction of boas genes during viral infection, roni/ cells were infected with sinv or sendai virus (sev). while the relative mrna level of boas increased by -to -fold during sinv or sev infection, boas mrna was only weakly ( -to -fold) induced and boas mrna was induced somewhat more ( -or -fold) during sinv or sev infection, respectively ( fig. b and c), similar to induction by ifn-␣ ( fig. a to c) . amino acid sequence alignment indicates that the egyptian rousette boas , boas , boas , and boasl proteins are oligoadenylate synthetases with potential catalytic activity. the human and mouse oas genes and proteins have been extensively studied ( , ) . to investigate the bat oas gene family, we aligned the predicted amino acid sequences of bat, human, and mouse oas and oasl proteins. while boas protein sequences are highly homologous to human and mouse proteins, in each case boas , boas , and boas share more homology with human than with mouse proteins (table ) . we aligned the catalytic domain of each boas protein (full-length boas , domain ii of boas [boas . ], and domain iii of boas [boas . ]) with the corresponding protein or domains of human oas , oas , and oas (fig. ). based on sequence homology and previous studies of the structure of human ( ) and porcine ( ) oas , bat oas catalytic domains are composed of three parts, the n-terminal lobe (blue line in fig. ), the linker (red dotted line), and the c-terminal lobe (black line). three aspartic acid residues (asp , asp , and asp of hoas ) indicated with diamonds are essential for enzymatic function of oass and conserved among all the bat and human oass ( ) . the p-loop which contributes to the formation of the catalytic cavity is also conserved. the arg or lys residues (indicated by stars) participate in dsrna binding and are conserved among all bat or human oass. these results predict that boas , boas . , and boas . have the potential to recognize dsrna and synthesize - a. besides three oas genes, the egyptian rousette genome has two oas-like genes (oasl and oasl ). the boasl protein shares high sequence similarity with hoasl ( %) and moasl ( %), suggesting that, like these human and mouse proteins, it does not have oligoadenylate synthetase activity (table ; see also fig. s in the supplemental material). however, boasl is similar to moasl ( %) ( table ) and when aligned with human oas has a conserved catalytic domain, suggesting that it may have oligoadenylate synthetase activity (fig. s ) . egyptian rousette (er) rnase l shares high amino acid similarity to human rnase l with conserved - a binding sites and endoribonuclease catalytic sites. by sequence alignment, we found that brnase l shares % amino acid similarity with the human protein (fig. ). similar to human or porcine rnase l, brnase l contains three predicted domains, an n-terminal ankyrin-repeat domain (blue line in fig. ) , a pseudo-protein kinase domain (red dotted line), and an rnase domain (black line) ( ) . the amino acids which are responsible for - a binding (stars) and catalysis residues (diamonds) are conserved among bat and human rnase l sequences. protein expression levels of oas and oas in bat cells are upregulated by ifn treatment. we generated rabbit polyclonal antibodies against domain ii of boas (anti-boas ) or domains ii and iii of boas (anti-boas ). (attempts to raise antisera against boas were unsuccessful.) we used these antisera to determine both basal and induced oas protein expression levels in bat cells derived from several species of bats. activation of bat rnase l depends on oas but not mavs ® interestingly, antisera against either boas or boas detected both oas proteins. basal expression of oas was detected in roni/ cells and eptesicus fuscus skin fibroblasts, and the expression was upregulated upon ifn-␣ treatment in both cell types (fig. a ). while no to low basal oas expression was detected in myotis lucifugus skin fibroblasts, myotis lucifugus embryonic fibroblasts, and myotis velifer embryonic fibroblasts, the oas protein expression level became detectable upon ifn treatment (fig. a ). while basal oas protein expression was observed in eptesicus fuscus skin fibroblasts and weakly in myotis lucifugus skin fibroblasts, expression of boas was detected in all of these bat cells following ifn treatment (fig. a) . no oas -oas fusion protein was detected in these cells, consistent with our cdna cloning data. to assess activation of rnase l in these cells, we infected these bat cell lines with sinv; rnase l was activated in roni/ cells and eptesicus fuscus skin fibroblasts but not in other bat cells (fig. b) , likely because sinv failed to replicate. we next sought to determine whether the activation of rnase l in bat roni/ cells is dependent on expression of one or more oas genes. thus, we deployed the clustered regularly interspaced short palindromic repeat (crispr)-cas geneediting technology to ko expression of boas , boas , boas , and brnase l, each separately, from roni/ cells. the kos of boas and boas were confirmed by western blotting, while the disruptions of the oas and rnase l genes for which we lacked antibodies were confirmed by sequencing of the crispr-induced insertion (boas ) or deletion (rnase l) ( fig. a and b ). upon infection with sinv, degradation of rrna, as assessed by bioanalyzer, was detected in wild-type (wt) and boas -ko and boas -ko cells but not in boas -ko and brnase l-ko cells (fig. c) , indicating that the activation of rnase l during sinv infection in roni/ cells is dependent on boas expression, similar to our previous findings in human cells. we assessed viral replication in this set of ko cells at several time points postinfection. at and h postinfection (hpi), both brnase l-ko and boas -ko cells showed approximately -to -fold higher titers than the parental wt cells (fig. d ). viral titers from boas -ko and boas -ko cells were more modestly elevated than in wt cells at and h postinfection, -to -fold for boas -ko and -to -fold for boas -ko (fig. d ). when the second boas -ko and activation of bat rnase l depends on oas but not mavs ® boas -ko clones were infected with sinv, similar data were obtained for activation of rnase l by bioanalyzer assay (fig. s a ) and for virus replication ( fig. s b and c) . the second boas -ko clone examined displayed lower levels of virus replication than the clone originally used, closer to levels observed in wt cells (fig. ), but neither clone showed increased rnase l activation as assessed by bioanalyzer, compared to wt cells (fig. s ). it is important to note that while boas and boas mrna and protein expression levels are upregulated during sinv infection ( fig. a and fig. e ), this is not sufficient to activate rnase l in the absence of boas expression (fig. c) . to verify that oas -ko cells were competent to activate rnase l, we introduced flag-tagged human oas into boas -ko roni/ cells. (we used human oas rather than bat oas because they are about % homologous and we did not have a full-length boas clone assembled.) expression of ϫflag-hoas in boas -ko roni/ cells was verified by western blotting (fig. a) . wt, boas -ko, and ϫflag-hoas expressing cells were infected with sinv. degradation of rrna, as assessed by bioanalyzer, was detected in wt and ϫflag-hoas cells but, as expected, not in boas -ko cells (fig. b) , confirming that these cells are competent to activate rnase l when they express oas . we assessed viral replication in this set of cells at several time points postinfection. at , , and h postinfection, ϫflag-hoas -ko cells produced the same titers as parental wt cells, approximately -to -fold lower than boas -ko cells (fig. c) , confirming as expected that hoas expressed in boas -ko cells restores the antiviral effects of rnase l activation and thus restricts virus replication to the same extent as in wt cells. to determine to what extent activation of rnase l is dependent on ifn signaling, we investigated the role of mavs in oas-rnase l activation. thus, we used crispr/cas editing to generate bmavs-ko roni/ cells. the ko was validated by sequencing (fig. a ) in the roni/ mavs gene. (fig. b) , consistent with rnase l activation. while sinv replicated to more than -fold-higher titers in brnase l-ko cells than in the parental wt cells (fig. d and fig. c ), replication in bmavs-ko cells did not show significant differences at h postinfection and showed a slightly higher titer ( -fold) at h postinfection (fig. c) . similar data were obtained for activation of rnase l by rrna degradation assay by bioanalyzer (fig. s a) and for virus replication when a second bmavs-ko clone was infected with sinv (fig. s b) . while as expected ko of mavs from roni/ cells reduced boas and boas induction during sinv infection (fig. d) , there was no effect on the activation of rnase l. similar results were observed when we used human a cells. during sinv infection, rrna degradation was observed in mavs-ko a cells (fig. e ). in addition, no significant titer differences were observed at or h postinfection in mavs-ko a cells, while rnase l-ko cells showed -fold-higher titers than wt cells (fig. f) at h postinfection. rna was harvested from cells at h postinfection for rrna analysis. degradation of rrna, as assessed by bioanalyzer, was observed in the infected wt, boas -ko, boas -ko, and bmavs-ko cells but not in infected boas -ko or brnase l-ko cells (fig. a ). in addition, cells were lysed at h postinfection for determination of viral replication by plaque assays. consistent with the rrna degradation data, viral titers were -and -fold higher in the brnase l-ko or boas -ko cells, respectively, than in the boas , boas , bmavs, and wt cells (fig. b) . thus, activation of rnase l during vacvΔe l infection of bat roni/ cells was dependent on rnase l and oas but not oas , oas , or mavs, similar to our findings with sinv. since rna viruses are often lethal in humans but relatively apathogenic in the natural bat host, it is important to understand host-virus interactions and how these may differ among hosts. we have focused on understanding expression and activation of the dsrna-induced antiviral oas-rnase l system, a pathway our group has investigated in detail in human and murine cells, but which had not been characterized in the bat. when we analyzed the annotated egyptian rousette genomic sequences in genbank, we noted three genes, boas , boas , and boas , homologous to human oas genes, each with one or two isres in their promoter regions indicative of isgs. we cloned and sequenced the open reading frames (orfs) from the mrnas encoding boas , boas , and boas . our findings are in contrast to the annotated egyptian rousette genomic sequences, which indicate two boas genes, boas , which is predicted to encode boas , a homologue of human oas , and a boas -like gene which was predicted to encode a long oas -oas fusion protein. interestingly, oas -oas fusion genes and proteins are predicted also by annotated genbank sequences for two additional bat species, eptesicus fuscus and myotis brandtii, and three other nonbat species (donkey, ferret, and marmot). using antiserum to detect oas and oas expression from bat cells, we did not detect a fusion protein for any of the bat species. it is likely that the oas -oas fusion genes and resulting fusion protein predicted arise from annotation mistakes in genbank. in addition to the oas genes, two oasl genes encoding oasl and oasl proteins were detected from genomic sequencing. alignment of the boas and boasl amino acid sequences with human and mouse protein sequences showed that the bat proteins are more similar to human homologues than to mouse proteins (table and fig. ) . the crystal structure of human ( ) and porcine ( ) oas has been well studied, and essential amino acids which correspond to dsrna binding and - a production have been identified. all three boas proteins contain highly conserved dsrna binding amino acid residues as well as catalytic amino acids, suggesting that the bat oass have the ability to recognize dsrna and synthesize - a. bats encode boasl and boasl , with homology to mouse and human oas proteins. boasl shares high sequence similarity with mouse oasl (see fig. s in the supplemental material), suggesting that activation of bat rnase l depends on oas but not mavs ® like the mouse protein, boasl may have catalytic activity. in contrast, humans encode only one enzymatically inactive oasl protein. a recent study suggested that human oasl (enzymatically inactive) and mouse oasl (enzymatically active) suppress the function of cgas-sting pathways ( , ) . this is surprising since hoasl is more similar to mouse oasl than to mouse oasl (fig. s ). nevertheless, it will be interesting to determine if boasl or boasl has a similar function. xie et al. showed that a point mutation of sting from many bats species attenuates the function of sting ( ). thus, it is possible that bats may use two mechanisms to suppress the cgas-sting pathway to make the host become more tolerant of viral infection. analysis of the promoter region of the boas genes revealed isre sequences in all the three genes, consistent with our qpcr data, showing that each boas mrna is induced by ifn treatment and during viral infection ( fig. and ). bat and human oas genes have one similar isre, and oas mrna expression is moderately inducible by ifn or virus infection ( -to -fold) in bats (fig. ) , similar to our findings in human cells ( ) . interestingly, while the boas gene has two adjacent isres and boas mrna expression is induced only -to -fold (fig. ) , the human oas has one isre and yet the expression of oas mrna is highly induced ( , -fold) during infection of human cells ( ) . similarly, while boas has one isre, human oas has two isres, and oas mrna induction was about -to -fold in both bat and human cells with ifn. these findings suggest that the number of isres by itself does not have a major influence on the fold induction of oas gene expression, with the caveat that extent of gene induction will vary with basal expression levels in different cell types. interestingly, we detected basal oas protein expression in roni/ bat cells and basal oas and oas protein expression in eptesicus fuscus skin fibroblasts. it is important to note that we cannot directly compare basal expression levels of oas and oas among cells from the various species of bats because the antisera were raised against roni/ cell proteins and may preferentially react to the homologous proteins. since we were unable to detect boas protein, it is possible, albeit unlikely, that oas is not expressed in roni/ cells. however, it is more likely that the oas and oas antibodies just do not cross-react with oas . this is not surprising as boas shares lower sequence homology ( %) with the catalytic domains of boas and boas than they do with each other. importantly, further evidence that oas protein is expressed is that the oas gene is intact (fig. ) , the mrna is clearly expressed and induced by ifn or infection (fig. ) and contains an orf encoding a protein very similar to human oas with predicted active site intact (fig. ; table ), and oas -ko cells have increased sinv replication (fig. ) . in contrast to bat cells, we did not detect basal oas protein expression in any human cells in our previous studies (fig. a) ( , ) . while this may suggest that basal oas could be important in enhancing bat resistance to viruses, it could also be due to the different cell types examined and differences in the sensitivity of detection of bat and human oas proteins by western blotting. there are clearly many more unanswered questions about the regulation of boas gene expression. infection of a series of roni/ cells engineered using crispr/cas to ko each boas gene and brnase l showed that boas is essential for activation of rnase l during sinv or vacvΔe l infection as assessed by rrna degradation. (please note that oas is induced in boas -ko cells as well as in wt cells.) these data are similar to our previous findings in several human cell lines, with diverse viruses. this was due to a higher affinity of oas for long dsrna compared to the shorter oas and oas proteins ( , ) , which is likely the case for the bat homologues as well. also consistent with our previous findings in human cells, sinv replication is markedly increased ( -to -fold in the boas -ko cells as well as brnase l-ko cells compared to wt cells). interestingly, in boas -ko or boas -ko cells sinv also replicates to a higher level than in wt cells, although not to the extent observed in boas -ko or brnase l-ko cells, and rrna degradation was observed in cells lacking expression of either boas or boas . these data suggest that boas and boas may exert some antiviral activity by activating rnase l in the absence of boas . in human cells, we previously observed increased sinv replication in oas -ko cells at some time points. however, we observed rrna degradation in oas -ko cells, as well as accumulation of - a to the same extent as in wt human cells ( , ) , suggesting that this antiviral activity mediated by oas in human cells may be at least in part independent of rnase l activation. consistent with this observation, previous studies concluded that human oas has an antiviral function which is rnase l independent ( ) . however, during infection of roni/ cells with vacvΔe l, as we previously found in human a cells, degradation of rrna occurred to a similar extent in the absence of oas and oas as in wt cells and virus replicated to the same extent in oas -ko, oas -ko, and wt roni cells (fig. ) , suggesting that only oas played a role in antiviral activity. further investigation needs to be carried out to understand the individual roles of oas or oas with different viruses and specifically whether there may be oas-dependent antiviral activities independent of rnase l in bat or human cells. oas genes are isgs, and as such, it has been generally thought that activation of rnase l is a downstream effect of ifn signaling. in this model, viral dsrna would first need to be recognized by pattern recognition receptors (prrs), such as rig-i/mda , leading to ifn induction and subsequent upregulation of oass, which would then be activated by viral dsrna ( ) . however, we found previously that the oas-rnase l pathway can be activated in the absence of murine coronavirus-induced ifn in mouse bone marrow-derived macrophages ( ) . here, we show that activation of rnase l during infections with either sinv (fig. ) or vacvΔe l (fig. ) is independent of mavs expression in bat roni/ cells, similar to findings for sinv in human a cells, indicating that basal oas levels which are weakly induced in the absence of mavs (fig. d) are sufficient for activation of rnase l. we recently reported similar results during zika virus infection of a cells ( ) . in addition, mavs plays little if any role in limitation of sinv replication in bat or human cells (fig. b and e) or of vacvΔe l in bat cells (fig. b ). this is in contrast to rnase l, which, as discussed above, dramatically limits viral replication (fig. , , and ) ( ) . these results imply that the oas-rnase l pathway plays a significant role in limiting viral infection before ifn induction or in the absence of ifn by a virus that antagonizes ifn induction or signaling. indeed, a recent study showed robust rnase l activation as early as h after pic transfection in human a cells ( ) . thus, whereas ifn induction of oass may enhance the activation of the pathway, it is not always required. these studies suggest that oass act in parallel to other prrs, including rig-i-like receptors, and the oas-rnase l system is a separate pathway rather than a secondary pathway of ifn induction/signaling. importantly, oas-rnase l is a potent antiviral pathway, activated by infection in bat roni/ cells. egyptian rousette (rousettus aegyptiacus) kidney-derived roni/ cells ( ) ( ) ( ) were obtained from marcel müller (charité-universitätsmedizin, berlin, germany), and myotis lucifugus skin fibroblasts ( ) , myotis lucifugus embryonic fibroblasts, myotis velifer embryonic fibroblasts, and eptesicus fuscus skin fibroblasts ( ) were obtained from cedric feschotte (cornell university). the cells were cultured in dulbecco's modified eagle's medium (dmem; gibco catalog no. ) supplemented with nonessential amino acids, % fetal bovine serum (fbs), u/ml of penicillin, and mg/ml streptomycin. african green monkey kidney vero cells (atcc ccl ) were cultured in dmem (gibco ), supplemented with % fbs, mm hepes, mm sodium pyruvate, u/ml of penicillin, mg/ml streptomycin, and g/ml gentamicin. baby hamster kidney (bhk- ) cells were cultured in dmem supplemented with % fbs, % tryptose phosphate broth (sigma-aldrich), u/ml penicillin, and mg/ml streptomycin. human hek t cells were cultured in dmem supplemented with % fbs and mm sodium pyruvate. human a cells were cultured in rpmi medium (gibco) supplemented with % fetal bovine serum (fbs), u/ml of penicillin, and mg/ml streptomycin. human hek t and a cells have been authenticated by the atcc. the roni/ cells were authenticated by cloning and sequencing of oas and rnase l mrnas, which matched the sequences derived from the egyptian rousette (rousettus aegyptiacus) bat genes deposited in genbank. rnase l or mavs-ko a cells were generated by crispr/cas technology and were described in previous studies ( , ) . sindbis virus girdwood g (sinv), expressing mcherry, was obtained from mark heise (university of north carolina, chapel hill) and was prepared in bhk cells as previously described ( ) . sendai virus (sev) strain cantell ( ) was obtained from carolina b. lopez (university of pennsylvania). mutant vaccinia virus (vacvΔe l) was obtained from bertram jacobs (arizona state university, tempe, az) and was grown as described previously ( ) . cloning and sequencing of bat oas cdnas. boas , boas , and boas cdnas were cloned from roni/ cells. briefly, the cells were treated with , u of ifn-␣ overnight, and total cellular rna was extracted using an rneasy plus mini kit (qiagen). cdna was synthesized by reverse transcription using superscript iii (invitrogen) and mrna-specific reverse primers. boas -rev and boas -rev (table ) primers were used for reverse transcription to synthesize boas and boas cdna, respectively. three cdna fragments were cloned to assemble the full length of the boas open reading frame. boas -f rev, boas -f -rev, and boas -f -rev (table ) were used for reverse transcription reactions to synthesize three cdna fragments (boas -f , boas -f , and boas -f , respectively). the dna fragments of boas , boas , boas -f , and boas -f were amplified by using accuprime dna polymerase (invitrogen) with the forward primers and reverse primers (see table s in the supplemental material), and were cloned into pcr . topo vectors (invitrogen). the dna fragment of boas -f was amplified by using q dna polymerase (neb) and was cloned into pcr ii blunt-topo vectors (invitrogen). the cloned dnas were sequenced and analyzed. the oas sequence matched the predicted mrna sequence in genbank (id ). construction of plasmids and pseudolentivirus. the oligonucleotide sequences to be used for generation of small guide rnas (sgrnas) for kos of boas , boas , boas , brnase l, and bmavs genes are shown in table s . a pair of forward and reverse oligonucleotides for generation of each sgrna (synthesized by idt) were annealed by published methods ( ) and were inserted into plenti-crispr (addgene) between bsmbi restriction sites. the resulting plasmids are named plenti-sgbo (targeting the boas gene), plenti-sgbo (targeting the boas gene), plenti-sgbo (targeting the boas gene), plenti-sgbrl (targeting the brnase l gene), and plenti-sgbma (targeting the bmavs gene). for packaging of pseudolentiviruses, ϫ hek t cells were plated in one well of a -well plate, and the next day were transfected with g plenti-crispr (with sgrna), . g pspax , and . g of pcmv-vsv-g (obtained from paul bates, university of pennsylvania) using lipofectamine (invitrogen) ( l in l of dmem). the supernatants were harvested at and h posttransfection and stored at Ϫ °c, and the -h supernatants were used for further ko experiments. construction of boas , boas , boas , brnase l, and bmavs gene knockout roni/ cells. for construction of roni/ ko cells using lenti-crispr, ϫ cells were plated into one well of a -well plate and were transduced with l of pseudolentiviruses. forty-eight hours postransduction, cells were cultured in medium containing . g/ml of puromycin for days. the resistant cells were further cultured for week in medium without puromycin before being cloned by limited dilution. briefly, cells were diluted to cells/ml and l of cells was added to one well of -well plates. single cells were selected for further amplification and genotyping. genomic dna was extracted using a dneasy blood and tissue kit (qiagen). dna fragments covering the region targeted by sgrna were amplified by pcr using gene-specific primers (table s ) mixed with gotaq master mix (promega), and the pcr products were sequenced. cells with frameshift mutations (deletion or insertion) in targeted genes were selected for further experiments. generation of ؋flag-hoas knock-in cells. boas -ko cells were electroporated (btx) with plasmid pcmv - xflag-hoas ( ) and plated without antibiotics. at h postelectroporation, cells were selected with g/ml of g (gibco) for weeks and then cloned by limiting dilution. clones were screened by western immunoblotting using anti-flag m antibodies. cell clones expressing ϫflag-hoas were maintained in medium containing g/ml of g . antibody. rabbit anti-boas and -boas antibodies were generated by immunizing rabbits with purified mbp-boas -d and mbp-boas -f / . briefly, boas domain (catalytic domain) was amplified by pcr using primers boas -d -for (acgtcgactacccccgggcatcttctggataaattc) and boas -d rev (gctctagatcactcgaggagcccccaacttctgaac), and boas fragment f -f was amplified by pcr using primers boas -f / -for (acgtcgactgatctgtctcagatccccgccaatgag) and boas -f / rev (gccaagctttcagacgtgtccaaggggagggaccacatg). the fragments were cloned into pmal (neb) vectors between sali and xbai restriction sites. pmal-boas -d and pmal-boas -f / were transformed into neb-express competent escherichia coli, and iptg was added to the bacterial cultures to induce expression of the proteins. the proteins were purified by elution from the protein bands cut from polyacrylamide gels with the pierce zinc reversible stain kit (thermo fisher). the eluted proteins were concentrated and showed a single band on a polyacrylamide gel by staining. rabbits were injected with g protein each times over about months before bleeding. mouse anti-gapdh ga r ( : , ; thermo fisher) was used to detect bat gapdh. mouse anti-flag m antibodies ( : , ; sigma-aldrich) were used to detect ϫflag-hoas . secondary antibodies goat anti-mouse antibody ( : , ; santa cruz) and goat anti-rabbit antibody ( : , ; cell signaling), conjugated to horseradish peroxidase (hrp), were used to detect mouse-or rabbit-derived primary antibodies. western blotting. confluent cells in -well plates were treated or mock treated with , u of ifn-␣ overnight or infected with sinv at an moi of at h postinfection. cells were harvested, washed in pbs, and lysed with np- buffer with protease inhibitor cocktail (roche). cell lysates were mixed with ϫ laemmli buffer, boiled at °c for min, and analyzed by electrophoresis on to % or to % gradient sds gels. proteins were transferred to polyvinylidene difluoride membranes, which were treated with % nonfat milk in tbst (tris-hcl-buffered saline with . % tween ) blocking buffer for h, followed by incubation overnight at °c with antibodies diluted into tbst. membranes were then washed three times with tbst, incubated with secondary antibodies for h at room temperature, again washed three times with tbst, and then incubated with supersignal west dura extended-duration substrate (thermo fisher), and the signal was detected using an amersham imager (ge). virus growth kinetics. cells were plated in -well (roni/ ) or -well (a ) plates, ϫ cells per well. the next day, cells were infected with the indicated viruses at an moi of , three parallel wells per virus. at , , and h postinfection, l of supernatant was harvested and stored at Ϫ °c for titration. plaque assays. sinv was diluted in dmem, and l was added to confluent vero monolayers in -well plates. the plates were incubated for h at °c and were rocked at -min intervals. cells were then overlaid with ml warm dmem containing % fbs and . % agar. vaccinia virus (vacvΔe l) titers were determined in bhk- cells as described previously ( ) . rrna cleavage assay. cells were infected with sinv at an moi of (bat cells) or an moi of (a cells). at (bat cells) or (a cells) h postinfection, cells were harvested in rlt buffer (rneasy minikit; qiagen). bat cells were infected with vacvΔe l at an moi of and at h postinfection were harvested in rlt buffer (bio basic, inc.). total rna was extracted and was resolved on rna chips using an agilent bioanalyzer ( ) . mrna quantification by quantitative reverse transcriptase pcr (qrt-pcr). cells were infected with sinv (moi ϭ ) or sev (moi ϭ ) or treated with , u of ifn-␣ in triplicate in -well plates. cells were lysed at (sinv) or (sev) h postinfection (hpi) or h after ifn treatment in rlt plus rna lysis buffer (qiagen), and rna was isolated using the rneasy plus mini kit (bio basic, inc.) as previously described ( ) . cycle threshold (c t ) values were normalized to ␤-actin. oas mrna levels were calculated relative to ␤-actin mrna and expressed as fold over mock-treated or mock-infected with the formula ϪΔ(Δct) (Δc t ϭ c tgene of interest Ϫ c tactin ). primer sequences are listed in table s in the supplemental material. software and statistical analysis. sequences were analyzed by vector nti. sequence alignment figures were created by vector nti. all other figures were created by graphpad. all analyses were performed in graphpad prism version . a. plaque assay data were analyzed by two-way anova. significance is shown as follows: ns, not significant; *, p Ͻ . ; **, p Ͻ . ; ***, p Ͻ . ; ****, p Ͻ . . data availability. the nucleotide sequences of boas and oas orfs were deposited in genbank with accession numbers mk and mk , respectively. supplemental material for this article may be found at https://doi.org/ . /mbio . - . oral shedding of marburg virus in experimentally infected egyptian fruit bats (rousettus aegyptiacus) experimental inoculation of egyptian fruit bats (rousettus aegyptiacus) with ebola virus virological and serological findings in rousettus aegyptiacus experimentally inoculated with vero cells-adapted hogan strain of marburg virus human betacoronavirus c emc/ -related viruses in bats, ghana 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oligoadenylate-synthetase-family protein oasl inhibits activity of the dna sensor cgas during dna virus infection to limit interferon production viruses in bats and potential spillover to animals and humans replication defective viral genomes exploit a cellular pro-survival mechanism to establish paramyxovirus persistence extracellular =- = oligoadenylate synthetase stimulates rnase l-independent antiviral activity: a novel mechanism of virus-induced innate immunity interferon-inducible antiviral effectors activation of rnase l by murine coronavirus in myeloid cells is dependent on basal oas gene expression and independent of virus-induced interferon zika virus production is resistant to rnase l antiviral activity real-time - a kinetics suggest that interferons beta and lambda evade global arrest of translation by rnase l comparative analysis of ebola virus glycoprotein interactions with human and bat cells differential sensitivity of bat cells to infection by enveloped rna viruses: coronaviruses, paramyxoviruses, filoviruses, and influenza viruses establishment of fruit bat cells (rousettus aegyptiacus) as a model system for the investigation of filoviral infection comparative biology of mammalian telomeres: hypotheses on ancestral states and the roles of telomeres in longevity determination identification of adult mouse neurovirulence determinants of the sindbis virus strain ar the ebola virus vp protein inhibits activation of interferon regulatory factor blockade of interferon induction and action by the e l double-stranded rna binding proteins of vaccinia virus genome engineering using the crispr-cas system antagonism of the interferon-induced oas-rnase l pathway by murine coronavirus ns protein is required for virus replication and liver pathology research reported in this publication was supported by the national institute of allergy and infectious diseases of the national institutes of health (nih) under award r ai to s.r.w. and r.h.s. and r ai to s.r.w. z.w. was supported in part by the chinese scholarship council.we thank marcel muller for roni/ cells and cedric feschotte for myotis lucifugus skin fibroblasts and eptesicus fuscus skin fibroblasts (originally from woody wright, university of texas southwestern), myotis lucifugus embryonic fibroblasts (originally from mario capecchi, university of utah), and myotis velifer embryonic fibroblasts (originally from david ray, texas tech university). we also thank cedric feschotte and rachel cosby, as well as ravi sachidanandam, for bat transcriptome analysis. we also thank melanie hoffner and earl poptic in the hybridoma core (lerner research institute) for making the rabbit anti-bat oas polyclonal antibodies and christina gaughan for expert technical assistance. key: cord- - gv yfh authors: cho, hae-wol title: enemy at the gate date: - - journal: osong public health res perspect doi: . /j.phrp. . . . sha: doc_id: cord_uid: gv yfh nan individuals at a higher risk of transmitting wfb communicable diseases as they may have come from areas where the population is living in a high-density, poor, social environment. in addition, medical care may be minimal, and access to safe drinking water and clean food may be limited. providing improved essential information on good personal hygiene practices to international travelers from/to korea maybe a simple and effective measure for minimizing the possibility of spreading wfb communicable diseases. world health organization. guidelines for drinking-water quality world health organization [internet]. foodborne diseases foodborne and waterborne diseases joint external evaluation of ihr cope capacities of the republic of korea mission report risk of water and food-borne communicable diseases in travelers entering korea key: cord- -rmxin da authors: de clercq, erik title: new nucleoside analogues for the treatment of hemorrhagic fever virus infections date: - - journal: chem asian j doi: . /asia. sha: doc_id: cord_uid: rmxin da eight different compounds, all nucleoside analogues, could presently be considered as potential drug candidates for the treatment of ebola virus (ebov) and/or other hemorrhagic fever virus (hfv) infections. they can be considered as either (i) adenine analogues ( ‐deazaneplanocin a, galidesivir, gs‐ and remdesivir) or (ii) guanine analogues containing the carboxamide entity (ribavirin, eicar, pyrazofurin and favipiravir). all eight owe their mechanism of action to hydrogen bonded base pairing with either (i) uracil or (ii) cytosine. four out of the eight compounds (galidesivir, gs‐ , remdesivir and pyrazofurin) are c‐nucleosides, and two of them (gs‐ , remdesivir) also contain a phosphoramidate part. the c‐nucleoside and phosphoramidate (and for the adenine analogues the ′‐cyano group as well) may be considered as essential attributes for their antiviral activity. abstract: eight differentc ompounds, all nucleoside analogues, could presently be considered as potential drug candidates for the treatment of ebola virus (ebov) and/oro ther hemorrhagic fever virus (hfv) infections.t hey can be considered as either (i)adenine analogues ( -deazaneplanocin a, galidesivir,g s- andr emdesivir) or (ii)guanine analogues containing the carboxamide entity (ribavirin, eicar, pyrazofurin and favipiravir). all eight owe their mechanism of action to hydrogen bondedb ase pairing with either (i)uracil or (ii)cytosine. four out of the eight compounds (galidesivir, gs- , remdesivir and pyrazofurin) are c-nucleosides,a nd two of them (gs- , remdesivir) also contain ap hosphoramidate part. the c-nucleoside and phosphoramidate (and for the adenine analogues the '-cyano group as well) may be considered as essential attributes for their antiviral activity. the hemorrhagic fever virus (hfv) infections, including lassa and ebola, that we highlighted as potential targets for antiviral agents in [ ] have essentially remained unchanged,a nd now, years later,w eare still awaiting the first antiviral drug(s)t ob ef ormally approved for the treatment of hfv infections.t he first antiviral compound ever to be reported to be effective in the treatment of any hfv infection, that is, lassa, that could be used at any point in the illness as well as for postexposure prophylaxis, was ribavirin. [ ] ribavirin (virazole, -b-d-ribofuranosyl- , , -triazole- -carboxamide) ( figure ) was the first synthetic nucleoside analogue ever reported (in ) to be active against ab road spectrumo fb oth rna and dna viruses. [ ] in their final remarks, sidwell et al. ( ) stated, rightfully that "the antiviral spectrum of virazole was the broadest ever reported for any syntheticm aterial that does not induce interferon", although the eventual clinicalu se of virazole would eventually be limited to rna virus infections. [ ] one of the structural analogues mentioned in the latter article wase icar ( - eicar had originally been reported as an antileukemic agent in mice. [ ] in its antiviral activity,i ts howed as imilar spectrum as ribavirin, being active against pox-, toga-, arena-, reo-, orthomyxo-and paramyxovirus infections, with ap otency that was -to -fold greater than that of ribavirin. [ ] as originally ascertained for ribavirin, [ ] eicar also provedt ob eapotent inhibitor of imp dehydrogenase, thus blocking the biosynthesis of gmp. [ ] the depletion of the intracellular gtp and dgtp pools, resulting from the decreased biosynthesis of gmp,m ay also explain variouso ther effects of eicar, such as its potentiating effect on the anti-hiv activity of ddi ( ', '-dideoxyinosine) [ ] and its inhibitory effects on the replication of av ariety of viruses such as the double-stranded rna virus, ipnv (infectious pancreatic necrosis virus). [ , ] in the latter publication, yet another antiviralc ompound, pyrazofurin, was mentioned to be as pecific inhibitor or ipnv. [ ] pyrazofurin [ -(b-d-ribofuranosyl)- -hydroxypyrazole- -carboxamide] (figure ) is an inhibitor of omp decarboxylase, a key enzyme in the de novo pyrimidine mononucleotide biosynthesis:i th as provent ob ea ctivea gainst both (+ +)rna viruses [picorna (polio, coxsackieb ), toga (sindbis) and flavi (yellow fever)] and (À)rna viruses [paramyxo( measles, rsv), orthomyxo (influenza), rhabdo( vsv) and arena (junin, tacaribe)]. [ , ] pyrazofurinh as been found to be an exquisitely potent inhibitor of vsv (vesicular stomatitis virus), which belongs to af amily (rhabdoviridae), closely relatedt ot he family of the filoviridae to which ebola virus (ebov) and marburg virus belong. vsv can be handled in conventionals afety conditions, whereas ebov and marburgv irus require biosafety level ; vsv could therefore be recommended as ap aradigm for predicting antiviral activity against ebov. [ ] pyrazofurinh as so far not been evaluated for its potentiala ctivity against ebov;i t should be done so, as has already been done with the neplanocin aa nalogues. these analogues are knownt ob et argeted at the s-adenosylhomocysteine hydrolase. [ ] the prototype of this class of compounds is -deazaneplanocina( figure ). two decades ago, -deazaneplanocin aw as shownt ob ee ffective against al ethal ebov infection in mice. [ , ] the compound induced massively increased interferon-a production in ebov-infected mice, [ ] as tartling observation that was never followed up. when reviewing in possible therapeutics trategiest o block ebov infections, im entioned -deazaneplanocin a, besides bcx andt - (favipiravir). [ ] later added to the list were gs- (remdesivir) and zmapp. [ ] the response to zmapp, am ixture of three monoclonal antibodies directed against the surfaceg lycoprotein of ebov,w as beneficial but did not meet the prespecified statistical threshold for efficacy [death occurred in of patients ( %) who received the current standardo fc are alone as compared to o f p atients ( %) who received the current standard of care plus zmapp]. [ ] apparently the outbreak of ebola in - ended before any incontrovertible evidenceo fa ny intervention could be assessed. in this review iw ill focus on the potentialo ff avipiravir (t- ), bcx (galidesivir) and gs- (remdesivir) on the treatment of ebov and other hfv infections. the imino-c-nucleoside bcx (galidesivir)w as first reported by warrene tal. [ ] to be activea gainst aw ide range of viruses, including filo-, toga-, bunya-, arena-, paramyxo, corona-, flavi-, orthomyxo-andp icornaviruses. it was found to completely protectc ynomolgus macaques from marburg virus infection when administered as late as hours after infection. [ ] it effectively blocked yellow fever virus infection in ah amster model. [ ] it is now under development for the treatmento f ebov infection. [ ] bcx (galidesivir) (figure ) has also been found effective against zika virus in cell culture and in a lethal mousem odel. [ ] antivirala ctivity of bcx has also been reported against west nile virus, at ypical mosquitotransmitted flavivirus, and against tick-bornef laviviruses, kyasanur forest disease virus (kfdv). [ ] it inhibits infection of rift valley fever virus (rvfv), am osquito-borne pathogen that causes severe disease in humansa nd livestock in sub-saharan africa and the arabian peninsula, in syrian golden hamsters. [ ] the phosphoramidatep rodrug of the pyrrolo[ , -f][triazin- amino]a denine c-nucleoside gs- (remdesivir) ( figure ) was first reported to protect %o fe bov-infected rhesus monkeys against al ethal ebov infection. [ ] it wasa lso found active against other emerging viruses such as respiratory syncytial virus (rsv) and hepatitis cv irus (hcv),a nd the presence of the '-cyano group in remdesivir wasf ound to be critical in providing selectivity toward the viral (rna) polymerases. [ ] gs- was then found to be highly inhibitory to various viruses other than filo, that is, pneumo-, paramyxo-and coronaviruses. [ ] that gs- inhibited both epidemic and zoonotic coronaviruses, and might prove effective against emerging coronaviruses in the future was emphasized by sheahane tal. [ ] the viral rna polymerase and the proofreading exoribonuclease were suggested as being responsible fort he coronavirus susceptibility to remdesivir. [ ] the viral rna polymerase has also been identified as the targete nzyme of paramyxoviruses (i.e. nipah virus) for the antiviral activity of gs- . [ ] for both ebov rna-dependentr na polymerase (rdrp) and rsv rdrp, chain termination was delayeda nd predominantly seen at position i + . [ ] the first newborn baby to have survived congenital ebov infection, received remdesivir in addition to zmapp and ab uffy coat transfusion from an ebola survivor. [ ] favipiravir (t- ) ( figure ) is ap yrazine derivative, that is, -fluoro- hydroxy- -pyrazinecarboxamide, sharing ac ommon structural feature, that is ac arboxamide entity,w ith ribavirin, eicar and pyrazofurin, andw hich is also an essential (hydrogen bonding) part of guanine( -hn -c o-). [ , ] as already mentioned, ribavirin and eicar would primarily owe their antiviral activity to interference with the imp dehydrogenase, ak ey enzyme in the biosynthesis of gmp and gtp.y et, eriksson et al. [ ] reported inhibition of influenzav irus rna polymerase by ribavirint riphosphate,a nd when furuta et al. [ ] had revealed the in vitro and in vivo activities of t- against influenza virus, they attributed the mode of action of t- to as pecific inhibition of the influenzavirus rna polymerase by the t- rtp (ribofuranosyl triphosphate. [ ] to this end (scheme ), t- had first to be converted to its ribofuranosyl monophosphate( rmp) by a phosphoribosylt ransferase, and then further processed by kinase(s) to its diphosphate( rdp) and triphosphate (rtp), before the latter would interact in ag tp-competitive manner with the viral rna synthesis. that t- (favipiravir), as av iral rna polymerase inhibitor, offers great potential in the treatment of aw idev ariety of rna virus infections, has been reviewed repeatedly. [ ] [ ] [ ] [ ] sidwell et al. [ ] demonstrated that t- inhibited al ethal avian influenza a( h n )v irus infectioni nm ice. this was confirmed by kiso et al.. [ ] it was ascertainedt hat in the influenzaa(h n ) virus-infected cells t- was metabolized to t- rtp. [ ] the latter is incorporated into the nascent rna strand as ap urine nucleotide analog (reminiscent of gmp) and inhibits strand extension. [ ] the rtp of t- acts as ag tp-competitive inhibitor of the influenza viral rna polymerase. [ ] favipiravir inhibits in vitro replication of aw ide range of influenzav iruses, including those resistantt oc urrently available drugs [ ] andt hose isolated from patients who had previously received favipiravir. [ ] favipiravir has been approved, as avigan, in japan for the treatment of influenza. [ ] lethal mutagenesis hasb een proposed to be ak ey mechanism in the activity of t- (favipiravir) againsti nfluenzaa (h n )v iruses in vitro. [ ] lethalm utagenesis has also been suggested for the activity of favipiravir against norovirus repli-cation [ ] and hepatitis cv irus. [ ] such mechanism of antiviral activity was originally proposed for ribavirin. [ , ] however,t he activity of ribavirin against yellow fever virus could not be explained by an error-prone mechanism. [ ] favipiravir has provene ffective againstv irtually all hemorrhagic fever virus infections:a rena-and bunyaviridae, [ ] pichinde virus (an arenavirus), [ , ] lassa fever virus, [ ] yellow fever virus, [ ] western equine encephalitis virus, [ ] west nile virus, [ ] punta to ro, ap hlebovirus, [ ] the hantavirus maporal, [ ] rift valley fever virus [phlebovirus (bunyaviridae)], [ ] crimean-congo hemorrhagic fever virus [nairovirus (bunyaviridae)], [ ] severef ever with thrombocytopenia syndrome virus (sftsv), ar ecently identified emerging virus, [ ] and norovirus (caliciviridae). [ , ] resistance to t- has never been reported, except for chikungunya virus (alphaviridae) which acquired resistance due to the k r mutation in the rna-dependentr na polymerase (rdrp). [ ] favipiravir proveds uccessful in the treatment of ebov infection in mice. [ , ] in nonhuman primates, treated intravenously with favipiravir,f ive of six animals ( %) survived al ethal marburg virus infection. [ ] in patients with the ebov infection in www.chemasianj.org sierra leone, favipiravir was found to increase the survival rate from . %( / ) to . %( / ). [ ] another study carried out in guinea ended with the statement that favipiravir monotherapy merits furthers tudy in patients with mediumt oh igh viremia but not in those with very high viremia. [ ] guedj et al. [ ] concluded that favipiravir mayh ave ap otentialr ole at high doses in the treatment of ebov infections in humans. ta king into account that rhabdo-and filoviruses are closely related,i ti sn ot surprising that favipiravir has also been advocated for the postexposure prophylaxis of rabiesvirus, as ap otential alternative to rabies immunoglobulin. [ ] the compounds described here can be considered as either adenine derivatives ( -deazaneplanocin a, galidesivir,r emdesivir or guanine derivatives (ribavirin), eicar, pyrazofurin,f avipiravir). this is immediatelyo bvious for the adenine derivatives, which can be expected to interferew ith viral rna synthesis at the level of the rdrp (rna-dependentr na polymerase), where they can serve as competitive inhibitors with respectt o atp( based on the hydrogen bonds formed between adenine and uracil) ( figure ). in addition, the adenine analogues can also serve as s-adenosylhomocysteine (sah) hydrolase inhibitors, thus interfering with the s-adenosylmethionine (sam)-dependentmethylationr eactions. that ribavirin, eicar, pyrazofurin and favipiravir would be able to act as guanined erivatives thus competing with gtp at the rdrp level is less obvious. yet, all c ompounds share a carboxamide group, reminiscent of the carboxamide (-c ( ) o-n ( ) h-) part of guanine, which could explain the hydrogen bondingw ith cytosine ( figure ). in addition to their action at the rdrp level, the carboxamide-containing derivatives could also interfere with the imp dehydrogenase activity,t hus suppressing the biosynthesis of gtp,a sh as been specifically demonstrated for ribavirin and eicar. the exact mechanism of action of favipiravir remains to be assessed;i tm ay well vary from one virus to another.a sf ar as the activity of favipiravir rtp against influenzaavirus polymerase is concerned, this has been ascribed to "ambiguous base-pairing". [ ] in , ip roposed that "c-nucleosidess hould be revisited". [ ] this proposal was prompted by the advent of two new c-nucleosides, bcx (figure ), which has in the meantime, been further pursued (as galidesivir) for the treatment of ebov infections (see supra), and gs- (compound , containing a '-c-methyl group imparting specific activity against hepatitis cv irus (gcv). [ ] gs- (figure ) also contained a '-cyano group, which later on was found to be critical in providing selectivity towardv iral (rna) polymerases. [ ] gs- has only been reported for its potentiala sa na nti-hcv agent. [ ] [ ] [ ] it has not been thoroughly explored for its potential activity against ebov or any other hemorrhagic fever virus (hfv) infections. according to literatured ata, [ ] it would only have weaka ctivity against ebov. remdesivir (gs- ) is at present aleadingd rug candidate for the treatment of ebov infections. its chemical attributes are that it is ac -nucleoside, extendedb yaphosphoramidate and equippedw ith ac ng roup. galidesivir (bcx ) is also acnucleoside, but what would happeni fi tw ould also contain a cn group and would be converted to ap hosphoramidate prodrug?f or favipiravir,i ts n-nucleoside t- is more effective than the free -pyrazinecarboxamide against yellow fever virus infection in hamsters, [ ] but less so against punta to ro virus in mice. [ ] what would happen if t- would be convertedt o its c-nucleoside, and, furthermore, extended by ap hosphora- www.chemasianj.org wiley-vch verlag gmbh &co. kgaa, weinheim midate entity?a lso, the forgotten c-nucleoside, pyrazofurin, [ ] could be converted to its phosphoramidate, and eicar, which containsa ne thynyl, reminiscent of the cyano group, could be first transformed to its c-nucleoside before being converted to its phosphoramidate (scheme ). any of these chemical interventions may provide potential clues in the design of new medicines against ebovand other hfv infections. the (candidate) antiviral compoundsc urrently availablef or the treatment of ebov and other hfv infections are -deazaneplanocin a, galidesivir,g s- , remdesivir,r ibavirin, eicar, pyrazofurin andf avipiravir.they are boxed in (scheme ). in attempts to increase their efficacy and potentially lower their toxicity, they could, where applicable, first be converted to their c-nucleoside, and, subsequently,t ot heir phosphoramidate (scheme ). for gs- and remdesivir,w hich,i na ddition, also contain ac yanogroup, these manipulations have already been achieved. the authordeclares no conflict of interest. keywords: antivirals · ebola · hemorrhagic fever viruses · nucleoside analogues proc. natl. acad.sci.u sa antiviral res currentc hemotherapy:p roceedings of the th international congress of chemotherapy proc. jpn. acad. ser.b proc. natl. acad.s ci proc. natl. acad. sci revised manuscript received key: cord- -tluc ck authors: kuiken, thijs; breitbart, mya; beer, martin; grund, christian; höper, dirk; van den hoogen, bernadette; kerkhoffs, jean-louis h; kroes, aloys c m; rosario, karyna; van run, peter; schwarz, matthias; svraka, sanela; teifke, jens; koopmans, marion title: zoonotic infection with pigeon paramyxovirus type linked to fatal pneumonia date: - - journal: j infect dis doi: . /infdis/jiy sha: doc_id: cord_uid: tluc ck the characteristics and risk factors of pigeon paramyxovirus type (ppmv- ) infection in humans are poorly known. we performed virological, pathological, and epidemiological analyses of a dutch case, and compared the results with those of a us case. both infections occurred in transplant patients under immunosuppressive therapy and caused fatal respiratory failure. both virus isolates clustered with ppmv- , which has pigeons and doves as reservoir. experimentally inoculated pigeons became infected and transmitted the virus to naive pigeons. both patients were likely infected by contact with infected pigeons or doves. given the large populations of feral pigeons with ppmv- infection in cities, increasing urbanization, and a higher proportion of immunocompromised individuals, the risk of severe human ppmv- infections may increase. we recommend testing for avian paramyxovirus type , including ppmv- , in respiratory disease cases where common respiratory pathogens cannot be identified. changes in socioeconomic, environmental, and ecological factors in recent decades are probably responsible for the significant increase of emerging infectious diseases, the majority of which are zoonotic [ ] . of particular concern are emerging respiratory viruses of the families orthomyxoviridae [ , ] , coronaviridae [ , ] , and paramyxoviridae [ , ] . the use of novel molecular techniques has made it possible to identify previously unsuspected or unknown viruses [ ] . however, it is important to assess the clinical and public health relevance of these viruses by determining their origin and establishing their impact in the human population. the impetus for the current study was the identification of a virus related to avian paramyxovirus type (apmv- ) from a fatal human case of unknown cause in the netherlands by viral metagenomics analysis [ ] . apmv- is classified in the genus avulavirus, family paramyxoviridae [ ] . infection with apmv- is mainly restricted to birds, but occasionally causes mild disease-usually transient conjunctivitis-in humans [ ] . so far, there is only one case report, from new york state, of a person who died with apmv- infection [ ] . in this study, we fully characterized the dutch clinical isolate of apmv- -like virus, determined its phylogenetic relationship to other apmv- strains, and correlated presence of this virus with lesions in tissues obtained from the patient at autopsy. we also examined virulence and excretion pattern of this virus in chickens and domestic pigeons. a -year-old woman with a history of multiple myeloma received an allogenic bone marrow transplant in september , following preparation with a nonmyeloablative regimen (cyclosporin) and while on antifungal therapy (intravenous itraconazole). she initially did well, but was hospitalized weeks after transplantation with symptoms of malaise, anorexia, fever, and abdominal complaints (including abdominal pain, diarrhea, and food intolerance). she lived in the country near to a city, but was not employed in agriculture, and there was no evidence of contact with animals in her clinical history. gastroscopic examination showed no abnormalities. infiltrative lesions in the thorax were detected by radiological and computed tomographic examinations, and there was progressive swelling of cervical lymph nodes. no pathogens were detected in bronchoalveolar lavage (bal) specimens (virus culture on hel, llc-mk , hep- , and human rhabdomyosarcoma cells; bacterial culture on blood agar, chocolate agar, columbia blood agar containing colistin and nalidixic acid, and sabouraud agar; cytological examination for bacteria, fungi, and pneumocystis carinii), and a biopsy of the peripheral right lower lung lobe was histologically normal. epstein-barr virus (ebv) antigen expression and lymphoproliferation were detected in a biopsy of the right jugular lymph node, and a blood sample was positive for ebv dna by polymerase chain reaction (pcr). reactivation of ebv and associated lymphoproliferation were treated successfully with rituximab (anti-cd ) and discontinuation of cyclosporin therapy, but other clinical signs remained. enteroscopic examination of the small intestine showed no abnormalities, and no pathogens were detected in fecal samples by bacterial culture. five weeks after hospitalization, the patient developed high fever and progressive worsening of respiratory signs. treatment with cotrimoxazole was initiated for suspected p. carinii infection, and with prednisone for suspected bronchiolitis obliterans organizing pneumonia or graft-vs-host disease of the lung. two weeks later, the patient developed respiratory failure. there was crepitation over both lung fields and bilateral, progressively expanding, blotchy infiltrates in the thorax by radiological examination. gram-negative, rod-shaped bacteria (pseudomonas species) were detected by microscopic examination of bal fluid, and treatment with ceftazidime and tobramycin was initiated. despite intubation and artificial respiration at increasing pressure, respiratory function deteriorated further and circulatory failure developed. artificial respiration was stopped after days, after which the patient died. autopsy was performed day after death. there were extensive areas of bronchopneumonia associated with pseudomonas species infection in both lungs by bacterial culture. in addition, there was marked diffuse alveolar damage, characterized by abundant hyaline membranes lining the alveolar septa. the mucosa of the colon ascendens was lined by a hemorrhagic exudate, but autolysis was too advanced to confirm or rule out graft-vs-host disease, and no satisfactory explanation was found for the abdominal complaints. in conclusion, the progressive respiratory failure was explained by severe diffuse alveolar damage of undetermined cause, and extensive bilateral bronchopneumonia caused by terminal pseudomonas species infection. as part of an in-depth investigation, archived cell culture samples with an unexplained cytopathic effect were subjected to metagenomic analysis as described previously [ ] . these samples included human rhabdomyosarcoma cells inoculated with a bal specimen collected in from the above patient. metagenomic analysis revealed the presence of short nucleotide fragments with homology to apmv- . to complete the genome of the clinical isolate, fragments obtained through whole transcriptome amplification were assembled against the apmv- genome with the highest similarity (supplementary methods). in brief, the assembly was performed using sequencher software (version . ; gene codes). because the whole-transcriptome amplification fragments did not overlap into a complete genome, the assembly was used to design primers for genome walking, pcr assays were performed with these primers, and products were sequenced. these efforts resulted in the genome completion of the dutch clinical virus isolate. nucleotide sequences were aligned with the clustal w program running within the bioedit software package, version . . [ ] . with the nucleotide sequence alignment, the best-fit model of nucleotide substitution was determined by jmodeltest [ ] . maximum-likelihood (ml) phylogenetic trees were generated using the gtr+Γ +i model of nucleotide substitution and the phyml package version . [ ] . the reliability of all phylogenetic groupings was determined through a bootstrap resampling analysis of a replicates with phyml. trees were visualized through the figtree program version . . (http://tree.bio.ed.ac. uk/software/figtree/) and rooted on the apmv- with the highest blast similarity in genbank (apmv- /nl/ / ). the following tissue biopsies and autopsy tissue specimens had been fixed in % neutral-buffered formalin and embedded in paraffin blocks: heart, lung, jugular lymph node, esophagus, thyroid gland, stomach, pancreas, liver, kidney, skin, diaphragm, and muscle. these specimens were used for detection of apmv- rna by real-time reverse-transcription pcr (rt-pcr), of apmv- antigen by immunoperoxidase method, and of histological lesions by hematoxylin and eosin stain (supplementary methods). chickens were inoculated intracerebrally with the dutch clinical virus isolate to determine the intracerebral pathogenicity index (supplementary methods). domestic pigeons were inoculated intratracheally with the dutch clinical virus isolate to determine infectivity and transmissibility, clinical signs, and pathological changes (supplementary methods). the sequence of the full-length genome of the dutch clinical virus isolate displayed % nucleotide sequence homology with an apmv- isolated from pigeons in belgium (apmv- / belgium/ - / ; jx ). phylogenetic analysis of fulllength genomic sequences of closely related apmv- isolates demonstrated that the dutch clinical isolate clustered among belgian and chinese avian strains in genotype vib/ ( figure ). viruses in this genotype are also called pigeon paramyxovirus type (ppmv- ), because pigeons and doves are the animal reservoir. therefore, the dutch clinical virus isolate was named human ppmv- (hppmv- /nl/ / ; accession number kj ). comparison of predicted amino acid sequences revealed that all of the hppmv- /nl/ / encoded proteins displayed > % identity with those of other ppmv- . in general, the fusion protein (f) cleavage site of medium/high virulent apmv- s contain the sequence rq(k/r)r*f , while those of low virulent apmv- s usually have the sequence (k/r) q(g/e)r*l . the cleavage site of hppmv- /nl/ / ( rqkr*f ) was consistent with a virulent pathotype, similar to that observed in all ppmv- ( r(q/k) kr*f ) [ ] [ ] [ ] . based on phylogenetic analysis of sequences of partial f protein sequences, ujvári et al identified sublineages of ppmv- (ie, apmv- genotype vib/ ) strains. these subgroups, designated iraqi (iq), early european (eu/ea), north american (na), with related avian paramyxovirus type (apmv- ) strains based on analysis of full-length genomes. assignment of apmv- genotypes (vib/ or vii) and sublineages (eure , eure , euea , or euea ) was based on the studies of ujvári et al [ ] and czegledi et al [ ] and the sequence comparison shown in table and recent european (eu/re), corresponded partly to the times of isolation and/or geographical origin [ ] . alignment of the f protein amino acid sequences from hppmv- /nl/ / with those of ujvári and others revealed that it had identical amino acids substitutions as isolates belonging to sublineage eur/re (table ) . no sequence information was available for this partial f protein sequence of the only other apmv- clinical isolate, from a fatal human case in new york state [ ] . the available partial sequence for this new york clinical virus isolate demonstrated the closest relationship with a ppmv- isolated from pigeons in new york in (kc . ), and clustering in sublineage vib/ na (data not shown). real-time rt-pcr analysis revealed the presence of high levels of ppmv- rna in the left and right lung ( table ) . lower levels were detected in liver, kidney, and bone marrow, while the remaining tissues tested negative. immunohistochemistry analysis revealed the expression of ppmv- antigen, visible as red-brown granular staining, in the same tissues that tested positive with real-time rt-pcr ( table ). the exact localization of these granules was not clear, although some aggregates of granules appeared to be located in the cytoplasm of degenerate cells (figure ). ppmv- antigen expression was present in the positive control tissue and absent in the isotype control and negative control tissues. histopathological examination demonstrated that ppmv- antigen-expressing sections of lung had diffuse alveolar damage, characterized by loss of alveolar epithelium, rare hypertrophic type ii pneumocytes, widened alveolar septa, and fibrin thrombi in alveolar capillaries ( figure ). no histological lesions were apparent in other tissues that expressed ppmv- antigen, or in ppmv- -negative tissues. based on intracerebral inoculation into -day-old chickens, the intracerebral pathogenicity index of hppmv- /nl/ / was . (supplementary table ). this corresponds with a lentogenic pathotype. in pigeons, all intratracheally inoculated animals developed a productive infection, with shedding from the pharynx between and dpi, and from the cloaca between and dpi (supplementary figure ) . virus was transmitted to of the naive pigeons. none of the pigeons displayed clinical signs at any time point during the -week observation period. no gross lesions were observed in any pigeons at autopsy at dpi. however, histopathological analysis showed that all intratracheally inoculated pigeons plus the infected naive pigeon had diffuse interstitial nonpurulent nephritis, which was absent in negative control pigeons from the same flock (supplementary figure ) . also, inoculated pigeon had multifocal nonpurulent pancreatitis. real-time rt-pcr analysis revealed that hppmv- /nl/ / was present in the kidney sample of inoculated pigeons with nephritis, and in the pancreas sample of inoculated pigeons, including the with pancreatitis (supplementary figure ) . discussion we diagnosed ppmv- infection as the cause of diffuse alveolar damage in the dutch female patient, based on colocalization of ppmv- antigen, high loads of ppmv- rna, and characteristic histological lesions in autopsy specimens ( figure and table ). the ppmv- -associated diffuse alveolar damage was exacerbated by bronchopneumonia due to pseudomonas species infection, probably from intravenous catheterization. the combination of these pulmonary diseases likely explains the severe respiratory failure leading to the death of this immunosuppressed patient. it is rare to diagnose ppmv- infection as the cause of severe respiratory disease in humans. to our knowledge, the only other known case was reported by the new york state department of health in [ ] . it is not clear whether this paucity of reports is because the disease is rare or because apmv- , including ppmv- , is not suspected in such cases. we suggest that apmv- , including ppmv- , should be included in the differential diagnosis of cases of respiratory disease that test negative for more common respiratory pathogens. samples could be tested by various molecular methods, such as a family-wide rt-pcr assay for paramyxoviruses or metagenomics approaches [ , ] . when they became infected with ppmv- , the dutch and new york patients were receiving immunosuppressive therapy to improve the success of peripheral blood stem cell or bone marrow transplantation. there is an increase in the proportion of immunocompromised people in the population [ ] . as these immunocompromised people are at high risk of severe disease from other opportunistic infections [ ] , they also may be at risk of severe disease from infection with apmv- , including ppmv- . ppmv- appears to target the lower respiratory tract epithelium in humans. this is based on the demonstration of ppmv- antigen expression in the alveolar epithelium of autopsy lung samples from both human cases (figure ) [ ] . the tissue tropism of ppmv- in these human cases resembles that of other emerging zoonotic respiratory viruses-h n influenza virus and the severe acute respiratory syndrome and middle east respiratory syndrome coronaviruses-that also target the lower respiratory tract [ , [ ] [ ] [ ] . there was evidence of extrarespiratory spread of ppmv- in the dutch case. this was based on evidence of ppmv- infection in liver, kidney, and bone marrow ( table ). this is consistent with the new york case, where evidence of ppmv- infection in feces and urine also suggested extrarespiratory pigeon paramyxovirus-linked pneumonia • jid : ( october) • table spread [ ] . the immunosuppressive state of the dutch and new york patients may have allowed spread of ppmv- beyond the respiratory tract. pigeons and doves are the animal reservoir of ppmv- ( figure and table ). in europe, these viruses are circulating predominantly in domestic pigeons (columba livia domestica) or their wild relatives (rock pigeons, c. livia), and other members of the family columbidae, especially eurasian collared doves (streptopelia decaocto) and european turtle doves (streptopelia turtur) [ ] . the tropism of hppmv- / nl/ / for the kidney and pancreas of experimentally infected pigeons ( supplementary figures and ) matches the tissue tropism of ppmv- in naturally infected pigeons [ ] . the low pathogenicity of this isolate in chickens fits with the phenotype of other ppmv- strains [ ] . the number of feral pigeons-free-living birds that have descended from domestic pigeons-in european and north american cities increased substantially from the s to the s as a result of changes in agricultural practices and the rapid human population increase after world war ii. feral pigeon numbers then stabilized, likely due to reaching the carrying capacity of the urban environment. however, population growth of feral pigeons is still likely to occur in recently colonized cities and at the newly built outskirts of cities [ ] . first reported in italy in [ ] , ppmv- subsequently spread across europe and has become endemic in feral pigeons, with regular spread to wild pigeons and doves [ ] . in north america, ppmv- was introduced in the s and now is maintained endemically in pigeons and doves [ ] , including the eurasian collared dove [ , ] . this invasive species, first reported in florida in the s, has rapidly spread across most of north america (http://www.audubon.org/field-guide/bird/ eurasian-collared-dove), and could facilitate virus dispersal throughout north america [ , ] . the clinical histories of the dutch and new york cases do not readily explain how they became infected. the dutch patient lived in a rural area near a city, but was not employed in agriculture, and there was no evidence of contact with animals (this study). the new york patient was an urban dweller, but it is unknown whether he had pets or was exposed to birds in other settings [ ] . based on phylogenetic analysis of the viruses, the most probable route of transmission was contact with infected pigeons or doves. ppmv- is environmentally stable in bird feces and can be spread by direct contact or by windborne dust [ ] . the route of transmission of other zoonotic pathogens from pigeons to humans may be instructive. five pathogen species (chlamydia psittaci, histoplasma capsulatum, aspergillus species, candida parapsilosis, and cryptococcus neoformans) have been reported to be routinely transmitted from feral pigeons to people [ ] , mostly by inhalation of airborne excreta, including dried feces, ocular discharges, and crop milk. contact was sometimes brief, and patients did not always recall any bone marrow (n = ) + heart, esophagus, stomach, pancreas, right kidney, skin, diaphragm, and muscle tested negative for ppmv- rna by real-time reverse-transcription polymerase chain reaction and for ppmv- antigen by immunohistochemistry; lung biopsy and lymph node biopsy tested negative for ppmv- antigen by immunohistochemistry. abbreviations: +, rare positive granules; ++, occasional positive granules; ct, cycle threshold; ppmv- , pigeon paramyxovirus type . encounters with birds. it is relevant for these ppmv- cases that the risk of pigeon-associated diseases-chlamydiosis and cryptococcosis-was largely a function of the immune status of patients, rather than contact with infected birds [ , ] . close contact between feral pigeons and humans commonly occurs in squares, public gardens, parks, markets, and railway stations in urban areas [ ] . therefore, people in urban areas are likely to have a higher rate of contact with feral pigeons than in rural areas. the proportion of the global human population living in urban areas is increasing. in , % of the world's population lived in urban areas; this increased to % in , and by , % of the world's population is projected to be urban [ ] . this suggests that the number of people at risk of contracting zoonotic infections, including ppmv- , from feral pigeons will increase in coming decades. the combination of the above factors-large populations of feral pigeons with endemic ppmv- infection, increasing urbanization, and a higher proportion of immunocompromised individuals-may increase the risk of severe human cases of ppmv- infection. supplementary materials are available at the journal of infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. global trends in emerging infectious diseases adaptive pathways of zoonotic influenza viruses: from exposure to establishment in humans limited airborne transmission of h n influenza a virus between ferrets the severe acute respiratory syndrome cross host transmission in the emergence of mers coronavirus nipah virus: transmission of a zoonotic paramyxovirus hendra virus ecology and 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of spread health hazards posed by feral pigeons chlamydial infections in feral pigeons in europe: review of data and focus on public health implications world urbanization prospects: revision, highlights (st/esa/ser. a/ ) the occurrence of five major newcastle disease virus genotypes we thank the department of pathology, leiden university medical center, the netherlands, for providing biopsies and autopsy specimens from the dutch patient.financial support. this work was supported by european fp programme antigone (anticipating the global onset of novel epidemics, project number ) and by the european commission h programme under contract number (www.compare_europe.eu).potential conflicts of interest. all authors: no reported conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord- -qh y authors: xu, puzhi; liu, ping; zhou, changming; shi, yan; wu, qingpeng; yang, yitian; li, guyue; hu, guoliang; guo, xiaoquan title: a multi-omics study of chicken infected by nephropathogenic infectious bronchitis virus date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: qh y chicken gout resulting from nephropathogenic infectious bronchitis virus (nibv) has become a serious kidney disease problem in chicken worldwide with alterations of the metabolic phenotypes in multiple metabolic pathways. to investigate the mechanisms in chicken responding to nibv infection, we examined the global transcriptomic and metabolomic profiles of the chicken’s kidney using rna-seq and gc–tof/ms, respectively. furthermore, we analyzed the alterations in cecal microorganism composition in chickens using s rrna-seq. integrated analysis of these three phenotypic datasets further managed to create correlations between the altered kidney transcriptomes and metabolome, and between kidney metabolome and gut microbiome. we found that genes and metabolites were deferentially expressed or accumulated in the kidney during nibv infection processes. these genes and metabolites were linked to nibv-infection related processes, including immune response, signal transduction, peroxisome, purine, and amino acid metabolism. in addition, the comprehensive correlations between the kidney metabolome and cecal microbial community showed contributions of gut microbiota in the progression of nibv-infection. taken together, our research comprehensively describes the host responses during nibv infection and provides new clues for further dissection of specific gene functions, metabolite affections, and the role of gut microbiota during chicken gout. gout is a urate crystal deposition disease that occurs when supersaturation of individual tissues with urate arises, leading to the construction of monosodium urate (msu) crystals in and around the joints, abdomen, and organs [ , ] . it is worth noting that in addition to its occurrence in humans, gout is one of the common diseases that plague poultry and causes huge economic losses to the poultry industry worldwide [ ] . avian gout is commonly divided into visceral gout and joint gout, and the typical clinical pathology of visceral gout is hyperuricemia. according to reports, various aviaries from all over the world have visceral gout, which has become one of the most commonly diagnosed causes of fatality in poultry [ , ] . in the poultry industry, visceral gout can be caused by many factors, including vitamin a deficiency, high dietary calcium, renal insufficiency, chicken diseased room (dis, n = ). birds in each experimental room were randomly divided into three subgroups ( birds for each subgroup), with ad libitum access to food and water. each chicken of dis groups was intranasally injected with . ml median embryo lethal doses of strain sx at days of age [ ] , while the con group intranasally received . ml of sterile physiological saline. at days of age, four chicken randomly chosen per subgroups were euthanized by carbon dioxide inhalation, then dislocated their cervical vertebra. the samples in a group were pooled and dead birds were not used for analysis. ten serum samples were randomly collected from surviving chickens in the con and dis groups before euthanasia that were used for uric acid test. six biological replicates of kidney samples were extracted from each group making a total of samples that were used for gc-tof/ms analysis. four biological replicates of kidney samples were collected from each group giving a total of eight samples that were used for rna-seq analysis. six biological replicates of cecal contents from each group were collected giving a total of samples that were used for s rrna gene sequencing analysis ( figure a shows the experimental design). the isolated kidney tissues were fixed by immersion in % neutral formalin at room temperature for over h. tissues were then routinely processed; h&e staining was performed and a section per chicken was observed under the optical microscope. metabolite extraction, metabolite derivatization, metabolite detection, and data analysis followed those of yang et al. [ ] . first, methanol (v methanol :v chlorofrom = : ) was used as an extraction liquid, and l- -chlorophenylalanine ( mg/ml stock in dh o) was added as an internal standard. the metabolites are then derivatized with the methoxy amination hydrochloride ( mg/ml in pyridine) and the stfa regent ( % tmcs, v/v). finally, gc-tof/ms analysis was performed using an agilent gas chromatograph system coupled with a pegasus ht time-of-flight mass spectrometer. the energy was - ev in electron impact mode. after . min of solvent delay, mass spectrometry data were acquired in full-scan mode with an m/z range of - at a rate of spectra per second. chroma tof . x software (leco corporation, st. joseph, mi, usa) and the leco-fiehn rtx database were used for raw peak exacting, data baseline filtering and calibration, peak alignment, deconvolution analysis, peak identification, and integration of the peak area. simca software package (umetrics, umea, sweden) was used for further data analysis, including principal component analysis (pca) and orthogonal projections to latent structures-discriminate analysis (opls-da). in addition, commercial databases including kyoto encyclopedia of genes and genomes (kegg, http://www.genome.jp/kegg/) and national institute of standards and technology (nist, http:// www.nist.gov/index.html) were utilized to search for metabolic pathways. metaboanalyst (http: //www.metaboanalyst.ca) was used for pathway enrichment analysis. rna preparation, library preparation, rna-sequencing, and data analysis followed those of yang et al. [ ] . first, total rna was extracted from each kidney sample individually using trizol reagent (invitrogen, burlington, on, canada). we further used the nanodrop (thermo, waltham, ma, usa) to measure the rna concentration, and the agilent bioanalyzer system (agilent technologies, ca, usa) to assess the rna integrity. library preparation was performed as described by li et al. [ ] . briefly, a total amount of µg rna per sample was used for the rna sample preparations. sequencing libraries were generated using nebnext ultratm rna library prep kit for illumina (neb, ipswich, ma, usa) and index codes were added to attribute sequences to each sample. the library fragments were purified with ampure xp system (beckman coulter, beverly, ca, usa). at last, pcr products were purified (ampure xp system) and library quality was assessed on the agilent bioanalyzer system. finally, the constructed cdna libraries were sequenced by an illumina hiseq xten platform. clean data of high quality were obtained from the raw data through in-house scripts by removing those containing adapters or poly-n sequences. all clean reads were aligned to the reference genome (https://www.ncbi.nlm.nih.gov/assembly/gcf_ . /) using tophat [ ] . cufflinks was used to calculate and analyze the gene expression levels, and fpkm (fragments per kilobase of exon per million fragments mapped) values of each gene were calculated based on the length of the gene and the fragments count mapped to this gene. differential expression genes (degs) analysis was performed by using the deseq r package ( . . ). only those genes with a fc (fold change) ≥ and fdr (false discovery rate) < . were defined as degs. furthermore, gene ontology (go) enrichment analysis of degs was implemented by the goseq r packages [ ] and the enrichment analysis of degs in kegg pathways was performed using kobas software [ ] . in addition, the target seven degs in response to nibv infection were chosen for validation using real-time quantitative pcr (rt-qpcr). the primer pairs for the selected genes were designed using primer and are shown in table s . total genome dna from samples was extracted from the cecal contents using ctab/sds method. dna concentration and quality were determined and diluted to ng/µl using sterile water. the s rrna genes of each sample v -v region were amplified. the primer set corresponding to primers f- r with the unique barcode was applied for amplification (forward f: cctaygggrbgcascag and reverse r: ggactacnngggtatctaat). all pcr reactions were carried out with phusion ® high-fidelity pcr master mix (new england biolabs, usa). the pcr products were detected on % agarose gel electrophoresis and bands of the desired size (approximately - bp) were chosen for further experiments. sequencing libraries were generated using trueseq dna pcr-free sample preparation kit (illumina, san diego, ca, usa) and index codes were added. next, sequencing was performed on an illumina hiseq platform (novogene company, beijing, china). based on their unique barcode, the paired-end reads were granted to samples. those files were demultiplexed and quality filtered with quantitative insights into microbial ecology (qiime) software (version . . ) [ ] . the chimera sequences were removed by using uchime algorithm and then the effective tags finally obtained [ ] . sequences were clustered to the same otus at % similarity by uparse software (uparse v . . ). moreover, the greengene database was used to annotate taxonomic information and the multiple sequence alignment was conducted using the muscle software (version . . ). further, otus abundance information was normalized and subsequent analysis were all performed based on this output normalized data including alpha diversity, beta diversity, and linear discriminant analysis (lda) effect size (lefse) analysis [ ] . heatmap for metabolomics, transcriptomics, and microbiomics data was generated using the pheatmap package. corrplot package in r was used to visualize the correlations coefficient (spearman method). availability of data and materials: rna-seq raw data are accessible through ncbi' database: bioproject: prjna ; s rrna gene sequencing raw data are accessible through ncbi' database: bioproject: prjna . the institutional animal care and use committee of jiangxi agricultural university approved these animal experiments and all animal experiments adhered rigorously to the animal care guidelines of jiangxi agricultural university (approval id: jxaull- ; approval date: march ). all the birds were sacrificed using carbon dioxide euthanasia, and all attempts were carried out to minimize the suffering of the animals. obvious enlargement and urate deposition in the kidney of a chick infected with nibv at dpi (right). (d) histopathological changes in the kidney of chickens infected with nibv (h&e staining). the black arrow shows the shedding of kidney tubular epithelial cells and the white arrow shows the interstitial expansion and prominent inflammatory cell infiltration. the black asterisk shows the brush border that was lost in some segments of proximal tubules. the black delimited area shows the loss of the kidney tubular structure. to explore the metabolic pathway alterations associated with nibv infection, we used a gc-tof/ms-based metabolomics method to examine metabolite alterations in the kidney. a total of valid peaks were identified in the total ion current profiles. to compare the metabolite composition of the con and dis groups, pca models were tested (figure a ). opls-da was conducted to determine whether nibv infection influenced the metabolic pattern ( figure b ) and a -fold cross validation was further applied to estimate the robustness and predictive ability to validate the model (figure c ). the results of pca and opla-da analysis showed that there was an obvious separation between the content of the con and dis groups, obvious enlargement and urate deposition in the kidney of a chick infected with nibv at dpi (right). (d) histopathological changes in the kidney of chickens infected with nibv (h&e staining). the black arrow shows the shedding of kidney tubular epithelial cells and the white arrow shows the interstitial expansion and prominent inflammatory cell infiltration. the black asterisk shows the brush border that was lost in some segments of proximal tubules. the black delimited area shows the loss of the kidney tubular structure. the chicks inoculated with strain sx showed obvious clinical signs from to "day post-infection" (dpi). the nibv-infected chickens were listless, huddled together, and displayed ruffled feathers, while no clinical symptoms similar to the above were observed in the con group. the dpi survival rate of all nibv-infected chickens under analysis was %, and the uric acid level in the serum of the dis group was much higher than that of the con group (~ vs.~ µmol/ml, n = , p < . , student's t-test, figure b ). we observed that kidney lesions were present in all dis group chickens infected with sx . at dpi (mortality peak), the kidney parenchyma of the dead birds were pale, swollen, and mottled ( figure c ). histological examination revealed remarkable injuries in the kidney, including tubular epithelial cell detachment, loss of the kidney tubular structure, as well as interstitial expansion and prominent inflammatory cell infiltration ( figure d ). these results indicate that the sx strain has strong renal tissue tropism and successfully replicates the chicken visceral gout model. to explore the metabolic pathway alterations associated with nibv infection, we used a gc-tof/ms-based metabolomics method to examine metabolite alterations in the kidney. a total of valid peaks were identified in the total ion current profiles. to compare the metabolite composition of the con and dis groups, pca models were tested (figure a ). opls-da was conducted to determine whether nibv infection influenced the metabolic pattern ( figure b ) and a -fold cross validation was further applied to estimate the robustness and predictive ability to validate the model (figure c ). the results of pca and opla-da analysis showed that there was an obvious separation between the content of the con and dis groups, revealing significant changes in the concentrations of metabolites in the kidney induced by nibv infection. all samples fell within the % (hotelling's t-squared ellipse) confidence interval. the differential metabolites between dis and con groups were the key to explaining the occurrence of gout in chickens under nibv infection. a total of metabolites displayed significantly different levels based on vip > (variable importance for the projection, opls-da model) and p-value < . (student's t-test). the lists of differential metabolites are shown in table s and its volcano plot are shown in figure d . there were annotated metabolites in all differential metabolites, and its categories are shown in figure e . as figure e shows, the differential metabolites were mainly classified as amino acids, carbohydrates, fatty acids, and conjugates. among those annotated differential metabolite profiles which were displayed in heat maps ( figure s ), metabolites were significantly upregulated and were significantly downregulated in the dis group compared to the con group. in addition, the pathway enrichment results are shown in figure f . the analysis revealed that the differential metabolites participated in six target pathways (p < . ), including valine, leucine and isoleucine biosynthesis, arginine and proline metabolism, alanine, aspartate and glutamate metabolism, d-glutamine and d-glutamate metabolism, aminoacyl-trna biosynthesis, and beta-alanine metabolism. among them, there are two pathways with an impact factor > , including valine, leucine and isoleucine biosynthesis (impact value = . ), and arginine and proline metabolism (impact value = . ). these pathways are related to amino acid metabolism and glycometabolism, which are involved in protein synthesis and energy production, respectively. moreover, metabolomics highlighted n-formyl-l-methionine, a well-known agent for kidney injury for its effects on the increase of the vascular tone/resistance and reduction of renal perfusion [ ] , as the most relevant metabolite alteration in nibv-infected chickens (vip = . ). undoubtedly, the uric acid level in the dis group was significantly upregulated compared with the con group (vip = . , . -fold). to study the gene expression alterations of chicken's kidney under nibv infection, cdna libraries from two groups were subjected to illumina sequencing. a total of . gb clean data was obtained, and the q base percentage of each sample was not less than . %. from the mapping results, the mapping efficiency between the reads and the reference genome of each to study the gene expression alterations of chicken's kidney under nibv infection, cdna libraries from two groups were subjected to illumina sequencing. a total of . gb clean data was obtained, and the q base percentage of each sample was not less than . %. from the mapping results, the mapping efficiency between the reads and the reference genome of each sample was between . % and . %. the detail of the sequencing and mapping results is provided in table s . pca analysis of rna-seq replicates from the kidneys of dis and con group revealed a great deal of variability (figure a) . a total of genes were differentially expressed in the chicken's kidney with nibv infection compared to con group, in which genes were upregulated and genes were downregulated. then, we screened seven degs for rt-qpcr analysis to validate the accuracy of the rna-seq data, and the result (figure b) showed that the general trends of the selected genes were consistent which proved the reliability of our rna sequencing profiling. go (http://www.geneontology.org) project was applied to explore the potential biological functions of degs. in this study, the most enriched go terms classified by molecular function (mf), cellular components (cc), and biological processes (bp) terms were listed in figure c . among them, heparin binding, oxidoreductase activity, external side of plasma membrane, extracellular space, membrane, integral component of membrane, oxidation-reduction process, and immune response were the significantly enriched go terms (p adj < . ) in chickens with nibv infection. to further study the biochemical metabolic pathway and signal transduction pathways related to nibv infection, kegg analysis and pathway enrichment analysis were conducted in the kegg pathway database. there were of the degs that were annotated to pathways in kegg analysis. the level- kegg classes are shown in figure d , and the significant enriched pathways (corrected p-value < . as determined by a fisher's exact test) are marked with red number. in environmental information processing pathways which include cytokine-cytokine receptor interaction and cell adhesion molecules (cams) were dramatically regulated. in cellular processes pathway, peroxisome was dramatically affected. in metabolism pathways, metabolism of xenobiotics by cytochrome p , drug metabolism-cytochrome p , pyruvate metabolism, arginine and proline metabolism, glycolysis/gluconeogenesis, and fatty acid degradation were significantly regulated. in organismal systems pathways, intestinal immune network for iga production and toll-like receptor signaling pathway were significantly regulated. furthermore, most of these pathways of the upregulated genes enriched were immune system-related pathways, and "cytokine-cytokine receptor interaction" was the most represented pathway. among these pathways we found upregulated, "toll-like receptor signalling pathway", "nod-like receptor signalling pathway" and "rig-i-like receptor signalling pathway" are pattern recognition signal receptor pathways involved in innate immunity. simultaneously, the transcriptome showed that the "peroxisome" and amino acid metabolite pathways were suppressed in chickens infected with nibv. based on the above results, we predicted a schematic diagram of important pathways for chickens in the nibv infection processes (figure e) . signalling is activated by monosodium urate (msu) and reactive oxygen species (ros). the activation of tlrs and the ros accumulation activates the nlrp inflammasome, resulting in proteolytic cleavage of caspase- and the maturation of il- and il- β, and msu has been identified to activate inflammasome complex. considering that the intestinal tract is an important organ for lowering serum uric acid concentrations, s rrna sequencing was performed and demonstrated marked alterations of the gut microbial communities in the dis group. the rarefaction analysis curves for each group were near saturation, revealing that the sequencing data had a great quality and that certainly few new species were present ( figure s ). nmds (nonmetric multidimensional scaling, figure a (figure g,h) . lefse was performed to identify significant microbiota composition differences between the groups. seven differentially represented core major groups were identified (figure i,j) . differentially abundant phylum detected showed that proteobacteria (lda = . ) phylum was most dominantly present in dis group. at the genus level, the microbiota of the con group was enriched with bacteroides (lda = . ) from the bacteroidetes phylum and lactobacillus (lda = . ) from the firmicutes phylum. at the species level, the microbiota of the con group was enriched with bacteroides vulgatus (lda = . ) from the bacteroidetes phylum. the above data describe that nibv infection alters kidney gene expression and metabolic pathways (figure a ). we filtered out five overlapping pathways in the transcriptome and metabolome pathway enrichment analysis, including valine, leucine and isoleucine biosynthesis, arginine and proline metabolism, taurine and hypotaurine metabolism, alanine, aspartate and glutamate metabolism, and glutathione metabolism. in addition, the degs in significantly enriched pathways associated with innate immunity and differential metabolites with vip > . were also chosen for correlation analysis (tables s and s , respectively). differences in those pathways-associated with degs and differential metabolites were shown with heatmap in figure b . then, we analyzed the spearman correlation between genes and metabolites in those pathways, and the result is shown in figure c . as shown in figure c , there was a positive correlation between tlr (tlr, toll-like receptor) and proline (r = , p = ), isoleucine (r = . , p = . ), threonine (r = . , p = . ), and valine (r = . , p = . ). conversely, strong negative correlations were found between glutathione synthetase (gss) and glutamine (r = − , p = ). interestingly, we observed that most genes related to innate immune responses had strong positive correlations with differentially abundant metabolites, such as amino acids and fatty acids. the gut microbiota is deliberated a massive "organ" able to perform complex functions and thereby produce a myriad of differentially abundant metabolites. to investigate the functional correlation between the gut microbiome changes and host metabolome alterations, a correlation plot was visualized by calculating spearman's correlation coefficients (figure d ). the result indicated that clear correlations could be identified between altered metabolic profiles and modulated gut microbiomes (table s ) . of particular note, some metabolites, including trans- -hydroxy-l-proline, guanine, and , -anhydro-d-galactose, which decreased in the kidney of nibv-infected chickens, were negatively correlated with the presence of the phylum proteobacteria. furthermore, other metabolites, including canavanine, , -diaminobutyric acid, , -dihydrouracil, malonamide, thymine, phenylalanine, , -diaminopropane, -acetamidobutyric acid, proline, threonine, isoleucine, valine, and oxalacetic acid, which increased in the kidney of nibv-infected chickens, were positively correlated with the presence of the phylum proteobacteria. these observations indicated that the significantly modulated gut microbiota was correlated with host metabolic disorders. in our study, the three pathways included in the activation of the innate immune system, the toll-like receptor signalling, rig-i-like receptor signalling, and nod-like receptor signalling pathways, that are the most important three parts of the pattern-recognition receptor (prr) signalling pathway are usually activated in response to infections to stimulate inflammatory responses [ ] [ ] [ ] . in the toll-like receptor signalling pathway (figure f, signal ) , a series of upregulated genes were noticed, including tlr , tlr , myd , irf , traf , and irf . it was already reported that tlr primarily recognizes single-stranded rna (ssrna) sequences of rna viruses that enter endosomes by endocytosis [ ] [ ] [ ] . nibv used in this experiment is a single-stranded positive sense rna virus. thus, the expansion of tlr is pivotal for the recognition of nibv and variable functions of the toll-like receptor signalling pathway. moreover, a number of viral glycoproteins have been shown to act as viral pamps (pathogen-associated molecular pattern) that bind to and activate tlr , leading to ifn-β and/or proinflammatory cytokine expression (such as sars coronavirus) [ ] . ru liu-bryan's study has established that host expression of tlr , tlr , and their intracellular adapter protein myd is a major mediator of msu crystal-induced inflammation [ ] . this explains the reason for the increase in tlr transcription levels in this experiment. in addition, the transcriptomic analysis showed that nibv infection also activated the rig-i-like receptor signalling pathway (figure f , signal ), which included the transcriptional upregulation of genes such as mda , ips- , traf , and iκb. this induction may be due to mda acting as a double-stranded rna (dsrna) sensor to trigger an innate immune response against viral infection [ ] [ ] [ ] , while coronaviruses can produce dsrna intermediates during replication [ ] . rlrs can not only be expressed in cells infected with various viruses but also directly recognize and perceive virus products and virus particles present in the cytosol outside of the endosomes [ ] . therefore, we suspect that the rig-i-like receptor signalling pathway has a greater role than toll-like receptor signalling in recognizing nibv infection. the activation of the toll-like receptor signalling and rig-i-like receptor signalling pathways results in the production of chemokines and other cytokines that induce a proinflammatory response and tissue destruction. in our study, several features that are familiar to chickens infected with nibv are consistent with the immunopathological disease. these features include the pathological damage of kidney and the presence of increased transcription of chemokines and other cytokines, such as il- (interleukin ), il- (interleukin ), and tgf-β (transforming growth factor β). peroxisomes act to eliminate a microbial infection by modulating the canonical innate immunity pathways through ros signalling [ ] . several enzymes with antioxidant activity in the peroxisome are involved in neutralizing ros to protect the cells from ros damage. among these enzymes, catalase (cat), superoxide dismutase (sod ), peroxiredoxin (prdx ), glutathione s-transferase kappa (gstk ), dehydrogenase/reductase member (dhrs ), and epoxide hydrolase (ephx ) are included [ , ] . multiple viruses have been shown to use different mechanisms to reduce peroxisome numbers or interfere with their functions. in our study, the "peroxisome" was the most important pathway according to the downregulated gene enrichment analysis, which includes cat, sod , dhrs , and ephx that belong to the peroxisome antioxidant defence system, while the catalase activity was decreased both in kidney and serum ( figure s ). thus, decreased abundance and/or impaired function of the peroxisome could potentially cause endogenous elevation of ros. ros may either straightly trigger nlrp inflammasome assemblage or be indirectly sensed through cytoplasmic proteins that modulate inflammasome activity (figure f, signal ) . the nlrp inflammasome senses pathogens or danger signals to promote the maturation of cytokines such as il- [ ] . the release of active il- engages il- receptor-harbouring cells and promotes inflammatory responses [ ] . in addition, uric acid has been reported as another well-established activator of nlrp that is usually generated via xanthine oxidase (xod), accompanying the generation of o •− [ , ] . consistently, in this study, we detected a significant increase in xod activity in serum ( figure s ) and a significant increase in serum uric acid levels (figure b) . these results indicate that nibv infection can activate the inflammatory response by inducing severe ros accumulation. the kidney is responsible for the elimination of % of the daily ua production [ ] . atp-binding cassette transporter, sub-family g, member (abcg ) is a high-capacity urate exporter that is located in the brush border membrane of kidney proximal tubule cells (s segment). abcg dysfunction results in extra-renal urate under-excretion and is a common mechanism of hyperuricemia [ ] [ ] [ ] . in the present study, the abcg mrna was downregulated in the model group chicken kidneys, partially explaining the significantly increased uric acid levels caused by nibv infection. in addition to being caused by insufficient urate excretion, hyperuricemia can also be caused by excessive production of uric acid [ ] . we found a high level of glutamine, which enter the purine metabolic pathway as a raw material for uric acid synthesis. the high level of glutamine can be explained by two reasons. first, the lack of mrna abundance of the gene gss in the kidney tissue of the model group inhibited the conversion of glutamine to glutathione. a significant negative correlation between gss and glutamine has confirmed this finding. second, the transcription level of the gene glutamic pyruvate transaminase (gpt ) in the model group chickens' kidneys increased significantly. gpt is a pyridoxal enzyme that promotes the conversion of α-ketoglutarate to glutamate [ ] . of note, α-ketoglutarate is an intermediate in the tca cycle, and glutamate can be reversibly converted to glutamine by glutamine synthetase. together, these data point to key genes and metabolites related to elevated uric acid synthesis, which provides us with new insights into host response to nibv infection. the present study highlights the correlation between differential expression of genes and differential abundance of metabolites in significantly enriched pathways, and the results showed that those differentially abundant metabolites that map in amino acid metabolism pathways have strongly positive correlations with degs related to innate immune responses. it is well known that organisms fuel or instruct the immune response to pathogen threats by modulating metabolic pathways [ ] . innate and acquired immune systems are regulated by a highly interactive network of chemical communications, which include the synthesis of the antigen-presenting machinery, immunoglobulins, and cytokines. this network is highly dependent on the sufficient availability of amino acids. thus, amino acids affect immune responses either directly or indirectly through their metabolites [ ] . in our study, the correlation analysis highlighted a significant positive correlation between tlr and proline, isoleucine, threonine, and valine. according to reports, isoleucine could maintain the development of immune organs and cells and stimulate the secretion of immune molecule substances in humans and animals [ , ] , valine could improve dendritic cell function [ ] , and it has been proved in vitro that threonine plays a key role in lymphocyte proliferation and immunoglobulin production [ ] . we observed an increase in the level of amino acids enriched in proline, leucine, and isoleucine biosynthesis pathway, including isoleucine, valine, and threonine. therefore, changes in these amino acid metabolism pathways may occur through the activation of innate immunity to enhance the body's antiviral response. although there are many studies on amino acids and immunity, the modulation of amino acid metabolism in innate immune responses is still poorly known and deserves further study. previous research has confirmed that the dysfunction of the gut microbiome is tightly associated with kidney and liver diseases, including liver cirrhosis [ ] , liver cancer [ ] , and chronic kidney disease [ ] . the gut microbiome incorporates a wide variety of commensal microbiota and has a large effect on the health of individuals. a community-wide effect involving the gain and loss of microbial populations and changes in general metabolic functions always occurs under pathological conditions. our results showed that the abundance of bacteroides, lactobacillus, and bacteroides vulgatus were significantly reduced in the cecal microbiota of nibv-infected chickens based on lefse analysis. similar to many other bacteroides species, especially bacteroides vulgatus which was shown to promote intestinal homeostasis, the bacteroides vulgatus-mediated induction of semimature dendritic cells is associated with inflammation silencing [ , ] . thus, decreased abundance of bacteroides vulgatus promotes intestinal flora imbalance and activation of the immune system. furthermore, it is well accepted that lactobacillus may lower serum uric acid levels by reducing intestinal absorption of purines in humans [ ] . therefore, a decrease in the abundance of lactobacillus in the caecum of the model group infected with nibv further promoted an increase in uric acid levels in serum. the results of our correlation analysis further confirm this point, that is, the negative relationship between the abundance of bacteroides vulgatus, lactobacillus and the uric acid concentration. however, the mechanism of nibv infection leading to a decrease in their abundance still needs further study. taken together, multitudinous studies to date endorse the concept that a bloom of proteobacteria in the gut reflects gut dysbiosis or an unstable gut microbial community [ ] . in view of balanced gut microbiota with high stabilization that has symbiotic relationships with the immune system of the host, which is capable of suppressing the uncontrolled expansion of proteobacteria, the increase in the abundance of proteobacteria in this experiment may be closely related to the immune and metabolic changes caused by nibv infection. this report is the first time that multi-omics approach was employed to profile the metabolic changes and immune responses in kidney, as well as the effects on the intestinal microbiome during nibv infection. our results showed that nibv significantly increased uric acid synthesis, inhibited the function of peroxisomes, and significantly elevated the pattern recognition receptor signalling pathways. in summary, our study comprehensively describes the host responses during nibv infection and provides new clues for further dissection of specific gene functions, metabolite affections, and the role of gut microbiota during chicken gout. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s . figure s : heat map of annotated significant differential metabolites. note: the increasing gradient of red color indicate higher upregulation, while an increasing gradient of blue color indicate higher downregulation. figure s : rarefaction curves of the species number in the con and dis groups. the curve in each group is nearly smooth when the sequencing data set is sufficiently large (n = per group). figure s : the catalase activity (a, b), xanthine oxidase activity (c, d) in kidney and serum. table s : primers of selected genes for rt-qpcr. table s : differentially abundant metabolites identified by gc-tof/ms. significant differences were declared at the level of p-value < . and vip > . table s : basic summary of sequence and sequencing reads mapping to reference genome. table s : degs in enriched pathways for correlation analysis. table s : differentially abundant metabolites in enriched pathways for correlation analysis. table s : the relative abundance of seven discriminative features (lda score ≥ ) identified by lefse analyse. author contributions: conceptualization, x.g.; methodology, x.g. and p.l.; software, c.z. and y.s.; validation, q.w., p.x., and y.y.; formal analysis, p.x., c.z., and p.l.; resources, g.l., p.l., and x.g.; data curation, p.x. and x.g.; writing-original draft preparation, p.x. and p.l.; writing-review and editing, x.g., p.l., g.l., and g.h.; visualization, p.x., c.z., and y.s.; supervision, g.h.; project administration, x.g.; funding acquisition, x.g. funding: this research was funded by the national natural science foundation of china, grant number . acknowledgments: all authors thank all members of the team for their help in the experimental process in clinical veterinary medicine laboratory in the college of animal science and technology, jiangxi agricultural university. we also thank vincent latigo for the language help rendered in reviewing the revised manuscript. the authors declare no conflict of interest. gout induced by intoxication of sodium bicarbonate in korean native broilers clinicopathology of gout in growing layers induced by high calcium and high protein diets nephropathogenic infectious bronchitis in 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gut-associatedbacteroides vulgatusmpk outer membrane vesicles blebbing contributes to b. vulgatus mpk-mediated immune response silencing evaluation of purine utilization by lactobacillus gasseri strains with potential to decrease the absorption of food-derived purines in the human intestine proteobacteria: microbial signature of dysbiosis in gut microbiota this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -w rw wak authors: burns, amy l; dans, madeline g; balbin, juan m; de koning-ward, tania f; gilson, paul r; beeson, james g; boyle, michelle j; wilson, danny w title: targeting malaria parasite invasion of red blood cells as an antimalarial strategy date: - - journal: fems microbiol rev doi: . /femsre/fuz sha: doc_id: cord_uid: w rw wak plasmodium spp. parasites that cause malaria disease remain a significant global-health burden. with the spread of parasites resistant to artemisinin combination therapies in southeast asia, there is a growing need to develop new antimalarials with novel targets. invasion of the red blood cell by plasmodium merozoites is essential for parasite survival and proliferation, thus representing an attractive target for therapeutic development. red blood cell invasion requires a co-ordinated series of protein/protein interactions, protease cleavage events, intracellular signals, organelle release and engagement of an actin-myosin motor, which provide many potential targets for drug development. as these steps occur in the bloodstream, they are directly susceptible and exposed to drugs. a number of invasion inhibitors against a diverse range of parasite proteins involved in these different processes of invasion have been identified, with several showing potential to be optimised for improved drug-like properties. in this review, we discuss red blood cell invasion as a drug target and highlight a number of approaches for developing antimalarials with invasion inhibitory activity to use in future combination therapies. malaria is a mosquito borne disease caused by parasites of the genus plasmodium. the majority of the ∼ malaria related deaths in were caused by p. falciparum and occurred in sub-saharan africa (murray et al. ; who ) . in addition, p. vivax, p. malariae, p. ovale (comprised of two different subspecies; p. ovale curtisi and p. ovale wallikeri (sutherland et al. ) ) and two zoonotic species, p. knowlesi and p. simium (singh et al. ; brasil et al. ) , are recognised as significant contributors to global malaria disease burden. while intervention against anopheles mosquito vectors and the success of artemisinin-based combination therapies have contributed to marked decreases in disease burden since the year , there is growing concern regarding the spread of p. falciparum strains throughout southeast asia which are resistant to artemisinin-based drugs and their partner drugs utilized in combination therapies (dondorp et al. ; ariey et al. ; ashley et al. ; tun et al. ; das et al. ) . resistance to other clinically used antimalarials, such as chloroquine and sulfadoxine-pyrimethamine, is also widespread globally (plowe et al. ; trape et al. ; mehlotra et al. ) . thus, there is an urgent need to bring to market new antimalarials with novel mechanisms of action which are active against all drugresistant strains, effective against all human pathogenic plasmodium spp. and can clear parasitemia rapidly for improved clinical outcomes (burrows et al. ) . targeting multiple lifecycle stages would improve the effectiveness of combination therapies across endemic areas and help slow the development of drug resistance. human infections begin with the bite of a mosquito vector and release of malaria sporozoites, with the sporozoite then traveling to the liver and invading hepatocytes (reviewed in aly, vaughan and kappe ) . after rapid multiplication of liver stage parasites, the mature hepatic schizont ruptures and releases red blood cell (rbc) invading merozoites into the blood stream. in the case of p. falciparum, after merozoite invasion of a rbc a hour cycle of growth, multiplication, rbc rupture and release of - new merozoites ensues (reviewed in white et al. ). the number of merozoites produced per cycle of growth and the length of the blood stage lifecycle varies between human malaria species. a small portion of blood stage parasites (< %) differentiate into sexual stage gametocytes, which are transmitted to anopheles vectors during blood meal feeding (reviewed in liu, miao and cui ) . as all malaria pathology is caused by blood stage parasites, and this is when infection is diagnosed and clinical symptoms occur, antimalarials used for treatment of clinical disease or clearance of parasitemia predominantly target this stage of the lifecycle. one emerging strategy to kill blood stage parasites is to target merozoite invasion of the rbc with antimalarials. rbc invasion is an extracellular step in the blood stage lifecycle which is essential for parasite proliferation. a model for the sequential process of invasion, from late stage merozoite development, priming of invasion ligands, merozoite release from the schizont (fig. ) , through rbc contact and invasion (fig. ) , is described: briefly (i) merozoites attach to the rbc, (ii) the apical tip of the egg-shaped merozoite contacts the rbc, (iii) invasion ligands from organelles situated at the apical tip (the rhoptry and micronemes) are secreted upon calcium signals and an irreversible interaction known as the tight junction is formed, (iv) the actin-myosin invasion motor engages, protease cleavage events are triggered as the rbc membrane is pulled around the parasite to form the parasitophorous vacuole and (v) the invasion pore is fused behind the invaded parasite (dvorak et al. ; gilson and crabb ; weiss et al. ). an important consideration in terms of drug-development is that rbc invasion requires a series of co-ordinated and often irreversible events to occur in sequence, with even small perturbations of this complex process likely to limit parasite survival in vivo. rbc invasion is the essential first step in the disease causing blood stage of the lifecycle and the extracellular merozoite is exposed directly to the bloodstream. vaccine development targeting merozoite invasion is well advanced with vaccines against merozoite surface protein (msp , phase b; genton et al. ; ogutu et al. ), msp (phase b; genton et al. ) , apical membrane antigen (ama , phase b; thera et al. ) , reticulocyte binding homologue (rh , phase a; payne et al. ) , erythrocyte binding antigen (eba- , phase a; koram et al. ) and others reaching clinical trials, but with limited efficacy demonstrated to date (reviewed in draper et al. ; beeson et al. ) . however, many essential proteins and protein/protein interactions required for invasion are unique to malaria parasites and are highly conserved between isolates, making them strong targets for antimalarial development. given this, there are several broad approaches that could be used therapeutically to block merozoite invasion. the importance of key parasite-parasite and parasite-rbc protein interactions on the merozoite surface highlights the possibility of blocking proteinprotein interactions directly; potentially by targeting rbc receptor(s). in addition, protease cleavage events, calcium signalling, the action of the invasion motor and structural changes are also key processes in rbc invasion which could be targeted by drugs. importantly, a drug that blocks a merozoite's ability to invade immediately and permanently ends the parasite's lifecycle, with this cidal activity potentially having benefits in terms of reducing the risk of tolerance leading to resistance and removing the parasites ability to transition to mosquito transmissible gametocytes. however, it has only been in recent years that protocols have been developed to test invasion-inhibitory compounds against p. falciparum merozoites directly in vitro), with the availability of these techniques now allowing improved screening and characterisation of new invasion-inhibitory chemotypes. this review will give an overview on compounds that inhibit invasion, either through inhibition of an upstream invasion priming event in the developing schizont ( fig. ) or during the merozoite invasion process (fig. ) , that have shown potential to be developed into antimalarial drugs. targeting rbc invasion has been established as a proof-of-concept through the demonstration of potent inhibitory activity of numerous compounds in vitro (summarised in supplementary table s ) and through several in vivo studies using animal models (xiao et al. ; zenonos et al. ; nasamu et al. ; pino et al. ) . numerous chemical starting points and targets have been identified that could be the basis for the development of potent inhibitors for therapeutic use, as described below. we compare this concept to the clinical use of inhibitors which block hiv entry into host cells (barre-sinoussi et al. ; gallo et al. ) to demonstrate that rbc invasion is a viable therapeutic target. in addition, we consider studies from the related apicomplexan parasite toxoplasma gondii, for which a number of optimisable inhibitors have been developed against targets analogous to proteins in malaria parasites. as resistance to frontline artemisinin-based combination therapies continues to spread (dondorp et al. ; ariey et al. ; ashley et al. ) , this review is a timely reminder that malaria invasion of rbcs provides an 'achilles heel' within the parasite's lifecycle that is of increasing interest for antimalarial development. viruses are highly successful and widespread lifeforms that require establishment of an infection inside a host cell in order merozoite formation is completed in mature schizonts. the pvm becomes permeable and pfpkg is activated, leading to activation and discharge of subtilisin-like protease (pfsub ) from the exonemes. the protease plasmepsin x (pmx) also resides in the exonemes and is required to process pfsub into an active form. (c) cleavage of pfsub by exoneme resident plasmepsin x leads to activation of the egress regulating papain-like proteases sera and sera , with loss of sera / activity preventing merozoite egress from schizonts. release and activation of pfsub also leads to the cleavage of a number of merozoite invasion ligands including msp , msp , msp , ama , with the rhoptry antigen rap processed by plasmepsin ix (pmix). whilst these ligands are largely not required until merozoite contact with the rbc and invasion commences (see fig. ), inhibition of these cleavage events around schizont egress is associated with loss of invasion. inhibitors have been labelled using a dual-colour system that allows their activity against merozoite development/egress (this figure) and their latter effects against invasion (fig. ) to be highlighted for inhibitors of: pmix (purple/green), pmx (purple/blue), pfsub (purple/orange). to replicate. as a consequence, the essential step of viral entry into the host cell has been targeted by peptide/protein mimics and small molecule entry inhibitors for a diverse range of viruses including hiv (kilby et al. ; dorr et al. ) , orthomyxoviruses (malakhov et al. ) , flaviviruses (wang et al. ), paramyxoviruses (lambert et al. ; rapaport et al. ) , filoviruses (watanabe et al. ) and coronaviruses (bosch et al. ) . the three-step process of viral entry, consisting of attachment, co-receptor binding, and fusion, precedes release of the viral genome into the host's cytoplasm where new virions are assembled before budding and release of the virions from the cell. hiv entry into memory cd + t-lymphocytes (klatzmann et al. ; ho et al. ) is reliant on a very small number of viral-protein/host-protein interactions and protease cleavage events. similar to malaria rbc invasion, hiv cell-entry is orchestrated by a multi-step process and is completed within a fraction of the total viral generation time (defined as time between virion release, infection of a new host cell and generation of daughter viral particles (excluding viral latency), estimated between and hours ( . - . days) (dixit et al. ; murray, kelleher and cooper ; puller, neher and albert ) ). host cell entry begins with virion attachment, binding to the host's coreceptor and entry; with inhibitors, both clinically approved and in development, identified to target each step (reviewed in kuritzkes ; henrich and kuritzkes ) . maraviroc, the first licenced chemokine receptor antagonist and first host targeting antiretroviral drug, specifically inhibits entry of hiv isolates by binding to the host cell receptor, c-c chemokine receptor type (ccr ), one of two chemokine receptors that hiv viruses use for entry into the cell (dorr et al. ; wood and armour ) . maraviroc specifically inhibits the entry of ccr -tropic (r ) viruses and is routinely used in second-line antiretroviral combination therapies against r hiv viruses (reviewed in perry ). after binding of the host cell receptors ccr or cxcr (cxc chemokine receptor type ), a conformational change occurs within the virion membrane which exposes the heptad repeat domains on viral envelope glycoprotein gp (heptad repeat (hr ) and hr ) (reviewed in klasse ) . enfuvirtide, the first antiretroviral fusion-inhibitor approved for hiv treatment, is a -amino acid synthetic peptide that mimics the hr region of gp that binds to hr , preventing the formation of the sixhelix gp bundle that is critical for viral fusion and entry (kilby et al. ) . enfuvirtide is typically active against hiv- isolates that are resistant to other classes of antiretroviral drugs and is reserved for second-line combination therapies of advanced stage hiv infections (kitchen et al. ) . from the apical tip organelles; the rhoptries and micronemes. the rhoptry antigen pfrh binds to its rbc receptor basigin in a key early interaction required for merozoite invasion. (d) an irreversible tight-junction is formed when the microneme-secreted protein ama binds to the rhoptry neck protein complex that is embedded on the rbc membrane. (e) entry of the parasite is powered by an actin-myosin motor that pulls the rbc around the invading merozoite while (f) the surface coat of msps is simultaneously shed. calcium signalling and phosphorylation by kinases are thought to play a key role in controlling the sequence of events required for invasion during this period. the vacuole membrane fuses behind the invading parasite forming a parasitophorous vacuole. (g) shortly after internalization, a large proportion of rbcs temporarily distort in a process known as echinocytosis. it has been postulated that echinocytosis is caused by rhoptry secretion and rapid entry of ca + discharged from the rhoptries into the rbc during invasion, but a more recent explanation suggests that it is incorporation of parasite rhoptry contents into the rbc membrane which leads to rbc membrane ruffling (dvorak et al. , gilson and crabb ) . examples of drug inhibitors which act at certain stages of the invasion process are labelled in purple. labels with two colours indicate the inhibitor also has activity around merozoite egress (see fig. ). although the cellular targets and kinetics of hiv entry into cd + t-lymphocytes differ to the requirements of plasmodium invasion into rbcs, these examples of clinically used hiv entry inhibitors for treatment of disease provide an informative comparison for considering the development of antimalarial drugs that target rbc invasion. the majority of current antimalarials target the blood stage of the lifecycle and work through various targets such as; (i) the parasites intracellular food vacuole (chloroquine, artemisinin) (fidock et al. ; klonis et al. ) , (ii) dna replication (pyrimethamine) (cowman et al. ), (iii) mitochondrion function (atovaquone (fry and pudney ) and proguanil (dickerman et al. ) ) or, (iv) the apicoplast, the parasite's remnant plastid organelle (doxycycline, azithromycin, clindamycin) (dahl and rosenthal ; goodman, su and mcfadden ) . currently, no clinically used antimalarial has activity against rbc invasion (wilson et al. ) , except azithromycin when used at higher concentrations (wilson et al. ) . in vitro live-cell filming of p. falciparum has shown that rbc invasion, from formation of the tight junction to completion of rbc entry, generally takes less than min (gilson and crabb ) . however, the time taken for a merozoite to commence invasion after egress from a schizont is variable, with one study finding that it took min for % of invasion events to be completed in vitro. depending on the drug target, key processes required for rbc invasion could be susceptible to an antimalarial during merozoite development and schizont egress (fig. ac) or during the process of invasion itself ( fig. a-g) . to be clinically useful, invasion-inhibitory drugs would need to have a long half-life so that the drug can be maintained in the blood at a high enough level to inhibit invasion as it occurs. a drug with a very short half-life (i.e. artesunate has a half-life of < minutes; dondorp et al. ) would not be suitable. of interest, the halflife of the hiv entry inhibitor maraviroc is ∼ hours (perry ) . the myriad of essential and unique targets required to work in a coordinated fashion to enable the rapid process of invasion, combined with the sensitivity of the invasion process to perturbation, provides a promising avenue for new antimalarial development. in this review, we highlight several essential processes targeted in invasion-inhibitory drug development (figs and ) and outline some of the compounds that have been tested to date (supplementary table s ). heparin, a member of the glycosaminoglycan family, is a known inhibitor of rbc invasion (butcher, parish and cowden ; boyle et al. ) . a diversity of other sulfated carbohydrates and heparin-like-molecules (hlms) have also been identified to inhibit invasion of p. falciparum merozoites, including curdlan sulfate (havlik, rovelli and kaneko ; evans et al. ), polyvinyl-sulfonate sodium salt (kisilevsky et al. ) , suramin (fleck et al. ) , carrageenans (adams et al. ) , sulfated cyclodextrins (crandall et al. ), fucosylated chondroitin sulfate (bastos et al. ) , k polysaccharides , inulin sulfate, xylan sulfate, tragacanth sulfate and scleroglucan sulfate (boyle et al. ) . furthermore, hlm invasion-inhibitory activity has also been reported for the zoonotic malaria parasite p. knowlesi (lyth et al. ) and p. berghei (xiao et al. ) , indicating that pan-species invasion inhibition of human malaria parasites is achievable with these molecules. although precise mechanisms of action for sulfated carbohydrates in inhibiting invasion are not fully understood, hlms have been reported to inhibit the earliest step in invasion, initial rbc attachment, and to bind msp , as well as to rhoptry and microneme proteins involved in reorientation and signalling steps of invasion ( fig. a-c) (baum et al. ; kobayashi et al. ; kobayashi et al. ) . therefore, it is likely that these compounds target multiple essential ligands in the invasion process, thus reducing the potential for developing drug resistance. indeed, attempts to generate heparin resistant parasite strains in vitro have been unsuccessful . although limited studies have been performed to evaluate the activity of hlms in vivo, there is data from animal models (xiao et al. ) and human clinical trials supporting their potential development (havlik et al. ; leitgeb et al. ) . heparin has been historically used as an adjunct treatment for disseminated intravascular coagulation that can occur in severe malaria (smitskamp and wolthuis ; munir et al. ; rampengan ) , but its use was stopped because its anticoagulative properties led to an increased risk of bleeding. recently, heparins with periodate oxidation of non-sulfated uronic acid residues that greatly reduced anticoagulation activity of heparin (pisano et al. ) were shown to be highly inhibitory to rbc invasion (boyle et al. ) . similar hlms have been tested for inhibition of lung cancer growth in mice with no anticoagulation activity reported across a range of tissue (yu et al. ) . of further promise, curdlan sulfate ) has a -fold reduced anticoagulative activity and testing in a small human trial suggested that treatment reduced malaria disease severity (havlik et al. ) . a recent phase i clinical trial of the nonanticoagulant hlm sevuparin, a negatively charged polysaccharide manufactured from heparin, limited parasite replication by blocking invasion (leitgeb et al. ). hlms such as sevuparin also disrupt pathogenic mediators such as rosetting and sequestration of infected rbcs, (udomsangpetch et al. ; carlson et al. ; rowe et al. ; barragan et al. ; vogt et al. ; kyriacou et al. ; skidmore et al. ; bastos et al. ; saiwaew et al. ) , potentially enabling hlms to provide dual protective mechanisms of action against severe malaria. current hlms that have been identified with antimalarial activity have relatively low potency (boyle et al. ) and they have also been reported to have relatively short half-lives when used clinically (i.e. heparin < hour (perry, herron and king ) , sevuparin ∼ hour (leitgeb et al. ) , curdlan sulfate ∼ - hours (gordon et al. ) ). however, the clinically used hlm fondaparinux has a longer half-life ( - hours) (donat et al. ) . work on improving oral availability of heparin derivatives (reviewed in neves et al. ) , as well as prolonging hlm drug activity and potency (hoffart et al. ; boyle et al. ) are ongoing and offer an avenue for hlms to be developed as antimalarials with invasion-inhibitory activity. initial interactions between the merozoite and the rbc membrane are low affinity, reversible and can occur irrespective of the parasite's orientation. these interactions are mediated by glycosylphosphatidyl inositol (gpi) anchored proteins present on the merozoite's surface gilson et al. ) . msp is the most abundant gpi anchored protein on the merozoite surface (gilson et al. ) and the proteolytic cleavage of msp to kda, kda, kda and kda fragments is essential for rbc invasion . the n-terminal msp - fragment binds to the rbc receptor glycophorin a and the cterminal msp - fragment has a role in binding to band- on the rbc surface. (baldwin et al. ) . the kda c-terminal cysteine rich epidermal growth factor (egf)-like domain of msp- is formed after secondary cleavage of msp - and this essential proteolytic event has been investigated as a potential vaccine and drug target (goel et al. ) . testing of egf domain inhibitors with anticancer properties against msp - identified a small-molecule glycan mimetic, -butyl- -chloro- -( -nitro-benzyl)- h-imidazole- -carbaldehyde (nic), as a specific inhibitor of msp - function and parasite invasion (fig. b, c) (chandramohanadas et al. ). the invasion-inhibitory activity of nic was confirmed using live filming of invasion and through the use of purified merozoites. nic not only inhibited the growth of p. falciparum isolates, it also inhibited p. falciparum expressing p. chabaudi rodent malaria msp - and p. vivax field isolates, with ic s ∼ μm; indicating the pan-species potential of these molecules against malaria parasite invasion. the authors highlight the possibility of targeting the egf domain of msp - using small molecule glycans that are being developed as anti-cancer agents ( fig. b ) (sugahara et al. ), but to date no examples of this have been published and further development of this strategy would be required before clinical applications could be assessed. a potential advantage of developing inhibitors that target msps, such as hlms and glycan mimetics, is that they can target merozoites throughout their extracellular phase; post release from schizonts through to resealing of the parasitophorous vacuole membrane. after binding to the rbc and apical re-orientation, the invading merozoite releases proteins residing within specialised apical secretory organelles, the micronemes and rhoptries, to establish an irreversible zone of attachment called the tight junction (aikawa et al. ; bannister and mitchell ) . this tight junction is formed as a result of ama (secreted from the micronemes) binding to the rbc bound rhoptry neck (ron) / / (secreted from the rhoptries) protein complex (alexander et al. ; collins et al. ; richard et al. ; tonkin et al. ) , with a known high affinity interaction demonstrated between ama and ron (srinivasan et al. ; tonkin et al. ) . the essential interaction between ama and ron has been targeted by vaccine induced antibodies (hodder, crewther and anders ; kennedy et al. ) , inhibitory peptides and drug development (reviewed in devine et al. ) (fig. d) . a phase b vaccine trial of children in mali demonstrated high anti-ama antibody titres and protection against clinical malaria caused by parasites harbouring vaccine-like alleles after months, but there was minimal efficacy against clinical malaria overall, highlighting the difficulties with targeting a polymorphic antigen such as ama (thera et al. ) . however, recent studies report substantial conservation of ama function between species, providing evidence that generating cross-species inhibitory activity may be possible (drew et al. ) . several invasion-inhibitory peptides that target ama /ron binding have been developed. the -amino acid r peptide, identified from a random phage display library , exhibits high binding affinity for the d parasite line ama /ron complex (kd ∼ . μm) . ron l mimics a conserved peptide region of ron and competes with native ron for the hydrophobic binding pocket of ama , blocking formation of the tight junction and inhibiting rbc invasion (srinivasan et al. ) . making use of the high binding affinity of peptides that block ama /ron interactions, a ron l(peptide)/ama binding inhibition assay was used to screen small-molecule inhibitors for activity against ama /ron (srinivasan et al. ) , with three hits suggested to directly inhibit rbc invasion. modification of the lead compound, nchc (a pyrrolopyrimidine; ic μm), yielded two analogues that showed a three ( . μm) and five ( μm) fold improvement in invasion inhibition (srinivasan et al. ) . however, subsequent studies failed to show binding of these compounds to ama with an affinity commensurate with their reported growth inhibitory activity (devine et al. ; pihan et al. ) , leading devine et al. ( ) to conclude that these compounds inhibited invasion through an ama /ron independent manner. nevertheless, the essential role of the ama /ron complex for invasion, the availability of complete protein structures for in silico screening and optimisation makes inhibitors of ama /ron complex function an attractive target for further development. after formation of the tight junction, the actin-myosin motor is engaged and the rbc membrane is pulled around the merozoite via treadmilling of short actin filaments (f-actin) which are pulled unidirectionally. invasion is powered by a myosin motor complex embedded in the merozoite's pellicle (inner membrane complex; fig. e ) (soldati, foth and cowman ) (reviewed in tardieux and baum ) . given the importance and complexity of the actin-myosin motor, a number of targets have been investigated for antimalarial development. a number of natural agents, such as cytochalasins (a fungal alkaloid) and latrunculins (from marine sponges) have been reported to disrupt actin polymerisation dynamics and ultimately arrest rbc invasion (fig. e) (miller et al. ; cooper ; johnson et al. ) . latrunculins bind to actin's monomeric form (gactin) near the adenosine triphosphate (atp) binding site and prevent polymerisation to filamentous actin (f-actin). a recent study identified key amino acid differences between human and plasmodium spp. actin within the atp binding pocket and sought to synthesise truncated latrunculin b analogues with improved activity against p. falciparum malaria and reduced toxicity against mammalian cells (johnson et al. ) . truncated latrunculin analogues achieved a -fold improved potency against in vitro parasite growth (to μm) and -fold higher selectivity over mammalian cell cytotoxicity (johnson et al. ) . to address whether these analogues had activity directly against parasite invasion, the authors used t. gondii invasion inhibition assays since this related apicomplexan parasite shares an identical amino acid sequence around the actin atp binding pocket as p. falciparum (johnson et al. ) . lead latrunculin analogues showed a > -fold improvement in invasion-inhibitory activity against t. gondii (to μm), indicating that latrunculin analogues target parasite actin during invasion and that activity against apicomplexan parasites is likely to be conserved (johnson et al. ) . but the high ic s of these compounds against both parasites highlights that further development is needed. myosin a (myoa) is the f-actin bound motor that powers apicomplexan gliding motility during invasion (meissner, schluter and soldati ) (fig. e) . the atp-powered protomotive movement of myoa is dependent on a conserved complex between myoa's c-terminal domain and the conserved nterminal domain of myoa tail interacting protein (mtip, called myosin light chain (mlc ) in t. gondii) (bosch et al. (bosch et al. , . not only is the interaction between myoa and mtip essential for parasite invasion of the rbc, but structural characterisation has also identified distinct differences between plasmodium spp. and human homologs, thus presenting a viable target for drug development (bosch et al. ) . modelling of the interaction between mtip and a growth inhibitory -amino acid c-terminal myoa peptide (bosch et al. ) was used to identify small molecule mtip/myoa binding inhibitors in a library of compounds (kortagere ) . a pyrazole-urea based compound (c ) demonstrated the best growth inhibitory activity (ic of . μm) (kortagere ) and further structure-based screening identified several analogues with improved activity over the original peptide (c - ic . μm; c - ic . μm). c - was investigated further and was found to inhibit gliding motility of mosquito stage sporozoites, a marker assay for actin-myosin based motor function that is shared between sporozoites and rbc invading merozoites (kortagere ) . comparative analysis identified that some compounds inhibited growth of both p. falciparum and t. gondii, thus suggesting a conserved target between the two divergent parasites. however, many analogues also showed variation in efficacy between p. falciparum and t. gondii, suggesting structural differences between the myoa/mtip interaction can be sufficient to reduce efficacy against different apicomplexan parasites (kortagere ; kortagere et al. ) . another high-throughput screen of , non-cytotoxic compounds against t. gondii tachyzoite host cell invasion identified compounds that inhibited parasite motility (carey et al. ). one of these hits, tachyplegin a, was found to covalently bind to tgmlc (mtip in plasmodium spp.) and the resulting post-translational modifications caused loss of myoa function and inability to drive the invasion motor (carey et al. ; heaslip ; leung et al. ). these studies have identified a diverse range of chemical scaffolds that inhibit function of the mtip/myoa driven invasion motor, a conserved complex that is essential for rbc invasion of apicomplexan parasites. invasion requires a co-ordinated series of proteolytic cleavage events to enable the correct function of essential proteins. the essential role of serine proteases in schizont rupture and rbc invasion have seen them become a significant target of invasion inhibitor drug development (reviewed in o'donnell and blackman ). the p. falciparum subtilisin proteases pfsub (blackman et al. ; yeoh et al. ) and pfsub are bacteriallike enzymes that have received significant interest because of the key role they play in the essential processing of proteins required for rbc invasion (figs. and ) (supplementary table s ). pfsub has been shown to cleave msps (msp , msp and msp ), invasion ligands released from the micronemes (ama ) and rhoptry (rap ) (yeoh et al. ; koussis et al. ; silmon de monerri et al. ) and is involved in priming the proteolytic cascade that leads to schizont rupture and merozoite egress (fig. c) (yeoh et al. ). comparison of the stage-specific efficacy of the pfsub inhibitor mrt indicates that the ic against p. falciparum in vitro invasion inhibition (∼ μm) was lower than that for schizont rupture inhibition (∼ μm) (yeoh et al. ) , highlighting the potential sensitivity of the invasion process to chemical inhibition compared to other stages of blood stage development. interestingly, a follow-up study identified that even partial inhibition of msp processing at invasion inhibitory concentrations of mrt was associated with invasion inhibition, indicating the sensitivity of the invasion process to chemical inhibition (koussis et al. ). more recent studies have begun to optimise inhibitors of pfsub from a range of chemical scaffolds (gemma et al. ; bouillon et al. ; giovani et al. ; kher et al. ) . plasmodium spp. sub are highly conserved and trials using recombinant proteins suggest that 'pan-species' inhibitors can be developed that target the sub of multiple species (withers-martinez et al. ) . indeed, an in silico screen using a d homology model of pvsub led to the discovery of cpd , a compound that inhibits the activity of both recombinant pvsub and pfsub (bouillon et al. ). furthermore, cpd had an in vitro ic of . μm against p. falciparum parasites and inhibited growth of p. berghei rodent malaria parasites in a dose-dependent manner, highlighting the pan-species potential of sub inhibitors (bouillon et al. ) . recently its been demonstrated that the aspartic proteases plasmepsin ix and x (nasamu et al. ; pino et al. ) have key roles in rbc invasion (fig. d , f) and invasion/egress (fig. b, c) , respectively. plasmepsin ix is located in the rhoptry organelle in merozoites and loss of this protease causes aberrant rhoptry formation and prevents cleavage of key invasion ligands (nasamu et al. ; pino et al. ) . plasmepsin x is located in merozoite exonemes (secreted from the merozoite prior to rupture) and is involved in activating sub (essential for invasion and schizont rupture), as well as directly processing ligands excreted from the microneme (nasamu et al. ; pino et al. ) . the activity of plasmepsin ix and x was effectively inhibited by the hydroxylethylamine aspartic protease inhibitor c (ciana et al. ) at low nanomolar concentrations, providing evidence that both essential proteases can be targeted by one drug (pino et al. ) . furthermore, c was effective in a p. berghei rodent model of malaria against multiple lifecycle stages, including liver stage parasites and gametocytes (pino et al. ) . recombinant plasmepsin x was found to be inhibited by the orally bioavailable aminohydantoins (meyers et al. ; nasamu et al. ) . further investigation revealed the aminohydantoins inhibited p. falciparum growth in vitro at submicromolar concentrations and growth of p. chabaudi rodent malaria parasites in vivo, providing a second starting point for drug development against plasmepsin x (meyers et al. ; nasamu et al. ) . since c is a potent inhibitor of plasmepsin ix (rhoptry biogenesis and invasion ligand processing) and both c/aminohydantoins inhibit plasmepsin x function (activation of the egress/invasion priming pfsub , invasion ligand processing), both chemical starting points offer activity against merozoite egress and invasion (nasamu et al. ; pino et al. ). the targeting of cellular signal transduction pathways has successfully been used to treat non-infectious diseases such as cancer and autoimmune diseases (aggarwal et al. ; croce et al. ; marciano and holland ) and is now of increasing interest for antimalarial development. one leading drug target involved in signalling during rbc invasion is calcium dependent protein kinase (cdpk ), a parasite kinase not present in the human host (harper and harmon ) that has key roles in microneme secretion, activation of the actin-myosin motor and other processes required for rbc invasion (fig. b-e) (green et al. ; bansal et al. ; bansal et al. ). pfcdpk has been targeted in several high throughput screens (hts) of compound libraries (green et al. ; kato et al. ; lemercier et al. ; chapman et al. ; ansell et al. ; chapman et al. ). , , trisubstituted purines such as purfalcamine inhibited p. falciparum parasite growth (ic of nm) as well as host cell invasion of related t. gondii tachyzoites, consistent with a cdpk in t. gondii having a key role in invasion (kato et al. ; lourido et al. ; kumar et al. ) . however, purfalcamine was unsuccessful in clearing p. yoelii rodent malaria parasites in vivo, possibly due to poor pharmacokinetics and reduced efficacy against pycdpk (kato et al. ) . a second screen identified , -disubstituted imidazopyridazines as compounds of interest, with modification of early leads reducing the growth inhibitory ic to < nm and providing superior pharmacokinetics to the initial hit compounds (chapman et al. ; chapman et al. ) . however, in vivo efficacy against p. berghei rodent malaria parasites was again limited (chapman et al. ; chapman et al. ) . further investigation revealed that a number of optimised imidazopyridazine pfcdpk inhibitors were more likely to be targeting p. falciparum cgmp dependent protein kinase g (pfpkg; green et al. ) . based on conflicting evidence for whether pfcdpk is essential for blood stage parasite growth, the limited efficacy in vivo and variable specificity of inhibitors, it has been suggested that pfcdpk may not be suitable for blood stage drug development bansal et al. )(reviewed in cabrera et al. ) . pfpkg has also been a focus for antimalarial development since it is expressed in multiple stages of the lifecycle and has different activation properties to mammalian kinases (mcrobert et al. ; alam et al. ; govindasamy et al. ) . inhibitors of pfpkg are potent inhibitors of merozoite egress from the developed schizont and are being developed as antimalarials (taylor et al. ) . studies have also identified that inhibition of pfpkg blocks invasion of mechanically released merozoites, with speculation that this invasion-inhibitory activity is due to preventing discharge of the invasion priming protease pfsub (fig. b , c) and interfering with phosphorylation of proteins thought to have a role in invasion including pfcdpk and invasion motor components (fig. b-e) (collins et al. ; alam et al. ; das et al. ) . recently, a highly potent series of compounds were developed based on an imidazopyridine inhibitor of pkg used for treatment of the apicomplexan parasite eimeria tenalla in chickens. the most active of these, ml , had an ic of nm against p. falciparum parasite growth in vitro. ml was also highly efficacious in a p. chabaudi rodent model of malaria, with twice daily doses of mg/kg for days reducing parasitemia to undetectable levels in a p. falciparum humanized mouse model of malaria (a promising outcome for development of a pkg inhibitor for inclusion in combination therapies. development of inhibitors against camp-dependent protein kinase a (pka), which has key roles in microneme secretion (dawn et al. ) , phosphorylation of the functional domain of ama (leykauf, ) and activation of the actin-myosin motor (lasonder et al. ) , has been less successful. general inhibitors of pka, such as h and kt , and its messenger molecule camp have been used as biological tools to block pfpka and to study its function, but these compounds have low potency (ic typically > μm) (syin et al. ; beraldo et al. ; leykauf et al. ; salazar et al. ) and we are currently unaware of any compounds that have been optimized for activity against pfpka and camp (buskes et al. ; cabrera et al. ). an alternative strategy to inhibit invasion is to target ', 'cyclic nucleotide phosphodiesterases (pdes) which regulate degradation of camp and cgmp into amp and gmp, respectively. increased camp and cgmp leads to activation of pka and pkg, respectively, making pdes significant regulators of signalling during egress and invasion (fig. c) (collins et al. ; . screening of human pde inhibitors identified zaprinast (growth inhibitory ic of μm) (yuasa et al. ) and a pyrazolopyrimidinone, termed bippo, (growth ic of . μm) (howard et al. ) as having activity against pfpdeα, an isoform that specifically inhibits cgmp. since pfpdeα has been demonstrated to be dispensable to blood stage parasite growth (wentzinger et al. ) and treatment with bippo causes activation of camp (pfpdeβ) and cgmp (pfpdeα) dependent pathways, it has been suggested that bippo may inhibit multiple pfpde isoforms due to conservation in active sites (wentzinger et al. ; howard et al. ) . modelling suggests that there are key similarities between human, plasmodium and toxoplasma pde orthologues that would support this cross-reactivity (howard et al. ) . indeed, bippo retains activity against isoforms of human pdes, including pde (ic = nm), and selectivity for plasmodium pdes would need to be greatly improved before this pde inhibitor could be developed as an antimalarial (howard et al. ) . although a number of kinases with key roles in rbc invasion have been identified, the development of effective inhibitors against these signalling molecules is still a work in progress with improvements in specificity and potency required for many early leads. however, the feasibility of targeting signalling effector molecules can be demonstrated by recent efforts to develop inhibitors against phosphatidylinositol -kinase (pi k), a key enzyme in protein trafficking required across multiple lifecycle stages, including merozoite development (mcnamara et al. ). this has led to the -aminopyrazine compound uct being taken forward into pre-clinical development (brunschwig et al. ). hts of small molecule or compound libraries have been used extensively to identify growth inhibitory compounds of the asexual blood stages of p. falciparum malaria. however, relatively few screens have been directly designed to identify inhibitors of rbc invasion. medicines for malaria venture (mmv) released a -compound library in , termed the malaria box, which contains a diverse set of compounds that display antimalarial properties (spangenberg et al. ) . subramanian et al. ( ) recently screened the malaria box for activity against p. falciparum blood stage egress and merozoite invasion inhibitors (subramanian et al. ) and identified out of hits that inhibited the schizont to ring stage transition at an ic of < nm. upon microscopic examination of blood smears, mmv and mmv treated schizonts ruptured normally, but free merozoites were found attached to rbcs and few successful invasion events were evident, a phenotype typical of invasion inhibitors (weiss et al. ) . further testing revealed that mmv and mmv were potent invasion inhibitors with up to % of invasion events inhibited at concentrations down to nm in assays using purified merozoites (subramanian et al. ) . of the compounds identified in this screen, of them have been characterised as inhibitors of pfatp , a sodium efflux pump on the parasite plasma membrane, indicating either pfatp is involved in egress and invasion, or that the compounds have targets additional to pfatp (lehane et al. ; subramanian et al. ) . screens of the related apicomplexan parasite t. gondii have opened up new starting points for invasion-inhibitory drug development against apicomplexan parasites. t. gondii in vitro motility and invasion assays were used as secondary screens against a library of putative kinase inhibitors (kamau et al. ) . of the lead compounds with growth inhibitory or enhancing affects, compounds c (ic . μm) and c (ic . μm) were found to irreversibly inhibit motility or motility and invasion, respectively. a second study using a fluorescence microscopy based assay screened covalent inhibitors directly for inhibition of t. gondii tachyzoite attachment and invasion, identifying invasion-inhibitory compounds. the leading compound, wrr- , demonstrated low micromolar (ic of . μm) invasion-inhibitory activity. biochemical and genetic analysis identified a homologue of human dj- (tgdj- ) as the target of wrr- , with inhibition of tgdj- linked to loss of microneme secretion and failure to invade (hall et al. ) . screening compounds for their invasion-inhibitory activity is providing starting points for the development of drugs with novel mechanisms of action and uncovering new insights into invasion biology of apicomplexan parasites. the majority of compounds identified that have invasioninhibitory activity against malaria parasites have no record of clinical use. recent identification of the invasion-inhibitory activity of the antibiotic azithromycin (wilson et al. ) marks one of the few clinically used compounds that have been shown to inhibit plasmodium spp. invasion of rbcs. macrolide antibiotics are known to target the malaria parasite's remnant plastid (the apicoplast) s bacteria-like ribosomal complex (sidhu et al. ; goodman et al. ) . inhibition of the apicoplast ribosome prevents replication of this essential organelle, resulting in the loss of isoprenoid pyrophosphate (ipp) precursor synthesis and parasite death a full two cycles of growth post treatment (termed delayed death) (dahl and rosenthal ; goodman, su and mcfadden ) . despite the limitations of a slow killing antimalarial for treatment of disease, azithromycin's safe clinical profile and long half-life (> hours) has led to the antibiotic being trialled as a partner drug in artemisinin combination therapies (cook et al. ; sykes et al. ). recently it was found that azithromycin could rapidly inhibit rbc invasion in vitro (ic , μm, in ethanol), which is independent of apicoplast-targeted delayed death (ic , . μm, in ethanol) activity (wilson et al. ) . although the speed of azithromycin's invasion-inhibitory activity for an otherwise slow acting drug provides a new avenue to develop the drug as an antimalarial, the requirement for a -fold higher concentration of azithromycin needed to inhibit invasion currently prevents clinical use of this drug as an invasion inhibitor. however, of note is the identification of several analogues that show > -fold improvement in invasion-inhibitory activity (wilson et al. , burns et al. unpublished data) , indicating that improved invasion-inhibitory potency is achievable. importantly, the most invasion-inhibitory azithromycin analogues also exhibit improved activity against short-term blood stage parasite development and retain activity against the apicoplast, suggesting that azithromycin can be developed to have both fast acting (rbc invasion inhibition and short-term parasite growth inhibition) and apicoplast-targeting delayed death properties (wilson et al. , burns et al. unpublished data) . given azithromycin's history of safety, proven activity, long half-life (> hours), availability of modified analogues and ease of modification, the identification of azithromycin's invasion-inhibitory activity opens up an attractive starting point to develop an invasion-inhibitory antimalarial with dualmechanisms of action. a focus of antimalarial development for treatment of clinical disease is on single-dose drug combinations that act broadly across blood-stage development to quickly kill parasites (burrows et al. ) . as a standalone drug, it is unrealistic to expect an antimalarial which only targets invasion will eliminate all parasites within a matter of hours. however, such a drug could be of benefit in a combination therapy and, as demonstrated in this review, many invasion inhibitors have activity against other lifecycle stages. combination therapies that have two (or more) safe and efficacious drugs with different mechanisms of action have significant potential advantages, including reducing the risk of developing drug resistance (hastings ) . importantly, the reported mechanisms of action for invasion inhibitors developed to date are not involved in the mechanisms of action of existing antimalarials, limiting the likelihood of cross-resistance. evidence from rodent models of malaria suggest that antimalarial monotherapy using drugs that target intracellular parasite growth may not prevent all parasites from progressing through to the next cycle (khoury et al. ). failure to rapidly inhibit blood stage replication may increase the risk of selecting for drug resistance and lead to higher numbers of mosquito transmissible gametocytes posttreatment, thereby contributing to transmission. targeting invasion directly, the first step in blood stage parasite growth, in a combination therapy would immediately stop progression of parasites into the next cycle of growth, thus limiting opportunities for drug resistance to develop and reducing the number of new gametocytes. combining a drug that targets intracellular parasite development (timing of action of current antimalarials) with one that inhibits rbc invasion (extracellular) has intrinsic appeal as targeting these two developmental stages could facilitate rapid clearance of disease causing blood stage parasites and increase drug efficacy. evidence from monotherapy drug efficacy studies of severe malaria patients suggests that rapid parasite clearance after treatment results in reduced mortality (dondorp et al. ) . complicating the speed of parasite clearance, studies have highlighted that a number of clinically used antimalarials have reduced efficacy as malaria parasites transition from mature schizonts, through invasion and into newly established ring stage infections (painter, morrisey and vaidya ; wilson et al. ; dogovski et al. ; khoury et al. ) . since each surviving p. falciparum schizont is capable of releasing - new rbc invading merozoites, providing additional cover through a drug combination that includes a potent invasion inhibitor has the potential to fast-track parasite clearance. since p. falciparum invasion occurs roughly every hours, this raises the question as to whether clinical treatment with a drug that has a short half-life risks being ineffective across one growth cycle if administered temporally distant from the next period of rupture and invasion. studies evaluating circulating and sequestered populations of parasites in infected subjects have generally found a wide developmental age range for parasite populations at the time of sampling; predominantly young parasites in peripheral blood and mostly mature stages for parasites sequestered in capillaries (but younger parasites can also be at high levels) in cerebral malaria, non-cerebral malaria and placental malaria cases (macpherson et al. ; oo et al. ; silamut et al. ; beeson et al. ; pongponratn et al. ) . these studies indicate that there is limited parasite synchronicity in vivo and it is likely that invasion inhibitors will encounter invading merozoites soon after administration and regularly across the next hours. in terms of ideal drug properties, invasion inhibitors should have: (i) a half-life that allows the drug to be maintained at effective concentrations with a dosing regimen no more frequent than daily, and (ii) an effective concentration well below that of each dose, allowing inhibitory concentrations of drug to be available over a time period equivalent to many cycles of parasite invasion and growth. as demonstrated by the clinical use of the hiv entry inhibitor maraviroc (half-life ∼ hours; perry ), maintaining drug concentrations to inhibit pathogen host cell entry over several replication cycles is clinically achievable. in terms of clinically used compounds with invasion-inhibitory antimalarial activity, the half-life is known for azithromycin (> hours) and several hlms (heparin < hour, sevuparin ∼ hour, curdlan sulfate ∼ - hours and fondaparinux - hours), and the half-life will need to be a consideration for any invasion inhibitors with higher potency. drug resistance models suggest that the increased selective window (when drug levels fall below the minimal inhibitory concentration) of long-lasting drugs can potentially increase drug resistance selection pressure posttreatment (stepniewska and white ; kay and hastings ) . in contrast, drugs with a short half-life, such as the artemisinins, have a much shorter selective window and are considered less likely to select for resistance due to minimal parasite exposure to sub-inhibitory concentrations (stepniewska and white ) ; but more frequent dosing is required to maintain treatment efficacy. therefore, there are several important considerations in selecting ideal drug combinations with the potential impacts on clinical efficacy, reducing the risk of drug resistance and reducing transmission all needing to be assessed for combination therapies that include an invasion inhibitor. despite the potential benefits of having an invasion-inhibitory drug in antimalarial combination therapies, the therapeutic efficacy of a drug combination featuring both an invasion inhibitor and an intracellular blood stage growth inhibitor in vitro or in vivo has yet to be assessed directly. thus, future studies will need to assess the potential synergies of using an invasion inhibitor in a combination therapy as well as model the therapeutic and resistance-proofing benefits of doing so. targeting rbc invasion is a promising antimalarial drug development strategy because: (i) extracellular parasites are exposed directly to drugs in the bloodstream, (ii) most parasite proteins required for invasion lack human equivalents, offering possibilities for selective inhibition and (iii) blocking invasion immediately stops multiplication of disease causing blood stage parasites. inhibition of host cell entry is a validated strategy for hiv combination therapies (kilby et al. ; dorr et al. ) and the predicted viral generation time of hiv (≥ hours; dixit et al. ; murray, kelleher and cooper ; puller, neher and albert ) is similar to the blood stage lifecycle of p. falciparum. therefore, the clinical use of hiv entry inhibitors provides a proof-ofconcept that inhibitors of rbc invasion can have a role in antimalarial combination therapies. the targets of invasion-inhibitory antimalarials under development are mostly essential, conserved and non-redundant (i.e. yeoh et al. ; kortagere ; wilson et al. ; pino et al. ) . the conservation evident in key, drug targetable, invasion machinery between malaria isolates, different plasmodium spp. and different lifecycle stages (i.e. sporozoite invasion) is leading to the development of pan-invasion inhibitors. therapeutic inhibition of invasion is likely to have profound effects on parasite viability since the merozoite has a short half-life and failure to invade immediately ends parasite growth and multiplication. this would mitigate the risk that parasites develop drug tolerance and persist as is the case for artemisinin resistance. merozoite invasion may be more sensitive to treatment than other intracellular rbc stages, as demonstrated by a lower ic for invasion inhibition (∼ μm) than achieved for rupture inhibition (∼ μm) for the pfsub inhibitor mrt (yeoh et al. ) . therefore, improving a drug's activity against the process of merozoite invasion could have a significant impact on parasite clearance and clinical effectiveness. to date, a number of diverse chemotypes with different targets have been identified to inhibit rbc invasion (supplementary table s ), but there is tremendous scope to develop new inhibitors of this essential step in parasite growth for use in combination therapies. the search for new invasion-inhibitory targets is helped by the availability of published mature schizont stage proteomic and phosphoproteomic resources (www. plasmodb.org) that highlight potential merozoite specific therapeutic targets for assessment (solyakov et al. ; lasonder et al. ) . encouragingly, the development of specific assays to quantify the invasion-inhibitory activity of compounds (wilson et al. ; wilson et al. ; weiss, crabb and gilson ) has led to the identification of new chemical scaffolds that inhibit invasion. although several compounds with invasion-inhibitory activity have achieved promising levels of potency in vitro and in vivo (gemma et al. ; bouillon et al. ; giovani et al. ; kher et al. ; meyers et al. ; nasamu et al. ; pino et al. ) , an important way forward for invasion inhibitor development is to optimise additional compounds with activity in the low nanomolar range to fast-track further development options. another future research priority is the evaluation of invasioninhibitory compounds in combination with currently used and new emerging therapeutics that target intraerythrocytic parasite development, including artemisinin. such studies would better define the properties and timing of action of drugs to be used in optimal combinations. while proof-of-concept for invasion inhibitors has been demonstrated in animal models, further in vivo studies are needed to better define the therapeutic potential of the different inhibitor classes alone and in combination. incorporation of mathematical modelling, as is increasingly being used in drug evaluation and clinical trials, to 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pan-malaria drug target the discovery of the ccr receptor antagonist, uk- , , a new agent for the treatment of hiv infection and aids sulfated polyanions inhibit invasion of erythrocytes by plasmodial merozoites and cytoadherence of endothelial cells to parasitized erythrocytes subcellular discharge of a serine protease mediates release of invasive malaria parasites from host erythrocytes pfpde , a novel cgmpspecific phosphodiesterase from the human malaria parasite plasmodium falciparum antitumor effect of butanoylated heparin with low anticoagulant activity on lung cancer growth in mice and rats basigin is a druggable target for host-oriented antimalarial interventions key: cord- -rdlinzrn authors: gralinski, lisa e.; sheahan, timothy p.; morrison, thomas e.; menachery, vineet d.; jensen, kara; leist, sarah r.; whitmore, alan; heise, mark t.; baric, ralph s. title: complement activation contributes to severe acute respiratory syndrome coronavirus pathogenesis date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: rdlinzrn acute respiratory distress syndrome (ards) is immune-driven pathologies that are observed in severe cases of severe acute respiratory syndrome coronavirus (sars-cov) infection. sars-cov emerged in to and led to a global outbreak of sars. as with the outcome of human infection, intranasal infection of c bl/ j mice with mouse-adapted sars-cov results in high-titer virus replication within the lung, induction of inflammatory cytokines and chemokines, and immune cell infiltration within the lung. using this model, we investigated the role of the complement system during sars-cov infection. we observed activation of the complement cascade in the lung as early as day following sars-cov infection. to test whether this activation contributed to protective or pathologic outcomes, we utilized mice deficient in c (c (–/–)), the central component of the complement system. relative to c bl/ j control mice, sars-cov-infected c (–/–) mice exhibited significantly less weight loss and less respiratory dysfunction despite equivalent viral loads in the lung. significantly fewer neutrophils and inflammatory monocytes were present in the lungs of c (–/–) mice than in c bl/ j controls, and subsequent studies revealed reduced lung pathology and lower cytokine and chemokine levels in both the lungs and the sera of c (–/–) mice than in controls. these studies identify the complement system as an important host mediator of sars-cov-induced disease and suggest that complement activation regulates a systemic proinflammatory response to sars-cov infection. furthermore, these data suggest that sars-cov-mediated disease is largely immune driven and that inhibiting complement signaling after sars-cov infection might function as an effective immune therapeutic. from coronaviruses circulating in animal markets in china ( ) . emergence of this novel virus led to a global outbreak of respiratory disease, with over , human cases and % mortality ( , ) . in , a new, related zoonotic coronavirus was identified in the middle east, designated middle east respiratory syndrome coronavirus (mers-cov), causing severe respiratory disease with greater than % mortality (www .who.int/emergencies/mers-cov/en) ( ) . both sars-cov and mers-cov cause a range of disease from asymptomatic cases to severe acute respiratory distress syndrome (ards) and respiratory failure ( ) . notably, metagenomics and synthetic virus recovery strategies have since revealed the existence of large pools of preepidemic sars-like bat coronaviruses which replicate in primary human airway epithelial cells. these viruses are poised for emergence because they both efficiently use human ace entry receptors and resist existing vaccines and immunotherapeutics ( , ) . due to the ongoing threat and continued emergence of new, highly pathogenic coronaviruses from animal reservoirs, a thorough understanding of the host-virus interactions that drive sars-cov pathogenesis will aid the public health response to current and future coronavirus outbreaks ( ) . the importance of complement in sars-cov pathogenesis is controversial. previous studies have investigated the role of known polymorphisms in the mannose-binding lectin (mbl) and mbl-associated serine protese- (masp ) genes in sars-cov infection outcome following the outbreak but with conflicting results. one retrospective analysis showed that people with low or deficient serum mbl levels were more likely to become infected with sars-cov ( ) than those with high mbl levels, suggesting that mbl and complement activation play a role in protecting the host from infection. however, a second study found no association between mbl haplotype and sars-cov infection status ( ) . additionally, it was shown mbl can bind to the sars-cov spike protein in vitro by some groups ( ) but not by others ( ) . examination of the role of the downstream complement gene masp found no association between genotype and sars susceptibility ( ) . together, the results leave a general uncertainty about the role of complement in response to sars-cov infection. despite the existing body of literature, the role of complement in sars-cov pathogenesis has never been directly assessed in vivo. the complement system is an ancient arm of the innate immune response comprised of multiple proteins whose reactive cascade of cleavage products can coordinate the inflammatory response at the sites of infection and can be directly antimicrobial. consisting of more than soluble and cell surface-associated proteins, complement is a major component of innate immunity that functions to recognize and eliminate invading pathogens ( ) . activation of the complement system occurs through multiple mechanisms that include three welldescribed pathways, the classical, lectin, and alternative complement activation pathways ( ) , and results in proteolytic processing of various components of the complement system, including c , c , and c . proteolytic processing of c generates an array of cleavage products that are involved in amplification of complement activity through formation of c and c convertases, opsonization of pathogens, and attraction and activation of leukocytes of both the innate and adaptive arms of the immune response. several studies, including a recent study showing that complement blockade results in reduced disease in a mers-cov human dpp transgenic (hdpp -tg) mouse model ( ) , have elucidated protective and pathogenic roles for the complement system following infection by a variety of viral pathogens ( ) . furthermore, the complement system has well-described roles in other pulmonary diseases ( ) , especially after influenza virus and respiratory syncytial virus infection ( ) ( ) ( ) . in this study, we assessed the role of the complement system in the pathogenesis of sars-cov infection. building from a systems biology analysis that suggested that complement was modulated during sars-cov infection, we confirmed that complement was activated upon sars-cov challenge. mice deficient in c (c -/-), the central protein of the complement signaling pathway, were protected from sars-cov-induced weight loss and had reduced pathology, improved respiratory function, and lower levels of inflammatory cytokines/chemokines in the lung and periphery. importantly, the kinetics and magnitude of virus replication in c -/and wild-type mice were the same, showing that complement does not play a role in controlling virus replication. we observed complement deposition in the lungs of sars-cov-infected mice, suggesting that complement activation results in immune-mediated damage to the lung. additionally, serum activation indicates that complement-mediated systemic inflammation may drive the pathogenic response to sars-cov infection. together, the results indicate that complement plays a critical role in sars-cov pathogenesis and that inhibition of the complement pathway might be an effective therapeutic to coronavirus-mediated disease. complement is activated in sars-cov ma -infected mice. while work by other laboratories has shown that c -deficient mice are extremely susceptible to both h n and h n influenza virus infection ( ) , the role of complement in sars-cov infection has not yet been evaluated in vivo. using a systems biology-based approach, we identified the complement pathway as a high-priority target for control of sars-cov ma (the mouse-adapted sars-cov) pathogenesis based on weighted gene correlation network analysis (wgcna) of rna transcripts in the lungs of mice infected with a lethal versus a sublethal dose of sars-cov ma ( ) . because the complement signaling cascade is activated through proteolytic cleavage events, we also assessed lung proteomics samples for complement protein abundance. c b, cfb, and c all had significantly higher abundances in the lungs of mice infected with a lethal dose of sars-cov ma than in those of mice infected with a sublethal dose (fig. a) . complement activation is measured by detection of pathway component cleavage products. c , the master regulator of complement signaling, is cleaved into c a and c b following creation of c convertase. c activation products (c fragments c a, c b, ic b, c dg, and c c) were detected by western blotting in lung tissue of sars-cov ma -infected mice, but not in control mice, as early as day postinfection (dpi) (fig. b) , confirming that sars-cov ma infection activates the complement pathway. to test the importance of the complement signaling pathway in sars-cov pathogenesis, we infected c -/mice and c bl/ j controls with sars-cov ma . control mice exhibited approximately % transient weight loss, with peak weight loss at day postinfection (fig. a) . in contrast, the c -/mice were significantly protected from infection, with no significant weight loss evident at any time point. surprisingly, viral titers in the lung were similar in c -/and c bl/ j controls (fig. b) , indicating that the lack of disease in c -/mice is uncoupled from viral replication efficiency and that complement signaling is not necessary for sars-cov ma clearance from the lung. we further measured sars-cov ma -induced disease by assessing respiratory function using whole-body plethysmography following infection of c bl/ j and c -/mice. enhanced pause (penh) is a calculated measure of airway resistance that we have associated with airway debris following sars-cov ma infection ( ) . the % exhalation force (ef ) measures the exhalation force midbreath, which increases as breathing becomes more difficult. finally, the ratio of peak expiratory flow (rpef) is the time to peak expiratory flow and has been associated with wheezing following infection. all three metrics have been shown to change significantly following sars-cov ma infection, with penh and ef increasing following infection and rpef decreasing. combined, these measurements show that sars-cov ma -infected animals have altered exhalation patterns in their breathing. c -/mice exhibit a decreased change in penh and ef levels following sars-cov ma infection relative to those of infected c bl/ j control mice; however, rpef values were similar between infection conditions ( fig. c to e). together, these data indicate that despite the lack of weight loss in c -/mice, the absence of the complement pathway did not alter host control of viral the respiratory function of sars-cov ma -infected c bl/ j and c -/mice and mock-infected mice was measured using a buxco whole-body plethysmography system for penh, a measure of calculated airway resistance (c), ef , midbreath expiratory flow (d), and rpef, the rate of peak expiratory flow (e). *, p Ͻ . between mock-infected mice and a given condition. (c to e) three mice were used for each infection group, and two mock-infected mice were used. replication or completely abolish respiratory disease following sars-cov ma infection. in order to determine which arm of the complement pathway contributes to sars-cov ma pathogenesis, we infected knockout mice lacking components upstream of c . c -deficient mice lack the ability to signal through both the classical and lectin pathways, while fb-deficient mice lack the ability to signal through the alternative pathway. both mouse strains showed reduced weight loss relative to that of infected control mice (see fig. s in the supplemental material) at days postinfection; however, neither c -/nor fb -/mice reproduced complete protection from weight loss observed in c -/controls. together, the results suggest that multiple arms of the complement pathway may be activated and contribute to sars-cov-mediated disease through c activation. reduced lung pathology in c -/mice. analysis of sars-cov ma -infected lung sections showed that the absence of c resulted in reduced, but still significant, lung pathology. at day postinfection, only minor effects on lung disease were observed with airway denudation and debris, the main histopathological phenotypes at this early time point; levels of pathology were similar between wild-type and knockout mice ( fig. ; table s ). c -/mice exhibited more airspace inflammation, including eosino- phils, at dpi than their wild-type controls, although this relationship was reversed later in infection. at dpi, c bl/ j mice displayed pronounced lung pathology, including inflammatory cells in the large airway and parenchyma, perivascular cuffing, thickening of the interstitial membrane, and low levels of intra-alveolar edema. in contrast, c -/mice showed reduced scores in these areas, consistent with the improved respiratory function observed in fig. . notably, the lung pathology results were not as pronounced as the complete absence of weight loss, suggesting a possible distinction between lung disease and overall pathogenesis. we also considered whether the decrease in sars-cov ma pathogenesis in c -/mice was due to reduced lung damage in the absence of complement pathway signaling. to investigate this possibility, we looked for signs of complement deposition on sars-cov ma lung tissue. at both and dpi, we observed scattered positive staining for complement in the lungs of sars-cov ma -infected mice, suggesting that local tissue damage might contribute to sars-cov pathogenesis (fig. ) . interestingly, staining was consistently found in the parenchyma of the lung and not in the large airways, which are the other main site of sars-cov ma replication. no positive staining was observed in the lungs of c -/mice. diminished infiltration of the lungs of a select immune population of infected c -/mice. in order to identify and quantitate inflammatory cells in the lung, we performed flow cytometry at dpi. in parallel with humans exhibiting lung pathology, c -/mice exhibited significant pulmonary infiltration following sars-cov ma infection, but this inflammation was reduced relative to that observed in wild-type mice. sars-cov ma -infected c bl/ j and c -/mice had similar total cell counts as well as similar percentages of cd -positive cells in their lungs (data not shown). consistently with what was observed in human sars-cov patients ( ) , lymphopenia was observed in the lungs of both sars-cov ma -infected c bl/ j and c -/mice with reduced percentages of b cells (fig. a ) and cd t cells relative to those in mockinfected mice following infection. despite similar overall lymphocyte levels, small but significant differences were observed in levels of t cell activation between infected c bl/ j and c -/mice; both cd and cd t cells in c -/mice expressed more ki- ( fig. c and d) , an intracellular marker of proliferation, than those in c bl/ j controls. analysis of myeloid cells in the lung showed that infected c bl/ j mice had significantly higher levels of neutrophils, particularly nonactivated neutrophils, in the lung than infected c -/mice ( fig. b and e) . furthermore, inflammatory monocytes, which have previously been associated with increased sars-cov ma pathogenesis ( ), were significantly increased in the lungs of wild-type mice but not c -/mice (fig. b) . finally, we observed significantly more dendritic cells and alveolar macrophages in the lungs of sars-cov ma -infected c -/mice than in the lungs of infected c bl/ j mice. together, although c -/mice produced a robust immune cell infiltration following sars-cov infection, they had significant reductions in both inflammatory monocytes and neutrophils relative to controls; both cell types that are associated with sars-cov pathogenesis ( ) . conversely, the presence of activated t cells is associated with recovery following infection ( ) . in addition to examining inflammatory cells, we evaluated the vascular integrity of the lung following sars-cov ma infection in the presence and absence of c . we observed no differences in the numbers of platelets present in the bronchoalveolar lavage (bal) fluid between c bl/ j and c -/mice at either or days postinfection, indicating that the absence of c does not appear to significantly alter vascular permeability following infection with sars-cov (fig. s b) . cytokine and chemokine levels are significantly decreased in the lungs of c -/mice. to further investigate the inflammatory response to sars-cov ma infection, we measured cytokine and chemokine protein levels in the lung in the presence and absence of complement signaling. multiple protein expression patterns were observed in response to infection. mip a, mip b, and mcp are all highly expressed in the lung following sars-cov ma infection of both c bl/ j and c -/mice (fig. a) , indicating that some inflammatory signaling remains intact in the absence of c . granulocyte colony-stimulating factor (g-csf), interleukin (il- ), tumor necrosis factor alpha (tnf-␣), and il- a comprised a group of cytokines and chemokines that were more highly produced in the lungs of c bl/ j mice than in c -/mice (fig. b) , all peaking at days postinfection. notably, these cytokines all have a role in the production, recruitment, or differentiation of neutrophils, consistent with the flow cytometry results in fig. b . with the exception of rantes, all inflammatory cytokines and chemokines were measured at the highest levels at dpi, indicating that the host immune response is triggered quickly following infection with sars-cov ma . together, these results indicate that the absence of complement has an impact on the magnitude of some cytokines and chemokines in the lung; however, robust production can occur in either the presence or the absence of c . sars-cov ma induces systemic complement activation. the absence of complement signaling resulted in reduced sars-cov ma pathogenesis, as measured by weight loss and a partial reduction of respiratory dysfunction, pathology, immune infiltration, and cytokine responses in the lung. we hypothesized that systemic disease coupled with no change in viral titer might also drive important elements of complement-mediated disease. therefore, we examined sera from wild-type and c -/mice for signs of systemic disease following infection. western blot analysis showed increased levels of c a-derived fragments in the serum, indicating systemic complement activation in sars-cov ma -infected mice at dpi (fig. a) . given this result, we next examined cytokine and chemokine protein levels for markers of inflammation in the sera of sars-cov ma -infected mice. both mcp- and rantes levels were elevated in the serum following infection, regardless of mouse genetic background (fig. b) . however, numerous cytokines and chemokines, such as il- , g-csf, and kc (keratinocyte chemoattractant or cxcl ) were present in significantly higher abundance in the lungs of c bl/ j mice than in those of c knockout mice (fig. c) . we further examined the possibility that sars-cov ma infection leads to complement deposition outside the lung and found no signs of increased complement staining in the kidney (fig. d) . although no complement deposition was seen, the presence of both activated complement and inflammatory cytokines in the sera likely contributes to a systemic inflammatory response that drives sars-cov ma -mediated weight loss following infection. the complement system is a critical part of the host immune response to bacterial and viral infection. originally identified in the s as a heat-sensitive, nonspecific complement to the more specific adaptive immune pathways ( ), the complement system is one way that the innate immune system detects and responds to foreign antigens. because of its potential to damage host tissues, the complement system is also tightly regulated through a number of inhibiting proteins that are constitutively present in the serum ( ). it has previously been shown that complement pathway signaling is critical for the protective host immune response to various bacterial infections ( ) as well as some influenza virus and flavivirus infections ( , , ) . furthermore, viruses, including herpesviruses, poxviruses, astroviruses, flaviviruses, and retroviruses, encode genes to help them evade detection by the complement system ( ) , strong evidence that complement is important in the host antiviral response. the host factors that drive protective ( ) or pathogenic ( ) complement-associated responses in viral infection are not well understood. of particular concern, the anaphylatoxins c a, c a, and c a are produced during activation of the complement signaling cascade; they have potent proinflammatory properties and can trigger inflammatory cell recruitment and neutrophil activation ( ) . c a and c a blockade has been proposed as a treatment for acute lung injury ( ) , and anti-c a antibody has been shown to protect mice from infection with influenza virus ( ) and, more recently, mers-cov ( ) . complement recognition is important for the control of paramyxoviruses ( ), dengue virus ( ) , and human t lymphotrophic virus type (htlv- ) ( ), and many more viruses have developed means of evading detection by the complement system ( ) . in contrast, the data presented here, in conjunction with recent findings for ross river virus ( , ) , influenza virus ( ) , and well-established autoimmune disease ( ) , demonstrate that complement system activation can also lead to exacerbated disease. previous reports clearly established the ability of mannose-binding lectin (mbl) to bind to the sars-cov spike protein ( ), dependent on an n-linked glycosylation site; however, the role of complement signaling in sars-cov pathogenesis was unclear ( , , , ) . in this study, we demonstrate that the complement system is activated following sars-cov ma infection (fig. b) . however, we did not observe any change in viral titer in c -/mice (fig. b) , indicating an important difference between in vivo and in vitro studies and the use of viral pseudoparticles. the absence of complement signaling resulted in protection from sars-cov ma -induced weight loss, as shown through the use of both c -deficient mice ( fig. a) and activation pathway-specific knockout mice (fig. s ). respiratory function in c knockout mice was improved relative to that of control mice, although significant changes in penh and rpef were still observed, indicating that the elimination of complement signaling did not completely remove the effects of sars-cov ma infection. while analysis of the cellular inflammatory response to sars-cov ma infection revealed modest changes in histopathology and overall inflammatory cell recruitment to the lungs, significant differences were observed in pathogenic inflammatory monocyte and neutrophil populations, indicating that complement signaling contributes to the broader immune response to infection. immunohistochemical staining revealed that sars-cov ma infection induced complement deposition in the lung (fig. ) , similar to that associated with pathogenesis in ross river virus-infected mice ( ) and some influenza virus infections ( ) , and it is likely that complement deposition contributes to pulmonary disease and inflammatory cell recruitment. the cytokines and chemokines il- , il- , kc (cxcl ), and g-csf have higher abundances in the lungs of sars-cov ma -infected wild-type mice than in c -/mice and are all known to promote neutrophil recruitment. indeed, significantly more neutrophils were observed in the lungs of sars-cov ma -infected c bl/ j mice than in the c -/mice (fig. b) . interestingly, while there were fewer neutrophils present, the neutrophils found in the lungs of c -/mice infected with sars-cov ma had significantly more staining of major histocompatibility complex class ii (mhc ii) and the costimulatory molecules cd and cd (fig. e) , indicating a state of activation ( ) . unpublished data from our laboratory have consistently demonstrated higher neutrophil counts in the lungs of mice with severe disease than in those of mice with only mild pathogenesis. additionally, neutrophilia in human sars-cov patients was associated with a poor outcome of infection ( ) , and studies of native rat coronavirus ( ) found both a protective and a pathogenic role for neutrophils following infection. combined, these data demonstrate that the absence of complement provides significant improvements in pulmonary disease following sars-cov ma infection and suggest that a nonpulmonary cause might also contribute to the lack of weight loss in c -/mice. importantly, our data demonstrate that sars-cov ma infection activates the complement system systematically as well as in the lung (fig. a and b) . wild-type c bl/ j mice exhibited an increased abundance of serum cytokines and chemokines in response to sars-cov ma infection (fig. c ) in comparison to c -/mice. in particular, the pyrogenic cytokine il- ( ) is present at higher abundance in the lungs and sera of c bl/ j mice than in those of c -/mice. il- ␣ and tnf-␣ are also more abundant in the lungs of c bl/ j mice, suggesting that wild-type but not c -/mice develop a fever response to infection that contributes to weight loss and respiratory dysfunction phenotypes. while the precise mechanism of complement activation following sars-cov ma infection is still unclear, it is likely through recognition of the viral spike glycoprotein and partially mediated by mbl (see fig. s in the supplemental material). the anaphylatoxins produced by the activated complement pathway, c a, c a, and c a, have important immunostimulatory roles in vascular permeability and inflammatory cell recruitment ( , ) . c a and c a in particular are noted for their roles in causing mast cell degranulation, initiating a cytokine storm, promoting vascular permeability, and contributing to acute lung injury ( , , ) . furthermore, a c a antibody blockade was recently shown to protect in a model of highly susceptible mers-cov mice ( ) . although there are no reports of mast cell activation following sars-cov or mers-cov infection, activation has been observed both in vitro and in vivo following infection with influenza virus ( ) and may occur following other severe respiratory infections, including coronaviruses. mast cells release cytokines, including il- , il- , and tnf-␣, consistent with the inflammatory profile observed following sars-cov ma infection. while this work cannot definitively conclude that the complement anaphylatoxins and mast cell activation contribute to sars-cov ma pathogenesis, the data are consistent with this possibility, and the concept warrants further investigation. complement pathway activation is a hallmark of bacterial infection, and genetic deficiencies in the complement pathway result in enhanced susceptibility to streptococcus pneumoniae, neisseria meningitidis, and haemophilus influenzae infections ( ), as well as to sepsis. interestingly, it has also been shown that sars-cov ma infection stimulates tlr ( , ) , which is classically known as the lipopolysaccharide (lps) receptor ( , ) and important for recognition of many bacterial infections. combined, these data suggest that host recognition of sars-cov ma infection may activate similar pathways recognizing a bacterial infection, leading to immune signaling cascades that cause systemic disease and enhance viral pathogenesis. while systemic activation of the complement pathway may be useful during a bacterial infection, it is less so during a localized acute viral infection, such as sars-cov. furthermore, complement activation, in conjunction with the presence of neutrophils, is known to cause increased vascular permeability, a condition that is also observed following sars-cov infection ( ) ( ) ( ) and was associated with poor outcome. baseline complement activation also increases with age ( , ), consistent with increased sars-cov morbidity and mortality in aged populations. finally, it has previously been reported that serum c a levels are predictive of ards development ( ) and that, in the absence of complement, animals are protected from bacterially induced "shock lung" ( ) , data consistent with the pathogenic role that we have found for complement following sars-cov ma infection. in this work, we demonstrate that sars-cov ma infection activates the complement pathway and that complement signaling contributes to disease following infection. this disease is likely mediated by complement protein deposition in the lung as well as systemic complement activation and inflammation. notably, the absence of c has no impact on viral titer, unlike what has been observed following influenza virus infection. despite these differences, it is notable that mers-cov and h n influenza virus-induced acute lung injury and pulmonary inflammation are reduced in mice that are treated with either a c a receptor (c ar) antagonist or antibodies to c a ( , ) . a similar treatment might be effective in mitigating sars-cov ma -induced disease, as sars, mers, and influenza have common disease manifestations, including development of acute lung injury. given the large array of zoonotic strains poised for cross-species transmission, broad-based inhibitors of emerging coronavirus infections are a high priority ( ) . pinpointing the precise arms of the complement pathway that contribute to sars-cov will help further identify therapeutic targets while minimizing unnecessary inhibition of the immune response. this work suggests that investigation of anticomplement drugs for treatment of coronavirus infections is warranted and would pair well with direct antiviral therapeutics. stocks of recombinant mouse-adapted sars-cov (ma ) ( ) were propagated and their titers were determined in vero e cells and stored as single-use aliquots at Ϫ °c as previously described ( ) . tissue titers of ma were determined by plaque assay on vero e cells as previously described ( , ) , with a limit of detection of pfu. all experiments using live virus were performed in a class ii biological safety cabinet in a certified biosafety level laboratory with negative air pressure and redundant exhaust fans; personnel wore personal protective equipment, including tyvek suits, hoods, and powered air-purifying respirators. mouse experiments. c bl/ j (stock number ) and c -/-(stock number ) mice were purchased from jackson laboratories. fb Ϫ/Ϫ mice were generously provided by charles jennette (unc), and c -/mice were provided by mark heise (unc). all animal husbandry and experiments were performed in accordance with all university of north carolina at chapel hill institutional animal care and use committee guidelines. age-matched ( -to -week-old) female mice were anesthetized with a mixture of ketamine-xylazine and intranasally inoculated with l of phosphate-buffered saline (pbs) or pfu of sars-cov ma diluted in pbs. mice were monitored for disease signs and weighed at -h intervals. microarray and proteomics analysis. microarray and proteomics analyses were performed on a time course and according to a dose-response study published by gralinski et al. by following the same methods ( ) . the proteomics data (experiment sm ) are publically available through the pnnl (http://omics.pnl.gov) web portal. briefly the mean intensity (abundance) for each protein was then graphed as an average ( mice for each infection, mice for mock infection) for each group at each time point. missing or absent values were not scored; however, if no value was observed in any of the samples at a time point, the sample was registered with a single , representing "not detected." histological analysis. at the times indicated in the figures, mice were euthanized using an overdose of isoflurane, and lung tissue was fixed in % formalin. tissues were embedded in paraffin, and -m sections were prepared. to determine the extent of inflammation and tissue pathology, tissues were stained with hematoxylin and eosin and scored in a blind manner from (no sign of phenotype) to (widespread and severe phenotype). platelet counts. for bronchoalveolar lavage (bal), immediately following euthanasia, ml of pbs was injected into the lung through the trachea by using a -gauge exel safelet catheter tip (fisher). this fluid was then drawn back out and used for subsequent analysis. two hundred fifty microliters of bal fluid was used for absolute counting of gross cell types using an abaxis vetscan hm analyzer. complement deposition staining. lung sections from sars-cov ma -, ross river virus-, or mock-infected mice were stained for the presence of c by the animal histopathology and laboratory medicine core at the university of north carolina. sars-cov ma lung samples were tested from -week-old mice at , , , and days after infection with pfu of virus. staining was performed using a goat anti-mouse c primary antibody (mp biomedicals). immunoblot analysis. mice were perfused with pbs, and then lung tissue was dissected and homogenized in lysis buffer ( mm tris [ph . ], mm nacl, % nonidet p- , . % deoxycholate, and . % sodium dodecyl sulfate [sds] supplemented with complete protease inhibitor cocktail [roche]). total protein concentrations were determined by using the coomassie plus assay kit (pierce). dilutions of serum or -to -g aliquots of protein were diluted in an equal volume of ϫ sds sample buffer, and sds-polyacrylamide gel electrophoresis was performed. proteins were transferred onto polyvinylidene fluoride membranes (bio-rad). membranes were blocked in ϫ pbs- % milk- . % tween and incubated with goat anti-mouse c antibody ( : , ; cappel) overnight at °c. membranes were washed in pbs- . % tween and incubated with rabbit anti-goat-horseradish peroxidase ( : , ; sigma) for h at room temperature. after a washing step, proteins were visualized by enhanced chemiluminescence (amersham) according to the manufacturer's instructions. flow cytometry. following euthanasia at days postinfection, mice were perfused with ml of pbs via cardiac puncture. lungs were dissected, minced, and incubated for min with vigorous shaking at , and cd -percp-efluor (clone e ; ebioscience). after being stained, cells were fixed in % paraformaldehyde overnight and then stored in pbs until acquisition within h. a minimum of , events were collected using an lsrii cytometer (becton, dickinson), and analysis was completed using flowjo software version (treestar). all samples were first evaluated through subsequent gates for (i) mononuclear cells, (ii) doublet exclusion, (iii) dead-cell exclusion based on uptake of a fixable live/dead cell discriminator (invitrogen), and cd -lca expression before downstream analyses. reported cell frequencies were normalized to the percentage of total cd -lca ϩ events, where appropriate. whole-body plethysmography. respiratory function was measured using whole-body plethysmography as described by menachery et al. ( ) . briefly, mice were loaded into individual chambers and allowed to acclimate for min before a -min measurement window. measurements were recorded every s for a total of measurements per time point per mouse. cytokine and chemokine protein analysis. the small center lung lobe of each mouse was homogenized in ml of pbs and briefly centrifuged to remove debris. fifty microliters of homogenate was used to measure cytokine and chemokine protein abundance using a bio-plex pro mouse cytokine -plex assay (bio-rad) according to the manufacturer's instructions. statistical analyses. percent starting body weights, viral titers, and inflammatory cell numbers were evaluated for statistically significant differences by the mann-whitney test or student's t test using graphpad prism software. accession number(s). the microarray data were previously deposited in the geo database under accession number gse ( ). supplemental material for this article may be found at https://doi.org/ . /mbio . - . research was supported by grants from the niaid of the nih (ai to r.s.b. and m.t.h., ai to r.s.b., ai to r.s.b., and k ag to v.d.m.). animal histopathology was performed by the animal histopathology and laboratory medicine core at the university of north carolina, which is supported in part by an nci center core support grant ( p ca - ) to the unc lineberger comprehensive cancer center. the unc flow cytometry core facility is supported in part by cancer center core support grant p ca to the unc lineberger comprehensive cancer center. the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. the content is solely the responsibility of the authors and does not necessarily represent the official views of the nih. molecular evolution of the sars coronavirus during the course of the sars epidemic in china characterization of a novel coronavirus associated with severe acute respiratory syndrome severe acute respiratory 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respiratory syndrome coronavirus host range expansion in human airway epithelium flow cytometric analysis of macrophages and dendritic cell subsets in the mouse lung key: cord- -yu oj v authors: kurskaya, olga; ryabichenko, tatyana; leonova, natalya; shi, weifeng; bi, hongtao; sharshov, kirill; kazachkova, eugenia; sobolev, ivan; prokopyeva, elena; kartseva, tatiana; alekseev, alexander; shestopalov, alexander title: viral etiology of acute respiratory infections in hospitalized children in novosibirsk city, russia ( – ) date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: yu oj v background: acute respiratory infections (aris) cause a considerable morbidity and mortality worldwide especially in children. however, there are few studies of the etiological structure of aris in russia. in this work, we analyzed the etiology of aris in children ( – years old) admitted to novosibirsk children’s municipal clinical hospital in – . methods: we tested nasal and throat swabs of children with upper or lower respiratory infection for main respiratory viruses (influenza viruses a and b, parainfluenza virus types – , respiratory syncytial virus, metapneumovirus, four human coronaviruses, rhinovirus, adenovirus and bocavirus) using a rt-pcr kit. results: we detected ( . %) samples were positive for at least one virus. the most frequently detected pathogens were respiratory syncytial virus ( / , . %), influenza virus ( / , . %), and rhinovirus ( / , . %). viral co-infections were found in out of the ( . %) positive samples. we detected significant decrease of the respiratory syncytial virus-infection incidence in children with increasing age, while the reverse relationship was observed for influenza viruses. conclusions: we evaluated the distribution of respiratory viruses in children with aris and showed the prevalence of respiratory syncytial virus and influenza virus in the etiological structure of infections. this study is important for the improvement and optimization of diagnostic tactics, control and prevention of the respiratory viral infections. we detected ( . %) samples were positive for at least one virus. the most frequently detected pathogens were respiratory syncytial virus ( / , . %), influenza virus ( / , . %), and rhinovirus ( / , . %). viral co-infections were found in out of the ( . %) positive samples. we detected significant decrease of the respiratory syncytial virus-infection incidence in children with increasing age, while the reverse relationship was observed for influenza viruses. we evaluated the distribution of respiratory viruses in children with aris and showed the prevalence of respiratory syncytial virus and influenza virus in the etiological structure of plos acute respiratory infections (aris) pose a significant public health problem worldwide, causing considerable morbidity and mortality among people of all age groups [ ] . children are on average infected two to three times more frequently than adults. [ ] . there are more than respiratory viruses that can cause aris. human respiratory syncytial virus (hrsv), human rhinovirus (hrv), human metapneumovirus (hmpv), human parainfluenza virus (hpiv), human enterovirus (ev), influenza virus (ifv), human coronavirus (hcov), adenovirus (hadv), and human bocavirus (hbov) are the most common viral agents associated with aris, accounting for around % of aris [ , ] . the frequency of mixed respiratory viral detection varies from % to % in hospitalized children [ ] [ ] [ ] . in addition, several new human respiratory viruses have been described in recent years, including human metapneumovirus [ , ] , human bocavirus [ ] , and novel human coronaviruses, including severe acute respiratory syndrome coronavirus (sars-cov) [ ] , human coronaviruses nl (hcov-nl ), hku (hcov-hku ) [ ] , and middle east respiratory syndrome coronavirus (mers-cov) [ ] . although the majority of aris are associated with respiratory viruses, antibiotics are often used in the clinical treatment of aris. as children with aris often have similar clinical symptoms, studying the clinical hallmarks of children with virus-related aris and the spectrum of respiratory viruses will help in developing more accurate treatments for aris [ ] . rapid diagnosis is important not only for timely treatment starting but also for the detection of a beginning influenza epidemic and the avoidance of unnecessary antibiotic treatment [ , ] . western siberia plays a key role in ecology, epizootiology and epidemiology of emerging diseases. this territory was involved in the circulation of a/h n and a/h n avian influenza viruses in - [ , ] . these viruses were spread by wild birds' migration. in western siberia migratory flyways of birds' wintering in different regions of the world: south east asia, central asia, middle east, hindustan, europe, and africa-cross. for this reason, there is high probability of the emergence of humans and animal influenza viruses reassortants, as well as emergence of local outbreaks of human morbidity caused by uncommon variants of influenza viruses. furthermore, novosibirsk is the largest transport hub in this part of russia with numerous international connections, that is important for the spread of aris [ , ] . the prevalence of respiratory viruses among children with aris differs in different regions and varies over time [ ] [ ] [ ] [ ] [ ] . thus, to better understand the epidemiology of acute respiratory infections in novosibirsk region, we investigated etiology of aris in children admitted to novosibirsk children's municipal clinical hospital in - . all aspects of the study were approved by the ethics committee of the federal state budgetary institution "research center of clinical and experimental medicine" ( - ). accordingly, written informed consent was obtained from parents prior to sample taking. children - years of age were enrolled in the study within days of illness onset and had at least two of the following symptoms: fever, sore throat, cough, rhinorrhea, nasal congestion, sputum, shortness of breath, lung auscultation abnormalities, tachypnea, and chest pain. paired nasal and throat swabs were collected from each patient admitted to novosibirsk children's municipal clinical hospital by hospital nurses. a total of samples collected during four epidemic seasons of - (october-april) were enrolled to the study. the epidemiological and clinical information including case history, symptoms, physical signs, and examination were included in a standardized questionnaire. the samples were placed immediately in viral transport medium (eagle mem, bsa and antibiotics) and stored at - ˚c prior transportation to the laboratory (not more than hours). detection of respiratory viruses was performed immediately after delivery to the laboratory. all specimens were tested for common respiratory viruses, including influenza virus types a, b (ifva and ifvb), human parainfluenza virus (hpiv) types - , human respiratory syncytial virus (hrsv), human metapneumovirus (hmpv), four human coronaviruses (hco), human rhinovirus (hrv), human adenovirus (hadv) and human bocavirus (hbov), using a real-time pcr assay-kit. viral nucleic acids were extracted from nasal and throat swabs using rna/dna extraction kit «ribo-sorb» (interlabservice, russia) according to the manufacturer's instructions. the extracted viral nucleic acid was immediately used to perform the reaction of reverse transcription using commercial kit "reverta-l" (interlabservice, russia). detection of respiratory viruses, including hpiv - , hrsv, hmpv, hcov-oc , hcov- e, hcovnl , hcov-hku , hrv, hadv, and hbov was performed using a rt-pcr kit «amplisens arvi-screen-fl» (interlabservice, russia), and ifva and ifvb virus detection was performed using a rt-pcr kit « amplisens influenza virus a/b-fl» (interlabservice, russia) according to the manufacturer's instructions. positive and negative controls were included in each run. two-tailed chi-square test (two by two table) was performed to compare the infection rates for respiratory viruses among different age groups. p-value < . was considered to be statistically significant. totally, samples collected from patients with aris during the period from december to april were enrolled in the investigation. there were males ( . %) and females ( . %), and the patient's ages ranged from months to years. the most numerous age group ( . %) was between and years old. the age distribution is shown in table . the incidence of respiratory viral infection between boys ( / ; . %) and girls ( / ; . %) (χ = . , p> . ). the respiratory virus positive rate appeared to decrease with age. the lowest positive rate was observed in the age group of more than years old ( / ; . %), while the positive rates in age groups less than years old were more than % (table ). statistically significant difference was observed between the age group of more than years old and other age groups (χ = . , p< . ). no statistically significant difference was observed among the age groups less than year old, - years, and - years. among all the samples, single infections accounted for . % ( / ), while co-infections accounted for . % ( / ) with the lowest rate of incidence in children more than years old compared to children younger than years (χ = . , p< . ) (fig ) . hrsv and ifv were the most frequently detected viruses with high incidence of . % ( / ) and . % ( / ), respectively, among all patients with aris. hrv was detected in . % ( / ), followed by hmpv, hpiv and hbov with the detection rates higher than . %. the positivity rates of hcov and hadv were lower than . % (fig ) . the data were analyzed with regard to the age and gender distribution of virus infection. no difference in the etiological distribution of viral pathogens was observed between males and females. among detected respiratory viruses, hrv, hpiv, hcov, and hadv did not have statistically significant difference in the distribution among the different age groups. hmpv was detected in age group less than year old much more frequently than in children - years old (χ = . , p< . ) and - years old (χ = . , p< . ). hbov was significantly more frequently observed in children younger than years old compare with children of - years old (χ = . , p< . ). the incidence of hrsv decreased significantly with increasing age (p < . ) dropping from . % in children younger than year old to . % in the school-age children ( - years old group), while the reverse relationship was observed for ifv (fig ) . the data were analyzed with regard to the seasonality. overall, virus detection positive rate did not differ significantly between seasons, ranging from . % to . %. co-infections with two or more viruses were detected in out of the ( . %) positive samples (table ) . dual infections accounted for . % ( / ) of all positive samples and acute respiratory infections are a serious health and economic problem, causing high morbidity and significant economic losses due to temporary disability payments and medical costs. children are the most vulnerable group to the development of the disease. as several authors mentioned previously [ ] , aris can lead to serious diseases such as bronchiolitis and pneumonia and sometimes even cause death in infants and children worldwide. nevertheless, most of the data on the epidemiological features and etiological structure of aris were from moredeveloped countries, and less is known about the etiology of aris in russia. in the present study, we examined the viral etiology of aris in hospitalized children in novosibirsk by real-time rt-pcr assay. we detected at least one of the tested viruses in . % ( / ) of the samples. in similar studies conducted in different regions of the world, the virus detection rate ranged from less than % to % [ ] [ ] [ ] . for example, in studies performed in china, the proportion of positive samples in children with aris ranged from . % to . % [ , , , ] . the previous study of respiratory infections among children in european part of russia revealed the . % detection rate of respiratory viruses [ ] . the percentage of the respiratory viruses' detection varies in different years in different regions, which may be associated with climatic and environmental factors, population distribution, economic status and diagnostic methods used [ ] . in addition, seasonality of sampling can also lead to differences in the level of viruses' detection in various studies. thus, ju x. et al. carried out a study continuously from july through july and found . % of samples to be positive for at least one respiratory virus [ ] . in contrast, we collected samples only during epidemic seasons of aris, so the positivity rate in our study was considerably higher. furthermore, acute respiratory infections can be caused by viruses that are not yet known, as well as bacteria that have not been included in these studies [ ] . we found that the prevalence of respiratory viruses did not differ between boys and girls ( . % and . % respectively), which confirms the absence of a gender-based susceptibility to respiratory viral infections [ ] . however, we observed a decrease in the respiratory viruses' detection rate with age, with the lowest detection rate in school-age children compared to children under years of age ( . % versus . %, respectively). these data are consistent with findings obtained in other regions of russia [ ] and it may be due to decreased sensitivity to respiratory virus infections in older children. etiology of aris and the respiratory virus prevalence varies in different studies. in the united states, ifv, hrsv and hpiv were the most frequently detected [ ] . studies conducted in france have shown that metapneumovirus and respiratory syncytial virus are the most common [ ] . ifv, hrsv and hrv were the most commonly detected respiratory viruses among children with aris in most regions of china [ ] . the study of aris etiologic structure showed that hrsv, hrv, hpiv and ifv were registered significantly often among children in western part of russia [ ] . in our study the most common viruses detected were hrsv and ifv, followed by hrv. the age distribution of aris indicated that children under years old were more likely to be infected by hrsv confirmed the importance of rsv in children with aris, especially in children < years of age [ ] [ ] [ ] [ ] . we observed a high rate of hrsv-detections in - ( . %), while in - it was less than % which could be due to the annual variation in the circulation pattern of hrsv. such year-to-year variation in the epidemiological patterns of viral infections confirms importance of the long-term study of the aris epidemiology [ ] . influenza virus is one of the major causative agents of respiratory disease in humans and may lead to serious illness [ ] . in temperate countries influenza outbreaks usually occur during the winter season. finally, in our study, ifv ( / , . %) was the second most frequent detected pathogen with markable seasonality in winter months. during the - epidemic season, influenza virus detection rate was low- . % of all respiratory viruses, which was in accordance with the official influenza surveillance results of ministry of health, russia [ ] . in our study in - , influenza viruses were detected in . % among all respiratory viruses. among those, the percentage of influenza a virus accounted for . % and influenza b virus made up . % of all detected influenza viruses. at the same time, in russia influenza b was the main etiological agent, accounting for . % of all detected influenza viruses. in - , influenza a(h n )pdm virus was predominant in russia, accounting for % of all influenza-positive samples consistent with our results ( %). in - influenza a(h n ) virus was dominant in russia detected in . % of all influenza virus cases [ ] . in our study influenza a and b viruses were detected with approximately the same frequency ( % and % respectively). with the introduction of molecular techniques, the detection of multiple co-infecting viruses has become common, though the prevalence of each virus varies between studies [ ]. in our study detection rate of viral co-infection was . % among the positive samples. according to the previous reports, the incidence of viral co-infection in children can reach % [ ] . co-infection is most often found in children under the age of , due to the immaturity of the immune system and, thus, greater susceptibility to infection [ ] . in our study, we significantly more frequently detected cases of simultaneous infection with two or more viruses in children under years of age compared with children of school age ( . % versus . %), while there was no significant difference in the incidence of viral co-infection between the age groups of - year, - years and - years. in conclusion, in our study we investigated the etiological structure of acute respiratory viral infections in hospitalized children in novosibirsk, russia, and evaluated age and seasonal distribution of the various respiratory viruses. systematic monitoring of respiratory viruses is necessary to better understand the structure of respiratory infections. such studies are important for the improvement and optimization of diagnostic tactics, as well as measures for the control and prevention of the respiratory viral infections. prevalence of respiratory viruses among children hospitalized from respiratory infections in shenzhen, china estimates of world-wide distribution of child deaths from acute respiratory infections role of respiratory viruses in acute upper and lower respiratory tract illness in the first year of life: a birth cohort study multiplex real-time pcr for detection of respiratory tract infections epidemiology of human respiratory viruses in children with acute respiratory tract infections in jinan, china respiratory virus infections in hospitalized children and adults in lao pdr. influenza other respir viruses single and multipathogen viral infections in hospitalized children with acute respiratory infections a newly discovered human pneumovirus isolated from young children with respiratory tract disease ten years of human metapneumovirus research cloning of a human parvovirus by molecular screening of respiratory tract samples identification of a novel coronavirus in patients with severe acute respiratory syndrome the novel human coronaviruses nl and hku coronaviruses: important emerging human pathogens epidemiological and clinical characteristics of respiratory viral infections in children in shanghai seasonal influenza in adults and children-diagnosis, treatment, chemoprophylaxis, and institutional outbreak management: clinical practice guidelines of the infectious diseases society of america reducing antibiotic use in influenza: challenges and rewards genetic and biological characterization of avian influenza h n viruses isolated from wild birds and poultry in western siberia novel reassortant clade . . . avian influenza a(h n ) virus in wild aquatic birds monitoring of influenza viruses in western siberia virological evaluation of avian influenza virus persistence in natural and anthropic ecosystems of western siberia high incidence of multiple viral infections identified in upper respiratory tract infected children under three years of age in viral etiology of acute respiratory infection in gansu province, china respiratory virus infections among children in south china aetiology of acute respiratory tract infections in hospitalised children in cyprus plos one epidemiology and seasonality of respiratory viral infections in hospitalized children in kuala lumpur, malaysia: a retrospective study of years molecular epidemiology of respiratory viruses in virus-induced asthma. front microbiol detection and typing by molecular techniques of respiratory viruses in children hospitalized for acute respiratory infection in rome, italy viral aetiology of influenza-like illness in belgium during the influenza a(h n ) pandemic etiology and seasonality of viral respiratory infections in rural honduran children the epidemiology and etiology of influenza-like illness in chinese children from to epidemiological analysis of respiratory viral etiology for influenza-like illness during in zhuhai the features of arvd etiological structure in different age and professional population groups in saint-petersburg during - epidemic season viral etiology of influenza-like illnesses in huizhou aetiology of influenza-like illness in adults includes parainfluenzavirus type nationwide surveillance of respiratory viruses in patients with influenzalike illnesses. a pilot feasibility study in the french sentinel network viral etiologies of acute respiratory infections among hospitalized vietnamese children in a -year prospective study of the epidemiology of acute respiratory viral infections in hospitalized children in shenzhen, china influenza other respir viruses relative frequency, possible risk factors, viral codetection rates, and seasonality of respiratory syncytial virus among children with lower respiratory tract infection in northeastern brazil. medicine (baltimore) respiratory viruses associated hospitalization among children aged< years in bangladesh viral aetiology influenza like illnesses in santa cruz understanding the symptoms of the common cold and influenza on the outcome of the epidemic season of influenza and aris viral co-infections in pediatric patients hospitalized with lower tract acute respiratory infections clinical disease severity of respiratory viral co-infection versus single viral infection: a systematic review and meta-analysis surveillance of respiratory viruses in patients with influenza-like illness in nanjing we thank the clinicians of novosibirsk children's municipal clinical hospital for their assistance with sample collection. we thank dr. galina skosyreva and elena timofeeva (department of propaedeutic of childhood diseases, novosibirsk state medical university, novosibirsk, russia) for their assistance with sample collection. key: cord- -spxgox authors: yu, jianhai; li, xujuan; he, xiaoen; liu, xuling; zhong, zhicheng; xie, qian; zhu, li; jia, fengyun; mao, yingxue; chen, zongqiu; wen, ying; ma, danjuan; yu, linzhong; zhang, bao; zhao, wei; xiao, weiwei title: epidemiological and evolutionary analysis of dengue- virus detected in guangdong during : recycling of old and formation of new lineages date: - - journal: am j trop med hyg doi: . /ajtmh. - sha: doc_id: cord_uid: spxgox the incidence of dengue is increasing in guangdong, china, with the largest outbreak to date in . widespread awareness of epidemiological and molecular characteristics of the dengue virus (denv) is required. in , we isolated the virus from patients and sequenced its genome. the sequences of denv isolated from guangdong and other countries screened since were studied to establish molecular evolutionary databases along with epidemiological data to explore its epidemiological, phylogenetic, and molecular characteristics. causes underlying the occurrence of the dengue epidemic included importation and localization of the virus. the number of indigenous cases significantly exceeded that of imported cases. dengue virus is the most important serotype and caused the long-term epidemic locally. based on the data available since , denv was divided into three genotypes (i, iv, and v). only genotypes i and v were detected in . in , an epidemic involving old lineages of denv genotype v occurred after years of silence. the genotype was previously detected from to . genotype i, which caused recent epidemics, demonstrated a continuation of new lineages, and a predictive pattern of molecular evolution since among the four lineages was present. the denv isolated from guangdong was closely related to those causing large-scale epidemics in neighboring countries, suggesting the possibility of its import from these countries. the lack of sufficient epidemiological data and evidence on the local mosquito-borne denv emphasizes the importance of studying the molecular evolutionary features and establishing a well-established phylogenetic tree for dengue prevention and control in guangdong. dengue virus (denv), a mosquito-borne flavivirus, is transmitted primarily by aedes aegypti and aedes albopictus, causing an acute infectious disease named dengue fever (df), which gives rise to public health problems in tropical and subtropical regions worldwide, such as china, singapore, and brazil. [ ] [ ] [ ] with the recent revision of the who dengue classification scheme, dengue patients are classified as having either dengue or severe dengue. the former refers to patients who recover without major complications, whereas the latter points to those who have any of the following conditions: plasma leakage resulting in shock, accumulation of serosal fluid sufficient to cause respiratory distress, or both; severe bleeding; and severe organ impairment. before , only nine countries had experienced severe dengue epidemics, but the disease is now endemic in more than countries. in recent decades, the spreading disease causes rapid upsurge in morbidity. one recent estimate indicates million dengue infections per year, of which million manifest clinically. , hence, wasting a lot of health resources and causing the growing global burden of disease, df is regarded as the most widely distributed vector-borne disease with the highest morbidity and great harm. the genome of denv is a single-stranded, positive-sense rna, and its single open reading frame encodes a polyprotein consisting of three structural proteins, which are as follows: the capsid, membrane-associated, and envelope (e) proteins. in addition, seven nonstructural proteins, ns , ns a, ns b, ns , ns a, ns b, and ns , are also present in its structure. four distinct denv serotypes (denv , denv , denv , and denv ) have been identified, and the extensive diversity within denv enables it to recognize different genotypes, such as genotype i (southeast asia and east africa), genotype ii (thailand), genotype iii (malaysia), genotype iv (south pacific), and genotype v (america/africa). dengue genotypes are phylogenetically distinct clusters of viruses, often associated with specific geographical regions, that are linked to epidemics of varying intensities and disease severity. , phylogeny is a science that makes use of a set of relationships among groups of genes or organisms and reflects their evolutionary history. the maximum likelihood method is used to describe and analyze biological sequences. alignment of the nucleic acid sequences of the denv followed by its phylogenetic analysis and subsequent generation of phylogenetic trees revealed information regarding its genetic evolution and epidemiology of the disease worldwide. [ ] [ ] [ ] the e protein of the denv is responsible for its tropism and virulence. the gene encoding the e protein has demonstrated its usefulness for decades for the phylogenetic reconstruction of the denv. thus, complete analysis of the coding region can help assign the correct denv genotype and infer the relationships within genotypes and lineages accurately. therefore, the e protein provides adequate resolution to characterize genetic relationship and evolution of the denv. guangdong province is located in the southern mainland of china and experiences tropical and subtropical monsoon climates with a hot and rainy environment that supports breeding of mosquitos, leading to epidemics and very high incidences of df. , the first outbreak of dengue in the foshan city of guangdong province occurred in , after which periodic infections and transmission of all four serotypes of dengue have occurred in the past years. the denv found in these areas may have different origins. there were two denv outbreaks in foshan in and , respectively. phylogenetic analysis revealed that isolates from the epidemic and denv from the epidemic and the epidemic were closely related to those from the epidemic in thailand, epidemic in indonesia, and the epidemic in the philippines, respectively. since , however, denv has been mainly isolated from the infected cases, and its continued existence in guangdong province indicated that endemic infectious agents of dengue may be circulating locally. sequence analysis of the viruses causing the epidemics at different time points revealed that the isolates were closely related to each other, implying that denv had probably circulated locally and caused the epidemics. since , all four serotypes were derived from autonomous patients from different outbreak localities in guangdong province. in , a total of , cases of dengue were reported, which exceeded the total number of cases reported over the previous years. although three serotypes of the denv (denv , denv , and denv ) were identified, denv was found to be the major causative agent responsible for . % of all , laboratory-confirmed cases diagnosed during this outbreak. a recent study revealed that the detected sequences belonged to viruses of multiple origins, but the strain isolated in possibly originated from the isolates of . it can be reasonably speculated that the infectious agents of denv from the endemic, which were circulating locally, played a crucial role in causing the dengue epidemic in guangdong province. however, the data mentioned previously are from the studies based on the outbreaks of the particular year and have the limitation of space and time. as a result, comprehensive evaluation of the epidemiological situation and molecular evolution of the viral agents is of significance to warn against their risks and establish preventive and control measures for df. based on denv isolated from the outbreak in , we systematically collected the e protein gene from to from genbank. with the epidemiological data since supplied by the guangdong provincial cdc, we studied phylogenetics, molecular characteristics, and epidemiology to strengthen the foundational research of denv for the prevention of large-scale dengue epidemics, providing preventive and control measures of df with important evidence. ethics statement. as this research involved human blood, the aims of our study were explained to all the dengue patients involved (all were adults) and all provided written informed consent. the collection methods of clinical samples and epidemiological data were reviewed and approved by the institutional ethics review board of southern medical university and were carried out in accordance with the approved guidelines. samples were selected randomly based on the laboratory diagnosis and clinical signs. sample collection and epidemiological data. dengue virus rna samples (n = ) were obtained from the guangdong provincial maternal and child care service. all samples were extracted from patients suspected of dengue and were confirmed by reverse transcription-polymerase chain reaction (rt-pcr) using specific primers. the steps included an initial denaturation ( °c, minutes); cycles of denaturation added to make a comprehensive evaluation of the epidemiological situation and molecular evolution in the large dengue outbreak in guangdong. at the same time, e gene sequences of denv since , comprising sequences from guangdong and , from other countries, were downloaded from genbank. after excluding several sequences with uncertain epidemiological data, representative epidemic strains in every lineage were screened using phylogenetic methods. since then, a molecular evolution database for the denv e gene in guangdong and other countries has been established. based on representative strains of the e gene in lineages of the outbreak, as well as the molecular evolution database, we analyzed molecular characterization and possibility of local circulation for denv since in guangdong. three-dimensional ( d) structure prediction of denv protein e and molecular docking with -kda glucoseregulated protein (grp ). amino acid (aa) sequences of denv protein e were translated by using dnastar. protein d structures were simulated by discovery studio . (ds . ) (http://accelrys.com/products/collaborativescience/biovia-discovery-studio/) through the homology modeling method based on the modeler program. the optimal protein d model was selected by combining the probability density function and discrete optimized protein energy, and the reliability of the model was evaluated using the ramachandran plot and profile- d. the optimal protein structure model was used to perform protein docking calculations with the grp protein (pdb number: ldp) using the zdock algorithm in ds . , and rdock was used to further optimize the docking configuration to minimize energy. finally, we analyzed aa sites in the binding interface of the optimal docking model. epidemiological findings. since , epidemics of dengue in guangdong have been characterized by periodic outbreaks, the coexistence of importation and localization, and the significantly increased number of indigenous cases compared with that of imported cases ( figure a and c). the incidence peaked in and . especially, during the large outbreak in , a total of , cases of dengue were reported. in , , cases were reported, with a continuous rising trend in comparison to cases in ( figure a ). more seriously, as of november , , a total of , cases were reported, an increase of . % over the same period in . among them, cases were imported, which was . % higher than those in the same period in ; , cases were local cases, which was . % higher than those in the same period in . based on the map of guangdong province, we diagrammed the distribution of the cumulative number of cases since . this showed that guangzhou, foshan, zhongshan, and chaozhou were the main epidemic areas. the trends of the epidemics were anastomotic, with periods of fluctuation in guangdong province ( figure ). after , the epidemic trend featured the gradual coexistence of various serotypes, with up to four serotypes emerging in recent years. each denv serotype was identified. dengue virus was the major cause of the outbreak of , and it continued to be detected in guangdong province as the primary serotype, except in . it is worth noting that the proportion of dengue virus in the total cases also gradually increased ( figure d ). data of samples were used to analyze the overall epidemiology. all patients who provided the samples had mild dengue and had no travel history (table ) . generally, the collection date distribution was concentrated in september and october ( figure b ). the endemic areas were primarily located in guangzhou, followed by zhongshan and chaozhou, accounting for . % of all samples. heyuan, huizhou, and shanwei were included in the collection areas ( figure e ). dengue virus was the major causative agent of this outbreak. the serotype detected through type-specific primers (table ) was denv , which is similar to the results from guangdong cdc that of df cases involved, cases were due to denv infection ( figure d , table ). phylogenetic analysis of denv outbreak in . all e genes were sequenced to construct phylogenetic trees. the dengue outbreak in involved asian genotype i and american/african genotype v. phylogenetic analysis showed that most of the sequences were clustered into a unified clade whose distribution differed in each city. among them, e gene sequences were identified as genotype v with . - % similarity. they clustered into the same clade, which was closely related to the denv sequences in malaysia, singapore, and india and evolved into a lineage of genotype v. during - , this lineage was involved in a co-epidemic in guangdong province, malaysia, and pakistan, with a continuous high-level local prevalence especially in singapore. significantly, this lineage was detected in six cities in our collection; sequences from shanwei, chaozhou, and huizhou all belonged to genotype v. in addition, another nine sequences of the e gene were identified as genotype i, with two different lineages. lineage i involved eight sequences in our collection and other sequences in singapore and thailand in recent years, with a . - % similarity. only one sequence in guangzhou contributed to lineage ii, which had been involved in local epidemics in malaysia, singapore, and indonesia for many years ( figure ). phylogenetic analysis of guangdong since . we downloaded e gene sequences since , comprising from guangdong and from other countries. databases of e gene molecular evolution were created via a screening process based on epidemiologic and phylogenetic methods. among them, strains in guangdong constituted the local database, whereas strains in other countries made up the imported database. based on the evolutionary lineage of denv detected in in guangdong, seven sequences (p , p , p , p , p , p , and p ) were added to the local we endeavored to clarify the relationship between guangdong and other countries concerning denv outbreaks. for this, we illustrated the origin of molecular evolution lineages and the potential of in situ evolution. representative strains from each lineage were added to the imported database for phylogenetic evolution analysis. in our collection, the molecular evolutionary lineage of the imported database proved to be highly consistent with the local database. the only distinctions were that the evolution time of lineages i and ii changed in genotype i, and denv in guangzhou, , formed a clade alone, named clade a ( figure a ). genotype i, which has extensively circulated in guangdong and has been detected in each year of the epidemics, displayed some differences with epidemic countries surrounding china. after , lineage i was not found in guangdong. lineage i was very homologous with lineages in singapore, thailand, and sri lanka in the same epidemic years. however, lineage i spread in neighboring countries, such as malaysia, myanmar, and new guinea, which contributed to cyclic epidemics ( figure b ). lineage ii in guangdong presented a more complicated epidemic situation in countries around china: for example, lineage ii extensively circulated in vietnam, singapore, thailand, malaysia, and cambodia, with vietnam and cambodia being hot spots. clade a only formed a cluster with denv in vietnam ( figure b ). recently, lineage iii in guangdong showed a complicated epidemic situation in surrounding countries as well. its sequence was related to sequences in thailand, laos, singapore, malaysia, and elsewhere. in addition, it was related to the co-epidemic in australia that occurred in and ( figure b ). there were few countries with lineage iv whose epidemic originated from the sequences in . only malaysia, singapore, and especially indonesia shared clustering in denv with long-term cyclic epidemics ( figure b ). in addition, the occurrence of genotypes iv and v was inconsistent in different years. genotype iv sequences in shared extensive homology with those in indonesia, whereasthe strains in were related to those in the philippines with long-term cyclic epidemics ( figure c ). genotype v was related to the strains in india and maldives and was divided into clade and and clade and ; the sequences shared long-term coepidemics with india and singapore, respectively ( figure d ). protein conformational changes caused by mutation of e protein-specific aas between denv genotypes i and v. a total of eight uniform aa substitutions in the ectodomain of the e protein were mainly concentrated in domains i (five substitutions) (di) and iii (two substitutions) (diii) between genotypes i and v, whereas domain ii (dii) has only one. among them, two substitutions caused the protein's secondary structure to change from β-sheet to coil, e (i → l) in dii and e (t → s) in di, whereas only e (d → n), e (s → t), e (t → i), and e (t → s) of di were observed on the d conformation surface of the protein ( figure a and b, table ). meanwhile, e (t → i) and e (t → s) of di were also found in the binding interface between the e protein of genotypes i and grp , and other substitutions were not observed in either serotypes ( figure e ). in the docking model with the grp protein, all three domains of genotype i were involved, whereas genotype v had only di and diii. we found three identical docking sites of diii in the binding interface of the two genotypes: a- p, t- d, and e- k ( figure c and d) . the distribution characteristic of denv in guangdong was determined from its long-term epidemic history. dengue virus was first detected in zhongshan, guangdong province. since then, denv epidemics have occurred sporadically in specific regions over - years. until , denv was the leading serotype, which triggered massive outbreaks in guangzhou. thereafter, denv was circulated continuously across multiple geographies in guangdong, and the isolated strains branched into several stable molecular evolutionary clades. , since , it has spread to different provinces of guangzhou, and imported cases were no longer primary. at indicated that although three denv serotypes (denv , denv , and denv ) were identified, denv was the major causative agent of this outbreak which had circulated continuously in multiple geographies. , so far, the results mentioned were consistent with those of our study. the term "lineage" has been used to denote the viruses clustered in clades at a taxonomic level beneath the genotype. the appearance, change, and reappearance of specific lineages are closely linked to the transmission of those viruses. [ ] [ ] [ ] here, strains of viruses differing in their e genes were obtained, of which nine and strains belonged to genotypes i and v, respectively. unlike the strains of genotype i which were linked to two lineages, each strain in genotype v branched into the same clade, forming a stable lineage. however, several lineages from different origins were responsible for the denv outbreak in . moreover, strains belonging to genotype v were detected in six areas evaluated in this study. all strains from shanwei, chaozhou, and huizhou gathered together also belonged to this genotype, illustrating that the same lineage was prevalent in multiple regions. coincidentally, some epidemiological data indicate that denv was restricted only to the local epidemics since , a phenomenon which was reported by guo et al. overall, the aforementioned evidence strongly opposes the conventional belief that df in guangdong was completely imported. lee et al. used the term "in situ evolution" to characterize denv in singapore through phylogenetic analysis. results of the analysis explained the correlation between its genetic and evolutionary aspects, suggesting that denv had lurked locally and reappeared after some time. rajarethinam et al. described that the dengue in singapore from to demonstrated cyclic epidemic patterns dominated by serotypes and . however, this in situ evolution, demonstrated by the step-ladder pattern of branching within each clade over time, has not been observed in guangdong since . another possible transmission and evolution pattern of denv broke out in parts (switch of the lineage), followed by silence (change of the lineage), and was then prevalent on a large scale (continuation of the lineage), achieving continuous evolution of a new lineage and the silent circulation of old lineages. in , when denv first became the leading serotype, it was due to a "switch" from the lineages in genotype i, which branched into lineages i-iii at the beginning. , from to , it was observed that the prevalence of denv was low and it frequently interchanged between the three lineages, leading to the occurrence of epidemics caused by denv belonging to each lineage. , however, since , the severity of dengue is increasing because of a continued epidemic caused by lineage iii. in , a switch in lineage iv, which was similar to the evolutionary process of lineage iii, was observed. the lineage of genotype iv was first formed in , which reappeared in , and was never detected in recent years. , it is reported that genotype v had circulated continuously from to , followed by an intermittent silence and further reappearance in . this type of molecular evolution was similar to the silent circulation prevalent figure . phylogenetic analysis of denv detected from the outbreak in guangdong. fifty-six envelope gene sequences were isolated in our study. these and reference sequences from genbank were used to construct the phylogenetic tree. asian genotype i and american/african genotype v were involved in this outbreak. forty-seven sequences identified as genotype v clustered into the same clade, whereas genotype i was divided into lineages i and ii. thirteen sequences from shanwei (blue circles), chaozhou (green circles), and huizhou (violet circles) all belonged to genotype v. this figure appears in color at www.ajtmh.org. in indonesia, brazil, etc., , , indicating that the same might happen in guangdong. castonguay-vanier et al. also described the active circulation of denv in laos and the concurrent multiple introductions of new strains from neighboring countries, whereas moore et al. claimed that the cocirculation of denv - in png provides molecular evidence of its endemic transmission. meanwhile, compared with denv , denv , and denv , denv , which is the leading serotype in guangdong, rarely caused severe df. [ ] [ ] [ ] in addition, because guangdong is the most densely populated province in southern china, which is surrounded by a large number of dengue-endemic countries and has a subtropical climate providing optimal environmental, social, and biological conditions for mosquito breeding and reproduction, a detailed evaluation of the current dengue epidemic is significant. nevertheless, there is still a lack of evidence on the local mosquito vector regarding its harboring of denv during epidemic and nonepidemic periods which can support the evidences of its molecular evolution revealed by the phylogenetic analysis. three genotypes (i, iv, and v) in denv from a variety of origins were identified. data obtained from the characterization of local or imported viruses with similarity in lineages found in guangdong demonstrated that these viruses, which were identified based on their molecular evolutionary analysis, were also present in many other countries, particularly those neighboring china. continuous occurrences of the epidemic in indonesia, malaysia, and singapore were consistent with the outbreaks in our country. the epidemiological data demonstrated that the early imported cases in guangdong had high correlation with those found in the neighboring countries, which indicated that these countries might be the source for the migration of denv to guangdong. , however, specific switches in different genotypes were observed, which are as follows: myanmar, thailand, vietnam, and laos for genotype i; the philippines for genotype iv; and india and maldives for genotype v. the outbreaks of severe epidemics over the years in the neighboring countries and frequent mobility of their populations to china may be able to explain the pandemic pattern of the spread of denv, which began with its spread to guangdong province and then broke out in other parts of the country. a similar conclusion was presented in the study conducted by sun et al. regarding the epidemiological characteristics and genetic diversity of denv in guangdong in . nevertheless, since , with an extremely high incidence of cases, denv spread more rapidly and affected a wider range of populations. the co-epidemic between other countries and guangdong and the dissemination of different genotypes and serotypes in multiple regions might not align totally with the theory that only the imported cases caused the epidemic. lee et al. reported a theory which stated that multiple factors are involved in addition to the ones described in in situ evolution that can explain this phenomenon. however, we still need more evidence to prove the applicability of this hypothesis to the epidemic outbreaks in guangdong province. a prediction of the algorithm of the secondary structure of proteins suggests that it is closely related to the distribution of protein epitopes. the high chemical bond energy of the α-helix and β-sheet enables folding of the protein, making it difficult to bind to the antibody, whereas the β-turn and coil, because of their loose structure, are easily displayed on the surface as antigenic epitopes, facilitating binding of the antibody. [ ] [ ] [ ] only two substitutions causing changes in the secondary structure from the β-sheet to coil were observed in our study, suggesting that its antigenicity was enhanced. however, e (t → s) located on the surface is more likely to be associated with protein function than e (i → l), which was located inside the protein. although di, the structure found in the central region of the e protein, was not significantly related to the protein function, in our study, majority of the aa substitutions were observed ( / ), and most of them were located on the surface of its d structure. even e (t → i) and e (t → s) were found in the binding interface between genotype i and grp but not genotype v, further suggesting that the substitution of e (t → s) may be playing a key role in the binding of the e protein of genotypes i and v to the receptor. moraes et al. found that, with changes in the ph, the specific interaction between di and diii of the denv e protein is destroyed, resulting in its conformational change during entry into the cell, whereas nayak et al. also observed the presence of a bundle structure consisting of four polar aa residues at the interface between di and diii, of which his- and his were unique to denv , implying that the change of di conformation will also affect the realization of diii function. domain iii has an immunoglobulin-like structure and a functional region where denv binds to a cellular receptor. drumond et al. predicted the structure of the denv e protein in brazil and observed that the substitution of e (s → f) can reduce its interaction with several residues (ser , lys , lys , val , val , ile , tyr , and gly ), resulting in a change in the folding of the area, whereas genotypes i and v of denv in guangdong have a unified mutation in e (i → v). the residue qhg at position e -e of the e protein of denv and is highly conserved, and docking by the zdock method showed that it possibly interacts with the membrane receptor protein tim- , although we found the same region conserved at e -e . meanwhile, three conserved regions ( a- p, t- d, and e- k) were concurrently marked in the binding interface of the two genotypes found in guangdong. chen et al. localized the neutralizing determinants of the inhibitory mabs demonstrating strong effects to a sequence-unique epitope on diii of the denv e protein, centered near residues t and d ( tqngrlitanpivtd ) which were highly conserved among different genotypes of denv but different from those of the denv , denv , and denv serotypes and other flaviviruses. currently, we believe that vaccination and vector control are the fundamental measures to control df. however, research on vaccines and mosquito-control measures have not made significant breakthroughs. we need additional information to understand the epidemic situation of df and evolution of its virus in guangdong to develop effective preventive and control measures. epidemiological analysis reveals information on the cities and months during which df was substantially prevalent and helps to develop mosquito-surveillance and killing strategies. the phylogenetic tree revealed that denv , which is the main serotype of the virus, has been prevalent in guangdong since a long time. the strains isolated from epidemic cases occurring during the same period are homologous, and genotype i has formed a stable evolutionary lineage in recent years. these results suggested that denv may be lurking and circulating in guangdong, although it cannot be stated with certainty. however, it is highly recommended that we detect the denv in local mosquito vectors urgently. at the same time, the phylogenetic tree of the input source suggests the possible countries and regions from which importation of df in guangdong can occur. this information is of great significance for the development of a plan to monitor the departure and entry of populations from regions with a high incidence of dengue. e-mails: chienhai@ .com, murong @outlook. com, hexiaoen @ .com, maiblume@ .com, xiyuxie @ .com, zhuli @ .com, @ .com, milyx @ .com, harpul@ sina.com, wenying@gdciq.gov.cn, and zhangb@smu.edu.cn. zhicheng zhong, and danjuan ma dengue fever: new paradigms for a changing epidemiology a local outbreak of dengue caused by an imported case in dongguan china dengue viruses in brazil guidelines for diagnosis, treatment, prevention and control world health organization refining the global spatial limits of dengue virus transmission by evidencebased consensus the global distribution and burden of dengue flavivirus genome organization, expression, and replication the origin, emergence and evolutionary genetics of dengue virus insights of the genetic diversity of denv- detected in brazil in years: analysis of the envelope domain iii allows lineages characterization microevolution and virulence of dengue viruses the role of models in reconstructing evolutionary trees molecular evolution and phylogeny of dengue- viruses phylogeny 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protein in the postfusion conformation and its implications for membrane fusion phylogenetic analysis of dengue virus isolated from south minas gerais, brazil epitopes based drug design for dengue virus envelope protein: a computational approach characterization and epitope mapping of dengue virus type specific monoclonal antibodies this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. key: cord- -m pur td authors: wang, changjian; wu, kangmin; zhang, xinlin; wang, fei; zhang, hongou; ye, yuyao; wu, qitao; huang, gengzhi; wang, yang; wen, bin title: features and drivers for energy-related carbon emissions in mega city: the case of guangzhou, china based on an extended lmdi model date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: m pur td based on the apparent energy consumption data, a systematic and comprehensive city-level total carbon accounting approach was established and applied in guangzhou, china. a newly extended lmdi method based on the kaya identity was adopted to examine the main drivers for the carbon emissions increments both at the industrial sector and the residential sector. research results are listed as follow: ( ) carbon emissions embodied in the imported electricity played a significant important role in emissions mitigation in guangzhou. ( ) the influences and impacts of various driving factors on industrial and residential carbon emissions are different in the three different development periods, namely, the (th) five-year plan period ( – ), the (th) five-year plan period ( – ), and the (th) five-year plan period ( – ). the main reasons underlying these influencing mechanisms were different policy measures announced by the central and local government during the different five-year plan periods. ( ) the affluence effect (g-effect) was the dominant positive effect in driving emissions increase, while the energy intensity effect of production (e-effect-production), the economic structure effect (s-effect) and the carbon intensity effect of production (f-effect-production) were the main contributing factors suppressing emissions growth at the industrial sector. ( ) the affluence effect of urban (g-effect-aui) was the most dominant positive driving factor on emissions increment, while the energy intensity effect of urban (e-effect-urban) played the most important role in curbing emissions growth at the residential sector. with anthropogenic releases of carbon emissions contributing toward climate change primarily characterized by global warming, many policymakers and scientific-researchers are seeking scales. therefore, comprehensive inventory and specific boundary are the basis for city-level carbon emissions accounting, as well as local emission mitigation strategies [ ] . the second strand of research focuses on the city-level driving factors and influencing mechanisms. decomposition analysis (e. g. index decomposition analysis (ida) and structural decomposition analysis (sda)) and regression method (e. g. stochastic impacts by regression on population, affluence, and technology (stirpat) model) are the most commonly applied methods for the scientific evaluation and quantitative analysis of factors influencing city-level carbon emissions, especially the logarithmic mean divisia index (lmdi) method based on the ida framework. as shown in table , the sda method based on the io technique applied in the city-level driving factors and influencing mechanisms research in china were mainly focused on the four municipalities (i. e. beijing, shanghai, tianjin and chongqing), because of the available io data base compiled by the municipality bureau of wang et al. [ ] - stirpat beijing wang et al. [ ] - lmdi beijing mi et al. [ ] io beijing wang et al. [ ] - sda beijing tian et al. [ ] - sda beijing wang et al. [ ] - lmdi and tapio index beijing, tianjin wang et al. [ ] - stirpat shanghai shao et al. [ ] - stirpat shanghai zhao et al. [ ] - lmdi shanghai shao et al. [ ] - lmdi shanghai kang et al. [ ] - lmdi tianjin li et al. [ ] - stirpat tianjin tan et al. [ ] - lmdi chongqing wang et al. [ ] - lmdi suzhou chen et al. [ ] - decoupling index macao liu et al. [ ] - lmdi beijing, shanghai, tianjin, and chongqing statistics. in other words, the io data sources were lacking in other provincial capital cities and medium-sized cities in china. consequently, the ida methods were used more frequently than the sda methods based on io technique [ ] [ ] [ ] [ ] , owing to its applicability in which each model can be applied to any available data at any aggregation level in a period-wise or timeseries manner [ ] [ ] [ ] , where the preferred one in the lmdi [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . by means of lmdi method, wang et al. [ ] investigated the main driver for industrial carbon emissions in beijing-tianjin-hebei economic band from to , and the results showed that economic output and energy intensity were both the most important influencing factors. wang et al. [ ] decomposed the influencing factors of residential carbon emissions in beijing and found that the rising per capita gdp and the declining energy intensity contributed the most to emissions changes during to . similar results, conducted by wang et al. [ ] , can also be found in suzhou city. zhao et al. [ ] decomposed the main drivers for industrial carbon emissions in shanghai and found that industrial output, energy intensity, energy and industrial structure were major determinants for emissions changes. shao et al. [ ] adopted an extended lmdi method and investigated the techno-economic drivers for industrial carbon emissions in shanghai, and the results showed that industrial output scale was mainly responsible for emission increments while industrial structure adjustment, r&d intensity, and energy intensity were most significant factors in mitigating emissions. kang et al. [ ] performed a multi-sectoral decomposition analysis of city-level greenhouse gas emissions in tianjin from to , including the agricultural, industrial, transportation, commercial and other sectors, and the results showed that economic growth was the most important driver for emissions increments while energy efficiency was primarily responsible for emissions reductions. hopefully, previous studies about driving factors and influencing mechanisms of city-level carbon emissions performed on china's megacities such as beijing, shanghai etc., have made great contributions to the realization of regional low-carbon road map. but, the previous lmdi studies mainly focused on the economic growth effect, the energy intensity effect, the population size effect, and the technology progress effect influencing the change of carbon emissions. it fails to distinguish the effects of economic structure, population structure, and energy structure on the change of carbon emissions. in fact, the strongest factors that offset china's energy consumption and carbon emissions have shifted from efficiency gains to structural changes [ , ] . structural indicators combined with other traditional and emerging factors should also be deeply considered and investigated in china's regional energy and carbon researches, which may perform different influencing mechanism in different regions during different development stages. compared with four municipalities, there were relatively few studies conducting on guangzhou city, one of china's first-tier cities, with the considerable gdp scale and total energy consumption amount (table ) . meanwhile, guangzhou has the highest level of per capita gdp and per capita energy consumption than the four municipalities. guangzhou is the provincial capital of guangdong province, the most developed coastal region in the southeast of china (fig ) . in addition, guangzhou city is an important manufacturing base in china as well as a pilot zone for low-carbon city announced by china's national development and reform commission (ndrc) in . therefore, guangzhou city may serve as a demonstration of how to realize the targets of low-carbon city development, thereby highlighting the importance and representativeness of studying its features and drivers for carbon emissions in detail. the innovation and contribution of this study compared with other references mainly lies in the following two aspects. firstly, city-level total carbon accounting approach based on the apparent energy consumption data from the comprehensive energy balance table was established. secondly, driving factors including structural indicators, e. g. economic structure, energy structure, and population structure, were investigated systematically by adopting the newly extended lmdi method based on the kaya identity and the ida theory. it was aimed to provide theoretical references for making more targeted policies on energy saving and emission reduction in china's mega cities. this study is organized as follows: after the introduction, section presents the methodology of city-level carbon accounting in guangzhou and the method to decompose the changes in carbon emissions during the whole research period. section addresses the case analysis and the main results of guangzhou city. finally, we conclude our study and provide some policy implications for low-carbon city development. city-level total carbon accounting as mentioned in table , urban emissions inventory is the key for city-level total carbon accounting. however, there are accounting discrepancies between the different types of methods [ ] . based on china's energy statistic and the reference approach of ipcc guideline for national greenhouse gas emission inventories, there are mainly three approach to account, i. e. primary energy consumption (e. g. coal, oil, natural gas, and renewables) [ ] , final energy consumption (e. g. coal, oil, natural gas, renewables, and electricity/heating) [ , ] , and total energy consumption from the "energy balance table" including the aggregate summary of energy production, energy transformation and final consumption [ , ] . compared with the primary energy consumption accounting and the final energy consumption accounting, apparent energy consumption data [ , ] from the comprehensive energy balance table was recommended for this study. as shown in the table , the comprehensive energy balance table is mainly based on three modules, namely, ) total energy available for local consumption as a result of interprovincial and international energy trades, ) input-output of energy conversion transformation for thermal power, heating, coking, petroleum refining, and gas production, ) final consumption due to primary industry, secondary industry, tertiary industry, and residential consumption. primary industry, namely, agriculture, mainly includes farming, forestry, animal husbandry, and fishery. secondary industry includes manufacturing industries and construction industry. tertiary industry includes transportation, storage, post and telecommunication services, wholesale, retail trade and catering services, and other service sectors. then, we calculated energy-related carbon emissions by energy types according to the ipcc guidelines for national greenhouse gas inventories based on the apparent energy consumption data from the comprehensive energy balance table, as follows the equation: greenhouse gas (ghg) in our case study is referred in particular to carbon emissions-that directly affect climate change, which didn't include other ghgs (i. e. ch , n o, etc.). where the subscript i is the various fuels in this case study, t means the time in years. c t represents total carbon emissions in year t (in million tons, mt). e i t represents the total energy consumption of fuel type i in year t (in million tons, mt), and ncv i is the net caloric value of fuel i. cc i t is the carbon content of the fuel type i, and o i is the oxidation rate of fuel i. the conversion factors, net caloric value, oxygenation efficiency and carbon content of the various fuels are listed in table . data sources, consisting of the gross domestic product including agriculture, production, construction, and service, the population including rural population and urban population, the total energy consumption based on industrial and residential perspective, covering the period from to , were all available. economic, population and energy data were collected from the guangdong province statistical yearbook ( - ) and guangzhou city statistical yearbook ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . economic data was measured by gdp in chinese yuan in time series. energy data includes physical quantity of total energy consumption by fuel types measured by tons of standard coal equivalent (tce), which were compiled by the guangdong province statistical bureau and guangzhou city statistical bureau. as mentioned and reviewed in table , the lmdi methods based on the ida theory, were used frequently to quantitatively analyze the drivers of carbon emissions at the city-level. the lmdi technique based on the ida theory and an extended kaya identity was conducted to uncover the main driving forces for energy-related carbon emissions in guangzhou city. the kaya identity [ , , , ] expresses carbon emissions as a product of four underlying driving factors: where p represents the population, g represents the gdp, e represents the total energy consumption; g p represents the per-capita gdp, e g represents the energy consumption intensity, and c e represents the carbon intensity of energy. then, the kaya identity was extended as follow: where x n k¼ c i (i = , , , , , ) represents carbon emissions from agriculture (i = ), production (i = ), construction (i = ), service (i = ), urban resident (i = ), and rural resident (i = ). k (k = , , , . . .n) represents the various energy types in this case study, mainly including coal, coke, crude oil, gasoline, diesel oil, kerosene, fuel oil, lpg, natural gas, and electricity. e i (i = , , , , , ) represents total energy consumption by different sectors. gdp i (i = , , , ) represents gross domestic product by different industries. p urban and p rural represent the total population of urban and rural residents, respectively. ti urban and ti rural represent the total income of urban and rural residents, respectively. ai urban and ai rural represent the average income of urban and rural residents, respectively. then, eq ( ) can be rewritten as follow: where p = p, represents the total population size, ur represents the level of urbanization. g ¼ gdp p , represents the per capita gdp. e i ¼ e i gdp i (i = , , , , , ), represents the energy consumption intensity of agriculture, production, construction, service, urban resident, and rural resident, respectively. , represents the energy carbon intensity of agriculture, production, construction, service, urban resident, and rural resident, respectively. s i ¼ gdp i gdp (i = , , , ), represents the economic structure of agriculture, production, construction, and service, respectively. then, the changes of regional carbon emissions between two years can be decomposed as: following the preferred lmdi method proposed by ang et al. [ , , ] , the various effects of each factor can be quantificationally calculated as: in the newly established lmdi method, the population effect (Δc p-effect ), the affluence effect (Δc g-effect ), the energy intensity effect (Δc e-effect ), the economic structure effect (Δc s-effect ), and the carbon intensity effect (Δc f-effect ). in addition, social and economic factors such as urbanization (Δc ur-effect ), family income in urban and rural households (Δc ai-effect ) were analyzed to further explain the changes of carbon emissions at the city level. economic development in guangzhou was described with gdp, which was converted into the constant prices (fig ) . according to the urban emissions inventory in guangzhou and eq ( ), we calculated the energy-related carbon emissions from to . total energy-related carbon emissions mainly include two parts, namely, carbon emissions generated in the boundary from fossil fuel combustion and out of the boundary embodied in the imported electricity (fig ) . carbon emissions embodied in the imported electricity need to be calculated by considering the total amount of imported electricity and the corresponding emissions factor of power generation the case of guangzhou based on extended lmdi model [ ] . as for china's power supply system, electricity is supplied by the main six regional grids five-year plan period, respectively.), which were issued by the central government and performed by the local government. but in fact, carbon emissions mitigation in the boundary mainly benefited from contributions of the imported electricity from the neighbors guizhou and yunnan provinces via the cross-province electricity trades. during the same period, carbon emissions embodied in the imported electricity dramatically increased . million tons in to . million tons in . rapidly increasing carbon emissions embodied in imported electricity indicated that there were large amounts of fossil energy were burned for power. final products-thermal power was consumed in guangzhou, while carbon emissions were discharged in its neighbor producers. if these carbon emissions embodied in the imported electricity were allocated to the final consumer, guangzhou would have paid more efforts and implemented more strict mitigation measures to achieve its energy saving and emission reduction targets. aiming to have a better understanding of the complex various influencing factors for carbon emissions in guangzhou city, we decomposed the total carbon emissions into two parts including the industrial sector and the residential sector. driving factors of carbon emissions in industrial sector in guangzhou. driving factors of carbon emissions in industrial sector were decomposed yearly first (table ). in order to have a better understanding of influencing factors in long time series, we divided the carbon emissions process into periods (fig ) , according to the regional five-year plans for socioeconomic development, combined with a certain historical background. during the th five-year plan period ( ) ( ) ( ) , guangzhou announced the overall objective in the th five-year plan that it would take the lead in realizing socialist modernization and building the modernization central city over china. guangzhou's industrial carbon emissions increased by . million tons mainly because the rapid economic development. the affluence effect (g-effect) was the dominant positive effect in driving carbon emissions increase, bringing in a . million tons increase, accounting for . % of the total industrial carbon emissions changes, which was followed by the energy intensity effect of service (e-effect-service), the carbon intensity effect of service (f-effect-service), and the economic structure effect (s-effect). in order to achieve the basic standards of modernization (i. e. per capita gdp is more than u.s. dollar, and the third industry added value accounted for more than % of gdp etc.), guangzhou paid more efforts to foster the three pillar industries (i. e. electronic information manufacturing industry, automobile industry, and petrochemical table period the case of guangzhou based on extended lmdi model economic structure effect (s-effect). the adjustment of industrial structure in guangzhou was mainly focused on upgrading the traditional advantage industries (i. e. iron and steel, shipbuilding, machinery and equipment, electricity, paper, and textile etc.) and fostering the new and high technology industries (i. e. software, new material, new energy, and digital industry etc.), announced in the th five-year plan for socioeconomic development in guangzhou driving factors of carbon emissions in residential sector in guangzhou. driving factors of carbon emissions in residential sector were decomposed yearly first (table ) and divided into periods (fig ) . although the residential carbon emissions in guangzhou accounted for a relatively small proportion of the total carbon emissions, it exhibited a rapid growth trend during the whole research period along with the improvement of urbanization level. the total residential carbon emissions increased from . million tons in accounting for . % of the total carbon emissions to . million tons accounting for . % in . furthermore, the residential carbon emission increments were mainly generated by the urban residents, which increased from . the case of guangzhou based on extended lmdi model was always two times of the average income of rural residents during the same period. the energy intensity effect of urban (e-effect-urban) played the most important role in curbing carbon emissions growth, resulting in a . million tons decrease, accounting for . % of the total residential carbon emissions changes in absolute value. during the th five-year plan period ( - ), guangzhou's residential carbon emissions increased by . million tons. the affluence effect of urban (g-effect-aui) and the carbon intensity effect of urban (f-effect-urban) were the most two dominant positive driving factors on the carbon emissions increment, bringing in . million tons and . million tons increase, accounting for . % and . % of the total residential carbon emissions changes, respectively. the energy intensity effect of urban (e-effect-urban) played the most important role in curbing carbon emissions growth, resulting in a . million tons decrease, accounting for . % of the total residential carbon emissions changes in absolute value. based on the apparent energy consumption data from the comprehensive energy balance table, a systematic and comprehensive city-level total carbon accounting approach was established and applied in one of the china's mega city-guangzhou. total carbon emissions including the fossil fuel combustion in the city's boundary and embodied in the imported electricity out of the boundary were still performing a rising tendency. carbon emissions embodied in the imported electricity played a significant important role in emissions mitigation in guangzhou, especially after . examining the effects of different boundaries on carbon accounting at city level is crucial for the emissions mitigation in urban china. then, the newly extended lmdi method based on the kaya identity was adopted to examine the main drivers for the carbon emissions increments both at the industrial sector and the residential sector in guangzhou city. quantitative analyses and time-series analysis were performed on the influencing mechanism for various influencing factors both at the industrial sector and the residential sector for the three different development periods, namely, the th five-year plan period ( ) ( ) ( ) , the th five-year plan period ( - ), and the th five-year plan period ( - ). the influences and impacts of various driving factors on industrial and residential carbon emissions are different in the three different development periods. overall, the affluence effect (g-effect) was the dominant positive effect in driving carbon emissions increase, while the energy intensity effect of production (e-effect-production), the economic structure effect (s-effect) and the carbon intensity effect of production (f-effect-production) were the main contributing factors suppressing the carbon emission growth at the industrial sector. considering the high urbanization level in guangzhou, the affluence effect of urban (g-effect-aui) was the most dominant positive driving factor on the carbon emissions increment, while the energy intensity effect of urban (e-effect-urban) played the most important role in curbing carbon emissions growth at the residential sector. in conclusion, affluence effect-economic growth, was still the most important contributor to the increases in carbon emissions in urban china. however, structural indicators, such as economic structure and energy intensity of different industrial sector, were playing significant negative effects on carbon emissions. in the future, economic structure adjustment and energy intensity improvement at the detailed production industries should be deeply investigated, in order to make these indicators play more negative effects on carbon emissions. considering the significant important role played by the emissions embodied in the imported electricity via the cross-province electricity trades, guangzhou should adjust its current emissions mitigation policies which only consider its emissions occurring within the city's boundary. guangzhou should make more joint efforts to help its neighbors improve the efficiency of thermal power generation. guangzhou might make efforts to buy more clean power such as hydropower from its neighbors yunnan and guangxi provinces to replace a certain amount of thermal power. in addition, carbon trading scheme [ ] about the cross-province electricity trades should be introduced and conducted on the imported electricity in guangzhou. in order to further promote the negative effects of the economic structure effect (s-effect), the energy intensity effect of production (e-effect-production), and the carbon intensity effect of production (f-effect-production), guangzhou should pay more efforts to the optimization of energy consumption structure and industrial structure at the industrial sector. ( ) coal consumption in energy mix should be further decreased, while relatively low carbon energy such as natural gas should be accelerated both in the industrial and residential sectors. renewable energy such as solar pv and hydropower should also be encouraged. ( ) efficiency of thermal power generation should be further improved in guangzhou, especially the major power plants such as the pearl river power plant and the huarun power plant in nansha district, the guangzhou power plant in liwan district, the hengyun power plant, the huangpu power plant, and the ruiming power plant in huangpu district. ( ) energy consumption structure and energy utilization technique should be also effectively optimized and improved, especially in the pillar industries (i. e. electronic information manufacturing industry, automobile industry, and petrochemical industry), the traditional advantage industries (i. e. iron and steel, shipbuilding, machinery and equipment, paper, and textile etc.) and the new and high technology industries (i. e. software, new material, new energy, and digital industry etc.) in guangzhou in the current and near future. urbanization level of guangzhou is relatively high than other cities in guangdong province and even in china. the rapid increase in the average income of urban residents along with the high level of urbanization has made the affluence effect of urban (g-effect-aui) become the most dominant positive driving factor on the residential carbon emissions increment during the whole research period. the residential energy consumption structure was mainly based on 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estimates from fossil fuel combustion and cement production in china examining the driving factors of energy related carbon emissions using the extended stirpat model based on ipat identity in xinjiang global and regional drivers of accelerating co emissions . ang key: cord- -aapg af authors: tambo, ernest; tang, shenglan; ai, lin; zhou, xiao-nong title: the value of china-africa health development initiatives in strengthening “one health” strategy date: - - journal: global health journal doi: . /s - ( ) - sha: doc_id: cord_uid: aapg af implementing national to community-based “one health” strategy for human, animal and environmental challenges and migrating-led consequences offer great opportunities, and its value of sustained development and wellbeing is an imperative. “one health” strategy in policy commitment, partnership and financial investment are much needed in advocacy, contextual health human-animal and environmental development. therefore, appropriate and evidence-based handling and management strategies in moving forward universal health coverage and sustainable development goals (sdgs) are essential components to the china-africa health development initiatives. it is necessary to understand how to strengthen robust and sustainable “one health” approach implementation in national and regional public health and disaster risk reduction programs. understanding the foundation of “one health” strategy in china-africa public health cooperation is crucial in fostering health systems preparedness and smart response against emerging and re-emerging threats and epidemics. building the value of china-africa “one health” strategy partnerships, frameworks and capacity development and implementation through leveraging on current and innovative china-africa health initiatives, but also, mobilizing efforts on climatic changes and disasters mitigation and lifestyle adaptations strategies against emerging and current infectious diseases threats are essential to establish epidemic surveillance-response system under the concept of global collaborative coordination and lasting financing mechanisms. further strengthen local infrastructure and workforce capacity, participatory accountability and transparency on “one health” approach will benefit to set up infectious diseases of poverty projects, and effective monitoring and evaluation systems in achieving african union agenda and sdgs targets both in africa and china. the china-africa partnership is one of the most important geopolitical and economic relationships of the st century that has ushered a new era of investment in mutual health development [ ] . china has become the world's second-largest economy and offered africa various based on win-win cooperation. traditionally, china is willing to work together with africa to achieve mutual benefits by taking advantage of its status as assistanceprovider in tackling infectious diseases of poverty [ , ] . furthermore, since africa is home to seven of world's ten fastest growing economies, chinese investments in the health sector in the continent can produce substantial financial gains and generate invaluable public health commodities and other goods that are much needed [ , ] . the need for an african centre for disease control and prevention (cdc) was recognized at the african union special summit on hiv and aids, tb and malaria held in abuja, nigeria, in july . the africa cdc has launched year with the establishment of an african surveillance and response unit, which will include an emergency operations center and exchanges on china's national disease surveillance and reporting system [ ] . currently, africa continent is experiencing a rapid economic growth, with a gross domestic product (gdp) of $ . trillion usd in and is estimated to increase to $ . trillion usd by . health-care spending rose from $ . billion usd in to $ billion usd in across african countries [ ] [ ] [ ] . the fact that sub-saharan africa accounts for % of the world's population and % of the global burden of infectious diseases caused by poverty, millions of people could be lifted out of poverty through bilateral trade and cooperation between china and africa. increasing and robust new commitments of outbreak and accounts for about % of all deaths recorded (a total of suspected cases, confirmed cases and deaths recorded since disease onset in august . in , us cdc estimated the yearly number of lf cases to be between , to , resulting in about , deaths across west africa. lf is endemic in most parts of west africa with sporadic cases occurring in other african countries every year. studies have predicted approximately % of liberia and sierra leone, % of nigeria, and % of benin to be at risk of lf through spatial analysis. in nigeria, approximately cases and deaths have been reported from to date. due to a paucity of data, the actual number of cases in other west african countries to date is still unknown. however, seroprevalence studies in the past have shown a prevalence of lassa igg antibodies in % to % of the general population in sierra leone and in guinea, and as high as % to % among inhabitants of tropical rain forest and % to % in hospital staff of gueckedou and lola prefectures in guinea. direct transmission from rodent to humans mainly occurs through inhalation of primary aerosols from infected rodent urine, ingestion of food contaminated with rodent excreta or by direct contact with broken skin. regional and nosocomial outbreaks of lf are commonplace in lf endemic countries and played a major role in recent outbreak. in nigeria, the lf outbreak has been estimated to have an overall case fatality rate of % and % in confirmed cases; the impact on healthcare workers due to inadequately equipped, weak preventive measure for hospital associated infections (hai) and well trained staff and facilities with poor laboratory and clinical management practices were the main reasons for a dearth of data. while there is no known vaccine for lf, early supportive care and treatment with ribavirin. prevention efforts include isolation of cases, implementing infection control measures such as barrier nursing supplies, rodent control and practicing adequate food hygiene (storing grain and other foodstuffs in rodent-proof containers) and personal hygiene. although treatment for lf is available, early diagnosis, prevention and prompt management of infection are necessary (table ) . these growing public health emergencies and challenges prompted a memorandum on building the africa cdc that was signed by the african union with two parties are including chinese and us govemrnents. this cooperation exploring ways of further cooperation and lessons learning from china's national disease surveillance and reporting system model [ , ] . based on a unified and integrated plan, china and us government are willing to leverage their respective strengths to support the african union in building this system, which will be the first regional disease surveillance system on the african continent from the ebola crisis. it is important to strengthen disease surveillance and monitoring efforts at the regional level in providing technical expertise and response coordination in future health emergencies, address complex health challenges, and build needed capacity responses, responsible for disease surveillance, investigations, analysis, and reporting trends and anomalies. this is a landmark event in african ownership of improving health across the continent. the us cdc looks forward to engaging in this partnership for many years to come to advance public health across africa and global health security [ , ] results from the first and second china-africa ministerial health development forum held in beijing, china and cape town, south africa in and , respectively, showed that china-africa health development partnership had entered a new collaborative paradigm with great global health opportunities [ , , ] . chinese and african health ministers have adopted a declaration to increase access to facilities, medication, health workers and training, linking chinese scholars with those in africa into shared responsibility and global solidarity [ ] . importantly, china-africa collaboration in health development will use "one health" approach to set the collaborative priorities, such as developing innovative information and communication technology for health, building regional surveillance systems, improving the core capacities of international health regulations and enhancing the using regulation of traditional medicines, etc [ , , ] . the significance of africa-china cooperation health development initiative milestone was the broad consensus mou aimed to support the establishment of africa cdc signed on april , as part of the agreement and of the pledge made at the summit that was held under focac in johannesburg, south africa december [ ] . this laudable mutual commitment was realized through the full operationalisation of the africa cdc in early supported by the chinese government, including providing infrastructure construction, equipment, information system, expertise, and professional training, etc. as well fostering continuous strengthening african states public health prevention and control system under the chinese supports are also provided through comprehensively capacity building (e.g., staff, postdocs and students) and providing technical assistance and technology transfering to africa cdc sub-regional centres. the benefits of the translation of the immense mutual public health priority aligns "africa union health vision " in the fields of infectious diseases of poverty surveillance and elimination, emergency preparedness timely response to early alert and risk communication capabilities against public health emergencies and disaster crises events. previously, china has already provided two million us dollars cash aid for the africa cdc in terms of capacity building and the on-site chinese experts visit for the regional collaboration with other partners' support [ ] . africa cdc has now developed a five year strategic plan to improve surveillance, emergency response, prevention and resilience against infectious diseases threats and outbreaks, man-made and natural disasters, antimicrobial resistance and chronic diseases public health events of regional and international concerns. africa cdc focus on strategic priority areas and innovative programs aiming at improving evidence-based decision making and practice in event-based capacity development for surveillance, disease prediction, and improved functional clinical and public health laboratory networks and actions in minimizing health inequalities, and promoting quality care delivery, public health emergency preparedness and response best practices in achieving regional [ , , ] . africa cdc collaborating sub-regional centres in five countries provides an opportunity for effective collaboration, integration and coordination in harnessing existing public health assets, epidemiological surveillance, strengthening existing networks of quality laboratories for early detection and response. infectious public health preparedness and emegergency response cannot deliver effectively if we do not implement "one health and biosecurity" approach bringing human, animal and environmental health. building evidence-based and adequate capacity building need to support integrated "one health" surveillance, laboratory systems and networks, emergency preparedness and response, and public health research for evidence-based heath programing and ample resource allocation. greater commitment to strengthen local and regional operationalization of integrated disease surveillance and response, public health systems and core capacities have been documented to critically address public health emergencies, biosecurity and disaster risk across the continent. national and regional public health emergencies, biosecurity surveillance, preparedness, rapid response, and recovery policy and strategies are robust and sustainable assets for socioeconomic transformation in line with africa health strategy ( - ), the africa union agenda and in attaining sdgs [ , , , ] . firstly, developing innovative information and communication technology for health will provide opportunities to avert thousands of deaths and disability by improving access to good-quality essential drugs, by increasing coverage of vaccines immunization and use of other pharmaceutical and medical commodities nationwide [ , , ] . in addition, leveraging on the unique "one health" approach to transform health care and health policy and to prioritize collaborative programs can be extended from infectious diseases to maternal and child health and health disparity in the poorest populations in africa [ , [ ] [ ] [ ] . secondly, building regional surveillance systems is another way to enhance the local health system. the importance of implementing a local and national "one health" policy and programs holds tremendous prospects, such as co-tackling the epidemiological and environmental challenges, and accelerating in the transition from control to elimination of infectious diseases under china-africa collaboration [ , ] . furthermore, it has potential to revolutionize national health systems, policies and strategic priorities and the patterns in health financing and resources allocation of african countries that require careful understanding of the local context of diverse stressors and drivers [ , ] . these will continue to dominate the performance and effectiveness of "one health" in threats and epidemics prevention strategies and policies on healthcare and health outcomes. thus, assessing health impact especially how greenhouse gas and ozone emissions, rising temperature and environmental pollution resulted in climate change impacts to health ecosystem, such as population movement, animal trading and ecology of vectorborne disease and ill health, aging, chronic disease, drug use and domestic violence, inequity and poverty [ ] . thirdly, improving the core capacities of international health regulations is the sustained efforts to improve the human welfare. for example, china's response impacting the global health fund (e.g., malaria, hiv/aids, schistosomiasis, ebola, influenza, tb, hepatitis, etc.) has shown robust global health leadership engagement [ , , ] . the leadership reflected in the strategic mobilization and investment of resources fostering more easily accessible, availability and cost effectiveness of prevention and treatment interventions to resources limited countries including african countries [ ] . the growing mutual china-africa win-win collaboration spans to technical expertise, technology transfer and capacity development using scientific and advanced methods to tackle the disease, and have enhanced their commitment to respect the dignity of the people such as chinese ebola outbreak emergency response in west africa. fourthly, enhancing the use and management of traditional medicines could improve the community involvement in health care and extend the trade among countries. so far, trade between china and africa is projected to reach $ million usd a year by . increasingly, embracing "one health" strategy to increase universal coverage of healthcare is significant as sharing china's rich expertise and lessons learnt in strengthening health systems and tackling public health burden both in china and africa communities. thus, africa has the opportunity to improve capacity of community health workers to reach remotes rural communities living beyond the margins of traditional health care systems [ , ] . therefore, china's advancements in research and development, technical and scientific capacity transferring can support african next generation of proactive scientists to develop more sensitive simplified diagnostic tools and reduce the costs of laboratory diagnosis and medical equipment. furthermore, research and development (r&d) is needed in examining the biological mechanisms of stressors or risk factors exposure and health effects, assessing evidence-based mitigation or adaptation interventions and benefits [ , , ] . innovative solutions and breakthroughs in human-animal-environment fields would not only enable africa to meet its own growing needs, but also support integrating health systems, including strengthening the capacities of laboratory diagnostics and medical care, as well as establishing the china-africa platforms that could generate evidence-based low-cost, available and easy-to-use health packages and solutions for the reduction of public health burden. the present paper has analyzed the values of implementing national to regional "one health" strategy for dealing with human, animal and environment related public health threats, diseases outbreaks emergencies and disaster risk challenges, and promote healthy mitigation measures and resilient management approaches in advancing targeted local, national and global health agenda. also, understanding how to develop, package and implement evidence-based and sustainable "one health" approach needs partnerships and investment for strategic priorities and resource mobilization. in addition, it also needs better financing mechanisms and participatory coordination in building capacity and technical assistance, monitoring, performance and effectiveness metrics evaluation for one health indicators. understanding the foundation of "one health" strategy in china-africa public health needs and challenges although significant progress has been made in improving health and safety of vulnerable population in low and middle-income countries (lmics), there is growing unprecedented public health emergencies crises due to natural disasters (such as disease outbreaks, floods, climate change, droughts and mud-/land-sliding) and man-made disasters including armed conflict and resulting forced refugees and displaced populations in lmics and mainly in africa as well as china. these have been resulting in significant direct and indirect health impacts including limited access to food, clean water, medicines, pre-existing mental health and other health services. conflict-affected countries have not achieved a single millennium development goal and have significantly higher maternal and infant mortality rates compared to stable and peaceful countries. natural disasters affect nearly million people each year, with a disproportionate effect on populations and environment. there is also limited quantity of highquality and integrated research to build evidence "one health" approach response. for example, recent emerging zika virus is known to be circulating in latin america, america, africa, asia-pacific and middle east regions due to climate change and rapid urbanization, intense regional and global travel and trade impact on zika virus risk transmission and documented congenital complications on fetal and maternal health. efforts to strengthen regional and global public health emergencies surveillance and preparedness should be maintained in order to better characterize the intensity of aedes and culex vectorial capacity, asymptomatic or syndromic viral circulation and geographical infection spread, epidemiology and laboratory monitoring of zika virus related complications in vulnerable settings. we found that most existing and emerging infectious diseases of poverty and chronic diseases public health programs are based more on top-down and anecdotal experiences rather than accurate research in fostering an integrated humananimal and environment or "one health" community practice in most vulnerable settings in africa and china (table ) . "one health" approach was officially adopted by international organizations and scholarly bodies in in response to the growing global human-animal and environment inter-dependence challenges and issues including climate change which needed new approaches. in such, "one health" broader interconnections understanding offers tremendous advantages and manifold benefits in tackling emerging zoonotic diseases and chronic diseases to disaster risk consequences, but also in improving safety health of people and animals, and safeguarding environment against pollutants and pollution. it aims to enhance across disciplinary and interagencies assessment complex including human-animal health systems vis-a-vis environmental and climatic determinants of health, development of contextual health or disease detection and surveillance-response systems, data sharing and communication; partnerships and mutual learning for positive transformation and behavioral changes outcomes. hence, strengthening firmer foundation in building evidence-based integrated healthy approach decision making, health programming and actions plans implementation, training and research practice to community-based programs ownership, shared values and experiences in integrated cost effective and beneficial china-africa "one health" strategy initiatives for mutual wellbeing and economic prosperity. prioritizing "one health" approach in emerging and current infectious diseases public health emergencies and disaster risk reduction is essential in attaining the regional africa union and global health agenda promises and benefits. it requires promoting and implementing evidence-based, effective and sustainable national "one health" strategy advocacy and mitigation strategies in most africa countries and worldwide [ ] [ ] [ ] . strengthening evidence-based, consistent and reliable community, national and regional 'one health' and biosecurity people's medical publishing house co., ltd. partnerships, leadership, road maps commitment, approaches and strategies is a crucial for zoonotic diseases threats and outbreaks public health emergencies and other disasters risks humanitarian crises. integration "one health" principles and frameworks in health and relating multisectorial units or agencies planning and actions plans in generating comprehensive, consistent and real time knowledge and information in guiding evidence-based decision-making policy and participatory commitment and investment [ , , ] . articulated interest and reliance of all stakeholders will cover communities and public articulated actions in preparedness and response to climate changes, infectious and zoonotic threat and epidemics public health burden has provided [ , , ] . the extent and nature of "one health" approach through political engagement and funding is critical in advancing community social mobilization and awareness on "one health" strategy integration in public health systems and primary health care. the needs and value is prerequisite in sustainable public emergencies and disaster risk reduction priorities, preparedness, preventive and control programs and activities. while, providing the enabling health-animal and environment interface biosecurity and protection of legislative and technical assistance support to policy makers, planners and implementers including the local vulnerable communities in transforming contextual positive knowledge, behavioural and attitudes changes [ , ] . understanding the climate change, global migration and country-specific complexities of emerging and current infectious diseases of poverty is needed in tackle operational programs challenges and bottlenecks, improved sustained control into elimination. for example national immunization programs hesitancy and resistance issues, such as misconceptions and mistrust or fear, weak coverage and non-adherence, persistent resurgence of zoonotic threats and emerging epidemics, continue to place a huge toll of maternal and child health morbidity and mortality on burden and coupled with the rising trend of chronic diseases related inequities and poverty vicious cycle [ , , , , ] . building china-africa "one health" strategy partnerships, frameworks and capacity development china's global health approach is an unique and distinctive path. this approach based on contextual policies and realities-based on their history, driveninter sectoral and multidisciplinary government related ministries strengthen health systems in different african countries [ , , , , ] . there is a steadily growth in depth and strength of china's global engagement and collective participation in fostering global health agenda through china-africa health development strategies. event-based preparedness and transparent support management and technical assistance on transferable chinese lessons in infectious diseases elimination and eradication including measles, filariasis, schistosomiasis, malaria, sars and ebola, etc. for example, the china-tanzania pilot project of community-based and integrated malaria control strategy and applications funded by china-uk partnership aimed at assessing the feasibility and transferability of chinese malaria skills in strengthening malaria health education, awareness knowledge and access to vector control interventions (e.g., rdt, llin, acts) to reduce the risk of malaria infection in tanzania [ , ] . moreover, in the absence of specific ebola infection treatment, the partners or organizations, including african governments, who, the gavi alliance and "ebola ça suffit ring vaccination trial consortium" should accelerate on joint consensus for the adoption and "expanded access" to proven efficacious and safe rvsv-zebov vaccine ring ebola immunization strategy implementation to boost immune response and protect vulnerable populations and global travelers from potential ebola outbreaks [ , , ] . china-africa mutual and comprehensive partnership in health and pharmaceutical has been encouraging and promoting the use of community-based health services; and increasing government investment in public health interventions [ , , ] . china has been very supportive on african countries' efforts in building medical facilities and health service. for example, in , the chinese government constructed medical facilities and provided batches of medical equipments and supplies to african countries. chinese enterprises and nongovernmental organizations have helped african people get quality medical services by means of building and running hospitals, investing in pharmaceutical factories and localizing medicine production in improving health management and well-being, including maternal and child health, and emerging pandemic threat programs, etc. moreover, chinese medical assistance to africa has been sustained and operative win-win mutual support tailored to local settings, which could enhance research priorities in dynamic mapping of vectors and infectious diseases transmission with interaction of human-animal-environment, and provide evidence-based strategies in national or regional diseases control programmes and effective response packages [ , , [ ] [ ] [ ] . good progress remains in developing and implementing these policies and strategies coupled with shared lessons learnt and experiences against unprecedented infectious diseases public health emergencies and rising non-communicable diseases (ncds) challenges, such as obesity, cardiac arrest and stroke, hypertension, diabetes, cancer, kidney disorders and mental health, etc. there is a shortage of qualified health professionals at grassroots health facilities. it is also shortage in accessing to basic health control and elimination packages and service delivery including vaccine preventable diseases immunization programs coverage inadequacies in most rural and remote settings across africa compared to china, insufficient public and private sector funding to r&d on safety and effective vaccines or drugs against most emerging coupled with unattended public health diseases threats and epidemics impact preparedness, and strategies mutually gains and economic benefits [ ] . remarkable results and outcomes have been documented from chinese medical assistance in of african countries, ranging from health workers, implementers and policy-makers. capacity development and skills acquisition were achieved in over health-related training courses to , health implementers and health workers since . chinese medical teams friendship and health cooperation, including construction of ophthalmic center where more than , free cataract surgeries were completed in four african countries and construction of more than six other chinese medical hospitals in the last decades [ , , ] . it is also worth noting the robust and efficient participation and contribution of twenty-seven chinese provinces, autonomous regions and municipalities with accumulated more than , chinese medical workers in medical centers since , and currently over , medical workers are working in african countries. continuous support in building medical facilities, africa cdc reference laboratories per excellence and health service capacity has been appraised in embarking on assessing public health emergencies needs, risk factors and determinants in understanding the perception, knowledge, attitude and practice in evidence-based promotion of integrated "one health" approach and biosecurity decision-making approach. this also provide priority and targets, methodologies and programs through effective indicators surveillance and monitoring. for examples, chinese government dispatched over medical facilities and over batches of comprehensive medical equipment for early diagnostics and prompt treatment or response, and supplies across africa since [ , ] . chinese partnerships with local firms and communities have helped medical services delivery to remote and rural vulnerable populations through joint activities in building and running hospitals, investing in improving and scaling up localized production in pharmaceutical and biotechnologies industries in africa. in addition, we also recorded the establishment of more than clinics of standard traditional chinese medicine (tcm) integrated to africa traditional medicine (atm) [ , ] . in achieving universal coverage and healthcare for all, upgrading china-africa mutual health development cooperation and collaboration through independent and joint institutional research project and capacity development in health services delivery and in promoting science and technology capabilities, joint projects and activities have been increasingly developed and implemented. these projects and activities aim at tackling the persistent and growing burden of infectious diseases of poverty, maternal and child morbidity and mortality, and responding timely to the global health concerns and emergency response called on emerging threats and epidemics in the continent. some examples of the landmark achievements include the china-zanzibar and china-tanzania projects on sharing chinese lessons and experiences in infectious diseases to support schistosomiasis and malaria prevention and control in african countries respectively, as well as chinese maternal-child heath safety and children nutrition, dissemination and transfer experiences in ghana [ , , ] . furthermore, china-comoros support to national malaria elimination that led to interruption of transmission and reducing in malaria mortality to zero in the last eight years in comoros [ , , ] . likewise, understanding strategic public health financing and human resources systems capacity is necessary in promoting uptake and efficiency of chinese global health initiatives and innovations in strengthening healthcare delivery system and quality outcomes in lmics including africa, asia-pacific, middle east and latin american countries. strikingly again, during the west africa ebola outbreaks in - , the chinese assistance in response valued at $ million usd and more than , experienced medical professionals were deployed in the frontline affected and neighboring communities to combat and contain the rapid spread of the ebola virus epidemics [ ] . in addition to the mobile biosafety laboratory, china also built permanent and well-equipped public biosafety laboratory in seirra leone and dr congo to improve the national capacity to detect, prevent and respond to future threats and epidemics. over batches of public health, clinical medicine and laboratory experts were dispatched in african countries in scaling up public healthcare delivery capacity and training of health workers and communities in risk assessment, communication, and response measures in effective ebola, malaria, schistosomiasis prevention and containment, amongst other shared responsibility and mutual commitment [ , , , , [ ] [ ] [ ] . future expansion of china-africa health development initiatives offers immense opportunities in increasing mutual benefits and growth to both continents' several domains not only limited to health, technology and trade. the scale and sustainability of existing and forthcoming programs and plan of actions will require aligning of national priorities and defining contextual performance and effectiveness indicators, but also mutual respect and trust, accountability and transparency with good governance and proactive stewardship. it is imperative that efforts should also be made in strengthening evidence-based translation to the benefits of vulnerable populations and global community through sharing of lessons learnt and care knowledge experiences and information for all generations in combating infectious diseases and rising burden of noncommunicable diseases. fostering health systems preparedness and smart response against emerging and re-emerging threats and epidemics chinese and african rapid economic growth and the importance of strengthening the local and national public health laboratory systems in both continents have been recognized in tackling the rising healthcare needs, challenges and issues [ , , ] . globalization of travel and trade is ever increasing local, national and global emerging and re-emerging infectious diseases threats and their impacts on human and animal health. resolving the persistent and unprecedented rising of emerging and reemerging epidemics, and new priorities of ebola, zika, hiv/aids, tuberculosis, malaria and neglected tropical diseases (ntds) requires collaborative and mutual cooperation with governments, bilateral and multilateral initiatives, including boosting private-public partnerships, regional and international organizations in achieving the global health security threat and agenda [ , , , , ] . for example, china has dispatched more than , medical experts to resist west africa ebola epidemics through contributions to coordinated international emergency response efforts that helped to contain ebola virus transmission dynamics and spread that retrieved lives of over , people [ , , ] . similarly, zika virus belongs to the family flaviviridae, genus flavivirus, and includes africa subtype and asia subtype. it is a mosquitoborne virus primarily transmitted by aedes aegypti mosquitoes, sexual transmission; blood transmission and mother-to-fetus transmission have been also reported. zika virus can go through blood-brain barrier and infect central nervous system. symptoms are generally mild and self-limited, but recent evidence suggests a possible association between maternal zika virus infection and adverse fetal outcomes, such as congenital microcephaly, as well as a possible association with guillain-barré syndrome. in absence of safe vaccine or effective antiviral zika medication for prevention and control zika virus infection, early laboratorial diagnosis includes nucleic acid detection, serological test, and isolation of virus and epidemiology and clinical risk assessment and syndromic surveillance is crucial [ , , ] . nevertheless, there remains a need to build a platform with function of effective surveillance, recovery, preparedness, consultation and communication, and to share surveillance based on the principle of sincerity, real time problem-solving and results-giving, and good faith towards collaborative global health solutions [ , [ ] [ ] [ ] . fostering surveillance capacity in laboratory, clinical, veterinary and allied health sciences in the africa continent are critical to overcoming the growing burden of diseases and ensuring a healthy future of its citizens [ , ] . meeting the urgent growing healthcare needs in africa requires strategic and technical approaches in the development and integration of sound and harmonized regulatory systems for diagnostic products, new drugs and vaccine r&d. while reinforcing the national and international public health laboratories networks are able to improve collaborative and participative early disease detection, early warning and surveillance research in guiding proactive vigilance and smart response activities. effective good governance and leadership coordination of sustainable strategies on emerging outbreak preparedness and response capacity is necessary towards the transformation from traditional to modernized digital laboratory systems in timely and effective quality service delivery. however, the need for laboratories quality improvements and accreditation of methods, tools and programs are critical in upholding the gains preparedness, and emergency response in various infectious diseases programs and strategies should be supported through both national and international initiatives. bilateral and multilateral cooperation with the world bank, un and who, global fund to fight aids, tuberculosis and malaria, worthy philanthropic individuals and organizations efforts can enable country to be ready and capable of early detection, prognosis, prevention, and smart response or management in any detrimental natural or man-made epidemics eventuality, while facing operational challenges and setting new research priorities [ , , , ] , for contextual "one health and biosecurity" programs, are also need to be supported with appropriate regulations and guidelines. there is an urgent need to invest in basic and operational research on climatic, ecological and evolutionary changes for understanding and forecasting persistent and future emerging threats dynamics and epidemics. timely evidence-based translation into policy programs and interventions is imperative to defeat the budding threat and burden through coordinate robust actions and better stakeholders leadership in response advocacy and mitigation in line with the paris climate change declaration in december, [ , ] . fostering integrated approaches with cuttingedge inter-sectoral and trans-disciplinary partnership is also needed evidence-based nationwide scaling up contextual surveillance and response capacity. moreover, with improvement of targeted strategies to deal with emerging outbreaks and infectious diseases of poverty elimination, understanding human-animal interface with increasing urbanization, globalization of trade and travel are necessary. hence, china-africa "one health" strategy sustainable implementation and alignment with local and national priorities hold great promises. integrating collaborative human-animal-environmental projects and programs have substantial prospects in increasing local and national food production and global food security [ , ] . this is critical in averting or reducing the persistent malnutrition, under-nutrition and related health complications and diseases (e.g. malnutrition linked kwashiorkor or rickets, obesity, typhoid, diarrhea, dysentery) resulted in children and youth developmental retardation, poor educational performance, poor quality of life and living including high daly and low qaly short life expectancy, worsening the vicious cycle of poverty and premature death documented in africa countries [ , , , , ] . similar high public health challenges and burden in africa were recorded in china before s, mainly in chinese rural communities circumventing with the implementation of the chinese rural cooperative medical insurance schemes. however, more investment is still needed in achieving food autosufficient and balanced food and nutrition/diet for all in both continents [ ] . developing and integrating climate changes resilience , mitigation and adaptation measures in allied health programs is vital in protection, conservation and management of the adverse socio-demographic, ecological, health and economic effects of greenhouse gas emissions and changes consequences, and in securing the future benefits of green and eco-friendly environment. the value of china-africa "one health" strategy implementation financial support from governments and various levels, advocacy and social mobilization to develop supportive community environment for infectious and emerging zoonoses threats and epidemics in population-based public health control and elimination interventions is imperative through implementation of evidence based and cost-effective "one health" surveillance and response strategy, in order to integrate human, animal, and environmental landscape, continue health education promotion, improve awareness and quality public health service delivery performance and effectiveness metrics across africa. enhanced disease surveillance response, community capacity development and strain capacity can provide significant opportunities in health education and promotion, shared responsibility, positive behavior changes and best practices by different health facilities, training health practitioners of diagnosis, treatment and rehabilitation services. identification of local and national health needs and evidence-based effectiveness of "one health" solutions are urgently needed to improve appropriate and sustainable resource development policies and strategic programs across africa. such new partnership initiatives linked to china belt and silk initiative action plan should attract more indigenous and international partners and stakeholders, more qualified multidisciplinary professionals to work, communicate and share experiences and lessons collectively. building local and national trans-disciplinary and trans-sectoral research teams towards improved understanding the genetic and molecular mechanisms of invasive pest and drug resistance, and control of complex disease systems and in strengthening continuous improvements of human, animal, ecosystem health and well-being [ ] [ ] [ ] [ ] . robust evidence in comprehensive control for multiple risk factors including health guidance on diet, fitness activity and promoting individual and community self-management model is important to services by general practitioners and mainly in translational research directed toward sustainable development activities and global environmental health. to support integrated veterinary, medical and ecosystem education, and to provide more professional career development opportunities, the governments need to continuously increase its investment in public health intervention programs and financial support to health insurance schemes. increased funding from both central and local governments needs to be directed to the underdeveloped regions and poorer rural areas to support global and national programes on infectious diseases of poverty and sustain control and elimination agenda for emerging epidemics tackle maternal and child health challenges, improve ncds mitigation interventions, and set up better health insurance schemes. in addition, it is equally important to strengthen monitoring of the use for public health interventions. "one health and biosafety" systems research projects development and implementation are also urgently needed in improving training programs and educational empowerment in guiding human-animal health and environment programming and technical assistance. addressing the existing and unprecedented public health emergencies or disaster risks requires optimizing the "one health and biosecurity" targets and interventions which will benefit indicators metrics monitoring in routine public health programs and humanitarian emergencies crises response [ , ] . there is need to promoting "one health and biosecurity" youths voices in healthy and ecofriendly "one health" community advocacy, engagement and participation. strengthening and sustaining "one health and biosecurity" strategy will improve the cost-effectiveness surveillance and communication interventions through continuous awareness, and knowledge improvements for the overall china-africa and global health security benefits [ , , , ] . robust and sustainable leadership commitment and investment is needed in integration of "one health" and global health security. advocacy and mitigation programs is needed in china-africa health development initiatives. to establish public emergencies indicators and metrics for early and timely community engagement and effective risk communication, following actions need to be handled to achieve sdgs and global health agenda: ( ) community-based partnerships and programs ownership, ( ) assessment for evidence based "one health", ( ) identification of the various stressors or risk factors, ( ) programmatic and proactive development and implementation of appropriate and sustainable "one health", ( ) resource mobilization mechanisms and solutions based on animal-humanenvironment interface challenges and impacts surveillance, preparedness, and timely collective response to public health threats and humanitarian emergency crises. china-africa health development initiatives: benefits and implications for shaping innovative and evidenceinformed national health policies and programs in subsaharan african countries enhancing collaboration between china and african countries for schistosomiasis control china-africa cooperation initiatives in malaria control and elimination establishing the africa centres for disease control and prevention: responding to africa's health threats tackling the challenges to health equity in china china's distinctive engagement in global health implementing a one health approach to emerging infectious disease: reflections on the sociopolitical, ethical and legal dimensions one health: an opportunity for an interprofessional approach to healthcare new orientation for china's health assistance to africa elimination of tropical disease through surveillance and response global implications of china's healthcare reform rift valley fever epidemic in niger near border with mali. the lancet inf diseases deciphering emerging zika and dengue viral epidemics: implications for global maternal-child health burden joint china-us call for employing a transdisciplinary approach to emerging infectious diseases china harvard africa network (chan) team. china, africa, and us academia join hands to advance global health china-africa cooperation initiatives in malaria control and elimination global health security: the wider lessons from the west african ebola virus disease epidemic one health: the hong kong experience with avian influenza genetic diversity and evolutionary dynamics of ebola virus in sierra leone building international genomics collaboration for global health security china's role as a global health donor in africa: what can we learn from studying under reported resource flows? the ebola threat: china's response to the west african epidemic and national development of prevention and control policies and infrastructure china's distinctive engagement in global health china takes an active role in combating an ebola outbreak: on-site observations and reflections from a chinese healthcare provider schistosomiasis control: experiences and lessons from china a strategy to control transmission of schistosoma japonicum in china schistosomiasis and water resources development: systematic review, meta-analysis, and estimates of people at risk surveillance-response systems: the key to elimination of tropical diseases rebuilding transformation strategies in post-ebola epidemics in africa evidence on public health interventions in humanitarian crises thanks to nipd, china cdc, shanghai, and universite des montagnes for the enabling environment to complete this review. data are freely available and accessible. the authors have no conflict of interests. not applicable. key: cord- -tac unnp authors: andré, nicole m; cossic, brieuc; davies, emma; miller, andrew d; whittaker, gary r title: distinct mutation in the feline coronavirus spike protein cleavage activation site in a cat with feline infectious peritonitis-associated meningoencephalomyelitis date: - - journal: jfms open rep doi: . / sha: doc_id: cord_uid: tac unnp case summary: this report describes a cat with chronic, progressive, non-painful, non-lateralizing multifocal neurologic clinical signs associated with feline infectious peritonitis (fip). the cat initially presented as underweight, despite a good appetite, and a complete blood count showed non-regenerative anemia. three months later the cat was returned having developed ataxia and paraparesis, which then progressed over months to tetraparesis, tail plegia, urinary and fecal incontinence, and titubation. histologic examination of the tissues with subsequent immunohistochemistry confirmed fip-associated meningoencephalomyelitis following necropsy. molecular analysis of the coronavirus spike protein within the tissues identified a specific, functionally relevant amino acid change (r m), which was only identified in tissues associated with the central nervous system (ie, brain and spinal cord). relevance and novel information: this case report describes an early presentation of a cat with primarily neurologic fip, with molecular characterization of the virus within various tissues. feline infectious peritonitis (fip) is caused by feline coronavirus (fcov) and is widely considered to be one of the most significant infectious diseases to affect the feline population. [ ] [ ] [ ] it is the most common infectious disease of the central nervous system (cns) of cats. fcovs have been reported to exist as two distinct serotypes: type i (more common) and type ii viruses, each with distinct biological properties. , both fcov serotypes have distinct 'biotypes'. these are typically classified as either feline enteric coronavirus (fecv) or feline infectious peritonitis virus (fipv), with the biotypes differing based on the severity of infection in cats. [ ] [ ] [ ] infection with fcov is common, especially in highdensity housing situations such as animal shelters and breeding facilities. the fecv biotype transmits readily and causes only a mild infection, with transmission occurring via fecal-oral and possibly other routes. , if the viral infection worsens and becomes systemic (typically infecting macrophages), then the virus is classified as the fipv biotype. such viruses are believed to contain an 'internal mutation' that accounts for the altered tropism, although the nature of this mutation is not well understood. clinical signs associated with the fipv biotype can be quite variable and non-specific, and can include fever, lethargy, anorexia, pica, vomiting and diarrhea. these clinical signs can be present in either the 'wet', 'dry' or 'mixed' presentations. the wet form of fip is characterized by an effusion in the abdominal and/or thoracic or pericardial cavities, and the 'dry' form by the presence of pyogranulomatous lesions. the 'mixed' form may present with an array of clinical signs. most commonly, neurologic clinical signs are associated with the 'dry' form but can occur with all presentations and may be the sole clinical sign observed. , clinical features of neurologic fip can include, but are not limited to, ataxia, head tremors, seizures and/or paresis. , , ocular lesions may be present with or without lesions in the cns. fip-associated pathologic changes to the cns include meningitis, encephalitis, ependymitis and choroid plexitis, often with concurrent vasculitis. , fip presenting with predominantly neurologic clinical signs provides a diagnostic challenge and definitive ante-mortem diagnosis is difficult. mri has been identified as a sensitive method of diagnosis in conjunction with clinical signs and cerebrospinal fluid analysis results such as elevated protein levels and neutrophilic pleocytosis. however, such findings are still not specific to fip and may be financially prohibitive. fip may also be considered a diagnosis of exclusion, following evaluation of clinical signs, history and physical examination findings and biochemical values. , this case report describes a cat with neurologic fip that progressed over several months. the observations and findings obtained in this case provide support that fip can present predominantly in the cns. when molecular techniques are applied to the virus, a propensity for certain mutations can be associated with specific clinical presentations or pathological changes. an intact female -week-old domestic shorthair cat was taken into a foster/rescue home and cohabited a house with approximately nine other cats. the facility had a periodic history of fip cases, including two deaths in the previous months. the cat was co-housed in a large open sunroom containing seven litterboxes, which were cleaned once daily. the diet consisted of commercially available dry and canned food, which was separately offered in individual dishes. the cat was not rabies vaccinated, but had obtained two feline viral rhinotracheitis, calicivirus and panleukopenia vaccinations. at weeks of age, the cat was presented to a general practitioner for evaluation of poor weight gain, soft stool and upper respiratory tract infection. the cat was underconditioned and weighed . lb ( . kg), with a body condition score (bcs) of / , despite being active, alert and having a good appetite. conjunctivitis and a yellow mucopurulent discharge from the nares were noted, and the cat had a fever of . °f ( . °c). a fecal flotation was performed owing to the soft but formed stool, and no ova or parasites were detected. a complete blood count (cbc) and chemistry profile were performed (tables and ). the chemistry profile showed marked elevations in alkaline phosphatase, alanine transferase and phosphorus levels. a decrease in creatinine and albumin was also noted (table ) , along with mild anemia and monocytosis. amoxicillin clavulanic acid (clavamox drops; zoetis) . mg/ml was dispensed and administered at . mg ( mg/kg) po q h for days. blood parameters were re-evaluated at weeks of age using a less defined panel and values were within the normal range (table ) . at approximately months of age, the cat returned to the general practitioner for evaluation of pelvic limb gait abnormalities that had progressed over the previous weeks. examination revealed symmetric pulses in both hindlimbs and the presence of a pain response; however, less of a response was noted on the right side. paresis was observed in the right hindlimb. when the forelimbs were lifted, the cat was able to walk minimally on the hindlimbs. the cat had severe non-ambulatory paraparesis, with more severe deficits on the right side. no information about spinal reflexes was available. a cbc and chemistry panel were performed (tables and ). the chemistry panel revealed hypoalbuminemia, a decrease in the albumin:globulin (a:g) ratio, low creatinine values and hyperphosphatemia. the cbc revealed a slight anemia, monocytosis and thrombocytopenia. platelet clumping was noted upon microscopic evaluation. meloxicam (metacam oral suspension) was dispensed and a single . mg dose was administered orally. at months of age, the cat was returned to the general practitioner due to progression of the paraparesis. the client noted further deterioration of the pelvic limb paresis, and now identified 'stiffness' in the thoracic limbs. there was no information about the pelvic limb reflexes; however, the cat had started to have occasional urinary and fecal incontinence. appetite seemed normal; however, the cat remained thin. physical examination revealed a temperature of . °f ( . °c), heart rate of beats per min and respiratory rate of breaths per min. bcs was / ; however, the weight was not noted. abdominal palpation revealed a large, easily expressible urinary bladder. neurologic examination findings revealed normal mentation with no involuntary movements such as tremors. the cat was still very ataxic and ambulatory but now tetraparetic, which was much worse in the pelvic limbs. there was no information about cranial nerve abnormalities, and the eyes and retinas were within normal limits. from a video provided by the owner (see supplementary material), tail paresis was identified. the lesion was considered to affect the cns and was localized as multifocal. a fecal flotation and direct smear were evaluated, with no ova or parasites seen. cryptococcus and toxoplasma antibody titers were performed and were negative. the tetra-ataxia and paresis significantly worsened over the next few months. additionally, the cat now had titubation, tail plegia (see video in the supplementary material) and consistent urinary and fecal incontinence. owing to the grave prognosis, the client elected humane euthanasia, at which time the cat was months ( weeks) of age. a necropsy was performed at the animal health diagnostic center, cornell university college of veterinary medicine, and this revealed no significant gross abnormalities outside of mild mesenteric lymphadenomegaly. representative sections of all organs, including the entire brain and spinal cord, were fixed in % neutral buffered formalin from which sections were cut, stained with hematoxylin and eosin, and analyzed via light microscopy. immunohistochemistry for fcov was carried out using monoclonal antibody fipv - ( : ), ap-anti-mouse igg and bond polymer refine red detection (leica microsystems). histologic examination revealed lesions typical of fcov infection within the cns. in the spinal cord, the leptomeninges were diffusely expanded by moderate numbers of predominantly plasma cells, admixed with fewer lymphocytes and macrophages, and surrounded by a moderate amount of edema. the underlying white matter was multifocally vacuolated with numerous dilated myelin sheaths, digestion chambers and rare spheroids (figure a) . at the level of the lateral aperture, the choroid plexus was expanded by large numbers of plasma cells, lymphocytes and macrophages (figure b) . the ependyma lining the ventricular system was effaced by a similar inflammatory population, admixed with fibrin, edema and was also forming thick perivascular cuffs often disrupting the sub-ependymal parenchyma (figure c) . immunohistochemistry revealed strong intracytoplasmic immunoreactivity within macrophages (figure d ). no fipassociated lesions were present in other organs. non-fcov comorbid histologic findings were chronic enteritis with mid-mucosal fibrosis and mesenteric lymphoid hyperplasia. molecular analysis of the viral spike protein was performed at several time points during the study. fecal samples were collected at months of age (feces # ) and at months of age (feces # ). following euthanasia (at months of age) tissue samples were collected, along with a fecal sample (feces # ). a central base pair region of the spike protein gene, including the critical s /s activation site of the virus, was pcr amplified and sequenced as described in licitra et al, with the following modifications: μl reverse transcription pcrs were performed with qscript xlt -step rt pcr kit (quantbio). pcr conditions were mins at °c, mins at °c and cycles of s at °c, s at °c, s at °c, then mins at °c. pcr products were purified using diffinity rapidtips (diffinity genomics). pcr and sequencing showed the presence of a type i fcov, based on a sequence alignment with reference genomes. the sequence information obtained from this cat is shown in figure . the viral sequences from the cns (brain and spinal cord) contained specific amino acid changes compared with other samples (feces, small intestine, mesenteric lymph node and kidney). the most notable change was an arginine to methionine (r-m) substitution at the critical p activation position, corresponding to residue . other changes that correlated with viruses present in the cns were present in two other positions: alanine to valine (a-v); and threonine to alanine (t-a). here we report clinicopathologic findings and molecular analysis of a cat with progressive neurologic clinical signs associated with fip. the cat initially presented to the referring veterinarian with respiratory signs and fever, and with abnormal liver enzyme function and anemia. at this time fip was not suspected. these initial signs resolved but were replaced by progressing neurologic signs, which led ultimately to euthanasia and submission to the study for evaluation of fcov involvement. upon euthanasia, fcov was found in various tissues in the cat, including the cns. however, histologic examination revealed fcov-associated pathology only within the cns, where there was meningoencephalomyelitis, ependymitis, choroid plexitis and vasculitis. histo logical lesions were compatible with a recent report describing meningoencephalitis in four cats with fip. molecular analysis of the viral spike protein within the tissues identified a specific, functionally relevant amino acid change (r m), which was only identified in tissues associated with the cns (ie, brain and spinal cord). the r m mutation in the spike protein s /s cleavage-activation site is a major chemical change from a basic to a hydrophobic residue, and is consistent with an elimination of furin-mediated proteolytic processing of the s protein, as seen by licitra et al, and a proposed change in the activation properties and entry pathway of the virus. it is interesting to note that the r m mutation was not present in other tissues tested in this cat at the time of necropsy but was found in our previous study (cat id # - ), where samples were of neural origin. while biological confirmation is not available, we consider that the other changes found in the viral spike protein from central nervous tissue of this cat (a v and t a) are not related to changes in the activation properties and entry pathway of the virus, as they are not in defined functional regions of the spike protein and are not markedly different in their chemistry. interestingly, all samples tested from this cat contained a distinct leucine residue at position (the p position of the furin cleavage site, typically serine or alanine as defined in licitra et al). the relevance of this is currently unclear. overall, our results provide evidence that mutation of the viral spike protein is linked to fip outcome, specifically in the s /s cleavage-activation site (residues - ). mutations leading to fip have also been linked to changes in other areas of the spike gene (position ) and in the c gene. , to compare our findings for the s /s region to other proposed fip-linked mutations, we performed additional sequencing, which is summarized in table . all fecal samples, as well as a kidney sample, contained methionine (m) at spike position , indicating that an 'enteric' form of fcov was present in the cat throughout our study. in contrast, samples from the brain and spinal cord contained leucine (l) at position , indicating an fip virus. an intact c gene was found in feces, with the c gene in neural and other tissues truncated and/or deleted depending on the sample tested. this case report describes a young cat with neurologic fip in which detailed clinical and molecular characterization of the associated fcov infection was performed. while the etiology of fip remains complex and likely involves multiple mutations in the viral genome, our results indicate that a specific mutation of the viral spike protein can be associated with infection of the cns, which may explain the tropism to the cns as opposed to other organ systems. an update on feline infectious peritonitis: diagnostics and therapeutics an update on feline infectious peritonitis: virology and immunopathogenesis cns disease in the cat: current knowledge of infectious causes prevalence of diseases of the spinal cord of cats fenner's veterinary virology improving virus taxonomy by recontextualizing sequence-based classification with biologically relevant data: the case of the alphacoronavirus species infectious diseases of the dog and cat a review of coronavirus infection in the central nervous system of cats and mice immunocytochemical demonstration of feline infectious peritonitis virus within cerebrospinal fluid macrophages practical overview of common infectious disease agents hagan and brunner's microbiology and infectious diseases of domestic animals diagnosis and clinical signs of feline infectious peritonitis in the central nervous system a retrospective study of the neuropathology and diagnosis of naturally occurring feline infectious peritonitis feline infectious peritonitis with neurologic involvement: clinical and pathological findings in cats feline infectious peritonitis with spinal cord involvement in two cats clinicopathologic features and magnetic resonance imaging findings in cats with histopathologically confirmed neurologic feline infectious peritonitis mutation in spike protein cleavage site and pathogenesis of feline coronavirus immunohistochemical studies on meningoencephalitis in feline infectious peritonitis (fip) spike protein fusion peptide and feline coronavirus virulence significance of coronavirus mutants in feces and diseased tissues of cats suffering from feline infectious peritonitis feline infectious peritonitis: insights into feline coronavirus pathobiogenesis and epidemiology based on genetic analysis of the viral c gene amino acid changes in the spike protein of feline coronavirus correlate with systemic spread of virus from the intestine and not with feline infectious peritonitis acknowledgements we thank wendy wingate for help with sample collection, all members of the whittaker lab for helpful comments and support, and dr john loftus for clinical consultation and critical reading of the manuscript. video of cat at months and months of age. the authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. center.ethical approval this study involved the use of clientowned animal(s) only, and followed internationally recognized high standards ('best practice') of individual veterinary clinical patient care. ethical approval from a committee was not therefore needed.informed consent informed consent (either verbal or written) was obtained from the owner or legal guardian of all animal(s) described in this study for the procedure(s) undertaken. for any animals or humans individually identifiable within this publication, informed consent for their use in the publication (verbal or written) was obtained from the people involved.orcid id nicole m andré https://orcid.org/ - - - key: cord- - s authors: norris, susan l.; sawin, veronica ivey; ferri, mauricio; raques sastre, laura; porgo, teegwendé v. title: an evaluation of emergency guidelines issued by the world health organization in response to four infectious disease outbreaks date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: s background: the production of high-quality guidelines in response to public health emergencies poses challenges for the world health organization (who). the urgent need for guidance and the paucity of structured scientific data on emerging diseases hinder the formulation of evidence-informed recommendations using standard methods and procedures. objectives: in the context of the response to recent public health emergencies, this project aimed to describe the information products produced by who and assess the quality and trustworthiness of a subset of these products classified as guidelines. methods: we selected four recent infectious disease emergencies: outbreaks of avian influenza a—h n virus ( ) and h n virus ( ), middle east respiratory syndrome coronavirus (mers-cov) ( ), and ebola virus disease (evd) ( to ). we analyzed the development and publication processes and evaluated the quality of emergency guidelines using agree-ii. results: we included information products of which were guidelines. these products demonstrated variable adherence to who publication requirements including the listing of external contributors, management of declarations of interest, and entry into who’s public database of publications. for guidelines, the methods for development were incompletely reported; who’s quality assurance process was rarely used; systematic or other evidence reviews were infrequently referenced; external peer review was not performed; and they scored poorly with agree ii, particularly for rigour of development and editorial independence. conclusions: our study suggests that who guidelines produced in the context of a public health emergency can be improved upon, helping to assure the trustworthiness and utility of who information products in future emergencies. in the context of the response to recent public health emergencies, this project aimed to describe the information products produced by who and assess the quality and trustworthiness of a subset of these products classified as guidelines. we selected four recent infectious disease emergencies: outbreaks of avian influenza a-h n virus ( ) and h n virus ( ), middle east respiratory syndrome coronavirus (mers-cov) ( ), and ebola virus disease (evd) ( to ). we analyzed the development and publication processes and evaluated the quality of emergency guidelines using agree-ii. we included information products of which were guidelines. these products demonstrated variable adherence to who publication requirements including the listing of external contributors, management of declarations of interest, and entry into who's public database of publications. for guidelines, the methods for development were incompletely reported; who's quality assurance process was rarely used; systematic or other evidence reviews were infrequently referenced; external peer review was not performed; and they scored poorly with agree ii, particularly for rigour of development and editorial independence. the world health organization (who) shapes public health policy for united nations member states through the development of technical guidance. more than a decade ago, in response to harsh external criticism of its guidelines, who instituted the guidelines review committee (grc) with the mandate to develop rigorous methods and procedures ensuring that each who guideline is evidence-based and meets the highest international standards so that guidelines are credible, trustworthy and relevant to end-users [ ] . who quality standards dictate that guidelines must address a critical public health problem, use transparent and explicit processes minimizing potential sources of bias such as conflicts of interest, include diverse perspectives in the guideline development group, reflect the current state of the evidence, and provide a clear link between the evidence and recommendations taking into consideration the balance of benefits and harms of interventions and other important considerations [ ] . who's scope of work includes producing guidelines and other information products in the context of public health emergency response such as natural and technological disasters, disease outbreaks, armed conflicts and other humanitarian crises. when relevant guidelines do not exist, either because the situation is new or because current guidelines are not applicable, it is extremely challenging to produce new guidelines. significant uncertainty in the field leads to urgent need, imposing short development timelines; scientific data are scarce and the collection of new data is hindered; health systems may be poorly functioning or nonexistent; and resources (both money and expertise) for guideline development may be scant. despite this challenging context, adherence to the principles and standards for high-quality, trustworthy and usable guidelines is essential such that end-users adopting and implementing recommendations in the field can optimally impact population health outcomes. the best way to apply traditional quality standards for guidelines to the context of emergencies remains unclear. who does not currently have an established approach with defined processes and tools to develop emergency guidelines. thus, guideline development groups at who and in other organizations must improvise and adapt standard methods on an ad hoc basis, potentially compromising the quality of the final product. to date there are no comprehensive evaluations of guidelines produced in the context of a public health emergency. the aim of this study is to describe the information products produced by who in recent emergencies and to assess the quality and trustworthiness of a subset of these products classified as guidelines. the overarching goal of this evaluation was to inform future processes and methods for producing these types of guidelines at who and by other organizations globally. we examined information products issued by who for four recent public health emergencies: outbreaks of avian influenza a-h n virus ( ), avian influenza a-h n virus ( ), middle east respiratory syndrome coronavirus (mers-cov) ( ) and ebola virus disease (evd) ( to ). there were other outbreaks, humanitarian emergencies, and natural disasters between and that were graded by the global emergency management team [ ] , however we selected these four emergencies as purposeful sample as they all focused on infectious diseases outbreaks, and involved a significant response by who including the production of a large number of information products. all occurred after the institution of the grc so that a standard approach to develop guidelines in the non-emergency context was in place at who. we included all information products published in any language that were specifically developed or adapted by who for the four selected emergencies. we excluded testimonials, media releases, situation reports and meeting reports. two independent reviewers assessed each document against study inclusion criteria. as per who's definition [ ] , we classified an information product as a guideline based on the content of that document (i.e., whether it contained a specific recommendation for the end-user), rather than by the process for development. guidelines included products that contained recommendations and did not reference an underlying, source guideline. these covered a wide range of topics including clinical care [ ] , water and sanitation [ ] , and occupational health and safety [ ] , among many other topics. all other information products were classified as non-guidelines, which included, for example, publications derived from existing guidelines [ ] , assessments of preparedness in countries [ ] , and tools for assessment or response [ ] . non-guidelines are not expected to contain detailed methods and other types of information that is expected in a high-quality guideline. we identified eligible information products available on april , from lists of technical documents related to each emergency assembled by the who department of communicable disease surveillance and response. we had difficulty identifying relevant documents and therefore searched multiple potential sources such as who's global digital library (institutional repository for information sharing (iris)), hand-searched who websites, consulted key who staff in technical units and reviewed who resource toolkits. two independent reviewers extracted information product characteristics and publication format, and identified translations. for guidelines, we extracted data on standard who guideline development processes (e.g., based on a systematic review of the evidence, involved external experts in the formulation of recommendations) and had two independent reviewers appraise each guideline using the appraisal of guidelines for research and evaluation (agree) ii instrument [ ] . this instrument, the most widely-used guideline quality appraisal tool, is comprised of items, six quality domains and a -point likert-like response scale. we report the prevalence of key characteristics for information products in each emergency. for continuous data, we report the mean and standard deviation (sd). we summarize agree ii scores for each domain (scaled to a percentage of the maximum score) across guidelines using the median value and the interquartile range, as scores were not normally distributed and sample sizes were small for some emergencies. all statistical analyses were conducted using stata . (statacorp lp, college station, texas, usa). we identified information products of which were classified as guidelines. table summarizes the characteristics of all information products. table outlines the methods and processes used in the development of guidelines specifically. for the h n emergency response, information products met our inclusion criteria: guidelines and other information products [ ] . systematic reviews were cited in only two guidelines ( %) and four other guidelines ( %) referred to a literature search. external experts were involved in % of guidelines, but only four named these contributors of which three reported that declaration of interests (doi) had been obtained. peer review rarely occurred. dates of expiration or planned update for the guidelines were provided in eight title contains: "guideline" or "guidance" "interim" documents ( %). h n -related guidelines received the highest scores in the agree ii domains of clarity of presentation (median %; interquartile range (iqr) %- %) and scope and purpose (median %; iqr %- %) (fig ) . the lowest scores were assessed in editorial independence (median %; iqr %- %), rigour of development (median %; iqr %- %), and applicability (median %; iqr %- %). for h n , we identified emergency information products, five emergency guidelines and nine other information products. all but one document was available only in english. one guideline referenced a systematic review; another cited evidence, but did not indicate whether the evidence review was systematic. of the three guidelines that reported involving external experts, only one listed those individuals and provided their doi. none of the guidelines provided a date of expiration or planned update, though one guideline indicated that recommendations would be updated as new information emerged. based on the agree ii instrument, h n emergency guidelines scored well in clarity of presentation (median %; iqr %- %) and scope and purpose (median %; iqr %- %). the lowest-scoring agree ii domains were rigor of development (median %; iqr %- %), applicability (median %; iqr %- %) and editorial independence (median %; iqr %- %) (fig ) . twenty-three information products, including emergency guidelines and seven other information products, met our inclusion criteria for mers-cov documents. eleven guidelines ( %) were available in at least two languages (english and arabic); we identified only an english version of the remaining information products. none of the mers-cov emergency guidelines referenced a systematic review. although five guidelines ( %) reported that external experts contributed to the recommendations, only four of these listed the names of individual experts and only one reported that doi had been collected. three guidelines ( %) provided a specific date for the expiration or update of the guideline. the agree ii domains that scored the highest across these mers-cov-related emergency guidelines were clarity of presentation (median %; iqr %- %) and scope and purpose (median %; iqr %- %). the lowest scoring domains were editorial independence (median %; iqr %- %) and rigour of development (median %; iqr %- %) (fig ) . we included evd information products, including emergency guidelines and other information products. overall, ( %) information products were available in english and french, including ( %) guidelines and ( %) non-guidelines; the remainder were available only in english. only two evd guidelines reported or referenced a systematic review, although a third guideline reported that a rapid review of the evidence had been performed. an additional five guidelines ( %) referenced evidence or indicated that a literature search had been performed, but it was unclear if the review methods were systematic. four guidelines ( %) reported that recommendations were based entirely on expert opinion. most guidelines ( %) cited other publications, including other who information products. four guidelines ( %) reported that doi had been collected from external experts. four guidelines provided a date for the expiration or update of the guideline; eight additional guidelines indicated that regular updates would be provided as new information became available. evd guidelines scored the highest in the agree ii domains of clarity of presentation (median %; iqr %- %) and scope and purpose (median %; iqr %- %). the lowest scores were assessed in editorial independence (median %; iqr %- %) and rigour of development (median %; iqr %- %) (fig ) . approximately half ( %) were published in (fig ) . guidelines were published an average of days (sd ) following the initial emergency grading of evd in sierra leone on july ; two documents ( %) were published prior to the assignment of the emergency grade. information products issued by who during four recent infectious disease epidemics were difficult to find, were often published at long intervals after outbreaks were graded, and demonstrated variable adherence to who publication and reporting requirements. few guidelines met international standards for the development and presentation of trustworthy and usable guidelines, as assessed with tools designed for standard (non-emergency) guidelines. the principles for trustworthy guidelines were developed in the context of standard (nonemergency) clinical guidelines where there is a much longer time-period for development, end-users' needs may be much clearer, there may be much more (high-quality) evidence, and the implementation setting and health systems may be much less fragile and chaotic [ ] . universally agreed-upon standards and methods for the optimal development of public health guidelines in the context of emergencies have not yet been established and thus our assessments using these standards must be interpreted with caution. nonetheless, the principles of transparency, minimization of risk of bias in the formulation of recommendations, and the presentation of implementable, impactful recommendations inarguably apply to all types of guidelines and recommendations. the difficulty we faced to identify and retrieve relevant documents suggests that end-users may have similar or greater difficulties during the emergency response. in addition, incomplete reporting of key information such as the responsible technical unit and publication date may prevent end-users from determining the relevance of a product to their specific context as the epidemic trajectory changes or new information emerges. there was no consistent format of the information products that we examined, including within specific emergencies. information products should vary in their content and appearance to meet the needs of the intended end-users, however who products should be readily identifiable with consistent appearance and branding, including the who logo. the labelling of information products varied across products of the same or similar types, even within emergencies. in particular, guidelines were referred to by a vast array of terms that did not appear to have a specific meaning. for example, "interim guidance" was relatively frequently used in evd-related documents, appropriately suggesting a limited shelf-life for these documents, however specific plans for updating were rarely presented. we noted incomplete implementation of who's mandatory policy on conflicts of interest of external contributors and the reporting of funding sources for guidelines. this is an important finding as the interests of both author and funder are correlated with the conclusions of systematic reviews and the recommendations in clinical practice guidelines [ ] . although doi does not in itself minimize the risk of bias associated with those interests, disclosure is widely regarded as the first and essential step in addressing competing interests and their potential effects on the conclusions of an evidence review or the recommendations in a guideline. despite the who grc's responsibilities, only three guidelines were approved by the quality assurance agency. the specific reasons were unclear, although the pressure to produce guidelines quickly could lead to ad hoc and variable processes and procedures. nonetheless, quality assurance and control processes must still be used for all who information products. although incomplete reporting likely impeded our assessment, the rigour of development of the guidelines examined in our study appears to be low, as assessed by agree ii and infrequent reference to systematic reviews. compressed timelines in response to urgent needs necessitate abbreviated methods, however an explicit description of the approaches used to develop a guideline is always possible. the information and considerations used to inform each recommendation must be clearly described and when direct evidence is scarce, it is even more important to be transparent about the basis for recommendations. the applicability of indirect evidence must be carefully assessed and the rationale for its inclusion provided (for example, the use of data from related viruses or other infectious agents with similar vectors). draft guidelines rarely underwent peer review by individuals and organizations distinct from the primary contributors to the guideline. this is an important gap in the development process, as peer review is a standard step in guideline development [ , ] , and has important benefits including stakeholder engagement, identification of errors and attention to key implementation issues. peer review can be executed within days and comments can be quickly evaluated with careful planning and ready access to a cohort of experts and stakeholders. a clear indication of plans for updating recommendations is a critical issue in the emergency context, as the evidence base and other considerations such as those related to implementation are continually evolving. all information products developed in response to an emergency, particularly those labelled "interim", should have a "review by" date and sufficient resources must be available to evaluate and update information products as required. high scores in the agree ii domains of clarity of presentation and scope and purpose may be attributable to the operational focus of most emergency guidelines, which typically requires a concise, clear format. other agree ii domain scores suggest significant room for improvement. for example, guidelines scored inconsistently in the domain of applicability, which includes criteria such as descriptions of barriers and facilitators to implementation of the recommendations, and provision of or reference to other tools to support implementation. although this evaluation examines a number of important measures, there are many additional questions that were not addressed. we focused on emergency information products for infectious disease outbreaks and it is important to examine information products developed in response to other types of emergencies such as natural disasters and humanitarian crises caused by displacement of populations secondary to conflict or climate change. we did not evaluate dissemination, utilization, perceived usefulness by end-users, implementation and uptake of recommendations, or most importantly, the impact on health outcomes and wellbeing. such impact evaluations are needed. nor did we try to identify gaps in the guidance covered by the available information products. despite our efforts to compile comprehensive lists of information products developed by who in response to h n , h n , mers-cov and evd, we may not have captured all relevant documents. we examined only the latest version available: some information products may have been updates which may have different characteristics than previous versions that were developed in shorter timelines or earlier in the emergency response. for practical reasons, we evaluated only information reported in the documents. we may have misclassified derivative products as guidelines if they failed to reference the underlying guideline. although widely used to assess the quality of reporting and conduct of guidelines, the agree ii instrument has limitations [ ] , and it was developed for clinical guidelines and not for public health or emergency guidelines. thus, the domain scores that we report should be interpreted with caution. the agree ii tool is also limited in its scope. for example, the domain of applicability examines the inclusion of tools for implementation and discussion of barriers, cost implications, and monitoring of the guideline's recommendations, but it does not address actual implementation or impact on health. in addition, we had only two assessors for the agree ii appraisals; although acceptable according to agree ii guidance, four reviewers would have enhanced the reliability of these assessments [ ] . this study did not intend to compare the quality of guidelines for the four responses with each other or with standard who guidelines developed in stable settings. it is challenging to compare guidelines across the four epidemics as each event differed with respect to incidence, mortality rate, rate of change in the burden of disease, geographic spread, prior experience with the disease, and the availability of resources. who guidelines produced in the context of a public health emergency can be improved upon, helping to assure the trustworthiness and utility of who information products in future emergencies. the principles of transparency, minimization of the risk of bias, and reliance on research and other evidence over expert opinion, apply to guidelines developed in any setting or context. with careful self-evaluation, thoughtful reflection on lessons learned in these emergencies, and a firm commitment to continuous improvement, who will continue to provide timely and high-quality guidance in the context of public health emergencies. supporting information s clinical practice guidelines we can trust world health organizaton. who handbook for guideline development- nd edition. geneva: world health organization emergency response framework (erf) world health organization. facilitation manual: psychological first aid during ebola virus disease outbreaks ebola virus disease: key questions and answers concerning water, sanitation and hygiene world health organization. ebola virus disease (evd): occupational safety and health: joint who/ilo briefing note for workers and employers infection prevention and control (ipc) guidance summary: ebola guidance package world health organization. ebola virus disease preparedness strengthening team: mali country visit - world health organization. facility questionnaire for ebola virus disease/viral haemorrhagic fever diagnosis capacity agree ii: advancing guideline development, reporting and evaluation in health care pandemic (h n ) guidance documents conflict of interest in clinical practice guideline development: a systematic review tools for assessing the content of guidelines are needed to enable their effective use-a systematic comparison the authors gratefully acknowledge the administrative support of ms myriam felber and dr martin mendelson. key: cord- -jso mbx authors: lee, sunhee; lee, changhee title: genomic and antigenic characterization of porcine epidemic diarrhoea virus strains isolated from south korea, date: - - journal: transbound emerg dis doi: . /tbed. sha: doc_id: cord_uid: jso mbx porcine epidemic diarrhoea virus (pedv) is a globally emerging and re‐emerging enteric coronavirus in pigs causing serious economic threats to the world swine industry. since the re‐emergence of massive pedv outbreaks in south korea in − , domestic pig farms have continued to experience ped epidemics or endemics. this study represents the molecular characterization of pedv isolates identified in diarrhoeic animals collected across the country in . initial sequencing analysis of the full‐length s genes revealed that % of the isolates ( / ) belong to the g b subgroup, while the remaining isolates were classified as g b. the data indicated that both variant g b and global epidemic g b strains were responsible for current ped outbreaks in south korea. the g b and g b isolates shared . %– . % and . %– . % amino acid sequence identity at the s gene level and . % and . %– . % nucleotide sequence homology at the genome level compared to the corresponding korean prototype g b and g b strains, respectively. in an interesting manner, one g b‐like knu‐ strain was found to possess a large ‐nucleotide deletion in the orf a region theoretically encoding nonstructural protein . phylogenetic analysis based on the entire genome and spike protein sequences indicated that the isolates were most closely related to other global g b or g b strains but formed different branches within the same genogroup. these results indicate that pedvs undergo continuous evolution in the field. in addition, one pedv strain, kor/knu‐ / , was successfully isolated and propagated in vero cells. the antisera raised against the korean prototype g b strain efficiently neutralized knu‐ virus infection, suggesting antigenic homology between the and pedv strains. our data advance the understanding of the molecular epidemiology and antigenicity of pedv circulating in south korea. polyadenylated tail and consists of seven canonical coronaviral genes, including open reading frame (orf) , in the following conserved order: untranslated region (utr)-orf a-orf b-s-orf -e-m-n- utr (kocherhans, bridgen, ackermann, & tobler, ) . the first two large orfs, orf a and b, encompass the -proximal two-thirds of the genome and code for nonstructural proteins (nsps). orf a translation yields a replicase polyprotein (pp) a, whereas orf b is expressed by a À ribosomal frame shift that c-terminally extends pp a into pp ab. these pp a and pp ab are proteolytically matured by internal viral proteases to generate processing end-products, named as nsp - . the remaining orfs in the -proximal region of the genome encode four canonical structural proteins, spike (s), envelope (e), membrane (m) and nucleocapsid (n), as well as one accessory gene, orf (duarte et al., ; kocherhans et al., ; lai, perlman, & anderson, ; lee, ; saif et al., ) . porcine epidemic diarrhoea was first observed in feeder and fattening pigs in england in (oldham, ) and caused widespread epidemics in multiple swine-producing countries in europe during the s (opriessnig, ) . a marked decrease in acute ped epizootics occurred in europe in the s and s, and only sporadic outbreaks have occurred in recent years (opriessnig, ) . in asia, ped was first reported in , and unlike in europe, it has since posed a huge economic threat to the asian pork industry (chen et al., ; kweon et al., ; li et al., ; puranaveja et al., ; takahashi, okada, & ohshima, ) . in may , ped outbreaks suddenly appeared in the united states and swiftly spread across the nation, as well as to adjacent countries. this outbreak caused the death of more than million newborn piglets in the united states alone during a -year-epidemic period, leading to annual losses in the range of $ million to $ . billion (langel, paim, lager, vlasova, & saif, ; mole, ; ojkic et al., ; stevenson et al., ; vlasova et al., ) . the us emergent strain-like viruses further reached east asian countries, resulting in nationwide ped disasters (lee, ; lin et al., ; maff, ) . during the - pandemics, ped rapidly swept across mainland south korea and jeju island, killing hundreds of thousands of piglets in domestic herds , . since then, ped epizootics or enzootics have regionally occurred through provinces in south korea with intensive swine industries. to investigate the diversity of pedvs responsible for the ongoing outbreaks in south korea, in this study, we determined the full-length sequences of the s proteins of field isolates and complete genome sequences of representative strains identified throughout . in addition, we isolated and serially propagated a kor/knu- / strain and assessed the antigenic cross-reactivity between and pedv field isolates. the small intestine or stool specimens were collected from piglets showing acute watery diarrhoea at various swine farms located in eight different provinces from march through december . intestinal homogenates were prepared as % (wt/vol) suspensions in phosphate-buffered saline (pbs) using a magna lyser (roche diagnostics, mannheim, germany) by three repetitions of s at a speed of , g. faecal samples were also diluted with pbs to % (wt/vol) suspensions. the suspensions were then vortexed and centrifuged for min at , g (hanil centrifuge fleta , incheon, south korea). the clarified supernatants were initially subjected to rt-pcr using a tge/ped detection kit (intron biotechnology, seongnam, south korea) according to the manufacturer's instructions. pedv-positive samples were filtered through a . -lm-poresize syringe filter (millipore, billerica, ma) and stored at À °c until subsequent sequencing analysis and virus isolation. the s glycoprotein gene sequences of the virus isolates were determined by the traditional sanger method. two overlapping cdna fragments spanning the entire s gene of each isolate were amplified by rt-pcr as described previously (lee, park, kim, & lee, ) . the individual cdna amplicons were gel-purified, cloned into a pgem-t easy vector system (promega, madison, wi) and sequenced in both directions using two commercial vector-specific t and sp primers and gene-specific primers. the full-length s sequences of pedv, designated knu- to - , were deposited in the gen-bank database under the accession numbers shown in figure a . in addition, the complete genomes of representative pedv field strains were sequenced by the traditional sanger method. ten overlapping cdna fragments spanning the entire genome of each virus strain were rt-pcr-amplified as described previously lee et al., , and each pcr product was sequenced as described above. the and ends of the genomes of individual isolates were determined by rapid amplification of cdna ends (race) as described previously . general procedures for dna manipulation and cloning were performed according to standard procedures (sambrook & russell, ) . the complete genomic sequences of the viruses were deposited in the gen-bank database under the accession numbers shown in figure b. the sequences of the fully sequenced s genes and complete genomes of global pedv isolates were independently used in sequence alignments and phylogenetic analyses. multiple sequence alignments were generated with the clustalx . program (thompson, gibson, plewniak, jeanmougin, & higgins, ) , and the percentages of nucleotide sequence divergences were further assessed using the same software program. phylogenetic trees were constructed from the aligned nucleotide or amino acid sequences using the neighbour-joining method and subsequently subjected to bootstrap analysis with , replicates to determine the percentage reliability values of each internal node of the tree (saitou & nei, pedv isolation was conducted from faecal suspensions on vero cells in the presence of trypsin (usb, cleveland, oh) as described previously . virus isolation was confirmed by cytopathic effect (cpe) observation, immunofluorescence assay (ifa) and nucleotide sequencing as described previously . the isolated pedv strain was propagated for serial passages in vero cells, and virus titres were determined as described previously . the cross-reactivity of antisera collected from sows inoculated with a korean pandemic g b strain knu- isolated in (baek et al., ) was evaluated by a serum neutralization (sn) test in -well microtiter plates against the past and present isolates as previously described oh, lee, choi, & lee, ) . the neutralization titre was calculated as the reciprocal of the highest dilution of serum that inhibited virus-specific cpe in all duplicate wells. the pedv s glycoprotein is a suitable viral gene for investigating genetic relatedness among isolates and the molecular epidemiology of pedv (chen et al., ; gerber et al., ; lee, ; lee et al., ; oh et al., ) . based on the s gene sequences, therefore, pedv can be genetically separated into two genogroup clusters, genogroup (g , classical and recombinant: low-pathogenic) and genogroup (g , field epizootic or panzootic: high-pathogenic), which are further divided into subgroups a and f i g u r e phylogenetic analysis based on nucleotide sequences of the spike genes (a) and full-length genomes (b) of porcine epidemic diarrhoea virus strains. a region of the spike protein and complete sequence of tgev were included as an outgroup in each tree. multiple sequence alignments were performed using the clustalx program, and the phylogenetic tree was constructed from the aligned nucleotide sequences using the neighbour-joining method. numbers at each branch represent bootstrap values greater than % of , replicates. names of the strains, countries, years of isolation, genbank accession numbers, and genogroups and subgroups proposed in this study are shown. the pedv isolates identified in this study are indicated by solid circles. scale bars indicate nucleotide substitutions per site lee and lee | b as well as a and b (lee, ; figure s ). in an interesting manner, the g b virus s genes were well-conserved, sharing . %- . % aa identity with each other, whereas the g b s genes were relatively variable, exhibiting . %- . % aa homology with each other (table ) (table s ). the number of nt/aa differences and percent identity shared between the isolates and genogroup representative strains is summarized in table s . to establish the genetic relationships involved, phylogenetic analyses were carried out using the nucleotide sequences of the s gene and full-length genome of the isolates, which were determined in this study and are available from genbank (figure ) . consistent with previous studies (lee, ; lee et al., ) , phylogenetic analysis based on the pedv s genes revealed clear separation among the g a, g b, g a and g b subgroups. all g b strains identified in were grouped within the g b clade; however, they were in different branches from the emergent us strains and past re-emergent korean field isolates (figure a) . the g b isolates were most closely clustered together, forming an independent branch within the g b subgroup. furthermore, a phylogenetic tree subsequently reconstructed from the complete genome showed the same grouping structure as the s gene-based tree (figure b) . as shown previously (lee, ; lee et al., ) , the entire genome-based phylogenetic tree revealed that the g b strains including knu- were grouped within the g clade because of the similarity between the g b and g b genomes, except for the n- the percent nucleotide identity was shown in the upper right and the percent amino acid identity was presented in the lower left. lee and lee | which were less than -log lower but not significantly different compared to those against knu- . taken together, our data indicate that the antisera cross-reacted well between the homologous g b field isolates, suggesting antigenic similarity between the and pedv strains. pedv has emerged or re-emerged as one of the deadliest and most contagious viral pathogens in swine, leading to large financial losses in the global swine industry. along with strict biosecurity, vaccination is a fundamental tool for managing and eradicating pedv during epidemic or endemic outbreaks. although g a-based vaccines against pedv were developed and used to combat this disease in south korea over the past decade, their efficacy in the field, as well as the advantages and disadvantages of their use, is continuously debated. furthermore, a growing body of evidence suggests that their incomplete effectiveness may result from antigenic, genetic (> % aa variation between respective s proteins) and phylogenetic (g versus g ) differences between vaccine and field epidemic strains (lee et al., ; oh et al., ; kim et al., ; lee et al., ; lee, ) . the advent of the - pedv pandemic led to a breakthrough in the development of g bbased vaccines phenotypically and genotypically homologous to field strains responsible for global ped epidemics, and these g b vaccines are currently applied to prevent pedv in south korea. another important policy for controlling pedv is to operate a monitoring and surveillance system (moss) to monitor genetic diversity among field isolates and surveil the emergence of novel variants in the field, which will contribute to preventing future outbreaks. to provide insight into the understanding of the current epidemiological status of pedv in south korea, the present study aimed to investigate the genetic, phylogenetic and antigenic characteristics of pedvs responsible for regional outbreaks in south korea in . nucleotide sequencing analysis revealed that two different pedv genotypes, low-pathogenic g b and high-pathogenic g b, caused regional outbreaks in south korea, with the latter genotype more % nucleotide sequence variations at the genome level with the - pandemic strains. however, field g b isolates with nearly % amino acid sequence divergence compared to previous g b strains at the s gene level were identified in the present study. furthermore, mutations within the s protein were randomly and extensively distributed in the s and s regions among the isolates ( figure s ). replication. however, this unique del is in the glu-rich acidic region, which does not affect the authentic roles of nsp and thus is nonessential for coronavirus replication (lei, kusov, & hilgenfeld, ) . although the virus can tolerate the large nsp -del which is dispensable for pedv replication as shown in figure conditions. therefore, the timeline of this situation is unclear and it is unknown whether antigenic differences among pedv epidemic strains will contribute to the failure of current g b vaccines. to counteract the prospective scenario, further studies are critical for securing culturable pedv epidemic strains that are genetically, phenotypically and antigenically characterized in the laboratory. in summary, genetic and phylogenetic analyses indicated that the epidemic-related isolates are closely related with corresponding global g b or g b strains identified in previous outbreaks and that the virus continues to evolve in its host environment. despite their genetic diversity, antigenicity currently seems to remain unchanged among g b strains, indirectly confirming the efficacy of g b-based vaccines against homologous g b pedvs responsible for current epidemics. because the virus is assumed to undergo an evolutionary process to accumulate mutations to ensure viral fitness in the field, new genotypes or variants of pedv, against which the current g b vaccine may provide partial protection, will eventually emerge. furthermore, this circumstance may advent earlier than expected if pedv outbreaks fade from our attention following sporadic or endemic outbreaks without serious economic problems. therefore, it is important to execute mandatory notification of ped-like outbreaks essentially followed by activating an moss, including early diagnosis, to survey forthcoming pedv strains that may emerge locally or globally through genetic drift (e.g., nonsilent point mutations) or genetic shift (e.g., recombination events) and obtain and characterize epidemic field isolates to predict and prepare for future epizootics or panzootics. we would like to acknowledge the swine veterinarians and choon- the authors declare that they have no conflict of interest. lee http://orcid.org/ - - - efficacy of an inactivated genotype b porcine epidemic diarrhea virus vaccine in neonatal piglets nidovirales: a new order comprising coronaviridae and arteriviridae isolation and characterization of porcine epidemic diarrhea viruses associated with the disease outbreak among swine in the united states molecular characterization and phylogenetic analysis of membrane protein genes of porcine epidemic diarrhea virus isolates in china sequence analysis of the porcine epidemic diarrhea virus genome between the nucleocapsid and spike protein genes reveals a polymorphic orf detection of antibodies against porcine epidemic diarrhea virus in serum and colostrum by indirect elisa nidovirales: evolving the largest rna virus genome genetic characterization of porcine epidemic diarrhea virus in korea from completion of the porcine epidemic diarrhoea coronavirus (pedv) genome sequence isolation of porcine epidemic diarrhea virus (pedv) in korea coronaviridae lactogenic immunity and vaccines for porcine epidemic diarrhea virus (pedv): historical and current concepts porcine epidemic diarrhea virus: an emerging and reemerging epizootic swine virus isolation and characterization of a korean porcine epidemic 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immunogenicity and protective efficacy of recombinant s domain of the porcine epidemic diarrhea virus spike protein the first case of porcine epidemic diarrhea in canada letter to the editor porcine epidemic diarrhea (ped) in europe and strategies to control outbreaks a new coronavirus-like particle associated with diarrhea in swine chinese-like strain of porcine epidemic diarrhea virus diseases of swine the neighbor-joining method: a new method for reconstructing phylogenetic trees molecular cloning: a laboratory manual emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences an outbreak of swine diarrhea of a new-type associated with coronavirus-like particles in japan mega : molecular evolutionary genetics analysis (mega) software version . the clustalx windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools distinct characteristics and complex evolution of pedv strains additional supporting information may be found online in the supporting information section at the end of the article. how to cite this article: lee s, lee c. genomic and antigenic characterization of porcine epidemic diarrhoea virus strains isolated from south korea key: cord- - ke vor authors: mittal, hemant; sharma, ashutosh; gairola, ajay title: a review on the study of urban wind at the pedestrian level around buildings date: - - journal: journal of building engineering doi: . /j.jobe. . . sha: doc_id: cord_uid: ke vor abstract urbanization is leading towards the change of local wind climate in the vicinity of tall buildings, which influences the pedestrian level wind environment to an uncomfortable or even dangerous level. therefore nowadays, building design should not be limited only for the consideration of wind load and indoor environment, but outdoor wind environment should also be considered. this study presents a review of the methods for the assessment of pedestrian level wind climate, different wind comfort criterion and various techniques to evaluate the wind speed at the pedestrian level. in later sections, brief review for the influence of different parameters related to building design and configuration on pedestrian level wind is presented. after analyzing previous literature it is suggested that there is a strong need for the homogenization of different wind comfort criterion, as it may lead to different consequences for the architects. among various wind tunnel measurement techniques, use of irwin probe is simple and accurate compared to hot-wire anemometry and it can be installed at numerous locations for simultaneous measurement of pedestrian level wind speed. for numerical simulation, reynolds averaged navier stokes based technique has been used by various researchers, although this technique is not accurate as much as large eddy simulation and detached eddy simulation. but this technique is cost effective and requires less computing resources. the socio-economic growth of a nation is majorly driven by urbanization, as it accommodates the increased demand for business and residential space. as an evidence of the current scenario, many cities of developed and developing nation like japan, hong-kong, china, malaysia, and india are moving towards the construction of cohesive skyline structures or mega tall buildings. on contrary to these advancements, mega tall buildings in the urban area affect the surrounding wind flow pattern and pedestrian level wind (plw) comfort. presence of tall building in the urban area tends to deflect the upper-level high-speed wind to the ground, which creates conditions that could be unpleasant or even dangerous to pedestrians. there are many such incidents which are reported due to strong winds. but nowadays modern megacities are packed with the high density of high rise buildings, which influences the air movement. the reduced air movement at pedestrian level causes weak natural ventilation and allow the pollutants to be accumulated at ground level which increases air pollution. such wind conditions are persistent in hong kong, tokyo and new delhi [ ] . many causalities have been reported due to the accumulation of air-borne sars virus (severe acute respiratory syndrome) because of low wind speed zone at a building site in hong kong [ ] . so it is inevitable to assess wind condition for pedestrian level comfort in the view of low wind speed as well as strong winds near buildings corners. many urban authorities have made it is essential to study the pedestrian level wind environment for large urban projects during initial design stage [ ] [ ] [ ] [ ] . initially, studies related to plw speed measurement had been conducted with on-site field measurement. as it is not viable to conduct full-scale testing for the initial design of a building project site, so wind tunnel measurements on the scaled model make it feasible to investigate the effect of changes in building design at the initial stage of the urban project. during early days, wind tunnel measurement for plws was conducted with hot wire or film anemometry at limited measurement points [ ] [ ] [ ] [ ] . later on, irwin [ ] devised a simple omni-direction probe for plw speed measurement. in which pressure difference between tubes at scaled pedestrian height and surface of the tunnel is calibrated with the corresponding velocity. recently the use of irwin probes has been paced up due to the availability of high precision simultaneous pressure measuring sensor. further, the use of sand erosion technique for such studies is limited as it provides qualitative information over the whole area under investigation [ ] . there are other measuring techniques which have been used to evaluate plw speed such as laser doppler anemometry (lda), particle image velocimetry piv, infrared thermography and thermistor anemometry. computational fluid dynamics (cfd) technique is also becoming a viable tool for plw studies with the advent of high-performance computational resources. till now steady reynolds averaged navier-stokes (rans) modelling approach was used successfully which requires less computing cost and time. but this technique is less accurate for predicting the flow in low wind speed region (deviation up to times) as compared to other high-cost techniques such as les and des. the present study comprehensively reviews the urban wind at the pedestrian level around buildings. the content of this paper is organized as follows: the second section presents the method for the assessment of plw climate with different wind comfort criteria. the third section describes the different techniques to evaluate the pedestrian level winds. the fourth section reviews effect of the various parameter related to building design on plw for generic building configuration. the last section presents different studies related to the actual urban environment, which comprises the effect of building design parameters and general guidelines for the urban planning in response to pedestrian comfort. the procedure for the assessment of favourable wind climate to pedestrians is comprised of ( ) statistical meteorological data of nearby weather station; ( ) aerodynamic information of the area and ( ) mechanical wind comfort criteria [ ] . the aerodynamic information helps to compute the statistical data at particular building site obtained from the weather station. then transformed data at this location is compared to wind comfort criterion. this procedure is schematically depicted in fig. . meteorological data from weather station consist of hourly mean wind speed (u ms , measured at m height) and wind direction in open terrain ( = y m . meteo , ). this wind speed data obtained from weather station is analyzed statistically using weibull distribution function [ ] [ ] [ ] to calculate the probability of exceedance of threshold wind speed as following eq. ( ). where > p u ( )represents the exceedance probability of the wind speed; u is the mean wind velocity magnitude at building site; c is the dispersion parameter and k is the shape parameter. these constants are obtained by fitting eq. ( ) to the meteorological data. then statistical information has to be transformed to the area of interest by the means of aerodynamic information using amplification factor r (eq. ( )). this amplification factor consists of design related contribution and terrain related contribution (eq. ( )) [ ] . the design related contribution comprises of modification of statistical wind climate information due to local building design. these modification can be obtained by either wind tunnel measurement or using cfd simulation. the whole research community in this area is devoted to evaluate the design related modification. the terrain related modification accounts for the differences in terrain roughness between the weather station and area of interest [ ] and can be obtained using eq. ( ) and eq. ( ). where u is the reference wind speed at certain distance upstream of area of interest or without the presence of building or at the inlet of computational domain; u* site and u * meteo are the friction velocity at building site and meteorological station respectively; y site , and y meteo , are the aerodynamic surface roughness length at building site and meteorological station respectively. the dependency of the probability of exceedance and amplification factor r is given by eq. ( ) [ ] . in wind comfort assessment, besides the wind speed, the frequency of its occurrence also matters. therefore the criteria for wind comfort involves threshold wind speed above which pedestrian will feel discomfort and its frequency of occurrence. a wide variety of wind comfort criterion, based on threshold mean wind speed and the probability of exceedance has been proposed earlier [ , , [ ] [ ] [ ] [ ] [ ] [ ] . details of different wind comfort criterion are shown in fig. , in which threshold mean wind speed (m/s) for different activities and its corresponding exceedance probability (%) is presented. most of the criterion is based on the same probability of exceedance and different threshold wind speed for different pedestrian activities. while nen [ ] considers same threshold mean wind speed and different exceedance probability. the comparison of different wind comfort criterion for different activities is shown in fig. , in which typical wind climate for indian city (palam airport, new delhi) is represented. it can be identified that, based on comparison with mentioned wind comfort criteria except by melbourne, ( ) [ ] , this place is vulnerable to high wind speed, which causes danger to pedestrians. since most of these criterion reports different threshold mean wind speed and exceedance probability, so it is difficult for developers, architects and planners to choose a general guideline on comfort criterion. with regard to this, various comparative assessments of wind comfort criteria have been proposed. based on discussion with developers and building managers, soligo et al. [ ] converted the different wind comfort criteria on the same scale of frequency of occurrence ( %) or for a particular activity. janssen et al. [ ] also focused on the standardization of different wind comfort criterion. however, all of the above-discussed criteria did not consider pedestrian discomfort due to weak wind conditions for the densely-built urban areas. du et al. [ ] proposed the wind comfort criteria for the urban areas of poor wind conditions such as hong kong, where the wind comfort becomes worst in hot and humid season. in this criterion overall mean velocity ratio (omvr) is used as threshold parameter (table ) , which represents the integration of direction values of mean velocity ratio ( = mvr u u / p r ), where u p is wind velocity at pedestrian level and u r is wind velocity measured at m height. to obtain design related contribution for the assessment of pedestrian level wind environment, generally field measurement on the real urban environment, wind tunnel testing on the scaled model of the urban area and cfd simulation are employed. but to make changes in the early design stage, it is not possible to conduct field measurement for urban developmental projects. the following sections describe the details of each method and comparison based on the accuracy of each method. this technique is regarded as a robust method to evaluate wind speed at limited points and was used successfully by several researchers [ ] [ ] [ ] [ ] [ ] . generally, a portable three cup-anemometer with wind vane system is used for this purpose. this instrument has very low onsetspeed of . m/s and light in weight [ ] . the output of this instrument is in the form of one electrical pulse per revolution of the rotor and this pulse rate is calibrated against the wind speed. a comparison of this technique with others is provided in the later section. in wind tunnel, the plw measurements are conducted by using hotwire/film anemometry (hwa, hfa), irwin probes, thermistor anemometer, sand erosion, laser doppler anemometry (lda), infrared thermography, and particle image velocimetry (piv). each of the mentioned technique is based on different working principle and unique experimental setup. since it is beyond the scope of this paper to discuss the working principle of each technique, only their application and capability is discussed in table . to simulate plw environment, cfd methods are gaining much popularity among researchers and industrialists recently owing to the development of the computer hardware and software. one of the major advantages of cfd over wind tunnel testing is that it gives detailed flow field data of associated parameters over the entire computational domain. in addition, similarity law requirements associated with wind tunnel testing is not a limitation of cfd simulation. most of the studies, to simulate wind environment are based on the use of different rans turbulence models e.g. std. − k ε, realizable − k ε and rng − k ε model with default values of model closure coefficients in cfd tools. however, the major issue for the acceptance of cfd results is related to the accuracy, which suffers badly in predicting the flow on leeward side of building. therefore cfd simulation results require verification and validation [ ] . zahid iqbal et al. [ ] mentioned that the simulation results for plw speed obtained by std. − k ε are also affected for different model closure coefficients. table wind comfort criteria based on overall mean velocity ratio (omvr) representing weak wind conditions by [ ] . threshold velocity (jun-aug) threshold velocity (dec-feb) exceedance probability (%) activity remark the accuracy of cfd simulation largely depends on the selection of turbulence model. the capability of each turbulence model and their accuracy is discussed in table . to evaluate plw environment on existing project site, it is suitable to conduct field measurements. but to assess the effect of changes in the initial design of developmental projects, wind tunnel measurements are preferred. comparison of these techniques is generally associated with practical difficulties such as variability inherent to the atmospheric phenomena, obstructions due to automobiles etc. isyumov and davenport [ ] obtained full-scale and wind tunnel measurement for mean wind speed at a site project in toronto, canada. they reported the agreement between full-scale and wind tunnel measurement within % for windy locations. dye [ ] compared the same in a sheltered urban area and found that wind tunnel test generally underpredicts the mean speed of % at most severe locations. while visser et al. [ ] reported the comparison of these two techniques to be dependent on the duration of full-scale data measurement, as the wind speed data of longer period will agree well with the model test. the use of cfd technique provides entire flow field data, whereas field measurement can be performed for few location only. however, it is a challenging task to simulate wind environment using cfd techniques accurately. blocken et al. [ ] compared the results of cfd simulation at a university campus. the simulation was performed using a realizable − k ε model with standard wall function and field measurement was performed with d ultrasonic anemometers. the authors clearly indicate that the short term measurement for few hours cannot serve as a validation data because the data suffers from intrinsic variability of meteorological conditions. large deviation of % between measured and simulated mean wind speed was reported at the locations where the gradient of wind speed is high. in addition, the comparison of the wind speed in the regions of high gradients can yield large deviation by a small shift in location. the cfd technique has attracted wide acceptability and strong support due to the establishment of several best practice guidelines [ ] . wind tunnel testing is often used to benchmark cfd models and simulation results. the use of rans was found to provide quite accurate results for wind speed ratio, however, it significantly underestimates wind speed in the regions of lower wind speed ratios [ ] . a possible [ , ] . hfa [ , , , , [ ] [ ] [ ] [ ] pros: less susceptible to fouling and fragility, easy to clean, shorter sensing length, the agreement between wind tunnel and fullscale measurement is within % [ ] . cons: intrusive technique, only suitable for moderate turbulence intensity, insensitive to direction changes. lda [ , ] pros: non-intrusive point-wise technique, allows measurement of high-turbulence intensity and calibration is not required. [ ] cons: this technique is costlier than hfa and hwa. infrared thermography [ , , ] pros: non-intrusive area technique, rms, peak and spectrum value can be measured [ ] cons: due to convection wind flow gets disturbed, sturdy and non-standard experimental set-up. no perfect correlation is obtained for temperature drop and wind speed [ ] piv [ , ] pros: non-intrusive area technique, high spatial resolution and directional sensitivity. cons: very expensive, sometimes dangerous, laser light shielding and reflection from buildings, not suitable for the cluster of buildings. erosion technique [ , , [ ] [ ] [ ] [ ] pros: area technique, results are comparable to hwa for high wind speed [ ] . for high turbulent flow, this technique agrees well with piv measurements of mean wind speed [ ] . cons: it is non-quantitative technique and difficult to ensure the repeatability. [ , [ ] [ ] [ ] [ ] [ ] pros: allows measurements at numerous locations. no re-alignment for different wind direction. simple in design and easy to operate [ ] . cons: less accurate for high turbulence intensity, it cannot accurately measure wind speed below . m/s [ ] . thermistor anemometer [ ] pros: small sensor size, susceptible to wind direction. the simple circuitry and low cost of thermistors make it economically feasible to operate the probes in large numbers. cons: only suitable for mean velocity measurements, fragile, require alignment for change in wind direction; nonlinear calibration of velocity. use of different cfd technique by various authors and their pros. and cons. std. − k ε [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] pros: computationally efficient and economically viable. agreement with wind tunnel measurement of wind speed is within % for regions with high wind speed ratio ( [ ] cons: underestimates wind speed notably five times or more for low wind speed regions [ ] . it cannot reproduce the reverse flow on the roof [ ] and overpredicts turbulent kinetic energy in separated flows around windward corners of buildings [ ] . realizable − k ε [ , , , [ ] [ ] [ ] [ ] pros: sensitive to flow separation, reattachment and recirculation. for high wind speed region accuracy further improves as compared to std. − k ε model [ ] . cons: less accurate compared to std. − k ε model for low wind speed region due to underestimation of tke in the wake region [ ] rng − k ε [ ] [ ] [ ] [ ] pros: for high wind speed region, accuracy improves as compared to std. − k ε model. [ ] cons: less accurate compared to std. − k ε model for low wind speed region due to underestimation of tke in the wake region [ ] . les [ ] [ ] [ ] [ ] pros: superiority of this method over steady rans is clearly reported [ ] . it can reproduce turbulence intensity and gustiness. cons: computationally very expensive as it requires more time and sensitive to many parameters such as sub-grid scale model, mesh resolution and time step size [ ] . des [ ] pros: capable of producing similar results as les with less computing time and lower mesh size. [ ] cons: sensitive to parameters such as sub-grid scale model, mesh resolution and time step size and sampling time. [ ] reason for high wind speed ratio is characterized by lower turbulence intensities and lower wind direction fluctuation, therefore better modelled by the statistically rans approach. richards et al. [ ] adopted erosion technique to investigate the plw environment downtown area of auckland and compared erosion contour with cfd simulation using std. − k ε model. the authors observed the difference at the location of highest wind contours. using cfd simulation, this location was immediately around the corner of the buildings, whereas the wind tunnel shows that the earliest erosion emanates from the corner and sweeps across the buildings. in past decade, various parametric studies related to the evaluation of pedestrian level wind environment around generic building configuration have been conducted, which considered the effect of building height, shape and pattern of a group of buildings. these studies are listed in table . in this section brief discussion about the effect of parameters related to the design of building configuration on pedestrian level flow is presented. as the height of building increases, the maximum wind speed ratio increases due to strong downwash effect, as taller building catches the more upper-level wind and directs it to pedestrian level. hence it poses high wind speed conditions but improves near-field air ventilation conditions as shown in fig. .(a) [ , , ] . whereas turbulence intensity is not influenced by height variation of building significantly [ ] . wider buildings increases the sheltering effect to the incoming wind, which enlarges the extent of low wind speed zone in the downstream side of the building. changes in cross-section of the building, such as tapering, rounding, and corner cut improve the wind environment around the building corners, as it reduces the extent of high wind speed zone near building corners due to less deviation of separated flow in comparison to the square building as shown in fig. (b) [ , ] . but root mean square value of wind speed distribution remains unaffected due to corner modification [ ] . circular and polygon shaped cross-section of the building also tends to have better wind climate in terms of reduced high wind speed zone as compared to the square-shaped building due to reduced downwash [ ] . till now this modification is investigated in detail by xu et al. [ ] for various building models and it was proposed that the change of building width at one-third height from the ground, majorly influences the plw environment. whereas the use of permeable floor at midheight of the building reduces the extent of high wind speed zone. however, it does not lower the maximum wind speed ratio [ ] , as this permeable floor provides a way for upper-level wind to pass through the building before reaching it to ground level. this design consists elevated main structure of the building from the ground by the central core and has a potential solution to improve wind conditions near the building. this design modification increases area averaged high wind speed ratio but decreases area averaged low wind speed zone on the leeward side of the building because of weak downwash [ , ] . to illustrate the effect of this building design, numerical simulation using cfd software ansys/fluent . was conducted. the governing equations of the flow are d steady-state reynolds-averaged navier-stokes with standard − k ε turbulence model. numerical simulation has been conducted using best practice guidelines [ ] for pedestrian level wind flow. all the details related to cfd simulation is not included as it is beyond the scope of this paper. the result of this simulation is presented in fig. , which represent the contours of mean wind speed at pedestrian height for lift-up and conventional square buildings. the qualitative results of the present simulation are similar to the experimental study by tse et al. [ ] which shows high wind speed near the lift up area and further lower values of low wind speed as compared to conventional building design. which helps in improving ventilation near the building in addition to this lift up area can be used for other purposes as the recreational area, parking, or shelter for pedestrians. besides this, use of lift up building design is limited due to unacceptable or high wind speed zone near the lift-up area [ ] . this unacceptable wind condition arises due to flow through openings of positive and negative pressure faces of the building. podium structure creates a sheltering effect at pedestrian level wind flow near the building and results in undesirable low wind speed zone at pedestrian level [ ] , as podium structure enhances the spatial extent of low wind speed zone near upstream and downstream of the building. in general podium structure is not recommended where the conditions for natural ventilation are required. besides this, it is believed that podium structure is used to protect pedestrians from high wind speed. the orientation of two buildings such as side by side, parallel, angular (converging, diverging and perpendicular) creates an adverse effect on plw speed differently as shown in fig. . when wind flow is perpendicular to the row of buildings, the windiest condition occurs in the upstream corners due to flow channelling and suppressed horseshoe vortices [ ] . as the separation between the buildings increases, channelling effect gets minimized and flow tends to be independent of the further increase in separation. for the converging arrangement, the windiest condition occurs for narrowest passage and decreases further for higher passage width with its location moving further away in the downstream direction as shown in fig. (a) . whereas for diverging arrangement wind speed ratio is often more adverse than converging passage as it does not provide shelter to the wind [ ] and its value decreases for higher passage width as shown in fig. (b) . in urban design, different arrangements of building govern the microclimate of the urban area differently. but orientation and shape of buildings majorly helps to improve plw climate. several studies [ , , ] propose the configuration with square central space with prevailing wind direction towards the windward opened side face offer better plw environment. as this configuration can effectively contain the air flow movements. in the lower part of the hilly regions (below m), wind profile gets twisted with large angles i.e. varying wind direction along the height, therefore wind-structure interaction in these conditions is different from interaction with the conventional profile. to investigate the effect of twisted wind flow on pedestrian level, tse et al. [ ] simulated such kind of twisted wind profile in boundary layer wind tunnel. the authors concluded that the spatial extent of low wind speed zone increases with the scattered type of flow conditions. the extent of low wind speed zone further increases with the increase of wind twist angle. the flow features around two parallel buildings were observed to be asymmetric. this section describes the general flow conditions around the actual complex urban area, the effect of various parameters affecting the plw speeds. in addition, the methodology to assess the pedestrian comfort is discussed and lastly, the key factors for the design of urban development projects in response to pedestrian comfort are discussed. when high rise building is located upstream of the low-rise building, the wind tends to flow over low rise building. the presence of tall building at downstream position intercepts high-speed winds and redirects to ground level [ ] . the wind conditions within space between tall buildings are accelerated due to so-called venturi effect, which results in high wind movement especially at pedestrian level [ ] . use of podium structure deflects the high wind to the upper level, thereby protects the pedestrian due to high-speed winds [ ] . these are the common flow condition, which arises due to different building configurations in an urban area and can be depicted in fig. . the wind flow over the ground surface is mostly affected by roughness element and building configuration, e.g. width, height, the arrangement of buildings and its density. it is considered that an increase in building density reduces the wind velocity in an urban area due to increased friction near the ground [ ] . stathopoulos & wu [ ] investigated the effect of spatial density of street blocks and the relative height of buildings. the authors concluded that the wind speed along streets decreases with blockage ratio (r b describe the percentage of oncoming air flow captured by building block) and proposed a simplified relation for wind speed up ratio as given in eq. ( ) . they also concluded that, the maximum wind speed ratio increases with the height difference between reference and surrounding buildings. in addition to this wu & kriksic [ ] reported that, the narrow streets and uniform building height results in low wind speed. most of the studies related to plw environment tackle the problem based on discrete point analysis [ , , ] . in this method, wind speed is evaluated at discrete locations. then this wind speed is compared to the suitable wind comfort criteria. but obviously, pedestrian wind environment varies spatially in an urban area. so this type of discrete analysis is not efficient to address the real needs of urban planner in design [ ] . in order to improve assessment method, shi et al. [ ] proposed that the practical pedestrian wind comfort for urban planning design must be space-based. the authors defined two parameters to analyze the pedestrian wind, the spatially averaged wind v avg (eq. ( )) and the maximum wind speed v max in a plane. in above equation a i is the proportion of i-th wind direction; v j is the wind speed in j-th mesh grid; n is the total number of mesh grid in the plane. the above calculated wind speed should be within the specified limit defined in comfort criteria. to achieve better outdoor pedestrian comfort, there are following considerations which needs to be incorporated while designing urban area. ( ) for hot and humid climate, the streets should be aligned parallel to the prevailing wind direction to remove accumulated pollution and improved air movements [ ] . ( ) in low wind speed and high-temperature areas, use of lift up building design [ ] and a cluster of buildings with central square space [ , ] promote the ground level natural ventilation. ( ) for cold climate regions, corner modified buildings [ , ] and a large stepped podium [ ] around the building are convenient to reduce the high wind speeds at the pedestrian level. ( ) modification in building width at one-third level from the ground influences the plw environment mostly [ ] . ( ) the positioning of taller buildings should be on the downside of a street to catch more winds at the pedestrian level [ ] . moreover, the addition of recreation spaces and wide streets, removing the obstacles to the flow throughout the urban area improves the pedestrian comfort level. ( ) for decision making in response to pedestrian comfort, suitable wind comfort criterion, improved terrain mapping and accurate design related modifications are required. the construction of high rise building in the vicinity of urban area alters the wind climate significantly. eventually, it becomes necessary to evaluate the wind environment at the initial design phase of urban projects so that suitable modification could be suggested to improve wind climate for pedestrians. while designing urban area, either it is considered to minimize high wind speed zone to reduce mechanical forces on pedestrians or to improve natural outdoor air ventilation. the present study reviews the different wind comfort criterion for pedestrians and measurement techniques to evaluate these wind speeds. further, the effect of various parameters related to building design is analyzed and at last, evaluation of urban wind at pedestrian level and urban design consideration are discussed. the findings from this review study are summarized as follows. ( ) wind climate statistics obtained from nearby weather station are obligatory for selecting the building shape, orientation and alignment of streets in urban planning and these wind statistics are compared with suitable wind comfort criterion. ( ) the probability of exceedance of threshold wind velocity at the particular site requires terrain and design related modifications. to obtain terrain related modification accurately, improved roughness mapping is required. otherwise, it may lead to wrong decisions in urban planning. ( ) to obtain design related modifications, irwin probes provide relatively accurate results and can be facilitated at numerous locations for simultaneous measurement of mean wind speed and turbulence intensity. ( ) to simulate plw environment, steady rans method is the best choice economically, however, les and des provide more accurate results. further to improve the accuracy of steady rans using std. − k ε model, the effect of different model closure coefficient needs to be investigated in detail. ( ) several parameters related to building design e.g. height & width of buildings have an adverse effect on plw environment, however, building depth does not affect plw environment significantly. moreover, studies based on different grouping of buildings suggest that configuration with square central space provides better plw environment. in addition, uses of lift up buildings podium, corner modified buildings offer a positive design approach. adopting "lift-up" building design to improve the surrounding pedestrian-level wind environment pedestrian level wind environment assessment around group of high-rise cross-shaped buildings: effect of building shape, separation and orientation evaluation of pedestrian wind comfort near "lift-up" buildings with different aspect ratios and central core modifications designing for pedestrian comfort in response to local climate sensitivity of inflow boundary conditions on downstream wind and turbulence profiles through building obstacles using a 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revisiting the "venturi effect" in passage ventilation between two non-parallel buildings cooperative project for cfd prediction of pedestrian wind environment in the architectural institute of japan comparison of various revised kε models and les applied to flow around a high-rise building model with : : shape placed within the surface boundary layer cfd evaluation of new second-skin facade concept for wind comfort on building balconies: case study for the park tower in antwerp use of cfd simulations to improve the pedestrian wind comfort around a high-rise building in a complex urban area air ventilation impacts of the "wall effect" resulting from the alignment of high-rise buildings modification of pedestrian wind comfort in the silvertop tower passages by an automatic control system numerical simulation of the wind field around different building arrangements assessment of pedestrian wind environment in urban planning design detached eddy simulation of pedestrian-level wind and gust around an elevated building prediction of building interference effects on pedestrian level comfort pedestrian level wind assessment through city development: a study of the financial district in toronto analysis of airflow over building arrays for assessment of urban wind environment evaluation of pedestrian winds in urban area by numerical approach large-eddy simulation of flow and dispersion around an isolated building: analysis of influencing factors cfd simulation of the wind environment around an isolated high-rise building: an evaluation of srans, les and des models aij guidelines for practical applications of cfd to pedestrian wind environment around buildings environment around building: a knowledge-based approach simulation of twisted wind flows in a boundary layer wind tunnel for pedestrian-level wind tunnel tests pedestrian-level wind environment around isolated buildings under the influence of twisted wind flows effects of lift-up design on pedestrian level wind comfort in different building configurations under three wind directions wind tunnel tests on the relationship between building density and pedestrian-level wind velocity: development of guidelines for realizing acceptable wind environment in residential neighborhoods cfd simulation of wind flow over natural complex terrain: case study with validation by field measurements for ria de ferrol this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. the authors thank the anonymous reviewers for their valuable comments and suggestions on the manuscript. key: cord- - voi y authors: han, hui-ju; liu, jian-wei; yu, hao; yu, xue-jie title: neutralizing monoclonal antibodies as promising therapeutics against middle east respiratory syndrome coronavirus infection date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: voi y since emerging in , middle east respiratory syndrome coronavirus (mers-cov) has been a global public health threat with a high fatality rate and worldwide distribution. there are no approved vaccines or therapies for mers until now. passive immunotherapy with neutralizing monoclonal antibodies (mabs) is an effective prophylactic and therapeutic reagent against emerging viruses. in this article, we review current advances in neutralizing mabs against mers-cov. the receptor-binding domain (rbd) in the spike protein of mers-cov is a major target, and mouse, camel, or human-derived neutralizing mabs targeting rbd have been developed. a major problem with neutralizing mab therapy is mutant escape under selective pressure, which can be solved by combination of neutralizing mabs targeting different epitopes. neutralizing mabs are currently under preclinical evaluation, and they are promising candidate therapeutic agents against mers-cov infection. middle east respiratory syndrome (mers) emerged in in saudi arabia with the death of a man with pneumonia; the causative agent was subsequently identified as mers-cov, which belonged to lineage c betacoronaviruses [ ] . with dromedary camels (camelus dromedarius, also known as arabian camel) as direct sources and bats as potential reservoirs [ ] , mers-cov has been frequently introduced into human populations. once mers-cov is introduced into a person, person-to-person transmission might occur, and is responsible for approximately % of mers cases globally [ ] . mers-cov has been a consistent threat to humans. as of october , mers-cov has caused laboratory-confirmed human cases, including deaths in countries, with the fatality rate as high as % (http://www.who.int/emergencies/mers-cov/en/). although mers cases are primarily reported in the middle east, facilitated by international travelling, mers-cov can also be a worldwide threat, which is well illustrated by the mers outbreak in south korea in [ ] . given the potential risk of causing worldwide public health emergencies and the absence of licensed vaccines and antiviral therapeutics, the world health organization has listed mers-cov in the "list of blueprint priority diseases" (http://www.who.int/blueprint/priority-diseases/en/). vaccines are the most important approach against viral infections, but usually take a long time to develop. they are also unable to provide either immediate prophylactic protection or treat ongoing viral infections. neutralizing monoclonal antibodies (mabs) have recently emerged as a powerful tool to provide prophylactic and therapeutic protection against emerging viruses [ ] . potent neutralizing mabs can be achieved by various technologies, such as hybridoma technology, humanized mouse, phage or yeast display, and single b cell isolation [ ] . mers-cov is a single, positive-stranded rna virus of about kb, which encodes four major viral structural proteins-including spike (s), envelope (e), membrane (m) and nucleocapsid (n)-as well as several accessory proteins [ ] . the s protein ( aa) plays an important role in virus infection and consists of a receptor-binding subunit s (aa - ) and a membrane-fusion subunit s (aa - ). s mediates viral attachment to host cells and s mediates virus-cell membrane fusion [ ] . the s subunit contains a receptor-binding domain (rbd) (aa - ) [ ] that can bind to cell receptor dipeptidyl peptidase (dpp , also known as cd ), and mediates viral attachment target cells [ ] . the rbd consists of a core subdomain and a receptor-binding motif (rbm) (aa - ). the schematic representation of mers-cov s protein is shown in figure a . neutralizing mabs binding to the s protein of mers-cov can prevent viral attachment to the cell receptor and inhibit viral entry [ ] . the s protein of mers-cov is a key target for antivirals, and rbd is the most popular focus. in this study, we review the current knowledge on neutralizing mabs targeting the rbd of mers-cov. stable hybridoma cell lines were generated by fusing myeloma cells with splenocytes of mice that were immunized with mers-rbd protein. two neutralizing mabs, c and e , had high affinity for the rbd of mers-cov and blocked both pseudovirus and live mers-cov entry into cells with high efficacy [ ] . humanized c showed similar neutralizing activity in cell entry tests. in vivo tests indicated that c could significantly reduce the virus titers in the lungs of ad -hcd -transduced mice which were infected with mers-cov, highlighting its potential application in humans not only for preventing but also treating mers-cov infection. crystallization of the c fab/mers-rbd complex showed that the c recognized conformational epitopes (y -n , k , l -k , p , v -s , w -e , and d -q ), which were partially overlapped the receptor-binding footprint in the rbd of mers-cov. the c complex interfered with mers-cov binding to dpp by both steric hindrance and interface-residue competition. e competed with c to bind to mers-rbd, indicating that they recognized proximate or overlapping epitopes [ ] . neutralizing mab mersmab was obtained by fusing myeloma cells with splenocytes of a mouse that was immunized with recombinant mers-cov s [ ] . mersmab effectively blocked the entry of pseudovirus and live mers-cov into cells. structural analysis showed that mersmab bound to the rbd of mers-cov through recognizing conformational epitopes, and all of the residues critical for mersmab binding were located on the left ridge of rbm. mersmab neutralized mers-cov by competitively blocking the binding of mers-cov rbd to dpp . based on escape mutant analysis of the key residues on the rbd, it was found that residue l , d , r , e , and w were critical for mersmab binding to the rbd, while mutation of e , d , or e did not affect the interaction of mersmab and the rbd at all [ ] . an ultra-large nonimmune human antibody-phage display library was constructed with b cells of unimmunized donors. with a unique spanning strategy, seven human neutralizing mabs with varying neutralization efficacy to mers-cov were identified [ ] . binding detection demonstrated that the epitopes of these mabs lay within aa - of the s protein, which overlapped a large part of the rbd of mers-cov. binding competition assays showed that these mabs recognized at least three distinct epitope groups, which was further confirmed by escape studies. with no cross-epitope resistance, these mabs neutralized mers-cov by competitively blocking the binding of the rbd of mers-cov to dpp . escape mutant assays showed that five residues were critical for neutralization of these mabs, namely l , t , y , r , and p . of the seven mabs, b exhibited the best neutralization activity against both pseudovirus and live mers-cov infectivity in cells. moreover, under the selective pressure of these mabs, the igg form of b was superior, since it did not induce neutralization escape [ ] . in vivo tests demonstrated that b reduced lung pathology in rhesus monkeys infected with mers-cov [ ] . with its high neutralizing activity and suppression of mutant escape, b in the igg form is a promising therapeutic mab against mers-cov. three human mabs-m , m , and m -were identified from a large naïve human phage display antibody library, which was constructed with peripheral blood mononuclear cells from healthy volunteers [ ] . the binding sties of the three mabs were within the rbd of mers-cov (aa - ), therefore they neutralized mers-cov by competing with dpp binding to the rbd. the three mabs also competed with each other to bind to the rbd of mers-cov, and mutant analysis showed that the three mabs possessed overlapping but distinct epitopes. of the three mabs, m neutralized both pseudovirus and live mers-cov infectivity in cells with exceptional potency (m inhibited % mers-cov pseudovirus infection at a concentration of . g/ml, and neutralized live mers-cov with ic of g/ml and ic of . g/ml). residues in the rbd crucial for m binding were l , d , e , d , w , and v [ ] . in vivo study demonstrated that prophylaxis with m reduced virus titers in the lung of rabbits infected with mers-cov [ ] , and m also provided transgenic mice expressing human dpp with full prophylactic and therapeutic protection from mers-cov [ ] . however, another study with a non-human primate, the common marmoset showed that m could only alleviate the severity of the disease, and did not provide complete protection against mers-cov [ ] . igg+ memory b cells were isolated from a mers patient, and were subsequently immortalized with epstein-barr virus. a neutralizing mabs, lca , was identified, and was the first fully human neutralizing mab with naïve heavy and light chain pairs [ ] . lca efficiently neutralize mers-cov infectivity in cells. in vivo study showed that lca provided balb/c mice transduced with adenoviral vectors expressing human dpp (hdpp ) with both prophylactic and postexposure protection against mers-cov. furthermore, the neutralizing efficacy of lca was evaluated in ifn-α/β receptor-knockout mice that were more stringent models of mers-cov infection. after transducing with hdpp , these mice showed more profound clinical symptoms when challenged with mers-cov [ ] ; administration of lca reduced mers-cov titer in the lungs of these mice more effectively (lung viral titer reduced by three logs in one day for ifn-α/β receptor-knockout mice vs. three days for balb/c mice) [ ] . with naïve heavy and light chain pairs, lca was more potent than b and comparable to m . cross-competition experiment demonstrated that lca competed with b to bind to the rbd. lca interacted with rbd residues around k , and the lca footprint on the rbd was partially overlapped with that of dpp . four residues in the rbd affected the binding of lca -namely t , k , e , and e -which were conserved in all mers-cov isolates. moreover, compared with dpp , the binding affinity of lca to rbd was significantly higher (~ -fold). therefore, one major neutralization mechanism of lca was to competitively inhibit the interaction of the rbd with dpp . interestingly, virus escape studies demonstrated that under the selective pressure of lca , a mutant variant (v a) in the n-terminal domain (ntd) of mers-cov s subunit was also generated [ ] . a gmp-approved cell line (lca . . ) that expresses lca in high concentrations has been established, highlighting its application as promising therapeutics against mers-cov infection [ ] . hybridoma b cells producing neutralizing mabs against the s protein of mers-cov were generated by immunizing humanized transgenic mice (velocimmune mice) with dna encoding the mers-cov s protein. two fully human neutralizing mabs, regn and regn , were obtained [ ] . the two mabs bound with high affinity to distinct epitopes on the rbd of mers-cov, which were conserved during the natural evolution of mers-cov. mutation as a result of selective pressure by one mab should not affect the binding of the other mab. regn neutralized a broad range of mers-cov isolates, the prototype emc/ strain and all clinical mutants including a p, s g, s f, a v, l f, d g, and v a. with the exception of v a variant, regn achieved similar neutralizing activity. in vivo study demonstrated that regn and regn reduced mers-cov replication in humanized dpp mice in both prophylactic and therapeutic settings [ ] . when evaluated in the common marmoset, both mabs seemed to be more effective for prophylaxis rather than for treatment of mers-cov infection [ ] . an anti-mers-cov phage display antibody library was constructed with the peripheral b cells of a mers survivor, and a human neutralizing mab against mers-cov, mca , was identified [ ] . mca showed potent neutralizing activity against mers-cov in cell entry tests. in vivo, mca completely inhibited the replication of mers-cov in common marmosets when administrated prophylactically or therapeutically. structure analysis of the mca fab-rbd complex showed that mca formed direct contacts with the receptor-binding site (rbs) subdomain on the rbd. epitopes on the rbs critical for mca binding were d , w , e , d , y , r , and q . superimposed structure analysis of mca -rbd and hdpp -rbd complexes showed that the binding interface of mca was largely overlapped with that of hdpp . therefore, the neutralizing mechanism of mca was achieved by competing with dpp for binding to the rbd [ ] . two potent human neutralizing mabs, mers- and mers- , were derived from a nonimmune human yeast display antibody library, which was constructed with spleen and lymph node polyadenylated rna from normal humans [ ] . [ ] . further structural analysis showed that mers- bound to unique epitopes and caused conformational changes in the rbd interface critical for accommodating dpp , therefore indirectly disrupting the interaction between the two. moreover, mers- also demonstrated synergistic effects with m and f (a ntd-specific mab). the special neutralizing mechanism made mers- a valuable addition for the combined use of mabs against mers-cov infection [ ] . thirteen ultrapotent neutralizing mabs, which all targeted the rbd of mers-cov were generated following a protocol for the rapid production of antigen-specific human mabs [ ] . briefly, antibody-secreting b cells were isolated from the whole blood of a mers patient, and the antibody genes were amplified and cloned into vectors to transfect human cell lines for mab production. of the mabs, mers-gd and mers-gd exhibited the strongest neutralizing activity against both pseudovirus and live mers-cov in cell infection tests. mers-gd directly competed with dpp to bind to the rbd to dpp , and the crystal structure of mers-gd showed that its epitopes were almost completely overlapped with dpp -binding sites. mers-gd and mers-gd recognized distinct epitopes on the rbd, and had a low level of competing activity. the combined use of the two mabs demonstrated synergistic effects in neutralization against pseudotyped mers-cov. mutant analysis demonstrated that residues l , d , v , e , and a on rbd were important for the neutralizing activity of mers-gd , and residue r was critical for mers-gd [ ] . moreover, in vivo study found that mers-gd could provide both prophylactic and therapeutic protection for hdpp -trangenic mice against mers-cov infection [ ] . dromedary camels exposed to mers-cov showed mild clinical signs but developed exceptionally potent neutralizing antibodies. camelid species naturally produced heavy chain-only antibodies (hcabs) [ ] , which are dimeric and devoid of light chains, and their antigen recognition region is solely formed by the variable heavy chains (vhhs) (also called nanobodies, nbs). vhhs or nbs have long complementarity-determining region (cdr ) loops and are capable of binding to unique epitopes not accessible to conventional antibodies [ ] . notably, camelid vhhs are relatively stable and can be produced with high yields in prokaryotic systems [ ] . because of their small size; good tissue permeability; and cost-effective production, storage, and transportation [ ] [ ] [ ] , vhhs or nbs have been gaining acceptance as antiviral agents. a vhh complementary dna library was constructed with the bone marrow of dromedary camels infected with mers-cov. four vhhs (vhh- , vhh- , vhh- , and vhh- ) with high neutralizing activity were identified by direct cloning and screening of the phage display antibody library [ ] . the four vhhs competed for a single epitope that partially overlapped with the rbd-dpp interface. mutant analysis showed that the four vhhs did not bind to the d n variant, which was a critical residue on the rbd for dpp binding [ , ] . therefore, these vhhs most likely neutralized mers-cov by blocking its binding to dpp . of the vhhs, vhh- showed the best neutralizing activity and epitope recognition. vhh- efficiently blocked the entry of mers-cov into cells, and it also prophylactically protected k transgenic mouse expressing hdpp from mers-cov infection. to extend the half-life of vhh- in serum, it was linked to a human fc domain lacking the ch exon to construct the chimeric camel/human hcab- , which showed similar neutralizing activity as vhh- . the chimeric camel/human hcab- was highly stable in mice and provided k mice with fully prophylactic protection against mers-cov infection [ ] . alpacas were immunized with recombinant mers-cov rbd-containing a c-terminal human igg fc tag, and vhhs were amplified from their peripheral blood mononuclear cells to construct a vhh phage display library. a neutralizing nb, nbms , which bound with high affinity to the rbd of mers-cov and blocked the binding of rbd to dpp , was identified [ ] . to extend its in vivo half-life, the human-fc-fused version, nbms -fc, was constructed. nbms competed with dpp to bind to rbd, indicating that the binding site of nbms on rbd overlapped with that of dpp . the binding site of the nbms on the rbd was mapped to be around residue d , which is part of a highly conserved conformational epitope at the receptor-binding interface in almost all the natural mers-cov published to date. nbms did not neutralize psuedotyped mers-cov bearing a mutation in d , confirming that residue d was critical for nbms binding. nbms efficiently neutralized the cell entry of live mers-cov. moreover, nbms showed potent prophylactic and therapeutic efficacy in protecting hdpp -transgenic mice against mers-cov infection [ ] . for their exceptionally high neutralization activity in vitro and in vivo, these newly identified neutralizing mabs are promising candidate therapeutics against the infection of mers-cov. however, the use of a single neutralizing antibody bears the risk of selecting escape mutants, a fact that has been observed for lca and other described antibodies [ , , ] . notably, the majority of these escape mutations had little impact on viral fitness and the interaction of dpp with the rbd [ ] . moreover, mutants of mers-cov during natural infection have also been reported [ ] . escape from neutralization is a major concern with therapeutic neutralizing mabs, however, this potential problem can be solved by combining mabs that target distinct epitopes and show different neutralizing mechanisms [ ] . this strategy can take advantage of the synergistic effects while decreasing the possibility of viral escape. currently, most of the mers-cov neutralizing mabs compete with dpp binding to the rbd, and residues on the rbd critical for mab neutralization are identified by mutant analysis. almost all of the residues identified critical for mab neutralization are located in rbm, and overlap with those critical for dpp binding ( figure b) . with the availability of crystal structure of mab fab-rbd complex, the neutralization mechanism of these mabs will be better illustrated. based on the crystal structure of rbd-dpp , it was found that several conserved residues in the rbd are critical for the interaction of the rbd with dpp (y , l , d , e , e , d , d , y , r , w , and v ) [ , ] . development of therapeutic neutralizing mabs targeting those critically conserved residues might be important for combating mers-cov. moreover, a study found a mouse-derived neutralizing mab, f , which bound to a possible linear epitope in the ntd of the mers-cov s subunit, exhibited efficient neutralizing activity against pseudovirus and live mers-cov in cell entry tests. this study highlighted the important role of ntd during the infection process of mers-cov. ntd might have significant implications for the development of prophylactic and therapeutic mabs against mers-cov infection [ ] . although the in vitro neutralizing potency of f was approximately -fold lower than that of the rbd-targeting neutralizing mabs [ ] , it may provide an alternative for the immunotherapy against mers-cov, once the virus mutates and is no longer susceptible to rbd-specific mabs. so far, there is a lack of appropriate animal models to mimic the pathology of merd-cov in humans. commonly-used laboratory animals-such as wild-type mouse, ferret, hamster, and guinea pig-are not susceptible to mers-cov infection due to differences in critical amino acids in the s-binding domain of their dpp [ ] [ ] [ ] . new zealand rabbits, hdpp -transduced/transgenic mice, camelids and non-human primates (rhesus macaque and common marmoset) are susceptible to mers-cov infection, however, rabbits showed asymptomatic infection [ ] ; dromedary camels displayed different clinical manifestations to that of humans [ ] ; rhesus macaque only showed transient lower respiratory infection [ ] , 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neutralizing monoclonal antibody inhibition of middle east respiratory syndrome coronavirus replication in the common marmoset potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein structural definition of a unique neutralization epitope on the receptor-binding domain of mers-cov spike glycoprotein ultrapotent human neutralizing antibody repertoires against middle east respiratory syndrome coronavirus from a recovered patient a novel human mab (mers-gd ) provides prophylactic and postexposure efficacy in mers-cov susceptible mice naturally-occurring antibodies devoid of light-chains molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies nanobodies: natural single-domain antibodies application of camelid heavy-chain variable domains (vhhs) in prevention and treatment of bacterial and viral infections nanobodies(r) as inhaled biotherapeutics for lung diseases generation and characterization of alx- , a potent novel therapeutic nanobody for the treatment of respiratory syncytial virus infection chimeric camel/human heavy-chain antibodies protect against mers-cov infection molecular basis of binding between novel human coronavirus mers-cov and its receptor cd a novel nanobody targeting middle east respiratory syndrome coronavirus (mers-cov) receptor-binding domain has potent cross-neutralizing activity and protective efficacy against mers-cov importance of neutralizing monoclonal antibodies targeting multiple antigenic sites on mers-cov spike to avoid neutralization escape severe respiratory illness caused by a novel coronavirus identification of residues on human receptor dpp critical for mers-cov binding and entry a novel neutralizing monoclonal antibody targeting the n-terminal domain of the mers-cov spike protein wild-type and innate immune-deficient mice are not susceptible to the middle east respiratory syndrome coronavirus the middle east respiratory syndrome coronavirus (mers-cov) does not replicate in syrian hamsters adenosine deaminase acts as a natural antagonist for dipeptidyl peptidase -mediated entry of the middle east respiratory syndrome coronavirus asymptomatic middle east respiratory syndrome coronavirus infection in rabbits experimental infection of dromedaries with middle east respiratory syndrome-coronavirus is accompanied by massive ciliary loss and depletion of the cell surface receptor dipeptidyl peptidase an animal model of mers produced by infection of rhesus macaques with mers coronavirus infection with mers-cov causes lethal pneumonia in the common marmoset multi-organ damage in human dipeptidyl peptidase transgenic mice infected with middle east respiratory syndrome-coronavirus funding: this study was supported by a grant from national natural science funds of china (nos. ). the authors declare that they have no conflict of interest. key: cord- - o t am authors: guo, xiao; guo, huifen; zhao, lei; zhang, yao-hua; zhang, jian-xu title: two predominant mups, obp and mup , are male pheromones in rats date: - - journal: front zool doi: . /s - - - sha: doc_id: cord_uid: o t am background: in rats, urine-borne male pheromones comprise organic volatile compounds and major urinary proteins (mups). a number of volatile pheromones have been reported, but no mup pheromones have been identified in rat urine. results: we used sodium dodecyl sulphate-polyacrylamide gel electrophoresis (sds-page), isoelectric focusing electrophoresis (ief), nano-liquid chromatography-tandem mass spectrometry (nlc-ms/ms) after in gel digestion of the proteins and quantitative real-time pcr (qrt-pcr) and showed that the levels of two mups, odorant-binding protein (obp ) (i.e. pgcl ) and mup (i.e. pgcl ), in urine and their mrnas in liver were higher in males than in females and were suppressed by orchidectomy and restored by testosterone treatment (t treatment). we then generated recombinant mups (rmups) and found that the sexual attractiveness of urine from castrated males to females significantly increased after the addition of either recombinant obp (robp ) or recombinant mup (rmup ). using c-fos immunohistochemistry, we further examined neuronal activation in the brains of female rats after they sniffed robp or rmup . both robp and rmup activated the accessory olfactory bulb (aob), medial preoptic area (mpa), bed nucleus of the stria terminalis (bst), medial amygdala (mea), posteromedial cortical amygdala (pmco) and ventromedial nucleus of the hypothalamus (vmh), which participate in the neural circuits responsible for pheromone-induced sexual behaviours. in particular, more c-fos-immunopositive (c-fos-ir) cells were observed in the posterior aob than in the anterior aob. conclusions: the expression of obp and mup was male-biased and androgen-dependent. they attracted females and activated brain areas related to sexual behaviours in female rats, suggesting that both obp and mup are male pheromones in rats. particularly, an obp excreted into urine was exemplified to be a chemical signal. pheromones play a crucial role in mediating socio-sexual interactions between conspecific members in rodents [ , ] . in mice, pheromone components comprise both volatile organic compounds and non-volatile proteins, such as exocrine gland-secreted peptide (esp ) secreted by tear glands [ ] and darcin (mup ), a member of the major urinary proteins (mups) [ ] . mups are low-molecularweight (approximately kda) members of the large lipocalin family and are produced by the liver, filtered by the kidneys and secreted into the urine in some rodent species, such as mice and rats [ ] . in rats, mups are also referred to as alpha- u globulin (i.e. α u-globulins) [ ] . mups are highly polymorphic and encoded by at least mup genes on chromosome in mice and approximately genes and pseudogenes on chromosome in rats [ , ] . mups are usually more prevalent in males than in females in rodents, implying a male-biased trait [ ] . rodents use voided urine to extensively mark their territories for chemosensory communication [ ] . the mups in the deposited urine bind to and slow the release of volatile compounds, which is critical for the longevity of scent marks [ ] [ ] [ ] . however, mups themselves function as chemosignals to convey socio-sexual information for conspecific receivers and thus regulate socio-sexual interactions. these two roles of mups are both related to urine-mediated chemical communication [ ] . in rodents, pheromones acting as sexually selected traits are associated with sex, reproductive readiness and the quality of the signallers [ ] . sex pheromones are released by animals to elicit a sexual interaction with a member of the other sex of the same species and particularly mediate female mate choice for male mates in rodents [ , ] . the identification of male pheromones has been a major focus of studies of sexual selection for many years. currently, a few organic volatile compounds have been chemically characterized from mouse and rat urine and preputial glands and identified to be male pheromone components [ , , , ] . several mups have been characterized from male urine in mice, and some of them have been demonstrated to promote male-male territorial aggression, female attraction, conditioned place preference, or play a role in signaling individual identity [ , [ ] [ ] [ ] . in rats, about mups have been found in urine, including obp (pgcl ), mup (pgcl ), and pgcl [ ] [ ] [ ] [ ] [ ] . since the genes of the mup family share high sequence homology, the purification of mups from urine and expression of a single specific protein in vitro is difficult [ ] , and the role in intraspecific communication of each mup isoform in male urine, is rarely experimentally verified in rats [ , ] . all identified mouse and rat male pheromone components, including volatile compounds and non-volatile proteins, are male-specific or more prevalent in males than in females and regulated by androgen [ , , , ] . male pheromone components partially or completely restore the sexual attractiveness of castrated male urine to female rodents [ , , , ] . male pheromones are primarily received by females via the vomeronasal organ (vno), which then sends neuronal signals to the accessory olfactory bulb (aob). the aob directly targets higher centres in the brain, including the medial amygdala (mea), bed nucleus of the stria terminalis (bst) and ventromedial nucleus of the hypothalamus (vmh), to process the sensory information [ ] . the hypothalamus acts as a major source of neuroendocrine hormones that influence reproductive behaviour, and the activation of the hypothalamus by male pheromones arouses female sexual behaviour [ ] . in the vno, vomeronasal type receptors (v rs) are specifically expressed in the apical cell layer projecting to the anterior portion of the aob (aaob) and are responsible for receiving signals from volatile pheromones; vomeronasal type receptors (v rs) are mostly expressed in the basal region projecting to the posterior portion of aob (paob) and receive signals from non-volatile pheromones, such as esp and mups [ ] [ ] [ ] . in rats, several urinary volatile compounds, such as heptanone, -heptanone and -hydroxy- -nonanone, have been definitely identified as male pheromones through a combination of chemical and behavioural studies [ , ] . researchers have not directly shown which single mup isolated from rat mups may act as a male pheromone candidate, although a few mups that have been purified from urine are sexually dimorphic and positively correlated with quality, sexual attractiveness and copulatory opportunities in male rats [ , ] . among rat mups, mup (also referred to as alpha- u globulin pgcl , uniprotkb accession number: p ) has been isolated from rat urine and confirmed to be a kairomone, inducing fear reactions in mice [ ] . obp (odorant-binding protein , also referred to as alpha- u globulin pgcl , uniprotkb accession number: q e ), is named due to its nasal expression and odorant-binding characteristics, and has been demonstrated to be a member of the mup family by sequence comparison [ ] . questions were raised about whether mup and obp function as male pheromones for conspecific communication in rats. volatile pheromones and mups are usually speciesspecific and widely documented in laboratory strains and wild populations of mice and rats [ , , , , ] . as one of laboratory inbred rat strains, lewis rats have clearer genetic background and lower individual variation than the outbred and wild-captured rats have, and also have higher levels of volatile pheromones than most other laboratory rat strains, providing a good model to study rat pheromones [ , , ] . in the current study, we analysed mups in lewis rats using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (sds-page), quantitative real-time pcr (qrt-pcr), isoelectric focusing electrophoresis (ief), in gel digestion and nano-liquid chromatography-tandem mass spectrometry (nlc-ms/ms), and then generated rmups. we further examined the activity of rmups by testing whether these proteins elicited behavioural and neuronal responses in female rats. according to the results of the bradford protein assay, males had approximately ten-fold higher concentrations of total urine protein than females (females, . ± . mg/ml; males, . ± . mg/ml; p = . , t = . , n = , independent t-test) (fig. a) . similarly, on sds-page, mup bands of approximately kda (between the kda and kda markers) were more abundant than the other bands in both male and female urine (fig. b) , and the total mup level was significantly higher in males than in females (p = . , t = . , n = , independent t-test) (fig. c) . based on the ief results, male urine contained more mup protein bands than female urine. in particular, bands - were the three most abundant mups (fig. d) , which respectively accounted for % (approximately . mg/ml), % (approximately . mg/ml), and % (approximately . mg/ml) of the total urine protein. mup bands were detected using sds-page (fig. a) . mup levels were significantly decreased by castration but restored by t treatment (p < . , f = . ; t-treated vs. castrated, p < . ; t-treated vs. shamoperated control, p = . ; sham-operated control vs. castrated, p = . ; n = for each group, oneway anova with tukey's post hoc honestly significant difference (hsd) test) (fig. b ). according to a b c d fig. comparison of mup levels between males and females. a concentrations of total urine proteins in males and females were assayed using the bradford protein assay (**p < . , n = , independent-sample t-test). b mup levels in the urine ( μl of diluted urine samples) from male and female lewis rats (n = for each sex) were detected using sds-page with a marker of molecular weight ( . kda- kda, cw s, beijing comwin biotech co., ltd., china). one male sample was loaded on every gel as a standard to normalize the intensities of samples on different gels. the gel shown is a representative sample of four females and four males. c mup abundance was quantified in sds-page gels using the imagej program, and the data were shown as the mean ± standard error (se), n = (**p < . , independent-sample t-test). ; remaining samples were on another gel which is not shown. one sham-operated male urine sample was run as a standard on every gel, and its intensity was used to normalize all values. b the relative abundance of mups on sds-page gels was analysed using the imagej program (mean ± se, n = , **p < . , one-way anova followed by tukey's post hoc hsd test). c each mup in the urine samples ( μl of a in dilution) from different groups was detected using ief with a marker of pi values in d gel. (pi - , , , serva, germany. s: sham-operated, c: castrated, c + t: castrated + t-treated). the bands - were the three most abundant proteins in mups the ief results, the testosterone (t)-treated and shamoperated control groups shared a very similar ief band pattern, and almost all single mup bands, such as the mup bands - , were present in these two groups but were absent in the castrated group (fig. c) . the isoelectric points (pis) of the three most intense protein bands in ief were approximately . - . . mass spectrometric analysis identified the three proteins as mup , alpha- u globulin pgcl and odorant-binding protein (obp ). the whole protein sequence was used as expasy entry and the sequence coverage was . % (fig. a) , . % (fig. b ) and . % (fig. c) using a sequence alignment of the three proteins by danman, mup and alpha- u globulin pgcl displayed high similarity ( %), with only three different amino acids, and the three proteins had a sequence similarity of approximately %, with divergent amino acids when their signal peptides were excluded ( fig. d and e) . hepatic levels of the obp and mup mrnas are related to sex and androgen levels female rats showed significantly lower levels of the obp (p = . , z = . , n = , mann-whitney u-test) and mup (p = . , z = . , n = , mann-whitney u-test) mrnas in the liver than did their male counterparts (fig. a) . hepatic levels of both the obp and mup mrnas were significantly downregulated in the castrated group and restored in the t-treated group (obp : p < . , f = . ; sham-operated control vs. castrated, p = . ; t-treated vs. castrated, p < . ; t-treated vs sham-operated control, p = . ; mup : p = . , f = . ; shamoperated control vs. castrated, p = . ; t-treated vs. castrated, p = . ; t-treated vs sham-operated control, p = . ; n = for each group; one-way anova followed by tukey's post hoc hsd test) (fig. b ). in order to test the sexual attractiveness of obp and mup , we produced the recombinant proteins. the rmups had clean bands of approximately kda in sds-page (fig. a) , suggesting similar molecular weight to the native mups, and exhibited high sequence coverage by lc-ms/ms ( . % for robp , fig. b for rmup , fig. c ), which was sufficient to confirm the expression of correct proteins. two-way choice tests revealed that females spent more time investigating the urine from males in the shamoperated control group than from males in the castrated group (p = . , t = . , n = , paired t-test). according to the ief results, urine from intact males contained approximately . mg/ml obp and . mg/ml mup . when robp or rmup was added to urine from castrated males at the same physiological level observed in intact males, female rats showed greater attraction to treated than untreated urine from castrated males (robp , p = . , t = . ; rmup , p = . , t = . ; n = , paired t-test). no preference was observed between urine from males in the sham-operated control group and urine from treated-castrated males (sham-operated control vs. castrated + robp , p = . , z = . , n = , wilcoxon signed-rank test; sham-operated control vs. castrated + rmup , p = . , t = . , n = , paired t-test). in addition, females responded equally to the two treated urines (castrated + robp vs. castrated + rmup , p = . , z = . , n = , wilcoxon signedrank test) (fig. ). the number of c-fos-ir cells significantly increased in the aob of females in both robp -and rmup exposed groups compared with those in their respective control groups (robp , p < . , t = . ; rmup , p < . , t = . ; n = for each group, independent ttest) (figs. b and b). specifically, c-fos expression was increased in both aaob (robp , p < . , t = . ; rmup , p < . , t = . ; n = , independent t-test) and paob (robp , p < . , t = . ; rmup , p < . , t = . ; n = , independent t-test) (figs. c and c). in addition, more c-fos-ir cells were observed in the paob than in aaob in both the robp -and rmup exposed groups (robp , p < . , t = . ; rmup , p = . , t = . ; n = , paired t-test) but not in the pbs-treated control groups (control for robp , p = . , t = . ; control for rmup , p = . , t = . ; n = , paired t-test) (figs. c and c). furthermore, females exposed to robp presented a greater number of c-fos-ir cells in the mea, bst, medial preoptic area (mpa), vmh and posteromedial cortical amygdala (pmco) than the pbs-treated control females the accession number was based on uniprot database. description: the protein identified in the uniprot database. score: ion score of the identified protein from the uniprot database. coverage: sequence coverage calculated by dividing the number of matching amino acid residues by the total number of residues in the observed protein. unique peptides: the number of sequences from the identified peptides that differed in at least amino acid residue aas number of amino acids, mw molecular weight, pi isoelectric point a b fig. comparison of the hepatic expression of the mup and obp mrnas between the two sexes and among the three groups in the castration experiments. a differences in mup and obp expression in female and male rats (mean ± se, n = for each sex, **p < . , independent-sample t-test). b expression patterns of mup and obp among groups in the castration experiment (mean ± se, n = for each group, *p < . , ** p < . , one-way anova followed by tukey's post hoc hsd test) (s: sham-operated, c: castrated, c + t: castrated + t-treated) (mea, p = . , t = . ; bst, p < . , t = . ; mpa, p < . , t = . ; vmh, p < . , t = . ; pmco, p < . , t = . ; n = , independent t-test) (fig. e ). females exposed to rmup also displayed more c-fos-ir cells in these brain regions than the pbs-treated control females (mea, p < . , t = . ; bst, p < . , t = . ; mpa, p < . , t = . ; vmh, p < . , t = . ; pmco, p = . , t = . ; n = , independent t-test) (fig. e ). based on our sds-page, ief and qrt-pcr results, the total mup concentration and the concentrations of the identified mup , alpha- u globulin pgcl and obp proteins in voided urine exhibited a male-biased sexual dimorphism and depended on androgen in rats [ , [ ] [ ] [ ] . sexual dimorphism was also observed for total mups and darcin in mice [ , ] . however, total mup levels were more than ten-fold higher in males than in females in the present study, consistent with previous findings that the dichotomy is much more pronounced in rats than in mice [ , ] . serum testosterone exerts a stimulatory effect on mups, indicating that male-biased mups might be regulated by male hormones [ , ] . therefore, rat mups have the necessary features of chemicals used as male pheromones in rodents [ , ] . we selected three predominant protein bands from ief for identification by nlc-ms/ms. the three proteins were putatively identified as mup , alpha- u globulin pgcl and obp . these proteins have also been found in the voided urine of sprague-dawley rats, wistar rats and wild rats [ ] [ ] [ ] [ ] ] . considering the possible technical difficulties in isolating mup members and acquiring a single specific mup [ ] , we first investigated obp and mup , for which purification has been reported [ , ] . we generated rmup and robp according to previous studies [ , ] , and studied their biological functions. the sequencing results of the clones confirmed that the genes we amplified were mup and obp . the identities of the two recombinant proteins were further confirmed by lc-ms-ms assay. the combination of gel-based separation and mass measurements is often used to identify information regarding protein sequences, where proteoform-specific unique peptides analysed by high-resolution mass spectrometry are required to discriminate homologous protein superfamilies at the amino acid level [ ] . ief resolves proteins that differ in their pi values by as little as . . some studies have efficiently separated mups and characterized mups related to kinship and individual recognition in mice using ief [ , ] castrated and castrated + robp -treated or castrated + rmup -treated males, sham-operated and castrated + robp -treated or castrated + rmup -treated males and castrated + rmup -treated males and castrated + robp -treated males (n = for each group, mean ± se, **p < . , *p < . , paired t-test or wilcoxon signed-rank test) flight/time of flight (maldi-tof/tof) mass spectrometry in outbred wistar rats [ ] , and a unique malespecific mup resolved by narrowed-range ief was also characterized by ms/ ms in mouse urine [ ] . inbred lewis rats may express fewer mup isoforms than outbred wistar rats, and thus, ief is more manageable for separating mups from lewis rats [ ] . although more than one mup is contained in a single band/spot on a -de gel [ ] , a greater number of specific unique peptides with high sequence coverage are contributed by the most abundant proteins [ ] and are used for ms-based quantification to successfully identify proteins [ , ] . in our study, the considerable sequence coverage of the matching peptides ( . %, . % and . % for mup , alpha- u globulin pgcl and obp , respectively) was sufficient to demonstrate that these three bands in ief were the predominant mups. female preferences for synthetic analogues of putative male pheromones in two-way choice tests are often utilized for the experimental verification of the sexual attractiveness and identity of male pheromones [ , , ] . volatile male mouse pheromones, such as farnesenes, hexadecanol acetate and hexadecanol, and non-volatile male mouse pheromones, including esp and darcin, and volatile male rat pheromones, such as -heptanone, heptanone and -hydroxy- -nonanone, were previously a d e b c fig. male urine treated with robp induced c-fos expression in females. the c-fos-ir cells were distributed in the aob (a) and higher brain areas (d). (b), (c) and (e) showed the statistical analyses of the number of c-fos-ir cells in different brain regions (n = , mean ± se, **p < . , *p < . , independent-sample t-test for five brain areas, aob, aaob and paob between the pbs-treated control and experimental groups; paired t-test or wilcoxon signed-rank test for aaob and paob in pbs-treated control or experimental rats. scale bars = μm) identified using this methodology [ , , , , , ]. in the current study, female rats exhibited consistent chemosensory preferences for mup-enriched urine from males compared with that from castrated males; female rats also exhibited a preference for urine from androgen-treated castrated males compared with that from castrated males. in particular, the urine from castrated males supplemented with rmup or robp showed the same attractiveness to females as urine from intact males, suggesting that both mup and obp in male urine aroused female attraction and were male pheromones. besides lewis, we have also found in sprague-dawley and wild-captured rats that the mup and obp exhibited male-biased sexual dimorphism and were associated with sexual attractiveness to female rats (unpublished data), suggesting mup and obp identified in lewis male rats might be common male pheromones across strains or populations of rats. female sexual attraction is organized by the mutual interaction of sensory and limbic systems, particularly hypothalamic and amygdalar neurons, and produces a collection of pheromones that activate discrete inherent behaviours [ ] . male protein pheromone signals are received by the vno and transferred to central structures that regulate the behavioural or neuroendocrine responses of female rodents [ , , , ] . it has been found in wistar rats that mixed mups induced attraction and activated a d e b c fig. increased c-fos expression in females after sniffing rmup . expression of c-fos in the aob (a) and five different higher brain centres (d); relative statistical analyses of the data presented in (b), (c) and (e) (n = , mean ± se, **p < . , *p < . , independent-sample t-test for brain areas, aaob, paob and aob in pbs-treated control and treated groups, paired t-test or wilcoxon signed-rank test for aaob and paob from the pbs-treated control and treated groups. scale bars = μm) neurons in the posterodorsal medial amygdala of females [ ] . here, mup and obp activated the characteristic neural pathways (i.e., paob, mea, bst and vmh) of female rats in response to male protein pheromones, providing neural evidence for the identity of male pheromones. in particular, the activation of the hypothalamus suggested that mup /obp stimuli may be male pheromone eliciting female sexual attraction. mup served as a male pheromone that reliably aroused female sexual preference and the defensive responses of prey [ ] . likewise, esp , a male pheromone in tears, not only induces sexually receptive behaviours in females but also enhances male-male aggression in mice [ , ] . the multiple effects of pheromones on behaviour may be attributed to mea. the mea, receives inputs from the aob and mob (main olfactory bulb) and is an important centre that integrates all kinds of information and governs social behaviour. accurate pheromone-behaviour responses, such as sex and aggression, are analysed by mea and then controlled by the vmh [ ] . the genes of mup family are expressed in a complex sex-and tissue-specific manner [ , , , ] . they are highly expressed in male liver and the submaxillary, lachrymal, preputial glands in both sexes and also expressed in female mammary glands [ , ] . in rats, the mrna of mup is expressed in liver, preputial gland, and spleen, and the mrna of obp was found in liver, salivary gland, submaxillary gland and nasal cavity [ ] . mups enter urine via hepatic biosynthesis [ ] . the hepatic expression of rat mups is under regulation of multiple hormone, and the hepatic expression is sex-dependent and under developmental control [ , , [ ] [ ] [ ] . in the present study, the hepatic mrna levels of both mup and obp are decreased by castration, consistent with previous findings that castration decreases the synthesis of mups, and the hormonal control of mups synthesis is associated with hepatic mrna level [ , ] . however, obp , but not mup , was significantly higher in t-treated group than in sham-operated control group, suggesting that the expressions of obp was slightly different from mup in response to testosterone. the mrna of obp , as well as the volatile pheromones which we reported in previous work, was overexpressed in t-treated group [ ] . the total testosterone levels in serum were measured, and no significant difference was found between sham-operated control and t-treated group ( . ± . ng/ml vs. . ± . ng/ml, mean ± standard error, n = for each group). it remains to be determined whether the free testosterone and other testis-related hormones are excess and related to the overexpression of obp and volatiles. here, we first demonstrated that obp and mup were male pheromones in rats. they were characterized by male-biased sexual dimorphism, under androgen control and sexual attractiveness to females. also, they could activate brain areas related to sexual behaviors in female rats. since the members of mup family may carry different behavioral information, it is worthwhile to investigate pgcl and the other mups in future studies. in mice, the males of the same inbred strain share the same mup patterns in urine; whereas the mup patterns between strains may different, and the wild-caught mice showed wide individual variations in mup types and amounts of mups [ ] . here, we found that obp , mup and pgcl were consistently present in each lewis male. moreover, small individual variations were found within inbred lewis males, possibly due to spontaneous mutations, stochastic and environmental events or epigenetic changes as we have evidenced for the quantitative changes of volatile pheromones among inbred c bl/ mice [ ] [ ] [ ] . previous studies on rat urinary mups were carried out mainly with wistar, sprague-dawley and lewis strains of rats [ ] [ ] [ ] [ ] . however, the differences between the current work and previous work in experiment procedures and techniques make it difficult to precisely compare them. the variation in mups among different strains and wild populations remains to be further investigated. in our study, obp , mup and alpha- u globulin pgcl , three of the most abundant mups, were identified in rat urine. these three mups showed malespecific sexual dimorphism and androgen dependency, and may be putative pheromones. the attraction of females and the activation of neural pathways by mup and obp confirm that these two mups are both pheromones. thirty-three male ( . ± . g) and female ( . ± . g) lewis rats at the age of weeks were sexually naive and purchased from the beijing vital river laboratory animal technology co., ltd., china. males were housed individually in standard plastic rat cages ( × × cm), and females were housed in groups of four animals per cage. the housing room had a reversed : h light: dark photoperiod (lights on at : ) and was maintained at a temperature of ± °c. after weeks of acclimatization, the rats were used for urine collection, behavioural tests, surgical operations and immunocytochemistry. we examined the vaginal cytology to assess the oestrous stages of females, and only oestrous rats were used in all behavioral and immunohistochemical experiments. twenty-one male rats were randomly selected and assigned to three groups (n = for each group) that underwent a sham operation (sham-operated control group), bilateral orchidectomy (castrated group) and castration + t treatments, respectively [ ] . for the castrated group, we anaesthetized the subjects via an intraperitoneal injection with sodium pentobarbital ( mg/ kg) and then removed the bilateral testicles following an incision of the scrotum and ligation of the blood vessels and vas deferens. for the sham-operated control group, subjects were treated with the same surgical procedures, but the bilateral gonads were not removed. for the ttreated group, subjects were surgically castrated and immediately received subcutaneous implantation of a silastic tubing capsule filled with crystalline testosterone (length: mm, calibre: . mm, from dow corning corporation, usa). the tubes were placed in the posterior scapular region of the rats, and the other two groups were implanted with empty tubes. after a week postsurgical recovery period, urine was collected from all animals. for urine collection, we individually caged urine donors in clean metabolic rat cages with standard rat chow and water provided and kept them continuously to collect the urine samples for h daily during the dark phase of the light cycle. the urine from each metabolic cage flowed into a tube immersed in an ice box. standard rat chow and water were freely available. urine samples were stored at − °c until use. metabolic cages were washed thoroughly with water and sterilized between urine collections. the assay reagent was prepared as follows: mg of coomassie brilliant blue (cbb) g- was dissolved in ml of % ethanol and mixed with ml of % phosphoric acid, and the solution was then diluted to l with distilled water. after estimating the concentration of urine samples, each male urine sample was diluted -fold with . % nacl, and female urine was not diluted according to the assay's detection limit. the urine samples and assay reagent were mixed at a : ratio, and a spectrophotometer (beckman du , beckman, usa) was used to assay the absorbance of the mixed solution at nm. bovine serum albumin (fraction v, genview, usa) was used to construct a standard curve. three technical replicates were used for each urine sample and the urine protein concentrations were calculated using this standard curve [ , ] . sds-page was performed using a mini-protean system (bio-rad, usa). each urine sample was diluted -fold by adding distilled water and then mixed : with × sds loading-buffer ( mm tris-hcl (ph . ), % (w/v) sds, . % (w/v) bromophenol blue, % (v/v) glycerol, and % (v/v) β-mercaptoethanol). the mixed protein samples were boiled at °c for min, slightly spun down and then resolved in sds-page. two microliters of the mixed samples and μl of protein markers were fractionated on % sds-page gels at a constant voltage of v. protein gels were stained with cbb and imaged using a chemidoc mp system (bio-rad, usa). the intensity of each band was measured as the relative abundance of the protein using the imagej program (national institutes of health, usa), according to the manual (http:// rsbweb.nih.gov/ij/docs/user-guide.pdf) [ , ] . one of the male was selected as a standard and carried along on every gel to correct for differences between runs, and all values of the samples on different gels were normalized to the detected intensity of the standard sample. ief was performed in a bio-rad model mini ief cell apparatus. a % solution of ampholytes at ph - and ief standards with pi ranging from to (both from serva electrophoresis gmbh, germany) were separated on polyacrylamide gels, according to the manufacturer's instructions. seventy microliters of rat urine were de-salted with zeba spin desalting columns (pierce, usa), freezedried in a vacuum freeze dryer (alpha - ld plus, martin christ, germany) and dissolved in μl of deionized water. two microliters of standards and μl of a : dilution of protein samples were focused at v for min, v for min, and v for h, and then visualized with cbb staining [ ] . the gels were imaged using a chemidoc mp system (bio-rad, usa). we excised each of the three predominant bands from an ief gel of urine samples, cut the gel slice into mm pieces and transferred them to . -ml microcentrifuge tubes. we washed each gel piece with μl of mm ammonium bicarbonate (nh hco ) for min and dehydrated the sample with % acetonitrile (acn) for min. we rehydrated the gel pieces by incubating them with μl of mm dithiothreitol at °c for min and then with μl of % acn for - min before the gel pieces were placed in μl of mm iodoacetamide and incubated in the dark for min. after discarding the supernatant, each gel piece was washed with μl of mm nh hco and then with μl of % acn for min. the gel pieces were dried in a speed-vac evaporator (thermo scientific, usa) for min and digested with ng/μl trypsin (sequencing grade, promega, usa) in mm nh hco overnight at °c. supernatants were collected in fresh . -ml tubes on the following day, and the remaining peptides were extracted from the gel pieces in μl of a solution comprising % acn, % trifluoroacetic acid and % deionized water via sonication for min. the tubes were centrifuged for s, and supernatants were collected. all supernatants from each band were pooled, dried in a speed-vac evaporator for min, and mixed with μl of a solution comprising . % acn, . % trifluoroacetic acid (tfa) and . % deionized water. the final supernatants were obtained, underwent sonication for min and centrifugation, and were stored at − °c until use. unless stated otherwise, all procedures were performed at room temperature [ , ] . the extracted peptides were analysed using an easy-nlc system (thermo scientific, usa) coupled to a q-exactive mass spectrometer through an easy-spray nano-electrospray ionization source (thermo scientific, usa). the peptide mixtures were loaded on a trap column (acclaim pepmap , μm × cm, nano viper c , μm, Å) and separated on an analytical column (acclaim pepmap rslc μm × cm, nano viper c , μm, Å) (both thermo fisher scientific, usa) using mobile phases containing solutions a ( . % formic acid in h o, chromatographic grade) and b ( . % acn and . % formic acid). the linear gradient of buffer b increased from % to % in the first min, to % in the next min and was then sustained for min at a flow rate of nl/min. the eluted peptides were electrosprayed into the mass spectrometer, and raw ms data were acquired in the data-dependent acquisition mode. the proteome discoverer software system (version . . . ; thermo scientific, usa) was used to analyse the raw data and score peptides for identification. a search of the ms/ms data against the rattus norvegicus database (swissprot, ) was performed using the built-in sequest-ht search engine. the mass deviation of the precursor ion, the fragment ion tolerance and the false discovery rate were set to ppm, . da and . , respectively. the minimal peptide length was amino acids, and two missed trypsin cleavage sites were allowed. the acrylamide modification of cysteines and methionine oxidation were set as fixed and dynamic modifications, respectively. when the search results were filtered, the matching proteins with a high peptide confidence for at least one unique peptide were considered significant [ ] [ ] [ ] [ ] [ ] . the pis of the identified proteins were calculated using expasy-compute pi/mw (https://web. expasy.org/compute_pi/), and danman software (version . , lynnon biosoft, usa) was used to align the protein sequences. the mass spectrometry proteomics data have been deposited to the proteomexchange consortium via the pride [ ] partner repository with the dataset identifier pxd . the trizol method (life technologies, usa) was used to extract the total rna from the rat liver, and the prime-script® rt reagent kit with a gdna eraser (takara bio, japan) was employed to reverse-transcribe cdnas, according to the manufacturer's instructions. then, qrt-pcr was conducted with the sybr green superreal premix plus kit (tiangen biotech co., ltd., beijing, china) and mx p quantitative pcr system (stratagene, la jolla, ca, usa). specific oligonucleotide primers were designed using the ncbi-primer-blast online tool (https:// www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?link_-loc=blasthome), and gapdh was chosen as a control gene. the primers were designed as follows: mup -f: ′-gggaacct cgatgtggctaa- ′, mup -r: '-gcac tctccattttccttaatacg- ′, obp -f: ′-gt tgcc gacaaaacagcaaag- ′, obp -r: ′-aaggatgaag aaatagat cttgccc- ′ and gapdh-f: '-gacaatg aatatggctaca gcaac- ′, gapdh-r: ′-tttattg atg gtattcgagagaagg- ′. the μl reaction included μl of × superreal premix plus, μl of cdna template, . μl of ( μm) of each primer, . μl of × rox reference dye△ and . μl of rnase-free water. the amplification reaction was performed under the following conditions: denaturation for min at °c, followed by cycles of °c for s, °c for s and °c for s. the temperature range for the melting curve analysis was °c to °c over s. the data were analysed using the −△△ct formula [ ] . the rat obp cdna (accession number: q e ) was amplified by pcr from male lewis liver using primer-f/ex/ndei: '-ggaattccatatggaagaagctagt ttcga gagag- ′ and primer-r/ex/xhoi: '-ttccg ctcgagtcaggcctggagacagcgatc- ′, containing ndei and xhoi restriction enzyme sites, respectively. this amplicon was double digested with ndei and xhoi and cloned into the pet a expression vector (novagen, madison, wi, usa). the plasmid construct was transformed into e. coli rosetta competent cells (novagen, usa). the positive clones were selected and sequenced using the universal primers (t promoter and t terminator primer). analysis of the clone sequences was performed by ncbi blast (https://blast.ncbi.nlm.nih.gov/blast.cgi). the robp protein was purified using hispur cobalt superflow agarose ( , , thermo fisher scientific, usa) according to the manufacturer's instructions [ ] . the rat mup cdna (accession number: p ) was purified using the same procedure and the following primers: f−/ex/ndei: '-gggtt tcatatgcatgcagaagaagctagttccacaaga g- ′ and r−/ex/xhoi: '-ccgctcgagtcctcgggc ctggagacag- ′. purified robp and rmup were pooled, dialyzed against × pbs buffer and stored at − °c until use. sds-page was used to assess the purification of robp and rmup , and nlc-ms/ms was used to verify the identity of these two recombinant proteins. twenty-four female rats were used as scent recipients and randomly assigned to two groups to test the sexual attractiveness of robp and rmup . all behavioural tests were double blind two-way choice tests and conducted during the dark phase of the photoperiod in a separate room with a dim red light [ ] . one female subject was left in the home cage while its cage mates were temporarily moved to another clean rat cage. each subject was allowed a min acclimation period prior to a trial and was used once every days in a balanced order. urine samples from individuals were mixed equally for each group. the mixed samples were used to prepare scent stimuli, and robp and rmup were added to urine from castrated males at the same levels observed in intact males. we painted a -μl scent sample on one end of the glass rod ( cm long, mm diameter) and held the other end with plastic gloves. two glass rods painted with different scent samples were simultaneously poked through the lid of the cage in the centre of the cage and were located . cm from each other to allow the subject to freely investigate each sample. the investigation time was recorded for min after the subject started to sniff or lick the rod. each female subject was caged in a clean rat cage and housed in a separate room with an air ventilation system for days of acclimation prior to use. we dissolved rmup and robp in × pbs at concentrations equal to the levels detected in urine from intact males to prepare scent stimuli. we painted a glass slide with μl of either rmup or robp for the experimental group or × pbs for the control group (pbs-treated control group). the slide was fixed on the wire bar lid of the cage. the protein product (fos) of the immediate early gene c-fos was used as a marker of neuronal activation and the number of c-fos-immunoreactive (c-fos-ir) cell indicated the activation levels [ ] . since the c-fos protein synthesis follows mrna accumulation and could be detected by immunohistochemistry at to min post stimulation [ , , ] , the subject was allowed to freely investigate the sample for min. the subject was anaesthetized with an intraperitoneal injection of sodium pentobarbital ( mg/kg) and perfused with ml of . % saline through the left ventricle, followed by - ml of % paraformaldehyde (pfa) in . m pbs via an infusion pump ( ml/min, longer, uk). the brain and olfactory bulb were removed, postfixed with % pfa overnight, dehydrated with % sucrose in pbs until they fell to the bottom of the container, respectively sliced into μm coronal sections and μm sagittal sections using the freezing microtome (leica, germany), and stored in . m pbs. brain sections including the bst, mea, pmco, mpa and vmh into which axons from the aob project [ ] , and sections of olfactory bulb containing the aob were selected after referencing the rat brain atlas [ ] . freefloating brain sections were selected at μm intervals and serial sections of the olfactory bulb were chosen for c-fos immunohistochemistry. briefly, sections were blocked with % normal goat serum in pbs-t ( % goat serum in . m pbs with . % triton x- ) for h and incubated with a primary antibody ( : dilution) against c-fos (ab , abcam, uk) for two nights at °c . we further incubated the sections with a biotinylated goat anti-rabbit secondary antibody ( : ) for h and with vector elite abc complex (both from vector laboratories inc., usa) for min. sections were stained with a dab kit (vector laboratories, usa) for - min. the number of c-fos-ir cells was counted using image-pro plus . software (media cybernetics, inc., usa) [ ] . we used the kolmogorov-smirnov test to examine the distribution of raw data and used either nonparametric tests or parametric tests in the subsequent analyses. independent-sample t-tests or mann-whitney u-tests were used to determine the differences in the density of c-fos-ir cells in each brain area between the control (pbs-treated) and mup-stimulated groups and to test the sex-specific differences in urinary mup levels and hepatic mrna levels. one-way anova followed by tukey's hsd tests were applied to examine the effect of the hormone status on mup protein and mrna levels. paired t-tests or wilcoxon signed-rank tests were used to determine the female preferences in the two-way choice tests and the differences in the density of c-fos-ir cells between the aaob and paob. all statistical analyses were conducted using spss (v . , spss inc.). significance was set to p < . . pheromonal communication in vertebrates the male mouse pheromone esp enhances female sexual receptive behaviour through a specific vomeronasal receptor darcin: a male pheromone that stimulates female memory and sexual attraction to an individual male's odour the major urinary protein system in the rat major urinary proteins, alpha ( u)-globulins and aphrodisin genomic organization of the rat alpha ( u)-globulin gene cluster species specificity in major urinary proteins by parallel evolution urinary proteins and the modulation of chemical scents in mice and rats individual recognition in mice mediated by major urinary proteins pheromone binding by polymorphic mouse major urinary proteins pheromones, binding proteins and receptor responses in rodents detection of alpha ( u)-globulin and its bound putative pheromones in the preputial gland of the indian commensal rat (rattus rattus) using mass spectrometry a male pheromone-mediated trade-off between female preferences for genetic compatibility and sexual attractiveness in rats pheromones and animal behavior: chemical signals and signatures dual role of preputial gland secretion and its major components in sex recognition of mice sex-and gonad-affecting scent compounds and male pheromones in the rat identification of protein pheromones that promote aggressive behaviour pheromonal induction of spatial learning in mice murine pheromone proteins constitute a context-dependent combinatorial code governing multiple social behaviors differentiation of protein species of alpha- u-globulin according to database entries: a half-theoretical approach temporal variations of the postnatal rat urinary proteome as a reflection of systemic maturation unveiling the rat urinary proteome with three complementary proteomics approaches proteomic identification of a large complement of rat urinary proteins the vomeronasal organ mediates interspecies defensive behaviors through detection of protein pheromone homologs regulation of highly homologous major urinary proteins in house mice quantified with label-free proteomic methods sexual attractiveness in male rats is associated with greater concentration of major urinary proteins positive identification of the puberty-accelerating pheromone of the house mouse: the volatile ligands associating with the major urinary protein pheromone detection mediated by a v r vomeronasal receptor olfactory receptors, vomeronasal receptors, and the organization of olfactory information divisions of the accessory olfactory bulb to the medial amygdala in the opossum, monodelphis domestica changes in male rat urinary protein profile during puberty: a pilot study identification of a third rat odorantbinding protein (obp ) limited variation in the major urinary proteins of laboratory mice exaggerated male pheromones in rats may increase predation cost preparation and characterization of a sex-dependent rat urinary protein. bba-gene subjects proof of the hepatic synthesis of a sex-dependent protein in the rat effects of sex hormones on the level of the messenger rna for the rat hepatic protein alpha u globulin differential, multihormonal regulation of the mouse major urinary protein gene family in the liver different forms of alpha u-globulin in male and female rat urine rodent urinary proteins: genetic identity signals and pheromones interspecific chemical signals and behavioral interactions between rattus tanezumi and r. norvegicus. msc thesis a label-free mass spectrometry method for the quantification of protein isotypes structural and functional differences in isoforms of mouse major urinary proteins: a male-specific protein that preferentially binds a male pheromone the direct assessment of genetic heterozygosity through scent in the mouse on the saliva proteome of the eastern european house mouse (mus musculus musculus) focusing on sexual signalling and immunity identification of mouse liver proteins on twodimensional electrophoresis gels by matrix-assisted laser desorption/ ionization mass spectrometry of in situ enzymatic digests sex-specific peptides from exocrine glands stimulate mouse vomeronasal sensory neurons genetic analysis of brain circuits underlying pheromone signaling segregated pathways to the vomeronasal amygdala: differential projections from the anterior and posterior divisions of the accessory olfactory bulb self-exposure to the male pheromone esp enhances male aggressiveness in mice a nose by any other name (should smell as sweetly) molecular evidence of complex tissue-and sex-specific mrna expression of the rat α u-globulin multigene family tissue-specific expression of the rat alpha u globulin gene family regulation of proteins by thyroid hormone and glucocorticoid: the responses of hepatic α u-globulin and pituitary growth hormone differ in adult male hypothyroid rats age-dependent regulation of the polymorphic forms of alpha u-globulin hydrocarbon-induced hyaline droplet nephropathy in male rats during senescence individuality and transgenerational inheritance of social dominance and sex pheromones in isogenic male mice genetic variation in coding regions between and within commonly used inbred rat strains genetic and epigenetic variation among inbred mouse littermates: identification of inter-individual differentially methylated regions social dominance-related major urinary proteins and the regulatory mechanism in mice rapid method for protein quantitation by bradford assay after elimination of the interference of polysorbate primary structural documentation of the major urinary protein of the indian commensal rat (rattus rattus) using a proteomics platform dynamics of the interaction between cotton bollworm helicoverpa armigera and nucleopolyhedrovirus as revealed by integrated transcriptomic and proteomic analyses mass spectrometry-based proteomics using q exactive, a high-performance benchtop quadrupole orbitrap mass spectromete dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc the q exactive hf, a benchtop mass spectrometer with a pre-filter, high-performance quadrupole and an ultrahigh-field orbitrap analyzer inhibition of melanization by serpin- and serpin- promotes baculovirus infection in cotton bollworm helicoverpa armigera update of the pride database and its related tools analysis of relative gene expression data using real-time quantitative pcr and the (−delta delta c (t)) method c-fos and related immediate-early gene-products as markers of activity in neuroendocrine systems the c-fos protein immunohistological detection: a useful tool as a marker of central pathways involved in specific physiological responses in vivo and ex vivo accessory olfactory bulb function is modulated by input from the main olfactory epithelium the rat brain in stereotaxic coordinates. qingchuan zhuge translate. beijing: people's medical publishing house agonistic encounters and brain activation in dominant and subordinate male greater long-tailed hamsters we thank jinhua zhang for providing animal care and assistance with behavioural tests, hongling guo for early assistance with the mup analysis, lianfeng zhao for correcting the figure layout, zuoxin wang for providing valuable comments on the data, wei yao for assistance with the c-fos immunohistochemistry and xiaowei qin for the use of the nlc-ms/ms system. the ms raw files and the search engine result files for the three ief bands are available in the pride repository (pxd , http://proteomecentral. proteomexchange.org). all the other data generated or analysed during this study are included in this published article.authors' contributions jxz and yhz conceived and designed the experiments. hfg initiated the mups' analysis. yhz performed the surgeries and collected behavioural data. xg, hfg and lz performed braford protein assay, sds-page, ief, in-gel digestion, nlc-ms/ms, qrt-pcr and c-fos immunocytochemistry. xg identified the proteins, analysed the data, and made the figures. xg, yhz and jxz wrote the paper. all authors read and approved the final manuscript. the procedures for animal care and use were performed in strict accordance with the guidelines of the animal use committee of the institute of zoology, chinese academy of sciences (ioz ). not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -bdpp qmf authors: lanzavecchia, antonio title: dissecting human antibody responses: useful, basic and surprising findings date: - - journal: embo mol med doi: . /emmm. sha: doc_id: cord_uid: bdpp qmf human memory b cells and plasma cells represent a rich source of antibodies that have been selected in response to human pathogens. in the last decade, different methods have been developed to interrogate the human memory repertoire and isolate monoclonal antibodies. i will discuss how a target‐agnostic approach based on high‐throughput screening of antibodies produced by cultured b cells and plasma cells has not only provided potent and broadly neutralizing antibodies against a range of pathogens, but has also advanced our understanding of basic aspects of the immune response, from host–pathogen interaction to the role of somatic mutations in affinity maturation and in the diversification of the antibody response. most surprisingly, this approach has also revealed a new mechanism of diversification based on templated insertion of non‐ig dna into antibody genes that we discovered in the context of the immune response to malaria infection. human memory b cells and plasma cells represent a rich source of antibodies that have been selected in response to human pathogens. in the last decade, different methods have been developed to interrogate the human memory repertoire and isolate monoclonal antibodies. i will discuss how a target-agnostic approach based on highthroughput screening of antibodies produced by cultured b cells and plasma cells has not only provided potent and broadly neutralizing antibodies against a range of pathogens, but has also advanced our understanding of basic aspects of the immune response, from host-pathogen interaction to the role of somatic mutations in affinity maturation and in the diversification of the antibody response. most surprisingly, this approach has also revealed a new mechanism of diversification based on templated insertion of non-ig dna into antibody genes that we discovered in the context of the immune response to malaria infection. a trove of antibodies m emory b cells represent the repository of the immune experience of an individual. they are selected in the germinal centres, where they undergo a process of somatic mutations and selection by antigen and t helper cells. memory b cells persist for a life time and can rapidly respond to a booster immunization by generating large numbers of plasma cells that are transiently present in the blood and localize to surviving niches in the bone marrow as long-lived plasma cells that are the main source of serum antibodies. the interrogation of memory b cell and plasma cell repertoires of humans offers unique opportunities. as compared to mice, humans are naturally exposed to their own pathogens, often through recurrent infections, and are therefore a better source of antibodies that target the most relevant antigens and can reveal mechanisms of host-pathogen interaction. furthermore, given the genetic variability of the human population and the idiosyncrasies of the immune response, some individuals may generate antibodies with unusual potency or breadth. finally, studies of the antibody response in humans can shed light on basic aspects of the immune response, such as the role of somatic mutations and the mechanisms that underpin the generation of broadly neutralizing antibodies or autoantibodies, and possibly uncover examples of such mechanisms that may be uniquely developed or better identified in humans. a target-agnostic approach to identify functional antibodies in the last decade, several methods have been developed to isolate human monoclonal antibodies. in principle, they come down to two basic approaches. the first approach consists of the isolation of single antigen-specific b cells using a fluorescently tagged antigen as a bait, followed by cloning of immunoglobulin genes and expression of the recombinant antibodies in a cell line. this method is very effective, but requires that the target antigen is known and available in a purified form. in my laboratory, we developed an alternative approach based on high-throughput screening of the antibodies produced by cultured b cells or plasma cells (fig a) . memory b cells from selected donors are immortalized with high efficiency (> %) by combining epstein-barr virus (that delivers signal and signal ) with a tlr or tlr agonist (that delivers signal required for potent activation of memory b cells) (traggiai et al, ) . the immortalized b cells proliferate and secrete large amounts of antibodies in the culture supernatant. antigen-specific plasma cells recovered following a booster immunization have lost proliferative capacity, but can be kept alive in cultures supplemented with il- and stromal cells. in these cultures that mimic the survival niches of the bone marrow, a single plasma cell secretes antibodies at a constant rate of pg/day (corti et al, ) . to facilitate the isolation of specific antibodies and the cloning of the vh/vl genes, both immortalized b cells and plasma cells are seeded in clonal conditions and cultures are handled with an automated liquid handling system. there are distinctive advantages in screening the repertoire of secreted antibodies. the first is that the approach is target-agnostic, since screening can be done using functional assays, such as virus neutralization, without a priori knowledge of the target antigens, using even whole bacteria or parasites. second, it is well suited to identify antibodies that cross-react with different antigens, since parallel screenings can be performed against multiple targets. third, it bypasses the need to sequence and express large numbers of antibody sequences since cells of interest are selected based on the initial screenings. a relevant example of the utility of the target-agnostic approach comes from a study of the antibody response to human cytomegalovirus (hcmv), a complex herpesvirus expressing different surface glycoproteins that causes serious pathology in the foetus and in immunocompromised patients. by screening for the capacity to neutralize the wild-type virus, we isolated antibodies that were , -fold more potent than antibodies to the fusion protein gb and identified their viral target as a pentameric complex formed by gh, gl, pul , pul and pul a. we then produced a soluble form of this pentamer and found that it can elicit, in mice, neutralizing antibody titres that exceeded by more than -fold those induced in humans by natural infection (kabanova et al, ) . these studies illustrate a general approach of "analytic vaccinology" where the neutralizing antibodies are used for the identification of the target antigen and for the quality control of the recombinant vaccine. the possibility of performing parallel screening against multiple targets was instrumental for the identification of broadly reactive antibodies capable of neutralizing multiple viruses. these include the antibodies fi and fy , which bind to conserved epitopes in the stem region of influenza hemagglutinin (ha) and neutralize all influenza a viruses (corti et al, ; kallewaard et al, ) , and mpe , an antibody that binds to a unique conserved site on the pre-fusion form of the f protein and neutralizes four different paramyxoviruses, including human rsv and mpv (corti et al, ) . these antibodies are promising candidates for prophylaxis and therapy of infections since they trigger effector functions and target conserved structures, thus limiting the possibility of selecting escape mutants. in addition, they represent useful tools for the design of stabilized pre-fusion glycoprotein vaccines. a straightforward application of the method lies in the rapid isolation of neutralizing antibodies against emerging pathogens, such as ebola virus, sars (traggiai et al, ) and mers coronaviruses. in the case of mers, it was possible, in only months, to go from a sample of memory b cells to preclinical validation of a neutralizing antibody and the generation of a cell line producing the antibody in grams/l amounts. similar studies on dengue and zika viruses defined different classes of antibodies endowed with neutralizing or infection-enhancing activity. the interrogation of the memory b-cell repertoire offers the opportunity to investigate, in biologically and medically relevant settings, fundamental aspects of the human immune response, such as the relative contribution of germ line v-gene polymorphism, vdj junctional diversity and somatic mutations to antigenic specificity. an interesting example comes from the study of antibodies that neutralize group influenza viruses and that are produced by most individuals following infection or vaccination. as reported by several laboratories, these "public" antibodies use vh - and bind, exclusively through the vh, to a conserved region in the ha stem. by isolating a large number of sister clones and by performing a genealogical analysis of different clonal families, we found that the unmutated common ancestors (uca) have low affinity for group ha and do not neutralize infection (fig b and c) . remarkably, the first branch points acquire through a single p aa mutation in hcdr , high affinity and neutralizing activity, comparable to that of the antibodies isolated from memory b cells that carried up to - amino acid substitution (pappas et al, ) . these findings demonstrate that affinity maturation can be achieved rapidly, through a single mutation, and that numerous and redundant somatic mutations continue to accumulate, providing an extensive diversification within the proliferating clone. interestingly, in these antibodies, a critical contact residue, f , is germline-encoded, but this position is dimorphic, with different alleles carrying either f or l. consequently, individuals who lack a f -encoding allele cannot produce the public vh - antibody response, while individuals that have a f allele have a high frequency of precursors, with the only additional requirement being a -amino acid hcdr with a y that makes a critical contact site with the antigen. these findings underline the impact that vh-gene polymorphism can have on the antibody response and suggest that certain alleles may play the role of immune response (ir) genes and become positively selected in the population exposed to a given pathogen. the genealogical analysis of the two paninfluenza neutralizing antibodies, fi and fy , showed that the corresponding ucas have already high binding affinity and neutralizing activity, but only on group viruses and acquire the capacity to neutralize group viruses through somatic mutations (kallewaard et al, ) . these findings are consistent with a model where pan-influenza neutralizing antibodies arise from the priming of high-affinity precursors by a group virus (in this case h n ), followed by somatic mutations and generation of a variant with broader reactivity to group viruses that can be boosted by a group virus such as h n (fig d) . importantly, the isolation of cells producing the identical fi antibody for consecutive years in response to vaccination or infection indicates that secondary antibody responses are to a large extent independent from germinal centres, a factor that allows the antibody to retain the original specificity. the genealogical analysis of the neutralizing antibody mpe revealed a similar mechanism, with the uca possessing high affinity for rsv only and mpe acquiring cross-reactivity to mpv through somatic mutations (corti et al, ) . the extensive antibody diversification observed in humans can increase antibody affinity and promote breadth, but comes at the price of generating many new specificities that may cross-react with self-antigens. the notion that autoantibodies can be generated by somatic mutations is supported in humans by the finding that the ucas of autoantibodies specific for desmoglein or gm-csf found in patients with severe autoimmune diseases do not bind to the respective self-antigens (di zenzo et al, ; piccoli et al, ) . thus, it is tempting to speculate that these autoantibodies may have been generated by somatic mutations in b-cell clones responsive to a foreign antigen in the absence of structural mimicry. importantly, the mechanisms of deletion or anergy that restrain naïve autoreactive b cells may not apply to activated/memory b cells, which have different triggering requirement and can traffic to peripheral tissues. as mentioned above, the target-agnostic approach is especially suited to dissect the antibody response to complex pathogens in the absence of a detailed knowledge of the target antigens. we were interested in identifying antibodies that could broadly recognize the variant surface antigens (vsas) of plasmodium falciparum, the parasite causing malaria, which are expressed on the surface of infected erythrocytes (ies). vsas mediate adhesion of ies to endothelia and are targets of antibodies that control disease, but their high number (> genes), their extensive polymorphism and their clonal expression provide the pathogen with a powerful chameleon-like escape strategy. out of a large kenyan cohort, we initially selected two individuals with serum antibodies that cross-agglutinated erythrocytes infected by different p. falciparum strains. from their memory b cells, we isolated a panel of broadly reactive monoclonal antibodies using staining of infected erythrocytes as a screening strategy. surprisingly, all the broadly reactive antibodies had a unique structure, since they carried a large insert between the v and the dj segments. the inserts, of approximately bp, comprised the exon encoding the extracellular domain of the collagen-binding inhibitory receptor lair /cd , encoded in the leucocyte receptor cluster on chr. (tan et al, ) . given the insertion between v and dj, the lair domain is positioned at the tip of the hcdr (fig a and b) . the target antigens of lair -containing antibodies were identified in some members of the rifins, a family of polymorphic vsas expressed on the surface of ies (fig c) . importantly, the inserted lair domain was both necessary and sufficient for binding to rifins, as demonstrated by its insertion into an irrelevant antibody or by the production of lair -ig fusion proteins. to investigate how frequently lair containing antibodies are produced, we screened two additional cohorts from mali and tanzania and found that up to % of the individuals have such antibodies in the serum (pieper et al, ) . this study led not only to the isolation of more lair containing monoclonal antibodies, but also to the finding of a new insertion modality where the lair exon with and intronic sequences is inserted into the immunoglobulin switch region. this insertion leads, by exon shuffling, to the production of the original antibody and of a bispecific antibody containing the lair domain precisely placed at the elbow between the vh and ch domains (fig a and b) . in one case, the lair insertion in the switch region was accompanied by the genomic deletion of vh and ch , generating a "camel-like" lair containing antibody. in each individual analysed so far, the lair -containing antibodies dominate the response to ies and are produced by a single b-cell clone that contains a unique insert and undergoes extensive expansion and somatic mutations, thus demonstrating an extraordinary fitness. somatic mutations in lair were more frequent when the insertion was at the v-dj junction, which is continually targeted by aid in germinal centres, and less frequent when the insertion was in the switch region, which is only transiently targeted by aid during switch recombination. interestingly, somatic mutations in the lair domain eliminate collagen binding and modulate affinity and cross-reactivity with different rifins. the finding that collagen binding was always lost, even when the somatic mutation mechanism was less effective, suggests that loss of self-reactivity is a prerequisite for expansion of b cells making lair -containing antibodies. the insertion of non-vdj sequences encoding structured domain into ig genes is a new and potentially general concept. our finding was facilitated by the huge expansion of lair -containing b cells and by the unique properties of the antibodies made. in an initial attempt to determine the generality of this phenomenon, we found frequent insertions in the switch region of memory b cells from european blood donors. interestingly, the inserts largely originate from genes that are transcribed in b cells and are encoded in virtually all chromosomes and, in rare cases, comprised exons with frames compatible with expression ( fig d) . malaria infection can cause chromosomal instability and translocations involving the switch region, suggesting that it may also play a causative role in the generation of templated insertions. however, the finding of frequent templated insertions in european blood donors is more consistent with a general mechanism that remains to be molecularly defined. the sites of insertion suggest a mechanism of patch-repair of dna double-strand breaks induced by either rag or aid during v-dj recombination or class switch. the integrity of the genomic lair loci in b-cell clones producing lair containing antibodies suggests a copy-andpaste mechanism. in view of the finding that the inserts contain intronic sequences and are derived from expressed genes, we favour the hypothesis that the templated dna may originate from the resolution of stalled replication forks caused by a clash between transcription and dna replication. our findings suggest that the insertion of templated dna represents a novel and general mechanism of antibody diversification that can provide a broad range of protein domains to be further diversified by somatic mutations. as exemplified by lair , the insertion of a domain encoding a pathogen receptor can generate public antibodies that are effective against the pathogen. distinctive advantages of these receptor-based antibodies lie in the broad recognition of all pathogen variants, leaving no room for the selection of escape mutants, and in the possibility of diversifying the receptor domain through somatic mutations to increase binding to pathogen and decrease self-reactivity. the new antibodies are reminiscent of ehrlich's "side-chain" theory of antibody production, where the antibody was essentially a secreted form of the receptor for the pathogen. we predict that other examples of receptor-based antibodies will be found in response to a variety of human pathogens. besides lair , other candidates for the generation of receptor-based antibodies to p. falciparum are icam , an adhesion molecule that binds to ies, and lilrb , an inhibitory receptor that, like lair , has been shown to bind to rifins. similarly, insertions of icam or slam domains may generate receptor-based antibodies that neutralize rhinoviruses or measles virus. as a final remark, we realize that, by uncovering the ingenuity of nature, we have learned new ways to engineer antibodies and, possibly, to edit antibodies in primary b cells using the endogenous aid activity. we also discovered how strong antibody responses can be generated by a single b-cell clone, raising a case for adoptive b-cell therapy. a neutralizing antibody selected from plasma cells that binds to group and group influenza a hemagglutinins cross-neutralization of four paramyxoviruses by a human monoclonal antibody pemphigus autoantibodies generated through somatic mutations target the desmoglein- cis-interface antibody-driven design of a human cytomegalovirus ghglpul l subunit vaccine that selectively elicits potent neutralizing antibodies structure and function analysis of an antibody recognizing all influenza a subtypes al is scientific founder of humabs biomed, has substantial interest in antibodies developed by humabs and is a shareholder of vir bio. key: cord- -kxhpdvri authors: grandvaux, nathalie; mccormick, craig title: csv : the nd symposium of the canadian society for virology date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: kxhpdvri the nd symposium of the canadian society for virology (csv ) was held in june in halifax, nova scotia, canada, as a featured event marking the th anniversary of dalhousie university. csv attracted attendees from across canada and around the world, more than double the number that attended the first csv symposium two years earlier. csv provided a forum to discuss a wide range of topics in virology including human, veterinary, plant, and microbial pathogens. invited keynote speakers included david kelvin (dalhousie university and shantou university medical college) who provided a historical perspective on influenza on the th anniversary of the pandemic; sylvain moineau (université laval) who described crispr-cas systems and anti-crispr proteins in warfare between bacteriophages and their host microbes; and kate o’brien (then from johns hopkins university, now relocated to the world health organization where she is director of immunization, vaccines and biologicals), who discussed the underlying viral etiology for pneumonia in the developing world, and the evidence for respiratory syncytial virus (rsv) as a primary cause. reflecting a strong commitment of canadian virologists to science communication, csv featured the launch of halifax’s first annual soapbox science event to enable public engagement with female scientists, and the live-taping of the th episode of the this week in virology (twiv) podcast, hosted by vincent racaniello (columbia university) and science writer alan dove. twiv featured interviews of csv co-founders nathalie grandvaux (université de montréal) and craig mccormick (dalhousie university), who discussed the origins and objectives of the new society; ryan noyce (university of alberta), who discussed technical and ethical considerations of synthetic virology; and kate o’brien, who discussed vaccines and global health. finally, because csv seeks to provide a better future for the next generation of canadian virologists, the symposium featured a large number of oral and poster presentations from trainees and closed with the awarding of presentation prizes to trainees, followed by a tour of the halifax citadel national historic site and an evening of entertainment at the historic alexander keith’s brewery. the canadian society for virology (csv) was founded in to provide support for the canadian virology research community, including basic, clinical, government, and industry researchers working on a broad range of viruses. to launch the new society, csv planned a satellite workshop in conjunction with the annual american society for virology (asv) meeting in blacksburg, virginia in june , which was supported by then-asv president and ex-pat canadian dr. grant mcfadden [ ] . the symposium opened with a keynote lecture from dr. david kelvin (dalhousie university and shantou university medical college) who provided a historical perspective on influenza on the th anniversary of the pandemic. dr. kelvin shared anecdotes from the pandemic and described how factors such as the impact of exposure to past circulating viruses may have influenced pathogenic outcome to the pandemic virus. he discussed the circumstances of other influenza pandemics of the past two centuries, and the unpredictable evolution of pandemic viruses, whereby some pandemic viruses can enter circulation as an endemic virus, whereas others, such as the h n virus, can disappear entirely. lessons learned from the study of these viruses could be applied to prevent or mitigate the impact of another influenza pandemic. the nd keynote address of the evening was delivered by dr. sylvain moineau (université laval) who transitioned from human hosts to bacterial hosts in a fascinating talk about crispr-cas antiviral systems. bacteria thrive in virus-rich ecosystems by using a combination of antiviral defenses including restriction endonucleases and crispr-cas enzymes that effectively act as bacterial intrinsic and adaptive immune systems, respectively. dr. moineau focused on bacterial crispr-cas type ii systems that function by archiving short dna "spacers" derived from invading phage genomes into a crispr array in their genome, providing a chronological record of past infections. the crispr array is then transcribed into a "guide" rna that associates with a cas the symposium opened with a keynote lecture from dr. david kelvin (dalhousie university and shantou university medical college) who provided a historical perspective on influenza on the th anniversary of the pandemic. dr. kelvin shared anecdotes from the pandemic and described how factors such as the impact of exposure to past circulating viruses may have influenced pathogenic outcome to the pandemic virus. he discussed the circumstances of other influenza pandemics of the past two centuries, and the unpredictable evolution of pandemic viruses, whereby some pandemic viruses can enter circulation as an endemic virus, whereas others, such as the h n virus, can disappear entirely. lessons learned from the study of these viruses could be applied to prevent or mitigate the impact of another influenza pandemic. the nd keynote address of the evening was delivered by dr. sylvain moineau (université laval) who transitioned from human hosts to bacterial hosts in a fascinating talk about crispr-cas antiviral systems. bacteria thrive in virus-rich ecosystems by using a combination of antiviral defenses including restriction endonucleases and crispr-cas enzymes that effectively act as bacterial intrinsic and adaptive immune systems, respectively. dr. moineau focused on bacterial crispr-cas type ii systems that function by archiving short dna "spacers" derived from invading phage genomes into a crispr array in their genome, providing a chronological record of past infections. the crispr array is then transcribed into a "guide" rna that associates with a cas endonuclease and allows it to cleave incoming foreign viral dna. bacterial viruses, known as phage, are not defenseless; they can bypass crispr-cas targeting by mutation or deletion of the crispr target in their genome, or by production of anti-crispr proteins that subvert the crispr-cas machinery. dr. moineau discussed current and potential applications of crispr technology and introduced the audience to a wide variety of newly discovered anti-crispr proteins that will undoubtedly be the subject of intense investigation for years to come [ , ] . he also described his efforts to bring crispr-cas technology to the undergraduate laboratory curriculum at université laval [ ] . the second day of csv started early for some, with a student-led fun run through south end halifax and point pleasant park. when fun run survivors arrived at breakfast, they were greeted by volunteers who gave them seating assignments that maximized potential interactions between pis and students. following this, the first scientific session of the day commenced, chaired by dr. hugo soudeyns (université de montréal), on "emerging viruses". to lead off, dr. vikram misra (university of saskatchewan, saskatoon, sk) presented his studies of bat viruses. in recent years several viruses that appear to cause no overt disease in their natural bat hosts have been transmitted to new mammalian hosts, causing serious and sometimes fatal disease. dr. misra's research program is focused on examining the apparently benign relationship between these viruses and their natural bat hosts, as well as factors that upset this relationship leading to increased viral replication and transmission to new mammalian hosts (recently reviewed in [ ] ). his team, in a comparison of bat and human host responses to viral infection, demonstrated that while both animals mount antiviral responses, inflammatory responses are diminished in bat cells [ ] potentially decreasing viral pathology. moreover, antiviral responses in bat cells were resistant to viral subversion mechanisms that normally operate in human host cells. the misra laboratory has identified a new bat coronavirus [ ] and a new bat herpesvirus [ ] that persistently infect two canadian bat species with no overt signs of disease. both viruses infect many, if not most, bats in long-term associations without causing overt disease. the stress of white-nose syndrome, a fungal infection that has decimated some north american bat species, leads to a dramatic increase in corona virus replication in little brown bats, potentially increasing the chances of transmission to other species [ ] . long-term viral persistence followed by stress-induced reactivation could be modeled in tissue-culture where the mers-coronavirus persistently infected bat cells and the suppression of innate antiviral responses led to increased viral replication [ ] . studying interactions between bat viruses and their natural host may provide key information to prevent future zoonoses. national microbiology laboratory (nml) researchers developed an ebola vaccine by pseudotyping vesicular stomatitis virus (vsv) with ebolavirus (ebov) glycoprotein precursor (gpc), yielding the rvsv-ebov-gpc vaccine. this vaccine was safe and efficacious in early clinical trials and was deployed in west africa in as an emergency measure to quell an ebola epidemic by ring vaccination (effectively, a phase iii clinical trial) [ ] . lassa virus is an arenavirus endemic across much of west africa. rodents are the natural host, and zoonotic transmission to humans can cause severe hemorrhagic fevers in % of infected individuals. no lassa virus vaccine is currently available because even though many candidates have performed well in clinical trials, none have yet shown efficacy and safety in humans. nml researchers developed a lassa fever vaccine using the same vsv platform previously used to create the successful ebola vaccine by inserting lassa virus gpc gene into the vsv platform [ ] . dr. michael drebot from the zoonotic diseases and special pathogens program at the nml summarized the pre-clinical studies conducted to date on the "made-in-canada" lassa fever vaccine, which performed well in guinea pig, rhesus and cynomolgus macaque models [ ] [ ] [ ] . dr. drebot also described work from dr. david safronetz and other colleagues at the nml who are working on the next steps required to bring this vaccine to clinical trials. the "emerging viruses" session also included presentations from the following trainees: md niaz rahim (university of manitoba) who discussed a pan-filovirus t-cell vaccine for ebola and marburg viruses, corina warkentin (university of ottawa) who discussed roles for kinases in filovirus entry pathways, and neda barjesteh (mcmaster university) who discussed links between viral infection and amyotrophic lateral sclerosis. the next session on "viral subversion of host cell processes" opened with a talk on herpesvirus assembly from dr. bruce banfield (queens university). herpesviruses replicate in the cell nucleus, where the viral genome is copied into branched concatemers that are trimmed and packaged into newly assembled procapsids. because these dna-containing capsids are too large to exit the nucleus via nuclear pores, herpesviruses must transit the nuclear envelope to access the cytoplasm where the final stages of virion maturation occur. this process, called nuclear egress, is achieved by budding of capsids into the inner nuclear membrane yielding an enveloped particle in the perinuclear space. the envelopes of these perinuclear virions then fuse with the outer nuclear membrane releasing dna-containing capsids into the cytoplasm. although this fusion mechanism is poorly understood, several groups have partially described the remarkable mechanism of budding, whereby two conserved viral proteins, pul and pul , assemble into an oligomeric nuclear egress complex (nec) on the inner nuclear membrane and facilitate capsid recruitment and budding into the perinuclear space [ , ] . dr. banfield described the herpes simplex virus type- (hsv- ) nec complex, which is sufficient to promote nuclear envelope vesiculation and drive nucleoplasmic invagination of the inner nuclear membrane in transfected cells. however, these nuclear envelope perturbations are not normally seen in cells infected with wild-type hsv- strains suggesting that these nec activities are negatively regulated during infection. the capacity of the nec to drive the formation of long inner nuclear membrane-derived tubules that extend deep into the nucleus suggest that capsid envelopment could, paradoxically, occur deeper in the nucleus rather than at the nuclear periphery where marginalized cellular chromatin and the nuclear lamina create formidable barriers to capsid envelopment. pul is a tegument protein that is required for efficient nuclear egress of hsv- capsids [ ] . new data from the banfield lab demonstrated that enveloped capsids accumulate in inner nuclear membrane bound structures deep within the nucleus of cells infected with ul mutant viruses suggesting that: ) pul negatively regulates the inner nuclear membrane invagination activity of the hsv- nec; and ) that inner nuclear membrane invaginations can serve as sites of capsid envelopment. mammalian orthoreoviruses selectively replicate in cancer cells and have been extensively studied as oncolytic agents in animal models and human clinical trials [ , ] ). dr. maya shmulevitz (university of alberta) described how knowledge of reovirus molecular genetics can be exploited to create mutant viruses that enter and kill cancer cells more effectively without compromising their selectivity towards transformed cells [ ] . for example, while virions bearing the full complement of twelve trimeric σ attachment protein complexes on their surfaces are relatively stable in the natural enteric environment, experimentally reducing the number of these attachment proteins on the virion accelerated uncoating and replication in cancer cells and increased oncolytic potency in a mouse melanoma model [ , ] . dr. shmulevitz also described how σ attachment protein cleavage by breast cancer metalloproteases dramatically lowered reovirus infectivity. mutagenesis of defined protease cleavage sites on σ prevented cleavage and inactivation of reoviruses by breast tumor metalloproteases. these studies suggest that reoviruses are quite amenable to repurposing for superior oncolytic properties. the "viral subversion of host cell processes" session also included presentations from the following trainees: nichole mcmullen (dalhousie university) who reported the unconventional egress mechanisms of non-enveloped reoviruses, justine sitz (université laval) who described interactions between a human papillomavirus protein and a host dna repair-specific e ubiquitin ligase, and quentin osseman (université de montréal) who described interactions between respiratory syncytial virus (rsv) and the host autophagy pathway. following this session, the csv conducted its first face-to-face business meeting, where members learned about the state of the society budget and adopted a set of by-laws. csv symposia and trainee-focused opportunities were discussed, as well as imminent plans for the first open election of a csv executive from the membership (which was completed in late ). meanwhile, in another theatre, many of the trainee attendees were treated to a writing workshop designed by science writer alan dove, plos pathogens editor karen mossman and plos one senior editor eileen clancy, who presented strategies to communicate scientific discoveries effectively in writing. particular attention was paid to making a compelling case for the importance of your research, articulation of a clear take-home message, and considering the audience for your research. due attention was paid to ethical issues in publishing, and open-access publishing was promoted as a mechanism to maximize the impact of research. the first afternoon session focused on "viruses of microbes" was chaired by dr. karen maxwell (university of toronto). in the first talk of the session, dr. maxwell described a new host defense mechanism against viral infection. bacteria have long been known to defend against bacteriophage infection using restriction endonucleases, cell surface modifications and abortive infection systems, and the past years have seen a rapid expansion of our understanding of diverse crispr-cas systems that provide a form of adaptive immune memory for bacteria [ ] . all these systems rely on bacterial proteins or protein/rna complexes to execute antiviral defense. dr. maxwell described the discovery of a new chemical defense against phage infection of the soil-dwelling bacteria streptomyces. these molecules include anthracyclines that act as dna-intercalating agents that disrupt the early stages of infection by phages with dna genomes, perhaps by blocking circularization of the incoming linear dna [ ] . dr. maxwell showed that streptomyces bacteria can secrete anthracyclines into the extracellular environment, which are then taken up by neighboring cells to block viral infection. in this way, these chemicals act as broad-spectrum antivirals that can protect and influence the evolution of bacterial communities. fecal microbiota transplants (fmts) are increasingly used to treat human intestinal diseases including ulcerative colitis [ ] and antibiotic-resistant clostridium difficile infections [ ] . however, documenting successful transplant of gut microbiota in fmt recipients remains challenging. fmt inevitably transfer bacteriophages as well [ ] , but little is known about how transferred phage affect the recipient microbiome. dr. alexander hynes (mcmaster university) described studies designed to address this directly by studying crassphage, the most abundant phage in humans. crassphage was recently discovered by metagenomic sequencing [ ] but its natural bacterial host had not yet been identified. using pcr and metagenomic analysis, dr. hynes's group tracked crassphage transfer from phage-positive human donors to human fmt recipients or germ-free mice. studying the bacteria transferred along with the crassphage into naïve recipients may provide opportunities to discover the elusive crassphage host. the "viruses of microbes" session also included presentations from the following trainees: casey jones (dalhousie university) who reported on the state of the gut virome in pediatric crohn's disease [ ] , jaclyn mccutcheon (university of alberta) who presented her discovery of type iv pili as receptors for bacteriophages on stenotrophomonas maltophilia [ ] , and nikhil george (university of waterloo) who presented his studies of crispr-cas-based warfare between phage and bacteria in a municipal landfill site. following an afternoon break, dr. matthias götte (university of alberta) chaired a session on "antivirals and vaccines". this session was kicked off in an electric fashion by four -minute thesis-style presentations from trainees. using only a single slide, ali zhang (mcmaster university), briti saha (university of ottawa) and mariel kleer and andrea monjo from dalhousie university clearly and concisely presented their respective research projects. the general consensus was that future csv symposia should feature more of these "flash" talks from trainees (and maybe the pis should be challenged to do the same!). this was followed by a panel discussion about the future of antiviral and vaccine discovery and development, chaired by dr. götte and featuring panel members following the "antivirals and vaccines" session, everyone had an opportunity to get some fresh air and participate in a preview of halifax's first soapbox science event, spearheaded by dalhousie university graduate student emma finlayson-trick. soapbox science is an international program that promotes the creation of events that showcase female scientists and provides opportunities for public outreach and science communication. for this preview, dr. maya shmulevitz from the university of alberta and drs. alyson kelvin and sarah wells and phd student krysta coyle from dalhousie university put on lab coats embossed with the purple soapbox science logo and stood on "soapboxes" built by volunteer engineers (figure ). they explained their research using simple props to engage and interact with the audience. of course, csv attendees were exactly the opposite of the laypeople that soapbox science is designed to target, but nevertheless this preview event provided a good example of how scientists can reach out and engage the public. on the saturday following the symposium, the actual soapbox science event launch at the halifax seaport farmers' market featured twelve scientists and attracted members of public over h. figure . soapbox science preview event. soapbox science is a science communication event in which female scientists stand on "soapboxes" (see above) and discuss their research with the lay public using simple props. these events are intended to boost the visibility of female scientists and inspire the next generation. in this photo, dr. maya shmulevitz (university of alberta) is explaining her research program using art supplies and candy; she is illustrating how scientists can transform a virus that causes disease, into a virus that treats disease. volunteer lucas jarche (dalhousie university) is seen here holding a "typical healthy human", which is composed of many human organs that each have their own unique features (e.g., bones are licorice, heart is a cherry gummy). the audience was asked to make a virus that targets a specific organ, using the corresponding candy. for example, a virus that homes to the heart would have evolved to recognize specific features of the heart/cherry gummy. additional features of viruses were then added; for example, viruses that cause disease often interfere with immune responses, so candies were added to represent these attributes. following a discussion of the features of cancer, the audience were instructed to modify their candy viruses so that they will target cancer cells rather than their natural host cells. following the soapbox science preview, the audience returned to the lecture hall for a keynote lecture from dr. kate o'brien, then from johns hopkins bloomberg school of public health, who had recently been awarded a prestigious canada research chair to support her recruitment to dalhousie university (dr. o'brien was subsequently recruited by world health organization in geneva, switzerland, where she is director of immunization, vaccines and biologicals). dr. o'brien's scientific and policy work is focused on vaccine-preventable illnesses in children and adults. she reported on results from the pneumonia etiology research for child health (perch) study, which is a collaborative study of populations in seven countries in africa and asia designed to determine the etiology of severe pediatric pneumonia in the context of widespread conjugate hemophilus influenzae type b (hib) and pneumococcal vaccination [ ] . the study included over hospitalized soapbox science is a science communication event in which female scientists stand on "soapboxes" (see above) and discuss their research with the lay public using simple props. these events are intended to boost the visibility of female scientists and inspire the next generation. in this photo, dr. maya shmulevitz (university of alberta) is explaining her research program using art supplies and candy; she is illustrating how scientists can transform a virus that causes disease, into a virus that treats disease. volunteer lucas jarche (dalhousie university) is seen here holding a "typical healthy human", which is composed of many human organs that each have their own unique features (e.g., bones are licorice, heart is a cherry gummy). the audience was asked to make a virus that targets a specific organ, using the corresponding candy. for example, a virus that homes to the heart would have evolved to recognize specific features of the heart/cherry gummy. additional features of viruses were then added; for example, viruses that cause disease often interfere with immune responses, so candies were added to represent these attributes. following a discussion of the features of cancer, the audience were instructed to modify their candy viruses so that they will target cancer cells rather than their natural host cells. following the soapbox science preview, the audience returned to the lecture hall for a keynote lecture from dr. kate o'brien, then from johns hopkins bloomberg school of public health, who had recently been awarded a prestigious canada research chair to support her recruitment to dalhousie university (dr. o'brien was subsequently recruited by world health organization in geneva, switzerland, where she is director of immunization, vaccines and biologicals). dr. o'brien's scientific and policy work is focused on vaccine-preventable illnesses in children and adults. she reported on results from the pneumonia etiology research for child health (perch) study, which is a collaborative study of populations in seven countries in africa and asia designed to determine the etiology of severe pediatric pneumonia in the context of widespread conjugate hemophilus influenzae type b (hib) and pneumococcal vaccination [ ] . the study included over hospitalized children to months of age, and analysis including molecular and culture methods, chest x-rays and descriptive etiology analysis. the perch study identified the frequency of etiologic association of different viruses with severe childhood pneumonia, which notably included rsv, widely recognized as a major cause of childhood mortality worldwide [ ] . after the lecture, the audience adjourned to an evening at second poster session, after which, many reunited at a beer garden on spring garden road. on friday morning, the first scientific session focused on "emerging methods in virology" was chaired by dr. marceline côté (university of ottawa) and was led off by dr. roger lippé (université de montréal) who described a powerful new technique called flow virometry. traditionally, flow cytometry has been a very useful approach to study virus infection on a single-cell basis, but viruses themselves have been too small to analyze due to the . µm resolution of most instruments. dr. lippé described how his research group prepares herpesvirus particles for analysis on these instruments by labelling with nucleic acid dyes that can penetrate viral particles or engineering viral particles with fluorescent fusion proteins (including capsid proteins, tegument proteins or glycoproteins) [ ] [ ] [ ] . this approach enabled characterization of individual hsv- particles harvested from different cellular compartments, as well as sorting to > % purity. flow virometry also enables the study of the impact of heterogeneity of viral populations on viral fitness and facilitates the analysis by mass spectrometry of highly enriched viral particles isolated along the egress pathway to monitor their maturation. because flow virometry can be performed on standard flow cytometers and flow sorters available at most institutions, these approaches can be rapidly adopted by others and applied to the study of other large viruses. low-cost dna synthesis has catalyzed a new revolution in synthetic biology, enabling efficient manipulation and re-coding of microbial and eukaryotic genomes. ryan noyce (university of alberta) described his efforts to use the principles of synthetic biology to create a synthetic poxvirus vector that could be used for a variety of therapeutic applications. edward jenner believed that his variolae vaccinae originated in horses, which has been corroborated by molecular analyses of modern vaccinia virus (vacv) isolates that share common ancestry with horsepox virus (hpxv) [ ] [ ] [ ] (incidentally, dr. noyce told us, this means that the term "vaccination" could be a misnomer; cows may have had nothing to do with it, and the term "equination" may be more accurate [ ] ). to improve on the safety and efficacy of vacv, dr. noyce and colleagues chose to reactivate the ancestral hpxv and use it as a template for safer and more efficacious vaccines and other therapeutics including oncolytic viruses. dr. noyce synthesized ten large ( - kilobase pair) fragments of dna based on the published hpxv sequence [ ] , along with vacv-derived terminal sequences [ ] . these sequences were recombined into a kilobase pair live synthetic chimeric (schpxv) virus in cells infected with shope fibroma virus (sfv), which served as a helper virus. schpxv was safe and effective in pre-clinical studies; it produced smaller plaques in cell culture and was less virulent in mice than vacv, but still provided protection against a lethal vacv challenge. dr. noyce discussed the technical challenges associated with using synthetic biology as a tool to build new viruses, as well as biosafety and ethical considerations. the "emerging methods in virology" session also included presentations from the following trainees: vanessa meier-stephenson (university of calgary and university of lethbridge) who presented her findings of complex dna structures in the hepatitis b virus (hbv) genome, yumiko komatsu (kyoto university) who presented her research on borna disease virus vectors, and brennan dirk (western university) who discussed his efforts to trace multiprotein complexes involved in hiv- immune evasion [ , ] . dr. andrew white (york university) chaired the next session on "rna in virus infection", which opened with a talk from dr. selena sagan (mcgill university). the liver-specific microrna- (mir- ) directly binds to the '-end of the hepatitis c virus (hcv) genome and promotes the accumulation of viral rna in cell culture and animal models, at least in part by protecting the genome from degradation by the host '- ' exonucleases xrn and xrn . because the hcv '-triphosphate structure is a molecular pattern expected to be recognized by pattern recognition receptors (prrs), the sagan (mcgill university) and wilson (university of saskatchewan) groups investigated whether mir- binding to the hcv genome affects prr binding and activation of innate antiviral signaling pathways. dr. sagan reported that mir- does not affect recognition of the hcv genome by protein kinase r (pkr), retinoic acid inducible protein i (rig-i)-like receptors, or interferon-induced protein with tetratricopeptide repeats (ifits) and [ ] . '-triphophate-containing rnas can be converted to '-monophosphate rna by the action of the cytoplasmic pyrophosphatases dom z and dusp ; they found that rna silencing of dom z or dusp rescued rna accumulation of hcv subgenomic replicons in the absence of mir- , suggesting that mir- normally protects the '-triphosphate structure on the viral rna and prevents pyrophosphatase activity and subsequent degradation by xrn / . using selective ' hydroxyl acylation analyzed by primer extension (shape), the sagan lab is exploring how mutations in the ' non-coding region of hcv genomes from patient isolates may help to promote genome stability and relieve hcv's dependence on mir- . however, there is evidence to support an additional role for mir- in the hcv replication cycle beyond genome stabilization. influenza a virus infection delivers eight (−)-sense single-stranded viral rna (vrna) genome segments to the cell nucleus, each bound to the tripartite viral rna-dependent rna polymerase (rdrp) complex. to initiate transcription, the rdrp complex simultaneously binds to host rna polymerase ii (pol ii) and '-cap structures on nascent host pre-mrnas. once in position, the polymerase acidic (pa) subunit of the rdrp complex catalyzes endoribonucleolytic cleavage of host pre-mrnas at a position - nucleotides downstream of the '-cap structure. this process, known as "cap-snatching", enables priming of viral mrna synthesis. it remains unclear whether there is some selectivity in this process, or the rdrp simply "snatches" these primers from the most abundant nearby host pre-mrnas. dr. martin pelchat (university of ottawa) described deep sequencing studies that support a more nuanced model of cap-snatching that integrates host pre-mrna abundance with selectivity, whereby each of the eight rdrp:vrna complexes acquire distinct subsets of host rnas due to differences in vrna untranslated region (utr) sequence [ , ] . importantly, he reported that host mrnas associated with antiviral responses are over-represented in the cap-snatched population. this suggests that cap-snatching may indeed play an important role in influenza a virus-host shutoff. the "rna in virus infection" session also included presentations from the following trainees: carolyn robinson (dalhousie university) who discussed kaposi's sarcoma-associated herpesvirus (kshv) modulation of rna granules, eric pringle (dalhousie university) who discussed how kshv mrnas are translated [ ] , and tyler mrozowich (university of lethbridge) who presented structural analysis of the japanese encephalitis virus rna genome. the "this week in virology" or "twiv" podcast was created in september by columbia university professors vincent racaniello and dickson despommier to discuss science in an informal and informative manner. ten years later, dr. racaniello and twiv co-host dr. alan dove accepted our invitation to record their th podcast at csv . after discussing the weather (as is their custom), the twiv hosts got down to business with an interview of drs. grandvaux and mccormick, who discussed their motivation in founding the csv, the current research funding climate in canada, and plans for the new society ( figure ). they also interviewed dr. ryan noyce from the university of alberta, who discussed his high-profile synthetic virology (or "synviro") research project, in which he used principles of synthetic biology to create a horsepox virus vector that could be used as the basis for new poxvirus vaccines and oncolytic viruses [ , ] . this project had been previously discussed on twiv , entitled "a pox on your horse" [ ] , and received scrutiny in the scientific and mainstream media due to concerns about ethics and potential "dual-use" of these new poxviruses. finally, the twiv team interviewed dr. kate o'brien from johns hopkins university, who discussed her global health research in vaccines, and in particular how certain viruses are etiologically linked to severe pneumonia in the developing world. the podcast was entitled "good virologists go to halifax" [ ] . viruses , , x for peer review of which he used principles of synthetic biology to create a horsepox virus vector that could be used as the basis for new poxvirus vaccines and oncolytic viruses [ , ] . this project had been previously discussed on twiv , entitled "a pox on your horse" [ ] , and received scrutiny in the scientific and mainstream media due to concerns about ethics and potential "dual-use" of these new poxviruses. finally, the twiv team interviewed dr. kate o'brien from johns hopkins university, who discussed her global health research in vaccines, and in particular how certain viruses are etiologically linked to severe pneumonia in the developing world. the podcast was entitled "good virologists go to halifax" [ ] . after the twiv live-taping, our program resumed on the topic of "viruses of flora and fauna", with dr. eric jan (university of british columbia) chairing and giving the first talk. internal ribosome entry sites (ires) can start translation in alternative reading frames. for example, dr. jan's lab previously showed that the ires of the honey bee dicistrovirus not only directs translation in the reading frame yielding a polyprotein, but also in the + reading frame [ , ] . dr. jan reported that the ires of the related dicistrovirus cricket paralysis virus (crpv) also promotes translation in the + reading frame, resulting in the translation of a new protein called orfx [ ] . however, unlike the honey bee ires, a subset of ribosomes recruited to the crpv ires can undergo a bypass mechanism to initiate translation downstream at the + frame th non-aug codon. dr. jan showed that mutagenesis of this downstream + frame orfx attenuates the virus in a drosophila infection model, suggesting that orfx plays a role in viral pathogenesis. these studies highlight the diversity of viral translation recoding mechanisms to increase coding capacity. in the next talk, hélène sanfaçon (summerland research and development centre, agriculture and agri-food canada) presented evidence for non-canonical proteases encoded by plant viruses. previously described plant virus proteases are grouped according to conserved catalytic active site residues: cysteine, serine or aspartic acid [ , ] . dr. sanfaçon described investigations of viruses of the family secoviridae that constitute a large group of picorna-like viruses [ ] and include strawberry mottle virus (smov). smov has a bipartite genome, with each rna encoding a large polyprotein. rna encodes a cysteine protease that is related to the canonical c protease of picornaviruses and cleaves at multiple locations on polyproteins generated from rna and rna [ ] . dr. sanfaçon and her team detected an additional proteolytic activity associated with the carboxy-terminal domain of the rna polyprotein [ ] . surprisingly, mutation of conserved serine, cysteine, histidine or aspartic acid residues did not affect this activity. instead, two glutamic acid residues were shown to be after the twiv live-taping, our program resumed on the topic of "viruses of flora and fauna", with dr. eric jan (university of british columbia) chairing and giving the first talk. internal ribosome entry sites (ires) can start translation in alternative reading frames. for example, dr. jan's lab previously showed that the ires of the honey bee dicistrovirus not only directs translation in the reading frame yielding a polyprotein, but also in the + reading frame [ , ] . dr. jan reported that the ires of the related dicistrovirus cricket paralysis virus (crpv) also promotes translation in the + reading frame, resulting in the translation of a new protein called orfx [ ] . however, unlike the honey bee ires, a subset of ribosomes recruited to the crpv ires can undergo a bypass mechanism to initiate translation downstream at the + frame th non-aug codon. dr. jan showed that mutagenesis of this downstream + frame orfx attenuates the virus in a drosophila infection model, suggesting that orfx plays a role in viral pathogenesis. these studies highlight the diversity of viral translation recoding mechanisms to increase coding capacity. in the next talk, hélène sanfaçon (summerland research and development centre, agriculture and agri-food canada) presented evidence for non-canonical proteases encoded by plant viruses. previously described plant virus proteases are grouped according to conserved catalytic active site residues: cysteine, serine or aspartic acid [ , ] . dr. sanfaçon described investigations of viruses of the family secoviridae that constitute a large group of picorna-like viruses [ ] and include strawberry mottle virus (smov). smov has a bipartite genome, with each rna encoding a large polyprotein. rna encodes a cysteine protease that is related to the canonical c protease of picornaviruses and cleaves at multiple locations on polyproteins generated from rna and rna [ ] . dr. sanfaçon and her team detected an additional proteolytic activity associated with the carboxy-terminal domain of the rna polyprotein [ ] . surprisingly, mutation of conserved serine, cysteine, histidine or aspartic acid residues did not affect this activity. instead, two glutamic acid residues were shown to be required for polyprotein processing. n-terminal sequencing by edman degradation method enabled precise mapping of two protease cleavage sites with the consensus sequence p↓xfp. together, these findings suggest that smov polyprotein processing requires two viral proteases, the c-like cysteine protease encoded by rna , and a novel protease encoded by rna that features two putative glutamic acid residues in the catalytic site. the novel protease domain is only found in the rna polyproteins of two other definite or putative members of the family secoviridae, suggesting that it was recently acquired. the "viruses of flora and fauna" session also included presentations from the following trainees: shuang yang (university of british columbia) who described a unique nuclear entry pathway for parvoviruses, gerard gaspard (dalhousie university) who presented mechanistic studies of the reptilian reovirus p fusion protein, and jared rowell (university of calgary) who presented studies of cell fate during porcine circovirus- infection. during the break following this session, everyone assembled in the courtyard for a group photo (figure ). viruses , , x for peer review of required for polyprotein processing. n-terminal sequencing by edman degradation method enabled precise mapping of two protease cleavage sites with the consensus sequence p↓xfp. together, these findings suggest that smov polyprotein processing requires two viral proteases, the c-like cysteine protease encoded by rna , and a novel protease encoded by rna that features two putative glutamic acid residues in the catalytic site. the novel protease domain is only found in the rna polyproteins of two other definite or putative members of the family secoviridae, suggesting that it was recently acquired. the "viruses of flora and fauna" session also included presentations from the following trainees: shuang yang (university of british columbia) who described a unique nuclear entry pathway for parvoviruses, gerard gaspard (dalhousie university) who presented mechanistic studies of the reptilian reovirus p fusion protein, and jared rowell (university of calgary) who presented studies of cell fate during porcine circovirus- infection. during the break following this session, everyone assembled in the courtyard for a group photo (figure ) . dr. karen mossman (mcmaster university) chaired the final symposium session on "antiviral innate immunity", which was led off by her former post-doctoral fellow dr. stephanie dewitte-orr (wilfrid laurier university) who discussed roles for class a scavenger receptors (sr-as) in innate antiviral immunity in lower vertebrates. in mammals the sr-as include five family members (sr-ai, marco, scara , scara and scara ) that play diverse roles in immunity, aiding macrophage clearance of pathogens and apoptotic cells, and acting as receptors for immune-stimulating molecules. they have also been shown to serve as virus receptors. notwithstanding how important these receptors are to host immunity, little is known regarding sr-a function in lower vertebrates. dr. dewitte-orr presented her findings to date regarding sr-as and their functions in fish and frogs. dr. dewitte-orr has identified marco, scara , scara and scara sequences from rainbow trout [ , ] . these sr-as appear to be functional in rainbow trout, and act as surface receptors for double-stranded rna (dsrna), a virus-derived innate immune stimulant [ ] . interestingly, marco does not appear to bind dsrna, a unique finding in vertebrates [ ] . sr-as appear to be surface receptors in amphibian cells as well, as dr. dewitte-orr presented her findings regarding sr-a mediated dsrna responses in the american toad cell line, bufotad [ ] . in addition to function studies, the dewitte-orr group has identified a cell line derived from chinook salmon (chse- ) that appears to lack functional sr-as and can be used as a model for sr-a/dsrna binding [ ] . finally, she presented evidence that sr-as could serve as surface receptors for the ranavirus, frog dr. karen mossman (mcmaster university) chaired the final symposium session on "antiviral innate immunity", which was led off by her former post-doctoral fellow dr. stephanie dewitte-orr (wilfrid laurier university) who discussed roles for class a scavenger receptors (sr-as) in innate antiviral immunity in lower vertebrates. in mammals the sr-as include five family members (sr-ai, marco, scara , scara and scara ) that play diverse roles in immunity, aiding macrophage clearance of pathogens and apoptotic cells, and acting as receptors for immune-stimulating molecules. they have also been shown to serve as virus receptors. notwithstanding how important these receptors are to host immunity, little is known regarding sr-a function in lower vertebrates. dr. dewitte-orr presented her findings to date regarding sr-as and their functions in fish and frogs. dr. dewitte-orr has identified marco, scara , scara and scara sequences from rainbow trout [ , ] . these sr-as appear to be functional in rainbow trout, and act as surface receptors for double-stranded rna (dsrna), a virus-derived innate immune stimulant [ ] . interestingly, marco does not appear to bind dsrna, a unique finding in vertebrates [ ] . sr-as appear to be surface receptors in amphibian cells as well, as dr. dewitte-orr presented her findings regarding sr-a mediated dsrna responses in the american toad cell line, bufotad [ ] . in addition to function studies, the dewitte-orr group has identified a cell line derived from chinook salmon (chse- ) that appears to lack functional sr-as and can be used as a model for sr-a/dsrna binding [ ] . finally, she presented evidence that sr-as could serve as surface receptors for the ranavirus, frog virus- (work described in this special issue). together, these findings provide the first glimpse into the role of sr-as in innate antiviral immunity in lower vertebrates. continuing with the theme on innate immunity in non-human hosts, dr. katharine magor (university of alberta) presented her studies of non-pathogenic and pathogenic influenza a virus (iav) infection of ducks. dr. magor shared a photo of herself as a child cradling her pet duck in yarmouth, nova scotia, and explained how this early love of animals influenced her career path. ducks are the natural iav reservoir host; the virus infects the gastrointestinal tract and typically does not cause overt disease. however, the emergence of avian h n iav strains with pandemic potential have disrupted this balance and can cause lethal infection in ducks. to better understand the pathogenic effects of lethal h n infection in ducks, dr. magor established an infection model of white pekin ducks with three similar h n viruses with known differences in pathogenicity and replication rate [ ] . viral replication and host responses were measured by rt-qpcr at different times post-infection. viral replication peaked and viral load declined in the spleen over time. the ducks rapidly mounted interferon and cytokine responses that peaked on the first day of infection and declined thereafter, with the strongest responses coming from the high-pathogenic viruses. further characterization of the duck immune responses to lethal and sub-lethal infections could advance our understanding of virus-host interactions in the reservoir host. the "antiviral innate immunity" session also included presentations from the following trainees: jiangyi he (memorial university) who described mechanisms of murine cytomegalovirus immune evasion, dacquin kasumba (université de montréal) who described roles for duox enzymes in host responses to respiratory virus infections, and hannah stacey (mcmaster university) who presented her work on iga immune complexes in infection and autoimmunity. csv closed with comments from dr. grandvaux thanking sponsors, the scientific advisory committee, and the local organizing committee. following an ovation for the small army of local volunteers who were instrumental to the success of the symposium, dr. grandvaux announced the travel awards ( figure ). we then gathered outside the lecture theatre where we were met by two members of the th highlanders pipe band, who marched us through the streets of halifax and ascended the slopes of the halifax citadel national historic site ( figure ). inside the walls of the star-shaped fortress, highlanders conducted historic tours in english and french. then pipe and drum sounded once again, and we marched out of the citadel and down sackville street to the historic alexander keith's brewery for an evening featuring local food, drink, and music. a good time was had by all, including drs. grandvaux and mccormick, who could be found relaxing in mr. keith's leather-bound office following a very successful symposium (figure ) , and perhaps twisting a few arms to encourage colleagues to assume leadership positions and host future csv symposia. pipe and drum sounded once again, and we marched out of the citadel and down sackville street to the historic alexander keith's brewery for an evening featuring local food, drink, and music. a good time was had by all, including drs. grandvaux and mccormick, who could be found relaxing in mr. keith's leather-bound office following a very successful symposium (figure ) , and perhaps twisting a few arms to encourage colleagues to assume leadership positions and host future csv symposia. pipe and drum sounded once again, and we marched out of the citadel and down sackville street to the historic alexander keith's brewery for an evening featuring local food, drink, and music. a good time was had by all, including drs. grandvaux and mccormick, who could be found relaxing in mr. keith's leather-bound office following a very successful symposium (figure ) , and perhaps twisting a few arms to encourage colleagues to assume leadership positions and host future csv symposia. author contributions: all authors contributed equally to the conception and writing of this paper. workshop of the canadian society for virology an anti-crispr from a virulent streptococcal phage inhibits streptococcus pyogenes cas widespread anti-crispr proteins in virulent bacteriophages inhibit a range of cas proteins crispr-cas in the laboratory classroom workshop of the canadian society for virology an anti-crispr from a virulent streptococcal phage inhibits streptococcus pyogenes cas widespread anti-crispr proteins in virulent bacteriophages inhibit a range of cas proteins crispr-cas in the laboratory classroom lack of inflammatory gene expression in bats: a unique role for a transcription repressor a persistently infecting coronavirus in hibernating myotis lucifugus, the north american little brown bat isolation, characterization and prevalence of a novel gammaherpesvirus in eptesicus fuscus, the north american big brown bat environmentally persistent pathogens present unique challenges for studies of host-pathogen interactions: reply to field generation and characterization of eptesicus fuscus (big brown bat) kidney cell lines immortalized using the myotis polyomavirus large t-antigen efficacy and effectiveness of an rvsv-vectored vaccine expressing ebola surface glycoprotein: interim results from the guinea ring vaccination cluster-randomised trial properties of replication-competent vesicular stomatitis virus vectors expressing glycoproteins of filoviruses and arenaviruses development of a new vaccine for the prevention of lassa fever vesicular stomatitis virus-based vaccines against lassa and ebola viruses a recombinant vesicular stomatitis virus-based lassa fever vaccine protects guinea pigs and macaques against challenge with geographically and genetically distinct lassa viruses will travel: structural basis of membrane budding during nuclear egress in herpesviruses. adv. virus res nuclear egress of herpesviruses: the prototypic vesicular nucleocytoplasmic transport the herpes simplex virus ul protein is essential for virus propagation reovirus therapy of tumors with activated ras pathway reovirus in cancer therapy: an evidence-based review potential for improving potency and specificity of reovirus oncolysis with next-generation reovirus variants reovirus variants with mutations in genome segments s and l exhibit enhanced virion infectivity and superior oncolysis reduction of virion-associated σ fibers on oncolytic reovirus variants promotes adaptation toward tumorigenic cells an updated evolutionary classification of crispr-cas systems a chemical defence against phage infection systematic review and meta-analysis: fecal microbiota transplantation for treatment of active ulcerative colitis clinical inquiries: how effective and safe is fecal microbial transplant in preventing c difficile recurrence bacteriophage transfer during faecal microbiota transplantation in clostridium difficile infection is associated with treatment outcome a highly abundant bacteriophage discovered in the unknown sequences of human faecal metagenomes multi-omics differentially classify disease state and treatment outcome in pediatric crohn's disease identification and characterization of type iv pili as the cellular receptor of broad host range stenotrophomonas maltophilia bacteriophages dlp and dlp the pneumonia etiology research for child health project: a st century childhood pneumonia etiology study global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis quantitative evaluation of protein heterogeneity within herpes simplex virus particles flow virometry: a powerful tool to functionally characterize viruses analysis of herpes simplex virus type i nuclear particles by flow cytometry genomic analysis, phenotype, and virulence of the historical brazilian smallpox vaccine strain ioc: implications for the origins and evolutionary relationships of vaccinia virus genome of horsepox virus an early american smallpox vaccine based on horsepox equination (inoculation of horsepox): an early alternative to vaccination (inoculation of cowpox) and the potential role of horsepox virus in the origin of the smallpox vaccine construction of an infectious horsepox virus vaccine from chemically synthesized dna fragments hiv- nef sequesters mhc-i intracellularly by targeting early stages of endocytosis and recycling where in the cell are you? probing hiv- host interactions through advanced imaging techniques mir- does not impact recognition of the hcv genome by innate sensors of rna but rather protects the end from the cellular pyrophosphatases, dom z and dusp influenza a virus cap-snatches host rnas based on their abundance early after infection insight into influenza: a virus cap-snatching. viruses mccormick, c. mtorc activity is dispensable for synthesis of kshv lytic proteins synthetic horsepox viruses and the continuing debate about dual use research a pox on your horse good virologists go to halifax global shape mimicry of trna within a viral internal ribosome entry site mediates translational reading frame selection functional insights into the adjacent stem-loop in honey bee dicistroviruses that promotes internal ribosome entry site-mediated translation and viral infection ires-dependent ribosome repositioning directs translation of a + overlapping orf that enhances viral infection plant viral proteases: beyond the role of peptide cutters. front expanding repertoire of plant positive-strand rna virus proteases identification of cleavage sites recognized by the c-like cysteine protease within the two polyproteins of strawberry mottle virus strawberry mottle virus (family secoviridae, order picornavirales) encodes a novel glutamic protease to process the rna polyprotein at two cleavage sites scavengers for bacteria: rainbow trout have two functional variants of marco that bind to gram-negative and -positive bacteria in vitro transcribed dsrna limits viral hemorrhagic septicemia virus (vhsv)-ivb infection in a novel fathead minnow (pimephales promelas) skin cell line class-a scavenger receptor function and expression in the rainbow trout (oncorhynchus mykiss) epithelial cell lines rtgutgc and rtgill-w class a scavenger receptors mediate extracellular dsrna sensing, leading to downstream antiviral gene expression in a novel american toad cell line chse- : a model for studying extracellular dsrna sensing in vitro duck innate immune responses to high and low pathogenicity h avian influenza viruses we are grateful to our colleagues who allowed us to communicate elements of their acknowledgments: we are grateful to our colleagues who allowed us to communicate elements of their workshop presentations in this report. we thank members of the csv scientific advisory committee (drs. marceline côté, darryl falzarano, matthias götte, karen maxwell, nelly pante and andrew white) who planned scientific sessions and reviewed travel award applications. we thank the csv local organizing committee (drs. lisa barrett, jeanette boudreau, jenn corcoran, roy duncan, may el-sherif, todd hatchette, alyson kelvin, denys khaperskyy, jason leblanc, chris richardson and rod russell) who raised funds and planned meeting logistics. we thank the csv student committee, led by dr. denys khaperskyy and including quinelle boudreau, katrina bouzanis, beth castle, becca chilvers, brett duguay, lucas jarche, mariel kleer, grant macneil, andrea monjo, rory mulloy, jacob nearing, carolyn robinson, eric pringle, jacob sicheri, gill singh, patrick slaine, iona stylianides and mackenzie thornbury, who provided essential on-the-ground logistical support for the symposium. we thank emma finlayson-trick for organizing the soapbox science preview and launch events. we thank drs. maya shmulevitz, alyson kelvin and sarah wells and phd student krysta coyle for their soapbox science presentations. we thank quentin osseman, gillian singh and denys khaperskyy for photography. we thank manon livernois (crchum) for administrative assistance. we thank lucas jarche for csv logo artwork. csv was supported by grants from the canadian institutes of health research (cihr-mpl- ) and the dalhousie medical research foundation. additional support was provided by dalhousie university, the li ka shing institute of virology, vido-intervac, plos pathogens, plos one, viruses, canadian journal of microbiology, imv™, zeiss, nikon and sanofi. the authors declare no conflict of interest. key: cord- -oj re k authors: zhou, haixia; chen, yingzhu; zhang, shuyuan; niu, peihua; qin, kun; jia, wenxu; huang, baoying; zhang, senyan; lan, jun; zhang, linqi; tan, wenjie; wang, xinquan title: structural definition of a neutralization epitope on the n-terminal domain of mers-cov spike glycoprotein date: - - journal: nat commun doi: . /s - - - sha: doc_id: cord_uid: oj re k most neutralizing antibodies against middle east respiratory syndrome coronavirus (mers-cov) target the receptor-binding domain (rbd) of the spike glycoprotein and block its binding to the cellular receptor dipeptidyl peptidase (dpp ). the epitopes and mechanisms of mabs targeting non-rbd regions have not been well characterized yet. here we report the monoclonal antibody d that binds to the n-terminal domain (ntd) of the spike glycoprotein and inhibits the cell entry of mers-cov with high potency. structure determination and mutagenesis experiments reveal the epitope and critical residues on the ntd for d binding and neutralization. further experiments indicate that the neutralization by d is not solely dependent on the inhibition of dpp binding, but also acts after viral cell attachment, inhibiting the pre-fusion to post-fusion conformational change of the spike. these properties give d a wide neutralization breadth and help explain its synergistic effects with several rbd-targeting antibodies. m iddle east respiratory syndrome coronavirus (mers-cov), a novel lethal human virus in the family of coronaviridae, was first identified in saudi arabia in june . infection by this pathogen causes an acute respiratory disease designated as mers, with symptoms that are very similar to those of sars . globally, mers-cov infections have been confirmed in countries causing deaths (http://www.who. int/emergencies/mers-cov/en/). interspecies transmission from dromedary camels to humans is considered to be one major route of transmission in the middle east region , . however, many infected patients without camel exposure and a recent mers outbreak in korea demonstrated that large-scale human-tohuman transmissions can occur through close contacts . due to its potential for mutating toward efficient human-to-human transmission and causing a pandemic, mers-cov was listed as a category c priority pathogen by the us national institute of allergy and infectious diseases. monoclonal antibodies (mabs) with potent neutralizing activity have become promising candidates for both prophylactic and therapeutic interventions against viral infections . on coronaviruses, the component primarily targeted by mabs is the homotrimeric spike (s) glycoprotein of the virion. as a typical class i fusion glycoprotein, the s trimer of highly pathogenic coronaviruses such as mers-cov and sars-cov, which mediates receptor recognition and membrane fusion during viral entry [ ] [ ] [ ] [ ] [ ] [ ] , undergoes protease cleavage into the s and s subunits, positional change of the receptor-binding domain (rbd) in the s subunit for receptor binding, dissociation of the s -receptor complex, and finally formation of a six-helix bundle by the s subunits. a series of rbd-targeting antibodies against mers-cov, which block the binding of the s trimer to the cellular receptor dpp , have been reported and characterized [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . these antibodies exhibited high potency in inhibiting the infectivity of pseudotyped and live mers-cov in cells and animal models. the neutralizing epitopes and mechanisms of antibodies including c , d , m , mers- , jc - , cdc-c , mers- , and mers-gd were further elucidated at the atomic level by structural and functional studies [ ] [ ] [ ] [ ] [ ] [ ] [ ] . sequence comparisons of different mers-cov strains have shown that most naturally occurring mutations of the s glycoprotein are located on the rbd of the s subunit and the s subunit. considering the rapid evolution and high genome variation of rna viruses, more mutations on the rbd may enable the new strains to escape neutralization by currently known rbd-targeting antibodies. therefore, new mabs targeting other functional regions of the mers-cov s glycoprotein and/or neutralizing by different mechanisms are important for developing effective prophylactic and therapeutic interventions against mers-cov infection. although several mabs targeting non-rbd regions have recently been reported, their neutralizing epitopes and mechanisms remain unclear , , . in this study, we isolated and characterized the mouse mab d by combining structural, biochemical, and functional studies. the d antibody recognizes the ntd of mers-cov s glycoprotein and neutralizes the infectivity of pseudotyped and live virus with a potency comparable to those of the most active rbd-targeting antibodies. we also found that the epitope and mechanism of d , which are different from those of rbdtargeting antibodies, enable it to have a better neutralizing breadth and to work synergistically with other antibodies against different mers-cov strains. all these results indicate that d is a very promising candidate for the future combined use of different antibodies in our battle against mers-cov. characterization of neutralizing mab d targeting the ntd. to generate mers-cov neutralizing mabs with epitopes outside the rbd, mice were immunized with recombinant mers-cov s protein (residues - ). subsequently, the spleenocytes were harvested and fused with sp / myeloma cells, and the hybridoma cell lines were screened for positive clones by elisa with the s protein . the positive clones were further tested for their reactivity to different s fragments, including the s subunit ntd (residues - ), rbd (residues - ), and the s subunit (residues - ). one ntd-specific mab, named as d , was finally isolated with an ec of approximately . μg ml − in elisa (fig. a) . it exhibited no crossreactivity with the rbd at a concentration of μg ml − (fig. b) . we further assessed the potential of d , in the form of crude extracts from mouse ascites, for inhibiting mers-cov entry into susceptible huh cells and vero e cells with either pseudotyped or infectious viruses. as expected, d was able to neutralize the infectivity of pseudotyped and live mers-cov (fig. c, d) . the neutralizing activity of d was dose-dependent, with an ic of approximately . μg ml − against pseudotyped virus and practically the same ic of approximately . μg ml − against live virus (emc strain) (fig. c, d) . images illustrating the reduced pfu formation, corresponding to the rate of neutralization of live mers-cov, are shown in fig. e . antibody isotyping showed that d belongs to the igg subtype. sequencing further determined that the heavy chain germline v and j segments are ighv - * and ighj * , while those of light chain are igkv - * , igkj * , and igkj * , respectively (supplementary table ) . we also generated a chimeric version of d ( d -h) by combining the v segments of d with the human igg backbone, which was efficiently expressed and purified in freestyle -f cells ( supplementary fig. a ). the bio-layer interferometry (bli) experiment showed that the affinity constant of the binding between d -h and ntd was approximately nm (table and supplementary fig. b) . the ic of the purified d -h against cell entry by pseudotyped mers-cov was approximately . μg ml − (supplementary fig. c ). we also investigated the protective efficacy of d -h against infection of pseudotyped mers-cov using r -hdpp mice model with a human dpp inserted into the rosa locus by crispr/cas , which could also been productively infected by high-titer mers-cov pseudovirus, with effects comparable to the authentic infection . bioluminescence of the fluc reporter showed that the pseudovirus infection in the mice was clearly prevented by d -h and rbd-specific mab mers- when both antibodies were administered by the intraperitoneal injection with a dose of μg per mouse ( supplementary fig. d ). the recombinant chimeric d -h, which retained the activities as the mouse d and protected r -hdpp mice against challenge of pseudotyped mers-cov, was utilized in subsequent binding and neutralization experiments. overall structure of the d scfv bound to the ntd. to structurally characterize the d and its binding to the spike protein, we determined the crystal structure of the antibody scfv ( d -scfv) in complex with the ntd at a resolution of . Å with a final r work of . and r free of . . statistics of diffraction data collection, processing, and structure refinement are listed in table . there were three complexes of d -scfv bound to ntd per asymmetric unit. the refined model contains residues tyr to ser of mers-cov ntd, glu to ser of the v h and asp to lys of the v l . n-linked glycans attached to asn , asn , asn , asn , asn , asn , asn , and asn of the ntd are also included in the model. it has been previously shown that the mers-cov ntd folds into a galectin-like structure, which can be separated into top, core and bottom subdomains (fig. a) . upon binding, the d -scfv contacts the top subdomain of the ntd and the asn -linked glycans with its heavy and light chains ( fig. a and supplementary fig. ). all three cdrs of the heavy chain and the cdr and cdr of the light chain participate in the binding (fig. a) . the buried surface between the d -scfv and the ntd encompasses approximately Å for the heavy chain and Å for the light chain. structural features of the interface between d and ntd. the binding interface between d -scfv and ntd consists of residues and asn -linked glycans from the ntd, as well as residues from all cdrs except for lcdr (fig. b, c) . the interacting residues from the ntd are tyr , asp , pro , asp , val , ser , glu , ser , asn , lue , arg , and asn . together with the asn -linked nag , nag and man , they form the conformational epitope recognized by d (fig. b) . the residues recognizing d are ser , tyr , asn from the hcdr , tyr , asn , and ser from the hcdr , arg , tyr , asn , tyr , and tyr from the hcdr , tyr , and tyr from the lcdr , and arg and asp from the lcdr (fig. c) . specifically, d hcdr residues ser , tyr , and asn interact with pro and asp from the ntd, and a formed hydrogen bond is from d asn to ntd asp ( fig. d and supplementary fig. crystal structure of d -scfv bound to ntd and the binding interface. a an overall structure of the ntd/ d -scfv complex in which the ntd, n -linked glycans on the ntd, d v l , and d v h are colored in blue, gray, magenta, and cyan, respectively. b epitope on the ntd recognized by d . the ntd is represented as blue surface, on which the protein region bound by d is displayed in orange and the n -linked glycans are displayed as gray sticks. c d residues that are involved in the binding. the v l and v h are colored in magenta and cyan, respectively, and the residues interacting with d are displayed in orange. d interactions between the d v h residues and the corresponding residues of ntd. e interactions between the d v l residues and the corresponding residues of ntd. f zoom-in view of interactions between n -linked glycans and d interacting with tyr , asp , pro , asp , and arg of the ntd (fig. d ). tyr and asn of d form two hydrogenbonding interactions with asp of the ntd (supplementary table ). for the light chain, the lcdr and lcdr residues tyr , tyr , arg , and asp interact with glu , ser , arg , and asn of the ntd, and a salt bridge is formed between arg of lcdr and glu of the ntd (fig. e and supplementary table ) . a prominent feature at the interface is the extensive recognition of asn -linked glycans by all three heavy chain cdrs ( fig. f and supplementary table ). specific hydrogen-bonding interactions occur between tyr and arg of d and the nag and man glycans, respectively ( fig. f and supplementary table ). confirmation of the neutralizing epitope. to confirm the epitope and its critical residues, we performed a mutagenesis study by introducing single mutations to all ntd recognized residues including trp , asp , pro , asp , val , ser , glu , ser , asn , asn , lue , arg , and asn . we first examined the effects of these ntd mutations on the binding by d -h. the d -h bound the wild-type ntd with an affinity of approximately nm (table and supplementary fig. ). by contrast, the d a and r a mutations dramatically reduced the binding, to a level that was undetectable by bli experiment (table and supplementary fig. ). the e a and n q mutations reduced by the binding affinity by -fold to . μm and -fold to . μm, respectively (table and supplementary fig. ). all the other nine mutations had variant unequal effects on the binding by reducing the affinity in the range of -to -fold (table and supplementary fig. ). the effects of these mutations on the neutralizing activity of d -h were in consistent with the changes of binding affinity. pseudotyped mers-cov bearing d a, e a, or r a mutation in the spike glycoprotein escaped the neutralization by d -h (table and supplementary fig. ). the ic values of d against pseudotyped mers-cov bearing d a, v a, or n q mutation were increased approximately by -, -, and -fold (table and supplementary fig. ). the binding and neutralization assays collectively revealed that asp , val , glu , arg , and asn -linked glycans are critical for recognition and neutralization of mers-cov by d . sequencing of multiple clinical isolates had revealed that the mers-cov s glycoprotein is evolving at an average rate of . × − substitutions per site per year . alignments of the deposited sequences in the ncbi identified naturally changing residues from the prototype emc sequence including v f, v i, v a, d y, l f, t i, a y, l f, d g, v l, v a, e k, d e, v i, q r, q h, r h, r q, a s, t i, g s, and v a, which are located in the ntd (residues - ) and rbd (residues - ) of the s subunit, and the s subunit (residues - ). several residue changes on the rbd, such as those occurring on d , d , and e , indeed enabled the mers-cov to escape the neutralization of antibodies targeting the rbd , . considering that most of the mutations are outside the ntd, we speculated that d -h would have a better tolerance for these naturally occurring mutations. we generated pseudotyped mers-cov bearing the emc strain s glycoproteins and its mutants harboring all the listed residue changes. the neutralization assays showed that d -h showed effective neutralizing activity against almost all pseudotyped mers-cov variants. only the two mutations v f and v a on the ntd increased the ic value of d -h by more than -fold and significantly reduced its neutralization activity (fig. a, b) , which confirmed the results of the structural and biochemical studies of the binding interface. all other naturally occurring mutations, most of them on the rbd and the s subunit did not affect the neutralization capability of d -h (fig. a, b) , indicating that d would have a wide neutralization breadth against different variants of mers-cov. combination of d with other rbd-targeting antibodies. the current available mers-cov antibody epitopes with solved structures are all on the rbd, which can be grouped into three categories: ( ) epitope of mers- ; ( ) epitopes of mers- , d , c , and jc - ; and ( ) epitopes of m , mca , cdc-c , and the newly reported mers-gd ( supplementary fig. ) . in our study of the rbd-specific mab mers- , we also found synergism with the ntd-targeting mab f . thus, the elucidation of the epitope targeted by d , which added a category outside the rbd ( supplementary fig. ), prompted us to study the combined effect of d together with the three representative antibodies mers- , mers- , and mers-gd in the neutralization of pseudotyped mers-cov by titrating the neutralizing potency of an equimolar mixture of the two antibodies and comparing the dose response with that observed in neutralization assays performed with the individual antibody alone. as shown in the fig. , the combination index (ci) values of mers-gd combined with d at fa values of effective dose %, %, %, and % (ed , ed , ed , and ed , respectively) were . , . , . , and . , respectively. as a ci value of indicates an additive effect, < indicates synergism, and > indicates antagonism, the combination of d and mers-gd worked in a clearly synergistic manner. meanwhile, the combination index (ci) values of combined mers- with d at fa values of effective dose %, %, %, and % (ed , ed , ed , and ed ) were . , . , . , and . , respectively. thus, the combination of mers- and d also demonstrated synergism, in particular at relatively lower concentrations. however, the percent neutralization obtained using combined mers- and d showed no obvious difference of half maximal inhibitory concentration (ic ) compared with that of d alone. the combination index (ci) values of combined mers- and d at fa values of effective dose %, %, %, and % (ed , ed , ed , and ed ) were . , . , . , and . , respectively. it indicated that the combination of d with mers- exhibited neither synergy nor antagonism. mechanism of d neutralization. a major reported mers-cov neutralization mechanism relies on inhibiting the binding of the s trimer with the cellular receptor dpp . the epitopes of these reported antibodies all reside in the rbd responsible for receptor binding. the fact that the d epitope is outside the rbd indicated that it may have a different neutralizing mechanism. we first examined if d is still able to inhibit the receptor binding by the s trimer. the facs analysis of cellsurface staining showed that the scfv and fab fragments of d -h did not inhibit the staining of huh cells by the s trimer, while the d -h slightly reduced the staining (fig. a , supplementary table and supplementary fig. ). by contrast, the rbdtargeting mab mers- was much more potent than d -h in inhibiting the binding of the s trimer to huh cells. moreover, the fab and scfv fragments of mers- retained nearly the same potency in the inhibition ( fig. a and supplementary table ). surface plasmon resonance (spr) analysis confirmed these conclusions by showing that d -h, and not its fab or scfv fragments, could interfere with the binding of the s trimer to chipcoupled dpp in a dose-dependent manner ( supplementary fig. ) , while the igg, fab, and scfv of mers- all inhibited the binding ( supplementary fig. ) . to investigate why the igg, fab, and scfv of d inhibit receptor binding differently, we constructed models of their binding to the s trimer. the mers-cov s trimer structure was determined by cryo-em with the rbd in standing or lying positions, and only the standing rbd could bind to the dpp receptor. after superimposing the ntd/ d -scfv crystal structure onto the s trimer, we observed no steric clashes between three ntd-bound scfv fragments and one or two rbdbound dpp receptors ( fig. b and supplementary fig. ). the s trimer with three rbd-bound receptors was not considered because the cryo-em study of the mers-cov s trimer only revealed conformations with one or two standing rbds. when the scfv was replaced with the fab, there were also no steric clashes between the fab and dpp receptor (fig. c ). it is more complicated to model the binding of d -h to the s trimer, considering that the igg form has two binding sites and the intrinsic flexibility. we found that binding of the d -h igg to the ntd in certain orientations could inhibit the binding of dpp due to steric clashes, while there were still no steric clashes with the d -h bound in some other orientations (fig. d, e) . these results provided a structural explanation for the inability of d -h scfv and fab to inhibit the binding of the s trimer to the dpp receptor. they may also explain why the d -h igg form is not as potent as the mers- igg, fab, and scfv which all directly bind to the rbd. in parallel with biochemical studies, we also examined the neutralizing activities of d -h igg, fab, and scfv. the d -h fab and scfv did not interfere with the binding of the s trimer to the dpp receptor. however, they were still able to inhibit the cell entry of pseudotyped mers-cov with ic value of . μg ml − and . μg ml − , respectively (fig. a) . although the d -h fab and scfv are less active than the igg in infection inhibition, they were still comparable to the fab or scfv fragments of several reported rbd-targeting antibodies such as mers- fab (ic : . μg ml − ) and mers- scfv (ic : . μg ml − ) ( supplementary fig. a ). these results collectively indicated that neutralization by d -h involves other mechanism besides interfering with the initial receptor binding. we tested and compared the neutralizing activity of d in pre-attachment and post-attachment settings. after the cell attachment, d was still able to inhibit infection by pseudotyped mers-cov with an ic of . μg ml − (fig. b) . in comparison, mers- , which is more potent than d in inhibiting receptor binding, exhibited very weak neutralization after receptor binding (supplementary fig. b) . the above results, especially the retaining activity of d after viral attachment indicated that d would also interfere with the prefusion to postfusion conformational transition of the s glycoprotein required for membrane fusion. this transition , . we showed that the mers-cov s glycoprotein in the prefusion state is sensitive to the digestion of proteinase k (fig. c) . previous studies have demonstrated that cleavage at the s /s site by trypsin and the binding with cellular receptor greatly enhanced the prefusion to postfusion transition of the spike glycoprotein . consistently, the amount of a kda and proteinase-k-resistant band of the s glycoprotein representing the postfusion six-helix bundle was at the maximum level in the presence of trypsin and dpp (fig. c) . and the addition of d -h fab obviously reduced the intensity of the band (fig. c) . meanwhile, we analyzed the full-length mers-cov s trimer embedded in the membrane of pseudotyped virus and the trigger we used to induce the conformational transition was the incubation with huh cells that endogenously expressing dpp receptor. after incubating the pseudotyped virus with huh cells for h at °c, a proteinase-k resistant band on the sds-page gel appeared and the addition of d -h, d -h fab, or d scfv all clearly decreased the intensity of this band ( supplementary fig. ) . thus, these biochemical results strongly suggest that d could also exert its neutralizing activity in the postattachment stage after receptor-binding by inhibiting the conformational transition of the s glycoprotein required for membrane fusion (fig. d) . since dpp , which is a critical step for viral cell attachment. in this study, we first isolated the neutralizing mouse antibody d targeting the ntd of the s glycoprotein. neutralization assays showed that d is highly potent and its activity is comparable to that of the most potent rbd-targeting antibodies. structural determination of d scfv bound to the ntd and mutagenesis studies revealed the epitope and key residues on the ntd for binding and neutralization at atomic level. comparisons of d scfv, fab, and igg forms in dpp -binding competition and neutralization assays indicated that its activity is not solely dependent on the inhibition of dpp binding. further experiments indicated that the neutralizing activity of d after cell attachment is through the inhibition of prefusion to postfusion conformational transition of the s glycoprotein trimer, which mediates the fusion of viral and cell membranes. we also showed that d has a wide neutralization breadth against mers-cov variants bearing naturally occurring mutations and exhibited synergistic effects with several rbd-targeting antibodies. these results collectively revealed an antibody epitope and neutralization mechanism on the s glycoprotein, which would contribute to the global efforts to control mers-cov infection and transmission by providing alternatives for mers-cov immunotherapy. similar the ntds of the s protein of other betacoronaviruses such as mhv, bcov and hku , that of mers-cov also folds into a galectin-like structure. although the galectin domain is a typical carbohydrate-recognition domain, the betacoronavirus ntds can include structural variations that enable more diverse functions in viral infection. the examples, include the ntd of bcov that retains the glycan-binding activity recognizing -n-acetyl- -oacetylneuraminic acid (neu , ac ) and the ntd of mhv that evolved specific protein-protein interactions with its cellular receptor ceacam , and both interactions are important for the viral cell attachment , . however, there is still no report on the glycan or protein-binding activities of the mers-cov ntd. in fact, crystallographic structure determination showed that the glycanbinding site on the mers-cov ntd is occupied by a short helix (residues - ) and the asn -linked glycan, indicating that it is not able to bind glycans in the same way as the ntd of bcov . notably, the asn -linked glycan is involved in the recognition by d , whereby nag and man undergoes specific hydrogen-bonding interactions with tyr and arg of d , respectively. the ntd n q mutation also dramatically reduced the binding and neutralization by d , but did not dramatically affect the cell infection of pseudotyped mers-cov ( supplementary fig. ) . therefore, the asn -linked glycan serves as an important anchor point for the binding of d to the mers-cov ntd. as the largest class i viral fusion protein, the coronavirus s glycoprotein is expected to undergo a prefusion to postfusion conformational transition to mediate the interaction between viral and cellular membrane proteins, although structural studies just began to shed light on this recently. the s glycoprotein of betacoronaviruses mhv and hku , whose structures have been determined by the cryo-em method, all adopt a similar prefusion homotrimeric architecture , . interestingly, in the prefusion architecture of the s trimer of highly pathogenic mers-cov and sars-cov, two major conformational states were observed. a d -h igg was tested for neutralizing activity against pseudotyped mers-cov before or after receptor binding. vrc mab was used as unrelated control. c the effect of d -h fab on the conformational change of the mers-cov s trimer was probed by western blotting using an anti-mers-cov s polyclonal antibody. refolding to the postfusion conformation was detected by the appearance of a proteinase-k resistant band. trypsin was used at μg ml − and proteinase k at μg ml − . digestion experiments and western blots were performed in triplicates, and a representative result is shown for each of them. d a cartoon representation designed by us showing the neutralization mechanism by which d blocks mers-cov entry. on the one hand, some virus particles can not bind to dpp due to steric hindrance caused by d binding. on the other hand, d still recognizes the particles when the up receptor-binding domain (rbd) binds to dpp , and may inhibit the prefusion to postfusion transition of the s subunit and the initiation of membrane fusion. source data are provided as a source data file major difference between them is the change of the rbd in the s subunit from a down to an up position, which was proposed to be a prerequisite for the binding of the s trimer to their respective cellular receptor dpp and ace . this proposal was recently confirmed by our cryo-em study of the sars-cov s trimer in complex with ace , and we also showed that ace -binding could induce the dissociation of the s subunit, which results in the falling apart of the prefusion s trimer and the transition to the prefusion state of the s subunit . a major neutralization mechanism of antibodies against mers-cov is to directly or indirectly compete with the cellular receptor dpp for binding to the rbd. in theory, antibodies that interfere with the coronavirus membrane fusion process other than receptor binding would also have a neutralizing activity, and the d mab targeting the ntd we studied is one such example. here, we showed that d neutralization is not solely dependent on dpp -binding competition, and its inhibition of the s trimer conformational transition after cell attachment also plays a significant role in the neutralization. we suggested that the binding of d may stabilize the prefusion architecture of the s trimer, even after the binding of dpp receptor. the stabilization of viral fusion protein at one conformational state for neutralization has also been observed and studied in other viruses such as hiv. a recent study revealed that the hiv env trimer is intrinsically dynamic with three major and distinct prefusion conformations . among them, the closed, ground-state conformation is dominant and could be remodeled to another two conformations by cd receptor binding, which is essential for the subsequent prefusion to postfusion transition . the binding of neutralizing antibodies, whether inhibiting the binding of the cd receptor (such as vrc ) or not (such as g and pgt ) all resulted in the stabilization of the ground-state conformation of the env, which finally disfavors its prefusion to postfusion state transition required for viral entry , . to the best of our knowledge, our study offers the first structural definition of the neutralizing epitope of an antibody targeting the s ntd of mers-cov. as we summarized in supplementary table , a total of six anti-ntd mabs have been reported , , , . all of them neutralize the infection of pseudotyped mers-cov emc strain with high potency except for mab . f . the mab f and our d showed the same neutralizing activity against live mers-cov in plaque reduction neutralization testing. notably, the mouse mab g can greatly relieve the symptom of dpp -transgenic mice infected following mers-cov infection and our d -h can inhibit the infection of pseudotyped mers-cov in r -hdpp mice. however, the specific neutralizing epitopes and mechanisms of f , g , jc - , and fib-h are largely unknown. in addition, the combination of different antibodies is supposed to be an effective strategy to combat mers-cov infection as it continues to spread among multiple animal species and to probe and adapt to the human population [ ] [ ] [ ] . an effective combination would require the candidate antibodies to bind to disparate epitopes or with distinct mechanisms and hence display additive or synergistic effects, as the mabs mers- and f we mentioned before . although the exact mechanism that leads to the synergy or additive is uncertain, our d -h with mers-gd or mers- antibodies demonstrated a synergy in inhibiting the infectivity of pseudotyped mers-cov, while d -h and mers- antibodies together had an additive effect. consequently, d is currently the most comprehensively studied ntd-targeting mab with a different epitope and working mechanism, which makes it an excellent candidate, in combination with other rbd-targeting neutralizing antibodies or alone, in our battle against mers-cov infection. three weeks after the initial immunization, these mice were boosted twice at -week intervals. cells collected from the spleens of sacrificed animals were fused with cultured sp / cells at a : ratio in the presence of peg (sigma). hat selection medium was used for the fused hybridoma cultures. after -weeks of incubation, the positive hybridomas were selected via s-coated elisa, and the positive clones were subjected to limited dilutions and downstream validation. for large-scale mab production, ascites fluid from mice inoculated with the hybridomas was collected and purified by the caprylic acid-ammonium sulfate precipitation method. protein expression and purification. the coding sequence of the mers-cov spike glycoprotein ectodomain (emc strain, spike residues - ) was ligated into the pfastbac-dual vector (invitrogen) with a c-terminal t fibritin trimerization domain and a hexa-his-strep tap tag to facilitate further purification processes. briefly, the protein was prepared using the bac-to-bac baculovirus expression system, purified by sequentially applying strep-tactin and superose column (ge healthcare) with hbs buffer ( mm hepes, ph . , mm nacl). fractions containing mers-cov s glycoprotein were pooled and concentrated for subsequent biochemical analyses. the sequence encoding mers-cov s ntd (residues - ) with a c-terminal hexa-his tag was inserted into the eukaryotic expression vector pvax. freestyle -f cells were transfected with the plasmid using polyethylenimine (pei) (sigma). after h, the supernatant was collected and the ntd was purified using nta sepharose (ge heathcare) and superdex high performance column (ge healthcare) with hbs buffer ( mm hepes, ph . , mm nacl). the sequence encoding the d v l and v h were separately cloned into the backbone of antibody expression vectors containing the constant regions of human igg . the chimeric antibody d -h was expressed in freestyle -f cells by transient transfection and purified by affinity chromatography using protein a sepharose and gel-filtration chromatography. the purified d -h was exchanged into phosphate-buffered saline (pbs), and was digested with papain protease (sigma) over night at °c. the digested antibody was then passed back over protein a sepharose to remove the fc fragment, and the unbound fab in the flow through was additionally purified using a superdex high performance column (ge healthcare). the gene encoding the d v l followed by v h with a connecting triple gggs linker and a c-terminal hexa-his tag was synthesized and cloned into the eukaryotic expression vector pvrc . freestyle -f cells were transfected the plasmid in the presence of pei (sigma). the cell-culture supernatant was collected h after the transfection, and the d scfv was collected and captured on nta sepharose (ge healthcare). the bound d scfv was eluted with hbs buffer containing mm imidazole and was then further purified by gel-filtration chromatography using a superdex high performance column (ge healthcare). complex preparation and crystallization. the mers-cov ntd and the scfv fragment of d were mixed at a molar ratio of : . , incubated for h at °c and further purified by gel-filtration chromatography. the purified complex concentrated to approximately mg ml − in hbs buffer ( mm hepes, ph . , data collection and structure determination. to collect the diffraction data, all crystals were flash-cooled in liquid nitrogen after being incubated in reservoir solution containing % (v/v) glycerol. the diffraction images were collected on the bl u beamline at the shanghai synchrotron research facility (ssrf) with the wavelength of . Å. all images were processed with hkl . the structure was solved by molecular replacement using phaser from the ccp suite . the search models were the mers-cov ntd structure (pdb id: vyh) and the structures of the variable domain of the heavy and light chains available in the pdb with the highest sequence identities. subsequent model building and refinement were performed using coot and phenix, respectively , . there are % of most favored, . % of allowed and . % of disallowed ramachandran plot in the final refinement model. all structural figures were generated using pymol . neutralizing assay of pseudotyped mers-cov. t cells cultured in mm dish were co-transfected with μg of pcdna . -mers-spike or its mutants and μg of pnl - .luc.re. the supernatants containing sufficient pseudotyped mers-cov were harvested - h post-transfection. subsequently, the % tissue culture infectious dose (tcid ) was determined by infection of huh cells. for the neutralization assay, tcid per well of pseudoytped virus were incubated with or serial : dilutions of purified antibodies, fabs or scfvs for h at °c, after which huh cells (about . × per well) were added. after incubation for h at °c, the neutralizing activities of antibodies were determined by the luciferase activity and presented as ic , calculated using the dose-response inhibition function in graphpad prism (graphpad software inc.) cell entry of pseudotyped virus. the concentration of the harvested pseudotyped virions was normalized by p elisa kit (beijing quantobio biotechnology co., ltd., china) before infecting the target huh cells. the infected huh cells were lysed at h after infection and viral entry efficiency was quantified by comparing the luciferase activity between pseudotyped viruses bearing the mutant-and wildtype mers-cov spike glycoproteins. postattachment neutralization assay. for the postattachment pseudotyped virus neutralization assay, huh cells, upon reaching a density of . × per well in a -well plate, were incubated with tcid per well of pseudotyped virus at °c for h. after removing the supernatant, μl of pbs was added twice to each well to wash the un-bond pseudotyped viruses. a total of serial : dilutions of purified antibodies in dmem ( % fbs) were then added to the huh cells with attached pseudotyped viruses, as well as dmem ( % fbs) alone as control. neutralization activities were determined based on the luciferase activity after incubation for h at °c and also presented as ic , calculated using the dose-response inhibition function in graphpad prism (graphpad software inc.) cooperativity of mabs for neutralization. synergistic, additive, and antagonistic interaction between d and mers-gd , d , and mers- , as well as d and mers- for virus neutralization were evaluated by the median effect analysis method using compusyn software as previously reported , . the measured neutralization values were input to the program as fractional effects (fa) ranging between . and . for each of the two antibodies and for both in combination. ci values were calculated in relation to fa values. a logarithmic ci value of indicates an additive effect, < indicates synergism, and > indicates antagonism. live mers-cov neutralization assay. the neutralizing activity of the mabs against live mers-cov was also determined in dpp -expressing vero e cells. upon reaching a density of × per well in a -well plate, cell monolayers were infected with - plaque-forming units (pfu) of live virus in the presence or absence of the mab. after three days of incubation at °c, the inhibitory capacity of the mabs was assessed by determining the numbers of plaques compared with the potent mers-cov anti-rbd and anti-n mabs. murine model of mers-cov pseudovirus infection. the mers-cov susceptible animal model hdpp -knockin mouse, which was established by inserting human dipeptidyl peptidase (hdpp ) into the rosa locus using crispr/cas , resulting in global expression of the transgene in a genetically stable mouse line , was used in this experiment. mice (n = ) were challenged by intraperitoneal injection (i.p.) with doses of . × . tcid of pseudotyped mers-cov. d -h and mers- were administered i.p. to r -hdpp mice at a dose of μg per mouse prior to challenge with pseudovirus. mice (n = for the pbs group and n = for the c group) were also administered pbs or control mab c (mab of anti-na of h n , at a dose of μg per mouse) and challenged using the same i.p. dose of pseudovirus. the ivis-lumina ii imaging system (xenogen, baltimore, md, usa) was used to detect bioluminescence. prior to measuring luminescence, the mice were anesthetized using an i.p. injection of sodium pentobarbital ( mg kg − ). the exposure time was s, and fluorescence intensity in regions of interest was analyzed using living image software (caliper life sciences, baltimore, md, usa). different wavelengths were used for detecting pseudovirus and tdtomato fluorescence. the substrate, d-luciferin ( mg kg − , xenogen-caliper corp., alameda, ca, usa), was injected i.p. and imaging was conducted min later. the relative intensities of emitted light were represented as colors ranging from red (intense) to blue (weak) and quantitatively presented as photon flux in photons s − cm − sr − . binding studies using bli. binding kinetics of mers-cov ntd and its mutants with d were studied using a fortébio octet htx instrument. assays with agitation set to rpm in hbs buffer ( mm hepes, ph . , mm nacl) supplemented with . % (v/v) tween were performed at °c in solid black tilted-bottom -well plates (greiner bio-one). d ( μg ml − ) was used to load anti-human igg fc capture probes for s to capture levels of . - nm. biosensor tips were then equilibrated for s in hbs buffer supplemented with . % (v/v) tween prior to binding assessment with different concentrations of wild-type or mutant mers-cov ntd for s, followed by dissociation for s. data analysis and curve fitting were performed using octet software, version . . binding competition assays by spr. real-time binding and analysis by spr were conducted on a biacore t instrument with cm chips (ge healthcare) at room temperature. for all the analyses, hbs buffer consisting of mm hepes, ph . , mm nacl and . % (v/v) tween was used, and all proteins were exchanged to the same buffer. the blank channel of the chip was used as the negative control. dpp ( μg ml − ) was immobilized on the chip at about response units. soluble mers-cov spike trimer (s) at the same gradient in the present or absence of the concentration gradient of iggs, fabs, or scfvs was flowed over the chip surface. after each cycle, the sensor surface was regenerated with . mm naoh. data were analyzed using the biacore t evaluation software by fitting to a : langmuir binding model. facs analysis of cell-surface staining. the binding between recombinant soluble mers-cov spike trimer (s) and human dpp expressed on the surface of huh cells was measured using fluorescence-activated cell sorting (facs). all cellsurface staining experiments were performed at room temperature. soluble mers-cov spike trimer (s) with strep-tag ( μg) was incubated with monoclonal antibodies (mabs) in advance at molar ratios of : , : , : , and : for h. huh cells were trypsinized and then incubated with s or s and mabs mixtures for h. after washing the un-bound s with pbs times, the huh cells were then stained with streptavidin apc (bd ebioscience) for another min. cells were subsequently washed with pbs times and analyzed by flow cytometry on a facs aria iii machine (bd ebiosciences). western blots. totally, μl pseudotyped mers-cov was thawed and mixed with μg of antibodies (igg, fab or scfv) for h. the virus alone or the mixture was incubated with μl of huh cell suspension for another h at °c. an equal volume of buffer and proteinase-k (final concentration of μg ml − ; thermo_fisher) was then added and incubated h at °c. for the soluble s, μg of the s trimer was incubated with μg of the dpp ectodomain or μg of d fab for h on ice. trypsin (final concentration of μg ml − ; thermo_-fisher) was then added to these samples and incubated min at °c. subsequently, the samples were supplemented with μg ml − proteinase-k and incubated min at °c. × sds-page loading buffer was then added to all samples prior to boiling at °c. samples were run on a - % gradient tris-mops-gel (genscript) and transferred to polyvinylidene fluoride membranes. an anti-s mers-cov s polyclonal antibody ( : dilution; thermo_fisher; cat#pa - ) and an hrp-conjugated goat anti-rabbit secondary antibody ( : dilution; huaxingbio; cat#hx ) were used for western blotting. ai was used to develop images. reporting summary. further information on research design is available in the nature research reporting summary linked to this article. the source data underlying figs. a-d, , , and a-c and supplementary figs. a-c, , , , - are provided as a source data file. crystal structures presented in this work has been deposited in the protein data bank (pdb) and are available with accession code j . isolation of a novel coronavirus from a man with pneumonia in saudi arabia the emerging novel middle east respiratory syndrome coronavirus: the "knowns" and "unknowns middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation human infection with mers coronavirus after exposure to infected camels, saudi arabia environmental contamination and viral shedding in mers patients during mers-cov outbreak in south korea history of passive antibody administration for prevention and treatment of infectious diseases structure of mers-cov spike receptor-binding domain complexed with human receptor dpp host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein cryo-electron microscopy structures of the sars-cov spike glycoprotein reveal a prerequisite conformational state for receptor binding immunogenicity and structures of a rationally designed prefusion mers-cov spike antigen cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains cryo-em structure of the sars coronavirus spike glycoprotein in complex with its host cell receptor ace a conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in middle east respiratory syndrome coronavirus spike protein potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein identification of human neutralizing antibodies against mers-cov and their role in virus adaptive evolution exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized 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multiple conformation-dependent epitopes that induce highly potent neutralizing antibodies a human dpp -knockin mouse's susceptibility to infection by authentic and pseudotyped mers-cov spread, circulation, and evolution of the middle east respiratory syndrome coronavirus two-step conformational changes in a coronavirus envelope glycoprotein mediated by receptor binding and proteolysis unexpected receptor functional mimicry elucidates activation of coronavirus fusion protective effect of intranasal regimens containing peptidic middle east respiratory syndrome coronavirus fusion inhibitor against mers-cov infection passive transfer of a germline-like neutralizing human monoclonal antibody protects transgenic mice against lethal middle east respiratory syndrome coronavirus infection prophylaxis with a middle east respiratory syndrome coronavirus (mers-cov)-specific human monoclonal antibody protects rabbits from mers-cov infection b -n, a monoclonal antibody against mers-cov, reduces lung pathology in rhesus monkeys following intratracheal inoculation of mers-cov jordan-n / crystal structure of bovine coronavirus spike protein lectin domain crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor pre-fusion structure of a human coronavirus spike protein cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer conformational dynamics of single hiv- envelope trimers on the surface of native virions broadly neutralizing antibodies and the search for an hiv- vaccine: the end of the beginning towards a solution to mers: protective human monoclonal antibodies targeting different domains and functions of the merscoronavirus spike glycoprotein hiv therapy by a combination of broadly neutralizing antibodies in humanized mice human monoclonal antibodies against highly conserved hr and hr domains of the sars-cov spike protein are more broadly neutralizing improving neutralization potency and breadth by combining broadly reactive hiv- antibodies targeting major neutralization epitopes automatic crystal centring procedure at the ssrf macromolecular crystallography beamline processing of x-ray diffraction data collected in oscillation mode phaser crystallographic software coot: model-building tools for molecular graphics phenix: building new software for automated crystallographic structure determination pymod . : improvements in protein sequence-structure analysis and homology modeling within pymol quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors drug combination studies and their synergy quantification using the chou-talalay method we would like thank dr. changfa fan (division of animal model research, institute for laboratory animal resources, national institutes for food and drug control) for help in providing the r -hdpp mouse model and experimental method. we thank dr. jianhua he and the staff scientists at the ssrf bl u beam line, as well as dr. shilong fan at the x-ray crystallography platform of the tsinghua university technology center for assistance in diffraction data collection. this work was supported by the national key plan for scientific research and development of china (grants yfd and yfd ), the national natural science foundation of china (grants and u ), and the national major project for control and prevention of infectious disease in china ( zx - ). h.z., w.t., l.z. and x.w. designed the experiments. y.c., k.q. and w.t. isolated the antibody d and sequenced the corresponding v l and v h genes. b.h. carried out the neutralizing assay with live mers-cov. h.z. and s.z. expressed, purified, and crystallized the protein, and h.z. carried out the bli and spr analysis. h.z. conducted all the neutralizing assays based on pseudotyped mers-cov with the help of w.j. h.z. conducted dpp -competition assays and the western blots analysis. p.n. performed the protection assay in mice. h.z. and j.l. collected the diffraction data. h.z. and x.w. processed the diffraction data, determined, and analyzed the structure. h.z. and x.w. wrote the paper with contributions from l.z. and w.t. supplementary information accompanies this paper at https://doi.org/ . /s - - - .competing interests: the authors declare no competing interests.reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ peer review information: nature communications thanks the anonymous reviewers for their contribution to the peer review of this work. peer reviewer reports are available.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/ licenses/by/ . /. key: cord- -a d dkv authors: hirsch, hans h title: spatiotemporal virus surveillance for severe acute respiratory infections in resource-limited settings: how deep need we go? date: - - journal: clin infect dis doi: . /cid/ciy sha: doc_id: cord_uid: a d dkv nan following the discovery of molecular cloning, nucleic acid sequencing, and polymerase chain reaction, the wonderland of molecular biology has opened its doors again to virtually unlimited possibilities, as next-generation/deep sequencing or, better, massive parallel sequencing allows for the comprehensive analysis of all genomic nucleic acids of any origin in any given sample in real time. initially applied to the bacterial microbiome, deep sequencing has similarly detected a multitude of viral genome sequences, and changed hitherto common views on persisting viral infections [ ] , putting forward the concept of the "virome" in general, and of the "human viromes" in particular [ ] . while the numeric summary of viral genome sequences is mind-blowing, even after careful annotation and curation through refined bioinformatic tools, the utility of massive parallel sequencing has been prominently demonstrated in epidemiologic cluster analysis, for example, leading to the identification of a novel arenavirus as the cause of donor-derived death of solid organ transplant recipients [ ] , or of a novel human polyomavirus causing lethal multiorgan failure in a pancreas transplant recipient [ ] . given the technical and bioinformatic advances as well as the declining laboratory costs, the application of deep sequencing to identify etiologic agents in clinical samples has been approached in different pathologies, including those caused by community-acquired respiratory viruses (carvs). despite the general enthusiasm for metagenomics, it remains important to realize that there are caveats. in fact, multiple technical and conceptual aspects influence the results of deep sequencing, may cause significant differences in the read-outs, and hence bias their interpretation. conceptually, it is clear that the human virome is different in different organ sites and body location (eg, the gastrointestinal, urogenital, or respiratory tract) and is subject to dynamic changes of the viral constituents, not only as a result of physiological changes (eg, dietary, hormonal, aging), but also due to complex pathological alterations such as trauma, inflammation, and immune responses [ ] , exemplified in inflammatory bowel disease or transplantation [ , ] . technically, nucleic acid extraction is presumed to be representative across all different agents, whereas in fact different sample preparations, buffers, purification, single fragment partitioning, and sequence analytic procedures may introduce an as-yet little-understood bias, which can lead to underrepresentation of certain viral genomes-for example, human coronavirus compared with paramyxovirus genomes (h. h. hirsch, unpublished observation). thus, every step of the wet and dry laboratory procedure is dependent on the methodology, and may yield potentially different results [ ] . independent confirmatory testing has become an essential part of the presumably hypothesis-free deep sequencing [ ] . these aspects are important when discussing the results of massive parallel sequencing to either rule in or rule out the involvement of a pathogen in a given pathology. with these caveats in mind, and given the significant global burden of viral respiratory tract disease in the very young and the very old [ ] [ ] [ ] oropharyngeal (np/op) samples of sari cases were identified through a national surveillance study conducted by the uganda virus research institute from through . the virome present in influenza virus-negative np/op samples was analyzed by deep sequencing enriching for viral sequences. the epidemiologic data were collected by sentinel hospitals in different geographic areas, and hence focused on sari (eg, pediatric and adult patients admitted to hospital for severe influenza-like illness), thereby combining feasibility and clinical significance. the numbers are impressive, as sari cases were identified, ( . %) of which were influenza-positive np/op samples. of the remaining cases of influenza-negative sari, had spatial and temporal data available to pinpoint clusters involving ( . %) influenza-negative sari cases. notably, the demographic characteristics of the ugandan sari cases indicated that mostly young children were enrolled, % of whom were < year of age, both within and outside of clusters. although coughing and shortness of breath were leading features of sari cases of both clustered and nonclustered cases, fever with temperatures > °c and stridor were significantly more frequent in cluster cases than in noncluster cases. in two-thirds of the sari cluster cases, a virus-capturing technique and massive parallel sequencing using the hiseq illumina system yielded informative genome sequences in %, identifying human rhinovirus in . %, human paramyxoviruses consisting of respiratory syncytial virus in . %, and parainfluenza viruses in . %, metapneumovirus in . %, followed by coronaviruses ( . %), adenoviruses ( . %), and coxsackie virus ( . %) and other enteroviruses ( . %). these numbers compare well with results from resource-rich settings testing symptomatic children and adults using multiplex nucleic acid testing (nat) [ ] [ ] [ ] , whereby the detection rate in children was around % and significantly higher than the % found in adults [ ] , and where human rhinoviruses/enteroviruses together accounted for > % of the detected carvs [ ] . while this suggests that, by and large, a significant part of the pathogens could have been detected by multiplex nat for carvs, the deep sequencing approach of this study revealed a number of remarkable surprises, which may help to close the gap on at least %- % of the carvnegative multiplex nat results [ ] . cytomegalovirus (cmv) was identified in . % of the sari clusters, and may either reflect reactivation as a bystander of significant inflammatory states including infections with other including viral pathogens [ ] [ ] [ ] , or may in fact reflect the manifestation of cmv primary infections as sari, a hypothesis viable in view of the high seroprevalence in developing countries [ ] and the very young age of % of the sari cluster patients described here. similarly, the discovery modus of deep sequencing identified more somewhat expected pathogens in that age group, such as human herpesvirus and parvovirus b , for which similar arguments could be made. by the same token, it is surprising that other viruses were not reported, some of which had been proposed to be associated with respiratory symptoms or febrile tonsillitis such as bk virus and other polyomaviruses [ ] . similarly, the complete absence of the apparently ubiquitous anelloviruses, which have received much attention in studies of respiratory samples from patients with inflammatory conditions or transplantation, is notable [ , [ ] [ ] [ ] . however, no samples from the lower respiratory tract were available for this analysis. the discovery potential of deep sequencing of this us-ugandan study is, however, highlighted impressively by the identification of picobirnavirus in cases, as well as measles, as the cause of sari clusters in cases, of which belonged to genotype b , which is endemic in eastern africa. remarkably, cases were caused by genotype b , and could be traced back to an unvaccinated tourist from england and an outbreak in manchester, united kingdom, in . thus, global travel and outbreak have been linked. even though undervaccination may play an important role in the ugandan setting, it is clear that measles outbreaks have been a problem in resource-rich settings such as in switzerland, where cases have occurred in the year (incidence of . per million inhabitants), causing outbreaks among mostly unvaccinated individuals, despite the current -dose vaccination coverage of % and % of -and -year-old children, respectively [ ] . of note, measles virus also belongs to the human paramyxovirus family and is transmitted via respiratory fluids, but was detected alone as well as together with other viral pathogens in the present study by cummings et al. taken together, this report from resource-limiting settings is also of relevance for resource-rich countries and raises the question about how to best expand current first-or second-line testing for respiratory viral pathogens including cmv, parvovirus b , and measles, and how to move to more deep sequencing virome analysis and comprehensive metagenomics in the near future. the spatiotemporal cluster analysis, combined with viral metagenomics presented in this study, is of interest also for other settings, and raises the question of how and when this is can be best delivered in real time. such efforts are under way for influenza virus vaccine prediction [ ] , the pacemaker of respiratory viral research, and will be of utility for characterizing vaccine failure and antiviral drug resistance. the average ugandan cluster consisted of cases within a -km radius and days, and viruses were detected in only some of the patients. not unexpectedly, certain factors were associated with the ugandan sari clusters, such as human immunodeficiency virus (hiv) infection, urban location, higher population density (eg, . vs persons/km ), or higher median annual rainfall of vs mm/year. these differences, though statistically significant, need to be put in perspective, as the study was anchored in sentinel hospitals, typically located in urban areas where hiv status is more likely to be known, whereas sari cases in rural areas may not have been equally able to seek hospital admission. given the > -fold higher average population density of boston ( persons/km ), or basel ( persons/km ), one might suspect that the associations in the ugandan setting may have been a surrogate for other factors such as general hygiene standards, other medical conditions, and socioeconomic factors in certain suburban quarters. similarly, the differences in annual rainfall of < % need to be balanced against larger seasonal changes in short-term higher rainfall, which may be more important for case clustering and should be addressed in future studies. despite a critical appreciation, this collaborative us-ugandan study combining epidemiology with state-of-the-art modeling and virology not only sets standards for resource-rich industrial settings, but provides next to hiv, ebola, and malaria another proof-of-concept of feasibility and impact of innovative joint projects and partnerships between advanced research groups and dedicated institutions in resource-limited countries. potential conflicts of interest. author certifies no potential conflicts of interest. the author has submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. redefining chronic viral infection translational aspects of the human respiratory virome a new arenavirus in a cluster of fatal transplant-associated diseases identification of a novel polyomavirus in a pancreatic transplant recipient with retinal blindness and vasculitic myopathy the human gut virome: inter-individual variation and dynamic response to diet intestinal domination and the risk of bacteremia in patients undergoing allogeneic hematopoietic stem cell transplantation temporal response of the human virome to immunosuppression and antiviral therapy full-length haplotype reconstruction to infer the structure of heterogeneous virus populations evaluation of unbiased next-generation sequencing of rna (rna-seq) as a diagnostic method in influenza virus-positive respiratory samples global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis mortality associated with influenza and respiratory syncytial virus in the united states global mortality estimates for the influenza pandemic from the glamor project: a modeling study comparing viral respiratory tract infections in symptomatic children and adults: multiplex nat from - comparing luminex nxtag-respiratory pathogen panel and respifinder- for multiplex detection of respiratory pathogens community-acquired respiratory paramyxovirus infection after allogeneic hematopoietic cell transplantation: a single-center experience comprehensive diagnostics for respiratory virus infections after transplantation or after potential exposure to swine flu a/h n : what else is out there? high incidence of active cytomegalovirus infection among septic patients sepsis and cytomegalovirus: foes or conspirators? viral impact on long-term kidney graft function human cytomegalovirus: clinical aspects, immune regulation, and emerging treatments bk virus: opportunity makes a pathogen reactivation of multiple viruses in patients with sepsis torque teno virus in children who underwent orthotopic liver transplantation: new insights about a common pathogen viral infection in acute exacerbation of idiopathic pulmonary fibrosis the evolution of seasonal influenza viruses key: cord- -u svz pf authors: li, jifen; eberwine, james title: the successes and future prospects of the linear antisense rna amplification methodology date: - - journal: nat protoc doi: . /nprot. . sha: doc_id: cord_uid: u svz pf it has been over a quarter of a century since the introduction of the linear rna amplification methodology known as antisense rna (arna) amplification. whereas most molecular biology techniques are rapidly replaced owing to the fast-moving nature of development in the field, the arna procedure has become a base that can be built upon through varied uses of the technology. the technique was originally developed to assess rna populations from small amounts of starting material, including single cells, but over time its use has evolved to include the detection of various cellular entities such as proteins, rna-binding-protein-associated cargoes, and genomic dna. in this perspective we detail the linear arna amplification procedure and its use in assessing various components of a cell's chemical phenotype. this procedure is particularly useful in efforts to multiplex the simultaneous detection of various cellular processes. these efforts are necessary to identify the quantitative chemical phenotype of cells that underlies cellular function. in the original cell or tissue are maintained. the assumption is that the relative abundances of the amplified rnas will reflect the biological state of the source, just as the endogenous rna abundances do. to avoid systematic errors of uneven amplification of different rnas that can occur with exponential amplification procedures (where error is also exponentially amplified), scientists developed linear nucleic acid amplification. linear rna amplification is an isothermal nucleic acid amplification system that rapidly amplifies target rna populations. there are two primary linear amplification procedures in use today: linear arna amplification and rolling circle amplification. in this perspective we concentrate on how the arna amplification methodology has been developed for a host of chemical analyses, so no further discussion of rolling circle amplification ensues. linear arna amplification is in vitro rna-transcription-mediated amplification, and is also referred to as amplified rna or simply arna. this technique was first developed and published in (ref. ) as a method to amplify rna populations, but since then it has been used for a number of other applications. in this procedure, van gelder et al. first used reverse transcription to create a cdna copy of the rna template, incorporating an rna polymerase promoter into each cdna molecule by priming cdna synthesis with a synthetic oligonucleotide (oligo(dt)-t ) that contained a poly(t) and phage t rna polymerase promoter sequence. the poly(t) portion of the primer is used to select for mrna species that contain a poly(a) tail, and the t promoter portion of the primer is used to direct t polymerase to bind to the promoter and synthesize an rna copy of the cdna template. this over the past ~ years, particular emphasis has been placed on understanding the transcriptome of cells, with the knowledge that the fundamental player in converting genomic information into biological function is rna. whether translated into protein or functioning in a regulatory manner, rnas underlie many aspects of cellular function, and it has become clear that their dysregulation is associated with various diseases. understanding rna biology requires detection, quantification, and sequencespecific characterization of the composite rnas. the technical evolution of cell biology methods has resulted in the relatively easy analysis of rna populations from organs and tissues where there are large amounts of rna. however, transcriptomics analyses become problematic when smaller amounts of tissue, such as punch biopsies, are used for human diagnosis, or in examinations of individual single cells where rna is present in small amounts and needs to be amplified to levels that can be experimentally analyzed. for example, there are ~ , expressed genes in most cells , giving rise to , total mrna molecules; thus the average number of rna molecules is per gene, with some genes transcribed into thousands of mrnas, and others into fewer than ten. one way to detect such low-abundance rnas is to amplify their numbers. the two most important considerations in the design of rna amplification methods are the yield of amplification and how well the relative abundances of the different rnas present the successes and future prospects of the linear antisense rna amplification methodology it has been over a quarter of a century since the introduction of the linear rna amplification methodology known as antisense rna (arna) amplification. whereas most molecular biology techniques are rapidly replaced owing to the fast-moving nature of development in the field, the arna procedure has become a base that can be built upon through varied uses of the technology. the technique was originally developed to assess rna populations from small amounts of starting material, including single cells, but over time its use has evolved to include the detection of various cellular entities such as proteins, rna-binding-protein-associated cargoes, and genomic dna. in this perspective we detail the linear arna amplification procedure and its use in assessing various components of a cell's chemical phenotype. this procedure is particularly useful in efforts to multiplex the simultaneous detection of various cellular processes. these efforts are necessary to identify the quantitative chemical phenotype of cells that underlies cellular function. in vitro transcription by t rna polymerase. if necessary, a third round of arna amplification can be performed after the secondround amplification procedure (fig. ) . after one round of amplification, ~ , -fold quantities of original mrna can be generated. after the second round, > × -fold rna amplification can be achieved , which is adequate for the generation of microarray probes or a sequencing library , . this procedure has proven useful for amplification of rna from single cells, which contain only ~ picograms of total rna [ ] [ ] [ ] . examination of expression profiles of single live cells has shown that linear arna amplification neither results in occurs after synthesis of double-stranded cdna, when t rna polymerase is added and arna is transcribed from the cdna template. there is then a second round of amplification, in which arna is amplified through conversion of arna into cdna, with random hexanucleotide primers used to intitiate cdna synthesis. random primers are used because of their random annealing property, which allows them to prime any rna species for reverse transcription into cdna. the oligo(dt)-t primer serves as a primer for second-strand cdna synthesis, in which the t promoter is added to the double-stranded cdna at the end of the second round, thereby allowing subsequent second-round arna in the first round, first-strand cdna synthesis by reverse transcription (rt) is primed from mrna after oligo(dt)-t primer anneals to the poly(a) tail of mrna. rnase h is then used to digest portions of the bound mrna to create rna fragments that serve to prime second-strand cdna by dna polymerase (pol). finally, arna is amplified via linear in vitro transcription by t rna polymerase, using the t rna polymerase promoter incorporated in the double-stranded cdna. in the second round, first-strand synthesis is primed by random primers instead of the oligo(dt)-t primer by reverse transcriptase using the arna as a template instead of mrna. after rna denaturation, second-strand synthesis is primed with the oligo(dt)-t primer, which binds to the poly(a) tail of the cdna created during first-strand synthesis by dna polymerase. finally, rna is again linearly amplified through the enzymatic activity of t rna polymerase acting on its promoter that is incorporated into the double-stranded cdna , . credit: marina spence/springer nature. after two rounds of linear arna amplification from isolated single cells, an illumina truseq library is generated as outlined. because of the short length of the arna amplified by the procedure, the step that breaks long rna into smaller parts in the original illumina library protocol can be omitted. for strand specificity, deoxyuridine triphosphate is incorporated into the second-strand cdna. after sequencing adaptor ligation, the cdna fragments with adaptors at both end are pcr amplified, and ready for sequencing , . (b) rna-binding protein (rbp)/rna cargo detection (apra). an antibody (ab) to rbp is conjugated to an oligonucleotide and applied to fixed cells. after antibody-rbp binding, the oligonucleotide (containing the t promoter sequence) is positioned closely enough to the rna to prime first-strand cdna synthesis in situ. after second-strand synthesis in vitro, the antibody is removed by restriction enzyme cleavage and the arna is linearly amplified by in vitro transcription using the t promoter incorporated in the cdna. the arna product is suitable for pcr, microarray, and next-generation sequencing analysis . (c) protein detection (idat). a detection antibody is first generated by conjugation of a target-protein-specific antibody to a double-stranded (ds) oligonucleotide containing t promoter. in a -well plate, a capture antibody then binds to the antigen (ag) of interest from a sample. after the addition of detection antibody to the sample, rna is linearly amplified by t rna polymerase from the double-stranded oligonucleotide template incorporating the t promoter. the amount of rna product is indicative of the original amount of antigen in the sample and can be used for fluorimeter detection, pcr. or sequencing . (d) whole-genome dna amplification (lianti). a lianti transposon is first created by joining of the t promoter site to a transposase-binding site. this transposon is then mixed with transposase to generate the lianti transposome. after the lianti transposome is mixed with dna isolated from single cells, the transposase mediates random insertion of lianti transposon into the dna and subsequent excision of genomic dna, which is followed by dna polymerase gap extension. after the addition of t rna polymerase, single-stranded arna is generated that is capable of self-priming on the ′ end. after reverse transcription, rnase treatment, and second-strand synthesis, double-stranded lianti amplicons tagged with unique molecular barcodes are formed and ready for dna library preparation and next-generation sequencing voltage-clamp techniques. using rna deep sequencing, the same group also detected more than , expressed genes in primary cells from live neurosurgically resected adult human brains, uncovering cell-type-specific and patient-specific transcriptional hierarchies . cel-seq is a modified arna method that allows for multiplexing of samples and high-throughput, next-generation sequencing . multiplexing is facilitated by alteration of the original arna primer so that it contains both a unique barcode and a ′ illumina sequencing adaptor between the anchored poly(t) and t promoter sequences. when arna is made via this procedure, it contains a barcode and a ′ sequencing primer, which enables many cells to be barcoded and sequenced simultaneously. the yanai group later adapted cel-seq for use on fluidigm's c system by adding unique molecular identifiers to the primer that allowed rna molecules to be counted. later, jaitin et al. also reported the same approach, which they renamed massively parallel single-cell rna sequencing (mars-seq). in an elegant study using cel-seq, hashimshony et al. studied the embryonic development of caenorhabditis elegans and reported differential distribution of transcripts between sister cells as early as the two-cell-stage embryo, identifying gene expression in a cell previously thought to be transcriptionally inert. jaitin et al. carried out multiplexing rna-seq (marsseq) to sample thousands of mouse hematopoietic cells from the spleen, and uncovered cell-type heterogeneity in steadystate gene expression levels and regulated changes in rna abundances after pathogen activation. most recently, indrop ('indexing droplets') sequencing technology was developed by klein and colleagues , . this microfluidic approach encapsulates single cells in reaction droplets and barcodes the cdna in each droplet during reverse transcription. droplets are then pooled, and the rna populations from all cells are linearly amplified and sequenced. this application of linear arna amplification was shown to exhibit low noise, be low-cost, and be effective in isolating and processing large numbers of individual cells simultaneously. linear arna amplification has been used to study many human diseases. its most prominent application has been to identify biomarkers, but it is also used to identify potential therapeutic targets. in many of the disease-associated studies, commercially available versions of the linear amplification technology have been used under trademarked names, including riboamp (thermo), ampliscribe (epicentre), and megascript (invitrogen). because arna is cell-type agnostic, it can be used in a variety of diseaseassociated cell types. for example, the transcriptome of platelets isolated from people with sickle cell disease has highlighted the role of metabolic pathways in the disease. transcriptomic insights into brain diseases such as schizophrenia , depression , parkinson's disease , alzheimer's disease , and even brain microvascular injury have provided many disease-associated targets for further analysis. the study of cancer has benefited preferential amplification of certain rna species (i.e., it shows no sequence preference) nor creates significant differences in the ratios of specific arnas in the amplified rna population compared with those of the initial host cell mrna abundances , . it should be noted that if there were bias in amplification, then the bias would be linearly amplified rather than exponentially as in pcr amplification. furthermore, arna has been shown to provide an accurate and precise amplification product [ ] [ ] [ ] [ ] . arna has been used extensively to generate probes for northern and southern blotting, in tandem with pcr , and microarrays , , , , and for sequencing of tissue-isolated rna , , , . the high yield, accuracy, and precision (i.e., similarity of same-sample technical repetitions) of this transcription-based amplification system have led to its use as a reporter system to detect chemical moieties other than rna. arna-associated reporter systems for proteins, rna-binding protein cargoes, and genomic dna sequences, as illustrated in figure , benefit from the linear nature of the procedure and are further described in the following sections. adaptations to the arna protocol that have enabled advances in the use of this technique to study other cellular chemicals are highlighted in table . the first single-cell rna analysis used arna (fig. a) to identify ribosomal rna in a single rat cerebellar purkinje cell . this was followed by the development of the first single-cell rna-sequencing library, where arna was used on individual rat hippocampal neurons and subsequently for differential display and sanger sequencing of hundreds of rnas from single neuronal dendrites . this showed that arna amplification was sufficiently sensitive to facilitate analysis of the rna content of a well-defined subcellular region of a neuron-for example, a dendrite-independently from the cell soma , , which represents a , × larger volume. a timeline highlighting some of the major scientific advances made with arna is presented in figure . microarray-based gene profiling was performed extensively before the advent of rna-seq and is still used as an experimental tool for the direct monitoring of large numbers of mrnas in parallel with rna sequencing. the first transcriptomic-level evaluation of linear amplified arna for gene expression analysis by microarray was done by lockhart et al. , and later wang et al. , conducted a statistical assessment of arna performance in microarray analysis. xu et al. developed a human transcriptome microarray for high-throughput and low-cost expression-profiling analyses in clinical studies. with a small amount of human tissue as starting material, they linearly amplified nanogram amounts of total rna, applied the arna to custom-designed . -millionfeature oligonucleotide arrays, and obtained reproducible and informative analyses of gene and exon expression. the use of arna in single-cell analysis increased dramatically after rna-seq was introduced. there have been many singlecell arna studies, but recently spaethling et al. identified > g-protein-coupled receptor mrnas, including mrnas for dozens of orphan g-protein-coupled receptors in serotonergic neurons, after three rounds of arna amplification and sequencing of rna libraries. they also validated the presence of the receptors by . one or two rounds of arna labeled with dye, incorporating a cleavage step during in vitro rna transcription. . arna modified for template switching for better ′-end representation in the arna products. . two rounds of arna adapted to amplify double-stranded chip-chip dna fragments. after the addition of poly(dt) to the dna template, oligo(da)-t is annealed. the resulting double-stranded dna fragment contains a functional t promoter that drives the in vitro transcription reaction, generating multiple rna copies of the dna template. . arna modified for long sage (serial analysis of gene expression) for detection of large-scale gene expression from microdissected primary tissue. after one round of arna, first-strand cdna is primed with sage random primer. second-strand cdna is then primed with dynabeads oligo(dt) primer and used for standard sage library generation. . arna modified for amplification on immobilized beads. after coupling of the oligo(dt)-t primer to magnetic beads, arna is synthesized directly on the beads to increase the yield. . modification of arna so that it produces sense rna. the use of random primers containing an upstream t promoter sequence to prime second-strand cdna synthesis enables in vitro transcription from t promoter sequences incorporated into the single-strand cdna, thus producing amplified rna in the sense orientation by t polymerase. high-resolution detection of biological processes such as stochastic copying of dna replication origins. the arna procedure has been adapted to identify mrna targets that are associated with specific rna-binding proteins (rbps) (fig. b) . miyashiro et al. , developed a technique called antibody-positioned rna amplification (apra), in which an rbp-specific antibody is conjugated to a singlestranded oligonucleotide containing a restriction enzyme site, a t rna polymerase promoter sequence at its ′ end, and a -base degenerate nucleotide sequence at its ′ end. when the antibody-oligo conjugate is applied to fixed cells, the oligonucleotide acts as an apra primer and is positioned near the rnas that are associated with the rbp, and the degenerate sequence of the antibody-bound oligonucleotide can prime first-strand cdna synthesis directly in situ in the fixed tissue section . the antibody-cdna complex is then removed from the fixed cells, and second-strand cdna is synthesized in vitro. the double-stranded cdna is removed from the antibody by restriction enzyme digestion, and arna is obtained by in vitro transcription followed by two rounds of arna amplification. using the apra procedure, the authors were able to isolate rna cargoes associated with fragile x mental retardation protein (fmrp) and identify hundreds of previously unidentified putative fmrp targets. among the fmrp apra targets that were tested, > % were confirmed as fmrp cargoes via filter binding or ultraviolet cross-linking assays to directly assess interaction with fmrp and mrna targets . from arna analysis of a variety of cancers, including breast cancer , ductal cancer , liver metastases , prostate cancer , and various brain cancers , . an interesting application of linear arna amplification to the study of pancreatic cancer involved the identification of pancreatic tumor markers in saliva, suggesting that tumor-derived exosomes can provide salivary biomarkers of disease . in addition, infection by pathogenic microorganisms has been identified in human samples by linear arna amplification, either directly through detection of microorganismderived rnas, or through biomarker surrogates in cases where changes in rna abundance for host genes known to be modulated by infection are detected. this is exemplified by the detection of hiv , influenza , and severe acute respiratory syndrome (sars) coronavirus infections. most recently, linear arna amplification was used by chen et al. for whole-genome sequencing in a method called linear amplification via transposon insertion (lianti) (fig. d) . this procedure enables single-cell whole-genome amplification to facilitate sequencing of % of the single-cell genomic dna, as well as detection of single-nucleotide variation with kilobase resolution. in lianti, the t rna polymerase promoter sequence is incorporated into dna associated with the t transposome, which, after insertion into the genomic dna, embeds the t promoter into the transposon-flanked dna. after the addition of t rna polymerase, multiple rna copies of the genomic dna are synthesized. the lianti-produced arna is reverse-transcribed and made into a sequencing library for subsequent analysis. the high fidelity and yield of arna permits linear rna coupled with microarray analysis ( ) linear rna used for protein detection (idat; ) linear rna used for genome sequencing ( ) detection of rna-binding protein cargoes ( ) detection of chip sequences ( ) linear rna coupled with pcr ( ) high-throughput arna cel-seq ( ) high-throughput arna indrop ( ) high-throughput arna mars-seq ( ) high-throughput arna microwell ( ) linear rna used for fixed-tissue transcriptomics ( ) arna on immobilized beads ( ) sensitive fluorescent arna for protein detection (factt; ) usually involves independent isolation of the additional chemical to be quantified, which risks differential loss of material and inaccurate interpretation of data. the linear arna reporting system, however, provides an opportunity to detect multiple biological entities in a single sample without specific isolation. this eliminates sample loss while harmonizing the detection output; because the rna-polymerase-promoter-containing dnas from the different detectors are transcribed, the resultant arnas can be used to deduce the proteome or other chemicals on the basis of the specific barcodes that are detected. for example, it may be possible to use apra to detect specific rbp-associated rna cargoes with unique barcodes in the same cell where the transcriptome is analyzed (both will be linearly amplified, but they will be distinct and distinguishable on the basis of different sets of barcodes). this would facilitate analysis of the dynamics of rna movement between different subcellular compartments. as another example, when coupled with the use of antibodies to specific post-translational modifications such as phosphorylation or glycosylation groups, idat could be used to assess the biology and dynamics of such protein modifications at the same time as the transcriptome analysis. careful use of barcodes in liantibased dna sequencing in conjunction with analysis of the transcriptome (using a distinctly barcoded oligo(dt)-t primer to prime amplification of the poly(a) rna population) would allow analysis of both genomic dna and the transcriptome in the same cell. one could take this even further by multiplexing more than two of these analysis procedures. for example, the transcriptome (assessed via the arna procedure), genomic dna (analyzed by lianti), and protein (evaluated by idat) could all be analyzed in the same cell. such combinatorial use of arna-amplificationbased methods should enable the quantitative analysis of many cellular components and their associated biologies from a single sample, down to and including subregions of a single cell. given the flexibility of the technique and the ability of different arna reporting methods to barcode different chemicals, it is reasonable to presume that linear rna amplification will continue to be used as a foundation for new technologies, providing deeper understanding of the multiple components and processes of cells and systems that contribute to their function. acknowledgments this work was supported in part by the nih (grants u mh , gm , mh , and mh to j.e.). we thank j.-m. eberwine and l. hoban for editing. we also thank our many colleagues who, over the years, have contributed their intellect and diligence in working with us to develop and refine these methods. author contributions j.l. and j.e. wrote, reviewed, and edited the manuscript. the authors declare no competing interests. the ability to detect antigens immunologically is often limited by the low affinity of many antibodies, the limiting amount of many antigens, and the high background often associated with highsensitivity enzymatic or fluorescence detection methods. these issues can be overcome through the use of arna amplification for antigen detection rather than the standard fluorescence detection, as the protein-detection sensitivity can be increased ~ -fold over that of conventional elisa methods. this arna proteindetection technique is termed immunodetection amplified by t rna polymerase (idat) (fig. c) . zhang et al. developed a method to facilitate arna detection of antibody-antigen interactions by covalently attaching a double-stranded cdna that contains a t rna polymerase promoter in front of a reporter sequence to a specific antibody. after immunostaining with the idat antibody, rna polymerase is added to the antigenantibody complex to linearly amplify rna from the cdna that is conjugated to the antibody. because the amplification is linear, the yield of arna is a direct measure of the amount of antibodyantigen interaction. a fluorescence-based modification of idat subsequently developed by the same group, termed fluorescent amplification catalyzed by t polymerase technique (factt), was successfully used to analyze low-abundance oncogene proteins in human serum . rather than using chemical crosslinking of the amplification primer to the antibody, factt uses streptavidin-labeled antibodies to bind the biotinylated amplification target sequence. because streptavidin binds many biotinylated molecules with higher affinity than antibody-antigen binding, the sensitivity of factt is greater than that of idat. ribogreen, a fluorescent rna intercalating dye, can be used to quantify the yield of amplified rna. idat and factt should be adaptable to automation and high-throughput protein screening. the coordinated activity of multiple cells gives rise to tissuespecific functioning. in this context the functional cell type is of utmost biological importance. quantitation of the subcellular components and processes that give rise to cellular functions such as genomic dna sequence variation, gene expression, methylation, and the creation of metabolites and proteins will improve the overall understanding of hierarchical cellular regulation and allow the construction of an 'architecture of cellular phenotype' . such an architecture would enable the codetermination of genomic dna sequence and expression state, as well as allow rna and protein abundances to complement each other, with variations in dynamics and absolute and relative amounts of each cellular chemical resulting in specific celltype designations. this will be possible only when all of these cellular constituents can be quantified in the same sample. the easy adaptation of the arna procedure to the analysis of different subcellular chemicals and biologies as illustrated above gives rise to the possibility of using this methodology in a multiplexed manner to simultaneously analyze multiple subcellular chemicals within a cell. such multiplexing is currently possible for two chemicals at a time (for example, transcriptome and protein analysis), but adding further levels of analysis deep sequencing reveals cell-type-specific patterns of single-cell transcriptome variation amplified rna synthesized from limited quantities of heterogeneous cdna analysis of gene expression in single live neurons molecular profiles of pyramidal neurons in the superior temporal cortex in schizophrenia altered expression of glutamate signaling, growth factor, and glia genes in the locus coeruleus of patients with major depression effects of gender on nigral gene expression and parkinson disease predominance of neuronal mrnas in individual alzheimer's disease senile plaques gene expression profiles of human breast cancer progression expression profiling of ductal carcinoma in situ by laser capture microdissection and high-density oligonucleotide arrays genome-wide screening of genes showing altered expression in liver metastases of human colorectal cancers by cdna microarray hepsin and maspin are inversely expressed in laser capture microdissectioned prostate cancer molecular profiling identifies prognostic subgroups of pediatric glioblastoma and shows increased yb- expression in tumors gene expression profile of glioblastoma multiforme invasive phenotype points to new therapeutic targets role of pancreatic cancer-derived exosomes in salivary biomarker development transcription-based amplification system and detection of amplified human immunodeficiency virus type with a bead-based sandwich hybridization format nucleic acid sequence-based amplification methods to detect avian influenza virus molecular signature of clinical severity in recovering patients with severe acute respiratory syndrome coronavirus (sars-cov) identification of rna cargoes by antibodypositioned rna amplification rna cargoes associating with fmrp reveal deficits in cellular functioning in fmr null mice in situ transcription: specific synthesis of complementary dna in fixed tissue sections protein quantification from complex protein mixtures using a proteomics methodology with single-cell resolution a sensitive and highthroughput assay to detect low-abundance proteins in serum improved genome-wide localization by chip-chip using double-round t rna polymerase-based amplification arna-longsage: a new approach to generate sage libraries from microdissected cells amplification of mrna populations using arna generated from immobilized oligo(dt)-t primed cdna a robust method for the amplification of rna in the sense orientation an automated microwell platform for large-scale single cell rna-seq single cell transcriptomics of hypothalamic warm sensitive neurons that control core body temperature and fever response: signaling asymmetry and an extension of chemical neuroanatomy intron retention facilitates splice variant diversity in calciumactivated big potassium channel populations analysis of mrna populations from single live and fixed cells of the central nervous system primary cell culture of live neurosurgically resected aged adult human brain cells and single cell transcriptomics serotonergic neuron regulation informed by in vivo single-cell transcriptomics cel-seq: single-cell rna-seq by multiplexed linear amplification assessing characteristics of rna amplification methods for single cell rna sequencing single-cell whole-genome analyses by linear amplification via transposon insertion (lianti) expression monitoring by hybridization to high-density oligonucleotide arrays dopamine receptor subtypes colocalize in rat striatonigral neurons embryonic neuronal markers in tuberous sclerosis: single-cell molecular pathology fidelity and enhanced sensitivity of differential transcription profiles following linear amplification of nanogram amounts of endothelial mrna advantages of mrna amplification for microarray analysis massively parallel single-cell rna-seq for marker-free decomposition of tissues into cell types on the nature and differential distribution of mrnas in hippocampal neurites: implications for neuronal functioning molecular characterization of the dendritic growth cone: regulated mrna transport and local protein synthesis cytoplasmic intron sequence-retaining transcripts can be dendritically targeted via id element retrotransposons highfidelity mrna amplification for gene profiling prospective molecular profiling of melanoma metastases suggests classifiers of immune responsiveness human transcriptome array for high-throughput clinical studies cel-seq : sensitive highly-multiplexed single-cell rna-seq single-cell barcoding and sequencing using droplet microfluidics droplet barcoding for single-cell transcriptomics applied to embryonic stem cells amplified expression profiling of platelet transcriptome reveals changes in arginine metabolic pathways in patients with sickle cell disease key: cord- - gq e f authors: staroverov, sergey a.; volkov, alexei a.; mezhenny, pavel v.; domnitsky, ivan yu.; fomin, alexander s.; kozlov, sergey v.; dykman, lev a.; guliy, olga i. title: prospects for the use of spherical gold nanoparticles in immunization date: - - journal: appl microbiol biotechnol doi: . /s - - - sha: doc_id: cord_uid: gq e f recent years have seen extremely fast development of new viral nanovaccines and diagnostic agents using nanostructures prepared by biological and chemical synthesis. we used spherical gold nanoparticles (average diameter, nm) as a platform for the antigen for swine transmissible gastroenteritis virus (tgev). the literature data demonstrate that immunization of animals with the tgev antigen coupled to gold nanoparticles (gnps) not only activates antigen-presenting cells but also increases the proliferative activity of splenic lymphoid (antibody-forming) cells. the contents of γ-ifn, il- β, and il- in animals immunized with gnp-antigen conjugates were found to be higher than those in intact animals or in animals given the antigen alone. the increased concentration of il- β in the immunized animals directly correlated with the activity of macrophages and stimulated b cells, which produce this cytokine when activated. the increased concentration of il- indicates that the injected preparations are stimulatory to cellular immunity. immunization with the tgev antigen conjugated to gnps as a carrier activates the respiratory activity of lymphoid cells and peritoneal macrophages, which is directly related to their transforming activity and to the activation of antibody generation. furthermore, the use of this conjugate allows marked improvement of the structure of the animals’ immune organs and restores the morphological–functional state of these organs. the microanatomical changes (increased number of follicles) indicate the activation of the b-dependent zone of the spleen and, consequently, the development of a humoral-type immunological reaction. the degradative processes observed in the animals immunized with tgev antigen alone are evidence of weak resistance to pathogen attack. these results can be used to develop vaccines against this infection by employing tgev antigen coupled to gold nanoparticles as a carrier. swine transmissible gastroenteritis is an acute, rapidly spreading, highly contagious enteric disease of pigs of all ages. it is characterized by vomiting, watery and yellow diarrhea, weight loss, and rapid dehydration, leading to a high mortality rate, especially in piglets (mortality decreases with age). swine transmissible gastroenteritis is caused by transmissible gastroenteritis virus (tgev) of the genus coronavirus in the family coronaviridae. the major route of virus transmission is fecal-oral. infected animals cannot digest food properly and die from dehydration. the disease causes great economic losses in pig farms all over the world, and the primary means of animal protection against it is vaccination (cheng and niu ; straw et al. ) . therefore, the development of new vaccines and the implementation of large-scale vaccination programs are important in pig breeding (moxley and olson ( ) . in several countries, vaccination against swine transmissible gastroenteritis is conducted effectively but only with inactivated monovalent and combined vaccines not recognized internationally. traditional inactivated vaccines have high contents of contaminant substances (up to %), causing side effects, and/or may contain, as inactivating compounds, toxic agents such as phenol and formaldehyde, normally used to inactivate microbial cells. moreover, traditional vaccines are usually low immunogenic, because killed microorganisms cannot induce fully functioning cellular immunity. therefore, there is a need to develop new, more effective, and less costly vaccines against swine transmissible gastroenteritis (mout et al. ) . recent years have seen extremely fast development of new viral nanovaccines and diagnostic agents using nanostructures prepared by biological and chemical synthesis. in particular, this can be attributed to the development of biocompatible inorganic nanomaterials-gold nanoparticles. gold nanoparticles are stable metallic nanoparticles which have excellent surface functional chemistry, high biocompatibility, and nil toxicity. in addition, gold nanoparticles can be easily synthesized, giving them shapes such as spheres, rods, cubes, stars, and pyramids. several reports have described the development and use of nanostructures for the delivery of biologically active agents to target cells (mout et al. ; bhattacharya and mukherjee ; staroverov et al. ). to reach their target cells in vivo, nanoparticles must bypass many barriers that protect animals against antigens (dreaden et al. ) . gold nanoparticles (gnps) have been used widely as carriers of antigens and therapeutic agents in human medicine, veterinary practice, and biology . of particular interest is the ability of gnps to induce humoral immune response to weakly immunogenic antigens and haptens (dykman et al. a; dykman et al. b ). m o r e o v er, g n p s c on j u ga t ed t o t g e v h a v e a n immunostimulatory effect (dykman et al. b) . we studied the immunological variable and morphological changes in the immune organs of guinea pigs immunized with tgev antigen conjugated to gnps as a nanocarrier. antigen tgev strain vn- was taken from the museum of strains of the laboratory of virology (scientific research veterinary institute, russian academy of sciences, saratov) and deposited in the all-russian state collection of microbial strains used in veterinary and animal husbandry by the number bvn- ^(patent ru a ). tgev strain vn- with a titer of . - . lg tcd ml − was used. test cultures were inoculated with the same strain at . lg tcd ml − after h of cultivation. tgev was purified in a sucrose gradient according to horzinek et al. ( ) . the resultant virus suspension was used for protein analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) at a constant voltage of v for min and then at v for h (maniatis et al. ) . for molecular m a s s d e t e r m i n a t i o n s , p r e c i s i o n p l u s p r o t e i n ™ kaleidoscope™ prestained protein standards no. were used. the results of protein separation were processed with the gel-imager and gel explorer, version . , programs (dna-technology, moscow). genome identity was checked by polymerase chain reaction (pcr) using a specific primera conservative part of tgev's s genome, containing base pairs (bp) (herrington and mcgee ) . rna was isolated from tgev by using a nucleos+tm suspension silicate sorbent (biokom, moscow) according to the manufacturer's instructions. pcr results were recorded on a transilluminator with a dna-analyzer video system. pcr products obtained from a preparation of the tgev strain vn- were analyzed by electrophoresis in agarose gel ( . %) using dna ladder as a molecular size marker and dna of bacteriophage λ as a negative control. as viral nucleic acid is responsible for virulence, it was inactivated, after being isolated from tgev and purified, with ribonuclease (rnaase), a hydrolase enzyme that catalyzes rna hydrolysis. the inactivation was done by the procedure described by fedorova et al. ( ) . the antigen resulting from nucleic acid inactivation was used for animal immunization. spherical gold nanospheres (average diameter, nm) were made by the citrate method of frens ( ) . for reduction, . ml of . % aqueous tetrachloroauric acid (haucl ; sigma-aldrich) was heated under reflux to °c on a magnetic stirrer in an erlenmeyer flask. this was followed by the addition of . ml of % aqueous sodium citrate to the flask. the particle diameter was found as described earlier (khlebtsov and dykman ) , by using a libra transmission electron microscope (carl zeiss, germany), a specord s spectrophotometer (analytik jena, germany), and a zetasizer nano-zs particle size and zeta potential analyzer (malvern). to prepare antigen-gold nanoparticle conjugates, we found the bgold number^(minimal amount of antigen that protects the sol against salt aggregation) for the antigen solution. to this end, μl of an antigen solution in pbs (initial concentration, mg ml − ) was titrated twofold on a -well microtiter plate. each well received μl of gnps and μl of . m nacl. the minimal stabilizing concentration for the antigen was . μg ml − . conjugation was done by simple mixing (without the use of coupling agents) (dykman et al. ). thirty six male guinea pigs, weighing - g at the time the experiment started, were used. the animals were cared for and handled in compliance with the guide for the care and use of laboratory animals, the european convention for the protection of vertebrate animals used for experimental and other scientific purposes, and the legislation of the russian federation. for immunobiological studies, the guinea pigs were divided into four groups of nine each. the animals were immunized subcutaneously along the spinal column by two injections with an interval of days between. the guinea pigs in group were injected with . ml of tgev antigen-gnps (antigen concentration, . μg per animal). group received ml of tgev antigen solution ( . μg per animal). group served as the control, which received ml of physiological saline. group served as the control gnps, which received ml of gnps solution. gnp-antigen group was in comparison with group which received only physiological saline (control group). the animals were killed days after the last injection, and blood serum samples were taken for immunological studies. immediately after removal, all pieces of tissues and organs were placed in a fixing solution ( % aqueous neutral formalin), and then -μm-thick histological sections were prepared on a freezing microtome, model (reichert wien, austria). all histological samples were stained with hematoxylin-eosin. for isolating peritoneal macrophages, the animals were killed and then fixed on their backs. an incision was made along the midline of the anterior abdominal wall, and the skin flap was carefully separated, with care taken to keep the peritoneum intact. after a puncture had been made with a needle connected to a syringe, ml of pbs, ph . , was injected into the peritoneal cavity. the anterior abdominal wall was then gently massaged, and after - min, peritoneal fluid was collected with a pasteur pipet through a cut made in the peritoneum and was filtered into a test tube through a nylon filter. the cells were washed three times by centrifugation in pbs at g, after which they were resuspended in ml of pbs and counted in a goryaev chamber. peritoneal macrophages were cultured by standard procedures (leiter ) . for isolating splenic lymphocytes, an incision was made along the white line of the peritoneum after peritoneal macrophages had been isolated, and the spleen was removed. the spleen was minced with scissors, and the tissue pieces were mashed through a fine sieve into a petri plate containing sterile pbs. the resulting suspension was subjected to ficoll-urografin density-gradient centrifugation. the lymphocyte ring was collected in a new test tube. the lymphocytes were washed three times by centrifugation in pbs, ph . , at g for min, and the cell sediment was resuspended in ml of pbs. the lymphocytic cells were counted with a haemascreenvet hematology analyzer (hospitex diagnostics, italy). the titer of antibodies in the sera was estimated by enzyme-linked immunosorbent assay (elisa) with horseradish peroxidase-labeled antibodies against guinea pig igg (jackson immunoresearch, uk). the synthetic peptide was used as the immobilized antigen. the reaction results were recorded on a plate screen microplate spectrophotometer. the interleukin concentration in the sera was measured with a plate screen analyzer (hospitex diagnostics, italy) and using reagents of il- β, il- , and inf-γ (vector-best, russia). respiratory activity was measured conventionally (bernas and dobrucki ) by the ability of cells to reduce nitrotetrazolium blue [ -( , -dimethylthiazol- -yl)- , diphenyl tetrazolium bromide, mtt (sigma-aldrich)] to formazan. briefly, suspensions of known concentrations of isolated animal cells (macrophages and lymphocytes) were centrifuged at g for min, and the sediment was resuspended in ml of . % mtt and incubated at °c for h. after incubation, the cells were centrifuged at g for min and the sediment was resuspended in . ml of dimethyl sulfoxide (fluka, switzerland). the amount of reduced formazan was measured with a genesys s uv-vis spectrophotometer at nm. to construct the calibration curve, we used commercial formazan (sigma-aldrich) at . , . , . , and mg ml − . for studying possible morphological changes in the spleen as an important part of the macrophage system, the animals were immunized with tgev antigen-gnps. the control group comprised of nonimmunized animals. we used the spleens of guinea pigs of the same physiological age. longitudinal and transverse spleen sections ( -μm-thick) were prepared on a freezing microtome, model (reichert wien, austria), by following standard procedures. histological sections were differentially stained with hematoxylin-eosin. the results were statistically processed by the standard procedures using student's t test to evaluate the reliability of the differences between samplings in the experimental and control studies. after the arithmetic mean and the standard deviation for a given data sample were found, we determined the standard error of the arithmetic mean and its confidence limits by student's t coefficient (n, p) at a significance level of % (p = . ). the obtained results were statistically processed by the standard procedures integrated in excel software (microsoft corp., usa). after the arithmetic mean and the standard deviation for a given data sample had been found, we determined the standard error of the arithmetic mean and its confidence limits with account of student's t coefficient (n, p) [number of measurements n = , significance level = % (p = . )]. these results are presented as histograms. the significance of differences between individual samples was evaluated by a two-sample unpaired student's t test with unequal variances. differences were considered significant when the experimentally found p exp value was ≤ . . the reliability of the changes recorded in the results of each of the experiments described above for the entire set of antigen formulations examined was assessed by one-way analysis of variance (anova) by using fisher's ratio test. the dependences found were considered significant at f > f crit , where the critical value f crit at n = and m = - (number of data sets) corresponded to p = . (with the number of degrees of freedom (df) lying between and ) and the effective value p eff was < . . swine transmissible gastroenteritis is caused by an rna virus belonging to group а of the genus alphacoronavirus in the family coronaviridae. the virion has a spherical shape with a diameter of - nm (martins et al. ) . the viral nucleocapsid is a flexible spiral containing single-stranded rna and a large number of nucleocapsid protein molecules (delmas et al. ; sturman et al. ) . the virus replicates in the cytoplasm of mature epithelial cells located at the ends of the intestine villi. a general scheme of the experiment is depicted in fig. . after the antigen was isolated and its protein composition was analyzed by sds-page, we found that the molecular masses of the separated proteins were identical to those of the tgev proteins. the antigen contained the following proteins: p protein (large surface glycoprotein s; molecular mass, - kda), n protein (basic nucleocapsid protein, - kda), and m protein (matrix membrane protein, - kda) (fig. ) . a protein fraction of - kda was also present. according to wesley et al. ( ) , kda is the size of an unglycated structural protein of tgev. thus, all known tgev proteins were separated. the tgev nucleic acid was identified by pcr using a specific primer-a conservative part of tgev's s genome, containing bp. analysis of the pcr products from a purified preparation of the tgev strain vn- , which was done by agarose gel electrophoresis with dna ladder , demonstrated sufficiently high separation of cdna (fig. ) . the presence of only a characteristic cdna fraction (size, bp) indicated the presence of the tgev genome in the sample. after the virus's nucleic acid was inactivated with ribonuclease, the resultant antigen (a mixture of viral capsid proteins) was used for conjugation with gnps and for subsequent animal immunization. the gnp diameter was measured by tem, the average particle size was . nm. analysis of the antibody titer data showed that the titer produced by immunization with gnp-antigen conjugates ( : . ; p = . ) was higher than that produced by immunization with unconjugated tgev antigen ( : . ). in immunology, the mononuclear phagocyte system (mps) (also known previously as the reticuloendothelial or macrophage system) is part of the immune system that consists of phagocytic cells located in reticular connective tissue. the cells are primarily monocytes and macrophages, which accumulate in lymph nodes and in the spleen. the effectiveness of interaction of the gnp-antigen conjugate with cells of the reticuloendothelial system was evaluated using the mtt test. on day after immunization, an increase in the respiratory activity of macrophages was noted (fig. ) . after immunization with the gnp-antigen conjugate, the activity increased by % (p = . ) as compared with the control and by % (р = . ) as compared with the immunization with tgev antigen and % (р = . ) as compared with gnp. a study of the respiratory activity of splenic lymphoid cells (fig. ) showed that after immunization with the conjugate, the activity increased . -fold, as compared to the control, whereas after immunization with tgev antigen alone, it did not change much. the data demonstrate that immunization of animals with tgev antigen-gnps not only activates antigen-presenting cells but also increases the proliferative activity of splenic lymphoid (antibody-forming) cells. this indicates that gnps can present antigens to the organs of the endothelial system. the effect of the conjugate on humoral immunity was assessed by comparing changes in the cytokine concentration between the control and experimental animals. there was a statistically significant increase in the content of the γ-ifn in animals immunized with gnp-antigen conjugates (fig. ) . these animals had the highest content of γ-ifn ( . ± . pgml - ; p = . ) this group was comparison with group which received only physiological saline (control group). we determined the p value error probability when deviating from the null hypothesis (errors of the first kind). we determined that for our research, p was ≤ . .when the animals were given the native antigen and gnps, no appreciable changes in γ-ifn content were found. the content of il- β in animals immunized with gnpantigen conjugate was . ± . pgml - (p = . ), which is higher than it was in intact animals ( . ± . pgml - ) or in animals given the antigen alone ( . ± pgml - ; p = . ) and gnps ( ± . pgml - ; p = . ) (fig. ) (p value for gnp-antigen group was comparison with group which received only physiological saline (control group). gnp-antigen group was comparison with group which received only physiological saline (control group). the increased concentration of il- β in the immunized animals directly correlated with the activity of macrophages and stimulated b cells, which produce this cytokine when activated. this point is so important, since it is desirable to avoid an inflammatory process during immunization, which results in the appearance of granulomas. similar data were obtained with il- ( fig. ) . this fact indicates that the injected preparations are stimulatory to cellular immunity. it is known that interferons, which possess protective properties, are produced in the body only when the virus enters or tumor diseases. that is why the analysis of the effect of immunization with the help of gold nanoparticles on the change in the level of interferon was made. it was found that in the immunized group, the concentration of interferon increased by % (compared to the control) against % for the group immunized with the antigen. when immunizing animals with native antigen, no significant changes in the concentration of interferon in blood serum of the immunized animals were observed. thus, an increase in the concentration of interferon indicates the stimulation of the introduced drugs of the cell link of immunity. the results obtained correlate with research in this area by other scientists. for example, in work (zhang et al. ) showed that tgev n protein induced er stress and activated nf-κb, which was responsible for the upregulation of il- markers protein markers (kda) protein p protein and bcl- expression. tao et al. ( ) also revealed an increase in the concentration of cytokines including interferon gamma and interleukin in response to immunization aunp-m e + scpg. but he mainly considered the reaction of splenocytes to a given complex (their reaction, which was expressed in the production of cytokines, on the effect of aunp) was studied. in addition, aunp-m e was administered together with scpg which is a strong immunomodulator. it was interesting for us to look at the production of these cytokines in the body as a whole and to reveal the general clinical changes that can develop on the introduction of antigen and aunp. one can speculate that gnps facilitate antigen expression with the major histocompability complex on the surface of antigen-presenting cells. this subsequence ensures that the viral peptides are effectively presented to cytotoxic t lymphocytes and natural killers. our data for the increase in γ-ifn activity are in agreement with those for the increase in the respiratory activity of splenocytes on day after the last injection, another indication that colloidal gold can stimulate the release of γ-ifn by t cells, thereby enhancing the activity of lymphoid immunity. in guinea pigs, the spleen forms leucocytes and removes old red blood cells (erythrocytes). the spleen framework is formed by trabeculae, which consist of reticular connective tissue, smooth muscle cells, and blood vessels (at the same time, the spleen contains only efferent lymphatic vessels). the reticular connective tissue includes both red and white pulp. the white pulp is a lymphoid tissue that produces immune and blood cells. the red pulp functions to filter blood for antigens, microorganisms, and defective red blood cells; therefore, it contains various blood corpuscles, including large and small lymphocytes, immature leucocytes, leucocytes at the final stage of graininess, and disintegrating erythrocytes. the spleen is also important for the development of humoral immunity and is a b-system organ. in the animals immunized with gnp-antigen conjugates, the lymphoid follicles had distinct boundaries and contained large number of densely spaced lymphoid cells (fig. c . the lymphoid tissue was well structured and was located in a compact manner. there was a clear boundary between the red and white pulp regions, and there were full of cellular elements. for comparison, we analyzed morphological changes in the spleens of the animals immunized with tgev antigen alone. the observed changes were characterized by the existence of perivascular edema (fig. d) , sometimes accompanied by proliferation and desquamation of the vessels' endothelium. moreover, the lymphoid tissue of the follicles became sparse, and the number and density of lymphoid cells decreased. in spleen tissue sections from the animals immunized with tgev antigen alone, the boundary between red and white pulp was less clear, the size and total number of follicles in the microscopic field decreased, and no germinative centers could be detected. these results point to degradative processes in the immune organs of the animals. in the control group of guinea pigs and gnps, injected with physiological saline, no morphological changes in spleen tissue were observed (fig. a, b) . gold nanoparticles are stable metallic nanoparticles which have excellent surface functional chemistry, high biocompatibility, and nil toxicity (pissuwan et al. ( ) . in addition, gold nanoparticles can be easily synthesized, giving them shapes such as spheres, rods, cubes, stars, and pyramids. this quality allows the use of gold nanoparticles for a variety of purposes, including vaccine delivery (versiani et al. ). there are a series of works devoted to the use of gold nanoparticles as carriers of antigens for pathogens such as the virus grippe (tao et al. ) , virus foot-and-mouth (chen et al. ) , tetanus toxoid (barhate et al. ) , and yersinia pestis (gregory et al. ) . in our work, we used spherical gold nanoparticles (average diameter, nm) as a platform for the antigen for tgev. the data demonstrate that immunization of animals with tgev antigen-gnps not only activates antigen-presenting cells but also increases the proliferative activity of splenic lymphoid a b c d fig. a spleen tissue section from the animals immunized gnps. hematoxylin-eosin staining; magnification, × . b spleen tissue section from the animals control group. hematoxylineosin staining; magnification, × . c spleen tissue section from the animals immunized with tgev antigen-gnps. hematoxylin-eosin staining; magnification, × . lymphoid follicles have distinct boundaries. d spleen tissue section from the animals immunized with tgev antigen alone. hematoxylineosin staining; magnification, × . perivascular edema and sparse lymphoid tissue in the follicles can be observed fig. concentration of il- in animals immunized with gnpantigen conjugates (tgev antigen р = . ; gnp-antigen conjugate р = . ; gnps р = . ) (antibody-forming) cells. the contents of γ-ifn, il- β, and il- in animals immunized with gnp-antigen conjugates were higher than those in the intact animals or in animals given the antigen alone. the increased concentration of il- β in the immunized animals directly correlated with the activity of macrophages and stimulated b cells, which produce this cytokine when activated. the increased concentration of il- indicates that the injected preparations are stimulatory to cellular immunity. several reports have described the development and use of nanostructures for stimulating cellular immunity. for example, xu et al. ( ) showed that gold nanorods associated with the dna vaccine may cause increased formation of γ-ifn in hiv-infected mice. assis et al. ( ) showed that gold nanorods, functionalized with cysteamine and associated with the protein rsm , are stimulatory to il- . in a work, tao et al. ( ) showed that the use of nanostructured vaccine led to an increase in the concentration of igg a, iga, and il- a, il- , il- , tnf-и rantes. our research correlates with these studies but in the present work we used gold nanoparticles for the first time as a platform for antigen for tgev. also, we did nonspecific adsorption of the antigen and did not perform a chemical surface functionalization. this made it possible to simplify the conjugation process, as well as to remove additional procedures related to the purification of the drug from toxic components. additionally we analyzed the morphological changes in the spleens of the animals immunized with gnp-antigen conjugates and tgev antigen. the microanatomical changes (increased number of follicles) indicate the activation of the b-dependent zone of the spleen and, consequently, the development of a humoral-type immunological reaction (galaktionov, ) . the degradative processes observed in the animals immunized with tgev antigen alone are evidence of weak resistance to pathogen attack. in conclusion, immunization with tgev antigen conjugated to gnps as a carrier activates the respiratory activity of lymphoid cells and peritoneal macrophages, which is directly related to their transforming activity and to activation of antibody generation. furthermore, the use of this conjugate allows marked improvement of the structure of the animals' immune organs and restores the morphological-functional state of these organs. the results of this study can be used in the further development of nanovaccines against swine transmissible gastroenteritis. the microanatomical changes (increased number of follicles) indicate the activation of the b-dependent zone of the spleen and, consequently, the development of a humoral-type immunological reaction (galaktionov, ) . the degradative processes observed in the animals immunized with tgev antigen alone are evidence of weak resistance to pathogen attack. funding information this study was funded by the russian science foundation (project no. - - ). conflict of interest the authors declare that they have no conflict of interest. ethical statement all applicable international, national, and/or institutional guidelines for the care and use of animals were followed. the use of gold nanorods as a new vaccine platform against schistosomiasis quillajasaponaria extract as mucosal adjuvant with chitosan functionalized gold nanoparticles for mucosal vaccine delivery: stability and immunoefficiency studies the role of plasma membrane in bioreduction of two tetrazolium salts, mtt, and ctc biological properties of bnakedm etal nanoparticles assessment of gold nanoparticles as a size-dependent vaccine carrier for enhancing the antibody response against synthetic foot-and-mouth disease virus peptide investigation on the porcine epidemic diarrhea prevalent on qinhai antigenic structure of transmissible gastroenteritis virus. ii. domains in the peplomer glycoprotein the golden age: gold nanoparticles for biomedicine gold nanoparticles in biomedical applications: recent advances and perspectives adjuvant properties of gold nanoparticles gold nanoparticles as an antigen carrier and an adjuvant. nova science publishers gold nanoparticles: synthesis, properties, biomedical applications inactivation of a non-enveloped rna virus by artificial ribonucleases: honey bees controlled nucleation for the regulation of the particle size in monodisperse gold suspensions conjugation of y. pestis f -antigen to gold nanoparticles improves immunogenicity diagnostic molecular pathology: a practical approach antigenic relationships among homologous structural polypeptides of porcine, feline, and canine coronaviruses optical properties and biomedical applications of plasmonic nanoparticles the nod mouse: a model for insulin dependent diabetes mellitus //current protocols in immunology diagnosis to detect porcine transmissible gastroenteritis virus (tgev) by optical and transmission electron microscopy techniques surface functionalization of nanoparticles for nanomedicine clinical evaluation of transmissible gastroenteritis virus vaccines and vaccination procedures for inducing lactogenic immunity in sows prospects for gold nanorodparticles in diagnostic and therapeutic applications usage of phage mini-antibodies as means of detecting ferritin concentration in animal blood disease of swine proteolytic cleavage of the e glyco protein of murine coronavirus by trypsin and separation of two different k cleavage fragments gold nanoparticle-m e conjugate coformulated with cpg induces protective immunity against influenza a virus consensus m e peptide conjugated to gold nanoparticles confers protection against h n , h n and h n influenza a viruses gold nanoparticles and their applications in biomedicine genetic analysis of porcine respiratory coronavirus, an attenuated variant of transmissible gastroenteritis virus surface-engineered gold nanorods: promising dna vaccine adjuvant for hiv- treatment transmissible gastroenteritis virus n protein causes endoplasmic reticulum stress, upregulates interleukin- expression and its subcellular localization in the porcine intestinal epithelial cell key: cord- - nlbu e authors: robinson, elektra k.; covarrubias, sergio; carpenter, susan title: the how and why of lncrna function: an innate immune perspective() date: - - journal: biochim biophys acta gene regul mech doi: . /j.bbagrm. . sha: doc_id: cord_uid: nlbu e next-generation sequencing has provided a more complete picture of the composition of the human transcriptome indicating that much of the “blueprint” is a vastness of poorly understood non-protein-coding transcripts. this includes a newly identified class of genes called long noncoding rnas (lncrnas). the lack of sequence conservation for lncrnas across species meant that their biological importance was initially met with some skepticism. lncrnas mediate their functions through interactions with proteins, rna, dna, or a combination of these. their functions can often be dictated by their localization, sequence, and/or secondary structure. here we provide a review of the approaches typically adopted to study the complexity of these genes with an emphasis on recent discoveries within the innate immune field. finally, we discuss the challenges, as well as the emergence of new technologies that will continue to move this field forward and provide greater insight into the biological importance of this class of genes. this article is part of a special issue entitled: ncrna in control of gene expression edited by kotb abdelmohsen. one of the most profound discoveries from the sequencing of the human genome is that over % of the genome is transcribed, yet < % encodes protein-coding genes [ ] . large consortiums such as encode and fantom have embarked on attempting to characterize all functional coding and noncoding elements in the genome and have compiled important regulatory data for these elements [ ] [ ] [ ] . long noncoding rnas (lncrnas) represent the largest group of non-coding rnas produced from the genome. lncrnas are defined as transcripts > nucleotides in length, lacking protein-coding potential. in the most recent gencode v release, there are , annotated lncrnas in the human genome [ ] . additionally, there are over , pseudogenes, that could fall under the description of long noncoding rnas which is simply based on them being nucleotides or greater in length. less than~ % of annotated lncrnas have ascribed functions. hence this class of rnas is greatly in need of further investigation [ ] . from those that have been characterized, it is clear that lncrnas can function through a variety of mechanisms to regulate gene expression both at the transcriptional and post-transcriptional levels [ ] . as we will discuss through this review lncrnas can mediate their functions through interactions with proteins, rna, dna, or a combination of these. furthermore, the function of lncrnas can often be dictated by their localization, sequence and/or secondary structure. there are many categories and sub-categories of lncrnas, but some of the major classifications include: antisense [ ] , bi-directional [ ] , enhancer-associated [ ] , intergenic lncrnas (lincrnas) [ ] , pseudogenes [ ] , while a full review of all classifications can be obtained from the recent review by jarroux et al. [ ] . lncrna function cannot be determined simply based on the lncrna classification. however, the classification can sometimes provide insight into its mechanism of action, such as antisense lncrnas impacting their neighboring genes. however, this same classification can also lead to erroneous assumptions about how the lncrna regulates gene expression. recently, some lncrnas have been demonstrated to actually encode small peptides indicating that these genes are misclassified as noncoding, although it is possible that they could also have functions as a noncoding rna in addition to their peptide coding capacity [ ] [ ] [ ] [ ] [ ] [ ] . it is therefore, important to have a logical methodology to study the biological importance of these genes. the innate immune system functions as a rapid initial response against specific pathogens, while also promoting the activation and development of the adaptive immune system [ ] . macrophages and dendritic cells are important innate immune cells that initiate the immune response through recognition of specific pathogen-associated molecular patterns (pamps) through their germline-encoded pattern recognition receptors (prrs) [ ] . these receptors couple pathogensensing to activation of downstream signaling cascades resulting in upregulation of numerous inflammatory pathways [ ] . while a robust immune response is crucial for eliminating pathogens, prolonged activation can be detrimental to the host [ ] . not surprisingly, many aspects of the inflammatory response are tightly regulated at both transcriptional and post-transcriptional levels allowing for a transient antimicrobial response while subsequently promoting a return to homeostasis [ ] . perturbations in this regulation can have significant consequences that can manifest in diseases, such as arthritis [ ] , multiple sclerosis [ ] and cancer [ , ] . while the role of coding genes in immune cell function has been well characterized, the role of lncrnas in these processes is just beginning to emerge [ ] (fig. ). here, we use the biological model system of macrophage activation as a framework to demonstrate how we approach the study of lncrna biology. we provide a step-by-step guide to consider when studying lncrnas. furthermore, we discuss the challenges, as well as the emergence of new technologies that are helping evolve the ways we study these genes. the lncrna field is in its infancy yet from what we do know we find that lncrnas play critical roles in a wide variety of biological processes and diseases from cell differentiation, tissue organ development, flowering in plants, to cancer metastasis to name just a few [ ] [ ] [ ] [ ] [ ] . we believe that lncrnas play regulatory roles in many biological processes and diseases. therefore, no matter what your research area is, there is a rich source of information to be obtained from the study of lncrnas in your field of interest. the bulk of lncrna studies to date have focused on the cancer field [ ] [ ] [ ] . meanwhile, studies of lncrnas in the context of innate immunity have lagged, making up only~ % of all lncrna papers to date (fig. ) . the innate immune system provides one of the first lines of defense against infection through the induction of inflammation [ , ] . the inflammatory response of murine macrophages offers a powerful system for applying genomic approaches for studying novel lncrnas within the framework of a pathway that has been studied for decades. macrophages are important mediators of inflammation and initiate this response through recognition of specific pathogen-associated molecular patterns (pamps) through their germline-encoded pattern recognition receptors (prrs). these receptors couple pathogensensing to activation of downstream signaling cascades resulting in activation of numerous transcription factors, including nf-kappab (nf-κb) and interferon regulatory factors (irfs) that can act in combination to both positively and negatively regulate the expression of thousands of genes [ , ] . there are different tlr genes in the human genome and tlr genes in mice [ ] [ ] [ ] , each binding a different pamp [ ] . using this extensively studied biological system, we identified the first example of a tlr-stimulated lncrna, lincrna-cox , which was capable of positively and negatively regulating distinct types of innate immune genes [ ] [ ] [ ] [ ] [ ] . knockdown of lincrna-cox resulted in impaired production of proinflammatory genes (i.e., il- ), while ifnrelated genes were hyperactivated in the absence of lincrna-cox [ ] [ ] [ ] [ ] [ ] . numerous other studies have made use of the tlr-signaling biological system, uncovering and characterizing dozens of novel lncrnas that act in a wide range of mechanisms to either positively or negatively regulate this pathway as reviewed in carpenter et al. and hadjicharalambous et al. [ , ] . as mentioned lncrnas are categorized into five main classes of long noncoding rnas based on their genomic location: antisense, bidirectional, intronic, enhancer-associated, and intergenic. intergenic and enhancer lncrnas contain their own promoters and are distinct from protein-coding genes. bidirectional lncrnas share a promoter and are transcribed from the opposite strand of a protein-coding gene, while intronic lncrnas are transcribed within an intronic region of a proteincoding gene ( fig. a) [ , ] . the specific class of lncrnas can often provide significant insight into how it may regulate gene expression. for example, lncrnas antisense to a coding gene have been demonstrated to be involved in transcriptional interference, negatively affecting the expression of their coding gene [ ] . while all categories of lncrnas will no doubt be important in various biological processes, the most targetable lncrnas are intergenic lncrnas. a benefit to studying an intergenic lncrna (lincrna) is the immense variety of molecular techniques that would not apply to other types of lncrnas, such as antisense, bidirectional and intronic lncrnas, which often overlap coding genes and their targeting could lead to possible unwanted interference of that coding gene. numerous studies have established that lncrnas can regulate the expression of their neighboring coding genes (cis regulation) [ ] . a recent study by engreitz et al. demonstrated that a significant portion of lncrnas had cis effects on their neighboring genes [ ] . interestingly, in most cases, the cis effect did not require the production of the lncrna transcripts themselves, but instead required the processes associated with their production, such as transcription and splicing [ ] . there are many examples of lncrnas that regulate their neighboring coding genes, for example: lnc-marcks, lnc-tnfaip , as-il α, lnc-il r, and il- β-erna [ ] [ ] [ ] [ ] [ ] . recently we used genetic mouse models to show that lincrna-cox can function as an enhancer rna in cis to regulate its neighboring gene ptgs [ ] . for these reasons, one aspect to consider when selecting a candidate, no matter the class, is to investigate the effect of the transcriptional expression on neighboring coding genes. this candidate selection approach is sometimes referred to as "guilt by association" [ , ] . this bioinformatic approach drives an initial hypothesis that the lncrna could be involved in the similar biological pathway as their neighboring protein-coding gene due to their co-expression. a large number of studies to date have also shown that lncrnas can regulate gene expression on different chromosomes (trans regulation) [ ] (fig. c ). the majority of lncrnas studied in immunity were initially identified following rna-sequencing to examine their expression profiles in specific cell lines or tissues during inflammatory activation. for example, lincrna-cox was initially identified as an up-regulated lncrna in murine dendritic cells following tlr stimulation [ ] , as well as murine bone marrow derived macrophages (bmdms) following tlr -depedent stimulation [ ] . studies have also highlighted the functions of lncrnas that are highly downregulated post inflammatory activation, such as lincrna-eps [ ] and lnc [ ] . both lincrna-cox and lincrna-eps were discovered in bmdms post tlr inflammatory activation and were chosen for further characterization based on their extreme expression profile. lincrna-cox is rapidly upregulated and regulates a large number of interferon stimulated genes (isgs) and nf-κb regulated genes [ ] . meanwhile, lincrna-eps is rapidly down-regulated during inflammation and acts as an inflammatory brake on all isgs during periods of homeostasis [ ] . these are just two examples of lncrnas that have provided critical insights into the roles of lncrnas in immunity. for more information on specific lncrnas that are involved in innate immunity we direct you to the following recent reviews on this topic [ , , ] . in addition to these bulk rna sequencing studies, a small number of single cell rna sequencing studies have been performed in both human and mouse that can be utilized to examine differential expression of lncrnas in basal versus treatment conditions or between cell types [ ] [ ] [ ] [ ] [ ] . numerous rna-seq (both bulk and single cell) datasets are available for a variety of primary cells, cell lines or tissues of interest either basally or under a multitude of inflammatory or cellular differentiation treatments. these datasets outlined in table [ , , - , , , , - ] and table [ , , - , - , - ] provide a rich source of lncrnas for further investigation. evlncrnas [ ] , noncode [ ] , or lncipedia [ ] are databases that categorize published information on all annotated lncrnas. these databases can be utilized to determine if a lncrna is experimentally validated within one or more studies. additionally, these databases can provide information on whether a lncrna possesses multiple isoforms, secondary structure, cross-species conservation and/or disease-association via presence of single nucleotide polymorphisms (snps). multiple studies have demonstrated that lncrna expression is more cell type specific compared to protein-coding genes [ ] [ ] [ ] . such specific expression patterns can often provide important clues into the specific biology that the gene could be involved in [ ] (tables and ) . a variety of consortiums exist for both human and mouse and can be utilized to determine cell type specificity of a lncrna candidate further. for instance, gtex [ ] and xena [ ] are two websites that include rna sequencing on healthy primary human tissue samples, in addition to samples from patients with diagnosed cancers. this will further assist the initial understanding of the expression of the lncrna in specific tissues as well as obtaining information on whether a lncrna is involved in cancer. in contrast, if a researcher is studying a mouse candidate lncrna, the mouse cell atlas (mca) [ ] , as well as tabula muris [ ] are excellent tools to assess specificity in cellular expression, as well as differential splicing isoforms amongst differentiated cell types. additional websites for mouse and human expression datasets can be found at the encode project [ ] , the european bioinformatics institute expression and the fantom projects [ ] . these sites are filled with raw and analyzed data sets from either single cell or bulk rna sequencing from primary cells, tissues or immortalized cells ready to use to determine the statistical significance of expression for any annotated lncrna candidate. transcriptional activation is depicted basally 'part i', as well as during inflammatory activation 'part ii' following stimulation of a pattern recognition receptor (prr). three lncrna examples genes are shown a, b and c. part iii depicts how a lncrna can undergo differential isoform expression which is regulated co-transcriptionally. during active transcription a lncrna can either undergo differential splicing or utilize a new transcriptional start site post inflammatory stimulation, such as lipopolysaccharides (lps). post-transcriptional regulation of a lncrna is broken up into three parts: iv, v and vi. after transcription is completed there is several processes that a lncrna can undergo. part iv depicts rna modifications, which can change the structure of a lncrna molecule. these modifications can be added or removed depending on the inflammatory state of a cell. part v depicts the process of mirna biogenesis, where a lncrna can be processed to a mature mirna. part vi shows that a lncrna can also be translated if it has a small open reading frame (smorf). (c) the lower right panel of the figure illustrates the regulatory function of a lncrna in the nucleus and the cytoplasm. during active transcription (basally 'part i' or inflammatory 'part ii'), a lncrna can function to repress genes (mrna gene a and c) or activate genes (mrna gene a and b). a lncrna can either be a scaffold for transcription factors to enhance activation, or a scaffold for chromatin remodeler proteins to open or close chromatin. a lncrna can also regulate a mrna transcript co-transcriptionally 'part iii' by affecting either the stability, change the splicing activity, editing of modifications, or even the capping of the mature mrna. finally, post-transcriptionally a lncrna can function to regulate the mrna in several ways. a lncrna can affect the stability of a mrna transcript: 'part iv and part v.' alternatively, a lncrna can function as a mirna sponge which indirectly de-represses the expression of a mrna that would be targeted by the mirnas. lastly, a lncrna can modulate the translation of a mrna by binding to ribosomes or mrna transcripts during translation. genome-wide association studies (gwas) have revolutionized the study of complex diseases by allowing quantitative disease-association of thousands of genetic loci [ ] . these studies include evaluation of single-nucleotide polymorphisms (snps) or deletions and determination of their association with a disease phenotype. diseases studied range from inflammatory bowel disease (idb) to schizophrenia [ , ] . until recently, most gwas studies focused on proteincoding genes, even though % of disease-associated snps lie in noncoding regions of the genome [ ] . there are several databases that summarize the plethora of published human sequencing studies, including uk biobank [ ] and gwas catalog -embl-ebi [ ] . other databases specifically focus on snps within lncrnas, such as lncrnasnp [ ] and lnc catlas [ ] . to date, a couple of studies have clearly shown how snps from gwas studies can be used to identify clinically relevant lncrnas. castellanos-rubio et al. identified a snp rs associated with celiac disease and showed it was located within a novel lncrna, lnc [ ] . lnc regulates inflammatory genes and mediates its function via an interaction with an hnrnp protein [ ] . furthermore, they showed that the snp disrupted the rna-protein interaction, thus making the lncrna dysfunctional [ ] . another fascinating study began by mining gwas for atherosclerosis disease-associated snps, which led to the discovery of a novel lncrna, lin , that had snps that were associated with atherosclerosis all located within an intronic region [ ] . the group went on to characterize linc in human primary and immortalized monocytes as a promoter of inflammation through activating the aryl-hydrocarbon receptor repressor (ahrr) -nf-κb pathway by directly binding to lipocalin- interacting membrane receptor (limr), acting as a scaffold to promote the interaction between limr and ahrr [ ] . both of these studies started by investigating clinically relevant snps that led to the discovery of disease-associated lncrnas, providing the groundwork for future studies on potential biomarkers or the development of novel therapeutic targets for a variety of inflammatory diseases. additionally, as previously mentioned, xena or lnc catlas are databases that combine rna sequencing of tissue samples from healthy and cancer patients, these tools can also be adapted to assess age, gender, tissue or even disease association of splice variants [ , ] . these disease-associated snps or splice variants will help guide further studies to determine therapeutic or biomarker potential. in summary, assessing disease association by a variety of databases can provide insight into the function of a lncrna [ ] . unlike lncrnas, coding genes are highly conserved across distal and related species. the requirement of coding genes to encode functional peptides likely constrains the variation within the open reading frame (orf) sequence [ ] . lncrnas by definition are not translated and often have poor sequence conservation across related species. many lncrnas display species-specific expression. thus, inferring function based on sequence similarity is a challenge (reviewed in [ ] ). a more useful conservation metric is to assess whether the lncrnas have conservation of synteny (location relative to flanking coding genes), along with expression conservation [ ] . the databases lncipedia and noncode have user-friendly interfaces, allowing assessment of both conservation of synteny and sequence for lncrnas. additionally, expression conservation can be useful to assess whether the specific lncrna has the same biological role in similar and divergent species [ ] . conservation of lncrna expression can also be indicative of conservation of regulatory regions, such as transcription factor binding sites within promoters [ ] . however, conservation of expression does not necessarily mean the rna product is important for lncrna function. enhancer rnas (ernas), for example, are thought to predominantly function by creating a localized, active transcriptional state, which can activate neighboring genes [ ] [ ] [ ] . it is unclear to what extent the specific rna sequence of ernas is important for their function [ ] . nevertheless, a couple of studies have provided examples of ernas for which the transcript sequence was necessary for their function [ , ] . to further investigate conservation of a candidate, blast (basic local alignment search tool) on ncbi (national center for biotechnology information) can be utilized to explore conservation across species [ ] or lncrnadb v . [ ] , by inputting the entire sequence of a lncrna of interest. if a lncrna has a known structure, inputting the shorter structured rna sequence can enhance conservation results. one can also, view the conservation track of the ucsc genome browser [ ] to assess if this specific sequence within the lncrna transcript is conserved across species. in summary, conservation of a lncrna is complicated to assess using current bioinformatic methods. however, understanding if there is a functional motif (therefore a shorter starting input sequence) within a lncrna can allow for an increased assessment of functional conservation across species. activation of inflammatory pathways result in both up and down regulation of specific lncrnas, which in turn can have either positive or negative regulatory effects on the pathway, such as activation of sequestered transcription factors [ , ] or enhanced or repressed expression of specific inflammatory cytokines [ , , ] . genes that are immediate regulators of immunity are poised for transcriptional activation, which can be assessed by defining the openness of promoter regions of a lncrna pre-and post-inflammatory stimulation. common methods for assessing chromatin accessibility of promoters include dnase-hyper-sensitivity (dnasehs) [ ] and the assay for transposase-accessible chromatin (atac) [ ] . dnase hs-seq and atac-seq datasets are available from a variety of tissues on encode for both mouse and humans (tables and ). if the promoter is open (accessible), this is indicative of either a poised or actively transcribed gene. accessibility of promoter regions in the hematopoietic cell lineage was assessed by lara-astiaso et al. through performing atac-seq for all cells in the hematopoietic lineage [ ] . this dataset provides insight into a gene's promoter accessibility, as well as cell type specificity. for instance, if the promoter is open in all cell types, it shows that it is ubiquitously accessible and possibly expressed, while if a promoter is only accessible in myeloid cells or terminally differentiated macrophages this provides insight into the cell type that could be most biologically relevant for a particular lncrna. another interesting data set from tong et al. have provided atac sequencing from bone marrow derived macrophages (bmdms) pre-and post-inflammatory time course stimulation [ ] . this dataset assesses both poised genes and genes that undergo promoter remodeling during inflammatory activation. if a promoter of a lncrna is inaccessible or accessible during inflammatory stimulation, this could provide insight to its regulation and biological significance during an immune response. once the accessibility of a promoter region is determined, defining post-translational modifications of histones on promoter regions will assess promoter activity in specific cell types or inflammatory states [ ] [ ] [ ] . a histone modification is a covalent post-translational modification (ptm) to histone proteins which includes methylation, phosphorylation, acetylation, ubiquitylation, and sumoylation (reviewed in [ ] ). posttranslational histone modifications do not affect dna nucleotide sequence but can modify chromatin availability to the transcriptional machinery [ ] . identifying the types of epigenetic histone marks will add additional layers of understanding if the candidate is active, poised, silenced or an enhancer. many publicly available datasets provide this information outlined in tables and . while the examples we outlined here are immune focused there is a vast array of additional primary and immortalized cell line for every histone mark available through the easily accessible encode database [ ] . finally, the transcriptional regulation of a lncrna can be defined by analyzing the transcription factor (tf) motif's that lie within the predicted promoter region [ ] [ ] [ ] . rnareg . [ ] or homer [ , ] are useful tools to predict tf binding sites by motif analysis. tf motifs can be indicative of biological pathway regulation indicating when a gene is expressed. these findings of predicted tf can then be put into a gene ontology tool (panther or david) to assess how a candidate lncrna is transcriptionally regulated. for instance, the presence of pioneering transcription factors could provide information on the cell type specificity of a lncrna. on the other hand, if the promoter motifs are enriched for p , interferon response factors (irfs) or activating transcription factors (atfs) this would be indicative of inflammatory specific expression. this finding can be further supported by chromatin immunoprecipitation (chip) sequencing data sets from a multitude of labs, as well as data from the encode project (outlined in tables and ). the categorization of lncrnas as "noncoding" is initially determined bioinformatically using the arbitrary cut-off of < codons [ ] . this leaves the possibility that some lncrnas may be mrnas. therefore, one of the first steps in characterizing a candidate lncrna is to confirm that it is noncoding. for example, in drosophila, a gene annotated as a lncrna fbgn , was shown to encode multiple small~ten amino acid peptides critical for development [ ] . the discovery of these germline-encoded biologically active peptides has opened the door into new and exciting levels of regulation. however, this discovery also shows that we should be cautious when characterizing a lncrna to confirm that indeed they are noncoding. several bioinformatic tools exist for predicting small orfs (smorf) but have noted that the predictive ability for smaller orf size is in general very poor [ ] . phylocsf uses codon substitution frequency, together with conservation across multiple species, to provide a score metric that can be used to determine the presence of a conserved orf [ ] . other approaches have sought to identify novel small peptides using mass spectrometry. however, a major challenge is determining whether the peptides identified correspond to novel smorfs or represent degraded intermediates of larger proteins [ ] . the development of ribosomal foot-printing coupled to next-generation sequencing (ribo-seq) has provided a powerful quantitative method for assessing global translation [ , ] . while ribosome profiling allows for ribosome nuclease-protected rna fragments to be mapped to transcripts to enable quantitative measurement of the translation efficiency [ ] . in addition, to mapping orfs, ribo-seq can be performed with a drug that stalls ribosomes at the start codon to globally map translation start sites-this revealed significant numbers of non-canonical translation initiation from ctg codons [ ] . riboseq has also found that many lncrnas appear to be translated, raising the possibility that some of these transcripts could be producing small peptides [ ] . guttman et al. developed the ribosome release score metric as part of the ribosome profiling analysis pipeline to more accurately predict translational efficiency. their findings show that ribosome occupancy of lncrnas and ′utrs does not always equate to translation [ ] . furthermore, guttman et al. concluded that most noncoding transcripts are not translated into peptides. several additional studies have examined lncrnas binding to ribosomes and have concluded that ribosome binding may not be functional and may serve as a quality control process to degrade transcripts with low coding potential via the nonsense-mediated decay (nmd) pathway [ , ] . a recent study by jackson et al. used ribo-tagging in lps-stimulated mouse macrophages to identify ribosome footprints within hundreds of annotated lncrnas, raising the possibility that they may be producing functional peptides [ ] . they characterized an aa peptide located within a previously annotated lncrna aw and showed it was produced by non-canonical "ctg" translation initiation [ ] . they demonstrated that this small peptide had a critical role in mucosal immunity in mice and was specifically required for the expression of il mrna [ ] . the exact mechanism of how this peptide drives il expression is yet to be elucidated. the laborious process of functionally characterizing lncrnas has remained a major limitation to lncrna functional discovery. for example, while~ , long noncoding rnas (lncrnas) have been identified in the human genome, only % of all validated lncrnas have an ascribed function [ , , ] . for more than two decades since its discovery, rna interference (rnai) has been the method of choice for loss of function studies. the ease and versatility of rnai make it appealing for use, requiring short complementary small rnas transfected into cells that can then utilize the endogenous cellular machinery to target specific transcripts [ , ] (fig. a) . additionally, short hairpin rnas (shrnas) can be expressed in a lentiviral context to accomplish stable rnai in cells [ ] . rnai is most active in the cytoplasm, which makes it most useful for targeting mrnas and lncrnas that reside in the cytoplasm [ ] . success in knocking down some lncrnas, for example lincrna-cox , has been attributed to the fact that this lncrna is expressed both in the cytoplasm and the nucleus (perhaps cycling between both compartments) and hence is susceptible to rnai [ ] . however, many lncrnas are thought to be nuclear-restricted where rnai has been demonstrated to have limited efficiency [ ] (fig. ) . antisense oligonucleotides (asos) can be either rna or dna-based and can be used to target complementary sequences within a transcript. unlike rnai, asos do not engage the cellular rnai machinery [ ] . instead, asos function by hybridizing to the target rna and inhibiting its function by either inducing the rnase h pathway or by steric inhibition. the rnase h enzyme is part of a cellular pathway that normally functions to resolve unwanted dna: rna interactions that can occur during replication and/or transcription [ ] . one of the most widely used types of asos for targeting lncrnas are gapmers which contain a "hybrid" modified/unmodified configuration consisting of nt dna core flanked by ′-o-methyl or lna-modified synthetic nucleotides (fig. b) . gapmers offer the benefit of modifications for stability and reduced toxicity, while still allowing engagement of the rnase h pathway (fig. b) [ ] . efficient depletion of nuclear lncrnas has been demonstrated using modified dna anti-sense oligonucleotides [ ] . ilott et al. used gapmers to successfully knock-down tlr-induced enhancer rna, as they were unable to knock-down these same ernas with different sirnas [ ] . recent in vivo applications for dna oligonucleotides have also proven successful as a possible treatment of a neural degenerative disease called angelman's syndrome. angelman's syndrome is a monogenic disorder caused by mutations in the e ubiquitin ligase a (ube a) [ , ] . as ube a is a paternally-imprinted gene, a mutation in the maternal allele is sufficient to lead to disease [ , ] . paternal imprinting of ube a requires the expression of an antisense lncrna called ube a-ats. therefore, targeting the paternal lncrnas ube a-ats using asos leads to de-repression of expression of paternal ube a, allowing the rescue of the defective maternal copy [ ] . in a mouse model, they showed that even partial restoration of the ube a protein expression ameliorated some cognitive deficits associated with the disease [ ] . mechanistically, it is important to dissect what features of a lncrna are important for its functionfor example, determining whether the lncrna transcript is required or whether the mere act of transcription is the important feature needed to mediate its function (discussed further below). sirnas and asos are thought to inhibit gene expression at the posttranscriptional level, albeit by different mechanisms [ , ] . nevertheless, there are examples of sirnas (reviewed in [ ] ) mediating transcriptional inhibition. targeting sequences near the ′ end of the transcript can induce the "torpedo-effect," resulting in pre-mature termination of transcription [ , ] . therefore, when targeting lncrnas with asos or sirnas, it's important to consider where along with the transcript you are targeting to ensure biological interpretation of the ablation is correct. the introduction of crispr/cas technology has revolutionized the field of functional genomics by providing a novel tool for interrogating gene function. crispr/cas is a deoxyribose nuclease (dnase) that can be specifically targeted to genomic regions via a guide rna (grna) [ , ] . targeting of cas to a region results in a blunt doublestranded dna break that engages the cellular non-homologous end-joining (nhej) dna repair pathway, which promotes imprecise repair, yielding small deletions in the repaired sequence. these small deletions result in a frame-shift that disrupts the orfs within coding genes, thereby disrupting protein synthesis. lncrnas do not contain orfs and span tens of kilobases of sequence in size. as such, targeting them with a single grna is thought to be insufficient to disrupt their function [ ] . an alternative application of cas that has proven effective for targeting lncrnas involves using two grnas flanking the lncrna region of interest to induce deletion of the entire locus (fig. e ). the main advantage of deleting lncrnas is obtaining complete loss-of-function, as demonstrated in a recent study that performed a crispr-mediated deletion-screen to identify lncrnas that positively and negatively regulate cancer growth [ ] . on the other hand, deletion of the dna sequence may result in an inability to resolve whether a phenotype is due to loss of lncrna production or loss of dna sequence (discussed below) [ ] . in an alternative approach, liu and colleagues recently performed a crispr screen targeting the splice sites of over -thousand lncrnas and identified lncrnas that proved essential for viability (fig. d) [ ] . targeting of splice sites has also been shown to induce exon skipping within coding genes [ ] . lncrnas have many of the same regulatory elements as coding genes. hence future studies may opt to target tf binding sites, secondary structure and/or polyadenylation sites as a way to more finely dissect the functional portions of lncrnas. the ease in which cas can be targeted to specific genomic regions sparked the development of a modified (catalytically inactivated) version of the protein fused to the krab (krüppel associated box) chromatin-silencing domain termed crispri [ , ] (fig. c) . crispri can be used to target both coding and noncoding genes (such as lncrnas), triggering localized heterochromatin-silencing at the transcription start site (tss) [ ] . unlike rnai, the transcription-based inhibition by crispri offers the ability to efficiently target lncrnas regardless of their localization in the cell. gilbert et al. have shown maximum knock-down efficiency when targeting regions + to + nucleotides relative to the transcription start site (tss) [ ] . however, crispri is limited by the availability of an accurately annotated tss, particularly for lncrnas. incorporating cage-seq, gro-seq and/or chip-seq data into grna design may improve efficient targeting of a lncrna of interest [ ] . nevertheless, a recent study from the weissman and lim groups utilized crispri to target , lncrna loci in diverse cell lines and identified hundreds of lncrnas required for cell growth [ ] . therefore, despite its caveats, it appears that crispri can be a useful and powerful tool for interrogating lncrna biology. an alternative approach to gene ablation is overexpression, gain-offunction. plasmid-based overexpression systems have been used for decades to overexpress specific coding genes [ ] . plasmid-based over-expression of lncrnas is possible but is limited by the size (max size: - kb) of the specific lncrna (unpublished observations). however, because lncrnas can function in cis, it's important that there is the significant mechanistic characterization of the candidate lncrna to justify its cloning into a plasmid. the same catalytically inactivated cas has also been fused to a transcriptional activation domain, for example, the vp , a strong transcriptional activator derived from herpesviruses [ , ] . this strategy allows crispr-based transcriptional gene activation that can be used to study gain-of-function phenotypes. the zhang group recently performed a crispr activation screen targeting over -thousand lncrnas [ ] . they identified lncrnas that upon activation, mediated braf inhibitor resistance in melanoma cells [ ] . one of the advantages of using a crispra library is that the same library can be used across different cell types, which makes it more cost effective. nevertheless, several caveats to the crispra system, include the possibility that high expression of lncrnas may create non-physiological conditions leading to incorrect conclusions of specific biology. lncrnas have been shown to mediate functions via binding to specific proteins. hence over-expression of lncrnas, without its protein partner, may result in the inability to identify important biology. in conclusion, it's important to understand the advantages and disadvantages of the loss-or gain-of-function methods used to modulate lncrna expression. all methods have their caveats, and these are important to consider when deciding which method to use. lncrnas can span large stretches of dna sequence and can contain important regulatory regions, such as enhancers, that are functionally independent of the lncrna product [ ] . lncrna promoters have been proposed to also function as enhancers, promoting the recruitment of transcriptional-activating factors that can affect the local nucleosome environment and ultimately the expression of neighboring genes [ , , ] . nevertheless, in vivo assessment of lncrna function has predominantly relied on assessing the consequence of deleting entire lncrna loci [ , ] . numerous studies involving deletion of lncrna loci have been unable to rescue the deletion phenotype using a transgene approach, making it difficult to attribute the phenotype to the lncrna product itself [ , ] . while deleting the entire lncrna is a useful first step to establish a phenotype, this approach can make it difficult to identify which component of the lncrna is important for the observed phenotype [ , , [ ] [ ] [ ] . there are many approaches to generating a lncrna knockout/ knockdown mouse. for instance, the complete knockout is the easiest first step to take, which can be designed to remove the entire gene. after this approach, one can use a more fine-tuned approach to reveal exactly how the gene is working, including deleting specific regions of the gene or inserting a poly-adenylation cassette before the exon of the gene. a well-documented example of this phenomena is fendrr, which was shown to have a lethal phenotype in two independent studies, but the importance of the lncrna in development differed due to the mouse ablation strategy. one in vivo study generated a knockout mouse by removing the genomic loci completely and replacing it with lacz while leaving the native promoter intact [ ] . this study identified fendrr as a key regulator of lung development and mesenchymal differentiation. however this study did not attempt to rescue the ablated fendrr allele via a transgene [ ] . an additional group generated a fendrr knockdown mouse using an alternative approach. instead of disrupting the chromatin architecture or removing any possible dna enhancer regions, they inhibited the transcription of fendrr through the insertion of a poly-a cassette into exon [ ] . using this lncrna knockdown mouse design, the scientists determined that loss of fendrr led to heart and body wall defects, which is slightly different from the phenotype in the ko mouse study. importantly, the heart and body wall defects caused by the terminator insertion were rescued by a transgene of fendrr in vivo, further confirming that the phenotype is due to the rna while the ko phenotype could be due elements within the genomic dna [ ] . fendrr is located~ kb downstream of foxf and~ kb upstream of irf . the observed phenotype of the ko mouse is possibly due to the deletion of an enhancer element that could impact the expression of protein-coding genes, which could have roles in lung development [ , ] . we recently used multiple genetic mouse models to dissect both cis and trans functions of lincrna-cox in vivo [ ] . working with a complete lincrna-cox knockout [ ] , in which the gene is replaced with a lacz cassette, we observed a strong cis defect on the neighboring protein-coding gene ptgs . from these studies, we concluded that lincrna-cox functions in cis through an enhancer rna mechanism to regulate its neighboring gene ptgs [ ] . in order to determine how lincrna-cox functions in trans to regulate genes independent of its cis effects, we generated a mutant/intronless mouse by targeting the splice sites of lincrna-cox using crispr/cas . this mouse represents a knockdown mouse, and because there is a low level of transcription of lincrna-cox , ptgs levels are the same as wt. lincrna-cox is not inducible in the mutant mouse, probably because of transcript instability due to the lack of splicing, enabling us to study its trans regulatory roles. similar to our early in vitro work we observed using an lps shock model that many genes are both up and downregulated in the serum of mutant mice indicating that lincrna-cox can indeed function in trans to regulate immune genes in vivo [ ] . to prove genetically that a lncrna is functioning in trans, a trans rescue experiment can be performed using transgenic mice that constitutively express a lncrna. this rescue strategy has been utilized in some studies including evf [ , ] , jpx [ ] , as well as pnky [ ] which both demonstrate successful rescue experiments where the phenotype from germline ablation of a lncrna is rescued through generation and crossing with a transgenic animal. understanding the mechanism of action of a lncrna lncrnas are immensely adaptable molecules that are capable of working through rna-rna, rna-dna, or rna-protein interactions. rna-directed technologies such as chromatin isolation by rna purification (chirp) [ , ] or rna antisense purification (rap) [ , ] , will help uncover lncrna interactomes for rna, genomic or protein partners for highly expressed candidates. if a candidate is lowly expressed, one can exogenously introduce a biotinylated form of the lncrna using the rna pull-down method, which we have successfully used to identify binding partners for lincrna-cox . this has been performed for many lncrnas [ , , ] . functions of lncrnas are associated with their subcellular fates. web servers can assist in quickly assessing experimentally determined or predictive rna-rna interactions [ ] or even rna-protein interactions [ , ] . depending on the subcellular or extracellular compartmental localization of a lncrna, this patterning will elude to the regulatory role of the gene, as well as how a lncrna might execute its function [ ] [ ] [ ] . some lncrnas that are localized to the nucleus or chromatin have been experimentally shown to function in cis to regulate the transcriptional expression of a neighboring gene, or in trans to regulate the transcriptional regulation of a subclass of genes through the interactions between heterogeneous nuclear ribonucleoproteins (hnrnps) [ , , , ] . in the cytosol, some lncrnas have been shown to interact with rnas and proteins to carry out their molecular functions [ , , , ] . cellular localization of lncrnas can be predicted using a publicly available user-friendly web server established at http://lin-group.cn/ server/iloc-lncrna [ ] . this can be the first step by a researcher to attempt to predict the localization of your candidate based off of its sequence. iloc-lncrna predicts subcellular location of a lncrna by utilizing the -tuple nucleotide features into the general pseknc (pseudo k-tuple nucleotide composition) and rigorous tests show the overall accuracy achieved by the new predictor is . %, which is over % better than previous algorithms [ ] . in addition to prediction methods, there are also publicly available sequencing datasets that have performed rna sequencing on fractionated cells. bhatt et al. utilized murine bone marrow derived macrophages, with and without inflammatory stimulation, and fractionated these cells into chromatin, nuclear, and cytoplasmic compartment to determine rna localization [ ] . this data set can now be utilized to investigate the localization of any murine candidate expressed in macrophages. a recent study took this question a step further by performing rna sequencing on nine separate locations with a cell including: nucleus, nucleolus, nuclear lamina, nuclear pore, cytosol, endoplasmic reticulum membrane (erm), outer mitochondrial membrane (omm), mitochondrial matrix (mito), and endoplasmic reticulum lumen [ ] . this exciting study utilized an apex-sequencing method, where the peroxidase enzyme apex was localized to these nine separate locations in nine separate human embryonic kidney (hek) t cell lines. apex can biotinylate nearby rna molecules allowing for streptavidin-based immunoprecipitation and rna sequencing. the apex-seq datasets will provide a powerful resource for referencing localization of specific lncrna candidates that are expressed in hek t cells [ ] . there are a few commonly used experimental approaches that can be used to validate and determine the localization of a lncrna. subcellular chromatin, nuclear and cytoplasmic fractionations of any primary or immortalized cells can be prepared using previously published procedures [ , ] , followed by rna isolation and rt-qpcr to assess the localization. if additional compartmental fractionation is desired other compartments can be enriched, such as mitochondria [ , ] . another standard gold technique is to visually determine cellular localization by rna fish [ ] . rna localization can also be directly visualized by microscopy [ ] . seqfish techniques have recently been pioneered for imaging thousands of cellular rnas at once using barcoded oligonucleotides [ ] . the drawbacks of these in-situ fluorescence hybridization (fish) based approaches, however, are the need for cell fixation and permeabilization, which can re-localize or extract cellular components [ ] . in addition to the difficulty of assigning rnas to specific organelles or cellular landmarks due to spatial resolution limits, some of these difficulties can be overcome with the addition of stains for markers of specific organelles. conventionally, lncrnas display poor sequence conservation across species, with the exception of finite regions of conserved bases surrounded by large seemingly unconstrained sequences [ ] . while sequence conservation does not constrain lncrna genes, lncrna function is found to be conserved across species when identifying motifs or structure. a great example of this phenomena is represented in the study of human maternally expressed gene (meg ), which utilized the computer program mfold and multiple ex vivo and in vitro chemical probing techniques to identify common motifs critical for retained function in orangutan, rat, mouse and pig [ , ] . another study highlighting the marsupial rsx lncrna was initially found to have no linear sequence similarity with the lncrna xist. however, it shared substantial levels of non-linear conservation within k-mer repeats that share functionally analogous protein-binding domains [ ] . publicly available web servers can be utilized to determine the rna structure of a lncrna depending on the size. the caveats to these web servers are they do not work efficiently with large transcripts. to overcome this, one can attempt to identify the critical sequence within the lncrna that could be functional using rip-sequencing databases, which allow you to input a gene id to identify possible rna binding proteins (rbps). additionally, if a lncrna has already been identified to bind to a protein (s), rip-sequencing databases can elucidate the specific binding location(s). knowing the location of rna-protein interaction narrows down the sequence input, that can be used for many web servers, which could enhance the elucidation of a predictive structure. if there is no information about potential protein binding partners, then these rna structure webservers will be of little use. an alternative approach to further investigate structure is to utilize bioinformatic tools which now include parameters for covariation. covariation analysis identifies the positions in an rna molecule that have similar patterns of variation and the purpose of this covariance is due to structural constraints initially shown for ribosomal rna [ ] and now also for lncrnas [ , ] . this study predicts structures for malat , using over vertebrate sequences, as well as lncrnas repa and hotair [ ] . these powerful tools allow scientists to predict structures in an rna molecule based on covariance, which in turn drive the next steps of experimentally validating these findings. fortunately, there are several in vitro and in vivo experimental techniques used to assess rna structure for any size, even up to , nt (xist). dimethyl sulfate (dms) probing uses a base-specific reagent that can bind and alter the methylation state of unpaired adenosine and cytosine nucleotides [ , ] . dms "footprinting" is optimized for structural analysis of rna. protein binding to rna will generate a "footprint" that can be traced due to alterations in the rna structure. the transcript size that can be evaluated is rather small (< nt) but this method can be performed both in vitro and in vivo as dms can easily penetrate the cell membrane, shown to work for lncrnas from nt, braveheart (brvht) [ ] . xue et al. utilized dms and shape to determine the multiple smaller order structures of brvht, including an agil motif and a -degree turn [ ] . this agil motif is critical for transcription factor binding, which specifies the cardiovascular lineage. targeting structure-seq relies on rna methylation by dma being performed in vivo. using this method, structural models of elements within xist were developed [ ] . shape (selective ′hydroxyl acylation by primer extension), as well as the modified shape-map [ ] , in-cell shape-seq [ ] and icshape-seq [ ] , can interrogate the rna structure both in vitro and in vivo using the chemical nmia and its derivatives to detect flexible regions in rna secondary structure [ , ] . this method has been proven valuable for xist [ ] , repa [ ] , and rox / [ ] . paris (psoralen analysis of rna interactions and structures) was recently developed to determine both rna structure and interactions in vivo [ ] . using this approach, a model for the higher order structure of xist was interrogated [ ] . these approaches are critical for identifying structural motifs and enhance conservation studies, as well as these identified elements, can be used as novel targets for further exploration in precise intervention suitable for therapeutic applications. alternative splicing (as) significantly impacts the diversity of rna isoforms produced, which in turn impacts the protein isoforms produced and can affect many aspects of the protein's biology including binding, intracellular localization, enzymatic activity, stability, posttranslational modifications [ ] . as also impacts lncrna genes which can have multiple isoforms depending on the cell/tissue, age, and disease state [ ] [ ] [ ] . the ucsc genome browser [ ] , as well as noncodev [ ] , have all the annotated isoform transcripts for each gene. these tools will identify annotated transcript isoforms while rna sequencing will provide information on which of these splicing events is utilized in a given cell or biological state. to date, there have only been a small number of papers focusing on the role that alternative splicing plays in controlling the immune system. one recent global study has shown that widespread shortening of ′ untranslated regions and increased exon inclusion are evolutionarily conserved features of innate immune responses in primary human macrophages following listeria monocytogenes and salmonella typhimurium infection [ ] . this is a transformative study for mrnas but can be reanalyzed to examine as and possible contributions from lncrnas. publicly available tools for tissue isoform expression specificity is available on gtex [ ] and xena [ ] for human genes. tabula muris [ ] , a murine specific dataset, is now available as a ucsc genome browser track on mm and can be used to view cell-type specific splicing events and isoform expression. in order to identify isoforms in illumina rna sequencing datasets, several tools can be utilized. miso (mixture of isoforms) is software for a probabilistic model for rna seq will identify specific the ′ splice sites used for each isoform [ ] . two other tools highly used for splicing analysis are juncbase [ ] , majiq-spel [ ] , and drimseq [ ] . these tools can be used to define if your candidate gene undergoes any alternative splicing (alternative start site, exon inclusion/exclusion or alternative last exon) during a specific biological process for example following inflammatory activation. these tools are limited because of their dependence on a fully annotated transcriptomes. therefore if a lncrna is unannotated or has unannotated transcriptional isoforms, these events will not be captured. to overcome the limitations of incomplete transcriptomes, researchers can perform de novo transcriptome assembly using short rna-seq reads [ ] [ ] [ ] . the future of rna sequencing is headed towards long read sequencing, which is being met by pacific biosciences single molecule real time sequencing technology (pacbio) and oxford nanopore technology (ont) [ , ] . while both powerful technologies perform long read sequencing, their platforms are very different. pacbio technology is dependent on sequencing-by-synthesis. a dna polymerase incorporates nucleotides that each have a corresponding conjugated fluorescent dye. the dna polymerase works at a rate of bp/s, which is beyond the capabilities of current technologies. however, by circularizing the dna pacbio has overcome this limitation through continuous long read sequencing, resulting in ability to generate k- million reads at an error rate of below % [ , ] . on the other hand, ont's approach relies on a pore embedded in a membrane. as a long cdna or rna strand translocates through the nanopore at single nucleotide precision from enzymatic regulation, the ionic current across the membrane is recorded. this technology can sequence full-length transcripts and can yield up to million reads on the minion or up to million reads on the promethion for cdna [ , ] . an initial limitation of ont was the - % per read error rate, which has been overcome with a new technology called rolling circle to concatemeric consensus (r c ) bringing the error rate down to . % by increasing the read coverage. overall, both of these technologies have overcome the transcriptome assembly and isoform identification limitations of short-read illumina sequencing [ ] . as stated above, ont and pacbio can perform long read cdna sequencing [ ] and even more exciting, both technologies can perform direct long read rna sequencing [ , ] . the beauty and simplicity that ont and pacbio offers is the ability to sequence the full expressed isoform (cdna and direct rna), without worries of misidentifying a complex splicing pattern, rna cleavage events and also not relying on transcript annotation files will lead to the identification of novel isoforms which are problems faced using short-read illumina sequencing. for lncrnas there have been a couple of studies that focus on isoform specificity and function for a given lncrna. neat [ ] and lncrna-pxn-as [ ] are two studies that show how one gene can have different functions depending on the rna isoform expressed. while these studies are not immunology specific, this field is still at the early stages and we anticipate it becoming more prevalent in future studies. rna modifications are widespread and diverse in chemical nature, as well as highly conserved in their occurrence and function throughout species. rna modifications function to affect rna stability, localization, alternative site of poly-adenylation, and more [ ] . since lncrnas can function as decoys and scaffolds, which are highly dependent on rna structure, a single modification can enhance or eradicate this rna-protein interaction. as you study a lncrna, the mechanism of this molecule could be dependent on a modified nucleotide. there are many techniques used to determine a single rna modification in a cell type and biology of choice. site-specific cleavage and radioactive-labeling followed by ligation-assisted extraction and tlc (scarlet) technology give scientists the ability to probe for n -methyladenosine rna (m a) modification status at single nucleotide resolution in mrna and long noncoding rna [ ] . the significance of rna modifications to the control of the immune response is beginning to be appreciated. a study by winkler et al. showed that m a modification controls the innate immune response to infection by targeting type i interferons [ ] . a few recent studies have shown that lncrnas do have rna modifications such as malat containing m a modifications [ , ] , hotair containing m c and m a [ , ] and xist containing ψ, m a and m c modifications [ ] . a study by zhou et al. showed that the rna modification, m a, acts as a structural 'switch' in malat . when there is a modification at site , it results in an increased ability to bind hnrnpg, while a modification at leads to an increase in binding to hnrnpc [ ] . in clinical research, lncrna rp - j . is highly expressed in colorectal cancer cells (crc), and this specific upregulation was controlled by m a methylation [ ] . the study showed that m a could regulate the lncrna, which in turn triggered the dissemination of crc cells via post-translation upregulation of the protein zeb . this novel study, connecting the interplay of rna modifications and lncrnas, has paved the way for a novel predictive biomarker or therapeutic target in crc [ ] . there are over identified rna modifications, while only a few have been studied to any extent [ ] . of these rna modifications, the way they are enriched for in analysis is through an assortment of techniques including methylated rna immunoprecipitation (merip), merip-iclip (crosslinking and immunoprecipitation), suicide enzyme trap and clickable chemicals (reviewed in [ ] ). these techniques have many limitations and biases, but hopefully, future studies using direct rna nanopore sequencing will overcome all these pitfalls. in a recent study, direct rna sequencing using nanopore technology showed detection of m a modifications with a % accuracy with the design of synthetic sequences [ ] . as the performance of the algorithm increases, use of this tool will be extremely insightful when the flow-chart provides a "beginning to end" guide to study lncrnas. not all suggested databases will be appropriate for all lncrnas being studied. a. selection of candidate lncrnas should factor in changes in expression, type of lncrna and nearby coding genes. b. bioinformatic characterization of lncrnas can be done using a variety of online databases including: lncipedia to assess conservation or using the mouse cell atlas (mca) to assess tissue specific expression. c. expression validation: this includes validation of expression and confirming that the lncrna is in-fact non-protein coding. d. functional validation dives into the final stage of mechanistic characterization of a lncrnas, which involved manipulating its expression, as well as uncovering the specific cis-elements within the transcript important for its function. analyzing myeloid or lymphoid primary cells with and without a treatment to understand how rna modifications are regulated in innate immunity and specifically as it relates to our long noncoding transcriptome. lncrnas, including xist and h , have been studied intensely for decades [ , ] . at the time we had no idea that these genes would represent the largest family of rna genes produced in the genome. as louis pasteur once said, "chance favors the prepared mind," and this is especially true following the development of next-generation sequencing. rna-sequencing provided an unprecedented insight into the human genome. we did not identify new proteins, instead we found a wealth of noncoding rna transcripts. the lncrna field is growing at a blistering pace with labs from all aspects of biology, and now immunology branching out to include questions about the regulatory impact of these pervasive long noncoding gene species. as detailed in this review, there are many publicly available datasets and web servers that will streamline how to begin a lncrna project, from how to pick a lncrna candidate by interrogating published rna-sequencing data, to determine the best tools to use to study the function and mechanism of a candidate (fig. ) . since this field is still at an early stage in its development, there are some shortcomings, including poorly annotated lncrna transcripts. however, this will be overcome with direct rna sequencing using ont and pacbio technology. these technologies will enable us to determine the exact isoforms of transcripts expressed in a particular cell and begin to catalog the different rna modifications that exist basally and during a biological process such as activation of inflammation. since lncrnas are cell-type specific in their expression patterns continued development of single-cell sequencing technologies will provide a complete catalog of lncrnas in the genome. as the list of annotated lncrnas grows, characterizing the function of all these genes has become a definite bottle-neck in the field. however, highthroughput crispr screening provides an approach to quickly identify functional lncrnas in a particular biological system. utilizing all the tools outlined here should enable researchers to develop this field rapidly. for our research focus, gaining a better understanding of the role of lncrnas in regulating immune responses will provide novel insights into the molecular mechanisms governing inflammation. this data will be critical for identifying new avenues for therapeutic intervention for infectious and inflammatory disease. the transparency document associated with this article can be found, in the online version. pervasive transcription of the human 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putative imprinting control element the rox genes encode redundant male-specific lethal transcripts required for targeting of the msl complex loss of the abundant nuclear non-coding rna malat is compatible with life and development targeted disruption of hotair leads to homeotic transformation and gene derepression the tissue-specific lncrna fendrr is an essential regulator of heart and body wall development in the mouse genomic and epigenetic complexity of the foxf locus in q . : implications for development and disease transcription factor irf plays a critical role in the development of murine basophils and mast cells balanced gene regulation by an embryonic brain ncrna is critical for adult hippocampal gaba circuitry evf (dlx as) lncrna regulates ultraconserved enhancer methylation and the differential transcriptional control of adjacent genes lncrna jpx induces xist expression in mice using both trans and cis mechanisms the long noncoding rna pnky is a trans-acting regulator of 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acid: isoform structure, expression, and functions structural characterization of maternally expressed gene rna reveals conserved motifs and potential sites of interaction with polycomb repressive complex non-linear sequence similarity between the xist and rsx long noncoding rnas suggests shared functions of tandem repeat domains structural constraints identified with covariation analysis in ribosomal rna covariation analysis with improved parameters reveals conservation in lncrna structures the existence of phylogenetic covariation in base-pairing is strong evidence for functional dms footprinting of structured rnas and rna-protein complexes dms footprinting of structured rnas and rna-protein complexes genome-wide probing of rna structure reveals active unfolding of mrna structures in vivo visualizing the secondary and tertiary architectural domains of lncrna repa a g-rich motif in the lncrna braveheart interacts with a zinc-finger transcription factor to specify the cardiovascular 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pacific biosciences sequel third-generation sequencing platforms in identification of agricultural and forest pathogens pacbio sequencing and its applications an evaluation of the pacbio rs platform for sequencing and de novo assembly of a chloroplast genome nanopack: visualizing and processing long-read sequencing data rna and gene expression analysis using direct rna and cdna sequencing improving nanopore read accuracy with the r c method enables the sequencing of highly multiplexed full-length single-cell cdna transcriptome-wide analysis of a baculovirus using nanopore sequencing native molecule sequencing by nano-id reveals synthesis and stability of rna isoforms transcriptome profiling using single-molecule direct rna sequencing approach for in-depth understanding of genes in secondary metabolism pathways of camellia sinensis structural analyses of neat lncrnas suggest long-range rna interactions that may contribute to paraspeckle architecture the mbnl splicing factor promotes hepatocellular carcinoma by increasing pxn expression through the alternative splicing of lncrna-pxn-as a majority of m a residues are in the last exons, allowing the potential for ′ utr regulation probing n -methyladenosine rna modification status at single nucleotide resolution in mrna and long noncoding rna m a modification controls the innate immune response to infection by targeting type i interferons n( )-methyladenosine modification in a long noncoding rna hairpin predisposes its conformation to protein binding m a modification of non-coding rna and the control of mammalian gene expression topology of the human and mouse m a rna methylomes revealed by m a-seq comprehensive analysis of mrna methylation reveals enrichment in ′ utrs and near stop codons m a-induced lncrna rp triggers the dissemination of colorectal cancer cells via upregulation of zeb modomics: a database of rna modification pathways. update detecting rna modifications in the epitranscriptome: predict and validate accurate detection of m a rna modifications in native rna sequences a gene from the region of the human x inactivation centre is expressed exclusively from the inactive x chromosome the product of the h gene may function as an rna support from : national institute of health ar and the tobacco related disease research program ip- h to susan carpenter. key: cord- - fle fpz authors: o’shea, helen; blacklaws, barbara a.; collins, patrick j.; mckillen, john; fitzgerald, rose title: viruses associated with foodborne infections date: - - journal: reference module in life sciences doi: . /b - - - - . - sha: doc_id: cord_uid: fle fpz foodborne pathogens cause acute and chronic health outcomes of very different durations, severity and mortality, resulting in high costs and burdens to society. the issues of food safety and food poisoning are being increasingly emphasised, particularly in developed countries. infection/contamination with many agents i.e., bacterial, parasitic and viral entities can result in foodborne illness. this article will focus mainly on viral agents of infection. a range of different viruses can cause food poisoning/foodborne infection, and infection can result in a myriad of symptoms, ranging from mild, acute disease to chronic, debilitating disease and even death. due to the inherent differences between bacteria and viruses, namely the fact that viruses do not replicate in food, while bacteria do, viruses are frequently difficult to detect. this is compounded by the fact that many of the viruses associated with enteric disease do not replicate in cell culture. these factors can lead to a lag between reporting, detection and analysis of foodborne viruses versus bacterial agents. despite these constraints, it is now evident that there are both well-established and emerging viruses implicated in foodborne infections, and the role of molecular detection and characterisation is becoming increasingly important. in recent years, there has been increasing awareness regarding food safety in terms of food poisoning that results in viral gastroenteritis i.e., stomach flu. it is important to clearly define these terms, for the purpose of this article, prior to focusing on the smaller group of diverse viruses associated with food borne illness. food safety encompasses the handling, preparation, and storage of food to prevent food-borne illness. food poisoning (also referred to as foodborne illness) is caused by eating contaminated food. infectious organisms, which include bacteria, viruses and parasites (or their toxins), are the most common causes of food poisoning. infectious organisms or their toxins can contaminate food at any point of processing or production. contamination can also occur in the home and eateries, if food is incorrectly handled, stored, or cooked. viral gastroenteritis (also known as stomach flu) is an intestinal infection with symptoms including watery (usually nonbloody) diarrhea, abdominal cramps, pain, nausea or vomiting, or both, and sometimes headaches, muscle aches and fever. the most common way to develop viral gastroenteritis (stomach flu) is through contact with an infected person or by ingesting contaminated food, water and water products such as ice. there is a huge annual cost of illness, disease burden and quality-adjusted life year (qaly) loss globally, caused by food-borne pathogens, which has been reported on by many investigators (scallan et al., ) . in the western world, the viruses frequently reported as having the highest total cost-of-illness, to date, are norovirus and rotavirus. these viruses will be discussed first. this profile could change, however, as new virus threats are constantly emerging. a variety of different foods and different agents are implicated in foodborne illnesses, and we will elaborate on these in the following sections. many agents cause gastroenteritis, the most common outcome being diarrhea. this can range from mild, self-limiting illness to fatal infection. diarrheal disease is the major cause of illness and death in children in developing countries, while in the developed world it is usually mild, except in the very young, the elderly and the immunocompromised. gastroenteritis (acute gastroenteritis, age) is caused by a variety of different pathogens, including parasites, bacteria and viruses ( table ) . every day we swallow large numbers of microorganisms in our food and beverages, and from contact with our environment e.g., from fingers. our body's defense mechanisms, [innate (non-specific) and adaptive (specific) defense systems] are very efficient, and the microorganisms rarely succeed in surviving passage to the intestine in sufficient numbers to cause infection. the body's innate (non-specific) defense is the first line of protection and does not distinguish between infectious agents. it includes physical (e.g., skin and mucous membranes) and chemical barriers (e.g., sweat, gastric juices), cellular (e.g., natural killer cells and phagocytosis) and modular defenses (e.g., interferon) and bodily responses (e.g., inflammation and fever). with regard to protection against potential enteric pathogens, the intestinal tract has an abundant microbiota that competes with invading pathogens for space and nutrients. the peristaltic action of the digestive tract encourages movement through the system, deterring colonisation by invading pathogens, with vomiting and diarrhea flushing harmful microbes and their chemical products from the digestive tract. the extremely acidic stomach environment discourages pathogen replication. however, these fortifications are not impenetrable e.g., the enteric virus hepatitis a has the ability to survive and penetrate the body through the gastrointestinal tract (campbell et al., ) . in addition, the epithelial barrier of the intestine is embedded with mucous-producing goblet cells. these simple columnar cells secrete gel-forming glycoproteins (mucins), to produce a transparent, impervious barrier (johansson and hannsson, ) on the luminal surface. the prevailing mucin of the intestinal tract is muc (mucin ), which is a major structural component of intestinal mucus layer. goblet cells emerge during foetal development at - weeks of gestation (kim and khan, ) and are continuously secreted, migrating from the intestinal glands; crypts of lieberkühn to the lumen of the intestinal tract via the apex of the digestive villi. non-bacterial gastroenteritis and diarrhea is usually caused by viruses. acute viral gastroenteritis (age) is seen in all parts of the world, especially in infants and young children. age ranges from mild and self-limiting to severe, debilitating diarrhea, occasionally resulting in mortality. these illnesses have a huge impact, particularly in parts of asia, africa and latin america, with over million fatalities recorded per annum. age also has major effects on nutritional status and growth. viruses appear to be the commonest causes of gastroenteritis in infants and young children but are not distinguishable clinically from other types of gastroenteritis. some of these viruses are specific for humans, while others are implicated in zoonotic disease. in most instances, infection follows the general rules for faecal-oral transmission. enteric viruses are transmissible by food and water and enter the body through the gastrointestinal tract, thus the viruses are commonly acquired after ingestion of sewage contaminated food or water or from poor hygiene when preparing food. the viruses are shed in high concentrations in faeces and vomit and can remain infectious in the environment for several months. many viruses are difficult or impossible to cultivate in vitro, and tests are unreliable or not developed, thus many cases of non-bacterial acute gastroenteritis viruses are under reported. well established viral pathogens will be discussed in this section. later in the article, emerging viral pathogens associated with food poisoning/foodborne illness/foodborne infections will also be discussed. there are several different viruses involved in foodborne outbreaks. some of these are human viruses that infect and cause illness following ingestion. virus particles are shed in the faeces. to date, the most notable examples in this category are noroviruses and hepatitis a virus. these viruses are different, belonging to different families ( table ) : noroviruses (formerly called small round structured viruses or srsvs) belong to the family caliciviridae and are small, non-segmented, positive sense, single stranded nonenveloped, icosahedral rna viruses. hepatitis a virus is also a small, non-segmented, positive sense, single stranded nonenveloped icosahedral rna virus, but belonging to the family picornaviridae. hepatitis a virus is slightly smaller and contains a linear genome. is one the five pathogens (namely, salmonella, campylobacter, listeria, toxoplasma, and norovirus) responsible for causing roughly % of mean loss due to foodborne illness in the united states (hoffman et al., ; scallan et al., ) . as outlined above, noroviruses are small, non-enveloped, positive sense rna viruses, within the family caliciviridae. noroviruses have been classified into seven genogroups (gi-gvii) and over genotypes. gii. has long been established as a predominant variant, but new variants are continually emerging (lennon et al., ) . transmission of the virus may occur through sewage contamination early in the food chain or lack of personal hygiene later in the food chain, particularly when 'ready-to-eat' (rte) food-items are involved. transmission can also occur via by person-to-person contact, environmental contamination, as well as via food-and water-borne transmission. typically, noroviruses cause problems in individuals who have consumed contaminated raw oysters. other implicated foods are fruit and vegetables that have, for example, been irrigated with contaminated water. outbreaks frequently occur in hospitals, nursing homes, restaurants, cruise ships, schools, summer camps, and even at family dinners. in these cases, large numbers of people often eat food handled or prepared by others. the symptoms of norovirus infection vary, but may include nausea, vomiting, abdominal pain or cramps, watery or loose diarrhea, malaise, low-grade fever, muscle pain. signs and symptoms usually begin - h after first exposure to the virus and last one to three days. shedding of virus can continue for up to two weeks post recovery. some people with norovirus infection may show no signs or symptoms but are still contagious and can spread the virus. there are several recommendations concerning food preparation, disinfection and decontamination, outlined on e.g., the centres for disease control and prevention (cdc) website (see "relevant websites section"). these measures involve practicing good hand hygiene, washing fruit and vegetables and cooking seafood, especially shellfish e.g., oysters, thoroughly, individuals who are ill avoiding contact with others, also food preparation, for at least h after symptoms abate, and decontamination using suitable disinfectants and detergents. some people are particularly vulnerable and should take measure to avoid norovirus infection, especially infants, older adults and people with underlying disease where vomiting and diarrhea can be severely dehydrating and require medical attention. preventative measures e.g., avoiding raw shellfish should be taken in these instances. for many years, rotavirus (rv) has been recognized as a major cause of gastroenteritis in the young of many animal species, including humans (bishop et al., ; estes and kapikian, ; flewett et al., ) . in humans, rv associated disease typically occurs in children less than years of age but rotavirus can infect all ages, including adults (anderson et al., ; collins et al., ; gunn et al., ) . in , an estimated , deaths worldwide were attributed to rotavirus infection, with most deaths occurring in resource-poor countries (o'shea et al., ; tate et al., ) . rotavirus belong to the family reoviridae and are medium sized, segmented, double stranded non-enveloped, icosahedral rna viruses. the segments encode six virion proteins (vp - , - ) and six non-structural proteins (nsp - ). rotaviruses are classified into antigenically distinct groups and one proposed group (a-i) (mihalov-kovács et al., ) , using immunological and phylogenetic analysis of the vp gene. rotavirus groups a, b, c, h and i have been identified in mammals, including humans, while rotavirus d-g to date have only been identified in non-human mammal and avian species. a tenth novel rotavirus species has been identified in schreiber's bats (miniopterus schreibersii), provisionally known as rotavirus j (bányai et al., ) . within the rva group, the viruses are classified antigenically and genetically, based on the main antigenic determinants, the outer capsid proteins, vp and vp , which specify the g and p serotypes/genotypes, respectively. a whole genome classification system has been adopted for classification of all segments of the rotavirus genome, applying nucleotide homology cutoff values to distinguish genotypes. transmission of rotavirus is predominantly faecal-oral. in humans, rotaviruses are ubiquitous, with % of children worldwide being infected by three to five years of age. in infants, prior to the introduction of rotavirus vaccines, rvas could be detected in up to %- % of all childhood hospitalisations due to acute gastroenteritis each year, were estimated to cause million cases of gastroenteritis annually, and , deaths in children o years of age living in developing countries. in other animals, rotavirus disease also occurs in the young of the species and in farm animals, leads to significant economic losses. the symptoms are usually characterised by watery dehydrating diarrhea and vomiting, often accompanied by abdominal cramps and low-grade fever, lasting - days. subsequent reinfections are associated with mild or subclinical presentations (bishop et al., ; velázquez et al., ) ; however, such reinfections are important in boosting immunity and maintaining long-term protection from rotavirus disease. due, in part, to the segmented nature of the genome, accumulation of point mutations (genetic drift) and re-assortment (genetic shift) are responsible for the huge genetic heterogeneity of rotaviruses. these mechanisms, in combination with the potential for interspecies (zoonotic and anthroponotic) transmission, can also lead to the emergence of "novel" strains in a given species, with a potential for epidemic or epizootic spread (martella et al., ; matthijnssens et al., ) . the zoonotic potential of rva has been documented on several occasions (martella et al., ) . uncommon human rva genotypes include g , g , g , g and g , in combination with p[ ], p[ ], p[ ], p[ ] and p[ ] p types, and are generally considered to be animal to human reassortment variants. however, certain animal-like genotypes have become established in human circulation, particularly in developing countries. since its discovery, many attempts have been made to produce effective rotavirus vaccines, with an emphasis on those directed to the prevention of human disease. today, two oral live attenuated vaccines are being used in rotavirus immunisation programmes globally; rotateq™, which is a pentavalent human-bovine reassortment containing human rva derived g to g and p [ ] types within the backbone of the wc bovine strain, and rotarix™, a monovalent human g p[ ] live attenuated vaccine. both vaccines are efficacious and endorsed by the who, which recommends the implementation of these vaccines in national immunisation programs (who, ) . the introduction of universal mass vaccination (umv) has resulted in a significant decrease in childhood rotavirus infection morbidity and mortality (curns et al., ; pendleton et al., ; tate et al., ; usonis et al., ; zeller et al., ) . hepatitis or inflammation of the liver can have a number of causes, including medications, toxins, alcohol use and viral infection. there are hepatitis viruses, a-e, but only a and e are transmitted by the oro-faecal route. both viruses are major causes of infectious disease with associated socio-economic consequences caused by morbidity and, in vulnerable groups, mortality. hepatitis a virus (hav), also known as hepatovirus a, belongs to the order picornavirales in the family picornaviridae and is the type species of the genus hepatovirus. it is an icosahedral, non-enveloped virus with a monopartite, positive, single-stranded rna genome. humans and vertebrates serve as natural hosts. there is a single serotype of hav but several genotypes: ia, ib, iia, iib, iiia, iiib primarily in humans and iv-vi, primarily found in non-human primates (cristina and costa-mattioli, ) . the virus infects hepatocytes and kupffer cells (liver macrophages). transmission: hav is spread in food, in the water system, by touching contaminated surfaces or by direct contact with an infected person. contamination of food can occur at any stage from farm to fork. according to the who, approximately . million people are infected each year, although the true infection rate is probably much higher due to the asymptomatic nature of many infections (hepatitis a fact sheet. in: world health organisation: media centre). due to the prolonged infection times, the economic consequences of an outbreak through absenteeism from work can be significant. in developing countries where sanitation is poor, most children are infected while young and are asymptomatic. this gives them immunity and, as such, they cannot become reinfected as adults. in such areas, epidemics of the virus are uncommon but unvaccinated adult visitors from areas where hav is not commonplace are vulnerable. in economically developed countries, epidemics are rare, because good hygiene practices will prevent person-to-person spread. however, in transitional economies, the introduction of improved sanitation may mean that the population is not exposed as children. as such, the introduction of the virus to these susceptible populations of adults can result in significant outbreaks (lemon et al., ) . the symptoms: the virus is normally mild without permanent repercussions and is typified by fever, fatigue, loss of appetite, nausea, vomiting, abdominal pain, dark urine, clay-coloured stools, joint pain and jaundice (yellowing of the skin and eyes). the development of fulminant hepatitis is rare (less than . % of cases) and can lead to more serious illness, including liver failure and death. severe illness is more common in older people, while in children around % are asymptomatic and in those with clinical manifestations the symptoms are generally mild. infected persons may shed infectious virus for several weeks before they show symptoms. in those individuals where symptoms occur, they usually start appearing weeks after exposure, but can occur as early as and as late as weeks following exposure. symptoms usually develop over a period of several days and last less than months, although some people ( %- %) with hepatitis a can have symptoms for as long as months (jeong and lee, ) . hea can survive outside a host cell for several weeks in groundwater and it has been shown to retain infectivity after days at °c in seawater. the virus is tolerant of desiccation and freezing and will survive on vegetables throughout the production process until consumption (a critical review of the effect of heat, ph and water activity on the survival of hepatitis a and e viruses -a report to the united kingdom food standards agency july ). hea is more resistant to heat than other picornaviruses and the centre for disease control states that temperatures of °c for a least min are required for inactivation. hep a is vaccine preventable (ott et al., ) . monovalent formalin killed vaccines are available worldwide and live attenuated versions are available in a number of countries. a double dose of vaccine can provide long-term protection and single dose immunisation strategies have been shown to be effective at least in the short and medium term. as well as for active immunisation, the vaccine can be used prophylactically in the first few weeks of infection. human immunoglobulins can also be used for both preventative and post-exposure prophylactic treatment (liu et al., ) . post-exposure prophylaxis provides high levels of protection ( %- %) if provided in the first weeks of exposure. otherwise standard methods for the prevention of food-borne infections apply: ensuring good sanitation and a clean water supply, hand washing and food safety. hepatitis e virus (hev) is a small non-enveloped icosahedral virus, - nm in size, with a single-stranded positive sense rna genome. the virus belongs to the family hepeviridae, genus orthohepevirus. there are four species in this genus, and the viruses that infect humans belong to the species orthohepevirus a. currently, eight genotypes of orthohepevirus a have been identified. hev and hev infect humans only, hev has a range of known host animals including humans and swine, hev infects humans and swine, hev and hev have been detected in wild boar and hev has been detected in camels (sridhar et al., ) . transmission: hepatitis e virus (hev) is the most common cause of acute viral hepatitis worldwide (sridhar et al., ) . the virus is responsible for endemics and epidemic outbreaks and the who estimate there are million infections and . million symptomatic cases worldwide per year; in these led to , deaths (world health organization, ). hev is primarily transmitted through the faecal-oral route via shedding in the faces of infected individuals and subsequent contamination of drinking water. other routes of transmission are consuming raw or undercooked foods from infected animals or shellfish, blood transfusions or vertical transmission from pregnant women to the foetus (wulffen et al., ) . the virus has a worldwide distribution but is most prevalent in south and east asia and is associated with areas where sanitation is poor and contaminated faeces can enter the water system. in regions where the virus is endemic, outbreaks are common, usually associated with contaminated drinking water, recurring at intervals of years and may involve thousands of people. such infections are predominantly genotype or (melgaço et al., ) . in non-endemic regions, infections were previously associated with international travel, but now the majority of cases are zoonotic genotype or infections from swine or deer. handling of swine and manure of porcine origin is now a public health concern and infectious hev has been isolated from meat products (yugo and meng, ) . the symptoms: while hev is primarily a disease of the liver, it has also been associated with non-hepatic diseases such as subacute and monophasic neurological disorders of the peripheral nervous system, acute pancreatitis, glomerulonephritis, mixed cryoglobulinemia, severe thrombocytopenia and haemolytic anaemia (pischke et al., ) . the hepatic symptoms of hev are indistinguishable from other forms of acute hepatitis and laboratory diagnosis is required for a definitive diagnosis. the incubation period is approximately - weeks. hev is characterised by fever, reduced appetite, nausea and vomiting, abdominal pain, and a slightly enlarged, tender liver (hepatomegaly) (kamar et al., ) . in rare cases, acute hepatitis e can be severe, resulting in liver failure and death. pregnant women and immunosuppressed people are at high risk of these severe symptoms. in pregnant women mortality rates of % have been recorded (perez-gracia et al., ) . like hav, infection of young children is often mild or asymptomatic. hev is self-limiting and most cases do not require treatment. for those with acute symptoms or for high risk individuals, hospitalisation is required. the antiviral drug ribavirin may be given, although in pregnant women this must be carefully considered, due to the teratogenic nature of this antiviral compound (krzowska-firych et al., ) . a recombinant subunit vaccine to prevent hepatitis e virus infection exists in china but is not licenced elsewhere (nan et al., ) . the family adenoviridae contains genera; atadenovirus ( species) infecting birds, lizards and mammals; aviadenovirus ( species) infecting birds; ichtadenovirus ( species) isolated from sturgeon; mastadenovirus ( species) infecting mammals only, including humans and; siadenovirus ( species), mostly infecting birds and frog species. virions are non-enveloped, - nm in diameter with a double-stranded dna genome and an icosahedral capsid. there are human species of human adenoviruses, a-g (lennon et al., ) . within these species there are at least subtypes distinguished either by serological differences of by genotypic classification (chen and tian, ) . transmission: adenovirus-associated gastroenteritis often occurs in clusters in schools, hospitals or military camps. adenoviruses are spread person-person contact, by coughing and sneezing, by touching contaminated surfaces or by the faecal-oral route (eckardt and baumgart, ). adenoviruses infections are not associated with contaminated food, but transmission can occur in water, through public water systems or in swimming pools; the latter are predominately associated with conjunctivitis (rodríguez-lázaro et al., ) . symptoms: adenoviruses in humans cause respiratory infections, gastrointestinal disease and conjunctivitis with hemorrhagic cystitis, hepatitis, hemorrhagic colitis, pancreatitis, nephritis, or encephalitis (lynch and kajon, ) . adenoviruses infections are often asymptomatic or mild and self-limiting. however, they can be associated with severe morbidity or mortality, with immunocompromised and the young being more susceptible. however, novel or emerging strains have been found to cause mortality in people of all ages such as ad , which was responsible for fatal respiratory illness in the usa (centres for disease control, ) . a number of human adenovirus subtypes can cause gastrointestinal symptoms, but subtypes and from species f are the most commonly associated with age and have been reported to be responsible for %- % of acute gastroenteritis in children (ziros et al., ) . adenovirus-associated gastroenteritis is most common in children under years old and is uncommon in adults, accounting for . %- . % of cases (eckardt and baumgart, ) . the incubation period is around - days after which diarrhea develops and, in some cases, mild vomiting. fever lasting - days may develop; severe dehydration is rare (wood, ) . currently vaccines against human adenovirus and are being used by the us military, but no vaccines are available for general use (chen and tian, ) . as the virus is usually self-limiting in most cases, treatment of adenoviral gastroenteritis is not required. in severe cases, hospitalisation and rehydration may be needed. prevention of adenovirus infection is by standard antiviral hygiene methods; avoiding sharing of eating and drinking utensils, hand washing and avoiding contact with sick individuals, especially in at risk environments such as hospitals. adenoviruses are susceptible to chemical disinfectants but can be resistant to uv irradiation (rodríguez-lázaro et al., ) ; in swimming pools, chlorine levels must be adequate. the family astroviridae consists of genera; avastrovirus and mamastrovirus infecting birds and mammals, respectively. virions are non-enveloped, approximately - nm in diameter with a single-stranded positive sense genome and an icosahedral capsid. the international committee for the taxonomy of viruses (ictv) currently identifies species in the genus mamastrovirus infecting felines, canines, cattle, cervids, rodents, swine, sheep, mink, bats, rabbits, sea lions and dolphins. the viruses that infect humans (referred to as classical hastvs) have traditionally been classified into serotypes, but recently a number of viruses have been detected in humans that are more similar to those from other animals, suggesting cross species transmission (bosch et al., ) . transmission: hastvs are predominantly transmitted through the faecal-oral route as well as through drinking water and in sewage. recreational activities in sewage contaminated water bodies is a risk of infection. hastvs are considered to be food-borne viruses with molluscs grown in, or fruits or vegetables irrigated with contaminated water, representing the biggest threat (vu et al., ) . as would be expected, poor food hygiene practices may play a role in outbreaks, particularly as asymptomatic carriers are more likely to contribute to infection in the food industry than symptomatic workers. contaminated fomites and surfaces can represent a threat in institutions such as schools and hospitals (abad et al., ) . as we are now aware that inter-species infections with astroviruses can occur, and, as such, the zoonotic route should be considered as a potential source of astrovirus infection (bosch et al., ) . the symptoms: classical human astroviruses are known to cause mild gastrointestinal disease in children, but symptoms and prevalence are serotype dependant. immunocompromised and elderly patients are also susceptible. serotype is the most common, with a reported level of seropositivity of %- % having been reported in children by years of age (koopmans et al., ) . the symptoms are usually watery diarrhea, vomiting, fever, abdominal pain, anorexia, and headache but these are usually mild in nature and self-limiting, typically lasting for - days (vu et al., ) . however, hastvs do cause asymptomatic infections (méndez-toss et al., ) and the association of these viruses with disease is not fully understood. while the symptoms are mild, hastvs do contribute to a large proportion of outbreaks of diarrhea, . %- % of all cases, and often occur as coinfections with other viruses such as noroviruses and rotavirus (de benedictis et al., ) . rarer astroviruses have been reported as being responsible for fatal cases of central nervous system infection (fremond et al., ) . as with most gastrointestinal infections control of hastvs is by prevention of contamination of food and water and prevention of person to person, or fomite to person spread through hygiene and handwashing. while survival of astroviruses in drinking water is known to be high, chlorination has been shown to be effective in reducing astrovirus viability by alteration of the capsid, resulting in non-infectious virions (abad et al., ) and % alcohol has been shown to be effective for decontamination of surfaces and hands (kurtz et al., ) . no vaccines are available for hastvs. as infections are usually mild and self-limiting, no treatment is normally required. however, those with severe gastroenteritis may require oral or intravenous rehydration. recent emerging epidemic and pandemic virus infections that cause severe disease in humans and that are associated with food production, preparation and food contamination include the coronavirus, severe acute respiratory syndrome (sars-cov), nipah virus, ebola virus and some of the highly pathogenic influenza virus strains, such as the h n subtype. transmission may be by a variety of routes, but often the emergent epidemic is started by contamination of a food source by saliva, urine or faeces from a wild reservoir species or use of a wild animal (bush meat) as the food source and infection through the capture and butchering process (e.g., hiv is thought to have entered the human population by this route). these infections have a low probability of occurring, but a very high impact if they establish infection in domestic animals and humans. the coronaviridae belong to the order nidovirales of single stranded, positive sense rna viruses. within the coronaviridae there are sub-families, of which the coronavirinae contain genera. phylogenetically, sars-cov is within the betacoronavirus genus (ictv, ) . in older literature, sars-like covs were assigned to group b coronaviruses (lau et al., ) . the viruses are roughly spherical ( - nm diameter), with an envelope that contains many surface glycoproteins that form a corona (crown) under electron microscopy (masters and perlman, ) . although enveloped, the virus is relatively stable especially in faeces and urine for - days (or longer) at room temperature, and up to days if stool is from diarrhea patients (the ph is higher) (see "relevant websites section"). however, they are sensitive to heat, lipid solvents, oxidizing agents and non-ionic detergents (markey et al., a) . many coronaviruses are associated with respiratory and intestinal disease and can cause severe epidemic gastrointestinal disease in agriculturally important species such as porcine epidemic diarrhea virus in pigs (masters and perlman, ) . transmission: is faecal-oral, respiratory by aerosolised secretions. sars-cov was first discovered in , after a pandemic of severe respiratory disease that originated in guangdong province, china. it caused disease in people and of these, died (see "relevant websites section"). it is now believed that chinese horseshoe bats are the original reservoir host of sars-like covs (lau et al., ; li et al., ) . bats transmit these viruses, probably via faecal contamination of food sources directly to humans or to intermediate hosts, most notably palm civets and raccoon dogs, and these then transmit virus to humans (guan et al., ) . these animals are traded in live animal markets as food and it is thought that aerosolisation of faeces and other body fluids caused respiratory infection of humans (wang et al., ) . once sars-cov entered humans, it was spread by respiratory droplets or contamination of surfaces by droplets that were then transferred to the mouth, nose or eyes by touching. there was also a cluster of cases in an apartment block in hong kong that probably arose from sewage aerosolisation into the ventilation system that proceeded to cause respiratory infection of many of the inhabitants (tilgner et al., ) . the incubation period is - days but can be up to days. infection results in symptoms of a high fever, chills, and headache. some people have mild respiratory symptoms from the beginning that progress to lower respiratory involvement with a nonproductive cough, which can lead to hypoxemia and pneumonia. a low proportion of patients ( %- %) had diarrhea. as stated above, approximately % of patients died from the severe respiratory complications, but these were usually older patients or those with other health problems. up to % of patients needed mechanical ventilation to survive. the severe sequela could take many days and weeks to develop. shedding in faeces occurs for a long time after symptoms are cleared so contact from soiled material from past patients can be a source of infection (masters and perlman, ) . there have been no known cases of sars since (see "relevant websites section"). however, sars-cov is not eradicated, as similar viruses are still present in their wild-life hosts. therefore, continued vigilance must be used to stop the spread of these viruses from their zoonotic hosts to humans. control of live animal markets and a ban on sales of exotic animals is required (see "relevant websites section"), but difficult to introduce due to the cultural background of the communities in which they are found. there is no vaccine or treatment against the virus (coleman and frieman, ) , although several drugs have been developed that could be used in the future (masters and perlman, ). nipah virus is a member of the viral family, paramyxoviridae in the genus henipavirus (ictv, ). it was originally identified in in malaysia during an outbreak of encephalitis and respiratory disease in pig farmers and those who had close contact with pigs. its virions are roughly spherical of about nm diameter but filamentous forms can be seen (wang et al., ) . it is a relatively unstable virion (markey et al., c) , but can survive well in ph-neutral fruit bat urine ( days at °c), on fruit (up to days) and in artificial date palm sap (de wit et al., ; fogarty et al., ) . it is however susceptible to desiccation (fogarty et al., ) . it is an enveloped virus that is inactivated by most common disinfectants, lipid solvents, and non-ionic detergents (markey et al., c) . transmission is respiratory and oral. the wild-life reservoir host of nipah virus are pteropus fruit bats (chua et al., ; yadav et al., ) and transmission may be by direct infection of humans, after the virus has passed through an intermediate host e.g., pigs, or from fomites. it is thought that bat saliva or urine is the major source of virus and so food such as partially eaten fruit contaminated with saliva or contamination by faeces and urine may be the source of infection for other hosts. in the malaysian outbreak of - , it is thought that contaminated fruit from trees adjacent to pig farms encroaching into forest were dropped into enclosures where they were eaten by the pigs or that there was direct contamination of enclosures with urine. the pigs amplified the virus (they also showed clinical respiratory and neurological disease), so that farmers and workers in direct/close contact were infected and were the population where the majority of cases was seen (parashar et al., ) . however, in the indian sub-continent, especially bangladesh, where nipah virus infection was first recognized in , the major risk factor for contracting nipah virus is drinking raw palm sap (luby et al., ) . there is photographic evidence of fruit bats licking sap from the cuts in the trees from which the sap is collected, and contamination by faeces is seen in or on the pots (khan et al., ; rahman et al., ) . human infection may be asymptomatic, but there can be acute respiratory disease with fatal encephalitis, leading to mortality rates of %- %. the incubation period is believed to be - days or longer (see "relevant websites section"). the malaysian outbreak saw more cases of encephalitis than respiratory disease and there was little evidence for human to human transmission. in the indian sub-continent, especially bangladesh, the symptoms of infection manifested more with respiratory disease with fewer cases of encephalitis (luby et al., ) . the aerosolisation of respiratory secretions (droplets) and close contact with the sufferer allows respiratory transmission to lead to human to human transmission chains (gurley et al., ; yadav et al., ) . nipah virus will be maintained in its bat reservoir and so it is unlikely to be eradicated. slaughter and burial of pigs in the affected regions controlled the malaysian and singapore outbreak. routine cleaning and disinfection on pig pens with detergents may reduce exposure of pigs but if an outbreak is suspected, quarantine and culling can reduce the spread of disease (wang et al., ) . in bangladesh, the use of bamboo skirts to restrict access of bats to the sites on trees where sap is collected reduces the risk of contamination (khan et al., ) . heat treatment of sap also inactivates the virus and this, along with stopping the feeding of raw palm sap to humans and domestic animals reduces the risk of infection and transmission (luby et al., ) . continual surveillance for re-occurrences must be in place to monitor possible incursions into humans. there is no vaccine and no specific treatment at present (see "relevant websites section"). influenza viruses are members of the orthomyxoviridae family, and the h n genotype is in the alphainfluenzavirus genus and is of the influenza a species (ictv, ). influenza viruses are negative stranded rna viruses that have a segmented genome (shaw and palese, ) . influenza a nomenclature uses the genomic segments that encode the envelope glycoproteins, haemagglutinin (h) and neuraminidase (n) because these proteins are major contributors to the pathogenicity of the viruses. the wildlife reservoir for these viruses is wild birds, especially waterfowl, in which a subclinical gastroenteric infection is usually seen. thus, these viruses are often called avian influenza. there are several high pathogenicity avian influenza a (hpai) virus genotypes but one of the most pathogenic for humans and other animals are those in the h n genotype (wright et al., ) . with widespread sequencing, the ability to group viruses as 'clades', i.e., those viruses that are derived from a common ancestor, has now allowed a numerical naming system to be instituted. the eurasian-african h n viruses that are circulating and causing human and animal disease can be split into several (≥ ) clades (see "relevant websites section"). within all the different clades, the original h genotype has remained, despite re-assortment of the other gene segments. the virions are spherical ( - nm diameter) or filamentous (up to µm long) (noda, ) . the virus is present in the faeces of wild waterfowl, water contaminated by their faeces, domestic poultry secretions (respiratory and faeces) and aerosolised respiratory secretions from infected animals. the virion is usually very labile (markey et al., b) but it has been shown that avian influenza virus is infectious for months in low temperature water and for over a week in water at °c (hinshaw et al., ; markwell and shortridge, ) with increased survival when water has neutral to basic ph ( . - . ) and low ammonia concentrations (keeler et al., ) . it is also stable in frozen lakes (shoham et al., ) , allowing maintenance in the environment and infection of waterfowl from year to year. the seasonality of epidemics in humans is affected by temperature and humidity with the virus surviving better at °c in low humidity conditions but at °c requiring higher humidity. thus, the cool, dry conditions of winter favour transmission in temperate zones and humid, rainy conditions favour transmission in tropical and sub-tropical zones. the presence of salts and proteins in respiratory droplets allows virus survival in aerosolised droplets for up to - hr (sooryanarain and elankumaran, ) . contamination of fomites may also occur allowing transfer by hands. transmission is via inhalation of small aerosol droplets, faecal contamination from poultry and water borne infection. in asia, contamination of the environment by the faeces of waterfowl leads to infection of domestic poultry including chickens, ducks and geese which are often kept in the same environment. this usually causes severe disease and high titres of virus to be secreted from the domestic birds promoting infection of those working with or in contact with them, or in the food chain. farming of poultry and pigs together increases the risk of transmission to pigs and they can amplify and increase the risk of human infection. influenza virus a causes seasonal outbreaks with occasional severe epidemics/pandemics in humans and animals. the asian-african h n lineage is thought to have originated from commercial geese in the guangdong province of china in . this has since been spread across the globe by wild birds and infected people. there is evidence that h n has infected humans directly by drinking duck blood or eating duck meat, however, aerosolisation and inhalation is the more common route (shao et al., ; tumpey et al., ) . this virus has evidence of direct bird to human transmission without the need for adaptation through a secondary species such as pigs (wright et al., ) . in humans, the symptoms it causes are an acute respiratory tract infection with fever and sore throat, cough and malaise. if the virus becomes systemic there may also be vomiting and abdominal pain. h n viruses are highly pathogenic and there is a high case mortality rate with these infections (wright et al., ) . reducing the exposure of domestic poultry to wild waterfowl i.e., increased biosecurity reduces the risk of infection of these poultry and so exposure of workers to the virus. quarantine and culling of premises as well as closing live bird markets also reduces the risk of spreading the infection (see "relevant websites section"). in humans, there is a killed vaccine against annual strains of influenza a and b, and a live attenuated influenza a and b nasal spray vaccine. however, none is specifically against the h n strains. there are several antiviral drugs (amantadine, rimantadine, zanamivir, oseltamivir) available to treat those infected with influenza a, but there is the risk of resistance occurring, with some of the circulating h n strains showing resistance to amantadine and rimantadine and oseltamivir resistant strains have been isolated from patients treated with the drug (wright et al., ) . infections by severe acute respiratory syndrome (sars) virus, nipah virus (niv), h n virus, hepatitis a virus (hav), hepatitis e virus (hev), adenovirus, astrovirus, norovirus (nov) and rotavirus (rva) in humans and animals are detected by nucleic acid amplification tests and serologic tests. for example, a standardised method based on quantitative real-time pcr (rt-qpcr) for the detection of nov and hep a in food has been developed by the european committee for standardisation (cen) working group (tc /wg /tag -detection of viruses in food) (lees and cen wg tag , ) and provides a tool to quantify nov concentration in shellfish. it has been used to demonstrate that the risk of gastrointestinal illness associated with consumption of oysters increases with increasing concentrations of nov genome copies present (lowther et al., ) . costantini et al. ( ) demonstrated that a commercially available nov enzyme immunoassay showed excellent specificity but low sensitivity both for outbreaks as well as for samples from sporadic cases. the assay detected of the genotypes evaluated and that at least virus particles g − of faecal sample were required for a positive signal. this assay may be useful for rapid screening of faecal samples collected during an outbreak of acute gastroenteritis. to detect and quantify hev virus present in environmental and food samples, a rt-qpcr method was developed by jothikumar et al. ( ) . this taqman assay was designed to target a conserved region in orf , allowing the detection of four different genotypes of hev. a number of commercially elisa (enzyme-linked immunosorbent assay) kits for the hev detection of igm and igg antibodies which can be used early diagnosis of patients suspected for infection with hev, for the screening of blood units and the follow-up of hev-infected patients are also available. a taqman ). this method, when applied to environmental samples, was able to detect rotavirus at a level .  and .  particles/ml in tap water and environmental water, respectively. a competitive rt-pcr sybr green assay was designed based on conserved regions of the vp gene of group a rotaviruses, producing a base pair fragment (schwarz et al., ) . an in vitro synthesised rna with a -base deletion with respect to the wild-type sequence of this fragment was used as an internal control. using these transcripts as templates, rna molecules were amplified and reproducibly detected. using this protocol, the assay could be used to investigate the presence of rotavirus in environmental, food and water samples. immunochromatography tests are also available for detecting rotaviruses in stool samples. a study undertaken by de grazia et al. ( ) compared the performance of two commercially available one-step chromatographic immunoassays that detect both rotavirus and adenovirus. both tests were able to detect the wide range of rva genotypes circulating over the study period (including g p[ ] , g p[ ] , g , g , g and g p[ ] ). the results of the present study showed a satisfactory efficacy of the two diagnostic tests analyzed using real-time pcr as a reference test. the case fatality rate for niv is estimated to be %- %. in addition, there is no treatment or vaccine available for either people or animals. the main tests used are real time rt-pcr from bodily fluids and antibody detection via enzyme-linked immunosorbent assay elisa (see "relevant websites section"). for example, a taqman assay as described by guillaume et al. ( ) for niv detected a wide range of virus concentrations from .  l pfu to . pfu per reaction, corresponding to a threshold of pfu/ml for rapid, accurate and quantitative diagnosis. the specificity of the niv taqman assay was determined by the absence of amplification using measles and hendra paramyxovirus rna. an antigen capture elisa was developed by chiang et al. ( ) for the viral detection of henipavirus and for the differentiation between niv and hendra virus. this assay allows for the rapid detection and differentiation between the henipaviruses and could be used in any future outbreaks of henipaviruses. astroviruses are classified into two genera: mammalian viruses (mamastroviruses, mastvs) and avian viruses (avastroviruses, aastvs). human astroviruses are found in four mastv species (mastv , , , ) (pérot et al., ) . an rt-pcr assay was designed by finkbeiner et al. ( ) , based on the astrovirus rna polymerase (orf b) that allows for the detection of the eight human astroviruses serotypes found in mastv , a common cause of viral gastroenteritis in children. this primer set also detects viruses in mastv . these two groups also contain astroviruses from cats, pigs, dogs and rabbits (pérot et al., ) . to date, there is no universal pan-astrovirus rt-pcr assay. immunochromatography (ic) tests are also available for detecting astroviruses. an ic test for the detection of astrovirus was evaluated in stool samples of pediatric patients with acute gastroenteritis in japan, during january to march , and it is a rapid method for the detection of astroviruses and may be useful for screening astroviruses during outbreaks of food-borne and person-to-person transmission (khamrin et al., ) . a broad-spectrum pcr assay was developed by sibley et al. ( ) for the detection of mastadenovirus (maadv) and atadenovirus (atadv), based on the adenovirus hexon gene. maadv, which comprises human and bovine adenoviruses and a large variety of mammalian adenoviruses. atadv includes bovine adenovirus (badv) as well as adenovirus infecting ducks, goats, sheep, deer, and reptiles (sibley et al., ) . this assay has been shown to demonstrate natural badv excretion in urine, badv detection in groundwater, and recombination in adv of livestock origin (sibley et al., ) and has the potential to be used in screening samples during outbreaks of food-borne disease. for the detection of h n influenza viruses, the world health organisation (who) (see "relevant websites section") provides updated information on the molecular detection/diagnostic protocols for the surveillance of influenza viruses in humans. for example, the who provides primers and probes sequences for real-time rt-pcr procedures for the detection of: ( ) influenza type a viruses (matrix gene). a taqman based assay was designed for the detection of sars and middle east respiratory syndrome (mers) coronaviruses (covs) by noh et al. ( ) , based on the conserved spike s region of human sars-cov, mers-cov, and their related bat covs. this assay can detect sars-cov and mers-cov in humans but also several bat covs that are closely related to these viruses in bats. a monoclonal antibody-based capture enzyme immunoassay for the detection of nucleocapsid antigen in sera from patients with sars was developed by che et al. ( ) . this assay used a mixture of three monoclonal antibodies for capture and rabbit polyclonal antibodies for detection of serum antigen. the sensitivity of the assay was . % in serologically confirmed sars patients with blood taken during the first days after the onset of symptoms ( of ). the specificity of the assay was . % in healthy individuals ( of ). there was no cross-reaction with other human and animal coronaviruses in this assay. • food can become contaminated by viruses at source and through contaminated food handlers and environments. • good food hygiene and personal hygiene, especially hand washing, are essential to help minimise the spread of these viruses within the food chain. • since foodborne viruses tend to be more resistant to physical and chemical treatments than bacteria, their control represents a challenge for the food industry. • ongoing research is required, for example, current laboratory detection methods can be modified to allow differentiation between e.g., infectious and non-infectious viruses. • there are a number of knowledge gaps in terms of how, for example, nov, hav and hev are transmitted through the food chain, the contribution they make to overall foodborne illness and their survival and elimination from food. • emerging trends indicate that viruses play an important role in foodborne illness. this has implications for the whole of the food chain. potential role of fomites in the vesicular transmission of human astroviruses astrovirus survival in drinking water rotavirus in adults requiring hospitalization virus particles in epithelial cells of duodenal mucosa from children with acute non-bacterial gastroenteritis human astroviruses acute respiratory disease associated with adenovirus serotype -four states alarmins: awaiting a clinical response sensitive and specific monoclonal antibody-based capture enzyme immunoassay for detection of nucleocapsid antigen in sera from patients with severe acute respiratory syndrome vaccine 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hepatitis e virus: infection beyond the liver date palm sap linked to nipah virus outbreak in bangladesh virus hazards from food, water and other contaminated environments foodborne illness acquired in the united states-major pathogens detection and quantitation of group a rotaviruses by competitive and real-time reverse transcriptionpolymerase chain reaction a brief review of foodborne zoonoses in china orthomyxoviridae persistence of avian influenza viruses in various artificially frozen environmental water types detection of known and novel adenoviruses in cattle wastes via broad-spectrum primers environmental role in influenza virus outbreaks hepatitis e: a disease of reemerging importance hepatitis e virus genotypes and evolution: emergence of camel hepatitis e variants estimate of worldwide rotavirus-associated mortality in children younger than years before the introduction of universal rotavirus vaccination programmes: a systematic review and meta-analysis world health organization environmental team characterization of a highly pathogenic h n avian influenza a virus isolated from duck meat the unpredictable diversity of co-circulating rotavirus types in europe and the possible impact of universal mass vaccination programmes on rotavirus genotype incidence rotavirus infection in infants as protection against subsequent infections novel human astroviruses: novel human diseases? epidemiology of classic and novel human astrovirus: gastroenteritis and beyond henipaviruses sars-cov infection in a restaurant from palm civet adenovirus gastroenteritis global use of rotavirus vaccines recommended. who orthomyxoviruses detection of nipah virus rna in fruit bat (pteropus giganteus) from india hepatitis e virus: foodborne, waterborne and zoonotic transmission rotavirus incidence and genotype distribution before and after national rotavirus vaccine introduction in belgium development and evaluation of a loop-mediated isothermal amplification assay for the detection of adenovirus and prevention and control: oie -world organisation for animal health who -first data on stability and resistance of sars coronavirus compiled by members of who laboratory network who information for the molecular detection of influenza viruses who -updated unified nomenclature system for the highly pathogenic h n avian influenza viruses key: cord- -mejd blb authors: lewnard, joseph a; reingold, arthur l title: emerging challenges and opportunities in infectious disease epidemiology date: - - journal: am j epidemiol doi: . /aje/kwy sha: doc_id: cord_uid: mejd blb much of the intellectual tradition of modern epidemiology stems from efforts to understand and combat chronic diseases persisting through the th century epidemiologic transition of countries such as the united states and united kingdom. after decades of relative obscurity, infectious disease epidemiology has undergone an intellectual rebirth in recent years amid increasing recognition of the threat posed by both new and familiar pathogens. here, we review the emerging coalescence of infectious disease epidemiology around a core set of study designs and statistical methods bearing little resemblance to the chronic disease epidemiology toolkit. we offer our outlook on challenges and opportunities facing the field, including the integration of novel molecular and digital information sources into disease surveillance, the assimilation of such data into models of pathogen spread, and the increasing contribution of models to public health practice. we next consider emerging paradigms in causal inference for infectious diseases, ranging from approaches to evaluating vaccines and antimicrobial therapies to the task of ascribing clinical syndromes to etiologic microorganisms, an age-old problem transformed by our increasing ability to characterize human-associated microbiota. these areas represent an increasingly important component of epidemiology training programs for future generations of researchers and practitioners. initially submitted september , ; accepted for publication november , . much of the intellectual tradition of modern epidemiology stems from efforts to understand and combat chronic diseases persisting through the th century epidemiologic transition of countries such as the united states and united kingdom. after decades of relative obscurity, infectious disease epidemiology has undergone an intellectual rebirth in recent years amid increasing recognition of the threat posed by both new and familiar pathogens. here, we review the emerging coalescence of infectious disease epidemiology around a core set of study designs and statistical methods bearing little resemblance to the chronic disease epidemiology toolkit. we offer our outlook on challenges and opportunities facing the field, including the integration of novel molecular and digital information sources into disease surveillance, the assimilation of such data into models of pathogen spread, and the increasing contribution of models to public health practice. we next consider emerging paradigms in causal inference for infectious diseases, ranging from approaches to evaluating vaccines and antimicrobial therapies to the task of ascribing clinical syndromes to etiologic microorganisms, an age-old problem transformed by our increasing ability to characterize human-associated microbiota. these areas represent an increasingly important component of epidemiology training programs for future generations of researchers and practitioners. infectious diseases; methods; modeling; surveillance abbreviation: ebov, ebola virus. the priority afforded to infectious diseases within epidemiologic research has been fluid over the past years or longer. despite the lasting prominence of early investigations into measles, cholera, plague, typhoid fever, malaria, and yellow fever ( ) ( ) ( ) ( ) ( ) ( ) , the intellectual tradition of modern epidemiology stems largely from studies of chronic diseases dating to the post-world war ii era, when such conditions came to surpass infectious diseases in morbidity and mortality in highincome countries amid improvements in living conditions and the introduction of numerous antibiotics and vaccines. this epidemiologic transition co-occurred with a shift in focus for epidemiologic research (and training programs) toward the multifactorial etiology of chronic conditions ( ) . in parallel, early th-century work on the "dependent happenings" of communicable diseases ( ) ( ) ( ) ( ) yielded to the development of today's core biostatistical methods for chronic diseases, premised on the independence of outcomes among subjects ( ) ( ) ( ) . since the late th century, the emergence of human immunodeficiency virus and acquired immunodeficiency syndrome and other infections has renewed interest in infectious diseases and their health, economic, and security implications ( ) . outbreaks of severe acute respiratory syndrome, pandemic influenza a h n , ebola virus (ebov), and zika virus have prompted international responses, whereas influenza a h n and h n , lassa fever virus, nipah virus, and middle east respiratory syndrome coronavirus, among other agents, have been a source of regional concern. the incidence and endemic range of "neglected" infections, including dengue and cholera ( , ) , have expanded, and antimicrobial resistance has threatened to derail the control of tuberculosis, typhoid, malaria, gonorrhea, yaws, and invasive bacterial infections ( ) ( ) ( ) ( ) ( ) ( ) . although highly effective vaccines are available, measles and yellow fever have resurged on multiple continents, due to gaps in vaccine coverage ( , ) , while short-lived vaccine-induced protection has facilitated unexpected resurgences in diseases once on the path to elimination, such as pertussis and mumps ( , ) . after decades of relative obscurity in the mid- th century, infectious disease epidemiology has experienced an intellectual rebirth in response to disease emergence. repopulation of this field by scientists trained not only in clinical medicine but ecology, demography, and quantitative sciences has led to the adoption of methods scarcely addressed in traditional public health training programs. cluster-randomized trial designs, for example, have become commonplace for evaluating infectious disease interventions, quantifying indirect effects resulting from contagion ( ) . classical models of ecological dynamics have been adapted to address the transmission and control of infectious agents, while our expanding ability to integrate such models with epidemiologic data through bayesian statistics has enhanced their relevance to policymaking ( ) ( ) ( ) . most recently, sequencing and phylogenetic analysis have afforded an unprecedented view into the population structure and dynamics of pathogens ( , ) . here, we review developments in infectious disease epidemiology together with their implications for research and practice, and for the training of future epidemiologists. we first consider the role of epidemiologic surveillance in the context of pathogen emergence and the integration of surveillance data into quantitative studies of transmission. next, we discuss challenges in causal inference, including the evaluation of public health interventions against emerging pathogens and the difficulties of attributing clinical syndromes to microbial agents. surveillance justifiably has been seen as a core public health function, with its important role articulated by langmuir in ( ) . in the united states and elsewhere, surveillance for diverse infectious (and noninfectious) diseases has typically relied on an essentially passive system of reporting by healthcare providers or laboratories, often mandated by public health laws. although such passive systems of disease reporting have proven invaluable, their limitations, such as incomplete, often inconsistent detection of cases and delayed detection of outbreaks, are well documented ( ) . as a result, active surveillance systems that do not depend on providers and laboratories to report have been developed and promoted. the centers for disease control and prevention-funded emerging infections program and its components (e.g., foodnet, abcs), initiated in , is notable example of such a system, as are international versions promoted through the centers for disease control and prevention's global health security initiative ( ) . such systems, while expensive to develop and maintain, are of great value, especially when they collect biological specimens (e.g., isolates of bacterial and viral pathogens) for typing and support analytic epidemiologic studies (e.g., casecontrol studies of vaccine effectiveness ( ) ). nevertheless, these systems, too, may have limitations, particularly in their ability to detect in a timely fashion outbreaks caused by novel microbial agents, prompting interest in alternative methods. in this article, we consider proposed alternatives, highlighting their benefits and challenges. beyond efforts to quantify the incidence of infectious diseases of known etiology, disease surveillance has been advocated as a means of mitigating the threat posed by novel pathogens. large-scale efforts to identify pathogens with the potential to spill over from animals to humans have received notable investment, for instance from the us agency for international development emerging pandemic threats program ( ) . more recently, metagenomic sequencing has enabled the number of known viruses to be multiplied in such studies ( ) . the aim of those working on the global virome project, launched in , to characterize within years all . million viruses thought to exist ( ) . however, the pathway for translating data sets produced by such activities into actionable threat-reduction programs remains unclear. that only approximately viruses are known to infect humans (and an even smaller handful to cause major epidemics) may constrain the value of large-scale virus discovery for identifying high-risk pathogens, as well as viral determinants of pathogenic potential ( ) . spillover events causing recent epidemics-including h n in mexico, middle east respiratory syndrome in saudi arabia, and ebov in west africa-have been poorly predicted by factors long believed to drive disease emergence ( ) . accordingly, interest in expanding our catalogue of potential pathogens should be weighed against our persisting need to enhance the detection and control of outbreaks of known pathogens ( ) . for instance, ebov epidemics in the democratic republic of the congo in took weeks to be identified, with dozens of suspected cases having already accumulated ( , ) . serological studies have received increasing enthusiasm for monitoring emerging pathogens of significance to humans ( , ) . the prevalence of antibodies indicating previous exposure may provide valuable information about the frequency of animal-human spillover events and the potential for person-toperson spread, overcoming reporting biases that favor detection of large outbreaks under traditional surveillance. moreover, the low cost of multiplex assays makes integrated surveillance of multiple pathogens plausible. although serosurveys have bolstered recent efforts to understand the geographic range and clinical spectrum of ebov and zika virus infections ( , ) , the enhancement of dengue hemorrhagic fever risk by prior exposure ( ) , and the role of immunologic history in influenza susceptibility and vaccine response ( ) , there remain few examples of public health programs undertaking serological studies for routine surveillance, at least in civilian populations ( ) . outside of laboratory-based surveillance, the increasing availability of passively collected "big data" on the health and behaviors of individuals has prompted enthusiasm about enhancing disease surveillance through alternative data streams. initiatives such as the promed-mail network and healthmap ( , ) compile and disseminate news about outbreaks from media and other sources, aiming to trigger investigation by public health organizations. data such as emergency department visits, medication sales, online search queries, and social media postings have also been suggested as real-time indicators of outbreak activity, although their integration into public health responses remains a subject of debate ( ) ( ) ( ) . the need to overcome reporting biases is a central challenge, because observations may be too nonspecific to distinguish between meaningful and spurious signals in settings with high technological capacity while also being insensitive to even high-risk events in resource-poor settings ( , ) . although nontraditional data sources have, in some applications, supported inferences about epidemic dynamics ( ) , limited information about cases from such sources remains a barrier. for instance, models fitted from news reports of recent measles and mumps outbreaks have yielded considerable underestimates of vaccine coverage ( ) ( ) ( ) , underscoring the importance of field investigations. forecasting the incidence of diseases has been a more successful application of these emerging data streams and dataanalytic approaches. although the acknowledged failure of google flu trends-a prediction approach based on internet search behavior-yielded important lessons about nonmechanistic forecasts, approaches based on machine learning and crowdsourced human judgment have provided the most accurate within-season predictions of us influenza activity in recent comparisons ( ) ( ) ( ) . given expanding interest in forecasting among researchers, funding agencies, and other stakeholders, there is a clear and compelling need to evaluate whether such forecasts can enhance the success and efficiency of public health response efforts. mathematical modeling as a means to understanding infectious disease spread dates to studies by sir ronald ross ( ) . although the use of models to connect data such as age of infection to transmission dynamics of endemic infections has longstanding precedent ( , ) , assimilation of outbreak data for near-term assessments of control priorities is a comparatively recent phenomenon. integration of modeling with the public health response to epidemics of bovine spongiform encephalopathy and foot-and-mouth disease in the united kingdom and the severe acute respiratory syndrome epidemic ( - ) has led to expectations for near real-time modeling studies during major outbreaks. in recent experience, models of the spread of pandemic influenza a h n ( ), cholera ( ), middle east respiratory syndrome ( ), ebov ( , ) , chikungunya virus ( ), zika virus ( , ) , yellow fever ( ) , and plague ( ) have all been published within weeks of the respective outbreak notifications. although the circumstances of particular epidemics dictate what data may be available and pertinent, methods for fitting models to data have generally focused on exponential growth rates in cases ( ) or the distribution of the serial interval ( ) . methods based on the latter class of data offer the advantage of illustrating real-time changes in reproductive numbers ( ) ; however, the requisite information from patient line lists is seldom available. reliance instead on ecological data exposes models to numerous vulnerabilities, notably the inability to discern individual risk factors (and thus the population meaningfully at risk). these shortcomings may prevent models from predicting reductions in transmission before depletion of the susceptible population. a challenge thus lies ahead in determining the role of models in outbreak response and the best practices for communicating modeling results. although the ability of models to evaluate prophylactic strategies may be considered a benefit, recommendations to act against remote future risks have sometimes triggered resistance among stakeholders ( ) . during the west african ebov epidemic, for example, attention to worst-case model-based projections prompted some to question the reliability of the models ( ), reflecting an important discrepancy between public understanding of modeling as a forecasting tool and the intended uses of models for scenario-based comparisons ( , ) . this use of modeling has been better understood in attempts to communicate the impact of interventions after the fact ( ). the ease of sequencing pathogen genomes has afforded a new view into transmission during outbreaks. use of sequence data to identify transmission clusters in the presence of unsure epidemiologic links dates to the early years of the human immunodeficiency virus and acquired immunodeficiency syndrome epidemic ( ) . in recent years, sequencing has aided efforts to track the sources of unexplained epidemics of cholera in haiti ( ) and ebov in west africa ( ) , and has shown increasing utility for reconstructing the geographic spread of pathogens ( , ) . a particular advantage of phylogenetic analysis is the possibility of estimating unobserved epidemiologic quantities, such as the reporting fraction ( ) and reproductive numbers for subcritical transmission ( ) , which remain difficult to assess from traditional case-notification data. beyond reconstructing the demographic history of pathogen lineages, recent years have seen progress toward joint analysis of epidemiologic and sequencing data ( ) . such "phylodynamic" approaches have shown particular relevance for emerging infections, including distinguishing the role of repeated introductions and subsequent local transmission ( ) ( ) ( ) ( ) . whereas most applications have been tailored to specific data sets and assumptions, the development of generalized methods for joint inference of epidemiologic and phylogenetic parameters remains a priority ( ) to support real-time analysis. the ability to rapidly develop and deploy countermeasures to mitigate the threat posed by emerging infections has received increasing recognition as a component of public health preparedness. however, outbreaks are difficult environments in which to evaluate interventions. during the west african ebov epidemic, the feasibility of a new paradigm for development and evaluation of interventions in emergencies was demonstrated by accelerated vaccine safety, immunogenicity, and efficacy studies ( ) . the coalition for epidemic preparedness innovations was established in , with an initial focus on vaccines against nipah virus, middle east respiratory syndrome coronavirus, and lassa fever virus, in addition to adaptable vaccine platforms for novel threats ( ) . lessons learned in ebov vaccine trials will have an influential bearing on evaluations during future emergencies. despite efforts to accelerate evaluation of candidate vaccines, incidence had reached low levels by the time phase iii efficacy trials were ready to begin, posing a threat to their statistical power: a planned trial in liberia was canceled due to declining transmission ( ) , and no cases of disease occurred in a second trial in sierra leone ( ), preventing efficacy assessments. in a stepped-wedge trial in guinea, clusters of primary and secondary contacts of ebov disease cases were randomly assigned to immediate or delayed vaccination; no cases were reported among vaccine recipients during the trial ( ) or in subsequent field deployments of the vaccine, supporting a conclusion of near % vaccine efficacy. debates surrounding design of these trials highlight methodological questions requiring additional attention. in the guinean trial, a "ring" vaccination scheme helped maximize power by enrolling contacts of known cases ( ) . however, the choice of individual-or cluster-level randomization within rings was debated. because members of a vaccinated cluster are exposed to direct protection through vaccination and indirect protection due to reduced transmission within their clusters ( ), cluster-randomized trials have weaker statistical power than individually randomized trials of the same size ( ) . moreover, the direct effect measured in individually randomized studies may be a preferred, transportable efficacy measure ( ) . uses of simulation helped in planning vaccine trials tailored to the real-world circumstances of the ebov outbreak ( ) and enabled trialists to compare alternative designs in terms of ethical mandates ( ) . simulation-guided design further presents the opportunity for applying adaptive trial methods ( ) in the context of infectious disease outbreaks, where dynamic trends in incidence may highlight the benefits of such approaches. in addition to efficacy trials for new interventions, observational studies are needed to assess licensed interventions against evolving and re-emerging pathogens. most commonly applied in evaluations of influenza vaccines, test-negative designs have become popular in routine ( ) and exploratory ( ) studies of vaccine effectiveness. by measuring vaccine effectiveness from the exposure odds ratio of vaccination among individuals seeking care who test positive or negative for a pathogen of interest, this design seeks to overcome associations of health-care seeking with vaccination status ( ) . however, it is uncertain whether health-care seeking and other sources of confounding are appropriately controlled for, and whether measures accurately capture vaccine direct effects ( , ) . uncertainty about the validity of estimates that routinely inform vaccine policy-making demonstrates the need for formal evaluations of such studies and strategies to reduce bias. time series analyses of public health surveillance data provide another approach to measuring the real-world impact of vaccination on disease incidence, with the advantage of identifying the overall effect of a vaccination program resulting from direct and indirect protection ( ) . although the ecological nature of such designs permits the introduction of biases from changes in diagnostic practices or health-care seeking, such studies nonetheless have offered important insights where other approaches failed. limited reductions in influenza-related deaths among elderly persons amid increases in influenza vaccine coverage during the s provided an important indication that the "healthy vaccinee" effect accounted for astonishing and implausible protection against all-cause mortality among elderly influenza vaccine recipients in cohort and case-control studies ( ) ( ) ( ) . newer methods continue to improve public health inferences obtained from time-series data. in a recent evaluation of invasive pneumococcal disease incidence, trends expected under continued use of -valent pneumococcal conjugate vaccine provided a counterfactual condition for measuring the impact of the switch to a -valent vaccine targeting emerging serotypes ( ) . bayesian averaging of models encoding differing pre-and postvaccination trends and change points provides a generalized strategy for defining such counterfactual comparisons ( ) . other signals of transmission intensity in surveillance data, such as age of infection and sub-or multiannual periodicity, may provide additional insights while reducing sensitivity to fluctuations in reporting effort ( , ) . the re-emergence of pathogens against which vaccines are widely deployed, such as varicella, pertussis, and mumps in the united states, poses additional challenges for conducting vaccine effectiveness studies. situational factors may undermine researchers' ability to establish the extent to which cases owe to primary or secondary vaccine failure in all or certain vaccine recipients, and whether emerging pathogen lineages are escaping vaccine-driven immune pressure. for instance, high compliance with vaccine schedules may limit variation in individuals' vaccination status and exposures, necessitating large samples to detect factors influencing vaccine performance ( ) . because re-emergence most likely reflects the expansion of or several pathogen clades, limited pathogen diversity may hinder the application of conventional approaches to identifying microbial determinants of vaccine escape ( , ) . novel methods to distinguish null from vaccine-driven mutations in antigencoding regions ( , ) may streamline efforts to identify vaccine escape, while mathematical modeling provides a basis for comparing candidate hypotheses with observations ( , ) . because vaccine studies are typically powered for primary clinical endpoints, long-term observational studies are needed to monitor for rare vaccine-attributable adverse events. such studies have been crucial to identifying safety concerns such as intussusception after rotavirus vaccination ( ) and to refuting spurious links, such as autism onset after measles-mumpsrubella vaccination ( ) . however, unique challenges arise in vaccine safety studies; at the individual level, vaccination and adverse-event detection may be confounded due to health-careseeking behavior, whereas at the population level, age-related confounding may occur when vaccine recommendations are based on the individual's age. ecological designs taking advantage of natural experiments have proven useful in numerous studies of vaccine safety ( , ) but inconclusive for certain classes of rare events, including those that also result from the vaccine-targeted infection ( , ) . recent years have seen growing interest in case-only methods offering the ability to reduce or rule out individual-level sources of confounding. case-crossover methods are common among such approaches ( , ) and resemble matched case-control studies by sampling "control" periods from the person-time contributed by case individuals before an adverse event. self-controlled case-series methods similarly benefit from the use of cases as their own controls, following a cohort logic in estimating the relative incidence of adverse events after vaccination within specified risk periods ( ) ; with adequate sample size, researchers may be able to use such analyses to eliminate or greatly reduce potential time-or age-related confounding. in response to the growing threat posed by antimicrobial resistance, the world health organization and national governments have prioritized bringing novel antimicrobial drugs to market ( ) . these plans will necessitate phase iii trials in which the efficacy of new therapeutic agents is addressed and possibly phase iv studies, in which the optimal use of new and existing drugs, either singly or in combination, can be determined. whereas patients traditionally have been enrolled in antimicrobial treatment studies on the basis of target bacterial species infections or clinical syndromes, it is uncertain within what strata such trials may yield transportable inferences. rather than merely the infecting pathogen's baseline resistance or susceptibility phenotype, strata may be defined by factors such as pathogen lineage, mutational barriers to resistance development ( ) , and presence of horizontally transferable resistance elements in cocolonizing agents or environmental sources ( ) . stratification based on interpatient and even intrapatient tumor heterogeneity is an emerging feature of cancer therapy trials and may provide a template for such designs ( ) . in addition to clinical endpoints, carriage of susceptible and resistant bacteria, including commensal agents not purposefully targeted by treatment, can inform the impact of treatment on resistance selection in targeted and bystander species ( ) . whereas between-group differences in the absolute prevalence of colonization with resistant organisms ( ) are tested for routinely, stratified measurements (see the reports of shrag et al. ( ) and feikin et al. ( ) , for example) of the effect of treatment on acquisition and clearance of susceptible and resistant pathogens are more informative of the underlying biology ( ) and may detect signals of selection masked by simpler betweengroup comparisons ( , ) . studies are also needed to address optimal deployment of new and existing antimicrobial drugs in clinical practice. the tradeoff between maximizing a drug's impact and minimizing resistance selection has led policymakers to ration certain new drugs as last-resort treatments. however, in recent experience, such decisions have ignited ethical debates ( ) . coupling mathematical modeling with field-based studies has proven useful for understanding the effectiveness of antimicrobial use policies, as highlighted in recent evaluations of the risk of resistance under population-wide access of the tuberculosis drug bedaquiline ( , ) and antimicrobial cycling to limit resistance selection in hospital settings ( , ) . whereas certain efforts we have discussed have been made to identify novel microorganisms able to cause human infection ( , ) , most epidemics caused by emerging pathogens have been recognized first by clusters of anomalous syndromes-such as cardiopulmonary syndrome caused by new world hantaviruses, severe acute respiratory syndrome caused by a coronavirus, and congenital abnormalities caused by zika virus -before the role or even existence of the etiologic microorganism had been characterized. the problem of ascribing a clinical syndrome to an etiologic agent is among the oldest in epidemiology, dating at least to the th century, when robert koch laid out criteria for such inference (i.e., koch's, or more correctly, henle-koch's, postulates ( ) ). however, these postulates have long been recognized as inadequate, particularly for illnesses caused by viruses, and thus of largely historical interest ( ) ; for instance, the notion that a pathogen should be absent from healthy individuals is incompatible with the prominence of carriage and asymptomatic infection in the natural history of numerous pathogens. a growing appreciation of the complexity of the human microbiome, and the likelihood that intricate mixtures of microorganisms at diverse body sites may be either the cause or consequence of both detrimental and beneficial physiological states, has further highlighted the difficulty of linking a given health outcome to infection by a single microorganism. the now-recognized critically important role of persistent infection and the inflammation it can produce in diverse cancers and possibly other chronic diseases has further diminished the relevance of koch's postulates and the age-old distinction between infectious and chronic diseases, as illustrated in the well-known example of human papillomavirus causing cervical, anal, and oral cancers ( ) . the best causal understanding of such relationships has come from randomized trials demonstrating that infection-preventing interventions are efficacious against downstream chronic illness, such as peptic ulcers due to helicobacter pylori and chronic wheeze due to respiratory syncytial virus ( , ) . natural experiments following the same intuition have provided additional evidence of such relationships, such as measles-induced immunosuppression ( ) , malnutrition and stunting due to enteric infection ( ) , and complex or chronic otitis media due to tissue damage from acute early-life disease ( ) . such relationships have proven difficult to probe in the absence of a randomized or natural experiment, because of the likelihood that confounding factors influence individuals' risk for initial infection as well as chronic sequelae. new paradigms for ascribing an etiologic role to microorganisms and resulting host responses are clearly needed and may prove important in efforts to quantify the health impacts of infectious disease interventions. the recognition in the s and s that infectious diseases were not, in fact, disappearing 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years of age in resourcepoor environments prevention of early episodes of otitis media by pneumococcal vaccines might reduce progression to complex disease the authors received no specific funding for this article. conflict of interest: none declared. key: cord- - lq r authors: hsu, tien-huan; liu, hao-ping; chin, chieh-yu; wang, chinling; zhu, wan-zhen; wu, bing-lin; chang, yu-chung title: detection, sequence analysis, and antibody prevalence of porcine deltacoronavirus in taiwan date: - - journal: arch virol doi: . /s - - -x sha: doc_id: cord_uid: lq r porcine deltacoronavirus (pdcov) was initially documented in hong kong and later in the united states, south korea, and thailand. to investigate if pdcov is also present in taiwan, three swine coronaviruses—pdcov, porcine epidemic diarrhea virus (pedv), and transmissible gastroenteritis coronavirus (tgev)—were tested using real-time reverse transcription polymerase chain reaction (rrt-pcr) in rectal swab samples from piglets exhibiting diarrhea between january and may on pig farms in taiwan. the rrt-pcr results were positive for pdcov ( / , . %), pedv ( / , . %), tgev ( / , . %), and coinfections ( / , . %). after cloning and sequencing, pdcov nucleocapsid genes were analyzed. phylogeny results indicated that the nucleotide sequences of all isolates were like those reported in other countries. to further trace pdcov in the period of to , an enzyme-linked immunosorbent assay (elisa) was used to detect antibodies against pdcov. the results showed that of , ( . %) sera were positive for the pdcov nucleocapsid protein, implying that pdcov might have existed in taiwan before . porcine deltacoronavirus (pdcov) is an enveloped, positive-sense single-stranded rna virus that belongs to the family coronaviridae. pdcov was first discovered in hong kong, china in [ ] . it was subsequently reported in the united states [ ] [ ] [ ] and south korea [ ] in , followed by thailand and mainland china in [ , ] . currently, there are at least three members of the family coronaviridae that can cause diarrhea in pigs: transmissible gastroenteritis virus (tgev), porcine epidemic diarrhea virus (pedv), and porcine deltacoronavirus (pdcov) [ ] . both tgev and pedv belong to the genus alphacoronavirus, whereas pdcov belongs to the new genus deltacoronavirus. pedv is an important enteric pathogen that causes piglet diarrhea worldwide, and it has caused significant economic losses in the swine industry in taiwan from to [ ] . however, pdcov and pedv share a similar clinical manifestation, and many studies have shown that coinfection with pedv and pdcov is common in piglets [ ] [ ] [ ] . thus, the purpose of this study was to identify the viruses responsible for causing diarrhea in piglets and to specifically investigate the prevalence of pdcov infection in taiwan. in this study, rectal swabs of piglets that suffered from diarrhea from pig farms located in central and southern taiwan were collected between january and may . all rectal swabs were transported in phosphate-buffered saline with % glycerol. the total nucleic acid content was then extracted from the rectal swabs using a labprep™ dna/rna mini kit (taigen biotechnology, taiwan). the isolated nucleic acid samples were tested for the presence of swine enteric coronaviruses-tgev, pedv, and pdcovusing the idexx™ realpcr ® test kits. pdcov-positive samples were further examined by traditional reverse transcription polymerase chain reaction (rt-pcr) with primers pdcov-n ( '-acc atc gct cca agt cat tcttg- ') and pdcov-n ( '-gag tgg agt tgg gtg ggt tta reverse-transcription reaction was performed at °c for minutes, and then a standard polymerase chain reaction was performed: cycles of °c ( seconds), °c ( seconds), and °c ( minute). after electrophoresis and gel elution, amplified products were cloned and sequenced. the nucleotide sequence data were analyzed using chromas lite, and the deduced amino acid sequences of the open reading frames were compared to other pdcov sequences using blast. the sequences of three complete pdcov nucleocapsid (pdcov-n) genes, taiwan , taiwan , and taiwan , were obtained and deposited in genbank (accession numbers ky to ky ). multiple sequence alignment and phylogenetic tree construction were then performed using the mega program [ ] . a pdcov-n clone (taiwan ) was further subcloned into the protein expression vector pet- a(+) using primers pdcov-n-f ( '-acc gga tcc atg gct gca cca gta gtccc- ') and pdcov-n-r ( '-cac aag ctt cta cgc tgc tga ttc ctgct- '). the pdcov-n protein was then expressed by adding isopropyl-β-d-thiogalactoside (iptg) (final concentration . mm) to a bacterial culture and was purified using immobilized metal affinity chromatography for a subsequent enzyme-linked immunosorbent assay (elisa). a total of , serum samples collected from pig farms in taiwan during to were tested, and specific-pathogen-free (spf) pig sera were obtained from the animal technology institute taiwan to be used negative controls. the elisa was conducted following the modified procedures described in a previous study [ ] . in brief, -well microtiter plates were coated with purified his-tagged pdcov-n ( ng/well) in mm carbonate buffer (ph . ) at °c for - hours and blocked with blocking buffer ( % bovine serum albumin in tris-buffered saline (tbs): mm tris-cl, ph . , mm nacl) at room temperature for hour. after washing three times with tbst (tbs containing . % tween- ), μl of the tested sera ( : dilution in blocking buffer) were added to the wells and incubated at room temperature for hour. after incubation, the plate was washed three times with μl of tbst and incubated at room temperature for hour with μl of goat α-porcine ig antibody conjugated with horseradish peroxidase at a concentration of : dilution in blocking buffer. after washing, μl of , ', , '-tetramethylbenzidine (tmb) substrate solution was added, and the plate was incubated at room temperature for minutes. the development reaction was stopped by adding μl of m h so , and the absorbance at nm wavelength was measured. all tests included a blank coated with antigen only, a second antibody control, and six spf sera as negative controls. if the detection value was lower than (mean neg + × sd), the serum was considered negative. on the other hand, a serum detection value larger than [(mean neg + sd) × ] was considered positive. of these tested samples, were positive for pdcov ( . %), were positive for pedv ( . %), and only two were positive for tgev ( . %). regarding coinfection rates, only one of the specimens ( . %) was positive for all three coronaviruses, one was positive for pedv and tgev ( . %), and of them ( . %) were positive for pdcov and pedv. based on the real-time rt-pcr (rrt-pcr) detection results, the percentage of pig farms that were positive for at least of one of the coronaviruses was % for pdcov ( / ), . % for pedv ( / ), and . % for tgev ( / ). we compared the sensitivity of the idexx rrt-pcr kit and the traditional rt-pcr used for cloning the n protein of pdcov in this study. all pdcov-positive samples were re-examined by conventional rt-pcr, which showed that only eight ( / , . %) were positive, accounting for . % ( / ) of total tested samples. the relatively low positive rate determined by conventional rt-pcr was comparable to that determined by a similar method in a recent study, in which pdcov was detected exclusively in nursing piglets, with an overall prevalence of approximate . % ( / ) in southern china [ ] . it is plausible that for the specimens containing pdcov genomic rna of low quantity or quality, most of the commercial rrt-pcr kits, which are designed to amplify short target sequences, can achieve higher sensitivity in detection compared with conventional rt-pcr. in addition, four of the eight positive samples were cloned and sequenced, and the sequence data matched the pdcov-n reference sequences. all of these four sequences covered the complete coding sequences of pdcov-n, but one of them, taiwan , had a nonsense mutation within the pdcov-n gene (data not shown). such a mutation might arise from an occasional change in rna sequence occurring during serial propagation of pdcov [ ] . despite the mutation, the result revealed the presence of the pdcov genome in the specimen in which taiwan was cloned, while it remained to be clarified if mutations occurred in other pdcov-encoded genes as well. phylogeny analysis of pdcov-n genes showed that pdcovs found in taiwan were highly similar in their nucleotide sequences to isolates from the united states, mainland china, and other countries (fig. ) . however, the aminoacid-based phylogeny results of the pdcov-n proteins revealed that taiwan isolates can be clustered into different groups (fig. ) . this might be due to missense mutations in the th (f → y), th (e → k), th (g → r/t), th (f → s), and th (g → e/d) amino acid residues of the pdcov-n protein (fig. ) . the charges of amino acids were changed in three residues ( th , th , and th ), and the remaining residue changes had altered polarity. the phylogeny results imply that pdcovs isolated in taiwan might have existed for a long time. to determine if pdcov had already existed in taiwan before , the pdcov-n protein was cloned, expressed, purified, and coated onto a -well microtiter plates for retrospective testing. swine sera collected between and were tested for their reactivity to a pdcov-n protein. the elisa results showed that of , ( . %) sera were able to react with the pdcov-n protein when [detected value > (mean neg +sd) × ] was used as positive threshold, but only of , ( . %) sera were positive when [detected value > (mean neg +sd) × ] was set as the threshold. the data indicated that pdcov has existed in taiwan since . similar elisa tests were also used to investigate the presence of pdcov in other studies [ , , , ] . based on our findings, we confirm that infection of pdcov and its coinfection with pedv in pigs have existed in taiwan between and . it is known that antibodies against pedv, tgev, or pdcov provide no crossprotection against either of the other two coronaviruses [ , [ ] [ ] [ ] [ ] . coinfection with pedv and pdcov might explain why some efforts have been ineffective in the pedv vaccination program. therefore, a divalent vaccine to control pdcov and pedv is desperately needed. discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus rapid detection, complete genome sequencing, and phylogenetic analysis of porcine deltacoronavirus detection and genetic characterization of deltacoronavirus in pigs porcine coronavirus hku detected in us states complete genome characterization of korean porcine deltacoronavirus strain kor/knu - porcine deltacoronavirus in mainland china pathogenecity of porcine deltacoronavirus strains in gnotobiotic pigs us-like strain of porcine epidemic diarrhea virus outbreaks in taiwan mega : molecular evolutionary genetics analysis version . for bigger datasets a recombinant nucleocapsid protein-based indirect enzyme-linked immunosorbent assay to detect antibodies against porcine deltacoronavirus occurrence and sequence analysis of porcine deltacoronaviruses in southern china isolation and characterization of porcine deltacoronavirus from pigs with diarrhea in the united states development and application of an elisa for the detection of porcine deltacoronavirus igg antibodies retrospective testing and case series study of porcine delta coronavirus in us swine herds antigenic relationships among porcine epidemic diarrhea virus and transmissible gastroenteritis virus strains two-way antigenic crossreactivity between porcine epidemic diarrhea virus and porcine deltacoronavirus evaluation of serological cross-reactivity and cross-neutralization between the united states porcine epidemic diarrhea virus prototype and s-indel-variant strains reactivity of porcine epidemic diarrhea virus structural proteins to antibodies against porcine enteric coronaviruses: diagnostic implications funding this study was supported by grants as- . . -bq-b and as- . . -bq-b from the bureau of animal and plant health inspection and quarantine (baphiq), council of agriculture in taiwan. tien-huan hsu has received grants from the bureau of animal and plant health inspection and quarantine. all authors declare no conflict of interest.ethical approval this article does not contain any studies with human participants or animals performed by any of the authors. rectal swabs were collected from clinically diarrheic piglets in pig farms in taiwan, while swine sera were obtained from the animal disease control centers of various counties and the animal technology institute taiwan. key: cord- -vql e j authors: wang, xinyi; tai, wanbo; zhang, xiaolu; zhou, yusen; du, lanying; shen, chuanlai title: effects of adjuvants on the immunogenicity and efficacy of a zika virus envelope domain iii subunit vaccine date: - - journal: vaccines (basel) doi: . /vaccines sha: doc_id: cord_uid: vql e j zika virus (zikv), a mosquito-borne flavivirus, has attracted global attention due to its close association with congenital zika syndrome and neurological diseases, and transmission through additional routes, such as sexual contact. currently there are no vaccines approved for zikv, and thus, there is an urgent need to develop an effective and safe zikv vaccine. domain iii (diii) of the zikv envelope (e) protein is an important vaccine target, and a vaccine developed using a mutant diii of e (ediii) protein protects adult and pregnant mice, and unborn offspring, against zikv infection. here, we have used immunocompetent balb/c mice treated with anti-interferon-α/β receptor (ifnar ) antibodies to investigate whether three adjuvants (aluminum (alum), monophosphoryl lipid a (mpl), and mf ), either alone or in combination, could improve the efficacy of this ediii subunit vaccine. our data show that, although vaccine formulated with a single adjuvant induced a specific antibody and cellular immune response, and reduced viral load in mice challenged with zikv, the combination of alum and mpl adjuvants led to a more robust and balanced immune response, stronger neutralizing activity against three recent zikv human strains, and greater protection against a high-dose zikv challenge. particularly, the combination of alum with mpl significantly reduced viral titers and viral rna copy numbers in sera and tissues, including the male reproductive organs. overall, this study has identified the combination of alum and mpl as the most effective adjuvant for zikv ediii subunit vaccines, and it has important implications for subunit vaccines against other enveloped viruses, including non-zikv flaviviruses. zika virus (zikv) is a mosquito-borne flavivirus first isolated from a rhesus macaque in uganda in [ ] . most zikv infections are asymptomatic or show mild clinical symptoms, such as fever, headache, or rash. however, some adults infected with zikv present with guillain-barré syndrome (gbs), a disorder of the nervous system that results in muscle weakness and/or paralysis [ , ] . in addition, infection during pregnancy may cause congenital zika syndrome (czs), which is associated laboratory animals, national research council committee [ ] . the protocols have been approved by the institutional animal care and use committee (iacuc) of the new york blood center (permit numbers: and ). this protein was prepared as previously described [ ] . briefly, zikv diii of e protein containing m n and e t mutations at residues and of e protein (hereinafter ediii) was constructed using a mutagenesis kit (thermo fisher scientific, waltham, ma, usa) and a zikv wild-type plasmid expressing residues - (diii) of e protein and a c-terminal fc tag of human (hfc) igg [ , ] . the recombinant ediii protein was transiently expressed in the culture supernatant of t cells, and purified by protein a affinity chromatography (ge healthcare, chicago, il, usa). the above purified zikv ediii protein was used to immunize mice in the presence or absence of various adjuvants as previously described [ ] . briefly, mice were intramuscularly (i.m., µl/mouse) immunized with ediii protein ( µg/mouse) and one of the following adjuvant(s): alum (i.e., aluminum hydroxide, µg/mouse, invivogen, san diego, ca, usa), mpl ( µg/mouse, invivogen), alum ( µg/mouse) + mpl ( µg/mouse), or mf ( µl/mouse) [ ] . mice injected with ediii protein or phosphate-buffered saline (pbs) only were included as controls. the immunized mice were boosted once with the same immunogens at three weeks, and sera were collected at days post-last dose to detect antibody responses and neutralizing antibodies, as described below. zikv e, ediii, or hfc-specific antibodies in immunized mouse sera were analyzed by elisa as previously described [ ] . briefly, elisa plates were coated with zikv ediii protein, zikv full-length e protein with a his tag (aviva systems biology, san diego, ca, usa), or a c-terminal hfc-fused control protein containing a receptor-binding domain (i.e., rbd-fc) of middle east respiratory syndrome coronavirus (mers-cov) spike protein [ ] ( µg/ml) overnight at • c, and blocked with % fat-free milk in pbst (pbs containing tween- ) at • c for h. the plates were washed with pbst for times, and sequentially incubated with serial dilutions of mouse sera and horseradish peroxidase (hrp)-conjugated anti-mouse igg ( : ) or igg-fab ( : ) (for anti-zikv-e or anti-hfc antibodies), igg ( : ), or igg a ( : ) antibodies (thermo fisher scientific) at • c for h. the reaction was visualized after addition of , , , -tetramethylbenzidine substrate (sigma, st. louis, mo, usa) and stopped with n h so . absorbance at nm was measured using an elisa plate reader (tecan, morrisville, nc, usa). three recent zikv human strains, including r ( /honduras), pan ( /panama), and prvabc ( /puerto rico), were used in the study. briefly, viruses were grown in vero e cells and detected for viral titers using a plaque-forming assay [ , ] . mouse sera (about µl) and tissues (about mg for eye, and mg for heart, spleen, muscle, and brain) collected days post-challenge were also detected for zikv titers as described above, and the detection limits were about plaque-forming unit (pfu)/ml for sera, pfu/g for eye, or pfu/g for heart, spleen, muscle, and brain tissues. neutralizing antibodies in immunized mouse sera were detected by the prnt as previously described [ , ] . briefly, pfu of zikv was incubated with -fold serial dilutions of mouse sera at • c for . h, which were added to vero e cells and incubated at • c for h. the cells were then overlaid with dmem containing % carboxymethyl cellulose and % fbs, cultured at • c for - days and further stained with . % crystal violet. the neutralizing titer based on the serum neutralizations at a % plaque reduction (prnt ) was calculated using the calcusyn computer program [ , , ] . the immunized mice were challenged with zikv as previously described [ , ] . briefly, nine days post-last immunization of zikv ediii protein with or without respective adjuvant(s), or pbs control, mice were pretreated with a blocking anti-interferon-α/β receptor (ifnar ) antibody ( mg/mouse, leinco technologies, fenton, mo, usa); and h later, they were intraperitoneally (i.p.) challenged with zikv (strain r , × pfu; µl/mouse). mouse splenocytes were isolated at days post-challenge and detected for cellular immune responses using flow cytometry analysis. mouse sera and tissues were collected as described above, and detected for zikv titers and rna copies using the plaque-forming assay and quantitative reverse transcriptase pcr (qrt-pcr), respectively. flow cytometry analysis was performed to detect zikv-specific cellular immune responses as previously described [ ] . briefly, splenocytes ( × cells/well) were isolated from immunized mice days post-zikv challenge, and treated with × red blood cell lysis buffer (biolegend, san diego, ca, usa), followed by incubation with a zikv ediii peptide mixture (final concentration of µg/ml) (table ) in the presence of brefeldin a ( µg/ml, biolegend), and cultured at • c for h. after stimulation, the cells were washed with pbs, and stained for surface markers using percp/cy . anti-mouse cd -positive (cd + ), fitc-anti-mouse cd -positive (cd + ), and af anti-mouse cd antibodies (biolegend). after fixation and permeabilization, the cells were further stained for intracellular markers using pe anti-mouse interferon-gamma (ifn-γ), brilliant violet tm anti-mouse interleukin (il- ), and brilliant violet tm anti-mouse il- antibodies (biolegend), which were analyzed using a flow cytometer (bd lsrfortessa system). this assay was performed to detect zikv rna copies in sera and tissues of challenged mice as previously described [ , ] . briefly, qiaamp minelute virus spin kit and rneasy mini kit (qiagen, germantown, md, usa) were used to extract rnas from sera and tissues, respectively, which were quantified by qrt-pcr using power sybr green pcr master mix, ambion rnase inhibitor, and multiscribe reverse transcriptase (thermo fisher scientific) in viia master cycler pcr system (thermo fisher scientific). the forward primer ( -ttggtcatgatactgctgattgc- ) and reverse primer ( -ccttccacaaagtccctattgc- ) were applied for amplification. a qrt-pcr standard curve was made via serial dilutions of a recombinant plasmid expressing the zikv membrane and e proteins, and a linear standard curve in the range of - rna copies (correlation coefficient: r value > . ; detection limit: rna copies) was selected for calculation of zikv rna copies in the test samples. mouse sera (about µl) and tissues, including eye, heart, spleen (about mg), muscle, and testis (about mg), were collected days post-challenge, and detected for zikv rna, so the detection limit was about rna copies/ml (for sera), × rna copies/g (for eye, heart, and spleen), and . × rna copies/g (for muscle and testis) [ ] . all values were expressed as a mean with a standard error of the mean (s.e.m). statistical significance of all data among different groups was calculated using one-way anova: tukey's multiple comparison test based on the graphpad prism statistical software. *, **, and *** indicate p < . , p < . , and p < . , respectively. comparison of the adjuvanticity of alum, mpl, mf , and in combination, for immunization with zikv ediii to compare the potency of adjuvants, balb/c mice were immunized with a zikv non-neutralizing epitope-masked, ediii subunit vaccine in the presence or absence of alum, mpl, and alum adjuvant in combination with the mpl adjuvant. in addition, mf was included as a control adjuvant. at seven days following the second dose, sera were collected and the antibody titers specific to zikv ediii (containing a c-terminal hfc tag) or e (without fusion with hfc) were determined ( figure ). when used without an adjuvant, ediii was immunogenic and induced ediii-specific igg antibodies (total igg), while inclusion of alum or mf adjuvant significantly improved the antibody response. although mpl did not appear to enhance the antibody response to ediii, when used in combination with alum, the antibody response was significantly higher than for ediii with alum or mf alone ( figure a ). since ediii protein was fused with the hfc tag, igg antibodies targeting hfc were also generated, showing a similar trend as those specific to ediii ( figure b ). nevertheless, when coating the elisa plate with a zikv full-length e protein without hfc, significantly high-titer igg antibodies were induced, particularly in the alum and mpl-adjuvanted ediii ( figure c ), suggesting that fusion of hfc to the ediii subunit vaccine did not affect the generation of zikv-specific igg antibodies. multiple comparison test based on the graphpad prism statistical software. *, **, and *** indicate p < . , p < . , and p < . , respectively. to compare the potency of adjuvants, balb/c mice were immunized with a zikv nonneutralizing epitope-masked, ediii subunit vaccine in the presence or absence of alum, mpl, and alum adjuvant in combination with the mpl adjuvant. in addition, mf was included as a control adjuvant. at seven days following the second dose, sera were collected and the antibody titers specific to zikv ediii (containing a c-terminal hfc tag) or e (without fusion with hfc) were determined ( figure ). when used without an adjuvant, ediii was immunogenic and induced ediiispecific igg antibodies (total igg), while inclusion of alum or mf adjuvant significantly improved the antibody response. although mpl did not appear to enhance the antibody response to ediii, when used in combination with alum, the antibody response was significantly higher than for ediii with alum or mf alone ( figure a ). since ediii protein was fused with the hfc tag, igg antibodies targeting hfc were also generated, showing a similar trend as those specific to ediii ( figure b ). nevertheless, when coating the elisa plate with a zikv full-length e protein without hfc, significantly high-titer igg antibodies were induced, particularly in the alum and mpl-adjuvanted ediii ( figure c ), suggesting that fusion of hfc to the ediii subunit vaccine did not affect the generation of zikv-specific igg antibodies. immunization and challenge schedule. mice were immunized intramuscularly (i.m.) with pbs (background control) or with zikv ediii without an adjuvant, or with aluminum salts (alum), monophosphoryl lipid a (mpl), alum + mpl, or mf adjuvant and boosted at day after the initial dose. sera were collected at seven days after the boost (day ) and used to determine zikv e or ediii-specific antibody, and neutralizing antibody levels. mice were injected with an anti-interferonα/β receptor (ifnar ) antibody (in order to become susceptible to subsequent zikv infection) [ , ] at nine days after the boost (day ) and subsequently challenged with zikv (strain r , × plaque-forming unit (pfu)) the following day (day ). sera and tissue samples were collected three days post-challenge (day ) and used to determine zikv titers and viral load (rna copies). splenocytes were also tested for the zikv ediii-specific cellular immune response. immunization and challenge schedule. mice were immunized intramuscularly (i.m.) with pbs (background control) or with zikv ediii without an adjuvant, or with aluminum salts (alum), monophosphoryl lipid a (mpl), alum + mpl, or mf adjuvant and boosted at day after the initial dose. sera were collected at seven days after the boost (day ) and used to determine zikv e or ediii-specific antibody, and neutralizing antibody levels. mice were injected with an anti-interferon-α/β receptor (ifnar ) antibody (in order to become susceptible to subsequent zikv infection) [ , ] at nine days after the boost (day ) and subsequently challenged with zikv (strain r , × plaque-forming unit (pfu)) the following day (day ). sera and tissue samples were collected three days post-challenge (day ) and used to determine zikv titers and viral load (rna copies). splenocytes were also tested for the zikv ediii-specific cellular immune response. combination of alum with mpl adjuvant induced significantly higher igg and igg a antibody titers than either alum or mpl alone, or when mf was the adjuvant ( figure d ,e). when used in combination, mpl and alum adjuvants induced a more balanced antibody response, indicated by a low igg (th )/igg a (th ) ratio. in contrast, alum generated a th -biased igg antibody response, while ediii alone, or when formulated with mf , showed a slight th bias ( figure f ). the above data demonstrate that the combination of alum with mpl adjuvant enhanced the levels of ediiispecific igg antibodies (igg and igg a) and produced a more balanced igg response. next, we compared the levels of anti-zikv neutralizing antibodies induced by ediii vaccine when formulated with the different adjuvants. to this end, sera collected at seven days after immunization with the second dose of ediii were tested for neutralization against three human zikv we also determined the levels of the igg and igg a antibodies induced by ediii at seven days after the second dose. ediii alone induced a good igg response while the igg a response was noticeably weaker ( figure d ,e). when used alone, alum induced higher levels of igg (but not igg a) antibodies than ediii without an adjuvant, while mpl induced significantly higher levels of igg a (but not igg ) antibodies. when used in combination, alum with mpl significantly improved the ability of ediii to induce the igg and igg a antibody responses ( figure d,e) . moreover, the combination of alum with mpl adjuvant induced significantly higher igg and igg a antibody titers than either alum or mpl alone, or when mf was the adjuvant (figure d ,e). when used in combination, mpl and alum adjuvants induced a more balanced antibody response, indicated by a low igg (th )/igg a (th ) ratio. in contrast, alum generated a th -biased igg antibody response, while ediii alone, or when formulated with mf , showed a slight th bias ( figure f ). the above data demonstrate that the combination of alum with mpl adjuvant enhanced the levels of ediii-specific igg antibodies (igg and igg a) and produced a more balanced igg response. next, we compared the levels of anti-zikv neutralizing antibodies induced by ediii vaccine when formulated with the different adjuvants. to this end, sera collected at seven days after immunization with the second dose of ediii were tested for neutralization against three human zikv strains (r , vaccines , , of pan , and pavabc ). for all three strains, alum and mpl as the adjuvants induced a significantly stronger neutralizing antibody response than ediii alone, while mpl or mf only enhanced the neutralizing antibody response for the r strain ( figure a-c) . importantly, for all three zikv strains, the neutralizing antibody titers induced by ediii were significantly higher when alum was used in combination with mpl than for alum, mpl, or mf as the sole adjuvant ( figure a-c) . in contrast, sera injected with pbs only induced a background-level neutralizing antibody ( figure a-c) . these data show that although single adjuvants increased the anti-zikv neutralizing antibody response, the combination of alum with mpl adjuvant consistently induced the highest level of neutralizing antibodies against all of the zikv strains investigated. vaccines , , x of strains (r , pan , and pavabc ). for all three strains, alum and mpl as the adjuvants induced a significantly stronger neutralizing antibody response than ediii alone, while mpl or mf only enhanced the neutralizing antibody response for the r strain ( figure a-c) . importantly, for all three zikv strains, the neutralizing antibody titers induced by ediii were significantly higher when alum was used in combination with mpl than for alum, mpl, or mf as the sole adjuvant ( figure a-c) . in contrast, sera injected with pbs only induced a background-level neutralizing antibody ( figure a-c) . these data show that although single adjuvants increased the anti-zikv neutralizing antibody response, the combination of alum with mpl adjuvant consistently induced the highest level of neutralizing antibodies against all of the zikv strains investigated. splenocytes isolated from immunized mice three days after zikv challenge (strain r ) were used to investigate if the adjuvants enhanced the zikv ediii-specific cellular immune response. specifically, we determined the levels of secretion of ifn-γ (th ), il- (th ) and il- (th ) by cd -positive (cd + )-cd + t cells, and ifn-γ and il- by cd + -cd + t cells. for both t cell subsets, only low levels of these cytokines were detected when mice were immunized with ediii alone ( figure a -e), suggesting that ediii without an adjuvant did not induce a strong ediii-specific t cell response. when used individually, alum or mf increased the secretion of il- by cd + -cd + t cells, while increased expression of il- was only seen for mpl ( figure b,c) . for cd + -cd + t cells, inclusion of alum significantly increased ifn-γ expression, while il- expression was increased by alum and mf ( figure d,e) . when used in combination, alum with mpl adjuvant significantly enhanced expression of ifn-γ and il- cytokines tested in both cd + -cd + and cd + -cd + t cells over that observed when the adjuvants (alum, mpl, and/or mf ) were used individually ( figure a,b,d,e) . these data indicate that although individual adjuvants enhanced the ediii-specific cellular immune response, the combination of alum with mpl had a greater effect, particularly in cd + -cd + t cells. figure . detection of neutralizing antibody titers in immunized mouse sera. sera collected at seven days following the second dose of ediii were tested by a plaque reduction neutralization test (prnt) assay for neutralizing antibodies against zikv human strains r (a), pan (b), and prvabc (c). pbs shows the background level of neutralizing antibodies. neutralizing activity for the indicated adjuvants is presented as the neutralizing antibody titer based on the serum neutralizations at a % plaque reduction (prnt ). data are expressed as the mean ± s.e.m (n = ). significant differences (*: p < . ; **: p < . ; ***: p < . ) among different groups are shown. splenocytes isolated from immunized mice three days after zikv challenge (strain r ) were used to investigate if the adjuvants enhanced the zikv ediii-specific cellular immune response. specifically, we determined the levels of secretion of ifn-γ (th ), il- (th ) and il- (th ) by cd -positive (cd + )-cd + t cells, and ifn-γ and il- by cd + -cd + t cells. for both t cell subsets, only low levels of these cytokines were detected when mice were immunized with ediii alone ( figure a -e), suggesting that ediii without an adjuvant did not induce a strong ediii-specific t cell response. when used individually, alum or mf increased the secretion of il- by cd + -cd + t cells, while increased expression of il- was only seen for mpl ( figure b,c) . for cd + -cd + t cells, inclusion of alum significantly increased ifn-γ expression, while il- expression was increased by alum and mf ( figure d,e) . when used in combination, alum with mpl adjuvant significantly enhanced expression of ifn-γ and il- cytokines tested in both cd + -cd + and cd + -cd + t cells over that observed when the adjuvants (alum, mpl, and/or mf ) were used individually ( figure a,b,d,e) . these data indicate that although individual adjuvants enhanced the ediii-specific cellular immune response, the combination of alum with mpl had a greater effect, particularly in cd + -cd + t cells. to investigate if the adjuvants increased protection against zikv infection, immunized mice were challenged with a high-dose zikv (strain r , × pfu/mouse) one day after administering an anti-ifnar antibody (figure ). viral load was determined by a plaque-forming assay and a zikv rna copy number determined by qrt-pcr assay using sera and tissues collected three days post-challenge, an optimal time point to detect zikv infection in anti-ifnar antibodytreated immunocompetent mice, including balb/c (our unpublished data) [ ] . similar levels of zikv were seen in sera and most tissues (eye, heart, spleen, and muscle) for mice immunized with ediii without an adjuvant or with pbs ( figure a -e), whereas only in brain tissue, a significant reduction in viral titers was observed for mice immunized with ediii alone ( figure f ). these results suggest that immunization with ediii is unable to elicit adequate protection against zikv challenge. mice immunized with ediii formulated with a single adjuvant (alum, mpl, and/or mf ) had significantly reduced viral titers compared to those immunized with ediii alone (sera, heart, spleen, muscle, and brain) ( figure a ,c-f) and those immunized with pbs (sera, eye, heart, spleen, muscle, and brain) ( figure a-f) , suggesting that inclusion of an adjuvant increased protection. importantly, in sera and all of the tissues tested, the combination of alum with mpl adjuvant resulted in significantly lower, or undetectable, viral titers than other immunization conditions with a single adjuvant (alum, mpl, and/or mf ) ( figure a-f) . these results demonstrate that, although single adjuvants can increase protection, the greatest level of protection against a high-dose zikv challenge is achieved when ediii vaccine is formulated with both alum and mpl adjuvants. to investigate if the adjuvants increased protection against zikv infection, immunized mice were challenged with a high-dose zikv (strain r , × pfu/mouse) one day after administering an anti-ifnar antibody (figure ). viral load was determined by a plaque-forming assay and a zikv rna copy number determined by qrt-pcr assay using sera and tissues collected three days post-challenge, an optimal time point to detect zikv infection in anti-ifnar antibody-treated immunocompetent mice, including balb/c (our unpublished data) [ ] . similar levels of zikv were seen in sera and most tissues (eye, heart, spleen, and muscle) for mice immunized with ediii without an adjuvant or with pbs ( figure a-e) , whereas only in brain tissue, a significant reduction in viral titers was observed for mice immunized with ediii alone ( figure f ). these results suggest that immunization with ediii is unable to elicit adequate protection against zikv challenge. mice immunized with ediii formulated with a single adjuvant (alum, mpl, and/or mf ) had significantly reduced viral titers compared to those immunized with ediii alone (sera, heart, spleen, muscle, and brain) ( figure a ,c-f) and those immunized with pbs (sera, eye, heart, spleen, muscle, and brain) ( figure a-f) , suggesting that inclusion of an adjuvant increased protection. importantly, in sera and all of the tissues tested, the combination of alum with mpl adjuvant resulted in significantly lower, or undetectable, viral titers than other immunization conditions with a single adjuvant (alum, mpl, and/or mf ) ( figure a-f) . these results demonstrate that, although single adjuvants can increase protection, the greatest level of protection against a high-dose zikv challenge is achieved when ediii vaccine is formulated with both alum and mpl adjuvants. , and brain (f). sera and tissues were collected at three days post-challenge with zikv (strain r ), and viral load determined by a plaque-forming assay. mice immunized with pbs served as the control. the detection limits for the samples were ~ pfu/ml (sera), ~ pfu/g (eye), and ~ pfu/g (heart, spleen, muscle, and brain). data are expressed as the mean ± s.e.m (n = ). significant differences (*: p < . ; **: p < . ; ***: p < . ) among different groups are shown. consistent with viral load, mice immunized with ediii alone had only slightly lower levels of zikv rna in sera and tissues (eye and spleen) compared to those immunized with pbs ( figure a ,b,d), confirming that an adjuvant is required to induce protection. similarly, inclusion of a single adjuvant (alum, mpl, and/or mf ) resulted in a significantly lower level of zikv rna in sera and tissues (eye, heart, spleen, or muscle) compared with immunization with ediii alone (figure a-e) . furthermore, the levels of zikv rna in sera and all of the tissues, when alum and mpl adjuvants were used in combination for immunization, were significantly lower than when a single adjuvant (alum, mpl, and/or mf ) was used ( figure a-e) . these results are consistent with the levels of viral load ( figure ) and confirm that protection against zikv is greatest when alum and mpl adjuvants are used in combination. zikv can replicate in the reproductive organs for long periods; therefore, we also determined the amount of zikv rna in testes of immunized and challenged mice. similar to the results in other tissues, inclusion of a single adjuvant reduced the level of viral rna more than immunization with ediii alone or with pbs ( figure f ). moreover, viral rna copies were the lowest in testis of mice receiving ediii formulated with alum and mpl in combination ( figure f) . importantly, our results show that formulation of ediii with a combination of alum and mpl adjuvants can reduce viral rna copies in male reproductive organs. figure . detection of zikv titers in challenged sera (a), eye (b), heart (c), spleen (d), muscle (e), and brain (f). sera and tissues were collected at three days post-challenge with zikv (strain r ), and viral load determined by a plaque-forming assay. mice immunized with pbs served as the control. the detection limits for the samples were~ pfu/ml (sera),~ pfu/g (eye), and~ pfu/g (heart, spleen, muscle, and brain). data are expressed as the mean ± s.e.m (n = ). significant differences (*: p < . ; **: p < . ; ***: p < . ) among different groups are shown. consistent with viral load, mice immunized with ediii alone had only slightly lower levels of zikv rna in sera and tissues (eye and spleen) compared to those immunized with pbs ( figure a ,b,d), confirming that an adjuvant is required to induce protection. similarly, inclusion of a single adjuvant (alum, mpl, and/or mf ) resulted in a significantly lower level of zikv rna in sera and tissues (eye, heart, spleen, or muscle) compared with immunization with ediii alone (figure a-e) . furthermore, the levels of zikv rna in sera and all of the tissues, when alum and mpl adjuvants were used in combination for immunization, were significantly lower than when a single adjuvant (alum, mpl, and/or mf ) was used ( figure a-e) . these results are consistent with the levels of viral load ( figure ) and confirm that protection against zikv is greatest when alum and mpl adjuvants are used in combination. figure . detection of zikv rna in challenged sera (a), eye (b), heart (c), spleen (d), muscle (e), and testis (f). sera and tissues were collected at three days post-challenge with zikv (strain r ) and viral rna copies determined by qrt-pcr. mice immunized with pbs served as the control. the detection limits were ~ rna copies/ml (sera), ~ × rna copies/g (eye, heart, and spleen), and ~ . × rna copies/g (muscle and testis). data are expressed as the mean ± s.e.m (n = ). significant differences (*: p < . ; **: p < . ; ***: p < . ) among different groups are shown. protein-based subunit vaccines are generally safer than vaccines based on live attenuated or inactivated viruses but usually have low immunogenicity and efficacy [ , ] . however, vaccine performance can be improved by structure-based designs via masking non-neutralizing or unfavorable epitopes, or via antigen conjugation [ , , ] . in addition, the choice of adjuvant can greatly influence the immunogenicity and efficacy of vaccines, including subunit vaccines [ , , ] . our previous studies demonstrated that masking of a non-neutralizing epitope (surrounding residue ) of a zikv diii of e protein subunit vaccine (i.e., ediii) improved immunogenicity and protection of adult and pregnant mice, and unborn offspring, against zikv infection [ ] . here, we extended these studies to investigate the influence of licensed adjuvants on the immunogenicity and protection of mice against high-dose zikv challenge following immunization with this ediii subunit vaccine. vaccines prepared with a single adjuvant (alum, mpl, or mf ) significantly improved the antibody response (total igg, igg , and/or igg a) over immunization without an adjuvant. however, the highest igg titers were obtained when alum and mpl were used in combination. as stated above, adjuvants influence the immune response to vaccines. alum is a potent inducer of the th (igg ) immune response [ , ] . similarly, mf generally induces a slightly-biased th response as seen for vaccines for influenza virus and mers-cov [ , ] . in contrast, mpl induces expression of ifn-γ and il- and, therefore, has a more th bias and generates a more balanced (th /th ) immune response [ , ] . moreover, a combination of mpl and alum adjuvants (i.e., as ) may shift the immune responses from th to th and generate a more balanced immune figure . detection of zikv rna in challenged sera (a), eye (b), heart (c), spleen (d), muscle (e), and testis (f). sera and tissues were collected at three days post-challenge with zikv (strain r ) and viral rna copies determined by qrt-pcr. mice immunized with pbs served as the control. the detection limits were~ rna copies/ml (sera),~ × rna copies/g (eye, heart, and spleen), and . × rna copies/g (muscle and testis). data are expressed as the mean ± s.e.m (n = ). significant differences (*: p < . ; **: p < . ; ***: p < . ) among different groups are shown. zikv can replicate in the reproductive organs for long periods; therefore, we also determined the amount of zikv rna in testes of immunized and challenged mice. similar to the results in other tissues, inclusion of a single adjuvant reduced the level of viral rna more than immunization with ediii alone or with pbs ( figure f ). moreover, viral rna copies were the lowest in testis of mice receiving ediii formulated with alum and mpl in combination ( figure f ). importantly, our results show that formulation of ediii with a combination of alum and mpl adjuvants can reduce viral rna copies in male reproductive organs. protein-based subunit vaccines are generally safer than vaccines based on live attenuated or inactivated viruses but usually have low immunogenicity and efficacy [ , ] . however, vaccine performance can be improved by structure-based designs via masking non-neutralizing or unfavorable epitopes, or via antigen conjugation [ , , ] . in addition, the choice of adjuvant can greatly influence the immunogenicity and efficacy of vaccines, including subunit vaccines [ , , ] . our previous studies demonstrated that masking of a non-neutralizing epitope (surrounding residue ) of a zikv diii of e protein subunit vaccine (i.e., ediii) improved immunogenicity and protection of adult and pregnant mice, and unborn offspring, against zikv infection [ ] . here, we extended these studies to investigate the influence of licensed adjuvants on the immunogenicity and protection of mice against high-dose zikv challenge following immunization with this ediii subunit vaccine. vaccines prepared with a single adjuvant (alum, mpl, or mf ) significantly improved the antibody response (total igg, igg , and/or igg a) over immunization without an adjuvant. however, the highest igg titers were obtained when alum and mpl were used in combination. as stated above, adjuvants influence the immune response to vaccines. alum is a potent inducer of the th (igg ) immune response [ , ] . similarly, mf generally induces a slightly-biased th response as seen for vaccines for influenza virus and mers-cov [ , ] . in contrast, mpl induces expression of ifn-γ and il- and, therefore, has a more th bias and generates a more balanced (th /th ) immune response [ , ] . moreover, a combination of mpl and alum adjuvants (i.e., as ) may shift the immune responses from th to th and generate a more balanced immune response [ ] . consistent with these observations, we found that mf and alum, when used individually as the adjuvant for ediii, induced a th -biased immune response, while, when mpl was the sole adjuvant or used in combination with alum, a more balanced (th /th ) immune response was generated. the humoral and cellular immune responses influence zikv pathogenesis and play key roles in controlling zikv replication [ ] . in particular, neutralizing antibodies, induced by zikv vaccines, may block infection [ , ] , while cd + and cd + t cells secrete ifn-γ, il (th ), il- , and il- (th ) cytokines to further promote antibody production or directly kill infected cells [ ] [ ] [ ] [ ] . cd + t cells are critical for protection against zikv infection in mice, non-human primates, and humans [ , , [ ] [ ] [ ] . in the current study, we observed that alum, mpl, and mf adjuvants enhanced production of anti-zikv neutralizing antibodies; however, the combination of alum with mpl generated significantly higher neutralizing antibody titers than the individual use of adjuvants, and these results were consistent for three emerging human zikv strains. in addition, alum and mpl, when used in combination, enhanced secretion of ifn-γ (th ) and il- (th ) by cd + , particularly cd + , t cells. viral vaccines (e.g., against hbv and hpv) formulated with a combination of alum and mpl adjuvants (i.e., as ) stimulate antigen-specific t and b cells, resulting in stronger antibody (including neutralizing antibodies) and cellular responses than vaccines formulated using alum alone [ , ] . using the zikv ediii protein as a model antigen, our study has confirmed these observations by showing that a combination of alum and mpl adjuvants enhanced the immunogenicity of ediii over that observed with single adjuvants (figures - ) , whereas the alum and mpl combination alone without ediii was unable to elicit zikv-specific immune response and protection against zikv infection [ , ] . zikv does not normally infect immunocompetent mice, including balb/c and c bl/ [ , ] , but does infect immunocompromised mice deficient for interferon receptors (interferon-α/β receptor (ifnar) or a , or interferon-α/β/γ receptor, ag ) [ , , ] . to carry out challenge studies, we used balb/c mice treated with anti-ifnar antibodies, which also render mice susceptible to zikv infection [ , ] . formulation of ediii with individual adjuvants improved protection against high-dose zikv challenge, and was consistent with our virus neutralization studies results, that is, the combination of alum with mpl significantly improved protection further. this was confirmed by showing that a combination of alum with mpl as the adjuvant significantly reduced viral titers and viral rna copies in sera and tissues compared to other immunization regimes. zikv can be transmitted through sexual contact, the virus can persist in female or male (testes and seminal vesicles) reproductive organs for a prolonged period, and viral rna can be detected in semen for up to one year after zikv infection [ , [ ] [ ] [ ] [ ] [ ] . thus, vaccines that reduce viral load in reproductive organs will likely be more effective in reducing virus transmission during sexual contact. importantly, our results show that ediii formulation using alum and mpl in combination significantly reduced viral rna in the testes of zikv-challenged mice, thereby confirming that this adjuvant combination is most likely to yield greater vaccine efficacy than single adjuvants alone. in contrast, ediii alone without adjuvant(s) resulted in the highest viral titers and viral rna copies in sera and tissues tested among all vaccination groups, including reproductive organs, which were only slightly lower than those in pbs-injected control mice. it has been shown that protection against zikv infection is positively correlated with serum neutralizing antibody titers [ , ] ; thus, the high-level zikv in the ediii alone-immunized mice identified above may be partially due to the relatively low serum neutralizing antibody titer induced in these mice. it should be noted that the ediii protein used in this study was fused with a c-terminal hfc tag. fc-fusion proteins have been approved by the fda for human therapy, and their safety and pharmacokinetic activity have been evaluated extensively [ ] [ ] [ ] [ ] . fusion of fc tag to recombinant proteins may promote the proteins to form dimeric structures, significantly improving their solubility, stability, and avidity, as well as increasing their immunogenicity and/or efficacy [ ] [ ] [ ] . fc-fused vaccine design has been applied in subunit vaccines against hiv, influenza virus, ebola virus, and other emerging or reemerging viruses [ ] [ ] [ ] [ ] [ ] . in terms of zikv vaccines, a fc-fused e protein dimer (ze-fc) has been shown to mimic native dimeric status of e protein and induce higher neutralizing antibodies than a monomeric e protein without fc (monze) [ ] . we have also found that hfc-fused zikv ediii proteins form dimeric structures capable of eliciting high-titer antibody responses, neutralizing antibodies, and protection against high-dose zikv infection (figures , , and ) [ ] , consistent with previous findings. in conclusion, using immunocompetent balb/c mice, we compared the immunogenicity, neutralizing activity, and protective efficacy (after injection with anti-ifnar antibodies) of a zikv ediii subunit vaccine when formulated with different adjuvants (individually or in combination). although ediii formulated with alum, mpl, or mf induced a specific antibody and cellular immune response, and reduced viral load, the greatest vaccine efficacy was achieved when alum and mpl adjuvants were combined in a single vaccine. overall, this study has identified alum combined with mpl as the adjuvant of choice for zikv subunit vaccines, and has important implications for subunit vaccines against other enveloped viruses, including non-zikv flaviviruses. author contributions: l.d. and c.s. initiated and designed the study. x.w., w.t., and x.z. performed the experiments and analyzed the data. y.z., l.d., and c.s. wrote, edited, and approved the manuscript. funding: this research was funded by the national institutes of health (nih) grant (r ai ) and intramural funds of the new york blood center (nyb ). zikv strains (r , pan , and pavabc ) were obtained from bei resources. the authors declare no conflict of interest. zika virus. i. isolations and serological specificity zika virus infection as a cause of congenital brain 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an approach to influenza vaccines ebola virus glycoprotein fc fusion protein confers protection against lethal challenge in vaccinated mice key: cord- -qjtifwhd authors: van diep, nguyen; sueyoshi, masuo; norimine, junzo; hirai, takuya; myint, ohnmar; teh, angeline ping ping; izzati, uda zahli; fuke, naoyuki; yamaguchi, ryoji title: molecular characterization of us-like and asian non-s indel strains of porcine epidemic diarrhea virus (pedv) that circulated in japan during – and pedvs collected from recurrent outbreaks date: - - journal: bmc vet res doi: . /s - - - sha: doc_id: cord_uid: qjtifwhd background: since late , porcine epidemic diarrhea virus (pedv) has reemerged in japan and caused severe economic losses to the swine industry. although pedv vaccines have been used widely, the disease has swept rapidly across the county, and is commonly observed in ped-vaccinated farms, and has recurred in domestic herds. to better understand pedvs responsible for the reemerging outbreaks in japan, full-length spike (s), membrane (m), and nucleocapsid (n) genes of pedvs collected in japan during – , were sequenced and analyzed. results: phylogenetic analysis based on s gene sequences revealed that all the recent field pedvs were genetically distinct from the classical japanese strains, and were classified into three genotypes: north american (na), s indel, and asian non-s indel. our data suggested a possibility that multiple parental pedv strains were introduced into japan from abroad at the same time or similar times. the newly identified japanese strains showed the closest relationship to the us strains. two sublineages of japanese strains circulating in japan were similar to two sublineages identified in the us, suggesting common ancestors for these strains. in comparison with two vaccine strains used in japan, the field strains had various changes in epitope regions, glycosylation sites, and phosphorylation sites. these substitutions, particularly observed in epitope regions of the s ( , , , and ), m ( ), and n ( , , and ) proteins, may have affected antigenicity and vaccine efficacy, resulting in an unsuccessful pedv control. sequence comparisons between pedvs collected from primary and secondary outbreaks in three herds revealed that the disease has developed to an endemic stage in which pedv could persist for nearly two years in the herds or local regions, causing subsequent epidemics. conclusions: these results elucidate the genetic characteristics, origin, and molecular epidemiology of pedvs circulating in japan, as well as the pedv strains causing recurrent outbreaks. this study provides a better insight into the pedvs responsible for recent outbreaks in japan, and could potentially help to develop measures for controlling and preventing the disease. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. porcine epidemic diarrhea virus (pedv) is the etiologic agent of porcine epidemic diarrhea (ped), an acute enteritis disease that is characterized by vomiting and watery diarrhea and usually leads to high morbidity and mortality, especially in piglets [ ] . the disease was first recognized in england in [ ] , and the prototype virus, which was designated as pedv cv , was identified in belgium [ ] . subsequently, pedv has been reported in several other european countries, causing only sporadic outbreaks. since the s, it has been widespread in asia and has become an economic concern for the swine industry in many asian countries such as japan, china, south korea, thailand, and taiwan [ , ] . in late , new chinese strains of pedv were detected. these new strains were clinically more severe than the classical strains, resulting in %- % illness among infected swine herds and a %- % mortality rate among infected suckling piglets [ ] . in april , a pedv outbreak was confirmed in the us for the first time. it swept across more than states in the country, causing deaths of more than million newborn piglets during the one-year-epidemic period, and subsequently spread throughout north america, including canada and mexico [ ] . afterward, us-like ped epidemics have been reported in south korea, taiwan, japan, germany, france, and belgium [ , ] . belonging to the coronaviridae family in the nidovirale order, pedv has a single-stranded positive-sense rna genome of approximately kb in size that encodes four structural proteins (s, e, m, and n) and nonstructural proteins ( a, ab, and orf ) [ ] . among the viral proteins, the s glycoprotein is the most diverse [ ] , and plays a critical role in the process of inducing neutralizing antibodies and binding specific receptors [ ] . at least two b cell epitopes (ss and ss ) and two regions containing neutralizing epitopes (coe and c ) have been identified on this protein [ ] [ ] [ ] . the s gene is also associated with growth adaptation in vitro and attenuation of virulence in vivo [ ] , and was used as an important component in studies for understanding genetic relatedness among pedv isolates, the epidemiological status, and vaccine development [ , ] . the m protein is the most abundant component of viral protein in the envelope, and is responsible for morphogenesis, assembly, and budding [ ] . the n protein of coronavirus interacts with viral genomic rna, serving as the critical basis for the helical nucleocapsid during the viral assembly [ ] . two antigenic epitopes (nep-d and nep-d ) have been identified in the n protein of pedv [ ] . in japan, the ped-like disease was observed from late to early , but was not reported again until the outbreaks that occurred between september and june [ ] . in an epidemic of diarrhea caused by a pedv infection in , more than , suckling pigs died [ ] . afterward, there were no ped cases reported from until the first half of [ , ] . ped outbreaks first reemerged in october from a southern location (okinawa island) and spread rapidly throughout the country. until may , pedv infections were reported from more than farms in of prefectures; the infections affected over . million pigs and led to the death of half a million pigs, according to the ministry of agriculture, forestry, and fisheries of japan (http://www.maff.go.jp). currently, pedv infections and piglet mortality has decreased significantly. however, ped still occurred and frequently recurred in pig farms in japan. therefore, better insight into the causes for the reemergence of ped outbreaks is required for more efficient control and prevention of the disease. toward this goal, we sequenced and analyzed the full-length s, m, and n genes of the japanese field pedvs in comparison with those of vaccine strains used in japan, classical japanese pedv strains, and isolates from other countries. sample collection, rna extraction, and pedv detection small intestine or stool specimens were obtained from suckling piglets, post-weaning piglets, and sows presenting acute watery diarrhea at pig farms in japan, between december and february . intestinal samples were collected from dead piglets, and fecal samples were non-invasively collected immediately after excretion. thus, no aggressive actions toward the pigs were carried out for sampling purpose. samples of two major vaccine strains that have been employed in japan, p -v (nisseiken co., ltd) and -p c (kaketsuken co., ltd., japan), were collected from commercial vaccine bottles used in the swine farms. sample preparation, rna extraction, and pedv detection were performed as previously described [ ] . japanese field samples may contain both pedv variants with large deletions in the ′terminus of the s gene and pedv strains with an intact s gene [ ] . due to the complexity in discriminating, isolating, and individually sequencing pedv strains, only specimens containing pedvs with an intact s gene were used in our study. finally, pedv-positive field samples were collected from farms, located in five different prefectures in japan. these samples, along with the two vaccine samples described above, were sequenced to determine the full-length sequence of the s, m, and n genes ( table ) . all these field pedvs were confirmed to possess an intact ′-terminus of the s gene, showing only the expected prominent single dna band from pcr products on a . % agarose gel [ ] . after producing cdna, as previously described [ ] , the full-length s gene of pedv was amplified using the primer pair fs-f/fs-r ( table ) and kod fx kit (toyobo co., japan). the pcr products were used as templates for nested pcr reactions that amplified five dna fragments spanning the entire s gene using primer sets (cs -cs ), as previously reported [ ] . the m and n genes of the pedv were also amplified from the cdna using two newly designed primer sets, fmf/fmr and fnf/fnr ( table ). emeraldamp max pcr master mix kit (takara bio, japan) was used for pcr amplifications of the cs -cs fragments, as well as the m and n genes. the amplified pcr products were purified using a fastgene gel/pcr extraction kit (nippon genetics co., ltd., japan) according to the manufacturer's protocol. all sequencing reactions were carried out in duplicate, and sequences were determined in both directions using bigdye® terminator v . cycle sequencing kits (applied biosystems, ca, usa). products were analyzed using abi prism xl genetic analyzers (applied biosystems). nucleotide (nt) and deduced amino acid (aa) sequences were edited, assembled, and aligned using geneious v . . software (http://www.geneious.com), and percentage sequence divergences at the nt and aa levels were further calculated by using the same software. the obtained nt sequences were deposited in genbank under the following accession numbers: ky -ky as shown in table . unrooted phylogenetic trees were constructed using molecular evolutionary genetics analysis (mega) software, version . [ ] , with the maximum likelihood method and bootstrap tests of replicates. the best-fit nt substitution models for analysis were assessed. phylogenetic trees based on the nt sequences of the full-length s and n genes were generated using the tamura-nei substitution model with a discrete gamma distribution (tn + g). a phylogenetic tree based on the nt sequences of the m gene was created using the kimura -parameter substitution method with a discrete gamma distribution (k + g). n-glycosylation sites were predicted using a service available on http://www.cbs.dtu.dk/services/ netnglyc. phosphorylation sites were predicted using the netphos . server (http://www.cbs.dtu.dk/services/netphos) and netphosbac . server (http:// www.cbs.dtu.dk/services/netphosbac- . ). prediction of antigenic regions was performed using the emboss protein analysis tool [ ] integrated into the geneious software. identical nt sequences of the s gene were distinguished and excluded, resulting in the identification of individual sequences from the total collected field strains. phylogenetic analysis based on the s gene sequences from the japanese strains and reference strains identified from various countries revealed two major clusters: genogroup g divided into subgroups g a and g b, and genogroup g divided into subgroups g a and g b (fig. ). all japanese field pedvs in the present study fell into subgroups g a, g b, and g b. the most dominant strains belonged to g b, which comprised of current japanese strains, along with the highly virulent (north american type) pedvs isolated in the us, canada, mexico, south korea, and taiwan. two pedv of the field pedvs, jm- from miyazaki and other strains from aomori, aichi, kagoshima, and miyazaki were grouped into a monophyletic branch designated as ped-j , and they shared high nt and aa sequence identities ( . %- % and . %- %, respectively) with each other. another subclade, designated as ped-j , including japanese strains collected in miyazaki were also clustered into a segregated branch as shown in fig. the sequence data revealed that s genes from the japanese field pedvs are of - nt long, and encode proteins with - aa residues. this consequence was due to the presence of several deletions or insertions that accumulated primarily in the n-terminus of the s protein. multiple alignments of the s gene demonstrated that jm- and jm- exhibited insertions and deletions ( -nt, -nt, and -nt deletions at positions , , and , respectively, and a -nt insertion between positions and ), which are typical for s indel strains [ , ] . compared to the two vaccine strains, all japanese field pedvs were highly conserved in epitopes c and ss , except for strain jm- that had an aa substitution (l i) within c (fig. ) . additionally, compared to the vaccine strains, all the japanese field pedvs had two aa changes (l s and d s) in the epitope ss , and three serine substitutions (a s, t s, and g s) in the neutralizing domain coe (excluding jm and jm- ). notably, the aa substitutions at positions and resulted in predictable phosphorylation sites in the s protein of the japanese field strains. furthermore, different amino acids were found at the sites within the coe domain (fig. ) . regarding the prediction of high-specificity n-glycosylation sites in the s protein, most japanese strains belonging to g b exhibited eight n-glycosylation sites over the entire s protein (additional file : table s ). jm- had one additional n-glycosylation site at residue , and jm- possessed an additionally unique n-glycosylation site at position of the aa chain. two japanese s indel strains ( jm- and jm- ) had seven n-glycosylation sites, while jm- had eight n-glycosylation sites. compared with the vaccine strain, -p c , most of the japanese non-s indel strains in g b lost three n-glycosylation sites (at , , and ) and gained another nglycosylation site (at ). compared with p- v, these g b japanese strains lost three n-glycosylation sites (at , , and ) and gained four other nglycosylation sites (at , , , and ). the m gene sequences from all the japanese field pedvs and the two vaccine strains consisted of nucleotides, encoding a protein of aa. identical nt sequences were distinguished and excluded, resulting in the identification of seven unique sequences from the total collected pedvs (table ) . a phylogenetic tree based on the m gene of the japanese strains and reference strains revealed that all the sequences were divided into groups (g and g ); g was further divided into two subgroups g a and g b (fig. a) . all the current japanese pedvs fell into g , and showed the closest relatedness to the na strains and emerging strains identified in europe, south korea, and china. these field pedvs were phylogenetically distant from the vaccine strains and a classical japanese strain (jme ) of g . the m genes of field pedvs had . %- % nt identity to each other and . %- . % nt identity with the vaccine strains. the b cell epitope, wafyvr [ ] , located at position - of the m protein, was conserved in all field and vaccine strains. compared with the vaccine strains, all japanese field strains had an aa substitution (a s) belonging to an antigenic region (aa - ), as predicted by the emboss protein analysis tool (additional file : figure s a ). strain jm- had a different aa (s n) leading to the loss of a predicted serine phosphorylation site. in another predicted epitope region (aa - ), the field pedvs had one different aa (v i) compared to p- v, and two aa substitutions (f s and q e) compared to -p c . these substitutions (f s) resulted in gaining a predicted high-specificity n-glycosylation site. the n gene of all the field pedvs and vaccine strains was nt in length encoding a protein of aa. identical nt sequences of the n gene were distinguished and excluded, resulting in the identification of unique sequences. phylogenetic analysis based on the n genes from the japanese strains and reference strains revealed the classification of pedv strains into three groups: g , g , and g (fig. b) . all japanese field strains were closely clustered together into subgroup g with other us and us-like strains. the japanese field pedvs shared . %- % n gene nt sequence identity to each other and . %- . % to the vaccine strains. strain -jm from the first outbreak in miyazaki had % nt identity to eight other field strains collected in miyazaki, aichi, and kagoshima, and this is likely to be the most dominant sequence of n gene among the field strains. compared to the vaccine strains, all the field strains in the present study had aa substitutions (additional file : figure s b ); the substitution k n resulted in forming a predicted n-glycosylation site on the n protein from the field strains, while the aa substitutions a t and n s observed in field strains formed two predicted phosphorylation sites. of note, the substitutions k n and n s belong to the reported antigenic epitopes nep-d (aa - ) and nep-d (aa - ), respectively [ ] . there were three swine farms where ped outbreaks first occurred in , and then recurred in , as shown in table . farm is located in aomori prefecture, while farms and are located in the same town in miyazaki prefecture. phylogenetic analysis based on the s gene sequences revealed that three pedv strains ( jm- , jm- , and jm- ) collected from two different outbreaks on farm were closest to each other and segregated into a distinct minor branch. pedvs from the primary ped outbreaks on farm ( jm- , jm- on farm , the s, m, and n genes of pedvs identified from the recurrent outbreak ( jm- and jm- ) shared the highest ( . %, %, and . % respectively) nt sequence identities to a strain ( jm- ) collected table information of primary and recurrent ped outbreaks occurred in the three pig farms in this study farm mortality rate of piglets less than two weeks-old; b mortality rate of the piglets less than one-week-old all the information for mortality and morbidity was kindly supplied by the veterinarians responsible for these farms from the primary outbreak. the s genes of pedvs from the primary outbreak on farms and had high ( . %- . %) nt identity to each other. these strains also had identical sequences for the m and n genes. in the secondary outbreaks, pedvs from farms and also shared highly homologous genes. the s and m genes of two strains ( jm- and jm- ) from farm shared % nt sequence identities with those of a strain ( jm- ) from farm . compared with the strain collected from the primary outbreak on farm , two strains from the recurrent outbreak had aa substitutions in their s proteins (additional file : figure s ) . notably, the substitution s n lead to the loss of a predicted serine phosphorylation site. these recurrent strains also had two aa substitutions (r s and t n) in the n protein; the r s substitution formed a predicted serine phosphorylation site. comparison to pedvs from the primary outbreak on farms and , pedvs from the recurrent outbreaks had aa substitutions in the s proteins, in addition to a single aa change (a v) in the m protein and two aa changes (l i and s f) in the n protein. notably, the substitutions l i and s f occurred in the reported epitope nep-d of the n protein. since late , massive ped outbreaks have recurred in japan causing tremendous financial losses in the swine industry. despite the nationwide use of available attenuated vaccines developed decades ago [ ] , pedv has rapidly swept across the county. our sequencing data revealed that the prevailing japanese pedvs are genetically heterogeneous and can be classified into genotypes: na, s indel, and asian non-s indel. both the na and s indel types, which have been identified in recent studies in japan [ ] [ ] [ ] ] were also responsible for recent outbreaks in the us and south korea. in our study, besides the two previously reported pedv types, we identified another type of japanese pedv (designated as asian non-s indel) that is closely related to the vietnamese, thai, and chinese strains. the asian non-s indel type was collected from an early ped epidemic that occurred in southern japan where the recent ped pandemic began spreading. moreover, the asian non-s indel type appeared at a similar time to the appearance of the na and s indel types in japan [ ] . together with the absence of ped cases from to in japan [ , ] , we speculated a possibility that the three types of japanese pedv originated from one source and emerged at the same or similar time in the southern region of japan before spreading across the country. the result of this study revealed that all the field pedv strains (except jm- ) were closely related to emerging strains identified in the us, south korea, taiwan, and in european countries. of note, the japanese pedvs were genetically most closely related to the us strains; some even had identical s gene. since the recent ped pandemics in japan started to reemerge six months after those in the us, the close genetic relationship indicated that the current japanese pedv might have been introduced directly from the us, or both the us and japanese pedvs were derived from one source of origin. since april , the na type of pedv has emerged in the us. the na strains were identified subsequently in south korea, taiwan, and japan from late . another pedv type possessing typical insertions and deletions in the n-terminus of the s gene when compared to the prototype na type was first discovered in ohio in the us [ ] . this pedv type, therefore, was designated as s indel. the s indel pedvs were also identified later in recent outbreaks in japan, south korea, canada, belgium, france, germany, portugal, slovenia, and the netherlands [ ] . both highly virulent and s indel pedvs prevailing in the us are supposed to be derived from chinese pedvs as a result of recombinant events [ , , ] . in accordance with these findings, our sequence analysis showed that the s indel strains, such as ch/hbqx/ and js , had already appeared in chinese ped outbreaks during - [ , ] . with the prevalence of both na and s indel strains in the us, south korea, and japan since , we suggested that there are two major possibilities of origin for the reemerging japanese pedv strains. first, us-like strains have been directly transmitted to japan from the us or south korea. second, the same source of the us strains could have introduced the pedv into japan, from china, shortly after the us outbreaks. however, our study revealed the prevalence of the asian none-s idel pedv strain in japan, which has not been reported in the us or south korea. thus, we incline toward the possibility that multiple parent pedv strains have been introduced into japan, from china or china's neighboring countries (such as the southeast asian region), causing the recent ped pandemic. further investigation and surveillance are required to specifically identify the source of origin for the reemerging japanese pedvs. sixteen pedv strains from four different prefectures including miyazaki, kagoshima, aomori, and aichi formed a well-supported subclade (ped-j ). fourteen pedv strains from various farms in miyazaki prefecture were also grouped into a distinct subclade (ped-j ). these results suggested that strains of those japanese sublineages (ped-j and ped-j ) may derive from two common ancestral pedvs whose progeny have spread in the regions. two genetic sublineages of us strains, namely ia -co/ and mn-ia [ ] , were identified from the us outbreaks in . notably, the japanese pedv of the japanese sublineage ped-j in this study showed a close relationship with the us sublineage ia -co/ , and pedvs of the japanese sublineage ped-j showed close relatedness with the us sublineage mn-ia . this data revealed the common pattern of genetic diversity as well as the close relationship between the japanese strains and the us strains, suggesting a common ancestor for these strains. in our previous study [ ] , by sequencing a partial s gene containing a neutralizing epitope region (coe domain), we suggested that jm- may belong to the s indel type that has been circulating in japan together with the highly virulent us-like strains. in this study, by sequencing the full-length s gene, we confirmed jm- to be an s indel strain. we also identified another strain that belonged to the s indel type, jm- , although it was not predicted to be an s indel pedv in the previous study. this discrepancy arose due to the region of the partial s gene utilized in the previous report that lack the typical insertions and deletions of the s indel type. our data revealed the circulation of s indel strains in ped outbreaks occurring in japan, but these strains compose only a minor population of the reemerging japanese pedvs. these results are consistent with two recent reports of japanese pedv [ , ] . phosphorylation of viral proteins is a reversible posttranslational modification that can have major impacts on viral infection, replication, and cytotoxicity in a host cell [ ] . for infectious bronchitis virus, belonging to the coronaviruses, the phosphorylation of the nucleocapsid protein has a dramatic influence on rna binding capability and the recognition of viral rna from nonviral rna in the process of viral replication [ ] . along with phosphorylation, glycosylation is another posttranslational modification that often strongly affects the protein functions of viruses. n-glycosylation sites in the spike gene of the severe acute respiratory syndrome coronavirus were demonstrated to be crucial for viral infection [ ] . on the other hand, mutations observed in the neutralizing regions have been proposed as presenting a viral evolution, allowing escape from antibodies developed against vaccine or classical pedv strains [ ] . in this study, we characterized differences in epitope regions, high-specificity n-glycosylation and phosphorylation sites, between the vaccine and recent field strains. various substitutions observed in the s ( , , , and ), m ( ) , and n ( , , and ) proteins may have affected the antigenicity, conferring the capacity for immune evasion in the pedv field strains, and consequently, influencing the efficacy of the vaccines. in fact, despite vaccination, pedv infections still spread rapidly and are commonly observed on ped-vaccinated farms in japan [ ] . vaccination showed very low efficacy and could not mitigate the severe losses caused by ped [ ] . based on the antigenicity analysis of the vaccine and field strains, as well as the recent performance of the ped vaccines on the japanese pig farms, we suggest that the development of novel vaccines based on reemerging pedvs is necessary to control current ped outbreaks. the first ped epidemic on farm was a severe occurrence in may (table ) . afterward, obvious clinical symptoms and death caused by ped were not observed in pigs of the herd until february when the subsequent outbreak recurred with less severity than the first. although the pigs did not show any obvious symptoms of ped, the presence of pedv on this farm was detected in piglets several times by rt-pcr during the period from june to january , according to the information supplied by veterinarians responsible for this farm. phylogenetic analysis based on the sequences of the s and n genes showed that pedv strains from the primary and secondary outbreaks formed monophyletic branches. they also shared with each other the highest sequence identity of the s, m, and n genes compared to other strains used in this study. additionally, ped outbreaks were not observed on other farms within a km radius of farm , and many strict measures for establishing a high level of security were implemented on this farm. taken together, we speculated that pedv strains causing the primary outbreak induced partial protective immunity but persisted in the herd for nearly two years. subsequently, the persisted pedvs caused the secondary epidemic. this is the first report of a two-year-long persistence of pedv in a pig herd. it has been proposed that the popular circulation of pedv variants with large deletions in the s gene is associated with the persistence of the pedv on the infected farms in japan [ ] . however, we did not detect the presence of the large s deletion variants in the fecal and intestinal samples collected from this farm. other factors, including the genetic variation of causative pedv strains, may play a more important role in the disease recurrence. on farms and , ped first occurred in may , despite all the sows receiving antepartum pedv vaccinations in . afterward, the disease disappeared until early when the subsequent outbreaks recurred. there was no detection of the pedv during the interval between these two outbreaks. phylogenetic analysis based on the s and n genes revealed that the strains collected from the first outbreaks on farms and formed a monophyletic branch. moreover, these two farms were located close to each other with a distance of . km (separated by a small mountain), and the outbreaks occurred at the same time. this suggests that the pedv strains introduced onto these farms that induced the first outbreaks were derived from a common source of pedv. likewise, in the secondary outbreaks, the strains collected from these two farms also have the highest genetic identity to each other and formed minor monophyletic branches, as shown in the phylogenetic trees for the s and n genes. all the strains collected from the primary and recurrent outbreaks on these farms were closely clustered into the subclade ped-j , together with other strains collected from other farms located in miyazaki prefecture (fig. ) . this data suggested that the pedv strains causing the subsequent outbreaks on farms and evolved from pedvs that had been circulating in the miyazaki region since . there are two possibilities for the origin of the strains that caused the subsequent outbreaks on farms and . first, pedvs may have persisted silently on these farms since , evolving, and then causing the subsequent outbreaks when suitable conditions arrived in early . another possibility is that pedvs may have persisted on other pig farms, circulating in the miyazaki region since the ped occurrence, after which the pedvs were introduced again onto farms and , causing the secondary epidemics. more data is needed to elucidate exactly the origin of these pedvs strains. the occurrence of ped in these herds has partially indicated the failure of using the current pedv vaccines, which are based on old seed stock of classical japanese strains. besides, the repeated outbreaks in these herds indicate that the disease has developed to an endemic stage. these results could be used to elucidate the prevalence of pedvs and to contribute to the prevention of ped in the region. in conclusion, our study revealed that japanese field pedvs causing ped outbreaks during - could be classified into three types, and these pedvs might have been introduced from overseas at the same time or during similar periods. the disease has developed to an endemic stage in which the pedv can persist for a long time in these herds or these local regions causing subsequent epidemics. the distant genetic relationships and various aa substitutions between the japanese field pedvs and vaccine strains may be responsible for the unsuccessful ped control in japan. our findings will be useful in understanding the origin and molecular epidemiology of the pedv, and helping to develop measures for the control of the disease. additional file : table s . highly-specific n-glycosylation sites in the spike protein of the vaccine and field strains. (docx kb) additional file : figure s . amino acid substitutions in the m and n proteins between the field and vaccine strains. porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines letter to the editor a new coronavirus-like particle associated with diarrhea in swine evolution, antigenicity and pathogenicity of global porcine epidemic diarrhea virus strains new variants of porcine epidemic diarrhea virus, china cell culture isolation and sequence analysis of genetically diverse us porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus detection and molecular diversity of spike gene of porcine epidemic diarrhea virus in china the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus the gprlqpy motif located at the carboxy-terminal of the spike protein induces antibodies that neutralize porcine epidemic diarrhea virus identification of two novel b cell epitopes on porcine epidemic diarrhea virus spike protein mutations in the spike gene of porcine epidemic diarrhea virus associated with growth adaptation in vitro and attenuation of virulence in vivo heterogeneity in spike protein genes of porcine epidemic diarrhea viruses isolated in korea genetic diversity of orf and spike genes of porcine epidemic diarrhea virus in thailand incorporation of spike and membrane glycoproteins into coronavirus virions the coronavirus nucleocapsid is a multifunctional protein the identification and characterization of two novel epitopes on the nucleocapsid protein of the porcine epidemic diarrhea virus an immunohistochemical investigation of porcine epidemic diarrhoea porcine epidemic diarrhea: its diagnosis and control molecular characterization of pig epidemic diarrhoea viruses isolated in japan from isolation and molecular characterization of porcine epidemic diarrhea viruses collected in japan in us-like isolates of porcine epidemic diarrhea virus from japanese outbreaks between novel porcine epidemic diarrhea virus (pedv) variants with large deletions in the spike (s) gene coexist with pedv strains possessing an intact s gene in domestic pigs in japan: a new disease situation mega : molecular evolutionary genetics analysis version . emboss: the european molecular biology open software suite emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences distinct characteristics and complex evolution of pedv strains new variant of porcine epidemic diarrhea virus identification of a conserved linear b-cell epitope in the m protein of porcine epidemic diarrhea virus isolation and experimental inoculation of an s indel strain of porcine epidemic diarrhea virus in japan origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states genetic variation analyses of porcine epidemic diarrhea virus isolated in mid-eastern china from to molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus field strains in central china during - outbreaks phosphorylation events during viral infections provide potential therapeutic targets role of phosphorylation clusters in the biology of the coronavirus infectious bronchitis virus nucleocapsid protein specific asparagine-linked glycosylation sites are critical for dc-sign-and l-sign-mediated severe acute respiratory syndrome coronavirus entry bioinformatics insight into the spike glycoprotein gene of field porcine epidemic diarrhea strains during - in guangdong, china measure to mitigate loss caused by ped (porcine epidemic diarrhea) using maternal immunity the work was conducted with funding from the university of miyazaki (grant number - ) and japan society for the promotion of science (kakenhi program, grant number h ). the funders had no role in the design of the study and collection, analysis, and interpretation of data and in preparation of the manuscript. data supporting the conclusions of this article are included within the article and its additional files table s , figure s , and figure s . sequence data supporting the conclusions of this article are available in the genbank repository under the accession numbers ky -ky .authors' contributions ry and nd designed the study. ry, ms, zn, and th provided the viral samples and other materials. nd, nf, at, ui, and om conducted experiments. nd designed the primers, interpreted the sequencing data and wrote the manuscript draft. ry, ms, zn, th, at, ui, nf, and om analysis the data and revise the manuscript. all authors prepared and approved the final manuscript. in this study, the intestinal samples were collected after the death of pigs due to the virus infection in the pig farms. no pig was culled for sampling purpose in this study the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -k rcpav authors: niespodziana, katarzyna; stenberg-hammar, katarina; megremis, spyridon; cabauatan, clarissa r.; napora-wijata, kamila; vacal, phyllis c.; gallerano, daniela; lupinek, christian; ebner, daniel; schlederer, thomas; harwanegg, christian; söderhäll, cilla; van hage, marianne; hedlin, gunilla; papadopoulos, nikolaos g.; valenta, rudolf title: predicta chip-based high resolution diagnosis of rhinovirus-induced wheeze date: - - journal: nat commun doi: . /s - - - sha: doc_id: cord_uid: k rcpav rhinovirus (rv) infections are major triggers of acute exacerbations of severe respiratory diseases such as pre-school wheeze, asthma and chronic obstructive pulmonary disease (copd). the occurrence of numerous rv types is a major challenge for the identification of the culprit virus types and for the improvement of virus type-specific treatment strategies. here, we develop a chip containing different micro-arrayed rv proteins and peptides and demonstrate in a cohort of pre-school children, most of whom had been hospitalized due to acute wheeze, that it is possible to determine the culprit rv species with a minute blood sample by serology. importantly, we identify rv-a and rv-c species as giving rise to most severe respiratory symptoms. thus, we have generated a chip for the serological identification of rv-induced respiratory illness which should be useful for the rational development of preventive and therapeutic strategies targeting the most important rv types. r espiratory viral infections are among the most common triggers of acute exacerbations of pre-school wheeze, asthma, and chronic obstructive pulmonary disease (copd) [ ] [ ] [ ] . asthma and copd are severe and disabling diseases of the respiratory tract and hence represent a serious global health problem affecting different age groups. acute pre-school wheeze and community-acquired pneumonia (cap) are other common causes of emergency visits with possible viral etiology. there is an increasing prevalence of these airways diseases, rising treatment costs, and therefore virus-induced respiratory illnesses are a heavy burden for patients and the community , . respiratory viral infections, mainly due to rhinovirus (rv), are responsible for approximately % of wheeze and asthma exacerbations in children , . moreover, infants with rhinovirus-induced wheeze have a significantly increased risk for subsequent development of recurrent wheeze and childhood asthma . since exposure to rv does not lead to wheezing illness in all children, additional factors such as the host genotype, defects of the respiratory epithelial barrier, and/or atopic predisposition have been suggested to play important roles in asthma [ ] [ ] [ ] . rv is genetically a highly diverse virus group with more than distinct rv types which have been divided into three distinct rv species, rv-a, rv-b, and rv-c , . rhinoviruses can also be classified according to which cellular receptor on human respiratory epithelial cells they use for entry . rv-b and most rv-a variants bind to the intercellular adhesion molecule- (icam- ) (i.e., major rv group), while a subset of rv-a species binds to the low-density lipoprotein receptor (i.e., minor rv group) , . more recently, a cadherin-related family member protein (cdhr ) has been reported as a one of the probable receptors for the rv-c species . the identification of the culprit rhinovirus species responsible for severe exacerbations of respiratory disease is an extremely important topic as certain rv species (e.g., rv-c) are suspected to be associated with more severe wheezing illnesses and acute asthma exacerbations in infants and children compared to others , . in fact, there are also several preventive and therapeutic strategies for rv infections under development which require a precise knowledge of the clinically relevant rv species to be targeted. for example, several approaches for developing vaccines based on polyvalent inactivated rv, synthetic rvderived peptides and recombinant rv proteins have been reported [ ] [ ] [ ] [ ] [ ] [ ] . the formulation of a broadly protective vaccine obviously requires the inclusion of the clinically most relevant and common rv species. furthermore, it has been shown that blocking of the viral receptor on respiratory epithelial cells (e.g., icam- ) can prevent rv infection . again therapeutic approaches targeting the viral receptors require knowledge which rv species are the most frequent and relevant ones. finally, it is important to investigate the role of the different rv species for exacerbations of severe bronchial obstruction in different populations and for different age groups and manifestations of respiratory illness (e.g., pre-school wheeze, asthma, copd, asthma-copd overlap: aco, cap). while rv is well established as an important trigger factor for childhood wheeze and asthma, less is known regarding the role of rv infections in exacerbations of copd and in respiratory disease exacerbations of older subjects . furthermore, the causal relationship of rv with cap is also unknown . currently, the detection of rv in the course of respiratory infections is mainly based on reverse transcription of viral rna and dna amplification by polymerase chain reaction (pcr) . such tests can demonstrate the presence of virus-derived nucleic acid but they do not necessarily indicate that the particular virus had caused an infection and is indeed responsible for clinical symptoms in the patient . in fact, rhinovirus rna has been found in a high proportion of asymptomatic infants and children [ ] [ ] [ ] . furthermore, little is known about levels and epitopespecificities of natural antibody responses capable of neutralizing rhinoviruses, and thus protecting individuals against rv infections. such information would be helpful for the development of new immunological strategies for the treatment and prevention of rv-induced exacerbations of respiratory diseases. therefore, there is a huge and so far unmet need for high-resolution serological detection of rhinovirus infections. we have previously identified the capsid protein vp and an n-terminal vp peptide as major target for the natural antibody response of rv-infected subjects . then we have demonstrated that in vivo inoculation of subjects with rv indeed induced increases of vp -specific antibody responses which were best detected with the vp protein of the corresponding species . similar increases of rvspecific antibodies were found in pre-school children after asthma attacks using complete recombinant rv capsid proteins . in a recent study performed in pre-school children with acute asthma attacks, we observed that increases of rv-specific antibody responses reflected the severity of respiratory symptoms . in the present study funded by the european union project "predicta" (https://cordis.europa.eu/project/rcn/ _en.html), we investigated if it is possible to generate a microarray-based serological test which can discriminate rv-a, rv-b, and rv-c as culprit species involved in childhood asthma attacks. development of a high-resolution predicta microarray. figure shows the arrangement of rv-derived proteins, peptides as well as control proteins on the predicta chip and the selection procedure for the n-terminal vp peptides. recombinant capsid proteins (vp -vp ) and fragments thereof representing rv-a, b, c species as well as non-structural proteins from rv were included (supplementary tables - ) . based on our previous finding that antibodies from rv-infected patients react preferentially with the n-terminus of vp (ref. ), we included synthetic n-terminal vp peptides from rv strains which were selected in a rational, multistep process to represent distinct rv strains (fig. a , supplementary table ) . starting with vp sequences retrieved from the ncbi database (rv-a: ; rv-b: ; rv-c: ), multiple sequence alignments were performed to identify clusters of peptides with high degrees of sequence identities (fig. a , supplementary table : clusters a -a ; b -b ; c -c ). in a next step, peptides were re-clustered into groups taking the chemical properties of amino acids into consideration (supplementary table : ai-axviii; bi-bix; ci-ciii). from these groups peptides with the most distantly related sequences were selected (fig. b, c; supplementary tables and ). for a further refinement of vp , vp , and vp antibody responses, vp and vp fragments as well as peptides spanning the complete vp sequence from rv were included (fig. d , supplementary tables and ). for control and calibration purposes we added recombinant allergens and control proteins (fig. d, supplementary table ) to the predicta chip. proteins and peptides were spotted in triplicates on a preactivated glass slide containing six microarrays surrounded by a teflon frame so that one chip allows testing of six serum samples (fig. d) . identities and purities of recombinant proteins and peptides were tested by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) followed by coomassie brilliant blue staining, western-blotting, and by mass spectrometry, respectively (supplementary tables - , supplementary fig. ). supplementary table shows that the predicta microarray allows reproducible measurement of igg levels to the antigens according to intra-and inter-assay variations. rv strain recognition is broader in older wheezing children. the predicta chip was tested with sera from pre-school children who were admitted to the hospital due to an acute wheezing episode . table summarizes demographic and clinical data of the children investigated in this study. to investigate if the spectrum of recognized rv peptides varies by age, children were grouped according to age (group i: < year, n = ; group ii: - years, n = ; group iii: > years, n = ). figure a tables - peptides followed by rv-a peptides whereas igg reactivity to rv-b peptides was less frequent and intense (fig. a) . similar results were obtained for iga responses which in general were lower than the igg responses (fig. b ). the analysis of igg reactivity to structural and non-structural proteins and to recombinant fragments and synthetic peptides spanning vp , vp , and vp from rv is shown in supplementary fig. a for all children and in supplementary fig. b for those children (n = ) who had shown increases of rv -specific antibody responses in follow-up serum samples taken after recovery. results obtained thus confirmed our earlier observations showing that the majority of rv-specific antibody responses are directed against the n-terminus of vp as represented by the peptides from the n-terminus of vp proteins from the different rv species . some children showed an igg response against vp and in particular to a fragment representing the middle portion of vp whereas vp and vp showed no relevant igg reactivity (supplementary fig. a, b) . among the non-structural proteins, c, a, and c showed some igg reactivity ( supplementary fig. ). the analysis of igg reactivity according to the age of children at the acute visit showed that children < year of age had much lower rv-specific igg levels compared to children who were older than year. there was almost no change of the rv strainspecificity pattern among the three age groups (fig. c ). however, we found a positive correlation between the number of recognized vp peptides and the age of the children (fig. d ). children between and months of age recognized a significantly lower number of n-terminal vp peptides than older ( - months) children. children older than years reacted with significantly more peptides than both groups of younger children (fig. e) . the patterns of antibody response against different rv peptides were also analyzed using an independent bioinformatics approach ( supplementary fig. ) . a phylogenetic clustering of all peptides was prepared and used to generate peptide groups according to sequence homology which represented to a large extent the rv subgroups a, b, and c. then an unsupervised computer algorithm was used to cluster the patterns of antibody responses. finally, the results of these analyses were superimposed. it turned out that there was a very strong correlation between the two groupings, one based on antibody reactivity and the other based on sequence identities among peptides made through the unsupervised analysis. thus antibody response patterns reflected very closely the peptide sequence similarities ( supplementary fig. ). identification of rv species-specific antibody increases. based on our previous observations that antibody increases specific for the n-terminal portion of vp can be detected in serum samples obtained from subjects after rv infection , the predicta chip was equipped with a vp peptide set which should allow detecting species-specific immune responses at high resolution ( fig. ). supplementary figure shows images of rv-specific antibody responses measured in sera from six representative children obtained at the time of the acute episode of wheeze and in sera at a follow-up visit - months later. in sera from the follow-up visit increased antibody responses to rv-a (sera # , # ), rv-c (sera # , # ), and rv-b (sera # , # ) peptides could be detected ( supplementary fig. ) . we then compared the peptide-specific igg antibody levels in sera obtained from the children at the time of the acute wheeze and at the follow-up - months later (i.e., median weeks later) ( table ). supplementary figure shows a color-coded map of the peptide-specific antibody responses for the acute phase and follow-up of each of the children demonstrating species-specific igg increases. next, we compared the peptide-specific increases in relative numbers for each child (fig. a) . according to these peptide-specific igg increases, children could be identified who responded preferentially to rv-a-derived peptides (n = ), rv-c-derived peptides (n = ), rv-b-derived peptides (n = ), and some with a mixed response pattern (n = ) (fig. a) . for children no increases of peptide-specific igg responses were found (fig. a, bottom) . the same analysis was performed for increases of antibody responses against complete recombinant vp proteins from the three rv-a strains (i.e., rv , , ), the rv-c strain yp, and the rv-b strain ( fig. a) but the results were less clear and negative for several children because only few strains were covered with the recombinant proteins. we have also included in fig. a (right column) results from the pcr testing performed using vp -vp specific primers in of the children , which showed that the nucleic acid-based detection of virus strains was negative for approximately % of children with increases of rv peptide-specific igg levels which may indicate a higher sensitivity of serology vs. pcr in these children. moreover, pcr results did not correspond well with the specificities of the antibody responses. there were also children without increases of rv peptide-specific antibody responses who had positive pcr results (fig. a, bottom) . tables and ) (x-axes: red: rv-a species; green: rv-b species; blue: rv-c species). antibody levels are color-coded and expressed as isac standardized units, isu-g and isu-a, respectively. c median igg levels (y-axis: isu-g) to vp peptides (x-axis) in children grouped according to age ( - months: squares; - months: triangles; - : circles). d spearman's rank correlation between the number of igg-reactive peptides (n, y-axis; median igg > isu) and age (x-axis: months). correlation coefficient (ρ) and p-value are shown. e comparison of the number of igg-reactive vp peptides (n, y-axis; median igg > isu) in children according to age (x-axis). horizontal lines indicate medians. statistically significant differences between groups are indicated (**p < . , ****p < . ) (mann-whitney u-test) # # # # # # # # # # # # # # # # yp c qpm yp # # # # # # # # # # # # # # # # antibody signatures associated with severity of wheeze. next, we investigated whether antibody responses to certain rv species were associated with the severity of rv-induced wheeze. for this purpose, we determined the number of days with respiratory symptoms and the number of days when medication was required in the period between the acute and follow-up visit. the number of days with respiratory symptoms but not with medication was significantly higher in subjects with an increase in rv-a > mixed > rv-c > rv-b specific signal (fig. , top and middle panels) . we then analyzed the sum of days with symptoms and medication and found that rv-a and rv-c antibody increases were associated with the highest number of days with symptoms and medication (fig. , bottom panel) . children with rv-a-or rv-c-specific antibody increases had significantly more days with symptoms and medication than children with rv-b-specific antibody increases (fig. , bottom panel) . within the european union-funded project predicta (https:// cordis.europa.eu/project/rcn/ _en.html) we developed the predicta chip which is based on micro-arrayed rv-derived proteins and peptides selected to represent the three rv species (rv-a, rv-b, and rv-c). using the predicta chip we could demonstrate in a cohort of pre-school children with acute wheeze and a follow-up visit that the rv-specific antibody response (igg > iga) is directed against an n-terminal peptide of the major capsid protein vp which confirms earlier results obtained by the mapping of rv -specific antibody responses . since the predicta chip was designed to contain a panel of synthetic peptides which represented the most diverse rv strains of the three genetic rv species in terms of sequence identity and physicochemical properties we were able to perform a high-resolution mapping of rv species-specific antibody responses by serology. in a cohort of clinically well-described swedish pre-school children from whom sera were available from an episode of acute wheeze requiring an emergency room visit and from a follow-up visit after convalescence, peptides from rv-a and rv-c species were most frequently recognized whereas rv-b species were much less commonly recognized. interestingly, we found that older children (i.e., children older than years of age) recognized peptides from more rv strains than younger children. this result would indicate that children encounter in their life different rv strains and thus may broaden their igg reactivity profiles later in life but this needs to be confirmed in longitudinal studies with samples taken from the same children at different ages, as has recently been done in the analysis of the evolution of ige reactivity profiles in allergic children in birth cohorts [ ] [ ] [ ] [ ] . one of the important findings of our study was that we could demonstrate that igg reactivity to peptides from certain rv strains increased in the children which may allow identifying the culprit rv species responsible for the acute wheeze by serology. furthermore, it turned out that increases of igg responses to rv-a and rv-c species were significantly associated with more severe illness as compared to igg increases to rv-b. the predicta chip thus seems to be not only suitable for identifying the culprit rv species responsible for an exacerbation of respiratory illness by simple serology but also allows to determine those rv species giving rise to severe symptoms. since we also had results from pcr-based testing of nasal swab samples from the same children we could compare the detection of strain-specific nucleic acid with antibody results. in fact, we found that all children without speciesspecific antibody increases were also negative in the pcr test. however, there was poor correlation between the pcr results from the nasal samples and the chip-based serological results. furthermore, with the exception of four children (# , # , # , # ) for which a positive pcr result has been obtained at the follow-up visit, all pcr results were negative at both the acute and follow-up visit in approximately % of the children for whom species-specific antibody increases could be clearly demonstrated. several possibilities for the discrepancies may be considered. for example, it may be possible that the time interval after acute infection chosen for serology was not optimal. however, we have previously investigated the time interval required for the appearance of vp -specific antibody increases in a controlled infection study and it is therefore very unlikely that the time interval used in our study was too long and responsible for discrepancies between pcr testing and serology. we also do not think that cross-reactivity among strains or the original antigenic sin is responsible for the observed differences because we have taken care to include on the chip sequences from several different rv strains and the results in fig. a clearly show that the serological results obtained with the chip allow a bona fide discrimination of rv-a, rv-b, and rv-c infections because we have used a large panel of rv peptides on the chip. finally, we found that older children recognized more rv peptides from different strains than younger children (fig. c-e) , which indicates that the children develop antibodies against new viruses and thus the concept of the original antigenic sin does not seem to apply here. one limitation of our study is, however, that we cannot exclude that infections with additional rv strains have occurred in the time window between the first and second blood sampling and thus are responsible for the discrepancy between pcr results and serology. nevertheless we think that the discrepancy between pcr results and serology is rather due to the fact that not every virus detected by pcr causes an infection with a consecutive immune response. in fact, studies performed in young children report that up to % of asymptomatic subjects have positive pcr results , . one more likely possibility for the poor correlation of pcr results and antibody results could be that we used a pcr strategy based on primers specific for vp -and vp -encoding regions of the viral genome, which may be less specific than pcr strategies based on the amplification and sequencing of the vp -encoding region or of the complete rv genome , , , . in general, nucleic acid-based strategies for virus detection only demonstrate the presence of virus-specific nucleic acid but provide no evidence that an infection has taken place which gave rise to a specific immune response. we therefore think that the predicta chip and future versions of it containing an even larger repertoire of n-terminal vp peptides from more rv strains will be a complementary tool in addition to pcr testing and eventually turn out to be even superior. the antibody test is actually fast and economical: it takes only few hours and requires only microliter amounts of serum. furthermore, serological analysis is robust and can be easily performed without need for pcr cyclers and subsequent sequencing. however, more prospective studies will be needed to investigate the diagnostic sensitivity and specificity of the rv chip. the predicta chip may be extremely useful to determine rv species-specific antibody responses in serum samples from existing cohorts world-wide to define the most common and relevant rv species involved in respiratory illness. chip-based measurements will allow exploring in prospective studies the role of rv infections in a variety of respiratory disease exacerbations. for example, it will be possible to discriminate whether asthma exacerbations have been triggered by an rv infection or by allergen exposure because it has been shown that both factors (i.e., rv infections as well as allergen exposure) induce increases of specific antibody responses when serum samples collected at the acute visit and during a follow-up visit are compared , , , . the identification of the culprit factors triggering asthma attacks becomes increasingly important in respiratory medicine due to availability of selective treatments of allergic asthma such as anti-ige antibodies and a variety of other biologics such as anticytokine antibodies targeting different forms of asthma , . furthermore, it will be possible to use the predicta chip to study by serological analysis the possible contribution of rv infections in exacerbations of other respiratory diseases such as copd and aco. further studies are also necessary to perform a multiple monitoring of the presence of rv strains and other respiratory viruses by pcr and the immune reaction by serology in close intervals and for extended periods after exacerbation. the reliable determination of the most common rv species involved in triggering severe respiratory illness will ultimately provide a rational basis for the development of rv vaccines and rv species-targeting therapeutic approaches [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in conclusion, we developed and evaluated a high-resolution antibody assay based on micro-arrayed peptides and recombinant antigens from the most common rv strains to identify antibody signatures discriminating rv infections at the levels of different rv species and allowed to point towards the culprit species responsible for the triggering of acute pre-school wheezing. the predicta chip has the potential to be useful for a serological global mapping of rv infections, the identification of rv species involved in triggering different forms of severe respiratory illness, and for paving the road for rv-specific therapeutic and prophylactic treatment strategies, such as vaccines. selection and production of n-terminal vp peptides. vp amino acid sequences of rv strains representing the three rv species (rv-a: n = ; rv-b: n = ; rv-c: n = ) were retrieved from the ncbi database (https://www.ncbi. nlm.nih.gov/) (supplementary table ). multiple sequence alignments of the vp n-terminal peptides (aa - ) were performed using clustalw software available at the embl-ebi website (http://www.ebi.ac.uk/tools/clustalw ) to determine amino acid sequence identities among the peptides. peptide sequences showing sequence identities greater than . % (i.e. differences of ≤ aa) were grouped together into clusters (fig. ) . sequences among the clusters (supplementary tables and , a -a , b -b , c -c ) were re-aligned using genedoc software (http://iubio.bio.indiana.edu/soft/molbio/ibmpc/genedoc-readme.html) and each amino acid mismatch was analyzed regarding physicochemical properties of the amino acids. this procedure led to re-clustering of the peptides (supplementary table , ai-xviii, bi-bix; ci-ciii). from each re-clustered group one representative rv strain peptide was selected for printing onto the chip (fig. b, sup- plementary tables and : rv-a: n = ; rv-b: n = ; rv-c: n = ). an enterovirus-derived peptide was also included (fig. ) . for the set of peptides to be printed a multiple sequence alignment was performed by clustalw and a phylogenetic tree was constructed by the neighbor-joining (n-j) method using mega software (www.megasoftware.net) (fig. c) . the evolutionary distances between sequences were computed using the kimura -parameter model with bootstrap values calculated from replicates. additional peptides spanning the rv vp protein (fig. , supplementary table ) were selected to detect antibodies towards vp epitopes other than the n-terminal portion. the peptides as well as the non-structural b protein from strain (vpg: gpysgepkpksraperrvvtq) were produced by solid-phase synthesis with the -fluorenyl-methoxy carbonyl (fmoc)-method (cem-liberty, matthews, nc, usa and applied biosystems, carlsbad, ca, usa) on peg-ps preloaded resins (applied biosystems). after synthesis, peptides were washed with dichloromethane, cleaved from the resins using ml trifluoroacetic acid (tfa), . ml silane, and . ml h o and precipitated into pre-chilled tertbutylmethylether. peptides were purified by reversed-phase high-performance liquid chromatography in a - % acetonitrile gradient using a jupiter μm proteo Å, lc column (phenomenex, torrance, ca, usa) and an ultimate pump (dionex, sunnyvale, ca, usa) to a purity > %. their identities and molecular weights were verified by mass spectrometry (microflex maldi-tof, bruker, billerica, ma, usa) . for the unsupervised analysis of antibody responses to rv peptides and proteins, unsupervised k-means clustering (k = ) was used to define clusters of peptides with similar antibody response measurements. the k number of clusters was pre-determined in order to match the number of the peptide homology groups. the peptides' amino acid sequences were aligned with a gap open cost of . and a gap extension cost of . . based on the alignment, a homology distance cladogram was built using the neighbor-joining algorithm and bootstrap replicates. the peptides were then color-coded based on the antibody response cluster that they belonged to. data were processed using the clc genomics workbench (clc, clcbio, qiagen, hilden, germany). the heat map representing rv-specific antibody responses was generated by qlucore omics explorer (qlucore, lund, sweden). expression and purification of recombinant rv proteins. recombinant histagged structural (vp - ) proteins from five representative rv strains (rv-a , -a , -a , -b , and -cyp) and mbp fusion proteins containing fragments thereof (vp - ) as well as non-structural ( a, c, a, c, and d) proteins from rv strain were expressed in escherichia coli as previously described , . dna sequences coding for the complete genes or fragments thereof (accession numbers are shown in supplementary table ) were codon optimized for bacterial expression, synthesized with the addition of the ′ sequence coding for a cterminal hexa-histidine tag and cloned into the ndei and ecori sites of plasmid pet b (genscript, piscataway, nj, usa). transformed e. coli bl (de ) cells (agilent technologies, santa clara, ca, usa) were induced with mm isopropylβ-thiogalactopyranoside (iptg) and cells were harvested at time-points of maximal expression. recombinant proteins were purified by nickel-affinity chromatography under denaturing conditions as previously described (qiagen, hilden, germany). refolding of recombinant proteins was achieved by a stepwise dialysis against mm nah po for structural and non-structural proteins and mm tris-hcl, mm nacl, mm edta for mbp fusion proteins, respectively. the purity of recombinant proteins was verified by sds-page followed by coomassie brilliant blue staining and the identity by immunoblotting using a monoclonal mouse anti-his-tag antibody : diluted (cat: dia- , dianova, hamburg, germany). bound antibodies were detected with : diluted alkaline phosphatase-coupled rat anti-mouse igg antibodies (cat: ; bd biosciences, erembodegem, belgium). protein concentrations were determined using bca protein assay kit (thermo fisher scientific, rockford, il, usa). the secondary structure of the proteins was measured by circular dichroism spectroscopy on a jasko j- spectropolarimeter (japan spectroscopic, tokyo, japan) at a protein concentration of . mg/ml in mm nah po . detection antibodies and printing of microarrays. anti-huigg (cat: - - ; jackson immunoresearch laboratories, west grove, pa, usa) and anti-huiga (cat.: ; becton dickinson, franklin lakes, nj) were labeled with dylight (pierce, thermo fisher scientific, rockford, il, usa). customized printing of rv microarrays was done by phadia-thermofisher using immunocap isac (immuno solid-phase allergen chip) technology , . spotting was performed by slow pin mode printing using the aushon printer (aushon, billerica, ma, usa). stock solutions of peptides ( mg/ml) were diluted : in a phosphate buffer, ph . and then used for spotting. antigens were spotted in triplicates on a glass surface coated with an amino-reactive organic polymer, each spot containing - fg of microarray component, corresponding to - attomol. allergens used for the calibration and other control proteins spotted on the microarray are listed in supplementary table . cohort of pre-school children with acute wheeze. serum samples examined in this study were from a cohort of pre-school children who had been admitted to the paediatric emergency ward as a result of acute wheeze, at astrid lindgren children's hospital, stockholm, sweden (table ) . this cohort and the genotyping of rv strains in the nasopharyngeal swab samples of of the children by nested pcr and sequencing have been previously described . a molecular diagnostic platform for the rapid detection of respiratory strains was used in of the children. the following respiratory viruses were found: adenovirus: children; bocavirus: children; coronavirus: children; influenza a/b: child; metapneumovirus: children; parainfluenzavirus: children: rsv: children . for of the children nasopharyngeal swabs had been available for the rv pcr targeting vp / sequences. written informed consent was obtained from the parents or by the legal guardians and the study was approved by the regional ethics committee of karolinska institutet, stockholm, sweden. peripheral blood samples had been obtained within h of presentation in the emergency unit and sera were stored at − °c. in addition to blood samples, nasopharyngeal swab samples were obtained at the acute visit and again at the follow-up visit by the research nurse and stored in the biobank at the department of clinical microbiology, karolinska university hospital . follow-up samples were obtained between and weeks after the initial recruitment (median weeks) at a scheduled visit after recovery. although this study was not planned as a prospective study for the assessment of increases of rv-specific antibodies, the time interval of - weeks was suited for this purpose because we found in an earlier study that increase of rv-specific igg responses emerge days after experimental inoculation . at the follow-up visit, the guardians also filled out a standardized questionnaire concerning the number of days the child had suffered from respiratory symptoms at home (i.e., 'cough and/or wheeze'), use of medication (i.e., β -agonists, inhaled corticosteroids, leukotriene receptor antagonist), as well as about any emergency visits between the acute and follow-up visits. the chip analysis of the anonymized sera was performed with the approval of the ethics committee of medical university of vienna (ek / ) , . microarray-based determination of antibody profiles. microarrays were washed in a washing buffer (phadia-thermo fisher) for min by stirring. after drying by centrifugation ( min, g, rt), µl of serum samples were applied on each microarray and the slides were incubated for h at gentle rocking (rt). for the detection of rv-specific igg and iga antibodies, serum samples were diluted : and : in a sample dilution buffer (phadia-thermo fisher), respectively. microarrays were then rinsed with washing buffer and washed for min as described above. after centrifugation, µl of fluorescence-labeled antibodies ( µg/ml) was added and the slides were incubated min at gentle rocking (in dark, rt). after further rinsing, washing and drying, microarrays were scanned using a confocal laser scanner (luxscan- k microarray scanner, capital-bio, beijing, people's republic of china) and the image analysis was evaluated by microarray image analyzer v . . software (phadia-thermo fisher) . for calibration and determination of background signals, a calibrator serum (i.e., a pool of allergic patients sera, diluted : ) and sample diluent were included in each analysis run. calibration and variability of the microarray. the phadia microarray image analysis software was used to process images, to calculate the mean fluorescence intensities (fi) of triplicate analyses and to calibrate the results. a calibration curve was generated by relating fluorescence intensities obtained by scanning the pre-dicta microarray with allergen-specific antibody levels measured by immunocap. results were reported in isac standardized units (isu) , . background reactivities of fluorescence-labeled α-huigg antibodies towards all microarray components were determined by testing six replicates of a sample diluent alone. for the characterization of the assay variability, one calibrator serum and three normal sera were profiled at : , : , : , and : in order to find a dilution at which the broadest spectrum of reactivity levels were covered. to assess intra-assay (i.e. within experiment) variability, six replicates of four serum samples were used to measure igg reactivities towards all microarray components on the same day. to assess inter-assay (i.e. between experiments) variability, four serum samples were evaluated in experiments conducted on five consecutive days. the evaluation of four different samples allowed determining whether the inter-assay variability was sample-dependent. for each microarray component, the mean isu-g, standard deviation (sd), coefficient of variation (cv % = sd/mean isu-g) across the six replicates and five different experiments, respectively, were calculated for each sample. microarray components were classified according to the isu-g level (> , and averages across all components within these groups on the array were also calculated for each of these quantities. data analysis. initial data processing was performed with microsoft excel. frequencies (i.e., the number of reactive sera) and intensities (i.e., isu-g levels) of peptide-specific igg and iga antibody levels were calculated using ibm spss statistics (version ; ibm corp., armonk, ny, usa). median values of peptidespecific igg levels (isu-g) were calculated using graphpad prism (la jolla, ca, usa). the cut-off for a positive igg reactivity was set at isu-g. the numbers of igg-reactive peptides in fig. d , e were calculated for specific igg levels > isu-g. increases of rv-specific antibody responses were calculated as differences between isu-g values measured at the follow-up (f) and the acute (a) visits (Δisu-g = isu-g f − isu-g a ) followed by subtraction of the double coefficient of variation ( × cv%) calculated from the baseline values (isu-g a ) for each of the antigens. for igg levels of - isu-g and > isu-g, % and . % were determined as cv% for intra-assay variability and subtracted from the data, respectively. rv-specific igm antibodies were not measured because we found in an earlier study that no 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human allergy via the nasal mucosa nasal application of rbet v or non-ige-reactive t-cell epitope-containing rbet v fragments has different effects on systemic allergen-specific antibody responses pharmacological therapy of bronchial asthma: the role of biologicals evolving concepts of asthma development and characterization of a recombinant, hypoallergenic, peptide-based vaccine for grass pollen allergy advances in allergen-microarray technology for diagnosis and monitoring of allergy: the medall allergen-chip hiv microarray for the mapping and characterization of hiv-specific antibody responses subnormal levels of vitamin d are associated with acute wheeze in young children data availability. primary data that support the findings of this study are available from the corresponding author on request. this study was funded by predicta, a fp -funded eu project (no. ), by the fwffunded projects f and p of the austrian science fund, by research grants from biomay ag and viravaxx, vienna, austria, by the swedish research council, the swedish heart-lung foundation, stockholm county council (alf project), the swedish asthma and allergy association´s research foundation, the king gustaf v´s -year foundation, the centre for allergy research at karolinska institutet and the swedish cancer and allergy foundation. supplementary information accompanies this paper at https://doi.org/ . /s - - - . novartis, faes farma, biomay ag, vienna, austria, hal, nutricia research, menarini, meda, abbvie, msd, omega pharma, danone, grants from menarini, outside the submitted work. r.v. reports grants from european union, grants and personal fees from biomay ag, vienna, austria, grants and personal fees from viravaxx, vienna, austria, during the conduct of the study; grants from austrian science fund (fwf), grants and personal fees from biomay ag, vienna, austria, grants and personal fees from viravaxx ag, vienna, austria, outside the submitted work. in addition, r.v. and k.n. are co-inventors in a patent application (pct/at / ) regarding the rhinovirus diagnosis reported in this paper. the remaining authors declare no competing interests.reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/ licenses/by/ . /. key: cord- -zmmnn b authors: lester, sandra; harcourt, jennifer; whitt, michael; al-abdely, hail m.; midgley, claire m.; alkhamis, abdulrahim m.; aziz jokhdar, hani a.; assiri, abdullah m.; tamin, azaibi; thornburg, natalie title: middle east respiratory coronavirus (mers-cov) spike (s) protein vesicular stomatitis virus pseudoparticle neutralization assays offer a reliable alternative to the conventional neutralization assay in human seroepidemiological studies date: - - journal: access microbiol doi: . /acmi. . sha: doc_id: cord_uid: zmmnn b middle east respiratory syndrome coronavirus (mers-cov) is a novel zoonotic coronavirus that was identified in . mers-cov infection in humans can result in an acute, severe respiratory disease and in some cases multi-organ failure; the global mortality rate is approximately %. the mers-cov spike (s) protein is a major target for neutralizing antibodies in infected patients. the mers-cov microneutralization test (mnt) is the gold standard method for demonstrating prior infection. however, this method requires the use of live mers-cov in biosafety level (bsl- ) containment. the present work describes the generation and validation of s protein-bearing vesicular stomatitis virus (vsv) pseudotype particles (vsv-mers-cov-s) in which the vsv glycoprotein g gene has been replaced by the luciferase reporter gene, followed by the establishment of a pseudoparticle-based neutralization test to detect mers-cov neutralizing antibodies under bsl- conditions. using a panel of human sera from confirmed mers-cov patients, the vsv-mers-cov particle neutralization assay produced results that were highly comparable to those of the microneutralization test using live mers-cov. the results suggest that the vsv-mers-cov-s pseudotype neutralization assay offers a highly specific, sensitive and safer alternative method to detect mers-cov neutralizing antibodies in human sera. middle east respiratory syndrome coronavirus (mers-cov) is a zoonotic pathogen that is associated with respiratory virus infections ranging in severity from asymptomatic to severe respiratory illnesses and death. mers-cov was first isolated from a -year-old patient who died with viral pneumonia and acute renal failure in in saudi arabia [ , ] . mers-cov cases have been identified in countries, although all cases outside of the arabian peninsula have been associated with exportations from the arabian peninsula [ ] [ ] [ ] [ ] [ ] . as of february , the world health organization (who) has reported confirmed cases of mers-cov infection globally, resulting in deaths (https://www. who. int/ emergencies/ mers-cov/ en/). genetically, mers-cov most closely resembles severe acute respiratory syndrome coronavirus (sars-cov), a betacoronavirus known as the first coronavirus to cause severe respiratory infections in humans. sars-cov emerged from an animal host in and caused a worldwide outbreak comprising an estimated cases with deaths [ ] [ ] [ ] [ ] . similar to sars-cov, mers-cov infections represent zoonotic transmission events [ ] [ ] [ ] . several sero-epidemiology studies have demonstrated that dromedary camels from the arabian peninsula and north africa have high titres of neutralizing antibodies against mers-cov, which can be detected in camel sera collected over years [ ] [ ] [ ] [ ] . in contrast to the sars outbreak, which was brief, mers-cov cases continue to occur years after its identification. the continuous spread of mers-cov serves as a constant reminder of the propensity of novel human coronaviruses to emerge from zoonotic reservoirs and cause severe disease in humans, and so continuous serological surveillance efforts remain essential for these viruses. the risk factors for camel-human transmission, humanhuman transmission and hospital outbreaks, and the roles of asymptomatic cases in human-human transmission are not fully understood. the clinical symptoms for mers patients overlap with those for other lower respiratory tract infections, further demonstrating the need to develop low biosafety-level laboratory-based diagnostic assays with high specificity and sensitivity. furthermore, countermeasures for the treatment or prevention of mers-cov infection, including vaccines, are unavailable. therefore, methods for measuring the potency and breadth of neutralization antibodies against mers-cov are essential to address these gaps in our knowledge, as well as to strengthen disease surveillance and disease preparedness platforms. the spike (s) protein of mers-cov is a surface glycoprotein that facilitates receptor binding, membrane fusion and viral entry into host cells via attachment to the cellular receptor dipeptidyl peptidase iv (dpp ), thus playing a pivotal role in mers-cov infection of the host cell [ ] . the s protein is a major immunogenic component of covs and is the major target for neutralizing antibodies [ ] [ ] [ ] [ ] . laboratory tests, such as immunofluorescence assays (ifas) and conventional enzyme-linked immunosorbent assays (elisas), are commonly used screening tools for detecting anti-mers-cov serum antibodies. however, these assays often yield false-positive reactions due to cross-reactivity with other common human coronaviruses, and so confirmatory tests such as neutralization tests are utilized [ ] . the most widely used confirmatory serological assays to detect and measure neutralizing antibody responses to mers-cov utilize neutralization of live virus, through either the plaque reduction neutralization test (prnt) or the microneutralization test (mnt) methods. however, due to the highly pathogenic nature of mers-cov and the absence of treatments and vaccines for mers-cov infection, prnt and mnt tests must be conducted under strict bio-containment procedures in a biosafety level (bsl- ) laboratory. this limits epidemiological and pathogenesis studies globally. the development of an alternative method that can be reliably and conveniently used to determine the prevalence of mers-cov antibodies at a population level is crucial to prevent the spread of mers-cov. a safe and convenient way to detect neutralizing antibodies against mers-cov in serum is the use of viral pseudotypes. viral pseudotype particles have proven to be very valuable tools for studying virus entry pathways, evaluating the efficacy of vaccines, serological surveillance and gene therapy studies [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . several reports have utilized mers-lentivirus pseudotyped viruses in sero-epidemiological studies and basic research investigations [ , [ ] [ ] [ ] [ ] [ ] . additionally, vesicular stomatitis virus (vsv) pseudotypes with mers-cov spike glycoproteins have been utilized to investigate the role of the mers s protein in virus-receptor-mediated entry, virus/host tropism and drug screening [ ] [ ] [ ] . however, systematic comparative equivalency analysis between vsv pseudotypes bearing mers-cov spike glycoproteins and the conventional mnt using human sera from confirmed mers-cov patients have not been performed. the purpose of this study was to develop a vsv pseudotypebased neutralization assay to detect mers-cov s protein neutralizing antibodies and calculate its equivalence to traditional neutralization assays, therefore circumventing the use of live virus and the need for widely unavailable high-level containment biosafety facilities. here, we validate a mers-cov neutralization assay using a membraned vsv pseudotype system, where its glycoprotein is replaced with a luciferase reporter gene and its membrane is decorated with mers-cov s proteins. additionally, we perform equivalence testing in comparison with the conventional mnt. we demonstrate that these pseudoparticles are neutralized by sera from mers-cov-infected patients. furthermore, most of the patient sera samples have neutralization activities, and these results were consistent with the neutralization activity that was measured by the conventional microneutralization assay using live mers-cov. these results demonstrate that the mers-cov-s protein pseudotyped vsv particle-based neutralization assay would serve as a safe, reliable and highly specific alternative method to detect mers-cov neutralizing antibodies to be used for future sero-epidemiological studies. baby hamster kidney- (bhk- ) cells were cultured and maintained in dulbecco's modified essential medium (dmem) containing % fetal bovine serum (fbs) (life technologies/gibco), and × penicillin/streptomycin (p/s) (sigma) at °c under a % co atmosphere. vero (african green monkey kidney epithelial) cells were cultured and maintained in dmem containing % fbs and × p/s °c under a % co atmosphere. different vsv-based pseudotypes bearing either mers-cov-s or vsv-g glycoproteins were produced in bhk- cells. the pseudoparticle titrations and neutralization assays were carried out in vero cells. pooled normal human serum (pnhs) was purchased from lee biosolutions and used as a negative control serum. human serum from a single patient with laboratory-confirmed mers-cov infection was used as the positive control serum. the positive control serum was collected from the first imported case of mers-cov in the usa during the case investigation, with a neutralizing titre of . a laboratory-confirmed sars-cov patient serum sample and a panel of human sera with confirmed high neutralizing antibody titres to human coronaviruses e, hku , oc and nl were used in this study to evaluate the vsv-mers-cov-s particle-based neutralization assay for potential cross-neutralization. a total of human sera samples from mers-cov-infected patients in saudi arabia were used to examine equivalences. anti-vsv-g monoclonal antibody was purchased from kerafast, inc. a codon-optimized s gene from the mers-cov florida isolate (genbank accession number: kj . ) was synthesized by genescript and sub-cloned into pcaggs . eukaryotic expression vector. vsv-mers-cov-s pseudoparticles were generated as previously described [ ] . the pseudoparticle titres were determined in vero cells. vero cells were seeded in -well black and white tissue culture-treated plates (perkin-elmer) and were inoculated with µl of pseudoparticles five fold serially diluted in serum-free dmem ( × p/s in triplicate in . after h adsorption, the inocula were removed, replaced with fresh dmem containing % fbs and × p/s, and incubated at °c. at h post-inoculation, luciferase activity was determined using the luciferase assay kit (promega, inc.) according to the manufacturer's instructions. the titre of the vsv-mers-cov-s pseudoparticles was defined as the highest dilution yielding relative luciferase units (rlu). for the titration of vsv-mers-cov-s pseudoparticles, the pcaggs plasmids encoding vsv recombinant g and the empty vector (ev) pcaggs-ev were used to generate vsv-g pseudoparticles and pseudoparticles without heterologous protein (ev) as positive and negative controls, respectively. ten fold serial dilutions of vsv pseudotyped with mers-s (vsv-mers-s) or negative control pcaggs empty vector containing no glycoprotein (vsv-ev) were used to infect vero cells in -well plates. rlu were measured h post-infection. the results are expressed as average rlu±standard deviation (sd) of triplicate wells from an experiment repeated three times with similar results. the error bars indicate the sd. briefly, vero cells were seeded in -well black and white tissue culture-treated plates day prior to infection. human sera were heat-inactivated at °c for min, and diluted as two fold serial dilutions with an initial dilution of : in serum-free dmem containing × p/s. two hundred microlitres of each serum dilution were mixed thoroughly with rlu-equivalent vsv-mers-cov-s pseudoparticles diluted to µl and incubated at °c under a % co atmosphere for h. positive and negative control sera were included with each assay run. in triplicates, μl of the pseudoparticleserum mixtures were transferred onto vero cell monolayers and incubated at °c under a % co atmosphere for h. after adsorption, µl of dmem containing % fbs p/s was added and incubated at °c and % co for h. at h post-inoculation, luciferase activity was determined using the luciferase assay kit according to the manufacturer's instructions. the results are expressed as the percentage neutralization ±sd of three parallel wells from three independent experiments. to calculate neutralization, the luciferase activity from each serial diluted human sera sample was normalized to that from the negative control, pnhs. neutralization was calculated as: (average rlu value of pnhs−average rlu value with test serum/average rlu value of pnhs)× . reciprocal endpoint titres were defined as the highest dilution of the test serum sample that decreased rlu values by % or more. serum samples that failed to show at least an % reduction at the : dilution were considered to be below the limit of detection. the slides were first coated with poly-l-lys and seeded with hek- t cells h prior to transfection. the cells were transfected with µg of pcaggs-mers-cov-s plasmid dna or mock transfection. at h post-transfection, the cells' expression of mers-cov-s was detected by convalescent sera from a mers-cov patient with a known neutralizing titre against live virus as a positive control (mers immune sera; : ), followed by fitc-labelled anti-human iga/igg/igm ( : ) (abcam). dapi stain was used and cells were imaged using a zeiss axioimager microscope at × magnification. all graphs were generated with the graphpad prism software. all results were calculated and presented as the means±sd obtained from duplicate or triplicate independent experiments. pearson's correlation analysis and the bland-altman method were used to assess the comparative analyses between the vsv-mers-cov-s pseudoparticle neutralization assay and the mnt assay. statistical analyses were performed using graphpad prism software. the specimens used in this study were determined to be nonhuman subject research by the centers for disease control and prevention (cdc) institutional review board (irb). to establish vsv-mers-cov-s pseudotype neutralization assays, we generated vsv-mers-cov pseudoparticles with surface s protein and a firefly luciferase reporter gene as previously described [ ] . the expression of mers-cov s proteins was confirmed by immunofluorescence at h post-transfection in hek t cells (fig. a) . it is known that bhk- cells do not have the s-binding mers-cov receptor, dpp , and are therefore not permissible to mers-cov infection, but vero cells do [ , ] . efficient packaging of mers-cov s on the surface of the vsv pseudoparticles and the ability of the pseudoparticles to enter target cells through receptor binding were confirmed by comparing the titrated inoculation of vero and bhk- cells of negative control vsv pseudoparticles containing no viral glycoproteins on their surface (vsv-ev) with that for vsv pseudotyped with mers-cov-s proteins, designated vsv-mers-s (fig. b and c) . as expected, in the vero cells the luciferase activity was proportional to the dilution of the mers-s pseudoparticle inoculum (fig. ) . in comparison to the control pseudoparticles, the mers-s pseudoparticles demonstrated an approximately six fold increase in luciferase activity (fig. ) . neither the mers-s pseudoparticles nor the control pseudoparticles induced high levels of luciferase activity in bhk- cells (fig. ) , further supporting the notion that bhk- cells are resistant to mers-cov infection [ ] . based on the rlu values observed at each serial dilution, it was decided to use a : dilution as the input pseudoparticle dose for further experiments for the mers-cov s protein pseudotyped vsv particle-based neutralization assay. to establish optimal parameters for the mers-cov s protein pseudotyped vsv particle-based neutralization assay, the cell density was optimized for maximum luciferase activity. no significant differences in luciferase activities were observed at - cellsper well, although maximum luciferase activity was reached at cells per well, and so cells per well was selected as the standard assay cell density (fig. s a , available in the online version of this article). next, we investigated the incubation time for optimal luciferase activity. the maximum rlu values peaked at h post-infection and the rlu values decreased with prolonged incubation times (fig. s b ). therefore, h post-infection was determined to be the most suitable infection time for mers-cov-s pseudoparticles. to investigate whether anti-mers-cov human sera neutralized vsv-mers-cov-s pseudoparticles, vsv-mers-cov-s pseudoparticles were preincubated with two fold serial dilutions of pnhs serum (negative control), convalescent sera from a mers-cov patient with a known neutralizing titre against live virus as a positive control (mers immune sera) or anti-vsv-g monoclonal antibody. luciferase activity was reduced when vsv-mers-cov-s was preincubated with positive control serum in a dose-dependent manner (fig. a) . in contrast, preincubation with the negative control pnhs or anti-vsv-g monoclonal antibody did not significantly reduce luciferase activity at any dilution factor, thus demonstrating that vsv-mers-cov-s pseudoparticles were specifically neutralized by serum from an individual with a confirmed prior mers-cov infection (fig. a) . as expected, at any dilution factor, preincubation with anti-vsv-g monoclonal antibody efficiently neutralized vsv-Δg pseudoparticles packaged with vsv-g, although neither the mers-cov positive control serum or the negative control pnhs neutralized vsv-Δg pseudoparticles packaged with vsv-g (fig. b) . neutralization may be assessed as the percentage reduction in infectivity at a single dilution. since our goal is to measure the endpoint neutralization titres of human sera that possibly contain mers-cov neutralizing antibodies, we evaluated the percentage neutralization reduction curve of vsv-mers-cov-s pseudoparticles when preincubated with a mers-cov immune serum with a known live-virus endpoint neutralization titre. the percentage reduction in rlu (% neutralization) was determined by calculating the difference between the average rlu number in triplicate wells with the anti-mers-cov serum and the average rlu number in triplicate wells with the negative control pnhs sample, dividing it by the average rlu number in triplicate wells with the negative control pnhs sample and then multiplying the result by . neutralization of vsv-mers-cov-s pseudoparticles was dependent on the serum dilution, reducing rlu between and % at the dilutions tested (fig. ) . using a target value of % reduction in rlu in comparison to the pnhs control was within the linear range of reduction. our standard positive control mers-cov immune serum crossed the % reduction line between and dilutions. this same serum has a known endpoint titre of approximately in neutralization assays using live virus (unpublished data), demonstrating relative equivalence in the two assays for this serum. we previously measured the endpoint neutralizing antibody titres of human sera collected from mers-cov patients using a live virus mnt. to determine if the vsv-mers-cov-s pseudoparticle neutralization test results in equivalent endpoint titres in comparison to the live virus mnt, we compared the endpoint titres from the live virus mnt to the endpoint titre necessary to achieve an % reduction in rlu with vsv-mers-cov-s pseudoparticles. neutralizing antibody titres ranging from to from human serum samples against mers-cov were successfully detected by both neutralization assays (fig. ) . sixty-seven per cent (n= ) of the serum samples had the same endpoint neutralization titres as those measured by either technique. we observed that approximately % (n= ) of the serum samples gave higher neutralizing titres in the vsv-mers-cov-s pseudoparticle neutralization assay than the conventional mnt. furthermore, five sera with neutralization titres below the limit of detection by mnt had neutralizing titres that were detectable by the vsv-mers-cov-s pseudoparticle neutralization assay, thus demonstrating that the vsv-mers-cov-s pseudoparticle neutralization assay may be more sensitive (fig. ) . the titres from both assays were then analysed statistically. pearson's correlation analysis of all data showed a strong correlation between the two neutralization assays, with r= . , p< . , % confidence interval (ci)= . to . (fig. a) . bland-altman method comparison analysis further demonstrated that these two methods are highly comparable. the average of the logarithmic difference of the two methods was . (fig. b) . collectively, these data indicate that the vsv-mers-cov-s pseudoparticle neutralization assay can be a reliable alternative to the live virus-based mnt. there are six known human coronaviruses that cause respiratory disease in humans. in order to test the specificity of our vsv-mers-cov-s pseudoparticles and their potential cross-reactivity with other common human coronaviruses, we tested serum samples from humans with no known anti-mers-cov exposure. when using the % reduction in rlu as the cut-off value, none of the serum samples neutralized vsv-mers-cov-s pseudoparticles at any dilution factor (fig. s ) . we also tested potential cross-neutralization of vsv-mers-cov pseudoparticles with sera from other humans with confirmed infections with the human coronaviruses e, hku , oc , nl and sars-cov. using the neutralization parameters of % or greater reduction in rlu, we found no cross-neutralization with e, hku , oc , nl or sars-cov anti-sera (fig. ) . these data confirm the specificity of the vsv-mers-cov-s pseudoparticles. mers-cov continues to circulate on the arabian peninsula and can cause severe infections, including fatalities. at present, there are no antiviral therapeutics or approved vaccines to combat mers-cov infections. the presence of serum neutralizing antibodies ≥ is a reliable indicator of prior mers-cov exposure. neutralization assays using live viruses are recognized as the gold standard serological assays to confirm the antibody responses to mers-cov infection. however, one major obstacle to performing the mnt assay is the requirement for access to high-level biocontainment laboratories, due to the use of live mers-cov. thus, only a limited number of facilities and researchers are able to perform such neutralization assays. additionally, determining the neutralization of infectious virus in this assay requires the assessment of cytopathic effect in -well plates, which is labour-intensive and time-consuming. furthermore, because of the large viral genome, reverse genetics for mers-cov is challenging, and still needs to be used under bsl- containment, thereby limiting molecular manipulation and studies. these limitations highlight the need to develop alternative methods for detecting mers-cov neutralizing antibodies. an alternative approach to overcome these shortcomings is the use of viral pseudotypes. generally, lentiviral and vsvbased pseudotypes are used for the study of enveloped viruses. several studies have utilized lentiviral and vsv pseudotype systems harbouring mers-cov spike glycoproteins to study viral entry, viral tropism and other basic science questions [ , , [ ] [ ] [ ] [ ] . in addition to being utilized to probe basic science questions, pseudoparticle-based neutralization assays have been used for serological surveillance of reservoir species for several other zoonotic viruses in addition to mers-cov [ , , [ ] [ ] [ ] [ ] [ ] [ ] . studies have employed lentiviral and env-deficient hiv- backbone vector pseudotype systems for mers-cov seroepidemiological surveillance in humans [ ] [ ] [ ] [ ] [ ] . using dromedary camel sera, hemida et al. demonstrated that lentiviral-based mers-cov s pseudoparticles detected antibodies specific to mers-cov. in our hands, lentiviral particles grew to lower titres than vsv particles, making it challenging to prepare high volumes of stock for use in multiple studies. standardizing many particles for use in multiple studies is important for making comparisons between studies in this context. barlan et al. utilized vsv pseudotyped with mers-cov-s and demonstrated that susceptibility to mers-cov infection is enhanced by cellular proteases that cleave the s protein [ ] . fukuma et al. demonstrated that infection with vsv-based pseudoparticles bearing truncated c-terminal mers-cov-s protein and non-truncated mers-cov-s protein was inhibited by rabbit anti-mers-cov s serum and that infection was dependent on the expression of dpp [ ] . this demonstrated that vsv-based mers-s pseudoparticles can be utilized for neutralization assays. nonetheless, studies featuring seroepidemiological surveillance in humans utilizing a vsv pseudotype system bearing mers-cov spike glycoproteins had not previously been performed. this report describes the first comparison between the neutralization assay using vsv-mers-cov-s pseudoparticles and the mnt using a panel of human sera. this study is limited by only utilizing specimens, which limits its statistical power. although analysing more specimens might yield a more powerful comparative analysis, the complexities of limited availability, diplomatic issues and biosafety concerns make acquiring mers-cov human specimens less feasible. despite this limitation, statistical analyses comparing mnt and vsv-mer-cov pseudoparticle neutralization indicate there was a positive correlation between the two methods. although there was a strong correlation between the two methods, the vsv-mers-cov-s pseudoparticle neutralization assay is slightly more sensitive than the conventional mnt. one potential explanation for this is the surface density of s protein. the generation of pseudoparticles by transient transfection in continuous cell lines, rather than producer cells that are more physiologically relevant, may lead to the production of large amounts of virus with less s protein on its surface. thus, the pseudoparticles may be more susceptible to neutralization because fewer neutralizing antibodies are required to block the entry of mers-cov. however, in corroboration with our data, fukushi et al. previously demonstrated that a neutralization test based on a vsv-based sars-cov-s protein bearing pseudoparticles was also more sensitive than the conventional virus neutralization test using live sars-cov [ ] . in comparison with the mnt method, the pseudoparticle neutralization assay required half as much time to test for mers-cov neutralizing antibodies. more importantly, this assay can be performed in a low-biosafety containment laboratory. in addition to being faster and more sensitive than conventional neutralization assays while maintaining specificity, our vsv-mers-cov-s pseudoparticle system offers a convenient approach for examining the antigenic and neutralizing epitope differences among different s proteins from divergent strains of mers-cov. the data we have presented here use one s plasmid, but the pseudoparticles can be packaged using the spike sequence from other virus strains. because s protein is provided in trans in a pcaggs vector, it can be easily mutated or swapped for any spike protein an investigator is interested in studying. there are existing reverse genetics systems for human coronaviruses, including mers-cov, but those systems are more difficult to generate and require bsl- facilities [ ] [ ] [ ] . using those full-virus reverse genetics systems are essential for viral replication and pathogenesis studies, but for neutralization studies, the presence of spike alone is sufficient. mers convalescent sera did not neutralize vsv particles packaged with vsv-g, demonstrating that neutralization was due to serum antibodies binding specifically to mers s. one of the main concerns in designing a mers-cov neutralization test is the presence of five other known human coronaviruses, four of which cause common infections. it had previously been shown that anti-coronavirus sera crossneutralize amongst similar coronaviruses [ ] . in this study we determined that vsv-mers-cov-s pseudoparticles were not neutralized by antisera from alpha-or betacoronaviruses, further demonstrating the assay's high specificity. in conclusion, we established a neutralization test using a vsv-based pseudotyped virus possessing mers-cov s protein and expressing a luciferase reporter gene. we used the vsv-mers-cov-s pseudoparticle neutralization assay at bsl- containment, improving the safety of mers-cov neutralization assays. additionally, we found the system to be more rapid and sensitive and easier to use than a conventional microneutralization assay. these characteristics make the vsv-mers-cov-s pseudoparticle neutralization assay a more attractive, convenient and suitable assay for mers-cov screening and confirmatory serology testing. it offers a safe, rapid alternative method to monitor the potency and breadth of neutralization antibodies against mers-cov exposure in future seroepidemiological studies. isolation of a novel coronavirus from a man with pneumonia in saudi arabia severe respiratory illness caused by a novel coronavirus middle east respiratory syndrome emerging diseases. soaring mers cases in saudi arabia raise alarms the global spread of middle east respiratory syndrome: an analysis fusing traditional epidemiological tracing and molecular phylodynamics an outbreak of middle east respiratory syndrome coronavirus infection in south korea middle east respiratory syndrome: an emerging coronavirus infection tracked by 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novel vesicular stomatitis virus pseudotype-based assay for detection of neutralizing antibody responses to sars-cov reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus transgene expression in the genome of middle east respiratory syndrome coronavirus based on a novel reverse genetics system utilizing red-mediated recombination cloning cross-reactive antibodies in convalescent sars patients' sera against the emerging novel human coronavirus emc ( ) by both immunofluorescent and neutralizing antibody tests the authors wish to acknowledge financial support from cdc lassi . the authors declare that there are no conflicts of interest. the findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the centers for disease control and prevention. names of specific vendors, manufacturers, or products are included for public health and informational purposes; inclusion does not imply endorsement of the vendors, manufacturers, or products by the centers for disease control and prevention or the us department of health and human services. the specimens used in this study were determined to be non-human subject research by the centers for disease control and prevention (cdc) institutional review board (irb). key: cord- -mzzvmyea authors: shumilak, geoffrey; sligl, wendy i. title: moving past the routine use of macrolides—reviewing the role of combination therapy in community-acquired pneumonia date: - - journal: curr infect dis rep doi: . /s - - - sha: doc_id: cord_uid: mzzvmyea purpose of review: despite advances in diagnostic microbiology and sepsis management, community-acquired pneumonia (cap) remains a significant cause of morbidity and mortality. current recommendations regarding the use of beta-lactams in combination with macrolides published in clinical practice guidelines are variable and based on low-quality evidence that is frequently retrospective, observational, and heterogeneous in nature. while population-based studies have historically suggested improved clinical outcomes with the routine use of macrolide combination therapy in hospitalized patients with cap, emerging evidence from recent randomized controlled trials has challenged this practice. in this article, we discuss the historical rationale and current evidence for combination macrolide therapy in the management of cap. recent findings: recent randomized controlled trials have assessed the non-inferiority of beta-lactam monotherapy compared to beta-lactam/macrolide combination therapy in adult patients hospitalized with cap. beta-lactam monotherapy was associated with equivalent clinical outcomes in patients with mild to moderate cap. patients with severe cap managed with beta-lactam monotherapy have demonstrated worse clinical outcomes when compared to patients treated with combination therapy. in addition, previous beta-lactam exposure prior to hospitalization has not been shown to negatively impact outcomes in patients managed with beta-lactam monotherapy in the hospital. summary: current evidence supports the use of beta-lactam monotherapy in adult patients hospitalized with mild to moderate cap. while existing evidence supports the use of combination therapy in patients with severe pneumonia, further large-scale randomized controlled trials are urgently needed to clarify the role of combination therapy in this population. community-acquired pneumonia (cap) remains a significant cause of morbidity and mortality, resulting in an estimated . million adult hospitalizations annually in the united states [ •, ] . despite advances in sepsis recognition and management and being a core measure by the centers for medicare & medicaid services, the in-hospital mortality of patients admitted with cap remains high at . % with all-cause mortality at days, months, and year estimated to be %, %, and %, respectively [ •, ] . the broad spectrum of pathogens causing cap remains unchanged and includes typical bacteria (streptococcus pneumoniae, haemophilus influenzae, moraxella catarrhalis), atypical bacteria (mycoplasma pneumoniae, chlamydia pneumoniae, legionella spp.), viruses (such as rhinovirus, influenza, parainfluenza, respiratory syncytial virus, coronaviruses, human metapneumovirus) and less frequently staphylococcus aureus, gram-negative bacilli, fungi, and mycobacteria [ ] . while diagnostic techniques to facilitate the accurate identification of causative agents have improved, including the rapid evolution of commercial multiplex pcr assays, an etiologic agent is identified in fewer than half of cap cases and frequently leaves clinicians to rely on empiric antimicrobials for management [ , •] . macrolide antimicrobials are commonly prescribed agents for the management of respiratory tract infections [ ] . as a class, macrolides are bacteriostatic and function by inhibiting bacterial protein synthesis through reversible binding to s ribosomal rna of the s ribosomal subunit, resulting in inhibition of rna-dependent protein synthesis [ ] . however, widespread resistance to macrolides due to point mutations in the s rrna binding site has significantly limited their utility [ , ] . s. pneumoniae resistance to macrolides has demonstrated regional variability with rates ranging from % in north america to over % in vietnam while m. pneumoniae demonstrates similar resistance ranging from % in the united states to over % in regions of japan [ ] [ ] [ ] [ ] . aside from antimicrobial properties, macrolides are also prescribed for their immune modulating effects, particularly in the setting of chronic inflammatory lung diseases. m a c r o l i d e s h a v e b e e n s h o w n t o s u p p r e s s p r oinflammatory cytokines [ ] , decrease polymorphonuclear cell recruitment [ ] , attenuate reactive oxygen species [ ] , and alter key transcription factors [ ] . while several small studies have shown that immune modulation from macrolides may also be beneficial in acute cap by reducing inflammatory mediators, supportive research linked to clinical outcomes remains limited and a clear benefit has not been established [ , ] . a recent effort to characterize causative cap pathogens through the use of modern diagnostic tests has been published in the center for disease control and prevention (cdc) etiology of pneumonia in the community (epic) study [ • ]. the cdc epic study was a prospective, multicenter, population-based active surveillance study of patients with radiographically confirmed pneumonia at five us hospitals between and . in addition to conventional diagnostic tests, extensive molecular and serologic testing was employed to characterize causative pathogens. in this cohort, human rhinovirus, influenza, and s. pneumoniae were the most common etiologic agents. atypical pathogens were infrequently identifiedwith the incidence of m. pneumoniae, c. pneumoniae, and l. pneumophila each representing less than . cases per , adults in the community. this study provides contemporary, comprehensive microbiologic cap data that is essential in informing guidelines and choosing empiric therapies wisely. the low incidence of atypical pathogens is of particular interest. published recommendations regarding the use of beta-lactams in combination with macrolides in current clinical practice guidelines are variable and based on relatively low quality evidence that is frequently retrospective, observational, and heterogeneous in nature. last updated in , the joint infectious disease society of america (idsa)/american thoracic society (ats) guidelines for cap recommend empiric combination therapy with a beta-lactam plus macrolide or monotherapy with a respiratory fluoroquinolone (e.g., moxifloxacin) for adult patients hospitalized with cap in a non-icu setting [ ] . combination therapy is recommended for all patients with severe cap (inpatient icu). in contrast, guidelines published by the british thoracic society (bts, ) and national institute for health and care excellence (nice, ) recommend beta-lactam monotherapy for lowseverity cap with consideration of combination therapy only in moderate to severe cap [ , ] . the body of evidence used to support current idsa/ats guideline recommendations that advocate for combination therapy with a beta-lactam plus macrolide in the management of hospitalized adult patients with cap originates from a series of large, retrospective cohort studies that showed improved clinical outcomes in patients treated with combination therapy. gleason et al. evaluated the relationship between initial antimicrobial therapy and clinical outcomes in a retrospective cohort of , medicare patients hospitalized with pneumonia [ ] . thirty-day mortality was lower when a macrolide was combined with a second-generation cephalosporin (hr, . ; % ci, . - . ) or third-generation cephalosporin (hr, . ; % ci, . - . ) compared to conventional beta-lactam monotherapy. houck et al. similarly evaluated a retrospective cohort of medicare patients hospitalized with pneumonia in western states during the years , , and [ ] . therapy with a macrolide in combination with a beta-lactam was associated with a significant reduction in mortality (aor, . ; % ci, . - . ) in but this effect was not observed during the other years of study. dudas et al. subsequently performed a prospective, observational cohort study at community hospitals to evaluate compliance with ats guidelines in patients with non-severe community-acquired pneumonia and compared outcomes between patients administered varying combinations of antimicrobials [ ] . compliance with ats guidelines was found to be % and the addition of a macrolide to a beta-lactam was associated with decreased mortality and reduced length of hospital stay. brown et al. further reinforced these findings by evaluating a retrospective cohort of , patients in a hospital claims database [ ] . in this cohort, macrolide combination therapy was associated with decreased mortality, length of hospital stay, and healthcare-associated costs overall. based on the findings of these large observational studies, many clinical practice guidelines recommend combination therapy with a beta-lactam plus macrolide or monotherapy with a respiratory fluoroquinolone as first-line therapy for hospitalized adult patients with cap. however, this recommendation for routine combination therapy for the management of cap has remained a contentious issue. despite the body of (predominantly retrospective, observational) evidence that demonstrates improved clinical outcomes, many clinicians regard this evidence (which grounds guideline recommendations) to be low quality and have called for further prospective, randomized-controlled trials to address this question. the routine use of macrolides in hospitalized adults to augment the spectrum of antimicrobial coverage in mild to moderate cap patients to include atypical pathogens has been questioned owing to the infrequent identification of these pathogens. m. pneumoniae and c. pneumoniae are also specifically known for producing relatively mild illness with minimal existing literature that targeted antibiotic therapy impacts patient mortality [ ] . additionally, any adjunctive benefit from the postulated anti-inflammatory effect of macrolides in the treatment of cap remains unproven. given the increasing resistance of s. pneumoniae and m. pneumoniae isolates to macrolide antibiotics, unnecessary exposure to potential harms associated with macrolide use (potential cardiotoxicity, clostridium difficile infection, etc.), and the economic costs of unnecessary combination therapy, many have called the routine use of combination beta-lactams plus macrolides into question [ ] [ ] [ ] [ ] [ ] . additional studies have attempted to bring clarity to this controversy. a systematic review and meta-analysis performed by the cochrane acute respiratory infections group explored if the addition of atypical coverage to an antimicrobial regimen would improve patient outcomes [ ] . in trials, including patients hospitalized with cap, there was no overall difference in mortality (rr . , % ci: . - . ) between the atypical and the typical coverage arms. a trend towards fewer clinical failures was observed in the atypical therapy arm, but this did not achieve statistical significance (rr . ; % ci: . - . ). of note, the cochrane review only included one study that specifically focused on the comparison of beta-lactam monotherapy to beta-lactam/macrolide combination therapy. asadi et al. also compared macrolides to other treatment regimens in adults hospitalized with cap in a systematic review and metaanalysis including studies and , patients [ ] . while overall macrolide use was associated with a statistically significant reduction in mortality when compared to nonmacrolide use, the survival advantage disappeared ( . vs . %; rr . ; % ci: . - . ) when analyses were restricted to rcts. in recent years, researchers have attempted to address the paucity of high-quality evidence with several prospective, randomized-controlled trials aimed at evaluating the utility of combination antimicrobial therapy in hospitalized adults with cap. garin et al. tested the non-inferiority of beta-lactam monotherapy compared with beta-lactam/macrolide combination therapy in moderately severe patients with cap in an openlabel, multicenter, non-inferiority randomized control trial [ ••] . the study was conducted in six acute care hospitals in switzerland from to . the primary endpoint in this trial was the proportion of patients not achieving clinical stability at day . after days of therapy, % in the monotherapy arm and % in the combination arm had not reached clinical stability ( % difference, p = . ), exceeding the predefined non-inferiority boundary. patients identified to have atypical pathogens or severe pneumonia (psi ≥ ) were less likely to reach clinical stability with monotherapy while patients infected with typical pathogens or mild to moderate cap severity (psi to ) had equivalent outcomes. secondary endpoints that included overall mortality, need for icu admission, complications, length of stay, and recurrence of pneumonia within days did not differ significantly between the two groups. postma et al. designed the cap-start trial, a multicenter cluster-randomized, crossover, non-inferiority trial conducted at hospitals in the netherlands to determine if empiric therapy for cap with beta-lactam monotherapy was noninferior to combination beta-lactam/macrolide therapy or a respiratory fluoroquinolone [ ••] . adult patients were included if they demonstrated radiographic evidence of pneumonia and compatible clinical findings. patients were excluded if they had cystic fibrosis, positive legionella antigen, recent hospitalization, or were from a long-term care facility. the unadjusted -day all-cause mortality was . % (betalactam monotherapy), . % (fq monotherapy), and . % (beta-lactam/macrolide combination). adjusted comparisons for -day all-cause mortality favored beta-lactam monotherapy with a . % lower risk of death when compared to macrolide combination therapy ( % ci: − . to . ). time to oral antibiotic de-escalation, length of hospital stay, and occurrence of complications during hospital stay were not significantly different between the groups. while criticisms of this study included a high rate of protocol deviation in the beta-lactam monotherapy group to include atypical coverage, subgroup analyses of strategy-adherent and antibiotic-adherent groups revealed no significant difference in outcomes. van werkhoven et al. further evaluated outcomes in a subset of patients from the cap-start trial that had previous beta-lactam exposure prior to hospitalization [ ••] . thirty-day mortality, hospital length of stay, and frequency of treatment escalations were compared between patients that were continued on beta-lactam monotherapy and patients that received beta-lactams and atypical coverage. despite the betalactam monotherapy group being older, having more medical comorbidities, and longer duration of symptoms prior to admission, the authors found no significant differences between groups with respect to -day mortality and hospital length of stay. while these recent trials performed by garin, postma, and van werkhoven provide evidence to support a strategy of beta-lactam monotherapy for patients presenting with mild to moderate cap, the role of combination beta-lactam/ macrolide therapy in critically ill patients with severe cap remains unclear. the use of macrolide combination therapy in severe cap is endorsed in various clinical guidelines (table ) . again, however, the data to support macrolide use is of relatively low quality and does not clearly explain whether improved outcomes in this population are derived from the antimicrobial or immune-modulatory properties of these agents. baddour et al. previously evaluated the impact of combination antibiotic therapy on adult patients with severe bacteremic pneumococcal pneumonia in a prospective, observational multicenter study [ ] . while overall -day mortality did not differ significantly in this cohort, combination therapy was associated with significantly reduced mortality among a subset of critically ill patients ( . vs . %, p < . ) regardless of nationality, level of icu support, class of antibiotics, and in vitro activity of the prescribed antibiotics. while this study was not limited to combination macrolide therapy, beta-lactam/macrolide therapy remained the most common regimen in the study population. multiple retrospective cohort studies have shown a similar effect in critically ill patients with severe pneumococcal pneumonia complicated by bacteremia [ ] . in addition, sligl et al. explored the impact of beta-lactam/macrolide combination therapy in critically ill patients in a systematic review and meta-analysis involving nearly , critically ill patients from observational studies and found that combination therapy including macrolides was associated with a significant reduction in mortality [ ] . a subgroup analysis of the cohort evaluated by garin et al. also supports this literature-demonstrating that patients with severe pneumonia were less likely to achieve clinical stability with beta-lactam monotherapy. despite the lack of randomized trials in severe cap, the available evidence is consistent with current recommendations published in the joint idsa/ats guidelines for management of cap in patients requiring icu admission. further research to characterize the potential immune-modulatory effects of macrolides in patients with severe cap is warranted, specifically because this population is more likely to have concomitant sepsis and deleterious systemic inflammation. the utility of macrolide combination therapy in the management of cap has been a source of significant controversy with a large body of conflicting evidence. considering the increasing resistance of typical bacterial isolates (specifically s. pneumoniae) to macrolides and the infrequent identification of atypical pathogens as causative agents of pneumonia, clinicians need to carefully consider the utility of macrolides in the treatment of cap. in addition, clinicians need to balance any potential incremental benefit from the anti-inflammatory effect that macrolides may possess against the urgent need for antimicrobial stewardship and responsible antibiotic use. while population-based epidemiologic studies have historically suggested a benefit of adjunctive macrolides in the management of cap, most of these studies are of relatively low quality. emerging evidence from several prospective studies has challenged this practice by failing to show a significant improvement in relevant clinical outcomes in patients hospitalized with mild to moderate cap. while existing evidence still supports the use of combination therapy in critically ill patients with severe cap, further randomized-controlled trials are urgently needed to clarify the role of combination therapy in this population. in particular, the potential immune modulatory effect of macrolides in sepsis remains an area of uncertainty. based on the current literature, we suggest a pragmatic approach pending further high quality evidence to guide therapy. for patients with mild-moderate cap requiring hospitalization, we suggest treatment with beta-lactam monotherapy (e.g., ceftriaxone) or a respiratory fluoroquinolone (e.g., moxifloxacin). for patients with severe pneumonia or those requiring icu-level care, we suggest beta-lactam/macrolide (e.g., ceftriaxone/azithromycin) combination therapy. empiric therapies, however, may differ based on local epidemiology and antimicrobial susceptibility patterns in each individual health region or institution. conflict of interest geoffrey shumilak and wendy sligl declare that they have no conflicts of interest. human and animal rights and informed consent this article does not contain any studies with human or animal subjects performed by any of the authors. prospective population-based cohort study evaluating incidence of cap in consecutive hospitalized patients at all adult hospitals in louisville, kentucky. calculation of annual population-based incidence of cap, mortality at , , and months following hospitalization national hospital ambulatory medical care survey: emergency department summary tables deaths: final for douglas and bennett's principles and practice of infectious diseases. chapter : acute pneumonia the canadian cap working group. summary of canadian guidelines for the initial management of community-acquired pneumonia: an evidence-based update by the canadian infectious disease society and the canadian thoracic society active population-based surveillance study of immunocompetent adults hospitalized with community-acquired pneumonia. in addition to conventional diagnostic testing, extensive molecular and serologic testing was employed to characterize causative pathogens and provide contemporary data on the causative agents of cap. despite extensive testing the value of macrolide-based regimens for community-acquired pneumonia structural basis for the interaction of antibiotics with the peptidyl-transferase centre in eubacteria the mechanism of action of macrolides, lincosamides and streptogramin b reveals the nascent peptide exit path in the ribosome antimicrobial resistance among streptococcus pneumoniae in the united states: have we begun to turn the corner on resistance to certain antimicrobial classes? antibiotic resistance threats in the united states high prevalence of antimicrobial resistance among clinical streptococcus pneumoniae isolates in asia (an ansorp study) macrolide-resistant mycoplasma pneumoniae, united states nationwide surveillance of macrolide-resistant mycoplasma pneumoniae infection in pediatric patients macrolide antibiotics as biological response modifiers effect of erythromycin on haemophilus influenzae endotoxininduced release of il- , il- and sicam- by cultured human bronchial epithelial cells membrane-stabilizing, antiinflammatory interactions of macrolides with human neutrophils molecular mechanisms of anti-inflammatory action of erythromycin in human bronchial epithelial cells: possible role in the signaling pathway that regulates nuclear factor-kappa b activation antiinflammatory effects of adjunctive macrolide treatment in adults hospitalized with influenza: a randomized controlled trial immunomodulatory effects of macrolides during community-acquired pneumonia: a literature review infectious diseases society of america/ american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults bts guidelines for the management of community acquired pneumonia in adults: update diagnosis and management of community and hospital acquired pneumonia in adults: summary of nice guidance associations between initial antimicrobial therapy and medical outcomes for hospitalized elderly patients with pneumonia empiric antibiotic therapy and mortality among medicare pneumonia inpatients in western states antimicrobial selection for hospitalized patients with presumed community-acquired pneumonia: a survey of nonteaching us community hospitals impact of initial antibiotic choice on clinical outcomes in community-acquired pneumonia: analysis of a hospital claims-made database antibiotics for communityacquired lower respiratory tract infections secondary to in children antibiotic treatment of moderate-severe community-acquired pneumonia: design and rationale of a multicenter cluster-randomized cross-over trial the controversy of combination vs monotherapy in the treatment of hospitalized community-acquired pneumonia does empiric therapy for atypical pathogens improve outcomes for patients with cap? azithromycin and the risk of cardiovascular death fluoroquinolone and macrolide exposure predict clostridium difficile infection with the highly fluorquinolone and macrolide-resistant epidemic c. difficile strain bi/nap / empiric antibiotic coverage of atypical pathogens for community-acquired pneumonia in hospitalized adults macrolide-based regimens and mortality in hospitalized patients with community-acquired pneumonia: a systematic review and meta-analysis evaluated the non-inferiority of beta-lactam monotherapy compared with beta-lactam/macrolide combination therapy in moderately severe cap in an open-label, multicenter, noninferiority randomized control trial. failed to meet primary outcome and demonstrate non-inferiority of beta-lactam monotherapy in moderately severe community-acquired pneumonia. subgroup analyses revealed equivalent outcomes in patients with mild to moderate cap (psi - ) but worse outcomes in patients managed with beta-lactam monotherapy and severe cap multi-center cluster-randomized, crossover, non-inferiority trial conducted at hospitals in the netherlands to determine if empiric therapy for cap with beta-lactam monotherapy was non-inferior to combination beta-lactam/macrolide therapy or a respiratory fluoroquinolone. -day all-cause mortality was lowest in the betalactam monotherapy arm. adjusted comparisons for -day all-cause mortality favored beta-lactam monotherapy with a . % lower risk of death when compared to macrolide combination therapy. time to oral antibiotic de-escalation evaluation of outcomes in a subset of patients from the cap-start trial that had previous beta-lactam exposure prior to hospitalization. compared thirty-day mortality, hospital length of stay, and frequency of treatment escalations between patients that were continued on beta-lactam monotherapy and patients that received beta-lactams and atypical coverage combination antibiotic therapy lowers mortality among severely ill patients with pneumococcal bacteremia macrolide therapy is associated with lower mortality in community-acquired bacteremic pneumonia macrolides and mortality in critically ill patients with community-acquired pneumonia: a systematic review and metaanalysis key: cord- - zu e y authors: piñeyro, pablo enrique; lozada, maria inez; alarcón, laura valeria; sanguinetti, ramon; cappuccio, javier alejandro; pérez, estefanía marisol; vannucci, fabio; armocida, alberto; madson, darin michael; perfumo, carlos juan; quiroga, maria alejandra title: first retrospective studies with etiological confirmation of porcine transmissible gastroenteritis virus infection in argentina date: - - journal: bmc vet res doi: . /s - - - sha: doc_id: cord_uid: zu e y background: in , a notification of porcine transmissible gastroenteritis virus (tgev) was made by the national services of animal health of argentina (senasa) to the world organization of animal health (oie). the notification was based on a serological diagnosis in a small farm with a morbidity rate of . % without enteric clinical signs. in order to determine if tgev was circulating before the official report, a retrospective study on cases of neonatal diarrhea was performed. the selection criteria was a sudden increase in mortality in - to -day-old piglets with watery diarrhea that did not respond to antibiotics. based on these criteria, three clinical cases were identified during – . results: all animals that were evaluated presented histological lesions consistent with enteric viral infection. the feces and ultrathin sections of intestine that were evaluated by electron microscopy confirmed the presence of round particles of approximately nm in size and characterized by finely granular electrodense nucleoids consistent with complete particles of coronavirus. the presence of the tgev antigen was confirmed by monoclonal specific immunohistochemistry, and final confirmation of a metabolically-active virus was performed by in situ hybridization to detect a tge mrna encoding spike protein. all sections evaluated in this case were negative for pedv and rotavirus a. conclusions: this is the first case series describing neonatal mortality with etiological confirmation of tgev in argentina. the clinical diagnosis of tgev infections in endemic regions is challenging due to the epidemiological distribution and coinfection with other enteric pathogens that mask the clinical presentation. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. there are five coronaviruses (covs) known to infect swine, and the clinical disease is mainly associated with neonatal diarrhea, but respiratory and neurological signs have also been reported [ ] [ ] [ ] [ ] . porcine transmissible gastroenteritis virus (tgev) and porcine epidemic diarrhea virus (pedv) belong to the coronaviridae family, coronavirinae subfamily, and genus alphacoronavirus [ ] . a new coronavirus genetically distinct from tgev and pedv, porcine deltacoronavirus (pdcov) (genus deltacoronavirus), has recently been associated with enteric disease in pigs [ ] . enteric porcine coronaviruses including tgev, pedv and pdcov are characterized by acute diarrhea and anorexia with rapid dissemination in naïve populations. the severities of clinical diarrhea, vomiting, and anorexia can vary based on the age of the affected pigs [ , ] . without adequate passive lactogenic immunity, the mortality rate in neonatal piglets can reach up to % [ , [ ] [ ] [ ] . etiological diagnosis relies mainly on molecular tools like pcr and serology, as the clinical signs and enteric lesions associated with tgev and pedv are indistinguishable [ , , ] . the epidemiology of tgev is rather complex, and infection in neonates can arise from multiple sources. in addition to swine, it has be documented that cats, dogs, and foxes can host tgev [ ] . the virus can be shed in feces for approximately months, and milk shedding from infected sows can result in vertical transmission. historically, tgev infection has followed a seasonal pattern, becoming more prevalent during winter months perhaps due to increased viral survival in colder temperatures and with less exposure to sunlight. tgev is susceptible to most commercial disinfectants, but resistant to digestive bile and stable at ph [ ] . only one tgev genotype has been described, however differences in pathogenicity among strains has been reported in field outbreaks, although not confirmed by an experimental study [ ] . infection with tgev has two different clinical presentations: epidemic and endemic. in epidemics, tgev enters a naïve herd and all pig categories are affected, particularly piglets that are - weeks old. the duration of the clinical presentation is short, approximately weeks, and in small, farrow-to-finish herd, the infection can be self-limiting [ , ] . endemic disease scenarios, those occurring after the epidemic phase, are observed in farms with incomplete aiao management or in breeding farms that have a continuous flow of naïve gilts. in breeding herds, the varying levels of humoral and lactogenic immunity lowers piglet mortality, but may lengthen the course of the disease [ ] . since the first description provided in the united states [ ] , tgev infections have been reported all over the world. in south america, it has been reported in colombia [ ] , venezuela [ ] , bolivia, and is currently seen in brazil [ ] . in argentina, an episode of high pre-weaning mortality related to isospora suis infection alone or in association with an unknown enteric virus was reported in [ ] . further studies using negative stain electron microscopy demonstrate the presence of viral particles consistent with coronavirus in feces of pre-weaning diarrheic ( . %) and post-weaning ( %) piglets [ ] . a retrospective histopathological study performed on cases of neonatal diarrhea at our laboratory during showed that % of neonatal diarrhea cases had lesions consistent with viral enteritis [ ] . in , a notification of tgev infection was reported by the national services of animal health of argentina (senasa) to the world organization of animal health (oie). it was detected by serology in a small farm with an apparent morbidity rate of . % without clinical signs [ ] . this is an unusual presentation of tgev infection and might be related to passed infection or interspecies transmission [ ] . in order to clarify the situation prior to the first official report of tgev infection in argentina, a retrospective study was performed on cases suspected of tgev-like disease recorded at the laboratory of special veterinary pathology at the college of veterinary sciences, la plata university. benchmarking analyses of epidemiological behavior and clinical histories were the criteria for herd selection. etiological diagnosis was confirmed by electron microscopy (em), immunohistochemistry (ihc), and in situ hybridization (ish-rna) of archived paraffin blocks. clinical, pathological and etiological findings case thirteen -to -day-old piglets with body weights ranging from to . kg were submitted for pathological investigation. pigs with clinical diarrhea were dirty, wet, had stained perinea, and showed moderate dehydration characterized by sunken eyes and diffusely pale mucosa. their small intestinal walls were thin with unremarkable mesenteric lymphatic vessels, and were distended by gas or occasionally contained yellow watery digesta with a ph of - ( fig. a) . their stomachs were empty or contained floccules of undigested milk. no other macroscopic findings were observed. the histopathological evaluation showed a shortening of intestinal villi with a crypt-villous ratio of : ( fig. b) , that villi were fused and lined by vacuolated cuboidal or attenuated epithelium, and that the lamina propria was expanded by moderate edema. microscopic lesions were limited to the jejunum and ileum. immunohistochemistry and ish-rna against tgev showed strong staining in the epithelium of sections that presented minimal epithelial villous changes (fig. c, d) . conversely, sections with the most severe epithelial damages were ihc negative. all sections evaluated in this case were negative for pedv and rotavirus. a gross evaluation of five piglets between and days old showed marked dehydration, wet and stool-stained perinea, and poor body conditions ( . kg average body weight) (fig. a) . the small intestine contained abundant yellow-watery diarrhea with a ph of - in two pigs and alkaline ph in three pigs (fig. b) . a histopathology examination of multiple sections of jejunum and ileum showed mild to moderate villous shortening and fusion (fig. c) . the villous enterocytes showed a marked cytoplasmic vacuolization (fig. d) . the lamina propria was minimally infiltrated by lymphocytes and plasma cells, and was expanded by edema. the superficial villous enterocytes in the ileum showed strong hybridization signals characterized by active-replicating tgev (fig. e) . no microscopic lesions were seen in the sections of colon. all sections evaluated in this case were negative for pedv and rotavirus a. in the ultrathin sections, rounded particles measuring approximately nm in diameter were located in the cytoplasm of the intestinal epithelial cells. the particles were characterized by finely granular electrodense nucleoids with electron lucent centers compatible with complete particles of coronavirus (fig. f ) . three neonatal piglets that died naturally presented with fecal stained perinea and were markedly dehydrated. the stomachs were empty, the small intestinal wall was thin/translucent, and scant yellow watery contents were sometimes apparent. the histopathology examination of the jejunum and ileum showed moderate villous fusion and shortening, and the villi were lined by low-cuboidal to flattened/attenuated epithelium (fig. a) . the lamina propria was infiltrated by numerous lymphocytes and plasma cells, was expanded by edema, and exhibited lymphangiectasia (fig. b) . the tgev antigen was detected by ihc in multiple sections of the jejunum and ileum (fig. c) . no significant lesions were observed in the section of colon. all sections evaluated in this case were negative for pedv and rotavirus a. the epidemiological and clinical presentations of outbreaks of neonatal mortality associated with enteritis and the detection of tgev started in the gestation units. both gilts and sows showed anorexia, diarrhea, and vomiting before enteric signs were observed in neonatal piglets. however, the prevalence of clinical signs in the breeding stock was low and no mortality was reported. when tgev enters in a naïve herds, an epizootic form characterized by a % mortality of pre-weaning piglets due to diarrhea and dehydration is normally observed [ , ] . although in the present study, the farms had a high prevalence of diarrhea in suckling pigs, only farms a and c showed almost % neonatal mortality, while in farm b had approximately % neonatal mortality. the clinical presentation and epidemiological pattern observed in farm b resembled the tge endemic form. although no other etiological diagnosis was confirmed, the low mortality associated with tgev that was observed in farm b might be the result of previous exposure to prcv. prcv infection can confer cross-protection against tgev, reducing the enteric clinical signs and pre-weaning mortality [ ] . therefore, herds concomitantly infected with prcv and tgev develop less severe clinical signs, making the clinical differentiation from other enteric infections such as rotavirus or e. coli infections more challenging [ , ] . another potential reason for the low mortality rate due to tge infection that was observed in farm b could be the intermittent viral exposure of the breeding stock that provided partial immunity to the neonatal piglets [ ] . in all herds in this study, it was suspected that the virus entered the farms through the subclinically infected replacement animals although the pre-weaning mortality rate in farm b was lower than that in farm a and c, the pre-weaning mortality was higher in this farm than the mortality rate seen in similar production systems due to other enteric causes such us i suis [ , ] , c. perfringes type a [ ] , or c. difficile [ ] . according to a previous study, the pre-weaning mortality due to diarrhea should not exceed % [ ] . usually, in porcine covs infections, the course of the clinical disease is short and normally does not exceed - weeks [ ] due to the establishment of a rapid herd immunity, as early as one week post-infection. however, in farm b the clinical presentation persisted for approximately months. potential reinfection due to poor husbandry and incomplete aiao management are just few potential causes of viral persistence in the environment that can predispose reinfection of the farrowing units. in clinically affected litters, most of the pigs are dirty, wet, and dehydrated, with diminished body weights. diarrhea is watery, yellowish, and with an acidic smell from the presence of undigested milk [ ] . the mortality rate is inversely correlated with the age of the piglets, reaching % in - day-old piglets. this predisposition is due to the slow replacement rate of villus epithelial cells (around days) in neonatal piglets compared with the replacement rate of -week-old piglets (around - days) [ ] . in this study, mortality varied from to %. the jejunum and ileum are the target segments of the small intestine for virus multiplication. however, tgev infection is segmental, so multiple segments should be included for histopathology or etiological diagnoses in situ such as ihc or ish-rna. due to the retrospective nature of this study, fixed tissue was used to confirm the presence of tgev and the morphological changes consistent with viral infection in the intestinal mucosa. it is important to highlight that although tgev was detected by different means in each farm, due to the segmental nature of the lesions and viral distribution, viral arn or viral antigen was not detected in all of the intestinal segments that were evaluated. in piglets that are less than weeks old, the reduction of villus length and the fusion of jejunum and proximal ileum are the main histological changes [ ] . the normal villous/crypt length ratio is : , however, after - h post-infection, the villous/ crypt ratio can be reduced to : or : [ ] . since tgev replicates in mature absorptive epithelial cells [ ] , a false negative diagnosis can be observed by ihc or ish in specimens that display villous shortening. few absorptive vacuolated cells are seen normally in the intestine, however, during tgev infection, they are found in great numbers with a cuboidal shape [ ] . in endemic tgev infection, histopathological diagnosis is more difficult because only % of pigs display typical tge lesions. in addition, immunofluorescence tests and ihc often fail in endemic farms due to a low number of enterocytes in the tgev-infected because of partial protection conferred by colostral antibodies [ , ] . different diagnostic techniques have been used to detect tgev infection such as ihc, ish, electron microscopy, and immunoelectron microscopy and pcr [ ] ; however, histopathology remains the most useful tool for screening diagnosis [ ] . in this study, although all cases were selected using clinical features and epidemiological information, the histological evaluation consistently showed lesions compatible with viral infection. the application of ihc and ish-rna on archived paraffin blocks from cases of neonatal diarrhea with high morbidity and mortality allowed retrospective identification of tgev infection. diagnosis of tgev infection in endemic regions, such as in argentina, is complicated due to the epidemiological distribution and clinical signs that might be masked with other enteric infections. further studies are necessary to determine the true prevalence of this pathogen and the correlation with neonatal enteric cases observed in confined production systems. case selection was based on the clinical history reported by the farm managers and referring veterinarians. the selection criteria was a sudden increase in mortality that included, significant increment of more than sd from average pre-weaning mortality and last for a period of a week. in addition reported mortality should be associated with the presence of watery diarrhea in -to -day-old piglets that did not respond to antibiotics. first screening of cases was done by histopathological evaluation, and only cases presenting features of viral enteritis with no other detected pathogen were included. based on these criteria, three clinical cases were identified from to . a -sow farrow-to-finishing herd located in buenos aires served as the first case. the farm produced its own replacement breeding stock, however, two months before the outbreak, gilts were introduced from a breeding company. the parity of the breeding stock was distributed as % gilts while the rest of the reproductive stock parity varied from to . on september , approximately % of the pregnant sows presented acute vomiting while % of the pregnant sows presented acute diarrhea. during the period when the sows showed gastro-enteric clinical signs, -to -day-old piglets presented vomiting ( - %) and diarrhea ( %), and the mortality rate of suckling pigs reached %. the course of the disease in both breeder stock and piglets lasted for approximately three weeks. case involved a one-site herd of sows with its own replacement gilts and the following parity distribution: % gilts, % parity - , and . % distributed amongst parity - . boars were purchased from a breeding company and were incorporated into the reproductive herd without quarantine. the farm was located within a few miles of a swine slaughterhouse in buenos aires. in february , the pregnant sows showed anorexia ( - %) and diarrhea ( %) associated with heat returns and abortions ( . %). in the farrowing houses, approximately % of the lactating sows presented with anorexia. pre-weaning mortality associated with the presence of diarrhea varied from . % at the beginning of the outbreak to . % to weeks after the initial clinical signs. an anatomopathological evaluation showed that . % of the total pre-weaning mortality was due to diarrhea. a one-site herd of sows was the subject of case . in july , two boars were located close to the gestation unit. a week later, gestation sows showed anorexia ( . %) and diarrhea ( . %). thereafter, in the gilts, diarrhea was evident in the nursery ( - %) and fattener ( - %). two-day-old piglets showed watery diarrhea ( %) with a mortality rate of %. affected piglets died from severe dehydration within two days of the onset of clinical signs. the course of the disease lasted approximately two months with an overall pre-weaning mortality of % during that period. at the onset of the outbreak, clinically affected suckling pigs were submitted for postmortem examination. tissue samples from different organs, including multiple segments of small intestine (duodenum, jejunum, and ileum) and large intestine, were fixed in % buffered formalin, processed for routine histopathologic examination, and stained with hematoxylin and eosin. etiological diagnosis in tissues and feces by immunohistochemistry, in situ hybridization, and electron microscopy immunohistochemestry ihc was carried-out briefly to differentiate tgev [ ] from pedv [ ] . monoclonal antibodies against tgev (osu: #. e - c) and pedv (osu c ) at dilutions of : were used. antigen retrieval was performed with humid heat and revealed with peroxidase (novocastra a , leica biosystems, il, usa). to rule out other potential viral enteritis, rotavirus a was evaluated in all sections by ihc [ ] . rotavirus ihc was performed using a monoclonal antibody against rotavirus a (santa cruz: sc- ) at a : dilution. antigen retrieval was performed with epitope retrieval solution for min, as programmed on leica bond iii, and revealed with powervision poly-hrp anti-mouse (leica pv ). all samples were tested in duplicate and sections were controlled appropriately for tgev, pedv and rotavirus a with positive and negative controls (additional file : figure s ). in situ hybridization ish-rna was developed through the rnascope platform (advanced cell diagnostics, inc., ca), targeting the specific reverse complementary nucleotide sequence of the tge viral mrna ( - region of spike gene, genbank: kc . ). therefore, positive hybridization signals represent a metabolically-active virus characterized by the tge mrna encoding spike protein. unstained paraffin tissue sections were processed as previously described [ ] . briefly, tissues were deparaffinized and treated with hydrogen peroxide at room temperature for min. the slides were hybridized using a hybridization buffer, and sequence amplifiers were added. the red colorimetric staining detected the tge hybridization signal, and counterstaining occurred with hematoxylin. representative sections of small intestine were fixed in . % glutaraldehyde and % paraformaldehyde in . m, ph . phosphate buffer (pbs) and post-fixed in % osmium tetroxide in pbs. after dehydration in an alcohol series, the fragments were embedded in epoxy resin, quetol (nisshin em co., ltd., tokyo). ultrathin sections were cut, double-stained with uranyl acetate-lead citrate, and observed under a jem- ex (jeol co. ltd., tokyo). diseases of swine new variant of porcine epidemic diarrhea virus hemagglutinating encephalomyelitis coronavirus infection in pigs porcine respiratory coronavirus: molecular features and virus-host interactions a comparative sequence analysis to revise the current taxonomy of the family coronaviridae pathogenicity and pathogenesis of a 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analysis platform for formalin-fixed, paraffin-embedded tissues additional file : figure s . panel of pathogens used as control for detection of tgev by immunohistochemistry. row one include immunostaining of tgev clinical cases against tgev, pedv, and rotavirus specific antibodies. a moderate to severe immunostaining is observe only reacting against tgev. in row two include positive controls for each pathogen detected by immunohistochemistry. row three present section tested negative by pcr for tgev, pedv, and rotavirus that were used as negative control of the immunohistochemistry techniques. ( the authors declare that all the data supporting the findings of this report is available in the article.authors' contributions pep, cjp, maq performed histological examination, data analysis and conclusion and were the major contributors in writing the manuscript; dmm, performed ihc; fv, performed ish; mil, rs, jac, emp, aa, lva field data collection and case identification. all authors read and approved the final manuscript.ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -z xbn authors: namvar, ali; bolhassani, azam; javadi, gholamreza; noormohammadi, zahra title: in silico/in vivo analysis of high-risk papillomavirus l and l conserved sequences for development of cross-subtype prophylactic vaccine date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: z xbn human papillomavirus (hpv) is the most common sexually transmitted infection in the world and the main cause of cervical cancer. nowadays, the virus-like particles (vlps) based on l proteins have been considered as the best candidate for vaccine development against hpv infections. two commercial hpv (gardasil and cervarix) are available. these hpv vlp vaccines induce genotype-limited protection. the major impediments such as economic barriers especially gaps in financing obstructed the optimal delivery of vaccines in developing countries. thus, many efforts are underway to develop the next generation of vaccines against other types of high-risk hpv. in this study, we developed dna constructs (based on l and l genes) that were potentially immunogenic and highly conserved among the high-risk hpv types. the framework of analysis include ( ) b-cell epitope mapping, ( ) t-cell epitope mapping (i.e., cd (+) and cd (+) t cells), ( ) allergenicity assessment, ( ) tap transport and proteasomal cleavage, ( ) population coverage, ( ) global and template-based docking, and ( ) data collection, analysis, and design of the l and l dna constructs. our data indicated the -epitope candidates for helper t-cell and ctl in l and l sequences. for the l and l constructs, combination of these peptides in a single universal vaccine could involve all world population by the rate of . % and . %, respectively. in vitro studies showed high expression rates of multiepitope l (~ . %) and l (~ . %) dna constructs in hek- t cells. moreover, in vivo studies indicated that the combination of l and l dna constructs without any adjuvant or delivery system induced effective immune responses, and protected mice against c tumor cells (the percentage of tumor-free mice: ~ . %). thus, the designed l and l dna constructs would represent promising applications for hpv vaccine development. www.nature.com/scientificreports www.nature.com/scientificreports/ charge and secondary structure. at first step, the conserved region sequences were analyzed by bepipred- server to predict potential b-cell epitopes (table ). in l protein, l [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (eatvylppvpvskvv-type ), l - (pppggtledtyrfv-type ) and l - (nfgvppppttslvd-type ) epitopes had the best b cell epitope identification scores. for l protein, l [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (kqsgtcppdvvpkv-type ), l - (psdpsivslveets-type ), l - (epvgptdpsivtli-type ) and l [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (glgigtgsgtggrt-type ) epitopes showed the highest epitope identification score between their own protein sequences. since a linear form of t-cell epitopes are bound to mhcs, the interface between t-cells and ligands can be accurately modeled. in this study, we used three different algorithms (published motifs, ann and quantitative matrix) for mhc-i and two algorithms for mhc-ii (ann and quantitative matrix). prediction of mhc-i. at first step, the l and l conserved regions were analyzed to find the most immunodominant peptides using netmhcpan . , syfpeithi and propred i. in each protein, peptides with the highest binding affinity scores were determined as high-potential ctl epitope candidates (tables and ). the analysis showed that l [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (ylppvpvskv-type and ylpppsvarv-type ), l - (dqfplgrkfll-type ) , l - (dqyplgrkflv-type ), l [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (krasatqlyk-type and krasvtdlyk-type ), l - (dpdfldivalhr-type ) and l - (dsdfmdiirlhr-type ) epitopes had the highest binding affinity among their own protein sequences. in general, the results of three different algorithms confirmed each other. conservancy and allergenicity analyses were done on the selected epitopes. the sequence of all the epitopes were well conserved among high-risk hpv types and none of them were allergens (tables and ). in addition, there was no cross-reactivity between peptide and human proteome. in this study, we used netmhciipan and propred servers for mhc-ii epitope identification analysis (table ). since a suitable t-cell epitope should be predicted to bind to different hla alleles, epitopes with the maximum number of binding hla-dr alleles were selected as high-potential helper t-cell epitope candidates. among predicted epitopes, l [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (eatvylppvpvskvv-type ) , l - (tqrlvwacvgvevgrgq-type and tqrlvwacagveigrgq-type ), l - (dtyrfvtsqaiacqk-type ) , l - (dtyrfvqsvaitcqk-type ), l - (dpsivtliedssvvtsgap-type ) , l - (pdfldivalhrpaltsr-type ) and l - (sdfmdiirlhrpaltsr-type ) had the highest scores of binding affinity. also, the sequence of all the epitopes were well conserved among high-risk hpv types and none of them were allergen (tables and ) . also, there was no cross-reactivity between peptide and human proteome. population coverage analysis. hla distribution varies among the diverse geographic regions around the world. thus, while designing an effective vaccine, population coverage must be taken into consideration to cover the maximum possible populations. in this study, population coverage was estimated separately for each putative epitope in specified geographic regions of the world (tables and ) . for ctl epitopes, the highest population coverage of world's population was calculated for l a clear band of ~ bp and ~ bp on agarose gel for l and l , respectively (data not shown). the recombinant endotoxin-free plasmids (i.e., pcdna-l and pcdna-l ) had a concentration range between . and . mg/ml. into the eukaryotic cell line (hek- t) was performed by turbofect as a transfection reagent. the levels of dna expression were evaluated using fluorescence microscopy and flow cytometry at h post-transfection. the data indicated that pegfp-l and pegfp-l can effectively penetrate into hek- t cells in vitro. the cellular uptake of the l and l genes into the hek- t cells was ~ . % and ~ . %, respectively. the delivery of pegfp-n as a positive control was detected in approximately ~ . % of hek- t cells (fig. ) . moreover, the spreading green regions were observed for l and l dna delivery using turbofect carrier by fluorescent microscopy in hek- t cells. on the other hand, western blot analysis indicated the successful expression of l and l proteins fused to gfp (i.e., l -gfp and l -gfp) using anti-gfp antibody. the data indicated the clear bands of ~ , ~ and ~ kda for l -gfp, l -gfp and gfp, respectively using dab substrate (fig. ). to evaluate the prophylactic effects of the designed l and l dna constructs, tumor growth and survival percentage were assessed in all groups for days after challenging with c tumor cells. as shown in fig. a , all test groups immunized with dna constructs (g , g & g ) demonstrated significantly lower tumor growth than that in control groups (pbs and empty vector, g & g , p < . ). our data showed progressive tumor growth in control groups on approximately - days (survival rate or tumor-free mice percentage: %). it was interesting that groups vaccinated with l dna, l dna and l + l dna constructs similarly reduced the tumor growth (p > . ). as shown in fig. b , group vaccinated with the mixture of l + l dna constructs showed a higher survival rate (g , ~ . %) than l and l dna constructs, alone (g & g , ~ . %). antibody assay. the levels of total immunoglobulin g (igg), igg a and igg b in mice immunized with the mixture of l + l dna constructs (g ) were significantly higher than other groups (p < . , fig. a ,c,d). moreover, our data showed that the levels of igg were similar in all groups vaccinated with dna constructs (g , g & g , p > . , fig. b ). there are no significant differences in the secretion of igg a and igg b isotypes between groups receiving the l and l dna constructs, alone (g & g , p > . , fig. c ,d). no significant anti-(l + l ) antibody responses could be detected in the sera of control groups, thus, the seroreactivities were completely l + l antigen-specific responses in mice. cytokine assay. the results of cytokine assay in each group showed that the levels of (l + l )-specific ifn-γ, il- and il- secretions in groups immunized with l (g ), l (g ) and l + l (g ) dna constructs were significantly higher than control groups (p < . , fig. www.nature.com/scientificreports www.nature.com/scientificreports/ fig. ) . furthermore, our data indicated that the ratios of ifn-γ/il- and ifn-γ/il- were higher in all test groups as compared to control groups; therefore, they could trigger th immune response. granzyme b secretion. the secretion of granzyme b in all test groups was significantly higher than the control groups (p < . , fig. ). the group immunized with the l + l dna construct (g ) produced significantly higher concentrations of granzyme b than other groups (g & g , p < . ). the level of granzyme b in group receiving l dna construct was similar to that in group receiving l dna construct (p > . ). in recent years, development of bioinformatics tools applied in vaccine researches could potentially save time and resources. indeed, the immunoinformatics tools help to identify antigenic domains for designing a multi-epitope vaccine. with sequence-based technology advancement, now we have enough information about the genomics and proteomics of different viruses . thus, using various bioinformatics tools, we can design peptide vaccines based on a neutralizing epitope. for example, in silico design of an epitope-based vaccine against human immunodeficiency virus , , coronavirus , dengue virus , and saint louis encephalitis virus has already been reported. while around high-risk hpvs were recognized, current vaccines just protect humans from few types. an important limitation of the current vaccines is their narrow coverage. the accessibility of fully sequenced proteome from high-risk hpv strains provides a prospect for in silico screening of reliable peptide-based therapeutic vaccine candidates among billions of possible immunogenic peptides. in silico approaches are intended to reflect the possibilities for overcoming the above-mentioned difficulties in hpv multi-type vaccine. gupta and coworkers designed prophylactic multiepitopic dna vaccine using all the consensus epitopic sequences of hpvs l capsid protein. they also evaluated how engineering cpg motifs by bioinformatics tools could increase immunogenicity of dna vaccines . hosseini et al. applied in silico analysis of l and l protein of hpv , , , and types to identify universal peptide vaccine in order to protect against mentioned types . in , singh et al. analyzed e , e , e and e proteins of high-risk hpv types to identify cd + t-cell epitopes. they suggested a pool of peptides ( to amino acids) to provide the protection against high-risk hpv types . www.nature.com/scientificreports www.nature.com/scientificreports/ panahi and colleagues used a two-step method (consist of molecular docking and sequence-based approach) to determine immunogenic epitopes for induction of immune system against the oncoproteins of hpv , , and types . in this research, we designed a framework for the comprehensive analysis of l and l conserved regions of high-risk hpv types containing both mhc-i and mhc-ii epitopes. the framework begins with conservancy analysis of all high-risk hpv strains following with ( ) b-cell epitope mapping, ( ) t-cell epitope mapping (cd + and cd + ), ( ) allergenicity assessment, ( ) tap transport and proteasomal cleavage, ( ) population coverage, ( ) global and template-based docking and ( ) data collection, analysis, and design of the l and l dna constructs. for experimental analysis, the final l or l dna constructs were cloned into mammalian expression vector with green fluorescent tag (pegfp vector) and their expression was evaluated in the eukaryotic cells using flow cytometry, fluorescent microscopy and western blotting. moreover, the l /l -specific antibody and t-cell immune responses induced by l and l dna constructs were assessed in mouse tumor model. at first, l and l sequences obtained from high-risk hpv types were aligned using muscle algorithms. conservancy analysis showed that five regions of hpv , l protein ( - , - , - , - and - ) and four regions of hpv , l protein ( - , - , - and - ) were more conserved among other subtypes and could be analyzed as an immunoinformatics input. in b-cell epitope prediction, l [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , l - , l - , l - , l - , l - and l - had the highest epitope prediction scores. unfortunately, a reliable method for prediction of b-cell epitope has not been revealed up to now and the sensitivity and specificity of existing methods were very low (the specificity and sensitivity of this method were . and . , respectively). in the case of t-cell epitope prediction, in silico analysis has been significantly improved, thus, the results are more reliable. in this study, for mhc-i epitopes, l - (ylppvpvskv-type and ylpppsvarv-type ), l - (dqfplgrkfll-type ), l - (dqyplgrkflv-type ), l - (krasatqlyk-type and krasvtdlyk-type ), l - (dpdfldivalhr-type ) and l - (dsdfmdiirlhr-type ) epitopes had the highest binding affinity scores. in addition, above-mentioned epitopes had the highest t-cell epitope prediction scores which were obtained from proteasomal cleavage and tap transport analysis. high degree of conservancy was observed between subtypes for these epitopes ( [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (ffgglgigtgsgtggr-type ) epitopes had the highest binding affinity scores. among them, l - had the greatest degree of conservancy (high similarity with all of the high-risk hpv types). one of the remarkable points is that l [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and l - epitopes are the same (or overlapping with little difference (among b-cell and mhc-ii selected epitopes. due to a limitation of mhc-peptide binding prediction such as the gap between the peptides that are predicted to bind to mhc and those that experimentally bind www.nature.com/scientificreports www.nature.com/scientificreports/ been employed to address this problem and raise the accuracy of mhc-peptide prediction. in the current study, template-based docking and also global docking were performed on the selected peptides to determine which peptide would get into the groove of mhc with the highest modeling scores. for mhc-i epitope, l - , l - and l - sequences had the highest interaction similarity and cluster density scores. for mhc-ii epitopes, l - , l - , l - and l - sequences had the highest docking scores. in this study, mhc-i-peptide docking scores confirmed mhc-i-peptide binding affinity scores because the same epitopes had the highest scores in both methods but in mhc-ii molecular docking, the results were slightly different. one of the reasons is the significant conformational changes during the process due to the longer epitope length. as a general rule: the longer the length of the query peptide, the more torsions and conformational flexibilities . herein, due to longer peptide sequences, docking results in mhc-ii were less accurate than mhc-i. for example, average similarity score in mhc-i was variable ( . - . ), but in mhc-ii was . - . after the completion of the analysis and according to all of the above-mentioned parameters, two separate constructs were designed. in addition, accumulative population coverage of helper t-cell and ctl epitopes for the designed constructs www.nature.com/scientificreports www.nature.com/scientificreports/ were estimated. for the l and l constructs, the combination of epitope candidates for helper t-cell and ctl in a single universal vaccine could involve all world population by the rate of . % and . %, respectively (fig. ) . in previous studies, ylppvpvskv (hpv l ) and krasvtdlyk (hpv l ) have been reported as potentially immunogenic epitopes. the ability of in vitro expression of the designed l and l dna constructs was determined in hek- t cells using flow cytometry and western blot analysis. the transfection efficiency of the l and l dna constructs was ~ . % and ~ . %, respectively indicating their high potency for delivery into the eukaryotic cells. as known, the use of a polytope dna vaccine containing multiple t-cell and b-cell epitopes is an attractive strategy for developing a therapeutic and prophylactic vaccine against hpv infections. after in vitro assay, immunological experiments were performed in mice to determine the efficiency of the designed l and l dna constructs without the use of adjuvant or delivery system for vaccine development. similarly, some studies used the pcdna vector harboring the gene of interest for immunization without any adjuvant , . our data indicated that the groups immunized with l , l and l + l dna constructs increased antibody and t-cell responses as compared to control groups. furthermore, the (l + l )-specific immunity in mice receiving the mixture of l + l dna constructs (g ) resulted in higher secretion of total igg, igg a, igg b, ifn-γ, il- and il- cytokines as well as granzyme b than other groups. the higher levels of igg a and igg b as well as ifn-gamma (as a th cytokine) in this group drive t-cell responses toward th -type immunity. the studies showed that immunoglobulin g (igg ) is related to a th -type response, while a th response is associated with the induction of igg a and igg b in mice . regarding to our observations in protective studies, this regimen (l + l dna construct: g ) could confer further protection against c tumor-challenged mice (survival rate: ~ . %) depending on stimulation of cd + t cell-dominated th responses as well as granzyme b secretion (indicating ctl activity) as compared to the l or l dna constructs, alone (survival rate: ~ . %). these data showed high potency of the combined l + l dna constructs versus each dna construct alone as a prophylactic hpv vaccine. taken together, immunoinformatics approaches have been emerged as a critical field for accelerating immunological researches. yet, the immunoinformatics techniques applied to t-cells have more advancement than those dealing with b-cells . moreover, recently, due to the limited options for choosing an adjuvant in clinical trials, bioinformatics analyses have been developed to predict the best adjuvant. in this way, in silico studies help researchers saving time and resources, and also can guide the experimental work with higher probabilities of finding the desired solutions and with fewer trial and error repeats of assays. the accessibility of hpv genomic sequences and functional characterization of the genes involved in the virulence has significantly improved our understanding of the molecular foundation for the pathogenesis of hpv and offered a wealth of data that can be used to design new plans for vaccine design. nowadays, powerful immune system simulators have been developed using bioinformatics tools which predict artificial immunity provided by the vaccine. these approaches could predict the best adjuvant for using in human vaccine studies. there is a multi-scale computational infrastructure approach which can stimulate the dynamics of the immune response induced by several vaccination formulations and predict optimal combination in terms of adjuvant type, dosage and timing. netlogo is an agent-based modeling of the immune system running different simulations with different parameter settings. it also can interact with different modeling strategies including the investigation of pathogen growth, life cycle modeling environment for simulation complex phenomena [ ] [ ] [ ] . therefore, using these methods can increase efficiency and reduce costs in vaccine studies. in this study, for the first time, comprehensively integrated methods (using sequence-based tools in combination with flexible peptide-protein docking) were used to design highly immunogenic and protective vaccine candidates which were able to boost both humoral and cellular table . mhc-ii -peptide docking scores of selected helper t-cell epitopes. *higher rate shows better quality of peptide-mhc interactions. www.nature.com/scientificreports www.nature.com/scientificreports/ immune responses against all high-risk hpv types. in addition, in vivo analysis demonstrated high potency of the designed l and l constructs as combined in dna-based vaccines without the use of adjuvant or delivery system. however, we will improve the efficiency of these dna-based vaccines using a delivery system and also will compare their efficacy with the designed peptide-based vaccines along with adjuvants in near future. table . physicochemical properties of l and l dna vaccine constructs. *higher rate shows high degree of peptide antigenicity. **higher rate shows high degree of peptide solubility. protein alignments and conservancy analysis. to determine conserved epitopes between different subtypes, l and l sequence datasets were first aligned using snapgene software . . (from gsl biotech; available at snapgene.com). after protein alignments analysis using muscle algorithms, the conserved epitopes of each protein were selected for immune-bioinformatics analysis such as b-and t-cell epitope prediction. also, to calculate the degree of variability and conservancy of each epitope, iedb epitope conservancy tools (http://tools.immuneepitope.org/tools/conservancy/) were used. linear b-cell epitope prediction. a successful vaccine must elicit a strong t-cell and b-cell immune response, but above all, provide protection against the disease being targeted. therefore, it is essential to show that constructed immunogens are able to induce protective cellular and humoral immunity. since the antibodies are induced against linear b-cell epitopes, it would be very difficult to synthesize long peptides with the native protein conformation resembling for the induction of protective antibodies. however, optimal peptide-based vaccines should be presented in a desired secondary structure of peptides in order to induce a specific humoral response , . for the b-cell epitope prediction of conserved regions in l and l proteins, bepipred- . server (http://www.cbs. www.nature.com/scientificreports www.nature.com/scientificreports/ dtu.dk/services/bepipred- . /) was employed. in this study, epitope threshold value was set as . (the specificity and sensitivity of this method are . and . , respectively) . t-cell epitope prediction. mhc-i epitope prediction: the initial step on applying bioinformatics to vaccine researches is to assess potentially immunoprotective epitopes. t-cell epitopes presented by mhc molecules are typically in a linear form containing to amino acids. this fact facilitates accurate modeling for the interaction of ligands and t-cells . thus, the most selective step in the presentation of antigenic peptide to t-cell receptor (tcr) is the binding of the mhc molecule . in this study, we tried to use three different algorithms including artificial neural networks (netmhcpan . server (http://www.cbs.dtu.dk/services/netmhcpan/), quantitative matrix (propred i (http://crdd.osdd.net/raghava/propred /) and published motifs (syfpeithi server (http://www.syfpeithi.de) to predict high-potential t-cell epitopes. for netmhcpan, percentile rank was set at . % for strong binders and % for weak binders and for propred i threshold was set at %. mhc-ii epitope prediction: for mhc class ii, netmhciipan . server (http://www.cbs.dtu.dk/services/ netmhciipan/) and propred (http://crdd.osdd.net/raghava/propred/) were employed to predict potential interaction of helper t-cell epitope peptides and mhc-ii. in this case, the threshold for strong and weak binders was set at % and %, respectively. prediction of mhc-i peptide presentation pathway. investigating the tap transport and proteasomal cleavage as well as affinity prediction of binding is essential in mhc-i presentation pathway. in this study, we used netctl . server combined with tap transport/proteasomal cleavage tools (http://www.cbs.dtu.dk/services/netctl/) to access the prediction of antigen processing through the mhc class i antigen presentation pathway. in this method, parameters of weight on the c-terminal cleavage, tap transport efficiency, and epitope identification were set to default ( . , . and . , respectively) . population coverage. since the response to t-cell epitopes is restricted by mhcs, the selection of epitopes with multiple hla-binding increases population coverage in defined geographical regions where the peptide-based vaccine might be employed. the coverage rate of population for each epitope was computationally validated using the iedb population coverage tool (/population/iedb_input). in this study, individual epitope and its binding to hla alleles were analyzed, and different geographic areas were also selected. allergenicity and cross-reactivity assessment. since proteins are very important in inducing allergenic reactions, the prediction of potential allergenicity is an important item in the safety assessment especially in the field of genetically modified foods, therapeutics, bio-pharmaceuticals etc. . the food and agriculture organization (fao) and world health organization (who) protocol includes three terms to evaluate the allergenicity of proteins which are defined as following: the term sensitivity refers to correctly predicted allergens (%), whereas www.nature.com/scientificreports www.nature.com/scientificreports/ specificity refers to correctly predicted non-allergens (%), and also accuracy refers to the proportion of correctly predicted proteins . the allergenicity of the epitopes was analyzed by the pa p (http://lpa.saogabriel.unipampa. edu.br: /pa p/pa p/pa p.jsp) using allergen online ( aa and wordmatch) and afds-motif algorithms based on amino acid composition. the specificity of these methods is . % ( aa), . % ( aa) and . % (adfs) . to assess cross-reactivity between peptide and human proteome, top-ranked epitope were analyzed by peptide matching program (https://research.bioinformatics.udel.edu/peptidematch/index.jsp) . peptide-protein flexible docking. computational docking methods have been known as an important tool for drug design . with the rapid development of peptide therapeutics in rational drug design, the use of new techniques such as protein-peptide docking is inevitable. in this study, two different algorithms (template-based docking and global docking) were performed by galexypepdock server (http://galaxy.seoklab.org/cgi-bin/ submit.cgi?type=pepdock) and cabs dock server (http://biocomp.chem.uw.edu.pl/cabsdock). to estimate the formation of mhc-peptide complex, the galaxypepdock server effectively models the structural d peptide-protein complexes from input peptide and protein sequences using the structure database and energy-based optimization (template-based docking). cabs-dock server performs global docking procedure which at first explicit fully flexible docking simulation and then clustering-based scoring. receptor flexibility was limited by default to small backbone fluctuation but could be increased to include selected receptor fragments , . this study presented an example of mhc-peptide docking performed by each individual epitope and available pdb file (table ) of hla alleles, separately. physicochemical properties of the designed l and l constructs. based on l and l top-ranked epitopes, two different constructs were designed. the physicochemical properties of top-ranked epitopes such as solubility, molecular weight, estimated half-time, instability index and antigenicity were determined by protparam (https:// web.expasy.org/protparam/) tools , vaxijen (http://www.ddg-pharmfac.net/vaxijen/vaxijen/vaxijen.html) and protein-sol (https://protein-sol.manchester.ac.uk/) server . www.nature.com/scientificreports www.nature.com/scientificreports/ experimental studies construction of the recombinant plasmids. after bioinformatics analysis, the selected peptides were assembled in two separated constructs (fig. ) . the puc -l and puc -l constructs were synthesized by biomatik company. for in vitro experiments, the puc -l and puc -l vectors were digested by xhoi/hindiii, and the l and l genes were subcloned into xhoi/hindiii sites of pegfp-n vector, individually (i.e., pegfp-l and pegfp-l ). all the recombinant vectors were transformed into escherichia coli (e. www.nature.com/scientificreports www.nature.com/scientificreports/ coli) dh α strain. after extraction of plasmids from single colonies using mini-kit (qiagen), the presence of inserted l and l fragments was confirmed by digestion with restriction enzymes and sequencing. for in vivo immunological assessment, the puc -l and puc -l vectors were digested by bamhi/hindiii and the l and l genes were subcloned into bamhi/hindiii sites of pcdna . (-) vector containing cytomegalovirus early promoter and enhancer sequence, individually (i.e., pcdna-l and pcdna-l ). indeed, we used the pcdna vector harboring cpg motif for in vivo studies. as a final point, the recombinant dna vectors harboring l and l genes were purified by an endotoxin-free plasmid extra ef kit (macherey nagel, germany). the concentration and purity of the recombinant l and l dna constructs were determined by nanodrop spectrophotometry . in vitro expression of l and l dna constructs in hek- t cells. human embryonic kidney cells (hek- t) were cultured in rpmi supplemented with % fetal bovine serum (fbs) at °c and % co atmosphere. after some passages, the cells were seeded in a -well plate. the optimal cell confluency for effective transfection was considered - %. for the generation of turbofect-plasmid dna complex, μl of turbofect (thermo scientific) and μg of each plasmid (pegfp-l , pegfp-l and pegfp-n as a positive control) were mixed and incubated for min at room temperature. then, the complex was added to each well in serum-free media. in addition, the non-transfected hek- t cells were used as negative control. after six hours, the media was replaced with the completed rpmi medium. finally, the cells were harvested, washed and resuspended in pbs buffer, to analyze the expression of l and l dna constructs using flow cytometry, fluorescent microscopy and western blotting at hr after transfection . western blot analysis. hek- t cells were scraped from their plates and washed with pbs x. after washing steps, the cells were lysed in whole-cell lysis buffer ( % glycerol, nm dtt, mm natrium fluoride, . % triton x- , . edta in pbs ph = . ). the extracted protein samples (l -gfp, l -gfp and gfp) were separated by sds-page in . % (w/v) polyacrylamide gel and transferred to nitrocellulose membrane (millipore). the membrane was equilibrated with tbst (tris-buffered saline tween- ) solution containing . % bsa (bovine albumin serum) overnight. the anti-gfp polyclonal antibody ( : v/v; acris antibodies gmbh) was used to recognize the expressed proteins under standard procedures. the immunoreactive protein bands were visualized by detection of peroxidase activity using a substrate named as , ′-diaminobenzidine (dab, sigma) . peptide constructs synthesis. for immunological assay (i.e., secretion of antibody, cytokine and granzyme b), two peptide constructs (l and l peptides, fig. ) were synthesized by biomatik co. with more than % purity. mice immunization. five groups of six female c bl/ mice (obtained from the breeding stocks maintained at pasteur institute of iran; mhc haplotype b/h- kb/h- db) were immunized on days , , and (i.e., three times with a -week interval) with µg of each plasmid dna (pcdna-l or pcdna-l : g or g ) or their combination (pcdna-l + pcdna-l : g ) at the right footpad as shown in table . the control groups (g and g ) received pcdna . and pbs, respectively. all mice were maintained under specific pathogen-free conditions . moreover, all of the animal experimental procedures were approved by animal care and use committee of pasteur institute of iran and carried out according to the animal experimentation regulations of pasteur institute of iran (national guideline) for scientific purposes (code: ). for in vivo protection assay, vaccinated mice were subcutaneously challenged in the right flank with c tumor cells ( × cells), two weeks after the last injection. the c tumor cells contain whole hpv genome, and the presence of l and l genes was confirmed in the previous studies . tumor growth and the percentage of tumor-free mice were monitored twice a week by palpation for days post-challenge. at each time, tumor volume was calculated by this formula: v = (a b)/ (a = the smallest diameter and b = the biggest diameter) . antibody assay secreted from b-cells. two weeks after the last injection, serum samples were collected from each group. the levels of goat anti-mouse immunoglobulin g (igg ), igg a, igg b and total igg antibodies (diluted : , in % bsa/pbs-tween, sigma) secreted from b-cells were measured in the pooled sera of each group by indirect elisa. the coated antigens were the mixture of l and l synthetic peptides ( μg/ml). moreover, mice sera were diluted : in % bsa/pbs-tween . cytokine assay secreted from t-cells. three mice from each group were sacrificed and the spleens were removed. the red blood cell-depleted pooled splenocytes ( × cells/ml) were cultured in -well plates for h in the presence of μg/ml of l + l peptides, rpmi % as negative control and μg/ml of concanavalin a (cona) as positive control in complete rpmi culture medium. the supernatants were harvested to assess the secretion of ifn-γ, il- and il- from t-cells using the sandwich-based elisa method (r&d systems) according to the manufacturer's instructions. all data were represented as mean ± sd for each sample . granzyme b assay (in vitro ctl activity). to measure granzyme b (grb) by elisa, the p target cells (t) were seeded into -well plates ( × cells/well) incubated with the mixture of l and l peptides (~ μg/ml) for h. then, the prepared splenocytes (effector cells: e, before section) were counted and added to the target cells at e: t ratio of : in complete rpmi culture medium for h incubation. finally, the supernatants were harvested to measure the concentration of grb by elisa (ebioscience kit) according to the manufacturer's instruction . table . immunization program for in vivo analysis. worldwide burden of cancer attributable to hpv by site, country and hpv type estimate of the global burden of cervical adenocarcinoma and potential impact of prophylactic human papillomavirus vaccination hpv vaccination: the promise & problems pros, cons, and ethics of hpv vaccine in teens-why such controversy? virus-like particles for the prevention of human papillomavirus-associated malignancies therapeutic human papillomavirus vaccines: current clinical trials and future 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multi-epitope vaccine the proteomics protocols handbook vaxijen: a server for prediction of protective antigens, tumour antigens and subunit vaccines protein-sol: a web tool for predicting protein solubility from sequence hpv l improves hpv l gene delivery as an important approach for vaccine design against cervical cancer whole recombinant pichia pastoris expressing hpv l antigen is superior in inducing protection against tumor growth as compared to killed transgenic leishmania immunogenicity of an hpv- l dna vaccine small heat shock protein : an effective adjuvant for enhancement of hiv- nef antigen-specific immunity recombinant leishmania tarentolae encoding the hpv type e gene in tumor mice model a.n. and a.b. conceptualized the work. a.n. performed the experiments. a.n., a.b., g.j. and z.n. analyzed the data. a.n. wrote the manuscript. a.b. edited the manuscript. all authors approved the final version of the paper. the authors declare no competing interests. correspondence and 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from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - syrrn authors: yang, yong-le; qin, pan; wang, bin; liu, yan; xu, guo-han; peng, lei; zhou, jiyong; zhu, shu jeffrey; huang, yao-wei title: broad cross-species infection of cultured cells by bat hku -related swine acute diarrhea syndrome coronavirus and identification of its replication in murine dendritic cells in vivo highlight its potential for diverse interspecies transmission date: - - journal: j virol doi: . /jvi. - sha: doc_id: cord_uid: syrrn outbreaks of severe diarrhea in neonatal piglets in guangdong, china, in resulted in the isolation and discovery of a novel swine enteric alphacoronavirus (seacov) derived from the species rhinolophus bat coronavirus hku (y. pan, x. tian, p. qin, b. wang, et al., vet microbiol : – , ). seacov was later referred to as swine acute diarrhea syndrome cov (sads-cov) by another group (p. zhou, h. fan, t. lan, x.-l. yang, et al., nature : – , ). the present study was set up to investigate the potential species barriers of sads-cov in vitro and in vivo. we first demonstrated that sads-cov possesses a broad species tropism and is able to infect cell lines from diverse species, including bats, mice, rats, gerbils, hamsters, pigs, chickens, nonhuman primates, and humans. trypsin contributes to but is not essential for sads-cov propagation in vitro. furthermore, c bl/ j mice were inoculated with the virus via oral or intraperitoneal routes. although the mice exhibited only subclinical infection, they supported viral replication and prolonged infection in the spleen. sads-cov nonstructural proteins and double-stranded rna were detected in splenocytes of the marginal zone on the edge of lymphatic follicles, indicating active replication of sads-cov in the mouse model. we identified that splenic dendritic cells (dcs) are the major targets of virus infection by immunofluorescence and flow cytometry approaches. finally, we demonstrated that sads-cov does not utilize known cov receptors for cellular entry. the ability of sads-cov to replicate in various cells lines from a broad range of species and the unexpected tropism for murine dcs provide important insights into the biology of this bat-origin cov, highlighting its possible ability to cross interspecies barriers. importance infections with bat-origin coronaviruses (covs) (severe acute respiratory syndrome cov [sars-cov] and middle east respiratory syndrome cov [mers-cov]) have caused severe illness in humans after “host jump” events. recently, a novel bat-hku -like cov named swine acute diarrhea syndrome cov (sads-cov) has emerged in southern china, causing lethal diarrhea in newborn piglets. it is important to assess the species barriers of sads-cov infection since the animal hosts (other than pigs and bats) and zoonotic potential are still unknown. an in vitro susceptibility study revealed a broad species tropism of sads-cov, including various rodent and human cell lines. we established a mouse model of sads-cov infection, identifying its active replication in splenic dendritic cells, which suggests that sads-cov has the potential to infect rodents. these findings highlight the potential cross-species transmissibility of sads-cov, although further surveillance in other animal populations is needed to fully understand the ecology of this bat-hku -origin cov. different animal species were tested for susceptibility to sads-cov treated with or without trypsin ( table ) . as a brief summary of the results, of the cell lines showed significant susceptibility to sads-cov infection, defined by efficient viral replication, antigen expression, and the appearance of cytopathic effect (cpe). the three cell lines that were not infected by sads-cov were mdck, bfk, and raw . . first, cpe was examined by inverted light microscopy at hours postinfection (hpi), and scores are shown in table . as the t, nih/ t , cho, brl- a, and nrk- e cell lines were sensitive to trypsin, they could not be tested for sads-cov infection in mmt cells. apart from that, cpe was visible in vero, st, and brl- a cell lines without trypsin, and prominent cpe appeared in or was enhanced with trypsin in marc- , cos- , bsc- , vero, st, pk , llc-pk , and bhk- cell lines ( table ) . as some cells did not display cpe after sads-cov infection, all cell lines were subsequently tested for viral m protein expression by immunofluorescence assay (ifa) fig. ) , revealing the same range as seen by cpe in the different cell lines (data not shown). syncytium formation was prominent in huh- , vero, and bhk- cells, whereas in mdck, bfk and raw . cells, the antigen expression was much less prominent than in the other cell lines (fig. a , c, and i). most cell lines tested showed evidence of productive infection, as indicated by expression of the m protein, while the inefficient antigen expression in marc- , llc-pk , and ipec-j cells suggested only a limited infection. next, viral load in the culture supernatants was detected over days postinfection (dpi) by reverse transcriptase quantitative pcr (qrt-pcr) ( fig. a to c). a higher mean viral load was detected by qrt-pcr after trypsin treatment in hepg , hela, marc- , cos- , bsc- , vero, llc-pk , ipec-j , bhk- , and df- cells. therefore, trypsin contributes to but is not essential for sads-cov propagation in these cell lines. there was no difference after trypsin treatment in the other cell lines, although huh- and tb- cells had high levels of sads-cov rna regardless of trypsin treatment. the progressive release of infectious sads-cov into the culture medium of six representative cell lines infected with sads-cov was determined by titration of super- to determine the effect of trypsin on sads-cov infection, each cell line was infected in the following three conditions: "no trypsin" treatment, inoculated with sads-cov diluted in maintenance medium (mm) for h and subsequently replaced with mm (a); "pretrypsin" treatment, inoculated with sads-cov diluted in mm containing g/ml trypsin (mmt) for h and subsequently replaced with mm (b); and "double-trypsin" treatment, inoculated with sads-cov in mmt and subsequently replaced with mmt (c). infection supernatants were collected at , , , , , and hpi for viral load detection by a qrt-pcr assay targeting the viral n gene. data are expressed as the mean viral load (log copies/l) Ϯ standard deviation (sd), and all experiments were performed in triplicate. the t, cho, brl- a, and nrk- e cell lines did not survive in the presence of trypsin. (d) infectious titers (tcid /ml) of sads-cov secreted from hela, vero, tb- , bhk- pk- , and mdck cells were determined on vero cells. natants in vero cells (fig. d ). unlike in mdck cells, sads-cov infection of hela, vero, tb- , bhk- , and pk- cells was productive, with hela cells showing the greatest susceptibility (fig. d) . wild-type c bl/ j mice can be infected by sads-cov via oral and intraperitoneal routes. with the observation that sads-cov could infect diverse rodent cell lines (from mice, rats, and hamsters as well as gerbil primary kidney cells), we hypothesized that mice may be susceptible to sads-cov. to test this, we inoculated -to -week-old wild-type b mice with ϫ % tissue culture infective dose (tcid ) of sads-cov by the per oral (p.o.) or intraperitoneal (i.p.) route and monitored them for days for clinical symptoms. the mice did not succumb to the infection nor did they develop diarrhea or experience weight loss during the incubation period (data not shown). to determine whether sads-cov infected the animals asymptomatically, tissue and fecal samples from inoculated mice were collected at , , , , , , and dpi to determine viral growth kinetics and shedding. analysis of tissue samples by qrt-pcr suggested that sads-cov replicated modestly in the stomach early after i.p. or p.o. infection, declining and reaching undetectable levels at or dpi and thereafter (fig. a) . a very limited viral replication was observed in each region of the small intestine, with the ileum via i.p. infection showing continuous and decent detectable viral rna (fig. b ). in the large intestine, i.p. infection also resulted in viral rna loads slightly above the limit of detection at each time point in the ceca, whereas it led to higher viral rna levels at to dpi, and much lower viral rna at to dpi in the colon than that of the p.o. route (fig. c) . however, this replication in the large intestine did not translate into higher shedding, as hardly any viral genomes were detected, even at dpi in the fecal samples collected from i.p.-infected mice (fig. f ). on the contrary, significantly more virus was detected in the feces of p.o.-infected mice at and dpi, indicating that i.p. inoculation does not lead to higher virus shedding. finally, sads-cov replicated more efficiently in the spleen following the i.p. route, with significantly higher viral rna loads at dpi (fig. d ). more importantly, the virus was not cleared from this tissue by dpi in the i.p.-infected group and by dpi in the p.o.-infected group, suggestive of a sads-cov prolonged infection in the spleen independent of inoculation route. in contrast to the spleen, only very low levels of viral rna were detected in the local lymphoid tissue of mesenteric lymph nodes (mlns) at to dpi, and no virus was detectable at later time points (fig. d ). we also looked for virus in other extraintestinal sites, including the heart, lungs, liver, kidneys, and blood, but they were all negative or had extremely low levels (fig. e ). igg antibody levels after days detected by sads-cov virion-based enzyme-linked immunosorbent assay (elisa) showed that the i.p. route could effectively elicit host immune responses (fig. g) . splenocytes support sads-cov replication. with the mouse infection model described above, our next step was to determine the cell tropism of sads-cov in vivo. thus, we performed immunohistochemistry (ihc) on sections of small and large intestine and spleen from mice infected i.p. with ϫ tcid of sads-cov at dpi. a monoclonal antibody against double-stranded rna (dsrna) was used to identify cells that supported active virus replication, as dsrna is an intermediate that only exists during intracellular viral replication. sads-cov dsrna signals were observed in the splenic white pulp in the marginal zone on the edge of lymphatic follicles and in the margins of the periarteriolar lymphocyte sheath (fig. a ). staining of tissue sections from mock-infected mice were used as a control (fig. b ). in addition to dsrna, we also used rabbit polyclonal antibodies (pabs) to detect the expression of the viral structural protein (m) or nonstructural protein (nsp -ac). at dpi, anti-m or anti-ac staining was observed in the white pulp around the lymphatic nodules (fig. c) , similar to the localization of dsrna staining (fig. a ). tissue sections from sads-cov or mockinfected mice probed with preimmune sera were negative, indicating the specificity of the sads-cov antibody. unfortunately, neither viral proteins (structural or nonstruc-tural) nor dsrna were detected in the intestine of infected mice, consistent with the detection of only very low levels of viral rna in these tissues by qrt-pcr (fig. ) . next, sads-cov infection was quantified in the spleen using flow cytometry. we inoculated b wild-type mice with ϫ tcid of virus either i.p. or p.o. and extracted the bulk immune cells from the spleen of infected animals at dpi. the flow cytometry method was first validated in vero cells infected with sads-cov at a multiplicity of infection (moi) of . , followed by staining with a pab against the n or ac protein at hpi (fig. d ). as the anti-ac pab exhibited optimal intracellular staining for viral signals (fig. d) , it was used to determine the percentage of infected splenocytes. there were approximately . -and . -fold increases of total splenocytes positive for virus replication after p.o. and i.p. inoculation, respectively (fig. e , left; fig. f ), with a significant increase in the total number of ac-positive splenocytes in i.p.-infected mice compared with that of p.o. (fig. e , right). these data are consistent with the significantly lower viral loads in the spleen at and dpi in p.o.-inoculated mice (fig. d ), suggesting better virus dissemination and replication and escape from mucosal immune clearance. we then evaluated the growth characteristics of sads-cov in splenocytes by assessing antigen production and replication kinetics ex vitro. splenocytes were first extracted from naive mice, plated in -mm dishes, and infected with ϫ tcid of sads-cov. we observed clusters of infected cells that appeared to have been engulfed by phagocytes (fig. g , middle), and the structural n protein was shown in the cytoplasm of infected cells by confocal microscopy (fig. g , middle and right). the percentage of infected cells was quantified by flow cytometry using anti-ac pab, revealing a nearly -fold increase in the splenocytes positive for viral signals (fig. h) , very similar to the percentage of infection observed in vivo. to further characterize the growth kinetic of sads-cov in primary splenocytes, cells were infected with ϫ tcid of sads-cov, and culture supernatants were harvested at , , , , and hpi. active viral replication was confirmed, with a . -log time-dependent increase in genomic rna equivalents, plateauing from to hpi (fig. i ). these data suggest that although only ϳ % of splenocytes were infected, and these cells supported a decent level of viral replication. together, these results indicate that sads-cov productively infects mouse splenocytes. splenic dcs support sads-cov replication. splenocytes were harvested from i.p.-infected mice at dpi, and the extracted cells were costained with antibodies against sads-cov-ac and each of four cell surface markers (anti-cd for b cells, anti-cd for t cells, anti-cd /c ϩ for dcs, and anti-f / ϩ for macrophages) using flow cytometry (fig. a ). the percentage of infected cd /c ϩ cells was significantly higher than the other cell subgroups, indicating that dcs are the major targets of sads-cov infection in the spleen. the phenotype was further confirmed by double-staining ifa with anti-dsrna, anti-m, or anti-ac antibody plus anti-cd /c ϩ in splenic sections. as expected, dsrna staining overlapped with the cd /c surface marker on the edges of lymphatic follicles (fig. b) , whereas no viral signals were seen in the mock-infected control (fig. c ). similar patterns of costaining were detected by m and ac antibodies (fig. d ). to gain insight into the relative quantity of dcs compared with other undefined target cells, cells positive for dsrna and cd /c were counted in to different microscope fields of spleens from infected mice (fig. e) , showing that . % of sads-covinfected cells were dcs (fig. f ). to our knowledge, these results reveal the most extensive cell tropism among known covs, suggesting the functional receptor(s) for sads-cov is likely to be a very common molecule. in order to test this hypothesis, it was first necessary to find a cell line that was refractory to infection only at the internalization step. mdck cells, which showed undetectable virus production in early infection tests ( fig. and ) , were chosen as a potential candidate. there are four known types of functional cov protein receptors, including angiotensin converting enzyme (ace ) for sars-cov ( ), dipeptidyl peptidase (dpp ) for mers-cov ( ) , aminopeptidase n (apn) for tgev ( ) and pdcov ( , ) , and mouse carcinoembryonic antigen-related cell adhesion molecule a (mceacam a) for mouse hepatitis virus (mhv) ( ) . to test whether one of these molecules serves as the sads-cov receptor, we attempted to inoculate nonsusceptible mdck cells overexpressing porcine apn, human dpp , mouse ceacam a, or human ace with sads-cov, but none of them allowed infection, as staining with anti-sads-cov-n pab was negative (fig. a) . meanwhile, the expression of each receptor in mdck cells was confirmed by ifa (fig. a) and western blot analysis (fig. b) using antibodies against the tags fused to the receptors. we confirmed that, as positive controls, lentiviruses pseudotyped with tgev, sars-cov, mers-cov, or mhv spike (i.e., pseudoviruses) efficiently entered mdck cells exogenously expressing the respective receptors (fig. c) . next, we demonstrated that mdck cells can confer sads-cov replication competency by transfection of a sads-cov/seacov infectious cdna clone established recently ( ) , as simultaneous expression of nsp -ac and n proteins were clearly detected by ifa (fig. d) . moreover, passaging of supernatants from psea-transfected mdck cells onto fresh vero cells resulted in progeny sads-cov infection, as evidenced by expression of the n protein (fig. e) , indicating that mdck cells can also support infectious sads-cov production without cell-to-cell spread. therefore, sads-cov apparently does not utilize any of the known cov receptors for cellular entry. the same conclusion was reached using hela cells overexpressing each of the four classical cov receptors followed by sads-cov inoculation by zhou et al. ( ) ; however, the hela cell line itself was most susceptible to sads-cov infection in the present study (fig. d) . in order to assess the potential species barriers of sads-cov infection, a cell line susceptibility study was first conducted using different cell lines. as sads-cov probably originated from a bat sadsr-cov ( ) derived from hku -cov identified in rhinolophus sinicus (chinese horseshoe bats) ( ), we commenced testing viral susceptibility in two available bat cell lines, namely, bfk from myotis daubentonii ( ) and tb- from tadarida brasiliensis. although bfk cells did not support sads-cov replication, it replicated efficiently in tb- cells (fig. a and fig. ) , suggesting that other bat species in addition to horseshoe bats are likely susceptible to sads-cov infection. interestingly, sads-cov protein expression was detected in almost all of the rodent cells (hamster, gerbil, mouse, and rat) including bhk- , which is not susceptible to other known human covs, such as sars-cov and mers-cov ( , ) , as well as three swine enteric covs, namely, pedv, pdcov, and tgev ( ) . given the fact that sads-cov infects both primary and passaged or primary cell lines originating from rodents, we hypothesized that rodents may be susceptible to sads-cov infection. to explore this possibility, we challenged wild-type b mice with sads-cov by two different inoculation routes. the challenged animals neither succumbed to infection nor manifested any signs of gastroenteritis. in fact, experimental infection of neonatal piglets with a higher dose of purified sads-cov in our laboratory resulted only in mild diarrheal signs or subclinical infection ( ) . also, there was a lack of robust viral replication in the intestines during infection, and no tissue damage was detected throughout the intestines ( fig. b and c) , reflecting the suboptimal infection by sads-cov in immunocompetent wild-type mice. on the contrary, the virus had more efficient replication within the spleen, which was reflected by a continuous detection of viral genomic rna in the immune cells at all time points over a -day period (fig. d) . the phenotype was also consistent with the replication kinetics in extracted splenocytes in vitro, in which viral genomic rna peaked and plateaued at hpi ( fig. g and i) . these data collectively led to speculation that sads-cov favors splenic cells over other tissues. the most logical explanation for these tissue-specific discrepancies in virus replication is (i) target cells are more concentrated in the spleen and more sporadic in the intestine or (ii) splenic immune cells have enhanced expression of the unknown receptor(s) over intestinal cells. the animals were more susceptible to i.p. infection, resulting in higher virus replication in the distal section of the small intestine, large intestine, and spleen, and perhaps a delayed clearance of viral infection in the cecum (fig. b to e) , suggesting the important role of mucosal immunity for controlling early infection in sads-cov in mice. it should be noted that mice (c bl/ j mice in this study) may not be the optimal rodent species for sads-cov infection, as wild rats are more commonly seen in chinese pig farms. in addition, other transmission routes may be considered. recently, pdcov has been shown to possibly spread via the respiratory route in addition to fecal-oral transmission ( ) . therefore, it will be interesting to try intranasal route for the inoculation in rats or the other rodent species to mimic sads-cov natural transmission in future studies. more interestingly, we identified dcs to be the precise cell population that supported sads-cov replication (fig. ) . there have been a few reports of immune cell tropism for covs. macrophages are susceptible to mhv infection, representing the largest group of innate immune cells that infiltrate the central nervous system after infection with neurotropic mhv strains ( ) . in addition, based on the fact that sars-cov spike-pseudotyped hiv-based vectors can efficiently transduce human dcs, kobinger ( ) . these previous evidences support our present results, showing that sads-cov can efficiently replicate in dcs. furthermore, this study gives us a novel inspiration that rodents may potentially serve as susceptible hosts for sads-cov in addition to bats and pigs. of note, the species rhinolophus bat ␣-cov hku , including sads-cov, possesses unique s genes closely related to the betacoronavirus (␤-cov), in a manner similar to some globally distributed rodent ␣-covs ( , , ) , implying an unknown evolutionary connection between the bat ␣-cov hku and rodent ␣-covs. under the field conditions of china, direct contact between pigs and flying bats is a low probability; however, rodents (especially rats) are frequently visible in the swine industry, causing great nuisance due to feed loss. it is possible that as bats prey on insects near pig facilities, they leave feces containing hku -like covs that contaminate pig feed, which is then eaten by pig and rodents that subsequently become carriers of sads-cov. rats and mice are increasingly implicated as external vectors for a wide range of different pig pathogens, such as lawsonia intracellularis ( ) . rodents not only spread pathogens but also harm the practitioners of the swine industry, as they are thought to be the major source of leptospirosis in pigs and piggery workers ( ) . future studies on identifying sads-covpositive samples in rodents near pig farms are warranted to test this hypothesis. in addition to rodents, we also measured the sads-cov susceptibility of cell lines from humans, monkeys, chickens, and dogs, revealing a remarkably broad spectrum of tropism (table and fig. ) . as for the ability of sads-cov to grow efficiently in human cell lines, we should not underestimate the risk that this bat-origin cov may "jump" from pigs to humans. it is noteworthy that camel workers with high rates of exposure to camel nasal and oral secretions had evidence of mers-cov infection ( ) . considering that sars-cov and mers-cov originated from bats and spread from one species to another through intermediate hosts (civets and camels, respectively), sads-cov may pose a similar risk to human health through transmission from pigs or other susceptible hosts. the cell susceptibility study and testing of the overexpression of four known cov receptors in nonsusceptible mdck cells (fig. ) demonstrated that sads-cov might use a new receptor molecule that is conserved in bats, pigs, rodents, chickens, monkeys, and humans, indicating a low barrier to cross-species transmission. this is in line with the unusual feature of broad species tropism of sads-cov. in summary, these results provide important insights into the ecology of this bat-origin cov, highlighting the possibility of its ability to jump interspecies barriers and the potential role of rodents as susceptible hosts in the field. identification of the unknown sads-cov cellular receptor and further surveillance of other animal populations are needed to fully understand the biology of sads-cov. the sads-cov isolate ch/gd- / at passage was used in all experiments and cultured in vero cells ( ) . the virus was passaged serially using the culture supernatant to infect fresh vero cells at a multiplicity of infection (moi) of . , and viral titers were determined in vero cells by endpoint dilution as the % tissue culture infective dose (tcid ). rabbit polyclonal antibodies (pab) against the membrane (m), nucleocapsid (n), and the nonstructural protein (nsp ) acidic domain (ac) of sads-cov were generated in-house and validated in sads-cov-infected vero cells ( ) . a mouse anti-sads-cov-n pab was also produced to allow double staining when mixed with the rabbit pab. a monoclonal antibody (mab) against dsrna (anti-dsrna mab j ; catalog number j - , scicons, hungary) was used to specifically detect viral replication of sads-cov. cell lines and cell culture. twenty-four cell lines derived from tissues of different species were used (table ) , including human (huh- , hepg /c a, t, a , and hela), monkey (marc- , cos- , bsc- , and vero), swine (st, pk , llc-pk , and ipec-j [ ] ), bat (bfk [ ] and tb- ), canine (mdck), mouse (nih/ t and raw . ), hamster (bhk- and cho), rat (brl- a and nrk- e), and chicken (df- ) cell lines and a primary kidney cell line from mongolian gerbils (prepared in-house). the bfk cell line was a generous gift from changchun tu at the institute of military veterinary medicine, changchun, china. each cell line was cultured in dulbecco's modified eagle's medium (dmem; hyclone) supplemented with % (vol/vol) fetal bovine serum (fbs; biological industries), u/ml penicillin, and u/ml streptomycin under °c, % co , and water-saturated humidity conditions. to determine viral susceptibility, each cell line was cultured at % confluence in -well plates with maintenance medium (mm) containing dmem, . % tryptose phosphate broth (tpb), and % penicillin-streptomycin or mm with addition of g/ml trypsin (mmt) (catalog number t - tab; sigma, st. louis, mo, usa). after cells were washed with phosphate-buffered saline (pbs), they were inoculated with sads-cov diluted in mm or mmt at an moi of . for h. nonattached viruses were removed by washing the cells three times with dmem, and cell monolayers were subsequently incubated in mm or mmt at °c for days. to determine the effect of trypsin on viral entry, cell monolayers were infected by sads-cov in the following three conditions: (i) no trypsin treatment, infected with sads-cov diluted in mm, subsequently incubated in mm; (ii) pretrypsin treatment, inoculated with sads-cov diluted in mmt, subsequently incubated in mm; and (iii) double-trypsin treatment, inoculated with sads-cov in mmt, subsequently incubated in mmt. supernatants from cells were collected at , , , , , and hours postinfection (hpi) for one-step quantitative rt-pcr analysis. cell cultures were examined for cytopathic effects (cpes) and immunofluorescence assay at to hpi. ifa for cell line susceptibility. different cells infected with sads-cov in -well plates were washed twice with pbs and fixed in % paraformaldehyde in pbs and then permeabilized with . % triton x- in pbs. cells were then incubated with the rabbit anti-sads-cov-m pab at : , dilution for h at °c, washed with pbs, and stained with the alexa fluor -conjugated goat anti-rabbit secondary antibody (thermo fisher scientific, usa) at : , dilution. after incubation for h at °c, the cells were washed with pbs, stained with =, -diamidino- -phenylindole (dapi) at : , dilution and visualized on a fluorescence microscope. one-step quantitative rt-pcr analysis targeting the n gene. the full-length sads-cov n gene was inserted into an appropriately digested pet- a vector using two unique restriction sites, namely, ndei and xhoi, and then linearized with xhoi. the n gene was in vitro transcribed using the t high-efficiency transcription kit (transgen biotech co., ltd., beijing china). standard curves were generated using dilutions of a known quantity of the n gene rna to allow absolute quantitation of sads-cov rna copy numbers in samples. total rna was extracted from culture supernatants or tissue homogenates using trizol (thermofisher scientific, usa) following the manufacturer's instructions. sads-cov rna titer was determined by one-step qrt-pcr (toyobo co., ltd.) targeting the n gene with the primers =-ctaaaactagccccaca ggtc- = and =-tgattgcgagaacgagactg- = and the probe -carboxyfluorescein (fam)-gaaacccaa actgaggtgtagcagg- -carboxytetramethylrhodamine (tamra). samples with a cycle threshold value of Ͻ were considered positive based upon validation data using the rna standards. mouse infections, tissue harvest, and viral load determination. wild-type c bl/ j mice (catalog number ; jackson laboratory) were purchased from the model animal research center of nanjing university and housed in animal facilities at the zhejiang university under specific-pathogen-free conditions. for sads-cov infections, -to -week-old female and male mice were inoculated with ϫ tcid (equal to ϫ genome copies) of sads-cov, either per oral (p.o.) infection with -l inoculum ( ϫ tcid /ml) or intraperitoneal (i.p.) infection with -l inoculum ( . ϫ tcid / ml). for viral load determination in specific tissues, mice were euthanized at , , , , , , and days postinfection (dpi), and tissues were harvested, including stomach, duodenum, jejunum, ileum, cecum, colon, mesenteric lymph nodes, spleen, kidney, liver, heart, lung, blood, and feces. tissues were weighed and homogenized in medium (dmem contained % fbs) by bead beating using sterile zirconium oxide beads (catalog number zrob ; midsci). total rna was extracted from tissue homogenates and tested by quantitative rt-pcr analysis targeting the sads-cov n gene, as described above. blood samples were collected from the heart, and serum was separated for virus-specific antibody detection. enzyme-linked immunosorbent assay. sads-cov virus particles were purified from infected cell culture supernatants by sucrose density gradient centrifugation, and protein concentration was determined by the bicinchoninic acid (bca) protein assay kit (beyotime biotechnology, shanghai, china). the optimal dilution of antigen was determined by square titration. the igg antibodies contained in serum at a : dilution were detected in wells coated with purified sads-cov virus particles ( . ng/well) as antigen. histopathology, immunohistochemistry, and immunofluorescence assay for murine spleen. mice were infected i.p. with sads-cov, and at dpi, spleens were harvested and fixed in % paraformaldehyde for h and embedded in paraffin. tissue sections were then deparaffinized and rehydrated in three changes of xylene, min each, dehydrated in two changes of pure ethanol for min, followed by rehydration in an ethanol gradient of % and % ethanol. after tissues were washed in distilled water, they were subjected to hematoxylin and eosin staining for histopathological examinations. for antigen retrieval, deparaffinized and rehydrated sections were immersed in sodium citrate antigen retrieval solution (ph . ) and maintained at a subboiling temperature for min, were left at °c for min, and then incubated again at subboiling temperature for min. after the sections were allowed to cool to room temperature (rt) and were washed three times with pbs (ph . ), endogenous peroxidase was blocked by immersion in % hydrogen peroxide at rt for min, and sections were again washed with pbs. tissue sections were blocked in % bovine serum albumin (bsa) at rt for min and then incubated with a : dilution of each primary antibody (anti-dsrna mab, anti-sads-cov-m pab, or anti-sads-cov-ac pab) overnight at °c. after slides were washed three times with pbs (ph . ), they were stained with appropriate secondary antibodies labeled with horseradish peroxidase at rt for min. freshly prepared diaminobenzidine chromogenic reagent was added and counterstained with hematoxylin and then dehydrated and visualized on a light microscope. spontaneous fluorescence quenching reagent (wuhan servicebio technology co., ltd., wuhan, china) was added to the tissue sections and incubated for min after antigen retrieval. the sections were then washed in running water, followed with blocking and antibody staining as described above. in addition, the primary antibody was supplement with a cd c antibody (wuhan servicebio technology) at a : dilution and then stained with appropriate secondary antibodies. finally, dapi was added, and sections were visualized on a fluorescence microscope; nuclei labeled with dapi appear blue, and positive cells are green by labeling with cd /c or red by labeling with a virus-specific antibody. preparation of murine splenocytes and flow cytometry. mice infected with sads-cov were euthanized at dpi, and spleens were removed and placed in ml complete dmem. after the excised spleen was ground through a -m cell strainer using the plunger end of a -ml syringe, cells were washed with an excess of dmem and centrifuged at ϫ g for min. after cells were resuspended in ml of red blood cell lysis buffer (solarbio life sciences, beijing, china) and incubated at rt for min, ml of dmem was added, and cells were passed through another strainer to remove clumps. after centrifugation at ϫ g for min, the supernatant was discarded, and cells were resuspended in ml fresh dmem for cell counting and viability checks using trypan blue and a hemocytometer. for flow cytometry, cultured cells were resuspended in fc block buffer (containing anti-mouse cd fc block antibody at : dilution) and incubated on ice. cells in fc block buffer were added to -well plates at ϫ cells/well. after a -min incubation, cells were centrifuged at ϫ g for min, the supernatant was discarded, and pellets were resuspended in a -l cytofix/cytoperm solution (cytofix/cytoperm soln kit; bd biosciences, san jose, ca, usa) to fix cells. after incubation on ice for min protected from light, cells were centrifuged at ϫ g for min at °c, supernatant was removed without disturbing cell pellets, and cells were washed twice in l of ϫ perm/wash buffer. after the addition of -l virus-specific primary antibody (anti-dsrna mab, anti-sads-cov-n pab, or anti-sads-cov-ac pab) diluted in ϫ perm/wash buffer with % bsa and incubation at °c for min, cells were centrifuged at ϫ g for min. cells were washed twice in l of ϫ perm/wash buffer followed by staining with appropriate secondary antibodies conjugated to alexa fluor (thermo fisher scientific, usa) at °c for min. after pellets were centrifuged at ϫ g for min at °c and washed with ϫ perm/wash buffer, they were resuspended in . ml fluorescence-activated cell sorter (facs) buffer and analyzed by flow cytometry. to detect replication of sads-cov in mouse splenic cells in vitro, splenocytes were extracted from naive mice, plated in -or -mm dishes, and infected with sads-cov at an moi of . . at hpi, splenocytes were harvested and placed in a -ml tube, centrifuged at ϫ g for min at °c, and analyzed by flow cytometry as described above. infected mouse splenic cells in -mm dishes were detected by immunofluorescence assay with anti-sads-cov-n antibodies, and infection supernatants were collected at , , , , , and hpi for one-step quantitative rt-pcr analysis. facs analysis of splenocytes with cell marker staining. mice infected with sads-cov were euthanized at dpi, and splenocytes were prepared for flow cytometry by staining with the following appropriate antibodies: anti-sads-cov-ac following secondary antibodies conjugated to alexa fluor (thermo fisher scientific, usa), anti-cd -fitc (catalog number ; ebioscience) for b cells, anti-cd -pe (catalog number ; ebioscience) for t cells, anti-cd /c-pe-cy (catalog number ; bd bioscience) for dcs, and anti-f / -pe/cy (catalog number ; biolegend) for macrophages. stained cells were resuspended in . ml facs buffer and analyzed by flow cytometry. production and transduction of s protein-pseudotyped lentiviruses. pseudovirions with various cov spike proteins were produced as described previously ( ) . briefly, each of the plasmids encoding tgev, sars-cov, mers-cov, and mouse hepatitis virus (mhv) s proteins were cotransfected into t cells with plenti-luc-green fluorescent protein (gfp) and pspax plasmids (kindly provided by zhaohui qian, chinese academy of medical sciences & peking union medical college) at a molar ratio of : : by using polyethylenimine (pei). the cells were fed with fresh medium in the next h, and the supernatant media containing pseudovirions were then collected and centrifuged at ϫ g for min to remove debris. to quantify s protein-mediated entry of pseudovirions, mdck cells were seeded at about % to % confluence in -well plates and transfected with either papn-flag, hdpp -flag, mceacam a-flag, hace -gfp (kindly provided by zhaohui qian) ( ) , or the control backbone vector by using lipofectamine (thermo fisher). the mdck cells overexpressing each receptor were inoculated with l of : diluted corresponding pseudovirions at h posttransfection. at hpi, cells were lysed at room temperature with l of medium with an equal volume of steady-glo (promega, madison, wi). the cell lysates were also used to confirm the expression of each receptor by using western blotting. transduction efficiency was monitored by quantitation of luciferase activity using a modulus ii microplate reader (turner biosystems, sunnyvale, ca). on the other hand, the mdck cells overexpressing each receptor were inoculated with sads-cov (moi, ) at h posttransfection. ifa was performed to test for sads-cov susceptibility using anti-n pab. the replication competency of sads-cov in mdck cells was further determined by a reverse genetics system. development of a dna-launched sads-cov (seacov) infectious cdna clone (named psea) and rescue of sads-cov by transfection of cultured cells with psea followed by passaging on vero cells have been described recently by our lab ( ) . ethics statement. all animal experiments were performed in strict accordance with the experimental animal ethics committee of zhejiang university (approval number zju ). recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans feline coronavirus antibody titer in cerebrospinal fluid from cats with neurological signs origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states bat-to-human: spike features determining "host jump" of coronaviruses sars-cov, mers-cov, and beyond a novel coronavirus associated with severe acute respiratory syndrome ecology, evolution and classification of bat coronaviruses in the aftermath of sars middle east respiratory syndrome coronavirus: 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hku in respiratory tract of pigs and first discovery of coronavirus quasispecies in =-untranslated region protective role of toll-like receptor -induced type i interferon in murine coronavirus infection of macrophages human immunodeficiency viral vector pseudotyped with the spike envelope of severe acute respiratory syndrome coronavirus transduces human airway epithelial cells and dendritic cells ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign shared common ancestry of rodent alphacoronaviruses sampled globally discovery, diversity and evolution of novel coronaviruses sampled from rodents in china colonisation and shedding of lawsonia intracellularis in experimentally inoculated rodents and in wild rodents on pig farms leptospirosis in piggery workers on trinidad high prevalence of mers-cov infection in camel workers in saudi arabia aminopeptidase-nindependent entry of porcine epidemic diarrhea virus into vero or porcine small intestine epithelial cells identification of the fusion peptide-containing region in betacoronavirus spike glycoproteins the professional editing service nb revisions was used for technical preparation of the text prior to submission. key: cord- - uzivsk authors: zhang, zheng; zhu, zhaozhong; chen, wenjun; cai, zena; xu, beibei; tan, zhiying; wu, aiping; ge, xingyi; guo, xinhong; tan, zhongyang; xia, zanxian; zhu, haizhen; jiang, taijiao; peng, yousong title: membrane proteins with high n-glycosylation, high expression, and multiple interaction partners were preferred by mammalian viruses as receptors date: - - journal: biorxiv doi: . / sha: doc_id: cord_uid: uzivsk receptor mediated entry is the first step for viral infection. however, the relationship between viruses and receptors is still obscure. here, by manually curating a high-quality database of pairs of mammalian virus-host receptor interaction, which included unique viral species or sub-species and virus receptors, we found the viral receptors were structurally and functionally diverse, yet they had several common features when compared to other cell membrane proteins: more protein domains, higher level of n-glycosylation, higher ratio of self-interaction and more interaction partners, and higher expression in most tissues of the host. additionally, the receptors used by the same virus tended to co-evolve. further correlation analysis between viral receptors and the tissue and host specificity of the virus shows that the virus receptor similarity was a significant predictor for mammalian virus cross-species. this work could deepen our understanding towards the viral receptor selection and help evaluate the risk of viral zoonotic diseases. systematic analysis of the characteristics of the viral receptor could help understand the mechanisms under the receptor selection by viruses. the virus-receptor interaction was reported to be a principal determinant of viral host range, tissue tropism and cross-species infection [ , , ] . the existence and expression of the virus receptor in a host (or tissue) should be a prerequisite for viral infection of the host (or tissue) [ ] . usually, a virus mainly infects some particular type of hosts or tissues. for example, the influenza virus mostly infects cells of the respiratory tract [ ] . however, the virus-receptor interaction is a highly dynamic process. some viruses can recognize one or more receptors [ , , ] , which can also differ among virus variants or during the course of infections [ , , ] . in some cases, a few amino acid mutations in the viral protein or the receptor could abolish or enhance viral infection [ ] [ ] [ ] . besides, the virus-receptor interaction is under we firstly investigated the structural characteristics of mammalian virus receptor proteins. as expected, all the mammalian virus receptor protein belonged to the membrane protein which had at least one transmembrane alpha helix ( figure s a ). twenty-four of them had more than five helixes, such as -hydroxytryptamine receptor a (htr a) and npc intracellular cholesterol transporter (npc ). the receptor protein was mainly located in the cell membrane. besides, more than one third ( / ) of them were also located in the cytoplasm, and thirteen of them were located in the nucleus. then, the protein domain composition of the mammalian virus receptor protein was analyzed. the mammalian virus receptor proteins contained a total of domains based on the pfam database, with each viral receptor protein containing more than two domains on average ( figure s b ). this was significantly more than that of human proteins or human membrane proteins (p-values < . in the wilcoxon rank-sum test). some viral receptor proteins may contain more than domains, such as complement c d receptor (cr ) and low density lipoprotein receptor (ldlr). the human viral receptors were observed to have ten or more n-glycosylation sites, such as complement c b/c b receptor (cr ) and lysosomal associated membrane protein (lamp ). figure b displayed the modeled d-structure of htr a, the receptor for jc polyomavirus (jcpyv). five n-glycosylation sites were highlighted in red on the structure, which were reported to be important for viral infection [ ] . for comparison, we also characterized the n-glycosylation level for the human cell membrane protein, human membrane proteins and all human proteins (figure a ). it was found they had a significantly lower level of n-glycosylation than that of human and mammalian virus receptors (p-values < . in the wilcoxon rank-sum test), which suggests the importance of n-glycosylation for the viral receptor. enriched, such as "regulation of leukocyte activation" and "lymphocyte activation". for the go molecular function (table s ) , besides for the enrichment of terms related to the virus receptor activity, the human virus receptor was also enriched in terms of binding to integrin, glycoprotein, cytokine, and so on. consistent with the enrichment analysis of go cellular component, the kegg pathways of "cell adhesion molecules", "focal adhesion" and "ecm-receptor interaction" were also enriched. besides, the pathway of "phagosome" was enriched (table s ) , which may be associated with viral entry into the host cell. interestingly, some pathways associated with heart diseases were enriched, including "dilated cardiomyopathy", "hypertrophic cardiomyopathy", "arrhythmogenic right ventricular cardiomyopathy" and "viral myocarditis". we next analyzed the protein-protein interactions (ppis) which the mammalian virus receptor protein took part in. as the reason mentioned above, we only used the human (table s ) . when looking at the interactions between viral receptors, we found that of viral receptors interacted with themselves. this ratio ( / = %) was much higher than that of human proteins ( %), membrane proteins ( %) and human cell membrane proteins ( %). however, we found the viral receptor tended not to interact with each other ( figure s d since the virus has to compete with other proteins for binding to the receptor, proteins (table s ) . since the viral receptor determines the host specificity of the virus to a large extent, it is expected that the closer between the viral receptor and its homolog in a species, the more likely the virus which used the receptor would infect the species. to validate this hypothesis, we firstly calculated the sequence identities between the viral receptor and their homologs in mammal species ( figure and table s ). for clarity, only mammal species, which were frequently observed, were presented in figure . then, table s . what's the relationship between glycosylation and viral receptor selection? as we know, glycosylation of proteins is widely observed in eukaryote cells [ ] . it plays an important role in multiple cellular activities, such as folding and stability of glycoprotein, immune response, cell-cell adhesion, and so on. glycans are abundant on host cell surfaces. they were probably the primordial and fallback receptors for the virus [ ] . to use glycans as their receptors, a large number of viruses have stolen a host galectin and employed it as a viral lectin [ , ] . for example, the sjr fold, which was mainly responsible for glycan recognition and binding in cellular proteins, was observed in viral capsid proteins of over one fourth of viruses [ ] . thus, during the process of searching for protein receptors, the protein with high level of glycosylation could provide a basal attachment ability for the virus, and should be the preferred receptor for the virus. secondly, our analysis showed that the viral receptor protein had a tendency to interact with itself and had far more interaction partners than other membrane proteins. besides the function of viral receptor, the receptor protein functions in the host cell by interacting with other proteins of the host, such as signal molecules and ligands. therefore, the virus has to compete with these proteins for binding to the receptor [ ] . the protein with less interaction partners are expected to be preferred by the virus. why did the virus select the proteins with multiple interaction partners as receptors? one possible reason is that the receptor proteins are closely related to the "door" of the cell, so that many proteins have to interact with them for in-and-out of the cell. this could be partly validated by the observation that for the interaction partners of human viral receptors, six of top ten enriched terms in the domain of go biological process were related to protein targeting or localization (table s ) . for entry into the cell, the virus also selects these proteins as receptors. another possible reason is that viral entry into the cell needs cooperation of multiple proteins which were not identified as viral receptors yet. besides, previous studies show that the virus could structurally mimic native host ligands [ ] , which help them bind to the host receptor. (table s ). the number of transmembrane alpha helix of the mammalian virus receptor was derived from the database of uniprotkb and the web server tmpred [ ] . the location for the viral receptor was inferred from the description of "subcellular location" for the receptor protein provided by uniprotkb, or from the go annotations for them: the viral receptors annotated with go terms which included the words of "cell surface" or "plasma membrane" were considered to be located in the cell membrane; those annotated with go terms which included the words of "cytoplasm", "cytosol" or "cytoplasmic vesicle", or shown to be in the cytoplasm in uniprotkb, were considered to be located in the cytoplasm; those annotated with go terms "nucleus" (go: ) or "nucleoplasm" (go: ) were considered to be located in the [ ] in r (version . . ) [ ] . to identify the homolog of the mammalian virus receptor in other mammal species, the protein sequence of each viral receptor was searched against the database of mammalian protein sequences, which were downloaded from ncbi refseq database [ ] on october th , , with the help of blast (version . . ) [ ] . analysis showed that in the database of mammalian protein sequences, there were mammal species which were richly annotated and had far more protein sequences than other mammal species (table s ) . therefore, only these mammal species were considered in the evolutionary analysis. based on the results of blast, the homolog for the viral receptor was defined as the hit with e-value small than e- , coverage equal to or greater than % and sequence identity equal to or greater than %. only the closest homolog, i.e., the best hit, in each mammal species was used (table s ). similar methods as above were utilized to calculate the indicators of conservation level for these proteins. for analysis of co-evolution between viral receptors, firstly for each viral receptor, a phylogenetic tree was built based on the protein sequences of the receptor and its homologs in mammal species with the help of phylip (version . ) [ ] . the neighbor-joining method was used with the default parameter. then, the genetic distances between the viral receptor and their homologs were extracted from the phylogenetic tree with a perl script. finally, for a pair of viral receptors, the spearman correlation coefficient (scc) was calculated between the pairwise genetic distances of viral receptors and their homologs, which was used to measure the extent of co-evolution between this pair of viral receptors. the set of housekeeping gene in human was adapted from the work of eisenberg et al [ ] . a total of genes were identified as the housekeeping gene. (table s ) the mammalian virus-host interactions were primarily adapted from olival's work [ ] . one hundred and fifteen viruses in our database and of richly annotated mammal species could be mapped to those in olival's work (table s ). these viruses used a total of viral receptors. the sequence identities of these viral receptor proteins to their related homologs in the corresponding mammal species were presented in table s . for comparison, we also extracted genetic distances (host relatedness) between the mammal species and the viral host with reported receptors based on olival's work (table s ) . then, the genetic distance of the mammal species to the viral host with reported receptors, and the sequence identity of the receptor homolog in the mammal species to the viral receptor protein, was respectively used to predict whether a mammal species could be infected by the virus which infected the host with reported receptors. the method of receiver operating characteristic (roc) curve was used to evaluate and compare their performance with the functions of roc(), auc(), roc.test() and plot.roc() in the package of "proc" [ ] in r (version . . ). statistical analysis all the statistical analysis was conducted in r (version . . ) [ ] . the wilcoxon rank-sum test was conducted with the function of wilcox.test(). clusterprofiler: an r package for comparing biological hz, tj and yp conceived and designed the study. zz and zzz did the computational key: cord- -dn t qa authors: salehi, bahare; sener, bilge; kilic, mehtap; sharifi-rad, javad; naz, rabia; yousaf, zubaida; mudau, fhatuwani nixwell; fokou, patrick valere tsouh; ezzat, shahira m.; el bishbishy, mahitab h.; taheri, yasaman; lucariello, giuseppe; durazzo, alessandra; lucarini, massimo; suleria, hafiz ansar rasul; santini, antonello title: dioscorea plants: a genus rich in vital nutra-pharmaceuticals-a review date: journal: iran j pharm res doi: . /ijpr. . . sha: doc_id: cord_uid: dn t qa dioscorea species, known as “yams,” belong to family dioscoreaceae. this genus consists of more than species distributed from africa, asia, the caribbean’s south america, and the south pacific islands. their organoleptic properties make them the most widely used carbohydrate food and dietary supplements. the underground and/or aerial tubers represent valuable sources of proteins, fats, and vitamins for millions of people in west africa. this review gives a shot of secondary metabolites of dioscorea plants, including steroids, clerodane diterpenes, quinones, cyanidins, phenolics, diarylheptanoids, and nitrogen-containing compounds. this review collected the evidence on biological properties of description dioscorea, including in-vitro and in-vivo studies. dioscorea species contain promising bioactive molecules i.e. diosgenin that support their different biological properties, including antioxidant, hypoglycaemic, hypolipidemic, anti- antimicrobial, inflammatory, antiproliferative, androgenic, estrogenic, and contraceptive drugs. indeed, besides its nutrient values, dioscorea is a potential source of bioactive substances of interest in the prevention/treatment of several diseases, and thus represents a great challenge in developing countries. however, ethnomedicinal potential should be validated and further researches on pharmacological properties and phytochemical composition should be carried out. particularly, doing some studies to convert the preclinical results to clinical efficacy should be guaranteed. dioscorea, food plant, traditional use, phytochemistry, pharmacological activities dioscorea belongs to the family dioscoreaceae, sub-family dioscoreoideae, consisting of species. dioscorea species are native to the old-world including high temperatures tropics and subtropic regions of the world. the major part of species occur in west africa, southern asia, and tropical america: they are herbaceous climbers with rhizome or tubers dioscorea species and, being usedin the formulation of pharmaceutical products, have a high medicinal, industrial and commercial importance ( ) . indeed, dioscorea species account for the most important dietary supplements ingredients used in cosmetics and pharmaceutical industries. it has also been commonly used by local people, mostly those who are engaged in the trade of medical plants worldwide. indeed, tubers of different dioscorea species are used as a cure for different diseases and ailments (cough, cold, stomach ache, leprosy, burns, fungal infections, dysentery, skin diseases, rheumatism, arthritis, etc.) in several formulations, and even for birth control ( ) . it is well known the potential of this plant is related due to the different phytochemical compounds found in dioscorea. tubers and roots contain steroidal sapogenins, mostly diosgenin as well as volatile compounds ( , ) . chemical substances like diosgenin found in dioscorea are commercially used in the pharmaceutical industry. the high demand for the medicinal use of this plant in different parts of the world suggests a strong need for conservation strategies. however, the conservation might not be easy or simple as it must involve plant protection and wellcontrolled access to its resources. there still a huge need for more research and information on the food, nutraceutical, and medicinal value worldwide of this plant species. therefore, this review tends to fill this gap by summarizing data on the current medicinal importance of dioscorea species. researches have been brought new knowledge about chemical compounds in tubers of dioscorea species and possible medicinal usage. the most common secondary metabolites are saponins, and more than steroidal saponins (based on aglycon part as stigmastanol, furostanol, spirostanol, cholestanol, ergostanol, and pregnanol glycosides (figure )) were isolated from various dioscorea species. besides these steroids, clerodane diterpenes, phenolics, cyanidins, quinones, diarylheptanoids, and nitrogen-containing compounds in the tubers of dioscorea species were quantified ( ) . steroidal saponins mainly consist of furostane, a pentacyclic ring system with a sixth open ring; spirostane, a hexacyclic ring system; and pregnane, a tetracyclic ring system. the sugar part is mainly composed of glucose and rhamnose in various proportions and linkages. steroidal saponins are glycosides consisting of an aglycone (diosgenin) and several glycosyl moieties. the most common sugars encountered in saponins are pentoses (arabinose, xylose, etc.), hexoses (glucose, galactose, etc.) and -deoxyhexose (rhamnose, etc.). the saponins of this plant species are both water-soluble and water-insoluble steroid saponins. dioscorea species are well known for containing steroidal saponins, which were used as marker compounds for quality control of the botanical products. as reported by jesus et al., , diosgenin ( -β-hydroxy- spirostene) is the primary furostanol saponin found in several plants, including dioscorea species, and is described as a promising bioactive compound with several medicinal properties, i.e. hypolipidemic, antioxidant, anti-inflammatory, hypoglycaemic, and antiproliferative activities ( ) . diosgenin obtained by hydrolysis of yam saponin were the main raw material for the industrial production of steroid drug i.e. anti-inflammatory, androgenic, estrogenic, and contraceptive drugs, by underlying its potential in the prevention/treatment of several diseases. dioscorea villosa l. roots and rhizomes, also are known as "wild yam", is a rich source of diosgenin. today, dietary supplements containing wild yam extracts are popular among women for the alleviation of menopausal symptoms and are widely used as alternatives to hormone replacement therapy ( ) . dioscorea alata l. dioscorea alata l. is one of the most popular varieties of yams. dioscorea alata (d. alata) were implicated in the promotion of the health of postmenopausal women. wild mexican yam was marketed for the treatment of irritability, hot flashes, insomnia, and depression. cheng et al., , reported the isolation and identification of two new compounds, hydro-q chromene, and γ-tocopherol- ; together with four known compounds (rrr-α-tocopherol, coenzyme q , cycloartane, and -feruloylglycerol): these compounds had phenolic hydroxyl group in common with the known phytoestrogens and have also antioxidant activity. moreover, their estrogenic activity was evaluated based on the ligand-dependent transcription activation assay ( ) . dioscorea antaly jum. and h. perrier is originated from madagascar, diffused in the west and north-west regions. the study of rakatobe et al., reported two clerodane diterpenoids, antadiosbulbins a and b and two furanoid -norclerodane diterpenes, -epidiosbulbins e and g along with the known diterpenoid diosbulbin e as well as nine known phenolics including five phenanthrenes and four flavonoids in the ethyl acetate soluble part of the methanolic extract of the tubers ( ) . dioscorea bulbifera l. dioscorea bulbifera l. (d. bulbifera l.), commonly known as air potato, is originated from africa and southern asia. the rhizome of d. bulbifera l. is called as "huang yao zi" in traditional chinese medicine and used for the treatment of thyroid diseases and cancers. in the northern districts of bangladesh, this herb is used for the treatment of cancers and leprosy. this plant has two types of storage organs, namely bulbils in the leaf axils of the stem and tubers. bulbils have been used as a folk remedy to treat diarrhea, conjunctivitis, and the extracts of the plant exhibit anti-tumor activity. the mannose-binding lectin from bulbils of d. bulbifera was studied for its clinical potential in hiv and cancer research ( ) . phytochemical studies reported as main bioactive compounds of this plant: flavonoids, clerodane diterpenoids, and steroidal saponins and phenolic compounds. as instance, liu et al. reported the isolation and identification of two new compounds, bibenzyl type- , , ′, ′-tetrahydroxy- ′methoxybibenzyl and diarylheptanone containing a dihydrophenanthrene moiety named as diobulbinone a along with sixteen known compounds ( ) . nine norclerodane diterpenoids were isolated from the tubers of d. bulbifera l. including two new compounds (diosbulbin n and diosbulbin p), and a naturally occurring compound (diosbulbin o) along with six known diosbulbins a-d, f, and g ( ) . another study also showed the molecular changes of liver dysfunction and reveal overall metabolic and physiological mechanisms of the subchronic toxic effect of dioscorea bulbifera rhizome ( ) . lam.-holl known as "yellow yam" is native to tropical west africa and its rhizomes are used as food and as a remedy for treating burns and fevers. a new furostanol glycoside namely -o-β -d-glucopyranosyl- β, -dihydroxy- , -seco- (r)-furost- en- , -dione- -o-α-l-rhamnopyranosyl-( → )-α -l-rhamnopyranosyl-( → )-[ α -l -r h a m n o p y r a n o s y l -( → ) ] -β -dglucopyranoside was isolated from the methanolic extract of the rhizome of dioscorea cayenensis growing in cameroon, together with the known spirostanol saponins described as methyl protodioscin, asperoside and prosapogenin a of dioscin ( ) . prosapogenin a of dioscin having a spirostan skeleton showed antifungal activity against candida species with mic values between . and mg/ml ( ) . two new fatty acidspirostan steroid glycoside esters, progenin iii palmitate, and progenin iii linoleate, were isolated from the methanol extract of the rhizomes of dioscorea cayenensis ( ) . it is worth mentioning the recent studies on the nutrient and antinutrient composition of yellow yam (dioscorea cayenensis) and their products; as instance raw dioscorea cayenensis tubers contained . g moisture, . g crude protein, . g lipid, . g fiber, . g ash, . g carbohydrates, . mg potassium, . mg magnesium, . mg iron, . mg zinc, and yielded . kcal energy with insignificant vitamin content/ g edible portion ( ) . dioscorea esculenta (lour.) burk. dioscorea esculenta also called as a "lesser yam" of dioscorea species, is widely distributed southern asia and the pacific. the tubers of d. esculenta were used traditionally in the treatment of various diseases. moreover, fiteen steroidal saponins were isolated from its tuber powder, and from the leaf of dioscorea esculenta (d. esculenta); four of them belonged to furostanol-type saponin,eleven as spirostane-type saponins ( ) . the rhizome of dioscorea japonica thunb., known as "san yak" in korea, is used as food and medicine. the plant was used in traditional chinese medicine to control/eliminate diarrhea, dilute sputum, improve anorexia, and moisturize skin ( ) . phytochemical studies on dioscorea japonica (d. japonica) showed the presence of active hypoglycemic compounds (dioscorans a-f), sesquiterpene, and acetophenone along with the steroidal constituents, including spirostane, furostane, and cholestane types ( ) . the rhizomes of dioscorea membrancea (d. membranacea) pierre have been used in thai traditional medicine. as instance, thongdeeying et al. isolated from the cytotoxic chloroform fraction of the rhizomes a new steroid (epi-panthogenin b), a known steroid (panthogenin b), two napthofuranoxepins dioscorealide a and dioscorealide b, phenanthraquinone (dioscoreanone) and three phenanthrene; the same authors reported that also, three steroids (β-sitosterol, stigmasterol, and β-dsitosterolglucoside) and two steroidal saponins [(diosgenin- -o-α-l-rhamnopyranosyl ( → )-β-d-glucopyranoside and diosgenin- -o -β -d -g l u c o p y r a n o s y l ( - ) -β -dglucopyranoside)] were obtained from d. membranacea ( ) . makino is bitter-sweet in taste and warm in nature according to the traditional chinese medicine. the rhizomes of d. nipponica were traditionally used in china as herbal medicine and food supplement for more than four thousand years; particularly, these plant species mainly act on the liver, kidney, and lungs, displaying anti-rheumatic, analgesic, blood circulation-stimulating, lung channeldredging, digestive, anti-diuretic, anti-tussive, panting-calming, and phlegm-dispelling activities. medicinally, it is commonly used for the treatment of rheumatoid arthritis, pain in the legs and lumbar area, kashin-beck disease, bruises, sprains, chronic bronchitis, cough and asthma and communication, and adherence to therapy is one of the main concerns ( , and ) . recently, it has been used as an important industrial raw material for the synthesis of steroidal drugs. concerning the phytochemical profile, twelve cyclic diarylheptanoids were isolated from the rhizomes of dioscorea nipponica ( ) , among which two new cyclic diarylheptanoids, diosniponol a and b; moreover, as reported by the same authors, these compounds were evaluated for their effects on nitric oxide production without cell toxicity in lipopolysaccharide-activated bv- cells. therefore, diarylheptanoid derivatives from d. nipponica may be potential candidates for the treatment of various neurodegenerative diseases associated with neuroinflammation ( ) . another study described the watersoluble non-saponin components [benzyl -o-β-d-glucopyranoside, leonuriside a, icariside d , pyrocatechol- -o-β-dglucopyranoside, (+) syringaresinol- -oβ-d-glucopyranoside, cyclo-(leu-tyr), and adenosine], and phenolic acid (piscidic acid) from the rhizomes of d. nipponica ( ) . on the other hand, -oxodioscin a new spirostan-type steroid along with known dioseptemloside g, ( r)-dracaenoside g, orbiculatoside b, dioscin, progenin iii, gracillin were determined. the presence of ( β, α, r)- - were also confirmed as furastan-type steroids ( ) . it is worth mentioning the study of that explore the chemical basis of the rhizomes and aerial parts of dioscorea nipponica makino by a combination of cheminformatics and bioinformatics, particularly their potential therapeutic and toxicity targets were screened through the drugbank's or t db's chemquery structure search: the compounds in the rhizomes possessed potential therapeutic targets and potential toxicity targets ( ) . dioscorea opposita thunb. dioscorea opposita thunb. has been cultivated in china, japan, and korea as a food, and widely used as traditional medicine, for a very long time. there are about yam cultivars that are originated in china, including the so-called "trueborn" chinese yam. the important ingredient of the yam is sugar, which provides energy and sweetness and adds to the general flavor of foods. also, the chinese yam assists in food digestion and is beneficial in cases of stomach disorders ( ) . phytochemical investigations of dioscorea opposite (d. opposita) have revealed many chemical components such as purine derivatives, phenanthrenes, stilbenes, sapogenins, and saponins. phytochemical studies of the chloroform soluble fraction of d. opposita thunb. have resulted in the isolation of four new compounds, two dihydrostilbenes: , -dihydroxy- -methoxybibenzyl, , ′, trihydroxy- ′-methoxybibenzyl; and dibenzoxepins , -dihydro-dibenz[b,f]oxepin- , diol and , -dihydro- -methoxy-dibenz[b,f] oxepin- -ol, together with an additional fifteen known compounds. all the nineteen isolated compounds were tested in the dpph, superoxide anion radical scavenging assays and cyclooxygenases (coxs) inhibition assay. among them, , ′, -trihydroxy- ′methoxybibenzyl showed the most potent cox- inhibitory activity ( ) . bioassayguided fractionation of d. opposita extracts led to the isolation and identification of phenolic compounds. of them, compounds reduced porcine pancreatic lipase activity at ic values of less than mm and from them , ′, -trihydroxy- ′-methoxybibenzyl and ( e, e)- , -bis( -hydroxyphenyl)- , -heptadien- -one showed the highest inhibition with an ic value of . mm and dose-dependently in the concentration range - mm ( ) . these findings provide that phenolic compounds with stilbenoids are considered to play important roles in the lipase inhibition of the d. opposita extract. especially, the stilbenoids possessing , -dihydroxybibenzyl moiety showed higher inhibitory potencies than the others. the lipase inhibitory activities of stilbenoids depend on the presence of the hydroxyl group in the c- position. the structural difference also influences the inhibitory effect of diarylheptanoids on pancreatic lipase. dihydrostilbene phenanthrene and diarylheptanoid structures were deemed to be responsible for lipase inhibition activity of this species ( ) . d. opposita thunb. is also rich in starch, water-soluble polysaccharides, and mucilage defined as a polysaccharide with unique viscosity characteristics are widely used in the pharmaceutical and food industries as a thickening agent and emulsion stabilizer. these agents are important in the food industry as they improve the sensory quality, flavor, texture, palatability, mouthfeel, and general appearance of the final products. therefore, the mucilage of this plant species is a potential candidate for food emulsifier resource of a natural emulsifier, especially under alkaline conditions obtained from industrial processing waste such as gum arabic, yellow mustard, and chia (salvia hispanica l.) ( ) . dioscorea preussii pax d. preussii pax collected in bambui, cameroon, was reported to have in-vitro cytotoxic, antileishmanial and antifungal activities. three new steroidal saponins, named as diospreussinosides a, b, c, along with two known compounds were determined as ( r)- α-hydroxyspirost- - r h a m n o p y r a n o s y l -( → )α -l -r h a m n o p y r a n o s y l -( → ) ] -β -dglucopyranoside were isolated from rhizomes of d. preussii ( ) . dioscorea pyrifolia kunth d. pyrifolia kunth is called also as "akar kemeyan paya" in malaysia. these plants grow wild and are diffused particularly in peninsular malaysia. sharlina et al. reported that the starch extracted from d. pyrifolia was characterized by the high amylose content ( . ± . %); the same authors marked how d. pyrifolia kunth represents a very important source of starch for food and non-food applications, such as crispy food, coating materials, hydrogels, films, bio-membranes, and adhesives ( ) . cholestane glycosides, dioseptemlosides a and b, together with six spirostane glycosides, dioseptemlosides c-h, were isolated from the rhizomes of d. septemloba. among these, spirostane aglycones containing hydroxyl group at c- were reported in the family dioscoreaceae. spirostane-type glycosides showed strong activity on nitric oxide production ( ) . on the other hand, three new diarylheptanoids, dioscorol a, dioscorosides e , e ; two new stilbenes, dioscorosides f and f were isolated from the rhizomes of dioscorea septemloba. besides, , -bis( -hydroxyphenyl)hepta- e, e-dien- -one, , -bis( -hydroxyphenyl)- , , -heptatrien- -one, , -dihydroxy- , -bis( -hydroxyphenyl)heptane, ( r, r)- , -dihydroxy- , -bis( -hydroxyphenyl)heptane -o-β-d-glucopyranoside, ( r, r)- , -dihydroxy- , -bis( -hydroxy- were also obtained as known compounds. they have also shown a significant increase in the glucose consumption in differentiated l myotubes and displayed triglyceride inhibitory effects in hepg cells . eight new compounds namely, dioscorosides g, h , h , dioscorol b, dioscorosides i, j, k , and k , together with twelve known ones were isolated from the rhizomes of dioscorea septemloba. moreover, the authors showed that some of these compounds were found to display significant inhibition of nitrite production evaluated in-vitro anti-inflammatory potential using lps-stimulated raw . murine macrophages ( ) . currently, he et al. have identified and elucidated the structure of a new phenanthropyran, dioscorone b, and a new phenanthrene, together with seven known compounds, is from the % ethanol extract of dioscorea septemloba rhizomes. moreover, the authors reported that new isolated phenanthropyran, tested for antioxidant activity, showed excellent activities with ic values of . ± . μm and . ± . μm, respectively ( ). dioscorea tokoro makino d. tokoro is one of those wild yams, and its rhizome was generally used for daily food.; in particular in the northern part of japan, the rhizome was used as health-promoting food. d. tokoro was characterized by steroidal saponins, such as dioscin and gracillin, but no data were found about its health-promoting effect. the acetonitrile extract of d. tokoro rhizome fractionation led to protodioscin as an antiproliferative compound to hl- leukemic cells ( ) . the most well-known species of this genus is dioscorea villosa, also called "wild yam." two furanostane type saponins, namely methyl parvifloside, and protodeltonin, were isolated from d. villosa as well as two spirostane types deltonin and glucosidodeltonin (zingiberensis i), whereas minor saponins were methylprotodioscin, disoscin, and prosapogenin ( ) . the same authors reported that two fla-van- -ol glycosides were also isolated from d. villosa and fifteen steroidal compounds, including two new metabolites, were isolated from the tubers of d. villosa; these compounds were characterized as cholestane type steroidal glycosides named as dioscoreavillosides a and b ( ). dong et al., described how the rhizomes were also afforded diarylheptanoids, five of which were new compounds containing a tetrahydropyran ring in the heptane portion of the molecule ( ) . in another study, three furostanol saponins, parvifloside, methyl protodeltonin, and trigofoenoside a- were isolated from the n-butanol soluble extract of d. villosa by using hsccc; moreover the authors reported that subsquent normal-phase hsccc separation also led to the identification of four spirostanol saponins identified as zingiberensis saponin i, deltonin, dioscin, and prosapogenin a of dioscin ( ) . on the other hand, two series of lipidated steroid saponins were isolated from d. villosa ( ) . the series a was identified as a mixture of five lipidated steroid saponins: the series b was characterized as a mixture of the following five compounds: -en-clionaster- these findings revealed two classes as new biomarkers: the diarylheptanoids and the lipidated steroid saponins. the rhizome of dioscorea zingiberensis c.h. wright is known as ''huang jiang" and represents an important source of diosgenin. d. zingiberensis were used for the treatment of lung heat, pyretic stranguria, anthracia, coronary heart disease, and swelling; in particular, the rhizome of dioscorea zingiberensis is extensively used for the extraction of diosgenin sapogenin and its glycoside dioscin, used for the synthesis of sex hormones and corticosteroids. it is worth mentioning the current study of zhang et al., that give comprehensive overview of the traditional usage and phytochemistry of the dioscorea zingiberensis c.h. wright: -more than compounds have been identified; -several of these have been tested in preclinical assays and clinical trials; -a wide spectrum of biological effects including cardiovascular, anti-thrombosis, hyperlipidemia, neuroprotection, anti-inflammatory, and anthelmintic effect has been verified ( ) . wang et al. reported how spirostanol based aglycon containing steroidal saponins are the representative steroidal saponins, and their quantity is higher than that of furostanol steroidal saponins in d. zingiberensis ( ) . apart from the steroidal saponins, other kinds of constituents were also identified from d. zingiberensis. until now, four steroids have been isolated from this plant including β-sitosterol, sterol, zingiberenin f, and ( r)- β-hydroxyspirost-Δ , ( ) in addition to the common steroidal saponins, other constituents classified as alkaloid, phenyl-glycoside, and benzoic acid derivatives have also been determined. -aminomethyl-phenol, -o-β-d-glucopyranosyl- -hydroxybenzoic acid and another nonsteroidal saponin called -o-β-d-glucopyranosyl- -methoxybenzoic acid, were isolated and identified from the n-buthanol extracts of fresh rhizomes. following the regular phytochemical research procedure, five compounds were obtained from d. zingiberensis ( ) . they consisted of two new phenanthrene derivatives, namely , , -trimethoxy- , -dione- , -dihydrophenanthrene and , , -trihydroxy- , -dimethoxy- , -dihydrophenanthrene; a new anthracenedione, i.e., , , -trimethoxy- , -dione-anthracene; and two known , -dihydrophenanthrenes, called , -dihydroxy- , -dimethoxy- , -dihydrophenanthrene and , -dihydroxy- , , -trimethoxy- , -dihydrophenanthrene. one new bibenzyl ( , -dihydroxy- , -dimethoxybibenzyl.) and one new phenanthrene ( , -dihydroxy- , -dimethoxy- , -dihydrophenanthrene.), together with two known bibenzyls ( , -dihydroxy- -methoxybibenzyl , ′,-dihydroxy- ′, -dimethoxybibenzyl and four known diarylheptanoids (( r, r)- , -dihydroxy- , -bis( -hydroxyphenyl)heptane, ( r, r)- , -dihydroxy- -( , -dihydroxyphenyl)- -( -hydroxyphenyl)heptane, ( r, r)- , -dihydroxy- -( -hydroxy- -methoxyphenyl)- -( -hydroxyphenyl)-heptane and ( r, r)- , -dihydroxy- , -bis( -hydroxy- -methoxyphenyl)-heptane were isolated from the dichloromethane soluble fraction of the rhizomes of d. zingiberensis ( ) . all these phenolic compounds were evaluated for their anti-pancreatitic activities on sodium taurocholate-induced pancreatic acinar necrosis as inhibition of necrotic cell death pathway activation. among them, ( r, r)- , -dihydroxy- -( , -dihydroxyphenyl)- -( -hydroxyphenyl) heptane which bears a trihydroxy-diarylheptane skeleton was shown to protect against pancreatic acinar cell injury through mediating novel potential therapy for pancreatic necrosis ( ) . the tubers of different dioscorea species present a wide variation of bioactive compounds, responsible for their pharmacological activities. generally, the evaluation of bioactive components and the assessments of their interactions could be viewed as the first step for the determination of potential of a plant ( ) ( ) ( ) ( ) ( ) ( ) ( ) . also, their ethnopharmacological importance promotes further investigations of dioscorea metabolites as a source of potential therapeutic agents to ameliorate different diseases (table ) . cytotoxic activity d. bulbifera ethanolic crude extract and different fractions (hexane, ethyl acetate, and water) at concentrations of , , and μg/ml were found to possess a potent cytotoxic activity against human colorectal carcinoma (hct ), human colorectal adenocarcinoma (ht- ), human lung carcinoma (a ), human breast carcinoma (mcf- ), human tuber immune-modulating activity ( ) . anti-inflammatory activity ( ) anti-severe acute respiratory syndrome associated coronavirus ( ) . gastroprotective activity ( ) enhance murine splenocyte proliferation ex-vivo and improve regeneration of bone marrow cells ( ) immunomodulatory activity ( ) anti-inflammatory activity ( ) . neuroprotective activity ( ) antioxidant activity ( ) mediated synthesis of gold and silver nanoparticles with catalytic activity ( ) neuroprotective activity ( ) antidiabetic activity ( ) cell shrinkage, and dna fragmentation as shown by flow cytometry. the authors also revealed that dbeaf induces apoptosis effects on hct cells through externalization of phosphatidylserine and by promoting the loss of mitochondrial membrane potential (mmp), dysregulation of the bcl- family proteins and the downregulation of the expression of procaspase - , - , - and - and parp protein expression. furthermore, their results suggested that the inhibitory effect of dbeaf on erk / and the activation of jnk were closely associated with the apoptotic cell death induction in human colorectal carcinoma ( ). rhizome antioxidant activity ( ) antitumor activity ( ) anti-hypercholesterolemic effect ( ) anti-platelet aggregation activity ( ) antifungal activity ( ) antihyperlipidemic and antioxidative activity ( ) dioscorea pentaphylla l. antioxidant activity and antibacterial activity d. collettii var. hypoglauca and its active metabolite protoneodioscin, a furostanol saponin, was also evaluated for its cytotoxic activity against cell lines including human leukemia, melanoma, and cancers of lung, colon, brain, ovary, renal, breast, and prostate. protoneodioscin exhibited significant cytotoxicity with % growth inhibition (gi ) ranging from . to mm against all cell lines from leukemia and most solid tumors ( ) . in the following year, the same research team evaluated two other compounds, namely; methyl protoneogracillin and gracillin. the results revealed that methyl protoneogracillin exhibited significant cytotoxicity with (gi ) ≤ mm. leukemia, cns cancer, and prostate cancer were the most sensitive subpanels, while ovarian cancer was the least sensitive subpanels. on the other hand, gracillin lacked selectivity against human cancer diseases ( ). a different approach was undertaken by ashri and co-workers ( ) ( ), who evaluated the cytotoxicity of modified d. hispida starchbased hydrogels on small intestine cell line (fhs- int) to ensure the safety of their use. their results revealed that the prepared hydrogels did not show any cytotoxicity and could be employed for future pharmaceutical use. a mucopolysaccharide of yam (d. batatas decne) (ymb) was found to increase the cytotoxic activity of mouse splenocyte against leukemia cell at µg/ml concentration. however, it did not affect the viability of splenocytes. the production of ifn-γ was significantly increased in the ymp treated splenocytes. at µg/ml concentration, it increases the up taking capacity and lysosomal phosphatase activity of peritoneal macrophages. in addition, ymp ( - µg/ml) significantly increased the viability of peritoneal macrophages ( ) . dioscorin, the glycoprotein isolated from d. alata was reported to activate toll-like receptor (tlr ) and induce macrophage activation via typical tlr -signaling pathways at µg/ml concentration. it induces tlr downstream cytokine expression in bone marrow cells isolated from tlr -functional c h/hen mice but not from tlr -defective c h/hej mice. dioscorin also stimulated multiple signaling molecules (nf-jb, erk, jnk, and p ) and induced the expression of cytokines (tnf-a, il- b, and il- ) in murine raw . macrophages ( ) . different dioscorea species were used traditionally for the treatment of memoryrelated disorders and dementia, i.e. alzheimer disease and other neurodegenerative diseases ( ) . the chloroform extract from d. opposite rhizomes significantly reduced the glutamateinduced toxicity in a dose-dependent manner with a maximum neuroprotective effect at µg/ml ( ) . kim ( ) . moreover, the compounds isolated from d. nipponica rhizomes exhibited anti-neuroinflammatory and neuroprotective activities, the most active of which was , -dihydroxy- , , trimethoxy-phenanthrene. the later was the most potent nerve growth factor (ngf) inducer, with . % stimulation, and strongly reduced nitric oxide (no) levels with an ic value of . μm in lps-activated bv microglial cells. also, it significantly increased neurite outgrowth in mouse neuro a (n a) cells and therefore possessed a significant neuroprotective activity ( ) . antidiabetic activity different extraction procedures as ultrasonic-assisted extraction, cold water extraction, warm water extraction and hot water extraction significantly influenced the antidiabetic potential of the rhizomes of d. hemsleyi as determined via both alphaglucosidase and alpha-amylase inhibition assays, where the ic values of alphaglucosidase inhibition assay varied from . to . µg/ml while those of alpha-amylase inhibition assay varied from . to . mg/ ml ( ) . the antioxidant activity of the extracts of dioscorea species received much scientific attention ( , , and ) . different extracts of rhizomes of d. hemsleyi, d. hispida dennst, d. opposite thunb., d. nipponica makino, d. esculenta (lour). burkill, d. japonica thunb. var. pseudojaponica and d. pentaphylla l. were all studied for their antioxidant properties by different in-vitro assays namely; reducing power assay, ferric reducing antioxidant power assay (frap), dpph radical scavenging assay, hydroxyl radical scavenging assay, superoxide radical assay, self-oxidation of , , -phentriol assay and antioxidant activity by radical cation (abts) assay. generally, the studied extracts from different dioscorea species showed a significant antioxidant potential which may account for their involvement in the treatment of various disorders via radical scavenging mechanisms. lipopolysaccharide-stimulated raw . cells were employed to determine the antiinflammatory properties of the ethanolic extract of tubers of d. japonica thunb. var. pseudojaponica and individual steroid glycosides isolated from d. septemloba rhizomes ( , ) . d. japonica extract at doses of and µg/ml significantly inhibited lps-induced inos and cox- protein expressions, also, the nitrite relative concentration percentage ranged between . % to . % for the steroid glycosides isolated from d. septemloba rhizomes. similar to the total ethanol extract of d. membranacea presented a potent inhibitory activity, with an ic value of . µg/ml. its most potent metabolite included diosgenin- -o-alpha-lrhamnosyl ( --> )-beta-d-glucopyranoside that showed a powerful inhibitory activity with ic value as low as . µg/ml ( ) . the study of the mechanism of anti-inflammatory activity of the ethanolic extract from the bark of d. batatas decne showed that it inhibited both no and pge production with an ic of - µg/ml respectively. it was also suggested that the extract act suppressing dna-binding activity and reporter gene activity as well as translocation of the nf-jb p subunit. it also down-regulated ijb kinase (ikk), thus inhibiting lps-induced both phosphorylation and the degradation of ijba. in addition, it also inhibited the lps-induced activation of erk / ( ) . a current study of hwang et al. reported the anti-inflammatory effect of aerial bulblets of dioscorea japonica thunb extract through inhibition of nf-κb and mapk signaling pathway in raw . . antiallergic activity d. membranacea ethanolic extract and compounds showed antiallergic activities by inhibiting beta-hexosaminidase release as a marker of degranulation in rbl- h cells, with an ic value of . µg/ml. from this extract, dioscorealide b showed the highest activity with an ic value of . µg/ml ( ) . du et al. reported how several compounds were isolated from the rhizomes of d. zingiberensis i.e. ( r, r)- , -dihydroxy- -( , -dihydroxyphenyl) - -( -hydroxyphenyl) heptane at a concentration of . mm showed anti-pancreatitis activities on sodium taurocholate -induced pancreatic acinar necrosis with an inhibitory effect of . %. moreover, the same authors marked that this compound's protective effects were mainly dependent on atp inhibition and excessive ros production and thus saving the cells from mitochondrial dysfunction ( ) . another study reported that the steroidal saponins obtained from the rhizomes of d. zingiberensis showed anti-arthritic activities on the lps stimulated . macrophage cells through induction of both p and iκbα protein expressions ( ) . the methanolic extract of tubers of d. pentaphylla l. showed antibacterial activity against s. pyogenes, s. mutans, v. cholera, s. typhi, and s. flexneri. the best activity was: . mm and . mm inhibition zone diameter observed (at μg/disc and μg/ disc) against s. pyogenes using disc diffusion, agar well diffusion assays, respectively. the mic values were μg/ml for the extract against v. cholera, s. typhi and s. flexneri while it was μg/ml against s. pyogenes and s. mutans. similarly, rajendra et al. reported the antibacterial activities of % and % methanolic extract of d. deltoidea wall ex griseb rhizomes against s. aureus and e. coli, but this concentration does not show activity against s. typhi and p. aeruginosa. it could be concluded that, in general, dioscorea species do not exhibit a potent antibacterial activity ( ) . the study of cho et al. studied dioscin, isolated from wild yam d. nipponica root bark, for its potential as an antifungal agent via the propidium iodide assay and calceinleakage measurement, in addition to, its ability to disrupt the plasma membrane potential, using , ′-dipropylthiadicarbocyanine iodide and bis-( , -dibarbituric acid)-trimethine oxanol. as reported by cho et al. the results showed that dioscin exerts a considerable antifungal activity, where, the dye-stained cells showed a significant increase in fluorescent intensity after treatment with dioscin, demonstrating that dioscin possess an effect on the membrane potential. the auhors concluded that dioscin could act by disrupting the fungal membrane structure resulting in cell death ( ) . globally a large number of dioscorea plants have investigated in-vivo. data on dioscorea species potency on the animal model are summarized in table . the anti-constipation activity dioscorea opposita thunb. yam tuber is rich in starch, which has always been ignored and discarded during the isolation of bioactive compounds. huang et al., studied the anti-constipation effect of native yam starch (ns), dual enzyme-treated starch (des), and cross-linked carboxymethyl starch (ccs) in constipation model induced by loperamide in km mice. the modified starches (des and ccs) could benefit the bowel function by increasing stool frequency and defecation moisture as they have the good water-binding capacity and swelling ability, the modified starches could increase the water-holding capacity of stools to remain soft. des and ccs groups had improved small intestinal propulsion through the production of more short chain fatty acids (scfas) and decrease ph by increasing acetic acid and butyric acid concentration in the feces. the study done on mice with high-fat diet showed that both native and modified starch from yam had anti-hyperlipidemic activity through a significant decrease of both cholesterol and triglycerides and the liver index. the reduction of superoxide dismutase (sod) and increase malondialdehyde (mda) was observed in hyperlipidemic mice while they were both improved in all starches-treated mice proving their antioxidant effect (table ) ( ). four types of resistant starch (rs) including native (rs ), retrograded (rs ), crosslinked with sodium trimetaphosphate/sodium tripolyphosphate (rs ) and complexed with palmitic acid (rs ) resistant starches from winged yam (d. alata l.) showed the gastroprotective activity of in ethanol-induced gastric ulcer in mice. the increase in gastric emptying rate may help in reducing the contact time of ethanol and gastric mucosa and helps to prevent the gastric tissue damage. therefore, an increase in gastric emptying induced by rs is a possible mechanism of anti-gastric ulcer. moreover, rs and rs groups generated more short chain fatty acids which are produced through fermentation of the starch by the colon microflora. the fatty acids have many physiological and biological functions, such as increasing the immunity of the stomach. etoh-ulcer can result from the imbalance between the generated reactive oxygen species (ros) and the natural antioxidant power. thus, the free radical scavenging is one of the mechanisms for inhibition of gastric ulcer ( ). mao et al. reported the decrease of sod activity in ethanol-treated rats with an increase in mda levels in gastric mucosa indicating severe oxidation reaction in ethanol-induced gastric ulcer mice. rs ( . g/kg) and rs ( . g/kg) exhibited the best antioxidant effect. therefore, the inhibition of oxidative stress by the four types of rs is a possible mechanism that may assist in decreasing the gastric mucosal damage. generally, the high dose groups of rs and rs ( . g/kg) showed the best activity ( ) . a current study also reported the protective effects of dioscorea batatas flesh and peel extracts against an ethanol-induced gastric ulcer in mice ( ) . li et al. reported the significant antithrombotic and anticoagulation effect of the total steroidal saponin extract (tsse) from the rhizomes of d. zingiberensis c.h. wright on inferior vena cava ligation thrombosis rat model and pulmonary thrombosis mice model. oral administration of the extract significantly inhibited adenosine diphosphateinduced platelet aggregation (pag) in-vivo, which was more effective than xue-sai-tong, a commercial product of triterpenoid saponins from panax ginseng having antithrombosis activity in china. in addition, tsse inhibited the thrombosis in a dose-dependent manner (more than % inhibition rate) which was more powerful than the standard xue-saitong at . , and . mg/kg doses of tss, and markedly reduced thrombus weight. tsse also exhibited in a dose-dependent manner prolongation of thromboplastin time (aptt) (which is an intrinsic clotting index), prothrombin time (pt) (that evaluates the extrinsic clotting pathway), and thrombin time (tt) (which is a test of fibrin formation, the addition of thrombin directly induces that). the more pronounced effect of tsse on pt and tt than aptt indicates that it inhibits the extrinsic pathway of coagulation and fibrin formation, and cut down the thrombotic risk ( ) . the ethanolic extract of the tubers of d. japonica thunb. var. pseudojaponica (eedj) showed in-vivo anti-inflammatory activity by significantly inhibiting the development of carrageenan-induced paw edema in mice after the th and th hour of the treatment at . g/kg. it also decreased the levels of tnf-α, which is a mediator of carrageenan-induced inflammatory response and induced the release of kinins and leukotrienes. carrageenaninduced paw edema causes oxidative stress with the release of peroxidation products, including malondialdehyde (mda) and nitric oxide. mda increased levels of oxygen free radicals, which attack polyunsaturated fatty acids in cell membranes and causing lipid peroxidation. eedj was able to reduce nitric oxide which was excessively released after administration of carrageenan and also could reduce the levels of mda through increasing the enzymatic antioxidant defense systems such as catalase (cat), superoxide dismutase (sod) and glutathione peroxidase (gpx) in the paw oedema which are the natural protectors against lipid peroxidation ( ) . the anti-arthritic effect of the total saponin extract from the rhizomes of d. zingiberensis c.h. wright (tsrdz) in freund's complete adjuvant (fca) induced arthritis in rats ( ) . in this study, a significant increase in arthritis scores and ankle perimeter were observed after days from intraplantar administration of fca, which reaches its maximum on day . administration of tsrdz at and mg/kg produced substantial dose suppressive significant effect in the treated groups. tsrdz at and mg/kg also caused a marked reduction of fca-induced elevated indexes of the thymus and spleen. the high dose of tsrdz caused only mild synovial infiltration with few inflammatory cells and no obvious damage in cartilage bone erosion, which appeared in histopathological study. the efficient regulation of tsrdz on the inflammatory cytokines at mg/ kg, was almost equivalent to that caused by the standard methotrexate. moreover, the mitigation of tsrdz on the prostaglandins (pge ), at mg/kg, was almost equivalent to that of standard methotrexate. furthermore, administration of tsrdz at mg/kg resulted in attenuating the production of mda and no, while elevating the sod activity compared with the control group ( ) . the total % ethanol extract of the tubercles of d. trifida l.f. as well as its three subfractions, dichloromethane, butanol, and aqueous fractions showed anti-inflammatory activity in food allergy induced by ovalbumin in balb/c mice ( ) . ova-sensitized balb/c mice received the ova solution to develop local signs of inflammation characterized by eosinophil infiltration, edema, an increase in a number of mast cells in the intestinal submucosa, mucus secretion, an increase in serum anti-ova igg and ige, and weight loss because of hyper-catabolism caused by the production of inflammatory cytokines. the crude extract of d. trifida tested at the doses of and mg/kg/day corresponding to . and . mg/kg/day of allantoin, and . and . mg of diosgenin correlated substances/kg/ day, respectively did not affect the body weight, but they inhibited ige production compared to untreated groups. the inhibition tended to be more significant in aqueous fraction and butanol fraction, which contains allantoin, than dichloromethane, which contains diosgenin. the ethanolic extract and its fractions exerted a reduction in several mastocytes and edema, which was also probably due to the presence of diosgenin and allantoin ( ) . diosgenin was also reported to reduce the production of anti-ova ige and the inflammatory infiltrate in allergic balb/c mice intestines, resulting in a reduction in edema ( ) . balb/c mice having food allergy showed an increase in mucus production by the caliciform cells in the small intestine ( ) . an important allergic response that is induced by interleukins il- and il- is hypersecretion of mucus. mucus has a protective effect on the intestinal cells by limiting the antigen absorption. the presence of eosinophil peroxidase (epo) indirectly reflects the infiltration of eosinophils into intestinal mucosa, which was reduced by administration of the ethanolic extract of d. trifida. mollica et al. also showed that d. trifida extract and fractions reduced all of the inflammatory parameters associated with food allergies in ova-sensitised animals and this activity is correlated with the presence of allantoin and diosgenin correlated substances ( ) . the animals treated with ethanolic extract showed a reduction in mucus production, and this result was also observed for the other fractions. previous studies showed that diosgenin and allantoin activities reduce mucus production in animals allergic to ova ( ) . the crude extracts of d. membranaceae and d. birmania, which contain high amounts of sapogenins, inhibited the production of nitric oxide (no) most probably through inhibiting the nitric oxide synthetase (inos) and tnf-α, with consequent reduction of intestinal edema ( , ) . a study by olayemi and ajaiyeoba also associated the antiedematogenic activity of crude extract from d. esculenta to the presence of sapogenins ( ) . in-vivo studies have demonstrated that oral administration of allantoin attenuates the production of ige, il- , and il- , has an anti-inflammatory effect and promotes healing ( ) . the methanol extract of the rhizomes of d. deltoidea wall.ex griseb. at mg/ kg inhibited the rat hind paw carrageenaninduced edema. the maximum percentage of inhibition was achieved at h after the intraperitoneal administration of the extract ( ) . in a study made by yang et al., the chloroform soluble extract of the rhizomes of d. opposita (csdo) showed cognitive enhancing effects on spatial memory and learning function of mice against scopolamineinduced amnesic deficits via morris water maze and passive avoidance tests. in morris water maze test, both acute treatment by csdo ( mg/kg body weight, p.o.) and prolongedtreatment ( days' daily administration of mg/kg body weight, p.o.) exhibited significant shorter escape latencies in daily first trial than the scopolamine-administered group during a -consecutive-day training period, which suggested that csdo improves the impaired reference memory (long-term memory) induced by scopolamine. especially, the acute treatment group showed a marked decrease in escape latencies in each daily trial compared with the prolonged treatment group. in this study, csdo also showed significant enhancing effects on spatial memory retention in the probe trial. similar results were obtained in the passive avoidance test. csdo treatment significantly increased step-through latencies of scopolamine-induced amnesic mice compared to control untreated group. the results of the two animal behavioral tests demonstrated that csdo improves spatial learning and memory function against scopolamine-induced amnesia ( ) . the study of jeon et al., investigated the neuroprotective effect of herbal mixture from euphoria longana, houttuynia cordata, and dioscorea japonica, by testing the hypothesis that administration of herbs reverses memory deficits and promotes the protein expression of brain-derived neurotrophic factor (bdnf) in the mouse brain: these herbs demonstrated an inductive effect on bdnf, cyclic-amp response element-binding protein (creb) and phospho-creb (pcreb) ( ) . the study of the antinociceptive effect of the methanol extract of the bulbs of dioscorea bulbifera l. var sativa (medb) was performed by nguelefack et al. ( ) the effects of medb persisted for days after two administrations in cfa-induced hypernociception at and mg/ kg. medb significantly inhibited acute lipopolysaccharides (lps)-induced pain at mg/kg but did not affect thermal hypernociception and capsaicin-induced spontaneous nociception. the antinociceptive effects of medb in prostaglandin-e (pge ) model was antagonized by either nitro-l-arginine methyl ester (l-name) or glibenclamide. this indicated that the plant exerted its antinociceptive effect through partial activation of the no-cgmp-atpsensitive potassium channels pathway ( ) . the intraperitoneal administration of n-butanol extract of the d. nipponica rhizomes at mg/kg regulates the blood cholesterol, triglyceride, hdl and ldl than the chloroform ( mg/kg) and water extracts ( mg/kg) ( ) . wang et al. reported the potential activity of trillin, a steroidal saponin isolated from d. nipponica rhizomes, as antihyperlipidemic and anti-oxidative agent invivo. the intraperitoneal administration of trillin ( . mg/kg dissolved in . % dmso) enhanced the blood circulation and could increase the bleeding time for more than % and coagulation in the rats fed on a high-fat diet in which the blood viscosity would be changed resulting in shorter blood coagulation time. the improvement effect of trillin in restoring the blood coagulation abnormality in high-fat diet fed rat was due to its ability to reduce the levels of blood cholesterol, triglyceride and two critical lipoproteins (hdl and ldl). the level of lipid oxidation was increased in high-fat diet rats, causing oxidative stress. trillin exerted an anti-oxidative effect through lowering lipid peroxidation, via the enhancement of superoxide dismutase (sod) level in blood. thus, the combination of trillin and lovastatin in treating hyperlipidemia could represent a more effective therapy (wang et al. ). the authors of this research marked how these findings greatly support the significant role of d. nipponica in protecting the cardiovascular system in-vivo against hyperlipidemia ( ). tang ( ); the authors reported how high serum levels of lactate dehydrogenase (ldh), aspartate aminotransferase (ast) and creatine kinase (ck) indicated cell membrane damage and were thus elevated in the iso model group. intra-gastric injection of total saponins of the three species could restore the activities of myocardial injury marker enzymes to the same extent. oxidative stress plays an essential role in the pathogenesis of mi injury. sod, catalase (cat), gpx, and total antioxidant capacity (t-aoc) levels were reduced significantly in the iso model group with the increase in mda. the activities of these enzymes were normalized by intragastric injection of the total saponins of the three species. the study revealed that the anti-mi mechanism of dioscorea saponins is related not only to the enzymatic antioxidant, such as gpx and cat but also to nonenzymatic antioxidants and its effect on myocardial histology ( ) . the current studies reported the beneficial effects of purple yam (dioscorea alata l.) resistant starch on hyperlipidemia in high-fat-fed hamsters ( , ) . the total saponins from rhizoma dioscoreae nipponicae could effectively reverse potassium oxonate-induced alterations in renal mouse urate transporter , glucose transporter , organic anion transporter , and organic anion transporter mrna and protein levels, resulting in enhancement of renal urate excretion in mice ( ) . moreover, the authors concluded that the total saponins from rhizoma dioscoreae nipponicae had a uricosuric effect on the regulation of renal organic ion transporters in hyperuricemic animals. in a further paper, the same authors here was the potent uricosuric effect of tdn on hyperuricemic rats by decreasing the expressions of renal rurat while increasing the expressions of renal roat and roat ( ) . a double-blind, placebo-controlled, crossover study was conducted to evaluate the effects of a wild yam cream in healthy women suffering from troublesome symptoms of the menopause. every female was treated with the active cream and a placebo for months randomly. after months treatment with topical wild yam extract in women suffering from menopausal symptoms it was found that the cream is free from side-effects, and has little effect on menopausal symptoms ( ) . dioscorea species make a significant contribution both as root crops and vegetables to the diets around the world. despite their importance as a food source, dioscorea plant parts are quite useful in the treatment of various ailments and disorders due to the presence of several bioactive compounds such as diosgenin. however, several reported ethnomedicinal potential need to be validated and detailed investigations on dioscorea pharmacological properties and phytochemical composition should be carried out to standardize the formulations used. indeed, some pharmacological properties such as hypolipidemic, hypoglycaemic, antioxidant, anti-inflammatory, antimicrobial, antiproliferative, androgenic, estrogenic, and contraceptive have been reported. however, most of the active constituents have not been characterized. thus, authentication of all the secondary metabolites (alkaloids, saponin, flavonoids, tannins, and phenols) from dioscorea should be performed thoughtfully to standardize and validate its quality and biological potentials. moreover, investigations are warranted to address the poor conversion of the preclinical results to clinical efficacity. therefore, an attempt should be made to understand their mechanism of action, pharmacokinetics/pharmacodynamics, and bioavailability and to conduct clinical trials. overall, these findings suggested that dioscorea should not be ignored and should rather be considered as a good alternative source of active molecules that can prevent or alleviate both functional and infectious disease burden, representing a current big challenge in developing countries. the knowledge 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japonica the beneficial effects of purple yam (dioscorea alata l.) resistant starch on hyperlipidemia in high-fat-fed hamsters the beneficial effects of purple yam (dioscorea alata l.) resistant starch on hyperlipidemia in high-fatfed hamsters effects of wild yam extract on menopausal symptoms, lipids and sex hormones in healthy menopausal women key: cord- - wwgdtp authors: stadnicka, katarzyna; sławińska, anna; dunisławska, aleksandra; pain, bertrand; bednarczyk, marek title: molecular signatures of epithelial oviduct cells of a laying hen (gallus gallus domesticus) and quail (coturnix japonica) date: - - journal: bmc dev biol doi: . /s - - - sha: doc_id: cord_uid: wwgdtp background: in this work we have determined molecular signatures of oviduct epithelial and progenitor cells. we have proposed a panel of selected marker genes, which correspond with the phenotype of oviduct cells of a laying hen (gallus gallus domesticus) and quail (coturnix japonica). we demonstrated differences in characteristics of those cells, in tissue and in vitro, with respect to different anatomical and functional parts of the oviduct (infundibulum (inf), distal magnum (dm, and proximal magnum (pm)). the following gene expression signatures were studied: ( ) oviduct markers (estrogen receptor , ovalbumin, and spink - ovomucoid), ( ) epithelial markers (keratin , keratin , and occludin) and ( ) stem-like/progenitor markers (cd glycoprotein, lgr , musashi- , and sex determining region y-box , nanog homebox, oct /cpouv gene encoding transcription factor pou f ). results: in chicken, the expression of oviduct markers increased toward the proximal oviduct. epithelial markers keratin and occludin were high in distal oviduct and decreased toward the proximal magnum. in quail oviduct tissue, the gene expression pattern of oviduct/epithelial markers was similar to chicken. the markers of progenitors/stemness in hen oviduct (musashi- and cd glycoprotein) had the highest relative expression in the infundibulum and decreased toward the proximal magnum. in quail, we found significant expression of four progenitor markers (lgr gene, sry sex determining region y-box , oct /cpouv gene, and cd glycoprotein) that were largely present in the distal oviduct. after in vitro culture of oviduct cells, the gene expression pattern has changed. high secretive potential of magnum-derived cells diminished by using decreased abundance of mrna. on the other hand, chicken oviduct cells originating from the infundibulum gained ability to express ovm and oval. epithelial character of the cells was maintained in vitro. among progenitor markers, both hen and quail cells expressed high level of sox , lgr and musashi- . conclusion: analysis of tissue material revealed gradual increase/decrease pattern in majority of the oviduct markers in both species. this pattern changed after the oviductal cells have been cultured in vitro. the results can provide molecular tools to validate the phenotype of in vitro biological models from reproductive tissue. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. avian species are excellent biological models in reproduction and tumorigenesis [ ] as well as efficient source of secreting cells for use in bioreactors [ ] [ ] [ ] . both hen and quail oviduct cells secrete human therapeutic proteins after genetic modification [ ] . therefore the oviduct epithelium is a useful and fast in vitro model to test for the efficiency of viral [ ] and nonviral genetic constructs [ ] to study the modified secretome. both quail and hen produce cellular substrates for the development of vaccines [ ] . genetic markers, including markers of stemness, are useful to identify mechanisms of malignant changes in a fallopian tube, because the somatic stem cells contribute to a population of tumorinitiating cells [ ] . recently, the knowledge about cell differentiation, physiology, and cancerogenic changes in avian oviduct has been extrapolated to women fallopian tube and uterine tract [ , ] . however, in avian species, markers of stemness in oviduct cells have not been reported yet. there is a knowledge gap regarding distinctive features of the epithelial cells in in vitro conditions vs. their status in tissue, which limits full understanding and characterization of this cellular model. in this paper, we have made initial attempts to confirm progenitor molecular signatures in oviducts of laying hen (gallus gallus domesticus) and quail (coturnix japonica), both in tissue and in cultured oviduct epithelial cells (in vitro assay). we have addressed the following questions: what is the location of progenitor cells in avian oviduct tissue? what is an individual molecular characteristic of distal oviduct tissue compartments? is this distinctive characteristic stable once the cells are plated in in vitro condition? is the molecular pattern shared between these two model species (laying hen and quail) used for oviduct studies? altogether, this study aims to provide a new understanding of molecular characteristic of oviduct epithelial cells in avian species. in adult tissue, epithelial progenitor cells have limited potential to divide and they can develop only into few differentiated cell types. they express stem cell markers and can differentiate into epithelial cells with various phenotypes. mucous epithelium of an avian oviduct is composed of simple columnar cells equipped with cilia to move the ovum from distal to proximal oviduct and of nonciliated secreting cells. both cell types require sustained renewal from the stem cell compartment and a high proliferation and maturation activity from the progenitor compartment. those compartments are putatively based under the luminal epithelium as cellular niches [ ] . in a mammalian fallopian tube, stem cells niches were tracked using antibodies and genetic markers and were found to be localized in the distal fallopian tube [ , ] . in our earlier research, we determined faster proliferation of cultivated hen oviduct cells derived from infundibulum/ distal magnum compared to the cells that were sourced from a proximal magnum. we have also determined that distal oviduct compartments were positively immunostained against cd and p , which are known to be epithelial stem/progenitor markers [ ] . thereby, we have hypothesized that distal segments of avian oviduct contain progenitor gene expression signatures. characterization of oviduct cells using molecular markers for epithelial progenitors contributes to the understanding of differentiation and regeneration processes, which occur in the oviduct epithelium. as reported earlier, the self-renewal activity of cells in the fallopian tube occurs in its distal part [ , ] . thereby, in this paper, we have focused on distal parts of the oviduct (the closest to ovaries and abdomen) to follow the molecular characteristics of the cells in tissue and in vitro. we propose a panel of epithelial genetic markers to determine the progenitor/epithelial cell pattern in selected compartments of the oviduct (fig. ) . in particular, we have aimed to reveal which of the avian oviduct compartments (infundibulum (inf), distal magnum (dm), or proximal magnum (pm)) carry known progenitor signatures. in this study, all the procedures involving experimental animals were approved by the local ethics committee for animal research (http://lke.utp.edu.pl) located at the faculty of animal breeding and biology, utp university of science and technology in bydgoszcz (study approval reference number / , in accordance with the _ _ue_pl directive). the hybrid tetra sl laying hens (n = , weeks old) were obtained from a commercial farm (nowosc, pradocin, poland). laying japanese quails (n = , weeks old) were obtained from a commercial producer (k. drazek, wyzne, poland). all birds laid eggs at a daily rate and no hormonal stimulation was applied for this study. immediately upon transportation, the animals were sacrificed by cervical dislocation. only the oviducts after egg passage, with ovum position in a shell gland, were used for the experiments. each oviduct was rinsed twice in tube filled out with ml physiological buffered saline (pbs) w/o mg, w/o ca (lonza biosciences, celllab, warszawa, poland), which was gently mixed with penicillin-streptomycin solution at : (v:v; life technologies, warszawa, poland). the epithelial cells were isolated from the oviduct tissue using the methodology described earlier [ ] . immediately after tissue collection, three oviduct fragments were dissected: infundibulum, distal magnum, and proximal magnum, each cm long. each fragment was cleaned off the mesentery tissue and minced with a scalpel blade on a petri dish. the minced fragments were digested in a solution of mg/ml collagenase p (sigma-aldrich, poznan, poland) in advanced dulbecco's modified eagles medium-f (dmem/f- ; life technologies, warszawa, poland) for min at °c, on a shaker. due to the size of oviductal tubes, the amount of minced tissue was~ % less in quail than in hen. thus, adequately lower volumes of digestion solution were applied to process the quail oviduct tissue. the cells were counted manually using neubauer hemocytometer and seeded at a density of × cells/cm into cm vented bd type primaria flasks (becton dickinson, diagmed, warszawa, poland). the cells were incubated in % co atmosphere at °c. the oviduct cells were maintained in dmem-f supplemented with % (v/v) fetal bovine serum (fbs; life technologies, cat. , - , batch no. g k, warszawa, poland), % (v/v) nonessential amino acids (sigma-aldrich, poznan, poland), mm l-glutamine (sigma-aldrich, poznan, poland), ng/ml human epidermal growth factor (human egf; r&d bioscience, biokom, janki, poland), % (v/v) antibioticantimycotic solution (life technologies, warszawa, poland), . μg/ml hydrocortisone (sigma aldrich, poznan, poland) and μg/ml insulin-transferrinselenium (its; sigma-aldrich, poznan, poland). the viability and the proliferation of oviduct cells were measured by a real-time cell analyzer (rtca) supplied by xcelligence system (roche applied science, basel, switzerland). the measurements of proliferating cells were conducted at . h intervals through h post seeding, in accordance with the producer's manual. the cells intended for sampling were cultivated for - days prior to harvesting and analysis. every second day, the epithelial colonies were counted and photographed under an objective with phase contrast (zeiss axiovert ) equipped with a digital camera (canon eos ). the cells were harvested upon reaching % of growth confluence. cultured oviduct epithelial cells were referred as chicken oviduct epithelial cells (coec) or quail oviduct epithelial cells (qoec) in further parts of this paper. rna isolation from oviduct tissue, coec, and qoec rna was isolated from three different sections of the oviduct tube (inf, dm, and pm) and cultivated oviduct cells, derived from the respective birds. for in vivo assay, inf, dm, and pm fragments, each cm long, were cut off aseptically and put separately into eppendorf tubes containing . ml rnafix (eurx, gdansk, poland). tissue samples were kept for h at °c and subsequently stored at − °c until isolation of rna. for rna isolation from coec, confluent cells were detached using accutase® solution (a&e life sciences, gentaur, sopot, poland) and centrifuged at ×g for min at room temperature (rt). cell pellets were resuspended in . ml rnafix (eurx, gdansk, poland) to preserve cells prior to rna isolation. rna was extracted using the universal rna purification kit (eurx, gdansk, poland) according to manufacturer's recommendation. rna was quantified using spectrophotometry and rna quality by gel electrophoresis. reverse transcription was performed with maxima first strand cdna synthesis kit for rt-qpcr (thermo scientific/fermentas, vilnius, lithuania). cdna was diluted to a final concentration of ng/μl and stored at − °c. reverse transcription-quantitative polymerase chain reaction (rt-qpcr) was performed in a total volume of μl, which included maxima sybr green qpcr master mix (thermo scientific/fermentas, vilnius, lithuania), μm of each primer (forward and reverse), and μl of diluted cdna ( ng). primer sequences (table ) were derived from the literature or designed with ncbi primer blast, based on cdna reference sequences [ ] . thermal cycling was conducted in lightcycler ii (roche applied science, basel, switzerland). qpcr thermal profile consisted of initial denaturation at °c for min, followed by cycles of amplification including s of denaturation at °c, s of annealing at °c, and s of elongation at °c. after completion of the amplification reaction, a melting curve was generated to test for the specificity of rt-qpcr. for this purpose, the temperature was gradually increased to °c with continuous fluorescence measurement. relative gene expression analysis was performed for each experimental group with ΔΔct method [ ] , using primer sequences reported in this study were designed based on the cdna reference sequence and ncbi primer blast [ ] . oligonucleotide primers spanned exon-exon boundaries to avoid unspecific gdna amplification. genome achicken (g. gallus), bquail (c. japonica) ubiqutin c (ub) and β-actin (actb) as reference housekeeping genes. geometric means of ct value of both reference genes was used in calculations. for tested samples, Δct was calculated by subtracting mean ct values of the reference genes from ct values of the target gene. a base sample (calibrator) was defined by an origin different from the reproductive system. for in tissue study, muscle samples from the same birds were used. for in vitro study, the chicken macrophage-like cell line [ ] was used as a calibrator. ΔΔct was then calculated using the equation: Δct sample -Δct calibrator. fold change of the gene expression was calculated as: r = -ΔΔct . rt-qpcr results were statistically analyzed using sas enterprise guide . (sas institute, cary, nc, usa). all tests were conducted on Δct values. first, shapiro-wilk test was used to assess the normality of data distribution. then the significance of changes in the gene expression (in comparison to calibrator samples) was conducted by student's t-test (p < . ). finally, multiple comparisons for all pairs (e.g., oviduct fragments or donor species) were performed with one-way anova followed by tukey's hsd post hoc test. standard error of the mean (sem) was used as a parameter of variability within the group. cultivated oviduct cells of hen (coec) and quail (qoec) reached the confluence after - days after seeding. the coec and qoec isolated from the infundibulum region typically occurred as cellular spheres, which attached to the polystyrene culture vessel after days post seeding and were consequently creating epithelial-like colonies, which spread on the surface of the culture vessel. once the small epithelial colonies appeared beneath the spheres, they enter a high proliferation phase to rapidly form a confluent monolayer. typical cultures from the infundibulum region were characterized by numerous compact epithelial islands, oval in shape, surrounded by elongated cells of mesenchymal or fibroblast-like phenotype (fig. a, b) . epithelial cells isolated from the region of distal magnum (a transition region between infundibulum and proximal magnum) formed visible epithelial islands days after seeding, which was sooner compared to the infundibulum region. in the case of distal magnum, spheres that formed epithelial-like colonies in vitro were half the size of those isolated from the infundibulum and about two times less colonies were initiated, compared to those from the infundibulum region on day (fig. c, d) . in most cases, the epithelial colonies from the distal magnum proliferated fast and were ready for passage by - days after seeding. the cells from the region of proximal magnum usually did not form spheres in the beginning of the cultivation (fig. e, f ) . typically, in proximal magnum, the proliferating epithelial colonies were observed in - days post seeding. the microscopic observations of growing colonies were in line with the measurements acquired from the xcelligence real-time cell monitoring system. the peak of the proliferation was determined for the cells from all oviduct compartments after days post seeding: at . (fig. e, f ) . motile cilia, which are characteristic for oviduct ciliated cells, were observed in the cultivated primary colonies, but only until the first passage. a movie file shows this in more detail (additional file ). in this study, we have used three gene panels to characterize oviduct fragments of hen and quail, and the respective primary epithelial cell cultures that were derived from them. those panels were comprised of oviduct (esr , oval, and ovm), epithelial (krt , krt , and ocln), and stem-like/progenitor (lgr , msi , sox , nanog, and oct /cpouv) gene expression signatures. table presents the overview of the gene function and the sequence similarity between a hen and a quail. the overall gene expression of the markers analyzed in both species (hen and quail) and sample types (tissue and in vitro) is presented in table . all twelve genes were expressed only in coec. ten out of twelve genes were expressed in oviduct tissues-sourced from both hen and quail. in the hen tissue, two progenitor markers (lgr and oct /cpouv) were at a level too low to be detected. in the quail tissue, one epithelial marker (ocln) and one progenitor marker (lgr ) were not detected. in qoec, oval and ovm (oviduct markers) were not expressed as well as ocln (epithelial marker). in both species, lgr , a progenitor marker, was absent in the oviduct tissue, but then we detected it in the oviduct epithelial cell culture. ocln was not expressed in quail oviduct-neither in the tissue, nor in the cell culture. hereby we have characterized gene expression profile in different parts (inf, dm, and pm) of hen and quail oviduct tissue (fig. ) . in hen oviduct (fig. a) , the expression of oviduct markers (esr , oval and ovm) increased spatially, from distal to proximal part of the oviduct with a peak in pm (p < . ). reversely, the expression of epithelial markers, krt and ocln, was high in inf and it decreased toward pm (p < . ). krt was expressed at much lower level and only in inf (p < . ). as for progenitor markers, sox was uniformly expressed at high level across all fragments of the oviduct in hen (p < . ). expression of msi and cd was the highest in inf and it gradually decreased toward pm (p < . ). expression of nanog was detected, but was not significant (p > . ). in quail oviduct (fig. b) , we had to use chicken primer sequences to show the expression pattern of oval, ovm, krt , krt , and oct /cpouv. we have determined a similar pattern of the gene expression for two oviduct markers oval and ovm, which were expressed in all the studied oviduct compartments (inf, dm and pm). whereas, oviduct marker for esr was significantly expressed in inf and dm compartments of a quail oviduct (p < . ). among epithelial markers, the expression of krt and krt was high and increased toward inf, but expression of ocln did not reach the significance threshold. in quail, we found significant expression of as much as four progenitor markers (lgr , oct /cpouv, sox , and cd ) (p < . ). lgr and oct /cpouv were most abundant in inf and dm compartments of the quail oviduct. expression of nanog was detected but it was not significant (p > . ). gallus sequence with human lgr , but the same protein sequence shows % identity with human vav gdp/gtp exchange factor. for a quail, only proteins are annotated in existing uniprot databases. thus, when a gap in quail database [ ] limits the interpretation of a sequence, a relevant genomic alignment onto chicken was performed [ ] . depending on the database used (ensembl, ncbi, and/or uniprot), sequences of the genes selected for this study had %- % similarity. thereby, gene expression assays developed were comparable between both species after having determined gene expression signatures in three specific fragments of chicken and quail oviducts, we have established the respective cell cultures, which were analyzed for the presence of the same markers. results of the relative gene expression analysis in chicken and quail oviduct epithelial cells are presented in fig. a . in coec (fig. a) only few markers were numerically and significantly upregulated, namely oval, ovm, krt , and sox (p < . ). in the case of qoec, we routinely found the abundance of ovalbumin in quail oviduct cell culture using antichicken ova antibody and western blot detection. msi was upregulated statistically (p < . ), though it did not have high numerical values of fold induction. in both, coec and qoec, oct /cpouv was significantly downregulated (p < . ). we did not determine any significant differences between coec derived from different fragments of the oviduct, apart from the expression of ocln (epithelial marker) and lgr (progenitor marker) that was high in the inf compartment (p > . ). in qoec, a similar significant expression, as in coec, was found for progenitor markers sox and msi as well as epithelial marker krt . the remaining epithelial and stem-like/progenitor markers were significantly expressed in the cultivated quail cells derived from all studied compartments of oviduct (p > . ). among avian species, laying hen (gallus gallus domesticus) and japanese quail (c. japonica) provide two excellent experimental oviduct models to study the immunology and reproductive biology [ ] . particular properties of oviductal cells include hormonal regulators as well as biosynthetic and secretive activity, which can be used for biomedical applications. firstly, the secreting function of an oviduct epithelium makes it an ideal natural bioreactor to obtain human therapeutic proteins by using genetic manipulation of the oviduct secretome. the product is accumulated in the egg white and is easily harvested [ ] . secondly, both hen and quail are recognized to reflect the development and chemoprevention of spontaneous leiomyoma, also known as fibroids of the oviduct in relation to human cancer [ ] . thirdly, the development of new oviduct cell lines would allow selectively propagating and studying important pathogens including campylobacter and salmonella strains or influenza and coronaviruses. such cell lines offer new in vitro substrates for pathogens originating from a reproductive tract. for this purpose, we have attempted to provide a utility set of molecular markers to characterize the avian oviduct tissue in hen and quail and in vitro-derived oviduct epithelial cell culture. for a quail, only proteins are annotated in the existing uniprot databases. thus, when a gap in quail database [ ] limits the interpretation of a sequence, a relevant genomic alignment onto the chicken is performed [ ] . depending on the database used (ensembl, ncbi, and/or uniprot), sequences of the genes selected for this study had %- % similarity. thereby, gene expression assays developed were comparable between both species. in our study, all analyzed genes were expressed in both hen and quail. in the first part, we have characterized gene expression signatures in three compartments of the oviduct tissue in hen and quail. the mrna abundance of the oviduct markers (esr , oval, and ovm) increased toward proximal parts of the oviduct. those differences between infundibulum and magnum compartments were significant only in hen earlier, but we have determined a clear numerical pattern also in quail. such a pattern of the oviduct markers reflects physiological functions of distinct compartments, e.g., oocyte transport and sperm storage in the infundibulum vs. egg white protein production in the magnum. for this reason, esr , which encodes the estrogen receptor , whose major function is binding estradiol-a major sex hormone of laying birds, was expressed in all parts of the oviduct. on the other hand, oval and ovm, which encode major egg white proteins, were expressed only in the magnum. such a pattern of the gene expression across the avian oviduct has been widely reported in the literature [ , ] and it validates the functional setup of this experiment. in a panel of epithelial markers characterized in tissue, we have determined a reverse pattern, i.e., decrease of mrna abundance from distal toward proximal parts of oviduct, in particular of krt , which was strongly expressed in the infundibulum of both, hen and quail. krt appeared to be more abundant in quail and ocln was significantly expressed in chicken oviduct. keratins encode for cytoskeletal proteins of highly proliferating basal epithelial cells [ ] . infundibulum is lined with ciliated epithelia, which are highly used by the frequent transportation of the oocyte and protein secretion. they require constant renewal from the basal epithelium, which is intensively proliferating. strong induction of keratin genes is related with this function of the infundibulum. previously, we have detected cytokeratins in chicken infundibulum by using immunohistochemistry technique, both in tissue and in vitro [ ] , which is in line with the results of the current study. as for stem-like/progenitor markers analyzed in tissue, chicken expressed high mrna abundance of cd and sox ; moderate abundance of msi and low of nanog. oct /cpouv and lgr were not expressed in the chicken oviduct tissue. in quail, we determined a high-fold induction of lgr and oct /cpouv and a moderate abundance of cd and sox . cd is a cell surface glycoprotein and an established progenitor/stemlike cell marker in fallopian tube in mammals. cd positive cell population showed the capacity for clonal growth and differentiation into tubal epithelial cells, particularly in the distal region of the tube [ , ] . we pairwise t-test was conducted to determine the significant modulation of the gene expression in the oviduct as compared to the external calibrator (breast muscle) (p < . ). an asterisk (*) indicates that the gene is differentially expressed, compared to the calibrator. letters a, b, and c in brackets indicate results of one-way anova multiple comparisons between different fragments of the oviduct (p < . ) earlier showed a high immunochemical stain of cd in the distal oviduct of a hen [ ] . sox is a transcription factor in early epithelial lineage [ , ] . it is involved in the organogenesis of different tissues and its main function is to maintain a population of undifferentiated somatic stem cells. sox was recently announced as a novel cancer stem cell marker [ ] . in our study, we consider this gene as a marker for precursor epithelial oviduct cells of avian species. musashi- is expressed in intestinal crypts and human endometrium, where it maintains multipotent potential for epithelial cells emerging from müllerian duct (precursor of oviduct in vertebrates) [ ] . oct /cpouv and nanog are chicken stem cell markers [ , ] , while lgr is recognized as marker stem cells in tubal epithelia [ ] . in our study, lgr and oct /cpouv were detected at high level in quail oviduct. overall, the pattern of expression of progenitor markers supports the designation of distal oviduct compartments as the source of progenitor epithelial cells. after being transferred to in vitro conditions, phenotypes of coec and qoec have changed in some aspects. secretive potential of magnum-derived cells was retained as reflected by the expression of ovm and oval in coec and esr in qoec. primary cultured cells, such as highly specialized oviduct epithelial cells, are prone to rapid differentiation in vitro. this way, they may easily lose their original phenotype, for example, the ability for protein secretion. on the other hand, inf-derived coec gained secretive potential after being cultured in vitro, which was reflected by changing the downregulation of ovm and oval to upregulation of those genes as compared to donor tissues. stimulation with estrogen was reported as necessary to maintain responsiveness of hen oviduct cells to this sex hormone [ ] . however, in this experiment, neither the birds were fig. expression of oviduct, epithelial, and progenitor markers in chicken (a) and quail (b) oviduct epithelial cells. relative gene expression analysis was performed with rt-qpcr method in three oviduct fragments: infundibulum (inf), distal magnum (dm) and proximal magnum (pm). pairwise t-test was conducted to determine the significant modulation of the gene expression in the oviduct as compared to the external calibrator (breast muscle) (p < . ). an asterisk (*) indicates that the gene is differentially expressed, compared to the calibrator. letters a, b, and c in brackets indicate results of one-way anova multiple comparisons between different fragments of the oviduct (p < . ) stimulated with the estrogen prior to tissue harvesting, nor the cultivated cells were treated with estrogen, which might explain the lack of esr- mrna in the cultivated coec. epithelial character of both coec and qoec was maintained, especially in krt (coec) and other epithelial markers (qoec) mrna abundance. expression of progenitor markers of early epithelial lineage (sox , msi , and lgr ) in both oviduct epithelial cultures was determined. lgr was significantly upregulated in cultivated cells, and has been proven to mark the stem cells in murine oviduct/fimbria [ ] . precursor character of certain populations of cultured cells allowed for their proliferation and differentiation in vitro. inf-derived coec gained gene expression signatures of oviduct secretive cells (ovm and oval). population of progenitor cells is required for the establishment of a primary cell culture [ ] . in our study, we have confirmed progenitor gene expression signatures in proliferating cultures. based on the morphological assessment, a subpopulation of the cultured cells displayed epithelial character of ciliated and secreting cells. but there was also a large subpopulation of differentiated mesenchymal and fibroblast-like forms in both coec and qoec, after passaging. with these observations, a stable oviduct epithelial cell line could be probably established from both in vitro models, with the prior purification of progenitor cells from the heterogeneous starting cell populations. in this study, we have characterized the expression of oviduct, epithelial, and stem/progenitor markers in the oviduct tissue and cell culture of two avian species, the hen and the quail. analysis of the oviduct tissue and cultured cells allowed for characterizing the molecular makeup of those cells in tissue, in relation to the source of the oviduct compartment (infundibulum, distal magnum, and proximal magnum). further analysis from in vitro-cultivated cells showed molecular pattern that was different from noncultivated oviduct cells. in conclusion, the analysis of tissue material revealed a gradual increase/decrease pattern in majority of the markers in both species. this pattern changed after those cells had been cultured in vitro. a progenitor marker, oct / cpouv was strongly downregulated in both in vitro models, whereas the expression of sox and the epithelial marker krt were not changed compared to the calibrator (fc~ ). cultivated hen cells (coec) gained the expression of lgr progenitor marker, which could indicate a shift toward a more specific epithelial progenitor cell type. these results can contribute to further research on creating new biological models from reproductive tissue and the characterization required to develop new avian cell lines. additional file : visualization of a typical phenotype of cultivated oviductal epithelial cells. the recording of the cultivated oviduct epithelial cells allows one to follow the typical cobble-like structure of lining epithelial cells and the rotatory movement of cilia on the nonsecreting ciliated cells, which are coisolated with the secreting tubular gland cells. estrogen receptor subtype expression is altered in the hen model of ovarian cancer advances in genetic engineering of the avian genome expression of recombinant human lysozyme in egg whites of transgenic hens transgenic quail production by microinjection of lentiviral vector into the early embryo blood vessels 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end of the fallopian tube: a site for injury and serous cancer initiation in vitro optimization of the gallus domesticus oviduct epithelial cells culture primer-blast: a tool to design target-specific primers for polymerase chain reaction analysis of relative gene expression data using real-time quantitative pcr and the -ÄÄct method chicken hematopoietic cells transformed by seven strains of defective avian leukemia viruses display three distinct phenotypes of differentiation normal ultrasonographic images of reproductive organs of female japanese quails (coturnix coturnix japonica): a laboratory animal model the effects of selenium supplementation on the spontaneously occurring fibroid tumors of oviduct, -hydroxy- ′-deoxyguanosine levels, and heat shock protein response in japanese quail next-generation sequencing reveals genomic features in the japanese quail integrated maps in quail (coturnix japonica) confirm the high degree of synteny conservation with chicken (gallus gallus) despite million years of divergence ovalbumin expression in the oviduct magnum of hens is related to the rate of egg laying and shows distinct stress-type-specific responses biochemical and functional characterisation of eggshell matrix proteins in hens derivation of keratinocytes from chicken embryonic stem cells: establishment and characterization of differentiated proliferative cell populations identification of quiescent, stem-like cells in the distal female reproductive tract sex determination and sexual differentiation in the avian model nucleotide sequence and embryonic expression of quail and duck sox genes sox is a novel cancer stem cell marker surrogated by osteopontin in human hepatocellular carcinoma expression of a putative stem cell marker musashi- in endometrium the oct homologue pouv and nanog regulate pluripotency in chicken embryonic stem cells chicken embryonic stem cells as a non-mammalian embryonic stem cell model characterization and application of oviductal epithelial cells in vitro in gallus domesticus progenitor cells are responsible for formation of human prostate epithelium primary cultures differential expression of the mhm region and of sex-determining-related genes during gonadal development in chicken embryos dietary inulin supplementation modifies significantly the liver transcriptomic profile of broiler chickens identification and validation of housekeeping genes as internal control for gene expression in an intravenous lps inflammation model in chickens interaction of estrogen and progesterone in chick oviduct development. . tubular gland cell cytodifferentiation cdna cloning and mrna expression of estrogen receptor alpha in japanese quail ovalbumin messenger rna of chick oviduct: partial characterization, estrogen dependence, and translation in vitro fine structural observations on magnum mucosa in quail and hen oviducts regulation of protein synthesis in chick oviduct. i. independent regulation of ovalbumin, conalbumin, ovomucoid, and lysozyme induction chicken ovomucoid: determination of its amino acid sequence, determination of the trypsin reactive site, and preparation of all three of its domains correlation between musashi- and c-hairy- expression and cell proliferation activity in the developing intestine and stomach of both chicken and mouse the stem-cell profile of ovarian surface epithelium is reproduced in the oviductal fimbriae, with increased stem-cell marker density in distal parts of the fimbriae the data supporting the conclusions of this article are included within the article and its additional file. any additional datasets which was used and/or analyzed during the current study are available from the corresponding author on reasonable request. authors' contributions ks and as made substantial contribution to conception and design of this manuscript. ks and ad acquired and interpreted the data reported. ks supervised the project and writing. ks, as, and ad provided the original draft. mb bp and ks reviewed and edited the final manuscript. all authors read and approved the final manuscript. the study was approved by the local ethics committee for animal research (http://lke.utp.edu.pl) located at the faculty of animal breeding and biology, utp university of science and technology in bydgoszcz (study approval reference number / ). not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -fxxceffl authors: razanajatovo, norosoa harline; guillebaud, julia; harimanana, aina; rajatonirina, soatiana; ratsima, elisoa hariniaina; andrianirina, zo zafitsara; rakotoariniaina, hervé; andriatahina, todisoa; orelle, arnaud; ratovoson, rila; irinantenaina, judickaelle; rakotonanahary, dina arinalina; ramparany, lovasoa; randrianirina, frédérique; richard, vincent; heraud, jean-michel title: epidemiology of severe acute respiratory infections from hospital-based surveillance in madagascar, november to july date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: fxxceffl background: few comprehensive data exist regarding the epidemiology of severe acute respiratory infections (sari) in low income countries. this study aimed at identifying etiologies and describing clinical features of sari-associated hospitalization in madagascar. methods: it is a prospective surveillance of sari in hospitals for years. nasopharyngeal swabs, sputum, and blood were collected from sari patients enrolled and tested for viruses and bacteria. epidemiological and clinical information were obtained from case report forms. results: overall, patients were enrolled in the study, of which . % ( / ) were tested positive for at least one pathogen. viral and bacterial infections occurred in . % ( / ) and . % ( / ) of tested samples, respectively. among all detected viruses, respiratory syncytial virus (rsv) was the most common ( . %; / ) followed by influenza virus a (flua, . %; / ), rhinovirus (rv, . %; / ), and adenovirus (adv, . %; / ). among bacteria, streptococcus pneumoniae (s. pneumoniae, . %, / ) was the most detected followed by haemophilus influenzae type b (hib, . %; / ), and klebsiella ( . %; / ). other streptococcus species were found in . % ( / ) of samples. compared to patients aged less than years, older age groups were significantly less infected with rsv. on the other hand, patients aged more than years (or = . ) were at higher risk to be infected with flua, while those aged – years (or = . ) and – years (or = . ) were more likely to be infected with flub (influenza virus b). conclusion: the frequency of influenza viruses detected among sari patients aged years and more highlights the need for health authorities to develop strategies to reduce morbidity amongst at-risk population through vaccine recommendation. amongst young children, the demonstrated burden of rsv should guide clinicians for a better case management of children. these findings reveal the need to develop point-of-care tests to avoid overuse of antibiotics and to promote vaccine that could reduce drastically the rsv hospitalizations. samples. compared to patients aged less than years, older age groups were significantly less infected with rsv. on the other hand, patients aged more than years (or = . ) were at higher risk to be infected with flua, while those aged - years (or = . ) and - years (or = . ) were more likely to be infected with flub (influenza virus b). the frequency of influenza viruses detected among sari patients aged years and more highlights the need for health authorities to develop strategies to reduce morbidity amongst at-risk population through vaccine recommendation. amongst young children, the demonstrated burden of rsv should guide clinicians for a better case management of children. these findings reveal the need to develop point-of-care tests to avoid overuse of antibiotics and to promote vaccine that could reduce drastically the rsv hospitalizations. severe acute respiratory infections (sari) are among the leading cause of hospitalization and deaths worldwide [ ] . it is estimated that about . million of deaths are attributed to sari annually [ ] . estimation in have shown that million of children aged less than years were admitted for sari in developing countries, with an estimated case fatality ratio of . %, compared to cases and a case fatality ratio of . % in developed countries [ ] . in low income countries, the annual incidence of pneumonia is estimated at million children of whom % are severe enough to warrant hospitalization [ , ] . nearly million children die from pneumonia every year, % of which occur in africa and southeast asia [ , ] . sari may be caused by various pathogens. while bacterial infections play a critical role in causing life-threatening pneumonia [ , [ ] [ ] [ ] , viral infections are associated with significant proportion that range from mild to severe infections [ , ] . however, due to the lack of gold standard methods to rapidly differentiate between viral and bacterial infections, most of the patients may be treated empirically with antibiotics [ ] . rapid etiologic diagnosis is therefore needed to select the most appropriate treatment protocol and to avoid the development of bacterial resistance. following the a/h n / influenza pandemic that was associated with a high morbidity and an increased risk of mortality among particular groups [ ] , a number of countries have strengthened vigilance for the surveillance of severe diseases and deaths in order to rapidly detect new viruses and to provide information in assessing the impact on the population and having operational preparedness plans. so far, only few sub-saharan african countries are collecting data on hospitalization associated to acute respiratory illness [ ] [ ] [ ] . in madagascar, the sentinel syndromic surveillance has been collecting sari data since . however, hospitals only collect few data that are not exploitable for analysis. thus, data are still scarce regarding the epidemiology of sari in this country. during the last influenza pandemic in , an excess of mortality was observed among inhabitant of the main capital city of antananarivo [ ] . following the pandemic, health authorities requested to develop an active surveillance of sari in order to better understand the epidemiology of this disease in the context of madagascar. the present study aimed to identify etiologies of sari and to assess clinical features of hospitalization linked to sari in hospitals during years of surveillance. a prospective hospital-based sari surveillance was conducted from november to july in selected sites: the soavinandriana hospital of antananarivo and the secondary level public hospital (chd ii) of moramanga. the cenhosoa is a national referral hospital and is among the largest hospitals that deserve antananarivo, the main capital city of madagascar which has around . million inhabitants. the chd ii in moramanga is the only local referral hospital for the health district of moramanga that has inhabitants. it is located km east from the capital city. antananarivo and moramanga present the same climatic profile: hot and rainy in summer and cold and dry in winter, but moramanga encompasses both semiurban and rural areas. all patients presenting sari symptoms at admission were enrolled. sari case definition was defined according to the who case definition as fever (t� ˚c) or history of fever and cough that required hospitalization. for children less than years, the eligibility criteria were suspected sepsis or sari diagnosed by physician including bronchiolitis, pneumonia, bronchitis, pleural effusion, and cough or difficult breathing as previously published [ ] . the onset of illness should be less than days before hospitalization. for each consented patient, demographic, socio-economic, clinical, and epidemiological data were recorded in case report forms (crfs). nasopharyngeal, blood, and sputum specimens were collected for each patient enrolled from monday to friday and shipped the same day to laboratories located at the pasteur institute of madagascar where they were immediately processed or stored at ˚c until testing (test were performed within hours post-sampling). all procedures for the biological analyses have been previously described [ , ] . briefly, nasopharyngeal swabs were screened for respiratory viruses using in-house multiplex real-time pcr [ ] . sputum was collected for cytobacteriologic testing while blood samples were used for cell blood count. single infection was defined as an infection caused by one pathogen (virus or bacteria) and multiple infection as an infection caused by at least pathogens (virus/virus, virus/bacteria or bacteria/bacteria) in a single sample. patients were divided into age groups: infants and young children (� years old); children ( - years old); adolescent and young adults ( - years old); adults , and elderly (� years old). demographic, clinical characteristics, and etiologies were compared by study sites and by age groups. univariate analysis and logistic regression were performed using r software. in univariate analysis, qualitative variables were compared with fisher's exact test and quantitative variables by non-parametric tests (wilcoxon test). furthermore, logistic regressions were performed to adjust odds-ratio (or) found via maximum-likelihood estimation according to age group for the different variables as dependent variables and to compare each of them according to age group by wald test. the age group less than years has been considered as reference group. statistical differences were considered significant for two-sided p-values < = . . although sari surveillance in madagascar is not considered as research, this prospective study requested extra sampling and the collection of personal information. for that reason, the present study was submitted and approved by the national ethics committee from the ministry of health in madagascar (authorization n˚ -msanp/ce). adult participants or parents were fully informed of the study's objectives and procedures. written informed consent was obtained from patients enrolled in the study. young participants aged from to years old were asked to sign an informed assent form if they were willing to take part in the study. for all patients aged less than years, consent was obtained from the parents or legal guardians. after obtaining the consent/assent form, the team survey proceeded to collect samples and fill out the crfs. refused consent and prior hospitalization within weeks before consultation were reasons for exclusion. from november to july , hospitalized patients completed the established sari case definition of whom cases ( . %) were from cenhosoa and cases ( . %) from chd ii moramanga. the mean age was . years ranging from day to years and . % of patients were male cases. the majority of recruited patients were children aged less than years and accounting for % of total inclusion. adult aged more than years represented % of cases ( table ) . differences of mean age ( . years vs. . years; p< . ; wilcoxon test) and age repartition (p< . ) of included patients were observed between the study sites with patients hospitalized in chd ii moramanga being younger than those hospitalized in cenhosoa (table ) . with exception of dyspnea and fever which are among the principal inclusion criteria, runny nose ( . %), intercostal recessions ( . %), productive cough ( . %), movement of nose wings ( . %), and anorexia ( . %) were the most recorded at the time of examination. bronchiolitis ( . %) was the most clinical diagnostic made by clinicians at admission followed by bronchopneumonia ( . %) and pneumonia ( . %). other low respiratory tract infections (bronchoalveolitis/exacerbation of chronic obstructive pulmonary disease (copd), pleuropneumonia, and acute lobar pneumonia) were observed in . % of inpatients (s table) . comparison of clinical symptoms between the sites showed that dry cough (p = . ), asthenia (p = . ), runny nose (p< . ), and anorexia (p = . ) were statistically frequently observed in patients hospitalized in chdii moramanga whereas productive cough (p = . ), sore throat (p< . ), headache (p = . ), thrill (p = . ), and cyanosis (p< . ) were significantly associated with patients hospitalized in cenhosoa (table ) . at least one pathogen was found in . % ( / ) of tested patients of which . % ( / ) were single infection and . % ( / ) were multiple infections. the highest rate of infection was obtained in patients aged less than years ( . %; / ). for this age group, single and multiple infections were reported in . % ( / ) and . % ( / ) of cases, respectively. the type of infection (mono-infection vs. multiple infection) differed significantly between age groups (p = . , fisher's exact test). indeed, patients aged - years were significantly less likely at risk for being co-infected (or = . ; p = . ) compared to those aged less than years (s table) . overall, viral and bacterial infections occurred in . % ( / ) and comparison of the prevalence of pathogens by study sites revealed that flua (p = . ), s. pneumoniae (p = . ), and hib (p = . ) were statistically relatively common in patients hospitalized in cenhosoa (s table) . in respect to multiple infections, flua (or = . ) . surprisingly, patients were co-infected with , , and different pathogens ( , , and , respectively) ( table ) . comparison of demographic and clinical characteristics of sari patients by age group. analysis of data by the age group showed that the frequency of clinical symptoms significantly varied across groups. indeed, by multivariate analysis adjusted by age, patients aged � years appeared to suffer chest pain compared to patients aged < years while dry cough and gpd were more often observed in the elderly. dyspnea and cyanosis were significantly more common in adults aged - years. headache, thrill, sweats, anorexia, weight loss, and asthenia were more often observed in the older age groups ( - years, - years and � years). sore throat was frequently reported in the age groups - years, - years and - years. runny nose and intercostal recession appeared to be relatively infrequent in the older age groups ( - years, - years and � years) ( table and s table) . regarding infection, prevalence of rsv (p< . ) and influenza viruses (p< . ) substantially varied depending the age group. as expected, rsv accounted for the highest proportion of sari in young children ( . %; / ). in fact, the older age groups were significantly less at risk to develop an rsv infection compared to the age group less than years (table ) ) and between - years (or = . ) were more likely to catch flub infection. by multivariate analysis, the age group between - years were less likely to suffer from s. pneumoniae infection (or = . ) than the age group less than years ( table ) . pattern of circulation of respiratory pathogens. sari cases were detected all year around during the years of surveillance with distinct peaks of detection observed each year: between january and march in and and between may and july in (fig ) . these peaks coincided with an active circulation of rsv and influenza viruses. nevertheless, peaks of sari appear to be more correlated to rsv circulation. unlike other respiratory pathogens that are detected all year along, rsv seems to circulate once a year and being responsible of an epidemic every year. to the best of our knowledge, this study is the first description of etiologies associated to hospitalized sari patients from both urban and peri-urban areas in madagascar. during the years of surveillance, consented patients meeting the established case definition of sari were enrolled. the observed different clinical spectrum across age groups may be helpful to avoid misclassification of patients presenting with respiratory illness at the triage level when no standardized protocol is available. the finding that about % of inpatients were tested positive for a pathogen is consistent with those reported elsewhere which showed etiologies ranging from % to % of hospitalized sari cases depending on case definition or methodology design [ , , ] . most of the included patients were children less than years old ( . %). this study confirms that young children are the most vulnerable group to suffer from sari [ , ] . nevertheless, these results could also be explained by a socio-economic behavior of the malagasy population. indeed, parents are keener to seek care for their children while older individuals try to cure themselves at home using self-medication or traditional medicine. in the present study, about % of inpatients presented multiple infections by at least pathogens. here, viral infections were commonly found in . % of tested patients which is similar to what was observed within the community during the influenza-like illness surveillance [ ] . taken together, the present study clearly demonstrates that respiratory viruses are the leading cause of severe acute respiratory illnesses in madagascar. overall, rsv was the most commonly detected followed by s. pneumoniae and influenza viruses. similar to other studies, substantial rate of rsv infection was observed in young population [ , , ] . a meta-analysis of data from africa reported that the incidence of rsv in lower acute respiratory infections that required hospitalization ranged from - per person year for infants and - per person year for children under years of age [ ] . the increased circulation of rsv that coincided with the peak of sari cases may be informative for clinicians to predict an epidemic due to rsv and to choose the most appropriate care for patients in order to avoid overuse of antibiotics. in addition, the epidemic pattern of this virus reveals the need to develop rapid diagnostic tests to rapidly manage patients and reduce the number of hospitalization, and to promote effective vaccine to prevent severe infections in high-risk populations. the role of influenza viruses in sari-associated hospitalization is increasingly defined because of available data from active influenza sentinel surveillance system initiated in several countries including sub-saharan africa [ ] [ ] [ ] [ ] . the proportion of influenza viruses in young population obtained here ( . %) is much higher than in other african countries were sari attributable to influenza are around % [ ] . as seen elsewhere, the highest rate of hospitalization is obtained with the subtype a/h n virus during its circulation [ , ] . in madagascar despite decades of reliable influenza surveillance, no national policy is implemented regarding vaccination. moreover, influenza vaccines are not widely accessible and affordable for the whole population. in the present study, about . % of sari cases were likely due to bacterial infection. nevertheless, it is not clear what this high proportion of bacteria means due to the notion of carriage. the substantial rate of s. pneumoniae detected might be partly explained by the very low pneumococcal vaccination rate among the younger population (estimated at . %) because most of the positive cases were detected before the introduction of pneumococcal vaccine into the expanded program on immunization (epi) in madagascar in [ ] . this vaccine is believed to reduce substantially the burden of pneumococcal disease. thus, it would be interesting to evaluate the proportion of sari associated to s. pneumoniae more than years after the introduction of the vaccine in madagascar. on the other hand, the role of atypical bacteria in hospitalized ari is poorly understood. one assumes that certain bacteria predispose to bacterial super-infections [ ] . while rv is considered as the common cold virus, the present study showed that this virus accounted for % of total sari cases. this finding highlights the potential role of rv in public health like other authors already postulated [ ] [ ] [ ] . our study presents some limitations. first, data were collected only from sites that are not representative of the whole population preventing us to real estimate the prevalence at the national level. indeed, prevalence of each pathogen may differ for regions having different bioclimatic or demographic patterns, access to healthcare, and connectivity. since children are more at risk to develop severe infection, our sampling was skewed toward the younger group and likely underestimate the prevalence in adult. indeed, few adult cases had been identified at the adult ward mainly because the duration of symptoms prior to admission exceeded the days limit of our sari case definition. it was also observed that severe cases were treated on external consultation or admitted at the emergency department for a few hours and then discharged. patients that did not recover, worsened at home, and returned to the hospital were then admitted resulting in prolonged duration of illness (usually more than days) at the time of admission. another limitation is to attribute the pathogen(s) that is (are) the main cause that lead to severe illness. indeed, we could not exclude that some pathogens may be present due to carriage. to conclude, the high prevalence of etiology associated to severe infections of relevant pathogens highlights the importance of sustaining national surveillance of sari to clearly estimate the role of associated pathogens and establish the burden of disease. further challenges in such program include efforts to implement strategies that capture adult cases as it is relevant to measure the impact of sari in this population. since vaccines play an important role in preventing sari, vaccination against important pathogens, including rsv, should be accessible at least for high-risk population in developing countries. indeed, although use of synthetic antibodies against rsv can be provided to certain at-risk infants, this treatment only provides a short-term protection, is not always effective, and is also expensive, putting it beyond the reach of developing countries like madagascar. supporting information s table. clinical diagnosis of patients hospitalized for sari, madagascar, november to july . � lrti: low respiratory tract infection including bronchoalveolitis/exacerbation of chronic obstructive pulmonary disease (copd), pleuropneumonia, and acute lobar pneumonia; �� other: neonatal infection, asthma, laryngitis, influenza-like illness etc. . . n = number of included patients. n = number of patients that responded by "yes" or "no" for a given symptom. only bronchiolitis, pneumonia, and bronchopneumonia were statistically analyzed. statistical analyses were performed using fisher's exact test. (docx) s table. univariate analysis of the distribution of monoinfection and multiple infection detected in sari adjusted by age groups, november to july . the age group less than years was considered as reference group. 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influenza-associated hospitalizations in the united states the burden of acute respiratory infections in crisis-affected populations: a systematic review epidemiology and clinical profile of pathogens responsible for the hospitalization of children in sousse area epidemiology of multiple respiratory viruses in childcare attendees we would like to express our gratitude to all clinicians and nurses involved in this study: emma rasoanirina, roland randriamirado, harimboahangy rakotoarison, lucien radozahana, rani razafindrakoto, domohina rakotovao, durandalle ny hanitrin'ny ala razana, dominique razafimandimby, françois de paul razanakoto, and saholiarisandy ndremihavana. we are deeply indebted to all the staff involved in the hospital surveillance program on a daily basis. we also would like to thank the laboratory technicians at the virology unit and the cbc for their tremendous work. the views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the ministry of health or of the us centers for disease control and prevention. key: cord- -nku kt authors: hoang, van-thuan; gautret, philippe title: infectious diseases and mass gatherings date: - - journal: curr infect dis rep doi: . /s - - - sha: doc_id: cord_uid: nku kt purpose of review: mass gatherings (mgs) are characterized by a high concentration of people at a specific time and location. infectious diseases are of particular concern at mgs. the aim of this review was to summarize findings in the field of infectious diseases with a variety of pathogens associated with international mgs in the last years. recent findings: in the context of hajj, one of the largest religious mgs at mecca, saudi arabia, respiratory tract infections are the leading cause of infectious diseases in pilgrims with a prevalence of – %. the most commonly acquired respiratory viruses were human rhinovirus, followed by human coronaviruses and influenza a virus, in decreasing order. haemophilus influenzae, staphylococcus aureus, and streptococcus pneumoniae were the predominant bacteria. the prevalence of hajj-related diarrhea ranged from . to . % and etiologies included salmonella spp., and escherichia coli, with evidence of acquisition of antimicrobial-resistant bacteria. in other mgs such as muslim, christian, and hindu religious events, sports events, and large-scale open-air festivals, outbreaks have been reported less frequently. the most common outbreaks at these events involved diseases preventable by vaccination, notably measles and influenza. gastrointestinal infections caused by a variety of pathogens were also recorded. summary: because social distancing and contact avoidance are difficult measures to implement in the context of many mgs, individual preventive measures including vaccination, use of face mask, disposable handkerchief and hand hygiene may be recommended. nevertheless, the effectiveness of these measures has been poorly investigated in the context of mgs. the who defines mass gatherings (mgs) as a "concentration of people at a specific location for a specific purpose over a set period of time which has the potential to strain the planning and response resources of the country or community" [ ] . mgs can be either planned or spontaneous and recurrent or sporadic [ ] . planned mgs may include sporting, social, cultural, religious, and political events. examples include music festivals, the olympic games, and the hajj [ ] . spontaneous mgs, given their nature, are more difficult to plan for and may include events, such as funerals of religious and political figures [ , ] . mgs may also include the gatherings of displaced populations due to natural disasters, conflicts, and wars [ ] . diverse health risks are associated with mgs, including transmission of infectious disease, non-communicable disease, trauma and injuries (occupational or otherwise), environmental effects (such as, heatrelated illnesses, dehydration, hypothermia), illnesses related to the use of drugs and alcohol, and deliberate acts, such as terrorist attacks [ ] . infectious diseases are of particular concern at mgs [ ] . in this review, we summarize recent findings in the field of infectious diseases associated with international mgs. the hajj (table ) the hajj, an annual muslim pilgrimage to mecca, saudi arabia, is one of the largest religious mgs in the world with about two million pilgrims from countries [ ] . as part of the hajj rituals, pilgrims visit various sacred places around the city of mecca. most of them also travel to the city of medina to visit the second holiest site of islam, the prophet's mosque containing the tomb of the prophet muhammad. the presence of a large number of pilgrims from different countries of the world and overcrowded condition considerably increases the risk of occurrence of infectious diseases, particularly respiratory and gastrointestinal diseases [ ] . furthermore, a vast majority of pilgrims are elderly people with a high prevalence of chronic diseases. in the past, hajj-related cholera has been a public health problem and the main cause of morbidity and mortality among pilgrims, leading to major epidemics and international spread. due to improved sanitary conditions in saudi arabia in general and at religious sites, large-scale cholera outbreaks have not been recorded during the last decades [ , ] . similarly, invasive meningococcal disease has been a hajj-related public health concern with its last outbreaks (serogroup w- ) in the s. however, with the strengthening of prevention through mandatory vaccination, no case of meningococcal disease has been reported in mecca since [ , ] . while gastrointestinal diseases and diarrhea have changed towards a lower prevalence, respiratory tract infections (rtis) now account for the vast majority of health problems during the hajj [ , ] . the inevitable overcrowding conditions at the grand mosque in mecca and the accommodation in tents in mina with an average of to people per tent are likely responsible for the high rate of respiratory infections among hajj pilgrims [ ] . over the last years, a significant number of publications from different countries based on both syndromic surveillance and pcr-based investigation of respiratory pathogen carriage were made available. studies were conducted in out-and inpatients at health structures in saudi arabia or on return in pilgrim's country of origin and in cohorts of pilgrims regardless of symptoms (table ) . rtis are among the leading causes of admission to hospitals in mina, mecca, and medina during the hajj period (table ). most cases are upper respiratory tract infections [ ] [ ] [ ] [ ] [ ] [ ] [ ] , but severe respiratory tract infections [ ] and pneumonia are not uncommon among pilgrims [ , , •] . respiratory diseases were the second cause of mortality in indonesian pilgrims during the hajj (following cardiovascular diseases) [ ] . among pathogens detected by pcr methods in ill pilgrims, the most common viruses were human rhinovirus (hrv), followed by human coronaviruses (hcov) and influenza a virus (iav). haemophilus influenzae, staphylococcus aureus, and streptococcus pneumoniae were the predominant bacteria isolated by culture [ , ] . cross-sectional and longitudinal cohort studies have recorded - . % prevalence of rti symptoms among hajj pilgrims [ - , •, , , •] . the rate of ili varied from . to . % [ , , •, , , - ] . cohort surveys allow evaluating the acquisition rate of respiratory pathogens regardless of symptoms. the most commonly acquired viruses were human rhinovirus (hrv) ( . - . %), followed by human coronavirus e (hcov-e ) ( . - . %) and influenza virus (iav) ( . - . %) [ , , , , , •, ] . the most commonly acquired bacteria were s. pneumoniae ( . to . %) and s. aureus ( . to . %) and h. influenzae ( . %) [ , , , •, •] . bordetella pertussis, mycoplasma pneumoniae, and chlamydia pneumoniae have not been detected in pilgrims in recent studies [ , , •] . middle east respiratory syndrome coronavirus (mers-cov) that emerged in the arabian peninsula in is associated with severe acute respiratory infection with high [ - , , , , , , , , - ] . tuberculosis (tb) transmission is another concern at the hajj, but there are no large-scale, specific studies to determine its prevalence among pilgrims [ ] . a prospective crosssectional study was conducted in mecca, during the hajj period in september . one thousand one hundred sixty-four pilgrims with cough were selected from five countries in africa and south asia that are endemic for tb and . % had active previously undiagnosed tb [ [ ] . in the latter study, escherichia coli was the predominant pathogen isolated from pilgrims by pcr. enteropathogenic e. coli, enteroaggregative e. coli, and shiga-like toxin-producing e. coli were acquired by . %, . %, and . % pilgrims, respectively [ ] . among persons infected during the - hajj and hospitalized in saudi hospitals, the pathogens responsible for enteric infection were mostly bacteria, with a prevalence of salmonella spp. of . %, while that of diarrhea associated e. coli ranged between . and . % according to pathotypes [ ] . two cases of tropheryma whipplei were recorded in a cohort of french pilgrims during the hajj [ ] . the frequency of infectious diseases during the hajj results in a significant demand for antibiotic use. [ ] . a prospective study conducted among pilgrims from marseille, france, during the periods of hajj in - showed that . % of the population used antibiotics because of respiratory diseases and . % because of diarrhea [ ] . although the dispensing of antibiotics without a prescription has been banned in saudi arabia for more than years [ ] , % of australian pilgrims used antibiotics either delivered in saudi arabia without prescription or purchased in australia before traveling [ ] . the predominance of bacterial pathogens in hajj-related gastrointestinal infections poses a major risk to public health through the potential emergence and transmission of antimicrobial-resistant bacteria [ ] . methicillin-resistant s. aureus had been isolated in % of pilgrims with acute sinusitis in [ ] and % of pilgrims with communityacquired infections hospitalized during the hajj in [ •] . one study addressed the carriage of resistant s. pneumoniae in a multinational cohort of pilgrims and showed that % of isolates were resistant to multiple antibiotics (resistant to three or more classes of antibiotics) [ ] . extended spectrum beta-lactamase enterobacteriaceae are also common among hospitalized pilgrims. during the - hajj, % of pilgrims attending hospitals for urinary tract infections showed blactx-m genes in e. coli isolates [ ] . during the and hajj seasons, studies were conducted using rectal samples obtained before and after the hajj in cohorts of french pilgrims to assess the carriage of the blactx-m gene. acquisition rates of . - . % were observed [ , ] . there was also a significant increase in the number of pilgrims harboring e. coli resistant to ceftriaxone and ticarcillin-clavulanic acid [ ] . the prevalence of c g-resistance was observed in . % acinetobacter baumannii isolates in a cohort of french pilgrims in [ ] and in . % of isolates obtained from hospitalized pilgrims suffering from community-acquired infections in [ •] . two french pilgrims carried s. enterica, resistant to ceftriaxone, gentamycin, and colistin after the hajj [ ] . mrc- resistance gene screening from rectal swabs was conducted in french pilgrims in - and found an acquisition rate of . % after hajj [ ] . risk factors for the spread of antibiotic-resistant bacteria at the hajj include international travel, misuse of antibiotics, and availability of over-the-counter antibiotics [ ] . however, gastrointestinal diseases and diarrhea continue to occur in pilgrims, outbreaks of food poisoning are reported, and the acquisition of multi-resistant bacteria is emerging. the ongoing monitoring of these diseases is part of the public health response regarding the hajj [ •, ] . currently, meningococcal vaccination (a, c, y, w- ) is mandatory for all pilgrims, national and international, as well as local residents of holy cities and workers in contact with pilgrims; however, polysaccharide vaccine which does not prevent meningococcal carriage is still in use in many countries. mandatory oral ciprofloxacin prophylaxis is provided upon arrival to all the pilgrims coming from the "meningitis belt" of sub-saharan africa [ , , , ] . a cross-sectional study among pilgrims arrived at king abdul aziz international airport, in jeddah for the hajj in showed antibody titers under the level of protection against serogroups a, c, w, and y of only . %, . %, . %, and . %, respectively. most of them ( . %) had received meningococcal vaccination in the three previous years [ ] . in a prospective cohort study conducted in turkish hajj pilgrims during , the carriage prevalence of neisseria meningitides, assessed by culture method, was % before and . % after the hajj with the majority being serogroup w- [ ] . in a prospective culture-based cohort study conducted among iranian pilgrims in , . % acquired n. meningitides at the hajj [ ] . a prospective study conducted in among international pilgrims at king abdul aziz international airport showed . % n. meningitides carriage by culture method upon arrival and . % upon departure, with the majority of typable isolates being serogroup b [ ] . outbreaks of the disease including those due to serogroups not included in the required vaccines, such as serogroups b and x, are therefore possible at the hajj. despite the wide use of polysaccharide vaccine, it does not prevent the carriage of serogroup w- and subsequent transmission to unvaccinated individuals by returning pilgrims. the grand magal of touba, the largest muslim pilgrimage in senegal, has specific features. besides its setting in a tropical environment, its population is characterized by a large range of age groups since most pilgrims travel with their family, including young children. a preliminary survey in has showed a high rate of febrile systemic illnesses and malaria ( . %), diarrheal diseases ( . %), and rtis ( . %) among ill pilgrims consulting at health care structures during the pilgrimage. the overall hospitalization rate was . % including gyneco-obstetric cases ( . %) and confirmed malaria ( . %) [ ••] . the kumbh mela in india is the largest mg in the world with about million visitors. it posed an exciting challenge to the provision of healthcare services. increased population density, reduced sanitation, and exposure to environmental pollutants open the way for easy transmission of pathogens [ ] . during kumbh mela in , , patients consulted at hospitals. respiratory infections accounted for % of illnesses and diarrheal diseases for %. in total, ( . %) were hospitalized. gastrointestinal disease risk, including cholera, is high because of potential contamination of water and food. in addition, vaccination against cholera is no longer considered adequate or even feasible in this context [ ] . the ashura mg at karbala is an increasingly popular religious event in iraq with about three to four million muslims from within and outside iraq. in , a cross-sectional study conducted in three public hospitals at karbala city showed that about % of the , consultations were at emergency rooms. febrile illness was recorded seven times more frequently during this event compared to previous events, in relation to an eight-fold increase in the population in the area during the event [ ] . other notable events include the moulay abdellah amghar moussem, an -day annual gathering in morocco, that documented an increase of gastrointestinal diseases from to % between and [ ] . during the anniversary of the death (urs) of baba farid, an annual mg in pakpattan, pakistan, % of people seen at healthcare facilities were affected by communicable diseases, including % gastrointestinal illnesses and % rtis [ ] . also in , a cross-sectional study of , attendees to the -day eid al adha holiday, aqaba (one of the largest muslim mgs in jordan), identified % and % increases in emergency department attendance and hospital admissions, respectively; however, no food poisoning outbreaks were reported [ ] . unlike the syndromic surveillance data mentioned above that lacked reliable identification of the responsible pathogen, s. enterica serotype typhimurium was determined to cause cases of gastrointestinal illness among participants in a christian religious festival in hamilton county, ohio; the outbreak was associated with the consumption of pulled pork prepared in a private house and sold at the festival [ ] . (table ) although numerous gastrointestinal and respiratory outbreaks have been documented at large-scale open-air festivals, particularly music festivals, with thousands of participants, these events are probably neglected, in terms of public health attention, as well as surveillance and prevention of infectious disease strategies, compared to other categories of mgs [ ] . since this review was published, several outbreaks were reported in the context of festivals. between july and , , during the annual independence celebrations in kiribati, the kiribati syndromic surveillance system reported an increase in children presenting with severe diarrhea due to rotavirus. in total, cases of gastroenteritis were reported and ( . %) died among ( . %) hospitalized. most of them ( . %) were younger than years of age [ ] . an outbreak of measles with cases identified at an international dog show occurred in november in slovenia, where measles virus had not been circulating for many years. twenty-three persons were infected there and were presumable secondary and tertiary cases. most cases ( ) were adults. five were unvaccinated children [ ] . also, a multistate measles outbreak that caught global attention occurred at the disney theme parks in california, usa [ ] . the rd world scout jamboree (wsj) in yamagushi, japan, from july to august , , was a mg attended by more than , participants from countries. the event is designed for scouts aged to years to live together, experience diverse cultures, and take part in recreational activities. in this event, six cases of invasive meningococcal disease related to the wsj were reported, affecting . per , wsj attendees, far exceeding the annual incidence rate in japan in ( . per , population) [ •] . finally, an outbreak of measles ( cases) was reported at music and art festivals in england and wales between june and october . almost half of the cases occurred in participants aged to years. several people who contracted measles at a festival later attended another festival when they were contagious, resulting in multiple, interconnected outbreaks. only one confirmed case was fully vaccinated. forty-two were not vaccinated. nine cases were not fully vaccinated, or their immunization status was unknown [ ] . an epidemic of measles occurred during the xxi olympic winter games that were held in february - , , in vancouver, canada, with cases [ ] . another epidemic of measles was noted during the th edition of the italia super cup, international junior football tournament in rimini, italy, from june nd to th, . most ill individuals had not been vaccinated [ ] . during the london olympic and paralympic games, no major public health incidents occurred. only a few outbreaks of gastrointestinal and respiratory infections were recorded during this period. no food-borne illness was directly linked to a games venue, despite the tendency for those reporting them to label them as such [ ] . during this event, olympic visitors were followed for sexually transmitted infections (sti), new sti diagnoses were made including non-specific genital infection, eight chlamydia, and eight genital warts (first episode) diagnoses. there were no new hiv or syphilis diagnoses [ ] . during euro european football, according to national data from ukraine, cases of acute gastroenteritis occurred in host cities, but daily notifications remained consistently below the epidemic threshold determined by ukraine. similarly, measles cases were reported in the host cities during the tournament, only one of which occurred in a foreign visitor. this number represented about % of the new cases reported throughout ukraine during the same period [ ] . during the european youth olympic festival in utrecht, the netherlands, in , a prospective cohort study was conducted among participants from countries. forty-six cases of diseases were reported. infection was the most commonly reported cause of illness ( . % overall) with . % patients reporting gastrointestinal symptoms and . % respiratory symptoms [ ] . among the athletes in the sochi olympic winter games, a total of illnesses were reported, resulting in an incidence of . illnesses per athletes ( % ci . to . ). most ill athletes suffered from respiratory symptoms ( . %), followed by gastrointestinal symptoms ( %) with % caused by infections [ ] . only three cases of dengue fever were confirmed at the fifa world cup [ ] . a recent multinational salmonella outbreak was reported at an international youth ice hockey competition in riga, lativa in [ ] . among , athletes from countries participating to the rio de janeiro olympic summer games, illnesses were reported, resulting in . illnesses per athletes. two hundred two individuals ( %) presented with respiratory symptoms and (n = ; %) gastrointestinal symptoms with % (n = ) due to infections [ ] . dengue case count was negligible and no case of zika virus was detected [ , ] . more recently, the pyeong ghang winter olympiad may have been hindered by a norovirus outbreak days before the event commenced. this outbreak affected mainly security staff for the games rather than athletes or visitors [ ] . this review has some limitations. it was limited to articles written in english, which may have been a source of bias. there was a significant heterogeneity in the studies in relation to the populations studied, the clinical criteria for syndromic surveillance and the diagnostic methods applied. infectious diseases at mgs are dominated by respiratory tract and gastrointestinal infections. meningitis outbreaks were also reported in some instances. inter-human transmission of airborne diseases is favored by the temporal and spatial concentration of people. because social distancing and contact avoidance are difficult measures to implement in the context of many mgs, individual preventive measures such as cough etiquette, the use of face mask and disposable handkerchiefs and hand hygiene may be recommended. nevertheless, the effectiveness of these measures has been poorly investigated in the context of mgs. most available data come from hajj studies and results are contradictory [ ] . non-compliance with hygiene rules and inadequate sanitation are responsible for fecal-oral transmission of gastrointestinal infections. public health measures aiming at provision of safe water and food supplies with rigorous quality control are likely the best way to limit the occurrence of gastrointestinal outbreaks at mgs. planned organization by highly specialized teams of staff is a key element. it should be noted that many mg-associated diseases are vaccine-preventable, including influenza, measles, mumps, meningococcal, and pneumococcal disease. mandatory vaccination against meningitis has proven effective in the context of the hajj. measles and mumps and meningococcal vaccination status should certainly be verified and updated if needed in young people attending mgs. influenza and pneumococcal vaccination should be recommended in at-risk individuals participating to mgs. this particularly applies to elderly people participating to religious mgs. finally, because of the evidence of circulation of resistant bacteria, at least in the hajj context, rationalization of antibiotic consumption should be promoted. unfortunately, official recommendations for prevention at mgs are lacking, with the exception of the hajj [ ] . conflict of interest philippe gautret and van-thuan hoang declare that they have no conflict of interest. human and animal rights and informed consent this article does not contain any studies with human or animal subjects performed by any of the authors. world health organization. public health for mass gatherings: key considerations world health organization. communicable disease alert and response for mass gatherings: key considerations mass gatherings and infectious diseases: prevention, detection, and control hajj: infectious disease surveillance 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study of athletes from countries zika virus and the rio olympic games no zika cases reported during rio olympics multiple outbreaks threaten the winter olympics. the disease daily non-pharmaceutical interventions for the prevention of rtis during hajj pilgrimage expected immunizations and health protection for hajj and umrah -an overview key: cord- -i soyabu authors: wabe, nasir; li, ling; lindeman, robert; yimsung, ruth; dahm, maria r; clezy, kate; mclennan, susan; westbrook, johanna; georgiou, andrew title: the impact of rapid molecular diagnostic testing for respiratory viruses on outcomes for emergency department patients date: - - journal: med j aust doi: . /mja . sha: doc_id: cord_uid: i soyabu objective: to determine whether rapid polymerase chain reaction (pcr) testing for influenza and respiratory syncytial viruses (rsv) in emergency departments (eds) is associated with better patient and laboratory outcomes than standard multiplex pcr testing. design, setting: a before‐and‐after study in four metropolitan eds in new south wales. participants: consecutive patients tested by standard multiplex pcr during july–december , and tested by rapid pcr during july–december . main outcome measures: hospital admissions; ed length of stay (los); test turnaround time; patient receiving test result before leaving the ed; ordering of other laboratory tests. results: compared with those tested by standard pcr, fewer patients tested by rapid pcr were admitted to hospital ( . % v . %; p < . ) and more received their test results before leaving the ed ( . % v . %; p < . ); the median test turnaround time was also shorter ( . h [iqr, . – . h] v . h [iqr, . – . h]). the proportion of patients admitted to hospital was also lower in the rapid pcr group for both children under ( . % v . %; p < . ) and patients over years of age ( . % v . %; p < . ). significantly fewer blood culture, blood gas, sputum culture, and respiratory bacterial and viral serology tests were ordered for patients tested by rapid pcr. ed los was similar for the rapid ( . h; iqr, . – . h) and standard pcr groups ( . h; iqr, . – . h; p = . ). conclusion: rapid pcr testing of ed patients for influenza virus and rsv was associated with better outcomes on a range of indicators, suggesting benefits for patients and the health care system. a formal cost–benefit analysis should be undertaken. the health and economic burdens associated with acute respiratory infections by influenza and respiratory syncytial viruses (rsv) are significant in australia and overseas. - polymerase chain reaction (pcr) testing is effective for confirming respiratory viral infections. multiplex pcr can detect numerous respiratory viruses, including influenza and parainfluenza viruses, rsv, adenovirus, rhinovirus, human metapneumovirus, enterovirus, bocavirus and coronavirus with very high sensitivity and specificity. although the results of standard multiplex pcr are accurate and comprehensive, it has traditionally been performed in a central laboratory with a lengthy turnaround time, which may be inconvenient in settings where test results are urgently required, including emergency departments (eds). rapid, easy-to-use pcr-based respiratory virus diagnostic tests have been introduced in recent years; , the genexpert system (cepheid), for instance, was introduced in new south wales in july . rapid pcr tests were expected to facilitate timely and appropriate initiation of treatment, improve outbreak prevention and infection control measures, and expedite the assessment of patients in eds. in this study, we analysed routinely collected data to determine whether rapid pcr testing for influenza and rsv infections in eds is associated with improved patient and laboratory outcomes. we compared data for patients tested for influenza a/b viruses and rsv immediately after rapid pcr diagnosis was introduced (july-december ) with data for patients tested with a standard multiplex pcr system during july-december . we undertook a before-and-after study in four metropolitan public hospital eds in sydney, nsw: three general hospitals (eds a, b and c; , and annual ed presentations respectively) and one children's hospital (ed d; annual ed presentations; all data for january-december ). the four hospitals were served by a single pathology laboratory provider. we analysed data for all patients tested for influenza virus or rsv. during july-december , patients were tested with the standard pcr system, a central laboratory-based multiplex pcr test for sixteen respiratory viruses (including rsv and influenza viruses a and b), available as a referral test at the central laboratory in hospital b. during july-december , patients were tested with the rapid pcr system, a hospital laboratory-based test specific for rsv and influenza viruses a and b. hospitals a, b and d have onsite laboratories that perform rapid pcr testing; hospital c sends samples to the nearby hospital a. all tests were conducted in virology laboratories by trained staff, and test results were entered into laboratory information system datasets. we obtained relevant patient characteristics and the proportion of patients admitted to hospital was also lower in the rapid pcr group for both children under ( . % v . %; p < . ) and patients over years of age ( . % v . %; p < . ). significantly fewer blood culture, blood gas, sputum culture, and respiratory bacterial and viral serology tests were ordered for patients tested by rapid pcr. ed los was similar for the rapid ( . h; iqr, . - . h) and standard pcr groups ( . h; iqr, . - . h; p = . ). conclusion: rapid pcr testing of ed patients for influenza virus and rsv was associated with better outcomes on a range of indicators, suggesting benefits for patients and the health care system. a formal cost-benefit analysis should be undertaken. the known: rapid polymerase chain reaction (pcr) testing for influenza and respiratory syncytial viruses (rsv) was introduced in new south wales in july . its impact on outcomes for emergency department (ed) patients has not been investigated. the new: compared with standard pcr testing, rapid pcr was associated with significantly fewer hospital admissions, more rapid test turnaround, more patients receiving test results before leaving the ed, and reduced numbers of some other common microbiology tests. it did not significantly affect ed length of stay. the implications: rapid pcr testing of ed patients for major respiratory viruses can benefit patients and reduce resource use. mja ( ) ▪ april laboratory data by linking the ed and laboratory information system datasets. detailed information about the datasets and the linkage process have been described previously. the primary outcomes were admission to hospital, ed length of stay (los), test turnaround time, and the patient receiving their test result before leaving the ed. ed los was defined as the time from arrival in the ed to discharge or admission to hospital. turnaround was defined as the time from when the sample was received by the laboratory to when the test result was available in hospital electronic records. as secondary outcomes, we compared ordering of typical biochemistry and haematology tests (full blood count; electrolyte, urea, creatinine levels; liver function test; blood gas analysis; c-reactive protein level) and microbiology tests (blood culture; urine microscopy, culture and sensitivity analysis; sputum culture; respiratory bacteria and virus serology) during the two study periods. analyses were conducted in stata (statacorp). descriptive statistics are reported (medians with interquartile ranges [iqrs], means with standard deviations [sds], numbers with proportions). baseline characteristics were compared in χ tests (categorical outcomes) or wilcoxon rank-sum tests (continuous outcomes). logistic regression analyses of binary outcomes (eg, hospital admission: yes/no) and quantile regression analyses of continuous outcomes (eg, ed los) were undertaken. all analyses were adjusted for baseline characteristics. sensitivity analyses limited to data for children (under years of age) or older patients (over years of age) were conducted. p < . ( -tailed) was deemed statistically significant. ethics approval for the investigation was granted by the human research ethics committee of the south eastern sydney local health district (reference, hrec/ /powh/ ). we analysed data for patients presenting to the four eds during two periods: consecutive patients during july-december (standard pcr) and during july-december (rapid pcr). baseline characteristics for the two groups were similar in terms of sex, triage category, and arrival day of the week, but differed significantly for age, arrival time, and mode of arrival (box the overall numbers of tests per patient were similar in the standard pcr (mean, . tests; sd, . ) and rapid pcr groups (mean, . tests; sd, . ). the mean number of microbiology tests per patient was significantly lower for the rapid pcr group ( . tests; sd, . ) than for the standard pcr group ( . tests; sd, . ; p < . after controlling for baseline characteristics). the biochemistry/haematology and microbiology tests comprised . % of the other tests (that is, not including pcr virus testing) ordered for patients in the study. after adjusting for baseline characteristics, the proportions of patients for whom full blood count, electrolyte/urea/creatinine levels, liver function, or c-reactive protein were assessed were similar, as were the proportions for urine microscopy, culture and sensitivity tests. significantly fewer blood culture, blood gas, sputum culture, and respiratory bacterial and viral serology tests were ordered for patients in the rapid pcr group (box ). information, figure ). ed los was similar for the standard pcr and rapid pcr groups in both age-based subgroups (supporting information, figure a ). the differences in test turnaround time identified in the main analysis were also evident for each age-based subgroup (supporting information, figure b ). in this before-and-after study, we found that rapid pcr testing of ed patients for major respiratory viruses was associated with significantly fewer admissions to hospital, more rapid delivery of test results, more patients receiving their test results before leaving the ed, and less frequent ordering of some common microbiology tests. other studies have also reported that hospital admission numbers were significantly lower when rapid influenza virus testing was used in eds. an analysis of outcomes for more than adults at a tertiary care centre in new york found that early diagnosis of respiratory infections was associated with significantly fewer hospitalisations of influenza-positive patients. in a small irish study ( patients), the hospital admission rate for obstetric patients declined from % to % after on-site rapid influenza pcr testing was introduced. the differences in clinical setting and patient group may explain the smaller decline in our study (from % to %). non-pcr-based rapid diagnostic tests for respiratory viruses have also been associated with lower hospital admission rates. , the main reason for fewer hospital admissions of patients tested by rapid pcr may be that the earlier availability of results enables clinicians to quickly diagnose or exclude respiratory infections and to make timely and informed decisions about whether to discharge the patient or admit them to hospital. when standard primary outcomes for patients at four metropolitan emergency departments (eds) tested for influenza and respiratory syncytial viruses by standard or rapid polymerase chain reaction (pcr) after adjusting for baseline characteristics (box ): * p = . ; ** p < . . ◆ mja ( ) ▪ april pcr was used, in contrast, our findings suggest that these decisions were made before the test results were available. the possible benefits of not admitting patients to hospital, beyond those for individual patient management, include better infection control and outbreak prevention, as well as reduced demands on hospital resources. , the impact of rapid pcr testing on outbreak prevention and infection control measures should be evaluated. rapid influenza virus testing may also have practical implications for hospital bed management. , ed los was similar in our study before and after the introduction of rapid pcr methods. this finding was not unexpected, as test turnaround time is not the only rate-limiting factor for decision making in eds. before rapid pcr methods were introduced, the long turnaround time of multiplex pcr did not necessarily extend a patient's stay in the ed, as they were usually admitted to hospital or discharged home before the results were available. consequently, more rapid delivery of test results alone would not reduce ed los. reports on the effect of rapid influenza virus testing and los have been conflicting. while evidence for an association between rapid testing and shorter overall inpatient los has been reported, , findings regarding ed los are inconsistent. , , for example, a cochrane review based on three randomised controlled trials did not find a statistically significant association of rapid viral diagnosis with lower mean ed los. in a study in children, ed los was actually minutes longer with rapid pcr; inpatient los did not differ between the two groups, but was significantly shorter when the analysis was limited to patients with positive test results. we found that ordering of some other microbiology tests, including blood culture, sputum culture, and respiratory bacterial and virus serology, was significantly less frequent for patients tested by rapid pcr. the effect of pcr-based rapid testing on the ordering of other laboratory tests has not previously been reported, although some studies of antigen-based pointof-care testing have examined this outcome. consistent with our finding, several investigators have reported fewer blood culture tests , and basic biochemistry and haematology tests, including full blood count, , c-reactive protein testing, and urinalysis, , when point-of-care testing was used. the higher rate of positive results for patients tested by rapid pcr than for those tested by standard pcr may reflect a higher prevalence of influenza during than in . the rapid pcr system in our study accurately detects influenza viruses a/b and rsv but, unlike the standard multiplex pcr, cannot detect other clinically relevant respiratory viruses, such as rhinovirus, coronavirus, human metapneumovirus, parainfluenza virus, adenovirus, enterovirus, and bocavirus. if infection with other respiratory viruses is suspected, patients may therefore need further investigations. standard multiplex pcr can provide broader information, as it can detect multiple respiratory viruses in a single run, although the long turnaround time restricts its suitability for urgent clinical decision making. improving the turnaround time of multiplex pcr analysis may achieve better outcomes. the strengths of our study include its relatively large sample size; further, unlike many similar investigations, ours was a multicentre study in four hospital eds, enhancing the generalisability of our findings. however, our analyses were not adjusted for comorbid conditions, as this information was not available. because our study was an uncontrolled before-and-after study, our results cannot be interpreted as indicating causal relationships. as with all non-randomised trials, we could not fully account for all potential confounding variables, nor for temporal trends and other unmeasured factors, such as changes in clinician testing practices or differences in the prevalence and severity of disease during the two study periods. for example, a shortage of inpatient beds caused by a high prevalence of influenza could influence decisions in a busy ed about admitting patients to hospital. however, we attempted to reduce seasonal effects by selecting similar time frames for the two study periods, to reduce selection bias by including all ed patients tested for influenza virus and rsv, and to control for differences in baseline patient characteristics by applying multivariate modelling. as medications data were not available to us, we were unable to assess the impact of rapid pcr testing on antibiotic and antiviral drug use. similarly, the cost-benefit balance of rapid testing was not evaluated because relevant data were not available. the cost per patient of rapid pcr testing is generally higher than for central laboratory testing, but our findings suggest potential savings through lower numbers of hospital admissions and reduced resource use. this question could be evaluated in a further study. rapid pcr testing for influenza virus and rsv infections in patients attending eds was associated with significant improvements in a range of patient and laboratory outcomes, suggesting potential benefits for both the patients and the health care system. a cost-benefit analysis could examine the impact of rapid pcr testing on bed management and antimicrobial drug prescribing. impact of introduction of xpert flu assay for influenza pcr testing on obstetric patients: a quality improvement project utility of early influenza diagnosis through pointof-care testing in children presenting to an emergency department the impact of commercial rapid respiratory virus diagnostic tests on patient outcomes and health system utilization economic impact of a new rapid pcr assay for detecting influenza virus in an emergency department and hospitalized patients economic evidence and point-of-care testing rapid testing for respiratory syncytial virus in a paediatric emergency department: benefits for infection control and bed management laboratory turnaround time effect of the influenza virus rapid antigen test on a physician's decision to prescribe antibiotics and on patient length of stay in the emergency department rapid viral diagnosis for acute febrile respiratory illness in children in the emergency department accuracy and impact of a point-of-care rapid influenza test in young children with respiratory illnesses a national study of the impact of rapid influenza testing on clinical care in the emergency department influence of rapid influenza test on clinical management of children younger than five with febrile respiratory tract infections the project was part of a partnership project funded by a national health and medical research council of australia partnership project grant (app ), in partnership with nsw health pathology and the australian commission on safety and quality in healthcare. key: cord- - h yyqj authors: zheng, xue-yan; xu, yan-jun; guan, wei-jie; lin, li-feng title: regional, age and respiratory-secretion-specific prevalence of respiratory viruses associated with asthma exacerbation: a literature review date: - - journal: arch virol doi: . /s - - -y sha: doc_id: cord_uid: h yyqj despite increased understanding of how viral infection is involved in asthma exacerbations, it is less clear which viruses are involved and to what extent they contribute to asthma exacerbations. here, we sought to determine the prevalence of different respiratory viruses during asthma exacerbations. systematic computerized searches of the literature up to june without language limitation were performed. the primary focus was on the prevalence of respiratory viruses, including adv (adenovirus), bov (bocavirus), cov (coronavirus), cmv (cytomegalovirus), env (enterovirus), hsv (herpes simplex virus), ifv (influenza virus), mpv (metapneumovirus), piv (parainfluenzavirus), rv (rhinovirus) and rsv (respiratory syncytial virus) during asthma exacerbations. we also examined the prevalence of viral infection stratified by age, geographic region, type of respiratory secretion, and detection method. sixty articles were included in the final analysis. during asthma exacerbations, the mean prevalence of adv, bov, cov, cmv, env, hsv, ifv, mpv, piv, rv and rsv was . %, . %, . %, . %, . %, . %, . %, . %, . %, . % and . %, respectively. env, mpv, rv and rsv were more prevalent in children, whereas adv, bov, cov, ifv and piv were more frequently present in adults. rv was the major virus detected globally, except in africa. rv could be detected in both the upper and lower airway. polymerase chain reaction was the most sensitive method for detecting viral infection. our findings indicate the need to develop prophylactic polyvalent or polyvirus (including rv, env, ifv and rsv) vaccines that produce herd immunity and reduce the healthcare burden associated with virus-induced asthma exacerbations. electronic supplementary material: the online version of this article ( . /s - - -y) contains supplementary material, which is available to authorized users. asthma is a chronic inflammatory airway disease that is susceptible to triggering factors, such as aeroallergens, air pollutants, and viral infection [ ] . despite major advances in prevention and management, asthma remains a considerable global healthcare burden [ ] . from to , the crude prevalence of asthma has increased by . % [ ] . currently, there are . million cases of asthma globally. despite dramatic reductions in age-standardized mortality, . million people died from asthma in [ ] . the management of asthma exacerbations, which have been consistently linked to upper and/or lower airway infections, is an important health issue [ ] . among school-age children, viral infection reportedly accounts for %- % of asthma exacerbations, and viruses are more frequently isolated from symptomatic patients than from asymptomatic patients [ ] . although rhinovirus (rv) has frequently been detected in asthmatic patients [ ] , asthma exacerbations have also been associated with infection with other respiratory communicated by tim skern. xue-yan zheng and yan-jun xu shared co-first authorship. the online version of this article (http s://doi.org/ . /s - - -y) contains supplementary material, which is available to authorized users. * wei-jie guan battery @ .com * li-feng lin @qq.com viruses, including adenovirus (adv), bocavirus (bov), coronavirus (cov), cytomegalovirus (cmv), enterovirus (env), herpes simplex virus (hsv), influenza virus (ifv), metapneumovirus (mpv), parainfluenzavirus (piv), and respiratory syncytial virus (rsv) [ ] . unfortunately, the rate of association of viruses with asthma exacerbations remains controversial. a hypothetical explanation might be substantial differences in study design, populations, geographic regions, detection methods, and the type of respiratory tract secretions examined. despite our increased understanding of the roles that viruses play in asthma exacerbations, it is less clear which viruses are involved and to what extent they contribute to asthma exacerbations in different subgroups of subjects. we conducted a systematic review of the prevalence of viral infection in patients with asthma exacerbations, with subgroup analyses to identify the determinants of variations. our findings may offer a better overview for developing preventive interventions to minimize virus-induced asthma exacerbations. studies were searched comprehensively in embase, pub-med, cochrane central register of controlled trials and ebm reviews-cochrane database of systematic reviews, web of science, ovid and highwire up to june (no date-of-start was specified). references were checked for additional data. we restricted our search list to publications in the english language. when the same population was used for analysis in several publications, only the largest and most complete study was included. we adopted combined keywords related to virus detection (adenovirus, bocavirus, metapneumovirus, coronavirus, epstein-barr virus, influenza virus, parainfluenza virus, rhinovirus, enterovirus and respiratory syncytial virus) and the outcomes of asthma exacerbations ((((((("pulmonary disease, chronic obstructive"[mesh])) and ("disease progression" [mesh]))) or (((copd)) and (exacerbation)))) and (((("viruses"[mesh])) or (respiratory viral infections)) or (respiratory virus))) and ((("polymerase chain reaction"[mesh])) or (virus pcr)). cross-sectional, prospective studies, cohort studies and case-control studies detailing the prevalence of respiratory virus(es) in asthma exacerbations were included. all eligible asthmatic patients were diagnosed as having viral infection based on polymerase chain reaction (pcr)/enzyme-linked immunosorbent assay (elisa)/cell culture/direct fluorescent antibody assay (dfa)/ indirect fluorescent antibody assay (ifa), which were evaluated during exacerbations. these studies investigated asthma exacerbations in children and adults. we excluded animal studies, ex vivo and toxicological studies, summaries, commentaries and editorials, case reports and case series, duplicate publications, samplerelated or laboratory-based studies, studies in intensive care settings, patients with concomitant chronic obstructive pulmonary disease, bronchiectasis or immunosuppression, non-peer reviewed articles (a potential source of bias), nonoriginal data, nosocomial infections (hospitalization within four weeks, or delayed sample collection [> h after hospitalization]). authors were contacted via e-mail if data were incomplete. studies were excluded if no reply was obtained despite repeated contacts with the corresponding authors. this study complied with preferred reporting items for meta-analysis [ ] . the quality of individual studies was assessed based on the revised quality assessment of diagnostic accuracy studies- tool [ ] . the highest total score was , with a higher score indicating a lower risk of bias. two independent reviewers (x.y.z. and y.j.x.) screened abstracts and titles. full texts were reviewed to determine eligibility. disagreement was resolved by discussion. if consensus was not reached, another reviewer (w.j.g.) was consulted to vote for final decisions. a standardized form was used for data extraction, including the main characteristics (author, year of publication, sample size, age, definition of exacerbation, quality, detection method, study design and season), primary outcome (the prevalence of viral infection during asthma exacerbations), and secondary outcomes (the prevalence of viruses in different strata). data extraction was done by two independent reviewers (x.y.z. and l.f.l.). disagreement was resolved by consultation with the third reviewer (w.j.g.). we did a meta-analysis to obtain point estimates of the rate of asthma exacerbations associated with viral infection across the whole age spectrum globally [ ] . the der-simonian and laird random-effect model [ ] was applied because the heterogeneity among the studies was greater than %. the variance of raw proportions reported in each study was stabilized by using the untransformed proportion, logarithmic transformation, logit transformation, arcsine transformation or freeman-tukey-type arcsine square-root transformation, according to shapiro-wilk's test for normality of data distribution. we also did a subgroup analysis to assess the weight of viral infection on asthma exacerbations with respect to geographic region, population, type of respiratory tract secretion examined, and detection method. if only one study was available, we calculated the % confidence interval ( %ci) using the clopper-pearson exact test. because difference in the geographic regions, age, study population, type of respiratory tract secretion, and detection method significantly confound the determination of the prevalence of individual viruses, heterogeneity was not assessed in this study. analyses were conducted with r software (version . . ). of articles relevant to our objectives, were excluded. no additional studies were identified through citations within articles. figure summarizes the selection process. sixty articles ( studies) were analyzed to assess the association of virus infection with asthma exacerbations. the characteristics of the patient population, study design, country, sample size, asthma definition, quality score, detection method, study design, type of respiratory tract secretion examined, and viruses involved varied substantially (e-table ). of the eligible studies, were cross sectional studies, were case-control studies, were cohort studies, and seven prospective studies. the studies were conducted between and , with the duration varying between one month and years. asthma was diagnosed by a physician in studies in which the data reported on the burden of asthma. the mean age of asthmatic patients varied among the different studies, and therefore, children and adults were dichotomized for reporting. upper/lower airway specimens were screened by different methods, including pcr, dfa assay, ifa assay, virus culture, elisa, rapid enzyme immunoassay and virus neutralization test. the criteria for quality assessment and risk of bias of individual studies are summarized in e- table . the mean score of quality assessment was . (range, to ). all included studies were, at least partially, prone to reporting bias. overall, the mean prevalence ( %ci) bov and env were most frequently detected in oro-/ naso-pharyngeal aspirates, whereas adv was primarily detected in induced sputum. cov, ifv, mpv and piv were most frequently detected in oro-/naso-pharyngeal lavage. rv and rsv were the viruses most frequently detected in spontaneous oro-/naso-pharyngeal secretions. (table ) overall, pcr was the most sensitive technique for detecting adv, bov, env, ifv, mpv and piv, but not for other respiratory viruses, when compared with ifa, elisa, dfa and virus culture. elisa yielded the highest detection rates for cov and rsv (table , fig. d ). in this study, we gathered the latest data on the prevalence of viruses detected during asthma exacerbations. asthma exacerbations were mainly associated with rvs ( . %), highlighting the priority for interventions to reduce the substantial healthcare burden caused by these viruses. this finding has been echoed by the high prevalence of rvs (~ %) among unselected populations residing in north america [ ] . in the netherlands, rvs were most prevalent among symptomatic children during wheezing episodes ( %) and convalescence ( %), as well as in healthy children ( %) [ ] . a possible explanation is that rvs are the most common resident pathogens and are prone to cause upper airway infections (e.g., common cold). consistently, rvs were isolated in . % of pharyngeal exudate samples from mexicans with acute upper respiratory tract infections [ ] . the prevalence of viruses varied considerably among different studies. for example the range was - % for rsv and - % for env) [ ] (for details, see the online supplement). intriguingly, the mean estimated prevalence of envs, hsv, ifv and rsv was greater than %, whereas other individual respiratory virus had a mean prevalence of less than %. in patients with other respiratory diseases, the prevalence of these viruses partially mirrored our findings. rsv was reportedly found in % of elderly patients with acute respiratory illnesses [ ] and in . % in mexicans with acute upper respiratory tract infection [ ] , env was isolated from . % of young infants with sepsis-like illnesses [ ] , and bov was detected in . % of nasopharyngeal aspirates from hospitalized children in spain [ ] . however, the prevalence of mpv was higher ( . %) in mexicans with acute upper respiratory tract infection [ ] , and the prevalence of adv was also higher ( . %) among hospitalized children in spain [ ] than that in our pooled analysis. the substantial heterogeneity of the prevalence data was probably due to differences in study design, ethnicity, geographic region, the site of sample collection, and detection methods. therefore, direct comparisons remained challenging, and therefore subgroup analysis was done to identify possible determinants of variation. we next stratified the prevalence data by age. env, mpv, rv and rsv were more prevalent in children, whereas adv, bov, cov, ifv and piv were more prevalent in adults. env and mpv were associated with asthma exacerbations in children [ , ] , rv were frequently associated with asthma exacerbations associated with upper respiratory tract infection in children, and rsv was commonly linked to onset of wheezing in childhood [ , ] . the significant variation in the viruses involved probably reflected a major shift during the development of host-defense mechanisms. the association with specific viruses varied considerably across geographic regions. rv was the most common virus in asia, europe, america and oceania. rv is probably the dominant resident virus colonizing the upper and lower airways (online supplement), irrespective of meteorological factors such as temperature and humidity. rsv has been reported to be more common among populations in africa, which could be partially related to its generally higher prevalence in low-income countries [ ] . however, the reasons for the variation in the prevalence of other common respiratory viruses are not known. further research is warranted to determine whether ethnicity-related susceptibility or meteorological factors play a significant role in shaping the prevalence of viral infections. consistent with literature reports (online supplement text), rv can be detected in both the upper and lower airways. for rsv, apart from the greater likelihood of initial infection of the nasopharyngeal mucosa, followed by spreading of the virus to the lower airways [ ] , the extensive use of nasal swabs, but not spontaneous or induced sputum for sample collection from children might also have resulted in a higher apparent prevalence in upper airway secretions. furthermore, the higher prevalence of bov and env detected in upper airway secretions might be associated with fecalto-oral transmission [ ] or transmission from oral mucosa or salivary glands [ ] , which would be detected more frequently in infants and toddlers. in accordance with literature reports [ , ] , pcr was the most sensitive method for detecting viruses in asthma exacerbations. it is a simple, rapid and cost-effective technique that has become the mainstay for viral detection. whilst virus isolation in cell culture remains the gold standard, it is time-consuming and labor-intensive, rendering it unsuitable for rapid batch detection in clinical settings [ ] . due to their limited sensitivity and specificity, other techniques such as dfa and ifa are less frequently applied clinically. our findings confirm that viruses of various species are closely associated with asthma exacerbations. the mechanisms are multifaceted, including ) necrosis of airway epithelium, ciliostasis and loss of cilia [ ] [ ] [ ] , ) excessive release of pro-inflammatory cytokines and chemokines [ ] , ) airway hyper-responsiveness and remodeling due to epithelial injury [ ] , and ) mucus hypersecretion, which impedes mucociliary clearance [ ] . unfortunately, there are few effective treatment options (e.g., immunoglobins, nebulized antivirals) for direct elimination of viral infections [ , ] . preemptive approaches should be directed to the prevention of viral infection, particularly in susceptible populations (e.g. children, pregnant women, the elderly, and patients with comorbidities). this has been exemplified by the vaccine development for prevention of rsv and influenza virus infection [ , ] . priority should be given to the most common viruses (e.g., rv, rsv, hsv, env, and ifv), which have caused a significant healthcare burden. recently, a polyvalent vaccine for rv was developed in animal models [ ] , indicating the possibility of establishing herd immunity using polyvalent or multiple-virus vaccines in future community-based practice, which might help to achieve greater reductions in the prevalence of virus-induced asthma exacerbations. our results may not be readily extrapolated to all countries. for instance, documentation of the prevalence of multiple viruses in africa is still lacking, and little is known regarding the distribution of specific viral strains. due to the small number of studies relevant to some of our subgroup analyses (e.g., cell culture and dfa assay) even slight variations in individual studies might have had a significant effect on modified our global estimates. we have summarized the prevalence of respiratory viruses associated with asthma exacerbations. rv, env and rsv are the most prevalent of these, and therefore preemptive prophylaxis with effective vaccines or novel antiviral agents is needed to minimize the healthcare burden of asthma exacerbation resulting from viral infection. fig. the prevalence of the five most common viruses in the stratified analysis a. the prevalence of the five most common viruses when stratified by age (adults vs. children). b. the prevalence of the five most common viruses when stratified by geographic region (from top to bottom: africa, oceania, american, asia, and europe). c. the prevalence of the five most common viruses when stratified by the type of respiratory tract sample analyzed (lower vs. upper airway). d. the prevalence of the five most common viruses when stratified by method of detection (from top to bottom: culture, ifa, elisa, dfa, and pcr). the horizontal axis represents the prevalence (%) of individual viruses. pcr, polymerase chain reaction; ifa, indirect fluorescent antibody assay; dfa, direct fluorescent antibody assay; elisa, enzyme-linked immunosorbent assay; rv, rhinovirus; adv, adenovirus; bov, bocavirus; cov, coronavirus; cv, cytomegalovirus; env, enterovirus; hsv, herpes simplex virus; ifv, influenza virus; mpv, metapneumovirus; piv, parainfluenzavirus (piv); rsv, respiratory syncytial virus asthma exacerbations: pathogenesis, prevention, and treatment global initiative for asthma (gina) program. the global burden of asthma: executive summary of the gina dissemination committee report global, regional, and national deaths, prevalence, disabilityadjusted life years, and years lived with disability for chronic obstructive pulmonary disease and asthma, - : a systematic analysis for the global burden of disease study community study of role of viral infections in exacerbations of asthma in - year old children preferred reporting items for systematic reviews and meta-analyses: the prisma statement quadas- : a revised tool for the quality 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ghulam; li, guangxing title: antiviral activity against infectious bronchitis virus and bioactive components of hypericum perforatum l. date: - - journal: front pharmacol doi: . /fphar. . sha: doc_id: cord_uid: sbtigcfr hypericum perforatum l., also known as saint john’s wort, has been well studied for its chemical composition and pharmacological activity. in this study, the antiviral activities of h. perforatum on infectious bronchitis virus (ibv) were evaluated in vitro and in vivo for the first time. the results of in vitro experiments confirmed that the antiviral component of h. perforatum was ethyl acetate extraction section (hpe), and results showed that treatment with hpe significantly reduced the relative messenger ribonucleic acid (mrna) expression and virus titer of ibv, and reduced positive green immunofluorescence signal of ibv in chicken embryo kidney (cek) cells. hpe treatment at doses of – mg/kg for days, reduced ibv induced injury in the trachea and kidney, moreover, reduced the mrna expression level of ibv in the trachea and kidney in vivo. the mrna expression levels of il- , tumor necrosis factor alpha (tnf-α), and nuclear factor kappa beta (nf-κb) significantly decreased, but melanoma differentiation-associated protein (mda ), mitochondrial antiviral signaling gene, interferon alpha (ifn-α), and interferon beta (ifn-β) mrna levels significantly increased in vitro and in vivo. our findings demonstrated that hpe had significant anti-ibv effects in vitro and in vivo, respectively. in addition, it is possible owing to up-regulate mrna expression of type i interferon through the mda signaling pathway and down-regulate mrna expression of il- and tnf-α via the nf-κb signaling pathway. moreover, the mainly active compositions of hpe analyzed by high-performance liquid chromatography/electrospray ionization–mass spectroscopy (esi-ms) are hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin, and a combination of these compounds could mediate the antiviral activities. this might accelerate our understanding of the antiviral effect of h. perforatum and provide new insights into the development of effective therapeutic strategies. the infectious bronchitis virus (ibv) is a prototype coronavirus containing a single-stranded positive-sense rna genome (cook et al., ) . ibv is the etiologic agent of infectious bronchitis (ib), which is a highly contagious, acute viral respiratory disease of chickens. ibv has been reported by many researchers all over the world (jungherr and terrells, ; jungherr et al., ; fabricant, ; yu et al., ; benyeda et al., ; sjaak de wit et al., ; westerbeck and machamer, ; wu et al., ) . ibv has led to severe losses in the poultry industry (jordan, ) , the direct losses are due to highly mortality, poor egg quality, and meat production, and the indirect losses result in increased costs and challenges in ibv prevention (liang et al., ) . at present, live attenuated vaccines are widely used for the prevention and control of ib. however, due to extensive genetic diversity of ibv strains, the vaccines are becoming increasingly inefficient, with poor cross-protection effects among different serotypes of vaccines (mo et al., ; chen et al., ; lin and chen, ; yan et al., ) . meanwhile, due to the lack of coordinated effort to prevent the ibv, and the lack of proper surveillance plus the introduction of foreign strains to combat the ibv in certain regions, the prevention and control of ibv has become very difficult. therefore, it is imperative to find an effective antiviral drug or agent for the prevention of ibv. in order to control drug residues, the chinese government has banned the use of antiviral drugs in food animals in china. therefore, the use of traditional antiviral herbs with no obvious side effects on the human body is still a major focus. some reports have confirmed that traditional chinese herbs could effectively inhibit the infection and replication of various viruses (li et al., ; wang et al., ; choi et al., ; sun et al., ; choi et al., ; yin et al., ; yi et al., ; luo et al., ) . hypericum perforatum l. belongs to the genus guttiferae, which contains approximately species all over the world. the extract of h. perforatum contains several active compounds, including flavonoids, naphthodianthrones, and phloroglucinol derivatives (napoli et al., ; barnes et al., ) . several reports have shown that h. perforatum extract had antiviral effects, such as influenza a virus, porcine respiratory and reproductive syndrome virus (prrsv), and hiv (barnes et al., ; birt et al., ; pu et al., a; pu et al., b; pu et al., ) . like influenza a virus and prrsv, ibv also belongs to rna virus, but these prrsv and ibv belong to different viral families. since h. perforatum could resist influenza a virus and prrsv, could it resist ibv? in this study, we investigated the antiviral effects of h. perforatum extract against ibv utilizing several approaches in vitro and in vivo for the first time. moreover, the purpose of this work was to point out the antiviral active ingredients of h. perforatum and its anti-ibv mechanisms. for this purpose, the relative messenger ribonucleic acid (mrna) expression levels of ibv in cek cells, tracheas, and kidneys were measured. the positive green immunofluorescence signal of ibv in ceks was observed. in addition, hematoxylin-eosin (he) staining of tracheas and kidneys was performed, and the relative mrna expression of il- , tumor necrosis factor alpha (tnf-α), ifn-α, ifn-β, mda , mavs, and nuclear factor kappa beta (nf-κb) in vitro and in vivo were analyzed. finally, the antiviral principal chemical composition of h. perforatum extract was analyzed by high-performance liquid chromatography (hplc)/esi-ms. in summary, our findings for the first time showed that h. perforatum extract had significant antiviral effect on ibv, and it might up-regulate mrna expression levels of type i interferon via mda pathway and down-regulate mrna expression levels of il- and tnf-α through the nf-κb pathway. the hpe was found to be composed mainly of hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin, and a combination of these compounds could mediate the antiviral activities. the ibv m strain (genbank: fj . ) was provided by key laboratory for laboratory animals and comparative medicine, northeast agricultural university and propagated in -dayold specific pathogen-free (spf) chicken embryos (harbin veterinary research institute, caas). µl ibv m strain was inoculated into spf chicken embryos under aseptic operation. the survival status of chicken embryos was observed every h, and the chicken embryos that died within h were abandoned. then allantoic fluid from infected embryos was collected at h post-inoculation and stored at − °c. the crude extract of h. perforatum was prepared in the laboratory. in brief, the drug was pretreated with carbon dioxide supercritical extraction method, and the supercritical extraction extract (see) was obtained with a yield of . % (wt/wt). then, g of the pretreated medicinal material was weighed, . l of % methanol was added (raclariu et al., ) , ultrasonic extraction was performed twice for min, and ultrasonic power was w. the two extract solutions were combined and filtered, and concentrated under reduced pressure to obtain the crude extract with a yield of . % (wt/wt). approximately g of the crude extract of h. perforatum was dissolved in ml of distilled water. an equal volume of ethyl acetate was added before shaking vigorously and separating in a separating funnel. the ethyl acetate layer (upper layer) and the water layer (lower layer) were obtained. it was then extracted with ethyl acetate until the upper layer had no color. all ethyl acetate extractions and water extractions were combined and evaporated to obtain extract of h. perforatum ethyl acetate (hpe) and extract of h. perforatum water (hpw). a stock solution of hpe ( mg/ml), hpw ( mg/ml) and see ( mg/ml) was prepared with dimethylsulfoxide (dmso, sigma), respectively. immediately before each experiment in vitro, the working solution of h. perforatum extract was diluted in m medium without serum to obtain a final concentration. the hpe was dissolved in % dmso, while ribavirin (rt) was dissolved in physiological saline solution (pss, . % nacl) in vivo experiments. cek cells were primary cultured from -day-old spf chicken embryo (harbin veterinary research institute, caas) and prepared according to the standard technique (schat and purchase, ; ghetas et al., ) . the cells were cultured in m medium (thermo fisher scientific, usa) supplemented with penicillin and streptomycin, and % serum at °c with % co . then, ibv m strain was passaged in cek cells for adaption. µl ibv m allantoic fluid was inoculated into cek cells in ml cell culture flask, and the cytopathic effect (cpe) was observed. when the cells showed obvious typical cpe, the time and degree of the lesions were recorded and the first generation of ibv was collected by freezing and thawing cells repeatedly. then, the collected first generation ibv was inoculated into cek cells according to the above method, and the time and lesion degree of cpe were also recorded. the second generation ibv was collected according to the above method. after times of passages of ibv in cek cells, there appeared typical cpe of cell exfoliation and fusion at same time post inoculation regularly. ibv was collected and stored at − °c until use. the results of adaptation and replication in cek cells, such as cpe, reverse transcription polymerase chain reaction (rt-pcr), and ibv growth curve determined by tissue culture infective dose (tcid ) at different time were tested. the cytotoxicity test was carried out according to the published method of thiazolyl blue tetrazolium bromide (mtt), and minor modifications were made (sui et al., ) . the monolayer of cek cells plated on -well culture plate were washed with d-hanks solution three times, and then hpe, hpw, and see with concentrations of . , . , . , . , and . µg/ml were added to the hole (three repeated holes per concentration). the control cells were incubated in the absence of experimental compounds, but incubated with the same concentration of dmso. the cek cells were cultured at °c for h, then μl mtt with concentrations of mg/ml in m medium were added, and then the cells were incubated at °c for h. after washing the cek cells with d-hanks, μl dmso was added to each well, then the cek cells were incubated at °c for min, and shaken gently at room temperature for min to dissolved formazan precipitates, and od was determined. using the mean values, the cell survival rate is calculated according to the following formula: in order to analyze the effects of hpe, hpw, and see on cells, cek cells cultured on well plates were incubated with hpe, hpw, and see solutions at concentrations of . , . , . μg/ml at °c for h, respectively, and then washed with d-hanks for three times. the cek cells were then infected with tcid ibv and cultured at °c for h. the cell samples were frozen and thawed repeatedly for three times, and the total rna was extracted and reverse transcribed into complementary deoxyribonucleic acid (cdna). the relative mrna expression of ibv n gene was detected by real-time quantitative rt-pcr (qrt-pcr). in addition, according to the conventional method, the virus titer of cell samples was determined by tcid . the specific operations were as follows: cek cells were cultured in -well tissue culture plates, and when the cells were full of monolayer, ibv virus was diluted to − , − , − …, and − times with serum-free m , respectively. then the monolayer cek cells were washed with d-hanks solution three times, and the d-hanks solution was discarded. then the µl ibv virus diluent as mentioned above was added to each hole, and each virus diluent repeated eight holes. the cells were treated by the same method with m culture as normal control. the culture plate was cultured in % co and °c for h. the cytopathic effect (cpe) was observed every h, and the number of cpe pores and no cpe pores were recorded. according to the reed-muench method as previously described (guo et al., ; yang et al., b) , the tcid was calculated according to the number of cpe holes recorded. at the same time, the infected cek cells treated with μg/ml rt and cek cells infected with ibv as control. in order to determine the impact of hpe, hpw, and see on ibv-infected cells, the cek cells cultured on well plates were infected with tcid ibv at °c for h, and then treated with hpe, hpw, and see solution at concentrations of . , . , . μg/ml at °c for h. according to the above description, the relative mrna expression level and virus titer of ibv were detected. in order to study the direct effects of hpe, hpw, and see on the virus, tcid ibv was incubated with . , . , . μg/ ml hpe, hpw, and see solution at °c for h, respectively. then cek cells were infected with the drug-treated ibv at °c for h. as described above, the relative mrna expression levels and the virus titer of ibv were detected. in the above three experimental designs, the best antiviral way was selected for indirect immunofluorescence assay. after washing cek cells with pbs, fixed with % paraformaldehyde, then incubated with . % glycine and quenched with % triton-x for min. after washing cek cells with pbs for times, the cek cells were incubated with rabbit anti-ibv antibody ( : ) (prepared and stored in laboratory of pathology and anatomy, northeast agricultural university) for h, and then incubated with fluorescent labeled goat antirabbit igg ( : ) (zhongshan, china) for min in the dark. the fluorescent images were examined with a fluorescence microscope (ti-s, nikon, japan). at the same time, the infected cells treated with μg/ml rt, and cek cells infected with ibv, and the mock cek cells were set as controls. one hundred and forty four -week-old spf chicks were randomly divided into hpe treated high dose group (hpe-th), hpe treated middle dose group (hpe-tm), hpe treated low dose group (hpe-tl), the ibv infected group (ibv), the ribavirin treated (rt) group, and normal control (nc) group with chicks in each group. each group was completely isolated from the other groups, and housed in negative pressure-filtered air isolators under pathogen-free conditions. chicks from in three hpe-treated groups, the ibv-infected group, and the rt group were inoculated by nasal drops with . ml of eid of ibv m allantoic fluid per chick. chicks in the control group were inoculated with . ml sterilized negative allantoic fluid in the same manner. the chicks of the three hpe groups and the rt group were orally administered varying doses of three hpe ( mg/kg, mg/kg, mg/kg) and ribavirin ( mg/kg), respectively. both agents were administered twice daily for days, beginning at h after exposure to the virus. at the same time, the chicks of the ibv-infected group and the normal group were orally administered with pss. at , , , and days postinfection, six chicks in each group were bled before euthanasia and necropsy at each designed day. the trachea and kidney tissues were collected and immediately extracted to obtain the total rnas. at the same time, tracheal and kidney in each group of chickens at days postinfection were fixed in % buffer formaldehyde for histopathological assay. after washing in phosphate-buffered saline (pbs), the trachea and kidney tissues were fixed in % formaldehyde solution for days, embedded in paraffin wax, cut into -μm-thick sections (leica rm , germany), and stained with he using standard histological staining procedure. the slides were examined by light microscopy (nikon, japan). the tissue slides were examined and evaluated according to the previous histopathologic scoring method for trachea (levisohn et al., ; todd et al., ) and kidney (reinhard et al., ; laudert et al., ; canales et al., ) with minor modification. for the trachea, the following morphological changes were included: cilia loss of mucous epithelium, extrusion of balloon-like cell and necrotic exfoliation of mucous layer, lymphocyte and heterophil infiltration in submucosal layer, hyperemia, and/or hemorrhage. for the kidney as followed: glomerular atrophy and fragmentation, tubular degeneration (granular and/or vacuolar degeneration) and necrosis (detached and broken epithelium), interstitial infiltration of lymphocytes and heterophils, hyperemia, and/or hemorrhage. the distribution and extent of the aforementioned lesions in the trachea and kidney of each chicken were scored as followed: , absence of lesion; , lesion represented in fewer than % of the involvement of tissue; , lesion represented in - % involvement; , lesion represented in - % involvement; and , lesion represented in more than % involvement. the pathological lesions of trachea and kidney were scored by experienced veterinary pathologists (dr. guangxing li, ruili zhang, and xiaodan huang, northeast agricultural university, harbin, pr china), which were blinded to the identities of the experimental groups. the pathological injuries of trachea and kidney were quantitatively analyzed, and the statistical results were given in tables and . treated cell samples were repeatedly frozen and thawed three times, and the equiponderant trachea and kidney tissues from each chick at various time points were prepared. then, the total rna was extracted using a universal rna extraction kit (thermo fisher scientific, usa) according to the manufacturer's instructions. all the concentration of rna and the a /a ratio were determined with nanodrop spectrophotometer (thermo fisher scientific, usa), and the integrity of the extracted rna was detected by agarose gel electrophoresis. the total rnas were reverse transcribed into cdnas by pcr instrument (takara, japan) for qrt-pcr, and the cdnas were stored at − °c. the relative quantification analyses was used to determine the levels of mrna expression of target genes, including n gene of ibv, il- , tnf-α, il- β, ifn-α, ifn-β, mda , mavs, and nf-κb by an applied lightcycler real-time pcr system (roche, switzerland). all primer pairs for real-time pcr detection are listed in table . these primers were synthesized by genewiz biological technology co., ltd. (suzhou, china). the relative mrna expression levels of target genes were calculated by using the −ΔΔct method (yu et al., ). the amplification system was μl cdna, . μl forward and reverse primers, μl universal sybr green (rox) and . μl nuclease-free water, and the final volume of each system was μl. the amplification reaction of qrt-pcr assays was conducted according to the following thermal profile: pre-incubation cycles at °c for s, followed by step amplification: cycles at °c for s and °c for s, followed by melting: cycles at °c for s, °c for s, and °c for s, followed by cooling cycles at °c for s. chromatography/diode array detector/ electrospray ionization-mass spectroscopy analysis of the hypericum perforatum ethyl acetate hplc/diode array detector/esi-ms analysis was performed on a waters hplc equipped with diode array detector and waters micromass zq esi-electrospray (waters, usa) operating in positive and negative ion mode. according to relevant reports (seger et al., ; jesionek et al., ) , the hplc condition was determined with a minor modification. hpe analysis was performed using an eclipse xdb-c column ( mm x . mm, μm, agilent technologies inc. usa). the mobile phase consisted of water (a) and acetonitrile (b) at a constant flow rate ( . ml/min). the solvent gradient elution method was as follows: - min, - % b and - min, % b. the detection wavelength was - nm, the column temperature was room temperature, and the injection volume was μl. the operating conditions of the mass spectrometer were dry gas temperature, °c, flow rate, l/min; nebulizer pressure, psi; sheath gas temperature, °c, flow rate, l/min; fragmenter voltage, v; capillary voltage, , v; mass range, - , d. determination of hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin in the hypericum perforatum ethyl acetate the sample solution was prepared by dissolving . mg dried hpe in ml mobile phase and filtered by . μm filter before hplc analysis. the content of hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin in the hpe was determined by hplc instrument and the chromatographic conditions described above. in the ml analytical solvent, the hyperoside standard of . mg, or the quercitrin standard of . mg, or the quercetin standard of . mg, or the pseudohypericin standard of . mg, or the hypericin standard of . mg are dissolved to prepared stock solution, respectively. according to the guide of international conference on harmonisation of technical requirements for registration of pharmaceuticals for human use (singh, ; armutcu et al., ; hsi et al., ), the signal-to-noise ratio (s/n) of and were defined as the detection limit (lod), and the quantitative limit (loq), respectively. in order to detect the s/n, the stock solution of hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin were diluted to different concentrations, respectively. in order to obtain the standard curve, the stock solution of the above standard compounds was diluted into six appropriate dilution concentrations. quantification was conducted by using a six-point standard curve and an external standard method. intra-day and inter-day precision for hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin were used to evaluate the repeatability and reproducibility of the established method. statistical analysis the number of the experiment repetition in all the experiment was three times. the experimental data was analyzed with spss . software (spss inc., chicago, il, usa). the results are expressed as the means ± standard deviation (sd). differences between groups were evaluated using the one-way analysis of variance (anova) of two tailed test. p < . were considered as statistically significant, and p < . were considered as highly significant. staining showed cell was survival (figure a) . at the same time, the cell survival rate measured by mtt method was close to %, which was further explained that μg/ml of the drug had no significant effect on the cells (figure b ). when ibv was propagated in cek cells to the tenth generation, stable typical cytopathic effect (cpe) appeared at h after ibv infection (figure a) . at the beginning, cells infected with ibv became round and refractive, then some cell exfoliated from the flask and left empty hole, some cells fused together and became multinuclear giant cells, indicating that ibv adapted to cek cells. the results of virus growth curve showed that ibv could infect and replicate in cek cells and reached the highest titer of − . tcid per μl at h post-inoculation (figure b ). at the same time, the existence of ibv m was identified by rt-pcr ( figure c ). antiviral effect of supercritical extraction extract, hypericum perforatum ethyl acetate, and hypericum perforatum water in vitro the relative mrna expression level of ibv-n gene was detected by qrt-pcr and the virus titer of ibv was determined by tcid to analyze the antiviral effect of hpe, hpw and see (figure ) . it could be seen from figure that under the maximum non-toxic concentration of the drug, the inhibition of hpe on ibv was significantly greater than that of hpw and see. hpw had a very weak effect on ibv and see had no effect on ibv. therefore, it was determined that the antiviral part of h. perforatum extract was ethyl acetate layer. as can be seen from figure a , in the three experimental designs, with the increase of hpe concentration, the mrna level of ibv decreased significantly in a dose-dependent manner. in addition, at the same drug concentration, the relative expression level of virus mrna was the lowest when hpe directly treated the infected virus cells, followed by hpe pre-treated cells before infection, and then hpe pre-treated virus before infection. it can be known from the figure b , with the increase of hpe concentration, the titer of ibv decreased gradually in a dosedependent manner. while, at the same drug concentration, the virus titer was the lowest when hpe directly treated the infected virus cells, followed by hpe pre-treated cells before infection, and followed by hpe pre-treated virus before infection. at the same time, the antiviral impact of hpe directly treated the ibv-infected cells at the concentration of . µg/ml, was similar to rt at the concentration of µg/ml. therefore, hpe was selected for subsequent experiments in vivo and in vitro, furthermore the hpe directly treated the ibv-infected cells was selected in vitro experiments. the results of reverse transcription polymerase chain reaction identification of ibv m in cek cells. in which, m stands for deoxyribonucleic acid marker, n stands for polymerase chain reaction product for ibv n gene, w stands for negative water control. frontiers in pharmacology | www.frontiersin.org october | volume | article in order to further confirm the inhibitory effect of hpe on ibv-infected cells, the fluorescent signal of virus was detected by ifa (figure ) . from the figure , it can be seen that the cek cells infected with ibv produced a strong fluorescence signal at h after infection. on the contrary, the fluorescence signal of cek cells infected with ibv and treated with hpe was weakened, and the fluorescence signal decreased in a dosedependent manner with the increase of hpe concentration. this further confirmed that hpe has a very good inhibitory effect on ibv infected cells. in order to study the effect of hpe on gene mrna expression induced by ibv infection in cek cells, the mrna expression level of related genes were determined, including mda , mavs, ifn-α, ifn-β, nf-κb, il- , and tnf-α. as can be seen from figure , hpe treatment significantly affected the mrna levels of mda , mavs, ifn-α, and ifn-β after ibv infection at and h, and the mrna expression levels of these genes changed similarly. these data suggested that hpe could increase mrna expression level of type i interferon in the late stages of ibv infection, which is possibly related to mda signaling pathway. in addition, hpe treatment significantly reduced the mrna expression levels of nf-κb, il- , and tnf-α, which were up-regulated after ibv infection at and h. these data demonstrated that hpe could decrease the mrna expression of pro-inflammatory genes, which is possibly related to nf-κb signaling pathway. at days after infection, the symptoms that appeared in the ibv infected group were sneezing, tracheal wet rales, mouth breathing, coughing, ruffled feathers, and frequently shaking. but the symptoms that appeared in the hpe and rt treatment group such as sneezing, tracheal wet rales, mouth breathing, coughing, feather wrinkling, and frequent trembling were mild. and the trachea and kidneys of different groups of spf chickens were conducted for histopathology assay. as can be seen from figure , most of the tracheal cilia in the ibv infected group fell off, the mucosal epithelial cells exfoliated and disappeared, the submucosal structure was loosely arranged, and there was a certain amount of serous exudation, inflammatory cells, and red blood cells. in the hpe treatment group, with the gradual increase of the dosage, the shedding of tracheal cilia and epithelial cells was gradually decreased, there were a small number of inflammatory cells and erythrocytes, and the exudation of inflammatory serous fluid decreased. as shown in figure , in the ibv infected group, a large number of renal tubular epithelial cells were degenerated and necrotic, the nucleus were concentrated, fragmented and dissolved, the renal tubular epithelial cells fell off into the lumen, dissolved or disappeared, the structure of renal tubules was figure | the effects of hypericum perforatum ethyl acetate on the messenger ribonucleic acid (mrna) expression level of related genes in vitro. chicken embryo kidney (cek) cells were infected with tcid infectious bronchitis virus (ibv) at °c for h, and then incubated with . μg/ml hpe. the cek cells were treated with m , tcid ibv, and . µg/ml hpe as the control, respectively. subsequently, total rna was extracted from cek cell samples at , , and h after treatment. the relative mrna expression of melanoma differentiation-associated protein (a), mitochondrial antiviral signaling gene (b), interferon alpha (c), interferon beta (d), nuclear factor kappa beta (e), il- (f), and tumor necrosis factor alpha (g) were determined by quantitative reverse transcription polymerase chain reaction. the differences between means were considered significant at *p < . and highly significant at **p < . when compared with the ibv-infected cell. frontiers in pharmacology | www.frontiersin.org october | volume | article incomplete, and there were a certain number of inflammatory cells and red blood cells in the interstitium. the wall of renal capsule was ruptured and a certain amount of inflammatory cells and serous exudation could be seen in the lumen of renal capsule. in the hpe treatment group, with the increase of the dosage, the exfoliation and dissolution of renal tubular epithelial cells decreased gradually, the structure of renal tubule was gradually complete, the number of red blood cells and inflammatory cells in the stroma was less, and the structure of renal capsule wall was more complete. there was little infiltration of inflammatory cells in the renal vesicle cavity. as can be seen from tables and , the pathological scores of trachea and kidney in ibv group were highly significantly higher than those in nc group (p < . ). compared with the ibv group, the pathological scores of trachea and kidney in the hpe-th group and the hpe-tm group decreased highly . ± . . ± . . ± . . ± . . ± . ** hpe-tl . ± . . ± . . ± . . ± . . ± . hpe-tm . ± . . ± . . ± . . ± . . ± . ** hpe-th . ± . . ± . . ± . . ± . . ± . ** the difference was considered highly significant at **p < . vs. ibv infection group. frontiers in pharmacology | www.frontiersin.org october | volume | article significantly (p < . ), and the score was similar to that of the rt group. at the same time, with the increase of hpe dose, the score decreased in a dose-dependent manner. this indicated that hpe had better anti-ibv effect. to confirm the inhibitory effect of hpe, ibv mrna levels in trachea and kidney of different groups were measured by realtime qrt-pcr. as shown in figure , compared with nc group, the expression level of mrna in trachea and kidney of spf chicks infected with ibv increased significantly. compared with ibv group, the expression level of mrna in trachea and kidney of spf chicks treated with hpe decreased significantly. with the decrease of hpe concentration, the expression level of mrna increased in a dose-dependent manner. with the increase of the days of ibv infection, the effect of hpe-th and hpe-tm group on the level of mrna expression was similar to that of rt group. these data indicated that infected chickens treated with hpe exhibited an overall reduction in viral mrna levels. it is well known that type i interferon, including ifn-α and ifn-β, plays a very important role in antiviral activity. as can be seen from figures and , the relative mrna expression of il- , tnf-α, ifn-α, ifn-β, mda , mavs, and nf-κb in the trachea and kidney were up-regulated after ibv infection, which was consistent with previous studies (he et al., ; chhabra et al., ) . in hpe treatment group, the mrna expression levels of ifn-α and ifn-β in the trachea were up-regulated in the early stage of ibv infection, and the changes of ifn-α and ifn-β were consistent with the mrna expression of mda and mavs in the trachea. meanwhile, in the hpe-treated group, the mrna expression levels of ifn-α and ifn-β in the kidney were up-regulated in the late stage of ibv infection. in addition, the changes of ifn-α and ifn-β were similar to the mrna expression levels of mda and mavs in the kidney. these data suggested that hpe may up-regulate mrna expression levels of ifn-α and ifn-β in trachea and kidney via the mda signaling pathway. . ± . . ± . . ± . . ± . . ± . nc . ± . . ± . . ± . . ± . . ± . ** rt . ± . . ± . . ± . . ± . . ± . ** hpe-tl . ± . . ± . . ± . . ± . . ± . hpe-tm . ± . . ± . . ± . . ± . . ± . ** hpe-th . ± . . ± . . ± . . ± . . ± . ** the difference was considered highly significant at **p < . vs. ibv infection group. figure | the effects of hypericum perforatum ethyl acetate (hpe) on infectious bronchitis virus (ibv) messenger ribonucleic acid (mrna) expression levels of trachea (a) and kidney (b). spf chickens were infected with ibv, followed by oral administration of hpe and ribavirin, respectively. total rna were subsequently extracted from trachea and kidney at , , , and day post-infection. the relative mrna expression of ibv was determined by quantitative reverse transcription polymerase chain reaction. the differences between means were considered significant at *p < . and highly significant at **p < . . frontiers in pharmacology | www.frontiersin.org october | volume | article at the same time, it is well known that inflammatory factors play an important role in inflammation. it also plays a very important role in ibv infection. as can be seen from figures and , in the hpe-treated group, the mrna expression levels of il- and tnf-α in the trachea and kidney were down-regulated, and the changes were consistent to nf-κb mrna expression levels, indicating that hpe may be down-regulated the mrna expression levels of il- and tnf-α by nf-κb signaling pathway. therefore, hpe may exert an anti-ibv effect by increasing the mrna expression levels of type i interferon and reducing mrna expression levels of proinflammatory factors il- and tnf-α, and this may be related to the mda signal pathway and nf-κb signal pathway. (figure ) . the mass spectra of these compounds were carefully examined (figure ) and compared with the standard and reference data (seger et al., ; cao et al., ) and found to have five peaks in the hpe (table ) . these peaks corresponded to hyperoside (peak ), quercitrin (peak ), quercetin (peak ), pseudohypericin (peak ), and hypericin (peak ). determination of hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin in the hypericum perforatum ethyl acetate the calibration curves of hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin were as followed: y = , x + , , y = , x + , , y = , x− , , y = , x + , , and y = , x + , . , where y is peak area, x is sample concentration, and the concentration ranges were - , - , - , - , and - μg/ml, respectively. and the calibration curve was in accordance with a linear regression (r = . - . ). the lod (s/n = ) of hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin was in the range of . to . μg/ml. the loq (s/n = ) of hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin was in the range of . to . μg/ml. by measuring the peak area and retention time of standard compounds to evaluate the intra-day and inter-day precision, their relative sd was less than %. the results showed that the hpe contained . % hyperoside, . % quercitrin, . % quercetin, . % pseudohypericin, and . % hypericin. ibv has a variety of known strains, these strains continue to mutate, and more mutants continue to recombine, resulting in more diverse and complex genotypes and serotypes of ibv (bande et al., ; feng et al., ; laconi et al., ) . due to the poor cross-protection of vaccines between different serotypes, it is difficult to prevent and control ibv infection. therefore, development of an effective antiviral therapy is a crucial strategy for treating ibv infection. in this study, the anti-ibv effect of the drug was studied by three kinds of methods, qrt-pcr, determination of tcid , and indirect immunofluorescence (ifa). qrt-pcr is a method of measuring the total amount of products after each reaction cycle with fluorescent chemicals in dna amplification. ifa is a method to detect the presence of specific antigens in cells after the cells were treated with fluorescent-antibodies. tcid is the amount of infection in which % of the cells are infected by the virus. the anti-virus effect of drugs is often detected by qrt-pcr (schnepf et al., ; li et al., ; zhang et al., ) , ifa (xu et al., ; zhang et al., a) and tcid (xu et al., ; zhang et al., ) in antiviral research. and these antiviral drug reports are similar to our research, so we also used these methods to detect the anti-ibv effect of h. perforatum. although qrt-pcr and ifa could not detect live viral particles, they were used as a supplement in this study, and in this study we used the method of tcid , which is a general method for the detection of live virus (leclercq et al., ; petiot et al., ) . therefore, the results of antiviral studies in vitro are also credible in this study. in addition, although the best way to culture ibv is to use chicken embryos or chickens, the ibv replication in cek cells was tested in this study. because if the virus doesn't replicate well, viral particles can also be reduced or even die, which in turn affects the antiviral effect of the drug. in this study, we showed that ibv can replicate well in cek cells (figure ) , which is consistent with the research report (zhang et al., ) , and hpe had potential utility as antiviral agents against ibv. our results showed that the hpe can reduce virus mrna expression level (figures a and ) and virus titer ( figure a) . additionally, the see had no inhibitory effects and the hpw had poor inhibitory effects (figure ) . the results suggest that the hpe, which contains an enrichment of the main effective substances, was the bioactive fraction of h. perforatum extracts. moreover, the hpe had anti-ibv activity, such as reducing the fluorescence signal produced by ibv (figure ) , and alleviating the pathological injury of trachea and kidney caused by ibv in chickens (figures and ) . when came to evaluation of antiviral effects, ribavirin was used as control drugs. the inhibitory effect of . µg/ml hpe was comparable to that of µg/ml ribavirin in vitro, and mg/kg hpe was comparable to that of mg/ kg ribavirin in vivo. in the efficacy of antiviral effects, as in the reporting of others herbs (choi et al., ; sun et al., ; choi et al., ; yi et al., ; luo et al., ) , the effects of the herbal extract were lower than ribavirin even under several times higher dosage against ibv mrna level, virus titer, and histopathological injury caused by ibv. however, it is logical since ribavirin is pure compared to the extracts containing many inactive compounds. some studies have shown that h. perforatum extract have an antiviral effect on influenza a virus and hiv (barnes et al., ; birt et al., ; pu et al., ) , suggesting that hpe have the potential to be developed and used as antiviral drugs. in this study, we found that h. perforatum extract had significantly antiviral effect on ibv in vitro and in vivo, respectively. in addition, anti-ibv effect of hpe might correlate figure | effect of hypericum perforatum ethyl acetate (hpe) on the messenger ribonucleic acid (mrna) expression of genes in trachea. spf chickens were infected with ibv, followed by oral administration of hpe and ribavirin, respectively. total rna were subsequently extracted from trachea at , , , and day postinfection. the relative mrna expression of (a) melanoma differentiation-associated protein , (b) mitochondrial antiviral signaling gene, (c) interferon alpha, (d) interferon beta, (e) nuclear factor kappa beta, (f) il- , and (g) tumor necrosis factor alpha were determined by quantitative reverse transcription polymerase chain reaction. the differences between means were considered significant at *p < . and highly significant at **p < . . frontiers in pharmacology | www.frontiersin.org october | volume | article figure | effect of hypericum perforatum ethyl acetate (hpe) on the messenger ribonucleic acid (mrna) expression of gene in kidney. specific pathogen-free (spf) chickens were infected with infectious bronchitis virus (ibv), followed by oral administration of hpe and ribavirin (rt), respectively. total rna were subsequently extracted from kidney at , , , and day post-infection. the relative mrna expression of (a) melanoma differentiation-associated protein , (b) mitochondrial antiviral signaling gene, (c) interferon alpha, (d) interferon beta, (e) nuclear factor kappa beta, (f) il- , and (g) tumor necrosis factor alpha were determined by quantitative reverse transcription polymerase chain reaction. the differences between means were considered significant at *p < . and highly significant at **p < . . frontiers in pharmacology | www.frontiersin.org october | volume | article with mda and nf-κb signaling pathway. belonging to acid inducible gene i (rig-i)-like receptors (rlrs) in pattern recognition receptors (prrs), it is well known that mda plays a crucial role in avian respiratory disease progression (yu et al., ) , whose function is to identify various pathogenassociated molecular patterns (pamps), and activates mitochondrial antiviral signaling gene (mavs, also called ips- /visa/cardif) (lin et al., ) , activated mavs can recruit downstream interferon regulatory factor- / (irf / irf ) (shi et al., ) , leading to the rapid production of type i ifns (xu et al., ; soulat et al., ) . nf-κb system is a master transcription factor in the recognition signaling and host responses to immune attacks (deng et al., ) . it is well known that nf-κb mediates inflammation, and several cytokine are also linked to nf-κb signaling to enhance the inflammatory responses, such as tnf-α, il- , and il- β (salminen et al., ) . several reports have pointed out the mda signaling pathways and innate immune cytokines were activated after infection with ibv m strain (he et al., ) . mda signaling pathway was disrupted by cleavage of the adaptor protein mavs in the js/ / strain of ibv infection (yu et al., ) . the type i ifn response plays a critical role in resisting saibk strain of ibv (yang et al., a) . mda signaling pathways and innate immune cytokine (nf-κb and irf ) were induced after ibv-m strain infection (zhang et al., b) . on the other hand, the report had shown that h. perforatum extract significantly downregulated the concentration of il- and tnf-α in lung tissue for mice infected with an influenza a virus (pu et al., ) . in this study, we found that mrna expression levels of mda , mavs, ifn-α, ifn-β, nf-κb, tnf-α, and il- were significantly up-regulated after ibv infection in vitro and in vivo (figures , , and ) . meanwhile, we found that hpe may be up-regulate mrna levels of ifn-α and ifn-β by mda signaling pathway, and down-regulate mrna expression levels of il- and tnf-α through the nf-κb signaling pathway (figures , , and ) , suggesting that hpe may be inhibit ibv by affecting innate immune cytokines. because of remarkable antiviral effect on ibv, we analyzed the active components of hpe. the mass spectrum of hyperoside contained a negative quasi-molecular ion at . m/z [m-h] − (figure b ), in accordance with the previous literature (cao et al., ) . the mass spectrum of figure b figure c) . in addition to a positive quasi-molecular ion at . m/z [m+na] + ( figure d ) and a negative quasi-molecular ion [m-h] − at . m/z (figure e) , confirming the presence of the pseudohypericin. finally, the mass spectrum of hypericin shows a positive quasi-molecular ion [m+h] − at . m/z ( figure f ) and a negative quasi-molecular ion [m-h] − at . m/z ( figure g) . according to the results in figure , the hpe is the main active fraction, containing abundant levels of hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin (table and figure ). our study provides, for the first time, clear evidence that the extract of h. perforatum, containing hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin, possess anti-ibv activities. furthermore, its anti-ibv effect may be associated with reduced mrna expression levels of the proinflammatory cytokines il- , tnf-α by nf-κb signaling pathway, and related to up-regulate mrna expression levels of type i interferon through the mda signaling pathway, and could be useful for the development of new antiviral agents. however, further studies are required to elucidate its detail mechanism of action. all datasets generated for this study are included in the article/ supplementary material. determination of ochratoxin a traces in foodstuffs: comparison of an automated on-line two-dimensional high-performance liquid chromatography and off-line immunoaffinity-highperformance liquid chromatography system progress and challenges toward the development of vaccines against avian infectious bronchitis st john's wort 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classical swine fever virus replication by sirnas targeting npro and ns b genes attenuation, safety, and efficacy of a qx-like infectious bronchitis virus serotype vaccine induction of innate immune response following introduction of infectious bronchitis virus (ibv) in the trachea and renal tissues of chickens porcine epidemic diarrhea virus-induced epidermal growth factor receptor activation impairs the antiviral activity of type i interferon andrographolide inhibits mechanical and thermal hyperalgesia in a rat model of hiv-induced neuropathic pain frontiers in pharmacology | www.frontiersin.org october chinese herbal medicine compound yi-zhi-hao pellet inhibits replication of influenza virus infection through activation of heme oxygenase- characterization of three infectious bronchitis virus isolates from china associated with proventriculus in vaccinated chickens avian infectious bronchitis virus disrupts the melanoma differentiation associated gene (mda ) signaling pathway by cleavage of the adaptor protein mavs in vitro inhibition of vesicular stomatitis virus replication by purified porcine mx protein fused to hiv- tat protein transduction domain (ptd) chicken mannose binding lectin has antiviral activity towards infectious bronchitis virus cellular immune response in chickens infected with avian infectious bronchitis virus (ibv) astragalus polysaccharides inhibit avian infectious bronchitis virus infection by regulating viral replication the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © chen, muhammad, zhang, ren, zhang, huang, diao, liu, li, sun, abbas and li. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -oxjd z authors: stern, zachariah; stylianou, dora c.; kostrikis, leondios g. title: the development of inovirus-associated vector vaccines using phage-display technologies date: - - journal: expert rev vaccines doi: . / . . sha: doc_id: cord_uid: oxjd z introduction: inovirus-associated vectors (iavs) are derived from bacterial filamentous viruses (phages). as vaccine carriers, they have elicited both cellular and humoral responses against a variety of pathogens causing infectious diseases and other non-infectious diseases. by displaying specific antigen epitopes or proteins on their coat proteins, iavs have merited much study, as their unique abilities are exploited for widespread vaccine development. areas covered: the architectural traits of filamentous viruses and their derivatives, iavs, facilitate the display of specific antigenic peptides which induce antibody production to prevent or curtail infection. inoviruses provide a foundation for cost-efficient large-scale specific phage display. in this paper, the development of different applications of inovirus-based phage display vaccines across a broad range of pathogens and hosts is reviewed. the references cited in this review were selected from established databases based on the authors’ knowledge of the study subject. expert commentary: the importance of phage-display technology has been recently highlighted by the nobel prize in chemistry awarded to george p. smith and sir gregory p. winter. furthermore, the symbiotic nature of filamentous viruses infecting intestinal f(+) e. coli strains offers an attractive platform for the development of novel vaccines that stimulate mucosal immunity research regarding the potential to use viruses to combat disease has been ongoing for over years [ , ] . while viruses have often been considered pernicious as they co-opt a host for their own survival, often killing the host in the process, new work with viruses that can be exploited as bacteriophages but without the harmful effects has arisen [ ] . these viruses, which can be easily manipulated and employed for phage display ability, are being increasingly used for a variety of potent biomedical tools [ ] . these filamentous bacterial viruses, which make up the genus inovirus in the family inoviridae, are thread-like viruses containing single-stranded dna genomes know as filamentous bacteriophages [ ] [ ] [ ] . over different species of filamentous viruses are known, of which a majority can infect gram-negative bacteria. although inoviruses are now being used for their phage display capabilities, these filamentous viruses have a relationship with the cell that they infect that is more similar to symbiotic non-pathogenic animal viruses than classical phages. unlike phages, which term comes from the greek word φάγος for destroyer, inoviruses do not kill their host and only slightly affect cell growth despite yielding titers of up to virions per milliliter of liquid culture. progeny virions are assembled in the host cell's membrane where single-stranded dna binding proteins are replaced by major capsid protein subunits before being released into the cell, resulting in opaque plaques on bacterial lawns [ , ] . receptor organelles in the bacterial host that are encoded by transmissible plasmids facilitate the interactions between inovirus and cell [ , ] . the functional architecture of inoviruses provides the foundation for their application in vaccine-related projects since inoviruses do not cause harm. a great number of inovirus species across the world have been isolated and characterized [ ] . despite variation by species, they have the same general physical characteristics. the virions are flexible, thin cylindrical filaments [ , ] under nm in diameter and approximately nm in length (see figure (a) for details). most of a single virion is composed of several thousand major capsid or coat protein subunits. these surround a circular single-stranded dna molecule. at the proximal end of the virion there are a few minor proteins which attach to the cell to initiate infection. at the distal end, there other minor proteins which are used for nucleation and assembly on the host membrane. the structures and life cycles are conserved across different species of inoviruses, resulting in similar functional applications. research into inovirus structure and application has been dominated by studies of ff [ ] , which infect male (f + ) strains of e. coli. the most used and best understood of the ff inoviruses are the closely related fd, f , and m types (for reviews see [ ] [ ] [ ] ) for which extensive information regarding their life cycle and genetics is known. these nearly identical viruses almost perfectly share all structural motifs, including dna and protein sequences and gene organization [ ] [ ] [ ] , although there are slight variations between the genomes. there are genes and a non-coding intergenic region which are conserved across the ff species [ ] [ ] [ ] as well as dna replication and transcriptional machinery (for a recent review see [ ] ). proteins are encoded by genes in the virion (g , g , g , g , and g ). gene proteins (gp ), the major coat protein, make up the vast majority of the virion, occurring in fivefold axial symmetry. gene proteins and (gp and gp ) are located on the proximal end of the virion and are involved in stabilization and infecting the host cell. gene proteins and (gp and gp ) are on the distal end of the virion and perform initiation assembly [ ] [ ] [ ] . as shown in the end-to-end model in figure (a), minor coat protein subunits have fivefold axial symmetry. while much research has been done into the structure of the ff virus over the past years, a conclusive structure has not been determined since the viruses cannot be crystallized. x-ray fiber diffraction studies and physiochemical measurements have revealed the five-star helical symmetry ( fold rotation axis) of the gp subunit and are referred to as class i [ ] [ ] [ ] . however, the structure of the ssdna and its relationship to the protein sheath is poorly understood, due to the small amount of dna in individual virions. however, the architecture is sufficiently understood to take advantage of the structures and capabilities of the virus throughout its life cycle. the life cycle of ff filamentous viruses begins when an adsorption structure on the proximal end of the virus is adsorbed to the tip of the f + specific pilus of e. coli. following binding between the virus and the bacterial cell (for a recent review, see [ ] ), the major coat proteins of the article highlights • inoviruses are easily manipulated viruses which coexist with their host while causing minimal harm and are highly reproducible. • simple genetic manipulation makes the insertion of random or specific oligonucleotides into the inovirus genome. these genetically modified inoviruses can display corresponding oligopeptides as fusion proteins on their surface and are referred to as inovirusassociated vectors (iavs). • iavs displaying randomly generated oligopeptides are used to identify the epitopes of specific antibodies through biopanning. by isolating recombinant inoviruses displaying mimotypes resembling an antibody discontinuous target epitope, the dna and amino acid sequence of the oligopeptides of interest can be determined. • iavs have bene used to do create vaccines against a variety of infectious and non-infectious diseases, being used both to the screen for immunogenic peptides and directly present immunogenic peptides to the organism to elicit proper antibody production. • iavs are highly immunogenic, can stimulate both humoral and cellular responses in a variety of diseases, pose no known health risks, and are cost-efficient to produce. this box summarizes key points contained in the article. virus become associated with the inner membrane of the cell [ ] [ ] [ ] . the virion's circular single-stranded dna (cssdna) is then ejected into the cytoplasm where it is converted to a parental double-stranded replicative form (rf). using a rollingcircle mechanism, ff inoviruses replicate their genome. the new virion is assembled through a complex set of interactions that binds the protein subunits to the ssdna [ ] [ ] [ ] and embeds newly synthesized coat proteins into the bacterial membrane [ ] [ ] [ ] . ssdna is passed through the mature coat protein, spanning the bacterial membrane, and additional coat proteins are added on the internal edge of the membrane. additional proteins are used to package the inovirus and release it into the cell [ , ] . inovirus assembly on the inner membrane of the bacteria is a harmonized sequential process with both viral-encoded and host proteins playing important roles. for more extensive reviews on inovirus life cycle and replication, see reviews [ , ] . importantly, this process, which requires both virus and host to complete, does not kill the host, making inovirus replication a sustainable process. inoviruses are useful for vaccine development because it is possible to insert random oligonucleotides into their genome. this easy genetic manipulation is the basis for inovirus display (phage display) technology [ , ] . inoviruses that have been genetically modified to display these oligopeptides as fusion proteins on their surface are referred to as inovirus-associated vectors (iavs). oligopeptides can be displayed on any capsid protein (gp , gp , gp , gp , and gp ) following genetic modification. a specific oligonucleotide sequence can be inserted into the viral genome to display the desired oligopeptide as a fusion with capsid proteins gp , gp , gp , or gp . this results in the oligopeptide's display on every copy of the target capsid protein. however, mosaic inovirus particles can be created where the specific capsid proteins display a mix of wild type and recombinant proteins with the desired oligopeptide [ ] . this is done using a phagemid vector which has an extra copy of a capsid protein fused to the specific oligonucleotide. a host exposed to both the phagemid vector and a wild type capsid protein from a deficient helper phage produces mosaic iavs displaying both wild type and oligopeptide fused capsid proteins. work using the capsid protein gp has resulted only in the production of mosaic iavs (for reviews see [ ] [ ] [ ] ) while both mosaic and non-mosaic iavs have been produced using gp and gp (for a review see [ ] ) and gp and gp (for reviews see [ ] [ ] [ ] ). as a result of the different locations, structures, and abundances of the capsid proteins in iavs, they have differential abilities to display different oligopeptides. figure (b) shows how each of the five capsid proteins of an iav can display antigens, as have been demonstrated in published literature. the capsid protein that can display the most copies of the desired oligopeptide is gp due to the large amount of gp in each inovirus. a non-mosaic iav can display a peptide on each of the approximately , copies of gp . however, doing so with large peptides distorts the virus; only peptides of up to amino acids can be displayed in such great quantity without affecting the structure of the inovirus. however, producing a mosaic iav with oligopeptides on far fewer gp units will not cause distortion [ ] . while it is theoretically possible to display an entire protein on gp [ ] , presenting on all copies per virion, studies have demonstrated that at most one copy of gp will display the desired protein [ ] . the ability of iavs to display different random oligopeptides on their surface has many applications, and is especially useful in for vaccine development. the creation of random peptide libraries (rpl), where random oligopeptides are fused to major capsid proteins (gp or gp ) and displayed on individual inovirus clones creating a random variety of iavs which can be used for vaccine design via epitope mapping using monoclonal or polyclonal antibodies. these random iavs, whose oligopeptide diversity increases with increased peptide length, can be used to identify the epitopes of specific antibodies through a process called biopanning. this allows for the isolation of iavs displaying mimotopes which mimic the antibody discontinuous target epitope. by isolating the recombinant inoviruses bearing mimotopes, the dna and amino acid sequence of the oligopeptides of interest can be determined. through this breakthrough technology which was the subject matter of the nobel prize in chemistry (see 'expert commentary' below), inovriuses displaying oligopeptides mimicking antigens (or specific epitopes of an antigen) can be used to vaccinate hosts thus inducing the desired antibody production. vaccines of this sort have been developed in a variety of organisms against many different diseases (see tables and in [ ] for a list of inovirus-based vaccines), as the introduction of the inoviruses can induce the production of specific antibodies. while there have already been various successes, this method can potentially open the door to the development of many novel vaccines and vaccine methodologies. iavs have been successfully used as vaccine carriers for a variety of species and diseases across a range of vaccine studies (as shown in [ ] ). these vaccines have been effective against infections agents such as viral, protozoan, and worm parasites as well as non-infectious diseases including alzheimer's and a variety of cancers. inovirus display technology has been used in two different ways to develop vaccines. the first method employs inovirus display technology to screen rpls with monoclonal antibodies to determine which peptides can be used. these immunogenic peptides are used as vaccines either with carrier proteins or in their soluble forms to elicit an immune response [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the second method not only employs inoviruses for epitope mapping, but also utilizes inoviruses to serve as the carrier for the isolated immunogenic peptide . directly using iavs to present the peptide has been more consistently successful in producing the production of the proper antibody than the introduction of soluble peptides. inovirus-bound peptides (on iavs) consistently retain their d structure and are more stable than soluble peptides, which do not always reflect the desired antigen epitope [ ] . iavs are structurally simple, which allows the immune system to respond to the fused peptides rather than the viral coat. combined with ability to display many copies of the desired peptide, iavs are highly immunogenic, creating effective vaccines [ ] . because the inoviruses replicate in e. coli cultures, the production of large numbers of vaccines is cost-efficient. iav's structural simplicity, high immunogenicity, and economical production make them an efficient and attainable system for creating a variety of effective vaccines. much of the research involving inovirus-based vaccines has been to target infectious diseases in animals. studies have shown that iavs have been constructed to protect vaccinated animals against a variety of infectious diseases by eliciting both humoral [ ] and cellular [ ] immune responses. to test the efficacy of iav vaccines against target pathogens, studies were conducted in which animals were challenged with a specific pathogen following iav vaccination. in these studies, iavs were shown to mitigate or prevent infection from viruses and parasites. in one study, mice were completely vaccinated against human respiratory syncytial virus (rsv) by binding a -mer linear epitope to gp , which induced a humoral response [ ] . monoclonal antibodies against various viral diseases have also been successfully screened against rpls to produce recombinant inovirus vaccines against herpes simplex virus type (hsv- ) using a -mer peptide [ ] and against neurotropic murine coronavirus sing a mer peptide [ ] . these inovirus vaccines induced humoral responses in mice, greatly reducing mortality and providing protection relative to the dose of the vaccine [ ] . iav vaccines have also been developed against fungal parasites in animals, providing both individual protection and large scale vaccination success. [ , ] . not only did vaccination lower the burden of infection, but it also increased the lifespan in the infected mice [ ] . in , manoutsarian et al. used iavs to vaccinate pigs against taenia solium, a parasitic worm which causes neurocysticercosis in humans but uses pigs as intermediate hosts. unlike previous studies, which used a single specific peptide fused to a inovirus, four different antigenic peptides were displayed by inoviruses in a cocktail of recombinant iavs. the induction of a cellular response completely vaccinated / of the pigs in the study and reduced the number of cysticerci in all other pigs [ ] . following the success of this study, a large-scale vaccination of pigs in mexico was undertaken in . the pigs in a natural environment were successfully immunized, reducing parasite presence in the vaccinated pigs, and providing a more costefficient alternative to vaccination with synthetic peptides [ ] . many additional vaccines have been developed that induced both humoral and cellular responses against parasites in animals providing partial protection against the infection and reducing infection burden. many of these studies were done by screening monoclonal and polyclonal antibodies against large rpl (for a review of more inovirus vaccine studies see [ ] ). in addition to their success against infectious diseases, inovirus display technology has been successfully used to design vaccines which prevent or mitigate the progression of non-infectious diseases. iavs have been used to display antigen epitopes which elicit immune responses against tumors and other non-infectious diseases. an epitope of the melanoma antigen a (mage a ) displayed on gp induced a cellular immune response against the melanoma tumor in vaccinated mice. not only did vaccination inhibit tumor growth, but it reduced mortality in the targeted mice [ ] . induction of a cellular response, decreased tumor growth, and higher survival rate was observed in mice vaccinated against murine mastocytoma p in a later study [ ] . the highly immunogenic characteristics of inovirus display technology has also been used to activate immune responses against colorectal cancer tumors, which often evade antitumor immune responses, and to reduce tumor growth [ ] . inovirus-based vaccines have also been used against diseases such as alzheimer's. the same methods have been used to induct antibodies against β-amyloid plaques by displaying a specific amino acid antigenic epitope on the surface of the inovirus. multiple studies in mice using recombinant inoviruses have induced a humoral response and reduced βamyloid plaque burden [ ] [ ] [ ] [ ] . while not used preventatively as has been done with infectious diseases, iavs can vaccinate animals against further disease progression in non-infectious diseases. these findings suggest that there will be future development of inovirus-based vaccines against non-infectious diseases that mitigate the damage done to both animals and humans. despite the advances in the use of iavs to combat disease, the consistent development of inovirus-based vaccines still faces challenges. even when inoviruses have been screened with specific antibodies to bear the desired peptide mimotopes, immunization using iavs does not always produce the expected immune response. keller et al. in were unable to induce the production of broadly neutralizing antibodies against human immunodeficiency virus type- (hiv- ) in rabbits despite having screened for the proper mimotopes [ ] . dorgham et al. in screened a -mer rpl using a broadly neutralizing antibody and, using the selected inoviruses, were able to induce antibodies in vaccinated mice. however, the induced antibodies did not exhibit neutralizing activity against hiv- [ ] . other rpls screened against monoclonal and broadly neutralizing antibodies have produced antibodies that do not have neutralizing capabilities [ , ] . in one such case, crystallization of the specific mimotope revealed that the oligopeptide borne by the inovirus was structurally different that the natural antibody epitope [ ] . this structural issue is likely at the foundation of many of the issues involving ineffectual inovirus-based vaccines. when iavs fail, it is probable that the chosen mimotopes poorly resemble the original antibody epitopes and cannot function in the desired fashion, either failing to induce antibodies or inducing of antibodies without neutralizing capabilities. despite the efficacy of these inovirus-based vaccines against a variety of infectious and non-infectious diseases, there are still many diseases for which effective vaccines have not been developed using phage display or other methods. for example as discussed above, despite extensive work with inoviruses beginning in by keller et al. [ ] , there has been very little success developing a (hiv- ) vaccine [ ] . while four major epitopes on the hiv- envelope gp and gp glycoproteins have been identified [ ] [ ] [ ] , inducing the production of effective antibodies has not been successful [ ] . while current techniques have not succeeded, both humoral and cellular responses to hiv- through inovirus-based vaccines are being researched [ , ] . however, the groundwork for future work involving hiv- and other diseases has been laid out, paving the way for a multitude of various new inovirus-based vaccines. while there have been many vaccine studies targeting hiv- using iavs, none have been completely successful. studies involving inovirus based-vaccines targeting hiv- initially only used the broadly neutralizing monoclonal antibodies f , g and b [ , [ ] [ ] [ ] [ ] [ ] . however, as discussed above, these studies have either failed to induce antibodies or induced non-neutralizing antibodies, despite often inducing a humoral response. in other studies, polyclonal sera from hiv- -infected individuals have been used to screen rpls to search for new broadly neutralizing monoclonal anti-hiv- antibodies [ ] [ ] [ ] [ ] [ ] ] . these studies have used iavs to induce the protection of neutralizing antibodies in mice [ ] and macaques (against simian-human immunodeficiency virus) [ ] . inovirusbased vaccines targeting hiv- have been able to control viral load in a variety of non-human mammals after a challenge by hiv- or induce neutralizing antibodies, but not inhibit novel infection or induce broadly neutralizing antibodies. additionally, inovirus-based hiv- vaccines have often failed due to the autoreactivity of the monoclonal antibodies that can hopefully be avoided with the new 'next generation' broadly neutralizing antibodies [ ] [ ] [ ] [ ] [ ] . inoviral vectors have been used extensively in the development of vaccines against a variety of infectious and noninfectious diseases over the past two decades. as shown above, the iavs have successfully induced a humoral or cellular response, or both. in the studies, the vaccines could provide partial or complete protection against pathogens causing infectious disease or merely lower the burden of infection. against non-infectious diseases, inovirus-based vaccines have been shown to be effective at mitigating disease development by initiating productive immune responses. there have already been many applications of vaccines against infectious and non-infectious diseases using inoviral vectors and iavs have distinct characteristics that make them more applicable for the development of new vaccines than other viral vectors. inoviruses and by extent iavs are highly immunogenic and do not require adjuvants, facilitating the ease and simplicity of usage as vaccines. they can display multiple (from few to thousands) copies of a peptide on their surface while still maintaining the desired structure and conformation as well as infectivity with their bacterial hosts. iavs, by displaying only particular peptides, allow the immune system to interact with a specific epitope rather than a larger, more complex protein, yielding a more targeted response. they are capable of stimulating humoral and cellular immune responses and do not pose known health risks to animals or humans. iavs can be administered safely in high doses through multiple routes, enhancing their usage in a variety of contexts [ ] . with their cost-efficient production, structural simplicity, and record of success in a variety of contexts, iavs have the potential to be exploited for many new vaccines in humans and animals against a variety of diseases. advances in inovirus research and phage display technology are making the development of new vaccines for a variety of diseases feasible in the near future. currently, there are many diseases without vaccines or for which treatment is highly invasive. further development involving iavs will be likely to introduce more, better, and increasingly cost-effective vaccines. as the structure and tools for genetic manipulation of inoviruses are being better developed, iavs will likely begin to be more extensively introduced into animal and human use. the current research is only revealing the tip of the iceberg of the extensive ways in which inovirus-based vaccines will be applied in future use. accordingly, there are aspects of the genetic engineering of phage-display that need to be further expanded. specifically, further studies should be conducted to evaluate the maximum antigenic load capacity of the gp coat protein without jeopardizing the architectural integrity and infectivity of the iav (ff.g , see figure ). furthermore, additional research about the potential of using the other capsid proteins (gp , gp , gp , and gp ) or combinations of any of the capsid proteins for antigen display is needed. additionally, future studies could elucidate the nuances of the uses of individual capsid proteins for specialized applications such as antigen display, penetration, targeting, etc. an additional aspect of iavs that needs to be further explored is how antigenic display on the surface of iavs induces immunogenicity. this predictive work needs to be undertaken by experimental and computational (in silico) research. further research will involve the development of new vaccines and improving the efficacy of current vaccines. additional undertakings should be done into identifying the structure of the viruses, since this will better be able to inform how the phage-display technology will best be applied as more complex work is being done using monoclonal and polyclonal antibodies to create more nuanced vaccines. the future has a dual focus: the development of vaccines for infectious diseases and the induction of immune responses in non-infectious diseases to limit the harm that they do (or eliminate them). the underlying principle of future iav-based vaccine development could be based on the unique natural symbiosis, which is known to exist between humans, non-pathogenic bacteria such as e. coli, and inoviruses. it is well established that humans or other warm-blooded animals acquire e.coli, including f+ strains, during their first few days in life or even before birth and that they are never thereafter without it. fspecific inoviruses (ff inoviruses) cannot infect humans, but they propagate at high viral loads in specific strains of e. coli including non-pathogenic strains which are symbiotic to humans. future iav-based vaccines within the next five to ten years could take advantage of the triple symbiotic nature between humans, enteric f + e. coli strains and f+-specific inoviruses to target the induction of mucosal immunity. this approach will be particularly effective against sexually transmitted pathogens such as hiv- . much future study lies the further development of iavs and how they can induce immune responses on a broad scale. research regarding the development and advantages of phage display technology was recently recognized by the nobel committee. george p. smith and gregory p. winter were awarded the nobel prize in chemistry for the 'elegant method known as phage display, where a bacteriophagea virus that infects bacteriacan be used to evolve new proteins … [this] revolution … is bringing and will bring the greatest benefit to humankind' [ ] . the development of more and better inovirus-based vaccines will continue to advance preventative treatment against diseases which have not been able to be combatted. papers of special note have been highlighted as either of interest (•) or of considerable interest (••) to readers an investigation on the nature of ultra-microscopic viruses sur un microbe invisible antagoniste des bacilles dysentériques phage display as a promising approach for vaccine development beyond phage display: non-traditional applications of the filamentous bacteriophage as a vaccine carrier, therapeutic biologic, and bioconjugation scaffold • a review of novel future applications of phage-display technology mahy bwj, regenmortel mhvv structure and assembly of filamentous bacteriophages dna packing in filamentous bacteriophages replication of coliphage m- . i. effects on host cells after synchronized infection filamentous bacterial viruses filamentous bacteriophage: biology, phage display and nanotechnology applications a recent comprehensive review about the molecular biology and life cycle of ff viruses (f + -specific filamentous viruses) filamentous phage: structure and biology nucleotide sequence of bacteriophage fd dna nucleotide sequence of bacteriophage f dna nucleotide sequence of the filamentous bacteriophage m dna genome: comparison with phage fd genetic analysis of the filamentous bacteriophage packaging signal and of the proteins that interact with it functional analysis of bacteriophage f intergenic region filamentous phage morphogenetic signal sequence and orientation of dna in the virion and gene-v protein complex a specific dna orientation in the filamentous bacteriophage fd as probed by psoralen crosslinking and electron microscopy morphogenesis of filamentous bacteriophage f : orientation of extrusion and production of polyphage orientation of the dna in the filamentous bacteriophage f the symmetries of filamentous phage particles helical viruses three-dimensional structure of a cloning vector. x-ray diffraction studies of filamentous bacteriophage m at a resolution chemical modification of the coat protein in bacteriophage fd and orientation of the virion during assembly and disassembly bacteriophage f infection: fate of the parental major coat protein the fate of the protein component of bacteriophage fd during infection detection of prokaryotic signal peptidase in an escherichia coli membrane fraction: endoproteolytic cleavage of nascent f pre-coat protein isolation of mutants in m coat protein that affect its synthesis, processing, and assembly into phage conserved residues of the leader peptide are essential for cleavage by leader peptidase filamentous phage are released from the bacterial membrane by a two-step mechanism involving a short c-terminal fragment of piii architectural insight into inovirus-associated vectors (iavs) and development of iav-based vaccines inducing humoral and cellular responses: implications in hiv- vaccines a detailed review of the architecture of inoviruses and major inovirus-associated vectors (iavs) and related vaccines libraries of peptides and proteins displayed on filamentous phage phage display • an early overview of the phage-display and its applications eliminating helper phage from phage display identification of peroxisomal proteins by using m phage protein vi phage display: molecular evidence that mammalian peroxisomes contain a , -dienoyl-coa reductase phage display of cdna repertoires: the pvi display system and its applications for the selection of immunogenic ligands surface expression and ligand-based selection of cdnas fused to filamentous phage gene vi multivalent display system on filamentous bacteriophage pvii minor coat protein membrane insertion and assembly of epitopetagged gp at the tip of the m phage next generation phage display by use of pvii and pix as display scaffolds phage display: concept, innovations, applications and future vaccination with cetuximab mimotopes and biological properties of induced anti-epidermal growth factor receptor antibodies identification and characterization of protective epitope of trichinella spiralis paramyosin mimotopes selected with neutralizing antibodies against multiple subtypes of influenza a induction of humoral immune response against plasmodium falciparum sporozoites by immunization with a synthetic peptide mimotope whose sequence was derived from screening a filamentous phage epitope library high-molecular-weight melanoma-associated antigen mimotope immunizations induce antibodies recognizing melanoma cells differential immunogenicity of two peptides isolated by high molecular weight-melanoma-associated antigen-specific monoclonal antibodies with different affinities vaccination with a human high molecular weight melanoma-associated antigen mimotope induces a humoral response inhibiting melanoma cell growth in vitro targeting melanoma cells with human high molecular weight-melanoma associated antigen-specific antibodies elicited by a peptide mimotope: functional effects specificity of mimotopeinduced anti-high molecular weight-melanoma associated antigen (hmw-maa) antibodies does not ensure biological activity a mimotope peptide-based anti-cancer vaccine selected by bat monoclonal antibody peptide mimotopes recognized by antibodies cetuximab and matuzumab induce a functionally equivalent anti-egfr immune response mimotope vaccination for epitope-specific induction of anti-vegf antibodies peptide mimics of the group b meningococcal capsule induce bactericidal and protective antibodies after immunization peptides selected from a phage display library with an hiv-neutralizing antibody elicit antibodies to hiv gp in rabbits, but not to the same epitope protective immune responses induced by the immunization of mice with a recombinant bacteriophage displaying an epitope of the human respiratory syncytial virus immunisation with phage displaying peptides representing single epitopes of the glycoprotein g can give rise to partial protective immunity to hsv- characterization of murine coronavirus neutralization epitopes with phage-displayed peptides prophylactic vaccination with phagedisplayed epitope of c. albicans elicits protective immune responses against systemic candidiasis in c bl/ mice protective immune responses against systemic candidiasis mediated by phage-displayed specific epitope of candida albicans heat shock protein in c bl/ j mice recombinant bacteriophage-based multiepitope vaccine against taenia solium pig cysticercosis schistosoma japonicum: isolation and identification of peptides mimicking ferritin epitopes from phage display library protective immunity induced by phage displayed mitochondrial related peptides of schistosoma japonicum induction of immunity in sheep to fasciola hepatica with mimotopes of cathepsin l selected from a phage display library trichinella spiralis: characterization of phagedisplayed specific epitopes and their protective immunity in balb/ c mice identification of hiv vaccine candidate peptides by screening random phage epitope libraries identification and characterization of a peptide that specifically binds the human, broadly neutralizing anti-human immunodeficiency virus type antibody b immunogenicity of hiv type gp cd binding site phage mimotopes a peptide inhibitor of hiv- neutralizing antibody g is not a structural mimic of the natural carbohydrate epitope on gp constrained peptide models from phage display libraries highlighting the cognate epitope-specific potential of the anti-hiv- mab f selection of hiv-specific immunogenic epitopes by screening random peptide libraries with hiv- -positive sera collection of phage-peptide probes for hiv- immunodominant loop-epitope mimotopes selected with antibodies from hiv- -neutralizing long-term non-progressor plasma hiv- v loop crown epitope-focused mimotope selection by patient serum from random phage display libraries: implications for the epitope structural features protection of rhesus macaques against disease progression from pathogenic shiv- . pd by vaccination with phage-displayed hiv- epitopes variable epitope library-based vaccines: shooting moving targets variable epitope libraries: new vaccine immunogens capable of inducing broad human immunodeficiency virus type -neutralizing antibody response a general strategy to identify mimotopes of pathological antigens using only random peptide libraries and human sera induction of hepatitis b virus-specific cytotoxic t lymphocytes response in vivo by filamentous phage display vaccine phage display for sitespecific immunization and characterization of high-risk human papillomavirus specific e monoclonal antibodies peptide mimotopes of rabies virus glycoprotein with immunogenic activity immunogenicity and epitope mapping of foreign sequences via genetically engineered filamentous phage multiple display of foreign peptides on a filamentous bacteriophage. peptides from plasmodium falciparum circumsporozoite protein as antigens random-peptide libraries and antigenfragment libraries for epitope mapping and the development of vaccines and diagnostics developing strategies to enhance and focus humoral immune responses using filamentous phage as a model antigen phage display of peptide epitopes from hiv- elicits strong cytolytic responses inexpensive anticysticercosis vaccine: s pvac expressed in heat inactivated m filamentous phage proves effective against naturally acquired taenia solium porcine cysticercosis the potential of phage display virions expressing malignant tumor specific antigen mage-a epitope in murine model phage display particles expressing tumor-specific antigens induce preventive and therapeutic antitumor immunity in murine p model a filamentous bacteriophage targeted to carcinoembryonic antigen induces tumor regression in mouse models of colorectal cancer active immunization against alzheimer's beta-amyloid peptide using phage display technology immunization against alzheimer's beta -amyloid plaques via efrh phage administration reduction of betaamyloid plaques in brain of transgenic mouse model of alzheimer's disease by efrh-phage immunization efrh-phage immunization of alzheimer's disease animal model improves behavioral performance in morris water maze trials hiv- vaccine strategies utilizing viral vectors including antigen-displayed inoviral vectors hiv- neutralizing antibodies: understanding nature's pathways antibodies in hiv- vaccine development and therapy broadly neutralizing antibodies and the search for an hiv- vaccine: the end of the beginning profound early control of highly pathogenic siv by an effector memory t-cell vaccine immune clearance of highly pathogenic siv infection inducing cross-clade neutralizing antibodies against hiv- by immunofocusing cardiolipin polyspecific autoreactivity in two broadly neutralizing hiv- antibodies the role of antibody polyspecificity and lipid reactivity in binding of broadly neutralizing anti-hiv- envelope human monoclonal antibodies f and e to glycoprotein membrane proximal envelope epitopes specific phospholipid recognition by human immunodeficiency virus type- neutralizing anti-gp f antibody the broadly neutralizing anti-human immunodeficiency virus type antibody g recognizes a cluster of alpha -> mannose residues on the outer face of gp antibody polyspecificity and neutralization of hiv- : a hypothesis immunocontraception: filamentous bacteriophage as a platform for vaccine development the nobel prize in chemistry we thank the university of cyprus for its support. the authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. this includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. peer reviewers on this manuscript have no relevant financial or other relationships to disclose. key: cord- -awkkper authors: bakri, faris ghalib; alqadiri, hamzah m.; adwan, marwan hmoud title: the highest cited papers in brucellosis: identification using two databases and review of the papers' major findings date: - - journal: biomed res int doi: . / / sha: doc_id: cord_uid: awkkper citation classics represent the highest impact work in a given field. we aim to identify and analyze the most frequently cited papers on brucellosis. we used the databases scopus and web of science to determine the most frequently cited papers. the most cited fifty papers in each database were identified. we then ranked the papers according to the highest citation count recorded from any of the two databases. the most frequently cited paper received citations and was by delvecchio vg et al. reporting the complete genomic sequencing of brucella melitensis. the papers were published in journals led by the “infection and immunity” journal and the “veterinary microbiology” journal (each had papers). citation classics in brucellosis were all in english except one in french and were mostly of basic science type. in addition, we noticed that articles that were identified among the highest fifty articles in one database were missed by the other database and vice versa. therefore, we suggest that searching in more than one database would detect additional citation classics. brucellosis is a zoonotic granulomatous disease that can affect any organ. it is caused by brucella species which are small, gram-negative, and coccobacilli bacteria. clinical presentation varies from an acute, nonspecific febrile illness to chronic, debilitating forms with features of osteoarticular and neuropsychiatric abnormalities [ ] . brucellosis was first described in by david bruce, a british surgeon, who isolated gram-negative coccobacilli from the spleens of five british soldiers who died of fever in malta. in , zammit, a maltese bacteriologist, showed that infected goats transmitted brucellosis and that banning the use of their milk would be effective in eliminating the disease. the observation that apparently healthy goats could be carriers of the disease has been termed one of the greatest advances ever made in the study of epidemiology [ ] . the disease has wide geographic distribution and it is one of the most economically important zoonosis. in a review of diseases of animals, brucellosis lies within the top in terms of impact on poor people [ ] . in low-income countries, brucellosis is endemic and neglected. it also causes large disease in animals and people and lacks effective control [ , [ ] [ ] [ ] . accurate epidemiological data are not available for many endemic areas, but it has been estimated that more than , new human cases occur annually [ ] . in , garfield listed the "top " best cited articles ever published in jama and named them "citation classics" [ ] , and these classics represent the highest impact work in a given field [ ] . citation analysis in the field of infectious diseases and microbiology was reported for tuberculosis [ ] , nontuberculous mycobacteria [ ] , anthrax [ ] , severe acute respiratory syndrome (sars) [ ] , jc virus [ ] , herpes simplex virus [ ] , ebola virus [ ] , schistosomiasis [ ] , sepsis [ ] , and neglected infectious diseases [ ] . here, for the first time to our knowledge, we identify and analyze the citation classics for brucellosis. two electronic databases, scopus and web of science (wos), were searched for the most cited articles using the keyword "brucell * ." for the search in scopus, we selected the "title, abstract, keyword" choice. for the search in wos, we selected the "topic" and "all database" choices. the search in both databases was performed on january , , for papers published in all times. textbooks were excluded. the most fifty cited papers were identified in both databases. the articles' abstracts were read by the two study investigators (fgb and mha) to determine whether the articles were specific to brucellosis [ ] . we recorded the citation count from the two databases for each selected article. for articles that were among the top fifty articles in one database but not in the other, the citation count in the other database for that article was looked up and recorded. we then ranked the articles according to the highest citation count obtained from any of the two databases. we analyzed the papers according to number of citations, publication year, authors, journal impact factor, country of origin, and article type (basic science, observational study, interventional clinical trial, and review) [ ] . basic science articles included genetic studies [ ] , in vitro studies, animal studies, or in vivo studies that focused on physiology [ ] . observational studies included case-control studies, case series, and cohort studies. to classify the article type, two study investigators (fgb and mha) reviewed all articles independently and in cases of disagreement, they discussed the article until consensus was achieved [ ] . the most recent impact factor, year , from journal citation report was used for analysis. in cases where the journal has continued as a new title, the impact factor of the new title was used in the analysis [ ] . the list of the most cited articles found in the scopus and wos searches is shown in table . the list included articles. of the total articles, articles appeared in both databases within the highest cited articles. however, among the highest cited articles that were identified by the scopus search, ( %) articles were not among the top articles within the wos search. similarly, among the highest articles that were identified by the wos search, ( %) articles were not among the highest articles within the scopus search. all articles eventually appeared in both databases except for one article (position ) which appeared only in wos. the mean number of the highest citation count per article from any of the two databases was . citations (sd = . ) and the median number was . (interquartile range = to . ). articles that were identified within the highest articles in wos but not in the highest article in scopus were at positions , , , , , , , , , , - - - - - - - our results provide a clear picture of the main cited articles in brucellosis research publications history. for example, in the group of genome sequencing, we find at positions , , , , and the articles that reported the complete genome sequence for brucella melitensis, brucella suis, brucella abortus strain - , b. abortus strain , and brucella ovis, respectively. at [ , ] . in the group of articles on molecular diagnostic tests, we find the following: bricker et al. (position , in ) described a pcr assay that can identify and differentiate most brucella species and biovars found in the united states. prior to this assay, pcr assays did not discriminate among species. baily et al. (position , in ) developed the first pcr assay around the brucella cell surface protein (bcsp ). this target became one of the most popular targets used in molecular assays [ ] . romero et al. (position , in ) published a brucella s rrna based pcr assay, and although similar assay was previously described by herman and de ridder in [ ] , the assay by romero et al. was taken up more widely [ ] . in the group of articles on vaccination, we find the following: at position , schurig et al. in produced a live attenuated rb strain for vaccination. "r" stands for "rough," "b" for brucella, and " " for an internal laboratory nomenclature used at the time it was derived. the vaccine has become one of the most commonly used vaccines [ ] . first to identify a new member of type iv secretion system family encoded by virb operon in b. suis during a screen for virulence factors. they also showed that the system is essential for the intracellular growth during infection [ ] . the system is one of few classical virulence factors identified to date [ ] . the type iv secretion system is a pumping system that selectively transports proteins or other macromolecules through membranes [ ] . after brucella is taken up by vesicles in macrophage, acidification is thought to induce virb expression. the virb system interacts with components of the endoplasmic reticulum, neutralising the ph and allowing the brucella to undergo regulated cell division [ ] . other classics that further the oldest citation classic article was published in and was at position . it was by harris who described the side effects associated with the use of aureomycin and chloramphenicol in treatment of brucellosis. prior to the development of these treatments, chemotherapy of brucellosis yielded unsatisfactory results [ ] . the list of classics did not include any article on outbreaks. we suggest the following explanations: (a) papers on brucella outbreaks receive lower citations compared to articles in basic science: in both databases (scopus and wos) the highest cited article on brucellosis outbreaks was "canine brucellosis: outbreaks and compliance, theriogenology, " ( citations in scopus and citations in wos); (b) outbreaks in brucella have been recognized at very early time; therefore, their findings might have become well known: we found reports of outbreaks as early as (water-borne outbreak of brucella melitensis infection. am j public health nations health, ); (c) identifying brucella outbreaks could be difficult: brucella is difficult to detect and identify [ ] ; and (d) brucella species are genetically homogeneous, and thus, the typing of brucella species for epidemiological purposes by conventional molecular typing methods has remained elusive [ ] . we also observed the lack of papers on brucellosis in animal health and for this we suggest two explanations: (a) journals in the categories of agriculture and food sciences receive fewer citations than those in basic and clinical sciences as evidenced by the impact factor in these categories. for example, in the wos, in the categories of "agriculture, dairy, and animal sciences" and "food science and technology," the highest impact factor for a journal was . and . , respectively. while in the category of "medicine, general and internal" and "microbiology," the highest impact factor for a journal was and . , respectively. (b) the possible low productivity of research that is performed on brucella as evidenced by the lower number of articles on brucella in agricultural journals. for example, a combined search for the word "brucell * " and the journals "veterinary research" and "journal of dairy science" yielded and papers, respectively, while the same search in the journals "clinical infectious diseases" and "journal of bacteriology" yielded and papers, respectively. furthermore, we doubt that our search missed important journals from the agricultural fields because the databases scopus and wos include large collection of agricultural journals. scopus has journals included under the "agricultural and biological sciences" subject area and wos has journals included under the category "agriculture, dairy, and animal sciences" and journals under the category "food science and technology." studies on citation classics that used more than one databases are few and have ranked the articles according to the mean of the citation counts in the databases [ ] [ ] [ ] [ ] . here, we ranked the articles according to the highest obtained citation count from any of the two databases. we believe that our method is more accurate because relying on the mean for ranking might lower the rank of a given article. this is because the databases differ in reporting the citation count for a particular article. the variation in citation count between databases results from differences in journal coverage and quality [ ] . scopus includes a more expanded spectrum of journals than wos, and its citation analysis is faster and includes more articles than the citation analysis of wos [ ] . however, scopus tends to miss older citations which results in omission of studies before [ , ] . here, we identified articles that were listed in the highest articles in one database but were not identified within the highest articles in the other database and vice versa. articles that were identified by wos and not by scopus tended to be older and of basic science type, while articles identified by scopus and not by wos were more recent and mostly of observational and review type. we found that many countries had contributed to the classics including american, european, african, and mediterranean countries (table ). this might reflect the epidemiological distribution of brucella. in addition, the finding that the most recent classic article was in indicates that brucellosis is a dynamic field of study [ , [ ] [ ] [ ] [ ] . our study has several limitations that are similar to other studies in citation classics [ ] . these limitations include the presence of inherent problems in the citation process itself, for example, incomplete or inappropriate citations, biased citation [ , , , ] ; changes in the list of citation classics with time making it a snapshot of the current state of research [ ] ; absence of articles with languages other than english which is mostly because authors are more likely to cite articles in their own language, and english articles are more likely to be cited overall [ ] ; and finally, missing of important studies because their findings became well known [ ] . the latter point is relevant here because brucellosis was discovered in and it is possible that some important studies were not indexed in current database but their findings are now considered well known. despite these limitations, the study provides a picture for the main cited articles in brucellosis research publications since the discovery of brucella years ago. in conclusion, the citation classics in brucellosis were (a) all in english except one in french, (b) contributed by authors from several countries where brucellosis was or is still endemic, (c) mostly of basic science type, and (d) published in relatively high numbers in recent years indicating a dynamic field of study. in addition, we suggest that performing the search in more than one database would detect additional articles. the authors declare that they have no conflicts of interest. brucellosis in low-income and middle-income countries how themistocles zammit found malta fever (brucellosis) to be transmitted by the milk of goats global burden of human brucellosis: a systematic review of disease frequency neglected tropical diseases of the middle east and north africa: review of their prevalence, distribution, and opportunities for control neglected zoonotic diseases-the long and winding road to advocacy economics of brucellosis impact and control in low-income countries the new global map of human brucellosis citation classics from the journal of the american medical association the most cited works in epilepsy: trends in the "citation classics the top-cited tuberculosis research studies the most-cited articles on non-tuberculous mycobacterial infection from to the seminal literature of anthrax research the highly cited sars research literature mapping the history and current situation of research on john cunningham virus -a bibliometric analysis citation classics of herpes simplex virus tracing the scientific outputs in the field of ebola research based on publications in the web of science analysis of highly cited schistosomiasis related papers from the top cited clinical research articles on sepsis: a bibliometric analysis bibliometric assessment of european and sub-saharan african research output on poverty-related and neglected infectious diseases from top-cited articles in digestive system disease from to citation classics in chronic granulomatous disease: a bibliometric analysis types of study in medical research: part of a series on evaluation of scientific publications evaluating research in cardiovascular medicine: citation counts are not sufficient brucella microti sp. nov., isolated from the common vole microtus arvalis from the discovery of the malta fever's agent to the discovery of a marine mammal reservoir, brucellosis has continuously been a reemerging zoonosis brucella abortus s rrna and lipid a reveal a phylogenetic relationship with members of the alpha- subdivision of the class proteobacteria brucella, a monospecific genus as shown by deoxyribonucleic acid hybridization recent advances in molecular approaches to brucella diagnostics and epidemiology identification of brucella spp. by using the polymerase chain reaction brucellosis vaccines: past, present and future on the biochemical mode of action of levamisole: an update type iv secretion system of brucella spp. and its effectors the brucella virb type iv secretion system human brucellosis the two-component system bvrr/bvrs: a master regulator of brucella virulence clinical manifestations and complications in cases of brucellosis: a retrospective evaluation and review of the literature present knowledge of brucellosis; a summary dna genotyping suggests that recent brucellosis outbreaks in the greater yellowstone area originated from elk evaluation of a multilocus variable-number tandem-repeat analysis scheme for typing human brucella isolates in a region of brucellosis endemicity analysis of the most-cited articles in periodontology the most-cited original articles in cardiac computed tomography: a bibliometric analysis the top articles on minimally invasive spine surgery an audit of top citations published in pediatric emergency care coverage and quality: a comparison of web of science and scopus databases for reporting faculty nursing publication metrics comparison of pubmed, scopus, web of science, and google scholar: strengths and weaknesses three options for citation tracking: google scholar, scopus and web of science top cited articles in cardiovascular magnetic resonance: a bibliometric analysis top-cited articles in rehabilitation the most cited papers in osteoporosis and related research the top cited articles in clinical orthopedic sports medicine bibliometric analysis of the top cited cardiovascular articles citation classics in anesthetic journals citation classics in critical care medicine the most cited works in essential tremor and dystonia citation classics in obstetrics and gynecology: the most frequently cited journal articles in the last years key: cord- -tydil d authors: wannier, s. rae; worden, lee; hoff, nicole a.; amezcua, eduardo; selo, bernice; sinai, cyrus; mossoko, mathias; njoloko, bathe; okitolonda-wemakoy, emile; mbala-kingebeni, placide; ahuka-mundeke, steve; muyembe-tamfum, jean jacques; richardson, eugene t.; rutherford, george w.; jones, james h; lietman, thomas m.; rimoin, anne w.; porco, travis c.; kelly, j. daniel title: estimating the impact of violent events on transmission in ebola virus disease outbreak, democratic republic of the congo, – date: - - journal: epidemics doi: . /j.epidem. . sha: doc_id: cord_uid: tydil d introduction: as of april , the current ebola virus disease (evd) outbreak in the democratic republic of the congo (drc) is occurring in a longstanding conflict zone and has become the second largest evd outbreak in history. it is suspected that after violent events occur, evd transmission will increase; however, empirical studies to understand the impact of violence on transmission are lacking. here, we use spatial and temporal trends of evd case counts to compare transmission rates between health zones that have versus have not experienced recent violent events during the outbreak. methods: we collected daily evd case counts from drc ministry of health. a time-varying indicator of recent violence in each health zone was derived from events documented in the who situation reports. we used the wallinga-teunis technique to estimate the reproduction number r for each case by day per zone in the – outbreak. we fit an exponentially decaying curve to estimates of r overall and by health zone, for comparison to past outbreaks. results: as of april , the mean overall r for the entire outbreak was . . we found evidence of an increase in the estimated transmission rates in health zones with recently reported violent events versus those without (p = . ). the average r was estimated as between . and . in regions not affected by recent violent events, and between . and . in zones affected by violent events within the last days, leading to an increase in r between . and . . within zones with recent violent events, the mean estimated quenching rate was lower than for all past outbreaks except the – west african outbreak. conclusion: the difference in the estimated transmission rates between zones affected by recent violent events suggests that violent events are contributing to increased transmission and the ongoing nature of this outbreak. since , of the over reported ebola virus disease (evd) outbreaks have been in the democratic republic of the congo (drc) (world health organization regional office for africa, ; anon, ) . the current - evd outbreak in northeastern drc is, as of april , the second largest evd outbreak in history and the first to occur in a conflict setting (world health organization regional office for africa, ). although its magnitude substantially trails the - evd outbreak in west africa, the current outbreak has surpassed epidemic forecasts, particularly mathematical modelling studies that used historical data from prior outbreaks kelly et al., ; enanoria et al., ; ebola haemorrhagic fever in sudan, ; breman et al., ) . these studies had projected a final outbreak size of up to cases as of february while the current outbreak now has reported cases kelly et al., ) . since the evd outbreak began, there have been reports of violent events that have ranged from destruction of ebola care facilities and injured and murdered healthcare workers to events unrelated to ebola care, linked to elections and local unrest (world health organization regional office for africa, ; christopher dickey, ; branswell, branswell, , a branswell, , b branswell, , c branswell, , d . following many of these violent events, ebola response activities have been disrupted. in some cases, new evd case counts have increased in the district affected following these events, despite intensive public health interventions comprising the deployment of rapid diagnostic tests, novel therapeutics, contact tracing, and ring vaccination using a vaccine approved for emergency use with an estimated . % efficacy (elsherif et al., ; world health organization regional office for africa, ; who, ). there has been a growing sentiment that such events may be contributing to evd transmission, but quantitative analysis is lacking. here, we hypothesized that during the current outbreak to date, there had been higher transmission rates in zones that had recently experienced violent events than in zones that had not experienced such events. furthermore, we also hypothesized that shannon entropy computed with respect to space, a measure of the spatial spread of cases and the uniformity of their distribution across districts in this epidemic, has increased over time. a time series of case counts were collected from situation reports presented by the drc ministry of health and were confirmed using situation reports posted by the world health organization (who) (world health organization regional office for africa, ). evd cases were classified as suspected, probable, or confirmed. suspected cases underwent diagnostic testing and were subsequently classified either as confirmed or not confirmed. cases reported post-mortem were classified as probable based upon their epidemiological history. the symptom onset date and the reporting date of probable, confirmed, and suspected cases were collected from the beginning of the outbreak on may through april . beginning on august , data by health zone were also collected for probable and confirmed cases. as the outbreak has continued, cases were reassigned by the drc ministry of health from their initially reported health zone to the health zone where epidemiological evidence pointed to evd acquisition. symptom onset data were later made available going back to the beginning of the outbreak. given the dynamic nature of violence in the region and the relatively short generation time of evd, we undertook a time-varying, health zone-level analysis of the outbreak comparing transmission in zones that had experienced recent violence to those without recently reported violence. we considered a violent event as one reported within the who situation reports until april (world health organization regional office for africa, ). we assigned each health zone as having been exposed to violent events or not. after a violent event, we considered the following three weeks as the exposure period. we modeled the effect of violent events within the previous week, two weeks, three weeks, four weeks, and five weeks to determine the sensitivity to the time period chosen. we used the wallinga-teunis technique to estimate the number of secondary cases r for each case in the - outbreak (wallinga and teunis, ) . we defined the serial interval as the interval between disease onset in an index case and disease onset in a person infected by that index case. we used both symptom onset and case report data in our analyses given the limitations of each dataset. historical outbreaks used case report data, though case report data in this dataset is subject to case reclassification between zones, reflecting epidemiologic knowledge on the place of transmission rather than place of report, that can show up as transmission. in the current outbreak, case report data were updated nearly daily from the official daily totals reported by the drc ministry of health and revised as needed. thus, while the symptom onset data may be the fundamental definition of the serial interval, these data were not always revised as more accurate information became available. we employed a gamma distribution with a mean of . days and a standard deviation of days for the serial interval distribution. this models a serial interval of evd cases approximately ranging from to days, with mean to days (aylward et al., ; wong et al., ; chowell and nishiura, ) . our application of the wallinga-teunis method assumes there are no missing cases. to compare transmission in the current outbreak to past outbreaks, we applied the same estimation technique to reported cases by date from prior outbreaks (ebola haemorrhagic fever in sudan, ; breman et al., ; baron et al., ; georges et al., ; khan et al., ; cdc, ; anon, ; boumandouki et al., ; world health organization abonnement annuel, ; nkoghe et al., ; rosello et al., ) . we estimated the initial reproduction number r initial and exponential decay (or quenching) rate τ for each outbreak by fitting an exponentially decaying curve r(d) = r initial e −τd to the outbreak's estimates of r by day d. this exponential decay "quenching" parameter approximates the often observed reduction in r over the course of an outbreak that may be due to phenomena such as formal control efforts including case finding and quarantine as well as less formal responses including individual behavioral changes or local depletion of susceptibles. this equation approximates temporal change in transmission rate by a smoothed quenching pattern in which r decreases exponentially over time at a rate defined by the quenching parameter. the estimates r initial and τ obtained by this fit are reported for comparison to historic outbreaks and not used for further modeling. we repeated the wallinga-teunis procedure to estimate the reproduction number r separately for each health zone, creating a time series of estimated reproduction numbers for each zone. when estimating r for each zone, we considered the possibility of transmission between regions. the probability of an inter-zone transmission relative to intra-zone transmission is denoted ω, representing the reduced probability of a case transmitting outside of its health zone as compared to their probability of transmitting within their own health zone. we compared estimates based on values of the inter-zone transmission-mixing parameter ω across its range from (no transmission allowed between regions) to (all cases in all regions transmit equally to all other cases, regardless of region). confidence intervals for the wallinga-teunis estimated r by health zone were simulated using the calculated probabilities for each case i transmitting to case j to probabilistically assign a transmission link for every case, based on simulations (wallinga and teunis, ) . using the distribution of r per day per zone, we then calculated the negativebinomial confidence interval for each day per zone. statistical inference on the resulting estimates of r by day per zone was conducted by regressing estimated r on the presence of a recently reported violent events (bartlett, ; politis, ) . to adjust for autocorrelation, the standard errors of the estimates were estimated using a time-series bootstrap with blocks of days over replications. the shannon entropy of the cumulative number of cases was computed based on all health zones and standard errors were computed from the proportion in each geographic region (from, ) . time series bootstrap based on a fixed length of was used to compute standard errors of the time trend, based on ordinary least squares linear regression of entropy on days since the first case. we conducted all analyses in r (v. . for macintosh, r foundation for statistical computing, vienna, austria). as of april , a total of evd cases had been reported. thus far during the outbreak period, a total of cases have been reported in the seven health zones that have experienced violent events whereas cases have been reported in zones without violent events. the outbreak has been centered in health zones of katwa ( %), beni ( %), mabalako ( %), butembo ( %). butembo and beni are the health zones with the most violent events (fig. ). since the beginning of the outbreak in may , the average reproduction number (mean r estimate ) was . . using case report data, after august , , the average reproduction number was between . and . in the days following a violent event in a district, and between . and . in all other cases (fig. ) . the difference between reproduction numbers with and without violent events was statistically significant and increased as the mixing between zones was assumed increasingly limited (transmission mixing parameter ω decreased). even at very high levels of inter-zone transmission mixing, corresponding to relatively homogenous transmission within and across health zones, (ω = . , p = . ), the difference was still statistically significant (fig. ) . we compared the initial estimated r and the quenching parameters of past outbreak to the current outbreak and its respective health zones (fig. ) . the inter-zone transmission mixing parameter ω was varied over a range from to (fig. ) . the log of the estimated quenching parameter was (− . ) for the current outbreak was closest, though slightly higher, to that estimated for the - outbreak in west africa (− . ). this was paired with a slightly higher estimated r initial = . than for the west african outbreak where r initial = . . the (r initial ) reported here for the west african outbreak is consistent with the previous literature (barbarossa et al., ; lau et al., ) . these numbers are consistent with the long trajectory and continued transmission of the outbreak as a whole, although the trend of observed r in the current outbreak is rather different from the west african outbreak where the mean overall r was only above . early in the outbreak before dropping below . as the outbreak continued, where here it can be seen to fluctuate with three peaks above r estimate = . and below r estimate = . (fig. ) (lau et al., ) . the estimated r initial and quenching parameters clustered around the current outbreak estimates, some more extreme (lower quenching) and other more like smaller outbreaks of the past. each health zone was also estimated. health zones at the center of the outbreak appeared on average to have lower quenching. mabalako, butembo, and katwa, for example, often had weaker quenching than the west african outbreak; however, this was not consistent because beni consistently reported a higher level of quenching from the estimates, indicating the complex geographic distribution of transmission among health zones. in evaluating a probable mixing parameter to use for evaluating the results from this outbreak, we can look at the evidence from the time series of r estimate by health zone and examine their behavior (figure ). both extremes of no mixing (ω = . ) or full mixing (ω = . ) are unrealistic and lead to inconsistent results. when no mixing is allowed (ω = . ) this causes false spikes in r estimate in health zones with low levels of transmission following an increase in transmission in a neighboring health zone as the neighboring cases are unable to account for the spread of transmission to the low transmission zone, and this creates a false apparent spike in their own health zone to compensate for a lack of inter-zone transmission. even low levels of mixing (ω = . ) are enough to remove false spikes in r estimate that we see when no mixing is allowed (ω = . ). when full mixing is allowed (ω = . ) this leads to all health zones having identical r estimate at each time point. this herding behavior is strongly seen still when (ω = . ) at unrealistically high levels. even at a middle level of mixing (ω = . ) it appears that health zones herd too strongly to the health zone driving transmission, as spikes in transmission at days , and cause all health zones reporting cases to spike as well, when it is most probable the increase in transmission is being driven only by a few health zones and the others only responding to the initial change in transmission. although we do not make an effort to formally estimate a probable mixing parameter, it would be reasonable to consider the estimates taken with ω = . to ω = . as these are the probable limits of the range for the mixing parameter in this outbreak. using case report data, at ω = . to ω = . we see a difference in the r estimate by violent events (fig. ) . looking only at ω between . and . , we see an increase in r estimate following violent events of . ( % ci: . , . , p < . ) to . ( % ci: . , . , p < . ). however, when considering the symptom onset data the strength of the effect of recently reported violence was reduced, though still significant overall with violent events leading to an increase in r estimate of . ( % ci: . , . , p = . ) to . ( % ci: . , . , p < . ) (fig. ) . to assess the sensitivity of the lag time chosen after a violent event, we looked at the effect of violent events reported in increments of increasing weeks. looking at the case report data with ω = . , the overall effect size is fairly constant whether we consider a period of days or days, with an increase in r estimate of . ( % ci: . , . , p < . ) and . ( % ci: . , . , p < . ), respectively. when looking at the onset data with ω = . , the strength of the effect of recent violent events is strongest when it is evaluated over a full - days, as r estimate increases from . ( % ci: − . , . , p = . ) to . ( % ci: . , . , p < . ) as the lag increases to days and then becomes relatively stable. wannier et al. page epidemics. author manuscript; available in pmc july . we did not consider event times longer than days apart because many of the affected health zones have events that occur less than one month apart, sometimes occuring as little as one week apart. for these health zones, increasing duration beyond this point does not increase the period of time considered as being impacted by violent events, and thus we lose much of our ability to further distinguish between transmission in zones with recent violent events. fig. shows the estimated shannon entropy over the course of the epidemic. the estimated entropy was . ± . on august , rising to . ± . by april . we find evidence of an increasing trend (p < . , time series bootstrap), showing less concentration of cases over time. some increase in entropy is expected early in an epidemic, as cases begin to appear in new health zones. later in an epidemic, a decrease in entropy could occur if there were an increased concentration of cases in a few of the affected health zones. it is possible that the continued spatial spread of the current outbreak, which is second only to the west african outbreak, is contributing to the difficulty in controlling this outbreak. among health zones situated within the evd outbreak in drc, we found that the evd transmission rate (reproduction number r) was higher following violent events. the outbreak was subcritical (r < . , non-sustaining transmission) in zones without violent events reported by who (world health organization regional office for africa, ), while it was supercritical (with estimated r > . , continued transmission) in zones with reported violent events, suggesting that ongoing violence is likely perpetuating an otherwise declining outbreak. our findings suggest that violent events increased transmission in the weeks following a violent event, and that this effect may be sustained for many more weeks. after the destruction of the ebola care facility in katwa, for example, over one month was needed before ebola virus disease relief efforts were fully resumed. our time series regression found an effect of violent events on estimated r across both symptom onset data and case report data, across all plausible levels of inter-region transmission (fig. ) , and lagged follow-up periods of or more days. the consistency of the effect of recent violent events across data sets strongly supports the idea that violence is indeed contributing to the increased transmission and ongoing nature of this outbreak (richardson and fallah, ; frankfurter et al., ; richardson et al., a richardson et al., , b richardson et al., , a richardson et al., , b . more research is needed to confirm and further understand how the frequency and intensity of events may affect the contributions of violence to evd transmission. there are several limitations in our analysis. cases may have escaped detection and reporting (richardson et al., a (richardson et al., , b . if cases were missing in a biased way that systematically under-reported cases in certain areas or at certain times, there could have been a biased estimate of the effect, though the direction of the bias would be unclear. additionally, if there was heterogeneity in the reporting delays, this could have biased the effect estimates made using the case report data, though this objection would not apply to the symptom onset data. the nature and causes of violent events can be quite different. moreover, such events can affect different numbers of people, can vary in geographic scope as well as the duration of their impact. other non-violent events not considered in this analysis, such as strikes, may also be contributing to ongoing transmission. unmeasured causes or determinants of violence in the region may also drive variation in transmission or reporting in an unmeasured way. the epidemic curve in each health zone describes relatively few cases, making interpretations of specific features of their epidemic curves unwise. our analysis relied on who reporting of violent events, and more accurate quantification of these events may be possible. note that the assumption of exponential decay (quenching) of r initial may not accurately characterize this epidemic; however, this was only used for comparisons to past outbreaks, and the estimates of the effect of violent events are not affected by this. while we have shown that the ongoing violence has likely hampered control and contributed to this becoming the second largest epidemic, our efforts to quantify the role of violence should be interpreted with caution. these considerations also suggest that meaningful interventions must consider the sociopolitical determinants of armed conflict in the drc, including the legacies of colonialism, and other forms of historical and ongoing violence (sweeney, ; nzongola-ntalaja, ; council on foreign relations, ; richardson and fallah, ; frankfurter et al., ; richardson et al., a richardson et al., , b richardson et al., , a richardson et al., , b . on april , the democratic republic of the congo's minister of health addressed the worsening outbreak and indicated that with "the difficult security situation, this epidemic had gone beyond public health" (rdc, ) . while this may have been the first time an evd outbreak occurred in an active conflict zone, it is unlikely to be the last time that violent conflict contributes to the prevention of epidemic decline. as evd surges in drc, a polio outbreak surges in a conflict area of nigeria. ebola virus and other infectious disease outbreaks have the potential to become neglected crises in conflict settings if left unchecked. despite immense security challenges, ebola responders and the people in the affected area work tirelessly under dangerous conditions and deserve great respect, gratitude, and protection for their ongoing work to contain this public health disaster. estimated initial reproduction number r initial and quenching rate τ for current and past outbreaks. r initial and τ were estimated for the current outbreak as an overall summary measure, as well as independently in health zones with over a range of inter-zone transmission mixing parameters ω. epidemics. author manuscript; available in pmc july . wallinga-teunis estimated r per day and violent events by health zone for symptom onset and reporting dates, allowing for mixing between regions. r was estimated over a range of inter-zone transmission mixing parameters ω. violent events are marked using triangles with colors matching the affected district(s). epidemics. author manuscript; available in pmc july . outbreak(s) of ebola haemorrhagic fever outbreaks chronology. ebola virus disease the impact of control strategies and behavioural changes on the elimination of ebola from lofa county transmission dynamics and final epidemic size of ebola virus disease outbreaks with varying interventions ebola virus disease in southern sudan: hospital dissemination and intrafamilial spread on the theoretical specification and sampling properties of autocorrelated timeseries prise en charge des malades et des défunts lors de l'épidémie de fièvre hémorragique due au virus ebola d'octobre à décembre ebola response suffers another setback, as who evacuates some staff after attack s redfield: it could take another year to control ebola in drc ebola response teams scrambling to care for patients after attacks set back efforts ebola response is working, who director-general says, amid criticism and violence on a knife edge': ebola outbreak threatens to escalate as violence rises discovery and description of ebola zaire virus in and relevance to the west african epidemic during - outbreak of ebola hemorrhagic fever -uganda transmission dynamics and control of ebola virus disease (evd): a review what's worse than ebola? fighting it in a war zone violence in the democratic republic of congo, global conflict tracker canadian immunization research network. assessing the safety and immunogenicity of recombinant vesicular stomatitis virus ebola vaccine in healthy adults: a randomized clinical trial evaluating subcriticality during the ebola epidemic in west africa indirect rule redux: the political economy of diamond mining and its relation to the ebola outbreak in kono district confidence intervals for gini's diversity measure and shannon's entropy using adjusted proportions ebola hemorrhagic fever outbreaks in gabon, - : epidemiologic and health control issues anatomy of a hotspot: chain and seroepidemiology of ebola virus transmission projections of ebola outbreak size and duration with and without vaccine use in Équateur the reemergence of ebola hemorrhagic fever, democratic republic of the congo spatial and temporal dynamics of superspreading events in the - west africa ebola epidemic a limited outbreak of ebola haemorrhagic fever in etoumbi the congo from leopold to kabila: a people's history. zed books, london. world health organization abonnement annuel the impact of bootstrap methods on time series analysis ebola doit être vaincu le plus tôt (tshibuyi) the genesis of the ebola outbreak in west africa minimally symptomatic infection in an ebola 'hotspot': a crosssectional serosurvey biosocial approaches to the - ebola pandemic the ebola suspect's dilemma the symbolic violence of outbreak: a mixed methods, quasi-experimental impact evaluation of social protection on ebola survivor well-being drc: conflict and displacement in nord kivu and ituri different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures preliminary results on the efficacy of rvsv-zebov-gp ebola vaccine using the ring vaccination strategy in the control of an ebola outbreak in the democratic republic of the congo: an example of integration of research into epidemic response a systematic review of early modelling studies of ebola virus disease in west africa realtime projections of epidemic transmission and estimation of vaccination impact during an ebola virus disease outbreak in the eastern region of the democratic republic of congo health topics: ebola virus disease. (online; accessed . world health organization regional office for africa we thank the ebola responders for their efforts in the - evd outbreak in north kivu and ituri provinces, democratic republic of congo. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. key: cord- - yd hr e authors: li, fei; liu, junjie; ren, jianlin; cao, xiaodong title: predicting contaminant dispersion using modified turbulent schmidt numbers from different vortex structures date: - - journal: build environ doi: . /j.buildenv. . . sha: doc_id: cord_uid: yd hr e air pollutant transmission has significant influences on indoor air quality (iaq). it is crucial to study mechanisms involved with airborne contaminant dispersion indoors. however, relationship between pollutant diffusion coefficient and viscosity in enclosed spaces has not been fully understood. in this study, turbulent schmidt number (sc(t)) was modified as a function of turbulent kinematic viscosity rather than a constant value to better simulate dispersion of airborne contaminant in two typical enclosed spaces: an aircraft cabin and an office room. an experiment for airborne contaminant transmission was conducted in an aircraft cabin mockup. combining with experimental data in the office room with an under floor air distribution (ufad) system from literature, sc(t) was modified based on airflow vortex structures. the performance of rng k-ε model using the modified sc(t) was found to be obviously better than that using the default sc(t) value in both the two enclosed spaces. in addition, model applicability to different enclosed spaces was analyzed based on the airflow vibration frequency. some confined vehicle cabins, such as aircraft cabins, provide an easy way for pathogens to spread. the virus laden bio-aerosols like severe acute respiratory syndrome (sars) and h n -a influenza generated by infected occupants through coughing or sneezing can be transported throughout the cabin and infect other occupants, as reported by wilder-smith [ ] and baker et al. [ ] . in residential buildings and other indoor environments, people are continuously exposed to different air pollutants. aerosol particles are regarded as major pollutant sources, and they are closely related to such adverse health effects [ ] . other pollutants, such as formaldehyde and volatile organic chemicals (vocs) can increase the risk of chronic toxicity and even cancer due to prolonged and high-dose exposure [ ] . therefore, it is important to study mechanisms involved in spreading of airborne contaminants, in order to prevent transmission and make a quick respond for chemical and biological attacks in enclosed environments as well. for airborne contaminant transport, turbulent schmidt number (sc t ) which represents the ratio between turbulent kinematic viscosity and turbulent diffusion coefficient is a key parameter for contaminant concentration prediction in turbulent airflow. previous researchers have investigated the effect of sc t on predicted contaminant dispersion for different situations. sc t values ranged from . to . in computational fluid dynamics (cfd) simulations for urban diffusion problems [ ] [ ] [ ] . for jet flow simulations, sc t values were suggested to be . for jet-in-cross flow [ ] and . for free round jet [ ] . riddle et al. [ ] reported reducing the sc t from its ''standard value of . ″ to . could improve the predicted ground level concentrations. from these studies, sc t varies in value for different local flow properties, indicating its value should be performed based on the airflow structures. shi et al. [ ] proposed a new dynamic turbulent schmidt number model based on local velocity gradient and density gradient, while their study focused on sc t in the boundary layer. mokhtarzadeh-dehghan et al. [ ] found sc t should be associated with richardson number in order to model the experimental spreading rates of heavier-than-air gases correctly in an atmospheric boundary layer. however, they didn't consider local flow properties based on the airflow structures. for the aircraft cabin and built environment, some researchers have investigated performances of different cfd models for airborne contaminant transport. zhao et al. [ ] and zhang et al. [ ] evaluated different eulerian and lagrangian models in enclosed spaces. wang et al. [ ] provided advanced turbulence models for predicting particle transport by integrating different numerical models. zhao et al. [ ] proposed an improved drift flux model to predict particle dispersion. however, these cfd models performed relatively poorly for concentration simulation. some experimental studies were also conducted in aircraft cabins. wang et al. [ ] studied airborne contaminant transport using co as a tracer gas inside an aircraft cabin. sze to et al. [ ] studied dispersion of expiratory droplets with mixing ventilation inside an aircraft cabin mockup. zhang et al. [ ] measured and predicted contaminant distributions in a cabin mockup. they used sf and mono-dispersed particles with diameter of . μm to simulate airborne contaminants. li et al. [ ] conducted an experiment for gaseous (sf ) and particulate ( μm particle) contaminant dispersion in an actual functional md- aircraft cabin. they stated that in narrow cabin space, source location and thermal buoyancy had a significant effect on airborne contaminant distributions. these research made a great contribution to understanding of contaminant transport in enclosed spaces, however, they did not analyze effect of different airflow structures on the sc t value. airflow in enclosed spaces is a kind of interaction of different flow structures, such as vorticity and deformation; therefore, the sc t value should not be a constant number, but be varying based on airflow structures. the objective of this study is to modify sc t numbers to quantify the relationship between turbulent kinematic viscosity and turbulent diffusion coefficient. an experiment for airborne contaminant dispersion is conducted in a well-controlled aircraft cabin mockup. combining with experimental data in an office room from literature, we establish new equations of the sc t value based on different vortex structures in airflow. the improved models are then validated by laboratory experiments. in addition, through airflow frequency analysis, the reason for inconsistent equations of sc t for different environments is explored. two typical cases of enclosed space environment were selected for sc t modification. as show in fig. (a) , the first case is in the seven-row aircraft cabin mockup [ ] . the cabin had heated manikins with sensible heat production of w for each. air was supplied through two rows of diffusers on cabin shoulders and was exhausted at outlets located on side wall near the cabin floor, and the air change rate was ach. the second case is in the office with an under floor air distribution (ufad) system [ ] as shown in fig. (b) . the room had four heated human simulators. air was supplied from two floor inlets and exhausted from a ceiling outlet, and the air change rate was . ach (air change per hour). since modification of sc t is associated with vortex structure, a numerical model was used to identify vortex structure firstly. . in this study, three different turbulence models: rng k-ε model, rsm model, and sst k-ω model were employed to calculate airflow distributions and compared with each other. the rng k-ε model has been widely used for predicting indoor airflows with many successes [ ] . the rsm turbulence model is more complex and more accurate with turbulent velocity fluctuations, but it needs more computing resource [ ] . martinho et al. [ ] reported the sst model typically gave better results for heat transfer between the heating body and thermal environment. the governing equation of all these turbulence models equations can be written in a general form: where ϕ is flow variables (velocity, enthalpy, turbulence parameters and mass fraction), Γ ϕ eff , is the effective diffusion coefficient, and s ϕ is the source term. details about coefficients Γ ϕ eff , and s ϕ for different variables ϕ can be found in the ansys theory guide [ ] . based on eq. ( ), the species transport equation can be written as: where c is local mass fraction, ν t is turbulent kinematic viscosity, d is the mass diffusion coefficient. airflow distribution was solved by eq. ( ) first, then it was frozen, and the concentration field was simulated by eq. ( ) . default sc t is . in most numerical simulations. however, as mentioned in the introduction part, sc t should be determined by considering the flow structures [ ] . the airflow field was simulated under steady state, and the gravity was considered. simple algorithm was applied to couple pressure and velocity, and standard and second-order were used for pressure discretization and all other variables. residual was below − for energy and − for all other variables. boussinesq approximation which is a common approach for indoor airflow simulations was employed to consider buoyancy effect. standard wall treatment was applied to model near wall turbulence. for the boundary conditions of the aircraft cabin, the inlet velocity direction was measured by ultrasonic anemometers (uas), while the velocity magnitude was measured by hot-sphere anemometers (hsas). thermal boundaries of the walls and heating manikins were measured by an infrared camera. the measured data were assigned into the diffusers which had same effective areas as the real model through userdefined functions (udfs). the pressure outlet boundary condition was applied on the exhausts, and the reference pressure value was set to pa. tetra-mesh was used in this study, with total grid number of nine million, and the meshes closed to diffusers and manikins were refined. details of boundary and mesh grid conditions for the office and aircraft cabin simulations can be found in zhang et al. [ ] and li et al. [ ] respectively. in this study, the relationship between ν t and sc t was established based on vortex structure of airflow. li et al. [ ] used the okubo-weiss parameter, q introduced by okubo and weiss to identify the type of vortex structure in an aircraft cabin. the parameter, q is differential of deformation and vorticity square and its expression is: where s n represents stretching deformation, s s represents shearing deformation, and w represents vorticity as follows: the interval of q is always divided into three parts by . δq, where δq is standard deviation of q in the entire fluid domain. when q > . δq, the region is dominated by deformation; when q < − . δq, the region is dominated by vorticity; and when − . δq < q < . δq, it is an ambient field. this standard was adopted to identify "deformation dominated region" and "vorticity dominated region" in the aircraft cabin and office room. sc t and ν t values in different regions were determined based on experimental data. for the study on contaminant transport, airflow fields should be predicted correctly. experimental data from literature was used to validate the numerical model. fig. shows comparison of experimental data from particle image velocimetry (piv) measurement [ ] with simulated airflow patterns in cs of the aircraft cabin ( fig. (a) ) by three turbulence models. as shown in fig. , decay of the simulated velocity is slower than the experiment due to underestimated turbulence kinetic energy [ ] . the predicted airflow fields from all turbulence models were similar and all captured the trend of piv measured airflow. jets from right and left diffusers merged in the middle, and the jet from the left side was a little stronger due to the asymmetry diffuser conditions in the cabin. in addition, velocity magnitudes were lower in top and side regions. however, remarkable differences exist if we look at the two sets of vectors at certain positions, especially in the middle where the jets from right and left merged. the inlet velocity direction was measured by uas, whose probes could not be placed very close to the diffusers [ ] . this maybe resulted in some errors of measured velocity direction. the experimental data may unavoidably have some instrumental errors and thereby lead to some deviations in the center of the cabin where the velocity was very small (< . m/s). fig. shows comparison of predicted airflow velocities with experimental data [ ] at poles v to v for the office room ( fig. (b) ) by different turbulence models. poles v to v were in the vicinity of the selected cross section. from fig. , the simulated data by rng k-ε model agrees best with the measured data. while the rsm model performs worst, which may be caused by its convergence problem in threedimensional buoyant flow [ ] . the simulation agreed better with measured data at lower and top part of pole v and v , but worse in the middle part of pole v and v . for pole v , predicted and experimental velocities were all lower in the middle part, but discrepancies were significant at its top and lower parts. rrmse (relative root mean square error) was used to conduct quantitative comparison between different turbulence models: where c and c exp i sim i , , are experimental and simulated concentration at sampling point i. c exp is average experimental concentration. table shows the rrmse values for three turbulence models. for the aircraft cabin, the rng k-ε model performs slightly better. considering overall low velocities and complex turbulent flow in the cabin, the difference between these turbulence models is not observable. for the office room, performance of the rng k-ε model is much better than other models. overall, the rng k-ε model performed the best in both spaces, and its predicted airflow fields were also acceptable. taking the computing efforts into consideration, it was employed to modify the turbulent schmidt number in this study. goldman et al. [ ] investigated sc t values for different working gases in an air pipe. in the pipe under steady condition, the concentration distribution can be expressed as eq. ( ) as follows: where c and u is concentration and average axial fluid velocity. m is source strength, and = + Γ d eff ν sc t t is the effective diffusion coefficient. with a known m, through measuring concentration (c), velocity (u ) and coordinate (x, r) in the pipe, an effective diffusion coefficient (Γ eff ) can be calculated through eq. ( ) . ν t in the pipe can be also calculated by empirical formulas [ ] . accordingly, corresponding to one ν t value, one sc t value can be obtained. goldman et al. [ ] used this method to study the relationship between ν t and sc t in the air pipe. they developed a fitting equation to express their relationship: where ν l and sc are laminar kinematic viscosity and laminar schmidt number. they are dependent on air and released working gas property. in enclosed space environment, the situation was much more complicated than that in the air pipe. sc t cannot be calculated through analytic equations like eq. ( ) , and no empirical formula can be used to calculate the ν t value as well. however, cfd provided a useful tool to solve this problem. in the rng k-ε model, ν t values can be calculated as follow: where c u is a constant number, k is turbulence kinetic energy, and ε is turbulence dissipation. through repeated numerical trial calculation, sc t values in different regions can also be obtained. referring to eq. ( ), relationship between ν t and sc t can be expressed as a unified equation for a certain working gas: where b and c are undetermined parameters. in the laminar layer, ν t approaches zero, sc t should approach unity, and mass diffusion coefficient controls the dispersion. in the turbulent core region, ν t is high, sc t approaches unity as well, and turbulent diffusion coefficient governs the dispersion. unlike goldman's study, only one pair of working gases (air -sf ) was used in our study, and b and c were constant. therefore, it needed two sets of sc t and ν t data to determine these two parameters. fig. shows the predicted q value in section cs of the aircraft cabin ( fig. (a) ). blue regions are dominated by vorticity, red regions in the figure are dominated by deformation, and green regions are ambient fields. the q value was higher in the vicinity of diffusers and at the aisle where two air jets from diffusers merged. while, the q value was lower in the sides of the cabin due to two large flow re-circulations on each side. two regions < d > and < m > with different vortex structures (fig. ) were selected to reveal the relationship between ν t and sc t in the aircraft cabin. in region < m > , source m was elected in the middle of the aisle dominated by deformation. contaminant released from source m was transported downward by airflow and directly exhausted from the cabin. in region < d > , source d was at the breathing level of a passenger dominated by vorticity. contaminant released from source d could travels repeatedly in region < d > and was locked up by eddy airflow. mixed tracer gas ( % sulfur hexafluoride-sf , % n balance) was released from a rubber bulb recommended by li et al. [ ] . diameter of the rubber bulb was cm, and holes with diameter of mm were distributed on the bulb surface to make an almost zero momentum release. volume flow rate of the mixed tracer gas released from each source was set to l/min and was controlled through two gas rotameters with relative errors of % (fig. ) . fig. shows the predicted q value in the cross section of the office room ( fig. (b) ). the q value was higher near the middle of the floor, and lower in the vicinity of manikins. two regions with different vortex structures (fig. ) were selected. source was in the vorticity dominated region of the cross section (x = . m), and source was in the deformation dominated region of the cross section (x = . ). particles with mean diameter of . μm were used in their study. murakami [ ] and li et al. [ ] reported particles smaller than μm have the similar concentration distribution as tracer gas in the room and aircraft cabin respectively. therefore, concentration distributions for . μm particles could be used as a surrogate for that of sf . concentration distributions with each source working in the aircraft cabin were analyzed with a photo-acoustic multi-gas analyzer (innova , lumasense technologies) whose lower detection limit is . ppm for sf . twelve sampling points including sampling points in region < d > and < m > were employed to cover cs , cs , and cs ( fig. (a) ), totally sampling points. sampling time of each point was set to be τ (τ is the time constant which is the volume of the cabin divided by the ventilation rate, and it is s in this study). this is because, under the hypotheses of well mixed, air in a cabin can be exchanged fully after τ [ ] , and concentration volatility and uncertainty can be captured in this period. accordingly, for each sampling point, measured data in s were used to determine its averaged concentration and confidence interval. for the office room, a particle counter (pc- h qcm impactor, california measurements inc.) was used to measure particle concentration in six locations (poles p to p ) as shown in fig. (b) at five fig. . comparison of the predicted airflow velocities with the experimental data from zhang et al. [ ] . different heights, totally sampling points. sampling in the vorticity dominated region of the cross section (x = . m) was elected to be paired with source (fig. ) . in zhang's study [ ] , no sampling point was set in the deformation dominated region of the cross section (x = . ) closed to source ; therefore, sampling on streamlines from the region was elected to match with source . for the aircraft cabin, the averaged ν t value was about . m /s in region < d > , and . m /s in region < m > . through adjusting sc t in numerical simulation and comparing predicted data with experimental data in the region, sc t in each region was got by repeated trial calculations. taking region < m > for example, the repeated trial calculation started from sc t = . , and the sc t value with the smallest relative root mean square error (rrmse, eq. ( )) between predicted and experimental data in region < m > was . , as illustrated in fig. . similarly, the sc t value for the region < d > was . . by inputting ν t and sc t of these two regions < d > and < m > into eq. ( ), b is . , and c is . . therefore, the relationship between ν t and sc t in the aircraft cabin can be expressed as: for the office room, through the similar method, averaging ν t values in the two regions were calculated by eq. ( ) based on the rng k-ε model; and the sc t value in each region was got by repeated trial calculation as well. the relationship between ν t and sc t in the office room can be expressed as: to test effectiveness of the improved sc t model in different enclosed spaces, comparison of improved model with existing solution (sc t = . ) was conducted. it should be noted that the data employed to develop the model were not used to validate the model. for the aircraft cabin, the data in the region < d > and < m > (fig. ) were used to improve the model, while all the data in cs , cs and cs were used to validate the model. for the office room, the data in the two regions were used to develop the model, while all the data in poles p -p were used to validate the model. normalized concentrations for source d from the simulation and experiment in cs , cs and cs were plotted against each other in fig. . the solid line represents perfect agreement, the dotted lines represent the region with errors ranging from − % to %, and the error bars presented % confidence intervals. rrmse and another index, correlation coefficient (r) were employed to quantify concentration data comparison. the correlation coefficient (r) [ ] is defined as follows: where experiment and simulation. the correlation coefficient (r) describes the linear correlation of the experimental and predicted data. it ranges from − to , with − meaning strong negative relationship, meaning no relationship and meaning strong positive relationship. as shown in fig. and table , for the aircraft cabin, the simulated result using the modified sc t model was obviously better than that using the default sc t , and it has a lower relative root mean square error and higher correlation coefficient. for the office room with an ufad system, normalized concentrations from the simulation and experiment in poles p -p were also plotted against each other in fig. . as shown in fig. , more points from the improved sc t model fall in the region with errors ranging from − % to % and are closed to the solid line represents perfect agreement. this also can be confirmed in table , in which rrsme is reduced from . to . . however, the correlation coefficient does not have improvement; and it is almost identical for default sc t and modified sc t . this may be due to the fact that the correlation coefficient was already high relatively, and it was difficult to be improved further. in this study, relationship between sc t and ν t for different vortex structures was revealed. when comparing table with table , it is found that the rrmse of predicted velocity is smaller than that of concentration. this is because the species transport equation is solved based on momentum equations, and a little discrepancy for the simulated velocity fields will lead to large discrepancy for the predicted concentration distributions. therefore, discrepancy for the simulated velocity fields may result in lack of improvement of the proposed procedure. additionally, because pipe flow and air cabin flow significantly differed, application of the equation deduced from pipe flow may also result in some limited improvement. however the modified sc t model was still better than that using the default sc t , because the modified sc t can compensate underestimation of turbulent diffusion in the rng k-ε model to some extent. for other turbulence models, because predicted ν t may be different with that from the rng k-ε model, the sc t expression function may have different parameters and need further investigation. bazdidi-tehrani et al. [ ] proposed various non-linear eddy viscosity turbulence models and reported the traditional k-ε model using the boussinesq's isotropic linear eddy-viscosity concept could overproduce turbulence kinetic energy at impingement zone and failed to predict complex flow structures around buildings. however, their study focused on flow and concentration fields on and around an isolated cubical building within the neutral turbulent boundary layer. different with the turbulent core region in enclosed spaces, it is well known that, in the boundary layer, the turbulence is assumed to be anisotropic. in addition, some turbulence models, such as the v -f model [ ] , were also proposed to predict anisotropic turbulence near the wall and solve overestimated turbulence fluctuation perpendicular to the wall. fig. illustrated the modified sc t equations and ranges of ν t values for the selected different vortex structures in the aircraft cabin and office room. in these two spaces, most of the ν t values fall within these ranges, but are not limited to these ranges. because the equations of the modified sc t are nonnegative and have specific physical meaning mentioned above, their effective ranges can cover all of the ν t values in these two spaces. as shown in fig. , equations of the modified sc t are observably different for the aircraft cabin (eq. ( )) and office room fig. . comparison of the experimental data with predicted sf concentrations by: (a) default sc t ( . ), (b) modified sc t (eq. ( ) ) in the aircraft cabin. (eq. ( )). we thought this may be caused by different airflow vibration frequencies. according to prandtl's theory, vibration frequency is ′ u l / , where ′ u is mean fluctuation velocity, and l, per shu's suggestion [ ] , should be the kolmogorov scale as follows: where ν is kinetic viscosity, ε is turbulence eddy dissipation. as it is well expected that ε and ′ u is the highest at air inlet, where the vibration frequency should be also highest there, we use ′ u and ε at the inlet boundary to calculate vibration frequency. the inlet dissipation ε can be estimated as [ ] : where u is mean velocity, k is turbulence kinetic energy, i is turbulence intensity, ′ l is the turbulence length scale (m), equals to . d, d is the hydraulic diameter of the inlet plane and ≈ c . μ . for the aircraft cabin, mean velocity of its inlets is . m/s [ ] , and turbulence intensity is % [ ] . for the room, mean velocity of its inlets is . m/ s, and turbulence intensity is - % [ ] . accordingly, vibration frequency of airflow in the aircraft cabin is hz, while it is hz in the office room with an ufad system. the remarkable difference of airflow frequency between the aircraft cabin and office room may lead to different modified sc t expression functions. higher frequency may result in stronger diffusion ability corresponding to a lower sc t value, and the relationship between airflow vibration frequency and schmidt number need further research. nevertheless, the proposed modification method expressing sc t values as a function of ν t associated with vortex structures can improve contaminant concentration prediction in turbulent flow. for other researchers in the field, if the enclosed spaces which they investigated have similar airflow structures and frequencies with ours, they can refer to our new schmidt number; otherwise they can still use our method to modify the turbulent schmidt number for their research. in this paper, a method to modify sc t as a function of turbulent kinematic viscosity based on airflow vortex structures was introduced to better simulate dispersion of airborne contaminant in two typical enclosed spaces: an aircraft cabin and an office room. this study was focused on gaseous pollutants, which are common in indoor environments. three turbulence models were evaluated. the rng k-ε model performed the best and was employed to modify sc t . during the modification, different airflow vortex structures including deformation and vorticity were taken into consideration. comparing with the experimental data, performance of the rng k-ε model using modified sc t was found to be better than that using default sc t value in both the two enclosed spaces. for the aircraft cabin environment, the improved model reduced the relative root mean square error from . to . , and increased the correlation coefficient from . to . . for the office room with an ufad system, the improved model reduced the relative root mean square error from . to . , but no observable increase was found in correlation coefficient. the application of proposed modified method for different turbulence models and environments with different airflow vibration frequencies needs further research. the severe acute respiratory syndrome: impact on travel and tourism transmission of pandemic a/h n influenza on passenger aircraft: retrospective cohort study review of the national ambient air quality standards for particulate matter: policy assessment of scientific and technical information formaldehyde, -butoxyethanol and -tert-butoxypropan- -ol, iarc monographs fig. . turbulent schmidt numbers for different enclosed spaces on the evaluation of carcinogenic risks to humans, world health organization international agency for research on cancer determination of plume capture by the building wake progress and challenges in the development of physically-based numerical models for prediction of flow and contaminant dispersion in the urban environment cfd simulation of airflow over a 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ansys, . theory guide, ansys inc turbulent schmidt numbers for cfd analysis with various types of flowfield study on the carbon dioxide lockup phenomenon in aircraft cabin by computational fluid dynamics global airflow field distribution in a cabin mock-up measured via large-scale d-piv numerical simulation of airflow and airborne pathogen transport in aircraft cabins-part i: numerical simulation of the flow field evaluation of various categories of turbulence models for predicting air distribution in an airliner cabin turbulent schmidt numbers turbulent heat transfer and friction in the entrance regions of smooth passages diffusion characteristics of airborne particles with gravitational setting in an convection-dominant indoor flow field building ventilation: theory and measurement mathematical statistics and data analysis investigation of various non-linear eddy viscosity turbulence models for simulating flow and pollutant dispersion on and around a cubical model building modifications of the ú -model for computing the flow the tracking behavior of scattered particles in turbulence hwa measurement and analysis of md- aircraft cabin environment flow field (in chinese) the research presented in this paper was supported financially by the national science foundation of china (nsfc) through grant no. and no. . key: cord- - jtfc authors: van nguyen, dung; van nguyen, cuong; bonsall, david; ngo, tue tri; carrique-mas, juan; pham, anh hong; bryant, juliet e.; thwaites, guy; baker, stephen; woolhouse, mark; simmonds, peter title: detection and characterization of homologues of human hepatitis viruses and pegiviruses in rodents and bats in vietnam date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: jtfc rodents and bats are now widely recognised as important sources of zoonotic virus infections in other mammals, including humans. numerous surveys have expanded our knowledge of diverse viruses in a range of rodent and bat species, including their origins, evolution, and range of hosts. in this study of pegivirus and human hepatitis-related viruses, liver and serum samples from vietnamese rodents and bats were examined by pcr and sequencing. nucleic acids homologous to human hepatitis b, c, e viruses were detected in liver samples of ( . %) of bats, ( . %), and ( %) of rodents, respectively. hepacivirus-like viruses were frequently detected ( . %) in the bamboo rat, rhizomys pruinosus, while pegivirus rna was only evident in ( . %) of rodent serum samples. complete or near-complete genome sequences of hbv, hev and pegivirus homologues closely resembled those previously reported from rodents and bats. however, complete coding region sequences of the rodent hepacivirus-like viruses substantially diverged from all of the currently classified variants and potentially represent a new species in the hepacivirus genus. of the viruses identified, their routes of transmission and potential to establish zoonoses remain to be determined. unlike many other communicable diseases, the burden of viral hepatitis has substantially increased over the last two decades to recently become the seventh leading cause of mortality worldwide. viral hepatitis now causes more deaths than tuberculosis, aids or malaria each year. hepatitis c virus (hcv) and hepatitis b virus (hbv) are responsible for > % ( % in ) of viral viruses , , of hepatitis-related mortality and disability. as such, these hepatitis viruses are the targets of efforts to combat viral hepatitis [ ] , including hbv vaccination, development of hcv vaccines, and highly effective drugs. in contrast, hepatitis e virus (hev) is endemic in many low-income countries [ ] but usually causes self-limiting hepatitis. infection with hev occasionally results in liver failure [ ] . hbv, hcv, and hev are members of virus families hepadnaviridae, flaviviridae, and hepeviridae, respectively. hbv has a partially double-stranded dna genome with overlapping open reading frames (orfs) [ ] , whereas hcv and hev have a single-stranded rna genome [ , ] . while the origins of these human viruses are unknown, rodents and bats are putative reservoir hosts because they host a diverse array of hepadnaviridae [ ] [ ] [ ] , hepeviridae [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , and genera pegivirus [ ] [ ] [ ] and hepacivirus [ , , ] of the flaviviridae family including homologues of the human hepatitis viruses under question. among these, it is of concern that a bat hepadnavirus may possess the ability to infect human liver cells [ ] . several factors may contribute to the risk of zoonotic rodent or bat virus transmission. rodent meat is a popular source of protein for human consumption in vietnam, particularly in the mekong delta, where rats (rattus spp. and bandicota indica) are commonly trapped and sold live in wet markets [ ] . the total annual consumption of rat meat in vietnam is estimated at - tonnes [ ] . hoary bamboo rats (rhizomys pruinosus) are additionally farmed for human consumption. moreover, bat faeces collected from bat caves or farms is used as natural fertilizer ("guano") in vietnam. as rodents and bats are reservoirs or carriers of a significant number of zoonotic pathogens [ ] and viruses with unknown zoonotic potential, there are health risks that are associated with exposure to these animals. however, a previous study [ ] showed none of the surveyed rat catchers or processors were aware of infection risks from contact with live rats. consequently, no precautions were taken for the handling of rodents. in the search for viral diversity and zoonotic viruses, novel paramyxovirus and coronavirus in vietnamese bats and rats were detected in a previous study [ ] . here, we report the detection pegivirus and human hepatitis-related viruses in these mammals. rodent and bat samples were collected within the vizions (vietnam initiative on zoonotic infections) framework [ ] for the screening of zoonotic microorganisms [ , [ ] [ ] [ ] . rodent samples. as it is important and essential to understand the risk associated with rodents, including those sold in the markets, a total of rats purchased from markets in five of twelve provinces in the mekong delta during - and farmed bamboo rats purchased from a market in dak lak in - were enrolled. in addition, trapped rats were also included. rat trapping was carried out in different locations (pig and poultry farms, rice fields, fruit groves, tropical forests, markets, slaughter-house) in the provinces of dong thap during march and dak lak in april , as previously described [ ] . serum and liver samples were collected post-mortem. species identification of rats was carried out on the basis of morphological characteristics and sequencing of a conserved housekeeping gene [ ] . all of the samples were stored in sterile tubes with rna later at − • c until nucleic acid extraction. special precautions were taken to avoid cross-contamination. bat samples. a total of bats were trapped at six sites in the provinces of dong nai (in cat tien national park) and quang ngai in may using mist nets and harp traps as described [ ] . trapped bats were speciated according to morphology [ ] , and liver samples were collected and stored as described above for rats. rna was manually extracted from rodent serum samples using qiaamp viral rna mini kit (qiagen, manchester, uk) and following instructions from the manufacturer. liver samples from bats and rodents were subjected to nucleic acid extraction using allprep dna/rna mini kit (qiagen, manchester, uk). briefly, about mg of liver per sample was first lysed and homogenised using tissuelyser (qiagen, manchester, uk). the lysate was applied to an allprep dna spin column for dna to bind onto the column. ethanol was added to the flow-through and rna and bound to the membrane when the sample was passed through an rneasy spin column. after washing steps, dna and rna was eluted separately in µl of nuclease-free water. extracted nucleic acid was used in screening for the targeted hepatitis viruses. in order to minimize contamination, pcr mastermix preparation, and the addition of templates were carried under separated laminar flow hoods and lab spaces. all of the surfaces, tubes, and equipment were uv irradiated between each pcr. reverse transcription using superscript iii reverse transcriptase (invitrogen, paisley, uk) was performed according to the manufacturer's instruction. synthesized cdna was screened for hepaciviruses and pegiviruses using a semi-nested pcr protocol with broad spectrum degenerate primers, which can detect all known hepaciviruses and pegiviruses. amplification conditions (using gotaq from promega, southampton, uk) included • c for min, and cycles of denaturation ( • c, s), annealing ( • c, s) and elongation ( • c, s). similarly, hev was screened using broadly reactive primers targeting viral rna-dependent rna polymerase as described in drexler et al., [ ] . dna extracted from liver samples was used for screening of hbv. degenerate primers targeting a highly conserved region of the polymerase gene of sequences from all known hbv hosts were designed for a nested pcr protocol using the above amplification conditions. primers for screening are listed in table s . the length of the sequenced screening fragments (excluding primer sequences) of homologues of hbv, hev, hcv, and pegivirus was , and nucleotides, respectively. for rodent hepacivirus, hev and pegivirus genomes, extracted rna from representative positive samples was subjected to deep sequencing using an illumina platform. libraries were prepared from total extracted rna using the nebnext ultra directional sequencing kit (new england biolabs, hitchin, uk) with omission of actinomycin d, then pooled and sequenced on a hiseq instrument (illumina, nr saffron walden, uk). quality control and trimming of reads were performed before genome mapping using clc genomics workbench (qiagen bioinformatics, redwood city, ca, usa) with the default affine gap cost parameters. the closest related virus genomes (genbank numbers kc , jx and kj for hepacivirus, hev and pegivirus, respectively) were used as templates for genome mapping. additional primers were designed using the obtained contigs for gap fillings. for bat hbv, primers were designed using sequences available from genbank and the obtained sequences from screening. these primers amplified amplicons, with overlapping regions as presented in table s . all of these nested or hemi-nested pcr protocols used superscript iii one-step rt-pcr system with platinum taq dna polymerase (invitrogen, paisley, uk) for rna viruses or q high-fidelity dna polymerase (new england biolabs, hitchin, uk) for hbv in the first round pcr, according to instructions from the manufacturers. q high-fidelity dna polymerase was also used in the second round pcr. two real-time pcr primer sets (table s ) in the untranslated region of bamboo rat hepaciviruses and the screening fragment of other rat hepaciviruses were designed using sequences available from this study. the targeted regions were amplified from positive samples and cloned into pgem-t easy vector (promega, southampton, uk) for in vitro transcription, as previously described [ ] . the obtained rna transcripts were used to generate standard curves of the real-time pcr assays for measurement of rodent hepacivirus rna titers using superscript iii reverse transcriptase (invitrogen, paisley, uk) and powerup sybr green master mix (thermo fisher scientific, northumberland, uk). sequences were imported into sse (simmonic sequence editor) [ ] for the alignment and calculation of sequence distance values from reference sequences of known viruses from which sequence identities were inferred. sequence distances instead of sequence identities in the regions used for classification of hepaciviruses and pegiviruses were presented to easily compare with the species p-distance thresholds set in the proposed update to the taxonomy of the genera hepacivirus and pegivirus [ ] . maximum-likelihood phylogenetic trees were reconstructed using the mega . software package [ ] with bootstrap resamples. the best-fitting model for each sequence dataset (as shown in figure captions) was first determined and used for phylogenetic reconstruction. sequences obtained in this study have been assigned the following genbank accession numbers mg -mg . nucleic acid that was extracted from liver samples of bats ( species; table s ) and rodents (six species) was screened for pegivirus and human hepatitis b, c, e viruses and their homologues ( table ) by nested and semi-nested pcr assays with degenerate primers. hepaciviruses were the most commonly detected ( . % of rodent samples, from three species), followed by hepatitis e related virus ( % of rodent samples, from four species) while hepatitis b related viral dna was only detectable in three bats ( species). most of the hepacivirus positive samples were from farmed hoary bamboo rats in dak lak province although the predominantly sampled rat species was rattus argentiventer. coinfection with hepacivirus and hev was observed in a sample from rattus losea. all liver samples from bats were negative for hepacivirus and hepatitis e related virus and no sample was positive for pegivirus. and other rats whose liver samples were screened for hepacivirus as above. hepacivirus rna was only detected in serum samples of bamboo rats with positive liver samples. pegivirus rna was detected in two samples from rattus tanezumi. two real-time pcr assays specific for bamboo rat hepaciviruses, and other hepaciviruses were used to determine viral rna concentration in the positive samples. the concentration of rna ( figure ) was high in both liver tissue (median, . × copies/gram; range, . × - . × ) and sera (median, . × copies/ml; range, . × - × ). viruses , , x of ( figure ) was high in both liver tissue (median, . × copies/gram; range, . × - . × ) and sera (median, . × copies/ml; range, . × - × ). amplicons derived from pcr screening experiments were all successfully sequenced and complete or near complete genome sequences were determined for representatives of the viruses by pcr or deep sequencing. after exclusion of primers, the obtained screening sequences from each targeted virus clustered in one or two clades of those with high identity and were most closely related to sequences previously reported from rodents or bats from the us [ , ] , china [ ] and vietnam [ ] . rodent hepacivirus sequences ( nucleotides) formed two well supported clades (figure a) . clade included all of sequences from rhizomys pruinosus which shared . - % pairwise nucleotide identity while three sequences (nucleotide identity of - %) from rattus losea and rattus argentiventer grouped in clade . these clades differed from each other by mean distances of . % and . % at nucleotide and amino acid levels, respectively. while the four vietnamese bamboo rat hepacivirus genomes were highly similar to each other (< % nucleotide and < % amino acid distances in the complete coding region (cds)), they were remarkably different from the closest sequence (kc ) with the corresponding distances of % and %, respectively ( table ). the amino acid p-distances of the obtained hepacivirus sequences and kc in the regions - and - (numbered relative to m ) were % and %, respectively. the newly identified hepaciviruses therefore could be provisionally classified as members of a new hepacivirus species (figure b ) [ ] . the other bamboo rat hepaciviruses in clade may also belong to this new virus species on the basis of the high sequence identity in this clade. similarly, although complete genome sequences were not determined for hepaciviruses in clade , they likely belong to species hepacivirus g due to their low amino acid p-distances ( . - . %) in the screening region to kj . the ' untranslated region sequences of these hepaciviruses contain two mir- seed sites (cacucc), which were located nucleotides from each other, as consistent with the suspected hepatotropism of the viruses. in contrast to host specificity of rodent hepaciviruses, the hev sequences ( nucleotides) from four rodent species were . - . % identical to each other and phylogenetically interspersed with each other and with reference sequences from other rat species (figure a) . the obtained complete genome of rat hev comprised of nucleotides excluding the poly a tail. its concatenated orf and orf differed by . % (table ) at amino acid level to the closest match (jx ) and these two sequences therefore belong to a same genotype in the species orthohepevirus c (figure b ), according to the latest proposal for classification of hepeviruses [ ] . amplicons derived from pcr screening experiments were all successfully sequenced and complete or near complete genome sequences were determined for representatives of the viruses by pcr or deep sequencing. after exclusion of primers, the obtained screening sequences from each targeted virus clustered in one or two clades of those with high identity and were most closely related to sequences previously reported from rodents or bats from the us [ , ] , china [ ] and vietnam [ ] . rodent hepacivirus sequences ( nucleotides) formed two well supported clades (figure a) . clade included all of sequences from rhizomys pruinosus which shared . - % pairwise nucleotide identity while three sequences (nucleotide identity of - %) from rattus losea and rattus argentiventer grouped in clade . these clades differed from each other by mean distances of . % and . % at nucleotide and amino acid levels, respectively. while the four vietnamese bamboo rat hepacivirus genomes were highly similar to each other (< % nucleotide and < % amino acid distances in the complete coding region (cds)), they were remarkably different from the closest sequence (kc ) with the corresponding distances of % and %, respectively ( table ). the amino acid p-distances of the obtained hepacivirus sequences and kc in the regions - and - (numbered relative to m ) were % and %, respectively. the newly identified hepaciviruses therefore could be provisionally classified as members of a new hepacivirus species (figure b ) [ ] . the other bamboo rat hepaciviruses in clade may also belong to this new virus species on the basis of the high sequence identity in this clade. similarly, although complete genome sequences were not determined for hepaciviruses in clade , they likely belong to species hepacivirus g due to their low amino acid p-distances ( . - . %) in the screening region to kj . the ' untranslated region sequences of these hepaciviruses contain two mir- seed sites (cacucc), which were located nucleotides from each other, as consistent with the suspected hepatotropism of the viruses. in contrast to host specificity of rodent hepaciviruses, the hev sequences ( nucleotides) from four rodent species were . - . % identical to each other and phylogenetically interspersed with each other and with reference sequences from other rat species (figure a) . the obtained complete genome of rat hev comprised of nucleotides excluding the poly a tail. its concatenated orf and orf differed by . % (table ) at amino acid level to the closest match (jx ) and these two sequences therefore belong to a same genotype in the species orthohepevirus c (figure b) , according to the latest proposal for classification of hepeviruses [ ] . the three hbv variants (from bat species hipposideros pomona and hipposideros larvatus) clustered with known bat hbv sequences (figure ). the two bat hbv complete genome sequences comprised and nucleotides. as with other hepadnaviruses, the bat hbv genomes have four open reading frames (orfs) encoding the surface (s), polymerase (p), core (c), and x proteins. a detailed comparison of the orfs of these viruses with their closest sequences is shown in table . bat consistently showed greatest sequence identity to ky in all orfs. in contrast, bat shared highest identity to ky in the p and s orfs, but was more similar to ky and ky in the orfs encoding for x and c, respectively. the two vietnamese pegivirus sequences from rattus tanezumi grouped with a sequence previously reported from rattus norvegicus (figure a ). the amino acid p-distance between the obtained rodent pegivirus sequence and kj in the region - (numbered relative to u ) was . % and the two viruses could be classified as members of species pegivirus j (figure b) , according in the update to the taxonomy of the pegivirus genus [ ] . the three hbv variants (from bat species hipposideros pomona and hipposideros larvatus) clustered with known bat hbv sequences (figure ) . the two bat hbv complete genome sequences comprised and nucleotides. as with other hepadnaviruses, the bat hbv genomes have four open reading frames (orfs) encoding the surface (s), polymerase (p), core (c), and x proteins. a detailed comparison of the orfs of these viruses with their closest sequences is shown in table . bat consistently showed greatest sequence identity to ky in all orfs. in contrast, bat shared highest identity to ky in the p and s orfs, but was more similar to ky and ky in the orfs encoding for x and c, respectively. the two vietnamese pegivirus sequences from rattus tanezumi grouped with a sequence previously reported from rattus norvegicus (figure a ). the amino acid p-distance between the obtained rodent pegivirus sequence and kj in the region - (numbered relative to u ) was . % and the two viruses could be classified as members of species pegivirus j (figure b) , according in the update to the taxonomy of the pegivirus genus [ ] . the present study reports the findings of pegivirus and human hepatitis-related viruses in vietnamese rodents and bats. the detection of hepacivirus, hev homologue and pegivirus in a number of rodent species, and hbv homologue in hipposideros larvatus indicates wider host ranges of these viruses. whereas, the identified rat hev and bat hbv showed relatively high sequence identity to previously characterized viruses infecting rattus rattus, rattus tanezumi and hipposideros pomona, the rodent pegivirus and hepacivirus showed substantial sequence distances to their closest sequences and represent new pegivirus variants and a new hepacivirus species. this highlights the incomplete genetic characterization of these viruses. thus far, only one complete genome and two complete coding sequences (including the one from this study) of rodent pegivirus are available on genbank. more highly divergent hepacivirus and pegivirus sequences are expected to be discovered from rodents in future studies. the absence of bat hev, hepacivirus, pegivirus, and low detection frequency of bat hbv are consistent with their low prevalence ( - %) reported in previous studies [ , , , ] . contrastingly, the prevalence of hepacivirus in the farmed bamboo rats was unprecedentedly high ( . %). the inbred nature of farmed bamboo rats that were investigated in this study may have contributed to susceptibility to infection and likelihood of persistence [ , ] . the existence of relatively homozygous individuals may play a key role in the maintenance of pathogens in a population [ ] . the high prevalence of hepacivirus in bamboo rats also indicates the need to reconsider transmission routes of hepaciviruses. among hepaciviruses, the transmission route of hcv has been relatively well studied, while those of other hepaciviruses are mostly speculative [ , ] . as a bloodborne virus, hcv is thought to be mainly transmitted through injections or blood transfusion. however, this does not explain how a range of divergent hcv strains have been maintained for centuries in some rural populations in central africa and southeast asia [ ] . equine hepacivirus has been shown to be transmittable via direct inoculation [ ] and via vertical transmission [ ] . rodent hepacivirus may utilize a similar transmission route as experimental intravenous injection of the supernatants of homogenised liver tissues from infected rats lead to viraemia [ ] . the high prevalence of hepacivirus in bamboo rats (in this study) and commensal rattus norvegicus ( . % in firth et al. [ ] ) indicates other more efficient transmission routes may exist such as via saliva the present study reports the findings of pegivirus and human hepatitis-related viruses in vietnamese rodents and bats. the detection of hepacivirus, hev homologue and pegivirus in a number of rodent species, and hbv homologue in hipposideros larvatus indicates wider host ranges of these viruses. whereas, the identified rat hev and bat hbv showed relatively high sequence identity to previously characterized viruses infecting rattus rattus, rattus tanezumi and hipposideros pomona, the rodent pegivirus and hepacivirus showed substantial sequence distances to their closest sequences and represent new pegivirus variants and a new hepacivirus species. this highlights the incomplete genetic characterization of these viruses. thus far, only one complete genome and two complete coding sequences (including the one from this study) of rodent pegivirus are available on genbank. more highly divergent hepacivirus and pegivirus sequences are expected to be discovered from rodents in future studies. the absence of bat hev, hepacivirus, pegivirus, and low detection frequency of bat hbv are consistent with their low prevalence ( - %) reported in previous studies [ , , , ] . contrastingly, the prevalence of hepacivirus in the farmed bamboo rats was unprecedentedly high ( . %). the inbred nature of farmed bamboo rats that were investigated in this study may have contributed to susceptibility to infection and likelihood of persistence [ , ] . the existence of relatively homozygous individuals may play a key role in the maintenance of pathogens in a population [ ] . the high prevalence of hepacivirus in bamboo rats also indicates the need to reconsider transmission routes of hepaciviruses. among hepaciviruses, the transmission route of hcv has been relatively well studied, while those of other hepaciviruses are mostly speculative [ , ] . as a bloodborne virus, hcv is thought to be mainly transmitted through injections or blood transfusion. however, this does not explain how a range of divergent hcv strains have been maintained for centuries in some rural populations in central africa and southeast asia [ ] . equine hepacivirus has been shown to be transmittable via direct inoculation [ ] and via vertical transmission [ ] . rodent hepacivirus may utilize a similar transmission route as experimental intravenous injection of the supernatants of homogenised liver tissues from infected rats lead to viraemia [ ] . the high prevalence of hepacivirus in bamboo rats (in this study) and commensal rattus norvegicus ( . % in firth et al. [ ] ) indicates other more efficient transmission routes may exist such as via saliva and bites, the zoonotic potential of the detected viruses is unknown and requires further investigation. while the identified rodent hepaciviruses appear host species specific, four rodent species were infected with highly similar hev homologues, which were phylogenetically interspersed, indicative of low host species specificity. this is a characteristic that may lead to their establishment and emergence in new hosts. understanding the receptor usage for cell entry of hev in rodents and other host species would potentially help predict the host range of the virus. furthermore, a surrogate assay with pseudotyped viruses carrying surface/envelope proteins of the identified viruses may be useful in assessing their potential to infect human liver cells. such an assay was used to show that a bat hbv could infect primary human hepatocytes [ ] . in summary, this study demonstrated the wide circulation of diverse pegivirus and human hepatitis-related viruses in new rodent and bat species. the presented findings form a framework for future investigations of human transmission risk, now that the rodent and bat species infected with these viruses have been identified and the human contact groups are better defined (e.g., bamboo rat farmers, rat catchers, rat sellers, and bat farmers). the transmission routes of the identified viruses are to be determined. the following are available online at http://www.mdpi.com/ - / / / / s . the global burden of viral hepatitis from to : findings from the global burden of disease study the global burden of hepatitis e virus genotypes and in hepatitis b: the virus and disease genetic organization and diversity of the hepatitis c virus hepatitis e virus: molecular virology, clinical features, diagnosis, transmission, epidemiology, and prevention bats carry pathogenic hepadnaviruses antigenically related to hepatitis b virus and capable of infecting human 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for novel hepaciviruses in rodents rodents and risk in the mekong delta of vietnam: seroprevalence of selected zoonotic viruses in rodents and humans. vector-borne zoonotic dis market study of meat from field rats in the mekong delta aciar monograph rodent-borne diseases and their risks for public health detection of potentially novel paramyxovirus and coronavirus viral rna in bats and rats in the mekong delta region of southern viet nam the vietnam initiative on zoonotic infections (vizions): a strategic approach to studying emerging zoonotic infectious diseases bartonella species and trombiculid mites of rats from the mekong delta of vietnam. vector borne zoonotic dis how important are rats as vectors of leptospirosis in the mekong delta of vietnam? vector borne zoonotic dis a guide to the mammals of southeast asia large-scale screening and characterization of enteroviruses and kobuviruses infecting pigs in vietnam sse: a nucleotide and amino acid sequence analysis platform proposed update to the taxonomy of the genera hepacivirus and pegivirus within the flaviviridae family mega : molecular evolutionary genetics analysis version . for bigger datasets consensus proposals for classification of the family hepeviridae parasite-mediated selection against inbred soay sheep in a free-living, island population disease susceptibility in california sea lions consanguinity and susceptibility to infectious diseases in humans hepacivirus cross-species transmission and the origins of the hepatitis c virus viraemic frequencies and seroprevalence of non-primate hepacivirus and equine pegiviruses in horses and other mammalian species the virus whose family expanded experimental transmission of equine hepacivirus in horses as a model for hepatitis c virus vertical transmission of hepatitis c virus-like non-primate hepacivirus in horses mouse models of acute and chronic hepacivirus infection key: cord- -wbb h zu authors: walker, gregory j.; stelzer‐braid, sacha; shorter, caroline; honeywill, claire; wynn, matthew; willenborg, christiana; barnes, phillipa; kang, janice; pierse, nevil; crane, julian; howden‐chapman, philippa; rawlinson, william d. title: viruses associated with acute respiratory infection in a community‐based cohort of healthy new zealand children date: - - journal: j med virol doi: . /jmv. sha: doc_id: cord_uid: wbb h zu acute respiratory infections (aris) are a major cause of morbidity among children. respiratory viruses are commonly detected in both symptomatic and asymptomatic periods. the rates of infection and community epidemiology of respiratory viruses in healthy children needs further definition to assist interpretation of molecular diagnostic assays in this population. children otherwise healthy aged to years were prospectively enrolled in the study during two consecutive winters, when aris peak in new zealand. parents completed a daily symptom diary for weeks, during which time they collected a nasal swab from the child for each clinical ari episode. a further nasal swab was collected by research staff during a clinic visit at the conclusion of the study. all samples were tested for respiratory viruses commonly causing ari using molecular multiplex polymerase chain reaction assays. there were aris identified from children completing the study, at a rate of . per child‐month. swabs collected during an ari were positive for a respiratory virus in . % ( of ), compared with . % ( of ) of swabs collected during asymptomatic periods. the most common viruses detected were human rhinovirus, coronavirus, parainfluenza viruses, influenzavirus, respiratory syncytial virus, and human metapneumovirus. all of these were significantly more likely to be detected during aris than asymptomatic periods. parent‐administered surveillance is a useful mechanism for understanding infectious disease in healthy children in the community. interpretation of molecular diagnostic assays for viruses must be informed by understanding of local rates of asymptomatic infection by such viruses. metapneumovirus (hmpv), human enterovirus (ev), and adenovirus (adv), are the most common viral aetiological agents associated with aris. although most respiratory viral infections are mild, they can potentially cause life-threatening illness, particularly in high-risk groups such neonates, the elderly, and those with chronic respiratory disease. advances in molecular diagnosis have made it possible to easily and quickly identify viruses in people with aris, with quantitative polymerase chain reaction (pcr) now considered by most to be the reference standard for detection of respiratory viruses over traditional methods such as viral culture. however, the clinical significance of virus detection is not always clear, as respiratory viruses are also detected in asymptomatic subjects. , further understanding of the role of viral detection in aris may improve clinical decision-making around infection control and therapy. direct comparisons between studies of respiratory virus epidemiology can be difficult for a number of reasons. climatic and social factors affect the survival and transmission of respiratory viruses, as well as host mobility and susceptibility. , this results in many viruses having characteristic seasonal patterns that vary by geographical location. reported epidemiology is also affected by study design. only one-fifth of aris result in a health care visit, were recruited for the study. this was made up of children from a previous study, while the remaining participants replied to advertising via staff email for the local district health boards, via posters and through referral from family and friends. there were of participants deemed ineligible for various reasons including that they slept in more than one bedroom, the bedroom was already heated, or they were going away for more than weeks during the study period. before commencement of the study of participants withdrew, while of participants commenced the program but were later excluded after failing to comply with study conditions. the remaining cohort for analysis was children, pcr, respectively, as previously described. sequences were compared to the ncbi nonredundant nucleotide (nr/nt) database using the basic local alignment search tool (blast) on the ncbi website (https://blast.ncbi.nlm.nih.gov/blast.cgi) and genotype assigned according to the closest identifying sequence. details of primers used in detection of respiratory viruses are included in the supplementary material (table s ). any period in which participants had a multi-day cumulative symptom score ≥ , with a single day symptom score ≥ was defined as an ari episode. the end of an ari episode was marked by three consecutive symptom-free days. a symptom score correction (daily baseline score of − ) was applied to participants who were considered to have background rhinitis. this included children who had (i) rhinitis symptoms (runny nose or sneezing) for ≥ of the study days, (ii) no more than days with no symptoms, or (iii) or more days of only rhinitis symptoms. in cases where multiple swabs were taken during a single ari episode, only the first swab taken was included in analysis. the exception to this was if a new virus was detected in subsequent swabs, or if swabs were taken > days apart. risk ratio (rr) was calculated as the proportion of samples positive for a virus with ari symptoms divided by the proportion of samples negative for a respiratory virus with ari symptoms. there were child-days of symptom observed and a total of nasal swabs collected from the participants. there were aris identified (table ) . nasal swabs were received from of of these episodes ( . %). in addition, swabs were taken when children were asymptomatic, most of which were performed during the clinical visit by researchers at the conclusion of the study. due to inclusion of children from a previous study of wheezing, those with asthma were highly represented within our cohort ( . %). overall, children with asthma displayed a small increase in the rate of aris per child-month ( . vs . ) compared with nonasthmatics (table s ), but this increase was not significant (p = . ). of the swabs taken during an ari, ( . %) were positive for a virus. during an ari, detection of respiratory viruses in nasal swabs was highest amongst the youngest age group ( year old), and decreased to age ( table ). during aris, multiple viruses were codetected in ( . %) swabs. the most commonly detected viruses in samples collected during ari were hrv ( . %), hcov ( . %), parainfluenza virus (pif) ( . %), ifv ( . %), rsv ( . %), and hmpv ( . %). these were all significantly more likely to be associated with ari episodes than asymptomatic detection (table ). adv was detected with similar frequency in both ari ( . %) and asymptomatic samples ( . %), while detection of hbov was relatively low in both groups. detection of any virus and codetection of viruses were both significantly associated with swabs collected during ari episodes. sequencing was performed on of a possible picornavirus positive samples ( . %). rhinovirus strains hrv-a and hrv-c were significantly associated with ari episodes (table ) . overall, detection of individual viruses was similar between asthmatic and nonasthmatic children in swabs taken during an ari (table s ) . hrv-b (p = . ) and rsv (p = . ) were significantly over represented in asthmatics during aris. aris were most frequent in may ( . ), which was also the month in which the least data was collected. this was followed by june ( . ) august ( . ), and july ( . ). the rate of aris steadily decreased following the winter season ( figure ). hrv was the predominant virus detected in each month, and was most prevalent in late-winter and spring (figure ). hcov and rsv had peaks during early winter, while adv peaked in spring. ifv and hmpv were most prevalent in july, but were also detected in other months. pif was detected consistently throughout study months, although not in may. there is a need for improved population data regarding respiratory viruses in children, particularly in the southern hemisphere. this is the first study assessing the epidemiology of an extended range of respiratory viruses in a community cohort of healthy children in new zealand. infection with respiratory viruses was common in both symptomatic and asymptomatic children, and this study demonstrated these relatively high frequencies were true of children with asthma ( . ) and those without asthma ( . ). the proportion of aris associated with a virus-positive swab in this population declined with age. children year of age were positive for a respiratory virus in . % of samples taken during an ari, compared to . % in participants aged greater than or equal to years age. a similarly designed household surveillance study showed that weekly detections of viruses in young (aged < ) and older (aged - ) children were significantly more frequent than in adults. in the current study swabs were taken at the onset of illness, when viral load reportedly peaks for some respiratory viruses. it is possible that this method is more likely to detect viruses associated with ari than other studies obtaining weekly swabs. sample sizes of each virus detected here were too small to compare individual viruses between age groups. however, a multi-country community surveillance study has previously shown that with the exception of influenza, prevalence of individual respiratory viruses was highest in younger children. the rates of ari per child-month were similar between age groups, suggesting that other factors such as development of allergy may play a more significant role than age in acute respiratory health. the study was conducted during the winter/spring seasons of consecutive years. consistent with previous reports in the southern hemisphere rsv peaked in this pediatric population in early winter. , interestingly, we found a similar pattern for detection of hcov. it has recently been reported in china that while overall hcov infection is most prevalent in winter, hcov subtypes display different epidemic patterns. as only a small number of hcovs were detected (n = ), we did not distinguish between subtypes when reporting our results. influenza was detected from june to october and peaked in july. a limitation was that no data were collected for the summer and autumn periods, capturing only the times of highest respiratory virus activity. this may account for the low detection of adv, hbov, and ev in comparison to other year-round epidemiological studies. a yearround analyses may also allow for investigation of phenomena such as the february back to school hrv peaks. our study differs from a previous nz-based analysis of viruses in hospitalized children with lrti, where relatively high rates of rsv ( %) and hmpv ( %) were detected. in our community-based study, incidence of these viruses was considerably lower. previous groups have reported that rsv and hmpv rarely occur in asymptomatic subjects, , , suggesting that detection of these viruses is almost always of clinical relevance. ifv may be present in people with few or no symptoms. however, the duration and copy number of viral shedding is considerably lower in these cases, and further research is needed to understand the contribution of asymptomatic detection to community transmission. in the present study, we found these viruses in very few samples of asymptomatic subjects, and demonstrated these three viruses (and others) to be significantly associated with aris. respiratory viruses were detected in a large proportion of swabs taken during asymptomatic periods ( . %), with the vast majority of these detections caused by hrv. this is unsurprising as hrvs were the most frequently detected virus overall, and have been commonly detected in asymptomatic children before. it has previously been unclear whether subtypes of hrv have varying clinical impact on their hosts, with studies of healthy, and asthmatic children reporting no differences in symptom risk between hrv subtypes. our data, along with another recent study suggest that hrv-a and hrv-c subtypes are significantly more likely to cause symptoms related to ari. in our study hrv-b, adv, and hbov were detected similarly in samples from both ari episodes and asymptomatic periods. while their corresponding risk ratios are not considered significant, the number of detections of these viruses is relatively small, and a larger analysis would be required to rule out the clinical significance of detecting hrv-b, adv and hbov in aris. the effect of viral coinfection on respiratory disease severity in children has not been well established. a number of studies have found increased hospitalizations, longer length of stay in hospital, and higher mortality when two or more respiratory viruses were detected. 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- - key: cord- -yq k authors: schubert, gena; haverland, josh; wyst, craig vander; mcgreevy, jon title: how much drool is too much?() date: - - journal: clin pediatr emerg med doi: . /j.cpem. . sha: doc_id: cord_uid: yq k a -year-old boy presented to the emergency department with a chief complaint of “lethargy” and was found to have ptosis with eventual respiratory failure and need for emergent intubation. there is a broad differential for a patient with respiratory failure, and careful physical examination and history are imperative to reduce morbidity and prevent mortality. after further evaluation and workup, the diagnosis is ultimately revealed. -year-old boy with a history of allergic rhinitis and persistent asthma presented to a pediatric emergency department (ed) in arizona with parental concern for congestion and drooping red eyes at : pm. his father noted that earlier in the day, his speech was more difficult to understand but returned to normal after a nap. vital signs on arrival to the ed were temperature . °c, heart rate beats per minute, respiratory rate breaths per minute, and pulse oximetry of % on room air. on physical examination, there were bilateral conjunctival erythema and swollen nasal turbinates, and the patient was noted to be rubbing his eyes. he was breathing comfortably on room air with clear breath sounds, and his heart had a regular rate and rhythm. cranial nerves - were intact, and he ambulated appropriately for age and followed commands. there were no focal neurologic deficits on examination. the patient was continued on home cetirizine, started on tetrahydrozoline eye drops, and discharged home with the diagnosis of allergic rhinitis and viral conjunctivitis. the patient returned to the same ed with his father the next day at around : am for a right forehead laceration after hitting his head on the edge of the bed. the injury occurred while in the care of his grandparents. there was no report of vomiting or loss of consciousness. both the father and the provider felt the patient's behavior was at neurologic baseline for a -year-old at the time of arrival to the ed. similar to his previous visit, he was afebrile to . °c with a heart rate of beats per minute, respiratory rate of breaths per minute, and pulse oximetry of % on room air. the physical examination noted a -cm linear forehead laceration over frontal bone with surrounding hematoma. there was no mention of any other abnormal physical examination findings including eye findings. he met low-risk criteria according to the pediatric emergency care applied research network (pecarn) head trauma algorithm and a -year-old boy presented to the emergency department with a chief complaint of "lethargy" and was found to have ptosis with eventual respiratory failure and need for emergent intubation. there is a broad differential for a patient with respiratory failure, and careful physical examination and history are imperative to reduce morbidity and prevent mortality. after further evaluation and workup, the diagnosis is ultimately revealed. myasthenia gravis; respiratory distress; ptosis; myasthenic crisis thus did not receive imaging or observation. his laceration was repaired with skin adhesive, and he was discharged home with his father. the patient returned hours later to the same ed for his third visit within hours. this time, he presented with his mother and with a chief complaint of lethargy. the patient lives in a split household and had been in the care of the mother since : pm. over the last hours, she had noted new-onset drooling, garbled speech which was difficult to understand, difficulty swallowing water, and increased fatigue. she also felt his eyes appeared more erythematous and watery with droopy eyelids. she denied a history of any of the above symptoms prior to this visit. since the patient had been in her care, he had episodes of urinary incontinence despite previously being fully potty-trained. the mother denied any recent illness including cough, fever, ear pain, diarrhea, or vomiting. the father arrived and recalled the child having increased secretions under his care as well. the patient pointed to back of his throat when asked if he was in pain, which the father thought may be related to molar teeth eruption. the initial vital signs on his third ed presentation were as follows: temperature . °c, heart rate beats per minute, respiratory rate breaths per minute, pulse oximetry %, and blood pressure of / mm hg. on examination, the patient was awake and calm in his mother's arms with no acute distress. he appeared appropriate for a -year-old and was interactive and did not appear altered. the provider was able to understand some of his speech, although some speech was garbled, making it difficult to understand fully. his head was atraumatic other than the previously repaired laceration. his pupils were of normal size, equal, round, and reactive to light bilaterally without significant conjunctival injection. he had ptosis bilaterally. the tympanic membranes were pearly. there was no rhinorrhea, but he did have drooling and clear oral secretions. no oropharyngeal erythema, exudates, palatal petechiae, uvular deviation, or tonsillar hypertrophy or asymmetry was appreciated. there was normal work of breathing and clear lungs sounds bilaterally. when agitated, he had intermittent stridor, but at rest, he did not exhibit stridor, tracheal tugging, accessory muscle use, wheezes, or crackles. he had tachycardia but no murmurs, rubs, or gallops. there was no lymphadenopathy. his abdomen was soft, nontender, nondistended, and without hepatosplenomegaly. neurologically, the patient had bilateral ptosis but no other gross cranial nerve deficits. he had / strength bilaterally in his upper and lower extremities. gross sensation was intact. his patellar reflexes were + bilaterally, and providers were unable to reliably elicit other reflexes. anal sphincter tone was normal. he was able to ambulate without difficulty and had no ataxia. because of the history of head injury earlier in the day with new-onset neurologic complaints, a computerized tomography (ct) study of the head without contrast was ordered in addition to soft tissue neck radiographs due to the increased drooling and stridor. a respiratory pathogen polymerase chain reaction panel (rpp) nasopharyngeal swab; complete blood count; complete metabolic panel; urine drug screen; and ingestion laboratory tests including aspirin, acetaminophen, and alcohol levels were sent. an electrocardiogram (ecg), as part of the toxicological workup, was also performed. the result of the ct head was normal. the neck radiographs showed "mild narrowing of the subglottic airway with loss of normal shouldering, suggesting croup" (figure ). complete blood count and complete metabolic panel were unremarkable, and aspirin, acetaminophen, and alcohol levels were negative. rpp was positive for coronavirus. ecg result was likewise normal. approximately hours after initial evaluation, the providers were called back into the room by his nurse who had noticed labored breathing, tachycardia to beats per minute, and a decreased respiratory rate to , and he was now on . l oxygen flow via nasal cannula to maintain oxygen saturations above %. the patient was less active and alert than his previous examination and had more copious clear oral secretions, labored pattern of breathing, diffuse crackles throughout bilateral lung fields, and inspiratory stridor. he was tachycardic but without any murmurs, rubs, or gallops and no appreciable hepatomegaly. other than being less active, there were no other acute changes in his neurologic examination. the decision was made to intubate the patient for airway protection, and he underwent rapid sequence intubation with fentanyl, midazolam, and rocuronium. he was easily intubated on the first attempt with a . fr microcuffed endotracheal tube (ett), which was downsized intentionally because of concern for increasing stridor and potentially narrowed airway. no caustic burns or other airway abnormalities were appreciated during direct laryngoscopic visualization. the patient oxygenated and ventilated without difficulty following intubation, although when the ett cuff was deflated for repositioning of the ett following radiograph, copious amounts of clear secretions from the patient's mouth were noted. the rapid urine drug screen was negative, and the patient was admitted to the pediatric intensive care unit for further care. the differential for a patient presenting to the ed with acute respiratory distress is broad and includes infection, ingestion, trauma, envenomation, muscular disorders, and autoimmune etiologies. scorpion envenomations along with other insect bites are common in arizona and can be a cause of drooling and increased secretions. this was at the top of the differential upon evaluation and the need for emergent intubation. scorpion envenomation often presents with respiratory distress along with increased drooling, abnormal eye movements, muscle twitching, and agitation. the patient did have respiratory distress, drooling, and ptosis of his eyes, but no abnormal eye movements were appreciated. additionally, the patient initially presented without respiratory distress and progressed, which is not characteristic of envenomation. grading and treatment of scorpion envenomation (table ) are based on location of pain, location of paresthesia, cranial nerve involvement (oral secretions, blurry vision, tongue fasciculations, nystagmus), and skeletal neuromuscular dysfunction (abnormal movements or flailing of the extremities). additionally, with stridor and drooling, infectious etiologies were on the top of the differential. most common bacterial etiologies include streptococcus species and staphylococcus aureus. there are many viral etiologies as well, including rhinovirus, adenovirus, enterovirus, coronavirus, and influenza. the patient was coronavirus positive on rpp. additionally, the patient had stridor along with an radiograph concerning for subglottic narrowing, most consistent with croup. croup, also referred to as laryngotracheitis, is an upper respiratory illness caused by parainfluenza or . the other etiologies for croup include influenza a and b, measles, adenovirus, and respiratory syncytial virus. croup is diagnosed based on signs and symptoms including stridor, barking cough, or hoarseness. a neck radiograph will show the traditional narrowing of the trachea known as a steeple sign. however, a radiograph is not routinely performed for diagnosis because croup is usually a clinical diagnosis. although the patient had coronavirus and concern for croup, this did not explain his ptosis and other neurologic findings. ingestion along with trauma is often also at the top of the differential as it typically presents acutely, and prompt diagnosis and treatment are critical. in additionally, poisoning with selenium and mercury can present similarly. treatment includes administration of atropine along with pralidoxime. drugs that can cause hypersalivation include morphine, pilocarpine, methacholine, haloperidol, and clozapine. cocaine and phencyclidine can also cause increased secretions. there was no history to suggest ingestion in the patient. muscular disorders can also cause respiratory distress and failure. muscular dystrophy, polymyositis, and myasthenia gravis should be on the differential. the most common muscular dystrophies in children are duchene and becker. both are x-linked recessive and caused by a mutation in the dystrophin gene which clinically presents with progressive weakness. weakness often affects proximal limb muscles before distal, and lower extremities before upper extremities. the classic finding is gowers sign, where a child uses their hands and arms to walk up their body to get to an upright position from sitting because of the lack of hip and thigh muscle strength. physical examination findings include pseudohypertrophy of the calf, lumbar lordosis, waddling gait, shortening achilles tendon, hypotonia, and hyporeflexia. the patient did not have any secondary symptoms of the above, and the patient had a normal gait. polymyositis, on the other hand, is a rare autoimmune myopathy with direct t-cell invasion of the muscle fibers. the hallmark of this disease is weakness, primarily proximal muscle weakness. lastly, myasthenia gravis is an antibody-mediated autoimmune disease that affects postsynaptic neuromuscular junction. usually, symptoms are gradual, but patients can present in myasthenic crisis leading to respiratory failure. most common symptoms include ptosis and diplopia which are worse with activity and improved with rest. myasthenic crisis is a life-threatening condition defined by worsening weakness and requires noninvasive ventilation or intubation. the patient arrived to the pediatric intensive care unit intubated on a ventilator with a rate of breaths per minute, tidal volume of ml ( ml/kg), peep of , pressure support of , and fio of %. he was sedated but had clear and equal breath sounds bilaterally. his heart had a regular rate and rhythm, and he had good perfusion throughout. no abnormal physical examination findings were found. his orogastric tube was set to low intermittent suction, and he remained on a dexmedetomidine drip at . μg/kg/h and ampicillin/sulbactam mg/kg intravenously every hours pending culture results. a ct of the neck was obtained to rule out other etiologies for respiratory distress, which was negative for peritonsillar or retropharyngeal abscess. an extubation trial was done on day of admission. because of inspiratory stridor and increased work of breathing, he was given inhaled dexamethasone and racemic epinephrine with minimal improvement. he continued with poor pharyngeal tone, drooling, dysarthria, and hypoxia with oxygen saturations %, and he was reintubated for airway protection. during extubation, the patient was moving all his extremities, but there was concern for hypotonia. the patient was on sedating medications, which were a confounding factor. neurology was consulted at that time because of concern for a neuromuscular process. their recommendations included to obtain negative inspiratory force studies, magnetic resonance imaging of the thoracic and lumber spine with gadolinium contrast, acetylcholine receptor antibody, ganglioside (gm , gd b, gq b) antibodies, lumbar puncture with standard indices plus cerebrospinal fluid (csf) igg, oligoclonal bands, igg/albumin ratio, igg index, and electromyography. csf studies were normal with wbc , rbc , protein , and glucose . csf west nile ab igg and igm were negative. csf oligoclonal bands were negative, and the patient had a normal igg to albumin ratio. a comprehensive drug screen was negative. negative inspiratory force was reported at − . electromyography with -hz repetitive stimulation was abnormal, which showed a greater than % decrement for the left deltoid/axillary, left tibialis anterior/peroneal, and left adduction digiti minimi/ulnar. ganglioside (gm , gd b, gq b) igg and igm antibodies were negative. initial acetylcholine binding and block antibodies were negative, but positive for acetylcholine receptor muscle binding ( . nmol/l, normal b . ), negative striated muscle ab titer, and achr muscle modeling ab %, therefore % loss of ach receptor. given these results, the diagnosis of myasthenia gravis was made. after diagnosis, the patient was treated with ivig and prednisone for days with improvement of symptoms. he was successfully extubated on day of hospitalization after treatment and remained stable throughout his -day hospitalization. he did not require plasma exchange. he was placed on pyridostigmine along with tapering doses of steroids prior to discharge. after discharge, the patient was followed by neurology, physical therapy, occupational therapy, general pediatrics, and immunology. he regained his baseline strength, his ptosis resolved, and he returned to his normal behavior and activities. he was tapered off steroids in the year after discharge from the hospital and remained on pyridostigmine. myasthenia gravis is the most common disorder of neuromuscular transmission. the hallmark of the disorder is fluctuating degrees and variable combination of weakness in ocular, bulbar, limb, and respiratory muscles. the weakness is from the antibody-mediated t-cell response to acetylcholine receptors or receptor-associated proteins at the postsynaptic neuromuscular junction. the incidence of myasthenia gravis is about per in the united states, which includes children and adults, and a prevalence of roughly patients affected. it is estimated that the risk of myasthenic crisis in myasthenia gravis patients may be as high as to %, and to % of myasthenia gravis presents with myasthenic crisis as the first manifestation. , precipitating factors include infection, thymectomy, pregnancy, childbirth, and tapering of immunosuppressive medications. medications most often associated are antibiotics (aminoglycosides, fluoroquinolones, erythromycin, azithromycin), cardiac medications (β-blockers, procainamide, quinidine), and magnesium. in the case of our patient, no specific cause or precipitating factor was found except for potentially an upper respiratory illness with coronavirus and signs and symptoms of croup. the diagnosis of myasthenia gravis is complex. bedside tests including the ice pack test and edrophonium test are sensitive but have major limitations with false-positive results. thus, a confirmatory test with antibodies and electrophysiological studies (repetitive nerve stimulation studies and single-fiber electromyography) are needed. an immunologic assay to detect the presence of circulating acetylcholine receptor antibodies is the first step in the laboratory confirmation of myasthenia. on the other hand, muscle specific kinase antibodies and receptor tyrosine kinases are found in less than % of generalized myasthenia gravis without acetylcholinesterase receptor antibodies but are rare in ocular myasthenia gravis. , the treatment for myasthenia gravis begins with respiratory support including intubation or noninvasive ventilation. once disabling and often fatal, myasthenia gravis can be managed effectively with therapies that include anticholinesterase agents, rapid immunomodulatory therapies, chronic immunosuppressive agents, and thymectomy. treatment varies based on the age of the patient, severity of disease, respiratory involvement, bulbar involvement, and progression of the disease. goals of treatment are to minimize adverse effects of the drug while having improvement in symptoms and return to baseline function. symptomatic treatment with acetylcholinesterase inhibition increases the amount of acetylcholine at the neuromuscular junction, thereby improving symptoms. pyridostigmine is often the treatment of choice and sometimes is used as monotherapy. with generalized myasthenia gravis, steroids and nonsteroidal immunosuppressive agents are used to target immune dysregulation. likewise, plasma exchange or ivig is an immunomodulating treatment used for bridge therapy or during acute exacerbations of myasthenia gravis. approximately % of patients with myasthenia gravis are found to have a thymoma. surgery with complete resection is indicated for these patients when possible. myasthenic crisis is an uncommon yet important diagnosis to keep on the differential of a patient with respiratory distress, especially in a patient with ptosis and sialorrhea. it is also imperative to take a closer look at patients who present for multiple visits to the ed in a short time period even if they appear to be unrelated or benign. our patient presented times to the ed in a -hour period. he was diagnosed with allergic rhinitis and a facial laceration in his first visits. individually, these were not concerning, but retrospectively, they were red herrings. when assessing a child with respiratory distress requiring intubation, the differential should remain broad, but the physical examination and history are paramount for diagnosis and differentiating infectious vs other etiologies. early recognition and consultation with specialists can help with diagnosis, treatment, and management. goldfrank's toxicologic emergencies clinical courses of croup caused by influenza and parainfluenza viruses the usefulness of lateral neck roentgenograms in laryngotracheobronchitis annual report of the american association of poison control centers' national poison data system (npds): th annual report diagnosis and management of duchenne muscular dystrophy, part : diagnosis, and neuromuscular, rehabilitation, endocrine, and gastrointestinal and nutritional management monoclonal antibody analysis of mononuclear cells in myopathies. i: quantitation of subsets according to diagnosis and sites of accumulation and demonstration and counts of muscle fibers invaded by t cells myasthenia gravis on the concept of myasthenic crisis myasthenic crisis the management and outcome of patients with myasthenia gravis treated acutely in a neurological intensive care unit medications and myasthenia gravis (a reference for health care professionals) detection and characterization of musk antibodies in seronegative myasthenia gravis anti-musk myasthenia gravis presenting with purely ocular findings key: cord- - nfukrfl authors: al-ahmadi, khalid; alahmadi, sabah; al-zahrani, ali title: spatiotemporal clustering of middle east respiratory syndrome coronavirus (mers-cov) incidence in saudi arabia, – date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: nfukrfl middle east respiratory syndrome coronavirus (mers-cov) is a great public health concern globally. although % of the globally confirmed cases have emerged in saudi arabia, the spatiotemporal clustering of mers-cov incidence has not been investigated. this study analysed the spatiotemporal patterns and clusters of laboratory-confirmed mers-cov cases reported in saudi arabia between june and march . temporal, seasonal, spatial and spatiotemporal cluster analyses were performed using kulldorff’s spatial scan statistics to determine the time period and geographical areas with the highest mers-cov infection risk. a strongly significant temporal cluster for mers-cov infection risk was identified between april and may , . most mers-cov infections occurred during the spring season ( . %), with april and may showing significant seasonal clusters. wadi addawasir showed a high-risk spatial cluster for mers-cov infection. the most likely high-risk mers-cov annual spatiotemporal clusters were identified for a group of cities (n = ) in riyadh province between and . a monthly spatiotemporal cluster included jeddah, makkah and taif cities, with the most likely high-risk mers-cov infection cluster occurring between april and may . significant spatiotemporal clusters of mers-cov incidence were identified in saudi arabia. the findings are relevant to control the spread of the disease. this study provides preliminary risk assessments for the further investigation of the environmental risk factors associated with mers-cov clusters. middle east respiratory syndrome coronavirus (mers-cov) is an emerging human viral respiratory infectious disease caused by a novel coronavirus. it was first reported in saudi arabia in [ ] , and since then, it has spread to several other countries, resulting in global public health implications. from april through february , a total of laboratory-confirmed mers-cov cases (with deaths, . %) were reported to the world health organization (who) by countries, with the majority ( cases, . %) being reported by saudi arabia (with deaths, . %) [ ] . the risk assessment of mers-cov infection, transmission and severity is crucial in predicting and preventing further outbreaks of human infections and in enhancing control measures. recent studies have advised that dromedary camels (camelus dromedarius) serve as a reservoir host for mers-cov, and camel-to-human transmission can occur through sporadic zoonotic infections associated with exposure to infected dromedary camels and their products [ ] [ ] [ ] . the risk factors were identified for primary mers-cov infection in persons with either direct or indirect exposure to camels. in particular, higher heterogeneity was more prominent in zoonotic than in human-to-human transmission in the middle east region; this result emphasizes the importance of the environmental component of the epidemic [ ] . although approximately % of the globally reported mers-cov cases are found in saudi arabia, spatial patterns and clusters of the occurrence of this disease have not been addressed, leaving a wide gap in knowledge on this important issue. this study aimed to examine the spatiotemporal clustering of the mers-cov incidence in saudi arabia between and using spatial scan statistics and gis. all laboratory-confirmed mers-cov cases reported between june , and march , were compiled from the official websites of the saudi ministry of health (smoh) [ ] and the who [ ] . we undertook a detailed review of the mers-cov data and performed a range of checks for data consistency, completeness and fitness for the study purpose. we then developed a dataset with variables of interest for each individual with mers-cov. the dataset included the following: diagnosis date, gender, age, nationality, healthcare, employment status and source of infection, as well as the city, governorate and province of residence. a confirmed case is defined as a suspected case that has a laboratory confirmation of mers-cov infection. a suspected case is defined as either (i) an adult patient presenting with severe pneumonia or acute respiratory distress syndrome, based on clinical or radiological evidence, or (ii) an adult patient presenting with an unexplained deterioration of a chronic condition, such as congestive heart failure or chronic kidney disease being treated with hemodialysis, or (iii) a child or an adult patient exposed to a confirmed case of mers-cov or who has visited a healthcare facility where a mers-cov patient was recently identified, or has had a history of contact with dromedary camels or consumption of camel products within days before symptoms and who presents with either (a) acute febrile illness (temperature ≥ • c) with or without respiratory symptoms, or (b) gastrointestinal symptoms and leukopenia or thrombocytopenia. laboratory testing for mers-cov is performed at approved regional smoh and selected non-smoh governmental laboratories to confirm a clinically suspected case and to screen contacts by using validated, commercial, real-time, reverse-transcription polymerase chain reaction (rrt-pcr) assays. the laboratory confirmation of mers-cov infection requires either a positive rrt-pcr result for at least two specific genomic targets, or a region upstream and open reading frame a (upe and orf a) [ ] . a primary case is defined as a person with a laboratory-confirmed mers-cov infection with no evidence of contact with infected individuals but is known or believed to have had direct or indirect exposure to camels or camel habitats. exposure to camels includes direct physical contact with camels or their surroundings (milking and handling excreta), drinking raw camel milk or other unpasteurized products derived from camel milk and handling raw camel meat. indirect contact includes casual contact with sites where camels have been (e.g., camel markets or farms) but without direct physical contact with camels, or living with a household member who has had direct contact with camels. by contrast, a secondary case is defined as a person who has shared the same enclosed space (e.g., a room or office) for frequent or extended periods with an individual with a symptomatic mers-cov infection. mers-cov is believed to spread between humans mainly through contact and respiratory droplets. however, transmission through small particle droplet nuclei (aerosols) may occur. environmental contamination during outbreaks in healthcare facilities can be extensive and might contribute to outbreak amplification, if adequate disinfection procedures are not followed [ ] . the spatial database of the mers-cov incidence in saudi arabia was created in the format of an esri file geodatabase on the three spatial levels of city, governorate and province. saudi arabia consists of administrative provinces, governorates and more than cities. mers-cov cases were grouped and aggregated to be represented by cities, governorates and provinces. the prevalence of intrinsic variance instability in estimating incidence rates as a result of the variation in populations across spatial units, which can possibly identify outliers, has received broad attention in the disease mapping field [ ] . to address this issue, we used geoda [ ] software for generating eb smoothed rate maps for mers-cov incidence. the number of mers-cov incidence cases for the governorates was used as an event variable, and the populations of governorates were estimated from the census [ ] and used as base variables. we analyzed the spatiotemporal clustering of the mers-cov incidence in saudi arabia between and at the city level by using kulldorff's spatial scan statistics via satscan . [ ] . we used purely temporal, seasonal, purely spatial and spatiotemporal retrospective analyses to scan, detect and evaluate the periods and geographical areas with the highest mers-cov risk incidence clusters. the purely spatial scan statistic is defined by a circular window on the map. the window is sequentially centered on each of several possible cities that are positioned throughout the study area. the spatiotemporal scan statistic imposes a cylindrical window with a circular geographic base and height corresponding to time. the temporal scan statistic uses a window that moves in one dimension, time, defined in the same way as the height of the cylinder used by the spatiotemporal scan statistic. the key feature that distinguishes the seasonal scan statistic from the purely temporal scan statistic is that the former ignores the year of the observation and retains only the day and month [ ] . the number of mers-cov cases by city was used as the case file, the city population estimated from the census [ ] was used as the population file and the latitude and longitude of each city were used in the coordinates file. in satscan, an analysis was conducted by progressively scanning a window across time and/or space through a comparison of the number of observed and expected cases of mers-cov incidence, assuming random distribution, inside the window at each city. the null hypothesis is that the incidence of mers-cov is randomly distributed, and the alternative hypothesis is that the incidence increases more inside the window than in areas outside it. the log likelihood ratio (llr) is the hypothesis-testing statistic estimated based on monte carlo randomization. the window with the maximum likelihood ratio is the most likely cluster; that is, it identifies the cluster that is least likely to occur by chance. in addition to the most likely cluster, satscan also designates secondary clusters for purely spatial and spatiotemporal analyses and ranks them in relation to their estimated llr statistic. satscan scans for clusters by using different criteria; the criterion recommended by satscan is the percentage of the population at risk, with a value of % [ ] . we tested the percentage of the population at risk from % to %, and from the result, % performed best; that is, the value of % did not include neighboring cities that have a non-elevated risk. the four types of analyses (purely temporal, seasonal, purely spatial and spatiotemporal) were conducted using the poisson discrete-based model with monte carlo permutations to test for statistical significance. only clusters with significance levels of . and only scans of cities with high rates were reported. for temporal analysis, values of day, month and year were set as the time aggregation units for the daily, monthly and annual clusters, respectively, whereas for the seasonal cluster, month was set. for spatiotemporal analysis, month and year were set for the monthly and annual spatiotemporal analyses, respectively. a total of laboratory-confirmed human mers-cov cases reported in saudi arabia during the period between june , and march , were included in this study. the primary cases accounted for . % (n = ) of the total number of confirmed cases; of these, . % (n = ) involved (direct and indirect) exposure to camels. secondary cases accounted for . % (n = ) of the total number of confirmed cases, whereas missing and unknown cases accounted for . % (n = ) and . % (n = ) of the total number of confirmed cases, respectively. on the incidence of mers-cov infection was mostly reported from riyadh (n = , . %), jeddah (n = , . %) and alahsa governorates (n = , . %), figure . the incidence in wadi addawasir, buraydah, taif, alkharj, najran, madinah and makkah governorates ranged from to cases. these were followed by five governorates in northern saudi arabia (hafr albatin, sakaka, hail, dumat aljundal and tabuk) and two governorates in eastern saudi arabia (alkhubar and dammam) with - cases. for the eb smoothed incidence rate of mers-cov infection, the wadi addawasir governorate showed the highest rate across the country, with . cases per , people ( figure ). dumat aljundal and najran governorates followed with . and . cases per , people, respectively. alkharj, alhinakiyah and afif governorates exhibited an incidence rate in the range of . - . cases per , people. in riyadh, the capital of saudi arabia, the the incidence of mers-cov infection was mostly reported from riyadh (n = , . %), jeddah (n = , . %) and alahsa governorates (n = , . %), figure . the incidence in wadi addawasir, buraydah, taif, alkharj, najran, madinah and makkah governorates ranged from to cases. these were followed by five governorates in northern saudi arabia (hafr albatin, sakaka, hail, dumat aljundal and tabuk) and two governorates in eastern saudi arabia (alkhubar and dammam) with - cases. for the eb smoothed incidence rate of mers-cov infection, the wadi addawasir governorate showed the highest rate across the country, with . cases per , people ( figure ). dumat aljundal and najran governorates followed with . and . cases per , people, respectively. alkharj, alhinakiyah and afif governorates exhibited an incidence rate in the range of . - . cases per , people. in riyadh, the capital of saudi arabia, the incidence rate of mers-cov infection was . cases per , people. in buraydah, alahsa and jeddah governorates, the incidence rates were . , . and . cases per , people, respectively. temporal cluster analysis generated from the spatial scan test identified the years , and , the months of april and may of , and the period from april to may , as the strongly significant clusters of annual, monthly and daily mers-cov incidence, respectively (table ) . seasonal cluster analysis revealed that april and may show strongly significant seasonal clusters of mers-cov incidence (table ) . temporal cluster analysis generated from the spatial scan test identified the years , and , the months of april and may of , and the period from april to may , as the strongly significant clusters of annual, monthly and daily mers-cov incidence, respectively (table ) . seasonal cluster analysis revealed that april and may show strongly significant seasonal clusters of mers-cov incidence (table ) . the results of the purely spatial cluster analysis of mers-cov incidence from to revealed the most significant and secondary clusters at the city level (table and figure ). wadi addawasir in riyadh province had the most likely high-risk cluster, followed by a secondary significant cluster in alkharj and aldilm cities in the same province. spatial clusters in single cities were identified across the country; alhofuf (east), dumat aljundal (north), najran (south), alqunfidhah (southwest), alhinakiyah (west) and buraydah (center) represented the third, fourth, fifth, sixth, seventh and eighth secondary clusters, respectively. the results of the spatiotemporal cluster analysis of mers-cov infection, using years and months as the time aggregates from to , showed significant most likely and secondary clusters in saudi arabia (table ; table and figure ; figure ). spatial variations existed between the annual and monthly spatiotemporal clusters. for the annual spatiotemporal clusters, a group of cities (n = ) located in riyadh province was identified as the most likely high-risk cluster for mers-cov incidence between and . this was followed by a secondary cluster that was found for three cities (jeddah, makkah and taif) in makkah province between and . spatiotemporal clusters in single cities were also observed and varied in space and time across the country. wadi the results of the spatiotemporal cluster analysis of mers-cov infection, using years and months as the time aggregates from to , showed significant most likely and secondary clusters in saudi arabia (table ; table and figure ; figure ). spatial variations existed between the annual and monthly spatiotemporal clusters. for the annual spatiotemporal clusters, a group of cities (n = ) located in riyadh province was identified as the most likely high-risk cluster for mers-cov incidence between and . this was followed by a secondary cluster that was found for three cities in this study, we examined the spatial pattern of mers-cov risk at the governorate level and the temporal, seasonal, spatial and spatiotemporal clustering of mers-cov incidence at the city level over a seven-year period. to the authors' knowledge, this is the first study that aims to analyze the spatiotemporal pattern and clustering of mers-cov in saudi arabia. a total of laboratory-confirmed mers-cov cases were reported in saudi arabia, representing approximately % of the global cases. overall, the majority of mers-cov cases were secondary infections ( . %). this result indicates that secondary infections, either hospital or community acquired, remain a major challenge for the saudi healthcare system in the prevention and control of mers-cov outbreaks, despite the significant improvement in mers-cov surveillance. on the other hand, the primary cases accounted for only . % of the total confirmed cases. although compared with the general population, people in close contact with dromedary camels have a higher risk of developing mers-cov infection via a primary source [ ] [ ] [ ] , our results indicated that only . % of the total primarily infected cases were associated with direct or indirect contact with camels. this result is consistent with previous findings [ ] regarding the ambiguity of primary mers-cov infection transmission. in saudi arabia, camel milk and meat production has increased by . % and . % per year, respectively [ ] . however, intensified animal production has epidemiological consequences, including increased risk of disease. recent trends in saudi arabia have indicated a tendency towards dromedary camel husbandry intensification since the s, as evident in increased production in nearby cities by providing enhanced supplemental diets for the animals and improving camel health management [ , , ] . these areas of intensified camel production are probable hotspots for the transmission and spread of mers-cov. in addition, dealing with camel products, consuming raw unpasteurized milk and conducting slaughter processes have been documented as risk factors for primary mers-cov transmission to humans [ ] . the epidemic curve of mers-cov has varied significantly each year from to , and it has exhibited variance in monthly peaks. a combination of sporadic and epidemic patterns as a result of animal-to-human, human-to-human and unknown exposure was observed. by contrast, the epidemic curve in south korea, where the largest outbreak outside of the arabian peninsula occurred, had a clear nosocomial epidemiological pattern [ ] . purely temporal cluster analyses of mers-cov infection illustrated significant clusters in april and may of . this finding is consistent with previous results [ ] , which showed significant peaks in mers-cov incidence between march and may during a similar period. seasonal cluster analysis identified april and may as a strongly significant seasonal cluster of mers-cov infection. in accordance with our findings, it was reported in [ ] that mers-cov infection occurred markedly in june, followed by may and april, and the lowest rates were seen in january. one possible reason for this trend is the seasonal variations in zoonotic infections in camels during the breeding season [ , ] , when camel farms are considered an important potential source of mers-cov transmission [ ] . moreover, a recent serological study in saudi arabia found a higher risk of mers-cov infection in camels in winter than in summer [ ] . however, the low daily frequency and sporadic cases of mers-cov indicate a reduced likelihood of zoonotic-to-human transmission and increased possibility of human-to-human transmission, which is consistent with other findings [ ] . the main mers-cov outbreaks in and were closely followed by human influenza a epidemics [ ] . no coincidence was found between the peak of influenza occurrence and mers-cov occurrence, which suggests that the seasonal characteristics of mers-cov infections may vary from those of human influenza viral infections. in this study, no epidemics of mers-cov were observed during mass gatherings of pilgrims in the hajj season, which is consistent with previous findings [ ] . this indicates another knowledge gap regarding the mode of transmission that needs further investigation. spatial clusters of mers-cov cases were mainly found in a group of cities in the provinces of riyadh, qassim, hail and najran located on the outskirts of larger deserts, which is the natural habitat for camels. however, significant spatial clusters of mers-cov cases were also reported from small general hospitals in single cities, such as wadi addawasir, dumat aljundal, alhinakiyah and alqunfidhah; in these settings, delays in the isolation of suspected patients, inadequate infection and control measures, and late case diagnosis and management are expected. the annual spatiotemporal high-risk mers-cov clusters occurred mainly in the early periods of the mers-cov epidemic (between and ) in major cities, such as riyadh, jeddah, taif, alhofuf and buraydah, whereas recent clusters (between and ) were observed in relatively small cities, such as dumat aljundal and wadi addawasir. this result can be explained by infection prevention and control practices, which were, to some extent, more effective in major cities than in small cities and remote areas. for the monthly spatiotemporal high-risk mers-cov clusters, the cities of jeddah, makkah and taif were identified as a part of the most likely high-risk mers-cov cluster for april and may of , when the number of cases represented the largest accumulation of cases reported since the beginning of the mers-cov outbreak. the probable source of infection in the majority of the cases in this outbreak was secondary human-to-human transmission in jeddah that took place in healthcare facilities as a result of overcrowding and inadequate infection control measures, rather than a sudden increase in primary cases in the community [ ] . the spatiotemporal cluster detected in riyadh between march and october could be attributed to several outbreaks, with the most prominent one occurring in a single healthcare setting in riyadh in august [ ] . in addition, the most recent clusters of mers-cov incidence were identified in wadi addawasir in february , in dumat aljundal in august and in buraydah in march ; according to [ ] [ ] [ ] , the majority of the cases were associated with healthcare-acquired infections. this indicates persistent challenges related to nosocomial transmission, which require a thorough investigation of compliance to infection control measures by healthcare workers. mers-cov infection has global public health implications and has been labelled as an epidemic in saudi arabia. this study examined the spatial pattern and spatiotemporal clusters of mers-cov incidence in saudi arabia for the first time by using the latest publicly available mers-cov data. the results of this study provide initial risk assessments that can be used as the basis for the further investigation of the potential environmental risk factors that can explain the spatiotemporal clusters of mers-cov infection. the immediate isolation of suspected patients, adequate infection control measures and early case diagnosis and management remain the principal elements in controlling the spread of the disease, especially in small hospitals and in remote areas. future research investigating the effects of age, gender and occupation on mers-cov infection and mortality is needed. isolation of a novel coronavirus from a man with pneumonia in saudi arabia emergencies: middle east respiratory syndrome coronavirus (mers-cov) 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and space-time scan statistics guide for version camel sciences and economy in the world: current situation and perspectives systems of camel management in saudi arabia awad-acharari, f. genetic and nongenetic effects for milk yield and growth traits in saudi camels mers-cov in upper respiratory tract and lungs of dromedary camels comparative epidemiology of middle east respiratory syndrome coronavirus (mers-cov) in saudi arabia and south korea a pandemic risk assessment of middle east respiratory syndrome coronavirus (mers-cov) in saudi arabia global seasonal occurrence of middle east respiratory syndrome coronavirus (mers-cov) infection emergence of mers-cov in the middle east: origins, transmission, treatment, and perspectives infection control and mers-cov in health-care workers the prevalence of middle east respiratory syndrome coronavirus (mers-cov) infection in livestock and temporal relation to locations and seasons interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk differences in the seasonality of middle east respiratory syndrome coronavirus and influenza in the middle east a systematic review of emerging respiratory viruses at the hajj and possible coinfection with streptococcus pneumoniae increase in middle east respiratory syndrome-coronavirus cases in saudi arabia linked to hospital outbreak with continued circulation of recombinant virus response: disease outbreak news: middle east respiratory syndrome coronavirus (mers-cov)-the kingdom of saudi arabia ministry of health saudi arabia world health organization. who mers-cov global summary and assessment of risk this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors wish to express their gratitude to king abdulaziz city for science and technology and king faisal specialist hospital and research centre for supporting this study, as well as the smoh and the who for providing the data. the authors declare no conflict of interest. key: cord- -so v fy authors: chan, isabelle y.s.; liu, anita m.m. title: effects of neighborhood building density, height, greenspace, and cleanliness on indoor environment and health of building occupants date: - - journal: build environ doi: . /j.buildenv. . . sha: doc_id: cord_uid: so v fy the influences of indoor environment quality on occupant health have long been one of the main focuses in built environment and public health research. however, evidence to this effect has been inconsistent. furthermore, previous urban studies have indicated the interaction between urban morphology and indoor environment. this study thus goes beyond indoor environment to investigate: i) the effects of neighborhood environment on occupant health; and ii) the mediating roles of indoor environment on the neighborhood environment and occupant health relationships. to achieve this aim, buildings located in different neighborhood environment in hong kong are selected. data are collected by post-occupancy evaluation (occupant health), indoor environment assessment (thermal comfort, indoor air quality, ventilation, visual comfort, and acoustic comfort) and neighborhood environment assessment (neighborhood building density, building height, cleanliness and greenspace) through questionnaire survey. through correlation analysis, regression modelling and sobel test, it is found that: i) occupant health is significantly affected by neighborhood building height, building density and cleanliness; ii) the relationships between neighborhood environment and occupant health are significantly mediated by indoor environment, in terms of visual and acoustic comfort; and iii) neighborhood greenspace affects occupant health indirectly through influencing indoor air quality. to cross validate the results of the survey study, which is conducted using subjective data, objective measurements and analyses are further conducted. the objective study, echoing the survey study results, indicates that buildings with lower neighborhood building density and height, and cleaner neighborhood environment have better visual (higher illuminance level) and acoustic (lower noise level) performances. buildings are often designed and developed based on various regulations and guidelines established with an attempt to maintain occupants' comfort within an indoor environment [for instance, compliance with design requirements for ventilation, sustaining indoor air temperature at design values, and maintaining background noise levels within prescribed criteria]. however, building occupants are not isolated from its neighborhood environment. buildings serve not only to shelter occupants from adverse outdoor environment and weather; but also bring favorable natural elements, such as natural lighting and fresh air, into occupants' work and life. permeability is one of the key features in any buildings (e.g., [ ] . it is this permeability nature which puts occupants of a building and its neighborhood environment into connection. therefore, the impact of neighborhood environment is a key factor which cannot be overlooked when studying occupant health and indoor environment. in fact, the effect of indoor environment quality on occupant health has long been an important topic in built environment research and practices. however, the results to these effects have been inconsistent. for instance [ ] , indicated that occupants' asthma and respiratory allergies are affected by indoor air quality, temperature, humidity and ventilation of an indoor building environment. similarly [ ] , and [ ] found that occupants' respiratory and asthmatic symptoms are predicted by poor indoor air quality and ventilation. however [ ] , found that indoor air quality can have both positive and negative effects on occupants' wheeze [ ] . found that air quality is not correlated with students' health problems in terms of respiratory symptoms, headache and gastrointestinal symptoms. the above inconsistent findings, to certain extent, indicate that the relationship between indoor environment and occupant health may be subject to other key factors. given that neighborhood environment can influence indoor environment (e.g., the influence of neighborhood building density on indoor temperature; [ ] , it is reasonable to postulate that indoor environment can be the mediator of the relationships between neighborhood environment and occupant health. however, it is unclear what and how neighborhood environment factors affect indoor environment and occupant health. hence, this study goes beyond indoor environment to identify what neighborhood environment factors affect occupant health and indoor environment quality; investigate the influence of these neighborhood environment factors on occupant health; and to examine the impact and interplay of indoor building environment and neighborhood environment on health of building occupants. it is hypothesized that: i) occupant health is significantly affected by a building's neighborhood environment; and ii) the impact of neighborhood environment on occupant health is mediated by indoor environment. previous studies have identified various indicators for indoor environmental quality, including indoor air quality (iaq), thermal comfort, ventilation, visual condition, and acoustic condition. iaq refers to the air quality within and around buildings and structures, and it is especially related to health and comfort of building occupants. it can be determined by the concentration of different air pollutants, such as carbon monoxide, nitrogen dioxide, sulphur dioxide, volatile organic compounds, ozone, nonmethane hydrocarbons, particulates sulphates and nitrates, formaldehyde and radon, in an indoor environment [ ] . previous studies have indicated that poor iaq can cause bronchoconstriction, asthma symptoms, lung cancer, irritation to eyes, visibility problems, headaches, dizziness and even fatal poisoning in occupants (e.g., [ , ] . since people spend around % of their time indoors, iaq has long been a key focus in different building performance assessments [ ] . when comparing with other indoor environment quality indicators, such as acoustic comfort, visual comfort, and iaq, thermal comfort has been ranked by building occupants as of greater importance [ ] . thermal comfort refers to the state of mind that expresses satisfaction and subjective evaluation of the thermal environment [ ] . human body has a thermoregulatory system which serves to maintain a constant internal body temperature [ ] . mediated by the physics of heat and mass transfer in the process of heat balance, people respond physiologically to any thermal imbalance between the body and the surrounding environment. previous studies have indicated that thermal environment is associated with occupants' well-being, in terms of asthma and respiratory allergies [ ] . ventilation, referred as the air movement within a building, is closely related to iaq and occupants' thermal comfort [ ] . poor ventilation has been identified as an antecedent of various respiratory diseases, such as severe acute respiratory syndrome (e.g., [ , ] . there are three main types of ventilation, namely mechanical, natural, and hybrid. mechanical ventilation can cause energy efficiency problem; while natural ventilation is constrained by neighborhood environment. hybrid ventilation functions to exploit the benefits of both natural and mechanical ventilation methods. visual comfort is defined as "a subjective condition of visual wellbeing induced by the visual environment" [ ] , which can usually be affected by two components, namely natural and artificial lighting. proper control of glare and shading is needed to minimize the impact of excessive or inadequate lighting on occupants' health, including fatigue and eye health, such as watery eyes, dry eyes, eye ache and tired eyes [ ] ; [ ] . in addition, acoustic comfort refers to the subjective noise annoyance experienced by an individual, which may further affect one's health and cognitive performance [ ] . though individual's acceptance and response to sound pressure and acoustic patterns is subjective, previous studies have proven the impact of noise on individuals' psychological health and memory [ , ] . the above section indicates the intimate associations between indoor environment and occupants' health. however, indoor environment quality are intimately associated with its neighborhood environment. for instance, previous studies have indicated the impact of urban neighborhood characteristics, in terms of land-use ratio and thermal mass, on indoor air temperature of buildings [ ] . on the other hand, air pollutants emitted from vehicles in busy district may cause greater indoor air pollution through permeable building façade [ , ] . hence, it can be postulated that health of occupants should not only be affected by the indoor environment. it is essential to investigate the interplay of indoor and outdoor environment and their impacts on occupants' health. according to the previous studies on built environment at neighborhood scale, built environment can generally be categorized into four fields, namely buildings, open spaces (e.g., greenspaces, sidewalks, parking, etc.), mobility (e.g., passenger car, train, bus, etc.) and networks (e.g., electricity, water, wastewater, gas networks, etc.) [ ] . while neighborhood networks and mobility are mainly associated with energy consumption, this study, focusing on occupant health, covers the first two fields, buildings and open spaces. the building category refers to both height and density of the buildings nearby, while open spaces refer to neighborhood greenspace and cleanliness of the surrounding area. due to the rapid business development and the issue of land scarcity, various modern cities, like hong kong, tokyo, singapore, and shanghai, have undergone land use intensification and adopted vertical development strategy in the past decades [ , ] . this has resulted in an increase in building density (site coverage of buildings over a certain area), increase in building height (from the mean formation level of the land on which the building stands, up to the top of the highest roof slab of the main roof of a building), and reduction in open spaces and pollution in urban areas. to prevent adverse urban environmental and social problems, many countries have adopted different regulations on building density and height, such as restrictions on lot size zoning, building height and plot ratio (e.g., [ , ] . however, along with the rapid development of construction and building services technologies, many cities have increased the maximum permitted building density and height [ , ] . in fact, previous studies have indicated the impact of neighborhood building height on the access of sunlight and solar radiation (e.g. [ ] , indoor temperature [ ] , dispersion of atmospheric pollutants [ ] , etc., of a building. on the other hand, high neighborhood building density can cause heat island effect, resulting in lower wind speeds and higher ambient temperatures inside a building [ ] . the existence of greenspace in neighborhood environment, such as tree canopy, parks and forests, has found to be associated with better physical health, reduction in morbidity in some disease categories, lower level of depression, lower level of stress, and so on (e.g., [ ] ; [ ] . in fact, the roles of greenspace have found to be especially significant in protecting building occupants from health hazards related to air pollution and extreme temperature (e.g. [ ] , and in promoting healthy behaviors amongst building occupants, such as physical activities (e.g., [ , ] . in addition, neighborhood cleanliness has been recognized as one of key issues facing policy makers when planning and developing cities [ ] . it can cover cleanliness of streets, sidewalks, and footpaths surrounding a building (e.g., existence of debris and graffiti; [ ] . previous studies found that neighborhood cleanliness can affect occupants' satisfaction and health through various factors, like influencing people's willingness to conduct physical activities (e.g., [ , ] . based on the above, the conceptual model of the study is developed in fig. . as illustrated in the figure, neighborhood environment is hypothesized to predict occupant health (h ); and the influence of neighborhood environment on occupant health is hypothesized to be mediated by indoor environment quality (h ). to achieve the research aim, we conducted a questionnaire survey study targeting occupants of four academic buildings located in different neighborhood environment in hong kong. these four buildings are strategically selected to involve those located in high (two) versus low (two) neighborhood building density, high (two) versus low (two) neighborhood building height, and large (two) versus small (two) neighborhood greenspace. please refer to figs. and for the locations of the buildings. academic buildings are selected mainly for two reasons. firstly, unlike commercial buildings which only accommodate the working age groups and residential buildings which accommodate residents with similar social background (e.g., housing affordability), academic buildings accommodate a good mix of occupants who come from different age (e.g., teenage students, senior students, middle aged staff, senior staff, etc.) and social (e.g., students needing financial aid have the same right as students who come from higher income families to enjoy education at universities) groups. on the other hand, previous studies have indicated the intimate relationships between property values and neighborhood environment, such as greenspace (e.g., [ , ] . in other words, occupants of buildings located in a greener environment may have a higher housing affordability, and thus social background, in which these have found to have impact on individuals' health [ ] . to ensure a good mix of respondents, this study targets academic buildings which accommodate occupants with different age and social background, and are located in areas with different neighborhood environment. previous studies, using academic buildings as the bases of data collection for investigating interactions between occupants and built environment, tend to sample students only (e.g., [ ] . in view of the potential impact of respondents' age on health and satisfaction, this study targets not only students, but also academic and administrative staff. purposive sampling is adopted, in which respondents are recruited only if they are academic or administrative staff working or students studying in the target academic buildings. in sum, valid responses are collected. students account for . % of the total sample, while academic and administrative staff account for . %. the respondents age from or below ( . %), - ( %), to or above ( . %), in which . % are male and . % are female. amongst the respondents, % spent - h in the building a week, . % spent more than h and . % spent h or less. to control the impact of building age on occupants, the target buildings are - year-old (built in the past decade), with % aged , % aged , and % aged . the post-occupancy evaluation survey is designed to have four main parts, namely, background information, indoor environment quality (indicated by occupants' satisfaction towards indoor air quality, ventilation, thermal comfort, lighting, and acoustics; [ ] , neighborhood environment quality (indicated by occupants' satisfaction towards neighborhood building density, building height, greenspace and cleanliness [ ] , and building-related health symptoms (frequency of occupants suffering from dry eyes, itchy or watery eyes, blocked or stuffy nose, runny nose, dry throat, lethargy or tiredness, headaches, dry, itching or irritated skin, sneezing, and breathing difficulties; [ ] . adopting the health measurement scale developed by ref. [ ] ; respondents were asked to rate the frequency of the symptoms. occupant health is then calculated by taking an average of the scores of these items. respondents were invited to answer the questions based on a -point likert measurement. statistical analyses are then conducted, using the software of spss, to investigate the hypothetical relationships between neighborhood environment, indoor environment, and health of occupants. since health differs according to individual occupants' background characteristics, this study, making references to previous studies on occupant health, statistically controls for gender and age of occupants in the correlation and regression analyses (e.g., [ ] . to preliminarily investigate the relationships between neighborhood environment, indoor environment and occupant health, pearson correlation analysis was conducted (see table ). the results indicate that all four neighborhood factors correlate significantly with the five indoor environment factors and occupant health. all correlation coefficients are significant at p < . level. the results act as a solid foundation for further testing the predicting effect of neighborhood environment on occupant health, and the mediating effects of indoor environment on neighborhood environment-occupant health relationships. to further investigate the predicting effects of neighborhood environment on occupant health, multiple regression modelling was conducted. based on the result of pearson correlation, all four neighborhood environment factors are significantly correlated with occupant health. they are thus all selected as independent variables in the multiple regression analysis with occupant health as dependent variable. as shown in model of table , neighborhood building height, neighborhood building density and neighborhood cleanliness are found to predict occupant health significantly (p < . ). the model explains . % of variance to occupant health. h is thus supported. the results also act as a basis for the following mediation tests. a mediator is referred to as a variable which accounts for the relation between an independent variable and a dependent variable. to measure the mediating effects of indoor environment (i.e., thermal comfort, indoor air quality, ventilation, visual comfort and acoustic comfort) on the relationships between neighborhood environment (i.e., neighborhood building height, neighborhood building density, neighborhood greenspace and neighborhood cleanliness) and occupant health, the classic sobel test is adopted [ ] . the sobel test involves three main steps [ ] : step a-to show that the independent variable (i.e., neighborhood environment) significantly affects the dependent variable (i.e., occupant health) in the absence of the mediator; step be to show that the independent variable significantly affects the mediator (i.e., indoor environment); and step ce to show that the independent variable and the mediator have significant effects on the dependent variable. while step a is done as shown in model for the first hypothesis of the current study, steps b and c are done in the following regression analyses. step b is then conducted as shown in models - in table , where the five indoor environment factors are included in each model as a dependent variable, and the four neighborhood environment factors are added as independent variables in each model respectively. the results indicate that neighborhood building density significantly predicts thermal comfort, ventilation, and acoustic comfort (p < . ). neighborhood building height significantly predicts visual comfort and acoustic comfort (p < . ). neighborhood greenspace significantly predicts thermal comfort, indoor air quality, and ventilation (p < . ). lastly, neighborhood cleanliness predicts visual comfort significantly (p < . ). model is further developed to investigate, with the absence of the effects of the neighborhood environment, the predicting effects of the five indoor environment factors on occupant health. the results indicate that occupant health is significantly predicted by acoustic comfort, indoor air quality and visual comfort (p < . ). the model explains . % of variance to occupant health. then, step c is conducted as shown in models - in table . models - are developed with occupant health as dependent variable and with a different combination of an indoor environment and a neighborhood environment as independent variables for each model. the combinations are determined based on the significant associations found in models - as shown in table . as shown in table , occupant health is significantly predicted by both indoor environment (i.e., visual comfort or acoustic comfort) and neighborhood environment (i.e., neighborhood building density, neighborhood building height and neighborhood cleanliness) in the four models respectively (p < . ). the regression coefficient estimates and the standard error of the paths from independent variable to mediator (i.e. 'a' and 'ta' from models - ) and from mediator to dependent variable (i.e. 'b' and 'tb' from models - ) are then obtained. then, the sobel z-value are calculated through dividing ab by the square root of b (a/ta) + a (b/tb) . the mediating effect is considered to be significant at the . level if the z-value is larger than . in absolute value. as shown in the last column of table , all four mediation effects are found to be significant (i.e., > . ). thus, h is also supported. the abovementioned significant associations are illustrated in fig. . fig. illustrates that health of occupants is influenced by neighborhood building density, neighborhood building height and neighborhood cleanliness, and these influences are mediated by indoor environment quality, in terms of acoustic comfort and visual comfort. however, previous studies indicate that human comfort level in a built environment can be affected by various psychological parameters, such as individuals' desired condition [ ] and environmental beliefs [ ] . to validate whether neighborhood environment does contain objective effects on indoor acoustic and visual levels, a field measurement study is further conducted. firstly, the four target buildings are categorized into two groups, in which group represents buildings located in a neighborhood environment with lower building density, lower building height, and better neighborhood cleanliness; while group represents buildings located in a neighborhood environment with higher building density, higher building height, and poorer neighborhood cleanliness. the differences in neighborhood building density and height of to further explore whether significant differences in neighborhood cleanliness exists between groups and buildings, one-way betweengroups analysis of variance (anova) was conducted using the survey data. respondents from group buildings (mean = . ) are found to have significantly higher satisfaction towards neighborhood cleanliness when compared with that of the respondents from group (mean = . ) buildings [f = . , p < . ]. hence, the following comparative analyses are conducted using groups and buildings as analysis units, representing buildings with different levels of neighborhood building density, height and cleanliness. the survey study unveils that neighborhood environment influences occupant health via two indoor environment factors, namely acoustic and visual comfort. therefore, in this section, the performance of the two groups of buildings in these two dimensions are measured objectively on site. since acoustic and luminance levels deviate from time to time throughout a day, measurements were conducted on an hourly basis, from : am to : pm. minolta lux meter was used to measure the illuminance level, and ono sokki la- precision integrated sound level meter was used to measure the noise level. in general, noise level in office buildings is recommended to be lower than ∼ db. for instance, the chinese code for sound insulation design for civil buildings recommended that the noise level in office buildings should be lower than db. the building environmental assessment method (beam) plus noise performance criteria for office premises recommends db for office areas where privacy is important. furthermore, a previous study found empirical support that office note: all analyses are controlled for age and gender. note: ** -significant at . level. all analyses are controlled for age and gender. building occupants are satisfied when noise level is below . db [ ] . however, as illustrated in fig. , during the work hours, the noise levels of groups and buildings are all above db. for group , the noise levels range from . db to . db. for group , the noise levels range from . db to . db. on average, the noise level of group is db, just meeting the upper limit as recommended by the chinese code; while that of group is . db, which is far above the upper limits as suggested by various code or guidelines. the above, conforming to the results of the survey study, indicates that the acoustic performance of group (buildings located in a neighborhood environment with lower building density, lower building height, and better neighborhood cleanliness) is better than group (buildings located in a neighborhood environment with higher building density, higher building height, and poorer neighborhood cleanliness). previous studies have found empirical support that the higher the illumination intensity, the higher the occupants' satisfaction level of the luminous environment (e.g., [ ] . furthermore [ ] , indicates that occupants start to feel satisfied when the illumination intensity is above lux, and the satisfaction level increased to 'quite satisfied' when the light level reached lux. as illustrated in fig. , during the work hours, the illuminance levels of groups and buildings are all above lux. on average, the illuminance level of group is lux, while that of group is lux. the above, conforming to the results of the survey study, indicates that the illuminance performance of group (buildings located in a neighborhood environment with lower building density, lower building height, and better neighborhood cleanliness) is better than group (buildings located in a neighborhood environment with higher building density, higher building height, and poorer neighborhood cleanliness). (refer to tables and for the coefficients of each relationship) . note:-→significant mediating effects (refer to table )-(xxx) contribution of the bracketed variable being taken into account in the mediating process-nbd -neighborhood building density-nbh -neighborhood building height-nh -neighborhood cleanliness-ac -acoustic comfort-vc -visual comfort fig. . comparison of noise level between groups (lower building density, building height, and better neighborhood cleanliness) & (higher building density, building height, and poorer neighborhood cleanliness) buildings. to investigate whether statistically significant differences exist between health of occupants from the two groups of buildings, an independent-samples t-test was conducted based on the survey data using spss. significant differences are found in occupant overall health (p = . ), and various health symptoms, namely dry eyes (p = . ), itchy or watery eyes (p = . ), blocked or stuffy nose (p = . ), runny nose (p = . ), dry throat (p = . ), lethargy or tiredness (p = . ), dry, itching or irritated skin (p = . ), and sneezing. the mean values are shown in fig. . the results of this study indicates that health of occupants are directly affected by neighborhood building density, neighborhood building height and neighborhood cleanliness, and these effects are significantly mediated by occupants' acoustic and visual comforts in the indoor environment. meanwhile, even though neighborhood greenspace is found to have no direct impact on occupant health, it has found to have indirect impact on occupant health through its influence on indoor air quality (refer to fig. ) . the survey results are further confirmed by the objective study which indicates that the acoustic and illuminance performance of buildings with lower neighborhood building density, lower neighborhood building height and cleaner neighborhood fig. . comparison of illuminance level between groups (lower building density, building height, and better neighborhood cleanliness) & (higher building density, building height, and poorer neighborhood cleanliness) buildings. fig. . comparison of health between occupants of groups (lower building density, building height, and better neighborhood cleanliness) & (higher building density, building height, and poorer neighborhood cleanliness) buildings. note: a star denotes significant difference found in t-test (p < . ). area are better that of their counterparts. the impact of neighborhood building height on occupant health is significantly mediated by occupants' visual comfort in the indoor environment. occupants' visual comfort can be affected by indoor lighting quality (e.g. [ ] , natural lighting, and bright reflection of visible light from concrete roof and /or from the facades of neighborhood buildings (e.g., [ ] . high-rise neighborhood buildings can act as obstructions, resulting in insufficient natural lighting in the indoor environment. hence, as indicated in the objective measurement study, the illuminance level of group buildings is higher than that of group (refer to fig. ). on the other hand, a low-rise neighborhood area can also mean lesser reflection of light from the roofs and facades of neighborhood buildings, reducing the impact of outdoor glare on indoor lighting quality. extreme light levels can cause eye health problems to building occupants [ ] , however, its impact can be reduced if a building is equipped with an effective design strategy and lighting system, such as the adoption of façade design with visible light transmittance glazing and smart lighting system with light sensors, which enhance occupants' visual comfort (e.g., [ , ] . on the other hand, the impact of neighborhood cleanliness on occupant health is also significantly mediated by occupant's indoor visual comfort. previous studies have found that people living nearby pollution sources, like industrial areas, have a higher risk on air-quality related diseases, such as lung cancer (e.g., [ ] . however, neighborhood cleanliness is found to have no direct association with indoor air quality or ventilation in the current study. perhaps, it is the dissatisfactory visual appearance of pollutants, such as debris and graffiti, in the neighborhood environment, which causes poor health of occupants [ ] .'s study indicated how occupants managed to address visual discomfort resulting from excessive sunlight due to an inappropriate building design through various informal workspace modifications, such as using umbrella to block the connection with outdoor lighting. hence, it is recommended to manage visual discomfort caused by poor neighborhood cleanliness using different space modifications, such as curtain blocking outside views to a certain level. the impacts of neighborhood building density and height on occupant health are significantly mediated by occupants' acoustic comfort in the indoor environment. excessive noise can cause building occupants to heart diseases, lower concentration level, and so on [ , ] . in developed cities, like hong kong, transport noise, such as road traffic and railway noise, is the major source of noise affecting building occupants [ ] . in this case, neighborhood buildings can act as obstructions to the free propagation of noise from street and road traffic, attenuating its sound level [ ] . in current study, since the two group buildings are located right next to two main roads, with the absence of neighborhood buildings serving as sound obstructions, group buildings are found to have higher noise levels than that of group buildings. enhancing sound insulation level of a building can reduce the level of sound energy emitted from the neighborhood environment entering its inner space, thus, enhancing occupant acoustic comfort and relieving the significant impact of outdoor noise to occupants. various previous studies indicated that neighborhood greenspace affects building occupant health. researchers tend to associate this result with the opportunity provided to occupants to walk and exercise (e.g., [ , ] . in current study, even though half of the buildings are located in areas surrounded by large greenspace, majority of these green areas are not accessible (fall outside the premises area) (refer to fig. ). as such, neighborhood greenspace is found to have no direct impact on occupant health in the current study. however, it significantly affects indoor air quality, which further influences occupant health. a larger area of neighborhood greenspace can, to certain extent, mean a lower number of neighborhood buildings. neighborhood buildings can act as obstacles to fresh air moving into a building. this blocking effect would be higher if a building is surrounded by denser and taller neighborhood buildings. the reduced level of indoor air ventilation can slowdown the transfer rate of indoor air pollutants when the indoor pollution concentration is higher than that of the outdoor [ ] , causing respiratory diseases, eye problems, headaches, and even fatal poisoning (e.g., [ , ] . hence, the need of an effective ventilation system is essential in fostering occupant health, especially when neighborhood greenspace is not satisfactory. previous studies tend to focus on the influence of indoor environment on occupants and to study the impact of urban environment on building performance (e.g., the impact of neighborhood building morphology on energy consumption of a building; [ ] . the intimate and intertwining relationships between neighborhood environment, indoor environment and occupant health are not clear. further developed from the results of these previous studies, the current study provides empirical support on the extended effects of neighborhood environment, when interacting with indoor environment, on occupants' health. the findings, to certain extent, indicate that consideration of indoor environment alone does not guarantee a better indoor environment, nor better occupant health. this sheds light to the importance of taking neighborhood environment and its interaction with respective indoor environment indicators into account in building assessment process. based on the findings of the current study, building designers and engineers are recommended to put more emphases and weighting on indoor air quality, acoustic comfort and visual comfort of occupants in building design and assessments processes, because these factors are found to have direct effects on occupant health. more importantly, considerations and assessments have to be extended to neighborhood building density (acoustic comfort), neighborhood building height (acoustic and visual comfort) and neighborhood greenspace (indoor air quality) when the above indoor environment quality issues are concerned. the sample size of the survey study is comparable to or even larger than some of the published works in the built environment field which use similar mixed method approach (e.g., survey samples collected by ref. [ ] ; survey samples collected by ref. [ ] ; survey samples collected by ref. [ ] . meanwhile, the data collection is strategically designed to include respondents with various background (i.e., age, occupation, gender, etc.), occupying in buildings located in different neighborhood environment (i.e., high versus low neighborhood building density and height, large versus small neighborhood greenspace, and good versus poor neighborhood cleanliness). the survey study adopts a self-report measurement approach, which could have resulted in common method variance. however, it should be noted that the scales in this study are adopted from the extensive literature on built environment and post-occupancy evaluation. in addition, the respondents are all staff and students who have direct, longterm occupancy experience in the case buildings. furthermore, the current study confirms the significant mediating effects of indoor environment quality on the relationships between neighborhood environment quality and occupants' building-related health symptoms. four neighborhood environment factors are included in this study. even though all of them are found to have significant impact on occupant health and /or indoor environment, it is recommended to include one more neighborhood factor, that is the neighborhood traffic, in the further study. the associations between acoustic comfort and occupant health are found to be affected by neighborhood building density. even though neighborhood building density can somehow reflect the traffic condition nearby, traffic flow has long been identified as the major source of noise for building occupants. hence, a further detailed study is recommended to investigate the impact of traffic on the indoor environment quality and occupant health. focusing on environment (neighborhood and indoor) and human (satisfaction and health) interactions, the results of the current study provide empirical support on the intertwining relationship between neighborhood environment, indoor environment and occupant health. based on the study results, further study is recommended to take into account the impact of building configuration and design (e.g., envelops, ventilation system, hvac system, sound insulation system, etc.) on the environment and human variables. in sum, the study provides empirical support that: i) occupant health is significantly affected by neighborhood building height, neighborhood building density and neighborhood cleanliness; ii) the relationships between neighborhood environment and occupant health are significantly mediated by indoor environment, in terms of visual comfort and acoustic comfort; and iii) even though neighborhood greenspace is found to have no direct impact on occupant health, it affects occupant health indirectly through influencing indoor air quality. the results lay solid platform on the importance of taking neighborhood environment into considerations during building design and assessment stages. furthermore, the study results also push forward the development of academic research in the field. researchers have conducted various studies on the impact of indoor environment quality on occupant satisfaction and health. however, evidence to this effect has been inconsistent. this study goes beyond indoor environment to develop the concept of outdoor and indoor environment interaction for revealing the intertwining relationships between neighborhood environment, indoor environment, and health of occupants. shade trees reduce building energy use and co emissions from power plants the moderator-mediator variable distinction in social psychological research: conceptual, strategic, and statistical considerations analysis of industrial contaminants in indoor air: part . volatile organic compounds, carbonyl compounds, polycyclic aromatic hydrocarbons and polychlorinated biphenyls exposure to neighborhood greenspace and mental health: evidence from the survey of the health of wisconsin strength of noise effects on memory as a function of noise source and age 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area: a numerical study green space as a buffer between stressful life events and health assessment of pollutant dispersion in the re-entrance space of a high-rise residential building, using wind tunnel simulations correlation analysis of lung cancer and urban spatial factor: based on survey in shanghai who guidelines for indoor air quality: selected pollutants, the who european centre for environment health evaluation of the impact of the surrounding urban morphology on building energy consumption greenspace in urban neighborhoods and residents' health: adding quality to quantity thermal comfort and building energy consumption implications-a review building environmental assessment schemes for rating of iaq in sustainable buildings automated derivation of urban building density information using airborne lidar data and object-based method the authors acknowledge the contribution of mr. felix t. h. tom in collecting the data for the study. key: cord- - yhxnvoh authors: guan, de-long; ma, li-bin; khan, muhammad salabat; zhang, xiu-xiu; xu, sheng-quan; xie, juan-ying title: analysis of codon usage patterns in hirudinaria manillensis reveals a preference for gc-ending codons caused by dominant selection constraints date: - - journal: bmc genomics doi: . /s - - -x sha: doc_id: cord_uid: yhxnvoh background: hirudinaria manillensis is an ephemeral, blood-sucking ectoparasite, possessing anticoagulant capacities with potential medical applications. analysis of codon usage patterns would contribute to our understanding of the evolutionary mechanisms and genetic architecture of h. manillensis, which in turn would provide insight into the characteristics of other leeches. we analysed codon usage and related indices using , coding sequences (cdss) retrieved from h. manillensis rna-seq data. results: we identified four highly preferred codons in h. manillensis that have g/c-endings. points generated in an effective number of codons (enc) plot distributed below the standard curve and the slope of a neutrality plot was less than . highly expressed cdss had lower enc content and higher gc content than weakly expressed cdss. principal component analysis conducted on relative synonymous codon usage (rscu) values divided cdss according to gc content and divided codons according to ending bases. moreover, by determining codon usage, we found that the majority of blood-diet related genes have undergone less adaptive evolution in h. manillensis, except for those with homologous sequences in the host species. conclusions: codon usage in h. manillensis had an overall preference toward c-endings and indicated that codon usage patterns are mediated by differential expression, gc content, and biological function. although mutation pressure effects were also notable, the majority of genetic evolution in h. manillensis was driven by natural selection. electronic supplementary material: the online version of this article ( . /s - - -x) contains supplementary material, which is available to authorized users. codons are the basic genetic code of mrna in all living organisms. there are types of codons that encode different amino acids; thus, codon degeneracy exists. in nuclear genomes, almost all amino acids, except for methionine and tryptophan, are encoded by two to six synonymous codons [ ] [ ] [ ] . for different genes or genomes, the choices of synonymous codons are non-random, which is known as codon bias. codon biases are specific to a given organism and can be influenced by gc content, gene expression levels, and gene lengths [ , ] . additionally, codon usage patterns may affect the mrna biosynthesis, translational elongation speed, protein folding, and other biological functions downstream of expression [ ] [ ] [ ] [ ] [ ] . given the substantial biological effects of different codon patterns, identifying these patterns in a given gene or genome is important for understanding the molecular mechanisms of expression and for revealing the effects of long-term evolution on a genome. currently, the most popular and widely accepted hypothesis that explains codon usage bias is mutation-selection balance, which proposes that codon usage reflects the combined effects of three evolutionary forces: mutation pressure, selection constraints, and genetic drift within a population [ , , ] . in general, mutation pressure tends to shuffle a/u and g/c pairs to cause nucleotide composition bias, selection constraints lead to preferences for codons that maximise protein production efficiency in highly expressed genes, and genetic drift eliminates codon changes across generations as a result of immigration and emigration at the population level. the codon usage pattern of a given organism reveals the balance between these three forces and by employing widely used methods, such as effective number of codons (enc) plots and neutrality plots, the driving forces and approximate contribution of each force to codon usage can be determined. previous studies have shown that codon biases in microbes are generally driven by mutation pressure, as exemplified in xanthophyllomyces dendrorhous, zika virus, and escherichia coli [ ] [ ] [ ] . among invertebrate animals, selection constraints are the major influence on codon usage, as demonstrated in taxa such as drosophila melanogaster, bemisia tabaci, and taenia pisiformis [ , , ] . these studies demonstrate that characterising codon usage and other relevant indices provides simple and intuitive strategies for examining complex evolutionary forces in many different species. hirudinaria manillensis (hirudinidae: arhynchobdellida: annelida) is an ephemeral, blood-sucking leech of medium body size that parasitizes mammals [ ] . an adult h. manillensis may consume more than ml of blood per host and can cause substantial and ongoing pain. even after the leech abandons the host, this pain can persist as bleeding continues due to the anticoagulants (e.g., hirudin) secreted by the salivary glands of the leech [ ] [ ] [ ] . the remarkable anticoagulant characteristics of h. manillensis secreted proteins have attracted much interest and the animal is considered a useful model for designing natural coagulation inhibitors and anticoagulant drugs [ , , ] . progress in molecular biology has generated interest in the analysis of h. manillensis mrna with the aim of revealing details about their nutritional requirements and mechanisms of expression of anticoagulants. it would be of great value to generate more reliable coding sequences for new gene discovery; furthermore, the genetic processes revealed by their codon usage patterns will further contribute to determining the effects of long-term evolution on the genetically diversified and unique physiological behaviour of medical leeches. such information on codon usage could be applied to biological research as a molecular tool to edit or optimise mrna sequences as a way to control gene expression. because of the interest in leech biology and codon usage patterns and the paucity of related research, we conducted transcriptome sequencing of h. manillensis with the objective of providing the first detailed genomic information of the species. all identified unigenes and their annotations have enriched the current genomic sequences and can serve to further identify potential anticoagulant genes. we also attempted to determine the general driving evolutionary forces and to describe in more detail the differential evolutionary status for the identified anticoagulant genes. all high confidence unigene coding sequences (cdss) were included to analyse codon usage bias, and the related indices and functional classifications. the general pattern of codon usage reflects the evolutionary process, and the differences in evolution between different metabolic mechanisms can be assessed by comparing these patterns. the results reported here will improve our understanding of the genetic architecture and mechanisms in h. manillensis, and will link the unique codon usage patterns of h. manillensis to specific genetic or genomic functions to help build a genetic profile for future research on the species. the error-corrected transcriptomic data retrieved from h. manillensis samples measured . gb and was comprised of more than million raw reads. from these data, we assembled , raw transcripts and obtained , high-confidence transcripts. using these , transcripts, we then identified , unigenes and , cdss longer than bp. the average lengths of the transcripts, unigenes, and cdss were . , . , and . bp, respectively. overall, the four nucleotides were asymmetrically represented in the cdss. adenine (a) was the most represented ( . %), cytosine (c) was the least represented ( . %), and there was little difference between guanine (g) ( . %) and thymine/uracil (t/u) ( . %) content. the gc content of the cdss was . %. to better understand the nucleotide base composition in h. manillensis, we measured the number of cdss with different gc content levels. almost all cdss contained - % gc content (fig. a) ; thus, cdss having gc contents lower than % or higher than % were considered as cdss with extreme gc content. we further divided the - % range into three smaller intervals and measured the number of cdss attributable to each interval. the - % interval contained the most cdss, with numbers approaching or exceeding , followed sequentially by the - % and - % intervals. this result corresponds with and to some extent explains the overall nucleotide composition of all h. manillensis cdss measured. we also measured the gc content at different codon positions (first, second, and third) in the cdss. the composition at the first codon position was similar to that of the overall nucleotide composition. the average gc content and the range between upper and lower quartiles were the lowest in the second codon position, and the average gc content and the range between upper and lower quartiles and boundaries were the highest in the third codon position (fig. b) . we performed a dinucleotide analysis on four cds datasets at different codon positions ( st & nd, nd & rd, rd & st) to define the lower order patterns in the cdss. all start and stop codons were removed because they are constrained by other rules. the ratios calculated using the observed frequencies over their theoretical frequencies, which are the mathematical products of the corresponding nucleotide content, are shown (fig. c) . for all four datasets, the dinucleotides were largely variable and unrelated to the expected unbiased frequency line. in the dataset that contained all cdss, eight dinucleotides (aa, ag, ca, cu, ga, uc, ug, and uu) were more prevalent than the rest, with ua having the lowest frequency. similar results were found in the nd & rd and rd & st databases, with only slight differences between them, while the ratios varied dramatically and lacked cg, ua, and ug pairs in the st & nd dataset. from the dinucleotide plot results, we observed that the various compositions of the sequences were not artefacts of different combinations of asymmetric nucleotide content. the sequences were formatted on the upper functional scale. among the synonymous codons identified (stop codons were excluded), were more frequently represented with an rscu > ( table ). the rscu value of the codon gcc, which encodes ala, was the highest. we divided these codons by their ending bases and found that they ended with a ( ), u ( ), c ( ), and g ( ) in differing degrees. h. manillensis transcripts clearly preferred codons with gc-endings, particularly those ending with c, over the other bases. furthermore, we wanted to locate the preferred/avoided codons and, therefore, emphasized those codons that had extremely high (> . ) or low (< . ) rscus. we found that codons such as gcc, cuc, and guc were highly preferred and codons such as cua, uua, and aua were particularly avoided in the cdss that we evaluated. these highly preferred/avoided codons deviated from the neutral frequency value (equal to ), which showed a preference for codons that ended with the gc dinucleotide. we also analysed the enc values in h. manillensis collected from the cdss. the enc values varied, ranging from . - . , which indicated different trends in (see figure on previous page.) fig. base composition of h. manillensis. a distribution of cdss with different gc content levels in of h. manillensis; (b) box plot indicates gc content variation in different codon positions ( st, nd and rd represent the first, second and third codon position, respectively) and overall genome (all). c dinucleotide frequency plot in different combination of different codon positions ( st& nd, nd& rd and rd& st) and overall genome (all), unbiased frequency means the theoretical frequency when all dinucleotides were presented equal, the curve located at . ( / ) was ubiquitous in h. manillensis and compositional constraint was not the determining factor driving codon preference, indicating that the pattern of codon usage in h. manillensis was caused by multiple factors as well as the composition of the ending dinucleotides. these other independent factors could be related to natural selection such as expression level or protein length, which may be more important than those related to mutation pressure. to provide a more accurate assessment of the deviation between observed and expected enc values, the formula (encexp − encobs)/encexp was used to calculate the ratios and to describe the degree of variation. ratios between . and . were the most frequent in our dataset, while ratios between . and . were relatively uncommon, as determined by calculations using permutations (fig. b ). this result showed that, in most cases, the observed enc values were slightly lower than the expected enc values and differed by less than %. therefore, other factors have influenced codon usage bias formation in h. manillensis, which has caused a reduction in usage complexity of approximately %. although it has been reported in analyses of mutation pressure that gc and au occur in pairs at third codon positions, our results indicate that the proportions of g and c (or a and u ) are in fact unequal in h. manillensis. evaluating the direction of such bias within au and gc pairs can provide more detail about the forces that drive deviation from neutral mutations. we used a parity rule (pr ) plot to measure these biases in h. manillensis cdss (fig. c) . most of the points in our plot fell between . and . on both axes, indicating an overall low bias toward either a /u or g /c in h. manillensis. furthermore, we divided the points into four quadrants centred on . to compare the quadrants and found that the third quadrant (in which the ratio of a /au and g /gc < . ) contained substantially more points than the other quadrants, whereas the first quadrant contained the fewest points. these results indicated that cdss in h. manillensis had a slight yet noticeable preference for u and c at the third codon position. therefore, the balance between au and gc has been disrupted in h. manillensis. to further clarify the driving forces behind, and the precise proportions of mutation or natural selection pressures contributing to, codon usage bias in h. manillensis, a neutrality plot was constructed using gc and gc values with a mean gc content at the first and second codon positions (fig. d ). many points in the graph were distributed away from the regression curve and the r of the equation was low. these results indicated that the overall correlation between gc and gc was not strict, and that gc and gc had different degrees of modification. additionally, the slope of the estimated equation revealed that mutation pressure accounted for a minority of the effects observed ( %), whereas selection constraints accounted for the majority (approximately %). enc values and gc content are unrelated to differential gene expression one of the advantages of analysing transcriptome data is that the expression level of all genes can easily be obtained; therefore, earlier methods that used codon adaption index (cai) values to predict expression levels among genes are no longer necessary. to explore the relationship between codon usage and gene expression levels, we selected cdss of different genes that had fragments per kilobase million (fpkm) values above , and we plotted their log values against their enc values. the purpose of calculating log values in this situation is to condense the broad range of fpkm values. to further investigate the effects of genetic composition bias, we also labelled different points that represented the gc content of these cdss. the upper and lower quartiles of the overall gc content in h. manillensis were . and . % ( fig. b; above) , respectively; therefore, we set three intervals for categorising different cdss: gc < . %; . % < gc < . %; and gc > . % (fig. ) . the three cds groups with mostly overlapping distributions were found near the abscissa axis. the non-overlapped regions indicated that most genes with enc < were from the gc < . % group, and the genes with enc > were from the . % < gc < . % group. effects of selection were observed, indicating that the nucleotide substitution favoured gc and led to a stronger codon usage bias. however, on the vertical axis, neither the enc values nor the gc content were fig. gc content and codon usage bias variation among expressed unigenes. all cdss with observed fpkm values above were taken and transformed using log , respectively. dots with different colors represent their different gc content, while red represent gc > . %, green represent . % < gc < . %, purple represent gc > . % separated from the fpkm values. this result indicates that the gene expression levels were generally unrelated to codon modifications, and that selection constraints of expression efficiency were not present. these results seemingly contradict each other, and to overcome this conflict we inferred that the overall expression profile was responsible. we reasoned that, to maintain general biological homeostasis, there must be many genes that are highly expressed but are not codon-modified due to their vital functions. under selection, these genes would maintain their functions and eliminate codon changes that would jeopardise these functions. therefore, genes with codon modifications would have higher expression efficiency due to adaptive selection forces, but the effects would be hidden among other genes. principal component analysis (pca) reveals that rscu patterns are related to gc and codon-end binucleotide content in our pca of the rscu values, the first two axes accounted for . and . % of the total variation. the primary axis (axis ) accounted for much more variation than axis , indicating that the overall codon usage pattern described by rscu was straightforward. to investigate the effects of genetic composition, the labelling intervals of gc < . , . % < gc < . %, and gc > . % were also used to divide the cdss into three groups (fig. a) . the three cds groups were distributed along axis , indicating that the primary axis was the only characteristic that could account for the differences between the groups. considering the positions of the groups, and following the direction from negative to positive along axis , the three groups were distributed sequentially and discretely (i.e., gc < . %, then . % < gc < . %, followed by gc > . %). taken together, these results indicate that the different gc content usages generated distinctly different distributions of rscu values, and that effects of nucleotide composition were clearly present in the codons. to investigate rscu usage patterns among codons, codon positions were also analysed using pca. all codon triplets, excluding stop codons and codons encoding met and trp, were divided into four groups according to the ending base (fig. b) . distribution regions were overlapped between codons ending in a and u, or codons ending in g and c. the au and gc pairs had similar rscu usage patterns, indicating general effects of mutation pressure on codon usage. further comparisons within each group indicated that pairs ending in au were more concentrated than pairs ending in gc. additionally, g-ending codons were extensively distributed across three quadrants, which is the most varied among all codons. rscu usage values for c-and g-ending codons occurred independently of codon type, indicating that the cor g-favouring usage bias was driven predominantly by selection constraints. rscu usage patterns are formed according to complex evolutionary forces; considering that the rscu analysis showed that c-ending codons were generally preferred by evolutionary pressures (table ), these results suggest that the direction of these forces may inherently favour g and c bases. usage trends in amino acid composition were analysed using pca with axes (fig. c) . the accumulative variance in the first six axes explained over half of the total amino acid variation and, accordingly, they were considered to be the principle components. the first axis accounted for . % of the total variation and the second axis accounted for . %. the amino acid primary axis was not correlated with either the enc value (r = . ; p > . ) or the rscu primary axis (r = . ; p > . ); thus, amino acid composition does not affect codon usage patterns in the h. manillensis transcriptome. weak linear correlations were found between protein length and enc (r = . , p > . ), and between protein length and rscu primary axis (r = . , p > . ). unlike amino acid composition, we observed two patterns when we analysed the variation between different proteins: short proteins had varying enc values, and there was more variation in protein length with higher enc values. codon usage bias is affected by protein length, and we have inferred that they might be negatively related. to test our hypotheses, we plotted enc against protein length, yielding results that supported our hypothesis (fig. d) . there was a gaussian distribution between protein length and enc. short protein products that contained less than amino acids had the greatest variation in enc values. the majority of data points were distributed between enc values of and . of the factors used in this analysis, length explained the greatest variation in enc values, with results ranging by a factor of between the lowest and highest values. these observations indicated that protein length affected codon usage as a result of selection constraints. although an overall weak codon bias was not exclusive to proteins of certain lengths, longer protein products had a weaker codon usage bias. conversely, shorter proteins did not particularly affect the codon usage; therefore, their usage patterns were predominantly shaped by other selective forces. hydrophobicity and aromaticity of the amino acids for each protein were not correlated with enc (r = . and . , respectively; p > . ) or with the primary rscu axis (r = . and . , respectively; p > . ). this result agreed with our finding that amino acid composition had little impact on codon usage. thus, it is unlikely that codon usage bias in h. manillensis is influenced by hydrophobicity or aromaticity. furthermore, because hydrophobicity and aromaticity determine the way proteins fold and how structures are configured in living organisms, we inferred that selection pressure during the protein folding process has little genomic effect. thus, protein length affects codon usage, albeit only through its effects on translation. in our study, unigenes were annotated by at least one database, which was helpful for analysing their various functions. h. manillensis has a blood-rich diet and its anticoagulant factors are a scientifically and medically interesting feature; therefore, we selected cdss from genes reported to encode anticoagulant products. their codon usage patterns, which were calculated as gc /gc ratios and enc values, were compared to the average values obtained from all cdss to measure the effects of different evolutionary states of anticoagulant-related mechanisms. in particular, we aimed to examine whether the anticoagulant genes had more adaptive modifications, which would help in understanding the evolutionary mechanism that drives anticoagulant characteristics. the results showed that unigenes that encode genes such as hirudin, heparin, and eglin have plotted data points that are above the average gc /gc ratios and enc values, indicating generally weaker codon usage than average (fig. ) . the effects of modifications on the codons for most direct anticoagulant genes were relatively relaxed, indicating that they were less adaptively evolved than were other parts of the genome, and that the genomic profile for the blood diet in h. manillensis matured before this species evolved from its ancestor. moreover, we found that the four genes that encode serpin, filcolin-like , thrombospondin type , and low-density lipoprotein receptor-related protein had lower enc values than average. in particular, thrombospondin type and low-density lipoprotein receptor-related protein also had a gc /gc ratio below average. interestingly, for these genes, a homologous sequence can be found in the genome of the host species, indicating an evolutionary force that has driven these genes to emerge and correspond to their host-specific adaption. transcriptome sequencing enriches our understanding of the h. manillensis genome and identifies codon usage patterns next generation transcriptome sequencing technology can greatly increase the quantity of known mrna sequences for a given organism [ , ] . to date, fewer than mrna sequences from the h. manillensis genome have been reported to ncbi. thus, our work, which retrieved , transcripts, greatly expands the molecular database for h. manillensis. after strictly filtering these transcripts, the identified cdss provide an overview of exons and the proteins that they produce, effectively identifying molecules with direct biological functions. our results demonstrate that at(u)/gc nucleotide usage differs between the three codon positions, and that these composition differences likely drive the overall codon usage bias in h. manillensis. however, codon bias was limited overall. because many codons are used during translation, we further hypothesised that extreme codon usage bias may only develop in particular or limited conditions. additionally, our results show that g and particularly c are more common in third codon positions than expected. third-position codon bases are directionally substituted, possibly because of effects from natural selection or mutation pressure. identifying which of these forces drive the preference for c-ending codons helps elucidate the roles different forces can play in the evolution of h. manillensis. because synonymous codons are not equivalent by their nature, the preference for ending bases may be caused by directional substitution, as a result of selection constraints acting against a background of mutation pressure. our results indicate that overall expression levels regulate genomic composition and modify codon usage bias. the direction of this bias manifests as increased expression that leads to stronger codon usage bias and more mutational substitution toward gc. thus, selection plays a dominant role in shaping the codon usage bias in h. manillensis. future efforts to identify factors that lead to codon usage bias via base selection, including expression levels, protein lengths, and amino acid proportions, will provide greater understanding of the formation of particular codon usage patterns. transcriptome data have additional advantages in the analysis of codon usage [ ] ; for example, the expression levels of genes can be detected and described in terms of fpkm values. according to the mutation-selection-drift theory, selection constraints can direct a preference toward codons that promote translation efficiency. values such as fpkm can be essential for these types of analyses; thus, previous studies that were unable to obtain absolute results were obliged to use one of several theoretical indices to measure expression rates, such as cai, codon bias index (cbi), or frequency of optimal codons (fop) [ ] [ ] [ ] [ ] . although these indices are statistically valid and have helped describe the formation of codon usage patterns in several species [ , , , ] , their use remains problematic because of their dependence on the correct identification of optimal codons, which vary between genes and organisms and do not always promote translation efficiency. for instance, in humans, cell-and tissue-specific trna composition is determined solely by translational elongation rates and optimal codons have no effect on this determination [ ] [ ] [ ] , which limits the ability to make certain predictions from transcriptional data. accordingly, we conclude that using fpkm values (actually, the log -transformed values) for genes obtained from transcriptome data generate more fig. comparisons of gc /gc ratio and enc values between different functional categories. the average enc and general gc /gc values were used to illustrate the differences of these cdss from the overall tendency, the average enc value is obtained as . and general gc /gc ratio is calculated to be . accurate results and that these values also help refine existing theories for the calculation of codon usage indices. additionally, transcriptome data allow for detailed identification of unigenes and cdss at various magnitudes, which in turn assists in identifying genomic effects on biological functions. we identified unigenes by comparing homology with nr, go, kegg, and several other databases, and removing those that lacked annotations to ensure that each cds was described in at least one unigene database [ ] [ ] [ ] [ ] . these cdss could be divided into different categories and comparison of these categories could be used to measure the differential effects of the relevant selection pressures in h. manillensis. we plan to use these comparisons in future studies to identify and characterise economically valuable components of the h. manillensis genome, such as genes that contribute to anticoagulant production. the results of these future analyses could lead to viral-mediated gene therapy to harness their potential to benefit human health. characterising codon usage patterns in h. manillensis was one of the principle aims of our study and the data provide the basis for future analyses that will detect evolutionary pressures. the overall codon usage bias that we detected in h. manillensis was not extreme, but did show a preference for gc-ending codons, particularly c-ending codons. similar biases have been detected in other species, but mostly invertebrates [ , , , , [ ] [ ] [ ] . in the leafhopper homalodisca coagulata, out of preferred codons ended in c [ ] , and in the cestode t. pisiformis, out of preferred codons were gc-ending, with ending in c [ ] . similar patterns across species that suggest long-term evolution in codon usage could be regulated by common biological mechanisms in invertebrates. the factors affecting codon bias in other species are likely to contribute to the codon bias that we characterized in h. manillensis as well. the uniqueness of codon usage in h. manillensis could be related to the different evolutionary forces that contributed to the preferences for specific usage effectors, including nucleotide composition, gene expression, and protein length. almost all previous studies on metazoans that used enc and neutrality plots support the conclusion that selection has a greater effect on codon usage than mutation pressure [ - , , , , - , - ] . in these cases, enc values deviated from expectations. gc was positively correlated with gc , and gc was lower than gc , which are consistent with the results we presented for codon usage patterns in h. manillensis. the enc values for the cdss we examined did not follow the standard expenc curve, but gc did correlate with the shallow but linear slope ( . ) of gc . although some enc values fell on the expected curve, we believe that they do not refute the overall pattern of random codon usage in this species; the random codon usage that we identified was most likely caused by strong selection over long periods of evolution, which is the driving force behind existing codon usage bias in h. manillensis. in addition to identifying selection as the driving force behind codon usage bias in h. manillensis, we measured the effects of the factors that produce selection pressure on codon usage patterns. the overall effect of gene selection is the maintenance of efficient gene expression; however, selection pressure from previous translation processes, also known as the translation elongation rate, or certain subsequent protein products could also contribute to this selection process. previous studies have shown that both conditions occur in different species. in many viruses such as bovine coronavirus or zika [ , ] , protein products are not contributing factors, whereas in plants like zea mays [ ] the effects of proteins products are significant. in our study, we investigated factors such as nucleotide content, expression levels, amino acid composition, protein length, hydrophobicity, and aromaticity to evaluate their relationships with codon selection. of these factors, only protein length had selective effects on codon usage; however, the effects of protein length were strictly determined by cds length and overall, attributable to the translation elongation rate. thus, we conclude that codon usage bias in h. manillensis is shaped only by translational mechanisms, the final protein products of which may be sufficiently conserved to be excluded from evolutionary processes. in h. manillensis, we identified preferred codons that used all four bases; however, the distribution of ending bases was uneven and favoured gc-ending codons. one possible explanation for this result is that preferred codons undergo selection that modifies ending bases from au to gc over long-term evolution. according to the mutation-selection-drift theory, preferred codons are selected because they better match the trna genes that promote translation efficiency [ ] . because we identified selection as the dominant force in h. manillensis evolution, we hypothesised that gc-ending codons would increase in frequency with increased gene expression. this speculation was largely supported by our comparative analysis of enc and gc content between the highest and lowest expressed genes in h. manillensis. we suggest that richer and more stable gc content in codons facilitates a more efficient use of trna in h. manillensis, as trna is the foundation of genetic mechanisms. as in many invertebrates [ , , , ] , preferred codons tend to include gc, and we suggest that this tendency is exhibited by other species and could be strengthened further by evolution. understanding these preferences facilitates the editing and promotion of the in vivo expression of target genes in h. manillensis. many previous studies have examined the molecular mechanisms and identified several functional genes in different types of leeches [ , , [ ] [ ] [ ] . the genes that encode hirudin, heparin, bdelin, and eglin have been characterised and are well known as typical anticoagulant-related sequences in medical leeches [ ] [ ] [ ] . the results of our gene annotations were largely similar to those reported in previous studies, which confirmed these as functional genes. using these genes as examples enabled us to evaluate their evolutionary status in h. manillensis. codon usage is predominantly affected by environmental forces and can be used to infer the differential effects of long-term evolution. the overall codon usage pattern could be representative of the current genetic nature of this species, while the draft status of these genes is more or less modified by differentiation during adaptation to specific environmental conditions. thus, we inferred the existing adaptions in these functional anticoagulant genes and found that relaxed selection likely occurred. the majority of blood-diet-related genes have undergone less adaptive evolution in h. manillensis, except for those with homologous sequences in the host species. based on these findings, we further suggest that the basic evolutionary mechanisms of genes in h. manillensis, especially genes common in other phylogenetically related leeches, have promoted genes that have undergone less adaptive selection than their genome due to their mature molecular mechanism. only genes that have newly participated in evolution will be modified by stronger selection, as revealed in their codon usage. this study characterised the transcriptome of h. manillensis, a leech known for its medicinally significant anticoagulant properties. the nature and functional pathways of all genes were identified in this species, and the effects of different evolutionary factors on codon usage were characterised and measured. as with other invertebrates, we found that in h. manillensis there is a preference for gc-ending codons, particularly those ending in c, and concluded that the primary contributing force to this preference is selection rather than mutation. these conclusions contribute to our knowledge of the h. manillensis genome, improve our understanding of invertebrate genomics, and provide further validation of genomic tools for indirectly measuring evolutionary processes. the h. manillensis samples used in this study were obtained from ponds in hechi city, guangxi province, china. h. manillensis is an invertebrate and is not endangered; there are no restrictions on the capture of h. manillensis individuals. following taxonomic identification, samples were stored in liquid nitrogen until rna extraction. to obtain the transcriptome, an illumina hiseq high throughput sequencing platform was used for sequencing (illumina, san diego, ca, usa). two cdna libraries, designed t and t , were derived from the extracted total rna of two h. manillensis individuals and were used for sequencing. libraries were prepared with a nebnext ultratm dna library prep kit (new england biolabs, ipswich, ma, usa) following the manufacturer's instructions. a total of . gb of sequence data was collected for t and t , comprising , , clean reads with q and q values higher than . %. reads were deposited in the ncbi database (project number: prjna , accession numbers: srx and srx ). sequence assembly was conducted in trinity . . (http://trinityrnaseq.github.io/) using default settings [ ] . transcripts were identified using transdecoder, which is a function within trinity [ ] . to avoid instrumental errors, identified transcripts were confirmed using blast (https://blast.ncbi.nlm.nih.gov/blast.cgi) and only those that were present in both t and t were retained [ ] . blast results of the common sequences were saved into one file and , transcripts were obtained. further, to exclude all possible contaminants such as bacterial or other types of rna (e.g., trnas and rrnas) that could bias the results, we built a high confidence dataset from the transcripts by blasting our results to the transcriptome data of poecilobdella javanica (srr ), a species that is phylogenetically close to h. manillensis. with the blastx e-value set to e- , we obtained , transcripts with an average length of . bp. the unigenes were identified using these highly confident transcripts and the final number of unigenes that we identified was , . prior to identifying and annotating the cdss, all obtained unigenes were first annotated using blast in the following databases: nr (ncbi non-redundant protein sequences) [ ] , pfam (protein family) [ ] , cog/egg-nog (clusters of orthologous groups of proteins) [ , ] , kegg (kyoto encyclopedia of genes and genomes) [ ] , and go (gene ontology) [ ] . the go database can be used to identify the biological functions of genes in different functional categories, and the kegg pathway database can be used to identify the series of interactions caused by genes that produce new molecules or lead to other changes within cells. the blast parameters x, p, and r were set under e-value = e- . unigenes that were not annotated were excluded from further analysis. the remaining unigenes were used in the identification of cdss using orffinder (http://www.geneinfinity.org/sms/sms_orffinder.html). to ensure the quality of these coding sequences, low quality sequences that were smaller than bp, contained internal n gaps, or included too many internal stop codons were removed. finally, a total of , qualified cdss were identified and annotated in at least one database. [ ] were used with the default parameters to calculate the essential raw values used in this study. for rscu values, a standard codon table was chosen. the enc values, which represent the capacity for codons to encode amino acids and are negatively related to codon bias, were calculated automatically in codonw while calculating rscu. these two parameters were used to describe codon usage patterns. several indices such as the overall gc content, gc content at the first, second, and third codon positions (gc , gc , gc ), amino acid composition, protein length, aromaticity (aromo) values, and general average of hydrophobicity (gravy) values were also calculated. in particular, reliable nucleotide content for single nucleotides and dinucleotides, amino acid composition, and protein lengths were conducted in dambe and validated using codonw. gravy and aromo values represent the mean frequency of hydrophobic and aromatic amino acids among gene products, respectively, and were calculated in codonw. all these results can be accessed in additional files , , and . additional file contains the values of enc, expected enc, gc content, protein length, gravy, and aromo. additional file contains the rscu values of each sequence and the first pca factors. additional file contains the proportion of amino acids in each sequence and the first pca factors. enc plots are scatter diagrams that compare observed enc against expected enc (expenc) for different gc (g or c in the third codon position) values. when observed enc values fall on the curve of expected enc values for a given sequence, mutation pressure is the sole force acting on third-position bases in that sequence. to make this plot, we submitted strictly increasing gc values from . to . to dambe to obtain their expenc values. these values were then used to draw a standard curve; a smooth curve was selected to connect these value points. we then plotted all of the points that represented the observed enc and gc values from each sequence to the curve to make the final plot. the obtained gc and gc (mean gc and gc ) values were plotted on the lateral and vertical axes, respectively, to produce a scatter diagram for the neutrality plot. the formula between the gc and gc was estimated with a linear model using spss . . neutrality plots are primarily used to assess the mutation pressure bias that affects codon usage bias. when the slope is , codon usage bias is driven solely by neutrality. the effects of other forces such as selection constraints can generally be inferred when the slope deviates from . the parity rule -bias plot was constructed using the values obtained from the formulae a /(a + u ) and g /(g + c ). these data were visualised in a scatter diagram with a plot root of , and thus all data points were distributed into four quadrants. rscu values are particularly useful for describing codon usage bias because they indicate the precise frequency of the appearance of each synonymous codon in a given coding sequence. however, using a matrix of rscu values directly can cause difficulties in the identification of the major driving force of codon usage bias, and the connections between factors involved. pca can be used as a dimensionality reduction tool to significantly reduce the data complexity by using principal components to reflect overall patterns. with this method, a matrix comprising complex rscu values can be transformed into just a few major axes. furthermore, by combining and illustrating other codon usage-related factors, the effects of these target factors are shown more intuitively at the macro level. pcas were defined using rscu values and the proportion of amino acids as factors in spss . (spss inc. software, chicago, illinois, usa). we included all codons (excluding three stop codons) and amino acids in the cdss to compare gc content and codon usage with the rscu values. we selected four principal components among the rscu values and two major groups were correlated with gc content to assess whether gc was a contributing factor to rscu values. the pca of rscu values in the cdss of unigenes was used to separate codons, whereas the pca of the proportion of amino acids was used to measure cumulative inertia and identify patterns in amino acid usage. pca initially produces a series of orthogonal axes that can classify trends that represent variation in the data, with each subsequent axis contributing a smaller proportion to the variation. pca locates each codon or amino acid on these axes and the location is the combination of columns (codons or amino acids) and rows (genes) and is highly distinguishable between different groups. evolution of synonymous codon usage in 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functional annotations for eukaryotic, prokaryotic and viral sequences analysis of codon usage dambe : a comprehensive software package for data analysis in molecular biology and evolution this work was financially supported by the excellent doctor innovation project of shaanxi normal university (s yb ), the fundamental research funds for the central universities ( ts ), and the fundamental research funds for the central universities (gk ; gk ; gk ). this work was supported in part by the national natural science foundation of china (no. ). these funding bodies had no role in the design of the study, collection, analysis, and interpretation of data, or in writing the manuscript. the rna sequence dataset supporting the results of this article is available in the repository of ncbi sequence read archive (sra) with the genbank accession no.: srx and srx . the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ethics approval and consent to participate mature h. manillensis samples were collected from ponds in hechi city, guangxi province, china. as prescribed by "law of people's republic of china on the protection of wildlife" and "regulations for the implementation of the people's republic of china on the protection of terrestrial wildlife" (state council decree [ ] no. ), the specimen collection did not ask for any ethical or institutional approvals, because h. manillensis is not endangered or protected by any current law. the care and treatment of animals in this study were performed according to the guideline for the care and use of laboratory animals in china. not applicable. key: cord- - iqjjema authors: wang, wei; ma, yuanhui; wu, tao; dai, yang; chen, xingshu; braunstein, lidia a. title: containing misinformation spreading in temporal social networks date: - - journal: chaos doi: . / . sha: doc_id: cord_uid: iqjjema many researchers from a variety of fields including computer science, network science and mathematics have focused on how to contain the outbreaks of internet misinformation that threaten social systems and undermine societal health. most research on this topic treats the connections among individuals as static, but these connections change in time, and thus social networks are also temporal networks. currently there is no theoretical approach to the problem of containing misinformation outbreaks in temporal networks. we thus propose a misinformation spreading model for temporal networks and describe it using a new theoretical approach. we propose a heuristic-containing (hc) strategy based on optimizing final outbreak size that outperforms simplified strategies such as those that are random-containing (rc) and targeted-containing (tc). we verify the effectiveness of our hc strategy on both artificial and real-world networks by performing extensive numerical simulations and theoretical analyses. we find that the hc strategy greatly increases the outbreak threshold and decreases the final outbreak threshold. many communications platforms, e.g., twitter, facebook, email, whatsapp, and mobile phones, allow numerous ways of sharing information [ ] [ ] [ ] [ ] [ ] [ ] . one task for researchers is developing ways to distinguish between true and false information, i.e., between "news" and "fake news" [ ] . this task is important because access to true information is essential in the process of intelligent decisionmaking [ ] [ ] [ ] . for example, when the severe acute respiratory syndrome (sars) spread across guangzhou, china in , the chinese southern weekly published a newspaper article entitled "there is a fatal flu in guangzhou." this information was forwarded over million times by tv news and in other newspapers [ , ] . individuals receiving this true information could adopt simple, effective protective measures against being infected (e.g., by staying at home, washing hands, or wearing masks). misinformation, on the other hand, encourages irrational behavior and reckless decision-making, and its spread can undermine societal well-being and sway the outcome of elections [ ] [ ] [ ] [ ] . bovet and makse [ ] analyzed million tweets sent during the five months prior to the us presidential election and found that misinformation strongly affected the outcome of that election. to contain the spread of misinformation we must understand the dynamic information spreading mechanisms that facilitate it [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . vosoughi et al. [ ] examined true and fake information on twitter from to and found that misinformation spreads more quickly than true information. using the spreading mechanisms common in real-data analysis, researchers have proposed several mathematical models to describe the spreading dynamics of true and fake information [ ] [ ] [ ] [ ] [ ] [ ] . moreno et al. [ ] developed mean-field equations to describe the spread of classical misinformation on static scale-free networks that enables a theoretical study not requiring extensive numerical simulations. borge-holthoefer and moreno [ ] found that although there are no influential spreaders in the classical misinformation model presented in ref. [ ] , nodes with high k-cores and ranking values are more likely to be the influential spreaders of true information and also of infectious diseases [ ] [ ] [ ] [ ] [ ] . when we include the burst behavior of individuals in the misinformation model, hubs emerge as influential nodes [ ] . using real-world data, researchers found that social networks evolve with time, and thus evolving temporal networks more accurately represent the topology of real-world networks than static networks [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . researchers have found that the temporal nature of networks strongly affect their spreading dynamics. perra et al. [ ] found that in susceptible-infected-susceptible (sis) epidemic spreading a temporal network behavior suppresses the spreading more effectively than a static integrated network. researchers have also found that the sis and susceptible-infected-recovered (sir) models on temporal networks exhibit the same outbreak threshold [ ] [ ] [ ] . nadini et al. [ ] found that tightly connected clusters in temporal networks inhibit sir processes, but accelerate sis spreading. pozzana et al. found that when node attractiveness-its role as a preferential target for interactions-in temporal networks is heterogeneous, the contagion process is altered [ ] . karsai et al. [ ] found that strong ties between individuals strongly inhibit the classical spreading dynamics of misinformation. several strategies for containing the spread of misinformation in temporal networks have been proposed [ ] [ ] [ ] [ ] . liu et al. [ ] examined epidemic spreading on activity driven temporal networks and developed mean-field based theoretical approaches for three different control strategies, i.e., random, targeted, and egocentric. the egocentric strategy is most effective. it immunizes a randomly selected neighbor of a node in the observation window. other effective approaches using extensive numerical simulations have been proposed [ ] [ ] [ ] . for example, holme and liljeros [ ] take into consideration the time variation of nodes and edges and propose a strategy for containing the outbreak of an epidemic based on the birth and death of links. because there is still no theoretical approach to containing the spread of misinformation in temporal networks, we here systematically examine its spread in activity-driven networks. the rest of the paper is organized as follows. section ii describes the misinformation spreading dynamics on temporal networks and develops a theory to describe the spreading dynamics. section iv proposes three containment strategies. section v describes the results of our extensive numerical stochastic simulations, which show that our suggested theory agrees with the numerical simulations. section iv presents our conclusions. we here introduce our model for the spreading dynamics of misinformation in temporal networks. the widely used approaches mathematically describing temporal networks tend to be either eventbased or snapshot representations [ ] . the event-based representation approach describes de-scribes temporal networks using ordered events {u i , v i , t i , ∆t i ; i = , , · · · }, where node u i and v i are connected at time t i in the time period ∆t i . the snapshot approach describes temporal networks using a discrete sequence of static networks g = {g( ), g( ), · · · , g(t max )}, where g(t) is the snapshot network at time t, and t max is the number of snapshots of the temporal network. each snapshot network g(t), contains n nodes, where n is fixed, and m t edges. thus the average temporal degree of snapshot network g(t) is k t = m t /n. using the adjacency matrix, we here adopt the snapshot approach to describe temporal networks. as the meaning of the adjacency matrix in static networks, a uv (t) = when nodes u and v are connected at time t, for undirected temporal networks. the average degree of node u in the temporal network g is knowing the adjacency matrix a we obtain the eigenvalues of a. here Λ (a) Λ (a), · · · , and Λ n (a) are the eigenvalues of a in decreasing order. the spectral radius is thus Λ (a), which quantifies the threshold outbreak of epidemics in temporal networks. we use the classical activity-driven network [ ] to model a temporal network with n nodes. we build the activity-driven network using the following steps. . we assign to each node i an activity potential value x i according to a given probability density distribution f (x). the activity of node i is a i = ηx i , where η is a rescaling factor, i.e., at each time step, node i is active with probability a i . the higher the value of η, the higher the average degree of the temporal network. the higher the value of a i , the higher the degree of node i. we assume that f (x) follows a power-law function, i.e., f (x) ∼ x −γ , where γ is the potential exponent. we make this assumption in order to generate a heterogeneous degree distribution temporal network. after further calculations we find and ǫ is the minimum value of the activity potential x i . each active node has m edges, and each edge randomly links to a network node. an edge connects the same pair of nodes with probability m/n. note that in the thermodynamic limit of a sparse temporal network, there are no multiple edges between nodes and non-local loops . at the end of time step t, we delete all edges in network g(t). ( ) and ( ) until t max in order to generate temporal network g. we use an ignorant-spreader-refractory model to describe the spreading dynamics of misinformation [ ] . here nodes are classified as either ignorant, spreader, or refractory. ignorant nodes are unaware that the information is false but are susceptible to adopting it. spreader nodes are aware that the information is false and are willing to transmit it to ignorant nodes. refractory nodes receive the misinformation but do not spread it. the misinformation spreading dynamics on temporal networks evolves as follows. we first randomly select a small fraction ρ of spreader nodes to be seeds in network g(t ), where ≤ t ≤ t max . we designate the remaining − ρ nodes to be ignorant. at time step t each spreader i transmits with probability λ the misinformation to ignorant neighbors in network g(t). in addition, each spreader i becomes a refractory node with a probability where µ is the intrinsic recovery probability, and n(t) is the number of nodes in the spreader and refractory states of node i. the dynamics evolve until there are no spreader nodes. note that when t reaches t max , the misinformation spreads on g( ) in the next time step. figure shows misinformation spreading on a temporal network. we here develop a generalized discrete markovian approach to describe the misinformation spreading dynamics on temporal networks [ ] . we denote i i (t), s i (t), and r i (t) to be the fraction of nodes in the ignorant, spreader, and refractory states, respectively, at time t. because a node can only be in one of the three states, an ignorant node i, becomes a spreader with probability p i (t) at time t, where here is the probability that node i has not received any misinformation from neighbors at time t in g(t). at time step t + , node i remains ignorant with a probability the decrease of i i (t) is equal to the increase of s i (t), because an ignorant node will become a spreader when it obtains the information from neighbors in state s. in addition, spreader i becomes . thus the evolution of node i in the spreader state is the evolution of node i in the refractory state is using eqs. ( )-( ), we obtain the fraction of nodes at time t in each state, where h ∈ {i, s, r}. note that in the steady state there are no spreader nodes, only refractory and stifler nodes. the fraction of nodes that receive the misinformation in the final state is r(∞) = r. here r is the order parameter of a continuous phase transition with λ. if the misinformation transmission probability λ is larger than the critical threshold, i.e., λ > λ c , the size of the global misinformation is of the order of the system size. otherwise, the global misinformation r = for λ ≤ λ c is in the thermodynamic limit. at shorter times, a vanishingly small fraction of nodes receive the misinformation, i.e., s i (t) ≈ and r i (t) = − s i (t) − i i (t) ≈ . the recovery probability in eq. ( ) of a spreader node i is µ i (t) ≈ µ, since node i must connect to a spreader that supplies the misinformation, and there is a low probability that it will connect to other spreader or refractory neighbors. thus eq. ( ) can be rewritten where δ ij is the kronecker delta function, i.e., δ ij = if i = j, and zero otherwise. we define the transmission tensor m to be we mask the tensorial origin of the space through the map inserting eq. ( ) into ( ), we have where s(τ ) is the probability that a node is in the spreader state at each time step t during here s(τ ) increases exponentially if the largest eigenvalue of m, denoted Λ , is larger than . thus the misinformation spreads, and the threshold condition is [ ] Λ (m) = , in an unweighted undirected network g, the largest eigenvalue Λ (m) of m is where p = tmax t= ( − µ + λa(t)). because misinformation spreading on social networks can induce social instability, threaten political security, and endanger the economy, we propose three strategies-random, targeted, and heuristic-for containing the spread of misinformation in temporal networks using a given fraction of containing nodes f . we first immunize a fraction of f nodes using a static containment strategy. the misinformation then spreads on the residual temporal network. if node i is "immunized," it cannot be infected (transmit) by the misinformation received from neighbors (the misinformation to neighbors). mathematically the immunized node set is v, and the number of immunized nodes equals the number of elements in v, i.e., |v| = ⌈f n⌉. we set v i = if node i is immunized, otherwise v i = . after immunization, eqs. ( )-( ) can be written and respectively. in an effective containing strategy the misinformation spreading dynamics is suppressed for a given fixed fraction f of immunized nodes, i.e., the objective function is where the constraint conditions are eqs. • strategy i: random containment (rc). the most used strategy for containing the spread of misinformation is randomly immunizing a fraction of f nodes [ ] . • strategy ii: targeted containment (tc). another intuitive way is to immunize the nodes with highest average degree k in the temporal network g. specifically, we first compute the average degree of each node i as k i = tmax tmax t= n j= a ij (t). we then rank all nodes in descending order in the vector w according to the average degree of each node. finally we immunize the top ⌈f n⌉ nodes of w. • strategy iii: heuristic containment (hc). using the tc, we apply an hc strategy. because tc is much better than rc, we perform the hc strategy by replacing the immunization nodes. when the repeat time is very large, the immunized nodes reach an optimal value. (i): we initialize a vector w according to the descending order of the average degree of nodes. the first ⌈f n⌉ nodes of w are immunized, the final misinformation outbreak size is r o , and w is denoted a set. the remaining nodes w = w\w are denoted a set. (ii): we randomly select nodes in w and w , denoted v and v , respectively. we switch their order in vector w and denote the new vector w n . we immunize the first ⌈f n⌉ nodes w n and compute the final misinformation outbreak size r n . (iii): when r n > r o , we update vector w, i.e., w → w n . otherwise, there is no change. (iv): we repeat steps (ii) and (iii) until ts ts i= |w −w n | < ǫ ′ . in the simulations we set t s = and ǫ ′ = n − . for the activity-driven network, we set n = , t max = , η = , m = , γ = . , and ǫ = − . for real-world networks, we use the data collected by the sociopatterns group [? ] , which records the interactions among the participants at a conference. the time resolution of the signal is sec. because the temporal network is sparse, it is difficult for the information to spread in the original network. we thus aggregate the temporal network using four windows, w = min, min, min, and min. we average all simulation results more than times. we use variability to locate the numerical network-sized dependent outbreak threshold [ , ] , where r is the relative size of misinformation spreading at the steady state. at the outbreak threshold λ c , χ exhibits a peak. when λ ≤ λ c , the global misinformation does not break out, but when λ > λ c the global misinformation does break out. figure shows the misinformation spreading on activity-driven networks. note that the final misinformation outbreak size r increases with λ. the larger the recovery probability µ, the lower the values of r because spreader nodes are less likely to transmit the misinformation to stifler neighbors [see fig. (a) ]. note that our theoretical and numerical predictions of the final misinformation outbreak size r agree. figure (b) shows the variability χ as a function of λ. there is a peak at the misinformation outbreak threshold λ c . figure shows λ c versus µ in which λ c increases linearly with µ. the theoretical predictions of λ c obtained from eq. ( ) agree with the stochastic simulations. figure shows misinformation spreading in real-world temporal networks. as in fig. , r increases with λ and decreases with µ. as in sir epidemic spreading [ ] , the effective outbreak threshold (λ/µ) c is a constant value. in addition, when the value aggregating window w is small, there are fewer opportunities for spreaders to transmit the misinformation to stifler neighbors, thus the misinformation does not break out globally, i.e., there are smaller values of r for smaller w. once again our theoretical results agree with the numerical simulations. we next examine the performances of our proposed strategies for mitigating misinformation spreading on artificial and real-world temporal networks. figure shows r versus λ for different values of the fraction of containing nodes f . note that r decreases with f because no more nodes receive the misinformation. note also that the tc strategy performs much better than the rc strategy because the higher degree nodes k are contained, and spreaders can no longer transmit the misinformation to stiflers. thus when we immunize the same fraction of containing nodes, the values of r for the tc strategy are smaller than those for the rc strategy. for example, when f = . , using the rc strategy ≈ % of the nodes are informed by the misinformation, but using the tc strategy none are informed by the misinformation. in addition, the outbreak threshold λ c to contain the misinformation the fraction of nodes informed by the misinformation is finite, i.e., r ≈ . . our theoretical results agree with the numerical simulation results. an effective containing strategy with a fraction of immunized node f and a small outbreak threshold λ c greatly decreases the final misinformation outbreak size r. figure shows the effective outbreak threshold (λ/µ) c versus f on activity-driven networks for the rc, tc, and hc strategies. here (λ/µ) c increases with f , and (λ/µ) c is the largest using the hc strategy when f is fixed. when f is sufficiently large, no λ value can induce a global misinformation outbreak. we denote f c the critical probability that at least a fraction of f c nodes must be containing to halt misinformation speading in temporal networks. we find that the values of f c for the hc containing strategy are the smallest of all containing strategies. the f c value for the rc strategy is times the f c value for the tc strategy, and the f c value for the tc strategy is . times the f c value for the lines and symbols are the theoretical and numerical predictions of (λ/µ) c , respectively. the vertical line represents the critical probability f c . hc strategy. thus the hc strategy is the most effective. we finally examine real-world networks to varify the effectiveness of our proposed three strategies. figure compares the performances of the tc and hc strategies by examining r versus f for given values of λ. as in fig. , the hc strategy most effectively contains the misinformation spreading on temporal networks irrespective of the values of λ. in addition, fig. shows that the effective outbreak threshold (λ/µ) c is the smallest when using the hc strategy. thus our theory accurately predicts the numerical simulation results. we have systematically examined the dynamics of misinformation spreading on temporal networks. we use activity driven networks to describe temporal networks, and use a discrete marko- the lines and symbols are the theoretical and numerical predictions of r, respectively. vian chain to describe the spreading dynamics. we find that the global misinformation outbreak threshold correlates with the topology of temporal networks. using extensive numerical simulations, we find that our theoretical predictions agree with numerical predictions in both artificial and real-world networks. to contain misinformation spreading on temporal networks, we propose three strategies, random containing (rc), targeted containing (tc), and heuristic containing (hc) strategies. we perform numerical simulations and a theoretical analysis on both artificial and four real-world networks and find that the hc strategy outperforms the other two strategies, maximizes the outbreak threshold, and minimizes the final outbreak size. our proposed containing strategy expands our understanding of how to contain public sentiment and maintain social stability. this work was partially supported by the china postdoctoral science foundation (grant no. m ), and fundamental research funds for the central universities. lab thanks unmdp and con-icet weekly releases ieee th international conference on data mining complex spreading phenomena in social systems temporal networks a guidance to temporal networks temporal network epidemiology journal of physics: conference series key: cord- -adaow w authors: asensio martín, m. j.; hernández bernal, m.; yus teruel, s.; minvielle, a. title: infecciones en el paciente crítico date: - - journal: medicine - programa de formación médica continuada acreditado doi: . /j.med. . . sha: doc_id: cord_uid: adaow w resumen introducción las infecciones son muy frecuentes en los pacientes que se encuentran ingresados en los servicios de medicina intensiva, siendo unas veces motivo de ingreso y en otras la infección se adquiere durante el ingreso. epidemiologia las causas más frecuentes de infección adquirida en la comunidad que precisa ingreso en la uci son las infecciones respiratorias, infecciones urinarias y las infecciones del sistema nervioso central. dentro de las infecciones adquiridas en la uci, las asociadas a dispositivos son las más frecuentes. etiología los gérmenes más frecuentes en la uci son los gram negativos. etiopatogenia. en el paciente crítico se aúnan factores, haciéndolos especialmente vulnerables a las infecciones. manifestaciones clínicas dependerán de la localización de la infección. diagnóstico debe ser precoz dada su alta mortalidad. pronóstico las infecciones nosocomiales se asocian con un aumento de la mortalidad y la estancia. tratamiento el retraso en el tratamiento se asocia con un aumento de la mortalidad. abstract introduction infections are very frequent in patients who are admitted to intensive care units, sometimes being a reason for admission and in others the infection is acquired during icu stay. epidemiology the most frequent causes of acquired infection in the community that require admission to the icu are respiratory infections, urinary tract infections and infections of the central nervous system. among the infections acquired in the icu, devices-associated infections are the most frequent. etiology the most frequent in icu are gram negative pathogens. etiopathogenesis in the critical patient, several factors are combined making them especially vulnerable to infections. clinical manifestations depends on the location of the infection. diagnosis it must be early due to its increased mortality. prognosis nosocomial infections are associated with an increase in mortality and in the length of stay. treatment the delay in treatment is associated with an increase in mortality. la prevalencia de la patología infecciosa en los servicios de medicina intensiva (smi) es elevada, siendo unas veces el motivo de ingreso en las unidades de cuidados intensivos (uci) y, en otras, la infección se adquiere durante su estancia en la misma. no obstante, existen marcadas diferencias, tanto en la frecuencia como en la etiología y la patogenia entre estas dos entidades infecciosas: la patología infecciosa comunitaria grave que requiere ingreso en la uci y la infección nosocomial (in) adquirida durante la estancia en la uci. la infección comunitaria grave es una de las principales causas de ingreso en los smi, tanto por las necesidades terapéuticas inherentes a la gravedad intrínseca del cuadro infeccioso como por el requerimiento de una adecuada monitorización del proceso. dentro del amplio campo de infecciones comunitarias que pueden requerir ingreso en los smi, destacan por su gravedad y frecuencia las neumonías, las infecciones del sistema nervioso central (snc) y las infecciones del tracto urinario (itu). la neumonía adquirida en la comunidad (nac) es una enfermedad infecciosa respiratoria aguda con una incidencia que oscila entre y casos por . habitantes por año, aumentando esta incidencia con la edad y las comorbilidades. el % de los pacientes con nac requiere ingreso hospitalario, y alrededor del % necesitan ser admitidos en una uci . la mortalidad global de la nac se sitúa alrededor del %, si bien en el subgrupo de pacientes que precisan ingreso en la uci la mortalidad es del - % . la prevalencia de los diferentes microorganismos causantes de la nac va a depender de diversos factores como la edad, la presencia de determinadas comorbilidades, los criterios diagnósticos utilizados o las pruebas diagnósticas empleadas. en la mayoría de los estudios, en cerca del % de los casos de nac no se puede demostrar la etiología. de los organismos aislados, el más frecuente es streptococcus pneumoniae, seguido de legionella pneumophila (cuya incidencia ha disminuido en los últimos años probablemente debido al uso extendido de las nuevas quinolonas y los macrólidos) y hamophilus influenzae. el tabaquismo y el tratamiento con corticoides son factores de riesgo para nac por legionella. las enterobacterias (sobre todo kebsiella pneumoniae) suelen provocar nac en pacientes con comorbilidades o antibioterapia previa. pseudomonas aeruginosa se identifica en un - % de las nac y generalmente en relación con la presencia de bronquiectasias, fibrosis quística, enfermedad pulmo-nar obstructiva crónica (epoc) grave, neoplasia o neutropenia y staphylococcus aureus se suele presentar tras infecciones víricas. los microorganismos atípicos, mycoplasma pneumoniae y clamydophila pneumoniae, a menudo son patógenos y pueden ocasionar cuadros graves . la mejoría en las pruebas diagnósticas con técnicas de biología molecular ha dado lugar a un aumento en el aislamiento de virus como causa etiológica, principalmente rinovirus e influenza . generalmente en adultos se presentan como copatógenos asociados a neumococo, h. influenzae y s. aureus. hay que resaltar que en una tercera parte de los casos de nac se aíslan dos o más patógenos, generalmente una combinación de bacterias y virus. en los últimos años, nuevos patógenos han emergido como causa de nac. el metapneumovirus, aislado por primera vez en el año , aunque típicamente se asocia con una enfermedad más leve, se ha relacionado con casos mortales de neumonía. los coronavirus también han surgido como grandes amenazas epidémicas, primero como un síndrome respiratorio agudo severo (sras) y, más recientemente, con el síndrome respiratorio de oriente medio (mers). el virus influenza también continúa siendo una amenaza, con la posibilidad de que varias cepas de influenza aviar, particularmente h n y h n , muten lo suficiente como para permitir una transmisión sostenida de ser humano a ser humano con el resultado de pandemias . en pacientes que precisan ingreso en la uci como patógenos causantes de la nac son frecuentes el neumococo resistente, s. aureus y legionella pp.. las manifestaciones clínicas habituales son fiebre mayor de ºc y afectación del estado general, acompañada de tos, expectoración, dolor torácico, disnea o taquipnea. el diagnóstico se basa en la existencia de clínica compatible, junto con presencia de ocupación del espacio alveolar en la radiografía de tórax. el hallazgo de un infiltrado en la radiografía es el patrón oro para establecer el diagnostico ( fig. ). para el diagnóstico etiológico es necesaria la realización de hemocultivos, tinción de gram y cultivo de esputo determinación de antígeno en orina de s. pneumoniae y l. pneumophila. la serología está indicada para el diagnóstico de neumonía por m. pneumoniae y clamydophila pneumoniae (título elevado de anticuerpos igm en el suero de la fase aguda y/o seroconversión del título de igg en el suero de la fase de seroconversión). igualmente la serología es la técnica diagnóstica en caso de que por contexto epidemiológico se sospeche infección por coxiella burnetti (fiebre q) o francisella tularensis (tularemia). las técnicas de biología molecular están indicadas en nac graves, en las que no se ha logrado establecer el diagnóstico etiológico por los medios habituales. asimismo, en determinados periodos epidémicos está indicada la detección de virus respiratorios, como el virus de la gripe en aspirado nasofaríngeo mediante inmunofluorescencia e inmunocromatografía, aunque el patrón oro sigue siendo el cultivo vírico. la toracocentesis diagnóstica con cultivo para aerobios y anaerobios está indicada en pacientes hospitalizados con derrame pleural significativo, no solo para el diagnóstico sino también porque el desarrollo de empiema es uno de los principales factores asociados a mala evolución de la nac . en pacientes con nac retrasar el ingreso en la uci en los pacientes que lo precisan se acompaña de un incremento considerable de la mortalidad. la sociedad americana del tórax (ats) y la sociedad americana de enfermedades infecciosas (idsa) proponen el ingreso en la uci de los pacientes que presenten shock séptico o insuficiencia respiratoria que precise ventilación mecánica (un criterio mayor), así como de los pacientes que presenten al menos de los criterios menores de la tabla . recientemente, las recomendaciones de la european respiratory society and european society for clinical microbiology and infections diseases , aconsejan el ingreso en la uci ante la presencia de al menos dos de las siguientes circunstancias: presión arterial sistólica menor de mm hg, presencia de insuficiencia respiratoria con pao /fio < o la afectación de o más lóbulos en la radiografía de tórax, o bien uno de los siguientes: necesidad de ventilación mecánica o necesidad de vasopresores durante más de horas (shock séptico), con un nivel de recomendación a . la administración precoz y efectiva de antibióticos puede disminuir la mortalidad de la nac grave, por lo que debe ser prioritario su inicio precoz. el espectro del tratamiento antibiótico debe ser amplio, capaz de cubrir los gérmenes etiológicos más probables, valorando la gravedad del cuadro clínico, factores de riesgo para microorganismos específicos, los patrones epidemiológicos propios de cada área geográfica y los patrones de resistencia (tabla ). etiología. la incidencia de meningitis bacteriana comunitaria es de - casos por . habitantes, siendo streptococcus pneumoniae y neisseria meningitidis responsables del % de los casos. son factores de riesgo la asplenia (neumococo y meningococo), agammaglobulinemia (neumococo) y el déficit del complemento (meningococo). en personas mayores de años, alcohólicos o jóvenes con alteración de la inmunidad celular, listeria monocytogenes suele ser el agente etiológico. en cuanto a las meningitis víricas, suelen tener una incidencia estacional, verano y otoño, y suelen estar causadas por enterovirus . manifestaciones clínicas. la clínica habitual cursa con fiebre, dolor de cabeza, rigidez de nuca y alteración de la conciencia, pero hasta un % presenta focalidad neurológica o convulsiones ( %). diagnóstico. la base del diagnóstico es el análisis del líquido cefalorraquídeo (lcr). en caso de existir focalidad neurológica, papiledema o convulsiones, debe realizarse una tomografía computadorizada (tc) craneal previa a la punción para descartar la existencia de efecto masa. en las meningitis bacterianas se observa pleocitosis ( . - . ) de predominio polimorfonuclear ( - %), glucosa baja (menos de mg/dl) y una ratio glucosa en lcr/glucosa en sangre igual o inferior a , y proteínas elevadas (más de mg/dl). por el contario, en las meningitis víricas, la pleocitosis es menor ( - células/mm ) de predominio linfocitario, glucosa normal y proteínas elevadas . ante la sospecha de meningitis debe realizarse de forma urgente tinción de gram del lcr, ya que permitirá una rápida identificación del germen en el - % de los casos, aunque su sensibilidad se reduce en pacientes que están recibiendo tratamiento antibiótico. tratamiento. el retraso en el tratamiento aumenta la morbimortalidad, por lo que este no debe demorarse por la realización de pruebas diagnósticas. se recomienda en las últimas guías clínicas administrar el tratamiento en la primera hora . el tratamiento debe incluir una cefalosporina de tercera generación (ceftriaxona g/ horas por vía intravenosa) asociada a vancomicina ( g por vía intravenosa/ horas) debido a que el neumococo presenta un % de resistencia a las penicilinas y un % a las cefalosporinas. si existen factores de riesgo para listeria se añadirá al tratamiento anterior ampicilina g por vía intravenosa cada horas y, en caso de sospecha de etiología vírica, añadir aciclovir mg/kg por vía intravenosa cada horas en adultos. debe administrarse en caso de sospecha de meningitis bacteriana, previo o de forma simultánea al tratamiento antibiótico, dexametasona en dosis de , mg/kg cada horas (adultos mg cada horas) durante días . tras los resultados del cultivo del lcr se modificará el tratamiento según antibiograma. etiología. hasta en un % de los casos de encefalitis no se logra identificar el agente etiológico, siendo los virus, sobre todo enterovirus, herpex simple tipo i y virus del nilo occidental, los gérmenes principalmente implicados. en las encefalitis existe afectación del parénquima cerebral, por lo que a la fiebre y la ce-falea se asocia una alteración del nivel de conciencia, junto con convulsiones y déficit motor. diagnóstico. el análisis del lcr es la base del diagnóstico, mostrando pleocitosis mononuclear moderada, glucosa normal y un aumento moderado de las proteínas, pero hasta en un % de los casos el lcr es normal. para el diagnóstico etiológico, la determinación de reacción en cadena de la polimerasa (pcr) en el lcr es la base del diagnóstico para herpes virus, virus de la varicela zoster (vvz), citomegalovirus (cmv) y virus de epstein-barr. la neuroimagen es el segundo pilar diagnóstico. la presencia en la rm cerebral de hemorragia y edema cerebral a nivel del lóbulo temporal es muy sugestiva de encefalitis por herpes simple. tratamiento. un retraso en el tratamiento se asocia a un mal pronóstico, por lo que ante su sospecha debe iniciarse el tratamiento de forma precoz, con aciclovir mg/kg cada horas en caso de sospecha de infección por el virus herpes o vvz. en caso de sospecha de infección por cmv, el tratamiento de elección es ganciclovir mg por kilo cada horas. tras - días de tratamiento sin respuesta positiva se recomienda nueva pcr del lcr, prolongando el tratamiento en caso de ser positivo. las itu constituyen una de las patologías infecciosas más frecuentes tanto en la comunidad como en el ámbito hospitalario, siendo responsables del - % de las sepsis graves que requieren ingreso en la uci. asimismo, son la causa más frecuente de bacteriemia secundaria de origen comunitario. los gérmenes que colonizan la uretra anterior o el introito vaginal ascienden hasta la vejiga dando lugar a la infección. la diseminación hematógena desde otros focos es rara, siendo en este caso stafilococcus aureus el germen más frecuente, seguido de cándida spp.. existen factores patogénicos que modulan el riesgo de itu que dependen del huésped y/o del germen responsable. los factores dependientes del huésped son: alteraciones del flujo urinario, alteraciones químicas u hormonales del epitelio uretral o genital, hábitos higiénicos, cateterismos y manipulación urinaria previas, embarazo y diabetes. en cuanto a los factores dependientes del germen, está la capacidad de adhesión de las bacterias al epitelio mediante los pili o fimbriae, la resistencia a los factores bactericidas del suero y la producción de hemolisinas, dando lugar a una gran virulencia. infección del tracto urinario inferior. aunque habitualmente son de curso benigno, la cistitis enfisematosa puede cursar con un cuadro de shock fulminante. el diagnóstico se realiza mediante prueba de imagen, observándose la presen-cia de gas en la pared vesical. los microorganismos responsables más frecuentes son e. coli y klebsiella, siendo rara la infección por anaerobios. urosepsis altas. suele darse en pacientes jóvenes y, en ocasiones, evoluciona de manera fulminante a shock séptico, con una mortalidad del %. en la pielonefritis aguda, infección del parénquima renal y/o sistema pielocalicial, suele caracterizarse clínicamente por la presencia de fiebre y escalofríos asociados a dolor en fosa renal, habitualmente acompañados o precedidos de síndrome miccional y, con menor frecuencia, náuseas y vómitos. no es infrecuente que en pacientes ancianos la única manifestación sea confusión o letargo. mediante prueba de imagen, ecografía de abdomen o tc se deberá descartar una causa obstructiva subyacente. los gérmenes habituales por orden de frecuencia son: e. coli, proteus mirabilis, klebsiella pneumoniae y enterococcus spp.. en los últimos años se ha producido un aumento en el número de aislamientos de bacterias productoras de betalactamasas de espectro extendido (blee) en las itu comunitarias, principalmente por k. pneumoniae y e. coli. los factores de riesgo para la presencia de blee son: sexo mujer, edad superior a años, diabetes mellitus, itu previas recurrentes, antecedentes de procedimiento invasivo urológico (cateterismo/sondaje) y el tratamiento previo con aminopenicilinas, cefalosporinas y fluorquinolonas . se realizará mediante el análisis del sedimento y la realización de tinción gram y cultivo de la orina. deberá realizarse de forma empírica en espera de los resultados del urocultivo. en infecciones no complicadas: ceftriaxona - g por vía intravenosa cada horas o tobramicina mg por vía intravenosa por kg cada horas. en caso de alergia, ciprofloxacino mg por vía intravenosa cada horas. la pielonefritis aguda deberá tratarse con un carbapenem (imipenen g por vía intravenosa cada horas) y en caso de alergia tigeciclina mg por vía intravenosa cada horas. si existen factores de riesgo para blee, el tratamiento de elección es un carbapenem (imipenen g intravenoso cada horas). las in son infecciones adquiridas durante la estancia en un hospital, no estando presentes ni en periodo de incubación al ingreso del paciente. las in constituyen un problema grave en los smi, conllevando una mayor morbimortalidad, junto con un aumento de los costes (debido a la prolongación de la estancia, gastos de tratamiento, pérdida de productividad del paciente por un retraso en su incorporación a la vida laboral, costes de desplazamiento de los familiares, etc.), junto con el riesgo de generación de gérmenes multirresistentes. el paciente crítico es especialmente vulnerable a la in, estimándose que entre el % y el % de los pacientes sufrirá una infección durante la estancia en la uci, frente a una frecuencia del % en los pacientes ingresados en planta de hospitalización [ ] [ ] [ ] . esta mayor susceptibilidad en los pacientes críticos a la in es debida a la convergencia de varios factores predisponentes: . factores de riesgo dependientes del paciente, como es la inmunosupresión, bien congénita o secundaria a fármacos (quimioterapia, corticoides, tratamiento inmunosupresor, etc.). . alteración de la inmunidad ligada a la patología aguda que motivó el ingreso en la uci. . la necesidad de uso de dispositivos invasivos (intubación, vía central, sonda vesical, etc.) que al romper la barrera natural de defensa facilitan la entrada de microorganismos. . la gran complejidad de estos pacientes, precisando numerosas cuidados y procedimientos, favoreciendo las oportunidades de transmisión cruzada. los gérmenes responsables más frecuentes de la in en la uci, aunque dependiendo del tipo de infección predominarán uno u otro germen, son los microorganismos gram negativos ( , %), seguidos de los gram positivos ( %), hongos ( , %) y otros microorganismos ( %). las infecciones más frecuentes adquiridas en la uci son las asociadas a dispositivos: la bacteriemia relacionada con catéter, la neumonía asociada a la ventilación mecánica (nav) y la infección relacionada con el sondaje uretral. estas tres entidades por su frecuencia y gravedad son motivo de seguimiento en el registro anual de estudio nacional de vigilancia de la infección nosocomial en servicios de medicina intensiva (envin-helics) en las uci españolas . la infección relacionada con dispositivos de derivación externa de líquido cefalorraquídeo es una complicación grave y frecuente en las uci neuroquirúrgicas, por lo que también se abordará en esta actualización. en la figura , se muestran los datos del estudio envin-helics de las infecciones asociadas a dispositivos en las uci españolas . se denomina bacteriemia a la presencia de microorganismos en la sangre. cuando la causa de una bacteriemia es un catéter (bien de origen central o periférico) hablamos de bacteriemia relacionada con catéter. la bacteriemia relacionada con catéter venoso central (brcvc) es la tercera in en frecuencia en el registro envin-helics. en el año supuso el , % de todas la in, con una densidad de incidencia (di) de , bacteriemias por . días de uso de catéter venoso central (cvc). la brcvc puede originarse a partir de tres vías patogénicas que enumeramos a continuación. vía exoluminal. los gérmenes que colonizan la piel penetran a partir del punto de inserción por la superficie exoluminal formando una biopelícula, con posterior diseminación hematógena. este mecanismo es más frecuente en la primera semana tras la inserción del catéter y está relacionada con la esterilidad en el proceso de inserción. la localización del cvc se relaciona con la frecuencia de infección, siendo mayor el riesgo para la localización femoral, seguida de yugular, subclavia y por último en las venas basílicas . los patógenos implicados suelen ser los saprófitos cutáneos, mayoritariamente gram positivos. vía endoluminal. la colonización del catéter se produce a través de las manipulaciones de las conexiones, migrando vía endoluminal formando una biopelícula. la bacteriemia es más tardía, generalmente a partir de las dos semanas de implantación del catéter y está relacionada con la higiene en la manipulación de las conexiones. en estas bacteriemias tardías o cuando la vía es de localización femoral suele haber mayor proporción de bacilos gram negativos y hongos. vía hematógena. es la menos frecuente, produciéndose la colonización del catéter a partir de un foco distal de infección vía hematógena. los patógenos más frecuentes en las brc son los gram positivos ( , %), seguidos de los bacilos gram negativos ( , %) y los hongos ( , %). dentro de los gram positivos los más frecuentes son el staphylococcus epidermidis ( %) y staphylococcus coagulasa negativo ( %). dentro de los bacilos gram negativos, klebsiella pneumoniae es el más frecuente ( , %), seguido de e. coli ( , %) y pseudomonas aeruginosa ( , %). las bacteriemias por hongos son fundamentalmente especies de candida (albicans y parapsilosis con la misma frecuencia y en menor proporción glabrata) . las manifestaciones clínicas pueden ser focales con induración, eritema y dolor en el punto de inserción o trayecto del catéter o sistémicas acompañándose de fiebre, escalofríos e hipotensión. no obstante, las manifestaciones locales son inespecíficas y pueden existir en ausencia de brcvc (sensibilidad menor al %). el diagnóstico de brcvc necesita la evidencia microbiológica de que el catéter es el origen de la bacteriemia y en ello reside la mayor dificultad diagnóstica. la retirada de un catéter implica la necesidad de una nueva canalización, técnica que no está exenta de riesgo. en los pacientes ingresados en la uci es frecuente la presencia de coagulopatía o trombopenia (riesgo elevado de hematoma y sangrado) o accesos vasculares limitados (quemados o accesos trombosados), por lo que en la práctica habitual con frecuencia es necesario mantener el catéter hasta confirmar que este es el foco de la bacteriemia. por tanto, la aproximación diagnóstica se divide en grupos . tras retirada del catéter venoso central. se define bacteriemia o fungemia relacionada con el catéter como el aislamiento del mismo microorganismo en los hemocultivos extraídos por punción en una vena periférica y en el cultivo cuantitativo o semicuantitativo de la punta del cvc retirado, en un paciente con clínica de sepsis, y sin otro foco de infección aparente. en caso de aislamiento de estafilococos coagulasa negativos se requerirá el aislamiento en al menos frascos de hemocultivos periféricos. se considera positivo el cultivo semicuantitativo (técnica de maki) de la punta del catéter si crecen más de unidades formadoras de colonia (ufc) por mililitro de sangre o en el cultivo cuantitativo de la punta (tras sonicación) crecen más de ufc. si no se ha retirado el catéter. se define bacteriemia o fungemia relacionada con el catéter como el aislamiento del mismo microorganismo en hemocultivos simultáneos cuantitativos en una proporción : en las muestras extraídas a través del catéter respecto a las obtenidas por venopunción. dado que este procedimiento es caro y costoso, en la práctica el método más usado es comparar el tiempo de diferencia hasta la positivación, considerándose significativo si la muestra extraída a través del cvc positiviza al menos horas antes que la extraída por venopunción. un % de las brcvc cursan con sepsis y un % con shock séptico. un estudio de casos-controles con datos extraídos del registro envin-uci demostró en pacientes críticos que la adquisición de una brc produce un significativo incremento de la mortalidad y la estancia, con una mortalidad iu-su bod-cvc atribuible del , % y una prolongación de la estancia de días . estos datos han sido corroborados por otros estudios, resaltando la mayor mortalidad y estancia en las bacteriemias por staphylococcus aureus resistente a meticilina . en , peter provonost publicó la experiencia de aplicar un paquete de medidas preventivas en el estado de michigan, consiguiendo una reducción de las brcvc de , por mil días de catéter a , durante los - meses de seguimiento. estos resultados impulsaron la puesta en marcha de un programa a nivel nacional denominado bacteriemia zero . el proyecto se implantó en enero de hasta junio en uci españolas, siendo el objetivo una reducción en la di a o menos episodios de bacteriemias por mil días de cvc (reducción del % respecto a la tasa media de los últimos años). el proyecto contaba con dos ramas: stop-brc y el plan de seguridad integral con el objetivo de crear una red de uci seguras en españa. la rama stop-brc incluía medidas de obligado cumplimiento relacionadas con la inserción y manejo de los cvc: . higiene de manos tanto durante la inserción como en la manipulación de los catéteres para evitar la colonización de la piel, conexiones o el propio catéter. . uso de máximas barreras durante la inserción con medidas de esterilidad máxima, incluido el uso de bata, gorro, guantes estériles y paño que cubra totalmente al paciente. . desinfección de la piel con clorhexidina. . preferencia a la hora de usar la localización en la subclavia. . retirada de catéteres innecesarios. la presencia de un catéter es el principal factor de riesgo para el desarrollo de bacteriemia, por lo que si se puede utilizar otra vía debe retirarse. no se recomienda la sustitución sistémica de los cvc ni el recambio con guía metálica. . manejo higiénico de los catéteres: antes de manipular un catéter (administrar medicación, cambiar sueros, etc.) debe realizarse la higiene de manos y la desinfección de las conexiones con clorhexidina. cambio de las líneas de infu-sión a los días, salvo en las perfusiones con lípidos o hemoderivados en las que se realizará diariamente. el proyecto fue un éxito, consiguiendo una reducción en la di a , bacteriemias por . días de cvc (se pretendía bajar a ), manteniéndose estos buenos resultados a lo largo en la actualidad. así, en el año la tasa de brcvc es de , bacteriemias por mil días de cvc, recomendándose que bz deba ser de obligado cumplimiento en las uci españolas. el tratamiento empírico dependerá de la situación del paciente y del tipo de catéter (fig. ) . los cvc de inserción periférica y los cvc no permanentes se han de retirar siempre que se sospeche que son el foco de origen de la bacteriemia. la existencia de signos locales de infección, aun sin bacteriemia relacionada, también es un criterio absoluto de su retirada. los cvc de uso permanente, sean o no tunelizados, se han de retirar en caso de: . infección persistente del punto de inserción. . signos de infección a nivel del túnel subcutáneo. . bacteriemia producida por ciertos microorganismos: s. aureus, bacilos gram negativos no fermentadores, hongos filamentosos, levaduras y micobacterias. . bacteriemia o candidemia persistente a pesar de haber transcurrido días desde el inicio de un tratamiento antimicrobiano adecuado. la terapéutica antimicrobiana tendrá que basarse en la identificación del agente causal, mediante hemocultivos y según antibiograma. en situación de shock séptico es necesario administrar un tratamiento empírico que ha de incluir antimicrobianos activos frente a los microorganismos gram positivos y gram negativos que más a menudo causan estas infecciones. la flora predominante en el hospital o en una determinada área del mismo y la existencia de patógenos multirresistentes pueden condicionar la elección de la terapia empírica. la cobertura empírica para bacteriemia por hongos deberá realizarse ante la existencia de shock séptico en un paciente crítico con colonización por cándida múltiple previa o en enfermos con procesos hematológicos y neutropenia asociada. la elección de fluconazol (en dosis de mg/día) o de una equinocandina (caspofungina, anidulafungina o micafungina en las dosis habituales) depende de la existencia o no de exposición previa a los azoles por parte del paciente (tabla ). una vez obtenido el germen, la antibioterapia irá dirigida según el antibiograma. en bacteriemia por estafilococo coagulasa negativa, se recomienda la retirada del catéter y valorar tratamiento - días. en las bacteriemias por s. aureus se recomienda la retirada del catéter, si esta opción no es posible, realizar un tratamiento local (sellado con vancomicina) y sistémico durante semanas, pero si persisten los síntomas a las horas se debe retirar el catéter y descartar la presencia de endocarditis o tromboflebitis séptica que, de estar presente, prolongará el tratamiento durante mes. en la bacteriemia por gram negativos se recomienda retirar el catéter y realizar tratamiento durante una semana. en la candidemia se debe retirar el catéter, y el tratamiento debe prolongarse hasta semanas tras el último hemocultivo negativo. debe realizarse un estudio del fondo de ojo para descartar la presencia de endoftalmitis (tratamiento durante semanas). la nav es la segunda infección más frecuente asociada a dispositivos, siendo responsable del % de las infecciones adquiridas en la uci. un % de los pacientes que precisan ventilación mecánica desarrollará una neumonía. en españa, en el año , según datos del registro envin-helics , la di fue de , neumonías por mil días de ventilación mecánica. se define como nav aquella que se desarrolla tras - horas de intubación endotraqueal. la patogenia de la nav h/ivtiene una estrecha relación con el tubo endotraqueal (tet). la nav es más frecuente en los primeros días tras la intubación y existen determinadas comorbilidades (epoc, paciente inmunodeprimido o enfermedad de base grave) o patologías agudas (traumatismo craneoencefálico, coma, sedación profunda o pacientes quemados con síndrome de inhalación) que favorecen el desarrollo de nav . existen tres vías en la patogenia de la nav que se enumeran a continuación. vía aspirativa. por macro-o microaspiración de secreciones procedentes de orofaringe y/o estómago. en el espacio subglótico se acumulan secreciones procedentes de la orofaringe o del tracto gastrointestinal, existiendo una colonización posterior de estas secreciones por gérmenes endógenos o exógenos. la colocación del tet mantiene las cuerdas vocales abiertas y permite el paso de las secreciones subglóticas. además, la pérdida de presión del neumotaponamiento permite el paso de dichas secreciones a la vía aérea inferior. a través del tet, durante la aspiración de secreciones, fibrobroncoscopias o nebulizaciones debido a una inadecuada higiene de manos, deficiente limpieza de los equipos o por condensación de agua en los circuitos del respirador. otras vías. por translocación bacteriana, al existir una disfunción de la mucosa intestinal que habitualmente actúa como barrera de protección entre los gérmenes de la luz intestinal y el torrente sanguíneo, se favorece el paso de bacterias y productos inflamatorios a la sangre. vía hematógena a partir de un foco infeccioso extrapulmonar. el momento del inicio de la neumonía va a influir en el tipo de patógeno y en el pronóstico. en las de inicio precoz (menos de días desde el inicio de la ventilación mecánica), el inóculo bacteriano se implanta en el momento de la intubación, suelen tener mejor pronóstico al ser producida por bacterias sensibles a los antibióticos. en las neumonías de inicio tardío (más de días desde el inicio de la ventilación mecánica) suele producirse por patógenos multirresistentes (bmr) y se asocia con un aumento de la morbimortalidad . no obstante, pacientes con neumonía precoz, pero que han recibido tratamiento antibiótico en los días previos o han estado hospitalizados, tienen un elevado riesgo de estar colonizados o infectados por bmr y deben ser considerados como si tuvieran neumonía tardía . los factores de riesgo para bmr se describen en la tabla . en general, en la nav los gérmenes más frecuentes son los gram negativos ( %), seguidos de gram positivos ( %), hongos ( %) y otros ( %), siendo pseudomonas aeruginosa el patógeno más frecuente ( %), seguido de staphylococcus aureus ( %) y klebsiella pneumoniae ( %). en la neumonía precoz, el patógeno más frecuente es staphylococcus aureus y en las tardías pseudomonas aeruginosa. la clínica consiste en un aumento de secreciones, de aspecto purulento, acompañado de fiebre y leucocitosis, junto con imagen de consolidación en la radiografía de tórax, dando lugar a un empeoramiento en la oxigenación. puede existir hipotensión si se acompaña de sepsis ( %), precisando un soporte hemodinámico si evoluciona a shock séptico ( % de las nav) ( fig. ) . la adquisición de una nav tiene un importante impacto sobre el pronóstico del paciente, asociándose a un aumento de la mortalidad y de la estancia: la mortalidad atribuible es de un %, prolongación de la ventilación mecánica entre y días y de la estancia media hospitalaria de días con un gasto total por cada episodio de nav de . dólares [ ] [ ] [ ] . el diagnóstico requiere la existencia de un diagnóstico clínico junto con la confirmación microbiológica. diagnóstico clínico. presencia de al menos signos (purulencia de las secreciones, temperatura > ºc y leucocitosis > . c/mm o leucopenia < . c/mm ), junto con la aparición de una opacidad en la radiografía de tórax. confirmación microbiológica. cultivo cuantitativo de en el lavado alveolar o de en una muestra de aspirado bronquial. la primera medida es retirar la intubación en cuanto sea posible junto con una estricta higiene de manos y asepsia durante la intubación y posteriores manipulaciones del tubo en ventilación mecánica. además, en la nav existen unas medidas preventivas específicas dirigidas a disminuir la producción, colonización o entrada en el árbol bronquial de las secreciones subglóticas: . cabecero de la cama a º para dificultar el reflujo de las secreciones gastrointestinales y su acumulación en el espacio subglótico. . aspiración de las secreciones del espacio subglótico mediante un tet con canal de aspiración a nivel subglótico, siendo la medida con mayor evidencia científica. . descontaminación de las secreciones subglóticas mediante el uso de antibióticos no absorbibles. su uso se asocia a una disminución de la incidencia de nav y de la mortalidad. el riesgo de seleccionar gérmenes multirresistentes ha dificultado su uso generalizado. . evitar su entrada en el árbol bronquial manteniendo una presión del neumotaponamiemto entre y cm h o. . disminuir el inóculo administrando en pacientes que se intuban en coma un tratamiento antibiótico profiláctico - días. en el año , se puso en marcha el proyecto neumonía zero (nz) , en las uci españolas, con el objetivo de disminuir la di de nav a episodios por mil días de ventilación mecánica (reducción del % respecto a la tasa media de los años anteriores). el proyecto contaba con unas medidas de obligado cumplimiento y otras altamente recomendables (tabla ). el proyecto nz, al igual que fue bz, ha sido un éxito, consiguiendo disminuir (y mantener la disminución en el tiempo) las tasas de nav. actualmente se considera un estándar de calidad smi con tasas por debajo de neumonías por mil días de ventilación mecánica. factores de riego de neumonía asociada a ventilación mecánica por patógenos multirresistentes tratamiento antibiótico en los últimos días nav que cursa con shock séptico sdra precediendo a la nav ingreso hospitalario de ≥ días previo a la nav técnicas de trr previo al inicio de la nav tratamiento antibiótico en los días previos tratamiento antibiótico en los días previos nav: neumonía asociada a ventilación mecánica; sdra: síndrome de distrés respiratorio del adulto; trr: técnica de reemplazo renal. el tratamiento empírico debe cubrir s. aureus, pseudomonas aeruginosa y otros bacilos gram negativos. el antibiótico inicial dependerá de si existe riesgo de infección por patógenos multirresistentes (tabla ): . sin factores de riesgo para patógenos multirresistentes: monoterapia con piperazilina-tazobactan ( g/ horas vía intravenosa) o cefepime o ceftazidima ( g/ horas vía intravenosa) o carbapenen (meropenem g cada horas o imipenen mg/ horas vía intravenosa) o levofloxacino ( mg/ horas vía intravenosa): si existe alergia a betalactámicos (aztreonan g/ horas vía intravenosa o levofloxacino). . si existe riesgo de samr el tratamiento debe incluir vancomicina mg/kg/ - horas o linezolid mg/ horas. . si existe riesgo de pseudomonas multirresistente asociar doble cobertura. . si hay riesgo de blee el tratamiento debe incluir un carbapenem. una vez obtenido el germen y el antibiograma, se desescalará y se realizará antibioterapia dirigida. en los pacientes ingresados en la uci, la itu relacionada con sonda uretral (su) es la infección asociada a dispositivos más frecuente, representando el % del total de las infecciones. su di es de , infecciones por mil días de sondaje . la permanencia de una su altera los mecanismos naturales de defensa del organismo. la su pone en contacto una zona intensamente colonizada como es el periné con una zona estéril como es la vejiga, proporcionando una puerta de entrada de los microorganismos a través de su superficie interna y externa. por otra parte, la su facilita el anidamiento de los microorganismos en forma de biopelícula, proporcionando un entorno favorable y de resistencia a los antibióticos. por tanto, la iu-su se puede adquirir por vías: . vía extraluminal. es la más frecuente ( %), produciéndose a partir de microorganismos endógenos que colonizan el periné o el tracto gastrointestinal. . vía intraluminal ( %). se adquiere por reflujo de microorganismos a través de desconexiones del sistema de drenaje o contaminación de la orina en la bolsa colectora. . la diseminación hematógena al tracto urinario es rara, siendo habitualmente producida por staphylococcus aureus. el factor de riesgo más importante para el desarrollo de iu-su es la permanencia de la sonda. este riesgo aumenta un - % por cada día de sondaje, estimándose que el % de los pacientes sondados desarrollan una iu-su a los días de sondaje. otros factores como sexo femenino, gravedad de la enfermedad de base, cirugía, edad mayor de años, diabetes mellitus, creatinina de m/dl y sondaje tras días de estancia en el hospital se asocian con un mayor riesgo para su desarrollo . cerca de un % de las iu-su se complican con bacteriemia secundaria, siendo factores de riesgo para su desarrollo la presencia de neutropenia, el tratamiento inmunosupresor, el sexo masculino, la infección por serratia y los días de hospitalización previa al desarrollo de la iu-su. los gérmenes más frecuentes son los gram negativos ( %), seguido de gram positivos ( %) y hongos ( %). escherichia coli es el patógeno más frecuente ( %), seguido de pseudomonas aeruginosa ( %), enterococcus faecalis ( %) y candida albicans ( %). los síntomas son variados e incluyen fiebre de nueva aparición, escalofríos, cambios en las características de la orina, hematuria, dolor en flanco o suprapúbico y alteración del estado mental en mayores de años. un % puede cursar con shock séptico y en un % existe bacteriemia secundaria. se basa en la presencia de signos o síntomas compatibles con infección urinaria, sin otro foco infeccioso, y el hallazgo de o más ufc/ml de una o más especies bacterianas en una muestra de orina obtenida a través de la sonda vesical o muestra de orina obtenida a mitad de la micción en un paciente al que se le retiró la sonda en un plazo de horas . entre un - % de las iu-su son evitables. si bien la tasa de mortalidad asociada es muy baja, los costes por episodio son de dólares, ascendiendo a . si se asocia bacteriemia. la mejor prevención es limitar su uso a los casos en los que exista verdadera indicación y su retirada lo antes posible. el tratamiento empírico dependerá del estado clínico del paciente y del riesgo de presentar bmr. en caso de shock séptico o riesgo de multirresistentes, el tratamiento es un carbapenem (meropenem g/ horas intravenoso) más ami-tabla formación y entrenamiento apropiado en el manejo de la vía aérea higiene estricta de manos en el manejo de la vía aérea control y mantenimiento de la presión del neumotaponamiento por encima de cm h o higiene bucal cada - horas utilizando clorhexidina ( , - , %) evitar, siempre que sea posible, la posición de decúbito supino a º favorecer todos los procedimientos que permitan disminuir de forma segura la intubación y/o su duración evitar los cambios programados de las tubuladuras, humidificadores y tubos traqueales descontaminación selectiva del tubo digestivo aspiración continua de secreciones subglóticas antibióticos sistémicos durante la intubación en pacientes con disminución del nivel de conciencia noglucósido (tobramicina mg/ horas intravenoso). en ausencia de las dos premisas anteriores estaría indicada la monoterapia con fosfomicina o un betalactámico antipseudomonas (cefepime o ceftacidima - g/ horas intravenoso-; piperacilina-tazobactan - g/ horas intravenoso-). se debe recambiar la sonda siempre en la infección por cándida y en el resto en caso de que la sonda lleve más de semanas de permanencia. la infección asociada a los sistemas de derivación de lcr externos (dve) es una grave complicación con una elevada morbimortalidad. su incidencia se sitúa entre el - %. la infección puede adquirirse por vía retrógrada intraluminal o por colonización tras la manipulación del catéter por vía extraluminal. son factores de riesgo para la infección las manipulaciones repetidas, los sistemas abiertos y los drenajes de más de días , . las bacterias gram positivas (s. aureus y estafilococos coagulasa negativos) suelen ser la etiología más frecuente, aunque los bacilos gram negativos (pseudomonas aeruginosa, klebsiella pneumoniae, enterobacter y acineobacter baumanii), incluidos los multirresistentes, van aumentando. cursa con un empeoramiento neurológico (alteración del nivel de conciencia o disfunción neurológica), junto con fiebre o febrícula. se basa en las manifestaciones clínicas y el estudio del lcr (citobioquímico y microbiológico). las características del lcr son pleocitosis moderada (con aumento de pmn), aumento de proteínas y lactato y disminución de la glucosa. deberá realizarse tinción de gram y cultivo tanto del lcr como de la punta del drenaje al retirarlo. el tratamiento implica la retirada del drenaje. si es preciso mantener un drenaje se colocará uno nuevo a ser posible por una vía de acceso diferente. la antibioterapia debe ser precoz debido a la elevada morbimortalidad, inicialmente guiada por el gram y posteriormente por el cultivo. si no se dispone de gram; la terapia empírica se realizará con vancomicina más meropenem ( g/ horas intravenoso) o ceftazidima ( g/ horas intravenoso) y en casos de alergia: linezolid ( mg/ horas intravenoso) más aztreonan ( g/ horas intravenoso). la adición al tratamiento sistémico de antimicrobianos por vía intratecal es controvertida, no estando de momento aprobado su uso por la food and drugs administration (fda). en principio se recomendaría su uso en: . infecciones producidas por gérmenes multirresistentes. . el antibiótico penetra mal o no alcanza una adecuada concentración en lcr. . cultivos de lcr persistentemente positivos. . cuando no es posible retirar el dve. . los antibióticos intratecales más utilizados son: vancomicina ( mg/día), aminoglucósidos (amikacina mg/día, gentamicina - mg/día) y colistina ( mg/día) disueltos en ml de suero fisiológico al , %, retirando previamente la misma cantidad de lcr, y manteniendo el drenaje cerrado - minutos. el tratamiento antibiótico deberá mantenerse - días o hasta la negativización de - cultivos de lcr. los autores declaran no tener ningún conflicto de intereses. muy importante ✔ metaanálisis ✔ artículo de revisión ✔ ensayo clínico controlado ✔ guía de práctica clínica ✔ epidemiología grupo de la guía multidisciplinar para el manejo de la neumonía adquirida en la comunidad. guía multidisciplinar para la valoración pronóstica, diagnóstico y tratamiento de la neumonía adquirida en la comunidad neumonía comunitaria grave. estudio descriptivo de años y utilidad de los criterios de la infectious diseases society of america y la american thoracic society en la identificación de los pacientes que requieren ingreso en una unidad de cuidados intensivos infecciones comunitarias que requieren ingreso en uci community-acquired pneumonia requiring hospitalization among u.s. adults advances in the causes and management of community acquired pneumonia in adults managing cap in the icu joint taskforce of the european respiratory society and european society for clinical microbiology and infectious diseases. guidelines for the management of adult lower respiratory tract infections infections in neurocritical care. neurocrit care escmid study group for infections of the brain (esgib). escmid guideline: diagnosis and treatment of acute bacterial meningitis febrile urinary tractinfection in the emergency room european centre for disease prevention and control. point prevalence survey of healthcare associated infections and antimicrobial use in european acute care hospitals estudio nacional de vigilancia de la infección nosocomial en servicios de medicina intensiva. envin helics. informe estudio de prevalencia de las infecciones nosocomiales en españa. informe prevention of intravascularcatheter-relatedinfections central venous accesssites for the prevention of venous thrombosis, stenosis and infection in patients requiring long-term intravenous therapy european centre for disease prevention and control. european surveillance of healthcare-associated infections and prevention indicators in intensive care units. hai-net icu protocol, versión . . stockholm: ecdc morbidity and mortality associated with primary and catheter-related bloodstream infections in critically ill patients the increased risks of death and extra lengths of hospital and icu stayf rom hospital-acquired bloodstream infections: a case-control study an intervention to decrease catheter-related bloodstream infections in the icu reducción de bacteriemias relacionadas con catéteres en los servicios de medicina intensiva de españa mediante una intervención multifactorial. proyecto bacteriemia zero. ministerio de sanidad y consumo impact of a national multimodal intervention to prevent catheter-related bloodstream infection in the icu: the spanish experience infecciones relacionadas con el uso de los catéteres vasculares neumonía asociada a la ventilación mecánica guidelines for the management of adults with hospitalacquired, ventilator-associated, and healthcare-associated pneumonia. the american thoracic society and the infectious diseases society of america management of adults with hospital-acquired and ventilator-associated pneumonia: clinical practice guidelines by the infectious diseases society of america and the attributable mortality of ventilator-associated pneumonia: a meta-analysis of individual patient data from randomised prevention studies mortality, attributable mortality, and clinical events as endpoints for clinical trials of ventilator-associated pneumonia and hospital-acquired pneumonia economic impact of ventilatorassociated pneumonia in a large matched cohort prevención de la neumonía asociada a ventilación mecánica en las ucis españolas preventing catheter-associated urinary tract infections in the intensive care unit infectious diseases society of america. diagnosis, prevention, and treatment of catheter-associated urinary tract infection in adults: international clinical practice guidelines from the infectious diseasessociety of america nosocomial infections in the neurointensive care unit key: cord- - b w l authors: alanazi, khalid h.; killerby, marie e.; biggs, holly m.; abedi, glen r.; jokhdar, hani; alsharef, ali a.; mohammed, mutaz; abdalla, osman; almari, aref; bereagesh, samar; tawfik, sameh; alresheedi, husain; alhakeem, raafat f.; hakawi, ahmed; alfalah, haitham; amer, hala; thornburg, natalie j.; tamin, azaibi; trivedi, suvang; tong, suxiang; lu, xiaoyan; queen, krista; li, yan; sakthivel, senthilkumar k.; tao, ying; zhang, jing; paden, clinton r.; al-abdely, hail m.; assiri, abdullah m.; gerber, susan i.; watson, john t. title: scope and extent of healthcare-associated middle east respiratory syndrome coronavirus transmission during two contemporaneous outbreaks in riyadh, saudi arabia, date: - - journal: infect control hosp epidemiol doi: . /ice. . sha: doc_id: cord_uid: b w l objective: to investigate a middle east respiratory syndrome coronavirus (mers-cov) outbreak event involving multiple healthcare facilities in riyadh, saudi arabia; to characterize transmission; and to explore infection control implications. design: outbreak investigation. setting: cases presented in healthcare facilities in riyadh, saudi arabia: a tertiary-care hospital, a specialty pulmonary hospital, an outpatient clinic, and an outpatient dialysis unit. methods: contact tracing and testing were performed following reports of cases at hospitals. laboratory results were confirmed by real-time reverse transcription polymerase chain reaction (rrt-pcr) and/or genome sequencing. we assessed exposures and determined seropositivity among available healthcare personnel (hcp) cases and hcp contacts of cases. results: in total, cases were identified, involving patients, hcp, and family members across hospitals, an outpatient clinic, and a dialysis clinic. at each hospital, transmission was linked to a unique index case. moreover, cases were associated with superspreading events (any interaction where a case patient transmitted to ≥ subsequent case patients). all of these patients were severely ill, were initially not recognized as mers-cov cases, and subsequently died. genomic sequences clustered separately, suggesting distinct outbreaks. overall, ( %) of hcp cases and ( %) of hcp contacts of cases were seropositive. conclusions: we describe distinct healthcare-associated outbreaks, each initiated by a unique index case and characterized by multiple superspreading events. delays in recognition and in subsequent implementation of control measures contributed to secondary transmission. prompt contact tracing, repeated testing, hcp furloughing, and implementation of recommended transmission-based precautions for suspected cases ultimately halted transmission. facilitate early recognition of suspect cases, followed by implementation of appropriate infection prevention and control (ipc) measures. mers-cov may be transmitted by close contact that likely includes respiratory droplets, but transmission routes are still not fully understood. both the saudi arabia ministry of health (moh) and the world health organization (who) recommend mers-cov-specific precautions for healthcare settings to reduce the risk of healthcare-associated transmission. , from may through june , , hospitals in riyadh, saudi arabia, reported mers cov outbreaks; epidemiologic links between the hospitals were not apparent, and the extent of circulation was unknown. the outbreaks were initially reported by who in july . the moh and us centers for disease control and prevention (cdc) conducted an investigation to describe the scope of healthcare-associated transmission using epidemiologic, molecular, and serologic methods. the investigation was conducted at healthcare facilities in riyadh, saudi arabia, where cases presented: ( ) hospital a, a , -bed tertiary-care moh hospital with intensive-care unit (icu) beds and a busy emergency department (ed); ( ) hospital b, a -bed moh specialty pulmonary hospital; ( ) clinic c, an outpatient clinic; and ( ) an outpatient dialysis unit. we defined a case as any patient with laboratory-confirmed mers-cov infection and an epidemiologic connection to the affected healthcare facilities as a patient, hcp, visitor or family member of a patient from may through june , . laboratory confirmation was performed either at the moh using a rrt-pcr assay targeting both the region upstream of the e gene (upe) and open reading frame (orf) a , or at the cdc by genome sequencing. an indeterminate rrt-pcr result was defined as positive result on only of the gene targets required for confirmation. we defined a superspreading event as any interaction in which a mers case transmitted to ≥ subsequent cases. patients with symptoms consistent with mers and contacts exposed to identified cases were tested. contact investigations were performed by hospital infection control personnel, local public health authorities, and moh personnel. moh recommends mers-cov testing of hcp identified with prolonged, close contact with a mers case (ie, > minutes within . m) if not properly wearing personal protective equipment (ppe). in addition to recommended testing, ad hoc testing of hcp contacts with various levels of exposure and ppe use occurred. we reviewed available medical and public health records for all cases and conducted key-informant interviews with hcp. we collected sera and interviewed available hcp cases and hcp identified as rrt-pcr-negative contacts. interview forms included questions related to demographics, occupation, exposures, ppe use, symptoms, and underlying medical conditions. available mers-cov rrt-pcr-positive samples from confirmed cases collected from may through june , , were stored at − °c and shipped to the cdc for further molecular analysis. sample aliquots ( µl) were extracted on a nuclisens easy-mag (biomerieux, marcy-l'Étoile, france), and µl of total nucleic acid was recovered. the specimen extracts were retested by mers-cov n and/or n real-time rrt-pcr assays, and genome sequencing was performed on positive samples with sufficient viral load using the previously described primer sets and protocol. , the nucleotide sequences were first aligned in mafft version . multiple-sequence alignment software. phylogenetic trees were inferred using the maximum likelihood (ml) method with phyml version . software, assuming a general time-reversible (gtr) model with a discrete γ-distributed rate variation among sites (γ ) and an spr tree-swapping algorithm and visualized using mega version software. serology serum samples were tested at the cdc for anti-mers-cov antibodies using indirect elisas for nucleocapsid (n) and spike (s) proteins followed by a confirmatory microneutralization test (mnt) as previously described. at the optical density cutoffs used by our laboratory, the n elisa has a sensitivity of . % and a specificity of . %, and the s elisa has a sensitivity of . % and a specificity of . % (unpublished data). mers-cov seropositivity was defined as having of positive assays, including n-elisa, s-elisa, and mnt, or positive by mnt alone. indeterminate seropositivity was defined as s elisa positive, but n elisa and mnt-negative. this investigation was determined by moh and cdc to be public health response, not research, and therefore was not subject to institutional review board (irb) review. signed consent was obtained from seroepidemiologic investigation participants. interviews were conducted in arabic, english, filipino, or malayalam. we identified mers cases, including linked to hospital a and linked to hospital b ( fig. and table ). at both hospitals, transmission was traced to a single introduction by the respective index cases (fig. ) . index patient a presented at hospital a on may , and index patient b presented at hospital b on june . no epidemiologic link was established between these cases. respiratory specimens from mers-cov cases were received by the cdc: were confirmed positive by rrt-pcr and positive specimen could not be confirmed by mers-cov n and/or n rrt-pcr assays but was confirmed by sequencing the spike gene. phylogenetic analysis of mers-cov genomes, including complete or nearly complete genomes in this study, showed clustering of the outbreak sequences in lineage within clade b , (fig. ) . the outbreak sequences from each hospital formed a monophyletic group and separated into distinct clusters, suggesting distinct outbreaks. the hospital a cluster appears to have been closely related to camel mers-cov (kt ) and human mers-cov (mg ) sampled at riyadh in and respectively. the hospital b cluster appears to have been more age, y, median (range) ( among cases linked to hospital a, were patient cases, were hcp cases, and were family members (table ) . index patient a was a -year-old factory worker with no history of contact with camels or camel products. he presented to the ed on may with cough, shortness of breath, and chest pain. although er triage was in place, the patient was not initially considered a suspected mers case, and he remained in the ed for > hours prior to transfer to a medical ward (ward a). index patient a was directly linked to subsequent cases: ambulance driver exposed in the ambulance, likely exposed in the ed, and on ward a, where index a was intubated without airborne precautions in place. on hospital day , index patient a was suspected of mers and was transferred from ward a to a negative-pressure room with recommended isolation precautions. index patient a was confirmed rrt-pcr positive for mers-cov on may , and contact tracing began the same day. all secondary transmission at hospital a likely occurred before suspicion of mers in individual cases and the subsequent implementation of recommended transmission-based precautions. may june in addition, secondary cases, cases and , overlapped with index patient a in the ed and were themselves associated with subsequent superspreading events (table ). initially, case was not identified as a contact of index patient a and was suspected to have had community exposure. however, later medical record review demonstrated overlap with index patient a during his initial ed visit for non-mers illness on may . he subsequently visited an outpatient dialysis unit, followed by a second ed presentation with admission to hospital a on june . case was directly linked to secondary cases: patient at hospital a, patients and cleaner at the outpatient dialysis unit, and household contacts. molecular evidence showed that case clustered with index a and other subsequent cases at hospital a. case was a known contact of index patient a in the ed, where he stayed for days before transfer to a medical ward (ward b). he remained on ward b for days, where he developed respiratory distress and was intubated on june . on june , mers was suspected, airborne precautions were implemented, and a sample was obtained for testing. mers-cov was confirmed on june , and the patient died the same day. case was linked to subsequent cases on ward b, including hcp (fig. ) , of whom were present during the intubation procedure on case . of hcp cases linked to hospital a, were available for interview and serum collection. all interviewed hcp cases reported ≥ symptom when tested for mers-cov, with most hcp cases reporting mild upper-respiratory symptoms and/or diarrhea; none developed severe illness, and all survived. of these available hcp, reported prolonged, close contact with an unrecognized patient case before implementation of mers-cov ipc measures and with limited ppe use ( table ). the remaining hcp case cared for a non-mers patient in the same room as a mers patient case. of these hcp cases, reported having been in the same room as a patient case during intubation, and none reported wearing an n mask or a powered air purifying respirator (papr). among the interviewed hcp cases, the time from first positive mers-cov result to serum collection was - days, and was seropositive: a -year-old female who had reported headache, muscle aches, and productive cough. additionally, we interviewed and collected serum from hcp contacts of cases; none were seropositive. among all hcp cases identified at hospital a and the ambulance driver, tested positive on their first rrt-pcr test, and among these , the median time from likely exposure to positive sample collection was . days (range, - days). the hcp cases who did not test positive on their initial test, tested positive on a second or later test, with a median time from likely exposure to first positive sample collection of days (range, - ) (fig. ) . ten cases were identified at hospital b; index patient b and hcp cases who reported direct contact with him. index patient b was a -year-old butcher who slaughtered camels and contacted camel products. on may , he developed fever, cough, and rhinorrhea and presented to clinic c. he was discharged home but returned to clinic c times over days with worsening respiratory symptoms. on june , he was diagnosed with pneumonia and cardiomegaly and was referred to hospital b, where he presented to the ed on june . he was not initially suspected to have mers; however, a chest radiograph revealed bilateral infiltrates and additional history indicated camel contact. he was then placed on isolation precautions, and specimens were collected for mers-cov testing. he was intubated later that day after ipc measures for mers-cov had been implemented, including transfer to a negative pressure room. he died on june . at clinic c, index patient b had hcp contacts, including with close, prolonged contact; no rrt-pcr confirmed hcp-cases were documented at clinic c. of hcp contacts of index b, ( %) were interviewed and had serum collected. among these, hcp were seropositive; both were physicians with initial indeterminate rrt-pcr test results. subsequent rrt-pcr testing was negative, and neither was recorded as a mers case. both cared for index b during multiple clinic visits and reported being within . m of index patient b for < minutes. one reported no ppe use, and the other reported wearing gloves and a surgical mask. both had diabetes, and reported hypertension and smoking. both developed symptoms within - days of caring for index b, including fever. at hospital b, healthcare contacts of index patient b were identified. among them, hcp ( %) tested rrt-pcr positive for mers-cov; reported contact with index patient b before isolation, and reported contact following implementation of ipc measures for mers-cov in a negative-pressure room. of these latter , participated in aerosolizing procedures, including intubation, open suctioning of airways, and/or cardiopulmonary resuscitation. all reported wearing full ppe, including gloves, gown, n mask, and face shield. also, of these hcp, reported visible contamination of gloves or gown by bodily fluids during care of index patient b, who was reported to have had copious respiratory secretions. no transmission to patients or visitors at hospital b was identified. of the hcp cases, were interviewed and had serum collected; all reported close, prolonged contact with index patient b. time from symptom onset to serum collection was - days. among these hcp, were seropositive, and had an indeterminate result. among the seropositive hcps, had been diagnosed with pneumonia, of whom also had diabetes mellitus. the third reported productive cough, dyspnea, and diabetes mellitus. among the with an indeterminate result, reported rhinorrhea and nonproductive cough, and the other had fever and upper respiratory tract and gastrointestinal symptoms; neither had comorbidities. the seronegative hcp-cases reported mild upper-respiratory-tract symptoms; also had fever and gastrointestinal symptoms. all survived, and none were critically ill. at hospital b, of mers-cov rrt-pcr-negative hcp contacts of cases ( %) were interviewed and provided serum. one was seropositive, a physician who had close, prolonged contact with index b after isolation and while wearing recommended ppe; however, he had previously tested rrt-pcr positive for mers-cov in . of hcp cases identified at hospital b, tested positive by rrt-pcr on their first test, tested negative then subsequently tested positive, and had an initial indeterminate rrt-pcr test result (fig. ) . one hcp case with an initial indeterminate result was subsequently confirmed by rrt-pcr, the other was confirmed by genome sequencing. for the hcp cases with a positive rrt-pcr test, the median time from known exposure to positive sample collection was . days (range, - days). a large mers-cov transmission event occurred in riyadh during may-june , with cases initially reported from hospitals. our molecular and epidemiologic investigation demonstrated separate virus introductions at the facilities, each by a single index case. similar to previous outbreaks, , , transmission was characterized by early superspreading events, which led to a rapidly escalating number of cases. during these outbreaks, delays in the recognition and isolation of early cases, along with emergency intubation (sometimes precluding recommended airborne precautions), were associated with superspreading events. cases linked to superspreading events included index cases and secondarily infected hospitalized cases; all had severe illness, low cycle threshold values suggesting high viral loads, and all died. these results are consistent with prior evidence that length of patient hospitalization before isolation and high viral loads have been linked to transmission. although of the cases associated with superspreading events were contacts of index a, they were not detected via contract tracing before developing symptoms and were associated with additional healthcareassociated transmission. the delay in recognition of index patient a due to the patient's comorbidities and complex presentation has been previously described. this case patient was admitted to the ed without respiratory precautions despite initial triage, highlighting the need for strengthening triage practices. the presentation of index patient b before hospitalization is notable; this -year-old male had no known comorbidities and initially presented to clinic c with a mild illness, followed by further visits with worsening respiratory symptoms. furthermore, physicians at clinic c tested seropositive after an indeterminate rrt-pcr test, suggesting transmission at clinic c. thus, increased testing for mers-cov in an outpatient setting for individuals with known risk factors and worsening respiratory symptoms might facilitate early recognition of mers cases. transmission from patient cases to hcp participating in aerosolizing procedures prior to airborne precautions was likely, despite taking recommended airborne precautions and wearing appropriate ppe. mers-cov has been detected in large quantities in respiratory secretions and live virus isolated from environmental surfaces. it is possible that inappropriate use of ppe (eg, insufficient fit testing) or contamination of ppe and inappropriate doffing resulted in transmission. transmission to hcp wearing isolation gowns and n respirators during intubation has been observed previously. , hcp should ensure appropriate fit testing and donning and doffing of ppe to prevent mers-cov transmission. among the hcp cases tested by serology, ( %) had no detectable antibodies. the seropositive hcp cases ( %) each had either evidence of pneumonia or symptoms suggestive of lower respiratory tract infection, consistent with previous evidence that hcp cases with lower-respiratory-tract symptoms are more likely to have detectable antibodies. the use of serologic testing to detect unrecognized infections in asymptomatic or mildly symptomatic individuals may be limited. in both outbreaks, rapid identification of contacts, symptom monitoring, and repeated testing allowed for efficient detection of secondary hcp cases and provided information to guide outbreak management. of the hcp-cases, were detected on initial rrt-pcr testing and by repeated rrt-pcr testing, including multiple hcp cases who initially tested rrt-pcrnegative up to days after known case exposure, indicating that asymptomatic or mildly symptomatic hcp may require repeated screening to rule out infection. although we found no evidence of transmission from hcp to hcp, rapid furlough of mers-cov positive hcp is important to avoid exposing susceptible individuals, particularly patients, to mers-cov positive hcp. our investigation had several limitations. complete medical records were not available for all patients. seropositivity may have been a result of unknown exposures outside of this outbreak. although hospitalized patients have been shown to develop mers-cov antibody responses after weeks, mers-cov antibody kinetics over time are not fully understood, particularly in asymptomatic or mildly ill individuals. genome sequencing was limited by sample quality, and full-genome sequences were not available from all patient samples. hcp ppe use was assessed via interview, so errors in recollection may have been incorporated into our data. due to the retrospective nature of this investigation, ipc practices during potential transmission events could not be confirmed by observation. the introduction of mers-cov into healthcare facilities continues to occur, resulting in substantial morbidity and mortality. in these contemporaneous but epidemiologically unrelated outbreaks, superspreading events were associated with extensive transmission and disruptions to hospital operations, including large-scale furloughing of exposed hcp. early recognition of cases, rapid implementation of recommended ipc measures, and aggressive contact tracing and repeated testing are necessary to effectively prevent and interrupt transmission of mers-cov. 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respiratory syndrome coronavirus deletion variant kinetics of serologic responses to mers coronavirus infection in humans acknowledgments. the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the us centers for disease control and prevention.financial support. no financial support was provided relevant to this article. supplementary material. to view supplementary material for this article, please visit https://doi.org / . /ice. . key: cord- - enteev authors: brisse, morgan; ly, hinh title: comparative structure and function analysis of the rig-i-like receptors: rig-i and mda date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: enteev rig-i (retinoic acid-inducible gene i) and mda (melanoma differentiation-associated protein ), collectively known as the rig-i-like receptors (rlrs), are key protein sensors of the pathogen-associated molecular patterns (pamps) in the form of viral double-stranded rna (dsrna) motifs to induce expression of type interferons (ifn ) (ifnα and ifnβ) and other pro-inflammatory cytokines during the early stage of viral infection. while rig-i and mda share many genetic, structural and functional similarities, there is increasing evidence that they can have significantly different strategies to recognize different pathogens, pamps, and in different host species. this review article discusses the similarities and differences between rig-i and mda from multiple perspectives, including their structures, evolution and functional relationships with other cellular proteins, their differential mechanisms of distinguishing between host and viral dsrnas and interactions with host and viral protein factors, and their immunogenic signaling. a comprehensive comparative analysis can help inform future studies of rig-i and mda in order to fully understand their functions in order to optimize potential therapeutic approaches targeting them. rig-i (retinoic acid-inducible gene i) encoded by the ddx gene in the human genome ( , ) and mda (melanoma differentiation-associated protein ) encoded by the ifih gene ( , ) are known as important protein initiators of earliest immune responses to viral infection. a relatively large body of work has focused on understanding their roles in triggering the same innate immune pathway as they indeed share many similarities at a structural and functional level. however, it is becoming increasingly clear that there are unique differences between rig-i and mda , such as their activation mechanisms and contextual functionalities, that need to be considered in order to fully appreciate their individual function. a comprehensive analysis of multiple aspects of rig-i and mda from their evolutionary origins and behavior among different species to their structures and molecular signaling will allow for a more nuanced understanding of their functional purposes. the innate immune response is a combination of non-specific defense mechanisms by the host that are critical for early detection and inhibition of pathogen growth before the adaptive immune response has time to produce proper cell-mediated immunity, such as the development of antibodies and cytotoxic t-lymphocyte responses (ctl) against the invading pathogen and/or the pathogen-infected cells ( ) . cells of the innate immune arm, such as leukocytes and epithelial cells, are able recognize general components of the microbes (e.g., viruses) that are shared among related microbes. these microbial structures are called pathogen-associated molecular patterns (pamps) (e.g., viral dsrna) that are specifically recognized by the cellular pattern recognition receptors (prrs) (e.g., rig-i, mda , or tolllike receptors tlrs) which are then activated (figure ) . the specific signaling mechanisms of rig-i and mda activation will be discussed in detail below. here, the cascade of event leading to ifn production is briefly summarized. upon binding to pamp (e.g., dsrna), the activated rig-i and mda interact with the mitochondrial antiviral signaling proteins (mavs), which forms a multilayered protein complex contain several different proteins ( ) ( ) ( ) ( ) . the mavs complex then catalyzes the interaction of inhibitor of nuclear factor kappa-b kinase subunit epsilon (ikkε) and the serine/threonine-protein kinase (tbk ) ( ) ( ) ( ) , which phosphorylate the transcription factors interferon regulatory factors and (irf and irf ) ( ). phosphorylated p-irf ( ) and -pirf ( ) factors then dimerize and translocate into the nucleus, where they activate the expression of the type interferon genes (ifn : ifnα and ifnβ). ifn proteins are then exported out of the cell to activate ifn signaling cascade by binding to their receptor (the ifnα/β receptor or ifnar) either on the same cells or neighboring cells in an autocrine or paracrine fashion. this results in the production of more ifn (in a positive feedback loop) and a variety of interferon-stimulated genes (isgs), which mediate vasodilation near the site of the pathogen infection and uptake of fluid, recruitment of innate immune cells, such as macrophages, neutrophils, and dendritic cells to the site of the infection that is aided by chemokine gradients to mediate innate immune cell-mediated killing of the infected cells ( ). rig-i and mda appear to differentially induce ifn in response to different viral pathogens ( ), with rig-i generally responding most potently to negative-strand rna viruses, such as influenza viruses ( , ) , bunyaviruses ( , ), filoviruses ( ), and rhabdoviruses ( , ) as well as the positive-stranded japanese encephalitis virus ( ), while mda is activated during infection by positive-strand picornaviruses ( , , ) and arteriviruses ( , ) as well as by hepatitis d virus ( ), kaposi's sarcoma-associated herpesvirus (kshv) ( ). rig-i and mda may also play a role in recognizing non-viral pathogens, as mda has been found to respond to malaria ( ) (figure ) . neither are individually critical in reovirus ( ) and in dengue virus infection ( , ) but the presence of either in combination with toll-like receptor (tlr ) is critical to have effective anti-viral repsonses ( ). each serves an additive role during west nile virus infection ( ), which is likely mediated by the production of multiple pamp species in the infected cells ( ). indeed, rig-i and mda have also been shown to recognize different sections of the same viral genome due to their differing preferences for rna binding ( ), illustrating how rig-i and mda can act both independently and synergistically. this has also been shown functionally in viruses where both rig-i and mda have been found to be essential to induce the necessary levels of ifnβ signaling for antiviral control against paramyxovirus ( , - ) and rotavirus infections ( ). while rig-i and mda participate in the ifn signaling pathway ( ), it is clear from animal modeling that they might be functionally distinct. while c bl/ mda ko mice exhibit no obvious phenotypes ( ), c bl/ rig-i ko have high embryonic lethality as they don't live past weeks of birth and experience growth retardation and liver degeneration ( , ) . furthermore, when rig-i ko mice are back crossed onto the more genetically flexible s strain ( ), these mice can spontaneously develop colitis symptoms ( ). clinical cases with mutations in rig-i and mda have distinct autoimmune presentations, with rig-i mutations being associated with atypical singleton-merten syndrome, while mda mutations have been linked to classical singleton-merten syndrome, aicardi-goutières syndrome, systemic lupus erythematosus, type diabetes and graves disease ( , ) (figure ). there is growing evidence that overt innate-immune interferon signaling plays a critical role in the development of other forms of autoimmune conditions ( ). taken together, this suggests that rig-i and mda may differ significantly in their roles during development as well as in responding to different types of viral infection that is partially dependent on the pamps that are available in any given context. there is also increasing evidence that rig-i and mda have additional distinct molecular functionalities in immune signaling ( ). it is well-established that the interferon regulatory factor (irf) and innate immune nfκb cytokine signaling pathways have many areas of cross-regulation and expression ( ). accordingly, both rig-i and mda have been shown to activate nfκb signaling during rsv infection, but only rig-i appears to act upstream of the canonical iκbα-nfκb pathway ( , ) (figure ) . while both are known to activate nfκb mediated expression of il- and pro-il- β through the interaction of card with bcl ( , ), the independence of mda from the iκbα pathway suggests that it influences nfκb signaling in other as yet uncharacterized ways ( ). a possible explanation for mda 's independence from the iκbα pathway may be that mda -mediated nfκb (but not irf) signaling requires trim , which activates rig-i by ubiquitination (to be discussed in detail below). this potentially implicates trim in other mechanisms besides activating rig-i ( , ). rig-i (but not mda ) also induces inflammasome assembly-mediated cleavage and maturation of pro-il- β by caspase ( , , ) . finally, rig-i has been shown to inhibit rnai complexes mediated by the endoribonuclease dicer, which is encoded by the dicer gene and cleaves dsrna and pre-micro rna into short single-stranded rna fragments known as small interfering rna (sirna) and microrna figure | rig-i/mda signaling pathway rig-i and mda are first activated by recognition of pamp dsrna, which causes them to interact with mavs. following the activation of mavs by rig-i/mda , a molecular cascade involves the interaction of ikkε and tbk , which is followed by phosphorylation of the transcription factors irf and irf , ensure to translocate the phosphorylated p-irf and p-irf into the nucleus, where they dimerize and bind to transcription factor binding sites of the ifnα and ifnβ genes to activate their transcriptions. expression and exportation of these genes into the cellular milieu trigger the ifn signaling cascade in an autocrine or paracrine fashion to induce expression of hundreds of interferon stimulated genes (isgs) and inflammatory genes to confer antiviral resistance. rig-i and mda also activate the nf-κb pathway. rig-i appears to act upstream of the canonical pathway, which results in the translocation of the two functional nf-κb units (p and p ) into the nucleus, while mda appears to affect nf-κb expression independently from this pathway. figure created using biorender software. ( ), by interacting with the probable atp-dependent rna helicase dhx (also known as the laboratory of genetics and physiology lgp protein), which inhibits dicer ( ) as well as the dicer-complex protein trbp ( ) . lgp has been shown to exhibit conflicting effects on rig-i and mda signaling ( ) ( ) ( ) , and future studies are needed in order to clarify these regulatory mechanisms. rig-i and mda are expressed in all cell types ( ) , but are most well-known for their functions inside innate immune cells, such as macrophages, neutrophils, and dendritic cells, as well as in other cells like mucosal epithelial cells. they are classified as atp-dependent dexd/h box rna helicases. their structure figure | venn diagram comparing the signaling and functional similarities and differences between rig-i and mda . is highly helical and consists of two caspase activating and recruiting domains (card) at the n terminus of ∼ amino acids each, followed by a flexible hinge region and the helicase domain that consists of the reca-like hel and hel domains with an atp binding and hydrolyzing domain at their interface ( figures a,b) . in particular, the structure of the atp binding site distinguishes rig-i and mda from other helicase proteins, such as dicer. unlike other dexd/h box helicases where rna binding catalyzes the atp binding site to become structurally organized, the atp binding site in rig-i and mda remains comparatively open and structurally dynamic following rna binding. this is aided by the atp binding site being formed by an interface between the two hel domains, which are relatively far apart ( ) . these structural features are connected by another flexible hinge region to the unique and predominantly β-sheet c terminal domain (ctd), which recognizes and binds to rna ( ) . the ctd in rig-i and mda contains a zinc binding domain that is related to those of the gdp/gtp exchange factors ( ) . each protein also contains a positively charged groove within this domain that recognizes dsrna and this groove is structurally unique in each protein, potentially explaining their different rna binding preferences ( ) . rig-i primarily recognizes short double-stranded rna with ′ triphosphate groups ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , while mda primarily recognizes long double-stranded rna ( ) ( ) ( ) ( ) (to be discussed in detail below.) it is notable in this regard that the hel-ctd motifs adopt different orientations relative to dsrna in rig-i and mda . specifically, the rig-i hel-ctd domain is tilted relative to dsrna with the ctd interacting with the ′ and ′ ends of the dsrna ( ), whereas the mda hel-ctd domain runs parallel to the rna strand (figures c,d) . the series of steps required for rig-i and mda activation have been described in depth elsewhere ( ) ( ) ( ) ( ) ( ) . briefly summarized, these proteins endogenously exist in the cytoplasm of the cell in a phosphorylated and inactivated conformation when they are not activated by pamp (dsrna) ( ) ( ) ( ) (figures a,f) . phosphorylation is mediated at the n terminal card domains (s and t ) of rig-i by pkc-α/β ( , ) and at the c terminal rna interaction domain (s , s , and t ) by ckβ ( ) . on the other hand, mda is phosphorylated at s by riok ( ) as well as by other yet unknown kinases ( , ) . rig-i is also acetylated at k in its c terminal domain that requires deacetylation by hdac to be able to recognize rna in its activated form ( ) . upon recognition of pamp (dsrna), rig-i unfolds into an open and activated state that is mediated by the flexible hinge regions between the card domains and the helicase domain, and between the helicase and the c terminal domain ( , , ( ) ( ) ( ) ( ) (figure b) . on the contrary, there is evidence to suggest that mda has a more dynamic structure ( ) . unlike a model of rig-i activation described above, mda exists in a conformational equilibrium between close and open forms, with close forms favored in the dsrna unliganded state. while not yet formally demonstrated, it is possible that mda may be inhibited in the absence of the dsrna ligand by its structural dynamics, which may prevent strong protein-protein interactions ( figure f) . however, upon binding to dsrna ligand, mda adopts an open and activated form, which is perhaps more conducive for protein-protein interactions (figures g,h) . once the c terminal domains have been de-phosphorylated, the e ubiquitin ligase riplet attaches ubiquitin peptides onto the c terminal domain of rig-i at residues k and k ( , ) . it was previously shown that ubiquitination by riplet was necessary for opening rig-i and for ubiquitination of the card domain ( ) . however, in-situ studies found that dsrna was sufficient to weaken the interaction between purified rig-i c terminal domain and rig-i card domains ( ) and that dsrna was necessary for riplet ubiquitination ( ) , calling into question the sequential order for rig-i activation ( figure c ). following de-phosphorylation of the card domain by the phosphatase pp -α/γ ( ) , this domain is polyubiquinated at k by the e trim ubiquitin ligase ( ) , which itself is activated by caspase ( ) (figure d ). trim interacting with rig-i may also be mediated by their mutual interactions with certain host long non-coding rna (lncrna), which occurs outside of the dsrna recognizing domain in the ctd of rig-i ( ) . a recent study showed that riplet rather than trim was primarily responsible for ubiquitinating and activating rig-i ( ). however, there are several factors to take into consideration with this study. these recent results were obtained using ko t and mouse embryonic fibroblast (mef) cells and that it was not clear whether k ubiquitination occurred at other known lysine sites in rig-i. the question remains whether riplet can ubiquitinate other lysine residues in the absence of trim . additionally, in-situ experiments comparing rig-i ubiquitination by riplet and trim utilized an e enzyme ( ) that had been found to be specific for riplet ( ) . while the e that utilizes trim has not yet been identified, trim has been shown to ubiquitinate rig-i in-situ when a general mixture of e proteins was used ( ) . the protein levels of trim may also have to be at a certain level in order for it to productively ubiquitinate rig-i, as the ubiquitin protease usp deubiquitinates trim at later time points in viral infection ( ) . finally, trim has been found to be essential for rig-i activation and ifn signaling in-vitro and in-vivo. for the former, sirna-mediated knock-down ( , ) , cellular knockout ( ) and inhibition by viral protein ( , ( ) ( ) ( ) ( ) conditions for trim in multiple cell types have been shown to change rig-i cellular localization ( ) and to negatively affect rig-i k ubiquitination, association with mavs and ifn signaling [when the constitutively active rig-i card domain was overexpressed ( , ( ) ( ) ( ) ( ) ( ) or during viral infection ( , , ) ]. viral inhibition of trim may even be a source of a positive selection during the evolution of certain viruses, as ns iav proteins have been found to interact with species specific trim ( ) . for the latter, mefs from trim ko mice have significantly downregulated ifn production upon viral infection ( ) and ko mice for nlrp , which is a competitive interactor with trim to rig-i, show increased interferon production and more resistance to viral infection ( ) . the known contributions of trim to innate immunity have recently been summarized elsewhere ( ) . it is clear that both riplet and trim can mediate k linked polyubiquitination. however, it has also been found that in-situ incubation of purified rig-i card domains with ubiquitin can be activated by free and unlinked k polyubiquitin chains ( ) , calling into question whether trim only attaches k -linked ubiquitin motifs to rig-i-card or if it also catalyzes the formation of unlinked k polyubiquitination chains ( ) . a possible explanation for these differing results is that rig-i has been shown to be covalently k ubiquitinated by trim when analyzed by mass spectrometry from cells ( ) , while experiments that demonstrate non-covalent k ubiquitination are those involve primarily interactions with purified proteins. it has also been recently found that rig-i is k ubiquitinated at k and that it may be functionally redundant to k ( , ) , with their ubiquitination possibly upregulating the k ubiquitination of the other lysine residues in rig-i ( ). however, it is unknown whether trim ubiquitinates k or any of the other rig-i lysine residues. notably, these additional lysine residues in the card and c terminal domains of rig-i and mda are known to be k and k ubiquitinated [which are associated with degradation of rig-i ( , ) and mda ( ) ], but the four listed above appear to be the essential residues for activation of rig-i ( , ) . the presence of k ubiquitin modifications on mda is more controversial. independent studies have found that mda is ( , ) or is not ( ) k polyubiquitinated. it has also been independently found that trim does not affect ubiquitination of mda (without distinguishing between k and k polyubiquitination) ( ) and for trim to increase k ubiquitination ( ) , the only apparent difference in the experimental models being the usage of hek t ( , ) vs. hek ( ) cells. trim has also been recently found to be essential for mda activation by k polyubiquitination at k ( ) . it is clear that additional studies are needed in order to clarify the ubiquitination mechanisms of mda . upon binding to pamp (dsrna), rig-i oligomerizes with other rig-i/dsrna complexes to form helical oligomers ( ) in a : complex using the purified rig-i protein ( ) , where the activating ubiquitin motifs serve as a scaffold to link the oligomers together ( ) . these oligomers have been found to be necessary under normal conditions to activate rig-i. this may be due to the helical structure of the rig-i oligomers closely matching those formed by mavs ( ) , which is known to form filaments in-vitro ( , ) mediated by its own card domains ( , ) . a structural model of mavs activation by rig-i has been proposed of stacking mavs card domains on top of rig-i card domains to extend the rig-i helix ( ) . the minimum length of dsrna found to activate rig-i is base pairs, which is equivalent to the minimum length to facilitate the formation of a -rig-i/dsrna dimer ( ) . that being said, shorter (∼ bp) ′ ppp stem loop dsrna complexes that have previously been used to obtain x-ray crystallographic structures of rig-i interacting with dsrna ( , , ) (figure c ) can also activate ifnβ signaling in cells ( , ) and in mice ( ) . furthermore, a cells that were transfected with rig-i plasmid h prior to rna transfection had a minimum dsrna length of only - bp required for activation ( ) . this indicates that rig-i oligomerization may not be necessary for activation of the ifnβ pathway under some experimental conditions, which need to be further investigated. mda has also been shown to oligomerize to form long rnaassociated filaments in vitro ( , , ) ( figures d, h) , which may be aided by chaperone proteins ( ). given that the k residue found to have been ubiquitinated by trim ( ) is located on the surface of hel , it is possible that k ubiquitin residues may also help stabilize mda -dsrna filaments ( ). however, mda also spontaneously forms filaments and induce mavs to form filaments independently of ubiquitin in-situ. it is also thought that the formation of longer filaments by mda may be mediated by a longer linkage region between card and hel than in rig-i by amino acids (the length of which is wellconserved across species), allowing for the association of more card domains in an oligomer ( ) . the formation of longer filaments by rig-i has been more controversial, giving rise to two alternate models of rig-i activation: formation of individual single unit of rig-i with short dsrna monomers (leaving a free dsrna end, such as a hairpin loop), which then oligomerizes via card tetramerization that is linked by their ubiquitin chains, or filamentation on longer dsrna. like mda , rig-i can form filaments in-situ independent of ubiquitin ( , ) and induces mavs to also form filaments ( ) , and mavs is known to form filaments in-vitro ( , ) mediated by its own card domains ( , ) . however, rig-i filamentation on an rna template (forming "beads on a string") as opposed to smallerscale oligomerization hasn't yet been shown to occur in-vitro. part of the reasons for the suggestion that rig-i was strongly activated by shorter dsrna was based the comparison on mass equivalents of rna species as there were less ′ triphosphorylated ends for longer dsrnas with greater mass than shorter dsrnas with more ′ triphosphorylated ends ( ) . however, when rna species were normalized by molar equivalence, dsrna length appeared to be positively correlated with rig-i signaling ( ) ( ) ( ) , which became insignificant at around bp ( , ) . it is significantly shorter than the length of dsrna that activates mda , which forms filaments on , bp dsrna ( ). the kinetics of rig-i and mda interacting with dsrna (which will be discussed in detail below) might possibly explain the decrease in dsrna length efficiency to activate rig-i as compared to mda , as rig-i seems to first recognize the ′ ppp end before sliding down the length of the dsrna ( ), whereas mda dynamically associates and disassociates along the length of long dsrna ( ). meanwhile, it is still unclear whether rig-i can preferentially be activated by longer dsrna independently of its unknown ability to form filaments in-vitro ( ) . once fully activated and oligomerized, the rig-i card domain can then interact with mavs ( ) ( ) ( ) ( ) (figures e,i) , which is part of a protein complex containing a variety of other cellular proteins ( ) ( ) ( ) ( ) . while the mda card domain has much weaker direct association with mavs than the rig-i card domain, it is sufficient to lead to its activation and potentiates activation of mavs by rig-i ( ), the mechanisms of which have yet to be determined. the activated mavs complex then initiates a molecular cascade which eventually results in expression of ifn ( ) (figure ) . interestingly, full length rig-i, when overexpressed, has been found to associate with mavs in the absence of activating dsrna and the interaction can be ablated by phosphorylation at s and t ( ) , suggesting that the card phosphorylation sites function at least in part to prevent association of the inactive form of rig-i with mavs. furthermore, the crystal structure of the interaction between the rig-i card and mavs card domains shows the rig-i card domain interacting with mavs card domains on the outside of the tetramer and the rig-i card ( - ) domain facing toward the center of the tetramer ( ) (figure e ). nmr solution structures of rig-i card also shows that t (which is required for dephosphorylation by pp -α/γ) is largely buried within the card domain in a section that would be in closer contact with the helicase domains, suggesting that dephosphorylation of t affects an interaction domain between card and the c terminus ( ) . furthermore, nmr of a c terminal construct of rig-i with the card domain shows stable interactions of card and the c terminal domain ( ) . what all this may mean is that, while the card domain of rig-i is somewhat exposed in its inactivated form and therefore can be shown to interact with mavs, full exposure and engagement of both rig-i card domains (card and card ) with the card domain of mavs is necessary in order to induce ifn signaling. the card domains of rig-i also appear to be generally structurally stable, as electron microscopic structures have been obtained of the full length rig-i bound to blunt-ended dsrna showing both card domains exposed ( ) . on the contrary, the card domains of mda may be comparatively more flexible than those of rig-i in order to mediate long mda -dsrna filament formation ( ) . the activated mavs complex induces association of the inhibitor of nuclear factor kappa-b kinase subunit epsilon (ikkε) and the serine/threonine-protein kinase (tbk ) ( - ), which collectively phosphorylate the interferon regulatory factors and (irf and irf ) ( ) (figure ) . ikkε and tbk also interact with a number of other co-factors ( , ) , such as the deadbox helicase (ddx ) ( ) . the activated p-irf ( ) and p-irf ( ) then translocate into the nucleus and dimerize, where they then act as the primary transcription factors for ifnα and ifnβ, respectively. existing evidence suggests that ifnα is more primarily produced in the earliest time points following rig-i/mda activation, while ifnβ is produced later and is responsible for more robust anti-viral control throughout the innate immune response period ( ) . there is also a distinction between innate immune cell types for ifn production, as cells like fibroblasts and conventional dendritic cells produce ifnα and ifnβ ( , ), while neutrophils only produce ifnβ ( ) and plasmacytoid dendritic cells only produce ifnα primarily through the tlr signaling pathways ( , ). signaling through rig-i is also known to be essential for the process of tlrmediated phagocytosis by macrophages ( ) . interferons are then secreted out of the cell, where they bind to their own receptor (ifnar) and activate the janus kinase/signal transducer and activator of transcription proteins (jak/stat) signaling pathways, which result in a positive feedback signaling loop to further increase rig-i/mda expression and activation ( ) and ifn production ( , ) . expression levels of rig-i and mda have consistently been found to be upregulated downstream of type i ( , ) and type ii ( , ) ifn signals. mda upregulation has additionally been found to occur independently of cytokine expression at least during picornavirus infection ( ). one of the most obvious distinctions between rig-i and mda is in the rna species to which they bind for activation (figures , ) . rig-i has the highest affinity for short dsrna that is tri-phosphorylated at the ′ end ( - ), with rig-i having been found to directly interact with the ′ tri-phosphate group of the dsrna ( , ) . while rig-i can bind to ss- ′ tri-phosphorylated rna ( ), rig-i cannot be activated by it ( , , ) , likely due to a conformational need to recognize double-stranded rna. as a result, rig-i is greatly attenuated by a ′ overhang as well as those with a ′ overhanging the ′ tri-phosphate end ( ) . in fact, a single unpaired ′ triphosphorylated nucleotide is sufficient to competitively inhibit rig-i, which has been exploited by rna viruses to evade rig-i recognition and ifn signaling ( ) . the unique preference of rig-i for ′ tri-phosphorylated rna can be explained by the specific orientation that the rig-i c terminus adopts when directly interacting with the ′ tri-phosphate group of the ′ tri-phosphorylated dsrna ( , ) as compared to unphosphorylated blunt-ended dsrna ( ) . the minimally required and exclusionary features of the ′ and ′ dsrna ends for rig-i activation have proven to be complex. certain studies suggest that a ′ diphosphate group is the minimum feature required for rig-i binding and activation, with ′ monophosphate dsrna failing to productively activate rig-i as compared to ′ di and tri-phosphate dsrna ( ) . frontiers in immunology | www.frontiersin.org additionally, rig-i poorly distinguishes between dsrnas with either ′ tri-phosphate and ′ diphosphate group. when the free energies of each interaction are calculated, the affinity for ′ triphosphate being lowered by disassociation of magnesium from the rig-i/dsrna complex. both are significantly more favorable for binding rig-i monophosphate dsrna ( ) . this similarity in affinity appears to be important in the context of infection with viruses that produce ′ diphosphate rnas, such as reoviruses ( ) . likewise, the difference of energic binding between monophosphate dsrna and bi-and triphosphate dsrnas is likely important for distinction between self (host) and non-self (foreign) rna, the mechanisms of which will be discussed in detail below. the atp hydrolysis functions of rig-i have been shown to drive rapid disassociation from certain rna features, such as ′ monophosphate dsrna ( , ) and ′ oh rna ( , ) , which is particularly important for ′ monophosphate dsrna because it is found in mrna after decapping during the mrna degradation process ( ) . on the other hand, other studies have shown that rig-i can interact with monophosphate dsrna to a certain degree, as has been found to be the case for short synthetic dsrna with a ′ and ′ monophosphate group ( ), poly(i:c) digested with rnase iii ( ) [which generates ′ mono-phosphate/ ′ -oh dsrna ( ) ] and hcv rna ( ) and mitochondrial rna [in the p deficient mice ( ) ] digested with rnase l [which produces ′ oh and ′ mono-phosphate dsrna at subnanomolar levels ( ) , as has been found to be the case for hcv rna ( ) .] it appears that the ′ monophosphate is the determinate feature for rig-i activation independently of the ′ or ′ oh group in all these cases. a possible explanation for the discrepancy between the studies was that higher order rna structures might compensate for the less optimal ′ and ′ ends, as monophosphate dsrna that did not contain stem-loop structures did not activate rig-i and rna regions repetitive in certain nucleotides had been found to be critical for rig-i activation ( ) . future studies are required to further characterize the behavior of rig-i with these rna species. as previously mentioned, mda preferentially associates with long dsrna ( ) ( ) ( ) ( ) . the crystal structure and molecular modeling of mda /dsrna complex suggest that it can recognize the entire first turn of the blunt-ended dsrna ( ) in a similar way as lgp can ( ) . like rig-i and mda , lgp belongs to the atp-dependent dexd/h box rna helicases ( ) , which is structurally similar to rig-i and mda but lacks the card domains at the n terminus ( ) . mda has also been found to be activated by the digested products of rnase l specifically from parainfluenza virus ( ) . the presence of certain repetitive rna elements appears to be another contributing factor in determining interaction of rna with rig-i and mda , which has recently been described in detail elsewhere ( ) . while rig-i and mda are mostly implicated in the immune response to rna viruses, it has also been found to be activated by ′ tri-phosphorylated dsrna intermediates generated by cellular rna polymerase iii from at-rich dna sequences ( ) and during infection with epstein-barr virus (a dna virus) ( ) . rig-i has additional binding preferences for certain nucleotide motifs, such as uridine-rich ′ tri-phosphorylated hairpin rna ( ) , synthetic au-rich hairpins ( ) and those naturally found in the genomes of sendai virus defective-interfering (di) particles ( ) , measles ( ) , influenza a virus (iav) ( ) and in kshv rna transcripts ( ) , and poly (u/uc) regions ( ) and poly (a/ag) regions ( ) in the antisense hepatitis c virus (hcv) genome. it is of particular interest that the poly (a/ag) hcv regions are located significantly downstream of the ′ triphosphate group ( ) , thus potentially implicating other parts of rig-i (e.g., helicase domain) as potential rna interacting domains. repetitive rna elements may also be important in allowing for interaction of inhibitory rnas that do not have ′ or ′ features needed for full activation of rig-i, as has been shown to be the case with ga-rich regions in circular longnon-coding rna lnc-lsm b ( ) . these specific interactions explain their primary role as anti-viral receptors, as these viral motifs are mostly not found in cellular rnas ( ) . rig-i and mda have been particularly implicated in their response to rna genomes of viral defective interfering (di) particles, as these defective viral genomes (dvgs) have originally been found to induce interferon signaling ( ) . di particles are produced by many viruses during infection, and while they are similar in many regards to standard viral particles, such as in appearance and composition, they cannot productively infect cells ( ) . this is largely thought to be due to the presence of large and deleterious deletions in the dvg of di particles ( ) . some dvg rnas have also been noted to have "copy-back" motifs in which one end of the genome can base pair with an inverted copy at the opposite end of the genome, which may be due to stalled and aberrant replication ( , ) . copy-back rna motifs specifically seem to be important for rlr activation in that they tend to contain hairpin motifs and ′ tri-phosphate groups, as has been found for sendai ( ) ( ) ( ) , measles ( , ), and chikungunya ( ) dvg rnas in activating rig-i. in the case of iav, dvg rnas might even be more potent activators of rig-i than the full-length viral genome. cells that were blocked from viral protein synthesis experienced rig-i mediated ifn expression when infected with iav stocks grown in chicken embryonic eggs (which produced higher relative quantities of di particles with dvg rnas) but not with iav grown in cell culture, indicating that rig-i activation by the genomes from primarily non-di iav particles may require active viral rna synthesis ( ) . a potential explanation to this observation is that rig-i appears to be activated by the full viral genome via its panhandle structure, the affinity of which is lowered by the presence of mismatched and unpaired nucleotides in this region of the viral genome that is conserved across influenza virus strains ( ) . however, the overall panhandle structure is conserved between dvgs ( ) and the full length viral genome ( ) , and deletions within dvgs are monogenic and internal ( ) . the specific molecular mechanisms of enhanced rig-i signaling by iav dvgs have yet to be elucidated, although the level of exposure of the panhandle may play a role. while the full extent of mda interacting with di rna is currently unknown, mda appears to be more predominantly activated by dvg rna than rig-i specifically in dendritic cells early in the viral infection cycle ( ) , which may be a contributor toward the phenomenon of di particles enhancing dendritic cell maturation ( ) . the comparative abilities for di particles vs. infectious virions to activate rig-i and mda have important implications for understanding viral pathogenesis and for vaccine development. there is a burgeoning interest in this regard, especially in populations which are typically more challenging to achieve successful preventative vaccination, such as elderly populations with iav vaccination ( ) . elderly populations in general do not develop as strong of memory immune responses to vaccines as their younger counterparts ( ) ( ) ( ) ( ) and have been found to have decreased rig-i mediated ifn signaling ( ) . correspondingly, the influenza vaccine has been shown to decrease in effectiveness in older populations as the influenza season progresses ( ) . a di-vaccine that strongly activates innate immune cells and increases the adaptive immune response could therefore potentially boost the immune responses to vaccines in more vulnerable populations. additionally, di particles have shown to be an important contributor of viral persistence ( , , ) . this raises the question of whether a viral infection may alternate between producing primarily infectious virions which eventually activates the innate immune response and producing primarily di particles which requires less cellular activity but may initiate an even stronger innate immune response ( ) ( ) ( ) . taken altogether, di particles provide yet another layer of distinction between rig-i and mda in terms of how each recognizes different species of dsrna. the preference for specific rna species by rig-i and mda allow for them to distinguish between viral rna and host rna in most circumstances ( ) , although the specific mechanisms of distinction are not as clear for mda as for rig-i. studies from clinical cases of mda mutations provide contradictory models, with certain mutations found in aicardi-goutières syndrome (ags) increasing mda avidity for self rna ( ) with alu retroelements found to be significantly enriched for interaction with ags mda mutations ( ) . the modification of dsrna by host cells may be a primary inhibitor of mda activation by host rna as knockout of adenosine deaminase (adar ), which weakens dsrna structures, allows wild-type mda to be activated by alu retroelements ( ) . however, other mda mutations decrease affinity for known mda ligands and atpase activity, yet still demonstrate increased ifnβ expression ( , ) . for rig-i, a highly conserved residue in the c-terminal rna binding pocket (h ) has been found to sterically exclude canonical self-rna by the means of the n - ′ o-methyl self-rna motif, also known as cap rna ( , ) . this results in a low binding affinity of rig-i to cellular cap rna and decreased atpase activity as compared to pamp (dsrna) ( , ) . flaviviruses take advantage of this precise discrimination by encoding a viral ′ -o-methyltransferase capable of n - ′ o-methylating its positive-strand rna genome in order to evade rig-i recognition and ifn activation ( ) . conversely, the mutations e a and c f found in the rig-i protein in patients with auto-immune disorder singleton-merten syndrome confer the ability of the protein to recognize cap rna and become activated by atp dependent and independent mechanisms, respectively ( ) . furthermore, the e q mutation of rig-i, which was designed to constitutively bind atp, was found to increase the affinity of rig-i with ribosomal rna ( ) . it is noteworthy that host rna contains additional internal rna modifications and non-watson-crick base pairing which can inhibit activation of the other known dsrna-sensing protein, the interferon-induced double-stranded rna-activated protein kinase (pkr) ( ) , and it is known that synthetic ′ triphosphorylated rna containing pseudouridine, -thiouridine or ′ -o-methylated uridine has significantly decreased ability to activate rig-i ( ), which has been demonstrated to occur by preventing rig-i filament formation in-situ ( ) . n- -methyladenosine (m a) nucleotides, which are well-known nucleotide modifications among viruses ( ) , have also been found to ablate dsrna binding to rig-i ( ) . it has been demonstrated that certain rna-dna hybrid constructs with ribonucleotides at positions and of the dna strand can bind to rig-i and activate its atpase activity ( ) . atpase activity is necessary for full activation of rig-i and expression of ifnβ ( , ) , so the minimum requirement of a motif not found in host rna for atpase activity has significant implications for the distinction between self and non-self rnas. expanding on this observation, exogenous atpase activity may also be sufficient to potentiate rig-i and mda , as lgp atpase mutant mice are significantly more susceptible to viral infection even in the presence of functional rig-i and mda ( ) . however, this model is further complicated by certain rna-dna hybrids that are able to bind rig-i and activate atpase activity, but don't induce ifnβ expression ( ) . it is currently undetermined whether such hybrids can sterically inhibit rig-i due to the presence of mostly dntps or whether they inhibit rig-i in a yet undescribed way. recent kinetic studies of rig-i and mda activation by pamp (dsrna) help illustrate how atpase activity is critical for their function and distinction between host (self) and foreign (non-self) rna. rig-i binding to atp is sufficient for interaction with dsrna ( , ) . rig-i atpase activity is inhibited in the absence of pamp (dsrna) by a helical arm that blocks the atpase site ( ) . upon interaction with pamp (dsrna), the helical arm shifts and the two helicase domains are brought together to form an active atpase site ( ) . rig-i then catalyzes atp to break the ′ ppp dsrna interactions within seconds. atp is then rapidly hydrolyzed to facilitate translocation of rig-i to the opposite dsrna end, after which the rig-i oligomers can form ( ) . on the other hand, atp hydrolysis drives rapid disassociation of rig-i from host rna features. these features include dsrna with a ′ monophosphate group ( , ) that is found in mrna after decapping during the mrna degradation process ( ) and ′ overhang rna ( , ) found in mirna ( ) as well as other rna motifs, such as ′ oh rna ( , ) found in bacteria ( ). furthermore, an impaired atpase functionality increases the promiscuity of rig-i binding these host rna motifs ( , , ) . similar atpase functions have been found during mda filamentous formation. the c terminus of mda is critical to form organized helical filaments ( ) and atp binding drives association and hydrolysis and disassociation from foreign dsrna [with little coordination being observed between neighboring mda proteins ( )] in a manner that involves mda twisting along its flexible and hydrophobic interface domains ( ) . taken together, atpase activity may be directed toward rapid disassociation from host dsrna and degradation of rna-dna hybrids, but primarily act on the translocation pathway upon interaction with pamp (dsrna). it is also possible that host and hybrid dsrnas could inactivate rig-i independently of their ability to bind the c-terminus and activate atpase activity. this has been shown, for example, for a hybrid rna that has one strand consists mostly of dna except at positions and , which appears to bind rig-i and activate its atpase activity but doesn't activate ifn signaling ( ) . future studies are needed in order to determine these differential interaction mechanisms. contrary to the traditional paradigm, there is increasing evidence to suggest that rig-i and mda interact with certain host rna motifs, resulting in auto-activation or auto-inhibition of the irf pathway ( figure ) . one of the most strongly supported models is activation by host and viral circular rnas (circrna). originally found in a variety of pathogen genomes, circrnas in eukaryotic cells were first thought to be byproducts of the pre-mrna splicing process. however, they have later been found to be produced by a non-canonical "backsplicing" process and there is increasing evidence to suggest that they play some important regulatory roles ( ) , suggesting that they may have specifically evolved for this purpose. rig-i was first found to interact with circrna produced in situ ( ) . interestingly, the minimum component required for rig-i activation is an intron of pathogenic origin to be spliced out during the circularization process. as human introns have been found to be associated with many rna binding proteins, it is speculated that these proteins may have prevented circularization of this particular synthetic circrna used in this study ( ) and that host rna binding proteins normally prevent endogenous circrnas from being detected by the innate immune system. nevertheless, some viral infections can potentially expose these endogenous circrnas for immune detection, as has recently been found to be the case for a novel host-derived circrna (lnc-lsm b) that is ifn-inducible and shows a down-regulation of its binding to host proteins during viral infection and therefore appears to compete with viral dsrna as an inhibitor of the rig-i signaling feedback loop ( ) . similar inhibitory mechanisms have also been noted for rna products of the exonuclease skiv l ( ) . finally, recent studies have found that hepatitis c virus (hcv) infection increases the expression of certain cellular rnas that can inhibit rig-i function. hcv infection increased the mrna levels of hepatic selenoprotein, which was able to bind to rig-i through a hairpin structure and inactivated it during viral infection ( ) . infection by hcv, vesicular stomatitis virus (vsv), or sendai virus, or direct exposure of cells to type and interferons increases expression of the cellular long non-coding rna (lncrna), namely lncatv, which similarly inhibits rig-i function by directly interacting with it in order to promote virus replication ( ) . in addition to the greatly increased implications of rig-i and mda modulation, these findings also have significant implications in characterizing new biomarkers of disease, as increased serum selenoprotein level has been found to significantly associate with treatment failure of anti-viral drugs in hcv patients, and can possibly explain the increased prevalence of type diabetes in hcv patients ( ) . cellular rna has also been found to activate rlr signaling during viral infection. vault rnas, which are transcribed from four genes and are normally found in large ribonucleoprotein complexes in cytoplasmic "vaults, " are significantly enriched for binding to rig-i during infection with kshv ( ). this may be due partly to viral infection-induced reduction in the level of cellular triphosphatase dusp , which dephosphorylates the ′ ppp group on the vault rnas, as they could only be immunogenic (in the absence of viral infection) by the addition of the ′ ppp group. rig-i and mda have also been found to be activated by rna microparticles produced in situ by rolling circle transcription, generating tandem repeat rna strands ( ) . retrotransposons may also be able to activate both rig-i and mda , as both can be activated by line rna independently of dna sensing mechanisms and retrotransposition ( ) . viral infections can also induce recognition of host rnas. herpes simplex virus (hsv ) infection, for example, has been shown to induce translocation of the host pseudogene rna sp ribosomal rna into the cytosol to bind to rig-i. knockdown of rna sp decreased cytokine signaling during infection with hsv and ebv as well as influenza a virus (iav) ( ) . rig-i has also been found to be activated by hairpin rna structures generated by cleavage of rna by rnase l, which has been demonstrated to occur during hcv infection ( ) as well as from mitochondrial dsrna produced in p deficient mice ( ) . the mitochondria, in particular, may be an important source of immunostimulatory host dsrna. viral infections are well-known to cause mitochondrial damage ( ) . knockdown and hepatocyte-specific conditional ko of mitochondrial rna degrading enzymes resulted in the increase of cytoplasmic mitochondrial dsrna which was able to activate mda ( ) . additionally, extracellular vesicles (ev) secreted by apoptotic endothelial cells were found to contain long interspersed nuclear element (line) and short interspersed nuclear element (sine) rnas that are products of rna polymerase iii and were able to activate rig-i signaling ( ) . collectively, these findings demonstrate the many unique ways by which cellular rnas can modulate rig-i and mda functions as well as the potential implications of rig-i activation by pharmaceuticals as an anti-viral or generalized immunotherapy, though much caution and studies would still be needed to determine the appropriate levels of rig-i and mda activation. given that rig-i and mda are critical for activating expression of ifn during viral infection, there is much interest in studying the interactions of these cellular proteins with viral factors (rnas or proteins), as the ability to modulate interferon expression is a major evolutionary driving force in viral evolution ( , ) . there are many mechanisms viruses have evolved to evade rig-i and mda signaling, which have been discussed at length elsewhere ( , ) . such mechanisms are of particular importance to segmented rna viruses, providing potentially more dsrnas for rig-i and mda activation ( ) . iav and the other orthomyxoviruses are unique in that they replicate in the nucleus of the cells ( ) , preventing the viral rna from being detected by the prrs. however, recent preliminary evidence seems to suggest that rig-i may also endogenously be present in the nucleus and performs similar viral rna binding and activation of the ifn pathway ( ) , yet this finding has yet to be replicated by other laboratories. there is also increasing evidence to suggest that rna processing is another mechanism of immune modulation. certain bunyaviruses can cleave the ′ tri-phosphate group from their genomic rna ( ) in order to avoid immune detection. rig-i has also been found to be subjected to negative modulation by rnai during iav infection ( ) . on the contrary, nucleoproteins from the sendai virus ( ) regulate the number of di particles being produced, and iav nucleoproteins also regulate the production of abortive replication rna ( ) , mini viral rnas ( ) and dvg rna ( ) , all of which are immunostimulatory. the semliki forest virus (sfv) polymerase has even been found to convert host rna into ′ -ppp dsrna to induce ifn expression ( ) . this raises an intriguing possibility that induction of ifn may actually benefit some viruses under certain circumstances despite ifn signaling negatively regulating viral replication. the viral rna levels and localization throughout the viral life cycle might also play an important role in immune evasion ( ) . control of viral rna levels by viral exoribonucleases in particular illustrates the complicated balance between viral production and immune evasion for optimal viral propagation, as has found to be the case for arenaviral nucleoproteins (nps) ( , ) and nonstructural proteins found in coronaviruses ( , ) . finally, viral infection has the capability to disrupt processes of the cell's basic functions, such as transcription and translation, thereby affecting viral replication and immune signaling in complicated ways ( ) . one of the most significant ways viruses modulate rig-i and mda signaling is through their viral proteins ( ) (figure ) . the respiratory syncytial virus (rsv) non-structural protein (ns ) protein and the z matrix proteins of pathogenic arenaviruses interact with the rig-i card domains to block its interaction with mavs ( , ) . the hsv deamidase ul specifically targets rig-i through its helicase domain, abrogating its ability to bind to rna ( ) . the iav polymerase components also interact directly with rig-i ( ), though their biological significance has yet to be determined as they don't significantly affect ifn production. on the other hand, rna binding appears to be an important bridge between the interaction of rig-i with other viral proteins, as the nucleoproteins (nps) of iav ( ) and arenaviruses ( , ) both interact with rig-i through viral rna. the ns protein of rotaviruses targets rig-i for degradation that is independent of proteasomes ( ) . the v protein of paramyxoviruses inhibits mda ( ) by targeting a unique feature of the atp binding pocket in mda ( ) and by inhibiting mda card dephosphorylation ( ) , but can also inhibit rig-i by interacting with the card domain to prevent its ubiquitination by trim ( ) . finally, the us protein of hsv ( ) and the arenaviral z matrix proteins ( ) directly interact with and inhibit rig-i and mda in a similar fashion. there are also many other viral proteins that can regulate proteins in the rig-i and mda pathways, which have been discussed in detail elsewhere ( , , , , , ) . it is important to consider the different regulatory mechanisms of rig-i and mda when considering their different functionalities (figures , ) . one of the key differences between these proteins is in their post-translation modifications ( ) . ubiquitination of rig-i is necessary for its activation ( ) and is a point of negative regulation by host proteins ( , , ) , viral proteins ( , , ) and ubiquitin mimics ( ) as well as positively regulated by influenza b ns protein ( ) and another ubiquitin mimic ( ) . on the contrary, mda is more well-known to be negatively regulated by ubiquitination ( ) , with positive regulation by k ubiquitination being more controversial. while the deubiquitinase usp inhibits mda as well as rig-i, it is thought that this may be due to usp directly binding the mda card domain to prevent rna filamentation ( ) . this raises the question of how rig-i can maintain its stability outside of the proteasome, as ubiquitination at other lysine residues in rig-i besides k induces proteasomal degradation ( ) ( ) ( ) . this proteasomal degradation may be mediated by a p autophagic complex that associates with lrrc /isg ( ) and sqstm ( ) and also mediates mitophagy and downregulation of mavs signaling during measles virus infection ( ) . one key observation is that, while both rig-i and mda are cleaved during picornavirus infection, this cleavage is mediated by the viral proteinase c pro ( ) and is independent of the proteasome ( ) for rig-i, whereas it is mediated by cellular caspases and the proteasome for mda ( ) . mda is also cleaved by caspases during apoptosis ( ), though it hasn't been shown whether this is mediated by mda 's ubiquitination sites. the ubiquitin linkage site may be a determinate of function, as the ubiquitin ligases rnf ( ) and stub ( , ) have been shown to negatively regulate rig-i catalyzed k linked ubiquitination as opposed to the known k -linked ubiquitination at the k , k and k activating sites, and rnf has also been proposed to k ubiquitinate rig-i ( ) (though it hasn't been shown directly) ( ) . trim has also been shown to negatively regulate rig-i and mda by k and k ubiquitination ( ) . substantiating the possibility that k ubiquitination on rig-i may be functionally distinct from its other ubiquitination sites by protecting it from degradation is the finding that the ns protein of west nile virus (wnv) targets both rig-i and mda for degradation by proteasomes. additionally, ns inhibited k ubiquitination of rig-i, but mda was not found to be k ubiquitinated ( ) . heat shock protein -alpha (hsp ) has been found to protect rig-i from proteasomal degradation, but it is unknown which type of ubiquitination that is inhibited by hsp ( ) . taken together, the experimental evidence suggests that rig-i may be protected from proteasome degradation despite its activating ubiquitin moieties ( ) . this warrants further studies for mechanistic elucidation. rig-i and mda additionally interact with different cellular co-factors, contributing to their differential regulations of function. rig-i is well-known for being potentiated by proteins that also bind dsrna, such as (pact) ( , ) , which was first discovered as a protein activator of pkr, the serine/threonineprotein kinase (tbk ) ( ) ( ) ( ) ( ) ( ) and the oligoadenylate synthetase l (oasl) ( ) . pact in particular has some functional similarities to rig-i, as they each contain three distinct rna binding domains ( ) and interact with many of the same cellular co-factors, such as pkr ( ) and dicer ( , ) . because of the important role of pact in augmenting rig-i function, it is a prime target for inhibition of rig-i signaling by several viral proteins from diverse families of viruses ( ) ( ) ( ) , the molecular mechanisms of pact inhibition by these viral proteins can vary and still need to be characterized in detail in future studies. similarly, the host ribonucleoprotein raver can increase affinity of mda for dsrna ( ) , and the zinc-finger protein zcchc has recently been found do so for both rig-i and mda ( ) in similar mechanisms to the other known rna-binding proteins. on the contrary, the human hemoglobin subunit beta (hb) has recently been suggested to decrease mda signaling by competing for long dsrna, while hb can enhance rig-i signaling by increasing k ubiquitination on rig-i ( ) . several host factors interacting with rig-i and mda do so by yet undescribed mechanisms. pkr [which is also activated by pact ( , ) and is sequestered by the cellular helicase dhx protein to form stress granules ( , ) along with rig-i ( , ) and trim ( )] appears to have a novel and yet uncharacterized function in enhancing mda -dependent mavs signaling that is dependent on the kinase activity of pkr ( ) . additionally, the porcine interferon-inducible oligoadenylate synthetase-like protein (poasl) has also been found to interact with and inhibit mda by an unknown mechanism ( ) . the rig-i card domain interacts with mavs to induce interferon signaling, so proteins that disrupt this interaction [as it has been proposed for the atg and atg autophagy proteins ( ) ] can specifically inhibit rig-i signaling. however, other cellular proteins, such as the complement protein gc qr ( ) and tarbp ( ) that interact directly with mavs, inhibit both rig-i and mda . lactate and hexokinase have also recently been found to inhibit rig-i and mda by interacting with mavs, which may be significant in explaining the interplay between metabolism and immune signaling as glycolysis was found to be greatly decreased upon rlr signaling ( ) . likewise, cellular proteins, such as nlrc ( ) that interacts with the rig-i and mda card domains have been shown to block interaction of both rig-i and mda with mavs. contrarily, dhx has been identified as a rig-i cofactor that interacts with the rig-i card domains and with pamp (dsrna), thereby increasing rig-i atpase activity ( ) . additionally, adp-ribosylation factor proteins can block rig-i and mda from interacting with pamps and thereby inhibit their activation ( , ) . lastly, the green tea molecule egcg has also been shown to inhibit the atpase function of rig-i ( ) . the similarities and differences between rig-i and mda modulations and signaling are complex and will need to be elucidated further in future studies. despite their structural and mechanistic differences, it is important to emphasize that existing phylogenetic analysis indicates that rig-i and mda come from a common origin that is also shared among several other protein families (figure ) . the linkage of the helicase and dexd/h box protein appear to be ancient, as orthologs of these proteins are found in the archaea kingdom ( , ) . mda orthologs are found in most vertebrates ( ) , while rig-i orthologs are only found in mammals, ducks, geese and some selected fish and reptiles ( , ( ) ( ) ( ) ( ) ( ) ( ) ( ) (figures , ) . it is therefore likely that mda evolved first, perhaps from a common ancestor with the closely related lgp helicase family ( ) , which is structural similar to rig-i and mda but lacks the card domains at its n terminus ( ) . lgp orthologs are also only found in vertebrates while the next closest related family of proteins (dicer) are more ancient proteins. it has therefore been proposed that the rig-i helicase-dexd/h complex may have been duplicated from mda in the common ancestor of vertebrates ( ) . the association of the two card domains appears to have followed, as individual card domains are found in a variety of vertebrates that also encode caspases ( , ) , but only rig-i, mda , and certain members of the nacht family of ntpases ( ) have two card domains. phylogenetic analysis has shown that the helicase-dexd/h and card have strong co-evolution history ( , ) , while card has evolved more independently ( ) . card appears to have been grafted onto the rig-i helicase-dexd/h complex first, with the card -mda being duplicated from this event. finally, card was grafted onto the card -helicase-dexd/h complex in separate events for rig-i and mda ( ) . in mammals, positive selection can be seen in the flexible hinge region connecting the card domains to the helicase in rig-i and mda . rig-i contains an additional site of positive selection within the hel structural motif (n ), while most of the unique positive selection sites for mda are in regions specific to it, including a amino acid insertion in hel ( ) . while rig-i and mda may both originate from common ancestors of vertebrates, there is increasing evidence to suggest , and related dexd/h-box helicases are shown as a phylogenetic tree, along with their lowest level of biological taxonomy that these proteins are found in present day. in short, the precursor of the mda helicase-ctd likely originated from a common ancestor with the precursor for lgp , which was then duplicated to create the helicase-ctd precursor of rig-i in the common ancestor of vertebrates. card was then grafted onto the helicase-ctd protein, and this protein was duplicated to create the card -helicase-ctd precursor of mda . finally, card was grafted onto these proteins in separate events, forming the modern-day rig-i and mda . that proteins with similar functions may have evolved separately in other species from ancient helicase-dexd/h proteins, implicating rna-mediated defense responses as a potentially universal biological function. a rig-i homolog has recently been found in a planarian that is able to activate downstream inflammatory genes in the absence of the traditional card domains ( ) , and a similar homolog in caenorhabditis elegans has been proposed to mediate anti-viral rnai by complexing with dicer and catalyzing their translocation on the viral genome ( ) . additionally, insects have been found to primarily respond to rna viruses by rnai mediated by dicer proteins ( ) . dicer may potentially mediate dsrna-activated anti-viral signaling pathways that is independent of rnai pathways, as has been found to be the case for the expanded cag-repeat dsrna ( ) . pattern recognition receptors (prrs) that respond to viral rna have not yet been found outside of the animal kingdom, as rlrlike proteins in prokaryotes do not have card domains and the prrs in plants found so far are surface-receptor kinases that respond to external molecular elements of bacteria ( ) [similar to the mammalian toll-like receptors (tlrs)]. however, rna silencing has been demonstrated to be an important anti-viral strategy in plants ( , ) and certain arabidopsis mutants appear to be more susceptible to infection by rna viruses ( ) . rig-i ( ) and mda ( , ) are known to influence antiviral signaling in zebrafish (danio rerio) and other fish species ( , ( ) ( ) ( ) through the canonical mavs signaling pathway. fish rig-i like receptors (rlrs) have been shown to be regulated by the expression of alternate splicing isoforms ( , ) , which have also been found to occur with a dominant-negative splice variant of the human rig-i ( ). rig-i and mda have also been found to participate in anti-viral signaling in ducks ( ) ( ) ( ) ( ) and geese ( , , ) , and mda alone in chickens ( ) ( ) ( ) and other birds ( ) . the observation across species of rlr's performing compensatory mechanisms when a function or a pathway protein is absent is reiterated in birds, as mda has been found to sense short and long dsrna in chickens ( ) and in the chinese shrew ( ) , both of which lack rig-i. additionally, trim activates rig-i in ducks ( ) and in the chinese goose ( ) in the absence of the k activating ubiquitin binding site that is conserved in primates and some rodents ( ) . finally, the rainbow trout (oncorhynchus mykiss) has been found to express a lgp variant in addition to the canonical lgp that contains an incomplete c-terminal domain of rig-i ( ). the differential presence of prrs may also influence viral evolution. a mutation in the iav polymerase subunit pb found in avian-adapted h n strains decreases the inhibition of human rig-i function by iav nucleoproteins, which may indicate a differential selective pressure for viruses that propagate in species that don't contain rig-i ( ) . the evolutionary pattern and compensatory mechanisms of rlrs across species implicate them as critical for anti-viral function, and that evolutionary forces drive the available pathway proteins to meet these functional needs. future studies need to be done to further differentiate rlr function among the different species, as this will provide critical information concerning the various methods of disease control by targeting the pathogen by these important host proteins. there is also increasing evidence for other rna-sensing dexd/h helicases serving important roles in anti-pathogen immune sensing, which have recently been reviewed elsewhere ( ) . some rna helicase (ddx) proteins appear to serve as complex proteins upon interacting with viral rna. ddx is a well-known example, being suspected of being a transcription factor for ifn-β ( ), associating with spliceosomes and the stress-induced p-bodies to influence mrna splicing and decay, respectively ( , ) , and interacting with the mavs complex during viral infection conditions ( , ) . in particular, ddx associating with mavs has been found to be important for anti-viral control against several viruses ( ) ( ) ( ) , and since the two ddx homologs are found on the x and y chromosomes, they may contribute to immunological differences between genders ( ). this is a repeated theme, as dhx ( ), dhx ( ) , and a complex consisting of ddx /ddx /dhx ( ) have also been found to associate with the mavs complex to enhance ifn signaling, while dhx interacts with mavs independently of viral infection ( ) . ddx proteins can also activate other proteins in the irf pathway. multiple ddx proteins can interact with ikkε, with ddx being phosphorylated by ikkε to induce irf interaction with the tbk -ikkε complex ( ) , and ddx blocking this interaction to inhibit ifn signaling ( ) . similar control mechanisms have been demonstrated for ddx interacting with viral proteins. for example, ddx has recently been found to associate with arenaviral nps to increase viral rna synthesis and ifn expression ( ) . additionally, the np of the h n iav pandemic strain has been shown to target ddx for degradation as a potential mechanism of virulence ( ) . dhx ( ) and dhx have also been found to activate nfκb and mapk signaling pathways. finally, ddx has been shown to act as a cofactor for rig-i ( , ) and dhx for mda ( ) . taken altogether, these cellular proteins have likely evolved to regulate rig-i and mda signaling from their common dexd/h helicase predecessors. as our capacity to study the molecular mechanisms and to purposefully modulate immune responses increases in specificity, so will our needs to characterize the differences between related immune signaling proteins. the concept of personalized medicine derives from the idea that we can therapeutically intervene in a situation that is designed around the individual's unique characteristics. while this is an achievable realm of medicine in the future, an immediate step is to determine the functions of some critical proteins, such as the rig-i and mda of the innate immune arm. examining their structural and functional similarities and differences at multiple levels will allow for a deeper level of appreciation of these proteins, which may be exploited therapeutically to differentially modulate rig-i and mda signalings by different rna ligands ( , , , ) or other pharmaceutical compounds ( ) rig-i, a human homolog gene of rna helicase, is induced by retinoic acid during the differentiation of acute promyelocytic leukemia cell retinoic acid-inducible gene-i is induced in endothelial cells by lps and regulates expression of cox- mda- : an interferon-inducible putative rna helicase with double-stranded rnadependent atpase activity and melanoma growth-suppressive properties overexpression of helicard, a card-containing helicase cleaved during apoptosis, accelerates dna degradation interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures mechanisms of mavs regulation at the mitochondrial membrane mavs coordination of antiviral innate immunity assembly of the whip-trim -ppp c mitochondrial complex promotes rig-i-mediated antiviral signaling zyxin stabilizes rig-i and mavs interactions and promotes type i interferon response. sci rep ikkε and tbk are essential components of the irf signaling pathway multiple functions of the ikk-related kinase ikke in interferon-mediated antiviral immunity trim in the regulation of the antiviral innate immunity. front immunol molecular mechanisms of dicer: endonuclease and enzymatic activity the rig-i-like receptor lgp inhibits dicer-dependent processing of long double-stranded rna and blocks rna interference in mammalian cells virus sensor rig-i represses rna interference by interacting with trbp through lgp in mammalian cells lgp : positive about viral sensing mda and lgp : accomplices and antagonists of antiviral signal transduction negative regulators of the rig-i-like receptor signaling pathway tissue-based map of the human proteome structural basis for m g recognition and '-o-methyl discrimination in capped rnas by the innate immune receptor rig-i structural basis for dsrna recognition, filament formation, and antiviral signal activation by mda molecular imprinting as a signal-activation mechanism of the viral rna sensor rig-i parts, assembly and operation of the rig-i family of motors the rna helicase rig-i has an essential function in doublestranded rna-induced innate antiviral responses the c-terminal regulatory domain is the rna '-triphosphate sensor of rig-i ′ -triphosphate rna is the ligand for rig-i rig-i-mediated antiviral responses to single-stranded rna bearing '-phosphates nonself rna-sensing mechanism of rig-i helicase and activation of antiviral immune responses cytosolic viral sensor rig-i is a '-triphosphate-dependent translocase on doublestranded rna the structural basis of ' triphosphate double-stranded rna recognition by rig-i c-terminal domain rig-i detects viral genomic rna during negative-strand rna virus infection structural and functional insights into '-ppp rna pattern recognition by the innate immune receptor rig-i the thermodynamic basis for viral rna detection by the rig-i innate immune sensor rig-i atpase activity and discrimination of self-rna versus non-self-rna length-dependent recognition of doublestranded ribonucleic acids by retinoic acid -inducible gene-i and melanoma differentiation -associated gene activation of mda requires higher-order rna structures generated during virus infection mda detects the double-stranded rna replicative form in picornavirusinfected cells visualisation of direct interaction of mda and the dsrna replicative intermediate form of positive strand rna viruses a structure-based model of rig-i activation toward a crystal-clear view of the viral rna sensing and response by rig-i-like receptors pattern recognition and signaling mechanisms of rig-i and mda antiviral rna recognition and assembly by rlr family innate immune sensors post-translational control of intracellular pathogen sensing pathways essential role of the n-terminal domain in the regulation of rig-i atpase activity structural basis for the activation of innate immune pattern-recognition receptor rig-i by viral rna structural and biochemical studies of rig-i antiviral signaling negative role of rig-i serine phosphorylation in the regulation of interferon-beta production conventional protein kinase c-α (pkc-α) and pkc-β negatively regulate rig-i antiviral signal transduction phosphorylation of rig-i by casein kinase ii inhibits its antiviral response riok -mediated phosphorylation of mda interferes with its assembly and attenuates the innate immune response dephosphorylation of the rna sensors rig-i and mda by the phosphatase pp is essential for innate immune signaling antagonism of the phosphatase pp by the measles virus v protein is required for innate immune escape of mda hdac regulates cellular viral rna sensing by deacetylation of rig-i the mechanism of atp-dependent rna unwinding by dead box proteins mechanisms of rig-i-like receptor activation and manipulation by viral pathogens the catcher in the rig-i viral rna detection by rig-i-like receptors mda cooperatively forms dimers and atp-sensitive filaments upon binding double-stranded rna riplet/rnf , a ring finger protein, ubiquitinates rig-i to promote interferon-beta induction during the early phase of viral infection reul is a novel e ubiquitin ligase and stimulator of retinoic-acid-inducible gene-i a distinct role of riplet-mediated k -linked polyubiquitination of the rig-i repressor domain in human antiviral innate immune responses ubiquitindependent and -independent roles of e ligase riplet in innate immunity trim ringfinger e ubiquitin ligase is essential for rig-i-mediated antiviral activity caspase- controls west nile virus infection via the viral rna receptor rig-i the long noncoding rna lnczc h a promotes a trim -mediated rig-i antiviral innate immune response ube d and ube n are essential for rig-i-mediated mavs aggregation in antiviral innate immunity ndr promotes the antiviral immune response via facilitating trim -mediated rig-i activation in macrophages the ubiquitin-specific protease usp promotes rig-imediated antiviral signaling by deubiquitylating trim the mitochondrial targeting chaperone - - ε regulates a rig-i translocon that mediates membrane association and innate antiviral immunity trim modulates type i interferon induction and cellular antiviral response by targeting rig-i for k -linked ubiquitination mechanism of trim catalytic activation in the antiviral rig-i pathway influenza a virus ns targets the ubiquitin ligase trim to evade recognition by the host viral rna sensor rig-i species-specific inhibition of rig-i ubiquitination and ifn induction by the influenza a virus ns protein the human papillomavirus e oncoprotein targets usp and trim to suppress rig-i-mediated innate immune signaling molecular mechanism of influenza a ns -mediated trim recognition and inhibition nlrp regulates anti-viral rig-i activation via interaction with trim reconstitution of the rig-i pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity emerging role of ubiquitination in antiviral rig-i signaling a hierarchical mechanism of rig-i ubiquitination provides sensitivity, robustness and synergy in antiviral immune responses stratified ubiquitination of rig-i creates robust immune response and induces selective gene expression ubiquitin-mediated modulation of the cytoplasmic viral rna sensor rig-i the e ubiquitin ligase trim attenuates antiviral immune responses by targeting mda and rig-i. cell rep regulation of rig-i activation by k -linked polyubiquitination. front immunol the zincfinger protein zcchc binds rna and facilitates viral rna sensing and activation of the rig-i-like receptors west nile virus ns antagonizes interferon beta production by targeting rig-i and mda trim -catalized ubiquitination is essential for mda -mediated antiviral innate immunity structural basis for ubiquitinmediated antiviral signal activation by rig-i mavs self-association mediates antiviral innate immune signaling mavs forms functional prion-like aggregates to activate and propagate antiviral innate immune response structural basis for the prion-like mavs filaments in antiviral innate immunity solid-state nmr resonance assignments of the filament-forming 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noncoding rna neat exerts antihantaviral effects by acting as positive feedback for rig-i signaling differential viral induction of distinct interferonalpha genes by positive feedback through interferon regulatory factor- the interferon response circuit: induction and suppression by pathogenic viruses h n influenza virus-induced mediators upregulate rig-i in uninfected cells by paracrine effects contributing to amplified cytokine cascades fundamental properties of the mammalian innate immune system revealed by multispecies comparison of type i interferon responses retinoic acidinducible gene-i is induced by interferon-γ and regulates the expression of interferon-γ stimulated gene in mcf- cells expression of retinoic acid-inducible gene-i in vascular smooth muscle cells stimulated with interferon-γ ′ -triphosphate rna requires base-paired structures to activate antiviral signaling via rig-i unpaired ' ppp-nucleotides, as found in arenavirus double-stranded rna panhandles, are not recognized by rig-i recognition of ' triphosphate by rig-i helicase requires short blunt double-stranded rna as contained in panhandle of negative-strand virus short double-stranded rnas with an overhanging ' ppp-nucleotide, as found in arenavirus genomes, act as rig-i decoys crystal structure of rig-i c-terminal domain bound to blunt-ended double-strand rna without ' triphosphate antiviral immunity via rig-i-mediated recognition of rna bearing ′ -diphosphates energetics of preferential binding of retinoic acid-inducible gene-i to double-stranded viral rnas with ′ tri-/diphosphate over ′ monophosphate rig-i selectively discriminates against '-monophosphate rna establishing the role of atp for the function of the rig-i innate immune sensor a mammalian pre-mrna ' end capping quality control mechanism and an unexpected link of capping to pre-mrna processing rnase iii: genetics and function; structure and mechanism rnase l releases a small rna from hcv rna that refolds into a potent pamp 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regulate the budding activity of the lassa virus matrix protein rigorous detection: exposing virus through rna sensing gain-of-function mutations in ifih cause a spectrum of human disease phenotypes associated with upregulated type i interferon signaling breaching self-tolerance to alu duplex rna underlies mda -mediated inflammation aicardi-goutières syndrome is caused by ifih mutations autoimmune disorders associated with gain of function of the intracellular sensor mda a conserved histidine in the rna sensor rig-i controls immune tolerance to n - ′ o-methylated self rna highresolution hdx-ms reveals distinct mechanisms of rna recognition and activation by rig-i and mda hdx-ms reveals dysregulated checkpoints that compromise discrimination against self rna during rig-i mediated autoimmunity atp hydrolysis by the viral rna sensor rig-i prevents unintentional recognition of self-rna a brilliant disguise for self rna: '-end and internal modifications of primary transcripts suppress 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inhibit type i interferon production rotavirus non-structural protein antagonizes innate immune response by interacting with retinoic acid inducible gene i paramyxovirus v proteins disrupt the fold of the rna sensor mda to inhibit antiviral signaling paramyxovirus v proteins interact with the rig-i/trim regulatory complex and inhibit rig-i signaling herpes simplex virus tegument protein us downmodulates the rlr signaling pathway via direct interaction with rig-i and mda- host and viral modulation of rig-i-mediated antiviral immunity. front immunol usp inhibits type i interferon signaling by deubiquitinating rig-i-like receptors usp promotes k -linked rig-i deubiquitination and suppresses antiviral immune responses mechanism of inhibiting type i interferon induction by hepatitis b virus x protein porcine deltacoronavirus nucleocapsid protein suppressed ifn-β production by interfering porcine rig-i dsrna-binding and k -linked polyubiquitination ubiquitin-like modifier fat attenuates rig-i mediated antiviral signaling by segregating activated rig-i from its signaling platform robust lys -linked ubiquitination of rig-i promotes cytokine eruption in early influenza b virus infection antiviral activity of human oasl protein is mediated by enhancing signaling of the rig-i rna sensor negative regulation of the rig-i signaling by the ubiquitin ligase rnf induction of siglec-g by rna viruses inhibits the innate immune response by promoting rig-i degradation cytoplasmic stat promotes antiviral type i ifn production by blocking chipmediated degradation of rig-i kai lrrc inhibits type i ifn signaling by targeting isg -associated rig-i for autophagic degradation lrrc modulates type i interferon signaling by restraining the sqstm /p -mediated autophagic degradation of pattern recognition receptor ddx /rig-i mitophagy enhances oncolytic measles virus replication by mitigating ddx /rig-ilike receptor signaling rig-i is cleaved during picornavirus infection foot-andmouth disease virus viroporin b antagonizes rig-i-mediated antiviral effects by inhibition of its protein expression mda- is cleaved in poliovirus-infected cells rnf suppresses antiviral type i interferon production by targeting rig-i cards to mediate rig-i degradation mll suppresses antiviral innate immune response by facilitating stub -mediated rig-i degradation the levels of retinoic acid-inducible gene i are regulated by heat shock protein -alpha pact, a double-stranded rna binding protein acts as a positive regulator for type i interferon gene induced by newcastle disease virus the double-stranded rna-binding protein pact functions as a cellular activator of rig-i to facilitate innate antiviral response tbk -associated protein in endolysosomes (tape)/cc d a is a key regulator linking rig-i-like receptors to antiviral immunity cell type-specific subcellular localization of phospho-tbk in response to cytoplasmic viral dna zika virus disrupts phospho-tbk localization and mitosis in human neuroepithelial stem cells and radial glia zebrafish mvp recruits and degrades tbk to suppress ifn production the role of optineurin in antiviral type i interferon production structural and functional analysis reveals that human oasl binds dsrna to enhance rig-i signaling reversal of the interferon-sensitive phenotype of a vaccinia virus lacking e l by expression of the reovirus s gene human trbp and pact directly interact with each other and associate with dicer to facilitate the production of small interfering rna approaching the rna ligand for rig-i? viral inhibitions of pact-induced rig-i activation hemorrhagic fever-causing arenaviruses: lethal pathogens and potent immune suppressors porcine deltacoronavirus nucleocapsid protein antagonizes ifn-β production by impairing dsrna and pact binding to rig-i raver is a coactivator of mda -mediated cellular antiviral response human hemoglobin subunit beta functions as a pleiotropic regulator of the rig-i/mda -mediated antiviral innate 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signaling pathway via targeting duba in miiuy croaker after poly(i:c) stimulation mda and lgp acts as a key regulator though activating nf-κb and irf in rlrs signaling of mandarinfish higher antiviral response of rig-i through enhancing rig-i/mavs-mediated signaling by its long insertion variant in zebrafish roles of rig-i n-terminal tandem card and splice variant in trim -mediated antiviral signal transduction activation of duck rig-i by trim is independent of anchored ubiquitin expression of immune genes rig-i and mx in mallard ducks infected with low pathogenic avian influenza (lpai): a dataset duck rig-i restricts duck enteritis virus infection in vivo cellular and molecular study on duck spleen infected by duck tembusu virus identification and expression profiling analysis of goose melanoma differentiation associated gene (mda ) gene goose mavs functions in rig-i-mediated ifn-β signaling activation characterization of chicken mda activity: regulation of ifnβ in the absence of rig-i functionality chicken cells sense influenza a virus infection through mda and cardif signaling involving lgp chicken mda senses short double-stranded rna with implications for antiviral response against avian influenza viruses in chicken cloning, expression and bioinformatics analysis of a putative pigeon melanoma differentiation-associated gene loss of rig-i leads to a functional replacement with mda in the chinese tree shrew trim identification in the chinese goose: gene structure, tissue expression profiles, and antiviral immune responses in vivo and in vitro expression and functional characterization of the rig-i-like receptors mda and lgp in rainbow trout (oncorhynchus mykiss) influenza virus adaptation pb - k modulates nucleocapsid inhibition by the pathogen sensor rig-i multiple functions of ddx rna helicase in gene regulation, tumorigenesis, and viral infection rna helicase ddx : at the crossroad of viral replication and antiviral immunity hiv- blocks the signaling adaptor mavs to evade antiviral host defense after sensing of abortive hiv- rna by the host helicase ddx the rna helicase ddx x is an essential mediator of innate antimicrobial immunity dhx pairs with ips- to sense double-stranded rna in myeloid dendritic cells dhx senses doublestranded rna in myeloid dendritic cells ddx , ddx , and dhx helicases form a complex with the adaptor molecule trif to sense dsrna in dendritic cells the interaction between the helicase dhx and ips- as a novel pathway to sense double-stranded rna and rna viruses in myeloid dendritic cells ddx inhibits type i interferon production by disrupting tbk -ikkε-irf interactions and promoting tbk and ikkε degradation ddx suppresses type i interferons and favors viral replication during arenavirus infection codegradation of interferon signaling factor ddx by pb -f as a basis for high virulence of pandemic influenza the deah-box rna helicase dhx activates nf-κb and mapk signaling downstream of mavs during antiviral responses ddx , a dexd/h box helicase, is a novel antiviral factor promoting rig-i-like receptor-mediated signaling ddx is involved in rig-i-dependent and independent antiviral responses, and its function is attenuated by virus-induced egfr activation dhx functions as an rna cosensor for mda -mediated emcv-specific antiviral immunity activation of rig-i signaling to increase the pro-inflammatory phenotype of a tumor immunotherapeutic effects of intratumoral nanoplexed poly i:c rig-i-like receptors as novel targets for panantivirals and vaccine adjuvants against emerging and re-emerging viral infections the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © brisse and ly. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms.